The USDA trypsin inhibitor study. II. Timed related biochemical changes in the pancreas of rats

A total of 600 rats was distributed equally among 15 diets formulated from mixtures of raw and toasted soy flour and casein so as to provide 5 different levels of trypsin inhibitor (TI) at 3 levels of protein (10%, 20%, and 30%). Animals were sacrificed at the end of 6 months and at 3-month intervals thereafter over a period of 22 months. Increases in the secretory activity of the pancreas, as measured by trypsin and chymotrypsin activities in the pancreas and small intestines, were associated with increasingly higher levels of TI and protein but did not change appreciably with time. Hypertrophy and hyperplasia of the pancreas, as reflected by changes in pancreas weight and RNA and DNA content, developed at an early stage (6 months) and were likewise positively correlated with the levels of TI and protein in the diet. Although the hypertrophic response remained unchanged with time, hyperplasia became more pronounced as the period of exposure to TI was prolonged. The peptide test confirmed the stimulatory effect of TI on the pancreas, but some functional impairment was noted in those animals which had been fed raw soy flour for 18 months of or longer. The appearance of nodules on the pancreas was observed by gross inspection after 15 months, and these became larger and more numerous with time. The incidence of nodules was highly correlated with the level of TI in the diet and was highest when the diet contained 20% protein.     

Effects of raw soy flour feeding in weanling pigs: comparison with rats and monkeys

Feeding of raw and heated soy flour (RSF & HSF) for 4 or 8 weeks to weanling swine was compared with feeding of casein diets. There was essentially no growth in pigs fed RSF, whereas body weight increased 4.5 fold over 8 weeks with either casein or HSF. No change in pancrease weight as percent body weight was found in any group in either experiment. Both total pancreatic RNA and DNA were decreased in RSF groups, although RNA and DNA/mg pancreas were increased by the RSF diets. Total pancreatic protein, as well as protein/mg, were decreased in RSF-fed pigs compared to casein and HSF. Pancreatic trypsin, chymotrypsin, lipase, and amylase, and fecal trypsin were decreased in RSF-fed pigs to varying degrees compared to either HSF or casein. HSF and casein performance was equivalent in all parameters measured. Results of these experiments are compared with results of feeding similar diets to rats and monkeys.     

The USDA trypsin inhibitor study. IV. The chronic effects of soy flour and soy protein isolate on the pancreas in rats after two years

In two year feeding trials, histologic changes in the pancreas of the Wistar rat were evaluated after chronic dietary exposure to raw and heated, dehulled, defatted soy flour and soy protein isolates which provided a range of trypsin inhibitor (TI) concentrations from 93 to 1271 mg/100 g diet. Also investigated was the nutritional interaction of level of dietary protein with the development of pancreatic pathology. Graded levels of TI were achieved from mixtures of raw and heated soy flour or protein isolate. Dietary protein levels were 10%, 20%, and 30%; the two higher levels obtained in some diets through casein supplementation. A total of 26 diets, including casein controls, were fed to groups of 40 male rats. Growth rates with these diets were commensurate with protein quality and level. Mortality rates tended to be slightly greater in the higher protein diets, and rats fed only raw soy as a source of protein survived well.Two lesions, nodular hyperplasia (NH) and acinar adenoma (AA) constituted the major pathological findings in the pancreas, and the incidence rates of both were positively associated with the concentration of dietary TI. The incidences of pancreatic NH and AA compared to those of the corresponding casein control were significantly elevated in rats fed heated soy flour supplying 10% protein and the lowest level of TI investigated (93 mg/100 g diet). At low levels of TI (215 mg/100 g diet), supplementation by casein to raise the concentration of protein from 10% to 20% and 30% reduced the frequency of NH and AA. When the dose-response data for NH and AA was linearized by plotting the probit of percent incidence against log of TI concentration per gram of dietary protein, the response to soy flour and soy protein isolate was not significantly different. Numerous lesions commonly found in the aging rat were diagnosed, and modulation of their rate of occurrence by nutritional factors associated with the diets was apparent in some instances.Reference to a company and/or product named by the Department is only for purposes of information and does not imply approval or recommendation of the product to the exclusion of others which may also be suitable.     

