SOME PROTEIN-RICH INFANT FOODS BASED ON RICE Thesis for the Degree of M. S. MICHIGAN STATE UNIVERSITY Ruth Wen Chen 159626 met: LIBkARY _ Mighiganc-n , . I R093? USE C722.” ISIC ABSTRACT SOME PROTEIN—RICH INFANT FOODS BASED ON RICE by Ruth Wen Chen Several enriched foods, taking rice flour as the basic ingredient (75-80%), were designed and tested for food efficiency and protein efficiency ratio (PER) at a protein level of 15% total protein before and after storage at 100% relative humidity and 95°F. for 145 days, and, fresh, at 10% total protein, by feeding these diets to male weanling rats. The proposed diets are dry flour-like products pre- pared with water in porridge form, similar to rice flour in appearance, and contain in addition to a basic supple- ment of yeast, sweet potato, CaCO3 and salt, various amounts of codfish flour and soybean flour. Eaten in amounts of 250-300 g per day by one year old (9 kg) infants, they will supply all the basic nutrients except vitamin C and vitamin D. These proposed diets, as Judged by PER and food ef— ficiency, are better than either soybean or skim milk. A prOper combination of vegetable and animal protein can give the same benefit as an animal protein, such as cod- fish, alone. Ruth Wen Chen The proposed diets after storage were slightly darker, lost up to 50% B—carotene, and developed off- flavors and odors which were, depending on the particular diet, marginally acceptable or unacceptable, although they still gave good PER values. SOME PROTEIN—RICH INFANT FOODS BASED ON RICE By Ruth Wen Chen A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Food Science 1966 ACKNOWLEDGMENT The author would like to express her deep appre- ciation to Dr. R. C. Nicholas for his kind, valuable, and patient guidance throughout the preparation of this thesis; to Dr. C. L. Bedford for letting her use the laboratory facilities; and to Miss Dee Fang for her skillful typing of the original manuscript. Thanks also to the Michigan State University International Programs--Ford Foundation, through its partial financial assistance, the author has been able to achieve this study. 11 TABLE OF CONTENTS Page ACKNOWLEDGMENT . . . . . . . . . . . . . ii LIST OF TABLES . . . . . . . . . . . . . iv LIST OF FIGURES. . . . . . . . . . . . . v INTRODUCTION . . . . . . . . . . . . . 1 LITERATURE REVIEW . . . . . . . . . . . . 4 Nutrition Requirements for Infants . . . . u Schemes to Prevent or Cure Malnutrition. . . 12 Summary . . . . . . . . . . . . . l9 DESIGN OF THE EXPERIMENTAL DIETS . . . . . . . 21 EXPERIMENTAL. . . . . . . . . . . . . . 26 Preparation of Ingredients . . . . . . . 26 Chemical Analysis of Ingredients . . . . . 29 Bioassay . . . . . . . . 35 Storage and Palatability. . . . . . . . A3 RESULTS AND DISCUSSION . . . . . . . . . . 51 SUMMARY AND CONCLUSIONS . . . . . . . . . . 56 REFERENCES . . . . . . . . . . . . . . 63 iii Table 10. ll. 12. LIST OF TABLES Comparison of two recommended amino acid patterns with those of several foods (mg/g total essential amino acid). Composition of diets, g/100 g Nutrients in the diets, per 100 g. Proximate composition of the ingredients, g/100 g . . . . . . . . . . . Test 3—-Performance of male weanling rats on fresh test diets . . . . . . . Test 5——Performance of male weanling rats on stored test diets Test u--Performance of male weanling rats on fresh test diets . . . . . . . . Physical and chemical determinations of pro— posed and standard diets before and after storage . . . . . . . . . . . Summary of performance of male weanling rats on test diets before and after storage Summary of performance of male weanling rats on diets containing soybean flour. . Summary of performance of male weanling rats on diets containing codfish flour. Performance of weanling rats on various diets. iv Page 22 2A 31 39 42 N5 “7 53 57 58 Figure LIST OF FIGURES Protein score, based on three different standards (human milk, FAO 1957, and whole egg), as a function of the main protein content at 15% . . . . . Growth curves of male weanling rats on fresh diets (Test 3—-15% total protein, except CASEIN at 10%) . . . . . . . . Growth curves of male weanling rats on stored diets (Test 5-—Stored for 145 days at 100% R. H. and 95°F,15% total protein, except. CASEIN at 10% and Dog Chow at 2 5%) . . . . . . . Growth curves of male weanling rats on fresh diets at 10% total protein, except Dog Chow at 24.5% (Test 4) . . . . . . Page 23 38 141 44 INTRODUCTION Malnutrition is the number one world health problem. People have a less adequate diet today than they did at the beginning of this century (Brown, 1963). Mortality rates are twenty to forty times higher for infants and pre-school children in less developed areas than in more favored countries (FAO, 1963a). In 1963, according to an FAO survey (Brown, 1963), 20% of the people in developing countries were undernourished and 60% were malnourished; that means 10-15% of the world population is undernourished and an additional 50% are suffering from hunger, mal- nutrition or both (Belden et al., 1964). United Nations officials estimated that almost one-third of the children of Latin America, Asia, and the Middle East will die this year from hunger and diseases associated with malnutrition, and more than one-fifth from tainted water (Ross, 1966). In most types of malnutrition, the children's growth is retarded (Jackson, 1966); they have low resistance to disease (Wills and Waterlow, 1958; Scrimshaw et al., 1959), poor psychomotor development, and high mortality rates, particularly children less than five years of age (FAO, 1963a). With outside capital and technical assistance, many less developed countries have attempted to increase food production, but thus far they have not corrected malnutrition. The food available per person is still decreasing despite crop increases because the population has increased much faster than food production (FAO, 1963a). Most