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BIOTECHNOLOGICAL DEVELOPMENTS: POTENTIAL FOR IMPROVEMENTS IN FOOD FORMULATION NUTRIENT DELIVERY, AND SAFETY John E. Kinsella This overview describes some biotechnological developments in the food production and processing sector, with emphasis placed on nutrients, and discusses these in the context of optimizing diet composition. Most nutrients consumed in the United States are derived from foods that have been processed to some extent. The food processing industry is immense, with retail sales of foods approaching $400 billion in 1987, and becoming increasingly sophisticated while it undergoes consolidation via mergers and acquisitions. Currently, some 50 companies account for approximately 60% of consumer food sales in the United States (Messenger, 1987a). The modern food processing industry increasingly influences what consumers eat, that is, nutrient intake, although the availability of many products is largely determined by what the majority of consumers buy. Because overall domestic market expansion is limited by population growth to only about 2% per annum, food companies must be very competitive to be successful (Behnke, 1983; Pehanich, 19871. Hence, progressive food companies must be responsive to consumer concerns and needs in order to capture a larger market share, while at the same time they must adopt new technologies to cut costs and facilitate innovation. Nutrition has therefore become an integral criterion in product development, and the current problem areas of nutrition are of interest in terms of developing consumer products that provide a more balanced array of nutrients. This interest encompasses all facets of food from producer to consumer 42 .

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NUTRITIONAL STATUS OF THE AMERICAN POPULATION Vital statistics indicate that the American population is generally healthy and well nourished but aging, with overt symptoms of nutritional deficiencies being relatively rare (Harper, 1987; McGinnis, 1987~. However, a number of diet-related chronic degenerative diseases that reduce the quality of life afflict a significant number of people and cause extensive morbidity and mortality (McGinnis, 1987~. The major contemporary nutrition-related health problems include obesity, hyperlipidemia, hypertension, coronary heart disease, atherosclerosis, thrombosis and stroke, diabetes, arthritis, and cancer. Genetic factors may play a complicating role in many of these health problems, for example, hypertension and hyperlipidemia (Motulsky, 1987~. According to the Joint Nutrition Monitoring Evaluation Committee, many of these nutrition-related health problems may result from cons,,mption of excess calories, fat, saturated fatty acids, cholesterol, and sodium. There is evidence that certain subgroups of the population (especially young females) may consume inadequate levels of iron and vitamin C and that dietary calcium may be deficient, resulting in osteoporosis in older women (Federation of American Societies for Experimental Biology, 1984; National Institutes of Health, 1986a). Data summarizing the incidence of nutrition-linked diseases in various segments of the U.S. population and the magnitude ot the excesses or inadequacies in intake of nutrients (compared with the generally recommended levels) have been collated recently (NRC, 1988~. The relationships between dietary fats and several chronic diseases have been reviewed (NRC, 1989 Visek, 1983~. _ Coronary Arterial Diseases ; Perkins and Hyperlipidemia is associated with an increased incidence of atherosclerosis, heart disease, and thrombosis and stroke, which are the major causes of morbidity and mortality in the United States (American Heart Association, 1986; Levy et al., 1979~. When excess calories are consumed as fat, especially as saturated fatty acids (SFAs), low-density lipoproteins (LDLs), which exacerbate these diseases, are increased (Keys, exacerbate these diseases, are increased (Keys, 1970; 43

