Development and Processing of Vegetable Oils For Human Nutrition

Development and Processing of Vegetable Oils for Human Nutrition
Copyright 1995 AOCS Press
Development and Processing of Vegetable Oils for Human Nutrition
Editors Roman Przybylski Bruce E. McDonald

Department of Foods and Nutrition University of Manitoba Winnipeg, Canada
Champaign, Illinois
AOCS Mission Statement To be a forum for the exchange of ideas, information, and experience among those with a professional interest in the science and technology of fats, oils, and related substances in ways that promote personal excellence and provide high standards of quality. AOCS Books and Special Publications Committee E. Perkins, chairperson, University of Illinois, Urbana, Illinois T. Foglia, USDAERRC, Philadelphia, Pennsylvania M. Mossoba, Food and Drug Administration, Washington, D.C. Y.-S. Huang, Ross Laboratories, Columbus, Ohio L. Johnson, Iowa State University, Ames, Iowa J. Lynn, Lever Brothers, Edgewater, New Jersey G. Maerker, Oreland, Pennsylvania G. Nelson, Western Regional Research Center, San Francisco, California F. Orthoefer, Riceland Foods Inc., Stuttgart, Arkansas J. Rattray, University of Guelph, Guelph, Ontario A. Sinclair, Deakin University, Geelong, Victoria, Australia G. Szajer, Akzo Chemicals, Dobbs Ferry, New York L. Witting, State College, Pennsylvania B. 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 Data

Development 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 664.3dc20
95-33314 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 saturated fat to 10 percent of their total energy intake has had a tremendous effect on the food industry, particularly the fats and oils industry. Other major developments that have affected the edible fats and oils industry include the findings that (i) monounsaturated fatly acids are as effective as polyunsaturated fatty acids in lowering blood cholesterol; (ii) hydrogenated fats, or more precisely the trans fatty acids 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 are under development in response to these findings. The development of novel oilseed varieties has produced a scramble among regulatory agencies to develop guidelines governing the licensing and release of these new crops. An added problem for governmental agencies, particularly in light of new agreements covering the international movement of food products, is the need to develop and standardize food labeling regulations. These developments were major factors in the decision to organize a conference on the development and processing of vegetable oils for human nutrition. The Canadian Section of the AOCS was invited to organize the conference in conjunction with its Annual Meeting on October 24, 1994. The Conference was a success thanks to the efforts of the Organizing Committee and its chairman James Daun, the support of sponsors and donors, and the distinguished group of speakers. Current nutrition issues and the contributions of processing, genetic engineering, and plant breeding were reviewed, as well as the role of government agencies in the development of novel oilseed crops. This monograph covers all of these issues, beginning with an up-to-date coverage of nutritional issues, followed by a discussion of current developments in processing vegetable oils for human consumption and the modification of traditional oilseed sources by genetic manipulation. The monograph concludes with a synopsis of the regulatory requirements in Canada, the United States, and Europe for the registration 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 in providing us with manuscripts. We are especially grateful to Angela Dupuis for willingly and patiently transcribing the manuscripts to a common format and
her very significant efforts toward the success of this publication. We are grateful for the unique satisfaction that comes with having contributed to the knowledge on this subject. Roman Przybylski and Bruce E. McDonald Department of Foods and Nutrition University of Manitoba Winnipeg, Canada
Contents
Preface Chapter 1 Chapter 2 Food Fats and Fatty Acids in Human Nutrition Joyce L. Beare-Rogers Nutrition and Metabolism of Linoleic and Linolenic Acids in Humans E.A. Emken Trans Fatty Acids in Canadian Breast Milk and Diet W.M.N. Ratnayake and Z.Y. Chen Food Industry Requirements for Fats and Oils: Functional Properties T.K. Mag Hydrogenation: A Useful Piece in Solving the Nutrition Puzzle Robert C. Hastert and Robert F. Ariaansz Interesterification: Current Status and Future Prospects Suresh Ramamurthi and Alan R. McCurdy Sources of Oilseeds with Specific Fatty Acid Profiles W.A. Keller Production of Oilseeds with Modified Fatty Acid Composition Rachael Scarth Classification of Oils with Modified Fatty Acid Compositions as Novel Foods Frank W. Welsh
Chapter 3 Chapter 4
Chapter 5 Chapter 6 Chapter 7 Chapter 8 Chapter 9
Chapter 10 Food Labeling in Canada Ian Campbell Chapter 11 Safety Evaluation and Clearance Procedures for New Varieties of Oilseeds in the United States and Canada Donna 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 has long been appreciated that a caged experimental animal given a high-fat diet eventually becomes obese. An excellent demonstration in humans showed the interaction of the level of fat, provided covertly in an ad libitum diet, and level of physical activity (1). At the lowest level of fat to maintain energy balance, physical activity produced a negative energy balance. The intermediate level of dietary fat caused a positive shift in balance with a pronounced difference between sedentary and active individuals. At the highest level, 60 en%, both groups of individuals had a positive energy energy balance, but the energy storage was greater in inactive individuals. Particularly within the range of usual fat intake, there is a tradeoff 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 in the same way that it does to carbohydrate (2). Individuals tend to be insensitive to the level of fat in a meal and are consequently apt to overeat. Excess carbohydrate is stored only to a limited extent and is then converted to fat. The cost of metabolic conversion is relatively high for carbohydrate and protein, but fatty acids are easily added to stored energy. Therefore, for sedentary individuals the recommendation 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 certain stages of life, particularly infancy. Most human milk provides fat in which the total proportion of saturated fatty acids shorter than 18 carbon atoms is approximately equal to the monounsaturated fatty acids, principally oleic (35). Since the fat in human milk is 4555% of the total dietary energy, the saturated component provides about 18% of the energy, considerably more than the ceiling of 10% that
is frequently recommended. Questions about the fatty acid composition of infant formula have usually revolved around the essential fatty acids and the role that docosahexaenoic acid plays in neural membranes. Here it is important that the n-6 fatty 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 correlated with the long-chain n-3 and n-6 fatty acids (6). This apparent interference with the conversion of essential fatty acids was therefore thought to involve the desaturases, but placental receptors also may be sites of influence.
Lipoproteins
The greatest debate about dietary fatty acids revolves around their effects on blood 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 of the particles in which it is carried), and the concentration of Lp(a) that limits plasmin production and promotes clotting and vascular smooth muscle proliferation. The saturated fatty acids, although frequently considered together, do have different degrees of influence on the concentration of LDL. Laurie and myristic acids, which are usually found in the same oils, are more hypercholesterolemic than palmitic 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 depends upon the reference oil. Thus, palm oil is hypocholestcrolemic with respect to coconut 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 in plasma cholesterol was proportional to twice the amount of energy supplied by saturated fatty acids minus the amount of energy supplied by polyunsaturated fatty acids plus a small factor for dietary cholesterol (11, 12). In all later regression lines, the greatest adverse effect on plasma cholesterol levels was also associated with the intake of saturated fatty acids. The attempts to reduce the consumption of saturates have sometimes led to extreme proposals, the idea apparently being that since high amounts are bad, intermediate amounts must be barely tolerable and low amounts must be best. The quest for extremely low dietary levels of saturated fatty acids seems futile because if there are insufficient dietary saturated fatty acids to occupy the 1-position of membrane phospholipids, they have to be synthesized by the body. The effect of linoleic acid in reducing plasma cholesterol is thought to be nonlinear, plateauing at about 5% of energy, and having a range of 310% according to
an individuals responsiveness (13). Below the so-called threshold, an increase of 3% of energy as linoleic acid decreased plasma cholesterol by 35 mg/dL. A similar change in plasma cholesterol above the threshold required 13% of energy as linoleic acid. In this range, there is relative insensitivity to changes in dietary fatty acids, although the lowest concentrations of blood cholesterol occurred with high intakes of linoleic acid (14). The fact that individuals have different thresholds helps 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-cholesterol may have been related to high thresholds for dietary linoleic acid (15). Linoleic acid appeared to reduce HDL-cholesterol, but the high intake of linoleic acid in this study would be difficult to attain or maintain with ordinary foods. In another study without a group fed a high level of linoleic acid, Grundy et al. showed that a diet high in oleic acid was preferable to a low-fat diet (high carbohydrate) in sustaining HDL-cholesterol while decreasing LDL-cholesterol (16), Test fats consisting of butterfat, beef fat, cocoa butter, and olive oil produced no differences in HDL-cholesterol (17). Low-density lipoprotein cholesterol was highest with butterfat and significantly lower with cocoa butter, indicating that the position of the fatty acids on the acylglycerols was important. It appears that in at least some situations, saturated fatty acids in the 2-position are the most hypercholesterolemic.
-Linolenic Acid and Postinfarct Patients

A comparison was made between the usual postinfarct prudent diet and a Mediterranean diet that used a margarine made from canola oil (18). Improved mortality after a first myocardial infarction was attributed to the increased intake of -linolenic acid. Although this observation is encouraging for canola oil, it must be remembered that many dietary features differed between the two dietary groups, and that more definitive work is required.
Trans Fatty Acids and Lipoproteins

The impact of dietary fats containing trans monounsaturated fatty acids, as determined in Trappist monks, has stood the test of time (19). In the presence of dietary cholesterol, the trans fatty acids were associated with serum cholesterol levels that were higher than those obtained with oleic acid and slightly lower than those obtained with a mixture of lauric and myristic acids. The much quoted paper of Mensink and Katan (20), was the first to show that trans monounsaturated acids increased LDL-cholesterol and decreased HDL-cholesterol, worsening the LDL/HDL ratio. Although this 3-week study was criticized for 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 in the study were similar to those found in partially hydrogenated soybean oil; the positional cis isomers had one type, the 8-octadecenoic acid, that was higher than ordinarily found (21), but no significance has been attached to it. Another study from the same laboratory (22), had a lower level of trans fatty acids, 7.7% of dietary energy instead of 11%. The results of the two separate studies suggested a doseresponse to trans fatty acids. The finest precision yet seen in the determination of lipoprotein levels appeared in Judd 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 the study of Mensink and Katan. Again, the trans fatty acids were associated with increased LDL-cholesterol and decreased HDL-cholesterol when compared with oleic acid. It must be remembered that the original purpose of these experiments was to determine how trans fatty acids should be regarded, given that saturated fatty acids were already designated as hypercholesterolemic. Also, products promoted as being low in saturated fatty acids were sometimes high in trans fatty acids. At issue is whether saturated fatty acids should be replaced by trans fatty acids. More sensibly, both should be reduced in the total diet. The average intake (50th percentile) of any substance gives no indication of the risk to vulnerable individuals. Information on at least the 90th percentile of intake and the associated food patterns is required. Investigations of trans fatty acids should therefore provide estimates of possible human exposure along with intake guidelines for essential fatty acids, particularly for pregnant and lactating women. The assessment of intake of trans fatty acids loses accuracy when a part of the diet is self-selected. For many foods, the fatty acid composition is not accurately known, and the possible combinations are considerable. The most reliable data on fatty acid consumption are obtained from the analysis of all foods given to the participants of a study. Values for the coefficients of variation of total cholesterol for example, calculated from papers dealing with dietary trans fatty acids (20,2227), are given in Table 1.1. Flynn et al. tested margarine versus butter with two eggs/day in an otherwise self-selected diet. Judd et al., Lichtenstein et al., Mensink and Katan, and Zock and Katan provided all foods to the test subjects. Wood et al. supplied test fats that 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 because of the control in subject selection and in the analytical procedures. The selection of subjects for this study raised questions about the general applicability of the results. The subjects had normal levels of all blood lipids, were free of any disease, and maintained their usual exercise program without gaining weight, in spite of a mean energy consumption of 3227 kcal/day for the men and 2025 kcal/day for the women. These healthy athletic subjects did not reflect the larger community, and
TABLE 1.1
Precision in Human Studies on Trans Unsaturated Fatty Acids
Study Flynn et al. Judd et al. Lichenstein et al. Mensink and Katan Wood et al. Zock and Katan
a
Foods provided + + +
Foods selfselected +
+ +
Coefficient of variationa % 19.3 4.7 10.7 15.3 14.6 14.7
For total cholesterol. Sources: Mensink and Katan (21), Zock and Katan (22), Judd et al. (23), Flynn et al. (24), Lichenstein el al. (25), and Wood et al. (26,27).
might be expected to represent a group with a well-regulated cholesterol metabolism. They constituted such a finely tuned bioassay that as little as 3% of energy from trans fatty acids produced a statistically significant result. Another effect of trans fatty acids on lipoproteins pertains to the risk factor of Lp(a) involved in thrombogenesis. Investigators in Australia and The Netherlands 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 situation will 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 heart disease, the adipose tissue fat had increased trans fatty acids and decreased shorter chain fatly acids (30, 31). It was concluded that the victims had consumed more hydrogenated fat and less ruminant fat than the controls. Also, in patients undergoing coronary angiography, the level of trans fatty acids was 1.38% versus 1.11% of fatty acids in controls (32). Such results were said to be consistent with the hypothesis that dietary trans fatty acids are a risk factor. Recent studies (33, 34), however, raise questions with this hypothesis, although issue also has been taken with the results and conclusions of these studies (35). More controversial estimates of exposure to trans fatty acids came from semiquantitative, food-frequency questionnaires. Answers to questions about how often over the previous year a given portion of a specified food had been consumed became the source of data. Clinical studies in which dietary variables are known and controlled exhibit a scientific rigor that is unfortunately lacking in the responses to semiquantitative questionnaires. In adult men (mean age 62 yr; range 4385 yr) assessed by a food-frequency questionnaire, total fat was given as 60 g/day and the trans fatty acid intake as 2.1 g at the 10th percentile and 4.9 g at the 90th percentile (36). These low values are inconsistent with other data. Nevertheless, the energy-adjusted intakes of trans fatty acids were reported to be positively correlated with LDL-cholestcrol and inversely correlated with HDL-cholesterol.
In the Nurses Health Study (37), data on consumption came from the same type of questionnaire, with average values used in the assessment for such foods as margarine, cookies, biscuits, cake, and white bread. The intake of trans fatty acids was reported to have varied from 2,45.7 g/day or 1.33.2% of energy, and to be correlated with the risk of cardiovascular disease. Another paper claiming an association between the intake of trans fatty acids and the risk of cardiovascular disease involved questionnaires administered 8 weeks after patients had been discharged from hospital after a first myocardial infarction (38). The patients were matched with residents of the same town. The average consumption of trans fatty acids was 1.5% of energy for men and 1.7% of energy for women. These levels were about one-half of that calculated for the average trans fatty acids in the American diet (39). The relative risk of myocardial infarction for each 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 quintile appeared to be the best. Overall, the epidemiological studies emphasize the need for additional research on the physiological effects of trans fatty acids and that, in the interim, prudence be exercised in the consumption of these fatty acids.
Idealized Dietary Fat

Biotechnologists have challenged nutritionists to provide them with the fatty acid profile of the ideal vegetable oil. What is important is the lipid content of the total diet. For one oil to have an impact, it would have to be an appreciable contributor to the dietary fat. This does happen with some types of food patterns, but in most mixed diets there is some trade-off between foods high and low in a particular fatty acid. It is the ultimate blend that counts. For the total fatty acids in an adult diet, the saturated fatty acids (mostly palmitic) could be 1025%, linoleic acid could be 1020%, -linolenic could be about 2%, and the rest could be oleic acid. The only virtue of a very low level of saturated 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 an ideal salad oil, an ideal spread, an ideal cooking fat, and so on. To propose a fatty acid composition for an ideal vegetable oil, one would need information about the other foods to be consumed. It is the total dietary fatty acids that are important in nutrition.
References
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Chapter 2
Nutrition and Metabolism of Linoleic and Linolenic Acids 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), that Eskimos had little or no cholesterol deposits in their coronary arteries and a low incidence of coronary heart disease because of the n-3 fatty acids in their diet, was largely ignored by public health and medical organizations. In fact, Sinclairs theory was termed imaginative by Keys, who was a leading authority on heart disease and diet (2). A dramatic change in the health and medical communitys perception of the nutritional importance of n-3 fatty acids occurred when Bang et al. reported in 1971 that the high intake of n-3 fatty acids from fish was a key factor in the low mortality rate from coronary heart disease observed in Greenland Eskimo populations (3). Since those early times, there has been a growing accumulation of evidence that indicate n-3 long-chain fatty acids (LCFA) are associated with various antiatherogenic properties and a number of other health benefits, although this issue is still controversial (46).
Biological Properties
It is now appreciated that n-3 and n-6 fatty acids have very different physiological effects. One reason is the difference in the physiological properties of the eicosanoids produced by the lipoxygenase and cyleooxygenase pathways from 20:5n-3, 20:3n-6, and 20:4n-6. In most cases, in vitro studies have shown that the physiological effects of the 1- and 3-series of prostaglandins formed from 20:3n-6 and 20:5n-3 are opposite the effects of the 2-series of prostaglandins formed from 20:4n-6. These results have led to the hypothesis that a balance between the various eicosanoids and their n-6 and n-3 precursors is necessary to regulate many physiological functions. These observations for the n-3 and n-6 LCFA have raised several questions concerning the nutritional importance of linolenic acid present in plant sources. A
1 Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable. Portions of this paper have been published in the Proceedings for the Scientific Conference on -3 Fatty Acids in Nutrition, Vascular Biology and Medicine, April 1719, 1994, Houston, Texas, American Heart Association.
basic question is whether the conversion of linolenic acid (18:3n-3) to eicosapentaenoic acid (20:5n-3) and docosahexaenoic acid (22:6n-3) in humans has quantitative importance. Of practical concern is whether linolenic acid from plant sources is a viable alternative to dietary sources containing preformed n-3 LCFA.
Requirements
Evidence from animal studies indicates that competition between fatty acids from the n-3 and n-6 families influences the incorporation of these fatty acids into tissue lipids and mediates their biological effects (46). These results raised the question of whether the actual amounts of linoleic acid (18:2n-6) and linolenic acid (18:3n-3) or the 18:2n-6/18:3n-3 ratio in the diet has more nutritional importance. It is difficult to determine exactly what the best 18:2n-6/l8:3n-3 ratio for the human diet is. Examples of some of the 18:2n-6/18:3n-3 ratios recommended are 6:110:1 (7), 5:1 (8), 4:16:1 (9). An interesting recent study reported that rats fed diets with a 4:15:1 ratio of 18:2n-6 to 18:3n-3 were smarter, healthier, and tougher than rats fed diets with an n-6 to n-3 ratio of 3:1 or 6:1 (10). The estimates given in Table 2.1 for a hypothetical diet provide some guidance for the actual amounts of dietary 18:2n-6 and 18:3n-3 required to meet essential fatty acid recommendations (1116).
Metabolism and Effect of Diet

