r ”L i. {7‘23” ‘ .. in T“- ‘. [Y 1:; . . O 0 4.4.2": I‘ "4-7;. {ti 3“ ' *1 . ._ . , _ l m;.¥~‘¢§i ' ‘. < . 95*- ‘9’... '.- , WW _I mg. . '. "on. -w nos—q. W-..” -Q. _ ;-J --- pod u..- ' . .. - u: . . I 4 ‘ -. Diw-J‘ " - -. ' I. '4... '~ ~ aw h—aa-v- -m.m ' ¢_.. - o . < 1 . . .. '. - A J 71 ~- ~. 9...... *-.:|;;v1;1 .4 1" . 1 11 M 11% 11”, ‘1); 111.!!flséh1‘mwl? Win 1:: 1,11: 11'1111JM11' 1111.3!!1i1'i'lfi1 ”rm d‘z‘l'tz'hr' ”('1 W191! 'l‘ I { 'v'. .‘1| 1“" i, 5:,- ”Wm S“ \!l I ' -:-:5¢.-.-‘ 1,. .1. . .3- . «guy: . - v n Y. . .2,- _ ‘ ... . I 2 . A“ . . . . - .. .a—r. ,.,- v . . —- or H - - .v-t.-:..'~.. (Mu- ~— I o . o _.. . . 1n . "I 1 :."“¢ oooo.m ooa mma ~.m m.~ maflcwumwm mamm.a mm mm ~.H s.a cmsmoudsue mvmm.a vm vw H.v m.v ocflcomuca oooo.~ ooa Hoa ~.> H.n ocflam> mamm.a mm mm m.m m.m mcaosoaomH oooo.~ OOH HHH m.m m.m wcfloswq oooo.m ooa maa 5.0H m.m mcamouma paw maficmamawamnm vmmn.a mm mm m.m m.m mcwumwo can wcacownumz oooo.m ooa eoa H.m m.h scammq monumm osnmm oaumm xz mmaxmv 12 sma\mv manna cassa Umuomunoo pouomuuoo mom Gammmo camuoum mo mmoq mom oaonz ll .xmch paod oases adducmmmm cmflmapoz can mo cosponsmEoull.¢ mamas 25 The geometric mean of the corrected egg ratio (values above 100 are corrected to this value) is computed by taking the logarithm of each egg ratio, averaging these logarithms and then obtaining the antilogarithm of this value. In the example, the average logarithm was 1.9603; and its antilogrithm, 91, is the modified EAA index for casein. Protein Scores by the FAO[WHO Procedure In 1963, the FAQ/WHO Expert Group modified the computation of the chemical score in order to obtain a better agreement with the experimental biological value. The whole egg provides more than double the requirements of each essential amino acid necessary to maintain nitrogen equilibrium in the adult man (Allison, 1958). The calculation of the protein score is done as follows: a. Add the amounts of all the essential amino acids together with those of cystine and tyrosine. b. Calculate the percentage contributions of the potentially limiting amino acids to this total. c. Compare these percentages with the corresponding ones for the reference pattern. The concentrations of essential amino acids in whole egg protein and the new reference pattern (FAQ/WHO, 1973), which was used as the reference, in this study are given in Table 5. 26 TABLE 5.--Essential Amino Acid Composition of the Whole Egg Protein and the New Reference Pattern (FAQ/WHO, 1973). FAQ/WHO Reference Whole Egg Pattern Amino Acids (g/l6g N) (9/169 N) Lysine 7.8 5.4 Methionine and Cystine 5.3 3.5 Phenylalanine and Tyrosine 9.3 6.1 Leucine 8.8 7.0 Isoleucine 5.9 4.0 Valine 7.1 4.9 Threonine 4.9 4.0 Tryptophan 1.4 1.0 Histidine 2.6 -- Total 53.1 36.0 Biological Value (BV) Protein quality bioassays with rats are subdivided into those based on weight gain and those based on N retention in the whole body either by direct carcass analysis or by N balance techniques. Thomas (1909) introduced the concept of biological value (BV) and the method of assessing BV with humans. Since that time, the term biological value has been synonymous with protein quality. He used adult subjects, but Mitchell (1923) adopted the method to both growing and adult rats. 27 Biological value is usually expressed by the following equation: N retained BV x 100 N absorbed The second equation expresses more meaningfully the biological value: I-(F-FK)-(U-UK) B-(BK-BO) or I- (F-FK) I- (F-FK) BV A = Absorbed nitrogen [I-(F-FK)] (:1 II Body nitrogen (at the end of test period) B = Body nitrogen at zero nitrogen intake (at the end of test period on animals fed a nonprotein diet) B0 = Body nitrogen at zero time (measured on a control group of animals at the beginning of the test period) F = Fecal nitrogen '11 ll Metabolic nitrogen (endogenous fecal nitrogen) I = Nitrogen intake U = Urinary nitrogen U = Endogenous urinary nitrogen Mitchell (1923) reported that the biological value was affected by the level of protein in the diet. He provided data which showed that an increase in the protein content of diets, when the protein sources were milk, corn, oat and potato, from five percent to ten percent, resulted 28 in a decrease in BV on the average by eleven percentage points. Even though the customary equation for BV looks simple, the method is too involved to be used as a routine procedure for protein quality. Net Protein Utilization (NPU) Net protein utilization (NPU) was derived from the Thomas-Mitchell procedure (Mitchell, 1923) for the deter- mination of biological value. NPU is defined by the FAQ/WHO Expert Group (1965) as the proportion of nitrogen intake which is retained, i.e., the product of biological value and digestibility. A I-(F-FK) Digestibility = —~= I I B I-(F-FK)-(U-UK) B-BK NPU = — = or I I I Protein Efficiency Ratio (PER) Osborne, Mendel and Ferry (1919) defined "a method of expressing numerically the growth-promoting value of proteins involving the determination of the gain in body weight per gram of protein consumed at level of dietary protein associated with the highest protein efficiency ratio (PER)." 29 Gain in body weight (9) PER = Protein intake (9) Protein efficiency ratios appear to be related reasonably well to other methods of evaluating proteins. Block and Mitchell (1946) found that there was a good relation between PER's and biological values and also between PER's and chemical score based on amino acid composition. Bender (1956) reported a good correlation between PER and net protein utilization. Since protein efficiency ratios are well correlated with several other methods of protein quality evaluation and since it is probably more frequently used than any other method and it is still the only official method for protein quality determination (AOAC, 1970), it was decided to use this method for evaluating protein quality in this work. Determination of PER requires strict adherence to certain conditions. It has been shown that several factors influence the PER determination: a. Strain and sex of rats (Morrison and Campbell, 1960): It appears, however that there is as much difference between strains of rats as there is between sexes. b. Species differences: Hegsted et al. (1947) and Mitchell (1954) reported that the results of growth trials in rats could be generally applicable to the assess- ment of human diets. Flodin (1959) found a good correlation 30 between PER values in rats and biological values in the adult man. Hegsted (1957) reported that the amino acid requirements of man and of the rat are, generally speaking, the same. Allison (1957) found remarkable correlations in the nitrogen balance indices of six different proteins in man, dog and the rat. c. Age of the rat and assay period: Chapman et al. (1959) have shown that significant differences could be obtained in PER values between rats put on test at 22, 36 or 45 days of age. In Osborne's (1919) original method, the assay period was eight weeks. According to Chapman et al. (1959) and Morrison and Campbell (1960), the coefficient of variation of PER's had a tendency to drOp after the first week and would be generally lower at the end of the third or fourth week, which indicates that the assay is becoming more stable at that point. d. Protein level: obviously comparisons will be valid if the only variable between one diet and another is the nature of the protein, i.e., all diets should have the same nitrogen content. Different levels of protein in the diet have been proposed in the literature. The standard AOAC method recommends 10% protein. Since such discrepancies would make impossible the comparison of data obtained by different researchers, it was felt necessary to standardize the method. The official method finally adOpted is the one proposed in 31 1960 by the Association of Official Analytical Chemists (AOAC) which is described in detail in the Materials and Methods section of this thesis. Net Protein Ratio (NPR) The protein efficiency ratio method has been criticized because it does not take into account the protein required for maintenance, since only gain in weight is used in calculation. Bender and Doell (1957) introduced the net protein ratio method in which a group of animals fed a protein- free diet is included in each test. Net Protein Ratio (NPR) is defined as the overall difference in gain (gain in weight of the test group plus loss in weight of the protein-free group) divided by the amount of protein eaten. NPR was shown to be highly correlated with NPU: Y = 3.3 + 15.5 X (Y = NPU, X = NPR); r = 0.986; P < 0.01 (Bender and Doell, 1957). Slope Ratio Technique The Relative Protein Value (RPV) or slope ratio technique was proposed by Hegsted and associates (Hegsted and Chang, 1965; Hegsted and Worcester, 1966; Hegsted et al., 1968; Hegsted and Neff, 1970). In this method the slope of the regression line relating body weight or body protein or body water of young rats fed a standard 32 protein (egg protein (n: lactalbumin), assumed to have maximal nutritive value, was compared to that of rats fed the test protein at various protein levels. It was originally assumed, as in the estimation of BV and NPU, that the dose-response lines should have a common inter- cept at zero dose. However, using several levels of intake, this assumption can be statistically tested; and it is shown, for most proteins, the regression lines do not have a common intercept. But, when young rats are fed diets of varying protein content, up to levels that permit substantial increases in body weight, the slope of the regression line may not be greatly influenced, whether or not one forces the regression through a common zero point. However, since it is certain that the regression lines do not meet at a common point, the slope of the regression line relating dose to response should be calculated for each protein individually and then compared to that of the standard protein (Hegsted, 1974). This method is claimed to be especially appropriate for evaluating protein quality when the protein content of the food is 10% or less (Hegsted, 1974; Elias et al., 1974). These authors evaluated protein quality in such foods as rice, corn and cassava by using regression equation between protein intake and weight gain. They considered the method to be appropriate for poor quality protein at 33 higher levels of intake, and in fact for any situation where the response (growth) is low whether it is due to protein quality or quantity. Microbiological Methods The microbiological procedures for the determination of the nutritive value of proteins and the availability of individual amino acids have passed through several stages. First, the production of lactic acid was used as a measure of growth of bacteria in a medium in which one amino acid was limiting. Next was to assay the overall value by measuring the growth response of microorganisms such as Leuconostoc mesenteroides and Streptococcus faecalis after ‘hydrolysis of the protein. In this technique, the results were compared with those of a casein standard and termed "relative nutritive value." The next development was to use microorganisms that would themselves hydrolyze the protein and so measure the available amino acid as distinct from total amino acid content. The organisms used included Streptococcus gymogenes, Pseudomones aeruginosa and the protozoan, Setrahymena. In general, the results correlated well with rat PER and NPU assays, which indicated a similarity between the microbes and rat requirements. 34 Methionine Bioassay Schweigert and Guthneck (1954) investigated the utilization of methionine with the young growing rat and the protein-depleted adult rat. Hydrogen peroxide-treated casein (oxidized casein), with appropriate supplements, was used as the source of amino acids in the methionine deficient ration. The rate of gain for the l4-day test period for groups receiving the graded levels of methionine was followed. They studied the methionine utilization in several animal and plant proteins. It was concluded that less variation among individual animals within each group was observed with the weanling rat than with the adult rat. The percentage of methionine utilized for weight gain varried for the different foods from 48 (unheated soybean flakes) to 83 (fresh pork) with the weanling rat. The methionine utilization from casein was high in both tests, 81% for weanling rat and 84% for the adult rat. They reported that 80% of the methionine from soybean oil meal was utilizable for the growth of weanling rats. DeMuelenaere and Feldman's (1960), using the rat ‘ as the test animal, reported an estimate of 95% for the availability of methionine in corn. Evans et al. (1974) using a balance study with growing rats showed that the availability of bean methionine and cystine was poor when compared to that found for the same amino acids from soybeans. They also indicated that 35 the degree of availability of these amino acids correlated with the PER values obtained for either beans or soybeans. Several investigators have used chicks as the test animal for bioassays of methionine. Miller et a1. (1965) showed by direct determination of available methionine by a growth procedure with chicks, that a large proportion of methionine of animal protein concentrates can be present in an unavailable form. Le umes Leguminosae comprise approximately 600 genera with around 13,000 species. Today, only a few species, about 20, are used for human food. Eight of these are extensively grown, but even within this group there are striking differences in the area of adaptation and use. Roberts (1970) has grouped them into four major classes depending primarily upon climate requirements. A. Low humid tropics 1. Pigeon Peas (Cajanus cajan) 2. Cow Peas (Vigna sinesis) B. Semi-dry or seasonal tropics 1. Ground nuts or peanuts (Arachis hypogaea) C. Tropical intermediate elevations to temperate zones 1. Soybeans (Glycine max) 2. Dry Beans (Phaseolus vulgaris) 36 D. Cool weather, high elevation zone 1. Chick Peas (Cicer arietinum) 2. Peas (Pisum satinum) 3. Broad beans (Vicia faba) Next to soybeans and ground nuts, peas and dry beans are the most extensively grown legume crops. Data on production and acerage of the major food legume crops and wheat, rice and corn (FAO, 1972) are given in Table 6. The data indicate that of the total area planted with legumes, which includes legume foods, soybean and peanut, close to 54% of it is in legume foods (pulses). This statistic also demonstrates that the area for cultivation of legume foods is about half of that allotted to rice and almost one-fifth of wheat and corn. Legumes are nutritionally important among vegetable foods because of their relatively high protein content. The protein located primarily in the cotyledons and embryonic axis, with only small amounts present in the seed coat (Singh et al., 1968). The seed coat of the Navy bean seed contains 4.8% crude protein, while the cotylecons and the embryonic axis have 27.5 and 47.6%,respectively. Since the cotyledons represents the greater part of the whole seed, they contribute the major amount of protein to the whole seed (Singh et al., 1968; Varner and Schidlowsky, 1963). 