Frontiers in the Nutrition Sciences: Proceedings of a Symposium (1989)

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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 .

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

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

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

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

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

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

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

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

~. ~ . 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

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

function continuously is being explored in cattle by using autoimmunization with conjugated ST (Spencer and Mallett, 1985~. Also being evaluated is the feasibility of immunizing animals against preadipocytes to prevent their differentiation into mature adipocytes and thus to reduce fat deposition. Further research to characterize endocrinological and humoral peptide regulators of cell metabolism and growth should provide new methods for manipulating animal growth and controlling body composition. Transgenic animals that contain exogenous functional genes for desirable functions or antisense RNA for unwanted functions may be possible in the future. The concept of incorporating genes coding for growth-promoting agents, for example, somatotropin, or antisense genes (Green et al., 1986) to fatty acid synthetase or acyltransferase into animals warrants investigation. Such developments (Greenberg, 1987) would be compatible with current methods for both meat and milk production. The intensive methods used for poultry production favor the fast-growing lean birds that contain about 15% (mostly subcutaneous) fat that is easily removed during processing or cooking or prior to consumption. The fatty acid composition of poultry and swine reflects dietary fatty acids, and thus can be manipulated to provide a more appropriate fatty acid composition for human consumption (NRC, 1988~. Aquaculture (e.g., of catfish, trout, Atlantic salmon, tilapia, crayfish, and shrimp) is a burgeoning new technology that is providing increasing quantities of lean muscle foods with a desirable lipid content and fatty acid spectrum. Dietary manipulation can increase the n-3 PUPA content of cultured fish (Kinsella, 1988~. Postharvest Modification of Animal Products Beef cuts may contain from 5 to 20% fat, depending on the cut and grade. Research has shown that the palatability of beef cuts containing from 3 to 7% fat is equally acceptable (NRC, 1988), suggesting that much of 53

the fat can be trimmed from prime cuts without a loss in quality. Trimming can be most easily accomplished at the time of slaughter (hot trimming). This is undesirable for the producer or processor, however, because it would alter the yield, and under current regulations of the U.S. Department of Agriculture (USDA), a carcass must have a yield grade to receive a quality grade. If a carcass is trimmed at slaughter, it is disqualified from receiving yield and quality grades. Because hot trimming can remove 80 to 90% of the fat and provide a leaner cut, the American Meat Institute has petitioned the USDA to allow, at the buyer's option, the separation of the yield and quality grades for beef. Approval of this now provides more lean mean cuts for consumers. The meat industry is currently providing a much wider selection of trimmed, low-fat meat cuts and is combining this with an informative educational effort on the nutrient contents of different cuts of meat. Several advances in meat processing technology are facilitating fat reductions. These include the successful development of low-fat, restructured meat products (e.g., turkey hams). These are made by vigorously mixing pieces of meat in the presence of salt to solubilize the myofibrillar proteins that act as adhesive agents and bind the pieces together in an appropriate mold. Other proteins (e.g., egg white) can be added, and polysaccharides (e.g., alginates) can be used to improve adhesion and reduce the salt levels in re-formed meat products (Mittal and Usbonne, 1985~. Surimi (a crab meat analog) is a gelled, restructured muscle food made from insoluble myofibrillar proteins, usually from fish (pollack), by "elation in the presence of binder proteins, starch flavors, and sorbitol. This low-fat product (<1% fat) can be colored and flavored to simulate a range of seafood products (Lee, 1986~. Comminuted and processed meat products (e.g., hamburgers and frankfurters) tend to be high-fat products, but there is interest in reducing the fat levels in these products. Advances in the knowledge of meat emulsions and gels are facilitating the formulation of acceptable products with reduced fat and increased amounts of water or in which fat is partly replaced by polysaccharides or other proteins (Kinsella, 1987a). 54

Milk Milk has approximately 3.5% fat, of which 0.2% is cholesterol (approximately 10 mg/100 ml). The fat exists in globules and can be readily removed by centrifugal separation (Webb et al., 1974~. This is now routinely practiced to reduce the fat content of fluid milks and products manufactured therefrom. Most fluid dairy products have relatively low amounts of fat and cholesterol. Traditional hard cheeses (Cheddar and Colby' for example) contain about 30% fat, and dairy creams represent products with relatively high amounts of fat. Research to develop low-fat cheeses is being pursued; however, it is a major challenge to simulate the flavor, texture, body, and mouth-feel of normal cheeses. More information concerning the physical and chemical contributions of milk fat to cheese quality is needed in order to produce high-quality, low-fat cheeses. With respect to cultured dairy products (cheeses, yogurts, sour creams, etc.), the potential to reduce fat and cholesterol and modify fatty acid composition by using strains of genetically engineered culture microbes is In one ~n~a' s cages or exploration (Harlander, 1987~. Thus, cloning of genes coding for desirable enzyme systems (e.g., §-oxidation, cholesterol oxidation, and fatty acid desaturation) into microorganisms in starter cultures represents a possibility for the future. Such research is warranted because of the importance of milk and dairy foods in the diet as sources of calcium, protein, and vitamins. Edible Oils Vegetable oils represent a major dietary source of calories in the U.S. diet, mostly as visible fats (salad oils, shortenings, frying oils, spreads), but in addition, they are used extensively in food formulations. Edible oils are composed mostly of soybean oil, are generally rich in linoleic acid, and contain limited amounts of SFAs. Olive oil contains 70% oleic acid, while palm oil and especially lauric oils (coconut and palm kernel oils) are rich in SFAs. Advances in oil processing technology, especially better selectivity and control of hydrogenation, have resulted in a reduction of trans-fatty acid isomers in shortenings and margarines 55

made from processed vegetable oils (Erickson, 1980~. Improvements in hydrogenation coupled with a reduction of autoxidat~on (by removing traces of catalytic transition metals, by using antioxidants and packaging under inert gas) has facilitated much greater use of high vegetable oils with levels of linoleic acid in the U.S. food supply. The apparent beneficial effect of n-3 PUFAs has stimulated commercial interest in the use of oils containing these fatty acids. Vegetable oils containing a-linolenic acid (LNA), that is, rapeseed oil with low levels of erucic acid (Canola oil), which has 10% LNA, and refined unhydrogenated soybean oil, which has 6 to 7% LNA, may be useful in this regard. Linseed oil, with approximately SO' LNA, is a potential rich source if the LNA can be stabilized against autoxidation. The new Canola rapeseed oil contains 60% oleic acid, 20% linoleic acid, and 10% LNA. This may represent a desirable ratio of dietary unsaturated fatty acids. , _ , _ _ _ a Fish oils represent a good source of long-chain _-3 PUFAs, usually containing 10 to 20% of these (mostly eicosapenteenoic and docosahexaenoic acids (Kinsella, 1989~. Effective stabilization of these fatty acids in foods and edible oils remains a major challenge, and currently, their use in foods is limited by their chemical instability (Kinsella, 1987b). The development of new information concerning the metabolic interrelationships of dietary unsaturated fatty acids (Lands, 1986a,b), the beneficial hydrolipidemic effects of oleic acid (Grundy, 1987), the potent effects and actions of eicosanoids, and the modulatory effects of _-3 PUFAs (Lands, 1987) may suggest that changes in the amounts and mixtures of unsaturated fatty acids used in food formulation and processing should be reassessed. As a result of developments in oil processing and stabilization together with the increasing capacity (especially via biotechnology) to produce edible fats and oils with desirable composition, a range of oils varying in physical and nutritional properties should become available. In this regard, the production of nonabsorbable food-grade edible oil substitutes is of significance (Haumann, 1986~. 56

