PROTEIN SUPPLEMENT ATION 9F NAW BEANS WITH BRAZIL NUTS Dissertation for the Degree of Ph. D.. MICHIGAN STATE UNIVERSITY ALOISIO JOSEANTUNES 1975 ' This is to certify that the thesis entitled Protein supplementation of Navy beans with Brazil nuts presented by f Aloisio Jose Antunes has been accepted towards fulfillment of the requirements for Ph.D. Food Science degree in /) ’l' .1 717 M. /-H 4A mfltacqj Aggi/u? 1 Major professor 4 -1 / 1, Date 52341152 /’} 0/15 0-7 639 ABSTRACT PROTEIN SUPPLEMENTATION 0F NAVY BEANS WITH BRAZIL NUTS 8y Aloisio José Antunes Rat feeding experiments were conducted which showed that the protein quality of Navy beans (a poor source of sulfur-containing ' amino acids) can be largely improved by mixing the beans with Brazil nuts (a rich source of methionine). Compared to a PER (protein efficiency ratio) of 2.50 for casein, the PER for beans was found to be 1.53. The PER for diets in which bean protein and Brazil nut protein were present at the ratios 80:20, 90:10 and 95:5 were 2.42, 2.16, and 1.93, respectively, at 10% total protein content in the diet. Two lots of Brazil nut defatted flour were used in this study and their selenium contents were 63.2 and 17.3 ppm. Brazil nut flour with 63.2 ppm Se was toxic to rats at the 10% protein level (11.3 ppm Se in the diet). However, when the diet was made to contain 20% pro- tein (10% from Brazil nut and 10% from casein) no toxic effect was ob- served. The mixed diets (Navy beanszBrazil nut) were prepared with the Brazil nut flour which contained 17.3 ppm Se. The supplementary effect of Brazil nut protein on Navy beans was also evaluated by a microbiological method, utilizing Streptococcus zymggenes as the assay organism. The relative nutritive value (RNV) Aloisio Jose Antunes of the mixtures were compared to PER values for the same mixtures. The following linear regression equation was calculated: Y = 0.0699 + 0.0253 X, where Y is the RNV and X is the PER. The correlation coefficient was 0.89. Protein scores and modified essential amino acid indices (MEAA) were also computed for Navy beans, Brazil nut defatted flour, and for the mixtures Navy beans:Brazil nut at the protein ratios 95:5, 90:10 and 80:20. The protein scores were 27, 66, 33, 42, and 58, respectively. The MEAA indices were 82, 74, 83, 84 and 86, respectively. The trypsin inhibitory activity of Navy beans and five varieties of Brazilian beans, Carioca, Goiano Precoce, Pirata, Rosinha, and Rico-23 varied from 48.3 (Goiano Precoce) to 86.3 (Rico-23) TUI/mg protein. Beans contain considerable amounts of phytic acid. In bean diets containing 0.34% phytic acid Zn was added, 55 ppm, as well as 0.1% and 0.2% D,L-methionine. No improvement of rat growth was obtained by the zinc supplementation, but the addition of 0.1 and 0.2% D,L-methionine improved the growth of the animals. PROTEIN SUPPLEMENTATION OF NAVY BEANS WITH BRAZIL NUTS By Aloisio José Antunes A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY DEPARTMENT OF FOOD SCIENCE AND HUMAN NUTRITION 1975 To Aparecida, Eduardo and Marcelo. ii ACKNOWLEDGEMENTS The author wishes to express his sincere gratitude to his major professor, Dr. Pericles Markakis, for his encouragement, advice and excellent guidance during the doctoral program and for his aid in the preparation of the thesis manuscript. Appreciation is also extended to Dr. J. R. Brunner and Dr. G. Borgstrom of the Department of Food Science and Human Nutrition: to Dr. H. A. ' Lillevick of the Department of Biochemistry, and to Dr. D. R. Dilley of the Department of Horticulture for serving on the guidance committee and for critically reviewing the thesis. The author feels grateful to the Instituto de Tecnologia de Alimentos (ITAL) and to the Fundacao de Amparo a Pesquisa do Estado de 550 Paulo (FAPESP) for the financial assistance granted to him during his doctoral program at this university. A very special acknowledgement goes to my father, Dr. José Antunes, for his invaluable and continuous help, and to my wife, Aparecida, for her patience, understanding, and encouragement during the course of the graduate program. iii TABLE OF CONTENTS Page ACKNOWLEDGMENTS ......................... iii LIST OF TABLES .......................... vi LIST OF FIGURES ......................... viii INTRODUCTION .......................... l ’ REVIEW OF THE LITERATURE .................... 3 Amino Acids and Protein Requirements ............ 3 Evaluation of Protein Quality ............... 9 Chemical Scores ...................... 11 Protein Scores by the FAO/WHO Procedure .......... 12 Oser's Essential Amino Acid Index ............. 12 Biological Value (BV) ................... 13 Net Protein Utilization (NPU) ............... 16 Protein Efficiency Ratio (PER) ............... l6 Microbiological Methods .................. 18 Legumes .......................... 20 Phytic Acid ........................ 31 Brazil Nut (Bertholletia excelsa) ............. 32 Selenium ......................... 33 MATERIALS AND METHODS ...................... 36 Navy Beans ........................ 36 Germination ........................ 36 Brazil Nut ......................... 37 Amino Acids Analysis .................... 37 Sulfur Containing Amino Acids ............... 39 Tryptophan ......................... 40 Determination of Biological Value with Streptococcus zymogenes ........................ 41 Culture Medium ....................... 42 Maintenance of Stock Culture ................ 43 Preparation of Inocula for Protein Assay .......... 44 Preparation of Samples for Test .............. 44 iv Fluorometric Selenium Analysis .............. 45 Standard Selenium Solution ................ 46 Total Protein ....................... 47 Crude Fiber ........................ 47 Oil ............................ 47 Ash ............................ 47 Moisture ......................... 48 Phytic Acid Determination ................. 48 Biological Evaluation of Protein Quality ......... 49 Zinc Supplementation Study ................ 50 Net Ashing Procedure ................... 50 Preparation of Standard Solution ............. 50 Instrument Parameters ................... 52 Determination of Trypsin Inhibitory Activity ....... 52 Protein Determination .................. 54 RESULTS AND DISCUSSION ..................... 55 Sample Material ...................... 55 Total Amino Acids of Brazil Nut and Bean Flours ...... 55 Feeding Experiments with Brazil Nut as Source of Protein . 58 Effect of Protein Intake on Selenium Toxicity ....... 62 Supplementation of Navy Bean Protein with Brazil Nut . . . 65 Protein Scores ...................... 78 Modified Essential Amino Acid Index (MEAA Index) ..... 81 Evaluation of Protein Quality by Microbiological Method. . 81 Trypsin Inhibitor Content of Dry Beans .......... 84 Zinc Supplementation of Navy Beans ............ 88 SUMMARY AND CONCLUSIONS ..................... 95 BIBLIOGRAPHY .......................... 99 APPENDIX ............................ 114 Table IO II 12 l3 I4 15 LIST OF TABLES Page Classification of Amino Acids ............... 4 Obligatory N losses in adult men on a protein-free diet . . 9 Est;m:ted amino acids requirements of adults and growing 10 Essential amino acid composition in the whole egg protein . 13 Computation of the Modified Essential Amino Acid Index. . . 14 Total world acreage and production of the major food legume crops and wheat, rice and corn ............. 