Asian countries are politically unstable and are still developing economically (FAO, 1963b). The Asian income is so low that the people live almost entirely on vegetables and cereals (WHO, l965). Tradition says that rice gruel alone will make babies healthy and strong. Moreover, from the economic standpoint, the parents cannot afford to feed babies animal protein (Do-Quang-Oanh, 1963). Typically, 80% of the calories in the infant diet is from rice and 20% from sugar, which means about 13 g pro— tein (protein score 72) and 900 calories per day. Conse- quently, the highest incidence of protein malnutrition in children is in these areas, as reported by WHO (1965). Schemes to solve the nutrition problems in the developing countries at a price within the reach of the population are needed. Supplementation with high quality animal proteins-—meat, egg, fish, milk and dairy products-- is not now possible because supplies are inadequate and/or too costly (Belden et al., 1964). Attention has been focused on making well balanced vegetable mixtures or on adding small amounts of animal proteins to the existing diets. Any food designed for developing countries must conform to certain principles. Milner (1962) suggests, among others, the following properties: capable of local production; within the economic means of the population; easily transportable and capable of non-refrigerated storage in the local climate; and acceptable to the pop- ulation. Low quality protein food can be improved by adding protein concentrates or supplements, as defined by the FAO‘WHO Protein Advisory Group, to the basic rice diet. The purpose of the present work is to design some protein- rich foods, based on rice, which are low in cost, easily kept and transported, acceptable, and which have a nutri— tive value sufficient to prevent malnutrition of infants in rice-eating countries. LITERATURE REVIEW Nutrition Requirements for Infants Nutritional requirements for infants are similar to those for adults in many respects, but different in certain important respects. These differences will be noted where information is available on the subject. Protein and Amino Acids The quantity of protein required depends upon the actual amino acid pattern of the diet and the extent to which protein is spared by carbohydrate and fat. A recent approach to protein requirements was reported by the WHO—FAO Joint Expert Group in 1965 (WHO, 1965). They con- cluded that 6- to 9-month old infants require 1.5 g protein/kg body weight from breast milk, cow's milk, or from any source of equal quality; 9- to 12-month old children require 1.2 g protein/kg body weight. Protein quality depends upon the amino acids of the protein, primarily the essential amino acids, through non-essential amino acids may be involved (Snyderman et al., 1962). Although the FAO Committee on Protein Requirement (FAO, 1957) did give an ideal amino acid pattern for adults and children, the Joint Expert Group (WHO, 1965) simply suggested human milk and whole egg patterns as guides for protein quality evaluation. This suggested pattern was not given specifically for children. However, a committee on amino acids has worked on infant amino acid require- ments (NAS/NRC, 1959). Two of these patterns are given in Table l. Histidine is considered essential for the young infant (Snyderman et al., 1963) but it is not considered in other patterns except the NAS/NRC (1959) recommended pattern. Cysteine and tyrosine, two non-essential amino acids available to spare phenylalanine and methionine (Holt) and Snyderman, 1965), are not considered in NAS/NRC (1959) pattern. It is hard to determine which pattern, if any, is the best because experiments on amino acid requirements give results which vary with experimental conditions. Holt and Snyderman (1965) concluded only that with more knowledge of amino acid interrelations and assistance from a computer could amino acid patterns of diets be accurately evaluated. Better clinical and chemical criteria of protein malnutrition also need to be established. The design of adequate diets from "book" values depends, of course, on a well-established standard. Vitamins Bitamin B1 or thiamine.-—Thiamine deficiency disease, beriberi, is prevalent in areas where polished white rice is the staple food. It causes the death of an unknown number of infants and children in Burma, Thailand, the Philippines, and other parts of Asia (NAS/NRC, 1966). The m>m£ pom manna pmgp CH pooAQOocH on on Lmoddm mozam> xHHE swag: Ammmav omz one .zaco mpcmh2a pom .opmc UopmSWUmmp Como U .unmfioz hoop mx\oaom OQHEm we mm co>Hw dump chflwfino on» Eonm mofiom oQHEm Hmfipcommo HMpOp w\oHom ocHEw we on oophm>cooo .mpcmmcfi new mpasom nomm a flea :mH ema mmfi :mH mcHHm> am am mm om m: canothLB mm mm Hm mm mm mcficomsce Ho :3 m: bad a» ocficoanpmz m: m: NH mm ocampmmo eoa mm mm mma mcficfimpcoo Inzmazm Hmpoe Hm mad OOH mm mcfimosse :HH mm Am :NH mm mcfiemamazcmsm man mom amfl med oapmsosm Hmpoe mma mma mmfl 32H :mH mcflmmq mea mma mmfl mom mmH mcfiosmq mma mmH ANH :NH :mfi msfiosmfiomH u I u s: u mcaefipmflm wwm m_cmm xafis cassm xfifis m.zoo o.ommmfl.omz\mom mo mmozp spa: menoppmo ofiom ocfiem cmocoEEoowh ozo mo comHEmQEoouu.H mqm¢e approximate daily requirement is 0.2 mg of thiamine per 1000 calories (Holt et al., 1949) but 0.4 mg per 1000 calories is recommended (NAS/NRC, 1964). Food prepara- tion, heat, acidity, and moisture will influence the degree of thiamine destruction. As temperature and mois- ture increase, more thiamine is destroyed (Heinz, 1963). Niacin.--Fatigue, irritability, and weight loss are early symptoms of niacin deficiency, followed by dementia, diarrhea, dermatitis and death (Heinz, 1962). The requirement for infants is 6 mg equivalent niacin per day (NAS/NRC, 1964). In vivo, 60 mg tryptophan can con- vert to 1 mg niacin (Heinz, 1963). Although rice is a poor source of niacin, examples of niacin deficiency in rice-eating areas have not been reported, but probably exist. Vitamin B2 or riboflavin.