37 TABLE 6.--Total World Acreage and Production of the Major Food Legume Crops and Wheat, Rice and Corn.a Area Production Products (1000 hectares) (1000 metric tons) Legume foods 67,619 43,700 Soybeans 38,489 53,024 Peanuts 19,665 16,887 Total legumes 125,773 113,611 Wheat 213,494 347,610 Rice 131,230 295,380 Corn 108,208 301,390 The essential amino acid content of beans has been the subject of numerous investigations. Two points are of particular interest. First, the lysine content of beans is rather high. Cereal products, on the other hand, tend to have a low lysine content. This fact is responsible for the complementarity of cereals and legumes. Second, beans have a low methionine and cystine content. The amino acid content of legume grains depends on species, varieties, localities and management practices. Landon et al. (1957) studied the factors influencing proteins, methionine, lysine and tryptophan content of 25 varieties of beans. Differences in nitrogen and tryptophan content among varieties and between localities were highly significant. The fertility of the land did not affect the content of nitrogen, methionine, lysine and tryptophan. 38 The essential amino acid content of selected beans is given in Table 7 and compared with the FAO pattern of amino acid requirements (FAQ/WHO Energy and Protein Requirements, WHO tech. Rep. Ser. No., 522-1973). TABLE 7.--Essential Amino Acid Content of Selected Beans Compared with the FAQ/WHO Pattern (g/16g N). ENWMHD Etienaxe: bhwy Kflhmx Enack Red Amino Acid Pattern? Beans BeansC BeansC Histidine —- 2.4 2.6 2.9 3.2 Lysine 5.4 5.7 6.7 6.7 7.2 Mbthionine and Cystine 3.5 1.7 1.9 3.0 2.2 Phenylalanine and Tyrosine ' 6.1 8.4 9.8 9.6 9.9 Leucine 7.0 6.7 8.1 7.7 7.7 Isoleucine 4.0 3.7 4.2 4.1 3.8 ‘Valine 4.9 4.4 5.1 4.9 5.0 Threonine 4.0 4.1 4.2 3.1 3.5 Tryptophan 1.0 1.2 1.5 1.5 1.7 aTAD/WHO (1973) reference pattern. bThfisstuhn cEwmmsandl&nfibmer. The amino acid differences within Phaseolus vulgaris cultivars reported by King (1964) and Lantz et a1. (1958) indicate that, through selection and hybridization, improved cultivars might be develoPed. .39 According to Kelly (1971) the level of methionine in mature seeds of the common bean is determined genetically and sufficient variation exists within the species to permit improvement through hybridization and selection. Kakade and Evans (1965) found some Navy bean varieties to vary in their protein content as well as in their methionine content. While inorganic sulfur occurs in relative abundance in the earth, the ocean and the air, organic sulfur com- pounds such as methionine and cystine are in short supply (Rose et al., 1955; Borgstrom, 1964). Only plants have the necessary mechanism to reduce sulfate, an oxidized form of sulfur, and synthesize methionine and cystine (Thompson et al., 1970; Allaway and Thompson, 1966). Animals depend on plants to obtain the methionine and cystine they need. Dietary cystine provides a methionine sparing effect. It is able to replace 80 to 89% of the methionine requirement (Rose and Wixom, 1955). Methionine is an important source of methyl groups in the mammalian metabolism being very important in trans- methylation reactions. Vitamin B12, epinephrine, ergosterol and lecithin are end products of transmethylation (White et al., 1968). Compounds such as S-adenosyl-methionine, lypoic acid, co-enzyme A, thiamin and biotin, have organic S-compounds as their building blocks. In bacteria, and also in the mitochondria of eucariyotic cells, the 40 synthesis of proteins starts with N-formylmethionyl-t-RNA, a formulated methionine t-RNA (Clark and Marcker, 1968; Lehninger, 1970). The amount of protein nitrogen in relation to nonprotein nitrogen seems to be affected by the availa- bility of sulfur. According to Stewart and Porter (1969), the protein nitrogen represented less than 25% of the total nitrogen found in sulfur deficient plants of wheat, corn and beans. However, 75% of the total nitrogen was protein when the sulfur content was adequate in these plants. Methionine and cysteine are very unstable during acid hydrolysis. This causes a problem for the deter- mination by the usual procedure for amino acid analysis which involves, as a first step, an acid hydrolysis of the proteins. These two amino acids are especially unstable during acid hydrolysis when carbohydrates are present (Blackburn, 1968). As a result of the acid hydrolysis, methionine is partially oxidized to sulfoxide, and cysteine is destroyed almost completely. Schram et a1. (1954) developed a procedure which overcomes the problem of destruction of these two amino acids during acid hydrolysis. They have proposed a prior oxidation of cystine and cysteine residues with performic acid to cysteic acid which is stable to acid hydrolysis. After this step, the acid hydrolysis of the protein is carried out, and the quantitative 41 determination of other amino acids in the hydrolysate is done by ion-exchange chromatography. Hirs (1956) used the same performic and oxidation procedure to oxidize both methionine and cystine in ribonuclease to methionine sulfone and cysteic acid, respectively. The oxidation is followed by hydrolysis and determination of all amino acids, including the two derivatives, methionine sulfone and cysteic acid by ion- exchange chromatography. In cases where there is no serious interference from carbohydrates, the colorimetric method of McCarthy and Sullivan (1941) can be used with success for the determination of methionine. The biological value--the amount of absorbed nitrogen retained in the body--has been found to be low in beans. This has been attributed to the low sulfur containing amino acids in the legumes. Jaffé (1950) and Patwardhan (1962) showed a variation in the biological value of beans from 32% to 78%. Values presented by 0 these authors for the Black bean show a variation of 62-68% for the biological value. The beneficial effect of methionine has been demonstrated by several investigators (Patwardhan, 1962; Kakade and Evans, 1965; Russell et al., 1946), not only tested by biological value, but also through protein efficiency ratios. 42 The problem of essential amino acid deficiency in plant proteins is usually approached through mutual supple- mentation between plant proteins, by amino acid supplements and supplementation with protein rich concentrate such as animal proteins. ’RThe supplementation of legume proteins with cereals and millets has been very successful. Since legume proteins have high amounts of lysine and threonine, they complement to a marked extent those of cereals and millets, which are characteristically low in these amino acids. On the other hand, the low methionine content of legumes is compensated by a higher content of this amino acid in cereals and millets. It has been shown that mixtures of cereals and legumes contain proteins superior to those of cereals or legumes alone. Phansalkar et a1. (1957) showed a good supplementary effect of chick peas, Black gram, Green gram and Red gram proteins on those of wheat, soy beans, and pearl millet. These authors emphasize that there is a point in the combination where an Optimum supplementation is achieved; in general, this maximum effect occurs when about 50% of the legume protein is replaced by cereal protein. Bressani et a1. (1962) reported that the best combination of cooked Black beans and lime-treated corn was one where each component contributed 50% of the total. protein of the diet. Several combinations were reported by Bressani and Elias (1969) of cooked Black beans and 43 Opaque-2 corn. The best results were obtained when 50% of the protein in the diet was derived from each one of the components. Improved nutritive values were obtained by Bressani and Scrimshaw (1961) and Bressani and Nalarbinte (1962) when polished rice and cooked Black beans were combined in the range of 50-80% of rice protein and 50- 70% for Black beans. A combination of 19% rice, 80% legumes and 1% of green vegetables, compared well to the stock rat diet which consisted of one of the vegetable rations supplemented with milk and meat (Baptist, 1956). Amino acid supplementation has been another approach to improve the nutritional value of plant proteins. Supplementation of wheat proteins with lysine has been found to cause significant improvement in its PER (Hutchinson et al., 1959). Whole wheat gave a PER of 0.93, (vs 2.50 for casein) but, when supplemented with 0.2%, L-lysine HCl, the PER increased to 1.45, and for wheat + 0.2% L-lysine HCl + 0.2% D, L-threonine, the PER value was improved to 2.00 (Howe et al., 1965). Methionine supplementation markedly improved the PER of cowpeas, peas, kidney beans, chick peas, green gram, black gram and Navy beans (Russell et al., 1946; Bressani et al., 1963; Richardson, 1948; Kakade and Evans, 1965). An addition of 0.2% methionine to autoclaved Navy beans gave a PER value similar to that obtained for casein. 44 Another way in which plant protein can be utilized more efficiently, particularly when it is of lower nutri- tional quality, is by supplementation of the staple food with small amounts of vegetable-protein concentrates or isolates or with animal proteins. This method has been tested extensively for wheat but has been applied only in limited areas of the world. This method has been shown repeatedly to be very useful not only as a means of improving protein quantity and quality but also as a means of introducing other necessary nutrients as well (Bressani and Marenco, 1963). The protein efficiency ratio (PER) of lime-treated corn was increased from about 1.0 to 2.25 by adding small amounts of egg protein, 3%, to the diet (Bressani and Marenco, 1963). Reports of several investigators indicate that \\ \ \ smalliamounts of good quality protein improve significantly the quality of wheat flour and other wheat products. Extensive reviews on this subject is written by Hegsted et al. (1954) and by Moran (1959). Legumes, besides the described deficiency in _sulfur containing amino acids, are known to contain antinutritional factors (Pusztai, 1967; Jaffé, 1968) such as trypsin inhibitors, hemagglutinins, cyanogenic glycosides, goitrogenic factors, flatulence factors and lathyric factors. 45 Amont the anti-nutritional factors found in legumes, the ones which are more commonly present in the common bean are the trypsin inhibitors, hemagglutinins and flat- ulence factors (Liener, 1962). Perhaps the best studied of all the antinutritional factors is the trypsin inhibitor which was first isolated from the soy bean by Kunitz (1945). Trypsin inhibitors have been isolated from a variety of plant materials as well as from various animal tissues. The reaction between trypsin and an inhibitor is one of the few known cases of pure protein interaction (Green and Work, 1953). Several trypsin inhibitors have been isolated and purified. Examples are the inhibitors from soy bean (Kunitz, 1945); Rackis et al., 1959), the lima bean (Fraenkel-Conrat et al., 1952) and the Navy bean (Bowman, 1948; Wagner and Riehm, 1967). Another factor usually associated with the low nutritional value of legumes in general and beans in particular, is their well-known low digestibility. Un- fortunately, information on this particular problem is limited. It is not known whether these effects are caused by a more rapid movement of the cooked legumes through the intestinal tract or by resistance to proteinhydrolysis by the gastrointestional enzymes (Bressani et al., 1973). In 1944, Everson and Heckert found that raw Navy beans were injurious to rats when fed a diet at 10% protein 46 level. They also reported that heating the beans destroyed the injurious effect. This was confirmed by Kakade and Evans (1963) who observed that rats given 10% protein diet based on raw Navy beans lost weight and died during the experiment. Kakade and Evans (1965) studied the effect of heating Navy beans on the growth of rats. They auto— claved the Navy bean flour for 5, 10, 15, 30, 60 and 240 minutes and found a better growth when the flour was autoclaved for 5-10 minutes at 121°C. Sesame Sesame, Sesamum indicum L., a member of the Pedaliacene family, has been called the "queen of the oilseed crops" because of the high yield of oil obtained and the good qualities of the seed, oil and meal (Eckey, 1954). The plant, cultivated in India for several thousand years, is grown extensively in tropical and subtropical areas of Asia, Mediterranian countries, and South America. During the last decade, imports of sesame seed by the U.S., largely for baked goods and confectionery products, have increased from 24 to 40 million pounds (Agricultural Statistics, 1970). The valuable components of sesame seed are the oil and protein and the contents of these have been deter- mined for several varieties of sesame which were grown in the southern and southwestern parts of the U.S. (Kinman 47 et al., 1954). Oil contents varied from 45-63% and averaged 54%. Protein contents varied from 17-32% and averaged 26%. The average protein content of the oil- free meals was 57%. Sesame protein is rich in sulfur containing amino acids, particularly methionine. It is deficient only in lysine and somewhat in isoleucine. Being a good source of methionine, sesame