Bionic ~r~G,CAL DEVELOPMENTS - ~POTENTIAL IMPACT ON NUTRIENTS Developments in biotechnology may facilitate the production and processing of designed edible oils, food materials and ingredients by plant, animal, and microbial processes. This should permit the formulation of foods with balanced nutrients for optimum nutrition and health. Some of these developments that may have an impact on such things as production, commodity composition and properties, and processing (Figure 1) are surveyed below PRODUCTION 1 Processing COMMODITIES Separations Fractionation FUNCTIONAL COMPONENTS 1 I DESIGNATED FABRICATED FOODS 1 CONSUMERS * Nutrient Balance * Quality * Safety * Price Or , Recombinant DNA Fermentations FIGURE 1 Major points or phases in food production and processing that can be affected by ongoing innovations in technology. Recombinant DNA and genetic engineering can improve production efficiencies and facilitate the rational design of components, and fermentations using genetically engineered microbes can become a significant source of functional ingredients. These developments will facilitate the production of formulated food products that more closely meet consumer criteria (i.e., nutrient balance, quality, costs, etc.). In that context the consumer increasingly may influence the sources of food components. S7

i Biotechnology, that is, the application of modern techniques of molecular biology, biochemistry, and chemical engineering to the production of plant, animal, and microbial materials, can have a major impact on conventional food production and the nutrient content of foods. Genetic engineering connotes the successful transfer of DNA or genes coding for specific compounds from one cell to another. Considerable success has been achieved in producing pharmaceutical~products (insulin, interferon, somatotropin, and vaccines), enzymes; and diagnostic agents by recombinant DNA techniques in microbes; and some limited success has been achieved in plants and animals. Plants Extensive research is being conducted to improve the efficiency of photosynthesis and to impart the capacity for nitrogen fixation into various food crops. Success in these areas should contribute to improved food production (Frawley et al., 1986~. The traditional breeding and selection techniques for new plant cultivars with greater resistance to pests and diseases and improved yield and quality characteristics are rapidly being superseded by more rapid tissue culture and protoplast fusion techniques to obtain genetic diversity (Frawley et al., 1986~. In addition, recombinant DNA is being new genetic materials from other plant, animal, or microbial sources. The success of this approach depends on the regeneration of plants from cultured cells, which is a challenging obstacle with many plant species. Genetic engineering in plants is more complex than it is in prokaryotes because plants contain nuclear, mitochondrial, and chloroplast genetic material that interacts and controls different traits; cloning and vector systems are very limited; cell replication is slower; and finally, the transformed cell must regenerate into a plant. In addition, gene regulatory mechanisms and rate-limiting enzymes in important metabolic pathways are not yet well understood (Frawley et al., 1986; Zaitlin et al., 1985~. used to introduce into plants r 58

Cell culture techniques have greatly accelerated plant cultivar development. The newer techniques (Table 4) being applied to the selection of new plants include TABLE 4 The Major Techniques Used to Improve the Quality of Edible Plants via Plant Biotechnology Method Example Genetic selection Tissue culture, somaclonal propagation Gametoclonal variation (anther culture) Protoplast fusion Recombinant DNA technology Rapeseed corn Potato, palm oil, tomato, asparagus Cereals (corn, wheat, rice) Hybrid plants Herbicide and insect resistance somaclonal variation in plants regenerated from cells, gamete culture, protoplast fusion, and gene transfer (transgenic plants) (Evans, 1985; Sharp, 1986~. These methods are mostly being used to select favorable production traits but can also be used to improve the functional and nutritional properties of the products, with transgenic modification being most appropriate in this respect (Sharp and Evans, 1986; Zaitlin et al., 1985). Routine clonal propagation of plants from cell culture is now used in plant breeding to propagate crop plants (Zaitlin et al., 1985~. Somaclonal variation in plants regenerated from cells has been widely exploited in 59

establishing new cultivars of tomatoes, carrots, and potatoes with different phenotypic characteristics (Whitaker and Evans, 1987~. In addition, this method can be manipulated to obtain products with altered compositions (fruits, tubers, or seeds), for example, carrots with higher levels of p-carotene, tomatoes with more solids, red peppers with more pigment, or soybeans with a different range of fatty acids (Evans, 1985; Sharp, 1986~. Intensive research on methods that can be used to i . . . . . develop strains of oilseeds, especially soybean and rapeseed with different levels of oil and containing different fatty acids (i.e., more oleic acid, less palmitic acid, and zero to 15% linolenic acid), is in progress (Haumann, 1987; Horsch et al., 1987; Nielsen, 1987; Sharp, 1986~. The dramatic successes in developing Canola oil (rapeseed), that is, replacing erucic acid by oleic acid (presumably from the loss of oleyl elongase) and sunflower cultivars with high levels of oleic acid derived from linoleic acid-rich cultivars (presumably from the loss of a desaturase) indicate that there is considerable genetic flexibility in the types of oil that can be synthesized by oilseeds. Green (1987) recently developed a flaxseed in which the LNA was reduced from 60% to approximately 1%, and linoleic acid was concomitantly increased to greater than 50%. Backcrosses of these flaxseeds gave intermediate levels of these two fatty acids. This crop could become a significant source of LNA for use in foods and as an edible oil. The various cell culture techniques in use should facilitate selection of cells with altered fatty acids. However, rapid methods are needed to diagnose desirable changes in the original cell rather than depending on plant regeneration and seeding (Frawley et al., 1986~. ~_ _ ; _ _ Gametoclonal variation in cultured anther cells (haploid and double haploid) provides a novel approach for producing new potato, wheat, rice, rye, pepper, barley, and corn genotypes in a short time (Whitaker and Evans, 1987~. This method is extremely promising for plant breeders and could be exploited more effectively if rapid screening methods were available. Protoplast fusion techniques can be used to produce somatic hybrids and to introduce new gene combinations from different species and genera. Hybrid plants have 60