—-Deficiency of vitamin B2 causes corneal vascularization, ulceration cataract, and ' abnormal pigmentation of the iris (Heinz, 1963). The re- quirement of riboflavin for infants is 0.4 mg per day (Snyderman et al., 1949). Vitamin B5.--The most prominent symptom of vitamin B6 deficiency in infancy is convulsive seizures, hemosi- derosis and hypochromic anemia might also be caused by insufficient vitamin B6 (Heinz, 1963). The requirement of pyridoxine for infants is 0.5 mg per day (Tenth M and R Pediatric Research Conference, 1953). Folic acid and B .——Folic acid and B 2, which can 12 1 be synthesized in the large intestine, are not a problem in the infant's diet (Holt and Snyderman, 1964). Other vitamin B factors.--Pathothenic acid, biotin, and inositol are not demonstrated as normal dietary essentials (Holt and Snyderman, 1964). Vitamin C or ascorbic acid.--In vitamin C deficiency, scurvy occurs mostly between ages of 5 and 24 months, with a peak incidence at 5 to 11 months (Woodruff, 1964). In a well-nourished population, the amount of ascorbic acid received from the mother is apparently sufficient to pre- vent scurvy up to about 5 months of life. But artificially fed infants or infants past the age of breast-feeding need a certain amount of ascorbic acid, about 25 mg/day. Ascor- bic acid is stable in dry form but is easily oxidized in aqueous solution in the presence of air. The rate of oxidation is greatly increased by a trace of copper or by high pH. Processing methods and storage generally reduce the amount of vitamin C in a food. Citrus fruits and their Juices and raw or minimally cooked vegetables are good sources of vitamin C (Holt and Snyderman, 1964). Vitamin A.--Night blindness, xerophthalmia, and kerotomalcia are symptoms of vitamin A deficiency. It is prevalent throughout Asia, the Middle East, parts of Africa, and Latin America. The mortality rate among mal— nourished children with xerophthalmia is very high because of the many other health factors adversely affected by vitamin A deficiency. The incidence of xerophthalmia is increasing (McLaren, 1966) and fortification of the diet with vitamin A is required in these developing areas. The vitamin A requirement for infants is not accurately known yet, but 1500 I.U./day or 0.9 mg B-carotene/day is definitely protective (NAS/NRC, 1964; Heinz, 1959). Vitamin D.-—Infants require 400 I.U. vitamin D/day. Since 7-dehydrocholesterol (vitamin D3) can be converted by provitamin D in the skin under ultraviolet light (Heinz, 1963), the requirement for this vitamin can be met by adequate exposure of the body to sunlight. Vitamin E and vitamin K.--The requirement of vitamin E for normal infants has not been established (Holt and Snyderman, 1964). Vitamin K deficiency in infants occurs only in clinical conditions from malabsorption from the intestine or from liver disease (Holt and Snyderman, 1964). Minerals Calcium.—-The requirement of calcium increases in the stage of skeletal growth from 250 mg/day to 700 mg/day in the first year (NAS/NRC, 1964). Milk-free diets need to be fortified with calcium (Holt and Snyderman, 1964). Iron.--Iron deficiency anemia, which may or may not be associated with protein-calorie malnutrition, is wide- spread throughout the developing areas (Finch, 1966). The requirement of iron for infants is 1 mg/kg bOdy weight (Moore, 1964). Iron deficiency anemia is observed more 10 often in breast-fed infants than in artificically fed infants since milk is a poor source of iron (Holt and Snyderman, 1964). Sodium.--Sodium deficiency could be a problem in children fed_cereal only since cereal grains are a poor source of sodium. Infants require 120 mg/day (Holt and Snyderman, 1964). Other minerals.--Infants require per day 420 mg potassium, 30 mg manganese, 330 mg chloride, and 200 mg phosphorous to maintain health and to promote growth. Usually infants can get enough intake of these minerals from most diets (Holt and Snyderman, 1964). Iodine deficiency is prevalent in mountain areas of the world. In many villages, most people have endemic goiter. Cretinism and dwarfism are the symptoms of iodine deficiency in children. The requirements of iodine for infants is not known yet, but the addition of iodine to the diet is required to cure or prevent this deficiency where it exists (Follis, 1966). m Infants are peculiarly susceptible to a_1ack of water. Water loss through the kidneys and skin is much greater than that of adults because of a larger surface to volume ratio (Holt and Snyderman, 1964). Water re- quirements are influenced by weather; for instance, hot weather increases the water requirement, but not that of 11 other nutrients (Norman and Pratt, 1958). In a temperate climate, infants require 150 c.c. per kilogram body weight, but under subtropical or tropical conditions they may need 175 c.c./kg body weight or more (Holt and Snyder— man, 1964). M Some evidence is reported that infants require a small quantity of polyunsaturated fatty acids (Wiese, Hansen, and Adam, 1958; Adam, Hansen, and Wiese, 1958; Hansen et al., 1958), but what the Optimum intake is has not been resolved (Holt and Snyderman, 1964). However, in the absence of further data, it seems safe and possibly desirable to include some unsaturated fatty acids in infant diets. EnergyrRequirements Energy is required for basal metabolism, physical exercise, and the specific dynamic effect of food. The daily energy need for infants is 120 cal/kg body weight (Hytten and MacQueen, 1954) or as Holt and Snyderman (1964) suggested, 70 cal/kg body weight for placid infants and 130 cal/kg body weight for crying babies. In diet design, there are a number of nutritional factors involved, some more critical than others. If a child eats the right amount of rice to meet his calorie requirement, then he may suffer from thiamine, niacin, l2 