Experiments with animal models have provided most of the information on the effect of varying the balance between 18:2n-6 and 18:3n-3 (46,1718). Studies with radioisotopelabeled substrates have been particularly useful for investigating
TABLE 2.1
Estimated Recommendations for Essential Fatty Acids Translated for 2400 KcalBased Diet Containing 90 g (34% Energy) of Total Fat
Fatty acid Linoleic acid Adult/infant Pregnant mother Lactating mother Adult Linolenic acid Adult Infant Adult 20:5n-3 plus 22:6n-3 Adult Adult Total calories (%) 23 4.5 6.0 2.4 1.0 23 0.3 0.13 0.27 Total fat (g) 58 12 16 6.4 2.7 0.8 0.30.4 0.7 (%) 69 13 18 7.1 3.0 0.9 0.4 0.8 Reference 11 11 11 12 13 11 14 15,16 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 22:6n3 (46,1921). By contrast, experiments in humans using isotope-labeled n-6 and n-3 fatty acids are limited. Results for the conversion of 18:2n-6 in vitro have been reported for human liver microsomes (2223) and human leukocytes (24). In vivo data have been published for one study with deuterium-labeled 20:3n-6 (25), two studies with C-labeled l8:2n-6 (2627) and two studies with deuterated 18:2n-6 (2829). We have recently reported results that directly compare the metabolism of deuterium-labeled linolenic acid and linoleic acid in young adult male subjects that 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 dietary 18:2n-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 (3.03.5 g) and 18:3n-3 (3.03.5 g). Three additional subjects were fed a deuterated TAG mixture that contained 2.2 g of deuterated 18:3n-3 as the only polyunsaturated fatty acid. In addition to labeled linoleic acid and linolenic acid, the mixtures of deuterated fats contained 2 or 3 of the following deuterated fatty acids: 16:0, 18:0, or 18:1. The deuterated TAG mixtures were fed after the subjects had fasted for 12 hr. Blood samples were collected over a 48-hr period. Methyl esters of the plasma lipids were analyzed by gas chromatograph-mass spectrometry methods (31). The subjects were fed control diets for 12 days prior to being fed the deuterated TAG mixtures. The composition of the control diets provided 3536% of calories from fat, 4344% from carbohydrates, and 21% from protein. The saturated 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-6 and 1.0 g 18:3n-3 (n-6/n-3 ratio = 30; P/S = 0.85). The amounts of 18:2n-6 in the diets 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].
14
Incorporation of Fatty Acids into Body Lipids Results for the chylomicron triglyceride samples showed that the deuterated 18:2n-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 indicates that 18:3n-3 may be absorbed slightly more efficiently than 18:2n-6, but the difference was not significant. Differences between subjects were relatively small for the concentrations of deuterated l8:2n-6 (range 17.1-19.4 g/mL) and l8:3n-3 (range 18.523.8 g/mL) in the chylomicron TAG samples. These results indicate that the fatty acid composition of the prefed diets had no significant effect on absorption of 18:2n-6 and 18:3n-3. Examples of time course curves for incorporation of deuterated l8:2n-6 and 18:3n-3 into plasma phosphatidylcholine (PC) are plotted in Figure 2.1.
Fig. 2.1. Examples of time course curves for incorporation of deuterated 18:2n-6 and 18:3n-3
into plasma phosphatidylcholine samples from male subjects fed diets containing 15 g (SAT diet) and 30 g (PUFA diet) linoleic acid. Source: Proceedings for the Scientific Conference on -3 Fatty Acids in Nutrition, Vascular Biology and Medicine, April 1719, 1994, Houston, Texas, American Heart Association.
Qualitatively, these curves illustrate that phosphatidylcholine acyltransferase is more selective for 18:2n-6 than 18:3n-3. The mean values for the integrated areas of the time course curves for plasma PC samples from the subjects from the PUFA diet group were 363 52 g/mL 18:2n-6 and 58.5 42 g/mL 18:3n-3. Mean plasma PC values for subjects from the SAT diet group were 600 6.5 g/mL 18:2n-6 and 66.0 21.7 g/mL 18:3n-3. These results indicated that the higher 18:2n-6 content of 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
in the ratio of deuterated 18:2n-6 to 18:3n-3 concentrations in plasma PC indicated that phosphatidylcholine acyltransferase is six to nine times more selective for 18:2n-6 than 18:3n-3. The mean concentrations for deuterated 18:2n-6 and 18:3n-3 in plasma TAG are compared in Figure 2.2 to concentration data for the deuterated 16:0, 18:0, and 18:1 fatty acids that were also part of the mixtures of deuterated TAG fed to these subjects. The general pattern for the deuterated fatty acids incorporated arc similar for subjects fed the SAT and PUFA diets. However, the concentration for the 18-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 Figure 2.3. The total lipid data show an overall preferential (ca. threefold) incorporation of 18:2n-6 relative to l8:3n-3. The higher 18:2n-6 content of the PUFA diet reduced the incorporation 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 the PUFA diet increased fatty acid oxidation by about 30%, which is reasonably consistent with the 9% increase in fat oxidation (based on use of O water methods) when a diet 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 evidence that incorporation of deuterated 18:2n-6 and 18:3n-3 in the major lipid classes were
18
Fig. 2.2. Concentration (g/mL plasma) of deuterated fatty acids in plasma triacylglycerol samples 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 data are significantly different (P < 0.05). Source: Proceedings for the Scientific Conference on -3 Fatty Acids in Nutrition, Vascular Biology and Medicine, April 1719, 1994, Houston, Texas, American Heart Association.
Fig. 2.3. Concentration (g/mL plasma) of deuterated 18:2n-6 and 18:3n-3 in plasma total lipid
samples 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 diet means. Source: Proceedings for the Scientific Conference on -3 Fatty Acids in Nutrition, Vascular Biology and Medicine, April 1719, 1994, Houston, Texas, American Heart Association.
significantly depressed by increased dietary 18:2n-6 intake. Why do dietary 18:2n6 levels influence both the amount of the deuterated 18:2n-6 and 18:3n-3 incorporated 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 the deuterated l8:2n-6 and 18:3n-3 was diverted into the -oxidation pathway when dietary 18:2n-6 levels were increased (33). Higher oxidation percentages would result in a general reduction of the concentration of deuterated fatty acids in plasma lipids which, in turn, would reduce the amount of 18:2n-6 and l8:3n-3 available for conversion to LCFA metabolites. An explanation for why 18:2n-6 increases fatty acid oxidation is that dietary 18:2n-6 reduces acyltransferase activity by reducing the synthesis of the mRNA, that codes for synthesis of the acyltransferase enzymes (20). Reduction in acyltransferase activity could allow a larger portion of the fatty acid pool to be diverted into the -oxidation pathway. A general reduction in the incorporation of nonn-6 and n-3 deuterium-labeled fatty acids (16:0, 18:0, and 18:1) that were fed to these subjects at the same time was also observed. This observation is consistent with the possibility of a general nonselective increase in fatty acid 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-3 and 18:2n-6 are shown in Figure 2.4. The concentration of all the individual n-3
Fig. 2.4. Concentration (g/mL) of individual deuterated n-3 and n-6 long-chain fatty acid metabolites in plasma total lipids from subjects fed diets containing 15 g (SAT diet) and 30 g (PUFA diet) linoleic acid. Bars indicate high and low values. Source: Proceedings for the Scientific Conference on -3 Fatty Acids in Nutrition, Vascular Biology and Medicine, April 1719, 1994, Houston, Texas, American Heart Association.
and n-6 LCFA metabolites were consistently lower for subjects that were prefed the high 18:2n-6 (PUFA) diet. Variability between subjects for the concentration of individual n-3 and n-6 LCFA metabolites was fairly large. However, when sums for the various n-3 and n-6 metabolites were compared (Figure 2.5), the variability between subjects was much smaller. The variability between the concentration data for the individual n-3 and n-6 LCFA metabolites indicates a considerable subject-dependent difference in the rate of conversion of the deuterated 18:2n-3 and 18:3n-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 individual subjects along with the means for subjects fed the SAT and PUFA diets are compared in Figure 2.5. These results demonstrate that conversion of 18:3n-3 to n-3 LCFA metabolites was considerably higher (ca. 3.7 times) than conversion of 18:2n-6 (P < 0.001) and that dietary 18:2n-6 significantly reduced (P < 0.01 for 18:3n3 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 conversion data by dividing the n-3 LCFA metabolite data by the total for 18:3n-3 plus n-3 LCFA metabolites, Percent conversion data for 18:2n-6 can be calculated in a similar manner. The results are shown in Figure 2.6. The average percent conversion was about 40% lower for l8:3n-3 and 56% lower for l8:2n-6 when the subjects were fed the diet enriched in 18:2n-6. Expression of the g/mL data as percent data distorts the conversion data because of the large difference between the amount of 18:3n-3 and 18:2n-6 incorporated into plasma lipids due to the high selectivity for 18:2n-6 discussed earlier.
Fig. 2.5. Concentration (g/mL) of the sums for n-3 and n-6 long-chain fatty acid metabolites
in 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 of deuterated fats fed to subjects 5, 6, and 7. Source: Proceedings for the Scientific Conference on -3 Fatty Acids in Nutrition, Vascular Biology and Medicine, April 1719, 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 from
subjects fed diets containing 15 g (SAT diet) and 30 g (PUFA diet) linoleic acid. For SAT versus PUFA, 18:3n-3 means (P < 0.07) are significantly different. Means for 18:2n-6 are not significantly different. Source: Proceedings for the Scientific Conference on -3 Fatty Acids in Nutrition, Vascular Biology and Medicine, April 1719, 1994, Houston, Texas, American Heart Association.
The sum of the concentrations for the deuterated n-6 LCFA metabolites was much lower 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 of deuterated l8:3n-3 was greater than for deuterated 18:2n-6. Deuterated 20:5n-3 (34.3 g/mL) and 22;6n-3 (29.8 g/mL) represent 6.0% and 3.8% of the total amount of labeled 18:3n-3 in total plasma lipids, respectively. In contrast, deuterated 20:4n-6 (7.2 g/mL) represents 0.5% of the labeled 18:2n-6 in total plasma lipids. Average total percent conversion of deuterated 18:3n-3 for all subjects (15.3%) was higher than that of deuterated l8:2n-6 (1.6%). This low percent conversion of deuterated 18:2n-6 is consistent with both in vivo and in vitro data from other human studies (2229). The difference in the amounts of deuterated 18:2n-6 and l8:3n-3 converted to long-chain polyunsaturated fatty acid metabolites is not easily explained. A higher amount of n-6 LCFA metabolites would be expected, since the concentration of deuterated 18:2n-6 in plasma total lipids (1260 g/mL) is about three times higher (P < 0.001) than the concentration of deuterated 18:3n-3 (450 g/mL). If one accepts that -6 desaturase is the rate-limiting step in the conversion pathway and if the rate constant is similar for both 18:2n-6 and 18:3n-3 (5,19), then the concentrations of deuterated n-6 and n-3 LCFA should be proportional to the concentrations 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 is a plausible explanation for the difference in conversion observed in this study. These in vivo data for deuterated n-6 and n-3 LCFA metabolites indicate that -6 desaturase is about four times more selective for 18:3n-3 than l8:2n-6. This selectivity is somewhat higher than the difference in -6 desaturation for 18:2n-6 and 18:3n-3 of 1.53.0 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 SAT and PUFA diets are illustrated by the concentrations of deuterated 18:2n-6 and 18:3n3 and their deuterated n-3 and n-6 LCFA metabolites in plasma total lipids (Figures 2.4 and 2.5). The concentrations of the deuterated fatty acids were clearly lower for the subjects fed the PUFA diet. These results indicate that the metabolism of both the 18:3n3 and 18:2n-6 was altered when subjects were fed diets containing different levels of 18:2n-6 (15.1 g vs. 29.8 g). This effect of dietary 18:2n-6 is consistent with animal data showing that 18:2n-6 competes with itself and with 18:3n-3 (4,5,18,20). The approximate twofold difference in dietary 18:2n-6 content lowered deuterated 18:2n6 and 18:3n-3 concentrations in plasma total lipids by 3739% and deuterated n-6 and n-3 LCFA metabolite concentrations by 6570%. The ratio of deuterated 18:2n-6 to 18:3n-3 and deuterated n-6 to n-3 LCFA metabolites were not influenced by the 18:2n-6 content of the diets. These results suggest that the absolute amounts of dietary 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-6 in a typical U.S. diet can be estimated from the deuterated LCFA data. Based on a total plasma volume of about 3000 mL (39 mL/kg body wt) and the concentration of deuterated n-3 LCFA metabolites (Figure 2.5), the total amount of deuterated n-3 LCFA metabolites in plasma lipids was 351 mg or 127 mg/g of deuterated 18:3n-3 fed (SAT diet) and 126 mg or 43 mg/g deuterated 18:3n-3 fed (PUFA diet). By extrapolation from the metabolite weight data, the 2 g of 18:3n-3 in a typical U.S. diet is estimated to provide 186 mg/day of n-3 LCFA. Based on a similar calculation, 537 mg/day of n-6 LCFA is estimated to be synthesized from the 21 g of dietary l8:2n-6 in typical U.S. diets, Estimates based on plasma concentration data indicate that dietary 18:3n-3 provides about 50% of the n-3 LCFA daily requirement for adults. The estimates based on the total weight of deuterated LCFA metabolites are believed to be the most reliable, although they underestimate conversion of both l8:3n-3 and 18:2n6 because the plasma data do not include the amounts of deuterated LCFA metabolites that were incorporated into tissue lipids. Alternatively, the amount of long-chain n-3 and n-6 fatty acids synthesized from dietary 18:3n-3 and 18:2n-6 can be calculated from the percent conversion data in Figure 2.6. The percent conversion calculated for a typical U.S. diet is about 15% for 18:3n-3 and about 1.8% for 18:2n-6. Thus, about 300 mg of n-3 LCFA metabolites/day is estimated to be synthesized from 2 g of l8:3n-3 in a typical U.S. diet and 378 mg of n-6 LCFA metabolites/day is estimated to be synthesized from 21 g of 18;2n-6. Based on the percent conversion data, the 18:3n-3 in a typical U.S. diet is estimated to provide 7585% of the 350400 mg of longchain 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 fatty acids in humans, it is clear that most U.S. and Canadian diets contain a large surplus of 18:2n-6, but diets do not contain a surplus of n-3 fatty acids. If the 18:3n3 provided by soybean and canola oils are not included in dietary estimates, both the U.S. and Canadian diets would be deficient in 18:3n-3. Therefore, the concern is that the development of the new low (23%) 18:3n-3 soybean and canola oils may have a negative nutritional and health impact if they were to replace the conventional soybean and canola oils that contain 710% 18:3n-3.
References
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6. Malasanos, T.H., and P.W. Stacpoole, Diab. Care 14: 1160 (1991). 7. Lasserre, M., F. Mendy, D. Spielmann, and B. Jacotot, Lipids 20: 227 (1985). 8. Crawford, M.A., Polyunsaturated Fatty Acids and Eicosanoids, edited by W.E.M. Lands. The American Oil Chemists Society, Champaign, Illinois, 1987, pp. 270295. 9. Galli, C, and A.P. Simopoulos (eds.) General Recommendations on Dietary Fats for Human Consumption, Dietary -3 and -6 Fatty Acids: Biological Effects and Nutritional Essentiality. NATO Series A, Life Sciences, Plenum Press, New York, 1989, pp. 40304. 10. Yehuda, S., and R.L. Carasso, Proc. Nat. Acad. Sci. 90: 10345 (1993). 11. Dietary Fats and Oils in Human Nutrition. A Joint FAOWHO Report, Food and Agricultural Organization of the United Nations, Rome, 1977, pp. 2330. 12. Bourre, J.M., M. Piciotti, O. Dumont, G. Pascal, and G. Durand, Lipids 25: 465 (1990). 13. Simopoulos, A.P., J. Nutr. 119: 521 (1989). 14. Bourre, J.M., O. Dumont, G. Pascal, and G. Durand, J. Nutr. 123: 1313 (1993). 15. Bjerve, K.S., I.L. Mostad, and L. Thoresen, Am. J. Clin. Nutr. 45: 66 (1987). 16. Bjerve, K.S., S. Fischer, F. Wammer, and T. Egeland, Am. J. Clin. Nutr. 49: 290 (1989). 17. Hagve, T.A., and B. Christophersen, Biochim. Biophys. Acta 796: 205 (1984). 18. Vamecq, J., L. Vallee, P. Lechene de la Porte, M. Fontaine, D. de Craemer, C. van den 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. Acta 1123: 18 (1992). 20. Sprecher, H., in Dietary 3 and 6 Fatty Acids: Biological Effects and Nutritional Essentiality, edited by C. Galli and A.P. Simopoulos, NATO Series A, Life Sciences, Plenum Press, New York, 1989, pp. 6979. 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. 4579. 22. de Gomez Dumm, I.N.T., and R.R. Brenner, Lipids 10: 315 (1975). 23. Poisson, J.-P., R.-P. Dupuy, P. Sarda, B. Descomps, M. Narce, D. Rieu, and A.C. de Paulet, Biochim. Biophys. Acta 1167: 109 (1993). 24. Cunnane, S.C., P.W.N. Keeling, R.P.N. Thompson, and M.A. Crawford, Brit. .J. Nutr. 51: 209 (1984). 25. El-Boustani, S., J.E. Causse, B. Descomps, L. Monnier, F. Mendy, and A. Crastes de 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 (1987). 29. Emken, E.A., R.O. Adlof, D.L. Hachey, D. Garza, M.R. Thomas, and L. Brown-Booth, J. 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).
32. Nichaman, M.Z., Nutrition Monitoring in the United States: An Update Report on Nutrition Monitoring, DHHS Publication 891255, Life Science Research Office, Hyattsville, Maryland, (1989). 33. Peter, J.H., and D.A. Schoeller, Metabolism 37: 145 (1988). 34. Hrelia, S., M. Celadon, C.A. Rossi, P.L. Biagi, and A. Bordoni, Biochem. lnt. 22: 659 (1990). 35. Brenner, R.R., R.O. Peluffo, A.M. Nervi, and M.E. De Tomas, Biochim. Biophys. Acta 176: 420 (1965).
Chapter 3
Trans Fatty Acids in Canadian Breast Milk and Diet

W.M.N. Ratnayakea and Z.Y. Chenb
a Nutrition 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 countries are prepared by the process of partial hydrogenation. This process converts liquid oils to solid fills that have the elasticity and texture desired for many food preparations. Negative aspects of this practice are the substantial reduction in the proportion of essential fatty acids in the dietary fats with a concomitant formation of trans and cis isomers of oleic and linoleic acids. The content of trans fatty acids (TFA) in dietary fat varies. The average daily intake of TFA for the U.S. population has been estimated to be at least 8 g or 3.7% of total energy (1,2). A recent estimate of TFA intake for the Canadian population is not available, nevertheless many of the food items in the Canadian retail market contain significant amounts of TFA. For example, Canadian margarines may contain up to 50% TFA (3). Cookies, biscuits, donuts, deep-fried foods, and many other common snacks made from partially hydrogenated vegetable oils also contain substantial amounts of TFA (4). It is well established that the fatty acids in breast milk reflect those of the maternal diet (514). The presence of TFA in human milk is a concern, because of their possible negative nutritional and physiological effects on the recipient infant. Human infants absorb and metabolize trans isomers and incorporate them into plasma and tissue lipids (15). Negative effects of TFA, such as perturbations of essential fatty acid and prostaglandin metabolism (16), and formation of unusual long-chain polyunsaturated fatty acids were observed in rodents (1719). In human infants, TFA seem to impair the biosynthesis of n-6 and n-3 long-chain polyunsaturated fatty acids (LCP) and the individuals growth (20). During late fetal and early postnatal growth, considerable amounts of n-6 and n-3 LCP are accreted in neural and other tissues (21). Phospholipids of the central nervous system and of retinal photoreceptor cells are particularly rich in arachidonic (20:4n-6, AA) and docosahexaenoic (22:6n-3, DHA) acids (22). Studies with infant animals have indicated that a deficiency of DHA in the brain and retina may impair development of visual acuity, and possibly also discrimination of learning (2326). Although the presence of TFA in human milk has been recognized for a long time, the literature data are not complete and generally may not be accurate, due to the difficulty of analyzing TFA. Recent reviews on human milk fatty acids have not
mentioned the content of TFA and other unusual isomeric fatty acids (2730). There is also a lack of information about these fatty acids in human breast milk from Canadians. Therefore, using a combined procedure of silver nitrate-thin layer chromatography (AgNO3-TLC) and gas-liquid chromatography (GLC), we analyzed the fatty acids of mature breast milk of 198 women across Canada. The TFA data were then 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 t18:1 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 Health Protection Branchs ongoing monitoring program of chlorinated hydrocarbon contaminants in the breast milk of Canadian women (unpublished work of W.H. Newsome, Health Protection Branch, Ottawa). Samples of mature milk (34 weeks of parturition) were collected from lactating women from across Canada (2025 samples per province), except from Prince Edward Island and the two territories. Donors were requested to express about 34 mL of their milk manually during each feeding, starting from the very first feeding to the last feeding of the day. The sample from each donor thus represented the accumulated milk collection per day. A total of approximately 2550 mL from each mother was collected in brown bottles with polytetrafluoroethylene-lined screw caps. Mothers were requested to refrigerate the milk samples between collections. The day following the 24-hr collection, the samples were shipped in dry ice to Ottawa and stored at -24C 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 triheptadecanoin (1 mg/mL) as an internal standard to quantitate total milk fat by GLC. The extracted fat was methylated with BF3-MeOH and analyzed by GLC using an SP-2560 flexible fused silica capillary column (100 m 0.25 mm i.d., 20 m film thickness). Column temperature was programmed from 150 to 180C at a rate of 0.5C/min, and then to 210C at a rate of 3C/min. A typical GLC trace of a human milk fatty acid methyl ester (FAME) profile is shown in Figure 3.1. Single step, direct GLC analysis cannot accurately determine the total t-18:1 due to overlap of high delta 18:1 trans isomers (12t-16t) with c-18:1 isomer peaks (32). In the human milk of this study, 20.8% (range 930%) of the total t-18:1 isomers overlapped with c-18:1 isomers. Therefore, the total t-18:1 and c-18:1 levels in the milk samples were determined using AgNO3-TLC in conjunction with capillary GLC. Silver nitrate thin-layer chromatography was performed as described previously (33). The t-18:1 band was isolated and analyzed by GLC. The proportion of t-isomers that overlapped with the c-18:1 isomer peaks was calculated by comparing the 18:1 region of the GLC chromatogram of the isolated 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 2627 in Figure 3.1) that were 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-18:1 isomers (12t-16t; peaks 28-30, and 34 in Figure 3.1) that overlapped with the cis isomers and the well-separated t-18:1 isomers. The t-18:1 and c-18:1 isomer distribution was determined by oxidative ozonolysis (34). The positional and geometrical isomers of linoleic acid were determined and identified as described previously (33).
Results and Discussion

No significant regional differences in the average fat content and fatty acid profile data of Canadian human breast milk samples were observed in this study. Therefore, only the mean values, standard deviation, and the ranges are presented for 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 level of C10C14 saturated fatty acids was higher in the milk of human vegans and vegetarians than in that of omnivores (30). The mean levels for 10:0, 12:0, and 14:0 in human milk of this study were remarkably similar to the levels found in vegan and vegetarian human milk in the United Kingdom (30). This might reflect that intake of meat by the mothers in the present study was low, although dietary records were not available. Sanders and Reddy have hypothesized that the origin of C10C14 saturated fatty acids in human milk is not dietary, but most likely derived by de novo synthesis from carbohydrates in the mammary gland (30). Intake of these fatty acids is low in vegans compared with the intake of omnivores, since vegan and vegetarian diets contain more carbohydrates and less fat than those of omnivores (35). The levels of linoleic, -linolenic acids, and their C20 and C22 metabolites in human milk are of special interest, because of their important physiological significance (22). The levels of linoleic (10.5%) and -linolenic (1.2%) acids found in this study are similar to those reported in studies of mature human milk from women 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 lower than for those in other countries (27,28,30,36) but similar to levels reported for vegans or vegetarians (30). The lower levels of n-6 and n-3 LCP further suggest that a large segment of the lactating women in the present study were vegans or vegetarians. In some of the samples of this study, only trace amounts (<0.005% of total 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 requirements of LCP for infants is not known, but the extremely low levels of LCP in some
capillary 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, 9c16:1; 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, -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, 20:5n3; 70, 24:0; 71, 24:1; 72, 22:4n-6; 73, 22:5n-6; 74, 22:5n-3; and 75, 22:6n-3.
Fig. 3.1. A typical GLC trace of FAME from human breast milk fat analyzed on an SP-2560
TABLE 3.1
Fatty Acid Composition (wt% of total fatty acids) of Pooled 24-hr Collection of Mature Breast Milk from 198 Canadian Mothers
Fatty acid Saturated fatty acids 10:0 11:0 12:0 13:0 14:0 15:0 16:0 17:0 18:0 20:0 14:0 brb 16:0 brb Cis-monounsaturated fatty acids 9c-14:1 9c-16:1 7c-16:1 10c-17:1 8c-18:1 9c-18:1 10c-18:1 11c-18:1 12c-18:1 13c-18:1 14c-18:1 15c-18:1 16c-18:1 11c-20:1 13c-20:1 13c-22:1 n-6 Polyunsaturated fatty acids 18:2n-6 18:3n-6 20:2n-6 20:3n-6 20:4n-6 22:4n-6 22:5n-6 n-3 Polyunsaturated fatty acids 18:3n-3 20:4n-3 20:5n-3 22:5n-3 22:6n-3 Trans fatty acids t-14:1 t-16:1 Meana (SD) 1.39 (0.59) 0.01 (0.02) 5.68 (2.01) 0.03 (0.03) 6.10 (1.73) 0.37 (0.12) 18.30 (2.25) 0.32 (0.08) 6.15 (0.97) 0.15 (0.09) 0.14 (0.06) 0.14 (0.06) 0.28 (0.08) 2.27 (0.56) 0.41 (0.13) 0.21 (0.06) 0.34 (0.07) 30.65 (2.66) 0.49 (0.06) 1.91 (0.17) 0.74 (0.23) 0.24 (0.08) 0.23 (0.07) 0.15 (0.08) 0.10 (0.07) 0.39 (0.13) 0.14 (0.09) 0.02 (0.03) 10.47 (2.62) 0.08 (0.06) 0.17 (0.09) 0.26 (0.09) 0.35 (0.11) 0.04 (0.05) 0.02 (0.02) 1.16 0.06 0.05 0.08 0.14 (0.37) (0.06) (0.05) (0.06) (0.10) Rangeb 0.46 tr 2.32 tr 2.26 0.12 12.90 0.03 3.49 tr tr 0.04 0.06 1.11 0.20 tr 0.15 23.55 0.32 1.26 0.21 0.07 0.06 0.05 0.01 0.13 tr tr 0.58 tr tr tr 0.05 tr tr 0.58 tr tr tr tr 4.42 0.08 11.77 0.14 11.68 0.67 24.06 0.44 9.85 0.36 0.26 0.45 0.66 3.88 0.70 0.44 0.48 40.64 0.68 2.33 1.01 0.31 0.20 0.21 0.20 0.65 0.42 0.11 1.90 0.21 0.46 0.46 0.69 0.18 0.16 1.90 0.26 0.25 0.45 0.53
0.09 (0.05) 0.18 (0.08)
tr 0.47 tr 0.42
TABLE 3.1 (continued)