Interaction of Dietary Fat and Soybean Isolate (SBI) on Azaserine-induced Pancreatic Carcinogenesis

Raw soybean flour contains high levels of both soybean trypsin inhibitor and unsaturated fat. The chronic ingestion of this flour enhances pancreatic carcinogenesis in rats that have been treated with pancreatic carcinogens. In these studies we have examined the separate and combined effects of trypsin inhibitor in purified soybean isolate (SBI) and a high level of unsaturated fat fed during the postinitiation phase of pancreatic carcinogenesis. Male Wistar rats were injected with the known pancreatic carcinogen azaserine (30 mg/kg, ip) 14 days after birth and were weaned to the test diets. No further exposure to carcinogen occurred. Dietary composition (% by weight) of fat (corn oil) was either 5%, or for the high fat group 20%. Dietary protein (20% in all diets) was supplied as either casein, or heated or raw SBI. All diets were fedad libitum for the postinitiation phase. The number and size of transections of azaserine-induced putative, preneoplastic foci and the volume of pancreas occupied by these foci were determined by light microscopy. High fat diet increased the number and transectional size of the azaserine-induced foci as compared to the control (5% corn oil) group. The volume of pancreas occupied by foci doubled. Heated SBI had no effect upon the foci. Raw SBI, however, significantly increased the number and size of foci. The combination of raw SBI plus the high fat diet elicited an increase in focal growth that was greater than that produced by either component alone. These observations indicated that both trypsin inhibitor and the high unsaturated fat content of raw soy flour contribute to the enhancement or promotion of pancreatic carcinogenesis.     

Impact of microwave heating on hemagglutinins,trypsin inhibitors and protein quality of selected legume seeds

Selected legume seeds (dry and soaked) including faba beans, peas, chickpeas, soybeans, lentils and common beans containing 8 and 25% moisture, respectively, were subjected to microwave heating, and to a conventional cooking method to determine the heating effect on toxic compounds and protein quality. Trypsin inhibitors, hemagglutinins and available lysine were analyzed, and laboratory rats were used to determine digestibility and protein efficiency ratio (PER). Results indicated that microwaving destroyed trypsin inhibitors to a similar degree to that observed in beans cooked using the conventional method without affecting PER for raw seeds with low antinutrients content (faba beans, peas, chickpeas and lentils). Microwave-heated soaked soybeans had a higher amount of destroyed trypsin inhibitors, along with a higher PER, compared with microwave-heated dry soybeans. Microwave heating of common beans failed to destroy hemagglutinins and trypsin inhibitors, and consequently their digestibility and PER values were poor. Finally it was concluded that microwave heating constitutes an adequate method for destroying hemagglutinins and trypsin inhibitors without affecting protein quality of most legume seeds, except for common beans that despite of this process retained the antinutritional substances.     

Protein and starch digestibilities and mineral availability of products developed from potato,soy and corn flour

A technique for development of potato flour was standardized. Five products viz. cake, biscuit, weaning food, panjiri and ladoo were prepared incorporating potato flour, defatted soy flour and corn flour. Baking and roasting were the major processing techniques employed for the development of these products. Protein, ash and fat contents of potato flour were almost similar to those of raw potatoes. Significant differences in protein, ash and fat contents of all the products were observed. Protein and starch digestibility of potato flour was significantly higher than that of raw potatoes. Protein digestibility increased by 12 to 17 percent on baking or roasting of products. Processed products had significantly higher starch digestibility and mineral availability compared to raw products. Thus, it can be concluded that roasting and baking are effective means of improving starch and protein digestibility and mineral availability of products.     