been regenerated from carrots, rapeseeds, and potatoes (Evans, 1985~. These techniques are of value as they allow integration and combination of cytoplasmic (chloroplast and mitochondrial) genetic materials into the hybrid, and hence, they may be useful in manipulating cytoplasm-linked factors (Frawley et al., 1986~. Gene transfer or gene modification is the most precise approach for introducing desired traits or product synthesis into plants. Genetic material can be introduced into many plant cells by using a plasmid from Agrobacterium tumefaciens as a vector to transfer and insert foreign DNA into the plant chromosome. This system has been successfully used to transfer foreign genes into a number of dicotyledonous plants (Jaworski, 1987~. Thus, single-function genes for insect luciferase, soy conglycinin, and other proteins have been successfully transferred and expressed in other species of plant cells. The cloning of the gene for the insect toxin from Bacillus thuringiensis has been successfully cloned into plants and is expressed, as evidenced by insect resistance. This can have a major impact in increasing the production of plants used for food, but it may also enable a wider range of plant cultivars to be cultivated (Lawrence, 1987~. However, the safety of this Bacillus toxin in human foods needs to be ascertained. A number of tranegenic plants containing foreign genes (mostly for herbicide resistance) have been propagated experimentally. However, more information concerning gene promoter sequences, transcription, and expression factors is needed. In recent years crop plants (e.g. rapeseed, tomatoes, potatoes, and lettuce) have been successfully transformed by using the A. tumefaciens plasmid vector (Frawley et al., 1986; Lawrence, 1987~. More recently, the direct microinjection of foreign DNA into plant protoplasts has been achieved (Jaworski, 1987~. This method is particularly relevant to monocotyledonous plants (e.g., cereals) that are not amenable to transfection by A tumefaciens and with the . exception of rice, have been difficult to regenerate from cells (Cocking and Davey, 1987~. Recently, the direct incorporation of DNA (which was injected into tillers of rye plants) into progeny cells and its expression in the 61

progeny plants was reported (de la Pena et al., 1987). This method circumvents the problem of regenerating plants from protoplasts and should be a boon to the improvement of cereals. Of the oilseeds, rapeseed, which readily regenerates from somaclonal, gametoclonal, or protoplast cells, currently most promising in terms of oil modification (i.e., increasing its LNA content) (Knauf, 1987~. Recombinant DNA techniques hold promise for modifying the nutrient content (e.g., increasing the essential amino acid content) of many food plants. The insertion of the appropriate base sequences corresponding to essential amino acids into genes coding for seed protein synthesis is feasible, and these genes can be inserted into protoplasts or cells by direct microinjection or by the A. tumefaciens system. The plants regenerated from these cells can thus be modified. For example, the proteins of cereals (e.g., rice and corn) can be enriched in lysine, and legumes (e.g., soybean) can be enriched in methionine by using multicopy insertions if necessary. In addition, the insertion of foreign genes coding for proteins rich in essential amino acids or amplifying the transcription of endogenous genes for proteins rich in amino acids may become feasible (Bruening et al., 1987~. Recently, Jaynes et al. (1986) successfully introduced a synthetic gene rich in essential amino acids into potato cells by using plasmids of A. tumefaciens and AYroh^~nrium rhizc,~enes as vectors Similar strategies can be used in other plants (e.g., corn) to provide proteins with the appropriate amino acid balance (Messing, 1983; Rao and Singh, 1986~. The content of other important nutrients, e.g., p-carotene, tocopherol, and certain B vitamins, should be amenable to manipulation. However, multigenic traits are still difficult to transfer (Rao and Singh, 1986; Zaitlin et al., 1985~. Detailed knowledge of the rate-limiting enzymes in biosynthetic pathways (for oil, protein) in plants is necessary in developing and applying a strategy for modifying products. Most pathways (e.g., fatty acid synthesis) require many enzymes, and hence, multigene manipulation may be required to alter a product. If one particular enzyme is rate limiting, however' the introduction of the gene for that enzyme may be sufficient to enhance synthesis of the product. 62 On the

other hand, if a particular product (e.g., trypsin inhibitor or lipoxygenase) is unwanted, then tranagenic modification with an antisense gene for the rate-limiting step in the synthesis of that product can be used (Ecker and Davis, 1986~. Elimination of lipoxygenase from soybeans has been shown to improve the flavor quality of soy products (Nielsen, 1987~. Antisense genes (Ecker and Davis, 1986) eventually may be used to suppress the synthesis of undesirable multigene products (e.g., tannins in sorghum, trypsin inhibitors, saponins, thiocyanates, and goitrogens) in food plants. In addition to the use of biotechnology to improve the nutrient content of plant products, increasingly it will be used to improve the physical and functional properties of plant (and animal) components to meet the exacting needs of food manufacturing. As the food industry fabricates new foods de nova, ingredients such as starches, proteins, polysaccharides, and fats with particular physical properties will be required in particular products (Kinsella, 1982~. This will require knowledge of the relationships between the structure and physical properties of these macromolecules (Table 5) and rather sophisticated genetic engineering of such TABLE 5 Intrinsic Factors That Affect Protein Structure, Function, and Behavior in Food Systems Amino acid composition (major functional groups) Amino acid sequence (segments or polypeptides) Secondary or tertiary conformation Surface charge, hydrophobicity, polarity Size, shape (topography) Intramolecular stabilizing forces Disulfide and sulfhydryl content Quaternary structures Secondary interactions (intra- and interpeptide) Substituent groups (phosphorylated, glycosylated) Bound or prosthetic groups 63

multigenetic functions. Thus, rational modification of plants to produce more soluble polysaccharides and fiber more heat-stable proteins, or a stronger gluten will be pursued as the food processing industry seeks more consistent functional ingredients (Lawrence, 1987~. Animals Much of the emphasis in animal biotechnology has focused on reproduction (superovulation, embryo . manipulation, multiple embryo generation, culture and transfer, and sex determination), the development of animal health care products (vaccines, interferon, hormones, and somatotropin), and veterinary diagnostic products by monoclonal and recomb-inapt DNA techniques (Evans and Hollander, 1986; Ratafia, 1987; Smith et al., 1986~. These can all markedly enhance the productivity of animal products and should thereby improve the quality and safety of the food supply. The use of growth hormone (somatotropin) has been very effective in growth stimulation and in enhancing muscle growth in steers and milk yields in cows (Bauman et al., 1985; Evans and Hollander, 1986~. The recent successful transfer and expression of exogenous genes in animals has greatly enhanced the potential of developing transgenic animals with improved growth, health, etc., that can also be manipulated to produce better quality meat and, possibly, milk containing desirable products of cloned genes that are activated by lactogenic hormones. The use of milking animals for the cloning of genes activated by lactation-specific regulators is particularly practical because the product is secreted and can be recovered or left in the milk for consumption. The successful cloning of foreign DNA into the mammalian genome was developed in mice. Usually, DNA is microinjected into the pronucleus. of a fertilized unicellular egg that is allowed to develop in the - oviduct. Some 10 to 20% of the eggs survive, and following implantation some 20% of the neonates carry one or more.copies of the original microinjected DNA, which is capable of tissue-specific expression for the production of a protein (Brinster and Palmiter, 1986~. Other less successful methods involve the use of retroviruses to transfect animals (Van der Fatten et al., 64