Fatty Acid Composition (wt% of total fatty acids) of Pooled 24-hr Collection of Mature Breast Milk from 198 Canadian Mothers
Fatty acid 8t-18:1 9t-18:1 10t-18:1 11t-18:1 12t-18:1 13t-18:1 14t-18:1 15t-18:1 16t-18:1 17t-18:1 tt-18:2 9c-13t/18t,12c-18:2 8t,13c-18:2 9c,12t-18:2 9t,12c-18:2 9t,15c-18:2 t-18:3 Unusual polyunsaturated fatty acids 9c,15c-18:2 8c,14c-20:2 5c,8c,11c-20:3 5c,8c,14c-20:3 5c,11c,14c-20:3 Othersc Total saturated fatty acids Total n-6 LCP Total n-3 LCP Total TFA Total (-18:1 Fat content (g/L)
a b
Meana (SD) (0.12) (0.32) (0.44) (0.49) (0.37) (0.25) (0.19) (0.17) (0.15) (0.01) (0.08) (0.14) tr 0.29 (0.15) 0.24 (0.12) tr 0.11 (0.08) 0.07 (0.05) 0.19 (0.08) 0.05 (0.06) tr (0.00) 0.02 (0.02) 2.24 (0.09) 38.50 (2.94) 0.83 (0.28) 0.33 (0.24) 7.19 (3.03) 5.87 (2.52) 31.58 (9.37) 0.15 0.88 1.22 1.31 0.80 0.63 0.37 0.29 0.21 0.01 0.05 0.36
Rangea tr 0.06 0.03 0.02 0.01 tr tr tr tr tr tr tr 0.00 tr tr 0.00 tr tr tr tr 0.00 tr 1.25 36.92 tr tr 0.10 0.10 9.29 0.69 2.71 3.39 3.61 2.44 2.07 1.26 0.88 0.70 0.02 0.28 0.76 tr 0.59 0.59 tr 0.34 0.15 0.49 0.18 tr 0.12 2.54 42.59 1.2 1.4 17.21 15.42 57.53
tr = trace amounts (<0.005% of total fatty acids). br = branched. c Others include fatty acids <10:0, very minor branched-chain fatty acids, 18:2 conjugated fatty acids, and unknowns.
Canadian human milk samples should be a concern, because the ability to metabolize linoleic and -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 levels in human milk. A dose-dependent increase in DHA content in milk was demonstrated in lactating mothers given fish oil concentrates rich in n-3 LCP (38). On the other hand, the LCP level of human milk does not appear to be correlated with the mothers intake of the parent fatty acids (14,36,39). This was confirmed in the present study (Figure 3.2). These findings suggest that milk LCP originate from the
Fig. 3.2. Scatter plots of % AA vs. % linoleic acid
and DHA vs. % -linolenic acid in human milk.
mothers diet and cannot be elevated by increasing the dietary levels of linoleic and -linoienic acids. This might explain the low LCP levels in vegan and vegetarian milk compared to milk from omnivores (30). Vegan and vegetarian diets contain very little LCP, because the latter are not plant products and are present only in animal tissues. Breast milk from lactovegetarians may contain small amounts of LCP derived from dairy products. Recent analysis in our laboratory shows that cows 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 dairy products 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 a wide variation in the TFA content (i.e., the sum of all unsaturated fatty acids with one 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 69% TFA range, while 22% contained more than 9% TFA. Only 6 samples contained very low levels (<3%) of TFA.
Data on TFA content in human milk from other countries is scarce. This is the only study that examined such a large number of samples collected across a country. Nevertheless, the limited data available in the literature indicate that the TFA level in the Canadian human milk is 1.5 to 3.6 times higher than that in other countries (711, 1315). 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), United States (8), and Germany (14), respectively. These differences may reflect both dietary and methodological differences between the studies. As mentioned earlier, t-18:1 isomers partially overlap with cis isomers in GLC analyses, and thereby total TFA level may be underestimated in studies that used direct GLC methods for analysis of TFA (32). In some hydrogenated fats, the underestimation of the total t-18:1 level by direct GLC can be as high as 32% (40). The limited number of samples analyzed in other studies (711,1315) also could account for differences in TFA content. The range for TFA reported in the present study compares well with results reported in 1976 by Beare-Rogers and Nera for some Canadian human milk samples (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 technique (41). The similarity in the TFA levels between the two studies may indicate that the TFA level in the Canadian diet has not changed over the last 18 years, in spite of a recommendation made in 1980 by an ad hoc Committee of Health and Welfare Canada to reduce the TFA level in the Canadian diet (42). As in partially hydrogenated vegetable oils, t-18:1 was the major trans group in Canadian human milk (Table 3.1). The average t-18:1 isomer distribution for the 198 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, but it differed from that of cows milk (Figure 3.4). This suggests the major source of TFA in the Canadian diet is partially hydrogenated vegetable oils, whereas the contribution from dairy products is relatively minor. Trans isomers of linoleic acid were the other important trans group found in human 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 6492), partially hydrogenated soybean oil (PHSO; blend of six base stocks of iodine value varying from 63109), and cows milk (n = 10).
0.9% milk fatty acids. This group was primarily composed of 9c,13t-18:2/8t,12c18:2 (0.4%), 9t,12c-18:2 (0.2%), and 9c,12t-18:2(0.3%). These fatty acids are also likely to have originated from partially hydrogenated vegetable oils (33). Canadian margarines contain up to 7.9% 18:2 isomers (3), and many bakery products also contain high levels of 18:2 isomers (4). The close similarity of both t18:1 and t-18:2 distribution patterns of human milk and partially hydrogenated vegetable oils implies that dietary isomeric fatty acids are incorporated into the mammary gland with little or no alteration of their relative proportions or structures. This observation supports the view that milk fatty acids reflect the fatty acid profile of the diet (43). However, under energy deficient conditions milk fatty acids are reported to resemble the fatty acid pattern in adipose tissue (43). In addition to the isomeric fatty acids originating from partially hydrogenated vegetable oils, our detailed analysis revealed the presence in Canadian human milk 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 presence of these two fatty acids has not been reported in dietary fats. However, we recently found these two fatty acids in various tissues of rats fed partially hydrogenated canola oil and suggested that they were derived from dietary 12c-18:1 by alternative desaturation and chain-elongation (19). The 12c-18:1 isomer is formed during partial hydrogenation of vegetable oils and is present in appreciable quantities in Canadian human milk (Table 3.1). The presence of the two C20 metabolites in human milk suggests that the metabolic pathway of 12c-18:1 described for rats is also operative in humans. In the human milk of the present study, the total TFA and t-18:1 levels were inversely related to linoleic and -linolenic acids (Figure 3.5; data for total TFA not shown), suggesting that the elevation of TFA in human milk is at the expense of essential fatty acids. This is to be expected, because food items made with partially hydrogenated fat contained higher levels of TFA and lower levels of essential fatty acids compared to food products made from unhydrogenated oils (4).
Fig. 3.5. Inverse correlation of % linoleic acid vs. t-18:1 and, -linolenic acid vs. t-18:1 in human milk.
From the present study, it is apparent that a substantial proportion of Canadian human milk samples contain large amounts of trans and other isomeric fatty acids. The consequences of this situation on the recipient infants health and physiology are unknown. Nevertheless, several lines of evidence indicate that large amounts of dietary isomeric fatty acids may have some negative effects on infants. Koletzko has reported that TFA may impair the essential fatty acid metabolism and the early growth of human infants (20). Since desaturase and elongase enzyme activity may be limited in infants (37), the impairment of essential fatty acid metabolism by TFA could considerably influence the availability of LCP in infants receiving breast milk containing high proportions of TFA. Another concern with large amounts of isomeric fatty acids in diets, is their possible chain-elongation and desaturation to unusual C20 polyunsaturated fatty acids. Several unusual polyunsaturated fatty acids were shown to occur in various tissues (1719), including the developing brain (18,44), and retina (45) of rats fed diets containing partially hydrogenated vegetable oils or synthetic isomeric fatty acids. The physiological effects of the presence of isomeric fatty acids in various tissues are not known, although the incorporation of a trans isomer of DHA into the retina of developing 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 production of C20 polyunsaturated fatty acid metabolites (19). In rats, these four isomers metabolized to C20 polyunsaturated fatty acids even with an adequate dietary supply of essential fatty acids (19). Whether these unusual metabolites could occur in humans is not known, but as discussed previously, trace levels of 8c,14c-20:2 and 5c,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-20:4 isomers in neonates. Future studies should investigate the possible incorporation of the isomeric fatty acids into various tissues, especially brain and retina, and evaluate the 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 depends mainly on the mothers recent dietary intake (8,11). This is particularly applicable to t-18:1 which fluctuates in human milk according to the content in the diet (11). CraigSchmidt et al. (11) found a strong linear correlation (r = 0.91) between the previous days 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 and dietary fat, respectively) the t-18:1 levels in the diet of lactating women could be estimated from human milk t-18:1 levels. Application of this equation indicates that intakes of t-18:1 by the lactating women of the present study ranged from 0.6 to 32.3% 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 of 58% 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 appeared to ingest more than 30% of the total dietary fatty acids as t-18:1. The total TFA in the diet is difficult to estimate, because a simple correlation for total TFA between diet and breast milk is not available. It would be expected to be about 20% higher than that of t-18:1, since t-18:1 accounted for only about 80% of the total TFA in human 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 fat consumption data. According to the recently published Nova Scotia Nutrition Survey free-living women in the child-bearing age of 1835 years consume on average 67.1 g fat and 1721 kcal of total food energy/day (46). Thus, for this group, the mean t-18:1 content in the diet could be estimated to be 7.0 g/person/day or 3.7% of the total dietary energy. Assuming that all the members in a given household eat the same type of food, but that total food intake among the individual household members may vary, the dietary t-18:1 content for both males and females of all adult age groups (from 1874 years) could be estimated based on data reported in the same survey (46). As shown in Table 3.2 the average t-18:1 intake could be 8.4 g/person/day or 3.7% of the total dietary energy for Canadian adults. Because of high fat intake (46), diets of young male adults (1834 years) could contain extremely high levels of 1-18:1 (mean 12.5 g, range 0.738.9 g). These data also indicate that a wide range of intake (0.338.9 g or 0.211.7% total energy) 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 lactating
women in Canada. Data were calculated using Craig-Schmidt equation (11) and the percentage of t-18:1 level in human milk fat.
TABLE 3.2
Estimates of Dietary t-18:1 Intake for Canadian Adults (1874 years of age)a
Age (years) 1834 3549 5064 6574 1834 3549 5064 6574 1874 Calories (kcal) 3020.5 2343.4 2229.7 2025.0 1720.8 1571.3 1571.3 1476.2 2070.2 Fat intake (g) 120.5 91.9 87.9 75.3 67.3 61.5 54.2 49.7 85.8 g t-18:1 person/day Mean Range 12.5 9.6 9.1 7.8 7.0 6.4 5.6 5.2 8.4 0.738.9 0.629.7 0.528.4 0.524.3 0.421.7 0.419.9 0.317.5 0.317.5 0.526.1 t-18:1 %energy/person/day Mean Range 3.7 3.7 3.7 3.5 3.7 3.7 3.4 3.4 3.7 0.211.6 0.211.4 0.211.5 0.210.8 0.211.3 0.211.4 0.210.7 0.211.3 0.211.3
Sex Male
Female
Averageb
a
Estimates 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 0.632.3%), which was calculated using the Craig-Schmidt et al. equation and t-18:1 levels found in Canadian human milk. b Average for both male and female adults (1874 years). Source: Craig-Schmidt el al. (11), and Nova Scotia Dietary Survey (46).
The average value estimated for Canadian adults is remarkably similar to the value of 8.1 g/person/day reported by Hunter and Applewhite for the U.S. population (1). The range is also similar to that estimated for the U.S. population (0.738.7 g/person/day) by Enig et al. (2). From the distribution pattern given in Figure 3.6 and the fat intakes shown in Table 3.2, it could be estimated that over 58% of Canadians would have t-18:1 intakes above 3.7% of energy, the level termed 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) is similar to that used by Mensink and Katan (48) in their clinical study in which they demonstrated that TFA adversely affect the LDL/HDL cholesterol ratio. Thus it is apparent that the results of recent clinical studies (4749) on the hypercholesterolemic effect of TFA are relevant to the Canadian population.
Conclusion
The fatly acid composition of 198 human breast milk samples collected in 1992 across Canada was determined by AgNO3-TLC and gas chromatographic procedures. Trans fatty acids ranged from 0.117.2% of the total milk fatty acids, with a mean value of 7.2 3.0%. Twenty-two percent of the samples contained more than 9% trans fatty acids whereas only 3% of samples had low levels (<3%). Using an equation based on the relationship between trans-octadecenoic (t-18:1) fatty acids in human milk and dietary fat, t-18:1 consumption in Canada was estimated. The estimate predicts a wide range of intakes from 0.338.9 g/person/day. Analyses of the t-18:1 isomer distribution of the human milk samples, indicated that partially
hydrogenated vegetable oils are the major source of isomeric fatty acids in the Canadian diet, whereas contribution from dairy products is relatively minor. The linoleic acid content in human milk varied from 5.821.4% of total fatty acids with an average value of 10.5%. On average, -linolenic acid accounted for 1.2%, with a range of 0.61.9%. The linoleic and -linolenic acid levels were inversely related to the total trans fatty acids, indicating that the elevation of trans fatty acids in Canadian human milk is at the expense of n-3 and n-6 essential fatty acids. The content of the physiologically important arachidonic and docosahexaenoic acids showed a wide variation and did not correlate with their parent fatty acids, linoleic and -linolenic. This finding suggests that it may be difficult to elevate the levels of n-6 and n-3 C20C22 polyunsaturated fatty acids in breast milk by increasing the levels of linoleic and -linolenic acids in the mothers diet. In summary, these findings suggest that breast milk in Canada has high levels of TFA and other isomeric fatty acids. This apparently is a reflection of the widespread use of hydrogenated vegetable oils in Canadian foods. The high level of isomeric fatty acids in the Canadian diet and human milk should prompt thorough investigation 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 are indebted to the mothers in this study for their cooperation and interest; and to the Bureau of Field Operations and H.W. Newsome for collecting and sharing the samples with us. R. Hollywood and G. Pelletier are thanked for analyses of fatty acids in milk samples.
References
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12. Chappell, J.E., M.T. Clandinin, and C. Kearny-Volpe, Am. J. Clin. Nutr. 42: 49 (1985). 13. Finley, D.A., B. Lonnerdal, K.G. Dewey, and L.E. Grivetti, Am. J. Clin. Nutr. 41: 787 (1985). 14. Koletzko, B., M. Mrotzek, and H.J. Bremer, Am. J. Clin. Nutr. 47: 954 (1988). 15. Koletzko, B., M. Mrotzek, and H.J. Bremer, in Human Lactation. Effect of Human Milk on the Recipient Infant, edited by A.S. Goldman, S. Atkinson, and L.A. Hanson, Plenum Publishing, New York, 1987, vol. 3, pp. 323333. 16. Kinsella, J.E., G. Bruckner, J. Mai, and J. Shimp, Am. Clin. Nutr. 34: 2307 (1981). 17. Holman, R.T., F. Pusch, B. Svingen, and H.J. Dutton, Proc. Natl. Acad. Sci. USA 88: 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. 6586. 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. OGrady. 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 and Health 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 used in 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) and palmitic (C16:0) are no longer viewed negatively; oleic (C18:l) is viewed positively in fat nutrition; linoleic (C18:2) is considered essential but should be somewhat limited; and linolenic (C18:3) is a precursor of EPA/DHA. These considerations place significant restrictions on the choice of fats and oils that should be used in making edible oil/fat products for the consumer, and also present serious obstacles to the optimal performance of fat products. The functional aspects that are of importance and influence the choices in making fat and oil products can be summarized very briefly as melting behavior, crystal habit and stability, and stability against breakdown reactions while avoiding synthetic antioxidants. Table 4.1 lists the functions that the different fat products have to fulfill.
TABLE 4.1
Technical Functions Required of Fats and Oils
Fat or oil Margarines Required quality Semisolid, spreadable Smooth texture Melt rapidly near body temperature Oxidative stability Fat crystals to pick up and hold air Semisolid Incorporate into batter Smooth texture Provide lubrication on eating Oxidative stability Heat stability (breakdown, polymerization) Oxidative stability Melt rapidly near body temperature No solid fat at refrigerator temperature Good oxidative stability at room temperature
Baking shortenings
Frying fats
Salad oils
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 been replaced by soft margarine. With either of these two products, good mouthfeel and good melt in the mouth is essential, but it should not be pourable. This can only be achieved with fats that have very small crystals, such as is achieved when the crystal form is . The stability of the polymorphic crystal form over time requires a variety of fatty acids composing the triglycerides in the crystal matrix of a fat product. This variety in the fatty acid composition is very important and will be mentioned repeatedly. Experience has shown that soft margarines may have crystals when there is a high dilution of the crystal mass with liquid oil, as is found in soft margarines with a very low solid fat content. But this does not produce excellent margarines, because such 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 several months. The liquid component, which is particularly high in soft margarine and prone to oxidation, must have some resistance to oxidation.
Baking Shortenings
For cakes and breads, certain fat is required for the development of texture and volume. The function of the fat is to introduce a large volume of finely dispersed air into the batter by the creaming effect, incorporating a large volume of air. Secondly, the fat 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 there be crystalline fat, and that most of the crystalline fat be in the polymorphic form, that is very small crystals that are stable over time. Also, crystalline fat is required to produce the proper cake and bread texture and structure when it is worked into the batter. The crystal structure prevents the fat from coalescing, as would occur with liquid oil. Fat coalescence results in poor microdispersion of the fat in the batter and, hence, poor texture and structure. These two requirements, air incorporation and the avoidance of fat coalescence in the batter, are the main reasons why liquid oils do not make good shortenings. Melt-in-mouth properties are not very important for shortenings because of the very fine dispersion of the solid fat in the baked product. In most instances, it is important that the shortening have a relatively high amount of the solid tat melting at 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. They are 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 be noted, however, that to bake good cakes with a minimum of fat requires optimal shortening performance. This means using crystalline (saturated) fat in the crystal habit as well as an appropriate emulsifier system. Puff pastry fats are a special case. The function of the fat in this application is primarily to separate the layers of dough to produce the flaky texture. To achieve this, a fat with a waxy texture and large amounts of high melting triglycerides is needed. This texture is achieved with fats that can be in the 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 temperatures used in frying. The oil is subjected to temperatures in the range of 150195C, open to the air, and in the presence of moisture and a host of other compounds that are introduced with the fried foods. Good stability means low unsaturation for oxidative and heat stability, and triglycerides that do not easily hydrolyze. The latter quality eliminates short-chain fatty acid oils. Crystal habit is not usually a significant consideration. Secondarily, mouth-feel, or melt-in-mouth is an important aspect. For this quality, large amounts of high melting triglycerides, must be avoided, In some applications, such as doughnut frying, a relatively high amount of solid fat is desirable 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 oil be 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 containing linolenic acid, such as canola and soybean oils, are usually less expensive than sunflower, corn, and cottonseed oils. It is relatively easy to make salad oil products 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 that occur in commonly available fats and oils. Also, the ease of oxidation, the fatty acid
composition, and the melting behavior of the various hydrogenated and unhydrogenated oils are of interest. Table 4.2 lists the melting points of fatty acids. The data are organized in two groups: the more common C18:0 fatty acids, and other fatty acids from C 16:0 to C6:0. Most of the vegetable oils are high in C18 acids. Canola and soybean oil are the most important representatives of this group of oils. Within the C18 acids, the data illustrate the well-known fact that melting temperature is a function of unsaturation. Melting is also a function of other aspects of molecular structure, such as positional and geometric isomerization. All of these are affected in partial hydrogenation. The melting temperatures indicate the range of changes in melting behavior that can be produced by hydrogenation. Partial hydrogenation produces considerable isomerization and yields fats that melt near body temperature, that is, fats that have steep melting profiles. Full saturation, or near-full saturation of these oils, produces relatively high melting fats (stearic acid melts at 69.6C) and removes isomerization. The other fatty acids in Table 4.2 show a similar range of melting temperatures as the C18 acids. Palmitic acid melts at 61.3C; lauric acid melts closer to body temperature at 44.2C; and caproic acid, included for comparison with low melting point C18 acids, melts at 1.5C. Fats that are predominantly made up of these fatty acids can be expected to have somewhat similar properties to partially hydrogenated C18 fatty acid oils, that is, melting close to body temperature. The fatty acid composition of fats is the main factor influencing their melting behavior. Another factor of importance is the distribution of fatty acids in the triglyceride. It modifies the previously indicated melting behavior and makes it more complex.
TABLE 4.2
Melting Temperatures of C18 and Other Fatty Acids
Fatty acid C18 fatty acids Linolenic cis 9,12,5 Linoleic cis 9,12 Oleic cis 9 Elaidic trans 9 Stearic C18:0 Other fatty acids Palmitic C16:0 Myristic C14:0 Lauric C12:0 Capric C10:0 Caprylic C8:0 Caproic C6:0 Melting point (C) 12.8 6.5 16.2 43.7 69.6 61.3 54.4 44.2 31.6 16.5 3.4 Occurrence Canola, Soybean Corn, Sunflower Olive, Canola Partially hydrogenated fats Tallow, coconut butter Lard, tallow, palm Palm kernel, Coconut Coconut Coconut Butter
Sources: Perry (4), and Eislor and Hagemann (5).
TABLE 4.3
Relative Oxidative Stability of C18 Fatty Acids
Stearic acid Oleic acid Linoleic acid Linolenic acid
Source: Swern (6).
1 10 100120 1 60250
Oxidative Stability In connection with C18 fatty acids, stability is an important aspect of the function of these fats and oils. Table 4.3 gives the relative oxidative stability of C18 fatty acids. It shows that linolenic acid and linoleic acid are about 20 times and 10 times more easily oxidized than oleic acid, respectively. Crystallization Behavior It was mentioned earlier that the crystallization habit of solid fat is an important aspect of a fats functionality. The tendency of a fat to be stable in the or in the form is a function of the heterogeneity of the fatty acid composition. It determines how well, or how poorly the triglycerides fit into a crystal matrix. Fats that have a fairly homogeneous fatty acid composition in their triglycerides tend to crystallize in the form. This means that large crystals are formed more or less quickly. As already pointed out, this is not desirable in margarines and in most uses of shortenings. Table 4.4 presents a classification of fats and oils according to their crystallization behavior. It can be seen that most of the oils used today are tending. The main reason is that many of these oils are high in C18 acids, lauric acid in the case of palm kernel and coconut oils; or a particular triglyceride predominates, such as for 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 4.4
Classification of Fats and Oils According to Crystallization Behavior
Type Soybean Safflower Sunflower Sesame Peanut Corn Canola Olive Coconut Palm kernel Lard Cocoa butter
Source: Wiedermann (7).
Type Cottonseed Palm Tallow Herring Menhaden Whale Rapeseed (HEAR) Butter fat Interesterified lard
TABLE 4.5
Methods to Prevent Crystallization in Fat Products
Blending with tending fats Interesterification (lard) Interesterification of a blend Partial hydrogenation (trans isomers) Using a crystal inhibitor (sorbitan tristearate)
are composed of significant amounts of palmitic or erucic acids, along with C18 acids, tend towards the structure. For this reason, palmitic acid occupies an important position in the formulation of functional oil products. In the case of the highly unsaturated vegetable oils, the crystallization habit refers to the partially and fully hydrogenated versions of these oils. It is apparent that the crystallization habit of many readily available oils is a problem in present edible oil product manufacture. Present methods to overcome the tendency of fat products to crystallize in the form are shown in Table 4.5. Blending of oils with different crystallization habit is the most widely practiced method to control crystal formation. Interesterification is not widely used, mostly because of cost. Trans isomers in partially hydrogenated fats represent desirable heterogeneity in fatty acid composition to reduce tendency. Using a crystallization inhibitor, such as sorbitan tristearate, is an option. It is classed as an additive in 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 are reviewed. These are the fats that can supply crystalline fat in fat products in place of the partially hydrogenated fats based on canola and soybean oil. They are the oils listed earlier that contain significant amounts of palmitic acid. It can be seen that lard, tallow, and especially palm oil and its fractions are high in C16:0. Cottonseed oil with 22% palmitic acid is not listed, because it is not a semisolid fat. The nutritional effect of palmitic acid is still not sufficiently clear to
TABLE 4.6
Fatty Acid Composition of Nonhydrogenated, Semisolid Fats
Fat Lard Tallow Palm Palm stearin Palm olein Palm kernel PK olein PK stearin Coconut C8/C10 8 9 5 11 C12 48 42 57 49 Fatty acid (%) C14 C16 25 4 26 44 63 40 15 8 12 8 22 8 19 9 C18 15 27 5 5 5 2 2 2 3 C18:1 44 36 (t5) 40 25 43 15 23 5 7 C18:2 10 3 10 5 11 3 3 1 2
Abbreviation: PK, palm kernel. Source: Palm Oil Research Institute of Malaysia data.
TABLE 4.7
Melting Profiles of Various Nonhydrogenated, Semisolid Fats
Fat Lard Tallow Palm P stearin P olein Palm kernel PK stearin PK olein Coconut 10.0 30 36 27 55 25 50 85 35 55 21.1 22 28 15 45 4 33 80 8 27 Solid fat index at C 26.7 33.3 16 5 26 22 12 9 44 43 0 0 13 0 60 0 0 0 0 0 40.0 3 13 5 41 0 0 0 0 0
Abbreviations: P, palm; and PK, palm kernel.
remove concerns about its presence in fat products, but this acid is very important in the formation of crystalline structure of fat products. Palm kernel and coconut oils have significant amounts of lauric and myristic acid, that according to present nutrition precepts should be avoided. Very small amounts of palm kernel oil are used in zero-trans margarines, because it helps to provide the kind of melting profile that is achieved with the trans isomer from hydrogenated fats. Palm kernel and coconut oils are used in cream fillings and chocolate coatings for their melting properties, but to a significant extent they have been replaced by high trans isomer partially hydrogenated canola and soybean oils, that have achieved similar melting behavior. Table 4.7 gives the melting profiles of the semisolid oils. It can be seen that they represent a wide variety of melting behaviors; from very steep, for palm kernel and coconut oils, to very flat, as is the case with tallow and palm stearin. The oils with flat melting behaviors are suitable for baking shortenings. Table 4.8 compares the fatty acid compositions of typical partially hydrogenated canola and soybean oils with the nonhydrogenated oils. These oils contain
TABLE 4.8
Fatty Acid Composition of Partially Hydrogenated Canola and Soybean Oils
C16:0 Canola oils NH, IV 115 IV 90 IV 80 IV 70 Soybean oils NH, lV 130 IV 110 IV 85 IV 70 4 4 4 4 11 11 11 11 C18:0 2 3 10 18 4 5 5 13 Fatty acids (%) C18:1 61 81 83 75 25 45 73 74 C18:2 Trans
21 (+9%C18:3) 9 26 2 35 0 50 52 (+8% C18:3) 36 16 11 30 2 47
Abbreviations: NH, nonhydrogenated; and IV, iodine value.
predominantly C18 acids. Hence, they exhibit the crystallization habit when hydrogenated. This is reduced somewhat by the presence of trans acids, and in the case of soybean oil by the presence of about 11% of palmitic acid. The nutritional concern with these oils stems from the trans isomer content. The nonhydrogenated canola and soybean oils are characterized by 812% 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 conditions and limits the shelf stability when used in margarines, shortenings, and, to some extent, when used as salad oils. They are poor frying oils. For these reasons, canola and soybean oils are usually lightly hydrogenated to lower the linolenic acid to 3% (canola, IV 90; soy, IV 110), to obtain fats of greater stability with a variety of melting profiles. After partial hydrogenation, the trans isomer content can range from 1550% of total fatty acids depending on the degree of hydrogenation. Nutritionally, nonhydrogenated soybean oil is considered less desirable than canola oil because of its high content of saturated fatty acids. Also, its high content of 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 a precursor of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Table 4.9 gives the melting profiles of typical partially hydrogenated canola and soybean oils at different iodine values, along with their fully hydrogenated counterparts, to compare with the semisolid fats in Table 4.7. The melting profiles are relatively steep, as one would expect from the presence of the trans and positional isomers listed in Table 4.2. Flatter profiles (not included here) can be produced, but not without significant trans isomerization, unless the oils are fully saturated, as shown at the bottom of Table 4.9. If fully saturated, the oils can supply very large amounts of solid fat. Their crystallization habit will not be suitable for most purposes, unless oils with different fatty acids are also used in the edible fat product, or they are interesterified with another oil component.
TABLE 4.9
Melting Profiles of Typical Partially Hydrogenated Canola and Soybean Oils
10.0 Canola oils IV 90 IV 80 IV 70 Soybean oils IV 110 IV 85 IV 70 Note Canola and Soybean, IV <10 3 21 52 3 19 53 21.1 0 5 34 0 6 36 Solid fat index at C 26.7 0 2 27 0 2 30 33.3 0 0 13 0 0 13 40.0 0 0 1 0 0 0
>90
>90
>90
>90
>90
Abbreviation: IV, iodine value.
From the previous discussion on nutritional and functional requirements in the production of fat and oil products, the most prominent issues that emerge are the need to have palmitic acid as part of solid fat component in margarine, and especially in shortenings, and to avoid trans acids. The need to reduce palmitic acid is particularly difficult. Its elimination is, in fact, not possible. Therefore, it is essential to clarify its role in cardiovascular disease. The avenues available for the reduction of palmitic acid and elimination of trans acids are listed in Table 4.10. The process of interesterifying fully hydrogenated canola and soybean oils to produce hard fats with zero-trans fatty acids that consist mostly of tristearate, and blending with nonhydrogenated oils for margarine and shortening applications still requires a great deal of work. Nevertheless, this approach is of interest if stearic acid is in fact not a problem in cardiovascular disease. It has been shown by various workers that the melting profile of interesterified blends of fully saturated soybean oil mixed with nonhydrogenated soybean oil is not a problem (1). The presence of 11% of palmitic acid in the interesterified blend is probably sufficient for crystal stability. Thomas interesterified fully hydrogenated vegetable oils with canola oil and showed that satisfactory melting profiles were obtained (2). There is also the question of the effect of Interesterification on fatty acid position on glycerol, and the fact that position to some extent determines if a fatty acid is hyper- or hypocholesteremic (3). This is a complication in the use of Interesterification that cannot be ignored. As the use of fully hydrogenated fats increases, it is probably important to change labeling regulations to make it mandatory to declare trans fatty acids. This will make it possible to use fully hydrogenated fats without engendering the present association of trans fatty acids with hydrogenation in the perception of consumers. The reduction of trans isomer formation in hydrogenation by the development of economical catalysts and process conditions that suppress these reactions is possible. This is discussed extensively in the chapter by R. Hastert. An important potential avenue to reduce the need for partial hydrogenation is the development of low linolenic acid and high oleic acid oils. Commercial use of such oils would eliminate a large volume of partially hydrogenated fat from edible oil products. This would reduce trans fatly acids (by restricting the use of partially
TABLE 4.10
Ways 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 isomers Development 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 considered insignificant. The fatty acid composition of the new oils in this category are shown 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 very high amounts of linoleic acid. They can be expected to have the oxidative and heat stability of sunflower oil. They would make stable salad oils and could be used along with the present standard canola and soybean oil. These oils would still be of 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 low amounts of linoleic acid. Their stability is essentially on par with lightly and moderately hydrogenated canola and soybean oils. This makes them good zero-trans replacements for these partially hydrogenated oils, except that they are not contributing solid fat to margarine and baking shortenings. In some cases involving Interesterification, blending the low linolenic and high oleic oils with fully hydrogenated fats from canola, soybean, cottonseed, and palm oils can satisfy the functional requirements of margarines and shortenings with respect to solid fat content, crystallization habit, and oxidative stability. When using Interesterification, the position of the fatty acid on glycerol must be considered to avoid negative nutritional effects. Avoidance of partially hydrogenated fats in margarine and shortening formulations will require greater use of semisolid fats containing palmitic acid, most notably palm oil. Consequently, interest in the development of canola and soybean oils 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, to produce crystal stability after partial or full hydrogenation. But, as indicated earlier, the nutritional effect of palmitic acid requires further clarification before deciding that high palmitic canola and soybean oils are useful developments.
TABLE 4.11
Fatty Acid Composition of Some New Oils
CI6:0 Low 18:3 Canola Low 18:3 Flax Low 1 8:3 Soy Trisuna Sunolab
a b
C18:0 3 5 3 4 2
Fatty acids (%) C16:1 64 24 22 80 90
C18:2 24 61 61 10 3
C 18:3 3 3 2 <1 <1
4 7 11 4 3
High oleic sunflower oil. High 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. 3-243-42. 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 the claims and counterclaims regarding either positive or negative aspects of various triglycerides in the human diet. Rather, it will be an examination of the chemical facts of the composition of commercial fats and oils products, both present and historical. Using these facts, an outline will be proposed as to how hydrogenation might be used in the future to attain triglyceride composition objectives that may be nutritionally desirable. Whether long-term human testing will eventually prove these objectives to be scientifically valid is not of immediate concern to the processor and marketer of oil and fat products. In the short term, the processor has no alternative but to respond positively to whatever the marketplace is requesting at any particular time. This paper will principally focus on margarine, as that is the product most discussed 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 Margarine Feeding Tests Show Blood Cholesterol Increase When Eating Hydrogenated Fat Noted Nutritionist Speaks Out About Fats in American Diet
Of course, the younger readers may be surprised to hear that the same headlines were in our newspapers in 1960. In other words, to anyone more than 50 years old, the current almost hysterical discussion concerning the nutritional effects resulting from the composition of fats in the diet seems a bit of deja vu. However, there is an important difference between 1960 and now. At that time, its relevance to ordinary persons was considerably more direct. That was because there truly appeared to be an epidemic out there. People, particularly men, were being felled by heart attacks, not only with increasing frequency, but also at younger and younger ages. The health professions responded to the public outcry by proclaiming cholesterol to be 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 of Time magazine (2). While the 1960s experience is recalled with some irony, its central thesis of a relationship between fats in the diet and heart disease cannot be considered unfounded. Even though everyone now agrees that the causes of heart disease are considerably more complex than was thought 30 years ago, the fact that the incidence 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 was done right to turn around a very alarming situation. How much of the reduction was due to eating an overall better diet, how much to more exercise, how much to a significant reduction in smoking, and how much to the ingestion of fats with modified triglyceride composition, can never be precisely known. However, the obvious conclusion is that they probably all contributed to some extent, and it would be ill-advised to discontinue an emphasis on any of them. The experience certainly 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 saturated fat/heart attack scare of almost 35 years ago can give considerable insight into the tools to be employed and the paths pursued to surmount the current very similar attack on trans-isomers in the diet. First and foremost, while the fats and oils industry felt unjustly accused of harmful actions in 1960, it did promptly respond to the accusations. The result was a reduction in the amount of completely saturated triglycerides, particularly in relation to polyunsaturated triglycerides, in margarine, shortenings, and salad oils. What were the motivations, technical approaches, and economic aspects of how this happened? It is particularly important to remember that it was accomplished without any perceptible change in the appearance and functionality of the end-products. For instance, margarine continued to look, taste, feel, and perform as it had previously. There was actually a functional improvement in that the increasingly popular tub 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 polyunsaturate content. The driving force, as mentioned previously, was the health professions accusation that the amount of saturated triglycerides in the then-conventional fats and oils products was causing the deaths of a very large number of people. Not surprisingly, this accusation made persons in the industry feel ethically uncomfortable. It also frightened companies with the prospect that sales of their products might decrease. Those same driving forces arc with us today. Economics was a big incentive to make the formulation conversions. Oil hydrogenated to a lesser degree meant some cost savings by reducing the amount
of hydrogen, catalyst, and processing time. The somewhat later incorporation of unhydrogenated oil into formulations meant even greater cost reduction. By-passing the hydrogenation step on a portion of the formulation completely eliminated the costs of catalyst, hydrogen, and labor associated with it. It also eliminated their overhead costs in conventional accounting methods. There was even a cost saving through yield gain by not incurring product loss in the black (posthydrogenation) filter press. All in all, considering the economic advantages, it is not surprising that manufacturers were very willing to accept the modest conversion costs necessary to adapt their physical facilities to utilize the new formulations. There is an obvious parallel economic advantage associated with the current enthusiasm of margarine manufacturers to market the spreads and light margarines that contain less fat. Selling water for the price of oil has got to be good for the bottom line. Not surprisingly, this has resulted in the reappearance of still another 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 importance of economics to the overall discussion must always be kept in mind. Economics, of course, also works in reverse. In other words, higher cost (price) is a marketing disincentive. It has repeatedly been shown that, while most consumers will give a positive response when asked whether they are willing to pay a higher price for a more nutritionally suitable margarine, shortening, or salad oil, in actuality the number 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-moreexpensive sunflower salad/cooking oil. A current example is olive oil. While olive oil 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 processors to incorporate lesser-hydrogenated and unhydrogenated oils into formulations, was the base stock system. Briefly, it involved the blending of two or more different oils, each having specific melting characteristics, rather than hydrogenating one feedstock into a single margarine base. Blending relied on the phenomenon that a mixture of a harder and a softer fat has physical characteristics closer to the harder one. While the base stock system came into wide use in the early 1960s, it was not described in the literature until Latondress did so in 1982 and again in 1985 (3,4). In the United States it utilized unhydrogenated and/or very lightly hydrogenated soybean oil, several partially hydrogenated ones, and stearin. OBrien further defined the approach at an AOCS Colloquium in 1986 (5). He illustrated the melting characteristics of the bases, as depicted in Figure 5.1.
Fig. 5.1. Soybean oil base stocks.
Calculations of the triglyceride composition of several of the base stocks are shown in Table 5.1. For clarity and illustrative purposes, all trans-isomers in this and other tables and figures were calculated as C18:1. While not exactly accurate analytically, comparisons among them are bona fide since the same procedure was used in all cases. For making various margarines, OBrien suggested formulations utilizing the base stocks (5), as shown in Table 5.2. A contemporary squeeze bottle liquid formulation has been added, as has a single stock formulation utilizing OBriens 75 IV base. While this 75 IV base is slightly softer than was actually the case with commercial products of that era (6), it is close enough to illustrate a valid comparison with contemporary products. From Figure 5.2, which illustrates melting curves obtained when using the previously mentioned formulations, it is apparent they have been flattened considerably in the progression from the old single stock base through the more lightly hydrogenated formula and into formulas incorporating unhydrogenated oil. The
TABLE 5.1
Composition of Base Stocks
Fatty acids % Saturates C 18:1 C 18:1 C 18:1 C 18:2 C 18:3 Unhydrogenated 16 21 21 0 54 9 108 16 44 21 23 36 4 Iodine value 85 75 18 20 68 77 26 28 42 49 13 3 1 0 66 27 73 21 52 0 0 Stearin 100 0 0 0 0 0
Total cis trans
TABLE 5.2
Margarine Formulations
Stick Single base 0 0 0 100 0 0 All hydrogenated 0 42 0 20 38 0 Highly unhydrogenated 60 0 0 25 15 0 Tub All High hydroliquid genated oil 0 50 80 30 0 0 0 0 20 20 0 0 Squeeze bottle
Base (%) Unhydrogenated 108 IV 85 IV 75 IV 66 IV Stearin
Soft 50 0 0 0 50 0
Liquid 97.5 0 0 0 0 2.5
advent of tub margarine, especially with a formula utilizing unhydrogenated oil, flattened the curves even more. Finally, the liquid squeeze bottle formulation practically eliminates the solids melting curve. While the melting curves in Figure 5.2 very strikingly illustrate what can be seen and felt, the scientist always asks, why? The seemingly logical assumption has always been that the new multibase stock formulations significantly reduced the amount of saturates, probably with a corresponding increase in trans-isomers. However, calculating the amount of saturates and trans-isomers, as listed in Table 5.3, and depicting them separately and added together, as shown in Figure 5.3, produces unexpected results. Surprisingly, the saturates content, as shown in the left bars of Figure 5.3, did not decrease appreciably over the entire range of formulations. However, even more surprisingly, the trans-isomers (middle bars) were significantly reduced, as was the sum of the two (right bars). In other words, whether by intent or accident, the
Fig. 5.2. Margarine melting curves.
amount of trans-isomers in stick and tub margarines has been cut by approximately one-half when compared to the pre-1960 single base stock. While it is easy to complain that this fact has been ignored by the media, they have the reasonable excuse of not possessing an adequate technical background to appreciate it. What is disconcerting is that it also seems to have been either unknown or ignored by persons 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 fat industry bear the primary blame for not having known and promulgated the significant trans-isomer reduction. Accepting these conclusions means abandoning the previously held thesis that the saturate content of margarine was greatly reduced by utilizing the base stock formulation procedure. The question then remains, what was the change in ester composition that contributed to the significant reduction in heart disease referred to earlier? A plausible answer may be the lowering of trans-isomer content, as is illustrated in Figure 5.3. Another possibility, that was also referred to earlier, is the significant increase in polyunsaturates, especially as related to saturates. This is illustrated in Figure 5.4, which show the amount of polyunsaturates and saturates in each formulation, along with their polyunsaturated to saturated ratio-referred to as P/S. (The P/S ratio has been multiplied by 10 in Figure 5.4 for illustrative purposes.) Since the saturate level is substantially the same in all the formulations, it is obviously the varying level of polyunsaturates among the formulations that causes the significant difference in P/S ratios.
Fig. 5.3. Saturates and trans-isomers for margarine formulations.
Fig. 5.4. Polyunsat urate to saturate relationship.
A summary of observations from this examination of the ester composition of various margarine bases, is as follows: 1. Beginning about 1960, fat products began to be formulated from two or more base stocks. This significantly changed their triglycerides composition by allowing the incorporation of unhydrogenated oil possessing a minimum of saturates and no trans-isomers. 2. The resulting effect(s) of either less trans-isomers and/or more polyunsaturates may have contributed to the quite astounding reduction in arteriosclerosis. 3. The increasing popularity of tub margarine facilitated the formulation changes. Future increases in the popularity of the squeeze bottle would move further in this direction. Pressure and Temperature Regulation Effective utilization of formulations utilizing two or more base stocks is dependent upon the effective control and monitoring of the production of those base stocks that are hydrogenated. Assuming uniform purity of the feedstocks, the conditions of hydrogenation that influence the formation of both saturates and transisomers are pressure, temperature, type of catalyst, catalyst concentration, and degree of mixing. Pressure and temperature have historically been the hydrogenators principal means of controlling both preferential and trans-isomer selectivity. They can be
Fig. 5.5. Nonselective vs. selective hydrogenation.
readily monitored and controlled within narrow limits. Their very significant effect on formation 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. 5.6. Effect of processing conditions on trans-isomer formation.
Fig. 5.7. Influence of process conditions on the solid fat melting profile of soybean oil with
a 70 IV.
Use of Catalysts Nickel catalysts possessing various degrees of selectivity and having good uniformity are commercially available. While catalyst concentration can have a small effect on selectivity, economics dictates the use of only a minimum quantity. Mixing can be very important in the hydrogenation reactions selectivity, as has been well documented in the literature (710). However, it has been largely ignored by processors, probably because it is difficult to measure in commercial converters of various sizes, configurations, and mixing devices. It is anticipated that it will receive much greater attention in the future. The previously mentioned parameters have been discussed only as they relate to batch-slurry hydrogenation using conventional nickel catalyst. Alternate modes and other metals, either separately or in combination, also offer advantages and will surely receive greater attention in the future than they have in the past. The natural conservatism embodied in the WNDITWB (Weve Never Done It That Way Before) philosophy usually results in slow changes in manufacturing technology. However, unusual circumstances can speed the rate of change. The current amount of attention being given to fat and nutrition could be the circumstance that hastens adaptation to what have previously been regarded as radical approaches in catalysis and processing technology. For instance, nickel has been the catalyst of choice ever since the inception of commercial hydrogenation almost a century ago. No other metal has been found to be comparable, either in performance or economics. Copper, based on its exceptional linolenic selectivity, was looked at seriously about 15 years ago. However, after having been proven industrially viable, it was abandoned for several reasons.
A principal one was that it required its own isolated system. Also, since the hydrogenated oil still required winterization to pass a cold test, processing costs were higher. Even more important, improvements in other processing techniques permitted the use of unhydrogenated oil, both in salad/cooking applications and as a base in formulating margarines and shortenings. Precious Metal Catalysts. Laboratory investigations over the years have shown precious metal catalysts to possess exceptional hydrogenation activity and unique selectivity characteristics, and at temperatures far lower than those required for nickel. These are attractive attributes. The principal stumbling block to serious consideration of the use of precious metals as fats and oils hydrogenation catalysis has always been cost. Their exceptional activity only requires an extremely low metal concentration, which makes reclamation and recycling very difficult. Oncethrough usage cannot be economically justified. Assuming the precious metal reclamation problem could be dealt with to an endurable extent, a paper authored by Berben deserves serious attention (11). It offers a significantly greater selectivity advantage for a modified platinum on carbon catalyst than anything reported previously. To establish a base, Berben first compared ester composition results when partially hydrogenating soybean oil using a current state-of-the-art very selective nickel catalyst to the results from various precious metal catalysts. As detailed in Table 5.5 and illustrated in Figure 5.8, they essentially duplicated the findings of previous investigators, that is in those cases where trans-isomers were reduced, saturates were elevated, and the reverse. Berben then chose 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 various precious metal catalysts.
TABLE 5.5
Comparison of 100 IV Constituents when Hydrogenating with Nickel and Various Precious Metal Catalysts
Nysosel 325 140 10 0.1 6.5 100.5 18.8 47.8 29 18.8 31.7 1.7 18.8 18.8 37.6 5% Pt/C 60 10 100 42 101.2 24.7 37.3 28.8 8.5 35 3 24.7 8.5 33.2 5% Pt/Al 60 10 200 90 101.8 29.3 28.9 23.5 5.4 37.6 4.2 29.3 5,4 34,7 5% Pd/C 60 10 100 44 101 .6 17.2 50.6 30.6 20 31.2 1 17.2 20 37.2 5% Ru/C 60 10 200 48 100.2 21.6 43.7 27.8 15.9 32.5 2.2 21.6 15.9 37.5
Temperature, C Pressure, bar Ni, % PM, ppm Time, min Iodine value % Saturates % C 18:1 total % C 18:1 cis % C 18:1 trans % C 18:2 % C18:3 % Saturates % trans % Saturates + trans
on selectivity of modifying the catalyst through incorporation of a small amount of ammonia into the hydrogen, as had been reported earlier by J. Kulper (12). The results were astounding. They are listed in column 4 of Table 5.6, along with results from an unmodified Pt/C, the nickel tested concurrently, and nickel results extrapolated from earlier work by OBrien (5). Table 5.7 contains data extracted from Table 5.6 and compares the saturate and trans-isomer contents at 100 IV and 70 IV between the modified platinum and the nickels. As can be seen, while the saturates are similar, with the modified platinum there is a dramatic reduction in trans-isomers. Figures 5.9 and 5.10 illustrate the phenomenon graphically. Also, as can be seen in Table 5.7, preferential polyunsaturate selectivity was not affected. While formulations having suitable functionality would need to be devised to make margarine and/or shortenings by utilizing base stocks hydrogenated using modified platinum catalyst, from a trans-isomer, saturate, and polyunsaturate standpoint, the results look exceedingly promising. Berbens paper is important because it quantifies the degree of formation of saturates and trans-isomers for a number of catalysts, including an entirely new onethe modified platinum on carbon, as Figure 5.11 illustrates (11). Another important point about Berbens paper is its reminder to never discount new adaptations of old knowledge, which may significantly alter what has been accepted as unalterable. It also can spur the probability of still additional adaptations. In this particular case, it makes fixed bed hydrogenation attractive, since metal reclamation from fixed bed catalysts is considerably more feasible than from the batchslurry mode. Rosen and Lee Poy have previously demonstrated the viability of fixed bed partial hydrogenation of vegetable oil with nickel catalyst (1316).
TABLE 5.6
Effect of Ammonia in Hydrogen on Preferential Selectivity and Trans-lsomer Formation at 100 and 70 IV
Nysel Temperature, C Pressure, bar Nickel, % PM, ppm NH3/PM molar ratio Time, min Iodine value % Saturates % C 18:1 total %C 18:1 cis %C 18:1 trans %C 18:2 %C 18:3 Time, min Iodine value % Saturates %C 18:1 total %C 18:1 cis %C 18:1 trans %C 18:2 %C 18:3 200 0.7 0.02 Nysosel 325 140 10 0.1 5% Pt/C 60 10 100 400 42 101.2 24.7 37.3 28.8 8.5 35 3 86 70 46.2 46.1 33.5 12.6 16.1 0.5 5% Pt/C Mod. 60 10 200 50 101.4 19.1 45.9 39.3 6.6 33.8 1.2 335 69.4 27.2 65.1 54.7 10.4 7.7 0
18 100 17 52 23 29 28 3 45 70 24 75 24 51 2 0
6.5 100.5 18.8 47.8 29 18.8 31.7 1.7 11 71.2 25.9 66.9 36.1 30.8 7.2 0
TABLE 5.7
Saturate and Trans-lsomers Comparison of Modified Pt/C with Nickel at 100 IV
Ni-OBrien % Saturates % Trans % Saturates + trans 17 29 46 Ni-Berben 19 19 36 Pt/C Mod. 19 7 26
Saturate and Trans Comparisons at 70 IV