riboflavin, vitamin C, vitamin D, vitamin A, and protein deficiency. Present knowledge is good enough to design more adequate diets for infants. Schemes to Prevent or Cure Malnutrition Up to the time of weaning, babies are adequately nourished by their mothers' milk (Gopalan and Belavady, 1961). But in the weaning and after weaning periods, the child is not strong enough to fend for himself in the family circle; protein-calorie deficiency occurs frequently (WHO, 1965). Shortages of milk and other animal proteins, where they occur, and the likelihood that these shortages will persist, has focused attention on vegetable proteins which are relatively inexpensive (Teply and Gy5rgy, 1962). It was reported by several groups that the mixture of a relatively small proportion of skim milk powder (Nicholo and Phillips, 1961; Gopalan, 1961) or of fish flour (Sure, 1957; Seneal, 1961; Graham, Baertle, and Cordans, 1962, 1965) be combined with vegetable proteins to provide ade- quate protein for infants. There are many schemes and projects, all at different stages of progress, for differ- ent areas of the world. The following is a summary of some of these programs. Asia In Indonesia, the incidence of vitamin A deficiency is high. In 1963, red—palm oil was distributed, in one village, to mothers to be fed to children for prevention 13 of xerophthalmia, and the incidence of the disease dropped significantly (GySrgy, 1966). The United Nations Chidren's Fund (UNICEF) provided assistance in producing an infant diet called Saridle, a product based on whole dehulled beans as the sole protein source, originally based on soy _ extract. The standardization of this product is still not well established (FAQ/UNICEF, 1965). The formula consisting of skim milk : sugar : mixture of oils (coconut, peanut, and red-palm oil) in the proportions 45 : 50 : 30 is under study (Gy5rgy, 1966). In India, the Central Food Technological Research Institute, CFTRI, at Mysore, has produced multipurpose foods for infants and pre-school children. These foods have been fortified with vitamins A, D, and riboflavin, and calcium and iron salts. They can be classified into the following groups: (a) High-protein foods (40% protein or more), used as dietary supplements for pre-school chil- dren, based on blends of oil-seed meals, legumes, skim milk powder and fish flour; (b) Cereals and biscuits (18 to 26% protein), used as supplements or complete foods; (c) High-protein foods (60% protein or more), used as a supplement in the treatment of protein malnutrition in children, based on peanut-protein isolate, chickpea, and skim milk powder; (d) A dried milk substitute, used for infants, is based on soybean, ground nut, and buffalo milk, and when used for weaning infants, is based on micro- atomized oil-seed meal (Subrahmanya et al., 1957; l4 Venkatachalam, and Srikantic, 1960; Bhatia and Swaminathan, 1963; Doraiswamy et al., 1963; Parpia, 1966). In Lebanon, evaluation of protein in sweet almond, which is used for infant feeding, was done by Cowan e£_a1. (1963). Methionine, threonine, and tryptophan are three limiting amino acids. The supplementation of sesame, a good source of lysine and threonine, and chickpea, a good source of lysine and threonine, might improve the quality of almond as Cowan suggested. But he believed that within the existing dietary pattern in the Middle East, such a food would not be accepted. In the Philippines, the infant mortality rate in 1962 was 2.7 times higher than in the United States; the death rate in children aged 1 to 4 years was forty- five times higher (Department of Health, 1962; U. N., 1963). In 1962, the Department of Health of the Philips pines reported that beriberi was one of the leading causes of infant death (Department of Health, 1962). In 1948, the National Rice and Corn Cooperation, NARIC, manufactured and distributed to Bataan province a premix which was added to rice and which contained thiamine, niacin, and iron. The mortality from beriberi was reduced to zero after two years of using enriched rice in that province. The price of this enriched rice to the consumer is 2% higher (Furter et al., 1946). The Philippine experi— ence is a good but discouraging example of a food which will solve the nutritional problems, but 15 has not, for various reasons, been widely used. As Williams (1964) said, ". . . the extent to which rice enrichment is practiced in the Philippines is virtually negligible." In Taiwan, 4-month old infants from lower middle class families were fed a test diet based on soybean, to which rice flour, sugar, minerals, a vitamin mixture, and soybean oil were added. It was found, based on a 12-month feeding test, that these full-fat soy products were prac-- tical and satisfactory dietarydalternatives to animal milk provided that they were fed at levels which are more than critical in proteins and calories (DeMaeyer, 1965). Africa For infants in Uganda, the British have tried a - w”“ fortified biscuit made of peanut, cereal flour, and sucrose (FAD/UNICEF, 1965). In Senegal, the French have tested a millet, peanut, and fish mixture for infants. It was hoped that by 1965 a precooked weaning food, couscous, made from grain sorghum, peanut protein concentrate, skim milk powder, and sugar would be introduced on the market (Teply, 1964). In Ethiopia, it is planned that the Children's Nutrition Uhit in Addis Ababa will provide basic facts and outline general policy for infant feeding. A basic health service would then be the institution responsible for an ". . . integrated combat against malnutrition" (Mannheimer, 1966). 