22 Acreage (1,000 ha) and production (1,000 m.t.) of dry beans by continent in 1972 ................... 23 Essential amino acid content of selected beans compared with the FAD/WHO pattern. (g/l6g N) .......... 25 Methionine content of selected vegetables and animal products ......................... 33 Composition of basal medium ................ 43 Composition of basal diet ................. 51 Composition of defatted Brazil nut flour, and Navy bean flour .......................... 56 Total amino acid composition of Brazil nut and bean flours (expressed in gram amino acid per 16 grams of total nitrogen and as mg amino acid per gram total nitrogen). 57 Effect of a standard diet containing 10 percent casein protein and a standard diet containing 10 percent B.N. protein on growing rats ................. 60 Growth of rats fed on diets containing casein and casein: Brazil nut mixture .................... 63 vi Table Page 16 Growth of rats fed on standard diets containing casein, autoclaved beans, germinated and autoclaved beans and a mixture of beanszBrazil nut (80:20) .......... 67 17 Growth of rats fed on standard diets containing casein, and mixtures of autoclaved beans + Brazil nut with the ‘protein ratios 95:5 and 90:10 .............. 72 18 Effect of supplementary methionine and selenium on the growth of rats given autoclaved beans as compared to casein and a mixture of autoclaved beans + Brazil nut with the protein ratio 80:20 ............... 76 19 Essential amino acid composition of whole egg. Navy bean flour, Brazil nut flour and mixtures of Navy bean:Brazil nut flours (expressed as mg amino acid per gram total nitrogen ........................ 79 20 Protein scores of Navy bean flour, Brazil nut flour, and mixtures of Navy beanzBrazil nut flours based on the essential amino acid pattern of whole egg ........ 80 21 MEAA indices and protein scores for Navy beans, Brazil nut (BN), and mixtures of Navy bean + Brazil nut at different protein ratios ....... 1 .............. 82 22 Growth of S, zymogenes as determined by the absorbance at 580 nm and RNV o the different samples compared to PER values ........................ 83 23 Trypsin inhibitor activity ................. 85 24 Zinc supplementation of bean diets fed to growing rats. . . 90 25 Zinc balance study (five days study) ............ 93 vii LIST OF FIGURES Figure Page 1 N 10 Pattern of essential amino acids (mg/g total N) of Brazil nut, Navy bean and whole egg ........... 59 Growth curves of rats fed on standard diets containing casein and Brazil nut flour ............... 61 Growth curves of rats fed on casein and casein:Brazil nut mixture ....................... 64 Weight gain of weanling rats fed on standard diets con- taining casein, autoclaved beans, germinated and auto- claved beans, and a mixture of autoclaved beans + Brazil nut with the protein ratio 80:20 ............ 68 Food intake of weanling rats fed on standard diets contain- ing casein, autoclaved beans, germinated and autoclaved beans and a mixture of autoclaved beans + Brazil nut (80:20 protein ratio) .................. 69 Weight gain of rats fed on standard diets containing casein, and mixtures of autoclaved beans + Brazil nut with the following protein ratios, 95:5 and 90:10 ......... 73 Food intake of rats fed on standard diets containing case- in, and mixtures of autoclaved beans + Brazil nut (95:5 and 90:10 protein ratios) ................ 74 Weight gain of rats fed on casein, autoclaved beans diets supplemented with methionine and selenium and a mixture of autoclaved beans + Brazil nut (80:20 protein ratio). . 77 Trypsin inhibitor activity of dry beans, determined by the BAPA method in relation to the level of crude bean extracts ...................... 86 Growth curves of rats fed on bean diets supplemented with zinc ........................ 9'I viii 1.. !‘£§.R lbs BEE-1'1. ; . 4. i. it INTRODUCTION Protein malnutrition is one of the major nutritional problems in the world today. The available animal protein does not suffice to meet the needs of the world population. Two-thirds of the protein consumed by human beings come from cereals (Borgstrom, 1967), which are known to be deficient in lysine, an essential amino acid. A significant proportion of the protein in the diet of large ‘segments of the world population come from legumes. Common beans (Phaseolus vulgaris) are among the most important legumes in human nutrition in Latin America, Far East, parts of Africa and India. They constitute an important source of protein for the general pop- ulation in developing countries and for some special groups in the more affluent countries. The Latin American production of dry beans represent 45 percent of the world legume production. Brazil, my homeland, produces 59 percent of the beans grown in Latin America. It is estimated that Brazilians have an intake of 66 grams of dry beans per person per day, which represents a daily intake of 224 cal- ories and 14.5 g of protein. The total protein intake of the Brazilian population is estimated to be 64 g per person per day, from which 41.5 9 comes from vegetable sources and 22.5 g is of animal origin. The total intake of bean protein represents almost 23 per- cent of the total protein intake and 35 percent of the vegetable pro- teins consumed by Brazilians. In general, vegetable proteins lack one or more of the essen- tial amino acids, but experiments have provided evidence that the nutritional value of several vegetable proteins can be largely improved by appropriate supplements of amino acids or by combining them with proteins that have complementary amino acid patterns. Two major nutritional problems have been associated with beans: the first is a deficiency in sulfur-containing amino acids and the second is the presence of anti-nutritional factors, such as trypsin inhibitors and hemagglutinin. Moderate heat treatment usually inactivates these deleterious substances and the nutritive value is ' improved. Brazih nuts (Bertholletia excelsa) have an unusually high level of sulfur-containing amino acids, and it is probably the rich- est food source of these essential amino acids. On the other hand, Brazil nuts are low in lysine, which is their first limiting amino acid. The objective of this research was to study the supplementary effect of Brazil nut protein on Navy bean protein through rat feeding experiments and a microbiological method. Protein scores and mod- ified essential amino acid indices were also determined. In parallel, trypsin inhibitory activity of several cultivars of common beans was determined, and the effect of zinc supplementation of Navy beans was studied. REVIEW OF THE LITERATURE Amino Acids and Protein Requirements The purpose of dietary protein is to provide amino acids in the appropriate pattern for efficient synthesis of protein and other nitrogeneous substances essential to the organism. The credit goes to Rose (1949) for the important information as to which amino acids can be synthesized within the organism and which cannot. The latter must be provided with the food and are called essential amino acids. Those amino acids that can be synthe- sized in the body are considered nonessential. Classification of amino acids in this way applies only to dietary needs since all are essential for the synthesis of proteins. Table 1 shows the classification into essential and non- essential amino acids (Rose, 1957). Histidine is generally considered essential for the infant (Snyderman et_al, 1963), but not for the adult man. However, Kopple and Swenseid (1975) recently reinvestigated this problem, comparing the metabolic response to deletion of histidine from the diet of normal and of chronically uremic adult males. These authors presented some evidence of a dietary histidine requirement. When the subjects were fed a histidine-free diet, a situation of negative N balance was observed, but it was reversed by adding histidine. Some amino acids may be dispensable for one species, but essential for another. Arginine is dispensable for man, even for 3 4 TABLE l.