Development and Processing of Vegetable Oils for Human Nutrition

Development and Processingof Vegetable Oils

for Human Nutrition

Copyright © 1995 AOCS Press

Development and Processingof Vegetable Oils

for Human Nutrition

Editors

Roman PrzybylskiBruce E. McDonald

Department of Foods and NutritionUniversity of Manitoba

Winnipeg, Canada

Champaign, Illinois


AOCS Mission StatementTo be a forum for the exchange of ideas, information, and experience among those with aprofessional interest in the science and technology of fats, oils, and related substances inways that promote personal excellence and provide high standards of quality.

AOCS Books and Special Publications Committee

E. Perkins, chairperson, University of Illinois, Urbana, IllinoisT. Foglia, USDA—ERRC, Philadelphia, PennsylvaniaM. Mossoba, Food and Drug Administration, Washington, D.C.Y.-S. Huang, Ross Laboratories, Columbus, OhioL. Johnson, Iowa State University, Ames, IowaJ. Lynn, Lever Brothers, Edgewater, New JerseyG. Maerker, Oreland, PennsylvaniaG. Nelson, Western Regional Research Center, San Francisco, CaliforniaF. Orthoefer, Riceland Foods Inc., Stuttgart, ArkansasJ. Rattray, University of Guelph, Guelph, OntarioA. Sinclair, Deakin University, Geelong, Victoria, AustraliaG. Szajer, Akzo Chemicals, Dobbs Ferry, New YorkL. Witting, State College, PennsylvaniaB. Szuhaj, Central Soya, Ft. Wayne, Indiana

Copyright © 1995 by AOCS Press. All rights reserved. No part of this book may be reproduced or transmitted in any form or by any means without written permission of the publisher.

The paper used in this book is acid-free and falls within the guidelines established to ensure permanence and durability.

Library of Congress Cataloging-in-Publication DataDevelopment and processing of vegetable oils for human nutrition/

editors, Roman Przybylski and Bruce E. McDonald.p. cm.

Includes bibliographical references and index.ISBN 0-935315-66-7 (alk. paper)1. Oils and fats, Edible. 2. Vegetable oils. 3. Nutrition.

I. Przybylski, Roman. II. McDonald, B.E. (Bruce Eugene), 1933– . TX407.034D49 1995 95-33314664′.3—dc20 CIP

Printed in the United States of America with vegetable oil-based inks.

00 99 98 97 96 95 5 4 3 2 1


Preface

The recommendation that consumers reduce total fat to 30 percent and saturatedfat to 10 percent of their total energy intake has had a tremendous effect on thefood industry, particularly the fats and oils industry. Other major developmentsthat have affected the edible fats and oils industry include the findings that (i)monounsaturated fatly acids are as effective as polyunsaturated fatty acids in low-ering blood cholesterol; (ii) hydrogenated fats, or more precisely the trans fattyacids found in hydrogenated fats, may have an undesirable physiological effect;and (iii) n-3 fatty acids are important dietary constituents in health and disease.Several new oilseed varieties have already been developed and many others areunder development in response to these findings. The development of noveloilseed varieties has produced a scramble among regulatory agencies to developguidelines governing the licensing and release of these new crops. An added prob-lem for governmental agencies, particularly in light of new agreements coveringthe international movement of food products, is the need to develop and stan-dardize food labeling regulations.

These developments were major factors in the decision to organize a confer-ence on the development and processing of vegetable oils for human nutrition. TheCanadian Section of the AOCS was invited to organize the conference in con-junction with its Annual Meeting on October 2–4, 1994. The Conference was asuccess thanks to the efforts of the Organizing Committee and its chairman JamesDaun, the support of sponsors and donors, and the distinguished group of speak-ers. Current nutrition issues and the contributions of processing, genetic engi-neering, and plant breeding were reviewed, as well as the role of governmentagencies in the development of novel oilseed crops.

This monograph covers all of these issues, beginning with an up-to-date cover-age of nutritional issues, followed by a discussion of current developments in pro-cessing vegetable oils for human consumption and the modification of traditionaloilseed sources by genetic manipulation. The monograph concludes with a synopsisof the regulatory requirements in Canada, the United States, and Europe for the reg-istration of novel oilseed crops and the nutrition labeling of these new oils.

As the editors, we would like to thank the speakers for their cooperation inproviding us with manuscripts. We are especially grateful to Angela Dupuis forwillingly and patiently transcribing the manuscripts to a common format and


her very significant efforts toward the success of this publication. We are gratefulfor the unique satisfaction that comes with having contributed to the knowledgeon this subject.

Roman Przybylski and Bruce E. McDonaldDepartment of Foods and Nutrition

University of ManitobaWinnipeg, Canada


Contents

Preface

Chapter 1 Food Fats and Fatty Acids in Human NutritionJoyce L. Beare-Rogers

Chapter 2 Nutrition and Metabolism of Linoleic and Linolenic Acids inHumansE.A. Emken

Chapter 3 Trans Fatty Acids in Canadian Breast Milk and DietW.M.N. Ratnayake and Z.Y. Chen

Chapter 4 Food Industry Requirements for Fats and Oils: FunctionalPropertiesT.K. Mag

Chapter 5 Hydrogenation: A Useful Piece in Solving the Nutrition PuzzleRobert C. Hastert and Robert F. Ariaansz

Chapter 6 Interesterification: Current Status and Future ProspectsSuresh Ramamurthi and Alan R. McCurdy

Chapter 7 Sources of Oilseeds with Specific Fatty Acid ProfilesW.A. Keller

Chapter 8 Production of Oilseeds with Modified Fatty Acid CompositionRachael Scarth

Chapter 9 Classification of Oils with Modified Fatty Acid Compositions as Novel FoodsFrank W. Welsh

Chapter 10 Food Labeling in CanadaIan Campbell

Chapter 11 Safety Evaluation and Clearance Procedures for New Varieties of Oilseeds in the United States and CanadaDonna Mitten, Keith Redenbaugh, and Julianne Lindemann


Chapter 1

Food Fats and Fatty Acids in Human Nutrition

Joyce Beare-Rogers

41 Okanagan Drive, Nepean, Ontario, K2H 7E9, Canada

This paper will deal principally with the fatty acids in food fats.

Total Dietary Fat

A first consideration should be the amount of fat or fatty acids in the diet. It haslong been appreciated that a caged experimental animal given a high-fat diet even-tually becomes obese. An excellent demonstration in humans showed the interac-tion of the level of fat, provided covertly in an ad libitum diet, and level ofphysical activity (1). At the lowest level of fat to maintain energy balance, physi-cal activity produced a negative energy balance. The intermediate level of dietaryfat caused a positive shift in balance with a pronounced difference between seden-tary and active individuals. At the highest level, 60 en%, both groups of individu-als had a positive energy energy balance, but the energy storage was greater ininactive individuals. Particularly within the range of usual fat intake, there is a trade-off with physical activity where the effect of fat is offset by the utilization of energy.

Energy Storage

Another aspect of fat ingestion is that appetite regulation fails to respond to fat inthe same way that it does to carbohydrate (2). Individuals tend to be insensitive tothe level of fat in a meal and are consequently apt to overeat. Excess carbohydrateis stored only to a limited extent and is then converted to fat. The cost of meta-bolic conversion is relatively high for carbohydrate and protein, but fatty acids areeasily added to stored energy. Therefore, for sedentary individuals the recommen-dation for an upper range of fat intake has been 30% of energy.

Fatty Acids for Infants

Fat and saturated fatty acids supply the energy consumed in cellular growth at cer-tain stages of life, particularly infancy. Most human milk provides fat in which thetotal proportion of saturated fatty acids shorter than 18 carbon atoms is approxi-mately equal to the monounsaturated fatty acids, principally oleic (3–5). Since thefat in human milk is 45–55% of the total dietary energy, the saturated componentprovides about 18% of the energy, considerably more than the ceiling of 10% that


is frequently recommended. Questions about the fatty acid composition of infantformula have usually revolved around the essential fatty acids and the role thatdocosahexaenoic acid plays in neural membranes. Here it is important that the n-6fatty acids be considered along with the n-3 fatty acids.

Of course, the maternal diet is the main source of fatty acids for the fetus.Koletzko reported that the trans fatly acids in infant blood were inversely corre-lated with the long-chain n-3 and n-6 fatty acids (6). This apparent interferencewith the conversion of essential fatty acids was therefore thought to involve thedesaturases, but placental receptors also may be sites of influence.

Lipoproteins

The greatest debate about dietary fatty acids revolves around their effects onblood lipoproteins, that is, the concentration of high-risk, low-density lipoproteins(LDL), the ratio of LDL to HDL (not just total cholesterol, but the distribution ofthe particles in which it is carried), and the concentration of Lp(a) that limits plas-min production and promotes clotting and vascular smooth muscle proliferation.The saturated fatty acids, although frequently considered together, do have differ-ent degrees of influence on the concentration of LDL. Laurie and myristic acids,which are usually found in the same oils, are more hypercholesterolemic thanpalmitic acid (7,8). Palmitic acid is more hypercholesterolemic than stearic acid (9),which is considered neutral in terms of modifying cholesterol levels. However,stearic acid may not be neutral in thrombotic tendency or in its effect on arrhythmia.

Whether a vegetable oil is considered hypo- or hypercholesterolemic dependsupon the reference oil. Thus, palm oil is hypocholestcrolemic with respect tococonut oil but hypercholesterolemic with respect to corn oil (10).

Prediction by Equations

The early equations of Keys et al. and Hegsted et al. emphasized that a change inplasma cholesterol was proportional to twice the amount of energy supplied bysaturated fatty acids minus the amount of energy supplied by polyunsaturatedfatty acids plus a small factor for dietary cholesterol (11, 12). In all later regres-sion lines, the greatest adverse effect on plasma cholesterol levels was also asso-ciated with the intake of saturated fatty acids.

The attempts to reduce the consumption of saturates have sometimes led toextreme proposals, the idea apparently being that since high amounts are bad,intermediate amounts must be barely tolerable and low amounts must be best. Thequest for extremely low dietary levels of saturated fatty acids seems futile becauseif there are insufficient dietary saturated fatty acids to occupy the 1-position ofmembrane phospholipids, they have to be synthesized by the body.

The effect of linoleic acid in reducing plasma cholesterol is thought to be non-linear, plateauing at about 5% of energy, and having a range of 3–10% according to


an individual’s responsiveness (13). Below the so-called threshold, an increase of3% of energy as linoleic acid decreased plasma cholesterol by 35 mg/dL. A simi-lar change in plasma cholesterol above the threshold required 13% of energy aslinoleic acid. In this range, there is relative insensitivity to changes in dietary fattyacids, although the lowest concentrations of blood cholesterol occurred with highintakes of linoleic acid (14). The fact that individuals have different thresholdshelps to explain some of the disparity in experimental results.

High Intakes of Oleic Acid

The reported equivalence of oleic acid and linoleic acid in reducing LDL-choles-terol may have been related to high thresholds for dietary linoleic acid (15).Linoleic acid appeared to reduce HDL-cholesterol, but the high intake of linoleicacid in this study would be difficult to attain or maintain with ordinary foods. Inanother study without a group fed a high level of linoleic acid, Grundy et al. showedthat a diet high in oleic acid was preferable to a low-fat diet (high carbohydrate) insustaining HDL-cholesterol while decreasing LDL-cholesterol (16), Test fats con-sisting of butterfat, beef fat, cocoa butter, and olive oil produced no differences inHDL-cholesterol (17). Low-density lipoprotein cholesterol was highest with butter-fat and significantly lower with cocoa butter, indicating that the position of the fattyacids on the acylglycerols was important. It appears that in at least some situations,saturated fatty acids in the 2-position are the most hypercholesterolemic.

α-Linolenic Acid and Postinfarct Patients

A comparison was made between the usual postinfarct prudent diet and aMediterranean diet that used a margarine made from canola oil (18). Improvedmortality after a first myocardial infarction was attributed to the increased intakeof α-linolenic acid. Although this observation is encouraging for canola oil, itmust be remembered that many dietary features differed between the two dietarygroups, and that more definitive work is required.

Trans Fatty Acids and Lipoproteins

The impact of dietary fats containing trans monounsaturated fatty acids, as deter-mined in Trappist monks, has stood the test of time (19). In the presence of dietarycholesterol, the trans fatty acids were associated with serum cholesterol levels thatwere higher than those obtained with oleic acid and slightly lower than thoseobtained with a mixture of lauric and myristic acids.

The much quoted paper of Mensink and Katan (20), was the first to show thattrans monounsaturated acids increased LDL-cholesterol and decreased HDL-cho-lesterol, worsening the LDL/HDL ratio. Although this 3-week study was criticizedfor the high level of trans fatty acids (11% of dietary energy) and the means of


production by chemical isomerization rather than by commercial hydrogenation,these initial findings have been confirmed. The positional trans isomers used inthe study were similar to those found in partially hydrogenated soybean oil; the posi-tional cis isomers had one type, the 8-octadecenoic acid, that was higher than ordi-narily found (21), but no significance has been attached to it. Another study fromthe same laboratory (22), had a lower level of trans fatty acids, 7.7% of dietaryenergy instead of 11%. The results of the two separate studies suggested a dose-response to trans fatty acids.

The finest precision yet seen in the determination of lipoprotein levels appeared inJudd et al. (23). The levels of trans fatty acids tested for 3 and 6 weeks were 3 and 6%of dietary energy. Unfortunately, only the data from the longer period were published;data from 3 weeks would have facilitated comparison with the results obtained in thestudy of Mensink and Katan. Again, the trans fatty acids were associated with increasedLDL-cholesterol and decreased HDL-cholesterol when compared with oleic acid.

It must be remembered that the original purpose of these experiments was todetermine how trans fatty acids should be regarded, given that saturated fattyacids were already designated as hypercholesterolemic. Also, products promotedas being low in saturated fatty acids were sometimes high in trans fatty acids. Atissue is whether saturated fatty acids should be replaced by trans fatty acids. Moresensibly, both should be reduced in the total diet.

The average intake (50th percentile) of any substance gives no indication ofthe risk to vulnerable individuals. Information on at least the 90th percentile ofintake and the associated food patterns is required. Investigations of trans fattyacids should therefore provide estimates of possible human exposure along with intakeguidelines for essential fatty acids, particularly for pregnant and lactating women.

The assessment of intake of trans fatty acids loses accuracy when a part of thediet is self-selected. For many foods, the fatty acid composition is not accuratelyknown, and the possible combinations are considerable. The most reliable data onfatty acid consumption are obtained from the analysis of all foods given to the par-ticipants of a study. Values for the coefficients of variation of total cholesterol forexample, calculated from papers dealing with dietary trans fatty acids (20,22–27), aregiven in Table 1.1. Flynn et al. tested margarine versus butter with two eggs/day in anotherwise self-selected diet. Judd et al., Lichtenstein et al., Mensink and Katan, andZock and Katan provided all foods to the test subjects. Wood et al. supplied test fatsthat were one-half of the total fat in diets that were rotated every 6 weeks.

The precision of the experiment of Judd et al. (23) stands out, partly becauseof the control in subject selection and in the analytical procedures. The selectionof subjects for this study raised questions about the general applicability of theresults. The subjects had normal levels of all blood lipids, were free of any dis-ease, and maintained their usual exercise program without gaining weight, in spiteof a mean energy consumption of 3227 kcal/day for the men and 2025 kcal/day forthe women. These healthy athletic subjects did not reflect the larger community, and


might be expected to represent a group with a well-regulated cholesterol metabo-lism. They constituted such a finely tuned bioassay that as little as 3% of energyfrom trans fatty acids produced a statistically significant result.

Another effect of trans fatty acids on lipoproteins pertains to the risk factorof Lp(a) involved in thrombogenesis. Investigators in Australia and TheNetherlands found Lp(a) to be elevated in persons consuming trans fatty acids(28,29). In North America, where this effect has not been observed, the situationwill have to be clarified with the most sensitive methods available.

Trans Fatty Acids in Epidemiological Studies

In an analysis of tissue fatty acids of individuals who died from ischemic heartdisease, the adipose tissue fat had increased trans fatty acids and decreasedshorter chain fatly acids (30, 31). It was concluded that the victims had consumedmore hydrogenated fat and less ruminant fat than the controls. Also, in patientsundergoing coronary angiography, the level of trans fatty acids was 1.38% versus1.11% of fatty acids in controls (32). Such results were said to be consistent withthe hypothesis that dietary trans fatty acids are a risk factor. Recent studies (33,34), however, raise questions with this hypothesis, although issue also has beentaken with the results and conclusions of these studies (35).

More controversial estimates of exposure to trans fatty acids came from semi-quantitative, food-frequency questionnaires. Answers to questions about “howoften over the previous year” a given portion of a specified food had been con-sumed became the source of data. Clinical studies in which dietary variables areknown and controlled exhibit a scientific rigor that is unfortunately lacking in theresponses to semiquantitative questionnaires.

In adult men (mean age 62 yr; range 43–85 yr) assessed by a food-frequencyquestionnaire, total fat was given as 60 g/day and the trans fatty acid intake as 2.1g at the 10th percentile and 4.9 g at the 90th percentile (36). These low values areinconsistent with other data. Nevertheless, the energy-adjusted intakes of transfatty acids were reported to be positively correlated with LDL-cholestcrol andinversely correlated with HDL-cholesterol.

TABLE 1.1Precision in Human Studies on Trans Unsaturated Fatty Acids

Foods Foods self- Coefficient of Study provided selected variationa %Flynn et al. + 19.3Judd et al. + 4.7Lichenstein et al. + 10.7Mensink and Katan + 15.3Wood et al. + 14.6Zock and Katan + 14.7aFor total cholesterol.Sources: Mensink and Katan (21), Zock and Katan (22), Judd et al. (23), Flynn et al. (24), Lichenstein el al. (25), andWood et al. (26,27).


In the Nurses’ Health Study (37), data on consumption came from the sametype of questionnaire, with average values used in the assessment for such foodsas margarine, cookies, biscuits, cake, and white bread. The intake of trans fattyacids was reported to have varied from 2,4–5.7 g/day or 1.3–3.2% of energy, andto be correlated with the risk of cardiovascular disease.

Another paper claiming an association between the intake of trans fatty acidsand the risk of cardiovascular disease involved questionnaires administered 8 weeksafter patients had been discharged from hospital after a first myocardial infarction(38). The patients were matched with residents of the same town. The average con-sumption of trans fatty acids was 1.5% of energy for men and 1.7% of energy forwomen. These levels were about one-half of that calculated for the average transfatty acids in the American diet (39). The relative risk of myocardial infarction foreach quintile of energy-adjusted intake of trans fatty acid was 1.0, 0.89, 0.52, 0.93,and 2.28, respectively; that is, only the last value exceeded the first. The third quin-tile appeared to be the best. Overall, the epidemiological studies emphasize the needfor additional research on the physiological effects of trans fatty acids and that, inthe interim, prudence be exercised in the consumption of these fatty acids.

Idealized Dietary Fat

Biotechnologists have challenged nutritionists to provide them with the fatty acidprofile of the ideal vegetable oil. What is important is the lipid content of the totaldiet. For one oil to have an impact, it would have to be an appreciable contributorto the dietary fat. This does happen with some types of food patterns, but in mostmixed diets there is some trade-off between foods high and low in a particularfatty acid. It is the ultimate blend that counts.

For the total fatty acids in an adult diet, the saturated fatty acids (mostlypalmitic) could be 10–25%, linoleic acid could be 10–20%, α-linolenic could beabout 2%, and the rest could be oleic acid. The only virtue of a very low level ofsaturated fatty acids in a vegetable oil would be to dilute those from other sources.

Since food preparation involves fats used in different ways, there might be anideal salad oil, an ideal spread, an ideal cooking fat, and so on. To propose a fatty acidcomposition for an ideal vegetable oil, one would need information about the otherfoods to be consumed. It is the total dietary fatty acids that are important in nutrition.

References

1. Stubbs, R.J., and A.M. Prentice, Am. J. Clin. Nutr. 62: 330–337 (1995).2. Flatt, J.P. in Obesity, edited by P. Bjorntorp and B.N. Brodoff, J.P. Lippincott Co.,1992,

pp. 100–116.3. Sanders, T.A.B., F.R.Ellis, and J.W.T. Dickerson, Am. J. Clin. Nutr. 31: 805 (1978).4. Carlson, S.E., P.G. Rhodes, and M.G. Ferguson, Am. J. Clin. Nutr. 44: 798 (1986).5. Chen, Z.-Y., G. Pelletier, R. Hollywood, and W.M.N. Ratnayake, Lipids, in press.6. Koletzko, B., Ada Paed. 81: 302 (1992).


7. McGandy, R.B., D.M. Hegsted, and L.M. Meyers, Am. J. Clin. Nutr. 23: 1288 (1970).8. Sundram, K., K.C. Hayes, and O.H. Siru. Am. J. Clin. Nutr. 59: 841 (1994).9. Bonanome, A., and S.M. Grundy, N. Eng. J.Med. 318: 1244 (1988).

10. Kris-Etherton, P.M., J. Derr, D.C. Mitchell, V.A. Mustad, M.E. Russel, E.T. McDonell, D.Salabsky, and T.A. Pearson, Metabolism 42: 121 (1993).

11. Keys, A., Anderson, J.T., and F. Grande, Lancet 2: 959 (1957).12. Hegsted, D.M., R.M. McGandy, M.L. Myers, and F.J. Stare, Am. J. Clin. Nutr. 17: 281 (1965).13. Hayes, K.C. and P. Kosla, Fed. Am. Soc. Exp. Biol. J. 6: 2600 (1992).14. Hegsted, D.M., L.M. Ausman, J.A. Johnson, and G.E. Dallal, Am. J. Clin. Nutr. 57: 875

(1993).15. Mattson, F.H., and S. Grundy,.J. Lipid Res. 26: 194 (1985).16. Grundy, S.M., L. Florentin, D. Nix, and M.F. Whelan, Am. J. Clin. Nutr. 47: 965 (1988).17. Denke, M.A., and S.M. Grundy, Am. J. Clin. Nutr. 54: 1036 (1991).18. De Lorgeril, M., S. Renaud, N. Mamelle, P. Salen, J.-L. Martin, I. Monjaud, J. Guidollet,

P. Touboul, and J. Dclaye, Lancet 343: 1454 (1994).19. Vergroesen, A.J., and J.J. Gottenbos, in The Role of Fats in Human Nutrition, edited by

AJ. Vergroesch, Academic Press, London, 1975, pp. 1–32.20. Mensink, R.P., and M.B. Katan, N. Eng. J. Med. 323: 429 (1990).21. Mensink, R.P., and M.B. Katan, N. Eng. J. Med. 324: 339 (1991).22. Zock, P.L., and M.B. Katan, J. Lipid Res. 33: 399 (1992).23. Judd, J.T., B.A. Clevidence, R.A. Muesing, J. Wittes, M.E. Sunkin, and J.J. Podczasy, Am.

J. Clin. Nutr. 59: 861 (1994).24. Flynn, M.A., G.B. Nolph, G.Y. Sun, M. Navidi, and G. Krause, J. Am. Coll. Nutr. 10: 93

(1991).25. Lichtenstein, A.H., L.M. Ausman, W. Carrasco, J.L. Jenner, J.M. Ordovas, and E.J.

Schaefer, Arter. Throm. 13: 154 (1993).26. Wood, R., K. Kubena, B. O’Brien, S. Tseng, and G. Martin, J. Lipid Res. 34: 1 (1993).27. Wood, R., K. Kubena, S. Tseng, and G. Martin, J. Nutr. Biochem. 4: 286 (1993).28. Nestel, P.J., M. Noakes, G.B. Belling, R. McArthur, P. Clifton, E. Janus, and M. Abbey, J.

Lipid Res. 33: 1029 (1992).29. Mensink, R.P., P.L. Zock, M.B. Katan, and G. Hornstra, J Lipid Res. 33: 1493 (1992).30. Thomas, L.H., and R.G. Scott, J. Epid. Comm. Health 35: 251 (198 I).31. Thomas, L.H., J.A. Winter, and R.G. Scott, J. Epid. Comm. Health 37: 22 (1983).32. Siguel, E.N., and R.H. Lerman, Am. J. Card. 71: 916 (1993).36. Troisi, R., W.C. Willet, and S.T. Weiss, Am. J. Clin. Nutr. 56: 1019 (1992).33. Aro, A., F.M. Kardinaal, I. Salminen, J.D. Kark, R.A. Riemersma, M. Delgardo-

Rodriquez, J. Gomez-Aracena, J.K. Huttunen, L.Kohlmeier, B.C. Martin-Moreno, V.P.Mazaev, J. Ringstad, M. Thamm, P. van’t Veer, and F.J. Kok, Lancet 345: 273 (1995).

34. Roberts, T.L., D.A. Wood, R.A. Riemersma, P.J. Gallagher, and F.C. Lampe, Lancet 545:278 (1995).

35. Letters to Editor, Lancet 345: 1107 (1995).37. Willet, W.C, M.J. Stampfer, J.E. Mason, G.A. Colditz, F.E. Speizer, B.A. Rosner, L.A.

Sampson, and C.H. Hennekens, Lancet 341: 581 (1993).38. Ascherio, A., C.H. Hennekens, J.E. Buring, C. Master, M.J. Stampfer, and W.C. Willet,

Circulation 89: 94 (1994).39. Hunter, J.E., and T.H. Applewhite, Am. J. Clin. Nutr. 54: 363 (1991).


Chapter 2

Nutrition and Metabolism of Linoleic and LinolenicAcids in Humans

E.A. Emken

USDA1, ARS, NCAUR, 1815 N. University Street, Peoria, illinois, 61604, USA.

Introduction

The importance of the early observations reported in 1956 by Sinclair (1), thatEskimos had little or no cholesterol deposits in their coronary arteries and a lowincidence of coronary heart disease because of the n-3 fatty acids in their diet, waslargely ignored by public health and medical organizations. In fact, Sinclair’s the-ory was termed imaginative by Key’s, who was a leading authority on heart dis-ease and diet (2). A dramatic change in the health and medical community’sperception of the nutritional importance of n-3 fatty acids occurred when Bang etal. reported in 1971 that the high intake of n-3 fatty acids from fish was a key fac-tor in the low mortality rate from coronary heart disease observed in GreenlandEskimo populations (3). Since those early times, there has been a growing accu-mulation of evidence that indicate n-3 long-chain fatty acids (LCFA) are associ-ated with various antiatherogenic properties and a number of other healthbenefits, although this issue is still controversial (4–6).

Biological Properties

It is now appreciated that n-3 and n-6 fatty acids have very different physiologi-cal effects. One reason is the difference in the physiological properties of theeicosanoids produced by the lipoxygenase and cyleooxygenase pathways from20:5n-3, 20:3n-6, and 20:4n-6. In most cases, in vitro studies have shown that thephysiological effects of the 1- and 3-series of prostaglandins formed from 20:3n-6and 20:5n-3 are opposite the effects of the 2-series of prostaglandins formed from20:4n-6. These results have led to the hypothesis that a balance between the vari-ous eicosanoids and their n-6 and n-3 precursors is necessary to regulate manyphysiological functions.

These observations for the n-3 and n-6 LCFA have raised several questionsconcerning the nutritional importance of linolenic acid present in plant sources. A

1Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the stan-dard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others thatmay also be suitable.

Portions of this paper have been published in the Proceedings for the Scientific Conference on ω-3 Fatty Acids inNutrition, Vascular Biology and Medicine, April 17–19, 1994, Houston, Texas, American Heart Association.


basic question is whether the conversion of linolenic acid (18:3n-3) to eicosapen-taenoic acid (20:5n-3) and docosahexaenoic acid (22:6n-3) in humans has quanti-tative importance. Of practical concern is whether linolenic acid from plantsources is a viable alternative to dietary sources containing preformed n-3 LCFA.

Requirements

Evidence from animal studies indicates that competition between fatty acids fromthe n-3 and n-6 families influences the incorporation of these fatty acids into tis-sue lipids and mediates their biological effects (4–6). These results raised thequestion of whether the actual amounts of linoleic acid (18:2n-6) and linolenicacid (18:3n-3) or the 18:2n-6/18:3n-3 ratio in the diet has more nutritional impor-tance. It is difficult to determine exactly what the best 18:2n-6/l8:3n-3 ratio forthe human diet is. Examples of some of the 18:2n-6/18:3n-3 ratios recommendedare 6:1–10:1 (7), 5:1 (8), 4:1–6:1 (9). An interesting recent study reported that ratsfed diets with a 4:1–5:1 ratio of 18:2n-6 to 18:3n-3 were smarter, healthier, andtougher than rats fed diets with an n-6 to n-3 ratio of 3:1 or 6:1 (10). The estimatesgiven in Table 2.1 for a hypothetical diet provide some guidance for the actualamounts of dietary 18:2n-6 and 18:3n-3 required to meet essential fatty acid rec-ommendations (11–16).

Metabolism and Effect of Diet

Experiments with animal models have provided most of the information on theeffect of varying the balance between 18:2n-6 and 18:3n-3 (4–6,17–18). Studieswith radioisotope–labeled substrates have been particularly useful for investigating

TABLE 2.1 Estimated Recommendations for Essential Fatty Acids Translated for 2400 Kcal–Based DietContaining 90 g (34% Energy) of Total Fat

Total calories Total fatFatty acid (%) (g) (%) ReferenceLinoleic acid

Adult/infant 2–3 5–8 6–9 11Pregnant mother 4.5 12 13 11Lactating mother 6.0 16 18 11Adult 2.4 6.4 7.1 12

Linolenic acidAdult 1.0 2.7 3.0 13Infant 2–3 — — 11Adult 0.3 0.8 0.9 14

20:5n-3 plus 22:6n-3Adult 0.13 0.3–0.4 0.4 15,16Adult 0.27 0.7 0.8 13

Source: Dietary Fats and Oils in Human Nutrition (11).Bourre et al. (12,14), Simopoulos (12), and Bjerve et al. (15,16).


the oxidation and conversion of 18:2n-6 to 20:4n-6 and 18:3n-3 to 20:5n-3 and 22:6n-3 (4–6,19–21). By contrast, experiments in humans using isotope-labeled n-6 and n-3fatty acids are limited. Results for the conversion of 18:2n-6 in vitro have beenreported for human liver microsomes (22–23) and human leukocytes (24). In vivo datahave been published for one study with deuterium-labeled 20:3n-6 (25), two studieswith 14C-labeled l8:2n-6 (26–27) and two studies with deuterated 18:2n-6 (28–29).

We have recently reported results that directly compare the metabolism ofdeuterium-labeled linolenic acid and linoleic acid in young adult male subjectsthat had been previously fed diets containing two different levels of linoleic acid(30). The results were used to address the question of whether an increase in dietary18:2n-6 intake influences incorporation and desaturation of 18:3n-3 and l8:2n-6.

The experimental design consisted of feeding four subjects a triacylglycerol(TAG) mixture containing both deuterated 18:2n-6 (3.0–3.5 g) and 18:3n-3(3.0–3.5 g). Three additional subjects were fed a deuterated TAG mixture that con-tained 2.2 g of deuterated 18:3n-3 as the only polyunsaturated fatty acid. In addi-tion to labeled linoleic acid and linolenic acid, the mixtures of deuterated fatscontained 2 or 3 of the following deuterated fatty acids: 16:0, 18:0, or 18:1. Thedeuterated TAG mixtures were fed after the subjects had fasted for 12 hr. Bloodsamples were collected over a 48-hr period. Methyl esters of the plasma lipidswere analyzed by gas chromatograph-mass spectrometry methods (31).