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Levy et al., 1979; Shaefer and Levy, 1985~. Reduction of SFAs or their substitution by polyunsaturated fatty acids (PUFAs) results in decreased plasma cholesterol and reduced incidence of heart disease (American Heart Association, 1986; Federation of American Societies for Experimental Biology, 1984; Keys, 1970~. Current intake of SFAs are about 13-14% of calories (National Institutes of Health, 1985~. A reduction in total fat, SFAs, and cholesterol intake has been recommended consistently (Federation of American Societies for Experimental Biology, 1984~. Obesity Increased consumption of fat and sugar are associated with increased obesity and about 30% of the U.S. population between 27 and 74 years is overweight (National Center for Health Statistics, 1983~. Obesity may adversely affect health and longevity and is frequently associated with hypertension, hypercholesterolemia, non-insulin-dependent diabetes, increased incidence of certain cancers, and other health-related problems (National Institutes of Health, 1985~. A reduction in caloric intake, especially fat, is recommended for people who are 20% or more overweight (National Institutes of Health, 1985~. Non-insulin-dependent diabetes (insulin resistance) is frequently associated with obesity. In rats, dietary PUFAs of the n-6 family (safflower oil) reduced insulin potency, whereas dietary n-3 PUFAs in fish oil potentiated responsiveness to insulin (Storlien et al., 1987~. This observation may be relevant to human diabetes and suggests a role for dietary n-3 PUFAs in modulating the insulin receptor. Cancer Diet plays an important role in cancer, the second major killer disease in the United States (Pariza and Simopolous, 1987~. There is a strong correlation between caloric intake, especially from fat, and the incidence of many common cancers (American Cancer Society, 1984; Doll and Peto, 1981; Wynder, 1976~. Available evidence suggests that fat intake may be more relevant and 44

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variable than calorie intake (National Research Council, 1982; Willett and MacMahon, 1984~. In addition, there appears to be an association between unsaturated fatty acids, especially _-6 PUFAs, and growth of certain tumors. This may be partly related to increased production of the prostanoid PGE2, which can exert immunosuppressive effects. In animal studies this is counteracted by n-3 PUFAs, which also reduce the growth of certain tumors (Karmali et al., 1987; Welsch, 1987~. This underscores the potent and pervasive physiological effects of dietary n-6 unsaturated fatty acids via eicosanoids and the need to recognize that in making recommendations to reduce the risk of one particular disease that others are not being exacerbated. Hypertension Hypertension afflicts approximately 20% of the population and has genetic, environmental (stress), and dietary etiologies. Excess dietary sodium has been strongly implicated (Federation of American Societies for Experimental Biology, 1979~. Consumption of sodium varies widely, with average intakes generally exceeding - the recommended daily intake of 3 g (Federation of American Societies for Experimental Biology, 1979~. - Because of the linkage between sodium intake and hypertension, efforts to reduce the use of sodium in foods are being made. Sodium chloride enhances flavor perception (Gillette, 1985), but in addition, it performs a number of important functions in conventional food processing (Dunail and Khoo, 1986~. In meats, dairy products, and soups, it controls microbial growth and may be a selective inhibitor of pathogenic or toxigenic microbes; in cheeses, it affects the activity of ripening enzymes and controls the microflora; and in processed meats, it functions in solubilizing myofibrillar proteins, which are required in the formation of the final product (Federation of American Societies for Experimental Biology, 1979~. Hence, sodium chloride cannot be summarily replaced or substituted. However, its use in processed foods can be reduced, and potassium chloride (which imparts a bitter taste to foods) can be substituted in limited amounts (<25%~. The reduction of sodium chloride in certain foods can be facilitated by 45

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adding herbs, spices, and other strong flavors such as monosodium glutamate. Certain hydrophobic dipeptides possess a salty taste. The dipeptides ornithine-glycine, lysine-glycine, ornithine-~-alanine, and ornithine-y -aminobutyric acid are approximately twice as salty as sodium chloride, while ornithine-taurine and lysine-taurine are equivalent to salt (Tada et al., 1984~. These dipeptides warrant further study as possible substitutes for salt in the diet of subjects with hypertension. DIETARY FAT AND FATTY ACIDS The genetic basis of human nutrient requirements evolved millions of years ago, presumably reflecting the nutrients available during the evolution of metabolic pathways (Eaton and Konner, 1985~. The adaptability of the human metabolic system to dietary changes is considerable, but it has limits, as evidenced by the need for around 45 essential nutrients. While the metabolic system has built-in feedback regulators, they can be overridden; it is conceivable that excesses or imbalances in the intake of certain nutrients can perturb the system and induce pathophysiologies to which humans are predisposed. Leaf and Weber (1987) suggested that relatively recent dietary changes (especially the increased fat intake and changes in types of fatty acids) exceed the biochemical (genetic) capacity of the system to adjust, and consequently, they are conducive to some of the familiar chronic diseases discussed above. These degenerative diseases become more obvious with increased longevity and are exacerbated by dietary imbalances. With the reduction in manual labor, much less dietary energy is needed; hence, less fat is required in the diet (American Heart Association, 1986~. In addition, with the elucidation of the important roles of eicosanoids in a number of chronic inflammatory and immune diseases, a reassessment of the quantitative importance of dietary _-6 PUFAs in the human diet may be warranted (Lands, 1986a,b). A number of chronic diseases (arthritis, asthma, psoriasis, allergies, and immune and inflammatory diseases) that afflict an enormous number of Americans, 46