Ni-OBrien % Saturates % Trans % Saturates + trans 24 51 75 Ni-Berben 26 31 57 Pt/C Mod. 27 10 37
Saturates and Polyunsaturates Comparisons at 100 IV

Ni-OBrien % polyunsaturates % Saturates P/S ratio 31 17 1.8 Ni-Berben 33 19 1.8 Pt/C Mod. 35 19 1.8
Fig. 5.9. Saturate and trans-isomer comparisons of modified Pt/C with nickel at 100 IV.
Other Methods of Hydrogenation More radical approaches to vegetable oil hydrogenation have also been proposed. Yusem and Pintauro investigated the electrolytic hydrogenation of soybean oil using an active Raney type nickel catalyst (17,18). While the reaction was shown to be very nonpreferentially selective, it produced few trans-isomers. Smidovnik et al. hydrogenated soybean oil with palladium on carbon catalyst (19). In both cases,
Fig. 5.10. Saturate and trans-isomer comparisons of modified Pt/C with nickel at 70 IV.
the reaction proceeded at low temperature (6070C). These novel approaches merit further study. The interesterification process has been utilized commercially since the mid1950s. Although initially intended to make lard more suitable for use in shortening, its principal application has been in the manufacture of coating fats (hard butters). While interesterified margarine has long been available in Europe and Canada, up to this time it has not made any inroads in the United States. This could change if the furor over trans-isomers continues. However, because of the prejudice that persists in the United States against ingesting either tropical oils or meat 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 for Agricultural Utilization Research in Peoria (20), could point the way. In it, completely hydrogenated soybean or canola oil was interesterified with unhydrogenated oil. While the saturate content was similar to other formulations, there were no trans-isomers.
Conclusion
While the future may show present catalysts used in novel ways, novel catalysts in present ways, and novel catalysts in novel ways, there seems no doubt that hydrogenation 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, The American 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. OBrien, 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 and Utilization, 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: 379382 (1995)
Chapter 6
InteresterificationCurrent Status and Future Prospects

