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.     

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 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.     

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.     

Influence of dietary soybean trypsin inhibitors and DL-ethionine on sulfur amino acid adequacy of diets for young rats

Weanling male Wistar rats were fed 20% protein diets based on casein or either of two combinations of soy protein isolate and ground raw soy providing three levels of soybean trypsin inhibitors (SBTI; 0,448 and 808 mg of trypsin inhibited per 100g of diet respectively). DL-ethionine was included at three levels (0,0.05% and 0.10%) with each level of SBTI. After 4, 8 and 12 weeks ofad libitum feeding, diets containing SBTI without DL-ethionine were associated with decreases in weight gain, feed efficiency, serum cholesterol and serum urea nitrogen. Higher levels of triglycerides, glutamate pyruvate transaminase (SGPT) and altered serum free amino acid levels were also found. Increased dietary levels of DL-ethionine also resulted in deficits in growth and feed efficiency, decreased serum cholesterol, increased SGPT and similar alterations in serum free amino acids. Combination of dietary SBTI with DL-ethionine resulted in even greater growth deficits and serum cholesterol decreases as well as increases in SGPT and serum triglycerides and changes in serum free amino acid levels. Methionine deficiency in the young rats fed SBTI and DL-ethionine was indicated by the changes in serum amino acids and growth deficits. Moderation of some effects over the 12 week test period suggested decreased methionine requirements in the older rats.     

Nutritional studies on rats and fish (carp Cyprinus carpio) fed diets containing unheated and heated Jatropha curcas meal of a non-toxic provenance

Unheated and heated (121 ^°C, 66% moisture; 15, 30 and 45 min) Jatropha meals of non-toxic provenance from Veracruz state in Mexico were evaluated using rats and fish. With rats, the weight gain was highest for the casein diet followed by heated (30 min; only this treatment was studied using rats) and unheated Jatropha meal containing diets. The protein efficiency ratio (PER) for unheated and heated Jatropha meal containing diets was 37 and 86%, respectively, of the casein diet. On the other hand, the body weight gain, PER and feed conversion ratio of fish were statistically similar for unheated and heated (15, 30 and 45 min) Jatropha meal containing diets fed for a period of 35 days. Although these parameters were statistically similar for the unheated and heated Jatropha meal containing diets, the body weight gain, PER and protein productive value were highest and the feed conversion ratio lowest with 15 min heated Jatropha meal, suggesting that the heat treatment for 15 min is optimal for the meal. Trypsin inhibitor and lectin activities decreased drastically (>83 and 99%, respectively) after 30 and 45 min of heat treatment and after 15 min, the residual lectin activity was negligible and the residual trypsin inhibitor activity was 34%. These results, together with the nutritional parameters investigated, imply that Jatropha trypsin inhibitors and lectins do not have any adverse effects on carp at least up to 35 days of feeding. The nutritional value of Jatropha meal of the non-toxic provenance is high, and potential exists for its incorporation into the diets of monogastrics, fish and possibly humans.     

The USDA trypsin inhibitor study. III. Sequential development of pancreatic pathology in rats

Weanling male, Wistar rats (40 per diet) were maintained on 15 diets which provided 4 graded trypsin inhibitor (TI) levels (93, 215, 337, and 577 mg/100g diet) repeated at 3 levels of protein (10%, 20%, and 30%). Raw and heated (toasted) soy flour provided 10% protein in the diet, while casein was added to increase the protein level to 20% and 30%. Three diets containing only casein as the protein source, at 10%, 20%, and 30% were included. Histologic changes occurring in the pancreas were evaluated in approximately 5 rats from each diet at 3 month intervals, beginning at 6 months, for 22 months. Criteria for the diagnosis of the observed histologic changes in the pancreas are discussed. Ultrastructure of the endocrine cells in the gastrointestinal mucosa was also examined.Pancreatic acinar cell density, as determined morphometrically, was reduced throughout the study in rats fed the 577 mg TI/100 g diet, indicating hypertrophy at the cellular level. Nodular hyperplasia was observed in the first sacrifice group at 6 months. Incidence of the lesion was positively related to both time of exposure and level of dietary TI. Acinar adenoma was first observed at 18 months and was most prevalent in rats fed the highest concentration of TI.Endocrine cells of the duodenal mucosa which contain cholecystokinin (so-called I cells) contained a denser population of secretory granules in animals fed the high TI diet compared with I cells from control animals, leading to the speculation that production and secretion of cytoplasmic hormones was increased in TI treated rats.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.     