16 In South Africa, ProNutro, a supplement containing 21.5% protein, 265 cal/100 g, has been produced in Durban by a commercial firm. The initial product, Incumbe, was made in 1938, and since then has been much modified and improved to the present product, ProNutro. The final formula consists of skim milk, whole soybean and peanuts, fish protein concentrate, food yeast, wheat germ, whey powder, bone meal with addition of iron, iodized salt, thiamine, riboflavin, and niacin; sugar is added for palatability. ProNutro was clinically tested successfully in the prevention of kwashiorkor and other Severe states of under or malnutrition in pre-school children (Simson and Mann, 1961) and other age groups. Two products are available; standard flaked ProNutro is 14 U. S. cents per pound, soup powder is 15 U. S. cents per pound (Belden et al., 1964). Central and South America In Central America, Incaparina, a dietary supplement for preeschool children, was developed at the Institute of Nutrition of Central America and Panama (INCAP). Incaparina is the name applied to vegetable protein mixtures, in which there is at least 25% protein of quality comparable to milk protein. In Guatemala, Mexico and Colombia, Incaparina formula9B is in commercial production. This formula con- sists of 29% ground whole maize, 29% ground whole sorghum grain, 38% cottonseed flour, 3% torula yeast, 1% calcium l7 carbonate, 4,500 I.U. vitamin A. per 100 grams, protein content 27.5%. Some choice in the main calorie compon- ents is allowed if local products are more available and cheaper. The nutritive value of this mixture has been proved by amino acid analysis, many feeding trials in rats, chicks, and swine, and clinical tests in children, includ- ing treatment of kwashiorkor and nitrogen balance studies. Incaparina can be drunk as an £3913, which is made by adding water (one glass of water for 25 g of incaparina), some sugar, and cooking for 25 minutes. It can also be incorporated into bread, puddings, and soups. Incaparina is being sold in polyethylene bags containing 75 g at a cost of 3 U. S. cents per bag (Shaw, 1964). In Brazil, with the assistance of UNICEF and tech- nical advice from FAO and WHO, Fortifex, a 30% protein- rich food, is produced by the Nestle Company of Brazil. The formula consists of corn starch, soy flour, methionine at 0.2%, calcium barbonate, vitamin A, riboflavin, and thiamine. The results of rat and child feeding experi- ments demonstrated that Fortifex is a useful child feeding supplement and as a weaning food. The price is about 2 U. S. cents per 100 grams. Distribution is now being initiated on a large scale (Teply, 1964). In Peru, the evaluation of fish protein concentrate, FPC, alone and in combination with wheat has been done by 18 Graham (Graham, Baertle, and Cordans, 1965). The combin- ation of FPC with wheat is superior to FPC alone. Wheat noodles enriched with 10 percent fish protein concentrate were used as a dietary supplement in experiments and gave good results (Graham et al., 1966). In Chile, there has been work at the University of Chile on an adaptation of fish flour for human nutrition and especially for infants. They have tested bread, some forms of pasta, and soup enriched with FPC and got en- couraging results on the small groups tested (Mancksberg, 1966). Europe In Yugoslavia, a project to achieve the production of a low cost, cereal based, dry food mixture including vegetables for infants is underway with the assistance of UNICEF and technical advice from FAO and WHO (UNICEF, 1964). U.S.A. and Canada At Fayetteville, Sure (1957) added a small amount of defatted fish flour to milled wheat flour, corn meal, and rice, and found fish flour had high protein content and high biological value and improved the nutritive value of mixtures which can be used for infant feeding. Sabby, Middleton, and Morrison (1962) found that the procedure used for defatting can markedly influence 19 the nutritive value of fish flour to be used as a protein supplement to infant foods. Do-Quang—Oanh (1963) proposed fortified foods for Vietnamese babies, in which rice at 70-80% of the diet was the basic ingredient. The protein content was brought up to 15% by the addition of soy, cod, egg, pork or a com— bination of soy and cod. The addition of sweet potato provided vitamin A, yeast provided vitamin B complex (except B12), calcium carbonate and salt were added. From PER measurements, the cod and soy supplement appeared to be the best. Summary There are more plans, projects, and programs than action. Generally, large scale application of test results to improve the nutritional status of developing countries has not been accomplished. With two exceptions, all schemes have in common improved protein quantity and quality. Most schemes were designed primarily for pre—school children. Some used a mixture of vegetable and animal proteins, others used only vegetable proteins to increase the degree of utilization of proteins. A11 add one or more vitamins and minerals, and in one case a pure amino acid is added. Proteins for these schemes must come, in many cases, from supplies which are locally produced, but are already in short supply, other proteins must be imported (e.g. milk). 20 An exception is Incaparina which uses cottonseed protein, not formerly used for human consumption. All schemes have one or more (and usually more) ingredients which require s0phisticated processing operations. DESIGN OF THE EXPERIMENTAL DIETS It seems clear that the nutritional problem of infants in the rice-eating countries of Asia (FAO, 1948; FAO, 1956; FAO, 1963) is many-fold. Improvements must be made in protein quantity and quality; vitamins must be added. In this study the main emphasis was placed on protein potency, the amino acid pattern of the diet. The solution was to try to find the best combination of vegetable and animal protein. Rice, the staple food in these areas, was used as the basic ingredient, the main calorie source. The protein level in the diets was raised to 15% as Davidson suggested (Davidson, Meiklejohn, and Passore, 1961) by the addition of soybean flour and cod fillet flour or egg powder. The nutritive values of these three animal proteins have been established (Patward- hon, 1961; Steinkraus, Van Buren, and Hand, 1961; Teply and Gyorgy, 1962; Standal, 1963; DeMaeyer, 1965; Peeler et al., 1951; Morrison, Sabby and Middleton, 1962; Pretorius and Wehmeyer, 1964; and WHO, 1965). Two per cent sweet potato was added as a source of provitamin A, 4% yeast as the source of vitamin B complex (Hock, Horn, and Dast, 1956; Scrimshaw and Behar, 1961); 0.5% calcium carbonate and 1.0% sodium chloride (see Table 2). The theoretical protein scores and nutritive values of the 21 22 proposed diets were calculated from tables of McCance and Widdowson (1960), Orr and Watt (1957), and Watt and Merill (1963). Nutritive values of the proposed diets were about the same and met infant requirements, except for vitamin C (see Table 3). TABLE 2.