Suresh Ramamurthi and Alan R. McCurdy Department of Applied Microbiology and Food Science, University of Saskatchewan, 51 Campus Drive, Saskatoon, Saskatchewan, S7N 5A8, Canada.
Introduction
Fats and oils are essential components of the diet; they are a concentrated source of energy, essential fatty acids, and fat-soluble vitamins. They also contribute to the flavor, texture, and palatability of the food that we consume. The natural oils and fats can be used directly or after blending. Frequently, to obtain a fat with specific physical properties, such as melting range, solid content, and so on, it becomes necessary to modify them more than just by blending (1). Hydrogenation, fractionation, and interesterification of oils are the three techniques commonly adopted by the processor to obtain a desirable end-product (2). The physical properties of fats and oils are dependent upon the distribution of fatty acids on the glycerol backbone, the chain length of the fatty acids, and the degree of unsaturation. The application of an oil or fat in food products is limited by its physical properties that in turn are controlled by the previously mentioned factors (3). Some of the properties of oils and fats, such as melting temperature, crystallization, and recrystallization form, are affected by changes in any of the three factors. For example, shortening and tailor-made fat compositions are formulated to obtain a specific solid fat index profile. This is done to obtain proper mouthfeel 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 onethird unsaturated fatty acids. The triacylglycerol structure of cocoa butter consists mainly of a molecule in which the unsaturated fatty acid is located in the sn-2 position while the saturated fatty acids are in the outer positions. The triacylglycerols 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). This unique triacylglycerol structure and composition is responsible for the physical properties of cocoa butter and make it suitable for confectionery applications. Fats and oils derived from natural animal and plant sources are endowed with a unique combination of fatty acids and their distribution on the glycerol backbone is nonrandom (5). Fats from animal sources and tropical plants, such as coconut and palm, 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 from soybean and sunflower contain mostly polyunsaturated fatty acids. In vegetable oils, unsaturated fatty acids are preferentially located at the sn-2 position of the triacylglycerol molecules. This distribution pattern is important for the slope of the melting curve of cocoa butter and for the crystal structure of lard. The oils and fats processor, due to the limitations presented by nature, resorts to the use of some modification techniques to obtain a product with desirable physical properties.
Interesterification
Interesterification also refers to the exchange of fatty acyl moiety between an ester and 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 mixed completely until an equilibrium is reached. At equilibrium, all possible combinations of fatty acids on the glycerol backbone are found. This is true for chemical interesterification and enzymatic interesterification using nonspecific lipase (4). Hence this process is also called randomization. There are some exceptions to the randomization effect, as in directed interesterification and enzymatic interesterification (discussed separately). Prior to the development of interesterification processes, hydrogenation was the major fat-modification process. Hydrogenation always results in an increase in the melting point and the solid phase content of the raw material (6). This is due to the decrease in the unsaturated fatty acid content and a change in the triacylglycerol composition. Fractionation refers to the process in which the raw material is heated above its melting point and allowed to cool under conditions that stimulate 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 for changing the properties of oils without altering the structure and composition of fatty acids originally present is interesterification. This process is unique in that it only alters the distribution of fatty acids on the glycerol backbone. Thus, interesterification is clearly distinguished from hydrogenation, in which the level of unsaturation of the fatty acids are reduced, and fractionation, where unmodified triacylglycerols or previously modified triacylglycerols are separated into two or more fractions through crystallization. In the United States, hydrogenation is preferred, while companies in Europe use interesterification to a large extent to modify their raw material (6). During the hydrogenation process some of the cis-fatty acids are converted to trans-fatty acids. Though not entirely confirmed, trans-fatty acids are associated with an increase in the risk of heart disease. Todays consumer is more aware of the type and nature of fatty substance in the food. Presently, there is a consumer trend toward oils from plant sources and 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 with hydrogenation and fractionation to provide products that can perform and satisfy the nutritional requirements. Chemical Interesterification Interesterification can be carried out at high temperatures (>300C) in the absence of any catalyst. However, it requires very long reaction times, and the final product is not of desired quality due to polymerization and decomposition products. Numerous catalysts have been employed to increase the reaction rate and to carry it out at lower temperatures (around 80100C). The most widely used catalysts are metallic sodium, sodium/potassium alloys, alkylates of sodium, and hydroxides of sodium or potassium in combination with glycerol (3). The sodium alkylates are less costly and are easily dispersible in the fat. They are active at lower temperatures (5070C) and the reaction can be carried out under atmospheric pressure. Depending on the quality of the starting material, catalyst dosage ranges from 0.20.4% of the fat. The sodium metal requires special handling care and it reacts with water or hydroxyl group very easily. It is usually used in the range of 0.050.1%. The alloy (Na/K) is a liquid and is much easier to handle. The least expensive catalyst is sodium hydroxide, used in combination with glycerol that results in the in situ formation of sodium glycerate (8). Sufficient sodium hydroxide is added to neutralize any free fatty acids already existing in the starting material. All the previously mentioned catalysts are very sensitive to the nature of the feed stock and are easily inactivated by moisture. Therefore, it is necessary to pretreat the fat to reduce the free fatty acids to less than 0.1% and to lower the water content to below 0.01%. Bleaching the fat to reduce the peroxide value is also recommended. The interesterification reaction can be carried out batchwise or in a continuous fashion. A typical batch chemical interesterification reaction vessel is shown in Figure 6.1. The reaction is carried out at 100C for 30 min. At the end of the reaction, the catalyst is inactivated with either water or acid, and the product is refined. A continuous Interesterification process using sodium hydroxide and glycerol as the catalyst has been patented by Unilever (9). A flow sheet of the process is presented in Figure 6.2. The catalyst is premixed with the preheated fat and spray dried under vacuum. The actual reaction takes place in a short period of time within a reactor coil at 130C. The reaction product is treated with water and acid to inactivate the catalyst, and then washed thoroughly. The extent of interesterification is usually monitored by evaluation of the melting point, solid content index, or some suitable standardized crystallization test. The mechanism of the reactions occurring during interesterification have not been completely confirmed. There are actually two processes at work in the randomization of fatty acid moieties. Intraesterification refers to the shuffling of fatty acids within the same molecule, producing isomers of the same acylglycerol. This initial reaction soon gives way to interesterification, a random arrangement within
Fig 6.1. Typical batch interesterification vessel. Source: Haumann (6).
all triacylglycerol molecules present. A detailed description of the two reaction mechanisms, one through formation of enolate ion and the other through the formation of an addition complex (carbonyl addition) of the catalyst is presented by Sreenivasan (3). The common factor between the two mechanisms is that the reaction proceeds via a keto ester. Both mechanisms require the formation of an ionic intermediate
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 formation of a dark brown color within 5 min of reaction initiation. Chemical interesterification proceeds to equilibrium, at which point all of the fatty acids are randomly distributed on the glycerol. It is possible to calculate the exact proportion of different types of triacylglycerols formed when the raw materials used are fully characterized. When equimolar concentrations of two monoacid triacylglycerols (RRR and RRR) are interesterified as shown in Figure 6.3, various triacylglycerols are formed according to statistical calculations. The parent triacylglycerols are each present in the final product at the 12.5% level, triacylglycerols containing two R fatty acids and one R fatty acid constitute 37.5%, and triacylglycerols containing two R fatty acids and one R fatty acid constitute 37.5%. Randomization of a model system consisting of trioleoyl triacylglycerol and tristearoyl triacylglycerol was found to follow this statistical distribution (10). The proportion of any given triacylglycerol can be calculated in a chemically interesterified mixture on the basis of random distribution theory if the fatty acid composition of the starting material is known (11). If X, Y, and Z are three fatty acids present, then the percentage of triacylglycerols containing three different fatty acids is %XYZ = %X in total oil %Y in total oil %Z in total oil 0.6 l04 The percentage of triacylglycerols containing two fatty acids is %XXY= (%X in total oil)2 %Y in total oil 0.3 104 The percentage of triacylglycerols containing one fatty acid is %XXX= (%X in total oil)3 104 Triacylglycerols containing short-chain fatty acids and long-chain fatty acids have been prepared using sodium methoxide Catalyzed interesterification of short-
Fig 6.3. Chemical interesterification of equimolar mixtures of two monoacid triacylglycerols (R and R 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 agree with the analytical data. An example of Na/K Catalyzed interesterification of high erucic acid rapeseed oil (HEAR) is presented (13). Native HEAR oil consists of 41% erucic acid that 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 fatty acid composition content for monoacylglycerols, diacylglycerols, and triacylglycerols. Thus, the HEAR oil that originally did not contain any erucic acid in the sn2 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 presence of trierucoyl triacylglycerol in the interesterified HEAR oil indicates randomization. Upon randomization the number of triacylglycerol peaks on the HPLC chromatogram increased in comparison to the chromatogram of HEAR oil, indicating the formation of different combinations of fatty acids in the triacylglycerol molecules. To our best knowledge, selectivity through chemical interesterification has been shown in only one study. Konishi et al. (14), studied the sodium methoxide Catalyzed interesterification reaction between soybean oil and methyl stearate. When the reaction was carried out at 30C in hexane, it was observed that after 24 hrs the acyl exchange at sn-1,3 position progressed 1.7 times faster than at sn2 position of the triacylglycerol. However, the reaction proceeded slowly at this temperature.
TABLE 6.1
Positional Fatty Acid Composition (mole %) of Native High Erucic Acid Rape-seed (HEAR) Oil and Randomized HEAR Oil
Fatty acid C16:0 C18:0 C18:1 C18:2 C18:3 C20:0 C20:1 C22:0 C22:1 C24:0 C24:1 Other sn-2 03 0.1 36.3 36.2 25.7 0.0 0.3 0.0 1.2 0.0 0.0 0.0 Native HEAR oil sn1,2 (2,3) 2.8 1.2 25.4 20.0 12.2 0.6 6.0 03 30.6 0.1 0.2 0.2 TAG 3.3 1.2 19.1 14.4 9.5 0.8 8.5 0.5 41.4 0.2 03 0.4 sn-2 4.1 1.4 20.5 14.3 8.6 0.8 8.4 0.5 40.9 0.0 0.0 0.0 Randomized HEAR oil sn-1,2 (2,3) 3.6 1.3 20.4 13.8 7.6 0.8 8.7 0.4 42.0 0.2 0.4 0.4 TAG 3.3 1.2 18.9 14.4 9.5 0.8 8.4 0.5 41.6 0.3 0.5 0.5
Abbreviation: sn, stereospecific number; and TAG, triacylglycerol. Source: Grewal (13).
Fig 6.4. HPLC chromatograms of native HEAR oil and randomized HEAR oil. Abbreviation:
TETAG, trierucoyl triacylglycerol. Source: Crewal (13).
Directed Interesterification Chemical interesterification leads to random distribution of fatty acids if the reaction is carried out in a single phase (8). However, when this reaction is carried out at temperatures below the melting point of the highest melting triacylglycerol, the reaction equilibrium is shifted to the synthesis of the highest melting triacylglycerol (5). This is because the crystallized triacylglycerol, does not participate in the reaction once it is out of the solution. This process proceeds until all possible high melting triacylglycerols are produced at that temperature. Thus, a liquid oil containing significant amounts of saturated fatty acids can be converted to a product of desired consistency. The idea of directed interesterification can be extended to a system in which a desired fatty acid can be added to increase its incorporation in the triacylglycerol produced. The undesirable fatty acids can be removed, either by distillation (if sufficiently volatile) or through solvent extraction. Application of directed interesterification has some limitations. Since the reaction temperature is 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 catalyst activity due to coating of the catalyst is frequently encountered. Lipase-Catalyzed Interesterification As an alternative to chemical catalysts, enzymes have been used to catalyze interesterification reactions. In aqueous systems, lipases (acylglycerol ester hydrolase EC 3.1.1.3) are hydrolytic enzymes that break down triacylglycerols into free fatty acids and glycerol (15). They constitute a ubiquitous group of enzymes that do not require 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-
sis reaction catalyzed by the enzyme can be reversed (Figure 6.5 [16]). Under these conditions, lipases can catalyze a wide variety of reactions, many with industrial potential. Among these reactions, the use of lipase to modify interesterification 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 is possible to produce triacylglycerols with specific fatty acids in specific sn-positions that would otherwise be impossible to prepare with chemical catalysts that produce randomized products. Typically, the enzyme-catalyzed reactions are carried out at lower temperatures. This is suitable from two viewpoints: lower energy costs and higher quality of end-product. With lipases as catalysts, fatty acids, fatty acid esters, or triacylglycerols can be used as reactants. With chemical catalysts it is not possible to use fatty acids, as they tend to form soaps. Unlike the chemical catalysts, lipases are not highly sensitive to the presence of moisture and other contaminants in the starting material. However, the presence of a large amount of water is deleterious for an enzymatic reaction, as the hydrolysis reaction is favored rather than the acyl exchange reaction. Lipases perform well in a wide variety of organic solvents and also in phases such as supercritical carbon dioxide. The reaction phase could be an organic solvent or solvent-free, biphasic, microaqueous, reverse-micellar, or even with immobilized substrates/enzyme (18). The use of immobilized lipases is advantageous, especially
Fig 6.5. Lipase-catalyzed ester hydrolysis and synthesis. Source: Miller et al. (16).
in interesterification reactions where a low water activity is required to depress hydrolytic reactions. Immobilized enzymes have increased stability, thereby increasing 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 are produced utilizing enzymes are considered natural by consumers, giving added marketing value (6). Lipase Specificity Lipase specificity can be broadly classified (17,20) into five groups: 1. 2. 3. 4. 5. Lipid class; Positional; Fatty acid; Stereochemical; and 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 display preference towards monoacylglycerols in comparison to di- and triacylglycerols (21). Lipases can be positionally specific with respect to the sn-position of the triacylglycerol molecule. They can be positionally nonspecific or can be sn1,3 specific. Nonspecific lipases, such as from Candida cylindracea, do not have any specificity for any of the sn-positions in the triacylglycerol molecule. During an interesterification reaction with a nonspecific lipase, the products would be randomized in a fashion similar to a chemically catalyzed reaction (as shown in Figure 6.3). When sn-1,3 specific lipases are used to catalyze an interesterification reaction, the action of the lipase is substantially confined to the sn-1 and.sn-3 positions of the triacylglycerol molecule. A lipase from Mucor miehei has been shown to be sn-1,3 specific (2).
TABLE 6.2
Specificities of Some Lipases
Source Penicillium cyclopium Candida cylindracea Pscudomonas fluorescens Rhizopus arrhizus Aspergillus niger Geotrichum candidum Mucor miehei Human lipoprotein lipase
Abbreviation: sn, stereospecific number. Source: Sonnet (17) and Malcata et al. (20).
Selectivity Monoacylglycerol sn-nonspecific sn-nonspecific sn-1,3 specific sn-1,3 specific sn-nonspecific, C18 (cis 9) fatty acid sn-nonspecific, C4 and C6 (hydrolysis) Fatty acid and stereoselective (hydrolysis)
Some lipases show selectivity toward long-chain unsaturated fatty acids that have 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 are preferentially hydrolyzed. esterified, or interesterified. Such lipases exhibit reduced activity on substrates that have an additional double bond between the carboxyl group and the 9-position and on triacylglycerols containing medium-chain fatty acids. 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 to steric factors (23) that prevent the access of the sn-2 carbon moiety to the active site of the lipase rather than due to stereospecificity. Recently, lipases have been used rather extensively for the preparation of chiral esters and alcohols (17). Lipase stereoselectivity has been observed only with nontriacylglycerol molecules. Combinations of fatty acid specificity and stereoselectivity has been observed in human lipoprotein lipase preparation (24). Examples of some of the lipase-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 interesterification reactions, it has not been mathematically modeled as has been done for lipase-catalyzed hydrolysis and ester synthesis (20). Information on the kinetics of reaction sheds light on the reaction mechanism and also is useful for future scale-up. Interesterification reactions proceed through intermediate stages involving both hydrolysis and esterification. The Candida cylindracea lipase-Catalyzed interesterification reaction was studied using a model system consisting of lauric acid, dilauroyl glycerol, and trilauroyl glycerol in cyclohexane (25). It was suggested that the hydrolysis step in the interesterification process may be the rate-limiting step, as the enzymatic turnover for the esterification step was found to be three times faster than the hydrolysis step. The esterification step was assumed to follow a ping-pong bi-bi mechanism that was supported by experimental data. When studying the esterification of oleic acid and methanol in hexane that was catalyzed by an immobilized lipase from Candida antarctica (26), the kinetics of the reaction were observed to follow a ping-pong bi-bi mechanism in which methanol was a substrate inhibitor of the lipase. A similar mechanism was observed in transesterification reactions catalyzed by lipase (27,28). Knowing that the hydrolysis step is the ratelimiting step in the interesterification process, it would be advantageous for industrial applications 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
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) Fatty acid specific lipase-catalyzed (specific for A and B) interesterification of triacylglycerol and free fatty acids. A, B, and C are fatty acyl groups. Source: Macrae and Hammond (1).
areas include preparation of zero-trans or low-trans fatty acid shortening or margarine, synthesis of cocoa butter-like fat, modification of butterfat, modification of palm oil to increase its utilization, and preparation of structured lipids containing medium-chain fatty acids and polyunsaturated fatty acids.
Zero-Trans or Low-Trans Margarines Complete hydrogenation accomplishes the total saturation of unsaturated fatty acids. However, with partial hydrogenation, trans-fatty acids arc formed that are geometric isomers of the cis-fatty acids. The metabolic effects of these fatty acids are under investigation. Interesterification of a liquid oil with a hard fat has long been used in Europe as an alternative to hydrogenation to prepare plastic fat suitable for use in margarines. Such a plastic fat has been prepared by List et al. through interesterification of soybean oil with completely hydrogenated soybean oil (29). Randomization was found to have no detrimental effect on the flavor and oxidative stability of the product. Sodium methoxide catalyzed interesterification of refined, unhydrogenated soybean oil (60%) and edible beef tallow (40%) resulted in a product that was similar to commercial tub margarine oils (30). The interesterified fat contained 3% trans fatty 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 fully hydrogenated soybean oil (31). A decrease in the amount of triunsaturated and trisaturated triacylglycerols and an increase in the amount of mono- and diunsaturated triacylglycerols was observed. An increase in the relative stability of the crystalline form (desired in margarine) was also observed in such margarine fats (32). Zeitoun et al. (33) prepared chemically interesterified fats from mixtures of fully hydrogenated soybean oil and commonly used vegetable oils, such as coconut, cottonseed, peanut, soybean, corn, sunflower, safflower, and canola oil. They suggest the preparation of zero-trans margarines from the interesterified mixtures. The physical properties of the interesterified product are strongly affected by the type of liquid oils used. Oils that contained significant amounts of palmitic acid were found to form a product with favorable melting characteristics and 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, and has good sensory properties, Schmidt et al. have combined both interesterification and fractionation processes (34). They randomly interesterified a mixture of a saturated fat and unsaturated oil. The olein fraction obtained on fractionation of the interesterified product was mixed with a portion of a nonhydrogenated or a partially hydrogenated oil (that was substantially free from crystallized fat at 10C) and a portion of the interesterified product. Processors can combine any of the three processes, interesterification, hydrogenation, and fractionation, and start with a blend of oils and fats to obtain products of desired nutritional, physical, and sensory properties. An example is presented in Figure 6.7, where all three processing techniques are used in the preparation of a margarine-fat blend based on sunflower and rapeseed oil (7). A mixture is prepared with part of the oil and completely hydrogenated oil. Due to the presence of a large percentage of unsaturated fatty acids, it has a high solid phase content. The solid
Fig 6.7. Interesterification and fractionation of mixtures of nonhydrogenated and fully hydrogenated vegetable oils. Source: Rozendaal (7).
phase content is reduced through interesterification and further reduced through the fractionation process to remove saturated triacylglycerols. Schmidt from Unilever has patented the preparation of low-trans fats and emulsion spreads combining hydrogenation and interesterification (35). The raw material includes vegetable oil (soybean, sunflower, rapeseed, etc.), fully hydrogenated oil (sunflower, soybean, rapeseed, etc.), saturated oil (coconut, palm kernel, babassu, etc.), and palm oil or partially hydrogenated palm oil. This raw material is interesterified with 0.1% sodium methylate at 90C for 20 min. The purified 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 of a margarine oil containing low-trans fatty acids and with low intermediate chain (C8C16) fatty acid content (36). They interesterified a mixture of vegetable oil and a stearic acid source material in hexane using a sn-1,3 specific lipase. The remaining fatty acid mixture was hydrogenated to replenish the stearic acid for further reaction. They also developed a countercurrent process using a supercritical gas to separate the products from the reaction zone once the reaction was completed. This patent covers a wide variety of products that can be prepared using interesterification. Cocoa ButterLike Fats Cocoa butter is largely used in the manufacture of chocolate and confectionery products in which its unique consistency and melting properties are desired. Among
all of the commercial oils and fats, cocoa butter demands the highest price, but the price fluctuates widely. Cocoa butter-like fats are produced for economic reasons and also to offset the supply uncertainties (4,37). Chong et al. have prepared cocoa butter-like fat through fractionation of interesterified palm olein (38). Palm olein was interesterified with stearic acid using an sn-1,3 specific lipase. The interesterified product was initially steam distilled to remove the free fatty acids and further fractionated using the solvents hexane and acetone. The product was mixed with hexane and left to stand at 4C for 24 hr; this was followed by the separation of the crystallized fat from the mother liquor. The liquid fraction was further fractionated with acetone at 4C yielding 25% cocoa butterlike triacylglycerols. Chang et al. have prepared cocoa butterlike fat through enzymatic interesterification of hydrogenated cottonseed oil and olive oil using a similar process, except they only used acetone for the fractionation step (39). Lipases can catalyze reactions in organic solvents and also in phases, such as supercritical carbon dioxide (40). Lipases can perform admirably well in supercritical carbon dioxide (4143). Lipase-Catalyzed Interesterification of trioleoyl triacylglycerol and stearic acid has been carried out in supercritical carbon dioxide to prepare a cocoa butter substitute (44). The versatility of lipases offer numerous potential possibilities. Microbial sources have been lapped for the production of cocoa butter equivalents (45,46). This attempt used yeasts in a substrate of whey; however, it proved unsuccessful because the price of cocoa butter on the world market fell. This process can be applied if the price of the cocoa butter increases, or if a less expensive substrate for the yeast is found. Davies et al. from Unilever have filed a patent for the preparation of symmetric triacylglycerol that contains two saturated fatty acids and one unsaturated fatty acid (SUS-type [47]). This process involves the fractionation (from 5C to 10C) of the raw material to remove stearin and olein fractions and is followed by further fractionation (5C to 30C) of the olein fraction to obtain a stearin rich in triacylglycerols of SUU-type. This stearin fraction is interesterified using sn1,3 specific lipase to obtain SUS-type triacylglycerols that can be used as cocoa butter 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 parenteral nutrition and in infant formulas. Christensen and Holmer interesterified (at 22C) a mixture of butter oil and concentrates of polyunsaturated fatty acids, oleic acid, and linoleic acid in hexane using a commercially produced enzyme. The polyunsaturated fatty acid composition of the product closely resembled that of human milk as did the positional distribution of n-3 and n-6 polyunsaturated fatty acids 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 separately. It was observed that hexane-chloroform was the most suitable for the interestedfication of butter fat. Safari and Kermasha have interesterified butterfat in a microemulsion system using four commercial sn-1,3 lipases in order to alter the location of oleic acid on the triacylglycerol molecule (50). They found that three of the lipases increased the palmitic acid content at the sn-2 position of the butter fat triacylglycerol while one of the lipases increased the proportion of oleic acid at the sn-2 position. Yu et al. (51) have interesterified canola oil and anhydrous milk fat in supercritical carbon dioxide using lipase from Candida cylindracea. The final product contained triacylglycerols with carbon numbers ranging from C42C50 and C54 while the starting material contained predominantly C52C56 triacylglycerols from canola oil and C32C38 triacylglycerols from anhydrous milk fat. Using supercritical carbon dioxide they have also successfully esterified ethanol, oleic acid, and fatty acids from anhydrous milk fat. Marangoni et al. have prepared a modified fat for use as an edible plastic fat from trioleoyl triacylglycerol and tripalmitoyl triacylglycerol (52). The interesterification reaction, catalyzed by lipase from Rhizopus arrhizus, was carried out in reverse micelle formed with canola lecithin and hexane. The application of this process 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 acids are being prepared to replace conventional fats. These synthetic fats provide the same functional properties but have additional nutritional properties in comparison to the fats they replace. The nutritional value and absorbability of triacylglycerols depend not only on their fatty acid composition but also on their distribution on the glycerol backbone (53,54). There is a lot of interest in the preparation of structured lipids in which specific fatty acids arc located on specific positions in the glycerol. These structured lipids are difficult or impossible to prepare using chemical means, therefore enzymatic approaches are resorted to. The nutritional properties of the medium-chain fatty acids (metabolized rapidly, like carbohydrates) and very long chain fatty acids (poorly absorbed) are different from the other fatty acids normally present in vegetable oils. Interesterification, catalyzed by sn-1,3 lipases, is a key step in the preparation of medium-chain triacylglycerols. These triacylglycerols are used to provide a dense form of calories to patients with pancreatic deficiencies and malabsorption problems. These triacylglycerols are easier to hydrolyze and are absorbed more efficiently. But these triacylglycerols do not contain any essential fatty acids. This problem has been overcome by using structured triacylglycerols containing 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 triacylglycerol (56). The triacylglycerol contains both a long-chain fatty acid, such as behenic 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 kernel oils, while the source for behenic acid is rapeseed (probably fully hydrogenated) oil. Tremendous potential is foreseen in the synthesis of specific triacylglycerols and lipids that can be targeted to improve health or cure a particular disorder. Shishikura et al. have studied the incorporation of long-chain polyunsaturated fatty acids, such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), into medium-chain triacylglycerols (57). They combined sn-1,3 specific lipase-Catalyzed interesterification with simultaneous extraction of the released medium-chain fatty acids with supercritical carbon dioxide. Their reaction scheme is illustrated in Figure 6.8. It was possible to incorporate up to 62 wt% EPA with an overall 8592% triacylglycerol recovery by using a continuous medium-chain fatty acid recovery system. It was observed that the lipase was activated in the presence of glycerol. The interesterification reaction was dependent on the removal of the released medium-chain fatty acids. The mild enzymatic modification offers several advantages when preparing triacylglycerols containing high amounts of polyunsaturated fatty acids, such as EPA and DHA, as these fatty acids, and oils containing these fatty acids, readily oxidize. Homogeneous triacylglycerols containing either EPA or DHA have been prepared by Haraldsson et al. using a nonspecific lipase from Candida antarctica (58). The reaction involved the direct esterification of glycerol and interesterification of tributyroyl triacylglycerol with stoichiometric amounts of 99% EPA or DHA (either as free fatty acids or as ethyl esters) under vacuum without the use of a solvent. The volatile products (either butyric acid or its ester) were removed through condensation in a cold trap to push the reaction toward synthesis of triacylglycerols containing EPA and DHA. Yields of the crude product in excess of 93% were obtained by this process.
Fig 6.8. Preparation of triacylglycerols containing medium-chain fatty acids and polyunsaturated fatty acids. Abbreviations: MCT, medium-chain triacylglycerol; EPA, eicosapentaenoic 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 nutritional properties of palm oil and its fractions, Graille et al. have interesterified palm oil or palm stearin with oils, such as coconut, palm kernel, soybean, rice bran, 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 were essentially complete in about 5 hr (flow rate 3.5 mL/hr). Products with different characteristics could be obtained by withdrawing the product at different time intervals. A mixture of palm stearin and palm kernel oil (30:70, w/w) after a 30 min 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 oil that was virtually fluid at 20C that could be used as salad oils in tropical countries. Porcine pancreatic lipase-Catalyzed interesterification of canola oil and mixtures of canola oil with lauric acid, or trilauroyl triacylglycerol, or fully hydrogenated high erucic acid rapeseed oil was studied by Thomas et al. (61). The solidification points of interesterified products were considerably lower than the starting mixtures as shown by cloud point temperatures in Table 6.3. Interesterification resulted in the formation of triacylglycerols that were not originally present in either of the starting materials. Specialty triacylglycerols, such as high erucic acid triacylglycerols, have been prepared using direct esterification and interesterification approaches (62,63). Triacylglycerols with high levels of erucic acid were first prepared using direct chemical esterification of erucic acid and glycerol (62). The synthetic triacylglycerol (containing >90% erucic acid) was interesterified with native HEAR oil (containing 45% erucic acid) to obtain triacylglycerols containing varying proportions of erucic acid (ranging from 4592%). These triacylglycerols were then studied to gather information on their physical properties. Development of a sound understanding of the bioengineering aspects is essential for marketing new products using new techniques. Izumoto et al. have optimized the production of a product of constant triacylglycerol composition from a
TABLE 6.3
Cloud Point of Starting Mixtures and Interesterified Mixtures
Mixture Canola + Canola + Canola + Canola + Canola + Canola + Cloud point (C) Starting mixture Interesterified mixture 8 13 10 16 40 11 45 30 48 34 52 40
5% lauric 5% trilauroyl triacylglycerol 10% HEAR 20% HEAR 30% HEAR 40% HEAR
Abbreviation: HEAR, high erucic acid rapeseed oil. Source: Thomas et al. (61).
microaqueous bioreactor using filamentous immobilized fungus from Rhizopus chinensis (64). For the continuous interesterification reactor, a feedforward/feedback controller with an on-line enzyme activity estimator was used. The ratio of distearoyl monooleoyl triacylglycerol to the total triacylglycerols was estimated to determine the enzyme activity. The usefulness of the controller was justified by running simulations along with the experiments. The interesterification process has been used to prepare modified fat that can be used as a fat substitute and as nutritional ingredients in food. Cooper has prepared a reduced calorie admixture of a substantially digestion resistant esterified alkoxylated polyol and a digestible triacylglycerol (65). As an example, equal parts of esterified propoxylated glycerine product (containing C6C18 fatty acids) and fully hydrogenated rapeseed oil are mixed. The mixture is interesterified at 150C with 0.6% sodium methoxide for 3 hr. The crude product is refined to obtain a final product that contributes only 3.5 calories/g. Charton et al. from Unilever have produced a new lipase from Geotrichum candidum (66). This lipase, called lipase B, has very high specificity for 9-cis fatty acids and can be used for the production of oleic acid through the hydrolysis of high oleic acid sunflower oil. It can also be used for the preparation of specific triacylglycerols for nutritional purposes through interesterification of symmetrical acylglycerols containing oleic acid with polyunsaturated fatty acids, such as linoleic and linolenic acids. The inventors predict that these products will be desirable ingredients in food products, since they combat high levels of cholesterol in human blood. The specificity of the lipase offers some advantage when products of desired characteristics are needed. Foglia et al. used two lipases that have different specificities, one that is sn-1,3 specific and the other that is cis-9, C18 specific, for the interesterification of high oleic sunflower oil and soybean oil with tallow and butterfat (67). Different products were formed with the two lipases showing the potential of lipase specificities. The melting point of a tallow-rapeseed oil (LEAR) mixture was lowered by altering its triacylglycerol composition through sn-1,3 specific lipase (from Mucor miehei)-Catalyzed interesterification without the use of an organic solvent in the reaction media (68). After a 24 hr reaction time, the melting point of the interesterified product was 30C in comparison to 42C of the starting material. To improve the fluidity of palm oil, Kurashige et al. interesterified mixtures of palm oil and canola oil or soybean oil with an sn-1,3 specific lipase from Rhizopus delemar (69). They found canola oil to enhance the fluidity more than soybean oil. Interestingly, chemical interesterification of the same blends of palm oil with canola did not alter the fluidity of the product. The increase in fluidity was probably due to decrease in trisaturated and disaturated triacylglycerols in the enzymatically 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 of researchers (70,71). It is believed that the rate of autoxidation of triacylglycerols is
dependent on the fatty acid composition and on their distribution. Both chemical and enzymatic interesterification techniques have been utilized to study the effect of the positional distribution of fatty acids on the rate of autoxidation. There exists conflicting results in the literature regarding the effect of randomization on the oxidative stability of the oil. Randomization caused decreased stability in cocoa butter, tallow, corn, and soybean oil (71); and lard (72); and increased stability in palm oil (72). In some studies no difference in the stability of native and randomized oils was observed (73,74). A possible explanation is that methylesters and soaps formed in the reactions where sodium methoxide is used as a catalyst are more likely to oxidize faster than the original triglycerides. Tautorus and McCurdy compared the oxidative stability of mixtures of trioleoyl triacylglycerol and linseed oil that were either blended together or enzymatically randomized (75). They observed that when the ratio of unstable to stable triacylglycerols is low, the randomized mixture was more stable than the blended mixture, 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 dilution of less stable fatty acids. Marine oils rich in the polyunsaturated fatty acids EPA and DHA, such as whale, sardine, cod liver, and skipjack oils, are assuming importance due to their nutritional value and are widely used for the preparation of specialty lipids. Kimoto et al. have studied the relationship between the triacylglycerol structure of marine oils and their oxidative stability and have also studied the effect of chemical and enzymatic interesterification (76). Enzymatic interesterification resulted in
Fig 6.9. Oxidative stability (measured as absorbance at 234 nm and indicative of conjugated diene content) of mixed and randomized trioleoyl triacylglycerol/linseed oil blends (wt/wt) as a function of storage at 52C. Source: Tautorus and McCurdy (75).
increased stability of fish oils, while chemical interesterification resulted in the increased stability of all oils except sardine oil and whale blubber oil.
Safety
New products (or novel products), such as those produced by chemical or enzymatic interesterification, have to be declared safe by the concerned agencies in their respective countries. These products have to be proven safe by the manufacturer. An enzymatically interesterified oil produced by the Fuji Oil Company, Ltd., from safflower oil or sunflower oil and ethyl stearate has been cleared by the Advisory Committee on Novel Foods and Processes in the United Kingdom with some 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 the Environment (COT) for use in food in United Kingdom. The modified oil is to be used as a substitute for cocoa butter in chocolate and confectionery products. This product has been produced in Japan since 1986 and was accepted for use in food in Japan. Unilever has also received clearance from the same committee for an interesterified product from vegetable oils and foodgrade fatty acids (78). The reaction in this case is carried out in hexane. The product is intended for use in chocolate and confectionery fat and as an ingredient at levels of up to 20% in frying fats as a replacement for saturated fats. The specifications for the product are listed in Table 6.4. The manufacturers also provide information 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 be approved and declared safe (79). Safety evaluation for an enzyme is focused on the presence of toxic contaminants (raw material, foreign microorganisms, additives and preservatives used, etc.) in the commercial enzyme preparation and, if the enzyme 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 fat substances when ingested, at present we do not entirely understand the metabolic significance of the positional distribution of fatty acids in the triacylglycerol molecules on plasma lipids. A metabolic balance study was carried out with infants that were fed with infant formulas containing either natural lard (which contains palmitic acid primarily in the sn-2 position) or randomized lard (80). The rest of the infant formula composition was the same. It was observed that infants fed with the formula containing natural lard excreted 0.3 g fat/kg/day while the infants fed with
TABLE 6.4
Specifications for Enzymatically Interesterified Product
Total acylglycerols Triacylglycerols Diacylglycerols Monoacylglycerols Free fatty acids Oxidation products Heavy metals Color Odor/taste Residual hexane Residual acetone Lipase activity Nitrogen
Source: U.K. Ministry of Agriculture (77).
>99% >93% <6% <0.1% <0.1% <1% 0.2 mg/kg Colorless Bland <1 mg/kg <2.5 mg/kg None <5 mg/kg
the formula containing randomized lard excreted 1.79 g fat/kg/day. It was postulated that the greater absorption of natural lard was due to the greater extent of micellization of sn-2 monopalmitoyl acylglycerol. In a more recent study by Innis et al. (81), it has been shown that palmitic acid is absorbed from human milk as sn-2 palmitoyl acylglycerol. Infants were exclusively fed with human milk (containing 21.0% palmitic acid with 54.2% of the total palmitic acid residing in the sn-2 position) or formula milk (containing 22.3% palmitic acid with 4.8% of the total palmitic acid residing in the sn-2 position). They found that the plasma triacylglycerols of infants fed with human milk had 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 as corn oil (83). The connection between dietary triacylglycerols and diseases has to be studied closely in order to apply new techniques to prepare the desired fat product. In this case, the demand for a particular product with nutritional benefits would motivate researchers and processors.
Future Prospects and Conclusions