The protein quality of lupins (Lupinus mutabilis) alone and in combination with other protein sources

The chemical compositions, of raw and treated lupine flour were determined and compared with other plant protein sources. The protein content in the dry matter was 47.7% in untreated seeds ofLupinus mutabilis and about 56% in debittered seeds. The oil-cake contained 65.3% protein. The amino acid analysis showed that lupine protein is characterized by a low level of S-amino acids, the content of which amounts to only 50% of that of the FAO standard reference pattern. The protein quality was measured using the biological tests PER (Protein Efficiency Ratio), NPU (net protein utilization), and BV (biological value) in rats on diets with and without DL-methionine supplementation. PER determinations gave low values for the non-supplemented lupine proteins (1.34, semi-sweet variety; 1.53, water-extracted seeds; 1.19, oil-cake; 3.09, casein), but the PER's were improved by the addition of 0.2% DL-methionine to the diets (3.05, 2.69, 2.81, respectively). Raw as well as processed lupine protein showed an excellent apparent digestibility (80.0–85.8%; casein, 87.1%). The observed NPU and BV values confirmed the importance of methionine supplementation. The true digestibility of 92% was equivalent to that of casein. The complementation effects of mixing lupine protein with proteins from wheat, oat, barley, rice, maize, potato, quinua or fish were investigated by determination of the PER values of the respective mixtures. Feeding lupine protein with cereal proteins resulted in PER values that exceeded by far those of the proteins fed separately (true complementation). This result was not observed for the mixture of potato and lupine. High quality proteins like quinua and fish protein also showed no complementation effect with lupine protein but did improve the quality of the lupine protein to a great extent. The best results could be obtained with combinations of three different plant proteins, in which lupine protein always contributed one third of total protein. These mixtures resulted in PER values equal to those for casein or other animal proteins and may be applied as an economical way to prevent and combat malnutrition.     

Relationship between amino acid scores and protein quality indices based on rat growth

Protein efficiency ratio (PER), relative PER (RPER), net protein ratio (NPR) and relative NPR (RNPR) values, and amino acid scores were calculated for 20 food products (casein, casein + Met, beef salami, skim milk, tuna, chicken frankfurters, sausage, heated skim milk, peanut butter, rolled oats, soy isolate, chick peas, pea concentrate, kidney beans, wheat cereal, pinto bean, lentils, rice-wheat gluten cereal, macaroni-cheese, and beef stew). In most cases, PER, RPER, NPR or RNPR ranked the products in the same order and positive correlations among the protein quality methods were highly significant (r=0.98–0.99). Amino acid scores (based on the first limiting amino acid, Lys-Met-Cys, Lys-Met-Cys-Trp or lys-Met-Cys-Trp-Thr) were positively correlated to the PER, RPER, NPR or RNPR data (r=0.61–0.75). Inclusion of the correction for true digestibility of protein improved the correlations between amino acid scores and the indices based on rat growth. The correlations were especially high between Lys-Met-Cys scores (corrected for true digestibility of protein) and PER, RPER, NPR or RNPR (r=0.86–0.91). Inclusion of the correction for true digestibility of individual amino acids did not result in further improvements of the correlations in most cases. It is concluded that adjusting amino acid scores for true digestibility of protein would be sufficient and further correction for digestibility of amino acids would be unnecessary in mixed diets.This paper was presented in part at the 71st annual FASAB meeting, Washington, DC, March 29–April 2, 1987.     

Trypsin inhibitors and nutritive value in cereals

Chemical assays demonstrated that rye and barley cultivars contained relatively high levels of trypsin inhibitor activity as compared to oat and wheat cultivars, and there was a low degree of stability to prolonged wet heat treatment. In feeding trials with broiler chicks, incorporation of 67% raw barley or 50% raw rye in the rations enhanced feed intake and weight gains, and the marginal increases in pancreas weight were not reversed by feeding autoclaved cereals. Raw rye cultivars fed at the 75% level in mouse diets reduced weight gains, feed efficiency, protein digestibility, protein efficiency ratio and biological value. Autoclaving to inactivate trypsin inhibitors, or ether extraction to remove the resorcinols, failed to improve the nutritive value of rye diets for mice. It appeared that the protease inhibitors in the four cereals were relatively weak inhibitors of trypsin in the digestive system despite stability to dry heat and acid pH.     