The subjects were fed control diets for 12 days prior to being fed the deuter-ated TAG mixtures. The composition of the control diets provided 35–36% ofcalories from fat, 43–44% from carbohydrates, and 21% from protein. The satu-rated fat (SAT) diet contained 15.1 g 18:2n-6 and 1.9 g 18:3n-3 (n-6/n-3 ratio = 8;P/S = 0.35) and the polyunsaturated fat (PUFA) diet contained 29.8 g of l8:2n-6and 1.0 g 18:3n-3 (n-6/n-3 ratio = 30; P/S = 0.85). The amounts of 18:2n-6 in thediets were chosen to bracket the 21 g of l8:2n-6 estimated for a typical U.S. diet(n-6/n-3 ratio = 11; P/S = 0.59) [32].

Incorporation of Fatty Acids into Body Lipids

Results for the chylomicron triglyceride samples showed that the deuterated18:2n-6 to 18:3n-3 ratio in the chylomicron TAG samples was slightly lower (ca.8%) than for the 18:2n-6 to 18:3n-3 ratio in the mixture fed. This difference indi-cates that 18:3n-3 may be absorbed slightly more efficiently than 18:2n-6, but thedifference was not significant. Differences between subjects were relatively smallfor the concentrations of deuterated l8:2n-6 (range 17.1-19.4 µg/mL) and l8:3n-3(range 18.5–23.8 µg/mL) in the chylomicron TAG samples. These results indicatethat the fatty acid composition of the prefed diets had no significant effect onabsorption of 18:2n-6 and 18:3n-3.

Examples of time course curves for incorporation of deuterated l8:2n-6 and18:3n-3 into plasma phosphatidylcholine (PC) are plotted in Figure 2.1.


Qualitatively, these curves illustrate that phosphatidylcholine acyltransferase ismore selective for 18:2n-6 than 18:3n-3. The mean values for the integrated areas ofthe time course curves for plasma PC samples from the subjects from the PUFA dietgroup were 363 ± 52 µg/mL 18:2n-6 and 58.5 ± 42 µg/mL 18:3n-3. Mean plasmaPC values for subjects from the SAT diet group were 600 ± 6.5 µg/mL 18:2n-6 and66.0 ± 21.7 µg/mL 18:3n-3. These results indicated that the higher 18:2n-6 contentof the PUFA diet reduced the amount of deuterated 18:2n-6 incorporated (P <0.02) but not the amount of 18:3n-3 incorporated into plasma PC. This difference

Fig. 2.1. Examples of time course curves for incorporation of deuterated 18:2n-6 and 18:3n-3into plasma phosphatidylcholine samples from male subjects fed diets containing 15 g (SATdiet) and 30 g (PUFA diet) linoleic acid. Source: Proceedings for the Scientific Conferenceon ω-3 Fatty Acids in Nutrition, Vascular Biology and Medicine, April 17–19, 1994,Houston, Texas, American Heart Association.


in the ratio of deuterated 18:2n-6 to 18:3n-3 concentrations in plasma PC indicatedthat phosphatidylcholine acyltransferase is six to nine times more selective for18:2n-6 than 18:3n-3.

The mean concentrations for deuterated 18:2n-6 and 18:3n-3 in plasma TAGare compared in Figure 2.2 to concentration data for the deuterated 16:0, 18:0, and18:1 fatty acids that were also part of the mixtures of deuterated TAG fed to thesesubjects. The general pattern for the deuterated fatty acids incorporated arc simi-lar for subjects fed the SAT and PUFA diets. However, the concentration for the18-carbon fatty acids were consistently lower for the subjects fed the PUFA diet.

Concentration data for total plasma lipids for each subject are compared in Figure2.3. The total lipid data show an overall preferential (ca. threefold) incorporation of18:2n-6 relative to l8:3n-3. The higher 18:2n-6 content of the PUFA diet reduced theincorporation of deuterated 18:2n-6 and 18:3n-3 by about 40%. The combined TAG,PC, and total lipid data suggest the possibility that the higher 18:2n-6 content of thePUFA diet increased fatty acid oxidation by about 30%, which is reasonably consis-tent with the 9% increase in fat oxidation (based on use of 18O water methods) when adiet with a P/S ratio of 1.65 was fed in place of a P/S 0.24 ratio diet (33).

The plasma TAG and total lipid data in Figures 2.2 and 2.3 provide evidencethat incorporation of deuterated 18:2n-6 and 18:3n-3 in the major lipid classes were

Fig. 2.2. Concentration (µg/mL plasma) of deuterated fatty acids in plasma triacylglycerolsamples from subjects fed diets containing 15 g (SAT diet) and 30 g (PUFA diet) linoleic acid.Bars indicate high and low values. For the unsaturated fatty acids, the SAT vs. PUFA diet dataare significantly different (P < 0.05). Source: Proceedings for the Scientific Conference onω-3 Fatty Acids in Nutrition, Vascular Biology and Medicine, April 17–19, 1994, Houston,Texas, American Heart Association.


significantly depressed by increased dietary 18:2n-6 intake. Why do dietary 18:2n-6 levels influence both the amount of the deuterated 18:2n-6 and 18:3n-3 incorpo-rated into plasma lipids and the amount converted to n-6 and n-3 LCFA metabolites?A possibility consistent with fatty acid oxidation data is that a larger portion of thedeuterated l8:2n-6 and 18:3n-3 was diverted into the ß-oxidation pathway whendietary 18:2n-6 levels were increased (33). Higher oxidation percentages wouldresult in a general reduction of the concentration of deuterated fatty acids in plasmalipids which, in turn, would reduce the amount of 18:2n-6 and l8:3n-3 available forconversion to LCFA metabolites. An explanation for why 18:2n-6 increases fattyacid oxidation is that dietary 18:2n-6 reduces acyltransferase activity by reducingthe synthesis of the mRNA, that codes for synthesis of the acyltransferase enzymes(20). Reduction in acyltransferase activity could allow a larger portion of the fattyacid pool to be diverted into the ß-oxidation pathway. A general reduction in theincorporation of non–n-6 and n-3 deuterium-labeled fatty acids (16:0, 18:0, and18:1) that were fed to these subjects at the same time was also observed. This obser-vation is consistent with the possibility of a general nonselective increase in fattyacid oxidation or storage in tissues when 18:2n-6 intake is increased.

Desaturation-Elongation of 18:2n-6 and 18:3n-3

Concentration data for the individual n-3 and n-6 LCFA metabolites of 18:3n-3and 18:2n-6 are shown in Figure 2.4. The concentration of all the individual n-3

Fig. 2.3. Concentration (µg/mL plasma) of deuterated 18:2n-6 and 18:3n-3 in plasma total lipidsamples from subjects fed diets containing 15 g (SAT diet) and 30 g (PUFA diet) linoleic acid. P< 0.05 for the 18:3n-3 SAT versus PUFA diet means. P < 0.17 for the 18:2n-6 SAT vs. PUFA dietmeans. Source: Proceedings for the Scientific Conference on ω-3 Fatty Acids in Nutrition,Vascular Biology and Medicine, April 17–19, 1994, Houston, Texas, American Heart Association.


and n-6 LCFA metabolites were consistently lower for subjects that were prefedthe high 18:2n-6 (PUFA) diet. Variability between subjects for the concentrationof individual n-3 and n-6 LCFA metabolites was fairly large. However, when sumsfor the various n-3 and n-6 metabolites were compared (Figure 2.5), the variabil-ity between subjects was much smaller. The variability between the concentrationdata for the individual n-3 and n-6 LCFA metabolites indicates a considerable sub-ject-dependent difference in the rate of conversion of the deuterated 18:2n-3 and18:3n-3 to the major metabolites (20:4n-6, 20:5n-3, and 22:6n-3).

Concentration data for sums of the n-3 and n-6 LCFA metabolites from indi-vidual subjects along with the means for subjects fed the SAT and PUFA diets arecompared in Figure 2.5. These results demonstrate that conversion of 18:3n-3 ton-3 LCFA metabolites was considerably higher (ca. 3.7 times) than conversion of18:2n-6 (P < 0.001) and that dietary 18:2n-6 significantly reduced (P < 0.01 for 18:3n-3 and P < 0.09 for 18:2n-6) total conversion (ca. 68%) of both 18:2n-6 and 18:3n-3.

The concentration data shown in Figure 2.5 can be converted to percent con-version data by dividing the n-3 LCFA metabolite data by the total for 18:3n-3plus n-3 LCFA metabolites, Percent conversion data for 18:2n-6 can be calculatedin a similar manner. The results are shown in Figure 2.6. The average percent con-version was about 40% lower for l8:3n-3 and 56% lower for l8:2n-6 when the sub-jects were fed the diet enriched in 18:2n-6. Expression of the µg/mL data aspercent data distorts the conversion data because of the large difference betweenthe amount of 18:3n-3 and 18:2n-6 incorporated into plasma lipids due to the highselectivity for 18:2n-6 discussed earlier.

Fig. 2.4. Concentration (µg/mL) of individual deuterated n-3 and n-6 long-chain fatty acidmetabolites in plasma total lipids from subjects fed diets containing 15 g (SAT diet) and30 g (PUFA diet) linoleic acid. Bars indicate high and low values. Source: Proceedings forthe Scientific Conference on ω-3 Fatty Acids in Nutrition, Vascular Biology and Medicine,April 17–19, 1994, Houston, Texas, American Heart Association.


Fig. 2.6. Percent of n-3 and n-6 long-chain fatty acid metabolites in plasma total lipids fromsubjects fed diets containing 15 g (SAT diet) and 30 g (PUFA diet) linoleic acid. For SATversus PUFA, 18:3n-3 means (P < 0.07) are significantly different. Means for 18:2n-6 are notsignificantly different. Source: Proceedings for the Scientific Conference on ω-3 Fatty Acidsin Nutrition, Vascular Biology and Medicine, April 17–19, 1994, Houston, Texas, AmericanHeart Association.

Fig. 2.5. Concentration (µg/mL) of the sums for n-3 and n-6 long-chain fatty acid metabolitesin plasma total lipids from subjects fed diets containing 15 g (SAT diet) and 30 g (PUFA diet)linoleic acid. For SAT versus PUFA, 18:2n-6 means (P < 0.09) and 18:3n-3 means (P < 0.01)are significantly different. Note: deuterated 18:2n-6 was not included in the mixture ofdeuterated fats fed to subjects 5, 6, and 7. Source: Proceedings for the Scientific Conferenceon ω-3 Fatty Acids in Nutrition, Vascular Biology and Medicine, April 17–19, 1994,Houston, Texas, American Heart Association.


The sum of the concentrations for the deuterated n-6 LCFA metabolites was muchlower than the sum of the concentrations for the deuterated n-3 fatty acid metabolites(Figure 2.5). Comparison of these data clearly show that desaturation-elongation ofdeuterated l8:3n-3 was greater than for deuterated 18:2n-6. Deuterated 20:5n-3 (34.3µg/mL) and 22;6n-3 (29.8 µg/mL) represent 6.0% and 3.8% of the total amount oflabeled 18:3n-3 in total plasma lipids, respectively. In contrast, deuterated 20:4n-6 (7.2µg/mL) represents 0.5% of the labeled 18:2n-6 in total plasma lipids. Average totalpercent conversion of deuterated 18:3n-3 for all subjects (15.3%) was higher than thatof deuterated l8:2n-6 (1.6%). This low percent conversion of deuterated 18:2n-6 isconsistent with both in vivo and in vitro data from other human studies (22–29).

The difference in the amounts of deuterated 18:2n-6 and l8:3n-3 converted tolong-chain polyunsaturated fatty acid metabolites is not easily explained. A higheramount of n-6 LCFA metabolites would be expected, since the concentration ofdeuterated 18:2n-6 in plasma total lipids (1260 µg/mL) is about three times higher(P < 0.001) than the concentration of deuterated 18:3n-3 (450 µg/mL).

If one accepts that ∆-6 desaturase is the rate-limiting step in the conversionpathway and if the rate constant is similar for both 18:2n-6 and 18:3n-3 (5,19), thenthe concentrations of deuterated n-6 and n-3 LCFA should be proportional to the con-centrations of 18:2n-6 and 18:3n-3 in plasma lipids. A difference in the selectivity of∆-6 desaturase and/or the rate constant for 6-desaturation for 18:2n-6 and 18:3n-3 isa plausible explanation for the difference in conversion observed in this study. Thesein vivo data for deuterated n-6 and n-3 LCFA metabolites indicate that ∆-6 desaturaseis about four times more selective for 18:3n-3 than l8:2n-6. This selectivity is some-what higher than the difference in ∆-6 desaturation for 18:2n-6 and 18:3n-3 of1.5–3.0 times reported for in vitro studies with rat liver microsomes (20,34,35).

Effect of Dietary Linoleic Acid

The influence of the rather large difference in dietary linoleic acid levels in the SATand PUFA diets are illustrated by the concentrations of deuterated 18:2n-6 and 18:3n-3 and their deuterated n-3 and n-6 LCFA metabolites in plasma total lipids (Figures 2.4and 2.5). The concentrations of the deuterated fatty acids were clearly lower for the sub-jects fed the PUFA diet. These results indicate that the metabolism of both the 18:3n-3 and 18:2n-6 was altered when subjects were fed diets containing different levels of18:2n-6 (15.1 g vs. 29.8 g). This effect of dietary 18:2n-6 is consistent with animaldata showing that 18:2n-6 competes with itself and with 18:3n-3 (4,5,18,20). Theapproximate twofold difference in dietary 18:2n-6 content lowered deuterated 18:2n-6 and 18:3n-3 concentrations in plasma total lipids by 37–39% and deuterated n-6 andn-3 LCFA metabolite concentrations by 65–70%. The ratio of deuterated 18:2n-6 to18:3n-3 and deuterated n-6 to n-3 LCFA metabolites were not influenced by the18:2n-6 content of the diets. These results suggest that the absolute amounts ofdietary 18:2n-6 and 18:3n-3 have a greater influence than the 18:2n-6/18:3n-3 ratio.


Nutritional Implications

The amounts of n-3 and n-6 LCFA synthesized per day from 18:3n-3 and 18:2n-6in a typical U.S. diet can be estimated from the deuterated LCFA data. Based ona total plasma volume of about 3000 mL (39 mL/kg body wt) and the concentra-tion of deuterated n-3 LCFA metabolites (Figure 2.5), the total amount of deuter-ated n-3 LCFA metabolites in plasma lipids was 351 mg or 127 mg/g of deuterated18:3n-3 fed (SAT diet) and 126 mg or 43 mg/g deuterated 18:3n-3 fed (PUFAdiet). By extrapolation from the metabolite weight data, the 2 g of 18:3n-3 in atypical U.S. diet is estimated to provide 186 mg/day of n-3 LCFA. Based on a sim-ilar calculation, 537 mg/day of n-6 LCFA is estimated to be synthesized from the21 g of dietary l8:2n-6 in typical U.S. diets,

Estimates based on plasma concentration data indicate that dietary 18:3n-3provides about 50% of the n-3 LCFA daily requirement for adults. The estimatesbased on the total weight of deuterated LCFA metabolites are believed to be themost reliable, although they underestimate conversion of both l8:3n-3 and 18:2n-6 because the plasma data do not include the amounts of deuterated LCFAmetabolites that were incorporated into tissue lipids.

Alternatively, the amount of long-chain n-3 and n-6 fatty acids synthesizedfrom dietary 18:3n-3 and 18:2n-6 can be calculated from the percent conversiondata in Figure 2.6. The percent conversion calculated for a typical U.S. diet isabout 15% for 18:3n-3 and about 1.8% for 18:2n-6. Thus, about 300 mg of n-3LCFA metabolites/day is estimated to be synthesized from 2 g of l8:3n-3 in a typ-ical U.S. diet and 378 mg of n-6 LCFA metabolites/day is estimated to be synthe-sized from 21 g of 18;2n-6. Based on the percent conversion data, the 18:3n-3 ina typical U.S. diet is estimated to provide 75–85% of the 350–400 mg of long-chain n-3 fatty acids/day that has been estimated to be required by adults (15,16).

From these and other data used to estimate the requirements for essential fattyacids in humans, it is clear that most U.S. and Canadian diets contain a large sur-plus of 18:2n-6, but diets do not contain a surplus of n-3 fatty acids. If the 18:3n-3 provided by soybean and canola oils are not included in dietary estimates, boththe U.S. and Canadian diets would be deficient in 18:3n-3. Therefore, the concernis that the development of the new low (2–3%) 18:3n-3 soybean and canola oilsmay have a negative nutritional and health impact if they were to replace the con-ventional soybean and canola oils that contain 7–10% 18:3n-3.

References

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The American Oil Chemists’ Society, Champaign, Illinois, 1987, pp. 270–295.9. Galli, C, and A.P. Simopoulos (eds.) General Recommendations on Dietary Fats for

Human Consumption, Dietary ω-3 and ω-6 Fatty Acids: Biological Effects andNutritional Essentiality. NATO Series A, Life Sciences, Plenum Press, New York, 1989,pp. 403–04.

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Branden, H. Lafont, R. Grataroli, and G. Nalbone, Biochim. Biophys. Acta 1170: 151(1993).

19. Yamazaki, K., M. Fujikawa, T. Hamazaki, S. Yano, and T. Shono, Biochim. Biophys. Acta1123: 18 (1992).

20. Sprecher, H., in Dietary ω–3 and ω–6 Fatty Acids: Biological Effects and NutritionalEssentiality, edited by C. Galli and A.P. Simopoulos, NATO Series A, Life Sciences,Plenum Press, New York, 1989, pp. 69–79.

21. Brenner, R.R., in The Role of Fats in Human Nutrition, 2nd edn., edited by A.J.Vergroesen and M. Crawford, Academic Press Inc., London, 1989, pp. 45–79.

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Paulet, Metabolism 38: 315 (1989).26. Nichaman, M.Z., R.E. Olson, and C.C. Sweeley, Am. J. Clin. Nutr. 20: 1070 (1967).27. Ormsby, J.W., J.D. Schnazt, and R.H. Williams, Meta. Clin. Exptl. 12: 812 (1963).28. Emken, E.A., W.K. Rohwedder, R.O. Adlof, H. Rakoff, and R.M. Gullcy, Lipids 22: 495

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LipidRes. 30: 395 (1989).30. Emken, E.A., R.O. Adlof, and R.M. Gulley, Biochim. Biophys. Acta 1213: 277 (1994).31. Rohwedder, W.K., S.M. Duval, D.J. Wolf, and E.A. Emken, Lipids 25: 401 (1990).


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Chapter 3

Trans Fatty Acids in Canadian Breast Milk and Diet

W.M.N. Ratnayakea and Z.Y. Chenb

aNutrition Research Division, Food Directorate, Health Protection Branch, Health Canada,Ottawa, Ontario, K1A 0L2, Canada; and bDepartment of Biochemistry, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong.

Introduction

Many commercial dietary fats available to the consumers in industrialized countriesare prepared by the process of partial hydrogenation. This process converts liquidoils to solid fills that have the elasticity and texture desired for many food prepara-tions. Negative aspects of this practice are the substantial reduction in the propor-tion of essential fatty acids in the dietary fats with a concomitant formation of transand cis isomers of oleic and linoleic acids. The content of trans fatty acids (TFA) indietary fat varies. The average daily intake of TFA for the U.S. population has beenestimated to be at least 8 g or 3.7% of total energy (1,2). A recent estimate of TFAintake for the Canadian population is not available, nevertheless many of the fooditems in the Canadian retail market contain significant amounts of TFA. For exam-ple, Canadian margarines may contain up to 50% TFA (3). Cookies, biscuits, donuts,deep-fried foods, and many other common snacks made from partially hydrogenatedvegetable oils also contain substantial amounts of TFA (4).

It is well established that the fatty acids in breast milk reflect those of the mater-nal diet (5–14). The presence of TFA in human milk is a concern, because of theirpossible negative nutritional and physiological effects on the recipient infant.Human infants absorb and metabolize trans isomers and incorporate them intoplasma and tissue lipids (15). Negative effects of TFA, such as perturbations ofessential fatty acid and prostaglandin metabolism (16), and formation of unusuallong-chain polyunsaturated fatty acids were observed in rodents (17–19). In humaninfants, TFA seem to impair the biosynthesis of n-6 and n-3 long-chain polyunsatu-rated fatty acids (LCP) and the individual’s growth (20). During late fetal and earlypostnatal growth, considerable amounts of n-6 and n-3 LCP are accreted in neuraland other tissues (21). Phospholipids of the central nervous system and of retinalphotoreceptor cells are particularly rich in arachidonic (20:4n-6, AA) and docosa-hexaenoic (22:6n-3, DHA) acids (22). Studies with infant animals have indicatedthat a deficiency of DHA in the brain and retina may impair development of visualacuity, and possibly also discrimination of learning (23–26).

Although the presence of TFA in human milk has been recognized for a longtime, the literature data are not complete and generally may not be accurate, due tothe difficulty of analyzing TFA. Recent reviews on human milk fatty acids have not


mentioned the content of TFA and other unusual isomeric fatty acids (27–30). Thereis also a lack of information about these fatty acids in human breast milk fromCanadians. Therefore, using a combined procedure of silver nitrate-thin layer chro-matography (AgNO3-TLC) and gas-liquid chromatography (GLC), we analyzed thefatty acids of mature breast milk of 198 women across Canada. The TFA data werethen utilized to estimate the trans-octadecenoic (t-18:1) content in the Canadian diet.These estimates were calculated using an equation based on a relationship between t-18:1 in milk and dietary fat (11). Part of this study has been published elsewhere (31).

Materials and Methods

The human milk samples used in this study were from the 1992 collection of HealthProtection Branch’s ongoing monitoring program of chlorinated hydrocarbon con-taminants in the breast milk of Canadian women (unpublished work of W.H.Newsome, Health Protection Branch, Ottawa). Samples of mature milk (3–4 weeksof parturition) were collected from lactating women from across Canada (20–25samples per province), except from Prince Edward Island and the two territories.Donors were requested to express about 3–4 mL of their milk manually during eachfeeding, starting from the very first feeding to the last feeding of the day. The sam-ple from each donor thus represented the accumulated milk collection per day. Atotal of approximately 25–50 mL from each mother was collected in brown bottleswith polytetrafluoroethylene-lined screw caps. Mothers were requested to refriger-ate the milk samples between collections. The day following the 24-hr collection,the samples were shipped in dry ice to Ottawa and stored at -24°C until analysis.

Fat from a 5 g milk sample was extracted using 25 volumes of CHCl3-MeOH(2:1, v/v) containing 0.02% butylated hydroxy toluene as an antioxidant and tri-heptadecanoin (1 mg/mL) as an internal standard to quantitate total milk fat byGLC. The extracted fat was methylated with BF3-MeOH and analyzed by GLCusing an SP-2560 flexible fused silica capillary column (100 m × 0.25 mm i.d., 20µm film thickness). Column temperature was programmed from 150 to 180°C ata rate of 0.5°C/min, and then to 210°C at a rate of 3°C/min.

A typical GLC trace of a human milk fatty acid methyl ester (FAME) profile isshown in Figure 3.1. Single step, direct GLC analysis cannot accurately determinethe total t-18:1 due to overlap of high delta 18:1 trans isomers (12t-16t) with c-18:1isomer peaks (32). In the human milk of this study, 20.8% (range 9–30%) of thetotal t-18:1 isomers overlapped with c-18:1 isomers. Therefore, the total t-18:1and c-18:1 levels in the milk samples were determined using AgNO3-TLC in con-junction with capillary GLC. Silver nitrate thin-layer chromatography was per-formed as described previously (33). The t-18:1 band was isolated and analyzedby GLC. The proportion of t-isomers that overlapped with the c-18:1 isomer peakswas calculated by comparing the 18:1 region of the GLC chromatogram of the iso-lated t-18:1 with that of the parent FAME mixture prior to AgNO3-TLC fractionation.


For this purpose, the t-18:1 isomer peaks (6t-11t; peaks 26–27 in Figure 3.1) thatwere well separated from the c-18:1 isomer peaks served as the internal standard.The total t-18:1 was then calculated, by summing up the proportions of the t-18:1isomers (12t-16t; peaks 28-30, and 34 in Figure 3.1) that overlapped with the cisisomers and the well-separated t-18:1 isomers.

The t-18:1 and c-18:1 isomer distribution was determined by oxidative ozonoly-sis (34). The positional and geometrical isomers of linoleic acid were determinedand identified as described previously (33).

Results and Discussion

No significant regional differences in the average fat content and fatty acid pro-file data of Canadian human breast milk samples were observed in this study.Therefore, only the mean values, standard deviation, and the ranges are presentedfor the 198 samples (Table 3.1).

Usual Saturated and Polyunsaturated Fatty Acids

The major fatty acid group was represented by saturated fatty acids (38.5%),approximately one-half of which was 16:0. Sanders and Reddy found that the levelof C10–C14 saturated fatty acids was higher in the milk of human vegans and veg-etarians than in that of omnivores (30). The mean levels for 10:0, 12:0, and 14:0 inhuman milk of this study were remarkably similar to the levels found in vegan andvegetarian human milk in the United Kingdom (30). This might reflect that intakeof meat by the mothers in the present study was low, although dietary records werenot available. Sanders and Reddy have hypothesized that the origin of C10–C14 sat-urated fatty acids in human milk is not dietary, but most likely derived by de novosynthesis from carbohydrates in the mammary gland (30). Intake of these fatty acidsis low in vegans compared with the intake of omnivores, since vegan and vegetariandiets contain more carbohydrates and less fat than those of omnivores (35).

The levels of linoleic, α-linolenic acids, and their C20 and C22 metabolites inhuman milk are of special interest, because of their important physiological signifi-cance (22). The levels of linoleic (10.5%) and α-linolenic (1.2%) acids found inthis study are similar to those reported in studies of mature human milk fromwomen following ad libitum diets in different regions of the world (27,28,30,36).However, the levels of C20 and C22 n-6 (0.8%) and n-3 (0.3%) LCP were lowerthan for those in other countries (27,28,30,36) but similar to levels reported forvegans or vegetarians (30). The lower levels of n-6 and n-3 LCP further suggestthat a large segment of the lactating women in the present study were vegans orvegetarians. In some of the samples of this study, only trace amounts (<0.005% oftotal fatty acids) of AA (6 out of 198 samples) and DHA (17 out of 198 samples),physiologically the most important LCP, were detected. The optimal requirementsof LCP for infants is not known, but the extremely low levels of LCP in some


Fig. 3.1. A typical GLC trace of FAME from human breast milk fat analyzed on an SP-2560capillary column (100 m × 0.25 mm i.d.). Peak identifications (peak no., fatty acid): 1, 8:0;2, 10:0; 3, 12:0; 4, 13:0; 5, 114:0; 6, 14:0; 7, 115:0; 8, t-14:1; 9, A115:0; 10, 9c-14:1; 11,15:0; 12, 116:0; 13, 16:0; 14, 117:0; 15, t-16:1; 16, t-16:1; 17, 7c-16:1; 18, A117:0; 19, 9c-16:1; 20, 11c-16:1; 21, 17:0; 22, 118:0; 23, c-17:1; 24, c-17:1; 25, 18:0; 26, (6t10t)-18:1;27, 11t-18:1; 28, 12t-18:1; 29, 13t-18:1; 30, (8c-10c)-18:1 + (14t-15t)-18:1;:31, 11t-18:1;32,12c-18:1;33, 13c-18:1; 34, 16t-18:1;35, 14c-18:1; 36, 15c-18:1; 37, tt-18:2; 38, 9t,12t-18:2;39, (9c,13t + 8t,12c)-18:2; 40, 8t,13c-18:2; 41, 16c-18:1 + 9c,12t-18:2; 42, 9t,12c-18:2; 43,9t,15c-18:2; 44, linoleic (18:2n-6); 45, 9c,15c-18:2; 46, 20:0; 47, 18:3n-6; 48, unknown; 49,unknown; 50, monot-18:3n-3; 51, 11c-20:1; 52, α-linolenic (18:3n-3); 53, 13c-20:1; 54,18:2 conjugate; 55, 18:2 conjugate; 56, 18:2 conjugate; 57, 8c,14c-20:2; 58, 20:2n-6; 59,20:3n-9; 60, 22:0 + 5c,8c,14c-20:3; 61, 5c,11c,14c-20:3; 62, 20:3n-6; 63, 13c-22:1 +unknown; 64, unknown; 65, 20:4n-6; 66, unknown; 67, unknown; 68, 20:4n-3; 69, 20:5n-3; 70, 24:0; 71, 24:1; 72, 22:4n-6; 73, 22:5n-6; 74, 22:5n-3; and 75, 22:6n-3.


TABLE 3.1 Fatty Acid Composition (wt% of total fatty acids) of Pooled 24-hr Collection of Mature Breast Milk from 198 Canadian MothersFatty acid Meana (SD) Rangeb

Saturated fatty acids10:0 1.39 (0.59) 0.46 – 4.4211:0 0.01 (0.02) tr – 0.0812:0 5.68 (2.01) 2.32 – 11.7713:0 0.03 (0.03) tr – 0.1414:0 6.10 (1.73) 2.26 – 11.6815:0 0.37 (0.12) 0.12 – 0.6716:0 18.30 (2.25) 12.90 – 24.0617:0 0.32 (0.08) 0.03 – 0.4418:0 6.15 (0.97) 3.49 – 9.8520:0 0.15 (0.09) tr – 0.3614:0 brb 0.14 (0.06) tr – 0.2616:0 brb 0.14 (0.06) 0.04 – 0.45Cis-monounsaturated fatty acids9c-14:1 0.28 (0.08) 0.06 – 0.669c-16:1 2.27 (0.56) 1.11 – 3.887c-16:1 0.41 (0.13) 0.20 – 0.7010c-17:1 0.21 (0.06) tr – 0.448c-18:1 0.34 (0.07) 0.15 – 0.489c-18:1 30.65 (2.66) 23.55 – 40.6410c-18:1 0.49 (0.06) 0.32 – 0.6811c-18:1 1.91 (0.17) 1.26 – 2.3312c-18:1 0.74 (0.23) 0.21 – 1.0113c-18:1 0.24 (0.08) 0.07 – 0.3114c-18:1 0.23 (0.07) 0.06 – 0.2015c-18:1 0.15 (0.08) 0.05 – 0.2116c-18:1 0.10 (0.07) 0.01 – 0.2011c-20:1 0.39 (0.13) 0.13 – 0.6513c-20:1 0.14 (0.09) tr – 0.4213c-22:1 0.02 (0.03) tr – 0.11n-6 Polyunsaturated fatty acids18:2n-6 10.47 (2.62) 0.58 – 1.9018:3n-6 0.08 (0.06) tr – 0.2120:2n-6 0.17 (0.09) tr – 0.4620:3n-6 0.26 (0.09) tr – 0.4620:4n-6 0.35 (0.11) 0.05 – 0.6922:4n-6 0.04 (0.05) tr – 0.1822:5n-6 0.02 (0.02) tr – 0.16n-3 Polyunsaturated fatty acids18:3n-3 1.16 (0.37) 0.58 – 1.9020:4n-3 0.06 (0.06) tr – 0.2620:5n-3 0.05 (0.05) tr – 0.2522:5n-3 0.08 (0.06) tr – 0.4522:6n-3 0.14 (0.10) tr – 0.53Trans fatty acidst-14:1 0.09 (0.05) tr – 0.47t-16:1 0.18 (0.08) tr – 0.42


Canadian human milk samples should be a concern, because the ability to metabolizelinoleic and α-linolenic acids to LCP may be low in infants (37) and therefore,preformed LCP may be required from the diet.