Several factors are involved in the development and marketing of a new fat product, especially if it is a designer fat that replaces already existing natural products. The development of tailor-made triacylglycerols is motivated and mainly driven by health questions concerning fat components, which in turn either stimulate or reduce consumer demand for that product. Manufacturers need to prepare specialty fats that satisfy niche markets, and processors that require new materials to substitute for or improve upon the performance of the fat to be replaced. In addition to these factors, there is the question of cost feasibility.
Lipid chemists, food scientists, and food regulators have to address several questions regarding the definition of natural fat or oil products. There is a question regarding authenticity, if a processor separates a particular fatty acid from say, an animal fat or a tropical oil (the use of these fats was questioned in the past decade), and utilizes these fatty acids to prepare a designer triacylglycerol of desirable 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? Todays consumer is more knowledgeable and has more understanding of the food components than the consumer of the past. More research is necessary to ascertain the effect (both beneficial and detrimental) of different fatty acids (short-and long-chain saturated fatty acids, medium-chain fatty acids, and polyunsaturated fatty acids) on absorption and metabolism in the human system. Much work needs to be done on the effect on the human metabolism of the distribution of fatty acids on the triacylglycerol that is ingested, on the end-products of these fatty acids in the human body, and their positional distribution in body triacylglycerols. It is also necessary for the bioprocess engineer to be able to devise continuous reactors for development of these designer fats. Mathematical modeling of the reaction system under consideration is necessary to predict the production of specific triacylglycerols. Such reactors also would need on-line detectors to monitor product formation and estimate biocatalyst residual activity. Research in the area of interesterification is being carried out in research laboratories around the world. Large companies with huge research and development facilities are developing new processes and these are eventually patented. In conclusion, the interesterification (both chemical and enzymatic) reaction is a useful tool in the hands of the processor. Currently, there is high interest in the application of lipases for the manufacture of designer oils and fats. The Unilever Group in cooperation with Novo Nordisk has recently built an enzymatic interesterification plant for the synthesis of specialty triacylglycerols using an .sn-1,3 specific lipase (84). Lipases with new characteristics (heat stable, high activity) and specificities have enormous potential in this field. Currently, lipase applications are limited to the manufacture of products that demand a high price in the market. The cost of 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 engineering 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 the industrial 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 with diets high in saturated fats. Consequently, dietary modification to reduce intake of saturated fatty acids has been recommended as a major step to lower the prevalence of heart disease (1). Research has also shown that diets high in monounsaturated 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 cholesterol (HDL-C [3]). High levels of HDL-C are positively correlated with reduced levels of heart disease. More specific studies have documented the effectiveness of oleic acid in reducing LDL-C but maintaining HDL-C levels (4). Oleic acid has therefore been recommended as the major fatty acid in the human diet (5). Oil stability, particularly during frying, is an important consideration in relation to acceptability. Oils high in polyunsaturated fatty acids will produce more oxidative products 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 example, oleic acid is 10 times more stable than linoleic acid and 25 times more stable than linolenic acid in terms of the rate of oxidative breakdown (7). The previously mentioned studies have contributed to the identification of desirable fatty acid compositions in vegetable oils for human consumption (8). The optimal vegetable oil should contain a predominance of oleic acid, with reduced levels of saturated fatty acids (i.e., palmitic and stearic acids) and polyunsaturated fatty acids (i.e., linoleic and linolenic acids). An exception to these objectives would be the development of specialty oil types with elevated levels of saturated fatty acids for direct use in margarine manufacture, thereby avoiding or greatly reducing the production of trans fatty acids associated with hydrogenation. It has been reported that dietary trans-fatty acids may increase the risk of heart disease (9). Genetic modification strategies employed in the development of new crop cultivars have been, and will continue to be, used to alter the composition of vegetable oils, thereby improving their value in human nutrition. This chapter presents an overview of four major genetic approaches to altering fatty acid composition in vegetable oils. These include
1. 2. 3. 4.
Utilization of desirable genetic variation present within a crop species, Application of mutagenesis procedures to induce desirable genetic traits, Interspecific and intergeneric transfer of traits into the target crop, and Alteration of oil composition through genetic transformation technologies.
Attention will be given to major crops cultivated in Canada for the production of oils that are consumed by humans. These include canola (Brassica napus and Brassica rapa), soybean (Glycine max), flax (Linum usitatissimum), and sunflower (Helianthus annuus). Emphasis will be placed on the generation of fatty acid modifications relevant to the objectives identified previously.
Modification of Fatty Acid Profiles in Oilseeds Using Genetic Variation Occurring Naturally in the Species
Plant breeders have primarily depended on genetic variation within a species in their continuing efforts to develop new cultivars. Such naturally existing variation has also been used to alter fatty acid profiles in the development of edible oilseed crops. An excellent example of intraspecies genetic variation that has had major impact on Canadas vegetable oil industry was the identification of genetic lines with reduced levels of erucic acid (2%) in B. napus and B. rapa (10,11). These lines were used by Canadian plant breeders to develop low erucic acid cultivars as the first major step leading to the development of canola (12). Another example is the identification of a genotype of B. rapa with elevated levels of palmitic acid (in the range of 911%) in its seed oil that may be useful in developing specialty cultivars for margarine manufacture (13). Substantial variation in fatty acid profiles also exists in sunflower. Breeders have taken advantage of this variation and have developed specialty oil cultivars containing reduced levels of saturated fatty acids (14, K. Fitzpatrick, Western Seed Corp, Saskatoon, Saskatchewan, personal communication). However, with the few exceptions described previously, the amount of genetic variation existing naturally within crop species has been limited. Therefore, additional genetic modification strategies are required to generate variations with desired fatty acid profiles in the target crop species. Such variants can then be incorporated into plant-breeding programs in order to develop agronomically acceptable cultivars/strains. The additional genetic strategies that could be employed for developing altered 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 plants involves treatment of large populations of imbibed seeds with a chemical mutagen,
such as ethyl methyl sulfonate (EMS). This approach has been successfully employed in altering fatty acid profiles of a number of oilseed crops. Brassica Oilseeds. Mutagenesis procedures have been successfully employed to reduce linolenatc and increase oleate in Brassica seed oils. In 1973, Rakow generated a mutant line of B. napus, with reduced levels of linolenic acid in its seed oil (15). This material has been utilized by researchers at the University of Manitoba to develop Stellar and Apollo, low linolenic acid cultivars, with the latter cultivar possessing linolenic acid levels of less than 2% (16, also see Chapter 8). Intermountain Canola (now owned by Cargill) developed a number of specialty lines of B. napus through seed mutagenesis. These contain elevated levels of oleic acid and reduced levels of linolenic acids (Table 7.1). These lines are grown under contract for specific uses in food processing. Pioneer Hi-Bred also established a mutagenesis program and produced lines with 8088% oleic acid, compared to 62% 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 identification of a mutant line with levels of linoleic acid and linolenic acid reduced to 2.1 and 3.0%, respectively, compared to the control having 11,9 and 8.6%, respectively (18). This mutant line was subsequently used in genetic crosses with low erucic acid lines of B. rapa to generate lines having less than 6% polyunsaturated fatty acids and more than 88% oleic acid (18). Sunflower. Several organizations are engaged in the development and/or marketing of specialty sunflower seeds and/or oil products with elevated levels of oleic acid (14). Apparently all lines with elevated oleic acid are derived from a single mutant line produced in the U.S.S.R. through seed mutagenesis (19). Western Grower Seed Corp. researchers have intentionally selected for high oleic acid content and have combined this trail with low saturate levels as well as the dwarf, early maturing character to produce a specialty purpose high oleic/low saturate strain (Table 7.2).
TABLE 7.1
Fatty Acid Profiles of Specialty Canola, (B. napus) Varieties Developed Via Mutagenesis
Variety IMC01 IMC129 IMC130 IMC02 508 18:1 (%) 61.9 793 77.0 67.9 87.3 18:2 (%) 23.9 8.7 11.9 20.7 2.0 18:3 (%) 4.9 4.8 3.9 2.1 3.4 16:0 (%) 4.1 3.7 4.0 4.1 3.1 18:0 (%) 1.9 1.8 1.9 1.9 1.6 Commercial Production 1989 1991 1991 1993 Under development
Source: W.H.-T. Loh, Cargill, unpublished data.
TABLE 7.2
Fatty Acid Profile of HO/LS Sunola
Strain HO/LS Sunola Sunola Sunflower (U.S.) 18:1 (%) 88 14 17 18:2 (%) 5 74 70 18:3 (%) 0 0 0 Saturateda (%) 7 12 13
a Refers to total saturated fatty acids. Source: K.C. Filzpatrick, Western Grower Seed Corp., unpublished data.
Soybean. Mutagenesis procedures have been used to produce stable genetic lines, independently containing reduced levels of linolenate (20), reduced levels of saturates (21), and elevated levels of stearic acid for potential use in margarine manufacture (22). These lines are being utilized in the development of commercial varieties that will compete effectively with canola and sunflower. Flax. Pioneering work by Green in Australia resulted in the identification of two mutant lines of flax that when combined yielded progeny with low linolenic acid and elevated linoleic acid levels that were stable, thereby providing a seed oil with a similar fatty acid profile to that found in sunflower (23). Lines derived from the original mutants were used in a breeding program sponsored by United Grain Growers (UGG) to develop an edible oilseed flax. An independent mutagenesis program established at the Crop Development Center at the University of Saskatchewan has also resulted in the isolation of mutants with reduced linolenic levels (Table 7.3 [24]). In a collaborative venture between the Crop Development Center and the Saskatchewan Wheat Pool, an edible oilseed flax is being developed for commercial production. The Crop Development Center Program has also generated 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 produce seed oil with reduced linolenate be referred to as Solin. The Solin cultivars developed by the UGG program are trademarked as Linola (25). Linola has been commercially cultivated in Canada with more than 30,000 acres grown in Manitoba in 1994 and more than 200,000 acres projected for 1995 (J. Dean, United Grain Growers, Winnipeg, Manitoba, personal communication).
TABLE 7.3
Fatty Acid Profiles of Three Genetic Lines of Flax and the Parent Cultivar
Line E67 E1747 E1929 McGregor 16:0 (%) 27.8 9.5 9.5 9.4 16:1 (%) 4.8 tr tr tr 18:0 (%) 1.8 4.6 3.4 5.1 18:1 (%) 17.5 15.6 51.7 18.4 18:2 (%) 6.0 65.3 16.3 14.6 18:3 (%) 42.0 2.1 16.2 49.5
Source: Rowland et al. (54).
Mutagenesis of Haploid Cells In Vitro The development of isolated microspore culture technology offers the possibility of a novel and unique approach to mutant isolation. A microspore culture system possesses distinct advantages in mutant selection: the haploid genome of the microspore should make mutagenesis procedures more effective, very large populations can be easily handled in small areas, and the production of chimaeras can be avoided. Microspore mutagenesis has been successfully employed to produce B. napus mutants expressing elevated levels of oleic acid (17). In the case of B. rapa, a line capable of producing large numbers of embryogenic microspores has been identified (26); this line is now being utilized to generate mutants with altered fatty acid profiles (27).
Modification of Fatty Acid Profiles in Oilseeds Via Interspecific Hybridization

