Modifying Lipids for Use in Food

Modifying lipids for use in food Edited by Frank D. Gunstone

CRC Press Boca Raton Boston New York Washington, DC

WOODHEAD

PUBLISHING LIMITED

Cambridge, England

Published by Woodhead Publishing Limited, Abington Hall, Abington, Cambridge CB1 6AH, England www.woodheadpublishing.com Published in North America by CRC Press LLC, 6000 Broken Sound Parkway, NW, Suite 300, Boca Raton, FL 33487, USA First published 2006, Woodhead Publishing Limited and CRC Press LLC © 2006, Woodhead Publishing Limited The authors have asserted their moral rights. This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. Reasonable efforts have been made to publish reliable data and information, but the authors and the publishers cannot assume responsibility for the validity of all materials. Neither the authors nor the publishers, nor anyone else associated with this publication, shall be liable for any loss, damage or liability directly or indirectly caused or alleged to be caused by this book. Neither this book nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming and recording, or by any information storage or retrieval system, without permission in writing from Woodhead Publishing Limited. The consent of Woodhead Publishing Limited does not extend to copying for general distribution, for promotion, for creating new works, or for resale. Specific permission must be obtained in writing from Woodhead Publishing Limited for such copying. Trademark notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation, without intent to infringe. British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library. Library of Congress Cataloging in Publication Data A catalog record for this book is available from the Library of Congress. Woodhead Publishing ISBN-13: 978-1-85573-971-0 (book) Woodhead Publishing ISBN-10: 1-85573-971-2 (book) Woodhead Publishing ISBN-13: 978-1-84569-168-4 (e-book) Woodhead Publishing ISBN-10: 1-84569-168-7 (e-book) CRC Press ISBN-13: 978-0-8493-9148-4 CRC Press ISBN-10: 0-8493-9148-2 CRC Press order number: WP9148 The publishers’ policy is to use permanent paper from mills that operate a sustainable forestry policy, and which has been manufactured from pulp which is processed using acid-free and elementary chlorine-free practices. Furthermore, the publishers ensure that the text paper and cover board used have met acceptable environmental accreditation standards. Typeset by Replika Press Pvt Ltd, India Printed by T J International Limited, Padstow, Cornwall, England

Contributor contact details (* = main point of contact)

Chapters 1, 2 & 7 Professor Frank Gunstone 3 Dempster Court St Andrews Fife KY16 9EU Scotland UK

Chapters 4, 14 & 24 Professor Gudmundur G Haraldsson Science Institute University of Iceland Dunhaga 3 107 Reykjavik Iceland

Tel: +44 1334 479 929 Email: [email protected]

Tel: +354 525 4818 Fax: +354 552 8911 Email: [email protected]

Chapters 3 & 13 Dr Martin RL Scheeder ETH Zurich Institute of Animal Sciences ETH Zentrum LFW 56.1 CH-8092 Zurich Switzerland

Chapters 5 & 23 Professor Colin Ratledge Department of Biological Sciences University of Hull Hull HU6 7RX UK

Tel: +41-44-632 32 78 Fax: +41-44-632 11 28 Email: [email protected]

Tel: +44 1482 465 243 Fax: +44 1482 465 822 Email: [email protected]

xiv

Contributor contact details

Chapter 6 K Warner US Department of Agriculture National Center for Agricultural Utilization Research Peoria, IL USA

Chapter 11 Xeubing Xu BioCentrum-DTU Building 221 Technical University of Denmark DK – 2800 Lyngby Denmark

Email: [email protected]

Tel: 45-45252773 Fax: 45-45884922 Email: [email protected]

Chapter 8 Dr Alejandro G Marangoni Department of Food Science Guelph Ontario, N1G 2W1 Canada Email: [email protected]

Chapter 9 Gary R List 1815 N. University Street Peoria, IL 61604 USA Tel: 309-681-6388 Fax 309-681-6340 Email: [email protected]

Chapter 10 Dr Véronique Gibon Project Manager R&D De Smet Techologies and Services Da Vincilaan, 2, Bus G1 1935 Zaventeus Belgium Tel: + 322 716 1390 Email: [email protected]

Chapter 12 Professor Denis J Murphy Biotechnology Unit School of Applied Sciences University of Glamorgan Treforest CF37 1DL Wales UK Tel: +44 1443 483 747 Email: [email protected]

Chapter 15 Professor Jan Pokorný Department of Food Chemistry and Analysis Institute of Chemical Technology Prague Czech Republic Email: [email protected]

Contributor contact details Chapter 16 Dr HM Premlal Ranjith* and Miss Upuli Wijewardene Diotte Consulting and Technology The Conifers 36 Bishops Wood Nantwich Cheshire CW5 7QD UK

xv

Chapter 19 Dr Earl Hammond Department of Food Science and Human Nutrition Food Sciences Building Iowa State University Ames, Iowa 50011 USA Email: [email protected]

Email: [email protected]

Chapter 17 Dr Gerrit van Duijn* and Eckhard Flöter Unilever Research PO Box 114 3130 AC Vlaardingen Holland Email: [email protected] [email protected]

Chapter 18 Professor William E Artz Dept Food Science and Human Nutrition 382 Agricultural Engineering Sciences Bldg University of Illinois 1304 West Pennsylvania Avenue Urbana, Illinois 61801-4726 USA Tel: (217) 333-9337 Fax: (217) 333-9329 Email: [email protected]

Chapter 20 Staffan Norberg AarhusKarlshamn (AAK) SE-374 82 Karlshamn Sweden Tel: +46 454 829 77 Email: [email protected]

Chapter 21 Dr Christian Gertz Chemiedirektor Chemisches Untersuchungsamt Hagen Pappelstrasse 1 D-58099 Hagen Germany Tel.: +49-2331-2074726 Fax.: +49-2331-2072454 Email: [email protected]

Chapter 22 Kaustuv Bhattacharya International Food Science Centre A/S Herredsvej 60C, Apt 12 8210, Aarhus V Denmark Email: [email protected]

1 Introduction: Modifying lipids – why and how? F. Gunstone, Scottish Crop Research Institute, UK

1.1

Introduction

Annual production of oils and fats was 136 million tonnes in 2004/05 and is forecast to be 141 million tonnes in 2005/06. In the quarter-century 1976– 2000 consumption (virtually the same as production) rose at an average rate of 3.7 %, equivalent to doubling every 20 years or so (Anon, 2005). This marked increase has come mainly from five vegetable oils (soybean, the two products of the oil palm – palm oil and palmkernel oil – rapeseed/canola and sunflower) and to a lesser extent from eight other vegetable oils (cottonseed, groundnut/peanut, sesame, corn, olive, coconut, linseed and castor) and four animal fats (butter, lard, tallow and fish oil). These increases result partly from larger areas being devoted to their cultivation and partly from rising yields. The composition of these oils is described in selected chapters in Part I of this book. It is estimated that ~ 80 % of total oil and fat production is used for food and the balance for animal feed and by the oleochemical industry. The major food uses include frying oils, baking fats, cooking fats, shortenings, spreads, salad oils, mayonnaise, confectionery fats and ice cream. However, there are additional dietary fats not counted by market analysts in their assessment of commodity oils and fats. For example, figures for groundnut (peanut) oil do not include the commodity eaten as nuts, figures for butter do not include milk consumed as such or as cheese, and figures for lard, tallow and fish oil do not include fat consumed when eating meat (beef, lamb, pork, chicken) or fish. The consumption of oils and fats varies considerably between developed, developing and under-developed countries and demand is expected to grow. This is illustrated in the figures for selected affluent and non-affluent countries/

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Modifying lipids for use in food

regions (Table 1.1). Increasing consumption in highly populated countries such as China and India will fuel the demand for oils and fats for many years to come. Apart from low levels of minor oils described in Chapter 2, consumption is confined to fewer than 20 commodity oils. These are not always ideal for food purposes, and have to be modified for their end use. It is useful in this preliminary chapter to consider the important properties required of an oil or fat for its use in foods, and to review briefly the major modifying procedures. The latter will be detailed in chapters in Part II and the application of modified lipids in foods is covered in Part III.

1.2

What properties are desired?

An oil or fat should have the optimum physical, chemical and nutritional properties dictated by its end use. The more important of these properties are indicated below, and some of the topics will be developed in later chapters. However, these factors are not always mutually compatible and compromises have to be made. The best physical and chemical properties may be achievable only at the sacrifice of some nutritional excellence. This provides an important challenge for food technologists, and this book will show how these challenges have been met in the past, how they are being met today and how they may be met in the future. Because nutrition is a developing science new problems appear, and what is practised and accepted today may be less so in the future. This is illustrated in the present concern about fats containing unsaturated acids with trans configuration. These are produced (among other ways) by partial hydrogenation, a process developed and exploited throughout the 20th century. Only in recent years have there been nutritional concerns about such acids that are now being addressed with greater or lesser urgency.

Table 1.1 Consumption of oils and fats for food and non-food purposes in selected countries in 1990/2000 and 2004/05. World

USA

EU-25

China

India

Personal (kg/person/year) 1999/00 2004/05

18.4 21.0

49.9 49.0

43.2 50.2

13.6 19.6

11.4 11.7

Total (million tonnes/year) 1999/00 2004/05

113.4 136.4

15.7 15.5

17.1 17.4

14.7 18.0

6.6 8.0

Source: Anon (2005).

Introduction: Modifying lipids – why and how?

3

1.2.1 Physical properties The most important physical properties of oils and fats for the food industry are thermal properties associated with crystallization and melting, with the formation of solids and liquids and with the behaviour of plastic fats that are mixtures of solid and liquid components. It is mandatory, for example, that salad oils do not contain lipids that will crystallize during storage in a refrigerator. Most frying oils and oils used as food coatings (and lubricants) should also be free of solid components. Few natural oils meet this requirement and appropriate modification has to be carried out. The successful production of spreads, on the other hand, depends on having appropriate levels of solid fat at refrigerator temperature, at ambient temperature and at mouth temperature. The solid fat content at 4 °C should not exceed 30–40 % if the fat is to be spreadable from the refrigerator; at 10 °C it should be 10–20 % so that the fat will ‘stand up’ (not collapse to a puddle of oil); and at mouth temperature the spread should melt completely to avoid a waxy mouth feel. Crystalline triacylglycerols, such as those present in spreads, show polymorphism. They exist in different crystalline forms with differing thermal properties and may change – quickly or slowly – from a physically less stable to a more stable form. It is important that solid triacylglycerols in a spread are in the most appropriate form (β′) and that this will not change during storage to the more stable β-form. β′-Crystals are relatively small, can incorporate large volumes of oil (liquid) and give the product a glossy surface and a smooth lustre. β-Crystals, on the other hand, though initially small, grow into needle-like agglomerates less able to incorporate oil and producing a grainy texture. Oils comprising acids of mixed chain length (generally C16 and C18) are more likely to remain in the β′ form while those containing almost entirely C18 acids are β-tending. 1.2.2 Chemical properties Food lipids are not usually considered to require defined chemical properties, but without oxidative stability of their lipid components foods would quickly become rancid and have a short shelf life. For this reason they should be oxidatively stable. An account of the deteriorative oxidation processes and of conditions under which these changes may be inhibited is an important part of Chapter 7.

1.2.3 Nutritional properties The selection and use of oils and fats in foods is strongly influenced by a range of nutritional properties. •

The total level of fat in a food with its marked effect on calorific value is important to consumers and has led to a demand for foods with lower fat content or for the use of fat or fat substitutes with reduced calorific value.

4

Modifying lipids for use in food

•

An appropriate balance between saturated, monounsaturated and polyunsaturated acids is desirable. There are many dietary recommendations given in terms of numbers (energy %) of total fat and of these three types of acids. This makes for simple messages that can be understood by the consumer and for relatively simple labelling. It has been argued, however, that this message is too simple and can be misleading (Gurr, 1999; Taubes, 2001). Saturated acids represent a group of acids that are of concern because they raise serum cholesterol levels. They can be described as cholesterolraising. The human diet contains saturated acids with 4–24 carbon atoms of which palmitic acid (16:0) dominates. Starting with Ancel Keys in 1957 (he died in November 2004, just two months short of his 101st birthday) several equations have been produced to correlate changes in serum cholesterol levels with changes in fatty acid dietary intake. Early equations took account only of changes for total saturated acids and total polyunsaturated acids, but the most recent include information for changes in (only) three individual saturated acids (lauric, myristic and palmitic), oleic acid, trans acids from partially hydrogenated vegetable oils and from partially hydrogenated fish oils and linoleic and linolenic acids taken together (Pedersen et al., 2001). This suggests that saturated acids outwith the 12:0–16:0 range are not cholesterol-raising. Among monounsaturated acids a clear distinction must be made between cis and trans isomers with the former being cholesterol-lowering and the latter cholesterol-raising. As is apparent in later chapters, much effort is now being put into producing high quality spreads with levels of trans acids approaching zero. There is also a growing concern about the quota for polyunsaturated fatty acids. In particular, the ratio of omega-6 to omega-3 acids should be between 5 and 10 to 1 (or less) but exceeds 25 in many countries. Since the presence of α-linolenic acid leads to oxidative instability and reduced shelf life, there is a need for development of more efficient antioxidant systems. Related to this is the efficiency or otherwise of the elongation–desaturation systems required to metabolize linoleic acid and linolenic acid to their very important C20 and C22 metabolites – arachidonic, eicosapentaenoic, and docosahexaenoic acids. Are materials containing these C20 and C22 polyunsaturated fatty acids a necessary part of the human diet or will the C18 members suffice? This question is discussed further in Chapter 7.

•

•

•

1.3

Methods of modifying oils and fats

It is convenient to divide the techniques for modifying oils and fats into technological and biological groups. In the former we accept what nature provides and seek to change fatty acid and/or triacylglycerol composition,

Introduction: Modifying lipids – why and how?

5

thereby modifying nutritional, chemical (mainly oxidative stability) and physical (mainly melting behaviour) properties to make them more suitable for their end-use. In the latter, we interfere at an earlier stage and either seek new and better sources or take plants which already produce large quantities of oil efficiently and try to modify the composition of the oils by conventional methods of seed breeding or by exploiting newer methods based on increasing genetic understanding.

1.4

Technological methods

Lipid technologists have developed several methods of modifying oils and fats. None of these is very new, but they are subject to incremental improvement either based on a better scientific understanding of the process or through the development of improved equipment. The oldest method is blending. This is a simple mixing of existing oils to provide a mixture with improved qualities. Examples include: • • • •

adding a small proportion of an oil with high oxidative stability (such as sesame oil or rice bran oil) to a less stable commodity oil to enhance its stability; mixing oils to get a fatty acid composition believed to have optimum nutritional value – an increasing number of such blends are appearing in the market place (see Chapter 22 on speciality oils); blending oils prior to interesterifacation to get a product with different triacylglycerol composition; blending oils to give a product with desired properties at minimum cost (Block et al., 1997).

Fractionation is a procedure by which a commodity oil or one that has already been modified is divided into two or more fractions differing in fatty acid and triacylglycerol composition. This can be achieved without loss of material and without need for further refining. The two fractions extend the range of usage of the original oil. This technique is used mainly for palm oil but has other applications also (Chapter 10). It is not always easy to predict what happens to minor components during fractionation, and this may have unforeseen consequences for oxidative stability. Hydrogenation of an unsaturated oil gives a product of higher melting point (more suitable for spreads and cooking fats) and of enhanced oxidative stability through having less polyunsaturated fatty acid. These benefits are achieved only at some nutritional cost. The level of essential fatty acid is lowered and acids with trans configuration are produced. These modifications follow the molecular changes resulting from partial hydrogenation which include saturation of some unsaturated centres, stereomutation of unsaturated centres (conversion of cis to trans isomers), double bond migration and conversion of linoleate mainly to trans 18:1 isomers (Chapters 7 and 9).

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Modifying lipids for use in food

Interesterification is a procedure for rearranging the fatty acids of an oil or a blend of oils so that triacylglycerol composition is changed. When an alkaline catalyst is used, fatty acids are randomly distributed in the product. This is in contrast to the natural vegetable oils produced by (enzymaticallycatalysed) biological processes where the fatty acids are not randomly distributed. These changes affect thermal behaviour and may also have nutritional consequences (Chapters 7 and 11). Interesterification can also be carried out with lipases. The enzymatic processes have several advantages in that they occur under milder conditions, may require less costly equipment and produce less by-product so that there is less waste and less effort is required to purify the product. However, the major benefit of using a lipase is the added control over the nature of the product as a consequence of the specificity shown by many lipases. Fatty acidspecific lipases relating to chain-length or double bond position can be used to confine changes to a particular group of acids, while other lipases are specific for glycerol esters (mono-, di- or triacylglycerols) or distinguish between the different glycerol ester groups. Thus many lipases are described as being 1,3specific implying that changes can be made at positions 1 and/or 3 but not at position 2 where the ester group remains unchanged (Chapters 7 and 11). Interesterified products are generally less stable than the original oils, probably through changes – not yet fully understood – in the balance of pro- and antioxidants. This holds with both chemical and enzymatic catalysts.

1.5

Biological methods

1.5.1 Domestication of wild crops The oil and fat business is based almost entirely on a limited number of commodity oils which vary in their fatty acid composition, but there are many other plant species with fatty acid composition not very different from the commodity oils. These could be used as food lipids, but there would have to be a special reason for developing them through the long chain of events from agronomical improvements to retail marketing. A few of these will be discussed in Chapter 2 under the section on minor oils. There are also plants producing uncommon acids such as epoxy acids, acids with conjugated unsaturation, or oils with a very high level (> 80 %) of a single acid. Attempts to domesticate and commercialize such plants and their seed oils have taken longer than originally thought, with some niche products of this kind taking 20 and more years to develop. Most, but not all, of these oils are of interest to the oleochemical rather than the food industry.

1.5.2 Oilseeds modified by conventional seed breeding or by genetic engineering As a consequence of the difficulties in domesticating wild plants, greater

Introduction: Modifying lipids – why and how?

7

effort has been directed to the modifying of plants that are already sown and harvested on a commercial scale and where the best agronomic procedures are well known. This has the disadvantage of minimizing the range of important plant species (limiting biodiversity). The changes to be sought are partly agronomic but, of greater interest for this book, they include changes in fatty acid composition, triacylglycerol composition and minor components. These changes should be achieved without sacrifice of yield and must be biologically stable from season to season. They have to be accompanied by procedures of identity preservation. The modified seed must be kept separate at all times from its more conventional form. This has consequences for harvesting, transporting and extracting the seed and for the subsequent handling of the oil. Such changes may be effected by conventional seed breeding or by newer procedures of genetic engineering. It is important to know which method has been used because of the concerns expressed by some about procedures involving transgenic modification. Modifications to fatty acid composition which have been sought include: reduced levels of saturated acid for nutritional reasons; reduced levels of linolenic acid and/or higher levels of saturated acids to avoid hydrogenation (with consequent formation of undesirable trans acids); and higher levels of oleic acid. These are detailed in Chapter 12. One exciting possibility is to develop plant systems that will produce long-chain polyunsaturated fatty acids such as arachidonic acid, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). There have been interesting developments in a number of research laboratories but such plants, which probably have to be genetically modified, are 10–20 years from commercial development (Chapter 12) (Drexler et al., 2003; Green, 2004; Qi et al., 2004).

1.5.3 Animal fats modified through nutritional changes From a nutritional viewpoint land animal depot fats are perceived as having several disadvantages. They are generally rich in serum cholesterol-raising saturated acids, frequently contain acids with trans unsaturation, and have high levels of cholesterol. Their level of essential fatty acids is low, and they contain little if any antioxidant. Further, animal fats are not acceptable to vegetarians and to some ethnic groups. Nevertheless animal fats contain low levels of long-chain PUFA (polyunsaturated fatty acid) and are a valuable source of such acids in the human diet. Some of the perceived disadvantages in ruminant animals are the consequence of biohydrogenation processes taking place within the rumen, and dietary regimes have been proposed to circumvent these changes. There is also an interest in modifying the fatty acid composition of chicken eggs and meat by appropriate changes to the diet of the chicken. This has been seen as a way of enhancing the (human) dietary intake of CLA (conjugated linoleic acid) and of EPA and DHA (long-chain omega-3 acids) (see Chapter 13).

8

Modifying lipids for use in food

1.5.4 Single-cell oils As indicated at the beginning of this chapter, oils and fats produced by the agricultural supply industry come mainly from plant sources and also to a minor extent from animals. An alternative approach is to seek new lipid sources from micro-organisms. Some of these can be made to produce high levels of lipids with an interesting fatty acid composition. While it is unlikely that these will replace the more conventional commodity oils for traditional use, nevertheless, they are already providing supplies of high quality longchain PUFA for infant formula and other special purposes (Chapters 5 and 23).

1.6

References

(2005), Oil World Annual 2005, Hamburg, ISTA Mielke GmbH. and GOMIDE F A C (1997), Blending process optimisation into special fat formulation by neural networks, J Am Oil Chem Soc, 74, 1537–1541. DREXLER H, SPIEKERMANN P, DOMERGUE F, ZANK T, SPERLING P, ABBADI A and HEINZ E (2003), Metabolic engineering of fatty acids for breeding of new oilseed crops: strategies, problems and first results, J Plant Physiol., 160, 779–802. GREEN A G (2004), Producing essential fatty acids in plants, Nat Biotechnol, 22, 680–682. GURR M I (1999), Lipids in Nutrition and Health – a Reappraisal, Bridgewater, Oily Press. PEDERSEN J I, MULLER H and KIRKHUS B (2001), Serum cholesterol predictive equations with special emphasis on trans and saturated fatty acids. An analysis from designed controlled studies, Lipids, 36, 783–791. QI B, FRASER T, MUGFORD S, DOBSON G, SAYANOVA O, BUTLER J, NAPIER J A, STOBART A K and LAZARUS C M (2004), Production of very long chain polyunsaturated omega-3 and omega-6 fatty acids in plants, Nat Biotechnol, 22, 739–745. TAUBES G (2001), The soft science of dietary fat, Science, 291, 2536–2545. ANON

BLOCK J M, BARRERA-ARELLANO D, FIGUEIREDO M F

Part I Understanding food lipid structure and composition

2 Vegetable sources of lipids F. Gunstone, Scottish Crop Research Institute, UK

2.1

Introduction

This chapter is devoted to the most important source of lipids, namely those derived from vegetable sources. The major commodity oils are described first, covering their fatty acid and triacylglycerol composition and their minor components. This is accompanied by some indication of the ways in which each oil is modified and the major food uses. There follows a shorter account of some significant minor vegetable oils after which the procedures by which all these oils may be extracted and refined are detailed. Finally there is a brief discussion of future trends in the supply of vegetable-based lipids. Dietary lipids may be ingested as part of a food as when nuts are eaten or when green vegetables are consumed. Although these last contain only low levels of lipids, they are frequently of high nutritive quality and are accompanied by valuable minor components. All living material contains membrane lipids that are mainly phospholipids. However, larger in quantity are those oils and fats of vegetable or animal origin consumed as frying oils, salad oils, spreads, baked goods or chocolate. Over the last half-century the availability of vegetable oils has increased much more than that of animal fats. One important commodity analyst reports weekly on 17 oils and fats of which 13 are of vegetable origin and make up 80 % of total supply while four of animal origin (butter, lard, tallow and fish oil) provide the balance. It is estimated that ~ 80 % of the total supply is used for food and the remainder as animal feed (~ 6 %) or by the oleochemical industry (~ 14 %). Details of the nature of the vegetable oils are given in Section 2.2 but it is appropriate to provide some production data in order to give balance to the ensuing discussion (Table 2.1). The major oils (soybean, palm and palmkernel,

12

Modifying lipids for use in food

Table 2.1 Commodity oils and fats: production levels (million tonnes) and major producing areas in 2004/05. Oil/fat

Production

Food use (%)a

Major producing countries/regionsb

Soybean

32.57

90

Palm Rape/canola

32.50 16.11

70 75

Sunflower

9.08

95

Cottonseed

5.00

95

Groundnutc Palmkernel Coconut Olive Corn Sesame Linseed Castor Cocoa butterd Four animal fatse World total

4.50 3.80 3.01 2.73 2.05 0.77 0.60 0.52 1.20

95 70 70 95 95 95 0 0 95

USA 8.60, Br 5.74, Ch 5.11, Arg 5.03, EU-25 2.63 Mal 15.16, Indon 12.90 EU-25 5.47, Ch 4.56, Ind 2.08, Can 1.36 CIS 3.20, EU-25 1.76, Arg 1.41 Cent Eur 0.72 Ch 1.55, Ind 0.69, Pak 0.51, CIS 0.44, USA 0.42 Ch 2.05, Ind 1.13, Nig 0.32 Mal 1.83, Indon 1.31 Pp 1.27, Indon 0.74, Ind 0.40 EU-25 2.06, Syria 0.20, Tun 0.13 USA 1.10, EU-25 0.23 Ch 0.20, Ind 0.14, My 0.11 EU-25 0.15, Ch 0.13, USA 0.13 Ind 0.32, Ch 0.11 Ivory Coast, Ghana, Indon, Br, Mal

23.16 136.4

(80)

a

These values are estimates made by the author based, in part, on information provided by USDA for 2001/02 [www.fas.usda.gov/oilseeds/circular/2000/oilstats.html]. b These are not necessarily the countries in which the plants are grown. Oils may be produced from imported seeds as well as from domestic supplies. c Also called peanut oil. d Not included in Oil World publications – information added by the author. e Total of tallow (8.19), lard (7.43), butter (6.47) and fish oil (1.07). Abbreviations: Arg = Argentina, Br = Brazil, Can = Canada, Cent Eur = Central Europe, Ch = China, CIS = Commonwealth of Independent States (Former Soviet Union), Indon = Indonesia, Mal = Malaysia, My = Myanmar (Burma), Nig = Nigeria, Pak = Pakistan, Pp = Philippines, Syr = Syria, Tun = Tunisia Source: Anon (2005).

rapeseed and sunflower seed) total 94.1 million tonnes (69 % of total production) (Anon, 2005). They represent an even larger proportion of trade (imports/exports) as many of the oils produced at lower levels are consumed predominantly in the country/region where they grow. Table 2.1 shows the total production of each oil in 2004/05, an estimate of the share of each oil used for food purposes and the major countries of production of the oil from domestic and/or imported oilseeds. Gunstone (2005) has reworked these production figures and calculated the annual production (from 13 vegetable oils, cocoa butter and four animal fats) of the major fatty acids. These total figures have been adjusted for the 80 % consumed as human food and for the changes that occur during partial

Vegetable sources of lipids 13 hydrogenation. Adjustment has not been made for wasted and discarded oil nor for the lipid consumed in forms not counted by market analysts, such as fat in nuts or cheese, or consumed as meat or fish. The results suggest that production of linoleic is in excess of our dietary needs and that production of linolenic is much below the desirable level leading to an omega-6/-3 ratio of ~ 30 that is far too high (Table 2.2).

2.2

Major vegetable sources of food lipids

The major vegetable oils are pressed and/or extracted from seeds or pressed from fruits such as olive or oil palm (Section 2.4). Crude oils are mainly triacylacylglycerols (usually > 95 %) accompanied by lower levels of free acids, monoacylgycerols, diacylglycerols, phospholipids (1–3 %), free and/ or acylated sterols (1000–5000 ppm in total), tocols (300–2000 ppm) and hydrocarbons such as alkanes, squalene and carotenes. Oils from different sources differ in fatty acid and triacylglycerol composition and in the detailed composition of the various minor components. Most oils used in the food industry have been subject to refining processes to raise the triacylglycerol level and to reduce the levels of other components. Ideally, refining (Section 2.4) should remove undesirable components as efficiently as possible but leave suitable levels of the desirable minor components. In some refining procedures materials such as free acids, phospholipids, sterols and tocols are recovered as valuable by-products and find further use in the food, feed, cosmetics, pharmaceutical and oleochemical industries.

Table 2.2 Annual production (million tonnes) for seven major fatty acids in the total oils and fats produced by the agricultural industry. Column 1 relates to total production in 2004/05, column 2 to that used for human food and column 3 after adjustment for changes accompanying partial hydrogenation of soybean oil and rapeseed oil. Fatty acid

1

2

Lauric Myristic Palmitic Stearic Oleic Linoleic Linolenic Other Total n-6/n-3

3.4 2.6 27.4 7.2 47.8 37.5 4.5 6.0 136.4 8.3

2.0 1.8 21.7 5.5 38.4 31.9 3.5 3.9 108.7 9.1

*After hydrogenation this figure includes significant quantities of trans isomers. Source: Gunstone (2005).

3 2.0 1.8 21.7 5.9 48.8* 23.8 0.8 3.9 108.7 29.8

14

Modifying lipids for use in food

2.2.1 Soybean oil Soybeans provide two important materials: oil extracted from the beans and a residual meal containing high quality protein. Whole beans contain protein (40 %), carbohydrate (34 %), oil (21 %), and ash (5 %) (Wang, 2002) with the levels of oil and meal produced commercially being 18 and 79 % respectively. Soybean meal is such an important commodity for animal feed and, to a lesser extent, for human food that the oil has been described as a byproduct. However, as already indicated, soybean oil is produced at a higher level than any other oil and is better described as a co-product. The major fatty acids in soybean oil are linoleic (53 %), oleic (23 %), palmitic (11 %), linolenic (8 %) and stearic (4 %) (Wang, 2002). The widelyaccepted view is that this is a healthy oil, low in saturated acids and rich in polyunsaturated fatty acids (PUFA), especially linoleic acid. However, attempts to modify this fatty acid composition by partial hydrogenation or by plant breeding suggest that it would be better to have less saturated acids and less linolenic acid. Opinion on linolenic acid is contentious. This triene acid is easily oxidized, and food products with linolenic acid have a short shelf life, which is a matter of concern for the food industry, the retailer, and the consumer. However, there is also a matter of nutritional concern. There is a growing awareness that omega-6/omega-3 ratios (Section 7.5) at levels exceeding 20:1 in some countries are far too high and that values of 5:1 or lower (Crawford, 2004) are desirable. It has been argued (Gunstone, 2005) that, compared with nutritional recommendations, the agricultural supply industry as a whole (plant and animal) provides too much linoleic acid and too little linolenic acid. According to Gunstone’s calculations the only significant commodity sources of linolenic acid for food purposes are soybean oil and rapeseed oil, and this is largely destroyed by partial hydrogenation (Table 2.2). He argues that more effort should be devoted to developing antioxidant packages that will allow the safe use of linolenic acid. Because of its high level of linoleic acid (> 50 %), over half the triacylglycerols in soybean oil contain two or three linoleic chains, and most of the remainder have one linoleic chain. In a typical analysis, triacylglycerols exceeding 4 % were LLL 17.6 %, LLO 15.3 %, LLP 10.2 %, LLLn 7.9 %, LLSt 4.2 %, PLO 6.9 %, OLO 6.3 %, LnLO 4.8 %, other 26.8 %. (These three-letter symbols stand for all the isomeric triacylglycerols containing the three acyl groups indicated, where Ln = linolenic acid, L = linoleic acid, O = oleic acid, St = stearic acid and P = palmitic acid.) Soybeans producing oil with a different fatty acid composition have been developed both through traditional seed breeding and by introduction of genes from other plant species. These important developments are discussed in Chapter 12. Crude soybean oil contains several valuable minor components that are recovered in some measure during refining. Degumming produces lecithin – a fraction rich in phospholipids which serves as the main industrial source of these materials. Deodorizer distillate contains tocopherols (vitamin E) (0.15– 0.21 % in the oil raised to ~ 11 % in the distillate) and phytosterols (~ 0.33 %

Vegetable sources of lipids 15 in the oil raised to ~ 18 % in the distillate). Both of these are valuable byproducts. For more details see Wang (2002). Soybean phospholipids (1.5–2.5 %) are removed during the degumming stage of refining. The crude lecithin obtained by this process is a mixture of triacyglycerols (35–40 %), phosphatidylcholines (10–15 %), phosphatidylethanolamines (9–12 %), phosphatidylinositols (8–10 %) and other minor components. The phospholipid content can be increased by ‘deoiling’ with acetone to remove triacylglycerols, and individual phospholipid classes can subsequently be concentrated by dissolution in ethanol. The desired amphiphilic properties of natural phospholipids can be further improved by chemical manipulation, such as partial lipolysis/hydrolysis to produce some lysolecithin or partial hydroxylation of double bonds to increase the polarity of the molecule (Chapter 15). Wang (2002) further reports that the difference between crude and refinedbleached-deodorized (RBD) oil is such that tocopherols are reduced by 36 %, sterols by 32 % and squalene by 38 %. However, the ‘lost’ materials can be trapped in the deodorizer distillate and be recovered for further use. The major sterols are β-sitosterol (125–236), campesterol (62–131) and stigmasterol (47–77) out of a total of 235–405 mg/100 g. Tocopherols in solvent-extracted soybean oil are given as 1370 ppm divided between the α (11 %), β (1 %), γ (63 %), and δ (25 %) compounds. Soybean oil in its native but refined form or in some partially hydrogenated form is widely used for food purposes such as frying and salad oils, margarine and shortening, and mayonnaise and salad dressing. It is used universally for these purposes, especially in those countries where soybeans are grown and extracted. For example, it is reported that in the USA 86 % of all food lipids are derived from soybean oil and that no other oil attains a level above 3 %.

2.2.2 Palm oil The oil palm produces two different oils – palmkernel oil (Section 2.2.5) and palm oil. The latter is extensively fractionated into palm olein and palm stearin. These differ in composition and so extend the range of uses for palm oil. The plant grows in wet tropical regions. Malaysia and Indonesia are the major producing and exporting countries (Table 2.2). Supplies of palm oil have increased rapidly in the last 20–30 years, mainly through increasing the area under cultivation. World average annual production of palm and palmkernel oils together for selected five-year periods is: 6.6 million tonnes in 1981–85 and 15.1 million tonnes in 1991–95 (Anon, 2002), and 31.0 million tonnes in 2001–05 (Anon, 2005). Though second to soybean oil in total production, palm oil exceeds it in volumes traded. Current figures for palm oil production and exports are given in Table 2.3. The average production of palm oil (3.5 tonnes/hectare) and of palmkernel oil (0.4 tonnes/hectare) makes the oil palm the most productive source of oil/

16

Modifying lipids for use in food

fat. These are average figures, and significantly higher yields are obtained from the best plantations. The fatty acid composition, triacylglycerol composition and iodine value for Malaysian palm oil and its fractions are listed in Table 2.4. Palm oil is unusual in containing about 5 % of diacylglycerols. In terms of molecular species, the major triacylglycerols in palm oil are given as POP 29 %, POO 23 %, PLO 10 %, PLP 9 % (Siew Wai Lin, 2002). Palm oil is considered as a saturated fat in comparison to the more highly unsaturated vegetable oils. With almost the same levels of saturated and unsaturated acids, the description saturated can be misleading. The oil is rich in nutritionally important minor components. Because of its fatty acid composition and its minor components which act as antioxidants, palm oil has high oxidative stability. Palm oil is not much used in North America, but it makes a significant contribution to dietary fat intake in the rest of the world. Table 2.3 Production and exports (million tonnes and percentage of world total) of palm oil and palmkernel taken together in 2004–05.

World Malaysia Indonesia Other

Production

Exports

36.30 16.99 13.91 5.40

27.44 14.57 9.93 2.94

(46.8 %) (38.3 %) (14.9 %)

(53.1 %) (36.2 %) (10.7 %)

Source: Anon (2005).

Table 2.4

Compositional data for palm oil and selected fractions.

Major fatty acids 16:0 18:0 18:1 18:2 othera

Oilc

Oleind

Stearind

Mid-fractiond

44.1 4.4 39.0 10.6 1.9

40.9 4.2 41.5 11.6 1.8

47–74 4–6 16–37 3–10

41–55 5–7 32–41 4–11

12–56 34–50 5–37 0–8 22–40

1–11 45–74 19–42 2–8 34–55

Major triacylglycerols by carbon numberb C48 8.1 3.3 39.9 39.5 C50 38.8 42.7 C52 C54 11.4 12.8 Iodine value 52.1 56.8 a

Other acids present at low levels include 12:0, 14:0, 20:0, 16:1 and 18:3. These tricacylglycerols consist mainly of 3, 2, 1, and 0 palmitic acid chains with 0, 1, 2, and 3 C18 chains respectively. c Mean of 244 Malaysian samples. d Mean of many Malaysian samples. Source: Siew Wai Lin (2002). b

Vegetable sources of lipids 17 Palm oil, palm olein and palm stearin contain 500–700, 600–760 and 380–540 ppm, respectively, of mixed carotenes (almost entirely a mixture of the β and α forms) when crude, but this is lost in RBD oils. Red palm oil, obtained by an alternative refining process, and still containing ~ 80 % of the carotenes originally present, is marketed as an oil with added nutritional value because of the carotenes which serve as pro-vitamin A. Crude palm oil is rich in tocols (600–1000 ppm), with the level of tocotrienols (~ 70 %) exceeding that of tocopherols. Refined oil still has ~ 70 % of the original tocols. Some loss occurs during deodorization, and palm oil fatty acid distillate (PFAD, equivalent to deodorizer distillate) is enriched in tocols (750–8200 ppm) and can be used as a source of tocotrienol-rich tocols. Many health claims have been put forward for these compounds. Palm oil contains sterols (200–600 ppm in crude oil) that are mainly β-sitosterol, campestrol and stigmasterol. These are present at lower levels in refined oil (70–300 ppm) and concentrate in PFAD at an average level of 6500 ppm. The acyclic C30 hydrocarbon squalene is present in crude palm oil (200–500 ppm) and concentrates in PFAD during refining (5000–8000 ppm). Palm oil is used for a wide range of food purposes including frying, in spreads and vanaspati and in shortenings. Palm olein is a major frying oil, and palm stearin finds increasing use as hard stock in fat blends which are interesterified to produce spreads with no hydrogenated oil and therefore containing little or no trans acids. The presence of palm oil or palm stearin in a spread helps to stabilize the β′-crystal form because of the mixed C16 and C18 chains. 2.2.3 Rapeseed/canola oil Rapeseed production is second only to soybeans, and rapeseed/canola oil is third after soybean and palm. Production levels have grown steadily in recent years and peaked in 1999/2000 at 14.5 million tonnes. Thereafter production declined somewhat through reduced planting and/or poor weather that reduced yield. After falling to 12.3 million tonnes in 2002/03 production is now rising steadily with figures of 14.4 and 16.1 million tonnes for 2003/04 and 2004/05, respectively (Anon, 2005). Rapeseed and canola are terms describing the seed and extracted oil from Brassica species including B. napus (formerly B. campestris), B. rapa and B. juncea. The seed oil from these species was typically rich in erucic acid (22:1), and the seed meal had an undesirably high level of glucosinolates. These components reduced the value of both the oil and the protein meal, but they have been bred out of the modern rapeseed, now known as double zero or canola. It is grown mainly in Western Europe, China, India and Canada (where the canola varieties were developed). Typically it contains palmitic (4 %), stearic (2 %), oleic (62 %), linoleic (22 %) and linolenic (10 %) acids and has less saturated acids than any other commodity oil. In one example its major triacylglycerols were LnLO (8 %), LLO (9 %), LnOO (10 %), LOO

18

Modifying lipids for use in food

(22 %), LOP (6 %), OOO (22 %) and POO (5 %) (Przybylski and Mag, 2002). With its low level of saturated acids, its high level of oleic acid and the presence of linoleic and linolenic acids at a favourable ratio (~ 2:1) rapeseed oil rates highly in the classification of healthy oils. Also, the plant system lends itself to genetic modification, and rapeseed varieties with modified fatty acid composition have been developed, although it is still not clear how many of these will be economically viable. Rapeseed oils with less linolenic acid, or enhanced levels of lauric acid, stearic acid, oleic acid, or with unusual acids such as γ-linolenic acid, ricinoleic acid or vernolic acid have all been developed for commercial exploitation. Oleic-rich varieties have about 84 % oleic acid. For more details see Przyblski and Mag (2002), Gunstone (2004b) and Chapter 12. Rapeseed oil contains ~ 3 % of phospholipid which is removed during degumming. Rapeseed oils contain sterols and sterol ester together at levels between 0.45 and 1.13 %. These are mainly β-sitosterol (47–52 % of total sterols) and campesterol (28–34 %). All brassica seeds are characterized by the presence of brassicasterol (12–16 %), a sterol virtually absent from other seed oils. Tocopherols occur in rapeseed oils at levels of 430–2680 mg/kg oil with the γ- (60–74 % of total tocols) and α-compounds (26–35 %) predominating. Brassica oils, particularly those grown during short seasons in Canada and Scandinavia, frequently contain chlorophyll (and related compounds) at levels up to 50 ppm, although RBD oil usually has a chlorophyll (and derivatives) specification of less than 25 parts per billion. High-erucic rapeseed is still grown with the high-erucic oil being used for a range of industrial purposes and particularly for the preparation of erucamide used in ‘clingfilm’ (Temple-Heald, 2004). Rapeseed oil is generally accepted as a healthy oil by virtue of its fatty acid composition, and is widely used for food purposes, sometimes after brush hydrogenation to reduce the level of linolenic acid and sometimes after partial hydrogenation in spreads. The oil is used as a salad oil and salad dressing and mayonnaise, in margarine and other spreads, as a frying oil, and in many minor food applications. The oil is widely used, particularly in China, EU-15 and India – all countries where the plants are grown extensively. Canada and Australia are major exporting countries of seed and/or oil. Both these countries have modest populations and therefore limited local demand.

2.2.4 Sunflower seed oil Oil obtained from sunflower seeds (Helianthus annuus) occupies the fourth position in the ranking of vegetable oils by production levels (Table 2.1). It became popular as the major lipid constituent of many margarines when animal fats were first replaced by linoleic-rich vegetable oils, but in recent years it has had to compete with increasing use of soybean oil.

Vegetable sources of lipids 19 Sunflower oil is available in three ranges of fatty acid composition. The traditional and still major sunflower oil is linoleic-rich, but two other forms have been produced by conventional seed breeding; these are a high oleic oil and a mid-oleic oil. The latter (NuSun™ oil – National Sunflower Association, USA) is much favoured in the USA where attempts are being made to replace the traditional oil. The fatty acid composition of these sunflower types is given in Table 2.5. Their oxidative stability increases as the ratio of oleic to linoleic acid increases. The major triacylglycerols of the traditional linoleicrich oil are typically LLL (14 %), LLO (39 %), LLS (14 %), LOO (19 %), LOS (11 %) and other (3 %) (S = saturated) (Gunstone, 2004a). Crude sunflower oil contains phospholipids (0.7–0.9 %), tocopherols (630– 700 ppm), sterols (~ 0.3 %) and carotenoids (1.1–1.6 ppm). The phospholipids removed during refining are mainly phosphatidylcholines (55–64 % of total phospholipids), with lower levels of phosphatidylinositols (15–24 %) and phosphatidylethanolamines (17–20 %). The tocols are almost entirely αtocopherol which makes sunflower seeds and the extracted oil good sources of vitamin E. The seeds are also a rich source of selenium compared to most other seeds and nuts. Sunflower seed oil contains some wax (esters of longchain alcohols and long-chain acids) coming from an outer protective seed coat. This makes the oil appear cloudy on standing and is usually removed by winterization if the oil is to be used as a salad oil. This is achieved by filtering oil that has been held at 7–8 °C for 12–24 hours. 2.2.5 Lauric oils (coconut, palmkernel) Coconut oil and palmkernel oil are similar to one another in their fatty acid composition and differ so much from other commodity oils that they can be considered together. Both have high levels of medium-chain saturated acids, especially lauric acid (12:0), hence the term lauric oils. As a consequence they have only low levels of unsaturated C18 acids and low iodine values. They are used extensively both as food and as non-food oils and serve as the major source of C8 (caprylic or octanoic) acid and C10 (capric or decanoic) acid. These short-chain acids are concentrated by distillation of the hydrolyzed oils. The production of coconut oil has generally exceeded that of palmkernel Table 2.5

Typical fatty acid composition of sunflower oils.

Saturated Oleic Linoleic Linolenic Iodine value (approx) Source: Gupta (2002).

Traditional

Mid-oleic

High oleic

11–13 20–30 60–70 <1 128

< 10 55–75 15–35 <1 108

9–10 80–90 5–9 <1 79

20

Modifying lipids for use in food

oil, but the latter has increased along with the production of palm oil. It now exceeds that of coconut oil and is likely to continue to do so. Details of fatty acid and triacylglycerol composition are presented in Table 2.6. The two lauric oils differ in that coconut oil contains slightly higher proportions of the C8 and C10 acids while palmkernel oil has higher levels of oleic acid. As a consequence, palmkernel oil has the higher iodine value (Codex range of 14–21 for palmkernel oil and 6–10 for coconut oil) and accordingly will show the greater change on hydrogenation. Coconut oil is the preferred source of octanoic and decanoic acids. These differences in fatty acid composition are reflected in triacylglycerol composition. As expected of such saturated oils, the content of tocopherols in the crude oils is low (coconut: a range 0–44 and mean of 10 mg/kg of oil, palmkernel: a range of 0–257 and a mean of 34 mg/kg of oil). Sterols are around 1000 ppm in both oils. Both oils may be subject to fractionation, hydrogenation and interesterification (often in a blend with a conventional C16/18 oil) to extend their range of use. The two oils have been reviewed by Pantzaris and Yusof Basiron (2002).

2.2.6 Other vegetable food oils (groundnut, cottonseed, corn, olive, sesame, rice bran, linseed) Sections 2.2.1 to 2.2.5 have covered the four dominant vegetable oils and the two lauric oils. There remain a number of other oils produced and traded at lower levels (see Table 2.1). Their fatty acid compositions are detailed in Table 2.7. Linseed differs from the others in its high level of linolenic acid. Table 2.6

Fatty acid and triacylglycerol composition of coconut and palmkernel oils.

Fatty acid compositiona

8:0 10:0 12:0 14:0 16:0 18:0 18:1 18:2

a

Triacylglycerol composition by carbon number

Coconut

Palmkernel

7.3 6.6 47.8 18.1 8.9 2.7 6.4 1.6

3.3 3.5 47.8 16.3 8.5 2.4 15.4 2.4

Trace fatty acids include 6:0 and 20:0. Source: Pantzaris and Basiron (2002).

C30 C32 C34 C36 C38 C40 C42 C44 C46 C48 C50 C52 C54

Coconut

Palmkernel

3.5 13.4 17.1 19.1 16.5 10.2 7.3 4.1 2.5 2.1 1.5 1.2 0.8

1.4 6.5 8.5 21.6 16.4 9.8 9.1 6.6 5.4 6.1 2.6 2.7 2.7

Vegetable sources of lipids 21 All the other oils are rich in oleic and/or linoleic acid. Cottonseed and rice bran oils have higher levels of palmitic acid than is usual in vegetable oils. Linseed is used mainly as an industrial oil. Its use as a food oil is discussed as a minor oil in Section 2.3. Groundnut (peanut), cottonseed and corn oils are used for the usual range of foods (frying, baking, salad oils, etc.) and may be partially hydrogenated, fractionated or interesterified to improve their properties for these purposes. Olive, sesame and rice bran oils are more usually used without modification as liquid oils. Sesame and rice bran oils are marked by high oxidative stability and are sometimes added to other oils or oil blends to enhance this property. In addition to the tocol mixtures found generally in vegetable oils, these two oils and also olive oil contain other types of powerful antioxidants. Sesame oil contains sesamin, sesamolin, and related lignans, rice bran oil has several oryzanols (sterol esters of ferulic acid) and olive oil is rich in a range of polyphenols. Further details for cottonseed, groundnut (peanut), olive, corn and sesame oils sare available in appropriate chapters of a recent book on vegetable oils (Gunstone, 2002).

2.2.7 Cocoa butter Cocoa butter is not generally cited by market analysts covering the usual range of oils and fats, perhaps because it has virtually only one use – the production of chocolate and related confectionery. A small amount is used in the cosmetics industry. Annual production is about 3 million tonnes of beans providing about 0.7 million tonnes of cocoa butter (Timms, 2003). The cocoa bean (Theobroma cacao) is the source of two important ingredients of chocolate: cocoa powder and a solid fat called cocoa butter. The usefulness of cocoa butter for this purpose is related to its fatty acid and triacylglycerol composition. It consists mainly of palmitic (26 %), stearic (34 %) and oleic acids (35 %). The major triacylglycerols are symmetrical disaturated oleic glycerol esters of the type SOS and include POP (18–23 %), POSt (36–41 %) and StOSt (23–31 %). This mixture shows characteristic Table 2.7

Groundnuta Cottonseed Corn Olive Sesame Rice bran Linseed a

Fatty acid composition of selected vegetable oils (typical values). 16:0

18:0

18:1

18:2

18:3

11 23 13 10 9 20 6

2 2 3 2 6 2 5

52 17 31 78 41 42 17

29 56 52 7 43 32 14

– tr 1 1 tr 1 60

Also known as peanut – this oil also contains C20 – C24 acids at a combined level of 6 %. tr (trace): levels < 0.6 %. Source: Gunstone (2004a).

22

Modifying lipids for use in food

melting behaviour with a sharp melting point just below mouth temperature. Cocoa butter generally commands a premium price and cheaper alternatives have been developed (see Section 2.3.2) These may or may not have similar chemical composition to cocoa butter, but they must display similar melting behaviour (Timms, 2003).

2.3

Minor vegetable sources of food lipids

In addition to the commodity vegetable oils detailed in Section 2.2, there are many minor seed oils which find a use in niche markets. Some used for food purposes are selected for discussion here, but those used industrially will not be covered. Many are found in the ‘health’ market and may be pressed oils rather than solvent-extracted. Some are not refined (beyond filtering) and others are refined only by the mildest possible procedures so as not to modify any of the fatty acids and to retain the greater part of the nutritionally important minor components, especially the antioxidants. Such oils are generally not modified by the procedures discussed in this book but are used in their native state as salad oils or for shallow frying. Some, for which health claims are made, are supplied as supplements in encapsulated form. 2.3.1 Oils containing γ-linolenic acid and/or stearidonic acid γ-Linolenic acid (∆-6,9,12-18:3) and stearidonic acid (∆-6,9,12,15-18:4) are metabolic intermediates in the conversion by animals of dietary linoleic acid to arachidonic acid and of dietary α-linolenic acid to eicosapentaenoic acid (EPA) and dodosahexaenoic acid (DHA) (Section 7.5). They occur only rarely among vegetable oils. It is thought to be beneficial to include γlinolenic acid and stearidonic acid in the diet in situations where ∆-6-desaturase is not operating effectively. This is usually achieved by supplying appropriate oils in capsule form. The oils most used for this purpose are borage seed oil, evening primrose seed oil and blackcurrant seed oil for γ-linolenic acid and echium oil for stearidonic acid. The fatty acid compositions of these oils are given in Table 2.8.

2.3.2 Cocoa butter alternatives Because of the high cost of cocoa butter, other tropical fats with similar physical properties are sometimes used as alternatives. There are strict rules (legal rather than technical) about what changes can be made if the product is still to be called chocolate (Chapter 20). Chocolate-like products that do not meet the full specifications for chocolate are described as confectionery. According to a recent EU directive, chocolate may contain up to a limited amount of palm mid-fraction or of one or more of five tropical oils. These have the compositions indicated in Table 2.9. This illustrates their resemblance

Vegetable sources of lipids 23 Table 2.8 Component acids of oils containing γ-linolenic acid (γ-18:3) and stearidonic acid (18:4) (typical results, % wt).

Evening primrose Borage Blackcurrant Echium plantagineum

16:0

18:0

18:1

18:2

γ-18:3

18:4

Other

6 10 7 6

2 4 2 3

9 16 11 14

72 38 47 13

10 23 17 12

tr tr 3 17

1 9a 13b 35c

a

Including 20:1 (4.5), 22:1 (2.5), and 24:1 (1.5). Including α-18:3 (13). Including α-18:3 (33). tr (trace): levels
Table 2.9 Typical values for major triaylglycerols in tropical fats used as partial replacements for cocoa butter. Common name

Cocoa butter Palm mid-fraction Borneo tallow (Illipe) Kokum butter Mango kernel stearin Sal stearin Shea stearin

Botanical name

Theobroma cacao Elaies guinensis Shorea stenoptera Garcinia indica Mangifer indica Shorea robusta Butyrospermim parkii

Major triacylglycerols (%) POP

POSt

StOSt

16 57 6 1 2 1 1

38 11 37 5 13 10 7

23 2 49 76 55 57 71

Abbreviations: O = oleic acid, P = palmitic acid, St = stearic acid. Copied with permission from Gunstone F D, The Chemistry of Oils and Fats, Oxford, Blackwell Publishing (2004), 13.

to cocoa butter in that they are all rich in SOS triacylglycerols, although the proportion of palmitic and stearic esters varies (Stewart and Kristott, 2004).

2.3.3 Other minor oils The fatty acid compositions of a selection of minor seed oils are listed in Table 2.10. As is common in seed oils, the major fatty acids are palmitic, oleic and linoleic sometimes accompanied by linolenic. They attract interest because of their convenient availability as a by-product of some other material or process or because they contain a high level of oleic acid (almond, avocado, hazelnut, macadamia, safflower), linoleic (hemp, passionfruit, pumpkin, high oleic safflower, wheatgerm, linola/solin) or linolenic acid (camelina, hemp, perilla), or because they contain other acids of interest. In many cases their value is enhanced by the minor components that are also present, such as

24

Modifying lipids for use in food

Table 2.10

Typical fatty acid composition of selected minor oils. 16:0

Almond (Prunus dulcis) Avocado (Persea americana) Camelina (Camelina sativa) Hazelnut (Corylus avellana) Hemp (Cannabis sativa) Macadamia (Macadamia integrifolia) Nigella sativa (also called black cumin) Passionfruit (Passiflora edulis) Perilla (Perilla frutescens) Pumpkin (Cucurbita pepo) Safflower (Carthamus tinctorius) Wheatgerm (Triticum aestivum)

18:1

10–20 4–9 10

65–70 60–70 10–20 65–75 8–15 35 8–12 13–15 21–47 14 60

4–14

18:2 10–15 16–24 16–23 53–60 55–65 45 13–20 14–18 35–59 75

18:3

Other

30–40

a

15–25

b c

65–75 57–64 35–59 5

d

a

C20 acids (mainly 20:1) 15–23. γ-18:3 0–5, 18:4 0–5. 16:1 16–23, 20:1 1–3. d Saturated 10, also a variety containing ~ 74 % of oleic acid. Source: adapted from Gunstone (2006) b c

Table 2.11 Fatty acid composition (% wt mean of 21 samples) of seed oil and berry oil from sea buckthorn.

Seed oil Berry oil

16:0

9–16:1

18:0

9–18:1

11–18:1

18:2

18:3

7.7 23.1

– 23.0

2.5 1.4

18.5 17.8

2.3 7.0

39.7 17.4

29.3 10.4

Source: Yang et al. (2003).

Table 2.12

Fatty acid composition of linseed oil and of linola.

Linseed Linola Solin High palmitic High oleic

Saturated

18:1

18:2

18:3

10 10 10 31 19

16 16 14 11 49

24 72 73 7 22

50 2 2 44 9

Source: Adapted from information given by Oomah and Mazza (1998).

tocopherols (vitamin E) or carotene. Sea buckthorn yields different oils from the berry and the seed, although these are not usually kept separate (Table 2.11). The oil is a rich source of palmitoleic acid (16:1) and of carotenoids (up to 1 % w/w of oil), tocopherols (0.3–0.5 %) and phytosterols (2–3 %) (Yang et al., 2003). Linseed has been modified to give a linoleic-rich oil (Table 2.12).

Vegetable sources of lipids 25

2.4

Extraction and uses

The commodity oils are generally extracted by pressing, by solvent extraction, or by both of these processes. Pressing produces a ‘virgin’ oil which may only need to be filtered. Further refining may not be necessary or is only carried out under very mild conditions. Pressed oils are perceived as being premium products. Pressing is appropriate for endosperm oils (palm, olive, avocado). Oil-rich seeds may also yield oil by pressing, but this is less efficient as an extraction process than solvent extraction and oil is left in the pressed cake. This is particularly true for soybeans and for cottonseeds which contain only low levels of oil (18 and 15 %, respectively). The residual cake may be fed to animals as a high-energy (expensive) product or may subsequently be extracted with solvent to give a second batch of oil. These batches may be kept separate and sold in two grades, but more usually they are combined and refined together. There are technological reasons why it is sometimes more efficient to carry out the extraction in two stages of pressing followed by solvent extraction. Refining occurs in several stages and is designed to make the crude oil more appropriate for its end use. The objective is to combine the removal of undesirable components such as free acids, phospholipids, oxidized material, coloured materials, metals and undesirable flavours with the retention of desirable minor components such as antioxidants. Several refining steps are combined in processes described as chemical refining and physical refining. The latter has several advantages but is not appropriate for oils rich in phospholipids (like soybean oil) nor for cottonseed oil containing gossypol that is not removed by physical refining. Both refining sequences furnish side streams containing valuable by-products which are recovered for use elsewhere. Chemical refining requires four major steps. Degumming involves reaction with water, dilute phosphoric acid or citric acid to remove phospholipids and trace metals. The former are powerful emulsifying agents that increase refining losses. Neutralization with sodium hydroxide solution or other alkali then removes free acids (and phospholipids, pigments, trace metals and sulphur compounds), and this is followed by water washing to remove soap. Bleaching is the most expensive refining step because of lost oil and the cost of obtaining and disposing of bleaching earth. Oil is heated with bentonite or other bleaching earth at 80–180 °C to remove coloured impurities. Finally, deodorization requires treatment with steam at reduced pressure to remove volatile oxidation products responsible for undesirable flavour and odour. During this process, the oil may be heated at temperatures up to 250 °C which may cause stereomutation of polyunsaturated fatty acids – especially linolenic – to trans isomers. It is desirable, therefore, not to exceed 220 °C and to compensate for this by further reducing the pressure. In physical refining, applied to oils with low phospholipid levels, there are only two steps – bleaching and deodorization – with the latter removing free acids, mono- and di-acylglycerols, oxidation products and pesticides.

26

Modifying lipids for use in food

This is now the usual way of refining palm oil and increasingly other oils also. It has environmental advantages, not least because it avoids the large aqueous waste stream characteristic of chemical neutralization that raises disposal problems. In order to preserve the oil from oxidative rancidity during transport and storage it may be necessary to add some antioxidant at an appropriate stage in the refining sequence (see Chapter 7). A fuller account of extraction and processing has been provided by Hamm and Hamilton (2000). The commodity oils, usually in the form of RBD oils and after appropriate modification, are ready for a wide range of food uses. These include frying oils, cooking oils, baking fats, shortenings, salad oils, mayonnaise, salad dressings, spreads, confectionery goods, ice creams and other minor uses. Most of these are described in Part III of this book.

2.5

Future trends

Future trends are likely to be related to changing ideas of the nutritional role of fats – which fatty acids need to be limited in consumption and which should be increased? There is general acceptance of the link between diet and health/disease and it is known that fat plays an important role in this link. There are many factors – dietary and otherwise – which have to be considered, but this book is concerned only with fats and not with other important factors such as smoking and exercise levels. In the media, fat has a bad image and this has to be countered. We need to emphasize the essential role of fat in our diet and, through collaboration with the food industry, provide foods containing fat in the appropriate quantity and of the optimum quality. To achieve these aims, there will be developments in both the technological and biological methods outlined in Chapter 1 and developed in Part II of this book. As discussed elsewhere, we need to change the omega6/omega-3 ratio in favour of the latter and, at the same time, to improve our antioxidant systems (see Chapter 7). Much of what is written about fat is written from the viewpoint of the affluent, over-fed, under-exercised members of the developed world. It must not be forgotten that there are many in the under-developed world as well as elsewhere who suffer from lack of calories. This need is best met by fat and, for many years to come, there will be a growing demand for fats of all kinds to feed the millions who are still hungry. Agronomists must, therefore, continue the search for increased yields.

2.6

Sources of further information and advice

Information on vegetable oils is available in several books included in the

Vegetable sources of lipids 27 reference list. Data on production of commodity oils is available from Oil World (www.oilworld.biz) and from USDA (www.fas.usda.gov/oilseeds). Websites for most of the commodity oils are listed in Gunstone (2002). Other useful websites include: www.lipidlibrary.co.uk, www.cyberlipid.org and www.bagkf.de/sofa.

2.7

References

(2002), The Revised Oil World 2020, Hamburg, ISTA Mielke GmbH. (2005), Oil World Annual 2005, Hamburg, ISTA Mielke GmbH. CRAWFORD M A (2004), Docosahexaenoic acid and the evolution of the brain – a message for the future, Lipid Technol, 16, 53–57. GUNSTONE F D (2002), Vegetable Oils in Food Technology, Oxford, Blackwell Publishing. GUNSTONE F D (2004a), The Chemistry of Oils and Fats, Oxford, Blackwell Publishing. GUNSTONE F D (2004b), Rapeseed and Canola Oil, Oxford, Blackwell Publishing. GUNSTONE F D (2005), Fatty acid production for human consumption, INFORM, 16, 736– 737. GUNSTONE F D (2006), ‘Minor Specialty Oils, in Shahidi F, Nutraceutical and Speciality Lipids and Their Co-products/New York, Marcel Dekker, Inc., 91–125. GUPTA M K (2002), Sunflower oil, in Gunstone F D, Vegetable Oils in Food Technology, Oxford, Blackwell Publishing, 128–156. HAMM W and HAMILTON R J (2000), Edible Oil Processing, Sheffield, Sheffield Academic Press. OOMAH B D and MAZZA G (1998), Flaxseed products for disease prevention, in Mazza G, Functional Foods – Biochemical and Processing Aspects, Lancaster, PA, Technomic, 91–138. PANTZARIS T P and BASIRON Y (2002), The lauric (coconut and palmkernel) oils, in Gunstone F D, Vegetable Oils in Food Technology, England, Blackwell Publishing, 157–202. PRZYBYLSKI R and MAG T (2002), Canola/rapeseed oil, in Gunstone F D, Vegetable Oils in Food Technology, Oxford, Blackwell Publishing, 98–127. SIEW WAI LIN (2002), Palm oil, in Gunstone F D, Vegetable Oils in Food Technology, England, Blackwell Publishing, 59–97. STEWART I and KRISTOTT J (2004), European Union Chocolate Directive defines vegetable fats for chocolate, Lipid Technology, 16 11–14. TEMPLE-HEALD C (2004), High-erucic oil: its production and uses, in Gunstone F D, Rapeseed and Canola Oil, Oxford, Blackwell Publishing, 111–130. TIMMS R E (2003) Confectionery Fats Handbook, Bridgewater, Oily Press. WANG T (2002), Soybean oil, in Gunstone F D, Vegetable Oils in Food Technology, Oxford, Blackwell Publishing, 18–58. YANG B, GUNSTONE F D and KALLIO H (2003), Oils containing oleic, palmitoleic, γ-linolenic and stearidonic acids, in Gunstone F D, Lipids for Functional Foods and Nutraceuticals, Bridgewater, Oily Press, 263–290. ANON ANON

3 Lipids from land animals M. R. L. Scheeder, ETH Zurich, Switzerland

3.1

Introduction

There are two major sources of land animal derived lipids used for human nutrition: the lipids in animal body tissues, commonly referred to as animal fats or meat fats, and the milk lipids. Eggs contribute to the supply with very long-chain polyunsaturated fatty acids but only a little to total fat intake. The origin and means of obtaining lipids from body tissues and milk, as well as their composition and characteristics, differ in principle. On the other hand, there are common basic endogenous mechanisms and external, mainly dietary, influences which affect animal lipids in a similar way. Furthermore, due to their common animal origin, animal lipids often share a similar public perception and image. This image and the role of land animal derived lipids in human nutrition seems somewhat paradoxical. In a historical context, meat fats and, later on, milk lipids were for long the only readily available and highly appreciated source of fats that was available in substantial amounts for human nutrition. With changes in lifestyle and consumer preferences, arising from health-related concerns about saturated fats and cholesterol on one side, and advances in the technology for obtaining and processing plant oils on the other side, the image of animal lipids changed dramatically, and the food industry is currently endeavouring to replace animal lipids in their products. Animal lipids, nevertheless, possess some desirable, and even beneficial, characteristics. Therefore, it still seems worth while to give an overview on production, characteristics, and use of animal lipids in order to provide some background for assessing potential trends in the use of animal lipids in food.

Lipids from land animals 29

3.2

Animal fats

3.2.1 Historical considerations Adipose tissue of animals has probably been used as food since the dawn of mankind. Meat already made up a big portion in the diets of early hominids (Sponheimer and Lee-Thorp, 2003; Plummer, 2004) and there is strong evidence that foods of animal source, obtained by scavenging or hunting, providing a dense packet of energy and functional nutrients, in the long run promoted human evolution. It is interesting to speculate that with the use of stone tools to crack bones and skulls of mammal carcasses left by larger predators from their kill, early hominids gained access not only to nutritious bone marrow but were also provided with the physiologically functional fatty acids arachidonic (ARA) and docosahexaenoic acid (DHA), which are practically absent from terrestrial plants, but which are the predominant polyunsaturated fatty acids and indispensable building blocks in the brain. An increased consumption of animal derived foods and the resulting improved dietary quality obviously supported the evolutionary development of a large brain (Cordain et al., 2001). Meat made and makes up a major part of hunter-gatherer diets (Cordain et al., 2000), and in these societies the choicest parts of the animals are the fatty tissues (Stefansson, 1945). It has been observed that, particularly under conditions of scarcity, hunter-gatherers may refuse to eat certain parts or even whole animals when they are too lean. This seemingly irrational practice can be explained by the fact that living on a lean meat diet only will actually lead to starvation (Harris, 1985). Stefansson vividly described this phenomenon observed under arctic conditions as ‘rabbit starvation’: when transferred from a diet normally rich in fat to one consisting wholly of rabbit, which contains very little fat, signs of starvation and protein poisoning will appear after a few days and, if no additional fat can be provided, diarrhoea will start and death will occur within weeks (Stefansson, 1945). The human hunger for meat, therefore, can actually be interpreted as hunger for meat fat (Harris, 1985), and it may be assumed that producing fat animals to gain animal fat was originally a major goal of planned livestock production. However, with ongoing industrialization (also in agriculture and animal production) and the already mentioned concomitant changes in affluent societies, the image of animal fats turned from a highly appreciated foodstuff to a by-product of the livestock industry. The technical term ‘animal fats’ or ‘meat fats’ actually refers to fat obtained from adipose tissues of land animals by a process called rendering (Love, 1996). At one time, animal fats also served purposes other than food, e.g. as fuels for torches (and later candles), cosmetics, and even as early oleochemicals for lubricants and soaps (Dugan, 1987). The Celts are known to have manufactured soap from boiling animal fats and ashes, and they probably transmitted their knowledge of fat processing to the Romans (Tufft, 1996). The variety of uses of rendered animal fats grew into manifold areas, and an

30

Modifying lipids for use in food

impressive list of non-food uses of animal fats or fatty acids produced therefrom is given by Dugan (1987). The major edible animal fats are tallow, derived from ruminant species (mainly cattle but also sheep and goat), lard, which is derived from pigs, and poultry fat, derived from poultry offal. More detailed descriptions of named animal fats are given in the Codex Alimentarius Standard 211–9 (FAO/ WHO, 1999). It has to be mentioned that ‘edible’ in this context means fit for human consumption since ‘inedible’ grades still are used as feed for animals.

3.2.2 Sources of animal fats Anatomical sites of fat depots In contrast to plants, fat is found throughout all tissues in animal organisms, although there are specific sites were fat is preferentially stored as so-called depots when meat animals are continuously fed above energy maintenance levels. Fat is typically deposited in the abdominal cavity, particularly around the kidneys and the stomach (internal fat), peripheral under the skin (subcutaneous adipose tissue), between muscles and between muscles and bones (intermuscular fat) and within skeletal muscles (intramuscular fat). In the muscle, lipids are also present as intracellular lipid droplets (particularly in red fibres) and in the form of phospholipids as important components of cell membranes (Wood, 1990). The fatty tissues obtained in the course of ‘dressing’, the part of the slaughter process in which the animal is separated into carcass, offal, and byproducts, are mainly the internal fats from the abdominal cavity. These fats are called slaughter fats, killing-floor fats, or killing fats. Adipose tissue obtained later during the butchering process, when the chilled carcasses are divided into prime cuts and the meat is further being prepared for final consumption, is subcutaneous and intermuscular fat. These are called butcher fats, cutting-floor fats, or simply cutting fats. The intramuscular fat is perceived as marbling when a fat level of about 2 % is exceeded. The degree of marbling is still a major element of the US Department of Agriculture (USDA) beef grading scheme with higher quality grades demanding higher marbling scores, although a direct and relevant cause and effect relation with sensory beef quality traits like tenderness is to be questioned (Savell et al., 1987) and the amount necessary to ensure a good eating quality is often over-estimated (Kempster, 1990). The intramuscular fat, however, is the only fat eaten in carefully trimmed lean-only cuts, and its dietetic value and contribution to the supply of specific fatty acids will be discussed later in Chapter 15. Beside these major sources of edible animal fats, animal lipids can be obtained from bones and rind (e.g. from gelatine production). Bone stock may contain 10–15 % fat (Williams and Hron, Sr, 1996), while bone marrow itself is reported to be the fatty tissue with the highest fat content (about

Lipids from land animals 31 97 %) in its dry matter (Foures, 1996). It is not only a rich but obviously also a tasty source of energy. Stefansson (1945) pleasantly described that Inuit, although they are very fond of their dogs, keep the best parts of hunted animals for human use and he states: ‘one thing dogs never get is the marrow bones’. Under industrial conditions, however, bone fats are rarely extracted and refined for use in food for human consumption, but, ironically, used as feeding-stuff for enhancing the palatability of pet foods (Foures, 1996). Another specific product extracted from bones is neat’s-foot oil, which is used as preferred leather dressing and fine lubricant. Bone marrow is harder the farther it is from the hoof. Toward the toes it is a liquid oil (Stefansson, 1945), because of the high proportion of monounsaturated fatty acids (MUFA), which have melting temperatures below 20 °C. In the bovine digital cushion (specific anatomical structures, which serve as shock absorbers) in the claws, MUFA levels as high as 82 % were found (Raeber et al., 2005). Proportion of adipose tissue and fat in the carcasses The amount of fat in the whole body or carcass depends on genetic factors like species, sex, breed, or even progeny group (i.e. genotype), as well as age and, of course, nutrition. At birth, most animals have only little fat but, when well nourished, body composition will markedly change towards a higher fat content with increasing age and weight. Fat accretion particularly increases in later stages of advancing age. When compared at a comparable physiological age and level of nutrition, females (heifers) are fatter than castrated males (steers), which are fatter than intact males (bulls) in cattle while the order in hogs is castrated male (barrow), female (gilt), male (boar) and in poultry female, male. Species also differ considerably in the distribution of fat deposits. Summarized data from various studies show that in young bulls of about 550 kg live weight internal fat makes up 25–30 % of total fat, subcutaneous fat 15–20 %, intermuscular fat 50–55 %, and intramuscular fat 2–3 %. For barrows of 100 kg live weight the respective figures are 5, 70–75, 20, and 1–2 % (Goutefongea and Dumont, 1990), which means that the major fat depot in pigs is subcutaneous fat while in cattle it is internal fat. The partition of fat, however, does not differ much between sexes, although it is reported that females have slightly more subcutaneous and less intermuscular fat than males (Fisher, 1990). Breeds, in contrast, can differ largely in fat partition. Traditional British beef breeds, for example, have relatively high proportions of subcutaneous and are low in internal fat compared with typical dairy breeds (Fisher, 1990). Selection for animals able to withstand feed shortage during wintertime by using fat depots in the case of traditional British beef breeds on the one hand and the necessity of getting rid of the heat produced by high metabolic rates, demanding a thin subcutaneous fat layer in dairy cattle, on the other may explain these differences. Similarly it has been reported that under influence of high ambient temperatures, subcutaneous fat depots will be diminished in favour of internal fats in pigs (Lefaucheur et al., 1991).

32

Modifying lipids for use in food

Considerable differences in total amount of fat deposits exist between breeds. The 179 German Landrace boars tested in Germany in 2003, for example, showed an average backfat thickness of 12.1 mm at 120 kg live weight, while 2207 Piétrain boars tested under the same conditions averaged 5.9 mm only (Gatzka, 2004). Young bulls of the Limousin breed, a typical continental beef breed, have been reported to contain about 12 % fat in the carcass while Herefords, a typical British beef breed, have on average 20.9 % fat in the carcass (Goutefongea and Dumont, 1990). For young cows (< 5 years) of the same breeds the values were 16.9 and 26.5, respectively. However, a major impact on the amount of fat in meat animals has been exerted by consumer demand for lean meat and the efforts undertaken by animal breeders to meet this demand. The lean-to-fat ratio in meat animals steadily decreased due to successful breeding for leaner carcasses during the last decades. According to reports of the Swiss pig performance testing station (SUISAG), the proportion of dissectible subcutaneous fat in the animals tested decreased from about 17.5 % in 1978 to about 12 % in 2003. It seems worth mentioning that the intramuscular fat (IMF) content had been introduced in the breeding index (because of its assumed positive effect on eating quality) and Swiss pig breeding stock has been selected also for IMF since 1987. From then on IMF increased from about 0.9 % to 2 % in 2003 (Schwörer, 2004). The genetic correlation between the amount of subcutaneous fat and intramuscular fat, therefore, must not be as narrow as one might expect. On the other hand, a linear relation between dissectible carcass fat and lipids in the lean have been reported by Kempster (1986, cited by Wood, 1990) with an increase of 4 % or 6 % dissectible fat for 1 % more lipids in lean beef or pork, respectively. Increasing the IMF content by increasing the feed intensity will, therefore, go along with an excessive increase of the other fat depots. In scientific investigations, very detailed separation between muscle and adipose tissues, nearly anatomical dissection, is often performed to gain precise information about the adipose tissue content in the carcass. Under commercial conditions, some of the bigger muscles might be cleaned from all adhering connective and adipose tissue (peeled, denuded), but some fat will remain with several other meat cuts. Furthermore, parts of the cutting fats are used in processed meat products and not delivered to renderers or fat processors. According to records of commercial slaughter and cutting plants in Switzerland, the proportions of killing fats removed during the slaughter process amounted roughly to 3.25 % of carcass weight for pigs and 4.5 % for cattle. Cutting fats left to fat processors were 7.4 % of cattle carcass weight, 5.5 % for lamb, and only 0.5 % for veal, while the cutting fat from pork was completely used in processed products. For poultry we found in several experiments with broiler chicken an overall average of about 1.75 % abdominal fat in the carcasses. According to records of a Swiss poultry slaughter plant, the actually collected and

Lipids from land animals 33 commercialized amount of fat was about 1.4 % of the chicken carcass weight. Part of this fat is not rendered but directly processed into poultry meat products. On a global scale, however, the proportion of poultry fat available for fat processing might be higher (Section 3.2.3).

3.2.3 Production and supply figures The total production of animal fats depends of course on the number of animals slaughtered and the proportion of fat in the carcasses. Global meat production doubled during the last quarter of a century from 127 million tonnes in 1978 to 257 million tonnes in 2004 (faostat.fao.org). Production of all varieties of meat increased, but the growth rate differed considerably between the meat animal species. Calculated on a per capita base, production of beef and veal even slightly declined while poultry meat production has been growing fastest and the per capita production meanwhile exceeds beef and veal (Fig. 3.1). The biggest share of meat, however, is still coming from pigs. The non-ruminant species therefore clearly dominate meat production. Given the pressure on available grazing areas and the reduced proportion of non-grain biomass of modern hybrid crops, this trend is likely to be continued. While meat consumption in the wealthy, industrialized nations has obviously reached a level of saturation and will hardly grow further, it is predicted that livestock production, and particularly meat animal production, will grow with rather high rates in transition and developing countries (Delgado et al., 1999). This might be particularly interesting in terms of a potential local use of animal fats.

25

50

Beef and veal Pigmeat Chicken meat Mutton and lamb Meat, total

20

45 40

15

kg

30 25

10

20 15

5

10 5

0

2004

2002

2000

1998

1996

1994

1992

1990

1988

1986

1984

1982

1980

1978

0

Years

Fig. 3.1

Changes in annual per capita meat production (faostat.fao.org).

kg, total meat

35

34

Modifying lipids for use in food

In line with the growth in meat animal production, tallow and grease and lard production steadily increased from about 6300 and 4180 thousand tonnes (kt) in 1978 to 8000 and 7270 kt in 2004, respectively. The production per capita, however, slightly declined for tallow and grease from 1.46 to 1.26 kg per year. Lard production only slightly increased from 0.97 to 1.14 kg (Fig. 3.2), although pork production increased from 10.6 to 15.8 kg per person per year during the same period (Fig. 3.1). The breeding and feeding of leaner pigs surely is one explanation. Figure 3.2 also reveals that animal fats are next in importance after the big four plant oils (soybean, palm, canola, and sunflower oil). However, when looking at the share of animal fats relative to the summarized production of the big four plant oils plus animal fats, it becomes evident that animal fats are successively replaced, particularly by palm oil (Fig. 3.3). Poultry production also increased enormously from 23 million tonnes in 1978 to 81 million tonnes in 2005 or, expressed as per capita production, from 5.3 to 12.6 kg per year (faostat.fao.org). Despite this growing importance of poultry, data on the development of poultry fat production during the last decades is not readily available. Recent data from the European Fat Processors and Renderers Association (EFPRA) indicate that poultry fat production in the EU increased from 181 kt in 2001 to close to 200 kt in 2003 (Table 3.1), 18

Soyabean oil Palm oil Rapeseed oil Sunflower oil Tallow and grease Lard Accumulated

7 6 5

16 14 12

kg

10 4 8 3 6 2

kg, accumulated

8

4

03/04

01/02

99/00

97/98

95/96

93/94

91/92

89/90

87/88

85/86

83/84

0

81/82

0

79/80

2

77/78

1

Years

Fig. 3.2 Development of the global production per capita of the four major plant oils, the two major animal fats and the total of all these items (reported on the righthand axis) [sources: oil and fat production: ISTA Mielke GmbH, Oil World (www.oilworld.biz/app.php); world population: US Census Bureau (www.censusgov/ ipc/www/world.html)].

15

10

10

5

5

0

0

93/94

89/90

03/04

15

01/02

20

99/00

20

97/98

25

95/96

25

91/92

30

87/88

30

85/86

35

83/84

35

81/82

40

79/80

40

77/78

[%]

Lipids from land animals 35

Years Soybean oil Palm oil Rapeseed oil Sunflower oil Tallow and Grease Lard

Fig. 3.3 Changes in the proportion of the major oils and fats relative to the annual production of the total of these items [ISTA Mielke GmbH, Oil World (www.oilworld.biz/app.php)]. Table 3.1 Annual production of the different categories of animal fats in EU-15 between 2001 and 2003 (× 1000 tonnes).

2001 2002 2003

Mixed animal fats

Lard

Tallow

Poultry fats

Bone fats

1427 1421 1378

543 518 430

368 378 343

181 186 198

106 127 118

Source: Woodgate (2004).

which is about 7.6 % of all animal fats produced (Woodgate, 2004). According to figures from the US Census Bureau (provided by the National Renderers Association in the USA – www.renderers.org), poultry fat production was 405 and 417 kt in 2003 and 2004, respectively, which is 9.5 % and 9.9 % of total animal fats and greases produced in the USA. These figures imply that poultry fat obtained in the EU and the USA constantly makes up about 2.3 % of poultry meat production. Tallow and grease expressed as proportion

36

Modifying lipids for use in food

of beef and veal produced is, on a global scale, about 13.5 %, quite constant over the last 25 years, while lard obtained from pork production declined from about 9.2 % in 1978 to 7.2 % in 2004.

3.2.4 The rendering process Rendering is the general term for a variety of processes applied to obtain animal fats from fatty animal tissue by separating the fat from proteins (mainly connective tissue) and water. In a more general meaning, the term rendering applies to all the processes of animal by-product processing and is often linked to the ‘invisible industry’ (Tufft, 1996) valorizing inedible animal by-products. Woodgate and Veen (2004) therefore recommend clear differentiation between processing of inedible and edible grade by-products. They suggest using the term ‘fat processing’ as an alternative to rendering when food grade products are produced. This is considered in the name of the European Fat Processors and Renderers Association. The principle of rendering is a rather old technique which has been applied for hundreds or thousands of years. A simple example of rendering (or better fat processing) on a household kitchen scale is the preparing of goosedripping (in this case, however, the proteinaceous ‘residues’, i.e. cracklings or greaves may be the choicest part for some and are often left with the spread). In general, the raw material is first mechanically cut or ground into small pieces with a heat treatment subsequently applied. The main objectives of the heat treatments are to denature the proteins and to achieve cell rupture in order to release the fats. Furthermore, the viscosity of the fats is lowered, facilitating the separation from the solids. The various rendering methods can be classified according to the basic system (continuous or batch), the fat level (natural fat, added fat, de-fatted), process conditions (atmospheric, vacuum, pressure) (Woodgate and Veen, 2004), and, depending on additionally added water or steam, into wet or dry processes. Williams and Hron (1996) describe four general methods in more detail: wet rendering, dry rendering, slurry rendering, and digestive rendering. Dry rendering is the simplest method and can be performed in continuous or batch processes. Fat and solid residue are both dehydrated in heated (steam-jacketed) vessels and then separated by passing over a drainage screen and subsequent pressing of the residues. After settling, centrifuging, and filtering, the fats are ready for market. For slurry rendering, the material is ground to a slurry and pumped through an evaporator for moisture removal under vacuum. The fat is then obtained by two steps, centrifugation and pressing of the solid residue in a screw press. Digestive rendering promotes the release and separation of the fat by adding chemicals or enzymes to hydrolyze or dissolve the connective tissue. Digestion by sodium hydroxide at 85–95 °C and following washing steps is described to recover fat at least equivalent to steam rendering and to yield fat of better quality. However, if the raw material is not fresh, already hydrolyzed fatty acids may form soaps in the aqueous phase. Several further approaches

Lipids from land animals 37 using enzymes are described but are rarely applied commercially, mainly due to the high cost. When lard and edible tallow are produced, wet rendering methods are preferably applied, because the milder conditions yield fats which are light in colour, mild in flavour, and low in free fatty acids compared to dry rendered fats. Water is commonly added in the form of live steam to the cut or ground fatty tissue. The use of water in wet melting processes provides several advantages. The steam displaces the air in the system, reducing oxidative stress. Elevated moisture helps to ensure proper denaturation and hardening of the proteins and rupture of the fat cells. Solid residues, which are mainly proteins, have a greater affinity for the ‘polar solvent’ water than to fat, thus less proteins will migrate into the fat in the presence of water. When quality and source of the raw material are appropriate, the fats resulting from wet melting may be suitable for use as food without further processing. Specific regulations, however, may require further heat treatment; e.g. in Switzerland rendered animal fat must be heated to 133 °C for 20 minutes when used as animal feeding stuff to comply with the regulations (VTNP, 2004). The yield from steam rendering of the usual killing and cutting fats is reported to be 80 and 70 % lard, respectively (Williams and Hron, Sr, 1996), but these figures may vary depending on the raw material and the processes applied. To give an example, the proportion of fats ready for market rendered from fatty tissue has been 71 % for premier jus from beef kidney fat, 46 % rendered fat from beef cutting fats, 61 and 57 % from pork killing and cutting fats, respectively, according to production figures from a Swiss fat processing plant, applying a continuous wet melting process with temperatures slightly below 100 °C (Widmer, 2005). 3.2.5 Composition and properties of animal fats The fatty acids deposited in animal tissues are derived from various sources: from endogenous synthesis or directly from the feed, and from microbial synthesis or modification in the digestive tract. Endogenous synthesis results predominantly in saturated C16 and C18 fatty acids, which in a second step can be desaturated to monounsaturated fatty acids. Dietary fatty acids can be absorbed and deposited as such. In particular, the essential fatty acids, linoleic acid (18:2n-6) and linolenic acid (18:3n-3) with a double bond at the 6th or 3rd carbon from the methyl end (which cannot be introduced by the animal organism and which is the actual essential component) can be further desaturated and elongated to very long chain (C20–C24) highly unsaturated fatty acids like arachidonic acid (20:4n-6), eicosapentaenoic acid (EPA, 20:5n3), and docosahexaenoic acid (22:6n-3). Part of the dietary polyunsaturated fatty acids (and in ruminants it is generally by far the biggest part of the essential fatty acids ingested) will undergo partial or complete microbial biohydrogenation resulting in a variety of conjugated, trans-unsaturated and saturated fatty acids. Branched fatty acids with the methyl group attached to

38

Modifying lipids for use in food

the n-1 (iso) or n-2 (anteiso) carbon, or odd-chain fatty acids originate from microbial synthesis in the rumen or the intestines. The fatty acid composition of animal fats is, therefore, far more complex and variable than in the major plant oils. Tallow, in particular, contains hundreds of different fatty acids, although most of them occur only in trace amounts. In the Codex Alimentarius Standard 211 (FAO/WHO, 1999), characteristic ranges of the fatty acid composition of lard and tallow are defined. Table 3.2 gives these ranges together with the ranges for 1993 (Love, 1996), indicating that the ranges accepted for compliance have been reduced between 1993 and 1999. Due to ruminal biohydrogenation and microbial synthesis, the proportions of saturated, branched, and odd-chain fatty acids are higher in tallow than in lard, while the proportion and range of polyunsaturated fatty acids are lower. In non-ruminants such as pigs and poultry the dietary fatty acids more directly influence the body fat composition, making nutrition an effective tool to manipulate animal lipid composition (as discussed in Chapter 13). Another factor is the amount of body fat. When animals grow fatter, either due to genetic disposition or feeding intensity, the endogenously synthesized fatty acids, i.e. saturated and monounsaturated, will ‘dilute’ the polyunsaturates of dietary origin. Vice versa, the leaner an animal is, the higher the proportion of polyunsaturated fatty acids, with the consequence of lower oxidative stability and consistency. As mentioned above, farm animals are bred and selected for lean body mass. This already causes problems with soft pork fat in the meat processing industry. Another general observation is that animal fats are softer the closer the adipose tissue is situated to the periphery of the body. Internal fats are more saturated than intermuscular fat, which is somewhat more saturated than subcutaneous fat. The lower temperature at the body surface obviously requires a lower melting temperature of the fats, which is achieved by a higher proportion of unsaturated fatty acids. Indeed, it was shown that the ambient temperature directly affects the fatty acid composition of backfat in pigs, most likely by regulating the activity of the enzyme stearoyl-CoA-desaturase, which is the endogenous tool to desaturate stearic to oleic acid and thus adapt fat consistency to the ambient conditions. There is even a difference between the inner and the outer backfat layer, but this might be of more academic interest than practical importance. In modern pigs, the proportion of polyunsaturated fatty acids can easily reach 20 % in leaf fat when plant oils are included in the diet (Eder et al., 2001), and in the subcutaneous backfat linoleic acid can reach 12 %, which is the upper level in the current Codex standard for lard and rendered pork fat, even when no additional oil or fat is supplemented to the feed (Gläser et al., 2002). Poultry fat, as shown in Table 3.3, is even higher in polyunsaturated and lower in saturated fatty acids than pork fat (Foures, 1996). Therefore, although animal fats are often generally blamed as saturated fat, it has to be emphasized that lard clearly contains less than 50 % saturated fatty acids and poultry fat hardly exceeds 30 %. In a survey on rendered animal fats in

Lipids from land animals 39 Table 3.2 Ranges of fatty acid composition (expressed as percentages) and definitions of rendered, named animal fats according to Codex Alimentarius.

6:0  8:0   10:0  12:0  14:0 14:ISO 14:1 15:0 15:ISO 15:ANTI ISO 16:0 16:1 16:ISO 16:2 17:0 17:1 17:ISO 17:ANTI ISO 18:0 18:1 18:2 18:3 20:0 20:1 20:2 20:4 22:0 22:1

Lard1/Rendered pork fat2

Premier jus3/Tallow4

19995

19936

19995

19936

< 0.5 in total

< 0.5 in total

< 0.5 in total

< 2.5

1–2.5 < 0.1 < 0.2 < 0.2 < 0.1 < 0.1 20–30 2–4 < 0.1 < 0.1 <1 <1 < 0.1 < 0.1 8–22 35–55 4–12 < 1.5 <1 < 1.5 <1 <1 < 0.1 < 0.5

0.5–2.5

2–6 < 0.3 0.5–1.5 0.2–1 < 1.5 in total

1.4–7.8 < 0.3 0.5–1.5 0.5–1 < 1.5 in total

20–30 1–5 < 0.5 <1 0.5–2.0 <1 < 1.5 in total

17–37 0.7–8.8 < 0.5 <1 0.5–2 <1 < 1.5 in total

15–30 30–45 1–6 < 1.5 < 0.5 < 0.5 < 0.1 < 0.5 < 0.1 not detected

6–40 26–50 0.5–5 < 2.5 < 0.5 < 0.5

< 0.2 < 0.1 < 0.1

      

20–32 1.7–5 < 0.1 < 0.5 < 0.5       

5–24 35–62 3–16 < 1.5 <1 <1 <1 <1 < 0.1

< 0.5

Note: Samples falling within the appropriate ranges specified below are in compliance with this standard. 1 Pure rendered lard is the fat rendered from fresh, clean, sound fatty tissues from swine (Sus scrofa) in good health, at the time of slaughter, and fit for human consumption. The tissues do not include bones, detached skin, head skin, ears, tails, organs, windpipes, large blood vessels, scrap fat, skimmings, settlings, pressings and the like, and are reasonably free from muscle tissues and blood. 2 Rendered pork fat is the fat rendered from the tissues and bones of swine (Sus scrofa) in good health, at the time of slaughter, and fit for human consumption. It may contain fat from bones (properly cleaned), from detached skin, from head skin, from ears, from tails and from other tissues fit for human consumption. 3 Premier jus (oleo stock) is the product obtained by rendering at low heat the fresh fat (killing fat) of heart, caul, kidney and mesentery collected at the time of slaughter of bovine animals in good health at the time of slaughter and fit for human consumption, as well as cutting fats. 4 Edible tallow (dripping) is the product obtained by rendering the clean, sound, fatty tissues (including trimming and cutting fats), attendant muscles and bones of bovine animals and/or sheep (Ovis aries) in good health at the time of slaughter and fit for human consumption. Sources: 5FAO/WHO (1999); 6 Love (1996).

40

Modifying lipids for use in food

Table 3.3 Average proportion of major fatty acids in poultry fat as well as maximum and minimum mean values reported in a total of seven studies.

14:0 16:0 16:1 18:0 18:1 18:2 18:3

Average

Maximum

Minimum

21.7 6.4 6.2 41.7 18.4 0.9

6 26.7 10.2 7.7 44.7 22 1.8

0.3 19.4 4.1 4.5 39.3 10.1 0.7

Source: Foures (1996).

Germany, a proportion of saturated fatty acids from 36–55 % saturated fatty acids was found (Bahadir et al., 2004). The fatty acid composition of various uncommon animal fats is given by Foures (1996). Horse fat, for example, contains extraordinarily high proportions of linolenic acid, which may be explained by the fact that the predominant polyunsaturated fatty acid in green forage is linolenic acid, and the horse, being a hind-gut fermenter (and not a ruminant), is therefore able to absorb and deposit this fatty acid unmodified and in quite large quantities. The triacylglycerol (TAG) structure also shows some species specificity. During TAG synthesis in pigs, the acyltransferases arrange a very typical positional distribution of fatty acids in TAG with predominantly 16:0 at the central sn-2 position (Christie and Moore, 1970), while the most abundant fatty acid in the sn-1 and sn-3 positions is oleic acid. In subcutaneous fat of cattle 16:0 is located with 41, 17, and 22 mol% in all of the positions 1, 2, or 3, respectively, while 18:1 makes up 41 mol% in the sn-2 position and 20 or 37 mol% at positions 1 and 3 (Christie, 1986). This pattern is similar for poultry fat, where only 15 % of all fatty acid at position 2 is 16:0, but 43 % is 18:1. In both position 1 and position 3 about 25 % is 16:0 and 34 % is 18:1 (Foures, 1996). The ratio of 16:0/18:1cis9 in sn-2 position of lard TAG is about 5, while it is about 0.4 in tallow. The analysis of fatty acids at the sn2 position would, therefore, allow for detecting lard in tallow if lard exceeds 5 % in the blend (Kirk and Sawyer, 1991, cited in Love, 1996). Another unique, even distinctive, property of animal tissues and fats is its cholesterol content. Cholesterol is not found in plants, except for very small traces e.g. in olive oil. Lard, tallow, and poultry fat contain about 86, 89, and 107 mg cholesterol per 100 g fat, respectively; butter contains about 238 and butter fat 286 mg/100 g (Souci et al., 2005). Cholesterol is controversially discussed as a health risk factor and there is widespread belief that dietary cholesterol causes coronary heart diseases (CHD). This belief goes back to Anitschkow’s studies (Anitschkow and Chalatow, 1913) with cholesterol-fed rabbits, in which atherosclerotic lesions were found. The current state of knowledge, however, reveals that dietary

Lipids from land animals 41 cholesterol has no effect on serum LDL:HDL cholesterol ratio, and there is little if any evidence that cholesterol increases the risk of CHD (Parodi, 2004). On the other hand, cholesterol is an inevitable constituent of cell membranes (Mouritsen and Zuckermann, 2004). Its importance for eukaryotic membrane functions is also emphasized by the fact that the biggest part of the cholesterol body pool is endogenously synthesized while dietary cholesterol makes only a modest contribution to it (Parodi, 2004). Cholesterol might nevertheless be of concern because heat treatment in the presence of oxygen may form cholesterol oxidation products, which are considered to promote CHD and cancer. The Codex standard also defines further quality characteristics and maximum levels of undesirable constituents which may result from improper handling, storage, or processing. A low content of free fatty acids (FFA) and a low peroxide value (POV) indicate appropriate raw material and fresh product. Typical commercial specifications, as described by Woodgate and Veen (2004), require a FFA content below 0.5 %, POV below 4 meg/kg, moisture below 0.2 % and insoluble impurities below 0.02 %, which is stricter than the Codex definitions. It is interesting to mention that commercial specifications, particularly for animal fats destined for use in calf milk replacers and pet food, traditionally are more rigorous than legal specifications. Physical characteristics of animal fats One of the most important properties of fats in terms of its technological use is the melting and crystallization behaviour (the dietetic value of animal source fats will be discussed in Chapter 13). The melting behaviour and related traits such as solid fat content and plasticity as well as the crystal habit are determined by the fatty acid composition and the TAG structure. The more saturated and the longer fatty acids are, the higher the melting temperature will be. The melting point is usually measured in an open tubecapillary and given as temperature when all solid disappears. Characteristic melting temperatures for animal source fats are given in Table 3.4. Butter, despite the rather low iodine value (i.e. high degree of saturation), shows a comparably low melting point due to the high proportion of short- and mediumchain fatty acids (high saponification number). Within the animal fats with a comparable saponification number (i.e. mean chain length), the melting point is negatively correlated with the iodine number. Neat’s-foot oil, due to its high quantity of monounsaturated fatty acids, is still liquid below the freezing point of water. This also underlines the fact that it is not only the number of double bonds but the proportion of unsaturated fatty acids which determines the melting behaviour (Gläser et al., 2004). Furthermore, fatty acids with double bonds in trans configuration have melting temperatures above those of the corresponding cis fatty acids. In lard, trans fatty acids normally occur only in trace amounts, but when fed to pigs, trans fatty acids not only accumulate in adipose tissue but also inhibit the endogenous

42

Modifying lipids for use in food

Table 3.4

Characteristics of typical animal derived fats.

Butter Lard Neat’s-foot oil Beef tallow Mutton tallow

Iodine value

Saponification number

Melting point [°C]

25–42 53–77 69–76 40–48 35–46

210–233 190–202 190–199 190–199 192–197

28–35 33–46 –4 40–48 44–51

Source: Dugan (1987).

desaturation of stearic to oleic acid, which tremendously increases firmness of the lard (see Chapter 13). The melting point, however, is not the temperature at which all solid turns to liquid. Even at temperatures fairly below the melting points, the fats contain some liquid phase. Meat and milk fats, therefore, appear to be solid at room temperature, but actually only part of the fat is crystallized while the other part is trapped as liquid oil in the crystal network. This is one of three requirements for a plastic fat. When, additionally, there is a proper proportion between the two phases and a sufficiently fine dispersion of the solid phase then a plastic fat results (Metzroth, 1996). Satisfactory plasticity is achieved when the solid fat resists small stress but, when sufficient forces are applied to cause deformation, it acts like a viscous liquid (as when spreading a ‘solid’ fat on bread or toast the fat is deformed by the pressure applied). Therefore, to maintain the desired solid fat content over a wide temperature range, flat melting curves are desired. Due to their high variety of fatty acids and TAG, animal derived fats generally have flat melting curves. Fats solidify in several crystalline polymorphic forms. It is agreed that at least three forms exist, α, β and β ′ (beta-prime), with β and β ′ being the two desirable stable forms. β ′ forms small, uniform, needle-like crystals, exhibits smooth texture and is able to enmesh a higher amount of liquid phase than the β polymorphic form, which is more coarse textured with large granular crystals. For use as shortening with high plasticity, the preferred polymorphic form is β ′. Animal derived fats typically tend to crystallize in β ′-form (Table 3.5). Only unmodified lard tends to crystallize in β-form, building large and grainy crystals, although lard contains a high proportion of palmitic acid, which usually promotes β ′-type crystallization (Wiedermann, 1978). This is due to the specific TAG structure of lard, with palmitic acid preferentially attached to the sn-2 position, while shorter fatty acids at the sn-1(3) positions and symmetrical TAG promote β ′ polymorphic form. On the other hand, unmodified lard is specifically used to give pie crusts a desirable flaky texture (Metzroth, 1996). Modifying lard by interesterification will result in a β ′-type shortening.

Lipids from land animals 43 Table 3.5

Classification of fats and oils according to crystal habit.

Beta-type ( β )

Beta-prime-type (β ′ )

Soybean Safflower Sunflower Sesame Peanut Corn Canbra (canola) Olive Coconut Palmkernel Lard Cocoa butter

Cottonseed Palm Tallow Herring Menhaden Whale Rapeseed Milk fat (butter oil) Modified lard

Source: Wiedermann (1978).

3.2.6 Use of animal fats in food Rendered or processed animal fats fit for human consumption have traditionally been used as shortenings in baked goods, spreads and margarine, and as (deep-) frying fats – in industry or the catering business as well as in private households. Traditionally, yellow grease acquired by renderers from food service establishments was of animal origin (Kiepper, 2001). Although they are still commercially offered for these purposes, the use of animal fats in food has declined. The major part of rendered animal fats is currently used as livestock feeds, cosmetics and industrial oils, and fats for producing fuel, lubricants and soaps. Only 10 % of the 2.77 million tonnes of animal fats in Europe were used as food in 2002 (Woodgate, 2004). In 2003 the amount of animal fats produced decreased slightly to 2.6 million tonnes and the proportion used for food fell considerably to 6 %, which was below the amount used for pet food (7 %). The share used as feed remained constant at 25 %. About 31 % was used for soap and oleochemicals and another quarter (24 %) as energy substitute. According to figures from the US Census Bureau (M311K series for Fat and Oils: Production, Consumption and Stocks), provided by the National Renderers Association (www.renderers.org), only a small and decreasing part of edible tallow produced is actually consumed for edible use (from 17.3 % in 1998 to 11.8 % in 2004) in the USA. The proportion of lard consumed for edible uses increased in the same time from about 55 to 73 % but, due to the overall decrease in lard production, the total amount of lard used for edible purposes declined from 135.9 to 84.2 kt. The share of lard and tallow consumed as food, therefore, decreased from 6.5 % of all animal fats and greases produced in the USA in 1998 to 4.4 % in 2004. However, an important contribution to the intake of animal lipids is its consumption as part of meat and meat products. The contribution of fresh meat to the consumption of fat might nevertheless be over-estimated because

44

Modifying lipids for use in food

fat is lost during cooking and visible subcutaneous and intermuscular fat is often discarded on the plate. Cooking loss and leftovers can reduce the actual fat intake by 35–65 % of the original fat content of the raw cut (Sheard et al., 1998; Gerber et al., 2004). Processed meat products, in contrast, may contain a fair bit of added cutting fats, particularly from pork, and killing fat (abdominal fat) in the case of poultry, which is directly used in products like salami or frankfurter type sausages. The composition of animal fat is, therefore, still of relevance for human nutrition.

3.2.7 Legal issues A useful overview of fat processing and rendering, including regulations and directives concerning meat by-product processing, is given by Woodgate and Veen (2004). The most relevant regulations concerning animal fats fit for human consumption or use in feed are the ‘Meat Products Directive’ 77/ 99/EEC (EU, 1977; EU, 1992) and the ‘Animal By-products Regulation’, ABPR, 1774/2002/EC (EU, 2002). Health scare issues, in the first instance transmissible spongiform encephalopathies e.g. BSE and scrapie, also led to intensive scientific investigation of tallow as a potential transmission vector. Woodgate and Veen (2004) summarized the state of knowledge concisely, concluding that there is no scientific evidence supporting the hypothesis that tallow might be risk factor for BSE transmission. However, the fact that in Germany the use of even edible grade animal fats as feeding stuff is prohibited leads to the conclusion that political and marketing decisions rather than scientific evidence will, to a large extent, determine the current and future use of animal fats in feed and food industry.

3.3

Milk fat

Milk is secreted by the mammary gland of female mammals after parturition to nourish the progeny. This biological concept evolved a great compositional variety of milk (Oftedal, 2005). The content of lipids, which is the milk’s most energy dense constituent, can be as low as 1.4 % for example in equines (ass, horse), and as high as 54 % in seals (Kirk and Sawyer, 1991). Milk became available as another valuable food source with the domestication of animals in the course of the neolithic revolution, and the first signs indicating the use of sheep, goats, and cattle for milking date back to 5000 BC (Teuber, 2000). The first documented evidence for dairy products was found in Sumerian bookkeeping documents from about 3200 BC, and it can be assumed that butter oil was among the earliest processed products because of its preferable storage characteristics due to microbiological stability (Teuber, 2000).

Lipids from land animals 45 3.3.1 Sources of milk fat The synthetic and secretory tissue of the mammary gland is composed of millions of alveoli connected and drained via terminal ducts to progressively larger ducts and ultimately to the collecting spaces, the gland and the teat cisterns. The alveoli are lined with a single layer of mammary epithelial cells that synthesize and secrete the milk. The TAG secreted with the milk are esterified in the mammary epithelial cells and originate either from fatty acids derived from de novo synthesis via acetyl-CoA carboxylase and fatty acid synthase in the cells or exogenously supplied as plasma lipids. The exogenously supplied fatty acids in plasma can be non-esterified fatty acids, which primarily originate from adipose tissue stores, or from the TAG in lipoproteins that originate from enterocytes of the gut [chylomicrons and very low-density lipoprotein (VLDL)] or from the liver (VLDL). The TAG in lipoproteins have to be hydrolyzed extracellularly by lipoprotein lipase to release the fatty acids. Since ruminant diets commonly contain only little fat, VLDL and chylomicrons from the gut contribute only modestly to the TAG synthesized in the epithelial cells compared with conditions prevailing in non-ruminant mammals (Clegg et al., 2000). The mechanism by which intracellular lipid droplets, the precursors of milk lipid globules, are formed is still a matter of conjecture. It has been hypothesized that TAG accumulate in between the bilayer membrane of the endoplasmic reticulum (ER) and are released surrounded by the outer, cytoplasmic membrane of the ER (Keenan and Patton, 1995). These droplets with a TAG-rich core fuse to larger cytoplasmic lipid droplets. It is suggested that the lipid droplets become surrounded by the apical membrane of mammary epithelial cells as they bud from the epithelial cells and are released as milk lipid globules. The size of the globules in raw bovine milk ranges from 0.1 to 15 µm (with about 90 % of all globules ranging between 1 and 8 µm). These globules make up about 95 % of total milk lipids, present as an oil-in-water emulsion (Tong and Berner 1994). The term ‘milk’ commonly refers to bovine milk, because the bulk of the milk produced for human consumption originates from cows (Bos taurus or Bos indicus). Milk from other species is described by adding the species of origin, e.g. buffalo milk. According to Food and Agriculture Organization (FAO) statistics, 84 % of global milk production in 2004 was from cows, 12.5 % from buffalo, 1.3 % from sheep, 2 % from goats, and 0.2 % from camels (faostat.fao.org). While the share of production from sheep and camels remained quite constant and goat milk only slightly increased its share over the last decades, a clear shift from cow milk, which made up 91 % of total milk production in 1978, to buffalo milk (5.7 % in 1978) can be observed. Total milk production increased during that time from about 452 to 613 million tonnes. The production per capita, however, declined from about 96 kg cow milk per year to 81 kg (Fig. 3.4). Concomitantly the supply of butter decreased, while, according to the increased share of buffalo milk, production of ghee from buffalo milk constantly increased from 1986 on.

46

Modifying lipids for use in food 2.0

100

80

60 1.0 Butter of cow milk Ghee, buffalo Butter and ghee, total Cow milk, fresh

0.0

20

1978 1979 1980 1981 1982 1983 1984 1985 1986 1987 1988 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004

0.5

40

Fresh milk [kg]

Fat [kg]

1.5

0

Years

Fig. 3.4 Development of the annual production per capita of milk, butter (cow milk) and ghee (buffalo) and total milk fat from 1978 to 2004 (faostat.fao.org).

3.3.2 Composition of the milk lipids The milk lipids mainly consist of TAG (about 97 %) and much smaller proportions of phospholipids and sterols, which are mainly associated with the membrane of milk lipid globules (Table 3.6). Mono- and diacylglycerols, free fatty acids, cholesteryl esters, lipid-soluble vitamins, carotenoids, and squalene also occur in minor amounts as well as trace amounts of hydrocarbons (Bitman and Wood, 1990; NDC, 2005). The proportions of cholesterol, and to a certain extent also phospholipids, tend to decline during lactation (Table 3.6), while the proportions of TAG and also of the phospholipid classes remain quite constant (Bitman and Wood, 1990).

Table 3.6

Lipid class composition of cows’ milk during lactation. Percentage at day of lactation

Lipid class

3

7

42

180

Phospholipid Cholesterol Triacylglycerol 1,2-Diacylglycerol Free fatty acids Monoacylglycerol Cholesteryl esters Fat content [g/dl]

0.72 0.53 97.35 1.01 0.26 0.06 0.05 3.45

1.06 0.41 97.11 1.16 0.19 0.06 0.03 3.97

1.11 0.46 95.8 2.25 0.28 0.08 0.02 3.25

0.56 0.3 97.17 1.72 0.18 0.03 0.04 3.13

Source: Bitman and Wood (1990).

Lipids from land animals 47 The fatty acid composition and TAG structure of milk fat is exceptionally complex. To give an example of the major fatty acids in milk fat, the composition of the EU BCR-Standard for anhydrous milk fat is given in Table 3.7. However, over 400 different fatty acids have been described in milk fat, ranging from chain length of four to 28, including odd- and branchedchain fatty acids, unsaturated fatty acids with one to six double bonds, and various positional and geometrical isomers including conjugated fatty acids (Jensen and Newburg, 1995). As a consequence of this impressive number of fatty acids, thousands of different TAG may occur. The structure and proportion of more than 220 TAG have been investigated and described in detail (Gresti Table 3.7 Fatty acid composition of the EU Food and Agriculture Anhydrous milk fat standard (BCR-164). Fatty acids

Mass fraction fatty acid (g/100 g)

Butyric acid

3.49 ± 0.06

Other 6:0 8:0 10:0 10:1 ω1 12:0 12:1 ω9 14:0 iso 14:0 14:1 ω5 15:0 iso 15:0 anti-iso 15:0 16:0 iso 16:0 total 16:1 ω7 16:1 total 17:0 iso 17:0 anti-iso 17:0 17:1 18:0 iso 18:0 total 18:1 total 18:2 ω6 cis1 18:2 conj 18:2 total 18:3 ω3 cis 20:0 20:1 ω9 cis Other

Mass fraction fatty acids (g/100 g) 2.36 ± 0.19 1.36 ± 0.10 2.89 ± 0.12 (0.3) 4.03 ± 0.10 (0.1) (0.1) 10.79 ± 0.35 (1.1) (0.3) (0.5) (1.0) (0.2) 26.91 ± 0.84 (1.5) (1.5) (0.5) (0.4) (0.5) (0.3) (0.1) 10.51 ± 0.40 24.82 ± 0.61 (1.8) (0.9) 2.68 ± 0.40 0.51 ± 0.04 (0.1) (0.2) (2.5)

Note: Values in brackets are not certified. 1 Includes isomers.

48

Modifying lipids for use in food

et al., 1993; Jensen and Newburg, 1995). None of the TAG exceeded a proportion of 5 %, indicating the high variability, but the proportion of observed TAG deviated significantly from the proportions expected from a random distribution of fatty acids to the three positions in the TAG. Obviously TAG with one short-chain fatty acid (4:0 or 6:0) and two fatty acids in the range C12 to C18 are preferentially synthesized by the mammary epithelial cells. This highly diverse composition and structure give milk lipids their characteristic properties. Melting of milk fat occurs over a very wide range starting at around –30 °C and being completed at about 37 °C (Hettinga, 1996), although some TAG with melting points up to 75 °C might be present (Tong and Berner, 1994). Together with the tendency to crystallize in the β ′ polymorphic form (Table 3.5), milk fat fulfils the preconditions for a plastic fat, making it a desirable shortening, which moreover may provide a characteristic flavour. Mimicking the flavour of milk fat remains a challenge to food chemists. Oxo- and hydroxy fatty acids are precursors of flavouractive ketones and lactones. Aldehydes, oxidation products of unsaturated fatty acids, also contribute to the flavour as do volatile short-chain fatty acids and branched fatty acids directly (Jensen and Newburg, 1995).

3.3.3 Obtaining milk fat The short-chain fatty acids, on the other hand, can produce a highly unpleasant flavour in raw milk when milk fat globules are destroyed and the released TAG left prone to the hydrolyzing action of lipase. Severe pumping or agitation of the raw milk and unfavourably designed pipelines causing turbulent flow should therefore be avoided. The milk fat globule membrane also prevents instant flocculation. For obtaining butter, the milk fat globules have to be destabilized to break the oil-in-water emulsion and to form fat granules. In early times this was achieved by churning the milk. Commonly, the first basic step in butter manufacture is the separation of cream from milk, which is possible due to differences in the specific gravity between fat globules and milk serum. Globules below 0.8 µm are considered as non-separable (Hettinga, 1996). The next steps are churning, aggregation of the fat granules, separating the buttermilk, formation and stabilization of the water-in-oil emulsion, and finally packaging. Butter manufacture can be performed in batch systems but, except for small butter plants, butter is today commonly produced in continuous systems (Hettinga, 1996; Tong and Berner, 1994). According to the Codex standard for butter [Codex Alimentarius Standard A-1 (FAO/ WHO, 1971)], butter must be derived exclusively from milk and/or products obtained from milk and must contain at least 80 % fat and a maximum of 16 % moisture. Spreadability is an important trait for butter, and several approaches to achieving the desired characteristics have been described (Hettinga, 1996), including mechanical treatment, temperature profiling of the cream, blending

Lipids from land animals 49 winter and summer butter, fractionation, interesterification, and the diet of the cow. To further concentrate butter or cream to anhydrous milk fat it is necessary to break the emulsion and to remove non-fat solids and water. For this purpose, disruption of the milk fat globule membrane has to be achieved mechanically by homogenization or with citric acid as a chemical measure to destroy the membranes. In several steps, applying elevated temperatures up to 95 °C under vacuum, the butter oil is then separated and dehydrated to a minimum fat content of 99.8 %. The physical properties may than be adapted by applying the fractionation technique. Ghee is another product obtained exclusively from milk or fat-enriched milk products, which, may however, originate from various species. Water and non-fat solids are removed as with anhydrous milk fat but at higher temperatures. This also leads to a strong (desired) buttery flavour. Ghee is also produced from anhydrous milk fat by adding ethyl butyrate or alternate synthetic flavours (Hettinga, 1996).

3.3.4 Uses of milk fat in foods Butterfat is used as ingredient in a variety of foods such as pastry, cake and biscuit products, ice cream, and confectioneries. Its major role is probably to provide the highly esteemed organoleptic sensations like flavour and texture. As a plastic fat, it serves as shortening in bakery applications, therefore helping to achieve the desired texture in the products. Its melting behaviour (modified by fractionation if required) is favourable for use in confectioneries and it is able to inhibit fat bloom in chocolate (see Chapter 20). Milk fat, therefore, fulfils nutritional, technological, and organoleptic functions in processed non-dairy foods. Milk fat is of course also consumed as such in milk and dairy products. Regarding recent trends to replace milk fat in ‘yoghurt’ by plant oils and fats, it should be mentioned that according to the Dictionary of Dairy Terminology (IDF, 1996), dairy or milk products must be derived ‘…exclusively from milk including added substances necessary for the manufacturing process, provided that these substances are not intended to take place in part or in whole of any milk constituents.’ Yoghurt with the milk fat replaced by sunflower oil may, therefore, no longer seriously be called a dairy product. Such approaches by the food industry obviously aim to meet public health concerns related to milk fat, particularly its content of saturated fatty acids and cholesterol. As the issue of nutritional health cannot be discussed here in detail, it will suffice to refer to the excellent review given by Parodi (2004). In brief, metabolic control of endogenous synthesis of cholesterol compensates for dietary intake in healthy subjects. Only for people with the inherited defect familial hypercholesterolaemia might dietary cholesterol intake be of concern. The impact of dietary cholesterol has been over-emphasized (Parodi, 2004). Potential health benefits related to the fat composition of milk fat and fats of animal origin in general will be discussed later (Chapter 13).

50

3.4

Modifying lipids for use in food

Future trends

A basic assumption for speculating about future trends in the use of animal derived fats is that animal production will grow, particularly in developing countries (Delgado, 2003). Fats of animal origin will, therefore, be available in growing quantities. The prevailing conditions might further support meat production with monogastric animals and, to a lesser extent, meat and milk from ruminants. In the recent past, a clear decline in the use of animal source fats in food has been observed. In a situation where marketing rather than scientific evidence is assumed to rule decisions in the food industry, it is expected that the amount of animal fat used in food will remain on a low but constant level in the developed countries, indicating the growing importance of ways to valorize meat fats via animal feed or even oleochemical and technical uses. The use of meat fats in animal feed – from an economical point of view the second best solution – will surely require the same, very high standards as for animal fats destined for use in human nutrition. In view of the overall increasing demand for food of animal origin (particularly meat), recycling of animal fat as feeding-stuff is surely a reasonable way to contribute to the sustainability of animal production systems. Separate processing of bovine, porcine, and poultry material will help the industry to be prepared for potential restrictions concerning intra-species recycling. Intraspecies recycling of processed animal protein as feed component is already prohibited. This will also help to cope with religious taboos on nutrition, an aspect which should not be under-estimated. Processed food products containing lard as ‘hidden fat’ will be rejected by members of Jewish and Islamic communities. On the other hand, a certain revival of meat and milk fat for use in food could be expected for several reasons. The major drawback for animal derived fats, dietary health concerns, may be weakened by results of ongoing reevaluations of the actual role of saturated fatty acids in diseases of affluence and a more differentiated view on the effects of specific saturated fatty acids. Upcoming production and marketing approaches making use of feeding strategies to improve the nutritional value of meat and milk fats by modifying the fatty acid composition may further help to change the reputation of animal source fats. Modern trends towards so-called low-carb-diets are also a sign of a changing perception of the role of fats. In the case of meat fats, high quality assurance and traceability standards in combination with potential control tools (like use of staining or/and invisible (chemical) markers in banned material) to avoid incorporation of banned products in the feed or food chain could increase safety and confidence (Woodgate and Veen, 2004). At the same time, the pressure on partly hydrogenated oils, which have replaced animal fats in several applications, is likely to increase due to the content of trans fatty acids, which are identified as a potent health risk factor. The discussion about the relevance of the origin of trans fatty acids (ruminal biohydrogenation in animals vs technological hydrogenation) will continue and probably trigger scientific work. There is

Lipids from land animals 51 at least some evidence that the intake of trans fatty acids from ruminant products generally is too low to exert negative effects in comparison to trans-intake from technological sources (Weggemans et al., 2004). It may also be expected that outside Europe, North America and Oceania, the use of animal fats for food will face less aversion and restrictions. However, the supply of animal derived fats directly by consuming animal source food (meat, milk, and their products) will be much more relevant for human nutrition than the supply by ‘hidden fats’ as ingredients in, for example, bakery products, frying fats, etc. Measures to improve the composition of animal lipids will, therefore, be of lasting relevance.

3.5

Sources of further information and advice

The present chapter can give only a brief summary and selected topics in the field of lipids of animal origin. For more detailed information about specific aspects of history, production, processing, nutrition, etc. the reader is advised to refer to more detailed and focused sources, some of which are listed and briefly introduced below. As a general source of further information and advice, Bailey’s Oil and Fat Products, the comprehensive reference work on the food chemistry and processing technology related to edible oils and fats and their non-edible by-products, has to be mentioned (now available as a sixth edition, 2005). Those who are interested in more specific information about meat and meat fats can find details in The Science of Meat and Meat Products (Price and Schweigert, 1987). This book provides timeless basic knowledge by combining fundamental scientific information about meat biology, physics, and chemistry with applied aspects in production and processing, including an extensive chapter about meat animal by-products also covering meat fats with a lot of basic data, definitions, and explanations (Dugan, 1987). In the context of meat science, of course, the standard work Lawrie’s Meat Science has to be mentioned (Lawrie, 1998). A great deal of specific information about the historical background of breeding leaner animals, fat metabolism, and measures which can be taken to control fat accretion in farm animals is given in Reducing Fat in Meat Animals (Wood and Fisher, 1990). This book also covers marketing aspects and implications for human nutrition and health. For a deeper insight in the evolutionary background of human nutrition in terms of physiological as well as social adaptation and how ‘meat eating and human evolution’ are related, an entertaining collection of essays can be recommended (Stanford and Bunn, 2001). Vividly written explanations about the background of more recent nutritional taboos and habits and the historical and socio-economic reason why some foods are ‘good to eat’ and some apparently are not are given by Marvin Harris (Harris, 1985).

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Exhaustive information about milk composition, not only from bovine origin but also human milk can be drawn from the Handbook of Milk Composition (Jensen, 1995)

3.6

References

ANITSCHKOW N,

and CHALATOW S (1913), Ueber experimentelle Cholesterinsteatose und ihre Bedeutung für die Entstehung einiger pathologischer Prozesse, Centralbl Allg Pathol Pathol Anat, 24, 1–9. BAHADIR M, BOCK R, DETTMER T, FALK O, HESSELBACH J, JOPKE P, MATTHIES B, MEYER-PITTROFF R, SCHMIDT-NÄDERL C, and WICHMANN H (2004), Chemisch-analytische Charakterisierung technischen tierischen Fettes aus einer Tierkörperbeseitigungsanstalt, Umweltwissenschaften und Schadstoff-Forschung: Zeitschrift für Umweltchemie und Oekotoxikologie, 16 (1), 19–28. BAILEY A E, (1996), Bailey’s Industrial Oil and Fat Products, 5th edn, New York, Wiley Interscience. BAILEY A E, (2005), Bailey’s Industrial Oil and Fat Products, 6th edn, Hoboken, NJ, Jossey-Bass / John Wiley and Sons, Inc. BITMAN J, and WOOD D L (1990), Changes in milk fat phospholipids during lactation, J Dairy Sci, 73 (5), 1208–1216. CHRISTIE W W, (1986), The positional distribution of fatty acids in triglycerides, in Hamilton R. J. and Russel J. B, Analysis of Fats and Oils, New York, Elsevier Science Publishing Co., Inc., 313–339. CHRISTIE W W, and MOORE J H (1970), A comparison of the structures of triglycerides from various pig tissues, Biochim Biophys Acta, 210 (1), 46–56. CLEGG R A, BARBER M C, POOLEY L, ERNENS I, LARONDELLE Y, and TRAVERS M T (2000), Milk fat synthesis and secretion: molecular and cellular aspects, Livestock Prod Sci, 70, 1–2, 3–14. CORDAIN L, MILLER J B, EATON S B, MANN N, HOLT S H, and SPETH J D (2000), Plant-animal subsistence ratios and macronutrient energy estimations in worldwide hunter-gatherer diets, Am J Clin Nutr, 71 (3), 682–692. CORDAIN L, WATKINS B A, and MANN N J (2001), Fatty acid composition and energy density of foods available to African hominids – evolutionary implications for human brain development, World Rev Nutr Dietet, 90, 144–161. DELGADO C L (2003), Rising consumption of meat and milk in developing countries has created a new food revolution, J Nutr, 133 (11), 3907S–3910. DELGADO C L, ROSEGRANT M W, STEINFELD H, EHUI S, and COURBOIS C, (1999), Livestock to 2020. The next food revolution, 2020 Vision for Food, Agriculture, and the Environment, Discussion Paper No. 28, Washington, DC, International Food Policy Research Institute. DUGAN L R JR, (1987), Meat animal by-products and their utilization, Part 1. Meat fats, in Price J. F. and Schweigert B. S., The Science of Meat and Meat Products, 3rd edn, Westport, CT, Food and Nutrition Press, Inc., 507–530. EDER K, NONN H, and KLUGE H (2001), The fatty acid composition of lipids from muscle and adipose tissues of pigs fed various oil mixtures differing in their ratio between oleic acid and linoleic acid, Eur J Lipid Sci Technol, 103, 668–676. EU (1977), Council Directive 77/99/EEC of 21 December 1976 amended and updated into council directive 92/5 EEC of February 10,1992 on health problems affecting intraCommunity trade in meat products, Official Journal, L026, no. 31/01/1977, 85–100. EU (1992), Council Directive 92/5/EEC of 10 February 1992 amending and updating Directive 77/99/EEC on health problems affecting intra-Community trade in meat products and amending Directive 64/433/EEC, Official Journal, L057, no. 02/03/ 1992, 1–26.

Lipids from land animals 53 (2002), Regulation (EC) No 1774/2002 of the European Parliament and of the Council of 3 October 2002 laying down health rules concerning animal by-products not intended for human consumption, Official Journal, L273, no. 10/10/2002, 1–95. FAO/WHO (1971), Codex Standard A1 Butter, revised 1999, amended 2003, Rome/Geneva, FAO, WHO, available at: www.codexalimentarius.net/web/index_en.jsp. FAO/WHO (1999), Codex Standard 211 Named Animal Fats, Rome/Geneva, FAO/WHO, available at: www.codexalimentarius.net/web/index_en.jsp. FISHER A V (1990), New approaches to measuring fat in the carcasses of meat animals, in Wood, J. O. and Fisher, A. V., Reducing Fat in Meat Animals, London and New York, Elsevier Science Publishing Co., Inc., 255–343. FOURES C (1996), Animal tissues – fats from land animals, in Karleskind, A and Wolff, J P, Oils and Fats Manual, vol. 1, Paris, Lavoisier Publishing, 247–266. GATZKA E M (2004), Zahlen aus der Deutschen Schweineproduktion 2003, Alfter-Witterschlick, Druckerei Martin Roesberg. GERBER N, WENK C, and SCHEEDER M R L (2004), Influence of cooking and trimming on the actual intake of nutrients from meat, in British Society of Animal Science, Pig and Poultry Meat Quality Genetic and Non-genetic Factors, Nottingham, Nottingham University Press, 59. GLÄSER K R, WENK C, and SCHEEDER M R L (2002), Effect of dietary mono- and polyunsaturated fatty acids on the fatty acid composition of Pigs’ Adipose Tissues, Arch Anim Nutr, 56, 51–65. GLÄSER K, WENK C, and SCHEEDER M R L (2004), Evaluation of pork backfat firmness and lard consistency using several different physicochemical methods, J Sci Food Agric, 84 (8), 853–862. GOUTEFONGEA R, and DUMONT J P (1990), Developments in low-fat meat and meat products, in Wood, J. D. and Fisher, A. V., Reducing Fat in Meat Animals, Elsevier Science Publishing Co., Inc., 398–436. GRESTI J, BUGAUT M, MANIONGUI C, and BEZARD J (1993), Composition of molecular species of triacylglycerols in bovine milk fat, J Dairy Sci, 76 (7), 1850–1869. HARRIS M, (1985), Good to Eat. Riddles of Food and Culture, Long Grove, IL, Waveland Press, Inc. HETTINGA D, (1996), Butter, in Hui, Y.H. Bailey’s Industrial Oil and Fat Products, 5th edn, vol. 3, New York, John Wiley and Sons, Inc., 1–63. IDF (1996), Dictionary of Dairy Terminology, 2nd edn, Amsterdam, Elsevier Science B V. JENSEN R G, (1995), Handbook of Milk Composition, San Diego, CA, Academic Press. JENSEN R G, and NEWBURG D S (1995), Milk Lipids. B. Bovine milk lipids, in Jensen R. G. Handbook of Milk Composition, San Diego, CA, Academic Press, 543–575. KEENAN T W, and PATTON S, (1995), The structure of milk: implication for sampling and storage. A. The milk lipid globule membrane, in Jensen, R. G., Handbook of Milk Composition, San Diego, CA, Academic Press, 5–49. KEMPSTER A J, (1990), Marketing procedures to change carcass composition, in Wood J. D. and Fisher, A. V., Reducing Fat in Meat Animals, London and New York, Elsevier Science Publishing Co., Inc. 437–458. KIEPPER B, (2001), Characterization of the generation, handling and treatment of spent fat, oil, and grease (FOG) from Georgia’s food service industry, available at: http:// www.engr.uga.edu/service/outreach/FOG%20Report%20Final.pdf. KIRK R S, and SAWYER R, (1991), Dairy products I, in Kirk, R.S. and Sawyer, R., Pearson’s Composition and Analysis of Foods, Harlow, Longman Scientific and Technical, 530– 574. LAWRIE R A, (1998), Lawrie’s Meat Science, 6th edn, Cambridge, Woodhead Publishing. LEFAUCHEUR L, LE DIVIDICHE J, MOUROT J, MONIN G, ECOLAN P, and KRAUSS D (1991), Influence of environmental temperature on growth, muscle and adipose tissue metabolism, and meat quality in swine, J Anim Sci, 69 (7), pp. 2844–2854. LOVE J A, (1996), Animal fats, in Hui, Y. H., Bailey’s Industrial Oil and Fat Products, 5th edn, vol. 1, New York, John Wiley and Sons, Inc., 1–18. EU

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METZROTH D J, (1996), Shortening: science and technology, in Hui, Y. H., Bailey’s Industrial

Oil and Fat Products, 5th edn, vol. 3, New York, Wiley Interscience., 115–160. and ZUCKERMANN M J (2004), What’s so special about cholesterol?, Lipids, 39 (11), 1101–1113. NDC (2005) Newer knowledge of dairy foods, available at http:// www.nationaldairycouncil.org/NationalDairyCouncil/Nutrition/Products/table18.pdf. OFTEDAL O T, (2005), Milk composition, species comparison, in Pond, W. G. and Bell, A. W., Encyclopedia of Animal Science, New York, Marcel Dekker, Inc., 625–628. PARODI P W, (2004), Milk fat in human nutrition, Aust J Dairy Technol. 59, 1, 3–59. PLUMMER T, (2004), Flaked stones and old bones: biological and cultural evolution at the dawn of technology, Yearbook of Physical Anthropology, 47, 118–164. PRICE J F, and SCHWEIGERT B S, (1987), The Science of Meat and Meat Products, 3rd edn, Westport, Food and Nutrition Press, Inc. RAEBER M, SCHEEDER M R L, OSSENT P, LISCHER CH. J, and GEYER H (2005), The content and composition of lipids in the digital cushion of the bovine claw with respect to age and location – a preliminary report, The Veterinary Journal, vol. in press. SAVELL J W, BRANSON R E, CROSS H R, STIFFLER D M, WISE J W, GRIFFIN D B, and SMITH G C (1987), National consumer retail beef study: palatability evaluations of beef loin steaks that differed in marbling, J Food Sci, 52, 517–519. SCHWÖRER D, (2004), Lipide im Schweinefleisch – Herausforderung für die Zucht, in Kreuzer M., Wenk C. and Lanzini T., Lipide in Fleisch, Milch und Ei – Herausforderung für die Tierernährun, Schriftenreihe Institut für Nutztierwissenschaften, ErnährungProdukte-Umwelt, ETH Zürich, 25, 22–36. SHEARD P R, WOOD J D, NUTE G R, and BALL R C (1998), Effects of grilling to 80 °C on the chemical composition of pork loin chops and some observations on the UK National Food Survey estimate of fat consumption, Meat Sci, 49 (2), 193–204. SOUCI FACHMANN, and KRAUT (2005), Food composition and nutrition tables. Deutsche Forschungsanstalt für Lebensmittelchemie, Garching, available at: http://www.sfkonline.net/cgi-bin/sfkstart.mysql?language=english. SPONHEIMER M, and LEE-THORP J A (2003), Differential resource utilization by extant great apes and australopithecines: towards solving the C4 conundrum, Comp Biochem Physiol A Mol Integr Physiol, 136 (1), 27–34. STANFORD C B, and BUNN H T (2001), Meat Eating and Human Evolution, Oxford, Oxford University Press. STEFANSSON V, (1945), Arctic Manual, New York, The Macmillan Company. TEUBER M, (2000), Fermented milk products, in Lund, B. M., Baird-Parker, T. C. and Gould, G. W., Microbiological Safety and Quality of Food, Gaithersburg, MD, Aspen Publishers, Inc., 535–589. TONG P S, and BERNER L A, (1994), Dairy processing and products, in Arntzen C. J. and Ritter, E. M., Encyclopedia of Agricultural Science, San Diego, CA, Academic Press, 557–574. TUFFT L A, (1996), Rendering, in Hui, Y. H., Bailey’s Industrial Oil and Fat Products, 5th edn, vol. 5, New York, Wiley Interscience, 1–20. VTNP (2004), Verordnung über die Entsorgung von tierischen Nebenprodukten, available at: www.admin.ch/ch/d/Sr/916–441–22/. WEGGEMANS R M, RUDRUM M, and TRAUTWEIN E A 2004, Intake of ruminant versus industrial trans fatty acids and risk of coronary heart disease – what is the evidence?, Euro. J Lipid Sci Technol, 106, 390–397. WIDMER B, (2005), Nutriswiss AG, Lyss, Switzerland, Personal Communication. WIEDERMANN L H, (1978), Margarine and margarine oil, formulation and control, J Am Oil Chem Soci, 55, 823–829. WILLIAMS M A, and HRON R J, SR. (1996), Obtaining oils and fats from source materials, in Hui, Y. H., Bailey’s Industrial Oil and Fat Products, 5th edn, vol. 4, New York, Wiley Interscience, 61–156. MOURITSEN O G,

Lipids from land animals 55 WOOD J D,

(1990), Consequences for meat quality of reducing carcass fatness, in Wood, J. D. and Fisher, A. V., Reducing Fat in Meat Animals, Elsevier Science Publishing Co., Inc. 344–397. WOOD J D, and FISHER A V, (1990), Reducing Fat in Meat Animals, Elsevier Science Publishing Co., Inc. WOODGATE S, (2004), Overview of the European animal by-products industry in 2003, EFPRA congress 2004; Industry Information. WOODGATE S, and VEEN J V D (2004), The role of fat processing and rendering in the European Union animal production industry, Biotechnologie, agronomie, société et environnement, 8 (4), 283–294.

4 Fish oils and lipids from marine sources B. Hjaltason, EPAX AS, Iceland and G. G. Haraldsson, University of Iceland, Iceland

4.1

Introduction

Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and other omega-3 polyunsaturated fatty acids (PUFA) are characteristic of marine fat. Fish oils are by far the most important sources of these fatty acids. For centuries the Icelandic and Scandinavian populations have used various types of fish oil as a household remedy to fight against malnutrition, night blindness, etc. Fish oil was also an important energy supplement in the diet. It is still a common practice among Norwegians and Icelanders to take cod liver oil as a daily spoonful or in soft gelatin capsules. Fish oils were also used as animal feed and to treat various animal diseases (Hjaltason, 1989; Haraldsson and Hjaltason, 2001). Fish oil served as an essential illuminant in indoor lamps for centuries in Iceland and Scandinavia and it was used in Copenhagen to light the streets. The Icelandic name for fish oil, lysi, meaning light, refers to this important role. In the 14th century cod liver and shark liver oils were the second most valuable exports from Iceland (Hjaltason, 1989). The first known clinical test on fish oil was from a report of a study by Samuel Key, a physician at the Manchester Infirmary (Stansby, 1990). It was carried out between 1752 and 1784 and demonstrated that cod liver oil gave relief to patients suffering from rheumatoid arthritis. It was not until the beginning of the 20th century that doctors realized that cod liver oil cured rickets, which was a common disease at that time. In the 1920s it became evident that cod liver oil was a major source of vitamins A and D. Soon after, there was a strong demand for cod liver oil as a vitamin source, which peaked during and after the Second World War for the war suffering countries.

Fish oils and lipids from marine sources 57 When scientists managed to produce vitamins A and D synthetically, this meant a dramatic setback for fish liver oil consumption. That situation changed considerably in the 1980s when the beneficial health effects of fish oil and fish consumption on heart diseases were observed. Fish body oil from industrial fish has been available since the beginning of the 20th century for various industrial purposes, such as in the leather industry and for making soaps and manufacturing paints. During the early decades of the 20th century, whale oil was the most valuable marine oil, but it was replaced in the 1950s by fish oil. In the 1940s fish oil became commercially available for human consumption when process technology became more sophisticated, and food manufacturers started to use partially hydrogenated fish oil in a variety of shortenings, margarines and fats for the baking and confectionery industry (Bimbo, 1987, 1989; Hjaltason, 1989; Haraldsson and Hjaltason, 2001). During the 1970s, epidemiological studies were conducted with Inuit and Japanese living by the sea. The studies showed that both populations had a relatively low incidence of coronary heart disease. This gave fish and fish oil consumption a boost in the 1980s when it became evident that the omega-3 PUFA might have beneficial effects on the heart and on cardiovascular diseases (Stansby, 1990; Simopolous, 1991; Nettleton, 1995; Lands, 2005). Several associations, such as the International Society for the Study of Fatty Acids and Lipids (ISSFAL) (www.issfal.org.uk) have now recommended intakes of marine-based omega-3 fatty acids. The American Heart Association (AHA) recommends that those that do not eat a fish meal at least two times a week should take EPA and DHA as dietary supplements (www.americanheart.org). The Food and Drug Administration (FDA) in the USA also approved a qualified health claim for reducing the risk of coronary heart disease for food and supplements containing omega-3 PUFAs (www.fda.gov). The new five-year dietary guidelines from the US Department of Agriculture (USDA), issued in January 2005, advise consumers to double their intake of oily fish consumption, citing evidence linking fish consumption with a reduction in cardiovascular diseases (www.health.gov/dietaryguidelines/). Finally, very recently, the Ministry of Health and Welfare in Japan issued new dietary guidelines stating minimum content of EPA and DHA that should be consumed on a weekly basis (www.mhlw.go.jp/houdou/2004/11/h1122-2.html#betu). During the 1980s scientists focused on the beneficial effects of EPA on heart diseases and inflammatory diseases. Consequently, consumers demanded concentrates with high EPA content, placing less emphasis on DHA. During the 1990s the focus turned to DHA for its beneficial effects during pregnancy and its importance in neonatal development and on development of the brain and the nervous system (Jumpsen and Clandinin, 1995; Haumann, 1997; Oski, 1997; Alexander, 1998; Newton, 1998) and various mental disorders (Soderberg et al., 1991; Horrobin, 1998). Three large epidemiological studies have clearly demonstrated that people with a regular intake of omega-3 PUFAs from seafood do not develop Alzheimer

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dementia (AD) to the same extent as those who do not eat fish (Kalmijn et al., 1997; Barberger-Gateau et al., 2002; Editorial, 2003; Morris et al., 2003). In patients with AD the concentration of DHA is significantly lower compared with age-matched controls. Concentrated DHA has been proven to have a positive effect in clinical studies, and a number of these are still ongoing (Calon et al., 2004; Lim et al., 2005; Lukiw et al., 2005). There have also been studies indicating that DHA can be effective in weight management (Kunesova et al., 2004; Ruzickova et al., 2004). It is believed that the DHA molecule increases the oxidation of fat by switching on genes responsible for breaking it down in the body. In the last few years, new clinical studies have shown the importance of EPA to the brain. Joseph Hibbeln at National Institutes of Health (NIH) conducted a survey comparing the prevalence of severe mental depression with intake of seafood (Hibbeln, 1998). There was a clear correlation between low intake of seafood and being diagnosed with depression. When the content of omega-3 PUFAs was measured in the blood of depressive patients, it was observed that their values of EPA were significantly lower than in mentally healthy controls. Several clinical studies on the effect of EPA in treating psychotropic diseases have been published or are in progress (Tanskanen et al., 2001; Nemets et al., 2002; Peet and Horrobin, 2002; Su et al., 2003; Frasure-Smith et al., 2004). In this chapter, fish oils as a source of EPA and DHA will be discussed. The most common industrial fish oil types will be described, together with the raw material they are made from, their quantities and quality, how they are produced and refined, their availability, the reliability and stability of their sources, their stability, composition, characteristics and pollutants and their current and past utilization, future development and prospects.

4.2

Fish oils, their main characteristics and composition

Three characteristic features of fish oil make it unique compared to other commercially available oils. These are (i) the high degree of unsaturation and number of carbon atoms of the constituent fatty acids; (ii) the high content of the long-chain omega-3 type PUFAs; (iii) the great number and variety of fatty acids present in the triacylglycerols (Ackman, 1982; Ackman and Lamothe, 1989). There are more than 50 different fatty acids present in a typical fish oil. Many of them are listed in Table 4.1. They range from C14 to C24 and include saturated, monounsaturated, polyunsaturated, omega-3, omega-6, branched, odd-numbered, etc. members. In fact, several more fatty acids may be included in the table because the monounsaturated fatty acids normally exist as a mixture of several positional isomers. The omega-3 long-chain PUFAs originate in the lipids of photosynthetic micro-algae that constitute phytoplankton (Haraldsson and Hjaltason, 2001).

Fish oils and lipids from marine sources 59 Table 4.1

Fatty acids found in fish oils.

12:0 14:0 14:1 15:branched 15:0 16:0 16:1 16:2 ω-7 16:2 ω-4 16:3 ω-4 16:3 ω-3 16:4 ω-4 16:4 ω-1

17:branched 17:0 17:1 18:branched 18:0 18:1 18:2 ω-9 18:2 ω-6 18:2 ω-4 18:3 ω-6 18:3 ω-3 18:4 ω-3 19:branched

19:0 19:1 20:0 20:1 20:2 20:2 20:3 20:3 20:4 20:4 20:5 21:0 21:5

ω-9 ω-6 ω-6 ω-3 ω-6 ω-3 ω-3

22:0 22:1 22:2 22:3 22:4 22:5 22:6 23:0 24:0 24:1

ω-3 ω-3 ω-3 ω-3

ω-2

They are passed up the food chain through zooplankton to the fish (Sargent et al., 1995; Henderson, 1999). Until recently, scientists believed that 22:5 omega-3 arising from the elongation of EPA was converted to DHA by the direct action of a ∆4 desaturase. Now it has been shown that in mammals and fish the 22:5 omega-3 is actually elongated to 24:5 omega-3, which in turn is ∆6 desaturated to 24:6 omega-3. Peroxisomal chain-shortening finally yields DHA (Sprecher et al., 1995). There has also been discussion about the conversion rate of α-linolenic acid (ALA) from flax seed oil into EPA in the human body. It is now clear that only a relatively small amount of ALA can be converted into EPA (and even less to DHA) and therefore ALA cannot replace fish oil as a source of EPA (Emken et al., 1994). Fish oils are virtually only triacylglycerols (TAG), but generally they contain small amounts of mono- and diacylglycerols (MAG and DAG) and minor amounts of various other non-triacylglycerol substances (Breivik and Dahl, 1992; Bimbo, 1998). Some of these minor substances may influence the flavour and odour quality of the oil and affect the stability of the oil as well as its safety. Refining of the oil aims at removing these substances while retaining desirable features. Undesirable substances include moisture, insoluble impurities, free fatty acids, trace metals, oxidation products, sulfur, halogen and nitrogen compounds, pigments, sterols and organic contaminants from the environment such as polychlorinated biphenyls (PCBs) and dioxins. Several different chemical parameters are used to judge the quality of fish oil. In the European Pharmacopoeia there are monographs for different fish oils as well as fish liver oils. There are monographs for plain fish oils as well as omega-3 PUFA concentrates, either as TAG or as ethyl esters (European Pharmacopoeia, 2005). In 2001, the Council for Responsible Nutrition (CRN) in the USA formed an omega-3 working group consisting of 18 ingredient suppliers and manufacturers of omega-3 PUFA. The first action of this group was to define a quality standard which led to a CRN voluntary (unofficial and not obligatory) monograph on fish oil (www.crnusa.org). The US

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Pharmacopoeia is now planning to adopt fish oil and fish oil concentrate monographs similar to the EU monographs. These give maximum values on quality-related issues such as colour, acid value and oxidative parameters as well as content of environmental pollutants such as organochlorine pesticides and heavy metals. Fish oils contain unsaponifiable matter which varies largely with the type of fish oil and includes sterols, hydrocarbons, glyceryl ethers and fatty alcohols as well as traces of pigments, vitamins and oxidized oil. Total cholesterol (free cholesterol and its fatty esters) is often a major part of the unsaponifiables of fish oils. It is possible to remove the free cholesterol by vacuum stripping of the oil, but this does not remove the esterified cholesterol. The potential use of cholesterol esterase to convert the esterified cholesterol into free cholesterol, that can be removed, is under investigation by the industry. Fish oils from different sources have different ratios of esterified cholesterol and free cholesterol, although the total cholesterol is usually of comparable content (de Koning, 1992). Besides environmental pollutants which are defined in the Pharmacopoeias, new pollutants have been coming up, although maximum limits have not yet been defined. This applies to brominated compounds used as fire retardants. When released into the marine environment, these slow-degrading fat-soluble compounds accumulate in fat tissues of fish and other marine organisms. Other pollutants include trace metals such as iron and copper which are considered as pro-oxidants in fish oils, but these are removed during degumming and refining. Heavy metals include arsenic, lead, mercury, selenium and cadmium which are present in trace amounts but are largely removed by refining. Most fish oils used for human consumption now undergo molecular distillation in order to remove all environmental pollutants down to negligible levels. It is usual to distinguish between industrial fish body oil and fish liver oil such as cod liver oil. Lean fish species such as cod and pollack possess a large liver from which fish liver oil is produced. This is usually a high quality oil used as medicinal fish oil, cod liver oil being one example (Hjaltason, 1992). Sprat and blue whiting belong to the same family as cod. Fatty fish species such as herring, capelin, sardine, mackerel, anchovy, sand eel, menhaden, sprat and blue whiting usually have a small liver and the bulk of the fatty oil is present in the fatty flesh of the fish. This is the main source of commercial fish oil. A minor source is the scrap which remains after edible fish has been filleted, the most important products being tuna oil and salmon oil. The bulk of the fish body oil is produced in association with fish meal by the wet-rendering process (Hjaltason, 1989; Bimbo, 1998; Haraldsson and Hjaltason, 2001). There is a considerable variation of fatty acid composition of fish oils among different fish species. This is apparent from Table 4.2, which shows the typical fatty acid composition of most common types of commercially available fish and other marine oils. For the sake of interest, typical whale

Fish oils and lipids from marine sources 61 Table 4.2

Typical fatty acid composition of various commercially available fish oils.

Fatty acid

Anchovya

Jack Menhadena mackerela

Sardine/ pilcharda

Capelina

Herringa

14:0 15:0 16:0 16:1 17:0 18:0 18:1 18:2 18:3 ω-3 18:4 ω-3 20:1 22:1 20:5 ω-3 22:5 ω-3 22:6 ω-3 Others

9 1 17 13 1 3 10 1 1 2 1 1 22 2 9 7

8 1 18 8 1 3 16 1 1 2 2 1 13 2 15 8

9 1 19 12 1 3 11 1 1 3 1 – 14 2 8 14

8 1 18 10 1 3 13 1 1 3 4 3 16 2 9 7

7 – 10 10 – 1 14 1 1 3 17 15 8 – 6 7

7 – 17 6 – 2 14 1 2 3 15 19 6 1 6 1

Fatty acid

Mackerel

Norwaya pout

Sand eela

Sprata

Tunaa

Cod liver oilb

14:0 15:0 16:0 16:1 17:0 18:0 18:1 18:2 18:3 ω-3 18:4 ω-3 20:1 22:1 20:5 ω-3 22:5 ω-3 22:6 ω-3 Others

8 – 14 7 – 2 13 1 1 4 12 15 7 1 8 7

5 – 12 4 – 3 10 1 1 3 13 17 9 1 14 7

7 1 13 5 – 2 7 2 1 5 12 18 11 1 11 4

– – 17 7 – 2 16 2 2 – 10 14 6 1 9 14

3 1 22 3 1 6 21 1 1 1 1 3 6 2 22 6

3 – 10 7 – 2 24 1 1 2 13 10 8 1 11 7

Continued overleaf

oil, seal oil and shark liver oil have been included in the table. Earlier, certain types of shark liver oil together with the liver oils of various other species of elasmobranch fish such as dogfish were important sources of squalene which was hydrogenated into squalane for the cosmetics industry. Shark and dogfish livers also contain glyceryl ethers which are of interest to the health industry (Mangold and Palthauf, 1983; Kayama and Mankura, 1998; Sargent, 1989). With increased farming of fish types such as salmon

62

Modifying lipids for use in food

Table 4.2 Continued Fatty acid

Minke whalec

Harp sealc

Greenland sharkd

Farmed salmond

Blue whitingb

14:0 15:0 16:0 16:1 17:0 18:0 18:1 18:2 18:3 ω-3 18:4 ω-3 20:1 22:1 20:5 ω-3 22:5 ω-3 22:6 ω-3 Others

7 – 10 10 – 2 21 – 1 – 16 10 4 2 5 10

7 – 9 13 – 1 18 – 1 – 13 6 8 3 10 11

1 – 7 5 – 2 27 1 – 1 23 14 3 2 6 8

5 – 12 6 – 3 20 3 1 2 10 9 7 3 11 7

4 – 12 6 – 2 16 1 1 2 14 16 9 1 10 6

a

Bimbo (1998) with kind permission of A P Bimbo J Ogmundssen, Lysi hf, Iceland (unpublished, 2004) c Myrnes et al. (1995) d S Viglundsdottir and B Hjaltason, Lysi hf quality control laboratory (unpublished). b

and cod, new sources of salmon oil and cod liver oil are available. The composition of fish oil made from farmed fish may be quite different from that made from wild fish, and this reflects the different fatty acid composition of their feed. New monographs for farmed fish oils and fish liver oils are in preparation (European Pharmacopoeia, 2005). Although the fatty acid composition of each species in Table 4.2 should not be looked upon as absolute, the profiles of each species can usually be used as a fingerprint characteristic of the type of fish oil (European Pharmacopoeia, 2005). When fish oil is sold based on fish types it is made from, it is not uncommon to mix in cheaper low quality fish oils to match the fatty acid composition of the original oil. This applies particularly to more expensive liver oils such as cod liver oil. One way of investigating the origin of fish oils is to employ 13C NMR (nuclear magnetic resonance) analysis technique to determine the distribution of certain fatty acids including EPA and DHA into the sn-1/3 and sn-2 positions of the triacylglycerols (Aursand et al., 1994). This distribution can be quite characteristic of certain species of fish oil and may be used together with the fatty acid composition as a protection against adulteration. The new monograph for farmed salmon oil in the EU Pharmacopoeia includes this method to minimize the risk of blends (European Pharmacopoeia, 2005). In some respects, the fatty acid profiles of these oils differ considerably, in other respects there is a strong resemblance between them. This has been discussed in detail by Ackman (Ackman, 1986; Ackman et al., 1988). There

Fish oils and lipids from marine sources 63 is a very high content of 20:1 and 22:1 in some of the fish oils from the North Atlantic, such as capelin, blue whiting and herring, while fish oils originated from South American fish are instead higher in EPA and DHA. This makes anchovy and sardine oils the best starting material to make concentrated omega-3 PUFA. These high levels of monounsaturates are believed to be of dietary origin (Ackman, 1982). Generally in fish the mid-position of the glycerol moiety is more enriched with the PUFA, especially DHA. It is interesting that in the TAG oil of marine mammals, including whale oil and seal oil, this is reversed and the mid-position is less enriched with the PUFA. This was established by Brockerhoff and published in numerous papers and has been summarized in a concise form (Ackman and Lamothe, 1989; Ackman and Ratnayake, 1989; Hölmer, 1989). In the light of the fact that the Inuit ate seal fat, not fish, as pointed out by Ackman (Ackman and Ratnayake, 1989; Ackman, 1988), this may be significant. It may also become interesting with regard to structured lipids. Another interesting fact about the marine mammal oils is their high content of docosapentaenoic acid, 22:5 omega-3 (DPA), which is almost equal in quantity to DHA. Possible beneficial health effects of that fatty acid have been pointed out in relation to the Inuit diet (Ackman and Eaton, 1988). In fish the omega-3 fatty acids are not only confined to the TAG, but can be also found as phospholipids (PL) (Vaskovsky, 1989), which are major constituents of the cell membranes. In fish they are much more highly enriched with EPA and particularly DHA, than the TAG, with 40–55 % EPA plus DHA content not uncommon. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are two of the main phospholipid constituents in fish. The former usually accounts for 60–70 % of the phospholipids whereas the latter counts for 20–30 %. Phosphatidylinositol (PI) and phosphatidylserine (PS) are usually far less abundant. Based on body weight, the total PL content of fish is much lower than the TAG, usually around 1–1.5 % as compared to 10–15 %, roughly. The percentage fatty acid composition of cod phospholipids as typical fish phospholipids (Haraldsson et al., 1993) is shown in Table 4.3. It is noteworthy that only four fatty acids account for most of the fatty acids in fish phospholipids, 16:0, 18:1, EPA and DHA, with 18:0 very high in the PI/ PS fraction. It is of particular interest to notice that the DHA content of PC is 30 % and nearly 47 % in PE. It is unlikely that the marine derived phospholipids will be viable as a bulk source of EPA and DHA due to their low content in fish. Today, there is a small commercial production of marine-based phospholipids extracted from krill, fish roes and squid (www.neptunebiotech.com; www.bizen.co.jp; www.nof.co.jp; www.phosphotech.com; www.eximo.no). This is used as an ingredient in special feed formulas for fish larvae and also to some extent as a dietary supplement. In the latter case, the marine-based PLs are then blended with other types of oil, due to high price of the phospholipids and also to make them more fluid since pure PLs are in the form of a paste. Marine

64

Modifying lipids for use in food

Table 4.3 Fatty acid composition of total phospholipids and individual phospholipid classes from cod fillets. Fatty acid

Total PL

PC

PE

PS/PI

14:0 16:0 16:1 18:0 18:1 18:2 18:4 ω-3 20:1 20:4 ω-6 20:5 ω-3 22:1 22:5 ω-3 22:6 ω-3 Others

1.2 20.9 1.7 2.6 9.2 1.4 0.4 1.5 2.3 17.2 0.1 1.2 34.6 5.7

1.5 27.5 1.7 0.8 9.9 0.2 0.2 1.1 1.7 18.6 0.1 1.0 29.6 6.1

0.5 7.8 0.8 5.0 14.2 0.7 0.1 3.6 1.4 10.6 0.2 1.7 46.8 6.6

1.0 3.5 1.1 30.5 12.8 0.3 0.3 3.5 3.3 7.3 0.6 0.9 27.3 7.6

Abbreviations: PC = phosphatidylcholine, PE = phosphatidylethanolamine, PL = phospholipids, PS/ PI = phosphatidylserine/phosphatidylinositol.

phospholipids have been observed to play an important role as a feed ingredient in halibut and fish aquaculture. When such marine PL were included in the lipid feed constituent, the survival rate of halibut larvae going through the complicated metamorphosis phase to healthy halibut juveniles increased dramatically (Hjaltason et al., 2005). There might also be an interesting application of marine-based PL for use in infant formula since DHA in the mother’s milk is in the PL form.

4.3

Production of fish oil

Almost all the fish oil produced today can be considered as a by-product of the fish meal industry. The main emphasis is on the meal production which is usually more valuable and has higher demand than fish oil. The quality of the crude fish oil has improved considerably in recent years. The main reason for this is a demand from the fish feed industry that fish meal produced for aquaculture feed should be produced only from very fresh fish. Some of the farmed fish species such as salmon are sensitive to meal produced from stale raw material. The use of fresh raw material also offers a better quality of fish oil. Crude fish oil is produced in the following manner (Hjaltason, 1989; Bimbo, 1998; Haraldsson and Hjaltason, 2001): after the fish have been caught and landed, they are stored in holding pits at the factory until fed into the cooker. The raw fish material is heated to 100°C by steam as it passes through the cooker. Next, the liquid consisting of water, oil and fine solids

Fish oils and lipids from marine sources 65 is removed. A decanter removes the small particles from the oil and water before going into a centrifuge which separates the oil from the water. Occasionally, an extra centrifugation step is added to purify the oil, since traces of protein and moisture affect the stability of the oil during storage. Finally the oil is cooled and stored. The oil from this process is crude fish oil. Figure 4.1 illustrates the crude fish oil production. Most of the fish oil required for fish and animal feed is used in the crude form. If used for human consumption, such as raw material for concentrated omega-3 products, or as an ingredient in functional food, it goes through classical processing. The crude oil is occasionally pre-treated with acid to remove any gum material or phospholipids that might interfere with the quality of the final product, although this step is often eliminated for fish oil. The next step is neutralization, where the free fatty acids are removed from the oil. The amount of free fatty acids varies, mainly in accordance with the freshness of the raw material used in the fish oil production. If they are not removed, the taste of the oil will be affected. The most common way to remove the free fatty acids from the fish oil is to add caustic soda to the oil in order to neutralize the free acids. The soap is then removed with water in a centrifuge. Finally, the oil is dried before bleaching. The bleaching process is used to remove undesirable colour compounds from the oil as well as other impurities and, to some extent, pollutants. This is done by adding powdered clay or earth that adsorbs the undesirable compounds. Afterwards, the oil is filtered for removal of the bleaching material. This process is also used to standardize the colour of the oil. In recent years, it has become more common to use silica material and activated carbon besides the bleaching earth. The activated carbon removes dioxins and, to some extent, PCBs from the oil while selected silica material removes metals such as iron and copper. In some cases the oil is winterized. This process removes the higher melting triacylglycerols (stearin) from the oil. The oil is slowly cooled to aid formation of suitable crystals of higher melting glycerol esters that are then separated from the liquid. After this process the oil can be stored at low temperature without becoming cloudy. The final step in classical processing of fish oil is deodorization. This process removes the volatile compounds that are the main cause of the strong fishy odour and flavour of the oil. The concentration of these compounds is usually between 200 and 1000 ppm before deodorization. During this process the oil is heated up to 150–190 °C, depending on the degree of unsaturation of the oil, at a vacuum of 1–3 Torr. Then, the oil is stripped with steam which removes the unwanted components. Afterwards, the oil is cooled and is ready for use. In recent years another processing step has been added to the fish oil that is used (non-hydrogenated) for human consumption or as starting material for the concentration process of omega-3 fatty acids. This is molecular distillation. Due to increased pollution of the oceans, fish oils contain some

Cooker Raw fish tank

Strainer Press

Conveyer Press cake Press liquid

Raw material Decanter

Weighing

Sludge Soluble Evaporator

Centrifuge Oil

Concentrate

Oil tank

Mixer

Oil centrifuge

Meal tanks

Dryer

Meal Screener

Fig. 4.1

Tank

Stickwater

Mill

Meal cooler

Flow sheet of crude fish oil production from fish meal process (courtesy of Professor Sigurjon Arason).

Fish oils and lipids from marine sources 67 pesticides and other organic pollutants (MAFF UK, 1997). This includes dioxins, PCBs, hexachlorobenzene (HCB), hexachlorocyclohexane (HCH), lindane and DDT. With better analytical methods and more concern about the long-term effect on human health of those compounds, the authorities are declaring new and lower maximum limits for such contaminants. Unfortunately new threats are on the horizon, such as brominated compounds used as flame retardants. These are now found in crude fish oil, but no maximum values have yet been defined for them. They are also efficiently removed by molecular distillation. Some companies have developed their own purification technology such as Pronova Biocare’s based on molecular distillation (Breivik and Thorstad, 2005) and Lysi hf that holds a patent on purification technology based on adsorption instead of distillation (Hjaltason, 2002). This method needs less investment than that based on molecular distillation. Croda in the UK is using a method based on super-refining to purify their oils (Coupland and Langley, 1995). Some of these methods remove not only the pollutants, but also certain oxidation products. In most cases there are also some losses of oil. The cost varies between methods, and it limits what kind of oils these techniques can be applied to. High vacuum distillation is a very effective way to remove all contaminants towards detection limits. However, investment in equipment is rather high and in addition both vitamins and natural antioxidants are very often removed from the oil. Table 4.4 shows the maximum limits on pollutants in fish oil according to the CRN voluntary fish oil monograph (www.crnusa.org) and the European Pharmacopoeia. Crude fish liver oil such as cod liver oil is processed in a very similar manner as crude fish oil. The raw material is the liver of the fish. Formerly, most of the fish was gutted on land and the liver separated from the viscera Table 4.4 Limits on pollutants according to the European Pharmacopoeia (PhEur/EU) and the USA voluntary monograph (USA vol. mono.). Pollutant

Units

PhEur/EU

USA vol. mono.

As Cd Hg Pb DDD1 DDE2 DDT3 HCB PCBs Dioxins

mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg pg/g

NA NA 0.5 0.1

0.1 0.1 0.1 0.1 0.05 0.05 0.05 0.05 0.09 2

1

2

Sum: o,p′+ p,p′ p,p′ 3 Sum o,p′+ p,p′ Abbreviations: DDD = dichlorodiphenyldichloroethane, DDE = dichlorodiphenyldichloroethene, DDT = dichlorodiphenyltrichloroethane, DCB = dichlorobenzene, HCB = hexacholorobenzene. 2

68

Modifying lipids for use in food

and trucked to the rendering plant. Nowadays, more and more fish are gutted at sea and the livers collected in containers that are then transported to the rendering plant after the boats have landed their catches. This is a very fresh material. After arriving at the rendering plant the liver is ground and then heated to a certain temperature in order to release the oil from the liver residue. Then, the liver mass is put through a decanter that removes solid parts before it goes to the centrifuges. The centrifuges are three-phase, separating the liver mass into crude oil, water and sludge. The crude oil is taken for further processing, but the residue can be dried and used in feed. Today, most of the cod liver meal is used in Southeast Asia as a chemoattractant for the shrimp feed industry due to its strong smell and taste. Omega-3 oils can also be extracted from oil-rich algae (Haumann, 1998; Henderson, 1999). The algae have been modified to increase the oil content and, subsequently, the amount of omega-3 fatty acids. The oil contains almost exclusively DHA which is used in infant formula. This is a rather costly ingredient and can be used only in expensive products. The American-based company Martek has a patented technology to produce DHA-rich algae oil that is now used in at least 70 % of the global infant formula market (www.martek.com). With improved technology and lower production costs, the functional food and dietary supplement market is starting to use algaebased oil to some extent. The whole algae can also be used as a feed ingredient for animals. Omega-3 eggs are today produced by hens given feed that includes omega-3 rich dried algae (Haumann, 1998).

4.4

Fish oil stability and protection

Antioxidants are very important to prevent both the crude as well as the fully-processed oil from being oxidized. Crude fish oil always contains some natural antioxidants. The most common is vitamin E, but it also contains colorants such as astaxthanthin which has been shown to be effective as an antioxidant. Eight different substances that have vitamin E activity occur in nature. They belong to two familes with the generic names tocopherols and tocotrienols. The members of each family are designated with the names α, β, γ or δ, depending on the position and number of the methyl groups attached to the chromane ring (Schuler, 1990). α-Tocopherol displays the highest vitamin E activity. It is also most effective in vivo as a radical scavenger but offers limited protection against oxidation in the oil. The most effective in vitro protectant is the δ-tocopherol. Fish oil and fish liver oil have different amounts of each type of tocopherol. Cod liver oil contains more than 90 % of its vitamin E as α-tocopherol. During processing, some of the natural antioxidants are removed. Figure 4.2 shows how fish oil becomes less and less stable after each processing step (Bragadottir M, Thorisson S and Hjaltason B, unpublished results).

Fish oils and lipids from marine sources 69

Oxygen uptake (µg/mol)

200 180

Crude Bleached

160

Deodorized

140

Neutralized

120 100 80 60 40 20 0 0.000

0.050

Fig. 4.2

0.100

0.150 0.200 Time (h)

0.250

0.300

0.350

The effect of processing on capelin oil stability.

Crude capelin oil was taken through the processing stages of refining, bleaching and deodorization. At each step, a sample was taken and the oxygen uptake measured with a Warburg apparatus. Oxidative stability was considerably lower in the fully-processed oil compared to the crude oil. This indicates the importance of the oil being well protected during processing and stabilizing the final product. Traces of metal ions such as copper and iron are strongly pro-oxidant. They are partly removed during bleaching, but it is possible to bind these ions to compounds such as EDTA that inactivate them. Therefore, it is always important to use equipment made of stainless steel, work as much as possible under an inert nitrogen atmosphere, and keep the temperature low during processing where possible. Antioxidants are always added to processed fish oil for human consumption to protect the oil against oxidation and keep the taste and smell of the oil as good as possible. Natural antioxidants are preferred. Blends of α-, β-, γ- and δ-tocopherols are most commonly used. Other natural antioxidants used include ascorbic acid, spice extracts, such as rosemary, and citric acid (Rajalakshmi and Narasimhan, 1996). Many producers of fish oil use their own antioxidant formulas based on different blends of the available natural antioxidants. In most cases they are looking for synergistic effects of the individual antioxidants. Some of the antioxidants also seem to have different degrees of efficiency in keeping down the undesirable smell and taste of the oil caused by compounds such as volatile ketones and aldehydes. The synergistic effect permits the reduction of the level of antioxidants added to food formulation considerably (Coppen, 1989). Synthetic antioxidants are also available and are, in most cases, more effective than natural antioxidants. Although some of them have been available

70

Modifying lipids for use in food

for almost half a century, very few have gained widespread approval or popularity. The most commonly used synthetic antioxidants are butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), tertbutylhydroquinone TBHQ and propyl gallate (Frankel, 1998). TBHQ has been shown to be a very effective antioxidant for fish oils. Unfortunately, TBHQ is not approved for use in Europe so its use is confined to the USA. Uses of synthetic antioxidants are in decline due to the perception that natural food ingredients are better and safer than synthetic. There are other ways to protect the oil from oxidation that are based on eliminating access of oxygen to the oil. One is soft gelatine capsules, which are commonly used in the dietary supplement industry. This is also a convenient dosage form. Studies have shown that the oil is stable for at least three years based on analysis of oxidative parameters (Nutrition Industry Executive, 2005: www.niemagazine.com). Recently, smaller gelatin beadlets are gaining popularity since they can be mixed into food products. Microencapsulation technology has advanced very much in recent years (Newton, 1996; Shelke, 2005). This technique provides an oxidatively stable free-flowing powder. The main problem has been that the microencapsulated product picks up a fishy smell and taste during storage, giving it a relatively short shelf life. Therefore, microencapsulated fish oil products are used in products that have a short shelf life in the shops such as bread, milk and yoghurt. Another limiting factor has been the bioavailability of the powder. If the coating material or matrix used is too ‘strong’, the bioavailability is less but the product has a longer shelf life. So far, this technology has mainly been used on non-concentrated fish oil, but technological improvements have opened up the potential to microencapsulate highly concentrated omega3 fish oils. There are also other methods available to protect PUFAs against oxidation, such as liposomes (Haumann, 1995). Emulsions are also promising for uses in fresh and pasteurized milk as well as enrichment of UHT (ultra high temperature) milk (www.dsmnutritionalproducts.com).