--C1assification of Amino Acids. Essential Non-Essential Lysine Glycine Tryptophan Alanine Phenylalanine Serine Leucine Cystine Isoleucine Tyrosine Threonine Aspartic Acid Valine Glutamic Acid Methionine Proline Histidine Hydroxyproline Arginine lthe growing infant (Holt, 1967). On the other hand, it is essential for the young rat (Rose et_al,, 1948). Another example is glycine, which is essential for the chick, but not for mammals (Meister, 1965). Some of the nonessential amino acids can be synthesized only from specific essential amino acids. If the former are provided in the diet, the need for the amino acid from which they are derived is reduced. Cystine can be formed only from methionine; and when cystine is present in the diet in adequate amounts, less methionine is required (Rose and Wixom, 1955). Tryosine can be formed only from phenylalanine and when tyrosine is supplied by the diet, less phenyla- lanine is required (Rose and Wixom, 1955). The other nonessential amino acids can be synthesized in the body from organic acids that are intermediates in carbohydrate metab- olism, e.g., o-ketoglutarate and pyruvate (Steele, 1952), nitrogen from surpluses of individual amino acids and from such compounds as ammonium citrate (Rogers et_al,, 1970). As far as the maximum growth of the young rat is concerned, most individual sources of nonspecific nitrogen are inferior to a mixture of all the nonessential amino acids, and diets containing only the essential amino acids, even if present in amounts well above the requirements, do not support normal growth (Stucki and Harper, 1962). If this is true for infants it is not known. Nitrogen for adult man can be supplied in large part by gly- cine and diammonium citrate (Terroine et_al,, 1930; Rose and Wixom, 1955). However, nitrogen balance in adult man is adversely affected if only one or two sources of nonspecific nitrogen make up a large 'part of the nitrogen in an amino acid diet. Possibly because the rate of synthesis of the nonessential amino acids is not adequate (Swendseid et 11., 1960: Anderson et_§l,, 1969). There are indications that intact proteins are superior over protein hydrolysates as a source of nitrogen for the growing rat. Evidence indicates that if the young rat is fed a diet containing amino acids instead of proteins from about 18 days of age, it requires another factor, not yet identified, that is associated with proteins, but is not a component of them (Schwartz, 1970). Because protein is the source of nitrogen in the human diet, it is convenient to speak of the protein requirement of man, but the true requirement is not for protein as such, but rather for specific amounts and proportions of the essential amino acids and of nonessen- tial amino acid nitrogen. \- Protein requirements for humans has been a controversial sub- ject for many years. Carl Voit (1876) concluded that a man weighing 70 kg required 118 g protein per day. Rubner (1902) suggested a value of 127 g of protein per day and Atwater (1903) advised 125 g per day for a sedentary man and 150 g per day for a working man. Siven (1900) and Landergren (1903) demonstrated that nitrogen equil- ibrium could be maintained on much lower intakes of protein than those previously suggested in the literature. Chittenden (1904) using himself as a subject showed that it was possible to maintain nitrogen equilibrium at an intake of 36-40 g protein per day. Sherman et_al, (1920), reviewing the literature on the protein requirements to maintain nitrogen balance, found it to vary from 21 to 65 g per day with an average of 44 g per day for a 70 kg man (0.63 g protein/kg body weight). According to his own studies, largely on cereal grains, he advised a mean value of 0.5 g of protein/kg body weight/day as the requirement for balance. This value, with an allowance for safety, was adopted by the Food and Nutrition Board (1945) in its Recommended Daily Allowance. It was recommended an intake of 1.0 g protein/kg body weight/day. Since the Recommended Allowance assumed that some of the proteins in the diet come from animal sources, the recommended intake seemed to be higher than the minimal need. Hegsted et_al, (1946) derived a value of 32.4 g protein/70 kg man/day on a diet of mixed vegetables and 27 9 when the diet supplied one-third of the protein as meat. These protein intakes would provide 0.46 g and 0.38 g protein/kg body weight/day, respectively. In 1957, an FAO Committee on Protein Requirements took account of N balance studies in man to arrive at an average minimum requirement for adults of 0.35 g of protein per kg of body weight, when the protein consisted of a reference protein of high nutritive value, such as whole egg protein. For optimal growth of infants, an intake of 2 g of reference protein per kg of body weight was recom- mended (FAO, 1958). The needs of other groups (e.g., adolescents, lactating women) were also estimated. Since the nutritive value of the mixed proteins of the diet is less than that of the reference protein, a further correction based on the chemical scores of the dietary protein was recommended. In a country with a high standard of living the allowance is 0.66 g f of dietary protein per kg of body weight, whereas in a country where proteins consumed have a lower chemical score, the allowance should be as high as 0.84 g per kg of body weight for adults. Further cor- rections were recommended when the utilization of the protein in the diet is determined by NPU. The Committee on Amino Acids of the Food and Nutrition Board (1959) estimated that an intake of 0.31 to 0.34 g/kg/day would meet the minimal adult need. V These values were calculated in terms of a protein of high nutri- tive value, such as those contained in milk, eggs and meat (FAO, 1957). The minimum nitrogen (N) requirement for an ideal dietary pro- tein in healthy adults has been assumed to be the sum of their urinary and fecal N losses, estimated after adaptation to an essentially N- free diet, plus integumental minimal sweat losses (FAO/WHO, 1965). In the 1968 edition of the Recommended Deitary Allowances (N.A.S.-N.R.C., 1968), the recommended intake is 0.9 g of protein per kg of body weight per day for the 70-kg reference man. The Joint FAO/WHO Expert Group (FAD/WHO, 1965) established the requirement of 0.59 g of protein per kg body weight per day for an adult man. According to the latest edition of the Recommended Dietary Allowances (N.A.S.