There is some evidence to show that dietary levels of LCP affect the LCP lev-els in human milk. A dose-dependent increase in DHA content in milk was demon-strated in lactating mothers given fish oil concentrates rich in n-3 LCP (38). On theother hand, the LCP level of human milk does not appear to be correlated with themother’s intake of the parent fatty acids (14,36,39). This was confirmed in the pres-ent study (Figure 3.2). These findings suggest that milk LCP originate from the

TABLE 3.1 (continued)Fatty Acid Composition (wt% of total fatty acids) of Pooled 24-hr Collection of MatureBreast Milk from 198 Canadian MothersFatty acid Meana (SD) Rangea

8t-18:1 0.15 (0.12) tr – 0.699t-18:1 0.88 (0.32) 0.06 – 2.71

10t-18:1 1.22 (0.44) 0.03 – 3.3911t-18:1 1.31 (0.49) 0.02 – 3.6112t-18:1 0.80 (0.37) 0.01 – 2.4413t-18:1 0.63 (0.25) tr – 2.0714t-18:1 0.37 (0.19) tr – 1.2615t-18:1 0.29 (0.17) tr – 0.8816t-18:1 0.21 (0.15) tr – 0.7017t-18:1 0.01 (0.01) tr – 0.02

tt-18:2 0.05 (0.08) tr – 0.289c-13t/18t,12c-18:2 0.36 (0.14) tr – 0.768t,13c-18:2 tr 0.00 – tr9c,12t-18:2 0.29 (0.15) tr – 0.599t,12c-18:2 0.24 (0.12) tr – 0.599t,15c-18:2 tr 0.00 – trt-18:3 0.11 (0.08) tr – 0.34

Unusual polyunsaturated fatty acids9c,15c-18:2 0.07 (0.05) tr – 0.158c,14c-20:2 0.19 (0.08) tr – 0.495c,8c,11c-20:3 0.05 (0.06) tr – 0.185c,8c,14c-20:3 tr (0.00) 0.00 – tr5c,11c,14c-20:3 0.02 (0.02) tr – 0.12Othersc 2.24 (0.09) 1.25 – 2.54

Total saturatedfatty acids 38.50 (2.94) 36.92 – 42.59Total n-6 LCP 0.83 (0.28) tr – 1.2Total n-3 LCP 0.33 (0.24) tr – 1.4Total TFA 7.19 (3.03) 0.10 – 17.21Total (-18:1 5.87 (2.52) 0.10 – 15.42Fat content (g/L) 31.58 (9.37) 9.29 – 57.53

atr = trace amounts (<0.005% of total fatty acids).bbr = branched.cOthers include fatty acids <10:0, very minor branched-chain fatty acids, 18:2 conjugated fatty acids, and unknowns.


mother’s diet and cannot be elevated by increasing the dietary levels of linoleicand α-linoienic acids. This might explain the low LCP levels in vegan and vege-tarian milk compared to milk from omnivores (30). Vegan and vegetarian dietscontain very little LCP, because the latter are not plant products and are presentonly in animal tissues. Breast milk from lactovegetarians may contain smallamounts of LCP derived from dairy products. Recent analysis in our laboratoryshows that cow’s milk fat contains approximately 0.4% n-6 LCP and 0.2% n-3 LCP.Nevertheless, because of the current popularity of low-fat milk, and other dairyproducts low in fat, intake of LCP by lactovegetarians may not be that significant.

Trans Fatty Acids and Other Unusual Isomeric Fatty Acids

Trans fatty acids were found in ail the human milk samples in the present study.Although there were no regional differences in the fatty acid profiles, there was awide variation in the TFA content (i.e., the sum of all unsaturated fatty acids withone or more trans ethylenic unsaturations) within the 198 samples (Figure 3.3)ranging from 0.1 to 17.2% of the total milk fatty acids with a mean of 7.2 ± 3.0%.The most frequent occurrence (48% of the samples) was in the 6–9% TFA range,while 22% contained more than 9% TFA. Only 6 samples contained very low levels(<3%) of TFA.

Fig. 3.2. Scatter plots of % AA vs. % linoleic acidand DHA vs. % α-linolenic acid in human milk.


Data on TFA content in human milk from other countries is scarce. This is theonly study that examined such a large number of samples collected across a coun-try. Nevertheless, the limited data available in the literature indicate that the TFAlevel in the Canadian human milk is 1.5 to 3.6 times higher than that in othercountries (7–11, 13–15). Mean TFA levels of 0.9%, 2.0%, 3.5%, 3.7%, and 4.4%were reported for breast milk of women from Africa (15), Australia (36), UnitedStates (8), and Germany (14), respectively. These differences may reflect bothdietary and methodological differences between the studies. As mentioned earlier,t-18:1 isomers partially overlap with cis isomers in GLC analyses, and therebytotal TFA level may be underestimated in studies that used direct GLC methodsfor analysis of TFA (32). In some hydrogenated fats, the underestimation of thetotal t-18:1 level by direct GLC can be as high as 32% (40). The limited numberof samples analyzed in other studies (7–11,13–15) also could account for differ-ences in TFA content.

The range for TFA reported in the present study compares well with resultsreported in 1976 by Beare-Rogers and Nera for some Canadian human milk sam-ples (6). In that study, the total TFA content was measured by infrared spectroscopy,which gives results comparable to that of the combined GLC/AgNO3-TLC tech-nique (41). The similarity in the TFA levels between the two studies may indicatethat the TFA level in the Canadian diet has not changed over the last 18 years, inspite of a recommendation made in 1980 by an ad hoc Committee of Health andWelfare Canada to reduce the TFA level in the Canadian diet (42).

As in partially hydrogenated vegetable oils, t-18:1 was the major trans groupin Canadian human milk (Table 3.1). The average t-18:1 isomer distribution for the198 milk samples in this study closely resembled that of partially hydrogenated soy-

Fig. 3.3. Frequency of occurrence of TFA in human milk.


bean oil and, to a lesser extent, the pattern in partially hydrogenated canola oil, butit differed from that of cow’s milk (Figure 3.4). This suggests the major source ofTFA in the Canadian diet is partially hydrogenated vegetable oils, whereas thecontribution from dairy products is relatively minor.

Trans isomers of linoleic acid were the other important trans group found inhuman milk (Table 3.1). Several isomers were detected, and the total amounted to

Fig. 3.4. The average t-18:1 isomer distribution in human milk fat (n = 198), partially hydro-genated canola oil (PHCO; blend of six base stocks of iodine value varying from 64–92),partially hydrogenated soybean oil (PHSO; blend of six base stocks of iodine value varyingfrom 63–109), and cow’s milk (n = 10).


0.9% milk fatty acids. This group was primarily composed of 9c,13t-18:2/8t,12c-18:2 (0.4%), 9t,12c-18:2 (0.2%), and 9c,12t-18:2(0.3%). These fatty acids are alsolikely to have originated from partially hydrogenated vegetable oils (33).Canadian margarines contain up to 7.9% 18:2 isomers (3), and many bakery prod-ucts also contain high levels of 18:2 isomers (4). The close similarity of both t-18:1 and t-18:2 distribution patterns of human milk and partially hydrogenatedvegetable oils implies that dietary isomeric fatty acids are incorporated into themammary gland with little or no alteration of their relative proportions or struc-tures. This observation supports the view that milk fatty acids reflect the fatty acidprofile of the diet (43). However, under energy deficient conditions milk fattyacids are reported to resemble the fatty acid pattern in adipose tissue (43).

In addition to the isomeric fatty acids originating from partially hydrogenatedvegetable oils, our detailed analysis revealed the presence in Canadian humanmilk of two other minor, but unusual, polyunsaturated fatty acids, 8c,14c-20:2(0.19%) and 5c, 8c,14c-20:3 (<0.005%). To the best of our knowledge, the pres-ence of these two fatty acids has not been reported in dietary fats. However, werecently found these two fatty acids in various tissues of rats fed partially hydro-genated canola oil and suggested that they were derived from dietary 12c-18:1 byalternative desaturation and chain-elongation (19). The 12c-18:1 isomer is formedduring partial hydrogenation of vegetable oils and is present in appreciable quan-tities in Canadian human milk (Table 3.1). The presence of the two C20 metabo-lites in human milk suggests that the metabolic pathway of 12c-18:1 described forrats is also operative in humans.

In the human milk of the present study, the total TFA and t-18:1 levels wereinversely related to linoleic and α-linolenic acids (Figure 3.5; data for total TFAnot shown), suggesting that the elevation of TFA in human milk is at the expense ofessential fatty acids. This is to be expected, because food items made with partiallyhydrogenated fat contained higher levels of TFA and lower levels of essential fattyacids compared to food products made from unhydrogenated oils (4).

Fig. 3.5. Inverse correlation of % linoleic acid vs. t-18:1 and, α-linolenic acid vs. t-18:1 inhuman milk.


From the present study, it is apparent that a substantial proportion of Canadianhuman milk samples contain large amounts of trans and other isomeric fatty acids.The consequences of this situation on the recipient infants’ health and physiology areunknown. Nevertheless, several lines of evidence indicate that large amounts ofdietary isomeric fatty acids may have some negative effects on infants. Koletzko hasreported that TFA may impair the essential fatty acid metabolism and the earlygrowth of human infants (20). Since desaturase and elongase enzyme activity may belimited in infants (37), the impairment of essential fatty acid metabolism by TFAcould considerably influence the availability of LCP in infants receiving breast milkcontaining high proportions of TFA.

Another concern with large amounts of isomeric fatty acids in diets, is their pos-sible chain-elongation and desaturation to unusual C20 polyunsaturated fatty acids.Several unusual polyunsaturated fatty acids were shown to occur in various tissues(17–19), including the developing brain (18,44), and retina (45) of rats fed diets con-taining partially hydrogenated vegetable oils or synthetic isomeric fatty acids. Thephysiological effects of the presence of isomeric fatty acids in various tissues are notknown, although the incorporation of a trans isomer of DHA into the retina of devel-oping rat was shown to substantially alter the electroretinographic response (45).

Of the various isomers in partially hydrogenated vegetable oils, 12c-18:1, 9c,13t-18:2, 9c, 12t-18:2, and 9t,12c-18:2 are the most active substrates for the produc-tion of C20 polyunsaturated fatty acid metabolites (19). In rats, these four isomersmetabolized to C20 polyunsaturated fatty acids even with an adequate dietary supplyof essential fatty acids (19). Whether these unusual metabolites could occur inhumans is not known, but as discussed previously, trace levels of 8c,14c-20:2 and5c,8c,14c-20:3, probably derived from 12c-18:1, were detected in human milk. Thus,it is conceivable that the 18:2 isomers present in appreciable quantities in human milk(1.7% of total fatty acids, Table 3.1) could also be desaturated and elongated to t-20:4isomers in neonates. Future studies should investigate the possible incorporation ofthe isomeric fatty acids into various tissues, especially brain and retina, and evaluatethe physiological effects of such incorporation.

Estimation of TFA Content in the Canadian Diet

It has been clearly established that the trans fatty acid content in human milk dependsmainly on the mothers’ recent dietary intake (8,11). This is particularly applicable tot-18:1 which fluctuates in human milk according to the content in the diet (11). Craig-Schmidt et al. (11) found a strong linear correlation (r = 0.91) between the previousday’s dietary intake of t-18:1 and its level in breast milk. Using their linear equation,Y = 1.49 + 0.42X (where Y and X represent the percentage t-18:1 in total milk fat anddietary fat, respectively) the t-18:1 levels in the diet of lactating women could be esti-mated from human milk t-18:1 levels. Application of this equation indicates thatintakes of t-18:1 by the lactating women of the present study ranged from 0.6 to32.3% of total dietary fat, with a mean value of 10.4 ± 2.5%.


The t-18:1 distribution pattern shown in Figure 3.6 indicates that the diets of58% of the lactating women contained high concentrations of t-18:1 isomers(>9% of total dietary fatty acids) and about 2% of the lactating women appearedto ingest more than 30% of the total dietary fatty acids as t-18:1. The total TFA inthe diet is difficult to estimate, because a simple correlation for total TFA betweendiet and breast milk is not available. It would be expected to be about 20% higherthan that of t-18:1, since t-18:1 accounted for only about 80% of the total TFA inhuman milk (Table 3.1).

The fat intakes of the lactating women were not recorded in the present study,nevertheless estimates of per capita intake of t18:1 can be made from known fatconsumption data. According to the recently published Nova Scotia NutritionSurvey free-living women in the child-bearing age of 18–35 years consume onaverage 67.1 g fat and 1721 kcal of total food energy/day (46). Thus, for thisgroup, the mean t-18:1 content in the diet could be estimated to be 7.0 g/person/dayor 3.7% of the total dietary energy. Assuming that all the members in a givenhousehold eat the same type of food, but that total food intake among the individ-ual household members may vary, the dietary t-18:1 content for both males andfemales of all adult age groups (from 18–74 years) could be estimated based ondata reported in the same survey (46). As shown in Table 3.2 the average t-18:1intake could be 8.4 g/person/day or 3.7% of the total dietary energy for Canadianadults. Because of high fat intake (46), diets of young male adults (18–34 years)could contain extremely high levels of 1-18:1 (mean 12.5 g, range 0.7–38.9 g).These data also indicate that a wide range of intake (0.3–38.9 g or 0.2–11.7% totalenergy) of t-18:1 is possible from Canadian diets.

Fig. 3.6. Distribution pattern of the estimated percentage of t-18:1 in dietary fat of lactatingwomen in Canada. Data were calculated using Craig-Schmidt equation (11) and the per-centage of t-18:1 level in human milk fat.


The average value estimated for Canadian adults is remarkably similar to thevalue of 8.1 g/person/day reported by Hunter and Applewhite for the U.S. popula-tion (1). The range is also similar to that estimated for the U.S. population(0.7–38.7 g/person/day) by Enig et al. (2). From the distribution pattern given inFigure 3.6 and the fat intakes shown in Table 3.2, it could be estimated that over58% of Canadians would have t-18:1 intakes above 3.7% of energy, the leveltermed as moderate in a study that examined the cholesterolemic effect of TFA(47). The upper estimated dietary level of t-18:1 (11.6% total dietary energy) issimilar to that used by Mensink and Katan (48) in their clinical study in whichthey demonstrated that TFA adversely affect the LDL/HDL cholesterol ratio. Thusit is apparent that the results of recent clinical studies (47–49) on the hypercho-lesterolemic effect of TFA are relevant to the Canadian population.

Conclusion

The fatly acid composition of 198 human breast milk samples collected in 1992across Canada was determined by AgNO3-TLC and gas chromatographic proce-dures. Trans fatty acids ranged from 0.1–17.2% of the total milk fatty acids, with amean value of 7.2 ± 3.0%. Twenty-two percent of the samples contained more than9% trans fatty acids whereas only 3% of samples had low levels (<3%). Using anequation based on the relationship between trans-octadecenoic (t-18:1) fatty acidsin human milk and dietary fat, t-18:1 consumption in Canada was estimated. Theestimate predicts a wide range of intakes from 0.3–38.9 g/person/day. Analyses ofthe t-18:1 isomer distribution of the human milk samples, indicated that partially

TABLE 3.2Estimates of Dietary t-18:1 Intake for Canadian Adults (18–74 years of age)a

g t-18:1 t-18:1Age Calories Fat intake person/day %energy/person/day

Sex (years) (kcal) (g) Mean Range Mean Range

Male 18–34 3020.5 120.5 12.5 0.7–38.9 3.7 0.2–11.635–49 2343.4 91.9 9.6 0.6–29.7 3.7 0.2–11.450–64 2229.7 87.9 9.1 0.5–28.4 3.7 0.2–11.565–74 2025.0 75.3 7.8 0.5–24.3 3.5 0.2–10.8

Female 18–34 1720.8 67.3 7.0 0.4–21.7 3.7 0.2–11.335–49 1571.3 61.5 6.4 0.4–19.9 3.7 0.2–11.450–64 1571.3 54.2 5.6 0.3–17.5 3.4 0.2–10.765–74 1476.2 49.7 5.2 0.3–17.5 3.4 0.2–11.3

Averageb 18–74 2070.2 85.8 8.4 0.5–26.1 3.7 0.2–11.3aEstimates were based on fat and calorie intakes reported in the Nova Scotia Dietary Survey and assuming 10.4% of the total fat as t-18:1 (range 0.6–32.3%), which was calculated using the Craig-Schmidt et al. equation and t-18:1levels found in Canadian human milk.bAverage for both male and female adults (18–74 years).Source: Craig-Schmidt el al. (11), and Nova Scotia Dietary Survey (46).


hydrogenated vegetable oils are the major source of isomeric fatty acids in theCanadian diet, whereas contribution from dairy products is relatively minor.

The linoleic acid content in human milk varied from 5.8–21.4% of total fattyacids with an average value of 10.5%. On average, α-linolenic acid accounted for1.2%, with a range of 0.6–1.9%. The linoleic and α-linolenic acid levels wereinversely related to the total trans fatty acids, indicating that the elevation oftrans fatty acids in Canadian human milk is at the expense of n-3 and n-6 essen-tial fatty acids. The content of the physiologically important arachidonic anddocosahexaenoic acids showed a wide variation and did not correlate with theirparent fatty acids, linoleic and α-linolenic. This finding suggests that it may bedifficult to elevate the levels of n-6 and n-3 C20–C22 polyunsaturated fatty acidsin breast milk by increasing the levels of linoleic and α-linolenic acids in themothers’ diet.

In summary, these findings suggest that breast milk in Canada has high lev-els of TFA and other isomeric fatty acids. This apparently is a reflection of thewidespread use of hydrogenated vegetable oils in Canadian foods. The high levelof isomeric fatty acids in the Canadian diet and human milk should prompt thoroughinvestigation of their potentially adverse effects on both fetuses and young infants.

Acknowledgments

Z.Y. Chen was a T.K. Murray postdoctoral fellow of the National Institute of Nutrition. We areindebted to the mothers in this study for their cooperation and interest; and to the Bureau of FieldOperations and H.W. Newsome for collecting and sharing the samples with us. R. Hollywood andG. Pelletier are thanked for analyses of fatty acids in milk samples.

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4830 (1991)18. Beyers, E.C., and E.A. Emken, Biochim. Biophys. Acta 1082: 275 (1991).19. Ratnayake, W.M.N., Z.Y. Chen, C. Pelletier, and D. Weber, Lipids 29: 707 (1994).20. Koletzko, B., Acta Paediatr. 81: 302 (1992).21. Svennerholm, L., J. Lipid Res. 9: 570 (1968).22. Sastry, P.S., Prog. Lipid Res. 24: 69 (1985).23. Clandinin, M.T., Chappel. J.E., and J.E.E. van Aerde, Acta Paediatr. Scand. 79 (Suppl

351): 63 (1990).24. Lamptey, M.S., and B.L. Walker, J. Nutr. 102: 86 (1976).25. Neuringer, M., W.E. Connor, C. van Petten, and L. Barstadt, J. Clin. Invest. 73: 272

(1984).26. Yamamoto, N., M. Saito, A. Moriuchi, M. Nomura, and H. Okuyama, J. Lipid Res. 28:

144 (1987).27. Innis, S., J. Pediatr. 120: S56 (l992).28. Koletzko, B., J. Pediatr 120: S62 (1992).29. Innis, S., in Nutritional Needs of the Preterm Infant, edited by R.C. Tsang, A. Lucas. R.

Uauy, and S. Zlotkin, Williams & Wilkins, Baltimore, 1993. pp. 65–86.30. Sanders, T.A.B., and S. Reddy. J. Pediatr. 120: S71 (1992).31. Chen, Z.Y., G. Pelletier. R. Hollywood, and W.M.N. Ratnayake, Lipids 30: 15 (1995).32. Ratnayake, W.M.N., and J.L. Beare-Rogers, J. Chromatogr. Sci. 28: 633 (1990).33. Ratnayake, W.M.N., and G. Pelletier, J. Am. Oil Chem. Soc, 69: 95(1992).34. Ackman, R.G., J.L. Sebedio, and W.N. Ratnayake, Methods Enzymol 72: 253 (1981).35. Roshanai, F., and T.A.B. Sanders, Human Nutr. Appl. Nutr. 38A: 345 (1984).36. Gibson, R.A., and G.M. Kneebone, Am. J. Clin. Nutr. 34: 252 (1981).37. Crawford, M.A., A.G. Hassam, and P.A. Stevens, Prog. Lipid Rev. 20: 31 (1981).38. Harris, W.S., W.E. Connor, and S. Lindsey, Am. J Clin. Nutr. 40: 780 (1984).39. Gibson, R.A., and G.M. Kneebone, Lipids 19: 469 (1984).40. Ratnayake, W.M.N., R. Hollywood, E. O’Grady. and J.L. Beare-Rogers, J Am. Oil Chem.

Soc. 67: 804 (1990).41. Wolf, R.L., J. Am. Oil Chem. Soc. 71: 277 (1994).42. Davignon, J., B. Holub, J.A. Little, B.E. McDonald, and M. Spence, Report of the Ad Hoc

Committee on the Composition of Special Margarines. Cat. No. H44-46/1980E, 70 pp.43. Insull, W., T.J. Hirsh, T. James, and E.H. Ahrens, J. Clin. Invest 28: 443 (1959).44. Cook, H.W., Can. J. Biochem. 58: 121 (1980).45. Chardigny, A., B. Bonhomme, J.L. Sebedio, P. Juaneda, M. Doly, and A. Grandgirard,

INFORM 5: 472 (1994).


46. Report of the Nova Scotia Nutrition Survey, Nova Scotia Department of Health andHealth and Welfare Canada, 1993. 120 pp.

47. Judd, J.L., BA. Clevidence, R.A. Muesing, J. Witts, M.E. Sunkin, and J.J. Podczasy, Am.J Clin. Nutr. 59: 861 (1994).

48. Mensink. R.P., and M.B. Katan, N. Eng. J. Med. 323: 439 (1990).49. Zock, P.L., and M.B. Katan, J. Lipid Res. 33: 399 (1992).


Chapter 4

Food Industry Requirements for Fats and Oils:Functional Properties

T.K. Mag

T. Mag/Associates, Consulting lnc., 35 Old Church Road, King City, Ontario, L7B 1 K4,Canada

Introduction

In recent years, there have been increasing public demands that certain fats usedin the manufacture of fats and oils products no longer be used. To review briefly,current thinking includes the following points: avoid lauric (C12:0), myristic(C14:0) saturated fatty acids, and trans isomer fatty acids; stearic (C18:0) andpalmitic (C16:0) are no longer viewed negatively; oleic (C18:l) is viewed posi-tively in fat nutrition; linoleic (C18:2) is considered essential but should be somewhatlimited; and linolenic (C18:3) is a precursor of EPA/DHA. These considerations placesignificant restrictions on the choice of fats and oils that should be used in mak-ing edible oil/fat products for the consumer, and also present serious obstacles tothe optimal performance of fat products.

The functional aspects that are of importance and influence the choices inmaking fat and oil products can be summarized very briefly as melting behavior,crystal habit and stability, and stability against breakdown reactions while avoid-ing synthetic antioxidants. Table 4.1 lists the functions that the different fat prod-ucts have to fulfill.

TABLE 4.1Technical Functions Required of Fats and OilsFat or oil Required qualityMargarines Semisolid, spreadable

Smooth textureMelt rapidly near body temperatureOxidative stability

Baking shortenings Fat crystals to pick up and hold airSemisolidIncorporate into batterSmooth textureProvide lubrication on eatingOxidative stability

Frying fats Heat stability (breakdown, polymerization)Oxidative stabilityMelt rapidly near body temperature

Salad oils No solid fat at refrigerator temperatureGood oxidative stability at room temperature


Margarines

Margarines were first developed to mimic the melting behavior of butter.Approximately 75% of the original market for this type of margarine has now beenreplaced by “soft” margarine. With either of these two products, good mouthfeel andgood melt in the mouth is essential, but it should not be pourable. This can onlybe achieved with fats that have very small crystals, such as is achieved when thecrystal form is ß’. The stability of the polymorphic ß’ crystal form over timerequires a variety of fatty acids composing the triglycerides in the crystal matrixof a fat product. This variety in the fatty acid composition is very important andwill be mentioned repeatedly.

Experience has shown that soft margarines may have ß crystals when there is ahigh dilution of the crystal mass with liquid oil, as is found in soft margarines witha very low solid fat content. But this does not produce excellent margarines, becausesuch a margarine is more prone to oiliness and the development of a sandy texture.

With block margarines, it is essential that the melting profile be fairly steep.A steep melting profile will give a fast melt-away at body temperature.

Regardless of the type of margarine, it should not become rancid over severalmonths. The liquid component, which is particularly high in soft margarine andprone to oxidation, must have some resistance to oxidation.

Baking Shortenings

For cakes and breads, certain fat is required for the development of texture and vol-ume. The function of the fat is to introduce a large volume of finely dispersed air intothe batter by the “creaming” effect, incorporating a large volume of air. Secondly, thefat is required to lubricate and tenderize the structure of the baked product.

The incorporation of air into the fat to a satisfactory degree requires that therebe crystalline fat, and that most of the crystalline fat be in the ß’ polymorphicform, that is very small crystals that are stable over time. Also, crystalline fat isrequired to produce the proper cake and bread texture and structure when it isworked into the batter. The crystal structure prevents the fat from coalescing, aswould occur with liquid oil. Fat coalescence results in poor microdispersion of thefat in the batter and, hence, poor texture and structure. These two requirements, airincorporation and the avoidance of fat coalescence in the batter, are the main rea-sons why liquid oils do not make good shortenings.

Melt-in-mouth properties are not very important for shortenings because of thevery fine dispersion of the solid fat in the baked product. In most instances, it isimportant that the shortening have a relatively high amount of the solid tat meltingat temperatures beyond body temperature to produce good baking performance.This is known, technically, as a “flat solid fat index curve” and as “good plasticity.”

Emulsifiers are used to improve dispersion of the shortening in the batter. Theyare to some extent a means of getting good baking performance with a minimum of


fat and are increasingly used to produce cakes with low fat content. It should benoted, however, that to bake good cakes with a minimum of fat requires optimalshortening performance. This means using crystalline (saturated) fat in the ß’crystal habit as well as an appropriate emulsifier system.

Puff pastry fats are a special case. The function of the fat in this applicationis primarily to separate the layers of dough to produce the flaky texture. To achievethis, a fat with a waxy texture and large amounts of high melting triglycerides isneeded. This texture is achieved with fats that can be in the ß’ polymorphic form,but have been worked and tempered after crystallization to prevent graininess.

Frying Shortenings

Frying fats must, above all, have good stability because of the high temperaturesused in frying. The oil is subjected to temperatures in the range of 150–195°C,open to the air, and in the presence of moisture and a host of other compounds thatare introduced with the fried foods. Good stability means low unsaturation foroxidative and heat stability, and triglycerides that do not easily hydrolyze. The lat-ter quality eliminates short-chain fatty acid oils. Crystal habit is not usually a sig-nificant consideration.

Secondarily, mouth-feel, or melt-in-mouth is an important aspect. For thisquality, large amounts of high melting triglycerides, must be avoided, In someapplications, such as doughnut frying, a relatively high amount of solid fat isdesirable to avoid greasiness in the fried food from an aesthetic point of view.

Salad Oils

Salad oils have the least complexity. The primary functional issues are that the oilbe free of crystalline components, such as triglycerides or trace amounts of waxes,mostly for appearance reasons and to produce emulsion stability in dressings.Secondly, the oil should have good oxidative stability.

Historically, salad oil prices have reflected relative stability. The oils contain-ing linolenic acid, such as canola and soybean oils, are usually less expensive thansunflower, corn, and cottonseed oils. It is relatively easy to make salad oil prod-ucts that satisfy fat nutrition precepts.

Properties of Fats and Oils

Fatty Acid Composition and Melting Behavior

At this point, it is well to review the melting points of the various fatty acids thatoccur in commonly available fats and oils. Also, the ease of oxidation, the fatty acid


composition, and the melting behavior of the various hydrogenated and unhydro-genated oils are of interest. Table 4.2 lists the melting points of fatty acids. Thedata are organized in two groups: the more common C18:0 fatty acids, and otherfatty acids from C 16:0 to C6:0.

Most of the vegetable oils are high in C18 acids. Canola and soybean oil arethe most important representatives of this group of oils.

Within the C18 acids, the data illustrate the well-known fact that melting tem-perature is a function of unsaturation. Melting is also a function of other aspectsof molecular structure, such as positional and geometric isomerization. All ofthese are affected in partial hydrogenation. The melting temperatures indicate therange of changes in melting behavior that can be produced by hydrogenation.Partial hydrogenation produces considerable isomerization and yields fats thatmelt near body temperature, that is, fats that have steep melting profiles. Full sat-uration, or near-full saturation of these oils, produces relatively high melting fats(stearic acid melts at 69.6°C) and removes isomerization.

The other fatty acids in Table 4.2 show a similar range of melting temperaturesas the C18 acids. Palmitic acid melts at 61.3°C; lauric acid melts closer to body tem-perature at 44.2°C; and caproic acid, included for comparison with low meltingpoint C18 acids, melts at –1.5°C. Fats that are predominantly made up of these fattyacids can be expected to have somewhat similar properties to partially hydrogenatedC18 fatty acid oils, that is, melting close to body temperature. The fatty acid com-position of fats is the main factor influencing their melting behavior. Another factorof importance is the distribution of fatty acids in the triglyceride. It modifies the pre-viously indicated melting behavior and makes it more complex.

TABLE 4.2Melting Temperatures of C18 and Other Fatty AcidsFatty acid Melting point (°C) OccurrenceC18 fatty acids

Linolenic cis 9,12,5 –12.8 Canola, SoybeanLinoleic cis 9,12 –6.5 Corn, SunflowerOleic cis 9 16.2 Olive, CanolaElaidic trans 9 43.7 Partially hydrogenated fatsStearic C18:0 69.6 Tallow, coconut butter

Other fatty acidsPalmitic C16:0 61.3 Lard, tallow, palmMyristic C14:0 54.4 Palm kernel, CoconutLauric C12:0 44.2 CoconutCapric C10:0 31.6 CoconutCaprylic C8:0 16.5 ButterCaproic C6:0 –3.4

Sources: Perry (4), and Eislor and Hagemann (5).


Oxidative Stability

In connection with C18 fatty acids, stability is an important aspect of the functionof these fats and oils. Table 4.3 gives the relative oxidative stability of C18 fattyacids. It shows that linolenic acid and linoleic acid are about 20 times and 10 timesmore easily oxidized than oleic acid, respectively.

Crystallization Behavior

It was mentioned earlier that the crystallization habit of solid fat is an importantaspect of a fat’s functionality. The tendency of a fat to be stable in the ß’ or in theß form is a function of the heterogeneity of the fatty acid composition. It deter-mines how well, or how poorly the triglycerides fit into a crystal matrix. Fats thathave a fairly homogeneous fatty acid composition in their triglycerides tend tocrystallize in the ß form. This means that large crystals are formed more or lessquickly. As already pointed out, this is not desirable in margarines and in mostuses of shortenings.

Table 4.4 presents a classification of fats and oils according to their crystal-lization behavior. It can be seen that most of the oils used today are ß tending. Themain reason is that many of these oils are high in C18 acids, lauric acid in the caseof palm kernel and coconut oils; or a particular triglyceride predominates, such asfor lard and cocoa butter. This illustrates that apart from the fatty acid composition,triglyceride composition is the important factor in crystallization behavior. Oils that

TABLE 4.3Relative Oxidative Stability of C18 Fatty AcidsStearic acid 1Oleic acid 10Linoleic acid 100–120Linolenic acid 1 60–250Source: Swern (6).