4.5

Commercial fish oils

Compared to the worldwide production of fats and oils, global production of fish oil is rather small (1–2 %). Since 1995, the average world production of fish oil has been around 1.14 million tonnes (ISTA Mielke GmbH, Oil World, Hamburg, www.oilworld.biz). There have been some fluctuations in fish oil production, basically due to the El Niño phenomenon (www.elnino.noaa.gov) which changed conditions in the ocean along the coastline of Peru and Chile, causing the fish to move to deeper waters. This caused a 34 % reduction in pelagic fisheries and fish oil production in South America between 1997 and 1998 (Oil World Annual, 1998; www.gafta.com/fin/finfacts3.html). This can be clearly seen in Figure 4.3 showing the world fish oil production 1990–

Fish oils and lipids from marine sources 71 1600

1505

Thousand tonnes

1200

1417

1336 1381

1400 1264 1237

1354 1194

1142

1065

1042 1000 865

993 904

887

800 600 400 200 0 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 Year

Fig. 4.3

World fish oil production in 1990–2004.

2004 (Stuart Barlow and Ian Pike, personal communication; ISTA Mielke GmbH, Oil World, Hamburg, www.oilworld.biz). Very little is known about what causes the El Niño phenomenon and, therefore, it is very difficult to predict when it may happen. Peru and Chile are the leading producers of fish oil in a normal year, followed by the USA, Iceland, Denmark and Norway. In the 1980s Japan was a large producer of fish oil, but sardines disappeared in 1992 from Japanese waters. Today, Japan is an importer of fish oil instead of being one of the biggest exporters as it was only 20 years ago. The largest users of fish oil are Chile and Norway, since major application of crude fish oil is in salmon feed. Chile has now to import fish oil from Peru in order to meet its needs and Norway is now the largest single importer of fish oil in the world. Peru is by far the largest exporter of fish oil due to low domestic uses of fish oil. The fish oil export from Peru is around 39 % of worldwide export of fish oil for 2004 (ISTA Mielke GmbH, Oil World, Hamburg, www.oilworld.biz), but the average fish oil production for the last 20 years has ranged between 1.0 to 1.7 million tonnes, except for 1992 and 1998, when the effect of El Niño caused reduced production (www.gafta.com/ fin/finfacts3.html). Fish oil is produced mainly from pelagic fish and is a by-product of the fish meal industry. The landings of industrial feed grade fish have according to the Food and Agriculture Organization (FAO) been fairly stable since 1985 at around 20–25 million tonnes per year apart from the years when the effects of El Ninjo reduced these numbers considerably. In general, feed grade fish are short-lived and fast growing and are therefore less vulnerable to overfishing compared to other species. Due to the rapid growth of aquaculture which creates a greater demand for fish oil for feed production, there has been an increase in focus and

72

Modifying lipids for use in food

discussion on sustainability of these fisheries. Sustainability was defined by the World Commission on Environment and Development in 1987 as ‘Development that meets the needs of the present without compromising the ability of future generations to meet their own needs’ (Shepherd et al., 2005). Table 4.5 shows the status of feed grade fish stocks in Europe and South America based on information from the International Council for the Exploration of the Sea (ICES), Institutio del Mar del Peru (IMARPE) and FAO’s report on the ‘State of World Fisheries and Future Sustainability Issues’ (see Shepherd et al., 2005). The European and South American feed grade fish stocks are monitored by government agencies. Overfishing from these feed grade fish stocks is prevented by fishing quotas as well as periodic fishing bans during spawning. There are, however, cases where fish stocks have been overfished, as is the case with blue whiting in Europe, due to disagreement between governments and consequent failure to set up official quotas. In late 2005 the governments involved finally agreed on official quotas which it is hoped will help the stock of blue whiting to recover. Fish liver oil, where the raw material is only the liver, is also produced. This is produced in relatively small quantities for speciality uses such as vitamins A and D. Today, cod liver oil and shark liver oil are the most common commercially available fish liver oils on the market. Earlier, halibut liver oil was commercially produced as a vitamin A supplement, but it is no longer economical to collect and produce that oil. The world production of cod liver oil today for human uses is around 10000 tonnes, but figures for shark liver oil are not available. Historically, marine mammal oils, such as whale oil and seal oil, were produced in large quantities. Today, there is no commercial supply of whale oil due to the ban on whale hunting and the sale of whale products, but seal oil is still produced in small amounts. With increasing production of farmed salmon, farmed salmon oil has become available. It is produced from waste from the salmon processors and is of high quality due to the freshness of the raw material. Using farmed salmon oil in fish feed is not allowed so the main application has been in the pet food industry. There is some interest in finding an application for farmed salmon oil as a food ingredient, an area in which Norway is in lead. Although the wild salmon catch is rather large in areas like Alaska, commercial salmon oil from wild salmon has never been produced to any great extent. Recently, tuna oil has also become commercially available. However, production remains rather small, mainly based on tuna heads and trimmings from the tuna canning factories. Thailand and Australia are two of the biggest producers of tuna oil today. The basic use of tuna oil is in infant formula and as a food ingredient. The main interest in tuna oil is related to its high DHA and low EPA content. Companies with products based on omega-3 fatty acids from fish oil have also started to look for supplies other than fish oil for these fatty acids. The

Table 4.5

Status of feed grade fish stocks.

Europe Capelin Blue whiting Sandeel

Whether used for human consumption

Commentary on status of fish stocks at December 2004

Source of status info

Roe used for human consumption. Frozen capelin for specific markets. Mainly used for fish meal. Potential use for human consumption although there are processing difficulties. Mainly used for fish meal. Not used for human consumption.

Icelandic – present state not known. Barents Sea – reduced reproductive capacity – no fishing recommended Spring 05. Stock classified as having full reproductive capacity, but harvested unsustainably. Spawning stock has remained stable at a high level 2002–2004. Stock classified as having reduced reproductive capacity. 21 % reduction in quota on 2005 over 2004. State of the stock is unknown. Indications of a good 2003 class recruiting to the 2004 fishery. 4 % increase in quota. There are five herring stocks. Three have full reproductive capacity, harvested sustainability, one has full reproductive capacity but harvested unsustainably and the 5th has very low abundance.

ICES

Sprat

Potential uses for human consumption, but mainly used for fish meal.

Herring

Primarily used for human consumption, but non-food grade fish and trimmings may be used for fish meal.

Trimmings

Trimmings generally comprise small pelagic species (50 %), i.e. mackerel and herring and trimmings from the food fish processing sector i.e. cod (50 %).

South America Anchovy

Very small amount for human consumption. Majority used for fish meal.

Jack mackerel

Used for human consumption (Africa). Majority used for fish meal.

Horse mackerel Sardine

Primarily used for human consumption. Primarily used for human consumption.

Biomass increasing following 97/98 El Niño. Controlled by licensing, satellite tracking, closed seasons, minimum landing size. Biomass increasing following 97/98 El Niño. Controlled by catch limits, satellite tracking, closed seasons and minimum landing size. Closed seasons, company catch limits. Closed seasons, company catch limits.

ICES ICES ICES ICES

FAO/IMARPE FAO/IMARPE FAO/IMARPE FAO/IMARPE

Abbreviations: FAO = Food and Agriculture Organization, ICES = International Council for Exploration of the Seas, IMARPE = Peruvian Institute of the Sea. Source: European Aquaculture Society Special Publication No 35, June 2005, 59–66 (www.easonline.org/home/en).

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Modifying lipids for use in food

most interesting are the marine organisms that produce omega-3 fatty acids (Haumann, 1998; Henderson, 1999). Bacteria, eukaryotic micro-algae, fungi and zooplankton are all interesting options. In terms of alternatives to fish oil as a source of omega-3 fatty acids, single-cell marine organisms offer the most realistic potential in the near future. Major US companies have also laid out their plans to genetically modify plant seeds in order to accumulate significant levels of EPA and DHA in the seed (Napier, 2004; Qi et al., 2004; Wu et al., 2005). Such oils might reduce the dependence of the fish feed industry on fish oil as a feed ingredient, although no cost structure is available yet. It is likely that such oils will be available commercially in 5–10 years’ time. Although such oils might be acceptable in the USA, consumers in Europe might reject them due to hostility towards the idea of genetic modification.

4.6

Fish oil application

There have been dramatic changes in the uses of fish oil in the last ten years. In 1990 around 76 % of the fish oil was used as raw material in margarines (after partial hydrogenation). Due to new information concerning negative health aspects of consumption of trans fatty acids (Katan, 1995; Ascherio et al., 1999) produced during the hydrogenation process, their use in margarine has fallen dramatically. Some countries have also enacted legislation stating maximum amounts of trans fatty acids in products such as margarines. The USA has set up a regulation from 1st of January 2006 that trans content must be declared on labels (Federal Register, 2003). In 2002 only 14 % (Barlow S and Pike I, personal communication) of fish oil was hydrogenated for uses in margarine and this is expected to fall to less than 1 % in the year 2012. This has, however, not affected the fish oil market too badly since at the same time the aquaculture industry is demanding more and more fish oil as a fish feed ingredient. The fish oil is not only used as an inexpensive energy source, but also as an omega-3 source for the farmed fish. In 1990 the aquafeed industry used 16 % of the fish oil production and in 2002 this number was up to 81 % (Stuart Barlow and Ian Pike; International Fish Meal and Fish Oil Organization: www.iffo.net). In 2012 it might rise to 88 %. Today, Norway is the biggest user of fish oil in their salmon feed. Their estimated annual need is around 190 000 tonnes. China is, however, the biggest producer of farmed fish. So far, their fish farming is not very industrialized and is based very much on fresh water fish species or species that do not need fish meal or oil in their diet, such as tilapia. However, China is becoming a larger buyer of fish meal and fish oil which might affect the balance between supply and demand of fish oil in near future. Figure 4.4 compares the use of fish oil for the years 1990, 2002 and 2012. The market for non-hydrogenated fish oil as functional food or food ingredient as well as for dietary supplements, fish oil concentrates and

Fish oils and lipids from marine sources 75 2002

1990 Aquafeed 16 %

Edible 76 %

Edible 14 %

Industrial 8%

Total production 975 TT

Industrial 8%

Total production 1.3 MT

Aquafeed 78 %

2012 Industrial + other animals 7%

Edible 5% Total production 1.1 MT

Aquafeed 88 %

Fig. 4.4 Summary of global fish oil use for the periods 1990, 2002 and 2012 (TT = thousand tonnes, MT = million tonnes).

pharmaceuticals is still very small. Only 1–2 % of the world production of fish oil goes into this application. So far, the main application of fish oil as an omega-3 source has been in the dietary supplement industry. At present most of the oil sold for this application is non-concentrated oil containing 18 % EPA and 12 % DHA. In recent years, more concentrated omega-3 products have been on the market with specific amounts and ratios of EPA and DHA intended to treat certain medical conditions such as arthritis and CVD or to enhance mental health. The fastest growth is likely to be in the food ingredient sector or as an ingredient for functional food. Organizations and government bodies are now giving specific recommendations and advice about intake of omega-3 fatty acids. Different bodies (Hornstra, 2004), such as British Nutrition Foundation, Nordic Council of Ministers, Health and Welfare in Canada, American Heart Association as well as the Ministry of Health and Welfare in Japan (www.mhlw.go.jp/houdou/2004/11/h1122-2.html#betu) have issued guidelines regarding intake of omega-3 fatty acids and also, in numerous cases the omega-6/omega-3 ratio. Generally, 0.6–1.0 g of omega-3 fatty acids in the form of EPA and DHA is recommended as a daily intake, but if the omega-3 fatty acids are used to treat specific diseases, a higher dosage is needed. Today, the situation is such that a ‘normal’ diet in the Western world only supplies about 10–15 % of the recommended daily intake of the omega-3 fatty acids. The challenge for the oil and fats industry is to close this ‘nutritional gap’ by increasing the consumption of omega-3 fatty acids, either by adding omega-3 oil into food products or by dietary supplements. With improved quality, new technology to protect the oil and increasing consumer awareness of the health benefits of the omega-3 fatty acids, it is probable that a larger

76

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part of the fish oil will be used for food application as an omega-3 source in the near future. Government regulations that limit the use of health claims might slow down the use of omega-3 in fortified food products. Research into the biological and clinical effects of omega-3 fatty acids has created several pharmaceutical products. Most of them are highly concentrated products, but one of the first was MaxEPA® (Sevenseas Healthcare Ltd, UK, www.sseas.com) containing 18 % EPA and 12 % DHA, which was registered as a drug for hyperlipidemia. Epadel was developed by the Japanese company Mochida in cooperation with Nippon Suisan Kaisha (www.nissui.co.jp). This is a highly concentrated EPA product. It was first approved in 1990 for arteriosclerosis obliterans (ASO) and in 1994 for hyperlipidemia. Recently, Pronova Biocare from Norway gained approval for their highly concentrated EPA pharmaceutical product named Omacor® (www.pronova.com). This is an 85 % EPA + DHA ethyl ester concentrate. This drug is now sold in Europe for secondary myocardial infarction and recently gained marketing approval in the USA for lowering blood fat. Clinical trials on the effects of highly purified EPA on schizophrenia and against Hodgkinson’s disease are now going on. Although these products are highly concentrated, they will not need large volumes of crude fish oil for their production. Makers of fish oil for the food, dietary supplement and pharmaceutical industries will always be able to compete in price with the feed makers. Since sustainability of pelagic fisheries is also responsibly managed, we should not face any shortage of fish oil in the coming years. Also, new sources as from algae or genetically modified plant seeds will be more commercially available and feasible in coming years.

4.7

References

(1982), Fatty acid composition of fish oils, in Barlow S M and Stansby M E, Nutritional Evaluation of Long-Chain Fatty Acids in Fish Oils, New York, Academic Press, 25–88. ACKMAN R G (1986), Perspectives on eicosapentaenoic acid (EPA), n-3 News, 1(4), 1–4. ACKMAN R G (1988), Some possible effects on lipid biochemistry of differences in the distribution on glycerol of long-chain n-3 fatty acids in the fats of marine fish and marine mammals, Atherosclerosis, 70, 171–173. ACKMAN R G and EATON C A (1988) Docosapentaenoic acid in blubber of dam and pup grey seals (Halichoreus grypus): implications in the Inuit diet and for human health’, Can J Zool, 66, 2428–2431. ACKMAN R G and LAMOTHE F (1989), Marine mammals, in Ackman R G, Marine Biogenic Lipids, Fats and Oils, Vol. II, Boca Raton, FL, CRC Press, Inc., 179–381. ACKMAN R G and RATNAYAKE W M N (1989), Fish oils, seal oils, esters and acids – are all forms of omega-3 intake equal?, in Chandra R K, Health Effects of Fish and Fish Oils, St John’s, Newfoundland, ARTS Biomedical Publishers, 373–393. ACKMAN R G, RATNAYAKE W M N and OLSSON B (1988), The ‘basic’ fatty acid composition of Atlantic fish oils: potential similarities useful for enrichment of polyunsaturated fatty acids by urea complexation, J Am Oil Chem Soc, 65, 136–138. ACKMAN R G

Fish oils and lipids from marine sources 77 (1998), Immunonutrition: the role of n-3 fatty acids, Nutrition, 14, 627–633. and WILLETT W C (1999), Trans fatty acids and coronary heart disease, New England J Med, 340, 1994–1998. 13 AURSAND M, RAINUZZO J R and GRASDALEN H (1994), Quantitative high resolution C high1 resolution and H nuclear magnetic resonance of ω-3 fatty acids from white muscle of Atlantic salmon (Salmo salar), J Am Oil Chem Soc, 70, 971–981. BARBERGER-GATEAU P, LETENNEUR L, DESCHAMPS V, PERES K, DARTIGUES J F and RENAUD S (2002), Fish, meat and risk of dementia: cohort study, BMJ, 325, 932–933. BIMBO A P (1987), The emerging marine oil industry, J Am Oil Chem Soc, 64, 706–715. BIMBO A P (1989), Fish oils: past and present food uses, J Am Oil Chem Soc, 66, 1717– 1726. BIMBO A P (1998), Guidelines for characterizing food-grade fish oil, INFORM, 9, 473–483. BREIVIK H and DAHL K H (1992), Production and control of n-3 fatty acids, in Frölich J C and von Schacky C, Fish, Fish Oil and Human Health, Clinical Pharmacology, Vol. 5, München, W. Zuckschwerdt Verlag, 25–39. BREIVIK H and THORSTAD O (2005), Removal of organic environmental pollutants from fish oil by short-path distillation, Lipid Technol, 17, 55–58. CALON F, LIM G P, YANG F, MORIHARA T, TETER B, UBEDA U, ROSTAING P, TRILLER A, SALEM JR. N, ASHE K H, FRAUTSCHY S A and COLE G M (2004), Docosahexaenoic acid protects from dendritic pathology in an Alzheimer’s disease mouse model, Neuron, 43, 633–645. COPPEN P P (1989), The use of antioxidants, in Allen J C and Hamilton R C, Rancidity in Foods, 2nd edn, London, Elsevier Applied Science, 83–104. COUPLAND K and LANGLEY N (1995), Modifying natural oil by adsorption chromatography, in Haraldsson, G G, Gudbjarnason S and Lambertsen G, Proc of the 18th Nordic Lipid Symposium, Reykjavik, Iceland, June 1995, Bergen, Norway, Lipidforum, 67–72. DE KONING A J (1992), The cholesterol content of South African fish oil and its seasonal variations, Fat Sci Technol, 94, 60–63. EDITORIAL (2003), Fish consumption and the risk of Alzheimer disease. Is it time to make dietary recommendations?, Arch Neurol, 60, 923–924. EMKEN E A, ADLOF R O and GULLEY R M (1994), Dietary linoleic acid influences desaturation and acylation of deuterium-labeled linoleic and linolenic acids in young adult males, Biochem Biophys Acta, 1213, 277–288. EUROPEAN PHARMACOPOEIA, 5th Edn, 2005, Strasbourg, Council of Europe. FEDERAL REGISTER (2003), July 11, Volume 68, No. 133. FRANKEL E N (2005), Antioxidants, in Lipid Oxidation, Bridgewater Oily Press, 209–258. FRASURE-SMITH N, LESPÉRANCE F and JULIEN P (2004), Major depression is associated with lower omega-3 fatty acid levels in patients with recent acute coronary syndromes, Biol Psychiatry, 55, 891–896. HARALDSSON G G and HJALTASON B (2001), Fish oils as sources of polyunsaturated fatty acids, in Gunstone F D, Structured and Modified Lipids, New York, Marcel Dekker, Inc., 313–350. HARALDSSON G G, KRISTINSSON B and GUDBJARNASON S (1993), The fatty acid composition of various lipid classes in several species of fish caught in Icelandic seawaters, INFORM, 4, 535. HAUMANN B F (1995), Liposomes offer hope as medical tools, INFORM, 6, 793–802. HAUMANN B F (1997), Nutritional aspects of n-3 fatty acids, INFORM, 8, 428–447. HAUMANN B F (1998), Alternative sources for n-3 fatty acids, INFORM, 9, 1108–1119. HENDERSON R J (1999), The production of n-3 polyunsaturated fatty acids in marine organisms, Lipid Technol, 11, 5–10. HIBBELN J R (1998), Fish consumption and major depression, Lancet, 351, 1213. HJALTASON B (1989), New frontiers in the processing and utilization of fish oil, in Somogoyi J C and Muller H R, Nutritional Impact of Food Processing, Bibl Nutr Dieta No. 43, Basel, Karger Publishing, 96–106. HJALTASON B (1992), Fish oils as vitamin sources, AOCS Short Course Manual on Modern Applications of Marine Oils, May 8–9, Toronto, Canada. ALEXANDER J

ASCHERIO A, KATAN M B, ZOCK P L, STAMPFER M J

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(2002), Method for producing marine oils with reduced levels of contaminants, Patent, EP 1303 580 A1. HJALTASON B, HARALDSSON G G and HALLDORSSON O (2005), Rearing of aquatic species with DHA-rich prey organisms, Patent US 6 959 663 B2. HORNSTRA G (2004), LCPUFA in Maternal Nutrition, Basel, DSM Nutritional Products Ltd. Available at [email protected]. HORROBIN, D F (1998), The membrane phospholipid hypothesis as a biochemical basis for the neurodevelopmental concept of schizophrenia, Schizophr Res, 30, 193–208. HÖLMER G (1989), Triglycerides, in Ackman R G, Marine Biogenic Lipids, Fats and Oils, Vol. I, Boca Raton, FL, CRC Press, Inc., 139–173. JUMPSEN J and CLANDININ M T (1995), Brain Development: Relationship to Dietary Lipid and Lipid Metabolism, Champaign, IL, AOCS Press. KALMIJN S, LAUNER L J, OTT A, WITTEMAN J C, HOFMAN A and BRETELER M M (1997), Dietary fat intake and the risk of incident dementia in the Rotterdam study, Ann Neurol, 42, 776– 782. KATAN M (1995), Exit trans fatty acids, Lancet, 346, 1245–1246. KAYAMA M and MANKURA M (1998), Natural oleochemicals in marine fishes, INFORM, 9, 794–799. KUNESOVA M, BRAUNEROVA R, HLAVATY P, MATOULEK M, KOPECKY J, SKRHA J, HAINER V, PARIZKOVA J and SVACINA S (2004), Weight reduction with VLCD and n-3 PUFA lead to higher decrease of weight and BMI in severely obese women, NAASO’s 2004 Annual Meeting, November 14–18, Las Vegas, NV, Obesity Research, 12, October 2004, Supplement Program Abstracts, A66. LANDS W E M (2005), Fish, Omega-3 and Human Health, 2nd edn, Champaign IL, AOCS Press. LIM G P, CALON F, MORIHARA T, YANG F, TETER B, UBEDA O, SALEM JR. N, FRAUTSCHY S A and COLE G M (2005), A diet rich with the omega-3 fatty acid docosahexaenoic acid reduces amyloid burden in an aged Alzheimer mouse model, J Neurosci, 25, 3032–3040. LUKIW W J, CUI J-G, MARCHESELLI V L, BODKER M, BOTKJAER A, GOTLINGER K, SERHAN C N and BAZAN N G (2005), A role for docosahexaenoic acid-derived neuroprotectin D1 in neural cell survival and Alzheimer disease, J Clin Invest, 115(10), 2774–2783. MAFF UK (1997), Dioxins and polychlorinated biphenyls in fish oil dietary supplements and licensed medicines, MAFF UK Food Surveillance Information Sheet, no. 106, London, Ministry of Agriculture, Fisheries and Food. MANGOLD H K and PALTHAUF F (1983), Ether Lipids. Biochemical and Biomedical Aspects, New York, Academic Press. MORRIS M C, EVANS D A, BIENIAS J L, TANGNEY C C, BENNETT D A, WILSON R S, AGGARWAL N and SCHNEIDER J (2003), Consumption of fish and n-3 fatty acids and risk of incident Alzheimer disease, Arch Neurol, 60, 940–946. NAPIER J A (2004), Production of long-chain polyunsaturated fatty acids in transgenic plants: a sustainable source for human health and nutrition, Lipid Technol, 16, 103– 107. NEMETS B, STAHL Z and BELMAKER R H (2002), Addition of omega-3 fatty acid to maintenance medication treatment for recurrent unipolar depressive disorder, Am J Psychiatry, 159, 477–479. NETTLETON J A (1995), Omega-3 Fatty Acids and Health, New York, Chapman and Hall. NEWTON I S (1996), Food enrichment with long-chain n-3 PUFA, INFORM, 7, 169–177. NEWTON I S (1998), Long-chain polyunsaturated fatty acids – the new frontier in nutrition, Lipid Technol, 10, 77–81. NUTRITION INDUSTRY EXECUTIVE (2005), The soft sell, September, 51–52. OIL WORLD ANNUAL (1998), Hamburg, ISTA Mielke GmbH. OSKI F (1997), What we eat may determine who we can be, Nutrition, 13, 220–221. PEET M and HORROBIN D F (2002), A dose-ranging study of the effects of EPA in patients with ongoing depression despite apparently adequate treatment with standard drugs, Arch Gen Psychiatry, 59, 913–919. HJALTASON B

Fish oils and lipids from marine sources 79 QI B, FRASER T, MUGFORD S, DOBSON G, SAYANOVA O, BUTLER J, NAPIER J A, STOBART A K

and (2004), Production of very long chain polyunsaturated omega-3 and omega-6 fatty acids in plants, Nature Biotechnol, 22, 739–745. RAJALAKSHMI D and NARASIMHAN S (1996), Food antioxidants: sources and methods of evaluation, in Madhavi D L, Deshpande S S and Salunkhe D K, Food Antioxidants. Technological, Toxicological, and Health Perspectives, New York, Marcel Dekker, Inc., 65–159. RUZICKOVA J, ROSSMEISL M, PRAZAK T, FLACKS P, SPONOROVA J, VECKA M, TVRZICKA E, BRYHN M and KOPECKY J (2004), Omega-3 PUFA of marine origin limit diet-induced obesity in mice by reducing cellularity of adipose tissue, Lipids, 39, 1177–1185. SARGENT J R (1989), Ether-linked glycerides in marine animals, in Ackman R G, Marine Biogenic Lipids, Fats and Oils, Vol. I, Boca Raton, FL, CRC Press, Inc., 175–197. SARGENT J R, BELL M V, BELL J G, HENDERSON R J and TOCHER D R (1995), Origins and functions of n-3 polyunsaturated fatty acids in marine organisms, in Cevc G and Paltauf F, Phospholipids: Characterization, Metabolism and Novel Biological Applications, Champaign, Ill, AOCS Press, 248–258. SHELKE K (2005), Hidden ingredients take cover in a capsule, available at: www.foodprocessing.com/articles/2005/421.html. SCHULER P (1990), Natural antioxidants exploited commercially, in Hudson B J F, Food Antioxidants, London, Elsevier Applied Science, 99–170. SHEPHERD C J, PIKE I H and BARLOW S M (2005), Sustainable feed resources of marine origin, European aquaculture society special publication No 35, June 59–66. SIMOPOULOS A P (1991), Omega-3 fatty acids in health and disease and in growth and development, Am J Clin Nutr, 54, 438–463. SODERBERG, M, EDLUND C, KRISTENSSON K and DALLNER G (1991), Fatty acid composition of brain phospholipids in aging and Alzheimer’s disease, Lipids, 26, 421–425. SPRECHER H, LUTHRIA D L, MOHAMMED B S and BAYKOUSHEVA S P (1995), Reevaluation of the pathways for the biosynthesis of polyunsaturated fatty acids, J Lipid Res, 36, 2471– 2477. STANSBY M E (1990), Nutritional properties of fish oil for human consumption – early development, in Stansby M E, Fish Oils in Nutrition, New York, van Nostrand Reinhold, 268–288. SU K-P, HUANG S-Y, CHIU C-C and SHEN W W (2003), Omega-3 fatty acids in major depressive disorder. A preliminary double-blind, placebo-controlled trial, Eur Neuropsych, 13, 267–271. TANSKANEN A, HIBBELN J R, TUOMILEHTO J, UUTELA A, HAUKKALA A, VIINAMÄKI H, LEHTONEN J and VARTIAINEN E (2001), Fish consumption and depressive symptoms in the general population in Finland, Psychiatric Services, 52, 529–531. VASKOVSKY V E (1989), Phospholipids, in Ackman R G, Marine Biogenic Lipids, Fats and Oils, Vol. I, Boca Raton, FL, CRC Press, Inc., 199–242. WU G, TRUKSA M, DATLA N, VRINTEN P, BAUER J, ZANK T, CIRPUS P, HEINZ E and QIU X (2005), Stepwise engineering to produce high yields of very long-chain polyunsaturated fatty acids in plants, Nature Biotechnol, 23, 1013–1017. LAZARUS C M

5 Lipids from microbial sources C. Ratledge and S. Hopkins, University of Hull, UK

5.1

Introduction

It is only since the 1980s that the commercial production of microbial oils has taken place, in spite of the fact that detailed knowledge of the occurrence of oils and fats in various micro-organisms has been available for well over a century. The major factor which has changed this perception has been the appreciation of the key roles of particular polyunsaturated fatty acids (PUFAs) in human nutrition (Carlson et al., 1993a,b) which has created a specific demand for such materials. The supply of these desirable PUFAs has then posed additional problems as the main acids, arachidonic (ARA, 20:4n-6) and docosahexaenoic (DHA, 22:6n-3), are not available from any plant source and can only be isolated from animal materials at considerable cost. Arachidonic acid is obtainable from egg yolk, but thousands of eggs are needed to produce a gram of pure ARA; similarly, DHA can be obtained from various marine sources, including a number of fish liver oils, but it always occurs along with eicosapentaenoic acid (EPA, 20:5n-3) which then necessitates extensive and expensive fractionation, eventually involving large-scale high-performance liquid chromatography (HPLC), to produce DHA without the presence of EPA or any other PUFA. Thus, as the nutritional information concerning the requirement for these two PUFAs for the development of brain and eye functions in newly born infants appears irreproachable (Huang and Sinclair, 1998), a clear need for a commercial source of these fatty acids has arisen. (The nutritional aspects of oils rich in ARA and DHA are discussed in greater detail in Chapter 24.) Identifying possible commercial sources of PUFA has not been a major problem as it has been known for many years that numerous micro-organisms,

Lipids from microbial sources

81

including most marine micro-algae (that is algae growing as simple, singlecelled organisms as opposed to the macro-algae which are also known as seaweeds and grow accordingly), do produce such materials. A few species, moreover, produce ARA and DHA as their sole PUFA making them ideal starting materials from which oils rich in these materials could be produced. However, before considering the current production processes used for the synthesis of these PUFAs, a short background to the concept of using microorganisms for lipid production needs to be given to allow present-day developments to be seen within their appropriate context.

5.2 Oleaginous micro-organisms, fatty acids and lipid accumulation Table 5.1 gives the fatty acid profiles of a number of micro-organisms to illustrate the diversity and variation that can occur in lipid composition in these unicellular organisms. Micro-organisms can be divided into bacteria, yeasts, fungi and algae, although in some cases the boundaries can be uncertain. Bacteria are prokaryotes without a defined nucleus and include some algae formerly known as the blue-green algae but now referred to as the cyanobacteria. Yeast and fungi, together with the algae proper, are eukaryotic phyla as they have defined nuclei; yeasts are non-filamentous whereas the fungi are mostly filamentous and may have complex life-cycles that can include differentiation. Algae are essentially water-borne organisms, the majority of which (but not all) are capable of carrying out photosynthesis to obtain energy which they can then use to fix CO2 from the atmosphere. There are both micro-algae, i.e. unicellular algae similar in size to yeasts and fungal cells, and macro-algae, which are readily identified as various seaweeds in the marine environments. It is only the micro-algae that will be of interest in this context of lipid production. Those micro-organisms capable of accumulating intracellular lipids as storage oils are confined to the eukaryotic group; bacteria do not in general accumulate useful amounts of lipid although some can produce small amounts of PUFAs in their membrane lipids. These bacteria may, therefore, be potentially useful, not as a source of oil per se, but as a source of genes which could be isolated from the bacterial DNA and transferred, by genetic engineering, into another microbe or even plant (see Section 5.7). These inserted genes would cause the recipient organism to synthesize PUFA of its own volition. This prospect of using genetic engineering taking genes from micro-organisms and inserting them into plants is discussed in the final section of this chapter. Micro-organisms that accumulate more than about 20 % of their own weight as lipid – usually in the form of triacylglycerols to act as a store of carbon and energy – are known as oleaginous species. These represent a small minority of the total number of species and are able to do this because they possess biochemical mechanisms not found in the other non-oleaginous

Table 5.1

Lipid content and fatty acid profiles of selected oleaginous yeasts, fungi and micro-algae

Species

Maximum lipid content [% (w/w)]

Major fatty acyl residues [relative % (w/w)] 14:0

16:0

16:1

18:0

18:1

18:2

18:3 n-6

20:4 n-6

20:5 n-3

22:6 Important n-3 others

Yeasts1 Cryptococcus albidus var. aerius Cryptococcus curvatus D Lipomyces starkeyi Rhodosporidium toruloides

65

–

12

1

3

73

12

–

–

–

–

–

58 63 66

– – –

32 34 18

– 6 3

15 5 3

44 51 66

8 3 –

– – –

– – –

– – –

– – –

– – 23:0 (3 %)

Fungi1 Mortierella elongata Mucor alpina-peyron Aspergillus terreus Tolyposporium ehrenbergii

> > > >

– 10 2 1

7 15 23 7

– 7 – –

2 – Tr 5

18 30 14 81

12 9 40 2

25 1 21 –

16 – – –

15 – – –

– – – –

– – – –

NA NA NA NA

8 12 12 –

63 13 10 10

2 21 11 21

– – – –

4 1 3 1

9 2 2 4

12 – – 1

– 3 <1 –

– 45 25 33

– – 11 4

– – – –

NA NA NA

– – –

– 12 34

– 1 1

– – –

– 2 2

– 1 12

– 3 –

– 20 40

– – 7

– 24 –

– 18:4n-3 (19 %) –

Micro-algae Spirulina maxima2 Chlorella minutissima2 Isochrysis galbana2 Phaeodactylum tricornutum2 Nitzschia laevis Amphidinium carterae3 Porphyridium cruentum3 1

25 25 25 25

% % % %

Yeast data from Ratledge (1991). Selected micro-algae from Ratledge (2001). Lipid contents are not available (NA) but are typically less than 10 % (w/w). 3 Selected micro-algae from Cohen and Khozin-Goldberg (2005). 2

Lipids from microbial sources

83

species (for further information on this aspect of fatty acid biosynthesis the interested reader is referred to the review of Ratledge and Wynn, 2002). Oleaginous micro-organisms, however, will only accumulate lipid when they are grown in a culture medium which has a limiting amount of a key nutrient, other than carbon. Usually, this is nitrogen in the form of NH4. When this is exhausted, the cells can no longer synthesize proteins and nucleic acids, but any carbon source still present continues to be assimilated and, instead of being used to make new cell material (which is now no longer possible because of the lack of nitrogen), it is converted into lipid. The accumulated lipid, usually in the form of triacylglycerols, is therefore a reserve storage material and will be mobilized by the cells should they be subsequently starved of a supply of carbon. An outline of this process of lipid accumulation is shown in Fig. 5.1. Clearly, when the accumulated lipid in the cell contains commercially desirable fatty acids, and especially if these are present as triacylglycerols, then the organism becomes a potential target for commercial development. The oil extracted from such sources is referred to as a single cell oil (SCO), which is something of a euphemism to avoid mentioning microbe, yeast or fungus which might have negative connotations to a potential consumer Balanced growth phase

Lipogenic phase

40

Microbial biomass Glucose + NH4

Lipid (% dry wt)

Biomass (wt l–1) Nutrients (wt l–1) Arbitrary units

60

20

Lipid

0

10

20

30 40 Time (hours)

50

60

0 70

Fig. 5.1 Diagram showing the course of lipid accumulation in an oleaginous organism. The organism is grown in a culture medium which is so formulated that one nutrient, usually nitrogen in the form of NH4, becomes quickly exhausted but with an excess of carbon, usually glucose or another sugar, remaining in plentiful supply. On nitrogen exhaustion, cells can no longer multiply but continue to assimilate the glucose which is then converted into a storage material. In the oleaginous organism, this is in the form of a triacylglycerol oil.

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of the oil. The term SCO is now in general use (see Kyle and Ratledge, 1992; Cohen and Ratledge, 2005).

5.3 Large-scale cultivation of oleaginous micro-organisms, costs, oil extraction and refinement Micro-organisms, of course, have to be grown on a large scale if they are going to produce tonne quantities of oils and fats at affordable prices. Although many algae might be useful for PUFA production – see Table 5.1 – most of them are photosynthetic and will only grow as if they were miniature plants; that is in sunlight (as the source of energy) and in air with CO2 being the source of carbon. Although this sounds a cheap system, it is in fact very expensive as such organisms need to be grown in sealed culture systems (or photobioreactors) to prevent contamination from air-borne organisms. In such systems, the biomass yield is low because the penetration of light into the bioreactor is severely limited by the self-shading effect of the cells and, consequently, the cell density rarely exceeds 3–5 g dry wt/l. Even this low yield may take three to four weeks to achieve. The algae also have an unfortunate tendency to attach themselves to surfaces which then makes light penetration into a photobioreactor even more difficult. Large areas of land need to be covered with these photobioreactor systems (which may be nothing more than kilometres of polythene tubing, of no more than 2 cm diameter, stretched out on racks) in order to achieve even a modicum of production. There is also a requirement for these reactors to be located in warm, sunny climates where, preferably, the ambient temperature is about 30 °C and sunshine is available 12 hours per day for an average of 250 days per year. A supply of clean water is also a requirement. All these factors restrict the possible locations for large-scale algal culture to just a few places around the world (Israel, Australia, southern California, North Africa, perhaps southern Italy, Spain and Portugal, Mexico, Namibia and parts of South America). The cost of oil production is higher than that of cultivating a micro-organism heterotrophically. A heterotrophic organism is one which requires a fixed source of carbon, e.g. glucose or sucrose also acting as a source of energy, coupled with a supply of fixed nitrogen (usually an ammonium salt, nitrate or urea) and other essential nutrients such as phosphate, sulphate, K+, Mg2+, etc. The technology of large-scale microbial cultivation is now extremely well developed as such systems are used extensively in the production of many materials, ranging from citric acid to antibiotics. Therefore, although such systems are clearly energy-intensive and require the carbon source (or feedstock) to be purchased, their overall costs are within an affordable limit. The scale of operation for modern fermentation processes is to use stirred tank reactors up to 500 m3, although both larger and smaller units may be employed. Very often, groups of four or more fermentors are used as they

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85

can then be run sequentially and the product can be recovered using the same downstream equipment (e.g. harvesting systems, dryers, etc.) which otherwise might stand idle in between runs when only a single fermentor is used. A typical large-scale fermentation system for microbial oil production is shown in Fig. 5.2, and the various unit operations that go to make up the whole process are shown in Fig. 5.3. The process invariably begins with the transfer of the desired organism from a storage vial into a culture flask – as would be used in any microbiological laboratory. Cultivation is then scaled up by a series of transfers of the organism into increasingly large fermentors. The final production fermentor is usually inoculated with about 10 % of its volume of a rapidly growing culture so that full growth and lipid accumulation can be accomplished in the shortest possible time. The scale-up protocols are usually kept as industrial know-how but may involve the organism being grown in the earlier, seed fermentors in medium that contains a balanced source of carbon and nitrogen so as to achieve maximum cell numbers as quickly as possible. It would then be only in the final fermentor that the medium would be nitrogen limited in order to effect lipid accumulation – as shown in Fig. 5.1. It is clearly desirable to produce as much oil as possible in the fermentor in the shortest possible time, and this means producing the highest density of cells that the fermentor can achieve. Whilst cell densities of up to 100 g dry cells per litre are not unusual, and in some cases up to 200 g/l has been recorded (see below), this may mean that the necessary amount of glucose or sucrose that has to be added into the fermentor may be considerable. If a final biomass density of about 100 g/l is to be achieved, this will require about 250 g sugar/l to be added. However, many micro-organisms would find such a concentration too high for them to grow. Consequently, the sugar has to be gradually fed into the fermentor during growth in what is known as a fed-batch process. The rate of adding the glucose is carefully monitored to ensure that it does not exceed the concentration at which cell growth would be retarded. Some of the nitrogen (usually an ammonium salt) may also be added along with the glucose feed in the initial stages of the fermentation although, towards the end of the process when sufficient numbers of cells have been created, the supply of nitrogen should be stopped and only the sugar then added. The cells enter the final lipid storage phase as depicted above (see Fig. 5.1). Various aspects of the large-scale cultivation of SCO-producing micro-organisms have been covered in a recent monograph on this topic (Cohen and Ratledge, 2005). Costs of SCO production are, of course, closely guarded industrial secrets, although some idea of the likely costs may be gathered by looking at other biotechnologically derived materials. Microbial products, such as citric acid and some amino acids, can be produced as cheaply as $2000 per tonne (figures in dollars could be converted into Euros without change), including the cost of the starting substrate. Biomass material can probably be produced

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Temperature control

Continuous sterilization

Glucose Other nutrients, e.g. nitrogen

Acid Alkali pH control

Compressed air (01–0.2 vvm)

Cooling/ refrigeration

Seed fermenter (10–20 m3)

Vial

Shake flask

Waste treatment and disposal

Fermentation scale-up

Main fermenter (100–200 m3)

Centrifuge

Holding tank with heating

Moist heatstabilized cells

Oil

Downstream processing

Spray dryer Hexane extraction

Fig. 5.2 Diagram showing a typical fermentation process for SCO production. Cultivation of the organism begins with transfer of a culture from cryovial into a small shake flask which is then transferred through a series of ever-larger fermenters until the final production fermenter is reached. The final fermenter is usually inoculated at about 10 % of its volume with a rapidly growing culture of cells. It is only in the final fermenter that the medium needs to be formulated with a high C:N ratio to commence lipogenesis (see Fig. 5.1). Cells need to be heat-stabilized before harvesting. They may be extracted as a screw-pressed (moist) biomass or be spray-dried prior to extraction. Oil extraction, refining and deodorization follow conventional process technology.

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Fermentation

Heat stabilization (pasteurization)

Harvesting (holding tank with heating)

Centrifugation

OR

Filtration

Spent fermenter liquor

Moist cells Disposal Spray drying

Dry cells

Solvent extraction (e.g. hexane)

Disposal or animal feed?

Refining

Removal of coloured materials

Deodorization

Oil

Fig. 5.3

Outline of the various unit processes involved in an SCO production.

for not much more than this and thus, if the biomass contains oil at, say, 40 % of its weight then, theoretically at least, a tonne of oil might cost no more than $5000, or $5 per kilogram. To this has to be added the cost of oil extraction and refinement (see below). Also, the process of oil accumulation

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may be slightly more costly than just growing a micro-organism for optimum biomass yield rather than optimum oil yield as a longer growth period would be needed to allow lipid accumulation to be complete. For an oil-producing micro-organism grown to a density of 50 g dry mass/l with an oil content of 40 %, the ensuing oil might reasonably be expected to cost no more than $10/kg. This value would be increased if the oil content of the cells was less than 40 %. For example, if the oil content of the cells was only 25 %, then four tonnes of cells would be needed to yield one tonne of oil; if one tonne of cells then cost, say, $3000 to produce, the oil would cost $12/kg with extraction and refining costs still to be added. However, these costings, it must be emphasized, are very much estimations and final costs may well exceed this value if a slow-growing organism is being used, or if any of the unit processes (such as harvesting or drying the cells – see Fig. 5.3) are found to require specialized equipment. Again, waste disposal of the spent fermentor broth may add a significant cost as it will probably have a high biological oxygen demand and will not be dischargeable into any river without infringing local environmental laws. Some simple, but cost incurring, process for its disposal may have to be incorporated into the overall unit operations and therefore added to the final costs of the oil. In the final stages of the fermentation process, the culture broth is normally heated (pasteurized) to about 60–65 °C to inactivate lipases, either within the cells themselves or secreted into the medium. This is needed so that during the subsequent harvesting of the cells, degradation of the oil within the cells is minimized. It is during the harvesting process that the cells become removed from their carbon source (or this may have been entirely consumed just prior to terminating the fermentor run) and, consequently, the cells then mobilize their accumulated lipids to meet this imposed starvation. If left unchecked, the lipases would cause deterioration in the quality of the lipid and would result in the appearance of free fatty acids in the final product. These would not only be undesirable but would impart unwanted odours and tastes into the final oil. Thus it is essential to inactivate lipase activity prior to commencing the final harvesting of the cells. In some processes, the entire culture may be discharged from the fermentor into a holding tank (see Fig. 5.2) as this then enables the principal fermentor to be cleaned and prepared for the next run with the minimum of delay. Heating of the culture may occur, therefore, either in the main fermentor, or during the transfer of cells (using a heat exchanger) or in the holding tank itself. The heat-stabilized biomass in the holding tank can be processed over a number of hours (if not a day or two) without deterioration of the oil. Cells may be harvested by centrifugation or more likely by rotary drum filtration – the choice will largely depend on what equipment is already available to the company carrying out the fermentation work. The freshly harvested cells can be either screw-pressed to give a moist mass of material (perhaps with a 30 % water content) or subjected to spray-drying to give a powder. The method used will largely depend on the proximity of the oil extraction process

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to the fermentation plant; moist cells can be directly extracted with solvent (see below), but if some time is expected to elapse before oil extraction commences then spray-dried cells may be the preferred option. Also, if some of the oil-rich biomass is to be used as such, then spray-dried cells would be the final product as these would have long-term stability with respect to their oil content. Extraction of oil from oleaginous micro-organisms has been covered in two recent reviews (Ratledge et al., 2004, 2005). In spite of the microbial biomass representing a complete novel source of lipids, and one which had never hitherto been processed for oil extraction, it was found that conventional solvent extraction systems, using existing commercial machinery, were sufficient to effect good oil recoveries. However, because the amount of microbial cells being processed at any one time would not exceed, perhaps, 20 tonnes dry wt, conventional oil extraction equipment used in conjunction with plant oilseeds would be entirely inappropriate. Here several hundreds of tonnes of plant seed can be processed in one day; a mere 20 tonnes of microbial biomass would literally be lost in the extraction machinery and the ensuing oil severely adulterated with plant oils already in the system. Consequently, small-scale extraction equipment has to be employed such as may be used in the extraction of essential oils and fragrances from various plants. Here, the emphasis is on complete extraction of the very valuable oils from plant tissue material and not just plant seeds. It is, therefore, a matter of choosing the correct size of existing solvent extraction unit rather than having to construct a purpose-built extraction facility to handle microbial oils. Extraction of microbial oils is usually by solvent extraction, with hexane being the preferred solvent. Isohexane may also be used. Moist, pressed cells may be directly extracted if these are immediately available from the fermentor. Where there may be a storage period involved before the cells can be extracted then it may be prudent to use spray-dried cells which will be stable much longer than moist cells, even though these will have been heat treated. Following oil extraction, the solvent is removed and the crude oil is then purified using both conventional techniques and process equipment for refinement, bleaching (removal of coloured materials) and the final deodorization step (see Fig. 5.3). At all stages, because the SCO is one that contains a valuable PUFA, care must be taken to prevent unwanted oxidation reactions. The oil should preferably be kept chilled whenever possible and exposure to air is minimized. To help in such measures, an antioxidant, such as one of the tocopherols (for example vitamin E), is usually added. However, microbial oils have, almost without exception, been found to have a high natural content of antioxidant coming from the original microbial cells. This serves to stabilize the oil during the initial stages of extraction but, as this may be removed from the oil during one of the final refining stages, it is advisable that extra antioxidant is added to the final oil to ensure its stability. The nature of the microbial antioxidants associated with the SCO is not

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known – apart from them being oil-soluble; they are probably related to the tocopherols themselves and this could include them being a related flavanoid material (Shahidi et al., 1997). Oil-extraction from microbial biomass using supercritical CO2 has been considered (Wisniak and Korin, 2005) and, although this appears to be effective both in efficiency of oil-extraction and in maintaining the structural integrity of the PUFA material, it is nevertheless probably too expensive to use in comparison with the standard solvent-extraction protocols.

5.4 The production of an SCO rich in γ-linolenic acid (Oil of Javanicus) The first commercial production of a microbial oil was one containing γ-linolenic acid (GLA, 18:3n-6) as the major PUFA. This process was developed in the late 1970s and early 1980s as an alternative oil to evening primrose (Oenothera biennis) oil which, at that time, was the sole commercial oil available that contained GLA. GLA was considered to be useful for the alleviation of multiple sclerosis, a claim which is no longer made, and also for the treatment of a variety of other ailments, including pre-menstrual tension. A range of other benefits arising from the ingestion of GLA-containing oils is still recognized (Huang and Mills, 1996; Huang and Ziboh, 2001). Perhaps the least controversial of these benefits of GLA is in the prevention of diabetic retinopathy (Horrobin, 1996) and in the treatment of atopic eczema (Horrobin and Morse, 1995). It is, however, the possible prevention of premenstrual tension that leads to its greatest volume of present-day consumption in the UK and in some other European countries. As the price of evening primrose oil in the mid-1970s was in excess of $50 per kilogram, and as it contained only 9–10 % GLA in its total fatty acids, an opportunity was recognized for producing an oil from a microorganism that would be superior in quality and less expensive. The occurrence of GLA in the oils of the group of organisms known as the lower fungi, or Zygomycetes and sometimes referred to as the Mucorales, had been known since 1948 (Bernard and Albrecht, 1948). A survey of the occurrence of GLA in filamentous fungi by Shaw (1965, 1966b) had revealed that GLA rather than the n-3 isomer of 18:3 occurred throughout the zygomyces fungi. From this prior knowledge, it was then a matter of screening a large number of possible organisms to find one which grew quickly and could achieve a high cell density in a laboratory fermentor and had a high content of lipid and a high content of GLA in the fatty acids. This work was carried out in the authors’ laboratory and led to finding that, of some 300 organisms, the best one appeared to be a strain of Mucor circinelloides (formerly known as Mucor javanicus). This fungus had an oil content of approximately 25 % with a GLA content of between 18 and 20 % of the total fatty acids. Other Zygomycetes fungi were found that had higher

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lipid contents and some with higher GLA contents but never together. Consequently, a compromise had to be reached between oil content and GLA levels and the chosen organism appeared to be the best available, bearing in mind the need for the organism to grow quickly and to a high cell density. Production of the oil using M. circinelloides was undertaken by J. & E. Sturge Ltd at Selby, North Yorkshire, UK and the first oil was released for sale in 1985 (Meers and Prescott, 1987). The fungus was grown in a nitrogenlimiting medium (see Fig. 5.1) and used stirred tank fermentors of 220 m3. These fermentors were normally used for the production of citric acid using the filamentous fungus, Aspergillus niger. The technology for the large-scale production of the first SCO product was, therefore, already in place within this company who used their expertise to develop a successful protocol within about two to three years. This time included carrying out all safety checks on the oil (see also Chapter 23). The fungus was grown to a cell density in excess of 50 g/l in about three days; the cells had a final oil content of about 25 % with oil containing about 18 % GLA (see Table 5.2). Higher cell densities could be attained in the fermentor, but this led to difficulties in removal of the cells from the fermentor due to limitations in the pumps and other ancillary downstream process equipment. A pasteurization step was included in the final stages of the fermentation process (see Fig. 5.3) in order to stabilize the cells. Extraction was carried out using the pressed but still moist cells. The oil was marketed under the brand name of Oil of Javanicus and was sold to various health companies as an alternative, over-the-counter nutraceutical to evening primrose oil. A full account of the development of the GLA-SCO process has been given elsewhere (Ratledge, 2005) and this should be referred to for further information. The specification of the refined oil is given in Table 5.2 together with a comparison of the major plant sources of oils rich in GLA. It is relevant to point out that, although a number of surveys of microorganisms have been carried out subsequently to find better GLA producers (Ratledge, 2005), there has only been one report of a significant improvement over M. circinelloides. This was the discovery by Weete et al. (1998) of an unusual species of zygomycetous fungus, Syzygites megalocarpus, that could produce an oil with up to 62 % GLA. This is the highest GLA content ever recorded for any micro-organism or plant. However, the oil content of this fungus was less than 10 % of the biomass and there were difficulties in growing it above 3.5 g/l, and even this level was only attained after six days. When compared with M. circinelloides, which can reach over 50 g/l in about three days with 25 % oil and approximately 20 % GLA, the overall productivity (that is grams GLA produced/litre of fermentor/day) is still in favour of the Mucor process. Nevertheless, this unusual fungus is worthy of note as a potential alternative source of a GLA-SCO. The fatty acid profile of the oil from this fungus is also given in Table 5.2. A rival process to that of Oil of Javanicus was developed by Idemitsu Kosan Co. Ltd, Tokyo, Japan and the ensuing oil was released for sale in the

Table 5.2

Fatty acid profiles of various fungi and plants used, or considered, for GLA production. Relative % (w/w) of major fatty acids

Mucor circinelloides1 Mortierella isabellina2 Mortierella ramanniana2 Syzgites megalocarpus3 Evening primrose1 Borage1,4 Blackcurrant 1

Oil content (% w/w)

16:0

16:1

18:0

18:1

18:2 (n-6)

18:3 (n-6) GLA

18:3 (n-3)

20:1

25 ~ 50 ~ 40 10 16 30 30

22 27 24 14 6 10 6

1 1 – – – – –

6 6 5 1 2 4 1

40 44 51 12 8 16 10

11 12 10 1 75 40 48

18 8 10 62 8–10 22 17

– – – – 0.2 0.5 13

– 0.4 – – 0.2 4.5 –

Ratledge (1992) and Sinden (1987) Ratledge (2001) 3 Weete et al. (1998) 4 Borage oil also contains small amounts of 22:1 (~ 2%) and 24:1 (~ 1%). Abbreviation: GLA = gamma-linolenic acid. 2

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late 1980s, five or six years after the launch of Oil of Javanicus. The Japanese process used mainly Mortierella isabellina although it would appear that Mortierella ramanniana may also have been used (Nakahara et al., 1992). Both organisms produced substantially less GLA in the final oil than M. circinelloides (see Table 5.2) although the oil content was about twice that of the Mucor. Sales of the Mortierella oils appear to have ceased by the early 1990s shortly after Oil of Javanicus ceased production. Production of Oil of Javanicus ceased in 1990 due to the falling prices of evening primrose oil and to the advent of borage oil (also known as starflower oil). Borage oil was developed as a further alternative to evening primrose oil and, being an agricultural crop and therefore enjoying EU subsidies as a non-edible plant, could be produced for less than the equivalent biotechnological product. It also had a slightly higher content of GLA than Oil of Javanicus (see Table 5.2). Thus, by 1990 the SCO process was no longer economical and, with the take-over of the production company, J. & E. Sturge, by RhonePoulenc, it was decided that the oil no longer fitted with the new company’s product profile and no further production was carried out. It was clearly evident, however, that SCO could be produced in high tonnage quantities and represented a product to rival the highest quality plant oils: SCO were oils that could be produced to an unvarying specification; their production did not depend on the vagaries of the climate, or even political changes in some of the countries producing rival plant oils. They could be produced all round the year and did not have to be stored for up to 12 months as did the plant products which, of course, are only harvested at one time of the year. Also, they could be classed as ‘organically grown’: they were not sprayed with herbicides, pesticides or fungicides. The content of pesticide residues in Oil of Javanicus was miniscule and far below the levels found in rival oils (the levels of pesticide residues in these plant oils were, however, below the levels permitted by law and did not represent any hazard to health). In spite of these numerous advantages, it was clear that a microbial oil, no matter how good, cannot compete economically with a plant oil – even rather exotic ones such as evening primrose oil or borage oil where volumes of production are relatively low and costs of harvesting and processing are high. When SCOs have been successful, as discussed below, it has been to provide PUFAs which are not available from plant sources and which cannot be satisfactorily produced from animal sources. Selection of the PUFA to be produced microbiologically therefore depends upon the absence of alternative sources.

5.5

Production of arachidonic acid-SCO

The occurrence of arachidonic acid (ARA, 20:4n-6) in zygomycetes fungi was noted during the extensive survey by Shaw (1966a) for PUFA in microorganisms. A number of subsequent surveys of this family of fungi identified

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Mort. alpina as probably the most prolific producer of ARA (Totani and Oba, 1987, 1988; Higashiyama et al., 2002). Processes for the production of ARA were developed in the early 1990s by Suntory Ltd and Lion Corporation in Japan, although only the former process seems to have gained any significant market. It remains in operation in Japan today. ARA was originally produced in Japan as a nutraceutical fatty acid but without any well-defined role and, consequently, sales of the SCO-ARA do not appear to have been particularly high during the early years of production. It was only when it was appreciated that ARA was an important and essential fatty acid in infant nutrition, especially in neonatal babies and ones born slightly premature (Carlson et al., 1993, 1994; Lanting et al., 1994, Crozier et al., 1996), that serious attempts were made to develop production of ARA for infant nutrition (Kyle, 1996). A commercial process was developed in the mid-1990s by Martek BioSciences, Maryland, USA, in conjunction with the Dutch company of Gist-brocades, now DSM (Dutch State Mines), for the production of an SCO rich in ARA using Mort. alpina as production organism. This process has been described by a number of authors including both Kyle (1997) and Streekstra (2005) who have been directly involved in the production, and it currently accounts for more than 95 % of the world’s production of ARA. However, a rival process has been developed in China by Wuhan Alking Bioengineering Co. Ltd also using Mort. alpina (Yuan et al., 2002). The company (http://www.alking.com.cn/english) has now entered into an agreement with Cargill Co. of the USA for marketing and sales of the oil in countries not covered by the various patents taken out by Suntory and Martek. Both the Martek and Wuhan Alking processes, and probably the Suntory one too, produce an oil with a content of ARA of over 40 % of the total fatty acids and, consequently, after being refined and deodorized, it is mixed with a vegetable oil, usually sunflower oil, to bring the ARA content down to a standardized level of 40 %. The fatty acid profiles of both these oils are given in Table 5.3. Because of the rapidly-growing demand for ARA (in conjunction with DHA) for the infant formula market (see Chapter 23 and also Wynn and Ratledge, 2005), expansion of the current DSM process has led to a new production unit being opened in Belvidere, NJ, at the end of 2004. Although this doubles the production of ARA, further expansion in its production, as well as that of DHA, can be anticipated during the next two or three years to keep pace with the demand for these fatty acids. The DSM/Martek oil is used exclusively by Martek in a 2:1 combination with the DHA oil from Crypthecodinium cohnii (see Section 5.6) and is sold under the trade name of Formulaid® (Martek Biosciences Corp, USA) exclusively for the infant formula market. This is now sold in over 60 different countries. The exact details of the commercial processes remain industrial secrets, although Mort. alpina grows more slowly than M. circinelloides (see Section

Table 5.3

Fatty acid profiles of oils rich in arachidonic acid available commercially. All oils are produced by strains of Mortierella alpina. Relative % (w/w) of major fatty acids

Alking1 Martek/DSM

2

14:0

16:0

16:1

18:0

18:1

18:2

18:3

20:0

20:3

20:4

22:0

24:0

0–2

4–15

0–3

4–15

5–20

3–10

1–5

0–3

3–10

40–45

0–2

0–5

0–2

3–15

0–2

5–20

5–38

4–15

1–5

0–1

1–5

38–44

0–3

0–3

Note: Analysis taken from (FSANZ, 2003). Product specifications from Wuhan Alking Bioengineering Co., Ltd. See http://www.alking.com.cn/english/AA%20OIL%2040%25%20SPEC.pdf. 2 Currently Martek Biosciences Corporation’s ARASCOTM is produced by DSM under licence. 1

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5.3) and it probably takes five to six days before the cells reach their final lipid levels. Also, because these cells are somewhat sensitive to high concentrations of glucose, the glucose, together with NH3, is added to the medium in a fed-batch system so that the cells are able to grow at their maximum rate without inhibition by excess glucose. In the fermentation process itself, it is very important that clumping, or aggregation, of the cells is prevented. The cells should grow preferably as small discrete pellets rather than as dispersed hyphae which makes oxygen transfer into the culture broth extremely difficult. These constraints mean that it is imperative that the aeration and mixing in the fermentor are closely monitored and also controlled to ensure maximum cell formation with maximum ARA content. The concentration of glucose in the fermentor is also another factor that may influence morphological changes in the cells, and it has been suggested that optimum growth (and lipid accumulation) may be achieved by maintaining the glucose no higher than 5 g/l (Park et al., 2002), thereby showing the importance of using a fed-batch system for production. The nature of the nitrogen source may also be a significant factor influencing growth and ARA production, with soy bean meal seemingly giving higher productivities than yeast extract (Park et al., 1999) but, whatever nitrogen source is used, it seems evident that the C:N ratio is of key importance (Koike et al., 2001), with values within the range 15–32:1 being the best for lipid accumulation and ARA production. Because of the unstable nature of the ARA oil, pasteurization of the culture broth is carried out prior to harvesting and this effectively kills all the cells as well as inactivating hydrolytic enzymes, including lipases, in the cells and broth. The cells are extracted with hexane without using a spray-drying step (see Fig. 5.2), but probably much excess water is first removed from the biomass using a screw press. The final oil ‘… is a translucent yellow oil with a distinctive but not strong flavour’ (Wynn and Ratledge, 2005). This latter review, together with that of Streekstra (2005) should be consulted for further details about the Martek ARASCO® process.

5.6

Production of DHA-rich SCOs

With the realization in the early 1990s that DHA was of crucial importance in infant nutrition for both neural and retinal membrane development, a clear demand for an oil rich in this PUFA was created. (Further information concerning the nutritional and functional role of DHA is given in Chapter 23.) The only reasonable source of DHA at this time was from fish oils where it occurs together with eicosapentaenoic acid (EPA 20:5n-3) in varying proportions. However, to produce an oil containing only DHA starting with fish oil was prohibitively expensive; in addition, EPA as an additional n-3 PUFA was thought to be inadvisable for infant nutrition, partly due to its lessening the effectiveness of DHA and partly due to doubts

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about the intrinsic safety of using fish oils. For these reasons alternative sources were required.

5.6.1 DHA-SCO production using Crypthecodinium cohnii Micro-algae have long been known to contain very long-chain PUFAs (see reviews by Shifrin, 1984; Wood, 1988; Yongmanitchai & Ward, 1989) and, indeed, these represent the origin of most DHA and EPA present in fish as they are the major feeding materials of many fish in their natural environments (Ackman, 1982). Of key relevance was the knowledge that the marine dinoflagellate, known as Crypthecodinium cohnii (also called Gyrodinium cohnii and Gymnodinium simplex which are now regarded as synonyms for C. cohnii) produced lipids in which the sole PUFA was DHA (Harrington and Holz, 1968; Beach and Holz, 1973; Henderson et al., 1988). This organism appears to be one of very few organisms that produces DHA as its sole PUFA (Yongmanitchai and Ward, 1989) but, initially, being a dinoflagellate of which no example of biotechnological exploitation had taken place, it did not seem an attractive proposition for becoming an SCO producer. However, with the increasing interest in finding an oil rich in DHA, research was carried out by Martek Biosciences Corp. of Maryland, USA to identify a possible amendable strain of C. cohnii and develop it as a source of DHA oil. Some of this initial work was reported by Kyle and Ratledge (1992) where the organism was only described as ‘microalga MK8805’ and no mention was made of it even being a dinoflagellate. This early work involved a thorough examination of the organism’s growth requirements and, somewhat surprisingly, it was found to be an obligate heterotrophic organism of a rather robust character allowing it to be grown without too much difficulty in stirred tank fermentors using glucose as sole source of carbon and energy. A nitrogen source other than NH4, which was toxic even in low concentrations to C. cohnii, was also needed. Although a high salt concentration would have been required for its growth in the initial phases of the work (because of the marine origins of the organism), this requirement seems to have been partially overcome either by adaptation to a low salt medium or by substituting salts with chloride ions by various sulphates (Wynn et al., 2005). The use of a high salt medium is regarded as detrimental to large-scale cultivations in stainless steel fermentors which suffer corrosion in the presence of high amounts of chloride ions; using sulphate salts in place of the chlorides does not cause the same problems, although clearly a low salt medium is preferred to a high salt medium even if Cl– is at a low concentration. In the C. cohnii process, a high grade of stainless steel is used in the construction of the fermentors to minimize corrosion as, although only a fraction of the original chloride ion content of seawater is used in the production medium, it is nevertheless still included in the medium, and this would still be detrimental to low-grade stainless steel.

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Once an appropriate strain of C. cohnii had been identified, it was grown successfully in stirred tank fermentors with biomass yields after six days of up to 10 g/l having an oil content of about 15% and with DHA at about 30 % of the total fatty acids (Kyle and Ratledge, 1992). Four years later, Kyle (1996) was reporting cultivation of the organism in 120 m3 fermentors yielding an oil with over 40 % DHA; at this stage, the other details of the process were not declared, but production of the first SCO since the advent of Oil of Javanicus was underway. The oil that was accumulated by C. cohnii during its lipid accumulation phase (see Fig. 5.1; with the organism behaving as a typical oleaginous one) was principally in the form of triacylglycerols; consequently, the oil was ideally placed to be an edible oil as it could be extracted and purified by standard protocols. Had the DHA been found to be mainly present as phospholipids (as occurs with the lipids in many microalgae) these would not have been such an acceptable source of DHA. Triacylglycerol oils are, of course, the form of all commercial plant oils and are the desirable type of oil destined for human consumption. It is this organism and this process which today provide over 95% of the total DHA market, and it is exclusively produced by Martek. As mentioned above, the DHA-SCO is used in conjunction with ARA-SCO as the source of these key fatty acids in infant formulae. The two oils are mixed at 2:1 (ARA:DHA) and sold under the name of Formulaid®. Some further details of the production process have been provided in a recent review by Wynn et al. (2005), although information concerning the final cell density, the time of the fermentation process itself and the oil content of the cells is not disclosed. From other work, the cells would appear to take between five and seven days to reach their maximum density which is probably in excess of 50 g/l. The oil content is about 50 % of the cell dry wt (Wynn et al., 2005) with a DHA content of between 44 and 50 %. The oil is extracted with hexane (Ratledge et al., 2005) and the final material is diluted with a vegetable oil, sunflower oil or high oleic sunflower oil, to give a standardized content of the oil of 40 % DHA. The fatty acid profile of the oil, known as DHASCO® (Markek Biosciences Corp, USA), is shown in Table 5.4. Although an antioxidant is added to the oil, it is nevertheless quite stable without such addition (Kyle, 1996), but the nature of the endogenous antioxidant of the organism has not been published. Like that of other SCOs, it is probably a derivative of tocopherol or a flavanoid-like material. Sales of this SCO dominate the total SCO market (see Table 5.5). The demand for the oil appears to have doubled every year since 2003. Martek currently has over 1000 m3 of fermentation capacity dedicated to DHASCO production (Wynn et al., 2005), and this will clearly need to be increased several times more if demand for the oil is to be met over the next few years. The oil is sold in over 60 countries for use in infant formulae. The Food and Drug Authority of the USA have accepted Martek’s submission that this is a Generally Recognized As Safe (GRAS) oil suitable for incorporation into infant formulae along with the ARA-SCO. The DHA-SCO has been shown

Table 5.4

Fatty acid composition of DHA-rich oils available commercially. Relative % (w/w) of major fatty acids 14:0

16:0

16:1

18:0

18:1

18:2

18:3

20:0

20:3

20:4

22:5

22:6

Martek1 (DHASCO®)

10–20

10–20

0–2

0–2

10–30

0–5

–

0–1

–

–

0–1

40–45

OmegaTech2 (DHASCO-S®)

–

13

29

12

1

1

1

2

3

12

25

Nutrinova3 (DHActive®)

3

30

0

1

0

0

0

0

1

12

46

1 2 3

0

Oil produced by Crypthecodinium cohnii by Martek Biosciences Corporation. Fatty acid analysis data was taken from FSANZ (2003). Formerly OmegaTech but is now part of Martek Biosciences Corporation. Analysis data from Barclay et al. (2005) and Ratledge et al. (2005). Nutrinova DHActive® is possibly Ulkenia sp. For fatty acid analysis data see Kiy et al. (2005).

100 Modifying lipids for use in food Table 5.5

Worldwide single cell oil (SCO) production.

Single cell oil

Percentage of SCO production total SCO (tonnes) (1980–2002) (1980–2002)

Percentage of SCO production total SCO (tonnes) (2003 estimated) (2003 estimated)

ARASCO® DHASCO® DHA-S GLA-SCO Other Total

52.1 26 7.3 7.3 7.3 100

64 32 2 0 2 100

360 180 50 50 50 690

360 180 10 0 10 560

Source: Adapted from Kyle (2005).

to be completely non-hazardous and free of all deleterious materials; the oil is probably the most stringently tested oil of any origin and has been shown to be of the highest quality in every single respect (see also Chapter 23). Some small amounts of the oil are sold as a neutraceutical for adults under the trade name of Neuromins® (Martek Biosciences Corp, USA), as there are strong suggestions that DHA may be important in relieving such conditions as hypertension, arthritis, arteriosclerosis, depression and thromobosis (Horrocks and Yeo, 1999a,b).

5.6.2 DHA-SCO production using Schizochytrium sp. Such has been the demand for DHA that other processes using alternative organisms have been developed since the start of the Martek process. The major rival process was developed by OmegaTech Ltd in Boulder, Colorado, USA and was based on using a marine organism known as a species of Schizochytrium. This genus, together with several related genera including Thraustochytrium, Japonochytrium, Ulkenia and Aplanochytrium (Mo et al., 2002), forms a group of organisms known as the thraustochytrids or, more correctly, the family of the Thraustochytriidae. They were originally thought to be fungi because of their lack of any photosynthetic machinery, but subsequent molecular biology protocols have identified them as heterotrophic micro-algae known as stramenopiles (Honda et al., 1999; Kumon et al., 2003). They are now regarded as part of a larger phylum referred to as the Labyrinthulomycota which comprises just two families: the Thraustochytriidae and the Labyrinthulidae (also referred to as the labyrinthulids). The taxonomy of organisms in this group is still poorly understood but, overall, in their marine environments, it is believed that they may constitute a greater amount of the phytoplankton biomass than previously suspected (Kimura and Naganuma, 2001; Barclay et al., 2005). They all produce DHA in their lipids. However, as far as current information allows, this DHA appears to be always accompanied by a significant amount of docosapentaenoic acid (DPA, 22:5n-6). [Note that this fatty acid is of the n-6 series and how it fits in with

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the biosynthesis of the n-3 series of PUFA is still uncertain (Ratledge et al., 2004).] The first report of the occurrence of DHA and also of DPA in these organisms was by Ellenbogen et al. (1969) who considered these PUFAs of possible phylogenetic significance. The process developed by OmegaTech (initially called Phycotech, which was part of the Kelco group of companies) was first patented in 1991 (Barclay, 1991; also Barclay, 1992) and then described, though somewhat vaguely in key areas regarding yields of biomass, etc., by Barclay et al. (1994). A more recent account of the development of the entire process, including reasons for exploring the thraustochytrids as possible DHA producers, has been given by Barclay et al. (2005). Schizochytrium species and the related Thraustochytrum spp. had been independently investigated by Bajpai et al. (1991a,b) and by Kendrick and Ratledge (1992), but with the conclusion that this group of organisms were an unlikely source of SCOs because of their poor growth performances and low oil contents. The work of Barclay et al. (1994), however, had started by isolating organisms directly from the marine environment and had not relied upon microbial culture collections for providing stock cultures. It would seem, in retrospect, that the work of Bajpai et al. (1991 a,b) and of Kendrick and Ratledge (1992) had failed to achieve good cell growth and good oil production because the organisms being used were much less vigorous than the original strains and clearly had lost much of their potency during their time in storage. By going directly to the marine environment for new isolates, Barclay avoided this problem and was able to identify rapidly growing strains that exhibited high levels of lipid accumulation (Barclay, 1992; Barclay et al., 1994). Production of the first batches of oil from Schizochytrium sp. commenced in the mid-1990s. The specifications of the oil are given in Table 5.4. Although this oil has a high DHA content, it also contains docosapentaenoic acid (DPA, 22:5n-6). Because at the time the role of DPA in human metabolism was uncertain, this appears to have deterred OmegaTech from trying to incorporate it into infant formula as an alternative to the Martek oil; instead, the oil was incorporated into poultry feed with the resulting incorporation of DHA but not DPA into the eggs. These eggs were then marketed as SeaGold® eggs and the oil itself was named as DHAGold® (Martek Bioscience Corp, USA). It is now known (see Chapter 23) that DPA is not contra-indicated in being included with DHA in various neutraceuticals as it does not interfere with the incorporation of DHA into neural or retinal lipids in either infants or adults. This new information has enabled its direct use for human consumption. The DHA-rich oil (now referred to as DHASCO-S® – Martek) was accorded GRAS status by the FDA of the USA in 2004 for its incorporation into a number of foods; similar approval was given in Australia and New Zealand in 2002 and in Europe in 2003 (Barclay et al., 2005). Its incorporation into various functional foods is, therefore, under active development. In addition, the entire biomass, without oil-extraction, is being used extensively

102 Modifying lipids for use in food in aquaculture for feeding to fish larvae and shrimp (www.aquafuana.com/ diets&feeds.htm). It thus competes directly with fish meal for this purpose, although it probably costs about three to four times as much to produce. Schizochytrium biomass production is, in fact, one of the success stories of SCO. It grows very rapidly and to very high cell densities: far greater than any other oleaginous organism. Using a fed-batch mode of production, the production organism of OmegaTech has been grown to cell densities in excess of 200 g/l in 72 hours (Bailey et al., 2003; Barclay et al., 2005) with an oil content of about 35 % and a DHA content of the lipid of between 35 and 45 %. Strains of the organism have been selected that no longer require a high salt concentration to grow which is particularly important to keep the total Cl– content as low as possible (see also Section 5.6.1), thereby preventing corrosion of the stainless steel fermentors used in the large-scale production process. Although the size of the OmegaTech production process has not been declared, it is reasonable to assume that this will be using fermentors of at least 100 m3 and, if the current plans for expanding production both of the DHASCO-S® oil and the DHA-rich biomass are to be fulfilled, the overall scale could be expected to double and double again over the next few years. The company itself was taken over by Martek in 2002 and, consequently, the applications for the DHASCO-S® are kept separate from those of the DHASCO from C. cohnii. Because of the very high productivities that can be attained by the Schizochytrium process, this oil is by far the cheapest SCO available; the biomass too is highly competitive with other biomass productions and, if further economies of scale can be realized, it may well prove to be the material of choice for fish feeding.

5.6.3 Other processes for the production of DHA-rich SCOs Such has been the success of the Martek and OmegaTech processes that rival ones have subsequently been developed. These have also explored the applications of the thraustochytrid and labyrinthulid organisms (see previous section). Of significant note is the process developed by Nutrinova GmbH, Frankfurt am Main, Germany. This uses an organism known as Ulkenia sp. and has recently been described by Kiy et al. (2005). The organism is now grown commercially in large-scale stirred tank fermentors in the same way as other SCO-producing micro-organisms. The fatty acid profile of the oil is shown in Table 5.4. This, like the Schizochytrium sp. oil, also contains a substantial proportion of DPA. The oil was initially sold under the trade name of DHActive but this has now been changed to NutrinovaDHA® (Nutrinova, Germany) (see also Chapter 23). Applications of the Ulkenia oil, which do not appear to include its addition into infant feeding formulae, are mainly as an additive to a variety of foods and drinks. Such DHA-enriched foods appear to be stable and the DHA does not apparently oxidize nor does it impart any fishy after-taste (Kiy et al.,

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103

2005). With the specification of the Ulkenia oil being similar to that of the Schizochytrium oil (see Section 5.5.2), it is perhaps not surprising to find that the two companies producing these oils are in dispute about possible patent infringement (www.martekbio.com/corporate/company_profile.asp). It may, though, be some time before this issue is resolved. Another recent patent that has been filed for DHA production using a related organism is that of Yokochi et al. (2002) which features an organism belonging to the genus of Labyrinthula. This is grown with oleic acid, linoleic acid or linolenic acid, or even with an oil such as soybean oil to produce an SCO which contains ARA, EPA, DPA and DHA as well as some docosatetraenoic acid (22:4). However, such a complex spectrum of fatty acids would inevitably limit the applications of the oil. In addition, this organism has fastidious growth requirements and needs a bacterium to be included in the medium for the Labyrinthula sp. to grow. Consequently, this would make the entire process completely unrealistic for commercialization. An account of this work has also been published conventionally (Kumon et al., 2002). A patent has also been filed by Suntory Ltd and Nagase & Co. Ltd (Tanaka et al., 2003) for the production of a DHA and/or DPA oil using a species of Ulkenia which is similar to the process already underway by Nutrinova GmbH. The profile of this oil would appear to be similar to the oils described in Table 5.4. It is uncertain at the time of writing (Spring 2006) whether this represents a potentially serious rival process for the production of these oils to that which uses the thraustochytrid/labyrinthulid group of organisms.

5.7

Prospects for other PUFA-SCOs

5.7.1 Eicosapentaenoic acid The major outstanding PUFA which is currently in demand, but is not easily available from any source, is eicosapentaenoic acid (20:5n-3). This fatty acid has strong anti-inflammatory properties and it has been suggested that it could be useful for the treatment of a number of clinical disorders including schizophrenia, bipolar disorder (manic depression), and possibly conditions such as Alzheimer’s disease as well as certain types of cancer (see Chapter 23). The only present source of this PUFA is from fish oils, but these always contain a mixture of EPA and DHA and are not suitable for clinical applications as DHA is contra-indicated for many of the effects that are brought about by EPA on its own. Thus, to produce an EPA-only oil (or methyl or ethyl ester of it), expensive and extensive purification protocols, eventually involving preparative-scale HPLC, are required. Although there would appear to be a clear commercial need for an EPAonly oil, searches for an EPA-producing, heterotrophic micro-organism, which would seem the best prospect for realising this aim, have so far not revealed any likely candidate for large-scale production. Possible candidates all appear

104 Modifying lipids for use in food to be photosynthetic micro-algae (Gill and Valivety, 1997; Vazhappilly and Chen, 1998a,b; Cohen and Khozin-Goldberg, 2005; Wen and Chen, 2005). Many produce EPA along with either ARA or DHA, but none produces EPA on its own. The large-scale cultivation of phototrophic micro-algae, moreover, is very expensive, as has been discussed in Section 5.3, and even the best ones are not realistic propositions for SCO production (see Cohen et al., 1992; Yongmanitchai and Ward, 1992; Wen and Chen, 2000a, 2001, 2002; Cohen and Khozin-Goldberg, 2005). Even when attempts have been made to grow algae under heterotrophic conditions (i.e. in the dark with glucose as carbon and energy source), these have not met with success. For example, Wen and Chen (2000b) grew the diatom Nitzschia laevis under heterotrophic conditions and phototrophically and also mixotrophically, providing both light and glucose simultaneously. Under heterotrophic conditions and mixotrophic conditions, about 2.5 g cells were produced over five to six days but less than 0.5 g was produced under phototrophic conditions. Lipid contents of the former cells were about 12 % with between 15 and 19 % EPA. Thus improvements by two orders of magnitude would be needed before such processes would rival the other SCO processes in terms of productivity. Admittedly, phototrophic processes enjoy a free carbon source (CO2) and free energy (sunlight) but these do not make up for the intrinsic very low productivities of growth and lipid production. We still await, therefore, the advent of a good, heterotrophic organism for an EPA-SCO that would rival either C. cohnii or Schizochytrium sp. being used for DHA production in terms of productivity, efficiency and cheapness. Although it has been suggested that Mort. alpina, as used for ARA production (see Section 5.2), could also be used to produce EPA (Shimizu et al., 1988, 1989), this does not seem to be a realistic proposition. The organism needs to be cultivated with α-linolenic acid (18:3n-3) or linseed oil and at a lower temperature (~ 12 °C) than is normally used for ARA production (~ 26– 28 °C). At the end of growth, the culture is allowed to stand for several days during which some lipolysis occurs and the level of EPA in the final oil goes up to about 12–15 %. The total growth period is now about two weeks but, even in this case, the EPA is still produced along with ARA so that it is still not an EPA-only oil.

5.7.2 Other PUFAs Other PUFAs that might be produced by micro-organisms have been recently reviewed by Sakuradani et al. (2005). These fatty acids include stearidonic acid (18:4n-3), dihomo-γ-linolenic acid (DHGLA, 20:3n-6), eicosatrienoic acid (20:3n-3; also known as Mead acid), and eicosatetraenoic acid (20:4n3). All these can be produced using various mutants of M. alpina (Certik et al., 1998) but, apart from DHGLA, yields are rather low (see also Ratledge, 2001) and, without further development work, are unlikely to represent realistic commercial sources. For DHGLA, however, the content of this fatty acid

Lipids from microbial sources

105

reaches about 23–25 % of the total fatty acids, and this might represent a reasonable route to obtaining oils rich in this particular PUFA. The main problem, however, with all of these PUFAs lies in their nutritional applications not being sufficiently clear, even although it has been stated that oils rich in DHGLA and Mead acid are in current production in Japan (Sakuradani et al., 2005). Docosapentaenoic acid (DPA, 22:5n-6) has already been mentioned as a component of the DHA oils produced by Schizochytrium sp. and other related organisms (see Table 5.4). It is thought that this fatty acid may be produced in Japan by Suntory Ltd (see Tam et al., 2000), possibly as a by-product of the purification of a DHA-enriched oil from one of the production organisms (see Section 5.5) already mentioned. Interestingly, DPA as the sole PUFA has been reported in a new isolate of a labyrinthulid (Kumon et al., 2003), but it is unlikely to be the commercial source of DPA as the organism can only be grown on agar plates with a dead bacterium added as a food source. As with other PUFAs mentioned above, nutritional applications of DPA are uncertain, although it has been suggested that it may be useful for it to be included with ARA and DHA as it can serve to maintain DHA at a higher circulating concentration in the blood stream (at least in rats) thereby allowing more DHA to be taken up by neural tissues (Tam et al., 2000). It would seem unlikely that there is a specific process for the production of DPA itself when the main market still remains for DHA.

5.8

Future trends

Without doubt, the very high level of current interest in DHA and other PUFAs of nutritional value will make the next decade of SCO production very exciting. In the short term, micro-organisms for the production of DHA and ARA are likely to hold centre stage probably for the next six to eight years and possibly more. A lot will depend on how successful the genetic engineers will be in producing genetically modified crops that could produce these desirable PUFAs in their seed oils as these would be cheaper than the SCOs. Martek Biosciences Corp., Maryland, USA are likely to remain the major player in this field, particularly following their acquisition of OmegaTech in 2002. However, other industrial companies will not let this position go unchallenged, especially when the various key patents begin to run out in the next five years or so. These activities will be primarily focussed, one would imagine, on establishing alternative microbial routes to DHA and ARA. The infant formula market for these oils is already established, but the market in general is capable of considerable expansion as more people become aware of the advantages of their inclusion in the diet. The incorporation of DHA into various functional foods is also likely to see considerable expansion over the next decade. Here, the benefits of DHA for the prevention of cardiac

106 Modifying lipids for use in food problems is likely to be the major driving force. To meet these demands it is likely that the Schizochytrium oils will be to the fore as these oils can be produced even more cheaply than the C. cohnii DHA-SCO and could therefore be expected to compete with the highest quality fish oils on an equal price basis. Their incorporation into a wide variety of foods and drinks should, therefore, allow considerable market penetration of these oils in the next few years. After ARA-SCO and DHA-SCO, the next likely target is probably an EPA-SCO (see Section 5.7.1). There are clear reasons why oils high in this fatty acid would find a ready and lucrative market as its major uses are in various clinical applications (see also Chapter 23). This means that high purity oils with a high content of EPA will be needed. These will probably have to be produced by fractionation of an appropriate microbial oil so that levels of EPA in the final oil would exceed 90 %. This will make the EPAoil extremely concentrated and allow consumption of multiple gram quantities without difficulty; also, any contra-indicated PUFA will be removed in such fractionation protocols. It may be anticipated that the high costs of producing and processing such an oil will not detract from its sales as products with clear medical benefits are likely to be in considerable demand and not limited by price. All that is needed to realize this target of an EPA-SCO is to find an appropriate organism, preferably one which can be grown efficiently in stirred tank fermentors, and produce an oil with at least 40 % EPA as the sole PUFA. Such an organism would then parallel the existing commercially used microorganisms for ARA and DHA oils. However, as various groups have been searching for such an organism for a number of years, it may be that such an organism does not exist and would then have to be created by genetic manipulation. This may be a formidable task, although the simplest option would appear to start with Mort. alpina which produces ARA (20:4n-6) in abundance and then clone into this organism a gene coding for a ∆17-desaturase (perhaps better termed an n-3 desaturase) so that the ARA is then converted into EPA (20:5n-3) in a single step (see Fig. 5.4). Until recently, a gene coding for an n-3 desaturase has been very elusive and difficult to find in any organism. Khozin-Goldberg et al. (2002) considered that EPA synthesis in the alga Monodus subterrraneus was via n-6 fatty acids which involved a non-specific desaturase that did act at the n-3 position but which probably could not be described as being strictly an n-3 desaturase. Huang et al. (2004) has indicated that these n-3 desaturases have a limited distribution occurring only in a few plants, some lower eukaryotic organisms (such as Mortierella) and cyanobacteria, and, moreover, may only act on C18 substrates. It is, therefore, of considerable importance in this connection to note the recent finding of Pereira et al. (2004) who reported the presence of a novel n-3 fatty acid desaturase in the EPA-producing lower fungus, Saprolegnia diclina. When the gene for this desaturase was cloned into the yeast, Saccharomyces cerevisae (which only has one desaturase, the ∆9 one

Lipids from microbial sources n-6 Series 18:2 (9, 12) (linoleic acid)

n-3 Series n-3 DS

18:3 (9, 12, 15) (α-linolenic acid)

∆6 DS

∆6 DS

18:3 (6, 9, 12) (GLA)

n-3 DS

18:4 (6, 9, 12, 15) (stearidonic acid) EL

EL 20:3 (8, 11, 14) (DHGLA)

n-3 DS

n-3 DS

n-3 DS

22:5 (7, 10, 13, 16, 19) ∆4 DS

∆4 DS 22:5 (4, 7, 10, 13, 16) (DPA)

20:5 (5, 8, 11, 14, 17) (EPA) EL

EL 22:4 (7, 10, 13, 16)

20:4 (8, 11, 14, 17) ∆5 DS

∆5 DS 20:4 (5, 8, 11, 14) (ARA)

107

n-3 DS

22:6 (4, 7, 10, 13, 16, 19) (DHA)

Fig. 5.4 Pathway for the biosynthesis and interconversion of polyunsaturated fatty acids of the n-3 and n-6 series. EL = elongase enzyme system; n-3DS = n-3 desaturase. This is the ‘conventional’ route of formation, although an alternative route known as the polyketide synthetase pathway exists in some bacteria, in Schizochytrium sp. (Metz et al., 2001) and Ulkenia sp. (Kiy et al., 2005) for the formation of EPA and DHA (see also Ratledge, 2004). In the pathways shown, the participation of the n-3 desaturases for some substrates is uncertain (see text).

for synthesizing oleic acid), and the yeast was fed with arachidonic acid, a 26 % conversion took place to EPA. The amount produced, however, was extremely small. Nevertheless, the opportunities for genetically engineering a micro-organism, such as Mort alpina, in order to achieve EPA production are now opened up. It is, however, much more likely that genetic modification of organisms will be with plants rather than with micro-organisms where there is now intense activity. These are the long-term goals of the key industrial groups active in this area. Such companies include BASF, Ross Foods, Monsanto, DuPont and others. Plants, of course, represent the ultimate cheapest source of oils and fats and thus would be the preferred route to obtain DHA, ARA, EPA and, indeed, any other desirable long-chain PUFA. Unfortunately no plant produces any PUFA longer than C18, with the best producer probably being Echium that produces stearidonic acid (18:4n-3). Other plants only produce PUFAs up to 18:3. Therefore, genetically-modified (GM) plants will be necessary to achieve these target oils.

108 Modifying lipids for use in food The problems of gene cloning into plants are considerable. Not only have the right genes to be identified and cloned, and we have seen how difficult this was with the n-3 desaturase gene mentioned above, but more than one gene will need to be put into a plant to achieve long-chain PUFA production. Even for plants such as sunflower, which is rich in linoleic acid (see Fig. 5.6), or flax, which produces α-linolenic acid (ALA), several genes would have to be inserted into them. For example, to convert ALA into EPA, two desaturases would be needed, a ∆6 and a ∆5, as well as an elongase for increasing the chain-length from C18 to C20 (see Fig. 5.4). These genes must then be expressed (i.e. the DNA must be transcribed into messenger RNA and then translated into a protein) and the resultant protein must be enzymatically active. This, in itself, may not be without difficulty as many recombinant proteins fold incorrectly and are inactive. Moreover, the genes have to work in conjunction with the existing plant fatty acid biosynthetic system and work only in plant seeds during their development and oil accumulation. The genes, thus, need to be spatially and temporally controlled. If the genes were to produce PUFAs throughout the plant, then the plant would probably be unable to function properly. To achieve this control over the expression of these foreign genes will require other genes to be inserted alongside them to ensure the correct location of the new proteins and the operation of them (switching on) at the correct time. This is a formidable task. The reports which have been issued to date (Huang et al., 2004; Napier, 2004; Napier et al., 2004; Qi et al., 2004; Sayanova and Napier, 2004) have indicated some small measure of success in getting some PUFA formation to occur in plants, but a careful inspection of the data indicates that the amounts being produced are very small indeed and would suggest that significant advances are still need to achieve even a modicum of success. An alternative strategy to cloning multiple genes from various sources is, however, possible by the discovery of a new pathway for EPA and DHA biosynthesis. EPA is synthesized in a few bacteria of marine origin by an alternative route to the conventional fatty acid synthetase system using a polyketide synthetase (PKS) (Metz et al., 2001). The same PKS route is also used in Schizochytrium sp. to produce DHA. This pathway has also been reported in Ulkenia sp. (Kiy et al., 2005), and the PKS route may be a common property of all the thraustochytrids. Whether DHA synthesis follows a similar route in C. cohnii is not yet known, but all the indications are that this is so (see for example Sonnenborn and Kunau, 1982). The PKS is an aggregate of five different proteins which together carry out 11 different reactions (Metz et al., 2001). The PKS system still uses acetyl-CoA units as the building units for fatty acid biosynthesis, but it produces a series of unsaturated intermediates where the double bond is not reduced in situ but is retained in the correct configuration so that the final product, DHA, is produced intact (see Ratledge, 2004). No further desaturation is required. No desaturases are needed and no elongases. The prospect of then cloning the intact genetic sequence coding for the

Lipids from microbial sources

109

entire PKS system thus opens up opportunities for achieving PUFA biosynthesis in a plant without having to clone in a number of different and disparate genes: however, this task is likely to be formidable. The genetic sequence of the PKS system is likely to be a number of times larger than even the largest genes that have been successfully cloned to date and may, therefore, pose problems for which solutions may not yet exist. Again, the PKS genes will need to be spatially and temporally expressed so that DHA is produced only in the maturing plant seed. How long this is likely to take is impossible to predict. When, or perhaps, if, this happens then it would be the virtual end of SCO production. Just as was seen with GLA production (see Section 5.4), it is not possible for even the cheapest SCO process to compete against plant oils. However, we should not forget that the costs of producing the Schizochytrium oil (DHASCO-S®), because of the very high productivity of the process, are likely to be reasonably low and so this will be the bottom line against which any GM-plant DHA oil will have to compete. Finally, and not insignificantly, there is always going to be the problem of the acceptance of any GM plant product by the general public. It will thus be an interesting sociological dilemma as to whether the public would prefer to consume a product from dead animals, i.e. fish, or take something from a GM crop. Perhaps, though, they may prefer to stay with SCOs – organically produced using only the purest of ingredients!

5.9

References

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112 Modifying lipids for use in food PEREIRA S L, HUANG Y S, BOBIK E G, KINNEY A J, STECCA K L, PACKER J C

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6 Methods of analysis to determine the quality of oils K. Warner, National Center for Agricultural Utilization Research, USA

6.1

Introduction

As discussed in other chapters in this book, the compositions of oilseeds are modified for various reasons, such as improving oxidative stability or nutritional quality. For these purposes, the fatty acid composition is the commonly altered attribute of oilseeds, although geneticists are working on changing the profiles of tocopherols too. The fatty acid composition unique to each oilseed type is most often not optimum for its end use. For example, traditional rapeseed oil contained erucic acid that was not nutritionally acceptable, so work was undertaken to decrease the erucic acid content from a range of 2– 60 % to less than 2 % in order to produce a low erucic acid rapeseed oil (also known as canola oil in North America). This oil still contained high levels (5–14 %) of linolenic acid, so additional work was done to decrease the linolenic acid to approximately 3 % to improve oxidative stability. The result was low linolenic acid canola oil. Geneticists based their early fatty acid composition targets on results from research studies on improving the oxidative stability of oils. Increasing oleic acid and decreasing linolenic and linoleic generally enhance the stability of oil compared to the unmodified oil. Examples of modified oilseeds include high oleic acid sunflower, high oleic acid safflower, high oleic acid peanut, high oleic acid soybean, and high oleic acid canola (rapeseed). Sunflower, corn and soybean have been modified to have moderate levels of oleic acid. Linolenate-containing oils such as soybean, canola and flax have cultivars with reduced linolenic acid levels. Many of these oils are suitable to replace hydrogenated vegetable oils for high-stability applications such as frying. Hydrogenated oils are also important for functionality in margarines and

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shortening, so oilseeds such as soybean have been bred with higher levels of palmitic and stearic acid. No matter what modifications are made to oilseed compositions, there are basic tests that need to be conducted with the resulting oil. Not only do plant geneticists want to know how successful they were in modifying the oilseeds, but also the oil processor and food manufacturer want to know the composition and characteristics of the oil before deciding to process the oilseeds or to use the oil in food applications. Finally, when fatty acids are modified, other attributes and compositional characteristics such as minor constituents of the oils may change, so it is important to evaluate the oil for more than fatty acids. As compositional changes in oilseeds become more and more intricate, accurate and reproducible methods of determining quality and composition will be important. This chapter will review the analytical methods used to measure the compositional changes from oilseed modification and to evaluate oil quality. Complete analytical details will not be provided in this chapter; however, methods will be referenced.

6.2

Methods to measure compositional attributes

6.2.1 Oil extraction Using authentic samples of seeds that are extracted by solvent or pressing without further processing provides the purest form to measure oilseed composition. In fact, Codex Alimentarius requires that oil composition analyses be conducted on crude, unprocessed oils from authentic seed samples. If crude oil is used without knowing the complete history of the sample, then contamination of the oil with unknown oil can give misleading results for the oil composition. Therefore, oil should be extracted from a known lot of seeds. In addition, processing steps can alter some compositional factors. For example, deodorization can remove various amounts of tocopherols depending on the conditions of deodorizing. No further processing beyond extraction is necessary if only the effects of oilseed modification are needed. Oil may be collected from the seeds by pressing or solvent-extraction. If oil yield information is needed then a solvent-extraction of ground seeds is necessary (AOCS, 1998).

6.2.2 Fatty acids and trans fatty acids Fatty acid composition is the primary attribute measured in altered oilseeds. Two methods for fatty acid composition by gas–liquid chromatography (GLC) of vegetable oils are ISO 6321:1991 (ISO, 2005) plus Amendment 1:1998 (ISO, 2005) and AOCS Ce 1e-91(97) (AOCS, 1998). The AOCS has method Ce 1e-91(97) (AOCS, 1998) for fatty acid composition by GLC of vegetable oils. If the oil is hydrogenated or refined, then AOCS Method Ce 1f-96 (97) (AOCS, 1998) or ISO 15304:2002 (ISO, 2005) can be used to determine cis

116 Modifying lipids for use in food and trans fatty acids by capillary GLC. This method is designed to identify and quantify the trans fatty acid isomers in vegetable oils using a capillary column with a highly polar stationary phase, according to their chain-length, degree of unsaturation and geometry and position of the double bonds. Data on all other fatty acids can be obtained at the same time. A spectrophotometric method to determine trans fatty acids is available as AOCS Method Cd 1495(97) (AOCS, 1998) and is appropriate for natural or processed long-chain acids, esters and triglycerides with trans levels greater than 0.5 %. It is not applicable for oils with over 5 % of conjugated unsaturation. The Codex Alimentarius Committee on Fats and Oils has a Named Vegetable Oil Standard (Codex Alimentarius, 2001) with the primary compositions of 21 oils including four modified oils (low erucic acid rapeseed, high oleic safflower, high oleic sunflower and mid-oleic sunflower). The fatty acid compositions of the modified oils and their corresponding traditional oils are shown in Tables 6.1 and 6.2. Two soybean oils (low linolenic acid soybean and mid-oleic soybean) were proposed for inclusion in the standard at the February, 2005 meeting of the Fats and Oils Committee. Codex Alimentarius quotes minimum and/or maximum limits for some of the fatty acids of modified oilseeds. For example, low erucic acid rapeseed oil must not contain Table 6.1 oils.

6:0 8:0 10:0 12:0 14:0 16:0 16:1 17:0 17:1 18:0 18:1 18:2 18:3 20:0 20:1 20:2 22:0 22:1 22:2 24:0 24:1

Fatty acid compositions of crude regular and modified soybean and rapeseed

Soybean

Soybean (low linolenic acid)

Soybean (mid-oleic acid)

Rapeseed

Rapeseed (low erucic acid)

ND ND ND ND–0.1 ND–0.2 8.0–13.5 ND–0.2 ND–0.1 ND–0.1 2.0–5.4 17.7–28.0 49.8–59.0 5.0–11.0 0.1–0.6 ND–0.5 ND–0.1 ND–0.7 ND–0.3 ND ND–0.5 ND

ND ND ND ND–0.1 ND–0.2 8.0–13.5 ND–0.1 ND–0.1 ND–0.1 2.0–5.4 22.0–33.0 48.0–60.0 0.5–4.5 ND ND–0.5 ND–0.1 ND–0.7 ND–0.3 ND ND–0.5 ND

ND ND ND ND–0.1 ND–0.2 5.0–13.5 ND–0.2 ND–0.1 ND–0.1 2.0–5.4 45.0–70.0 15.0–40.0 0.5–4.5 ND ND–0.5 ND–0.1 ND–0.7 ND–0.3 ND ND–0.5 ND

ND ND ND ND ND–0.2 1.5–6.0 ND–3.0 ND–0.1 ND–0.1 0.5–3.1 8.0–60.0 11.0–23.0 5.0–13.0 ND–3.0 3.0–15.5 ND–1.0 ND–2.0 > 2.0–60.0 ND–2.0 ND–2.0 ND–3.0

ND ND ND ND ND–0.2 2.5–7.0 ND–0.6 ND–0.1 ND–0.1 0.8–3.0 51.0–70.0 15.0–30.0 5.0–14.0 0.2–11.2 0.1–4.3 ND–0.1 ND–0.6 ND–2.0 ND–0.1 ND–0.3 ND–0.4

ND = not detectable Source: Codex Alimentarius (2001).

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Table 6.2 Fatty acid compositions of crude regular and modified safflowerseed and sunflower oils. Fatty acid

Safflowerseed

Safflowerseed (high oleic acid)

Sunflower

Sunflower (mid-oleic acid)

Sunflower (high oleic acid)

6:0 8:0 10:0 12:0 14:0 16:0 16:1 17:0 17:1 18:0 18:1 18:2 18:3 20:0 20:1 20:2 22:0 22:1 22:2 24:0 24:1

ND ND ND ND ND–0.2 5.3–8.0 ND–0.2 ND–0.1 ND–0.1 1.9–2.9 8.4–21.3 67.8–83.2 ND–0.1 0.2–0.4 0.1–0.3 ND ND–1.0 ND–1.8 ND ND–0.2 ND–0.2

ND ND ND ND–0.2 ND–0.2 3.6–6.0 ND–0.2 ND–0.1 ND–0.1 1.5–2.4 70.0–83.7 9.0–19.9 ND–1.2 0.3–0.6 0.1–0.5 ND ND–0.4 ND–0.3 ND ND–0.3 ND–0.3

ND ND ND ND–0.1 ND–0.2 5.0–7.6 ND–0.3 ND–0.2 ND–0.1 2.7–6.5 14.0–39.4 48.3–74.0 ND–0.3 ND ND–0.3 ND 0.3–1.5 ND–0.3 ND–0.3 ND–0.5 ND

ND ND ND ND ND–0.1 4.0–5.5 ND–0.05 ND–0.05 ND–0.06 2.1–5.0 43.1–71.8 18.7–45.3 ND–0.5 ND 0.2–0.3 ND 0.6–1.1 ND ND–0.09 0.3–0.5 ND

ND ND ND ND ND–0.1 2.6–5.0 ND–0.1 ND–0.1 ND–0.1 2.9–6.2 75.0–90.7 2.1–17.0 ND–0.3 ND 0.1–0.5 ND 0.5–1.6 ND–0.3 ND ND–0.5 ND

ND = not detectable Source: Codex Alimentarius (2001).

more than 2 % erucic acid as a percentage of the total fatty acids. High oleic acid safflower oils cannot contain less than 70 % oleic acid as a percentage of the total fatty acids, and high oleic acid sunflower oil cannot contain less than 75 % oleic acid as a percentage of the total fatty acids. When oleic acid content of mid-oleic sunflower was added to the Named Vegetable Oil Standard (Codex Alimentarius, 2001) its composition for oleic was between that of sunflower oil and high oleic acid sunflower oil. Oleic acid ranges are for sunflower 14–39.4 %, 43.1–71.8 % for mid-oleic sunflower oil and 75–90.7 % for high oleic sunflower oil. Although the ranges of fatty acids for these oils for the Codex standard provide valuable information, they are the result of hundreds of crude oil samples. Several researchers have published fatty acid compositions of modified oilseeds that have been processed into edible oils through the traditional processes of refining, bleaching and deodorizing (Fuller et al., 1971; Purdy, 1985; Warner and Knowlton, 1997; Petukhov et al., 1999; Pokorny, 2000; Tompkins and Perkins, 2000; Warner et al., 2003; Hosseinian, 2004). The fatty acid composition data in these papers is for individual oils rather than many samples as in the Codex tables.

118 Modifying lipids for use in food 6.2.3 Tocopherols and tocotrienols It is important to measure the tocol content of an oil because of both nutritional quality and oxidative stability. In addition, the tocopherol profile is sometimes used as an identity characteristic for an oil. Tocopherols and tocotrienols are important bioactive constituents in vegetable oils. They are vitamin-E-active substances derived from a chromanol structure. In vivo, α-tocopherol is the primary precursor to vitamin E; however, δ- and γ-tocopherol are the most potent antioxidants in vitro. Although plant geneticists are currently modifying some oilseeds to optimize the amounts, types and ratios of tocopherols, the effect of fatty acid modification on tocopherol contents is the present objective in measuring tocopherols. Mounts (1996) compared the tocopherol contents of 12 soybean oils with modified fatty acid compositions. The soybeans with high palmitic acid and high stearic acid had higher levels of β-tocopherol; however, few differences in tocopherol contents were reported in modified and unmodified canola oils (Abidi et al., 1999). AOCS method Ce 8-89 (AOCS, 1998) describes a procedure to determine tocopherols and tocotrienols in vegetable oils and fats by high-performance liquid chromatography (HPLC). Calibration curves are determined for each tocopherol from tocopherol standards, and the calibration factors for tocotrienols are considered to be equivalent to that of the corresponding tocopherols. The ISO 9936:1997 (ISO, 2005) method can also be used. Normal-phase HPLC techniques are most commonly used to separate tocol peaks because β-tocopherol and γ-tocopherol can be resolved. However, reverse-phase (RP)HPLC methods with an octadecylsilica (ODS) column cannot resolve the β- and γ-tocopherols. Abidi (2003) found that a polar RP-HPLC column (pentafluorophenlylsilica) system could separate these homologues. The tocopherol contents of crude low linolenic acid soybean, mid-oleic acid soybean, low erucic acid rapeseed, high oleic acid sunflower, mid-oleic acid sunflower and high oleic acid safflower oils are given in Tables 6.3 and 6.4 along with the original oils. Few differences in the ranges of the compositions are seen between the regular and modified oils with the exception of the αtocopherol contents of the sunflower oil. The range of α-tocopherol is wider for sunflower oil and high oleic acid sunflower oil than for mid-oleic sunflower oil, probably because the seeds for the mid-oleic sunflower were only collected from the USA but the seeds for sunflower and high oleic sunflower were obtained from various parts of the world. Tocotrienols are important nutritionally as well as for improving oxidative stability. They are found at only low levels in most vegetable oils with higher levels in palm, maize (corn), and rice bran oils.

6.2.4 Sterols Desmethylsterols are often measured for the purposes of oil identity because oils have distinct patterns of sterols. Sterol contents can be measured by methods ISO 12228:1999 (ISO, 2005) or AOCS Ch 6-91 (97) (AOCS,

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1998). Gas chromatography (GC) is the most frequently used technique for the analysis of sterols. Fused-silica capillary columns contain chemically bonded stationary phases of variable polarity and are widely used because of their durability and flexibility. When high-temperature capillary GC columns (e.g. phenylmethylsilicone) are used, sterol sample assays can be conducted with high sensitivity and good resolution. In a typical analysis, GC is coupled with flame ionization (FID) to monitor analytes in the column effluents or with mass spectrometry (MS) for structural identification and quantitation. Other detectors such as electron capture and thermal conductivity detectors are less commonly used because FID has the best GC features in terms of detection sensitivity, response linearity, and response generality (Abidi, 2001). Mounts et al. (1996) found no correlations of sterol content changing with changes in fatty acids of 13 crude soybean oils. On the other hand, Abidi et al. (1999) reported that sterol composition was affected by modification of fatty acids in canola oil. For example, brassicasterol, campesterol, and β-sitosterol levels were consistently lower in high oleic/low linolenic acid canola oils than in regular canola oils. The phytosterol contents of crude modified soybean, rapeseed, sunflower, and safflower are given in Tables 6.3 and 6.4.

6.2.5 Phospholipids Phospholipids are important minor constituents of crude oils that are removed to varying degrees during oil processing. Mounts et al. (1996) found little effect on the phospholipid contents of 13 crude soybean oils as fatty acid compositions were altered compared to a control with traditional composition. In contrast, Abidi et al. (1999) found that crude high oleic/low linolenic acid canola oils had higher amounts of phosphatidylcholine and phosphatidic acid than regular canola oil. In another type of soybean that was bred to be resistant to glyphosate-based herbicides, List et al. (1999) reported no differences in amounts of phospholipids in crude glyphosate-tolerant soybean oil vs regular crude soybean oil. Phospholipids can be analyzed by AOCS method Ja 7b-91 (AOCS, 1998).

6.3

Methods of measuring characteristics of edible oils

6.3.1 Chemical and physical characteristics A group of chemical and physical characteristics that may vary with fatty acid modification include relative density, refractive index, saponification value, iodine value, and unsaponifiable matter. The values for crude sunflower oil and its two modified fatty acid composition counterparts are given in Table 6.5. There are few differences in the ranges of values for the various characteristics with the exception of iodine value for which differences would be expected. Appropriate methods for these procedures are available from

Table 6.3

Compositions of tocopherols and sterols in crude regular and modified soybean and rapeseed oils. Soybean

Soybean (low linolenic acid)

Soybean (mid-oleic acid)

Rapeseed

Rapeseed (low erucic acid)

Tocopherols (mg/kg) Alpha tocopherol Beta tocopherol Gamma tocopherol Delta tocopherol Alpha tocotrienol Gamma tocotrienol Delta tocotrienol Total tocols

9–352 ND–36 89–2307 154–932 ND–69 ND–103 ND 600–3370

84–138 ND–30 356–424 262–392 ND–45 ND–85 ND 740–945

139–168 ND–30 271–324 266–303 ND–45 ND–85 ND 676–778

234–660 ND–17 ND–12 ND ND ND–12 ND 240–670

100–386 ND–140 189–753 ND–22 ND ND ND 430–2680

Phytosterols (% total) Cholesterol Brassicasterol Campesterol Stigmasterol Beta sitosterol Delta 5-avenasterol Delta 7-stigmasterol Delta 7-avenasterol Total sterols (mg/kg)

0.6–1.4 ND–0.3 15.8–24.2 14.9–19.1 51.0–60.0 1.9–3.7 1.4–5.2 1.0–4.6 1800–4100

0.2–0.5 ND–0.2 22.4–25.7 19.6–20.4 47.5–51.8 0.6–1.6 0.3–1.9 0.2–1.1 1967–2997

0.2–0.5 ND–0.2 22.2–24.1 20.0–25.2 44.1–45.4 1.8–2.0 1.3–1.9 0.3–0.5 1569–2568

ND–0.7 ND–0.4 24.7–38.6 ND–0.9 45.1–57.9 3.1–6.6 ND–1.3 ND–0.8 4800–11 300

0.5–1.3 5.0–13.0 24.7–38.6 ND–0.9 45.1–57.9 3.1–6.6 ND–1.3 ND–0.8 4800–11 300

ND = not detectable Source: Codex Alimentarius (2001).

Table 6.4

Compositions of tocopherols and sterols in crude regular and modified safflowerseed and sunflower oils. Safflowerseed

Safflowerseed (high oleic acid)

Sunflower

Sunflower (mid-oleic acid)

Sunflower (high oleic acid)

Tocopherols (mg/kg) Alpha tocopherol Beta tocopherol Gamma tocopherol Delta tocopherol Alpha tocotrienol Gamma tocotrienol Delta tocotrienol Total tocols

234–660 ND–17 ND–12 ND ND ND–12 ND 240–670

234–660 ND–13 ND–44 ND–6 ND ND–10 ND 250–700

403–935 ND–35 ND–34 ND–7 ND ND ND 440–1520

488–668 19–52 2.3–19.0 ND–1.6 ND ND ND 509–741

400–1090 10–35 3.0–30.0 ND–17.0 ND ND ND 450–1120

Phytosterols (% total) Cholesterol Brassicasterol Campesterol Stigmasterol Beta sitosterol Delta 5-avenasterol Delta 7-stigmasterol Delta 7-avenasterol Others Total sterols (mg/kg)

ND–0.7 ND–0.4 9.2–13.3 4.5–9.6 40.2–50.6 0.8–4.8 6.5–24 2.2–6.3 0.5–6.4 2100–4600

ND–0.5 ND–2.2 8.9–19.9 2.9–8.9 40.1–66.9 0.2–8.9 7.7–7.9 ND–8.3 4.4–11.9 2000–4100

ND–0.7 ND–0.2 6.5–13.0 6.0–13.0 50.0–70.0 ND–6.9 6.5–24.0 3.0–7.5 ND–5.3 2400–5000

0.1–0.2 ND–0.1 9.1–9.6 9.0–9.3 56.0–58.0 4.8–5.3 7.7–7.9 4.3–4.4 5.4–5.8 2200–4500

ND–0.5 ND–0.3 5.0–13.0 4.5–13.0 42.0–70.0 1.5–6.9 6.5–24.0 ND–9.0 3.5–9.5 1700–5200

ND = not detectable Source: Codex Alimentarius (2001).

122 Modifying lipids for use in food Table 6.5 Chemical and physical characteristics of crude regular and modified sunflowerseed oils. Sunflowerseed oil

Sunflowerseed oil (mid-oleic acid)

Sunflowerseed oil (high oleic acid)

Relative density (x°C/water at 20°C)

0.918–0.915 x = 20°C

0.914–0.916 x = 20°C

0.909–0.915 x = 25°C

Refractive index

1.461–1.468

1.461–1.4671

1.467–1.471

Saponification value (mg KOH/g oil)

188–194

190–191

182–194

Iodine value

118–141

94–122

78–90

Unsaponifiable matter (g/kg)

≤15

≤15

≤15

ND = not detectable Source: http://www.codexalimentarius.net (Official Standards: CXS_210_2003e [1].pdf).

ISO or AOCS. Refractive index can be conducted according to ISO 6320:2000 (ISO, 2005) or AOCS Cc 7-25 (02) (AOCS, 1998). Saponification value procedures are available as ISO 3657:2000 (ISO, 2005) or AOCS Cd 3-25 (02) (AOCS, 1998). Unsaponifiable matter can be conducted according to ISO 3596:2000 (ISO, 2005), or ISO 18609:2000 (ISO, 2005), or AOCS Ca 6b-53(01) (AOCS, 1998). Oils intended for margarines and shortenings must have suitable fatty acid composition either present naturally or obtained by processing/treatments such as hydrogenation and interesterification. Slip point and solid fat index can provide information as to the suitability of an oil for margarines and shortenings. List et al. (1996) used solid fat index (AOCS method Cd 10-57) (AOCS, 1998) to show that some soybean oils modified by plant breeding to have increased amounts of stearic acid could be used for margarines. The melting point of oils for margarines and shortenings is also of interest. The slip point can be measured using ISO 6321 (1991) (ISO, 2005) or AOCS method Cc 3b-92 (AOCS, 1998), which is adopted from the ISO method. Triacylglycerol (TAG) composition is an additional compositional analysis that can provide information on the potential functionality of an oil as well as its potential oxidative stability. Reversed-phase HPLC with various detection methods such as FID, refractive index, evaporative light scattering, or atmospheric chemical ionization (coupled with mass spectrometry) can be used to determine TAG composition (Neff et al., 1994, 2001).

6.3.2 Quality characteristics According to the Codex Alimentarius Committee’s Named Vegetable Oil Standard (Codex Alimentarius, 2005), there are quality characteristics that each oil must meet. For example, each oil should be characteristic of the

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designated product and be free of foreign and rancid odor and taste. Maximum levels of matter volatile at 105 °C [ISO 662:1998 (ISO, 2005)] should be 0.2 % m/m, insoluble purities [ISO 663:1998 (ISO, 2005)] 0.05 % m/m, and 0.005% m/m soap content [AOCS Ce17-95 (97) (AOCS, 1998)]. Limits for metals are 1.5 mg/kg iron (Fe) in refined oils and 5.0 mg/kg Fe in virgin oils, whereas only 0.1 mg/kg of copper (Cu) are allowed in refined oils and 0.4 mg/kg Cu in virgin oils. Methods to use for measuring Fe and Cu include ISO 8294:1994 (ISO, 2005) or AOCS Ca 18b-91 (97) (AOCS, 1998). Limits for oil deterioration include peroxide value and acid value. Codex allows up to 10 meq/kg oil for refined oils and up to 20 meq/kg of cold pressed or virgin oil. Acid value limits range from a low of 0.6 mg/KOH/g oil for refined oils to 4 mg/KOH/g cold pressed or virgin oils. There are two quality characteristics specific for low erucic acid rapeseed oil. The Crismer value is used to identify oils through their miscibility with a standard reagent [AOCS Cb 4-35 (97) (AOCS, 1998)]. The value for low erucic acid rapeseed (canola) oil should be in the range of 67–70. Also, the brassicasterol content should not be greater than 5 % of the total sterols [ISO 12228:1999 (ISO, 2005) or AOCS Ch 6-91 (97) (AOCS, 1998)].

6.3.3 Oxidation The oxidative stability of oil can be determined in the crude stage, although it is much more common to evaluate the oil after complete processing through the deodorization step. AOCS has a recommended practice (AOCS method Cg 3-91) (AOCS, 1998) for assessing oil quality and stability. The practice lists procedures that measure either directly or indirectly some of the primary and secondary oxidation products. For example, peroxide value analysis (AOCS method Cd 8-53) (AOCS, 1998) determines the hydroperoxide content and is a good analysis of primary oxidation products. To determine secondary oxidation products, the procedure recommends p-anisidine value (AOCS Method Cd 18-90) (AOCS, 1998), volatile compounds by GC (AOCS Method Cg 4-94) (AOCS, 1998), and flavor evaluation (AOCS Method Cg 2-83) (AOCS, 1998). The anisidine value method determines the amount of aldehydes (principally 2-alkenals and 2,4-dienals) in fats and oils, by reaction in an acetic acid solution of the aldehydic compounds in oil and the p-anisidine. The mixture is examined at an absorbance of 350 nm. The volatile compound analysis method measures secondary oxidation products formed during the decomposition of fatty acids. These compounds can be primarily responsible for the flavors in oils. The flavor evaluation of oils by tasting is the best method for determining oil quality and stability, although the procedure must be done using trained sensory judges. The Oil Stability Index (OSI) is another method to measure oil stability that can be conducted using AOCS Method Cd 12b-92 (AOCS, 1998) with a Rancimat instrument or an Oxidative Stability instrument. In this method for determining an induction period for oxidation, a stream of purified air is passed through a sample of oil held in

124 Modifying lipids for use in food a heated bath. The effluent air from the oil is then bubbled through a vessel with deionized water and the conductivity of the water is continually monitored. The effluent air contains volatile organic acids such as formic acid, swept from the oxidizing oil, that increase the conductivity of the water as oxidation continues. The OSI is defined as the point of maximum change of the oxidation. The OSI may be run at temperatures of 100, 100, 120, 130, and 140 °C. It can be used to analyze crude oils or fully processed oils.

6.4

Future trends

Plant geneticists have revolutionized the vegetable oil industry with modified fatty acid composition oils. This is an opportune time for these new oils as we are looking for more healthful oils that are stable without the need for saturated fatty acids and/or hydrogenation. Some oils with modified fatty acid compositions such as low linolenic soybean oil and low linolenic acid canola oil are potential substitutes for hydrogenated oil; however, these oils are not as stable as hydrogenated oil. On the other hand, some vegetable oils such as rice bran have enhanced stability, probably because of naturally occurring phytochemicals. The next stage of oil modification will probably be focused on altering the types and amounts of minor oil constituents in oilseeds. Although plant geneticists relied on oil stability research conducted in the 1950s and beyond to decide which fatty acids to target for change, they now need information on optimum phytochemical compositions to breed oilseeds with altered levels and ratios of these natural antioxidants. Not enough information is available on how these antioxidant phytochemicals affect product quality, stability, and end use performance. Basic research is needed to determine the relationship between product composition and desired flavor quality and stability attributes in soybean, sunflower, corn, and other oilseeds. An ideal substitute for hydrogenated oil would combine the positive attributes of both modified fatty acid composition and phytochemicals. Methods of evaluating oil quality and stability also must evolve and change with the need to measure minor oil constituents more accurately and reproducibly. Oil processors and food manufacturers are always interested in rapid measurements of oxidation. The oxidative stability index is the newest technique to measure stability of oil quickly; however, the high temperature that the procedure is conducted at may not be relevant to ambient temperature at which most oils are stored. Frankel (1993) has suggested that the variation in results at 110 °C with the rapid analysis and ambient temperature storage may be because of differences in the oxidation mechanisms at the two temperatures. New procedures that are rapid but do not have this problem must be developed. The ability to distinguish between the seeds modified by genetic engineering and those modified by traditional plant breeding may be of concern to lipid chemists. Presently, a molecular geneticist can distinguish

Methods of analysis to determine the quality of oils

125

between seeds using molecular analysis of the sequences of DNA bases to detect the presence or absence of the gene in the DNA of the plant cell’s nucleus. Distinguishing between oils derived from a transgenic plant versus a traditonally developed plant was not thought to be possible because oil does not usually have any DNA in it. However, if some plant cells are present in the oil, the DNA in the nucleus can be analyzed to find out what plant produced that oil – either a transgenic plant or a traditionally developed plant.

6.5

Sources of further information and advice

Original research papers in scientific journals and review papers in books are a primary source of information about methods used to measure oil quality. Official methods and recommended practices are available from AOCS, ASTM, IUPAC, AOAC, and ISO. In addition, the Codex Alimentarius Committee on Fats and Oils has developed compositional tables of crude vegetable oils. Their Standard for Named Vegetable Oils is available through the Codex Alimentarius web site as listed in the reference section. In addition, extensive information about lipid structure, composition, and methods of analyses is available in The Lipid Handbook (Gunstone et al., 1994). The third edition of W.M. Christie’s Lipid Analysis: Isolation, Separation, Identification and Structural Analysis of Lipids (2003) is a good resource for analyzing classes of lipids. In Trans Fatty Acids in Human Nutrition by Sebedio and Christie (1998), Ratnayake discusses several methods for measuring trans fatty acids. For extensive information on measuring lipid oxidation, the second edition of Lipid Oxidation (Frankel, 2005) is recommended.

6.6

References

(2001), Chromatographic analysis of plant sterols in foods and vegetable oils, J Chromatogr A, 935, 173–201. ABIDI S L (2003), Tocol-derived minor constituents in selected plant seed oils, J Am Oil Chem Soc, 80, 327–333. ABIDI S L, LIST G R and RENNICK K R (1999), Effect of genetic modification on the distribution of minor constituents in canola oil, J Am Oil Chem Soc, 76, 463–467. AOCS (1998), Official Methods and Recommended Practices, Champaign, IL, AOCS Press, 5th edn. CHRISTIE, W M (2003), Lipid Analysis: Isolation, Separation, Identification and Structural Analysis of Lipids, Bridgewater, Oily Press. CODEX ALIMENTARIUS (2001), Codex Standard 210 Named Vegetable Oils, amended 2005, available at: www.codexalimentarius.net. FRANKEL E N (1993), In search of better methods to evaluate natural antioxidants and oxidative stability in food lipids, Trends Food Sci Tech, 4, 220–225. ABIDI S L

126 Modifying lipids for use in food (2005), Lipid Oxidation 2nd edn, Bridgewater, Oily Press. and APPLEWHITE T H (1971), High-oleic safflower oil stability and chemical modification, J Am Oil Chem Soc, 44, 266–271. GUNSTONE F D, HARWOOD J L and PADLEY F B (1994), The Lipid Handbook, London, Chapman and Hall. HOSSEINIAN F S, ROWLAND G G, BHIRUD P R, DYCK J H and TYLER R T (2004), Chemical composition and physicochemical and hydrogenation characteristics of high-palmitic acid Solin (low-linolenic acid flaxseed) oil, J Am Oil Chem Soc, 81, 185–188. ISO (1994), ISO Standard 8294, Animal and vegetable fats and oils – Determination of copper, iron and nickel contents – Graphite furnace atomic absorption method, Geneva, ISO. ISO (1997), ISO Standard 9936, Animal and vegetable fats and oils – Determination of tocopherols and tocotrienols contents – Method using high-performance liquid chromatography, Geneva, ISO. ISO (1998), ISO Standard 662, Animal and vegetable fats and oils – Determination of moisture and volatile matter content, Geneva, ISO. ISO (1999), ISO Standard 12228, Animal and vegetable fats and oils – Determination of individual and total sterols contents – Gas chromatographic method, Geneva, ISO. ISO (2000a), ISO Standard 6320, Animal and vegetable fats and oils – Determination of refractive index, Geneva, ISO. ISO (2000b), ISO Standard 3596, Animal and vegetable fats and oils – Determination of unsaponifiable matter – Method using diethyl ether extraction, Geneva, ISO. ISO (2000c), ISO Standard 18609, Animal and vegetable fats and oils – Determination of unsaponifiable matter – Method using hexane extraction, Geneva, ISO. ISO (2000d), ISO Standard 663, Animal and vegetable fats and oils – Determination of insoluble impurities content, Geneva, ISO. ISO (2002a), ISO Standard 6321, Animal and vegetable fats and oils – Determination of melting point in open capillary tubes (slip point), Geneva, ISO. ISO (2002b), ISO Standard 3657, Animal and vegetable fats and oils – Determination of saponification value, Geneva, ISO. ISO (2002c), ISO Standard 15304, Animal and vegetable fats and oils – Determination of the content of trans fatty acid isomers of vegetable fats and oils – Gas chromatographic method, Geneva, ISO. LIST G R, MOUNTS T L, ORTHOEFER F and NEFF W E (1996), Potential margarine oils from genetically modified soybeans, J Am Oil Chem Soc, 73, 729–732. LIST G R, ORTHOEFER F, TAYLOR N, NELSEN T and ABIDI S L (1999), Characterization of phospholipids from glyphosate-tolerant soybeans, J Am Oil Chem Soc, 76, 57–60. MOUNTS T L, ABIDI S L and RENNICK K A (1996), Effect of genetic modification on the content and composition of bioactive constituents in soybean oil, J Am Oil Chem Soc, 73, 581–586. NEFF W E, MOUNTS T L, RINSCH W, KONISHI H and EL-AGAIMY M A (1994), Oxidative stability of purified canola oil triacylglycerols with altered fatty acid compositions as affected by triacylglycerol composition and structure, J Am Oil Chem Soc, 71, 1101–1109. NEFF W E, BYRDWELL W C and LIST G R (2001), A new method to analyze triacylglycerol composition of vegetable oils, Cereal Foods World, 46, 6–10. PETUKHOV I, MALCOLMSON L J, PRZYBYLSKI R and ARMSTRONG L (1999), Frying performance of genetically modified canola oils, J Am Oil Chem Soc, 76, 627–632. POKORNY J (2000), Resistance of high-oleic acid peanut oil against autoxidation under storage and deep frying conditions, Czech J Food Sci, 18, 125–126. PURDY R H (1985), Oxidative stability of high oleic sunflower and safflower oils, J Am Oil Chem Soc, 62, 523–525. SEBEDIO J L and CHRISTIE W W (1998), Trans Fatty Acids in Human Nutrition, Dundee, Oily Press. FRANKEL E N

FULLER G, DIAMOND M J

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and PERKINS E G (2000), Frying performance of low-linolenic acid soybean oil, J Am Oil Chem Soc, 77, 223–229. WARNER K and KNOWLTON S (1997), Frying quality and oxidative stability of high oleic corn oils, J Am Oil Chem Soc, 74, 1317–1322. WARNER K, VICK B, KLEINGARTNER L, ISAAK R and DOROFF K (2003), Compositions of sunflower, NuSun (mid-oleic sunflower) and high oleic sunflower oils, available at: http:// www.sunflowernsa.com/research_statistics/research_workshop/documents/107.PDF, 1–7. TOMPKINS C

7 Selected topics in the chemistry and biochemistry of lipids F. Gunstone, Scottish Crop Research Institute, UK

7.1

Introduction

This book is not a primer devoted to the chemistry and biochemistry of fatty acids. However, the concepts described in several chapters require some basic information, and selected topics are presented in this chapter to facilitate understanding elsewhere.

7.2

Partial hydrogenation

Following the invention of a replacement spread for butter by Mège Mouriés in 1869, there was an increased demand for semi-solid fat to replace the beef tallow olein used in the original recipe. This requirement was met by the development and use of partially hydrogenated vegetable oils following the discovery of catalytic hydrogenation of fatty oils by Normann in 1902. Vegetable oils subjected to partial hydrogenation are mixtures of triacylglycerols (Chapter 2). Most of these are liquid at room temperature. During hydrogenation the proportion of solid triacylglycerols increases and eventually a plastic solid is produced. This is a mixture of solids and liquids that appears to be solid but deforms under pressure as when spreading with a knife. During partial hydrogenation there is a fall in the level of polyunsaturated acids and a rise in the level of monounsaturated and saturated acids. The lower levels of linoleic and linolenic acid result in a product with higher oxidative stability (Section 7.3). However, these benefits are gained at a price. The partially hydrogenated oil has lower nutritional value compared with the original vegetable oil since it contains less essential fatty acids (linoleic and α-linolenic acid) and has unsaturated acids with trans configuration.

Selected topics in the chemistry and biochemistry of lipids

129

These changes are a consequence of molecular alterations that occur during partial hydrogenation viz. saturation of double bonds (reduction) and double bonds changing position in the carbon chain and configuration (isomerization). The control and balance of saturation and isomerization during partial hydrogenation are important to get a good quality product and to get it repeatedly. Dijkstra (2002) has proposed a modification of the Horiuti–Polanyi mechanism to explain the changes that occur during partial hydrogenation of fatty acids and their esters. In the following sequence the horizontal line shows the conversion of diene to monoene and of monoene to saturated acid/ ester via the half-hydrogenated states DH and MH. The steps shown vertically are the reverse processes whereby DH goes back to D and MH goes back to M. It is during these stages that trans and positional isomers are formed. There are six stages altogether, and it is important to understand their relative rates. In the conversion of D to M the first step is rate-determining and the second step is fast. Accordingly, the concentration of DH is never high, and the conversion of DH back to D is slow and is only important in the unusual situation that hydrogen is present in very low concentration. In the conversion of M to S the final stage is slow and rate-determining, thus making it more likely that there will be considerable recycling of M and MH leading to formation of stereochemical and positional isomers. D → DH → M → MH → S ↓ ↓ D M On a commercial scale the catalyst most commonly employed is nickel. This is usually deposited on an inert support and is supplied encased in a hardened fat. Reaction is generally conducted at 180–200 °C and 3 bar pressure in vessels containing up to 30 tonnes of oil. In a batchwise operation 8–10 batches can be hydrogenated in a 24 hour period. Forty years ago catalyst was used at a level of 0.2 %, but now this is generally 0.025–0.05 %. This reduction has been achieved through improved catalyst and through increasing awareness of the need to use highly refined oils and pure hydrogen (Berben, 2002). During partial hydrogenation, competition between reduction (saturation) and isomerization is controlled by the relation between the supply and demand of hydrogen at the catalyst surface. A good supply of hydrogen leads to reduction while isomerization is more significant when hydrogen supply is inadequate. Hydrogen supply and demand depend on reaction temperature and pressure, agitation, catalyst quantity and quality, and the level of unsaturation of the oil being hydrogenated. Agitation is linked to transfer of hydrogen through gas, liquid (dissolved in oil) and solid phases (adsorbed on the catalyst). Attempts are being made to modify hydrogenation procedures so that less

130 Modifying lipids for use in food trans acids are formed, such as the development of catalysts based on platinum or palladium, but these are not yet being used on an industrial scale. A major purpose of partial hydrogenation is to harden fats, and this is presently achieved through a small rise in the level of saturated acids and a larger rise in trans acid content. It is difficult to see how, if trans acids are to decline, the necessary hardening can be achieved without raising levels of saturated acids which is also considered to be undesirable (Kellens, 2000).

7.3

Autoxidation, photo-oxidation and antioxidants

The unsaturated components in oils and fats and in materials such as rubber and paint films deteriorate as a consequence of their reaction with oxygen. In food lipids these changes are noticeable, even at modest levels, because they lead to rancidity which is apparent to the olfactory senses and is a matter of concern for the food industry, the retailer and the consumer. Most food systems contain some built-in protection against oxidation, but once this barrier is overcome oxidation proceeds steadily. The period of time during which oxidation is slow is called the induction period, and ideally this should be as long as possible. It is important to understand what causes oxidation, what are its consequences and how it can be inhibited. In any of the modification procedures discussed in this book, it is important to consider what effect the changes might have on potential oxidation resulting either from alterations in the nature of the fatty acids or in the (generally unassessed) levels of pro- and antioxidants. Two processes of oxidative deterioration occur in unsaturated lipids. Autoxidation involves reaction with normal triplet oxygen and photo-oxidation occurs through reaction with the more reactive singlet oxygen formed in the presence of light and a sensitizer such as chlorophyll. Although similar in many respects, these two reactions are not identical. In both, olefinic esters react with oxygen to give unsaturated hydroperoxides (RCH=CHCH(OOH)R′). These are not themselves the direct cause of off-flavour, but they are unstable molecules which break down and yield, among other products, short-chain compounds, particularly aldehydes, responsible for undesirable odours and flavours. The situation is complicated in that at certain concentrations and in certain mixtures a flavour may be considered to be desirable, but under other circumstances it is seen to be undesirable. Oxidative deterioration is associated with olefinic unsaturation, but there are significant differences between monoene and polyene acyl chains because the latter, but not the former, contain one or more reactive doubly allylic sites. Oxidation is promoted by heat, light, metals and several initiators (all classed as pro-oxidants) and inhibited by a range of compounds acting in different ways and described as antioxidants. Natural lipids, whether crude or refined, may contain both prooxidants and antioxidants.

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131

–CH2CH=CHCH2–

monoene with two monoallylic CH2 groups –CH2CH=CHCH2CH=CHCH2– diene with one diallylic CH2 group and two monoallylic CH2 groups For a more detailed account of oxidation see Gunstone (2004a).

7.3.1 Autoxidation Autoxidation is a radical chain reaction occurring through initiation, chainpropagation and termination processes. The first product is usually a mixture of allylic hydroperoxides which may be oxidized further to more complex products. The hydroperoxides break down to compounds of lower molecular weight. When a C18 chain in a glycerol ester is fragmented in this way, one portion is volatile but the other fragment remains as part of the former triacylglycerol and may affect its subsequent behaviour. The initiation step is not clearly understood, but a hydrogen atom is removed from an acyl chain (represented here as RH) to produce the alkyl radical R• by breaking a C–H bond. The ease of removing the hydrogen atom is given by the sequence: doubly allylic as in linoleate > allylic as in oleate > non-allylic as in saturated esters. The propagation sequence involves two steps; a quick reaction of R• with triplet oxygen to give a peroxy radical ROO• followed by a ratedetermining hydrogen abstraction to produce the hydroperoxide and another radical. Normally this hydrogen will come from another olefin molecule, and the resulting alkyl radical can enter another cycle of reactions. This process will continue until interrupted by a termination sequence which generally involves union of two radicals to produce a dimer that does not react further (Fig. 7.1). As will be discussed later, this reaction cycle can be hindered by reducing the rate of initiation and/or by shortening the propagation sequence, usually by addition of molecules which act as antioxidants reacting with R• to give species that cannot continue the cycle. (Porter et al., 1995). One important initiation step involves the reaction of pre-formed hydroperoxide with metals, and in oxidatively stable systems it is desirable to avoid these compounds – which may be difficult – or to keep them apart Initiation

RH

R•

Propagation

R• + O2

resonance-stabilized alkyl radical RO2•

RO2• + RH Termination

R• + R•

Fig. 7.1

RO2H + R• rate-determining step

RO2• + RO2• RO2• + R•

fast reaction to a peroxy radical

stable products (dimers) stable products (dimers)

stable products (dimers)

Olefin autoxidation. RH represent an olefinic compound in which H is attached to an allylic carbon atom.

132 Modifying lipids for use in food by control of the surface charge on emulsion droplets which may be possible in some emulsions (Min Hu et al., 2004). Interesting and surprising results have shown that the order of reactivity of olefinic esters differs between bulk systems (for which most studies have been made) and emulsions such as commonly exist in foods (Miyashita, 2002). 7.3.2 Photo-oxidation Photo-oxidation involves reaction between olefin and singlet oxygen. The latter is a highly reactive but short-lived species (half life 50–700 µs), produced when the more common triplet oxygen is activated through reaction with light and a sensitizer such as chlorophyll or a range of coloured substances. The mechanism of photo-oxidation differs from the radical autoxidation reaction. Singlet oxygen is a powerful electrophile and reacts with an electronrich double bond at either olefinic carbon atom. The reaction is accompanied by double bond migration and stereo conversion to the (mainly) trans configuration (Fig. 7.2). The product is an unsaturated hydroperoxide, but its detailed structure may differ from that obtained by autoxidation. There is also a marked difference between the reaction rates for the two processes (Table 7.1). Photo-oxidation is dependent on the presence of olefinic groups and less dependent on the presence of the 1,4-dienes so important for autoxidation. Once formed, photo-oxidized hydroperoxides may initiate the autoxidation process. Most antioxidants that inhibit autoxidation have little effect on photo-oxidation which is inhibited mainly by singlet oxygen quenchers such as carotenes.

7.3.3 Hydroperoxide structure In photo-oxidation, methyl oleate and other oleate esters give C18 products with oxygen attached to C-9 or C-10. The double bond shifts along the chain and is mainly trans. In autoxidation, because of resonance in the radical R•, the oxygen may appear on carbon atoms 8, 9, 10 or 11. The distinction cis RCH==CHCH2R′ + 1O2

Fig. 7.2

trans RCH(OOH)CH==CHR′

Reaction of olefin with singlet oxygen to give allylic hydroperoxides with double bonds in different position and of changed configuration.

Table 7.1 Relative rates of autoxidation and photo-oxidation of oleate, linoleate and linolenate. Reaction

Oxygen

18:1

18:2

18:3

Autoxidation Photo-oxidation Ratio

triplet singlet

1 3 × 104 30 000

27 4 × 104 1500

77 7 × 104 900

Selected topics in the chemistry and biochemistry of lipids

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between these two sets of hydroperoxides is important because each will break down to a different set of volatile aldehydes with different flavours and different threshold values. With linoleate, photo-oxidation gives four major hydroperoxides associated with each olefinic carbon atom (C-9, 10, 12 and 13). Double bond shift and steroisomerism accompany the oxidation. Some of the resulting dienes are conjugated and some are not. With autoxidation there are two major products resulting from the radical formed at the doubly allylic center (C-11). Because of resonance the initially formed hydroperoxides are mainly 9-hydroperoxy 10t12c-18:2 and 13-hydroperoxy 9c,11t-18:2, although sometimes these change to yet other isomers. (13)

(9)

–CH=CHCH 2 CH=CH– → linoleate ( ∆ 9,12)

–CH(OOH)CH=CHCH=CH– and –CH=CHCH=CHCH(OOH)– hydroperoxides with conjugated diene systems

7.3.4 Cholesterol oxidation The possible link between cholesterol oxidation products and coronary heart disease and other disease states makes it appropriate to discuss the source and formation of such compounds. Cholesterol (Fig. 7.3) contains a cyclic double bond (∆5) and some tertiary carbon atoms in its side chain (C-20 and C-25); all sites where oxidation may occur. Cholesterol oxides are produced as part of the normal metabolism of cholesterol, but at higher levels they affect human health by contributing to the development of atherosclerosis. When cholesterol oxides replace cholesterol in the cell membrane they alter its fluidity, permeability, stability and other properties. Oxidised animal-based foods represent a primary source of oxidized cholesterol. Such products are not present in fresh foods but are formed during handling prior to consumption, mainly through autoxidation. Between 0.5 and 1.0 % of dietary cholesterol may be oxidized, and the levels increase with unsaturation of associated phospholipids. The primary oxidation products include 7-α-hydroxy-, 7-β-hydroxy- and 7-keto-cholesterol, cholesterol αand β-epoxides, 3,5,6-trihydroxycholesterol and 20- and 25-hydroxycholesterol (Cuppett, 2003).

H

HO

Fig. 7.3

Cholesterol.

134 Modifying lipids for use in food 7.3.5 Decomposition of hydroperoxides to short-chain products Hydroperoxides contain a weak O–O bond which is easily broken to give an alkoxy radical. This unstable intermediate furnishes aldehydes by a series of processes shown in Fig. 7.4. As typical examples, the two major linoleate hydroperoxides give hexanal (from the 13-hydroperoxide) and decadienal (from the 9-hydroperoxide) as volatile aldehydes.

7.3.6 Antioxidants Because of the ease with which fats undergo oxidation and become rancid, it is important to develop systems which will slow this process down when it occurs in food, feed, cosmetics and pharmaceuticals. In addition to avoiding access to air and, where possible, keeping products in the dark and at a reduced temperature, this requires the addition of chemicals known as antioxidants which inhibit oxidative deterioration. Rancidity is caused mainly by short-chain aldehydes and, since these are formed as a consequence of a series of processes, it is possible to tackle the problem at each stage. There are thus antioxidants which act at different stages of the total process. These are often classified as primary and secondary antioxidants. Mixtures of antioxidants are often more effective than single substances, either because they operate at different stages of aldehyde production or because one component is able to convert reacted antioxidant back to its reactive form. For food use, antioxidants themselves must be nutritionally acceptable, and that applies also to the quinones and dimers which are formed from them during the antioxidative process. Among synthetic antioxidants, only permitted material may be used and then only at levels below a prescribed maximum. Regrettably not all countries have made the same decision in these matters. For example TBHQ (tert-butylhydroquinone) is not permitted in EU countries although it may be used elsewhere. Some antioxidants act twice because the first-formed oxidised molecule can act a second time in this capacity. Sometimes a compound is added to an antioxidant which can regenerate its effectiveness. Antioxidant molecules act sacrificially, and when all the antioxidant has been expended then unprotected unsaturated lipid will oxidise rapidly. This stage represents the end of the induction period. Autoxidation and photo-oxidation occur by different mechanisms and are inhibited in different ways. To inhibit photo-oxidation, materials should be kept in the dark with added singlet oxygen quenchers such as carotene. This RCH==CHCH(OOH)R′ (allylic hydroperoxide) RCH==CHCH(O•)R′

RCH==CH• + R′CHO and RCH==CHCHO + R′•

RCH==CH2 or RCH2CHO

R′H or R′OH

Fig. 7.4 Homolytic breakdown of allylic hydroperoxides to short-chain compounds. The alkyl radicals are converted to hydrocarbons or alcohols by reaction with H• or HO•. The aldehyde on the last line is a 2-enol written in ‘keto’ form.

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last substance can also inhibit autoxidation, but compounds used to inhibit autoxidation do not generally influence photo-oxidation. It is important to restrict photo-oxidation because the reaction is so rapid and because it leads to hydroperoxides which can themselves initiate autoxidation. Oxidative rancidity through autoxidation requires initiation, propagation, the possibility of isomerization of the hydroperoxides first formed and cleavage of hydroperoxides to short-chain aldehydes. It should be possible to inhibit each of these stages with appropriate compounds. Primary antioxidants (also known as radical acceptors or radical scavengers) inhibit both initiation and propagation (the latter by promoting termination) by trapping active radicals R• or RO2•. They do this by providing a ready source of hydrogen and furnishing a product which does not react further. Many phenols act in this way. Molecules with extended conjugated unsaturation show the same effect through formation of an addition product. RO2• + AH → ROOH + A• → products RO2• + B → ROOB• → products AH represents a phenolic molecule and B a molecule like carotene Secondary antioxidants are mainly metal chelators such as ethylene diamine tetra-acetic acid (EDTA), citric acid, phosphoric acid, and certain amino acids. These remove the metal ions (mainly iron and copper) that promote initiation by interaction of existing hydroperoxide with metal ion (Min Hu et al., 2004). In emulsions it may be possible to keep these species apart by control of pH or by use of appropriate surfactants. Vitamin C (ascorbic acid) acts as an oxygen scavenger, removing traces of residual oxygen in a packed and sealed product. It is a water-soluble molecule but can be supplied in a lipid-soluble form as ascorbyl palmitate. Phospholipids show ill-defined antioxidant activity, possibly acting as a chelating agent and/or as an emulsifier. Antioxidants may be natural or synthetic compounds with the latter often cheaper and more effective. There is, however, a growing demand for natural antioxidants, even although the quantities available are insufficient and even these are frequently subject to minor chemical manipulation. Most vegetable oils contain tocopherols which serve as powerful antioxidants. In addition, some seed oils contain other antioxidants such as sesamol and its derivatives in sesame oil and oryzanols (sterol esters of ferulic acid) in rice bran oil. Many herbs and spices also show antioxidant properties, and this may be part of their long-known preservative properties. Some natural antioxidants may be lost during refining, but it is often possible to trap them in a side stream which then becomes a source of these valuable commodities. Deodorizer distillate from soybean oil refining and palm fatty acid distillate both serve as commercial sources of tocol mixtures with both antioxidant and vitamin E properties. Synthetic antioxidants are mainly phenolic compounds such as BHA (butylated hydroxyanisole, E320), BHT (butylated hydroxytoluene, E321),

136 Modifying lipids for use in food PG (propyl gallate, E310), and TBHQ (tertiary butylhydroquionone, no E number) with the structures shown in Fig. 7.5. The best-known and most widely used natural antioxidants are the tocols (tocopherols and tocotrienols) which are widely distributed in plant products but not in those which are animal-derived. There are eight natural tocols – four tocopherols with a phytyl side chain and differing from one another in the number and disposition of methyl groups, and four tocotrienols which are similar to the corresponding tocopherols but have a tri-unsaturated side chain (Fig. 7.6). Natural tocol mixtures are usually used at levels up to 500 ppm along with ascorbyl palmitate (200–500 ppm) which has a sparing activity on vitamin E. Above ~ 1000 ppm α-tocopherol acts as a pro-oxidant. Since most vegetable oils already contain tocols at levels of 200–800 ppm, further addition shows little effect. In contrast, the oxidative stability of lard, with little or no natural antioxidant, is markedly enhanced with tocopherol. Lard has an induction period of only 2.5 hours when heated to 100 °C with blown air, but this is extended to 18 hours with added tocopherol (0.01 %). In comparing antioxidant activity several factors have to be considered. 1. Effects vary with different oils and fats because of their varying fatty acid composition and the differing levels of antioxidants already present. 2. Results obtained at different temperatures may not be directly comparable because mechanisms of hydroperoxide formation and breakdown change with temperature, as does the volatility of the antioxidants. 3. Results vary with the method of assessment: some measure primary OH

OH But

But

OMe

Me

BHA (E320)

OH But

BHT (E321)

HO

OH But

OH

COOC3H7 PG (E310)

OH TBHQ (no E number)

Fig. 7.5 The structures and E-numbers of synthetic antioxidants. TBHQ has no E number because it is not a permitted antioxidant in EU-15. R HO

5 7

8

R

O R

Fig. 7.6 Tocopherols and tocotrienols. Tocopherols have a saturated C16 side chain, tocotrienols have three double bonds at the positions indicated by the arrows, R = H or CH3. α = 5,7,8-trimethyltocol, β = 5,8-dimethyltocol, γ = 7,8-dimethyltocol, δ = 8-methyltocol.

Selected topics in the chemistry and biochemistry of lipids

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products (hydroperoxides) and others measure secondary products (carbonyl compounds and/or volatile compounds). 4. Mixtures of antioxidants are influenced by synergistic effects, and it is difficult to disentangle these. 5. Solubility factors have to be considered especially when there is a distribution between aqueous and lipid phases.

7.4

Reactions of esters and other acyl compounds

In its simplest form esterification is the reaction of a carboxylic acid (RCOOH) with an alcohol (R′OH) to produce an ester and water. The ester (RCO–OR′) contains an acyl group (RCO) derived from the carboxylic acid and an alkoxy group (OR′) derived from the alcohol. RCOOH + R′OH ===` RCOOR′ + H2O

`

The reaction is reversible and the four reactants establish an equilibrium. There are standard methods of shifting the equilibrium in the desired direction. Those most commonly employed involve using an excess of one of the reactants and/or removal of one of the products. For example, the reaction can be carried out with an excess of alcohol, under conditions where a volatile product is removed, or in a two-phase system such that one product is transferred from the phase in which reaction occurs to the second phase where reaction does not take place, thereby disturbing the equilibrium in the reaction phase. Establishment of the equilibrium state is generally attained only slowly so a catalyst is required to achieve a useful speed of reaction. Depending on the acylation process being conducted the catalyst may be acidic such as H2SO4 or BF3, basic such as NaOH or NaOMe or an enzyme (generally a lipase). The union of an acyl group (RCO) with an alkoxy group (OR′) to produce the ester RCOOR′ can be achieved in a variety of ways, depending on the source of these two groups and on the fact that the reaction is reversible. The more important of these processes are listed in Table 7.2. For example, when an ester is mixed with an alcohol in the presence of an appropriate catalyst there is an exchange of alkoxy groups between that in the alcohol and that in Table 7.2

Acid Ester Ester Ester

Esterification and related reactions. Reagents

Reaction types

Alcohol Alcohol Acid Ester

Esterification Alcoholysis Acidolysis Interesterification

All the reactions using an ester as a reagent are also called transesterifications.

138 Modifying lipids for use in food the ester. Two examples of alcoholysis widely used with lipids have their own specific names (methanolysis and glycerolysis). Reaction with methanol (methanolysis) to convert glycerol esters to methyl esters is carried out on a mg scale as a preliminary to gas chromatographic analysis and on a tonne scale for the production of methyl esters used as solvents, as biofuels or as intermediates in the production of alcohols. When natural oils and fats interact with free glycerol there are not enough acyl groups to react with all the hydroxyl groups present and the final product is a mixture of partial glycerol esters – mainly monoacyglycerols and diacylglycerols (glycerolysis). An exchange of acyl groups by reaction of triacylglycerols with acids is termed acidolysis. This may be used, for example, to introduce lauric acid into oils and fats which do not contain this acid. When two esters react together then new equilibria are established between all the acyl groups and all the alkoxy groups. When an oil/fat is used then the reaction starts with a complex mixture of glycerol esters. This process is usually catalysed by alkali, but the nature of true catalyst is still a matter of debate (Dijkstra, 2004; Liu, 2004). There is a growing interest in using enzymes (lipases) to catalyze these reactions. Such processes occur under milder process conditions so there is a saving of energy. There is also less by-product and therefore less loss in recovering the major product. However, of greater importance is the fact that many lipases show specificities which make it possible to carry out these reactions with an element of control which would be difficult to achieve with normal chemical catalysts. Since the reaction being catalyzed involves an acyl function and an alkoxy function, specificity may appear as a consequence of the interaction of the lipase with either or both of these and as a consequence of the conditions under which the reaction is carried out. Acyl components vary according to the structure of the fatty acid [in terms of chain length and the position and configuration of the unsaturated centre(s)] and according to whether the acylating agent is a free acid or an ester (of which there are many kinds). The alkoxy component can be a free alcohol or be present in an ester. For example, among hydroxy compounds free glycerol, monoacyglycerols and diacylglycerols may react differently. In any particular reaction more than one selectivity may be operating leading to further complications. Examples of interesterification by chemical catalysts or by lipases are detailed in Chapter 11 (see also Kellens, 2000).

7.5

Metabolism of linoleic and linolenic acids

The polyunsaturated fatty acids generally contain 1,4 patterns of unsaturation with two to six cis olefinic groups and are frequently described as methyleneinterrupted. They are grouped into families depending on the position of the first unsaturated centre with respect to the end methyl group. The two most

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139

important families are the omega-6 family based on linoleic acid and including arachidonic acid (20:4) and the omega-3 family based on α-linolenic acid and including eicosapentaenoic acid (EPA, 20:5) and docosahexaenoic acid (DHA, 22:6). The C20 and C22 members of each family are metabolically linked with the first (C18) member by a series of enzymatically catalyzed and controlled desaturation and elongation processes. Animals, including humans, cannot make linoleic or α-linolenic acid which must themselves have a plant origin. An animal in good health can then usually metabolize the C18 acids to long-chain polyunsaturated fatty acids (PUFA) (Fig. 7.7) Animals are not generally able to interconvert omega-6 and omega-3 acids and therefore it is important to consume dietary omega-6 and omega-3 acids in an appropriate ratio. But what should that be? The omega-6/omega-3 ratio in the human diet has changed through history. It is widely accepted that when humans were hunter-gatherers the ratio was between 1:1 and 3:1. The polyunsaturated acids are important in lipid membranes, particularly in the brain where the ratio is around 2:1. It is further accepted that the ratio in the diet has changed markedly since the 1950s. Prior to that time fats were largely animal-derived, but since then linoleic-rich vegetable oils have become more dominant (Chapter 2) and one authority has written of a thousand-fold increase in supplies of linoleic acid (Crawford, 2004). Virtually the only dietary omega-6 acid is linoleic acid present in unsaturated Omega-6 family 18:2 (9, 12) linoleic ∆6-desaturase 18:3 (6, 9, 12) γ -linolenic elongation 20:3 (8, 11, 14) ∆5-desaturase 20:4 (5, 8, 11, 14) arachidonic

Omega-3 family 18:3 (9, 12, 15) α-linolenic ∆6-desaturase 18:4 (6, 9, 12, 15) stearidonic elongation 20:4 (8, 11, 14, 17) ∆5-desaturase 20:5 (5, 8, 11, 14, 17) eicosapentaenoic elongation 22:5 (7, 10, 13, 16, 19) elongation 24:5 (9, 12, 15, 18, 21) ∆6-desaturse 24:6 (6, 9, 12, 15, 18, 21) β-oxidation 22:6 (4, 7, 10, 13, 16, 19) docosahexaenoic

Fig. 7.7 The omega-6 and omega-3 families of polyunsaturated fatty acids and their metabolic relationships. The most significant acids in these sequences are linoleic and arachidonic in the omega-6 family and α-linolenic, eicosapentaenoic and docosahexaenoic acid in the omega-3 family. The two C20 acids are precursors of an important group of eicosanoids including the prostaglandins and leukotrienes. The numbers in parenthesis indicate the positions of the double bonds, all of which have the cis configuration.

140 Modifying lipids for use in food oils such as soybean, sunflower, cottonseed and corn oils. Supplies of omega3 acids are confined almost entirely to α-linolenic acid derived from soybean and rapeseed (canola) oils. Unfortunately much of this is destroyed by hydrogenation before it is consumed. Gunstone (2005) has calculated that out of 136 million tonnes of oils and fats produced by the agricultural supply industry in 2004/05, 109 million tonnes was used for human food, and this provided 31.9 million tonnes of linoleic acid and 3.5 million tonnes of αlinolenic acid – a ratio of 9.1:1. After allowing for hydrogenation, these figures change to 23.8 and 0.8 million tonnes giving a ratio of ~ 30:1. This predominance of linoleic acid in the diet leads to concern over the effect that this has on the efficiency of metabolism of α-linolenic acid and over the consequences of this for health and disease. It has long been considered that the omega-6/omega-3 ratio should be between 5 and 10 to 1. There is a growing preference for the lower end of this range, but some have argued for a figure of 2:1 or even lower (Okuyama, 2000). The figures cited above suggest that it is difficult to attain these low levels, although it should be remembered that the ratio can be lowered by increasing the intake of αlinolenic acid and/or by lowering the intake of linoleic acid. The second target may be the easier to achieve. The omega-6 and omega-3 acids and their long-chain metabolites have important physiological functions. Omega-3 acids are major components of biological membranes. DHA, for example, is present in high concentrations in the retina, the brain and in sperm. They can also alter gene expression – down-regulating some enzymes and up-regulating others – and EPA has an important role in the regulation of eicosanoids produced from arachidonic acid through competition for the metabolising enzymes. Eicosanoids from EPA and AA show different properties. However, α-linolenic acid is not converted efficiently to EPA and, more particularly, to DHA. Feeding α-linolenic acid leads to some increase in tissue α-linolenic acid, to small increases in EPA (20:5) and DPA (22:5) and to a still smaller increase or to a decrease in DHA. The best way to increase levels of DHA in tissue is to consume DHA itself from fish and for most people intakes of long-chain PUFA are considerably below recommended levels. Other routes to the C20 polyunsaturated acids have been identified as for example in the production of arachidonic acid from linoleic acid by elongation followed first by ∆-8 desaturation and then by ∆-5 desaturation (Drexler et al., 2003; Qi et al., 2004).

7.6

Sources of further information and advice

These topics are reported in most books devoted to lipid chemistry and technology and lipid biochemistry, such as those of Hamm and Hamilton (2000), Akoh and Min (2002) and Gunstone (2004a).

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7.7

141

References

and MIN D B (2002), Food Lipids: Chemistry, Nutrition, and Biotechnology, New York, Marcel Dekker, Inc. BERBEN P (2002), Hydrogenation of fats and oils: trends in catalyst development, Lipid Technol Newsletter, 8, 133–137. CRAWFORD M A (2004), Docosahexaenoic acid and the evolution of the brain – a message for the future, Lipid Technol, 16, 53–57. CUPPETT S L (2003), Cholesterol oxides: sources and health implications, Lipid Technol Newsletter, 9, 9–13. DIJKSTRA A J (2002), Hydrogenation and fractionation, in Rajah K H, Fats in Food Technology, Sheffield, Sheffield Academic Press, 123–158. rd DIJKSTRA A J (2004), The interesterification mechanism revisited, Lecture at the 3 Euro Fed Lipid Congress, Edinburgh, September. DREXLER H, SPIEKERMANN P, DOMERGUE F, ZANK T, SPERLING P, ABBADI A and HEINZ E (2003), Metabolic engineering of fatty acids for breeding of new oilseed crops: strategies, problems and first results, J Plant Physiol, 160, 779–802. GUNSTONE F D (2004a), The Chemistry of Oils and Fats, Blackwell Publishing, Oxford. GUNSTONE F D (2004b), Agricultural supplies of oils and fats do not meet nutritional demands, 9th Tan Sri Dato’ B Bek-Nielsen Foundation Lectureship, delivered in Kuala Lumpur, Malaysia, September, 2004. See also Inform 2005, 16, 736–737. HAMM W and HAMILTON R J (2000), Edible Oil Processing, Sheffield, Sheffield Academic Press. KELLENS M (2000), Oil modification processes, in Hamm W and Hamilton R J, Edible Oil Processing, Sheffield, Sheffield Academic Press, 129–173. LIU L (2004), How is chemical interesterification initiated: nucleophilic substitution or α-proton abstraction? J Am Oil Chem Soc, 81, 331–337. MIN HU D, MCCLEMENTS J and DEKKER E A (2004), Emulsion droplets engineered to improve the oxidative stability of n-3 acids in functional food emulsions, Lipid Technol, 16, 79–82. MIYASHITA K (2002), Polyunsaturated lipids in aqueous systems do not follow our preconceptions of oxidative stability, Lipid Technol Newsl, 8, 35–41. OKUYAMA H (2000), Prevention of excessive linoleic acid syndrome, Lipid Technol Newsl, 6, 128–132. PORTER N A, CALDWELL S E and MILLS K A (1995), Mechanisms of free radical oxidation of unsaturated lipids, Lipids, 30, 277–290. QI B, FRASER T, MUGFORD S, DOBSON G, SAYANOVA O, BUTLER J, NAPIER J A, STOBART A K and LAZARUS C M (2004), Production of very long chain polyunsaturated omega-3 and omega-6 fatty acids in plants, Nat Biotechnol, 22, 739–745. AKOH C C

8 Structure and properties of fat crystal networks S. Martini, T. Awad and A. G. Marangoni, University of Guelph, Canada

8.1

History and introduction

Fats provide fundamental structural and textural attributes to a wide range of consumer products including lipstick, chocolate, and everyday products such as butter and margarine (Milosav, 1985; Halmos, 1997). Within these fatbased products, certain ‘textural’ properties are required to meet desirable sensory attributes, in order to gain consumer acceptance (de Bruije and Arjen, 1999). This has led to an increase in research efforts on the physical properties of fats, particularly their rheology. Over the past 50 years, most of the research on fat texture has concentrated on the examination of triacylglycerol (TAG) composition, polymorphism, solid fat content (SFC), and their individual effects on macroscopic properties. Unfortunately, none of these components in isolation can fully describe the macroscopic properties of a fat. Therefore, the ultimate goal is to examine the parameters and any potential interactions that may exist between them to determine how they affect the physical properties of fats. A model that can be used to describe the formation and interaction of the elements that affect the final properties of a three-dimensional fat crystal network is shown in Fig. 8.1. This hierarchical model suggests that when fats are crystallized, lipid composition, directly under the influence of the processing and storage conditions, will affect the SFC polymorphism and microstructure of the fat crystal network. The model also suggests that polymorphism, SFC and microstructure may have interactive effects. All of these properties affect the final three-dimensional fat crystal network, and in turn influence the macroscopic properties and sensory perception. The objective of this chapter is to review the properties of fat crystal

Structure and properties of fat crystal networks Processing conditions

Molecular/lipid composition

143

Storage conditions (time and temperature)

Crystallization

Solid fat content

Polymorphism

Morphology

Microscopic properties

Fat crystal network

Macroscopic properties

Fig. 8.1 Model used to describe the formation and interaction of the elements that affect the physical properties of a three-dimensional fat crystal network.

networks and their relationship with microstructure, rheology and processing conditions in order to optimize the quality of the final product.