meal offers great advantage as a natural supplement to many legume proteins deficient in this amino acid. The supplementary value of sesame to soya, groundnut, chick pea or mixtures of these legumes in different proportions has been demonstrated in several investigations using rats as experimental animals (Krishnamurthy et al., 1960; Tasker et al., 1960; Guttikae et al., 1965). Evans and Bandemer (1967) demonstrated the effect of fortification on the nutritive value (relative to casein) of sesame meal. Sesame alone had a protein nutritive value of 47%. Fortification with 0.2% lysine raised this to 94%. The protein nutritive value of a 1:1 mixture of sesame and soybean protein was about the same as that of casein. Soybean protein has an abundance of lysine but is deficient in methionine. In related studies (Joseph et al., 1962; Joseph et al., 1958), it was demonstrated that incorporation of 25% of sesame meal raised the PER of a 2:1 mixture of 48 peanut and bengal gram protein from 1.79 to 2.03. In human feeding tests (GOpalorn, 1961), the sesame-peanut- bengal gram protein mixture, which is somewhat deficient in lysine, was nearly as effective as skim milk in controlling the clinical manifestations or protein malnutrition but was inferior to skim milk with regard to serum albumin regeneration. High protein vegetable mixtures for human feeding containing 35% sesame flour, were developed (Scrimshaw et al., 1961) by the Institute of Nutrition of Central America and Panama (INCAP). These low-cost mixtures were readily accepted and well-tolerated as the chief protein source of a needy population. .Z_i_n_c_ Zinc was first shown to be essential for growth of the rat by Todd et a1. (1934). However, the essentiality of zinc under practical conditions was not demonstrated until Tucker and Salmon (1955) showed that zinc would prevent or cure parakeratosis in swine fed diets composed of natural foodstuffs. The fact that zinc deficiency develops in animals fed diets based on soybean protein and corn whereas it is not seen when diets, containing the same amount of zinc, are based on animal protein has led to the hypothesis that zinc in certain plant proteins is not readily available to animals. The decreased availability of zinc from plant-seed was attributed to 49 phytate (O'Dell et al., 1960). This has been confirmed several times in many species (Likuski et al., 1914; Oberleas et al., 1966), and a detailed discussion on the mechanism of phytate action was published (Oberleas et al., 1966). The hypothesis was tested by adding phytic acid to a casein-based diet and the growth response of chicks were compared to those fed soybean protein as the source of protein (O'Dell and Savage, 1970). Phytic acid decreased the availability of zinc in the casein diet and produced symptoms similar to that observed among animals fed soy- bean protein containing a comparable level of phytate. Oberleas and Prasad (1969) studied the effect of supplementation of zinc to soya protein diets at several levels of dietary protein on growth of the rat over a 10 week feeding experiment. Chemically pure phytic acid was added to all diets to equalize the phytic acid content to one percent. They concluded that zinc supplementation of plant proteins will make them as good as that of animal proteins. Rats fed casein or egg white as the source of protein required approximately 12 PPM of zinc in the diet, while those fed soybean protein required 18 PPM (Forbes et al., 1960). The apparent absorption of zinc by rats fed casein was 84%, compared to 44% by those fed soybean protein. Similar results were obtained in balance studies performed on growing chicks (Savage et al., 1964). MATERIALS AND METHODS Navy Beans Navy beans of the Sanilac variety were grown in Michigan and obtained from the Department of Crop Science at Michigan State University. The finely ground beans (60—mesh) were autoclaved in shallow pans at 121°C for 10 minutes. After autoclaving, the samples were placed in a ventilated hood, allowed to dry at room temperature, and reground to a fine meal (60-mesh). Germination Prior to germination, the dry beans were washed with soap and rinsed thoroughly, and soaked in water for five hours at room temperature. After soaking, the beans were spread in a 3 cm thick layer of vermiculite (W. R. Grace and Co., Mass.). Sufficient distilled water was added in order to keep the seed bed always moist without immersing the beans in water. Germination was continued at room temperature for approximately 60 hours. At the end of this time, almost all of the beans had sprouts varying in length from two to ten mm. They were ground in'a meat grinder. The resulting flour was autoclaved in 50 51 shallow pans at 121°C for 10 minutes and then dried as described previously. They were then reground to a fine meal (60-mesh) in a Wiley mill. Sesame Seeds Sesame seeds were obtained from food stores at Michigan State University. They were already dehulled. The flours were prepared by extracting the oil with commercial hexane. The seeds were ground with twice as much hexane in a fireproof blender. After blending, the slurry was filtered under vacuum in a Buchner funnel. The extraction with hexane was repeated four times and after the last extraction the flour was left as a thin layer in a ventilated hood for 24 hours at room temperature for a complete removal of the solvent. Amino Acids Analysis The analysis for amino acids in the Navy bean and sesame flours was performed on 22 hour hydrolysates of the protein in the flours. These analyses were carried out on a Beckman Model 120C Amino Acid Analyzer in which the amino acids were separated by column chromatography and quantitatively determined by automatically recording the intensity of the color produced by their reaction with ninhydrin (Moore and Stein, 1948, 1951, 1954; Moore et al., 1958; Spackman et al., 1958). Samples were prepared according to the method described in the Beckman manual for amino acid analysis (Toeffer, 1965). 52 About 25 mg portions of Navy bean flour and 8 mg of sesame flour were weighed into 10 ml ampoules. Five milliliters of glass-distilled 6 N HCl were added to the ampoules. The contents of the ampoules were frozen in a dry-ice alcohol bath, evacuated with a high vacuum pump, and allowed to melt slowly under vacuum to remove gases. After evacuation, the ampoule contents were again frozen in the dry-ice alcohol bath and then sealed with a pin- point air-propane flame. The evacuated and sealed ampoules were placed in an oil bath at 110:1°C oven. After hydrolysis for 22 hours, the ampoules were removed from the oven and cooled to room temperature. The top of the ampoules was cut and one milliliter of 2.5 uM norleucine solution was added to each ampoule as an internal standard to measure mechanical losses during transfer. The contents of each ampoule were transferred to a pear-shaped evaporating flask and evaporated to almost dryness under vacuum on a rotary flask evaporator immersed in a 45-50°C water bath. After evaporation, a small volume of distilled water was added and the evaporation was repeated. Addition of distilled water and evaporation was repeated twice more in order to eliminate completely all remaining hydrochloric acid. The dry hydrolysate was transferred from the pear-shaped flask into a 5 m1 volumetric flask with a citrate -HC1 buffer, pH 2.2. In order to separate solid 53 particles, the 5 ml hydrolysate was filtered and the filtrate was transferred into a small vial and stored at 4°C. An aliquot of 0.2 ml was then applied to the analyzer for amino acid analysis. The amino acid analyzer was operated at 57°C. The running time was four hours, 55 minutes for the short and 185 minutes for the long column. The chromatograms were compared to the one obtained from a standard amino acid calibration mixture. Sulfur Containing Amino Acids Methionine and cysteine are known for their instability during acid hydrolysis when carbohydrates are present (Schram et al., 1953). In order to protect these amino acids during acid hydrolysis, a preliminary oxidation with performic acid was carried out, cysteine being oxidized to cysteic acid and methionine to methionine sulfone as described by Schram et al. (1954) and Lewis (1966). The performic acid solution was prepared by mixing one volume of 30% (w/w) hydrogen peroxide with nine volumes of 8.8% (w/w) formic acid. This mixture was allowed to stand for one hour at room temperature. Eight milligrams sesame flour and 25 milligrams of Navy bean flour were weighed into a special pear-shaped flask and cooled to 4°C. Then, 10 m1 of performic acid, which was previously cooled to 0°C, was added to the samples. Oxidation was carried out at 4°C for 16 hours. 54 After the oxidation, the performic acid was evaporated on a rotary evaporator, with a cold finger trap for the very corrosive performic acid. After evaporation, the residues were hydrolyzed, as described previously, with 5 m1 of 6 N HCl for 22 hours at 11011°C. Following the hydrolysis, the norleucine standard was added and the sample was treated exactly the same as in the procedure described previously. The chromatograms were compared to those of the standard methionine sulfone and cysteic acid. TryptOphan Tryptophan is very labile during acid hydrolysis, and after prolonged hydrolysis of a protein, little or none of the amino acid is left. Therefore, it must be determined separately. Tryptophan was determined colorimetrically after hydrolysis with the enzyme pronase as described in Procedure W by Spies (1967): a. A 10-30 mg sample was weighed directly into a 2.0 ml glass vial with a screw cap. b. To each vial 0.1 ml (100 pl) of pronase hydrolytic solution and a drop of toluene, as a preservative, was added. Pronase hydrolytic solution was pre- pared daily by adding 100 mg pronase (Calbiochem, activity 45,000 PUK/g, lot 101185) to 10 m1 of 0.1 M phosphate buffer, pH 7.5. The suspension was shaken gently for 15 minutes, then clarified by centrifugation for 15 minutes at 10,000 RPM. c. The vials were closed and incubated for 24 hours at 40°C. After incubation, 0.9 m1 of 0.1 M phosphate buffer, pH 7.5, were added to each vial. The uncapped vials were placed into 50 m1 Erlenmeyer 55 flasks containing 9.0 m1 of 21.2 N sulfuric acid and 30 mg of dimethylaminobenzaldehyde (DAB). The vials were tipped over and the contents were quickly mixed by rotating the Erlenmeyer flasks. Samples were cooled to room temperature and kept in the dark at 25°C for six hours. d. 0.1 m1 of 0.045% sodium nitrate solution was added to each Erlenmeyer flask. After gentle shaking, the flasks were left standing for 30 minutes for the development of the color. The absorbance was measured at 590 nm, using a Beckman DU spectroptoto- meter. Duplicate blanks of the pronase hydrolytic solution were treated similarly and the tryptophan content of pronase was subtracted from the total tryptophan content. A standard curve from zero to 120 pg of tryptophan was prepared according to the Procedure E described by Spies and Chambers (1948). D, L-tryptOphan (2.4 mg) was dissolved in 200 m1 21.2 N sulfuric acid containing 600 mg of dimethylyamino- benzaldehyde (DAB). 0, l, 2, 4, 6, 8 and 10 ml of this solution were made up to 10 ml with solutions of 21.2 N sulfuric acid containing 600 mg DAB/200 ml, and placed in 50 ml Erlenmeyer flasks. The mixtures were kept in the dark at 25°C for six hours, then 0.1 m1 of 0.045% sodium.nitrite was added to each flask. The flasks were allowed to stand for 30 minutes for color development, and absorbance was measured at 590 nm, using a Beckman DU spectrophotometer. A straight line relationship was obtained between absorbance and tryptophan content. 56 Total Protein The "total protein" content (N x 6.25) of the beans and defatted sesame seeds was determined according to the AOAC (1970) micro-Kjeldahl method, after grinding the samples to pass a loo-mesh sieve. The "total protein" content (N x 6.25) of the diets was determined according to the AOAC (1970) macro- Kjeldahl method. . Crude Fiber The crude fiber content of the beans and sesame flour was determined by the AOAC (1970) method. Oil The oil content of the beans and sesame flour was determined according to the AOAC (1970) method. Ash The ash content of all samples was determined according to the AOAC (1970) method. Moisture The moisture content of all samples was determined according to the AOAC (1970) method. 57 Biological Evaluation of Protein Quality A. The Protein Efficiency Ratio (PER) Method Weanling male rats of the Sprague Dawley strain, 21 days of age, 10 for each diet (unless otherwise specified) were used throughout the experiments. Rats were housed individually in stainless steel cages in a room at 23°C. Assay diets and water were offered ad libitum. The animals were weighed twice a week and their food intake and waste measured every three days. The total experimental period for PER determination was 28 days. Prior to the 28 days test period, the animals were fed a standard rat diet for three days in order to adapt the animals to the new environmental conditions. The materials under test were fed as the sole source of protein at the 10% protein level. The composition of the basal diet is shown in Table 8. The average 28 days weight gain and protein (N x 6.25) intake per rat for each group were calculated. The Protein Efficiency Ratio (weight gain/Protein intake) was, then determined for each group. The ratios x 100 of PER for each assay group to PER for ANRC casein reference group was also calculated. B. Net Protein Ratio (NPR) NPR values were calculated in the same way as the PER values, except the weight loss of one group of four 58 TABLE 8.--Compositions of Basal Diet for the PER Method. .A Ingredient Amount % Protein source1 10 Corn oil 8 Salt mixture2 5 Vitamin mixture3 1 Non-nutritive fiber 1 Corn starch To complete 100 l Vitamin-free casein; purchased from Tecklad Test Diets., P.O. Box 4220, Madison, Wisconsin, Protein content: 83.6 (N x 6.25). All rations contained the equivalent of 10% protein (N x 6.25). 