TABLE 4.4Classification of Fats and Oils According to Crystallization Behaviorß Type ß’ Type

Soybean CottonseedSafflower PalmSunflower TallowSesame HerringPeanut MenhadenCorn WhaleCanola Rapeseed (HEAR)OliveCoconut Butter fatPalm kernelLard Interesterified lardCocoa butterSource: Wiedermann (7).


are composed of significant amounts of palmitic or erucic acids, along with C18acids, tend towards the ß’ structure. For this reason, palmitic acid occupies animportant position in the formulation of functional oil products. In the case of thehighly unsaturated vegetable oils, the crystallization habit refers to the partiallyand fully hydrogenated versions of these oils.

It is apparent that the crystallization habit of many readily available oils is aproblem in present edible oil product manufacture. Present methods to overcomethe tendency of fat products to crystallize in the ß form are shown in Table 4.5.Blending of oils with different crystallization habit is the most widely practicedmethod to control crystal formation. Interesterification is not widely used, mostlybecause of cost. Trans isomers in partially hydrogenated fats represent desirableheterogeneity in fatty acid composition to reduce ß tendency. Using a crystalliza-tion inhibitor, such as sorbitan tristearate, is an option. It is classed as an additivein Canada. Its use is limited to a maximum of 1% in margarine or shortening.

In Table 4.6, the fatty acid composition of nonhydrogenated semisolid fats arereviewed. These are the fats that can supply crystalline fat in fat products in placeof the partially hydrogenated fats based on canola and soybean oil. They are the oilslisted earlier that contain significant amounts of palmitic acid.

It can be seen that lard, tallow, and especially palm oil and its fractions arehigh in C16:0. Cottonseed oil with 22% palmitic acid is not listed, because it is nota semisolid fat. The nutritional effect of palmitic acid is still not sufficiently clear to

TABLE 4.5Methods to Prevent ß Crystallization in Fat ProductsBlending with ß’ tending fatsInteresterification (lard)Interesterification of a blendPartial hydrogenation (trans isomers)Using a crystal inhibitor (sorbitan tristearate)

TABLE 4.6Fatty Acid Composition of Nonhydrogenated, Semisolid Fats

Fatty acid (%)Fat C8/C10 C12 C14 C16 C18 C18:1 C18:2Lard — — — 25 15 44 10Tallow — — 4 26 27 36 (t5) 3Palm — — — 44 5 40 10Palm stearin — — — 63 5 25 5Palm olein — — — 40 5 43 11Palm kernel 8 48 15 8 2 15 3PK olein 9 42 12 8 2 23 3PK stearin 5 57 22 8 2 5 1Coconut 11 49 19 9 3 7 2Abbreviation: PK, palm kernel.Source: Palm Oil Research Institute of Malaysia data.


remove concerns about its presence in fat products, but this acid is very importantin the formation of crystalline structure of fat products.

Palm kernel and coconut oils have significant amounts of lauric and myristicacid, that according to present nutrition precepts should be avoided. Very smallamounts of palm kernel oil are used in zero-trans margarines, because it helps toprovide the kind of melting profile that is achieved with the trans isomer fromhydrogenated fats. Palm kernel and coconut oils are used in cream fillings andchocolate coatings for their melting properties, but to a significant extent theyhave been replaced by high trans isomer partially hydrogenated canola and soy-bean oils, that have achieved similar melting behavior.

Table 4.7 gives the melting profiles of the semisolid oils. It can be seen thatthey represent a wide variety of melting behaviors; from very steep, for palm ker-nel and coconut oils, to very flat, as is the case with tallow and palm stearin. Theoils with flat melting behaviors are suitable for baking shortenings.

Table 4.8 compares the fatty acid compositions of typical partially hydro-genated canola and soybean oils with the nonhydrogenated oils. These oils contain

TABLE 4.7Melting Profiles of Various Nonhydrogenated, Semisolid Fats

Solid fat index at °CFat 10.0 21.1 26.7 33.3 40.0Lard 30 22 16 5 3Tallow 36 28 26 22 13Palm 27 15 12 9 5P stearin 55 45 44 43 41P olein 25 4 0 0 0Palm kernel 50 33 13 0 0PK stearin 85 80 60 0 0PK olein 35 8 0 0 0Coconut 55 27 0 0 0Abbreviations: P, palm; and PK, palm kernel.

TABLE 4.8Fatty Acid Composition of Partially Hydrogenated Canola and Soybean Oils

Fatty acids (%)C16:0 C18:0 C18:1 C18:2 Trans

Canola oilsNH, IV 115 4 2 61 21 (+9%C18:3)IV 90 4 3 81 9 26IV 80 4 10 83 2 35IV 70 4 18 75 0 50

Soybean oilsNH, lV 130 11 4 25 52 (+8% C18:3)IV 110 11 5 45 36 16IV 85 11 5 73 11 30IV 70 11 13 74 2 47

Abbreviations: NH, nonhydrogenated; and IV, iodine value.


predominantly C18 acids. Hence, they exhibit the ß crystallization habit whenhydrogenated. This is reduced somewhat by the presence of trans acids, and in thecase of soybean oil by the presence of about 11% of palmitic acid. The nutritionalconcern with these oils stems from the trans isomer content.

The nonhydrogenated canola and soybean oils are characterized by 8–12%linolenic acid; soybean oil is also characterized by its high content of linoleic acid(50%). This produces stability problems in any use that does not have optimal con-ditions and limits the shelf stability when used in margarines, shortenings, and, tosome extent, when used as salad oils. They are poor frying oils.

For these reasons, canola and soybean oils are usually lightly hydrogenated tolower the linolenic acid to ‹3% (canola, IV 90; soy, IV 110), to obtain fats ofgreater stability with a variety of melting profiles. After partial hydrogenation, thetrans isomer content can range from 15–50% of total fatty acids depending on thedegree of hydrogenation.

Nutritionally, nonhydrogenated soybean oil is considered less desirable thancanola oil because of its high content of saturated fatty acids. Also, its high contentof linoleic acid is no longer considered an advantage when compared to canola.Both oils supply linolenic acid, which may be significant in human nutrition as aprecursor of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA).

Table 4.9 gives the melting profiles of typical partially hydrogenated canolaand soybean oils at different iodine values, along with their fully hydrogenatedcounterparts, to compare with the semisolid fats in Table 4.7. The melting profilesare relatively steep, as one would expect from the presence of the trans and posi-tional isomers listed in Table 4.2. Flatter profiles (not included here) can be pro-duced, but not without significant trans isomerization, unless the oils are fullysaturated, as shown at the bottom of Table 4.9. If fully saturated, the oils can sup-ply very large amounts of solid fat. Their crystallization habit will not be suitablefor most purposes, unless oils with different fatty acids are also used in the ediblefat product, or they are interesterified with another oil component.

TABLE 4.9Melting Profiles of Typical Partially Hydrogenated Canola and Soybean Oils

Solid fat index at °C10.0 21.1 26.7 33.3 40.0

Canola oilsIV 90 3 0 0 0 0IV 80 21 5 2 0 0IV 70 52 34 27 13 1

Soybean oilsIV 110 3 0 0 0 0IV 85 19 6 2 0 0IV 70 53 36 30 13 0

NoteCanola andSoybean, IV <10 >90 >90 >90 >90 >90

Abbreviation: IV, iodine value.


From the previous discussion on nutritional and functional requirements in theproduction of fat and oil products, the most prominent issues that emerge are theneed to have palmitic acid as part of solid fat component in margarine, and espe-cially in shortenings, and to avoid trans acids. The need to reduce palmitic acid isparticularly difficult. Its elimination is, in fact, not possible. Therefore, it is essen-tial to clarify its role in cardiovascular disease. The avenues available for the reduc-tion of palmitic acid and elimination of trans acids are listed in Table 4.10.

The process of interesterifying fully hydrogenated canola and soybean oils toproduce hard fats with zero-trans fatty acids that consist mostly of tristearate, andblending with nonhydrogenated oils for margarine and shortening applicationsstill requires a great deal of work. Nevertheless, this approach is of interest ifstearic acid is in fact not a problem in cardiovascular disease.

It has been shown by various workers that the melting profile of interesteri-fied blends of fully saturated soybean oil mixed with nonhydrogenated soybean oilis not a problem (1). The presence of 11% of palmitic acid in the interesterifiedblend is probably sufficient for °’ crystal stability. Thomas interesterified fullyhydrogenated vegetable oils with canola oil and showed that satisfactory meltingprofiles were obtained (2).

There is also the question of the effect of Interesterification on fatty acidposition on glycerol, and the fact that position to some extent determines if a fattyacid is hyper- or hypocholesteremic (3). This is a complication in the use ofInteresterification that cannot be ignored.

As the use of fully hydrogenated fats increases, it is probably important tochange labeling regulations to make it mandatory to declare trans fatty acids. Thiswill make it possible to use fully hydrogenated fats without engendering the presentassociation of trans fatty acids with “hydrogenation” in the perception of consumers.

The reduction of trans isomer formation in hydrogenation by the developmentof economical catalysts and process conditions that suppress these reactions ispossible. This is discussed extensively in the chapter by R. Hastert.

An important potential avenue to reduce the need for partial hydrogenation isthe development of low linolenic acid and high oleic acid oils. Commercial use ofsuch oils would eliminate a large volume of partially hydrogenated fat from edibleoil products. This would reduce trans fatly acids (by restricting the use of partially

TABLE 4.10Ways to Reduce the Use of Undesirable Fatty Acids

Greater use of Interesterification (crystallization habit, melting behavior)Full hydrogenation (zero-trans fatty acids to supply solid fat)Hydrogenation catalysts and conditions to minimize trans isomersDevelopment of low linolenic and of high oleic canola, soybean, sunflower oils (stable oils

for margarines, shortenings, and frying fats without hydrogenation)Use of more palm oil (crystallization habit, if C16:0 is nutritionally innocuous)Development of canola, soybean and other oils with higher palmitic content (crystallization

habit, if C16:0 is nutritionally innocuous)


hydrogenated fats) in fat products to levels that might come to be consideredinsignificant. The fatty acid composition of the “new” oils in this category areshown in Table 4.11. Some of these oils are already available commercially.

The three low linolenic oils at the top of the table still contain about 3%linolenic acid and with the exception of low linolenic canola, also contain veryhigh amounts of linoleic acid. They can be expected to have the oxidative and heatstability of sunflower oil. They would make stable salad oils and could be usedalong with the present standard canola and soybean oil. These oils would still beof interest, even if linolenic acid is indeed a significant precursor of EPA/DHA,because of the greater stability of these oils.

The two high oleic acid oils have essentially no linolenic acid and lowamounts of linoleic acid. Their stability is essentially on par with lightly and mod-erately hydrogenated canola and soybean oils. This makes them good zero-transreplacements for these partially hydrogenated oils, except that they are not con-tributing solid fat to margarine and baking shortenings.

In some cases involving Interesterification, blending the low linolenic andhigh oleic oils with fully hydrogenated fats from canola, soybean, cottonseed, andpalm oils can satisfy the functional requirements of margarines and shorteningswith respect to solid fat content, crystallization habit, and oxidative stability.When using Interesterification, the position of the fatty acid on glycerol must beconsidered to avoid negative nutritional effects.

Avoidance of partially hydrogenated fats in margarine and shortening formu-lations will require greater use of semisolid fats containing palmitic acid, mostnotably palm oil. Consequently, interest in the development of canola and soybeanoils with a higher palmitic acid content has increased. For crystallization habit,this is desirable. It would eliminate blending with other oils, such as palm oil, toproduce ß’ crystal stability after partial or full hydrogenation. But, as indicatedearlier, the nutritional effect of palmitic acid requires further clarification beforedeciding that high palmitic canola and soybean oils are useful developments.

TABLE 4.11Fatty Acid Composition of Some “New” Oils

Fatty acids (%)CI6:0 C18:0 C16:1 C18:2 C 18:3

Low 18:3Canola 4 3 64 24 3

Low 18:3Flax 7 5 24 61 3

Low 1 8:3Soy 11 3 22 61 2

Trisuna 4 4 80 10 <1Sunolab 3 2 90 3 <1

aHigh oleic sunflower oil.bHigh oleic short stem sunflower oil.Sources: Personal data and data published in product sheets by SVO Specialty Products, and Western Grower Seed Corp.


References

1. Zeitoun, N.A.M., W.E. Neff, G.R. List, and T.L. Mounts, J. Am. Oil Chem, Soc. 70:467(1993).

2. Thomas, K.C., J. Can. Food Sci. Tech. 21: 167 (1988).3. Elson, C.E., Crit. Rev. Food Sci. Nutr. 31: 79 (1992).4. Perry, (ed.), Handbook of Chemical Engineering, 4th edn., 1963, pp. 3-24–3-42.5. Eislor, R.L., and J.W. Hagemann, in Fatty Acids, edited by E.H. Pryde, The American Oil

Chemists’ Society, Champaign, Illinois, 1979, pp. 180.6. Swern, D., in Autoxidation and Antioxidants, edited by W.O. Lundberg, Interscience, New

York, 1961, pp. 10.7. Wiedermann, L.H., J. Am. Oil Chem. Soc. 55: 825 (1978).


Chapter 5

Hydrogenation: A Useful Piece in Solving the Nutrition Puzzle

Robert C. Hastert and Robert F. Ariaansz

Hastech, 10485 Manderson Pz., Omaha, Nebraska 68134, USA.

Introduction

This paper will only peripherally touch upon the nutritional justification for theclaims and counterclaims regarding either positive or negative aspects of varioustriglycerides in the human diet. Rather, it will be an examination of the chemicalfacts of the composition of commercial fats and oils products, both present and his-torical. Using these facts, an outline will be proposed as to how hydrogenation mightbe used in the future to attain triglyceride composition objectives that may be nutri-tionally desirable. Whether long-term human testing will eventually prove theseobjectives to be scientifically valid is not of immediate concern to the processor andmarketer of oil and fat products. In the short term, the processor has no alternativebut to respond positively to whatever the marketplace is requesting at any particulartime. This paper will principally focus on margarine, as that is the product most dis-cussed in technical papers and presently receiving the most media attention.

Nutritional Effects of Fat in the News

Do the following newspaper headlines look familiar?

Medical Report Indicts “Bad Fats” in MargarineFeeding Tests Show Blood Cholesterol Increase When Eating Hydrogenated FatNoted Nutritionist Speaks Out About Fats in American Diet

Of course, the younger readers may be surprised to hear that the same headlineswere in our newspapers in 1960. In other words, to anyone more than 50 years old,the current almost hysterical discussion concerning the nutritional effects resultingfrom the composition of fats in the diet seems a bit of deja vu. However, there is animportant difference between 1960 and now. At that time, its relevance to ordinarypersons was considerably more direct. That was because there truly appeared to bean “epidemic” out there. People, particularly men, were being felled by heartattacks, not only with increasing frequency, but also at younger and younger ages.The health professions responded to the public outcry by proclaiming cholesterol tobe the villain, saturated fat to be its precursor, and hydrogenation to be its cause.


Overnight, heart surgeons became superstars and nutritionists became gurus (1).Ancel B. Keyes, a noted nutritionist from Minnesota, was even on the cover ofTime magazine (2).

While the 1960s experience is recalled with some irony, its central thesis of arelationship between fats in the diet and heart disease cannot be consideredunfounded. Even though everyone now agrees that the causes of heart disease areconsiderably more complex than was thought 30 years ago, the fact that the inci-dence of death from heart disease in the United States has fallen 50% since then(more than 500,000 fewer deaths/year) is irrefutable evidence that something wasdone right to turn around a very alarming situation. How much of the reductionwas due to eating an overall better diet, how much to more exercise, how much toa significant reduction in smoking, and how much to the ingestion of fats withmodified triglyceride composition, can never be precisely known. However, theobvious conclusion is that they probably all contributed to some extent, and itwould be ill-advised to discontinue an emphasis on any of them. The experiencecertainly convinced the public of a link between fats in the diet and health.

Industry Response

An examination of how technologists and industry successfully responded to the sat-urated fat/heart attack scare of almost 35 years ago can give considerable insight intothe tools to be employed and the paths pursued to surmount the current very similarattack on trans-isomers in the diet. First and foremost, while the fats and oils industryfelt unjustly accused of harmful actions in 1960, it did promptly respond to the accu-sations. The result was a reduction in the amount of completely saturated triglycerides,particularly in relation to polyunsaturated triglycerides, in margarine, shortenings, andsalad oils. What were the motivations, technical approaches, and economic aspects ofhow this happened? It is particularly important to remember that it was accomplishedwithout any perceptible change in the appearance and functionality of the end-prod-ucts. For instance, margarine continued to look, taste, feel, and perform as it had pre-viously. There was actually a functional improvement in that the increasingly populartub margarine products were more spreadable when taken from the refrigerator.

There was both a driving force and an incentive behind manufacturers’acceptance of the formulation changes required to increase the polyunsaturatecontent. The driving force, as mentioned previously, was the health professions’accusation that the amount of saturated triglycerides in the then-conventional fatsand oils products was causing the deaths of a very large number of people. Notsurprisingly, this accusation made persons in the industry feel ethically uncom-fortable. It also frightened companies with the prospect that sales of their productsmight decrease. Those same driving forces arc with us today.

Economics was a big incentive to make the formulation conversions. Oilhydrogenated to a lesser degree meant some cost savings by reducing the amount


of hydrogen, catalyst, and processing time. The somewhat later incorporation ofunhydrogenated oil into formulations meant even greater cost reduction. By-pass-ing the hydrogenation step on a portion of the formulation completely eliminatedthe costs of catalyst, hydrogen, and labor associated with it. It also eliminated theiroverhead costs in conventional accounting methods. There was even a cost savingthrough yield gain by not incurring product loss in the “black” (posthydrogena-tion) filter press. All in all, considering the economic advantages, it is not sur-prising that manufacturers were very willing to accept the modest conversioncosts necessary to adapt their physical facilities to utilize the new formulations.There is an obvious parallel economic advantage associated with the currententhusiasm of margarine manufacturers to market the spreads and light mar-garines that contain less fat. Selling water for the price of oil has got to be goodfor the bottom line. Not surprisingly, this has resulted in the reappearance of stillanother 1960s newspaper headline:

Consumer Activist Accuses Corporations of Greed:Using Cheaper Ingredients But Not Lowering Prices.

While competition will no doubt eventually rectify the prices, the importanceof economics to the overall discussion must always be kept in mind. Economics, ofcourse, also works in reverse. In other words, higher cost (price) is a marketingdisincentive. It has repeatedly been shown that, while most consumers will give apositive response when asked whether they are willing to pay a higher price fora more nutritionally suitable margarine, shortening, or salad oil, in actuality thenumber who will and/or the amount they will pay, has not been found to be large.A past example was the marketplace failure of a higher quality, but slightly-more-expensive sunflower salad/cooking oil. A current example is olive oil. While oliveoil sales on a percentage basis are booming, the increase is from a very small base,not significant in overall market share.

Manufacturing Techniques

The manufacturing technique so successful in the 1960s, that enabled processorsto incorporate lesser-hydrogenated and unhydrogenated oils into formulations,was the base stock system. Briefly, it involved the blending of two or more differ-ent oils, each having specific melting characteristics, rather than hydrogenatingone feedstock into a single margarine base. Blending relied on the phenomenonthat a mixture of a harder and a softer fat has physical characteristics closer to theharder one. While the base stock system came into wide use in the early 1960s, itwas not described in the literature until Latondress did so in 1982 and again in1985 (3,4). In the United States it utilized unhydrogenated and/or very lightlyhydrogenated soybean oil, several partially hydrogenated ones, and stearin.O’Brien further defined the approach at an AOCS Colloquium in 1986 (5). Heillustrated the melting characteristics of the bases, as depicted in Figure 5.1.


Calculations of the triglyceride composition of several of the base stocks areshown in Table 5.1. For clarity and illustrative purposes, all trans-isomers in thisand other tables and figures were calculated as C18:1. While not exactly accurateanalytically, comparisons among them are bona fide since the same procedure wasused in all cases.

For making various margarines, O’Brien suggested formulations utilizing thebase stocks (5), as shown in Table 5.2. A contemporary squeeze bottle liquid for-mulation has been added, as has a single stock formulation utilizing O’Brien’s 75IV base. While this 75 IV base is slightly softer than was actually the case withcommercial products of that era (6), it is close enough to illustrate a valid com-parison with contemporary products.

From Figure 5.2, which illustrates melting curves obtained when using thepreviously mentioned formulations, it is apparent they have been flattened con-siderably in the progression from the old single stock base through the more lightlyhydrogenated formula and into formulas incorporating unhydrogenated oil. The

Fig. 5.1. Soybean oil base stocks.

TABLE 5.1Composition of Base Stocks

Fatty Unhydro- Iodine valueacids % genated 108 85 75 66 StearinSaturates 16 16 18 20 27 100

C 18:1 Total 21 44 68 77 73 0C 18:1 cis 21 21 26 28 21 0C 18:1 trans 0 23 42 49 52 0C 18:2 54 36 13 3 0 0C 18:3 9 4 1 0 0 0


TABLE 5.2Margarine Formulations

Stick Tub Squeeze bottleAll Highly All High

Single hydro- unhydro- hydro- liquidBase (%) base genated Soft genated genated oil LiquidUnhydrogenated 0 0 50 60 0 50 97.5108 IV 0 42 0 0 80 30 085 IV 0 0 0 0 0 0 075 IV 100 20 0 25 0 0 066 IV 0 38 50 15 20 20 0Stearin 0 0 0 0 0 0 2.5

Fig. 5.2. Margarine melting curves.

advent of tub margarine, especially with a formula utilizing unhydrogenated oil,flattened the curves even more. Finally, the liquid squeeze bottle formulation prac-tically eliminates the solids melting curve. While the melting curves in Figure 5.2very strikingly illustrate what can be seen and felt, the scientist always asks,“why?” The seemingly logical assumption has always been that the new multibasestock formulations significantly reduced the amount of saturates, probably with acorresponding increase in trans-isomers. However, calculating the amount of sat-urates and trans-isomers, as listed in Table 5.3, and depicting them separately andadded together, as shown in Figure 5.3, produces unexpected results.

Surprisingly, the saturates content, as shown in the left bars of Figure 5.3, didnot decrease appreciably over the entire range of formulations. However, evenmore surprisingly, the trans-isomers (middle bars) were significantly reduced, as wasthe sum of the two (right bars). In other words, whether by intent or accident, the


amount of trans-isomers in stick and tub margarines has been cut by approxi-mately one-half when compared to the pre-1960 single base stock. While it is easyto complain that this fact has been ignored by the media, they have the reasonableexcuse of not possessing an adequate technical background to appreciate it. Whatis disconcerting is that it also seems to have been either unknown or ignored bypersons in the health professions, who should be more technically knowledgeable.Bringing it even closer to home, those of us closely associated with the oil and fatindustry bear the primary blame for not having known and promulgated the sig-nificant trans-isomer reduction.

Accepting these conclusions means abandoning the previously held thesisthat the saturate content of margarine was greatly reduced by utilizing the basestock formulation procedure. The question then remains, what was the change inester composition that contributed to the significant reduction in heart diseasereferred to earlier? A plausible answer may be the lowering of trans-isomer con-tent, as is illustrated in Figure 5.3. Another possibility, that was also referred toearlier, is the significant increase in polyunsaturates, especially as related to satu-rates. This is illustrated in Figure 5.4, which show the amount of polyunsaturatesand saturates in each formulation, along with their polyunsaturated to saturatedratio-referred to as P/S. (The P/S ratio has been multiplied by 10 in Figure 5.4 forillustrative purposes.) Since the saturate level is substantially the same in all theformulations, it is obviously the varying level of polyunsaturates among the for-mulations that causes the significant difference in P/S ratios.

Fig. 5.3. Saturates and trans-isomers for margarine formulations.


A summary of observations from this examination of the ester composition ofvarious margarine bases, is as follows:

1. Beginning about 1960, fat products began to be formulated from two ormore base stocks. This significantly changed their triglycerides compositionby allowing the incorporation of unhydrogenated oil possessing a minimumof saturates and no trans-isomers.

2. The resulting effect(s) of either less trans-isomers and/or more polyunsatu-rates may have contributed to the quite astounding reduction in arteriosclerosis.

3. The increasing popularity of tub margarine facilitated the formulationchanges. Future increases in the popularity of the squeeze bottle wouldmove further in this direction.

Pressure and Temperature Regulation

Effective utilization of formulations utilizing two or more base stocks is depend-ent upon the effective control and monitoring of the production of those basestocks that are hydrogenated. Assuming uniform purity of the feedstocks, the con-ditions of hydrogenation that influence the formation of both saturates and trans-isomers are pressure, temperature, type of catalyst, catalyst concentration, anddegree of mixing.

Pressure and temperature have historically been the hydrogenator’s principalmeans of controlling both preferential and trans-isomer selectivity. They can be

Fig. 5.4. Polyunsat urate to saturate relationship.


readily monitored and controlled within narrow limits. Their very significant effect onformation of saturates is illustrated in Figure 5.5 and on trans-isomers in Figure 5.6.Their combined resulting effect on a solids melting curve is depicted in Figure 5.7.

Fig. 5.5. Nonselective vs. selective hydrogenation.

Fig. 5.6. Effect of processing conditions on trans-isomer formation.


Use of Catalysts

Nickel catalysts possessing various degrees of selectivity and having good unifor-mity are commercially available. While catalyst concentration can have a smalleffect on selectivity, economics dictates the use of only a minimum quantity.

Mixing can be very important in the hydrogenation reaction’s selectivity, ashas been well documented in the literature (7–10). However, it has been largelyignored by processors, probably because it is difficult to measure in commercialconverters of various sizes, configurations, and mixing devices. It is anticipatedthat it will receive much greater attention in the future.

The previously mentioned parameters have been discussed only as they relateto batch-slurry hydrogenation using conventional nickel catalyst. Alternate modesand other metals, either separately or in combination, also offer advantages andwill surely receive greater attention in the future than they have in the past. Thenatural conservatism embodied in the WNDITWB (We’ve Never Done It ThatWay Before) philosophy usually results in slow changes in manufacturing tech-nology. However, unusual circumstances can speed the rate of change. The currentamount of attention being given to fat and nutrition could be the circumstance thathastens adaptation to what have previously been regarded as radical approaches incatalysis and processing technology.

For instance, nickel has been the catalyst of choice ever since the inception ofcommercial hydrogenation almost a century ago. No other metal has been foundto be comparable, either in performance or economics. Copper, based on its excep-tional linolenic selectivity, was looked at seriously about 15 years ago. However,after having been proven industrially viable, it was abandoned for several reasons.

Fig. 5.7. Influence of process conditions on the solid fat melting profile of soybean oil witha 70 IV.


A principal one was that it required its own isolated system. Also, since the hydro-genated oil still required winterization to pass a cold test, processing costs werehigher. Even more important, improvements in other processing techniques per-mitted the use of unhydrogenated oil, both in salad/cooking applications and as abase in formulating margarines and shortenings.

Precious Metal Catalysts. Laboratory investigations over the years have shownprecious metal catalysts to possess exceptional hydrogenation activity and uniqueselectivity characteristics, and at temperatures far lower than those required fornickel. These are attractive attributes. The principal stumbling block to seriousconsideration of the use of precious metals as fats and oils hydrogenation cataly-sis has always been cost. Their exceptional activity only requires an extremely lowmetal concentration, which makes reclamation and recycling very difficult. Once-through usage cannot be economically justified.

Assuming the precious metal reclamation problem could be dealt with to anendurable extent, a paper authored by Berben deserves serious attention (11). It offersa significantly greater selectivity advantage for a “modified” platinum on carbon cat-alyst than anything reported previously. To establish a base, Berben first comparedester composition results when partially hydrogenating soybean oil using a currentstate-of-the-art very selective nickel catalyst to the results from various preciousmetal catalysts. As detailed in Table 5.5 and illustrated in Figure 5.8, they essen-tially duplicated the findings of previous investigators, that is in those cases wheretrans-isomers were reduced, saturates were elevated, and the reverse. Berben thenchose the best of the precious metals (50% Pt/C) for a test to determine the effect

Fig. 5.8. Comparison of 100 IV constituents when hydrogenating with nickel and with vari-ous precious metal catalysts.


on selectivity of “modifying” the catalyst through incorporation of a small amountof ammonia into the hydrogen, as had been reported earlier by J. Kulper (12). Theresults were astounding. They are listed in column 4 of Table 5.6, along with resultsfrom an unmodified Pt/C, the nickel tested concurrently, and nickel results extrap-olated from earlier work by O’Brien (5).

Table 5.7 contains data extracted from Table 5.6 and compares the saturateand trans-isomer contents at 100 IV and 70 IV between the modified platinum andthe nickels. As can be seen, while the saturates are similar, with the modified plat-inum there is a dramatic reduction in trans-isomers. Figures 5.9 and 5.10 illustratethe phenomenon graphically. Also, as can be seen in Table 5.7, preferentialpolyunsaturate selectivity was not affected. While formulations having suitablefunctionality would need to be devised to make margarine and/or shortenings by uti-lizing base stocks hydrogenated using modified platinum catalyst, from a trans-iso-mer, saturate, and polyunsaturate standpoint, the results look exceedingly promising.

Berben’s paper is important because it quantifies the degree of formation ofsaturates and trans-isomers for a number of catalysts, including an entirely “new”one—the modified platinum on carbon, as Figure 5.11 illustrates (11). Anotherimportant point about Berben’s paper is its reminder to never discount new adap-tations of old knowledge, which may significantly alter what has been accepted asunalterable. It also can spur the probability of still additional adaptations. In thisparticular case, it makes fixed bed hydrogenation attractive, since metal reclama-tion from fixed bed catalysts is considerably more feasible than from the batch-slurry mode. Rosen and Lee Poy have previously demonstrated the viability offixed bed partial hydrogenation of vegetable oil with nickel catalyst (13–16).

TABLE 5.5Comparison of 100 IV Constituents when Hydrogenating with Nickel and Various PreciousMetal Catalysts

Nysosel325 5% Pt/C 5% Pt/Al 5% Pd/C 5% Ru/C

Temperature, °C 140 60 60 60 60Pressure, bar 10 10 10 10 10Ni, % 0.1PM, ppm 100 200 100 200Time, min 6.5 42 90 44 48Iodine value 100.5 101.2 101.8 101 .6 100.2% Saturates 18.8 24.7 29.3 17.2 21.6% C 18:1 total 47.8 37.3 28.9 50.6 43.7% C 18:1 cis 29 28.8 23.5 30.6 27.8% C 18:1 trans 18.8 8.5 5.4 20 15.9

% C 18:2 31.7 35 37.6 31.2 32.5% C18:3 1.7 3 4.2 1 2.2% Saturates 18.8 24.7 29.3 17.2 21.6% trans 18.8 8.5 5,4 20 15.9% Saturates + trans 37.6 33.2 34,7 37.2 37.5


TABLE 5.6Effect of Ammonia in Hydrogen on Preferential Selectivity and Trans-lsomer Formation at100 and 70 IV

Nysel Nysosel 325 5% Pt/C 5% Pt/C Mod.

Temperature, °C 200 140 60 60Pressure, bar 0.7 10 10 10Nickel, % 0.02 0.1PM, ppm 100 200NH3/PM molar ratio 400Time, min 18 6.5 42 50Iodine value 100 100.5 101.2 101.4% Saturates 17 18.8 24.7 19.1% C 18:1 total 52 47.8 37.3 45.9

%C 18:1 cis 23 29 28.8 39.3%C 18:1 trans 29 18.8 8.5 6.6

%C 18:2 28 31.7 35 33.8%C 18:3 3 1.7 3 1.2Time, min 45 11 86 335Iodine value 70 71.2 70 69.4% Saturates 24 25.9 46.2 27.2%C 18:1 total 75 66.9 46.1 65.1

%C 18:1 cis 24 36.1 33.5 54.7%C 18:1 trans 51 30.8 12.6 10.4

%C 18:2 2 7.2 16.1 7.7%C 18:3 0 0 0.5 0

TABLE 5.7Saturate and Trans-lsomers Comparison of Modified Pt/C with Nickel at 100 IV

Ni-O’Brien Ni-Berben Pt/C Mod.