8.2

Crystallization and melting

Crystallization is an important process during the manufacture of edible fatrich products such as margarine, butter, chocolate, whipped cream and ice cream. In food emulsions where the oil droplets are partially or fully crystallized, the degree of crystallinity of the oil in the droplets affects the rate of creaming (Dickinson and McClements, 1996). In margarine, the properties of melting, spreadability, texture, and consistency are dominated by the fat crystals, which control the quality, shelf life and stability of the product. The consistency of fat spreads is derived from interactions between fat crystals, which determine network structure and rheology (Heertje, 1993; deMan and deMan, 2001). By controlling crystallization kinetics, it is possible to modify fat crystal network structure and control macroscopic rheological properties. Crystallization occurs through three stages; achievement of supercooling, nucleation and crystal growth (Boistelle, 1988). Below the equilibrium melting point of a liquid, crystallization is favored because the free energy of the solid phase is lower than that of the melt phase. The degree of supercooling (∆T) is defined as the difference between the temperature of the material (T ) and its melting point (Tm): ∆T = Tm – T. Nucleation involves the transition

144 Modifying lipids for use in food from a metastable to a stable phase in which crystal formation is controlled by temperature and cooling rate. By increasing the supercooling of the melt, molecules start to form clusters that eventually transform into an ordered crystallite. Other molecules continue to incorporate into the new nucleus until it reaches a critical size (n*) and then the nucleus can develop into a three-dimensional and/or two-dimensional ordered aggregate (Aquilano and Squaldino, 2001). The rate of formation of critical nuclei formed in the melt per unit time is called the nucleation rate (J). There are two types of nucleation, namely homogeneous and heterogeneous. The total change in the free energy associated with the formation of the new solid phase is different between these two nucleation types. While homogeneous nucleation occurs in the bulk of the mother phase (the melt), heterogeneous nucleation occurs onto substrates such as catalyzing impurities, substrates and oil/water (O/W) interface. Interfacial heterogeneous nucleation can occur in O/W emulsions where droplets are surrounded by high melting surfactant molecules and other additives. The O/W emulsion interface may itself act as a catalytic impurity (McClements et al., 1994; Awad et al., 2001; Awad and Sato, 2002). In both cases, nucleation can be initiated at lower degrees of supercooling than homogeneous nucleation. Hence, the total free energy change for heterogeneous nucleation (∆Ghet) is less than that of homogeneous nucleation (∆Ghom). After the formation of a stable nucleus, molecules move through the supercooled melt towards the stable solid–liquid interfaces. The growth rate of the crystal is mainly based on the melt viscosity, which retards the mobility of molecules (diffusion). A molecule must have a specific size, shape and orientation to be incorporated into crystal surfaces. The growth rate is also temperature dependent. As the degree of supercooling increases, the growth rate also increases to a maximum. Below a certain temperature, the rate decreases due to the increasing melt viscosity, which slows down molecular diffusion. This leads to a decrease in crystal growth (Timms, 1994). Incorporation of specific emulsifiers (P-170, S-170 and DAS-750) strongly enhances the rate of nucleation but retards the rate of crystal growth in emulsions with palm mid-fraction (PMF) and palmkernel oil (PKO), mainly due to a poisoning effect (Awad and Sato, 2001 and 2002a, respectively). PMF is a product of the double fractionation of palm oil that is used in its bulk state as shortening or in the emulsion state as margarine and vegetable oil-based creams. PMF rich in POP (where P is palmitic acid and O is oleic acid) is suitable for blending with SOS (where S is stearic acid) fractions to prepare cocoa butter equivalents (Sontag, 1979). PKO is a co-product of the palm oil industry and is regarded as high quality oil for food use and a valuable component for margarine formulation, giving rapid meltdown in the mouth. Figure 8.2 shows the effect of the addition of sucrose oligoester emulsifier additive on the nucleation (i) and crystal growth (ii) of PMF (Awad and Sato, 2001). Additionally, several authors studied the effect of processing conditions

Structure and properties of fat crystal networks

145

1530 1520 1510

V (m/s)

1500 1490 1480 1470 1460 1450 0

200

400 600 Time (min)

800

1000

800

1000

(a) 1500 1490

V (m/s)

1480 1470 1460 1450 1440 1430 0

200

400 600 Time (min) (b)

Fig. 8.2 Ultrasonic velocity measurements showing the effect of a sucrose oligoester (S-170) on (a) nucleation of PMF/water emulsion at 10 °C and (b) crystal growth of PMF melt supercooled and left to crystallize at 25 °C after adding seed crystals of PMF. ■: no additives, 䉭: with S-170 (0.5 wt %), 䊊: with S-170 (1.0 wt %).

on the crystallization behavior of different bulk fat systems with and without the addition of emulsifiers. For example, Herrera and Marquez Rocha (1996) found that the addition of P-170 delayed the nucleation of hydrogenated sunflower oil and also delayed the polymorphic transformation from β′ to β. Martini et al. (2002c) together with Puppo et al. (2002) and Cerdeira et al. (2003) found that the addition of sucrose esters (0.5 % of S-170 and P-170) to high melting milk fat fraction/sunflower blends (HMF/SFO) mixtures influenced the crystals’ microstructure and rheology. The addition of emulsifiers also delayed the crystallization kinetics (nucleation and growth); which was

146 Modifying lipids for use in food evidenced by longer induction times of crystallization and lower maximum solid fat contents (SFCmax), especially at higher crystallization temperatures. The effects of emulsifiers on the crystallization kinetics of different fat systems in bulk and emulsions are due to differences in the chemical structure of the emulsifiers and fats. Two mechanisms have been reported in the literature to interpret the effects of emulsifiers on fat crystallization in bulk systems. First, the emulsifiers can act as heteronuclei, accelerating nucleation through the catalytic actions of such impurities. During crystal growth, the emulsifiers are adsorbed at steps or kinks on the surface of the growing fat crystal and thereby inhibit crystal growth and modify crystal morphology. Second, fats and emulsifiers are able to co-crystallize because of their somewhat similar chemical structures. However, the structural dissimilarities between triacylglycerols and emulsifiers can delay nucleation and inhibit growth. In general, an emulsifier with a high molecular weight and the above-mentioned chemical characteristics has the potential to be a good inhibitor of crystallization (Garti, 1988). The crystallization behavior of oils depends on their chemical composition as well as on processing conditions (Herrera and Hartel, 2000b). When the oil becomes dispersed as in oil-in-water emulsions (e.g. whipped-cream) or forms the continuous phase as in water-in-oil emulsions (e.g. margarine), the crystallization process becomes much more complicated due to the effect of other additional factors such as emulsifiers, droplet size and distribution and droplet–droplet interactions (Awad and Sato, 2002b), as shown in Table 8.1. Emulsifiers strongly affect the stability of the emulsion interface as well (Goff and Jordan, 1989; Dalgleish et al., 1995). Temperature, cooling rate and shear are the major factors that influence the crystallization kinetics of fats during processing. After production, temperature fluctuation during transport and consumption becomes a significant factor that must be taken into account. Product mistreatment and/or improper storage will thus cause many problems, which leads to a decline in product quality such as blooming in chocolate, graininess in margarine and hardness in ice cream. A very common phenomenon that occurs during post-processing storage is Ostwald-ripening, which consists in the coarsening of crystals. Table 8.1 Factors affecting crystallization of oil in bulk and emulsion states. Bulk

Emulsion

Temperature Cooling rate Impurity Polymorphism

Temperature Cooling rate Impurity Polymorphism Droplet size and distribution Droplet–droplet interaction Emulsifiers

Structure and properties of fat crystal networks

147

This process may occur spontaneously or due to temperature cycling, e.g. during transportation or during use, as a result of the lower surface free energy of bigger crystals. The process is greatly accelerated by temperature cycling, because the re-dissolution of crystallized fat in oil at higher temperatures followed by subsequent crystallization at lower temperatures helps the transport of fat molecules from the smaller crystals to the bigger ones. Therefore, a specific temperature–time profile is usually applied to control crystallization during the manufacture of margarine. When crystallization conditions are tightly controlled, it is relatively easy to achieve good and well-defined margarine texture. The effect of cooling rate is also important both in margarine and in bulk systems. Slowly crystallized fats will continue to crystallize after packaging, resulting in a firm product, while a fast crystallization rate results in a soft and overworked product (Heertje, 1993; Herrera and Hartel, 2000a). The control of crystallization is a key factor in the processing of W/O emulsions. Control of heat, mass and momentum transfer rates is the main tool to manipulate crystal size, mainly by balancing crystal nucleation and growth processes. This balance can be influenced through changes in supersaturation, typically by the degree of supercooling, or changes in flow conditions by shear. Other means to stimulate nucleation, though less common in spread manufacture, are ultrasonic radiation or seeding. Application of shear and ultrasound is also used during the processing of fat spreads, which offers good emulsification and controls the size of the crystal/aggregates. Thus, rapid cooling followed by intense stirring (which promotes secondary nucleation) leads to small crystals, e.g., margarine; slow cooling with gentle stirring leads to large crystals, e.g. fractionation of palm oil (Timms, 1994). Recently, our group has found that shear accelerates the rate of polymorphic transformation and can cause the orientation of fat crystallites in milk fat, palm oil and cocoa butter (Mazzanti et al., 2003; 2004a,b). Melting and crystallization are reversible. The melting behavior of fats in foods is important for both consumer perception and product stability. For example, the melting of margarine is an endothermic process where the release of heat from the mouth to the product melts the fat crystals, imparting a pleasant cooling sensation. The steeper the melting profile of a fat (e.g., coconut and palm kernel fats), the greater the cooling sensation. Complete melting of the fat phase will result in a rich mouth feel product, but fat components that melt at or above mouth temperature will impart a waxy mouth feel.

8.2.1 Chemical composition The complexity of the crystallization process of natural fats and oils may be due to the multi-component nature of the material and the molecular interactions between the different fat molecules. Fats are mainly composed of TAGs together with diacylglycerols (DAGs), monoacylglycerols (MAGs), free fatty acids (FFA) and minor lipid components such as phospholipids and glycolipids.

148 Modifying lipids for use in food A TAG consists of a glycerol backbone and three fatty acids. Each fatty acid has its own chain length, degree of unsaturation and position of the double bond. Figure 8.3 shows the basic structure and the structural characteristics of TAGs. Fats with high content of long-chain fatty acids will crystallize at high temperatures; meanwhile, the presence of unsaturated fatty acids in TAG molecules will result in lower crystallization temperatures. The molecular diversity of the TAGs, including the symmetry (mono-acid or mixed-acid) and the number, position and the configuration of the double bond (i.e., cis or trans) will also result in different crystallization and polymorphic behaviors (Sato, 2001b). Several authors have studied the effect of chemical composition on the crystallization behavior of the fats and on their microstructure. Rousseau et al. (1996a,b,c) studied the melting behavior of interesterified butterfat mixed with canola oil together with the microstructure, polymorphism and rheology of these blends. Wright et al. (2000) studied the effect of minor components on the crystallization of milk fat. The effect of DAG on milk fat crystallization, microstructure and rheology was also studied by Wright and Marangoni (2002, 2003) and by Herrera et al. (1999). In addition, Martini et al. (2001, 2002a,b) have studied the effect of processing condition on the nucleation, growth and microstructure of mixtures of high melting milk fat fractions and sunflower oil blends. Independently of the type of fat used to perform the experiments, all the studies mentioned above found that chemical composition has a very significant influence on the crystallization behavior, the microstructure and the rheology of the fat systems.

8.2.2 Polymorphism TAGs exhibit polymorphism, which is defined as the ability of a compound to form different crystalline structures with various states of molecular conformation and molecular packing. The crystallization rate, crystal size, crystal morphology and degree of crystallinity are directly influenced by TAG molecule

H

H

H

H

C

C

C

O R

1

O R

Chain length structure

H

O

2

R3 double

R = Fatty Acids Monoacid: R1 = Mixed acid: R1 ≠ R1 = R2 ≠

Fig. 8.3

R2 R2 R2 R1

= R3 (PPP, SSS) ≠ R3 (POS) ≠ R3 (PPO, OOP) = R3 (POP, SOS)

triple

Degree of un-saturation Monounsaturated (SOS, SSO) Polyunsaturated (OSO, PLO)

Basic structure and the structural characteristics of triacylglycerols (TAGs).

Structure and properties of fat crystal networks

149

polymorphism (Sato, 2001a). Figure 8.1 shows the interrelationship between polymorphism and the physical properties of fats. As shown in this figure, the polymorphism phenomenon relates to molecular properties such as TAG composition, presence of DAGs, MAGs or other minor components and molecular interactions. Crystallization conditions influence the polymorphism and polymorphic transitions, which in turn affect morphology. All of these parameters will influence fat crystal network structure, its texture and rheological properties. Three basic polymorphs of mono-acid TAGs have been identified by powder X-ray diffraction, on the basis of their subcell structure (Larsson, 1966). A subcell is defined as the cross-sectional packing mode of the aliphatic chains in TAGs. Ten types of subcells have been identified in crystalline lipids (Small, 1986; Hernqyist, 1988). Among those, the three major polymorphs namely, α, β′ and β, have been identified (Larsson, 1966), as shown in Fig. 8.4. The α form is metastable, with hexagonal packing (H). The β′ form is more stable with orthorhombic perpendicular packing (O⊥) and β is the most stable form, with triclinic parallel packing (T//). TAGs often crystallize initially in the α or the β′ forms, although β is the most stable form. This can be explained by the fact that the form has a higher free energy of activation for nucleation ( ∆G *n ) compared with the other two forms. The melting temperature increases from the less stable to the more stable form (i.e., in the order of α- β′-β), owing to the difference in their molecular packing densities (Fig. 8.5a). The polymorphic transformation is an irreversible process from the least to the most stable form, depending on temperature and time (monotropic phase transformation). At constant temperatures, the α and β′ forms transform, as a function of time, to the β form through solid–solid or solid–liquid–solid transformation mechanisms (Fig. 8.5b) (Herrera and Marquez Rocha, 1996). The polymorphism of fats described here influences the textural characteristics of fats and emulsions. The polymorphic behavior of the TAG components in margarine is important for improving the functional properties of a spread. The β′ form is the most functional form in many food emulsions because it usually comprises of a large number of small crystals that are needle or platelet-like in shape. For margarine it is desirable to form crystals Hexagonal (H)

α

Orthorhombic (O⊥)

Triclinic (T// )

β′

Fig. 8.4 TAG major polymorphic forms.

β

150 Modifying lipids for use in food (a)

∆Gc#

Melt α

∆Gc

β′ β (b)

Melt

α

β′

β

Fig. 8.5 Free activation energy of nucleation change (∆Gn ) for the three major polymorphs. (∆Gn# ) is the free energy barrier that must be exceeded before stable nuclei can be formed (a). Polymorphic transformation pathways (b).

in the β′ polymorph during processing; however, the transformation towards the most stable and undesired β form is usually favored during storage and distribution. The presence of large grains (graininess) in margarine is associated with the crystallization of the most stable β polymorph. In addition, the surface sheen of margarine becomes dull because the β crystals are less able to incorporate liquid oil, and when the crystals become very large the surface appears mottled and the texture becomes brittle (deMan and deMan, 2001). It has been demonstrated that the presence of certain TAGs retards the formation of large crystals in margarine by modifying the structure of crystals and retarding their polymorphic transformation (Heertje, 1993; Herrera and Marquez Rocha, 1996). In mixed TAG systems, the re-crystallization behavior is more complex, because different TAG molecules have to be incorporated in the crystal lattice. This incorporation occurs less readily at higher packing densities of the crystal, which implies that the transition from a less stable to a more stable polymorphic form has to involve a change in composition of the crystalline phase. It has been empirically established that the rate of a polymorphic transition is a function of temperature and, in particular, of the molecular composition of a fat mixture. However, the details of this process at a molecular level are still unclear, and may involve either melt-mediation

Structure and properties of fat crystal networks

151

or a solid-state transformation, or a simple competitive crystallization process.

8.2.3 Fat crystal network A fat crystal network is the product of the aggregation process of molecules into particles, and of particles into larger clusters, until a space-filling threedimensional network is formed. Work by Heertje and coworkers originally demonstrated that fat crystal networks of fat spreads can display such structural hierarchy (Heertje et al., 1987; Juriaanse and Heertje, 1988; Heertje, 1993). When triacylglycerols are cooled from the melt to a temperature below their melting point, i.e. when they are supercooled, they undergo a liquid–solid transformation to form primary crystals with characteristic polymorphism. These primary crystals aggregate, or grow into each other, to form clusters, which further interact, resulting in the formation of a continuous threedimensional network. The macroscopic rheological properties of networks formed by lipids are of great importance in food products that contain significant amounts of fats. Such products include margarine, butter, chocolate, peanut butter, many spreads such as cream cheese, and ice cream. Many sensory characteristics such as spreadability, hardness, appearance and mouth feel are dependent on the mechanical strength of the underlying fat crystal network (Marangoni and Rousseau, 1996, 1998; Narine and Marangoni, 1999a; Marangoni, 2002). Therefore, the firmness and solid-like properties of manufactured plastic fats, such as margarine, are established during the formation of a fat crystal network.

8.2.4 Mesoscale structure The mesoscale structure of fat crystal networks includes elements in the length range between 0.5 and 500 µm. Note that the term ‘mesoscale structure’ will be used interchangeably with the term ‘microstructure’. At the lower range of this mesoscale structure, one may encounter crystallites; whilst at the upper ranges, one decidedly is observing aggregates of microstructural elements (clusters of crystallites). This level of structure has an enormous influence on the macroscopic rheological properties of the network, noted as early as 1987 by deMan and Beers. Other researchers have also noted the importance of the mesoscale level on the rheological properties of the network, and the fact that the microstructure is properly changed with processing conditions of crystallization (Heertje et al., 1987, 1988; Herrera and Hartel, 2000c; Martini et al., 2002b), as well as with interesterification (Marangoni and Rousseau, 1996). Figure 8.6 shows the different levels of structure present in a cocoa butter crystal network, from primary crystallites (Fig. 8.6a) to microstructural elements – or particles (Fig. 8.6b) to microstructures – or flocs or clusters (Fig. 8.6c). These images were acquired using an atomic force microscope (AFM) operated in tapping mode.

152 Modifying lipids for use in food

(a)

(b)

(c)

Fig. 8.6 Tapping-mode atomic force micrograph of cocoa butter at different magnifications. The micrographs on the left-hand side correspond to height view and those on the right-hand side to the phase view.

In order to truly understand, and eventually predict, the macroscopic properties of soft materials, it is necessary to characterize and define the different levels of structure present in the material and their respective relationship to a macroscopic property. Knowledge of the relationships between molecular composition and phase behavior, solid-state structure, growth mode, static structure and macroscopic properties will eventually allow for the rational design of specific macroscopic properties.

8.3

Mechanical properties and structure

Fat crystal growth is dictated by external processing conditions and TAG composition. The TAGs crystallize from the melt into certain polymorphic

Structure and properties of fat crystal networks

153

states that then aggregate into larger microstructures or crystal aggregates. Aggregation continues until a three-dimensional network is formed, with a structure that can be described using fractal geometry principles (Narine and Marangoni, 1999a,b, 2002; Marangoni, 2000, 2002). The formation of a fat crystal network is of key importance in the manufacture of plastic fats because it provides firmness or solid-like properties (viscoelasticity). This network can be visualized as being built from aggregates rather than straight chains of fat particles, and can be thought of as a colloidal aggregate, analogous to a protein gel. Each of the fat particles in turn comprises several aggregated fat crystals. The quantitative description of such a complex and ‘random’ system is difficult. Recently, fractal geometry has proven to be extremely helpful in the characterization of these fractal objects (Marangoni and Rousseau, 1996; Narine and Marangoni, 1999c).

8.3.1 Fat crystal network as a particle gel Figure 8.7 shows the formation of a crystal network in a cocoa butter (CB) sample highly diluted with canola oil (CO) when crystallized statically at 5 °C. We can observe how the flocs, size and the interfloc links increase with time. Originally suggested by van den Tempel (1979), Vreeker and coworkers (1992) proposed that the structure of a fat crystal network resembles that of a flocculated colloid, i.e., a colloidal gel. Subsequently, our group developed this concept further (Marangoni and Rousseau, 1996, Narine and Marangoni, 1999a). The solid-like macroscopic properties of materials structured as flocculated colloids, in particular elasticity, are highly dependent on their structure at the nanometer range (nanoscale), as well as micrometer range (mesoscale). Early developments of a theory to explain the elastic properties of colloidal gels from their structure were carried out by Brown and Ball (1985). They proposed a power law dependence of the shear elastic modulus (G) of a colloidal network on the volume fraction (Φ) of network mass G ~ Φµ

[8.1]

where the exponent of the volume fraction term (µ) is related to the mechanism of particle aggregation. Based on the seminal work of Kantor and Webman (1984), Brown and Ball (1985), Shih et al. (1990) outlined the development of a scaling theory to explain the elastic properties of colloidal gels by again considering the structure of the gel network as a collection of close-packed fractal flocs of colloidal particles (Fig. 8.8). However, these authors also defined two separate rheological regimes depending on the relative strength of the inter-floc links vis-à-vis those within the flocs. Their formulation of the strong-link regime (applicable at low volume fractions, Φ < 0.1), where the flocs yield under an applied stress, was identical to that of Brown and Ball (1985). Their formulation of the weak-link regime (applicable at high volume fractions, Φ > 0.1), where the inter-floc links yield under an applied stress, differed from that

154 Modifying lipids for use in food (b)

(a)

(d)

(c)

200 µm

Fig. 8.7 Polarized light images of crystalline structures of a mixture of cocoa butter and canola oil at 5 °C after (a) two hours; (b) six hours; (c) 12 hours and (d) 20 hours. Cluster

Particle

ξ

Link

a

Fig. 8.8 Idealized structure of a gel network, where ξ is the diameter of the microstructure and ‘a’ is the diameter of each particle that forms the microstructure.

Structure and properties of fat crystal networks

155

suggested by Brown and Ball (1985). The forms of the equations for the strong-link and weak-link regimes are, respectively: d+x

GSLR ~ Φ d – D

[8.2]

and 1

GWLR ~ Φ d – D

[8.3]

where d is the Euclidean dimension, D the fractal dimension and x the chemical length exponent, backbone fractal dimension, or the tortuosity of the effective chain of stress transduction within a cluster of particles yielding under an externally applied stress. The parameter x is difficult to estimate and is usually assumed to be in the range [1–1.3]. The fractal dimension is a parameter that describes the spatial distribution of mass within the network. It is possible to judge whether a system is in the weak-link or strong-link rheological regimes by determining the dependence of the strain at the limit of linearity (γo) on the solids’ volume fraction (Φ). According to the model of Shih et al. (1990), γo increases as a function of Φ for the weak-link regime (γo ~ Φ1/(d–D)), while γo decreases as function of Φ for the strong-link regime (γo ~ Φ–(1+x)/(d–D)). In all these treatments, the macroscopic elastic constant of the network is merely the product of the elastic constant of a basic mechanical unit (the flocs, the links between flocs, or a combination of both) and the number of these units present in the direction of an externally applied force (Shih et al., 1990). In the weak link regime, the fractal dimension merely defines the size of the cluster. Thus, for example, a higher fractal dimension will result in a larger cluster size. A larger cluster size translates to less cluster–cluster interactions per unit volume, resulting in a decrease in the elastic modulus of the material. At higher volume fractions, the average cluster size decreases, thus increasing the number of cluster–cluster interactions, which leads to an increase in the value of the elastic constant.

8.4

Fat crystal networks and microstructure

The physical properties of fats are influenced by all levels of structure, but particularly by microstructure, since this is the level of structure closest to the macroscopic world. Microstructure includes the spatial distribution of mass, particle size, inter-particle distance and particle shape. Two methods for characterizing microstructure in fat systems have been developed by our group. These methods include small deformation rheology and microscopy techniques, employing a fractal approach (Marangoni and Rousseau, 1996; Narine and Marangoni, 1999c).

156 Modifying lipids for use in food 8.4.1 Determination of the fractal dimension of fat crystal networks by microscopy The determination of the fractal dimension of a fat crystal network can be carried out using light scattering techniques (Vreeker et al., 1992), but only at very low volume fractions of solid fat. Microscopy is another technique that can be used to determine the fractal dimension of fat crystal networks. Polarized light microscopy (PLM) is by far the most popular and practical microscopy technique used in studies of fat microstructure. Particle counting method Our group has developed a PLM-based technique for the determination of the fractal dimension of fat crystal networks. Lipid films of about 20 µm thickness are imaged using a standard light microscope equipped with crossed polarizers using 4x, 10x or 40x objective lenses. The choice of magnification will depend on the scale at which a non-homogeneous distribution of mass is observed. This region should theoretically correspond to the intra-cluster region of the fat crystal networks. As stated above, mass will be distributed in a homogeneous fashion at scales above the size of a particle cluster. Having said this, the growth of fat crystals crystallized as a thin film on a microscope slide is somewhat restricted to two-dimensional growth and most probably does not correspond to the bulk situation. This does not decrease its usefulness in detecting relative changes. The particle-counting procedure is based on counting the number of crystal reflections or ‘particles’ within boxes of increasing size placed over a properly thresholded and inverted image (Fig. 8.9). Thresholding converts a grayscale image into a binary image, a necessary step in any image analysis, while inversion (white crystals become black) is necessary since features to be analyzed are considered to be black. The relationship between the number of particles (Np) and the fractal dimension (Dp) is: N p ~ ( L / a ) Dp

[8.4]

where L corresponds to the diameter of the region of interest (ROI) and a to the diameter of the particle. Therefore, the slope of the log–log plot of the number of particles versus the box size corresponds to the fractal dimension of the network. In this procedure, it is necessary to count the number of particles within a ROI, including as well as excluding particles that touch the edge of the boxes. The estimate of the fractal dimension of a network is obtained by averaging the fractal dimensions obtained from counts including and excluding particles touching the edge of the boxes. In our experience, values will range from approximately 1.5 to 2.5 (Marangoni, 2002). Box counting method The box dimension is defined as the exponent Db in the relationship:

N (d ) ≈

1 d Db

[8.5]

Structure and properties of fat crystal networks

157

6

Log (N)

5 4 3 2 3.5

D = 2.01 (0.06) r2 = 0.0994 4.0

4.5 5.0 Log (length)

5.5

6.0

Fig. 8.9 The particle-counting method for the determination of mass fractal dimension from polarized light micrographs of fat crystal networks. The grayscale image of milk fat crystallized at 15 °C is inverted and thresholded. The slope of a log– log plot of the number of ‘particles’ counted in the different regions corresponds to the mass fractal dimension in d = 2 space.

where N(d) is the number of boxes of linear size d necessary to cover a data set of points distributed in a two-dimensional plane. The basis of this method is that, for objects that are Euclidean, equation [8.5] defines their dimension. In practice, to measure Db count the number of boxes of linear size d necessary to cover the set (i.e., the microstructures) for a range of values of d; and plot the logarithm of N(d) on the vertical axis versus the logarithm of d on the horizontal axis. If the set is indeed fractal, this plot will follow a straight line with a negative slope that equals –Db. Depending on the fat structure, the relationship is sometimes not entirely linear and the progression line deviates near the small boxes range, as shown in Fig. 8.10. The ‘Benoit’™ software package (Trusoft Int’l, USA, www.trusoftinternational.com) is one of the best tools on the market available for performing box counting determinations. Features to be analyzed have to be white, rather than the usual black. We also recommend users to threshold their images prior to the analysis using Adobe® Photoshop® (Adobe, USA) or other similar software to make sure that the thresholded image actually represents what is depicted in the grayscale image.

158 Modifying lipids for use in food

100 000

Number of occupied box

Db = 1.853 10 000

1000

100

10

1 1

10 100 Box side length

1000

Fig. 8.10 The box dimension method for the determination of mass fractal dimension from polarized light micrographs of fat crystal networks. The slope of a log–log plot of the number of occupied boxes ‘particles’ to the box side length corresponds to the mass fractal dimension in d = 2 space.

8.4.2 Determination of the fractal dimension of fat crystal networks by rheology (Dr) Most rheological investigations of fats involved empirical tests designed for quality control and attempt to simulate consumer sensory perception (de Bruijne and Arjen, 1999; Wright et al., 2001). These experiments involve large deformation techniques that measure hardness-related parameters, which are then directly compared to attributes evaluated by a sensory panel (de Bruijne and Arjen, 1999; Rousseau and Marangoni, 1998). Large deformation

Structure and properties of fat crystal networks

159

techniques have included penetrometry using cone, pin, cylinder and several other geometries (Haighton, 1959; Prentice, 1972; Davey and Jones, 1985; deMan and Beers, 1987; deMan et al., 1989; Fearon and Johnston, 1989; Rousseau et al., 1996c; de Bruijne and Arjen, 1999), compression (Kawanari et al., 1981), extrusion (Kawanari et al., 1981; Rohm and Ulberth, 1989), spreadability (Huebner and Thomsen, 1957; Pompei at al., 1988), texture profile analysis (Halmos, 1997) and shear tests (Kawanari et al., 1981). Each of these methods offers analytical benefits and rapid evaluation and utilizes relatively inexpensive equipment (Lawless and Heymann, 1999). The majority of these tests are based on the breakdown of structure and single parameter measurement of the rheological properties of ‘hardness’, ‘yield stress’, ‘relative spreadability’, etc. (Szczesniak, 1966; Corey, 1970; Mortensen and Danmark, 1982; Lawless and Heymann, 1999). The elastic properties of fats are not solely influenced by the amount and spatial distribution of network mass, but also by particle properties, including size, shape and particle–particle interactions. A general formulation for the relationship between the shear elastic modulus (G′) of networks of particle clusters in the weak-link regime was recently derived by our group (Marangoni and Rogers, 2003). For spherical clusters interacting exclusively via Van der Waals forces, the expression is: 1

G = λΦ d – D λ is a constant, where A λ~ 2 πad o2 ε*

[8.6]

[8.7]

or

λ~

2δ a ε*

[8.8]

In these expressions, A corresponds to Hamacker’s constant, a to the diameter of the primary particles and do, to the inter-cluster separation distance; ε* is the extensional strain at the limit of linearity and δ is the crystal-melt interfacial tension. In order to estimate the fractal dimension of a colloidal gel-like material, it is necessary to determine the elastic modulus (G′) at different volume fractions of network material. This can be done by diluting the material with an appropriate solvent in a temperature range where solids are not significantly solubilized by the solvent and the structure is not altered by the dilution process. The slope of a log–log plot of G′ versus Φ can then been used to determine the fractal dimension of a network assuming a particular rheological regime (strong-link, weak-link). The pre-exponential term (λ), which is influenced primarily by particle properties and their interactions, can be determined from the y-intercept [8.6]. In all of our work, we have considered that fat crystal networks, at relatively high solid fat contents, are in the

160 Modifying lipids for use in food weak-link rheological regime. This is based on the fact that crystal clusters are relatively ‘hard’ structures, not likely to yield at small deformations. In addition, in our system we observed a positive dependence between the solids’ volume fraction and the strain at the limit of linearity. It is more likely that the links between these clusters yield instead. As well, the value for the fractal dimension obtained assuming a weak-link regime agrees with our physical model of such networks. Figure 8.11 shows one such plot for cocoa butter (CB). Molten CB at 80 °C was mixed with canola oil at different ratios, ranging from 17 % to 50 % CB, and allowed to crystallize at 5 °C for 24 hours prior to measurement (Awad et al., 2004). The effects of cooling rate on Dr are shown in Table 8.2. In general, an increase in the cooling rate results in a decrease in the Dr value (Herrera and Hartel, 2000a). Previous work in our laboratory has also established that higher fractal dimensions occur in networks that are more ordered; thus it can be expected that lower cooling rates would display this trend. The higher the Dr value, the more ‘ordered’ the crystal packing is said to be. For this reason the value for Dr is expected to vary in the order of Dr–0.1°C/min > Dr–1°C/min > Dr–5°C/min. When observed by time-lapse microscopy, the samples cooled slowly (0.1 °C/min) yielded less numerous (Fig. 8.12) and more sporadically formed nuclei relative to the more rapidly cooled samples. This allows for a more ordered crystal growth. The 1 °C/min sample initially 8

12 %

Log G ′

7

SFC range

6

y = 4.1338x + 8.9167 r2 = 0.9622

40 %

5 Slope (s) = 1/3 – D D = 3 – (1/s) = 3 – (1/4.1338) = 2.75 4 –0.3

–0.4

–0.5

–0.6 –0.7 Log (SFC/100)

–0.8

–0.9

–1

Fig. 8.11 Log–log plot of the storage modulus (G′) vs the solids volume fraction (Φ) used to determine the fractal dimension (D) of cocoa butter network. The fat was mixed with different canola fractions to achieve 12–40 % SFC (solid fat content) values.

Structure and properties of fat crystal networks

161

Table 8.2 Rheological determination of fractal dimension Dr, λ, yield force and storage modulus G′ values for 80 % milk fat in canola cooled to 5 °C at different cooling rates after one and seven days. Cooling rate (°C/min)

Storage time (days)

SFC (%)

Dr

λ (Pa)

0.1 0.1 1 1 5 5

1 7 1 7 1 7

34.2 36.3 40.1 39.6 39.6 39.6

2.82 2.77 2.67 2.59 2.57 2.50

1.15E 3.74E 9.12E 6.50E 6.53E 5.20E

+ + + + + +

09 08 07 07 07 07

Yield force (N)

G′ (Pa)

14.7 18.2 26.7 28.7 37.4 31.9

3.72E 3.61E 4.82E 5.26E 7.21E 8.94E

+ + + + + +

06 06 06 06 06 06

Abbreviation: SFC = solid fat content.

(a)

(b)

(c)

(d)

(e)

(f)

Fig. 8.12 Polarized light micrographs of AMF crystallized to 5 °C at cooling rates of (a, b) 0.1, (c, d) 1 and (e, f) 5 °C/min. Micrographs were taken immediately (a, c, e) and after storage for one day (b, d, f).

162 Modifying lipids for use in food demonstrated sporadic nucleation until reaching 13 °C. Below 13 °C, a burst in nucleation took place and crystalline mass rapidly filled the space, resulting in a more disordered system. Finally, samples cooled at 5 °C/min immediately nucleated extensively and spontaneously at 16 °C, resulting in an even more disordered system, with lower fractal dimension Dr than at lower cooling rates. The values for λ shown in Table 8.2 were also affected by cooling rate and storage time at 5 °C. As with the Dr value, the values for λ decrease with increasing cooling rate. It may be possible that significant changes in the Hamaker’s constant occur as a result of differences in crystal composition due to different cooling rates. During storage at 5 °C the λ-values decrease moderately at each cooling rate then appear to equilibrate at seven days, with only minor differences detectable between the seven and 14 days values. Recently, the fractal dimensions of fats calculated by image analyses and rheology were compared (Awad and Marangoni, 2004). For CB, the Dr was 2.75, while the Dp was 1.88 (± 0.06) and the one calculated by box dimension was 1.64 (± 0.003) for the same CB sample. These D values belong to twodimensional Euclidean space and by adding ‘1’ to the values we can thus translate them to three-dimensional space (Russ, 1984). However, the validity of this procedure still remains to be proven.

8.5

Structuring fat crystal network using processing

The aggregation state of fat crystal networks as a collection of microstructures similar to the structuring of particles in colloidal gels suggests that the mechanical properties of fats are dependent on microstructural properties. Early work by Heertje and co-workers (1987, 1988) has shown that characterization of the microstructure can facilitate the choice of composition and processing conditions necessary for the production of functional fat spreads. The mechanical properties of fats and fat-structured materials are mainly controlled by the amount of solids (SFC) and the microstructure or mesoscale structure of their fat crystal network, including both size and shape of flocs and particles and spatial distribution of mass. The SFC and microstructure can be controlled by altering the composition (blending), varying temperature as well as varying cooling and shear rates. Figure 8.13 shows a SFC-temperature diagram that demonstrates the manufacturing process of a typical margarine. First, the mix is quenched (rapidly cooled) to a temperature where crystallization occurs in the α polymorph. As shown in the figure, crystals will be formed upon further cooling, which lead to an increase in SFC along the α SFC line. Adiabatic (re)-crystallization from α to β modification leads to an increase in temperature. Since it is usually impossible to extract all the thermal energy from the crystallizing emulsion, repeated cooling (A) and resting steps (C) can be combined. The optimal process will depend always on the fat blend used, to give a final product with the desired final microstructure, plasticity and droplet size distribution. Recent

Solid fat content (%)

Structure and properties of fat crystal networks

163

C

A C α

A

β′

Tα Temperature (°C)

Fig. 8.13 Schematic representation of a crystallizing fat during margarine processing conditions in a SFC vs temperature diagram. The solid line labeled with α refers to the conditions under which α crystallization occurs. The dashed path describes a system which is cooled below the α crystallization temperature, Tα in an A-unit. Subsequently, adiabatic re-crystallization to the β′ modification in a C-unit results in an increase in temperature, but new crystallites are formed as long as the system remains below the β′ crystallization temperature. The A–C sequence is repeated once more in this diagram.

work in our lab has shown that varying the SFC can lead to changes in the crystallization kinetics and network structure in three natural fats (anhydrous milk fat, palm oil and cocoa butter) (Awad et al., 2004). Figure 8.14 shows the different types of microstructures obtained for cocoa butter diluted with different amounts of canola oil as a function of SFC at 5 °C. 8.5.1 Effect of shear The effect of shear rate at different temperatures on the fractal dimension of milk fat is shown in Fig. 8.15. The fractal dimension obtained by rheology (Dr) decreased by decreasing the shear rate (Vanhoutte, 2002). The linear relationship at 26.5 °C is quite significant and indicates a strong correlation between the mass spatial distribution and shear rate. However, this behavior is likely to change under different temperatures and storage conditions. Temperature will influence the crystallization kinetics while storage may induce polymorphic transitions, which entail molecular rearrangements and restructuring of the fat crystals causing a decrease in the microstructural parameters as shown in Table 8.2. Therefore, the control of processing is not an easy task but a critical one.

8.5.2 Effect of cooling rate The effects of cooling rate on the spatial distribution of mass (D) and the particle properties (λ) of AMF are shown in Table 8.2 (Rye, 2003). The D and λ values were obtained by rheology from the slope of the log–log linear relationship between G′ and SFC. As shown in the table, the microstructural

164 Modifying lipids for use in food (a)

(b)

(c)

(d)

(e)

(f)

Fig. 8.14 Polarized light micrographs of cocoa butter diluted with canola oil crystallized in 20 µm films at 5 °C for 24 hours. The SFC values are (a) 16 %, (b) 22 %, (c) 39 %, (d) 49 %, (e) 57 % and (f) 67 %.

3.0 26.5 °C 21 °C

2.8

Dr

2.6

D = 2.8–29 · SR–1 2.4 2.2 2.0 0.000

D = 3.0–50 · SR–1 0.005

0.010 0.015 0.020 1/Shear rate (RPM–1)

0.025

Fig. 8.15 Effect of shear rate at different temperatures on the fractal dimension of milk fat.

Structure and properties of fat crystal networks

165

parameters (i.e., D and λ) decreased upon increases in the rate of cooling. For example, the D value for AMF samples cooled to 5 °C at cooling rates of 0.1, 1 and 5 °C/min and then stored for one day at that temperature were 2.82, 2.67 and 2.57, respectively. Figure 8.12 demonstrates the difference in microstructure after applying different cooling rates immediately and after storage for one day. Large microstructures were shown at low cooling rate (0.1 °C/min) while small homogeneous microstructures were obtained when the cooling rate was increased to 5 °C/min. Small deformation rheology data obtained at different cooling rates showed that the loss modulus (G″) and the tangent of the phase angle tan (δ) decreased while the storage modulus (G′) increased by increasing the cooling rate (Rye, 2003). In a brief description, G′ is a measurement of the elastic or solid component of the energy applied to the system, G″ is a measure of the viscous dissipation of energy of the system and tan (δ) indicates whether the system behaves like a solid, liquid or viscoelastic structure. In addition, large deformation rheology measurements indicated that the cooling rate affected the hardness and that the harder sample was more brittle than the softer one. From a rheological point of view, a higher cooling rate will result in a more solid-like behavior and thus more rigid and harder fats. It was suggested that the links between larger particles will yield more than those between smaller particles (Alberola et al., 1994).

8.6

Future trends

One of the most important applications of structured fats is margarines. Margarines have come a long way since their invention for the French army in the late 1860s. It has been possible for many years to make margarines having a neutral effect on blood cholesterol by balancing the ratio of polyunsaturated, monounsaturated and saturated fatty acids in the fat blend. Nowadays, the latest generation of healthy margarines is actively lowering blood cholesterol levels significantly with repeated use. Additionally, there are now fortified (containing vitamins and essential minerals) variants focused on delivering a balanced nutrition. A recent successful development to lower the saturated fatty acid content (the structuring component in the fat network) is the introduction of liquid margarine. We cannot call this product a particle gel, but rather a liquid emulsion. The consumer has also expressed an increasing interest to move away from processed foods to natural or organic products. Whilst margarine at first sight may appear to be the archetypal processed food, there is a very difficult challenge in the establishment of a good sustainable raw material supply chain to develop an organic margarine. This pushes the technology to its limits since the raw materials should not have fertilizers or pesticides, oil has to be free of solvents or chemical processing and no chemically derived emulsifiers are allowed. Despite these severe limitations, the first high fat level products are already on the market. A much greater

166 Modifying lipids for use in food challenge lies ahead to make the first low fat variants of such organic products. Proper structuring via modulation of fat crystal network structure will play a central role in the manufacture of such products.

8.7

References

ALBEROLA N, BAS C

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and JOHNSTON D E (1989), A comparison of three instrumental techniques to evaluate butter spreadability, J Food Quality, 12, 23–38. GARTI N (1988), Effects of surfactants on crystallization and polymorphic transformation of fats and fatty acids, in Garti N and Sato K, Crystallization and Polymorphism of Fats and Fatty Acids, New York, Marcel Dekker, Inc., 267–303. GOFF H D and JORDAN W K (1989), Action of emulsifiers in promoting fat destabilization during the manufacture of ice cream, J Dairy Sci, 72, 18–29. HAIGHTON A J (1959), The measurement on the hardness of margarine and fats with cone penetrometers, J Am Oil Chem Soc, 36, 345–348. HALMOS A L (1997), Food texture and sensory properties of diary ingredients, Food Australia, 49(4), 169–173. HEERTJE I (1993), Microstructural studies in fat research, Food Structure, 12, 77–94. HEERTJE I, LEUNIS M, VAN ZEYL W J M and BERENDS E (1987), Product microscopy of fatty products, Food Microstructure, 6, 1–8. HEERTJE I, VAN EENDENBURG J, CORNELISSEN J M and JURIAANSE A C (1988), The effect of processing on some microstructural characteristics of fat spreads, Food Microstructure, 7, 189–193. HERNQVIST L (1988), Crystal structures of fats and fatty acids, in Garti N and Sato K, Crystallization and Polymorphism in Fats and Fatty Acids, New York, Marcel Dekker, Inc., 97–137. HERRERA M L and HARTEL R W (2000a), Effect of processing conditions on physical properties of a milk fat model system: rheology, J Am Oil Chem Soc, 77, 1189–1195. HERRERA M L and HARTEL R W (2000b), Effect of processing conditions on crystallization kinetics of a milk fat model system, J Am Oil Chem Soc, 77, 1177–1187. HERRERA M L and HARTEL R W (2000c), Effect of processing conditions on physical properties of a milk fat model system: microstructure, J Am Oil Chem Soc, 77, 1197–1204. HERRERA M L and MARQUEZ ROCHA F J (1996), Effects of sucrose ester on the kinetics of polymorphic transition in hydrogenated sunflower oil, J Am Oil Chem Soc, 73, 321– 326. HERRERA M L, DE LEON GATTI M and HARTEL R W (1999), A kinetic analysis of crystallization of a milk fat model system, Food Res Int, 32, 289–298. HUEBNER V R and THOMSEN L C (1957), Spreadability and hardness of butter. I. Development of an instrument for measuring spreadability, Am Dairy Sci, 40, 834–838. JURIAANSE A C and HEERTJE I (1988), Microstructure of shortenings, margarine and butter – a review, Food Microstructure, 7, 181–188. KANTOR Y and WEBMAN I (1984), Elastic properties of random percolating systems, Phys Rev Lett, 52, 1891–1894. KAWANARI M, HAMANN D D, SWARTZEL K R and HANSEN A P (1981), Rheological and texture studies of butter, J Texture Studies, 12, 483–505. LARSSON K (1966), Classification of glyceride crystal forms, Acta Chemica Scandinavia, 20, 2555–2260. LAWLESS H T and HEYMANN H (1999), Texture evaluation, in Heymann H and Lawless H, Sensory Evaluation of Food, Gaithersburg, M D, Aspen Publishers Inc., 379–405 MARANGONI A G (2000), Elasticity of high volume fraction fractal aggregate networks: a thermodynamic approach, Phys Rev B, 62,13951–13955. MARANGONI A G (2002), The nature of fractality in fat crystal networks, Trends Food Sci Techol, 13, 37–47. MARANGONI A G and ROGERS M (2003), Structural basis for the yield stress in plastic disperse system, Appl Phys Lett, 82, 1–3. MARANGONI A G and ROUSSEAU D (1996), Is plastic fat rheology governed by the fractal geometry of the fat crystal network?, J Am Oil Chem Soc, 73, 991–994. MARANGONI A G and ROUSSEAU D (1998), The influence of chemical interesterification on the physicochemical properties of complex fat systems. 3. Rheology and fractality of the crystal network, J Am Oil Chem Soc, 75, 1633–1636. FEARON A M

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and HARTEL R W (2001), Effect of cooling rate on nucleation behavior of milk fat–sunflower oil blends, J Agric Food Chem 49, 3223–3229. MARTINI S, HERRERA M L and HARTEL R W (2002a), Effect of cooling rate on crystallization behavior of milk fat–sunflower oil blends, J Am Oil Chem Soc, 79, 1055–1062. MARTINI S, HERRERA M L and HARTEL R W (2002b), effect of processing conditions on microstructure of milk fat fraction/sunflower oil blends, J Am Oil Chem Soc, 79, 1063–1068. MARTINI S, PUPPO M C, HARTEL R W and HERRERA M L (2002c), Effect of sucrose ester and sunflower oil addition on crystalline microstructure of a high melting milk fat fraction, J Food Sci, 67, 3412–3418. MAZZANTI G, GUTHRIE S E, SIROTA, E B, MARANGONI A G and IDZIAK S H (2003), Orientation and phase transitions of fat crystals under shear, Crystal Growth and Design, 3 (5), 721– 725. MAZZANTI G, GUTHRIE S E, SIROTA E B, MARANGONI A G and IDZIAK S H (2004a), Novel shearinduced phases in cocoa butter, Crystal Growth and Design, 4(3), 409–411. MAZZANTI G, GUTHRIE S E, SIROTA E B, MARANGONI A G and IDZIAK S H J (2004b), Crystallization of bulk fats under shear, in Dutcher J R and Marangoni A G, Soft Materials – Structure and Dynamics, New York, Marcel Dekker, Inc., 279–299. MCCLEMENTS D J, DUNGAN S R, GERMAN J B, SIMONEAU C and KINSELLA J E (1994), Droplet size and emulsifier type affect crystallization and melting of hydrocarbon-in-water emulsions, J Food Sci, 58, 1148–1151. MILOSAV K (1985), Microstructure of dairy foods. 2. Milk products based on fats, J Dairy Sci, 68, 3234–3248. MORTENSEN B K and DANMARK H (1982), Consistency characteristics of butter, Milchwissenschaft, 36, 393–395. NARINE S and MARANGONI A G (1999a), Relating structure of fat crystal networks to mechanical properties: a review, Food Res Int, 32, 227–248. NARINE S and MARANGONI A G (1999b), Mechanical and structural model of fractal networks of fat crystal at low deformations, Phys Rev E, 60, 6991–7000. NARINE S and MARANGONI A G (1999c), Fractal nature of a fat crystal network, Phys Rev E, 59 (2), 991–994. NARINE S and MARANGONI A G (2002), Structure and mechanical properties of fat crystal networks, Adv Food Nut Res, 44, 33–145. POMPEI C, CASIRAGHI E, LUCISANO M and ZANONI B (1988), Development of two imitative methods of spreadability evaluation and compression with penetration tests, J Food Sci, 53(2), 597–602. PRENTICE J H (1972), Rheology and texture of dairy products, J Texture Studies, 3, 415– 458. PUPPO M C, MARTINI S, HARTEL R W and HERRERA M L (2002), Effect of sucrose ester on crystallization and rheological behavior of blends of milk fat fraction sunflower oil, J Food Sci, 67, 3419–3426. ROHM H and ULBERTH F (1989), Use of magnitude estimation in sensory texture analysis of butter, J Texture Studies, 20, 409–418. ROUSSEAU D and MARANGONI A G (1998), The effect of chemical and enzymatic interesterification on the physical and sensory properties of butterfat-canola oil spreads, Food Res Int, 31, 381–388. ROUSSEAU D, FORESTIERE K, HILL A R and MARANGONI A G (1996a), Restructuring butterfat through blending and chemical interesterification. 1. Melting behavior and triacylglycerol modification, J Am Oil Chem Soc, 73, 963–972. ROUSSEAU D, HILL A R and MARANGONI A G (1996b), Restructuring butterfat through blending and chemical interesterification. 2. Microstructure and polymorphism, J Am Oil Chem Soc 73, 973–981. ROUSSEAU D, HILL A R and MARANGONI A G (1996c), Restructuring butterfat through blending and chemical interesterification. 3. Rheology, J Am Oil Chem Soc, 73, 983–989.

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Part II Modifying lipids for use in food

9 Hydrogenation of lipids for use in food G. R. List, National Center for Agricultural Utilization Research, USA and J. W. King, University of Arkansas, USA

9.1

Introduction

An edible oil quality triangle has been described as having functionality, oxidative stability and health/nutrition as the three vertices (Lui, 1999). An ideal oil would have enough solid fat for use in spreads and shortenings, be resistant to oxidation at frying temperatures and contain no saturated or trans fatty acids (TFA). Thus, in order to satisfy these requirements, edible oils require modification most usually by catalytic hydrogenation. Since the 1970s, the health/nutrition leg has received considerable attention since both saturated and trans fatty acids are formed during hydrogenation (Sebedio and Christie, 1998). Trans fatty acids were reported as cholesterol-elevating agents raising low-density lipoproteins (LDL) (bad cholesterol) and lowering high-density lipoproteins (HDL) (good cholesterol) in humans (Mensick and Katan, 1990; Judd et al., 1994). Epidemiological studies have suggested a link between TFA consumption and coronary heart disease (CHD) risk (Hennekens and Willett, 1997; Hu et al., 1997). Other workers have questioned the results of CHD risk reports (Hegsted, 1998; Ockene and Nicolosi, 1998). Some reports, however, reject the link between TFA consumption and CHD risk (Kris-Etherton et al., 1995; Anon, 1996). Nonetheless, despite being a controversial issue, the US Food and Drug Administration enacted rules for trans fatty acids labeling to meet requirements for the Nutrition Labeling and Education Act passed by the US Congress in 1990. Since January 1 2006, food labels must contain TFA content as a separate line. A product containing less than 0.5 g TFA/serving may be declared as zero (Anon 1999, 2003). In 1995, the International Margarine Association of the Countries of Europe

174 Modifying lipids for use in food (IMACE) introduced a code of practice recommending a level of > 5 % TFA (equivalent to butter) that, by 2002, was reduced to > 1 % in spreads, while processed foods should not exceed the 5 % previously recommended. Denmark has enacted the most stringent regulation with a maximum of 2 % TFA with a trans-free claim only below 1 % TFA and including all C14–C22 trans isomers (Robinson, 2004). Under US regulations, conjugated linoleic acid is excluded for labeling purposes. Worldwide consumption of fats and oils in 2004–2005 (Anon, 2006) accounted for about 108 million tonnes, of which 57 % can be accounted for by soybean oil (29.6 million tonnes) and palm oil (31.6 million tonnes). While soybean oil is relatively low in saturated acids (15 %), it represents a major source of trans fatty acids in our food supply because it must be hydrogenated to achieve functional properties and oxidative stability in food oils. On the other hand, palm oil, while containing no trans acids, contains about 50 % saturated acids, making it attractive for use in these products, particularly when modified by fractionation and/or interesterification. Thus, just two fats and oils dominate and dictate oil processing worldwide and strategies to reduce trans and saturated acids must focus on soybean and palm oils. This chapter will review the hydrogenation process with emphasis on trans fatty acid formation and their occurrence in food oils.

9.2

Trans fatty acid contents of food oils

Reviews of margarine/spread formulations in North America have been published by Mag (1994), Chrysam (1996) and Pelloso (2001). European practices have been reviewed by Moran (1994) and Podmore (1994). A survey of the trans content of soft tub margarine/spreads taken over a seven year period in the USA is shown in Table 9.1 (List et al., 2000a). The samples reported are premium products taken from grocery store shelves and, according to their label, were formulated from hydrogenated and liquid soybean oil. Over the seven year period, trans reduction ranged from about Table 9.1 Brand

Trans content of soft margarines/spreads by year [US samples, List (2000a)]. Year

Overall

1992

1995

1999

reduction (%)

1 2 3 4 5 6 7

19.4 10.3 12.2 31.4 11.6 24.6 29.5

15.9 18.2 7.8 16.2 19.5 14.7 20.2

14.5 7.9 2.6 14.6 6.1 10.5 5.3

25.3 23.4 79.7 53.6 47.5 57.4 82.1

Average

19.9

16.1

8.8

55.8

Hydrogenation of lipids for use in food

175

25 % to 82 % with an average reduction of nearly 56 %. The 1992 data indicates an average of nearly 20 % trans, whereas the 1999 data shows less than 9 % trans in soft tub products. Stick/spreadable products have shown decreased trans acid contents over the past decade. In 1989 (Postmus et al.) trans acids averaged 26.8 %, but by 1999 the average had dropped to 16.9 % or a 37 % overall reduction (List et al., 2000a). Thus, the domestic edible oil industry has made concerted efforts to reduce trans acids in edible products. This has been accomplished through reformulation methods in which a multiple basestock system, employing three hydrogenated oils, has been replaced with a single component. Typically, soybean oil hydrogenated to an iodine value of about 65 (40 % trans) is blended with liquid soybean oil (25–50 %) to yield components suitable for a wide variety of spreads, including soft tub, spreadable stick or stick products. The trans acid contents of margarines and spreads taken from the literature over a seven year period (1995–2002) are shown in Table 9.2 (Bayard and Wolff, 1995; Oveson et al., 1998; Ratnayke et al., 1998; Tsanev et al., 1998; Alonzo et al., 2000; List et al., 2000a; Tekin et al., 2002). Low trans products are arbitrarily defined as those having 5 % or less while zero trans products may have smaller amounts of trans acids (1–2 %). Of the 228 samples reported, 87, or 38.2 %, may be considered zero or low trans. The largest number of samples reported include those from Canada and the USA, where 10 of the 126 are zero/low trans type. These results indicate that margarine/ spreads formulated in North America are formulated primarily from hydrogenated components rather than by interesterification. The products produced in Denmark are formulated from fractionated or interesterified oil components and represent the only country surveyed where hydrogenation has been replaced entirely. However, if the 59 samples from Denmark are excluded, 28 of 169, or 16.6 %, are of the low/zero trans composition. Thus, it would appear that hydrogenation remains the technology of choice to formulate margarine/spread products throughout most of the world. Table 9.2

Zero/low trans margarines formulation trends across the world. Zero/low trans*

Year

Country

No. samples reported

No

%

1995 1998 1998 1998 2000 2000 2002

France Denmark** Bulgaria Canada USA Spain Turkey

12 59 5 109 17 12 14

5 59 3 8 2 6 4

41.7 100 60 73 11.8 50 28.6

228

87

38.2

Total

*5 % or less. **Excluding Denmark, only 16.6 % are zero/low Trans.

176 Modifying lipids for use in food A recent report (Matsuzaki et al., 2002) analyzed margarines marketed in Scandinavia and 11 other European countries and in the USA for trans fatty acids and other compositional data. Their data confirm that any decrease in trans fatty acids is achieved at the expense of increased saturated acid content. Compared to the rest of the world, stick margarines produced in the USA had the lowest trans/saturated acid content (41.4 %) and the highest ratio of PUFA (polyunsaturated fatty acids)/trans fatty acids (TFA) + saturated acids (SA). The same trend was observed for soft tub margarines. Extensive surveys of the TFA content of European fats/oils, bakery products and dietary fats have been published by ARO et al. (1998), Van Erp-Baart et al. (1998) and Van Poppel et al. (1998). An extensive compilation of the TFA content of 117 foods manufactured in the USA has recently appeared along with saturated and unsaturated fatty acid data. Eight food groups, including breads and cakes, margarines, cookies and crackers, frozen potato products, salty snacks, vegetable oil/shortenings, salad dressings and breakfast cereals were reported (Satchithanandam et al., 2004). Worldwide trends in trans acid consumption have been extensively covered (Craig-Schmidt, 1998).

9.3 Formulation of food oils by hydrogenation (soybean based) A detailed discussion of hydrogenation is beyond the scope of this chapter. For further information the reader should consult reviews listed in the bibliography (Patterson, 1994; Erickson and Erickson, 1995; Hastert, 1996). Temperature, pressure, agitation and catalyst concentration are the most important factors governing the course and speed of hydrogenation. However, with other factors being equal, temperature has the largest effect on trans acid formation (Stingley and Wrobel, 1961; Puri, 1980). Industrially, hydrogenation is carried out under selective conditions favoring reduction of polyunsaturated groups over that of monoenic acids. Typically, selective conditions involve high temperature (160–220 °C), low hydrogen pressure (10–40 psi) in the presence of 0.02–0.04 % nickel metal (Table 9.3). Typically the catalysts are supplied as 25 % nickel on a support. Although selective conditions promote trans fatty acid formation, it is imperative that the amount of stearic acid formed at lower iodine values be kept at a minimum since any tristearin formed in the reaction will unduly raise the melting point of the finished oil such that the sharply melting trans functionality is compromised. 9.3.1 Margarine basestock and formulation of spreads Excellent reviews of basestock systems have been published by O’Brien (1998), Latondress (1980) and Erickson and Erickson (1995). Margarine oil basestocks require ‘selective conditions’ where stearic acid formation needs

Table 9.3

Fatty acid content and properties of partially hydrogenated soybean oils.

Oil

Original soybean oil Hydrogenated soybean oila Stearine HW SBO Margarine oilb Shortening oilc

Fatty acid composition C16

C18

C18:1

C18:2

C18:3

11.2 11.4 12.8 10.5 11.3 11.3

3.7 4.7 6.5 4.4 13.6 5.1

22.1 40.3 48.4 42.0 75.2 72.6

55.0 40.5 30.3 40.0 0.0 11.0

6.8 3.0 1.9 2.9 0.0 0.0

160 °C, 40 psi H2 pressure, 0.02 % nickel. 216 °C, 20 psi H2 pressure, 0.03 % nickel. c 221 °C, 20 psi H2 pressure, 0.03 % nickel. Abbreviation: HW SBO = hydrogenated, winterized soybean oil. a

b

Iodine value 132.0 112.6 97.2 109.5 64.7 81.5

% Trans

0.0 8.7 13.3 9.1 39.7 31.8

Melting Solid fat content at temperature (°C) point (°C) 10 21.1 26.7 33.3 40 –14.3 16.7 27.6 –7.5 41.3 29.3

0.0 0.0 14.2 0.9 73.7 36.3

0.0 0.3 5.5 0.0 54.1 13.7

0.0 0.0 1.7 0.1 44.7 4.6

0.0 0.2 0.0 0.0 22.3 0.0

0.0 0.2 0.0 0.0 3.3 0.0

178 Modifying lipids for use in food to be controlled very carefully. Generally, low pressure and high temperatures are used. Typically, the preparation of multi-purpose margarine basestocks involves reducing the iodine value (IV) of soybean oil from about 130 to 65– 70 in the presence of a nickel catalyst at temperatures of about 220 °C and low hydrogen pressures. The solid fat content of such a product is shown in Fig. 9.1.

9.3.2 Partially hydrogenated winterized soybean oil (PHWSBO) Partially hydrogenated winterized soybean salad/cooking oil (PHWSBO) has been produced commercially in the USA since the early 1960s (Evans et al., 1964). Typically, PHWSBO is produced by hydrogenation under selective conditions (160–170 °C, low hydrogen pressure and 0.02 % nickel) where the iodine value is reduced from approximately 130–132 to 110–115. This feedstock is then chilled slowly to allow the higher melting triacylglycerols to crystallize. The liquid oil is then recovered by filtration. The higher melting fraction, or stearin, may be incorporated into margarine/spread or shortening products. The composition and properties of PHWSBO are given in Table 9.3, along with hydrogenation conditions used in its manufacture. PHWSBO (IV 110–112) will remain clear at refrigerator temperatures and is superior to unhydrogenated oil for light frying in the home (Gooding, 1957; Evans et al., 1964; List and Mounts, 1980). Formulations of spreads by blending hydrogenated soybean oil (IV 65) with liquid soybean oil 5% 10 % 15 % 20 % 25 % 30 % 40 % 50 % 100 %

60

Solid fat index

50

40

30

20

10

0 10

15

20

25 30 Temperature (°C)

35

40

Fig. 9.1 Solid fat index curves for blends of IV 65 soybean oil and liquid soybean oil.

Hydrogenation of lipids for use in food

179

9.3.3 Margarine/spreads/oils Margarine oils typically show the following solid fat content (SFC): tub products 10 °C 8–14, 21.1 °C 4–8 and 33.3 °C 1–2; stick products 10 °C 20– 25, 21.1 °C 10–12 and 33.3 °C 2–4 (List et al., 2001a). The hydrogenated soybean oil, when blended with 25–50 % liquid oil, results in SFC suitable for tub and stick products, respectively (List et al., 2000a). The melting points of the blended oils will usually be from 32–35 °C. The trans isomer content of the basestock is about 40 % which, after blending, yields final trans values for tub and stick products of 10 and 20 %, respectively. It will be noted from Fig. 9.1 that trans-containing triacylglycerols are sharp-melting materials at higher temperatures. They provide functionality for spreadability and resistance to oil off (separation of oil and water) at room temperatures, yet they melt very quickly at body temperatures to yield a pleasant, cooling sensation in the mouth.

9.3.4 Shortening basestock and formulation of baking shortenings A shortening basestock is also illustrated in Fig. 9.2. Typically shortening basestocks are prepared by hydrogenation of soybean oil to an iodine value of about 80. Lower hydrogenation temperatures and higher hydrogen pressures are used compared to the preparation of margarine basestocks. To formulate shortenings, liquid oil is blended with the IV 80 oil to decrease the 10 °C SFC, and addition of completely hydrogenated hardstock (10–12 %) raises the SFC values for 33.3° and 40 °C. Added cottonseed flakes

Added soybean flakes 40

40

0% 2% 4% 6% 8% 10 %

35 30

35 30 25

SFI

SFI

25 20

20

15

15

10

10

5

5

0

0 10

Fig. 9.2

0% 2% 4% 6% 8% 10 % 15 %

15 20 25 30 35 40 Temperature (°C) (a)

10

15 20 25 30 35 Temperature (°C) (b)

40

Effect of added soybean and cottonseed flakes on solid fat indexes of IV 80 soybean oil.

180 Modifying lipids for use in food The baking industry employs a number of shortenings (Suhan, 1980; Johnson, 1999; Wainwright, 1999) and their trans acid contents are shown in Table 9.4. The trans acid contents vary from about 12 % to nearly 25 %. Reduction in the trans acid contents of shortenings is possible by alternative processing techniques such as interesterification. However, no/low trans shortenings, while equivalent in performance to hydrogenated products, may be more expensive and will have elevated saturated acid contents. Fluid baking shortenings (Holman and Quimby, 1950; Mitchell, 1950; Andre and Going, 1957; Linteris and Thompson, 1958; Baldwin et al., 1972) have been used for many years where high solids are not required, such as in fillings, cakes and bread (Moncrief, 1970; Jackel, 1980; Herzing, 1996). Fluid shortenings consist of small amounts of hard fat suspended in either liquid or hydrogenated oils. Some reduction of TFA may be achieved by substituting liquid oil in formulations where hydrogenated oils have been traditionally used. They serve the same purpose as solid shortenings by imparting tenderness and lubricity as well as serving as carriers for emulsifiers needed for aerating cake batter and imparting strength to bread.

9.3.5 Frying fats Excellent reviews of the formulation, composition and properties of frying shortenings have been given by Erickson (1996), Bessler and Orthoefer (1983) and Robertson (1966). Both liquid/fluid and plasticized frying shortenings are used in deep fat frying with composition and properties dictated by the particular end use. Liquid high-stability frying shortenings can be formulated from partially hydrogenated soybean oil under selective conditions followed by winterization. Typically, these products have iodine values in the 85–100 range and contain about 21 % TFA. Since they are liquid at room temperature, they can be easily handled, and are typically packaged in 35 lb plastic containers. These products have excellent oxidative stability since they contain antioxidants, metal inactivators and anti-foaming agents. Other vegetable oils, including safflower and cottonseed, can be used to prepare high-stability liquid shortenings by selective hydrogenation. Typically, oils are hydrogenated to an iodine value of 80, followed by agitation at 13–18 °C for several hours. The liquid oil recovered by filtration in yields of about 50 % has very low solids at 10 or 21 °C and contains about 35 % TFA (Table 9.5) (Simmons et al., 1968). Fluid opaque grilling and frying shortenings can be prepared by blending a 90–100 IV hydrogenated oil with small amounts of low IV hard fat and passing the mixture through a scraped surface heat exchanger, followed by a crystallizing unit and finally to a tempering tank. Typically, these products contain added silicones, antioxidants and lecithin (Widlak, 2001).

9.3.6 Functional properties of trans lipids in food oils Triacylglycerols can be classified by melting point into four groups, with

Table 9.4

Composition and trans acid content of commercial baking shortenings. Solid fat content (°C)

Drop % melting Trans point (°C) acids

Sample

10

21.1

26.7

33.3

40

45

50

55

Pie shortening Cake Cake and icing Puff pastry Veg./butter, all purpose All purpose butter flavor Icing Cake and icing All purpose shortening

27.2 22.9 37.7 40.1 32.7

20.3 15.9 21.9 29.5 18.1

17.2 12.9 19.5 27.7 15.2

10.3 8.9 14.4 21.5 9.5

3.2 3.0 8.1 11.4 3.8

0.2 0.6 3.7 5.0 0.8

0.4 0.8 0.8 1.1 0.0

0.0 0.2 0.0 0.0 0.3

41.3 41.1 45.8 47.7 42.4

30.0

17.8

16.5

14.4

9.4

6.0

2.0

0.5

38.6 37.1 37.0

23.1 18.9 18.8

20.7 16.2 15.5

15.9 10.7 10.4

8.9 5.8 6.0

3.2 3.6 3.8

0.6 0.9 1.6

0.3 0.5 0.0

Fatty acids by FAME-GC 14:0

16:0

18:0

18:1

18:1 trans

18:2

18:2 trans

18:3

12.8 11.8 24.4 18.0 11.6

0.18 0.00 0.22 0.20 4.77

13.93 11.56 15.14 13.59 21.75

11.50 18.16 14.06 17.23 14.85

30.14 36.98 36.36 32.51 29.39

11.87 9.15 19.23 16.19 11.17

27.99 18.34 7.29 16.75 15.32

1.45 4.34 7.51 2.83 0.81

2.94 1.47 0.19 0.70 1.95

48.4

20.4

0.19

12.87

13.31

44.32

17.49

7.06

4.43

0.34

45.6 45.0 45.6

22.7 24.7 24.9

0.20 0.00 0.09

14.77 12.07 11.64

13.39 11.94 11.24

40.58 43.61 44.57

20.50 22.26 22.78

7.00 6.62 6.52

3.27 3.20 2.83

0.29 0.31 0.34

Note: 18.1 cis had high amounts of C11 and C13 double bonds, up to 10 % of total fatty acid. Abbreviation: FAME–GC = fatty acid methyl ester–gas chromatography.

Table 9.5

Liquid shortenings by hydrogenation/fractionation.

Fractionation condition Temp. (°C)

15 15 18 15 15 15 15 18 13 15 13 13 15 15 15 15 15 18

Agitator, R. P. M.

40–45 24 24 40 24 24 24 24 24 24 24 24 24 12 24 36 48 24

Hours at Temp.

2 1 1 1, 5 1 2 4 1 1 1 1 1 1 1 1 1 1 1

Abbreviation: IV = iodine value. Source: Simmons (1968).

Starting stock Hydrogenation, Temp. (°C)

49–54 49–54 49–54 71–85 71–85 71–85 71–85 71–85 71–85 71–85 71–85 71–85 71–85 71–85 71–85 71–85 71–85 71–85

IV

82.6 82.6 82.6 80.8 80.8 80.8 80.8 80.8 80.8 83.9 83.9 75.2 75.2 81.1 81.1 81.1 81.1 83.9

Oil portion

Solids at (°C) 10

33

20.0 20.0 20.0 29.8 29.8 29.8 29.8 29.8 29.8 25.4 25.4 45.9 45.9 30.5 30.5 30.5 30.5 25.4

0.5 0.5 0.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 3.4 3.4 0.2 0.2 0.2 0.2 0.0

Melt point

IV

Percent trans

Solid at (°C) 10

30.9 30.9 30.9 29.7 29.7 29.7 29.7 29.7 29.7 29.8 29.8 35.6 35.6 30.2 30.2 30.2 30.2 29.8

89.1 90.3 88.5 85.4 85.3 85.7 85.7 84.4 85.9 88.4 89.3 81.9 81.4 85.7 86.0 85.7 85.0 87.3

31.8 28.8 33.1 35.5 37 36.9 37.3 38 36.6 35.3 35.3 46.2 47.5 39.4 38 39.9 38.1 36.4

0.2 0.1 13.0 8.1 4.7 1.5 0.7 16.5 0.2 0.1 0.0 29.0 31.6 7.1 4.2 4.6 5.5 11.7

Percent Yield

33

0.7 0.2 0 0.4 0.2 0.2 0.1 0.6 4 0.3 0.2

58.3 50.8 70 50.4 47.8 49 48.3 54.6 39 54 47.3 32.4 25.8 49.3 49.6 48.6 49.2 64.7

Hydrogenation of lipids for use in food

183

each group furnishing functional properties to margarines/spreads, baking/ frying fats and salad oils (Bessler and Orthoefer, 1983). Group I consists of those triacylglycerols whose melting points range from –13 to 1 °C and include simple triacylglycerols found in common vegetable oils including soybean, corn, cottonseed, peanut and canola. Examples include trilinolein, oleo dilinolein and palmito dilinolein. The low melting points of group I triacylglycerols ensure liquidity at refrigerator temperatures and that they are pumpable during processing, handling and storage. In addition, triacylglycerols high in linoleic acids are desirable from a nutritional standpoint, since they furnish essential fatty acids. Group II triacylglycerols melt from 5.5 to 23 °C and remain liquid only if stored at ambient temperatures, i.e. 25 °C. Examples include triolein (OOO) and monosaturated isomers SOL, OOP and SOO, where S = stearic, P = palmitic and L = linoleic. Group III includes triacylglycerols melting near or at body temperature, i.e. 27–42 °C. Examples include disaturated monounsaturated isomers of palmitic, stearic and oleic/linoleic acids, i.e. PPL, SSL, PPO, SPO and SSO. An excellent example of functional/structural relationships in fats can be illustrated by cocoa butter, whose composition consists of over 80 % SOS, SOP and POP. Cocoa butter is a hard, brittle material at room temperature, yet melts quickly at body temperature and, for this reason, is an excellent confectionary fat. Group IV triacylglycerols are high-melting materials (56–65 °C) and most usually do not occur naturally to any great extent in common vegetable or animal fats and oils. Examples include SSP, PPP, SSP and SSS. Triacylglycerols from this group result primarily from the complete or nearly complete hydrogenation of common vegetable oils, i.e. corn, soybean, cottonseed and palm. A margarine/spread should be spreadable at refrigerator temperature, hold together at room temperature and melt sharply at body temperature. Normally, these properties can be approximated by the amount of solid fat at 10, 21 and 33 °C, as well as the melting point of the liquid components. Although spreads with solid fat index (SFI) values of less than 30 at 10 °C will be spreadable directly out of the refrigerator, optimum spreadability is observed at much lower SFI values at 10 °C, often as low as 8–10 for soft table spreads and 18–20 for spreadable stick products. Margarines formulated from interesterified soybean oils have SFI values at 10 °C as low as 5–6 and tend to be less spreadable than those formulated from hydrogenated components (Kok et al., 1999; Chrysam and Pelloso, 2000; List, 2001b). Other factors influencing the spreadability of margarine include processing conditions used in the chilling, crystallization and working of the emulsion through scraped surface heat exchangers (Haighton, 1976). Structure at room temperature or the ability of the spread to resist separation of oil and water is also governed by the solid fat content at 21 °C. Typically, hydrogenated components have SFC ranging from 4–8 (tub) up to 9–14 in stick products. In the USA and Canada spreads have SFI values in the 1–3 range to ensure sharply melting properties that, when placed in the mouth at

184 Modifying lipids for use in food body temperature (37 °C), the spread melts quickly to give a pleasant, cooling sensation. High-melting triacylglycerols will impart a waxy sensation and poor mouth feel. Spreads manufactured in the USA are formulated from hydrogenated components such that the final melting point (approximately 32 °C) is only about 9 °C less than the harder trans-containing component (41.3 °C). The high solubility of trans fatty acids of groups I, II and III in liquid oils, coupled with their sharp-melting properties, render them as highly functional materials. On the other hand, group IV triacylglycerols are virtually insoluble in liquid oils over temperature ranges required of a baking shortening. Pie shortenings are loosely dispersed into the dough because over-mixing causes absorption of the fat into the dough, thereby creating a tougher crust and shrinkage. Absorption is controlled by mixing the dough at low temperatures. To ensure adequate rolling and formation, the shortening must have adequate plasticity at low temperatures and sufficient solids for flaky texture. In addition, pie shortenings should melt quickly for adequate mouth feel. Lard and hydrogenated lard perform well in pie crust (Kincs, 1985). Typically, pie crust shortenings contain about 13 % TFA and have melting points of about 41 °C. Cake shortenings function to incorporate air into the batter which, in turn, governs cake volume, grain, moistness and storage life (Howard, 1972). Often, emulsifiers are added to allow high levels of sugar to be incorporated into the cake. Cake shortenings generally have less solids than pie shortenings and slightly less TFA, but similar melting points. Icing and crème fillers require shortenings allowing incorporation of sugar, flavorings and water (Brody and Cochran, 1978). Prime functional considerations include creaming ability, body, texture and mouth feel. Mono/ diacylglycerols are used to emulsify the shortening. Typically, icing shortenings contain high solids and trans acids. The solids profile of icing and all purpose baking shortenings are similar. Melting points are about 45 °C with TFA contents of 23–25 %. Puff pastry shortenings have been discussed by Colburn and Pankey (1964). They usually contain about 18 % TFA.

9.3.7 High-stability oils High-stability oils (HSO) were developed over 30 years ago (Gooding, 1972) (see also Carrick and Yodice, 1993 and Miller, 1993). Compared to commodity oils, they are expensive, extremely stable, yet fill definite needs for the food industry. They are liquids at ambient temperature and perform well as spray oils and in applications in products with large surface areas and/or where long shelf life is required. HSOs are at least four times more resistant to oxidation and hydrolysis than commodity salad oils which translates into slower development of off-flavors, and color stability shows marked improvements. Typical applications include roasting of nuts, as carriers of flavors, use as moisture barriers, as viscosity modifiers, glass enhancers, lubricating/releasing agents, anti-dusting agents and frying operations. The market for HSO use was estimated at 45–57 thousand tonnes in the year

Hydrogenation of lipids for use in food

185

2000 with a breakdown as 70 % low-end HSO, 10 % mid-range and 20 % high-end. These designations refer to their relative stability under active oxygen method conditions (100 °C) or hours to reach a peroxide value of 100. Low, mid-range and high-end oils have values of 50–100, 100–300 and > 300, respectively (Lampert, 2000). HSOs can be processed from both commodity and genetically/plant breeding modified oils. High oleic corn, soybean and sunflower oils meet requirements for low-end HSO without processing beyond the customary refining, bleaching and deodorizing, whereas soybean oil requires partial hydrogenation and dry fractionation as well. Both medium and high-end HSO can be prepared from both commodity and genetically/structurally modified oils. However, light, partial or heavy hydrogenation and dry fractionation must be employed with the usual refining, bleaching and deodorization steps. Low-end HSO prepared from hydrogenated, dry-fractionated soybean oil contains 15 % saturates and 32 % trans, whereas high oleic sunflower contains 9.5 % saturates and 1 % trans. Canola and high oleic canola-based HSO offer opportunity for trans reduction in mid-range applications compared to soybean oil. Canola HSO shows trans values of 18–29 % compared to 51 % for soy-based oil. Similarly, high oleic-based, high-end oils have 33 % trans compared to 48 % for cottonseed/soybean-based oils (Lampert, 2000). HSOs are liquids at ambient temperature, highly functional and convenient to use. They are used at low levels, often between 0.2 and 1 %, and are most commonly sprayed onto the surface of the food or ingredient. Although more expensive than commodity oils, processing costs are reduced making the final pricing competitive. It is expected that increased use of HSOs will occur in the future to achieve fat reduction in foods, to improve product shelf life and to improve health and nutrition.

9.4

Source oils for trans fatty acids

9.4.1 Canola oil Hydrogenation of canola oil has been studied extensively in the laboratory (Deman et al., 1981; Deman and El-Shattory, 1981; El-Shattory et al., 1981; Bansal and Deman, 1982) and has been reviewed by Koseoglu and Lusas (1990). Although canola oil is widely used in margarines/spreads and shortenings, the trans isomer content of these products tends to be somewhat higher than soybean-based products (Postmus et al., 1989). Compared to soybean oil, hydrogenation of canola oil results in more trans acids (TFA) at a given IV level. For example, canola oil (IV 113), when hydrogenated to an iodine value of 80 for shortening stock, contains about 45 % TFA compared to 31–32 % for soybean at the same IV. Canola margarine stock (IV 75) contains 50 % trans compared to 40 % for soybean oil at IV 65. Lightly hydrogenated canola (IV 90) used as a frying fat contains 20 % trans, whereas soybean oil at IV 110–116 contains about 10–12 % trans

186 Modifying lipids for use in food (Eskin et al., 1996). These properties can be accounted for, in part, by the differences in fatty acid composition of the two oils. Canola oil contains 60 % oleic acid and about 32 % polyunsaturated acids (linoleic acid 22 %, linolenic acid 10 %), whereas soybean oil contains about 22 % oleic acid and 63 % polyunsaturated acids. Sulfur-containing glucosinolates found in canola oil tend to poison the catalyst, thus promoting TFA formation. Canola oil tends to crystallize in the undesirable β form owing to the low levels of palmitic acid found in the oil. To improve the crystal habit, the addition of palm oil prior to hydrogenation reportedly stabilizes the oil in the β′ form desired for margarines and shortenings (Eskin et al., 1996).

9.4.2 Cottonseed oil Excellent reviews of food uses and processing of cottonseed oil have been published by O’Brien and Wan (2001) and Jones and King (1996). Cottonseed oil has an iodine value of about 110 and contains about 25 % saturated acids. A basestock system for formulation of shortenings, margarines and spreads consists of five hydrogenated oils along with a hard stock or completely hydrogenated component (IV > 5). Oils hydrogenated to an IV of 75–80 under non-selective conditions (177 °C, 30–45 psi, 0.02 % nickel catalyst) possess flat SFI curves suitable for shortenings, whereas the 58, 65 and 70 IV oils hydrogenated under selective conditions (227 °C, 11–15 psi, 0.04– 0.08 % nickel catalyst) possess steep SFI curves needed for margarine/spreads. The IV 58 oils have the solid fat and melting properties very similar to IV 65 soybean oil and are similar in trans acid content. In 1997 only 3628 tonnes of cottonseed oil was used in margarines/spreads in the USA (O’ Brien and Wan, 2001).

9.4.3 Corn oil During the period 1950–1980 corn oil usage in margarine/spreads increased from about 454 tonnes to 101000 tonnes, but, by 2000, had dropped to about 25 400. (O’Brien, 2004). Corn oil contains about 13 % saturated acids and has an iodine value of approximately 127. Soft and stick margarines formulated with hydrogenated soybean oil and liquid corn oil are available commercially. Soybean oil basestocks are produced by hydrogenation of the oil to iodine values of 65–67 under selective conditions and contain about 48 % trans acids. The 65 IV oil, when blended with 25–30 % liquid corn oil, is suitable for soft products while the 67 IV oil, when blended with 45–47 % liquid corn oil, is suitable for stick products. Trans acid content of soft and stick products ranges from 12 to 14 % and from 20 to 24 %, respectively.

9.4.4 Sunflower oil Sunflower oil is produced in many parts of the world and ranks fourth in

Hydrogenation of lipids for use in food

187

worldwide consumption behind soybean, palm and canola oils. It contains about 10 % saturated acids and 73 % linoleic acids. Sunflower oil behaves much like soybean oil during hydrogenation. Under selective conditions (227 °C, 10 psi, 0.02 % nickel) reduction of the iodine value from 132 to 66 produces basestocks very similar to soybean oil at the same iodine value. Shortening stocks are normally prepared using lower temperatures. Melting points and SFI are comparable to soy oil basestocks. The TFA contents of these basestocks are of the order of 60 %, or about 1.12 % trans/IV unit. Soybean oil shows a value of 0.61 % trans/IV unit. Tub margarines are formulated from IV 75–80 (50 %) and 50 % liquid oil. Stick products require 35 % IV 98 and 35 % IV 65 oil with the balance liquid sunflower oil. All purpose shortening can be formulated from 92 % IV 86 oil and 8 % fully hydrogenated cottonseed or palm oil (Davidson et al., 1996). Other sunflower cultivars include the high oleic (~ 84 %) and mid-oleic (50–65 %, NuSun™ – National Sunflower Association, USA). As discussed previously, the high oleic oil qualifies as a high stability oil without hydrogenation. NuSun™ also requires no hydrogenation, therefore offering a trans-free fat for deep fat frying. A major snack food manufacturer has switched its premium brand potato chips from cottonseed oil to NuSun™ with very satisfactory results (Gupta, 2001).

9.5

Mechanism of hydrogenation/isomerization

The theory of edible oil hydrogenation was outlined in a series of papers by A. E. Bailey (Bailey and Fisher, 1946; Bailey, 1949a, b) and was advanced by Allen and Kiess (1955), who proposed a mechanism to account for positional and geometric isomerization. Albright (1963) and Eldib and Albright (1957) reviewed factors affecting rate, selectivity and isomerization of triacylglycerols occurring during catalytic hydrogenation. Transfer of the reactants to the catalyst surface and of the products away from the catalyst are important steps. Both hydrogenation (saturation) and isomerization (both geometric and positional) occur simultaneously on the catalyst surface. Mass transfer steps include transfer of the hydrogen from gas to liquid phase, transfer of hydrogen from the liquid phase to the catalyst surface, transfer of the unsaturated triglyceride from the liquid phase to the catalyst surface and transfer of the reaction products to the main body of liquid. A mechanism to account for both hydrogenation and positional isomerization is shown in Fig. 9.3 (Allen and Kiess, 1955). Hydrogen adsorbed onto the catalyst surface is dissociated into two hydrogen atoms (H*) resulting in either isomerization or saturation. In a recent review of the hydrogenation reaction, Dijkstra (1997) proposed the following mechanism to account for both hydrogenation (saturation) and isomerization (trans acid formation) (Fig. 9.4). The extent of isomerization occurring during hydrogenation is controlled by two factors referred to as

188 Modifying lipids for use in food 1

2

3

4

CH2

CH

CH

CH2

CH2

CH2

CH2

+ H*

CH2 + H*

CH2 + H*

CH

CH

CH2

CH2 + H*

Geometrical isomers formed

Fig. 9.3

CH2

CH

CH2

Positional and geometrical isomers formed

CH2

CH2

CH2

CH2

Hydrogenation step

Isomerization during hydrogenation, from Allen and Kiess (1955). Reprinted with permission of the American Oil Chemists’ Society.

D+H DH DH + H M+H MH MH + H

K1 K2 K3 K4 K5 K6

DH D+H

(slow)

M

(relatively fast)

MH M+H

(trans-formation)

S

(slow)

Fig. 9.4 Hydrogenation reaction scheme from Dijkstra (1977) (D = diene, H = hydrogen, M = monoene, S = saturate). Reprinted with permission of the American Oil Chemists’ Society.

hydrogen availability and hydrogen demand. Hydrogen demand may be defined as the potential rate at which the unsaturated fatty acids combine to form saturated acids and is controlled by kinetics when mass transfer resistance at the gas–liquid and liquid–solid interface is negligible. However, hydrogen availability is dependent on the mass transfer rates at the two interfaces and, thus, controls the course of the hydrogenation reaction. For any given hydrogenation system, the hydrodynamics of the gas–liquid and liquid–solid phase is fixed through design of the vessel, agitation and gas sparger at any given level of catalyst activity. This variable is also fixed and temperature, pressure and catalyst concentration are the only process variables that govern hydrogen availability and demand during a hydrogenation reaction. The effects of process variables on trans isomer formation is summarized in Table 9.6 (Puri, 1980). Stingley and Wrobel (1961) reported the effects of hydrogenation on the physical properties and of soybean and cottonseed oils. Temperatures ranging from 127 °C to 252 °C were used at hydrogen pressures of 10–70 psi. Iodine values were reduced from 132.5 and 109, respectively, to values of 90, 80,

Hydrogenation of lipids for use in food Table 9.6

189

Effect of process variables on trans-isomer formation. Effect on

Increase of

Hydrogen concentration

Trans-isomer formation

Temperature Pressure Catalyst

– + –

+ – +

Source: Puri (1980).

70 and 60. Their data clearly shows that, at a constant pressure, the rate of isomerization increases with temperature. For example, at 10 psi and 127 °C, the rate of isomerization was 0.34 % trans/unit iodine value drop, while at 252 °C the rate was doubled to 0.70 % trans/iodine value, and the rate at 171 °C was intermediate. The same trends were observed at 40 and 70 psi. Evans et al. (1964) observed a 0.73 % trans/iodine value drop for soybean oil hydrogenated at 170 °C. The aforementioned studies were conducted under selective conditions favoring the reduction of polyunsaturates over monoenoic acids. There are few studies on the rates of isomerization under commercial conditions. However, it is general commercial practice to use selective conditions for soybean and canola oils since they contain 7–8 % linolenic acid, a component which should be removed in order to achieve flavor and oxidative stability. Trans fatty acid content increases linearly with iodine value until trans double bonds begin to hydrogenate, which is in the area of about IV 70 and Wrobel’s data indicate that the rates of isomerization on soybean and cottonseed oil are virtually identical. The properties of soybean oil hydrogenated under selective conditions (220 °C, 25–40 psi hydrogen pressure, 0.02 % nickel in a dead end batch converter) are given in Table 9.7. Under these conditions, soybean oil shows an isomerization rate of 0.8 % trans/unit iodine value drop which compares well with the Evans et al. (1964), Stingley and Wrobel (1961) and data of 0.73 and 0.7 % trans/unit iodine value drop. During the course of heterogeneous catalytic hydrogenation, two distinct, competing reactions occur: positional/geometric isomerization and saturation of double bonds. Factors affecting the course of these reactions include pressure, temperature, hydrogen dispersion and catalyst concentration. Generally speaking, conditions promoting isomerization are classified as ‘selective’, whereas ‘non-selective’ conditions promote saturation. Thus, selective conditions promote isomerization and minimize saturation of monoenoic fatty acids to stearic acid. The kinetics of edible oil hydrogenation under selective conditions can be depicted as: Trienes Slow → Dienes   → Monoenes Slow → Saturates Fast Trienes (linolenic acid) being reduced to dienes is a slow reaction since the iodine value must be reduced from about 130 to 90 in order to eliminate this

190 Modifying lipids for use in food Table 9.7 Properties of soybean oil hydrogenated under commercial selective conditions. Iodine value

∆IV

% Trans

Melting point (°C)

Solids @ 50 °C (NMR)

SII

133.7 124.6 121.9 114.2 109.7 103.6 100.3 90.5 85.4 83.0 75.6 69.1

0.0 9.1 11.8 19.5 24.0 30.1 33.4 43.2 48.3 50.7 58.1 64.6

>1 6.4 7.3 13.1 16.9 21.4 23.8 29.7 37.5 36.2 47.8 51.9

– – – 16.6 17.7 19.6 21.4 24.0 28.4 27.9 34.4 41.2

0.0 0.0 – 3.0 4.4 8.5 11.7 18.9 35.7 34.8 45.9 88.3

– 70.3 61.9 67.2 70.4 71.1 71.3 68.8 77.6 71.4 82.3 80.3

Abbreviations: NMR = nuclear magnetic resonance, SII = specific isomerization index (% trans/IV) × 100.

component (7–8 %) from soybean oil. Dienes being converted to monoenes is a relatively fast reaction. Various workers have described the rate of trans isomer formation during hydrogenation of soybean, cottonseed and canola oils, and this has given rise to hydrogenation index (Stingley and Wrobel, 1961) and specific isomerization index (Puri, 1978). Essentially, both are determined from the amount of trans fatty acids measured at a given iodine value and the drop in iodine value and are expressed as the number of trans double bonds formed per unit rate of iodine value reduction. Other investigators have described methods to predict TFA formation from experimental data obtained in stirred batch reactors (Allen and Covey, 1970; Puri 1978). A typical hydrogenation flow diagram is shown in Fig. 9.5.

9.6

Future trends

A recent report suggests that existing limitations on current equipment is a major obstacle limiting the production of low trans oils via hydrogenation (Beers and Mangus, 2004a). They indicate that pressures of 50–60 bars (735–882 psi) are needed, while most existing equipment can handle pressures only up to five bars (73.5 psi) Hydrogenation at very high pressure, 500– 1000 psi (34–60 bars), has been shown to be disadvantageous because significant amounts of high-melting triacylglycerols are formed under these conditions which produce high, flat SFI curves (List et al., 2000c). The insolubility of these highly saturated triglycerides in soybean or other liquid oils renders them unsuitable in spreads and of limited use in baking shortening formulations. Most likely, such fats might find use in frying operations (King

Measuring tank

Steam

Catalyst

Vacuum unit

Steam H2 Pl

PT

Pl FT

Reaction heat recovery Reactor

Hydrogen dosing

Water

PLC

Oil

TI

Black filter

Oil-oil heat exchanger Spent catalyst Drop tank

Hydrogenated oil

to bleaching & refining

Fig. 9.5

Hydrogenation flow scheme from List (2004a). CFT = flow transmitter, PI = pressure indicator, TI = temperature indicator, PT = pressure transducer, PLC = pressure lowering controller. Reprinted with permission of Food Technology, Walter Farr.

192 Modifying lipids for use in food et al., 2001). More recent studies conducted in the author’s laboratory have shown, at hydrogenation temperature ranging from 140–170 °C and at pressure of 13.6 bars (200 psi), the trans fatty acid content can be reduced significantly. At an iodine value of 65, about 17 % TFA are produced compared to nearly 40 % in commercial hydrogenation. Blending of the IV 65 oil with 70 % liquid oil results in spread oils meeting FDA requirements for TFA labeling in the USA (Eller et al., 2005). Refitting commercial hydrogenation connectors to handle 200 psi pressures should not be difficult. Since the mid 1990s, catalyst manufacturers have studied trans reduction in edible oils (Berben et al., 1994, 2005; Hasman, 1995; Ariaansz and Okonek, 1998; Beers and Mangus, 2004a; Beers et al., 2004) using both conventional nickel and noble metals such as platinum, palladium and ruthenium. Platinum modified/ammonia catalysts show promise in both TFA reduction and minimization of saturated acids (Berben et al., 2005). The use of noble metal catalysts may be limited by cost. Hasman (1995) showed that significant trans reduction can be obtained with both canola and soybean oils. However, the rather high catalyst levels required may discourage commercial acceptance along with pressure limitations on equipment. Several other technologies offer the potential to reduce TFA during hydrogenation. Harrod and Moller (1999, 2001) and King et al. (2001) have reported that hydrogenation in critical fluids such as propane and carbon dioxide produces oils with lowered TFA content. Electrochemical hydrogenation also shows promise for TFA reduction (Warner et al., 2000). About a decade ago it was estimated that about 90 % of the world’s edible oils were processed by hydrogenation with the remainder by interesterification, fractionation or a combination thereof (Haumann, 1994). Recent TFA labeling requirements in the USA will most likely result in increased use of alternatives to hydrogenation or modifications of existing hydrogenation methods (Hunter, 2004). Since the mid 1990s, a number of low/zero trans shortenings (Scavone, 1995; Roberts et al, 2000) and spreads have appeared in US markets. Some have been produced by random chemical interesterification of canola oil and fully hydrogenated soybean oil. Others have been produced by enzymatic interesterification of soybean and cottonseed oils. At least one line of products has been produced by modified hydrogenation technology (Anon, 2004; Higgens, 2004). A number of functional cholesterol lowering spreads have been introduced based on hydrogenation or simple blending of liquid vegetable oils with tropical fats (List, 2004b). Thus, it would appear that hydrogenation will continue to be a prominent oil processing technology.

9.7

Alternatives to hydrogenation: low and zero trans fats

A number of alternatives to hydrogenation for producing margarine/spread, shortenings and frying fats are available, including interesterification, fractionation of tropical fats and, more recently, the development of oilseed

Hydrogenation of lipids for use in food

193

crops with modified fatty acid composition. Reviews of these technologies can be found in the reference section (Gillies 1974; Deffense, 1985; Timms, 1995, 1997; Krishnamurthy and Kellens, 1996; Rozendall and Macrae, 1997; Tirtiaux, 1998; McDonald and Fitzpatrick, 1999; Gunstone, 2001). Random interesterification of 80 % liquid soybean oil with 20 % completely hydrogenated soy provides a route to soft margarine oil (List et al., 1977). Further work, in which the interesterified oil was formulated into soft margarine, showed that the 80:20 blend resulted in a product that was more difficult to spread than hydrogenated controls. However, an additional 20 % liquid oil was required in the formulation to achieve suitable spreadability and softness (List et al., 1995a). Random interesterification of other liquid oils, including cotton seed, corn, canola and peanut, with either soybean or cottonseed flakes also leads to basestocks suitable for formulation of zero trans margarines and shortenings (List et al., 1995b). Rapid growth of the palm industry during the 1970s prompted development of improved fractionation technology. Historically, solvent and detergent processes had been used, but today physical or dry fractionation is the industry standard. Palm oil (IV 51–53) can be fractionated into olein (IV 56–59) and stearin (IV 32–36) which, in turn, can be fractionated into mid, super and top oleins. Palm mid-fractions (IV 42–48) can be fractionated into IV 32–36 fractions. Fractionation of the stearin IV 32–36 yields soft (IV 40–42) and super stearines (Tirtiaux, 1998). These developments in fractionation technology, combined with interesterification, have led to numerous studies reporting food uses for palm and palmkernel oils (Duns, 1985; Traitler and Dieffenbacher, 1985; Majumdar and Bhattacharya, 1986; Noraini et al., 1989, 1998; Berger, 1990, 1993, 1998; Ong et al., 1995; Achaya, 1997; Ozay, et al., 1998; Petrauskaite et al., 1998; Yusoff et al., 1998) Beginning in the late 1980s, a number of oilseeds with modified fatty acid compositions have been developed and commercialized. Most have resulted from traditional plant breeding techniques (Lui 1999; Wilson, 1999; Loh, 2000; Gunstone, 2001). These include high and mid-level oleic sunflower oil (Gupta, 1998, 2001; Kleingartner and Warner, 2001; Kiatsaichart et al., 2003), high oleic corn, soybean and safflower oil, low linolenic canola and soybean oils and high oleic/low linolenic canola oil. A number of laboratory frying studies have demonstrated superiority over commodity oils (Warner and Mounts, 1993; Mounts et al., 1994; Warner and Knowlton, 1997; Warner et al., 1997; Sohelli et al., 2002; Su et al., 2003; Warner and Gupta, 2003). A frying study comparing low linolenic soybean, hydrogenated low linolenic soybean and high oleic sunflower against liquid, opaque and heavy duty oils formulated with hydrogenated soybean oils showed that the former group compared well in both fry life and flavor evaluations. Other tests showed that high oleic sunflower and low linolenic soybean oils compare well to hydrogenated oils in spray oil applications and non-dairy creamer formulations. Other applications include fluid margarines/spreads and dressings (Erickson

194 Modifying lipids for use in food and Frey, 1994). Although low linolenic canola and soybean oils have reached commercialization, costs and production problems have impeded their success in the marketplace (Krawczyk, 1999). However, the new labeling regulations may provide new markets and demands for modified composition oils. The A90, A6 and HS-1 cultivars developed by Pioneer, Iowa State University and Hartz Seed Company, respectively, represent the high saturate soybean oils in which the normal 15 % saturated acids have been elevated to as high as 40 %. In their natural states these oils lack sufficient solids at temperatures required of a margarine/spread oil. Addition of harder components such as palm oil, interesterified palm oil or cottonseed/soybean stearins shows potential in soft margarine applications (List et al., 1996, 2000b). Owing to the symmetrical nature of their triacylglycerol structures, the high saturate lines are low, sharply melting materials that, upon random interesterification, melt over a wide range rendering them more amenable to utilization in soft margarine formulation (List et al., 1997, 2001b; Kok et al., 1999). Typically, soft margarines formulated from hydrogenated and liquid soybean oils contain about 10 % trans fatty acids and about 20 % saturated acids. Studies have shown that approximately 25–30 % saturated acids are required to formulate zero trans soft margarine oils from soybean oil-based components (List et al., 1995a; List 2001b; Kok et al. 1999). Thus, any reduction in the trans fatty acids is likely to be achieved at the expense of increased saturated acid content.

9.8

References

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HOLMAN, G. W. and QUIMBY, O. T. (1950) Process of preparing suspensions of solid triglyceride

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(1980) Shortenings and Their Uses in Practical Baking, Westport, CT, AVI, 5–10. TEKIN, A., CIZMECI, M., KARABACAK, H. and KAYABAN , M. (2002) Trans fatty acids and solid contents of margarines marketed in Turkey, J Am Oil Chem Soc, 79, 443–445. TIMMS, R. E. (1995) Crystallization of fats, in Hamilton R J, Developments in Oils and Fats, and Professional Press, London, Blackie Academic, 204–223. TIMMS, R. E. (1997) Fractionation, in Gunstone F D and Padley F B, Lipid Technologies and Applications, New York, Marcel Dekker, Inc., 199–222. TIRTIAUX, A. (1998) Dry fractionation – The beat goes on, in Koseoglu S S, Rhee K C and Wilson R F, Proc World Conference on Oilseed and Edible Oils Processing, Champaign, IL, AOCS Press, 92–98. TRAITLER, H. and DIEFFENBACHER, A. (1985) Palm oil and palm kernel oil in food products, J Am Oil Chem Soc, 62, 417–421. TSANEV, R., RUSSEVA, A., RIZOV, T. and DONTCHEVA, I. N. (1998) Content of trans fatty acids in edible margarines, J Am Oil Chem Soc, 75, 143–145. VAN ERP-BAART, M. A., CONET, C., CUADRADO, C., KAFTOS, C., STANLEY, A. and VAN POPPEL, G. (1998) Trans fatty acids in bakery products from 14 European countries: The trans fair study, J Food Comp Anal, 11(2), 161–169. VAN POPPEL, G., VAN ERP-BAART, M. A., LETH, T., GEVERS, E., VAN AMELSVOORT, J., PETITHARYLANZMANN, D., KAFTOS, A. and ARO, A. (1998) Trans fatty acids in foods in Europe: the trans fair study, J Food Comp Anal, 11(2), 112–136. WAINWRIGHT, B. (1999) Oils and fats for the baking industry, Cereal Foods World, 44(3), SR16–SR19. WARNER, K. and GUPTA, M. (2003) Frying quality and stability of ultra low and low linolenic acid soybean oils, J Am Oil Chem Soc, 80, 275–280. WARNER, K. and KNOWLTON, S. (1997) Frying oils and oxidative stability of high-oleic corn oils, J Am Oil Chem Soc, 74, 1317–1322. WARNER, K. and MOUNTS, T. L. (1993) Frying stability of soybean and canola oils with modified fatty acid compositions, J Am Oil Chem Soc, 70, 983–988. WARNER, K., ORR, P., PARROTT, L. and GLYNN, M. (1994) Effect of frying oil composition on potato chip stability, J Am Oil Chem Soc, 71, 1117–1121. WARNER, K., ORR, P. and GLYNN, M. (1997) Effect of fatty acid composition of oils on flavor and stability of fried foods, J Am Oil Chem Soc, 74, 347–356. WARNER, K., NEFF, W. E., LIST, G. R. and PINTAURO, P. (2000) Electrochemical hydrogenation of edible oils in a solid polymer electrolyte reactor. Sensory and compositional characteristics of low trans oils, J Am Oil Chem Soc, 77, 1113–1117. WIDLAK, N. (2001) Formulation and production of fluid shortenings, In Wilson R F, Proc of World Conference on Oilseed Processing Utilization, Champaign, IL, AOCS Press, 39–44. WILSON, R. F. (1999) Alternatives to genetically modified soybeans – the better bean initiative, Lipid Technol., 11, 107–109. YUSOFF, M., KIFLI, H., NOORLIDA, H. and ROZIG, M. P. (1998) Formulation of trans-free margarines, in Koseoglu S S, Rhee K C and Wilson R F, Proc World Conference on Oilseed and Edible Oils Processing, vol. 1 Champaign, IL, AOCS Press, 156–158.

10 Fractionation of lipids for use in food V. Gibon, De Smet Technologies and Services, Belgium

10.1

Introduction

Hippolyte Mège Mouriès (1817–1880) is generally thought to be the pioneer of fractionation with his famous patent concerning margarine: ‘Application for a patent of fifteen years for production of certain fats of animal origin’, accepted on October 20th 1869 in Paris (Patent number 86480). Based on the hypothesis that butter fat should first be formed in animal tissues, he processed beef tallow until he obtained a liquid fat, the oleo-margarine or tallow olein. After mixing with skimmed milk, oleo-margarine gave a solid product first named ‘beurre économique’ and later ‘margarine Mouriès’. In 1871, Mège sold his invention to the Dutch company Jurgens. On the other hand, Holde and Stange (1901) reported that olive oil, when cooled in an ether solution to – 40 °C, produced small quantities of solid triacylglycerols (principally oleo-dipalmitin). This publication is the first report of the use of low-temperature fractional crystallization for separation of triacylglycerols. In the past century, a great deal of progress has been made in terms of fractionation of edible oils and fats, the goal being always to add value to the starting material.

10.2 Different fractionation techniques The physico-chemical characteristics of edible oils and fats are closely linked to their triacylglycerol composition. Nature endows each fat with a particular

202 Modifying lipids for use in food composition and distribution of the fatty acids on the glycerol moiety. Because of this, their use in specific applications is limited. Modification techniques such as hydrogenation, interesterification, fractionation or combinations of these make it possible to go beyond these limitations leading to a wide range of new products. Fractionation (also called fractional crystallization) refers to a separation process in which the fat material is crystallized, after which the liquid phase is separated from the solid. The fractional crystallization process is based on differences of solubility of the solid triacylglycerols in the liquid phase depending on their molecular weight and degree of unsaturation. In addition to fractional crystallization, lipids can be separated according to other physical principles: fractional distillation, short path distillation, supercritical extraction, liquid–liquid extraction, adsorption, complexation and membrane separation are the main techniques practised.

10.2.1 Fractional distillation Fractional distillation is based on the difference in volatility of the different lipid components and is largely applied to the fractionation fatty acids and methyl esters for the oleochemical industry according to their chain-length (Gervasio, 1996). Distillation is carried out under high vacuum, low temperatures and with the shortest possible residence time.

10.2.2 Short path distillation Triacylglycerols have high boiling points and undergo decomposition or polymerization when heated to high temperatures. This problem can be minimized by reducing the time of thermal exposure using wiped-film evaporation or centrifugal stills. Fractionation of anhydrous milk fat has been described by Arul et al. (1988) and, more recently, by Campos et al. (2003). Different fractions are reported showing a lower solid fat content (SFC) profile in the distillate and a higher SFC in the retentate, relative to native milk fat.

10.2.3 Supercritical extraction Supercritical fluids, mainly carbon dioxide, under pressure (100–400 bars) and near room temperature (30–60 °C) behave as very selective non-polar solvents in which the separation of triacylglycerols is based on their solubility in the supercritical fluid. This technique was of interest for the fractionation of milk fat (Bhaskar et al., 1998), but its very high cost and complexity made it unsuitable for industrial application. The technique is, nevertheless, suitable for refining some specific oils, de-oiling and fractionating lecithins, purifying polyunsaturated fatty acids, concentrating acylglycerols and extracting vitamins from vegetable oils (Perrut, 1999; Brunner, 2000; Barth, 2004; Linder et al., 2004).

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10.2.4 Liquid–liquid extraction Liquid–liquid extraction is used in the pharmaceutical industry, but it has no applications in the fats and oils industry. The principles are based on differences in solubility of components to be separated between two immiscible solvents.

10.2.5 Adsorption Adsorption on solid followed by several extractions with solvents is able to fractionate triacylglycerol mixtures, but the development of this technology in the oils and fats industry is very slow.

10.2.6 Complexation Urea complexation is a simple method for fractionating fatty acids from various seed (Hayes et al., 1998), fish (Gamez-Meza et al., 2003) and other oils (Robles-Medina et al., 1995; Guil-Guerrero and Belarbi, 2001). It is easy to scale up and considered to be ecologically friendly, despite large volumes of solvent and aqueous waste. In highly polyunsaturated systems, the goal is mainly to increase EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid) concentration. The method cannot be applied to triacylglycerols.

10.2.7 Membrane separation A patent has been taken out by Parmentier et al. (1993) describing the possibility of using membrane separation to fractionate anhydrous fats. Two separation mechanisms are claimed. At a temperature not too far from their solidification point, fats can present lamellar structures stable within the liquid. Such ‘micellar’ structures make the filtration possible as they artificially increase the mass dispersion of aggregates and improve filterability. The second mechanism refers to specific molecular interactions between fat and membrane; ‘tuning-fork’ and ‘trident’ conformations are described for the fat, depending on the relative lipophilicity between membrane and triacylglycerol components (Parmentier et al., 2003). First attempts have been carried out (Bornaz et al., 1995a) on anhydrous milk fat using stainless steel membranes. Further developments with different kinds of membranes (hydrophobic and lipophilic) have followed (El’Amac et al., 2000). The results show good and stable divergence between permeate and retentate relative to milk fat. Industrial scale-up has been tested with moderate success.

10.2.8 Fractional crystallization The fractional crystallization process is mainly based on the ability of fats to produce crystals. On an industrial scale, crystals can be obtained according

204 Modifying lipids for use in food to three main technologies: detergent fractionation, solvent fractionation and dry fractionation. In 1905, Lanza patented the adding of a detergent to ‘wet’ the crystals which are consequently transferred in the aqueous phase; the mixture is then easily separated by centrifugation. This process is known as the Lipofrac concept. Detergent fractionation is based on aqueous crystallization using surfactant and electrolyte followed by centrifugal separation. The surfactant (usually sodium lauryl sulphate) and the electrolyte (usually magnesium sulphate) allow crystals to be easily suspended in the aqueous phase; the purpose is to facilitate the agglomeration of oil droplets formed during the mixing process. After centrifugation, the light liquid oil is separated from the heavy water phase containing crystals; this heavy phase is heated and melted solid phase is recovered in a second centrifuge. Interest has diminished because of negative aspects such as the high costs of the chemicals, effluent problems and contamination in the end products. In solvent fractionation, the fat is dissolved in a solvent like acetone or hexane (other solvents such as isopropyl alcohol can also be used) and the dilute solution is cooled to initiate the crystallization of the highest melting triacylglycerols. Crystals are consequently separated by filtration and the fractions are recovered by solvent evaporation. Solvent fractionation is interesting because of high separation efficiency and high purity of the finished products. In diluted conditions, the presence of solvent hinders liquid oil occlusion in the solid phase. On the other hand, a high degree of crystallization can be obtained in one single operation. Hexane is used when a high quality liquid phase is desired as diacylglycerols responsible for the cloudiness of this phase concentrate in the solid phase. When the goal is to produce POPrich fractions (P is palmitic acid and O is oleic acid), acetone is preferred (clear separation and low diacylglycerol content in the solid phase) (Timms, 1983). The process with isopropyl alcohol is quite different: when the temperature is lowered, there is a separation between the alcohol and the fat, and crystals grow in the solvent (Koslowski, 1972; Hani, 1999). Alternatively, a solvent can be added to partially crystallized oil thereby improving the separation between the two fractions. This becomes very close to the detergent fractionation concept (Ong et al., 1983). The investment required in a solvent fractionation plant is unfortunately high: it has to handle large volumes of solvents and has to be explosion proof; operating costs are also high because cooling to low temperatures and solvent evaporation require high energy consumption. Today, most of the plants still in operation produce speciality products such as cocoa butter replacement fats; the production of palm midfractions and shea stearin are good examples. Dry fractionation is the simplest and cheapest fractional crystallization process, well known as ‘natural’ or ‘green’ technology (no effluent, no chemicals, no losses). In contrast to detergent or solvent fractionation, dry fractionation does not require any additional substance. It simply consists of a controlled crystallization of the melted oil, conducted according to a specific

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cooling program followed by separation of solid from liquid fraction (Tirtiaux, 1989; Kellens, 1998; Deffense, 2000; Gibon and Tirtiaux, 2002). Since the liquid phase is not diluted as in the solvent fractionation process, dry fractionation tends to be less selective, and increasing the selectivity of the dry fractionation operation is a necessity. The use of membrane filter presses to squeeze out as much liquid occlusion as possible is one way; combining proper crystal development with a highly efficient separation process is state-of-the-art. Due to continuous developments taking place in the dry fractionation process, a whole variety of products normally produced by solvent fractionation can now be obtained with a high degree of selectivity with dry fractionation. As the crystallization operates in the bulk, viscosity problems limit the degree of crystallization in one step and multi-step operations are currently used, giving rise to a wide range of fractions suitable for different applications. Besides solvent and detergent fractionation, palmkernel oil is also largely fractionated using a special dry process called ‘panning and pressing’ (Rossell, 1985; Krishnamurthy and Kellens, 1996). This oil is poured into pans and stored in cold rooms to crystallize. The cakes are wrapped in filter clothes and stacked between plates in hydraulic presses operating at very high pressure (more than 100 bars). Good quality palmkernel stearins can be obtained in a reasonable yield. Although the fact that no solvent or detergent is required reduces the capital cost, this process is highly labour-intensive. The introduction of continuous belts instead of the pans and of an automated high-pressure membrane filter-press instead of the hydraulic system has considerably reduced the manual operations with comparable process results. The advantages and disadvantages of each processing technique (solvent, detergent and ‘panning and pressing’) applied to palm kernel oil have been fully discussed by Wong Soon (1987). Recently, a new process has been developed resulting in an overall higher yield of good quality palmkernel stearin from which a major part could be used as CBS without additional hydrogenation (Hendrix and Kellens, 2001; Calliauw et al., 2005).

10.2.9 Winterization While winterization and fractionation are based on the same principles and are similar processes, nevertheless industry considers them subtly different because winterization focuses only on producing liquid oil with improved cold-tolerance while fractionation concentrates on separating and recovering any desired oil portion for any of several needs. Winterization is a process whereby high-melting triacylglycerols are crystallized and removed from the oil by filtration to avoid clouding of the liquid fraction at cold temperatures. The term winterization was originally applied when cottonseed oil was simply subjected to winter temperatures to perform this process (Jones, 1990). Dewaxing is a similar process used to clarify oils containing small amounts of clouding constituents or waxes (Bloch, 1998; Strecker et al., 1990).

206 Modifying lipids for use in food

10.3 Crystallization of fats One of the most fascinating properties of oils and fats is their ability to form crystals. Although information about crystal structures of fats and complex lipids is fundamental for various fields, the structural analysis of those compounds often reveals difficulty in preparing single crystals suitable for X-ray diffraction analysis and for full three-dimensional determination (Larsson, 1964; Jensen and Mabis, 1966; Doyne and Gordon, 1968; Gibon et al., 1984; Culot et al., 2000; Sato et al., 2001a; Van Langevelde et al., 2000). 10.3.1 Polymorphism As described in other chapters, fats are generally considered to have three basic crystal forms also called polymorphs, α, β′ and β forms, in order of increasing melting point and thermodynamic stability. Additional forms (or sub-forms) are also claimed (γ, pseudo-β′, β1, β2 …) especially in the case of cocoa butter components (Hageman, 1998). Crystal modifications can be classified according to their powder X-ray diffraction pattern. The lateral packing between acyl groups gives rise to diffraction peaks: one single peak is observed for the α form (hexagonal packing), two peaks are detected for the β′ form (orthorhombic perpendicular packing) and mainly three for the β form (triclinic parallel packing). Longitudinal organization between layers is indicated by a suffix: double (L-2), triple (L-3), quadruple (L-4) and eventually sextuple (L-6) chain-length arrangements are reported. The α form is obtained after rapid cooling of the fat; transitions to β′ and β are possible on heating (these transitions are irreversible). Crystallization of the β′ form is possible by slow cooling above the melting point of the α form. Very slow cooling above the melting point of the β′ form will induce crystallization in the β form. Crystallization from solvent in diluted conditions usually also gives rise to solidification usually in the β form. 10.3.2 Intersolubility Because of closely linked structural properties, triacylglycerols can produce co-crystals by intersolubility; they most frequently show solid solutions, monotectic interactions, eutectic systems, molecular compounds, etc. Binary systems have been extensively studied (Rossell, 1967; Gibon, 1984; Desmedt et al., 1990; Wesdorp, 1990; Ollivon, 1992; Sato, 2001). The case of edible oils and fats is more complex: they are made of numerous triacylglycerols that have very similar chemical structure but variable chain-lengths, degree of unsaturation and positional isomers. Depending on their chemical structure or their polymorphic form, some triacylglycerols will be very soluble when mixed and form solid solutions; others will crystallize separately being immiscible in the solid state and giving rise to monotectic or eutectic interactions. A representation in terms of phase diagrams of binary mixtures of triacylglycerols is of great help in understanding the intersolubility properties. The cases of PPP/PStP and PPP/POO binary systems (Fig. 10.1) have been

Fractionation of lipids for use in food

207

selected as reference situations (Gibon, 1984). PPP and PStP are relatively similar (St is stearic acid): the difference is only two carbons on a single acyl group. PPP and POO differ by two acyl groups differing in chain-length and in the presence of a cis unsaturated centre. The most stable polymorphic form of PPP is β-2, although PStP stabilizes in β 1′ -2. PPP and PStP form solid solutions over a large part of the binary phase diagram. They are very miscible in their most stable form ( β 1′ ) as well in the unstable forms (α and β ′2). There is a large melting point difference between PPP and POO. When mixed, they form eutectic interaction as far as the most stable polymorphic form is considered (β); they are not miscible. Nevertheless, in their unstable polymorphic forms (sub-α, α and β′), PPP and POO are very soluble and crystallize as solid solutions. Molecular structure PPP and PStP miscible in β′-2 (the most stable form) Solid solution

PPP and POO: not miscible in β-2 (the most stable form) Monotectic interaction

L

L

β-2 + L

β1′ -2

50

50

β 2′ -2

T (°C)

T (°C)

α-2

0

β-2 + L

β′-2

0

α-2

sub-α-2 PPP

0.50

PStP

PPP

PPP and PStP: miscible in unstable polymorphic forms

0.50

POO

PPP and POO: miscible in unstable polymorphic forms Polymorphism

Fig. 10.1 Binary phase diagrams (temperature/composition) of PPP/PStP and PPP/ POO (Gibon, 1984). Binary phase diagrams have been established by mixing and melting pure triacylglycerols. The samples are afterwards quenched at –40 °C and heated at constant rate (5 °C/min): transition (squares) and melting (circles) peaks are detected by differential scanning calorimetry. Powder X-ray diffraction is used in similar thermal conditions to determine the polymorphic behaviour.

According to those data, it can be stated that: (i) similar triacylglycerols will tend to make co-crystals, (ii) rapid cooling that induces crystallization in an unstable form (mainly α) will favour co-crystallization of even very different molecular structures. A slow cooling is always required to ensure the best selectivity of the crystallization.

208 Modifying lipids for use in food 10.3.3 Palm oil The case of palm oil is interesting (Smith, 2001). This oil is rich in PPP, POP and OOO; the consequence is the formation of an eutectic made of two solid solution phases. The differential scanning calorimetry thermogram of palm oil clearly indicates the presence of two endothermic peaks in the range of –20–15 °C and 18–40 °C, respectively (Fig. 10.2). The higher melting peak roughly corresponds to PPP and POP (SSS and SUS components – S is saturated fatty acid, U is unsaturated fatty acid) while the lower is mainly made from POO and OOO (SUU and UUU components). Both PPP and POP are stable in β form (β-2 and β-3, respectively), although OOO stabilises in β-2. Nevertheless, due to phase interactions, palm oil crystals will solidify in β′ during the cooling phase of a classical dry fractionation process. Only very careful cooling will induce β crystallization.

10.4 The dry fractionation process The dry fractionation process consists of two steps: the crystallization stage that produces solid crystals in a liquid matrix and the separation stage where the liquid phase is separated from the crystals.

0.2

Heat flow (mW)

0.0

– 0.2

– 0.4

– 0.6 –40

–20

0 20 Temperature (°C)

40

60

Fig. 10.2 Differential scanning calorimetry thermogram of palm oil. After melting, the fat is quenched at –40 °C and heated at constant rate (5 °C/min.).

Fractionation of lipids for use in food

209

10.4.1 Crystallization stage The fat is melted in order to destroy the memory effect (Van Maalsen et al., 1996): presence of seeds or lamellar structures within the melted state could affect the process and its repeatability. Classically, a temperature of some degrees above the melting point of the highest melting triacylglycerol is recommended; in most cases, a temperature of 30 °C above the melting point of the fat is satisfactory. Crystallization is only possible when the concentration of triacylglycerols reaches the saturation (or normal solubility) curve (Timms, 1997). Below the solubility temperature, the fat enters into metastable conditions: crystallization does not occur spontaneously. At still lower temperatures, the fat comes into unstable conditions and crystallization is immediate. Crystal nuclei will be formed when the energy due to heat of crystallization exceeds the surface energy; they are formed in metastable conditions, i.e. when the temperature has been decreased below the solubility limit required to get small crystals (supercooling conditions). Excessive supercooling conditions give rise to large variations in solubility and to nuclei of smaller critical size (minimal size to be stable). As seen above, a fat is characterized by several polymorphs (mainly α, β′ and β) each having different stabilities and solubilities. The competition between heat of crystallization and surface energy of the polymorphs leads consequently to different nucleation rates (Boistelle, 1998). This phenomenon explains why rapid cooling leads to unstable forms (α or β′), even if the most stable form is thermodynamically favoured because of its lower solubility. Secondary nucleation can occur if small pieces of nuclei are removed from the growing crystals due to an external cause such as agitation. If the critical size is obtained, those nuclei become stable and grow independently from the first nuclei. Crystal growth is permitted by diffusion of surrounding molecules that will settle on the formed nuclei. It depends on the degree of supercooling and is inversely proportional to viscosity. Moderate supercooling is required for perfect crystallization. In excessive supercooling, molecules will not have sufficient time to attach in good conditions giving rise to dislocations in the crystal lattice and to formation of co-crystals, possibly made of very different triacylglycerols: the result is a loss of selectivity of the crystallization. When crystallization is pushed too far, high viscosity will affect the rate of diffusion and crystal growth is hindered. As in the case of nucleation, polymorphs are characterized by different growth rates: the most stable form will grow more rapidly (Van Putte and Bakker, 1987). Local increase of temperature due to heat of crystallization is unwanted; in such conditions, the nucleation/growth sequence is perturbed leading to imperfect crystals and loss in selectivity. If the increase in temperature during crystallization is not controlled, it can give rise to re-dissolution of part of the already formed crystals, and lamellar structures could occur. In the worst cases, the result is the formation of a milky mass of micro-particles of all sizes, which will be very difficult to separate (Gibon and Tirtiaux, 2000a).

210 Modifying lipids for use in food

Oil cooling curve based on fixed water cooling profile Toil = f (Twater)

Oil cooling curve based on oil temperature dependent water profile Twater = f (Toil)

Temperature

Temperature

Perfect control of heat removal and agitation speed during the crystallization cycle is necessary to avoid this unwanted situation. Practically, nucleation and growth occur simultaneously. The rate of nucleation increases more rapidly than the rate of growth, essentially in excessive supercooling conditions. The result is the formation of very small and imperfect crystals which are very difficult to separate. In consequence, industrial crystallization plants must be designed to operate at slow cooling rates during the crystallization cycle. Several cooling modes (Fig. 10.3) are possible that will mainly depend on the supplier of the dry fractionation technology (Krishnamurthy and Kellens, 1996). The oil cooling curve can be based on fixed water cooling profile; in this case, the temperature of the oil will depend on the temperature of the water. Other designs are based on ∆-T principles; in those cases, the temperature of the water is regulated by the temperature of the oil itself (Tirtiaux, 1968). Those differences in the cooling scheme result in technological variations essentially expressed in the geometry of the cooling surfaces and in the type of agitation (Fig. 10.4). Some suppliers operate according to the ∆-T concept, with specially designed propeller agitation and a large cooling heat transfer area of the fin type. Concentric crystallizers provided by other suppliers are based on the fixed water concept; concentric cooling surfaces are combined with sweeping surface agitation. Weber et al. (1998) present a crystallizing system principally made of eccentric moving cooling coil bundles without any extra agitator. In any case, crystallization cycles are affected by the technology used, keeping in mind that slow cooling time (where the crystallization takes place in conditions close to equilibrium) is always recommended for the best control of the operation. However, it must be stated that cooling surfaces alone do not determine the crystallization times; some oils are characterized

Oil ∆T1 Water

∆T2

∆T3

Time (a)

∆T4

Oil

T1 T2

∆T5

T3

Water

T4

Time (b)

Fig. 10.3 Schematic representation of (a) an oil temperature related profile and (b) an independent water cooling profile, in a dry fractionation operation (Krishnamurthy and Kellens, 1996). ∆T1 to ∆T5: fixed difference between temperature of oil and temperature of water. T1 to T4: fixed temperature of water.

Fractionation of lipids for use in food

(a)

(b)

211

(c)

Fig. 10.4 Technical variations of dry fractionation equipments (crystallization stage) (Weber et al., 1998): (a) fins at the walls and propeller agitation; (b) concentric jackets and sweeping surface agitation; (c) stirring cooling surfaces.

by slower crystallization rates (polyunsaturated oils), whereas others solidify more rapidly (saturated and/or hydrogenated oils). In the first case, stirring is often not sufficient to induce nucleation: seeding is a necessary option to start the crystallization.

10.4.2 Separation stage As seen before, the efficiency of dry fractionation is greatly determined by the quality of the crystallization: the best separator fed with bad crystals may become a nightmare. The method of cooling is of great importance in controlling the nucleation and growth; a perfect control will make the crystallization more selective in terms of co-crystals. Nevertheless, in some operating conditions, growing crystals can agglomerate, trapping liquid within the solid particles; this phenomenon becomes critical in high-viscosity systems. In those cases, removing this liquid phase requires more sophisticated separation systems. The goal of the separation is to produce a solid (stearin) and a liquid (olein) phase, each having its proper physico-chemical characteristics and its particular applications. At the end of the crystallization process, triacylglycerols are distributed in three locations: (i) as solids in the form of co-crystals, (ii) as liquids (non-crystallized oil), and (iii) as liquids physically trapped on the surface of the crystals. Based on this, different types of separation equipment are available, depending on the efficiency of the separation required. Vacuum filters will leave the crystals coated with some trapped olein. Two types of vacuum filters are in use: rotary drums and belt filters which operate in two stages. The first stage consists of the separation of the crystals from the mother oil and the second stage permits a ‘drying’ of the cake by sucking under vacuum in order to reduce entrained liquid oil. Such filters are still in operation in fractionation plants when the market favours soft stearins.

212 Modifying lipids for use in food Indeed, it is sometimes more economical to leave a good proportion of olein in the stearin cake to produce some shortenings such as the South American ‘manteca’, vegetable ghee or vanaspati. Nowadays, many users of fractionation plants favour the automatic filterpress, fitted with airtight membranes (Fig. 10.5). Although it does not have the benefit of continuous operation of vacuum filters, its advantage lies mainly in the higher percentage of liquid it yields, by applying a pressure to the cake during each filtration cycle. The result is to expel the liquid physically trapped on the crystals. The filtration is made through a sequence of mechanical operations, each carefully controlled and monitored via a computer. During the filling and filtration, the important point is to make sure all chambers are evenly filled with crystals. The squeezing follows by applying a pressure behind each membrane by means of a gas or a liquid. The third operation takes care of draining the feed core as well as the olein channels. Finally, the filter is opened and carefully controlled for an orderly discharge of the stearin cakes into a melting hopper. Once a day, the filter is washed by a flow of hot olein to melt away any stearin traces smeared on the filter cloths. If the crystallization is conducted carefully, the quantity of olein physically trapped on the crystals is relatively low and squeezing pressures of 5–10 bars (pneumatic design) are generally satisfactory. Higher pressures, up to 30 bars (hydraulic design), become indispensable for the separation of highb

a

Air Olein

Olein

Feed

Olein

Air

Air

Olein

Fig. 10.5 Automatic filter-press fitted with airtight membranes used in dry fractionation (separation stage). Schematic view of membranes: a: in filling and filtration step; b: in squeezing step + draining.

Fractionation of lipids for use in food

213

viscosity crystallized fats; this is generally the case for the production of special products such as POP-rich fractions. Increase of squeezing pressure corresponds to a stronger design of the filter frame and plates. The olein is normally not affected by the filtration pressure, unless stearin passes through the filter cloths. This could eventually arise if the size and the thickness of the crystals are not adequate; imperfect crystallization may be the reason. However, some oils produce weak crystals, whatever the care taken during the crystallization phase. Crystals break down and fluidize during the squeezing sequence: in this case, low squeezing pressure is recommended. An alternative to vacuum and to membrane filter-press systems is the use of centrifugal separators. Westfalia (Wieking and Dolle, 1998) patented a special nozzle centrifuge and its use in the dry fractionation process (Fig. 10.6). A nozzle separator operates in clarification mode for separation and concentration of solids out of the liquid phase. The operation is fully continuous due to the discharge of the concentrate through nozzles at the periphery of the rotating bowls. This system has the advantage of being compact and is especially valuable when space is limited. Unstable fats subject to polymerization in open devices can be easily processed. Additionally, the special hygienic construction of bowl, sludge catcher and separator frame is used to achieve optimum cleaning in place (CIP). Applied on palm oil Feed Olein Cold/hot water Disc stack

Centripetal pump Segment-holding insert

Nozzle Sludge catcher

Cold/hot water frame discharge Pressurized air seal

CIP

Stearin

Horizontal bowl section

Fig. 10.6

Westfalia nozzle centrifuge for separation of stearin and olein in dry fractionation (CIP: cleaning in place) (Wilp, 2001).

214 Modifying lipids for use in food (first fractionation step), the yield in olein is claimed to be between those obtained by the vacuum filter and the membrane filter-press procedures (Wilp, 2001).

10.5

Applications

10.5.1 Winterization Winterization is the term used when the goal is to remove from oils small quantities of solids that normally cause cloudiness when kept at refrigeration temperature. These substances can be waxes or saturated triacylglycerols. The principle is close to the one used for dry fractionation: the oil is cooled slowly in crystallizer tanks and kept at low temperature (below 10 °C) for long time periods (> 12 hours), to allow waxes or saturated triacylglycerols to crystallize. Filter aids can be added during crystallization or before filtration to permit easier filtration and reduce oil losses. Sunflower and corn oils For sunflower or corn oils, winterization is used to reduce the wax content to below 50 ppm and ensure a cold test of at least 5.5 hours at 0 °C; in many cases, a cold test of 24 or even 48 hours minimum at 0 °C can be required. This is normally made according to the principle described above. An improvement of the SOFT® degumming process, initially used to reduce the phospholipids content in physical refining, simultaneously permits removal of the waxes leading to a cold test of 48 hours minimum at 0 °C (Gibon and Tirtiaux, 2000b). The principle of the SOFT® degumming/dewaxing process is to cool (below 10 °C) an emulsion made from oil and water (containing complexing substance and detergent) in a crystallizer tank and maintain it for several hours under smooth agitation; the whole is slightly heated for viscosity reasons and subsequently centrifuged. Cottonseed oil (Table 10.1) Historically, winterization has always been associated with cottonseed oil. During winter months, cottonseed oil stored in large tanks would cloud as the higher melting triacylglycerol components settled out to the bottom of the tanks. The goal here is to remove saturated triacylglycerols in order to make the oil suitable for salad, mayonnaise and salad dressing applications. Properly winterized cottonseed oil will pass a cold test of at least 10 hours at 0 °C. The stearin is often blended with non-winterized cottonseed oil for frying applications or is hydrogenated for use in shortenings, puddings, cocoa butter replacers, icings and other food uses; the higher palmitic content of the stearin assists proper crystal formation in shortenings and icings (Jones and King, 1993). In some applications, cottonseed oil can be slightly hydrogenated

Table 10.1

Characteristics of some winterized oils and fractions.

Hydrogenated cottonseed oil

Feed oil

Olein

Stearin

IV

IV

IV

100

– a

–

107

9c a

–

87

Yield (% olein)

Stepl single

–

32b

65k

b

82

j

Cottonseed oil

109

–5

–

115

–8

–

80

–

22

Virgin olive oil (Stearin)

78 74

– –

8b 19b

82 77

– –

4b 9b

74 63

– –

17b 25b

86j 77j

single double

115 124 111 114

– – – –

31b 28b 27b –

129 135 114 117

–2a –4a – –

– – – –

84 105 105 107

– – – –

37b 34b 32b 26b

70j 65j 70k 50k

single single single double

198 146 172 163 214

2a 5a 12d 12d –

– – 13e 10e –

208 156 – 226

–8a –5a 13d 14d 3.2f

160 119 – – 182

14a 19a 9d 9d –

– – 11e 8e –

80j 70j 77j 72j 65j

single single single single single

105 110 84 77 115 75

h

b

108 112 – – 119 84

7c 14c 0.9i 0.4i –11a –5a

91 102 – – 98 68

19h 12h 0.6i 0.4i – –

30b 25b 32b 36b 34b 37b

80k 83k 68k 72k 81j 44j

single single single single single single

Hydrogenated fish oil (Olein) Crude fish oil

Hydrogenated soybean oil

a

Cloud point (°C) Dropping point (°C) c Cold test at 0°C (hours) d EPA content (%) e DHA content (%) f Increase in EPA + DHA content (%) b

17 11h 0.8i 0.4i 2a 21a

18 14b 24b 31b – –

– – 15e 12e –3.5g 15h 10h 16b 27b > 24c < 2c g

Decrease in saturated content (%) Trans content (%) i 18:3 content (%) j Filtration on membrane filter press k Filtration on vacuum belt l Fractionated in a single or double step h

Abbreviation: IV = iodine value Sources: Tirtiaux et al., (1984), Tirtiaux (1990), Krishnamurthy and Kellens (1996), Kellens and Hendrix (2000), Gibon (2001).

single

216 Modifying lipids for use in food to IV 100; the result is formation of crystals of better quality that can be more easily filtered. Olive oil (Table 10.1) Virgin olive oil is obtained from olives by mechanical or other physical means only. The IOOC (International Olive Oil Council) and EC (European Community) have developed standard definitions for olive-based oils: extra virgin, virgin, ordinary and lampante virgin olive oils; pommace oil is solventextracted from mechanical extraction residue (Mordret et al., 1997). The best quality extra virgin olive oil is characterized by good sensorial evaluation, low acidity (1 % max) and low content of minor components like waxes and free alcohols. Olive oil is the richest traditional source of monounsaturated oleic acid which contributes to its health benefits. Under normal transport conditions, olive oil is liquid; however, if temperatures fall within the solidification range, the oil has to be heated during transportation to achieve pumpability and reduce oil losses; however, long heating is not recommended, mainly for oxidative stability reasons. Winterization of olive oil prior to transportation is a means of reducing the problem, especially in the case of low iodine value products; crystallization of extra virgin olive oil can be performed below 5 °C in order to remove the most saturated fraction and improve its cold stability. The stearin, enriched in palmitic and stearic components, can be re-fractionated into a harder stearin; both fractions have potential applications in the margarine industry. Fish oil (Table 10.1) Fish oil has been used in food from time immemorial; rapid growth in world aquaculture has also created a market for fish oil in fish feed (Opstveldt et al., 1990). As a partially hydrogenated oil, it has long been a fat source for margarine production. Lightly hydrogenated and winterized fish oil for use in salad oils has a market only in certain South American countries. Practically, fish oil is hydrogenated to improve its oxidative stability and retard rancidity. Depending on its origin and on the hydrogenation conditions, fish oil is classically hydrogenated in the range IV 115–125 and consequently winterized. However, to eliminate all risks of fishy odours, it is sometimes recommended to start with lower IV (around 110) and to operate in a double-stage winterization process. Unhydrogenated fish oil is now considered to have health benefits with positive impact on heart, brain and normal growth and development. Fish oil can be used for dietary enrichment and pharmaceutical use, as natural oils or as EPA-DHA concentrates (in the form of fatty acids or esters). Nutritional complements made from neutralized, refined and selectively winterized fish oil are enriched in EPA/DHA up to 30–40 % concentrations. Winterized fish oil can be incorporated into a series of food products, such as salad oil dressings, spreads or table margarines. When the process is applied to triacylglycerols, increase in omega-3 acids

Fractionation of lipids for use in food

217

resulting from the winterization process of fish oil is lower than the levels obtained in concentrates. This increase is generally limited to 3–4 %, depending on the starting oil. Greater enrichments are reported only when the winterization is performed in presence of solvents at very low temperature (Lee and Foglia 2001). Soybean oil (Table 10.1) Soybean oil is a natural ‘winter salad oil’, but it has relatively short flavour stability because of its high degree of polyunsaturation (about 43–56 % linoleic acid and 5–11 % linolenic acid) (O’Brien, 1995). Partial hydrogenation significantly reduces the incidence of offensive odour and flavour development by decreasing levels of the linoleic and linolenic, but the process creates higher-melting disaturated triacylglycerols and trans isomers, responsible for cloudiness. Winterization of the partially hydrogenated soybean oil makes it possible to meet the requirements of a salad oil or a high-stability clear oil (liquid oils with active oxygen method (AOM) stabilities of 350 hours are available commercially). For such application, hydrogenation is generally set for an iodine value above 100, and the linolenic content is reduced to around 3 %. It is important to note that the production of trans isomers is strongly related to the performances of the hydrogenation process, leading to fractionated oleins of different grades in terms of cold test stabilities (Krishnamurthy and Kellens, 1996). For cooking oil applications, hydrogenation is continued until the IV is below 90 to reduce the linolenic content to very low levels (with strong improvement of oxidative stability). Melting point is significantly increased making this oil unsuitable for liquid applications. Different feedstocks with different IV and fatty acid compositions can be processed in dry fractionation. The stearins can be used for shortening and margarine applications; depending on the quality, the corresponding oleins are suitable either for cooking or as semi-liquid shortening.

10.5.2 Dry fractionation One of the main differences between winterization and dry fractionation processes is the distinct change in physico-chemical properties of both the liquid and solid fractions produced by fractionation. The quantity of solids produced in the crystallization stage is generally higher which makes the control of crystallization more critical; multi-stage dry fractionation processes are often possible, and that makes the range of applications very large. Palm oil Palm oil is without doubt the most widely fractionated oil. Multi-step dry fractionation (Fig. 10.7) gives rise to soft fractions (oleins) that are used as cooking and salad oils, and to harder fractions (stearins and mid-fractions) finding applications in frying fats, margarines, shortenings and speciality

218 Modifying lipids for use in food Super stearin IV 17–21 Hard stearin IV 32–36 Soft stearin IV 40–42 Palm oil

Hard PMF

IV 51–53

IV 32–36 Soft PMF

CBE

IV 42–48 Olein IV 56–59

Recycling

Oleins

Super olein IV 64–66 Top olein IV 70–73 Step 1

Fig. 10.7

Step 2

Step 3

Multi-step dry fractionation of palm oil (olein route for the production of hard PMF) (IV = iodine value); CBE = cocoa butter equivalent).

fats. Fractionation is classically carried out on the refined oil, but the high vitamin content of crude oil makes the dry fractionation process an attractive route for the non-refined oil; as a matter of fact, carotenes, tocopherols and tocotrienols concentrate markedly in the liquid fractions. The single-stage fractionation process of palm oil is classically carried out to produce an olein still liquid below 24 °C; the stearin has a melting point ranging between 45 and 53 °C. Since 1990, olein has become a cheap primary commodity because it is formed in high proportions in single-stage fractionation and carries a low profit margin (especially in Malaysia). For this reason, attention has been focused on multiple-step dry fractionation to manufacture high added-value products, such as super oleins or top oleins and palm mid-fractions (PMF). Starting with IV 51–53 palm oil, IV 56–59 oleins are the targets of the single stage. In many satisfactory cases, IV 60–63 super oleins can also be produced in one single step. Higher IV (64–66) is obtained by re-fractionating the first olein; this route leads also to the production of soft PMF. When climate requires more specific oleins, IV 70–73 top oleins can be successfully produced from super oleins in a third stage fractionation process.

Fractionation of lipids for use in food

219

Palm oil is principally made up of trisaturated triacyglycerols SSS (8– 10 %, mainly PPP), disaturated triacylglycerols SUS (44–48 %, mainly POP and PLP), monosaturated triacylglycerols SUU (about 38–42 %, mainly POO and PLO) and unsaturated triacylglycerols UUU (about 6–8 %, mainly OOO and OOL) (L is linoleic acid). The goal of the first fractionation step is to reduce the SSS content of the olein as low as possible. When the crystallization is operated properly, this content falls to zero in favour of higher levels of SUU–UUU, while SUS content remains unchanged. If this first stage is not conducted carefully, oleins of IV 56–58 will contain traces of SSS that will considerably affect their cloud point. In good situations, i.e. when SSS is totally removed in the first step, SUS begins to decrease in the second step, leading to high IV super and top oleins. In consequence, considerable reduction in cloud point is observed (Fig. 10.8). During the two-stage process, SSS concentrates mainly in the hard stearin. This benefits the soft PMF that is considerably reduced in trisaturated triacylglycerols and consequently suitable for further fractionation. As seen before, palm oil is quite rich in POP, which after concentration is a very suitable source for cocoa butter equivalents (CBE). The physicochemical characteristics of CBE are relatively close to those of cocoa butter (CB) with regard to crystallization, texture and melting properties. This is due to similar triacylglycerol composition (SUS components) that makes CBE fully compatible with CB and in consequence usable in all types of applications to replace partially or totally the CB. Classically, two routes are described for producing the PMF: the olein route (more commonly followed in Asia) (Fig. 10.7) and the stearin route which is preferably used in South America, because of the need for high IV olein in the first fractionation step (Fig. 10.9). The best CBE are obtained by the olein route. Attempts have been made to explain this in terms of the POP/ 76 IV 74

Top oleins from third step

72 70 68

Super oleins from second step

66 64

(Super) oleins from first step

62 60 58 56 –4

Fig. 10.8

–2

0

2

4 6 8 Cloud point (°C)

10

12

14

Iodine value (IV) versus cloud point of liquid fractions in a multi-step dry fractionation process of palm oil.

220 Modifying lipids for use in food Hard stearin IV 22 Stearin

Hard PMF IV 36

IV 40 Soft PMF IV 47

CBE

Palm oil

Olein

IV 52

IV 54

Super olein IV 62 Step 1

Fig. 10.9

Step 2

Step 3

Stearin route for the production of hard PMF (Kellens, 2000) (IV = iodine value, CBE = cocoa butter equivalent).

OPP ratio in the final products and of the presence of traces of PPP not removed by the stearin route. In the olein route, SUS concentrate in the soft PMF at the second fractionation stage. The SUS enrichment can reach 72–73 % corresponding to an SUS/SUU ratio of 3–4, and a low SSS content. Re-fractionation of this soft PMF produces an excellent hard PMF made of 85–90 % SUS. This has SUS/SUU ranges between 9 and 12, less than a few percent of SSS and diacylglycerol content sufficiently low to avoid any adverse effect on the crystallization properties of the fraction (Deffense, 1995). One of the main characteristics of palm oil is its red colour due to its particularly high β-carotene content. Chemical neutralization followed by deodorizing at low temperature is able to produce a refined palm red oil still rich in carotenes, tocopherols and tocotrienols. Such specially refined oil can be fractionated according to the classical scheme (olein route), giving solid and liquid red fractions. The pigment concentration in the liquid fractions is particularly important: β-carotene in the top olein issued from the triple fractionation is nearly twice the content in the neutralized oil, giving this fraction very high oxidative and cold stabilities and permitting positive claims on the nutritional label (Table 10.2). Products such as Carotino® put on the market by Carotino Sdn. Bhd. or Nutrolein 64 produced by Aarhus Inc. in Malaysia are super oleins of high carotene and tocopherol/tocotrienol specifications. Those red super oleins are claimed to contain no less than 600 ppm of carotene and 800 ppm of vitamin E. They are used as high vitamin salad oils with high cold and oxidative stabilities or as frying oils in ‘low’ temperature applications; those fractions can benefit from an additional nutritional claim, due to their beneficial effect on cellular ageing and to their role as cancer preventer. The red stearins as well as red palm mid-fractions find applications in the production of margarines, shortenings and CBE rich in vitamins for dietetic use.

Fractionation of lipids for use in food

221

Table 10.2 Concentration of β-carotene in the fractions produced by multi step dry fractionation of chemically refined red palm oil (olein route). Neutralized and bleached red

β-carotene (ppm)

Palm Palm Palm Palm Palm Palm Palm

382 409 670 854 280 235 80

oil olein super olein top olein hard stearin soft PMF hard PMF

Source: Gibon et al. (2002).

Table 10.3

Physico-chemical properties of palm kernel oil and stearin fractions.

Fatty acid (%) 6:0–10:0 12:0 14:0 16:0 18:0 18:1 18:2 SFC (%)/°C 20 25 30 35

Palmkernel oil IV 17.5

Palmkernel stearin IV 9

Palm kernel stearin IV 7

Hydrogenated palmkernel stearin IV 0.4

7 48 16 9 2 15 2

6 53 21 9 2 8 1

4 55 22 9 2 7 1

4 55 22 9 9 0.5 0

44 20 0

70 48 8 0

82 68 29 0

95 90 50 5

Abbreviations: IV = iodine value, SFC = solid fat content. Source: Rossell (1985).

Palmkernel oil Palmkernel oil is fractionated to concentrate in the stearin the triacylglycerols containing medium-chain fatty acids (lauric and myristic). Concentration of triacylglycerols containing 12:0 and 14:0 into the stearin fraction to the detriment of short-chains (6:0–10:0) and of unsaturated C18 chains causes marked changes in the SFC profile (Table 10.3) (Rossell, 1985). Palmkernel stearin is an excellent cocoa butter substitute (CBS) generally used after hydrogenation (Tan et al., 1995). Its triacylglycerol composition makes it totally incompatible with cocoa butter. Palmkernel stearin is a material of choice in the manufacture of compounds for moulding and coatings. Quality is generally assessed in terms of high SCF levels at 20 and 25 °C and low levels at 35 °C, in order to ensure a good

222 Modifying lipids for use in food ‘snap’, good fingerprint resistance and good melting down. Hydrogenation of palmkernel stearin affects its melting point and SFC at 35 °C: 18:1 and 18:2 contents are lowered while the content of 18:0 is increased. The crystal structure of substitute chocolate manufactured with hydrogenated palmkernel stearin is finer than if it is made with the nonhydrogenated fraction. Chocolate products made with hydrogenated palmkernel stearin melt rapidly at mouth temperature and do not melt at finger temperature. Palmkernel olein is principally considered as a by-product. Nevertheless, few uses have been described, such as incorporation in the defatted meal for feed consumption, mixing with palmkernel oil for further hydrogenation, interesterification with palm stearins to produce shortenings, ice cream, biscuit filling creams, chocolate soft center, etc. (Rossell, 1985). Solvent fractionation can be used to produce CBS; the main advantage is the high yield of stearin with very low IV: 5–7; the main disadvantages are the capital and production costs as well as the risks and detriment to the environment. With the ‘panning and pressing’ method, the capital cost is lower; the yield and the stearin quality (IV: 6–8) are slightly inferior but still acceptable. The quality and yield of the stearins obtained by the detergent route are highly dependent on the specifications of the crude oil (acidity and IV) (Rossell, 1985). The dry fractionation route is still difficult due to high viscosity during crystallizing which makes the control of the crystallization difficult due to reduced heat exchange; transfer of the slurry to the filter is also not easy. These problems can be overcome either by blending the oil with olein (40–60 %) or by operating in multi-stage fractionation. The quality of the feed oil greatly affects the efficiency of the dry fractionation process; in addition, acceptable yields are only obtained after multi-stage (Wong Soon, 1987). As with fractionation technologies, static dry fractionation (Hendrix and Kellens, 2001) is a new, highly reliable technology for the consistent production of good quality palmkernel stearin for use as CBS. A new process route now permits the production of unhardened yet high quality CBS (Calliauw et al., 2005). Soybean oil Double-stage dry fractionation of hydrogenated soybean oil IV 77 (Fig. 10.10) is an excellent route for production of mid-fractions usable as feedstocks for cocoa butter replacers (CBR) (Kellens, 2000). The CBR fats are enriched in asymmetrical SUU (StEE, PEE, EPP, etc.) triacylglycerols principally made of trans isomers (E is elaidic acid) and are partially compatible with cocoa butter in the solid state; up to 25 % of CB is tolerated. Like CB, they posses a steep melting curve profile (SFC) but are characterized by rapid crystallization rates, making the product harder at low temperatures. Applications are wide-ranging, going from solid and filled chocolate to coatings with good gloss properties, long shelf life and high mechanical resistance. Unfortunately, the negative impact of trans fatty acids on health has led to the need for a marked reduction in the level of trans isomers in edible fats and, in consequence, alternatives must be found.

Fractionation of lipids for use in food

223

Stearin IV 61 MP 45 °C PH Soybean oil IV 77 MP 35 °C Mid stearin IV 73 MP 35 °C Olein

CBR

IV 81 MP 31 °C Mid olein

Step 1

IV 88 MP 18 °C Step 2

Fig. 10.10 Multi-step dry fractionation of hydrogenated soybean oil (IV) 77 for the production of CBR (Kellens, 2000). (IV = iodine value, MP = melting point, PH = partially hydrogenated, CBR: cocoa butter replacer).

Cocoa butter Unlike other fats, the composition of cocoa butter mainly consists of three symmetrical monounsaturated triacylglycerols: POP, POSt and StOSt. Cocoa butter greatly varies in hardness due to processing, origin, climatic and seasonal factors; variation is mainly sensitive in the SFC profile between 20 and 35 °C (Timms, 1999). Producing stearin and olein fractions and mixing them with native CB is a way either to standardize or to optimize the quality of the chocolate products (Weyland, 1992). Stearins and oleins are generally produced by solvent fractionation, but recent developments have proved the feasibility of single- and even multi-stage dry fractionation for the development of new fractions. Milk fat Among all oils and fats, milk fat has a most complicated triacylglycerol composition based on at least 40 different fatty acids and probably many more, distributed in at least 32 triacylglycerol groups with carbon numbers varying from 26 to 54 (Hartel and Kaylegian, 2001). Depending on the hardness of milk fat, which is highly dependent on the season (summer and winter), the feed and the breed of the animal, a double or triple fractionation process can be considered (Deffense, 1993; Gibon, 1999). In triple fractionation, milk fat is able to produce different grade stearins and oleins with dropping points varying between 45 and 6 °C (Fig. 10.11 and Table 10.4). Comparison of the triacylglycerol composition of hard stearin and native milk fat (gas chromatography) indicates a marked decrease in triacylglycerols containing short chains (carbon number less than 40) compensated by a strong increase in the long chains. Based on high-performance liquid

224 Modifying lipids for use in food Hard stearin DP 45 °C

Mid stearin

Milk fat

DP 32 °C

DP 34 °C Soft stearin DP 26 °C Olein

Recycling

Oleins

DP 21 °C Super olein DP 13 °C Top olein DP 6 °C Step 1

Fig. 10.11 Table 10.4

Step 2

Step 3

Multi-step dry fractionation of milk fat (DP = dropping point).

Melting profile (SFC) of milk fat and fractions.

SFC (%)/°C

Milk fat

Olein

Super olein

Top olein

Hard stearin

Soft stearin

Mid stearin

5 10 15 20 25 30 35 40

56 46 31 16 9 3 0

47 32 17 2 0

31 13 2 0

5 0

84 79 73 63 53 42 29 15

75 68 59 37 13 2 0

86 84 78 67 43 5 0

Abbreviation: SFC = solid fat content. Source: Gibon et al. (2000c).

chromatography (HPLC) examination it is apparent that trisaturated triacylglycerols based on myristic, palmitic and stearic fatty acids contribute to the hardness of this stearin. Top olein is almost free from trisaturated longchain molecules but particularly enriched in short chains and unsaturated long-chain acids (Gibon and Tritiaux, 2000c). The hard stearin can be used in the pastry making industry, integrated in puff pastry, croissant or brioche pastes. It finds applications as an ingredient in confectionery: its compatibility with cocoa butter makes it interesting as an inhibitor of bloom formation. It is also the basic material for ghee production and can be used in the reconstitution of hard butter in hot countries or as a constituent of spreadable butter. Hard stearin may also be used in blending with liquid oil as an

Fractionation of lipids for use in food

225

alternative to hydrogenated products. Depending on their dropping point, the oleins, super oleins or top oleins find an application in softening hard butter for creaming applications in the biscuit and cheese industry; they can be incorporated in the milk powder to improve the reconstitution or be used as constituents of spreadable butters. The production of spreadable butters has acquired a place of choice since the 1990s in Europe and New Zealand. The liquid fractions can be either incorporated into the cream itself or blended with the hard stearin, in proportions related to the dropping point of the fractions and to the specifications of the end products. Depending on the market, itself highly dependent on climatic conditions and the habits of the population, different kinds of spreadable butters (‘hard’ or ‘soft’) can be found in supermarkets (Table 10.5). Typically, ‘hard’ spreadable butters are available on the French market, French people being originally great consumers of intact butter. The Belgian population prefers a softer product: they have long been great consumers of margarine. A liquid butter-based product intended for cooking also appeared on the market recently, which complements the vegetable based cooking margarines. Edible beef tallow Edible beef tallow has some similarities with palm oil: the major saturated moiety in both materials is palmitate and the major unsaturated is oleate. Beef tallow has more stearate and less linoleate than palm oil which enhances its oxidative stability. Different possibilities of single, double and triple fractionation routes can be followed. In the triple fractionation scheme (Fig. 10.12), a wide variety of products, ranging from super stearin with dropping point 54 °C to top olein with very low cloud point, can be obtained. The stearin can be used to harden shortenings, table and puff-pastry margarines, without hydrogenation. The olein is a natural pourable deep frying shortening. Super olein and top olein can be used as ingredients of low-cost salad oils in some countries, blended with seed oils. The potential to re-fractionate the Table 10.5 origins.

Melting profile (SFC) of the fat fraction of spreadable butters from different

SFC (%)/ °C

French French butter market: spreadable, quality A

French market: spreadable, quality B

Belgian market: spreadable, quality A

Belgian English Liquid market: market: butter spreadable, spreadable quality B

5 10 15 20 25 30 35

60 49 34 16 10 4 0

50 37 21 11 5 0.5 0

39 25 11 5 1 0

35 23 11 6 3 0

49 37 22 11 5 2 0

Abbreviation: SFC = solid fat content. Source: Gibon et al. (2000c).

36 22 8 3 0.5 0

5 0

226 Modifying lipids for use in food Super stearin DP 54 °C Hard stearin DP 49 °C Olein Recycling

DP 45 °C

Beef tallow DP 43 °C Soft stearin DP 27 °C Olein

Recycling

DP 19 °C

Stearin DP 18 °C

Super olein DP 14 °C Top olein CP –3 °C Step 1

Step 2

Step 3

Fig. 10.12 Multi-step dry fractionation of edible beef tallow (CP = cloud point, DP = dropping point).

stearin in super hard stearin makes edible beef tallow an excellent basis to substitute hardened products at good price. Intermediate products with other melting characteristics can also be tailored; as an example, the olein issued from single-stage fractionation has received a great deal of attention as a frying oil due to its high content of polyunsaturated triacylglycerols. This product is still sold on the Belgian market for frying applications under the commercial name ‘Blanc de Boeuf/Ossewit’. Lard Lard is mainly used in shortenings and frying fats applications. It produces coarse crystals due to its β-tending crystallization behaviour. Randomization of lard makes it more suitable for shortening applications, due to the fact that the level of palmitic acid in position 2 drops from 64 to 24 %; interesterified lard fat is more stable in the β′ form with improved plastic range (DeMan et al., 1991). Dry fractionation of lard fat also suffers from this characteristic; its very sharp crystallization and melting range can be improved by randomization, making dry fractionation of lard easier. A series of interesterified lard-based fractions can be produced with multiple application ranges. Some top quality non-interesterified lard fats can nevertheless be fractionated (Table 10.6). In both situations, the result is a decrease in the saturated fatty acids

Fractionation of lipids for use in food Table 10.6

227

Melting characteristics of lard and interesterified lard fractions.

DP (°C)

Olein

Super olein

Hard stearin

Mid stearin

Step

Interesterified lard 33 35

24 30

– 16

48 51

– 38

Single* Double*

Lard 46 40 41 33

31 34 31 27

– – 27 20

55 59 57 49

– – 36 34

Single* Single* Double* Double*

*Fractionated in a single or double step. (DP = dropping point). Source: Kellens and Hendrix (2000), Gibon (2001).

and an increase in the levels of oleic and linoleic. Selectivity of this exchange seems to be better with the interesterified fat. Chicken fat Dry fractionation of chicken fat has been recently discussed in relation to nutritional and physico-chemical characteristics of the fractions (Arnaud et al., 2004). Chicken fat has lower stearic and higher linoleic acid contents, as compared to other animal fats; it is also a good source of α-linoleic acid. It is found to contain approximately 30, 44 and 26 % of saturated, monounsaturated and polyunsaturated fatty acids, respectively, with a ratio n-6/n-3 of 12; those characteristics confer on chicken fat high nutritional potential. The market for 100 % poultry products is now booming, especially since they are not prohibited by any religious group. Chicken fat fractions could be incorporated in several delicatessen meats with several new applications. Depending on practical applications, different fractions can be obtained by simply modifying the temperature of filtration. The result is a particular increase of mono- and polyunsaturated content in the oleins, especially when filtration is performed at very low temperatures in one single step (Table 10.7).

10.6 Future trends 10.6.1

Recent hot topics

Zero trans margarines and shortenings Prior to 1990, there was a tendency to consider that both cis and trans fatty acids (TFA) have the same effect on blood cholesterol. This opinion stimulated the use of trans instead of saturated fatty acids as hard fractions for margarines (Van Duijn, 2000). In recent years, TFA have received further attention; it is

228 Modifying lipids for use in food Table 10.7

Characteristics of chicken fat and fractions. Chicken fat

Olein

Stearin

Filtration temperature (°C)

SFA (%) MUFA (%) PUFA (%) IV

30 44 26 85

27 46 27 88

44 36 20 68

13.5

SFA (%) MUFA (%) PUFA (%) IV

33 51 16 72

29 54 17 77

41 45 14 63

10.0

SFA (%) MUFA (%) PUFA (%) IV

34 49 17 73

26 55 19 81

46 41 13 59

5.0

SFA (%) MUFA (%) PUFA (%) IV

34 49 17 73

24 57 19 84

43 44 13 62

0.0

Abbreviations: IV = iodine value, MUFA = monounsaturated fatty acids, PUFA = polyunsaturated fatty acids, SFA = saturated fatty acids. Sources: Gibon et al. (2002), Arnaud et al. (2004).

now clear that high levels in TFA in diet result in unfavourable effects on both low-density lipoprotein (LDL) cholesterol (‘bad’ cholesterol) and highdensity lipoprotein (HDL) cholesterol (‘good’ cholesterol) (Hunter, 2004). On 11 July 2003, the US Food and Drug Administration (FDA) published a final rule to require that trans fatty acids be declared in the nutrition label of conventional foods and dietary (List, 2004). This rule took effect on 1 January 2006. Recommendations from a number of authoritative bodies have been published. The consensus is that, although the risk to health of TFA intake at average consumption levels is small, the intake should not be increased. In consequence, food manufacturers should reduce the levels of trans isomers of fatty acids as far as possible. Complete elimination of TFA in margarines means reducing or eliminating the use of partially hydrogenated oils. Production of trans-free fats issued from combination of fractionation, interesterification and full hydrogenation of liquid and tropical oils is one way (Van Duijn, 2000; List, 2004). Products high in solids derived from fractionated palm, palmkernel and coconut oils or combinations of different fractions to meet specific needs offer good opportunity for zero and low trans formulation. Palm olein, PMF and palmkernel stearin could be used in combination with liquid oils to create no trans or low trans/low saturated shortenings and margarines (Hunter, 2004). Conjugated linoleic acids Not all trans fatty acids have the same physiological effect (Stanley, 2004).

Fractionation of lipids for use in food

229

Conjugated linoleic acid (CLA) is a term currently used to characterize 9cis, 11-trans octadecadienoic acid and other isomers which occur naturally in ruminant-derived fats such as dairy products. CLA has been shown to have a number of beneficial effects on disease in animal models. For example, CLA has unique chemoprotective properties against carcinogenesis, and its effects occur at low dietary concentrations, close to the human average intake. It may also have positive effects on cardiovascular risk factors (decrease in total cholesterol and LDL cholesterol). CLA is consequently a beneficial component of milk fat. An approach to CLA enrichment was examined on a laboratory scale as a potential alternative means of developing CLA-enriched milkfat fractions (O’Shea et al., 2000). The results indicate that the ‘soft fraction’ issued from a single dry fractionation step contains 63.2 % more CLA than original milk fat. This study clearly demonstrates that dry fractionation provides an alternative method to approaches involving dietary oil supplements.

10.6.2 Non-edible applications Beside edible applications, there is a large potential for dry fractionation in the non-food industry. Industrially, fatty acids are largely separated and purified according to the fractional distillation process (see Section 10.2.1). Due to the very close boiling point of saturated and unsaturated fatty acids such as stearic and oleic, solvent and detergent fractionation are usually used to separate those acids. For economic and ecological reasons, there is a strong pressure to replace existing plants with the dry fractionation process.

10.7

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11 Chemical and enzymatic interesterification of lipids for use in food X. Xu, Z. Guo, H. Zhang, A. F. Vikbjerg and M. L. Damstrup, Technical University of Denmark, Denmark

11.1

Introduction

Interesterification, as defined in a lipid glossary (Gunstone and Herslöf, 1992), is a general term for alkali-catalyzed reaction of, for example, triacylglycerols producing a random distribution of ester groups within the molecules. This definition has been widely accepted as a term for chemical randomization, typically between liquid oils and solid fats, in the oil and fat modification industry, in particular for the manufacture of margarine or shortening fat products. A number of papers relating to this concept have recently been presented and its use in industry has expanded because it provides a better alternative to hydrogenation in the coming trans-free era (Rozendaal and Macrae, 1997; Rousseau and Marangoni, 2002). The primary motivation for the modification of naturally occurring oils and fats by interesterification during the production of margarine and shortening fats stems from the increasing demand for lipids with specific melting behaviour and crystallization characteristics. This is because the supply of natural products capable of meeting these particular requirements is insufficient (Macrae, 1981; Gunstone, 1999; Baljit et al., 2002). It is not surprising that the triacylglycerol structure of natural oils and fats is the result of either a regular distribution of fatty acids on the glycerol backbone or a limited statistical distribution, because these distributions are the intrinsic response of living organisms in adjusting their triacylglycerol structure to biological functions and to the environment (Ucciani and Debal, 1996). The functional properties of oils and fats are closely related to both their fatty acid profiles and the distribution of different fatty acid species on individual triacylglycerol molecules. Interesterification allows the rearranging of existing acyl groups or the incorporation of other fatty acids to create novel properties.

Chemical and enzymatic interesterification of lipids for use in food 235 Interesterification in a chemical sense, as defined by Sonntag (1982), often refers to the reaction of triacylglycerols within or between the molecules or with other acyl donors or alcohol moieties, yielding new esters by an interchange of fatty acid groups. It can be classified into ester–ester interchange, acidolysis and alcoholysis, depending on the nature of the substrates. This definition covers more applications in terms of product development and process development, in particular with the boom in research on enzymes as catalysts. The subject is easily divided into chemical and enzymatic interesterification depending on the catalysts employed. Therefore, in this chapter, the concept of interesterification for lipid modification will be expanded to include acidolysis and alcoholysis (glycerolysis) in addition to the ester–ester interchange, to which it usually refers. Several good papers have been presented on chemical interesterification, and since this technology is now mature the emphasis in this chapter will be on enzymatic interesterification, which is currently close to industrial development. With this wider definition in mind, the chapter will present a few typical and important applications in the area of lipid modification and discuss development of new products or technologies. The writing is primarily focused on our own research.

11.2 Interesterification in general practice: an introduction of potential reactions and applications 11.2.1 Chemical interesterification Chemical interesterification has long been used to modify oils and fats into functional products (Marangoni and Rousseau, 1995). It modifies the physical properties of lipids by rearranging the distribution of fatty acids on the glycerol backbone without changing fatty acid profiles. During interesterification, fatty acids are exchanged within (intra) and among (inter) triacylglycerols until a thermodynamic equilibrium is reached. This equilibrium is based on the composition of the starting material and can be predicted from the laws of probability. The degree of difference depends upon the temperature, time and other conditions of the reaction (Marangoni and Rousseau, 1998; Rousseau et al., 1998; Gunstone, 1999). Early patents concerning chemical interesterification were filed by W. Normann between 1920 and 1930, and these contributed to the production of margarine with defined physical properties. The process was used in industrial applications from the 1940s. Typically, chemical interesterification can be carried out using sodium methoxide (0.2–0.3 %) or an alkali metal (0.1– 9.2 %) as a catalyst (Anderson, 1996). Oil blends need to be dried under vacuum at a temperature higher than 90 °C for 0.5–1 hour. The reaction can be initiated by the addition of catalyst after the blend is cooled to 60–70 °C. At the beginning of the reaction, the reaction mixture becomes brown, which indicates the start of the reaction. Once such colour change occurs, the

236 Modifying lipids for use in food reaction can be completed within 30 minutes. The reaction is stopped by the introduction of water, and subsequent processing usually includes washing, bleaching and deodorization to obtain the final randomized product. Figure 11.1 illustrates the difference between chemical and specific lipasecatalyzed interesterification in two triacylglycerols. For the chemical randomization of ACA and BBB governed by thermodynamics and statistics, theoretically all 18 isomers (27 if stereoisomers are included) might be detected in the end reaction mixture. For comparison, with 1,3-specific lipasecatalyzed reaction governed mainly by enzyme specificity and dynamics, only four new isomers (six if stereoisomers are included) are expected, apart from the original ACA and BBB. The formation of isomers with acyl group C at the 1- or 3-positions and A at the 2-position of the glycerol backbone is theoretically impossible if acyl migration can be ruled out. The randomization of triacylglycerols can also be driven by non-specific lipases, yielding similar results to chemical interesterification. In practice, only a very few lipases are strictly non-specific. Most lipases are specific to a certain extent (Matori et al., 1991). That is to say, the result of the reaction catalyzed even by a non-specific lipase will not be exactly the same as that with a chemical catalyst. The widely used chemical catalysts are Na-K alloy or sodium alcoholate (e.g. CH3ONa). However, they are believed not to be the real catalysts due to their ease of consumption during reaction. Glycerate is the real catalyst of interesterification. This mechanism explains the formation of both monoalcohol ester and diacylglycerol, the latter being formed by glycerate hydrolysis (Ucciani and Debal, 1996). Dijkstra et al. (2005) proposed a new mechanism in order to explain the experimental observations. This mechanism assumes that the reaction of a base (such as sodium methanolate) with the oil will eventually lead to the abstraction of an α-hydrogen from a fatty acid moiety and that the enolate anion thus formed acts as the catalytic intermediate. This enolate can re-abstract a proton from the hydroxyl group of a partial glyceride, whereupon the resulting alcoholate attacks the carbonyl group. This leads to a new ester and a glycerolate anion which then regenerates a new enolate anion. chemical catalyst or nonspecific lipase

A

B

C +

B

A

B 1,3-specific lipase

ABC AAB AAA A C B* B B B* AAC + + + BAC CCC A B A* A C A*

B B B B

BC B A* + AB C B*

C C C C

CA CB AC BC

A C +

B B +

A C +

A B +

B B +

B C

A

B

B

A

A

B

Fig. 11.1 Schematic illustration of the difference between randomization and 1,3specific interesterification, exemplified by the reaction between ACA- and BBB-type triacylglycerols. Underlined molecules are starting materials and those with star are possible products for 1,3-specific lipase-catalyzed reaction.

Chemical and enzymatic interesterification of lipids for use in food 237 The reaction between oils and fats could yield the required plastic property and crystal forms (Rousseau et al., 1998; Gunstone, 1999; Baljit et al., 2002). For example, the shortening property of lard can be improved by randomization, which changes its crystalline form from β to β ′ by reducing the content of palmitic acid at the 2-position. Trans-fatty acid free margarine can be manufactured by interesterification from palm stearin and sunflower oil in appropriate ratios. Chemical interesterification can also take place between triacylglycerols and glycerol, and this method is currently used for the industrial production of partial acylglycerols (see Section 11.3.4) (Bornscheuer, 1995). The reaction of triacylglycerols and methanol is now widely used for the preparation of methyl esters for gas chromatographic analysis of fatty acid composition. However, due to the predicted shortage of petroleum, this reaction has been utilized on a much larger scale to produce biodiesel (Korbitz, 1999; Ma and Hanna, 1999). Primary and secondary monohydric aliphatic alcohols having from one up to eight carbons can be used in the interesterification process. Among these, methanol and ethanol are used most frequently, especially methanol due to its low cost and its physical and chemical advantages. The reaction can be catalyzed by alkalis, acids or enzymes. Alkali-catalyzed interesterification is much faster than the acid-catalyzed reaction and is most often used commercially so long as the oil does not have a high acid value.

11.2.2 Enzymatic ester–ester exchange Ester–ester exchange can occur between two triacylglycerols (TAGs), or between triacylglycerol and methyl or ethyl esters employing either a chemical or an enzymatic catalyst. Enzymatic TAG–TAG interchange, in principle, gives different triacylglycerol composition in the end product compared with the chemical process, depending on the regiospecificity of the lipase (Fig. 11.1). This reaction has been applied to margarine or shortening fat production and the manufacture of other structured lipids with specific functions (Negishi et al., 2003; Zhang et al., 2004a,b). Recently, an enzymatic scaleup formulation of butter incorporating polyunsaturated fatty acids (PUFA) in varied proportions has been studied. Nutritional concerns and sensory evaluation were also examined (Rønne et al., 2005). Triacylglycerols of UPU type (U = unsaturated fatty acids, P = palmitic acid) are believed to be readily absorbed by infants. Loders-Croklaan in the Netherlands manufactures such a product as a constituent of infant formula from tripalmitin and a vegetable oil with high oleic acid content confining the acyl exchange to the sn-1 and sn-3 positions by 1,3-specific lipases (Kavanagh, 1997).