Nutritive value of soybean,rapeseed and wheat proteins,and various blends of these vegetable proteins and their fractions in rats

Blends of vegetable proteins were prepared to improve the nutritive value of these proteins. Soybean flour, rapeseed protein concentrate, whole wheat flour, soybean 2S+11S extract, wheat albumin-globulin + glutenin (AG+G) functions, and some of their blends were compared to casein for protein efficiency ratio (PER) and apparent digestibility coefficient (ADC). Autoclaving (1 h) soybean proteins was also studied. Casein and rapeseed protein concentrate gave the highest weight gains and PER. Other protein sources gave lower values for both PER (P<0.05) and weight gain. The digestibility of all vegetable proteins was lower (P<0.05) than that of casein. PER of soybean 2S+11S extract was significantly lower (P<0.05) than that of soybean flour. Autoclaving significantly (P<0.05) improved the PER of both soybean flour and 2S+11S extract. The ADC of autoclaved 2S+11S extract was similar (P<0.05) to that of autoclaved soybean flour and significantly higher (P<0.05) than that of 2S+11S extract. Soybean flour had the lowest (P<0.05) ADC. Heat treatment destroyed antinutritional factors, probably trypsin inhibitors and/or haemagglutinins of soybean. This was accompanied by improvement in the nutritional value of protein. The four blends were chosen on the basis of amino acid composition, chromatography of proteolyzates and nutritive value of each fraction. The PER of wheat albumin-globulin and glutenin (AG+G) blend was similar (P<0.05) to that of wheat flour and lower (P<0.05) than that of all other blends used. Wheat AG+G+rapeseed protein concentrate and wheat AG+G+soybean flour blends gave the highest (P<0.05) PER. Wheat AG+G improved the PER of 2S+11S extract and of soybean flour but decreased the PER of rapeseed protein concentrate. Wheat AG+G and wheat AG+G+rapeseed protein concentrate blends gave an ADC significantly higher (P<0.05) than that of wheat AG+G blend containing soybean flour of 2S+11S extract. However, blending wheat AG+G with either 2S+11S extract of soybean flour improved ADC.     

The USDA trypsin inhibitor study. I. Background,objectives, and procedural details

Short-term feeding studies have shown that raw soy flour and purified trypsin inhibitors (TI) cause pancreatic hypertrophy and hyperplasia in certain monogastric animals. Prolonged exposure to high levels of TI in raw soy ultimately leads to pancreatic nodular hyperplasia and acinar cell adenoma in rats exposed to low levels of pancreatic carcinogens. Such change has been observed even in the absence of any exposure to known carcinogens. These results emphasized the need for chronic (2 yr) feeding trials which would clearly reveal dose response relationships of alterations in pancreatic pathology to dietary TI and possible interactions with dietary protein and fat.Here we report on the objectives and designs of the first phase of the USDA TI Study, including composition of diets, preparation, and analyses of test substances. To provide the requisite TI and protein levels, raw and heated defatted soy flours, soy protein isolates, and lactic casein were used. The interrelationship of dietary level of TI (93–1270 mg/100 g diet) and that of protein (10%, 20%, and 30%) to pancreatic function and pathology will be reported in the following three papers. Both serial and chronic sacrifice regimes, respectively, were employed with 26 diets and 40 weaning male Wistar rats per dietary group.The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.     

The nutritive quality of maize-soybean (70:30) tempe flour

The nutritive quality of maize-soybean (70:30) tempe flour manufactured by fermentation withRhizopus oligosporus: Rhizopus orysae (1:1) was determined using weanling rats. Mould fermentation of maize-soybean mixture did not significantly affect its proximate composition. It increased the content of reducing sugars, total acids and aminonitrogen by about 43, 195 and 482 percent, respectively, and decreased phytate content by 46 percent. In vitro iron absorption for maize flour and maize-soybean tempe flour was 2.46 and 5.51 percent, respectively. Protein Efficiency Ratio (PER) and Net Protein Ratio (NPR) for maize-soybean tempe flour and skim-milk diets were 2.71 and 2.96, respectively, for PER, and 3.31 and 3.51, respectively, for NPR. In vivo protein digestibility of the two products was 95.0 and 98.0 percent, respectively.     