--Composition of diets, g/100 g. Ingredient Diet COD-SOY SOY-COD SOY-EGG Rice flour 83.7 74.8 73.0 Soybean flour 2.8 16.2 13.5 Whole egg powder 6.0 Cod fish flour 6.0 1.5 Sweet potato 2.0 2.0 2.0 Yeast 4.0 4.0 4.0 CaCO3 0.5 0.5 0.5 NaCl 1.0 1.0 1.0 Protein scores based on various standards (see Table 1) were plotted as a function of the amount of the main protein supplements as shown in Figure 1. The ob- Jective was to cover a range of combinations of an animal and vegetable protein with a limit on the total protein. Six percent and 1.5% cod fillet flour and 6% egg powder were selected as the amounts of animal protein for these 23 IOO I I 1 HUMAN MILK PROTEIN SCORE 70 ~ " CODFISH FLOUR —— — WHOLE EGG POWDER so ‘ 1 ‘ o 2 4 5 8 CODFISH FLOUR on WHOLE EGG POWDER,% Fig. l.-—Protein score, based on three different standards (human milk, FAO 1957, and whole egg), as a function of the main protein supplement, total protein content at 15%. (Amino acid content from McCance and Widdowson, 1960, and Orr and Watt, 1957.) 24 TABLE 3.——Nutrients in the diets,a per 100 g. Diet Ingredient a COD-SOY SOY-COD SOY-EGG Water, g 9.54 8.95 8.52 Protein, g 15.4 15.8 15.8 Fat, g 1.64 4.25 5.86 Ash, g 2.56 2.78 2.8a Crude fiber, g 0.75 0.99 0.93 Calcium, mg 240.0 260.0 270.0 Iron, mg 1.9 2.7 2.9 B—carotene, mg 0.97 0.99 ' 1.04 Thiamine, mg 0.68 0.79 0.82 Riboflavin, mg 16.0 16.0 17.0 Niacin, mg 4.4 4.0 3.0 aMinerals and vitamins, except Vitamin A, were calculated from Watt and Merrill (1963). The rest of the nutrients were measured. 25 feeding trials. The rest of the protein was from soybean flour and, of course, rice, yeast, and sweet potato; total protein was 15%. In a sense, the ideal pattern was being tested at the same time, because, if human milk represented the ideal pattern, then there should be little or no difference between 1-1/2% and 6% cod; on the other a hand, if FAO, 1957 or whole egg were the ideal, then 6% cod might be much better. . The proposed diets are intended to be cooked with )“ water and are expected to have a flavor, texture, and consistency similar to rice gruel, the traditional infant food. The proposed diets are intended to be cooked with water and are expected to have a flavor, texture, and consistency similar to rice gruel, the traditional infant food. EXPERIMENTAL Preparation of Ingredients Rice Flour Commercial rice flour of unknown history was used. Soybean Flour Chippewa soybeans, 1964 crop, partially cleaned, were soaked in cold water overnight. About 10% of the skins of the beans were removed. Then the beans were washed and autoclaved in a steam retort at 110°F for 30 minutes. They were initially dried at 160°F for 5-1/2 hours in a Proctor and Schwartz cabinet drier and kept in the drier for three days. Following this treatment, on the fourth day, the beans were dried at 150°F for another two hours. During the drying period, the beans were turned over and the tray positions were changed in the drier once an hour to obtain a more uniform drying temperature. While still warm, the dried beans were ground in a Wiley mill, first through a No. 20 sieve and then through a No. 40 sieve. The soybean flour was prepared about 20 weeks before the first feeding test (Test 3) in the present study. The flour was stored in sealed glass bottles and refrigerated until used. 26 27 Codfish Flour At 108 days before Test 3, frozen cod fillets were cooked in a steam kettle at boiling temperature for 20 minutes, including time to thaw, with occasional stirring. The cooked fish was drained by pressing lightly, through cheese cloth, and broken into coarse pieces by passing it through a food and meat chopper. Then the coarse pieces were placed on perforated trays, dried at 184-180°F for one hour and then at 158-162°F for two hours successively in the cabinet drier under cross air flow. The dried fish was ground in a Wiley mill first through a No. 20 sieve, then through a No. 40 sieve, and stored under refrigeration in sealed glass bottles. Whole Egg Powder A commercial product, glucose reduced and stablized, of unknown history was used. Yeast A commercial product, Anheuser-Busch chipped yeast, strain G was used. Sweet Potato The Goldrush, precooked dehydrated sweet potato flakes, provided by the Southern Regional Research Labora- tory of the U. S. Department of Agriculture were used. In their procedure, the potatoes were preheated, lye peeled, and made into a puree of 20% solids with water, to which 28 0.01% sodium sulfate, 0.04% sodium bisulfate, and 0.1% antioxidant Tenox VI (all dry basis) were added. The puree was dried on a drum drier for 17 seconds. The dehydrated sweet potato flakes were packed in tin cans and sealed in an atmOSphere of nitrogen (Deobald gt_al., 1962). Before use in these studies the flakes were ground in a Wiley mill through a 40 mesh sieve. Dog Chow A commercial food, Purina Dog Chow, ground in a food and meat chopper, was used. Calcium Carbonate A chemically pure grade was used. Sodium Chloride Diamond crystal salt, food grade, was used. CASEIN Diet A standard CASEIN diet suggested by Campbell (1963) was used in the animal feeding trials as a means of com— paring