Safety evaluation of water debittered andean lupins (Lupinus mutabilis): 12-week rat feeding study

In a 12-week feeding study, water debittered grains of Andean lupins (Lupinus mutabilis) were tested as unique protein source in growing male and female rats. Before the phsyiological study, the lupin flour was evaluated with regard to its possible content of anti-nutritive substances, such as alkaloids, cyanogenetic glucosides, trypsin inhibitor activity and hemagutanin activity. During the feeding study, parameters such as body weight, food consumption, hemoglobin and blood cells, glucose, urea, total protein, total lipid, cholesterol, uric acid, as well as weight and histopathological examinations of some organs (liver, kidneys, heart, spleen and brain) were controlled. The lupin flour did not show physiologically significant amounts of anti-nutritive substances. Rats fed on lupin diet did not show any statistically different growth performances as compared to the casein fed control animals. Food consumption as well showed no difference between the two diets, although food utilization of casein was slightly better during the first two months. All other observed parameters gave no indication of physiological differences of the performance of the diet. Results of the 12-week feeding test did not give any indication against the use of the water extracted grains of Andean lupins in human and animal nutrition.     

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.     

Growth,blood chemistry and histology of rats fed zincsupplemented rapeseed protein concentrates

Two rapeseed protein concentrates (RPC) supplemented with zinc oxide were fed to male and female rats at two levels providing approximately half or all of the protein in a purified basal diet containing 20% protein (N×6.25). Control groups were given either a purified basal diet with casein or a laboratory chow diet. After 16 weeks, there were no significant (P<0.05) differences in body weight except the male rats fed the casein control diet had higher body weights than did other male rats. No significant differences were observed in organ weights including thyroids, hematology, bone marrow differential counts, serum glucose, urea nitrogen, total protein, albumin/globulin ratio, cholesterol, transaminases or alkaline phosphatase. The levels of serum vitamins A and E of females fed some of the RPC diets were less than in those fed the caseincontrol diet, but equal to those in females fed laboratory chow diet. Females fed either RPC or casein in the semi-purified diets, which contained zinc oxide, exhibited kidney calcification.Bureau of Nutritional Sciences, No. 112; Food Research Institute, No. 420.     

Comparative effects of feeding a protease inhibitor-enriched potato protein concentrate and soy flour to rats

Rats were fed casein diets to which had been added a potato protein concentrate (PPC) at a level which provided trypsin- and chymotrypsin-inhibitor activities comparable to that present at the same level (10%) of protein derived from raw soy flour. Control groups consisted of the same diets in which the unheated PPC and raw soy flour were replaced by their heated counterparts in which over 90% of the inhibitor activities had been destroyed. After 5 weeks the following measurements were made: (1) growth, (2) PER, (3) pancreas weight, (4) protein digestibility, (5) a peptide test that reflects pancreatic function, and (6) enzyme activities (trypsin, chymotrypsin, amylase, and lipase) in the pancreas and small intestine. PPC was found to produce the same general pattern of effects as raw soy flour, namely, an inhibition of growth, a decrease in PER and protein digestibility, an increase in the size of the pancreas, an increase in the recovery of p-aminobenzoic acid in the peptide test, and a depletion of enzyme in the pancreas with concomitant increases in the intestines (except for trypsin). PPC was somewhat more effective than raw soy flour in causing an increase in trypsin and chymotrypsin activities in the faces.     

Protein and energy utilization of brown,undermilled and milled rices by preschool children

The protein and energy utilization of brown, undermilled and milled rices (variety IR32) were studied in 5–6 preschool children through diets in which 2/3 of N was from rice and 1/3 from casein or milk powder at 200 or 250 mg N/kg body weight. Brown rice was not significantly inferior to milled rice in terms of nitrogen absorption and retention by the children. Its energy and fat contents were, however, less utilized than those of milled rice. Children on the control casein or milk diets showed better apparent nitrogen absorption than those on the rice-based diets but nitrogen retention of all the diets was similar in all three experiments.     