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especially older people, may be affected by dietary components, and there -is accumulating evidence that dietary PUFAs may directly;and indirectly affect the etiology and severity of these diseases by influencing the production of eicosanoids,'both prostanoids (PG) and leukotrienes (LT) (Table 1~. High intakes of n-6 PUFAs , , 1 . TABLE 1 Some Diseases Associated with Disturbances in Eicosanoid Metabolism . Disease ~Eicosar~oida Cell or Tissue Hyperaggregatabili~y TXA2 Platelets Increased adherence TXA2 Platelets, of platelets macrophages Excess bleeding or PGI2, PGI3 Blood bruising Immunosuppression PGE2 Macrophages Asthma TXA2, LTs Lungs Inflammation LTs, PGE2 Polymorphonuclear i . leukocytes Psoriasis . . LTs, HETEs Skin Rheumatoid arthritis PGE2, LTs Monocytes Diabetes TXA2 Platelets .. . Autoimmune disorders PGE2,LTs Spleen Hypercholesterolemia TXA2 Platelets Chronic placental insufficiency PGI2 Umbilical cord aTXA2 - thromboxane; PG - prostaglandin; LTs leuRotriene; HETE ~ hydroxyeicosatetraenoic acid. .: .. . ~ , , may increase the"severity of some of these diseases, while consumption of n'-3 PUFAs may ameliorate them (Lands, 1986a,b, 1987~. The current high'consumption of n-6 PUFAs is a relatively recent phenomenon reflecting innovative oilseed processing technology and the promotion of _-6 PUFAs in high-fat diets to reduce plasma cholesterol. Historically, mankind was accustomed to a 47

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relatively low-fat diet rich in both ~-3 and n-6 PUFAs, mostly from plant foods (Crawford, 1987; Leaf and Weber, 1987~. Hence, the contemporary diet in which PUFAs are preponderantly of the n-6 family may be predisposing the system to a hypersensitive - proinfl~matory state by synthesis of excessive eicosanoids (Lands, 1986a,b). Some dietary ~-3 PUFAs may be desirable to modulate the metabolism of n-6 PUFAs and to down-regulate eicosanoid synthesis (German et al., 1988; Kinsella, 1987b; Lands, 1986a,b, 1987~. The apparent beneficial effects of dietary n-3 PUFAs of fish oils on numerous parameters (Table 2) are consistent with this (Lands, 1987; Simopoulos et al., 1986) and suggest that n-3 PUFAs from green leaves and seafoods should be included in the diet (Kinsella, 1989) ! TABLE 2 Some Common Diseases That May Be Ameliorated by Dietary n-3 PUFAs Arthritis Atherogenesis Autoimmune diseases Burns Hyperlipidemia Ischemic heart disease Thrombosis Vasospasm (Asthma?) Atherosclerosis Blood pressure resistance Diabetes (insulin) Inflammatory disease Psoriasis Tumor growth The minimum requirement for Iinoleic acid to prevent deficiency symptoms and apparently to provide ample arachidonic acid for normal eicosanoid synthesis is 1 to 2% of calories (Lands, 1986a). Hence, the current intake of linoleic acid (6 to 7% of calories) may be overtaxing to the human system. Because n-3 PUFAs are effective hypolipidemic agents and down-regulate eicosanoids, a mixture of n-6 and n-3 PUFAs may be desirable and perhaps should be present at levels below the Currently 48