PER data from different laboratories and of avoiding or correcting for variations in technique from one experiment to another. The 10% protein content special CASEIN diet was prepared by General Biochemicals, Chagrin Falls, Ohio. The diet consists of 67.7% corn starch, 12.3% casein, 10.0% corn oil, 5.74% non-nutritive fiver, 4.00% sale mix U.S.P. XIV, and 0.26% vitamin mix. 29 Salt mix U.S.P. XIV consists of 0.009% ammonium alum, 11.280% calcium biphosphate, 6.860% calcium carbonate, 30.830% calcium citrate, 0.008% cupric sulfate, 1.526% ferric ammonium citrate, 3.520% magnesium sulfate, 0.020% manganese sulfate, l2.470% potassium chloride, 0.004% potassium iodide, 21.880% dibasic potassium phosphate, 7.710% sodium chloride, and 0.050% sodium fluoride. The vitamin content in 100 pounds of CASEIN diet is 2.2700 g vitamin A, 0.1135 g vitamin D, 4.1270 g DL—alpha tocopherol, 0.2270 g menadione, 0.2270 thiamine hydrochloride, 0.4540 g riboflavin, 0.1816 g pyridoxine hydrochloride, 1.8160 g calcium pantothenate, 1.8160 g niacin, 90.8000 g choline chloride, 11.3500 g inositol, 4.5400 g para-amino benzoic acid, 0.9080 g vitamin B12 (0.1% triturationwith mannitol), 0.0091 g biotin, and 0.0908 g folic acid. The proximate analysis of the CASEIN diet was 7.45% moisture, 2.71% ash, 5.69% crude fiber, 10.35% protein, 11.84% fat. All ingredients, Dog Chow, CASEIN diet and the proposed diets were stored in a 3-5°C cool room before chemical analysis and feeding trials. Chemical Analysis of Ingredients The nutritive values of the proposed diets are de- pendent upon their ingredients. A proximate analysis of materials was carried out by the methods described below; 30 the results are summarized in Table 4 (all analyses except B—carotene followed AOAC, 1960, methods or modi- fications thereof). Moisture Duplicate 10-g, well mixed samples of all ingredi- ents were accurately weighed in cool moisture dishes, dried in an electric oven at 105 : 1°C for 5 hours, transferred to a desiccator, and weighed after half or one hour when they had cooled to room temperature. The residues are reported as total solids and loss in weight as moisture. The residues were used for ether extraction. _A_s_r_1 Duplicate 5-g samples were accurately weighed into ash crucibles, carbonized first under a Bunsen burner, then put into a muffle furnace which was preheated to 600°C, and held at this temperature for two hours. The crucibles were transferred directly into a desiccator, cooled to room temperature, and weighed immediately. The remaining weight is referred to as ash. Crude Fat Duplicate 10—g samples, residues from the moisture determination, were extracted with petroleum ether in a Soxhlet apparatus for 16 hours. The petroleum ether was evaporated under vacuum. The flasks were kept in a desiccator overnight and then weighed. The percentage of fat is referred to the original sample weight. TABLE 4.--Proximate composition of the ingredients, g/100 g. Ingredient Water Protein Fat Ash RISE: Rice flour 11.0 7.52 0.99 0.81 0.63 Soybean flour 4.92 35.6 18.3 4.24 1.90 Cod fish flour 3.24 91.1 1.08 4.35 0.00 Whole egg powder 4.28 46.8 41.9 3.56 0.00 Sweet potato 3.84 8.17 1.00 4.13 4.40 Yeast 5.11 52.1 1.92 6.45 3.28 Protein Duplicate 0.7—2.2 g samples were accurately weighed and put into digestion flasks to which 0.7 g HgO, 15 g anhydrous Na2SOu and 25 m1 H280” were added. Two or three "tamer tabs" were added for taming the boiling action. Flasks were placed in an inclined position and heated until frothing ceased. Samples were hydrolyzed at least 30 minutes beyond the time at which the solutions became clear. The solutions were cooled below 25°C, 200ml cool water and 25 m1 thiosulfate solution were added, and mixed to precipitate the mercury. A few zinc granules were added to prevent lumping. The flasks were tilted and 25 g solid NaOH were added without agitation. The 32 flasks were immediately connected to the distilling bulbs on the condensers and the tips of the condensers were immersed in standard acid solutions in the receivers. Then the flasks were rotated to mix the contents thoroughly, and heated until all NH had distilled (at 3 least 150 m1 distillate). The excess standard acid in the distillate was titrated with NaOH, using methyl red as an indicator. The results were corrected with a blank. Factors to convert g N to g protein were taken from Orr and Watt (1957). Crude Fiber Duplicate 2-g samples, residues from ether extrac- tion, were used. To the samples in the digestion flasks were added 0.5 g asbestos, and 3 drops dilute Dow Corning Antifoam A emulsion (l + 3) and 200 ml of boiling 1.25% H280“ solution. The flasks were immediately connected with the reflux condensers, rotated frequently until the samples were thoroughly wetted, and heated exactly 30 minutes. Samples were kept in contact with the solution. After 30 minutes, the flasks were promptly removed and the solutions were filtered through linen in fluted funnels; the samples were washed with boiling water until the washings were no longer acid. Then the samples were returned to the flasks to which 200 ml boiling 1.25% NaOH solution were added. The flasks were connected with the 33 reflux condensers again and boiled exactly 30 minutes. Then the flasks were removed immediately, and the solu- tions filtered through Gooch crucibles which were prepared with asbestos mats. The residues were washed with hot 10% K2804 solution whenever the filtration became dif- ficult. After being thoroughly washed with boiling water and 15 ml alcohol, the crucibles and contents were dried at 105°C for 5 hours to constant weight, then cooled in a desiccator and weighed. The contents of the crucibles were ignited in an electrical muffle furnace at 600°C, then cooled in a desiccator and weighed. The loss in weight was reported as crude fiber. Carbohydrate Carbohydrate was not determined, but the calorie content of the diets was