1985) and embryonic stem cells-(Bradley et al., 1984; Smith et al., 1986~. Clark et al. (1987) have recently described the successful development of transgenic animals by microinjection of DNA into the ova of farm animals (e.g., sheep). Their observations indicated that microinjection of the complete intact gene sequence ensures tissue-specific expression in the transgenic animal. Alternatively, the gene segments comprising the promoter, coding sequence, and some extra nucleotides can be fused with an endogenous tissue-specific gene (e.g., the casein gene for mammary tissue expression) prior to injection. Appropriate signals for splicing and polyadenylation are required for transport and stability of the messenger RNA (mRNA) (Clark et al., 1987~. In mammary tissue the mostly single-copy genes coding for milk proteins are expressed at very high levels, reflecting promoter sequences that are very active. Fusion of these sequences with exogenous genes should ensure high rates of synthesis and secretion into milk. The possibility of using this approach to modify the nutrient composition of milk has been discussed. This modified composition could include caseins with higher amounts of methionine (Kang et al., 1986), milk with greater concentrations of transferrin or lysozyme, low-lactose milk, and f-lactoglobulin with a rich array of essential amino acids and better functional properties. Eventually, it may be possible to clone additional acyl desaturases into bovine mammary tissue and increase the degree of unsaturation of fatty acids in milk, to introduce antisense RNA into fatty acid synthetase, and to reduce milk fat. This approach has already been used successfully to produce tissue plasminogen activator in the mammary tissues of transgenic animals (Clark et al., 1987~. More information is needed concerning appropriate signal sequences and posttranscriptional modifications for the successful production of proteins in animal cells. Microbial Fermentation The most promising biotechnology developments have occurred in the microbial fermentations arena (Table 6) although few products are in use because of cost and 65

TABLE 6 Examples of Some fo the Major Products That Can Be Produced for the Food Industry by Biotechnological Innovations Product Uses Amino acids Glutamic acid Aspartic acid Phenylalanine Methionine Lysine Tryptophan Enzymes a-Amylase Glucoamylase Glucose isomerase Amylogalactosidase Pullulanase Proteases Rennin, Lysozyme Improved organisms Yeasts Lactic acid bacteria starters ow-calorie sweeteners Aspartame Thaumatin Monellin Stevioside Modified triglycerides and fatty acids Microbial polysaccharides Flavors, fragrances, and colorants Food testing Monoclonal antibody kits DNA Hybridization Microbial proteins Vitamins B2, B:2, C, and E Food additive Aspartame (NutraSweet) Animal feed  High-fructose corn syrup Light beer Protein modification Cheeses Wine and beer Milk fermentations, cheeses Non nutritive sweeteners Cooking oil and food additives Thickeners and gelling agents Fabricated foods Salmonella, Listeria aflatoxins, clostridia Animal and human food supplements Human and animal dietary supplements 66

regulatory constraints. Recombinant DNA techniques are most advanced for prokaryotic systems and to a lesser extent for yeasts. Basically, the gene or DNA sequence coding for an enzyme is isolated (or in the case of eukaryotic genes, the complementary DNA [cDNA] made from isolated mRNA, lee., devoid of introns) is placed in a vector (usually a plasmid, phage, or virus) and is thereby incorporated into the microbial cell, which is then propagated (cloned); the transformants are then isolated and cultured for product generation (Lin, 1986~. For high levels of expression, the cloned DNA sequence should be placed after the promoter and ribosome-binding site sequence and an appropriate transcription terminator sequence should be inserted in the plasmid. Ideally, the host cell should not degrade the message or the product, which, for most purposes, should be secreted (Lin, 1986) Advances are being made in the application of recombinant DNA to the modification and improvement of functions of food-grade microbes used in food and beverage fermentations (Bats, 1986a,b; Follette and Sinskey, 1986; Harlander and Labuza, 1986; Knorr 1987; Stewart, 1984; Stewart and Russell, 1987~. However, knowledge of, for example, the genetics, plasmid-linked traits, suitable cloning vectors, effective gene transfer mechanisms, and expression in food microbes is limited compared with what is known about Escherichia colt. Nevertheless, extensive research is focusing on these food organisms to amplify and improve their metabolic efficiencies, to introduce additional stable traits, and to enhance the nutrient content of fermented foods (Kinsella, 1986b). There is a precedent for exploiting microbes approved for use in foods because several of the ingredients and nutrients currently used in the food industry are produced by microorganisms (Harlander and Labuza, 1986~. These normal endogenous products of selected microbes include amino acids, flavor enhancers (monosodium glutamate), acidulants (gluconic, lactic, and citric acids), flavor compounds (yeast extracts and nucleotides), pigments, sweeteners (fructose and aspartame), stabilizers, thickeners (xanthan gum), and a range of enzymes. Current research is focusing on extending the number of functional ingredients (colors, flavors, essences, enzymes, gums, antioxidants, fatty acids, n-3 PUFAs, and vitamins) and developing transformed microbial organisms and plant cell cultures to improve productivity (Harlander and Labuza' 1986; Knorr, 1987; Lin, 1986; Whitaker and Evans, 1987~. 67

Elucidation of the biochemical pathways identifying the rate-limiting enzymes and then cloning these into the cell or organism can greatly increase the production of multienzyme products (Lin, 1986~. By exploiting recombinant DNA techniques, microbial biotechnology has the potential to greatly supplement traditional sources of food materials and to manipulate the structure and physical properties of functional ingredients. Some examples are discussed below. Molecular Design: Proteins and Enzymes Computer modeling of functional macromolecules (proteins, enzymes, and polysaccharides) to elucidate structure-function relationships in foods and to assess the effects of genetic modification at specific sites is being increasingly exploited (Blundell and Sternberg, 1985; Lin, 1986~. This facilitates molecular engineering via a single point mutation or substitution and subsequent cloning of the modified gene. Because the functional properties of proteins in foods reflect particular structures and secondary interactions (Kinsella, 1982), the rational modification of proteins by altering specific genes for functional uses may now be feasible (Hoi, 1987~. Because they are mostly single gene products and many are unmodified posttranscriptionally (via glycosylation or phosphorylation), proteins and enzymes have been used most successfully in molecular cloning to date. Point mutations and cassette insertions or substitutions can be used to alter the amino acid sequence, structure, and functional properties of proteins and enzymes produced in transformed microorganisms. The thermal stability of proteins can be improved by introducing arginine, lysine, and new disulfide bonds. This approach has been successfully used to modify the stability and properties of enzymes (Perry and Wetzel, 1984; Pitcher, 1986~. The possibility of improving the nutritional value of proteins by genetically incorporating more essential amino acids while concurrently enhancing functional properties should also be feasible. In addition, proteins that can be used in conventional food processing may be produced. Thus, in hard cheeses (e.g., provolone and emmenthal), late blowing, that is, gas formation caused by Clostridium butyricum, causes 68