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recommended 10% of dietary calories. Low-fat diets containing both n-6 and _-3 PUFAs typical of vegetarian diets may be more representative of the desirable pattern of dietary PUFAs, especially as total fat intake is reduced (Crawford, 1987; Dyerberg, 1986; German et al., 1988; Leaf and Weber, 1987~. The gradual elucidation of the role of the immune system in health and disease and the potential role of food components in modulating the immune system will become of increasing importance to food scientists and nutritionists (Chandra, 1985; Pestka and Witt, 1985~. Recent research demonstrating the key roles of eicosanoids in intercellular signaling and modulation of lymphocyte, monocyte, and neutrophil functions strongly implicates dietary PUFAs in immunocompetence (Chandra, 1985; Kunkel and Chensue, 1986~. The increased tendency of macrophages to produce more immunosuppressive PGE2 in animals fed increasing amounts of n-6 PUFAs is significant (German et al., 1988~. The capacity of n-3 PUFAs of fish oils to suppress eicosanoid synthesis in cells of the immune system needs to be studied more extensively as n-3 PUFAs are introduced into foods (Kinsella, 1989~. Fat Consumption The current intake of fat is about 38% of calories, down from approximately 41% in 1977. Food intake data indicate that this is made up of approximately 15, 14, and 7% of calories from saturated, monoenoic, and polyunsaturated fatty acids, respectively. Actual consumption of dietary fat may be about 85 to 90 g/day (Rizek et al., 1983~. Approximately 50% is from animal sources, with 25, 12, 9, 5, 3, and 0.7% being derived from red meat, dairy foods, butter, lard, poultry, and seafoods, respectively. The remainder is derived mostly from salad, frying, and cooking oils; shortenings; spreads; and fat consumed as components of various prepared foods, for example, bakery products, fruits, and vegetables. Approximately 30, 25, 35, and 5% of saturated fatty acids are derived from dairy foods, meat, vegetable oils, and eggs, respectively. Dietary cholesterol is obtained preponderantly from animal (30% meat, 15% dairy) and avian products, with eggs being the major source (NRC, 1988). 49

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Data concerning quantitative intake of dietary fat and fatty acids, however, are questionable, and many calculations are based on supply, which overestimates intake. Data on muscle foods tend to be for raw untrimmed products and tend to overestimate the intake of animal fats and cholesterol. On the other hand, deep-fried foods (e.g., French fries, fish sticks, and doughnuts), and especially breaded products, are major sources of fat and contain up to 20 to 30% fat by weight (Kinsella, 1988~. In light of the association of dietary fat with many of the major chronic diseases, there is universal agreement that dietary fat intake should be reduced to match energy output and should not exceed 30% of total calories (Federation of American Societies for Experimental Biology, 1984; National Institutes of Health, 1986b; NRC, 1989; DHHS, 1988~. In addition, the fatty acid composition of dietary fat should meet certain guidelines. Thus, saturated fatty acids should not exceed 10% of total calories and PUFAs (not defined) should be included but should not be more than 10% of total calories. The remainder should be composed of monoenoic fatty acids. In addition, cholesterol intake should be 100 mg/1, 000 kcal (<300 mg/day), sodium intake should be <3 g/day, and dietary fiber intake should be about 25 g/day (National Institutes of Health, 1986b). To meet the guidelines (less fat, cholesterol, and saturated fatty acids), all actual and potential sources of dietary fat need to be considered. Thus, traditional commodity foods, meats, dairy foods, contemporary fabricated foods, franchise foods, prepared meals, shortenings, spreads, and fried foods should be examined in the context of reducing their dietary fat levels and modifying their fatty acid compositions. The replacement of SFAs and perhaps some _-6 PUFAs with hypolipidemic monoenoic fatty acids (Grundy, 1987) and the inclusion of _- 3 PUFAs as a partial replacement for n-6 PUFAs may be desirable (Kinsella, 1988b). With respect to nutrients or food components, the tendency to single out a specific compound and overemphasize its importance or overdramatize its potential danger results in confusion among the public. The necessity of consuming a range and variety of foods from different sources is generally recognized. However, 50