2USP X IX Salt mixture (Tecklad Test Diets., P.O. Box 4220, Madison, Wisconsin). Salt mixture composition (%): Sodium chloride (Na Cl), 13.93, Potassium iodide (KI), 0.079; Potassium phosphate monobasic (KHZPO4), 38.90; Magnesium sulfate (Mg 804), 5.73; Calcium carbonate (CaCoB), 38.14; Ferrous sulfate (FeSO4z7H20); Manganese sulfate (MnSO4:H20), 0.401; Zinc sulfate (ZnSo4.7H20), 0.548; Cupric sulfate (CuSo4.5H20), 0.0477; Cobalt chloride (C0C12.6H20), 0.0023. The nitrogen sources used in the diets contained different amounts of ash. To compensate for that, salt mixture was added to the diets at a level to make the sum of the mineral mixture and the ash in the test materials equal to five percent. 3The vitamin mixture contained (mg/100 g diet): Vitamin A, 2000 (IU); Vitamin D, 200 IU); Vitamin E, 10 (IU); Menadione, 0.5; choline, 200; P-Aminobenzoic acid, 10; Inositol, 10; Niacin, 4; Ca-D-Pantothenate, 4; Riboflavin, 0.8; Thiamine. HCl, 0.5; Pyridoxine. HCl, 0.5; Folic acid, 0.2; Biotin, 0.04; Vitamin B 12, 0.003. 59 weanling rats fed the non-protein diet was taken into account. The Net Protein Ratio (NPR) is defined as (gain in weight of test group + weight loss of non-protein group) divided by protein intake. C. Slope Ratio Technique Weanling male rats of the Sprague Dawley strain, 21 days of age, four for each diet were used. Rats were housed individually in stainless steel cages in a room at 23°C. Assay diets and water were offered ad libitum. The animals were weighed twice a week and their food consumption was recorded during a 3-week experimental period. Prior to the test period, the animals were fed a standard rat diet for three days as described previously. The lactalbumin was fed as the standard diet at the 4, 6, 8 and 10% protein level. All other test materials (were fed at the 6, 8, 10 and 12% protein level. Each protein level for each test diet used four rats equally distributed by weight. Four rats were also fed the non- protein diet. The composition of basal diet is shown in Table 9. ' Weight gain and protein intake were determined for each rat for the 21 days test period. For the Relative Nutritive Value (RNV) calculation a regression analysis 60 TABLE 9.--Composition of Basal Diet for the Slope Ratio Technique. Ingredient Amount % Protein source1 4, 6, 8, 10 Salt mixture2 4 Vitamin mixture3 1 Cod liver oil 1 Corn oil 9 Non-nutritive fiber 1 Corn starch To complete 100 1 Lactoalbumin--Purchased from United States Bio- chemical Co., Cleveland, Ohio, protein content: 77.4% (N x 6.25). 2Salt mixture was the same as Table 8. The nitrogen sources used in the diets contained different amounts of ash. To compensate for that, the mineral salt was added to the diets at a level to make the sum of the mineral mixture and the ash in the test materials equal to four percent. 3Vitamin mixture was the same as in Table 8. was performed between weight gain and protein intake including the data for the rats fed the nonprotein diet. Therefore, each line was calculated from 20 points (four animals per group fed one of four test levels of protein and four animals consuming nonprotein diet). The value for RNV was calculated for each test diet as the ratio of the slope of the regression line of the test diet to the slope obtained with lactalbumin in the same experiment, the value for lactalbumin being taken as 100. Relative 61 Protein Value (RPV) was calculated in the same way except that the zero data was omitted, the repression lines being calculated only from the 16 points i.e., four animals per group at four levels of protein. Evaluation of the Effect of Processing on the Navy Bean Protein This study was conducted to determine the effect of processing on the protein quality of Navy beans. Beans of Sanilac variety, obtained from the Department of Cr0p Science at Michigan State University, were subjected to different processing methods used at home and commercially (Bedford, C., personal communication, 1976). The processing methods evaluated were as follows: A. Canning Beans were washed and rinsed three times with water. They were then soaked in a 90°F water for half an hour. After soaking, the temperature of soaking water was elevated to 180°F and the beans were kept in the water for another half an hour. The beans were cooked in the same water for two minutes at 212°F. The water was discarded and approximately 7 ounces of beans were weighed in a 303 can. Plain hot water was added to fill the head space and after sealing, they were autoclaved in a retort for 45 minutes at 240°F. After retorting, the cans were water cooled to approximately 58°F in the retort. 62 The content of the cans was emptied into a Waring blender and blended for about 10 minutes. The slurry was dried in a cabinet drier Operating at 150°F for approxi- mately 8 hours. After drying, the samples were ground to a fine flour (60-mesh). B. Canning with Sugar The same procedures were followed as described previously except a 1.5% (w/w) sucrose solution was added to the cans to fill the head space before sealing. C. Home Cooking Beans were washed and rinsed three times with water. They were then soaked in a 90°F water for half an hour and slow-cooked at simmering temperature (about 200°F) in the same water until they were well-cooked. The water was added as needed to cover the beans during cooking. After the beans were well-cooked (orally tested), which took approximately two hours, they were blended, dried and ground as described before. D. Autoclaving The same procedures were followed as described in the preparation of the bean sample in the supplementa— tion experiments. 63 Lysine Availability A chemical method, using l-fluoro-2, 4-dinitro- benzene (DNFB), to measure lysine availability was first introduced by Carpenter and Ellinger (1955). In this technique, DNFB reacts with free e-amino groups in the protein, forming DNFB—e-amino lysine which is stable to acid hydrolysis the sample is then acid-hydrolyzed and the unavailable lysine is determined using an amino acid analyzer. In our study a modification of this method was used as described by Couch (1975). Approximately, one 350 mg sample was weighed and transferred to a 500 ml boiling flask along with 4-5 glass beads. Ten ml of freshly prepared 10% NaHC03 solution, 10 ml alcohol (ethanol) and 0.4 ml of a 90% dinitrofluorobenzene (DNFB) solution were added to the flask. The flask was stoppered and shaken for 73 hours using a mechanical shaker. The mixture was carefully acidified with approximately 2 m1 of 6NHC1, and evaporated to oily dryness at 40°C in a vacuum rotary evaporator. The vacuum was released very slowly to avoid disturbing the residue. Then, 50-75 m1 anhydrous ether was added, and re-evaporated, in a rotary evaporator at 40°C with- out vacuum. The washing with ether and evaporation were repeated three additional times. Approximately, 125 ml of 6NHC1 was added to the sample and a 5 ml aliquot was transferred to a 10 ml 64 glass vial with screw cap. From this point, the pro- cedures were the same as the total amino acid deter- mination, except only the short column in the Amino Acid Analyzer (Model 120C) was used to measure the un- available lysine content of the sample. The total lysine was determined on the untreated sample. The available lysine was calculated by difference. Methionine Bioassay There is no established method to determine the biological availability of the methionine for the rat. The following procedure is rather novel. The essential amino acid composition of a 7% protein diet in which casein is the only source of protein is very close to that of a 10% protein diet in which Navy beans is the only source of protein (Table 10). Cystine and tyrosine were also compared for their sparing effect on methionine and on phenylalanine, respectively. 3 Therefore, a 7% casein protein diet was prepared to which adequate amount of essential amino acids (in- cluding cystine) were added to match the 10% bean protein diet. Since in the amino acid analysis of the bean protein only 82% of the total N was recovered as amino N, 1.2% dispensible amino acids (equal amount of glycine and glutamine) were added to the casein diet to compensate for the total amino N difference. 65 .hosum menu scum muasmmm m .Amema .mooom «0 namuqoo whoa ocflsav can gonna so.o mo.o + No.0 manummo on.v oa.o + H.o Am.ov o.o + ~.o mcflcowsumz Hm.o mm.o mcflcwmnd NH.o HH.o :mcmoummua oa.o + Hm.o H¢.o ocwmoume mm.o ma.o mm.o mcwcmamamcmnm v¢.o mv.o OGHHM> av.o mo.o mm.o mcficomune hm.o mm.o mafioooaomH no.0 hm.o mcflocmq nm.o hm.o mcwmmq mwflod OGflEd pmpv¢ + ma new Nu .Q m H mofloa ocfls< amass + amen :ammmo cwod osflad deducommm .Asowumucmemammsm waflcownumz mo Hm>mq ccoomm m3onm mammaucoummv umwn m ooa\m mm pmmmmumxm .mcmmm so comma amen samuonm woa m can :Hmmmu no woman umwo aflououm an w No ucmucou cflod onwam HMflusmmmm mnall.oa mamma 66 This 7% protein diet from casein was used as the reference and the methionine utilization of the bean protein, relative to casein, was studied. To correct for the difference in the methionine content of the two diets, 0.1% L-methionine, assume to be 100% available, was added to the bean diet to make it equal to the casein diet (0.2% methionine). It was also decided to check the methionine utilization of the bean diet at another dietary level of methionine. Therefore, 0.3 and 0.4% L-methionine were added to the casein and bean diets, respectively, to make the methionine level 0.5% of the diet. To compensate for the sparing effect of cystine, 0.1% L-cystine was added to all diets. For this experiment, weanling male rats of the Sprague-Dawley strain, 21 days of age, 5 for each diet, were utilized. They were housed individually and fed §d_libitum throughout the test period which was 14 days. The animals were weighed twice a week and their food intake was recorded. Zinc Supplementation Study In this study weanling rats were individually housed in stainless steel cages, fed ad libitum; the rats were offered diStilled water which was also demineralized by passing through a mixed ion exchange 67 bed. The salt mixture was the same as used before except for zinc which was excluded. The zinc content of beans, casein, corn oil and corn starch was determined by atomic absorption spectrophotometry, using a Perkin-Elmer Atomic absorption Spectrophotometer Model 303. Instrument Parameters a. Lamp current: 15 ma b. Fuel: acetylene c. Support: air d. Wavelength: 214 nm Wet Ashing Procedure Approximately one 9 sample was weighed into a 100 ml Kjeldahl flask. Twenty-five m1 of HNO3: perchloric acid mixture (17:3) were added and the flasks and contents were heated for approximately two hours. When the digestion was completed, the digests were transferred to volumetric flasks and appropriate dilu- tions were made with distilled deionized water. Preparation of Standard Solutions 1.000 g of zinc granules (99.99% pure) was dissolved in 4.0 ml 1:1 hydrochloric acid and diluted with distilled deionized water to give 1000 mg zinc/ml solution. Further dilutions were made to prepare 68 solutions containing 0.2, 0.5, 1.0, 2.0 and 3.0 ppm zinc. These solutions were used to make a standard curve for zinc. RESULTS AND DISCUSSION Sample Materials The autoclaved Navy bean flour, and defatted sesame flour were analyzed for moisture, oil content, total protein (N x 6.25), crude fiber, ash and zinc. The results of these analyses are shown in Table 11. Total Amino Acids of Sesame and Bean Flours The total amino acid content of sesame and bean flours expressed as grams of amino acids per 16 grams of total N and as mg of amino acids per g of total N are given in Table 12. It is evident from Table 12 that the total sulfur containing amino acids in the sesame meal (methionine + cysteine = 4.7 g per 16 g of total N) is approximately 2.8 times that of the Navy bean flour (1.7 g per 16 gm total N). On the other hand, the lysine content of the protein in the Navy bean flour is 2.5 times higher than the lysine content of the protein in the sesame meal. Figure 1 shows the comparison of the essential amino acids composition (expressed as mg amino acid per gram of total N) of sesame meal, whole egg and Navy bean 69 70 TABLE ll.--Composition of Defatted Sesame Flour, and Navy Bean Flour. Sesame Navy Bean Flour Flour Moisture (%) 8.0 8.6 Oil (%) 1.5 1.84 Total Protein (N x 6.25) (%) 58.19 21.06 Ash (%) 3.01 3.92 Crude Fiber (%) 3.0 4.1 Zinc (PPM) 172.8 29.0 Carbohydrate (%)1 26.3 60.5 1Obtained by the difference. flour. It is reasonable to assume at this point that sesame flour and Navy beans could complement each other since each of them is deficient in an essential amino acid that the other one has in excess; whole egg protein could also supplement the Sulfur amino acid in which the protein of beans is deficient. Supplementation of Navy Bean Protein With Sesame Flour The first feeding experiment was carried out to test two mixtures of autoclaved Navy bean and sesame flour as a source of protein in the diet. A 10% protein diet was prepared in which: (a) 50% of the protein came from the Navy bean flour and 50% from the sesame flour; 71 m.mm m.H o.mh ~.H candoummua m.ms~ m.m m.Hmm m.m maacmamamcmnm m.HmH m.~ m.mma H.m mcnmoume m.nmm v.m m.mas p.o manosmu m.ema o.m m.Hmm n.m manosmaomH m.mma H.m m.am mm.o mafiaoasumz m.m¢~ m.m o.m>~ v.v mcflam> o.ooa m.a m.ms n.o unamumao m.mH~ m.m m.mo~ m.m maaamaa o.m- m.m m.mmH H.m manomao m.sma o.m m.mo~ m.m mzflaoum m.mooa H.5H o.oom m.~a anus oasmusao o.m- o.m m.mam H.m mcaumm m.hmH o.m m.mm~ H.¢ ocflcomusa m.mas h.o m.~mo o.oH choc onuummma o.m~h m.aa «.mam H.m mcacwmua m.nma ~.m o.oma ¢.~ magenumnm m.msa m.~ m.mmm a.m mcammn zm\ms 2 dance mma\m zmxms z Hmuoe moa\m onus canes unoam madmom Haoam cmom h>mz .Acmmouuflz Hmuoa Echo Mom cflod ocH54va mm was somouuaz Hence no macho ma mom pend ocflsg manna ca commmumxmv mnsoam comm can madmom mo cofluwmomsoo cao< ocfl84 Hmuoall.~H mange 72 .239 >>mz can one 2055 .50: 9:33. to A2 :28 395 coEmanoo mEom 05:5 32:33 42:9“. mi ._<> mE urn. 96 E: m5 8.. m... ‘ x .\ \ \ ‘ ‘ “ “ “ “ “ “ \ \ \ \ \ \ n m “ “ m m “ -oo. \. “ “ x \ \ “ “ “ “ “ .08 M. \ \ \ \ \ \ \ \ \ \. WV “ \ “ “ “ - v x “ \ \ \ oom ./ u r \ \ \ 5 . r r .. \ \ .08 O r “ m “ L m coma>>oz§ \ 00m 8... 22.3.”