% Saturates 17 19 19% Trans 29 19 7% Saturates + trans 46 36 26

Saturate and Trans Comparisons at 70 IV


% Saturates 24 26 27% Trans 51 31 10% Saturates + trans 75 57 37

Saturates and Polyunsaturates Comparisons at 100 IV


% polyunsaturates 31 33 35% Saturates 17 19 19P/S ratio 1.8 1.8 1.8


Other Methods of Hydrogenation

More radical approaches to vegetable oil hydrogenation have also been proposed.Yusem and Pintauro investigated the electrolytic hydrogenation of soybean oilusing an active “Raney” type nickel catalyst (17,18). While the reaction was shownto be very nonpreferentially selective, it produced few trans-isomers. Smidovnik etal. hydrogenated soybean oil with palladium on carbon catalyst (19). In both cases,

Fig. 5.9. Saturate and trans-isomer comparisons of modified Pt/C with nickel at 100 IV.

Fig. 5.10. Saturate and trans-isomer comparisons of modified Pt/C with nickel at 70 IV.


the reaction proceeded at low temperature (60–70°C). These novel approachesmerit further study.

The interesterification process has been utilized commercially since the mid-1950s. Although initially intended to make lard more suitable for use in shorten-ing, its principal application has been in the manufacture of coating fats (hardbutters). While interesterified margarine has long been available in Europe andCanada, up to this time it has not made any inroads in the United States. Thiscould change if the furor over trans-isomers continues. However, because of theprejudice that persists in the United States against ingesting either tropical oils ormeat fats, they are unlikely to be utilized in the near or mid-term future. Rather,the direction indicated in recent work by List et al. at the National Center forAgricultural Utilization Research in Peoria (20), could point the way. In it, com-pletely hydrogenated soybean or canola oil was interesterified with unhydro-genated oil. While the saturate content was similar to other formulations, therewere no trans-isomers.

Conclusion

While the future may show present catalysts used in novel ways, novel catalysts inpresent ways, and novel catalysts in novel ways, there seems no doubt that hydro-genation will continue to be, “a useful piece in helping to solve the nutrition puzzle,”

Fig. 5.11. Comparison of the performance of various catalysts.


References

1. Hastert, R.C., in Dietary Fats and Health, edited by E.G. Perkins and W.J. Visek, TheAmerican Oil Chemists’ Society, Champaign, Illinois, 1983, Chapter 4.

2. Time, January 13, 1961 (cover).3. Latondress, E.G., J. Am. Oil Chem. Soc. 58: 185 (1982).4. Latondress, E.G., in Handbook of Soy Oil. Processing and Utilization, edited by D.R.

Erickson et al., The American Oil Chemists’ Society, Champaign, Illinois, 1980, Chapter 10.5. R.D. O’Brien, in Hydrogenation: Proceedings of an AOCS Colloquium, edited by R.C.

Histert, The American Oil Chemists’ Society, Champaign, Illinois, 1980, Chapter 10.6. Bailey, A.E., and E.A. Kraemer, J. Am. Oil Chem. Soc: 254 (1944).7. Patterson, H.B.W., in Hydrogenation: Proceedings of an AOCS Colloquium, edited by

R.C. Hastert, The American Oil Chemists’ Society, Champaign, Illinois, 1987, Chapter 8.8. Bern, L., J.O. Lidefeldt, and N.H. Scheon,.J. Am. Oil Chem. Soc. 53: 463 (1976).9. Oldshue, J.Y., and A.K.S. Murthy, Chem. Eng. Progress, 76: 6 (1980).

10. Ariaansz, R.F., in Proceedings of the World Conference on Oilseed Technology andUtilization, edited by T.H. Applewhite, AOCS Press, Champaign, Illinois, 1993, p. 169.

11. Berben, P.H., B.H. Reesink, and E.G.M. Kuijpers, INFORM 5: 516 (1994).12. Kulper, J., European Patent Application 80200577.7, June 18, 1980.13. Rosen, B.I., U.S. Patent 3,123,626 (1964).14. Rosen, B.I., U.S. Patent 4,385,001 (1983).15. Rosen, B.I., U.S. Patent 4,510,091 (1985).16. Lee Poy, F.V., J. Am. Oil Chem. Soc. 64: 632 (1987).17. Pintauro, P.N., J. Am. Oil Chem.. Soc. 69: 399 (1992).18. Pintauio, P.N., U.S. Patent 5,225,581 (1993).19. Smidovnik, A., A. Stimac, and J. Kobe, J. Am. Oil Chem. Soc. 69: 405 (1992).20. List, G.R., F. Orthoefer, W. Neff, and T. Mounts,.J. Am. Oil Chem. Soc. 72: 379–382

(1995)


Chapter 6

Interesterification—Current Status and Future Prospects

Suresh Ramamurthi and Alan R. McCurdy

Department of Applied Microbiology and Food Science, University of Saskatchewan, 51Campus Drive, Saskatoon, Saskatchewan, S7N 5A8, Canada.

Introduction

Fats and oils are essential components of the diet; they are a concentrated source ofenergy, essential fatty acids, and fat-soluble vitamins. They also contribute to theflavor, texture, and palatability of the food that we consume. The natural oils andfats can be used directly or after blending. Frequently, to obtain a fat with specificphysical properties, such as melting range, solid content, and so on, it becomes nec-essary to modify them more than just by blending (1). Hydrogenation, fractionation,and interesterification of oils are the three techniques commonly adopted by theprocessor to obtain a desirable end-product (2).

The physical properties of fats and oils are dependent upon the distribution offatty acids on the glycerol backbone, the chain length of the fatty acids, and thedegree of unsaturation. The application of an oil or fat in food products is limitedby its physical properties that in turn are controlled by the previously mentionedfactors (3). Some of the properties of oils and fats, such as melting temperature,crystallization, and recrystallization form, are affected by changes in any of thethree factors. For example, shortening and tailor-made fat compositions are for-mulated to obtain a specific solid fat index profile. This is done to obtain propermouthfeel of the food formulation, aeration in case of cake and icing applications,and coating hardness in confectionery applications.

Cocoa butter contains approximately two-thirds saturated fatty acids and one-third unsaturated fatty acids. The triacylglycerol structure of cocoa butter consistsmainly of a molecule in which the unsaturated fatty acid is located in the sn-2position while the saturated fatty acids are in the outer positions. The triacylglyc-erols in cocoa butter are made up of 16% POP, 41% POS, and 27% SOS, where P,S, and O stand for palmitic acid, stearic acid, and oleic acid, respectively (4). Thisunique triacylglycerol structure and composition is responsible for the physicalproperties of cocoa butter and make it suitable for confectionery applications.

Fats and oils derived from natural animal and plant sources are endowed with aunique combination of fatty acids and their distribution on the glycerol backbone isnonrandom (5). Fats from animal sources and tropical plants, such as coconut andpalm, have a large percentage of saturated fatty acids, while oils from olive and


canola are monounsaturated due to the dominant presence of oleic acid. Oils fromsoybean and sunflower contain mostly polyunsaturated fatty acids. In vegetable oils,unsaturated fatty acids are preferentially located at the sn-2 position of the triacyl-glycerol molecules. This distribution pattern is important for the slope of the melt-ing curve of cocoa butter and for the crystal structure of lard. The oils and fatsprocessor, due to the limitations presented by nature, resorts to the use of some mod-ification techniques to obtain a product with desirable physical properties.

Interesterification

Interesterification also refers to the exchange of fatty acyl moiety between an esterand an acid (acidolysis), an ester and an alcohol (alcoholysis), or an ester and ester(transesterification [5]). When an oil or fat is subject to random interesterification,irrespective of the original nonrandom distribution of fatty acids, they are mixedcompletely until an equilibrium is reached. At equilibrium, all possible combina-tions of fatty acids on the glycerol backbone are found. This is true for chemicalinteresterification and enzymatic interesterification using nonspecific lipase (4).Hence this process is also called randomization. There are some exceptions to therandomization effect, as in directed interesterification and enzymatic interesterifi-cation (discussed separately).

Prior to the development of interesterification processes, hydrogenation wasthe major fat-modification process. Hydrogenation always results in an increase inthe melting point and the solid phase content of the raw material (6). This is dueto the decrease in the unsaturated fatty acid content and a change in the triacyl-glycerol composition. Fractionation refers to the process in which the raw mate-rial is heated above its melting point and allowed to cool under conditions thatstimulate the crystallization of triacylglycerols with the highest melting point (7).The liquid phase, olein, is then separated from the solid phase, stearin.

Of hydrogenation, fractionation, and interesterification, the only method forchanging the properties of oils without altering the structure and composition of fattyacids originally present is interesterification. This process is unique in that it only altersthe distribution of fatty acids on the glycerol backbone. Thus, interesterification isclearly distinguished from hydrogenation, in which the level of unsaturation of the fattyacids are reduced, and fractionation, where unmodified triacylglycerols or previouslymodified triacylglycerols are separated into two or more fractions through crystallization.

In the United States, hydrogenation is preferred, while companies in Europe useinteresterification to a large extent to modify their raw material (6). During the hydro-genation process some of the cis-fatty acids are converted to trans-fatty acids. Thoughnot entirely confirmed, trans-fatty acids are associated with an increase in the risk ofheart disease. Today’s consumer is more aware of the type and nature of fatty sub-stance in the food. Presently, there is a consumer trend toward oils from plant sourcesand products containing low amounts of trans-fatty acids. Thus, interesterification


of oils and fats can be used either as a stand-alone process or in combination withhydrogenation and fractionation to provide products that can perform and satisfythe nutritional requirements.

Chemical Interesterification

Interesterification can be carried out at high temperatures (>300°C) in the absenceof any catalyst. However, it requires very long reaction times, and the final prod-uct is not of desired quality due to polymerization and decomposition products.Numerous catalysts have been employed to increase the reaction rate and to carryit out at lower temperatures (around 80–100°C).

The most widely used catalysts are metallic sodium, sodium/potassiumalloys, alkylates of sodium, and hydroxides of sodium or potassium in combina-tion with glycerol (3). The sodium alkylates are less costly and are easily dis-persible in the fat. They are active at lower temperatures (50–70°C) and thereaction can be carried out under atmospheric pressure. Depending on the qualityof the starting material, catalyst dosage ranges from 0.2–0.4% of the fat. Thesodium metal requires special handling care and it reacts with water or hydroxylgroup very easily. It is usually used in the range of 0.05–0.1%. The alloy (Na/K)is a liquid and is much easier to handle. The least expensive catalyst is sodiumhydroxide, used in combination with glycerol that results in the in situ formationof sodium glycerate (8). Sufficient sodium hydroxide is added to neutralize anyfree fatty acids already existing in the starting material.

All the previously mentioned catalysts are very sensitive to the nature of the feedstock and are easily inactivated by moisture. Therefore, it is necessary to pretreat thefat to reduce the free fatty acids to less than 0.1% and to lower the water content tobelow 0.01%. Bleaching the fat to reduce the peroxide value is also recommended.

The interesterification reaction can be carried out batchwise or in a continu-ous fashion. A typical batch chemical interesterification reaction vessel is shownin Figure 6.1. The reaction is carried out at 100°C for 30 min. At the end of thereaction, the catalyst is inactivated with either water or acid, and the product isrefined. A continuous Interesterification process using sodium hydroxide andglycerol as the catalyst has been patented by Unilever (9). A flow sheet of theprocess is presented in Figure 6.2. The catalyst is premixed with the preheated fatand spray dried under vacuum. The actual reaction takes place in a short period oftime within a reactor coil at 130°C. The reaction product is treated with water andacid to inactivate the catalyst, and then washed thoroughly. The extent of inter-esterification is usually monitored by evaluation of the melting point, solid con-tent index, or some suitable standardized crystallization test.

The mechanism of the reactions occurring during interesterification have notbeen completely confirmed. There are actually two processes at work in the ran-domization of fatty acid moieties. Intraesterification refers to the shuffling of fattyacids within the same molecule, producing isomers of the same acylglycerol. Thisinitial reaction soon gives way to interesterification, a random arrangement within


all triacylglycerol molecules present. A detailed description of the two reactionmechanisms, one through formation of enolate ion and the other through the for-mation of an addition complex (carbonyl addition) of the catalyst is presented bySreenivasan (3).

The common factor between the two mechanisms is that the reaction proceedsvia a ß keto ester. Both mechanisms require the formation of an ionic intermediate

Fig 6.1. Typical batch interesterification vessel. Source: Haumann (6).

Fig 6.2. A simplified flowchart of a continuous interesterification process. Source:Rozendaal (8).


and that the fatty acyl exchange is homogeneously catalyzed by the active catalyst.During the reaction, the intermediate formation is associated with the formationof a dark brown color within 5 min of reaction initiation.

Chemical interesterification proceeds to equilibrium, at which point all of thefatty acids are randomly distributed on the glycerol. It is possible to calculate theexact proportion of different types of triacylglycerols formed when the raw mate-rials used are fully characterized. When equimolar concentrations of twomonoacid triacylglycerols (RRR and RıRıRı) are interesterified as shown inFigure 6.3, various triacylglycerols are formed according to statistical calcula-tions. The parent triacylglycerols are each present in the final product at the 12.5%level, triacylglycerols containing two R fatty acids and one Rı fatty acid constitute37.5%, and triacylglycerols containing two Rı fatty acids and one R fatty acid con-stitute 37.5%. Randomization of a model system consisting of trioleoyl triacyl-glycerol and tristearoyl triacylglycerol was found to follow this statisticaldistribution (10).

The proportion of any given triacylglycerol can be calculated in a chemicallyinteresterified mixture on the basis of random distribution theory if the fatty acidcomposition of the starting material is known (11). If X, Y, and Z are three fattyacids present, then the percentage of triacylglycerols containing three differentfatty acids is

%XYZ = %X in total oil × %Y in total oil × %Z in total oil × 0.6 × l0–4

The percentage of triacylglycerols containing two fatty acids is%XXY= (%X in total oil)2 × %Y in total oil × 0.3 × 10–4

The percentage of triacylglycerols containing one fatty acid is%XXX= (%X in total oil)3 × 10–4

Triacylglycerols containing short-chain fatty acids and long-chain fatty acidshave been prepared using sodium methoxide Catalyzed interesterification of short-

Fig 6.3. Chemical interesterification of equimolar mixtures of two monoacid triacylglycerols(R and Rı are fatty acyl groups). Source: Macrae and Hammond (1).


chain fatty acid triacylglycerols and long-chain fatty acid triacylglycerols (12).The triacylglycerols, containing either one, two, or three long-chain fatty acids,calculated by the statistical model for the random distribution was found to agreewith the analytical data.

An example of Na/K Catalyzed interesterification of high erucic acid rape-seed oil (HEAR) is presented (13). Native HEAR oil consists of 41% erucic acidthat is located almost exclusively in the sn-1,3 positions of the triacylglycerol(Table 6.1). Positional analysis of the randomized HEAR revealed a similar fattyacid composition content for monoacylglycerols, diacylglycerols, and triacylglyc-erols. Thus, the HEAR oil that originally did not contain any erucic acid in the sn-2 position of the triacylglycerol, on interesterification contained around 40%erucic acid in the sn-2 position.

Figure 6.4 shows the reverse-phase high-performance liquid chromatography(HPLC) profile of the HEAR oil before and after interesterification. The presenceof trierucoyl triacylglycerol in the interesterified HEAR oil indicates randomiza-tion. Upon randomization the number of triacylglycerol peaks on the HPLC chro-matogram increased in comparison to the chromatogram of HEAR oil, indicating theformation of different combinations of fatty acids in the triacylglycerol molecules.

To our best knowledge, selectivity through chemical interesterification hasbeen shown in only one study. Konishi et al. (14), studied the sodium methoxideCatalyzed interesterification reaction between soybean oil and methyl stearate.When the reaction was carried out at 30°C in hexane, it was observed that after24 hrs the acyl exchange at sn-1,3 position progressed 1.7 times faster than at sn-2 position of the triacylglycerol. However, the reaction proceeded slowly at thistemperature.

TABLE 6.1Positional Fatty Acid Composition (mole %) of Native High Erucic Acid Rape-seed (HEAR)Oil and Randomized HEAR Oil

Native HEAR oil Randomized HEAR oilFatty acid sn-2 sn–1,2 (2,3) TAG sn-2 sn-1,2 (2,3) TAGC16:0 03 2.8 3.3 4.1 3.6 3.3C18:0 0.1 1.2 1.2 1.4 1.3 1.2C18:1 36.3 25.4 19.1 20.5 20.4 18.9C18:2 36.2 20.0 14.4 14.3 13.8 14.4C18:3 25.7 12.2 9.5 8.6 7.6 9.5C20:0 0.0 0.6 0.8 0.8 0.8 0.8C20:1 0.3 6.0 8.5 8.4 8.7 8.4C22:0 0.0 03 0.5 0.5 0.4 0.5C22:1 1.2 30.6 41.4 40.9 42.0 41.6C24:0 0.0 0.1 0.2 0.0 0.2 0.3C24:1 0.0 0.2 03 0.0 0.4 0.5Other 0.0 0.2 0.4 0.0 0.4 0.5Abbreviation: sn, stereospecific number; and TAG, triacylglycerol.Source: Grewal (13).


Directed Interesterification

Chemical interesterification leads to random distribution of fatty acids if the reac-tion is carried out in a single phase (8). However, when this reaction is carried outat temperatures below the melting point of the highest melting triacylglycerol, thereaction equilibrium is shifted to the synthesis of the highest melting triacylglyc-erol (5). This is because the crystallized triacylglycerol, does not participate in thereaction once it is out of the solution. This process proceeds until all possible highmelting triacylglycerols are produced at that temperature. Thus, a liquid oil con-taining significant amounts of saturated fatty acids can be converted to a productof desired consistency. The idea of directed interesterification can be extended toa system in which a desired fatty acid can be added to increase its incorporationin the triacylglycerol produced. The undesirable fatty acids can be removed, eitherby distillation (if sufficiently volatile) or through solvent extraction. Applicationof directed interesterification has some limitations. Since the reaction temperatureis low, the reaction itself proceeds slowly. It normally takes a long time (>24 hr)for the crystallization of the highest melting triacylglycerol. Also, loss of catalystactivity due to coating of the catalyst is frequently encountered.

Lipase-Catalyzed Interesterification

As an alternative to chemical catalysts, enzymes have been used to catalyze inter-esterification reactions. In aqueous systems, lipases (acylglycerol ester hydrolaseEC 3.1.1.3) are hydrolytic enzymes that break down triacylglycerols into free fattyacids and glycerol (15). They constitute a ubiquitous group of enzymes that do notrequire a cofactor and exhibit maximum catalytic activity at an oil-water interface.It is now very well established that under conditions of limited water the hydroly-

Fig 6.4. HPLC chromatograms of native HEAR oil and randomized HEAR oil. Abbreviation:TETAG, trierucoyl triacylglycerol. Source: Crewal (13).


sis reaction catalyzed by the enzyme can be reversed (Figure 6.5 [16]). Underthese conditions, lipases can catalyze a wide variety of reactions, many withindustrial potential. Among these reactions, the use of lipase to modify interester-ification is the best known example of their use in organic media.

There are many advantages to the use of lipase in place of alkaline catalysts.Lipases offer a wide range of specificity in interesterification reactions (17). It ispossible to produce triacylglycerols with specific fatty acids in specific sn-posi-tions that would otherwise be impossible to prepare with chemical catalysts thatproduce randomized products. Typically, the enzyme-catalyzed reactions are car-ried out at lower temperatures. This is suitable from two viewpoints: lower energycosts and higher quality of end-product. With lipases as catalysts, fatty acids, fattyacid esters, or triacylglycerols can be used as reactants. With chemical catalysts itis not possible to use fatty acids, as they tend to form soaps. Unlike the chemicalcatalysts, lipases are not highly sensitive to the presence of moisture and othercontaminants in the starting material. However, the presence of a large amount ofwater is deleterious for an enzymatic reaction, as the hydrolysis reaction is favoredrather than the acyl exchange reaction.

Lipases perform well in a wide variety of organic solvents and also in phasessuch as supercritical carbon dioxide. The reaction phase could be an organic solventor solvent-free, biphasic, microaqueous, reverse-micellar, or even with immobilizedsubstrates/enzyme (18). The use of immobilized lipases is advantageous, especially

Fig 6.5. Lipase-catalyzedester hydrolysis and syn-thesis. Source: Miller et al.(16).


in interesterification reactions where a low water activity is required to depresshydrolytic reactions. Immobilized enzymes have increased stability, therebyincreasing their lifetime and allowing for the application of continuous processes(19). Also, the cost of the lipase/unit of product is reduced. New products that areproduced utilizing enzymes are considered “natural” by consumers, giving addedmarketing value (6).

Lipase Specificity

Lipase specificity can be broadly classified (17,20) into five groups:

1. Lipid class;2. Positional;3. Fatty acid;4. Stereochemical; and5. Combinations of any of the above.

Examples of some of the known lipase specificities are listed in Table 6.2.A lipase produced by a strain of Penicillium cyclopium has been shown to dis-

play preference towards monoacylglycerols in comparison to di- and triacylglyc-erols (21). Lipases can be positionally specific with respect to the sn-position ofthe triacylglycerol molecule. They can be positionally nonspecific or can be sn-1,3 specific.

Nonspecific lipases, such as from Candida cylindracea, do not have anyspecificity for any of the sn-positions in the triacylglycerol molecule. During aninteresterification reaction with a nonspecific lipase, the products would be ran-domized in a fashion similar to a chemically catalyzed reaction (as shown inFigure 6.3).

When sn-1,3 specific lipases are used to catalyze an interesterification reac-tion, the action of the lipase is substantially confined to the sn-1 and.sn-3 posi-tions of the triacylglycerol molecule. A lipase from Mucor miehei has been shownto be sn-1,3 specific (2).

TABLE 6.2Specificities of Some Lipases

Source SelectivityPenicillium cyclopium MonoacylglycerolCandida cylindracea sn-nonspecificPscudomonas fluorescens sn-nonspecificRhizopus arrhizus sn-1,3 specificAspergillus niger sn-1,3 specificGeotrichum candidum sn-nonspecific, C18 (cis ∆9) fatty acidMucor miehei sn-nonspecific, C4 and C6 (hydrolysis)Human lipoprotein lipase Fatty acid and stereoselective (hydrolysis)

Abbreviation: sn, stereospecific number.Source: Sonnet (17) and Malcata et al. (20).


Some lipases show selectivity toward long-chain unsaturated fatty acids thathave a cis double bond in 9-position from the carboxylate group of the fatty acid.Fatty acids, such as oleic, palmitoleic, linoleic, and linolenic acids, or their esters arepreferentially hydrolyzed. esterified, or interesterified. Such lipases exhibit reducedactivity on substrates that have an additional double bond between the carboxylgroup and the 9-position and on triacylglycerols containing medium-chain fattyacids. An example is a lipase produced by the mold Geotrichum candidum (22).

The specificity of the lipase to the sn-1,3 position is believed to be related tosteric factors (23) that prevent the access of the sn-2 carbon moiety to the activesite of the lipase rather than due to stereospecificity. Recently, lipases have beenused rather extensively for the preparation of chiral esters and alcohols (17).Lipase stereoselectivity has been observed only with nontriacylglycerol mole-cules. Combinations of fatty acid specificity and stereoselectivity has beenobserved in human lipoprotein lipase preparation (24). Examples of some of thelipase-catalyzed reactions are illustrated in Figures 6.6a, 6.6b, and 6.6c.

Kinetics of Lipase-Catalyzed Interesterification

Even though plenty of experimental data is available for lipase-Catalyzed inter-esterification reactions, it has not been mathematically modeled as has been donefor lipase-catalyzed hydrolysis and ester synthesis (20). Information on the kinet-ics of reaction sheds light on the reaction mechanism and also is useful for futurescale-up. Interesterification reactions proceed through intermediate stages involv-ing both hydrolysis and esterification.

The Candida cylindracea lipase-Catalyzed interesterification reaction wasstudied using a model system consisting of lauric acid, dilauroyl glycerol, and tri-lauroyl glycerol in cyclohexane (25). It was suggested that the hydrolysis step inthe interesterification process may be the rate-limiting step, as the enzymaticturnover for the esterification step was found to be three times faster than thehydrolysis step. The esterification step was assumed to follow a ping-pong bi-bimechanism that was supported by experimental data.

When studying the esterification of oleic acid and methanol in hexane that was cat-alyzed by an immobilized lipase from Candida antarctica (26), the kinetics of the reac-tion were observed to follow a ping-pong bi-bi mechanism in which methanol was asubstrate inhibitor of the lipase. A similar mechanism was observed in transesterifica-tion reactions catalyzed by lipase (27,28). Knowing that the hydrolysis step is the rate-limiting step in the interesterification process, it would be advantageous for industrialapplications to use a lipase in a solvent system that promotes faster hydrolysis.

Interesterification Applications

Numerous applications of interesterification of oils and fats have been reported.Some of the recent applications are mentioned in this chapter. The major application


areas include preparation of zero-trans or low-trans fatty acid shortening or mar-garine, synthesis of cocoa butter-like fat, modification of butterfat, modificationof palm oil to increase its utilization, and preparation of structured lipids contain-ing medium-chain fatty acids and polyunsaturated fatty acids.

Fig 6.6. a) Lipase-catalyzed (sn-1,3 specific) interesterification of triacylglycerols. b) Lipase-catalyzed (sn-1,3 specific) interesterification of triacylglycerol and free fatty acid. c) Fattyacid specific lipase-catalyzed (specific for A and B) interesterification of triacylglycerol andfree fatty acids. A, B, and C are fatty acyl groups. Source: Macrae and Hammond (1).


Zero-Trans or Low-Trans Margarines

Complete hydrogenation accomplishes the total saturation of unsaturated fattyacids. However, with partial hydrogenation, trans-fatty acids arc formed that aregeometric isomers of the cis-fatty acids. The metabolic effects of these fatty acidsare under investigation.

Interesterification of a liquid oil with a hard fat has long been used in Europeas an alternative to hydrogenation to prepare plastic fat suitable for use in mar-garines. Such a plastic fat has been prepared by List et al. through interesterificationof soybean oil with completely hydrogenated soybean oil (29). Randomization wasfound to have no detrimental effect on the flavor and oxidative stability of theproduct. Sodium methoxide catalyzed interesterification of refined, unhydrogenatedsoybean oil (60%) and edible beef tallow (40%) resulted in a product that was sim-ilar to commercial tub margarine oils (30). The interesterified fat contained 3% transfatty acids that were originally present in the tallow.

Low-trans fatty acid margarine fat has been prepared through lipase-catalyzed(both sn-1,3 specific and nonspecific) interesterification of cottonseed oil and fullyhydrogenated soybean oil (31). A decrease in the amount of triunsaturated and trisat-urated triacylglycerols and an increase in the amount of mono- and diunsaturatedtriacylglycerols was observed. An increase in the relative stability of the ß’ crys-talline form (desired in margarine) was also observed in such margarine fats (32).

Zeitoun et al. (33) prepared chemically interesterified fats from mixtures offully hydrogenated soybean oil and commonly used vegetable oils, such ascoconut, cottonseed, peanut, soybean, corn, sunflower, safflower, and canola oil.They suggest the preparation of zero-trans margarines from the interesterifiedmixtures. The physical properties of the interesterified product are stronglyaffected by the type of liquid oils used. Oils that contained significant amounts ofpalmitic acid were found to form a product with favorable melting characteristicsand crystallization behavior.

To prepare a margarine that contains low amounts of trans-fatty acid (<10%)that is easily spreadable, contains a high proportion of unsaturated fatty acids, andhas good sensory properties, Schmidt et al. have combined both interesterificationand fractionation processes (34). They randomly interesterified a mixture of a sat-urated fat and unsaturated oil. The olein fraction obtained on fractionation of theinteresterified product was mixed with a portion of a nonhydrogenated or a par-tially hydrogenated oil (that was substantially free from crystallized fat at 10°C)and a portion of the interesterified product.

Processors can combine any of the three processes, interesterification, hydro-genation, and fractionation, and start with a blend of oils and fats to obtain productsof desired nutritional, physical, and sensory properties. An example is presented inFigure 6.7, where all three processing techniques are used in the preparation of amargarine-fat blend based on sunflower and rapeseed oil (7). A mixture is preparedwith part of the oil and completely hydrogenated oil. Due to the presence of a largepercentage of unsaturated fatty acids, it has a high solid phase content. The solid


phase content is reduced through interesterification and further reduced throughthe fractionation process to remove saturated triacylglycerols.

Schmidt from Unilever has patented the preparation of low-trans fats andemulsion spreads combining hydrogenation and interesterification (35). The rawmaterial includes vegetable oil (soybean, sunflower, rapeseed, etc.), fully hydro-genated oil (sunflower, soybean, rapeseed, etc.), saturated oil (coconut, palm ker-nel, babassu, etc.), and palm oil or partially hydrogenated palm oil. This rawmaterial is interesterified with 0.1% sodium methylate at 90°C for 20 min. Thepurified product is blended with some liquid oil to obtain the desired final product.

Brown et al. from Kraft General Foods Inc. have patented the preparation ofa margarine oil containing low-trans fatty acids and with low intermediate chain(C8–C16) fatty acid content (36). They interesterified a mixture of vegetable oiland a stearic acid source material in hexane using a sn-1,3 specific lipase. Theremaining fatty acid mixture was hydrogenated to replenish the stearic acid forfurther reaction. They also developed a countercurrent process using a supercriti-cal gas to separate the products from the reaction zone once the reaction was com-pleted. This patent covers a wide variety of products that can be prepared usinginteresterification.

Cocoa Butter–Like Fats

Cocoa butter is largely used in the manufacture of chocolate and confectioneryproducts in which its unique consistency and melting properties are desired. Among

Fig 6.7. Interesterification and fractionation of mixtures of nonhydrogenated and fully hydro-genated vegetable oils. Source: Rozendaal (7).


all of the commercial oils and fats, cocoa butter demands the highest price, but theprice fluctuates widely. Cocoa butter-like fats are produced for economic reasonsand also to offset the supply uncertainties (4,37). Chong et al. have prepared cocoabutter-like fat through fractionation of interesterified palm olein (38). Palm oleinwas interesterified with stearic acid using an sn-1,3 specific lipase. The inter-esterified product was initially steam distilled to remove the free fatty acids andfurther fractionated using the solvents hexane and acetone. The product was mixedwith hexane and left to stand at 4°C for 24 hr; this was followed by the separationof the crystallized fat from the mother liquor. The liquid fraction was further frac-tionated with acetone at 4°C yielding 25% cocoa butter–like triacylglycerols. Changet al. have prepared cocoa butter–like fat through enzymatic interesterification ofhydrogenated cottonseed oil and olive oil using a similar process, except they onlyused acetone for the fractionation step (39).

Lipases can catalyze reactions in organic solvents and also in phases, such assupercritical carbon dioxide (40). Lipases can perform admirably well in supercrit-ical carbon dioxide (41–43). Lipase-Catalyzed Interesterification of trioleoyl tria-cylglycerol and stearic acid has been carried out in supercritical carbon dioxide toprepare a cocoa butter substitute (44). The versatility of lipases offer numerouspotential possibilities.