-N.R.C., 1974), the allowance for the mixed proteins of the United States diet is 0.8 g/kg of body weight per day. The allowance for a 70-kg man is 56 g of protein per day. It was estimated by the Joint FAO/WHO Ag_Hgg_Expert Committee (WHO, 1973) that an adult man (65 kg body weight) would need 0.57 g of protein per kg of body weight per day to meet nitrogen require- 'ments. Two approaches have been proposed to estimate the nitrogen requirements of man. The factorial method (FAO, 1957) is based on estimates of the obligatory N losses (the amount of N found in urine, feces, sweat, etc., when the diet contains no protein) and of the amounts of N needed for the formation of new tissue. Table 2 shows the nitrogen losses in adult men on a protein- free diet (FAO/WHO, 1973). Balance studies provide direct evidence on N requirements from measurements of the lowest protein intake at which N equilibrium can be achieved in adults or satisfactory growth and N retention in children. FAO/WHO Ag_Hgg_Expert Committee (1973) estimated that the average N intake to maintain balance in adults is 77 mg of N per kg per day when the N is derived from milk, egg, casein or mixed diets containing animal protein. The average for subjects consuming cereals or vegetable diets is 93 mg of N per kg. TABLE 2.--0b1igatory N losses in adult men on a protein-free diet. mg N per unit of Route mg N per kg of basal energy body weight kcal Urine 37 1.4 Feces 12 0.4 Skin 3 0.13 Miscellaneous 2 0.08 Total 54 2.0 Table 3 summarizes some of the information on the amino acids required by adult human beings and growing rats. In general, the criterion of adequacy was the attainment of a positive nitrogen bal- ance. Evaluation of Protein Quality The term protein quality means the usefulness for particular purpose of the protein in question. This might be in connection with human nutrition, growth of an infant, growth of maternal and fetal tissues, lactation, recovery from convalescence and maintenance of the body tissues. In animal nutrition, the purpose is a higher biological efficiency in terms of production of meat, milk, eggs, wool, etc., together with maintenance of the animal. When one wishes to know the usefulness of a protein food in fulfilling one of these purposes, the protein is fed to the animal or men and the function of interest is measured. Since most of the time such direct measures are lengthy and expensive, shorter biological and chemical methods have been devised. 10 TABLE 3.--Estimated amino acids requirements of adults and growing rats. Amino Acid Requirements Amino Acid Women(a) Men(b) Rat(c) glday g/day g/day Isoleucine 0.45 0.70 0.090 Leucine 0.62 1.10 0.138 Lysine 0.50 0.80 0.138 Methionine + Cystine 0.55 1.10 0.090 Phenylalanine + Tyrosine 1.12 1.10 0.138 Threonine 0.31 0.50 0.080 ’ TryptOphan 0.16 0.25 0.045 Valine 0.65 0.80 0.115 Arginine -- -- 0.045 Histidine -- -— 0.080 gLeverton, R.M. (1959). cAlbritton, E.C. (1954). Rose, W.C. and R.L. Wixon (1955). For practical purposes, the methods usually employed for the evaluation of protein quality can be divided into the following groups: 1. Methods which are based on chemical analysis of amino acids; chemical scores of Mitchell and Block (1946), pro- tein scores by the FAO/WHO procedure (FAO, 1957), Oser's essential amino acid index (Oser, 1951). 2. Methods based on enzymatic and microbiological procedures. 3. Animal bioassays. 11 Chemical Scores In 1946, after analysis figures about amino acid composition of proteins had become known, Mitchell and Block (1946) published a method for assessing the biological value of proteins, a method based on the law of minimum, a concept developed by Liebig (1840) for fertilizers. If this concept is applied to protein synthesis, it states: protein synthesis is limited by those essential amino acids which are in the smallest amount, expressed as a percentage, in re- lation to their requirement. Mitchell and Block (1946) used whole egg as their reference protein and the percentage of each individual ‘ amino acid content in the whole egg is put at 100; the essential amino acid contents of another protein are given as a percentage of that. The smallest percentage amount of the essential amino acid being dealt with determines the biological value of the protein in question. Mitchell and Block (1946) have called this percentage figure of the limiting amino "the chemical score" and look upon this figure as the biological value. The lower the lowest egg ratio the poorer the pro- tein as a source of amino acid, the larger the chemical score the better is the protein. Whole egg protein was chosen as standard be- cause it is almost perfectly utilized in digestion and in the metab- olism of the growing rat (Mitchell and Carman, 1926), the mature rat (Briker and Mitchell, 1947), the dog (Allison et al,, 1949) and the adult man (Hawley et_al,, 1948). The excellent protein quality of the whole egg was demonstrated by Mitchell (1950) when he showed that the growth-promoting value of this protein was not improved for the growing rat by supplementation with any of the essential amino acids 12 except lysine, which induced a three percent increase in body weight in a 28-day feeding period. Protein Scores bygthe FAD/WHO ProcedUre In 1963, the FAO/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 up the amounts of all the essential amino acids to- gether 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 concentration of essential amino acids in whole egg pro- tein, which is used as the standard, is given in Table 4. Oser's Essential Amino Acid Index Oser's (1951) index introduced the use of the geometric mean of the egg ratios to estimate the nutritive value of proteins. He incorporated the "egg ratio" concept of Mitchell and Block, but in- cluded all the essential amino acids, plus cystine, tyrosine, histi- dine and arginine. The essential amino acid index (EAA index) was de- fined as the geometric mean of the "egg ratios" (the ratio of the essential amino acids in a protein relative to their respective amounts in whole egg protein). 13 TABLE 4.--Essential amino acid composition in the whole egg protein. Amino Acids 33$g99593 Lysine 7.8 Methionine and Cystine 5.3 Phenylalanine and Tyrosine 9.3 Leucine 8.8 Isoleucine 5.9 Valine 7.1 Threonine 4.9 ’Tryptophan 1.4 Histidine 2.6 Total 53.1 This procedure was modified by Mitchell (1954) and is known as "Modified Essential Amino Acid'Index" (MEAA). The procedure is illustrated in Table 5 taking casein as an example. The geometric mean of the corrected egg ratios (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 average value. In the example, the average logarithm was 1.9603; and its antilogarithm, 91, is the modified EAA index for casein. Biological Value (BV)_ Biological methods with rats are usually subdivided into those based on weight gain and those based on nitrogen retention 14 pm u << oooo.m oo_ mNF N.m m.~ mcwuwumm: memm.p mm mm ~.P ¢.F canaopaxch m¢~m.~ cm em _.¢ m.¢ mcwcomcsh oooo.~ ooF _op N.~ F.“ mcwpm> Npmm.F mm mm m.m m.m . mcwoampomH oooo.~ cop PFP w.m m.m mcwuzmo oooo.~ cop mFF N.o_ m.m mammoczp can mcwcmmeA=mga ammu.~ No we m.m m.m mcwpmxu new mcwcowzumz oooo.~ 00— eoF _.w m.u mammao mowpma owpam owpam Az am_\mv A2 a m_\mv aumccou umuumccou mum cwmmmu :mmuoca muwu< ocws< mo moo; mom open: .xmu:~ apu< o=_s< Pawpcmmmm um_cpuoz 5;“ co cocpapaaeou--.m mam . cee o;d who so: ass zoo o_H oop com com ooc 4:44.. oom .. c c o .c. con". :mmm ...:.. 3m. 22.2 V\\\K as: _wuacm muuuuu coo .mmm open: can soon x>mz .uac prmcm mo 52 _muop m\mev mvwom ocwEm Fowpcmmmm mo :cmuumdii.~ mczmwu UBBOJllN 19101 6 / 'V'V 5H 60 .mm Egg N.mo umcwmpcoo umwu on» mo cowpmcmamca on“ cm now: LaoFm as: FPchm .Ezwcmpom mo sag m.- vmcwmpcoo porn psz Fwchm .<. Emmy op\o m.mmw o.om mn.o A m.mi ¢N.N H m.mm o¢.N H m.oo mm.m A o.