. @6085. L 000 73 (b) 75% of the protein of the diet was furnished by Navy bean flour and the rest, 25%, came from the sesame flour; (0) sesame flour was the only source of protein; and (d) bean flour provided all the protein in the diet. The results from this experiment are given in Table 13, and the growth rate and food intake, during the 28 day experimental period, are shown in Figures 2 and 3, respectively. Group A was fed a 10% casein protein diet and it was used as control to calculate the protein efficiency ratios. The approximate weight gain was 4.35 i 0.32 g per rat per day, and the feed intake was 13.2 i 0.75 g per rat per day. The PER for this group was adjusted to 2.5 as recommended by the National Academy of Sciences, National Research Council (1963). All the PER values obtained throughout this work were corrected for casein - 2.50. Group B was fed a 10% protein diet derived from autoclaved beans. The gain in body weight was 2.33 t 0.11 g per rat per day. The protein quality of the bean diet was 62% of that for the casein control group. The relatively low efficiency of the bean protein diet in promoting growth is attributed mainly to their low methionine content (Russell et al., 1946; Kakade and Evans, 1965). The protein efficiency ratio (PER) was 1.56 and it is similar to the value obtained by Kakade and Evans (1965). 74 .Hm>ma suflannmnoum mm map um ucoummmwo >HHMOHumHumum uoc mum Hmuuma wEmm mnu an ©o3oHHOm mosac> m .om.~ n cflmmmo How cmuomHHOUm .zmm H Ma pom No. H m~.~ mo. H mm.~ m.mv¢ o.m~ma Ammumhv m + m m omm so. A om.~ mo. H mo.m h.¢mv o.mova Aomuomv m + m o owe mo. n mH.H _HH. H nm.a o.mm~ o.nmm Amy Hoon mammmm 0 Amy mammm nmm mo. H mm.H so. H mo.m H.mHm 0.8mm pm>mHoou54 m wooa mo. H om.~ no. A m~.m m.mmm o.mama cwmmmu 4 mcflmmmu ~.Hmmm Hmmm Amy Amy uofin msouw mam w omumsn a mxmuaH urmflmz seem .c cwmuoum ca swam .woa .mumfio mo ucoucoo samuonm Hmuoa .Aowumm cflmuoum mmumhv Macaw mammmm + cmmm can Aofiumm :Hmuoum omuomv Macaw mammmm + mammm “macaw mEmwmm “mammm cm>maoous¢ xcflmmmo umcflcflmucou mpwwo cumccmum com mumm mo cp30u0|l.ma mummy 75 250- . B+S(50=50) A 8+8 (7525) o Casein A B o S 200- ‘ O A ,._7 l50- . 3 A >~. '0 I o (D IOOT z _ 2: ° 50 1 1 1 IO 20 30 Time, days Figure 2. Weight gain of weanling rats (av. of 10) fed standard diets containing: casein; autoclaved beans (8); sesame flour (S); B + S, 50:50 protein ratio; and B + S, 75:25 protein ratio. 76 £88 . Ammnmt m+m 6909 w+m . 6:9 529a mmnme .w + m was 6:8 £203 omnom .w + m 29 So: mEmmmm ”Ame mcmmn 33.023 ”Somme 6552.50 266 23.86 c2 SF .0 53 38 95:33 .0 9.25 too“. .m 2.6K 966 .85. ON o. \\ .00. 0 ‘9); mu; p003 77 Group C was fed a diet in which the sole source of protein was from sesame meal. The protein efficiency ratio of this group was 1.19 which represented 48% of that for casein control diet. This is exactly the same as the PER of 1.19 for solvent extracted dehulled sesame reported by Subramamian et a1. (1971). The relatively low protein quality of sesame meal is attributed to its deficiency in lysine and somewhat in isoleucine (Lyon, 1972). In a rat feeding study (Evans and Bandemer, 1967), the effect of fortification on the nutritive value (relative to casein) for sesame meal was demonstrated. Sesame alone had a protein nutritive value of 47%, and when fortified with 0.2% lysine it was raised to 94%. The average weight gain for Group D (Navy bean: sesame flour, 50:50 protein ratio) was 5.03 i 0.15 g per rat per day. The rate of growth and food intake were higher than those for the casein diet. The PER was 2.30 and was 92% of that for casein diet. Rats in Group E (Navy bean:sesame flour, 75:25 protein ratio) gained an average of 4.75 i 0.16 g per animal per day. The growth rate was higher than the one for casein and the animals consumed more food during the experimental period. The PER was 2.26 or 90% of the PER for casein, which was not statistically (P < .05) different from the one obtained for Group D (50:50 mixture). 78 Alquist and Grav (1944) conducted experiments to study the supplementary effect of sesame and soybean meals and showed that the best gains were made by chicks fed a ration in which the ratio of sesame-soybean protein approached 7:13. Mankernika et al. (1965) investigated the nutritive value of blends of different plant proteins and reported that protein foods based on 40:30:30 blend of ground nut, soya and sesame flours possessed a fairly high PER value of 2.41. Evans and Bandemer (1967) studied the supplementary value of sesame protein to that of soybean proteins. They concluded that a diet containing 5% protein supplied by Mexican sesame seed and 5% supplied by Chippewa soybeans promoted better growth than one containing 10% protein furnished by either Mexican sesame or Chippews soybeans. The protein quality of sesame alone was 47% (relative to casein) and the 1:1 mixture of sesame and soybean protein was almost the same as that of casein. The same authors reported that when only one-fourth of the protein was furnished by sesame seed and three-fourths by soybeans, growth was not as good as when soybeans were the sole source of protein. In the next experiment in this series, we had two objectives. First, we were interested to see if germina- tion of Navy beans, prior to autoclaving, would have any 79 beneficial effect on the growth of weanling rats, as compared to beans that had been just autoclaved. The second objective was to determine how low the amount of sesame flour in the mixture with Navy beans could be in order to observean improvement on the nutritive value of the beans. Results of these tests are presented in Table 14 and Figures 4 and 5. The mixture of Navy bean:sesame flour containing the protein ratios of 87.5:12.5 was also able to promote the growth of rats over that of beans alone. The average weight gain for Group C (Navy bean: sesame, 87.5:12.5) was 2.52 i 0.12 g per rat per day. The PER was 1.79 which represented 72% of that for casein and was significantly (P < .05) higher than the PER of beans alone. The food intake of this group was almost the same as beans alone, but they gained more weight. Group D was fed a diet in which the protein source was Navy beans that had been perminated prior to auto- claving. The average weight gain for this group was 1.35 i 0.14 g per rat per day and the protein efficiency ratio was 1.21. This represents 49% of the casein control group. The total food intake during the 28 days experi- mental period was 1ow as compared to the casein and autoclaved beans groups (Figure 5). Everson et al. (1943) reported no improvement of the nutritive value of soybeans, 80 .Hm>mH muaannmnona em can an udouwmmwp haamoapmflumum won one Houuma 65mm on“ an ©o3oHH0m mooam> m .om.m u :Hmmmo MOM cmuomHHOUm saw a ma mammm om>maoou5« was so.o H H~.H mo.o n ~m.a o.ae~ o.mem u emumcaenmo a Am.maum.nmv emu mo.o n as.a so.o H m~.~ m.mam o.moe m + m o mcmom nmo No.o n ~m.a mo.o n so.~ o.¢~m o.omo em>maoousm m mo3 mo.o n om.~ oo.o H sH.m n.mmm o.smoa seemed a menmmmo ~.Hmma Hmmm Ame Ame peso muons mam w seemsnca mxmueH unmamz zoom . :wmuoum cw demo .woa .ucmucoo cflmuoum Hence .mcmmm ©m>mHoousm i woumcwanmw cam Aoflumm cwmuoum m.maum.hmv unon mammmm ”mammm mo muouxwz m .memmm em>maoousa .cammmo manenmuaoo mumps enmsemum cam mumm «0 eusouoii.sa mamas 81 .029 £2.95 mdtmfim .50: 2:33 + 932. 8.6.02.6 .o 225:. .0 new .932. 8.8.0026 ocm umfiEEEo .0 .2me “092022... .m .533 .< ”9.2.2.50 £66 Emncflm no. 8? .0 $3 29 mazcmm; .o Emu 2935 .v 2:9“. mace .95... 0% CW 0.. 0 o .00 o mmv 8 o n O o . . H .8. .w m . m. o .D o . .8. 4 o 82 .0sz £82.... m.wtm.$ .So: mEmmom + 28: 86.02:: to 235:. .o :5 6:3: 85.03% ocm uBmEEEm .0 6:3: 83.02% .m .5023 .< 5:33:00 32: Emucflm cm. .3 .0 Se. 29 3::me .o 9.9:. coon. .m 2:9“. £6: .06.... 0.. 0.. e o -00. 4. O O D. .00N W m. Vol .9 -00m .0 83 as determined by PER, when soybeans were germinated prior to cooking. Chattopadhay and Banerjee (1953) demonstrated that germination improved the nutritional value of certain beans. However, Kakade and Evans (1966), who worked with Navy beans, did not find any beneficial effect of soaking and germination on the nutritive value of the beans. The low growth rate obtained in our study is understandable in view of the low food intake by the animals. Net Protein Ratios In order to calculate Net Protein Ratios (NPR), a group of 4 rats was fed a non-protein diet. In a 28 day experimental period, they lost an average 28 i lg body weight. It is suggested that this corresponds to the maintenance requirements of rats for protein (Bender and Doell, 1957). The NPR values were determined for casein, Navy bean, sesame and mixtures of beans and sesame flour (87.5:12.5, 75:25 and 50:50 protein ratios) and were in the same order as their PER values. Table 15 indicates the NPR values of the casein and the tested samples. It is again evident that the protein quality of Navy beans is greatly improved by supplementation of sesame flour. In addition, there was no significant difference (P < .05) in NPR values between the bean: sesame mixtures with protein ratios of 75:25 and 50:50. Bender and Doell (1957) determined the NPR, PER and NPU of some animal and plant proteins including 84 .Hm>ma muwaanmnoum mm 650 um ucmuomwflc madmowumfiumum uoc mum Hmuuoa mean on» ma cm3oa~0m mosam> N sum “.mH ii iii: o.m~ i o.o :wmuoumicoz mmm mo.o H mo.m o.mova + h.vm¢ .omuom. m + m mom mo.o H vo.m o.m~ma + m.mv¢ Ammumn. m + m 0mm wo.o n mm.~ o.mon + m.mam .m.~aum.>mv m + m can 50.: « mm.a o.:mm + m.mmm Am. Hooam mesmmm Amy mcmmm >>mz new mo.o a vH.m o.¢mm + H.mam om>maoous< nco... aa.o H nm.m o.mH~H + m.mom sawmmo mmmz w maz .m. .m. page unmflmz atom mxmucH omcmco cwmuoum .woa .mumao umm Ham mo ucmucoo :flmuoum Hmuoa .omuom 0cm mmumh .m.~aum.pm «0 moans: :Hmuoum fiflz HSOHh @fimmmuflmmm HO mmhfiflxflz GEM sHDOHh mammmm .mfimwm U¢>MHUO¥5¢ ~GHmmMU mcficflmucoo mumwa ousccmum mo AooH Gammmuv mmz w 000 Ammzv Oflumm GHOuOHm umZII.mH MAMflB 85 sesame meal. They reported the NPR of 3.16 and PER of 0.69 for sesame meal. This is not in agreement with our result (NPR = 1.69; PER = 1.19) which might be due to the fact that their experiment lasted only 14 days but ours, 28 days. Furthermore, we found the NPR value of sesame meal to be higher than its PER value. This is because NPR takes into account some allowance for mainten- ance requirements and proteins low in lysine appear to be much more efficient in maintaining than in promoting growth (Said and Hejsted, 1969 and 1970; Said et al., 1974). Protein Scores The essential amino acid composition of Navy bean flour, sesame flour, the three mixtures of Navy bean: sesame flour (87.5:12.5, 75:25 and 50:50 protein ratios) and the new 1973 essential amino acid pattern (FAQ/WHO, 1973) were compiled and expressed as milligrams of essential amino acid per gram of total nitrogen (Table 16). In Table 17 the protein scores of the tested samples are shown. This new FAO/WHO (1973) provisional scoring pattern was derived from estimates of amino acid require- ments for the normal growth of young children. This pattern has been recently shown by Kaba and Pellet (1975) to be superior to all others tested in correlating with the NPU values obtained with young rats and in predicting the true limiting amino acids. 86 .mpsum man» Scum muasmom H meow ooam Homm mama momm ommm .<.a Hoauommmm Hence oom pom Hem «om mom can meaam> mm om he so me om anacondaue mmm mmm mam mos omm omm maaeomuns mes oom mam mmo mmm omm anemones a mcflcmHmamgmnm .¢.¢ Owumeoud Hmuoa com «ma mNH «mm moa omm msflmumwo a coacOwnumz .<.¢ mcflcwmucou insmasm Houoa omm mom omm sea omm oom monmsq mom mam moo mmm mas coo monsoon cam omm own was Hmm omm meaosmHOmH .omuom. .mmumh. .m.~a.m.em. .m. .m. .mhma. .mm. modem cased m + m m + m m + m Hannah Hmcomm om3\o¢m mammom mbmz .Acomouuflz Hmuoe Educ Mom owed ocwE¢ me we ommmmnmxmv mofiudm :wmuoum ucoummman um musoam manmmmucmom >>mz mo monouxaz can Hooam cashew .Hdon comm m>mz .AMNmH .om3\0¢m0 cuouumm moamummmm on» no coauflmomfiou owom Oddsd HMHucmmmmll.mH mummy 87 Ha om mm mm mm moaao> ooH ooa ooH ooH ooH ooaoouamua so am ooH mm ooH mongoose: ooH ooH ooH ooa ooa .:.« oaoosoua Hence so me oo ooa ms .<.a moaoaoucoo uuomaom Hence we mm mm on ooH enemas no so mo om om moaoooq so No no mm mm ooaoooaoma .omuom. .mmume. .m.~a.m.em. .m. .m. ..s.¢. monom cease mum mum mum Hoon mammm mammom >>mz .Am>ma romz\o¢m. owed ocwfid Hugucwmmm mo cumuumm mucoummmm 3oz on» no oommm moflumm :wmuonm acouwmmwo um muoon madmmm ”comm m>mz mo mmHoMXHz can Hnoam cannon .Hsoam comm mbmz mo mouoom aflououmii.>a mamas 88 The amino acids cysteine and tyrosine were included in the calculation since they have sparing actions on methionine and phenylalnine, respectively. Based on the protein scores, the Navy bean flour has methionine as the first limiting amino acid, followed by valine and isoleucine. Sesame flour has lysine as its first limiting amino acid, followed by isoleucine and threonine. -Table 17 shows that the protein score for Navy bean protein is 48 and it is improved by addition of sesame flour. The protein scores for the bean + sesame with 87.5:12.5 and 75:25 protein ratios are 60 and 70, respectively. These two mixtures still have methionine as their first limiting amino acid, but the degree of deficiency_is smaller. However, in the combination of Navy bean:sesame flours with a protein ratio of 50:50, methionine is no longer the limiting amino acid and the score is 78. The sesame flour protein (score:50) was largely improved by the Navy bean flour protein. This is due to the supplementation of lysine, which is the first limiting amino acid in sesame protein, by the Navy bean protein which is rich in this essential amino acid. The protein score, based on lysine, improved from 50 for sesame alone to 78, 93, and 99 for mixtures with protein ratios of 50:50, 75:25 and 87.5:12.5, respectively. 