Microbial sources have been lapped for the production of cocoa butter equiv-alents (45,46). This attempt used yeasts in a substrate of whey; however, it provedunsuccessful because the price of cocoa butter on the world market fell. Thisprocess can be applied if the price of the cocoa butter increases, or if a less expen-sive substrate for the yeast is found.

Davies et al. from Unilever have filed a patent for the preparation of sym-metric triacylglycerol that contains two saturated fatty acids and one unsaturatedfatty acid (SUS-type [47]). This process involves the fractionation (from –5°C to10°C) of the raw material to remove stearin and olein fractions and is followed byfurther fractionation (–5°C to –30°C) of the olein fraction to obtain a stearin richin triacylglycerols of SUU-type. This stearin fraction is interesterified using sn-1,3 specific lipase to obtain SUS-type triacylglycerols that can be used as cocoabutter equivalents. Such a product has been prepared from shea fat, sal fat, palm,soybean, and high oleic acid sunflower oils. Among the different raw materials,shea fat was found to be highly suitable as it resulted in greater yields.

Butter Fat

There is considerable interest in preparing structured triacylglycerols for use in par-enteral nutrition and in infant formulas. Christensen and Holmer interesterified (at22°C) a mixture of butter oil and concentrates of polyunsaturated fatty acids, oleicacid, and linoleic acid in hexane using a commercially produced enzyme. Thepolyunsaturated fatty acid composition of the product closely resembled that ofhuman milk as did the positional distribution of n-3 and n-6 polyunsaturated fattyacids and monounsaturated fatty acids.


Modification of triacylglycerols in butter fat has been studied by Safari et al.using a commercial immobilized lipase (49). Hexane, hexane-chloroform (70:30,v/v), and hexane-ethyl acetate (70:30, v/v) were used as the reaction medium sep-arately. It was observed that hexane-chloroform was the most suitable for theinterestedfication of butter fat. Safari and Kermasha have interesterified butterfatin a microemulsion system using four commercial sn-1,3 lipases in order to alterthe location of oleic acid on the triacylglycerol molecule (50). They found thatthree of the lipases increased the palmitic acid content at the sn-2 position of thebutter fat triacylglycerol while one of the lipases increased the proportion of oleicacid at the sn-2 position.

Yu et al. (51) have interesterified canola oil and anhydrous milk fat in supercriti-cal carbon dioxide using lipase from Candida cylindracea. The final product con-tained triacylglycerols with carbon numbers ranging from C42–C50 and C54 whilethe starting material contained predominantly C52–C56 triacylglycerols from canolaoil and C32–C38 triacylglycerols from anhydrous milk fat. Using supercritical carbondioxide they have also successfully esterified ethanol, oleic acid, and fatty acids fromanhydrous milk fat. Marangoni et al. have prepared a modified fat for use as an edi-ble plastic fat from trioleoyl triacylglycerol and tripalmitoyl triacylglycerol (52). Theinteresterification reaction, catalyzed by lipase from Rhizopus arrhizus, was carriedout in reverse micelle formed with canola lecithin and hexane. The application of thisprocess is being directed toward the modification of butter fat.

Structured Triacylglycerols Containing Medium-Chain Fatty Acids and Polyunsaturated Fatty Acids

Triacylglycerols containing both short-chain fatty acids and long-chain fatty acidsare being prepared to replace conventional fats. These synthetic fats provide thesame functional properties but have additional nutritional properties in compari-son to the fats they replace. The nutritional value and absorbability of triacylglyc-erols depend not only on their fatty acid composition but also on their distributionon the glycerol backbone (53,54).

There is a lot of interest in the preparation of structured lipids in which spe-cific fatty acids arc located on specific positions in the glycerol. These structuredlipids are difficult or impossible to prepare using chemical means, therefore enzy-matic approaches are resorted to. The nutritional properties of the medium-chainfatty acids (metabolized rapidly, like carbohydrates) and very long chain fatty acids(poorly absorbed) are different from the other fatty acids normally present in veg-etable oils. Interesterification, catalyzed by sn-1,3 lipases, is a key step in the prepa-ration of medium-chain triacylglycerols. These triacylglycerols are used to providea dense form of calories to patients with pancreatic deficiencies and malabsorptionproblems. These triacylglycerols are easier to hydrolyze and are absorbed more effi-ciently. But these triacylglycerols do not contain any essential fatty acids.

This problem has been overcome by using structured triacylglycerols contain-ing medium-chain fatty acids in the sn-1 and sn-3 positions and an essential fatty


acid in the sn-2 position (55). Procter and Gamble has patented a structured triacyl-glycerol (56). The triacylglycerol contains both a long-chain fatty acid, such asbehenic acid (C22:0), and medium-chain fatty acids, capric and caprylic acid(C10:0 and C8:0). The sources of medium-chain fatty acids are coconut or palm ker-nel oils, while the source for behenic acid is rapeseed (probably fully hydrogenated)oil. Tremendous potential is foreseen in the synthesis of specific triacylglycerols andlipids that can be targeted to improve health or cure a particular disorder.

Shishikura et al. have studied the incorporation of long-chain polyunsaturatedfatty acids, such as eicosapentaenoic acid (EPA) and docosahexaenoic acid(DHA), into medium-chain triacylglycerols (57). They combined sn-1,3 specificlipase-Catalyzed interesterification with simultaneous extraction of the releasedmedium-chain fatty acids with supercritical carbon dioxide. Their reaction schemeis illustrated in Figure 6.8. It was possible to incorporate up to 62 wt% EPA withan overall 85–92% triacylglycerol recovery by using a continuous medium-chainfatty acid recovery system. It was observed that the lipase was activated in the pres-ence of glycerol. The interesterification reaction was dependent on the removal ofthe released medium-chain fatty acids.

The mild enzymatic modification offers several advantages when preparingtriacylglycerols containing high amounts of polyunsaturated fatty acids, such asEPA and DHA, as these fatty acids, and oils containing these fatty acids, readilyoxidize. Homogeneous triacylglycerols containing either EPA or DHA have beenprepared by Haraldsson et al. using a nonspecific lipase from Candida antarctica(58). The reaction involved the direct esterification of glycerol and interesterifi-cation of tributyroyl triacylglycerol with stoichiometric amounts of 99% EPA orDHA (either as free fatty acids or as ethyl esters) under vacuum without the useof a solvent. The volatile products (either butyric acid or its ester) were removedthrough condensation in a cold trap to push the reaction toward synthesis of tria-cylglycerols containing EPA and DHA. Yields of the crude product in excess of93% were obtained by this process.

Fig 6.8. Preparation of triacylglycerols containing medium-chain fatty acids and polyunsat-urated fatty acids. Abbreviations: MCT, medium-chain triacylglycerol; EPA, eicosapen-taenoic acid; and DHA, docosahexaenoic acid. Source: Shishikura et al. (57).


Other Applications

It is predicted that the world production of palm oil will catch up with soybean oil(59), presently the major vegetable oil. To improve the functionality and nutri-tional properties of palm oil and its fractions, Graille et al. have interesterifiedpalm oil or palm stearin with oils, such as coconut, palm kernel, soybean, ricebran, borage, and Myrianthus arboreus (contains about 90% linoleic acid) oils,using sn-1,3 specific lipase in a fixed catalyst bed reactor (60). The reactions wereessentially complete in about 5 hr (flow rate 3.5 mL/hr). Products with differentcharacteristics could be obtained by withdrawing the product at different timeintervals. A mixture of palm stearin and palm kernel oil (30:70, w/w) after a 30min reaction gave a firm margarine fat and after 3 hr gave a soft margarine fat.Interesterification with oils such as soybean resulted in either a plastic fat or an oilthat was virtually fluid at 20°C that could be used as salad oils in tropical countries.

Porcine pancreatic lipase-Catalyzed interesterification of canola oil and mix-tures of canola oil with lauric acid, or trilauroyl triacylglycerol, or fully hydro-genated high erucic acid rapeseed oil was studied by Thomas et al. (61). Thesolidification points of interesterified products were considerably lower than thestarting mixtures as shown by cloud point temperatures in Table 6.3.Interesterification resulted in the formation of triacylglycerols that were not orig-inally present in either of the starting materials.

Specialty triacylglycerols, such as high erucic acid triacylglycerols, have beenprepared using direct esterification and interesterification approaches (62,63).Triacylglycerols with high levels of erucic acid were first prepared using directchemical esterification of erucic acid and glycerol (62). The synthetic triacylglyc-erol (containing >90% erucic acid) was interesterified with native HEAR oil (con-taining 45% erucic acid) to obtain triacylglycerols containing varying proportionsof erucic acid (ranging from 45–92%). These triacylglycerols were then studied togather information on their physical properties.

Development of a sound understanding of the bioengineering aspects is essen-tial for marketing new products using new techniques. Izumoto et al. have opti-mized the production of a product of constant triacylglycerol composition from a

TABLE 6.3Cloud Point of Starting Mixtures and Interesterified Mixtures

Cloud point (°C)Mixture Starting mixture Interesterified mixtureCanola + 5% lauric –8 –13Canola + 5% trilauroyl triacylglycerol –10 –16Canola + 10% HEAR 40 11Canola + 20% HEAR 45 30Canola + 30% HEAR 48 34Canola + 40% HEAR 52 40Abbreviation: HEAR, high erucic acid rapeseed oil.Source: Thomas et al. (61).


microaqueous bioreactor using filamentous immobilized fungus from Rhizopuschinensis (64). For the continuous interesterification reactor, a feedforward/feed-back controller with an on-line enzyme activity estimator was used. The ratio ofdistearoyl monooleoyl triacylglycerol to the total triacylglycerols was estimated todetermine the enzyme activity. The usefulness of the controller was justified byrunning simulations along with the experiments.

The interesterification process has been used to prepare modified fat that canbe used as a fat substitute and as nutritional ingredients in food. Cooper has pre-pared a reduced calorie admixture of a substantially digestion resistant esterifiedalkoxylated polyol and a digestible triacylglycerol (65). As an example, equalparts of esterified propoxylated glycerine product (containing C6–C18 fattyacids) and fully hydrogenated rapeseed oil are mixed. The mixture is interesteri-fied at 150°C with 0.6% sodium methoxide for 3 hr. The crude product is refinedto obtain a final product that contributes only 3.5 calories/g. Charton et al. fromUnilever have produced a new lipase from Geotrichum candidum (66). Thislipase, called “lipase B,” has very high specificity for ∆9-cis fatty acids and canbe used for the production of oleic acid through the hydrolysis of high oleic acidsunflower oil. It can also be used for the preparation of specific triacylglycerolsfor nutritional purposes through interesterification of symmetrical acylglycerolscontaining oleic acid with polyunsaturated fatty acids, such as linoleic and ∂-linolenic acids. The inventors predict that these products will be desirable ingredi-ents in food products, since they combat high levels of cholesterol in human blood.

The specificity of the lipase offers some advantage when products of desiredcharacteristics are needed. Foglia et al. used two lipases that have different specifici-ties, one that is sn-1,3 specific and the other that is cis-∆9, C18 specific, for the inter-esterification of high oleic sunflower oil and soybean oil with tallow and butterfat(67). Different products were formed with the two lipases showing the potential oflipase specificities. The melting point of a tallow-rapeseed oil (LEAR) mixture waslowered by altering its triacylglycerol composition through sn-1,3 specific lipase(from Mucor miehei)-Catalyzed interesterification without the use of an organic sol-vent in the reaction media (68). After a 24 hr reaction time, the melting point of theinteresterified product was 30°C in comparison to 42°C of the starting material.

To improve the fluidity of palm oil, Kurashige et al. interesterified mixturesof palm oil and canola oil or soybean oil with an sn-1,3 specific lipase fromRhizopus delemar (69). They found canola oil to enhance the fluidity more thansoybean oil. Interestingly, chemical interesterification of the same blends of palmoil with canola did not alter the fluidity of the product. The increase in fluidity wasprobably due to decrease in trisaturated and disaturated triacylglycerols in theenzymatically interesterified product in comparison to the original blend.

Oxidative Stability of Interesterified Fat

The oxidative stability of interesterified mixtures has been studied by a number ofresearchers (70,71). It is believed that the rate of autoxidation of triacylglycerols is


dependent on the fatty acid composition and on their distribution. Both chemicaland enzymatic interesterification techniques have been utilized to study the effectof the positional distribution of fatty acids on the rate of autoxidation. There existsconflicting results in the literature regarding the effect of randomization on theoxidative stability of the oil. Randomization caused decreased stability in cocoabutter, tallow, corn, and soybean oil (71); and lard (72); and increased stability inpalm oil (72). In some studies no difference in the stability of native and random-ized oils was observed (73,74). A possible explanation is that methylesters andsoaps formed in the reactions where sodium methoxide is used as a catalyst aremore likely to oxidize faster than the original triglycerides.

Tautorus and McCurdy compared the oxidative stability of mixtures of tri-oleoyl triacylglycerol and linseed oil that were either blended together or enzy-matically randomized (75). They observed that when the ratio of unstable to stabletriacylglycerols is low, the randomized mixture was more stable than the blendedmixture, and when the ratio was increased the stabilizing effect was not seen(Figure 6.9). They hypothesized that the stabilizing effect was due to the dilutionof less stable fatty acids.

Marine oils rich in the polyunsaturated fatty acids EPA and DHA, such aswhale, sardine, cod liver, and skipjack oils, are assuming importance due to theirnutritional value and are widely used for the preparation of specialty lipids.Kimoto et al. have studied the relationship between the triacylglycerol structure ofmarine oils and their oxidative stability and have also studied the effect of chemicaland enzymatic interesterification (76). Enzymatic interesterification resulted in

Fig 6.9. Oxidative stability (measured as absorbance at 234 nm and indicative of conjugateddiene content) of mixed and randomized trioleoyl triacylglycerol/linseed oil blends (wt/wt)as a function of storage at 52°C. Source: Tautorus and McCurdy (75).


increased stability of fish oils, while chemical interesterification resulted in theincreased stability of all oils except sardine oil and whale blubber oil.

Safety

New products (or novel products), such as those produced by chemical or enzy-matic interesterification, have to be declared safe by the concerned agencies intheir respective countries. These products have to be proven safe by the manufac-turer. An enzymatically interesterified oil produced by the Fuji Oil Company, Ltd.,from safflower oil or sunflower oil and ethyl stearate has been cleared by theAdvisory Committee on Novel Foods and Processes in the United Kingdom withsome restrictions (77). The restrictions include the following:

1. The raw material used must be food-grade quality;2. Only food-grade solvents can be used in the process;3. The product should only be used as a substitute for cocoa butter, and the

intake of saturated fatty acids should not be increased; and more importantly,4. The lipase has to be declared safe by the Food Advisory Committee (FAC) and

the Committee on Toxicity of Chemicals in Food, Consumer Products, and theEnvironment (COT) for use in food in United Kingdom.

The modified oil is to be used as a substitute for cocoa butter in chocolate andconfectionery products. This product has been produced in Japan since 1986 andwas accepted for use in food in Japan. Unilever has also received clearance fromthe same committee for an interesterified product from vegetable oils and food-grade fatty acids (78). The reaction in this case is carried out in hexane. The prod-uct is intended for use in chocolate and confectionery fat and as an ingredient atlevels of up to 20% in frying fats as a replacement for saturated fats. The specifica-tions for the product are listed in Table 6.4. The manufacturers also provide informa-tion related to the toxicity of the product, such as LD50 values and mutagenicity.

The lipases that are used in the preparation of novel products also have to beapproved and declared safe (79). Safety evaluation for an enzyme is focused onthe presence of toxic contaminants (raw material, foreign microorganisms, addi-tives and preservatives used, etc.) in the commercial enzyme preparation and, if theenzyme is immobilized, on the safety of the carrier (leakage under forced conditions).

Even though we have some information on the fate of the fatty acids from fatsubstances when ingested, at present we do not entirely understand the metabolicsignificance of the positional distribution of fatty acids in the triacylglycerol mol-ecules on plasma lipids. A metabolic balance study was carried out with infantsthat were fed with infant formulas containing either natural lard (which containspalmitic acid primarily in the sn-2 position) or randomized lard (80). The rest ofthe infant formula composition was the same. It was observed that infants fed with theformula containing natural lard excreted 0.3 g fat/kg/day while the infants fed with


the formula containing randomized lard excreted 1.79 g fat/kg/day. It was postu-lated that the greater absorption of natural lard was due to the greater extent ofmicellization of sn-2 monopalmitoyl acylglycerol.

In a more recent study by Innis et al. (81), it has been shown that palmitic acidis absorbed from human milk as sn-2 palmitoyl acylglycerol. Infants were exclu-sively fed with human milk (containing 21.0% palmitic acid with 54.2% of thetotal palmitic acid residing in the sn-2 position) or formula milk (containing22.3% palmitic acid with 4.8% of the total palmitic acid residing in the sn-2 posi-tion). They found that the plasma triacylglycerols of infants fed with human milkhad 26.0% palmitic acid with 23.3% of the total palmitic acid in the sn-2 position,while the infants fed with formula milk had the same total palmitic acid content,but the sn-2 position of the triacylglycerols contained only 7.4% palmitic acid.

In one study, native peanut oil was found to be more atherogenic than corn oil(82). But when peanut oil was randomized, it was found to have the same effect ascorn oil (83). The connection between dietary triacylglycerols and diseases has tobe studied closely in order to apply new techniques to prepare the desired fat prod-uct. In this case, the demand for a particular product with nutritional benefitswould motivate researchers and processors.

Future Prospects and Conclusions

Several factors are involved in the development and marketing of a new fat prod-uct, especially if it is a designer fat that replaces already existing natural products.The development of tailor-made triacylglycerols is motivated and mainly drivenby health questions concerning fat components, which in turn either stimulate orreduce consumer demand for that product. Manufacturers need to prepare spe-cialty fats that satisfy niche markets, and processors that require new materials tosubstitute for or improve upon the performance of the fat to be replaced. In addi-tion to these factors, there is the question of cost feasibility.

TABLE 6.4Specifications for Enzymatically Interesterified Product

Total acylglycerols >99%Triacylglycerols >93%Diacylglycerols <6%Monoacylglycerols <0.1%Free fatty acids <0.1%Oxidation products <1%Heavy metals 0.2 mg/kgColor ColorlessOdor/taste BlandResidual hexane <1 mg/kgResidual acetone <2.5 mg/kgLipase activity NoneNitrogen <5 mg/kg

Source: U.K. Ministry of Agriculture (77).


Lipid chemists, food scientists, and food regulators have to address severalquestions regarding the definition of natural fat or oil products. There is a ques-tion regarding authenticity, if a processor separates a particular fatty acid fromsay, an animal fat or a tropical oil (the use of these fats was questioned in the pastdecade), and utilizes these fatty acids to prepare a designer triacylglycerol ofdesirable properties. Should the label actually carry the source of the fatty acid?Is the fatty acid different if it is obtained from a fully hydrogenated vegetable oil,or does it really matter where the fatty acids are from, if they are pure?

Today’s consumer is more knowledgeable and has more understanding of thefood components than the consumer of the past. More research is necessary toascertain the effect (both beneficial and detrimental) of different fatty acids(short-and long-chain saturated fatty acids, medium-chain fatty acids, and polyun-saturated fatty acids) on absorption and metabolism in the human system. Muchwork needs to be done on the effect on the human metabolism of the distribution offatty acids on the triacylglycerol that is ingested, on the end-products of these fattyacids in the human body, and their positional distribution in body triacylglycerols.

It is also necessary for the bioprocess engineer to be able to devise continu-ous reactors for development of these designer fats. Mathematical modeling of thereaction system under consideration is necessary to predict the production of spe-cific triacylglycerols. Such reactors also would need on-line detectors to monitorproduct formation and estimate biocatalyst residual activity. Research in the areaof interesterification is being carried out in research laboratories around theworld. Large companies with huge research and development facilities are devel-oping new processes and these are eventually patented.

In conclusion, the interesterification (both chemical and enzymatic) reaction isa useful tool in the hands of the processor. Currently, there is high interest in theapplication of lipases for the manufacture of designer oils and fats. The UnileverGroup in cooperation with Novo Nordisk has recently built an enzymatic interester-ification plant for the synthesis of specialty triacylglycerols using an .sn-1,3 specificlipase (84). Lipases with new characteristics (heat stable, high activity) and speci-ficities have enormous potential in this field. Currently, lipase applications are lim-ited to the manufacture of products that demand a high price in the market. The costof the lipase is prohibitive for applications where the end-product is inexpensive.However, it is expected that in the near future, through the use of genetic engineer-ing techniques and improved fermentation processes, that the price will come down.The change in price coupled with the demand for new products could speed up theindustrial development of designer oils and fats.

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Chapter 7

Sources of Oilseeds with Specific Fatty Acid Profiles

W.A. Keller

Plant Biotechnology Institute, National Research Council of Canada, Saskatoon,Saskatchewan S7N 0W9, Canada.

Introduction

Numerous studies have documented the increased health risks associated withdiets high in saturated fats. Consequently, dietary modification to reduce intake ofsaturated fatty acids has been recommended as a major step to lower the preva-lence of heart disease (1). Research has also shown that diets high in monounsat-urated fatty acids reduce low-density lipoprotein cholesterol (LDL-C [2,3]).Elevated LDL-C levels have been associated with enhanced risk of heart disease.However, monounsaturated fatty acids do not lower high-density lipoprotein cho-lesterol (HDL-C [3]). High levels of HDL-C are positively correlated with reducedlevels of heart disease. More specific studies have documented the effectiveness ofoleic acid in reducing LDL-C but maintaining HDL-C levels (4). Oleic acid hastherefore been recommended as the major fatty acid in the human diet (5).

Oil stability, particularly during frying, is an important consideration in relationto acceptability. Oils high in polyunsaturated fatty acids will produce more oxidativeproducts under frying conditions, resulting in the production of off-odors and flavors(6). Oils with higher saturation levels possess a longer acceptable shelf life. For exam-ple, oleic acid is 10 times more stable than linoleic acid and 25 times more stable thanlinolenic acid in terms of the rate of oxidative breakdown (7).

The previously mentioned studies have contributed to the identification ofdesirable fatty acid compositions in vegetable oils for human consumption (8). Theoptimal vegetable oil should contain a predominance of oleic acid, with reduced lev-els of saturated fatty acids (i.e., palmitic and stearic acids) and polyunsaturated fattyacids (i.e., linoleic and linolenic acids). An exception to these objectives would bethe development of specialty oil types with elevated levels of saturated fatty acidsfor direct use in margarine manufacture, thereby avoiding or greatly reducing theproduction of trans fatty acids associated with hydrogenation. It has been reportedthat dietary trans-fatty acids may increase the risk of heart disease (9).

Genetic modification strategies employed in the development of new cropcultivars have been, and will continue to be, used to alter the composition of veg-etable oils, thereby improving their value in human nutrition. This chapter pres-ents an overview of four major genetic approaches to altering fatty acidcomposition in vegetable oils. These include


1. Utilization of desirable genetic variation present within a crop species,2. Application of mutagenesis procedures to induce desirable genetic traits,3. Interspecific and intergeneric transfer of traits into the target crop, and4. Alteration of oil composition through genetic transformation technologies.

Attention will be given to major crops cultivated in Canada for the production of oilsthat are consumed by humans. These include canola (Brassica napus and Brassicarapa), soybean (Glycine max), flax (Linum usitatissimum), and sunflower(Helianthus annuus). Emphasis will be placed on the generation of fatty acid mod-ifications relevant to the objectives identified previously.

Modification of Fatty Acid Profiles in Oilseeds Using GeneticVariation Occurring Naturally in the Species

Plant breeders have primarily depended on genetic variation within a species intheir continuing efforts to develop new cultivars. Such naturally existing variationhas also been used to alter fatty acid profiles in the development of edible oilseedcrops. An excellent example of intraspecies genetic variation that has had majorimpact on Canada’s vegetable oil industry was the identification of genetic lineswith reduced levels of erucic acid (‹2%) in B. napus and B. rapa (10,11). Theselines were used by Canadian plant breeders to develop low erucic acid cultivars asthe first major step leading to the development of canola (12). Another exampleis the identification of a genotype of B. rapa with elevated levels of palmitic acid(in the range of 9–11%) in its seed oil that may be useful in developing specialtycultivars for margarine manufacture (13).

Substantial variation in fatty acid profiles also exists in sunflower. Breedershave taken advantage of this variation and have developed specialty oil cultivarscontaining reduced levels of saturated fatty acids (14, K. Fitzpatrick, WesternSeed Corp, Saskatoon, Saskatchewan, personal communication).

However, with the few exceptions described previously, the amount of geneticvariation existing naturally within crop species has been limited. Therefore, addi-tional genetic modification strategies are required to generate variations with desiredfatty acid profiles in the target crop species. Such “variants” can then be incorporatedinto plant-breeding programs in order to develop agronomically acceptable culti-vars/strains. The additional genetic strategies that could be employed for developingaltered fatty acid profiles are described in the remainder of this chapter.

Modification of Oilseed Fatty Acid Profiles Via Mutagenesis

Mutagenesis of Seeds

The most commonly used method of inducing stable genetic mutations in plantsinvolves treatment of large populations of imbibed seeds with a chemical mutagen,


such as ethyl methyl sulfonate (EMS). This approach has been successfullyemployed in altering fatty acid profiles of a number of oilseed crops.

Brassica Oilseeds. Mutagenesis procedures have been successfully employed toreduce linolenatc and increase oleate in Brassica seed oils. In 1973, Rakow gen-erated a mutant line of B. napus, with reduced levels of linolenic acid in its seedoil (15). This material has been utilized by researchers at the University ofManitoba to develop Stellar and Apollo, low linolenic acid cultivars, with the lat-ter cultivar possessing linolenic acid levels of less than 2% (16, also see Chapter8). Intermountain Canola (now owned by Cargill) developed a number of specialtylines of B. napus through seed mutagenesis. These contain elevated levels of oleicacid and reduced levels of linolenic acids (Table 7.1). These lines are grown undercontract for specific uses in food processing. Pioneer Hi-Bred also established amutagenesis program and produced lines with 80–88% oleic acid, compared to62% normally present in B. napus (17, D. Charne, Pioneer Hi-Bred Production,personal communication).

A study on mutagenesis of B. rapa cv R500 has resulted in the identificationof a mutant line with levels of linoleic acid and linolenic acid reduced to 2.1 and3.0%, respectively, compared to the control having 11,9 and 8.6%, respectively(18). This mutant line was subsequently used in genetic crosses with low erucicacid lines of B. rapa to generate lines having less than 6% polyunsaturated fattyacids and more than 88% oleic acid (18).

Sunflower. Several organizations are engaged in the development and/or market-ing of specialty sunflower seeds and/or oil products with elevated levels of oleicacid (14). Apparently all lines with elevated oleic acid are derived from a singlemutant line produced in the U.S.S.R. through seed mutagenesis (19).

Western Grower Seed Corp. researchers have intentionally selected for higholeic acid content and have combined this trail with low saturate levels as well asthe dwarf, early maturing character to produce a specialty purpose high oleic/lowsaturate strain (Table 7.2).

TABLE 7.1Fatty Acid Profiles of Specialty Canola, (B. napus) Varieties Developed Via Mutagenesis

18:1 18:2 18:3 16:0 18:0 CommercialVariety (%) (%) (%) (%) (%) ProductionIMC01 61.9 23.9 4.9 4.1 1.9 1989IMC129 793 8.7 4.8 3.7 1.8 1991IMC130 77.0 11.9 3.9 4.0 1.9 1991IMC02 67.9 20.7 2.1 4.1 1.9 1993508 87.3 2.0 3.4 3.1 1.6 Under developmentSource: W.H.-T. Loh, Cargill, unpublished data.


Soybean. Mutagenesis procedures have been used to produce stable genetic lines,independently containing reduced levels of linolenate (20), reduced levels of sat-urates (21), and elevated levels of stearic acid for potential use in margarine man-ufacture (22). These lines are being utilized in the development of commercialvarieties that will compete effectively with canola and sunflower.

Flax. Pioneering work by Green in Australia resulted in the identification of twomutant lines of flax that when combined yielded progeny with low linolenic acidand elevated linoleic acid levels that were stable, thereby providing a seed oil witha similar fatty acid profile to that found in sunflower (23). Lines derived from theoriginal mutants were used in a breeding program sponsored by United GrainGrowers (UGG) to develop an edible oilseed flax. An independent mutagenesisprogram established at the Crop Development Center at the University ofSaskatchewan has also resulted in the isolation of mutants with reduced linoleniclevels (Table 7.3 [24]). In a collaborative venture between the Crop DevelopmentCenter and the Saskatchewan Wheat Pool, an edible oilseed flax is being devel-oped for commercial production. The Crop Development Center Program has alsogenerated a mutant line with elevated levels of palmitic acid (Table 7.3).

The Flax Council of Canada has recently proposed that all flax lines that pro-duce seed oil with reduced linolenate be referred to as “Solin.” The Solin cultivarsdeveloped by the UGG program are trademarked as Linola (25). Linola has beencommercially cultivated in Canada with more than 30,000 acres grown inManitoba in 1994 and more than 200,000 acres projected for 1995 (J. Dean,United Grain Growers, Winnipeg, Manitoba, personal communication).

TABLE 7.2Fatty Acid Profile of HO/LS Sunola

18:1 18:2 18:3 Saturateda

Strain (%) (%) (%) (%)HO/LS Sunola 88 5 0 7Sunola 14 74 0 12Sunflower (U.S.) 17 70 0 13aRefers to total saturated fatty acids.Source: K.C. Filzpatrick, Western Grower Seed Corp., unpublished data.

TABLE 7.3Fatty Acid Profiles of Three Genetic Lines of Flax and the Parent Cultivar

16:0 16:1 18:0 18:1 18:2 18:3Line (%) (%) (%) (%) (%) (%)E67 27.8 4.8 1.8 17.5 6.0 42.0E1747 9.5 tr 4.6 15.6 65.3 2.1E1929 9.5 tr 3.4 51.7 16.3 16.2McGregor 9.4 tr 5.1 18.4 14.6 49.5Source: Rowland et al. (54).


Mutagenesis of Haploid Cells In Vitro

The development of isolated microspore culture technology offers the possibility of anovel and unique approach to mutant isolation. A microspore culture system possessesdistinct advantages in mutant selection: the haploid genome of the microspore shouldmake mutagenesis procedures more effective, very large populations can be easilyhandled in small areas, and the production of chimaeras can be avoided. Microsporemutagenesis has been successfully employed to produce B. napus mutants expressingelevated levels of oleic acid (17). In the case of B. rapa, a line capable of producinglarge numbers of embryogenic microspores has been identified (26); this line is nowbeing utilized to generate mutants with altered fatty acid profiles (27).

Modification of Fatty Acid Profiles in Oilseeds Via InterspecificHybridization

Interspecific Sexual Hybridization

It is possible to make interspecific sexual crosses among Brassica species fol-lowed by backcrossing of the hybrid to the crop species as a method of transfer-ring desirable traits into oilseed Brassica. In some cases, in vitro culturetechniques are required to rescue hybrid embryos (28). Interspecific hybridizationapproaches have been used to develop B. napus lines with elevated linoleate andreduced linolenate (29). The University of Manitoba has established a program totransfer the low linolenate trait from B. napus, in which it was derived throughmutagenesis, to B. rapa through sexual hybridization (R. Scarth, Department ofPlant Science, University of Manitoba, personal communication).

In the case of sunflower, a number of wild, closely related, and sexually com-patible species are indigenous to large areas of North America. These wild rela-tives serve as a potential reserve of desirable genes, including modified fatty acidprofiles that can ultimately be transferred to cultivated sunflower (14).