¢n mN.o H m.m «~22 _euccm oF\oF 11 ii «v.6.w o.om+ P—.m H N.mo~ om.~ H N.mm om.“ H m.mo— em.o H m.F_ cwmmmo mco>w>cam Amav mamv Amv co _mooe Axon\mav pesto: seem a mswhmvccp cpgmwoz Amv FwoMW= dosso\s coco Longsz oxmucH ancmpmm cw mmcmzu : . 3 — .u meuwcH x “H c m .mumc mcwzocm co cwmuoca .z.m ucoocma op mcwcwmpcou umwu cceucmpm m can :wmpoca :wmmmo pcmucma oF mcwcwmucoo umwu ccmvcmum a do pomccmir.¢_ mom<~ 61 .cso—m as: —w~mcm vcm :wmmmo mcwcwmpcoo mpmwv ucmncmum co owe mum; co wm>c=o cpzocwii.m mesmwu mxmv .mewh m— op m . cm 33: p.595 0 or I . v A . on 8 O o p .A M .1 . .b . om :wmmmo . io__ 62 of 60 pg, which represented an ingestion of 839.5 Hg for the whole experimental period. One of the first consequences of selenium toxicity is a refusal of food which was reflected by a low food in- take. Effect of Protein Intake on Selenium Toxicity The aim of the next experiment was to study the possible effect of protein quantity and quality on the toxicity of selenium. A 20 percent protein diet was prepared, being 10 percent from Brazil nut flour and 10 percent from casein. Casein was included in the 'diet in replacement of starch in order to keep the selenium content in the diet exactly the same as in the previous experiment in which 11.3 ppm of Se was present. Results of this experiment are shown in Table 15 and Figure 3. Values reported are the average of replicates for each treatment. The two groups of animals were fed the casein and the experimental diet for a period of 23 days. Since the objective was not to compare growth efficiency but rather the effect of a higher percent of protein in the diet on the selenium toxicity, the casein control diet had the protein level maintained at a 10 percent level. Animals fed the mixture Brazil nut:casein grew at a normal pattern. The feed intake was similar to the casein fed group, however protein intake was doubled. The experimental group gained an average of 102.4 9 during the 23 days experimental period, which represents a daily increase of 4.5 gm. The animals were 45 days old when the exper- iment started, which explains a smaller gain in body weight than it is (53 m,_4 mo.mAF A.w A «.moA ~.AF A o.mc~ m.mP A o.mop o.p A c.NA P.m A ~.mom Am.o A m.mp Acwvocd Romy As: FAAAcm + :womuo -- -- A.A A ¢.ow A.AF A c.0mm A.Ap A N.oA~ mm.o A A.AM m.m A o.Pmm mm.o A “.mp Accvoca ao_v crmmou Amav AA a: m a Beta: 2 Amara“ 29%? AME: A“? as: so; as . . . . oxAA=~ good Esscvom Fawovcm cAquca .mcauxme an: FAchmucwmmmu new crammo mcwcwmucoo muowu co to» man; do zuzocuii.m~ m4mczo cwzocwii.m mcamwm op oo_ com 1 com 6 "1n Kpoa 65 normally expected when 21 days old rats are used. During the 23 days experimental period no indication of selenium toxicity was observed. It is interesting to note that, in spite of the fact that the animals fed the mixture (Brazil nut:casein) had ingested almost three times more selenium than the animals in the previous experiment, no harmful effect was observed. In the previous experiment, a daily intake of 60 ug Se/day was highly toxic to rats when the diet contained 10 per- cent protein from Brazil nut flour. In the second experiment, although the daily Se intake was 180 09, no toxicity was observed. Similar re- sults were obtained by Smith (1939) when he postulated that the toxicity of naturally occurring food selenium is largely determined by dietary factors. Supplementation of Nagy Bean Protein with Brazil Nut The Brazil nut flour used for the supplementation studies was the one which contained 54 percent protein (N x 6.25) and 17.3 ppm selenium. Feeding experiments were again carried out with the aim of determining the effectiveness of Brazil nut proteins in supplementing those of the Navy bean. The next experiment had two objectives. First, we were in- terested to see if germination of Navy beans, prior to autoclaving, would have any beneficial effect on the growth of weanling rats, as compared to beans that had been just autoclaved. The second objective was to test a mixture of autoclaved Navy bean and Brazil nut flours as source of protein in the diet. A 10 percent protein diet was 66 prepared, in which 80 percent of the protein came from the Navy bean flour and 20 percent from the Brazil nut flour. The results from this experiment are given in Table 16, and the curves for gain in body weight and for food intake, during the 28- day experimental period, are shown in Figures 4 and 5 respectively. Group A was fed a 10 percent casein protein diet and it was used as control to calculate the protein efficiency ratios. The approximate weight gain was 3.76 i 0.22 g per rat per day, and the feed intake was 12.7 i 0.56 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 through- out this work were corrected for casein = 2.50. Group B was fed a diet containing autoclaved beans. The rate of growth was not as fast as with casein. The gain in body weight was 2.30 i 0.10 g per rat per day. The total body weight gain was 61 percent of that of the casein control group. The low efficiency of the bean protein diet in promoting growth is attributed primarily to their low methionine content (Russell et_gl,, 1946; Kakade and Evans, 1965). The protein efficiency ratio (PER) was 1.57 i 0.03 and it is similar to the value obtained by Kakade and Evans (1965). Group C was fed a diet in which the protein source was beans that had been germinated prior to autoclaving. The average weight gain for this group was 0.97 i 0.11 g per rat per day and the protein efficiency ratio was 0.89 t 0.07. The total body weight gain repre- sents only 26 percent of the control group. The total feed intake during the 28 experimental period was low as compared to the casein 67 om.m u cwmmmu com umpomccoun 2mm A.Mm No.0 A ~¢.N o.w A m.pm¢ N.m A m.mm— o.o_ Aomuomv Azz _AAacm + AcAom o mo.o H mm.c n.m~ A n.w¢N ~.m A F.nm 0.0? mcwmm vo>mpuop=< Ucm UQHMCwELOO U mo.o A Am._ A.o_ A P.0Am m.~ A A.Ac o.o_ Acaom oo>A_ooA=< m mo.o A om.~ n.mp H m.mmm N.@ H m.mo~ o.op :mmmmo < nmfimmav mAmv mamv Hfimwm3 Afiv Hmwo QEOLG ompmx xocowowmmm :Awuoca wxmch comm xuom cw :wmw cwmuocm so Fm>m4 .Aomuomv pa: prmcmnmcmmn do mcspxwe a new mammn vm>mpoou=m use umuchEme .mcmmn um>mpoopam .cmmmmu mcwcwmpcoo mumwu ucmucmpm no em» mum; co zpzocoii.mp mom<~ 68 200 I Beans + . B.N.,80:20 Casein 150 1.. m C : ¥ 5; . "/,/” é? 4”’”"T’T” Cooked Beans 8 ’,/”’ o/ ‘ 100 - .////// /° ‘ / ./ Germ. 81 Cooked Beans //. 50 F 10 20 30 Time, days Figure 4.--Weight gain of weanling rats fed standard diets containing casein, autoclaved beans, germinated and autoclaved beans, and a mixture of autoclaved beans + Brazil nut with the protein ratio 80:20. Food Intake, 9 69 450 I ' Beans + B.N. . 80:20 3501. ' Casein * Cooked Beans 250 ’ Germ. & Cooked Beans 150” ’ 50 10 20 30 Time, days Figure 5.--Food intake of weanling rats fed standard diets containing casein, autoclaved beans, germinated and autoclaved beans and a mixture of autoclaved beans + Brazil nut with the protein ratio 80:20. 