PPP + UUU

1,3-specific lipase

PPU + UPU

238 Modifying lipids for use in food The reaction between triacylglycerols and fatty acid ethyl esters can be treated as an alternative to acidolysis of triacylglycerols for the production of, for example, fats or similar products with particular locations of specific fatty acids (see below). Therefore, the reaction will start from PPP and U-EE (ethyl esters of U) for the above product. After reaction, the remaining U-EE and released PEE have to be distilled to obtain the product (UPU + PPU).

11.2.3 Enzymatic acidolysis The acyl exchange reaction between triacylglycerols and fatty acids is called acidolysis. A particular interest in this area is that 1,3-specific lipases can selectively catalyze the acyl exchange at the sn-1 and sn-3 positions whilst leaving the sn-2 acyl group unchanged. This provides an opportunity to tailor some functional lipids with special requirements for the fatty acid types located in 1,3- or 2-positions. With this technology, a series of functional lipids, such as cocoa butter equivalents, human milkfat mimics or structured lipids containing medium- or short-chain fatty acids have been developed (Macrae, 1985; Balcao and Malcata, 1998; Gunstone, 1999; Xu, 2000c; Baljit et al., 2002). An important application of 1,3-specific lipase-catalyzed acidolysis is the enzymatic production of cocoa butter equivalents (Bloomer et al., 1990; Undurraga et al., 2001). For example, cocoa butter equivalents could be produced from palm mid-fraction (rich in POP) (O = oleic acid) or OOOtype oil by enzymatic incorporation of stearic acid (St) to the 1,3-positions: POP + St OOO + St

1,3-specific lipase 1,3-specific lipase

POSt + StOSt + P StOSt + St

An anti-blooming agent for chocolate, 1,3-dibehenoyl-2-oleoyl glycerol (BOB), has been prepared from triolein and behenic acid or its EE employing Lipozyme RM IM (Yoon et al., 1996): OOO + B

1,3-specific lipase

BOB + O

Structured lipids (MUM) with medium-chain fatty acids at 1,3-positions and unsaturated fatty acids at the 2-position are supposed to be an ideal energy and nutrition supplier, especially for patients suffering from malabsorption (Ikeda et al., 1991; Ingle et al., 1999). MUM are absorbed and digested more rapidly and the essential fatty acids at the sn-2 position meet the nutritional requirements. A substantial amount of work has been carried out in this area using oils with unsaturated fatty acids at the sn-2 position and medium-chain acyl donors (Xu, 2000c):

UUU + M

1,3-specific lipase

MUM + U

Chemical and enzymatic interesterification of lipids for use in food 239 Lipase-catalyzed modification of vegetable oils by introducing certain polyunsaturated fatty acids, such as γ-linolenic acid, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), etc. from micro-organism oils, unusual plant oils and fish oils, in order to increase nutritional values constitutes another important aspect for lipase applications in lipid processing (Macrae, 1985; Huang and Akoh, 1994; Gunstone, 1999; Baljit et al., 2002).

11.2.4 Enzymatic alcoholysis Alcoholysis is the reaction between an ester and an alcohol, resulting in an alcohol exchange. The alcoholysis of triacylglycerols with glycerol is called glycerolysis and is widely used for production of mono- and diacylglycerols: TAG+Gly

1,3-specific lipase

1,2-DAG+1-MAG

1,3-specific lipase Glycerol

2-MAG+21-MAG

The reaction of triacylglycerols with monohydric alcohols generates the simple alcohol esters of fatty acids, which have found applications in the production of biodiesel, as well as alternative acyl donors of fatty acids, e.g. fatty acid ethyl esters. The intermediate product of 1,3-specific lipase-catalyzed alcoholysis, 2-monoglycerides, can also be used as starting materials for the synthesis of ABA-typed structured lipids: 3DHA-EE+Gly

nonspecific lipase

DHA DHA +EtOH DHA

1,3-specific lipase

DH 2DHA-EE+ DHA DH

More attention has been devoted to the enzymatic production of partial glycerol esters. Recently, we demonstrated a novel and efficient reaction system for enzymatic glycerolysis of oils and fats to produce a good yield of monoacylglycerols in a tetra-ammonium-based ionic liquid (Guo and Xu, 2005, 2006). In general, many products and manifold process possibilities have been created with feasible reactions not only in theory but also in practice. Intensive demonstrations concerning important application areas are given in the following sections.

11.3

Typical interesterification in lipid modification

11.3.1 Enzymatic TAG–TAG interchange for plastic fat production Since the 1990s, there has been a strong debate regarding trans-fatty acids (TFA) consumption and their relationship with high total blood cholesterol and low-density lipoprotein cholesterol levels, and the increase in death through coronary heart disease (Kris-Etherton and Nicolosi, 1995). The content of TFA in oils and fats has become a global issue. Legislation to limit the

240 Modifying lipids for use in food TFA content of lipid and food products and to label the amount of TFA in fatcontaining products has been issued in several countries in recent years. On July 11, 2003, the FDA announced final regulations for TFA labelling of foods in the USA from January 1, 2006. Demark issued such a requirement as early as 2004. To avoid trans fats produced by partial hydrogenation, interesterification of fully hydrogenated fats and liquid oils (List et al., 1995) or some tropical oils (Lai et al., 1998; Zhang et al., 2004a, 2005) is frequently used for producing zero-trans plastic fats for industrial applications. Enzymatic interesterification is now much studied for plastic fat production, although it is not a new technology. Unilever obtained a patent in 1977 on ‘catalytic rearrangement of fatty acid groups in glycerol fats or oils – by contacting with an enzyme transesterification catalyst activated with water’ (Unilever, 1977). However, the industrial breakthrough of lipase-catalyzed interesterification for plastic fat production has been delayed, mainly because the major commercially available immobilized lipases have hitherto been too expensive (Christensen et al., 2001). Only high value-added oil and fat products have so far been processed in industry, such as cocoa butter substitutes (Chang et al., 1990), human milk fat substitutes (Betapol™ from Loders Crokelaan), polyunsaturated fatty acid (PUFA)-enriched marine oils (Shimada et al., 1997; Haraldsson and Kristinsson, 1998), functional 1,3-diglycerides and structured lipids (Soumanou et al., 1998; Xu et al., 1999a, 2000). However, in more recent years, the development of new cost-effective immobilized lipases is clearly providing more opportunities for the plastic fat industry to produce an economy-balanced product with better properties (Pedersen and Christensen, 2000). Lipozyme TL IM (Thermomyces lanuginosus lipase immobilized on silica granulates) is now successfully applied to bulk fat modifications (Novozymes A/S, 2005). Reactions for enzymatic TAG–TAG interchange Lipase-catalyzed interesterification involves water participation during the reactions and is accompanied by the formation of new triacylglycerol products as well as some diacylglycerol (DAG) and free fatty acid (FFA) by-products in the system (Wong, 1995). A simplified form, in which E stands for enzyme, instead of a detailed molecular structure is used to describe the process of lipase-catalyzed interesterification between triacylglycerols based on the formation of acyl enzyme complex (Zhang et al., 2001): TAG1 + E [ TAG1 · E [ DAG1 + FFA1· E

[11.1]

TAG + E [ TAG · E [ DAG + FFA · E

[11.2]

DAG1 + FFA2· E [ TAG3 · E [ TAG3 + E

[11.3]

DAG + FFA · E [ TAG · E [ TAG + E

[11.4]

TAG3 · E [ · · ·

[11.5]

TAG ·E [ · · ·

[11.6]

2

2

4

2

1

2

4

2

4

Chemical and enzymatic interesterification of lipids for use in food 241 FFA1 · E + H2O [ FFA1 + E

[11.7]

[11.8] FFA · E + H2O [ FFA + E The reaction will continue to reach equilibrium in the system involving production of new intermediates and formation of new TAG products (Eq. 11.1–6). If the water content increases in the system, the FFA content will increase (Eq. 11.7–8), whereas FFA1·E or FFA2·E decreases. As a consequence, the content of DAG will also increase (Eq. 11.3–4). The total yield of interesterified new TAG products will decrease. The free water is the main reason for the rise in the level of FFA in the system. One mole of free water in the reaction system generates one mole of FFA (Zhang et al., 2000). Hence it is very important to control water content in the system to reduce the contents of FFA and DAG in the final products. When an sn-1,3-specific lipase is used as a catalyst, the pathway for interesterification starts with the reaction of the TAG and an enzyme molecule (E), giving a 1,2(2,3)-DAG and FFA·E intermediate. A problem often encountered for a selective lipid modification is acyl migration, which leads to the non-specific distribution of fatty acids. Acyl migration is a non-enzymatic reaction catalyzed by acids, bases, ion exchange resin (carrier) and increased by heat and reaction time (Bloomer et al., 1991; Xu et al., 1998b). The mechanism of acyl migration has been studied (Fischer, 1920; Serdarevich, 1968) under the acidic condition. For enzymatic interesterification (ester– ester exchange), the acyl donors could be free fatty acids, which relate to the water content in the reaction system. Reaction temperature and reaction time are crucial for the control of acyl migration for enzymatic interesterification. The suggested pathway of acyl migration is shown in Fig. 11.2. Acyl migration is initiated by the nucleophilic attack of a lone pair of electrons of the free hydroxyl oxygen on the ester carbonyl carbon (sn-2 position), resulting in a five-membered ring intermediate called an orthoester. The ring is unstable and opened by the cleavage of the original ester carbon single bond resulting in the formation of the 1,3-DAG. The initial driving force for the acyl migration is the efficiency of the nucleophilic attack and the formation of the orthoester intermediate. The primary hydroxyl oxygen is a better nucleophile than the secondary hydroxyl and, therefore, the acyl shift from the secondary hydroxyl to the primary is favoured (Kodali et al., 1990). An equilibrium ratio between sn-1,3-DAG and sn-1,2(2,3)-DAG was reported to be between 3/2 and 2/1 (Jensen and Pitas, 1976; Zhang et al., 2000). 2

2

O OH O

OH OH

O

C

R2

O

C

R3

O

Fig. 11.2

H

O

O

C

R2

O

C

R3

O

H

OH

O

R2

OH

C

R2

C

R3

C O O

C O

R3

O

O

Mechanism of acid-catalyzed acyl-migration (adapted from Serdarevich, 1968).

242 Modifying lipids for use in food Bioreactors for enzymatic TAG–TAG interchange Enzymatic interesterification can be carried out in a batch or a packed-bed reactor (PBR). Most reactions in the laboratory are carried out in a batch reactor. It requires less enzyme dosage and it is easy to apply vacuum or add molecule sieves to reduce the water content in the system. However, it also has some drawbacks. Longer reaction times are normally required due to the low enzyme dosage. It also gives rise to a high degree of acyl migration (Xu et al., 1998c), which leads to non-specificity of the interesterified products. In a PBR, the ratio between enzyme and substrate is much higher and the reaction time much shorter than in a batch reactor. Therefore, the specificity of the enzyme can be better maintained since less acyl migration can occur. At the same time, it is suitable for continuous operation on an industrial scale. For a batch reactor (Levenspiel, 1999), the relationship between enzyme load, flow rate, reaction rate and conversion degree is wb t= Vb

∫

Xa

0

d Xa – ra

[11.9]

where wb is the enzyme dosage, Vb is the amount of oil and t is the reaction time, Xa is the substance conversion degree and ra is the reaction rate. For a PBR (Levenspiel, 1999), a similar relationship exists, which can be expressed as: wp = Vp

∫

Xa

0

d Xa – ra

[11.10]

where wp is the amount of enzyme in the PBR, Fp is the flow rate through the PBR. Assuming the reaction rate and conversion degree are the same because the same enzyme is used, the right sides of Eqs 11.9 and 11.10 are the same. Therefore, the following equation is obtained:

wp w = bt Fp Vb

[11.11]

If no deactivation occurs in the PBR, Eq. 11.11 can be written as:

Fp = w p

Vb wb ⋅ t

[11.12]

On the basis of experimental data in the batch reaction, Eq. 11.12 can be used to calculate the amount of enzyme and the flow rate in a PBR. Interesterified products with a desired conversion degree can be easily obtained by adjusting the flow rate for the PBR. Monitoring of enzymatic TAG–TAG interchange process During interesterification, the changes of chemical (sn-2 position, TAG composition) and physical properties [dropping point (DP), solid fat content

Chemical and enzymatic interesterification of lipids for use in food 243 (SFC), differential scanning calorimeter (DSC) etc.] can be used to monitor the reaction. For industrial applications, SFC is mostly used for control of feedstock quality for plastic fat production. Therefore, SFC is chosen to monitor the interesterification process. For a batch reactor (Levenspiel, 1999), the mass balance for the reaction is shown as: input = output + accumulated + disappearance

[11.13]

where input and output are zero. Therefore, based on the variation in SFC during the reaction, Eq. 11.13 can be written as: 0 = 0 + V · dSFC – W · r(SFC) · dt

[11.14]

where r is the reaction rate, V is the volume of reactor, W is the amount of lipase and t is the reaction time. Eq. 11.14 then becomes:

d SFC W = ⋅ r ( SFC ) dt V

[11.15]

Introducing a weight based reaction time, τ = (W/V) t, gives the following equation: [11.16] dτ = W ⋅ d t V combining Eqs 11.15 and 11.16, gives the following relationship: dSFC = r ( SFC ) [11.17] dτ Enzymatically catalyzed interesterification is assumed to be a first-order reversible reaction (Rozendaal and Macrae, 1997). Thus, Eq. 11.17 can be written as: [11.18]

r(SFC) = –k*SFCr

where SFCr is the reduced SFC content at reaction time τ and k is a constant. It is equal to: SFCr = SFC – SFC∞

[11.19]

where SFC∞ is the SFC value when reaction reaches equilibrium. Combining Eqs 11.17–19 gives the following relationship:

∫

SFC

SFC

d SFC = – SFC – SFC∞

∫

τ

k ⋅ dτ

[11.20]

0

After integration, Eq. 11.20 becomes: SFC – SFC∞ = e – kτ SFC0 – SFC∞

[11.21]

where SFC is the SFC value of the product at time τ and SFC0 is the initial SFC value. ∆SFC is introduced to represent the maximum changes in SFC as follows:

244 Modifying lipids for use in food ∆SFC = SFC0 – SFC∞

[11.22]

Therefore SFC can be written as: SFC = SFC0 – ∆SFC(1 – e–kτ)

[11.23]

This model (Eq. 11.23) has three parameters which have physical and chemical meanings. The k value is related to the reaction rate of the enzyme on the given blend. SFC0 and ∆SFC are related only to the types of blends and the blend ratios. The weight base reaction time (τ), adjusted for the volume of oil samples removed from the batch reactor, is expressed as follows:

τ n = t n–1 ⋅

we VToil

+ ( t n – t n–1 ) ⋅ n

we

[11.24]

n –1

VToil – Σ Si i =1

where t is the reaction time (min), n is the sampling times, we is the enzyme dosage in grams, VToil is the initial blend weight and Si is the amount of sample withdrawn in grams at a given sampling time. The advantage of using weight-based reaction time is the ease with which these data can be applied in different reaction systems or enzyme dosages to obtain the same products. If the sampling amount is sufficiently small compared to the total volume during the reaction process, this means that τ = t. Then the equation can be simplified: SFC = SFC0 – ∆SFC (1 – e–kt)

[11.25]

The model has been evaluated (Zhang et al., 2004a) using different oil blends and different analytical methods (Fig. 11.3) for monitoring the enzymatic interesterification process. 1.0

16 14

SFC (%)

10

0.6

SFC Peak ratio

8

0.4

6 4

Peak ratio

0.8

12

0.2

2 0

0.0 0

1

2

3 4 Reaction time (hr)

5

6

Fig. 11.3 Model fitting of experimental data (different signs) with exponential model (solid line) for an enzymatical interesterification process. (Reaction conditions: 70/30 blend of palm stearin and coconut oil; 4 wt % Lipozyme TL IM; 70 °C; three volumes of rapeseed oil were interesterified for 30 minutes at 70 °C to reduce the water content of Lipozyme TL IM, then Lipozyme TL IM was quickly washed by the blend. Finally, the blend was poured into the reactor and interesterified for three hours.) The peak ratio is the peak area between equivalent carbon number (ECN)44 and ECN48 analyzed by high-performance liquid chromatography (HPLC).

Chemical and enzymatic interesterification of lipids for use in food 245 During enzymatic interesterification, degree of conversion can be controlled by different reaction times.The conversion degree (X) is defined as follows (left side); it can also be derived from Eq. 11.21 and redefined by the relationship between reaction time t and reaction rate k (right side): X (%) =

SFC0 – SFC × 100 = (1 – e – kt ) * 100 SFC0 – SFC∞

[11.26]

where SFC0 is the SFC at time 0, SFC is the SFC at reaction time t and SFC∞ is the SFC at the equilibrium stage. Recent progress for enzymatic TAG–TAG interchange Current research work shows that Lipozyme TL IM-catalyzed interesterification presents no problem for the production of plastic fats. In general, the technology can easily be moved to the industrial sector for commercial exploitation. Both stirred tank reactors (Zhang et al., 2001) and PBR (Rønne et al., 2005) can be used for the production of plastic fats, and the system is generally straightforward. The control of water activity in the system presents no particular difficulty, as is often the case in other lipase-application systems, in which the lipase activity was not affected by the reduction of water content in the system (Zhang et al., 2001). The process indeed shows tremendous advantage for plastic fat production compared to the chemical process with its additional washing and bleaching procedure or other lipase processes in which water content has to be adjusted during reuse (Zhang et al., 2000). The exponential model (Eq. 11.23) is very useful for monitoring the enzymatic interesterification process. It has the potential to be implemented in routine industrial operations for practical control purposes. The degree of conversion of enzymatic interesterification has significant effects on triacylglycerol composition (Zhang et al., 2001, 2004a; Rønne et al., 2005), crystallization behaviour (Zhang et al., 2004b) and storage stability of the products (Zhang et al., 2005, 2006). Based on the enzymaticallyinteresterified blend of palm stearin and coconut oil (70/30, w/w), it was observed that the increased conversion reduced the crystal size and, consequently, the crystal surface was changed from a coarse and condensed leaf-like crystal (β crystal) to a fine needle ball-like crystal (β′ crystal, confirmed by X-ray analysis) (Fig. 11.4). At the same time, margarines made from enzymatically-interesterified fats became more physically stable with the increase in conversion degree. During a 12 week storage stability study at both 5 and 25 °C, the margarine made from enzymatically fully converted fat had the same physical properties as the margarine made from chemically interesterified fat. The margarines produced from enzymatically interesterified fats generally had much lower peroxide values compared to the margarine produced from chemically randomized fats stored at 25 °C (Zhang et al., 2006). A similar observation was also apparent in a pure oil system (Tautorus and McCurdy, 1990). This confirms that enzymatic interesterification has advantages for the oxidative stability of the products.

246 Modifying lipids for use in food Blend

31 % conversion

71 % conversion

58 % conversion

100 % conversion

Fig. 11.4 Morphology of the blend (palm stearin/coconut oil, 70/30) and enzymatically interesterified products at different degrees of conversion (measured by HPLC). (Samples with 50 % added rapeseed oil were melted at 70 °C, and one drop was put on a slide. They were then cooled to room temperature. The morphology was observed after one hour at room temperature by polar light microscopy) (adapted from Zhang et al., 2004b).

11.3.2 Enzymatic acidolysis for the production of structured lipids Structured lipids (SL), in this text, means fats that are modified or restructured from natural oils and fats, or the fatty acids therefrom, with regio-positional preference of each fatty acid or each group of fatty acids in the glycerol backbone. Structured lipids are designed for a particular functionality or nutritional property for edible or pharmaceutical purposes. This definition

Chemical and enzymatic interesterification of lipids for use in food 247 covers fats, particularly those produced by enzymatic methods using sn-1,3specific lipases, including cocoa butter equivalents (SOS; S = saturated fatty acids, O = oleic acid), anti-bloom agent (BOB; B = behenic acid), breast milk fat substitutes (UPU; U = unsaturated fatty acids, P = palmitic acid), MLM-type oils containing long-chain (L, usually polyunsaturated or essential) and medium-chain (M) fatty acids. The chain length of fatty acids is normally defined as long-chain (more than 12), medium-chain (8–12) and short-chain (less than 8). The nutritional, physical or chemical properties of SL have been widely discussed in countless publications (Akoh, 1998; Mu and Porsgaard, 2005). Acidolysis using enzyme technology has been more widely explored for the production of SL than other products. Chemical acidolysis has fewer practical applications due to its having no positional specificity. The reaction is between a triacylglycerol and a free fatty acid (FFA) with an sn-1,3specific lipase. The acid will be exchanged with the acid in the ester. In fact, the production of SL can only be carried out by enzymatic interesterification, especially in large quantities. The reaction is normally conducted between a substrate oil and acyl donors (fatty acids or their ethyl esters) with sn-1,3specific lipases as the biocatalysts, as demonstrated in industry for commercially available SL products (Quinlan and Moore, 1993). Our research group has been studying structured lipids since 1996. A national framework program has been run for about seven years with the name LipoTech (www.biocentrum.dtu.dk/lipotech/). Process technology was widely studied in the early stages. Most of the progress made has been reviewed in a few early papers or chapters (Xu, 2000a,b,c; Høy and Xu, 2001). Concerning the enzymatic production of structured lipids in general, a few recent review publications from different groups have further addressed the issue (Kim and Yoon, 2003; Xu, 2003, 2004; Iwasaki and Yamane, 2004). Acidolysis reaction with sn-1,3-specific lipases for production of structured lipids Enzymatic acidolysis reaction is commonly considered as a two-step reaction: hydrolysis and esterification with diacylglycerols as reaction intermediates (Xu et al., 1999b). Through these two steps, the new fatty acids are incorporated into triacylglycerols, and the reaction will finally reach equilibrium. This procedure for the reaction between a TAG (LLL) and a fatty acid (M) is depicted in Fig. 11.5. Enzymatic acidolysis is a reversible reaction, and equilibrium will eventually be reached. The product yield under reaction equilibrium is decided by the ratio between fatty acids and esters (Xu et al., 1998a). For the reaction between TAG and FFA, the incorporated fatty acids in the product can be calculated (Fig. 11.6). The higher the FFA to TAG substrate ratio, the higher the maximum incorporation of the FFA to TAG which can be expected. However, the time to reach equilibrium is related to many parameters, such as reaction temperature, enzyme dosage, water content and reaction system.

248 Modifying lipids for use in food M L L L

L

M

via diacylglycerols

M L L

Substrate oil

M L M

via diacylglycerols

L

M

L

M

Mono-incorporated structured triacylglycerols

L Di-incorporated structured triacylglycerols

Incorporation of acyl donors and contents of triacylglycerol components (mol%)

Fig. 11.5 Reaction principle of the lipase-catalyzed interesterification between LLL and M and the dynamic balance between LLL, sn-MLL/LLM and sn-MLM. L = longchain fatty acid, M = medium-chain fatty acid. 90 80

Inc

70

sn-MLM

60 50 40 30

sn-MLL/LLM

20 10

LLL

0 1

2

3

4 5 6 7 8 Substrate molar ratio

9

10

Fig. 11.6 The relationship between the substrate molar ratios (M/LLL) and the contents of substrate oil left (LLL), mono-incorporated SL (sn-MLL/LLM) and diincorporated SL (sn-MLM), and the incorporation of the acyl donor (Inc) in the sn-1,3 regiospecific lipase-catalyzed interesterification (L = long-chain fatty acid, M = medium-chain fatty acid, SL = structured lipids).

In particular, when substrate molar ratios increase, the effect of substrate inhibition on the reaction activity will also increase and lead to prolongation of the equilibrating time of the reaction. This often reduces the productivity of the process. It is very important to choose a suitable substrate molar ratio in terms of reaction efficiency (incorporation level of acyl donors per unit time) and productivity (product quantity per unit time) with a specific reaction system. The choice of substrate molar ratio is also related to the downstream processing cost and associated difficulties of separating free fatty acids or acyl donors by evaporation and/or distillation. A high substrate molar ratio may reduce the number of reaction stages needed to obtain a suitable product, but the purification of the product may also be more difficult.

Chemical and enzymatic interesterification of lipids for use in food 249 Depending on the products required, careful consideration should be given to the reaction system before embarking on the process design. There are many natural or synthetic oils and fats available for selection as substrate oils. In addition to the requirement of specific fatty acids at a specified position (usually at sn-2 position), purity and other characteristics should also be considered. Normally, free fatty acids are chosen because of their ready availability in large quantities, low price and high reactivity compared to the EE. Lipases differ in selectivity for various fatty acids (Halling, 1987). A screening of different lipases is necessary to find one appropriate for the specific reaction system. Commercially available lipases have been extensively documented in laboratory experiments (Xu, 2000c). A careful survey is necessary for the synthesis and production of the required products. Recent progress in enzymatic production of structured lipids Positional distribution in SL products is of particular concern, being one of the aspects in which enzyme process is important. However, during the reaction acyl migration occurs leading to non-specificity of the products. This issue has been widely discussed with respect to the effects of parameters and reactor designs (Xu et al., 1998a,b,c). The mechanism of acyl migration has been illustrated in Fig. 11.1. The possible acyl migration steps during the enzyme acidolysis are illustrated in Fig. 11.7. In one recent study we have evaluated the possibility of using temperature programming for the suppression of acyl migration in batch reactions (Yang et al., 2005a). Acyl migration is a thermodynamic process and one which is very difficult to fully stop in actual reactions. It seems likely that suppressing acyl migration by a programmed change of reaction temperature without loss of reaction yield is feasible. The model reactions were the acidolysis of tripalmitin (PPP) with conjugated linoleic acid (CLA) or with caprylic acid (CA, 8:0) intended for human milk fat substitutes. Acyl migration was considerably inhibited in the temperature-programmed acidolysis with only slight reduction of acyl incorporation. Temperature programming was more significant in solvent-

+L OH

L

L

M

M

M

L

M –M

–L

–L

+M L

OH M

OH

M

M

M

+L

–M M

OH

L

L

+M

M

M

Fig. 11.7 Schematic diagram of acyl migration and formation mechanism of nonspecific by-products during enzymatic production of specific structured lipids (L = long-chain fatty acids, M = medium-chain fatty acids).

250 Modifying lipids for use in food free systems for the reduction of acyl migration. The study suggests that it is feasible to reduce acyl migration by means of programmed change of acidolysis temperature without significant loss of reaction yield. Fats for infant formula are one group of typical SL, where the second position is mostly occupied by palmitic acid. In two recent studies (Yang et al., 2003b; Nielsen et al., 2006) concerning the production of such fats, the source of the materials has been taken into consideration in a comparison with current commercial products, Betapol™ (Loders-Croklaan, Netherlands). Taking into account the high quantity of palmitic acid in lard (much of it already present in the sn-2 position), this fat has been evaluated for the production of infant fat using enzyme technology. Lard and soybean fatty acids were subject to acidolysis in a solvent-free system in a batch reaction (Yang et al., 2003b). The reaction substrates for the production were specially chosen to mimic human milk fats. The characteristics of the product, produced in the scale-up acidolysis under selected conditions, were similar to the fat in mothers’ milk. In another study, reactions were conducted in a PBR (Nielsen et al., 2006). Residence time and production operational stability were investigated on the kg-scale with lard and soybean oil fatty acids as substrates. There was no significant effect of residence time and operation time on 18:2 and 18:3 incorporation or on acyl migration in the sn-2 position. Adding antioxidants to the enzyme processes has been evaluated for the production of SL (Xu et al., 2005) since, for various reasons, the products from enzymatic processes are generally less stable (Jacobsen et al., 2003; Timm-Heinrich et al., 2003a,b, 2004; Nielsen et al., 2004). In the production process for structured lipids, the influence of addition of antioxidants before enzymatic acidolysis was investigated. Eight different antioxidants were screened: butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), propyl gallate (PG) ascorbyl palmitate, citric acid, ethylene diamine tetraacetic acid (EDTA), a tocopherol blend and lecithin. As substrates, oils with different degree of unsaturation were used. Enzyme activity was not significantly influenced by the addition of antioxidants in either a batch process or a PBR operation. α-Tocopherol concentrations remained stable for those mixtures where tocopherols were added. Primary oxidation products were reduced after acidolysis in the PBR, possibly due to the adsorption in the enzyme bed. The study by Xu et al. (2005) shows that the addition of antioxidants before enzymatic reactions has no negative effects on the reaction process, but none of the antioxidants chosen had a significant positive effect on either the reaction process or the oxidative status of the structured lipid produced.

11.3.3 Enzymatic acidolysis for the modification of phospholipids Phospholipids (PL) are used for different applications in food, pharmaceutical and cosmetic products, where they function as emulsifiers, stabilizers and antioxidants. Most applications of PL are based on natural products having

Chemical and enzymatic interesterification of lipids for use in food 251 complex composition, such as lecithin, which is a by-product of vegetable oil refining. Chemical and enzymatic modifications of PL to alter emulsifying and dispersing properties are common practice in industry, as the range of application of PL is widely extended (Schneider, 1997). The aim of these modifications is to adapt PL for specific application requirements by providing technical or physiological properties that the natural substance does not posses. Chemical modification includes hydrolysis, hydroxylation, acylation and hydrogenation (Bueschelberger, 2004). Commercial use of enzymes for PL modification is significant only for partial hydrolysis to produce lyso-PL with improved emulsifying properties compared to natural PL (Schneider, 1997). However, since the 1980s interest in enzymatic acyl modification has increased continuously as enzymes are able to tailor PL with defined fatty acid composition at the sn-1 and sn-2 positions. Replacement of the existing fatty acids in original PL with desired fatty acids might also improve physical and chemical properties, or even nutritional, pharmaceutical and medical functions. Enzymatic synthesis of PL has some advantages compared to chemical synthesis, such as selectivity and specificity. Furthermore, the use of toxic and deleterious solvents is greatly reduced, which simplifies subsequent purification and reduces solvent residue in the final product. For those interested in this area, published data concerning the use of lipases and PLA2 for PL acyl modification with acidolysis reactions have recently been reviewed by Adlercreutz et al. (2003) and Guo et al. (2005). Enzymes for acidolysis of phospholipids Enzymes that utilize PL as their natural substrate are called phospholipases and are named according to hydrolytic action on the PL molecule. The general formula for glycerophospholipids, together with the enzymes which hydrolyze the different ester bonds in the molecules, is shown in Fig. 11.8. In principle, these enzymes can also catalyze the reverse reaction, i.e. the ester bond formation. Phospholipase A1 and A2 are acyl hydrolases acting on fatty acids at sn-1 and sn-2 positions, respectively. Phospholipase C and D are enzymes that cleave the phosphorus–oxygen bond between glycerol and Phospholipase A1 O O O

O O O Phospholipase A2

Phospholipase C

Fig. 11.8

P

O

N + Me3

O

Phospholipase D

1,2-Diacyl-sn-glycerophosphorylcholine molecule and phospholipases which can be used to hydrolyze or form different ester bonds.

252 Modifying lipids for use in food phosphate, or phosphate and head group, respectively. Head group exchange of PLs will also change the chemical and physical properties; however, it is beyond the scope of this text to describe all reactions and products possible for these enzymes. The focus will be on the enzymatic acyl modification. Besides phospholipases, there are many other enzymes that can be used for PL acyl modification, such as sn-1, 3-specific lipases, which work for the modification of acyl group in the sn-1 position of PL. Lipases are at a more mature stage of development than phospholipases in terms of theoretical and practical understanding, and have in practice been the most common enzymes used for PL acyl modification at the sn-1 position. Lipases are able to catalyze both esterification and interesterification reactions. Phospholipase A2 (PLA2) from porcine pancreas has been the most commonly used enzyme for the exchange of fatty acids on PL at the sn-2 position. Pancreatic PLA2 functions reasonably well for esterification, but cannot catalyze interesterification since no acyl-enzyme intermediate is formed (Adlercreutz et al., 2003). However, it is possible to use this enzyme to exchange the fatty acid in the sn-2 position in a one-step reaction. Hydrolysis and re-esterification then occur in parallel. For industrial applications, enzymes are preferred in an immobilized form because it is possible to reuse them. Only lipases can currently be obtained commercially in immobilized form for PL modification; phospholipases in immobilized forms are not yet available. The majority of lipases sold commercially come from different microbial sources. Screening of different lipases showed that Thermomyces lanuginosa lipase (TLL) had higher activity compared to Rhizomucor miehei lipase (RML) and Candida antarctica lipases (CAL) during lipase-catalyzed acidolysis between soybean lecithin and caprylic acid (Peng et al., 2002). CAL has in certain cases been claimed to be non-specific; however, in a recent study it has been shown that during PL modification it is specific for the sn-1 position (Lyberg et al., 2005). Although lipases show good performance, the stability of the enzyme carrier is important. Commercial silica granulated TLL can easily be removed from the reaction medium in the presence of solvent during batch operation; however, in solvent-free systems, the immobilized lipase is not easily removed. During reaction, silica granulates are crushed and do not easily precipitate in the solvent-free system due to the highly viscous reaction medium. RML on resin carrier is more mechanically stable and can more easily be removed after solvent-free acidolysis reaction. However, during continuous operation in the PBR, the stability of silica granulated TLL can be maintained. Lipase from Rhizopus oryzae has also been reported to work well for PL modification at the sn-1 position (Adlercreutz et al., 2002); however, this lipase cannot be obtained commercially in the immobilized form. Reaction elucidation for enzymatic acidolysis of phospholipids Product yield under reaction equilibrium during acidolysis of PLs is determined by the substrate ratio. Maximum incorporation of acyl donors to a phospholipid

Chemical and enzymatic interesterification of lipids for use in food 253 (Incmax) can be calculated at certain substrate ratios assuming no by-product formation. An equation is given below:

Inc max (mol%) =

50 ⋅ S r Sr + 1

[11.27]

where Sr is the molar ratio between fatty acids and PL. Theoretical maximum of new fatty acids to be incorporated into PC is expected to reach 50 % for an sn-1,3-specific lipase or a PLA2. In practice, lipases are not equally specific towards different fatty acids and side reactions are difficult to avoid. A diagram of reaction and principle of the lipase-catalyzed interesterification and side reactions for the production of specific structured PL is given in Fig. 11.9. Lipase-catalyzed acidolysis of PLs is considered to be a two-step reaction involving hydrolysis and esterification. Lysophosphatidylcholine (LPC) produced in the first step is reactant in the second step. This intermediate is not thermodynamically stable, and intermolecular transfer of the fatty acid moiety from the sn-2 to the sn-1 position may occur. This non-enzymatic acyl migration is the reason for decreased yield and purity of the final product (Haraldsson and Thorarensen, 1999). LPC with fatty acids at the sn-1 position can be further hydrolyzed by the lipase to form glycerophosphorylcholine (GPC) which can be reacylated with novel fatty acids in the sn-1 position. If the acyl group migrates from the sn-1 position to the sn-2 position, the lipase has the potential to incorporate yet another new fatty acid into the sn-1 position, which would give rise to PC with novel fatty acid at both positions. Very little consideration has been given to the distribution of by-products formed during the reaction. The formation of GPC and LPC containing the novel fatty acids is a direct consequence of acyl migration and should be L L X

–L +L

OH L

+M

L –M

X

L OH X

M

–L +L

X

OH OH X

M M X

+M

M OH

–M

+M –M

X

OH M X

Fig. 11.9 Reaction process and mechanism for the production of specific structured phospholipids by enzymatic interesterification with sn-1,3 specific lipase (L = longchain fatty acids, M = medium-chain fatty acids).

254 Modifying lipids for use in food minimized in order to achieve a high product yield and high product purity. We have conducted reactions with different lipases in solvent and solventfree systems. As is the case with the other enzymatic reactions mentioned in this chapter, the performance of enzymatic synthesis of structured PL depends on many factors involving type of reaction, enzyme, temperature, water content, composition of substrates, reaction time, mode of operation, etc. Incorporation and final yield are highly affected by the selected parameters. Process technology for enzymatic acidolysis of phospholipids The type of the acyl donors plays an important role in the reaction rate of lipase-catalyzed reactions. High incorporation can usually be obtained with saturated fatty acids; however, fatty acids with a high degree of unsaturation are usually more difficult to incorporate (Peng et al., 2002; Hossen and Hernandez, 2005; Reddy et al., 2005). Different lipases show discrimination of long-chain PUFAs. For example, RML, TLL and CAL give higher incorporation of EPA compared to DHA (Peng et al., 2002). Reactivity also depends on the phospholipid head groups. During interesterification with EPA in hexane with RML the following order of reactivity was observed: PC > PI > PE > PS (PC is phosphatidylcholine, PI phosphatidylinositol, PE phosphatidylethanolamine, PS is phosphatidyserine) (Mutua and Akoh, 1993). The nature of PL also affects the incorporation rates of caprylic acid by TLL in hexane in the following order PC > PE > PA > PI (Peng et al., 2002). PL can be obtained commercially at different purities. PC is the most abundant phospholipid in nature and has usually been the substrate selected for enzymatic modification. Incorporation of novel fatty acids has been made with both pure phospholipid compounds and de-oiled lecithin (Peng et al., 2002; Vikbjerg et al., 2005a). Purified compounds have considerably higher price compared to the de-oiled lecithin. Selection will depend on the purity requirements. In most cases, lipase-catalyzed acidolysis reactions have been conducted with the assistance of organic solvents such as hexane or toluene (Mutua and Akoh, 1993; Adlercreutz et al., 2002; Hossen and Hernandez, 2005). The use of solvents increases the capital investment when the process is scaled up. Furthermore, it has been reported that increasing the amount of solvent reduces the recovery of PL more strongly than it increases the incorporation during TLL-catalyzed acidolysis (Vikbjerg et al., 2005a). If possible, it is recommended that the reaction should be conducted under solvent-free conditions. A clear elucidation of side reactions is important for practical operation in order to minimize by-products during reactions. Recently we produced caprylic acid-containing PC in a batch reactor by RML-catalyzed acidolysis between PC and caprylic acid in a solvent-free system (Vikbjerg et al., 2005b). A typical time course of the acidolysis reaction can be seen in Fig. 11.10. By-products were formed due to parallel hydrolysis reactions and acyl migration in the reaction system. Usually there was a tendency for a decrease in yields along with an increase in acyl incorporation. Response surface design was used to evaluate the influence of

50

100

40

80

30

60

20

40

10

20

0

0

20 40 60 80 Reaction time (h)

PC recovery (mol%)

Incorporation of caprylic acid (mol%)

Chemical and enzymatic interesterification of lipids for use in food 255

0 100

Fig. 11.10 Time course for immobilized Rhizomucor miehei lipase-catalyzed acidolysis reaction between phosphatidylcholine (PC) and caprylic acid. Reaction conditions: substrate ratio, 6 mol/mol caprylic acid/PC; enzyme dosage, 30 % (wt. % based on substrate); reaction temperature, 50 °C; water addition, 2 % (wt. % based on substrate).

major factors (enzyme dosage, reaction time, reaction temperature, substrate ratio and water addition) and their relationships on a number of responses reflecting the turnover of main reactions as well as side reactions. Several parameters important for the main reaction also affect by-product formation resulting in lower recoveries. All parameters besides water addition had an effect on the incorporation of caprylic acid into PC and LPC. Increased reaction time and enzyme dosage showed increased effect on incorporation into PC, while increased substrate ratio and reaction temperature showed opposite effects. The PC content decreased with increase of all parameters except for substrate ratio. In the solvent system using immobilized TLL, the incorporation of the desired acid was seen to increase with increase in temperature and substrate ratio (Vikbjerg et al., 2005a). Clearly optimization must be individually performed in each case. The exchange of fatty acids in PL has mainly been performed batch-wise in small screw cap vessels or glass bottles with either orbital or magnetic stirring. For larger scale production, it would be more convenient to operate in PBR as this allows continuous operation. Furthermore, due to the high enzyme dosages usually applied for lipase-catalyzed PL modification (> 40 % based on substrate), the final product is not easily removed from the reaction mixture after batch operation. To obtain the same conversion degree with the same enzyme load in the PBR, a very long residence time is required. Trials have been made for the continuous operation of the acidolysis reaction (Vikbjerg et al., 2005c). If no water was added to the substrate during reaction in the solvent-free system, very low incorporation of novel fatty acids was observed. Operative stability was tested for several days. Incorporation was highest at the beginning and decreased in the first 30 hours, where it stabilized afterwards. Incorporation of novel fatty acids was slightly higher when the water content was increased. In the presence of hexane, incorporations into PL were considerably higher and increased continuously in the first two

256 Modifying lipids for use in food days, and thereafter were stable with only a slight decline in incorporation over several days. Using PBR with solvent-free systems, it seems that small amounts of water are beneficial for the incorporation of desired fatty acids. Due to the high PL concentration, the substrates probably remove bound water from the enzymes, thus reducing the catalytic activity. Increasing water content will at the same time decrease the yield. Therefore, some kind of compromise needs to be made.

11.3.4 Glycerolysis for partial glyceride production Partial glycerides, more commonly known as mono- and diglycerides, have been produced commercially for many years. Today, they are widely used in the food, cosmetic and pharmaceutical industries as well as in the textile, fibre and plastic industries (Bornscheuer, 1995; Coteron et al., 1998; ElfmanBorjesson and Harrod, 1999; Bellot et al., 2001; Ferreira-Dias et al., 2001; Kaewthong and H-Kittikun, 2004). Mono- and diacylglycerols of edible fatty acids are approved by the EU as food grade additives. They have been given ‘Generally Recognized As Safe’ (GRAS) status by the US Food and Drug Administration (FDA), and can be used quantis satis (no permitted maximum level is specified) according to the European Parliament and Council Directive (European Parliament Council, 2004). According to World Health Organization (WHO) and the EU directive, mono- and diglycerides of fatty acids are required to contain at least 70 wt % MAG + DAG (mono- + diacylglycerols), at least 30 wt % MAG and maximum 7 wt % glycerol (European Food Emulsifier Manufacturers’ Association, 2004; European Parliament Council, 2004). Pure monoacylglycerols have excellent emulsifying properties, superior to diacylglycerols and mixtures of partial glycerol esters (Bornscheuer, 1995; Peng et al., 2000; Kaewthong and H-Kittikun, 2004). This is due to the MAG molecular structure with a favourable distribution between one hydrophobic fatty acid moiety esterified to a hydrophilic glycerol moiety. This combination, as well as the dietary safety profile, makes monoacylglycerols and mono/diacylglycerol mixtures very popular additives for facilitating uniform quality of food products among other applications (Krog, 1997). Accordingly, mixtures of mono- and diacylglycerols and distilled monoacylglycerols contribute to a market estimated at around 75 % of the worldwide emulsifier production. This corresponds to approximately 200 000– 250 000 tonnes produced per year (Moonen and Bas, 2004). The main applications of mono-diacylglycerols in foods are typically in fat-based products such as margarine, spreads, bakery products, cake mixtures, confectionery and the like (Krog, 1997; Kaewthong and H-Kittikun, 2004). Mono/diacylglycerols are often added to industrial food formulations in combination with other more hydrophilic emulsifiers, for instance in combination with hydrocolloids in dairy emulsions such as ice cream (Krog, 1997).

Chemical and enzymatic interesterification of lipids for use in food 257 Recent studies on DAG nutritional properties and dietary effects suggest that oils with high DAG content play an important role in reducing levels of serum TAG (Flickinger and Matsou, 2003; Watanabe et al., 2005). Thus, to fight obesity and other lifestyle-related diseases, substitution of TAG oils with DAG oils has attracted much attention recently. The first DAG cooking oil entered the Japanese market after its introduction in February 1999. With more than 70 million bottles sold (year 2003) and launched in the USA in 2005, the interest in a global DAG-market has indeed begun (Flickinger and Matsou, 2003; Kristensen et al., 2005). Chemical glycerolysis Today, commercial mono-diacylglycerol mixtures are widely manufactured using a glycerolysis reaction between glycerol and fats or oils, as illustrated in Fig. 11.11. The currently used glycerolysis reaction is performed at high temperatures (220–260 °C) with inorganic alkaline catalysts, such as NaOH or Ca(OH)2. Approximately 30 to 60 minutes of processing leads to an equilibrium mixture with partial glycerol esters and excess glycerol. To remove impurities and achieve high-purity products, subsequent removal of excess glycerol by distillation followed by molecular/short path distillation (SPD) processing is often performed (Bornscheuer, 1995; Krog, 1997; Rosu et al., 1997; Elfman-Borjesson and Harrod, 1999; Xu et al., 2000, 2002; Xu, 2000b; Bellot et al., 2001; Kaewthong and H-Kittikun, 2004; Lee et al., 2004). O O C O O C OH O

O O C

OH

O C

OH Catalyst

OH

OH

O C

O

2

+2

O C O

O

O C

Trillinolein (TAG)

Glycerol OH O O C OH OH OH O O C

Mixture of partial acylglycerols (MAG + DAG)

Fig. 11.11

Reaction scheme for production of mono-diacylglycerols by glycerolysis.

258 Modifying lipids for use in food The reaction can, to a certain extent, favour the formation of certain glycerol esters depending on the reaction conditions such as glycerol to oil ratio, reaction time and pressure (Peng et al., 2000). The effect on the product distribution of MAG, DAG, TAG and glycerol at equilibrium conditions after glycerolysis of different blended glycerol to oil ratios is illustrated in Fig. 11.12. Current chemical glycerolysis processing is usually conducted with a relatively low molar ratio of glycerol to oil of about two (Rendon et al., 2001). This provides an amount of non-reacted glycerol of about 8 wt% (Fig. 11.12b) and a distribution between MAG, DAG and TAG of 45–55 %, 38–45 % and 8–12 %, respectively (Krog, 1997). Enzymatic glycerolysis Today, it is well known that the Western diet is characterized by a general low intake of essential n-3 polyunsaturated fatty acids (PUFAs) and an 100 %

(wt %)

80 % 60 %

MAG DAG TAG

40 % 20 % 0% 0

10

20 30 Molar ratio gly: oil (a)

40

50

100 %

(wt %)

80 % Glycerol MAG DAG TAG

60 % 40 % 20 % 0%

0

10

20 30 Molar ratio gly: oil (b)

40

50

Fig. 11.12 Calculated product distribution at equilibrium conditions after glycerolysis reaction with different molar ratios of glycerol to oil. (a): Distribution between MAG, DAG and TAG (fat phase). (b): Distribution in the complete reaction mixture (fat phase and glycerol). Calculations are based on binomial random distribution with probability calculations of ester formation between mole fatty acids (FA) and mole hydroxyl groups (OH) and expressed as weight percentages (adapted from Damstrup et al., 2006).

Chemical and enzymatic interesterification of lipids for use in food 259 overabundance of n-6 fatty acids compared to international recommendations (Volker and Garg, 2001). In Australia and the USA, the current intakes of n-3 PUFAs are for instance less than 100–200 mg/day (Peters and Nurmikko, 2002). The intake is far from the recommendations of 1–2 g/day from organizations such as WHO, FDA and American Health Association (AHA) (Anselmino, 2004). This has led to a great deal of commercial interest in producing health-promoting and functional lipids with specific fatty acid profiles to overcome the gap between actual and recommended n-3 PUFA intake. Vegetable oils are considered as easily accessible, relatively cheap and neutral flavoured triacylglycerol raw materials for carrying nutritionally important PUFAs. However, the current chemical glycerolysis process makes the use of raw materials based on PUFA-vegetable oils difficult due to their sensitivity to oxidation at high temperatures (Peng et al., 2000). In contrast, enzymatic glycerolysis requires only a low temperature, below approximately 80 °C, which makes the use of heat-sensitive MAG and DAG with PUFAprofile feasible. Furthermore, the gentle enzyme technology reduces some of the heat-accelerated problems with development of bad flavour, dark colour and unwanted side reactions (Bornscheuer, 1995; Elfman-Borjesson and Harrod, 1999; Bellot et al., 2001; Kaewthong and H-Kittikun, 2004). Accordingly, lipase-catalyzed glycerolysis of vegetable oils is believed to be a potential alternative/supplementary industrial method for the production of nutritional high-valued monoacylglycerols with important n-3 PUFAs. This opportunity for product and process development has led to a great interest in the enzymatic glycerolysis process, not only from academia but also from industry since the mid-1980s. Glycerolysis under enzymatic catalysis has similar reaction routes to chemical glycerolysis, as shown in Fig. 11.11. Reaction equilibrium is still a central determinant for the evaluation of the product yield as shown in Fig. 11.12. However, because of its use of low temperature and its possible use of solvents, reaction equilibrium is not a target, which is imperative to achieve through the enzymatic glycerolysis process. This is because the operating conditions make it possible for a product ‘fishing’ strategy. All possible effort is being made to speed up the procedure of reaching reaction equilibrium on the other hand. This is illustrated in Fig. 11.13. Possible methods for such ‘fishing’ have been tested using crystallization, chromatographic separation, extraction, etc. as a step to break the reaction equilibrium and force the reaction to form more product (Peng et al., 2000). Recent progress has been made with the idea of using solvent partitioning to push the reactions beyond reaction equilibrium (Guo and Xu, 2005; Yang et al., 2005b, Damstrup et al., 2005, 2006). In such systems with the selected media, the reaction efficiency and effectiveness have been dramatically improved, and higher yields of monoacylglycerols and diacylglycerols have been obtained. Figure 11.14 compares the reaction behaviour of glycerolysis of sunflower oil using ionic liquid and tert-butanol as system media with a solvent-free system. The first

260 Modifying lipids for use in food Lipases Fats and oils + glycerol

Monoglycerides + Diglycerides

• Membrane separation • Extraction • Fractionation • Adsorption • etc.

Illustration of product ‘fishing’ for the enzymatic production of partial glycerol ester glycerides.

Yield of monoglyceride (mole %)

Fig. 11.13

100 80

60 40

20 0 0

2

4

6 8 10 12 Reaction time (h)

22 24

Fig. 11.14 Different reaction behaviours of glycerolysis of sunflower oil in ionic liquid, tert-butanol and the solvent-free system (0.5 mmol sunflower oil and 5 × 0.5 mmol glycerol for reaction at
, 40 °C; ◊, 50 °C; 䉭, 60 °C; , 70 °C in 2.2 g ionic liquid; 䉱, 50 °C in 2.5 g tert-butanol; •, 2 mmol sunflower oil and 5 × 2 mmol glycerol for reaction at 70 °C in solvent-free system) (adapted from Guo and Xu, 2005).

two systems have been shown to have good potential for industrial implementation. Recent progress in enzymatic glycerolysis In general, the major challenge in using enzymes in low-temperature glycerolysis reactions is that the system comprises of three heterogeneous phases: a hydrophobic oil phase, a hydrophilic glycerol phase and a solid enzyme phase, as illustrated in Fig. 11.15. Since enzymes in their native forms have hydrophilic characteristics, glycerol often binds to the enzyme particles and makes the access of the hydrophobic oil molecules to the enzyme difficult (Kristensen, et al., 2005; Yang et al., 2005b). Poor miscibility of the glycerol and oil at low temperatures in combination with high reactant viscosity results in a reaction system with high mass transfer limitations. As a result, long reaction times and/or low conversion of reactants generally make the reaction inefficient.

Chemical and enzymatic interesterification of lipids for use in food 261 Enzyme (Solid)

Glycerol (Hydrophilic) Inhomogeneous system

Oil (Lipophilic)

Fig. 11.15

Illustration of the heterogeneous reactant mixture used for enzymatic glycerolysis.

However the sustainability of an effective system has, to a certain extent, relationships with the content of glycerol in the system. In the case of DAG production, the theoretical stoichiometric amount of glycerol in the reaction system is low. Although the reaction time remains long, it is possible to have a sustainable system for DAG production without the usage of any medium assistance (Kristensen et al., 2005). With a sufficiently low glycerol amount, a conversion of TAG up to 60–70 % can be achieved (Fig. 11.16). However, for MAG production, assistance of media or involvement of alternative techniques is normally required to improve the formation of MAG in reasonable time and amounts. Solid phase crystallization (McNeill et al., 1990; Bornscheuer et al., 1994; Rosu et al., 1997), glycerol adsorbed to silica gel (Aha et al., 1998; Elfman-Borjesson and Harrod, 1999; Rendon et al., 2001), usage of protected glycerol (Akoh, 1993) and reactions performed in different media such as supercritical CO2 (Jackson and King, 1997) or organic solvents (Bornscheuer et al., 1994; Kwon et al., 1995; Rendon et al., 2001) are among the many interesting approaches to improve reaction efficiency and/ or product quality. Many of the approaches work successfully on a laboratory scale, and a great deal of knowledge, revealed from 20 years of progressing research, has been accessible. However, due to practical difficulties, the big industrial breakthrough for MAG production has still not taken place. For instance, solid phase crystallization and glycerol adsorption are difficult to handle continuously, and the reaction time required is still lengthy from an industrial point of view. At present, the high cost of the enzyme also makes the use of lipases in industrial applications uneconomic (Yang et al., 2003a; Kaewthong et al., 2005). To overcome some of the problems with high enzyme costs, a widely used strategy is to employ the lipase in an immobilized form. Immobilization of the enzyme onto a solid support material allows easy separation and reutilization of the enzyme. Furthermore, the use of immobilized enzymes on porous supports eliminates some of the problems arising with the use of suspended enzyme powders, such as the tendency to aggregation and attachment to the wall of the reactor, as the enzyme spreads on a large surface area (Barros et al., 1998).

262 Modifying lipids for use in food Lipase AK

Lipase PS-D

100

Composition (wt %)

Composition (wt %)

100 80 60 40 20

80 60 40 20 0

0 0

5

10 15 20 Time (h)

25

0

30

5

30

25

30

100

Composition (wt %)

Composition (wt %)

25

Novozym 435

Lipase F-AP15 100 80 60 40 20 0

10 15 20 Time (h)

80 60 40 20 0

0

5

10 15 20 Time (h)

25

30

0

5

10 15 20 Time (h)

Fig. 11.16 Time courses for enzymatic glycerolysis with different lipases in batch reactors (䉭, TAG; 䊊, 1,3-DAG; 䊉, 1,2-DAG; 䊐, MAG; ■, total DAG (adapted from Kristensen et al., 2005).

Other benefits from immobilized enzymes compared to non-immobilized enzymes are improved activity and long-time stability in various organic media (Goto et al., 2005; Nakaoki et al., 2005). For instance, it has been shown that when lipases are immobilized onto a hydrophobic matrix, their activity increases 51-fold and exhibits superior heat resistance compared to the native lipase in esterification reactions of lauric acid in isooctane system (Goto et al., 2005). Since Zaks and Klibanov discovered that lipase enzymes act well in organic solvents (Zaks and Klibanov, 1984, 1985), many investigations of lipase-catalyzed interesterification reactions in solvents confirm the benefits of using non-conventional media. Among the useful solvents in interesterification reactions are dioxan, n-hexane, n-heptane, acetonitrile, acetone, isooctane and tert-butanol and tert-pentanol (Goto et al., 1995; Hess et al., 1995; Elfman-Borjesson and Harrod, 1999; Bellot et al., 2001; Rendon et al., 2001; Kaewthong and H-Kittikun, 2004; Yang et al., 2005b). In our experience, the usage of selected media is believed to provide potential approaches for glycerolysis with industrial applications (Guo and Xu, 2005; Damstrup et al., 2005). In such systems, commercially available immobilized CALB is shown to be one of the most stable enzymes. In spite

Chemical and enzymatic interesterification of lipids for use in food 263 of lowered productivity due to the presence of solvent, glycerolysis reaction in the tertiary alcohols, tert-pentanol (2-methyl-2-butanol) and tert-butanol (2-methyl-2-propanol), is found to have great potential for efficient glycerolysis (Damstrup, et al., 2005, 2006). These solvents offer benefits beyond the drawbacks related to lowered productivity. Amongst these advantages are high MAG yield/purity in the product mixture and very short reaction time together with a well-preserved PUFA profile of the MAGs (Damstrup et al., 2005, 2006; Yang et al., 2005b). In addition, fluid solvent systems make continuous operation in PBR feasible. The obvious benefits of using continuous PBR are: ease of separation between reactant mixture and catalyst; reuse of the enzyme without the need for a prior separation; a cost-effective reactor design; and long-term production due to high density loading of the immobilized enzyme into the reactor (Kaewthong et al., 2005; González Moreno et al., 2005). These advantages indeed promote the arguments for using solvents in enzymatic glycerolysis. Finally, a process design including reusability of the solvent minimizes the drawbacks from lowered space-time yield. A good review of lipase-catalyzed production of high-quantity monoacylglycerols by Peng et al. (2000) is recommended for further reading. In addition, a recently published and excellent patent review on lipid technology including patent literature involving monoglycerides by Lai and Lo (2005) is also recommended.

11.4

Remarks and future trends

The concept of interesterification, as defined in this chapter, with catalysis either by enzymes or chemicals, can be widely explored for unlimited product or process development. The subject is becoming more interesting and attracting more attention. Chemical interesterification, to a large extent, is a mature technology that offers the prospect of many new applications in industry. Chemical randomization of oils and fats for plastic fat production, although in practice for about a century, is gaining new momentum with the coming ‘trans-free’ ruling for the future market. The method has been widely used but the ‘share’ of industrial capacity using this method for plastic fat production is expanding. Chemical glycerolysis is a common method for monoacylglycerol production in the present food industry, and it is expected to dominate monoacylglycerol production in the foreseeable future. Another case of chemical interesterification, which is receiving more and more importance, is chemical methanolysis for the production of biodiesel. Energy crises in the past year have led to the expansion of biodiesel production capacity from oils and fats. Enzymes as catalysts for the interesterification of oils and fats, although far from mature and a long way from achieving widespread implementation

264 Modifying lipids for use in food in industry, have been a focus for extensive studies for decades. Even though nobody expects a fast increase in the applications of enzymatic interesterification in industry, interest in using enzyme processes and realization of their benefits have increased in the industrial sector. More and more industrial think-tanks are including this area in their development strategy. As an alternative to chemical randomization for plastic fat production, enzyme processes, even with such a low-priced product, have made an entry in the oils and fats industry in very recent years. Fats produced from the enzyme process in general can be used for margarine production without significant change of properties, though there are some unusual aspects compared to either blended or chemically randomized fats (Zhang, 2005). From the point of view of oxidation during storage, the enzymatically-produced fats have advantages over the chemically randomized product; the latter develops high peroxide values during storage. With regard to physical properties, the former can form the required crystal type and textures along with suitable solid fat profiles which are comparable to those of the chemically randomized fats. However, margarines produced from the enzymatically-interesterified fats become more physically stable with a higher conversion degree of the interesterification. The control of the conversion degree, therefore, becomes highly critical to achieving good quality products. Furthermore, the optimization of margarine formulation, such as the use of diacylglycerols to postpone crystal transformation and the optimization of process conditions for margarine production, needs more studies to cater for the new requirement of using the partially interesterified fats from the enzymatic processes. For the enzymatic production of structured lipids with structural specificity concerning the locations of fatty acids, enzyme processes are the only potential methods for large-scale production. Products of high value intended for functional foods and pharmaceutical applications provide only a small market. So far, a few products, such as fats for infant formula, fats for confectionery products, etc. have been available in the market. The initial criteria are the functional values of the products and their market demands. The economical balance of the process is the crucial point for industrial consideration. So far, technical issues for enzymatic interesterification concerning the production of structured lipids are not, in general crucial. The benefits of such products in human nutrition remain to be explored. Process optimization for a defined product needs a lot more work to reduce the cost of the process at engineering and management levels. Concept development for process engineering is a high priority for future research. A package of knowledge for building up a new plant is still far from available. For enzymatic modification of PL, an understanding of the reaction systems is just the beginning. Before these types of reactions can be implemented industrially, a lot more work will need to be done to increase efficiency. For these reactions to be applicable, their operative stability needs to be better understood and a longer lifetime of the immobilized enzyme is desired.

Chemical and enzymatic interesterification of lipids for use in food 265 Although solvent-free systems may seem to work well during batch operation, there is no information as to whether the lipase can actually be re-used. In terms of stability during continuous operation, it may be beneficial to use non-polar solvents as higher incorporation can be obtained for a prolonged time compared to solvent-free systems. Even though major by-product formation occurs during lipase-catalyzed acidolysis, it should be kept in mind that these by-products (LPC and GPC) are themselves valuable products having wide applications in the same area as the original material. These compounds can be purchased in purified form from different companies, and are usually sold at considerably higher price compared to natural PL. Concerning the production of partial glycerol esters, it is well documented that enzymatic glycerolysis has become a powerful method in terms of MAG/ DAG quality and functionality, which makes it possible to expand their fields of applications into functional foods, pharmaceuticals, etc. Of particular interest is the possibility of producing ‘new’ MAGs with PUFA profiles that differ significantly from MAGs produced by traditional chemical glycerolysis methods. With the introduction of efficient long-lasting commercially available immobilized lipases and the benefits of large-scale PBR, the industrial breakthrough for enzyme-catalyzed glycerolysis has moved closer. Accordingly, in the future, it is most likely that the fat and oil industry will, to a certain extent, adapt the enzyme-catalyzed glycerolysis into industrial plants, even although the chemical process will still occupy the main position for many years to come. As discussed in the above section, a medium, usually an organic solvent, has been an advantage in achieving a high degree of efficiency and effectiveness in the process and good product yields. The use of solvents, however, is contrary to the aim of an environmentally friendly and energy-reduced enzymatic process due to the extra processing required for solvent removal. From this point of view a solvent-free medium or the application of nonevaporative solvents such as ionic liquids could be a better solution. This certainly opens a new window for future visions.

11.5

Sources of further information and advice

Enzymatic interesterification has been attracting significant interest since the 1980s and chemical interesterification for even longer. A large amount of information has been collected and accumulated. A large number of recent review papers and chapters concerning each topic have been cited in the relevant sections. A number of company homepages are worth visiting, including technology providers (e.g. Desmet Ballestra, Alfa Laval), enzyme providers (e.g. Novozymes, Amano) and product providers (e.g. ADM, Unilever, Danisco, Degussa, Karlshmns, Aarhus United, Kao, Nisshin Olillio, Loders-Croklaan). These companies provide a real picture of interesterification in industry.

266 Modifying lipids for use in food

11.6

Acknowledgements

Financial support for our group research activity is acknowledged from the Research Council for Technology and Production, the Danish Dairy Foundation, Danisco, and the Centre for Advanced Food Studies as well as other assistance from industry and collaboration partners.

11.7

References

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268 Modifying lipids for use in food HOSSEN M and HERNANDEZ E (2005), Enzyme-catalyzed synthesis of structured phospholipids

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270 Modifying lipids for use in food ROSU R, UOZAKI Y, IWASAKI Y

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12 Plant breeding to change lipid composition for use in food D. J. Murphy, University of Glamorgan, UK

12.1

Introduction

In global terms, plant lipids are the second most important source of edible calories in the human diet (after carbohydrates). Plant lipids are also sources of several essential vitamins and nutrients. For example, plant lipids are the ultimate source of the so-called ‘essential fatty acids’ that are an obligatory component of the diet of all mammals – ever since that time many millions of years ago when our distant animal ancestors lost the ability to introduce double bonds beyond the ∆9 position in long-chain fatty acids. Since the dawn of agriculture, certain plant species have been cultivated specifically for their lipid compositions. For example, the earliest olive plantations have been dated to more than nine millennia before the present day, and maize may have been domesticated in Mesoamerica as early as ten millennia ago. In addition to their acyl lipid ingredients, plants are important dietary sources of a host of other lipophilic compounds, including vitamins A and E and a range of phytosterols. Most of our dietary plant lipid is derived from oil crops and is in the form of either ‘visible’ (e.g. oils, margarines, chocolate) or ‘invisible’ (cakes, confectionary, processed foods) fats. In the past, the lipid compositional requirements for these products have been provided by a variety of commodity plant oils that may be blended together and/or chemically modified (e.g. by hydrogenation) for a particular edible application. More recently, there has been a move towards a greater segmentation of the commodity plant oils market, with far more stress placed upon the initial composition of the plant oil itself. Hence, the increasing demand for plant oils that are enriched in monounsaturates, very long-chain ω-3 fatty acids, carotenoids, phytosterols

274 Modifying lipids for use in food and tocotrienols. With an increased willingness by buyers to pay a premium for such nutritionally enhanced oils, it is becoming more economic for growers and processors to segregate such value-added products. This in turn is driving plant breeders to select new varieties of oil crop designed for consumers who are becoming ever more aware of lipid-related nutritional issues, such as the presence of trans-fatty acids and saturates in foodstuffs of all kinds. In this chapter, I will describe how plants are being manipulated through various forms of breeding in order to supply this wide range of dietary lipids, with a special focus on fatty acid composition.

12.2 General perspective 12.2.1 Margarine – the beginnings of an industry for plant lipids Prior to the late 19th century, there was no plant lipids industry and the few dedicated oil crops that were grown tended to be consumed locally. In Northern Europe, rapeseed had been grown as an oil crop since Roman times, but the oil was mainly employed for lighting, as a supplement to tallow and beeswax. The few edible oils that were transported and traded, such as olive oil, were treated very much as luxury products away from their region of origin around the Mediterranean. In much of Europe and the New World, virtually all of the visible dietary fat was derived from dairy and meat products, and edible vegetable oils were a distinct rarity. Indeed, a few thousand years ago, Northern Europeans became so dependent on milk products that most of them still carry a mutation in the lactase gene that enables it to be expressed throughout life, rather then ceasing its activity after weaning as it does in most members of our species. This is the reason that 98 % of adult Thais and 100 % of Amerindians are lactose-intolerant, while in milk-drinking Sweden the figure is only 2 % (Enattah et al., 2002). Plant lipids were first propelled into mainstream food production by a technological innovation in the late 1860s. This happened when a French chemist called Hippolyte Mège Mouriès produced what we now know as margarine. Even in those days, plant lipid researchers had to respond to the needs of industry and government and the work of Mège Mouriès followed a call by French emperor, Napoleon III, for research into a possible alternative to butter as a high-calorie foodstuff for the French army. After a considerable amount of experimentation, the raw material that Mège Mouriès selected for the new product was a solid fatty acid fraction called margaric acid, because of the lustrous pearly drops of the crystalline form that are reminiscent of pearls, which are called margarites in Greek (this is also the derivation of the name, Margaret). The earliest forms of margarine were mixtures of animal and plant fats but, initially, this mixed lipid product was not a great commercial success. Two technical advances tipped the balance towards using only plant fats in margarine and allowed the new fatty spread to compete more effectively

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with butter. Firstly, improved refining methods allowed the purification of a greater variety of liquid oils and solid vegetable fats that could be blended to make a good spreadable margarine. Secondly, the process of hydrogenation, which was invented in 1901 by German chemist William Normann, enabled the large-scale conversion of relatively cheap plant oils into solid fats. Not only did the hydrogenation process produce a good, inexpensive butter substitute, it also significantly reduced the amount of oxidation-prone polyunsaturates in the solid margarine, which greatly extended its shelf life and therefore its utility for consumers. Margarine soon spread around the world and, a few years later, the American author, Mark Twain, overheard the following conversation between two businessmen aboard a Cincinnati riverboat: Why, we are turning out oleomargarine now, by the thousands of tons. And we can sell it so dirt-cheap that the whole country has got to take it – can’t get around it, you see. Butter don’t stand any shows – there ain’t any chance for competition. However, US dairy farmers soon mounted an effective counter-attack against a product that they believed would ruin their livelihoods. Thanks to their political clout, especially in strong dairy States like Wisconsin, the farmers managed to get margarine classified as a ‘harmful drug’ that was subject to restricted sales. Margarine was also heavily taxed; stores had to be licensed to sell it and, like alcohol and tobacco, it was promptly bootlegged. Ironically, in view of its origins as a food for the French army, the US government refused to purchase margarine for the use of its own armed forces. Finally, as a way of making it even less attractive, some states did not allow yellow margarine to be sold, so the shopper was faced with purchasing an unsightly, off-white slab of fat (van Stuyvenberg, 1969). Surprisingly, the punitive federal taxes on margarine were not abolished until 1950; yellow margarine could not be sold in Wisconsin until 1967; and, to this day, the sale of yellow margarine is prohibited in the Canadian Province of Quebec. Some of this hysteria about margarine might appear somewhat comical, but there are still echoes of similar attitudes to food innovation in some of the current debates about aspects of the use of genetic engineering for crop improvement, including the modification of fatty acids in oil crops.

12.2.2 Diversity of plant lipids Before looking at plant lipid modification in more detail, it will be useful to consider the historical role of these products as part of our diet and to examine the crops that are grown specifically as sources of dietary lipid. Globally, there are now just 15 major crops that supply most of the human diet and five of these are crops with relatively high oil contents, namely soybeans, oil palm, maize, peanuts and coconut (Harlan, 1992). About two-

276 Modifying lipids for use in food thirds of the 110 million tonnes of commercially produced plant oil is from soybean, palm and canola (genetically improved rapeseed). The major fatty acids from the world oil supply are palmitic, linoleic and oleic acids. In addition to these major edible fatty acids, many unusual fatty acids can accumulate in seed oils of other plant species, as is shown in Table 12.1. Sometimes these unusual fatty acids might comprise in excess of 90 % of the seed oil (Hildebrand et al., 2005). As we can see from Table 12.1, unusual fatty acid modifications include variations in carbon chain length and degree of unsaturation. Most naturally-occurring fatty acids have double bonds in the cis configuration, although occasionally trans double bonds are also found, e.g. in photosynthetic membrane lipids. There are also different positional isomers, conjugated unsaturated, acetylenic, hydroxy, epoxy and keto fatty acids. Cyclopropenoids, cyclopentenoids and even fluoro fatty acids add more diversity to the list of fatty acid species. These more exotic fatty acids are rarely used in edible products; rather they are part of the 20 % of plant oils that are used for non-food purposes, i.e. as oleochemicals in the manufacture of detergents, resins, paints, polymers and pharmaceuticals or as animal feedstuffs (Murphy, 1994).

Table 12.1

Accumulation of novel fatty acids by some oil-producing plants.

Fatty acida

Amountb

Plant species

Actual and potential uses

8:0 10:0 12:0 14:0 16:0 18:0 20:0 22:0 24:0 18:1∆6 cis 18:1∆9 cis 22:1∆13 cis 18:2∆9,12 cis α18:3∆9,12,15 cis γ18:3∆6,9,12 cis 18:1–hydroxy 18:1–epoxy 18:2 9c12a 18:3–oxo 18:3–conj 20:1/22;1wax

94 95 94 92 92 65 33 48 19 76 78 58 75 60 25 90 60 70 78 70 95

Cuphea avigera Cuphea koehneana Litsea stocksii Knema globularia Myrica cerifera Garcinia cornea Nephelium lappaceum Brassica tournefortii Adenanthera pavonina Coriandrum sativum Olea europaea Crambe abyssinica Helianthus annuus Linum usitatissimum Borago officinalis Ricinus communis Crepis palestina Crepis alpina Oiticica Tung Simmondsia chinensis

Fuel, food Detergents, food Detergents, food Soaps, cosmetics Food, soaps Food, confectionery Lubricants Lubricants Lubricants Nylons, detergents Food, lubricants Plasticisers, nylons Food, coatings Paints, varnishes Therapeutic products Plasticisers, cosmetics Resins, coatings Coatings, lubricants Paints, inks Enamels, varnishes Cosmetics, lubricants

a

% % % % % % % % % % % % % % % % % % % % %

Fatty acids are denoted by their chain length followed by the number of double bonds or nature of other functionalities. b Percentage of total seed or mesocarp fatty acids; data are taken from Murphy (2001).

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12.2.3 Markets for plant lipids Despite recent increases in the global consumption of animal products, for most people, plants are still the major source of dietary fats, although they are often unaware of exactly how much and what type of lipid they are consuming (this is especially true for ‘invisible’ fats). Plant lipids are normally obtained in the form of liquid vegetable oils either from oilseed crops like soy or rape, or from oil-rich fruits like oil palm or olive. In 2005, the ten most important commercial oil crops produced a total of 107 million tonnes of oil with a value of about $70 billion (Oil World, 2005). Therefore, plant sources supply about 80 % of the total global demand for traded fats and oils – the non-plant fats and oils are mainly obtained from animal, fish and dairy sources. Plant-derived oils tend to have relatively narrow fatty acid profiles, being mainly dominated by C16 and C18 saturates, and by the C18 mono-, di-, and tri- unsaturates. Such a profile has suited the treatment of plant lipids as generic commodity oils, to be produced and transported in bulk and to be blended and/or hydrogenated as necessary to fit a particular end use. This is especially apparent in the processed food sector where most plant lipids are used. Hence, different plant oils may be blended in different proportions to produce the various types of solid fat that are used in products such as spreads and shortenings. Since the 1980s, there has been an increasing segmentation of the plant oils market as food producers seek to highlight oils from particular crops, which may have special attributes that can add value to an end product. For example, high linoleate sunflower oil is favoured for certain ‘high polyunsaturate’ margarines, while cold-pressed, unprocessed ‘virgin’ olive oil is favoured for its organoleptic qualities. In contrast, other plant oils, such as soy and rape, have tended to remain as generic commodity products. In the case of rape oil, this is rather odd because the oil has a very high oleic acid content, which makes it suitable to be branded as ‘high in monounsaturates’. There are also varieties of oilseed rape that have less than 4 % linolenic acid, which avoids the need for hydrogenation and potentially allows the oil to be marketed as ‘low in trans-fatty acids’. Despite these favourable attributes, however, rape oil still tends to be treated as a low-cost commodity, rather than as a higher value, segmented-market product like olive oil. This brings us to an important point about the reasons for the manipulation of plant lipid composition. Since the early 1990s, many new types of plant oil crop have been developed, and many more are in the pipeline. However, many of these new plant oils have not been taken up by the market, or have not been exploited to their full potential. Part of the reason for this is that many of the modifications of plant lipid composition, especially by genetic engineering, have been technology-driven, rather than being market-led. This means that markets may be unprepared, unaware or simply unwilling to take on the new products.

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12.3 Plant breeding Plant breeding, and indeed any other form of breeding, is based on two mutually dependent processes: namely genetic variation and selection. A given population must exhibit some heritable variation in a character of interest to a breeder before selection is possible. Hence one cannot usefully breed from excessively inbred or clonal populations. Equally, it is vital to have a system that enables recognition of variability in a character, especially if such a character is cryptic. Therefore, while the early farmers could readily select for seed size or taste, a character such as fatty acid composition could not be selected for directly until the advent of modern techniques of lipid analysis in the 20th century.

12.3.1 Discovering variation There are at least 500 000 species of higher plants and many of these accumulate storage oils in either their seeds and/or fruits. Unlike membrane lipids, which are extremely constrained in their acyl chain lengths and functionalities, storage lipids appear to be able to contain virtually any type of acyl moiety with chain lengths extending from as little as C8 all the way up to C24 (see Table 12.1). From the 1960s, the regional research division of the USDA at Peoria, Illinois has been undertaking a survey of some of the enormous diversity in acyl lipid composition of oils from plants collected from around the world. It has been found that there are many hundreds of plants, which are currently not grown as crops, but which have oil-rich seeds that accumulate novel and potentially useful fatty acids. Since the 1990s, much of the focus on such plants has entailed the isolation of genes that regulate the formation of the exotic fatty acids and their transfer to mainstream oil crops like soy or rape. However, as we shall see below, this genetic engineering approach has not been without its problems. Quite apart from the technical problems of persuading an existing crop to accumulate novel fatty acids in the right place and in the appropriate quantity, there is the problem of managing their cultivation and processing. After all, a soy seed that contains regular soy oil looks exactly the same as one that has been engineered to contain a non-edible, and possibly toxic, industrial oil. An alternative strategy that is now beginning to receive more deserved attention is to domesticate the original plants that made the exotic fatty acids in the first place, so that they can be grown as commercial crops in their own right.

12.3.2 Domesticating new oil crops The great advantage of domesticating existing plants rather than developing transgenic crops is that these existing plants are already adapted to accumulate their exotic fatty acids in the appropriate cellular compartment, namely in the triacylglycerols of their storage oil bodies. In the native plants, these

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exotic fatty acids are hardly ever found in cell membranes or in any other acyl lipids where their presence could be damaging. A further advantage of novel oilseed crops is that the seed oils already contain accessory stabilizing agents, such as antioxidants, which prevent the breakdown of some of the more highly reactive fatty acids such as conjugated polyunsaturates and those containing acetylenic bonds. Although many of the potential new crops may already be excellent sources of useful products, such as novel fatty acids, they are often not suitable for large-scale agriculture. The reason for this is simple: these plants have not been optimized for agronomic performance over centuries or even millennia, as have some of our more familiar crops. They suffer from the usual characteristics of wild plants; for example, they tend to flower asynchronously throughout the summer and therefore do not produce their seeds at a single time, which makes harvesting very difficult. They often produce seed pods that are prone to shatter before or during harvest, resulting in a loss of many of the seeds. Often, the canopy architecture of the plant is not suitable for existing harvesting machinery. They may be susceptible to a variety of diseases or pests, including fungi and insects. Finally, in the case of oilseeds, although they may contain as much as 90 % of a novel fatty acid in their seed oil, the overall oil yield in tonnes per hectare (T.ha–1) may be relatively low. The improvement of these important agronomic characters requires the manipulation of numerous complex traits. Companies are often dismayed by the prospect of domesticating new species, citing the example of major crops such as wheat, which is still being improved after more than 10 000 years of domestication. Nevertheless, we can now be more optimistic about the prospects for crop domestication. Many of our newer commercial crops have been improved at a much more rapid rate than wheat since the mid-20th century, thanks to the use of modern breeding techniques. Examples of such crops include hybrid maize, rapeseed, sunflower and soybean, which have only been grown as mainstream commercial crops for a century or less. There is also now the prospect of using biotech methods, such as marker-assisted selection (see below) to accelerate the development of new oil crops.

12.3.3 Creating new variation The domestication of new crops is a relatively recent option for plant breeders. In the past, breeders had to rely on the existing portfolio of crops in order to select for useful variation. Remarkably, almost all of our major crops were domesticated many thousands of years ago and no new mainstream crops have been domesticated since Roman times. The major revolution in plant breeding over the past hundred years has been the development of techniques that enable breeders to create additional variation in populations of the existing domesticated crops, even ones that are relatively inbred. To this we can add the development of a huge range of technological tools that facilitate selection for genetic characters that were invisible to our forebears. Before the early

280 Modifying lipids for use in food 20th century, breeders had to rely on existing variation resulting from ‘spontaneous mutations’ caused by errors in DNA replication. Such spontaneous mutations may be due to environmental insults or to high-energy electromagnetic radiation (X- or γ-rays), normally from cosmic rays. However, the rate of such spontaneous mutations is very slow indeed. Even the discovery of hybridization in the 18th century only modestly improved the capacity of breeders to improve variation. By the early 20th century, two developments revolutionized the ability of breeders to effect such genetic manipulations and to greatly extend their ability to create more novel and useful variations in crop genomes. Firstly, it became possible for the first time to cause mutations to occur deliberately and thereby to increase the rate of mutagenesis in a population by many thousand-fold. This is the technique of induced mutagenesis. Secondly, the efficiency of hybridization was vastly increased in the mid-20th century by the invention of tissue culture and the use of plant growth regulators. These developments greatly extended the potential gene pool that could be accessed by the breeder of a particular crop. Novel genes could be acquired from very distantly related species, and transferred into an elite crop cultivar by interspecific hybridization, followed by repeated backcrossing, in order to create new genetic combinations that have been of great utility to farmers. This is the technique of wide crossing. Induced mutagenesis was first used for the genetic manipulation of crops in the 1920s when agents such as X-rays and chemical mutagens were successfully used in maize and wheat. The most commonly used chemical mutagens in plant breeding are alkylating agents that directly react with DNA bases and modify their structure. One of the best known of these alkylating agents is ethyl methane sulphonate while another useful mutagen is sodium azide. Since the 1950s, γ-radiation from cobalt-60 or caesium-137 sources has been used with considerable success in crop breeding, most particularly in developing countries. An example of induced mutagenesis being applied to oil crops is the conversion by a group at CSIRO, Australia, of the non-edible, high α-linolenate oilseed, linseed, to a new edible, high linoleate variety that has been called ‘Linola™’. In this case, many tens of thousands of seeds from a conventional high α-linolenate variety of linseed were subjected to treatment by the mutagen, ethyl methane sulphonate (Green and Marshall, 1984). The aim was to produce a few mutagenized seeds in which the gene(s) encoding or regulating the fatty acid desaturase responsible for the conversion of linoleate to α-linolenate had been disrupted. It was found that there were several mutants that produced about half of the normal amount of α-linolenate, implying that the character was controlled by two genes. By crossing some of these single mutants, a population of double mutants was created in which the α-linolenate level was as low as 2 % of seed fatty acids. This was a great improvement on the situation in normal linseed oil, where linolenate is typically about 50 % of total fatty acid.

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As a result of this process of mutagenesis-assisted breeding, linseed oil, which is non-edible and oxidation-prone and is used in the manufacture of paints and putties, was converted into a premium quality edible oil with a similar acyl composition to that of sunflower. Unfortunately for the breeders who developed Linola in the 1980s, the market for high-polyunsaturate oils was already well supplied by the likes of sunflower and safflower, and demand was already moving away from polyunsaturates towards highmonounsaturate, low-trans oils. This has meant that the new variety was not as much of a commercial success as originally hoped (global Linola crop areas are only about 100 000 ha, whereas sunflower and rapeseed areas are in the tens of millions of hectares). However, this example does illustrate how an existing non-edible crop can be genetically modified by induced mutagenesis to create a completely different oil profile for food use (note that such mutagenized genetic manipulations are not officially classified as ‘GM’ because the resulting plants are not transgenic). Another method for the introduction of novel variation into a plant is by so-called ‘wide crossing’. In this case, a crop is hybridized with another plant that carries the desired genetic characters in order to create a hybrid that resembles the original crop parent, but also has the novel traits from the other parent. Wide hybridization can be done between plants of different species and even different genera. Normally, the progeny of such crosses are sterile and often the embryonic plants do not develop into seeds. However, since the 1930s, breeders have used a variety of tissue culture methods, such as chromosome doubling and embryo rescue, to obtain fertile progeny from wide crosses. Wide crossing has been used successfully to transfer genes into crops from other species, most notably for characters like disease resistance and salt tolerance. Several efforts have also been made to use wide crosses for the manipulation of plant lipid compositions, but so far no commercial products have been developed. As I hope it is apparent from this brief account of modern plant breeding, the intrusive manipulation of crop genomes by humans, including the transfer of genes from other species and the use of chemical- and radiation-induced mutagenesis, has been underway for over a century. Since the 1930s, all of the principal crop species have been modified using these methods, and it is estimated that radiation mutagenesis alone has produced over 3000 new varieties that are grown in some 60 countries around the world (Maluszynski et al., 2000). It is a moot point as to whether such long-standing intrusive breeding techniques carry intrinsically greater or lesser risks, or are more or less ‘natural’, than the more recently developed technologies of direct DNA manipulation via transgenesis.

12.3.4 Selecting for variation One of the major problems confronting breeders who are trying to produce crop varieties with modified lipid profiles is the identification of the desired

282 Modifying lipids for use in food lipids in seed populations that may number in the tens of thousands. Lipid analysis is a destructive technique, unless a small fragment of the seed tissue can be accurately assayed without damaging the rest of the seed. The development of routine gas–liquid chromatography (GLC) in the 1960s, and the later automation of this technology to allow for round-the-clock analyses, has greatly facilitated the task of the oilseed breeder. Another innovation, made possible by the sensitivity of GLC, is the use of half-seeds for analysis. This involves the dissection of a small fragment of seed tissue for analysis, while the rest of the seed is retained for germination to produce a new adult plant. This combination of methods was used in the breeding of the most successful new oil crop of the past half century, namely the canola varieties of oilseed rape as discussed below in Section 12.4.2. Some idea of the improvement in selection efficiency represented by the combination of the half-seed method and GLC analysis can be gleaned from the following figures. Prior to the development of GLC, it required about 200 000 whole seeds (1 kg) and two weeks to perform just one fatty acid analysis. Now, it is possible to analyze a fragment of a single seed, weighing just 2 mg, in 15 minutes. Thanks to this 650 million-fold improvement in analytical efficiency, it became feasible to accurately screen many thousands of seeds in the search for that most rare of events; a spontaneous mutation in just one or two genes in a genome that might contain from 25 000 to over 100 000 genes. Similar automated methods of analysis have now been developed for the mass screening of variants in other lipidic molecules of nutritional interest, ranging from sterols and carotenoids to tocopherols, lycopenes and xanthins. However, not all selection methods need to be high-tech in order to be effective. In the case of the new high linoleic linola variety of linseed described in Section 12.3.3, the tens of thousands of mutagenized seeds were initially screened using a method that was simple, rapid and very cheap, i.e. permanganate staining. Each seed was pressed lightly onto a strip of filter paper so that some of the oil was absorbed onto the paper. Next, the filter paper was dipped in a solution of potassium permanganate, which oxidized any α-linolenate in the absorbed seed oil to yield a purple colour. Nearly all of the seeds produced bright purple spots. However, the tiny number of seeds in which the α-linolenate genes had been mutagenized, produced a much lighter coloured spot that was instantly recognisable. These seeds were then grown up and the seed oils of their progeny were analyzed by the more accurate and quantitative method of GLC. The use of the preliminary permanganate screen saved the breeders from tens of thousands of relatively expensive and time-consuming GLC analyses. These screening tools allow breeders to select variants from much larger populations than was previously possible, thereby increasing the chances of identifying rare mutations that result in sometimes rather subtle, but nevertheless very useful, changes in lipid composition in a crop. As we have seen, such methods of selection have resulted in the development of at least two new edible oil crops, linola and canola, but similar approaches are now used

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across the seed industry for the selection of improved lipid profiles in all the major oil crops.

12.4 Oil crop modification 12.4.1 Introduction Since the early 1960s, several established oilseed crops have been successfully modified in order to improve their edible qualities, while in other cases new crops have been more or less developed from scratch. Such developments have been facilitated by the sorts of advances in plant breeding that were outlined in the previous section. Another important set of tools has been provided by improved analytical techniques that have allowed for the rapid and accurate mass screening of thousands of plant samples for possible changes in lipid composition. All of the major analytical techniques are chromatography- or spectroscopy-based, beginning with relatively crude and insensitive (but often useful) methods like column and thin-layer chromatography and progressing to GLC and mass spectroscopy.

12.4.2 Canola – a new oilseed crop Probably the most impressive example of the manipulation of an oilseed to enhance its edible performance was the modification of the existing high erucic form of oilseed rape in the 1960s to produce the current high oleic canola oil. Prior to this time, oilseed rape, and indeed all the other brassica species, produced a seed oil that consisted mainly of the very long-chain C22 erucic acid. In Europe and North America, this oil was normally used for non-edible purposes and oilseed rape was very much a minor crop with a limited and not very profitable market. In the 1960s, the Canadians were looking for new crops to grow on their huge prairie farms and one possibility was to breed an edible form of oilseed rape. Plant breeder Keith Downey led a small team in Saskatoon that was looking for a way to reduce the amount of erucic acid and instead to increase the amount of a much more useful fatty acid such as oleic acid (18:1). Oleic acid is the main ingredient of olive oil and is the premium monounsaturated fatty acid recommended by nutritionists. Downey’s genetic and biochemical analysis of rapeseed plants had already indicated that the unwanted erucic acid was formed by elongation of much more desirable oleic acid. If the elongation pathway could be disrupted, the seeds should accumulate large amounts of oleic acid and would be readily marketable for their new edible oil. The genetic analysis showed that this elongation pathway was controlled by only a few genes and was thus potentially amenable to manipulation by a classical breeding approach (Downey and Craig, 1964). All Downey’s group needed was a plant that had one or two mutations that prevented it from elongating oleic acid so that it accumulated oleic acid instead.

284 Modifying lipids for use in food By using the mass-screening method outlined in Section 12.3.3, it was eventually possible to find spontaneous mutants of conventional high erucic rapeseed that had lower levels of erucic in their seed oil. Because several genes were involved, it was necessary to cross some of the mutants with intermediate amounts of erucic to produce varieties with very low levels of the fatty acid. Although this took a few years of hard work, by 1964 the project was eventually rewarded with success as the team developed the first zero-erucic acid variety of oilseed rape, which they christened ‘canola’. The remarkable achievement of these breeders has been highlighted by recent advances in molecular genetics, which have allowed us to discover the exact nature of the mutations that the Canadian group selected in order to create the low erucic acid phenotype of modern rapeseed/canola. Their canola plants contain single point-mutations in two genes encoding isoforms of the enzyme β-ketoacyl CoA synthase: this protein is part of the fatty acid elongase complex now known to mediate the formation of erucic acid from oleate (Fourmann et al., 1998). This means that they succeeded in the alteration of just two nucleotides in a genome that contains over 1.2 billion nucleotides. It also demonstrates the power of genetics as applied to plant breeding. Such a result would be the envy of any latter-day biotechnologist and is a useful reminder that genetic engineering is not the only way to achieve the precise genetic manipulation of a crop. Since the late 1970s, canola has been a mainstay of Canadian prairie agriculture and a major export earner for the country. Canola-standard oilseed rape has also been adopted enthusiastically as an edible oil crop around the world with an annual value in excess of $6 billion (Oil World, 2005). Thanks to this small team of Canadian breeders, oilseed rape is now a globally important crop that is used to make salad oil, cooking oil, margarine, as well as being a key ingredient in all manner of food products from biscuits and cakes to curries and pies. It is also interesting to reflect that this single rather modest new crop, developed over about a decade by breeders, has already earned far in excess of all the profits of the agbiotech industry in the two decades after 1985 (James, 2005). Since the development of high oleic canola varieties in the 1960s, rapeseed has been improved further as an edible oil crop by reductions in its content of oxidation-prone linolenic acid so as to avoid the need for hydrogenation and the accumulation of trans fatty acids, as we will now discuss.

12.4.3 Other modified oilseed crops The need to reduce levels of trans fats in foods has driven breeders to develop several new high oleic varieties of the major oil crops since the 1990s. For example there are now commercial high oleic varieties of the ‘big-three’ oilseed crops, soy, rape and sunflower, all of which have been produced by conventional breeding. Efforts are also underway to produce a high oleic version of oil palm, both by screening for existing variation and

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by transgenic methods but, with such a slow growing tree crop, this process may take several decades. A particular attraction of high oleic, plant oils is that they have applications both as premium edible oils and as high-grade feedstocks for the manufacture of many oleochemicals. An example of the latter market is the use of high oleic soybean oils as biodegradable lubricating fluids that have relatively long working lives and low susceptibility to oxidation at high temperatures (Cahoon, 2003). High oleic soybean varieties with as much as 83 % oleate and less than 3 % α-linolenate have been developed (Rahman et al., 2001) and are now being marketed by major seed companies. Breeders have also developed other lines of soybean that have high levels of stearic acid (Rahman et al., 2003) and other nutritionally relevant fatty acids. Several high oleic canola/ rape varieties have been developed that typically contain about 70–80 % oleate, 15 % linoleate and only 3 % α-linolenate. Major seed companies such as Cargill, Dow Agrosciences and Bayer are now developing such varieties for various end-use markets in the edible and non-edible sectors. By 2004, high oleic rape/canola was already being planted on about 250 000 ha in Canada, which is 5 % of the total area of canola cultivation (AgCanada, 2004). Sunflower oil, once the high polyunsaturate edible oil par excellence, is also steadily being rebranded as a high oleate oil. Traditional sunflower oil consists of about 68 % linoleic acid, 20 % oleic acid and 10 % saturates, which means that hydrogenation is still necessary for many food uses. During the 1990s in the USA, there was great interest in very high oleic sunflower varieties (with 80 % oleic acid, 10 % polyunsaturates and 10 % saturates) that had already been developed by breeders. However, these varieties were only available to farmers in very limited quantities because of a patent on hybrid planting seed with oleic levels at or above 80 %. The patent holders chose not to license their breeding material to other companies; thus high oleic sunflower production was very limited and the price of the oil was quite high compared to commercial oils. By 1999, these problems were resolved with the development of a new intermediate oleate hybrid variety called NuSun™ (National Sunflower Association, USA), which was commercially launched in that year. NuSun oil contains about 65 % oleic, 25 % polyunsaturates and 10 % saturates, which does not require hydrogenation and works especially well in commercial frying applications. By 2001, over 200 000 tonnes of NuSun™ oil were being produced and the hoped-for commercial breakthrough came in the same year with the announcement that Proctor & Gamble would be using NuSun™ oil exclusively in its popular Pringles® line of potato chips (Kleingartner, 2002).

12.4.4 Advanced breeding for oil crop modification During the mid–late 20th century oil crop breeding was driven by advances in analytical technologies and by a vastly improved knowledge of lipid

286 Modifying lipids for use in food metabolism. Since the 1980s, the task of the oilseed breeder has been facilitated further by new techniques of advanced breeding, such as the use of DNAbased molecular markers, greatly improved tissue culture methods and more recently by the application of genomics and proteomics (Murphy, 2005). DNA marker-assisted selection Plant breeding has always relied on the selection of agronomically favourable characters from the diverse gene pool that is present in any crop species, even if many elite commercial cultivars tend to be highly inbred. Often these agronomic characters are visible and easily identified, e.g. height or flower colour or resistance to fungal attack. In other cases, the characters can be much more subtle and sometimes can only be measured by sophisticated analytical techniques, e.g. the amounts of certain secondary products or the fatty acid composition of the seed oil. In all of these cases, it was formerly necessary for the breeder to grow up and analyze each new generation before it was possible to measure the character, or phenotype, and select the appropriate plants. The advent of marker-assisted selection has changed this as breeders can now select a few plants that are likely to express the required characters from amongst tens of thousands of progeny even before the plants have developed to maturity. The basis of the method is DNA-fingerprinting and it is in principle no different from the methods used with such great effect in modern medical diagnostics or in forensic science (Gill et al., 1985). Molecular markers such as microsatellites, RFLPs (restriction fragment length polymorphisms) and RAPDs (random amplified polymorphic DNA) have now been developed for many oil crops, including trees like oil palm. These markers can be assembled into genetic maps that have considerable utility both in basic biological research and in commercial breeding programmes. The markers can be used to track the presence of valuable characters in large segregating populations as part of a crop-breeding programme. For example, if a useful trait like disease resistance, improved nutritional quality or higher yield can be linked with a specific marker, many hundreds or even thousands of young plantlets can be screened for the likely presence of the trait without the necessity of growing all the plants to maturity or doing costly and time-consuming physiological or biochemical assays. While the earlier molecular markers like RFLPs were relatively expensive, newer markers like microsatellites and SNPs (single nucleotide polymorphisms) are considerably cheaper and easier to use. The use of molecular markers can decrease the timescale of crop breeding programmes by several years and can substantially reduce costs. Although largely limited to the major temperate crops at present, the same technology can be applied to assist the breeding of any crop and even to domesticate entirely new crops. A good example of the potential for marker-assisted selection can be seen with edible tree crops, many of which are major export earners for developing countries. Examples of such crops include oil palm, coconut, coffee, tea, cocoa and the many commercial fruit tree species, such

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as bananas and mangoes. In the case of oil palm, the fruits that are the source of the palm oil are not produced for 5–7 years after planting. This means that a breeder must wait for at least 5–7 years before being able to determine the oil composition of a particular experimental cross. In contrast, breeders of annual crops have to wait only a few months before a plant like soy or oilseed rape sets seed. However, by using DNA markers, the oil palm breeder can now (in theory) identify whether new plantlets carry the required gene when they are only a few weeks old. This type of molecular marker technology is reasonably well developed for the major annual oilseed crops like soy or rape (Quiros and Paterson, 2004), but is still very much under development for more complex crops like oil palm (Basri et al., 2004). Tissue culture and mass-propagation The use of modern techniques of cell, tissue and organ culture is central to many crop improvement programmes in both industrialized and developing countries. Indeed the limiting step to the successful development of transgenic varieties of the major edible crops has not been transgene insertion itself, but rather the regeneration of viable plants from the transgenic explant material. Tissue culture has been widely used in crop breeding programmes since the 1950s (Phillips, 1993). For example, the use of embryo rescue techniques has enabled the incorporation of characters like disease resistance from wild relatives of crops into elite breeding lines. It is now possible to make wide crosses between hexaploid wheat and barley, rye or diploid wheat. The hybrids of such crosses are sometimes sterile due to embryo abortion but can be ‘rescued’ by culturing or transplanting the embryos. Another important technique that is increasingly used in crop breeding programmes is the production of doubled haploids. The repeated selection of heterozygous materials in a breeding programme can increase uniformity, but many generations are required to reach homozygosity in loci associated with agronomic traits. The artificial production of haploid plants followed by chromosome doubling offers the quickest method for developing homozygous breeding lines from heterozygous parental genotypes in a single generation. Haploid gamete cells from anthers or ovaries can be converted into diploids after colchicine treatment and then regenerated to yield doubled haploid plants. This technique is now used widely for the improvement of many of our most important oil crops, including maize, rapeseed and soybean (Forster et al., 2000). Yet another useful application of tissue culture methods is the mass clonal propagation of certain crops, in particular trees. Clonal propagation has not always been commercially successful, however. In the 1980s, a scheme to mass propagate millions of oil palm plantlets from a superior breeding line foundered when many of the maturing trees were discovered to have an abnormality in their floral development (Corley, 2000). This led to a failure of fruit formation and, since the major products of the crop are fruit oils, the trees were effectively useless. The abnormality is now known to be due to a

288 Modifying lipids for use in food tissue culture effect whereby the expression of homeotic genes regulating meristem identity is disrupted. Although the problem is now in the process of being rectified by further research, commercial confidence in clonal propagation has not recovered and relatively little commercial planting of clonal oil palm has been done since the late 1980s (Corley, 2000). The continuing scope for crop improvement, following the identification of higher yielding germplasm and its multiplication by mass-propagation, can be exemplified once again by considering the case of oil palm. Oleic acid rich oil from palm mesocarp is the most important edible oil crop produced in Asia. Moreover, palmkernel oil is also the most widely used oleochemical feedstock for the manufacture of detergents and other lauricbased products. Since the early 1980s, the average yield of Malaysian palm oil on plantations has more or less stagnated at 3.5–4.0 T.ha–1 (USDA, 2004). This is despite the availability of new clonal lines that can yield as much as 7.5 T.ha–1 (Ginting et al., 1995). Malaysia currently produces about 13.5 MT.yr–1 of palm oil worth an annual $4 billion: this is in a country with a total GNP (gross national product) of $60 billion. To put this figure of $4 billion into context, the estimated entire revenue generated by the US agbiotech sector in 1999 was just $2.3 billion – and this included all the companies supplying inputs to the sector or its employees (Ernst & Young, 2000). Therefore, the effective doubling of the palm oil yield that could be implemented following a successful mass-propagation programme could contribute a significant 6.6 % extra to the overall gross national product of this single Asian country. The application of a similar strategy with other tree crops, or even relatively undomesticated annual crops, could also yield equally striking results that would particularly benefit developing countries. Genomics Genomics is the term given to the massively parallel study of the DNA and protein sequences in an organism and the specification of when and where such sequences are expressed. However, genomics is much more than the mere assembly of DNA or protein sequence information or gene expression catalogues. It can also be used a tool in crop breeding programmes and even for the domestication of new plant species as future crops. Many characteristics of agricultural importance in crop plants, including some fatty acid traits, appear to be regulated by a large number of genes and therefore do not segregate into simple Mendelian ratios, as would be expected if only one or two genes were involved. Examples of such complex traits include height, branching, seed oil and protein yield, oil quality and flowering time. The use of more sophisticated genomic tools since the 1990s has shown us that, although dozens of genes may underlie such complex traits, sometimes much of the variation in their phenotypic expression can be caused by a small number of key regulatory genes. These genes can now be identified and mapped based on sequence similarities, expression profiles and molecular markers. Genes that play a major role in regulating agronomically relevant

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complex traits in model plants like Arabidopsis, and also some crops like maize, are now being isolated at an ever-increasing pace. Examples include height, flowering time, vernalization, shattering of seed pods and stem branching. Many of these plant genes encode transcription factor proteins that in turn regulate the expression of large sets of other genes. For example, transcription factors can switch on entire metabolic pathways or patterns of cell division, resulting in the formation of new tissues or organs and the accumulation of new storage products (Murphy, 1998a). The application of information from genomics is enabling crop scientists to identify key genes that regulate the accumulation of specific lipids that breeders may wish to either eliminate (e.g. α-linolenate for trans-free foods) or up-regulate (e.g. oleate for high-monounsaturate oils). This information can then be used to develop DNA-based markers for marker-assisted selection as described above.

12.5 Transgenic oil crop modification Transgenesis is the addition of exogenous (i.e. externally derived) DNA sequences and their incorporation into the genome of a recipient organism, such as a plant or animal. In the case of plants, the DNA can be added to cells directly by propelling small gold particles coated with DNA into plant tissues. This technique, called biolistics, can be used for any plant, crop or otherwise, but is a rather hit-and-miss affair that does not always result in the incorporation of the DNA into the plant genome. Alternatively, the DNA can be added in a more controlled fashion by means of a bacterial vector, e.g. Agrobacterium tumefaciens, that is able to insert a specific region of DNA into the genome of the plant. Even with their uncertainties, both of these methods of DNA transfer, or transgenesis, are much more efficient than alternative methods of crop genetic manipulation, such as induced mutation or wide crosses. For example, as discussed in Section 12.3.3, the creation of new crop varieties via radiation, chemical or somaclonal mutagenesis normally involves the repeated treatment of tens of thousands of tissue explants or seeds. These extremely drastic procedures result in hundreds of mutated genes, nearly all of which will be undesirable, and perhaps even lethal, to the crop. It then takes many years of backcrossing and selection to obtain a plant that carries a mutation in the desired gene(s), but not in other essential genes. Even then, it is still possible that there may be undetected hidden, or cryptic, mutations that only manifest themselves in later generations as the crop is tested or grown in commercial cultivation. Another significant drawback of mutagenesis is that the breeder can only manipulate genes that already exist in the crop genome. Furthermore, nearly all mutations result in a loss of gene function so mutagenesis is nearly always about reducing the effect of unwanted genes, rather than increasing the expression of desirable genes.

290 Modifying lipids for use in food These drawbacks in the existing technology of creating variation in plants made the prospect of a new and more direct method very appealing to plant researchers. The first experimental transgenic plant cells were produced by several European and US groups in 1983 (Barton et al., 1983; Caplan et al., 1983). By 1987, the commercial utility of the technology was demonstrated when it was shown that copies of a bacterial gene could be transferred to plants and thereby confer resistance to certain insect pests. In 1992, the first transgenic crop, the Flavr Savr® (Calgene Inc, USA, now part of Monsanto) tomato, was released in the USA. This tomato variety was not successful, however, mainly due to the use of poorly performing breeding lines and commercial mismanagement (Martineau, 2001). As detailed below, several transgenic oilseeds were developed at this time, but were also commercial failures. However, the next group of transgenic crops, that have been released on a steadily increasing scale after 1996, proved to be much more successful. Four major crops, soybean, maize, oilseed rape and cotton, have been bred to express two groups of simple traits, namely herbicide tolerance and insect resistance. For the first five years or so, commercial cultivation of these transgenically bred crops was largely limited to North America, but the technology has now been adopted more widely, especially in South America and China.

12.5.1 The concept of ‘designer oil crops’ During the late 1980s and early 1990s, oilseeds like soy and rape were at the forefront of attempts to produce commercial transgenic (‘genetically-modified’) crops. These efforts were pioneered by researchers in small biotech companies, such as Calgene, as well as the large multinationals, such as DuPont, Monsanto and Zeneca (now Syngenta). Part of the rationale for these efforts was a belief that fatty acid biosynthesis was well understood at the biochemical level and that relatively few genes would be needed to effect substantial changes in oil profiles in a seed. This led to the concept of ‘designer oil crops’ as described in the book of the same name that appeared in 1994 (Murphy, 1994). As pointed out at that time, there are three major challenges that confront those who wish to use transgenesis to modify plant lipid composition. These are to ensure that the new transgene is only expressed in the appropriate place (normally the fruit or seed); to ensure that the novel fatty acid is segregated into storage lipids and away from membrane lipids; and to ensure that the transgenic crop varieties and their products are adequately segregated from other identical-looking varieties of the same crop that are producing a different oil. In those optimistic days of the early 1990s, oilseed rape was often talked about as an archetypical ‘designer oil crop’ that could be engineered to produce as many as a dozen different oils to supply products ranging from margarines and pharmaceuticals to bioplastics and lubricants (Murphy, 1994). Despite the initial optimism of many researchers (including the author),

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the use of agbiotech to manipulate the fatty acyl composition of oils has turned out to be more complex than was first thought. Indeed, recent findings suggest that our understanding of even the basic pathway of triacylglycerol oil biosynthesis is far from complete and that there are probably several alternative parallel biosynthetic pathways rather than just one (Murphy, 2004). The consequence of these complexities of plant lipid metabolism has been that, although there have been many impressive achievements in isolating oil-related genes and producing transgenic plants with modified oil compositions, it has not yet been possible to achieve the kind of high levels, i.e. 80–90 %, of novel fatty acids that will make possible their widespread commercial exploitation (Murphy, 1999). As of the 2006 season, there were no significant commercial plantings of transgenic oil crops with modified fatty acid profiles. Although there were over 90 Mha of transgenic oilseeds such as soybean, canola, maize and cotton planted in 2005, these varieties were all modified to carry input-related genes involved in herbicide tolerance or insect resistance traits, rather than alterations in seed oil content (James, 2005). The only commercially grown transgenic crop with modified seed oil is the ‘laurical’ variety of canola (rapeseed) originally marketed by Calgene in 1995. From an original level of 40 % lauric acid newer ‘laurical’ varieties have been produced with 40–60 % lauric acid by the insertion of several additional transgenes (Voelker et al., 1996). However, this crop remains far from being a commercial success and cannot compete with cheaper tropical lauric oils from coconut and oil palm. Many genes that regulate the formation and accumulation of other exotic, non-edible fatty acids were isolated during the 1990s, including hydroxylases, conjugases, desaturases and epoxidases, but so far it has not been possible to use any of these genes to effect the production of any commercially useful oils in transgenic plants. Two of the major challenges facing designer oil crops are to prevent the novel fatty acids from leaking into cellular membranes and to segregate the seeds and oils during cultivation and processing, as we will now consider.

12.5.2 Segregation of novel fatty acids from membrane lipids The cellular membranes of all organisms are crucial to their metabolism and survival. Biological membranes are made up of a lipid bilayer into which are embedded the many proteins that mediate such processes as transport, respiration, photosynthesis and signal transduction. The fatty acid composition of a given membrane is closely regulated and the presence of inappropriate acyl groups often leads to serious disruption of membrane, and hence cellular, function. In plants that accumulate fatty acids that differ significantly from those of membrane lipids, specific mechanisms have evolved that prevent the ‘leakage’ of unwanted acyl groups into membrane lipid pools. Biochemically speaking, this is not a trivial task, because storage lipids and many membrane lipids are assembled on the same organelle – the endoplasmic reticulum.

292 Modifying lipids for use in food As yet, we do not completely understand the mechanisms by which some plants are able to segregate unusual fatty acids away from membranes (Millar et al., 2000). We know that this mechanism is very important because the leakage of a novel fatty acid, namely the saturated species, stearic acid, resulted in very poor seed germination rates in a transgenic variety of oilseed rape that had been engineered to have an elevated stearate oil for use in the manufacture of edible spreads (Thompson and Li, 1997). In this study, it was found that a small amount of stearate had leaked into cell membranes, resulting in a reduction in membrane fluidity and impairment of function that affected the development of the entire plant. Although, in this case, the transgenic rape seeds only accumulated about 40 % stearate and just 3–5 % leaked into cell membranes, other oilseeds like mangosteen (Garcinia cornea) can accumulate over 65 % stearate in their seed oil without any detectable leakage into cell membranes. Two hypotheses have been proposed to explain the sort of lipid segregation that plants like mangosteen seem capable of, but which seems to be lacking in major oil crops like oilseed rape. The first hypothesis is that there is a compartmentation of membrane and storage lipid synthesis in specific membrane domains of the endoplasmic reticulum. The other hypothesis is that there is a selective accumulation of the novel fatty acids in triacylglycerols after synthesis (Millar et al., 2000). Research is currently underway to address these issues but, until we understand more about fatty acid segregation, the production of most exotic fatty acids in transgenic crops will remain more of an aspiration than a reality (Thelen and Ohlrogge, 2002).

12.5.3 Segregation of transgenic crops from other crops Unless a transgenic variety of an oil crop completely replaces all non-transgenic varieties of the same crop, it will require complete segregation at every stage of production from seed storage and planting to harvesting and downstream processing. This can add at least 10–20 % to costs and imposes considerable (and often overlooked or under-estimated) management problems. These challenges can be rather formidable, given the complexity of the supply chain from breeder to grower to crusher to processor and so on, all the way to the retailer and ultimately to the consumer. The difficulties in ensuring strict segregation of otherwise indistinguishable transgenic crops have been pointed out (Murphy, 1994, 1996) but have consistently been under-estimated by many in the industry. However, several well-publicized failures in the segregation of transgenic crops since 2000 have thrown this issue into much sharper focus. The latest episode in a growing list of such failures involved a variety of transgenic maize that was widely grown for several years in the USA (and exported for human consumption to Europe) although it had not been granted regulatory approval (Macilwain, 2005). Such incidents do not inspire confidence in the ability of sections of industry to regulate and manage themselves.

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They have also called into question the wisdom of growing crop varieties that contain therapeutically active compounds alongside major commodity varieties of the same crop. An example would be a maize variety engineered to produce high levels of omega-3 fatty acids growing in the same region as regular maize. However, these management problems can and should be resolved. After all, since the late 1970s, UK farmers have been growing two (non-transgenic) oilseed rape varieties that produce respectively a major edible commodity oil and an industrial oil that is prohibited for human consumption. Despite the potential for cross-pollination between the two crops and the fact that over 400 000 ha of rape is grown in the UK, careful management by farmers, seed crushers and oil processors has ensured that there have been no reported instances of cross-contamination. So, segregation can work, but everybody in the food chain must cooperate to ensure that strict standards are observed.

12.6 Plant lipid manipulation in the 21st century There are two apparently contradictory trends in the current development of plant lipids for edible production. First, there has been a huge consolidation and concentration of both the major oil crops and the suppliers of plant material for growing such crops. This trend leads to a more generic, commoditybased market. Second, however, there is a trend towards segmentation and identity preservation of individual oils that may carry a considerable price premium. These divergent tendencies are driven by different forces that favour the cheapest generic oils, especially for use in mass-produced processed food, on one hand, while also favouring the creation of niche products with specialized identity-preserved oil compositions that are often based on health claims on the other. The speed with which such dietary fads can come and go makes it difficult for breeders to produce appropriate varieties, especially given the decades-long timescale of most breeding programmes. For example, since the 1980s, we have gone from advice to reduce all dietary saturates, then to increase polyunsaturates, then monounsaturates were favoured, and now omega-3 acids are the ‘flavour of the month’ for fatty acid nutritionists. The most recent consumer interest seems to be in high-monounsaturate oils and in omega-3 acid oils, ideally with some lipophilic antioxidants like tocols or carotenoids thrown in for good measure. Plant oils with each of these profiles are currently being developed by breeders, as we will now see.

12.6.1 Low-trans oils The market for oils that contain reduced or zero levels of trans fatty acids is currently driven by health concerns that have led to the imposition of labelling requirements revealing whether a product contains over a given threshold of these fatty acids. Such labelling requirements were introduced into the USA

294 Modifying lipids for use in food on 1 January 2006, and are likely to be required in the European Union in the near future. Typical threshold levels of trans fatty acids that would trigger compulsory labelling are in the region of 0.5–1.0 %, whereas some existing foods can contain as much as 40 % trans fatty acids. The solution in most cases will be to develop high oleic oil crops and, as we have seen above, breeders have been gradually producing such varieties of the major oilseeds over the past decades. There are still challenges for breeders in attempting to reduce further, or to eliminate altogether, α-linolenate from seed oils and to ensure that the high oleic traits are crossed into their highest performing elite commercial lines. For most crops, it is possible to produce high oleic varieties by nontransgenic routes and, given the sentiment about GM crops in Europe, such a route is obviously preferable at the present time. Given the huge potential market for foodstuffs that are free of trans fatty acids, it is possible that high oleic oil crops will become the mainstream commodities over the next decade, rather than existing polyunsaturate-rich varieties. Such a development would certainly be welcome to processors as it would obviate the need to segregate the oil, but it would be less well received by farmers and seed suppliers who would lose their premium for a value-added variety. The types of product, e.g. a spread or a liquid oil, that would result from a high monounsaturate plant variety would also be potentially suitable for the delivery of omega-3 fatty acids, as long as they were kept down to about 10 % to avoid oxidation problems. However, if customers required higher levels of omega-fatty acids, e.g. in a neutraceutical format, it would be better to produce dedicated high omega-3 oil crops. We will now look at the rationale for, and recent progress towards, this objective.

12.6.2 Can plants substitute for ‘fish oils’? Oils rich in omega-3 fatty acids include the so-called ‘fish oils’ (or more correctly ‘marine oils’), which are characterized by relatively high levels of very long-chain polyunsaturated fatty acids (VLCPUFAs) such as eicosapentaenoic acid (20:5ω-3, EPA) and docosahexaenoic acid (22:6ω-3, DHA). These compounds are part of the group of omega-3 fatty acids that are essential components of mammalian cell membranes, as well as being precursors of the biologically active eicosanoids and docosanoids (Funk, 2001; Hong et al., 2003). There have been numerous reports concerning the importance of dietary supplementation with these fatty acids for human health and well being. For example, dietary VLCPUFAs have been shown to confer protection against common chronic diseases such as cardiovascular disease, metabolic syndrome and inflammatory disorders, as well as enhancing the performance of the eyes, brain and nervous system (Crawford et al., 1997; Spector, 1999; Benatti et al., 2004). It is worth mentioning here that none of these VLCPUFAs are strictly ‘essential’ in the diet in the same way that vitamins are. The only unequivocally essential fatty acid is cis 9, 12

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linoleic acid, an omega-6 fatty acid that mammals are unable to synthesize. All of the VLCPUFAs can be synthesized from linoleic acid in a wellnourished and healthy individual. Unfortunately, most Western diets do not provide an adequately balanced fatty acid intake and this often necessitates the inclusion of VLCPUFAs to maintain optimum health. Consumption of fish is therefore currently recommended in most Western countries to provide a balanced diet, and much of the nutritional benefit of the fish actually comes from the VLCPUFAs of the fish oils. These fatty acids are not only synthesized by the fish themselves, but are also derived from micro-organisms, especially photosynthetic micro-algae, that are ingested as part of their diet. As an alternative to fish consumption, therefore, it is possible to purchase VLCPUFA dietary supplements that are derived from cultured micro-algae or fungi. However, low oil yields and high costs of oil extraction have limited the scope for this production method, and everdwindling fish stocks are also threatening supplies of the main source of marine oils. This situation has led to renewed interest in the possibility of breeding oilseed crops that are capable of producing significant quantities of VLCPUFAs in their storage oils. Higher plants do not normally accumulate such fatty acids, but they can accumulate C22 and C24 monounsaturates and C18 polyunsaturates in their seed oils, so it seemed possible that C20 and C22 polyunsaturates might also be accumulated providing the plants were able to synthesize these fatty acids. The most serious of several technical challenges to the engineering of VLCPUFA production in plants is the number of enzymes that are needed for the conversion of a typical plant C18 PUFA, such as linoleate or linolenate, to the C20 and C22 VLCPUFAs with up to six double bonds that are characteristic of fish oils, as shown in Fig. 12.1. Other key challenges are similar to those that have confronted previous attempts to engineer transgenic oilseed, namely to ensure seed-specific expression of the transgenes and to channel the novel fatty acids towards oil accumulation and away from membrane lipids. During 2004 and 2005, there were several reports that encourage the view that the economic production of VLCPUFAs in transgenic plants might be possible (Abbadi et al., 2004; Qi et al., 2004; Wu et al., 2005). In one rather heroic experiment, no fewer than nine genes from various fungi, algae and higher plants were inserted into the oilseed, Brassica juncea, with the resultant accumulation of as much as 25 % arachidonic acid and 15 % eicosapentaenoic acid (Wu et al., 2005).

12.6.3 Transgenic oil palm – the emerging behemoth? Although the three major edible oilseed crops have been produced in steadily increasing amounts since the 1980s, their rate of growth is dwarfed by that of oil palm. From being a very minor crop in the 1960s, palm oil production has steadily increased until in the 2005–06 season it finally caught up with soy oil (the long-time industry leader) as the major global source of plant

296 Modifying lipids for use in food ω-6 (∆ ∆6) pathway Oleic acid ∆12 desaturase Linoleic acid

ω-3 (∆ ∆3) pathway

18:1 n-9 ∆15 desaturase 18:2 n-6

18:3 n-3

α-Linolenic acid

18:4 n-3

SDA

20:4 n-3

ETA

20:5 n-3

EPA

∆6 desaturase GLA

18:3 n-6

DGLA

20:3 n-6

AA

20:4 n-6

∆6 desaturase

∆6 desaturase

∆5 elongase 22:5 n-3

DPA

∆4 desaturase 22:6 n-3

DHA

Fig. 12.1 Biosynthetic pathways for very long chain polyunsaturated fatty acids. This figure shows the complexity of the various pathways involved in the synthesis of the kinds of omega-6-and omega-3-enriched ‘marine oils’ that various groups are attempting to engineer into oil crops. The starting substrates are linoleic acid and α-linolenic acid (ALA) in the omega-3 and omega-6 pathways, respectively. The ∆6 desaturation of LA and ALA gives rise to γ-linolenic acid (GLA) and stearidonic acid (SDA). Arachidonic acid (AA) and eicosapentaenoic acid (EPA) are synthesized by further ∆6 fatty acid elongation and ∆5 desaturation steps. An omega-3 desaturase interconnects the omega-6 and omega-3 pathways for more efficient EPA production. Finally EPA is elongated by a ∆5 fatty acid elongase and desaturated by a ∆4 desaturase to produce docosahexaenoic acid (DHA). Other abbreviations are: DGLA = dihomo-γ-linolenic acid, ETA = eicosatetraenoic acid and DPA = docosapentaenoic acid.

oils. Annual production of edible palm oil in 2005–6 was in excess of 34.6 MT (USDA, 2005), with China and Europe as the principal importers. An additional 3.7 MT of palmkernel oil was also produced in 2005. Although most of this high lauric oil is used in non-edible applications such as detergents, some of it is also used in high-energy sports drinks and in infant formulations. What is most impressive about oil palm, however, is not its current production figures but its future potential as the world’s main source of edible and nonedible oils. The average mature oil palm plantation currently yields about 3.5–4.0 T.ha–1, bears fruit throughout the year and lasts for 25–30 years of productive life. Moreover, there are higher yielding cultivars that already produce over 10 T.ha–1 and individual trees have been identified that could be clonally propagated, and which yield the equivalent of over 50 T.ha–1. In contrast, a typical oilseed rape or sunflower crop needs to be replanted each year, can only be harvested during a short period in summer and yields a

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paltry 0.5–1.0 T.ha–1. Yields of soy oil are even lower at about 0.3 T.ha–1. This means that if we satisfied the world’s annual plant oil requirement of 120 MT by only growing oil palm at 10 T.ha–1 instead of temperate oilseed crops, we could save as much as 70–80 million hectares for other uses. Oil palm is also a much more economic crop that has a lower environmental impact than the other oil crops. The main thing that is holding back oil palm from competing in the developing niche markets for added-value edible oils is the difficulty in manipulating its fatty acid profile. Palm oil has a relatively high content of the saturate, palmitic acid, as well as the more desirable oleic acid. This means that, to date, the food industry has treated palm oil as a relatively lowvalue generic commodity. For example, the April 2005 oil prices in Rotterdam (the main global spot market) showed palm oil trading at a discount of $100– $200 per tonne compared to its major rivals, soy, rape and sunflower (Oil World, 2005). However, unrefined palm oil is also characterized by high level of nutritionally desirable carotenoids and tocols that give this oil an attractive but, unusual (for a vegetable oil), red colour. The renewed interest in these antioxidant and otherwise desirable lipids has led to attempts to market red palm oil as a high-value niche product in the health food sector (see more on these non-acyl lipids in the next section). Despite these developments, palm oil is still something of a Cinderella product in some quarters. There are two factors that may enable breeders to turn this position around for the oil palm industry. First, worldwide sampling studies are beginning to show more genetic diversity in fatty acid profiles than had been suspected hitherto. Coupled with improving breeding and clonal propagation methods, this may mean that new varieties of oil palm, including high oleate genotypes, might be developed in the coming decades. The second development is the use of transgenesis to produce ‘designer’ oils from this crop. Although such studies are still at an early stage, efforts are proceeding to produce a new transgenic high oleate variety, as well as several varieties aimed at the market for renewable and biodegradable industrial feedstocks. These programmes may take several decades to come to fruition, but oil palm is certainly an interesting crop to watch for the future.

12.6.4

Modification of non-acyl lipids in plants

Golden rice Probably the best-known recent example of a nutritionally enhanced crop is the development of the transgenic ‘golden rice’ by a Swiss-based group (Ye et al., 2000). The grains of this GM rice variety are yellow because they have been engineered to accumulate the lipophilic pigment, β-carotene (pro-vitamin A), which is normally absent from rice grains. The transgenic rice contains three inserted genes encoding the enzymes responsible for conversion of

298 Modifying lipids for use in food geranylgeranyl diphosphate to β-carotene. It is claimed that consumption of this rice by at-risk populations may alleviate the vitamin A deficiency (leading to night blindness) that currently afflicts some 124 million children worldwide. Such claims are hotly disputed by anti-GM groups (e.g. Greenpeace http:// www.greenpeace.org/~geneng/) and the ‘golden rice’ has yet to prove itself in large-scale field and nutritional trials in the target developing countries. Interestingly, the rights for the commercial exploitation of ‘golden rice’ in developed countries, including the USA and Europe, have now been acquired by Syngenta. It is possible that this could lead to the marketing of ‘vitaminenhanced’ food products derived from golden rice, e.g. in breakfast cereals, which may be more acceptable to the public than the current generation of food from input trait modified GM crops. One of the reservations expressed about the original varieties of golden rice was the relatively low content of pro-vitamin A, which might require the daily consumption of several kilograms of rice to meet dietary requirements for vitamin A. More recently, this problem has been solved by replacing the daffodil phytoene synthase gene in the original varieties of transgenic golden rice with a similar gene from maize. Use of the maize transgene in rice led to a 23-fold increase in pro-vitamin A levels and grains of ‘golden’ rice that were bright orange, rather than an insipid yellow (Paine et al., 2005). This improved variety of golden rice still has many years of backcrossing into local varieties and field testing before it will be known whether it is a viable crop. Not the least of the challenges is to ensure that the pro-vitamin A is in a form that can withstand processing, storage and cooking, and is also completely bioavailable following consumption by people. There are many cases of vitamins and mineral nutrients that are either lost during postharvest treatments or pass through the digestive system, e.g. due to chelation or other forms of complexing. Probably the best-known example of this is spinach, where only 2 % of the iron is actually bioavailable due to the presence of oxalates – sadly, a real-life Popeye would not garner much strength from canned spinach! (Although spinach is not a good provider of dietary iron, it is an excellent source of omega-3 polyunsaturates, due to the α-linolenic acid in its abundant thylakoid membranes.) Notwithstanding the many challenges that face golden rice, the development of several new cultivars is well underway in Asia and this crop may yet have a modest impact on human nutrition in some parts of the world. The vitamin E group The vitamin E group of compounds includes four tocopherols and four tocotrienols, all of which have significant antioxidant properties. These lipophilic vitamins can be found in most non-processed seed oils but are often lacking in foods made from processed oils. There is interest in trying to increase the levels of this group of lipidic vitamins in plant oils using a variety of approaches. For example, transgenic plants that accumulate 10–15 fold higher levels of vitamin E compounds have been engineered by adding

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homogentisic acid geranylgeranyl transferase genes from several cereals to Arabidopsis plants (Cahoon et al., 2003). As described above, unrefined palm oil also contains significant amounts of vitamin E group compounds (Han et al., 2004). Since 2000, several varieties of oil palm have been identified that produce oil that is highly enriched in tocols to levels in excess of 1500 ppm, which would be of great interest as potential health food products. Phytosterols and stanols Another category of plant lipid of interest to the food industry is the phytosterols. Margarines enriched in phytosterols extracted from (non-transgenic) wood pulp or vegetable oils have recently been marketed and, despite an appreciable price premium compared to conventional margarines, they have enjoyed modest commercial success. The appeal of the phytosterol-enriched margarines is based on evidence that they may help to reduce blood cholesterol levels and hence combat heart disease. Such products could be made more cheaply if more of the phytosterols were synthesized in the same seeds as the oil from which the margarine is derived. Efforts are now underway to up-regulate phytosterol biosynthetic pathways in transgenic plants. The impact on human health of such products could be considerable. Indeed, it has been surmised that the widespread availability and consumption of low-cost, phytosterolenriched margarines could eventually lead to a quantifiable reduction in national rates of cardiovascular disease, which is still the most common cause of mortality, especially in low-income groups, in all industrial societies (Plat and Mensink, 2001).

12.7 Future trends We have seen that modern plant breeding technologies are capable of providing a wide spectrum of altered lipid profiles in plant oils that can potentially satisfy the demands of what has been a rather fickle and inconstant food industry. As the industry produces an ever wider range of foodstuffs that are targeted at different sorts of customer (e.g. health foods, organics, highquality ranges, cheaper ranges, etc.) it will become more economic for specific plant oils to be segregated and identity preserved for certain applications. This in turn will signal plant breeders to focus on satisfying such niche markets. It seems likely that we will gradually move away from the current low-value commodity oils that are bought in bulk and blended to order, and towards a more sophisticated segmented market that will include addedvalue oils from transgenic crops. Examples of the latter will include oils that are highly enriched in nutritionally desirable lipids, such as long-chain omega3 fatty acids and antioxidants such as carotenoids and tocols. There are two additional developments that may perturb the present market structure, namely biodiesel and oil palm. The potential impact of new high-

300 Modifying lipids for use in food yielding oil palm varieties has already been addressed in Section 12.6.4 and this may affect the industry very significantly in the long term. A shorter term trend that is already having an impact is the increasing diversion of oil crops away from food markets towards biodiesel production (Murphy, 1998b). This is beginning to affect the prices of plant oils and may have even more effect as Europe in particular seeks to use this renewable biofuel to help fulfil its obligations under the Kyoto agreement. In the longer term, however, it seems unlikely that society will countenance such a profligate waste by burning of a semi-refined product that can serve as either a nutritious food or a source of valuable oleochemicals. Besides, even if we used all of the available arable land on the earth for biodiesel crops, we would still only produce a fraction of the fuel needed to sustain current rates of usage for transportation. It is more likely that biodiesel will be a significant, but temporary, perturbation in the use of oil crops and that in the longer term, much of the production of such crops will shift to more efficient tropical systems such as oil palm.

12.8 Sources of further information and advice Links to databases and other resources for plant lipids (much of this information comes from the Lipid Analysis Unit site, as listed below) General lipids •

• • • • • •

The American Oil Chemists’ Society – the largest global society of oils and fats chemists, technologists and biologists with much useful information on nutrition and labelling (www.aocs.org). Of particular interest to the general reader is their monthly magazine, INFORM (http://www.aocs.org/press/inform/) and the journals JAOCS and Lipids. The European Federation for the Science and Technology of Lipids (http://www.eurofedlipid.org/index.htm) publishes the European Journal of Lipid Science and Technology (www.ejlst.de) and organizes conferences. Cyberlipid – a website containing much useful information on lipid chemistry, biochemistry and analysis (http://www.cyberlipid.org/). Lipid Nomenclature – this is the IUPAC guide http:// www.chem.qmw.ac.uk/iupac/lipid. Conjugated linoleic acid – Wisconsin Food Research Institute (http:// www.wisc.edu/fri/clarefs.htm). These pages give a comprehensive list of references to papers dealing with CLA. Compilation of trivial names of fatty acids (by RO Adlof and FD Gunstone) (http://www.aocs.org/member/division/analytic/fanames.htm). Lipidat – a relational database of thermodynamic and associated information on lipid mesophase and crystal polymorphic transitions, including lipid molecular structures (glycero- and sphingolipids) http:// www.lipidat.chemistry.ohio-state.edu/.

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Lipid Bank for Web – a database of information on lipid structures and properties with thousands of references (http://lipid.bio.m.u-tokyo.ac. jp/). Lipid Analysis Unit – this site is a general information resource on lipids supported by the Lipid Analysis Unit at the Scottish Crop Research Institute (http://www.lipid.co.uk).

Plant lipids •

•

• •

• •

On-line chemical data base for new seed crops produced by the New Crop Research Unit at NCAUR, Peoria, IL, USA (http:// www.ncaur.usda.gov/nc/ncdb/search.html-ssi/) (see Abbot et al., J Am Oil Chem Soc, 74, 723–726 (1997) and correction on p. 1181 for instructions) – chromatographic, physical chemical and spectroscopic information on oil seeds. Similar but more extensive database to the one previously provided by BAGKF (Institute for Chemistry and Physics of Lipids), Munster, Germany – SOFA (Seed Oil Fatty Acids) (www.bagkf.de/sofa) (see Aitzetmuller et al., Eur J Lipid Sci Technol, 105, 92–103 (2003)). A catalogue of genes for plant lipid biosynthesis at Michigan State University (http://www.canr.msu.edu/lgc/index.html). NPLC (National Plant Lipid Cooperative) (http://www.msu.edu/user/ ohlrogge/). A further source of links to web-based lipid information, includes: NPLC Directory of Plant Lipid Scientists, The NPLC Electronic Mailing List, The NPLC Database of Plant Lipid Literature. The Plant Lipids Home Page (http://blue.butler.edu/~kschmid/lipids.html). Maintained by Katherine Schmid this page contains many useful lipidrelated links. The Malaysian Palm Oil Board (MPOB) – website devoted to all aspects of oil palm biology, technology, food and non-food uses and commercial matters (http://www.mpob.gov.my).

Food and industry related •

• • •

Loders Croklaan, once a subsidiary of Unilever Ltd, is now part of the IOI group in Malaysia. The site covers science, technology and nutrition related to lipids in general and to oil palm products in particular (www.croklaan.com). ITERG – French Research Institute dealing with oils and fats research and technology (www.iterg.com). Natural of Norway – manufacturers of conjugated linoleic acid for health food and other applications (www.natural.no). The website of the British Nutrition Foundation carries information on lipids in addition to other food components (www.nutrition.org.uk).

302 Modifying lipids for use in food • • • • • • •

• • • •

USDA – US government site with information on fat-related nutritional advice (http://www.nalusda.gov/fnic/dga/dga95/lowfat.html). CTVO – a European group devoted to the chemical and technological utilization of vegetable oils (www.danet.de/fnr/ctvo). IENICA – an Interactive European Network for Industrial Crops and their Applications (www.csl.gov.uk/ienica/). ACTIN – a UK group devoted to non-food uses of oils and fats (www.actin.co.uk). European website for the American Soybean Association (www.asaeurope.org). International Food Science and Technology – contains information on various food problems including those involving lipids (www.ifst.org). Oil World is a German company producing data on a weekly basis for oilseeds, oils and fats and oil meals and covers production, imports, exports and disappearance. Information is based on different commodity oils and fats and is presented on the basis of individual countries (www.oilworld.de). FFA Sciences is a company manufacturing probes to measure free fatty acid levels in oils and clinical samples (www.ffasciences.com). Britannia Foods has some articles ‘By Invitation Only’ of interest to lipid technologists mainly – (www.britanniafood.com). Peter Lapinskas – consultant to the oils & fats industry – some interesting data on unusual seed oils (www.lapinskas.com). Plant Lipids – an Indian company specializing in products derived from a range of plant lipids (http://www.plantlipids.com).

Lipidomics-related websites • • • • •

http://www.ksu.edu/lipid/lipidomics http://hcc.musc.edu/research/shared_resources/lipidomics.cfm http://medschool.mc.vanderbilt.edu/brownlab/comlip.html h t t p : / / w w w. w i s s e n s c h a f t - o n l i n e . d e / g b m / h o m e p a g e / abstract_detail.php?artikel_id=265 http://www1.elsevier.com/gej-ng/29/50/lipids/119/47/26/article.pdf

12.9

References

ABBADI A, DOMERGUE F, BAUER J, NAPIER JA, WELTI R, ZAHRINGER U, CIRPUS P

and HEINZ E (2004), Biosynthesis of very-long-chain polyunsaturated fatty acids in transgenic oilseeds: constraints on their accumulation, Plant Cell, 16, 2734–2748. AGCANADA (2004), The United States Canola Industry: Situation and Outlook, Agriculture and Agri-Food Canada Bi-weekly Bulletin, February 27, 17(4), available at http:// www.agr.gc.ca/mad-dam/e/bulletine/v17e/v17n04_e.htm. BARTON KA, BINNS AN, MATZKE AJM and CHILTON MD (1983), Regeneration of intact tobacco plants containing full length copies of genetically engineered T-DNA, and transmission to R1 progeny, Cell, 32, 1033–1043.

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and HENSON IE (2004), Oil palm – achievements and potential, in New directions for a diverse planet, Proc 4th International Crop Science Congress, 26 Sep–1 Oct, Brisbane. BENATTI P, PELUSO G, NICOLAI R and CALVANI M (2004), Polyunsaturated fatty acids: biochemical, nutritional and epigenetic properties, J Am Coll Nutr, 23, 281–302. CAHOON EB (2003), Genetic enhancement of soybean oil for industrial uses: prospects and challenges, AgBioForum 6, 11–13, available at http://www.agbioforum.org. CAHOON EB, HALL SE, RIPP KG , GANZKE TS, HITZ WD and COUGHLAN SJ (2003), Metabolic redesign of vitamin E biosynthesis in plants for tocotrienol production and increased antioxidant content, Nat Biotechnol, 21, 1082–1087. CAPLAN A, HERRERA-ESTRELLA L, INZÈ D, VAN HEUTE E, VAN MONTAGU M, SCHELL J and ZAMBYRSKI P (1983), Introduction of genetic material into plant cells, Science, 222, 815–821. CORLEY H (2000), New technologies for plantation crop improvement, available at: http:/ /www.taa.org.uk/WestCountry/corley.html. CRAWFORD MA, COSTELOE K, GHEBREMESKEL K, PHYLACTOS A, SKIRVIN L and STACEY F (1997), Are deficits of arachidonic and docosahexaenoic acids responsible for the neural and vascular complications of preterm babies? Am J Clin Nutr, 66, 1032S–1041S. DOWNEY RK and CRAIG BM (1964), Genetic control of fatty acid biosynthesis in rapeseed (Brassica napus L.), J Am Oil Chem Soc, 41, 475–478. ENATTAH NS, SAHI T, SAVILAHTI E, TERWILLIGER JD, PELTONEN L and JARVELA I (2002), Identification of a variant associated with adult-type hypolactasia, Nature Genetics, 30, 233–237. ERNST & YOUNG (2000), The economic contribution of the biotechnology industry to the US economy, accessed, available at http://www.bio.org/news/publications/ ernstyoung.pdf, 15 April 2005. FORSTER BP, ELLIS RP, THOMAS WT, NEWTON AC, TUBEROSA R, THIS D, EL-ENEIN RA, BAHRI MH and BEN SALEM (2000), The development and application of molecular markers for abiotic stress tolerance in barley, J Exp Bot, 51, 19–27. FOURMANN M, BARRET P, RENARD M, PELLETIER G, DELOURME R and BRUNEL D (1998), The two genes homologous to Arabidopsis FAE1 co-segregate with the two loci governing erucic acid content in Brassica napus, Theor Appl Genet, 96, 852–858. FUNK CD (2001), Prostaglandins and leukotrienes: advances in eicosanoid biology, Science, 294, 1871–1875. GILL P, JEFFREYS AJ and WERRETT DJ (1985), Forensic application of DNA ‘finger prints’, Nature, 318, 577–579. GINTING G, SUBRONTO, HUTOMO T, FATMAWATI and LUBIS AU (1995), Early performance of oil palm clones produced by IOPRI, J Indonesian Oil Palm Res Inst, 3, 1. GREEN AG and MARSHALL DR (1984), Isolation of induced mutants in linseed (Linum usitatissimum) having reduced linolenic acid content, Euphytica, 33, 321–328. HAN NM, MAY CY, NGAN MA, HOCK CC and ALI HASHIM M (2004), Isolation of palm tocols using supercritical fluid chromatography, J Chromatogr Sci, 42, 536–539. nd HARLAN JR (1992), Crops and Man, 2 edn, Madison, WI, American Society of Agronomy. HILDEBRAND D, YU K, MCCRACKEN C and RAO SR (2005), Fatty acid manipulation, in: Murphy DJ, Plant Lipids: Structure, Biogenesis and Utilisation, Oxford, Blackwell, 67–102. HONG S, GRONERT K, DEVCHAND PR, MOUSSIGNAC RL and SERHAN CN (2003), Docosatrienes and 17S-resolvins generated from docosahexaenoic acid in murine brain, human blood, and glial cells. Autacoids in anti-inflammation, J Biol Chem, 278, 14677–14687. JAMES C (2005), Global Status of Commercialized Biotech/GM Crops: 2005, ISAAA Briefs, No 34, Ithaca, NY, ISAAA, available at http://www.isaaa.org. KLEINGARTNER LW (2002), NuSun sunflower oil: Redirection of an industry, in Janick J and Whipkey A, Trends in New Crops and New Uses, Alexandria, VA, ASHS Press, 135– 138. MACILWAIN C (2005), Stray seeds had antibiotic-resistance genes. Accidental release of genetically modified crops sparks new worries, Nature, 434, 548. MALUSZYNSKI M, NICHTERLEI K, VAN ZANTEN L and AHLOOWALIA BS (2000), Officially released mutant varieties – the FAO/IAEA database, Mutation Breeding Review, 12, 1–84.

304 Modifying lipids for use in food (2000), First Fruit – The Creation of the Flavr SavrTM Tomato and the Birth of Biotech Food, New York, McGraw Hill. MILLAR A, SMITH MA and KUNST L (2000), All fatty acids are not equal: discrimination in plant membrane lipids, Trends in Plant Science, 5, 95–101. MURPHY DJ (1994), Designer Oil Crops, Weinheim, VCH Press. MURPHY DJ (1996), Engineering oil production in rapeseed and other oil crops, Trends Biotechnol, 14, 206–213. MURPHY DJ (1998a), Impact of genomics on improving the quality of agricultural products, in Dixon GK, Copping LC and Livingstone D, Genomics: Commercial Opportunities from a Scientific Revolution, Cambridge, SCI Press, 199–210. MURPHY DJ (1998b), Biodiesel and its prospects, NRC-PBI Bulletin, Saskatoon, Canada, May, 8–10. MURPHY DJ (1999), Production of novel oils in plants, Curr Opinion Biotechnol, 10, 175– 180. MURPHY DJ (2001), New oil crops for the 21st century. What is in the pipeline and what is coming in the future? Proc Oils and Fats International Congress, September 4–8, 2000, Kuala Lumpur, Malaysia. MURPHY DJ (2004), Biotechnology, its impact and future prospects, in Barber J and Archer M, From Molecular to Global Photosynthesis, London, Imperial College Press, 649– 740. MURPHY DJ (2005), Plant Lipids: Structure, Biogenesis and Utilisation, Oxford, Blackwell. OIL WORLD (2005), Vol 48, 1 April, Hamburg, ISTA Mielke GmbH. PHILLIPS RL (1993), Plant genetics: out with the old, in with the new? Am J Clin Nutr, 58, 259S–263S. PLAT J and MENSINK RP (2001), Effects of plant sterols and stanols on lipid metabolism and cardiovascular risk, Nutr Metab Cardiovasc Dis, 11, 31–40. RAHMANA SM, KINOSHITA T, ANAI T and TAKAGI Y (2001), Combining ability in loci for high oleic and low linolenic acids in soybean, Crop Sci, 41, 26–29. RAHMANA SM, ANAI T, KINOSHITA T and TAKAGI Y (2003), A novel soybean germplasm with elevated saturated fatty acids, Crop Sci, 43, 527–531. QI B, FRASER T, MUGFORD S, DOBSON G, SAYANOVA O, BUTLER J, NAPIER JA, STOBART AK and LAZARUS CM (2004), Production of very long chain polyunsaturated omega-3 and omega6 fatty acids in plants, Nat Biotechnol, 22, 739–745. QUIROS CF and PATERSON AH (2004), Genome mapping and analysis, in Pua EC and Douglas CJ, Biotechnology in Agriculture and Forestry, Volume 54, Brassica, Berlin, Springer Verlag, 31–41. PAINE JA, SHIPTON CA, CHAGGAR S, HOWELLS RM, KENNEDY MJ, VERNON G, WRIGHT SY, HINCHLIFFE E, ADAMS JL, SILVERSTONE AL and DRAKE R (2005), Improving the nutritional value of Golden Rice through increased pro-vitamin A content, Nat Biotechnol, 23, 482–487. SPECTOR A (1999), Essentiality of fatty acids, Lipids, 34, 1–3. THELEN JJ and OHLROGGE JB (2002), Metabolic engineering of fatty acid biosynthesis in plants, Metabolic Eng, 4, 12–21. THOMPSON GA and LI C (1997), Altered fatty acid composition of membrane lipids in seeds and seedling tissues of high-saturate canolas, in Williams JP, Khan MU and Lem NW, Physiology, Biochemistry and Molecular Biology of Plant Lipids, Dordrecht, Kluwer, Netherlands, 313–315. USDA (2004), Malaysian Oil Palm, Production Estimates and Crop Assessment Division, Foreign Agricultural Service, available at http://www.fas.usda.gov/pecad/highlights/ 2004/11/mypalm/index.htm. USDA (2005), Oil Crops Outlook, September 13, Economic Research Service, USDA, available at http://usda.mannlib.cornell.edu/reports/erssor/field/ocs-bb/2005/ocs05hf.pdf. VAN STUYVENBERG JH (1969), Margarine: An Economic, Social and Scientific History, 1869–1969, Liverpool, Liverpool University Press. VOELKER TA, HAYES TR, CRANMER AM, TURNER JC and DAVIES HM (1996), Genetic engineering MARTINEAU, B

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13 Modifying fats of animal origin for use in food M. R. L. Scheeder, ETH Zurich, Switzerland

13.1

Introduction

Fatty acids in animal lipids originate from endogenous de novo synthesis, from dietary sources or from microbial synthesis and modification in the digestive tract. The essential polyunsaturated C18 n-6 and n-3 fatty acids cannot be synthesized by vertebrate organisms and are exclusively supplied by the diet. Subsequent modification of fatty acids occurs in the digestive tract by microbes or after absorption by endogenously produced enzymes. The activity of endogenous enzymes can in turn be affected by specific fatty acids. Therefore, genetic disposition for de novo synthesis and enzyme activity, diet composition, microbial activity and the interactions between these factors contribute to the highly complex fatty acid composition of animal lipids. These factors also provide manifold opportunities for considerable biomodification of animal lipids. However, desired objectives may be very different. One can be to increase the content of potentially health-beneficial bioactive fatty acids and to decrease saturated fatty acids which are still considered to be potential health risk factors. On the other hand, typical and desired technological properties of animal fats, such as oxidative stability, firmness and plasticity, depend on the presence of a certain amount of saturated fatty acids and may be adversely affected by (poly)unsaturated fatty acids. The challenge, therefore, is to increase the dietetic value of animal lipids and at the same time to maintain physical and sensory characteristics at an acceptable level. Technological methods of adjusting physico-chemical properties can be applied to extracted meat or milkfats (i.e. rendered animal fats or purified butter fat) as for any other fat or oil. However, the bulk of animal lipids is consumed in the form of meat and milk or their products and is ingested

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without being chemically modified. The focus of this chapter will, therefore, be on bio-modification measures that aim to change the lipid composition in the animal organism.

13.2 Motivation for bio-modification of animal lipids The perception of food of animal origin is somewhat inconsistent. Animal products are highly esteemed as food, but they have a bad image as nutrient. The great demand for animal derived food despite its rather high price reflects eating pleasure, sensory value and status. With increasing income in developing and transition countries, demand for and production of animal derived food are predicted to grow appreciably (Delgado, 2003) and food disappearance data clearly illustrate the correlation between increasing demand for animal derived food and increasing wealth in the industrialized countries during the last decades (faostat.fao.org, 2005) – at least until a certain level of affluence is reached. Although no cause and effect relationship is implied, it may be mentioned that during the same period life expectancy increased considerably in the wealthy nations. In contrast, food of animal origin is often denounced as a health risk factor, blamed for its supposedly high content of fat, particularly saturated fatty acids, and cholesterol. There is still a widespread belief and public perception that ‘fat is bad’ and dietary cholesterol a major cause of coronary heart disease (CHD). These associations, however, have been severely challenged. It is clear now that the type of fat rather than the total amount in the diet is important for human health (Calder and Deckelbaum, 2003) and it seems clear that the importance of dietary cholesterol as CHD risk factor has, despite public perception, obviously been over-emphasized (Hu et al., 2001; Parodi, 2004). The proportion of saturated fatty acids in major animal products, e.g. lean meat and lard, is below 50 % and, moreover, not all saturated fatty acids (SFA) detrimentally affect plasma lipids to the same extent; short to medium-chain SFA (C4–C10) are not associated with risk of CHD, and stearic acid also seems to be related to a far lower risk than C12– C16 (Kris-Etherton and Yu, 1997; Hu et al., 2001). Not only may the health risk of SFA and cholesterol be over-stated but animal products contain several beneficial components, including bioactive fatty acids with specific physiological functions (Macrae et al., 2004). In this context, polyunsaturated fatty acids (PUFA) of the n-3 family, particularly the long-chain C20 and C22 fatty acids (LC-) and the so-called conjugated linoleic acids (CLA), deserve specific attention. Additionally, the multibranched-chain fatty acid phytanic acid recently gained some interest as a potential anti-diabetes agent (McCarty, 2001) and promoter of fatty acid βoxidation (Ellinghaus et al., 1999). The natural and common sources of these fatty acids in human nutrition are predominantly or even exclusively of animal origin. Because of the

308 Modifying lipids for use in food importance granted to these fatty acids, alternative sources have been developed. CLA for example are produced semi-synthetically and are commercially available. However, the isomeric composition of these products is very different from CLA occurring naturally in meat and milk of ruminants (AFSSA, 2005). LC-PUFA, particularly arachidonic acid (ARA, 20:4n-6), eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3), are commercially produced by the use of micro-organisms (Gunstone, 1999), and ARA and DHA single-cell oils are now used as dietary supplements in infant formulas (Gunstone, 1999; Ratledge, 2004). Another possibile way to produce specific fatty acids would be to modify oil-seed crops through genetic engineering techniques (Huang et al., 2004). However, food not only fulfills nutritional demands but is also strongly related to tradition, taste and joy. The basic idea of modifying animal lipids is, therefore, to provide food with optimized compositional characteristics with the aim of meeting nutritional recommendations without the need to change traditional eating habits. Producing health-promoting animal products has indeed become a recommended strategy to maintain economics of future livestock production (Macrae et al., 2004). This is, however, a rather challenging aim due to conflicts between the requirements and recommendations of dieticians (nutritive value), the technological properties requested by food processors (physicochemical properties) and the sensory characteristics desired by the consumers (palatability).

13.3 Specific bioactive fatty acids in animal products 13.3.1 Polyunsaturated n-3 fatty acids Omega-3 or n-3 unsaturated fatty acids contain the first double bond at the third carbon from the methyl end of the chain. Vertebrates normally do not possess the ability to synthesize fatty acids with double bonds further to the methyl end of the acyl chain than the n-7 position (e.g. ∆9-desaturation of palmitic acid). The availability of n-6 and n-3 fatty acids in the organism therefore depends on the dietary supply of these essential fatty acids. The basic n-3 fatty acid, α-linolenic acid (ALA, 18:3n-3), is found mainly in photosynthetic active tissue of plants, while the predominant PUFA in storage organs (oilseeds, cereals) is linoleic acid (LA, 18:2n-6). It is only in few seed oils, such as linseed or rapeseed oil, that considerable amounts of ALA are found. ALA has several specific functions in mammalian organisms, although its major metabolic fate is to be used as fuel in β-oxidation (Sinclair et al., 2002). A major metabolic function of ALA is carbon recycling, e.g. for the lipid synthesis in the brain. It also plays a role in the protection of skin and fur and it is probably involved in regulating water homeostasis (Sinclair et al., 2002). However, much more attention has been given to the cardioprotective effects of ALA, which have been confirmed by epidemiological

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studies and secondary prevention trials (cited in Hauswirth et al., 2004). Another highly relevant role of ALA is to serve as precursor of the very long-chain n-3 polyunsaturated fatty acids EPA, DPA (docosapentaenoic acid, 22:5n-3) and DHA. These n-3 LC-PUFA can be endogenously synthesized from ALA in avian and mammalian organisms via a series of elongation and desaturation steps (Leonard et al., 2004). It is important to mention that the same enzymes are also involved in the elongation and desaturation of n-6 PUFA with consequent competition of LC-PUFA synthesis between these two fatty acid families. The dietary n-6/n-3 ratio, therefore, is considered an important nutritional issue, although there are indications for duplicate enzyme sets in mitochondria (Infante, J.P., 1997 cited by Stordy, 1999) and recently it has been concluded from results of an epidemiological study that n-3 fatty acids decrease the risk of CHD irrespective of the n-6 intake (Mozaffarian et al., 2005). The effectiveness of conversion from ALA to n-3 LC-PUFA has been a matter of considerable discussion (Gerster, 1998; Brenna, 2002), because of the fundamental and highly specific physiological functions of EPA and DHA. EPA, like its n-6 counterpart ARA, is a precursor of so-called eicosanoids, a series of endogenous mediators (prostaglandins, prostacyclins, thromboxanes and leukotrienes) which are involved in inflammatory response, regulation of blood pressure, platelet aggregation and further physiological reactions. Recently, DHA was also recognized as a precursor of endogenous mediators, called docosanoids (Hong et al., 2003; Serhan, 2005). However, a major function of DHA is its role as a vital building block in membranes of brain, synapses, retina and spermatozoa (Blank et al., 2002; Broadhurst et al., 2002). Moreover, an impressive list of further beneficial effects of DHA can be cited (Horrocks and Yeo, 1999). Overall, it may be concluded that EPA and DHA have specific and physiologically important functions while their production from ALA, which is quite readily available from plant sources, is limited (Burdge and Calder, 2005). This makes animal derived food as a potential source of LC-PUFA interesting because farm animals, when supplied with dietary ALA, may convert it to the more valuable n-3 LC-PUFA to a certain degree.

13.3.2 Conjugated linoleic acids CLA came into the focus of nutritional research when it was identified as an anti-carcinogenic compound found in fried beef and effective in animal tumour models (Ha et al., 1987). The dominant CLA isomer in ruminant products is the 18:2 cis-9, trans-11 compound, aptly named ‘rumenic acid’. This is formed in the mammary gland mainly by endogenous ∆9-desaturation of trans-vaccenic acid (18:1 trans-11), which in turn is an intermediate product of microbial PUFA biohydrogenation in the rumen (Griinari et al., 2000). In contrast, the best synthetic CLA products, often used in feeding experiments, mainly contain the cis-9, trans-11 (c9, t11) and the trans-10,

310 Modifying lipids for use in food cis-12 (t10, c12) isomers in approximately equal proportions. Meanwhile, an increasing number of studies raised evidence for impressive beneficial effects like anti-atherosclerotic and anti-diabetic effects, inhibition of carcinogenesis, modulation of immune functions and reduction of adipose tissue (Belury, 2002). In consequence, CLA has become an interesting feed supplement in animal nutrition in order to fortify CLA in food of animal origin and to make use of its effects in terms of enhancing feed conversion efficiency and proportion of lean in the carcasses (Jahreis et al., 2000), although these effects are not always consistent (Bee, 2000; Dugan et al., 2001; Scheeder et al., 2002b; Corino et al., 2003). However, the value of strategies to enhance the CLA content of animal products by adding synthetic CLA to the feed may be questioned in the light of the rather disappointing results from human studies, which have been far less convincing than might have been expected from the effects exerted in animal models and in vitro studies (Calder, 2002). Meanwhile some detrimental effects have been attributed mainly to the t10, c12 isomer (Belury, 2002; Wahle et al., 2004) which is generally found as one of the two major compounds in synthetic CLA (Jahreis et al., 2000). In an exhaustive report about health risks and benefits of dietary trans fatty acids, including CLA, the authors recommend that the use of synthetic CLA mixtures in animal nutrition not be authorized (AFSSA, 2005). As outlined below, there might nevertheless be reasonable and safe ways to use synthetic CLA mixtures as a tool to modify the fatty acid composition of adipose tissue in pigs.

13.3.3 Phytanic acid More recently, another fatty acid, which is originally also derived from ruminant products, the so-called phytanic acid, has raised interest mainly because of a hypothesized anti-diabetic effect (McCarty, 2001). Phytanic acid is a branched-chain fatty acid (3,7,11,15-tetramethylhexadecanoic acid), derived from phytol, a side chain of chlorophyll which can be cleaved by microbes in the rumen. Because of the first methyl group at position 3 in the acylic chain (the β-carbon), phytanic acid has to undergo peroxisomal αoxidation before being further subjected to β-oxidation. It is, therefore, a key player in rare, inherited disorders of α-oxidation in humans (e.g. Refsum’s disease), which cause phytanic acid accumulation in tissues and serum and lead to degenerative changes in the retina and the nervous system (Mukherji et al., 2003). On the other hand, phytanic acid was shown to increase insulin sensitivity and it is hypothesized that it may mimic or complement various effects of CLA including inhibition of tumour growth (McCarty, 2001).

13.4 Genetic effects on the composition of animal lipids A major genetic effect on the fatty acid composition of animal lipids is based on the genetic disposition of the animal to synthesize fat. This involves the

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partition of nutritional energy beyond that required for maintenance and work, between protein (i.e. muscle) and adipose tissue synthesis. The animal organism can synthesize de novo only SFA, which then can be desaturated to monounsaturated fatty acids (MUFA). Greater fat accretion or secretion of lipids from de novo synthesis will ‘dilute’ the PUFA, which can only originate from the diet. Thus, the fatter an animal, the more saturated will the lipids in adipose tissue be (Malmfors et al., 1978; Nürnberg et al., 1997; De Smet et al., 2004). Due to successful breeding for a high lean meat content and a reduced amount of adipose tissue in the carcasses, the proportion of PUFA is generally rather high in modern pigs (Scheper, 1982). Even a century ago, butchers complained about the impaired meat and fat quality in the ‘modern’ lean pigs (Herter and Wilsdorf, 1914), and the basic conflict between nutritional and technological quality became evident: PUFA are desirable in human nutrition, but high amounts in lard are undesired because of an increased susceptibility to oxidation (Monahan et al., 1992; Flachowsky et al., 1997) and an impaired (soft) consistency of adipose tissue (Enser et al., 1984; Whittington et al., 1986; Gläser et al., 2004). Similar consequences as in adipose tissue can be observed in muscle tissue or lean meat. The intramuscular lipids mainly consist of phospholipids (PL) and triacylglycerols (TAG). PL normally contain more PUFA than TAG, while TAG contain appreciably more MUFA and somewhat more SFA than PL (Table 13.1). The amount (not the composition) of PL, which are the major structural lipids forming the membranes, is quite constant in the muscle. An increasing amount of intramuscular fat therefore is nearly exclusively due to an increase of TAG. Consequently, the fatty acid composition of meat Table 13.1 Fatty acid composition of neutral and phospholipids in M. longissimus dorsi of pigs fed either a control diet or a diet containing extruded linseed. Neutral lipids

Phospholipids

Treatment

Control

Linseed

Control

Linseed

SFA 16:0 18:0 MUFA 16:1 18:1 PUFA 18:2n-6 18:3n-3 20:4n-6 20:5n-3 22:5n-3 22:6n-3

39.7 25.2 12.5 56.1 4.4 50.8 4.2 3.0 0.26 0.14 0.03 0.06 0.05

39.4 24.8 12.6 55.1 4.1 50.1 5.6 3.3 1.05 0.13 0.04 0.12 0.05

30.2 20.6 8.1 27.6 2.8 23.9 42.2 25.2 0.8 7.4 1.1 1.8 1.5

31.1 21.2 8.5 22.5 2.1 19.7 46.5 26.5 3.0 5.7 3.1 3.0 1.3

Source: Sottnikova and Scheeder (unpublished).

312 Modifying lipids for use in food changes to a higher proportion of MUFA and SFA at the expense of PUFA with increasing amount of intramuscular fat. Therefore, genetic differences between species and breeds can be related mainly to effects of the extent of fat accretion in muscle (De Smet et al., 2004). Further genetic effects may be attributed to differences in the activity of desaturases and elongases, synthesizing LC-PUFA from their essential C18 PUFA precursors or MUFA produced from palmitic or stearic acid. To our knowledge, not much work has been done in this area probably because measuring fatty acid composition in a huge number of individual animals as required for breeding is still laborious and expensive. This might also apply to dairy cows and milk fat, where some work has still to be done for a full understanding of lipogenesis, lipid accretion and modification in the mammary gland (Clegg et al., 2000). New technologies for efficient analyses, such as portable near-infrared devices to enable on-line measurement of fatty acid composition related traits, or modern breeding tools, such as marker-assisted selection, together with the increasing demand for optimization of the fat composition are likely to trigger future activity in this field. The fatty acids in milk fat are derived from (i) the diet and rumen microorganisms, (ii) adipose tissue stores, and (iii) de novo synthesis in the mammary gland. Because de novo synthesis in the mammary gland results predominantly in short- and medium-chain fatty acids but not in C18 fatty acids, genetic correlations of milk fat concentration and proportion of short-chain fatty acids are positive, while the correlations with long-chain fatty acids, derived either from body stores or from diet/micro-organisms, are negative (Palmquist et al., 1993a). Accordingly, milk from Jersey cows, a breed known for its high milk fat percentage, was higher in C6–C14 fatty acids than milk from Holstein cows, while it was the other way round for 18:1. Both 18:1 and short-chain fatty acids help to lower the melting temperature of milk fat, which is supposed to be necessary to successfully deliver the milk to the offspring (Gibson, 1991). Thus, it can be assumed that 18:1 compensates for a shortage in short-chain fatty acids (Palmquist et al., 1993a). The desaturation capacity might, therefore, be another genetic disposition important both for milk fat composition and for formation of cis-9, trans-11 CLA from transvaccenic acid (trans-11-18:1) (Griinari et al., 2000). Thus there are some starting points for the alteration of milk fat composition by means of breeding, but it has been questioned whether milk fat composition will become an important element of genetic improvement of dairy cattle, because of the gradual and slow changes which can be achieved. There may also be unfavourable antagonistic correlations between desired improvements in milk fat quality and other product traits leading to overall unclear economic incentives (Gibson, 1991). A highly interesting field of research opens at the interface of genetics and nutrition. Several fatty acids have been identified as signalling molecules and transcription factors which may affect gene expression (Crestani, 2004). A specific example is phytanic acid, which has been recognized as a natural

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ligand of nuclear receptors and was shown to act as transcriptional activator (Ellinghaus et al., 1999). It has also been hypothesized that phytanic acid could induce fatty acid catabolism (Ellinghaus et al., 1999) and may exert anti-diabetic effects (McCarty, 2001), making it a potentially interesting compound in animal feed and functional foods. Recently, it has been reported that phytanic acid is increasingly incorporated in liver, heart and muscle of finishing pigs with increased amounts of phytol up to 2 % in the diet (Raes et al., 2004a). Phytanic acid reached impressive levels of about 20 % of total fatty acids in liver and heart, predominantly replacing PUFA, while the incorporation in muscle tissue was much lower and no phytanic acid was found in lard. In a pilot study, we found a linear increase of phytanic acid in erythrocytes of finishing pigs from not detectable to 0.6, 1.2 and 1.86 g/100 g total fatty acid methylesters (FAME) with increased duration of exposure to 0.5 % phytol in the diet for 0, 20, 40 or 60 days prior to slaughter (Scheeder et al., 2005). In plasma TAG and PL, phytanic acid rose to remarkable 31.9 and 22.5 g/100 g FAME. The liver mass increased significantly with exposure time, whereas no consistent effect on liver glycogen content was detected. Despite the high phytanic acid levels in plasma TAG and PL, no effect on growth or adipose tissue accretion and no clear effects on medium(fructosamine) and long-term markers (glycosylated haemoglobin, HbA1c) of blood glucose levels were observed. The outcome could have been different in other, perhaps more appropriate, models than young, intensively growing pigs. However, phytanic acid can be increased in pork, depending on dose and duration of phytol intake, up to concentrations above those commonly found in beef. It must nevertheless be reported that phytanic acid is also suspected to promote prostate cancer (Mobley et al., 2003). Thus, further research in this field is certainly needed in order to elucidate the beneficial potential of fortification in animal products, and the drawbacks of phytanic acid. CLA is another, perhaps more promising, example of fatty acids potentially acting as transcriptional activator. The potential of CLA, administered as feed supplement, to modify lard composition, is outlined below. Genetic engineering would be another approach to modify the fatty acid composition in animal tissues. Spinach ∆12-desaturase has been successfully transferred and expressed in pigs, increasing LA from 9.9 to 11.6 % in adipose tissue (Saeki et al., 2004). The reported effect, although significant, seems quite limited compared to simple dietary interventions. Increasing n3 PUFA was achieved in mice engineered to carry a fat-1 gene from the roundworm Caenorhabditis elegans encoding for an enzyme that can add a double bond into an unsaturated fatty acid hydrocarbon chain and thus convert n-6 to n-3 fatty acids (Kang et al., 2004). Both groups argue that this approach could be a way to improve the dietetic value of animal products, however, there may be problems arising concerning the acceptance of genetically engineered farm animals.

314 Modifying lipids for use in food

13.5 Housing and temperature effects on adipose tissue composition Ambient temperature can affect distribution and fatty acid composition of adipose tissue in the animal organisms. Pigs held at 12 °C developed more subcutaneous fat than pigs held at 28 °C. The subcutaneous fat was at the same time more unsaturated in the pigs held at lower temperatures (Lefaucheur et al., 1991). The endogenous desaturases are obviously activated at low temperatures to decrease the melting temperature and keep the subcutaneous fat sufficiently mobile. Even rather moderate temperature differences or seasonal influences when pigs have access to outdoor areas can lead to significant differences in 18:1 proportion in the backfat (Lebret et al., 2003). Housing and temperature can therefore influence pig fat quality under commercial production conditions.

13.6 Methods of modifying animal fats by changes in diet Dietary effects on the composition of adipose tissue or milk lipids are manifold and can be very powerful. In principle, all factors which increase or inhibit fatty acid de novo synthesis will change the share of fatty acids from endogenous versus dietary sources and therefore lipid composition. Dietary compounds, among which are certain fatty acids themselves, may also affect the activity of enzymes involved in the modification of fatty acids, as well as the microbial modification of fatty acids in the digestive tract. However, a major effect is the direct influence of dietary fatty acids by incorporation into triacylglycerols of adipose tissue, milk fat and/or phospholipids of the cell membranes, e.g. in muscle. Particularly in non-ruminants, in which most of the dietary fatty acids are absorbed unchanged, adipose tissue well reflects the composition of dietary fats. In ruminants, however, the feed is ‘processed’ in the rumen like in a fermentation vat. Feeding ruminants, therefore, actually means feeding the rumen microbes and this has fundamental consequences for dietary effects on the lipid composition.