89 Modified Essential Amino Acid Index (MEAA Index) This method was originally proposed by Oser (1951) and was modified later by Mitchell (1954). It has an advantage over the protein score method since it rates the proteins based not only on their first limiting amino acids but it takes into account the total individual essential amino acid content of the protein. This approach seems logical since all the essential amino acid must be present at the site of the protein synthesis within a tissue in order to have this process going on efficiently. However, the computation of the MEAA index for Navy bean flour, sesame flour, and the mixtures of both (Table 18), failed to show the complementary values of the bean and sesame proteins. Only the sesame flour index showed an increase from 78 to 89 as a result of supplementation with Navy beans. This is because sesame protein has a lower concentration of most of the essential amino acid than Navy bean protein. .For the purpose of comparison, the protein scores are also shown in this table. Evaluation of Protein Quality by Slope Ratio Technique This technique was developed by Hegsted and his associates (Hegsted and Chang, 1965; Hegsted and WOrcester, 90 TABLE l8.--MEAA Indexes and Protein Scores for Navy Beans, Sesame Flour, and Mixtures of Navy Bean and Sesame Flour at Different Protein Ratios. thwy Saame .Eaut Fknmw B +53 B-tS B-tS (B) (S) 87.5:12.5 75:25 50:50 MEAA Indexes 88 78 89 89 88 Protein.Scores 48 50 60 72 78 1966; Hegsted et al., 1968; Hegsted and Neff, 1970). The Relative Nutritive Value (RNV) was defined as the slope of the dose-response curve of the protein under test divided by the slope of the dose-response curve obtained with the standard protein, lactalbumin. A modification of the original RNV has been considered as a potential replacement for the Protein Efficiency Ratio (PER) as the official protein quality determination method in the United States and Canada. The method is considered by its designers to be theoretically and methodologically superior to other protein quality evaluation procedures. The term Relative Protein Value (RPV) was used to describe a modification of the standard RNV procedure where the slope of the regression line linking weight gain with protein consumed was calculated without the non-protein group data. This modification was necessary because of the considerable deviation from linearity that has been found to occur at very low levels of protein intake both 91 with rats (Hegsted and Neff, 1970) and with human subjects (Young et al., 1975). This experiment was conducted to evaluate the protein quality of Navy beans, sesame flour and mixtures of bean:sesame flours (with 87.5:12.5, 75:25 and 50:50 protein ratios) using this technique. The gain in body weight was chosen to be the criterion of response since Hegsted et al. (1968) found that the results obtained with weight gain, body water and body nitrogen as measures of response were generally similar. They found that body weight measurements had less variation and the correlation coefficient from 0.89 to 0.91 (average 0.946) between weight gain and body nitrogen was obtained. One reason for the smaller errors when weight gain is utilized is probably because the calculation of weight gain automatically takes into account the variation in size of the animals at the start of the experiment whereas the values used for body water and body nitrogen do not. The mean weight changes and protein intake for the 24 groups of rats during the 21 days experimental period are shown in Table 19. An increase in food consumption was observed in all cases as the protein content of the diets improved, and the better the protein the more marked the elevation in food intake. The protein intake was well correlated with the average weight gain for all rats. There was, however, some loss 92 m.aun.mm o.mnm.NHH m.onm.om m.vum.o~ o.~wa.om a.mwm.m> NH m.anm.m~ m.mwm.am o.an~.mm n.0um.hm m.awv.a~ m.mwm.¢m ca H.H«m.>a :.mnm.mm H.HHH.5H h.mwo.o¢ m.oum.ma w.aum.m~ m m.onm.oa m.aum.mm m.o«c.oa m.ano.am m.oum.> m.~wm.m 0 .Il' .II- II- '.I III. 'II v .omuom.m+m .mmumh.m+m .m.~H"m.hm.m+m m.awn.om ¢.¢Hm.mm >.onm.mm o.a«m.ov Ii ii NH n.onm.ma m.mum.mm m.ono.ma w.mnm.om m.owm.m~ m.mwm.ooa 0H m.onh.~H m.anm.ma m.o«v.oa m.mwo.m m.owm.ma H.wnm.am m m.oum.n m.awm.m m.ona.b n.anm.mi m.onm.HH m.mwm.om o it in it It w.o«o.w m.~wm.mm w .msoo H~\o. .msoo H~\o. .mmoo H~\o. w oxmucH awmw mxmucH cflmw oxmucH GHMO uqu camuoum unmflmz cflmuoum usmflmz aflmuonm unmflmz cH cflmuonm umfio mEMmmm “can mammm umfla «A .moauom onooouo omuom one mmnme .m.~a.m.eo suw3 musoam madmmm can mammm mo mmusuxflz Ugo .Amv Macaw madmmm .Am. mammm m>mz .Amqv cflfisnamuomn mm Umflammsm cwmuoum mo mugsOE¢ mGHUflEwAinu3ouw com mumm mo moxmucH awmuonm can name unmflwz Azmmuv cmmzii.mH wands 93 in body weight for the rats consuming the bean diets at the 6% protein level. The Relative Protein Value (RPV), was calculated for each test diet as the slope of the regression line of the test diet, and are expressed as a percentage of the slope obtained with lactalbumin in the same experi- ment. The regression lines were obtained using the weight gain and protein intake of each individual rat for each diet rather than averaging them out as a group. The results are shown in Table 20 and Figure 6. The Relative Nutritive Value (RNV) calculations were performed in the same way except the regression analysis was done between weight gain and protein intake including the data for the rats fed the nonprotein diet. These animals lost an average of 22.5 9 during the experimental period. The correlation coefficients, regression equations, RNV and RPV for the tested samples as well as for the lactalbumin standard, are given in Table 21. The two sets of equations, with and without the zero protein data, were used to calculate the RNV and RPV data, respectively. The RPV values for Navy bean (B), sesame flour (8), B:S mixtures with 87.5:12.5, 75:25 and 50:50 protein ratios were 57, 52, 67, 71 and 74% (assuming lactalbumin has a RPV of 100), respectively. The RNV values for these diets were 54, 52, 66, 68, and 74% of that for 94 .caeonamuoma HOW cmcwmuno msHm> samuoum man no mwmusmmumm m mm ommmmumxm moam> camuoum may q .mxmucw Gammonm co ommmmummu ma :wmm cmnz pmcwmuno mafia man no macaw mas .o m.- umoa memos m no: .s m .wamfiacm HouucomIoumu man an pmumoflocw ucfloa may um mflmew may ummoumucw uo: can mcflmuoum ummu may you cmcwmuno mmgwa cowmmmummu mas z. umwo cwmuoumIoumn m omm massage mo moonm d N .ucmaowmmmoo coaumamuuou a on mm.m mm.m I mm.o 0H NH.0H~m.0 Aomuomv m+m an hm.m hm.hHI mm.o 0H NH.OH.m.0 Ammnmhv m+m hm ho.m oa.m I hm.o 0H NH.oa.m.m Am.NHumwhmv m+m Nm hm.N mo.oHI mm.o 0H NH.0H.m.o Amy mammmm hm N0.N mm.mHI wm.o 0H NH.oa.m~o Amv mammm 00H hm.v mh.m I mm.o 0H 0H.m .0.¢ dq Aw. msam> AmmonV ummoumusfllw m z A». samuoum mousom scflmuoum mmsHm> N H mo Hm>mq :Hmuoum m>flumamm cwmuoum .omuom one mmumh may nufi3 mnsoam memmmm cam mammm ..m. moomm m>mz ..aa. oasanaouooq mo .mumanm.hm "mowumm cwmuoum mcflsoaaom mo mmnsuxwz com Am. Macaw mammmm omaaaasm camuoum mo muaoosa monuasna I£u3ou0 0mm mumm mo mxmucH cflmuoum cam awmu unmflmz mo mwnmcoHHMHmm mnaII.o~ mamfia 95 I40, . 8+8 (5050) LA B+S(87.5=|25) Izo- //e+5(75=25) IOO- ‘ a a o 9‘ o i o I r' I 80- o B E n '3 u S 15‘ ° _ . A 8 60- O I’X’A ' .5 - ‘ .5 '~‘ - 3 40- A ,’ l l l l 20 3O 4O 50 Protein Intake, 9 Figure 6. Regression lines relating weight gain and protein intake for rats fed growth-limiting amount of proteins supplied by lactalbumin (LA), autoclaved Navy beans (8), sesame flour (S) and mixtures of beans and sesame flour with protein ratios of 87.5:12.5, 75:25 and 50:50. 96 v: mo.m Ixmm.muw mm.o v: no.wHIxmb.muw mm.o .omuomv mEommm + mcomm a: nm.>aixnm.mnw mm.o mm ah.naixmv.muw mm.o Ammumnv mEommm + mcomm mm H.@ Ixno.muw nm.o mm mm.omemm.muw mm.o .m.~aum.nmv meommm + mcomm mm mo.maixnm.mnw mm.o mm om.o~Ixmo.Nuw mm.o ucon mEommm am mm.maux~o.mn» so.o om oa.mmixoe.~um mm.o mooom >>oz ooa m>.m bem.¢uw mm.o ooa om.maixwa.muw mm.o cwacnaouoom >mm coauocvm .wmoo >zm coauocvm .mmou moucom chuoum c0flmmmcmmm .Hmuuoo cowmmmummm .Hmuuou ouom cflmuoum ocmN usocuwz omuoacoaou ouom camponm onow some omuoasomoo .mofiuom cwmuoum ucmHmHMHm no mucoam mEommm oco comm mo mmucpxflz oco Hcon mEommm .mcomm aboz .cflEcnaouoom an omofl>onm mo3 cfimuoum .muccoam mchHEwm cu3ou0 ca muom on Umm mumflm Mom A>mmv mmcHo> cfimuoum m>fluoamm oco A>zmv mmcHo> m>fluwmpoz m>Huonm .mcowuocvm cowmmmnmmmII.HN mamas 97 lactalbumin. The RPV determination puts the protein quality of the tested samples in the same order as the RNV estimation. However, the values for RNV are lower than the RPV values since by incorporating the zero protein data the slope of the regression line for lactalbumin is increased more than other diets. This is in agreement with the findings of Chavez and Pellet (1976). The slope of the lactalbumin line was 4.57 g gain in body weight per gram of protein eaten; and 2.62, 2.37, 3.07, 3.27 and 3.39 9 gain per gram of protein eaten for beans (B), sesame (S) and bean:sesame flour with protein ratios of 87.5:12.5, 75:25 and 50:50, respectively (Figure 6). It is also evident that the regression lines, relating the weight gain and protein consumed of the tested proteins did not have a common Y-intercept, nor did they meet at the point indicated by the zero protein group. The intercept of the lactalbumin line falls at -5.79 g, well above the mean of the animals fed the protein-free diet (-22.5 g). This is usually seen for most proteins (Hegsted and Juliano, 1974). Actually this is the main criticism of this technique. However, since it is certain that the regression lines relating dose to response do not go through the same intercept, the slope of the regression line for each protein was 98 calculated individually and compared to that of the standard lactalbumin protein (Hegsted, 1974). The slope-ratio technique also shows the great supplementary value of sesame protein for bean proteins. The RPV as well as RNV values of the beans diet improved from 57 and 54% to 74% (mixture 50:50) by addition of sesame flour. A greater improvement was observed for the sesame protein when bean protein was present in the diet (Table 21). The results of the different testing methods for the protein quality of the beans, sesame and their mixtures with three different protein ratios, are presented in Table 22. The linear regression equations and correlation coefficients between protein values of the tested diets estimated by the Relative Nutritive Value (RNV), Relative Protein Value (RPV), Protein Efficiency Ratios (PER), Net Protein Ratio (NPR), MEAA indexes and protein scores methods of estimating the protein quality are shown in Table 23. In general, the correlation between biological assays (RNV, RPV, PER and NPR) were all high with an average correlation coefficient of 0.96. The chemical assays (protein score and MEAA indexes), however, did not correlate with each other and had a low correlation coefficient (r = 0.50). Of the two chemical methods, 99 .OOH Hocwm cHEcHHouooH ou cOHuonu cH omuoHOOHoo xmchccomu oHuoHImQOHm Eoum mcHo> m>HuHHucc m>Huonmm .ooH Honoo annHHouooH on cOHuonH cH omuoHcoHoo .mchccomu OHHouImQOHm Eoum mcHo> chuocm m>HuonMo .OOH Hocmm chmoo op coHuonu cH ommmmumxm .oHuoH chuon umzm .OOH Hocvm chmoo op coHuonH cH ommmmumxm “moHuoH mocmHOHmmm chuoumN ..mnoH. nnounoa oononomon oms\onn on» no ommon oouoHnoHno H OOH OOH II II II II cHEcmHopooH II II OOH OOH II II chmoo on on mm mm mm m: Homuomvmum O mucutz mm H: OO om mm N: Ammum>0mum m mucuxHE no no mm mm mm Ow .m.~Hnm.:mvmum a mnnust mm mm on mo mm om Am. Hcon mEommm om hm om mm mm mo Am. mcomm m>oz om>oHooucm m>zm «Hm/mm mmmz Nmmm meUMH cwwwmww umwo noHuonHumm mo moonuoz .mOHuom chuon ucmHmHMHm no Hcon mEommm + comm >>oz mo mmucuxflz Uco HcOHm msommm .mnomm m>oz mo >zm ono >nm .mmz .mmm .mmxmonH moms .mmnoom nHmuonmII.m~ mamas 100 mo.oun so.oun oo.oun so.onn om.oun m~.o~+ HH.m~+ mi o~.ou Hs.mn+ onoom xnoo.oum nsoo.oum noo~.Hum mem.Hu> onH.ou> II nHouono oo.oun oe.oun ~5.oun m~.onn om.oun em.~mi e~.moi on.ooH- oo.ooHI Ho.me+ noonH mem.Hu> xmos.Hum noos.~u» none I- nopH.ous «an: so.oun no.onn oa.oun mn.oun so.oun oeH.oN+ oo.o~+ o~.o+ oo.ooHI o~.o- xmno.ous xmoo.oum xmoo.ous I- xmum mem.Hum can No.oun mo.oun mm.onn me.oun oo.oun o.o~+ as.msi o~.o+ oh.ooHu o.m- nos.ous ono.Hu» I- nmom.onm noon.~u» noom.Hum moz mo.onn mo.onn ao.oun oh.onn oo.onn «H.HI mo.moi so.o~+ s~.moi HH.m~+ noo.onm I- ono.Hns xmoo.ou> xmno.Hu» xnoo.ous >on oo.oun ~m.oun oo.ouo oo.ouv mm.oun oH.HI o.o~+ AH.om+ em.