70 and autoclaved bean group. Everson et_gl, (1943) reported on the im- provement of the nutritive value of soybean, as determined by PER, when soybeans were germinated prior to cooking. Desikachar and De (1947), Chattopadhay and Banerjee (1953) have demonstrated that ger- mination improved the nutritive value of certain beans. However, Kakade and Evans (1966), working with Navy beans, did not find any beneficial effect of soaking and germination on the nutritive value of these beans. The low growth rate observed in our work is under- standable in view of the low food intake by the animals. In Group D the rats were fed the Navy beanszBrazil nut mixture 'containing the following protein ratio: 80 bean protein to 20 Brazil nut protein. The highest gain in body weight was achieved within this group of animals. This diet contained 0.18 percent of methionine. The diet fed to Group B and C (autoclaved; germinated and autoclaved beans, respectively) contained only 0.07 percent methionine. According to Albritton (1955) the growing rat requires 0.090 g of methionine per day to achieve maximum growth. The group fed the Navy bean:Brazi1 nut mixture ingested 16.13 i 0.31 g of diet per day, providing the animals with 0.030 g of methionine per day. This would repre- sent one-third of the rat requirement for maximum growth. Rats under the autoclaved bean diet had an intake of 12.4 i 0.37 g of diet per day. This provides the rats with 0.009 g of methionine per day, and represents a value 10 times lower than the requirement for maximum growth. Since the feed intake for Group C was even lower, the amount of methionine ingested by the rats was very low indeed. 71 The protein efficiency ratio (PER) for the group fed the Navy beanzBrazil nut mixture (80:20) was 2.42 i 0.02, a value that is not statistically significant from the one obtained for casein. In the next experiment the objective was to determine how low the amount of Brazil nut flour in the mixture with Navy beans could be in order to observe an improvement on the nutritive value of the beans. Results of these tests are presented in Table 17 and Figures 6 and 7. The mixtures of Navy bean:Brazi1 nut containing the protein ratios of 90:10 and 95:5 were also able to promote the growth of rats ’over that of beans alone. The average weight gain for Group 8 (Navy beanzBrazil nut, 95:5) was 2.83 i 0.12 g per rat per day. The PER was 1.92 i 0.03. The rate of growth was lower than for casein and the feed intake was higher. Group C (Navy beanzBrazil nut, 90:10) showed a better re- sponse than Group B. Rats gained, in average, 3.43 to i 0.19 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 2.16 i 0.03. In the last experiment in this series, it was studied the effect of fortification of Navy beans with methionine. This amino acid was added to the bean diets in order to make them with the same methionine content as present in the mixture Navy bean:Brazil nut (80:20). The total methionine content of the Navy bean diet was 0.18 percent methionine. Selenium was also added in the form of Narselenite. 72 om.~ u cwmmmo com cmpumccou a 2mm A.Mm mo.o A c_.N N.AF A m._mm m.m A o.mm o.op Aopuomv mus: FAchm + mcmmm u mo.o A Nm._ A.AA A m.ocm m.m A _.me o.o_ Am mmv was: PAchm + mcmom m mo.o A om.~ o.o~ A m.mom m.m A o.~m o.o_ =Aomau < Amy Amy AAV gamma oxamcm ooou :m ”A ewes cwvocd co Po>oA Aowa daocu .opnom can mnmm mowumc cwmuocq osp sum: as: PPchm + mcmon um>mpoop=m mo mmcapst can .cwmmwo m:_cwma:oo mumwu ucmccmom co now mums co cuzocori.n_ mom<~ 170 150 130 Body Wt., g 90 7O 73 T Beans-+B.N.,90:10 Beans + B.N., 95:5 l 10 20 30 Time, days Figure 6.--Weight gain of rats fed on standard diets containing casein, and mixtures of autoclaved beans + Brazil nut with the following protein ratios, 95:5 and 90:10. 74 400t. Beans +B.N., 90:10 i Beans + B.N. , 95:5 300 - U) :5 Casein f6 4.) .5 hr '0 O O u. 200 - 100 ’ l I L 10 20 30 Time, days Figure 7.--Food intake of rats fed on standard diets containing casein, and mixtures of autoclaved beans + Brazil nut with the following protein ratios: 95:5 and 90:10. 75 The objective was to verify if the amount of-selenium present in the Navy bean:Brazil nut mixture (80:20) would have any beneficial or harmful effect on the PER value. The results are shown in Table 18 and Figure 8. Group A, casein fed animals, gained 3.56 i 0.35 g per rat per day and the feed intake on a daily basis was 10.31 i 0.78 g per animal per day. The bean fed animals, Group B, as happened before gained 2.27 t 0.17 g per rat per day, with a feed intake of 10.29 i 0.51 g per animal per day and the PER was 1.60 i 0.05. The group fed with Navy beans fortified with D,L-methionine, Group C, in order to meet the same methionine content as in the Navy bean:Brazi1 nut mixture (80:20) had an average gain in body weight of 5.62 i 0.21 g per rat per day and the average daily food intake per rat was 15.94 i 0.47 g. The PER was 2.57 i 0.04. Group D had methionine added as for Group C and the diet was made to contain 0.64 ppm of selenium added as sodium selenite as in the Navy bean:Brazil nut mixture (80:20). The average body weight gain per rat was 5.45 i 0.25 g and the feed intake also on a daily basis was 15.61 i 0.48 g per rat. The PER was 2.54 i 0.04. Rats in Group E, were fed the Navy bean:Brazi1 nut mixture (80:20). Average daily gain in body weight per animal was 4.93 t 0.18 g, the average daily feed intake was 14.84 i 0.59 g per rat and the PER was 2.42 i 0.03. The results of this experiment indicated that the Navy bean diet supplemented with methionine in such a way that the total om.~ u cwommo to» umuuoccou 76 a sum A.Mm mo.o A ~e.~ A.A. A A.AAA o.m A o.mm_ OP A.Aoz Aw_.ov m Aomuomv zm + Acaom Ao.o A Am.~ A.mp A A.Ama m.o A c.~m_ o_ om Eda Ao.o + .Aoz o Amp.o + Acaom Ao.o A Am.N ~.m_ A «.0AA m.m A A.Am_ o_ ocpeongoz u Amp.o + Acaom mo.o A om._ A.AA A A.AAN A.A A o.mm o_ Acaom m mo.o A om.N m._~ A A.AAN w.m A o.mm OF cAoAAU < mfimv mfimv Axv ‘ gamma AAAAcH coca cweo Aemwoz cAvoca co Ao>o4 Avo asocw .omuom ovum; cwmpoca on» gum: p3: PPchm + mcmmn um>m_oou=m co weapxms a wee :mmmmo ca umcmasoo mm memos um>m~oop=m cw>wm mum; mo nuzocm on» :o Ezwcmpmm use mcwcowcpoe acmucmsmpaaam co uomwcm11.m— mom<~ 200» 150~ a +3 3 )3 'U :8 IOOF // 50’ l 77 /; / / Beans + Me Beans + Met + Se Beans + B.N., 80:20 Casein i z”////66;ked * Beans ¢””””’T’ Time, days Figure 8.--Weight gain of rats fed on casein, autoclaved bean diets supplemented with methionine and selenium and a mixture of autoclaved beans + Brazil nut with a protein ratio 80:20. 78 methionine content equalized the one in the mixture 80:20 (Navy bean: Brazil nut) promoted a better growth of the rats and consequently a higher PER. This suggests that probably not all the methionine present in the Brazil nut protein is available to the rat. The difference in PER values between Groups C and 0 (Navy bean + methionine and Navy beans + methionine + selenium, respectively) are not significant. This indicates that, at least under the experimental conditions described, selenium at this amount (0.64 ppm Se in diet) was neither beneficial nor toxic in terms of rat growth. Figure 8 illustrates the growth pattern of the rats in Groups IA, B) C, D, and E. Protein Scores The essential amino acids of Navy bean flour, Brazil nut flour, the three mixtures of Navy beanzBrazil nut (95:5, 90:10, 80:20, re- spectively) and whole egg were compiled and expressed as milligrams of essential amino acid per gram of total nitrogen (Table 19). Protein scores were then calculated (Table 20) as described by the Joint FAO/ WHO Expert Group (1965). The amino acids cysteine and tyrosine were included in the calculations since they have sparing actions on methionine and phenylalanine, respectively. As far as the protein scores are concerned, the Navy bean flour has methionine as the first limiting amino acid, followed by isoleucine. Brazil nut flour has lysine as its first limiting amino acid, followed by isoleucine and threonine. Table 20 shows that the protein score 79 .Aomnowv as: Fwchmucmmn x>mz m .Aopnomv as: FwNmLm ”coma x>mz¢ .Amummv on: FAchmucmmn a>oz .zuaum mwzu Soc; mppammm .Ammmpv o:3\o mm am am oo_ em cop cmgaouazc» com mpm omm mup me Rpm mcwcomcgh mop mmp ooP mFm _w mom mcwmpmxu + mcwcowipwz mcwcwmpcoorcampsm wmwoh onm mmm mmm oma ooo mmm mcwmocxw + mcwcmpmpxcmcm .<.< owmeocm Fmpoh Foe owe mmc mom omo moo mcwmzb mum «mm mmm oma «cm mmm mcwoamo me mom mom Pmm mom mmq mcwoampomH mu meszwz em mczuxwz m< mcszwz mus: FAchm magma »>mz mem m_053 A.<.mz co mmcsuxwe use czopm as: Pwnmcm Aesopw camp >>mz .mmm m_ocz do cowpwmoaeoo ovum ocwsm pmwucmmmmrr.mp m¢m