irregular vacuoles. This can be inhibited by nitrate (which is not allowed) or it can be controlled by lysozyme, which is currently isolated from egg whites. Recently, Perry and Wetzel (1984) isolated and cloned the thermally unstable T4 lysosome. By site-directed mutagenesis, they replaced isoleucine with a cysteine that could form an extra intramolecular disulfide link to produce a stable lysozyme. This lysozyme may have antimicrobial applications in foods and perhaps can be exploited in fermentations. Similarly, the cloning of other proteins (e.g., bovine lactoferrin) is of interest for use in baby formulas and feeds as an antimicrobial agent. Scarce and expensive enzymes can now be cloned into microbes and produced in quantity (Table 7~. Successes TABLE 7 Examples of Colon Food Enzymes That Can Be Produced by Genetic Engineering. Enzyme Substrate o-Amylase Catalase Cellulase G}ucoamylase Glucose isomerase Glucose oxidase Invertase Lactase Lipase Pectinase Protease Remeet Starch Hydrogen peroxide (cellulose Dextri ns Glucose Glucose and oxygen Sucrose lactose Lip ids  Pectin Proteins Casein liquefaction to dextrins, brewing, baking Milk sterilization Juice clarif ication Hydrolysis to glucose High- fructose corn syrup Elimination of browning discoloration Production of invert sugar Hydrolys is of lactose for low- lactose milks Cheese ripening Wine or Jui ce clarification Meat tenderizer, sausage curing, dough conditioning, beer clarification Casein coagulation, cheese manufacture SOURCE: Harlander (1987) . 69

to date include chymosin (rennet for cheese making), Blase, glucose isomerase (for corn sweetener), alkaline proteases, glucoamylase, and a broad array of other enzymes used in small quantities (Lasure, 1986; Lin, 1986~. The microbial enzyme p-galactosidase is routinely used to hydrolyze lactose in milk and thereby render it suitable for consumption by lactase-deficient consumers. Cloning and site-directed mutagenesis to improve enzyme function is now feasible, but the relative costs and Food and Drug Administration approval for food uses are current obstacles (Pitcher, 1986~. In food processing, the traditional approach of trying to accommodate a process to suit the available ingredient or enzyme can now be changed so that ingredients or enzymes are redesigned to suit a particular process. Thus, enzymes that remain active in particular or unusual =~ri'^-m=-te i; ~ Impel=' m=~i=, lipids, etc.) can be generated. An example is a lipase that catalyzes the trans-esterification of fatty acids into glycerides in an organic or glyceride phase (Gillis, 1987~. Such enzymes can be exploited to make triglycerides with the appropriate fatty acid composition and physical properties to meet particular nutritional or functional criteria. ~& ~ _^ ~L~ ~ _ ~ ~ · ~ __ ~_~ Microbial polysaccharides Hydrocolloid gums are widely used in the food industry as viscosity modifiers, for gelling and emulsion, or as stabilizing agents that can impart desirable factual and sensory properties to foods (Table 8~. These are derived from seaweed, algae, plants, seeds, and microbial sources and show a wide range of properties. Most are nonmetabolizable, indigestible fibers that exert desirable physiological effects (filch, 1987~. Microbial sources of homo- and heteropolymers that are being developed as functional ingredients include xanthans (glucuronic acid), dextrans (glucose), pullulans (glucose), alginates (mannuronic acid), and curdlan (glucose) polymers. These are examples of the range of hydrocolloids with different properties (Sinskey et al., 1986a,b) that can be used to substitute for fats in some processed foods. 70

TABLE 8 Examples of Types, Sources, and Structures of Food Polysaccharides and Gums Used in Food Formulations Gums Sources Structures Agar Marine algae of the class ~hodoT>hYceae Alginate Brown seaweed (MacrocYstis ,pYrifera) Arab ic Exudate produced by acacia tree in response to in, ury Carrageenan Red seaweed such as B- (1, 3) -linked Chondrus crispus B-Glucan Aleurone cell wall and endospene of oats asked barley Cuar Pectin Xanth~ Endosperm of seeds from two annual leguminous plants (C`ramopsis te~cragonolobus and C . psoraloides ) Midlamella between cells of apple pomade and citrus peel Microorganism (Xanthomonas campestris) Heterogenous chains of B- (1, 3) -d-galactose and ~ - ( 1, 4 ) - 3, 6 - anhydro - 1- galac tose Linear polymers of S- (1,4) -linked d-mannuronic acid and a-(1,4)-linked 1- guluronic ac id Spiral chains of B-galactopyranose linked ~ - ( 1, 3 ) w! th s ide chains of ( 1, 6 ) - 1 inked galactopyranose, arab inopyranose, rhamnose, and glucuronic acid (molecular weight - 500,000) Linear chains of alternating B- d - galactopyranosyl and ( I, 4 ) -1 inked a-d-galactopyranosyl used ts sulfated at 2, 4, and 6 pos itions (molecular we ight - 200, 000 ) Linear polymer of k-(1,3)- and B-(1,4)- d-glucopyranosyl units (molecular weight - 30, 000 to 130, 000)  Linear chain of B-d-mar~s~opyranosyl units linked o- (1,4), with single members of o-d-galatopyranosyl linked (1, 6) as side branches (molecular weight - 220,000) Mostly an o- linked linear polysaccharide of d-galacturonic acid esterified with methoxyl groups from O to 85% (molecular weight - 50, 000 to 180, 000) Main chain of B- ( 1, 4 ) -1 inked B - d - glucose with side chains of mannose and glucuronic acids (molecular weight - 2,000,000) SOURCE: Ink and Hurt (1987). Vitamins Many vitamins are produced by microbial fermentation (Knorr, 1987~. The successful cloning of a critical enzyme in the synthesis of vitamin C from glucose was reported recently (Anderson et al., 198S); with the cloning of gluconolactone oxidase, the final enzyme in vitamin C synthesis, a microbial system for the complete synthesis of vitamin C may be available presently. With advances in molecular cloning, the synthesis and production of vitamins via bioengineering will increase. The possibility of eventually cloning vitamin C synthesis into microbes used in food fermentations might improve food stability, and in processed meats it could reduce the need for nitrite. 71