13.6.1 Effects of dietary fatty acids on the composition of milk and adipose tissue lipids from ruminants The fermentation process in the rumen and its interactive response to feed compounds is highly complex and only a brief, simplified description of the main mechanisms can be given here. For more detailed information and accurate descriptions topical reviews are available (Harfoot and Hazelwood, 1988; Sutton, 1989; Palmquist et al., 1993a; Chilliard et al., 2000; Walker et al., 2004). Dietary carbohydrates and protein can be fermented by rumen microbes to volatile fatty acids, which are predominantly acetate, propionate and butyrate. Acetate is derived mainly from fermentation of cellulose while

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rapidly degradable carbohydrates, e.g. from cereals, preferentially yield propionate. Acetate-CoA and 3-hydroxybutyrate (formed from butyrate in the rumen wall) are the starting substrates for fatty acid de novo synthesis, propionate is a precursor of lactose. It is, therefore, likely that effects on ruminal acetate production affect fatty acid de novo synthesis and thus milk fat concentration and the proportion of short- and medium-chain fatty acids in milk fat. Propionyl-CoA instead of acetyl-CoA or methylmalonyl-CoA instead of malonyl-CoA can serve as starting substrate for the synthesis of odd-chain fatty acids (15:0 and 17:0) or methyl-branched isomers. However, the contribution of these processes to odd- or branched-chain fatty acids in milk is assumed to be very small. The major source of these fatty acids is rumen microbes, which contain a large proportion of odd- and branchedchain fatty acids in their membrane lipids (Vlaeminck et al., 2005). Oddchain fatty acids are formed through microbial elongation of propionate or valerate, while branched-chain amino acids (valine, leucine and isoleucine) and branched short-chain carboxylic acids (isobutyric, isovaleric and 2-methyl butyric acid) are assumed primers of microbial (iso and anteiso) branchedchain fatty acid synthesis (Kaneda, 1991). Dietary lipids may decrease microbial fermentation and, furthermore, provide long-chain fatty acids, which may lead to a shift from C6–C14 to C16/ C18 fatty acids in milk fat with the C16/C18 ratio depending on the C16/C18 ratio in the diet (Palmquist et al., 1993a). Dietary lipids are rapidly hydrolyzed in the rumen leading to free fatty acids. Unsaturated free fatty acids then undergo severe microbial biohydrogenation and commonly only a small portion of PUFA escape the rumen unchanged and are available for absorption at the duodenum. On average 80 % of LA and 92 % of ALA are hydrogenated, although with high concentrate diets these proportions may drop to 50 and 65 %, respectively (Chilliard et al., 2000). Biohydrogenation pathways for LA and ALA are different, resulting in stearic acid as end product but also various intermediates including fatty acids with trans and conjugated double bonds (Harfoot and Hazelwood, 1988). Due to the specific pathways of microbial biohydrogenation also a specific distribution of double bonds in the 18:1-trans isomers with predominantly ∆11-trans-18:1 (trans-vaccenic acid; vacca: Latin for cow) occurs in ruminant fats. In partially hydrogenated plant oils, in contrast, elaidic acid (∆9-trans-18:1) is the predominant isomer (Aro et al., 1998; Wolff et al., 2000). Trans fatty acids (TFA) are identified as a highly relevant risk factor for ischemic heart disease and further deleterious effects (Stender and Dyerberg, 2004). Because trans-vaccenic acid can be desaturated endogenously to c9, t11 CLA, it is often argued that TFA of animal origin might be less harmful than technologically produced TFA. However, this hypothesis has not been approved yet (Weggemans et al., 2004), although it may be assumed from the epidemiological studies cited in the review of Weggemans et al. (2004) that the amount of trans fatty acids of animal origin seldom reaches levels likely to exert a health risk. Furthermore, a shift from the less harmful vaccenic acid to the more harmful 18:1-trans10

316 Modifying lipids for use in food may occur when higher proportions of concentrate are fed (Dannenberger et al., 2004). Thus the health risk potential might be different for meat and milk fats derived from different production systems, depending on type and amount of concentrates used. The extensive biohydrogenation of unsaturated fatty acids is the key factor for the high degree of saturation of ruminant fats, and the idea of increasing the polyunsaturated/saturated fatty acid ratio (P/S) to improve the dietetic value of ruminant fats has, therefore, been of interest for quite some time (McDonald and Scott, 1977) and remains so (Scollan et al., 2005). Various attempts to protect dietary lipids from microbial lipolysis and unsaturated fatty acids from biohydrogenation have been undertaken: the use of whole or only coarsely crushed oilseeds, formaldehyde-protein-protected fat and selected fatty acids in fat prills or as calcium soaps (Sutton, 1989). The two latter sources mainly contain saturated fatty acids, and formaldehyde treatment is often not permitted or accepted. Crushed oilseeds in contrast provide a natural source of partially protected (poly)unsaturated fatty acids and significantly alter the fatty acid composition of adipose tissue in fattening bulls (Casutt et al., 2000). Fatty acids typical for the respective oilseeds (oleic, linoleic, linolenic acid in canola, sunflower and linseed, respectively) were increased significantly, although to a rather limited extent. Due to the still high biohydrogenation, the most obvious effect was an increase in stearic acid. This also led to the apparently contradictory effect that supplementation with highly unsaturated fatty acids produced tallow with a higher solid fat content at 20 °C compared with the control (Casutt et al., 1999). The supplementation of oilseeds thus affected the melting properties of the tallow, but the effect was too small to exert significant effects on other physical consistency traits (Casutt et al., 1999) and did not markedly affect properties of beef patties produced therewith (Scheeder et al., 2001). This is somewhat different for milk fat. Because of the high desaturation capacity of the mammary gland, a high availability of dietary 18:0 increases 18:1 in milk fat. Together with the decrease of 16:0 the melting temperatures are concomitantly decreased, leading to a lower solid fat content at 5 °C and therefore improved spreadability of butter at refrigeration temperatures (Banks cited by Palmquist et al., 1993b). The ruminal biohydrogenation of PUFA is a severe obstacle when trying to fortify n-3 fatty acids in the products. Wachira et al. (2000) reported that the extent of hydrogenation and therefore the transfer efficiency is higher when the n-3 source is forage where ALA is esterified to glycolipids, which are less prone to lipolysis. Feeding grass-based diets indeed increased the proportion of n-3 fatty acids in milk fat compared with a diet containing maize silage and concentrates (Leiber et al., 2005). In cheese from Switzerland, where mainly grass-based diets are still common, clearly higher proportions of n-3 fatty acids have been found than in e.g. cheddar (Hauswirth et al., 2004). There was also a specific n-3 enhancing effect of alpine grazing observed, which could not be explained with a higher ALA content in the

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alpine flora but was probably due to a reduced ruminal hydrogenation of ALA as a potential consequence of nutritional energy shortage and/or inhibiting secondary plant metabolites in the alpine flora (Leiber et al., 2005). DHA and EPA were also higher in milk from grass fed cows, but did not specifically respond to alpine grazing. Similarly, intramuscular lipids of grass fed bulls contained more n-3 PUFA including EPA and DHA compared with concentrate fed bulls (Dannenberger et al., 2004), whereas it was reported that including ALA-rich linseed in cattle diets increased ALA and EPA, but not DHA (Scollan et al., 2005). Attention must also be paid to the sensory properties of the products, as the susceptibility of the highly unsaturated fatty acids to oxidative degradation may result in deleterious flavour (Scollan et al., 2005). Because the main CLA isomer in milk is mainly built by desaturation of trans-vaccenic acid in the mammary gland or adipose tissues, increasing the amount of ALA in the diet will also increase CLA in milk and tissues of ruminants. Besides the cis-9, trans-11 isomer, a number of other isomers are also naturally occurring in ruminant products, but to a much lower extent (Jahreis et al., 2000; Nürnberg et al., 2002). The trans-11, cis-13 isomer might deserve special attention, because the amount of this isomer in the milk fat seems to respond to the altitude at which the cows are grazing and it could therefore be a typical ‘alpine’ CLA-isomer (Collomb et al., 2004). This has been supported by results of a controlled feeding experiment and alpine sojourn of dairy cows (Leiber et al., 2005). Increasing CLA is still claimed to be beneficial to human health and strategies to enhance its content in milk fat have been described recently (Chilliard et al., 2000; Lock and Bauman, 2004). Interestingly, dietary EPA and/or DHA specifically increase the ruminal production of trans fatty acids and CLA by a mechanism unknown so far (Chilliard et al., 2000). Supplementation of EPA and DHA to cattle diets could, therefore, increase these valuable n-3 LC-PUFA as well as CLA. However, the transfer efficiency of EPA and DHA from diet into milk is very low (2–4 %), and the supplementation of these fatty acids to the diet can massively reduce milk fat synthesis (Lock and Bauman, 2004). More detailed background information about strategies to increase EPA and DHA in milk and explanations for the low transfer efficiency are given by Rymer et al. (2003). A more efficient way might be to add EPA and DHA formulations directly to the milk. Such fortified products are already commercially available, marketed for example as ‘Einstein milk’, referring to the claims that DHA helps brain development and improves learning ability.

13.6.2 Modifying fats from non-ruminant (monogastric) animals In non-ruminant (monogastric) animals there is less microbial modification of dietary fatty acids in the digestive tract, although traces of 18:1 trans and CLA (18:2 9c, 11t) can be found in adipose tissue of pigs fattened on diets not containing these fatty acids (Gläser et al., 2000). This indicates microbial

318 Modifying lipids for use in food biohydrogenation already at sites in the gut where absorption of long-chain fatty acids takes place. Nevertheless, in monogastric animals the bulk of dietary fatty acids is absorbed as such (which also applies to humans) and the body lipids may well reflect the fatty acid composition of the feed. This is well established knowledge, and scientific evidence for the considerable influence of the fatty acid composition in the feed on the animal’s body fat composition dates back to the first quarter of the last century (Ellis and Ishikawa, 1926). Feeding diets with different fatty acid composition to pigs is surely one of the most certain ways to conduct an experiment resulting in significant differences. In fact, lipids are the only macronutrients for which Feuerbach’s philosophical proverb ‘You are what you eat’ (‘Der Mensch ist was er isst’) (Lemke, 2004) also applies in a physiological manner and this provides vast opportunities for manipulating the lipid composition in animals – but also bears the risk of undesired effects on fat quality. Medium-chain fatty acids (MCFA), naturally occurring in milk fat, coconut and palmkernel oil, can be used in animal nutrition for different purposes: MCFA have been shown to decrease methane emission from ruminants (Soliva et al., 2004); they can be used as growth promoting feed additives, because of their antimicrobial effect (Dierick et al., 2002); and MCFA are known to increase firmness and oxidative stability of lard when fed to pigs (Jaturasitha et al., 1996). MCFA could, therefore, be used to improve lard firmness, which is desired by meat processors, but the concomitantly increased amount of SFA is undesirable in terms of dietetic value. Furthermore MCFA bear another risk, which has made industrial processors of poultry fat for use in dehydrated soup concentrates or stock cubes establish a restrictive upper limit of maximum 0.25 % lauric acid. These convenience products are nowadays usually produced without heat treatment, leaving lipases from included cereals and spices sufficiently active to hydrolyze triacylglycerols. MCFA are particularly prone to hydrolysis, and lauric acid produces an undesired ‘soapy’ flavour making the soups unacceptable. If the aim is to avoid lauric acid in poultry fat, the close and linear relationship between lauric acid in the feed and in the abdominal fat should be considered (Fig. 13.1). A concentration of 2.1 g lauric acid per kilogram feed already resulted in 1.35 % lauric acid in abdominal fat. Accordingly, only traces below 0.3 g lauric acid per kilogram feed would be tolerable to cope with the threshold of 0.25 % in poultry fat (Scheeder et al., 2002a). It seems worth mentioning that similar effects can cause feed intake refusal, when MCFA-containing oils are used as ingredient in animal feed and mixed with ground cereals. One of the most striking conflicts in pig production arises from the contradictory demand of meat processors for firm lard and lean meat and of dieticians for fat low in saturated fatty acids and high in PUFA. Because pigs readily incorporate dietary PUFA in adipose and muscle tissue, it would be easy to produce high-PUFA lard and pork. Additional PUFA, however, markedly increase the oxidative potential of muscle and adipose tissue lipids. Autoxidation of PUFA increases specific volatile flavour compounds and may lead to

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12:0 in abdominal fat (g/100 g FAME)

5 4 3 2 1 0

Fig. 13.1

y = 0.142x + 0.070 R2 = 0.997

0

5

10

15 20 25 Total 12:0 intake (g)

30

35

Correlation between lauric acid (12:0) intake and proportion in abdominal fat of broiler chicken fattened to 2.3 kg live weight.

deterioration of the flavour of animal products (Elmore et al., 2000). While the oxidative stability can be controlled to a certain extent by the use of antioxidants in the feed (Wood et al., 2004) or added to products, an undesirably soft consistency of adipose tissue can hardly be corrected by means of food technology. Therefore, controlling consistency seems to be the most important issue when fortifying pork with PUFA. To control the PUFA content in lard in order to ensure a sufficiently high processing quality of pork and pig adipose tissue, various guidelines have been developed independently limiting PUFA concentrations in diets for growing-finishing pigs (Warnants et al., 1996). The Swiss guideline is one of the most restrictive with a recommended maximum of 0.8 g PUFA/MJ digestible energy or 12 g PUFA/kg feed (Perdrix and Stoll, 1995). To our knowledge, Switzerland is the only country where fat quality is routinely measured in pig carcasses and plays a part in the payment system. For this purpose, the so-called ‘fat score’, a semi-automated on-line method to measure the amount of double bonds in backfat, has been established in Swiss slaughter plants (Häuser et al., 1989). The analytical principle of the fat score is based on the iodine value according to Margosches (Margosches et al., 1924), and pooled samples of each batch of slaughter pigs are analyzed (Scheeder et al., 1999). When the current threshold for tolerable fat quality is exceeded (fat score 62) price deductions will be applied to the whole batch. Because of the mentioned correlation between lean meat content of the carcass, which generally is a main breeding goal, and the PUFA content in pig fat, these are challenging conditions for pig breeders. As shown in Table 13.2, pigs in the highest lean class often exceed the threshold for acceptable fat quality (Schwörer, 2004). Lard consistency, however, is more dependent on the saturated to unsaturated fatty acid ratio than on PUFA content alone (Gläser et al., 2002a, 2004). In a feeding experiment with finishing pigs a low fat control diet or the control diet supplemented with either lard, olive oil or soybean oil to achieve a similar amount of double bond in the diet were fed (Gläser et al., 2002a).

320 Modifying lipids for use in food Table 13.2 Relation between the proportion of lean meat in pig carcasses and the degree of saturation in the backfat. Lean classes [% Proportion of valuable cuts]1

Fat score2

46.95–49.99 50.00–52.99 53.00–55.99 56.00–62.31

58.6 60.4 61.6 63.4

± ± ± ±

1.2 2.0 2.6 2.5

1

Trimmed prime cuts as proportion of cold carcass weight, a measure of lean meat content in the carcass. Fat quality criterion based on the iodine value. Source: Schwörer (2004). 2

The fatty acid composition of backfat clearly reflected the dietary fatty acid composition in the supplemented groups (Table 13.3), but only the diet high in MUFA decreased SFA below 30 %. PUFA from soybean oil were mainly incorporated at the expense of MUFA, because LA as well as ALA inhibit the activity of ∆9-desaturase (Kouba et al., 2003). Therefore, although the fat score (like the iodine value) is highest in the soybean oil group, firmness of lard is not lower than in lard of pigs fed olive oil and the crystallization time was highest for the lard high in MUFA and low in SFA. The impact of the dietary-induced difference in fatty acid composition can also be seen in the melting curves and the development of solid fat content (SFC) between –6 and 20 °C of typical lard samples from this feeding experiment (Figs 13.2 and 13.3). With increasing degree of saturation, the low-melting fractions (peak 1 and 2) decline and a very high-melting fraction (peak 5) appears. SFC of lard with less than 30 % SFA is already at 0 °C below 50 % and therefore much too soft for meat products (Gläser et al., 2004). The oxidative stability, however, was quite high, suggesting that antioxidants in the olive oil (Baldioli et al., 1996) might have been transferred to the adipose tissue (Wenk et al., 2000). The firmness of pig adipose tissue can be increased by feeding saturated fats, but even larger effects can be achieved with trans fatty acids. Lard of pigs fed partially hydrogenated canola oil (D in Figs 13.2 and 13.3; Table 13.4) developed melting characteristics close to tallow. When the pigs were fed diets supplemented with 6 % of pure high-oleic sunflower oil (HO) or HO plus increasing amounts of partially hydrogenated canola oil (HR; 1.85 %, 3.70 %, 5.55 %), containing high levels of 18:1 trans fatty acid isomers, 18:1 trans fatty acids and cis-9, trans-11 CLA increased linearly in backfat and firmness was boosted up to eight-fold while the proportion of PUFA even slightly increased (Gläser et al., 2002b). This clearly indicates that trans-vaccenic acid is transformed to cis-9, trans-11 CLA in pigs and demonstrates the inhibiting effect of trans fatty acids on the activity of ∆9desaturase.

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Table 13.3 Fatty acid concentration and amount of double bonds in the diet of finishing pigs and in the outer layer of their backfat. Control

Lard

Fatty acid composition of the diet [g FAME1/kg DM] SFA2 3.8 34.5 3.2 35.3 MUFA3 18:1n-9 cis 2.8 29.4 10.0 18.8 PUFA4 18:2n-6 9.1 15.7 18:3n-3 0.80 1.55 Double bonds 82 256 [mmol/kg DM]

Olive oil

Soybean oil

11.6 43.2 41.0 17.1 15.5 1.42 266

9.0 12.3 11.2 29.2 26.2 2.81 249

Fatty acid composition and fat quality traits of the outer backfat layer [g/100 g FAME] SFA2 16:0 18:0 MUFA3 18:1n-9 PUFA 18:2n-6 18:3n-3 Double bonds [mmol/g] Fat score4 Oxidation stability5 Firmness 6 RIC-Box7 [s]

38.8a 23.4a 13.3a 46.6c 39.6c 14.7b 12.1b 0.93b 2.64c 60.8c 4.33a 151a 205c

35.2b 21.3b 11.9b 49.9b 42.1b 15.0b 12.0b 0.94b 2.79bc 64.2b 4.10a 114b 366b

29.8c 19.3c 8.8c 56.2a 50.4a 14.0b 11.8b 0.92b 2.92b

35.0b 21.0b 12.1ab 39.7d 34.3d 25.4a 21.1a 2.00a 3.18a

66.8b 4.59a 56c 610a

70.1a 2.36b 53c 296bc

Note: Superscripts a–d identify the presence or absence of significant differences between least squares means, i.e. common superscripts indicate no significant difference (Scheffé, p < 0.05). 1 Fatty acid methyl esters. 2 Saturated. 3 Monounsaturated fatty acids. 4 A semi-automated measure according to the iodine value. 5 Induction time [h], measured with Rancimat. 6 Firmness of lard at 0 °C, penetration force [g] at 2.5 mm distance. 7 Rapid interesterification control (crystallization time). Source: Gläser et al. 2002 b.

A similar effect on ∆9-desaturase is achieved by a CLA isomer. In a series of feeding experiments with growing-finishing pigs, clear dose–response and exposure time-dependent effects of CLA supplements on lard firmness were observed (Fig. 13.4) and it was shown that the trans-10, cis-12 isomer is the active isomer in this respect (Scheeder et al., 2004). At the same time, both CLA isomers increased linearly in backfat and muscle TAG as well as PL with increased intake. The trans-10, cis-12 isomer, however, was incorporated to a lower level than the cis-9, trans-11 isomer. This raises the possibility of making use of the firmness-enhancing effect of CLA with only

5 ak Pe

ak 3 ak 4 Pe

Pe

ak Pe

Pe

ak

1

2

322 Modifying lipids for use in food

A

Heat flow (W/g)

B

C

Endotherm

D

– 50 – 40 – 30 – 20 – 10 0 10 20 Temperature (°C) A B SFA 29.9 36.1 cis-MUFA 54.6 39.2 trans-MUFA – – PUFA 15.5 24.7

30

40

50

C 38.5 45.8 – 15.6

D 44.8 41.7 2.0 11.5

Fig. 13.2 Melting profiles of lard, differing in fatty acid composition (Gläser et al., 2004; Copyright Society of Chemical Industry. Reproduced with permission granted by John Wiley & Sons Ltd on behalf of the SCI).

Solid fat content (%)

100 A B C D

80

60

40

20

0 – 10

–5

0

5 10 15 Temperature (°C)

20

25

Fig. 13.3 Development of solid fat content of lard samples, differing in fatty acid composition, with increasing temperature (for fatty acid composition of the samples see Fig. 3.2) (Gläser et al., 2004; Copyright Society of Chemical Industry. Reproduced with permission granted by John Wiley & Sons Ltd on behalf of the SCI).

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Table 13.4 Fatty acid composition (g/100 g FAME) and firmness (N) of extracted backfat of pigs fed different amounts of partially hydrogenated canola oil.

16:0 18:0 16:1 18:1 18:1 18:1 18:1 18:1 18:1 18:1 18:2 18:2 18:3 Firmness2

∆9 ∆6–∆8 ∆9 ∆10 ∆11 ∆9 ∆11 ∆13 ∆9, 12 ∆9, 11 ∆9, 12, 15

trans trans trans trans cis cis cis all cis cis, trans all cis

HO

HOHR

HRHO

HR

18.2 8.9c 1.38b –1 – – – 52.8a 2.30b 0.09d 11.0 0.08d 0.75 0.77c

18.2 10.0b 1.47b 1.77c 1.57c 0.88c 0.31c 46.5b 2.21b 0.20c 10.5 0.70c 0.71 1.60c

18.7 10.5b 2.21a 3.61b 3.12b 1.62b 0.62b 38.2c 2.46a 0.38b 10.7 1.31b 0.73 2.91b

19.2 11.8a 2.34a 5.25a 4.46a 2.54a 0.97a 30.2d 2.51a 0.49a 10.4 1.81a 0.72 6.17a

Note: Superscripts a–d identify the presence or absence of significant differences between least squares means, i.e. common superscripts indicate no significant difference (Student-Newman-Keuls, p < 0.05). Abbreviations: HO = 6 % high oleic sunflower oil (HOSO) in the diet, HOHR = 4 % HOSO, 1.85 % partially-hydrogenated canola oil (PHRO), 0.15 % sunflower oil (SO), HRHO = 2 % HOSO, 3.7 % PHRO, 0.3 % SO, HR = 5.55 % PHRO, 0.45 % SO added to a basal diet. 1 Concentration too low to detect and separate individual 18:1 trans isomers, 2 Maximum force needed to drive a 3.5 mm punch 15 mm into the extracted fat at 0 °C Source: Gläser et al., 2002a.

Penetration force (N)

5 4 3 2 1 0

0

200

400

600 800 1000 Total CLA intake (g)

1200

1400

Fig. 13.4 Firmness of lard extracted from pigs fed diets containing 0, 0.25, 0.5 or 0.75 % CLA from 66–106 kg live weight measured as penetration force of a 3.5 mm punch (Scheeder et al., 2004).

little accumulation of the trans-10, cis-12 isomer, which has been judged critically in a recently launched report about health effects of trans fatty acids (AFSSA, 2005). CLA was also reported to improve feed conversion ratio and to decrease carcass fat content and backfat thickness in pigs (Jahreis et al., 2000). Although these effects have not been found in other studies (Bee, 2000; Dugan et al.,

324 Modifying lipids for use in food 2001; Scheeder et al., 2002b, 2003; Corino et al., 2003), CLA might nevertheless be a promising feed additive in pig production, because it may compensate for the softening effect of PUFA. A higher amount of PUFA in the diet and consequently in backfat might, therefore, be acceptable when CLA is fed at the same time. Thus, CLA provides the possibility of moderating the conflict between technological quality and dietary value of pork to a certain extent and gives some opportunity for a higher proportion of valuable n-3 PUFA in pig feed and therefore products (Enser et al., 2000). For instance, the inclusion of extruded linseed in finishing pig diets, corresponding to an additional 4.5 g ALA/kg feed, decreased lard firmness by about 40 %, but led to an increase of n-3 PUFA in cooked pork loin and neck from about 90 to 250 or 170 to 490 mg/100 g edible portion, respectively (Sottnikova et al., 2004). Arachidonic acid (AA, 20:4 n-6) was slightly decreased and the n-6/ n-3 ratio was lowered from about 10.2 to 3.7 and from 8.8 to 2.8 in loin and neck, respectively. The amount of EPA was on a low level and increased from 7.4 to 17.6 and from 9.6 to 22.7 mg/100 g in cooked loin and neck, respectively, while DHA did not change at all. These findings are consistent with other studies (Kouba et al., 2003; Wood et al., 2004), leading to the assumption that fortifying meat by supplementing ALA to the feed is likely to improve EPA and DPA supply but will not increase DHA supply to the same extent. In contrast, feeding ALA to laying hens markedly increases DHA in the eggs (Bourre, 2005). Eggs, therefore, not only contribute to the supply of n3 LC-PUFA, particularly DHA (Meyer et al., 2003), at present but have great potential to increase the share of DHA provided by farm animal products. Another favourable aspect surely is that the susceptible, highly-unsaturated fatty acids are in that case provided in a naturally well-protected package. While the fatty acid composition can be changed greatly by dietary measures in monogastric animals, there seems to be no possibility to modify TAG structure by feeding fat differing in the positional distribution of fatty acids at the glycerol backbone but not in fatty acid composition (Scheeder et al., 2003a). Changing TAG structure at a given fatty acid composition can probably only be achieved technologically by chemical or enzymatic interesterification.

13.7 Technological modifications Animal derived lipids are used to a great extent directly as foods of animal origin, such as milk, cream and cheese or meat and sausages, without technological modification apart from processing steps such as grinding or churning. Rendered animal fats or butter fat as any other fats and oils may, however, undergo the same modification and refining processes as vegetable oils in order to modify the degree of saturation (hydrogenation), remove undesired colour or flavour compounds (bleaching and deodorization), achieve desired texture, plasticity and melting behaviour (plastication,

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interesterification), separate the fat into fractions with specific characteristics (fractionation) or manufacture special products (powders). These processes are described in detail elsewhere in this book and will only briefly be addressed here, with special regard to animal derived lipids. Fats derived from ruminants are seldom hydrogenated because they are generally already high in saturated fatty acids. Mild hydrogenation of tallow may be applied to reduce highly unsaturated tri- and tetraenoic fatty acids to prevent development of flavour active oxidation products (Dugan, 1987). In lard, fatty acid composition and, therefore, melting properties can vary appreciably due to genetic and dietary effects as described above. Hydrogenation can help to obtain a product with consistent characteristics. Lard can also be completely hydrogenated and blended with unhydrogenated lard to achieve the desired physical characteristics (Dugan, 1987). Interesterification (also known as randomization, ester interchange and transeserification) is applied to randomize the distribution of fatty acids in the triacylglycerols. This is most relevant to lard, in which palmitic acid is typically esterified predominantly to the central sn-2 position of the triacylglycerols. Positional randomization changes the crystal habit of lard from β to β′, which is the preferred polymorphic form for fats used as shortenings. Interesterification of a blend of hard fat (e.g. tallow) and oil can be applied as an alternative to hydrogenation to produce plastic fats (Love, 1996). Interesterification can also be applied to butter fat, achieving a significant change in the triacylglycerol composition, but during the process the typical flavour will be lost and commercial interest in using this process for milk fat is obviously low (Hettinga, 1996). The flavour of milk fat is commonly perceived as highly desirable and is a most important property making milk fat an attractive food ingredient. The physical properties of milk fat, however, often fail to meet the technological functionality required for applications where its use is desired because of the flavour. Milk fat melts over a very wide range from about –30 to 40 °C, but the rather steep melting curve from 0 to 20 °C with a high solid fat content at refrigeration temperatures makes cold butter too firm to spread easily while it is not firm and plastic enough at higher temperatures to be used as shortening in pastry. Several physical modification processes such as tempering, texturization and fractionation can be applied to improve melting behaviour and solid fat content as desired (Kaylegian et al., 1993): air or nitrogen is incorporated into so-called whipped butter to improve the spreadability. With this method the volume is increased by about one third. A thermal treatment to cream before churning was invented by the Alnarp Dairy industry in Sweden giving a name to the processes of temperature profiling of the cream (Alnarping) to decrease the firmness of butter produced in this way. With mechanical treatments (texturizing or working) the crystal network and primary crystal structure of the butter is disrupted and a new structure is formed. It is, however, important to let butter crystallize completely before applying the

326 Modifying lipids for use in food mechanical treatment, and a severe disadvantage of this method is that the new structure is rather weak and the butter will lose its properties when subjected to temperature fluctuations. Blending of high- and low-melting fractions of anhydrous milk fat is another method to achieve milk fat with specific melting properties (Kaylegian and Lindsay, 1992). Fractionation can be achieved by separating triacylglycerols according to their melting points (dry fractionation) or by their solubility in solvents. The crystals formed at different temperatures during a thermally controlled process are then physically separated, e.g. by filtration or centrifugation. Solvent fractionation can be more efficient than dry fractionation, also leading to a better separation of fractions. The use of solvents, however, is often undesired or even prohibited. An interesting approach to fractionation of anhydrous milk fat has recently been reported, using plant oils as solvent (Wright et al., 2005). Although interesting products may be drawn from such a process, the use for fractionation purposes may be questioned, because of an unsatisfactory separation of the solids from the liquid phase. More detailed reviews about these methods and further examples are given by Hettinga (1996) and Kaylegian et al. (1993). Another pragmatic approach to combining desirable characteristics of milk fat with the supposedly beneficial dietetic value of vegetable oils is blending these items to a spread. The International Dairy Federation introduced ‘Guidelines for Fat Spreads’ (IDF Standard 166:1993) to provide a framework for more specific definitions and standards. This standard suggested the use of terms such as Blend, Blended spread, Low fat blended spread, depending on the fat content, for mixed fat products containing 15–80 % milk fat of total fat. Butter–vegetable fat spreads were invented and first introduced in Sweden but are now established fat spreads in various countries (Mann, 1997). For example, it has been reported that butter–vegetable oil blends accounted for 17 % of total spreads used in 1996 in Finland (Lampi et al., 1997). Quite an effort has been undertaken to develop technologies for reducing cholesterol in milk fat. Spreads made from ‘designer fats’ like cholesterolreduced anhydrous milk fat were shown to be acceptable for people used to consuming margarine, but were less liked by butter eaters (Michicich et al., 1999). The potential and drawbacks of various approaches, such as vacuum steam distillation, short path molecular distillation, absorption, solvent and supercritical fluid extraction and enzymatic methods, are described and reviewed by Boudreau and Arul (1993). Industrial application of such methods, however, seems to be scarce, and most of the techniques for reducing cholesterol became irrelevant with growing scientific evidence that dietary cholesterol hardly influences serum cholesterol and – probably more important – when regulations in the USA restricted advertisements for low-cholesterol products to food with less than 2 g SFA per serving (Hettinga, 1996). Cholesterol may, however, become relevant again when discussing the use of animal fats as (deep) frying fats, because of the potential formation of

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327

cholesterol oxidation products (COP) (Zhang et al., 1991) and their potential role in atherogenesis (Leonarduzzi et al., 2002) and carcinogenesis (Linseisen and Wolfram, 1999).

13.8 Modified animal fats: the relevance of fortifying functional fatty acids in animal lipids It is widely accepted that decreasing the n-6/n-3 ratio in the human diet by a higher intake of n-3 PUFA is advantageous for health, and this is considered in nutritional guidelines (DACH, 2000). EPA and DHA in particular exert beneficial effects. A relevant question is, how far terrestrial animal products contribute to the supply with n-3 LC-PUFA and if strategies to enhance n-3 LC-PUFA content in animal products can substantially improve the supply. A recent survey on the contribution of various food sources to the dietary intake of n-3 LC-PUFA revealed that, under Australian conditions, where daily n-3 LC-PUFA intake of adults is around 150–220 mg, meat and meat products contribute about 20 % of EPA and 12 % DHA of the total intake (Meyer et al., 2003). Eggs provide another 6 % of n-3 LC-PUFA, mainly DHA. We estimated from food disappearance data in Switzerland a similar average daily intake of about 85 mg EPA and 110 mg DHA of which 10 and 9 %, respectively, were contributed by meat. According to these calculations, milk and milk products contribute about one third of the EPA consumption but less than 3 % of DHA. Eggs provide only a little EPA but about 6 % of DHA consumption. To attain a higher n-3 content in animal products by feeding ALA-rich feed-stuffs to farm animals could be one way to achieve a more preferable n6/n-3 ratio in the human diet, even without changing nutritional habits. This approach would be particularly promising with monogastric animals, because the n-6/n-3 ratio in their meat is generally on a high level, due to the high amount of concentrates fed and the high proportion of linoleic acid (18:2n6) in grains (Raes et al., 2004b). The depletion of the natural marine resources and the potentially high mercury contamination of sea fish, which may counteract the beneficial effects of its n-3 fatty acids (Guallar et al., 2002), make alternative n-3 LC-PUFA sources even more attractive. It may also be argued that fish oil should be directly applied to humans (e.g. as capsules) instead of being fed to animals, where it can also exert undesired effects such as increased oxidative damage and/or undesired flavour effects. Making use of the animals’ ability to convert ALA to n-3 LC-PUFA will surely improve the n-6/n-3 ratio and the amount of EPA and DPA in meat but, as outlined above, the amount of DHA will hardly be affected. Nevertheless, it was shown that a feeding strategy, using extruded linseed as feed supplement, to produce n-3 fortified animal products, effectively increased the n-3 fatty acids in blood plasma lipids of the consumers and brought the overall n-6/n-3

328 Modifying lipids for use in food ratio close to the recommended value (Weill et al., 2002). In France, there is already a label programme existing, which has successfully implemented this strategy (http://www.bleu-blanc-coeur.com/prodcat.htm). In ruminants, increased ALA supply is likely to also increase CLA in the products, which may be rated positive, because only the naturally occurring isomers will be enhanced. CLA could also easily be increased in meat of monogastric animals by supplementing synthetic isomers to the feed. However, because of the lack of confidence in the safety of certain CLA isomers, fortification of animal products by supplementing synthetic CLA mixtures cannot be recommended (AFSSA, 2005). A future field of application could nevertheless be supplementation to finishing pigs’ diets in order to improve the firmness of backfat and lard. Compensating in that way for the softening effect of PUFA could allow for a higher amount of beneficial n-3 PUFA in the diet and, consequently, in the meat products. CLA, therefore, provides the potential to moderate the conflict between technological quality and dietary value of pork to a certain extent.

13.9 Future trends The current change of view concerning the role of fat and carbohydrates in nutrition may exert some impact on livestock production strategies. Labelling programmes providing primary products (milk, meat, eggs) with an improved fat composition in terms of dietetic value already exist and are likely to expand. The incentive to further develop feeding strategies in order to improve the nutritional value of milk and animal fats by modifying the fatty acid composition to meet nutritional recommendations might become stronger in the light of current trends to low-carb diets of the Atkins type. Improved milk or animal fat might also be used for manufacturing of processed products like cheese and sausages and perhaps regain fields of application lost to hydrogenated plant oils. The discussion about trans fatty acids will go on. There is some evidence that the naturally (in ruminant products) occurring trans MUFA might be less harmful (i) because of the potential to desaturate trans vaccenic acid and/or (ii) because the intake from ruminant products generally is too low to exert negative effects and/or (iii) milk and animal fat might contain other protective or beneficial compounds. Regarding the rather critical reviews about synthetic CLA, it seems unlikely that CLA fortifying strategies will gain ground. However, when used appropriately, CLA might become a valuable tool to control fat composition in pork production, opening possibilities to cope with the demand for firm and at the same time healthy lard.

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13.10

329

Sources of further information and advice

Several valuable reviews are already cited in the text and listed below; however, the excellent review about milk fat and human nutrition should be particularly highlighted (Parodi, 2004). A very useful review on nutritional control of milk fat composition with detailed but concise physiological background information has been given by Chilliard et al. (2000). Concerning CLA, an enormous body of scientific literature is available together with several reviews and books. Most of the relevant literature might be cited in the recent review of Wahle et al. (2004). The proceedings of a Symposium by the British Society of Animal Science about ‘Milk Composition’ provide not only reviews and research papers about genetic and nutritional improvement of milk composition but also a useful introductory review about consumer requirements and future trends (BSAS, 2000). More general information about fats, however, with special emphasis on fats of animal origin is provided by the Weston A. Price Foundation. Although the point of view might be somewhat biased, the critical reader will find interesting and entertaining hints and background information (http:// www.westonaprice.org/knowyourfats/).

13.11

References

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14 PUFA production from marine sources for use in food G. G. Haraldsson, University of Iceland, Iceland and B. Hjaltason, EPAX AS, Iceland

14.1

Introduction

There is an increasing demand for eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) concentrates from the health food industry as food supplements and functional food and from the pharmaceutical industry for drugs. The n-3 polyunsaturated fatty acid (PUFA) concentrates may be roughly divided into three classes. First, ethyl esters of various enrichment levels of EPA or DHA or both, that have been developed into health supplements and drugs (Haraldsson and Hjaltason, 2001). Ethyl esters may also be used as starting material for glycerol derived lipids enriched with these fatty acids. Second, n-3 PUFA concentrates in the natural triacylglycerol (TAG) form or of high TAG content that are available in various enrichment levels where their fatty acid distribution in defined positions of the glycerol backbone is not of much concern (Haraldsson, 2000; Haraldsson and Hjaltason, 2001). Finally, structured TAG containing EPA or DHA located at the mid-position with medium-chain fatty acids (MCFA) at the end-positions of the glycerol moiety are currently the most sophisticated concentration form (Haraldsson, 2005). This chapter is intended to describe the best procedures for concentrating EPA and DHA in fish oil to high levels by physical means and by enzymatic methods involving lipases. Most emphasis will be placed on enzymatic methods to prepare TAG-based concentrates and structured TAG constituting EPA and DHA. Lipases are ideally suited as biocatalysts for esterification and transesterification processes involving the highly labile n-3 PUFA because of their efficiency and the mild conditions under which they act (Haraldsson and Hjaltason, 1992; Haraldsson, 2000). Based on their fatty acid selectivity,

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lipases have been widely used to enrich n-3 PUFA in fish oil by kinetic resolution in hydrolysis, transesterification and esterification reactions (Haraldsson and Hjaltason, 2001). They may be used to concentrate EPA together with DHA or they may offer discrimination between EPA and DHA to concentrate EPA or DHA individually. Since they can tolerate water-free organic media extremely well, they may also be exploited to introduce EPA and DHA into natural TAG. Finally, lipases, owing to their regioselectivity, are perfectly suited as biocatalysts for preparing structured TAG comprising n-3 PUFA at the mid-position and MCFA at the end-positions (Haraldsson, 2005).

14.2 Concentration of n-3 PUFA by non-enzymatic methods Numerous methods are available for concentrating EPA and DHA in fish oils or for separating and purifying EPA and DHA (Ackman, 1988; Breivik et al., 1997; Medina et al., 1998; Shahidi and Wanasundara, 1998; Haraldsson and Hjaltason, 2001). These methods are summarized in Table 14.1. Usually a combination of fractionation techniques is needed to obtain EPA and DHA in highly purified form. This relates to the complexity of marine oils which often contain more than 50 different fatty acids linked as esters into triacylglycerols (Haraldsson and Hjaltason, 2001). Winterization is a simple cooling that is used to separate the saturated TAG from residual oil in certain selected types of fish oils by precipitation (Ackman, 1986; Breivik and Dahl, Table 14.1

Methods to concentrate EPA and DHA in fish oils.

Fat type

Level of concentration (%)

Triacylglycerols Winterization Organic solvent crystallization

30 35–40

Free acids or monoesters Counter-current fractionation or crystallization Short-path distillation Supercritical fluid carbon dioxide extraction Lipase kinetic resolution Urea complexation

50 50 50–60 50–75 70–80

Separation of EPA and DHA as free acids or monoesters Lipase kinetic resolution HPLC Silver-ion chromatography Corey’s iodolactonization

> > > >

90 95 95 95

Abbreviations: DHA = docosahexaenoic acid, EPA = eicosapentaenoic acid, HPLC = high-performance liquid chromatography.

338 Modifying lipids for use in food 1992). This results in a slight enrichment in PUFA, up to 30 % levels. Crystallization of TAG from organic solvents at lower temperatures to freeze out more saturated TAG from fish oils has also been used to concentrate fish oils up to 35–40 % levels (Ackman, 1986; Breivik and Dahl, 1992). It is difficult to obtain higher levels on the natural TAG form and, in order to accomplish further concentration, the fatty acids need to be released from the TAG as free acids or monoesters. After the fatty acids are set free, various methods are available (Medina et al., 1998; Shahidi and Wanasundara, 1998; Haraldsson and Hjaltason, 2001). Short path distillation may easily be used to obtain enrichment levels up to approximately 50 % (Ackman, 1986; Breivik and Dahl, 1992; Shahidi and Wanasundara, 1998). Comparable concentration levels may be obtained by simple counter-current fractionation or by crystallization of free acids or esters (Brown and Kolb, 1955; Ackman, 1986; Breivik and Dahl, 1992; Shahidi and Wanasundara, 1998). The increased polarity of the long-chain n-3 PUFA gives them higher solubility in more polar solvents compared to saturated and monounsaturated fatty acids. Various extraction methods (Medina et al., 1998; Shahidi and Wanasundara, 1998; Haraldsson and Hjaltason, 2001) have been reported for the separation and concentration of long-chain n-3 PUFA up to high levels, including supercritical fluid carbon dioxide extraction (Krukonis, 1984; Mishra et al., 1993; Walker et al., 1999) and silver nitrate solution extraction (Kubota et al., 1997; Ozawa et al., 2001). Urea complexation is a simple and efficient technique to increase the enrichment of free acids or monoesters up to the 70–80 % levels with high EPA and DHA recovery (Haagsma et al., 1982; Ratnayake et al., 1988; Medina et al., 1998; Shahidi and Wanasundara, 1998; Wanasundara and Shahidi, 1999). The saturated or monounsaturated fatty acids complex easily with urea crystals that can accommodate aliphatic long straight-chain fatty acids and crystallize at appropriate reduced temperatures. The presence of several double bonds in long-chain PUFA alters their shape, rendering these molecules more bulky and causing these PUFA to resist complexing with urea. The drawback of this method, however, is the large amount of solvents, chemicals and by-products involved. This method has been scaled up to a multi-tonne production scale in combination with short path distillation by Norsk Hydro/Pronova Biocare in Norway to produce a concentrate of 85 % EPA plus DHA (Breivik and Dahl, 1992; Breivik et al., 1997). A combination of urea complexation and a subsequent supercritical fluid carbon dioxide extraction resulted in concentrates of EPA and DHA in purities exceeding 90 % (Nilsson et al., 1988). Further enrichment almost to 100 % purity level (≥ 95 %) is performed by chromatographic methods. As a result of their higher polarity, long-chain n3 PUFA can be easily separated by high-performance liquid chromatography (HPLC). Reversed-phase HPLC has been particularly useful to isolate longchain n-3 PUFA, and eventually highly pure n-3 PUFA were produced on a relatively large scale by that method (Tokiwa et al., 1981; Beebe et al.,

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1988). Silver ion resin as an absorbent was observed to be more effective than normal chromatography and the purity of the long-chain n-3 PUFA could be improved (Teshima et al., 1978; Adlof and Emken, 1985; GuilGuerrero and Belarbi, 2001). The iodolactonization method, first introduced by Corey and coworkers (Wright et al., 1987), was modified by Russian scientists to allow a further HPLC purification of the DHA iodolactone to accomplish highly pure DHA (Gaiday et al., 1991). Although chromatographic methods can be used to produce highly pure EPA and DHA, large amounts of organic solvents are needed and the production capacity is relatively low. Also, tedious methods involving several repeated HPLC steps are often required.

14.3 Concentration of n-3 PUFA by lipase Owing to their fatty acid selectivity and discrimination against n-3 PUFA, lipases can be used as an alternative means to concentrate EPA and DHA in fish oils (Medina et al., 1998; Haraldsson and Hjaltason, 2001). Compared with more traditional physical and chemical methods, the lipase-catalyzed methods offer numerous advantages. First, the catalytic efficiency of lipases is high, so a relatively low amount of lipase is needed for production on a large scale, and multiple re-use is possible with immobilized lipase. Second, the selectivity of lipase against PUFA is crucial for certain applications, and they may also discriminate between EPA and DHA. Third, the mild conditions of lipase-catalyzed reactions in terms of temperature, pH and pressure are very important when the highly labile long-chain n-3 PUFA are involved. Fourth, lipases retain their high activity under virtually water-free conditions remarkably well, shifting the thermodynamic equilibrium to favor esterification over hydrolysis. This makes them ideally suited to catalyze various esterification and transesterification reactions and for synthesis of highly pure TAG. Finally, as the lipase-catalyzed esterification can be conducted under solvent-free conditions, the bulkiness of the process and investment cost will be considerably reduced, and operators can work in a safer environment. As indicated in Table 14.1 lipases may be used to concentrate EPA and/or DHA up to the 50–70 % levels and also to separate and purify these acids to a large extent. Today there are approximately 70 preparations of lipases commercially available from animal, plant and microbial sources, with the last being most abundant (Bornscheuer and Kazlauskas, 1999). Numerous reports on lipase screening for marine oil fatty acid selectivity have revealed that there are significant variations among lipases depending on their origin and source (Hoshino et al., 1990; Tanaka et al., 1992; Zuyi and Ward, 1993; Shimada et al., 1994, 1997a; McNeill et al., 1996; Haraldsson et al., 1997; Shimada et al., 1997b; Wanasundara and Shahidi, 1998a; Halldorsson et al., 2004). Generally there is a clear-cut preference for the saturated and less unsaturated fatty acids. Many lipases display very low activity towards fish

340 Modifying lipids for use in food oils, and only a few are suitable for biotransformations involving marine oils and n-3 PUFA, all of microbial type. The lipases that have been successfully applied for enrichment of the n-3 PUFA are listed in Table 14.2. They include the Aspergillus niger, Candida rugosa (formerly named Candida cylindracea), Geotrichum candidum, Pseudomonas cepacia (supplied by Amano in Japan as Lipase PS, formerly classified as Pseudomonas fluorescens), Pseudomonas fluorescens (Amano’s Lipase AK, formerly termed Pseudomonas sp.), Rhizomucor miehei (supplied by Novozyme A/S in Denmark immobilized as Lipozyme RM IM, formerly named Mucor miehei), Rhizopus delemar and Rhizopus oryzae lipases. The lipases displaying sufficient activity may be divided into two categories (Haraldsson and Hjaltason, 2001). The first category comprises lipases discriminating against n-3 PUFA including both EPA and DHA offering potential to concentrate EPA and DHA together. The lipases belonging to this category include those from Geotrichum candidum, Pseudomonas fluorescens and Pseudomonas cepacia. The Aspergillus niger lipase also appears to belong to this class, although it may act too slowly to be of practical value (Hoshino et al., 1990; Halldorsson et al., 2004). Lipases offering a strong discrimination between EPA and DHA in favor of EPA belong to the second class. They include the Candida rugosa, Rhizomucor miehei and Rhizopus delemar lipases. The bulk of the prefered fatty acids including EPA devoid of DHA may then subsequently be used as a source for further EPA concentration (Haraldsson and Kristinsson, 1998). The immobilized Candida antarctica lipase (Novozym 435 from Novozyme A/S in Denmark) warrants a special comment. This lipase displays relatively high activity for both EPA and DHA and is of no value to concentrate EPA or DHA. However, it is ideally suited for various biotransformations involving n-3 PUFA of Table 14.2

Lipases successfully used for enriching n-3 PUFA in fish oil.

Lipase

Type concentrate

References

Aspergillus niger Candida rugosa Geotrichum candidum Pseudomonas cepacia Pseudomonas fluorescens Rhizomucor miehei

EPA + DHA EPA + EPA + EPA + DHA EPA DHA DHA

[1, 2] [1–8] [3, 7, 9] [1, 10–12] [1, 10, 11] [13–18] [13, 15] [4, 19, 20] [1, 6]

Rhizopus delemar Rhizopus oryzae

DHA DHA DHA DHA

References for [1]: Halldorsson et al., 2004; [2]: Hoshino et al., 1990; [3]: Shimada et al., 1994; [4]: Shimada et al., 1997a; [5]: Tanaka et al., 1992; [6]: Wanasundara and Shahidi, 1998a; [7]: McNeill et al., 1996; [8]: Wanasundara and Shahidi, 1998b; [9]: Shimada et al., 1995; [10]: Zuyi and Ward, 1993; [11]: Haraldsson et al., 1997; [12]: Maehr et al., 1994; [13]: Haraldsson and Kristinsson, 1998; [14]: Shimada et al., 1998; [15]: Takagi, 1989; [16]: Langholz et al., 1989; [17]: Hills et al., 1990; [18]: Halldorsson et al., 2003b; [19]: Shimada et al., 1997b; [20]: Shimada et al., 1997c. Abbreviations: DHA = docosahexaenoic acid, EPA = eicosapentaenoic acid.

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great importance. It has been used in esterifying glycerol with n-3 PUFA concentrates including pure EPA and DHA (Haraldsson et al., 1995). The Candida antarctica lipase has also been observed to display excellent regioselectivity toward the outer positions of the glycerol moiety in transesterification reactions of triacylglycerols (Irimescu et al., 2001a, 2002) and glycerol (Halldorsson et al., 2003a) and is a key lipase in the synthesis of positionally labeled symmetrically structured TAG of the MLM type. It is of great interest that crude hydrolytic enzyme mixtures from fish intestine display reversed fatty acid selectivity. Lie and Lambertsen demonstrated that the hydrolytic enzyme mixture from cod displayed preference for n-3 PUFA over saturated and monounsaturated fatty acids in fish oil hydrolysis (Lie and Lambertsen, 1985). Gellesvik isolated a bile-salt dependent lipase from cod intestines that likewise displayed preference for the longchain n-3 PUFA (Gellesvik, 1991). More recently, Halldorsson et al. reported similar reversed fatty acid selectivity for hydrolytic enzyme mixture from both salmon and rainbow trout intestines in fish oil TAG (Halldorsson et al., 2004) and astaxanthin diester (Halldorsson and Haraldsson, 2004) hydrolysis.

14.3.1 Lipase hydrolysis Lipase hydrolysis of a TAG oil results in the formation of selected free acids and a mixture of monoacylglycerols (MAG), diacylglycerols (DAG), TAG and glycerol depending on the degree of conversion. The higher selectivity of lipase towards the saturated and monounsaturated fatty acids results in their removal from the glycerol backbone of the oils. The less preferred long-chain n-3 PUFA remain in residual acylglycerol molecules. They may be released subsequently by traditional chemical or enzymatic hydrolysis or alcoholysis reactions into the corresponding free fatty acid (FFA) or monoester concentrates for further concentration (Breivik et al., 1997). Usually such lipase-promoted hydrolysis is performed on an oil-to-water (or buffered water) ratio between 1:1 and 1:1.5 as based on weight. The lipase-catalyzed fish oil hydrolysis reaction is illustrated in Fig. 14.1. Yamane and coworkers examined several lipases for selective hydrolysis of cod liver oil and sardine oil to concentrate EPA and DHA (Hoshino et al., 1990). The reactions were conducted O Fish oil

O

TAG

Lipase H2O O

O MAG/DAG/TAG

SMFA

OH

PUFA

O

Fig. 14.1 A simplified fish oil hydrolysis by lipase to afford saturated and monounsaturated free fatty acids (SMFA) and an acylglycerol mixture constituting triacylglycerols (TAG), diacylglycerols (DAG) and monoacylglycerols (MAG) enriched with n-3 PUFA, EPA and DHA or only DHA, depending on the lipase fatty acid selectivity.

342 Modifying lipids for use in food at 20 °C. With Candida rugosa and Aspergillus niger lipases, acylglycerols containing more than a two-fold increase in the n-3 PUFA content compared to the original fish oils were produced. Both lipases afforded concentration levels up to 50 %. Tanaka et al. described the use of the Candida rugosa lipase to concentrate DHA in fish oil constituting 13 % EPA and 9 % DHA by partial hydrolysis at 37 °C (Tanaka et al., 1992). With the Candida rugosa lipase at 70 % conversion, the DHA content in the acylglycerol product mixture was three times higher than in the original fish oil, whereas the EPA content was reduced to 70 % of that in the original oil. When DHA-rich tuna oil comprising 6 % EPA and 25 % DHA was hydrolyzed with that lipase, acylglycerols of 53 % DHA content were obtained (see Fig. 14.2) with the EPA content (4 %) remaining close to that in the original oil. The Candida rugosa lipase was observed to be ineffective with TAG comprising DHA (Tanaka et al., 1993). Maehr et al. used Pseudomonas cepacia lipase on fish oils comprising 30 % EPA + DHA to produce acylglycerols close to the 50 % EPA + DHA concentration level in 23–50 % weight recovery yields (Maehr et al., 1994). Concentration levels of 70 % were obtained at a higher conversion, but in much lower recovery of only 14–21 %. McNeill and coworkers reported on fish oil hydrolysis employing Candida rugosa and Geotrichum candidum lipases (McNeill et al., 1996; Moore and McNeill, 1996). At 60 % conversion the n-3 content had increased from 30 % in the initial oil to 45 % in the residual glyceride mixture that was enriched with both EPA and DHA. At 80 % conversion a DHA-enriched concentrate with an EPA-to-DHA ratio of 1:5 was afforded (7 % EPA and 40 % DHA) by the Candida rugosa lipase. Shimada and coworkers used the Geotrichum candidum lipase to concentrate EPA together with DHA in tuna oil containing 8 % EPA and 30 % DHA by selective hydrolysis (Shimada et al., 1994). The reaction was conducted at 30 °C and after 16 hours 34 % conversion was obtained resulting in production of glycerol esters of 10 % EPA and 39 % DHA content. A second hydrolysis resulted in acylglycerols comprising 11 % EPA and 47 % DHA in 82 % recovery of these fatty acids in 55 % yield. PUFA-rich TAG, especially those containing DHA, accumulate in the acylglycerol product indicating that TAGcontaining DHA are resistant to Geotrichum candidum lipase (Shimada et al., 1995). This reluctance of many lipases to act on DHA-enriched TAG is a major impediment to the synthesis of positionally labeled symmetrically structured TAG of the MLM type (Irimescu et al., 2001a).

Tuna oil TAG

CRL H2O

O

O MAG/DAG/TAG

SMFA/EPA

OH

DHA

O (53 % (DHA)

Fig. 14.2 Tuna oil hydrolysis by Candida rugosa lipase (CRL) by Tanaka et al. (1992) (for abbreviations see Fig. 14.1).

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343

Wanasundara and Shahidi investigated a number of lipases in their hydrolysis of seal blubber and menhaden oils to generate n-3 PUFA concentrates (Wanasundara and Shahidi, 1998a). The reactions were conducted at 35 °C using buffered water. The highest concentration levels of n-3 PUFA were obtained with the Candida rugosa lipase, 44 % for both oils. Wanasundara and Shahidi have optimized their n-3 fatty acid concentration using the Candida rugosa lipase (Wanasundara and Shahidi, 1998b). Enrichment levels of close to 54 % were obtained for both oil types.

14.3.2 Lipase alcoholysis Alcoholysis of fish oil TAG by a short-chain monohydric alcohol may be regarded as a simple modification of the hydrolysis process. In ethanolysis, where ethanol is used instead of water, the product is ethyl esters of the bulk of the more saturated fatty acids in the original fish oil instead of free fatty acids. As with hydrolysis, the residual acylglycerol mixture becomes enriched with EPA and DHA (illustrated in Fig. 14.3) or only DHA, depending on the nature of the lipase fatty acid selectivity. There are two reports on fish oil enrichment of EPA together with DHA by the TAG ethanolysis approach (Zuyi and Ward, 1993; Haraldsson et al., 1997). Zuyi and Ward investigated numerous lipases in alcoholysis of cod liver oil TAG using various primary and secondary short-chain alcohols (Zuyi and Ward, 1993). Their aim was to concentrate both EPA and DHA, and the best results were obtained with the Pseudomonas fluorescens lipase with isopropanol and ethanol. The reactions were conducted in the alcohol as a solvent at 30 °C in the presence of 5 % water by weight. The high water content resulted in high levels of free fatty acids, but the residual acylglycerol mixture comprised EPA + DHA levels close to 50 %. Pseudomonas lipases belong to the category of lipases that discriminate between the bulk of saturated and monounsaturated fatty acids and n-3 PUFA in fish oil as was confirmed by Haraldsson and coworkers (Haraldsson et al., 1997). They observed that two commercially available Pseudomonas cepacia and Pseudomonas fluorescens lipases afford concentrates of approximately 50 % EPA plus DHA in high recoveries, 80 and 90 % for DHA and EPA, respectively, and are highly efficient. The reactions were conducted on sardine oil containing 15 % EPA and 9 % DHA at room temperature, without a solvent, and with only a two-fold stoichiometric amount of ethanol. The O Fish oil TAG

PFL Ethanol

O MAG/DAG/TAG

SMFA

O

EPA/DHA

O

(50 % EPA + DHA)

Fig. 14.3 Fish oil ethanolysis by Pseudomonas fluorescens lipase (PFL) by Haraldsson et al. (1997) to produce saturated and monounsaturated ethyl esters and residual acylglycerol mixture enriched with EPA and DHA.

344 Modifying lipids for use in food reaction is demonstrated in Fig. 14.3. This resulted in a considerable reduction in bulk of the process, and the ethyl esters produced were distilled off by short path distillation from the residual acylglycerol mixture of EPA and DHA. The short path distillation was beneficial in that monoacylglycerols containing shorter-chain fatty acids were distilled off, whereas ethyl esters of EPA and DHA remained in the residue. This resulted in further increases in the EPA and DHA levels in the residual acylglycerol mixture after the distillation (Breivik et al., 1997). Ten per cent dosage of lipase as powder based on the weight of fish oil was used, but the activity of the lipase had already dropped significantly after the first run. Immobilization solved the productivity problem and far less lipase was needed, and it could also be reused more than ten times without any deterioration of the activity and results. These factors render this method highly feasible from an industrialization point of view (Breivik and Haraldsson, 1994; Breivik et al., 1997). Furthermore, the immobilized Candida antarctica lipase converted the acylglycerol mixture remaining after short path distillation into the corresponding ethyl ester concentrate by ethanolysis for further concentration. Only a two-fold stoichiometric amount of ethanol at room temperature was needed. Shimada and coworkers observed lipase to be highly sensitive to the amount of ethanol and the type of acylglycerols to be ethanolyzed (Watanabe et al., 1999). There are also reports on DHA enrichment in fish oil by lipase-catalyzed alcoholysis reactions. Haraldsson and Kristinsson obtained good separation of DHA by ethanolysis of tuna oil TAG comprising 6 % EPA and 23 % DHA under the ethanolysis conditions described above with immobilized Rhizomucor miehei lipase (Haraldsson and Kristinsson, 1998). Seventy per cent conversion into ethyl esters was obtained after 48 hours with the residual acylglycerol mixture containing 54 % DHA (and 6 % EPA) in 78 % recovery. The separation and the performance of the lipase, however, were dramatically improved after a modification based on esterification of free acids with ethanol as will be described later in this chapter (Table 14.3). Shimada and coworkers reported on the alcoholysis of fish oil monoesters with medium-chain fatty alcohols (Shimada et al., 1997c). They used Rhizopus delemar lipase immobilized on a ceramic carrier to effect a selective alcoholysis of tuna oil ethyl esters with lauryl alcohol to enrich DHA. The alcoholysis was conducted at 30 °C using a 1:3 molar ratio of tuna oil ethyl esters to Table 14.3 Comparison between ethanolysis of tuna oil TAG and esterification of tuna oil FFA using the immobilized Rhizomucor miehei lipase. Substrate

Reaction

Conversion (%)

Time (h)

DHA conc. (%)

DHA recov. (%)

Tuna TAG Tuna FFA

Ethanolysis Esterification

70 70

48 11

54 77

78 78

Abbreviations: DHA = docosahexaenoic acid, FFA = free fatty acid, TAG = triacylglycerol.

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345

lauryl alcohol. After 50 hours the DHA content in the residual ethyl esters increased from 23 to 52 mol% with 90 % DHA recovery. When ethyl esters comprising 60 % DHA were subjected to the alcoholysis reaction DHA was enriched to 83 %, with DHA recovery above 90 %. The productivity of the immobilized lipase was very high as indicated by studies showing that after nearly 50 runs, replacing the reaction mixture with fresh substrates every 24 hours, there was only a 15 % decrease in extent of alcoholysis. The problem with the Rhizopus delemar lipase was that the initial EPA composition was also elevated. When replacing that lipase with immobilized Rhizomucor miehei lipase after the first enrichment step, higher enrichment levels of DHA were obtained as well as a considerable decrease in the EPA content of the residual ethyl esters under similar conditions (Shimada et al., 1998). In a productivity study, a drop of only 17 % was observed after 100 24 hour reaction cycles. In an experiment involving ethyl esters constituting 60 % DHA, the DHA content of the residual ethyl esters was raised to 93 % with 74 % DHA recovery with EPA falling from about 9 to 3 %. That methodology offered higher concentration levels of DHA with the Rhizomucor miehei lipase, but lower yields of DHA in the residual ethyl ester fraction. These examples demonstrate the potential of lipase to separate and enrich DHA to high purity levels by a multi-step enzymatic treatment involving one or more lipase types.

14.3.3 Lipase esterification There are several reports on selective esterification of fish oil FFA with simple monohydric alcohols using the Rhizomucor miehei lipase to strongly discriminate between EPA and DHA, usually in hexane as a solvent. In one of the first reports, Takagi used methanol and a free fatty acid concentrate of EPA and DHA obtained by urea precipitation of Japanese sardine oil, comprising 30–40 % EPA and 25–30 % DHA (Takagi, 1989). Methyl ester product enriched with EPA (> 50 %) and a residual free fatty acid concentrate enriched with DHA (about 50 %) were obtained in yields of 60 and 40 %, respectively. Schmidtsdorff and coworkers investigated the fatty acid selectivity of the immobilized Rhizomucor miehei lipase toward fish oil fatty acids, also with methanol, to obtain residual free fatty acids comprising 48 % DHA (Langholz et al., 1989). Similar results were reported by Hills et al. on cod liver oil FFA with n-butanol using the same lipase (Hills et al., 1990). In attempts to separate EPA and DHA in fish oils by the immobilized Rhizomucor miehei lipase, Haraldsson and Kristinsson demonstrated that direct esterification of free fatty acids released from fish oil was much more efficient than ethanolysis of fish oil TAG (Haraldsson and Kristinsson, 1998). When tuna oil containing 6 % EPA and 23 % DHA was transesterified with ethanol under the same conditions as described above for the Pseudomonas lipases, 70 % conversion into ethyl esters was observed after 48 hours. The residual acylglycerol mixture comprised 54 % DHA and 6 % EPA by mol,

346 Modifying lipids for use in food with 78 % DHA recovery into the acylglycerol mixture and 75 % EPA recovery into the ethyl ester product. When the corresponding tuna oil free fatty acids were esterified with a two-fold stoichiometric amount of ethanol 70 % conversion was obtained after only 11 hours. The residual free fatty acids comprised 77 % DHA and only 2 % EPA by mol. The recovery of both DHA in the residual free fatty acid fraction and EPA in the ethyl ester product remained very high, 78 and 89 %, respectively. Table 14.3 compares the ethanolysis results of tuna oil TAG and esterification of tuna oil FFA with ethanol using the Rhizomucor miehei lipase. The reaction is illustrated in Fig. 14.4. A modification using free fatty acids with glycerol instead of ethanol under similar esterification conditions gave good results with the same lipase as reported by Haraldsson and co-workers (Halldorsson et al., 2003b). Haraldsson and Kristinsson also demonstrated by their ethanol-based procedure that a free fatty acid concentrate of 77 % EPA and 10 % DHA could be freed from DHA in a single step and purified to higher than 90 % EPA content at 50 % conversion with 60 % recovery of EPA (Haraldsson and Kristinsson, 1998). Shimada and coworkers used a highly efficient two-step esterification of free fatty acids from tuna oil to purify DHA up to nearly 90 % purity levels (89 % by weight) with high DHA recovery (71 %) based on the initial oil using the Rhizopus delemar lipase with lauryl alcohol as the acyl acceptor (Shimada et al., 1997a). The reaction was conducted at 30 °C using a 2:1 molar ratio of lauryl alcohol to tuna oil FFA without a solvent in the presence of 20 % water based on weight of substrates. In the first step, the DHA content rose from 23 to 73 % in very high DHA recovery (84 %) at 72 % conversion obtained after 20 hours. It is noteworthy that despite the high water content the lauryl esters were not susceptible for hydrolysis. This relates to lauryl esters being very poor substrates or non-substrates to lipase. The water content was observed to have profound effects on the DHA enrichment levels with the highest value obtained at 20 % water content. It is of interest that with this lipase the shorter-chain alcohols including ethanol and glycerol gave poor results with low or no fatty acid selectivity observed. After the second enzymatic treatment the EPA content remained very low (< 2 %). The reaction is illustrated in Fig. 14.5. Shimada and coworkers have also reported on a two-step enzymatic method to purify DHA from tuna oil consisting of a lipase-promoted tuna oil hydrolysis and a subsequent selective esterification of the resulting FFA (Shimada O Tuna

OH

RML Ethanol

O Tuna/EPA

O O

DHA

OH

(77 % DHA)

Fig. 14.4 Esterification of tuna oil FFA with ethanol by immobilized Rhizomucor miehei lipase (RML) by Haraldsson and Kristinsson (1998).

PUFA production from marine sources for use in food O Tuna

OH

RDL Lauryl alcohol

RDL Lauryl alcohol

O Tuna/EPA

347

O O

DHA OH (73 % DHA)

O DHA

OH

(89 % DHA)

Fig. 14.5

A two-step enzymatic esterification of tuna oil FFA with lauryl alcohol by Rhizopus delemar lipase (RDL) by Shimada et al. (1997a).

et al., 1997b). Pseudomonas fluorescens lipase was exploited to hydrolyze tuna oil at 48 °C using a 1:1 ratio of tuna oil to water by weight. At 79 % conversion 83 % of the DHA was recovered in the FFA fraction. The free fatty acid product obtained from the enzymatic hydrolysis was subjected to a selective esterification with lauryl alcohol catalyzed by the Rhizopus delemar lipase under the above-described conditions. The DHA content in the residual FFA fraction rose from 24 to 72 % by weight in 83 % recovery (69 % overall). After a second selective esterification treatment, the DHA content was elevated to higher than 91 % in 80 % recovery (60 % overall). The examples from the groups of Haraldsson and Shimada demonstrate that enrichment levels well beyond those obtained by urea crystallization can be obtained highly efficiently by lipases. The fact that an immobilized lipase can be re-used tens of times without much deterioration in performance suggests that the application of lipase in the field of marine oils is a highly feasible choice from an industrial point of view. Based on these results, there are reasons to believe that in terms of purifying EPA and DHA, lipase can be used as a powerful alternative to traditional separation techniques up to the chromatography levels by stepwise lipase reactions. Figure 14.6 illustrates how lipases may be introduced complementary to traditional separation techniques to separate and purify EPA and DHA to various purity levels (Haraldsson and Hjaltason, 2001).

14.4 TAG concentrates of n-3 PUFA Preparation of TAG up to the 30–35 % EPA + DHA concentration level can be brought about directly on fish oils without splitting the fat by a careful selection of fish oils and simple methods such as winterization, molecular distillation and solvent crystallization (Ackman, 1988; Breivik and Dahl, 1992). A good example of such a concentrate is MaxEPA™ containing 18 % EPA and 12 % DHA. This was the first dietary n-3 supplement in the natural TAG form to be introduced to the market in the early 1980s by Seven Seas in the UK (A History of British Cod Liver Oils, 1994). It was widely used for various clinical studies for over 15 years. Another example is a cod liver oil

348 Modifying lipids for use in food Fish oil triacylglycerols (20–30 % EPA + DHA) Ethanolysis or hydrolysis Ethyl esters or free fatty acids (20–30 % EPA + DHA)

Lipase

Short-path distillation

Lipase

Ethyl esters or free fatty acids (50 % EPA + DHA)

Urea inclusion Ethyl esters or free fatty acids (80–85 % EPA + DHA) HPLC

> 95 % EPA

> 95 % DHA

Fig. 14.6 A flow diagram illustrating how lipase may be used complementary to traditional separation techniques to separate and purify EPA and DHA in fish oil to various purity levels.

concentrate comprising 16–17 % each of EPA and DHA introduced to the market in Iceland in the late 1980s by Lysi Ltd. It was produced by solvent crystallization of refined cod liver oil using acetone-water as a solvent (Haraldsson and Gudbjarnason 1986, unpublished results) and remained on that local market for a few years. TAG concentration beyond that level requires splitting of the TAG into free acids or monoesters, concentration of EPA and DHA by the various physical methods and combination of methods described in Section 14.2 above and reintroduction of such free acid or monoester concentrates into TAG concentrates (Haraldsson, 2000; Haraldsson and Hjaltason, 2001). Although the resynthesis of pure TAG highly enriched with EPA and DHA is not easily carried out by traditional chemical esterification methods, there have been several producers of such EPA- and DHA-enriched TAG by such methods. Usually the esterification reactions involved are incomplete and result in products comprising only 50–70 % TAG, largely contaminated with MAG and DAG and some residual monoesters, usually ethyl esters. There is not much public information available about the details of the

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349

methods used, but there are strong indications of triacetin being used as a source of the glycerol backbone with ethyl ester concentrates as the source of the n-3 PUFA by transesterification. Sodium ethoxide or sodium methoxide are used as catalysts to bring about the transesterification reaction. The volatile co-produced ethyl acetate is then simply distilled off during or after the reaction (see Fig. 14.7). Another approach is based on glycerolysis of ethyl ester n-3 PUFA concentrates by reacting glycerol with the monoesters in the presence of sodium alkoxides as a catalyst. In this case the volatile coproduct is ethanol (see Fig. 14.8). In the late 1980s lipases were introduced to the n-3 field to solve these problems of producing TAG highly enriched with EPA and DHA. Haraldsson and coworkers were the first to report the use of lipase for the preparation of such TAG (Haraldsson et al., 1989; Haraldsson and Almarsson, 1991). They used immobilized Rhizomucor miehei lipase as a biocatalyst to bring about transesterification reactions of cod liver oil with EPA and DHA concentrates. Both acidolysis and interesterification reactions were conducted without a solvent, using 10 % dosage of lipase based on weight of fat at 60–65 °C and a three-fold excess of free acids or monoesters based on the number of mol equivalents of esters present in the fish oil TAG. TAG of high purity comprising 60–65 % EPA + DHA and well over 70 % total n-3 PUFA content were produced. This is illustrated in Fig. 14.9. Yamane and coworkers have also reported on a similar solvent-free methodology to enrich cod liver oil TAG up to similar levels by lipasecatalyzed acidolysis using the Rhizomucor miehei lipase and a two-stage acidolysis approach (Yamane et al., 1992, 1993). Adachi et al. reported a similar acidolysis of sardine oil by Pseudomonas sp. lipase in organic solvents (Adachi et al., 1993). The yield of TAG and enrichment levels of EPA and O PUFA

OAc O

OAc PUFA

O

O

PUFA O

O–Na+

O

O

OAc

Fig. 14.7

AcO

O PUFA

Transesterification of triacetin with PUFA ethyl esters using sodium ethoxide as a catalyst to produce TAG comprising PUFA. O

OH

PUFA O

OH PUFA OH

Fig. 14.8

O Na

O

O

PUFA O O

O O

HO PUFA

Glycerolysis of PUFA ethyl esters using sodium ethoxide as a catalyst to produce TAG comprising PUFA.

350 Modifying lipids for use in food O CLO

O

O

CLO

O

O O O

O

PUFA

RML

PUFA*

O

PUFA* O

OR

CLO

O R

O

O O

PUFA*

OR

PUFA*

– H (acidolysis) – Et (interesterification)

Fig. 14.9 Enrichment of cod liver oil (CLO) with PUFA by acidolysis (R=H) or interesterification (R=Et) reactions. CLO refers to cod liver oil fatty acid composition, but PUFA* to equilibrium composition.

DHA were strongly dependent on the water content. There are numerous reports describing treatment of various types of TAG oils with n-3 PUFA concentrates of both fish and single-cell origin in lipase-catalyzed transesterification reactions. They include incorporation of n-3 fatty acids into vegetable oils (Li and Ward, 1993a; Huang and Akoh, 1994), melon seed oil (Huang et al., 1994), trilinolein (Akoh et al., 1995), evening primrose oil (Akoh et al., 1996), borage oil (Akoh and Moussata, 1998; Ju et al., 1998; Senanayake and Shahidi, 1999), palm stearin (Osorio et al., 2001) and various TAG of medium-chain fatty acids (Lee and Akoh, 1996, 1998), trilaurin, tricaprin and tricaprylin. It appears that the most efficient lipases used acted preferably at the C-1 and C-3 positions to provide positionally labeled structured TAG. High incorporation levels of the n-3 fatty acids were obtained into these positions, although high (but lower) levels of n-3 fatty acids were also incorporated into the mid-position, depending on the reaction time. High levels of n-6 and n-3 fatty acids were produced with n-6-enriched oils such as borage and evening primrose oils. Most of these reactions were conducted in organic solvents and only a few without a solvent, but there are also reports on such reactions under supercritical carbon dioxide conditions (Shishikura et al., 1994). The fish oil TAG transesterification approach was obstructed by the excessive amounts of n-3 PUFA concentrates needed to obtain high enrichment levels of EPA and DHA into fish oil (Haraldsson, 2000). Haraldsson and coworkers developed a method to produce TAG with composition identical to that of the concentrate being used to avoid the above-mentioned limitations (Haraldsson et al., 1991, 1993, 1995). This procedure is based on a direct esterification of stoichiometric amount of free fatty acids with glycerol, and it enabled synthesis of TAG homogeneous with EPA or DHA, i.e. 100 % EPA or DHA. The immobilized Candida antarctica lipase was observed to offer superiority over the Rhizomucor miehei lipase in esterifying glycerol with free fatty acids of varying n-3 PUFA content (Haraldsson et al., 1991). That lipase was highly efficient in generating TAG of both 100 % EPA and DHA content using only stoichiometric amount of pure EPA and DHA (Haraldsson et al., 1995). No solvent was required and the reaction was performed at 65 °C under vacuum with 10 % dosage of the immobilized

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351

lipase based on substrate weight. The reaction is displayed in Fig. 14.10 for EPA. TAG homogeneous with both EPA and DHA of excellent purity were accomplished in virtually quantitative yields. The same methodology was used by Kosugi and Azuma to prepare nearly pure TAG (96 %) of EPA, DHA and arachidonic acid under similar conditions using the Candida antarctica lipase (Kosugi and Azuma, 1994). There are also reports on a similar direct esterification of glycerol with n-3 PUFA concentrates where the reaction was conducted in an organic solvent (Li and Ward, 1993b; Cerdan et al., 1998; He and Shahidi, 1998). TAG concentrate preparation by direct esterification of n-3 PUFA-enriched partial acylglycerols obtained from Candida rugosa lipase promoted hydrolysis of fish oil with n-3 PUFA as free acids has also been reported. Such an acylglycerol mixture obtained at 70 % hydrolysis level of tuna oil containing 4 % EPA and 53 % DHA was treated by Tanaka et al. with n-3 PUFA containing 23 % EPA and 57 % DHA to obtain TAG of higher than 90 % purity (Tanaka et al., 1994). The reaction was conducted at 50 °C with a three-fold molar excess of n-3 PUFA using 40 wt % molecular sieves as a dehydrating agent and an immobilized Chromobacterium viscosum lipase. Similar results were obtained by McNeill and coworkers in their treatment of an acylglycerol mixture from fish oil hydrolysis with stoichiometric amounts of DHA-enriched fatty acids (McNeill et al., 1996; Moore and McNeill, 1996). TAG of 95 % purity were obtained with both the immobilized Rhizomucor miehei and Candida antarctica lipases with continuous removal of water using vacuum at 55 °C. There is no doubt that the Candida antarctica lipase offers superiority over other lipases in terms of TAG synthesis involving n-3 PUFA. That lipase is highly efficient, tolerating the n-3 PUFA very well, and highly pure TAG were produced under the proper conditions with little or no contamination by MAG or DAG. No solvent is needed and only stoichiometric amounts of substrates. That lipase is suitable for the production of pure TAG of whatever desired composition identical to that of the starting free acids as was demonstrated by Haraldsson and coworkers (Haraldsson et al., 1991). This lipase is, therefore, highly feasible for industrialization (Haraldsson and Hjaltason, 2001), and there is little doubt that this is the future lipase for production of highly pure TAG comprising high levels of n-3 PUFA.

O OH

O

OH

EPA CAL

3 EPA OH

O O

EPA O

3 H2O

O

OH O

EPA

Fig. 14.10 Direct esterification of glycerol with pure EPA to prepare structured TAG homogeneous with EPA by immobilized Candida antarctica lipase (CAL).

352 Modifying lipids for use in food

14.5 Positionally labeled structured TAG derived from fish oil Structured TAG comprising certain types of fatty acids at the outer-positions and different fatty acids at the mid-position of the glycerol backbone have gained increasing attention as dietary and health supplements. Of particular interest from human nutritional point of view are structured TAG possessing biologically active long-chain PUFA located at the mid-position with MCFA at the end-positions (Miura et al., 1999). The MCFA located at the endpositions undergo a rapid hydrolysis by pancreatic lipase, are absorbed into the intestines and are rapidly carried into the liver where they are consumed as a quick source of energy. The remaining 2-monoacylglycerols (2-MAG), on the other hand, become a source of essential fatty acids after being absorbed through the intestinal wall (Christensen et al., 1995). They are accumulated as TAG in the adipose tissues or as phospholipids in the cell membranes from where they can be released upon demand for their desired biological functions. Various approaches have been designed to undertake the preparation of positionally labeled MLM structured TAG comprising n-3 PUFA and MCFA. The simplest method is to treat fish oil, of which the mid-position usually constitutes a significantly higher n-3 PUFA composition than the end-positions (Christie, 1986; Hölmer, 1989), with a regioselective lipase. The lipase acts preferably or exclusively at the outer-positions by promoting fatty acid exchange reactions with MCFA as free acids (acidolysis) or monoesters (interesterification). Shimada and coworkers reported on the production of such structured TAG enriched with DHA at the mid-position by exchanging fatty acids at the end-positions of tuna oil for caprylic acid (CA) using an immobilized 1,3regioselective Rhizopus delemar lipase (Shimada et al., 1996, 2000). The reaction was conducted at 30 °C using approximately a six-fold molar excess of CA based on the fish oil TAG. After 40 hours the incorporation level of CA into the fish oil had reached a steady state and remained at 43 mol%. The immobilized lipase could be used 15 times (over 30 days) without a significant decrease in the CA content. Regiospecific analysis indicated that the regioselectivity of the lipase was very high and that the extent of acyl migration was very low. The fatty acid composition of the mid-position was observed to hardly change during the reaction, whereas the fatty acid composition of the end-positions changed dramatically, apart from DHA being resistant to the lipase action. The reaction is shown in Fig. 14.11. Similar acidolysis was reported by Jennings and Akoh to incorporate capric acid into TAG highly enriched with EPA (41 %) and DHA (33 %) with and without organic solvent at 55 °C (Jennings and Akoh, 1999). The highest capric acid incorporation levels of 65 % were obtained in hexane with a molar ratio of 1:8 between the TAG and capric acid, but 56 % under solventfree conditions with a 1:6 molar ratio. Analysis of the mid-position suggests

PUFA production from marine sources for use in food O O FO

O

CA

O O O

O

O

PUFA

OH

CA

O O

PUFA O O

RDL FO

353

O

CA

Fig. 14.11 Production of fish oil derived positionally labeled structured TAG containing caprylic acid (CA) and PUFA by fish oil acidolysis using immobilized Rhizopus delamar lipase (RDL).

that some acyl-migration was taking place during this reaction by the presence of capric acid at that position. Yamane and coworkers enriched single-cell oil (SCO) of high DHA (35 %) and docosapentaenoic acid (DPA; 10 %) content with CA under acidolysis conditions using Rhizomucor miehei and Pseudomonas fluorescens lipases (Iwasaki et al., 1999). Both lipases required extended reaction time of several days and high ratios of CA to SCO TAG. Much higher incorporation levels were obtained for the Pseudomonas lipase with the final CA content of the TAG reaching 65 mol% after 168 hours at 18.8 CA/SCO molar ratio at 30 °C. Xu and coworkers studied the effects of water content and reaction time on production of such positionally labeled TAG of the MLM type under pilot batch conditions using the immobilized Rhizomucor miehei lipase on fish oil and capric acid (substrate ratio 6:1 FFA/TAG in mol) under solvent-free conditions at 60 °C (Xu et al., 1998). After 30 hours over 65 % incorporation of the MCFA had taken place into the end-positions together with 12 % acylmigration levels into the mid-position during the reaction. Distillation under vacuum was used to separate the structured TAG and free acid products when further acyl-migration was noticed to take place. They also reported the use of a packed-bed reactor with the same lipase as a biocatalyst to treat menhaden oil under acidolysis with caprylic acid (Xu et al., 2000). The task to generate such fish oil derived structured TAG was addressed differently by Bornscheuer and coworkers in their two-step strategy (Schmid et al., 1998; Soumanou et al., 1998). In the first step, 2-MAG enriched with n-3 PUFA were generated from fish oil TAG by lipase-catalyzed ethanolysis using a 1,3-regioselective lipase in organic solvent. The resulting 2-MAG were subsequently esterified in a second enzymatic step. This strategy worked well for less unsaturated TAG, but when fish oils containing n-3 PUFA were used less favorable results were accomplished. This relates to the low yield of the 2-MAG intermediate as a result of low activity of the lipases toward TAG comprising EPA and especially DHA, but also to complications in isolating and purifying the n-3 PUFA-enriched 2-MAG. The first step was improved when lipases that are not usually considered to be 1,3-regioselective, Pseudomonas fluorescens and Candida antarctica lipases, were used in the ethanolysis reaction (Irimescu et al., 2001b; Wongsakul et al., 2003). A

354 Modifying lipids for use in food further n-3 PUFA enrichment of the 2-MAG from tuna oil was effected by low-temperature crystallization by freezing out the saturated 2-MAG (Wongsakul et al., 2003). Yamane and coworkers followed the methodology of Bornscheuer and coworkers in producing structured TAG enriched with DHA at the midposition and CA residues at the end-positions (Irimescu et al., 2001b). They exploited the immobilized Candida antarctica lipase in a highly efficient and regioselective ethanolysis of bonito oil TAG to yield 93 % of the 2-MAG with 44 % DHA content in only two hours at 35 °C. The subsequent re-esterification was conducted directly on the crude reaction mixture in the presence of the ethyl esters produced from the end-positions of the original oil, after filtering off the enzyme and stripping off the excessive ethanol. The reaction was conducted for only one hour under vacuum at 35 °C using the immobilized Rhizomucor miehei lipase and excessive amount of ethyl caprylate (7–8-fold stoichiometric excess). Structured TAG comprising well over 40 % DHA at the mid-position and well over 90 % CA content at the endpositions were obtained. Their approach is illustrated in Fig. 14.12. This method offers various advantages over other reported methods. It is fast, the regioselectivity is high and acyl-migration is kept at a minimum. High yields of the 2-MAG intermediates are obtained with the original fatty acid composition in that position largely preserved. However, an efficient separation, presumably by short-path distillation and purification of the final product, needs to be demonstrated.

14.6 MLM type structured TAG comprising pure n-3 PUFA and MCFA Positionally labeled symmetrically structured TAG of the MLM type comprising pure homogeneous PUFA such as EPA or DHA and MCFA of absolute regioisomeric and chemical purity are ideally suited as libraries of pure compounds for various purposes. For example, they may be exploited to compare the effects of individual fatty acids by biological screening, as standards for analysis, fine chemicals and as potential drugs. Figure 14.13 O O FO

O

PUFA

O OH CAL

O O O

FO

OH O

OH

O

O

PUFA CA

O RML

O CA

PUFA

O O O O

CA

Fig. 14.12 Production of fish oil derived positionally labeled structured TAG containing caprylic acid (CA) and PUFA by fish oil ethanolysis using immobilized Candida antarctica lipase (CAL) and re-esterification of the resulting 2-MAG with CA by Rhizomucor miehei lipase (RML) (FO = fish oil fatty acids located at the end-positions of the glycerol moiety of the fish oil TAG).

PUFA production from marine sources for use in food

355

O O O O O O

O O O O O O

Fig. 14.13 The structure of homogeneous positionally labeled symmetrically structured TAG of the MLM type containing capric acid and EPA (top) and caprylic acid and DHA (bottom).

shows the structure of homogeneous positionally labeled symmetrically structured TAG of the MLM type comprising capric acid and EPA (top) and CA and DHA (bottom). Such structured TAG require total synthesis from glycerol with a full regioselectivity control. There are two alternative approaches: a fully enzymatic approach involving two or three enzymatic steps and a two-step chemoenzymatic approach. In the fully enzymatic approach, the first step involves the synthesis of homogeneous TAG containing the PUFA that is intended to be accommodated at the mid-position. In a two-step process this is followed by transesterification with an ethyl ester of MCFA by a 1,3-regioselective lipase (see Fig. 14.14). An alternative three-step procedure requires a 2-MAG homogeneous with the PUFA that is achieved by alcoholysis of the homogeneous TAG by a 1,3regioselective lipase. The pure MCFA is then introduced to the end-positions by lipase in the third step (see Fig. 14.15). In the first step of the chemoenzymatic approach a 1,3-regioselective lipase is exploited to prepare 1,3-DAG of a pure MCFA. This is followed by a subsequent chemical coupling reaction of pure EPA or DHA into the free mid-position (see Fig. 14.16 below). Both approaches have their drawbacks and limitations. The fully enzymatic approach is obstructed by the need of a three-fold excess of the pure EPA or DHA. Also, large excesses of MCFA are often required in order to reach satisfactory results in terms of yields and purity of the final structured TAG. The main advantage is that the overall process constitutes environmentally friendly processes, where no toxic and hazardous chemicals or organic solvents are involved. This approach is usually hampered by extreme difficulties in affording products of absolute regioisomeric and chemical purity that may need tedious purification processes involving organic solvents. Provided that a strict regiocontrol is maintained using a fully regioselective

356 Modifying lipids for use in food O

O

O

OH OH

EPA

OH

O

EPA

CA

O O

CAL

O

O

EPA

CA

O

O O

EPA O O

RML

OH

O

EPA

O

CA

Fig. 14.14 Synthesis of MLM type symmetrically structured TAG containing caprylic acid (CA) and EPA by a fully enzymatic two-step approach using immobilized Candida antarctica lipase (CAL) for direct esterification and Rhizomucor miehei lipase (RML) for interesterification. O O DHA

O

DHA

O

OH

OH

O O

O

CA

CAL

O

O

O

DHA

O OH

CA

DHA

O O O

RML

DHA

O

OH

CA

Fig. 14.15 Synthesis of MLM type symmetrically structured TAG containing caprylic acid (CA) and DHA by a fully enzymatic three-step approach using immobilized Candida antarctica lipase (CAL) for ethanolysis of TAG homogeneous with DHA and Rhizomucor miehei lipase (RML) for a subsequent esterification of the resulting 2-MAG. (The first step to produce the homogeneous TAG is not included.) O

O OH OH

CA

OH

RML

CA

O

O O

EPA OH DCC

OH O

O CA

EPA

O O O

OH O

CA

O

CA

Fig. 14.16 Chemoenzymatic synthesis of MLM type symmetrically structured TAG containing caprylic acid (CA) and EPA by immobilized Rhizomucor miehei lipase (RML) and dicyclohexylcarbodiimide coupling agent (DCC) by Yamane’s approach.

lipase and a suitable coupling agent, the chemoenzymatic approach has the advantages of offering chemically and regioisomerically pure products where only stoichiometric amounts of the pure fatty acids are needed. The drawbacks relate to use of chemicals and organic solvents, but that is widely practised in pharmaceutical synthesis and can be justified when the advantages in terms of purity are borne in mind. When homogeneous products of that purity are involved, analytical methods such as high-field 1H and 13C nuclear magnetic resonance (NMR) spectroscopy are ideally suited to monitor the regioisomeric purity and regioselectivity control.

14.6.1 MLM type structured TAG by fully enzymatic approach The first attempts involving a fully enzymatic process to produce regioisomerically pure structured TAG of the homogeneous MLM type were

PUFA production from marine sources for use in food

357

reported by Yamane’s group (Irimescu et al., 2000, 2001c). Their two-step approach was based on a lipase-promoted preparation of TAG containing pure EPA (EEE) and a subsequent interesterification with ethyl caprylate by a 1,3-regioselective lipase. This process is illustrated in Fig. 14.14. Immobilized Candida antarctica lipase was used to esterify glycerol with pure EPA to afford the homogeneous EEE in 90 % conversion yield. The crude reaction product mixture was then subjected to the second enzymatic step without purification, apart from removal of the lipase. That step was also conducted without a solvent using immobilized 1,3-regioselective Rhizomucor miehei lipase and a hundred-fold molar excess (50-fold stoichiometric amount) of ethyl caprylate (Yamane, 2000). The final reaction product mixture constituted 89 % of the intended CEC (where C is caprylic acid and E is EPA), but no attempts were made to isolate and purify the desired product from the bulk of the reaction product mixture constituting only 3 wt % of the desired structured TAG. Shimada and coworkers made an effort to simplify Yamane’s approach somewhat by treating TAG homogeneous with EPA in three successive acidolysis reactions using 15 mol parts of CA (Kawashima et al., 2001). The reaction was conducted for 48 hours each time at 30 °C using their immobilized Rhizopus delemar lipase. After the three successive acidolysis reactions, the CA content of the TAG product reached 66 mol%. The product was still a mixture constituting 86 wt % of the desired CEC structured TAG, but it was contaminated with 2 % of the undesired regioisomeric CCE. No attempts were made by the groups of Yamane and Shimada to isolate and purify the desired product or to fully characterize it, and the required purification by fractional multi-step molecular distillation and preparative column chromatography is expected to be tedious. The main impediment in the fully enzymatic synthesis of MLM-type structured TAG comprising MCFA such as CA and DHA (CDC) is the very low activity of 1,3-regioselective lipase on TAG containing DHA located at the end-positions. Such TAG are very resistant to lipase action, and DHA remains in place at the end-positions causing the final product to constitute a mixture of regioisomers. Yamane and coworkers managed to solve this by a three-step modification based on a highly 1,3-regioselective lipase-catalyzed ethanolysis of the homogeneous DHA TAG and a subsequent lipase-promoted esterification of the resulting 2-MAG with a different lipase (Irimescu et al., 2001a). The immobilized Candida antarctica lipase was observed to display excellent regioselectivity in ethanolysis of both tridocosahexaenoylglycerol (DDD) and trieicosapentaenoylglycerol (EEE) at 35 °C using a 33-fold stoichiometric excess of ethanol to TAG. For DHA a mixture was afforded comprising 93 % of the desired 2-MAG together with 2 % of starting TAG and 5 % of the 1,2-DAG intermediate. There was no noticeable formation of any of the undesired regioisomers such as 1,3-DAG or 1(3)-MAG during this step. The crude product mixture with the co-produced DHA or EPA ethyl esters

358 Modifying lipids for use in food present was introduced to the third enzymatic re-esterification reaction. This was transesterification with a 20-fold molar excess of ethyl caprylate using the immobilized Rhizomucor miehei lipase as biocatalyst under reduced pressure at 35 °C for one to five hours. The process is shown in Fig. 14.15. The product contains not only the structured TAG contaminated with other acylglycerols that need to be removed, but also two molar equivalents of EPA or DHA ethyl esters and excessive amounts of ethyl caprylate that need to be separated. Therefore, the desired structured TAG product constituted less than 15 % by weight in the crude reaction product mixture. As before, the structured TAG were not isolated nor were they fully characterized. Again, it is evident that tedious chromatography procedures will be needed for purifying these positionally homogeneously labeled structured TAG obtained from the fully enzymatic approach, demanding organic solvents. Yamane’s method has recently been modified and significantly improved by Hou and coworkers (Irimescu et al., 2002). They managed to improve the regiocontrol and yields in the second step by lowering the temperature to 25 °C and carefully controlling the ratio of the substrates to a molar ratio of 77:1 between ethanol and the homogeneous TAG of DHA and various other PUFA including EPA. For DHA the final acylglycerol reaction mixture constituted 97 % of the desired 2-MAG (together with 3 % of the corresponding 1,2-DAG) after seven hours in 93 % recovery yield as based on initial TAG but, evidently, some glycerol was formed during the reaction (varying from 3–20 % depending on type of PUFA). For the corresponding EPA synthesis, the purity of the 2-MAG was 98 % in 75 % recovery yield; 98 % pure 2-MAG of DHA was obtained from the reaction mixture after purification by selective extraction in 87 % yield. This treatment required the use of organic solvents (acetonitrile, hexane and chloroform) to remove excessive amounts of the PUFA, ethanol and the co-produced glycerol. The subsequent reesterification step was dramatically improved when the purified 2-MAG was directly esterified with a stoichiometric amount of CA at 25 °C with the immobilized Rhizomucor miehei lipase under vacuum. The desired regioisomerically pure DHA structured TAG adduct was obtained after eight hours in 96 % purity. Again, isolated yields of the purified structured TAG from these very successful processes were not reported and need to be demonstrated together with a full characterization of these products and their 2-MAG intermediates.

14.6.2 MLM type structured TAG by chemoenzymatic approach The chemoenzymatic approach is based on lipase regioselectivity to produce symmetric 1,3-DAG of a pure MCFA as a key intermediate. A subsequent chemical introduction of a long-chain PUFA into the mid-position results in a symmetrically structured TAG of the MLM type. The basis for the enzymatic part of the chemoenzymatic approach was laid by Schneider and coworkers in their lipase-promoted 1,3-DAG generation (Berger et al., 1992; Aha et al.,

PUFA production from marine sources for use in food

359

2000). A whole range of regioisomerically pure 1,3-DAG of medium- and long-chain saturated fatty acids was obtained in good yields (74–85 %) from glycerol adsorbed on silica gel using the immobilized Rhizomucor miehei lipase in organic solvents. Vinyl esters were superior to free acids and methyl esters as acyl donors in terms of reaction rate and product yields. The regioselectivity, however, was by no means perfect since there were clear indications of acyl-migration taking place under these conditions. Yamane and coworkers were the first to report a chemoenzymatic synthesis of an MLM type structured TAG containing pure EPA and caprylic acid (Rosu et al., 1999). In the first step, regioisomerically pure 1,3-DAG was prepared by modification of the procedure of Schneider and coworkers using Lipozyme as the biocatalyst. Yields comparable to those of Schneider and coworkers were obtained (75–80 %). Purified 1,3-DAG (95 %) was then subjected to the subsequent chemical esterification step, where dicyclohexylcarbodiimide (DCC) was used as a chemical coupling agent to introduce pure EPA into the mid-position of the 1,3-DAG in the presence of 4-dimethylaminopyridine (DMAP). This resulted in TAG of 98 % purity, but in only 42 % yield, and the product turned out not to be regioisomerically pure. The synthetic approach is illustrated in Fig. 14.16. Haraldsson and coworkers managed to improve dramatically on the chemical coupling step by replacing DCC with 1-(3-dimethylaminopropyl)-3ethylcarbodiimide (EDCI) as a coupling medium in the presence of DMAP. This was demonstrated in their synthesis of a structured TAG comprising pure stearic acid (Haraldsson et al., 2000) and caprylic, capric and lauric acid (Halldorsson et al., 2001) residues at the end-positions with pure EPA or DHA at the mid-position by the chemoenzymatic approach. The enzymatic step was based on the procedure of Schneider and coworkers (Berger et al., 1992; Aha et al., 2000) in ether to afford the regioisomerically pure 1,3DAG adducts in moderate to good yields after purification by crystallization. Pure EPA and DHA were then introduced to the mid-position using EDCI as a coupling agent in the presence of DMAP in dichloromethane in excellent yields (≥ 90 %), after purification by treatment on silica. Recently, Haraldsson and coworkers reported a modification of their chemoenzymatic approach towards synthesis of structured TAG of the above MLM type (Halldorsson et al., 2003a). A dramatic improvement of the regiocontrol and yields of the enzymatic step were described. This is based on a rapid, irreversible transesterification of glycerol using vinyl esters of the MCFA and the immobilized Candida antarctica lipase in dichloromethane at 0–4 °C. The Candida antarctica lipase acted exclusively at the glycerol end-positions and no acyl-migration took place. The yields (90–92 %; see Table 14.4) are based on pure material after re-crystallization. In the subsequent coupling reaction EDCI was used as a chemical coupling agent to introduce EPA and DHA into the mid-position of the 1,3-DAG adducts. The reaction was conducted at room temperature in dichloromethane in the presence of 30–50 % DMAP (as based on mol) using an exact

360 Modifying lipids for use in food Table 14.4 Yields and type of products and 1,3-DAG intermediates (1a,b,c) from the chemoenzymatic synthesis of MLM type structured TAG comprising pure MCFA and EPA (2a,b,c) or DHA (3a,b,c). Compound

MCFA

PUFA

Yield

1a b c 2a b c 3a b c

–C7H15 –C9H19 –C11H23 –C7H15 –C9H19 –C11H23 –C7H15 –C9H19 –C11H23

– – – EPA EPA EPA DHA DHA DHA

90 92 90 90 93 92 90 94 95

% % % % % % % % %

Abbreviations: DHA = docosahexaenoic acid, EPA = eicosapentaenoic acid, MCFA = medium-chain fatty acid, PUFA = polyunsaturated fatty acid.

stoichiometric amount of EPA or DHA as based on the 1,3-DAG adduct. The reactions were completed in 12–15 hours. Chemically and regioisomerically pure structured TAG were afforded as colorless and slightly yellowish oils, respectively, for the EPA and DHA adducts, in yields of 90–95 % (see Table 14.4) after chromatographic treatment on silica gel. No sign of any acyl-migration side-reaction was observed to take place during the coupling reaction. Their approach is demonstrated in Fig. 14.17. A whole range of structured MLM type TAG was synthesized by this methodology ranging from C8 to C16 saturated fatty acids. All C8–C12 products and intermediates are listed in Table 14.4 and all were obtained in excellent yields. The yields are based on isolated and purified material and all compounds were fully characterized and their regioisomeric and chemical purity established by modern methods. High-resolution 1H and 13C NMR spectroscopy were of great use to monitor the regiocontrol of both reactions and to establish the regioisomeric purity of all compounds involved.

14.7 Industrial aspects and future trends Lipase has been in use in the fats and oils field since the 1980s with the use of lipase in the n-3 PUFA area dating back before 1990. Despite the existence of a number of feasible processes, it has taken a long time for lipase to become accepted and commercialized within the n-3 PUFA industry. There is, however, little doubt that several Japanese industrial companies are using lipase for producing n-3 PUFA concentrates on an industrial scale both through kinetic resolution processes and a subsequent esterification into TAG. It is by no means easy to get detailed information on this from the literature. Due to regulatory issues in Japan, chemically concentrated ethyl esters and

PUFA production from marine sources for use in food

OH OH

MCFA

O

CAL OH

O

O

O MCFA

O

O O

MCFA OH O O

PUFA

OH

PUFA

O O O

EDCI/DMAP MCFA

361

O

MCFA

Fig. 14.17 Chemoenzymatic synthesis of MLM type symmetrically structured TAG comprising MCFA (C8–C12) and PUFA (EPA or DHA) by immobilized Candida antarctica lipase (CAL) and EDCI.

acylglycerols are not appproved for food or dietary supplement application. Therefore products for such application in Japan must be enzymatically concentrated. There are also companies in Europe and presumably North America that have started industrial-scale production of n-3 PUFA concentrates as TAG by the lipase technology. There is little doubt that this will develop further in the near future. Table 14.5 lists some of the main n-3 PUFA concentrate producers with indications of their main products in terms of composition and type (ethyl esters vs TAG). The names and homepages of the companies are provided. The concentrates are divided into three levels of EPA and DHA, 30–70 %, 70–90 % and 90–100 %, as well as ethyl esters and TAG (or acylglycerols). The table shows that a number of companies produce the 30–70 % and 70–90 % concentration levels both as ethyl esters and TAG. Some of these products are highly enriched with EPA, others with DHA and still others with EPA and DHA. In Europe TAG concentrates are dominant in the market while ethyl esters are widely used in USA. There appears, however, to be a trend in the USA that the market is moving from ethyl esters to TAG although this is a slow process. The highest purity category includes virtually pure EPA and DHA. Only one company supplies such purity levels as TAG, i.e. Chemport in Korea. A few Japanese suppliers provide such products as ethyl esters. Most of the Japanese companies making the highly concentrated n-3 ethyl esters are using the final product as an active pharmaceutical ingredient (API) and the bulk oil is not commercially available. Omacor® is a pharmaceutical ethyl ester product from Pronova Biocare in Norway that contains 460 mg EPA and 380 mg DHA per 1000 mg. This has a marketing license in EU and USA as well as other countries as a prescription drug. Likewise, Mochita in Japan has a highly concentrated EPA (higher than 96 %) approved as a pharmaceutical drug in Japan. It is anticipated that structured TAG will also be developed further where lipase will be utilized. There are already several such products on the marked. Marinol™ D-40 is a concentrated enzymatically produced fish oil from Lipid Nutrition (a lipid division of Loders Croklaan: www.lipidnutrition.com). It has a total of 40 % DHA in the form of glyceride. Marinol™ C-38 is a similar product comprising both EPA and DHA, total of 38 %. All of the marinol products can be used in dietary supplements and find application in

Table 14.5

Producers of EPA and DHA n-3 PUFA concentrates.

Company

Country

Web page

PUFA concentrates (%) 30–70

Pronova Biocare Ocean Nutrition Croda Chemport KD Pharma Napro Pharma Arjuna Naturals Maruha Nissui Lipid Nutrition Sepu DSM Nutr. Prod. Sanmark Bizen NOF Tama Biochemical Harima Chemicals

Norway Canada UK Korea Germany Norway India Japan Japan Malaysia Korea USA/Holl China Japan Japan Japan Japan

Abbreviation: PUFA = polyunsaturated fatty acid.

epax.com ocean-nutrition.com croda.com/europe/hc chemport.co.kr kd-pharma.de napro-pharma.no arjunanatural.com maruha.co.jp nissui.co.jp lipidnutrition.com sepufc.com nutraaccess.com sanmarkltd.com bizen.co.jp nof.co.jp tama-bc.co.jp harima.co.jp

70–90

90–100

EE

TG

EE

TG

x x x x x x x

x x x x

x x x x x

x x x x

x x x x x

EE

TG

x x

x

x x x x x

x

x x x x

x x

x

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363

functional food, medical food, enteral food and infant nutrition. Japanese producers of such structured TAG include Nissui and NOF. There are already strong indications that n-3 PUFA-enriched phospholipids will become important in the near future. Purified marine phospholipids highly enriched with DHA, but lower in EPA, were observed to play decisive roles in aquaculture involving the production of halibut juveniles from larvae (Hjaltason et al., 2005a,b). Krill oil from Antarctic krill has already been marketed as a source of DHA in the form of a mixture of marine phospholipids and marine TAG by Neptune Technologies and Bioresources Inc. in Canada (www.neptunebiotech.com) (Sampalis, 2005). A French company is also producing purified marine phospholipids on a relatively small scale (Phosphotech: www.phosphotech.com). The Japanese companies NOF and Bizen are also suppliers of such purified marine type phospholipids.

14.8

References

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15 Production, separation and modification of phospholipids for use in food J. Pokorný, Institute of Chemical Technology, Prague, Czech Republic

15.1

Introduction

15.1.1 Chemical structure of phospholipids Phospholipids are natural products containing fatty acids and phosphoric acid in their molecule. They also contain an alcohol; in phospholipids important for food purposes this almost exclusively glycerol (they are called glycerophospholipids). The 1- and 2-positions of the glycerol molecule are esterified with fatty acids and the 3-position with phosphoric acid. Saturated fatty acids, frequently palmitic acid, are preferentially bound at position 1, while position 2 is preferentially esterified with polyenoic fatty acids, most often linoleic acid. In the literature, phospholipids are often abbreviated as follows: PC = phosphatidylcholines; PE = phosphatidylethanolamines; PI = phosphatidylinositols; PS = phosphatidylserines; PA = phosphatidic acids; PG = phosphatidylglycerols; PDG = phosphatidyldiacylglycerols; SP = sphingolipids. In addition to the hydroxyl group of phosphoric acid esterified with a diacylglycerol residue, another hydroxyl group of phosphoric acid is esterified with choline, ethanolamine, serine, inositol, glycerol or diacylglycerol (Fig. 15.1). In many scientific texts, they are called by their group name, i.g. phosphatidylcholine. However, the phospholipid classes are not chemically individual as they contain different fatty acids; therefore, they may each be considered as a group of structurally related compounds. Nevertheless, they are usually called by their class name, e.g. ‘phosphatidylcholine’ means a group of all the phosphatidylcholines present. Some physiologically important phospholipids do not contain bound glycerol, but an acyl group is bound as an amide to the primary amino group of a long-chain aminoalcohol, such as

370 Modifying lipids for use in food O

O

R1

O

R1

O

R2

O

R2

O

O O

O

OH

P

O O

O

N

O

P O

O Phosphatidic acids

Phosphatidylcholines

O

O

R1

O

R2

O

1

O

R2

O

R O O

O

NH3

O

P

OH O O

O

P

OH O

O

O Phosphatidylethanolamines

Phosphatidylglycerols O

O R1

O

R2

O

O O

O

P

OH OH O HO

R1

O

R2

O

OH OH

O O

O

P

NH3

O

O

O

O

OH

Phosphatidylserines

Phosphatidylinositols

OH

O O

R

NH

P

O

N

O

O Sphingomyelins O O HO

R O

O

P

O

X

O Lysophosphatidyl derivatives

Fig. 15.1

Chemical structures of some important phospholipids (R – the respective fatty acid residues; X – residues bound to phosphatidic acid).

sphingosine or related compounds. Sialic acid or neuraminic acid may also be present. They are bound in mycophospholipids, containing also bound sugars.

Production, separation and modification of phospholipids 371 15.1.2 Sources of phospholipids Phospholipids are present in small amounts (up to 1 % dry weight) in all plant or animal tissues because phospholipids form the boundary layers (membranes) of all cells and sub-cellular particles. They are consumed with food but, for industrial purposes, only two sources are of importance, i.e. egg yolk and crude soybean oil. Neural tissues, such as brain, are also rich in phospholipids, but they are less readily available for industrial production. The phospholipid content in soybeans is not much different from the content in other oilseeds, but most oilseeds contain about 40 % oil, while soybeans contain only about 20 %. Therefore, crude oil produced by extraction of crushed soybeans contains about 3 % phospholipids. Other crude oils are not so rich in phospholipids as they are more diluted with co-extracted neutral lipids. Therefore, the yield of phospholipids from crude soybean oil is much higher than is case with other crude oils, and the product has an advantageous composition (Table 15.1). Phospholipid concentrates obtained by plant-scale operations are called lecithins. They are not pure phospholipids as they always contain 30–40 % triacylglycerols and other lipidic components. In older literature, lecithin often means phosphatidylcholines.

15.1.3 Industrial production of phospholipids Egg yolk is rich in lipids (about 31–36 % dry weight). The lipid fraction consists of triacylglycerols, of which 3 % are phospholipids and 5 % is cholesterol. The lipid fraction can be obtained from the dried raw material. As egg lecithin is a relatively expensive ingredient in food products, it is produced only for special preparations where the price is not so critical, such as in the pharmaceutical industry. Soybean lecithin is much more important than egg lecithin as an ingredient in the food industry, as it can be easily obtained as a by-product of soybean oil production. Soybeans are crushed and treated with steam. Proteins present in soybean meal lipoproteins are denaturated at high temperature, and Table 15.1 Composition of the most important phospholipid classes in egg yolk, soybean and other important sources. Phospholipid class

Egg yolk

Cows’ milk

Bovine brain

Rapeseed

Sunflower seed

Soybean

LP PA PC PE PI PS SP

3–8 trace 66 – 82 8 – 24 <1 1–3 1–3

trace trace 20 – 30 28 – 35 <1 1–8 15 – 29

1–2 1–2 18 – 43 18 – 36 1–7 9 – 18 15 – 22

1 – 10 0 – 28 20 – 38 14 – 31 11 – 28 1–4 <1

0–3 0 – 13 27 – 41 16 – 17 18 – 23 1–2 <1

1– 5 0 – 12 22 – 46 21 – 32 13 – 22 5– 6 <1

Abbreviations: LP = lysophospholipids, PA = phosphatidic acids, PC = phosphatidylcholines, PE = phosphatidylethanolamines, PI = phosphatidylinositols, PS = phosphatidylserines, SP = sphingomyelins.

372 Modifying lipids for use in food phospholipids bound in lipoproteins are liberated so that they can be extracted together with soybean neutral lipids. The extraction plants are continuous or semi-continuous with a high capacity of more than 800 tonnes daily. In absence of water, phospholipids are easily soluble in hexane or other hydrocarbons used as solvents. Crude oil is filtered in order to remove fine particles of soybean meal, and the solvent is removed by distillation. The last solvent residues are removed by steam. Phospholipids are obtained from crude oil in a process called degumming. During the degumming, crude oil is heated for at least 30 minutes with 2–3 % water or water mixed with phosphoric acid. Phospholipids are hydrated, thereby becoming insoluble in the non-polar oil phase. In the presence of phosphoric acid, calcium and magnesium salts of acidic phospholipids, such as phosphatidic acids or phosphatidylinositols, are decomposed, and the yield of lecithin increases. Another advantage is the lower phospholipid phosphorus content in the degummed oil, which makes it more suitable for further refining. In the process of super-degumming, used in the case of physical refining of soybean oil, citric acid and/or other polyvalent organic acids are added, and the heating is intensified. The yield of lecithin is higher and the degummed oil is better purified, but lecithin obtained in this way is less suitable for edible purposes in most food industry applications. Lecithins obtained in an analogous way from crude oils other than soybean are used mostly for non-edible applications.

15.1.4 Laboratory preparation of phospholipids On the laboratory scale, phospholipids are usually extracted after the wellknown procedure, proposed by Folch, using a mixture of a medium-polar solvent (such as chloroform, ethyl acetate or diethyl ether) and a polar solvent (such as methanol or ethanol), which is able to form hydrogen bridges. The polar solvent would also remove water from the material and, during the following treatment with the same solvent system, repeated a few times, both fewer polar neutral lipids and more polar phospholipids are quantitatively extracted. The solvents are removed by distillation, but some water always remains. Therefore, the extract is dissolved in hexane or cyclohexane, the solution is washed with saline and the organic phase is dried, e.g. with anhydrous sodium sulfate. Solvent is then distilled off and a mixture of neutral lipids and phospholipids remains in the residue. Phospholipids are obtained by fractionation of the concentrate with acetone.

15.2 Separation and purification of phospholipids 15.2.1 Industrial separation of phospholipid concentrates Phospholipid concentrates, obtained in the process of degumming, have variable composition, depending on degumming conditions. They always contain

Production, separation and modification of phospholipids 373 neutral lipids, too. A typical concentration of phospholipids in soybean lecithin is 60–70 %. The phospholipid content is higher at higher degumming temperature. Crude lecithin has a dark brown colour, due to the presence of natural pigments, such as carotenoids and chlorophylls, and brown substances formed by degradation reactions of sugars. For some purposes, lecithin should be bleached. About 1 % of a 30 % aqueous hydrogen peroxide is added, and the mixture is heated for up to 30 minutes at 50–60 °C. Benzoyl peroxide can be used instead of hydrogen peroxide. Bleached gums contain 30–50 % water. They are dried to about 0.3–0.8 % water, using a vacuum batch dryer or a thin-film continuous evaporator. During the drying operation, remaining peroxides are decomposed. Double bonds present in unsaturated fatty acids bound in phospholipids are partially converted into their hydroxylic derivatives. To obtain a fluid product, free fatty acids and soybean oil are added to crude lecithin; the content of acetone-insolubles is about 65–68 % in the resulting product, and the viscosities of about 104 cP (1 P = g/cm s) at 25 °C are obtained after distillation of the solvent from the acetone-insoluble fraction.

15.2.2 Plant-scale purification of lecithin Industrial lecithin contains soybean oil and is suitable for most applications. For special purposes, de-oiled products are required. As phospholipids are insoluble in acetone at refrigeration temperatures, cold acetone is used for fractionation. Large excess of acetone is necessary. Usually, however, lower excess is used, but the fractionation is repeated 2–4 times. Excess acetone is removed from the insoluble residue by drying. The acetone layer contains neutral lipids, which may be recovered after removal of acetone. Pure phospholipid mixture is obtained by low-presssure gas extraction of lecithin with propane (Weidner et al., 1993); the advantage is easy removal of the solvent. Phospholipids may be fractionated with ethanol also. The ethanol-soluble fraction is rich in phosophatidylcholines and is also enriched with phosphatidylinositols and phosphatidylethanolamines (Štrucelj et al., 1995). In the case of soybean lecithin fractionation with 90 % aqueous ethanol, two fractions were obtained: (i) the phosphatidylcholine-enriched fraction consisting of 73 % phosphatidylcholines, 24 % phosphatidylethanolamines and 3 % phosphatidylinositols; (ii) the phosphatidylinositol-rich fraction consisting of 39 % phosphatidylinositols, 35 % phosphatidylethanolamines and 26 % phosphatidylglycerols (Wu and Wang, 2003). Nearly 100 % pure phosphatidylcholine is obtained by pilot plant high-performance liquid chromatography (HPLC) fractionation of egg yolk lecithin.

15.2.3 Laboratory purification of lecithin At the laboratory scale, the same procedure is used as at the plant scale, i.e.

374 Modifying lipids for use in food repeated treatment with acetone at refrigeration temperature. Preparations containing up to 95 % phospholipids are obtained. The remaining neutral lipids can be removed by column chromatography packed with silica gel, by washing with hexane. Triacylglycerols are removed and phospholipids are then eluted by polar solvent mixtures. Further fractionation of phospholipids, e.g. into fractions of acidic and neutral phospholipids, can be achieved using preparation ion exchange chromatography (Elsner and Lange, 1995). Nearly pure phospholipid classes are obtained by HPLC of soy phospholipids.

15.3 Modification of phospholipids It is possible to modify lecithin in many ways. Properties of soy lecithin can be modified by addition of special edible oils, lanolin and emulsifiers, such as monoacylglycerols, modified monoacylglycerols and surfactants. Only the additives approved by state authorities can be used as edible phospholipids. Another method is the chemical modification of phospholipids, for instance by esterification, hydrolysis or oxidation. Other modifications, such as sulphonation, halogenation, acylation or ozonization, cannot be used for food products.

15.3.1 Hydrolysis reactions Phospholipids contain at least three or four ester groups in their molecule, which could be hydrolyzed in the presence of alkali or strong acids, but the chemical hydrolysis is not selective so that such reactions are not interesting. In contrast, the enzymatic hydrolysis is selective, and may be used for preparation of specific products. The ester bonds are cleaved by phospholipases, which are selective for cleavage of different ester bonds (Fig. 15.2). Fatty acids bound to positions 1 and 2 of the glycerol moiety are cleaved by reactions catalyzed by phospholipases A1 and A2, respectively. Some lipases are active, too. The hydrolysis of ester bonds between the diacylglycerol moiety and phosphoric acid is catalyzed by phospholipase C, and the bond between the phosphatidic acid group and choline, ethanolamine, serine or A1

B

O A2 R″

C O

C O

O

C

C

R′

O

C

O

P

O

X

O C

Fig. 15.2

D

Hydrolytic cleavage of phospholipids under catalysis of phospholipases (A, B, C, D – the respective phospholipases).

Production, separation and modification of phospholipids 375 other groups is hydrolyzed under the catalytic action of phospholipase D (Fig. 15.2). The hydrolysis catalyzed by phospholipase A2 is most important, and its mechanism was recently reviewed by Wilton (2005). Lysophospholipids are obtained as a result of this reaction. The fatty acids split off are mostly polyunsaturated fatty acids, from the sn-2 position, such as linoleic acid and linolenic acid. The same reaction is catalyzed by some lipases too, e.g. a lipase from porcine pancreas (EC 3.1.1.4), which is a competitor of phospholipases (Doig and Diks 2003a,b). Patatin, a lipid acyl hydrolase from potato tubers, selectively hydrolyzed unchanged phospholipids with formation of sn-2-lysophospholipids (Anderson et al., 2002). The activity of phospholipase A2 substantially depends on the water content in the reaction mixture (Wang et al., 2003). Immobilized lipases are more stable and more suitable for the hydrolytical reaction than free enzymes. The resulting 2lysophospholipids are very advantageous as they are more polar compounds than the original phospholipids. They are also more stable against oxidation as most polyenoic acids, located at position 2, have been removed. Because of these features, they are applied as emulsifiers for oil-in-water emulsions as their HLB (hydrophilic-lipophilic balance) value is about equal to 19 (a very high value), and they also improve the rheological properties of starch gels. They are thus useful as bread improvers or for other applications. Their emulsification properties are superior to those of the original phospholipids and they can also be applied in continuous processes (Morgado et al., 1995). Other enzyme preparations catalyze the phospholipid hydrolysis at position 1, e.g. phospholipase A from Rhizopus oryzae (Doig and Diks, 2003a). While lipases are less selective, cleaving both triacylglycerols and phospholipids, phospholipases A2 cleave only phospholipids at position 2 and triacylglycerols remain intact (Haas et al., 1995). Therefore, the phospholipase A2 (phosphatide 2-acylhydrolase) has been suggested as more suitable for membrane bioreactors, where it was active in reverse micelles in isooctane (Morgado et al., 1996). The optimum temperature was 35–40 °C and the optimum pH = 7.0 (Pahn, 1997). Yamashita et al. (2000) studied the effect of time and temperature, pH value and calcium ion concentration. Hara et al. (1997) compared the reactivity of 19 commercially available lipases and obtained high reactivity in the case of Mucor and Rhizopus spp. lipases. Phospholipase A1 was discovered in various animal organs, such as rat liver or rat brain, but the enzyme is less important for industrial applications. Phospholipase C converts phospholipids into diacylglycerol and phosphorylcholine. In the presence of non-specific lipases, both fatty acids are cleaved with formation of glyceryl-phosphoryl-choline (Mustranta et al., 1995). Phospholipase C from Bacillus sp. cleaved phospholipid classes at different rates in aqueous medium and in ether (Kamata et al., 1996). Phospholipid modification with use of phospholipase D (EC 3.1.4.4) is important for conversion of less useful phospholipids into phosphatidylcholines

376 Modifying lipids for use in food (Yamane, 1995) and for other transphosphorylations (Rich and Khmelnitsky, 2001; Doig and Diks, 2003b). For instance, phospholipase D isolated from cabbage leaves increased the content of phosphatidylserines, while the content of phosphatidylcholines decreased (Kamata et al., 1996). The formation of phosphatidic acids occurred under suitable reaction conditions (Doig and Diks, 2003b).

15.3.2 Transesterification of phospholipids The fatty acid composition of phospholipids is different from that of triacylglycerols present in the same material. Each phospholipid class has a unique fatty acid composition (Table 15.2), and the fatty acid composition at position 1 is different from that at position 2. Saturated fatty acids prevail at position 1, while polyunsaturated fatty acids prevail at position 2. The fatty acid composition is not always appropriate for the particular purpose so that it should sometimes be modified by transesterification. The transesterification means an exchange of fatty acids either within a phospholipid molecule (an intramolecular transesterification) or between two phospholipid molecules or a phospholipid and another lipid molecule (an intermolecular transesterification). The latter process is technically more important. Phospholipid transesterification is catalyzed by the respective phospholipases. The enzymic transesterification of phospholipids was recently reviewed (Hayes, 2004). The fatty acid, which should replace a fatty acid in a native phospholipid, is added to the reaction mixture. Water must be added to the system, too, as the reaction can proceed only in an aqueous medium. Phospholipases are hydrolases so that some lysophospholipids are always formed (Fig. 15.3). Therefore a substantially larger quantity of fatty acids is added than would be necessary for the esterification reaction (Svensson et al., 1992), but, nevertheless, the yield is never quantitative. For instance, in the transesterification of lecithin with methyl laurate, the product contained about 43 % lauric acid, incorporated, and the yield of fully acylated Table 15.2 Difference in the fatty acid composition of soybean and rapeseed phospholipids (compiled from various sources and from personal unpublished results). Source

Phospholipid Position in class the glycerol molecule

Saturated Monounsaturated Polyunsatufatty acids fatty acids rated fatty acids

Soybean

PC

Soybean

PE

Rapeseed

PC PI PE

42 2 32 2 64 56 65

1 2 1 2 total total total

19 8 6 9 32 39 30

38 89 61 80 4 6 5

Abbreviations: PC = phosphatidylcholines, PE = phosphatidylethanolamines, PI = phosphatidylinositols.

Production, separation and modification of phospholipids 377 H2C—O—OC—R1 R2—CO—O—CH

O

Phospholipids (X = groups as shown in Fig. 15.1)

H2C—O—P—O—X O H2O Catalyzed by phospholipase A2

R2COOH H2C—O—OC—R1 HO—CH

O

Lysophospholipids

H2C—O—P—O—X O R3COOH Catalyzed by phospholipase A2

H2O H2C—O—OC—R1 R3—CO—O—CH

O

Transesterified phospholipids

H2C—O—P—O—X O

Fig. 15.3

Transesterification of phospholipids via lysophospholipids.

phospholipids was only 28 %. Therefore, the transacylated phospholipids had to be concentrated by solvent partition. It was found that yields of transacylated phospolipids were higher if phospholipids were first hydrolyzed into lysophospholipids and then esterified with the desired fatty acid (Adlercreutz et al., 2003). Higher temperatures decrease the rate of hydrolysis, but the rate of transesterification is not affected; however, the temperature of 40 °C is considered as the optimum (Doig and Diks, 2003b). At higher temperatures, phospholipases catalyzing the transesterification could be denaturated. Immobilized phospholipases were found to be better than free enzymes as they were more stable and reaction rates were higher, for instance in the reaction of phosphatidylcholines with heptadecanoic acid (Hara and Nakashima, 1996). The effects of water and solvents depend on the source of enzymes as has been shown in systems catalyzed by enzymes from Aspergillus niger and Rhizomucor miehei (Mustranta et al., 1994).

378 Modifying lipids for use in food Two modifications of phospholipids by transesterification are important for the improvement of nutritional and functional properties, namely, to replace a fatty acid with fish polyunsaturated fatty acids, or to replace a fatty acid with medium-chain-length fatty acids. Many applications have been reported so that only a few will be mentioned. Phosphatidylcholines were transesterified with eicosapentaenoic acid (EPA) ethyl ester at 50 °C for 48 hours; the ratio of phosphatidylcholine to EPA was quite high, 1:10 (Park et al., 2000). Similarly, 2-lysophosphatidylcholine was esterified with EPA and DHA, using phospholipase A2 as a catalyst (Na et al., 1990). Other fatty acids could be incorporated under similar conditions. Totani and Hara (1991) transesterified soy lecithin with sardine oil in hexane with catalysis of microbial lipases, but the recovery was rather low. The incorporation of EPA and DHA was studied in a mixture of phosphatidylcholines and lysophosphatidylcholines (Haraldsson and Thorarensen, 1999). The transesterification was accompanied by hydrolysis. Water increased the hydrolysis, but increased the reaction rate; optimum results were obtained with 5 % water. Good results were reported when only small amounts of water were used (Na et al., 1990). Phospholipids were transesterified with medium-chain-length fatty acids to decrease the available energy during metabolism and to improve the digestibility. Phospholipids were transesterified with fatty acids in the sn-2 position in organic solvents, using the Rhizopus oryzae lipase as a catalyst (Adlercreutz et al., 2002). The yields were quite high (about 75 %). Soy lecithin was transesterified with methyl esters of saturated 10–14 carbon atom fatty acids at 60 °C for 48 hours using immobilized lipase of Mucor miehei as a catalyst (Ghosh and Bhattacharyya, 1997); the incorporation of saturated fatty acids affected the interfacial tension in emulsions. The fatty acids bound in position 1 were replaced by ricinoleic acid; the ratio of one part lecithin:five parts ricinoleic acid was necessary; the temperature of 50 °C, the reaction time of 24 hours, and the use of hexane as a solvent were found optimum for both egg and soy phospholipids (Vijeeta et al., 2004).

15.3.3 Hydrogenation of phospholipids Refined soy lecithin and purified phospholipid fractions are rich in polyunsaturated fatty acids and are, therefore, more unstable against oxidation even under storage conditions. For some purposes, more stable products are desirable. Fully hydrogenated phospholipids, containing saturated fatty acids bound both at positions 1 and 2, are very resistant towards atmospheric oxygen under storage conditions or on heating. Hydrogenated lecithin protects tocopherols against their oxidative decomposition more than natural lecithin. However, it is more difficult to obtain fully hydrogenated phospholipids by synthesis or transesterification than to obtain them by complete hydrogenation of natural phospholipids. Hydrogenated phospholipids are not only more resistant towards autoxidation than natural phospholipids, but they also protect

Production, separation and modification of phospholipids 379 oil against discolouration and decomposition of tocopherols (Kajimoto et al., 1987). It is much more difficult to hydrogenate phospholipids than triacylglycerols, the reason being that highly polar groups present in phospholipids become strongly adsorbed on active sites on the surface of hydrogenation catalyst. The active sites are thus deactivated by this process. Such small amounts as 5–10 mg/kg phospholipids are sufficient to deactivate nickel catalyst, and 50 mg/kg are sufficient to inhibit the hydrogenation completely (Szukalska, 2000). Phosphatidic acids are more active in the catalyst’s deactivation than other phospholipids or lysophospholipids. Phospholipids have no effect on isomerization reactions of double bonds during the hydrogenation. The rate of hydrogenation of refined vegetable oils decreased 2–6 times in presence of lecithin (Hye, 1995). Kajimoto et al. (1987) studied the effect of hydrogenation on tocopherols. Platinum or palladium catalysts are more resistant to deactivation by phospholipids, but they are very expensive. The feasible process is to use higher concentration of a catalyst and/or very high pressure of hydrogen (about 10 MPa) in order to prepare fully hydrogenated lecithin. There is always a danger that traces of nickel catalyst remain bound to phosphoryl groups, and sophisticated procedures are then necessary to obtain an acceptable product, free of contaminating metals.

15.3.4 Phospholipid oxidation Phospholipids may be oxidized by various agents, e. g. hydrogen peroxide or organic peroxides; epoxides (oxirane derivatives) are formed which, being very reactive, react with amine and other reactive groups (Fig. 15.4). During the reaction, epoxides are usually hydrated forming hydroxyl derivatives. The hydroxylation increases the polarity of phospholipids and improves their emulsifying properties. For industrial uses, they may be treated with ethylene or propylene oxides to further increase their polarity and, thus, the HLB value. Phospholipids contain a large amount of polyenoic fatty acids and are, therefore, easily oxidized by atmospheric oxygen (Pikul and Kummerow, 1991), either in the process of autoxidation or lipoxygenase-catalyzed enzymatic oxidation. Free radicals are obtained in both cases, mainly by abstraction of a proton bound to a methylene hydrocarbon group, adjacent to a double bond. Free radicals react with a molecule of oxygen, and a peroxy free radical results from the reaction (Fig. 15.5), which is converted in a molecule of phospholipid hydroperoxide, following the same mechanism as that which is known in the case of triacylglycerols. Phospholipid hydroperoxides are bound mostly at the polyenoic acid bound in the 2-position so that they can be removed from autoxidized phospholipids by hydrolysis catalyzed by phospholipase A2. However, secondary reactions may be different as hydroperoxides can react with a nitrogen group either of the same phospholipid molecule, or another phospholipid molecule. Phosphatidylcholines react with

380 Modifying lipids for use in food —CH

CH— Double bond of an unsaturated fatty acid bound in phospholipids H2O2

H2 O —CH—CH—

—CH(NHR) CH(OH)— Hydroxyl substituted secondary amine

O Epoxide

R–NH2 H2O

Hydroxylated phospholipids

—CH—CH— OH OH Ethylene oxide

—CH—CH— OH O—CH2—CH2—OH

Fig. 15.4 CH

CH

Ethylene glycol derivatives

Formation of polar hydroxylated phospholipids. Unsaturated site of a fatty acid bound in a lipid

CH2 O2

CH

CH

CH

Lipid hydroperoxide

O–OH R–NH2

Phosphatidylethanolamines

2H2O CH

CH

C

N

R

Imine (X = oxidized lipid residue)

X

Fig. 15.5

Reaction of a lipid or a phospholipid hydroperoxide with a nitrogen group of phospholipid.

lipid hydroperoxides by cleavage of the amine oxide group (Fig. 15.6). The reaction often proceeds in a non-radical way. Brown products are formed (Section 15.3.5). Other secondary products of hydroperoxide decomposition are similar to

Production, separation and modification of phospholipids 381

H2O

H+

Phosphatidyl–O–CH2–CH2–OH

R—OOH

Lipid hydroperoxides

R—OH

O

N(CH3)3

Trimethylamine oxide

Fig. 15.6 Reaction of a phosphatidylcholines with lipid hydroperoxides.

those of triacylglycerol hydroperoxides, ending with formation of aldehydes and other sensory active products. Dienals and related aldehydes cause offflavours, especially in such relatively flavour-neutral foods as fermented milk (Surlyaphan et al., 2001a). De-oiled soy lecithin increased the offflavour intensities in reduced-fat milk, due to the formations of 2, 4-alkadienals (Surlyaphan et al., 2001b). While lecithin enhanced off-flavour formation in milk, in contrast, phospholipids reduced the rancidity in oils (Pokorný et al., 1976a) and acted as moderately active antioxidants (see Section 15.4.3). Phosphatidylethanolamines are more active as antioxidants than phosphatidylcholines (Sugino et al., 1997). Soy lecithin present in the amount of about 10 % increases the stability of vegetable oil against rancidification (Judde et al., 2003). Synergism was observed between phospholipids and γand δ-tocopherols, but not with α-tocopherol. The antioxidant activity of lecithin is particularly pronounced in polyunsaturated oils such as fish oils, containing pentaenoic and hexaenoic fatty acids (Hara et al., 1991). The ratio of phosphatidylcholines and phosphatidylethanolamines is important for the resulting activity, while phosphatidic acids are not efficient as antioxidants. However, they may bind pro-oxidative heavy metals into relatively inactive salts. Phospholipids were also found active as antioxidants in emulsions containing fish oil (Hara et al., 2000). Phospholipids containing bound docosahexaenoic fatty acid in their molecule were substantially more stable against autoxidation than triacylglycerols or ethyl esters with the same fatty acid composition (Jin and Miyazawa, 1997).

15.3.5 Modification of phospolipids by Maillard reactions with reducing sugars Properties of phospholipids are modified by Maillard reactions with glucose, fructose, galactose and other reducing sugars. Sucrose is easily hydrolyzed in glucose and fructose so that it participates at Maillard reactions, too. Contrary to triacylglycerols, phospholipids participated at Maillard reactions of cysteine and ribose, which are both present in muscle foods and contribute

382 Modifying lipids for use in food to the formation of meat flavour. It has been observed (Farmer and Mottram, 1990) that phosphatidylethanolamines, a common component of muscle phospholipids, increased the meat-like odour in the course of heating. Phosphatidylcholines increased the formation of sensory active 1-alcohols and 2-alkylfurans (Farmer and Mottram, 1992), and the amount of aldehydes decreased by reaction with phosphatidylethanolamines in aqueous medium. Sensory-active 3-methylfurans and thiophenes were detected in model systems of cysteine, ribose and phospholipids, heated to 185 °C, simulating roasting (Mottram and Whitfield, 1995). The first steps of interactions between phosphatidylethanolamine and reducing sugars were obviously similar to those of Maillard reactions between amino acids and reducing sugars, as phosphatidylglucosylamines, Amadori compounds, 5-hydroxy-methylpyrrole-2-carboxaldehydes and other carboxymethyl or carboxyethyl derivatives were detected in the reaction mixture (Utzmann and Lederer, 2000). The reactions were studied in foods, such as infant formulas and chocolate, and Amadori-glycated phosphatidylethanolamines were identified there also (Jeong et al., 2002).

15.3.6 Phospholipid browning reactions Lipid hydroperoxides or their carbonyl degradation products react with nitrogen groups of phospholipids. Both reactions are concurrent as hydroperoxides can react directly with amines, or their carbonyl decomposition products react with phospholipids. Precursors of brown compounds are unsaturated imines (Fig. 15.4), formed by interactions of lipid hydroperoxides with primary amine groups of phosphatidylethanolamines or phosphatidylserines, but the mechanism is different in reactions with other phospholipids. The contribution of phosphatidylcholines is much lower. The intermediary products are colourless, but they are rapidly converted into yellow compounds which are polymerized to brown macromolecular products (Pokorný et al., 1973). The extent of browning is, naturally, particularly high in white fish muscle as the unsaturation degree of some fish fatty acids bound in triacylglycerols and phospholipids is high, and brown products are distinctly perceptible. This occurs only in presence of water, and it is accompanied by fluorescence of the reaction mixture (Smith and Hole, 1991). In packed dried egg powders, the degree of browning depends on the content of residual water and on the excess of air (Satyanarayana Rao, 1991), and it increases with increasing temperature in the range of 37–55 °C. As a consequence of the non-radical decomposition of lipid hydroperoxides, phospholipid concentrates are relatively resistant towards oxidation in spite of their high unsaturation. The content of browning precursors, i.e. polyenoic fatty acids and amine groups, is relatively low in ghee (anhydrous buffalo fat) but, nevertheless, the butter oxidation was accompanied by changes of phospholipid extractibility from 84–87 % in fresh ghee to 37–59 % in stored or heated ghee (Pruthi and Kumar, 1991). However, the main cause of browning reactions is

Production, separation and modification of phospholipids 383 polyunsaturated fatty acids, and not the degree of phospholipid degradation (Sono et al., 2001). During the microwave heating of beans under roasting conditions, the degree of browning was found to be related to the decrease of amine group content in systems containing low moisture (Yoshida et al., 1995). Phospholipids as liposome vehicles increased the rate of browning of liposome-encapsulated mixtures of amino acids and reducing sugars (Haynes et al., 1992). The browning kinetics of egg phosphatidylamines has been studied in the temperature range 20–100 °C (Phan-Trong et al., 1974). The transformation of polyenoic fatty acids, bound in phosphatidylethanolamines, into conjugated dienoic fatty acids followed the first order kinetics. Decomposition of hydroperoxides by reaction with amine groups was an apparent zero order reaction, resulting in the formation of colourless Schiff bases. The conversion of Schiff bases into brown pigments was a first order reaction. Browning of phospholipids could be caused not only by reaction with lipid hydroperoxides, but also by secondary reactions of antioxidant oxidation products. Antioxidants are converted into free radicals and into quinones by reactions with lipid hydroperoxides. Purple and violet condensation products were produced in a model system by reaction of phosphatidylethanolamines with 1,4-benzoquinone, converting the intermediary products by consecutive polymerization into macromolecular brown pigments, accompanied by a change of the absorption maximum from 450 to 490 nm (Pokorný et al., 1975). The browning was much less intensive in reaction mixtures with phosphatidylcholines. Brown products were bleached by reaction with hydrogen peroxide, similarly as in natural lecithin, both in solution and in emulsion. Dibenzoylperoxide or cumenehydroperoxide were less efficient. The reaction rate depended almost linearly on the temperature in the range of 20–100 °C, and resulting products possessed their absorption maximum at 400–450 nm (Pokorný et al., 1976b). A semi-logarithmic relationship was obtained between the concentration of hydrogen peroxide and the bleaching effect. A special technological process is the thermalization of lecithin (Weete, 1995). Lecithin is heated for 90 minutes to 180 °C, until it turns brown, and the product is then fractionated with acetone and ethanol. Preferential decomposition of non-phosphatidylcholine phospholipids occurs during the process. The degradation rate of phospholipid classes decreases in the following order: phosphatidylethanolamines > phosphatidylinositols > phosphatidic acids > phosphatidylcholines. The viscosity of the thermalized product decreases with increasing temperature.

15.4 Phospholipid functionality and uses in food processing Phospholipid concentrates are applied in many branches of the food or feed industry and for non-edible uses. Therefore, they will be discussed here only

384 Modifying lipids for use in food very briefly. More information may be obtained from the literature listed in Section 15.6.

15.4.1 Use of phospholipids as emulsifiers Lecithins are very good emulsifiers, suitable for water-in-oil emulsions. Margarines are a typical example of their application. Their efficiency depends on the composition of the phospholipid classes, which can be changed by enzymic modifications. More polar modified lecithin, such as lysophospholipids or hydroxylated phospholipids, have higher HLB values and are suitable for specific emulsions. They mainly form micro-emulsions, which are stable even without any stabilizers and even in an acidic medium. Fatty particles, such as oil droplets, are easily solubilized with lecithin and lecithins are, therefore, important in the production of instant foods and infant formulas. However, the relatively high price of phospholipids as food emulsifiers is a limitation, meaning that phospholipids are usually replaced by cheaper and more easily available emulsifiers, such as monoacylglycerols. Another main application is in the bakery industry. Emulsions formed with lecithin are stable even in aqueous ethanolic medium and, therefore, egg yolk is used in the preparation of special liqueurs.

15.4.2 Use of phospholipids to modify viscosity of food dispersions The most important application in this area is the use of lecithin to decrease the viscosity of chocolate coatings so that they become thinner, cutting down on the use of expensive chocolate mass.

15.4.3 Use of phospholipids as inhibitors of lipid oxidation The antioxidant and synergist activity of phospholipids has been known for more than 60 years and is based mainly on the reaction of lipid hydroperoxides with amine groups of phospholipids. They are active as synergists of tocopherols in edible oils (Pokorný et al., 1976a). Their activity was found to be lower at room temperature, but increased at temperatures higher than 80 °C (Dziedzic and Hudson, 1984). Phosphatidylcholine and phosphatidylethanolamine were more active in salmon oil than acidic phospholipids such as phosphatidylserines, phosphatidylinositols or phosphatidylglycerols (King et al., 1992). Trimethylamine oxide produced by action of lipid hydroperoxides with phosphatidylcholines (Fig. 15.7) also has a synergistic activity (Ishikawa et al., 1978). The synergistic activity of phospholipids is lower than that of ascorbyl palmitate, but phospholipids can be added at higher concentrations (of the order of 0.05–0.50 %) as they are relatively cheap (compared with other antioxidants) and considered as generally safe.

Production, separation and modification of phospholipids 385 A—H

A• R2CH—O•

R2CH—OOH

Reaction of lipid hydroperoxide with antioxidant

H2O O

N(CH3)3

HO—N•(CH3)3

R2CH—O•

R2C A•

HO—N•(CH3)3

Fig. 15.7

O

Reaction of lipid alkoxy radical with trimethylamine oxide

A—H O

N(CH3)3

Regeneration of antioxidant

Synergistic mechanism of trimethylamine oxide (A–H = antioxidant).

15.4.4 Use of phospholipids for fortification of foods with nutrients Many food preparations, declared as suitable for healthy nutrition, based on lecithin are available on the market, and are a source of essential fatty acids and of choline, serine or inositol. They are applied as food additives in the form of pills, capsules and emulsions and as additives to various food preparations or beverages. Salts of phosphatidic acids can be used as sources of potassium, magnesium and trace metals.

15.4.5 Application of phospholipids in cosmetic and pharmaceutical products Phospholipids are important for brain function, and both egg and soybean phospholipid concentrates are useful. Their efficiency may be enhanced by suitable modifications, e.g. by increasing the content of phosphatidylserines. They have many other metabolism-related activities so that their field of pharmaceutical applications is very broad. They are used as materials for liposome formation, advantageous for application on the skin. The main field of lecithin application is in cosmetic products, but products containing phospholipids are also important in health care. 15.4.6 Application of phospholipids for feeds and non-edible uses Phospholipid gums, obtained as by-products of crude oil refining, are often of poor sensory quality or functionality so that they are added back to extracted meals and used as feeds, especially for poultry. They thus improve the nutritional value of extracted meals. The literature on non-food uses of lecithins is enormous, concerning different functions and different applications. However, non-edible applications are not included in this chapter. Lecithins may be suitably modified for particular purposes. They are used for coatings, paints, inks, as corrosion inhibitors, leather emulsions, for printing and photocopying, as detergents, additives to

386 Modifying lipids for use in food pesticides, for modification of fuels, textiles and polymers and as wetting and dispersing agents in masonry. More information can be obtained from the sources listed in Section 15.6.

15.5 Future trends Phospholipid concentrates (lecithins) are very important for the food industry, but their only commercial source for food uses is soy lecithin, which is not available on the market in sufficient amount. The industry will thus seek to use phospholipid concentrates from oilseeds other than soybeans. Of course, the concentration of phospholipids in crude oils other than soybean oil is much lower so that their isolation will more expensive. Other crude oil degumming procedures will be developed, which could help in their isolation. Another disadvantage of lecithins other than soybean lecithin is that their phospholipid composition is less advantageous for some applications. Nevertheless, the prospects are not altogether hopeless as different phospholipid classes could be interconverted by enzymic transesterification. Another possibility is to manufacture semi-synthetic phospholipids on the plant scale. Diacylglycerols are converted in the respective phosphatidic acids by reaction with phosphorus pentoxide or concentrated metaphosphoric acid. They are then converted into the respective ammonium salts, which are already available on the market as emulsifiers, cheaper than the natural phospholipids. The procedures could probably be improved by replacing ammonium ions with choline or ethanolamine residues. Modifications of phospholipids using suitable transesterification reactions are prospective tools in the fortification of foods with fish oil polyunsaturated fatty acids and in the fortification of natural phospholipids with phosphatidylcholines or phosphatidylserines. The most important field of phospholipid application is their use as food emulsifiers. Improved emulsifying properties could be obtained by increasing their polarity, and thus their HLB value. Modification by lipolysis, conversion into hydroxy derivatives and reactions with ethylene oxide or propylene oxide are possible.

15.6 Sources of further information and advice Cevc C and Paltauf F (1995), Phospholipids: Characterization, Metabolism, and Novel Biological Applications, Champaign, IL, AOCS Press. Graille J (2003), Lipides et Corps Gras Alimentaires, Paris, Lavoisier, F. Gunstone F D (2004), The Chemistry of Oils and Fats, Boca Raton, FL, CRC Press, Inc. Kumpulainen J T (1999), Natural Antioxidants and Anticarcinogens in Nutrition, Health and Disease, Boca Raton, FL, CRC Press, Inc.

Production, separation and modification of phospholipids 387 Kumpulainen J T (1996), Natural Antioxidants and Food Quality in Atherosclerosis and Cancer Prevention, Boca Raton, FL, CRC Press, Inc. Sikorski Z E and Kolakowska A (2002), Chemical and Functional Properties of Food Lipids, Boca Raton, FL, CRC Press, Inc. Sipos E F and Szuhaj B F (1996), Lecithins, in Hui Y H, Bailey’s Industrial Oil and Fat Products, Vol. 1, 5th edn, New York, Wiley Interscience. Szuhaj B F (1988), Lecithins: Sources, Manufacture, Uses, Champaign, IL, AOCS Press. Zeisel S H and Szuhaj B F (1998), Choline, Phospholipids, Health and Disease, Champaign, IL, AOCS Press.

15.7

Acknowledgement

The book Lipid Glossary 2 by F D Gunstone (2000), Bridgewater, Oily Press, was used as a help for preparing the figures.

15.8

References

ADLERCREUTZ D, BUDDE H

and WEHTJE E (2002), Synthesis of phosphatidylcholine with defined fatty acid in the sn-1 position by lipase-catalyzed esterification and transesterification reaction, Biotechnol Bioeng, 78, 403–411. ADLERCREUTZ P, LYBERG A M and ADLERCREUTZ D (2003), Enzymatic fatty acid exchange in glycerophospholipids, Eur J Lipid Sci Technol, 105, 638–645. ANDERSON C, PINSIRODOM P and PARKIN K L (2002), Hydrolytic selectivity of patatin (lipid acyl hydrolase) from potato tubers toward various lipids, J Food Biochem, 26, 63–74. DOIG S D and DIKS R M M (2003a), Toolbox for exchanging constituent fatty acids in lecithins, Eur J Lipid Sci Technol, 105, 359–367. DOIG S D and DIKS R M M (2003b), Toolbox for modification of the lecithin headgroup, Eur J Lipid Sci Technol, 105, 368–373. DZIEDZIC S Z and HUDSON B J F (1984), Phosphatidylethanolamine as a synergist for primary antioxidants in edible oils, J Am Oil Chem Soc, 61, 1042–1045. ELSNER A and LANGE R (1995), Separation of phospholipids in macro-scale by ion exchange chromatography, in Cevc G and Paltauf F, Phospholipids, Champaign, IL, AOCS Press, 363–373. FARMER L J and MOTTRAM D S (1990), Interaction of lipids in the Maillard reaction between cysteine and ribose: the effect of a triglyceride and three phospholipids on the volatile products, J Sci Food Agric, 53, 505–525. FARMER L J and MOTTRAM D S (1992), Effect of cysteine and ribose on the volatile thermal degradation products of a triglyceride and three phospholipids, J Sci Food Agric, 60, 489–497. GHOSH M and BHATTACHARYYA D K (1997), Soy lecithin-monoester interchange reaction by microbial lipase, J Am Oil Chem Soc, 74, 761–763. HAAS M J, CICHOWICZ D J, JUN W and SCOTT K (1995), The enzymatic hydrolysis of triglyceride– phospholipid mixtures in an organic solvent, J Am Oil Chem Soc, 72, 519–525. HARA F and NAKASHIMA T (1996), Transesterification of phospholipids by acetone-dried cells of a Rhizopus species immobilized on biomass support particles, J Am Oil Chem Soc, 73, 657–659.

388 Modifying lipids for use in food HARA F, NAKASHIMA T

and FUKUDA H (1997), Comparative study of commercially available lipases in hydrolysis reaction of phosphatidylcholine, J Am Oil Chem Soc, 74, 1129– 1132. HARA S, OKADA N, HIBINO H and TOTANI Y (1991), Antioxidative behaviour of phospholipids in polyunsaturated fatty acids of fish oil, Yukagaku, 41, 130–135. HARA S, SONE T and TOTANI Y (2000), Antioxidative activity of nitrogen-containing phospholipids towards emulsified fish oils, J Oleo Sci, 49, 937–943. HARALDSSON G G and THORARENSEN A (1999), Preparation of phospholipids highly enriched with n-3 polyunsaturated fatty acids by lipase, J Am Oil Chem Soc, 76, 1143–1149. HAYES D G (2004), Enzyme-catalyzed modification of oilseed materials to produce ecofriendly products, J Am Oil Chem Soc, 81, 1077–1103. HAYNES L C, LEVINE H, OTTERBURN M S and MATTHEWSON P (1992), Microwave browning composition, Patent, US 5,089,278. HYE S K (1995), Changes of linolenic acid content and reactivity during partial hydrogenation of soybean oil with and without lecithin, Korean J Food Sci Technol, 27, 41–46. ISHIKAWA Y, YUKI E, KATO H and FUJIMAKI M (1978), The mechanism of synergism between tocopherols and trimethylamine oxide in the inhibition of the autoxidation of methyl linoleate, Agric Biol Chem, 42, 711–716. JEONG H O, NAKAGAWA K and MIYAZAWA T (2002), UV analysis of Amadori-glycated phosphatidylethanolamine in foods and biological samples, J Lipid Res, 43, 523–529. JIN H S and MIYAZAWA T (1997), Superior oxidative stability of docosahexaenoic acidenriched oils in the form of phospholipids, J Food Lipids, 4, 109–117. JUDDE A, VILLENEUVE P, ROSSIGNOL-CASTERA A and LEGUILLOU A (2003), Antioxidant effect of soy lecithins on vegetable oil stability and their synergism with tocopherols, J Am Oil Chem Soc, 80, 1209–1215. KAJIMOTO G, YOSHIDA H and SHIBAHARA A (1987), Effect of soy lecithin with different degrees of hydrogenation on the coloring and decomposition of tocopherol in heated oils, Nihon Eiyo Shokuryo Gakkai-shi, 40, 497–504. KAMATA M, HARA S and TOTANI Y (1996), Hydrolysis of soybean phospholipids by phospholipase C from Bacillus sp. J Jap Oil Chem Soc, 45, 1255–1259. KING M F, BOYD L C and SHELDON B W (1992), Antioxidant properties of individual phospholipids in a salmon oil model system, J Am Oil Chem Soc, 69, 545–551. MORGADO M A P, CABRAL J M S and PRAZERES D M F (1995), Hydrolysis of lecithin by phospholipase A2 in mixed reversed micelles of lecithin and sodium dioctyl succinate, J Chem Technol Biotechnol, 63, 181–189. MORGADO M A P, CABRAL J M S and PRAZERES D M F (1996), Phospholipase A2-catalyzed hydrolysis of lecithin in a continuous reversed-micellar membrane bioreactor, J Am Oil Chem Soc, 73, 337–346. MOTTRAM D S and WHITFIELD F B (1995), Maillard-lipid interactions in nonaqueous systems: volatiles from the reaction of cysteine and ribose with phosphatidylcholine, J Agric Food Chem, 43, 1302–1306. MUSTRANTA A, SUORTTI T and POUTANEN K (1994), Transesterification of phospholipids in different reaction conditions, J Am Oil Chem Soc, 71, 1415–1419. MUSTRANTA A, FORSELL P and POUTANEN K (1995), Comparison of lipases and phospholipases in the hydrolysis of phospholipids, Process Biochem, 30, 393–401. NA A , ERIKSSON O , ERIKSSON S G, ÖSTERBERG E and HOLMBERG K (1990), Synthesis of phosphatidylcholine with (n-3) fatty acids by lysophospholipase A2 in microemulsion, J Am Oil Chem Soc, 67, 766–770. PAHN S C (1997), Hydrolysis of phosphatidylcholine in aerosol-oil-isooctane reversed micelles by phospholipase A2, Korean J Food Sci Technol, 29, 26–31. PARK C W, KWON S J, HAN J J and RHEE J S (2000), Transesterification of phosphatidylcholine with eicosapentaenoic acid ethyl ester using phospholipase A2 in organic solvent, Biotechnol Lett, 22, 147–150. PHAN-TRONG T, POKORNY ´ J and JANÍŐEK G (1974), Kinetics of the oxidative browning of phosphatidylethanolamine, Z Lebensm Unters-Forsch, 156, 257–262.

Production, separation and modification of phospholipids 389 and KUMMEROW F A (1991), Thiobarbituric acid reactive substance formation as affected by distribution of polyenoic fatty acids in individual phospholipids, J Agric Food Chem, 39, 451–457. POKORNY ´ J, NGUYEN T L, SVOBODOVÁ H and JANÍŐEK G (1976a), Antioxidative effect of soybean phospholipids, Nahrung, 20, K 3–4. POKORNY ´ J, PHAN-TRONG T, HEGEDÜšOVÁ B and JANÍŐEK G (1976b), Bleaching of oxidized phosphatidylethanolamine with peroxides, Sb VSCHT Praze E, 46, 17–34. POKORNY ´ J, PHAN-TRONG T and JANÍŐEK G (1973), Autoxidation and browning reactions of phosphatidylethanolamine, Z Lebensm Unters-Forsch, 153, 322–325. POKORNY ´ J, ZWAIN H, HEGEDÜšOVÁ B, NGUYEN-THIEN L and JANÍŐEK G (1975), Reaction of phospholipids with 1,4-benzoquinone, Z Lebensm Unters-Forsch, 159, 287–292. PRUTHI T D and KUMAR S (1991), Effect of browning on recovery of phospholipids from clarified butter fat (ghee) by counter current distribution technique, Indian J Dairy Sci, 44, 532–534. RICH J O and KHMELNITSKY Y L (2001), Phospholipase D-catalyzed transphosphatidylation in anhydrous organic solvents, Biotechnol Bioeng, 72, 374–377. SATYANARAYANA RAO T S (1991), Changes in phospholipid browning of hens’ whole egg powder packed in different packing materials, J Food Sci Technol India, 28, 120–122. SMITH G and HOLE M (1991), Browning of salted sun-dried fish, J Sci Food Agric, 55, 291– 301. SONO R, BAN N, SAKAMOTO S, HAMAGUCHI N, TEBAYASHI S, KIM C, KOH H and HORLIKE M (2001), Heat deterioration of phospholipids. I. Decomposition of soybean lecithin and formation of new products by heating, J Oleo-Sci, 50, 905–911. ŠTRUCELJ D, MOKROVŐAK Ž, RADE D and STASTNY M (1995), Alcohol fractionation of soybean phospholipids, in Cevc G and Paltauf F, Phospholipids, Champaign, IL, AOCS Press, 248–356. SUGINO H, ISHIKAWA M, NITODA T, KOKETSU M, RAJ-JUNEJA L, KIN M and YAMAMOTO T (1997), Antioxidant activity of egg yolk phospholipids, J Agric Food Chem, 45, 551–554. SURLYAPHAN O, CADWALLADER K R and DRAKE M A (2001a), Lecithin associated off-aromas in fermented milk, J Food Sci, 66, 517–523. SURLYAPHAN O, DRAKE M A and CADWALLADER K R (2001b), Lipid oxidation of deoiled lecithin by lactic acid bacteria, Lebensm Wiss Technol, 34, 462–468. SVENSSON I, ADLERCREUTZ P and MATIASSON B (1992), Lipase-catalysed transesterification of phosphatidylcholine at controlled water activity, J Am Oil Chem Soc, 69, 986–991. SZUKALSKA E (2000), Effect of phospholipid structure on kinetics and chemistry of soybean oil hydrogenation with nickel catalyst, Eur J Lipid Sci Technol, 102, 739–745. TOTANI Y and HARA S (1991), Preparation of polyunsaturated phospholipids by lipasecatalyzed transesterification, J Am Oil Chem Soc, 68, 848–851. UTZMANN C M and LEDERER M O (2000), Identification and quantification of aminophospholipidlinked Maillard compounds in model systems and egg yolk products, J Agric Food Chem, 48, 1000–1008. VIJEETA T, REDDY J R C, RAO B V S K, KARUNA M S L and PRASA R B N (2004), Phospholipasemediated preparation of 1-ricinoleoyl-2-acyl-sn-glycero-3-phosphocholine from soya and egg phosphatidylcholine, Biotechnol Letts, 26, 1077–1080. WANG Y, WANG X, OU S, TANG S, FU L and LI A (2003), Hydrolysis of soy lecithin by phospholipase A2, J Chinese Cereals Oils Assoc, 18, 33–37, 49–51. WEETE J D (1995), Studies on the thermalization of lecithin, in Cevc G and Paltauf F, Phospholipids, Champaign, IL, AOCS Press, 357–362. WEIDNER E, ZHANG Z, CZECH B and PETER S (1993), Entölung von Rohlecithin mit Propan, Fat Sci Technol, 95, 347–351. WILTON D C (2005), Phospholipases A 2: structure and function, Eur J Lipid Sci Technol, 107, 193–205. WU Y and WANG T (2003), Soybean lecithin fractionation and functionality, J Am Oil Chem Soc, 80, 319–326. PIKUL J

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Part III Applications of modified lipids in food

16 Lipid emulsifiers and surfactants in dairy and bakery products H. M. Premlal Ranjith and U. Wijewardene, Diotte Consulting and Technology Limited, UK

16.1

Introduction

Food production today is a major industry that has been developed and mechanized over many years to meet consumer demand. Consumer preference and convenience are partly responsible for technological development of the methods used for food production. Milk originates in the basal regions of the mammary epithelial cells. The milk secretion process, the formation of lipid globules and the release of these into the milk, has been widely studied. The mammary epithelium of the udder cells of mammals undergoes remarkable changes to secrete the white biological fluid termed milk. Man has long utilized this wholesome food and has developed techniques to produce specific food commodities using some of its major components such as fat. Milks from other mammals (sheep, goat, camel, mares and yak) have also been utilized in many parts of the world as a major part of the food supply. Fat has been used for food applications for many years, especially in the baking industry. For example, lard, suet and oil have been widely used in home baking and other domestic cooking as well as in cottage industries prior to mechanization and technological development. Some of these developments include the refining of oil for palatability and use of vegetable oils to produce table spreads as a low-cost alternative for butter. An important technological development was the understanding of the structure and crystalline properties of lipids and their modified forms to achieve desired characteristics in the end products. The result of this development is apparent in baked products such as pastry, cakes and puff pastry. Understanding lipids as an ingredient helped to promote milk lipids as a source of consistent and distinctive organoleptic quality in baked products.

394 Modifying lipids for use in food These changes were brought about in the developed countries through consumer demand for nutritive value, convenience in use, shelf life and organoleptic quality. In developing countries the demand for modifications and improvements in lipid technology was met by adapting the available modern technology to achieve high levels of production output for a variety of food products. Synthetic emulsifiers entered the food chain in France in the late 19th century where in 1869 margarine was developed using tallow. As new formulations were developed using emulsions, various items of legislation were implemented in individual countries to limit their use in food products for consumer protection. Today a large amount of food emulsifiers and stabilizers are produced and distributed worldwide to meet the demands of the food industry and the consumer.

16.2 Food emulsions Food emulsions exist in two basic forms; the oil in water type (O/W) and the water in oil type (W/O). Other types of multi-phase composite systems also exist as a mixture of the two. Milk is a water-continuous emulsion where lipid component is dispersed as fat globules in the aqueous phase (water, protein, mineral, etc.). This is because the complex fat globule membrane has emulsifying components that hold the two immiscible phases together. Butter and creams are good examples of emulsions based on natural emulsifiers in the products.

16.2.1 Milk fat globule A unique difference between milk fat and vegetable fat is that the milk fat exists in globular form contained within a complex membrane. This membrane protects the fat inside the globule from chemical and lipolytic action by chemicals and enzymes present in the aqueous phase. The membranes also extend protection against mechanical damage during handling in the farm as well as the dairy and prevent the globules from flocculation and coalescence. Controversy exists regarding the mechanism by which each globule acquires the membrane materials prior to exiting from the cell. However, the globule membrane is a unique feature of milk lipid and its presence contributes towards specific product characteristics. The milk fat globule membrane is estimated to be about 5–10 nm thick (Mulder and Walstra, 1974). Scientists describe the fat globule as having two basic layers with an inner core of fat surrounded by an emulsifier system with a hydrophobic tail linked to fat and the hydrophilic head attached to the aqueous layer containing proteins and other components. Therefore, the fat globule membrane surface is hydrophilic and it maintains the fat in an emulsified state. Figure 16.1 shows a diagrammatic layout of milk fat globule

Lipid emulsifiers and surfactants in dairy and bakery products

Phospholipids Fat globule membrane

395

Water Proteins Phospholipids Minerals Enzymes

Water Crystallized fat

Liquid fat

Proteins

Fig. 16.1

Milk fat globule components.

components to highlight the key layers. The inner layers are triacylglycerols surrounded by the membrane. Chemical components such as lecithin hold the lipids and the aqueous material together. Other components such as casein units organize themselves round the aqueous layer to form the outermost layer. This biological membrane is formed during the milk secretion process in the mammary epithelium of the cow’s udder cells. Major constituents of the globule membrane are proteins, phospholipids and glycerol esters. Mulder and Walstra (1974) have given a detailed account of the composition of milk fat globule including that of the globule membrane. In addition to the lipid components, in the inner cavity of the globule there are fat-soluble vitamins such as A, D, E and K. The colour pigment in the milk lipids derives mainly from carotene (vitamin A), principally β-carotenes. The intensity of the colour pigment depends on the concentration levels. For example Jersey cows produce high-fat milk (5–7 % w/w) compared to Friesian (3–4.2 % w/w) and Jersey milk has a high concentration of the carotene pigment. In contrast buffalo milk contains about 6–7 % w/w) fat but the carotene (pigment material) is very low, causing the lipid fraction to be offwhite. Other factors that affect the colour are breed type, season and feed material. In most species the fat content varies throughout milking or suckling, generally increasing as the gland is emptied. Apart from this, the fat content in all species is influenced by the stage of lactation, age and breed type of the

396 Modifying lipids for use in food cow and diet. The fat is dispersed as minute globules ranging in diameter from 0.1 to 20 µm (Walstra, 1969). It is estimated that the mean fat globule diameter for cow’s milk is about 3 µm and that for goat’s milk is about 2 µm. In different breeds the size of the globules tends to vary with the fat content of the milk. Therefore, the mean for Holstein milk is 2.5 µm and that for Channel Island cattle is 3.5 µm (Ling et al., 1961). However, it was found that Ayrshires are an exception. Their milk has higher fat content than Holstein, but the size of the globules is the same. As lactation advances the fat globules tend to become smaller. The long-chain fatty acids of the membrane phospholipid are buried in the fat and the hydrophilic part of the molecule is directed outwards. It is likely that the carotenoids, vitamin A and cholesterol associated with the membrane are in the phospholipid layer. The characteristic nature of the globule surface is such that at normal temperatures (8–20 °C) the globules are grouped together in the form of clusters, which play an important part in the rising of cream. Milk fat begins to melt at about 28 °C but is not completely liquid until the temperature has reached 33 °C. As secreted, the fat globules are, therefore, liquid, but when milk is cooled the crystallization commences and this may take up to 24 hours to reach completion. The reasons for this lengthy period could be the crystallization process and that because the liquid fat is in the dispersed phase, there is no opportunity for the hastening effect of ‘seeding’ which usually operates when the melt is in a continuous phase. Like other fats, milk fat is soluble in fat solvents such as petroleum and ether, but it is not possible to extract milk fat by merely shaking it with these solvents due to the protection from the fat globule membrane. In quantitative solvent extraction the fat globule membrane is first removed by the action of acid or alkali. The membrane materials are constantly changing to keep the equilibrium with the components in the milk’s aqueous phase. This means that some membrane material may leave and join the aqueous phase and vice versa. Therefore, the thickness of the membrane is variable. Such clear demarcation of layers in the fat globule structure indicates that boundaries can be defined based on the components associated with each layer. The outer boundary is associated with the components outside the fat globule and belongs to membrane materials. The inner boundary is the triacylglycerols in the core of the globule and has links with the emulsifying materials at the outer periphery. The membrane is disrupted when subjected to homogenization during processing due to size reduction of fat globules, but it undergoes reformation immediately.