oo—A oo~A oopx oopx ooFA cegaopaagh oopx ooflx oopx Nu oopx mcwcomcnh AA AA mm oo_x AN .<.< mcmcwaocoo-c=cpam oopx ooFA oopx mm copA .<.< owumeocm Page? ooFA ooFA oopx on oopx mcwmz4 oopx oOFA OOFA oopx oopx ocwoamo mm cw em Nu mw mcwoszomH ownom ofiuom mumm Azmv zmucmmm zmncemm zmncmmm mcmmm A.<.mz we mmcapst vcm Aesopm on: _wchm .caopm cmmn >>mz co mocoom cwmpocaii.o~ m4m umpmponocpxm k. om.om mp.¢~ um.¢m mw.~— mmapm> :mmz m.om _o.mm m.om o.—~ mmioowm m.mm ¢_.m_ m.mm m.FF accwmom m.mo mm.mF m.- P.Np camera m.m¢ mm.p_ N.~N m.F_ ouoomcm ocmwow m.wm Np.mp e.o~ N.NP moomcmo o.em mp.m o.¢N m.—— ompwcmm A.Aoca me\H=ev AAAoecho PE\H=ev Amm.e x zv AAV ecoeeou xuw>wuo< couwnwccH :AmQALH cwmpoca page» wczpmwoz Pepe» “coucou comm .xpw>wpom copmnwccw :AmQXgpri.mm m4mmp 62A 6A eoAAAFoc ee eoere AAoe coeeeeeee eAAaAcA-.m oczaea AFEV uoacuxw mo oszpo> A_sv uumcuxo mo oe:~o> o.~ m; 3: N; o; m. c. e. N. o 5 A: e; A; A; o; m. e. A. N. o A nu aw - o i o . m .. m Lri11d1111M11|iiillulliiiilit¢ - o— L op ---- .. 2 mm.: AX 2 ---- . mm _F + x no or u > L e_ mp + x mm _- u > ¢P.mp ATiLu-ni 258 3 «1:52 . 2 FE\H=P _E\H=H AFEV uuocuxo we waspo> a—EV uumcuxm mo osaFo> o.~ m.~ o.P ¢._ ~.~ o.p m. m. e. N. o o.~ m.P m. ¢.p ~.F o._ m. m. e. N. o A A" . op . w r .N_ . op x 1 ep III! 1 NF . . - -----. . .- - 2.2 A-\- 32:37» B.2»\.2 22:23-» 3 87 p o.p ¢.p ~.p o.— w. o. d d mp.m + x mm.o: u > z> m. c. c. N. o o.m w._ e.p ¢.p N.~ o." —o.mm + x No.91 1 IdI Iqu d L . NF . ep . mp . mp 77. . ON I I -- AN SAN All-y _E\H:» 88 It is also evident that the total protein content in these six beans varieties do not correlate with the magnitude of the trypsin inhibitory activity. Zinc Supplementation of Navy Beans The availability of zinc in the diet is influenced by many factors. The presence in the diet of chelates influences the avail- ability. The phytate present in animal diets markedly interferes with the availability of zinc for absorption (Oberleas et_gl,, 1962). Zinc phytate complexes can be formed in the gastrointestinal tract making .zinc unavailable. The aim of this experiment was to study the effect of zinc supplementation of Navy beans on the growth of weanling rats. The animals were fed the following diets, all at a 10 percent protein level. A-Casein B-Autoclaved beans C-Autoclaved beans + 55 ppm zinc D-Autoclaved beans + 0.1% D,L-methionine E-Autoclaved beans + 0.1% D,L-methionine + 55 ppm zinc F-Autoclaved beans + 0.2% D,L-methionine G-Autoclaved beans + 0.2% D,L-methionine + 55 ppm zinc The analysis of Navy bean flour revealed a phytic acid content of 0.86% and a zinc content of 29 ppm. All diets in which autoclaved bean flour was the source of protein contained 0.36 percent of naturally occurring phytic acid. Analysis of the different diets for zinc gave the following results: 89 Qj§S§_ mg zinc/100 g diet A* f 0.80 B 1.22 C 6.65 D 1.19 E 6.59 F 1.20 G 6.60 *The zinc present in the casein diet was supplied by the regular min- eral salt mixture used as ingredient. The results of a four-week gg_libitum feeding are shown in Table 24 and Figure 10. The highest average gain in body weight was achieved by Group A, and the lowest was for Groups 8 and C. As expected, the supplemen- tation of the autoclaved Navy bean diet with 0.1% and 0.2% D,L- methionine (Groups D,E,F,G) improved the growth of the animals. However, no indication of improvement, as determined by gain in body weight and protein efficiency ratios, was observed when the diets were supplemented with 55 ppm of zinc. The only improvement noticed was due to the addition of D,L-methionine to the diets. It is known that the requirement of the rat for zinc is 12 mg/ kg of diet for maximum weight gain (Forbes and Yohe, 1960), when the animals are maintained in a zinc-free environment and fed a diet based on casein or egg white. According to the same authors, if the rats are housed in galvanized cages, no more than 2-4 mg/kg of diet are required. 90 N =~_.AomeN AA eoeee oeAA and mm o _ om.N u :Aommo coe umpooccoun 2mm A.mA mo.o A mm._ A.m_ A A.APM m.m A A.AA o_ _eN + AAN.ov .Aoz + Aeemm u eo.o A e_.~ N.c A _.AmN No.N A A.AA o_ AA~.ov .Aoz + Aeeom a Ao.o A _m.F m.a A A.AAN mm.m A m.oe o_ _eN + AA_.ov .Aoz + Aeeom m eo.o A AA.. mm.m A A.FAN AN.N A Ae.om o_ AA_.ov.Aoz + Aeeom a mo.o A mo., mm.m A e.m_N m~.N.A m.om o_ _eN + Aeeom u so.o A em.o em.~ A A.AON we.o A Am.em o_ Aeeom m N_.o A om.~ c.m_ A A.AAN mo.m A AP.~A o, geomeo < ee oxeAeH some ewes Aemwoz erAoce Amen agate mo Pm>m4 .mumc mcwzocm co to» mpmwu coon mo coepmucmEm—qaam o:AN-i.em m4mc=o guzocu-.o_ acumen when .osmh cc om om op och + comm - zoom. ,3 .iiiiiiiii. - cup Ad: up.o + eeom iiiiiiiiii 85+A£Aco+§£. w 6 ‘iufiian 4906 i ocp comm w um: am.o + N.o.+:mom ocpN + um: u . cop :mmmmu 92 The effect of supplementation of zinc to soya assay protein diets at several levels of dietary protein on growth of the rat was studied by Oberleas and Prasad (1969) over a 10 week feeding experi- ment. All diets had the phytic acid content equalized to one percent by addition of chemically pure phytic acid. Their results have shown that the diets containing added zinc promoted a more efficient growth than the ones without zinc. An 8 percent soya assay protein diet was as good in promoting growth of the animals as a 12 percent soya assay protein diet without zinc. Unfortunately these authors did not report the zinc content of the soya assay protein, but as a protein isolate 'it is likely to have none or an insignificant amount of zinc. Also, 1 percent phytic acid level in the diet seems to be a little unrealis- tic, and besides that this acid was added in a free form, thus in a form entirely favorable to form complexes with zinc and making it un- available. Phytic acid can be present in a food product in a variety of different forms, yet not known. Probably only a small fraction of the naturally present phytic acid in cereals and legumes is available to complex with dietary zinc. Fifty varieties and lines of mature dry beans were analyzed for phytic acid (Lolas and Markakis, 1975) and have shown a variation from 0.54 to 1.58 percent on a dry weight basis. The aim of this experiment was to investigate the effect of zinc supplementation to diets in which the phytic acid present was the one naturally occurring in the Navy bean flour. It seems that the zinc naturally present in the beans meets the requirements of the rats, at least during a 28 day feeding period. 93 During the last five days of the feeding experiment (28th to 33rd day) a zinc balance study was carried out. Feed intake was measured and feces were collected, dried and analyzed for zinc by atomic absorption spectrophotometry. The percent of zinc absorption was determined and the results are given in Table 25. TABLE 25.