Digestibilities of the protein in various foods as determinedin vitro by an immobilized digestive enzyme assay (IDEA)

The digestibility of the protein in various foods or food components was analyzed using an immobilized digestive enzyme assay (IDEA) system. The assay consists of two bioreactors, one containing pepsin and the other containing trypsin, chymotrypsin and intestinal mucosa peptidases. The fraction of the peptide bonds hydrolyzed during an extent of hydrolysis assay was correlated with independentin vivo determinations of the digestibilities. A correlation coefficient of 0.80 was obtained. The derived linear regression equation can be used to predict digestibility. The method is sensitive to structural modification of protein, as for example, those caused by effects of heat treatment.Paper No. 11032 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27695-7601. The use of trade names does not imply endorsement by the North Carolina Research Service of the products.     

Biological utilization of vegetable peas: Effect of cooking and varietal differences

The flour of raw as well as cooked vegetable pea cultivars (Arkal and Bonneville) was used for biological evaluation of their protein quality. Cooking did not have a significant influence on the food intake. There were some differences in the food as well as protein intake and gain in body weights of rats when fed uncooked or cooked pea flour diets. Varietal differences did not seem to influence on apparent digestibility, true digestibility, biological value, net protein utilization and utilisable protein but cooking brought a significant (p<0.05) improvement in all of above mentioned parameters. Varietal differences as well as cooking exhibited no influence or serum protein concentration.     

Protein quality of some Nigerian traditional diets based on the African yambean (Sphenostylis stenocarpa) and pigeon pea (Cajanus cajan)

The protein quality of Nigerian traditional diets based on the African yambean (AYB) and pigeon pea (PP) were performed by in vivo and in vitro bioassays. The individual foods were processed, cooked, dried and mixed to resemble the traditional diets. Weanling male Wistar rats (45–55 g) were used for the bioassay. The rats were fed ad libitum for 35 days of which 28 days were for growth and 7 days for N-balance. All the eleven diets including casein (control) provided 1.6 g N/100 g diet. The parameters tested include PER, growth, N-balance, BV, NPU and apparent digestibility (AD). The rats fed the AYB diets, except AYB: Agidi (Ag), showed superiority in most of the parameters tested. There were significant positive correlations between PER and NPU (r=0.925;p<0.001); PER and AD (r=0.908;p<0.001); PER and body weight gain (r=0.969;p<0.001). The in vitro protein digestibility of the diets was significantly correlated (r=0.80;p<0.01) with the in vivo apparent digestibility. These parameters were used to rank the various diets. Crayfish protein was a better supplement to legume/cereal or legume starchy staple mixtures than leguminous oil seed. However, supplementation of PP: steamed corn (SC) diet with crayfish did not prove beneficial. The result showed that diets based on the African yambean, an under-exploited legume, are nutritious. The need for its reintroduction into the fare of the populace through increased production and appropriate processing technology is stressed. The results of this study can form a base for the standardization of Nigerian diets based on these legumes.     

Compositional,functional and storage properties of flours from raw and heat processed African breadfruit (Treculia africana Decne) seeds

African breadfruit (Treculia africana Decne) seeds were either boiled or roasted and then milled into flour. Chemical composition, functional properties and storage characteristics of raw and treated flours and the effect of partial proteolysis on selected functional properties of the raw flour were determined. Raw flour contained 20.1% crude protein, 2.5% total ash and 13.7% fat. Heat processing significantly (p<0.05) improved in vitro protein digestibility, and water and fat absorption capacities but decreased bulk density, nitrogen solubility, emulsion and foaming properties, trypsin inhibitor, and phytic acid and polyphenol contents of the samples. Boiling proved more effective than roasting for improving protein digestibility, emulsion capacity and foam stability and reducing antinutritional factor levels. Partial proteolysis increased nitrogen solubility, bulk density and water and fat absorption capacities but decreased foam capacity at hydrolysis levels greater than 35%. Fatty acid and peroxide values of the samples increased during storage. Compared to raw samples, heat processed samples had significantly (p<0.05) lower and more acceptable peroxide values and free fatty acid contents and higher and more stable water (3.0 g/g sample) and fat (2.4 g/g sample) absorption capacities.     