Somatic Hybridization

By fusing isolated protoplasts to produce hybrid cells from which hybrid plants canbe regenerated, it may be possible to bypass barriers that prevent successful sexualhybridization and thereby introduce genes from sexually incompatible species intothe target crop (30). Of the major Canadian oilseed crops, protoplast technologythus far has only been established at a reliable level in B. napus type canola. A num-ber of somatic hybrids have been produced in which B. napus has been one of theparents (31). To date there have been no reports on the use of this approach to alterfatty acid profiles in oilseed crops. However, somatic hybridization of Arabidopsiswith Brassica could offer the possibility of transferring desirable fatty acid profilesfrom the former into the latter. Arabidopsis has been widely used in many genetic


studies, and a large number of mutants, including mutants with altered fatty acidprofiles, have been produced and characterized (32). Somatic hybrids betweenArabidopsis and B. napus have been successful generated (33), however low lev-els of fertility of such hybrids could make backcrossing to the crop speciesextremely difficult. It might be possible to employ asymmetric fusion strategies(34), in which the Arabidopsis genome is pulverized by chemical or irradiationtreatment, thereby significantly reducing the amount of genomic informationtransferred while enhancing the possibility for fertility in the hybrid.

Modification of Fatty Acid Profiles in Oilseeds Via Genetic Transformation

During the last decade major advances have been made in the development ofmethodologies for integrating foreign genes into the crop plant genome (35). Thetwo major methods for inserting genes into plant cells and tissue include co-culti-vation with Agrobacterium tumefaciens, and application of biolistic technologies. Interms of oilseed crops, methodology for genetic transformation has been publishedfor B. napus (36–38), B. rapa (39), soybean (40), flax (41,42), and sunflower (43).

To complement the technical advances in gene insertion methodology, a greatdeal of research activity has been focused on the characterization of the enzymaticsteps, and identification of genes, involved in triacylglycerol synthesis (44–6). Arecent review by Slabas et al. indicates that the following genes have been isolated andcharacterized: acyl carrier protein (ACP), acetyl CoA carboxylase, 3-ketoacyl-ACPsynthetase, enoyl-ACP reductase, 3-ketoacyl-ACP reductase, several desaturases (∆6,∆9, ∆12, ∆15), several ∆ acyl-ACP thioesterases, and glycerol-3-acyltransferase (47).

Molecular strategies available to biotechnologists for modification of fattyacid profiles include targeted expression of foreign genes in developing seeds aswell as reduced expression of native genes for oil synthesis through insertion ofantisense gene constructs. Through such targeted modifications in biosynthesisand triacylglycerol assembly, it is possible to substantially alter the fatty acid pro-file in oilseeds. Substantial progress has already been announced in this rapidlyemerging field (48–51), and significant advances should be anticipated in the nearfuture. The commercialization of genetic varieties produced through genetic trans-formation should be anticipated over the next few years. Although a great deal ofeffort is being devoted to the development of modified fatty acid profiles forindustrial applications, reference in this chapter will only be made to alterationsfor human nutritional/health objectives.

Brassica Oilseeds

Research by Calgene Inc. has demonstrated that it is possible to successfully uti-lize antisense RNA technology to underexpress a ∆9 desaturase gene in B. napus,


thereby producing an oil with elevated levels of stearic acid (52). Transgenic linesof high stearate B. napus are being field tested on multiple sites in North America.The commercial objective of this program is the development of specialty strainsfor use in the margarine industry eliminating the need for hydrogenation and theassociated generation of trans fatty acids.

Insertion of an antisense ∆12 desaturase by Dupont researchers resulted in theidentification of transgenic B. napus lines with oleic acid levels in the order of 83%(53). Introduction of an antisense ∆15 desaturase resulted in a reduction of linolenicacid levels to the range of 2% of total fatty acids (53). Genetic transformation hasalso been employed to reduce saturated fatty acid levels in B. napus (54).

Soybean

Using ballistic-based genetic transformation technologies, Dupont researchershave introduced an antisense ∆12 desaturase gene into soybean and have identi-fied transgenic lines with more than 70% oleic acid in their seed oils (53).

Flax

Collaborative research between the Crop Development Center, University ofSaskatchewan and the National Research Council of Canada has been undertakento develop a Canadian-based cocoa butter replacement oil (55). A high palmitic/lowlinolenic mutant line is being transformed with antisense stearoyl-ACP desaturase (∆9desaturase) with the aim of producing an oil with palmitic acid, stearic acid, and oleicacid at similar levels, simulating in composition cocoa butter.

Conclusions

Studies on human health and nutrition, as well as evolving consumer demands forfood products with improved nutritional characteristics, have dictated a continu-ing requirement for research to alter fatty acid profiles in vegetable oils. Plantbreeders have made significant advances by using genetic variation for fatty acidcomposition already existing in the species. The development of canola was pri-marily based on the use of such naturally occurring variation.

Additional alterations in oil composition in canola and in other oilseeds has to alarge extent been based on the application of additional genetic modification strate-gies. These strategies include mutagenesis, interspecific hybridization, and genetictransformation. Mutagenesis has been successfully used to alter fatty acid profiles inBrassica oilseeds, sunflower, flax, and soybean; a number of commercial strains/vari-eties have been developed from mutagenized lines of these crops. Interspecific sexualhybridization as a method of altering oil composition has thus far been employed onlyin oilseed Brassica because of the ability to intercross with related species.Somatic hybridization offers a potential approach to alter the fatty acid profile inoilseeds, but it is very dependent on the availability of protoplast technology that


is presently confined to B. napus. The large array of fatty acid mutations availablein Arabidopsis, a member of the same taxonomic family as Brassica spp, mightserve as a source of genetic variation for incorporation into oilseed Brassica.

The greatest long-term potential for modification of fatty acid profilesinvolves the application of biotechnology, specifically genetic transformationtechnology (genetic engineering). The approach is based on the insertion of mod-ified gene expression systems, thereby giving specific and predictable end-prod-uct alterations. The potential genetic transformation technology has already beendemonstrated with the generation of high stearate and high oleate B. napus lines,as well as soybean lines with modified fatty acid profiles. Significant commercialimpact of this technology should be anticipated over the next decade.

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Chapter 8

Production of Oilseeds with Modified Fatty Acid Composition

Rachael Scarth

Department of Plant Science, University of Manitoba, Winnipeg, Manitoba, R3T 2N2,Canada.

Introduction

As recently as 20 years ago, knowing the crop that produced the vegetable oil was suf-ficient to tell you the oil quality. Oilseed rape, for example, produced an oil high in along-chain fatty acid, erucic acid (C22:l). This oil could be successfully substituted forpetroleum lubricants and adhered well to metal surfaces even when wet. The oil qual-ity of this simple oilseed crop changed radically when Canadian plant breeders mod-ified the fatty acid composition of oilseed rape by reducing the C22:1 content from40% to nearly 0%. The change was motivated by a desire to enhance the quality of theoil for the edible oil market and to address the concern that long-chain C22 fatty acidsare poorly digested. The result was an oil low in C22:1 acid and high in the C18 fattyacids, in particular oleic acid (C18:1). The new oil, along with an enhanced quality inthe seed meal, was given a new commodity name “canola,” a term that is now used forthe oilseed rape crop that produces the canola products (1).

Production of canola in western Canada has increased to a record high of 14million acres in 1994 (2), challenging wheat for the leadership of the prairie crops.One of the reasons for canola oil’s increasing share of the edible oil market is itsvery favorable fatty acid composition. Canola oil has the lowest saturated fat con-tent of the major vegetable oils. Health Canada has recommended that allCanadians reduce the amount of saturated fat in their diet, and canola oil providesone means to this end.

Breeding for Low Linolenic Acid Content

Another distinction of canola oil that is not as favorable is the high content oflinolenic acid (C18:3). The high C18:3 content results in additional cost and effortto the processors of canola as well as soybean oil, which is also high in C18:3. Oneof the objectives of the canola breeding program at the University of Manitoba isto produce low C18:3 canola. This change has been accomplished through theapplication of mutagenesis followed by selection.

The target in the case of the low C18:3 mutation was the desaturation stepbetween linoleic (C18:2) and C18:3 in the seed oil biosynthetic pathway. The objec-tive was to disable the enzyme that adds the third double bond. The mutation work


was conducted by Rakow and Röbbelen at the University of Gottingen, Germany,and several low C18:3 mutations were identified (3,4).

One of these mutants, M11, was sent to Stefansson at the University ofManitoba. Several years of work were required to grow out the progeny, select fornormal growth, and generally clean up the mutation line while selecting for theblock in the desaturation pathway between C18:2 and C18:3. This was followedby crossing to Regent, the adapted cultivar that was suitable for production in thedesired area of western Canada. The objective of the crossing program was torecover all the attributes of Regent with the addition of the low C18:3 trait. Thisaim was accomplished after several generations of backcrossing and selection; theworld’s first low C18:3 cultivar, Stellar, was registered in 1987 and was followedby Apollo in 1992(5,6).

The result of the low C18:3 breeding program is a unique fatty acid compo-sition illustrated by the profile of the low C18:3 cultivar, Apollo (Table 8.1). Thelow levels of C18:3 are accompanied by a slightly higher content of C18:2 and, inparticular, a higher C18:1 content in comparison to the conventional canola oilquality of the cultivar Westar.

The reduction in C18:3 has produced the anticipated benefits in reducing thehydrogenation required, namely, an increase in stability, and an improvement inroom odor of the heated oil (7,8). The effect of the reduction in C18:3 on the oil’sstorage properties was the objective of a recent study by Przybylski and his col-leagues in the Department of Foods and Nutrition, University of Manitoba (9). Acomparison of odor development in conventional canola and low C18:3 oilshowed that the low C18:3 oil remained relatively stable over a period of 0-12days, while canola oil developed a progressively more intense unpleasant odor.This slower development of off-odors is especially critical for canola oil in theimportant application of domestic frying.

TABLE 8.1Fatty Acid Composition of Low Linolenic (C18:3) Canola, Flax, and Soybean

C16:0 + C18:0 C18:1 C18:2 C18:3(%) (%) (%) (%)

Canolaa

Apollo 5.7 67 24 1.7Westar 5.6 63 22 9.3

Soybeanb

A16 15.4 41.1 41.2 2.2Century 84 13.6 24.3 54.6 7.4

Flaxc

Linola 947 10.5 15.6 71.5 2.4AC Linora 8.4 16.6 18.1 56.9aSource: Scarth, McVeity, and Rimmer (6).bSource: Fehr el al. (10).cSource: Dribnenki and Green (12).


Similar methodology was used in the development of low C18:3 soybean cul-tivars, involving an initial mutation treatment followed by selection. The lowC18:3 strain, A16, was developed from a mutation line developed at Iowa StateUniversity. In comparison to the conventional soybean oil profile (Table 8.1), A16shows a low level of C18:3 with slightly lower C18:2 levels, an increase in C18:1and a slight increase in palmitic (C16:0) and stearic (C18:0) acids (10).

The third new low C18:3 crop, flax, was developed using a similar process ofmutagenesis and selection by Green in Australia (11). The block between C18:2and C18:3 resulted in a dramatic reduction of C18:3 levels from 57% to just over2% and was accompanied by an increase in C18:2 (Table 8.1) to produce a fattyacid profile similar to sunflower oil. In 1993, the first low C18:3 flax was regis-tered in Canada (12). Low C18:3 flaxseed oil has been approved for human con-sumption in Canada and similar approval is being sought in the United States. Theflax breeding program conducted by Rowland at the Crop Development Centre,Saskatoon, also has had success with mutagenesis in creating new low C18:3mutations and cultivar development is under way (13).

There are now three new low C18:3 crops, canola, soybean, and flax, all withlevels of C18:3 under 3% but otherwise with distinctive fatty acid compositions.To complete the description of new oil qualities in traditional oilseeds, we need toconsider two additional changes in the C18 fatty acid contents.

Breeding for Other Fatty Acid Characteristics

A high C18:1/low C18:3 line of canola has been developed by the researchers atPioneer Hybrid. Mutagenesis was applied to embryos produced by incubatingimmature pollen grains or microspores that were then cultured to produce plantsthat carried the mutation. The target was to produce a mutation earlier in the desat-uration pathway, to block the desaturation of C18:1 to C18:2. The desired muta-tion was selected and then crossed to incorporate the low C18:3. The result is anoil low in both C18:3 and C18:2, with 86% C18:1 (14). The further reduction indesaturation may result in additional stability during prolonged frying use.Industrial testing of these high C18:1 oils is now under way.

This high C18:l character also has been developed in sunflower by selectionfrom Russian sunflower germ plasm naturally high in C18:l acid. The SpecialtyVegetable Oil Division of Lubrisol is marketing the high C18:1 sunflower oil.

The other development is a canola germ plasm high in C18:0 that was developedusing genetic transformation techniques. The first generation of transformants produceup to 40% stearate, and subsequent generations have shown stable inheritance (15).

Combining New Oil Characteristics with Desired Agronomic Traits

The new oil qualities have been created using a number of different techniques andthe most advanced of these developments have reached the stage of field production.


It is timely to consider the key components required for successful production of aspecialty oil cultivar. These can be summarized as stability of expression, simpleinheritance of the specialty oil trait, productivity of the specialty oil cultivar,development of methods to segregate the specialty oilseed, and finally the pre-mium value of the specialty oilseed crop.

The question of whether a specialty oil trait has the required stability of expres-sion can only be answered after the new oil quality is transferred into adapted culti-vars. Field tests can then be conducted at different locations over several years todetermine the effect of environment on the expression of the specialty oil trait.

The effect of environment on oil quality in conventional sunflower is dra-matic, with variations of up to 30% in the C18:l acid content of sunflower oil pro-duced in the northern and southern United States (16). The high C18:l germ plasmis more stable. A similar stability of expression has been observed in tests of thehigh C18:1 canola germ plasm (14).

Sunflower is a long season crop, and production in Canada is limited by thelength of the growing season. Sunflower breeders at Western Grower Seed,Saskatoon, are developing a short stature sunflower with early maturity to allowproduction in western Canada, north of the usual limits for sunflowers, with theadded value of very high levels of C18:1 acid in the sunflower oil.

The low C18:3 mutation lines of flax produced by Green also have beentested under different temperatures (17). M1589 and M1722 have moderate reduc-tions in the level of C18:3. When the two lines were crossed, the result was a verylow level of linolenate represented by the genotype Zero. A conventional highC18:3 cultivar was also included in the study. In all four lines, exposure to highertemperatures resulted in oils with lower C18:2 and C18:3, and higher C18:l. Thepercentage of the saturated fatty acids C16:0 and C18:0 also increased underhigher temperatures. At the lower temperatures, C18:3 levels were under 3% andthe C18:l content of the Zero genotype was over 62%.

We have just completed a study of the effect of environment on the fatty acidcomposition of the low linolenic acid cultivar Stellar and the cultivar Regent thatwas used in the development of Stellar (18). The breeding method used in thedevelopment of Stellar resulted in the two cultivars having very similar geneticmake-ups with the exception of the low C18:3 trait (5). The cultivars were grownin isolated plots over several locations and years. There was a significant effect oflocation on the fatty acid composition of the two cultivars. In general, the loca-tions having the highest daily mean temperatures during seed developmentyielded seed oil containing the lowest level of C18:3 and the highest level of thesaturated fatty acids C16:0 + C18:0. Levels of C18:1 and C18:2 were more vari-able. The overall pattern was a fatty acid profile higher in saturated and monoun-saturated fatty acids and lower in polyunsaturated fatty acids when the seeddeveloped under higher temperatures.

The reliable production of seed with a composition low in C18:3 is necessaryfor the success of the low linolenate canola oil. To confirm the effect of tempera-ture on seed oil composition, another set of experiments was conducted under


controlled environment conditions that reproduced the stress of high temperaturesduring seed development. Under these conditions, the low C18:3 content of Stellarremained stable. However, when the low C18:3 plants were subjected to long dura-tion (20–30 days) of 30/25°C temperatures during seed development, the saturatedfatty acid content climbed above the critical 7% in both the low C18:3 cultivarStellar and the canola cultivar Regent. High temperature may retard desaturaseactivity or the rapid seed development may fix a greater amount of the fatty acidsin the saturated form. Seed maturity is 10–12 days faster under the long durationof high temperatures when compared to the control maintained at 20/15°C.

Elevated levels of saturated fatty acids provide an important consideration inthe production of the low linolenic acid oil. Production of low C18:3 canola andconventional canola cultivars should be carried out in areas that do not have pro-longed periods of high temperature during seed development. Fortunately, the areaof canola production in western Canada does have relatively low temperate grow-ing conditions.

Successful production also depends on completing seed development beforefrost. Early maturity is a key to high-quality seed production with high oil contentand low seed chlorophyll content. One alternative is to move the low C18:3 traitinto the other canola species, Brassica rapa, to provide an early maturing sourceof low C18:3 oil. The Brassica rapa crop currently produces an oil low in satu-rated fat that is useful for blending with canola oil higher in saturated fatty acidsto achieve the required 7% limit.

A similar study of the effect of extreme temperatures on soybean oil compo-sition was conducted by Rennie and Tanner at the University of Guelph (19). Thesoybean germ plasm had a range of fatty acid compositions including low C18:3,high C18:0, and the conventional soybean profile. The C18:3 levels in the lowlinolenic acid content lines ranged from 2.5 to 4.6% under field conditions. Thetemperatures in the controlled environments were lower (15/12°C) and higher(40/30°C) than those usually occurring during seed development in the field. The15/12°C environment produced high levels of C18:3 in the low C18:3 lines com-pared to those observed at 28/22°C. The high C18:1, low C18:2, and low C18:3values observed at 40/30°C were beyond the usual range of values of the lines inthe study. The high C18:0 line grown at 15/12°C produced C18:0 at the same levelas the conventional cultivars. Exposure to cold temperatures apparently alters thepattern of C18:0 metabolism. The highest expression was observed at 28/22°C.

Soybean breeders face a major challenge in bringing the low linolenic acidcontent oil into commercial production. The soybean-growing area is distributednorth to south over a wide range of environments. Any change in oil quality thatis sensitive to environmental influences, such as temperature, has to be introducedinto adapted soybean cultivars in each area of production. The method of intro-duction, which involves crosses and backcrosses for several generations toadapted cultivars with selection for the specialty oil trait, is dependable but slow.

For this reason the inheritance of the specialty oil trait is critical. Simple inher-itance involving a single gene or two genes with a simple method of selection


ensures rapid progress in the introduction of the new oil quality. Plant breeders areaiming at the development of a cultivar with a package of characteristics, in whichspecialty oil quality is just one. The simpler the inheritance of the change in oilquality, the easier it is to handle in a selection program. A trait controlled by sev-eral genes may require the use of molecular markers to tag the desirable lines fromcrosses without the requirement for seed production and analysis. Other savingsin time and effort could be made if the specialty oil trait is linked to another traitthat is easy to select at the seedling stage, for example herbicide resistance.

The choice of the cultivar to use as the adapted parent in crosses is critical.While the development of the specialty oil cultivar is progressing, so is the devel-opment of the conventional cultivars. For example, in 1985, the top-yieldingcanola cultivar, Westar, was the obvious choice as the adapted parent in crosses todevelop special quality cultivars. However, Westar is highly susceptible to the dis-ease blackleg, that is now well established in the canola-growing areas of westernCanada. The virulent form of blackleg can reduce yields by as much as 75% insusceptible cultivars and highly susceptible cultivars, such as Westar, are devas-tated by the disease. Specialty oil cultivars that are susceptible to blackleg alsowill suffer yield losses.

Disease resistance is just one part of the productivity package that appliesequally to specialty oil cultivars and conventional cultivars and includes yield, oiland protein content, and maturity. Stability of yield performance over environ-ments is critical for the success of the cultivar, as is early maturity to allow seeddevelopment to be completed under favorable conditions. High seed oil and pro-tein content and low chlorophyll levels are important quality factors influenced byseed development. Disease resistance provides an insurance policy that productivitywill be maintained if the year is conducive to disease development. All of these char-acteristics need to be in place for successful production of specialty oil cultivars.

During the development of specialty oil cultivars, the small scale of the pro-duction makes segregation of the specialty oil seed relatively easy. From the ini-tial tissue culture through single plants in the greenhouse, single row increases inthe field, and harvesting of the seed with the small plot combine, the plant breedercan carefully label the vials and seed packets to indicate the special oil quality.

The Commercialization of Specialty Oils

Once the production is on field scale, a new set of challenges must be met. Thespecialty oil character adds an additional factor in the need for isolation of pro-duction. Both canola species are cross-pollinating, and pollen from outsidesources can introduce variation in oil quality that is undesirable. Cross pollinationis a particular hazard in areas of high insect pollinator activity. Large-scale pro-duction of specialty oil cultivars will have to be carefully coordinated to avoidcontamination from adjacent fields of conventional canola.

The grain-handling system in western Canada is set up for volume—largeamounts of seed harvested, stored in bins on the farm site, delivered in large volumes


to the elevator, and transported in large containers to processors or to ship con-tainers at the ports for export. At each step, the specialty oil seed must be kept sep-arate to retain the value of the oil. Any factors that can ease this process ofsegregation in the transport system would be an advantage.

As an example, the low linolenic acid flax cultivar has been developed in ayellow-seeded background (12). The seed-handling system can use this character-istic to segregate the edible oil crop from the industrial oil crop and prevent mix-ing that would lower the value of both crops. There is similar variation for yellowseed coat color in canola. Unfortunately, the trait is multigenic and influenced bythe environment during seed development (20). The requirement to add this traitto the development of specially oil canola cultivars would demand considerabletime and effort.

The final factor in the success of specialty oil cultivars is premium value,determined by the market available for the oil and the value that the market placeson the oil. Before processors and manufacturers will commit to the purchase oflarge volumes of specialty oil, they must have two guarantees. The first is theguarantee of quality—the minimum standards for the specialty oil must be metconsistently. The second guarantee is a guarantee of continued supply.Manufacturers must be sure that production levels will allow a commitment of aparticular label or processing line to the specialty oil. Those volumes must beavailable to maintain the market for that product.

We can develop a checklist for the ideal specialty oil cultivars that includesgood stability of oil over different environment and years. Productivity must be atleast equal or, if not, the premium value must compensate for any losses in pro-ductivity. If there are any distinct production practices that must be followed, suchas isolation to prevent pollen contamination, these practices have to be veryclearly defined for the producers. Ease of segregation using seed characteristics,for example, would be an advantage.

Conclusion

Each specialty oil quality modification is aimed at creating the ideal oil for themarkets of the next century. Once the required variation has been created throughselection, mutation, or transformation, the next step is to ensure that agronomicperformance is sufficient for successful production. Market development is criti-cal to determine where the specialty oils will find a market and if the new oil qual-ity is what the market requires.

In 1994, the traditional oilseeds no longer have just traditional oil quality. Thereare low linolenic acid soybean, flax, and canola; high oleic canola and sunflower;and high stearic canola. The seed-handling system will have to adapt to these spe-cialty oil cultivars and develop means of handling the seed to ensure that specialtyoil quality is maintained. The successful production of specialty oil cultivarsrequires close coordination between all aspects of this industry—plant molecularbiologists to create the necessary variation to start this process, the plant breeders


to produce the adapted cultivars, then it is up to the producers and agronomists toensure the best quality seed production. Processors work with oilseed chemists,marketing analysts, and nutritionists to produce the finished oil product. The finalelement to ensure success would be a crystal ball to reveal the ideal oil quality forthe next century. I would welcome your ideas!

References

1. Downey, R.K. and G. Röbbelen, in Oil Crops of the World, edited by G. Röbbelen, R.K.Downey, and A. Ashri, McGraw-Hill, New York, 1989, p. 355.

2. Canola Digest, Canola Council of Canada, Winnipeg, Manitoba, Canada, November1994, p. 9.

3. Rakow, G., Z. Planzenzuchtg 69: 62 (1973).4. Röbbelen, G. and A. Nitsch, Z Pflanzenzüchtg 75: 93 (1975).5. Scarth, R., P.B.E. McVetty, S.R. Rimmer, and B.R. Stefansson, Can. J. Plant Sci. 68:

509(1988).6. Scarth, R., P.B.E. McVetty, and S.R. Rimmer, Can. J. Plant Sci. 75: 203 (1995).7. Eskin, N.A.M., M. Vaisey-Genser, S. Durance-Tod, and R. Przybylski, J. Am. Oil

Chem.Soc. 66: 1081 (1989).8. Prevot, A.J., L. Perrin, G. Laclcverie, P.H. Auge, and J.L. Coustille, J. Am. Oil Chem.

Soc.67: 161 (1990).9. Przybylski, R., L.J. Malcolmson, N.A.M. Eskin, S. Durance-Tod, J. Mickle, and R.Carr,

Lebensm.-Wiss. u.-Technol. 26: 205 (1993).10. Fehr, W.R., G.A. Welke, E.G, Hammond, D.N. Duvick, and S.R. Cianzio, Crop Sci. 32:

903 (1992).11. Green, A.G., Can. J. Plant Sci. 66: 499 (1986).12. Dribnenki, J.C.P., and A.R. Green, Can. J. Plant Sci. 75: 201 (1995).13. Rowland, G.G., R.S. Bhatty, J. Am. Oil Chem. Sac. 67: 213 (1990).14. Charne, D., in Program of Eight Crucifer Genetics Workshop, Saskatoon, Saskatchewan,

Canada, 1993, p. 19.15. Knutson, D.S., G.A. Thompson, S.E. Radke, W.B. Johnson, V.C. Knauf, and J.C. Kridl.

Proc. Natl. Acad. Sci. U.S.A. 89: 2624 (1992).16. Morrison, W.H., J. Am. Oil Chem. Soc. 52: 522 (1975).17. Green, A.G., Crop Sci. 26: 961 (1986).18. Deng, X. Effect of Temperature upon the Fatty Acid Composition during Seed

Development in Oilseed Rape, M.Sc. thesis, University of Manitoba, 1994, pp. 73–75.19. Rennie, B.D., and J.W. Tanner, J. Am. Oil Chem. Soc. 66: 1622 (1989).20. Van Deynze, A., and K.P. Pauls, Euphytica 74: 77 (1994).


Chapter 9

Classification of Oils with Modified Fatty AcidCompositions as Novel Foods

Frank W. Welsh

Bureau of Hood Regulatory, International and Interagency Affairs, Food Directorate, Health Canada

Introduction

Advances in food science and technology have contributed to the development offoods that previously were not available in the Canadian marketplace. Examplesof these developments include novel macro ingredients, functional foods, newfood-packaging technologies, and the use of genetic engineering (1). Some of theendpoints, such as the development of novel ingredients and the use of geneticengineering, can be observed with the development of novel oils. For example,the genetic engineering of oilseed crops to enhance pest control and diseaseresistance properties is well established. Similarly, research is underway withrespect to tailoring the fatty acid composition of the oil to address the specific needsof the food-processing industry, or to alter the nutritional or dietary characteristics ofthe food, by both genetic modification of the plant and postharvest modification of theoil (2). Such developments represent a significant change in the approach scientistshave used to develop new products for the marketplace.

Accompanying these developments have been questions regarding the impactof such modifications on the nutrient composition of the altered food and the pos-sibility of affecting the allergenic potential or introducing other unintended effectsinto the modified food. Compounding these issues is the development of an edu-cated, concerned, and increasingly activist population that wishes to better under-stand and have more control regarding the food they eat.

The challenge to government is to develop regulations that address the con-cerns expressed by scientists and the public without unduly hindering the growth,development, and competitiveness of this industry. The purpose of this paper is todiscuss the approach that Health Canada is taking to address safety issues regard-ing novel foods, in particular the ongoing technical developments in the fats andoils industry. This will be accomplished by: discussing proposed regulatory pol-icy for novel foods, the Guidelines for the Safety Assessment of Novel Foods (3),the relationship between Health Canada and Agriculture and Agri-Food Canadawith respect to the Seeds Act, and regulatory considerations regarding the devel-opment of novel oils.


Regulatory Policy for Novel Foods

The development of regulations for novel foods, including the products ofbiotechnology, have been the subject of intense activity by many organizationsand jurisdictions. The major documents that have resulted from these activities areidentified in Table 9.1. Two types of developments can be identified: the estab-lishment of criteria for the safety assessment of products of biotechnology thatwill be used as food; and the development of regulatory approaches to control theentry of novel foods into the marketplace. An example of the former activity is theOrganization for Economic Cooperation and Development (OECD) documentconcerning the safely evaluation of foods developed by modern biotechnology.The proposed regulation for novel foods of the European Union is an example ofthe latter development, while the United Kingdom’s Food Safely Act andGuidelines represent a voluntary approach to the regulation of novel foods.

The regulatory developments that are taking place in Canada are similar tothe international developments in that two distinct activities are being undertaken.The first activity is related to the development of regulations to require notificationprior to the sale of novel foods. The second step is the development of guidelines for

TABLE 9.1A Summary of Recent Developments for the Regulation of Novel Foods and Food ProcessesCountry or Organization Year Title

United Kingdom 1990 The Food Safety Act, and Guidelines for the Assessment of Novel Foods and Processes. Source: ACNFP (4).

International Food 1991 Assuring the Safety of Foods Biotechnology Council Produced by Genetic Modification.

Source: International Food Biotechnology Council (5).

Food and Agricultural 1991 Strategies for Assuring the SafetyOrganization, World of Foods Produced by Biotechnology.Health Organization Source: WHO (6).

Food and Drug 1992 Statement of Policy: Food Derived Administration from New Plant Varieties. Source: FDA (7).

European Union 1993 Proposal for a Council Regulation on Novel Foods and Novel Food Ingredients. Source: Council of the European Communities (8).

Organization for 1993 Safety Evaluation of Foods DerivedEconomic Cooperation by Modern Biotechnology. Conceptsand Development and Principles. Source: OECD (9).


the safety assessment of these products. The latter guidelines will be discussed ina subsequent section of this paper.

The development of regulatory policies in Canada began in 1990 with therelease of Information Letter (I.L.) No. 806 concerning novel foods and novelfood processes. This I.L. proposed the development of regulations that wouldrequire notification prior to the sale or advertising for sale of novel foods andfoods from novel processes, and identified the information requirements for sucha notification. A definition was provided for the term “novel food” that was basedon the definition developed by the United Kingdom (4).

Over 60 comments were received as a result of the publication of I.L. No.806. These comments were generally supportive of the proposal, but addressed anumber of issues, including the definition of a novel food, equivalency to interna-tional developments, competitiveness considerations, safety assessment concerns,and the labeling of genetically engineered foods.

The original proposal has been revised to reflect the issues raised as a resultof the I.L. and the ongoing consultations since its release. The actual text for theregulation is expected to be published for comment in Canada Gazelle, Part I dur-ing 1995. However, a description of the regulatory principles can be found inVolume I of the guidelines (3).

These regulatory principles continue to require notification to the FoodDirectorate prior to the sale of a novel food, but now encompasses a revised defi-nition for novel food, identifies the information requirements for a notification,and provides time frames for the review of a novel food notification. The proposeddefinition of a novel food is as follows:

A novel food includes:

• Products and processes that have previously not been used as food or toprocess food in Canada;

• Food containing microorganisms that have not previously been used as foodor to process food;

• Foods that result from genetic modification and exhibit new or modifiedcharacteristics that have previously not been identified in those foods, orthat result from production by organisms exhibiting such new or modifiedcharacteristics; and

• Food that is modified from the traditional product or is produced by aprocess that has been modified from the traditional process (3).