Interspecific Sexual Hybridization It is possible to make interspecific sexual crosses among Brassica species followed by backcrossing of the hybrid to the crop species as a method of transferring desirable traits into oilseed Brassica. In some cases, in vitro culture techniques are required to rescue hybrid embryos (28). Interspecific hybridization approaches have been used to develop B. napus lines with elevated linoleate and reduced linolenate (29). The University of Manitoba has established a program to transfer the low linolenate trait from B. napus, in which it was derived through mutagenesis, to B. rapa through sexual hybridization (R. Scarth, Department of Plant Science, University of Manitoba, personal communication). In the case of sunflower, a number of wild, closely related, and sexually compatible species are indigenous to large areas of North America. These wild relatives serve as a potential reserve of desirable genes, including modified fatty acid profiles that can ultimately be transferred to cultivated sunflower (14). Somatic Hybridization By fusing isolated protoplasts to produce hybrid cells from which hybrid plants can be regenerated, it may be possible to bypass barriers that prevent successful sexual hybridization and thereby introduce genes from sexually incompatible species into the target crop (30). Of the major Canadian oilseed crops, protoplast technology thus far has only been established at a reliable level in B. napus type canola. A number of somatic hybrids have been produced in which B. napus has been one of the parents (31). To date there have been no reports on the use of this approach to alter fatty acid profiles in oilseed crops. However, somatic hybridization of Arabidopsis with Brassica could offer the possibility of transferring desirable fatty acid profiles from 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 acid profiles, have been produced and characterized (32). Somatic hybrids between Arabidopsis and B. napus have been successful generated (33), however low levels of fertility of such hybrids could make backcrossing to the crop species extremely difficult. It might be possible to employ asymmetric fusion strategies (34), in which the Arabidopsis genome is pulverized by chemical or irradiation treatment, thereby significantly reducing the amount of genomic information transferred 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 of methodologies for integrating foreign genes into the crop plant genome (35). The two major methods for inserting genes into plant cells and tissue include co-cultivation with Agrobacterium tumefaciens, and application of biolistic technologies. In terms of oilseed crops, methodology for genetic transformation has been published for B. napus (3638), B. rapa (39), soybean (40), flax (41,42), and sunflower (43). To complement the technical advances in gene insertion methodology, a great deal of research activity has been focused on the characterization of the enzymatic steps, and identification of genes, involved in triacylglycerol synthesis (446). A recent review by Slabas et al. indicates that the following genes have been isolated and characterized: acyl carrier protein (ACP), acetyl CoA carboxylase, 3-ketoacyl-ACP synthetase, 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 fatty acid profiles include targeted expression of foreign genes in developing seeds as well as reduced expression of native genes for oil synthesis through insertion of antisense gene constructs. Through such targeted modifications in biosynthesis and triacylglycerol assembly, it is possible to substantially alter the fatty acid profile in oilseeds. Substantial progress has already been announced in this rapidly emerging field (4851), and significant advances should be anticipated in the near future. The commercialization of genetic varieties produced through genetic transformation should be anticipated over the next few years. Although a great deal of effort is being devoted to the development of modified fatty acid profiles for industrial applications, reference in this chapter will only be made to alterations for human nutritional/health objectives. Brassica Oilseeds Research by Calgene Inc. has demonstrated that it is possible to successfully utilize antisense RNA technology to underexpress a 9 desaturase gene in B. napus,
thereby producing an oil with elevated levels of stearic acid (52). Transgenic lines of 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 strains for use in the margarine industry eliminating the need for hydrogenation and the associated generation of trans fatty acids. Insertion of an antisense 12 desaturase by Dupont researchers resulted in the identification 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 linolenic acid levels to the range of 2% of total fatty acids (53). Genetic transformation has also been employed to reduce saturated fatty acid levels in B. napus (54). Soybean Using ballistic-based genetic transformation technologies, Dupont researchers have introduced an antisense 12 desaturase gene into soybean and have identified transgenic lines with more than 70% oleic acid in their seed oils (53). Flax Collaborative research between the Crop Development Center, University of Saskatchewan and the National Research Council of Canada has been undertaken to develop a Canadian-based cocoa butter replacement oil (55). A high palmitic/low linolenic mutant line is being transformed with antisense stearoyl-ACP desaturase (9 desaturase) with the aim of producing an oil with palmitic acid, stearic acid, and oleic acid at similar levels, simulating in composition cocoa butter.
Conclusions
Studies on human health and nutrition, as well as evolving consumer demands for food products with improved nutritional characteristics, have dictated a continuing requirement for research to alter fatty acid profiles in vegetable oils. Plant breeders have made significant advances by using genetic variation for fatty acid composition already existing in the species. The development of canola was primarily based on the use of such naturally occurring variation. Additional alterations in oil composition in canola and in other oilseeds has to a large extent been based on the application of additional genetic modification strategies. These strategies include mutagenesis, interspecific hybridization, and genetic transformation. Mutagenesis has been successfully used to alter fatty acid profiles in Brassica oilseeds, sunflower, flax, and soybean; a number of commercial strains/varieties have been developed from mutagenized lines of these crops. Interspecific sexual hybridization as a method of altering oil composition has thus far been employed only in oilseed Brassica because of the ability to intercross with related species. Somatic hybridization offers a potential approach to alter the fatty acid profile in oilseeds, 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 available in Arabidopsis, a member of the same taxonomic family as Brassica spp, might serve as a source of genetic variation for incorporation into oilseed Brassica. The greatest long-term potential for modification of fatty acid profiles involves the application of biotechnology, specifically genetic transformation technology (genetic engineering). The approach is based on the insertion of modified gene expression systems, thereby giving specific and predictable end-product alterations. The potential genetic transformation technology has already been demonstrated with the generation of high stearate and high oleate B. napus lines, as well as soybean lines with modified fatty acid profiles. Significant commercial impact 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 sufficient to tell you the oil quality. Oilseed rape, for example, produced an oil high in a long-chain fatty acid, erucic acid (C22:l). This oil could be successfully substituted for petroleum lubricants and adhered well to metal surfaces even when wet. The oil quality of this simple oilseed crop changed radically when Canadian plant breeders modified the fatty acid composition of oilseed rape by reducing the C22:1 content from 40% to nearly 0%. The change was motivated by a desire to enhance the quality of the oil for the edible oil market and to address the concern that long-chain C22 fatty acids are poorly digested. The result was an oil low in C22:1 acid and high in the C18 fatty acids, in particular oleic acid (C18:1). The new oil, along with an enhanced quality in the seed meal, was given a new commodity name canola, a term that is now used for the oilseed rape crop that produces the canola products (1). Production of canola in western Canada has increased to a record high of 14 million acres in 1994 (2), challenging wheat for the leadership of the prairie crops. One of the reasons for canola oils increasing share of the edible oil market is its very favorable fatty acid composition. Canola oil has the lowest saturated fat content of the major vegetable oils. Health Canada has recommended that all Canadians reduce the amount of saturated fat in their diet, and canola oil provides one means to this end.
Breeding for Low Linolenic Acid Content

Another distinction of canola oil that is not as favorable is the high content of linolenic acid (C18:3). The high C18:3 content results in additional cost and effort to the processors of canola as well as soybean oil, which is also high in C18:3. One of the objectives of the canola breeding program at the University of Manitoba is to produce low C18:3 canola. This change has been accomplished through the application of mutagenesis followed by selection. The target in the case of the low C18:3 mutation was the desaturation step between linoleic (C18:2) and C18:3 in the seed oil biosynthetic pathway. The objective was to disable the enzyme that adds the third double bond. The mutation work
was conducted by Rakow and Rbbelen 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 of Manitoba. Several years of work were required to grow out the progeny, select for normal growth, and generally clean up the mutation line while selecting for the block in the desaturation pathway between C18:2 and C18:3. This was followed by crossing to Regent, the adapted cultivar that was suitable for production in the desired area of western Canada. The objective of the crossing program was to recover all the attributes of Regent with the addition of the low C18:3 trait. This aim was accomplished after several generations of backcrossing and selection; the worlds first low C18:3 cultivar, Stellar, was registered in 1987 and was followed by Apollo in 1992(5,6). The result of the low C18:3 breeding program is a unique fatty acid composition illustrated by the profile of the low C18:3 cultivar, Apollo (Table 8.1). The low levels of C18:3 are accompanied by a slightly higher content of C18:2 and, in particular, a higher C18:1 content in comparison to the conventional canola oil quality of the cultivar Westar. The reduction in C18:3 has produced the anticipated benefits in reducing the hydrogenation required, namely, an increase in stability, and an improvement in room odor of the heated oil (7,8). The effect of the reduction in C18:3 on the oils storage properties was the objective of a recent study by Przybylski and his colleagues in the Department of Foods and Nutrition, University of Manitoba (9). A comparison of odor development in conventional canola and low C18:3 oil showed that the low C18:3 oil remained relatively stable over a period of 0-12 days, while canola oil developed a progressively more intense unpleasant odor. This slower development of off-odors is especially critical for canola oil in the important application of domestic frying.
TABLE 8.1
Fatty Acid Composition of Low Linolenic (C18:3) Canola, Flax, and Soybean
C16:0 + C18:0 (%) Canolaa Apollo Westar Soybeanb A16 Century 84 Flaxc Linola 947 AC Linora
a b c
C18:1 (%) 67 63
C18:2 (%) 24 22
C18:3 (%) 1.7 9.3
5.7 5.6
15.4 13.6
41.1 24.3
41.2 54.6
2.2 7.4
10.5 8.4
15.6 16.6
71.5 18.1
2.4 56.9
Source: Scarth, McVeity, and Rimmer (6). Source: Fehr el al. (10). Source: Dribnenki and Green (12).
Similar methodology was used in the development of low C18:3 soybean cultivars, involving an initial mutation treatment followed by selection. The low C18:3 strain, A16, was developed from a mutation line developed at Iowa State University. In comparison to the conventional soybean oil profile (Table 8.1), A16 shows a low level of C18:3 with slightly lower C18:2 levels, an increase in C18:1 and 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 of mutagenesis and selection by Green in Australia (11). The block between C18:2 and C18:3 resulted in a dramatic reduction of C18:3 levels from 57% to just over 2% and was accompanied by an increase in C18:2 (Table 8.1) to produce a fatty acid profile similar to sunflower oil. In 1993, the first low C18:3 flax was registered in Canada (12). Low C18:3 flaxseed oil has been approved for human consumption in Canada and similar approval is being sought in the United States. The flax breeding program conducted by Rowland at the Crop Development Centre, Saskatoon, also has had success with mutagenesis in creating new low C18:3 mutations and cultivar development is under way (13). There are now three new low C18:3 crops, canola, soybean, and flax, all with levels 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 to consider 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 at Pioneer Hybrid. Mutagenesis was applied to embryos produced by incubating immature pollen grains or microspores that were then cultured to produce plants that carried the mutation. The target was to produce a mutation earlier in the desaturation pathway, to block the desaturation of C18:1 to C18:2. The desired mutation was selected and then crossed to incorporate the low C18:3. The result is an oil low in both C18:3 and C18:2, with 86% C18:1 (14). The further reduction in desaturation 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 selection from Russian sunflower germ plasm naturally high in C18:l acid. The Specialty Vegetable 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 developed using genetic transformation techniques. The first generation of transformants produce up 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 and the 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 a specialty oil cultivar. These can be summarized as stability of expression, simple inheritance of the specialty oil trait, productivity of the specialty oil cultivar, development of methods to segregate the specialty oilseed, and finally the premium value of the specialty oilseed crop. The question of whether a specialty oil trait has the required stability of expression can only be answered after the new oil quality is transferred into adapted cultivars. Field tests can then be conducted at different locations over several years to determine the effect of environment on the expression of the specialty oil trait. The effect of environment on oil quality in conventional sunflower is dramatic, with variations of up to 30% in the C18:l acid content of sunflower oil produced in the northern and southern United States (16). The high C18:l germ plasm is more stable. A similar stability of expression has been observed in tests of the high C18:1 canola germ plasm (14). Sunflower is a long season crop, and production in Canada is limited by the length of the growing season. Sunflower breeders at Western Grower Seed, Saskatoon, are developing a short stature sunflower with early maturity to allow production in western Canada, north of the usual limits for sunflowers, with the added 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 been tested under different temperatures (17). M1589 and M1722 have moderate reductions in the level of C18:3. When the two lines were crossed, the result was a very low level of linolenate represented by the genotype Zero. A conventional high C18:3 cultivar was also included in the study. In all four lines, exposure to higher temperatures resulted in oils with lower C18:2 and C18:3, and higher C18:l. The percentage of the saturated fatty acids C16:0 and C18:0 also increased under higher temperatures. At the lower temperatures, C18:3 levels were under 3% and the C18:l content of the Zero genotype was over 62%. We have just completed a study of the effect of environment on the fatty acid composition of the low linolenic acid cultivar Stellar and the cultivar Regent that was used in the development of Stellar (18). The breeding method used in the development of Stellar resulted in the two cultivars having very similar genetic make-ups with the exception of the low C18:3 trait (5). The cultivars were grown in isolated plots over several locations and years. There was a significant effect of location on the fatty acid composition of the two cultivars. In general, the locations having the highest daily mean temperatures during seed development yielded seed oil containing the lowest level of C18:3 and the highest level of the saturated fatty acids C16:0 + C18:0. Levels of C18:1 and C18:2 were more variable. The overall pattern was a fatty acid profile higher in saturated and monounsaturated fatty acids and lower in polyunsaturated fatty acids when the seed developed under higher temperatures. The reliable production of seed with a composition low in C18:3 is necessary for the success of the low linolenate canola oil. To confirm the effect of temperature on seed oil composition, another set of experiments was conducted under
controlled environment conditions that reproduced the stress of high temperatures during seed development. Under these conditions, the low C18:3 content of Stellar remained stable. However, when the low C18:3 plants were subjected to long duration (2030 days) of 30/25C temperatures during seed development, the saturated fatty acid content climbed above the critical 7% in both the low C18:3 cultivar Stellar and the canola cultivar Regent. High temperature may retard desaturase activity or the rapid seed development may fix a greater amount of the fatty acids in the saturated form. Seed maturity is 1012 days faster under the long duration of high temperatures when compared to the control maintained at 20/15C. Elevated levels of saturated fatty acids provide an important consideration in the production of the low linolenic acid oil. Production of low C18:3 canola and conventional canola cultivars should be carried out in areas that do not have prolonged periods of high temperature during seed development. Fortunately, the area of canola production in western Canada does have relatively low temperate growing conditions. Successful production also depends on completing seed development before frost. Early maturity is a key to high-quality seed production with high oil content and low seed chlorophyll content. One alternative is to move the low C18:3 trait into the other canola species, Brassica rapa, to provide an early maturing source of low C18:3 oil. The Brassica rapa crop currently produces an oil low in saturated fat that is useful for blending with canola oil higher in saturated fatty acids to achieve the required 7% limit. A similar study of the effect of extreme temperatures on soybean oil composition was conducted by Rennie and Tanner at the University of Guelph (19). The soybean 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 low linolenic acid content lines ranged from 2.5 to 4.6% under field conditions. The temperatures in the controlled environments were lower (15/12C) and higher (40/30C) than those usually occurring during seed development in the field. The 15/12C environment produced high levels of C18:3 in the low C18:3 lines compared to those observed at 28/22C. The high C18:1, low C18:2, and low C18:3 values observed at 40/30C were beyond the usual range of values of the lines in the study. The high C18:0 line grown at 15/12C produced C18:0 at the same level as the conventional cultivars. Exposure to cold temperatures apparently alters the pattern of C18:0 metabolism. The highest expression was observed at 28/22C. Soybean breeders face a major challenge in bringing the low linolenic acid content oil into commercial production. The soybean-growing area is distributed north to south over a wide range of environments. Any change in oil quality that is sensitive to environmental influences, such as temperature, has to be introduced into adapted soybean cultivars in each area of production. The method of introduction, which involves crosses and backcrosses for several generations to adapted 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 inheritance 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 are aiming at the development of a cultivar with a package of characteristics, in which specialty oil quality is just one. The simpler the inheritance of the change in oil quality, the easier it is to handle in a selection program. A trait controlled by several genes may require the use of molecular markers to tag the desirable lines from crosses without the requirement for seed production and analysis. Other savings in time and effort could be made if the specialty oil trait is linked to another trait that 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 development of the conventional cultivars. For example, in 1985, the top-yielding canola cultivar, Westar, was the obvious choice as the adapted parent in crosses to develop special quality cultivars. However, Westar is highly susceptible to the disease blackleg, that is now well established in the canola-growing areas of western Canada. The virulent form of blackleg can reduce yields by as much as 75% in susceptible cultivars and highly susceptible cultivars, such as Westar, are devastated by the disease. Specialty oil cultivars that are susceptible to blackleg also will suffer yield losses. Disease resistance is just one part of the productivity package that applies equally to specialty oil cultivars and conventional cultivars and includes yield, oil and protein content, and maturity. Stability of yield performance over environments is critical for the success of the cultivar, as is early maturity to allow seed development to be completed under favorable conditions. High seed oil and protein content and low chlorophyll levels are important quality factors influenced by seed development. Disease resistance provides an insurance policy that productivity will be maintained if the year is conducive to disease development. All of these characteristics need to be in place for successful production of specialty oil cultivars. During the development of specialty oil cultivars, the small scale of the production makes segregation of the specialty oil seed relatively easy. From the initial tissue culture through single plants in the greenhouse, single row increases in the field, and harvesting of the seed with the small plot combine, the plant breeder can 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. The specialty oil character adds an additional factor in the need for isolation of production. Both canola species are cross-pollinating, and pollen from outside sources can introduce variation in oil quality that is undesirable. Cross pollination is a particular hazard in areas of high insect pollinator activity. Large-scale production of specialty oil cultivars will have to be carefully coordinated to avoid contamination from adjacent fields of conventional canola. The grain-handling system in western Canada is set up for volumelarge amounts 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 containers at the ports for export. At each step, the specialty oil seed must be kept separate to retain the value of the oil. Any factors that can ease this process of segregation in the transport system would be an advantage. As an example, the low linolenic acid flax cultivar has been developed in a yellow-seeded background (12). The seed-handling system can use this characteristic to segregate the edible oil crop from the industrial oil crop and prevent mixing that would lower the value of both crops. There is similar variation for yellow seed coat color in canola. Unfortunately, the trait is multigenic and influenced by the environment during seed development (20). The requirement to add this trait to the development of specially oil canola cultivars would demand considerable time 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 places on the oil. Before processors and manufacturers will commit to the purchase of large volumes of specialty oil, they must have two guarantees. The first is the guarantee of qualitythe minimum standards for the specialty oil must be met consistently. The second guarantee is a guarantee of continued supply. Manufacturers must be sure that production levels will allow a commitment of a particular label or processing line to the specialty oil. Those volumes must be available to maintain the market for that product. We can develop a checklist for the ideal specialty oil cultivars that includes good stability of oil over different environment and years. Productivity must be at least equal or, if not, the premium value must compensate for any losses in productivity. If there are any distinct production practices that must be followed, such as isolation to prevent pollen contamination, these practices have to be very clearly 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 the markets of the next century. Once the required variation has been created through selection, mutation, or transformation, the next step is to ensure that agronomic performance is sufficient for successful production. Market development is critical to determine where the specialty oils will find a market and if the new oil quality is what the market requires. In 1994, the traditional oilseeds no longer have just traditional oil quality. There are 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 specialty oil cultivars and develop means of handling the seed to ensure that specialty oil quality is maintained. The successful production of specialty oil cultivars requires close coordination between all aspects of this industryplant molecular biologists 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 to ensure the best quality seed production. Processors work with oilseed chemists, marketing analysts, and nutritionists to produce the finished oil product. The final element to ensure success would be a crystal ball to reveal the ideal oil quality for the next century. I would welcome your ideas!
References
1. Downey, R.K. and G. Rbbelen, in Oil Crops of the World, edited by G. Rbbelen, R.K. Downey, and A. Ashri, McGraw-Hill, New York, 1989, p. 355. 2. Canola Digest, Canola Council of Canada, Winnipeg, Manitoba, Canada, November 1994, p. 9. 3. Rakow, G., Z. Planzenzuchtg 69: 62 (1973). 4. Rbbelen, G. and A. Nitsch, Z Pflanzenzchtg 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. 7375. 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 Acid Compositions 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 of foods that previously were not available in the Canadian marketplace. Examples of these developments include novel macro ingredients, functional foods, new food-packaging technologies, and the use of genetic engineering (1). Some of the endpoints, such as the development of novel ingredients and the use of genetic engineering, can be observed with the development of novel oils. For example, the genetic engineering of oilseed crops to enhance pest control and disease resistance properties is well established. Similarly, research is underway with respect to tailoring the fatty acid composition of the oil to address the specific needs of the food-processing industry, or to alter the nutritional or dietary characteristics of the food, by both genetic modification of the plant and postharvest modification of the oil (2). Such developments represent a significant change in the approach scientists have used to develop new products for the marketplace. Accompanying these developments have been questions regarding the impact of such modifications on the nutrient composition of the altered food and the possibility of affecting the allergenic potential or introducing other unintended effects into the modified food. Compounding these issues is the development of an educated, concerned, and increasingly activist population that wishes to better understand and have more control regarding the food they eat. The challenge to government is to develop regulations that address the concerns expressed by scientists and the public without unduly hindering the growth, development, and competitiveness of this industry. The purpose of this paper is to discuss the approach that Health Canada is taking to address safety issues regarding novel foods, in particular the ongoing technical developments in the fats and oils industry. This will be accomplished by: discussing proposed regulatory policy for novel foods, the Guidelines for the Safety Assessment of Novel Foods (3), the relationship between Health Canada and Agriculture and Agri-Food Canada with respect to the Seeds Act, and regulatory considerations regarding the development of novel oils.
Regulatory Policy for Novel Foods