16.2.2 Milk lipid composition In milk fat the main components are triacylglycerols which are esters of glycerol and fatty acids found in plant and animal tissues (Fig. 16.2). They

Lipid emulsifiers and surfactants in dairy and bakery products CH

OH

R

COO

CH2

CH

OH

R′

COO

CH2

CH2

OH

R″

COO

CH2

Glycerol (propane-1, 2, 3-triol)

Fig. 16.2

397

Triacylglycerol (fat/oil)

A diagrammatic illustration of a glycerol and milk triacylglycerol molecule.

are essential to living matter and are stored in body tissues from which they can be made available for metabolic activities. Animals can synthesize most (but not all) fatty acids but, due to rumen bacterial biohydrogenation, some get transformed to trans fatty acids. Lipids are insoluble or only sparingly soluble in water but soluble in most organic solvents. Hydrolysis of the triacylglycerols produces fatty acids and glycerol. Fatty acids are monobasic and have the general un-ionized formula RCOOH. The main fatty acids in milk are given in Table 16.1. Milk lipid is a mixture of different fatty acids and glycerol as indicated in Fig. 16.2. The presence of a variety of fatty acids linked to glycerol gives rise to numerous permutations of triacylglycerols. The most abundant fatty acids are myristic (14:0), palmitic (16:0), stearic (18:0) and oleic (18:1) of which only oleic is liquid at room temperature. Other fatty acids that are liquid at room temperature are butyric, caproic, caprylic, linoleic, linolenic and arachidonic. The relative proportions of these fatty acids present in the glycerol esters can vary and that affects the melting point as well as the crystallization behaviour. For example, lipids with high-melting fatty acids such as palmitic acid will have a hard texture while a lipid containing oleic acid would give rise to a softer texture. Table 16.1 Fatty acid

Butyric acid Caproic Caprylic Capric Lauric Myristic Palmitic Stearic Oleic Linoleic Linolenic Arachidonic

Fatty acids in milk. % of total fatty acid Summer

Winter

9.60 4.50 2.20 4.20 4.10 11.50 2.90 27.60 10.10 17.80 1.40 0.80

12.00 4.50 2.30 4.20 4.00 10.80 2.20 22.00 13.10 21.50 0.70 0.30

398 Modifying lipids for use in food Fats and fatty acids exhibit polymorphism. This means that a solid fatty acid or triacylglycerol can exist in more than one structural form, each of which differs in stability and in melting point. The three common polymorphs in lipids have been represented by the Greek symbols α, β′ and β (Timms, 1994). The least stable form (α) has the lowest melting and β is the most stable with the highest melting point. The β′ form is intermediate in stability and in melting point. The crystallization and subsequent transformation of the glycerol esters takes place in increasing order of stability from α to β′ to β. Ranjith and Rajah (2001) reported the relative melting points for polymorphic forms of milk fat during butter making as 17–22 °C (the α type formed by rapidly cooling to 5 °C), 28–31 °C (the β′ form produced when cooled to 15– 18 °C) and 35–38 °C for the β form (when cooled to ≤ 21 °C). 16.2.3 Crystal formation and growth The starting point of crystallization of lipid is the formation of a nucleus during cooling of a liquid lipid phase. Nucleation involves energy in two ways: first heat of crystallization which encourages the process; and second the need for energy to overcome surface tension during crystal growth. The nucleation of high-melting triacylglycerols in crystal formation can be initiated by ‘active impurities’ such as dust and material on the container surface (Van den Tempel, 1968). The small crystals so formed then act as ‘active sites’ for other triacylglycerol molecules to be incorporated to an acceptable site. In milk lipid the presence of very similar triacylglycerols enables one to act as nucleus for other types. The crystal nucleus then starts to grow further by the incorporation of other triacylglycerol molecules taken from the adjacent surrounding liquid layers. The crystallization behaviour of milk fat can be altered by variations in processing conditions such as cooling rate, method and rate of agitation and cooling temperature. The crystal formation and growth due to process conditions adapted has a direct effect on the final size and shape of milk fat crystals (Vanhoutte et al., 2003). This means that the size and shape of milk lipid crystals vary considerably, based on the rate of crystallization (Kaylegian and Lindsay, 1995). Numerous small crystals of needle or platelet shape form if a rapid cooling method is adapted, and the crystal size ranges from about 0.1 to 3 µm in diameter (de Man, 1961; Muldor and Walstra, 1974; Mortensen, 1983; Foley and Brady, 1984). Slow cooling of milk fat causes few large crystals to form with diameters up to 40 µm. 16.2.4 Emulsion structure Prior to defining an emulsion it is important to be aware of descriptions of other similar systems such as solutions, colloidal solutions, suspensions and foams. A genuine solution is the simplest of all physico-chemical states, whether it is solid, liquid or gaseous. A simple example of a genuine solution

Lipid emulsifiers and surfactants in dairy and bakery products

399

is when sugar is dissolved in water. The particle size is less than 0.001 µm. In a colloidal solution particle size varies from about 0.001 to 0.1 µm. The complex calcium salts in milk exist in colloidal form together with albumin and most of the casein. There is very little difference between an emulsion and a suspension except that the particles of an emulsion are liquid, whilst those of the suspension are solid. In both systems the particle size is greater than 0.1 µm and could be as large as 1 mm. The fat in milk exists in small globular form as suspended particles. Foam can be considered to be a suspension where the size of the air cells in the foam is about 30–100 µm. An emulsion is defined as a mixture formed by combining two immiscible fluids in which one is uniformly distributed in the other without separation. Particle size ranges from about 0.1 µm to a visible size. In food emulsions the two main components are the oil and the water with other components dispersed either in the continuous or in the dispersed phase. As described before, these systems are either O/W or W/O and the semi-solid and solids could exist through crystallization as in ice cream or butter. Figure 16.3 shows a diagrammatic illustration of the basic structure of O/W and W/O emulsions. In milk the fat (approximately 4 %) is dispersed in an aqueous phase containing protein. High-fat emulsions such as creams also contain protein in the aqueous phase but to a lesser extent. Milk, creams and ice cream also differ from emulsions formed by mixing vegetable oil and water in that the milk fat structure is unique because the fat exists in a membranesurrounded globule. The aqueous phase may be described as a colloidal phase due to the presence of proteins, salts, carbohydrate, micro-components and other aqueous phase materials. Inside the cow udder, milk is a stable emulsion where the dispersed phase (fat) is uniformly distributed without separation. However, once the milk exits the udder the tendency is for the fat globules to rise because of the difference in density between fat globules and aqueous phase. This separation Water in oil

Oil in water

Oil Oil

Water

Water

(a)

(b) Hydrophilic head Lipophilic tail

Fig. 16.3

The role of emulsifiers in the formation of (a) oil in water and (b) water in oil emulsification.

400 Modifying lipids for use in food is also referred to as creaming, and milk and cream storage silos and tanks are kept in gentle agitation at regular intervals to minimize it. This natural separation of the fat phase in milk was used in earlier times to prepare highfat milk products.

16.2.5 Microbiological quality of raw milk Cow’s milk is a perfect food designed for calves and an almost perfect food for humans. Unfortunately, it is also a good source of food for micro-organisms. Milk from the udder of a healthy cow contains very few organisms (not more than about 300/ml), and these are of no danger to the consumer. Milk is contaminated generally by post-production handling methods including milking equipment and general hygiene of operatives. The keeping quality of raw milk is mostly determined by the initial number of micro-organisms present in the milk and by the temperature at which it is retained after production. Hygienic milk production has advanced in most countries and in the EU the standards are defined for the maximum allowable total viable counts per ml of raw cow’s milk. Raw milk viable counts are an important factor in modern dairy processing as the time interval between milk production and processing has increased. Other factors, such as alternate day collection and changes to factory operation methods, further extend this time interval. Delay in processing means that psychotropic bacteria can proliferate. Psychotropic spores in large numbers can undermine a heat treatment regime as microbial inactivation follows first order reaction kinetics. For example, if the heat treatment regime were capable of achieving four decimal reductions, an initial count of 105 cfu (colony forming units) per ml would leave 10 cfu per ml after processing. This is the scenario with heat-resistant organisms but not with the vegetative pathogens. Thermodurics are defined as those which survive 63 °C for 30 minutes, whereas endospores can survive 80 °C for 10 minutes (Lewis, 1999). Therefore, raw milk quality may vary depending on general production hygiene, equipment used, environment and organism population and type.

16.3 Lipid modification and processing Ancient writing refers to the use of butter in food preparation in India between 2000 BC and 1400 BC (McDowall, 1953). It was also reported (1480) that in Italy cream was recovered by skimming and converted to a butter-like product. Therefore, historically the effect of variation in density has been used to collect the cream and manufacture dairy products from fats in milk. This also included fat from other mammals than cow. Similarly, a denser material (such as protein) in milk will eventually settle to the bottom of the container holding the milk.

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16.3.1 Water-continuous emulsions The cooled stored raw milk is either filtered or clarified prior to processing and separation. A clarifier is used in almost all modern, medium to large dairy installations. A clarifier works on the same principle as the centrifugal separator. Centrifugal force is generated by rotational movement of material. The magnitude of the centrifugal force is dependent on the radius and speed of rotation and the density of the materials being rotated. Milk produced under good hygiene conditions will be substantially free from foreign matter as it passes through a filtering system in the farm. However, in all dairies milk passes through a filtering system (filter cloth, etc.) or a centrifugal clarifier. Clarification is conducted normally in the cold and the milk is not split into cream and skimmed milk, but heavier foreign bodies from milk are removed using centrifugal force. The insoluble matter is thrown to the rotating bowl from which it is discharged at regular intervals. Control of fat content in cream In early times the cream recovered from milk was used mainly for the manufacture of butter-type products. The development of the dairy separator provided a new opportunity to manufacture products based on high fat, the creams, as well as low-fat and ‘no-fat’ milks (semi-skimmed and skimmed milk). Figure 16.4 illustrates milk separation and cream standardization using a dairy separator. The method of preparation of creams included either using the skim fraction or whole milk to dilute the cream to obtain the desirable fat content of the final product. In these techniques, the fat content of each fraction must be analyzed in order to formulate the appropriate combination required. The dairy industry regularly standardizes creams and milks, and in order to accurately adjust the fat content the Pearson’s rectangle is used for calculations. In high-throughput separators with an in-line densitometer the fat adjustment is effected automatically (Fig. 16.4). The basic requirement is to use flow meters, densitometers and control valves to adjust the fat content by mixing the correct proportion of skim milk and cream. The actual fat content of the streams is continuously monitored by the densitometer and the information communicated back to the control unit for comparison with the pre-set parameters corresponding to the required fat content in the cream. The density transmitter is capable of detecting small changes in the density of cream when the fat content changes. Increase in fat percentage in cream results in its density being lower and vice versa. This means that a relationship exists between fat % and density; that is the fat content in cream varies inversely with density. Table 16.2 lists the categories of cream in use in the UK. The efficiency of separation is reflected in the quality of skimmed milk obtained and the free fat in the cream. Efficient separation produces skimmed milk with less than 0.05 % fat and a low level free fat in the cream. The free fat level increases due to damage caused to the fat globule membrane from mechanical and shear action in the circuit and pumps. Milk is normally

402 Modifying lipids for use in food Warm milk from heat exchanger Double centripetal pump unit

Cream

1

Skimmed milk

2

Separator 3 Creams 3 4 3 2 Standardized milk

3

Skimmed milk

1 2 3 4

Density transmitter Flow transmitter Flow control valve Isolating valve

Fig. 16.4 Milk separation and standardization circuit. Table 16.2

Cream categories in the UK.

Type

Minimum fat content (g 100 g–1)

Half cream Single cream Whipping cream Double cream Sterilized cream Clotted cream

12 18 35 48 23 55

warmed to at least 40 °C prior to separation, and the feed to the separator should be adjusted to ensure that it satisfies the designed specification by the manufacturer. Lower than optimum feed rate into the separator causes the fat fraction to stay in the separator longer than necessary, causing further damage to the fat globule membrane due to shear. Once the globule membrane is damaged, it allows the free fat to escape into the aqueous phase. High percentage of free fat leads to high free fatty acids (FFA) in cream resulting from lipolysis by indigenous lipase enzyme. Not all FFA are formed by this method; some are formed due to poor quality milk as well as poor handling practices. In poor quality milk, high concentration of the microbial enzyme lipase is available to hydrolyze the triacylglycerols and increase the FFA. Therefore, it is important to reduce the FFA in the raw milk originating from poor microbiological quality as well as due to handling methods.

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Recombined creams The formulations and procedure for recombined creams have been known to the food and dairy industries for quite some time, and Buchanan and Smith (1966), Zadow and Kieseker (1975) and Towler and Stevenson (1988) studied the manufacture and properties of these products. Originally, butter oil, skimmed milk powder and water were used to manufacture such products. However, the resulting cream had a very poor over-run and no foaming at all. In addition, such high-fat recombined cream should be pourable as well as thick enough to use as a dessert cream. In formulating a recombined cream, various ingredients are selected so that they enhance the desirable qualities and are cost-effective. Recombined creams are popular in countries where real cream is in short supply or not available, and they are popular in bakery applications. For bakery applications, high-fat products were specially formulated using stabilizer/emulsifier systems to minimize serum drain and improve foam quality. Some details of emulsifiers and stabilizers are given in Table 16.3. Emulsifiers such as distilled monoacylglycerols are found to produce acceptable overrun and lecithin induces the desirable stiffness to the foam. Zadow and Kieseker (1975) reported that with anhydrous milk fat, nonfat milk solids and an emulsifier such as glycerol monostearate good quality whipping cream (35 % fat) was obtained. A two-stage homogenization step was used at pressures between 1.4 and 2.1 MPa at the first stage and 0.7 MPa at the second stage. The optimum temperature for homogenization was found to be about 48 °C. In the formulation 0.1 % of the emulsifier was used and the product could withstand process conditions used in the UHT treatment. The steps involved in the preparation of recombined cream are: • •

dissolve non-fat milk solids and emulsifiers in water at about 40–50 °C using a high-speed mixer; melt fat (e.g., anhydrous milk fat, butter, etc.) at about 40–45 °C and add slowly to the liquid mix;

Table 16.3 HLB value

Common emulsifiers and their hydrophilic lipophilic balance (HLB) values. Application

Emulsifiers

3–6

W/O emulsion

8–14

O/W emulsions

Monoglycerides Glycerol lactopalmitate Propylene glycol monostearate Sorbitan esters Diacetyltartaric acid esters Polyoxyethylene sorbitan esters Sucrose esters Decaglycerol distearate Soaps Lecithin Decaglycerol monolaurate

14–18

Detergents

404 Modifying lipids for use in food • •

continue mixing to ensure that a homogeneous mix free from lumps and oil droplets is formed; heat treat and cool (i.e. pasteurization, UHT) – see Section 16.3.3.

16.3.2 Lipid-continuous emulsions Milk lipid is one of the most expensive sources of fat used in the food industry, but it is also the only major fat used for its desirable flavour. Use of milk fat is regulated in almost all major milk producing countries to prevent food manufacturers from taking unfair advantage by exploiting its intrinsic flavour and marketability. Historically butter and margarine dominated the lipid-continuous emulsions with butter at the luxury end and margarine becoming the alternative substitute. In recent years, due to technological advances as well as changes in lifestyles, a wide range of table spreads with varying fat content has entered the market. These include reduced fat, lowfat and very low-fat spreads based on milk fat, non-dairy fat or a mixture of both where milk fat is in the range 15–85 % in the fat phase. Complex emulsifiers and stabilizer systems have been used in these products in order to achieve the optimum organoleptic quality. Food emulsifiers Food emulsifiers should be described in terms of the properties required for specific applications. They promote the emulsification of oil and aqueous phase; they have the ability to complex starch, interact with proteins and modify fat crystallization and the viscosity characteristics of food ingredients; and they enable control of both foaming and anti-foaming effects, dispersion of solids in water and lubrication. This shows that, in addition to basic emulsification, emulsifiers have many other functions. These functional properties have been the key factors in determining their suitability for various applications. The use of an emulsifier increases the ease of formation and promotes the stability of emulsions by reducing the amount of work required to form a homogeneous mixture of two normally immiscible layers (e.g. oil and water) through reduction of interfacial tension between the phases. This function is possible when the molecule is amphiphilic, having both hydrophilic (water-soluble) and lipophilic (oil-soluble) groups. The emulsifier can then partially dissolve in both the phases and thus unite them in the form of a homogeneous emulsion (Fig. 16.3). Hydrophilic–lipophilic balance (HLB) HLB represents the oil and water solubility of an emulsifier and is used to classify emulsifiers. The emulsifiers are also described as amphiphilic. The balance between the hydrophobic and hydrophilic properties of the molecules determines the performance of an emulsifying agent, for instance the type of emulsion formed. The hydrophilic portion of an emulsifier could originate from a variety of groups, for example the ionized forms of SO4Na, COONa,

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and COOK. The lipophilic groups are typically saturated or unsaturated alkyl chains. The HLB number is usually on a scale of 0–20. Lower HLB values are an indication of high oil affinity. A high HLB value, on the other hand, indicates high water-solubility. The method to calculate the HLB value is given by Stauffer (2002) and Ranjith (2002). Table 16.3 indicates the HLB values of common emulsifiers. As the HLB value increases, the emulsifiers become more soluble in water and their function changes from being W/O emulsifiers to being O/W emulsifiers. HLB values are useful for selecting the most appropriate type of emulsifiers for food application. However, they give a one-dimensional description of the properties and those influenced by molecular weight and temperature are not considered. For example, it is difficult to calculate the HLB value for phospholipids, which form an important group of food emulsifiers. Butter Butter has been manufactured since ancient times and internationally traded since the 14th century. As for all dairy production, the handling and processing of raw milk is important if a good quality product is to be achieved. In the case of butter, extra care must be taken to prevent unnecessary damage to fat globules in milk and subsequently in cream. Both ripened and sweet creams are stored in refrigerated tanks below 5 °C (preferably 2–3 °C) for 18–36 hours before churning. During storage, fat crystallization takes place and at the same time the fat crystals grow. Fat crystallization is exothermic causing the cream temperature to rise so adequate cooling systems and cream agitation should be provided to absorb this heat. Cream treatment for functional properties Milk composition changes due to variation in • • • • •

animal species; lactation time and number; seasonal changes; feeding materials; health of the cow.

A well-known significant change in milk lipids is that the summer milk contains softer fat compared to the winter milk leading to softer and harder butters, respectively. Figure 16.5 shows the solid fat content (SFC) in summer and winter milks. Milk fat contains a large proportion of triacylglycerols (98 % w/w) and contains about 400 fatty acids of which only 15 are of great significance. As indicated before, various factors cause fatty acids present in milk to change. A large quantity of fresh pasture is consumed from spring to autumn (usually April to September but March to October in some regions), leading to an increase in unsaturated fatty acids (for example 18:1) causing the fat to be

406 Modifying lipids for use in food

70 60 50 Solid fat 40 content 30 (%) 20 10 0 5

10

15

20

25 Temperature (°C)

Fig 16.5

30 35

Solid fat content (SFC) of summer (unshaded area) and winter (shaded area) milk fat in the UK.

relatively soft. Other fatty acids are particularly associated with the unique flavour of butter, for example butyric acid (4:0). Butter manufacture Butter is an expensive commodity and its price remained unchanged even after large-scale production equipment was introduced. Both butter and margarine have a minimum fat content of 80 %, and for many diet-conscious consumers this fat level is too high irrespective of the nature of the fats. Butter manufacture involves four main processes. 1 2 3 4

concentration of the fat phase of milk or separation of cream; crystallization of the fat phase of milk; phase inversion of the oil in water emulsion; formation of a solidified water in oil emulsion.

In conventional butter manufacturing the above four processes are followed in that order, but it differs in some continuous butter making processes. Typically cream is concentrated to a fat content of about 38 % and about 42 % for batch and continuous butter making respectively. It is recommended to use hermetic separation to prevent aeration. These concentrations help to reduce the consumption of energy, and a relatively high separation temperature of 50–55 °C is used to reduce the problems arising from enzyme activities such as lipolysis. Cream is heat-treated using a plate heat exchanger to minimize damage to fat globules and to destroy vegetative micro-organisms. In high-temperature treatment, sulphydryl groups, for example from cysteine, are produced. These act as antioxidants and reduce lipid oxidation. The most common temperature range used for heat treatment is 85–95 °C for 10–30 seconds, but sometimes it can be 85–112 °C. Further increase of temperature may cause quality defects resulting from excess sulphydryl groups.

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Crystallization of the fat phase Crystallization of the fat phase occurs during ageing and ripening. Many physical properties of the butter are determined at this stage as a consequence of the ratio of solid to liquid fat and the size and shape of the crystals. Large numbers of very small fat crystals are required in butter and other yellow fats, and crystallization can be achieved by rapid cooling of the cream to 3– 7 °C. The tempering is used to increase the relative quantity of liquid fat and thereby improve the spreadability and consistency of the butter. Tempering is developed from the Alnarp process, and involves the cold–warm–cold cycles. Cream is warmed from 6–8 °C to 14–21 °C and then cooled again to 8–13 °C. The optimum temperature at each stage depends on the triacylglycerol content and the melting and solidification properties of the cream. Cream ripening (for ripened or cultured butter) Cream is ripened using a culture with a mix starter, which usually consists of Lactococcus lactis ssp. lactis or Lactococcus lactis ssp. cremoris in combination with Lactococcus lactis ssp. diacetylactis or Leuconostoc mesenteroides ssp. cremoris. Ripening involves incubation at 20 °C, until the desired pH value or diacetyl level has been reached and then cooling to below 10 °C. At this point fat crystallization commences. The Alnarp process is used to facilitate cooling of the viscous ripened cream and to produce butter of the desired texture. Ageing and culturing of cream is carried out in vertical silos fitted with agitators to ensure proper mixing and jackets to control the temperature. The consistency of cream is important in continuous butter making. The important key factors are pH and fat content and physical properties such as viscosity, fat crystallization and temperature. The optimum temperature is obtained by plate heat exchangers operated with a low pressure drop and a temperature differential of only 1–2 °C between cream and the hot water heating medium to minimize burning-on. In conventional batch butter making, the churning temperature is not dependent on the fat content of the cream, but is modified by the hardness of the butterfat, pH value of the cream and the size and design of the churn. Generally the churning temperature is in the range 5–7 °C. Churning and working Churning and working are related stages of butter making. The oil in water (O/W) emulsion is broken followed by an inversion of phase to water in oil (W/O) emulsion. These processes are done in the butter churn in conventional batch production or in a continuous butter make. In a batch churn, a large mass of cream is destabilized relatively slowly by rotating the churn so that cream is lifted up the ascending wall and splashed down to the base. This induces shear action on fat globules causing disruption of the globule membrane and release of fat.

408 Modifying lipids for use in food There are two main types of continuous production: • •

accelerated churning (Fritz-type) and phase inversion churning high fat (> 75 %) cream and phase inversion

There are other small variations adapted to the handling and processing of high-fat cream in the second method; some start as O/W emulsion (New Way and Alfa method); and there are others (Cherry-Burrell, CreameryPackage and Senn) where the cream is partially oiled off before separation to allow the production of a butterfat–serum mixture containing 87–99 % of fat (McDowall, 1953). In accelerated churning, butter grains are formed from cream by high-speed beaters. Then the butter milk is drained and the resulting grains are worked into butter by further concentrating the cream to 80 % fat. In this process the concentrated cream is phase-inverted from O/W to W/O emulsion. In the second type of continuous butter making, cream is further concentrated and the emulsion is broken. Fat, water and salt concentration are standardized, followed by re-emulsification, cooling and working. Higher speeds produce larger butter grains but can also result in retention of buttermilk and loss of fat. The mixture of buttermilk and butter grains formed during primary churning passes into the secondary churning section which consists of a large-diameter perforated drum rotating at a variable speed, usually 35 rpm. Butter grains undergo consolidation and aggregation during passage along the screen, and the buttermilk is drained. Butter grains pass from the secondary churning cylinder to the working section, which is normally inclined upwards to facilitate drainage of buttermilk. Working is an extremely important part of the butter making process, and it is at this stage that colour, appearance, consistency and spreadability are determined. Under-working produces a final butter having a crumbly consistency, while over-working results in a weak body resembling thick cream. In each case large water droplets or free moisture are likely to be present. Butter is then salted. Salting is an important process, necessary to prevent loss of salt in the buttermilk. AMF The raw material for anhydrous milk fat (AMF) or butter oil is milk from ruminants, for example cows and buffalos. According to WHO/FAO (1986) this product contains 99.8 % milk fat, 0.1 % moisture, with a peroxide value (PV) not greater than 0.3 meq/kg and free fatty acid (FFA) 0.3 % (maximum as oleic acid). The major fatty acids in AMF are similar to that found in other high-fat milk products and include oleic at about 30–40 %, 25–32 % palmitic, 10–15 % stearic and 11–12 % myristic. In addition, small amounts of linoleic 2–4 % and linolenic < 1 % are also present in the milk fat. It is also known that due to rumen bacterial digestive activities, conjugated linoleic acid (CLA), which mainly consists of cis-9, trans-11 isomer, is present at levels of 2– 30 mg g–1 of total fat in milk (Parodi, 1994).

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The polyunsaturated fatty acids are prone to oxidative rancidity and hence AMF suffer from shelf life limitations. In milk fat naturally-occurring antioxidants are present but only in small concentrations, 10–46 mg/kg in the form of α-tocopherol. Therefore, the unique milk fat flavour from diacetyl, butyric acid and δ-lactones is severely affected during processing, handling and storage of AMF due to insufficient antioxidants. Manufacture of AMF/butter oil There are two main methods available for the industrial manufacture of AMF. The initial step is to prepare cream from milk by centrifugal separation to 35–40 g 100 g–1 fat and then further process this to expel large quantities of moisture until AMF is obtained. The cream is kept in chilled storage (4 °C) in jacketed tanks with slow agitation for at least eight hours. This important ageing process allows milk fat in the globules to undergo partial crystallization which aids churning. AMF from cream In this method, the processing factory separates milk to obtain the total quantity of cream required for AMF production. However, it is also known for the manufacturers to buy cream from other dairies to meet the demand for production. This cream is further separated to remove the aqueous phase (skimmed fraction) and thereby increase the fat concentration. The separation is carried out at about 60–70 °C. An important step in this process is the conversion of the high-fat cream (75 % fat) which is oil-in-water emulsion (O/W) to water-in-oil emulsion (W/O) by phase inversion. This is done with a high-pressure homogenizer. After homogenization, further concentration is carried out by centrifugal separation to obtain 95.5 % butterfat. The main steps in AMF manufacture using sweet cream are given in Fig. 16.6. AMF from butter The industrial manufacture of butter has been described on p. 406. The phase inversion of cream (O/W) to butter (W/O) takes place during the churning stage of sweet cream. Unsalted butter is made using sweet cream. Butter blocks (25 kg) are transferred into a melter and then pumped into a holding tank where they are kept at about 60 °C. In the holding tank all the fat is in the liquid state, and protein aggregation also takes place by holding for about 30 minutes. From the holding tank it is separated by centrifugation to obtain butter oil containing 99.5 % fat. See Fig. 16.6 for the main steps in the manufacture of AMF starting from sweet cream unsalted butter. Fractionation of milk fat Milk lipids are a mixture of high-, medium- and low-melting triacylglycerols formed during crystallization. At elevated temperature, for example about 38–40 °C, the triacylglycerols are in a molten state having a high kinetic energy allowing individual particles (molecules) to be mobile. Such energy

410 Modifying lipids for use in food Milk

Centrifugal separation (50–55 °C) Skimmed milk

Cream 40–42 % fat (pasteurization) Centrifugal separation (60–70 °C)

Cream 75–80 % fat o/w

AMF from

Cold storage (3–6 °C) Crystallization (8–16 hr)

Churning

Butter milk

Phase inversion Butter AMF

Homogenization

cream

Phase inversion Skim fraction

Fig. 16.6

Cold storage (in 25 kg blocks)

from butter

Centrifugal separation

Melter (60 °C)

Butter oil AMF

Centrifugal separation

Skim fraction

Manufacture of anhydrous milk fat (AMF)/butter oil.

overcomes the intermolecular forces that hold the molecules together. However, during cooling the molecules come closer together and the stronger intermolecular forces restrict particle movement. The next stage is the crystal formation and growth. During fractionation, the initial step is to crystallize the high-melting acylglycerols from the melt and harvest after separation. Based on this method, Norris (1976) described a process of extracting highand lower-melting fractions and mixing to improve spreadability of butter. Therefore fractionation is a physical process where unmodified triacylglycerols are separated into mainly three basic fractions, for example, high-, middleand lower-melting. Other fractionation methods are also available, for example crystallization from solvent such as acetone. This method produces purer fractions compared to fractionation of melted lipid. The disadvantage would be the solvent residues left in the fractions. Alternatively, dry fractionation with a detergent is also available. This is a cheaper process than the solvent fractionation, but

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it also suffers from detergent residues in the fractions which have to be refined to remove. (See also Chapter 10 on Fractionation.) Margarine Margarine was introduced as a cheap substitute for butter, and its quality was judged on how closely it resembled butter. Requirements for spreadability and nutrition were met by manufacturing margarine with a high level of polyunsaturated fats. Later, margarine came to be preferred by consumers due to its healthfulness, rather than the price. The fat phase is important in determining the physical properties of margarine, especially the structure and its effect on consistency and plasticity. Margarine manufacture Margarine like butter is a W/O emulsion. The fat phase is important in determining the physical properties of margarine, especially its structure, consistency and plasticity which depend on the melting behaviour of the component triacylglycerols, the solid fat content at any given temperature, the distribution of solid fats over a temperature range and the polymorphic modification or crystal habit of the fat composition. An important physical attribute of margarines is that they are spreadable even when first lifted out of a refrigerator. A harder margarine is used in the bakery industry and a still harder margarine with a high melting point is used in puff pastry to facilitate rolling into the dough. A wide range of land animal, marine and vegetable fats and oils have been used in margarine manufacture. Olive oil, high in oleic acid, is used in making a healthy version of margarine, although in some cases flavour reversion due to autoxidation of linolenate is possible. This applies to margarine containing significant proportion of linolenate originating from soybean oil, marine oil and olive oil. Nowadays vegetable oils with large amount of polyunsaturated linoleic acid are widely used in soft margarine manufacture. The desired properties of margarine are achieved by using a blend of fats or by modification of fat. Hydrogenation is the most important way of modifying the fat and thereby raising its melting point. Blending of fats and oils is also used to achieve the required consistency of the end product. The aqueous phase of margarine was originally skim milk, although water itself could be used and usually a ‘milk’ consisting of water and a source of dry protein is employed. This latter may be skim milk powder or whey products. Mono- and diacylglycerols of fatty acids at levels of 0.1–0.3 %, usually in combination with lecithin at a level of about 0.1 %, are used to stabilize the emulsions. Lecithin encourages the reversion of the W/O emulsion to O/W emulsion under the shear forces occurring during chewing. The O/W emulsions enhance release of flavour compounds. The fats and oil used in margarine manufacture should be bland and have no effect on the flavour of the final product. The flavours used in margarine manufacture are derived from butter. Salt is added to enhance the flavour and to apply an anti-microbial effect.

412 Modifying lipids for use in food Margarine is generally fortified with vitamin A or vitamins A and D. Colourings such as β-carotene and palm oil concentrate are added to enhance the natural colour. Processing The processing of margarine is done either on a batch-continuous or fullycontinuous basis. Two phases are prepared separately. The oils and fats are blended and then the emulsifiers are added. ‘Milk’ is prepared from water, and dry protein source and other water-soluble ingredients such as salt and preservatives are incorporated. The two phases are then metered into an emulsifying unit at 45 °C and combined under conditions of vigorous agitation. Flavouring and a vitamin/colour premix are usually added at this stage. Pasteurization is carried out downstream of the emulsification stage at 80–85 °C for two to three seconds in a scraped surface heat exchanger. The emulsion is then passed to a stirred tank where crystallization is initiated. Depending on the hardness, margarine is either moulded into sticks or filled into plastic tubs. Crystallization is completed after packaging, but before dispatch. It is vital that the margarine has appropriate melting point and organoleptic characteristics such as texture and mouth feel. Reduced-fat and low-fat spreads Reduced-fat and low-fat spreads have fat content ranging from about < 30 to about 60 %. Spreads may be based on vegetable fats, a blend of vegetable and butterfat or butterfat alone. Table 16.4 indicates the main categories of yellow fat spreads based on the fat content. Special types of table spreads Many special types of table spreads have been developed to address the concerns of spreadablity and healthfulness. The intention of producing a spreadable ‘butter’ ended up with margarines and spreads with advantages of spreadability and healthfulness over butter. With the use of vegetable oil, the name ‘butter’ is not permitted and proprietary names such as Clover® (Dairy Crest UK) and Bregott (Arla Foods, Sweden) have been used. The manufacturing technology usually involves the butter making process, and the vegetable oil is blended with butterfat at any stage from milk before separation to the finished butter, but most commonly the two types of fat are blended immediately before butter making. Emulsifiers are added to assist Table 16.4

Yellow fat categories.

Fat content (%)

Description

<30 39–41 50–60 72–80

Very low fat Low fat Reduced fat Full fat

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churning and to stabilize the final product. Vegetable oil must be present at levels of 15–35 % to be effective, un-hydrogenized soybean oil is most common, but rapeseed and sunflower oil are also used. Texturization is also applied to improve the spreadability of conventional butter by kneading ready-churned butter vigorously. It is essential that crystallization has been completed with seven days resting period after churning for texturization. Spreadability is improved by liberating the liquid fat from the crystal network at refrigerator temperature in the texturization process. The higher spreadability decreases during use, due to temperature fluctuations. Spreadability of already churned butter may also be improved by whipping, hardness being reduced proportionately to the quantity of gas phase whipped in. The industrial application of fat modification techniques, notably fractional crystallization, to butterfat has enabled spreadable butter to be made by combining a hard fraction with a very soft fraction. High-fat dairy spreads can be made using a continuous butter maker and high-fat vegetable oil spreads using margarine technology. The aqueous phase becomes very important in products with reduced fat content. Most current products are of the W/O type, although some O/W types are also available. The fats and oil of the spreads may be vegetable, dairy or blend of the two. The vegetable fat such as sunflower seed oil is considered to be healthier due to its high content of polyunsaturates. Marine oils and oils from microbiological sources high in eicosapentaenoic and docosahexanoic acids are used in infant formula (human milk fat substitutes for infant feeding). When hydrogenated fat is used in reduced fat spreads it ensures the presence of fat crystals which are important in stabilizing the emulsion. High levels of mono- and diacylglycerol emulsifiers are used to stabilize high water content spreads. But the combined use of relatively low levels of emulsifiers and aqueous phase structuring agents is now commonplace, and this helps to minimize the coalescence of water droplets during processing and spreading. Milk proteins such as sodium caseinate and butter milk powder at levels of as much as 12 % and polysaccharide stabilisers such as carrageenan, alginates and pectins at lower levels and the use of gelatine are also widespread. The amount of the structuring agent is critical, and unstable products will result from use of lesser amounts. Poor organoleptic quality will require the use of extra structuring agents due to high residual viscosity after fat melting during consumption. In order to achieve very low fat in oil-based products and all types of oil in water-based products very high levels of structural agents are required. Spread-like properties depend on a gelled aqueous phase. Combinations of milk protein and modified starch, gelatine and monoacylglycerols are also suitable. Interactions between starch and milk proteins are important in stabilizing very low-fat dairy spreads such as St Ivel Gold Lowest® (Dairy Crest, UK), which contains only 25 % fat.

414 Modifying lipids for use in food Processing of spreads Most W/O spreads are processed using conventional margarine processing equipment. In general, processing conditions are more critical than with margarine. Shrouded propeller mixers are mostly used, and it is necessary to vary the rate of addition of the aqueous phase, applied shear rates and intensity of mixing as the viscosity of the emulsion increases. It is important to control the crystallization and working to gain desired body and consistency. At present, fewer yellow fats, O/W-based spreads are available. Formulation, with respect to fat level and choice of hydrocolloids as water structuring agents, requires very careful control and tends to negate the advantages. The most important manufacturing stage is homogenization, and altering the conditions allows a range of products of different rheological properties to be made. Spreads made with fat replacers are entirely aqueous systems. Some table spreads Utterly Butterly® Original (Dairy Crest, UK) is a buttery tasting dairy spread with only 63 % fat, compared to 80 % for butter and spreadable butter. Utterly Butterly Scandinavian (launched in October 2001) is a lighter, creamier tasting spread with a paler appearance, typical of the spreads enjoyed in Scandinavia. It is high in monounsaturates, low in saturates and low in salt. Unlike any other dairy spread, it is made by the traditional churning and uses fresh buttermilk to give the characteristic buttery taste. Clover® was launched in 1983 and was the very first spread that could be used straight from the fridge, yet had all the taste of butter. Vitalite® (Dairy Crest, UK) is ideal for growing families with active children because it is low in saturates and high in polyunsaturates. It is a source of vitamins A, B and D, and naturally rich in vitamin E. Willow® (Dairy Crest, UK) is a highly versatile creamy product suitable for cooking, baking and spreading. St Ivel Gold® was the UK’s first ‘half fat’ spread and immediately stood out on supermarket shelves and quickly claimed the leading position in the low-fat spreads market. St Ivel Gold is also the only spread made with semi-skimmed milk and, with just 35 % fat, contains half the fat of butter or standard spreads. Lipids in bakery products In bakery applications, the lipid ingredients were modified to meet specific requirements to bring out the desired functional properties. In addition, the lipids should be able to withstand a range of very high temperatures. As for other lipids the functions of milk lipid in bakery applications are: • • • • •

shortening ability and lubricity; batter aeration; emulsifying properties; shelf life; organoleptic quality. Butter and butter oil are not widely used in short pastry manufacture but,

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because of their superior flavour and the texture qualities, they have a place in high-quality bakery products. In many recipes, lard or shortenings are mixed with butter for economic reasons and to improve performance. In bakery terms ‘short’ means friable, easily broken in contrast to the opposite of tough, resilient or elastic. All fats are shortening agents in that they reduce the extensibility of gluten according to the amount used for a given weight of flour and according to the recipe and the method of production. A fat blend process is an important step to ensure that the correct solid/ liquid ratio of fat exists in the final mix. Typically, a relatively high proportion of solid crystalline form of the triacylglycerols at about 25–30 °C is desirable. However, care must be taken when formulating blends to avoid undesirable firmness (due to high level of solid fat) while at the same time achieving the desired level of solid fat at higher temperature. It is also important that the fat prepared by blending retains its plastic characteristics over a wide temperature range. The importance of such plastic characteristics has been particularly relevant in the preparation of cake batter where air bubbles are held initially in the fat phase. In modern industrial manufacture of bakery products, high-speed mixing units are used for batter preparation using plastic lipids and other ingredients. In this process, air is injected to the mixer head and allowed to distribute evenly in the batter. Even distribution of fine lipid particles and air bubbles produces cakes with better volume and crumb structure. The correct balance of solid/liquid lipid provides sufficient liquid portions to surround the air bubbles and trap them. It is equally important that the solid lipids at this stage stabilize the food system. The proportion of crystalline triacylglycerol has to be above a minimum value at the working temperature to be effective in stabilizing the system (Podmore, 1994). This was shown to be about 5 %, but most bakery fats contain about 20 % solid triacylglycerols. It is also reported that small β′ crystals are most effective in stabilizing the air bubbles. Once foam is created, it is important that the structure does not collapse under process conditions due to mechanical shear and it should resist softening.

16.3.3 Heat treatment of emulsions Creams are some of the two-phase O/W emulsions where water or the aqueous phase is continuous, and these emulsions are constantly under threat from possible destabilization caused by microbiological activity, chemical changes or physical changes. In cream, the pH is in the neutral range and that, together with the continuous aqueous material an provides ideal environment for a large cross-section of the micro-organism population. The aqueous phase in O/W systems contains a variety of nutrients which can be easily digested by bacterial cells and is therefore a good substrate. The primary objectives of a heat treatment process are to ensure food safety, to comply with hygiene requirements and to facilitate those ingredients that require heat to activate and initiate functional properties. This applies to water-continuous emulsions.

416 Modifying lipids for use in food Milk separation may be carried out as part of the heat treatment process (e.g. pasteurization). In many continuous heat treatment applications on farms, a separator is linked to the heat exchange unit, either in the heating cycle or after the final heating stage and during the cooling cycle. In either arrangement, the separation temperature used is in the region of 40–45 °C. In the former method, the separated fractions, skimmed milk and cream, were further heat treated to pasteurize and then cooled. The heat treatment regimes designated for example in the UK the Dairy Products Hygiene Regulations (1995) are used for heat treatment of milks, creams and ice cream (see below). Pasteurization The pasteurization method can be either batch type or continuous hightemperature short-time (HTST). Batch method • Heated to a temperature of not less than 65.6 °C and held for at least 30 minutes (batch method). HTST method • heated to not less than 72.0 °C and held for at least 15 seconds (continuous method); • using other temperature/time regimes which have an equivalent pathogen elimination effect. Some important design features in a HTST heat treatment plant are: • the pasteurization temperature sensor is located in the early part of the holding tube; • a divert valve is fitted at the end of the holding tube; • a continuous recording method for probes monitors temperatures of pasteurization, hot water and final product cooler; • a pressure differential measurement and indicating device for the raw and pasteurized products is fitted – if pressure differential is not monitored, a valid pressure test certificate should be available for inspection by the licensing authority (every 12 months). HTST extended shelf life process In the high-temperature pasteurization method, product temperatures in the range 115–125 °C for times between one and five seconds have been recommended as suitable regimes for high- and low-fat milk products. These conditions, together with an aseptic packing arrangement, extend the shelf life of the liquid products from about 10 days to more than 30 days under refrigerated storage. A detailed account of this process and investigation results are given by Ranjith (2002).

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Ultra high temperature treatment (UHT) Long-life creams have been produced successfully for many years in Europe. The UHT process was originally applied successfully to milk, and the same basic processing methods were later adapted for creams. The UHT process regimes used in UK are defined in the UK Dairy Product (hygiene) Regulations, 1995. The minimum heat treatment required for milk and cream is defined as: • •

heated to not less than 140 °C and held for not less than two seconds; or other temperature–time regimes having an equivalent lethal effect to eliminate vegetative pathogens and spores.

The normal heat treatment regimes used in commercial operations are 136– 145 °C for two to six seconds. The regulations may vary according to the food process control measures introduced in individual countries. Ranjith and Thoo (1984) described a procedure to produce fresh tasting milk and milk products after UHT treatment, and this process is in commercial production in UK. Burton (1988) has documented a comprehensive account of UHT processing. The heat treatment given to a product was originally quantified in terms of lethality values based on the work carried out in the food canning industry. For example, the lethality value given to food products is described in terms of F0 values with reference to the death rate of the organism Clostridium botulinum. F0 of 1 is given when a product receives a heat treatment of 121.1 °C for one minute. A simplified formula used in the calculation to derive the F0 values in high-temperature processes is given in the following equation:  T –121.1   Z

F0 = 10 

t

[16.1]

where T = temperature (°C) of the process, t = time in minutes and z = change in temperature (°C) required for thermal death time to transverse one log 10 cycle. Kessler and Horrak (1981) described alternative dimensionless values to quantify the lethal effects of a heat treatment. These are the B* and C* values. B* value of 1 refers to a heat treatment when the spores of B stearothermophillus were reduced by 109 log cycles. C* value of 1 refers to a heat treatment where vitamin B1 (thiamine) is reduced by 3 %. For milk and cream processing, it is necessary to achieve a higher B* value and the lowest possible C* value. These values can be calculated using graphical methods as described by Kessler (1981) and by Kessler and Horrak (1981). UHT treated products always end up in an aseptic filling arrangement without which a long shelf life at ambient temperature is not possible. Homogenization of emulsions A homogenizer is simply a high-pressure pump normally designed to operate with a three-piston arrangement invented in 1899 by a Frenchman, August

418 Modifying lipids for use in food Gaulin. The main components in a homogenizer are the piston driving unit, the high-pressure head and the homogenizing valve housing. A homogenization step in the process would be expected to produce a stable emulsion due to particle size reduction, smoother mouth feel from smaller fat globules, use of fewer stabilizers and decreased tendency to churn fat in water-continuous emulsions. In the manufacture of butter oil, a homogenizer is used to achieve phase inversion. The design of the homogenizing valve has been improved over the years, and Phipps (1985), Stistrup and Andreasen (1966) and White (1981) have reported on the performance of various commercially available systems. It is important to note that the homogenization procedure could be either single-stage or two-stage, depending on the design of the valve arrangement needed to achieve this particle reduction duty. Single homogenization Single-stage homogenization tends to encourage cluster formation and, depending on the homogenization pressure, gel formation in the cream is a possibility during storage. Second stage is used to prevent cluster formation. Table 16.5 indicates some homogenization conditions used for fresh creams and ice creams with flat valve homogenizer. In Table 16.5, a range is given which reflects the conditions used in the dairy industry, taking into consideration variables such as the throughput of the homogenizer, temperature used at different installations and the efficiency of the valves in the head. The product itself could determine the optimum conditions, as the fat to MSNF (milk solids non-fat) ratio is important to ensure that sufficient solids material is available to reduce the interfacial tension. Sommer (1944) reported that for cream a ratio of MSNF to fat of > 0.85 would prevent fat clumping. A ratio in the range 0.6–0.85 could lead to some clumping and < 0.6 significantly increases clumping of fat globules. The ideal homogenization conditions for creams are best established by conducting trials to select the most appropriate parameters producing desirable product characteristics. Double homogenization Information on double homogenization or multiple homogenizations is limited. Geyer and Kessler (1989) reported that double homogenization of 12 % fat Table 16.5 Homogenization pressures and temperatures used in commercial production of creams and ice creams. Product

Cream 12 % fat Cream 18 % fat Ice cream

Temperature (°C)

45–70 45–70 50–75

Homogenization pressure (MPa) Stage 1

Stage 2

15–20 10–32 16–20

3–6 3–8.5 3–5

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cream showed improvement in physico-chemical properties. Stistrup and Andreason (1966) were among the early investigators to establish the effect of single-stage, two-stage and double homogenization of ice cream mixes. Their results showed that fat globule dispersion in ice cream was better with liquid whirl valve design compared to flat and conical valve designs in single-stage homogenization. Two-stage homogenization did not improve the degree of dispersion in comparison with one-stage homogenization, whereas double-homogenization gives a higher degree of dispersibility than onestage or two-stage homogenization. Four linear relationships existed between logarithm of optical dispersion (a measure of light scattering) and the logarithm of pressure applied for all methods. In contrast to creating small fat globules to form a stable emulsion in creams, the homogenization step is used to disrupt fat globule membrane to achieve phase inversion in AMF manufacture.

16.4 Factors affecting lipid emulsions In water-continuous emulsions where the pH is in the neutral range, there is a good medium for the micro-organisms to grow. Therefore, milks, creams and ice cream mixes provide a good environment for the multiplication of bacteria, yeasts and moulds unless some control measures are taken to limit their numbers. In these emulsions, microbiological activity leads to changes in pH, mainly lowering it, depending on the extent of the activity. In milks and creams, such a change in pH, for example to less than about 6–6.2, would bring about emulsion instability as well as undesirable organoleptic characteristics.

16.4.1 Emulsion stability of high fat creams In milk stored under refrigeration at 3–5 °C, without agitation, the fat globules tend to form clusters. Therefore, milk silos are kept under slow agitation at regular intervals to minimize cluster formation and fat separation. The size of the fat globules affects efficiency of separation of cream from milk, and the optimum temperature for separation could vary depending on the globule size. Large proportion of smaller fat globules (< 2 µm) could reduce skimming efficiency by allowing the fat percentage in the skimmed milk to rise. Larger fat globules with distinct yellow colour (in contrast to milk from a Friesian herd) are normally present in Jersey herds. Rothwell (1966) and Foley et al. (1971) investigated various processing parameters that influence the emulsion stability of creams. These investigations highlighted the influence of milk separation temperature, cream pumping, pasteurization, cooling and fat percentage on the physical properties of cream and on damage to the integrity of the fat globules.

420 Modifying lipids for use in food Physico-chemical defects of fresh creams In many industrial installations the equipment designed for more robust products than cream (i.e., milk, skimmed milk and juice drinks) has been employed for handling creams. As stated before, the handling and processing circuit needs to be very gentle in order to minimize shear and mechanical damage to the cream. In the preparation of high-fat creams, for example 48 % fat cream (double cream in UK), there is a tendency for a solid plug of cream to develop and, in some instances, the viscosity may rise to unacceptably high levels under optimum process conditions. Defects in cream are associated with the fat percentage of cream and, dependent on their applications, these defects may come under strong criticism from the end user. Defects such as oiling off, cream plug and age thickening have been directly associated with the levels of free fat (or solvent-extractable fat) present in cream. An important factor associated with cream is the free fat level which has been known to increase in fat content, especially above about 40 % fat level. O/W emulsions prepared with increased levels of free fat show instability immediately after preparation or in the early part of the shelf life. This instability has been described as creaming, flocculation, coalescence and disruption. The process of fat globules rising to the surface due to difference in densities between the aqueous phase and the fat globules and forming a thick fat layer is termed creaming. Free fat tends to aggravate this as the crystallized and liquid fat exits from the globules due to damaged or missing globule membranes and forms a solid fat layer on the surface of the cream. The fat globules also come together and form floccules making them much larger particles which rise faster than individual globules due to floccules being less dense than the aqueous phase. However, the floccules are fairly re dispersible. In floccules, individual fat globules are bound together with the neighbouring globules by weak forces. Fat globules coming together and bound by strong forces, on the other hand, form clusters. These fat globules unite at their contact points and share the interfacial layers. The clusters can be re-dispersed with mechanical energy as in the case of the second stage of the homogenizer. The formation of clumps indicates serious destabilization of the emulsion as these are normally considered not to be re-dispersible. The fat globule membrane material of individual globules comes together and forms a continuous membrane round them, and the fat forms a continuous mass completing the clump formation. It is reported that clumps are formed from partly solid globules (Mulder and Walstra, 1974). These clumps will coalesce into one large globule when all the fat becomes liquid (due to rise in temperature). Once the cream is cooled and some of the high-melting fat fraction has been crystallized, the crystal formation generates physical forces within the globules. If the cold cream is subjected to mechanical forces due to harsh handling methods, the fat globules tend to get damaged. This again leads to further cluster formation. Te Whaite and Fryer (1975) found that the gel formation in cream is directly linked with the formation of free fat in creams.

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Physico-chemical defects in UHT creams The most widely manufactured UHT creams in UK are the half-cream (not < 12 % fat) and single creams (not < 18 % fat). The general term coffee cream is used for creams with fat content in the range 10–20 %, mostly for catering applications. Other high-fat creams (whipping, 35–38 % fat, and double, 48–50 % fat) are produced, but the demand tends to be seasonal. Since the mid-1980s demand for low-fat whipping cream increased in Europe and the USA. Mann (1987) reviewed whipping cream, including low-fat whipping cream. Anderson and Cawston (1975) reviewed the progress of research work under the heading ‘The fat globule membrane’ covering the details of milk fat globule membrane and composition. Stability of UHT coffee cream A common problem associated with some coffee creams is an instability known as ‘feathering’ which shows coagulated curd-like flocks floating on the surface when added to hot coffee. This phenomenon was first reported in 1929 (Doan, 1929, 1931). Recently Anderson et al. (1977a,b) and Geyer and Kessler (1989) also investigated the stability of UHT coffee creams with reference to shelf life, extending the shelf life and the influence of the manufacturing methods on feathering. The stability of cream to hot coffee is affected by homogenization pressure and temperature, homogenization position (i.e., upstream or downstream position in the circuit), the hardness of the water used to make coffee, acidity of the coffee and the temperature of the coffee. This shows that the feathering is partly due to changes brought about by changes to the structure of the milk proteins resulting from processing and partly due to harsh conditions in the coffee preparation. Normally these creams are stable to hot coffee immediately after processing (i.e., resist feathering), and the tendency is to become susceptible to feathering on storage at ambient temperature. The exact transformation of various components in the O/W emulsion leading to feathering was not fully understood. Early researchers suggested the possibility of changes to fat–water interface but Anderson et al. (1977a,b) confirmed that increase in fat phase casein:calcium ratio was associated with the feathering in UHT coffee cream, suggesting that casein is the more important factor. They also observed that susceptibility to feathering is associated with the tendency for adjacent fat globules to become linked by bridges of casein (Fig. 16.7). Most of the fat globules have casein micelles associated with them, and numerous sub-micellar casein particles appear to be attached to the surface. Therefore, the extent of feathering observed was accompanied by an increase in the calcium and casein levels and in the casein:calcium ratio in the fat phase of the cream. Ranjith (1995) investigated single cream prepared by various methods (Table 16.6). This included a new method of producing cream by adjusting the diffusible calcium (calcium in the aqueous phase). In this method, milk was initially subjected to membrane filtration (ultra filtration) so that the retentate has a total solids content of 35–38 % w/w.

422 Modifying lipids for use in food

Submicellar casein in membrane

Fat globule

Casein bridge Casein micelle

Fig. 16.7

Diagrammatic illustration of severe feathering in UHT single cream.

Permeate was subjected to an ion exchange process to remove calcium and magnesium (diffusible divalent ions). Deionized permeate was added back to the retentate to reconstitute the original whole milk. The whole milk was then separated to obtain diffusible calcium reduced (DCR) cream (single cream or coffee cream). Each cream preparation was divided into two equal portions and subjected to UHT treatment by the indirect plate method, with one portion being homogenized in the upstream position at 170/34 bar using an APV Manton Gaulin homogeniser. The cream was pre-heated to 55 °C prior to homogenization. In the downstream method, after UHT treatment at 140 °C for 3.5 seconds the cream was cooled to about 60 °C prior to homogenization (170/ 34 bar). The creams were cooled in both methods to 20 °C before aseptically filling into 150 ml plastic containers and sealed with an aluminium foil lid. The cream prepared according to this method was found to resist feathering when tested for up to six months at ambient storage. More importantly, this cream was very stable to alcohol in the alcohol stability test. It was stable to 95 % v/v ethanol when the cream was tested after six months at ambient storage. A cream liqueur prepared using this cream was stable to retort sterilization and the emulsion was stable for more than two years. Table 16.6

Methods used for the preparation of coffee cream (20 g 100 g–1 fat).

Cream no.

Composition

1 2 3 4

Control cream Cream having approximately 20 % of original Ca reduced As in 2 + 1.5 g 100 g–1 Na caseinate Diffusible Ca reduced (DCR) cream (see text)

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The viscosity of coffee cream made by the methods in Table 16.6 was tested over six months, and the results are given in Fig. 16.8. All cream samples made by adjustment to the calcium content (except the batch containing added caseinate) showed slight reduction in viscosity after six months at ambient storage. The control cream made by upstream homogenization developed a thick plug after two months. The control sample made by downstream homogenization showed thickening after six months (viscosity increased by 36 %) but did not form a plug. The sample containing added sodium caseinate showed gradual increase in viscosity, although the total calcium level was reduced by about 20 % of the original value. It is possible that calcium adjustment reduces the likelihood of forming casein bridges between fat globules, thereby keeping the viscosity low or even reducing viscosity during long-term storage and helping to maintain a uniform, homogeneous O/W emulsion. Increasing the casein could be responsible for the increase in viscosity during storage due to building casein bridges between fat globules. Other experimental work also indicated that the feathering problem could be minimized by immobilizing the calcium in cream using chemical additives such as phosphates, citrates and carbonates. 8.00 1

7.50

Viscosity (mPa.s)

7.00 6.50 6.00

2

5.50 5.00

4 5 7

4.50

6

4.00 Month 1

Month 3 Storage period of UHT cream

Month 6

Fig. 16.8 The change in viscosity of UHT single cream when stored at ambient temperature. Cream 1: control cream made by downstream homogenisation; creams 2 and 3: Ca reduced cream mode by upstream and downstream homogenisation respectively; creams 4 and 5: Ca reduced cream with added sodium caseinate, made by upstream and downstream homogenisation respectively; creams 6 and 7: diffusible Ca reduced cream made by upstream and downstream homogenisation respectively.

Stability of UHT whipping cream The primary objective of UHT treatment is to achieve a long shelf life of products at ambient storage with minimum heat damage and minimum change to organoleptic characteristics compared to fresh products. However, a homogenization step is essential in the manufacture of high-fat liquid dairy

424 Modifying lipids for use in food products to minimize fat separation. An optimum homogenization pressure is desirable as high pressure tends to be somewhat disadvantageous when attempting to create stable foam having about 100 % over-run. Kieseker and Zadow (1973) found that milk separation at 43 °C was ideal to manufacture UHT whipping cream (36 % fat) with desirable whipping properties. Whipping properties are assessed on the basis of over-run, serum leakage or seepage, low free fat content and whipping rate (assessed by using a scale 1–5 where 1 is very poor and 5 is excellent). The over-run may not be the same for different cream preparations, and each batch of cream must be treated as different to previous ones. Fine tuning of the adjustments is desirable to optimize the control parameters in order to achieve stable foam. Excessive shear in the worker unit could be detrimental and leads to poor over-run and other whipping properties. Foam formation and stability The formation of air bubbles when handling milk, skimmed milk and creams is a common problem in dairies and food factories. Based on the foaming ability, other aerated products such as whipping cream and ice cream established markets and became popular in the food sector. Lately, milk for cappuccino coffee became popular in many countries. As popularity increased, the consumer expected a good quality foam. For example, the foam in a cappuccino coffee is expected to stay stable until at least half the coffee is consumed. Similarly, whipped cream must produce stable foam after whipping to produce an overrun of about 100 %. Additives to improve aeration are not permitted in milk in many countries. Sometimes milk fails to produce good foam in cappuccino coffee. Whipping cream also sometimes produces a poor over-run or the foam collapses soon after it was formed. This means that the mechanism of foam formation must require a few basic steps to be satisfied before being able to achieve the desired properties of foam. In whipped cream the air bubbles in the foam are held in a three-dimensional matrix by the partially coalesced fat globules. The microstructure study of whipped cream also clearly shows that partly destabilized fat globules adsorb at the air–water interface. The gas phase in the foam provides specific textural characteristics, for example the lightness in whipped cream or scoopability in ice cream. In dairy creams and alternatives, the emulsions are stabilized by the proteins. These proteins should be soluble in the emulsion and rapidly diffuse to the oil–water or air–water interface. This is an important functional property of the protein, and poor solubility characteristics mean poor emulsion stability as well as poor foaming ability of the emulsion. Another equally important characteristic of the protein is that it should re-orient its molecular structure. Some degree of unfolding of the molecule is necessary to bring about intermolecular interactions leading to the formation of a coherent film. This continuous, cohesive film brings about considerable mechanical strength and viscoelastic properties, which play an important part in the stability of emulsions and foams.

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During aeration, the protein diffuses to the air–water interface and gets adsorbed. When more and more proteins are adsorbed, the surface tension in the interface is reduced. The protein structure orientation at the interface is important at this stage, as its hydrophobic part must unfold towards the air while the hydrophilic part associates with the aqueous part or water phase. Interaction and association between proteins is important to the integrity of the film. Emulsion temperature, acidity and ionic strength all affect the protein:protein interactions. Whey protein, especially β-lactoglobulin, is well known for its foaming properties in milk and milk products. It increases its adsorption rate with increase in ionic strength. This protein exists in globular form and, during pasteurization, unfolding of the structure causes more charged sites to be exposed to the emulsion which enhances the adsorption process. Anderson et al. (1987) examined the structure of whipped cream by surface electron microscopy (SEM), and their pictures show that the inner surface of air bubbles consists of a continuous air–serum interface through which individual fat globules protrude. In the whipping process, fat globules penetrate into the air–water interface and then attach to the air bubbles. At the same time, fat globules clump and form a network spreading some of the fat on the air bubble surface. A network of air bubbles and fat clumps finally entraps the liquid from the emulsion to provide rigidity and stability to the foam. Therefore, the foaming ability of milks and creams depends on: • • • • •

solubility of proteins and protein:protein interactions; ability of proteins to reorient and get adsorbed at air–water interface; formation of a strong and viscoelastic film to entrap air bubbles; formation of fat globule clumps and network over air bubbles and film; ability of the film to hold serum and provide rigidity to the foam.

Other factors such as high free fatty acids in the emulsion tend to act as an anti-foaming material and cause poor over-run as well as serum drain. Stabilizer helps to minimize the serum leakage from the foam, but it also reduces the over-run of the foam. In bakery applications, the air is dispersed into the batter, and retention of these bubbles is essential until the starch has swollen and the product structure is set. In traditional cake making, air has been entrained in the shortening (fat) phase during the first stage of mixing. It is not an efficient method as it relies on the ability of the plastic shortening to trap air bubbles during creaming. However, use of emulsifiers such as monoacylglycerols enables the baker to create manageable smaller air bubbles and to efficiently retain them by the shortening phase. This gives more uniform nucleation for leavening gases throughout the batter during baking.

16.5 Future trends There is wide recommendation to consume food containing polyunsaturated

426 Modifying lipids for use in food fat and reduce consumption of those with saturated fatty acids. However, legislation may restrict some products coming into the market due to health implications. In general, the drive towards healthy eating could well develop a new generation of products based on nutritive value and the wellbeing of the consumer. In the spreads market, the products made from polyunsaturated fats have become well-established. Inclusion of omega-3 fatty acids in spreads is getting popular due to its ability to reduce blood serum lipids and to lower blood pressure and other health benefits. Other lipids containing omega-6 and omega-9 fatty acids could be popular due to health benefits related to heart diseases. The technology in the production of table spreads is so well advanced that, together with the advances in lipid refining technology, a new generation of spreads could emerge and capture this specific market sector. More work is required to establish other economical methods to produce cis9, trans-11 CLA isomer for incorporation into more food products other than spreads. Further investigations into possible use of suitable CLA precursor for endogenous synthesis of CLA in humans would be beneficial, especially for those who suffer from dietary restrictions. It is also important to be conscious of adverse comments expressed in terms of calorie intake, trans fatty acids, cholesterol content, etc. The reduced fat spreads are more popular compared to 80 % fat spreads. Reduced fat spreads and low-fat spreads are also popular due to the fact that there is some perceived financial advantage as a significant proportion of fat is replaced with water. However, this perceived financial gain may be overshadowed by the cost involved in the advanced processing technology and additional ingredient cost. Milk is the starting material for a variety of food products including milk drinks, creams and table spreads. Milk composition is affected due to seasonal changes which have a direct effect on foaming characteristics, feathering in cream and spreadability of yellow fats. Modifications to cows’ feeding material and milk lipid tempering have been successfully adjusted so that acceptable results can be obtained to optimize the functional properties. Research work is yet to produce successful and commercially viable methods to improve foaming characteristics of milk and cream without having to use additives.

16.6 Sources of further information and advice Oils and Fats Volume 3: Dairy fats (2003), Barry Rossell, Leatherhead, Leatherhead Food Research Institute.

16.7

References

and CAWSTON T E (1975), Review of the progress of dairy science: the milk fat globule membrane, J Dairy Res, 42, 459–483.

ANDERSON M

Lipid emulsifiers and surfactants in dairy and bakery products ANDERSON M, BROOKER B E, CAWSTON T E

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and CHEESEMAN G C (1977a), Changes during storage instability and composition of ultra-heat-treated aseptically-packed cream of 18% fat content, J Dairy Res, 44, 111–123. ANDERSON M, CHEESEMAN G C and WILES R (1977b), Extending shelf life of UHT creams, J Soc Dairy Technol, 30, 229–232. ANDERSON M, BROOKER B E and NEEDS E C (1987), The role of proteins in the stabilisation/ destabilisation of dairy foams, in Dickinson E, Food Emulsions and Foams, London, Royal Society of Chemistry, 100–109. BUCHANAN R A and SMITH D R (1966), Recombined whipping cream. Proc XVII Int Dairy Congr, München E/F, 363–367. BURTON H (1988), Ultra High Temperature Processing of Milk and Milk Products, London, Elsevier Applied Science. DAIRY PRODUCTS (HYGIENE) REGULATIONS (1995) Statutory Instruments No. 1086, London The Stationery Office. DE MAN J M (1961), Physical properties of milk fat 2, Some factors influencing crystallisation, J Dairy Res, 28, 117–122. DOAN J F (1929), Some factors affecting the fat clumping produced in milk and cream mixtures when homogenised, J Dairy Sci, 12, 211. DOAN J F (1931), The homogenising process, J Dairy Sci, 14, 527. FOLEY J and BRADY J P (1984), Temperature-induced effects on crystallisation behaviour, solid fat content and the firmness values of milk fat, J Dairy Res, 51, 579–589. FOLEY J F, BRADY J and REYNOLDS J (1971), The influence of processing on the emulsion stability of cream, J Soc Dairy Technol, 24, 54–58. GEYER S and KESSLER H G (1989), Effect of manufacturing methods on the stability to feathering of homogenised UHT coffee cream, Milchwissenschaft, 44, 423–427. KAYLEGIAN K E and LINDSAY R C (1995), Handbook of Milk Fat Fractions, Technology and Applications, Champaign, III, AOCS Press. KESSLER H G (1981), Food Engineering and Dairy Technology (translated by M. Wotzilka), Freising, Verlag A. Kessler, 173–201. KESSLER H G and HORAK F P (1981), Objective evaluation of UHT milk heating by standardisation of bacteriological and chemical effects, Milchwissenschaft, 36, 129– 133. KIESEKER F G and ZADOW J G (1973), Factors influencing the preparation of UHT whipping cream. Aust J Dairy Technol, 15 (December), 165–169. LEWIS M J (1999), Microbiological issues associated with heat treated milks, Int J Soc Dairy Technol, 52, 121–125. LING E R, KON S K and PORTER J N G (1961), The composition of milk and the nutrition value of its components, in Kon S K and Cowie A J, Milk: The Mammary Gland and its Secretion, New York & London, Academic Press, Vol. 11, 195–263. MANN E J (1987), Whipping cream and whipped cream, Dairy Ind Int, 52(9), 15–16. MCDOWALL F H (1953), The Butter Making Manual, Vol. 1, Wellington, New Zealand University Press. MORTENSEN B K (1983), Physical properties and modification of milk fat, in Fox P F, Developments in Dairy Chemistry – 2 Lipids, New York, Applied Science Publishers, 159–194. MULDER H and WALSTRA P (1974), The Milk Fat Globule. Emulsion science as applied to milk products and comparable foods, Farnham Royal, Commonwealth Agricultural Bureaux, 101–226. NORRIS R (1976), Fractionating Fats: Butter Spreadable Over Wide Temperature Range, Patent N Z 172,101. PARODI P W (1994), Conjugated linoleic acid: an anticarcinogenic fatty acid present in milk fat, Aust J Dairy Technol, 49, 93–97. PHIPPS L W (1985), Technical Bulletin 6: The High Pressure Dairy Homogeniser, Reading, National Institute for Research in Dairying (NIRD).

428 Modifying lipids for use in food (1994), Fats in bakery and kitchen products, in Moran D P J and Rajah K K, Fats in Food Products, Glasgow, Blackie Academic and Professional, 213–253. RANJITH H M P (1995), Assessment of Some Properties of Calcium-reduced Milk and Milk Products from Heat Treatment and Other Processes, PhD Thesis, Department of Food Science and Technology, University of Reading, UK. RANJITH H M P (2002), Water continuous emulsions, in Rajah K K, Fats in Food Technology, Sheffield, Sheffield Academic Press, 69–122. RANJITH H M P and RAJAH K K (2001), in Tamime A Y and Law B A, Mechanisation and Automation in Dairy Technology, Sheffield, Sheffield Academic Press, 119–151. RANJITH H M P and THOO Y C (1984), Int. Patent, 85306220.6. ROTHWELL J (1966), Studies on the effect of heat treatment during processing on the viscosity and stability of high-fat market cream, J Dairy Res, 33, 245–254. SOMMER H H (1944), The Theory and Practice of Ice Cream Making, Madison, WI, Sommer. STAUFFER C E (2002), Emulsifiers and stabilisers, in Rajah K K, Fats in Food Technology, Sheffield, Sheffield Academic Press, 228–274. STISTRUP K and ANDREASEN J (1966), Homogenisation of ice cream mix, Proc. XVII Int Dairy Congr, München, E/F, 375–386. TE WHAITE I E and FRYER T F (1975), Factors that determine the gelling of cream, New Zealand J Dairy Sci. and Technol, 10, 2–7. TIMMS R E (1994), Physical chemistry of fats, in Moran D P J and Rajah K K, Fats in Food Products, Glasgow, Blackie Academic and Professional, 1–27. TOWLER C and STEVENSON M A (1988), The use of emulsifiers in recombined whipping cream, New Zealand J Dairy Sci and Technol, 23, 345–362. VAN DEN TEMPEL M (1968), Effects of emulsifiers on the crystallisation of triglycerides, surface-active lipids in foods, SCI Monograph No 32, London, 22–33. VANHOUTTE B, HUYGHEBAERT A, MAYER Z and HOUSKA M (2003), Physical properties of milk fat, in Rossell B, Oils and Fats Volume 3; Dairy Fats, Leatherhead, Leatherhead Publishing, 99–138. WALSTRA P (1969), Preliminary note on the mechanism of homogenisation, Netherlands Milk and Dairy J, 23, 290–292. WHITE G C (1981), Homogenisation of ice cream mixes, Dairy Inds Int (February), 29–36. WHO/FAO (1986), Codex standard A2 – Milk Fat Products, Geneva/Rome WHO/FAO. ZADOW J G and KIESEKER F G (1975), Manufacture of recombined whipping cream, Aust J Dairy Technol, September, 114–169. PODMORE J

17 Trans-free fats for use in food E. Flöter and G. van Duijn, Unilever Research & Development, Vlaardingen, The Netherlands

17.1

Introduction

When considering the functionality of fats and oils in food products, two main contradicting aspects come into play. These are nutrition and physical structure (of solids), related respectively to oils and fats. The term oil implies compositions essentially free of solid material at ambient temperature. Fats are at least semi-solid lipid materials. The physical functionality of fats is strongly related to the presence of saturated and trans fatty acids (TFA). These types of fatty acids do not contribute positively to the nutritional value of a food product except for the delivery of energy. Walter Willett’s paper from 1993 (Willet et al., 1993) is the starting point for the significant attention that the negative health effect of dietary TFA has received in recent years. As a result of subsequent studies and publications, it is widely agreed that a limitation of the intake of TFA is desirable. This chapter discusses the structuring functionality of different fat compositions with special attention to the role of TFA and their possible elimination.

17.1.1 The meaning of trans-free One has to admit that in industrial practice there is no such thing as fat completely free of TFA. This is because fat compositions, except in laboratory conditions, will practically always contain small amounts of TFA. These result either from natural processes or are due to configurational changes of the unsaturated bonds in fatty acids on exposure to elevated temperatures. However, these TFA are typically found at low levels, so their nutritional contribution can be neglected. Consequently the term ‘virtually trans free’

430 Modifying lipids for use in food (VTF), implying levels of TFA of less than 1 % in the fat phase, seems more appropriate in products that contain no deliberately generated or added TFA. The translation of the desire to restrict the consumption of TFA by legislators has so far resulted in quite divergent approaches. On one hand, the Danish Veterinary and Food Administration issued an order (Stender and Dyerberg, 2003) that limits the concentration of TFA in fat phases of food products to a level below 2 wt %. Naturally occurring TFA in animal fats and conjugated linoleic acid are excluded from this restriction. On the other hand, the labeling of ‘0 g Trans’ in the USA per January 1 2006 according to the Food and Drug Administration (Food and Drug Administration, 2003) is related to an uptake of TFA of less than 0.5 g per serving of the food product. Here, the official definition of TFA reads ‘all unsaturated fatty acids that contain one or more isolated double bonds in a trans configuration’. The FDA also excludes conjugated fatty acids from this definition. These different definitions obviously allow for different compliant technical solutions.

17.1.2 Functional benefits of trans fatty acids Looking back into the early 1990s, one finds that partially hydrogenated fats could be found in essentially all fat applications that involved some kind of challenge to the fat composition. Partially hydrogenated fats were an important working horse for fat technologists. In simple terms, this role was based on three distinct properties. • • •

the high chemical stability towards oxidation of the partially hydrogenated fats which corresponds to the significantly reduced levels of polyunsaturated fatty acids (PUFA) compared to native oils; the potential to manipulate the melting profile of fat compositions as a function of the degree of hydrogenation; the fact that partially hydrogenated fats have favorable crystallization properties as they crystallize quickly and that they effectively deliver structure to fat phases.

An additional benefit of the application of partially hydrogenated fats is the fact that the final fat functionality is more dependent on the hydrogenation process than on the actual native starting fat. This feature creates a fair amount of raw material flexibility with its known benefits – a phenomenon described as ‘interchangeability’. Typically in frying applications, mildly hydrogenated oils are preferred because their semi-liquid nature permits convenient handling while their chemical composition, in particular the absence of linolenic acid (18:3), ensures longevity of the frying medium. Utilization of partially hydrogenated fats as cocoa butter substitute or as an extremely steep-melting coating fat is related to high levels of TFA. Compared to alternative fats with a similar melting range, TFA-containing fats show a solidification behavior which is clearly superior in the manufacturing processes under quiescent conditions.

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(Quiescent indicates that no shear is applied during crystallization.) A number of other applications require long-term storage at ambient temperatures. Examples are bouillon cubes, cookies and the like. In these applications, aspects such as chemical stability, crystallization behavior for manufacturing and melting profile are of key importance. While the first two aspects are obvious, the melting profile in these applications is related to a compromise between the absence of solid fatty residue or waxy mouth feel when consumed and the integrity of the product over its shelf life. For this type of commodity, the storage conditions are in essence not controlled, and robust designs are necessary. Last but not least, the application of partially hydrogenated fats in spread products such as margarine is highly favored by the ease of manufacturing and the good structuring and melting behavior. In Europe, substitution of TFA in this product area has been widely achieved as the elimination process started in the mid-1990s. Before the detailed productspecific discussion of the conversion from partially hydrogenated fats to VTF fats, it is necessary to consider aspects such as crystallization behavior, product integrity or stability and melting behavior. Detailed discussions on the different aspects of chemical stability can be found elsewhere (Chan, 1987; Allen and Hamilton, 1994).

17.1.3 Crystallization behavior How far a fat composition is suited to supply the necessary structure in a certain product application depends on the combination of the structuring potential of the formulation and the manufacturing process. Without reiterating the discussion of fat structures in Chapter 8, a few basic comments on the structuring of fat phases must be made here. In a first rough approximation, the structure or hardness of a semi-solid fat mixture scales with the amount of solid fat present (Kloek, 1998; de Bruijne and Bot, 1999). Taking a more refined view, but without going into any details, the number of continuous connections through the bulk of the mass and the strength of these connections drive the bulk rheological properties. The first aspect can be directly linked to the size and shape of the crystals present in the system. This is in line with the general rule that smaller crystals are favorable. To get a coherent view of crystal–crystal interaction is far more complicated. As long as only secondary bonds are considered, one might assume that the van de Waals adhesive forces between the various crystals are similar. They will, however, be very different if primary bonds come into play. Primary bonds are related to socalled sintering. This is the formation of solid bridges between the crystals of the primary network due to additional material crystallizing on the original fat crystal scaffolding (Johansson and Bergenstahl, 1995a). The presence of primary bonds delivers much harder structures but is accompanied by a dramatic increase in the brittleness of the semi-solid material. For products that are meant to be plastic they have to be avoided (Haighton, 1965). The solid state of fats is characterized by monotropic polymorphism.

432 Modifying lipids for use in food Polymorphism is the ability to appear in different forms. These different crystal structures α, β′ and β relate to different molecular packing arrangements, and each structure has its specific set of physical properties. The term monotropic indicates that of the three basic polymorphic forms only one is thermodynamically stable. More detailed descriptions of the polymorphism of fats can be found elsewhere, e.g. Sato (1999, 2001). According to the basic principles of thermodynamics, the molecular composition of a fat defines how much solid material can at best exist at any given temperature. If one ignores that fats are complex multi-component mixtures, composed of numerous triacylglycerols (TAGs), the knowledge of the physical properties for a given composition makes it possible to calculate the equilibrium solubility of each polymorph (Wesdorp, 1990). The solubility is straightforwardly converted into the so-called solid fat content (SFC) at any given temperature. Typically fat compositions are characterized according to their SFC line. Figure 17.1 shows a few typical SFC lines. These are not a reflection of equilibrium states because reality is far more complicated due to kinetic influences. The various triacylglycerols that are supersaturated with respect to the system’s temperature form multiple mixed crystals. How this process evolves is strongly dependent on the actual crystallization conditions. The main parameters to classify the process are the supersaturation, the speed at which the supersaturation is generated and the shear the system is exposed to during the crystallization process. These parameters influence the essential processes of crystallization, nucleation and growth. The final size of the resulting crystals is strongly related to the management of these two processes. In a case of abundant nucleation, a large number of small crystals will evolve 100

SFC (%)

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50

25

0 10

25 Temperature (°C)

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Fig. 17.1 Solid fat content (SFC) versus temperature lines for selected fats: - - - -, partially hydrogenated rapeseed oil (slip melting point 32 °C); — — —, partially hydrogenated rapeseed oil (slip melting point 36 °C); 䊉, interesterified fat based on palm oil and palmkernel fat; 䉬, dry fractionated stearin of palm oil; 䉱, partially hydrogenated palm oil (slip melting point 44 °C); 䊏, fully hydrogenated palm oil (slip melting point 58°C).

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from the crystallization process. Nucleation increases with increasing supersaturation and so too does crystal growth. However, the polymorphism of fats complicates this picture. When substantial supersaturation is applied, the crystallizing material behaves according to Ostwald’s famous rule of stages (Ostwald, 1897). This rule implies that a less stable polymorph appears as an intermediate state in the crystallization process. Obviously this is subject to the necessary condition that this less stable polymorph is also supersaturated. Consequently, a high supersaturation crystallization process takes place in the following order: supersaturated liquid → crystallization α → transition into β′. The initial crystallization of the α polymorph can be controlled by the cooling process and occurs almost instantaneously once the solution is supersaturated with respect to the α form. In contrast to this, the solid-tosolid transition process into the more stable polymorphic form is primarily dependent on the fat composition. In the process description above it is referred to the β′ structure, as this more commonly persists over time in typical fat mixtures. In pure triacylglycerols, the β structure is considered the most stable polymorph due to the better crystal packing, but the β′ form is often energetically more favorable for crystals containing many different triacylglycerols. Even though the crystallization process outlined above is more complicated than a single-step process, it is preferably applied in industrial practice. This is due to the fact that this detour via the metastable crystal form is the fastest – and sometimes only – way to create solid fat in the preferred final polymorph. The main complication resulting from this process is the adjustment of the manufacturing processes to the kinetics of the polymorphic transition, which is primarily a function of fat composition. Depending on the molecular composition, triacylglycerols and also other minor components such as partial glycerides, the timescale of the polymorphic transition can vary between tens and thousands of seconds. To avoid the development of primary bonds, it is advisable to manufacture in such a way that the polymorphic transition is largely achieved prior to packaging of the product (Bot et al., 2003).

17.1.4 Product stability The stability or integrity of products always has to be seen in the light of the challenge the product is potentially exposed to. For the fat-based products discussed here, the main challenge is typically the fluctuation of the storage temperature. Other minor challenges are of a mechanical nature due to handling and transportation. Another driver for product disintegration is obviously gravity. In addition, the products obviously change because they are not found in their true equilibrium state. This is particularly true, as fat crystal structures tend to change over time even in the most controlled storage conditions. These changes are mainly due to relaxation processes in the

434 Modifying lipids for use in food network and Ostwald ripening (Bot and Pelan, 2000). Ostwald ripening describes the phenomenon that large crystals actually grow larger at the expense of small crystals. This process is driven by a difference in chemical potential that results from the higher surface energy contribution in the small crystals. Additionally, a typical industrial crystallization process, with high driving forces, results mostly in kinetically frozen non-equilibrium states (Bot et al., 2003). This is possible because the speed at which the mixed solid system approaches its equilibrium state is extremely low. At a macroscopic level, there are principally five types of instabilities. These are changes of the product hardness, oil exudation (meaning the separation of liquid oil out of the semi-solid mass), product inhomogeneities, product clumping for powders and the coalescence of droplets or change of the even distribution of the dispersed particles for emulsions and suspensions. The hardness of a product might increase or decrease on storage or through a temperature challenge. The reduction of hardness is accounted for by the coarsening of crystals, which is stimulated by temperature fluctuations since they involve a change in solubility. At higher temperatures some of the original solid material dissolves into the liquid oil and is re-deposited onto the solid material once the temperature is reduced again. Depending on whether the re-deposited solid is forming solid bridges on the original scaffolding or just grows the existing crystals, this process results in increased or reduced hardness, respectively. Furthermore, parts or the whole crystal structure can undergo a polymorphic transition. This is either accompanied by a steep loss of the product hardness or, in cases of a distinct segregation of specific triacylglycerols into a certain conformation, by the occurrence of large crystals or distinct crystal agglomerates. A well-known example of this type is the development of tropical graininess related to the separate crystallization of triacylglycerols of the type palmitic–oleic–palmitic (Watanabe et al., 1992) derived from palm oil or its fractions in spherulitic crystals. The diameter of these particles can be as big as 2 mm. Another problem is the socalled sandiness caused by large needle-like crystals that evolve from recrystallization of triacylglycerols made up from stearic and elaidic acid. The resulting particles are truly a product defect because their sizes are clearly above the threshold of oral perception, approximately 30 µm. The process of clumping together of particles of a free-flowing powder is clearly related to temperature fluctuations. Adjacent particles partially melt and become greasy at elevated temperatures, forming a joint liquid layer. On cooling, this joint layer reverts to a solid or semi-solid structure gluing the particles together. Through this process a free-flowing powder can be easily converted into a solid block. The other three defects are strongly related to the coarsening of the crystalline network. The capacity of the fat crystal network to hold oil is based on capillary forces and adhesion comparable to the function of a sponge. Smaller crystals generate much more surface and thus a finer sponge. On coarsening, it is less capable of holding oil. In most fat-based water-in-oil emulsion

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products the main mechanism to stabilize the oil–water interface is Pickering stabilization (Pickering, 1907). This means that solid particles, in our case fat crystals, wet and cover the oil–water interface and thus prevent the coalescence of the water droplets (Johansson and Bergenstahl, 1995b; Johansson et al., 1995a,b; Rousseau et al., 2003). On either crystal coarsening or depletion of the crystals due to higher temperatures, the coverage of the interface might become incomplete or crystals start bridging the individual droplets and thus permit coalescence. This becomes a macroscopical problem only when excessive coalescence changes the product appearance. However, relatively limited progression of coalescence might also yield detrimental effects on product quality. This is so because small droplets of diameters below 7 µm suppress microbiological growth through confinement (Verrips and Zaalberg, 1980; Verrips et al., 1980). At larger droplet sizes, microbiological stability can usually be ensured only by use of preservatives such as potassium sorbate. Consequently, the microbiological stability of emulsion products free of preservatives relies on the maintenance of the small droplets. Lastly, inhomogeneous distribution of solid material is most likely to occur in systems that are best described as viscous liquids. Here also the coarsening or partial dissolution of the fat crystal network is the main cause of the defective distribution of the particles.

17.1.5 Mouth feel The mouth feel of a product is a multi-dimensional oral sensation. Main aspects of the mouth feel are the structure breakdown on mastication, the release of flavor as a function of emulsion break-up, the cooling effect due to melting and preferentially the absence of waxy after-taste due to highmelting material. The fat crystals present directly influence these properties to a great extent. On mastication and heating, the fat crystals start to melt and to dissolve. Intense mixing with the saliva helps the heat transfer and also the dissolution. Additionally, kneading further improves heat transfer and softens the product, as fat crystal networks are sensitive to shear. The effects the fat crystals have on the oral sensation are consequently mainly linked to SFC and crystal size. The cooling effect of a disintegrating product depends on how much solid material is actually melting in the mouth. This correlates to the steepness of the SFC curve in the temperature range from 20–34 °C, the actual mouth temperature. This sensation is modulated by the speed of product disintegration, depending on the heat transfer and shear but also on the crystal size. Smaller crystals melt or dissolve much quicker in the mouth. The liberation of flavors from an emulsified water phase happens on product inversion (de Bruijn et al., 1993; de Bruijn and Bot, 1999; Bot and Pelan, 2000). This means during the emulsion breakdown induced by insufficient stabilization of the droplets by fat crystals (see previous section). Finally, if the amount of fatty material that remains solid in the mouth is too high, a few per cent, there is a risk that instead of the desirable lubrication, combined with

436 Modifying lipids for use in food beneficial flavor perception enhancement, a waxy sensation persists. This phenomenon is typically related to triacylglycerols melting above 50 °C.

17.2 Requirements for trans-free fat compositions There are two main requirements with respect to trans-free fat compositions: on the one hand, the functional specification relating to the actual product application; on the other hand, even although the TFA are eliminated, the formulation should not compromise the overall nutritional value of the composition. Furthermore, one has to take into account that for the successful elimination of TFA time is also an important parameter. Immediate solutions cannot afford to be registered as novel foods or suffer from long clearance procedures. Lastly, it is not very likely that the consumer is willing to pay a premium for TFA-free products because the elimination of TFA is not an additional benefit but more a correction of the status quo driven by general consensus.

17.2.1 Functional requirements for successful trans fatty acid elimination Every approach to substitute another fat for trans containing partially hydrogenated fats in product applications is doomed to fail if one tries only to match the properties of the fat. This is because only a limited set of the physical properties can be matched. Consequently, the substitution process has to be based on understanding the application at hand. A further consideration is the acceptability of any perceptible change in the product that accompanies the change in fat composition. Starting from the simple end, chemical stability is based mainly on the absence of polyunsaturated fatty acids (PUFA). Therefore, alternative highstability oils, as used in frying applications, should also have a limited amount of PUFA. Another condition related to chemical deterioration is related to products containing enzymes, such as those present in herb preparations. Products containing herbs and significant amounts of medium-chain fatty acids such as lauric or myristic acid tend to develop a soapy taste over time through lipolysis. In applications that rely on partially hydrogenated fats for structure, three aspects of the TFAs are key to their success. These are the excellent crystallization behavior, the steep melting profile and the reliable formation of small crystals with the consequent high structuring effectiveness of the solid material. Consequently, successful substitutes will in the first place need to form small crystals to maintain product structure and stability. For manufacturing processes under more or less quiescent conditions with relatively low supersaturation, as for example found for bouillon cubes, it is necessary that the crystallization from a slurry to a solid mass proceeds in a comparable

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timeframe to the TFA-containing reference. Crispness and form stability of the product and the absence of stickiness are important attributes in the further manufacturing or packing of products. Manufacturing processes with high supersaturation and high shear are related to high throughput and intrinsically to polymorphic transition. For this type of application, such as spreads, it is important that the substituting fat undergoes the polymorphic transition quickly. TFA-containing fats undergo the polymorphic transition very quickly, often in less than 100 seconds. As the throughput of the manufacturing process scales inversely with the transition time, unless the hardware configuration of the manufacturing equipment is changed, dramatic increases of the transition time are prohibitive. As the final consumer remains king, obviously no substitution scenarios should yield a deterioration in properties perceived by the consumer, such as mouth feel.

17.2.2 Nutritional constraints Next to the functional and cost restrictions in the substitution of partially hydrogenated fats, a few other aspects have to be taken into account. Trans fatty acids are considered to be more health-adverse than saturated fatty acids (SFA). However, it is not desirable that the elimination of trans fatty acids results in final fat compositions with levels of saturated fats that are substantially larger than the original combined levels of TFA and SFA. It should also be mentioned that consumer understanding around trans fatty acids is limited and that, therefore, the discrimination between partially hydrogenated fats, high-trans and fully hydrogenated fats, virtually zero-trans, is blurred. An illustration of this is readily available via an Internet search on ‘trans fatty acids’.

17.3 Production of trans-free fats and their application The main tools that we find at our disposal to fabricate suitable fats for the substitution of partially hydrogenated fats have been described in great detail in Chapters 9–12 of this book. These are hydrogenation, preferentially executed to iodine values close to zero, chemical interesterification, enzymatic interesterification, fractionation and the search for new raw materials, possibly executed via modern plant breeding (e.g. Bockisch, 1993). Another tool, which is suggested to play a successful role in substitution of partially hydrogenated fats, is the use of emulsifiers and other non-triacylglycerol ingredients. Full hydrogenation offers a simple answer to the search for chemically stable fatty materials, as for example in frying applications. However, replacing a trans-containing viscous liquid by a solid block of fully hydrogenated fat for frying applications might not be agreeable, the more so because fully hydrogenated oils have slip melting points above 65 °C and would quickly

438 Modifying lipids for use in food generate a solid fat layer around fried goods. In the recent past there has been a lot of activity from oil suppliers around the launch of new trans-free oils. In 2004, Dow AgroSciences, Bunge and DuPont all launched their various brands of zero- or low-trans oils, with Cargill and Bayer CropScience joining in 2005. Most of these oils are supposed to be an answer to the limited chemical stability of conventional oils as these new oils are high oleic variants of soybean, canola or other seed oils. The new traits have been developed by conventional breeding or by genetic modification techniques. Alternatively, one could attempt the procurement of more stable oils through fractionation of, for example, palm oil. In doing so, however, it has to be noted that even a double-fractionated palm olein is relatively rich in saturated fatty acids, approximately 30 %, as this is just the nature of the triacylglycerols present in palm oil. For applications that rely on the structuring function of TFA-containing triacylglycerols, the substitution can be much more difficult. While in the applications focussing on chemical stability the absence of PUFA is the key objective, here specific triacylglycerols that truly functionally substitute for the TFA-containing triacylglycerols have to be identified. This means that, depending on the specific application, tailor-made solutions have to be sought. Applications of fats where high temperature stability and manufacturability are key can be served by fat compositions rich in fully saturated triacylglycerols. These are most easily generated by full hydrogenation, producing a fat composition rich in stearic acid. If, for reasons of consumer preference, hydrogenation has to be avoided, stearin fractions of palm oil also offer the starting point for compositions rich in fully saturated triacylglycerols. Either wet (solvent-supported) fractionation or multiple-step dry fractionation deliver palm stearins with levels of saturated fatty acids of more than 80 %. Both routes outlined above create fat compositions rich in only a single triacylglycerol, typically tristearin in fully hydrogenated oils and tripalmitin in palm stearin. This may not however, deliver the functionality of mixed crystals, which tend to be small. To this end, one could either just mix these fats or subject them jointly to an interesterification process. If the melting behavior of a fat composition is important not only for the stability and integrity of a product, but also for the mouth feel or deposition behavior, then the fat has to satisfy a much narrower specification. Fully saturated triacylglycerols based solely on palmitic or stearic acid have to be used in very limited amounts on such occasions. The steep melting of partially hydrogenated fats and their good mouth feel are based on the physical properties of triacylglycerols containing both stearic acid and elaidic acid. These yield a range of individual triacylglycerol melting points well above body temperature but below 60 °C. Nature delivers triacylglycerols with melting points in this range very sparingly. These glycerol esters are composed of two saturated and one unsaturated fatty acid with the fatty acids typically arranged in a symmetric fashion (SUS). They can be found in cocoa butter, well appreciated for its melting behavior, and a range

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of other exotic fats, such as sal fat, kokum fat, shea nut oil, mango kernel oil and obviously also palm oil. A significantly increased use of palm oil and palm oil fractions is already anticipated by oil suppliers as they currently enlarge their production capacities. An alternative way to manufacture a fat composition rich in SUS- and SSU-triacylglycerols is currently promoted by ADM. Their enzymatically interesterified hardstock is based on fully hydrogenated soybean oil and native soybean oil. This is particularly interesting for the USA because of the relatively low acceptance of palm oil. Besides this approach, there have been numerous attempts to develop seed oils with elevated levels of stearic acid, rich in SUS-triacylglycerols, none of which is available on an industrial scale at the moment. The SUS triacylglycerols unfortunately have a melting point very close to body temperature, and typically show a complicated and slow crystallization. The relatively low melting point of SUS-triacylglycerols necessitates that for elevated temperature structuring high levels of these TAGs are present. The two features mentioned in combination with their price and limited availability make these TAGs less suited for robust commodity applications. Alternatively, triacylglycerols composed of saturated medium-chain and long-chain fatty acids also melt in the desired intermediate temperature range (see also Garti and Sato, 1988, 2001). Unfortunately these do not exist naturally. They can be fabricated by esterification of a mixture of fats containing adequate amounts of long-chain saturated fatty acids, derived from palm oil from full hydrogenation, and medium-chain fatty acids present in palmkernel or coconut fat. Since interesterification always delivers a statistical mix of triglycerides in accordance with the starting fatty acid mixture, the concentration of the targeted triacylglycerols, di-long mono medium-chain (HM-TAGs), is always limited. Alternatively, similar steep-melting fats with good crystallization properties can be fabricated by full hydrogenation of palmkernel fat. To further optimize the characteristics of this fat, highly suitable for coating and other cocoabutter like applications, it is often subsequently interesterified to randomize the distribution of its fatty acids. In spite of the suggestion that interesterified fully hydrogenated palmkernel fat is a good alternative for partially hydrogenated fats, its application in other products remains limited due its price and its interaction with enzymes. For the replacement of partially hydrogenated fats in spreads and similar applications, other constraints apply. In the first place, modern spreads, soft tub products, are typically designed to deliver high amounts of healthy liquid oils. This implies that the structuring fat, in general referred to as hardstock, is used in limited quantities. Fats similar to those discussed above qualify for use in spreads. As already outlined, for manufacturing processes under high supersaturation the kinetics of the polymorphic transition is of prime importance. It turns out that fats rich in triacylglycerols composed of medium- and long-chain saturated fatty acids (HM-TAGs) actually have short transition times. Furthermore, this type of triacylglycerol, possibly

440 Modifying lipids for use in food driven by the fairly complex packing at the molecular level into the crystal lattice, produces smaller crystals than, for example, fully saturated longchain fatty acid-based triacylglycerols. This makes the mixed saturated triacylglycerols particularly suitable candidates for the substitution of partially hydrogenated fats. It has to be noted here that in this substitution the melting profile of the products will also change according to the illustration in Fig. 17.1. Interesterified fats yield relatively straight solid fat content versus temperature lines that can be manipulated by the composition of the interesterification mixture. With straightforward application of interesterified fats, the limits of a high SFC at 20 °C in combination with very low SFC level at 35 °C are quickly reached. In order to create significantly steeper SFC-lines, either triacylglycerols of the SUS (SUS) type or HM-TAGs levels have to be optimized in the formulation. This can be achieved by combination of different hardstocks. However, in mixing for example a HM-TAGs hardstock with cocoa butter fat, economically not very attractive for spreads, one can find that, instead of a synergistic benefit, quite the opposite occurs. At certain mixing ratios, immiscibility of the TAG in the solid phase occurs and both SFC and structuring potential actually drop. This illustrates that the mixing behavior of the TAGs, which can be influenced by the processing conditions, is a key element in the design of functional fat compositions. In the attempt to fabricate highly functional hardstocks, fractionation plays an important role. There are two possible applications of fractionation; it can be applied either post or prior to interesterification. The economics of the application of fractionation is heavily dependent on the value and usage of the secondary fraction evolving from the separation process. For example, to increase the concentration of HM-TAGs in a fat one could improve the yield of the interesterification with respect to the HM-TAG concentration by optimizing the fatty acid composition of the starting materials towards two thirds of stearic plus palmitic acid mixed with one third of lauric acid. The elimination of unsaturated fatty acids from the interesterification mixture can be achieved by utilization of fully hydrogenated starting materials. However, for non-hydrogenated fat compositions, fractionation of the starting materials is the only tool available to move in this direction. The abundant use of palm stearin in interesterifications which, due to the good market value of palm olein is economically attractive, is the most prominent example for his process. Again, this supports the installation of increased palm oil manufacturing capacities as mentioned before. Higher yields of functional TAGs in the hardstock fats can be achieved by fractionation applied after interesterification. However, there are two downsides to this manufacturing approach. The TAGs one wants to concentrate are characterized by mixed crystal formation with related small crystal sizes. This feature has obvious adverse effects on the smooth execution of the fractionation process, as the separation of the stearin and the olein fractions will be negatively affected. Remedies to this drawback can either be the use

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of solvent fractionation with significant cost implications or redesign of the process. Secondly, the by-product from post-fractionation processes is less likely to be of high value, hence possibly creating prohibitive cost for the overall application. In general, it is fair to conclude that post-fractionation of hardstock fats is considered a last resort to substitute partially hydrogenated fats as it will add substantial cost. However, for other high-value applications, the process discussed might very well be suited.

17.4 Implementation of trans-free fats into the manufacturing and supply chain The nature of the successful replacement of partially hydrogenated fats as outlined above yields a number of specific solutions to specific product applications. The identification of the appropriate best set of solutions for a manufacturer is more complicated. On implementing a technical solution, the balance between utilization of a special fat composition, redesign of the manufacturing process and complexity of the supply chain, as well as more raw materials on site, has to be found. All three factors are related to additional costs. Consequently, they should all be subject to optimization after initial trans-free solutions are established. The complexity of a new trans-free raw material portfolio could be a ‘show stopper’ in cases where layout and tank capacity of a manufacturing site do not allow implementation. Either investment in hardware or preferably harmonization of raw materials can overcome this impasse. This harmonization of trans free raw materials needs to involve process optimization and the re-evaluation of product specifications. In retrospective consideration of the elimination of trans fatty acids in practically all European spreads, one finds that actually the industry as a whole has undergone such a process of harmonization towards an optimal raw material base. Subsequent to the original elimination of trans fatty acids in the mid1990s, initial solutions have been further optimized. Products have slowly changed, and it turns out that finally a wide range of products use a limited set of structuring fats. This industry-wide optimization process has obviously also been helped by the consolidation of the oils and fat suppliers.

17.5 Future trends Future developments with respect to the elimination of TFA will to a great extent depend on non-technical issues. The choice of the legal framework can drive the evolution of technology. The Danish legislation practically bans partially hydrogenated fats from food. In contrast to this, the FDAendorsed limitation of uptake of trans fatty acids per serving can, in some instances, be met with lower-fat products still based on partially hydrogenated

442 Modifying lipids for use in food fats. Finally, consumer preference will decide what product technology will prevail, for example fully accepting hydrogenation or not. The power of consumer preference is documented in the fact that canola oil is well appreciated as very healthy oil in Scandinavia but can practically not be sold in Spain because of its unhealthy image, due to malpractices involving canola oil in 1981. Similarly, Europeans use a vast amount of products based on palm oil, which is only sparingly found in products in the USA. The main future technology development relevant to trans fatty acid elimination beyond those already mentioned is the increased use of nontriacylglycerol structuring. This is already advocated by ingredient suppliers and is the subject of numerous research projects. However, these mainly emulsifier-based structuring techniques have not yet found widespread application. Among other reasons, this is based on the fact that known systems such as those based on monoglycerides or combinations of fatty acids and fatty alcohols are not suited for emulsion systems.

17.6

Conclusions

The replacement of partially hydrogenated fats in their role in aerating, emulsifying, lubricating and providing texture, structure and flavor characteristics to food products is a challenge for food developers. Due to the versatility and robustness of partially hydrogenated fats, their substitution is strongly application-specific. Understanding of the specific application of partially hydrogenated fats and the resulting functional specifications of alternative fats is a necessary pre-requisite for a successful substitution. This process eliminates some historical specifications and could result in the change of technological paradigms. Local consumer preference and legal frameworks have a strong influence on technology evolution with respect to the trans-free challenge. Finally, in the short term technological solutions are likely to be based on the smart combination of the conventional oil modification techniques as described elsewhere in this book.

17.7

References

and HAMILTON R J (1994), Rancidity in Foods, 3rd edn, Glasgow, Blackie Academic and Professional. BOCKISCH M (1993), Nahrungsfette und Öle, Handbuch der Lebensmitteltechnologie, Stuttgart, Ulmer Verlag. BOT A and PELAN E (2000), Food emulsions inside and outside the mouth, Food Ingredients and Analysis International, 22(6), 53–58. BOT A, FLÖTER E, LAMMERS J G and PELAN E (2003), Controlling the texture of spreads, in McKenna B M, Texture in Foods, volume 1: Semi-solid Foods, Cambridge, Woodhead Publishing, 350–372. CHAN H W-S (1987), Autoxidation of Unsaturated Lipids, London, Academic Press. ALLEN J C

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and BOT A (1999), Fabricated fat-based foods, in Rosenthal A J, Food Texture: Measurement and Perception, Gaithersburg, MD, Aspen Publishers, Inc., 185–227. DE BRUIJNE D W, HENDRICKX H C A M, ALDERLIESTEN L and DE LOOFF J (1993), Mouthfeel of foods, in Dickinson E and Walstra P, Food Colloids and Polymers: Stability and Mechanical Properties, Cambridge, Royal Society of Chemistry, 204–213. FOOD and DRUG ADMINISTRATION (2003), Food Labeling: Trans Fatty Acids in Nutrition Labeling, Nutrient Content Claims, and Health Claims, Federal Register – 68 FR 41433 July 11. GARTI N and SATO K (1988), Crystallization and Polymorphism of Fats and Fatty Acids, New York, Marcel Dekker, Inc. GARTI N and SATO K (2001), Crystallization Processes in Fats and Lipid Systems, New York, Marcel Dekker, Inc. HAIGHTON A J (1965), Worksoftening of margarine and shortening, J Am Oil Chem Soc, 42, 27–30. JOHANSSON D (1995), The influence of temperature on the interactions and structures in semi-solid fats, J Am Oil Chem Soc, 72, 1091–1099. JOHANSSON D and BERGENSTAHL B (1995a), Sintering of fat crystal networks in oil during post-crystallisation processes, J Am Oil Chem Soc, 72, 911–920. JOHANSSON D and BERGENSTAHL B (1995b), Wetting of fat crystals by triglyceride oil and water II. Adhesion to the oil/water interface, J Am Oil Chem Soc, 72, 933–938. JOHANSSON D, BERGENSTAHL B and LUNDGREN E (1995a), Wetting of fat crystals by triglyceride oil and water I. The effect of additives, J Am Oil Chem Soc, 72, 921–931. JOHANSSON D, BERGENSTAHL B and LUNDGREN E (1995b), Water-in-glyceride emulsions. Effect of fat crystals on stability, J Am Oil Chem Soc, 72, 939–950. KLOEK W (1998), Mechanical Properties of Fats in Relation to Their Crystallization, PhD Thesis, University of Wageningen, The Netherlands. OSTWALD W (1897), Studien uber die Bildung und Umwandlung Fester Korper, Zeitschrift der Physikalischen Chemie, 22, 289–293. PICKERING S U (1907), Emulsions, J Chem Soc, 91, 2001–2021. ROUSSEAU D, ZILNIK L, KHAN R and HODGE S M (2003), Dispersed phase destabilisation in table spreads, J Am Oil Chem Soc, 80, 957–961. SATO K (1999), Solidification and phase transformation behaviour of food fats – a review, Fett/Lipid, 101, 467–474. SATO K (2001), Crystallization behaviour of fats and lipids – a review, Chem Eng Sci, 56, 2255–2265. STENDER S and DYERBERG J (2003), The Influence of Trans Fatty Acids on Health, 4th edn, Danish Nutritional Council (now Danish Fitness and Nutrition Council), also available at www.ernaeringsraadet. dk/pdf/Transfedt_UK_ny.PDF. VERRIPS C T and ZAALBERG J (1980), The intrinsic stability of water-in-oil emulsions. 1. Theory, Eur J Appl Microbiol Biotechnol, 10, 187–196. VERRIPS C T, SMID D and KERKHOF A (1980), The intrinsic stability of water-in-oil emulsions. 2. Experimental, Eur J Appl Microbiol Biotechnol, 10, 73–85. WATANABE A, TASHIMA I, MATSUZAKI N, KURASHIGE J and SATO K (1992), On the formation of granular crystals in fat blends containing palm oil, J Am Oil Chem Soc, 69, 1077– 1080. WESDORP L H (1990), Liquid-multiple Solid Phase Equilibria in Fats, Theory and Experiments, PhD Thesis, TU Delft, The Netherlands. WILLETT W C, STAMPFER M J, MANSON J E, COLDITZ G A, SPEIZER F E, ROSNER B A, SAMPSON L A and HENNEKENS C H (1993), Intake of trans fatty acids and risk of coronary heart disease among women, Lancet, 341 (8845), 581–585. DE BRUIJNE D W

18 Reduced and zero calorie lipids in food W. E. Artz, University of Illinois, USA, S. M. Mahungu, Egerton University, Kenya and S. L. Hansen, Cargill Analytical Services, USA

18.1

Introduction

18.1.1 Satiety Satiation can be defined as the process of feeling full, followed by termination of food consumption, during a meal (Gerstein et al., 2004). Satiety is the sensation of fullness between eating episodes that tends to inhibit the resumption of eating. There is substantial evidence that dietary protein, rather than carbohydrate or fat, is the macronutrient most effective in increasing satiety. Fat is less satiating than protein but does influence satiety. Compared to foods high in protein or carbohydrates, high-fat foods tend to have the least effect on satiety, but they are generally more palatable. The combination of reduced efficacy with respect to satiety and higher caloric density in foods with moderate to high concentrations of fat generally results in more calories being consumed, when compared to high-protein and low-fat food products. The physiological control of food intake and appetite involves an elaborate series of signals emanating from the gastrointestinal tract, liver and adipose stores, as well as centrally generated hunger and satiety signals (French, 2004). The standard methodology for this research is that of oral preloading; a covertly manipulated food or drink is given at a predetermined time prior to ingestion of an ad libitum test meal. The production of chylomicrons following the consumption of fat will result in the formation of satiety factors, such as apolipoprotein A-IV, the peptide YY (3-36) and cholecystokinin (Tso and Liu, 2004). A greater number of voluntary reductions in energy consumption have been noted in subjects on a high-protein diet compared to individuals consuming high-carbohydrate meals ad libitum (Skov et al., 1999; Araya et al., 2000; Eisenstein et al.,

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2002). The primary effect of a high-protein, low-carbohydrate diet on weight reduction is the increase in satiety due to the high protein content of the diet (Johnstone et al., 1996; Blundell and Macdiarmid, 1997; de Castro, 1999; Marmonier, 2000). In their review of the energy density of foods Stubbs et al. (2000) noted that under normal circumstances in which fat contributes disproportionately more to energy density, protein, carbohydrate, and fat exert hierarchical effects on satiety in the order of protein > carbohydrate > fat; and there is additional research that appears to confirm this phenomenon (Johnstone et al., 1996; Blundell and Macdiarmid, 1997). However, other studies have suggested that this effect is decided almost exclusively by energy density (Raben et al., 2003). Three major areas associated with fat structure have been investigated with regard to their satiating effectiveness (French, 2004). Chain-length and degree of saturation of triacylglycerols have a substantial impact on their physico-chemical properties. Several studies have investigated the effect of these factors on appetite and eating behaviour. Medium-chain triacylglycerols (MCT), hydrolyzed to medium-chain fatty acids, are absorbed more rapidly than long-chain triacylglycerols. Medium-chain fatty acids are directly absorbed into the portal system, whereas long-chain fatty acids and their monoacylglycerols are transported in chylomicrons through the lymphatic system. In addition, MCT are preferentially oxidized and are able to cross the inner mitochondrial membrane without acylcarnitine transferase (Bremer, 1983; Langhans, 1996). MCT are more satiating than long-chain triacylglycerols (TAG). Furthermore, MCT added to a very low-energy diet will speed weight loss and enhance satiety in the first two weeks of weight loss (Krotkiewski, 2001). MCT have been suggested as potential agents for the prevention of obesity (St-Onge and Jones, 2002). Clearly, protein has the greatest effect on satiety, but research published in the last few years suggests that, with an equal caloric density, fat and carbohydrate have comparable effects on satiation, unlike the consensus during the 1990s.

18.1.2 Low-fat foods More than 1000 reduced- or low-fat products were introduced each year during the 1990s (USDHHS, 1990; ADA, 1998; Wylie-Rosett, 2002). The three most popular reduced-fat product categories included fat-free or lowfat milk, (Wylie-Rosett, 2002), salad dressing, sauces or mayonnaise (ADA, 1998) and cheese/dairy products (USDHHS, 1990). A survey conducted for the Calorie Control Council in 1998 (Calorie Control Council, 2004) indicated that these product categories are consumed by approximately one half of those who consumed low-fat products. In addition, consumption of fat-reduced or fat-free margarine/spreads, chip/snack foods, meat products and ice cream/ frozen desserts was reported by more than a third of the consumers that use reduced fat products. Since ~ 88 % of the adult population consumes low- or

446 Modifying lipids for use in food reduced-fat foods and beverages, the large majority of the population has demonstrated substantial interest in foods that are promoted as low in fat (Wylie-Rosett, 2002; Calorie Control Council, 2004).

18.1.3 Health issues In spite of the successful development and commercialization of low-fat food products and widespread acceptance and consumption of these products, as of 2001 obese and overweight adults comprised 58 % of the population in the USA (Mokdad et al., 2003). The obesity problem is not limited to the USA either. In 2000 the World Health Organization reported that more than one billion adults were overweight and over 300 million of these were clinically obese. Throughout the entire world, the problem of obesity is increasing, and not only in industrialized nations but also in urban areas of developing nations (WHO, 2004). A reduction in caloric intake and a simultaneous increase in energy expenditure via regular exercise is the primary solution recommended by knowledgeable nutritionists and medical experts. However, many are not listening to the experts, so the application of appropriate food technologies designed to reduce fat content and caloric density could help ameliorate the severity of the problem. The macronutrient composition of a diet can influence hunger, satiety, food intake, body weight and body composition (Rolls, 1995). Fat, rather than carbohydrates, has been the macronutrient most associated with overeating and obesity. Fat is often consumed in excess because palatability and caloric density of fat are high and satiety is low. Low-fat foods in combination with the appropriate fat substitute can potentially reduce caloric intake by making less palatable low-fat foods more desirable. Fats contribute to the appearance, taste, mouth feel, lubricity, texture and flavor of many food products, provide essential fatty acids and are carriers of fat-soluble vitamins (Akoh, 1995; Artz and Hansen, 1996a,b). The amount and type of fat present in foods determines the characteristics of that food and can affect consumer acceptance. An ideal fat replacer should be completely safe and physiologically inert, and achieve a substantial fat and caloric reduction while maintaining the desired functional and sensory properties of a conventional high-fat product (Grossklaus, 1996). Historically, dietary fats and oils have been considered a primary source of energy without regard to the health effects of their specific complement of fatty acids and sterols (Glueck et al., 1994). In 1995, fats accounted for ~ 38 % of the total calories in the diet of Western populations, particularly in the USA (Akoh, 1995). However, dietary fat intakes greater than 11 % of the total caloric intake only developed after the domestication of mammals and the subsequent selective breeding of genetically fatter animals (Garn, 1997), indicating that a high-fat diet has become the norm only relatively recently in the history of Homo sapiens. Although there are many nutrition recommendations that remain

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controversial, there is a consensus among health and nutrition professionals that North Americans should reduce their dietary fat intake and alter the composition of their dietary fat (Gershoff, 1995). The American Dietetic Association (ADA, 2005) has indicated that the majority of fat replacers, when used in moderation by adults, are safe and useful adjuncts to reducing the fat content of foods and may play a role in decreasing total dietary energy and fat intake. They suggest that it is not a good idea to consume low-fat and low-calorie products in unlimited amounts, particularly if these foods are consumed in place of nutrient dense foods. The moderate use of low-calorie, reduced-fat foods, combined with low total energy intake, could potentially promote dietary intake consistent with the objectives of Healthy People 2010 and the 2005 Dietary Guidelines for Americans. The relationship of dietary fat and cholesterol to coronary heart disease is supported by extensive and consistent clinical, epidemiological, metabolic and animal evidence. Studies strongly indicate that the formation of atherosclerotic lesions in coronary arteries is increased in proportion to the levels of total and low-density lipoprotein (LDL) cholesterol in blood which, in turn, are increased by diets high in total fat (Glueck et al., 1994). Reduction in the relative amounts of high-fat food products in the diet can be an effective means of reducing caloric intake and is consistent with public health goals to reduce the risk of chronic diseases (Borzelleca, 1992, 1996; Degraaf et al., 1996). Thus, dietary fat is one of the major nutrition concerns of Americans. In response to the rising consumer demand for reduced-fat foods, the food industry has developed a multitude of non-fat, low-fat and reduced-fat versions of regular food products (Calorie Control Council, 2004). To generate reducedfat or fat-free products that have the same sensory characteristics as the regular fat version, food manufacturers frequently employ fat substitutes (Miller and Groziak, 1996) made from carbohydrates, protein or fat, or a combination of these components. Many of the carbohydrate- and proteinbased fat substitutes have received GRAS (Generally Recognized As Safe) status from the Food and Drug Administration (FDA) (Artz and Hansen, 1996a). In January 1996, the US Food and Drug Administration approved olestra (currently termed Olean® – Proctor & Gamble Co, USA) for use in savory snacks (Akoh, 1996; Freston et al., 1997; Zorich et al., 1997). This fat substitute is sucrose polyester or sucrose fatty acid polyester whose Code of Federal Regulation (CFR) reference number is CFR 172.867 (US Government, 1997). There are other fat substitutes with the potential to partially replace some, but not all, calories from fat under development or in the market (Calorie Control Council, 2004). Fat substitutes could replace a significant proportion of dietary fat and become macronutrient substitutes (Borzelleca, 1992). Hence, the safety of these materials must be established via extensive safety testing prior to FDA approval and introduction into the food supply (Artz and Hansen, 1996b). Appropriate methods of safety evaluation must be used. Traditional methods for the safety evaluation of macronutrient substitutes are inappropriate, since

448 Modifying lipids for use in food an evaluation of concentrations high enough to provide a 100-fold safety factor is not feasible (Borzelleca, 1996).

18.2 Fat substitute chemistry 18.2.1 Synthesis and composition of fat substitutes This section will address the synthesis and/or preparation of some of the major fat substitutes in use or under development that have potential as fat substitutes or fat replacers. The terms fat substitutes and fat replacers will be used interchangeably in this chapter. While many of the fat substitutes are included in this chapter, the absence of a fat substitute from this chapter implies nothing about its utility, safety or potential. Some of the fat substitutes here discussed are those that contain fatty acids attached to a molecule other than glycerol, such as in olestra. In other examples, such as the esterified propoxylated glycerols, the attachment has been modified to reduce the susceptibility of the compound to fatty acid release via lipase. The other category of fat substitute discussed will be reduced calorie triacylglycerols.

18.2.2 Esterified propoxylated glycerols (EPGs) Fatty acid esterified propoxylated glycerols (EPGs) (ARCO Chemical Company, Newtown Square, PA) were developed for use as frying fat substitutes. Glycerol is propoxylated with propylene oxide to form a polyether triol (Fig. 18.1). Fatty acids are then esterified to the triol (Gillis, 1988; White and Pollard, 1988, 1989a,b,c; Dziezak, 1989; Anon, 1990; Cooper, 1990; Arciszewski, 1991; Duxbury and Meinhold 1991; Hassel, 1993) to R O R

O O R Triacylglycerol

R O

R O

O

O

O

O

O

O O

O

O R

Fatty acid esterified propoxylated glycerol

Fig. 18.1 Triacylglycerol and fatty acid esterified propoxylated glycerol. In this figure, R refers to a fatty acid or acylgroup, connected with an ester bond.

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form a series of oligomers. Figure 18.2 is a capillary chromatogram of a separation of a series of fatty acid esterified propoxylated oligomers containing from three to 13 molecules of propylene oxide per molecule of glycerol. The propoxylated triacylglycerol is similar to natural fats in structure and functionality. Fatty acid EPG is a low-to-non-caloric oil that is heat-stable, only very slightly digestible and non-toxic. Preparation of propoxylated glycerides for use as fat substitutes involved transesterifying propoxylated glycerol with fatty acid esters in a solvent-free system (ARCO Chemical Technology Limited Partnership, Wilmington, DE) (Cooper, 1990) to avoid substances unacceptable in food systems.

18.2.3 Fatty acid partially esterified polysaccharide (PEP) The ARCO Chemical Technology Limited Partnership (Wilmington, DE) has patented a polysaccharide (PEP) that is partially esterified with fatty acids (White, 1990). It is non-absorbable, non-digestible and non-toxic. Suitable oligo/polysaccharide materials include xanthan gum, guar gum, gum arabic, alginates, cellulose hydrolysis products, hydroxypropyl cellulose, starch hydrolysis products (n < 50), karaya gum and pectin. The preferred level of esterification involves one or more hydroxyl groups per saccharide unit with one or more fatty acids.

FID response (mvolts × 10)

18.2.4 Carbohydrate fatty acid esters The carbohydrate-fat combination fat substitutes include those derived from polydextrose, sugar alcohols, altered sugars, starch derivatives, cellulose and gums. They can also be made from components from rice, wheat, corn, oats, tapioca or potato, and they can replace from 50 to 100 % of the fat in foods (Glueck et al., 1994). The synthesis and analysis of carbohydrate fatty acid esters have been

3.80

3.60

3.40

3.20 5.50

6.00 6.50 7.00 Time (minutes × 10)

7.50

Fig. 18.2 Solid fat content separation of fatty acid EPG triacylglycerol oligomers (FID = flame ionization detector).

450 Modifying lipids for use in food reported by several groups (Akoh and Swanson, 1989a,b; Drake et al., 1994; Rios et al., 1994). Carbohydrate fatty acid polyesters with a degree of substitution (DS: number of hydroxyl groups esterified with long-chain fatty acids) of 4–14 are lipophilic, non-digestible, non-absorbable, fat-like molecules with the physical and chemical properties of conventional fats and oils and are referred to as low-calorie fat substitutes (Akoh, 1994a; 1995). Swanson has published work from his laboratory on carbohydrate fatty acid esters synthesized from a variety of carbohydrate sources under a range of catalytic conditions (Akoh and Swanson, 1990; Akoh, 1994a,b; 1995), including glucose, sucrose, raffinose, stachyose and verbascose fatty acid esters (Akoh and Swanson, 1989a). The synthesis of both trehalose octa-oleate and of sorbitol hexa-oleate (Akoh and Swanson, 1989b) have been reported. Oleic acid esters of erythritol, pentaerythritol, adonitol and sorbitol were prepared by transesterification with an excess of methyl oleate to form complete esters (Mattson and Volpenhein, 1972). The esters formed were erythritol tetra-oleate, pentaerythritol tetra-oleate, adonitol penta-oleate and sorbitol hexa-oleate. These esters were not susceptible to in vivo lipolysis by lipolytic enzymes of rat pancreatic juice, suggesting potential application as lowcalorie oils (Mattson and Volpenhein, 1972; Akoh and Swanson, 1989a). Chung et al. (1996) also reported the preparation of a sugar alcohol fatty acid ester made with sorbitol. Enzymatic methods for the synthesis of carbohydrate fatty acid esters have been discussed in detail by Riva (1994). One of the most promising enzymes tested, particularly for fatty acid esterification of the alkylated glycosides, was a lipase from the yeast Candida antarctica, which had been immobilized on macroporous resin beads. Mutua and Akoh (1993) have reported the synthesis of glucose and alkyl glycoside fatty acid esters in organic solvents using Candida antarctica as a catalyst.

18.2.5 Sucrose polyester (SPE) – olestra (Olean®) The most extensively studied and publicized of the low-to-non-caloric fat substitutes are the sucrose fatty acid esters (Fig. 18.3). Typically, sucrose fatty acid esters are prepared from the reaction of sucrose with long-chain OR

OR

OR

OR R = Fatty acid group OR

O O

RO

OR OR

OR

Triacylglycerol

O OR

OR

Olestra

Fig. 18.3 Triacylglycerol and olestra. In this figure, R refers to a fatty acid or acyl group, connected with an ester bond.

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fatty acid methyl esters (Gardner and Sanders, 1990). Depending upon the reaction conditions, anywhere from one to eight fatty acids can be attached. Olestra is the generic name for the mixture of hexa-, hepta- and octa-esters of sucrose formed with long-chain fatty acids. Digestive enzymes do not release the fatty acids, so olestra is non-caloric (Gershoff, 1995). Olestra was approved by the FDA for use in savory snacks in 1996 (Akoh, 1996). A review by Akoh (1995) has described various methods used to prepare sucrose polyesters. SPE have been prepared in 80–90 % yields by reacting sucrose octa-acetate (SOAC) with methyl palmitate. This solventfree method was improved by Volpenhein (1985). Yamamoto and Kinami (1986) reported another method to produce sugar fatty acid esters. The methods of Volpenhein (1985) and Yamamoto and Kinami (1986) required molecular distillation to remove unreacted fatty acid methyl esters. Akoh and Swanson (1990) reported a solid phase extractionbased synthesis procedure with yields between 96.6 and 99.8 % of the purified SPE.

18.2.6 Alkyl glycoside fatty acid esters Alkyl glycoside fatty acid esters are non-ionic, non-toxic, odorless and biodegradable compounds with emulsification properties. Direct esterification of reducing sugars such as glucose and galactose often results in excessive sugar degradation and charring. Therefore, alkylation to form, for example, the methyl glycoside, is necessary to convert reducing sugars with reactive C-1 anomeric centers to non-reducing, less reactive, anomeric C-1 centers (Akoh and Swanson, 1989a,b). The alkyl groups used were fatty acid methyl esters, primarily methyl oleate, although there was one example of peanut oil fatty acid methyl esters. Alkyl glycoside fatty acid esters can be used to replace fat in food products, such as frying oils and Italian salad dressings (Curtice-Burns, Inc., Rochester, NY) (Meyer et al., 1989). Alkyl glycosides can be formed by reacting a reducing saccharide with a monohydric alcohol, such as methanol. The hydroxyl groups of these alkyl glycosides are then esterified to form a lower acyl ester alkyl glycoside. The lower acyl ester alkyl glycoside is then admixed with a fatty acid lower acyl ester and an alkali metal catalyst to form the reaction mixture. Soybean, safflower, corn, peanut and cottonseed oils are preferred since they contain C16–C18 fatty acids that do not volatilize at the temperatures used for interesterification. Albano-Garcia et al. (1980) reported a solventfree synthesis of methyl glucoside esters of coconut fatty acids. Akoh and Swanson (1989a,b) have synthesized novel alkyl glycoside polyesters, such as methyl glucoside polyesters methyl galactoside polyesters, and octyl glucoside polyesters by solvent-free interesterification. To achieve high yields, the alkyl glycosides’ free hydroxyl groups were modified by acetylation prior to interesterification. Glucosides containing 1–50 alkoxy groups can be used as fat substitutes

452 Modifying lipids for use in food at substitution ranges of 10–100 % in low-calorie salad oils, plastic shortenings, cake mixes, icing mixes, mayonnaise, salad dressings and margarines (Procter & Gamble Co., USA) (Ennis et al., 1991).

18.2.7 Reduced calorie fat-based fat replacers The objective for these products is similar to that for the protein- and carbohydrate-based fat substitutes, a substantial reduction in calories, rather than a complete elimination of fat. These compounds cannot be used for frying. Examples for fat-based replacers include caprenin, captrin and salatrim. Caprenin is a reduced calorie triacylglycerol (Procter and Gamble) formed by the esterification with selected fatty acids: caprylic (8:0), capric (10:0) and behenic (22:0). Since behenic acid is only partially absorbed and shortchain acids produce fewer calories than the common C16 and C18 acids, the caprenin contains ~ 5 rather than the normal 9 kcal per gram. Caprenin has functional (melting) properties similar to cocoa butter and is intended to replace some of the cocoa butter in selected confectionery products. It is digested, absorbed and metabolized by the same pathways as other triacylglycerols. Captrin from Stepan Food Ingredients (Anon, 1994) is a randomized triacylglycerol made from linear saturated fatty acids, primarily caprylic and capric. Some of the proposed uses include baked goods, confections, dairy product analogs, snack foods and soft candy. Another fabricated triacylglycerol, similar to caprenin, is salatrim, a triacylglycerol comprising a mixture of long-chain (primarily stearic acid) and short-chain (acetic, propionic and butyric) fatty acids randomly esterified to glycerol (Smith et al., 1994). It contains approximately 5 kcal per gram rather than the 9 kcal/gram contained in regular fats and oils. An entire symposium on Salatrim was published in the February 1994 issue of the Journal of Agriculture and Food Chemistry. The research reported included structural characterization(s) of the oil, an analysis of the oil in food products and an extensive series of papers on the metabolism and toxicology of the oil in various animal and human model systems. Salatrim has the same utility as caprenin as a fat replacer in reduced-fat systems and could be used as a cocoa butter substitute in confectionery products and in baked products and filled dairy products. Caprenin and salatrim are of little use for deep fat frying applications, since release of the smaller molecular weight fatty acids is likely to cause undesirable flavor effects. Salatrim has been prepared by interesterification of saturated long-chain fatty acid (LCFA) triacylglycerols and short-chain fatty acid (SCFA) triacylglycerols (Klemann et al., 1994). The SCFA (triacetin, tripropionin, tributyrin) were reacted with LCFA (hydrogenated canola oil, cotton seed oil and soybean oil) using sodium methoxide as catalyst. An alternative to regular cooking oils, developed and marketed in Japan, has essentially the same calories as regular fats and oils, although the oil is metabolized differently. Diacylglycerol cooking oils have (mainly) a 1,3

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configuration. The taste and texture are similar to triacylglycerol cooking oils. However, 1,3-diacylglycerols are not hydrolyzed to 2-monoacylglycerols in the gut, so absorption and metabolism of 1,3-diacylglycerols differ from that of triacylglycerols. The physiological differences include lower postprandial lipemia and, in comparison to normal triacylglycerol cooking oils, an increased percentage of the fatty acids are oxidized rather than being stored as body fat. Preliminary studies suggest that these differences in the metabolism of diacylglycerols and triacylglycerols can be exploited to reduce the amount of body fat stored from the consumption of cooking oil and other food items with added fats and oils. It has been widely sold in Japan since its introduction in early 1999, and the product is being test-marketed in the USA. Increased consumption of diacylglycerol (DAG) oil may provide an additional means of reducing obesity, while concurrently achieving desirable food product characteristics and maintaining good food product quality (Flickinger and Matsuo, 2003; Katsuragei et al., 2004).

18.2.8 Fat-based systems designed to enhance satiety In 2000 and 2001 it was shown that a fat emulsion (Olibra® – Lipid Technologies Provider AB, Sweden) formulated from palm oil and oat oil fractions can affect the energy and macronutrient intakes in lean, overweight and obese subjects up to 36 hours post-consumption (Burns et al., 2000, 2001). It was suggested that the reason for the increase in satiety and reduction in food consumption was the ‘ileal-brake’ mechanism. There is also evidence that this effect is dose-dependent in lean adults, which is consistent with this mechanism (Burns et al., 2002). This product is now being marketed in USA. In 1998, Safeway began sale of Swedish yoghurt containing Olibra®. Safeway marketed the yoghurt product Skåne Dairy Maval which, since its release in southern Sweden in January, has achieved a 2 % share of the national fruit yoghurt market, despite selling at more than twice the price of rival brands. In 2005 the Swedish firm LTP, Lipid Technologies Provider AB, signed a license and supply agreement in the USA with General Nutrition Corporation (GNC), giving GNC an exclusive right in the USA to LTP’s satiety ingredient Olibra®, for the dietary supplement market.

18.3 Food applications Many factors must be considered when selecting a fat substitute, in addition to the obvious and critical sensory quality questions. Is any thermal processing applied to the product? How severe is the thermal processing (pasteurization versus sterilization)? How pH sensitive is the fat substitute? How long will the product be stored, and are there undesirable textural or flavor changes that occur during long-term storage or during excessively turbulent shipping? Will it be refrigerated? Must it be refrigerated? What home preparation steps

454 Modifying lipids for use in food are involved? Since there are generally water activity changes, is the product microbiologically stable? Are there ‘opportunities’ for abuse in the home, i.e. if opened and left on the counter overnight is food poisoning a possibility?

18.3.1 Baked goods Mono- and diacylglycerol emulsifiers can be used to replace as much as 50 % of the fat in baked goods, since they can be used as a 50/50 emulsifier/ water mixture (Frye and Setser, 1993; Vetter, 1993). Additional emulsifiers used similarly include sodium stearoyl lactylate, sorbitan monostearate and polysorbate 60 at high hydration levels. A hydrated blend of emulsifiers developed specifically for fat replacement includes stearyl monoglyceryl citrate, ethylene glycol monostearate and lactylated monoglycerides. Simplesse™ (CP Kelco Aps, USA) protein hydrolysates, Sta-Slim™ 143 (Tate and Lyle, UK) and sucrose fatty acid esters have been used as fat substitutes in yoghurt, sour cream, cream cheese, cheese spread and frozen dairy desserts. Additional applications are discussed by Frye and Setser (1993), and Sharp (2001) has discussed some of the technical limitations regarding baked goods and reduced-fat formulations. Low-fat shortbread cookies have been prepared using carbohydrate-based fat substitutes (Sanchez et al., 1995). A combination of carbohydrate-based fat substitutes (Litesse® – Danisco Sweeteners, Denmark, N-Flate® – National Starch and Chemical Co, USA, Rice*Trin ® – Weinberg Food, Inc, USA, Stellar™ – A E Staley Manufacturing Co, USA, or Z-trim™ – Fibergels Technologies, USA) and emulsifiers (such as diacetyl-tartaric esters of monoacylglycerols, glycerol monostearate or sodium stearoyl-2-lactylate) were used. The principal effects of fat substitutes on shortbread cookie attributes were an increase in moisture content, greater toughness and reduced specific volume. Zoulias et al. (2002) examined four different types of fat mimetics using cookies. A fat reduction of 35–50 % was achieved with acceptable cookies.

18.3.2 Dairy, frying fats and table spreads Olestra can be used as a partial fat substitute in shortening, margarines and frying oils (Hollenbach and Howard, 1983; Robbins and Rodriguez, 1984; Roberts, 1984). Cheddar cheese has been prepared with milk fat sucrose polyesters (Crites et al., 1997). No significant differences in moisture, pH or whey titratable acidity were observed between the control cheese and cheeses containing milk fat sucrose fatty acid polyester. Cheese containing milk fat sucrose polyester contained fat globules that were smaller and more uniform in size compared to a control cheese with no added sucrose polyester. Bachmann (2001) reviewed various types of cheese analogs that had been developed using fat substitutes. A patent was awarded for the incorporation of the alkyl glycoside fatty

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acid polyester into food products (Curtice-Burns, Inc., USA) (Winter et al., 1990). The fat substitute could replace fats in such products as shortening, margarine, butter, salad and cooking oils, mayonnaise, salad dressing and confectionery coatings or ‘invisible fats’ in such foods as oilseeds, nuts, dairy and animal products. Substitution at 10–100 % is possible, preferably in the range of 33–75 %.

18.3.3 Novel applications One creative use of fat substitutes was the suggestion that sucrose fatty acid esters could be used to inhibit lipoxygenase and thereby improve food quality (Nishiyama et al., 1993). There was an increase in the binding strength of the sucrose fatty acid monoester and soybean lipoxygenase-1 (L-1) as the fatty acid carbon chain-length was increased from eight to 12. Thermodynamic analysis of the binding constants indicated that the binding was hydrophobic in character. Sucrose fatty acid esters can also suppress lipase activity and even have an antibacterial effect in some cases.

18.4

Toxicology

As with any new food ingredient, fat substitutes must be tested on animals before they are tested on humans. Ordinarily in animal toxicological tests, food additives are fed at dietary levels several fold in excess of the concentrations that will occur in foods destined for human consumption (Gershoff, 1995). This is done to provoke potential toxic responses and to establish safety factors. Because the amount of a fat substitute that could occur in the human diet is very large relative to other food additives such as added colors or flavors, feeding the fat substitutes at very high levels could result in spurious results, since this would require reducing a large part of the nutrients in the diet. Munro (1990) has pointed out that responses that ‘at first glance may be considered to be of toxicological significance may on further investigation be the result of dietary nutrient imbalance or physiological perturbation induced by the test material when fed at excessive exposure levels’. Diets with a large component percentage of fat substitutes could become unpalatable with a poor consumption rate leading to poor growth that might be wrongly interpreted as a sign of toxicity. Measurements of growth, of blood and urine chemistry, plus gross and histologic examination of tissues are often made. In addition, when appropriate, carcinogenicity, genotoxicity and reproductive and developmental toxicity testing may also be performed. Even if animal testing proves negative, the FDA recognizes that confirmatory studies in humans are an important part of confirming the safety of macronutrient substitutes (Gershoff, 1995). In toxicological studies, potential effects of fat substitutes that may not be evident in standard toxicological tests also need to be considered, based on

456 Modifying lipids for use in food physiological effects that may be specific to the chemical or physical properties or the mechanism or site of action of the substitute. There is also a need for confirmatory human studies in normal as well as at-risk populations, such as people with diabetes or compromised gastrointestinal (GI) tracts, or abnormalities that could possibly be caused by the fat substitute under consideration. For non-absorbable fat substitutes, effects on GI epithelium, colonic microflora ecology, bile acid physiology, pancreatic function and laxation effects should be considered (Munro, 1990; Vanderveen and Glinsmann, 1992; Glueck et al., 1994; Gershoff, 1995). For fat substitutes that are absorbed, absorption, distribution, metabolism and elimination of the substitute should be considered. The most exhaustively studied fat substitute has been Olestra. Olestra is neither hydrolyzed nor absorbed (Mattson and Volpenhein, 1972; Miller et al., 1995). Olestra is not toxic, carcinogenic, mutagenic or teratogenic and when fed to animals at doses up to 10 % of the total diet, no toxic effects on weight gain, hematology, urinalysis or tissue pathology were noted (Bergholtz, 1992). Since it is not absorbed, the only organ that olestra contacts is the GI tract. It has no significant effect on gastric emptying, total transit time, fecal water or pH of pancreas, fecal bile acids or interohepatic circulation of bile acids. It was reported that for specific GI symptoms (gas, diarrhea, abdominal cramping), there were no significant differences between humans who consumed chips with either olestra or triacylglycerols (Freston et al., 1997; Cheskin et al., 1998). Gut microflora do not metabolize olestra under anaerobic conditions, but during waste treatment, it is degraded aerobically in sludgeamended soils (Haighbaird et al., 1997; Thomson et al., 1998).

18.5 Future trends Research and development on the heat-stable fat-based fat substitutes slowed substantially after the FDA’s ruling limited fat substitutes to the ‘savory snack’ category. Olestra is still the only heat-stable fat substitute approved for food use in the USA. Frito-Lay has over half the market in the ‘savory snack’ category. Only one fat substitute, olestra (Proctor & Gamble Co.), is used in Frito-Lay products, so the market possibilities for other fat substitutes are very limited. If, or perhaps when, other product categories are approved in the USA, a limited number of other fat substitutes, such as sorbestrin or fatty acid esterified propoxylated glycerol, have a relatively good chance of further development and eventual approval. Other technologies are also being investigated, such as lipase inhibitors and identification of the compounds that control satiety in the brain. These could be marketed in the future, as well. While the best solution to the adult and childhood obesity problem in the USA is clearly an increase in exercise and a reduction in the calorie intake, most consumers seem to prefer other options. Although many companies in the US food industry respond to consumer needs, most respond best to

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consumer demands. Companies continue to produce only the products that consumers purchase. If consumers continue to purchase ‘super-size’ portions, those products and similar products will be provided. It is very likely the market for fat substitutes will remain strong for the foreseeable future.

18.6

References

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and DOBARGANES M C (1994), Isolation and characterization of sucrose polyesters, J Am Oil Chem Soc, 71, 385–390. RIVA S (1994), Enzymatic synthesis of carbohydrate esters, in Akoh C C and Swanson B G, Carbohydrate Polyesters as Fat Substitutes, New York, Marcel Dekker, Inc., 37– 64. ROBBINS M B and RODRIGUEZ S S (1984), Low calorie baked products, Patent, US 4,461,782. ROBERTS B A (1984), Oleaginous compositions, Patent, US 4,446,165. ROLLS B J (1995), Carbohydrates, fat and satiety, Am J Clin Nutr, 61, S960–S967. SANCHEZ C, KLOPFENSTEIN C W and WALKER C E (1995), Use of carbohydrate-based fat substitutes and emulsifying agents in reduced-fat shortbread cookies, Cereal Chem, 72, 25–29. SHARP T (2001), Technical constraints in the development of reduced-fat bakery products, Proc Nutr Soc, 60, 489–496. SKOV A R, TOUBRO S, RONN B, HOLM L and ASTRUP A (1999), Randomized trial on protein versus carbohydrate in ad libitum fat reduced diet for the treatment of obesity, Int J Obes, 23, 528–536. SMITH R E, FINELY J W and LEVEILLE G A (1994), Overview of Salatrim, a family of lowcalorie fats, J Agric Food Chem, 42, 432–439. ST-ONGE M P and JONES P J H (2002), Physiological effects of medium chain triglycerides: potential agents in the prevention of obesity, J Nutr, 132, 329–332. STUBBS J, FERRES S and HORGAN G (2000), Energy density of foods: effects on energy intake, Crit Rev Food Sci Nutr, 40, 481–515. THOMSON A B R, HUNT R H and ZORICH N L (1998), Review article: olestra and its gastrointestinal safety, Aliment Pharmacol Ther, 12, 1185–1200. TSO P and LIU M (2004), Ingested fat and satiety, Physiology & Behavior, 81, 275–287. USDHHS (1990), Healthy People 2000: National Health Promotion and Disease Prevention Objectives (Nutrition 2.15 Availability of reduced fat processed food), Washington, DC, US Public Health Service, USDHHS. US GOVERNMENT (1997), Code of Federal Regulations, Foods and Drugs, Title 21, Parts 170–199, Office of the Federal Register, National Archives and Records Administration, Washington, DC, US Government Printing Office. VANDERVEEN J E and GLINSMANN W H (1992), Fat substitute: a regulatory perspective, Ann Rev Nutr, 12, 473–487. VETTER J L (1993), Low-calorie bakery foods, in Altschul A M, Low-Calorie Foods Handbook, New York, Marcel Dekker, Inc., 273–291. VOLPENHEIN R A (1985), Synthesis of higher polyol fatty acid polyesters using carbonate catalysts, Patent, US 4,517,360. WHO (2002), Controlling the global obesity epidemic, available at: www.who.int.nutrition/ topics/obesity/en/. WHITE J F (1990), Partially esterified polysaccharides (PEP) fat substitutes, Patent, US 4,959,466. WHITE J F and POLLARD M R (1988), Esterified epoxide-extended polyols as nondigestible fat substitutes of low-caloric value, Patent, EP 254,547. WHITE J F and POLLARD M R (1989a), Non-digestible fat substitutes of low-calorie value, Patent, US 4,861,613. WHITE J F and POLLARD M R (1989b), Non-digestible fat substitutes of low-calorie value, Patent, EP 325,010. WHITE J F and POLLARD M R (1989c), Low-calorific and non-digestive substitute of fat/oil, Patent, China 1,034,572. WINTER D B, MEYER R S, ROOT J M and CAMPBELL M L (1990), Process for producing low calorie foods from alkyl glycoside fatty acid polyesters, Patent, US 4,942,054. WYLIE-ROSETT J (2002), Fat substitutes and health – an advisory from the Nutrition Committee of the American Heart Association, Circulation, 105, 2800–2804.

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19 Filled and artificial dairy products and altered milk fats E. Hammond, Iowa State University, USA

19.1

Introduction

Milk fat sells for a higher price than most other fats and oils, and nearly all of it is used in foods (Hammond, 2000). Milk fat owes its favored economic value to its subtle flavor, the unique flavors it generates when heated and its melting profile. These characteristics are shaped by rumen fermentation and the mammary gland (Walstra and Jenness, 1984). In the rumen the polyunsaturated fatty acids typical of the animals’ diets are hydrogenated by micro-organisms. In the mammary gland short-chain saturated fatty acids are introduced. These factors give milk fat a unique composition. Like other animal fats, milk fat requires no deodorization to make its delicate flavor acceptable, although milk may acquire undesirable flavors from silage or plants such as garlic or onion in the cows’ diet. Many of the mild flavors unique to good quality milk are inherent in its biosynthesis and others are produced during pasteurization. Heating milk fat can release methyl ketones from 3-keto esters and γ- and δ-lactones from 4- and 5-hydroxy esters. These precursors are present in small amounts in milk fat (Hammond, 1989). These flavors are of particular importance in baked goods traditionally made with milk fat. For other dairy products, particularly ripened cheese and cheeses made from lipase-treated milk, the release of flavorful short-chain free fatty acids is vital to their flavor. The role of these flavor compounds limits the use of milk fat substitutes in many dairy applications. Milk fat progresses from a fairly hard solid at refrigeration temperatures to a liquid at slightly above human body temperature (Walstra and Jenness, 1984). The amount and distribution of fatty acids in the triacylglycerols are vital for this melting profile. For fat in many edible applications, it is important

Filled and artificial dairy products and altered milk fats

463

that the fats melt completely at body temperature, so this characteristic must be repeated in milk fat substitutes in filled dairy products. The hardness of milk fat at refrigeration temperatures is regarded as a disadvantage, especially in the USA where very soft textured bread is the norm (Brunner, 1974; Bobe et al., 2003; Chen et al., 2004). Finding acceptable ways to soften the texture of cold butter has long been a goal of the dairy industry. Conversely, it is relatively easy to control the texture of margarine to be spreadable at refrigeration temperatures. The texture and melting properties of milk fat vary with the breed and the cows’ diets and, thus, these properties typically vary with season of the year (Kurtz, 1974). Cows on pasture generally give softer milk fat than those on dry feeds. Lack of uniformity in foods is always a disadvantage, and this is another advantage of margarines. Milk fat from pasture-fed cows is also yellower, but this change in butter color and in milk fat substitutes can easily be controlled by the addition of natural pigments, such as annatto or βcarotene. Nutritional considerations also affect the consumption of dairy products and their substitutes. Milk fat, like other animal fats, contains significant amounts of cholesterol, and many people are interested in limiting their cholesterol intake (Walstra and Jenness, 1984). There is interest in minimizing the cholesterol content of milk fat, but many of these techniques require isolating the fat from the dairy product. Vegetable oil substitutes for milk fat do not have significant amounts of cholesterol, and plant sterols are sometimes regarded as conferring a nutritional advantage. Milk fat is a poor source of polyunsaturated fatty acids because of rumen hydrogenation, and this particularly affects its use in infant formulas. Milk fat is regarded as one of the most atherogenic fats that is consumed in large amounts in Western countries (Ulbricht and Southgate, 1991). It is rich in saturated fatty acids, especially palmitic, myristic and lauric acids that are considered particularly atherogenic. Milk fat appears to suffer unnecessarily from laws that require labels to state the saturated fat content because it is rich in stearic acid, which is generally regarded as not being atherogenic. Likewise, the saturated fatty acids of chain-lengths four through ten are metabolized differently from longer-chain fatty acids and are regarded as non-atherogenic; however, they are all saturated and are listed as such on labels.

19.2 Filled and imitation dairy products Dairy products made with the substitution of milk fat with other fats are termed ‘filled dairy products’, ‘imitation dairy products’ or ‘dairy product analogs’. Imitation products are generally made without milk protein or milk fat, although products made with sodium caseinate are also termed imitation dairy products because the caseinate is considered a chemically modified

464 Modifying lipids for use in food product (Winkelmann, 1974). The production of filled and imitation products has been driven by scarcity of dairy products and by economics. The economic advantage of a filled product obviously depends on the amount of fat that it contains, so the first instance of a filled product was margarine, which has become sufficiently successful to merit a chapter of its own in this volume (Chapter 17). During the 1960s and 1970s, there was a great upsurge in interest in making milk fat substitutes. Hydrogenation technology had matured to the point where vegetable fats could be tailored to fit a variety of needs. Also at that time, there was a real concern that the dairy industry could not expand enough to meet future demands for dairy products (Jonas, 1975). Statistics on the consumption of filled and imitation milk are scattered, and many of them are dated, but filled and imitation dairy products captured significant proportions of the market milk, ice cream, coffee creamer and whipped topping markets in these decades. In general, they gained most in products where the fat content was relatively high, where the physical properties of milk fat were not ideal, where the flavor difference caused by substituting milk fat was not noticeable or where the instability of the dairy product to off-flavor development was a problem. These market losses caused considerable concern in dairy circles in the USA, and counter measures were considered (Hedrick, 1969). Today in some countries a trend towards natural foods has dampened enthusiasm for filled and imitation dairy products, but in other countries reconstituted milk is flourishing. In 2002 the recombined milk industry was estimated to have had a product turnover worth $5–6 billion (Sanderson, 2004). This industry had grown especially in South and Central America, China and Southeast Asia and the Middle East with major imports in powdered whole milk, skim milk and whey (Davidson, 2004). Now dairy interests often take a more relaxed stance and look on dairy products as ingredients for all kinds of food, including filled ones. Trade in these products is dominated by New Zealand, Australia and the European Union (Sanderson et al., 2004). However, lactose intolerance in Chinese and Southeast Asian populations may limit the consumption of dairy products. In a recent study in Thailand (Sirichakwal and Puwastien, 2005), lactose intolerance was measured by observing digestive symptoms (diarrhea, abdominal cramps, flatulence, etc.) during the 24 hours following the consumption of milk. The results showed no lactose intolerance in children (5–6 years), but the incidence of lactose intolerance increased with the age of subjects to 18 % for adolescents (13– 16 years), 40 % for adults (18–45 years) and 64 % for subjects 45–60 years old. Measurement of lactose maldigestion, by the presence of > 20 ppm of hydrogen in the subjects’ breath within seven hours after consuming dairy products, yielded greater percentages than the intolerance study but similar trends. Reducing the lactose in the products by fermentation or other means greatly reduced the incidence of maldigestion and intolerance.

Filled and artificial dairy products and altered milk fats

465

The 1990s have seen the advent of materials with fat-like mouth feel but zero or reduced caloric content, such as sucrose polyesters, short-chain fatty acid-rich fats, micro-particulate proteins and carbohydrates (Smith, 1995; Lindsay, 1996). Plans to have food labels report the amounts of trans fatty acid they contain are likely to have a significant effect on the acceptance of hydrogenated foods in filled dairy products, and the fats used for these purposes may require significant reformulations. Regulatory agencies usually have been concerned that filled and imitation products are not missing important nutrients that are contained in the traditional product and that the label not be deceptive. However, decisions about the acceptability of the names of products can be affected by the political influence of those with an economic interest in the outcome (Weik, 1969; Winkelmann, 1974). Many of the products used for filled and imitation dairy products have been proprietary products, and information about their constituents, fatty acid compositions, melting points and solids content at various temperatures is not readily available. Frequently these products, produced under a particular proprietary name, are prepared from different starting materials depending on price and availability, so the fatty acid composition can vary from time to time. An example of this may be seen in the data of Horvath et al. (1971) who reported on the fat in several brands of filled milk and coffee whiteners over a six-month period. Also, the materials used can vary with geographical location, and a number of countries have encouraged the use of locallyproduced fat and oil sources in making filled and imitation dairy products (Winkelmann, 1974). Thus, the fatty acid composition of milk fat substitutes can vary considerably. Table 19.1 gives the composition of the fat content and fatty acid composition of a number of filled and imitation dairy products. Often more attention is given to keeping the melting point, stability and solid fat profile of milk fat substitutes within a narrow range. The solid fat index and other properties of a number of fats used as milk fat substitutes that were manufactured by the former Durkee Company are given in Table 19.2. One of the problems with filled products made with hydrogenated fats is that they developed a unique flavor called ‘hardening flavor.’ These are particularly noticeable in products with bland flavors. There are two types of hardening flavor, one generated when a fat is hydrogenated and one that develops in hydrogenated fats that have been deodorized and then allowed to oxidize (Kawada et al., 1966). The former seem to arise from hydrogenation of oxidation products produced during processing, and the second arises from the oxidation of the fatty acids that have been isomerized during hydrogenation (Merker and Brown, 1956). Attempts to identify specific flavor compounds that are responsible for the hardening flavors have led to conflicting results that may reflect differences in hydrogenation conditions (Kawase et al., 1970; Feenstra and Meijboom, 1971; Yasuda et al., 1975).

Table 19.1

Fat percentage and fatty acid percentage composition of some filled and imitation dairy products and fats used to make them. Fatty acid

Product

Fat%

8:0

10:0

12:0

14:0

16:0

18:0

16:1

18:1

18:2

18:3

Fat type

F milk 1 2 3 4 I milk Fl I milk Pd1 2 3 F concentrate I sour cream I creamer 1 2 I creamer Pd I topping Aer I topping Pd I topping gel Polawar® E31 Confao® 5

3.4 3.2 3.5 3.4 3.7 26.4 27.2 22.3 9.2 19.6 9.2 11.2 35.6 21.2 44.7 23.8 100 100

5.3 3.3 – 0.3 6.7 5.3 0.1 – – 5.6 3.7 0.1 4.0 2.1 1.8 2.6 3.5 –

4.1 2.6 – 0.3 5.5 5.3 0.1 – – 5.5 2.9 0.1 4.4 3.0 2.6 5.0 3.0 –

64.1 41.1 0.6 0.3 46.1 42.6 0.6 – – 44.2 70.5 0.7 40.6 38.0 38.4 36.1 41.5 –

12.5 7.9 0.9 – 18.8 18.2 0.8 0.1 – 18.3 10.7 0.2 17.9 15.4 15.2 15.4 12.0 –

5.9 9.9 14.8 9.8 9.6 10.9 12.9 11.0 10.8 10.8 4.6 9.3 11.2 11.4 14.4 12.7 9.0 7.0

4.7 7.2 6.9 6.7 8.1 11.3 9.8 5.5 3.6 12.1 6.4 10.2 19.0 19.7 24.9 18.8 17.5 13.0

– – – – – – 0.8 0.3 0.4 – – – – – – 1.0 – –

3.1 27.3 54.1 82.0 5.2 5.7 62.6 28.7 25.3 3.1 1.2 79.3 2.9 9.1 1.6 5.7 13.5 63.0

0.3 0.7 21.8 0.6 – 0.5 11.5 47.6 51.4 0.3 – 0.2 – 1.1 1.2 1.5 – 6.0

– – 0.9 – – – 1.2 6.8 8.4 – – – 0.3 – – 1.0 – –

PC PCHV V HV PC PC HV V V PC PC HV PC PC PC PC PC Ra

Note: Polawar®, Bears Co., Russia; Confao®, Aarhus/Karlshamn, Sweden. a Also contains 1 % 20:0, 3 % 20:1, 1 % 22:0 and 6 % 22:0. Abbreviations: Aer = aerosol, C = coconut, F = filled, Fl = fluid, H = hydrogenated, I = imitation, P = palmkernel, Pd = powder, R = rapeseed, V = vegetable. Source: Based on the data of Posati et al. (1975) and Lausten (1986).

Table 19.2

Properties of milk fat and some milk fat substitutes. Max

Max

Solid fat index (°C)

Product

Wiley melting pt (°C)

Lovibond color

Iodine value

Form

10

21

27

33

38

43

Paramount® C Paramount® X Kaomel® Kaola Dariplus® R Dariplus® S Hydrol 92 Milk fat Dariplus® L Durkex® 100 Betrkerme

38–39 44–46 35–38 31–34 38–41 36–38 33–36 35

2.0R 2.0R 3.0R 2.5R 2.0R 2.0R 1.0R 8.0R 2.0Ra 2.0Rb 4.5R

3 3 58–63

Flake Flake Flake Plastic Plastic Plastic Plastic Plastic Semi liquid Liquid Flakes

68 69 72 40 55 34 57 33

56 58 63 18 33 18 33 14

40 50 55 8 19 13 8 10

12 27 25 <2 7 5 <3 3

<4 14 <5 0 5 <4 0 3

0 6 0

44–46

l

Note: Paramount®, Kaomel® and Durkex®, Loders Croklaan, Netherlands; Dariplus®, Parmalat, Canada. a 16:0/18:0 = 1.15 ± 0.2, saturates < 25 %, polyunsaturates > 22 %. High stability to oxidation. Source: Johnson (2004). b

0 0 0

468 Modifying lipids for use in food 19.2.1 Filled and imitation milk Typical compositions of filled milk and filled chocolate drink are given in Table 19.3, and imitation milk is given in Table 19.4. The chocolate drink can use the same fat as the filled milk but has less fat and non-fat solids and more sugar. The imitation milk has less protein than whole or filled milk, and proteins from other vegetable sources are often used, particularly soy protein isolate (Winkelmann, 1974; Jonas, 1975). Milk has a very delicate flavor, so the primary consideration in selecting a fat for filled milk is low flavor. Coconut or palmkernel oils are frequently used. These oils require a very low free fatty acids content because their free acids with chain-lengths six to ten carbons affect milk flavor at low concentrations. Table 19.1 suggests that both laurate-rich and hydrogenated oils are used in filled milk. Of the products shown in Table 19.2, Paramount® C (Loders Croklaan, Netherlands) was recommended for filled milk. Like milk fat, Paramount® C was > 96 % melted by 37.8 °C, but at lower temperatures it had more solid fat than milk fat. In filled milk, the emulsion must be stable for long periods, so a suitable emulsifier must be selected. Arenson (1969) recommended glyceryl monostearate as an emulsifier. Cordon et al. (1994) studied four commercial combinations containing mono- and di-glyceride and a variety of stabilizers. Table 19.3 Percentage composition of a typical filled fluid milk and filled chocolate drink. Ingredient

Milk

Chocolate drink

Water Skim milk solids Fat Emulsifier Carrageenan Sugar Cocoa

86.0 10.0 3.5 0.2 0.3 – –

81.1 8.0 1.5 0.35 0.05 7.0 2.0

Source: Arenson (1969).

Table 19.4

Composition of a typical imitation fluid milk.

Ingredient

%

Source

Water Protein Carbohydrate Fat Emulsifier Stabilizer Buffer Vitamins

86.6 1.0 8.0 3.0 0.4 0.8 0.2 +

Soy or caseinate Corn syrup, sucrose Coconut oil Monoglyceride Carrageenen, alginate Sodium dihydrogen phosphate A and D

Source: Waite (1973).

Filled and artificial dairy products and altered milk fats

469

All gave satisfactory oxidative and emulsion stability. They concluded that coconut oil could be replaced by hydrogenated canola oil and any of the emulsifiers were acceptable. Hanson (1980) recommends ways that dried filled milk powders can be produced using a fat melting in the range of 40– 42 °C to avoid free fat in the powder and poor wettability. He also advocated a low drying temperature followed by fluidized-bed drying. In the USA filled milk sales were not allowed in inter-state commerce, so its popularity varied widely in the various states. In the 1969 to 1974 era Hedrick (1969) reported that filled milk had been discontinued for lack of sales in some states but had captured 10–25 % of milk sales in Hawaii, 8 % in Arizona and 0.2 % in California. In the Philippines, an estimated 3600 tonnes of oils were used in filled milk in 1961 (Anon, 1969), and filled milk had captured 85 % of the market (Winkelmann, 1974). In Thailand, 90 % of the fluid milk was made with vegetable fat, and in Cambodia blends of milk fat and vegetable oils were frequently used. The cost of producing filled milk was estimated to be $0.047/L less than whole milk (Moede, 1970). The soy milk ‘Vitasoy’® (Vitasoy, USA) was popularized in Hong Kong (Jonas, 1975), but its beany flavor limited its popularity in Western markets. Soy milks are enjoying a resurgence in recent years because of better ways to limit its beany flavor and claims that the consumption of soy protein or its attendant isoflavones protects from various chronic diseases (Munro et al., 2003). Soy milk usually contains natural soybean oil as its fat component. Consumer studies in North America (Gustafson, 1969; Hedrick, 1969; Russell and Sanderson, 1971) reported that 95 % of the purchasers of filled milk rated its flavor excellent. Their primary reason for using filled milk was a $0.048–0.083/L lower price. Both whole and filled milks were preferred to the imitation milk. In chocolate drink, imitation milk made with sodium caseinate was as good as that made with whole milk. Filled milk is generally the caloric equivalent of regular milk. The vegetable oils used in filled milk have the advantage of having no cholesterol, which is present in regular milk, but the coconut and palmkernel oils used in many filled milks now are believed to be even more atherogenic than milk fat (Ulbricht and Southgate, 1991). The use of hydrogenated fat in filled milk will introduce trans double bonds, which are now believed to be atherogenic. In contrast, imitation milk is usually inferior to dairy milk in calcium and protein content and protein value. Even when the milk-derived protein, sodium caseinate, is used, the imitation milk is often lacking in the nutritionally important whey proteins of milk. The lack of calcium in imitation milks can be attributed to the sensitivity of its proteins to precipitation by calcium ion.

19.2.2 Coffee creamers (whiteners) Typical compositions of coffee creamers are given in Table 19.5 for both filled and imitation products. Since the delicate flavors of milk have little impact on creamed coffee and tea, it is not surprising that imitation creamers

Table 19.5

Typical percentage compositions of some filled and imitation dairy products. Coffee cream

Heavy cream

Sour cream

Yogurt

Whipped topping

Filled1

Imitation2

Filled3

Filled2

Filled3

Imitation2

74.6 18.0 0.2 – 7.2 – – – – –

79.3 10.0 0.6 0.3 – 2.0 7.5 0.3 – –

60.8 32.0 0.2 – 7.2 – – – – –

76.2 14.0 0.5 0.3 9.0 – – – – –

87.7 3.0 –0.5 – 8.8

62.7 25.0 0.6 0.2 –

– – –

– – 1.5 10.0

Ingredient Water Fat Monoglyceride emulsifier Stabilizer Nonfat milk powder Protein Corn syrup solids 24 DE Buffer Sodium caseinate Sugar

Source: 1 Arenson (1969), 2 Hedrick (1969) average values, 3 Lausten (1986).

Filled and artificial dairy products and altered milk fats

471

have almost completely captured the market. These cream analogs have a lower cost and superior flavor stability compared with spray dried cream. Liquid creamers, which are used primarily in the restaurant market, are usually imitation formulas. The fat-soluble flavors of the coffee or tea preferentially distribute themselves in the creamer fat, which reduces their vapor pressure and flavor impact. Whitening is affected by both the protein and fat in the creamer, so good emulsification is important (Johnson, 2004). ‘Feathering’, an unsightly coagulation of the creamer protein as it is added to coffee, is a perennial problem with coffee creamers and is caused by the acid and tannins of the coffee or tea. Buffering the creamer with phosphates helps avoid this defect. Vanness and McManus et al. (1986) compiled labeling information from 25 cholesterol-free non-dairy creamers and reported that 22 contained sodium caseinate and three soy protein. Twenty of the product labels gave a ‘contains one or more of the listed oils’ statement. Of the five not making this statement, two contained only coconut oil, two contained only soybean oil and one contained a mixture of soybean and hydrogenated coconut oils. Of the remaining 20 products 20 listed coconut oil, 19 soybean oil, 15 palm oil, 12 palmkernel oil, 11 cottonseed oil, 1 safflower oil and 1 peanut oil. The creamers contain no cholesterol as advertised, but all are likely to contain very atherogenic oils. Table 19.1 gives the fat content and fatty acid composition of both liquid and powdered creamers. Of the products listed in Table 19.2, Paramount® X was recommended for the production of creamers because of its good freeze–thaw stability and good spray-drying characteristics. Dariplus® L (Parmalat, Canada) was recommended when a less saturated creamer was desired for good mouth feel (Johnson, 2004). In 1970 the ingredients costs for dairy creamer and an imitation liquid creamer were estimated to be $0.41 and $0.11/L, respectively. The ingredients for spraydried creamer were estimated to cost $0.19/kg (Moede, 1970).

19.2.3 Cream, sour cream and yogurt Table 19.5 gives typical compositions for cream, yogurt and sour cream. These products are often made with the same fats and emulsifiers used for filled milk (Lausten, 1986). Of the products in Table 19.2, Kaomel® (Coders Croklaan) was recommended for sour cream (Johnson, 2004). These products are usually acidified directly with citric or lactic acid to pH 4.4–4.7 and thickened by the addition of gums (Weiss, 1983). Table 19.1 gives the fat content and fatty acid compositions of an imitation sour cream. Moede (1970) estimated that the ingredients for dairy sour cream cost $0.45/L compared with $0.15/L for the imitation.

19.2.4 Whipped toppings Table 19.5 gives the composition of a typical whipped topping, although the

472 Modifying lipids for use in food composition can vary over a considerable range (Winkelmann, 1974). Typical fat compositions are given in Table 19.1. Foams are inherently unstable. The foam cells are sensitive to liquid fat, so if the solid fat fraction becomes too small to hold the liquid fat, the foam collapses. Thus, in terms of composition the fat used for whipped toppings should have very high solid fat at lower temperatures but, for good mouth feel, the fat should melt almost completely at body temperature. Of the fats in Table 19.2, Paramount® C and Dariplus® R are recommended for use in whipped toppings. A comparison of their solid fat profile with that of milk fat shows that milk fat has less solid fat in the room temperature range and thus makes a less stable topping. Whipped toppings are often stored frozen, so good freeze–thaw stability is also required. This can be achieved by the addition of vegetable gum stabilizers that bind water and minimize the growth of ice crystals. Emulsifiers are also needed and a variety has been used including monoacylglycerols and sorbitol esters and their derivatives. (Weiss, 1983). Over-runs (volume of air incorporated/ volume of mix) of 250 are typical. Because of the superior foam stability and lower price of imitation whipped toppings they rapidly displaced conventional whipped cream and by 1969 had 65 % of the US market (Hedrick, 1969). Whipped topping (25 % fat) was estimated to cost $0.20/kg compared with whipping cream (30 % fat) at $0.62/kg (Winkelmann, 1974).

19.2.5 Frozen desserts Table 19.6 gives the composition of typical filled frozen products. In frozen desserts the most important characteristics of the fat are mouth feel, meltdown and stability (Hansen, 1994). Monoacylglycerols typically are used as emulsifiers and alginates as stabilizers. Of the products in Table 19.2, Kaola and Dairyplus L were typically used in frozen desserts. Bäckman et al. (1989) analyzed the fatty acid composition of six samples of Finnish dairy ice cream and four samples of filled ice cream. The dairy ice cream’s composition was typical of milk fat while the filled ice cream contained Table 19.6 Typical percentage compositions of filled frozen deserts with various fat contents. Type Ingredient

A

B

C

Fat Water Skim milk solids Corn syrup solids Stabilizer-emulsifier

4.0 62.5 13.0 6.0 0.5

6.0 62.0 12.5 6.0 0.5

10.0 61.1 11.5 5.0 0.4

Source: Hedrick (1969).

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mostly oleate, linoleate, stearate and palmitate, in general order of decreasing amounts. Two of the samples contained significant amounts of laurate and myristate. In the USA the consumption of filled ice milk was stimulated by the proliferation of soft-serve shops that sell a semi-solid product direct from a freezer at about –6 °C. As early as 1955 Whitted found 50 % of consumers using filled ice cream and 75 % saying they found it acceptable. By 1969 filled ice cream had captured 10 % of the market in several states and nearly half the market in Texas (Johnson, 2004). In 1974 filled ice cream had 90 % of the market in Sweden, 50 % in Japan and the Netherlands and 45 % in Belgium (Winkelmann, 1974). Several studies showed that other fats could be substituted for milk fat and give ice cream with an acceptable flavor (Kosikowski and Mehran, 1971; El-Safty et al., 1978; Umesh et al., 1989; Cheema and Ahora, 1991a; Im and Marshall, 1998). Homogenizer pressures on the mix should be increased and freezer conditions may require changes to preserve over-run (the volume of air incorporated into the ice cream during freezing). Price savings were calculated to range from 13–40 % (Kosikowski and Meran, 1971; Cheema and Ahora, 1991b).

19.2.6 Ghee Patel and Gupta (1983) made a simulated ghee with hydrogenated vegetable oil boiled with skim milk solids. They found that adding a small amount of cultured cream improved the flavor of the ghee as well as filled milk and spreads made from the ghee.