estimated. The carbohydrate was assumed to be the difference between total weight and the sum of water, protein, fat, ash, and crude fiber. The calorie calculation was based on the Atwater system using energy values from Watt and Merrill (1963). The results were COD-SOY 360, SOY-COD 366, SOY-EGG 377, and CASEIN 401 cal/100 g. B—carotene (Wail and Kelly, 1943) Duplicate samples (10 g in the case of soybean flour, 1 g in the case of sweet potato flakes) were accurately weighed in 100 m1 beakers. Thirty milliliters of hot 34 distilled water were added and mixed well with the samples. After 20—30 minutes hydration, 75 ml 95% ethanol were added to the samples and then stirred thoroughly and left overnight in a dark room. Thirty milliliters Skellysolve B (normal hexane) was added to the beakers and stirred thoroughly. The solutions were transferred and passed through glass fritted filters, which were connected to suction flasks. The residues were washed with alternate portions of 30 ml alcoholand 30 ml Skellysolve B until colorless filtrates were obtained. The filtrates were transferred into 500 ml separatory funnels to which 100 ml H20 and 50 m1 5% NaBSOu were added. The funnels were swirled and turned upside down while the stoppers were held and the stopcocks were opened to release pressure. The lower aqueous alcoholic solution was extracted three times with 30 ml Skellysolve B and 100 ml water. The Skellysolve layer was filtered through a filter paper con— taining 1 teaspoonful of anhydrous sodium sulfate into an Erlenmeyer flask containing a small amount of anhydrous sodium sulfate. Then the contents were condensed to 25 m1 on a steam bath; the filtrate was transferred into an absorption column prepared with a mixture of three parts Hyflo Supercel and one part activated magnesium. After the sample had all been transferred to the column, the carotene was eluted by pulling a Skellysolve B-acetone 35 mixture (95:5) through the column. About 50-70 m1 of solvent was required after the first color came through. The elute was brought up to 100 ml with Skellysolve B and the transmittance was measured with an Evelyn colorimeter at 440 mu. The B-carotene content was determined from a standard curve. For dry egg powder, duplicate l g samples were weighed in 150 ml beakers to which 4 m1 H 0 was added. 2 The samples were stirred to a paste, and 50 ml acetone was added, a little at a time, starting with about 5 ml. After standing for 5 to 10 minutes, samples were filtered through Watman No. 4 filter paper and washed thoroughly with acetone. Filtrates were collected in 100 ml volumetric flasks and brought up to 100 ml with Skellysolve B. The transmittance was measured in an Evelyn colorimeter. Ingredients had been stored in a refrigerator for two months before analysis. The B-carotene in the dehy- drated sweet potato flakes was 50.5 mg/100 g, egg powder 2.15 mg/100 g, soybean flour 0.137 mg/100 g. Bioassay The proposed diets were evaluated before and after storage by bioassay in Test 31 and Test 5. Since 15% protein level of a high quality protein of proposed diets 1The first two feeding experiments in this project of which the present study is a part were presented by Do-Quang-Oanh (1963). 36 were evaluated at 10% protein in Test 4. The protein level was reduced to 10% by adding corn starch to the diets. In all feeding trials, male weanling rats of Holtz- man strain were used. First the diets were assigned randomly to the cages and some adjustments were made to avoid what seemed to be too many cages on a side or a tier for one diet. Each rat as it was removed from the shipping box was weighed and assigned to a cage at random. Some adjustments were made upon receipt and just before the rats were put on the test diet to get equal average weights among groups. The diets were prepared from ingredients by mixing them in a Patterson-Kelley Liquid-Solids Twin Shell Blender for 15 minutes. In all three testing periods, rats were kept in the same room in which the lights were turned on from 8:00 A.M. to 5:00 P.M. every day during the trial. All rats were kept in individual cages (9.5" x 8" x 7.5") with screen bottoms, and received food in Franke jars (capacity about 65 ml) and water ad libitum. Food consump- tion was recorded every other day; the amount of spilled food was estimated. In the large majority of cases, 1/2 g or less was spilled in any 2-day period. A few rats spilled as much as 6 g/day; when a rat had this habit, his spilled food was collected and weighed. CASEIN diet was subject to more spilling than any of the others. Each rat was weighed to the nearest half gram every four days. 37 They received fresh water every other day. In Tests 3 and 5, respiratory infection was widespread, but much less so in Test 4; however, there seemed to be no complete correlation between weight gain and infection. The rats were killed at the end of the test periods. Third Feeding Experiment Rats 23 days old were kept in a room maintained at 76-80°F. Upon receipt, the rats were fed Dog Chow for one day. There were 12 rats on each test diet and the control group which was fed with the standard CASEIN diet. The initial weights of the rats at zero days (September 23, 1965), average 57.7 g, range from 54-63 g, were not significantly different among groups. The growth response of the rats was tested for 28 days and is plotted in Figure 2. Performance is summarized in Table 5 from re- duced data, reduced by discarding the results from five very sick rats. There Was no difference (5% level) in PER among COD-SOY, SOY-COD and SOY-EGG (EMS = 0.026). All of these three diets were good as judged by PER and growth rate. Fifth (storage) Feeding Experiment The same diets used in the third test, COD-SOY, SOY-COD, SOY—EGG, and CASEIN, were stored at high humidity (100% relative humidity) and high temperature (35°C) for 145 days. 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