Growth,hormonal status and protein turnover in rats fed on a diet containing peas (Pisum sativum L.) as the source of protein

The inclusion of peas (Pisum sativum L.) as the source of protein in the diet of growing rats brings about a reduction in growth rate as well as the impairment in the liver, muscle and spleen weights as compared with casein fed controls. Also, a fall in plasma glucose, triglycerides and protein was observed in the legume fed animals, while no changes in cholesterol levels were found. Furthermore, the rats fed on the diet containing peas showed lower levels of plasma insulin, corticosterone, IGF-I and T₄ as compared with casein controls. Liver and muscle total protein (mg) and total DNA (mg) were markedly decreased in the legume fed animals, but DNA/g, protein/DNA and RNA/protein ratios were similar in both dietary groups. Likewise, liver and muscle fractional synthesis rates were similar in the casein and legume groups, while the whole body protein synthesis is assumed to be lower in the legume fed animals due to differences in body weights. It is concluded that animals fed on a diet containing peas (Pisum sativum L.) as the only source of protein showed less adverse effects than those found with other legumes such asVicia faba L. orPhaseolus vulgaris L., in which protein quality, antinutritional factors and nutrient availability could be involved.     

Organic and Genetically Modified Soybean Diets: Consequences in Growth and in Hematological Indicators of Aged Rats

The aim of this study was to evaluate the protein quality of organic and genetically modified soy by feeding specific diets to rats. Three groups of Wistar rats (n = 10) were used, and each group was named according to the food that they ate. There was an organic soy group (OG), a genetically modified soy group (GG), and a control group (CG). All animals received water and diet ad libitum for 455 days. At the end of this period, the weight of the GG group was the same as that of the OG, and both were higher than CG. Protein intake was similar for the OG and GG, which were significantly lower (p < 0.0005) than the CG. The growth rate (GR) of the rats, albumin levels, and total levels of serum protein were comparable for all groups. Hematocrit (p < 0.04) and hemoglobin (p < 0.03) for the OG and GG were less than the CG. Although the OG and GG demonstrated reduced hematocrit and hemoglobin, both types of soy were utilized in a way similar to casein. This result suggests that the protein quality of soy is parallel to the standard protein casein in terms of growth promotion but not hematological indicators.     

Effects of Blackcurrant and Dietary Fibers on Large Intestinal Health Biomarkers in Rats

This study examined the effects of anthocyanin-rich blackcurrant extract and dietary fibers individually and their combinations on biomarkers of large intestinal health in rats. After six weeks of feeding, rats fed diets with blackcurrant gained significantly less body weight and reduced their food intake resulting in a lower food efficiency compared with those rats fed control diets. Combining dietary fiber (apple or broccoli) with blackcurrant in the diet was more effective in reducing the body weight gain and food intake. Cecal bacterial populations and short-chain fatty acids differed between the experimental diets. Blackcurrants significantly altered the bacterial populations by increasing the abundance of Bacteroides-Prevotella-Porphyromonas group and Lactobacillus spp., while decreasing the abundance of Bifidobacterium spp. and Clostridium perfringens. Propionic acid concentrations were increased by the diets with blackcurrant. Butyric acid concentrations were increased by dietary fiber supplementation. Dietary fiber increased the number of goblet cells in the colon. Diets with blackcurrant were more effective in altering the biomarkers of large intestinal health than those without blackcurrant.     

Bioavailability of tryptophan in selected foods by rat growth assay

Tryptophan bioavailabilities were estimated in 16 protein sources using 10 day rat growth assays with casein as the reference protein. Growth responses of rats fed test food diets were compared to growth responses of rats fed basal diets with graded levels of tryptophan ranging from 50 to 100 mg of tryptophan/100 g diet. Estimates of tryptophan availabilities were 85–100% for all products except whole wheat cereal (73%) and pinto beans (59%). Results of a previous study on lysine availability indicated that poor response to pinto beans was due either to poor digestibility or to the presence of some unidentified growth inhibitor.     

Iodine metabolism and tissue mineral levels in rats fed rapeseed protein concentrates supplemented with zinc

A 16-week feeding test of Tower (Brassica napus) and Span (Brassica campestris) rapeseed protein concentrates (RPC) was carried out on male and female Charles River CD (Sprague-Dawley) weanling rats. The diets contained 10% or 20% protein (N × 6.25) from each RPC and were supplemented with 150 mg zinc/kg. The control groups received a purified diet with casein or a laboratory chow diet. The results of the test diet groups did not reveal any adverse effect due to RPC on thyroid metabolism or the metabolism of zinc iron, copper, manganese, calcium, phosphorus, and magnesium. Nephrocalcinosis was observed in all female rats that were fed the test diets and the casein control diet, and therefore it could not be attributed to dietary RPC.Bureau of Nutritional Sciences, no. 120. Food Research Institute, no. 448.     