--Zinc balance study (five days study). Groups Zinc($3§ake Zinc excrfigion (feces) % Absorption A 0.5700 0.1919 66.3 8 0.4710 0.3435 27.1 C 2.7261 1.967 27.8 0 0.6503 0.4753 26.9 E 3.7280 2.8073 24.7 F 0.8434 0.6171 26.8 0 4.1690 3.1441 24.6 These results indicate that the amount of zinc absorbed by the rats was essentially the same for the Navy bean diets with and without zinc, except for the casein fed groups. It seems that the zinc con- tent of the beans was sufficient to meet the requirements of the animals and the phytic acid naturally present in the beans did not increase the requirements for this element. The absorption of zinc is affected by the level of intake of the element, by the amount and proportion of several other elements and dietary components, and by the chemical form in which is ingested 94 (Underwood, 1971). Studies conducted with man on the Zinc 65 uptake has shown that one month after the intravenous administration, about 80 percent of the dose or more was still retained in the body (Spen- cer §£;_l:: 1966). When Zinc 65 was adninistered orally great varia- bility among individuals was observed. The absorption varied from 20 to 80 percent, the average absorption being 51 percent. The reasons for such variability are still not known. In 1957 it was reported that the zinc in soybean protein was less available to chickens than that in casein (O'Dell and Savage, 1957). The same phenomenon was also observed with pigs (Smith et_gl,, 1962) and rats (Forbes and Yohe, 1960). Forbes and Yohe (1960) reported that the absorption of zinc by rats fed casein was 84 percent. Values obtained in this work indi- cate an absorption of 66.3 percent when rats were fed a casein diet containing 8 ppm of zinc. Animal variabilities would explain this difference. For rats fed a soybean diet containing 18 ppm of zinc the absorption was 44 percent. N0 values for absorption of Zn by animals fed bean diets were found in the literature. SUMMARY AND CONCLUSIONS The amino acid composition of Navy bean (Phaseolus vulgaris L.) flour of the Sanilac variety and Brazil nut (Bertholletia excelsa L.) defatted flour was determined by the Beckman Model 120C amino acid analyzer. Two lots of Brazil nut flour were utilized in this study. Brazil nut flour Lot 1 was found to contain 55 percent protein (N x '6.25) and 63.2 ppm of Se, and Lot 2 contained 54 percent protein (N x 6.25) and 17.3 ppm of Se. Brazil nut flour Lot 1 was tested in a rat feeding experiment at a 10 percent protein level in the diet. The diet was found to contain 11.3 ppm of selenium and it was highly toxic to the rats. In a second rat feeding experiment the effect of a higher intake of protein on the selenium toxicity was investigated. It was found that a 20 percent protein diet (10 percent from Brazil nut flour and 10 per- cent from casein) containing the same amount of selenium as in the previous experiment, did not show any harmful effect on the rats, suggesting a protein protective effect against selenium toxicity. The supplementation effect of Navy bean protein with Brazil nut protein was studied in a series of experiments. Mixtures of Navy bean flour with defatted Brazil nut flour (Lot 2--17.3 ppm of selenium, 54 percent protein) were prepared. 95 96 All bean flours used in these experiments were autoclaved for 10 minutes at 121°C. Ten percent protein diets containing dif- ferent proportions of Navy beans and Brazil nut flour were tested against the casein control diet. The diets were prepared containing the following protein ratios: 95 bean protein to 5 Brazil nut pro- tein: 90 bean protein to 10 Brazil nut protein and 80 bean protein to 20 Brazil nut protein. The protein efficiency ratios were re- ferred to casein, which was corrected to 2.5, and compared with the PER's of a bean diet and a diet in which the beans were germinated prior to autoclaving. The PER for the bean diet was 1.57 i 0.03, for 'the germinated bean was 0.89 i 0.07, for the 95:5, 90:10 and 80:20 (protein ratios in the beanzBrazil nut mixtures) were 1.92 i 0.03, 2.16 2 0.03 and 2.42 t 0.02, respectively. In a subsequent experiment a study was made of the effect of supplementing Navy beans with an amount of methionine and selenium (added in the form of Na-selenite) equal to that present in the 80:20 beanzBrazil nut protein mixture. No differences in PER were observed due to the addition of selenium (0.64 ppm), but the addition of 0.18% of D,L-methionine increased the PER (2.57 t 0.04) over that observed for the 80:20 beanzBN mixture (2.42 i 0.03). Selenium, at the 0.64 ppm level in the diet, was neither beneficial nor toxic in terms of rat growth. The FAO/WHO Group (1965) procedure was used to calculate pro- tein scores based on the essential amino acid pattern of whole egg. Protein scores for the Navy bean, the Brazil nut, and the 95:5, 90:10 and 80:20 mixture were 27, 66, 33, 42 and 58, respectively. The 97 protein score of the Navy bean was considerably improved by addition of Brazil nut and the best combination (80:20) represents an increase of more than 100 percent. Protein scores verified that methionine is the limiting amino acid in Navy beans. The modified essential amino acid indices (Mitchell, 1954) for Navy bean, Brazil nut, and the 95:5, 90:10 and 80:20 mixtures were 82, 74, 83, 84 and 86, respectively. The protein quality was also determined by microbiological method using Streptococcus zymogenes as the assay organism. The rela- tive nutritive value (RNV), referred to a value of 100 for casein, were '69 for Navy beans, 75 for the 95:5 mixture, 76 for the 90:10 mixture, 82 for the 80:20 mixture, and 101 for the beans + methionine mixture. The RNV were compared to PER values as determined with rats, and a correlation coefficient of 0.89 was found between these two methods. Total protein (N x 6.25), moisture content and trypsin inhibi- tory activity were determined for six varieties of beans. One was Navy beans, and the others were Carioca, Goiano Precoce, Pirata, Rosinha and Rico-23, grown in Brazil. The highest trypsin inhibitory activity was shown by Rico-23 a variety of Black bean, and the lowest was found for Goiano Precoce. Our results indicated that there is not correlation of the protein content of these beans with the magnitude of the trypsin in- hibitory activity. In a final experiment a study was made of the effect of supple- menting Navy beans with zinc. Zinc at a 55 ppm level was added to diets which had as protein source Navy beans, Navy beans plus 0.1% 98 D,L-methionine and Navy beans plus 0.2% D,L-methionine. These diets were compared to a casein diet in order to determine PER values. The experiment was carried out for 33 days, the data from the first 28 days being used for PER calculations. A zinc balance study was carried out during the last five days of experiment. Results show improvement in growth only as a result of D,L-methionine addition. No beneficial effect was noticed from zinc supplementation. The diets contained 0.36% phytic acid and the beans had 29 ppm of zinc. It appears that the zinc naturally present in the beans was sufficient to satisfy the rat requirements in spite of the high phytic acid content of the diets. The zinc balance study showed a higher % absorption for the casein fed group (66.3%) while no significant differences were ob- served for the other treatments. 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