Specific role of endopeptidases in modulating the nature of protein digestion products

The impact of various endopeptidases on the nature of protein digestion products was measured with the digestion cell technique. After a 30 min pepsin pre-digestion, casein and rapeseed concentrates were hydrolyzed with various amounts of pancreatin, trypsin and/or chymotrypsin. This hydrolysis was performed in a dialysis tube (molecular weight cut-off 1000) with continuous collection of the digested material. The addition of pure trypsin or chymotrypsin to pancreatin (Enzyme:Substrate 125) did not change the digestibility of casein. Only a higher pancreatin level (Enzyme: Substrate 112.5) increased the total protein digestibility without affecting the amino acid spectra. Rapeseed digestibility was markedly increased by the addition of pure trypsin to pancreatin. Lysine and arginine, target amino acids of trypsin, were favored at the expense of chymotrypsin and elastase target amino acids. Supplementation of pancreatin with, chymotrypsin enhanced rapeseed digestibility without affecting the relative amino acid digestibility. The impact of a higher pancreatin ratio (Enzyme: Substrate 112.5) was similar to that of enriched pancreatin but the rate of amino acid release was modified. The differences between protein sources were mainly attributed to protein structure.     

Chemical composition and biological evaluation of Okra (Hibiscus esculentus) seeds and their kernels

Okra (Hibiscus esculentus) whole seeds and their kernels were analysed for their nutrient composition. They were rich in protein as well as fat. Most of the protein and fat of the seed is accumulated in the kernel while crude fiber is concentrated in the seed coat or hull. Its essential amino acid composition was nearly equal to that of the WHO/FAO reference pattern (1973), but lower than that of whole egg protein; its chemical score was 67. Diets containing whole seed and kernel protein at the 10% level were fed to weanling rats both in the raw and cooked forms for 4 weeks. Dry matter digestibility and protein digestibility of whole seed diets were significantly lower than the kernel diets. Cooking improved PER and NPU values of both the whole grain and kernel diets. These results indicated that dietary fiber present in the hull of Okra seed as well as heat labile antinutritional factors present in the seed hinder Okra seed protein utilization.     

Improved functional properties for soy–wheat doughs due to modification of the size distribution of polymeric proteins

Physical modification of soy flour was shown to greatly improve the dough and baking qualities of soy–wheat (1:1) composite doughs, compared to raw soy flour, giving better stability and R_max, although extensibility was still below that of the wheat dough.Reasons for improvements caused by the physical-modification process were sought by determining the relative size distribution of proteins in the soy–wheat composite doughs by size-exclusion high-performance liquid chromatography (SE-HPLC). Results were expressed as the proportion of ‘unextractable polymeric protein’ (%UPP)—the proportion of the protein that is over 100,000 Da and only extractable after sonication. Protein extracts from the soy–wheat dough were sampled at different stages of dough mixing and fermentation, and their molecular-size distributions evaluated.Unextractable soy proteins were lower in raw soy flour (only 8% UPP) than in two physically-modified soy flours (19 and 34% UPP, respectively). Unextractable polymeric protein was much greater for wheat flour (57% UPP). After mixing a 1:1 soy–wheat composite dough, the %UPP was 36 and 22 (for the two types) when made from physically modified soy flours, compared to 8 for a composite dough using raw soy flour, and 43 for a wheat-only dough. The higher proportion of UPP for the wheat-modified soy doughs was taken as a reason for this composite dough providing better dough and baking qualities. Prolonged fermentation time caused a decrease in UPP percentages for all composite doughs and for the wheat-only dough.     

Effects of extrusion cooking on functional properties of mixtures of full-fat soy and sweet potato

Full-fat soy and sweet potato were extruded at 0:100, 25:75, 50:50, 75:25 and 100:0 ratios in an Insta-Pro 600 single screw extruder operated at 180, 200, 220 rpm respectively. Functional properties: bulk density, expansion ratio, water absorption and solubility indices and trypsin inhibitor were assayed. Results showed that addition of soybeans to sweet potatoes considerably increased the protein, fat, ash and trypsin inhibitor levels of the raw material mixtures. It also led to decreases in viscosities of raw mixtures, with corresponding increases in pasting temperatures. Addition of soybean significantly (p<0.05) affected expansion ratio, water absorption and solubility indices and trypsin inhibitor but not bulk density. Variation in screw rotation speed (rpm) significantly (p<0.05) affected only water solubility index at increasing speed. Extrudates generally received severe heat treatment, rendering them applicable only in soup bases, flour mixes and breakfast foods.