Culture Organisms Biotechnology, as epitomized by food fermentations (i.e., the rational use of microbes for improving the stability, quality, safety, and indirectly, the nutritional value of food products), has been exploited for thousands of years to preserve milk and to produce bread and alcoholic beverages. The molecular biology and genetics of the microorganisms used in food fermentations are elucidated to improve their applications. Research to improve lactic acid starter cultures for cheese and dairy fermentations is in progress in several laboratories (Bats, 1986a,b; Chassy, 1985; McKay, 1986; Venema and Kok, 1987~. This includes research to enhance lactose metabolism to lactic acid, increase resistance to phage infection, and control proteolytic activity. In addition, the idea of incorporating additional plasmid-linked genetic capabilities into lactic acid streptococci is being explored. This includes, for example, the modification of cholesterol and the use of temperature-activated proteases and/or lipases to enhance flavor development and the ripening rates of cheese. The gene for the sweet protein thaumatin has been cloned and expressed in fermentative organisms (Kluyveromyces lactic), although it was not secreted (Edens and van der Waals, 1983~. Ideally, the cloning of genes for desirable functional ingredients and nutrients for human consumption should be conducted by using food-approved microbes. Hence, research on the genetics and molecular biology of lactic acid bacteria is particularly important because of their long history of use and safety. Information to improve their successful exploitation as host microbes for heterologous proteins should accelerate their use (Venema and Kok, 1987~. In addition, the transformation of starter microbes with an increased ability to synthesize B vitamins, ascorbic acid, etc., may be feasible in the future. The direct effects of viable food microorganisms on health is also of interest. The putative beneficial effects of Lactobacillus acidophilus in fermented dairy foods (acidophilus milk, yogurts, etc.) in reducing the production of enzymes (~-glucuronidase and nitroreductase) that are known to convert procarcinogens into carcinogens in the large intestine (Goldin and 72

Gorbach:, 1984) deserve study from a molecular genetics viewpoint. Elucidation of this effect may be of significant value to public health. Flavors In addition to nutrition, contemporary consumers are concerned about safety, and in this regard natural ingredients are perceived as being safer than ingredients produced by chemical methods. Hence, there is a keen interest in exploiting biotechnology as a source of natural food ingredients. This is particularly true of flavors for which there are limited supplies and costs are high. Hence, plant, animal, and microbial cell cultures are being exploited and enzymes are being used to transform flavors to meet the criteria of natural food products. Genetic engineering of generally recognized as safe (GRAS) microbes with the appropriate enzymatic pathways for the synthesis of organic flavor compounds is at a premium, even for compounds such as acetaldehyde, benzaldehyde esters, and vanillin, which can be synthesized inexpensively by chemical methods. Plant tissue culture techniques are being used as a source of flavors (e.g., vanilla, strawberry, tomato, and mint),~as a method for the biological transformation of flavor intermediates, and as a source of precursors that generate desired flavors when used in processing (Evans, 1983, 1985; Knorr, 1987~. Food Safety Advances in molecular biology have resulted in the development of a number of new sensitive methods for the rapid detection of pathogenic and toxicogenic food-borne microbes (Harlander, 1987~. This is particularly significant as new strains of infectious microbes are encountered in food systems. In view of the high priority of food safety, the improvement of rapid methods represents a major contribution to public health. Because of the need to monitor the safety of foods before they are released into the market, rapid and sensitive methods for the reliable detection of pathogenic or toxigenic microbes are at a premium. New methods based on enzyme immunoassays with monoclonal antibodies and, more recently, DNA hybridization techniques are becoming available (Table 9~. Immunoassays with monoclonal 73

antibodies have been used for the detection of Salmonella and Staphylococcus enterotoxins in foods DNA-DNA , hybridization probes provide a new technology for detecting various food-borne organisms. For example, Escherichia cold and Yersinia, Salmonella, and Listeria species (Fists, 1985) have greatly improved the specificity of these tests (Fists, 1985, 1986; Mattingly et al., 1985~. TABLE 9 Some Examples of DNA Probes Being Developed for Detecting Food-Borne Pathogens Organism Component Detected Staphylococcus aureus Clostridium botulinum Yersinia enterocolitica Vibrio parahaemolyticus Vibrio cholerae Salmonella spp. Enterotoxins A, B. and C1 Neurotoxins A, B. and E Invasiveness Thermostable hemolysin Enterotoxin, hemolysin, and cytotoxin Species specific Escherichia cold Heat-labile and heat-stable enterotoxins Listeria monocytogenes Hemolysin SOURCE: Harlander (1987) Algal Culture . Interest in algal culture as a source of nutrients (e.g., p-carotene and tocopherol) is burgeoning. This 74

has been accentuated by the ability of certain algal strains to produce very high concentrations of _-3 PUFAs, and some strains can produce either eicosapentaenoic acid or docosahexaenoic acid preponderantly (Kinsella, 1989~. Because of the therapeutic potential of _-3 PUFAs and the limited sources of relatively pure n-3 PUFAs (eicosapentaenoic and docosahexaenoic acids) produced by algae represent a potential source of these important nutrients (Pohl, 1982~. Comment on Biotechnological'Developments The potential of genetic engineering for revolutionizing food production by conventional (animal, plant, and avian) and nonconventional (microbial, algal, and marine) means is immense, and when coupled with advances in human nutrition, it should facilitate the formulation of a wide selection of high-quality foods with the appropriate balance of nutrients, calories, and fiber. The ability to design genetically macromolecules with the proper physical and nutritional properties, to simulate fats, to generate flavors, and to selectively eliminate antinutrients will accelerate automated food assembly and fabrication processes. Ongoing basic research should help to elucidate the mechanisms) underlying the regulation of gene expression and the factors modulating cell replication, differentiation, and tissue growth and development and to identify the rate-controlling enzymes in multienzyme'pathways. The lack of such information is currently retarding progress in the application of these new techniques. Biotechnology has great potential, but it will take time for the use of products because the path from success in the laboratory (i.e., detection of nanogram quantities of a product) through scaleup to production is a tortuous one with numerous technical, engineering, regulatory, and marketing challenges. There is already evidence of nascent success with drugs and vaccines in the medical arena. The potential is great and the promise is exciting; however, educating the public sector to ensure reasonable but prudent regulatory criteria is critically important. The new technologies are easily transferable 'and can be exploited worldwide to produce more 'foods and relieve people from hunger, malnutrition, and debilitating diseases. 75