~ m- o~.o~+ I- xmo.oum noo.oum xmns.ous nHmm.Hu> xsoo.ou> >2: >zm >mm mmz mmm xmocH muoom ucmHOHmmmoo cOHuonHHOU 4mm: chuon oco cOHuocwm conmmummm .mnoum nHouonn ono .nnmz. monoonc oHon oann HoHunommm oonHooz .Hmmmv moHuom mocmHOHmmm chuoum .Ammzv OHuom cwmuoum umz .A>mm0 mcHo> chuonm m>Huonm .A>zmv mcHo> m>HuHuacz m>Huonm an ompoEHumm mumwm mo mmcHo> chuoum cmmsvmm mccmHOHmmmOU cOHuonHHOU oco chHuocvm conmmHmmmII.m~ mHmmH 101 protein score had a good correlation (r = 0.94) with all bioassays tested, but MEAA index method had a relatively poor correlation (r = 0.74) with the biological methods. This was of no surprise, since MEAA indexes failed to show any differences between the protein quality of the diets (Table 22). It is claimed that the RPV method should be taken as the most accurate estimate available for the protein quality (Hegsted et al., 1968). If this is true, we found the best correlation between RPV and PER (r = 0.97) and between RPV and NPR (r = 0.95). Therefore, NPR was not superior to PER as it was claimed because of the nonprotein control group which makes some allowance for maintenance requirements (Bender and Doell, 1957; Chavez and Pellett, 1976). Hegsted (1971) claimed that the amino acid score is an inadequate estimate of the nutritive value of proteins. It may not predict protein quality as well as one would wish, but in most instances, prediction from amino acid data (protein score) is reasonably accurate (Chavez and Pellett, 1976). Hegsted (1971) has claimed further that the slope- ratio technique is superior to other protein quality bioassays. For relatively poor quality protein, this is probably true, beans and sesame alone nevertheless are shown to have a lower protein quality by all the 102 assays tested and the two mixtures of beans and sesame (with 75:25 and 50:50 protein ratios) were superior by all tests. Furthermore, the slope-ratio technique did not show as high a difference between the higher quality proteins (75:25 and 50:50 mixtures, Table 22) and lower quality proteins (87.5:12.5 mixture, beans or sesame alone) as did the PER and NPR methods; and the estimates of protein quality by standard procedures and by calcu- lation from amino acid data are very close to those obtained with the RPV lepe-ratio assay procedure. Chavez and Pellett (1976) came to the same conclusion after estimating the protein quality of different diets by RPV, RNV, PER, NPR, relative NPR and amino acid scores. Supplementation of Nayy Bean Protein with Whole Egg Powder The purpose of this study was to see if we could get any improvement in the growth of rats by supplementing the beans with small amounts of animal proteins such as egg protein. Therefore, three different protein ratios of a mixture of beans and whole egg powder were tested and the animals were fed the following diets at the 10% protein level: a. Casein b. Autoclaved beans c. Autoclaved beans and egg powder with bean:egg protein ratio of 90:10 103 d. Autoclaved beans and egg powder with bean:egg protein ratio of 95:5 e. Autoclaved beans and egg powder with bean:egg protein ratio of 97.5:2.5 The results of this experiment are presented in Table 24 and Figure 7. Protein scores of the tested diets were also calculated which are shown in Table 24. Group A, casein fed animals, gained 3.69:0.35 g rat per day and the food intake was ll.87iO.87 g per animal per day. The bean fed animals, Group B, gained 1.50i0.40 g per rat per day, with a food intake of 8.13:0.51 g per animal per day and the PER Value was 1.45. Supplementation of bean protein with whole egg proteins provided a better growth for the animals than beans alone (Figure 7). The Protein Efficiency Ratios (PER) of a mixture of beans and egg powder with the following protein ratios: 90:10, 95:5 and 97.5:2.5 were 1.71, 1.68 and 1.63, respectively, and protein scores were 56, 52 and 50, respectively. Even though the protein quality of the Navy bean flour improved (as indicated by the PER's and protein scores) by addition of egg protein to the diet, there was no significant difference (P < .05) between the PER values obtained for the mixtures of beans and egg with different protein ratios. This could be due to the small contribution of egg protein in preparation of the tested diets. 104 .Hmo. v m. ucmummmHo hHHooHumHuoum uoc mco HmuumH meow mcu an om3oHHom mmcHo> m .Om.m u chmoO How omuomncoum 2mm H MH om 0mm mo.OHmw.H v0.0H~O.~ O.~v~ O.Hmo .m.~"m.hm. mom + m m mm chm o0.0Hmm.H m0.0HmO.~ m.mv~ O.on .mnmmv mom + m O on 0mm o0.0HHn.H :0.0H~H.~ H.omm O.>m¢ .OHuomO mom + m 0 mo cmm mo.OHmv.H v0.0HHm.H O.mmH O.mmm Am. mcomm om>oHooucm m om oOOH m0.0wom.m m0.0HHH.m m.mmm O.mMOH chmoO m mnoom mchmou .Hmmm Hmmm .0. Am. umHm moonw nHouonn mmo m omumnflon oxounH uanoz chuon hoom cH cHoU .wOH mos mumHm HHo mo ucmucoo chuoum Houos .m.mum.>m oco mnmm "moHuom chuoum mcH3oHHom mcu cuHs .Hmozom mmm oco mcomm mo mchHtz mmuca oco mcomm om>oHooucm .chmoO co ommom mumHm mo muoom chuoum oco mmmII.om mHmmB .OHuom 105 200 i ' A I50 - O . I B i . c >~. I ‘ D 3 E m 0 lOO - ‘ //:/ 6° :6 26 3'0 Time, days Figure 7. Weight gain of weanling rats (av. of 10) fed standard diets containing: A, casein; B, autoclaved beans + egg, 80:20 protein ratio; 0, autoclaved beans + egg, 95:5 protein ratio; D, autoclaved beans + egg, 97.5225 protein ratio; and E, autoclaved beans. 106 Effect of Processipg on the Protein Quality of Navy Beans The following processing methods were evaluated in this experiment: a. Canning with no sugar added. b. Canning with 1.5% sugar in the brine. c. Home cooking. d. Autoclaving at 250°F for 10 minutes. The results of the moisture and protein deter- mination of the samples are shown in Table 25. TABLE 25.--Moisture and Protein (N x 6.25) Content of Navy Beans Processed by Different Methods, and Dried in Air Forced Drier. % Protein Sample % Moisture (moisture free basis) Canned Beans 6.2 23.9 Canned Beans (1.5% sugar in the brine) 7.0 23.6 Home Cooked Beans 7.4 23.2 Autoclaved Beans 8.7 23.0 The three criteria used to estimate the quality of the bean protein were: PER, lysine availability and methionine availability. The first feeding experiment in this series was carried out to test the growth of young rats fed diets based on Navy beans subjected to different types of 107 processing. The results of this experiment are presented in Table 26, and the growth rate and food intake, during the 28 day experimental period, are shown in Figures 8 and 9. Group A was fed a 10% protein diet based on casein and it was used as the reference diet to calculate the PER. The weight gain was 5.1010.55 g per rat per day. The PER value was 3.42 which was corrected to 2.50. Groups B,C,D and E were fed a 10% protein diet derived from Navy beans which had been subjected to processing. The gain in body weight and food intake of these groups were close as can be seen in Figures 8 and 9. The PER values of the beans subjected to various types of heating, ranged from 1.40 to 1.49 and they were not statistically different (P < .05). They represented between 56 to 60% protein quality of the control casein diet. In order to estimate the availability of lysine in the processed bean proteins, a chemical method using l-fluoro-Z, 4-dinitrobenzene (FDNB) was used (Couch, 1975). The results of this study are presented in Table 27. Apparently, the availability of lysine in the Navy bean proteins was not impaired by the evaluated processing methods. The availability ranged from 93.3 to 97.5% which indicates that lysine is highly available in the 108 mcu no ucmHmMMHc MHHoOHumHuoum uoc mco HmupmH mEom mcu an om3oHH0m mmcHo> .Ho>oH moHHHnononc on m om.m u chmoo How omuomuuoum 2mm H MH com M0.0Hmv.H o0.0HvO.m m.mmH 0.0mm m com mo.OHHo.H v0.0HHm.H m.>o~ O.hmm a com mo.OHO¢.H oo.onom.H m.mmH O.mom O mam N0.0Hov.H m0.0Hnm.H m.omH O.mmm m oOOH oo.onom.~ m0.0H~o.m m.hmm O.hHOH chmoO chmoO .Hmmm Hmmm Am. .00 mcouu men n o umnflon oxounH unmnos chuon moom cH cHow .mOH «mumHm HHo mo ucmucoo,chmoum Houoa .Am..mcomm om>oHooucm oco ADV mcomm omxooo mEom .AOV mcHHm mcu cH Homcm mm.H cuH3 mcomm omccou Am. mammm vmccoo .chmMU mcwcwmucou mumHQ QHMUGoum 0mm mumm MO nH3OH0II.mN mHmmB 109 ZZOF . A 200- or 2‘ .9’ 0: ISO- 3 . >. 8 m g /’ 7O lOO - 2%- Time, days Figure 8. Weight gain of weanling rats (av. of 10) led standard diets containing: A, casein; B, canned beans (no sugar added); C, canned beans (with 1 .5% sugar in the brine); 0, home cooked beans; E, autoclaved beans. 110 .330 00.6.0026 .w .9500 09.000 060: .n. .355 05 :. 500.... can. F 5.3. 0:80 00:58 .0 20000.... noose 0:. 0:80 858 .m 8.008 .< 655250 30.0 Bogota. 00. SP .0 $8 $9 05.503 .0 9.3:. 000“. .O 200.“. 9.00 .oEfi OM) ow o. o co. m ..\. 00m 0 w o O com 4 00v 6 ‘anquI p003 111 TABLE 27.--Lysine Availability of Bean Protein Processed as Follows: A, Canned with no Sugar Added; B, Canned with 1.5% Sugar in the Brine; C, Home Cooked; and D, Autoclaved. % Available Sample Lysine A 93.3 B 93.9 C 97.5 D 97.4 Navy bean proteins regardless of the methods of processing used in this study. The results of the bioassay which aimed at deter- mining the utilization of methionine in processed beans are shown in Table 28 and 29. Table 28 shows the growth of rats fed standard diets in which the methionine content of the diets was 0.2%. This level does not correspond to maximum growth rate for rats, which is close to 0.6% (NAS, 1972). The 0.2% methionine in the casein diet corresponds to the methionine content of casein itself. The bean diets contained 0.1% methionine originating in the beans and 0.1% L-methionine added as a crystalline compound. The bean methionine availability was calculated as follows: There are two assumptions made in this calculation: first, that casein methionine is utilized 81% according to 112 wm.o HON QHQMHflM>M QDHM> OZ .umHo chmoU o cH mchochmz .AvmmH. Homccucw oco uummHm3cOm Eoum cmxoa m .Hm>oH muHHHnononm mm mcu no ucmumMMHo mHHoUHumHuoum uoc muo HmuumH meow man an om3oHHoM mmcHo>H II mo.m> om.O ~.an m.Oh m.O HIm II Hm.mm mH.H o.om~ m.mm m.O HIchon oom mm.mmH vv.o ~.m- 0.0m N.O m owe O0.0mH m¢.O m.>H~ m.mh «.0 m omv m~.mnH mo.O m.mm~ O.mm ~.O 0 omo mm.HmH oo.O O.mHN «.0: N.O m NHm mO.mmH mo.O «.mmm «.mm m.O chmoO manoHnums omsnmnoo .0. .m. uanmz zoom m umHo nH uoHn anoHHo>m w .umz.0 omEcmcoo mxoucH cH mchochmz cHoo .u3.0 mchOchmz 000m mmoum>m cHoU mmoum>m mo Hm>mH .wm.o 0cm N.o Op unmunoo oanoHnuoz Hence one manm on muoHo on» on oooon .Amv mcomm om>oHooucm oco HOV mcomm omxoou mEom ..o. oanm one nH noonm om.H nuns mnoom oonnoo ..mo unmom oonnoo ”muonn .mouom nueono no: no oomom .mnomm oommooonn nH muHHHnnHHo>n oanoHnuosii.o~ momma mo3 mchochszH 113 mo.mh 0.0: 00.0 .umz 00.0 + mcomm mh.~hH «.00 000.0 .umz mm.0 + mcomm mm.mmH 0.00 00.0 .umz 0H.0 + mcomm No.0mm v.nm 000.0 mcomm .m\m. noHuoNHHHuO .mv nHoo .m. omenmnoo umHo manochmz .u3.o .umz.m mo sonononmmn .Acwmuoum wOHV mumfin HHo OH @mwflfl mo3 mcHummuIH 0H.0 .mchOchmz mo mHm>mH mcoHHo> co .m mo .>n. moon on own muono noon on nonnoNHHHuo oanonnooz mo monoHonmnii.o~ manna 114 Schwergert and Guthneck (1954); and second that crystalline L-methionine is utilized 100%. Casein diet had 0.2% methionine which corresponds to: 0.2% x I%%'= 0.162% available methionine; this resulted in 198.03 g Wt. gain per rat per g available methionine consumed. The 0.2% methionine in the autoclaved bean diet (H) resulted in 183.36 9 weight gain per rat per g methionine consumed. Some of this growth is due to the pure methionine added to the diet and the remaining growth is due to the bean methionine. The following calculation permits the portion of bean methionine utilized by the growing rats: 0.162% casein available methionine resulted in 198.03 g Wt. gain/g methionine consumed 0.1% added crystalline methionine resulted in X = 122.24 g Wt. gain/g methionine consumed Therefore: 183.36 - 122.24 = 61.12 g Wt. gain was due to the consumption of the bean methionine, and the availability of the bean methionine is: 61.129 (Wt.ga.i_n/rat/14 days caused by mnsuning bean methionine) x100 122.24g(Wt.gain/rat/14 days caused by oonsum'ng pure methionine) ==50% This 50% availability of the autoclaved bean methionine found by growth bioassay corroborates the results of Evans et a1. (1974), who investigated the 115 availability for the rat of methionine and cystine in Navy beans and soybean meal using a microbiological assay (Leuconostoc mesenteroids). Methionine and cystine balance studies were conducted with growing rats and it was reported that about 50% of the methionine and 25% of the cystine in beans were excreted with the undigested protein in the feces. Approximately, 26% of the methionine and 11% of the cystine of soybean meal were excreted in the feces. It was also reported that only small amounts of methionine (1%) or cystine (2-9%) were excreted in the urine. Our calculation was extended to estimate the methionine availability of beans subjected to other processing treatments (Table 28). Animals, which were fed canned beans with 1.5% sugar in the brine, consumed more methionine but gained less weight than rats fed canned beans with no sugar added. Therefore, the methionine availability was 42% for the former versus 49% for the latter. The methionine availability of the home cooked beans was 48%. When the total methionine content of the casein and autoclaved bean diets was raised to 0.5%, the weight gain per gram of methionine consumed decreased drastically. It must be noted that the term "availability" does not describe the phenomenon observed here. The pure methionine added to the diet is readily available to the animal, but 116 it is simply not utilized for growth. Schweighert and Guthneck (1954) had observed that the growth of protein- depleted rats levelled off when L-methionine was added to casein at levels higher than 200 mg L—methionine per 100 g diet. Efficiency of methionine utilization for growth would be a better term to describe the relationship between methionine levels in the diet and rat growth. From experiments conducted in this study the following effi- ciency ratios were obtained (Table 29). The efficiency of methionine utilization is reduced as the methionine level in the diet increases. It is, therefore, concluded that under the condi- tions of our experiments, the evaluated processing methods which included: canning with or without added sugar and home cooking did not impair the protein quality of the Navy bean as indicated by the PER, lysine availa- bility and methionine availability values. Johnson, et al. (1939), in studies of sulfur and nitrogen balances in rats fed diets containing raw, heated and solvent-extracted soybeans, found that equal amounts of sulfur and nitrogen disappeared from the digestive tract regardless of the previous treatment of the meal or beans. Guttridge (1961) developed a method for the bio- assay of methionine using White Leghorn chicks at 11-18 117 days of age. Using this technique, assays of biological available methionine was made on several soybean samples which had received varying heat treatments. He obtained a high available methionine value, an average of 90% for the tested samples. Guttridge and Lewis (1964) studied the chick bio- assay of methionine and cystine in soybean meals and groundnut meals. They reported an average methionine availability of 85% and 74% for soybean meal and ground- nut meal, respectively. Growth assays with 8-day old crossbred chicks were used to estimate the availability of sulfur amino acids in corn gluten and corn protein by the slope-ratio technique and standard curve method (Sasse and Baker, 1973). The availability estimates for corn gluten meal were 98.912.l% and 99.211.6% and for corn 96.5i6.5% and 93.918.7% using the slope ratio and standard curve methods, respectively. Miller et a1. (1965) measured lysine availability (DNFB method and chick assays) and methionine availability (Streptococcus zymogenes and chick assays) in freeze-dried cod muscle subjected to 16 different conditions of heat treatment, with temperatures from 45 to 116°C and time from 9 to 729 hours; in a few of the treatments the dried muscle was first mixed with 5 or 10% glucose. They reported that mild heat treatment in the presence of 5% 118 glucose reduced the available lysine by 18% although available methionine was unchanged. From chick and microbial trials it was