19.2.7 Cheeses There has been considerable interest in making filled and imitation cheeses, and more of this information is available in the literature. For filled milk cheeses, fats have been emulsified into skim milk and usually conventional cheese cultures and procedures have been used. For emulsification, homogenization has sometimes sufficed, but the use of emulsifying agents often has been advocated. Similar techniques have sometimes been used for imitation cheese, but more usually the ingredients have been blended into a dough with a minimum amount of added water and the mixture has been acidified by adding acids or cultures. Caseinates have been the primary protein in imitation cheese. Those interested primarily in cheese flavor have usually used filled milk and conventional cheese processing while those interested in cheese texture have used a dough method. Although the solids content of the fat used to make these cheeses seems to be critical for their texture (Marshall, 1990), most investigators used vegetable oils or hydrogenated oils whose solid fat content was unspecified. Several investigators made filled cheese by homogenizing fats into skim

474 Modifying lipids for use in food milk and using conventional cheese making procedures (Peters, 1956; Kristoffersen et al., 1971; Abo-El Naga et al., 1974; Foda et al., 1974; Mohamed et al., 1982). Generally, they found the texture of the filled cheese was comparable to but a little weaker than that of milk fat cheese. Peters (1956) recommended homogenizing at 35–70 kg/cm2 for optimum texture, and this has usually been followed by subsequent investigators. The filled cheese was criticized for having a bland or foreign flavor and scored lower than milk fat cheese. Foda et al. (1974) reported that cheese made from milk fat homogenized into skim milk did not taste as good as cheese made from the natural milk emulsion. The flavor of cheese made with milk fat could be improved by using gum acacia as an emulsifying agent for the milk fat. The effect of gum acacia was attributed to its effect on the fat–water interface. Cheese made with a bland mineral oil had a better flavor than cheese made with the hydrogenated fats Kaola and Kaomel® (see Table 19.2). Johnson (1991) and Whitehouse (1996) examined the flavors of emmenthaler cheese made with corn oil and high-oleic sunflower oil (HOSO), respectively. Short-chain fatty acids from butyric to lauric and in the appropriate ratio to simulate milk fat were esterified with glycerol using a toluenesulfonic acid catalyst and benzene or toluene distillation to remove water of esterification. The resulting triacylglycerols were interesterified into the cheese oils. The cheeses made with oils interesterified with short-chain fatty acids tasted better than that made with the unmodified oils but were inferior to cheese made with milk fat. The oils with short-chain acids had off-flavors that were difficult to remove completely. This flavor is believed to come from the incorporation of toluenesulfonic acid in the triacylglycerols (Jiang and Hammond, 2002). Yu and Hammond (2000a) avoided these problems with off-flavors with an improved deodorizer. Emmenthaler cheeses made with HOSO containing interesterified short-chain fatty acids was equal in flavor to that made with milk fat and significantly better than cheese made with unmodified HOSO (Yu and Hammond, 2000b). Thus, it appears that with proper controls it is possible to make filled emmenthaler cheese with excellent flavor, but so far this has not been commercialized. Iassonova and Hammond (2004) avoided the incorporation of toluenesulfonic acid into HOSO by interesterifying HOSO with ethyl esters of the short-chain acids. The residual ethyl esters of the short-chain acids and the long-chain ethyl esters formed by the interesterifcation were removed by molecular distillation. To make imitation cheese spreads based on soy protein Lee (2001) developed a dough method as well as a milk coagulation method based on the work of Hang and Jackson (1967a,b). Lee used milk fat and soy white flakes and ripened the spreads with conventional cheese starter cultures. The soy protein was more hydrophilic than casein, so she did not obtain a cheese-like texture, but the products made desirable cheese spreads. By using milk fat she was able to able to achieve a pleasant dairy-like flavor reminiscent of cream cheese. The products made by the dough method were inherently more lumpy

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and grainy than those made by milk coagulation, but the texture of cheeses made by both methods could be improved by adding small amounts of protease. Iassonova and Hammond (2004) found that substitution of milk fat with HOSO gave a bland flavored product, but when a synthetic mixture of free fatty acids, lactones and 2-ketones typical of milk fat was added, the flavor of HOSO cheese spread was indistinguishable from milk fat controls. Seemingly, unripened cheeses have little flavor development besides the release of short-chain fatty acids, ketones and lactones from milk fat. These flavor compounds can be added as synthetic mixtures or be produced from hydrolyzed milk fat. Thus, unripened cheeses make good candidates for filled and imitation cheeses. This is especially true of mozzarella, which is frequently used on pizza where strong flavors mask the flavors arising from the cheese. Foda et al. (1976) made high-salt white cheese from filled milk using corn oil. The fresh milk control tasted best, but they added no ketones, lactones or short-chain acids. Ghosh and Kulkarni (1996) made filled mozzarella using skim milk with several oils, and coagulated the milk by direct addition of citric or acetic acids. They found the flavors acceptable and the melting and stretching properties as good as those of conventional mozzarella. They noticed no texture difference with type of fat. There have been a number of texture studies on imitation mozzarella or mozzarella analogs. These usually use caseinate as the protein in order to have good melting and stretch characteristics. The cheeses are generally made on a small scale by making a dough of the ingredients. The dough is heated to melting and cast in a form for cooling (Swanson et al., 1984). This simple method has allowed many variables to be tested by microscopic, sensory and rheological tests. Jana and Upadhyay (2003) provided an excellent review about imitation mozzarella. They suggest the fat should melt at 32–43 °C and have an iodine value 65–90. Marshall (1990) studied the effect of the amounts of four fats (sunflower oil, milk fat and two hydrogenated palm oils) on a model fat system. As the amount of soft fat, sunflower oil and milk fat, increased, the stress in uniaxial compression decreased. However, with the hard, hydrogenated fats, the stress increased with increasing fat content. The fat content was correlated with many other rheological parameters, and the scores of these parameters were a function of the logarithm of the fat content. Lobato-Calleros et al. (1998) added soybean oil, milk fat and hydrogenated soybean fat mixtures to a caseinate-based analog and gave response surface diagrams for their effect on sensory and mechanical rheological properties. They found trends in agreement with Marshall’s (1990) generalizations. Narimatsu et al. (1985) suggested that the fat for imitation cheeses should be rapeseed oil or soybean oil mixed with coconut or palmkernel oil so that a solid fat index is 25–55 at 10 °C, 10–45 at 20 °C, < 25 at 30 °C and < 10 at 35 °C. Others have invariably found that fat content was an important texture variable but usually give little attention to the solid fat content (Yang and

476 Modifying lipids for use in food Taranto, 1982a,b; Emery and Pangborn, 1988; Stampanoni and Noble, 1991a,b). Guinee et al. (1999) studied the texture of dairy and analog pizza cheeses in a low-amplitude strain rheometer at various temperatures. As the temperature increased the elastic shear modulus decreased and the viscous component of texture increased. The changes with temperature were sharper in the dairy cheddar and mozzarella than in the analog pizza cheese. The detection of fat adulteration in dairy cheeses can be detected using gas chromatographic analysis of the triacylglycerols using equations proposed by Precht (1991), but Battelli and Pellegrino (1994) have shown that for cheeses with lipolysis exceeding 15 mmol/100 g fat, the suggested formula leads to inaccurate classification.

19.2.8 Infant formulas It is generally agreed that commercial infant formulas should give the same physiological responses as human breast milk, and as a result considerable attention has been given to the fat component of infant formulas (Gurr, 1997; Forsyth, 1998). Since the fat content and composition of human breast milk changes with stage of lactation, time of day, during an individual feeding and with the diet of the mother (Gurr, 1997; Forsyth, 1998; Ailhaud and Guesnet, 2004) this standard has significant variation. Evidence has been accumulating that docosahexaenoic acid should be present in infant formulas (Simopoulos, 2000; Gil et al., 2003) and the recommendations for polyunsaturated fatty acids in infant formula (Simopoulos et al., 2000) and are shown in Table 19.7. Particular attention should be given to the linoleate/linolenate ratio because these acids give rise to C20 acids which produce different prostaglandins. Ailhaud and Guesnet (2004) provided evidence that high linoleate levels may encourage childhood obesity. Telliez et al. (2002) showed that medium-chain fatty acids in infant formula can increase food intake, energy expenditure and sleep time. Breast milk triacylglycerols have palmitate on their sn-2 position whereas in most vegetable oils saturates are on the sn-1 or -3 positions (Forsyth, 1998). The distribution Table 19.7 Percentage of fatty acids in triacylglycerols to meet adequate intake recommendations for polyunsaturated fatty acids in infant formulas. Fatty acid Linoleic α-Linolenic Linoleic/linolenic Arachidonic acid Docosahexaenoic Eicosapentaenoic Source: Simopoulos et al. (2000).

10.00 1.50 6.67 0.50 0.35 < 0.10

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of the acyl groups may affect the digestion and nutritive value of the fat, but it is not clear how important this is to infant nutrition. Likewise, breast milk has considerably higher concentrations of cholesterol than most infant formulas, even those containing cows’ milk fat. It is suggested that cholesterol level during nursing may affect an individual’s ability to metabolize cholesterol later in life, but there is no consensus that cholesterol should be added to infant formulas. Table 19.8 shows the range of percentages of major fatty acids reported in infant formulas in various countries during the past 17 years and compares them with breast milk. The ranges are quite broad because of the various fats that are used. These include coconut oil, the medium-chain-length fraction of coconut oil, corn oil, high oleic sunflower oil, oleo oil from beef tallow, palm olein, soybean oil, safflower oil and sunflower oil. The major protein sources are casein hydrolysate, milk, soy protein isolate and whey. The compositions of the formulas vary with special dietary needs and whether the formula is liquid or powder. The differences in composition may reflect the effect of the date of the study and changes in dietary guidelines as well as the country of origin. Many of the formulas reflect a higher ratio of linoleic to linolenic acids than current recommendations. These studies did Table 19.8 Range of major fatty acid percentages in infant formulas in various countries compared with breast milk. Fatty acid/ location

India 1988a n=7

France 1996b n = 20

Canada 1997c n = 24

USA 2002d n = 25

Breast milke

4:0 6:0 8:0 10:0 12:0 14:0 16:0 18:0 14:1 16:1 18:1 18:2 18:3 20:4 20:5 22:6 CLA Trans Fat content

– – 0.8–6.8 1.2–4.9 1.2–35.3 8.7–15.7 11.7–41.7 2.6–10.5 1.9–2.5 0–3.5 11.9–26.0 0–11.8 0–7.4 – – – – – 6–24

0–3.59 0.21–2.41 0.82–18.6 1.35–14.04 1.56–12.01 3.86–12.05 14.55–33.29 4.19–12.34 0.28–0.99 0.07–1.54 16.75–29.97 1.33–17.54 0.52–1.63 – – – 0.15–0.97 1.97+0.28 –

– 0–0.63 0–34.79 0–18.42 0–18.19 0.03–10.05 3.48–22.88 1.41–7.54 0–0.23 0–2.00 7.46–34.53 14.48–55.09 0.69–5.32 – – – 0–0.21 0.09–3.10 –

– 0.03–0.81 0.09–12.55 0.08–10.19 6.72–17.09 3.10–6.52 6.62–0.78 1.56–7.06 0.01–0.29 0.07–1.16 28.51–40.72 13.66–21.53 1.10–2.76 – – – – 0.16–4.47 4.4–5.5

0.2 0.2 0.5 1.0 4.4 6.3 22.0 8.1 0.4 3.3 31.3 10.9 1.0 0.5 0.1 0.3 – 1.2 3.0–4.0

Abbreviation: CLA = conjugated linoleic acid. Sources: a Dood and Dutta (1988), b Chardigny et al. (1996), c Ratnayake et al. (2002), d Satchithanandam et al. (2002), e Gurr (1997).

478 Modifying lipids for use in food not measure eicosapentaenoic and docosahexaenoic acids, probably because they were completed before if was customary to fortify the formulas with these acids. Trans fatty acids were present in all the samples in which their presence was investigated. These isomers seemed to arise mostly from hydrogenation. The Life Science Research Office of the American Society for Nutritional Sciences recommends that hydrogenated fats not be used in infant formulas (LSRO 1998). Forsyth (1998) recommended the trans fatty acid not exceed 4 %.

19.3 Changing milk fat composition 19.3.1 Changing milk fat by feeding Karijord et al. (1982) analyzed the milk from 3500 cows and found that the composition was affected by stage of lactation and the herd to which they belonged. The fat percentage of the cows was positively correlated with short-chain fatty acids and negatively correlated with long-chain fatty acids. They concluded that it is possible to change the fatty acid composition of milk through cow selection. Palmquist et al. (1993) arrived at similar conclusions, but also noted that the fatty acid composition could be strongly influenced by the animals’ feed, especially the feed’s fatty acid composition. Beginning in the 1970s there was considerable interest in increasing the amount of polyunsaturated fatty acids in milk fat. This was very difficult to achieve because of rumen hydrogenation. Scott et al. (1970) showed this could be accomplished by protecting dietary fat from hydrogenation in the rumen but leaving it available for digestion in the intestinal tract. This was done by homogenizing oil with sodium caseinate followed by a treatment with formaldehyde. With this technique, it is possible to achieve percentages of linoleate in milk fat as high at 30 % compared with the range for normal milk fat of 1–4 %. But it was immediately apparent that milk and dairy products with elevated concentration of linoleate were highly susceptible to oxidized flavor development (Wong et al., 1973; Czulak et al., 1974; Badings et al., 1976). The addition of the antioxidant butylated hydroxy anisole (BHA) gave limited protection, but the addition of such additives is usually not allowed in dairy products. Feeding additional tocopherols to dairy cows was also helpful (Focant et al., 1998). Additional studies showed that more modest elevation of linoleate could be achieved by feeding oilseed that had been slightly flattened by passage between steel rollers (Middaugh et al., 1988; Stegeman et al., 1992) or extruded (Focant et al., 1998). Even modest increases in linoleate and other polyunsaturates may increase milk fat oxidation. Timmons et al. (2001) found oxidized flavor was correlated with milk linoleate, copper ion and dehydroascorbic acid concentrations. In general, an increase in milk linoleate was accompanied by a decrease in C6–C16 saturated fatty acids and an increase in stearate and oleate. These changes were accompanied by a decrease in the melting point of the milk fat and the percentage of solid

Filled and artificial dairy products and altered milk fats

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fat at various temperatures. This translated into a greater softness of dairy products and in cheese, at times, a ‘mealy’ texture. If the oxidized flavor problem was avoided, the flavor of high-linoleate products was usually blander than their regular milk fat controls, probably because of an accompanying decrease in short-chain fatty acids. More recently there has been less emphasis on increasing linoleate content and more on increasing oleate (Lin et al., 1996a) and conjugated linoleate (CLA) (Ramaswamy et al., 2001b). By feeding calcium salts of high oleic sunflower oil it is possible to partially prevent rumen hydrogenation. With this technique, it was possible to raise the oleate content of milk fat to 40 % compared with 25 % in controls. In these milks, linoleate declined slightly (Aigster et al., 2000). Butter made from such milk had normal stability to oxidation and a softer more spreadable texture, and ice cream mix was more viscous (Lin et al., 1996b; Gonzalez et al., 2003). Ramaswamy et al. (2001b) fed cows fish oil and found it slightly depressed milk fat content but increased milk fat CLA from 0.56 to 2.30 % and trans-vaccenate, a CLA precursor, from 0.86 to 4.08 %. Butter from cows fed fish oil was also softer. Consumers were not able to distinguish between milk and butter from cows fed fish oil and control milk (Baer et al., 2001; Ramaswamy et al., 2001a).

19.3.2 Changing milk fat by cow selection Bobe et al. (2003) and Chen et al. (2004) used the formula of Ulbricht and Southgate (1991) to calculate the healthfulness of milk fat as a ‘health promoting index’ (HPI). HPI = % unsaturated fatty acids / (% laurate + % palmitate + 4[% myristate]) They showed that it was possible to select cows that gave milk ranging in HPI from 0.21 to 1.65, and that cows were consistently low or high in HPI for as much as seven weeks. They made various dairy products from milk with HPIs ranging from 0.29 to 0.66 and showed that they had acceptable flavors and textures. For all the products, the texture of the high-HPI products was consistently weaker than that of the low-HPI products. High-HPI milk fat contained more stearate, oleate, linoleate and linolenate and less palmitate, myristate, laurate and shorter-chain fatty acids except butyrate. Chen et al. (2004) suggested that if a low-cost and rapid analysis of milk fatty acid composition could be developed, milk from high-HPI cows could be identified, segregated and sold for a premium to make more healthful dairy products. This could produce a market that could reward farmers and encourage the breeding and feeding of cows for more healthful milk fat composition. It appears that Fourier transform infra red spectrophotometry can provide an adequate measure of HPI (Bloomer, 2003). However, none of the technology to change fatty acid composition of milk fat by feed manipulation or genetic selection has achieved commercial production, partly because of the costs

480 Modifying lipids for use in food and diminished flexibility of feeding special diets to cows. More important has been the lack of infrastructure to analyze milk fat composition and a market that offered incentive to dairy farmers to produce milk that might be regarded as more healthful. Milk and butter with a lower saturated content improves the blood lipid profiles of humans (Noakes et al., 1996; Poppitt et al., 2002).

19.3.3 Changing milk fat composition by fractionation Milk fat can be fractionated in a number of ways: crystallization from solvents, crystallization from melted fat, critical carbon dioxide extraction, steam vacuum distillation and molecular (short path) distillation. Of these, only crystallization from melted fat has achieved commercial application, and most of this has been practised in Europe (Deffense, 1993; Kaylegian and Lindsay, 1995). Crystallization from solvents, such as hydrocarbons, alcohols, acetone, supercritical carbon dioxide and from melts give similar fractions. The solubility of triacylglycerols increases with temperature and unsaturation and decreases with triacylglycerol carbon number. In distillation, the volatility of the triacylglycerols increases with temperature and decreases with carbon number regardless of unsaturation. Generally, milk fat is separated into three to five fractions by crystallizing the fat at appropriate temperatures and separating the liquid phase. The ease of separation is affected by the rate of cooling, agitation of the fat while cooling and length of the equilibration time at the final temperature. These measures affect crystal size and polymorphic form. The separation is achieved by centrifugation or filtration using a pressure or vacuum difference across the filter. Centrifugation is often done with a water emulsion containing detergent. Re-crystallization and re-fractionation may also be practised. The fractions may then be blended to optimize physical properties for particular uses. Fractionation is monitored by refractive index, melting point, dropping point, differential scanning calorimetry, fatty acid composition and triacylglycerol composition. The dropping points of a five fraction separation were reported to be 46, 42, 24, 18 and 9 °C (Deffense, 1993). The typical dropping point of whole milk fat is 32 °C. Cholesterol and acyl groups that are precursors of lactones and ketones tend to be concentrated in lower melting fractions. Rizvi and Bhaskar (1995) outlined a scheme showing how continuous supercritical carbon dioxide processing of milk fat fractionation might be practised. This technique tends to give sharper fractionation of the liquid and solid phase because of the low viscosity of the supercritical fluid. Their calculations suggest that supercritical fractionation with carbon dioxide is economically feasible. The higher melting fractions of milk fat find their use in laminated pastries, shortenings and confectionary fat (Deffense, 1993). In pastries, they contribute lubrication between pastry dough layers and produce a layered, flaky crust.

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In shortenings, they provide a solid fat content that persists over a long temperature range and maintains aeration of the dough. In confectionary fat, they are compatible with cocoa butter and allow more milk fat to be incorporated in the confection. They also decrease the problem of chocolate bloom. High melting fractions are also suitable for use in ghee and butter in warm climates where they maintain spreadability. The lower melting fractions are used for making cookies and spreads and softening butter so it is spreadable at low temperatures. It is also possible to make butter with good spreadability by blending low and high melting fractions. To call these products butter often required a change in legislation that defines the composition of butter. Jacques et al. (1999) fed a milk fat modified by fractionation to men with normal blood lipids. The modified milk lowered the very low-density lipoproteins and total triacylglycerol contents significantly but did not change total or low-density lipoprotein cholesterol content.

19.3.4 Removing cholesterol from milk fat Atherosclerosis is often monitored by the amount of plasma cholesterol and cholesterol-containing lipoproteins. The public has been advised to limit consumption of high-cholesterol foods, and the amount of cholesterol is often given on food labels. This has stimulated interest in ways to lower the cholesterol content of animal fats. Several methods to remove cholesterol have been advocated and explored although none of them has achieved much commercial use. Schwartz et al. (1969) showed that it was possible to remove cholesterol completely from milk fat by passing the fat in solvent through a digitonindiatomaceous earth column. Courregelongue and Maffrand (1988) showed that cholesterol could be removed from fats by agitation of the melted fat with β-cyclodextrin and water. They achieved a 26–32 % removal depending on the amount of β-cyclodextrin used. Lee et al. (1999) applied the cyclodextrin process to homogenized milk, and after optimizing parameters achieved a 95 % reduction in cholesterol. Toyodo and Kihara (1989) proposed to hydrolyze the cholesterol in fat by incubating it with cells of Rhodococcus. They claimed complete removal of the cholesterol from pork fat. Martin (1989) patented several techniques for removing cholesterol that included washing, with and without solubilizers; adsorbents such as silica gel, carbon or alumina; fractional crystallization; steam distillation; and extraction with polar solvents. He considered silica gel treatment the most effective and reported 88 % reduction of cholesterol in milk fat. Short path distillation techniques have been patented in which milk fat was partially distilled alone (Mont Audoin and Rancurel, 1991) or in the presence of mono- and di-acylglycerol entrainment aids (Johnson and Conte, 1991). Cholesterol reduction of 89 % was reported. Roczniak et al. (1991) found that cholesterol forms a fat-insoluble complex with calcium or magnesium bromide that could be used to remove cholesterol from fats completely. Athnasios and Templeman (1992) reported removing

482 Modifying lipids for use in food 99 % of the cholesterol from fat by treatment with activated carbon. Fat components that have a free hydroxy group can be reacted with succinic or glutaric anhydride in the presence of acetic acid, and the hemisuccinates or hemiglutarates that result can be removed by alkali refining (Hammond and Chen, 1993; Gu et al., 1994). They achieved 60–70 % cholesterol removal. Wrezel et al. (1992) patented a similar process catalyzed with sulfuric acid. Kankare and Alkio (1993) used supercritical carbon dioxide coupled with silica gel adsorption and removed 99 % of the cholesterol from milk fat. Supercritical carbon dioxide fractionation can also concentrate lactone flavor precursors (Rizvi and Bhaskar, 1999). Kodali (1998) removed 50 % of the cholesterol from fats to an aqueous layer by mixing fats with water containing phospholipids followed by centrifugation. Beitz et al. (1999) and Li and Beitz (1996) advocated reducing the cholesterol in milk and other food by inoculating it with Eubacterium coprostanoligenes, which reduces cholesterol to coprostanol. The coprostanol is very poorly absorbed in human gastrointestinal tracts. The micro-organism can also colonize the consumer’s gastrointestinal tract and further reduce both the exogenous and endogenous cholesterol. Application of these techniques to milk fat has various limitations. Cyclodextran treatment can remove free fatty acids and vitamins along with cholesterol. Steam distillation tends to produce and remove ketones and lactones from their precursors in the milk fat. The succinic anhydride method removes lactone precursors and tocopherols as well as cholesterol. Methods that use adsorbants are likely to cause losses of vitamins and flavor precursors.

19.4

References

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and HASSAN H N (1974), Attempt in use of skim milk powder and filled milk for the manufacture of cheddar cheese, Sci Tec Latt Casearia, 25(5), 187–201. AIGSTER A, SIMS C, STAPLES C, SCHMIDT R and O’KEEFE S F (2000), Comparison of cheese made from milk having normal and high oleic fatty acid compositions, J Food Sci, 65(5), 920–924. AILHAUD G and GUESNET P (2004), Fatty acid composition of fats is an early determinant of childhood obesity: a short review and an opinion, Obes Rev, 5(1), 21–26. ANON (1969), Imitation milk and substitute milk products, Mont Bull Agric Econ, 18(9), 13–14. ARENSON S W (1969), Imitation dairy products – their formulation, processing and quality control, Food Eng, 41(4), 76–79. ATHNASIOS A K and TEMPLEMAN G J (1992), Removal of sterols and saturated fatty acids from fats with activated carbon, Patent, US 5, 061, 117. BÄCKMAN C, LILLEBERG L and ESKO U-R (1989), The fatty acid composition of ice creams sold in Finland, Finn J Dairy Sci, 47(1), 58–52. BADINGS H T, TAMMINGA S and SCHAAP J E (1976), Production of milk with a high content of polyunsaturated fatty acids 2. Fatty acid composition of milk in relation to the quality of pasteurized milk, butter and cheese, Neth Milk Dairy J, 30(2), 118–131. BAER R J, RYALI J, SCHINGOETHE D J, KASPERSON K M, DONOVAN D C, HIPPEN A R and FRANKLIN S

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T (2001), Composition and properties of milk and butter from cows fed fish oil, J Dairy Sci, 84(2), 345–353. BATTELLI G and PELLEGRINO L (1994), Detection of non-dairy fat in cheese by gas chromatography of triglyericdes, Ital J Food Sci, 4, 407–419. BEITZ D C, YOUNG J W, LI L and BUHMAN K K (1999), Oral administration of coprostanol producing microorganisms to humans to decrease plasma cholesterol concentration, Patent, US 5,972,685. BLOOMER S (2003), personal communication. BOBE G, HAMMOND E G, FREEMAN A E, LINDBERG G L and BEITZ D C (2003), Texture of butter from cows with different milk fatty acid compositions, J Dairy Sci, 86(10), 3122– 2127. BRUNNER J R (1974), Physical equilibria in milk: the lipid phase, in Webb B H, Johnson A H and Alford J A, Fundamentals of Dairy Chemistry, 2nd edn, Westport, CT, AVI, 474– 602. CHARDIGNY J M, WOLFF R L, MAGER E, BAYARD C C, SÉBÉDIO M L and RATNAYAKE W M N (1996), Fatty acid composition of French infant formulas with emphasis on the content and detailed profile of trans fatty acids J Am Oil Chem Soc, 73(11), 1595–1601. CHEEMA A S and AHORA K L (1991a), Manufacture of filled ice-cream, Indian J Anim Sci, 61(3), 316–323. CHEEMA A S and AHORA K L (1991b), Cost estimation for filled ice-cream, Indian J Anim Sci, 61(7), 742–746. CHEN G, BOBE G, ZIMMERMANN S, HAMMOND E G, LUHMAN C M, BOYLSTON T D, FREEMAN A E and BEITZ D C (2004), Physical and sensory properties of dairy products from cows with various milk fatty acid compositions, J Agric Food Chem, 52(11), 3422–3428. CORDON J, JEON I J, ROBERTS H A and SENECAL A G (1994), Effect of stabilizers and partially hydrogenated vegetable oils on the stability and quality of filled milk, J Food Process Preserv, 18(1), 61–72. COURREGELONGUE J and MAFFRAND J P (1988), Removal of cholesterol from animal fat by complexation with cyclodextrin, Patent Fr 2 601 959A1. CZULAK J, HAMMOND, L A and HORWOOD, J E (1974), Cheese and cultured dairy products from milk with high linoleic acid content, Aust J Dairy Technol, 29(8), 124–128. DAVIDSON R M (2004), Product trends, challenges and opportunities for recombined dairy products-South East Asia perspective, in Proc 4th International Symposium on Recombined Milk and Milk Products, Arlington VA, US Dairy Export Council, 60–65. DAY E A (1967), Cheese Flavor, in Schultz H W, Day E A and Libbey L M, The Chemistry and Physiology of Flavors, Westport, CN, AVI, 331–361. DEFFENSE E (1993), Milk fat fractionation today: a review, J Am Oil Chem Soc, 70(12), 1193–1201. DOOD N S and DUTTA S (1988), Fat content and fatty acid composition of infant and weaning foods, J Food Sci Technol, 25(5), 319–320. EL-SAFTY M S, KHALIFA M Y, ELHAMI M and NOFAL A A (1978), Use of hydrogenated oils in ice cream making, Egypt J Dairy Sci, 6(1), 81–90. EMERY D and PANGBORN R-M (1988), Influence of fat, citric acid and sodium chloride on texture and taste of a cheese analog, Sci aliments, 8(1), 15–32. FEENSTRA W H and MEIJBOOM P W (1971), 2-trans-Nonenal, the hardened flavor present in hydrogenated peanut oil, J Am Oil Chem Soc, 48(11), 684–685. FOCANT M, MIGNOLET E, MARIQUE M, CLABOTS B, BREYNE T, DALEMANS D and LARONDELLE Y (1998), The effect of vitamin E supplementation of cow diets containing rapeseed and linseed on the prevention of milk fat oxidation, J Dairy Sci, 81(4), 1095–1101. FODA E A, HAMMOND E G, REINBOLD G W and HOTCHKISS D K (1974), Role of fat in flavor of cheddar cheese, J Dairy Sci, 57(10), 1137–1142. FORSYTH J S (1998), Lipids and infant formulas, Nutr Res Rev, 11(2), 255–278. GHOSH B C and KULKARNI S (1996), Low cholesterol mozzarella cheese technology standardization, J Food Sci Technol, 33(6), 488–492.

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486 Modifying lipids for use in food MUNRO I C, HARWOOD M, HLYWKA J J, STEPHEN A M, DOULL J, FLAMM W G

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and PUWASTIEN P, personal communication. (1995), Labeling and the law, World Ingredients, (Sept/Oct), 20–25. STAMPANONI C R and NOBLE A C (1991a), The influence of fat, acid, and salt on the perception of selected taste and texture attributes of cheese analogs: A scalar study, J Texture Stud, 22(4), 367–380. STAMPANONI C R and NOBLE A C (1991b), The influence of fat, acid and salt on the temporal perception of firmness, saltiness and sourness of cheese analongs, J Texture Stud, 22(4), 381–392. STEGEMAN G A, BAER R J, SCHINGOETHE D J and CASPER D P (1992), Composition and flavor of milk and butter from cows fed unsaturated dietary fat and receiving bovine somatotropin, J Dairy Sci, 75(4), 962–970. SWANSON A M, WOHLT E E and SWANSON R J (1984), Method for making a process cheese analog, Patent, US 4,459,313. TELLIEZ F, BACH V, LEKE A, CHARDON K and LIBERT J-P (2002), Feeding behavior in neonates whose diet contained medium-chain triacylglycerols: short-term effects on thermoregulation and sleep, Am J Clin Nutr, 76(5), 1091–1095. TIMMONS J S, WEISS W P, PALMQUIST D L and HARPER W J (2001), Relationships among dietary roasted soybeans, milk components and spontaneous oxidized flavor of milk, J Dairy Sci, 84(11), 2440–2449. TOYODA K and KIHARA H (1989), Cholesterol-hydrolyzing enzyme and Rhodococcus producing the same for reducing cholesterol in fat, Patent, JP 01 229 097 A2. ULBRICHT T L V and SOUTHGATE D A T (1991), Coronary heart disease: Seven dietary factors, Lancet, 338(8773), 985–992. UMESH A R, ATMARAM K and JAYAPRAKASHA H M (1989), Utilization of vanaspathi in preparation of filled soft serve ice cream, Cherion, 18(3), 118–123. VANNESS A F and MCMANUS B M (1986), Cholesterol-free nondairy creamers: compositional conundrums and cardiovascular contradictions, New England J Med, 341(10), 651. WAITE R (1973), Manufactured milk, Nutr Food Sci, 1973(30), 6–8. WALSTRA P and JENNESS R (1984), Dairy Chemistry and Physics, New York, John Wiley and Sons, Inc. WEIK (1969), Food and Drug Administration attitudes on imitations, J Milk Food Technol, 32(11), 448–452. WEISS, T J (1983), Food Oils and Their Uses, West Port, CN, AVI, 295–302. WHITEHOUSE F K (1995), Factors affecting the flavor development of Swiss cheese, Ph.D. Thesis, Iowa State University. WHITTED S F (1955), Dairy substitutes in Missouri, Bull Missouri Agric Exp Sta, 658, [Dairy Sci Abst, 21, 1128]. WINKELMANN F (1974), Imitation Milk and Imitation Milk Products, Rome, FAO. WONG N P, WALTER H E, WESTAL J H and LACROIX D C (1973), Cheddar cheese with increased polyunsaturated fatty acids, J Dairy Sci, 56(10), 1271–1275. WREZEL P W, KRISHNMURTHY R G and HASENHUETT G L (1992), Method for removing cholesterol from edible oils, Patent, US 5,128,162. YANG C S T and TARANTO M V (1982a), Morphological and textural comparisons of soybean mozzarella cheese analogs prepared with different hydrocolloids, Food Microstruct, 1(2), 223–231. YANG C S T and TARANTO M V (1982b), Textural properties of mozzarella cheese analogs manufactured from soybeans, J Food Sci, 47(3), 906–910. YASUDA K, PETERSON R J and CHANG S S (1975), Indentification of volatile flavor compounds developed during storage of a deodorized hydrogenated soybean oil, J Am Oil Chem Soc, 52(8), 307–311. YU L and HAMMOND E G (2000a), The modification and analysis of vegetable oil for cheese making, J Am Oil Chem Soc, 77(9), 911–915. YU L and HAMMOND E G (2000b), Production and characterization of a Swiss cheese-like product from modified vegetable oils, J Am Oil Chem Soc, 77(9), 917–923. SIRICHAKWAL P SMITH R

20 Chocolate and confectionery fats S. Norberg, AarhusKarlshamn (AAK), Sweden

Modified lipids are used in the majority of chocolate and confectionery applications. This section covers the use of modified fats in areas such as chocolate compounds, filling fats in pralines, aerated products and cold products such as ice cream toppings. The first part of the chapter discusses lipid use in general and the effects of lipids on production capability, the quality of lipids required for different applications and their effect on the sensory properties of the end product. Production economics is often related to such issues as price, speed of production and equipment requirements, which in turn are related to the raw materials and their ability to crystallize rapidly. The quality is related to the capacity of the fat to remain stable in terms of appearance, texture and taste. Finally, the sensory properties can briefly be described as appearance, smell, taste and the role that fat plays in mouth feel with regard to texture and melt off properties. The second part of the chapter is focussed on special problems encountered in chocolate applications, such as bloom and migration. Fat bloom is still a problem in the industry. This shortens the shelf life of the end products and makes life difficult for product development. Fat migration is one of the causes of bloom, but it will also soften the products during storage.

20.1 Introduction: cocoa butter and the use of modified lipids in chocolate and confectionery Chocolate applications comprise several types of products and include everything from plain chocolate to pralines and coated biscuits. Plain chocolate

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is a suspension of sugar and cocoa solids in fat. Bars of plain chocolate are produced by moulding. Melted chocolate is added to moulds followed by a cooling step. The chocolate solidifies when the temperature decreases. Pralines, normally filled with interior products, are often called fillings and can be produced in different ways, such as moulding and coating (the same as enrobing). For the traditional praline, the shell (the chocolate) is produced in the mould. The filling is then added and finally a bottom layer of chocolate conceals the praline. In coated pralines, the filling is produced first followed by a coating step. One common practice is that the filling is transported through a curtain of liquid chocolate. The liquid chocolate covers the filling and continues to a cooling tunnel where the chocolate solidifies. In this chapter, discussions about coating are applicable for shells and vice versa. Over the years, cocoa butter alternatives have gained status as reliable and useful raw materials for chocolate. Cocoa butter alternatives have been divided into three classes according to the fat’s similarity to cocoa butter. These are CBE (cocoa butter equivalent), CBR (cocoa butter replacer) and CBS (cocoa butter substitute). The popularity of cocoa butter alternatives is primarily based on their ability to enhance end product properties in different applications as well as to improve the price structure and quality (Wilson and Pease, 1999). Fat accounts for one-third of the content of chocolate. It is of considerable importance for the quality of the chocolate as it influences processing conditions such as tempering and cooling. The type of fat used also makes a great deal of difference to the consumer. It has a major impact on the eating qualities of the final product, including melting behaviour, flavour release and consistency. Finally, the choice of fats in confectionery products is crucial for their shelf life. Factors such as fat bloom and fat migration in composite products are greatly influenced by the fat or combination of fats used in the product. Fat-based fillings, such as in pralines, often contain both nut oil and vegetable fats. The latter is used to accentuate the mouth feel and sensory properties of the filling. It can also help to improve both bloom stability and production economy.

20.2 Preparation and use of alternatives to cocoa butter 20.2.1 Coating and shells Most moulded and coated applications have cocoa butter as the original fat. Cocoa butter is thus the measure by which all cocoa butter alternatives are compared. From this point of view, it is natural to start the discussion with cocoa butter.

490 Modifying lipids for use in food Tempered systems Cocoa butter Cocoa butter has a very special triacylglycerol composition, which gives it its special texture and sensory properties. There are three dominant triacylglycerols in cocoa butter, POP, POSt and StOSt (where P = palmitic acid, O = oleic acid and St = stearic acid) (Wennermark, 2002). Despite the fact that these symmetrical triacylglycerols account for more than 80 % of the triacylglycerol composition, the crystallization behaviour is surprisingly complicated (Schlichter-Aronhime, 1988). It has been shown that cocoa butter can crystallize in six different polymorphic forms, called forms I–VI (van Malssen et al., 1999). Forms I and II are obtained with rapid cooling to a low temperature while forms III and IV are formed at moderate temperatures. In practice, the two most stable forms, forms V and VI, are not achieved directly from a complete melt, but only via one of the metastable polymorphic states. The melting points of the crystal forms are summarized in Table 20.1 (Loisel et al., 1998a). Oleic acid and the saturated fatty acids do not associate readily in the fat crystal due to steric factors; oleic acid is kinked while the saturated chains are straight. This lack of fit between saturated and unsaturated chains promotes a triple chain-length structure (TCL). The formation of the TCC polymorph is a slow process, facilitated by temperature cycling in order to drive the transformation. In practice, the tempering process is a combination of a temperature program and shearing. First, the cocoa butter is solidified into a β′, double-layer structure (a rapid process), normally between 26 and 28 °C. When enough crystals are obtained, the chocolate is heated to a temperature around 31–33 °C. At higher temperatures the β′ crystals melt. Some crystals are rearranged from β′ double chain-length into β triple chainlength, the so-called form V cocoa butter. When sufficient form V is formed (normally 0.5–1 %), the chocolate can be cooled and the rest of the fat crystallizes smoothly into form V. The tempering and subsequent crystallization process influences the quality of the chocolate with respect to bloom stability Table 20.1

Polymorphic forms of cocoa butter. Form

Melting point (°C) Polymorphism Long space

I

II

III

IV

V

VI

17 β′ – subα 2L

23 α 2L

25 β ′2 2L

27 β 1′ 2L

34 β2 3L

36 β1 3L

Note: The nomenclature is based on the nomenclature of Wille and Lutton. α represents the hexagonal, β′ the orthorhombic and β the triclinic unit cell. The indexes (β1 /β2 ) relate to the two different βforms found in chocolate. 2L/3L = double chain length (DCL) and triple chain length (TCL), respectively. Source: Loisel (1998a).

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and sensory properties such as texture, melting behaviour and cooling sensation. It has been shown that the cocoa butter triacylglycerols, POP, POSt and StOSt, crystallize in a high-melting fraction consisting mainly of StOSt and POSt, while the low-melting fraction contains more POP and POSt. The different triacylglycerols and combinations of them influence the properties. It has been reported that StOSt improves bloom stability. For instance, Timms described in an accelerated test how the StOSt-level was proportional to the number of cycles before bloom (Timms, 2003a). The storage temperatures in these accelerated tests follow a cycle of low temperature, normally around room temperature, and high temperature around 30 °C. Typical time for a complete cycle includes storage for 12 hours at each temperature. The fat then partly melts at the higher temperature but re-crystallizes at the lower temperature. Eventually bloom occurs, and the number of cycles is a measurement of bloom stability – the more cycles the better. The positive effect of shearing has been known for a long time. Stapley used shear rate and a temperature-controlled tempering device to study the effect of processing on milk chocolate (Stapley et al., 1999). A sufficiently high shear rate combined with a careful selection of holding times and temperatures facilitated the formation of the desired crystal forms. Loisel has shown that the crystallization during tempering occurs in two steps above 26 °C (Loisel et al., 1998b). In the first step, a fraction, rich in saturated triacylglycerols, crystallizes followed by a second step containing the bulk of the other triacylglycerols. The amount of trisaturated fat only affected the first part, while the bulk crystallization was influenced by minor components such as diacylglycerols, lecithin and other emulsifiers. CBE/CBI – Cocoa butter equivalent and cocoa butter improvers Cocoa butter equivalents and cocoa butter improvers contain the same symmetrical triacylglycerols as cocoa butter, POP, POSt and StOSt, and are thus miscible with cocoa butter (Table 20.2). CBE fats can be used in all applications, from plain chocolate and milk chocolate to pralines. Since CBE fats are similar to cocoa butter, any proportion with cocoa butter can be used (Fig. 20.1). CBEs and CBIs are either produced by enzymatic interesterification and/or are refined fractions of exotic plants such as illipe, palm, shea, mango, sal or kokum. The ratio between the three SOS (S = saturated acyl group) triacylglycerols varies between the different species, and thus makes it possible to optimize the properties sought and to overcome the natural variation of cocoa butter. The melting points of the β form for POP, POSt and StOSt are 37, 36 and 43 °C, respectively. This implies that StOSt-rich butters such as shea stearin can be used to improve the heat stability (Table 20.3). In turn, palm mid-fractions, containing mainly POP, are often used to give a softer texture. In milk chocolate with high amounts of milk fat, StOSt-rich CBEs, also named CBIs, can help to restore the softening effect of the milk fat. In other cases, CBEs are used to produce a

492 Modifying lipids for use in food Table 20.2

Triacylglycerol composition of different plant species.

Cocoa butter – West Africa Palm mid-fraction Shea stearin Illipe butter Sal fat Kokum Mango stearin

POP

POSt

StOSt

17 65 1 10

39 13 8 36 11 5 19

26 2 69 42 42 74 58

2

StOA 2 3 4 13 5

Other 16 20 19 8 34 8 16

Note: Typical triacylglycerol composition of cocoa butter and cocoa butter equivalents complying with the 5 % rule within the European Union. The chocolate directive allows the use of fats from the palm, shea, illipe, sal, kokum and mango to substitute cocoa butter as 5 % of the total formulation. Abbreviations: POP = 1,3-dipalmitoyl-2-oleoylacylglycerol, POSt = 1(3)-palmitoyl-2-oleoyl-3(1)stearoylacylglycerol, StOSt = 1,3-distearoyl-2-oleoylacylglycerol, StOA = 1(3)-stearoyl-2-oleoyl3(1)-arachidoylacylglycerol. Source: Wennermark (2002). According to the 5 % rule: EU (2000). 100

100 10 °C

90

20 °C 25 °C

80

Solid fat content (%)

70

90 80 70

27.5 °C 60

60

50

50 40

40

30 °C 30

30

20

20 32.5 °C

10

10 35 °C 0 100/0

Fig. 20.1

80/20

60/40 40/60 20/80 CBE hard/cocoa butter

0 0/100

Mixing diagram of cocoa butter equivalent (CBE) and cocoa butter (CB).

more economical recipe. Normally, CBEs and CBIs are supplied by speciality fat companies, which know how to optimize the application of these products. The recommended level to obtain improved heat stability is 15–30 % CBI of the fat phase (Table 20.4). In most countries chocolate is covered by ‘vertical food standards’, which tell what ingredients are permitted and to what extent they can be used. For

Chocolate and confectionery fats Table 20.3

493

Melting profiles of cocoa butter and cocoa butter equivalents.

Temp (°C)

Cocoa butter

CBE – soft

CBE – hard

CBI

10 20 25 27.5 30 32.5 35 40

89 82 75 68 50 17 0 0

94 85 72 62 38 8 0 0

89 85 77 71 58 30 5 0

92 83 79 76 69 48 27 0

Note: Typical solid fat content for typical cocoa butter from West Africa and common cocoa butter equivalents (CBE). CBE – soft is cocoa butter equivalents with a high level of palm fractions, while CBE – hard represents a product with higher level of shea stearin. Cocoa butter improver (CBI) contains mainly StOSt. The solid fat content is measured using nuclear magnetic resonance (NMR) for tempered fats at AarhusKarlshamns AB. Source: Petersson, (1985).

Table 20.4 Comparison of the solid fat content in cocoa butter alone or mixed with various cocoa butter equivalents. Temp (°C)

CB

CB and soft CBE

CB and hard CBE

CB and CBI

CB and MF (85/15)

CB, MF and CBI (70/15/15)

10 20 25 27.5 30 32.5 35 40

91 89 83 71 40 11 0 0

90 86 80 67 36 9 0 0

91 88 83 71 38 10 0 0

92 87 81 70 40 15 1 0

80 62 56 46 31 8 0 0

80 62 55 45 31 11 0 0

Note: These are typical mixtures of CB and CBE complying with the 5 % rule in Europe. The solid fat content is measured using nuclear magnetic resonance (NMR) at AarhusKarlshamns AB. Abbreviations: CB = cocoa butter, MF = milk fat, CBE = cocoa butter equivalent, CBI = cocoa butter improver. Source: Petersson (1985).

instance, to be called chocolate, a minimum level of cocoa solids is required. Cocoa solids include dry material originating from the cocoa bean, such as cocoa powder, cocoa butter and cocoa liquor. However, there can also be an upper limit for some ingredients. In Europe legislation allows 5 % of total formulation from defined species of vegetable fats as long as the cocoa solids and similar prime ingredients are in accordance with the legislated definition (EU, 2000). Similar regulations are found in other countries. Non-tempered systems Compound coatings, based either on CBR or CBS, have the advantage of high throughput production, which has a positive effect on the manufacturing

494 Modifying lipids for use in food economy. These fats have a completely different crystallization pattern compared to cocoa butter. In contrast to cocoa butter, these fats tend to crystallize in double-layer β′ crystals directly from the melted state on cooling. Since the crystals obtained are stable in the β′, and no re-crystallization occurs, no tempering is needed. In general, these non-tempered systems benefit from shock cooling, which favours the formation of small crystals. Small crystals in turn give excellent gloss and are usually associated with good bloom stability. CBR – Cocoa butter replacer Cocoa butter replacers have found favour among chocolate producers due to their bloom-stable properties and manufacturing economy. CBRs are mainly used in enrobed and coated biscuits and fillings. The improved heat stability and bloom stability achieved by CBR have proved to be particularly useful in warmer climates. These fats are traditionally based on hydrogenated oils from soya beans, rape, palm or cotton seeds, which have mainly 16:0, 18:0 and 18:1 cis and trans fatty acids. Recipes based on both cocoa powder and cocoa liquor are commonly formulated with CBR. In recipes based on cocoa powder, the fat phase consists almost entirely of vegetable fats. The cocoa powder contains small amounts of cocoa butter which makes up to around 6 % of the fat phase in a standard recipe. Cocoa liquor contains a high level of fat and is richer in flavour compared to cocoa powder. To formulate a compound coating rich in cocoa liquor flavour, some cocoa powder can be exchanged with up to 8 % of cocoa liquor of the total recipe. Such a recipe increases the amount of cocoa butter to about 20 % of the total fat phase. CBRs normally allow up to 20 % of cocoa butter in the fat phase without any problem (Fig. 20.2). The setting time is prolonged with increased amount of cocoa butter. The tolerance between CBR and cocoa butter allows them to be used in the same production units without any risk if mixed. Fractionated varieties of the hydrogenated vegetable fats have the same properties but with better mouth feel. The fractionation process allows formulation of CBRs with lower solid fat contents at 35 and 40 °C, which explains the better melting properties (Table 20.5). The sensory properties of chocolate are strongly related to the amount and type of fat in the recipe. The demand of the fat is that it should not melt when touched, i.e. at the temperature of the finger tips, but rapidly melt in the mouth. Triacylglycerols which do not melt in the mouth give a waxy mouth feel and thus poor melt off properties. The solid fat content can to some extent explain the texture and melt off properties of a chocolate recipe. Generally speaking, the solid fat content at room temperature is associated with the hardness of the product, while the amount of solids at temperatures above 30 °C relates to the melt off properties of the fat. Cocoa butter replacers are found in most chocolate applications. Recipes based on non-tempering CBRs have low viscosity, fast crystallization and, with their elastic properties, they are especially popular for coating, giving high gloss and gloss retention. Milk fat shows no eutectic effect with CBRs,

Chocolate and confectionery fats

495

100

100 10 °C

90

90 Solid fat content (%) 80

20 °C

80

70

25 °C

70

60

60

50

50

27.5 °C

40

40

30

30 30 °C

20

20

10

10 32.5 °C 0 100/0

Fig. 20.2

Table 20.5

35°C 80/20

60/40 40/60 CBR/cocoa butter

20/80

0 0/100

Mixing diagram of fractionated cocoa butter replacer (CBR) and cocoa butter.

Typical solid fat content values for non-tempered fats.

Temp (°C)

CBR type 1

CBR type 2

CBS type 1

CBS type 2

10 20 25 30 35 40

92 78 63 44 16 0

95 90 80 50 4 0

95 85 65 30 12 5

97 95 90 50 2 0

Note: Type 1 represents standard CBR/CBS quality based on hydrogenated fats. Type 2 represents high quality CBR/CBS based on hydrogenated and fractionated fats. The solid fat content is measured using NMR-IUPAC S (2.150b) at AarhusKarlshamns AB. Abbreviations: CBR = cocoa butter replacer, CBS = cocoa butter substitute.

but since it is soft, a practical limit is 20 %. The same reasoning goes for nut, almond and other common oils. To obtain even more flavour release, cocoa liquors are mixed in the formulations. Despite the eutectic effects of CBR/ CB systems, CBR allows 20 % of cocoa butter in the fat phase without problems.

496 Modifying lipids for use in food The direct crystallization into the β′ makes it possible to achieve a highly glossy appearance. By shock cooling of the product, small crystals are obtained which in turn improves the gloss and bloom stability. In moulded applications, shock cooling of CBR also improves the demoulding. When crystallizing a CBR in a cooling tunnel, the temperature should be around 6–8 °C. The temperature at which the CBR leaves the cooling tunnel should be close to the ambient temperature, for instance around 16 °C, to avoid condensation of moisture. CBS – Cocoa butter substitute Lauric fats, from palm kernel and coconut, are widely used as a substitute for cocoa butter. Both hydrogenated and fractionated products are found on the market. CBS is used in most applications where cost is important. The excellent melting properties and quick crystallization make CBS suitable for moulded products and where a thin coating is required. These fats contain a high amount of 12:0 (lauric acid), and are not miscible with cocoa butter. It is not recommended to include more than 5 % cocoa butter in the fat phase of a CBS-type of formulation (Fig. 20.3). Lauric fats have also been associated 100

10 °C

100

90

20 °C

90

Solid fat content (%) 80

80

70

70 25 °C

60

60

50

50 27.5 °C

40

40

30

30 30°C

20

10 0 100/0

Fig. 20.3

20

10

32.5 °C 35 °C 80/20

60/40 40/60 CBS/Cocoa butter

20/80

0 0/100

Mixing diagram with fractionated cocoa butter substitute (CBS) and cocoa butter.

Chocolate and confectionery fats

497

with soapy tastes related to hydrolysis to lauric acid. On the other hand, the melting properties of CBS are better than for CBR. The initial glosses of CBS formulations are good. However, their sensitivity to mechanical handling and development of bloom during storage below room temperature make them less robust than CBR.

20.2.2 Filling fats The appearance and the sensory properties of a filling need to be carefully selected in order to meet consumer expectations. Since fat is the main component affecting these properties in confectionery fillings, the choice of fat is of major importance. Furthermore, the shelf life and process parameters need to be considered when choosing the fat. It must be possible to produce the fillings in the equipment available and the fillings must remain unchanged during different storage conditions. The sensory properties relate to sensations such as hardness, texture, melting behaviour and flavour release. It is not merely the filling itself that will affect the final sensory properties. Besides adding to the flavour sensation, other components will contribute to building up the fat phase, for instance cocoa powder or liquor, milk powder, nuts and other fat containing ingredients. Normally the fillings contain considerable amounts of liquid oil (Table 20.6), which softens the shell or coating as well as increasing the risk for bloom. The relation between softening and bloom is discussed later. Fat-based confectionery fillings include various praline fillings such as nougats, truffles, yoghurt fillings or chocolate spreads. The amount of fat in the filling often constitutes 30–40 % of the filling, and the choice of fat contributes substantially in giving the filling either a hard and cooling effect or a creamy and soft eating sensation. Tempered systems Tempered fillings often contain fats based on palm fractions, shea butter or illipe. These fats are normally used in fillings where a cooling sensation is important and/or cocoa liquor is included in the recipe. The compatibility Table 20.6 Melting profile of soft, medium and hard filling fats for chocolate applications. Temp (°C)

Soft

Medium

Hard

20 25 30 35

35 20 7 2

45 30 12 3

60 40 20 3

Note: Typical values of the solid fat content for soft, medium and hard filling fats. The solid fat content was measured by NMR IUPAC S (2.150b) at AarhusKarlshamns AB.

498 Modifying lipids for use in food with cocoa butter is high since they contain similar triacylglycerols. Fillings based on these fats normally allow higher amounts of nut oil. To obtain the best results, tempering should be used. The tempering process enhances the sensory properties, with respect to the cooling sensation, increasing the hardness and improving the bloom stability as well as resulting in less waxy mouth feel (Fig. 20.4). Non-tempered systems Traditionally, hydrogenated fats have dominated non-tempered fillings. However, low- and non-trans filling fats are now being supplied by speciality fat companies. Also fillings based on lauric fats have properties suitable in non-tempering systems. Traditional filling fats are based on hydrogenated rapeseed, soya bean and palm oil. The hydrogenation of soya bean and palm oil makes them stable in the β′ form and they crystallize rapidly directly into this polymorph. Hydrogenated rapeseed oil is not stable in the β′ form, but by increasing the content of 16:0 in the system this form is stabilized. The features discussed ensure that these products have robust and easy to use properties, which have been widely appreciated. Both hard and soft fillings can be obtained. Low- and non-trans fillings The interest in avoiding trans fatty acids has increased every year since 2000, and this includes the chocolate and confectionery industry. The increased interest in low-trans products is based on the current health concern correlated to trans fatty acids, which is reflected in public opinion and in legislation, the latter especially in Denmark and the USA. In the USA the requirement to label trans fatty acid contents came into effect on January 2006. The Waxiness Tempered Non-tempered

Toughness

Thickness

Sticky

Hardness

Totally melted

Early melt start

Rapid melting

Fig. 20.4

Cooling sensation

Sensory comparison of filling fat.

Chocolate and confectionery fats

499

European Food Safety Authority (EFSA) have performed a study, ‘the transfair study’, and concluded that while the trans fatty acid intake is not a specific subject for concern, the amount of saturated and trans fatty acid as a whole should be decreased (EFSA, 2004). Denmark, however, has passed legislation to ban products containing trans fatty acid levels above 2 % of the total fat phase of the product. The trans fatty acids of milk fat origin are excluded from the 2 % rule. Many speciality fat companies have started to supply non- and low-trans filling fat solutions, often based on palm oil and its fractions. The first generation of non- and low-trans filling fats, on the market today, can offer the same qualities as traditional filling fats from the consumer point of view, e.g. sensory, appearance and stability. However, they show a different crystallization pattern compared to traditional filling fats. Three factors, the dependence on cooling conditions, the crystallization rate and the risk for post-hardening, are different compared to traditional filling fats. Solid fat content, often based on nuclear magnetic resonance (NMR) techniques, has become standard within the industry, both as part of the specification but also as a way to communicate sensory properties. Petersson has shown how to improve the NMR-technique for tempered systems (Petersson et al., 1985). In the future, the NMR techniques will remain the leading tool for quality control. During product development, however, the solid fat content cannot be used as the sole tool to evaluate sensory properties. Particularly when comparing different types of fats, the solid fat content and the mouth feel can diverge (Fig. 20.5). Post-hardening, e.g. increased hardness during Thickness

Waxiness

Sticky

Toughness

Early melt start

Hardness

Neutral taste

Rapid melting Cooling sensation

Fig. 20.5

Hydrogenated filling Non-trans filling

Totally melted

Sensory evaluation of fillings based on a hydrogenated fat and a non-trans fat.

500 Modifying lipids for use in food storage, is more evident in the case of non-trans filling fats. More knowledge within this area is needed to understand the mechanism. Palm-based filling fats are known for their slow crystallization rate, which becomes evident when compared with traditional filling fats. The hydrogenation of palm oil increases the crystallization rate and results in more complete crystallization (Yap et al., 1989). The reason is that hydrogenated fats crystallize more or less directly into the β′ form, and the crystallization is rapid. Palm-based products show a more complex crystallization pattern, with a slower crystallization, often via a quite stable α-polymorph, visible for several minutes. Lower temperature stabilizes the α form and prolongs the transition time to β′. This crystallization pattern is such that the effect of cooling is of less importance with respect to hardness (Fig. 20.6). Still, sufficient cooling is important to achieve small crystals, thus the recommended cooling temperatures remain quite low. Non-trans filling fats based on palm oil have quite good compatibility with cocoa butter, since they often include a relatively high amount of symmetric triacylglycerols. However, the amount of cocoa butter allowed in the fat phase, before tempering, necessarily varies with the filling fat.

20.2.3 Chocolate spreads Chocolate spreads are increasing in popularity. The products are normally stored at room temperature and are ready to be spread on sandwiches. Since many consumers keep the products in the refrigerator, an additional requirement for spreadability directly from the fridge is demanded. The fat content is often around 30 %. It is a fat-continuous system, where sugar and other particles are dispersed. As in all fat-continuous systems the properties of the fat have a major influence on the sensory behaviour. For spreads, the structure is preferably soft and gives a creamy sensation. The fat for spreads is normally very soft, which increases the risk for oiling out. The process often includes milling in the open air and therefore oxidation stability is important. 120

Hardness (gram)

100 80

Lauric Hydrogenated Palm

60 40 20 0

0

5 10 15 20 Cooling temperature (centigrade)

Fig. 20.6 The influence of cooling temperature for different filling fats.

Chocolate and confectionery fats

501

The risk for oiling out is severe. Thus all functional spread fat systems need to be efficient in entrapping the liquid oil in a crystal network. The crystal network should handle changes in temperature during storage and the expectation that the consumer can stir the product without releasing oil, so called oil separation. Normally fats, which crystallize rapidly into β′ stable crystals, are preferred. Small crystals enhance the oil entrapping properties. Therefore hydrogenated fats have traditionally been used in spreads, with excellent properties to entrap liquid oil. Other systems based on special high-melting triacylglycerols in liquid oil are common today. The highmelting triacylglycerols crystallize rapidly into small crystals, which entrap liquid oil.

20.2.4 Sugar confectionery The most important types of sugar-based fillings for confectioneries are toffee and fondant. Since both these types contain a high amount of water and go through high temperatures during the production, the fat used must tolerate these conditions without giving off-flavours due to oxidation and hydrolytic reactions. Toffee Toffee is based on sugar, water and milk ingredients. A Maillard reaction between the sugar and the milk proteins occurs upon heating, which gives the characteristic flavour and colour. Hardness and texture are mainly dependent on the water content. The fat’s role is to act as a shortening and to smooth the texture, making the toffee less sticky. Additionally, the fat is also included in toffee as a flavour carrier. Fondant During the cooling of sugar under constant agitation, small crystals are formed, transforming it to a white sticky mass. The amount of water has an important impact on the texture. Low levels are used in moulded products, while higher water content gives cream fillings. The role of the fat is similar to the fats used in toffees. It can smooth the texture and add creaminess to the fondant. The main reason for using the fat is its function as a flavour carrier. Since the fat is mixed in during the whipping process, the oxidative stability of the fat is vital, to avoid off-flavour of oxidized fats.

20.2.5 Cold applications Both chocolate and compounds are used in the coating of ice cream. For chocolate, the normal chocolate legislation is applicable, i.e. if vegetable fat is allowed, the same rules apply to the use of ice cream coatings. In the European Union coconut oil is added to the list of permitted vegetable fats. Compound coating then refers to coatings where other vegetable fats have

502 Modifying lipids for use in food replaced the cocoa butter and other allowed fats, but the product cannot be called chocolate coating. Today, an ice cream coating contains more than 50 % fats, often between 55 and 70 % of fat. Thus the properties of the coating are dependent on the properties of the fat. In ice cream applications, products based on coconut oil show many advantages compared to cocoa butter, especially when fast crystallization and thin layers are required. Typically these fats need to give a hard, snappy feeling and rapid melting in order to give a good mouth feel. The solid fat curves are often used as an indicator for these properties. The coating fat should melt rapidly from 20 to 30 °C, at which temperature it should be completely melted (Table 20.7). Fats based on lauric fats, as well as hydrogenated vegetable fats, usually have a much lower viscosity compared to coatings based on cocoa butter. Therefore, they are easy to handle when producing thin, non-transparent layers. For thick formulations there is a risk for uneven thickness of the layer, so-called ‘bleeding’. The ‘bleeding effect’ is unattractive from the consumer’s point of view, but also increases the risk for the product sticking to the wrappings. Lauric-based fats with lower viscosity decrease this risk of ‘bleeding’. Hydrogenated fats are somewhat more elastic in structure, which prevents the coating from chipping off the ice cream during eating. Ice dippings and ice toppings are other types of ice cream coatings, applied at the point of sale or at home. These fats should be liquid at room temperature but solidify immediately when in contact with the cold ice cream. To obtain these properties, low viscosity and rapid crystallization is needed. Lauric fats are often used in these applications. Since these fats are often kept at room temperature for a long time, stability against hydrolytic rancidity and oxidation is important. 20.2.6 Aerated products The incorporation of gas bubbles into a chocolate product is a traditional procedure, which can be performed in many ways. The most traditional way is whipping, whereby air is incorporated in the form of small and medium size bubbles in the product. Other techniques are based on aeration under pressure, gas expansion under vacuum and continuous aeration with special mixer heads. Air, CO2 or N2 are the most commonly used gases. Table 20.7

Solid fat content of compound chocolate fats for cold applications.

Temp (°C)

Lauric topping fat

Lauric coating fat

Non-lauric coating fats

10 20 25 30

40 0 0 0

55 18 2 0

65 20 5 0

Note: The solid fat content represents typical values for fats used in cold applications. The solid fat content was measured by NMR IUPAC S (2.150b) at AarhusKarlshamns AB.

Chocolate and confectionery fats

503

There are two major ways of aerating. In the bakery industry, precrystallized fats are mixed with other ingredients and whipped. In continuous processes, the fat is crystallized during the aeration. Completely melted fat is cooled down during the aeration, which means that good control of the temperature gradient during the process is required to obtain optimal effect. In continuous processes, viscosity and rapid crystallization of the fat is critical for maximized aeration. The reason is that to entrap the air bubbles a certain amount of crystals are needed. Thus, the most desirable property of the fat is a rapid crystallization start, enabling the formation of small crystals, followed by a gradual increase in viscosity. Even though most fats give some aeration properties, CBR fats crystallize quickly and have moderate setting times. These properties make them ideal for aerated products. The reason is that CBR fats create small β′ crystals that are able to retain the air bubbles during the aeration.

20.3 Improving the functionality of chocolate and confectionery with modified lipids 20.3.1 Bloom stability Fat bloom is still not fully understood in chocolate applications. The reasons for bloom are numerous, but some of the mechanisms behind blooming can be divided into the following categories: crystal growth control, meltingrecrystallization, dissolution-recrystallization or phase separation. In all cases, triacylglycerols migrate out to the surface and recrystallize into large crystals. The crystal growth controlled bloom is typical for bars of chocolate where larger crystals are growing at the expense of the smaller crystals, so called Ostwald ripening. Bloom caused by melt-recrystallization often occurs in warmer climates and during the summer when there is a risk that the product melts completely. When the temperature decreases again the fat starts to crystallize. During the recrystallization, unstable crystal forms are created, leading to bloom. Bloom due to phase separation typically occurs when incompatible fats are mixed. A typical example is when some lauric fats are mixed with cocoa butter. During storage these fats separate, resulting in the formation of bloom. Finally, bloom related to dissolution-recrystallization mechanism is common with soft confectionery fillings. The liquid oil of the filling migrates to the surface of the coating. At room temperature, mainly POP and POSt, but also some StOSt, dissolve into the liquid phase and start to migrate. On the surface of the coating the POP, POSt and StOSt recrystallize causing bloom. Ziegleder has shown that bloom crystals are to some extent enriched with POP and POSt (Ziegleder et al., 1994). The different mechanisms behind bloom formation described can be the result of both the fat composition and the process parameters. The focus in this chapter is on how to use lipids and modified lipids to avoid bloom or at least improve bloom stability.

504 Modifying lipids for use in food Bloom related to processing and storage is discussed elsewhere (Seguine, 1991). Coating To improve bloom stability of the coating, special lipids can be added to cocoa butter or the cocoa butter can be replaced with CBR-type fats. Tempered systems Milk fat is probably the most common additive to improve the bloom stability in chocolate. Milk fat, especially the high-melting fraction as well as hydrogenated milk fat, prevents the formation of large crystals and transformation of form V to form VI, according to Bricknell and Hartell, (1998). Small amounts of modified lauric fats and up to 5 % of palmkernel stearin in cocoa butter have been mentioned by Timms (2003b) to have antibloom properties. However, so-called ‘cold bloom’ during storage at 10– 15 °C has been reported with lauric-based solutions (Padley, 1997). Other products based on asymmetric triacylglycerols, such as StStO, have been reported to have anti-bloom properties in cocoa butter. The formation of bloom was also significantly delayed. The addition of 10 % StStO to cocoa butter retarded the transformation from form V to form VI, as explained by Arishima (Arishima and McBrayer, 2002). The action of asymmetric triacylglycerols is therefore different from the more commonly used symmetric triacylglycerols. Many CBE and CBIs with symmetric triacylglycerols, especially rich in StOSt, improve the bloom stability. In Europe, these products comply with the chocolate regulations if used at the 5 % level. Timms (2003b) has reported that the bloom stability as a function of the number of cycles in cyclic storage is directly related to the StOSt composition of the coating. These results are in line with the study performed by Pedersen, which showed that addition of StOSt-rich CBEs to milk chocolate is to be preferred compared to POP-rich fats (Table 20.8) (Pedersen, 2004). POP and POSt by themselves have a melting point around 36 °C. The melting point was decreased to Table 20.8 Bloom stability with respect to triacylglycerol composition in milk chocolate.

Ratio POP/POSt Ratio StOSt/POSt Number of cycles until bloom

Milk chocolate

Milk chocolate + StOSt-rich CBE

Milk chocolate + POP-rich CBE

0.42 0.63 22

0.45 0.73 38

0.52 0.63 14

Note: Bloom stability of milk chocolate (15 % of milk fat) alone or in combinations of CBE rich in either POP or StOSt. The bloom stability was accelerated using cyclic storage, 20 °C for 12 hours followed by 30 °C for 12 hours. The bloom stability is measured in terms of the number of tempering cycles before bloom appears. Abbreviation: CBE = cocoa butter equivalent. Source: In-house study at AarhusKarlshamns AB.

Chocolate and confectionery fats

505

34 °C when mixing them in the ratio POP/POSt ~ 1. StOSt has a much higher melting point around 43 °C, and has lower solubility at ambient temperature in the liquid phase of the cocoa butter. In addition to the improved heat stability effect, the crystals are stabilized, preventing bloom formation. Other symmetrical triacylglycerols such as BOB (1,3-dibehenoyl-2oleoylacylglycerol) are also known to improve the bloom stability of cocoa butter. According to Sato, BOB fats always crystallize in form V with a high melting point (Sato and Koyano, 2001). The effect is thought to be that the BOB triacylglycerols do not melt and act as seed crystals, promoting the recrystallization. This effect is particularly of interest in hot climates where chocolate is at risk of being completely melted. A few crystals of highmelting triacylglycerols, which do not melt, ensure a rapid re-crystallization in the right crystal form upon cooling (van Langevelde et al., 2001). The mechanism is believed to be the same for StOSt. It seems that StOSt is effective at a lower level than BOB fats. Finally the StOSt molecule improves the tempering properties, which in turn improves the bloom stability. Non-temperered systems Cocoa butter replacers are known for their superior bloom stability compared with cocoa butter. The reason for this is the stability of the β′ crystals obtained with hydrogenated fats. The β′ form is generally expected to be a metastable polymorph. However, some triacylglycerols, for instance trans fatty acid-containing triacylglycerols, are stable in the β′ polymorph. Both the β and the β′ form of cocoa butter crystallize in two forms. It is more and more evident that these are not two discrete crystal forms, but a variation of less perfect and less stable crystal forms. Nevertheless, the change itself constitutes a driving force for crystal growth. Both CBRs and CBSs are preferably shock cooled to obtain small crystals with uniform sized crystals. The reason is their ability to crystallize directly in the β′ form. The faster the supercooling the smaller the crystals. Small crystal size results in a glossy appearance. Normally, the quick crystallization also ensures a small crystal size distribution, which in turn is the key to slowing down any Ostwald ripening process. The Ostwald ripening process is the phenomenon in which larger crystals grow at the expense of the smaller crystals, i.e. the triacylglycerols of the small crystals dissolve in the liquid phase and diffuse into the larger crystals where they recrystallize. To inhibit the Ostwald ripening process, small evenly-sized crystals are preferable. Minor components such as STS (sorbitan tristearate) and diacylglycerols are often added to non-tempering systems to extend shelf life. Both STS and diacylglycerols decrease the rate of polymorphic conversion as well as crystal growth. The presence of non-co-crystallizing fractions, for instance trilaurin, in CBS causes bloom. Smith showed in 2004 that trilaurin was enriched in the bloom crystals formed when stored at higher temperatures, above 20 °C (Smith et al., 2004). However, the bloom crystals which developed during

506 Modifying lipids for use in food cold storage at 15 °C were enriched with symmetric triacylglycerols from the cocoa powder. However, by optimizing the triacylglycerol composition by using co-crystallizing triacylglycerols, the β′ form can be thermodynamically or kinetically stable for years.

Filling The compatibility between the coating and filling should be considered when choosing the filling fats, in order to decrease the risk for fat bloom (Table 20.9). The reason for the increased risk is based on the assumption that migration occurs during storage. The filling fats migrate to the surface of the praline. If the filling fats and the coating are incompatible, eutectic mixtures evolve. This increases the dissolution of medium-melting triacylglycerols which re-crystallize on the surfaces of the praline with visible bloom as the result. In general, tempered systems are highly compatible with cocoa butter and CBEs. Hydrogenated fats and non-lauric non-tempered filling fats have a medium compatibility with cocoa butter and CBEs. These types of filling fats work perfectly together with CBR fats. To avoid incompatibility, lauricbased filling fats are not usually used when shells and coatings are based on cocoa butter or CBEs. However, there are some lauric-containing anti-bloom fats on the market which claim to increase the bloom stability. Anti-bloom fats based on interesterified and enriched fractions of triacylglycerols containing mainly medium-chain and longer-chain fatty acids, such as 12:0, 16:0 and 18:0, have been available on the market for a couple of years. The triacylglycerols contain two medium-chain fatty acids and one longer-chain fatty acid (LM2) or one medium-chain fatty acid and two longerchain fatty acids (L2M). Filling fats enriched with these triacylglycerols have shown significant fat-bloom retarding effects on shells of cocoa butter. The exact mechanism is still not fully understood, but the triacylglycerols need to migrate out to the surface of the shell and praline in order to become ‘active’, i.e. preventing bloom formation. The main explanation of the Table 20.9

The compatibility between different groups of fillings and coatings. Tempered fillings

Tempered coatings Non-tempered coatings

Cocoa butter CBE CBR CBS

Non-tempered fillings Hydrogenated

Lauric

Non-trans and non-lauric

Excellent

Good

Poor

Good

Excellent Good Poor

Good Excellent Poor

Poor Poor Excellent

Good Good Poor

Note: The rating is based on experience of AarhusKarlshamns AB which is in line with the literature (Timms, 2003b). Abbreviations: CBE = cocoa butter equivalent, CBR = cocoa butter replacer, CBS = cocoa butter substitute.

Chocolate and confectionery fats

507

mechanism is summarized as follows. The L2M/LM2 triacylglycerols migrate to the surface. Meanwhile the symmetric triacylglycerols in form V cocoa butter dissolve in the liquid phase. Eventually the symmetric triacylglycerols reach the surface where they tend to re-crystallize. The L 2M/LM 2 triacylglycerols have a slight solubility in crystals containing POP/POSt/ StOSt, but hinder the formation of large crystals. Recently, other fats, based on enriched fractions of triacylglycerols with 22:0 have been found to have certain anti-bloom properties (Bach and Juul, 2003). The mechanism is assumed to be similar to that for L2M/LM2 fats, i.e. the need for migration to the surface to prevent bloom formation. In this chapter, four different mechanisms have been described to cause fat bloom. To summarize strategies to defeat bloom problems, the key is to identify which mechanism is causing the bloom. In the case where the bloom formation can be linked to the crystal growth mechanism, where recrystallization from form V to form VI takes place, the strategy is to retard polymorphic transformation and crystal growth (Table 20.10). As described earlier, asymmetric triacylglycerols or high-melting milk fat fractions can help. An alternative to reducing the crystal size distribution is to add StOSt or utilize seeding to improve the tempering. Bloom related to melting followed by re-crystallization is retarded by increasing the amount of high-melting triacylglycerols such as StOSt. The key is thus to increase heat resistance and keep some crystals as seeds. Phase separation as the cause of bloom requires a third alternative to stay bloom-free. The phase behaviour needs to be checked and controlled and in turn adjusted in the formulation. The most common and most difficult case is the dissolution–re-crystallizing process. This is often the case in pralines with a soft filling. The key is to retard crystal growth and/or control migration. Typically modified lipids that retard crystal growth are the L2M/LM2 triacylglycerols, which migrate to the surface and inhibit crystal growth. Table 20.10

Summary of various strategies to solve bloom problems. Cause of bloom

Strategy to solve problem

Crystal growth

Dissolution – recrystallization

Melt – recrystallization

Phase Unstable crystal separation form

Milk fat HPKS ATG

ABF ATG Increase SFC Minimise migration

StOSt BOB

Consider fat phase

Consider cooling, tempering, fat phase

Note: The summary is based on personal experience and the literature referred to in this chapter. Abbreviations: ABF = anti-bloom fat, like H2M/HM2, ATG = asymmetric triacylglycerols, BOB = 1,3dibehenoyl-2-oleylacylglycerol, HPKS = hydrogenated palmkernel stearin, O = oleic acid, SFC = soft fat content, St = stearic acid.

508 Modifying lipids for use in food 20.3.2 Migration Migration is universal, and chocolate products tend to move towards chemical and thermodynamic equilibrium. For instance, in a praline composed of a shell and a filling, the shell often consists of more triacylglycerols with melting points above 30 °C, while the filling contains mainly liquid triacylglycerols. During storage the triacylglycerols of the shell migrate into the filling and vice versa. It has been shown that all liquid triacylglycerols migrate with equal rate, meaning that triolein migrates as quickly as POO and StOO. Ziegleder has shown that the migration approximately follows the rate of diffusion (Ziegleder et al., 2001). M(t) = ∆C*√(D*t)/h where M(t) = migrated fat over time, ∆C = chemical potential, D = diffusion constant, h = distance (thickness of shell) and t = time. The equation proposes that the driving force for the migration is dependent on factors such as the difference in triacylglycerol composition between the shell and the filling, ∆C. The migration rate is also dependent on the rate of the molecular movement and on many other factors, including the amount of crystals, crystal network, cracks and possible pores. Loisel showed the possible presence of pores in chocolate (Loisel et al., 1997). In well-tempered chocolate roughly 1 % porosity was present while over-tempered chocolate had roughly 4 % porosity. Other scientists have also pointed in this direction. Ziegleder showed that milk chocolate is more porous than dark chocolate (Ziegleder et al., 2003). The mobility of liquid oil is dependent on the amount of crystals in the system but also on the network created by the crystals in the system. The strength of the network is dependent on a number of factors. Besides the amount of crystals, factors such as particle size, particle shape and the size of the agglomerates have a large influence on the structure. In general, small crystals with a needle-shape form give a harder structure than large round crystals. The size and shape is dependent on the cooling conditions as well as the polymorphism of the fat. Spontaneous polymorphic transition in general leads to larger crystals. Small needle-like crystals are often found in β′ stable fats, which crystallize directly into the β′ form, such as hydrogenated fats. Confectionery fat manufacturers have over the years supplied the market with structured fats to minimize oil migration. Since the 1990s understanding of the relationship between the fat crystal network and macroscopic properties such as structure as well as ability to entrap oil has improved (Marangoni, 2002; and see also Chapter 8). The concept of critical volume fraction has been used to explain how to virtually eliminate oil migration. The phenomenon was quantified and explained by Alander (Alander et al., 1994). To summarize, migration depends on temperature, storage time, how the fat was crystallized as well as inherent properties of the fat phase, for example the solid fat content and the polymorphism. In pralines and other two-

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compartment applications nature wants to level out differences in fatty acid compositions. Taking this into account, several means can be expected to minimize migration, such as: • • • • •

increase the solid fat content of the filling; minimize the difference in composition between the shell and the filling; use structuring fats to lock in oil; reduce the temperature during storage; use fats which are compatible with the shell.

Apart from these strategies, barrier fats can be used between coating and filling.

20.3.3 Barrier to hinder moisture and oil migration Barrier fats are commonly used to prevent moisture migration, for instance in ice cream or caramels, reaching the waffle or crunchy biscuit in a chocolate application. It has been suggested that thin layers of high-melting triacylglycerols between the filling and the shell reduce the rate of migration, thus reducing the softening and bloom formation. To prevent moisture being transported through the barrier, the structure and the water solubility in the barrier should be taken into consideration. The solubility of water depends on the type of lipid. Short-chain fatty acids are more polar than longer chain. The lower polarity of the long-chain fatty acids is favourable as a moisture barrier. In turn some types of waxes and mineral oils are more hydrophobic than long-chain triacylglycerols. However, mineral oil is not suitable in food applications. Waxes on the other hand have been used at low levels. Commercial barriers based on chocolate, containing water-soluble substances, have better moisture barrier effects. The reason is not clearly understood. Minor components such as lecithin, which contains polar groups, have the ability to entrap water molecules. In addition to their hydrophobic properties, fats also give structure to the barrier. The structure plays an important role in decreasing the mobility over the film. Thus normally solid fats are used, such as hydrogenated rapeseed, soya bean and palm oil. Lidefelt showed that at least 30 % of solid fat content is needed to obtain an improved moisture barrier (Lidefelt, 2002). Cracks and pores increase the mobility of water drastically. Contraction during fat crystallization is one of the factors inducing cracks. CBR fats based on hydrogenated rape seed, soya bean and palm oils contract less than fats based on cocoa butter or CBS and are thus preferred in these applications. To hinder oil migrating from a filling to the shell, the structure is just as important as when preventing moisture mobility. Cracks and pores need to be avoided. The technologies are often based on spraying a thin film on the shell or coating before the filling is applied. The fat needs to crystallize rapidly to cover the surface and be completely solidified when the filling is applied. Cracks and incomplete coverage should be avoided. Thus fats with

510 Modifying lipids for use in food little shrinkage which are flexible and have plastic properties, such as CBRs, are suitable. Another aspect to consider is that the mobility of the liquid oil is important as well as the distance the liquid triacylglycerol travels. The distance is related to the thickness of the film, but also to the amount and shape of the crystals. The mobility of the liquid oil is dependent on crystal network, see Section 20.3.2.

20.3.4 Crystallization rate Crystallization of fat is a multi-dimensional event. The first step for the crystallization from melted fat is nucleation, followed by the creation of crystals and crystal growth. Since fats generally are polymorphic, the polymorphic transition during this process has an impact on the crystallization. Besides the fat itself, other components such as emulsifiers and solid particles such as sugar and cocoa powder affect the crystallization rate. Solid particles often increase the nucleation rate. Differential scanning calorimetry (DSC) is commonly used to investigate crystallization rates. Three to 10 mg of fat is put in a cup, often based on aluminium. The fat is heated and cooled according to a program, where the heat of crystallization or melting is measured. The DSC is very accurate and gives valuable information. The interpretation is very easy in the case of hydrogenated and lauric fats, which crystallize directly into β′. In processes where agitation is used during the crystallization, a rheometer is a valuable instrument. The rheometer requires gram scale quantities and either oscillation or stirring conditions can be used. The cooling conditions in the production can be simulated. The rheometer shows the development of the structure, which is often relevant in production. From a production point of view, it is more relevant to discuss setting time, since that is related to the time needed before the product is ready for further treatment, such as additional filling and applying the bottom chocolate or ready for packaging. In most cases, fast and complete crystallization is required. The influence of fats The choice of fat has a very large influence on the crystallization rate. Fats with simple crystallization patterns such as hydrogenated or lauric-containing fats generally crystallize rapidly. They crystallize directly in the β′ form. Incompatible fats should be avoided when fat crystallization is critical, since they tend to disturb the crystallization. Palm oil has a very slow crystallization pattern. Minor components such as diacylglycerols are one of the factors explaining the slow crystallization rate of palm oil. Next to the crystallization pattern, the level of supercooling is important. Small amounts of high-melting triacylglycerols increase the onset of the crystallization. Fully hydrogenated fats based on rapeseed, soya bean and palm oil are often added in levels up to 5 % to increase the crystallization rate. Special palm stearin fractions are widely used as non-hydrogenated alternatives. However, too much of these fats gives a waxy mouth feel.

Chocolate and confectionery fats

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Seeding Seeding of chocolate has proved to increase the crystallization rate significantly. The crystallization of fat includes a number of steps, specifically for cocoa butter. The first step during the cooling is the creation of nuclei. The nuclei become a crystal with crystal growth as the third event. Cocoa butter crystals convert from unstable crystals to the stable V form. The addition of seeding crystals at levels between 0.5 and 1 % of cocoa butter replaces the nucleation step. It has been shown by Sato, Loisel and others that seeds in the right crystal form were needed to take full advantage of seeding. The reason is that the rate-limiting step is the conversion to form V. Studies with different seeds such as tristearin (StStSt, β, DCL), StOSt (β, TCL), BOB (β TCL) showed the importance of the right polymorphism in order to avoid tempering. The addition of StStSt did not improve the demoulding properties or the bloom stability of dark chocolate. Seeds based on StOSt were effective at the lowest, 0.1 % level, and both improved the demoulding properties and reduced fat bloom. BOB worked in a similar way to the StOSt and improved the bloom stability even further during ‘tropical’ conditions. Other studies verify the effect of StStSt, where increasing levels of tristearin were added to cocoa butter. The crystal onset was correlated to the amount of StStSt, with more rapid onset at higher levels. However, the cocoa butter started to crystallize independently of the level of tristearin added. For recipes based on CBR and CBS the type of crystallization starter is of less importance. The addition of seed based on tristearin increases the crystallization rate of CBR. Minor components In chocolate formulations surface-active substances are often used, for instance to reduce viscosity. Popular additives are sorbitan tristearate (STS), sorbitan monoesters, lecithin, mono- and diacylglycerols. Since roughly two-thirds of the chocolate recipe contains non-fat-soluble substances such as sugar and cocoa powder, the lecithin acts as a lubricant. The polar part of the lecithin covers the sugar particles, while the hydrophobic part faces the fat phase. Roughly 0.5 % is needed to cover the sugar and cocoa powder particles. The covered particles reduce the viscosity of the chocolate mass which is favourable. Lecithin itself is known to reduce the crystallization rate of fat indicating that the amount of lecithin should be controlled (Guth et al., 1989). Diacylglycerols also have a negative effect on the crystallization rate and on polymorphic transformation. However, there are several types of diacylglycerols each with different properties (Siew and Ng, 2000). For instance, it has been shown that 1.3-dipalmitin increases the melting point of the palm oil while 1.2-dipalmitin decreases the melting point. Sorbitan tristearate is a component often used in CBR and CBS applications to stabilize β′ crystals (Wilson, 1999). It is shown to be one of the most effective emulsifiers for improving both initial gloss as well as bloom stability (Weyland, 1994). However, STS also seems to have a negative effect on

512 Modifying lipids for use in food crystallization rate in these applications. Sorbitan monoesters and monoacylglycerols improve the crystallization rate in CBR and CBS systems because they are insoluble in the fat phase and act as nucleation agents. However, bloom stability does not seem to improve. In summary, the minor components in a fat play a crucial part in fat crystallization, yet there is inadequate understanding of the mechanisms behind their influence. The reason is that the levels are low and individual components often influence each other.

20.4 Future trends The major brands are today sold all over the world. The economic impact of any bad image on these super brands has led the major companies to focus on brand image. Moreover, in Western Europe and USA the alarming increase in obesity has focused on health aspects of foods in general but also on chocolate and confectionery products.

20.4.1 Health During 2004, Denmark banned trans fatty contents in food products above 2 % of the total fat phase, except for milk fat. In the USA, since 1 January 2006, all trans levels above 0.5 g per serving have been required to be labelled. The reason for these actions is that it has become evident that trans fatty acids in high levels increase the amount of low-density lipoprotein cholesterol and reduce levels of high-density lipoprotein cholesterol. These changes are related to increased risk for coronary heart disease. The European Community as a whole does not consider that the trans fatty acid levels consumed within the European Union is a health concern, but recommends that the sum of trans and saturated should be lowered. According to the study, the so-called ‘transfair’ study, which included 14 EU countries, the intake of trans fatty acids was close to 1.5 % compared to the recommended 1 %. The consumption of saturated fatty acids was around 15 % compared to the recommended maximum of 10 %. Despite the different attitudes between countries, the reduction of trans fatty acids will be a major focus within the confectionery industry during the next few years. The reasons are numerous, but avoiding bad publicity by changing to healthier fats is important, as well as keeping a clean label. The fat suppliers have started to supply the market with non- and lowtrans products for filling fats. Some suppliers have also proposed low-trans CBR solutions. Non-hydrogenated CBS fats have been suggested as well. The non-trans CBS will indeed have a clean label, but they are based on almost 100 % of saturated fats, thus not complying with the overall directive by the EU that the sum of trans and saturated should be decreased. CBE as the fat phase would reduce the sum of saturated and trans. The sensory

Chocolate and confectionery fats

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properties would be similar to cocoa butter, but it would be more price efficient. However, tempering is needed. It remains to be seen which way the consumer will move.

20.4.2 Food safety Next to trans fatty acids, governments as well as public opinion are putting more emphasis on food safety. Pesticides and other chemicals should be removed. EFSA – The European Food Safety Authority – is active in these areas and is about to enforce more legislation with respect to pesticides and other components. Other considerations, such as traceability of the raw materials used in consumer products, will become more important. Palm oil has received increased interest as an alternative to hydrogenated fats, and has become one of the largest vegetable oil crops in the world. However, the question of sustainability has been raised.

20.4.3 Application Machinery development is a strong driving force for optimizing and modifying fats. For instance, machines that seek to improve production speed such as seed masters, one-shot production and frozen cone will affect the properties of the fat needed. Seed masters is one of many different techniques where a small amount of fat crystals, often cocoa butter in form V or VI, is added to the chocolate mass. The tempering step is not needed with such procedure. Often these technologies also include a small reflux of crystallized chocolate feeding the non-crystallized mass with crystals in the right polymorphic form. The oneshot production of for instance pralines is a technique where both the coating and the filling are applied at the same time. The procedure increases the speed of production. Frozen cone is the third technique mentioned, and it gives almost identical features of the end products and fast production. The pralines’ shells are cooled on the inside at very cold conditions, often far below the freezing temperature. Seed masters and other seeding machines promise to make tempering obsolete. If that promise is fulfilled, cocoa butter alternatives become even more an economical choice for production of high quality products. Oneshot production, where both the coating and the filling are added simultaneously, increases the need for rapid crystallization and good compatibility.

20.5 Sources of further information and advice The chocolate and confectionery applications include most aspects of lipids chemistry, as described in this chapter. This chapter has focussed on a small,

514 Modifying lipids for use in food but important, part of the total application. In order to try to get a full picture of the field, a starting point could be to read the book by Stephen Beckett The Science of Chocolate, which explains many of the fundamentals of chocolate production (Beckett, 2000). He begins by briefly describing the history of the cocoa bean and its components, but then goes on to explain in detail the need and science behind each step in chocolate production. The book is not very focussed on the role of fats, but is a good starting point for the application as such. To better cover the role of vegetable fats in chocolate applications, Ralph Timms has written Confectionery Fats Handbook – Properties, Production and Application (Timms, 2003b). There are several chapters discussing the role of vegetable oils, but the processing is also well described. The book can give some more insight into the role of the processing and the choice of fat with respect to the final results. For instance there is a chapter with numerous diagrams showing which combination of those fats gives eutectic effects and which does not. Since fat crystallization is so important in chocolate and confectionery applications, the book edited by Garti and Sato, Crystallization Processes in Fats and Lipid Systems, can explain fat crystallization in detail (Garti and Sato, 2001). Even though the book is mainly a general text on fat crystallization, there are many chapters which are specifically related to chocolate applications and the effect of fats in these. There is a chapter specifically discussing the crystallization properties of cocoa butter. Finally, to get an overview of what has been written and discussed about fat bloom in chocolate applications, the review by Hartel can be recommended (Lonchampt and Hartel, 2004). This article summarizes most aspects of bloom formation in chocolate and chocolate compounds. The effects of different fats, minor components as well as processing conditions, are included.

20.6

References

ALANDER J, GEORGE P

and SANDSTROM L (1994), Prevention of oil migration in confectionery products, International Food Ingredients, 4, 27–30. ARISHIMA T and MCBRAYER T (2002), Applications of speciality fats and oils, Manufacturing Confectioner, 82(6), 65–76. BACH M and JUUL B (2003), Non-Lauric, Non-Trans, Non-Temper Fat Compositions, Patent application, WO03037095A1. BECKETT S (2000), The Science of Chocolate, Cambridge, Royal Society of Chemistry. BRICKNELL J and HARTEL R (1998), Relation of fat bloom in chocolate to polymorphic transition of cocoa butter, J Am Oil Chem Soc, 75(11), 1609–1615. EFSA (2004), Opinion of the Scientific Panel on Dietetic Products, Nutrition and Allergies on a request from the Commission related to the presence of trans fatty acids in foods and the effect on human health of the consumption of trans fatty acids (Request No EFSA-Q-2003-002), available at: http://www.efsa.eu.int/science/nda/nda_opinions/ 588_en.html EU (2000), Directive 2000/36/EC, Cocoa and chocolate products intended for human consumption, Official Journal, L197, 19–25.

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and SATO K (2001), Crystallization Processes in Fats and Lipid Systems, New York, Marcel Dekker, Inc. GUTH O, ARONHIME J and GARTI N (1989), Polymorphic transitions of mixed triacylglycerols, SOS, in the presence of sorbitan monostearate, J Am Oil Chem Soc, 66(11), 1606– 1613. LIDEFELT J-O (2002), Barrier fats in food applications. How to prevent migration of oil or water, ZSW, 55(3), 31–33. LOISEL C, LECQ G, PONCHEL G, KELLER G and OLLIVON M (1997), Fat bloom and chocolate structure studied by mercury porosimetry, J Food Sci, 62(4), 781–788. LOISEL C, KELLER G, LECQ G, BOURGAUX C and OLLIVON M (1998a), Phase transitions and polymorphism of cocoa butter, J Am Oil Chem Soc, 75(4), 425–439. LOISEL C, LECQ G, KELLER G and OLLIVON M (1998b), Dynamic crystallization of dark chocolate as affected by temperature and lipid additives, J Food Sci, 63(1), 73–79. LONCHAMPT P and HARTEL R (2004), Fat bloom in chocolate and compound coatings, Eur J Lipid Sci Technol, 106, 241–274. MARANGONI A (2002), The nature of fractality in fat crystal networks, Trends Food Sci Technol, 13(2), 37–47. PADLEY F (1997), Chocolate and confectionery fats, in Gunstone F and Padley F, Lipid Technologies and applications, New York, Marcel Dekker, Inc., 391–432. PEDERSEN M (2004), Internal work at Karlshamns AB, November 2004, Karlshamns AB. PETERSSON B SANDSTROM L and ANJOU K (1985), Pulsed NMR method for solid fat content determination, Part I: Cocoa butters and equivalents, Fette Seifen Anstrichtm, 87, 225–230. SATO K and KOYANO T (2001), Crystallization properties of cocoa butter, in Garti N and Sato K, Crystallization Processes in Fats and Lipid Systems, New York, Marcel, Dekker, Inc., 429–456. SCHLICHTER-ARONHIME J and GARTI N (1988), Solidification and polymorphism in cocoa butter and the blooming properties, in Garti N and Sato K, Crystallization and Polymorphism of Fats and Fatty Acids, New York, Marcel Dekker, Inc., 363–393. SEGUINE E (1991), Tempering – the inside story, in Proc 45th Annual Production Conference, Hershey, April 1991, PMCA, 21–29. SIEW W and NG W (2000), Differential scanning thermograms of palm oil triacylglycerols in the presence of diglycerides, J Oil Palm Res, 12, 1–7. SMITH K, CAIN F and TALBOT G (2004), Nature and composition of fat bloom from palm kernel stearin and hydrogenated palm kernel stearin compound chocolate, J Agric Food Chem, 51(17), 5539–5544. STAPLEY A, TEWKESBURY H and FRYER P (1999), The effects of shear and temperature history on the crystallization of chocolate, J Am Oil Chem Soc, 76(6), 677–685. TIMMS R (2003a), Interaction between fats, bloom and rancidity, in Timms R, Confectionery Fats Handbook – Properties, Production and Application, Bridgewater, Oily Press, 255–294. TIMMS R (2003b), Confectionery Fats Handbook – Properties, Production and Application, Bridgewater, Oily Press. VAN LANGEVELDE A, DRIESSEN R, MOLLEMAN W, PESCHAR R and SCHENK H (2001), Cocoa-butter long spacings and the memory effect, J Am Oil Chem Soc, 78(9), 911–918. VAN MALSSEN K, VAN LANGEVELDE K, PESCHAR R and SCHENK H (1999), Phase behavior and extended phase scheme of static cocoa butter investigated with real-time X-ray powder diffraction, J Am Oil Chem Soc, 76(6), 669–676. WENNERMARK B (2002), Chocolate and confectionery, in Lidefelt J-O, Handbook – Vegetable Oils and Fats, Karlshamn, Karlshamns AB, 112–162. WEYLAND M (1994), Functional effects of emulsifiers in chocolate, in Proc 48th Annual Production Conference, Hershey, PMCA, 32–38. WILSON E (1999), Emuslifiers and their effect on confectionery fats, in Proc 53rd Annual Production Conference, Hershey, PMCA, 74–79. GARTI N

516 Modifying lipids for use in food and PEASE J (1999), Confectionery fats from palm and lauric oils, revisited, in Leonard E, Proceedings of the World Conference on Palm and Coconut Oils for the 21st Century: Sources, Processing, Applications, and Competition, Denpasar, AOCS, 93–101. YAP P, DEMAN J and DEMAN L (1989), Crystallization rates of palm oil and modified palm oils, Fat Sci Technol, 91(5), 178–80. ZIEGLEDER G, GEIER-GREGUSKA J and GRAPLIN J (1994), HPLC analysis of bloom, Fat Sci Technol, 96(10), 390–394. ZIEGLEDER G, PETZ A and MIKLE H (2001), Fat migration in filled chocolates. The dominant influences, ZSW, 54(12), 23–25. ZIEGLEDER G, BALIMANN G, MIKLE H and ZAKI H (2003), Conching – new findings, Susswaren Technik und Wirtschaft, 48(3), 14–16. WILSON E

21 Developments in frying oils C. Gertz, Official Institute of Chemical Analysis, Germany

21.1

Introduction

Since time immemorial foods have been cooked to make them more digestible and provide desirable appearance and taste. The heat may be transferred from the medium such as air, water or oil to the product surface via conduction (pan-frying), convection (deep frying) or radiation (microwave). Deep frying is an ancient method of food preparation which has gained enormous popularity because of its speed, operational simplicity and ability to supply a good texture or crispness, golden color and good flavour. The success of deep-fried products is associated with progress in the frozen food distribution of par-fried products, such as French fries, chicken or coated vegetables. It is estimated that the total usage of frying fats and oils in restaurants, commercial frying and household is more than 20 million tonnes a year. Today, the world production of French fries exceeds 4 500 million kg (4.5 million tonnes) (Stier, 2004b).

21.2 The frying process Even though deep frying is an old process, it is poorly understood. Frying is basically a dehydration process in which oil serves as medium for heat and mass transfer. The oil becomes part of the food being fried and reacts with the proteins and carbohydrates in the food (Maillard reaction). Good understanding of the frying process helps in optimizing the manufacturing processes with regard to quality of food and fat and of energy consumption.

518 Modifying lipids for use in food

Temperature

100 °C

21.2.1 Heat and mass-transfer Foods to be fried differ in their thickness or shape, in their outer surface area, which interacts with the hot oil and participates in oil adhesion when the frying product is removed from the fryer and, finally, in the distance between the outer layer and the geometrical center (Vitrac et al., 2000). However, the physical mechanisms are not fundamentally different. In 1987, Blumenthal published a monograph first proposing his ‘Surfactant theory of frying’ to better understand and explain the basic principles of heat and mass transfer. For frying, heat must be transferred from the non-aqueous frying medium, the oil, into the mostly aqueous medium, the food. Because oil and water are immiscible, surfactants are needed to reduce the surface tension between these two immiscible materials. Perhaps surprisingly, French fries produced in fresh unused oil are uncooked, light in colour, with no gelatinization of the starch and little oil-pickup. The taste is not good. As the oil degrades, more surfactants are formed and taste and appearance improve (Pravisani and Calvelo, 1986). A food is a solid body with holes and pores filled with water and air. After the food is immersed into the hot oil, the frying process starts. Traces of free water at the surface of potato chips evaporate rapidly and the surface becomes dry (Gamble et al., 1987a) (Fig. 21.1). The inner moisture of the frying material is heated to boiling inducing the gelatinization of starch and denaturation of proteins. This layer develops a porous structure (the crust) with a number of small pores filled with air and water. In the second phase, as frying continues the pores containing water start to enlarge due to the increase in temperature and vapour expansion. Water vapour bubbles escaping from the food into the oil cause considerable turbulence in the oil. The higher gas pressure introduces stresses in the pore walls. Many pores get

150–180 °C

Frying oil

103–150 °C

Dried zone

100–103 °C

Vaporization region

100 °C

Migration region

75–100 °C 75 °C

Fig. 21.1

Liquid water region Moisture Food center

Heat and mass-transfer during frying process (Gertz, 2004).

Developments in frying oils

519

interconnected forming a network of pipelines suitable to conduct steam. Other pores filled with water or air remain isolated. This region of crust has totally different thermal and physical properties from the crumb. It acts as a barrier for heat transfer from the surface to the centre (crumb) of the product. It is important to retard the loss of moisture and not to overheat the product when the food is first immersed into the oil. Otherwise, the distribution of pores will be irregular and the pores will be too large or be destroyed due to the higher vapour pressure. After this boundary zone is dehydrated, water migrates from the central position of the food radially outward to the walls to replace that which is lost during heating. Excessive darkening and drying of the surface do not occur while vapour is leaving the crust. Behind this front the temperature within the food is 100–103 °C, representing the temperature of the change from water to steam (Farid and Chen, 1998). The vapour pressure within the food is constantly high (Vitrac et al., 2000), but it falls within the crust when vapour moves from the food into the surrounding oil. The crust/crumb interface starts moving towards the center of the food. Finally, no more moisture can leave the food and vapour pressure drops within the food. The temperature profile in the crust region is mainly a function of oil temperature, whereas the core region temperature profiles are not affected by oil temperature due to the presence of the 100–103 °C boundary in the crust. The final thickness of the crust is about 0.3–2.5 mm (Ngadi et al., 1997; Moreira et al., 1999). When frying thin potato chips (crisps) the crust region enlarges very quickly and the crumb disappears. High availability of heat, lack of available liquid water and the low thickness of the food material quickly raise the temperature of the material above the boiling point of water and induce chemical reactions such as darkening and forming of acrylamide as the vapour pressure within the food drops very quickly compared to food like French fries (Vitrac et al., 2000). In general, fried products can be classified according to their surface to volume ratios (Blumenthal, 1991), as chips (crisps) with no crumb and French fries with a larger outer layer.

21.2.2 Oil-uptake The mechanism and factors of oil-uptake are important to reduce the oil content of fried foods. Newer theories and tests support the idea that the microstructure of the crust region formed during the first moments of frying and the moving boundary at the crust/core interface determine the texture, oil-uptake, colour and crispiness of the food (Gamble et al., 1987a, Pinthus et al., 1995; Farid and Chen, 1998; Farkas et al., 1999), Moreira et al., 1999). In many cases, a lower frying oil temperature, a longer frying time, a higher moisture content of the food (Pinthus et al., 1993) or a high density of the crust (Gamble and Rice, 1988) are responsible for a higher oil-uptake. Nevertheless, oil-uptake is primarily a surface phenomenon involving an equilibrium between adhesion and drainage of oil during the post-frying

520 Modifying lipids for use in food process (cooling period). Most of the oil does not penetrate the product during frying but only after removal of the fried product from the fryer during the cooling period. Oil-uptake is controlled by the liquid mass flow within the connected pores due to capilllary forces (Banks, 1996; Moreira et al., 1997). Moisture loss and oil uptake are interrelated and both are linear functions of the square root of frying time (Gamble et al., 1987b). Berliners, doughnuts and tortilla chips fried in used oil have more oil accumulated at the surface due to the thin crust layer. In this case, a higher viscosity and/or reduced interfacial tension cause more oil to adhere to the product surface with a thin crust. However, a higher viscosity of the frying oil does not lead to increasing oil-uptake in products with a thick crust as it is more difficult for the degraded oil to flow into the centre of the chip during cooling. The temperature after the fried food has been removed from the fryer has the most influence on oil absorption. Oil does not penetrate the food during frying but does when the food is removed from the fryer due to capillary forces. As the post-frying time (cooling period) increases, the oil absorption increases and concentrates in the crust region (Yamsaengsung and Moreira, 2002). In general, process variables such as frying oil temperature, frying time, moisture content of the food and pre-frying treatment are the main factors affecting the microstructure of the crust, especially its porosity and thickness.

21.3 Chemical changes of fats and oils at frying temperature The deep-frying process is a very complex and dynamic system due to the combination of heat and mass transfer between food and frying medium. The kinetics of the chemical and physical alterations during deep frying are also affected by factors like oil/food ratio, composition of the food and frying oil, oil/surface area ratio, fryer construction and frying temperature. The system becomes more complicated as the frying operation continues because the composition of the food being fried and the frying medium change continuously due to the progressive deterioration of the frying medium. The frying oils themselves undergo extensive physical and chemical changes of which the most important are (aut)oxidation and polymerization forming isomerized, oligomerized, oxidized or cyclic fatty acids bound in a triacylglycerol molecule. The development of flavour in fried foods due to oxidation has been extensively reviewed by Gillat (2001), while the interactions between fat and food during the deep frying process are discussed by Dobarganes et al. (2000). 21.3.1 Autoxidation Initially the oil undergoes autoxidation during the storage and heating and cooling period, followed by dimerization and polymerization at frying

Developments in frying oils

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temperature and thermal oxidation in the presence of air and during aeration with the introduction of food into the hot oil. These reactions are often called oxidative polymerization with oxygen-containing dimers and polymers being formed through a radical route. Di- and trimerized triacylglycerols formed by oxidation at low temperatures are held together by oxygen bridges (C–O– C), whereas those formed during elevated temperatures result mainly through formation of intermolecular carbon–carbon bonds. Nearly all polar dimeric triglycerides found in used frying fats are linked by C–C and not C–O–C bonds (Dobarganes et al., 2000). C–O–C linkages would be the more expected if a free radical mechanism was predominating. It is assumed (Gertz and Kochhar, 2001) that during actual frying a non-radical mechanism for the formation of the dimeric and polymeric compounds is more important than autoxidation. Investigations with mixtures of triolein and trilinolein (Kamal-Eldin et al., 2003) and sunflower oil (Gertz, 2004) indicated that maximum peroxide values were obtained at intermediate temperatures while the formation of polar compounds and polymers was only significant at temperatures above 130–140 °C. Between 70 and 140 °C autoxidation is the predominant reaction forming hydroperoxides. Their decomposition leads to the formation of a wide range of carbonyl compounds via a free radical route. Secondary products like oxidized triacylglycerol monomers (TAG) containing different oxygenated groups, mainly hydroxy, keto and epoxy, as well as short-chain fatty acyl and short-chain n-oxo fatty acyl groups (Chang et al., 1978) have been identified. Ester-bound aldehydic C8:0, C9:0 fragments and ester-bound C9:0 acid fragments are the major compounds formed in thermoxidized TAG (Peers and Svoboda, 1982; Marquez-Ruiz and Dorbarganes, 1996; Berdeaux et al., 1999, 2002), besides lower amounts of free C7:0, C8:0 aldehyde and C8:0 acid, probably coming from breakdown of linoleate hydroperoxide along with further oxidative reactions resulting in the formation of hydroperoxide of oleic acid. The 2,2-diphenyl-1-picrylhydrazyl radical test was used to study lipid oxidation of oil under frying conditions by determining the antiradical power (ARP). The fact that ARP decreased faster when oil was heated indicates that a non-radical mechanism predominates at frying temperatures (Wil et al., 2004). Non-radical reactions like elimination (acid-catalyzed dehydration) or nucleophilic substitution most likely lead to the formation of C–C linked dimeric, polymeric or cyclic triacylglycerols occurring above 140 °C as described before (Gertz, 2000, 2004), which contribute to gumming, foaming, viscosity and colour darkening. The assumption of different routes of fat degradation at lower and elevated temperature can also provide a good explanation of the differing protective stabilizing effects of synthetic and natural components in frying oils. The extent of the radical catalyzed oxidative degradation reactions seems to be affected by the amount of surface area of the fat that is exposed to air (oxygen) and the temperature profile during the frying process. Fryer operators

522 Modifying lipids for use in food are often told to lower the temperature of frying oil when it is not in service. When the fryer is either heated up or cooled down, crossing temperatures between 60 and 140 °C (Gertz, 2004), increasing anisidine values and peroxide values are observed. Oils without fluctuations in temperature last longer during frying.

21.3.2 Hydrolysis After autoxidation, the hydrolytic decomposition of triacylglycerols is often considered as the most important reaction during the deterioration of frying fats (Fritsch, 1981). It is generally assumed that the presence of water during a frying process is disadvantageous because of hydrolytic decomposition of triacylglycerols forming free fatty acids, and mono- and diacylglycerol (Warner, 2004). However, no significant variations were found for diacylglycerol, monoglycerol or glycerol concentrations throughout the successive fryings, although an increase of the peak area of diglycerides is sometimes found in the size exclusion chromatographic separation of used frying fats. Octanoic and heptanoic acid and the corresponding aldehydes are mainly formed during the oxidation of triglycerides’ oleic and linoleic moieties and remain bound to the parent triacylglycerol (Peers and Svoboda, 1982; Marquez-Ruiz and Dorbarganes, 1996). The remaining triacylglycerols contain the oxidized fatty acid fragments at position sn-1 and sn-3, resulting in a lower molecular mass which is now similar to that of diacylglycerols. Only short-chain fatty acids (C6, C7, C8, C9) are volatile and leave the oil because of the high temperature and the influence of water-steam. Some heptanoic and/or octanoic acid were detected in the fried samples (Andrikopoulos et al., 2002). If free fatty acids (C10 and higher) are really hydrolysis products their amount must be correlated with the frying time and not be influenced by the nature of frying oil due to their higher boiling point. However, as an objective index the acid value is nearly meaningless. Many researchers (Mankel, 1970; Buxtorf et al., 1976; Pazola et al., 1985; Gertz, 2000) came to the conclusion that there is no direct relationship between the quality of a used frying fat and its acid value. This poor correlation (R2 < 0.4) was confirmed by analyzing 150 different used frying oils collected by food inspections. The hydrolysis of triacylglycerols, which is often supposed to provide one pathway of acrolein formation, is negligible (Umano and Shibamoto, 1987). Many researchers demonstrated that the heating of asparagine or ammoniumion as nitrogen source in combination with reducing sugars like glucose or fructose causes the formation of acrylamide (Gertz et al., 2003). Some practical aspects also contradict the theory of the deterioration during deep frying by hydrolytic reactions. Despite the fact that food contains moisture, it has been found that frying of food slows the degradation process in comparision with frying in the absence of food. The moisture seems to protect the frying fat (Dana et al., 2003; Gertz et al., 2003) and does not catalyze hydrolytic reactions increasing free fatty acids, di- and monoglycerides and glycerol.

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Water escaping from food creates a steam blanket above the oil surface which reduces the interaction of the heated oil with the atmospheric oxygen. An accelerated oxidation effect (high para-anisidine value) is only observed during frying when water vapour bubbles escaping from the food into the oil cause considerable turbulence in the oil and higher interaction with oxygen. Furthermore, these results suggest that the effect of water on oil quality is influenced by the drop size released during the frying process. Consequently, hydrolytic deterioration is probaby less important than is commonly believed today. The amount of ‘free fatty acids’ determined by the titration with sodium hydroxide does not really describe the formation of the liberated fatty acids. As explained above, there is no hydrolysis of the triacylglycerol molecule with carbon acids with a chain-length over 10. Obviously, the acidic bound fragments of the triacylglycerol as a result of oxidation are neutralized during the titration (Velasco et al., 2004). This may be the reason for the confusion between oxidative deterioration and hydrolytic degradation.

21.4 Factors affecting quality of frying oils 21.4.1 Unmodified and non-hydrogenated oils The choice of fresh oil or fat used for deep frying depends on preference and availability and differs from country to country. In European countries, refined rapeseed oil, semi-solid and solid vegetable fats like palm oil, partially hydrogenated rapeseed oil, palm oil blended with rapeseed or soybean oil and palm olein or ‘super’ olein are used. Sunflower, partially hydrogenated sunflower and animal fats like tallow and lard are employed for special applications. Olive oil or sunflower oil are used in Mediterranean countries. In North America, the frying industry and fast food restaurants employ frying fats and oils based on cottonseed oil. In the 1980s animal fats were linked to coronary heart disease due to their cholesterol content. These findings led to the removal not only of animal fats but also palm oil, palm olein and coconut oil from the US market because of their high content of saturated acids. Tropical fats were replaced with partially hydrogenated soybean oil containing high amounts of trans fatty acids and saturated fatty acids. Coconut and palmkernel oil are very oxidatively stable and have a melting point slightly above ambient temperature. They contain high levels of fatty acids with fewer than C14 carbon atoms. The moisture in the fried food causes hydrolysis of the glycerol esters and liberation of the short- and medium-chain fatty acids. A few of these are volatile and cause excessive smoke development. Other vegetable oils give no such smoke problem. Palm oil is the most widely produced semi-solid oil. It has the advantage of being low in polyunsaturated fatty acids (10 %) and consequently has good heat and oxidative stability. Its high proportion of saturated fatty acids may be a disadvantage from a nutritional point of view and may result in

524 Modifying lipids for use in food solidification in pipes and during storage. Palm oil is too high-melting to give the right appearance on cold served foods or snacks. On the other hand, this fact makes palm oil an trans-free alternative for higher-melting oils like hydrogenated peanut oil which are used in bakeries for the frying of yeast raised products like doughnuts or berliners. Palm oil and palmolein are used for frying salty snack food in almost every country, except the USA. Liquid oils such as soybean or rapeseed oil have a poor oxidative stability due to the presence of linolenic acid (6–10 %). Linolenic acid oxidizes 100 times faster than oleic acid (linoleic 40 times). Therefore, rapeseed oil is blended with other more stable oils or is partially hydrogenated. Both soybean and rapeseed oils are often used where storage of the fried food is not necessary. France and Belgium have established legal limits of 2 % maximum for linolenic acid in frying oils, as it was assumed that the cyclic fatty acids formed from this acid are toxic. This regulation stimulated a great deal of research activity. Cyclic dienoic fatty acids are formed from linolenic acid (Dobson et al., 1995), cyclic fatty acids from linoleate (Dobson et al., 1997) and bicyclic saturated fatty acids from oleate (Dobson et al., 1996). Fears concerning the cyclic acids have receded, but the limitations for linolenic rich oils are still applied. Groundnut oil has declined in popularity due to its cost and because the public worries about the peanut allergy and the aflatoxin problems. Groundnut oil is susceptible to oxidation. Hydrogenated groundnut oil is used in India for deep frying. Partially hardened groundnut oil is also used in German bakeries to produce a yeast raised cake called a berliner. These are like doughnuts but shaped as buns instead of rings, folding a filling of jam or pastry cream. Rice bran oil is increasingly popular as a frying oil due to its high smoke point and stability. High-oryzanol rice bran and refined bleached and deodorized rice bran oil used for French fried potatoes show an enhancement in stability with increasing orzyanol contents (Abidi and Rennick, 2003). Unrefined rice bran oil contains 2–20 % free fatty acids (FFA), depending on the quality of bran. Because of its high FFA and color (De and Bhattacharya, 1998) it is difficult to refine rice bran oil without reducing the amount of oryzanol. Sesame seed oil has a high potential for heat stability, but it is considered as an allergen. Cottonseed oil is more stable compared to other liquid oils but is less stable under frying conditions than palm oil or tallow. It must be winterized due to its content of longer-chain fatty acids (4 %). Cottonseed, corn, partially hydrogenated soybean, rapeseed and sunflower oils are used in the USA for frying snackfood and for French fries. New developments of trans-free frying oils rich in oleic acid and low in polyunsaturated fatty acids and saturated fatty acids are producing oils with good stability (Pethukov et al., 1999; Barrera-Arelanno et al., 2002; Warner and Gupta, 2003) (Table 21.1). Some, but not all, of these improvements have been achieved by the new technique of genetic engineering. The chemical and physical properties of conventional and high oleic sunflower (HOSO), palm olein and high oleic, high palmitic sunflower oil

Developments in frying oils Table 21.1

525

Fatty acid composition of non-hydrogenated oils. Fatty acid composition

Oil

16:0

18:0

18:1

18:2

18:3

Normal soybean High oleic soybean Low sat. soybean

10.4 6.4 4.3

4.1 3.3 2.9

22.9 85.6 19.7

52.9 1.6 61.8

7.5 2.2 8.6

Normal rapeseed High linoleic canola High oleic canola Low linolenic canola Natreon

4.0 3.4 3.6 4.0 4.4

2.0 1.7 2.3 2.0 2.0

58.0 75.2 78.8 63.0 73.2

20.0 13.4 5.1 23.0 14.8

9.0 3.6 5.2 4.0 2.8

Normal sunflower High oleic sunflower (HOSUN) Mid-oleic sunflower (NuSun) HOSO (Good-Fry®)

7.0 3.6

4.5 4.3

18.7 82.2

67.5 9.9

Traces Traces

4.6

4.2

61.3

27.3

Traces

4.7

3.7

78.6

12.0

Traces

2.0 2.6 4.6 4.4 3.2–4.3

25.4 18.6 39.3 42.5 43.2–49.2

59.6 54.4 10.7 11.2 10.7–15.0

1.0 0.7 0.4 0.4 Traces

Corn oil Cottonseed oil Palm oil Palm olein Palm super olein (Slip point: 12.9–16.6 °C)

10.9 21.6 42.9 39.8 30–37

Note: Natreon, Dow AgroSciences LLC, USA; NuSun, National Sunflower Association, USA; Good-Fry®, Good-Fry International BV, Netherlands. Sources: Rossel (2003), Tiffany (2004).

(HOHPSO) have been compared by Guinda et al. (2003). The HOHPSO was less susceptible to polymerization than the palm olein due to its lower content in linoleic acid and showed a higher thermo-oxidative stability than the unmodified commodity oils. Due to its unique fatty acid and triacylglycerol composition, the HOHPSO could be an alternative to the use of HOSO and palm olein in deep frying. The HOHPSO melted completely at 20.2 °C and palm oil at 29.6 °C. However, oils produced by traditional plant breeding techniques to give the desired fatty acid pattern have the marketing advantage in Europe and in some other countries that they can be labeled as ‘free from GMO’. The selection of frying fats and oils is more and more influenced by nutritional and health aspects. In many countries, the oil must be free of transgenic material and low in trans fatty acids. In the last ten years, trans fatty acids have been implicated as a risk factor for coronary heart disease. In 2004, Denmark legislated for a maximum 2 % trans fatty acids in edible vegetable oils, and since 1 January 2006, labelling of trans fatty acids has been mandatory in the USA. A direct relationship between fatty acids and increased risk for coronary heart diseases has been suggested (Koletzko, 1994; Enig, 1995; Mensink and Katan, 1995). It has been recommended that the intake of trans

526 Modifying lipids for use in food fatty acids be limited to a maximum of 1 % of the daily intake of fats and oils (DACH, 2001). Finally, the economics of the frying process depend on the stability of the oil and its availability. An oil at a lower price can prove more expensive in the long run because of the cost of discharging the oil and the shorter shelf life of the fried products. The resale value of waste frying oils has fallen as such frying oils cannot be used for animal feed because of legal regulations in the European market intended to avoid contamination of food with industrial transformer oil.

21.5 Modified frying oils Most of the natural oils and fats have only a limited application in their original forms. To extend their use in fat-based food products, therefore, oils often undergo a chemical or physical modification such as fractionation, interesterification, hydrogenation or blending. The addition of antioxidants and anti-polymerizing agents helps to improve their heat and oxidative stability. Hydrogenation The main purposes of hydrogenation are to improve the oxidative stability of the oil and to modifiy the physical state of naturally occurring oils and fats so they become more functional and suitable in, for example, margarines and deep-frying fats. Without a certain amount of saturated fatty acids, for example, par-fried products passing through a freezer will not solidify properly. Partial hydrogenation of vegetable oils is used to obtain fats with the required degree of plasticity and oxidative stability. However, this process generates nutritionally undesirable trans isomers of the unsaturated fatty acids. Too much saturated fat with a melting point above body temperature may result in an unpleasant waxy mouth feel of the fried products. Solid fats may be also responsible for grey, pale and dry looking surfaces. Additionally, solid fats are more difficult to handle in industry and restaurants with a view to avoiding partial crystallization or solidification and local over-heating during the start up of the fryer. For products such as cheese snacks or doughnuts, a certain amount of solid fat is required at ambient temperature to prevent a run-off of the coating from the surface and to provide a certain amount of fat melting in the mouth during chewing. Hydrogenated soybean, groundnut or rapeseed and palm oil are often used for this purpose. In the past, important characteristics of industrial frying oils were oxidative stability, high smoke point and low foaming. These days, a healthier fatty acid profile with no trans fatty acids is as important as good oxidative and heat stability. The importance of hardened fat with large amounts of trans fatty acids will decrease rapidly when suitable cheaper solid fats are available on the market.

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Interesterification Interesterification modifies the melting behaviour of a fat or blend due to the changes in triacylglycerol structure. Low trans fatty acid containing oils can be made by interesterification of different vegetable fats and oils (Table 21.2). The fatty acid composition pattern does not change through interesterifiation. Chemical interesterification can provide alternative fat products low in trans fatty acid with the desired solid fat content and slip melting point profiles. However, the data indicate that blends exhibited the best stability and that chemically interesterified blends of the same fatty acid composition were inferior to blends with regard to oxidative stability. During chemical interesterification with, for example, sodium methoxide, fatty acid methyl esters and fatty acid soaps, mono- and more specifically diglycerides are formed (Petrauskaité et al., 1998) as minor by-products and the tocopherol content is reduced up to 80 %. Nearly all partial glycerides are removed during deodorization. Traces of soaps, however, reduce heat stability and may cause emulsion problems and must, therefore, be removed carefully by adsorption or chemical means. Enzymatic interesterification has already been proven to work successfully (Macrae, 1983; Quinlan, 1993). As the interesterification can be specifically directed and the losses of tocopherols are lower, it could become an alternative for the production of trans-free solid frying fats. Econa Oil® (KAO, Japan), a special frying oil with a very high amount of diacylglycerols, is produced in this way. Interesterified fats and oils are still very expensive due to oil losses during deodorization. Compared to interesterification and hydrogenation, the cost of fractionation is considerably lower. Fractionation Palm oil is by far the most important fractionated oil. A whole variety of products can be produced from palm oil. Single-stage fractionation yields olein with a cloud point below 10 °C and a stearin with melting point of 44– Table 21.2

Melting point changes due to chemical interesterification.

Fat

Soybean oil Cottonseed oil Coconut oil Palm oil Cocoa butter Tallow Hydrog. cotton oil:coconut oil (40:60) Hydrog. palm oil:palm kernel oil (25:75) Soybean oil:hydrog. palm stearin (40:60) Source: MINAL (2004).

Melting point (°C) Before

After

–7 10.5 26 39.8 34.4 46.4 57.8 50 45

5.5 34 27.9 47 52.2 44.6 41.1 40.3 38.6

528 Modifying lipids for use in food 52 °C. There is an increasing tendency to use double and even triple fractionation of palm oil. Palm oil (IV 52) (IV = iodine value), palm olein (IV 56) and super olein (IV 70) are used as a substitute for soft oils in frying, cooking and salad oils. Hard fractions of palm oil (stearins) find application in frying fats, too (see Chapter 10 on fractionation). Effectiveness of additives Common antioxidants, including tocopherols, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), propyl, octyl or dodecyl gallate (PG, OG, DG) or tertiary butylated hydroquinone (TBHQ) retard oxidation at ambient temperature. To ensure a good shelf life for the product, phenolic antioxidants such as BHA or BHT–BHA mixtures are sometimes added into the frying oil, giving some carrythrough for the fried snack food on storage. These components are often applied in combination with chelating agents to get a synergistic effect. Citric acid chelates or combines with metal ions on the fryer’s steel wall. Adding citric acid to frying oils reduces the deposition of brown degradation products on the fryer steel wall. Metal ions, especially iron and copper, promote the radical formation of degradation products. These synthetic antioxidants become substantially less effective or even inactive when subjected to elevated temperatures, whereas ascorbyl palmitate, sterols (Gordon, 1989; Gertz et al., 2000) and many other natural ingredients provide the antioxidant effect only at elevated temperatures. There is a wide range of compounds like sterols, sesamolin or γ-oryzanol (Abidi and Rennick, 2003; Xu and Godber, 2001) found in nature which seem to have antioxidant or anti-polymerizing properties. Obviously, the acid-catalyzed polymerization of triacylglycerols during deep frying is retarded by a concurrent acid-catalyzed dehydration of the anti-polymerizing agent which needs less activation energy and happens at lower temperature than the dimerization of triacylglycerols (Table 21.3). Addition of unsaponifiable material isolated from olive, corn and wheat germ protects vegetable oils from oxidative deterioration during heating at frying temperature (Sims et al., 1972). Abdalla (1999) studied the protective effect of unsaponifiable matter from olive oil deodorizer distillate on the stability of sunflower oil during frying. A protective activity against dimer formation was observed for an oat extract (Tian and White, 1994), the ethanol extract from summer savory (Yanishlieva et al., 1997), the petroleum ether extract from oregano (Lolos et al., 1999), rosemary extract (Gordon and Kourisma, 1995) and sage extract (Che Man and Irwandi, 2000). The contribution of polyphenolic compounds to the oxidative stability is widely accepted, but not fully investigated. The Goodfry® Patent (Kamal-Eldin et al., 1998; Silkeberg, 1990) for a high quality deep-frying oil is based on the fact that sesamolin, a component of sesame oil, is transformed to the powerful antioxidant sesamol by isomerization and dehyration under the frying conditions. Strangely, if one attempts to measure the stabilizing effect at frying temperature of phytosterols

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Table 21.3 Anti-polymerizing agents and their reaction products at elevated temperature. Substance

Reaction products

Temperature of formation

Tocopherols

Dimeric tocopheryl-reaction products (C–O–C linked) (Gogolewski et al., 2003) Squalene hydroperoxides and squalene hydroxide (Assunta Dessi et al., 2002) Sterol oxides Sesamol Sesamin Sesaminol-isomers (Fukuda et al., 1986) Dehydro ascorbyl palmitate; Ascorbic acid Steradienes (Abou-Gharbia et al., 2000, Abidi and Rennick, 2003) Dimeric and trimeric compounds (C–C linked) (Gogolewski et al., 2003) Tetracyclosqualene

~ 80 °C

Squalene

Phytosterols Sesamolin

Ascorbyl palmitate Phytosterols, Oryzanol Tocopherols Squalene

~ 100 °C

~ 100 °C ~ 120 °C1

~ 130 °C ~ 150 °C ~ 150 °C ~ 170 °C

1

Abou-Gharbia et al. (2000).

or oryzanol-(sitosterol ferulate) by Rancimat or other oxidative stability index (OSI), the results of their antioxidative efficacy will be practically zero as these compounds do not act as radical scavengers. They are antipolymerizing agents whose effectivness is based on another mechanism. Frying oils are non-polar in nature. In fresh oil, the heat is not being transferred to the food due to the lack of polar compounds acting as emulsifying agents. Therefore, emulsifiers can be added to fresh frying oil to improve the contact between the ‘break in’ oil and the food. Thus, it may be possible to produce high quality fried products from the beginning. The emulsifiers which are best used for quickly lowering the interfacial tension are preferably selected from mono- and diacylglycerols and fatty acid esters of sorbitan, sugars and polyalcohols. They are added to frying oil in an amount not exceeding 4.0 wt %. Above that level, the emulsifier in the frying oil gives an adverse effect on taste and oil uptake. It is observed that the diacylglycerols present in palm olein (4–8 %) act as emulsifiers and improve crispness and heat transfer but also increase oil adsorption of food. Many heat-stabilizing liquid preparations containing emulsifiers, citric acid, natural ingredients and/or synthetic antioxidants and anti-polymerizing agents are already commercially available (Jaswir et al., 2000). Their effectiveness has been tested in practice by many authors (Gertz, 2004). The results confirm that natural ingredients and some formulations possess a strong stabilizing activity.

530 Modifying lipids for use in food The addition of these compounds may be an alternative to stabilizing deep frying oils rich in unsaturated fatty acids.

21.6 Quality control and safety of fresh frying oil Any fat or oil may be used for frying, but not every fat or oil will be suitable for all applications and produce the desired good flavour, texture and appealing appearance. Gupta et al. (2004) proposed to choose the frying oil for any given product on the basis of the following criteria: product flavour, product texture, mouth feel, aftertaste, availability, product shelf life, cost and nutritional requirements. Many practical tests are necessary to find the appropriate frying oil. For good frying performance, it is important to ensure that the composition and quality of the delivered oil correspond to the specification of the oil which has been used in the practical tests before. The manufacture of the test branded frying oil has to maintain its declared specification. It is therefore necessary that purity, authenticity and oxidative state of the frying fresh oil can be checked by simple and quick analytical methods. Gas-liquid chromatography of fatty acid methyl esters (FAME) is the traditional method of analysis for vegetable oils. Vegetable oils may contain more than 1000 triacylglycerols combining only ten different fatty acids. Fatty acid composition on its own is not enough to define triacylglycerol composition. Therefore, the gas chromatographic separation of the individual triglycerides is the method of choice to verify the authenicity of the frying oil. The analytical standards recommended for frying oils to check the oxidative and heat stability and the authenticity of the oil are listed in Table 21.4. The Table 21.4 Recommended specifications for fresh frying oils (see also Warner 2004). Parameter

Specification (limit)

Taste, flavor Free fatty acids (wt %) Monoacylglyceride (wt %) Peroxide value (meq O2/kg) Anisidine value Conjugated dienes (%) Phosphorus (mg/kg) Iron, copper (mg/kg) Magnesium (mg/kg) Calcium (mg/kg) Oxidative stability (Rancimat) Heat stability (OSET) Linolenic fatty acids

Bland < 0.05 < 0.4 < 0.5 < 4.0 < 1.0 < 0.5 < 0.5 < 0.5 (desired: < 0.2) < 0.5 (desired: < 0.2) High High <2%

Abbreviation: OSET = oxidative stability test at elevated temperature. Source: Warner (2004).

Developments in frying oils

531

limits of peroxide value, anisidine value, free fatty acids, phosphorus, metals and monoacylglycerols provide good information about shelf life, rancidity and oxidative resistance of the oil. A free fatty acid content above 0.05 % or higher phosphorus content in the fresh oil indicates incomplete refining. Good quality frying oil should have less than 0.4 % monoacylglycerols. Higher amounts cause excessive foaming. If the frying oil is not fully refined, it is essential that nearly all phospholipids are removed as these develop fishy flavors upon heating due to liberation of free amines. The fatty acid composition of the oil blends does not give realistic information about their stability under frying conditions. Minor components, which may be pro-oxidant or antioxidant, present in oil have strong influence on their stability, especially at frying temperatures. The recommended standard methods for oxidative stability, e.g. OSI or Rancimat Method, are carried out with an excess of oxygen at elevated temperatures, which are still different from frying conditions. These tests assume that thermal oxidative changes at 100 or 120 °C are not different from those at elevated temperatures and would follow the autoxidation radical mechanism. Nevertheless, they provide good information about the expected shelf life, rancidity and oxidative resistance of the fried product at normal temperature. These tests are not, however, suitable if one wants to check the behaviour of fats and oils at frying conditions. OSET (oxidative stability test at elevated temperature) (Gertz et al., 2000) is a new laboratory method to estimate the stabilizing activity of synthetic and natural food additives at simulated frying temperature. It does not need special equipment. The principle is to accelerate the dimerization of triacylglycerols catalyzed by acidic silica gel. The vegetable fat or oil is heated for two hours at a temperature of 170 °C after adding water-conditioned silica gel. Additionally, it is possible to measure the effectiveness of synthetic or natural compounds after adding 50 mg sample to 20 g of a vegetable oil. The determination of di- and polymeric triacylglycerols by size exclusion high-performance liquid chromatography (HPLC) was carried out for the estimation of the oxidative stability of the compounds at elevated temperature (OSET index = 100/amount of polymyeric triacylglycerols (%)). The test even makes it possible to measure the influence of a protective gas like nitrogen or of an anti-foaming agent. Results of the OSET test presented in Table 21.5 further show that there is a considerable effect on heat stability at 170 °C when vegetable oil is pre-oxidized. The fatty acid composition does not give realistic information about the stabilty of oils under frying conditions. The OSET test demonstrates that minor components, which may be pro-oxidant or antioxidant, have strong influence on oxidative stability, especially at frying temperatures. There is some evidence which supports the co-relationship between the unsaponifiable matter content and oxidative stability. Corn oil was more stable than soybean oil and rapeseed oil better than olive oil. It was also observed that non-refined oils proved to have a better stability at elevated temperature than refined oils. The behaviour of antioxidants like silicone or

532 Modifying lipids for use in food Table 21.5

Heat stability, measured by OSET, of commercially available frying fats and oils.

Vegetable oil

Almond oil, refined Corn oil, refined Grape seed oil, refined Groundnut oil, refined Olive oil. non-refined Palm oil, refined Pumkin seed oil, refined Pumkin seed oil, non-refined Rapeseed, refined Rapeseed, refined (Totox: 15)* Safflower oil Sesame oil, refined Sesame oil, non-refined Soybean oil, refined Sunflower, non-refined (Totox: 5)* Sunflower, refined (Totox: 6)* Sunflower, refined (Totox: 15)* Walnut oil, non-refined Walnut oil, refined

OSET

Fatty acid composition Saturated fatty acids

Monoenic fatty acids

Polyenic fatty acids

Trans fatty acids

36 51 27 32 45 48 62 112

8.3 14.0 11.3 17.7 12.5 44.8 18.3 19.4

65.2 30.4 17.5 42.1 78.8 43.6 26.9 25.8

26.3 55.4 70.1 38.6 8.7 11.1 54.7 54.7

0.2 0.2 1.1 1.6 0.0 0.5 0.1 0.1

51 32

8.1 8.3

64.8 65.1

26.8 26.3

0.3 0.3

25 38 78 39 46

11.4 16.6 16.2 16.4 11.8

17.3 40.7 40.2 24.7 18.6

70.8 42.6 43.5 58.3 69.5

0.5 0.1 0.1 0.7 0.1

27

12.9

21.6

65.4

0.1

22

11.1

26.6

61.5

0.1

109 52

9.4 10.4

17.7 17.1

72.8 71.2

0.1 1.3

*(Totox = 2 * peroxide value + para-anisidine value. Abbreviation: OSET = oxidative stability test at elevated temperature.

natural antioxidants like phytosterols and squalene could now be checked whereas the Rancimat/OSI failed (Gertz and Kochhar, 2001).

21.6.1 Monitoring fat degradation The nature and rate of decomposition products depend, among other things, on the composition of the oil (fatty acids, unsaponifiable matter content) and the food (moisture), the mode of frying (intermittent or continuous), frying temperature, length of frying process, type of food being fried, equipment, fresh oil replenishment and filtering systems. This fact reveals the difficulty in defining one single analytical parameter and prescribing exact limits of deterioration for these parameters. The organoleptic evaluation of used frying fats is difficult and subjective. Therefore, the sensory test must be confirmed by objective analytical criteria. To cover physical and chemical changes of frying fats and oils, many methods are available to monitor the degree of decomposition. Chemical tests are the determination of free fatty acids (acid value), iodine value, para-anisidine

Developments in frying oils

533

value, peroxide value, saponification value, polymerized triacylglycerols, petroleum–ether insoluble fatty acids and total polar compounds. None of these were found to be fully satisfactory. Nevertheless, the determination of polar compounds (Gertz, 1977; ISO, 2002) and of polymerized triacylglycerols (ISO, 2001) are recognized as the most reliable methods to quantify the degree of oil degradation worldwide. The following recommendation has been given during the 3rd International Symposium on Deep Fat Frying, March 2000 in Germany (DGF, 2000): ‘Analysis of suspect frying fats and oils should use two tests to confirm abuse. Recommended analyses should be: Total polar materials (TPM) (> 24 %) and, polymeric triglycerides (PTG) (< 12 %)’. Obviously, two different methods (Gertz, 2000) based on different chemical or physical properties give a more objective view of the degree of degradation. In most European countries, the limit of rejection for a frying oil has been regulated between 24 and 27 %TPA (Firestone, 1996). All these chemical methods require laboratory equipment. In the past, the main effort has been concentrated on detecting the point of discard. According to the recommendations of the symposium mentioned above, rapid tests should correlate with internationally recognized standard methods, provide an objective index, be easy to use, quantify the degree of oil degradation and be safe for use in food processing (avoid the use of glassware and chemicals). These requirements exclude qualitative test kits like Oxifrit® (Merck, Germany), LRSM® (3M Belgium), Rau-Test® or Fritest® (Merck, Germany) as they use chemicals and do not allow quantification of the degree of degradation. There are many quick tests and analytical methods measuring quantitative changes of physical properties such as density, viscosity, conductivity, refractive index, dielectric constant, smoke point, colour, foaming, smoking and UVabsorption. All test systems using one of these physical properties use the unheated fresh oil as reference for calibration. This calibration procedure is not correct as the composition of the oil in the fryer is changing all the time with input of par-fried products or the natural fat content as the fat of the food is fully replaced by the frying oil. The Food Oil Sensor® (FOS) (Northern Technologies International Corporation, USA) and newly developed instruments delivered by the German companies Ebro or Testo measure the dielectric properties of the oil, thus assessing the level of polar components as products of thermal deterioration. Fats containing short-chain fatty acids like coconut or animal fats enhance the polarity and change the dielectricity constant. Results were observed to be unduly sensitive to cold draughts of air and to the level of moisture in the oil (Gertz, 2000). Recent work indicates that the values obtained using Fri-Check® (Hulshout, Belgium) show excellent correlation with traditional methods for evaluating reduction of frying oil quality (Stier, 2004a). For good food processing controlling the formation of oxidation products, by measuring the anisidine value, is more important than determining the

534 Modifying lipids for use in food polar compounds or dimeric triacylglycerols or controlling the endpoint of discard of the used oil. A higher concentration of peroxides and secondary oxidation products accelerates the oxidation in the fried products and reduces shelf life, due to development of oxidized and rancid flavour even when the fried product is packed in nitrogen. Oxidation products are the main reason for a bad taste like rancidity and reduced shelf life. In a bakery, the doughnuts taste rancid when the oil reaches an anisidine value of 30 whereas the polar materials have only reached 12 % (Fig. 21.2). Additionally, it was observed that oxidative degradation of the product continues during storage at –5 to 10 °C. The basic decision on oil/fat throw away should always be made on the basis of the fried food quality.

21.7 Future trends Oils may someday be designed not only to resist formation of negative byproducts, but also to produce desirable flavours. The goal will be to provide consumer products with better shelf life and to provide more healthful, better tasting food. Another reason to modify fats and oils is to improve their nutritional properties, to change the fatty acid composition in the desired direction or to increase important minor components, such as certain phytosterols and antioxidants. If the application needs solid fats they will be replaced by trans-free fats produced by enzymatic interesterfication or by fractionation.

30

Fryer down

Fryer restart

Fryer down

Fryer restart

Fryer down

Fryer restart

25

PV or pAV

20

15

pAV PV

10

5 0 0

10

20

30 40 Frying time (h)

50

60

70

Fig. 21.2 Peroxide value (PV) and para-anisidine value (pAV) in fryer oil during frequent shutdowns (data source: Warner, 2004).

Developments in frying oils

535

21.8 Sources of further information and advice Boskou D and Elmadfa I (1999), Frying of Food, Lancaster, PA/Basel, Technomic. Gupta M K, Warner K and White P J (2004), Frying Technology and Practices, Champaign, IL, AOCS Press. Moreira R G, Castell-Perez M E and Barrufet M A (1999), Deep-fat Frying – Fundamentals and Applications. Gaithersburg, MD, Aspen Publishers, Inc. Perkins E G and Erickson M D (1996), Deep-frying – Chemistry, Nutrition, and Practical Applications, Champaign, IL, AOCS Press. Rossel J B (2001), Frying – Improving Quality, Cambridge, Woodhead Publishing.

21.9

References

(1999), Antioxidative effect of olive oil deodorizer distillate on frying oil and quality of potato chips, Fett/Lipid, 101, 57–63. ABIDI S L and RENNICK K A (2003), Non volatile components and frying performance of high-oryzanol rice bran oil, J Am Oil Chem Soc, 80, 1057–1062. ABOU-GHARBIA H A, SHEDATA A A Y and SHAHIDI F (2000), Effect of processing on oxidative stability and lipid classes of sesame oil, Food Res Int, 33, 331–340. ANDRIKOPOULOS N K, KALAGEROPOULOS N, FALIERA A and BARBAGIANNI M N (2002), Performance of virgin olive oil and vegetable shortening during domestic deep-frying and panfrying of potatoes, Int J Food Sci Technol, 37, 177–188. ASSUNTA DESSI M, DEIANA M, DAY B W, ROSA A, BANNI S and CORONGIU F P (2002), Oxidative stability of polyunsaturated fatty acids: effect of squalene, Eur J Lipid Sci Technol, 104, 506–512. BANKS D (1996), Edible oil technology: industrial frying, in Perkins E G and Erikson M D, Deep Frying: Chemistry, Nutrition, and Practical Applications, Champaign, IL, AOCS Press, 258–270. BARRERA-ARELLANO D, RUIZ-MENDEZ V, VELASCO J, MARQUEZ-RUIZ G and DOBARGANES C (2002), Loss of tocopherols and formation of degradation compounds at frying temperatures in oils differing in degree of unsaturation and natural antioxidant content, J Food Sci Agric, 82, 1696–1702. BERDEAUX O, MÁRQUEZ-RUIZ G and DOBARGANES C (1999), Selection of methylation procedures for quantitation of short-chain triacylglycerol-bound compounds formed during thermoxidation, J Chromatogr, A863, 171–181. BERDEAUX O, VELASCO J, MÁRQUEZ-RUIZ G and DOBARGANES C (2002), Evolution of shortchain glycerol-bound compounds during thermoxidation of FAME and monoacid TAGs, J Am Oil Chem Soc, 79, 279–285. BLUMENTHAL M M (1987), Optimum Frying: Theory and Practice, Piscataway, NJ, Libra Laboratories. BLUMENTHAL M M (1991), A new look at the chemistry and physics of deep-fat frying, Food Technol, 45, 68–71 BUXTORF U P, MANZ W and SCHUPBACH M (1976), Zur Praxisgerechten Beurteilung von Fritierfette, Mitt Gebiete Lebensm Hyg, 67, 429–432. CHANG S S, PETERSON R J and HO C T (1978), Chemical reactions involved in the deep-fat frying of foods, J Am Oil Chem Soc, 55, 718–727. ABDALLA A

536 Modifying lipids for use in food CHE MAN Y B and IRWANDI J (2000), Effect of rosemary and sage extracts on frying performance

of refined, bleached and deodorized (RBD) palm olein during deep-frying, Food Chem, 69, 301–307. st DACH (2001), Referenzwerte für die Nährstoffzufuhr (2001) 1 edn, Heidelberg, Umschau/ Braus-Verlag, 47–48. DANA D, BLUMENTHAL M M and SAGUY I S (2003), The protective role of water injection on oil quality in deep frying conditions, Eur Food Res Technol, 217, 104–109. DE B K and BHATTACHARYA D K (1998), Physical refining of rice bran oil in relation to degumming and dewaxing, J Am Oil Chem Soc, 75, 1683–1686. rd DGF (2000), Recommendations of the 3 International Symposium on Deep Fat Frying – Optimal Operation, Eur J Lipid Sci Technol, 102, 594. DOBARGANES C, MARQUEZ-RUIZ G and VELASCO J (2000), Interaction between fat and food during deep-frying, Eur J Lipid Sci Technol, 102, 521–528. DOBSON G, CHRISTIE W W, BRECHANY EY, SEBEDIO J L and LE QUÉRÉ J L (1995), Silver ion chromatography and gas chromatography – mass spectrometry in the structural analysis of cyclic dienoic fatty acids formed in frying oils, Chem Phys Lipids, 75, 171–182. DOBSON G, CHRISTIE W W and SÉBÉDIO J L (1996), Monocyclic saturated fatty acids formed from oleic acid in heated sunflower oils, Chem Phys Lipids, 82, 101–110. DOBSON G, CHRISTIE W W and SÉBÉDIO J L (1997), Saturated bicyclic fatty acids formed in heated sunflower oils, Chem Phys Lipids, 87, 137–147. ENIG M G (1995), Trans-fatty acids in the food supply, Silver Spring, MD, Enig & Associates. FARID M M and CHEN X D (1998), The analysis of heat and mass transfer during frying of food using a moving boundary solution procedure, Heat and Mass Transfer, 34, 68– 77. FARKAS B E, SINGH R P and RUMSEY T R (1996), Modelling heat and mass transfer in immersion frying. I. Model development, J Food Eng, 29, 211–226. FIRESTONE D (1996), Regulation of frying fat and oil, in Perkins E G and Erickson M D, Deep Frying Chemistry, Nutrition and Practical Applications, Champaign, IL, AOCS Press, 323–334. FRITSCH C W (1981), Measurement of frying fat deterioration: a brief review, J Am Oil Chem Soc, 58, 272–274. FUKUDA Y, NAGATA N, OSAWA T and NAMIKI M (1986), Chemical aspects of the antioxidative activity of roasted sesame seed oil, and the effects of using the oil for frying, Agric Biol Chem, 50, 875–862. GAMBLE M H and RICE P (1987), Effect of pre-fry drying on oil uptake and distribution in potato crisp manufacture, Int J Food Sci Technol, 22, 535–548. GAMBLE M H and RICE P (1988), Effects of initial tuber solids content on final oil content of potato chips, Lebensm Wiss u Technol, 21, 62–65. GAMBLE M H, RICE P and SELMAN J D (1987a), Relationships between oil uptake and moisture loss during frying of potato slices, Int J Food Sci Technol, 22, 233–241. GAMBLE M H, RICE P and SELMAN J D (1987b), Distribution and morphology of oil deposits in some fried products, J Food Sci, 52, 1742–1745. GERTZ C (1976), Zur Analytik der Fritierfette und –öle, Lebensmittelchemie und gerichtl. Chemie, 31, 65–68. GERTZ C (2000), Chemical and physical parameters as a quality indicator of used frying fats, Eur J Lipid Sci Technol, 102, 566–572. GERTZ C (2004), Optimising the baking and frying process using oil-improving agents, Eur J Lipid Sci Technol, 106, 736–745. GERTZ C and KOCHHAR S P (2001), A new method to determine oxidative stability of vegetable fats and oils at simulated frying temperature, Oléagineux Corps gras Lipides, 8, 82–88. GERTZ C, KLOSTERMANN S and KOCHHAR S P (2000), Testing and comparing oxidative stability of vegetable oils and fats at frying temperature, Eur J Lipid Sci Technol, 102, 543– 551.

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and KOCHHAR S P (2003) Deep-frying: the role of water from food being fried and acrylamide formation, Oléagineux Corps gras Lipides, 10, 297–303. GILLAT P (2001) Flavour and aroma development in frying and fried food, in Rossel J B, Frying – improving quality, Cambridge, CRC Press, Woodhead Publishing, 266–336. GOGOLEWSKI M, NOGALA-KALUCKA M and GALUBA G (2003), Studies on dimerisation of tocopherols under the influence of methyl linoleate peroxides, Nahrung/Food, 47, 74– 78. GORDON M H (1989), Plant sterols as natural anti-polymerisation agents, in Proc IUFoST International Symposium: New Aspects of Dietary Lipids, Benefits, Hazards & Use, SIK, Goteborg, Sweden, 23–34. GORDON M H and KOURISMA L (1995), The effects of antioxidants on changes in oils during heating and deep frying, J Sci Food Agric, 68, 347–353. GUINDA A, DOBARGANES C, RUIZ-MENDEZ M V and MANCHA M (2003), Chemical and physical properties of a sunflower oil with high levels of oleic and palmitic acids, Eur J Lipid Sci Technol, 105, 130–137. ISO (2001), ISO Standard 16931, Animal and vegetable fats and oils – determination of polymerized triglycerides by high-performance size-exclusion chromatography (HPSEC), Geneva, ISO. ISO (2002), ISO Standard 8420, Animal and vegetable fats and oils – determination of polar compounds content, Geneva, ISO. JASWIR I, CHE MAN Y B and KITTS D D (2000), Optimisation of physiochemical changes of palm olein with photochemical antioxidants during deep-fat frying, J Am Oil Chem Soc, 77, 1161–1168. KAMAL-ELDIN A, APPELQUIST L A, GERTZ C and STIER R (1998), Enhancing the frying performance of high oleic sunflower oil using a specially manufactured sesame and rice bran oil, J Foodservice Systems, 10, 130–157. KAMAL-ELDIN A, VELASCO J and DOBARGANES C (2003), Oxidation of mixtures of triolein and trilinolein at elevated temperatures, Eur J Lipid Sci Technol, 105, 165–170. KOLETZKO B (1994)‚ Trans-fatty acids and the human infant, World Rev Nutr Diet, 75, 82– 85. LOLOS M, OREOPOULOU V and TZIA C (1999) Oxidative stability of potato chips: effect of frying oil type, temperature and antioxidants, J Sci Food Agric, 79, 1524–1528. MACRAE A R (1983), Lipase-catalyzed interestification of oils and fats, J Am Oil Chem Soc, 60, 291–294. MANKEL A (1970), Zur Analytik und Beurteilung von Fritürefetten I., Fette Seifen Anstrichmittel, 72, 483–487. MARQUEZ-RUIZ G and DORBARGANES C (1996), Short-chain fatty acid formation during thermoxidation and frying, J Sci Food Agric, 70, 120–126. MENSINK R P and KATAN MB (1995), Commentary on the supplement trans-fatty acids and coronary heart risk, Am J Clin Nutr, 62, 518–519. MINAL J (2003), An introduction to random interesterification of palm oil, Palm Oil Developments, No. 39, 1–6. MOREIRA R G, SUN X and CHEN Y (1997), Factors affecting oil uptake in tortilla chips in deepfat frying, J Food Eng, 31, 485–498. MOREIRA R G, CASTELL-PEREZ M E and BARRUFET M A (1999), Oil absorption in fried foods, in Moreira R G, Castell-Perez M E and Barrufet M A, Deep-Fat Frying: Fundamentals and Applications, Gaithersburg, MD, Aspen Publishers, Inc., 179–221. NGADI M O, WATTS K C and CORREIA L R (1997), Finite element method modelling transfer in chicken drum during deep fat frying, J Food Eng, 32, 11–20. PAZOLA Z, GAECKI J, BUCHOWSKI M, KORCZAK J, JANKUN J and GRZESKOWIAK B (1985), Choice of simple methods for quality control of frying fat during deep frying of potato products, Fette Seifen Anstichmittel, 87, 190–193. PEERS K E and SWOBODA A T (1982), Deterioration of sunflower seed oil under simulated frying conditions and during small-scale frying of potato chips, J Sci Food Agric, 33, 389–395.

538 Modifying lipids for use in food PETHUKOV I, MALCOLMSON L J

, PRZYBYLSKI R and ARMSTRONG L (1999), Frying performance of genetically modified canola oils, J Am Oil Chem Soc, 76, 627–632. PETRAUSKAITÉ V, DE GREYT W, KELLENS M and HUYGHEBAERT A (1998), Physical and chemical properties of trans-free fats produced by chemical interesterification of vegetable oil blends, J Am Oil Chem Soc, 75, 489–493. PINTHUS E J, WEINBURG P and SAGUY I S (1993), Criterion for oil uptake during deep fat frying, J Food Sci, 58, 204–211. PINTHUS E J, WEINBURG P and SAGUY I S (1995), Deep-fat fried potato oil uptake as affected by crust physical properties, J Food Sci, 60, 770–772. PRAVISANI C I and CALVELO A (1986), Minimum cooking time for potato strip frying, J Food Sci, 51, 614–617. QUINLAN P and MOORE S (1993), Modification of triglycerides by lipases: process technology and its application to the production of nutritionally improved fats, Int News Fats Oils Relat Mater, 4, 580–585. ROSSEL J B (2003), Developments in oils for commercial frying, Lipid Tech, 15, 5–8. SILKEBERG A (1990), Edible fats and oils stabilized with sesame oil as a constituent, Patent, CA2052124, EP0477825. SIMS R J, FIORITI J A and KANUK M J (1972), Sterol additives as polymerisation inhibitors for frying oils, J Am Oil Chem Soc, 49, 298–301. STIER R F (2004a), Tests to monitor quality of deep-frying fats and oils, Eur J Lipid Sci Technol, 106, 766–771. STIER R F (2004b), Frying as a science – An introduction, Eur J Lipid Sci Technol, 106, 715–721. TIAN L L and WHITE P J (1994), Antipolymerisation activity of oat extract in soybean and cottonseed oils under frying conditions, J Am Oil Chem Soc, 71, 1087–1094. TIFFANY (2004), Transformation and modifications des gras et des huiles (vue d’ensemble), Oral presentation during the Séminaire Les matiéres grasses et les huiles, October 14– 15, Montreal. UMANO K and SHIBAMOTO T (1987), Analysis of acrolein from heated cooking oils and beef fat, J Agric Food Chem, 35, 909–912. VELASCO J , MARMESAT S, MÁRQUEZ-RUIZ G and DOBARGANES C (2004), Formation of shortchain glycerol-bound oxidation products and oxidised monomeric triacylglycerols during deep-frying and occurence in used frying fats, Eur J Lipid Sci Technol, 106, 728–735. VITRAC O, TRYSTRAM G and RAOULT-WACK A L (2000), Deep-fat frying of food: heat and mass transfer, transformations and reactions inside the frying material, Eur J Lipid Sci Technol, 102, 529–538. WARNER K (2004) Chemical and physical reactions in oil during frying, in Gupta M K, Warner K and White P J, Frying Technology and Practices, Champaign, IL, AOCS Press, 16–28. WARNER K and GUPTA M (2003), Frying quality and stability of low and ultra low linolenic acid soybean oils, J Am Oil Chem Soc, 80, 275–280. WIL A M, VAN LOON J P H, LINSSEN A, LEGGER A and VORAGEN G (2004), Personal communication. XU Z and GODBER J S (2001), Antioxidant activities of major components of γ-oryzanol from rice bran using a linoleic acid model, J Am Oil Chem Soc, 78, 645–649. YAMSAENGSUNG R and MOREIRA R G (2002), Modeling the transport phenomena and structural changes during deep fat frying, J Food Eng, 53, 11–25. YANISHLIEVA N V, MARINOVA E M, MAREKOV I N and GORDON M H (1997), Effect of an ethanol extract from summer savory (Saturejae hortensis L) on the stability of sunflower oil at frying temperature, J Sci Food Agric, 74, 524–530.

22 Speciality oils and their applications in food K. Bhattacharya, International Food Science Centre A/S, Denmark

22.1

Introduction

Oils and fats are consumed for caloric reasons and also for their non-caloric functions such as flavour, palatability, appearance, consistency and texture. They have the highest caloric density among foodstuffs and are carriers of important vitamins (A, D, E and K) and various other nutrients that are vitally important for normal physiological functions (Formo, 1979). Oils extracted from crops such as soybean, canola, sunflower, safflower, corn, palm, palmkernel, coconut, etc. are extensively used in food items such as baked and fried products, margarines and spreads, chocolate and other confectionery items, ice creams, salad dressings and mayonnaise, non-dairy coffee whiteners and baby foods. Individual oils may be used in these applications or two or more oils may be blended in defined proportions with or without modification. The processes commonly used for modification of fats and oils are fractionation, partial hydrogenation and interesterification. Some of the important objectives for modification include: (i) production of a fat or oil to meet certain characteristics not possible with its natural physical properties; (ii) selection of the cheapest blend that will provide desirable physical, chemical and nutritional properties; (iii) increase of oxidative stability; and (iv) development of nutritionally healthier products.

22.2 Speciality oils and fats Modification of an oil or fat changes the fatty acid composition and/or the regiospecific distribution of the fatty acids in the triacylglycerols and affects

540 Modifying lipids for use in food physical properties of oils and fats such as melting point, melting profile or melting behaviour, rate of crystallization. The final use of an oil or fat determines the range of values of these parameters. Thus modification provides a much wider range of raw materials. Modified oils and fats with specific characteristics and specific application areas are called ‘speciality oils and fats’ and are sometimes also referred to as ‘tailor-made fats’ or ‘recipe engineered fats’. Design and production of a speciality fat may involve one or more modifying steps depending on the availability of raw materials and the nature of the end product. A more recently developed procedure for modification involves genetic mutation and selective breeding to alter the fatty acid composition of commodity oils such as sunflower, safflower, peanut, soybean and canola oil. With reduced levels of linoleic acid and α-linolenic acids and enhanced levels of oleic acid, the ‘high oleic’ varieties have much higher oxidative stability than the commodity versions and can also be termed ‘speciality oils’. They are used for various commercial applications. For example, high oleic sunflower oil is now being used as spray oil for snacks, crackers and breakfast cereals (Gunstone, 2002a). Numerous publications, conferences and advertisements regularly highlight the link between dietary fats and health. As a result, the lipid composition and fat content of food products have become primary areas of consumer concern. With the introduction of the term ‘functional’, the food industry has entered a new sphere where greater attention is given to the ‘extra’ or ‘added’ benefits of the food ingredients. Although ‘neutraceuticals’ is another word commonly used, ‘functional’ is the term which seems to be most widely employed and acknowledged by consumers regarding the healthy nature of any food or food ingredient. In many cases, new ingredients of known beneficial health properties are being added into an existing formulation with the sole objective of labelling it as functional to increase its sales potential. In the same connection, dietary fats are also often described as functional. A modern day definition of speciality oils and fats should thus include oils and fats which, in addition to having specific physical characteristics, contain bio-active molecules that impart health benefits or restrict the proliferation of a disease, improve oxidative stability or have clinically proven health-friendly fatty acid composition or distribution in the triacylglycerol molecule.

22.3 Health benefits and claims for speciality oils The growing consumer demand for healthier products has stimulated the development of new products with reduced fat content and/or altered fatty acid profiles. With the ageing population concerned about diet and health, nutritional information and related health claims play significant and influential roles in the minds of the consumer. ‘Health-friendly’ food products often

Speciality oils and their applications in food

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claim to contain less of a particular ingredient, additive or component of concern. Apart from reduced fat, nowadays, low-trans (< 5 %) or no-trans (< 1 %), low saturates and higher polyunsaturated fatty acids (PUFA) are primary claims of various fat-based food items. Nutritional claims have two kinds of approaches: ‘less harmful’ or ‘health beneficial’. In terms of oils and fats, the former claim generally highlights the reduction of saturated fatty acids (SFA) and of trans fatty acids (TFA) while the second emphasizes the presence of polyunsaturated fatty acids (PUFA) in appropriate amounts and ratio between omega-6 and omega-3 acids and/or the presence of healthy ingredients, such as vitamins, antioxidants, cholesterol-lowering-compounds, etc. In the USA, the Food and Drug Administration (FDA) defines and regulates nutritional products which include Foods with Health Claims, Dietary Supplements, Foods for Special Dietary Uses and Medical Foods but not terms such as Functional Foods, Designer Foods, and Antioxidants, etc. However, health claims for other terms not defined by FDA may be supported or accredited by various research institutes and academic or industrial organizations (Kamarei and Trygstad, 2004). The FDA perceives claims in three different ways (Kamarei and Trygstad, 2004). These are: (i) health claims that are authorized by the FDA which include correlation between dietary lipids (fat) and cancer and dietary saturated fat and cholesterol and risk of CHD, (ii) qualified health claims permitted by the FDA which include long-chain omega-3 fatty acids [(eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)] and CHD and (iii) qualified health claim petitions received by the FDA. This last group includes, for example, claims associated with eggs enriched with omega-3 fatty acids and a balanced 1:1 ratio of omega-3/omega-6 fatty acids leading to reduced risk of heart disease and sudden death.

22.4 Dietary fatty acids and health effects The possible role of dietary fatty acids in health and diseases has received significant public attention in recent years, and most of the investigations have involved the trans, saturated and polyunsaturated fatty acids. The effects of these fatty acids on health are briefly mentioned in the following sections.

22.4.1 Trans fatty acids and health Blends of hydrogenated and non-hydrogenated oils and fats have been used to produce base stocks for margarine, frying oils and a variety of generalpurpose fats where solid and stable fats were required. Traditionally such blends have had high proportions of TFAs which are now considered to have significant detrimental health effects. The level of TFAs in a hydrogenated fat depends on the processing conditions like temperature, pressure, catalyst and duration of reaction (see Chapter 9). Small amounts of TFAs are also

542 Modifying lipids for use in food found in ruminant fats such as milk, butter and tallow as a result of biohydrogenation (Hunter, 2004). Trans fatty acids arising from partial hydrogenation have been extensively investigated in relation to their effect on health (Hunter, 1992). The risk of CHD increases proportionally with serum levels of total and low-density lipoprotein (LDL) cholesterol and decreases with increase in high-density lipoprotein (HDL) cholesterol (Castelli et al., 1986; Martin et al., 1986). Trans monounsaturated fatty acids raise LDL cholesterol concentrations (Katan et al., 1995) and decrease HDL cholesterol concentrations (Mensink et al., 2002) in contrast to intake of cis monounsaturated fatty acids. Researchers have also examined the correlation between TFAs and cancer, type-2 diabetes and allergies (Stender and Dyerberg, 2004). However, no report has yet conclusively demonstrated direct association of TFAs intake and tumour development (Hunter, 1992). Weggemans et al. (2004) investigated whether TFAs from ruminant sources differ from those resulting from partial hydrogenation with respect to CHD and concluded that below an intake level of 2.5 g/day there was no differences in effects on CHD between the two sources of TFAs, but that at total intake levels of above 3 g/day industrial TFAs cause bigger risk of CHD. However, insufficient data on intake of TFAs from ruminant sources at this level leads to an inconclusive comparison.

22.4.2 Saturated fatty acids and health Saturated fatty acids are reported to be cholesterol-raising but not all acids in this class show the same effect (Mensink et al., 2002). Alkanoic acids are divided into three classes: (i) fatty acids having less than 12 carbon atoms, (ii) fatty acids with 12, 14 or 16 carbon atoms and (iii) the 18 carbon homologue, stearic acid. Cater et al. (1997) have indicated that the first group slightly reduces LDL cholesterol relative to palmitic acid but raises this parameter when compared to oleic acid. From the second group, lauric acid has been reported (Denke and Grundy, 1992) to increase plasma total cholesterol and LDL cholesterol concentrations compared to oleic acid but to a lower extent relative to palmitic acid while effects on HDL cholesterol were not observed. However, Temme et al. (1996) reported an increase of total cholesterol due to an increase of HDL cholesterol as compared to palmitic acid. Zock et al. (1994) have shown that myristic acid has an increasing effect on both LDL cholesterol and HDL cholesterol and hence on total cholesterol concentration relative to palmitic acid. Despite being hypercholesterolaemic compared to stearic acid (Mensink et al., 2002), palmitic acid has not been labelled in all cases as a cholesterol-elevating saturated fatty acid (Ng et al., 1992; Choudhury et al., 1995). This holds true when dietary cholesterol intake is less than 300 mg/day and 6–7 % of daily energy comes from linoleic acid. Stearic acid had been shown not to elevate plasma total cholesterol concentration (Keys et al., 1965; Grande et al., 1970). In fact, later studies by Bonanome and

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Grundy (1988) reveal that stearic acid has a neutral effect on plasma lipoproteins similar to that of cis-monounsaturated oleic acid. Overall, it can be concluded that TFAs and lauric, myristic and palmitic acids raise the levels of both total and LDL cholesterol. The FDA announced changes to the Nutritional Labelling Education Act requiring the labelling of trans-fats from 1 January 2006 while the Danish government banned the selling of all food products containing more than 2 % TFAs from 1 January 2005.

22.4.3 PUFA and health benefits In contrast to the negative publicity of trans and saturated fatty acids, PUFAs and omega-3 fatty acids in particular are welcomed by the health-conscious. There are two major PUFA families: one based on linoleic acid (∆-9,12-18:2 omega-6) and the other on α-linolenic acid (∆-9,12,15-18:3 omega-3). The importance of PUFAs in human health and nutrition was postulated first in the 1920s. Linoleic acid and α-linolenic acid were termed essential fatty acids (EFA) (Burr and Burr, 1929) since they cannot be synthesized in vivo by animals including humans. They must, therefore, be consumed from plantderived dietary sources. Once consumed, both linoleic and α-linolenic acid are converted to other long-chain omega-6 and omega-3 fatty acids by metabolic pathways in mammals through enzymatic catalysis. These changes require chain-elongation and desaturation. The most important omega-6 metabolite is arachidonic acid (AA, 20:4) and the most important omega-3 metabolites are eicosapentaenoic acid (EPA, 20:5) and docosahexaenoic acid (DHA, 22:6). AA is the principal substrate for a series of enzymes that produce eicosanoids such as thromboxanes, prostaglandins, leukotrienes, prostacyclins and lipoxins (Yamamoto and Smith, 2002). Based on animal experiments, it is now acknowledged that α-linolenic acid, through its downstream longer-chain homologue DHA, is essential for normal visual functions (Sinclair et al., 2002). DHA has been shown to be vital in various physiological activities such as membrane fluidity which can influence the function of membrane receptors, regulation of membrane-bound enzymes, regulation of eicosanoid synthesis from AA and protection of neural cells from apoptotic death. Deficiency of α-linolenic acid in animal diets leads to a lowering of DHA in the brain leading to changes in the auditory, olfactory, learning and memory functions, appetite control, neuron size and nerve growth factor levels (Sinclair et al., 2002). Fish oils, rich in long-chain omega-3 fatty acids, have been found to reduce plasma triacylglycerols of hyperlipidaemic subjects, especially in patients with elevated triglyceride concentrations (Harris, 1989). Harris et al. (1983) showed that in the case of normocholesterolemic subjects, longchain PUFA from fish oils do not induce any changes in plasma LDL cholesterol or HDL cholesterol concentrations but have a lowering effect on plasma triacylglycerols and the concentration of cholesterol in very low-density lipoprotein (VLDL).