FOOD PROCESSING In addition to progress in material production, developments in food processing and handling are altering the quality and nutritional and safety characteristics of the food supply. The primary function of food-processing is to ensure safety, especially from pathogenic microbes and toxins, and concomitantly, to improve quality and storage stability, remove antinutrients (both natural and incidental), and improve the digestibility of proteins and starch and the bioavailability of nutrients (Tannenba~m, 1979~. In addition, contemporary food processing involves nutrient fortification and supplementation, product formulation on the basis of nutrient content and convenience, aseptic processing, and the production of new foods from ingredient blends. Thermal Processing Thermal processing results in the destruction of some thermolabile nutrients, especially thiamin, riboflavin, and vitamin C. The extent of destruction depends on the time, temperature, and pH of treatments, reflecting relative activation energies (Karel, 1979~. Water-soluble nutrients may be significantly reduced during the washing, blanching, and canning of fruits and vegetables. Thiamin is particularly alkali labile. Water blanching, which is necessary for vegetable preservation (to inactivate enzymes and reduce microbes), removes water-soluble vitamins; but this can be minimized by steam or air blanching (IFT, 1986~. New methods of microwave heating or quick blanching to reduce nutrient losses, especially.in products that are a major source of a particular nutrient, may warrant further exploration. The traditional methods of canning can result in the loss (10 to 90%) of vitamins unless heating times and temperature are carefully controlled (IFT, 1986~. Commercial sterilization also. causes.destruction, especially when it is done in traditional cans or glass containers. However, recent developments, that is, high temperature, short time (HTST) heat treatments and aseptic processing, both of which are facilitated by innovative packaging, can improve the retention of nutrients. HTST processing exploits the higher energy of activation of microbial lethality compared with that of 76

nutrient destruction; hence, microbial destruction is favored at higher temperatures (Karel, 1979~. These aseptic and ultra-high-temperature treatment methods are now being applied to the thermal processing of solid food particles in which the use of thinner films allows faster heat penetration and the more rapid destruction of microbes, with subsequent improved nutrient retention. Technologies for improving heat transfer efficiency or the generation of heat internally, for example, by microwave radiation, need to be explored further. ... . rraatatlon Interest in preservation, pasteurization, and sterilization by irradiation has been revived in the United States. Low dose irradiation (<1 kilogray [kGy]) has been approved for use in fruits and vegetables to control maturation, in wheat and flour to control insects, in fresh pork (<0.3 kGy) to reduce trichinosis, in potatoes to prevent sprouting, and in herbs and spices for use as a disinfectant. It is used to sterilize foods for immunocompromised patients. Irradiation commonly involves gamma-rays from cobalt-60 or cesium sources, although electron beams are effective for the surface sterilization of products. Limited up-to-date data are available concerning the effects of controlled irradiation in nutrients (IFT, 1983~. Irradiation is an effective method, but because of the public perception, its immediate future in food processing in the United States is uncertain. Microwave Microwaves can be used effectively for pasteurization and sterilization, microbial destruction, and enzyme inactivation in a variety of products. Microwave drying and pasteurization are efficient and are being applied to the drying of pasta and precooking of bacon for franchise and institutional trades (Svenson, 1987~. With the development of appropriate materials for use in packaging and containers, microwaves may be more widely and more efficiently used for the pasteurization of foods and for improving the safety of products. 77

The use of microwave ovens (now in more than 70% of households) is significantly changing the eating habits and food selection of Americans (Messenger, 1987b; Svens on, 1987~. Numerous new meals, many of them low in calories (lean or lite meals), are being successfully marketed, and microwave cooking has favored the grazing eating trend at the expense of the traditional family meal. This creates a new situation in terms of monitoring and controlling nutrient intake and underscores the need for manufacturers to provide complete nutrient data on their products. The need for water and ions to accentuate heat generation from microwave dishes and the energy loss problem with fats (with limited dielectric polarity, fat is a very poor heat-generating medium) may discourage the use of fats in microwaveable foods and encourage the use of more water, salt, and polar, water-soluble ingredients (Schiffman, 1986~. Because of the differences in the heating mechanisms (rates and directions) between conventional and microwave cooking methods and the markedly different microwaveable behavior of different food components (e.g., heating rate, energy diffusion, penetration, and radiation), the formulation of acceptable microwaveable foods in which all components cook evenly remains challenging to food processors (Best, 1987; Rosenberg and Bogt, 1987~. In this regard it is important that technical problems not be solved at the expense of good nutrition. foods has generated demands for new coloring and flavoring agents and an array of new functional ingredients (e.g., modified starches and gums) (Best, 1987; Messenger, 1987a,b). Biotechnology may be partly able to meet these needs. , _ Progress in microwaveable Freeze-Drying The use of improved drying techniques, especially large-scale freeze-drying, has grown rapidly and is used commercially for the preservation of such foods as diced vegetables, fruits, and spices. Separations and Fractionation Ultrafiltration, diafiltration, microfiltration, and reverse osmosis represent relatively new technologies That are changing a number of food processing operations 78

concerned with the concentration, separation, clarification, and deionization of liquid foods. It is widely used in the dairy industry, in beverage processing, and in concentrating fluid foods (Kosikowski, 1986; Paulson et al., 1984~. Ultrafiltration can be used to modify the composition of fluid foods and to fractionate foods (e.g., milk) for the preparation of functional ingredients. - Supercritical gas and solvent extraction technologies (Table 10) may provide additional options for selectively removing undesirable organic components from raw materials TABLE 10 Some Current Applications of Supercritical Fluid Extraction to Foods Decaffeination of coffee and tea Deodorization of oils and fats Extraction Vegetable oil and fats from seeds Food coloring from plant material Flavors, fragrances, aromas, and perfumes Hops and spices Fruit juices Drugs from plant material Oil from potato chips and snack foods and foods (Riz~i et al., 1986~. Supercritical carbon dioxide is currently used for decaffeination of coffee and extraction of hops flavor (Hoyer, 1985~. Supercritical carbon dioxide extraction is effective as a delipidating method; by manipulating conditions and reflexes, selective fractionation or removal of saturated fatty acids or cholesterol from fats is feasible. It is effective in removing cholesterol from muscle (Hardardottir and Kinsella, 19-88) and may eventually be applied to the selective removal of saturated fat and cholesterol from commodities and food products (e.g. egg yolks and organ meats). Supercritical technology also 79

provides an alternative method that avoids the use of organic solvents in the processing of edible oils. This method is very efficient in refining crude oils (e.g., fish or vegetable oils) and may provide an approach for the selective recovery of polysaturated fatty acid components from marine oils (Daniels et al., 1988~. Packaging Developments in food and beverage packaging, particularly the range and versatility of packaging materials, have greatly facilitated the development of new products and have improved the range and safety of foods available in the marketplace (Sacharow, 1986~. Aseptic barrier plastics, oxygen barrier plastics, and modified and controlled atmosphere packaging (based on selective barrier plastics composed of different laminated films of various polymers) are revolutionizing the food and beverage industry. Thus, storage of individual vegetables and fruits for retail trade in a controlled or modified atmosphere is being used increasingly to extend the shelf life of vegetables and fruits, which should increase their year-round availability. FOOD FORMULATION In addition to ongoing consolidation and globalization, the food processing industry has undergone major changes in the past 20 years--from mostly a commodity-handling industry to a more sophisticated industry manufacturing consumer food products. This has changed the relationships among the producers, processors, and consumers. The most notable are the concerns about nutrition and safety, the increased influence of consumers on producers via the processors, and the growing integration of the technical needs of the processors with production protocols. This reflects the evolution of a food processing industry that increasingly uses traditional food commodities (milk, cereals, etc.) as raw materials from which functional ingredients are fractionated and isolated for use in product reformulation (Kinsella, 1987a). 80