This definition is similar to one previously developed by the European Union (8).In addition, these guidelines identify the information requirements for a notifi-

cation. The basic requirements include the name under which the novel food will besold; the name and address of the principle place of business of the manufacturer orimporter, if applicable; a statement of the nature of the novel food, its process of man-ufacture, its intended uses, and history of consumption if used as food in anothercountry; information about the possible displacement of existing foods, and the


nutritional impact thereof, as applicable; the written text of all labels to be used inconjunction with the novel food; and the name and title of the person who signedthe notification and date of signature. Information demonstrating the safety ofsuch products as food may be requested by the Director. Although this safetyinformation would have to be provided at the request of the Director, it is antici-pated that companies developing novel foods would address food-safety issues aspart of ongoing research programs without the proposed regulation.

Volume 1 of the guidelines also indicates that the notification should be com-pleted within 90 days of receipt of a complete information package. However, thereview period would be stopped should additional information be requested. The90-day response time would be started over after receipt of the requested infor-mation. Safety information also may be requested after review of the productinformation is complete, should evidence become available indicating that theremay be a safety issue concerning the product.

Guidelines for Assessing the Safety of Novel Foods

During October 1993, the Food Directorate issued a draft document entitled“Guidelines for the Safety Assessment of Novel Foods” for comment. Over 30responses were received and the comments have been used to revise this document.The revision was released on September 27, 1994. The original text was divided intotwo volumes, with Volume 1 of the document providing a clarification of the proposedregulatory policies, as described previously. This volume also included decision treesthat further identify those products that may be considered novel foods (3). Figure 9.1contains the introductory decision tree that aids in identifying those substances that areaddressed by other sections of the Food and Drug Regulations. Figure 9.2 clarifiesthose products of biotechnology that require notification. Additional decision trees areprovided for identifying those foods from novel processes that may require notifica-tion and those cases when food additives may require notification as novel foods.

Volume 2 of the guidelines addresses the safety assessment of geneticallymodified plants and microorganisms. The safety assessment criteria described inthis volume encompass the concept of “substantial equivalence” that has beendeveloped by the OECD (9). This concept “embodies the idea that existing organ-isms used as food or as a source of food can be used as the basis of comparisonwhen assessing the safety of the human consumption of a food or food componentthat has been modified or is new.” The OECD document goes on to identify thosepoints that must be taken into consideration to demonstrate substantial equiva-lence, and the difficulty in demonstrating equivalence as experience with the sub-stance decreases, or if there is a lack of similarity with an established product.

The assessment of a novel food will be accomplished on a case-by-case basis,and will require information concerning the development and production of themodified plant; information regarding the product; and, as needed, information


regarding dietary exposure, nutritional data and toxicological data. These require-ments are summarized in Table 9.2. It should be noted that not all information willbe required in all situations, and that scientific discussion of the issues may besuitable for addressing some concerns. These questions should be addressed in astepwise fashion, until the safety concerns are addressed. The Food Directorateencourages developers of novel foods to come forward early in the developmentprocess so that both parties can better understand each other’s concerns, and toprevent the situation where unnecessary work is undertaken.

Fig. 9.1. Decision tree identifying those substances that are regulated under the authority ofthe Food and Drug Regulations and would not require notification as novel foods.


Health Canada, Agriculture and Agri-Food Canada and the Seeds Act

The necessity of providing for a food-safety assessment of a novel oil and the reg-istration of such crops under the Seeds Act may appear to be an overlap of author-ity. To address this concern, Agriculture and Agri-Food Canada (AAFC) andHealth Canada have cooperated to more clearly identify the activities of bothorganizations. An agreement has been reached that identifies the criteria forrequiring a safety assessment of a genetically modified food, describes the rolesof the two organizations, and provides time frames for completion of the food-safety assessment. Those crops, including oilseeds, that are registered under theSeeds Act that will require a food-safety assessment include varieties that resultfrom genetic engineering, or varieties where the breeding objective of the modifi-cation is the alteration of the historical compositional characteristic associatedwith the parent or related crop, or there is reason to suspect that the modificationmay have a negative impact on food safety or human health.

This agreement also identifies the role of the two organizations. Agricultureand Agri-Food Canada will be the primary contact for the agricultural industry, andwill receive the data necessary to conduct a food-safety assessment from the regis-trant. They are also expected to give equal consideration to any recommendation

Fig. 9.2. Decision tree that identified those products of biotechnology that would requirenotification as novel foods.


regarding the safety of a new variety during its consideration for registration.Health Canada is expected to provide an objective evaluation of the availablesafety data for the edible portion of the new variety according to the “Guidelinesfor Assessing the Safety of Novel Foods.” It is also expected that the evaluationswill be completed within 90 days of receipt of a complete information package,and that the registrant will be notified of an incomplete data package within 30days of its receipt.

Novel Oils

The preceding discussion has addressed the major topics of concern related to theconsideration of a modified oil or oilseed crop as a novel food. It should be real-ized that the characteristics of rapeseed oil have only been modified recently, byreduction of the erucic acid and glucosinolate concentrations, to produce canola oiland permit its sale as food. The alteration of the composition of existing oils canbe expected to permit the expanded use of certain oils as food, or to modify the

TABLE 9.2General Information Requirements for Genetically Modified PlantsArea Information RequiredDevelopment and Host and donor organism.production of the Modification process.modified plant. Modified host.

Methodology.

Product informationPlants used as food. Description of the plant material.

Information on its proposed use. Details on processing and quality control. Comparison of the composition of the novel food to that of the unmodified host.

Plant products used in food. Products identical to existing food additives should provide information to indicate substantial equivalence, as mentioned previously. Products that are novel food additives should be noted as such.

Dietary Exposure. Information to indicate the amount of product that may be found in the diet, both in the general population, and target population.

Nutritional data. Macro and micro nutrient composition. Nutrient bioavailability.

Toxicological data. Laboratory animal studies (if necessary). Allergenicity considerations.


composition of existing oils to better meet the needs of the food-processing andother industries. These modifications can be expected from the development ofmodified fatty acid compositions using conventional plant-breeding techniques ornucleic acid techniques to modify the plant, and the use of chemical and enzy-matic methods to directly modify the fatty acid composition of the oil.

These various approaches to modifying the fatty acid composition of oils willbe viewed separately under the proposed novel food regulations. It is clear that anovel oil that is developed using nucleic acid techniques to modify the plant willrequire notification, whether the oil composition is substantially modified or not.On the other hand, if the modified oil is developed using traditional breedingapproaches, notification would only be required if the oil composition was viewedas not being equivalent to that of the variety from which it was developed.

The use of chemical or enzymatic esterification techniques to modify thefatty acid composition of an oil would result in the modified oil being considereda food from a novel process. As such, these products would also require notifica-tion. To date, the Food Directorate has not been approached regarding a notifica-tion for food from such a process. However, the Advisory Committee on NovelFoods and Processes (ACNFP) of the United Kingdom has evaluated productsfrom two such processes and approved their use as food, subject to certain condi-tions specified by the ACNFP (10).

Conclusion

Over the last 2 years, the Food Directorate has come from developing the initialproposals for novel foods, to the point of having published guidelines for thesafety assessment of novel foods, and is developing regulatory proposals that willbe published in Canada Gazette, Part I in 1995. Thank you for your contributionsto date, and I look forward to receiving your comments when the regulatory pro-posals are published.

References

1. GAO, Food Safety and Quality. Innovative Strategies May Be Needed to Regulate NewFood Technologies, Report of the Chairman, Subcommittee on Oversight andInvestigations, Committee on Energy and Commerce, House of Representatives,GAO/RCED-93-142, 1993, p. 101.

2. U.S. Congress Office of Technology Assessment, A New Technological Era for AmericanAgriculture, OTA-F-474, Washington, D.C., Government Printing Office, 1992, p. 452.

3. Health Canada, Guidelines for the Safety Assessment of Novel Foods, Health Canada,Ottawa, Vols. 1 and 2, 1994, pp. 13 and 19.

4. Advisory Committee on Novel Foods and Processes, Guidelines on the Safety Assessmentof Novel Foods and Processes, Report on Health and Social Subjects, Department ofHealth, London, 1990, p. 30.


5. International Food Biotechnology Council, Reg. Toxicol. Pharmacol. 12 (1990), Part 2 of2, p. 196.

6. World Health Organization, Strategies for Assessing the Safety of Foods Produced byBiotechnology, Report of the Joint FAO/WHO Consultation, Geneva, 1991, p. 59.

7. Food and Drug Administration, Statement of Policy: Foods Derived from New PlantVarieties; Notice, Federal Register, Volume 57, Number 104, 1992, 22948-23005.

8. Council of the European Communities, Off. J. Eur. Com. C190: 3-6 (1992).9. Organization for Economic Cooperation and Development, Safety Evaluation of Foods

Derived by Modern Biotechnology. Concepts and Principles. Organization for EconomicCooperation and Development, Paris, 1993, p. 79.

10. Advisory Committee on Novel Foods and Processes, ACNFP Annual Report 1993,Ministry of Agriculture, Fisheries and Food, and the Department of Health, London,United Kingdom, 1994, p. 69.


Chapter 10

Food Labeling in Canada

Ian Campbell

Agriculture and Agri-Food Canada, 59 Camelot Drive, Nepean, Ontario, K1A 0Y9, Canada

General Labeling Requirements

In Canada there are five Federal Acts that deal in some manner with food labeling andfour federal departments that have a role in their administration. I will give a briefoverview of this as well as deal with some of the specifics of fat and fatty acid label-ing, both concerning developments on the Canadian scene and events taking placeinternationally. I also intend to touch on other developments that will have a bearingon the labeling of foods, and particularly edible oil products, in the future.

There are a number of matters to be concerned with when considering foodlabeling. These may be summarized as follows:

1. Adequate and accurate information is present to assist consumers in foodchoices relative to health, safety, and economic concerns.

2. Consumers and industry are protected from fraudulent or deceptive label-ing, packaging, and advertising practices.

3. Fair competition and product marketability is promoted and maintained.

The Department of Agriculture and Agri-Food Canada (AAFC) establishesbasic labeling policy and requirements for all foods. It assumed these responsibili-ties when they were transferred from the former Department of Consumer andCorporate Affairs on June 25, 1993. The department administers these requirementsat the manufacturing and import levels for foods other than fish and marine prod-ucts. The Department of Fisheries and Oceans administers requirements for suchproducts under the Fish Inspection Act.

Further complicating the picture is the presence of the new Department ofIndustry that administers labeling requirements at the retail level of trade for thoseproducts for which the retailer has a direct responsibility, that is, retailer-packagedproducts. Completing the picture are the activities of Health Canada as related tohealth and safety in labeling matters. Health Canada look the lead role in develop-ing the regulations and guidelines for nutrition labeling, and it established the crite-ria for such things as declaration of allergens. An example of this requirement is therecent regulatory amendment to require the specific listing of peanut oil when usedas an ingredient in other foods. Although it was recognized that peanut allergenswould not usually carry over into a refined peanut oil, the declaration was requiredas a precautionary measure.


The five Federal Acts that support departmental activities are as follows.

1. The Consumer Packaging and Labeling Act2. The Food and Drugs Act3. The Meat Inspection Act4. The Canada Agricultural Products Act5. The Fish Inspection Act

The Consumer Packaging and Labeling Act deals with basic labelingrequirements for prepackaged products of all sorts. The bilingual requirementsappear here as does the requirement to state a net quantity.

The Food and Drugs Act is the basic act in Canada controlling matters of foodsafety and fraud. This legislation deals with such food-labeling requirements asingredient listings and exemptions from ingredient declarations in specified cases.It deals with durable life information; common names; and with a substantialnumber of specific labeling matters, such as declarations of milk fat content,nutrition labeling, cautionary statements, among others.

The Meat Inspection Act, The Canada Agricultural Products Act, and The FishInspection Act contain product-specific labeling requirements associated withmeat, poultry, fruit, and vegetable products; processed dairy products; mapleproducts; honey; and fish products.

Labeling can be a very powerful tool in product promotion. In addition to reg-ulations on mandatory information, there are controls over the optional claims thatmanufacturers may want to apply to their labels. The department has a large bodyof policy and precedents that it relies on for rulings and interpretations in thisarea. A major reference document widely praised nationally and internationally isthe Guide to Food Manufacturers and Advertisers. Currently it is undergoing athorough review and revision.

Fat and Fatty Acid Labeling

Control over what may be said about fatty acids on food labels in Canada is ratherstrict. Specific mention may be made only for the following fatty acids or classesof fatty acids: linoleic acid, saturated fatty acids, monounsaturated fatty acids,polyunsaturated fatty acids, and cholesterol. If any information is provided forone, quantitative information in grams/serving is required for all of them. Therestrictions mean that canola or sunflower oils that have had their oleic acid con-tent increased through breeding may not be described on their labels using any ref-erence to this increased oleic acid content. However, under current rules they maybe described as high monounsaturated sunflower oil, for example.

There is much current debate over the labeling of trans fatty acids. Somemembers of The Expert Committee on Fats and Oils have for some time advocatedlabeling of this class of compounds. Current regulations prohibit their mention. The


Health Protection Branch maintains that the evidence is not conclusive regarding thehealth effects of these substances and has not agreed to change the current restrictions.

Although I am not able to debate the scientific question one way or another,I am very interested in the information about the levels of trans fatty acids pres-ent in human adipose stores and the considerable efforts currently underway byindustry on a number of fronts to reduce levels in edible oil products. I cannot helpbut conclude that labeling would be a significant factor to stimulate this action.

Additional Fatty Acid Claims

There were recommended nutrient intakes for both ω-6 and ω-3 fatty acids (1).Health Canada is of the view that more information on consumer use and under-standing of fatty acid labeling is needed in order to develop meaningful criteria toguide regulation development. They have signalled their willingness to entertainapplications for temporary marketing authorizations to test certain labelingschemes and generate consumer data. The regulation permitting temporary mar-keting is in place to allow products that do not conform to current regulationsaccess to the market when information is needed to determine what the mostappropriate requirements should be. It is not invoked to allow manufacturers toplace products violating the normal regulations on the market to test viabilityprior to taking any necessary corrective action to achieve compliance. It isexpected that there will soon be products carrying ω-6 and ω-3 fatty acid infor-mation on the market under this temporary marketing provision.

In the case of trans fatty acid labeling, it is possible that this approach mightbe considered. However, no announcement has been made in this regard.

Comparative Claims

Regulations and policies are well developed in the area of comparative fat and fatty acidclaims in Canada. The claim “low in saturated fatty acids” may be made for food prod-ucts that contain no more than 2 g of saturated fatty acids/serving and no more than15% of energy from fatty acids. The claim “lower in saturated fat” compared to a ref-erence food may be made for products that have reduced saturated fatty acids by 25%and contain an absolute reduction of at least 1 g/serving. There are criteria for claimsinvolving increased levels of polyunsaturates, for reduced levels of cholesterol, and forothers. There are criteria to be met in speaking of reduced levels of fat itself, and so on.

Canada–U.S. Harmonization Efforts

A major initiative in all aspects of food control is that dealing with harmonizationof regulations with the United States under the Canada–U.S. Free Trade Agreement(CUSTA) and now under the North American Free Trade Agreement also involving


Mexico (NAFTA). There has been some progress in the labeling area, and also whatmight be considered a major set-back. On January 6, 1993 the U.S. published itsfinal rules under the Nutrition Labeling and Education Act for nutrition labeling andfor nutrient content claims. The U.S. accepted certain of Canada’s recommendationsfor modification of their earlier proposals but in the end developed a scheme thatsignificantly digressed from the Canadian system introduced in November 1988 andthose of the European Union and the Codex Alimentarius Commission (the Rome-based organization established to administer the FAO/WHO International FoodStandardization Program). Canada’s overall preferred position was to follow asclosely as possible the international standards developed by the Codex.

In March 1993, Health Canada and (the former) department of Consumer andCorporate Affairs issued a consultation document soliciting Canadian views onthe U.S. requirements from a wide range of constituencies. The consensus was thatCanada should retain its own nutrition-labeling requirements. It was generally feltthat the U.S. system was too onerous and complicated.

On the question of nutrient content claims, however, there was a consensusthat we should work toward harmonization, so that such things as the criteria for“low in” or “light in” would be the same in both countries. Although separatelabels would be needed for each country, there would be no need to reformulatethe product. Health Canada is currently assessing specific recommendationsreceived on this subject.

Regulatory Review and International Developments

Health Canada and AAFC have recently completed Phase I of the review of theFood and Drugs Act and Regulations. In this consultation phase, a number of rec-ommendations were made with respect to labeling. The most important of theseinvolves the question of more specific and comprehensive lists of ingredients onfood labels. Since 1976, all food labels with some exceptions, require a list ofingredients. Foods such as standardized alcoholic beverages and foods prepack-aged by retailers are exempt. There is also a substantial list of foods that do notrequire a declaration of their components when used as ingredients in other foods.Additionally, some ingredients may be identified by class names. For example,vegetable oils, other than specified tropical oils and peanut oil, may simply bedeclared as “vegetable oil.” The reason for this is to allow manufacturers freedomof substitution in the face of price shifts, availability, and other factors.

At the international level, the Codex Committee on Food Labeling, at itsOctober 1994 meeting in Ottawa, again discussed the issue of food hypersensitivityand the need for more complete disclosure of ingredient information. There hasbeen a recommendation from the Nordic countries that the general labeling standardbe amended to require declaration of components of ingredients if that ingredient ispresent at levels in excess of 5% in the finished food. The current standard requires


such declaration only if the ingredient is present at levels in excess of 25%. The Nordiccountries also developed a list of substances that should always be declared. Other del-egations were of the view that the important issue is complete identification of sub-stances known to cause severe adverse reactions regardless of level. The committee’sconclusion at this meeting was that countries needed to better develop their positionson the home front in preparation for further discourse at future Codex meetings.

In Phase II of Regulatory Review, now underway in Canada, this matter will befully examined and regulatory proposals developed. In terms of more comprehensiveingredient disclosure, the primary motivating force for change will be one associatedwith substances that cause severe adverse reactions. We are aware of the interest ofcertain segments of the edible oil industry in more specific declarations of vegetableoils. Primary producers tend to adopt this position with marketing considerations inmind. Processors tend to favor continued flexibility. The United States does requirespecific vegetable oil declaration. Phase I of Regulatory Review did not produce anystrong position on further increased specificity in this area. If I could speculate on theoutcome I would say that there will continue to be the opportunity to use the classname vegetable oil when safety concerns are not present.

Biotechnology

As we have heard, there is a great deal of work going on in the tailoring of fattyacid profiles of domestic oilseed crops for a variety of reasons. Genetic engineer-ing is one of the techniques employed. The Canadian regulatory system is in theearly stages of developing public policy with respect to labeling these foods.

At a workshop held in Ottawa in November 1993, there was a wide range ofviews on whether labeling should be required. There were those who favoredlabeling simply as a “right to know” matter or for religious or ethical reasons.Others were concerned that labeling might have a negative impact on the viabilityof the technology. There was reasonable consensus that if there were safety issues,such as the introduction of a foreign allergen into the edible portion of the plantor animal, labeling should be considered.

At the October 1994 Codex Food Labeling meeting in Ottawa, there was a polar-ity of national views on the matter. A significant number of countries favored labelingin all circumstances in which recombinant technology had been used. Others favoreda more conservative approach, advocating labeling only when there might be a safetyconcern. Again, countries were asked to develop a more definitive national positionon the subject for further discussion at the next Codex labeling meeting.

The Department of Agriculture and Agri-Food along with its sister agencies isconvening a workshop at the end of November this year to develop principles andto provide focus for further assessment of the issue. There are some fundamentalissues that must be dealt with. Among these are the matters of right-to-know, andreligious or ethical concerns. For those advocating complete labeling, do they mean


only in the case where the edible food portion has been modified through geneticengineering or would they extend labeling to foods from plant products that hadbeen genetically modified for some agronomic feature, such as drought or herbi-cide resistance? Would they advocate labeling of meat or milk from cattle fedgenetically modified fodder, or to which a veterinary drug made using recombi-nant technology had been applied to control diseases or to enhance performance.

Once the question of whether or not to label is answered, the next question mightbe what type of information would be appropriate. If, for example, the composition ofcanola oil were significantly altered from the normal, and it was determined that label-ing of this fact was in the public interest, would it be sufficient to signal the differencewith a statement that genetic engineering had been employed. Some information onthe altered fatty acid composition may be more appropriate. But, would consumersalso demand information to the effect that genetic engineering had been employed?There arc a number of other questions that could be raised.

Single Access Label Review Service and Revenue Generation

I would be somewhat remiss if I did not mention a service initiated by the AAFCin October 1993. To overcome manufacturer and importer complaints that theyhad difficulty in determining where to get assistance with the design of theirlabels, a review service was established in 12 locations across Canada. Officers inthese locations will provide advice on labels of all products, except those subjectto the Fish Inspection Act. If consultation with officials in Ottawa or with HealthCanada is necessary in the resolution of issues, such as those involving the pro-priety of particular claims, this will be done by officials in the office. The idea isone-stop shopping and a maximum 10 working day turn-around.

The idea of sharing the cost of the department’s inspection program is becom-ing popular. Whether it is done through negotiations with the various industry seg-ments or through direct regulatory imposition, part of the inspection costs will beborne by industry in the future, on a sliding scale depending on the degree of pri-vate versus public good of the service, Label review and advisory services will notbe exempt from this process.

Conclusion

We have learned that the activities and developments in the edible oil industry arevery exciting indeed. Activities in the food-labeling business may be somewhatless dramatic but carry with them their own challenges. We are in an era of increas-ing consumer activism. Consumers are demanding more information and they aredemanding a more active part in the decision-making processes that ultimatelyaffect them. The trick will be to find a practical and equitable way to respond toconsumer demands and needs. We must balance these demands with the realities of


the day and the ability to enforce the demands. New developments must take placein an atmosphere of open, honest dialogue. The means will have to be found tokeep pace with new technologies now taking place at all levels in the food indus-try. Organizations, such as The American Oil Chemists’ Society, can assist in thisprocess.

Reference

1. Canadian Department of National Health and Welfare, Nutrition Recommendations, TheReport of the Scientific Review Committee, Canadian Government Printing Centre, 1990,pp. 40–52.


Chapter 11

Safety Evaluation and Clearance Procedures for NewVarieties of Oilseeds in the United States and Canada

Donna Mittena, Keith Redenbaugha, and Julianne Lindemannb

aCalgene, Inc., 1920 Fifth, Davis, California, 95616; and bLindemann Consulting, El Cerrito,California, 94530, USA.

Introduction

There are common elements for the clearance of new plant varieties derived usingrecombinant DNA techniques. Canada and the United States require the evaluation offood, feed, and environmental safety. In this paper, we provide background on thebasis of regulation in these two countries. We will discuss the issues that should beaddressed for new varieties of oilseeds and, using Calgene’s experience with modi-fied-oil rapeseed products, the types of data necessary to complete a safety evaluation.

Food and Feed Safety

To date, the United States Food and Drug Administration (FDA) has been operat-ing under a policy statement issued in May, 1992 (1). The policy outlined steps tobe taken to determine the safety of foods derived from genetically modified plantvarieties, and indicated that developers should confer with the FDA on issues ofsafety. Within the FDA, the Center for Food Safety and Applied Nutrition and theCenter for Veterinary Medicine were consulted for modified-oil rapeseed food andfeed products. The FDA has indicated that it intends to require premarket notifi-cation of foods derived from genetically modified plants, We expect that theamount of safety data Calgene will generate on any one oil will not be affected bya rule change.

According to policy at Health Canada, food and feed products require formalreview and approval. The criteria for evaluation Guidance Document was pub-lished in September 1994 (2). Health Canada is committed to a 90-day reviewperiod. Supporting data will be almost identical to that generated for the UnitedStates. Exact requirements are to be identified through consultation with HealthCanada. The safety of livestock feed is also under the purview of the Feed Sectionwithin Agriculture and Agri-Foods Canada. Guidelines are under discussion forlivestock feed. An advisory committee composed of experts from academia, indus-try, and government have convened to review safety issues and develop guidelines.

There is agreement on the concerns about modified oils and meal. Three areaswere identified by both Canadian and U.S. agencies:


1. Acceptable levels of nutrients, toxicants, and antinutritional factors;2. Safety of transformation markers; and3. Genetic characterization.

Compositional Analysis

Compositional analysis addresses the first point; acceptable levels of nutrients, tox-icants, and antinutritional factors. Oil and meal derived from transgenic plantsshould meet the standards of products currently on the market, the standards of com-merce. The analysis should include components of the edible oil and the seed meal.

Transformation Markers

The second issue is the transformation marker, a gene for resistance to the antibi-otic kanamycin. The FDA issued a Food Additive Regulation allowing the use ofthe kanamycin resistance gene product APH(3’)II (aminoglycoside phosphotrans-ferase) in cotton, tomato, and Brassica napus (3). Use of this gene product hadbeen shown to not compromise antibiotic therapy in humans, and also it does notreduce stability of neomycin in animal feeds. APH(3’)II is not toxic, it is not anallergen, and it is not active in the digestive tract where it is degraded.

Calgene tested the stability of neomycin in animal feeds. Neomycin wasmixed with fresh meal of two transgenic lines and the parent varieties. The mealwas stored under conditions near the optimum temperature for enzyme activity.The meal was sampled for stability of the added antibiotic, and no differenceswere found between transgenic lines and their parents. The study concluded thatthe stability of neomycin mixed with seed meal was not diminished even after 56days in storage (4).

Likelihood of horizontal gene transfer to soil microbes was addressed by aprobability model and found to not present a risk. Calgene addressed these issuesby constructing models of gene transfer, using data from the published literatureon in vitro transformation events and reasoned scientific judgment. Calgene didnot attempt to directly measure gene transfer, because background level ofkanamycin resistance in bacteria are fairly high and transformation events wouldbe so rare as to be undetectable relative to the background. Based on the results ofthe evaluations, Calgene concluded that there will be no significant increase inexposure to kanamycin-resistant bacteria from consumption; at most 1 new kanr

bacterial cell would be produced for every 750 billion that are already present inthe human gastrointestinal tract (4). The potential for transformation of soil bac-teria was also evaluated, by constructing a hypothetical model and using availablepublished literature and reasoned scientific judgment. At most 1 new kanr bac-terium would be produced via transformation for every 10 billion that are alreadypresent in the soil.


Genetic Characterization

Definition of genetic change requires a description of the function of the insertedDNA. Evidence of stability is provided by the study of Southern hybridizations ofDNA from several generations. The sequence of the DNA is defined and an openreading frame analysis is completed. A search of databases for homology withknown allergens and toxicants is conducted.

Environmental Safety

The United States Department of Agriculture (USDA) examines environmentalsafety under the Plant Pest Act. Key elements of clearance involve environmental safetyand absence of plant pest characteristics. Authority under the Plant Pest Act allowsUSDA to require permits for environmental introduction (field tests) of any organ-ism that may be a plant pest. Use of the plant pathogen, Agrobacterium tumefaciens,during transformation places virtually all transgenic plants under USDA’s authority.United States Department of Agriculture promulgated regulations in 1993 that allowit to issue a Determination of Nonregulated Status to exempt an organism from per-mit requirements after review of a Petition from the developer of the plant (4).Petition review emphasizes potential environmental effects and detrimental effectson nontarget, beneficial organisms. A separate determination is required for eachphenotype. Approvals are specific to genetic constructs in specific plant lines.Additional lines may be added by amendment. A determination of nonregulated sta-tus allows commercial production without further oversight by the USDA.

Agriculture and Agri-Food Canada (AAFC) is the lead agency responsible forthe regulation of agricultural products of biotechnology. Before any geneticallyengineered plant can be grown uncontained, it must be evaluated for environmentalsafety. Assessment Criteria for such a determination were issued by AAFC inSeptember 1994 (5). Under the Seeds Act, an agricultural crop variety to be sold inCanada must be registered. It must be recommended by the appropriate variety reg-istration recommending committee, based on performance testing to show “merit”of the new variety during 3 years of trials. Field-testing data are required from loca-tions where commercial production would take place. Environmental safety “sign-off ” will follow review of safety data by AAFC.

Canada and the United States agree on the environmental safety issues for mod-ified oilseed crops. An environmental safety evaluation should identify and exam-ine potential significant impacts on natural and agricultural systems. Potentialecological effects to be evaluated for oil-modified rapeseed include

1. Introduction of new pests;2. Worsening of an existing pest; and3. Displacement of a naturalized plant community.

Selective advantage is probably the most critical element of the risk assessmentfor an oil-modified canola. Without a selective advantage, a modified genotype will


not be able to persist to any greater extent than the unmodified type, and the con-sequences of gene flow will be neutral.

Relative Fitness

Data generated in agronomic field tests can be used to directly compare character-istics of the transgenic and parent lines. Studies conducted in nonmanaged settings,that is, without common agronomic practices may be required to evaluate the poten-tial for a crop or gene to move from a managed agricultural system into a naturalcommunity. The quantification of the net replacement value and ecological studiesfor the determination of persistence and invasiveness are parameters that may beused to measure the fitness advantage of the transgenic crop relative to its parent.

For example, in Calgene’s petition to the USDA for Laurate Canola (6), liter-ature review and data were presented to address relative fitness. Field and con-trolled environment studies were designed to assess the probability of theestablishment of feral populations of transgenic oil-modified canola lines relativeto that of the parent(s) or other cultivars. Studies were also designed to pay spe-cial attention to seed germination and seedling establishment, the phase of the lifecycle where changes in seed oil should show the greatest effect. Results of thestudies have not identified any cause for concern.

Gene Flow

The potential for gene flow via pollen in any oilseed crop can be assessed beginningwith a review of the botanical literature and floristic surveys. For example, descrip-tion of pollen movement, and outcrossing to wild Brassica relatives, as well as, stud-ies to identify the fate of hybrids with wild relatives were provided to the USDA inCalgene’s Laurate Canola petition (6). Wild relative by crop hybrids show no differ-ence in seed production in greenhouse crosses. Germination, seedling vigor, anddormancy tests of the hybrid seed showed the hybrid had neither the germinationcuing nor the dormancy characteristics of its wild maternal parent.

Conclusions

Although the regulatory oversight of new plant varieties of oilseed crops has a dif-fering legal basis, we see few differences in the issues identified and the types ofdata required for safety evaluation in Canada and the United States. As we cometo the end of 1994, the policy for safety review is in place and is being used tocomplete the safety assessment of the first modified oil products derived fromrecombinant DNA techniques for commercial sale.

References

1. Food and Drug Administration, Statement of Policy: Foods Derived from New PlantVarieties, Federal Register 57 (104): 22983–23005, Washington, D.C., May 29, 1992.


2. Health Canada, Guidelines for the Safety Assessment of Novel Foods, Vols, I and II, FoodDirectorate, Health Protection Branch, Ottawa, 1994.

3. Food and Drug Administration, Secondary Direct Food Additives Permitted in Food forHuman Consumption; Food Additives Permitted in Feed and Drinking Water of Animals;Aminoglycoside 3’-Phosphotransferase II, Federal Register 59: 26700-26711,Washington, D.C., 1994.

4. United States Department of Agriculture, Genetically Engineered Organisms andProducts; Notification Procedures for the Introduction of Certain Regulated Articles; andPetition for Nonregulated Status; Final Rule, Federal Register 58: 17043-17059,Washington, D.C., March 31, 1993.

5. Agriculture and Agri-Food Canada, Environmental Safety Assessment of BiotechnologyRelease Regulations under the Seeds Act, D94-03. Plant Industry Directorate, Ottawa,1994.

6. United States Department of Agriculture, Availability of Determination of NonregulatedStatus for Genetically Engineered Canola, Docket No. 94-052-2, Federal Register 59(213): 55250-55251, Washington, D.C., November 4, 1994.


Development and Processing of Vegetable Oils for Human Nutrition

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