Nutrient composition and biological evaluation of mesta (Hibiscus sabdariffa) seeds

Two varieties of mesta (Hibiscus sabdariffa) seeds were analysed for their proximate composition. Their protein (18.8–22.3%), fat (19.1–22.8%) and dietary fiber (39.5–42.6%) contents were found to be high. The seeds were found to be a good source of minerals like phosphorus, magnesium and calcium. Their lysine and tryptophan contents were also high. Sulphur containing amino acids were limiting in this seed protein and the chemical score of mesta seed protein was 40 and 57 for AMV-2 and Bhimili-1 varieties respectively. Mesta seed oil is rich in unsaturated fatty acids (70%), of which linoleic acid constituted 44%. Weanling rats were fed with 10% mesta seed protein before and after cooking for 4 weeks. Food intake of animals receiving raw mesta seed diets was significantly lower than those receiving cooked mesta seed diets as well as the casein control diet. Protein and dry matter digestibilities of raw and cooked mesta seed diets were lower than that of casein control diet. Cooking improved the food intake, gain in body weight, dry matter and protein digestibility of mesta seed diets. PER and NPU of cooked mesta seed diets were significantly higher than the corresponding raw diets. These results indicate that cooked mesta seed protein is of relatively good quality.     

Herring Milt and Herring Milt Protein Hydrolysate Are Equally Effective in Improving Insulin Sensitivity and Pancreatic Beta-Cell Function in Diet-Induced Obese- and Insulin-Resistant Mice

Although genetic predisposition influences the onset and progression of insulin resistance and diabetes, dietary nutrients are critical. In general, protein is beneficial relative to carbohydrate and fat but dependent on protein source. Our recent study demonstrated that 70% replacement of dietary casein protein with the equivalent quantity of protein derived from herring milt protein hydrolysate (HMPH; herring milt with proteins being enzymatically hydrolyzed) significantly improved insulin resistance and glucose homeostasis in high-fat diet-induced obese mice. As production of protein hydrolysate increases the cost of the product, it is important to determine whether a simply dried and ground herring milt product possesses similar benefits. Therefore, the current study was conducted to investigate the effect of herring milt dry powder (HMDP) on glucose control and the associated metabolic phenotypes and further to compare its efficacy with HMPH. Male C57BL/6J mice on a high-fat diet for 7 weeks were randomized based on body weight and blood glucose into three groups. One group continued on the high-fat diet and was used as the insulin-resistant/diabetic control and the other two groups were given the high-fat diet modified to have 70% of casein protein being replaced with the same amount of protein from HMDP or HMPH. A group of mice on a low-fat diet all the time was used as the normal control. The results demonstrated that mice on the high-fat diet increased weight gain and showed higher blood concentrations of glucose, insulin, and leptin, as well as impaired glucose tolerance and pancreatic β-cell function relative to those on the normal control diet. In comparison with the high-fat diet, the replacement of 70% dietary casein protein with the same amount of HMDP or HMPH protein decreased weight gain and significantly improved the aforementioned biomarkers, insulin sensitivity or resistance, and β-cell function. The HMDP and HMPH showed similar effects on every parameter except blood lipids where HMDP decreased total cholesterol and non-HDL-cholesterol levels while the effect of HMPH was not significant. The results demonstrate that substituting 70% of dietary casein protein with the equivalent amount of HMDP or HMPH protein protects against obesity and diabetes, and HMDP is also beneficial to cholesterol homeostasis.     

Bioavailability of lysine in selected foods by rat growth assay

Lysine bioavailabilities in reference protein and 16 test protein diets were estimated using 10 day rat growth assays. A standard growth curve was obtained by feeding 5 diets containing casein, zein and synthetic amino acids ranging in total lysine concentration from 0.3 to 0.7%. Experimental foods were added to the basal diet at the expense of zein and/or synthetic amino acids to provide 2 specific lysine concentrations, i.e., 0.4 and 0.6%. Availabilities were established by comparing growth responses from the test food diets to the regression line of the standard growth data. Availabilities were over 88% for 13 of 16 products. Utilization was poor in pinto beans (73%), rice-wheat gluten cereal (70%), and skim milk powder heated to 100°C for 12 h (66%). Addition of excess lysine (700 mg/100 g diet) to the pinto bean diet did not improve growth response; thus poor digestibility or some unidentified growth inhibitor is indicated.