The practice of new product development to meet market and consumer needs has placed a premium on the availability of a range of ingredients with precise physical and functional properties suitable for formulation into products designed to provide desirable physical, organoleptic, and nutritional properties; safety; and convenience to the modern consumer. The need for ingredients with precise physical properties is accentuated by the trend toward automation (Kinsella, 1987c). In this regard, biotechnology and genetic engineering, as applied to conventional ingredient production, are timely and will enable producers of both agricultural and nonagricultural products to supply market needs. The trend toward food fabrication will encourage the development and production of rationally designed molecules (proteins, polysaccharides, and lipids), processing aids (enzymes), ingredients (acidulants and flavors), and nutrients (amino acids and vitamins) by the most competitive production systems. The rapidity with which this will occur is greatly influenced by food regulations and standards of identity, both of which require continual updating. Fat Substitutes foods with reduced calories The fabrication of new foods, especially products low in fats and sugars, has been stimulated by the demand for ~ - . This is being facilitated by a range of new functional ingredients (sweeteners, low- calorie fat substitutes, and flavors). A consensus panel of the National Institutes of Health (1986b) recommended that the food industry actively develop new products that are lower in fat and cholesterol. A number of alternatives or substitutes for fat in foods have been promoted. Maltodextrins can be used to replace some of the oil (30 to 50%) in such foods as salad dressings, gravies, dips, frozen desserts, and ice cream (Kaper and Gruppen, 1987~. Gums, soluble fiber (Ink and Hurt, 1987) (see Table 8), and dextrins can be used to impart a smooth mouth-feel to foods in which fat has been partly replaced. Polydextrose, a synthetic polymer, is a low-calorie functional ingredient that can be used to replace fat and add body, viscosity (thickener), and mouth-feel. Effective flavoring is important in such products because the fat components are usually the principal carriers of desirable flavors in foods. _ - 81

Nonmetabolizable, nonabsorbable fats have been synthesized. These include acylated sucrose (sucrose polyesters), polyglycerol esters, glycerol ethers, and polycarboxylic acid esters, and they have physical properties similar to those of fats and oils in food products, but they are not absorbed (Haumann, 1986~. However, ingestion is limited by their laxative effects and the elusion of fat-soluble vitamins. Whereas triacyl esters of glycerol are readily hydrolyzed, the penta- and hexaacyl esters of polyhydric alcohols are not hydrolyzed and, hence, are not absorbed. Sucrose polyesters (olestra) consist of a mixture of hexa-, hepta-, and octaacyl esters of sucrose with fatty acids; are nondigestible; and reduce plasma cholesterol and low-density lipoproteins (Mattson et al., 1979~; they may exert laxative effects in some patients, however. These fats can be readily substituted in products, but the anal leakage problem and regulatory criteria are still major obstacles (Haumann, 1986~. Nevertheless, they may have a place in food products in limited amounts. Because oils absorbed by foods during deep frying in fat are a significant source of calories in the American diet, nonabsorbable fats can be useful as a cooking or frying media. - Sweeteners The total consumption of sweeteners in the United States is about 125 lbs/capita/ann'm. Sugar provides about 17% of calories in the U.S. diet. The reduction of dietary calories can be aided by using the wide array of sweeteners on the market. Sucrose consumption (approximately 85 lbs/annum) is decreasing, while corn sweeteners (high-fructose corn syrup) have increased (to approximately 35 lbs/annum), saccharin is consumed at about 10 lbs/capita/annum, and noncaloric sweeteners are approaching consumption levels of 20 lbs/capita/annum (Dziezak, 1986; Newsome, 1987~. The successful use of glucose isomerase facilitated a major breakthrough in enabling the production of fructose from glucose derived from cornstarch hydrolysates (the most successful example of enzyme biotechnology). Fructose is sweeter than sucrose per unit calorie, and its adoption by soft drink producers greatly expanded its 82

1 .' ,1 use and general tecl~pttabi}ity in the U.S. food supply. . . . . . . . The concern math palpries has resulted in the development a! ~ ,~ pr Tine of low-calorie foods for which there is a~c.~ept estimated market that approaches $2 billion. B'cq~,ei~f the continuing demand for low-calorie sweq~epest' Several new products are available Stable ..~.~:) :(G¢~prdi, 1987~. The dipeptide aspartylpheny~at~ni~rmpthylester (aspartame) has been adopted for w s;ipi~a~y foods, and current sales are burgeoning (:~$70~.~;~1-~top, or approximately . . . .. .. . . . . . . , ,, . · . ; TABLE 11 Woke- ~ Adeptly Available Food Sweeteners , . ~ ~ . .. .. . ,. . . Sucrose : ,; Fructose (highytrup*ose carp syrup tHF¢~) Aspartame Glucose Saccharin Xylitol Thaumatin Stalled ;; Acesulfame Stevioside Glycyrrhizin Neohesperidin Dihydrochalcone Phyllodulcin Monellin . , , · , .,;.. ; · . . . .. lbs/capitaiannuq). Aspartame is 200 times sweeter than sucrose, is $.~''t$ive to heat, and is somewhat unstable in solution a ~ e pH 5.0. Xylitol, which is as sweet as glucose, is q~t'b:~;ed (4 cal/g) but is chemically very stable and i' dot p~riog:enic. Thaumatin is a protein that is more than 2~000 times as sweet as sucrose and acts as a ft ~ enhancer. This protein has been cloned and can be gepertt'4 in fermented products by using transformed '~ig~o'b ~ (E4ens and Van der Waals, 1983~. Several other. s#eetenip:' agents, for example, stevioside, glycyrrhizin, ~ Win, and sucralose (chlorinated sugar), are blips sad for particular applications (Dziezak, i986, ~0£,~nly et al., 1983~. ~ . . . In addition, Arch pn the mechanism of taste perception i' progressing' and Schiffman (1987) has summarized `~$.~\ in this area. Certain compounds .. . . . . . ; . it, . . . 83

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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