concluded that heat conditions more severe than 85°C for 27 hours, achieved either by elevation of temperature or by heating for a longer time all brought significant loss of available sulfur amino acids. For the mildest treatment (85°C, 27 hours) the FDNB lysine value was not affected whereas for the others there was a significant fall. The long term treatment (85°C, 729 hours) caused as much apparent damage to methionine as did the short, high temperature treatment (115°C, 27 hours) but considerably less damage to the lysine. Segal and Motoc (1970) reported that during the heat treatment of green peas important losses of essential amino acids occurred and the amount of loss depended upon the heat treatment, with the largest losses found in arginine, lysine, histidine and methionine. They also found that the presence of glucose in the added brine increased the losses. However, sterilization of peas in cans did not affect the biological value of the proteins significantly because the losses in essential amino acids were low due to short duration of the high temperature process. Molina et al. (1975) investigated the effect of storage, soaking time (0,8,16 and 24 hours), and cooking 119 time (10,20, and 30 minutes at 121°C and 15 psi pressure) on the nutritive value of black beans. They reported that cooking time had a statistically significant (P < 0.05) effect in lowering the protein quality of the beans processed either immediately after harvesting or after 3 and 6 months of storage. They showed that the total protein content was unaffected either by storage or by any of the processes evaluated; but the methionine and available lysine content of the processed samples tended to increase with storage, independent of the process to which they were subjected. Braham et a1. (1965) found that the optimum cooking temperature for pigeon peas (Cajanus Cajan) was 20 minutes at 121°C and 16 pound pressure and a longer heating time lowered the nutritive value of the beans. With respect to cooking periods, Bressani et al. (1963) have recommended autoclaving at 16 psi and 121°C for 10 to 30 minutes for black beans (Phaseolus vulgaris). Heating for longer periods resulted in a decrease in the nutritive value of the protein, due to changes in the essential amino acid content of the bean, specially in the content of available lysine, which decreased pro- portionally to the increases in cooking time. Miller (1973) studied the effect of processing on the nutrient retention and the PER value of pinto bean products. Three processing methods were investigated: 120 (a) instant drum—dried bean powder processed in water (the regular method and by prior HCl treatment); (b) cooking for two hours at 210°F, and (c) retorting for 45-90 min. at 250°F (soak-blanched canned beans). They found no difference in the PER values of these bean products. Zinc Supplementation of Nagy Beans The statements have been made "that a relation- ship exists between zinc and the utilization of phytate containing plant seed proteins and that when properly supplemented with zinc these proteins were equal in quality to animal proteins" (Oberleas and Prassad, 1969); and "the reluctance on the part of many nutritionists to supplement adequate zinc in experimental diets has resulted in the belief that plant seed proteins are inferior--when in fact they may be quite comparable to animal protein" (Oberleas, 1973). There are many factors that can influence the availability of zinc in the diet. The presence of chelates in the diet influences the availability. The phytate present in animal diets reportedly interferes with the intestinal absorption of zinc (Oberleas, et al., 1962). Zinc phytate complexes can be formed in the gastrointestinal tract making zinc unavailable. 121 The purpose of this study was to test the effect of zinc supplementation of Navy beans on the growth of weanling rats. The animals were fed the following diets at a 10% protein level. a. casein b. autoclaved beans c. autoclaved beans + 3.8 ppm zinc d. autoclaved beans + 6.2 ppm zinc e. autoclaved beans + 16.2 ppm zinc f. autoclaved beans + 21.2 ppm zinc g. autoclaved beans + 0.5% D, L-Methionine h. autoclaved beans + 0.5% D, L-Methionine + 10.9 ppm zinc The analysis of Navy bean flour revealed a zinc content of 29 ppm. The casein used in this study contained 34 ppm zinc, the corn starch 7 ppm, and no zinc was detected in the corn oil. The results of a four-week ad libitum feeding are shown in Table 30 and Figure 10. The highest average gain in body weight was achieved by Group G and H, in which beans were supplemented with methionine and methionine plus zinc, respectively. The lowest weight gain was for Group B, in which no zinc. (as a pure salt) was added to the bean diet. The Protein Efficiency Ratio (PER) of the bean diet fortified with methionine was 2.80 which was statistically higher than the one for the casein diet (PER = 2.50). Further .Hm>mH moHHHnonono om mcp no ucmumMMHo aHHooHumeoum uoc mco HmuumH meow man an omsoHHOM mmcHo>m .0m.~ u chmoo cow cmuomuuoo 122 0 2mm “.mm .uHom mucm o no omoco uoc mos ocHNN .Omm 5.00m cN mo omooo mos ocHNH ooHH m0.0«om.~ 00.0Hmm.m 0.00 m.mmH m.:m cu Ema 0.0H+ .umz wm.0+ Hcomm .ucm m omHH 00.0H00.m 00.0Hm>.m H.00 0.~mH 0.0H .umz mm.0+ Ncomm .ucm 0 00m m0.0H00.H 50.0H00.H 0.0m N.Hm 0.>m Hcomm .ucm m 00m 00.0HHo.H 00.0HHO.H m.Hm m.00 0.~m Hcomm .ucm m 00m «0.0H0v.H 00.0H00.H 0.0m v.mm m.m~ Hcomm .ucm 0 ohm m0.0nmo.H h0.0Hm0.H 0.m~ m.mv 0.00 Hcomm .ucm 0 cmm no.0nmm.H 00.0HHO.H n.0m 0.00 0.oH Ncomm .ucm m o00H 00.0H0m.~ HH.0Hmm.m >.mm m.oHH . H.mH HchmoO m mchmoO . mmm mmm .00 .00 5mm moucom mcouw mmm m owumcnom m mxoucH cHoo ucmucou chuoum chuonm ucmHmz ocHN .muom mo cusoco ms» co mcomm m>oz om>oHoouc< mo cOHuoucmEmHmmcm ocHN oco mchochmz mo ummmmmII.Om mmmma 250, A 200- ° . Casein °j l50- E» g /21: 5‘ lOO- / /V 8 /:,/ % /./ 5O 0 I0 20 50 Time, days Figure 10. Growth curves of rats (av. of 10) fed bean diets supple— mented with Zn and D,L-methionine: A, beans + 0.5% D,L-methionine; B, beans + 0.5% methionine + 10.9 ppm Zn; C, beans + 16.2 ppm Zn; 0, beans + 6.2 ppm Zn; E, beans + 21.2 ppm Zn; F, beans + 3.8 ppm Zn; G, beans; and casein. 124 supplementation of this diet with zinc increased the PER value only one percent (PER = 2.84) which was not sta- tistically significant at the 5% probability level. The beans alone provided about 14 ppm zinc to the rat diet. An increase of 6% in PER was observed with an addition of about 6 ppm zinc to the diet. However, no indication of improvement, as determined by gain in body weight and PERs, was observed by addition of graded levels of zinc up to a total concentration of about 38 ppm. This can be seen in Figure 11 which shows the percent PER of bean proteins, relative to casein equal 100, as a result of increasing zinc content of the diets. It is apparent that maximum rat growth was obtained on a diet in which Navy beans were the only source of protein with a zinc concentration of 19.8 ppm. However, when the bean diet was supplemented with methionine the growth rate increase was 110% in terms of PER. Furthermore, the very modest additional growth obtained by supplementing the bean diet with zinc dis- appeared when the bean protein quality was upgraded by addition of methionine. Therefore, the contention that addition of zinc can upgrade the quality of a plant-seed protein to that of animal protein (Oberleas and Prasad, 1969) is invalid, at least in the case of Navy bean protein and when protein efficiency ratio is used as an index of protein quality. On the other hand, this 125 .03 no .00 no; no.0 5003 20.0.:00 o .0 mma 0:... .:N .0 mo:.Eo=0 000:? 5.3 025820000 $0.0 :omn _0 Ema. 00:8 55.0.0.0 £20.“. .3 200.“. 600 .:N on 00 ON . . . . O on % d 3 .8 cN 58 0.0. - mm 0 I! 0 w I m. U 126 experiment clearly demonstrates the great supplementation value of methionine for Navy bean protein which is severely deficient in this amino acid. Oberleas and Prasad (1969), on the basis of an experiment in which pure phytic acid was added to a soy protein diet, had concluded that "a relationship exists between zinc and the utilization of phytate containing plant seed proteins and that when properly supplemented with zinc these proteins were equal in quality to animal proteins." The aim of this study was to investigate the effect of zinc supplementation to the diets in which the phytic acid present was the one naturally occurring in the Navy bean flour. Our results agree with those of Forbes and Yoke (1960) who showed that 17.8 ppm zinc resulted in maximum growth rate when soy protein was the sole source of protein in rat diets. In fact, if the glucose used in the soy protein experiment contained zinc at a level close to that of the corn starch used in our experiment, the optimum zinc value of Forbes and Yoke would have been even closer to ours (19.8 ppm Zn). Antunes (1975) showed that the amount of zinc absorbed by the rats was essentially the same for the Navy bean diets with or without addition of 55 ppm zinc. The bean flour contained 1.3% phytic acid (Lolas and Markakis, 1975). All diets in which autoclaved bean 127 flour was the sole source of protein contained 0.6% of naturally occurring phytic acid. Assuming that the growing rat requirement for zinc to be 12 ppm in a 90% dry diet (NAS, 1972), the bean diet necessitated the presence of an additional 8 ppm zinc. If all of this additional zinc was chelated by phytic acid of the beans, and made un- available to the rat, only 0.002% of the phytic acid was used in the binding of zinc. SUMMARY AND CONCLUSIONS The amino acid composition of Navy bean (Phasealus vulgaris L.) flour of the Sanilac variety and sesame seed (Sesamum indicum L.) defatted flour was determined by the Beckman Model 120°C amino acid analyzer. The supplementation effect of Navy bean protein with sesame protein was studied in a series of experi- ments. Mixtures of Navy bean flour with defatted sesame flour were prepared. All bean flours used in these experiments were autoclaved for ten minutes at 250°F (121°C). Diets were prepared containing the following protein ratios: all bean protein; all sesame protein; 87.5 bean protein to 12.5 sesame protein; 75 bean protein to 25 sesame protein; and 50 bean protein to 50 sesame protein. Protein quality of the diets were evaluated by the Protein Efficiency Ratio (PER), Net Protein Ratio (NPR), and slope-ratio methods. The FAO/WHO group (1965) procedure was used to calculate protein scores based on the FAO/WHO reference pattern (1973). The Modified Essential Amino Acid (MEAA) indices (Mitchell, 1954) of the tested diets was also computed using the same reference pattern (FAQ/WHO, 1973). 128 129 The PER for the bean diet compared to 100 for that of casein, was 62, and 48 for the sesame meal. For the 87.5:12.5, 75:25, and 50:50 mixtures of beans:sesame flour, the PER's were 72, 90, and 92%, respectively. The NPR values for beans (B), sesame (S), B:S (87.5:12.5), B:S (75:25), and B:S (50:50) were 64, 50, 69, 90 and 92%, respectively, compared to 100 for casein. The Relative Protein Value (RPV) and Relative Nutritive Value (RNV) from slope-ratio techniques were claculated when the corresponding lactalbumin values were set at 100. The RPV for the tested diets were, 57, 52, 67, 71 and 74% respectively. The RNV were, 54, 52, 66, 68 and 74%, respectively. The protein scores (PS) of Navy beans, sesame meal and mixtures of Navy beans:sesame flours with protein ratios of 87.5:12.5, 75:25 and 50:50, were 48, 50, 60, 72 and 78, respectively. The MEAA indices were 88, 78, 89, 89 and 88, respectively. The protein values of the tested diets from rat bioassays and chemical methods were compared. The correlation coefficients between the evaluated biological methods were high with an average of 0.96. The correlation coefficient between the chemical methods (PS and MEAA indices) was low (r = 0.50), and of the two methods tested, only protein score gave a good correlation with the bio- logical assays (r = 0.94). 130 The supplementary effect of whole egg on the Navy bean protein was also studied, and the protein quality of the beans improved by the presence of egg protein in the diet. The mixtures of beans and egg powder with three different protein ratios were prepared. The PER values relative to casein 100, of the Beans (B), B:Egg (97.5:2.5), B:Egg (95:5) and B:Egg (90:10) were 58, 65, 67 and 68 respectively. In the second series of the experiments, the protein quality of the Navy beans subjected to different processing methods was estimated. The processing methods evaluated were: Canning with or without sugar, home cooking and autoclaving. The PER, methionine availability (growth assay with wealing rats) and lysine availability (reaction with l-fluro-2, 4-dinitr0phenol benzene) were used to estimate the protein quality of the processed beans. The PER values (compared to casein = 100) for the canned beans, canned beans with 1.5% sugar in brine, home cooked beans and autoclaved beans were, 58, 56, 56 and 60% respectively; the methionine availability (compared to crystalline methionine equal to 100) were, 49, 42, 48 and 50%, respectively; the lysine availabilities were, 93.3, 93.9, 97.5, and 97.4% respectively. It was con- cluded that the evaluated processing methods did not impair the protein quality of the Navy beans as indicated by the PER, methionine availability and lysine availability values. 131 A study was conducted to determine if zinc supple- mentation can upgrade the quality of the bean protein. Increasing quantities of zinc were added to 10% protein diets in which the sole source of protein was autoclaved Navy beans. The concentration of zinc in the diets varied from 16.6 to 37.8 PPM. These diets were compared to a standard casein diet in order to determine PER values. A very small increase in growth (6% in PER) was observed when the natural Zn content in the bean diet (16.6 ppm) was raised to 20 ppm zinc by adding ZnSO4 ' 7H20. Further supplementation of the bean diets with zinc did not promote the growth. Supplementation of the same diet with 0.5% D, L-methionine resulted in a much greater increase in growth rate (110% in PER). 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