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~. ~ . the emphasis on dietary cholesterol and saturated fat and the concomitant proscription of animal products as' sources of cholesterol and SFAs (which is not valid for such foods as trimmed meats, milks, and yogurt) may - contribute to problems emanating from inadequate iron, calcium, and vitamin B:2 intakes in certain''subgroups of the population. A balanced comprehensive perspective is important when dietary recommendations are made in the context of ameliorating chronic degenerative diseases that may be only marginally responsive to dietary modifications. Because animal products 'provide significant quantities of essential nutrients, appropriate modification of their fat content is a practical and prudent approach (Briggs, 1985~. APPROACHES FOR MODI FYING FATS IN FOOD PRODUCTS Reduction of the fat and cholesterol contents of foods and modification of the fatty acid composition is the goal of a number of current production and processing technologies in conventional agriculture that are being facilitated by developments in biotechnology. Animal Products Establishe'd"'pract~ces and pricing systems have traditionally placed a premium on fat production in animals for both meats and milk. In response to nutritional and marketing pressures, _ however, the situation is changing, and producers~and researchers are exploring various alternative approaches (Table 3) for reducing the'fat content of animal products. . . . . . The fat content of animal tissues can be reduced by restricting energy (starch) in the diet, especially during the"'finishing period and by selecting for leaner strains of'animals. selection for leaner animals, new techniques for measuring body composition and fat levels are important. Thus, ultrasonic methods, X-ray-based computerized automated tomography, nuclear magnetic resonance imaging, and total body electrical' conductivity are modern nondestructive methods being evaluated for sire selection and for determining the fat content and market readiness of animal's (NRC', 1988~. ~ To facilitate these measures and 51

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TABLE 3 Some New Technologies That Provide Options for Producing Leaner Meats Strain selection Nutritional plane or finishing diets (less starch) Noncastration of male animals Growth hormones (somatotropin) ,B-Agonists (epinephrine analogs) Immunological suppression of adipocytes, growth inhibitors, etc. Transgenic animals Advances in the knowledge of factors affecting muscle growth and partitioning of nutrients are being explored for use in altering the composition of animal products. Adrenergic amines, especially those that bind to f-receptors (~-agonists), mobilize fat from adipose tissue; enhance its oxidation; and favor the deposition of muscle proteins in swine, beef, sheep, and fowl. Analogs of {-agonists or repartitioning agents are effective in increasing muscle mass and reducing fat deposition by directing nutrient flow to muscle growth (Dalrymple et al., 1986~. The administration of somatotropin or growth hormone to animals markedly enhances muscle growth (Chung et al., 1985) and significantly increases milk yields in cows (Bauman et al., 1985) e These new agents are being investigated intensively, and bovine growth hormone is being produced routinely by recombinant DNA techniques. In addition, immunological techniques are being explored; for example, the generation of antibodies that might bind biologically active molecules such as somatostatin (ST) and that might allow somatotropin to 52