The development of regulations for novel foods, including the products of biotechnology, have been the subject of intense activity by many organizations and jurisdictions. The major documents that have resulted from these activities are identified in Table 9.1. Two types of developments can be identified: the establishment of criteria for the safety assessment of products of biotechnology that will be used as food; and the development of regulatory approaches to control the entry of novel foods into the marketplace. An example of the former activity is the Organization for Economic Cooperation and Development (OECD) document concerning the safely evaluation of foods developed by modern biotechnology. The proposed regulation for novel foods of the European Union is an example of the latter development, while the United Kingdoms Food Safely Act and Guidelines represent a voluntary approach to the regulation of novel foods. The regulatory developments that are taking place in Canada are similar to the international developments in that two distinct activities are being undertaken. The first activity is related to the development of regulations to require notification prior to the sale of novel foods. The second step is the development of guidelines for
TABLE 9.1
A Summary of Recent Developments for the Regulation of Novel Foods and Food Processes
Country or Organization United Kingdom Year 1990 Title The Food Safety Act, and Guidelines for the Assessment of Novel Foods and Processes. Source: ACNFP (4). Assuring the Safety of Foods Produced by Genetic Modification. Source: International Food Biotechnology Council (5). Strategies for Assuring the Safety of Foods Produced by Biotechnology. Source: WHO (6). Statement of Policy: Food Derived from New Plant Varieties. Source: FDA (7). Proposal for a Council Regulation on Novel Foods and Novel Food Ingredients. Source: Council of the European Communities (8). Safety Evaluation of Foods Derived by Modern Biotechnology. Concepts and Principles. Source: OECD (9).
International Food Biotechnology Council
1991
Food and Agricultural Organization, World Health Organization Food and Drug Administration European Union
1991
1992
1993
Organization for Economic Cooperation and Development
1993
the safety assessment of these products. The latter guidelines will be discussed in a subsequent section of this paper. The development of regulatory policies in Canada began in 1990 with the release of Information Letter (I.L.) No. 806 concerning novel foods and novel food processes. This I.L. proposed the development of regulations that would require notification prior to the sale or advertising for sale of novel foods and foods from novel processes, and identified the information requirements for such a notification. A definition was provided for the term novel food that was based on 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 a number of issues, including the definition of a novel food, equivalency to international 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 result of the I.L. and the ongoing consultations since its release. The actual text for the regulation is expected to be published for comment in Canada Gazelle, Part I during 1995. However, a description of the regulatory principles can be found in Volume I of the guidelines (3). These regulatory principles continue to require notification to the Food Directorate prior to the sale of a novel food, but now encompasses a revised definition for novel food, identifies the information requirements for a notification, and provides time frames for the review of a novel food notification. The proposed definition of a novel food is as follows: A novel food includes: Products and processes that have previously not been used as food or to process food in Canada; Food containing microorganisms that have not previously been used as food or to process food; Foods that result from genetic modification and exhibit new or modified characteristics that have previously not been identified in those foods, or that result from production by organisms exhibiting such new or modified characteristics; and Food that is modified from the traditional product or is produced by a process 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 notification. The basic requirements include the name under which the novel food will be sold; the name and address of the principle place of business of the manufacturer or importer, if applicable; a statement of the nature of the novel food, its process of manufacture, its intended uses, and history of consumption if used as food in another country; information about the possible displacement of existing foods, and the
nutritional impact thereof, as applicable; the written text of all labels to be used in conjunction with the novel food; and the name and title of the person who signed the notification and date of signature. Information demonstrating the safety of such products as food may be requested by the Director. Although this safety information would have to be provided at the request of the Director, it is anticipated that companies developing novel foods would address food-safety issues as part of ongoing research programs without the proposed regulation. Volume 1 of the guidelines also indicates that the notification should be completed within 90 days of receipt of a complete information package. However, the review period would be stopped should additional information be requested. The 90-day response time would be started over after receipt of the requested information. Safety information also may be requested after review of the product information is complete, should evidence become available indicating that there may 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 30 responses 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 into two volumes, with Volume 1 of the document providing a clarification of the proposed regulatory policies, as described previously. This volume also included decision trees that further identify those products that may be considered novel foods (3). Figure 9.1 contains the introductory decision tree that aids in identifying those substances that are addressed by other sections of the Food and Drug Regulations. Figure 9.2 clarifies those products of biotechnology that require notification. Additional decision trees are provided for identifying those foods from novel processes that may require notification and those cases when food additives may require notification as novel foods. Volume 2 of the guidelines addresses the safety assessment of genetically modified plants and microorganisms. The safety assessment criteria described in this volume encompass the concept of substantial equivalence that has been developed by the OECD (9). This concept embodies the idea that existing organisms used as food or as a source of food can be used as the basis of comparison when assessing the safety of the human consumption of a food or food component that has been modified or is new. The OECD document goes on to identify those points that must be taken into consideration to demonstrate substantial equivalence, and the difficulty in demonstrating equivalence as experience with the substance 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 the modified plant; information regarding the product; and, as needed, information
Fig. 9.1. Decision tree identifying those substances that are regulated under the authority of the Food and Drug Regulations and would not require notification as novel foods.
regarding dietary exposure, nutritional data and toxicological data. These requirements are summarized in Table 9.2. It should be noted that not all information will be required in all situations, and that scientific discussion of the issues may be suitable for addressing some concerns. These questions should be addressed in a stepwise fashion, until the safety concerns are addressed. The Food Directorate encourages developers of novel foods to come forward early in the development process so that both parties can better understand each others concerns, and to prevent the situation where unnecessary work is undertaken.
Fig. 9.2. Decision tree that identified those products of biotechnology that would 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 registration of such crops under the Seeds Act may appear to be an overlap of authority. To address this concern, Agriculture and Agri-Food Canada (AAFC) and Health Canada have cooperated to more clearly identify the activities of both organizations. An agreement has been reached that identifies the criteria for requiring a safety assessment of a genetically modified food, describes the roles of the two organizations, and provides time frames for completion of the foodsafety assessment. Those crops, including oilseeds, that are registered under the Seeds Act that will require a food-safety assessment include varieties that result from genetic engineering, or varieties where the breeding objective of the modification is the alteration of the historical compositional characteristic associated with the parent or related crop, or there is reason to suspect that the modification may have a negative impact on food safety or human health. This agreement also identifies the role of the two organizations. Agriculture and Agri-Food Canada will be the primary contact for the agricultural industry, and will receive the data necessary to conduct a food-safety assessment from the registrant. They are also expected to give equal consideration to any recommendation
TABLE 9.2
General Information Requirements for Genetically Modified Plants
Area Development and production of the modified plant. Information Required Host and donor organism. Modification process. Modified host. Methodology.
Product information Plants 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. 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. Information to indicate the amount of product that may be found in the diet, both in the general population, and target population. Macro and micro nutrient composition. Nutrient bioavailability. Laboratory animal studies (if necessary). Allergenicity considerations.
Plant products used in food.
Dietary Exposure.
Nutritional data.
Toxicological data.
regarding the safety of a new variety during its consideration for registration. Health Canada is expected to provide an objective evaluation of the available safety data for the edible portion of the new variety according to the Guidelines for Assessing the Safety of Novel Foods. It is also expected that the evaluations will 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 30 days of its receipt.
Novel Oils
The preceding discussion has addressed the major topics of concern related to the consideration of a modified oil or oilseed crop as a novel food. It should be realized that the characteristics of rapeseed oil have only been modified recently, by reduction of the erucic acid and glucosinolate concentrations, to produce canola oil and permit its sale as food. The alteration of the composition of existing oils can be expected to permit the expanded use of certain oils as food, or to modify the
composition of existing oils to better meet the needs of the food-processing and other industries. These modifications can be expected from the development of modified fatty acid compositions using conventional plant-breeding techniques or nucleic acid techniques to modify the plant, and the use of chemical and enzymatic methods to directly modify the fatty acid composition of the oil. These various approaches to modifying the fatty acid composition of oils will be viewed separately under the proposed novel food regulations. It is clear that a novel oil that is developed using nucleic acid techniques to modify the plant will require notification, whether the oil composition is substantially modified or not. On the other hand, if the modified oil is developed using traditional breeding approaches, notification would only be required if the oil composition was viewed as not being equivalent to that of the variety from which it was developed. The use of chemical or enzymatic esterification techniques to modify the fatty acid composition of an oil would result in the modified oil being considered a food from a novel process. As such, these products would also require notification. To date, the Food Directorate has not been approached regarding a notification for food from such a process. However, the Advisory Committee on Novel Foods and Processes (ACNFP) of the United Kingdom has evaluated products from two such processes and approved their use as food, subject to certain conditions specified by the ACNFP (10).
Conclusion
Over the last 2 years, the Food Directorate has come from developing the initial proposals for novel foods, to the point of having published guidelines for the safety assessment of novel foods, and is developing regulatory proposals that will be published in Canada Gazette, Part I in 1995. Thank you for your contributions to date, and I look forward to receiving your comments when the regulatory proposals are published.
References
1. GAO, Food Safety and Quality. Innovative Strategies May Be Needed to Regulate New Food Technologies, Report of the Chairman, Subcommittee on Oversight and Investigations, 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 American Agriculture, 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 Assessment of Novel Foods and Processes, Report on Health and Social Subjects, Department of Health, London, 1990, p. 30.
5. International Food Biotechnology Council, Reg. Toxicol. Pharmacol. 12 (1990), Part 2 of 2, p. 196. 6. World Health Organization, Strategies for Assessing the Safety of Foods Produced by Biotechnology, Report of the Joint FAO/WHO Consultation, Geneva, 1991, p. 59. 7. Food and Drug Administration, Statement of Policy: Foods Derived from New Plant Varieties; 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 Economic Cooperation 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 and four federal departments that have a role in their administration. I will give a brief overview of this as well as deal with some of the specifics of fat and fatty acid labeling, both concerning developments on the Canadian scene and events taking place internationally. I also intend to touch on other developments that will have a bearing on the labeling of foods, and particularly edible oil products, in the future. There are a number of matters to be concerned with when considering food labeling. These may be summarized as follows: 1. Adequate and accurate information is present to assist consumers in food choices relative to health, safety, and economic concerns. 2. Consumers and industry are protected from fraudulent or deceptive labeling, packaging, and advertising practices. 3. Fair competition and product marketability is promoted and maintained. The Department of Agriculture and Agri-Food Canada (AAFC) establishes basic labeling policy and requirements for all foods. It assumed these responsibilities when they were transferred from the former Department of Consumer and Corporate Affairs on June 25, 1993. The department administers these requirements at the manufacturing and import levels for foods other than fish and marine products. The Department of Fisheries and Oceans administers requirements for such products under the Fish Inspection Act. Further complicating the picture is the presence of the new Department of Industry that administers labeling requirements at the retail level of trade for those products for which the retailer has a direct responsibility, that is, retailer-packaged products. Completing the picture are the activities of Health Canada as related to health and safety in labeling matters. Health Canada look the lead role in developing the regulations and guidelines for nutrition labeling, and it established the criteria for such things as declaration of allergens. An example of this requirement is the recent regulatory amendment to require the specific listing of peanut oil when used as an ingredient in other foods. Although it was recognized that peanut allergens would not usually carry over into a refined peanut oil, the declaration was required as a precautionary measure.
The five Federal Acts that support departmental activities are as follows. 1. 2. 3. 4. 5. The Consumer Packaging and Labeling Act The Food and Drugs Act The Meat Inspection Act The Canada Agricultural Products Act The Fish Inspection Act
The Consumer Packaging and Labeling Act deals with basic labeling requirements for prepackaged products of all sorts. The bilingual requirements appear here as does the requirement to state a net quantity. The Food and Drugs Act is the basic act in Canada controlling matters of food safety and fraud. This legislation deals with such food-labeling requirements as ingredient listings and exemptions from ingredient declarations in specified cases. It deals with durable life information; common names; and with a substantial number 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 Fish Inspection Act contain product-specific labeling requirements associated with meat, poultry, fruit, and vegetable products; processed dairy products; maple products; honey; and fish products. Labeling can be a very powerful tool in product promotion. In addition to regulations on mandatory information, there are controls over the optional claims that manufacturers may want to apply to their labels. The department has a large body of policy and precedents that it relies on for rulings and interpretations in this area. A major reference document widely praised nationally and internationally is the Guide to Food Manufacturers and Advertisers. Currently it is undergoing a thorough review and revision.
Fat and Fatty Acid Labeling

Control over what may be said about fatty acids on food labels in Canada is rather strict. Specific mention may be made only for the following fatty acids or classes of fatty acids: linoleic acid, saturated fatty acids, monounsaturated fatty acids, polyunsaturated fatty acids, and cholesterol. If any information is provided for one, quantitative information in grams/serving is required for all of them. The restrictions mean that canola or sunflower oils that have had their oleic acid content increased through breeding may not be described on their labels using any reference to this increased oleic acid content. However, under current rules they may be described as high monounsaturated sunflower oil, for example. There is much current debate over the labeling of trans fatty acids. Some members of The Expert Committee on Fats and Oils have for some time advocated labeling of this class of compounds. Current regulations prohibit their mention. The
Health Protection Branch maintains that the evidence is not conclusive regarding the health 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 present in human adipose stores and the considerable efforts currently underway by industry on a number of fronts to reduce levels in edible oil products. I cannot help but conclude that labeling would be a significant factor to stimulate this action.
Additional Fatty Acid Claims

There were recommended nutrient intakes for both -6 and -3 fatty acids (1). Health Canada is of the view that more information on consumer use and understanding of fatty acid labeling is needed in order to develop meaningful criteria to guide regulation development. They have signalled their willingness to entertain applications for temporary marketing authorizations to test certain labeling schemes and generate consumer data. The regulation permitting temporary marketing is in place to allow products that do not conform to current regulations access to the market when information is needed to determine what the most appropriate requirements should be. It is not invoked to allow manufacturers to place products violating the normal regulations on the market to test viability prior to taking any necessary corrective action to achieve compliance. It is expected that there will soon be products carrying -6 and -3 fatty acid information on the market under this temporary marketing provision. In the case of trans fatty acid labeling, it is possible that this approach might be 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 acid claims in Canada. The claim low in saturated fatty acids may be made for food products that contain no more than 2 g of saturated fatty acids/serving and no more than 15% of energy from fatty acids. The claim lower in saturated fat compared to a reference 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 claims involving increased levels of polyunsaturates, for reduced levels of cholesterol, and for others. There are criteria to be met in speaking of reduced levels of fat itself, and so on. CanadaU.S. Harmonization Efforts A major initiative in all aspects of food control is that dealing with harmonization of regulations with the United States under the CanadaU.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 what might be considered a major set-back. On January 6, 1993 the U.S. published its final rules under the Nutrition Labeling and Education Act for nutrition labeling and for nutrient content claims. The U.S. accepted certain of Canadas recommendations for modification of their earlier proposals but in the end developed a scheme that significantly digressed from the Canadian system introduced in November 1988 and those of the European Union and the Codex Alimentarius Commission (the Romebased organization established to administer the FAO/WHO International Food Standardization Program). Canadas overall preferred position was to follow as closely as possible the international standards developed by the Codex. In March 1993, Health Canada and (the former) department of Consumer and Corporate Affairs issued a consultation document soliciting Canadian views on the U.S. requirements from a wide range of constituencies. The consensus was that Canada should retain its own nutrition-labeling requirements. It was generally felt that the U.S. system was too onerous and complicated. On the question of nutrient content claims, however, there was a consensus that 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 separate labels would be needed for each country, there would be no need to reformulate the product. Health Canada is currently assessing specific recommendations received on this subject.
Regulatory Review and International Developments

Health Canada and AAFC have recently completed Phase I of the review of the Food and Drugs Act and Regulations. In this consultation phase, a number of recommendations were made with respect to labeling. The most important of these involves the question of more specific and comprehensive lists of ingredients on food labels. Since 1976, all food labels with some exceptions, require a list of ingredients. Foods such as standardized alcoholic beverages and foods prepackaged by retailers are exempt. There is also a substantial list of foods that do not require 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 be declared as vegetable oil. The reason for this is to allow manufacturers freedom of substitution in the face of price shifts, availability, and other factors. At the international level, the Codex Committee on Food Labeling, at its October 1994 meeting in Ottawa, again discussed the issue of food hypersensitivity and the need for more complete disclosure of ingredient information. There has been a recommendation from the Nordic countries that the general labeling standard be amended to require declaration of components of ingredients if that ingredient is present 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 Nordic countries also developed a list of substances that should always be declared. Other delegations were of the view that the important issue is complete identification of substances known to cause severe adverse reactions regardless of level. The committees conclusion at this meeting was that countries needed to better develop their positions on 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 be fully examined and regulatory proposals developed. In terms of more comprehensive ingredient disclosure, the primary motivating force for change will be one associated with substances that cause severe adverse reactions. We are aware of the interest of certain segments of the edible oil industry in more specific declarations of vegetable oils. Primary producers tend to adopt this position with marketing considerations in mind. Processors tend to favor continued flexibility. The United States does require specific vegetable oil declaration. Phase I of Regulatory Review did not produce any strong position on further increased specificity in this area. If I could speculate on the outcome I would say that there will continue to be the opportunity to use the class name 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 fatty acid profiles of domestic oilseed crops for a variety of reasons. Genetic engineering is one of the techniques employed. The Canadian regulatory system is in the early 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 of views on whether labeling should be required. There were those who favored labeling 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 viability of 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 plant or animal, labeling should be considered. At the October 1994 Codex Food Labeling meeting in Ottawa, there was a polarity of national views on the matter. A significant number of countries favored labeling in all circumstances in which recombinant technology had been used. Others favored a more conservative approach, advocating labeling only when there might be a safety concern. Again, countries were asked to develop a more definitive national position on the subject for further discussion at the next Codex labeling meeting. The Department of Agriculture and Agri-Food along with its sister agencies is convening a workshop at the end of November this year to develop principles and to provide focus for further assessment of the issue. There are some fundamental issues that must be dealt with. Among these are the matters of right-to-know, and religious or ethical concerns. For those advocating complete labeling, do they mean
only in the case where the edible food portion has been modified through genetic engineering or would they extend labeling to foods from plant products that had been genetically modified for some agronomic feature, such as drought or herbicide resistance? Would they advocate labeling of meat or milk from cattle fed genetically modified fodder, or to which a veterinary drug made using recombinant 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 might be what type of information would be appropriate. If, for example, the composition of canola oil were significantly altered from the normal, and it was determined that labeling of this fact was in the public interest, would it be sufficient to signal the difference with a statement that genetic engineering had been employed. Some information on the altered fatty acid composition may be more appropriate. But, would consumers also 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 AAFC in October 1993. To overcome manufacturer and importer complaints that they had difficulty in determining where to get assistance with the design of their labels, a review service was established in 12 locations across Canada. Officers in these locations will provide advice on labels of all products, except those subject to the Fish Inspection Act. If consultation with officials in Ottawa or with Health Canada is necessary in the resolution of issues, such as those involving the propriety of particular claims, this will be done by officials in the office. The idea is one-stop shopping and a maximum 10 working day turn-around. The idea of sharing the cost of the departments inspection program is becoming popular. Whether it is done through negotiations with the various industry segments or through direct regulatory imposition, part of the inspection costs will be borne by industry in the future, on a sliding scale depending on the degree of private versus public good of the service, Label review and advisory services will not be exempt from this process.
Conclusion
We have learned that the activities and developments in the edible oil industry are very exciting indeed. Activities in the food-labeling business may be somewhat less dramatic but carry with them their own challenges. We are in an era of increasing consumer activism. Consumers are demanding more information and they are demanding a more active part in the decision-making processes that ultimately affect them. The trick will be to find a practical and equitable way to respond to consumer 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 place in an atmosphere of open, honest dialogue. The means will have to be found to keep pace with new technologies now taking place at all levels in the food industry. Organizations, such as The American Oil Chemists Society, can assist in this process.
Reference
1. Canadian Department of National Health and Welfare, Nutrition Recommendations, The Report of the Scientific Review Committee, Canadian Government Printing Centre, 1990, pp. 4052.
Chapter 11
Safety Evaluation and Clearance Procedures for New Varieties of Oilseeds in the United States and Canada
Donna Mittena, Keith Redenbaugha, and Julianne Lindemannb Calgene, Inc., 1920 Fifth, Davis, California, 95616; and bLindemann Consulting, El Cerrito, California, 94530, USA.
a
Introduction
There are common elements for the clearance of new plant varieties derived using recombinant DNA techniques. Canada and the United States require the evaluation of food, feed, and environmental safety. In this paper, we provide background on the basis of regulation in these two countries. We will discuss the issues that should be addressed for new varieties of oilseeds and, using Calgenes experience with modified-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 operating under a policy statement issued in May, 1992 (1). The policy outlined steps to be taken to determine the safety of foods derived from genetically modified plant varieties, and indicated that developers should confer with the FDA on issues of safety. Within the FDA, the Center for Food Safety and Applied Nutrition and the Center for Veterinary Medicine were consulted for modified-oil rapeseed food and feed products. The FDA has indicated that it intends to require premarket notification of foods derived from genetically modified plants, We expect that the amount of safety data Calgene will generate on any one oil will not be affected by a rule change. According to policy at Health Canada, food and feed products require formal review and approval. The criteria for evaluation Guidance Document was published in September 1994 (2). Health Canada is committed to a 90-day review period. Supporting data will be almost identical to that generated for the United States. Exact requirements are to be identified through consultation with Health Canada. The safety of livestock feed is also under the purview of the Feed Section within Agriculture and Agri-Foods Canada. Guidelines are under discussion for livestock feed. An advisory committee composed of experts from academia, industry, and government have convened to review safety issues and develop guidelines. There is agreement on the concerns about modified oils and meal. Three areas were identified by both Canadian and U.S. agencies:
1. Acceptable levels of nutrients, toxicants, and antinutritional factors; 2. Safety of transformation markers; and 3. Genetic characterization. Compositional Analysis Compositional analysis addresses the first point; acceptable levels of nutrients, toxicants, and antinutritional factors. Oil and meal derived from transgenic plants should meet the standards of products currently on the market, the standards of commerce. 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 antibiotic kanamycin. The FDA issued a Food Additive Regulation allowing the use of the kanamycin resistance gene product APH(3)II (aminoglycoside phosphotransferase) in cotton, tomato, and Brassica napus (3). Use of this gene product had been shown to not compromise antibiotic therapy in humans, and also it does not reduce stability of neomycin in animal feeds. APH(3)II is not toxic, it is not an allergen, and it is not active in the digestive tract where it is degraded. Calgene tested the stability of neomycin in animal feeds. Neomycin was mixed with fresh meal of two transgenic lines and the parent varieties. The meal was stored under conditions near the optimum temperature for enzyme activity. The meal was sampled for stability of the added antibiotic, and no differences were found between transgenic lines and their parents. The study concluded that the stability of neomycin mixed with seed meal was not diminished even after 56 days in storage (4). Likelihood of horizontal gene transfer to soil microbes was addressed by a probability model and found to not present a risk. Calgene addressed these issues by constructing models of gene transfer, using data from the published literature on in vitro transformation events and reasoned scientific judgment. Calgene did not attempt to directly measure gene transfer, because background level of kanamycin resistance in bacteria are fairly high and transformation events would be so rare as to be undetectable relative to the background. Based on the results of the evaluations, Calgene concluded that there will be no significant increase in exposure to kanamycin-resistant bacteria from consumption; at most 1 new kanr bacterial cell would be produced for every 750 billion that are already present in the human gastrointestinal tract (4). The potential for transformation of soil bacteria was also evaluated, by constructing a hypothetical model and using available published literature and reasoned scientific judgment. At most 1 new kanr bacterium would be produced via transformation for every 10 billion that are already present in the soil.
Genetic Characterization Definition of genetic change requires a description of the function of the inserted DNA. Evidence of stability is provided by the study of Southern hybridizations of DNA from several generations. The sequence of the DNA is defined and an open reading frame analysis is completed. A search of databases for homology with known allergens and toxicants is conducted. Environmental Safety The United States Department of Agriculture (USDA) examines environmental safety under the Plant Pest Act. Key elements of clearance involve environmental safety and absence of plant pest characteristics. Authority under the Plant Pest Act allows USDA to require permits for environmental introduction (field tests) of any organism that may be a plant pest. Use of the plant pathogen, Agrobacterium tumefaciens, during transformation places virtually all transgenic plants under USDAs authority. United States Department of Agriculture promulgated regulations in 1993 that allow it to issue a Determination of Nonregulated Status to exempt an organism from permit requirements after review of a Petition from the developer of the plant (4). Petition review emphasizes potential environmental effects and detrimental effects on nontarget, beneficial organisms. A separate determination is required for each phenotype. Approvals are specific to genetic constructs in specific plant lines. Additional lines may be added by amendment. A determination of nonregulated status allows commercial production without further oversight by the USDA. Agriculture and Agri-Food Canada (AAFC) is the lead agency responsible for the regulation of agricultural products of biotechnology. Before any genetically engineered plant can be grown uncontained, it must be evaluated for environmental safety. Assessment Criteria for such a determination were issued by AAFC in September 1994 (5). Under the Seeds Act, an agricultural crop variety to be sold in Canada must be registered. It must be recommended by the appropriate variety registration recommending committee, based on performance testing to show merit of the new variety during 3 years of trials. Field-testing data are required from locations where commercial production would take place. Environmental safety signoff will follow review of safety data by AAFC. Canada and the United States agree on the environmental safety issues for modified oilseed crops. An environmental safety evaluation should identify and examine potential significant impacts on natural and agricultural systems. Potential ecological effects to be evaluated for oil-modified rapeseed include 1. Introduction of new pests; 2. Worsening of an existing pest; and 3. Displacement of a naturalized plant community. Selective advantage is probably the most critical element of the risk assessment for 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 consequences of gene flow will be neutral. Relative Fitness Data generated in agronomic field tests can be used to directly compare characteristics of the transgenic and parent lines. Studies conducted in nonmanaged settings, that is, without common agronomic practices may be required to evaluate the potential for a crop or gene to move from a managed agricultural system into a natural community. The quantification of the net replacement value and ecological studies for the determination of persistence and invasiveness are parameters that may be used to measure the fitness advantage of the transgenic crop relative to its parent. For example, in Calgenes petition to the USDA for Laurate Canola (6), literature review and data were presented to address relative fitness. Field and controlled environment studies were designed to assess the probability of the establishment of feral populations of transgenic oil-modified canola lines relative to that of the parent(s) or other cultivars. Studies were also designed to pay special attention to seed germination and seedling establishment, the phase of the life cycle where changes in seed oil should show the greatest effect. Results of the studies have not identified any cause for concern. Gene Flow The potential for gene flow via pollen in any oilseed crop can be assessed beginning with a review of the botanical literature and floristic surveys. For example, description of pollen movement, and outcrossing to wild Brassica relatives, as well as, studies to identify the fate of hybrids with wild relatives were provided to the USDA in Calgenes Laurate Canola petition (6). Wild relative by crop hybrids show no difference in seed production in greenhouse crosses. Germination, seedling vigor, and dormancy tests of the hybrid seed showed the hybrid had neither the germination cuing nor the dormancy characteristics of its wild maternal parent.
Conclusions
Although the regulatory oversight of new plant varieties of oilseed crops has a differing legal basis, we see few differences in the issues identified and the types of data required for safety evaluation in Canada and the United States. As we come to the end of 1994, the policy for safety review is in place and is being used to complete the safety assessment of the first modified oil products derived from recombinant DNA techniques for commercial sale.
References
1. Food and Drug Administration, Statement of Policy: Foods Derived from New Plant Varieties, Federal Register 57 (104): 2298323005, Washington, D.C., May 29, 1992.
2. Health Canada, Guidelines for the Safety Assessment of Novel Foods, Vols, I and II, Food Directorate, Health Protection Branch, Ottawa, 1994. 3. Food and Drug Administration, Secondary Direct Food Additives Permitted in Food for Human 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 and Products; Notification Procedures for the Introduction of Certain Regulated Articles; and Petition 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 Biotechnology Release Regulations under the Seeds Act, D94-03. Plant Industry Directorate, Ottawa, 1994. 6. United States Department of Agriculture, Availability of Determination of Nonregulated Status 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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Copyright 1995 AOCS Press