544 Modifying lipids for use in food To improve the nutritional aspects of food products, there is a steady influx of oils and oil blends rich in omega-3 fatty acids (α-linolenic acid in the case of vegetable oils and DHA in the case of fish oils) into existing food items such as breads, spreads, yoghurts, cereals, dairy products, drinks, etc.

22.4.4 Monounsaturated oils and health benefits Plant breeding techniques have led to the commercialization of new varieties of traditional oils such as sunflower, safflower, canola and peanut oils with very high proportions of monounsaturated fatty acids (MUFA), particularly oleic acid (cis-9-octadecaenoic acid) (Sakurai and Pokorny, 2003). The term high oleic oil is applied to appropriately modified commodity oils which are more usually rich in linoleic acid such as high oleic sunflower oil. Oleic acid is not an essential fatty acid as animal tissues contain active ∆-9-desaturase activity and oleic acid is readily produced biologically from stearic acid (Gurr, 1998). Various researchers have investigated the effect of MUFA on health (Grundy, 1986; Mensink and Katan, 1987). Mensink and Katan argue that the term ‘neutral’ used to describe the effect of oleic acid on plasma total cholesterol concentration is frequently misinterpreted. They consider that ‘neutral indicates that monounsaturated fatty acids have the same effect on plasma total cholesterol as an isocaloric amount of carbohydrates’. They also noted that ‘increasing the intake of oleic acid at the expense of carbohydrates increased plasma HDL cholesterol concentrations and decreased those of triacylglycerols. The increase in HDL cholesterol was compensated by a decrease in VLDL cholesterol’. The comparative effects of high-MUFA diet, a low-fat diet, Step II lowfat diet recommended by the AHA (American Heart Association) (25 % fat, 7 % SFA, 12 % MUFA) and the average US diet (34 % fat, 16 % SFA, 11 % MUFA) on lipids and lipoproteins have been examined (Kris-Etherton et al., 1999). They conclude that high-MUFA diets and AHA Step II diets lowered total cholesterol by 10 % and LDL cholesterol by 14 % compared to the average US diet. There was a reduction of triacylglycerols by 13 % with the high-MUFA diets but an increase of 11 % on AHA Step II diets. The latter lowered HDL cholesterol by 4 % (Jewett, 2002). Studies at lower intakes (less than 28 % of energy) reveal that both oleic acid and linoleic acid have similar effects on the concentration of LDL cholesterol and HDL cholesterol (Mensink and Katan, 1989; Valsta et al., 1992; Wahrburg et al., 1992). Mensink et al. have concluded that ‘replacement of dietary saturated fatty acids in the diet by monounsaturated fatty acids causes the same favourable change in plasma lipoprotein cholesterol levels as their replacement by polyunsaturated fatty acids’ (Mensink et al., 2002). In contrast, Howard et al. consider that PUFAs have a greater cholesterol-lowering effect and a lesser triacylglycerol-elevating effect compared to MUFAs in a dose–response study in a multiracial group (Howard et al., 1995).

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Although several reports based on serum lipid and lipoprotein levels indicate beneficial effects of MUFA-rich diets, work by Rudel et al. (1998) on primates has indicated increased risk of cardiovascular diseases when the primates were subjected to MUFA diets compared to primates on PUFA diets. Their findings suggest that primates on MUFA diets had larger LDL cholesterol particle size, which is often linked to greater risk of cardiovascular diseases, compared to PUFA-enriched diets.

22.5 Designing and application of speciality oils Intake of oils and fats is primarily through cooking oils, baked items such as breads, flutes, croissants, etc. often enjoyed with butter, margarine or similar spreads, all forms of fried products, chocolate and sugar confectionery, dairy products and desserts, salad oils, mayonnaise and other dressings, and fat consumed when meat, poultry or fish are eaten. All these sources make up a complex matrix of various visible and invisible oils and fats that end up in the body. With increasing demand for low-saturate/MUFA oils and PUFA oils over saturated and hydrogenated fats, there is a new interest in designing food products and cooking oils to meet these desires. There is a further point to be addressed in designing healthy PUFA oils – the ratio of omega-6 to omega-3 fatty acids. The changes from the dietary unsaturated C18 acids to the long-chain PUFA involve several biochemical reactions in which the acids are elongated and desaturated. These reactions, occurring with the omega-6 and omega-3 acids, are catalyzed by the same enzymes, and there is competition between linoleic acid and its metabolites and α-linolenic acid and its metabolites for these. It is, therefore, important that the dietary intake of linoleic acid and αlinolenic acid be in an appropriate balance. The present dietary ratio of omega-6 to omega-3 acids is estimated to be within the range 7:1 to 25:1 (Djordjevic et al., 2005). Deficiency in α-linolenic acid intake is compounded by the fact that the 18:3 acid is not efficiently metabolized to EPA and DHA, especially in the presence of a large excess of linoleic acid. This high ratio has developed since the 1950s following the increased availability and popularity of vegetable oils rich in linoleic acid. These comments relate to total lipid intake and not to individual oils that are consumed. It seems desirable that our fat intake should contain less linoleic acid and more αlinolenic acid. Perhaps we should also increase our intake of EPA and DHA from fish or from fish oils, but improvement of the ratio will start for most of us by reducing the linoleic acid/α-linolenic acid ratio.

22.6 Use of MUFA and PUFA oils in food applications High oleic or MUFA oils are preferred to unmodified PUFA oils in applications

546 Modifying lipids for use in food like frying and cooking in which there is an exposure to air, light and high temperature which cause undesirable changes in PUFA, especially when used for long periods. However, use of high oleic oils is not restricted to frying oils. They have other applications. Table 22.1 shows the principal fatty acids (as percentage of total fatty acids) of both regular and high oleic varieties of some commodity oils. It is evident from Table 22.1 that in all the high oleic varieties, oleic acid has increased mainly at the expense of linoleic acid but also with marked reduction of α-linolenic acid in the case of soybean and canola oils. The decrease of α-linolenic acid to about one-tenth of its original value is greater than the effect of brush hydrogenation performed on the commodity versions of these oils to reduce α-linolenic acid. These high oleic varieties are also often referred to as ‘low linoleic’ or ‘low linolenic’ oils. These changes also have a beneficial effect on oxidative stability. It is well known that the greater the number of unsaturated sites, the greater is the tendency of oxidation. If the rate of oxidation for oleic acid (18:1) is 1, then the relative rates of oxidation for linoleic acid and α-linolenic acid are 12 and 25 respectively (Gunstone and Hilditch, 1946). Such big reductions of the diene linoleic acid and the triene α-linolenic acid have helped the high oleic oils to gain high oxidative stability. Comparative inherent oxidative stability ratings rank high oleic sunflower oil (1.894) and high oleic safflower (1.710) much below safflower oil (9.546), soybean (8.578), sunflower (8.489), corn (7.708) and cottonseed oil (6.890) (O’Brien, 2004). Higher oxidative stability of high oleic sunflower and safflower oil had previously been noted by Purdy (1985) from AOM (Active Oxygen Method) values (see Table 22.2). Various applications of such high oleic MUFA oils, alone or in conjunction with PUFA oils, are discussed in the following sections.

22.6.1 MUFA and PUFA oils as speciality frying and cooking oils New trends in development of frying oils include Nu-Sun®, by Archer Daniels Table 22.1 Principal fatty acids (as % of total fatty acids) of some regular oils and their high oleic varieties.

Saturates 18:1 18:2 18:3

Sunflower

Safflower

Regular High oleic

Regular

High oleic

Regular

High oleic

Regular

High oleic

13 22 65 <1

10 12 77 tr

8 77 13 tr

8 58 20 10

6 76 14 <2

15 22 54 8

11 83 4 1

tr = trace Source: Kristott (2003).

9 83 10 <1

Canola

Soybean

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Table 22.2 Principal fatty acid composition and AOM values of conventional and high oleic varieties of sunflower and safflower oil. Principal fatty acids (% of total fatty acids)

Conventional safflower High oleic safflower Conventional sunflower High oleic sunflower High oleic sunflower

AOM (hours)

16:0

18:0

18:1

18:2

7 5 7 3 3

2 tr 5 5 5

12 80 18 79 83

70 15 69 12 7

10 35 11 38 60

Abbreviation: AOM = active oxygen method. Source: Adapted from Purdy (1985).

Midland Company, USA (ADM). Nu-Sun™ (National Sunflower Association, USA) is a de-waxed mid-oleic sunflower oil grown mainly in the USA, produced by conventional seed breeding techniques (Gupta, 1998) and having about 64.5 % oleic acid, 22.1 % linoleic acid and less than 1 % α-linolenic acid. High oleic sunflower oils are now frequently used as frying oils, with or without blending with other oils. Some examples of commercially available high-oleic sunflower oils are Viola from Bulgaria, High-Oleic-Sunflower oil 80+ and High-Oleic-Sunflower oil 90±® (Dr Faische GmbH) from Germany, Clear Valley® from Cargill Inc. and Trisun® from Humko Oil, both from the USA. High oleic safflower oil, Montola high-oleic safflower from Montola Growers Inc. of the USA and Odyssey® brand of high oleic canola from Cargill Inc. are also available (Sakurai and Pokorny, 2003, anon: www.higholeic.de, anon: www.montola.com, anon: www.clearvalleyoils.com). Despite the improved oxidative stability of the high-oleic oils, there are reservations about the sensory properties of food products fried in them (Kochhar, 2001). This is mainly because of the low content of linoleic acid, the mild oxidation of which is essential for the production of low concentrations of oxidation compounds which contribute significantly and positively to deep-fat fried flavour (Perkins, 1996; Hamilton and Perkins, 1997). A 5– 10 % content of linoleic acid is recommended by Sakurai and Pokorny (2003) for optimum fried flavour development. Braddock et al. (1995) have noted that roasted high oleic, low linoleic peanuts have longer shelf life with more stable flavour, lower peroxide value and less hexanal when stored at 25 or 40 °C for up to 74 days. However, the high oleic peanuts had less intensive roasted taste mainly due to lower content of pyrazines (Baker et al., 2003). Comparative tests with potato crisps to evaluate high oleic oils against reference oils such as palm olein and partially hydrogenated rapeseed oils have led to interesting observations. While analytical parameters such as total polar compounds, free fatty acids, dielectric constant and colour of frying medium indicate performances of high oleic oils similar to and better than the reference oils, the sensory properties of the fried products were different. The

548 Modifying lipids for use in food shelf life of fried food was longer in the case of palm olein, probably due to its higher natural tocopherol content (Kristott, 2003). This aspect of shorter shelf life slightly undermines the use of high oleic oils for fried items that need a specified shelf life. Yet, from the view point of saturated and TFA intakes through snacks, high oleic oils provide a better lipid profile. Frito-Lay has been a pioneer snack food manufacturer in the shift to non-trans oils and declares trans fat information on the nutrition labels of their products in the USA (Juttelstad, 2004). Japan has increased its consumption of high oleic oils, in particular high oleic canola, over the last few years (Corbett, 2003) while in Canada, New York Fries was the first fast-food chain to offer transfree fries by the end of March 2004 (Anon, 2004). High oleic oils can be also used as base oils for blending with other oils to provide yet further benefits. One such example is the ‘Good-Fry®’ oil from Good-Fry International BV in the Netherlands which consists mainly of high oleic sunflower oil to which a small amount of specially refined sesame oil and specially produced rice bran oil have been added. Its fatty acid profile indicates 4.5 % palmitic, 3.7 % stearic, 78.7 % oleic acid, 10.8 % linoleic acid and 0.1 % α-linolenic acid, and it has been approved as ‘dietetic’ oil by the Federal Institute for the Health Protection of Consumers in Berlin, Germany (Kochhar, 2001). Frankel and Huang (1994) studied the oxidative stability of various blends of high oleic sunflower oil with soybean, canola and corn oil at 60 °C. Measurement of peroxide values and volatiles (hexanal and propanal) by static headspace capillary gas chromatography form the basis of their report. From peroxide values they concluded that ‘a partially hydrogenated soybean oil containing 4.5 % linolenate was more stable than the mixture of soybean oil and high oleic sunflower oil containing 4.5 % linolenate’. However, static headspace gas chromatographic data showed that ‘mixtures of soybean and high oleic sunflower oil containing 2.0 and 4.5 % linolenate were equivalent or better in oxidative stability than the hydrogenated soybean oil. Mixtures of canola oil and high oleic sunflower oil containing 1.0 and 2.0 % linolenate had the same or better oxidative stability than did hydrogenated canola oil containing 1.0 % linolenate’. Such experimental data suggest that by blending high oleic oils with common oils like soybean and canola one can avoid the conventional brush hydrogenation and produce trans-free oils with high oxidative stability. Two examples of cooking oils containing high oleic oils are Isio 4, a blend of high oleic sunflower oil [referred to as Oleisol® (Lesieur, France)], conventional sunflower oil, canola and grape seed oil and Croustidor having 40 % high oleic sunflower oil with the rest probably conventional sunflower oil (anon: www.lesieur.com). Both these oils are from the French company Lesieur. Vivola™ functional oil from Forbes Medi-Tech in Canada is also an example of multi-component cooking oil. It is a blend of tropical lauric oils, olive oil, coconut oil and flax seed oil and contains about 65 % of mediumchain triacylglycerols (Ohr, 2003).

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The preferred cooking oil in many countries is a function of the oil grown most in that geographical location. Gunstone (2002b) has reported annual average disappearance of oils in various countries in 1996/2000 and shown a clear pattern in which the locally produced oil represents close to or higher than 50 % of total disappearance. Examples include Indonesia (palm oil 89 %), Brazil (soybean oil 70 %), Malaysia (palm oil 60 %), Argentina (sunflower oil 56 %), Philippines (coconut oil 55 %), USA (soybean oil 52 %) and Canada (canola oil 47 %). However, these data do not relate only to consumption as frying or cooking oils but total edible and non-edible applications. Rice bran oil has gained interest due to its nutritional constituents and is used mainly as cooking oil in Japan, Korea, China, Taiwan, Thailand and Pakistan. It is also used in India as such or in combination with oils like safflower and sunflower. Apart from a fatty acid composition of about 20 % SFA, 45 % MUFA, 37 % linoleic acid and 1.5 % α-linolenic, rice bran oil contains tocotrienols and γ-oryzanols, which have beneficial physiological effects. Oryzanol is a group of sterol esters of ferulic acid such as ferulates of cycloartenyl, 24-methylene cycloartanyl, cycloartanyl, β-sitosteryl ferulate and campesteryl ferulate (Kochhar, 2001). Oryzanol lowers plasma and liver cholesterol concentrations, decreases arterial fatty depositions (McCaskill and Zhang, 1999), and also exhibits antioxidant properties (Diack and Saska, 1994). Rice bran oil and peanut oil have similar fatty acid compositions, and reports indicate that rice bran oil is equivalent to peanut oil in model generalpurpose frying operations. The Schaal oven test of potato chips fried in rice bran oil shows this oil to give products with flavour and odour stability between that of peanut and cottonseed oil (Orthoefer, 1996). With improvement of extraction and refining technology, rice bran oil has gained commercial importance in India and is widely available as cooking oil. Commercial brands in India containing rice bran oil include Sundrop Heart (Agro Tech Foods) and Saffola Gold (Manico Industries) with sunflower and safflower oil, respectively, as the second component.

22.6.2 MUFA and PUFA oils in margarine and spreads Margarine and related spreads can serve as important delivery systems for health beneficial ingredients which include PUFAs, antioxidants, cholesterolreducing components like phytosterols and phytostanols, vitamins, etc. Margarine is a legally defined food and products with less than 80 % fat must be described as spreads (Chrysam, 1996). Numerous researchers have examined the comparative health effects of butter and margarine (Zock et al., 1994; Chisholm et al., 1996; Judd et al., 1998) and suggested that replacing butter with soft and low trans fat margarines can significantly reduce the risk of heart disease, although other health benefits have been claimed for butter resulting from the presence of CLA and of sphingolipids. The description of margarines and spreads frequently contains terms like regular, diet, low-fat, trans-free, cholesterol-lowering, low salt or no salt, etc. and these make up a very wide range of products.

550 Modifying lipids for use in food The first ‘heart-healthy’ high-PUFA (linoleic acid) tub margarine was introduced as early as the mid-1960s (Rajah, 2004) and since then formulators have regularly used vegetable oils which are often rich sources of MUFA and PUFA in margarine formulation. These include rapeseed/canola, sunflower, soybean, corn and cottonseed sometimes after partial hydrogenation. Gunstone (1999) reported the approximate fatty acid composition of some spreading fats showing a high of 66 % monoenes and 35 % of polyenes in stick margarines based on vegetable oils only. SmartBalance Omega Plus™, a buttery spread available in the USA, is enriched with omega-3 fatty acids from marine sources and α-linolenic acid from soybean and canola. It also contains palm oil and olive oil. Camelina oil, also known as ‘false flax’, typically contains 38–42 % of α-linolenic acid and is a good alternative to flax seed oil as a vegetable source of omega-3 fatty acids. The Finnish food manufacturer Raisio has introduced camelina oil into its Beneviva margarine (anon:www.nutraingredients.com). OmegacranTM, an oil blend containing cranberry seed oil from Decas Botanical Synergies in the USA, has equal proportions of omega-6 and omega-3 fatty acids and could be an ideal component in spreads (anon: www.decasbotanical.com). Laboratory-based enzymatic interesterification of lard and high oleic sunflower oil Trisun® in the ratio 60:40 (w/w) by Seriburi and Akoh (Seriburi and Akoh, 1998) provided a fat blend similar to soft-type margarine fat. Such enzyme-catalyzed interesterification of saturated fats with MUFA-rich liquid oils provides ways to design plastic fats needed for margarine production without hydrogenation. Simple physical blends of the same starting oils were much harder and had less monounsaturation in the sn-2 position. Studies have shown that presence of mono- or polyunsaturation at the sn-2 position is important for better absorption in the body (Seriburi and Akoh, 1998).

22.6.3 Long-chain PUFA oils in margarine and spreads Traditionally, oils from cold water fish such as menhaden, sardine, herring, etc. have been perceived as basic sources of omega-3 fatty acids and have been used in margarines. Partial hydrogenation of fish oil is often a necessity to prevent oxidation and consequent development of a strong and undesired fishy smell. For table margarines, hydrogenated fish oils have been used in the ranges between 45 and 72 %, while for industrial cake and creaming margarines it falls between 5 and 10 % and for industrial puff or pastry margarines it varies between 50 and 70% of total fat content (Opstvedt et al., 1990). However, recent practices make use of concentrated and encapsulated forms of EPA and DHA, such as Nutrinova® DHA. Developed from vegetable sources, Nutrinova® DHA can be added to various spreads as well as to yoghurts, baked goods, snacks and fruit juices and products with high fat content or products with high viscosity (Ruessing, 2004). Supplementation with different levels of γ-linolenic acid (GLA) together with constant intake of EPA and DHA shows a synergistic health beneficial

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effect (Laidlaw and Holub, 2003). Measurements of plasma lipids and fatty acids of serum phospholipids after a supplementation period of 28 days showed that the plasma triacylglycerol concentrations were significantly lower in those receiving mixes of EPA, DHA and GLA compared to those using only EPA and DHA. The report also mentioned that the group which received a mixture of EPA + DHA (4 g) and of GLA (2 g) was estimated to have a 43 % reduction in the ten year risk of CVD. Thus addition of small portions of GLA oils in conjunction with EPA and DHA in a margarine or spread formulation can provide added benefits. Common plant sources of GLA are borage oil (21–25 %) and evening primrose oil (7–10 %). There is a third source, blackcurrant seed oil (BCO), which is one of the few oils to contain linoleic acid, α-linolenic acid, GLA and stearidonic acid (18:4 n-3), all together. Typically, BCO contains 6–8 % palmitic, 1–2 % stearic, 9–13 % oleic acid, 44–51 % linoleic acid, 15–20 % GLA, 12–14 % α-linolenic acid and 2–4 % stearidonic acid (Traitler et al., 1988). Due to such unique fatty acid composition, especially GLA and stearidonic acid, BCO can provide important fatty acids for both the omega6 and omega-3 metabolic pathways. BCO is reported to reduce hypertension, lower LDL cholesterol, raise HDL cholesterol, decrease platelet aggregation, improve atopic eczema, and help in fighting rheumatoid arthritis, etc. (Tuomasjukka, 2004). Seal oil is a good source of EPA, DHA and docosapentaenoic acid (DPA, 22:5). Although it is believed that EPA is the key in producing prostaglandins that keep the artery wall soft and free of plaque, DPA is considered to be much more powerful than EPA in this effect (www.omegaplus.nf.ca). DPA has been found (Kanayasu-Toyoda et al., 1996) to be up to ten times as effective as EPA in stimulating the migration of endothelial cells. This suggests that it is a powerful anti-atherogenic factor. In another study (Benistant et al., 1996) DPA was found to reduce prostacyclin production in endothelial cells. In seal oil, EPA, DHA and DPA are located at the primary positions of glycerol (sn-1 and sn-3) while they are in the middle position (sn-2) in fish oils (Ackman, 1997). In view of such findings, concentrated forms of EPA/ DPA/DHA can be added to margarine and spread formulations to further enrich their omega-3 profiles. Micro-encapsulation of fish oil with protective films of hydrocolloids provides protection from oxidation. The different techniques for encapsulation of oils include spray-drying, spray-chilling, fluid-bed encapsulation, extrusion encapsulation and encapsulation by complex coacervation. Fish oil concentrates, rich in EPA and DHA, are commercially available as triacylglycerols (both as oil and powder), ethyl esters and free fatty acids. Examples include Incromega from Croda Chemicals Europe, Marinol™ from Loders Croklaan in the Netherlands and Ropufa® from DSM in Switzerland. Such products have been recommended for use in functional foods, dietary supplements and pharmaceuticals. In bakery products, UK-based Warburtons Ltd have introduced DHA (30 mg/100 g bread) in their Good Health Loaf

552 Modifying lipids for use in food while Bioriginal Food & Science of Canada launched omega-3-rich flax flour BakOmega™ for use in baked products (Ohr, 2003). In Australia, TipTop®Up™ bread from George Weston is another example of bread fortified with micro-encapsulated HiDHA tuna fish oils from Nu-Mega Ingredients.

22.6.4 MUFA and PUFA in salad oils, dressings, mayonnaise and gourmet oils Leafy salad mixes, sandwiches and similar cold dishes are very often accompanied by salad oils, mayonnaise or some form of dressings. While salad oils are pure liquid oils (which may be spiced), mayonnaise and other dressings are emulsions of oil, water, vinegar, seasoning, egg yolk, salt, sugar and preservatives. Such products can be thick (spoonable) or fluid (pourable) and may contain up to 84 % oil. The oil phase provides the lubrication and the pleasant mouth feel of all kinds of dressings. Thus, such food items can be viewed as an important medium of oil intake and there is scope to provide nutrition through them. Oxidative stability and liquidity even during storage at refrigeration temperature of 4 °C are the two primary requirements for the oils used in all salad oils and dressings. Salad oils may need to be winterized to remove high-melting triacylglycerols and solid waxes which cause cloudiness at refrigeration temperature. Refined soybean, winterized cottonseed, dewaxed sunflower, corn, safflower and canola are the major oils used in such applications in the USA (O’Brien, 2004). Very often, soybean and canola oils are mildly hydrogenated to reduce the α-linolenic acid content, thus preventing oxidative flavour reversion and deterioration. They may also be winterized to remove high-melting triacylglycerols. High oleic oils provide the dual benefit of stability and liquidity. Herb and spice extracts may be added to the oils for dressings and mayonnaise. These provide oxidative protection and other benefits as mentioned in later sections. Alongside various cooking oils, supermarkets now also offer a large variety of gourmet oils. These oils are generally rich sources of PUFA and serve as important ingredients for culinary preparations. They are generally expeller pressed and often filtered, but not rigorously refined like other commodity oils which would strip off their characteristic flavours. For example, walnut, sesame, almond, hazelnut, pistachio, macademia nut oils provide a nutty flavour and are suitable for salads, pastas and sautés. Argan oil also provides a distinctive nutty flavour with fruity hint while almond oil provides its own unique flavour and aroma (Wright, 2004). The presence of a variety of phytochemicals in these unrefined oils accounts for their typical flavours. These oils are generally used for applications in salads, marinating and, in some restricted cases, stir frying. From the principal fatty acids of some of the commonly available gourmet oils as shown in Table 22.3 one can see the low content of SFA and predominance of oleic and linoleic acid.

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Table 22.3 Principal fatty acid (as % of total fatty acids) composition of some gourmet oils. Oil

16:0

18:0

18:1

18:2

18:3

Almond Avocado Argana Flaxseedb Grape seed Macadamia nutc Pistachio Pumpkin seed Sesameb English walnut

12.2 9.0 12.9 4.8 7.0 10.1 8.6 16.4 9.9 7.3

0.1 0.5 5.9 4.7 3.9 4.3 2.3 7.7 5.2 2.3

44.5 77.7 46.9 19.9 15.6 57.4 68.8 33.8 41.2 19.1

40.5 8.5 32.5 15.9 72.2 2.5 17.8 42.0 43.3 57.4

0.6 0.9 0.1 52.7 0.2 – 0.3 – 0.2 13.1

a

Commercial supplier Yinag GmbH, Austria. White (1992). c Also contains 16:1–20.1 %, 20:0–2.8 %. Source: Kamel and Kakuda (1992). b

Flaxseed and walnut oil contain appreciable quantities of α-linolenic and so need greater care while handling. International Food Science Centre A/S of Denmark have launched a blend of flaxseed, rapeseed and camelina oil called Nutridan which is recommended for various culinary preparations apart from bakery applications (Shukla and Bhattacharya, 2003a). Nutridan has a very low ratio of linoleic acid to α-linolenic acid. Typically the ratio is 0.6.

22.7 Use of spice extracts in gourmet oils Gourmet oils are often flavoured with extracts of Capsicum annum, chilli, garlic, onion, etc. which in addition to catering to savoury aspects provide added physiological benefits. Shon et al. have reported the antioxidant, antimutagenic and free radical scavenging activities of ethyl acetate extracts from white, yellow and red onions (Shon et al., 2004) and have attributed them to the phenols and flavonoids present in them. Harunobu et al. (2001) have demonstrated the cholesterol-lowering properties of oil-soluble sulfur compounds from garlic, e.g. diallyl sulfide, diallyl disulfide or diallyl trisulfide. Garlic oil is produced by steam distillation of crushed raw garlic and subsequent dilution with vegetable oils to reduce the powerful odour of the volatile oilsoluble sulfides. The antiplatelet activity of methyl allyltrisulfide, a component commonly present in steam-distilled garlic oil, has been demonstrated (Ariga et al., 2000). Chilli pepper extract contains terpenoids with antioxidative and antimicrobial activities (Draughon, 2004). β-Carotene, acyl derivatives of capsanthin and acyl derivatives of capsorubin were isolated from red paprika (Capsicum annuum) oleoresin and studied. Comparative studies of Cu2+-

554 Modifying lipids for use in food catalyzed oxidation revealed that the carotenoids efficiently suppressed the oxidation of LDL cholesterol, inhibited the formation of conjugated dienes from polyunsaturated fatty acids, lowered the content of the small dense LDL cholesterol sub-fraction, and inhibited the transformation of cholesterol to autoxidized products (Medvedeva et al., 2003). Among many producers of oil-soluble concentrates of onion, garlic and chilli paprika oleoresin, Kalsec® is widely known while Carl J. Nielsen & Søn A/S is prominent in Denmark. Nutridan is one salad oil available with added chilli and garlic extracts.

22.8 Use of natural antioxidants Various herb extracts, spices, teas, oilseeds and oils, cereals, legumes, fruits and vegetables contain minor components that act as natural antioxidants. The different types of natural antioxidants investigated include (i) tocopherols and tocotrienols, (ii) phenolic acids (carnosic acid and rosmarinic acid) found mainly in the Lamiaceae family of herbs, (iii) flavonoids (e.g. quercetin, kaemferol, luteolin, morin, myricetin) from plant sources and (iv) catechins or phenols (carnosol, rosmanol, epirosmanol) from tea and Labiatae family of herbs. Among the herb extracts, rosemary extract, consisting of a mixture of phenolic acids and diterpenes, has been most extensively studied (Cuvelier et al., 1996). The main antioxidative effect of rosemary extracts comes from three phenolic compounds, namely carnosic acid, carnosol and rosmarinic acid, of which over 90 % of the antioxidant activity is from carnosic acid and carnosol (Aruoma et al., 1992). Flavonoids, particularly flavones, have also been identified in rosemary extracts, and there are reports of antimicrobial, antiviral, antimutagenic and anticarcinogenic activities of rosemary extracts (Schwarz, 2003). Shukla and Bhattacharya (2003b) have described the antioxidative effect of rosemary extracts on various PUFA oils like evening primrose, borage, camelina, flaxseed oil and blackcurrant oils. Carnosic acid and carnosol, the main active ingredients of rosemary extracts, are effective radical scavengers for peroxyl radicals, the primary product of autoxidation, and interrupt the propagation steps (Marinova et al., 1991). Addition of rosemary extracts prevents oxidation and reduces oil darkening during deep-fat frying (Lalas and Dourtoglou, 2003) while the synergistic effects of rosemary, sage and citric acid on unsaturated fatty acid retention of palm olein during deep-fat frying have been reported (Jaswir et al., 2000). Sewalt et al. have shown that the addition of 0.1 % w/w of water-dispersible rosemary extract to mayonnaise or salad dressing produced lower peroxides and alkenal formation during storage (Sewalt et al., 2005). The Odyssey® brand of high oleic canola from Cargill Inc. is an example of commercially available oil containing rosemary extracts (anon: www.clearvalleyoils.com). Polar and non-polar fractions of oregano leaves inhibit oxidation of linoleic acid measured by ferric thiocyanate and thiobarbituric acid methods (Nakatani,

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1997). Addition of ground or ethanolic extract of oregano into frying oils (cottonseed) reduced the formation of polymerized and dimeric triacylglycerols and polar compounds and decreased the rate of formation of conjugated dienes (Dimitra et al., 2004) and of aldehydes responsible for the anisidine value. It has been shown that rosemary and sage inhibited oxidation in lard and mayonnaise and in French dressings and pie crusts during practical applications (Nakatani, 1997). Cinnamon, turmeric, mace, clove and nutmeg have also shown strong antioxidative properties in heated peanut oil, and Nakatani has concluded that spices like allspice, aniseed, basil, cardamom, cassia, ginger, etc. also display substantial antioxidative properties. Application of such spices in gourmet oils, mayonnaise and other salad dressings will serve the dual purpose of providing flavour and oxidative stability. Constituents of oilseeds such as sesame, soybean, cottonseed, etc. have been investigated for antioxidant activities. Sesame seed and its oil have been found to contain bisepoxylignans, sesamin and sesamolin (Kochhar, 2001) which serve as the precursors of powerful antioxidants. The high stability of sesame oil obtained from roasted sesame seeds stems from the synergistic combination of γ-tocopherol, tocotrienols, sesamol, Maillard reaction products formed during roasting, ∆5-avenasterol and other related ethylidene sterols (Shukla et al., 1997; Kochhar, 2001). Silkeberg and Kochhar had reported that addition of a blend of ‘dedicated’ refined sesame oil and specially produced rice bran oil’ (Good-Fry® constituents) can improve the stability of frying oil blends made from sunflower, soybean and canola. Such oil blends, protected with these natural antioxidants, are nutritionally better than partially hydrogenated frying oils.

22.9 Effect of dietary fatty acids in poultry and meat Several studies have been conducted with a view to increasing the PUFA content, particularly of the long-chain omega-3 fatty acids and of conjugated linoleic acid (CLA), in eggs, edible tissues of poultry and intramuscular fat of beef, lamb and pork meats. Fatty acids present in the adipose tissues of animals may originate from de novo synthesis or directly from the diet (Wiseman and Garnsworthy, 2004). The dietary approach provides a quicker route for the incorporation of unusual fatty acids into milk, eggs, or meat. Increase of omega-3 fatty acids can be achieved through diets containing flaxseeds, flaxseed oil, fish oil or fishmeal and forages. Diets rich in α-linolenic acid lead to increased levels of α-linolenic acid, EPA and DPA but not of intramuscular DHA which can be achieved only using dietary fish meal or fish oil already containing DHA (Raes et al., 2004). A review by Rymer and Givens (2005) shows that the concentration of α-linolenic acid in the edible tissues of poultry can be readily increased by increasing the proportion of α-linolenic acid in the birds’ diet. This is more so in meat with skin and dark meat than in white meat. The concentration of

556 Modifying lipids for use in food EPA in both dark and white meat also increases with inclusion of EPA supplements in the diet. However, such rise in EPA was not observed when its precursor α-linolenic acid was included in the diet. Incorporation of long-chain PUFA in poultry feed has led to the commercial availability of eggs containing high amounts of DHA. Omega-3 rich eggs were first introduced in UK by Columbus followed by the ‘Intelligent Eating Healthy Eggs’ from Stonegate Farms. The latter brand is a product arising out of the use of chicken feed enriched with DHA-rich tuna oil from NuMega while Columbus utilize omega-3 from flaxseeds. In the USA, Martek Biosciences feeds marine algae to chickens leading to high-DHA eggs which are sold under the brand of Gold Circle Farms. Columbus eggs are sold in the USA as Christopher eggs by Belero, a Belgium-based company. Body Egg™ from Chanteclair Farms in Australia is also a commercially available omega-3-rich egg. Typically, an egg from Stonegate Farms contains 0.11 g DHA (information taken from packaging) while a Columbus egg contains 0.7 g total omega-3 acids (anon: www.columbuseggs.com). DHA-rich eggs can act as important source of DHA for pregnant women (Smuts et al., 2003) whose dietary intake of DHA should be increased to meet the demands of the foetus, especially during the third trimester when the brain is developing rapidly. However, such long-chain omega-3 fatty acids need to be protected from oxidation. Cortinas et al. (2003) have shown that use of 100 ppm of α-tocopheryl acetate in chicken feed markedly reduces lipid oxidation as measured by thiobarbituric acid values in hard-boiled and scrambled eggs containing long-chain omega-3 acids from fish oils. Incorporation of CLA in eggs is also possible by feeding CLA-rich diets to egg laying hens, and a preferential higher deposition rate percent is observed for the c9 t11 isomer compared to the t10 c12 isomer (Raes et al., 2002). Thus, development of such enriched eggs can provide important dietary components without necessitating any change in food habits. The use of high oleic oils in animal feed has provided beneficial changes in the fatty acid composition of the adipose tissues of non-ruminant farm animals. Reports (Rhee, et al., 1990; Myer et al., 1992; Rentfrow et al., 2003) indicate that inclusion of high oleic sunflower oil and high oleic peanut oil in swine diet increases the ratio of MUFA to SFA in the pork depot fat. Use of high-oleic oleic sunflower oil in fish meal had led to the lowering of lipid oxidation products and weaker fishy aroma but with consequent reduction of omega-3 fatty acids, the very reason for which fish and fish oil are valued (Sakurai and Pokorny, 2003). This calls for a selective balance of choice of aroma against nutrition. Howe and co-workers have reported significant increase of long-chain omega-3 in pork, chicken and eggs by using PorcOmega™, a stabilized tuna fish meal formulation from Barlett Grain Pty Ltd in Australia, as a source of DHA for omega-3 supplementation (Howe et al., 2002). Fish meal and protected tuna fish diet of lambs have also been reported to increase EPA and DHA in the longissimus (Kitessa et al., 2001; Raes et al., 2004). In most of such

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supplemented diet studies, increase of omega-3 has been associated with lowering of omega-6 deposition, mainly due to reduced input of linoleic acid. Addition of dry pulverized herring to regular corn-based diet for cows led to significant amounts of DHA in cows’ milk (Hurteau, 2004). Neilsen Dairy of Canada markets a DHA-rich milk under the brand name Dairy Oh!™ containing 0.01–0.02 g (10–20 mg) of DHA per 250 ml serving. Although increased PUFA in animal tissues has its nutritional advantages, there are some limitations. In the absence of natural antioxidants, PUFAs could undergo in vivo oxidation within the animal causing metabolic problems leading to more unstable meat and generation of volatile components resulting from oxidation (Wiseman and Garnsworthy, 2004; Ziprin et al., 1990). Such oxidation would degrade meat quality and decrease consumer acceptance. An enhanced level of vitamin E is recommended to avoid such oxidation. PUFAs can also undergo extensive biohydrogenation in the rumen. The extent of biohydrogenation of linoleic acid and α-linolenic acid in vivo in ruminants has been estimated at 80 and 92 %, respectively (Doreau and Ferlay, 1994), leading to lowering of PUFA and increase in TFA moieties.

22.9.1 Effect of CLA in animal feed CLA has been acknowledged to have antioxidative and anti-carcinogenic properties in many animal models. It also enhances growth and feed efficiency and lowers body fat via fat-to-lean partitioning in some animal species (Joo et al., 2002). CLA is produced via bioconversion of linoleic acid in the rumen by the bacterium Butyrivibrio fibrosolvens and is an intermediate metabolite for the production of vaccenic acid and stearic acid. It is also formed by desaturation of vaccenic acid. Generally, the CLA content of beef ranges from 1.7 to 8.5 mg/g of fat (Mir et al., 2001). Direct enrichment of ruminant diets with CLA might be difficult due to the rapid biohydrogenation of CLA into stearic acid (Dugan et al., 1997), and supplementation with CLA has to occur in pre-ruminant stages. Alternatively, use of linoleic-rich safflower oil as a component of the diet has been successful in increasing the CLA content in lamb (Mir et al., 2001; Kott et al., 2003) and beef. King and co-workers reported the rise of CLA in pork adipose tissue by feeding CLA-enriched fat sources (King et al., 2004). Interestingly, use of fish oils for steers also resulted in the increase of CLA (c9, t11 isomer), although the exact mechanism or reason is yet to be established (Raes et al., 2004). Incorporation of CLA in pig diets is promising from a nutritional standpoint, but it is associated with increased deposition of SFA (myristic, palmitic and stearic acid). The SFAs were primarily positioned in the sn-1 and sn-3 position accompanied by a proportional decrease in MUFA (mainly oleic acid) and linoleic acid in these positions. As a result, the fat from the CLA fed pigs had higher slip melting points which is considered to be helpful for production of bacon (King et al., 2004). King has suggested that the effect of CLA on oleic

558 Modifying lipids for use in food acid content is proportional to the level and duration of CLA supplementation. Rise of SFA (especially myristic and palmitic acid) in pork fat due to supplementation of CLA can cause cardiac problems (Zock and Katan, 1994). The effect of CLA on lipoproteins in serum of fasting swine shows a trend for higher triacylglycerols, cholesterol and phosphatidylcholine in very lowdensity lipoproteins and in LDL, without marked changes in the HDL concentrations. This leads to a significant increase in the LDL/HDL cholesterol ratio (Stangl et al., 1999). Feeding CLA to pigs has the potential to improve animal performance, carcass composition and pork quality and to provide CLA-enriched pork for human consumption.Variations in CLA response and enrichment still need to be addressed, but uniform production practices could very well bring CLA-enriched pork to market.

22.10

Structured lipids

The distribution of the fatty acids in triacylglycerols can be rearranged and, if desired, new fatty acids can also be introduced through interesterification. Utilizing the principles of interesterification, short- (2:0 to 6:0), medium(8:0 to 12:0) or long-chain fatty acids (14:0 and above) can be preferentially esterified into a given position in the glycerol moiety. This leads to a ‘structured’ configuration of the triacylglycerol molecule forming a class of lipids called ‘structured lipids’. Simple physical blending does not produce such structured lipids. The rationale behind the development of structured lipids is based on the effects of dietary fatty acids and the importance of their relative position (sn1 or sn-3 and sn-2) in triacylglycerol molecules. Generally, in the case of vegetable oils, unsaturated fatty acids are situated in the sn-2 position while SFAs occupy the sn-1 and sn-3 positions (Hunter 2001). Triacylglycerols can be tailored to contain appropriate proportions of n-3, n-6, n-9 and SFAs which are beneficial in lowering serum LDL cholesterol and triacylglycerol levels, preventing thrombosis, enhancing the immune system, reducing the risk of cancer and improving nitrogen balance. Structured lipids are also often designed to obtain desired melting characteristics for application in margarine and spreads, confectionaries, snack foods, baked products or as low-fat or low-calorie fats (Akoh, 2002). Some of the commercially available structured lipids are Caprenin (Procter & Gamble), Betapol™ (Loders Croklaan, Netherlands), Benefat® (Danisco, Sweden), Neobee® (Stepan Company, USA), Captex® (ABITEC Corp., USA), IMPACT® (Novartis Nutrition, USA), Structolipid™ (Fresenius Kabi Germany), INFAT™ (Enzymotec, Israel), etc. (Akoh, 2002; Xu, 2000).

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22.10.1 Betapol Betapol™ and Betapol™ 45 from Loders Croklaan are designed for use in infant formulas in an attempt to make these correspond more closely to human milk fat. The key feature is the specific positioning of palmitic acid (16:0) at the sn-2 position as in human milk triacylglycerols. These differ from vegetable fats in the unusually high level of 16:0 in the β or sn-2 position. Betapol™ contains about 21–24 % palmitic acid of which 45 % is at sn-2 and 38–42 % oleic acid situated mainly at the sn-1 and sn-3 positions. The significance of this ‘structural’ aspect is that there is an increased fat absorption, increased calcium absorption, softer stools and reduced incidence of constipation in infants. The hypothesis of higher efficiency of fat absorption from the sn-2 position rather than from sn-1 and sn-3 is supported by studies by Filer et al. (1969) and Carnielli et al. (1995). The acyl chain in the sn-2 position is more resistant to the lipolytic action of pancreatic lipase. Consequently, the fatty acids in the sn-2 position remain intact as monoacylglycerols during digestion and absorption. In the presence of sufficient pancreatic lipase activity, 2monoacylglycerol and free fatty acids are the first products of triacylglycerol digestion. 2-Monoacylglycerols then form mixed micelles with bile salts and are better absorbed. Liberation of free palmitic acid is undesirable because it leads to undesirable loss of calcium as the insoluble palmitate (Lucas et al., 1997; Quinlan, 1996). Using formulas containing Betapol™, Lucas and Quinlan have demonstrated the validity of this hypothesis in the case of pre-term infants and it would hold true for full term infants as well. InFat from Enzymotec is a similar product with the predominance of palmitic acid in the 2-position recommended for use in infant formulas. 22.10.2 Salatrim Salatrim is an acronym for short- and long-chain acyl triglyceride molecules (Akoh, 2002). Salatrim is designed on the basis of (i) short-chain fatty acids (< 6 carbons) which contain fewer calories and (ii) stearic acid at the sn-1 and sn-3 positions which is only partially absorbed (only about 50 %) (Klemann et al., 1994). Salatrim is prepared by interesterification of heavily hydrogenated canola, soybean, cottonseed or sunflower oil with short-chain fatty acids like acetic, propionic and butyric (Smith et al., 1994). This causes the replacement of long-chain fatty acids with specific ratios of short-chain fatty acids, producing triacylglycerols containing two short and one long or with one short and two long-chain fatty acids on the glycerol. Benefat is the generic name for salatrims developed by Nabisco Foods Group (USA) and is commercially available under the brand name Benefat™, marketed by Cultor Food Science (USA). Benefat™ with a calorie content of only 5 kcal/g is recommended for confectionery, baking chips, baked and dairy products, salad dressings, etc. Based on the petition by Nabisco, the FDA granted Salatrim GRAS (Generally Recognized As Safe) status in June 1994.

560 Modifying lipids for use in food

22.11

Use of diglyceride oils as cooking oils

The role of dietary fat in obesity-related health problems has led to the development of various ways of reducing fat intake, for example developing fat substitutes such as Olestra or pancreatic lipase inhibitors like Orlistat. Another novel approach has been the introduction of diacylglycerol (DAG)based oils. These were developed in Japan by the Kao Corporation. Now they are also marketed in the USA by ADM Kao LLC, a joint venture company of Archer Daniels Midland Company (ADM) and Kao Corporation of Japan, under the brandname Enova™. Enova™ predominantly consists of more than 80 % DAG and is mainly the 1,3-isomer. Produced from a blend of soybean and canola through a patented enzymatic process that greatly increases the DAG content (Watkins, 2003), Enova™ has received GRAS status for applications like cooking, baking, spreads, soups, sauces, dressings, etc. An Israeli company, Enzymotec, has also launched a product based on natural oils enriched with phytosterols and DAG under the brand name Cardia Beat™ recommended for cooking, spreads, salad dressings, mayonnaise, etc. DAGs present in edible oils usually occur at the 1–10 % level and are mainly 1,2 DAGs. In contrast, DAG oils are mainly 1,3 DAGs (57 % by weight) along with 1,2-DAG and TAG (Flickinger Matsuo, 2003). Although DAG oils are digested and absorbed like TAG oils, they are metabolized differently. It is believed that DAGs are converted directly by the body to energy rather than accumulating as fat in the adipose tissues, especially in the vicinity of the internal organs (Watkins, 2003). DAG oils also lower serum triacylglycerols and remnant lipoprotein-cholesterol levels two and four hours after consumption. Reduction in glycosylated haemoglobin, indicating improved blood sugar control, has also been observed in diabetics during 12 week consumption as compared to diet based conventional oil of similar fatty acid composition (Flickinger and Matsuo, 2003). DAG oils are unique speciality fats. Such oils are likely to penetrate newer and bigger segments of the food industry and provide a safer, healthier yet tasty means of enjoying food.

22.12

Conclusion and future trends

In the absence of all the desired nutritional qualities in single oil, modifications of oils and fats will always be a tool for formulators. The future of speciality oils and fats will be dominated by the synergistic combination of structured lipids, natural antioxidants and other micronutrients. Simultaneously, conscious effort has to be made to translate scientific findings into a language understood by consumers who need to feel confident and comfortable about what they eat. This new millennium will enhance the scientific base for product development and expand collaboration among agricultural, nutritional and medical scientists. This should bring about a greater involvement of nutritionists

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and dieticians in industrial research and development to respond to an everincreasing consumer interest in the health attributes of food.

22.13

Sources of further information and advice

www.high-oleic.de www.montola.com www.clearvalleyoils.com www.lesieur.com http://www.nutraingredients.com/news/news-ng.asp?id=55613 www.omegaplus.nf.ca www.columbuseggs.com

22.14

References

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564 Modifying lipids for use in food KITESSA SM, GULATI SK, ASHES JR, SCOTT TW

and FLECK E (2001) Effect of feeding tuna oil supplement protected against hydrogenation in the rumen on growth and n-3 fatty acid content of lamb fat and muscle, Austr J Agric Res, 52, 433–437. KLEMANN LP, FINLEY JW and LEVEILLE GA (1994), Estimation of the absorption coefficient of stearic acid in salatrim fats, J Agric Food Chem, 42, 484–488. KOCHHAR SP (2001), The composition of frying oils, in Rossell JB, Frying: Improving Quality, Cambridge, Woodhead Publishing, 85–114. KOTT RW, HATFIELD PG, BERGMAN JW, FLYNN CR, WAGONER VAN H and BOLES JA (2003), Feedlot performance, carcass composition, and muscle and fat CLA concentrations of lambs fed diets supplemented with safflower seed, Small Ruminant Res, 49, 11–17. KRIS-ETHERTON P, PEARSON TA, WAN Y, HARGORE RL, MORIARTY K, FISHELL V and ETHERTON TD (1999), High-monounsaturated fatty acid diets lower both plasma cholesterol and triacylglycerol concentrations, Am J Clin Nutr, 70, 1009–1015. KRISTOTT J (2003), High-oleic oils – how good they are for frying?, Lipid Technol, 15, 29– 32. LAIDLAW M and HOLUB BJ (2003), Effects of supplementation with fish oil-derived n-3 fatty acids and γ-linolenic acid on circulating plasma lipids and fatty acid profiles in women, Am J Clin Nutr, 77, 37–42. LALAS S and DOURTOGLOU V (2003), Use of rosemary extract in preventing oxidation during deep-fat frying of potato chips, J Am Oil Chem Soc, 80 (6), 579–583. LUCAS A, QUINLAN P, ABRAMS S, RYAN S, MEAH S and LUCAS PJ (1997), Randomised controlled trial of a synthetic triglyceride milk formula for preterm infants, Arch Dis Child Fetal Neonatal, Ed, 77, 178–184. MARINOVA E, YANISHLIEVA N and GANEVA I (1991), Antioxidative effect of Bulgarian rosemary and inhibiting activity of its carnosol, Oxidation Commun, 14, 12–31. MARTIN MJ, BROWNER WS and HULLEY SB (1986), Serum cholesterol, blood pressure, and mortality: implications from a cohort of 361,662 men, Lancet, 2, 933–936. MCCASKILL DR and ZHANG F (1999), Use of rice bran oil in foods, Food Technol, 53 (2), 50– 52. MEDVEDEVA NV, ANDREENKOV VA, MOROZKIN AD, SERGEEVA EA, PROKOF’EV IUI and MISHARIN AIU (2003), Inhibition of oxidation of human blood low density lipoproteins by carotenoids from paprika, Biomed Khim, 49 (2), 191–200. MENSINK RP and KATAN MB (1987), Effect of monounsaturated fatty acids versus complex carbohydrates on high density lipoproteins in healthy men and women, Lancet, 1, 122–125. MENSINK RP and KATAN MB (1989), Effect of a diet enriched with monounsaturated or polyunsaturated fatty acids on levels of low-density and high-density lipoprotein cholesterol in healthy women and men, N Engl J Med, 321, 436–441. MENSINK RP, PLAT J and TEMME EHM (2002), Dietary fats and coronary heart disease, in Akoh CC and Min DB, Food Lipids, New York, Marcel Dekker, Inc., 603–636. MIR Z , RUSHFIELD ML , MIR PS, PATERSON LJ and WESELAKE RJ (2001) Effect of dietary supplementation with either conjugated linoleic (CLA) or linoleic acid rich oil on the CLA content of lamb tissues, Small Ruminant Res, 36, 25–31. MYER RO, JOHNSON DD, KNAUFT DA, GORBET DW, BRENDEMUHL JH and WALKER WR (1992) Effect of feeding high-oleic acid peanuts to growing-finishing swine on resulting carcass fatty acid profile and on carcass and meat quality characteristics, J Anim Sci, 70, 3734–3741. NAKATANI N (1997), Antioxidants from spices and herbs, in Shahidi F, Natural Antioxidants, Chemistry, Health Effects and Applications, Champaign, IL, AOCS Press, 64–75. NG TK, HAYES KC, DEWITT GF, JEGATHESAN M, SATGUNASINGAM N, ONG AS and TAN D (1992) Dietary palmitic acid and oleic acids exert similar effects on serum cholesterol and lipoprotein profiles in normocholesterolemic men and women, J Am Coll Nutr, 11, 383–390. O’BRIEN DR (2004), Fats and oils formulation, in O’Brien RD, Fats and Oils: Formulating and Processing for Applications, Boca Raton, FL, CRC Press, 235–289.

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(2003), Fats for healthy living, Food Technol, 57 (7), 91–96. and PETTERSEN J (1990), Fish oils – an old fat source with new possibilities, in World Conf. Proc., Edible Fats and Oils Processing: Basic Principles and Modern Practices, Champaign, Ill, AOCS. th ORTHOEFER FT (1996), Rice bran oil, in Hui YH, Bailey’s Industrial Oil & Fat Products, 5 edn, vol 3, New York, Wiley Interscience, 393–409. PERKINS EG (1996), Volatile odour and flavour components formed in deep frying, Perkins EG and Erickson MD in Deep Frying: Chemistry, Nutrition and Practical Applications, Champaign, Ill, AOCS Press, 43–48. PURDY RH (1985), Oxidative stability of high oleic sunflower and safflower oils, J Am Oil Chem Soc, 62 (3), 523–525. QUINLAN P (1996), Structuring fats for incorporation into infant formulas: fat in the diet, Proc 21st World Congress of the International Society for Fat Research (ISF), October, The Hague, Netherlands, Bridgewater, PJ Barnes & Associates, 21–26. RAES K, HUYGHEBAERT G, DE SMET S, NOLLET L, ARNOUTS S and DEMEYER D (2002), The deposition of conjugated linoleic acid in eggs of laying hens fed diets varying in fat level and fatty acid profile, J. Nutr, 132, 182–189. RAES K, DE SMET S and DEMEYER D (2004), Effect of dietary fatty acids on incorporation of long chain polyunsaturated fatty acids and conjugated linoleic acid in lamb, beef and pork meat: a review, Anim Feed Sci Technol, 113, 199–221. RAJAH K (2004), Innovations in the spreads market, Oils & Fats International, 20 (3), 18– 20. RENTFROW G, MADDOCK KR, STAHL CA, LINVILLE ML, SAUBER TE, ALLEE GL and BERG EP (2003) The influence of diets containing either conventional corn, conventional corn with choice white grease, high oil corn, or high oil high-oleic corn on belly-bacon quality, Meat Sci, 64, 459–466. RHEE KS, ZIPRIN YA and DAVIDSON TL (1990), Characteristics of pork products from swine fed a high monounsaturated diet. II. Uncured processed products, Meat Sci, 27, 343–357. RUDEL LL, PARKS JS, HEDRICK CC, THOMAS M and WILLIFORD K (1998), Lipoprotein and cholesterol metabolism in diet-induced coronary artery atherosclerosis in primates, Progr Lipid Res, 37, 353–370. RUESSING M (2004), Fortifying foods with fatty acids, Oils & Fats International, 20 (6), 18–19. RYMER C and GIVENS DI (2005), n-3 Fatty acid enrichment of edible tissue of poultry: a review, Lipids, 40 (2), 121–130. SAKURAI H and POKORNY J (2003), The development and application of novel vegetable oils tailor-made for specific human dietary needs, Eur J Lipid Sci Technol, 105, 769–78. SCHWARZ K (2003), Phenolic diterpenes from rosemary and sage, in Shi J, Mazza G and Lemaguer M, Functional Foods, 2, Biochemical and Processing Aspects, Boca Raton, FL, CRC Press, Inc., 189–211. SERIBURI V and AKOH C (1998), Enzymatic interesterification of lard and high-oleic sunflower oil with Candida antarctica lipase to produce plastic fats, J Am Oil Chem Soc, 75 (10), 1339–1345. SEWALT V, ROBBINS K and GAMBLE W (2005), Lipid-soluble antioxidant components of Rosmarinus officinalis, Lipid Technol, 17 (5), 106–111. SHON MY, CHOI SD, KAHNG GG, NAM SH and SUNG NJ (2004) Antimutagenic, antioxidant and free radical scavenging activity of ethyl acetate extracts from white, yellow and red onions, Food Chem Toxicol, 42, 659–666. SHUKLA VKS and BHATTACHARYA K (2003a), Nutridan bakes a recipe for health, Oils & Fats International, 19 (3), 26–27. SHUKLA VKS and BHATTACHARYA K (2003b), Extending the shelf life of cosmetic products through novel stabilisation of exotic butters and oils, Seifen, Öle, Fette, Wachse, 129 (9), 38–44. SHUKLA VKS, WANASUNDARA PKJPD and SHAHIDI F (1997), Natural antioxidants from oilseeds, OHR LM

OPSTVEDT J, URDAHL N

566 Modifying lipids for use in food in Shahidi F, Natural Antioxidants, Chemistry, Health Effects and Applications, Champaign, Ill, AOCS Press, 97–132. SILKEBERG A and KOCHHAR SP (2000), Refining of edible oil retaining maximum antioxidative potency, Patent US 6,033,706 March 7, EPA 98102528.1-2109, 13.02.98. SINCLAIR AJ, ATTAR-BASHI NM and LI D (2002) What is the role of α-linolenic acid for mammals?, Lipid, 37 (12), 1113–1123. SMITH RE, FINLEY JW and LEVEILLE GA (1994), Overview of salatrim, a family of low-calorie fats, J Agric Food Chem, 42, 432–434. SMUTS CM, BOROD E, PEEPLES JM and CARLSON SE (2003) High-DHA eggs: feasibility as a means to enhance circulating DHA in mother and infant, Lipids, 38 (4), 407–414. STANGL GI, MÜLLER H and KIRCHGESSNER M (1999), Conjugated linoleic acid effects on circulating hormones, metabolites and lipoproteins and its proportion in fasting serum and erythrocyte membranes of swine, Eur J Nutr, 38 (6), 271–277. STENDER S and DYERBERG J (2004), Influence of trans fatty acids on health, Ann Nutr Metab, 48 (2), 61–66. TEMME EHM, MENSINK RP and HORNSTRA G (1996), Comparison of the effects of diets enriched in lauric, palmitic, or oleic acids on serum lipids and lipoproteins in healthy women and men, Am J Clin Nutr, 63, 897–903. TRAITLER H, WILLIE HJ and STUDER A (1988), Fractionation of blackcurrant seed oil, J Am Oil Chem Soc, 65 (5), 755–760. TUOMASJUKKA S (2004), Physiological effects of blackcurrant seed oil, AgroFOOD Industry Hi-tech, September/October, 42–46. VALSTA LM, JAUHIAINEN M, ARO A, KATAN MB and MUTANEN M (1992), Effects of a monounsaturated rapeseed oil and a polyunsaturated sunflower oil diet on lipoprotein levels in humans, Arterioscler Thromb, 12, 50–57. WAHRBURG U, MARTIN H, SANDKAMP M, SCHULTE H and ASSMANN G (1992), Comparative effects of a recommended lipid-lowering diet vs a diet rich in monounsaturated acids on serum lipid profiles in healthy young adults, Am J Clin Nutr, 56, 678–683. WATKINS C (2003), Time for an oil change?, INFORM, 14 (2), 70. WEGGEMANS RM, RUDRUM M and TRAUTWEIN ELKE A (2004), Intake of ruminant versus industrial trans fatty acids and rise of coronary heart disease, Eur J Lipid Sci Technol, 105 (6), 390–397. WHITE PJ (1992), Fatty acids in oilseeds (vegetable oils), in Chow CKK, Fatty Acids in Foods and Their Health Implications, New York, Marcel Dekker, Inc., 237–262. WISEMAN J and GARNSWORTHY P (2004), Influence of dietary fats and oils on animal product quality, INFORM, 15 (5), 334–335. WRIGHT A (2004), Oil variety is the spice of life, INFORM, 15 (2), 134–136. XU X (2000), Production of specific-structured triacylgylcerols by lipase-catalysed reactions: a review, Eur J Lipid Sci Technol, 102 (4), 287–303. YAMAMOTO S and SMITH WL (2002) Molecular biology of the arachidonate cascade (second edition), Prostaglandins Other Lipid Mediat, 68–69, 1. ZIPRIN YA, RHEE KS and DAVIDSON TL (1990), Characteristics of pork products from swine fed a high monounsaturated diet III. A high fat cured product, Meat Sci, 28, 171–80. ZOCK PL and KATAN MB (1994), Butter, Margarine and Serum Lipoproteins, Atherosclerosis, 131, 7–16. ZOCK PL, VRIES DE JHM, and KATAN MB (1994), Impact of myristic acid versus palmitic acid on serum lipid and lipoprotein levels in healthy women and men, Arterioscler Thromb, 14, 567–575.

23 Applications and safety of microbial oils in food C. Ratledge and S. Hopkins, University of Hull, UK

23.1

Introduction

In Chapter 5, we described the current processes being used for the production of oils rich in polyunsaturated fatty acids (PUFA) using micro-organisms – the so-called single cell oils (SCO). The two fatty acids which are currently produced in this way are arachidonic acid (ARA; 20:4n-6) and docosahexaenoic acid (DHA; 22:6n-3). Neither of these fatty acids is readily available from other sources. Plant oils do not produce PUFAs beyond 18:4 (stearidonic acid) – see Chapter 2 – and, of animals, only fish oils are potentially useful as a source of DHA but not of ARA – see Chapter 4. Thus micro-organisms represent the only route by which both these fatty acids can be produced economically. The main applications of these oils are at present in infant nutrition where they are used in combination: two volumes of ARA-SCO plus one volume of DHA-SCO. Development of the oils, especially the DHA-rich ones, for adult consumption is currently underway. This chapter reviews the various uses, applications and safety aspects of SCOs in current production. This, therefore, does not include the discontinued process of producing an SCO rich in γ-linolenic acid (18:3n-6) which used Mucor circinelloides (see Chapter 5 for brief details and also Ratledge, 2005 for a more detailed account). Current supplies of GLA-rich oils are from two main plant sources: evening primrose (Oenothera biennis) and borage or starflower (Borago officinalis). Blackcurrant oil, which also contains α-linolenic acid (ALA, 18:3n-3), is also sold commercially as a mixed source of ALA and GLA. Readers wishing further information on the clinical applications of these GLA oils should consult the monograph edited by Huang and Ziboh (2001). There are also numerous websites covering various

568 Modifying lipids for use in food aspects of the GLA oils from plants, including sources and health applications, for example, www.pdrhealth.com; www.consumerlab.com; www.berkeley wellness.com and www.1stvitality.co.uk. However, it should be appreciated that many claims may be made for the efficacy of these oils in the treatment of various disorders and complaints that may lack scientific rigour. Caution therefore needs to exercised in accepting at face value any unsupported evidence for applications of these oils. It does though appear that GLA-rich oils are contra-indicated for consumption by pregnant females and nursing mothers.

23.2 Uses and applications of SCOs rich in arachidonic acid and docosahexaenoic acid Linoleic acid (LA, 18:2n-6) and α-linolenic acid (ALA, 18:3n-3) are essential fatty acids for human nutrition as neither can be synthesized by animals from oleic acid. These fatty acids must be acquired in the diet. They are the respective precursors of the n-6 and n-3 series of fatty acids culminating in the synthesis of arachidonic acid (ARA, 20:4n-6) [and possibly docosapentaenoic acid (DPA, 22:5n-6)] and docosahexaenoic acid (DHA, 22:6n-3) – see Fig. 5.4. These are the precursors of various prostaglandins and leucotrienes and other physiologically active lipid derivatives (see Fig. 23.1 and also below). ARA and DHA are found throughout the body but are particularly concentrated in the membrane phospholipids of brain tissue. ARA can account for up to 30 % of all fatty acyl groups attached to phosphatidylinositol in brain lipids with a further 10 % being attached to phosphatidylserine; DHA, on the other hand, is predominant in phosphatidylserine (~ 30 %) with only 2–5 % in phosphatidylinositol (Kyle, 1997a). Both PUFAs also occur at high levels in phospholipids of the retina and thus are involved in visual responses. The high amounts of ARA and DHA in brain phospholipids in numerous mammals were established by Sinclair (1975) and led to suggestions that they must be playing a vital role in the nervous system. Other earlier work (Wheeler et al., 1975) on feeding n-3 PUFAs to rats showed a doubling of the retinal response to visual stimulation compared to feeding n-6 PUFAs. The roles of ARA and DHA are outlined in Fig. 23.1. Sinclair et al. (2005) have summarized the roles of DHA as fulfilling crucial roles in: • • • •

membrane-related events including modulation of membrane receptors such as rhodopsin; regulation of dopaminergic and serotoninergic neurotransmission; signal transduction via various inositol phosphates, diacylglycerols and protein kinase C; alteration of ion flux through voltage-gated K+ and Na+ channels in membranes;

Applications and safety of microbial oils in food

569

Phospholipid membrane EPA

DHA

Phospholipase A2

Phospholipase A2

Phospholipase A2

Free ARA

Free EPA

ARA

COX PG2

TX2

LOX

LOX

5-HPETE

LTs

Free DHA

COX PG3

TX3

Resolvin E

LOX

COX

Docosatriene

Resolvin D

LT4

Fig. 23.1 Formation of physiologically active metabolites from the fatty acid precursors, arachidonic (ARA), eicosapentaenoic (EPA) and docosahexaenoic (DHA) acid. Important enzymes are indicated by ellipses, COX = cycloxygenase, LOX = lipoxygenase, PG = prostaglandin (series in subscript), LT = leukotriene (series in subscript), 5-HPETE = 5-hydroperoxyeicosatetraenoic acid).

• • • • • •

regulation of eicosanoid synthesis from ARA; use in the synthesis of novel anti-inflammatory mediators known as resolvins; regulation of gene expression; regulation of phosphatidylserine levels and protection of neural cells against programmed cell death (apoptosis); stimulation of neurite outgrowth in brain or neuron cells; development of neurons and neuron size by involvement with nerve growth factor.

The functional role of ARA appears to be either by the fatty acid itself being the active material or by acting through one of its key metabolites which include various prostaglandins, prostacyclins, thromboxanes and leucotrienes (Fig. 23.1). For example, transmission of impulses between nerve synapses may be regulated by ARA through its specific activation of glutamate receptors in these synapses. Metabolites of ARA are involved in a variety of physiological responses which include regulation of blood pressure, involvement in renal function, recognition of pain and in the inflammatory response; a discussion of such roles would extend this chapter beyond its remit and readers wishing for further information should consult reviews such as those by Rahman et al. (1997) and Brash (2001). Although in the normal, healthy adult ARA is considered to be adequately synthesized in the body from linoleic acid (LA, 18:2n-6), which is an essential fatty acid being unable to be synthesized in the body from oleic acid, there is strong evidence to suggest that both it and DHA (from linolenic acid) are not synthesized at

570 Modifying lipids for use in food an adequate rates in the newly born infant and, therefore, supplementation of their diet with both these long-chain PUFAs is now recommended.

23.3 Applications of ARA-SCO and DHA-SCO for inclusion in infant formulae As described in Chapter 5, the two principal SCOs in current production are those rich in ARA and DHA. The former is produced using the fungus, Mortierella alpina, and large-scale production (measured as more than 500 tonnes of oil per year) is carried out by DSM Food Specialities BV (Netherlands) at a purpose-built fermentation plant in Italy. The ensuing oil is sold exclusively to Martek Biosciences Corp., Maryland, USA. Related processes are carried out in Japan by Suntory and in China by Wuhan Alking Engineering Co using different strains of the same organism. For the production of oils rich in DHA, at least three different processes are used. The most abundantly produced oil is that from a non-photosynthetic dinoflagellate, Crypthecodinium cohnii. This process is run by Martek BioSciences, and the final oil is blended at a 1:2 ratio with the arachidonic acid oil for incorporation into infant formulae. Currently, this blend of oil, sold under the trade name of Formulaid® (Martek Biosciences Corp, USA), is used in over 60 countries and total sales of the combined oils are in excess of 1000 tonnes per year with a doubling in this quantity predicted over the next 12 months. The profile of the fatty acids of this oil (see Table 5.4) shows no other unsaturated fatty acid besides oleic acid, and the oil is thus unique for its very high content of DHA and the absence of all other PUFA. Other processes for DHA production use organisms known as thraustochytrids, which are non-photosynthetic marine organisms originally considered to be marine fungi. These oils are not, however, used in infant formula preparations. One of these processes is run in the USA by Martek Biosciences through the acquisition of OmegaTech Inc. in 2002. This uses an organism known as Schizochytrium sp. A second process is operated in Germany by Nutrinova and uses a related organism referred to as Ulkenia sp. The DHA-rich oils of both Schizochytrium sp. and Ulkenia sp. also contain docosapentaenoic acid (22:5n-6) which, though unusual, does occur in animals. The oils from both these thraustochytrids are under development for incorporation into various foods and drinks to provide supplementation of DHA in the diet and are discussed in the following section. For reasons that are not yet fully understood, synthesis of both ARA and DHA appears not to be fully developed in the newly born infant and possibly not at all in pre-term babies. Some researchers (see Krawczyk, 2001) believe that infants can synthesize sufficient ARA and DHA themselves provided that the precursors LA and α-linolenic acid (ALA, 18:3n-3) are present in the diet; other researchers consider that, even if these precursors are present in the diet, synthesis of ARA and DHA is insufficient to meet the further

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biosynthetic requirements of neural membranes. Certainly during the first year of life, there is a rapid uptake of DHA and ARA into the infant brain which strongly suggests a key role for these PUFAs in neural development (Sinclair et al., 2005). Whilst mothers’ milk contains small amounts of ARA and DHA, usually between 0.2 and 1 % of the total milk fatty acids, these two fatty acids are not found in cows’ milk or in infant formula unless they have been deliberately added. Thus, the argument has been advanced over the past 12–15 years (see for example Uauy et al., 1990; Hoffman and Uauy, 1992; Carlson, 1995; Huang and Sinclair, 1998) that both fatty acids should be added to all infant formulae. If newly born babies are fed with unsupplemented milk formula preparations then there is a steady decline in blood levels of DHA and ARA together with a loss of DHA, but not of ARA, from brain tissue (Gibson et al., 1994; Farquharson et al., 1995) which reinforces the claims for the beneficial additions of the two PUFAs to formula milk. Kyle (2001) has reported that since about 1986 there had been over 30 well-controlled clinical studies involving over 2000 infants showing that improvement in mental and visual acuities was greater in infants fed either with mothers’ milk or formula preparations with DHA supplementation than in those fed with standard infant formulae. Further studies since this report (see Sinclair et al., 2005) have consolidated this view. There is also very strong evidence that pre-term infants (i.e. those born prematurely) should receive both ARA and DHA supplementation (Crawford et al., 2003) as such infants have minimal fat stores and probably depend absolutely on a supply of both long-chain PUFAs. These authors have commented, ‘Just as preterm infants have been found to require intakes of protein, energy, and minerals that match the placental output, so also it is likely that they will require the placental output of lipids, i.e. ARA and DHA.’ These, in the view of the authors, are being denied to the infant by not being included in their feeding formulae. They are used almost invariably as such low body weight infants are generally unable to feed for themselves from their mothers. Crawford et al. (2003) pointed out that at the time of birth, which now may be anything up to 15 weeks before full-term, the premature infant is using something like 70 % of its energy for brain development and function, and the brain itself is 60 % lipid. ‘It is not surprising,’ these authors concluded, ‘that membrane-rich systems fail under the stress of rapid growth and a dearth of the very substances needed for their growth and integrity.’ There have now been a large number of deliberations at governmental advisory board level on the advantages of including DHA and ARA in the diets of infants being fed exclusively on formula preparations (see Watkins, 2004; and also FSANZ, 2003). The recommendations from advisory boards associated with the governments of the USA, Canada, the UK and other countries of the EU, Australia and New Zealand (see Table 23.1) are supportive of the inclusion of both fatty acids, knowing that the sources of both materials are, for the foreseeable future, from microbial

572 Modifying lipids for use in food Table 23.1 Selected websites covering the acceptance of single cell oils (SCO) in infant formulae around the world. SCO

Year

Acceptance1

References

ARASCO™ Europe US Canada New Zealand and Australia

1994 2001 2002 1998

‘Principle of mutual recognition’ GRAS (FDA) Health Canada FSANZ

[8] [1,2] [3,4] [5] [6,7]

DHASCO™ Europe US Canada New Zealand and Australia

1994 2001 2002 1998

‘Principle of mutual recognition’ GRAS (FDA) Health Canada approval FSANZ

[8] [1,2] [3,4] [5] [6,7]

DHASCO-S Europe US New Zealand and Australia

2003 2004 2002

EC/258/97 GRAS (FDA) FSANZ

[9] [10] [6,11]

DHA45 oil Europe New Zealand and Australia

2003 2005

German CA FSANZ

[12] [6,13]

1

Acceptance does not necessarily mean approval. Refer to the relevant references for specific status information. Abbreviations FDA = US Food and Drug Administration, FSANZ = Food Standards Australia New Zealand, GRAS = Generally Recognized as Safe, CA = Competent Authority. Sources: [1] Advisory Committee on Novel Foods and Processes (ACNFP) Annual Report 1996. Available from http://www.food.gov.uk/science/ouradvisors/novelfood/acnfpannreps/acnfp_report_1996 [2] Netherlands State Journal, Number 48 of 8.3.1995 [3] US Food and Drug Administration, Agency Response Letter, GRAS Notice No. GRN000041. Available from http://www.cfsan.fda.gov/~rdb/opa-g041.html [4] US Food and Drug Administration, Agency Response Letter, GRAS Notice No. GRN000080 Available from http://www.cfsan.fda.gov/~rdb/opa-g080.html [5] http://www.hc-sc.gc.ca/food-aliment/mh-dm/ofb-bba/nfi-ani/e_dhasco.html [6] http://www.foodstandards.gov.au/_srcfiles/fsc_1_3_4_Identity_&_Purity_v78.pdf [7] http://www.foodstandards.gov.au/_srcfiles/DHASCO%20and%20ARASCO% 20in%20infant%20formula.pdf [8] http://www.martekbio.com/Nutritional_Products/Milestones.asp [9] http://europa.eu.int/cgi-bin/eur-lex/udl.pl?REQUEST=Seek-Deliver&SERVICE=eurlex& COLLECTION=oj&LANGUAGE=en&DOCID=2003l144p00130014 [10] http://www.cfsan.fda.gov/~rdb/opa-g137.html [11] http://www.foodstandards.gov.au/_srcfiles/A428_FAR.pdf [12] http://europa.eu.int:80/comm/food/food/biotechnology/novelfood/notif_list_en.pdf [13] http://www.foodstandards.gov.au/_srcfiles/A522_DHA-rich_oil_FAR.pdf

sources. As already stated in Chapter 5, the microbial method is the only economic way in which these SCO could be produced.

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The reason why ARA is included in infant formulae along with DHA is that when DHA is administered on its own there can be a lowering of the ARA content in the blood and this, in turn, may be associated with reduced growth, although there is not overwhelming evidence to support this (R. Gibson, quoted by Krawczyk, 2001). As ARA is found in mothers’ milk, at between 0.3 and 0.55 % of the total fatty acids, it has been considered that adding ARA into infant formula will, at worse, do no harm and, at best, will help to maintain DHA at an appropriate level. It is not, however, known why ARA should decrease in infants if DHA alone is added to their diet, and one of the key nutritionalists in this field, Robert Gibson, has suggested that this is one of the key outstanding questions of infant nutrition with the exact role that ARA plays in infants still completely unknown (Krawczyk, 2001). There also appears to be a benefit of including DHA and ARA together in the diet of pregnant women, particularly in the second trimester (Otto et al., 2000), as such supplementation significantly increases the DHA level in the fatty acids of red blood cells and in the plasma whilst maintaining ARA at its original level. The argument would then be advanced that such an improvement in DHA levels in the mother would then ensure a more than sufficient supply of DHA and ARA to the foetus. A more recent survey (Forsyth et al., 2003), which followed up the development of children fed with an ARA/DHA-supplemented infant formula at birth into later childhood, found that such children had a lower blood pressure than children fed with an unsupplemented formula. Breast-fed infants were similar to those on the supplement feed. It was concluded that as the blood pressure of an individual tends to track from childhood into adult life, an early exposure to dietary long chain PUFAs, e.g. ARA and DHA, may then reduce cardiovascular risk in adulthood. As already stated above, the evidence for the benefits of including both ARA and DHA in infant formulae has been assessed by a number of national regulatory bodies (see Table 23.1). Of key significance was probably the acceptance by the US Food and Drug Administration (FDA) in 2001 of the claim made to them by Martek Biosciences Corp. (who were then the sole manufacturers of SCOs rich in ARA and DHA) for the inclusion of the company’s blend of ARASCO® and DHASCO® into infant formulae. This evaluation of the proposal took three years and was based on using the oils derived, respectively, from Mort. alpina and C. cohnii. Oils from other organisms were not considered. Thus an a priori case has been made, and approved by all other regulatory authorities who have been requested to evaluate these claims for infant feeding, for the use of the two oils being given in the ratio of 2:1 (v/v) where the content of both ARA and DHA is at 40 % of the total fatty acids in each oil. The combined oils are sold by Martek under the trade name of Formulaid® now available in over 60 countries. There is, therefore, a considerable body of evidence that suggests both immediate and long-term benefits of including ARA and DHA in the diets of newly born children. It may be anticipated that as further studies are carried

574 Modifying lipids for use in food out they will reinforce this view making the production of these SCOs of key significance for the nutritional well being of children everywhere.

23.4 Applications of DHA-rich oils for supplementation of adult diets Since the early pioneering work of Sinclair reporting on the low incidence of coronary diseases amongst Norwegians, Icelanders and Greenlanders and the almost entire absence of such diseases in Inuit (Sinclair, 1953; see also Ewin, 2001), there have been consistent and increasing claims for the inclusion of fish oils in the diet. This is because all the above nationalities are characterized by having a very high proportion of such oils in their diets. DHA and EPA (eicosapentaenoic acid, 20:5n –3) are, of course, the two major n-3 PUFAs in many fish oils, and the composition and uses of such oils are delineated in other chapters of this book (see Chapters 4 and 24) and readers should refer to these for further information. Briefly, however, it can be stated that since this early work of Sinclair’s, there has been a steady stream of reports concerning the improvement in heart condition of adults who have taken long-chain PUFAs, mainly in the form of various fish oil preparations. These surveys, which have been of increasing frequency over the past 7–10 years, have culminated in a detailed report issued (February 2005) by the Joint Health Claims Initiative (JHCI) of the UK (http://www.jhci.org.uk/approv/omega.htm) which has concluded that, on the basis of an examination of all the available evidence, eating 3 g weekly, or taking 0.45 g daily, long-chain n-3 PUFAs will help to maintain a healthy heart. The key surveys included those of Bucher et al. (2002) and Marchioli et al. (2001, 2002) as well as other information provided by N. Baldwin & R. Rice in their submission to the JHCI [JHCI Omega-3 dossier (pdf) May 2004 – see above web site]. There is also evidence that intake of n-3 long-chain PUFAs may help to protect the eye from retinal damage incurred either in old age or by damage from oxygen, light or an inflammatory cause (Sangiovanni and Chew, 2005). This would be in keeping with the known high levels of DHA in the retinal membranes and is an additional reason for the initial advocacy of including DHA-rich oils in the diets of newly born infants. For adults, DHA is also available as an over-the-counter dietary supplement. It is produced by Martek Biosciences and is sold under the trade name of Neuromins®. It is the same oil as is used in infant formulae being produced by C. cohnii. The specifications of the oil are, therefore, exactly the same as the infant formula oil and are given in Chapter 5. Information concerning this product is available on-line at http://www.martekbio.com/ Nutritional_Products/Introduction.asp. The oil is widely available in the USA, although outlets for it in other countries appear to be limited.

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575

In addition to the sales of DHA oil from C. cohnii for adult consumption, Martek also produce a DHA-rich oil using Schizochytrium sp. (see Section 23.3). This oil was originally sold under the trade name of DHA Gold and, more recently, as DHASCO-S®. It was used initially mainly as a feed material for poultry and other animals (see Section 23.5). Since the acquisition of OmegaTech by Martek in 2002, this oil has now received regulatory approval (see Table 23.1) for consumption by humans. It is now being incorporated into various food preparations and drinks being sold under a new name of Martek DHA®. A similar oil is also produced by Nutrinova GmbH, Frankfurt, Germany, using a micro-organism known as Ulkenia sp. (see Chapter 5). This oil was originally sold under the trade name of DHActive, although the current name appears to be Nutrinova®DHA (http://www.NutrinovaDHA.com). The oil is principally intended for adult consumption to provide DHA as a nutritional supplement. Like the Martek DHA oil, the Ulkenia oil is also being incorporated into various foods such as margarines and creamed cheeses. However, Kiy et al. (2005) have commented that the oil also works well in a variety of other applications including bread, biscuits, health bars and yoghurts. It can also clearly be incorporated into drinks, such as soy milk substitutes and even milk itself. These preparations of DHA-rich oils, therefore, provide new possibilities of the adult population achieving the desired intake of long-chain PUFAs for the maintenance of a healthy heart. It is perhaps not surprising that there is considerable interest by the food industry in the availability of these DHA-rich oils. Production using Schizochytrium sp. and Ulkenia sp., and indeed other related thraustochytrid organisms (see Chapter 5), has the advantage over the process using C. cohnii in that much higher productivities (grams DHA produced per hour per litre of fermentor volume) can be achieved with the consequent much cheaper price of the oil. This makes their widespread use very attractive and, because of their lack of fishy odours and taste, considerably superior to fish oils. Neither of these oils, however, is currently used in infant formulae, principally because the profiles of the fatty acids in the oils from Schizochytrium sp. and Ulkenia sp. are not the same as that of C. cohnii (see Table 5.3). Both the former oils contain docosapentaenoic acid (DPA, 22:5n-6) at about 40 % of the DHA content. Although somewhat unusual, this fatty acid also occurs in the lipids of humans and other animals, albeit at low concentrations (see, for example, Burdge et al., 2002). When a DHA/DPA oil (produced by an organism related to the Schizochytrium sp. of Martek but prepared by Suntory Ltd., in Japan) was administered to rats, it was found (Tam et al., 2000) that the DPA was converted into ARA (which of course is another n-6 PUFA but two carbon atoms shorter than DPA). This then served to increase the level of ARA which otherwise would have been decreased by administration of DHA alone. Thus, whilst giving an oil containing n-6-DPA as the sole PUFA would not be recommendable, as it would not provide any DHA, it is nevertheless an advantage to include it along with DHA. The retroconversion of DPA into ARA then serves the same metabolic purpose as administering

576 Modifying lipids for use in food a mixture of DHA and ARA oils, as is done with the mixed SCO preparation for infant formulae (see above). Whilst reviews of the health benefits of DHA (see for example Haumann, 1998; Horrocks and Yeo, 1999; Nordoy, 1999; Connor, 2000; Schmidt et al., 2001; Kris-Etherton et al., 2002; Leaf et al., 2003; Ruxton et al., 2004) tend to equate an adequate consumption of this long-chain PUFA with diets either rich in oily fish or, alternatively, supplemented with fish oils, there are, of course, two major PUFAs in fish oils: DHA and EPA. The ratio of these two fatty acids in fish oils (see Chapter 4) can vary widely. They do, however, fulfil different metabolic roles (see Fig. 23.1). Although the case for inclusion of fish oils in the diet is generally perceived to be mainly due to their contents of DHA rather than EPA, EPA is not without its benefits either. EPA acts mainly as an anti-inflammatory fatty acid (see Fig. 23.1). Recommendations for its specific administration (rather than consumption along with DHA in the form of fish oil preparations) are mainly associated with treatments of various neuropsychiatric disorders, including schizophrenia, depression and Huntington’s disease (see, for example, Babcock et al., 2000; Horrobin, 2002; Keller, 2002; Puri, 2004). There are also indications that it may be useful in the treatment of certain cancers (see, for example, Palakurthi et al., 2000; Siddiqui et al., 2004). However, as yet there is no microbial source of EPA commercially available even although searches for such a source have been extensive (e.g. Wen and Chen, 2005). Thus a detailed discussion of the possible nutritional benefits of this fatty acid is outside the scope of this review, but interested readers should consult the references provided for additional citations of relevant research papers and reviews.

23.5 Other applications of DHA-rich SCOs and biomass The production of a DHA-rich oil using the marine organism, Schizochytrium sp., by OmegaTech Inc. in 1994/5 led to an immediate problem of identifying possible nutritional applications of the oil. Whilst the infant formula market for a DHA-rich oil was being developed by Martek with its oil from C. cohnii, it was not apparent that the Schizochytrium oil could be used similarly. The principal problem was the occurrence of DPA within the oil (see above and also Chapter 5); this fatty acid had no prior history of being fed to babies, although it does occur at very low concentrations (~ 0.03 % of the total fatty acids) in mothers’ milk (Gibson et al., 1998). Consequently other outlets for the oil had to be identified and this meant looking outside human foods until the nutritional position of the DPA had been evaluated. Feeding trials were carried out using spray-dried biomass of Schizochytrium (Abril and Barclay, 1998; Barclay et al., 1998), known by its trade name of DHAGold® (Martek Biosciences Corp, USA). Evidently, there was no need to extract the oil from the cells. All that had to be done was to ensure that the spray-dried biomass was digestible and that the fatty acids within the microbial

Applications and safety of microbial oils in food

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cells would become available to the animal. This strategy had a firm scientific basis in that algae and algae-like micro-organisms have long been used as feed ingredients, but cost of production and the indigestibility of some species had always limited their applications. Of particular interest from this work was the finding that the feeding of the dry biomass to poultry led to the preferential incorporation of DHA, but not DPA, into the phospholipids of eggs (Abril and Barclay, 1998; Abril et al., 2000). The ratio of DPA:DHA went from 1:3 in the starting material to 1:20 in the hens’ eggs. There was also incorporation of DHA into the meat of cows and pigs (Marriott et al., 2002a,b) as well as into rotifers and shrimps (Barclay and Zeller, 1996) but not much into cows’ milk (Barclay et al., 1998). Although it is also possible to increase the DHA content of eggs by feeding poultry with fish oils (see Farrell, 1998), the use of the Schizochytrium biomass appears to be an attractive alternative, especially as no off-flavours are produced in the final egg and the DHA does not need to be encapsulated as needs to be done with fish oil. The Schizochytrium cell provides a stable environment for the DHA which remains unoxidized for as long as the microbial cell remains intact. The advantages of being able to market omega3 enriched eggs is clear. The consumption of such eggs leads to the incorporation of DHA into the body and other studies have shown positive benefits to expectant mothers who have consumed such DHA-enriched eggs with an increased birth weight of the baby and a greater proportion going to full term before delivery (Borod et al., 1999; Smuts et al., 2003). The eggs, it has been reported, were well-tolerated by the expectant mothers consuming 18 such eggs per week (Smuts et al., 2003). This work has now led to the take-up of the product by various egg producers. Such eggs are now being sold in the USA by Gold Circle Farms (http://www.goldcirclefarms.com) as Gold Circle Farm™ eggs and in the UK by Marks & Spencer™ eggs (http:// www2.marksandspencer.com). Doubtless other companies will follow suit in the near future which will then have a possible major influence on the way in which n-3 PUFAs may be presented in the diet. With the relative cheapness of the Schizochytrium sp. biomass and other similar micro-organisms, due to the high productivities of the fermentation process (see Chapter 5), the way has now opened up for these materials to be considered for fish feeding (Barclay et al., 2005). Fish farming is now big business in many countries of the world; however, the feed material that is used almost invariably contains fish meal as an essential source of PUFAs which are needed for larval development and growth of the small fish fry. It is claimed that some five tonnes of fish meal are need to rear one tonne of fish. Clearly this is an unsustainable position and active research programmes are now underway, including large EU-sponsored ones, which seek to develop non-fish containing feed material. Of major significance in this capacity is the use of DHA-rich biomasses of Schizochytrium and other related thraustochytrids. Earlier work by from the OmegaTech company (Barclay

578 Modifying lipids for use in food and Zeller, 1996; Barclay et al., 1998) showed that this biomass was effective as a nutritional ingredient in feed going to larval fish, rotifers (which are then fed to fish) and to shrimp (see Barclay et al., 2005). Other work (for example: Reitan et al., 1997; Lewis et al., 1999; Brown, 2002; Harel et al., 2002) has confirmed the value of both DHA-rich oils as well as the whole biomass as sources of DHA and other PUFAs for development and growth of fish in aquaculture systems. However, the success of the algal biomass as a replacement for fish meal is clearly dependent on the species of fish which is being used: halibut seem particularly responsive to Schizochytrium biomass, although other fish species (sea bream, sea bass and striped bass) appear to respond equally to the biomass from Schizochytrium, C. cohnii and also Mort. alpina (containing ARA but not DHA) (Harel et al., 2002). Hatching of larvae from striped bass appeared to be especially responsive to the Mort. alpina biomass. The development of fish feeding materials using the biomass material of SCO-producing micro-organisms would, therefore, seem to be an attractive proposition, although it is too early to conclude what will be the optimum way in which such biomass material can be used. Possibly it could be used as an entire replacement of fish meal or as a partial replacement, depending on the fish species being cultivated (Harel et al., 2002). Cost will be of key importance: at the present time the biomass cost of a fast-growing microorganism such as Schizochytrium sp. appears to be at least double that of fish meal. Therefore, there is currently no financial incentive for aquaculturists to switch feed material even though it is self-evident that harvesting one fish to feed to another fish is unsustainable. But, until the time comes when this unsustainability results in the price of fish meal becoming greater than microbial biomass, the present situation of using fish meal rather than microbial biomass will continue. When the price of fish meal exceeds that of SCO-biomass, and it seems to be a matter of ‘when’ rather than ‘if’ that happens, then microbial biomass could be produced at the level necessary to replace fish meal in all fish feeds throughout the whole of the fish feeding business, even though this would mean perhaps producing up to 100 000 tonnes per annum and possibly more if the markets in South-east Asia, China and Japan were to be included. Given that it is now possible to cultivate Schizochytrium sp. in fermentors with biomass yields in excess of 200 g dry cells/L in three days (see Barclay et al., 2005), then a simple calculation indicates that a 500 m3 fermentor would produce in excess of 10 000 tonnes of biomass per year. Only ten such fermentors would be needed to feed all the world’s farmed fish.

23.6 Safety assessments of SCOs Single cell oils represent novel sources of fatty acids. It has, therefore, been imperative to establish their safety and to ensure that they present no immediate

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or long-term hazard to health. This, of course, is particularly important where SCO is being fed to newly born infants and to premature babies (see Section 23.3). The first commercially produced SCO was that from M. circinelloides (see Chapter 5) which was an oil rich in GLA. Work carried out with this oil to establish its safety turned out to be of crucial importance in establishing the safety of other SCOs that were later produced. Toxicological trials of the GLA-SCO were carried out initially using brine shrimps and then subsequent feeding to various rodents and other animals (see Ratledge, 2005 for further information). These trials began with the first experimental batches of oil and started before full-scale production commenced in 1985. The data obtained were submitted to the UK Advisory Committee on Novel Foods and Processes for evaluation and approval. The final assessment of the oil was, however, still continuing when production of the oil ceased in 1990. Thus a lengthy and somewhat reiterative process had been used to evaluate this first SCO. Nevertheless, lack of formal approval did not prevent the oil being sold throughout the UK for human consumption as an alternative to evening primrose oil which, at that time, was the only other source of an oil rich in GLA. One of the principal reasons in establishing the intrinsic safety of the oil was that the producing organism, M. circinelloides, was itself a Generally Recognized As Safe (GRAS) organism. The fungus was a component of the oriental foods of tempeh and tapé which had been consumed for millennia by the indigenous populations of many countries in this region. It was, therefore, argued and accepted that as the organism itself was safe to eat then consumption of a component of the fungus, namely the oil, would also be safe. In the safety evaluation of a microbial oil, it is important to establish that the production organism has no known history of producing any toxin or biologically active molecule. This can usually be done by appraisal of the existing microbiological literature. If there is no history of toxin production, appropriate conclusions can be drawn that it is then highly likely that the organism being used for SCO production is similarly free from such materials. Additionally, any allergic properties of the organism need to be known: allergens may not be present in the final oil but, if they are present in the microbial cells themselves, then this would be a hazard during the fermentation production process where site workers may be exposed to the whole organism, particularly during the final stages of harvesting and processing the biomass. An additional factor in helping to establish the safety of microbial oils, which applies to all SCOs, is that the oil itself, after extraction, refining and deodorization (see Chapter 5), is capable of being analyzed down to the last 0.1 % of material, and possibly beyond this limit. Thus comparison of the fatty acids within a SCO with existing known fatty acids being consumed via plant or animal fats, or known to occur in human tissues, can be taken as a primary measure of safety. Other lipids, such as sterols or carotenoids, that

580 Modifying lipids for use in food might be present in the oil can also be identified and compared to what may be present in other commercial oils. The fact that most plant oils have never been toxicologically tested before being used for human consumption does not indicate any intrinsic hazard with them; but their acceptance by regulatory authorities is then based on decades if not centuries of safe consumption. Materials are regarded as safe to eat if they have been eaten over many years without causing problems in the vast majority of the population. However, no such long-term prior use of SCO exists, but the safety of the oils can be established by comparing them with existing oils: if these latter oils are safe to eat, and if the SCO contains the same fatty acids and other lipid components as might be found in oils already established to be safe, then the SCO may be deemed to be safe itself. Thus, as the number of different SCOs being produced around the world continues to increase, it becomes possible to compare any novel SCO with existing SCOs, as well as with plant and animal oils and fats. Each new acceptance of a SCO means that it is then slightly easier for the next oil to gain approval. Zeller (2005) has provided a definitive account of the manner in which SCOs have been evaluated for safety and also a detailed guide to the regulations and laws enacted by various countries (see Table 23.2) that govern the acceptance of SCOs. This article should be consulted for detailed information on the topic. It does, however, need to be emphasized that SCOs are probably the most thoroughly evaluated oils of any type. With regard to SCOs themselves, the safety of ARA-SCO from M. alpina was subjected to the closest of scrutinies as this oil was, from the very outset, destined for incorporation into infant formulae (Kyle, 1997a,b). The possible presence of mycotoxins in the fungus was monitored very carefully, but no record of such materials has ever been recorded in this fungus and nor could any toxicity of the biomass be established (Streekstra, 1997). Numerous safety assessments of the oil were carried out by independent groups and Streekstra (1997) listed over 10 such trials; subsequent trials have been listed by Zeller (2005). All trials of the ARA-SCO concluded that the oil was entirely safe when administered to a variety of animals including humans. Hempenius et al. (1997) similarly concluded that this oil was entirely safe with no obvious signs of toxicity arising from the oil even when administered to animals at the equivalent of 1 g ARA/kg body wt/day: this would amount to a human consumption of over 200 ml of oil per person per day. Subsequent trials and testing of this oil by long-term feeding tests have confirmed its complete safety (Burns et al., 1999; Arterburn et al., 2000; Hempenius et al., 2000; Huang et al., 2002). These trials and the establishment of the complete safety of ARA-SCO were particularly important as there had been an early suggestion (Seyberth et al., 1975) that a high level of ARA in the diet was undesirable due to its potentially adverse effects on blood clotting. However, no evidence to support these earlier concerns has even been found. With respect to the DHA-SCO from C. cohnii, similar and equally extensive

Table 23.2

Selected regulations of various countries governing use of single cell oils in human nutrition.

Region

Regulations

Coverage

Europe

Article 28 (ex Article 30) of the EC Treaty. See http://europa.eu.int:80/eur-lex/pri/en/oj/dat/2002/c_325/c_32520021224en00010184.pdf ‘Cassis de Dijon’ judgment: case 120/78 (Rewe -Zentrale v Bundesmonopolvwerwaltung für Branntwein) of 20 February 1979, European Court Reports (1979) p649. See http://www.ena.lu?lang=2&doc=6417 Regulation (EC) No. 258/97 of the European Parliament and of the Council (OJ L 43/1, 2.14.1997) Commission Regulation EC No. 1852/2001 (OJ L 253/17, 9.21.2001)

Principle of mutual recognition

Commission Recommendation No. 97/618/EC (OJ L 253/1, 9.16.1997)

US

Canada

New Zealand and Australia

Regulation (EC) No. 178/2002 of the European Parliament and of the Council (OJ L 31/1, 2.1.2002) FDCA (21 USC §301) See http://uscode.house.gov/download/pls/21C9.txt Substances Generally Recognized as Safe (GRAS) (21 CFR §170, §184, §186 and §570) Requirement for premarket notification (62 Fed. Reg. 49886-49892; 21 CFR §190.6) Food and Drugs Act Food and Drug Regulations (Division 28) See http://www.hc-sc.gc.ca/food-aliment/friia-raaii/food_drugs-aliments_drogues/actloi/e_index.html Australia New Zealand Food Standards Code • Standard 1.3.4 (Identity and Purity) • Standard 1.5.1 (Novel Food Standard) • Standard 2.9.1 (Infant Formula Products) See http://www.foodstandards.gov.au/foodstandardscode/

Novel foods and novel food ingredients Confidential information for novel foods submission Scientific aspects for novel foods General principles and requirements of food law Food additives GRAS Dietary supplements Food additives and novel foods Food standards guidelines

Abbreviations: CFR = Code of Federal Regulations, FDCA = Food, Drug and Cosmetic Act, Fed. Reg. = Federal Regulation, OJ = Official Journal of the European Communities, USC = United States Code.

582 Modifying lipids for use in food trials have been conducted. The oil has not only been tested on its own but also along with ARA-SCO® which is the combination used for incorporation into infant formula. Zeller (2005) has documented some 36 trials of these two oils administered both singly and together to humans and other animals. Studies with human volunteers ingesting up to 200 ml of oil per day found that the only problem was slight diarrhoea and occasional ‘fishy burps’ (Wynn and Ratledge, 2005). Similar extensive safety evaluations with DHASCO-S®, i.e. the oil derived from Schizochytrium sp., have been carried out (see, for example, Hammond et al., 2001a,b,c, 2002; Abril et al., 2003). A review of the safety of the oil produced by Ulkenia sp., referred to as DHA45-oil, has also been presented (Kroes et al., 2003). These studies have been particularly valuable in establishing the complete safety of these oils as both contain docosapentaenoic acid (DPA, 22:5n-6) as an unusual PUFA. However, although there were some initial concerns about the level of DPA in these oils, it has been found that this fatty acid can in fact be beneficial in providing additional ARA in the body by its retroconversion in situ (Tam et al., 2000). After all the trials and testing have been carried out on a novel SCO, the entire data is then submitted to one or more regulatory authorities (see Table 23.1) for approval for use in the manner being advocated by the production company. Thus, the final decision to offer a SCO for sale in a particular country or region is taken by independent bodies. Such permission for use, however, does not absolve the manufacturer from any subsequent claim for damages from a customer who feels that some detrimental effect has arisen by using the SCO. Finally, it is worth pointing out that the safety of the single cell oils as described here has been established on an absolute basis of examining each oil as if it posed some potential hazard. No such trials have been conducted, however, on the safety of ‘traditional’ sources of long-chain PUFAs, such as fish oils. The safety of fish oils, which are regarded as major sources of EPA and DHA, needs careful assessment (see Chapter 24) as many of these oils may be contaminated with various environmental pollutants including various dioxins and heavy metals. Further, fish oils can be very rich in their contents of the fat-soluble vitamins A and D, both of which can be damaging to health if taken in large doses over a long period of time. No such hazards are associated with SCOs. Single-cell oils remain the most intensively examined of all oils; they have an exemplary safety record and have now been consumed by infants and adults in over 60 countries for over ten years without any untoward problem being identified.

23.7

References

and BARCLAY W (1998), Production of docosahexaenoic acid-enriched poultry eggs and meat using an algae-based feed ingredient, World Rev Nutr Diet, 83, 77–88.

ABRIL R

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and ABRIL P G (2000), Safe use of microalgae (DHA goldtm) in laying hen feed for the production of DHA. Enriched eggs, in Sim J S, Nakai S and Guenter W, Egg Nutrition and Biotechnology, Wallingford, CAB International, 197–202. ABRIL R, GARRETT J, ZELLER S G, SANDER W J and MAST R W (2003), Safety assessment of DHArich microalgae from Schizochytrium sp. Part v: target animal safety/toxicity study in growing swine, Regul Toxicol Pharmacol, 37(1), 73–82. ARTERBURN L M, BOSWELL K D, LAWLOR T, CIFONE M A, MURLI H and KYLE D J (2000), In vitro genotoxicity testing of ARASCO and DHASCO oils, Food Chem Toxicol, 38(11), 971–976. BABCOCK T, HELTON W S and ESPAT N J (2000), Eicosapentaenoic acid (EPA): an antiinflammatory omega-3 fat with potential clinical applications, Nutrition, 16(11–12), 1116–1118. BARCLAY W and ZELLER S (1996), Nutritional enhancement of n-3 and n-6 fatty acids in rotifers and artemia nauplii by feeding spray-dried Schizochytrium sp, J World Aquacult Soc, 27(3), 314–322. BARCLAY W, ABRIL R, ABRIL P, WEAVER C and ASHFORD A (1998), Production of docosahexaenoic acid from microalgae and its benefits for use in animal feeds, World Rev Nutr Diet, 83, 61–76. BARCLAY W R, WEAVER C and METZ J (2005), Development of a docosahexaenoic acid production technology using Schizochytrium: a historical perspective, in Cohen Z and Ratledge C, Single Cell Oils, Champaign, Ill, AOCS Press, 36–52. BOROD E, ATKINSON R, BARCLAY W R and CARLSON S E (1999), Effects of third trimester consumption of eggs high in docosahexaenoic acid on docosahexaenoic acid status and pregnancy, Lipids, 34 (Suppl), S231. BRASH A R (2001), Arachidonic acid as a bioactive molecule, J Clin Invest, 107(11), 1339– 1345. BROWN M R (2002), Nutritional value of microalgae for aquaculture, in Cruz-Suárez L E, Ricque-Marie D, Tapia-Salazar M, Gaxiola-Cortés M G and Simoes N, Avances en Nutrición Acuícola VI. Memorias del VI, Simposium Internacional de Nutrición Acuícola, 3 al 6 de Septiembre del 2002. Cancún, Quintana Roo, México. BUCHER H C, HENGSTLER P, SCHINDLER C and MEIER G (2002), N-3 polyunsaturated fatty acids in coronary heart disease: A meta-analysis of randomized controlled trials, Am J Med, 112, 298–304. BURDGE G C, JONES A E and WOOTTON S A (2002), Eicosapentaenoic and docosapentaenoic acids are the principal products of alpha-linolenic acid metabolism in young men, Br J Nutr, 88(4), 355–363. BURNS R A, WIBERT G J , DIERSEN-SCHADE D A and KELLY C M (1999), Evaluation of single-cell sources of docosahexaenoic acid and arachidonic acid: 3-month rat oral safety study with an in utero phase, Food Chem Toxicol, 37(1), 23–36. CARLSON S E (1995), The role of PUFA in infant nutrition, INFORM, 6(8), 940–946. CONNOR W E (2000), Importance of n-3 fatty acids in health and disease, Am J Clin Nutr, 71(1 Suppl), 171S–175S. CRAWFORD M A, GOLFETTO I, GHEBREMESKEL K, MIN Y, MOODLEY T, POSTON L, PHYLACTOS A, CUNNANE S and SCHMIDT W (2003), The potential role for arachidonic and docosahexaenoic acids in protection against some central nervous system injuries in preterm infants, Lipids, 38(4), 303–315. EWIN J (2001), Fine wines & fish oil: The life of Hugh MacDonald Sinclair, Oxford, New York, Oxford University Press. FARQUHARSON J, JAMIESON E C, ABBASI K A, PATRICK W J, LOGAN R W and COCKBURN F (1995), Effect of diet on the fatty acid composition of the major phospholipids of infant cerebral cortex, Arch Dis Child, 72(3), 198–203. FARRELL D J (1998), Enrichment of hen eggs with n-3 long-chain fatty acids and evaluation of enriched eggs in humans, Am J Clin Nutr, 68(3), 538–544. FORSYTH J S, WILLATTS P, AGOSTONI C, BISSENDEN J, CASAER P and BOEHM G (2003), Long chain polyunsaturated fatty acid supplementation in infant formula and blood pressure in later childhood: follow up of a randomised controlled trial, BMJ, 326(7396), 953. ABRIL J R, BARCLAY W R

584 Modifying lipids for use in food (2003), DHASCO and ARASCO oils as sources of long-chain polyunsaturated fatty acids in infant formula: A safety assessment, Technical report series No. 22, Food Standards Australia New Zealand, available at; http://www.foodstandards. gov.au/_srcfiles DHASCO%20and%20ARASCO%20in%20infant%20formula.pdf. GIBSON R A, MAKRIDES M, NEUMANN M A, SIMMER K, MANTZIORIS E and JAMES M J (1994), Ratios of linoleic acid to alpha-linolenic acid in formulas for term infants, J Pediatr, 125(5 Pt 2), S48–55. GIBSON R A, NEUMANN M A and MAKRIDES M (1998), The effects of diets rich in docosahexaenoic acid and/or γ-linolenic acid on plasma fatty acid profiles in term infants, in Huang YS and Sinclair A, Lipids in Infant Nutrition, Champaign, Ill, AOCS Press, 19–28. HAMMOND B G, MAYHEW D A, HOLSON J F, NEMEC M D, MAST R W and SANDER W J (2001a), Safety assessment of DHA-rich microalgae from Schizochytrium sp, Regul Toxicol Pharmacol, 33(2), 205–217. HAMMOND B G, MAYHEW D A, NAYLOR M W, RUECKER F A, MAST R W and SANDER W J (2001b), Safety assessment of DHA-rich microalgae from Schizochytrium sp, Regul Toxicol Pharmacol, 33(2), 192–204. HAMMOND B G, MAYHEW D A, ROBINSON K, MAST R W and SANDER W J (2001c), Safety assessment of DHA-rich microalgae from Schizochytrium sp, Regul Toxicol Pharmacol, 33(3), 356–362. HAMMOND B G, MAYHEW D A, KIER L D, MAST R W and SANDER W J (2002), Safety assessment of DHA-rich microalgae from Schizochytrium sp, Regul Toxicol Pharmacol, 35(2 Pt 1), 255–265. HAREL M, KOVEN W, LEIN I, BAR Y, BEHRENS P, STUBBLEFIELD J, ZOHAR Y and PLACE A R (2002), Advanced DHA, EPA and ARA enrichment materials for marine aquaculture using single cell heterotrophs, Aquaculture, 213(1–4), 347–362. HAUMANN B (1998), Alternative sources for n-3 fatty acids, INFORM, 9(12), 1108–1119. HEMPENIUS R A, VAN DELFT J M, PRINSEN M and LINA B A (1997), Preliminary safety assessment of an arachidonic acid-enriched oil derived from Mortierella alpina: summary of toxicological data, Food Chem Toxicol, 35(6), 573–581. HEMPENIUS R A, LINA B A and HAGGITT R C (2000), Evaluation of a subchronic (13-week) oral toxicity study, preceded by an in utero exposure phase, with arachidonic acid oil derived from Mortierella alpina in rats, Food Chem Toxicol, 38(2–3), 127–139. HOFFMAN D and UAUY R (1992), Essentiality of dietary omega 3 fatty acids for premature infants: plasma and red blood cell fatty acid composition, Lipids, 27, 886–895. HORROBIN D F (2002), Cardiovascular disease, affective disorders and impaired fatty acid and phospholipid metabolism, in Chiu E A, Ames O and Katona, C, Vascular Disease and Affective Disorders, London, Martin Dunitz, 75–94. HORROCKS L A and YEO Y K (1999), Health benefits of docosahexaenoic acid (DHA), Pharmacol Res, 40(3), 211–225. HUANG M C, CHAO A, KIRWAN R, TSCHANZ C, PERALTA J M, DIERSEN-SCHADE D A, CHA S and BRENNA J T (2002), Negligible changes in piglet serum clinical indicators or organ weights due to dietary single-cell long-chain polyunsaturated oils, Food Chem Toxicol, 40(4), 453–460. HUANG Y-S and SINCLAIR A (1998), Lipids in Infant Nutrition, Champaign, Ill, AOCS Press. HUANG Y-S and ZIBOH V A (2001), γ-Linolenic Acid: Recent Advances in Biotechnology and Clinical Applications, Champaign, Ill, AOCS Press. KELLER J R (2002), Omega-3 fatty acids may be effective in the treatment of depression, Topics Clin Nutr, 17(5), 21–27. KIY T, RUSING M and FABRITIUS D (2005), Production of docosahexaenoic acid by the marine microalga, Ulkenia sp., in Cohen Z and Ratledge C, Single Cell Oils, Champaign, Ill, AOCS Press, 99–106. KRAWCZYK T (2001), Do infants need extra DHA, AA? INFORM, 12(11), 1064–1074. KRIS-ETHERTON P M, HARRIS W S and APPEL L J (2002), Fish consumption, fish oil, omega-3 fatty acids, and cardiovascular disease, Circulation, 106(21), 2747–2757. FSANZ

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and WILLIAMS G M (2003), A review of the safety of DHA45-oil, Food Chem Toxicol, 41(11), 1433–1446. KYLE D J (1997a), Safety and bioavailability of single cell oil enriched in arachidonic acid, Lipid Technol Newsl, 3(5), 100–103. KYLE D J (1997b), Production and use of a single cell oil highly enriched in arachidonic acid, Lipid Technol, 9, 116–121. KYLE D J (2001), The large-scale production and use of a single-cell oil highly enriched in docosahexaenoic acid, in Shahidi F and Finley J W, Omega-3 Fatty Acids: Chemistry, Nutrition and Health Effects, ACS Symposium Series 788, Washington, DC, American Chemical Society, 92–107. LEAF A, KANG J X, XIAO Y F and BILLMAN G E (2003), Clinical prevention of sudden cardiac death by n-3 polyunsaturated fatty acids and mechanism of prevention of arrhythmias by n-3 fish oils, Circulation, 107(21), 2646–2452. LEWIS T E, NICHOLS P D and MCMEEKIN T A (1999), The biotechnological potential of thraustochytrids, Mar Biotechnol (NY), 1(6), 580–587. MARCHIOLI R, SCHWEIGER C, TAVAZZI L and VALAGUSSA F (2001), Efficacy of n-3 polyunsaturated fatty acids after myocardial infarction: results of gissi-prevenzione trial. Gruppo italiano per lo studio della sopravvivenza nell’infarto miocardico, Lipids, 36 (Suppl), S119– 126. MARCHIOLI R, BARZI F and BOMBA E et al. (2002), Early protection against sudden death by n-3 polyunsaturated fatty acids after myocardial infarction, Circulation, 5(16), 1897– 1903. MARRIOTT N G, GARRETT J E, SIMS M D and ABRIL J R (2002a), Performance characteristics and fatty acid composition of pigs fed a diet with docosahexaenoic acid, J Muscle Foods, 13(4), 265–277. MARRIOTT N G, GARRETT J E, SIMS M D, WANG H and ABRIL J R (2002b), Characteristics of pork with docosahexaenoic acid supplemented in the diet, J Muscle Foods, 13(4), 253–63. NORDOY A (1999), Dietary fatty acids and coronary heart disease, Lipids, 34 (Suppl), S19– 22. OTTO S J, VAN HOUWELINGEN A A C and HORNSTRA G (2000), The effect of supplementation with docosahexaenoic and arachidonic acid derived from single cell oils on plasma and erythrocyte fatty acids of pregnant women in the second trimester, Prostaglandins Leukot Essent Fatty Acids, 63(5), 323–328. PALAKURTHI S S, FLUCKIGER R, AKTAS H, CHANGOLKAR A K, SHAHSAFAEI A, HARNEIT S, KILIC E and HALPERIN J A (2000), Inhibition of translation initiation mediates the anticancer effect of the n-3 polyunsaturated fatty acid eicosapentaenoic acid, Cancer Res, 60(11), 2919– 2925. PURI B K (2004), Monomodal rigid-body registration and applications to the investigation of the effects of eicosapentaenoic acid intervention in neuropsychiatric disorders, Prostaglandins Leukot Essent Fatty Acids, 71(3), 177–179. RAHMAN M, WRIGHT J T, JR. and DOUGLAS J G (1997), The role of the cytochrome p450dependent metabolites of arachidonic acid in blood pressure regulation and renal function: a review, Am J Hypertens, 10(3), 356–365. RATLEDGE C (2005), Single cell oils for the 21st century, in Cohen Z and Ratledge C, Single Cell Oils, Champaign, Ill, AOCS Press, 1–20. REITAN K I, RAINUZZO J R, OIE G and OLSEN Y (1997), A review of the nutritional effects of algae in marine fish larvae, Aquaculture, 155(1–4), 207–221. RUXTON C H, REED S C, SIMPSON M J and MILLINGTON K J (2004), The health benefits of omega3 polyunsaturated fatty acids: a review of the evidence, J Hum Nutr Diet, 17(5), 449– 459. SANGIOVANNI J P and CHEW E Y (2005), The role of omega-3 long-chain polyunsaturated fatty acids in health and disease of the retina, Prog Retin Eye Res, 24(1), 87–138. SCHMIDT E B, CHRISTENSEN J H, AARDESTRUP I, MADSEN T, RIAHI S, HANSEN V E and SKOU H A (2001), Marine n-3 fatty acids: basic features and background, Lipids, 36 (Suppl), S65–68.

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and OATES (1975), Increased arachidonate in lipids after administration to man: effects on prostaglandin biosynthesis, Clin Pharmacol Ther, 18(5 Pt 1), 521–529. SIDDIQUI R A, SHAIKH S R, SECH L A, YOUNT H R, STILLWELL W and ZALOGA G P (2004), Omega 3-fatty acids: health benefits and cellular mechanisms of action, Mini Rev Med Chem, 4(8), 859–871. SINCLAIR A, ATTAR-BASHI N, ANURA JAYASOORIYA, GIBSON R and MAKRIDES M (2005), Nutritional aspects of single cell oils: Uses and applications of arachidonic acid and docosahexaenoic acid oils, in Cohen Z and Ratledge C, Single Cell Oils, Champaign, Ill, AOCS Press, 182–201. SINCLAIR A J (1975), Long-chain polyunsaturated fatty acids in the mammalian brain, Proc Nutr Soc, 34(3), 287–291. SINCLAIR H M (1953), The diet of Canadian indians and eskimos, Proc Nutr Soc, 12, 69– 82. SMUTS C M, BOROD E, PEEPLES J M and CARLSON S E (2003), High-DHA eggs: feasibility as a means to enhance circulating DHA in mother and infant, Lipids, 38(4), 407–414. STREEKSTRA H (1997), On the safety of Mortierella alpina for the production of food ingredients, such as arachidonic acid, J Biotechnol, 56(3), 153–165. TAM P S Y, UMEDA-SAWADA R, YAGUCHI T, AKIMOTO K, KISO Y and IGARASHI O (2000), The metabolism and distribution of docosapentaenoic acid (n-6) in rats and rat hepatocytes, Lipids, 35(1), 71–75. UAUY R, BIRCH D, BIRCH E, TYSON J and HOFFMAN D (1990), Effect of dietary omega-3 fatty acids on retinal function of very-low-birth-weight neonates, Pediatr Res, 28, 485– 492. WATKINS C (2004), Infant formula update, INFORM, 15(6), 381–382. WEN Z and CHEN F (2005), Prospects for eicosapentaenoic acid production using microorganisms, in Cohen Z and Ratledge C, Single Cell Oils, Champaign, Ill, AOCS Press, 138–160. WHEELER T G, BENOLKEN R M and ANDERSON R E (1975), Visual membranes: specificity of fatty acid precursors for the electrical response to illumination, Science, 188(4195), 1312–1314. WYNN J P and RATLEDGE C (2005), Microbial production of oils and fats, in Shetty K, Food Biotechnology, Boca Raton, FL, CRC Press, 443–472. ZELLER S (2005), Safety evaluation of single cell oils and the regulatory requirements for use as food ingredients, in Cohen Z and Ratledge C, Single Cell Oils, Champaign, Ill, AOCS Press, 161–181. J A

24 Use of fish oils and marine PUFA concentrates B. Hjaltason, EPAX AS, Iceland and G. G. Haraldsson, University of Iceland, Iceland

24.1

Introduction

The increasing uses of marine PUFA concentrates for human consumption have been driven by strong and solid science. Between Spring 2004 and Spring 2005, more than 580 scientific papers were published dealing with marine-based omega-3 fatty acids. It is also interesting to note that more than half of those articles deal with human trials (Holub, 2005). This has increased consumer awareness of the health benefits of the omega-3 fatty acids. The application of fish oil and fish oil concentrates as an omega-3 source for human consumption can be divided into three categories: 1. as food ingredients or functional foods*; 2. as dietary supplements; 3. as pharmaceuticals. The fortification of omega-3 preparations into food products opens new possibilities to provide more omega-3 fatty acids in the public diet. Government agencies are also promoting more consumption of omega-3 fatty acids. As part of the Bush administration’s effort to make America healthier, the White House’s Office of Management and Budget (OMB) sent a letter to Health and Human Services (HHS) and the United States Department of Agriculture (USDA) requesting that departments promote the consumption of omega-3 * According to the Institute of Food Technologists (IFT) functional foods are defined as foods and food components that provide health benefits beyond basic nutrition (IFT Expert Report, 2005) (www.ift.org).

588 Modifying lipids for use in food fatty acids. OMB requested HHS to consider incorporating this advice in its Dietary Guidelines for America, which they did in their 2005 version. They also asked USDA to update its 1992 Food Guide Pyramid with this recommendation. In Europe, the growth of the market has been slow due to the fact that only limited health claims can be made and also because there has been no recommended daily intake (RDI) for omega-3 polyunsaturated fatty acids (PUFA). It is expected that by 2008 most of the uncertainties surrounding EU legislation will be removed and also that the RDI will be in place by 2010 at the latest. It is difficult to estimate the global spending on fish oil for human consumption. There have been retail numbers for 2004 of Euro 1.2 billion (www.euromonitor.com) or US$ l.5 billion (Nutrition Business Journal, 2005a) while the global fish oil ingredient trade stands between 350 and 400 million US$. This chapter provides an insight into use of fish oil and marine PUFA concentrates. It is by no means intended to be a comprehensive review, but rather an insight into the area, and it is anticipated that the reader will get a good overview of the current situation. First, the focus is put on the use of marine oils and omega-3 PUFA concentrates in food ingredients and functional foods. This is followed by a discussion of the use of marine oils and PUFA concentrates as dietary supplements and by a brief description of use of omega-3 PUFA as pharmaceuticals. Finally, there is a short conclusion on future trends and new types of omega-3 PUFA concentrates.

24.2 Food ingredients and functional foods Application of fish oil and especially fish oil concentrates as food ingredients has been restricted by the limited availability of high quality oils which have no smell and taste and a reasonable shelf life. Until Hoffman–LaRoche launched their ‘RoPUFA®’ product in the 1980s now marketed by ASM Nutritional Products in Switzerland, there was hardly any commercial fish oil available with sufficient stability to be used in food. The uses were limited to products with relatively short shelf life such as low-fat spread, etc. Several such products were launched in the 1980s, especially in the UK and Scandinavia, but most of them failed. Furthermore, the consumer was at that time not ready to pay a premium for omega-3 oil in the product. This problem was solved to some extent by using micro-encapsulation technology to protect the oil from oxidation and to mask the taste and smell of the fish oil. Micro-encapsulation is based on creating a barrier between the sensitive fish oil and the environment in order to protect it against oxidation during storage or further processing (Shelke, 2005). The process also protects the fish oil from interacting with other reactive ingredients. The coating can be made from sugars, proteins, natural and modified polysaccharides, synthetic polymers and fats. Ingredients can be encapsulated

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by various techniques, but spray-drying is still the most economical and most widely used method. The main disadvantage of the micro-encapsulation technology is how much carrier is needed. Sometimes 75 % of the weight is the carrier while the fish oil makes up only 25 %. This means that food processors are more inclined to use micro-encapsulated fish oil concentrates since these need less volume to deliver the omega-3 amount needed in each serving. This is also necessary when adding micro-encapsulated fish oil to products that have limited volume capacity such as nutritional bars. Bioavailability is also very important. The problem is that usually the ‘stronger’ the coating material, giving better protection to the oil, the lower the bioavailability. There is a great deal of work going on to improve the micro-encapsulation technology – to find better carriers or coating material as well as developing new technology. One of the first uses of micro-encapsulated fish oil was in bakery products. Micro-encapsulated fish oil is now used in infant formulas (www.martek.com), dairy products such as yoghurt, cereals, nutrition bars and even chewing gum (www.jolgum.com, www.olalafoods.com). The number of products launched is increasing every year. These products cater for the demand for products with convenient and immediate health benefits, as well as appealing to an ageing population in the Western world that is more concerned with long-term health and well being. Increasing consumer purchasing power makes this possible. One of the limiting factors in selling and promoting food products containing omega-3 fish oils has been the fact that authorities have not allowed any health claims. In early 2005, the FDA approved limited health claims for food products containing eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) as a factor for improved cardiovascular health (www.fda.gov). This has prompted the major food manufacturers to start incorporating omega3 oils into their products. The EU is still evaluating the potential health benefits of adding omega-3 oils to food products and this is slowing the progress of this development. In November 2005, the Australian and New Zealand food authorities (FSANZ) published new proposals for a health claim standard that would allow disease risk reduction claims for the first time (www.foodstandards.gov.au). One of the major concerns for the consumer has been the negative discussion about mercury content in fish and fish products as well as other environmental contaminants such as dioxins and PCBs. Consumers assume that if the fish contain heavy metals and contaminants, then they must also be found in fish oil and fish oil concentrates. Since the voluntary Council for Responsible Nutrition (CRN) fish oil monograph was first published (www.crnusa.org), as well as strict EU regulations about maximum content of these compounds, almost all fish oil and fish oil concentrate producers are purifying their oil to comply with the existing standard (European Pharmacopoeia, 2005). To make the concentrates, molecular distillation is performed and that is efficient in

590 Modifying lipids for use in food removing most of the dioxins and PCBs. Although certain fish species may have a high mercury content, it is usually in the form that does not go with the fish oil phase when the fish is processed into meal and oil. Also, relatively young fish are used, and fish species tend to accumulate pollutants and heavy metals only as they get older. The market potential for omega-3 fatty acids in functional food and dietary supplements is enormous as can be seen in Table 24.1 (The Health Industry News: www.kenko-media.com/media/health_idst/; Nutritional Supplement Japan 2005. Japan’s Nutritional Supplement Market Report 2005, Paul Yamaguchi & Associates Inc., Tarrytown, New York, USA; www.functional foodsjapan.com). Only a fraction of this value is due to products containing omega-3 fish oil. In the USA, functional foods make up slightly over 50 % of the sales compared to the dietary supplements, but it is expected that the main growth in the coming years will be in the area of functional food containing omega-3 products. Table 24.2 lists some of the products available on the market as well as potential products (Andersen, 1998; Haraldsson and Hjaltason, 2001).

24.2.1 Japan One of the most interesting and most advanced markets for omega-3 concentrates in functional food is Japan. The term functional food originated in Japan in 1988 and was used by the industry to describe foods fortified with specific ingredients imparting certain health benefits. In 1991, the Table 24.1 The 2003 nutrition market for combined dietary supplements and functional food. Countries

Value (US$ billion)

USA EU Japan Asia Others

45 35 27 12 9

Total Table 24.2 products.

Population (million) 293 450 126

128 Some of functional food products available on the market and potential

Cereal Beverages Dietetic products Dairy products Fats and oils

Bread, breakfast cereals, pasta, noodles, cookies/biscuits, nutritional bars Soft drinks, orange juice, soy milk Milk powder, infant formula, slimming powder, maternal nutrition, medical nutrition, sports nutrition Fresh milk, UHT milk, yoghurt, cheese Margarine, salad dressing, mayonnaise, edible oil

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government created a regulation called Foods For Specified Health Use (FOSHU) to enable functional food makers to claim health benefits (Nutritional Supplement Japan, 2005; Ministry of Health, Labor and Welfare (MHLW): www.mhlw.go.jp/english/index.html; National Institute of Health and Nutrition (NIHN): www.nih.go.jp/eiken/index.html). The FOSHU health claims are permitted for foods and beverages as well as supplements that contain physiologically active components. Companies must apply to MHLW for approval and submit data to support their application. Since this system was established, more than 400 products have been allowed to carry health claims. In Spring 2001, the Japanese government set up a new regulation that applies to food with health claims. This is the Foods with Health Claim Act (Nutritional Supplement Japan 2005; MHLW: www.mhlw.go.jp/english/index.html; NIHN: www.nih.go.jp/eiken/index.html). This was done in order to provide more information to the consumer about the health benefits of the product. FOSHU is still under this act, but also Foods with Nutrient Function Claims (FNFC). Products falling into these two categories are the only ones allowed to have health claims. New FOSHU regulation guidelines were issued in January 2005 to expand the category and make the approval process from MHLW easier. The new FOSHU regulations apply to all types of foods and dietary supplements and give companies new opportunities to use health claims for their omega-3 concentrates. The use of DHA as functional ingredient began in Japan in 1993. The Japanese government supported a program to find methods to isolate and concentrate DHA. This was a co-operation between industry, universities and research institutes. The aim was to fortify food with DHA since fish consumption had been decreasing among the younger generation. In 1997 the DHA and EPA Association was formed with the aim to promote the manufacture and supply of high quality products as well as offer information to consumers and develop the EPA and DHA industry. There are more than 34 company members as well as ten supporting members (Japan Marine Oil Association, 2005). The Japanese authorities have not allowed concentrated ethyl esters or chemically modified omega-3 concentrates to be sold as dietary supplements or health food. Only concentrated triacylglycerols produced with lipases are allowed for general sale. This has made it more difficult for European producers of concentrates to enter the Japanese market. It is interesting to note that the main emphasis in Japan today is on DHA, although health benefits of both EPA and DHA are well known in Japan. There is a strong belief in Japan that DHA improves memory and affects a person’s intelligence. In Japan, DHA-rich oil is often called ‘brain food’. Based on the fact that the average Japanese consumes only one-third of the amount of fish compared to 25 years ago, there is no wonder that mothers are eager to give their children food containing DHA. The use of fish oil as supplement was around 400 tonnes in 2004, which is a 20 % increase in

592 Modifying lipids for use in food volume, while the retail value went from US$ 42 million to 90 million or up by 114 %. In the functional food area, the volumes are estimated to be around 800–1000 tonnes, mainly high-DHA oil derived from tuna. The biggest volume of this oil is added to processed fish derived products in order to increase their DHA content. This also masks the smell and taste of the fish oil. Recently, there has also been an increased interest in EPA as protection against CVD. Several pharmaceutical products with a high EPA content are on the market, and a recent Japanese study showed that patients with history of coronary diseases who took high doses of concentrated EPA with statin therapy had a 19 % reduction in risk of major coronary events (Yokoyama, 2005; www.medscape.com/viewarticle/518574; www.americanheart.org). Also, a drink containing both EPA and DHA was recently launched in Japan by Nissui with advertisement in the main media (www.nissui.co.jp/product/ imaq/data.html).

24.2.2 Europe In the EU there has been a rapid development in launching new products that include omega-3 oils and concentrates. As in the USA, most of the products contain α-linolenic acid (ALA) as an omega-3 source – not the marine-based PUFA EPA and DHA. This is very confusing for the consumer who does not know the difference between these fatty acids and is only aware of the term omega-3, and it is a real threat to the fish oil industry. According to Global New Products Database (www.gnpd.com), 153 products were launched across Europe in 2004 that made reference to omega-3 content in their production description compared to 190 by early November 2005. However, only ten of the products from 2005 contained fish oil. These include Nestlé’s Petit Drinking Yoghurt with DHA, that was launched in late 2005 in Spain, Plus Omega-3 bread from Kohlberg in Denmark, IQ Brainstorm cereal bar developed by Biomedical Laboratories for the UK market and number of milks from dairies like Dairy Crest and Covap. Dairy Crest has also launched a low fat spread to the market. In 2006 Müller Vitality™ will launch a range of functional yoghurts and yoghurt drinks that have been boosted with the benefits of long-chain omega-3 acids. So far there have been only two worldwide successful brands containing marine-based omega-3 based oils. The first is Omega-3 milk which Puleva in Spain launched (www.puleva.com). It sold for more than 100 million US$ in 2005 in a country with population around 40 million. The second is the Australian George Weston Foods Tip Top® Up™ Brand which achieved 13 % share of the Australian bread market in 2005, only three years after it was launched. The reason for the success in Australia seems to be linked to the fact that Australians have amongst the highest level of awareness in the world of omega-3 fatty acids due to information from the local press. Education about the benefits of omega-3 fatty acids seems to be the driving force in selling products containing EPA and DHA (www.new-nutrition.com).

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In late 2005, Tetra Pak and DSM Nutritional Products announced that they could provide dairy customers with an easy solution to producing chilled milk with added omega-3 fish oil. This technology is called the FlexDose system, and the omega-3 that is supplied by DSM is in the form of an emulsion that is UHT treated for aseptic delivery and specially formulated for dairy application (www.ferret.com.au/Companies/Tetra-Pak). Until now most attempts to add omega-3 fish oil to chilled milk have failed due to oxidation of the oil giving a fishy flavour of the product. It is going to be interesting to see if this technology will open up new possibilities for the application of omega-3 fish oil into fresh milk. In recent years several companies have launched omega-3 enriched eggs (Schreiner, 2005). An omega-3 source is added to the feed of the hens so their eggs contain some DHA. The main omega-3 sources are fish oil (tuna oil), algae and flax seed. This is a very convenient way to enrich the eggs, but currently such eggs are sold at premium prices. Dr Bruce Holub from the University of Guelph has shown that an omega-3 egg product, Omega Pro, can reduce triacylglycerol levels by up to 25–30 % (www.canadafreepress.com/ 2004/health050304.htm). Attempts have also been made to enrich meat with omega-3 fatty acids by feeding the animals with fish oil but with only limited success (Pike, 1999). The omega-3 PUFA are hydrogenated in the ruminants’ rumen and cannot, therefore, be transferred to the meat fat. In cases like pigs and piglets, very small amounts of omega-3 fatty acids in the diet give undesirable flavours in the meat as well as changing the texture of the fat.

24.2.3 USA The sales of functional foods in 2004 increased by 6.8 % from 2003 and totaled US$ 24.3 billion. These are big numbers and the industry is increasing its share of the overall food market. In 1995, functional foods were just 2.6 % of the entire US food market, but this had doubled to 4.5 % in 2004 (Nutrition Business Journal, 2005b; www.nutritionbusiness.com). In the USA the omega-3 concept is also moving into the mainstream. The confusion regarding the difference between omega-3 fatty acids from flaxseed versus omega-3 PUFA derived from marine oils is even greater in the USA and especially Canada than in the EU. According to ProductScan Online, around 300 products were launched in the first 11 months of 2005 in the USA containing omega-3, whether from marine or vegetable sources. This is comparable to 171 products in 2004. The increase is linked to the increasing awareness of the health benefits of omega-3 fatty acids after FDA extended qualified health claims linking EPA and DHA to reduced risk of coronary heart disease from dietary supplements to foods. According to Prepared Food’s R&D Trends Survey in 2005 for the USA, omega-3 fatty acids were number 6 (41.8 %) among functional ingredients on the rise, following antioxidants (57.3 %), dietary fibre (55.8 %), organic ingredients (44.2 %), calcium (43.5 %) and soy protein (43.8 %) (www.preparedfoods.com).

594 Modifying lipids for use in food There are several products on the market in North America containing marine-based omega-3 fatty acids. One of them is a product called Brainiums DHA which was launched in August 2005 by Nutrilite. This is a lemonflavoured gum that contains both EPA and DHA and offers, therefore, a natural source of omega-3 fatty acids to children. George Weston Bakeries have also launched a bread enriched with micro-encapsulated fish oil. In March 2005, three US bread makers including George Weston Bakeries launched omega-3 breads with Ocean Nutrition’s (OCN) MEG-3® microencapsulated fish oil. This was followed by Farmers’ Choice Partly Skimmed Milk in Canada. Danone offered Cardivia® yoghurt in Canada. SmartBalance® Omega Plus™ margarine was launched containing liquid fish oil from Omega Protein® (Nutrition Business Journal, 2005b). PBM Products Inc. launched in 2004 the first diabetic nutrition drink containing DHA. This drink offers a combination of omega-3 polyunsaturated fatty acids, amino acids and other desirable nutrients for good health.

24.2.4 Rest of the world Consumers in Australia have a high awareness of the health benefits of marine-based omega-3 fatty acids. The company Nu-Mega, which belongs to the Clover Corporation, was a pioneer in this area. It was one of the first companies to start producing tuna oil for infant formula and today offers both liquid tuna oil and micro-encapsulated tuna oil for a broad spectrum of products. These two preparations are used in the number one omega-3 enriched bread in Australia sold by George Weston Foods (www.nu-mega.com; www.foodspec.com.au/). DSM and OCN from Canada have also entered the Australian market with micro-encapsulated and liquid products for functional food application. Australian UpBread launched a bread in 2005 containing the MEG-3® brand of micro-encapsulated fish oil from OCN (Runestad, 2005). Although there are a few companies in China making micro-encapsulated fish oil, the market for such products has developed only slowly. Foreign firms are facing registration and regulation restrictions and the retail channel is still largely under-developed. Since 2003, the regulation and registration processes have been streamlined and simplified and new types of retail formats have been created, partly because of the great amount of capital investment into those sectors. One company taking advance of this situation is OCN, but Trappist Dairy in Hong Kong has now launched OMU milk with their MEG-3® fish oil (Habiger, 2005).

24.3 Dietary supplements Until recently, the main application of fish oil and fish oil concentrates has been as dietary supplements. There is a long tradition of using fish liver oil

Use of fish oils and marine PUFA concentrates

595

as dietary supplement as a source of vitamins A and D, and with the discovery of health benefits of omega-3 fatty acids, a new market opened up for whole body fish oil as omega-3 source. The dietary supplement market has moved from promoting well being products into products with specific health indications. The biggest category in dietary supplements is vitamins followed by herbs/ botanicals, sport nutrition and minerals. Omega-3 fatty acids belong to speciality supplements as part of essential fatty acids (EFA). Global spending on fish oil supplements rose by 60 % in retail value between 1997 and 2004. The cod liver oil market grew 38 % while the other fish oils grew 82 %. Since 1999, the market has been dominated by other omega-3 fish oils rather than cod liver oil, with the biggest growth in the USA (www.euromonitor.com). 24.3.1 USA Speciality supplements grew 14 % in 2003 in the USA (Nutrition Business Journal, 2004; www.nutritionbusiness.com) compared to 6.8 % the year before. The speciality category is led by growth of fish oil which grew 43 % in 2003. Overall, the sales of fish oil supplements in the USA have climbed nearly ten-fold from 35 million US$ in 1995 to an estimated 310 million US$ in 2005. It is expected that the growth for the next five years will be a double digit or between 10 and 15 % annually. It is interesting that this goes in hand with increasing seafood consumption. In 2003 the USA set a record at 16.3 pounds per capita and the figure for 2004 was an even higher 16.6 (Fiorillo, 2005; www.intrafish.no/global/paper/;www.fishupdate.com). Sales rose 40 % in the mass market led by WalMart and Costco store labels which now represents around one-third of the market (Nutrition Business Journal, 2004; www.nutritionbusiness.com). The branded seafood giant Bumble Bee has announced they are also entering the market. In Spring 2005 they signed a marketing agreement with Leiner Health Products to produce and market fish oil supplements under the Bumble Bee brand name (Horovitz, 2005; www.usatoday.com). Most of the concentrates of marine-based omega-3 oils are sold as dietary supplements in soft gel capsules. In recent years, with improved quality, concentrates are now also being sold in bottles, often flavoured and blended with other oils. Emulsions are also available, but one of the most successful products in the USA has been a pudding-like flavoured emulsion that has been sold in sachets and can be mixed with yoghurt or blended with orange juice (www.coromega.com). DSM Nutritional Products Inc. did a study in 2002 among pharmacists who recommended fish oil as a dietary supplement (Vitamin consumption in the US 2003, DSM Nutritional Products Inc: www.nutritionalproducts.com). The question they asked was what health benefits pharmacists associated with fish oil. The results are shown in Table 24.3(a). It is interesting to note that the highest-rated symptoms are all related to the CVD.

596 Modifying lipids for use in food When the consumer was asked the same questions, the pattern was a little different. Only adults who were aware of the health benefits of fish oil were asked the questions, and this was around 30% of the survey. The results are shown in Table 24.3(b). The consumer seems to have a more diversified idea of the health benefits of fish oil, probably due to the many publications on the different health benefits of omega-3 fatty acids.

24.3.2 Europe Although there is a growing interest in the use of marine oil-based functional foods and beverages in Europe, the main market is in nutritional supplements. What is also special about the dietary supplement market in Europe is the role of cod liver oil which accounted for 23 % of the volume in 2004. This is due to the tradition of taking cod liver oil as a vitamin A and D source in Scandinavia as well as the UK. The main omega-3 ingredient in dietary supplements is 18:12 natural fish oil that contains 18 % of EPA and 12 %

Table 24.3(a) Results from a survey by DSM Nutritional Products Inc. in 2002 regarding health benefits associated with fish oil among pharmacists. Health benefits associated with fish oil

Frequency of mentioning (%)

Lowers cholesterol Prevents heart diseases Lowers triglycerides Promotes healthy hair/skin I do not know Improves circulation Prevents Alzheimer’s disease Improves mental health Eases arthritis Improves eye health

61 53 42 20 16 15 12 11 10 10

Table 24.3(b) Results from a survey by DSM Nutritional Products Inc. in 2002 regarding health benefits associated with fish oil among consumers. Health benefits associated with fish oil

Frequency of mentioning (%)

Lowers cholesterol Prevents heart attack Good for skin Reduces blood pressure Lowers triglycerides Relieves arthritis Good for infant brain and vision Reduces memory loss Improves mental function/attention deficit disorder Treatment of digestive tract inflammation

55 47 47 31 26 16 13 12 12 10

Use of fish oils and marine PUFA concentrates

597

DHA. This accounted for around 70 % of the market in 2004 while the rest was made up by concentrates (Frost and Sullivan, 2005: www.frost.com). If we look at the future trends, we see the use of cod liver oil still going down and being replaced mainly by highly concentrated products. This means that the market is moving away from selling low omega-3 content products intended to promote general well being and into highly concentrated products intended for specific health purposes backed up by clinical trials. Most of the fish oil sold as dietary supplement is packed in soft or hard gelatine capsules. Earlier cod liver oil was sold in Scandinavia and the UK in bottles, but the younger generation of consumers prefer the gelatine capsules to avoid the smell and taste of the oil. Today marketing companies both in Europe and the USA are offering chewable capsules for children as well as enteric coated capsules that will not open until in the small intestine, eliminating any regurgitation. Furthermore the oil in capsules can also be flavoured or aroma added to the gelatine to make their intake more palatable (www.cardinal.com)(www.capsugel.com)(www.swisscaps.com). In 2004 the European market for marine oils in food beverage and supplement application was valued at US$ 150 million with a volume of 11 400 tonnes. More than 90 % of the value and volume is from dietary supplements. It is expected that in the coming years the annual growth in revenue will be around 10 % slowing down to around 7 % due to lower prices leading to US$ 219 million in 2010 (www.frost.com). The highest growth rate of fish oil sales other than cod liver oil in the last seven years is in Italy and Germany with growth rates around 30 % during this period. This is followed by Norway and the UK (www.euromonitor.com). The main market driver in Europe, as in the USA, is the solid science that has been promoted by the media, increasing consumer awareness of the health benefits of the omega-3 PUFA. Also consumers are now more aware of the fact that they need more omega-3 in their diet to bridge the nutritional gap. Market growth has remained slow because regulations in the EU are not well defined in this area and because there is no RDI. In some EU countries, such as Finland and Denmark, selling the ethyl ester form of the omega-3 fatty acids as a dietary supplement is not permitted. In addition the public has been alarmed by negative discussion concerning pollution in fish and the possibility of contaminated fish oil products reaching the market.

24.3.3 Rest of the world Australia is a large market for marine-based dietary supplements. As in so many former British colonies, there is a strong tradition for taking cod liver oil. In recent years, some of the cod liver oil market has been replaced by the so-called 18:12 whole body fish oil that contains around 18 % EPA and 12 % DHA and no vitamins A and D. So far, Australian authorities have not allowed sales of concentrated omega-3 products as dietary supplements, but this decision is now under review.

598 Modifying lipids for use in food The market for marine-based dietary supplements in Japan is limited, partly due to the strength of the functional food market for such products and also since there are very few health food shops and health corners in the supermarkets. Most of the sales of the marine-based dietary supplements take place on the internet. One of the leading brand companies in this area is DHC (www.dhc.co.jp). Encapsulated fish oils in soft gelatine capsules are very popular in China. Most of them are imports from the USA, either imported by overseas Chinese or sold by multi-level companies such as Amway or Pharmanex/Nuskin. Originally the products were called ‘Alaskan’ fish oil, although there is no commercial fish oil production for human consumption in Alaska. US products are popular in China, and the image of a clean and cold environment makes these products very popular. In the 1990s the Chinese government put up strict control on sales of dietary supplements. Every product has to be registered at a cost between US$ 75 000 and 150 000 and this may take up to one year. This has increased the quality of the products on the market. The Chinese government also allows health claims, but the most common use is for reduction of cardiovascular diseases. In the last two years there have been changes in the classification of the products, consolidation of government offices for ease of application process and the creation of new multi-level marketing laws. The de-regulation along with greater purchasing power in China, from 3679 yuan per capita in 1994 to 8500 yuan in 2004, will drive a strong rise in consumption of dietary supplements (Habiger, 2005; www.sfda.gov.cn).

24.4

Pharmaceuticals

Documented clinical effects of omega-3 fatty acids have resulted in several pharmaceutical products. Most of them are highly concentrated, but one of the first was MaxEPA™ (Seven Seas Healthcare Ltd, UK), that only contained around 18 % of EPA and 12 % of DHA and which was later registered as a drug for hyperlipidemia (A History of British Cod Liver Oils, 1994). Japan has been a leading country in using highly concentrated fish oils as prescription drug. Epadel, a highly concentrated EPA product, was developed by the Japanese company Mochida in co-operation with Nippon Suisan Kaisha (www.mochida.co.jp). It was first approved in 1990 to treat arteriosclerosis obliterans (ASO) and in 1994 for hyperlipidemia. There are now several similar generic products on the market in Japan. Pronova Biocare from Norway (www.pronova.com) launched its pharmaceutical product Omacor® in Europe first for treatment of hypertriglyceridemia. This is an ethyl ester concentrate of approximately 55 % EPA and 30 % DHA. Omacor reduces triglycerides by up to 45 % and is complementary to statins. The second approval of Omacor was post-MI treatment where Omacor reduces all-cause mortality by 21 %, cardiovascular

Use of fish oils and marine PUFA concentrates

599

mortality by 30 % and sudden cardiac death by 44 %. This was based on the results from the Gissi Prevention study that was done in Italy and published in 1999 (GISSI-Prevenzione Investigators, 1999). A recent Japanese study has shown that omega-3 fatty acid supplements combined with statin therapy give a synergic effect that reduces major coronary events, particularly for those with established coronary artery disease. This has led to planning of a number of clinical trials where omega-3 concentrates are mixed with statins with the aim of launching them in the future as prescription drugs (www.clinicaltrials.gov). PBM Pharmaceuticals Inc. has announced prescription medication in the USA with omega-3 fatty acids. Animi-3® is prescribed by physicians as supplement therapy in rheumatoid arthritis (RA), cardiovascular disease and depression (www.animi-3.com). Mainland China has long produced highly concentrated EPA for pharmaceutical application. This has mainly been applied against hypertriglyceridemia and as a prescription drug (DUOXIKANG capsule, produced by Zhejiang Hailisheng Pharmaceutical Company).

24.5 Commercial fish oils for human consumption and marine PUFA concentrates There are a number of producers of non-concentrated fish oil for human consumption. Many of these companies are listed in Table 24.4 together with their webpages for further information and references. Their products are the main ingredients for the foods and functional foods discussed in Section 24.2 above. Many of these products are also used directly as dietary supplements. Table 24.4 Producers of commercial non-concentrated fish oils for human consumption. Company

Country

Web page

Lysi Ocean Nutrition Napro Pharma Maruha Nissui Lipid Nutrition DSM Nutrition Products Neptune Biotech BASF Berg Lipid Technology Omega Protein Maritex Denomega GC Rieber Oils Nu-Mega Ingredients

Iceland Canada Norway Japan Japan Malaysia USA/Holland Canada Germany Norway USA Norway Norway Norway Australia

www.lysi.com www.ocean-nutrition.com www.napro-pharma.no www.maruha.co.jp www.nissui.co.jp www.lipidnutrition.com www.nutraaccess.com www.neptunebiotech.com www.basf.com www.blt.no www.omegaprotein.com www.maritex.com www.denomega.no www.alnaes.no www.nu-mega.com

600 Modifying lipids for use in food Table 24.5 lists the names of the main producers of omega-3 PUFA concentrates, their homepages, type of concentrates (triacylglycerols or ethyl esters) and concentration levels. These products are the main ingredients for dietary supplements, and many of them are also used in the pharmaceutical area. This is discussed in more detail in Section 14.7.

24.6 New types of concentrates and future trends In recent years phospholipids have been introduced to the market as a new source of omega-3 fatty acids, in particular DHA, for application in food, supplements and even for the pharmaceutical ingredients industry. Phospholipids are major constituents of biological membranes of cells and organelles, providing the matrix and defining their boundaries. They have a structural and functional synergy with fatty acids and act as sites for the trafficking of molecules within and into/out of cells. The phospholipids are the most important storage of the biologically active PUFA, thus playing immensely important roles as signal and messenger molecules, for example in the complicated pathway of the eicosanoids. In fish and marine products, the omega-3 fatty acids are usually found both in triacylglyerol form and as phospholipids. When the fish oil is extracted from the fish, the phospholipids are left in the protein. Therefore taking omega-3 only as triacylglycerols does not give the same composition of the omega-3 fatty acids as eating fish (Hjaltason, 1989). There are no rich sources of marine-based phospholipids, but the most commonly used are roes and krill. A Canadian company has introduced krill oil containing more than 40 % of phospholipids with phosphatidylcholine making up more than 80 % of the phospholipids. The company has undertaken some human studies on evaluation of the effect of krill oil on hyperlipidemia, pre-menstrual syndrome and dysmenorrhea as well as the clinical course of osteoarthritis and rheumatoid arthritis (www.neptunebiotech.com; Bunea et al., 2004; Sampalis, 2005). There are other producers of marine-based phosphoplipids, but their products have been sold mainly as an ingredient in aquaculture feed formulas (Hjaltason et al., 2005a,b). An example is the French company Phosphotech (www.phosphotech.com) producing purified marine phospholipids on a relatively small scale. The Japanese companies NOF (www.nof.co.jp) and Bizen (www.bizen.co.jp) are also suppliers of such purified marine phospholipids. There is little doubt that marine phospholipids will become very important in the near future as a highly potent source of the omega-3 PUFA. There are already strong indications of that. Another potential use of such phospholipids is omega-3 PUFA enriched liposomes in the pharmaceutical area. It is also anticipated that omega-3 based structured lipids will be developed further

Use of fish oils and marine PUFA concentrates

601

based on lipase application. Finally, genetically modified plants may also soon become an important source of the long-chain omega-3 PUFA. Today, all the concentrates produced by respectable producers are of high quality. So far these products have been used as dietary supplements and as pharmaceutical ingredients but, with improved quality, the products will start to appear in the functional food market. The key to this is improvement in quality, especially improving smell and taste. This can be achieved with improved processing technology, improved antioxidant systems as well as new and better micro-encapsulation methods. Pronova Biocare has recently started to deodorize its triacylglycerol concentrates and DSM has offered its RoPUFA® 75EE for some time as a deodorized product with excellent sensory characteristics.

24.7

References

(1994), A History of British Cod Liver Oils. The First 50 Years with Seven Seas, Cambridge, Martin Books. ANON (2005), European Pharmacopoeia, 5th edn, Strasbourg, Council of Europe. ANDERSEN S (1998), Long-chain Omega-3 Fatty Acid Fortified Foods. Omega-3 Fatty Acids. Health issues and product applications, Leatherhead, Leatherhead Food RA, December 1, Book of Abstracts. BUNEA R, FARRAH K E and DEUTSCH L (2004), Evaluation of the effects of Neptune krill oil on the clinical course of hyperlipidemia, Alternative Medicine Review, 9, 420–428. FIORILLO J (2005), Are fish oil supplements a threat?, IntraFish, 3 (11), 18–19. FROST and SULLIVAN (2005), European Omega-3 and Omega-6 PUFA Ingredients Market 12 July 2004, available at: www.frost.com. GISSI-PREVENZIONE INVESTIGATORS (1999), Dietary supplementation with n-3 polyunsaturated fatty acids and vitamin E after myocardial infarction: results of the GISSI-prevenzione trial, The Lancet, 354(9177), 447–455. HABIGER S (2005), Opening the door to China, Functional Foods & Nutraceuticals, December, 16–22. HARALDSSON G G and HJALTASON B (2001), Fish oils as sources of polyunsaturated fatty acids, in Gunstone F D, Structured and Modified Lipids, New York, Marcel Decker, Inc., 313–350. HJALTASON B (1989), New frontiers in the processing and utilization of fish oil, in Somogoyi J C and Muller H R, Nutritional Impact of Food Processing, Bibl. Nutr. Dieta. No. 43, Basel, Karger Publishing, 96–106. HJALTASON B, HARALDSSON G G and HALLDORSSON O (2005a), Cultivation of DHA-rich prey organisms for aquatic species, Patent, US 6,789,502. HJALTASON B, HARALDSSON G G and HALLDORSSON O (2005b), Rearing of aquatic species with DHA-rich prey organisms, Patent, US 6,959,663. HOLUB B (2005), DHA/EPA update 2005: A look at the new evidence, Oral presentation at the Annual Banner Pharmacaps Educational Symposium, May 10–13, High Point, NC. HOROVITZ B (2005), Bumble Bee joins swarm in fish oil aisle, USA Today, Money Section B, Tuesday, May 10. IFT (2005), Expert Report on Functional foods: Opportunities and Challenges, available at: www.ift.org. JAPAN MARINE OIL ASSOCIATION (2005), Sheet of information (DHA & EPA Association), October 19, 2005. ANON

602 Modifying lipids for use in food Nutrition Business Journal (2004), NBJ annual industry overview IX, (5/6), May/June, available at: www.nutritionbusiness.com. Nutrition Business Journal (2005a), Nutrition industry raw materials and ingredients supply 2005, X (8/9), August/September, available at: www.nutritionbusiness.com. Nutrition Business Journal (2005b), Functional foods VIII: the emergence of healthy foods X (10/11), October/November, available at: www.nutritionbusiness.com. Nutritional Supplement Japan (2005), Japan’s Nutritional Supplement Market Report 2005, Tarrytown, New York, Paul Yamaguchi & Associates Inc. PIKE I H (1999), Health benefits from feeding fish oil and fish meal. The role of long chain omega-3 polyunsaturated fatty acids in animal feeding, IFOMA Technical Bulletin, No. 28, May. RUNESTAD T (2005), Novel health benefits for marine omega-3s, Functional Foods & Nutraceuticals, December, 36–39. SAMPALIS T (2005), The Healing Power of Neptune Krill Oil, New York, Rebus LLC. SCHREINER M (2005), Omega-3 enriched eggs: positional distribution of fatty acids in response to different dietary lipids, Lipid Technol, 17, 271–275. SHELKE K (2005), Hidden ingredients take cover in a capsule, available at: www.foodprocessing.com/articles/2005/421.html. YOKOYAMA M (2005), Effects of eicosapentaenoic acid (EPA) on major cardiovascular events in hypercholesterolemic patients: The Japan EPA lipid intervention study (JELIS), American Heart Association Scientific Sessions, Dallas, TX, 13–16 November, available at: www.medscape.com/viewarticle/518574; www.americanheart.org.

Index

acidolysis 137, 238, 246, 250, 254 acyl migration 241 additives in frying oils 528 adipose tissue dietary effects 314, 317 temperature effects 314 adsorption 203 aerated products 502 alcoholysis 137, 239, 343 almond 24 AMF 409 analytical methods 114 animal fats 306 blending 326 chemical characteristics 42 CLA 309, 323 composition 37 food use 43 fractionation 325 general 28 interesterification 325 legal issues 44 melting point 42 nutritional changes 7 omega-3 acids 308, 327 physical characteristics 41 phytanic acid 309 production of fat 34, 35 production of meat 33

antioxidants (see also tocopherols and tocotrienols) 130, 134 AOM values 547 arachidonic acid 93, 139, 568 atomic force micrograph 152 automated analysis 282 autoxidation 130, 131 avocado 24 baked goods 454 bakery products 414 barrier fats 509 betapol 559 BHA 136 BHT 136 biological methods of modifying fats 6 bioreactors for enzymic interesterification 242 biosynthetic pathway for PUFA 107 blackcurrant seed oil 23 bleaching 25 borage oil 23 borneo tallow 23 box counting 156 brain phospholipids 371 butter 223, 405 camelina 24 canola oil 185

604 Index canola oil – a new crop 283 canola oil: see rapeseed oil caprenin 452 captrin 452 carbohydrate fatty acid esters 449, 454 carotene in rice 297 carotenes in palm oil and its fractions 221 centrifuge 213 chain length structure 148 cheese 473 chemical changes during frying hydrolysis 522 oxidation 520 chemical properties 3 chicken fat 227 chocolate 488 fat migration 508, 509 seeding 513 chocolate drink 468 chocolate spreads 500 cholesterol 40, 326, 447, 471, 481 cholesterol oxidation 133 CLA 309, 323 CLA in animal feed 557 cloud point 219 cocoa butter 23, 152, 490 cocoa butter alternatives 22, 489 cocoa butter equivalents 491 melting profiles 493 solid fat content 493 triacylglycerol composition 492 cocoa butter improvers 491 cocoa butter replacer 494 cocoa butter substitute 496 coconut oil: see lauric oils Codex Alimentarius 39, 116, 117, 120, 121 coffee creamers 469 complexation 203 confectionery fats 488 conjugated linoleic acids 228 consumption figures 2 cooking oils 546 cooling curves 210 corn oil 21, 186 cottonseed oil 21, 186 cream 471 categories 402 churning 407

fat content 401 ripening 407 stability 419, 421, 423 Crypthecodinium cohnii 94 crystal formation 398 crystal growth 145, 398 crystal networks 142 crystallization 143, 146, 206, 431. 510 dairy products artificial 462, 463, 466, 467, 468 filled 462, 463, 466, 467, 468 degumming 25 deodorization 25 deterioration during frying 532 DHA from C, cohnii 96 DHA from marine sources 336, 337, 348 DHA from other SCO 102 DHA from Schizochytrium sp 100 DHA see also docosahexaenoic acid diacylglycerol 453 diacylglycerol oils 560 dietary supplements Europe 596 rest of the world 597 USA 595 differential scanning calorimetry 208, 243 dioxins 589 distillation 202 docosahexaenoic acid 139, 568 docosahexaenoic acid 568 domestication of wild crops 6 echium oil 23 egg yolk phospholipids 371 eggs 577 eicosanoids 569 eicosapentaenoic acid 139 El Niño 71 emulsifiers 144, 394, 399, 403, 404 emulsions 394, 398, 401, 404, 415, 417, 419 EPA from marine sources 336, 337, 348 EPA from SCO 103 EPA see also eicosapentaenoic acid ester-ester exchange 237 esterification 137 esterification with lipase 344, 345, 351

Index esterified propoxylated glycerols 448, 449 ethanolysis 243 evening primrose oil 23 extraction 25, 85, 89, 115 fat bloom 503 non-tempered systems 505 tempered systems 504 fat crystal networks 142, 151, 153, 155 fat migration 508 fat substitutes alkyl glycoside fatty acid esters 451 carbohydrate fatty acid esters 449, 454 esterified propoxylated glycerols 448, 449 Olean® 450 Olestra 450, 456 sucrose polyesters 450 fatty acid – annual production 13 fatty acid composition high-oleic varieties 546 regular oils 546 fatty acids uncommon structures 276 fermentation process 86 filling fats 497, 506 low and non-trans 498 sensory comparisons 498 filter press 212 fish oils 587 applications 74 composition 58 epidemiological studies 56 fatty acids 59, 61 oxidative stability 68 phospholipids 63 pollutants 60, 67 production levels 70 production procedures 64 major producers 599 fish stocks 73 foam formation 424 foam stability 424 fondant 501 food safety 513 FOSHU regulations 591 fractal dimensions 156, 158 effect of cooling rate 163 effect of shear 163, 164

605

fractional distillation 202 fractionated oils in frying 527 fractionation 193, 201, 203, 325 crystallization 208 beef tallow 225 chicken fat 227 cocoa butter 222 hydrogenated soybean oil 222 lard 226 milk fat 223 palm oil 208, 217 palmkernel oil 221 separation 211 fractionation – milk fat 480 fractionation of hydrogenated oils 182 fractionation of milk fat 409 frozen desserts 472 frying fats and oils 180, 518, 546 frying oils chemical changes 520 groundnut oil 524 heat transfer 519 high-oleic oils 524 hydrogenated oils 524 lauric oils 523 mass transfer 519 modified frying oils 526 oil uptake 519 palm oil 523 quality control 530 rice bran oil 524 specifications 530 the frying process 518 genomics 288 ghee 473 GLA: see linolenic acid (γ) GLA-oils 90, 92 glycerolysis 349 chemical 257 enzymatic 256, 258, 260 golden rice 297 gourmet oils 552 groundnut oil 21 groundnut oil in frying 524 hazelnut 24 health issues 446, 512, 540, 596 monounsaturated acids 544 PUFA 543

606 Index saturated fatty acids 542 trans fatty acids 541 heat treatment of emulsions 415 hemp 24 high stability oils 184 high-oleic oils in frying 524 HLB values 403, 404 homogenisation of emulsions 417 hydrogenated oils in frying 524 hydrogenated soybean oil 176, 177, 190, 191 hydrogenation 173 hydrogenation – isomerization 187 hydrogenation – mechanism 187 hydrolysis 374 hydrolysis of phospholipids 378 hydroperoxide decomposition 134 hydroperoxide structure 132 hydroxylation of phospholipids 379 ice cream 501 identity preservation 292 illipe butter: see Borneo tallow infant formula 476, 570 interesterification 137, 193, 234, 239, 325 chemical 234, 235 enzymatic 234, 239, 245 interesterified oils in frying 527 intersolubility 206 IP see identity preservation kjokum butter 23 lactose intolerance 274, 464 lard 226 lard see also pig fat lauric oils 19 lauric oils in frying 523 linola 24 linoleic acid – metabolism 138 linolenic acid – metabolism 138 linolenic acid (γ) 22 linseed oil 21, 24 lipases to enrich PUFA 340 lipases, 1,3-specific 247 lipolysis 341 liquid-liquid extraction 203 long-chain PUFA 550 low fat foods 445

low trans oils 293 lysophospholipids 377 macadamia 24 maillard reaction 381 mango kernel stearin 23 margarine 274, 411, 549 margarine basestock 176 mayonnaise 552 meat fats 555 mechanical properties 152 melting of fat crystals 143 membrane separation 203 mesoscale structures 151 metabolism 138 microbial lipids 80 microbial oils in foods 567 milk microbiological quality 400 milk fat 223 fatty acid composition 47, 312, 314 food use 49 general 44 lipid composition 46 recovery from milk 48 milk fat composition – changed by cholesterol removal 481 cow selection 479 feeding 478 fractionation 480 milk fat globule 394 milk fats – altered 462 milk fatty acid composition 397, 405 milk lipid composition 396 milk phospholipids 371 minor vegetable oils 22 MLM glycerol esters 354 modified fatty acid composition 193 modified frying oils additives 528 fractionation 527 hydrogenation 526 interesterification 527 modified oilseed crops 284 Mortierella oils 94 mouth feel 435 Mucor circinelloides 90 natural antioxidants in speciality oils 554

Index neutralization 256 nigella sativa 24 NMR – for solid fat content 499 nucleation 145, 150 nutritional properties 3 obesity 446 Oil of Javanicus 90 oleaginous yeasts 82 Olean® 450 Olestra 450, 456 Olibra® 453 olive oil 21 omega-3 acids 308, 327 omega-3 acids in plants 294 omega-3 market 590, 592, 593 oxidation – analytical 123 oxidative stability 547 palm mid-fraction 23, 144, 218 palm oil 15 palm oil in frying 523 palm olein 16, 218 palm stearin 16, 218 palmkernel oil and its stearins 221 palmkernel oil: see lauric oils partial hydrogenation 128 particle counting 156 passion fruit 24 Pasteurization 416 PCBs 589 peanut oil: see groundnut perilla 24 pharmaceuticals 598 phase diagrams 207 phosphlipases 251, 374 phospholipids 119, 600 antioxidants 384 browning reactions 382 chemical structure 369 cosmetic use 385 emulsifiers 383 enzymatic acidolysis 250, 252 feed use 385 functionality 383 hydrogenation 378 hydrolysis 374 hydroxylation 380 industrial production 371 industrial separation 372

607

laboratory preparation 372 maillard reaction 381 modification 374 non-edible uses 385 nutritional value 385 oxidation 379 pharmaceutical use 385 purification 373 sources 371 transesterification 376 photo-oxidation 130, 132 physical properties 3 phytanic acid 309 phytosterols 119, 120, 121, 122, 299 pig fat 311, 317 plant breeding for plant lipids 273, 278 plant lipids designer oil crops 290 diversity 275, 278 DNA marker-assisted selection 286 genomics 288 markets 277 mass propagation 287 modified oilseeds 283 mutagenesis 280 new oil crops 278, 283 new variants 279, 281 novel fatty acids 276 plant breeding 273 tissue culture 287 transgenic crops 289 plastic fats 239 polarized light images 154, 161, 164 pollutants in fish oils 60, 67 polymorphism 148, 149, 206, 490 poultry fat 40 poultry fats 555 production figures 12 propyl gallate 136 PUFA concentrates 587 food ingredients 588 functional foods 588 major producers 599 pharmaceuticals 598 phospholipids 600 PUFA from marine sources 336, 337, 348 by lipase alcoholysis 343 by lipase esterification 344, 345, 351 by lipase ethanolysis 343 by lipase hydrolysis 341

608 Index chromatographic methods 338 producers of concentrates 362 with lipase 339 PUFA TAG concentrates 347 by acidolysis 350 by glycerolysis 349 by transesterification 349 pumpkin 24 randomisation 236 rapeseed oil 17, 116, 120 rapeseed phospholipids 371 reduced calorie lipids 444, 452 refining 85 refining – chemical 25 refining – physical 25 rendering 36 resolvins 569 rheological properties 161 rice bran oil 21 rice bran oil in frying 524 safflower 24 safflower seed oil 117, 121, 122 sal stearin 23 salad oils 178, 552 salatrim 452, 559 satiety 453 seabuckthorn oils 24 seed breeding 6 sesame oil 21 shea stearin 23 shortenings 179, 181 short-path distillation 202 single cell oils 8, 80, 84, 87, 567, 572 regulations 581 safety assessments 578 solid fat content 242, 244, 432, 493, 495, 496, 502 NMR measurement 499 solid fat index 178, 179 soybean oil 14, 116, 120 soybean oil see also hydrogenated soybean oil soybean phospholipids 371 speciality oils and fats 539 applications 545 cooking oils 546 design 545 frying oils 546

gourmet oils 552 health issues 540 margarines 549 mayonnaise 552 monounsaturated acids 545 natural antioxidants 554 PUFA 545 salad oils 552 spreads 549 structured lipids 558 spices in gourmet oils 553 spreadable butter 223 spreads 176, 179, 412, 549 stanols 299 stearidonic acid 22 sterols 118 storage modulus 160 structural TAG chemoenzymatic process 358, 361 enzymatic process 356 from fish oil 352 MLM type 354 structured lipids 246, 249, 588 structured lipids in speciality oils 558 structured phospholipids 253 sucrose polyesters 450 sugar confectionery 501 sunflower oil 18, 117, 121, 186 sunflower seed phospholipids 371 super olein 218 supercritical extraction 202 supplements 574 surfactants 394 tallow 225 TBHQ 136 technological methods of modifying fats 5 tocopherols 118, 120, 121, 122, 136, 298 tocotrienols 118, 120, 121, 122, 136 toffee 501 top olein 218 toppings 471 toxicology 455 trans acids 115 trans acids – functional properties 180 trans acids in spreads 174, 175 transesterification 349 transesterification of phospholipids 376 trans-free fats 430, 436

Index crystallisation 431, 433 functional properties 430, 436 marketing 441 mouth feel 435 nutritional constraints 437 production 437 stability (physical) 433 transgenic oil palm 295 UHT creams 421 urea complexation 338 urea fractionation 203 vegetable oils 11 vitamin E 298 wheatgerm 24

whiteners 469 winterisation 205, 214, 337 corn oil 214 cottonseed oil 214 fish oil 216 olive oil 216 soybean oil 217 sunflower oil 214 winterised salad oil 178 yeasts 82 yogurt 471 zero calorie foods 444 zero trans fats 227

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