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1 i' that stimulate the activity of the sodium pump can elicit the sensation of sweetness. The growing knowledge of receptor mechanisms should accelerate developments in designing new no-calorie condiments and flavors. ., CONCLUSIONS .- , .. .; Improvements in the diet should ensue from the numerous developments that are occurring in molecular biology, food production technologies, fermentations, and food processing technologies. Technical advances will come at an accelerated pace, but their exploitation and application may only develop at a nlegp1 papas (i.e., slowly) unless the benefits of innovation are clearly communicated to the public and legislators and appropriate actions are taken to rationalize regulations and expedite their execution. ,} . ' . '? It should become more feasible provide optimum amounts of nutrients in high quality food products. However, continued research and closer attention to food safety, particularly the role of microbes and their products in the etiology of both acute and chronic diseases, is justified. The potential long-*erm effects of these products on the immune system and chronic diseases need more attention. The burgeoning developments in biotechnology, foods, and nutrition (Figure 2) underscore the integrated effort that is required in developing nutritious and safe .. . ~Ct3LTURE go .. . ~ FOOD PRICING to AND FaRM~TION 1 r - r I ' ^';;~' - - I REm4RTUAI" non 1 ~ I a ~ I `. ., Processing addicts ' I~redlents TatIored functional' components Fat ~Dodification Flavors Enzymes Colors Elimination of deleterious its Functional analogs Safety . ~AND '' BIO1~OIDGY ~' . Vernon ~ - ~ ~ v - . ^~. les4~nt`t,,,,a,L ~D,O acids I PUBLIC BEAL~ i ~Poly,saccharide 'Sweeteners ,, ,j Fibers ~ Ath-roscl; rosis , - !,~,` ~4 ,~, maturated ~`ty acids Cancer Saris Clbesity - ~ Cboles~iirol Infections lmtiDutricuts Foodbo~e dlaeas-e - '` Costs JUler~lea/senattivitles Nutrient l~ibalancea ~; FIGURE 2 Sch-ec howis~6 the aver ~ Mach bloteichnolo~r $~-.h~ ,.- Act on floriculture, food processl~u, nutrition Ad public health Ida 'food Ad nutrient Manipulation In addition genetic ~6iDe-r=6 can hav an Evict, on public health via gene therepgr Ad the production of vacclnea, therapeutic vents, and drugs Improvements day involve production of desired components or el4~4neSios~ of unwanted components by applying modern blotechnolotica1 techniques to food production and processing ., 84

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products and dramatize the innovations that are necessary in the education system to provide the appropriate and balanced training of scientists in the area of food science, nutrition, safety, diet, and public health. Research support to foster collaborative research in these areas is needed. The existence in academia of discrete departments that are each concerned with such areas as production, food processing, nutrition, and dietetics creates an atmosphere of reductionism and separateness and does not foster a complementary interdependence of these disciplines as they impinge on food, nutrition, and health. Finally, these developments may provide more options for eliminating hunger and malnutrition around the world. Implementation of new developments requires commitment, a sense of balance in priorities, and humanitarian motivation in providing aid and training for those less fortunate around the world. REFERENCES American Heart Association. 1986. Dietary guidelines for healthy adult Americans. Circulation 74: 1465A. American Cancer Society. 1984. cause and prevention. special report. Nutrition and cancer, An American Cancer Society Ca--A Cancer J. 34~2~. Anderson, S., C.B. Marks, R. Lazarus, J. Miller, K. Stafford, J. Seymour, D. Light, W. Rastetter, and D. Estell. 1985. Production of 2-keto-L-gulonate; an intermediate in ascorbate synthesis by genetically modified Erwinia herbicola. Science 230:144. Batt, C. 1986a. Use of recombinant DNA to improve lactic acid starter cultures. P. 151 in Proceedings Bio-Expo 1986. Butterworth, Stoneham, Mass. Batt, C. 1986b. Genetic engineering of Lactobacillus. Food Technol. 40:95. Bauman, D., J. Eisenmann, and B. Currie. 1985. Responses of high producing dairy cows to long term treatment with pituitary somatotropin and recombinant somatotropin. J. Dairy Sci. 68:1352. 8ehnke, J. 1983. Growth in a non-growth industry: Expanding our horizons. Food Technol. 37:22. Best, D. 1987. Microwave formulation: A new wave of thinking. Prepared Foods 70:70. 85

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Blundell, T., and M.J. Sternberg. 1985. Computer aided design in protein engineering. Trends Biotechnol. 3:229. Bradley, A., M. Evans, M.K. Kaufman, and E. Robertson. 1984. Nature 309:255. Bravo, J., and D.A. Evans. 1985. P. 193 in J. ed. Plant Breeding Reviews, Vol. 3. Briggs, G.M. 1985. Muscle Foods and Human Health. Food Technol. 38:54. Brinster, R.L., and R.D. Palmiter. 1986. Harvey Lect. 80:1. Bruening, G., J. Marada, T. Kosuge, and A. Hollander. 1987. Tailoring Genes for Crop Improvement. Plenum, New York. ~ Chandra, R. 1985. Effect of nutrient deficiency and excess on~3immune response. Food Technol. 39:91. Chassy, B. ;1985. Prospects for improving economically significant Lactobacillus strains by genetic technology. Tibtech 3:273. Chung, C.S., T. Etherton, and J. Wiggins. 1985. Stimulation of swine growth by porcine growth hormone. J. Anim. Sci. 60:118. Clark, A.J., P. Simons, I. Wilmut, and R. Lathe. 1987. Pharmaceutical from transgenic livestock. Tibtech 5:20. Cocking, E., and M.R. Davey. 1987. Gene transfer in cereals. Science 236:1259. Crawford, M.A. 1987. The requirements of long chain n-6 and n-3 fatty acids for the brain. In W. Lands, ed. Polyunsaturated Fatty Acids and Eicosanoids. American Oil Chem. Society, Champaign, Ill. Dalrymple, R., P. Baker, D. Ingle, J. Pensack, and C.A Ricks. 1986. A repartitioning agent to improve perfo~mance and carcass composition of broilers. Poult. Sci. 63:2376. Daniels, I., S. Rizvi, M. Black, and B. German. In press. Supercritical fractionation of herring oil. Food Sci. de la Pena, A., H. Lorz, and J. Schell. 1987. Transgenic cereals by direct injection of DNA into plants. Nature 325:274. DHHS (Department of Health and Human Services). 1988. The Surgeon General's Report on Nutrition and Health. DHHS (PHS) Pub. No. 88-50210. Public Health Service, U.S. Department of Health and Human Services. U.S. Government Printing Office, Washington, D.C. 712 pp. 86

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Doll, R., and R. Peto. 1981. The causes of cancer. ' Quantitative estimates of avoidable risks of cancer in the United States today. J. Natl. Cancer Inst. 66:1191-1308. Dunail, G., and C.S. Khoo. 1986. Developing low and reduced sodium products. Food Technol. 40:106. Dyerberg, J. 1986. Linolenate derived polyunsaturated fatty acids and prevention of atherosclerosis. Nutr. Rev. 44:125. Dziezak, J. 1986. Sweeteners and product development. Food Technol. 40:114. Eaton, S.B., and M. Konner. 1985. Paleolithic nutrition: A consideration of its nature and current implications. N. Engl. J. Med. 312:283. Ecker, J.R., and R.W. Davis. 1986. Inhibition of gene expression in plant cells by expression of antisense RNA. Proc. Natl. Acad. Sci. USA 83:5372. Edens, L., and H. Van der Waals. 1983. Microbial synthesis of sweet tasting plant protein thaumatin. Trends Biotechnol. 3:61. Erickson, D. 1980. Handbook of Soy Oil Processing and Utilization. American Oil Chem. Society, Champaign, Ill. Evans, D. 1983. Plant cell culture: Potential. Biotechnol. 1:253. Evans, D. 1985. Handbook of Plant Cell Culture, Vol. 4, Macmillan, New York. Evans, J.W., and A. Hollander. 1986. Genetic Engineering of Animals. Plenum, New York. Evans, D., and W.'Sharp. 1986a. Applications of somaclonal variation. Biotechnology 4:528. Evans, D., and W. Sharp. 1986b. Potential applications of cell culture in biotechnology. S. Harlander and T. Labuza, eds. Food Processing. Noyes, Park Ridge, N.J. Evans, D., and R.J. Whitaker. 1987. 'In D. Knorr, ed. Food Biotechnology. Marcel Dekker, New York. Federation of American Societies for Experimental Biology. 1979. Evaluation of the Health Aspects of Sodium Chloride and Potassium Chloride as Food Ingredients. Pub. No. PB 298139. Life' Sciences Research Office, Federation of American Societies for Experimental Biology, Rockville, Md. Federation of American Societies for Experimental Biology. 1984. Assessment of the Nutritional Status of the U.S. Population Based on Data Collected in the Second National Health and Nutrition Examination Survey, 1976-1980. Life Sciences Research Office, 87

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