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. 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.     

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.     

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.     

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.     

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 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.     

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.     

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.     

Effect of soy-fortification method on the fermentation characteristics and nutritional quality of fermented maize meal

Studies were conducted to develop an appropriate household/small-scale enterprise level technique for the production of soy-fortified fermented maize dough (or meal) by comparing different treatments, processing methods and fortification levels. The effects of fortification method of the Ghanaian traditional fermented maize dough with raw or heat-treated whole soybeans and full-fat soyflour at 0%, 10% and 20% replacement levels, on the rate of fermentation and product quality were investigated. Sensory characteristics, trypsin inhibitor activity, amino acid pattern, proximate composition and hot paste viscosity were used as the indices of quality. Addition of whole soybeans to maize before milling and fermentation reduced the fermentation time by 60% while increasing the protein content by 24% and 70% respectively for 10% and 20% levels of fortification. A significant improvement was also achieved in the amino acids pattern of the fortified dough. However, raw whole soybeans imparted an undesirable color and beany flavor and an appreciable concentration of trypsin inhibitor activity (TIA) to the dough. Boiling soybeans for 20 min before incorporation into the maize for milling and fermentation was found necessary for desirable flavor and low levels of TIA. Little or no changes in the pasting viscosity characteristics occurred in samples containing boiled soybeans, while the usual method of fortifying maize meal with soy flour was found to severely depress the pasting viscosity characteristics and drastically reduced the acidity of the fermented dough. Based on the findings of the study, the most appropriate technique for the production of soy-fortified high protein fermented maize dough has been suggested to involve incorporation of boiled whole soybeans in soaked maize before milling and fermentation for improved sensory characteristics, enhanced nutritive value and optimal functional properties.     

Quality attributes of fermented yam flour supplemented with processed soy flour

Pretreated soy flour was used to replace 10, 20, 30 and 40% of fermented yam flour as a protein supplement. The effect of the supplementation on the physicochemical and sensory properties of amala, a popular West African food made from rehydrated yam flour, was investigated. Blanching (10 min in boiling water) and subsequent fermentation (24 h) as pretreatment methods produced flour that was lighter in color than the unfermented samples. Protein content of the yam-soy mixture increased from 3.5% in the control to 19.7% for 40% soy fortification. Water binding capacity increased from 212.6 g/100 g for the yam flour control to 257.3 g/100 g for the blend with 40% soy flour. However, swelling capacity and solubility were adversely affected with increased soy flour addition as dough became sticky and soft. There was no significant difference in color, taste, flavor or overall acceptability when compared with the control up to 20% soy substitution. Dough with 20% soy flour is a possibility for increasing the protein content of yam flour in human feeding.     

Inactivation of trypsin inhibitors in sweet potato and taro tubers during processing

In order to understand the extent of elimination of trypsininhibitors during processing of sweet potato (Ipomoea batatas) andtaro (Colocasia esculenta) tubers, a detailed study was conductedusing tubers processed by oven drying, cooking, and microwavebaking. Between 80 and 90% trypsin inhibitor (TI) activity wasretained in sweet potato chips up to 2h at 70 ^°C. Among thefour cultivars of sweet potatoes, RS-III-2 trypsin inhibitors were more heatlabile. Heating at 100 ^°C led to rapid inactivation of TI of sweetpotatoes. Varietal differences in thermal stability were more pronouncedfor the trypsin inhibitors of taro than sweet potatoes. Taro inhibitors werealso more rapidly inactivated than sweet potato TI. Between 17 and31% TI activity was retained in cooked tuber pieces of sweet potatoes,while only 3–10% were retained in taro cultivars. Very effectiveinactivation of trypsin inhibitors of sweet potatoes and taro could beobtained through microwave baking. Flour prepared from taro was devoidof TI activity, while 5–12% TI activity was retained in the flour preparedfrom sweet potatoes. The study clearly established that among the fourtechniques used, microwave baking and flour preparation were the best methods to eliminate TI from sweet potatoes and taro.     

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.     

Long-term consumption of beef extended with soy protein by men,women and children: I. Study design,nutrient intakes,and serum zinc levels

The effect of long-term consumption, under practical conditions, of beef extended with soy protein on serum zinc levels of men, women and children was studied. Fifty-two families were randomly assigned to consume for 180 days 1 of 7 beef products: all beef, beef extended with either soy isolate, soy concentrate or soy flour (20% reconstituted soy product, 80% beef), or beef extended with each of the 3 soy products fortified with 60 mg Fe and 25 mg Zn/100g protein. The beef product was consumed by the subjects as their principal source of protein for 1 meal a day (children 1–8 yrs) or 1–2 meals a day (11 per week) for adult men and women. A control group consumed their usual self-selected diet. Diet records (4-day) of all foods and beverages consumed by the subjects were obtained pre-study and 63±7 and 126±7 days for calculation of nutrient intakes. No deleterious effects were found on the serum zinc levels when subjects consumed beef extended with soy protein for 180 days.Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture, and does not imply its approval to the exclusion of other products that may also be suitable.     

Incorporation of soy proteins into the wheat–gluten matrix during dough mixing

Farinograph methodology was used to evaluate the possible incorporation of soy proteins into a glutenin–soy complex during mixing and to study the contribution of soy proteins to the chemical and physical properties of the dough. To facilitate the interaction of soy and wheat proteins, a redox process was used, which allowed the partial reduction (using dithiothreitol, DTT) and subsequent reoxidation (using potassium iodate) of glutenin without changing its functionality in the dough (a composite of equal parts of wheat and soy flours, 300 g in total). Either raw soy flour (RSF) or physically modified soy flour (PMSF) was used as the soy component. Dough samples were taken at peak mixing time and at break time during mixing, and these were freeze dried for SE-HPLC analysis and capillary electrophoresis (Lab-on-a-chip).     

Long-term consumption of beef extended with soy protein by men,women and children: II. Effects on iron status

The iron status of men, women and children consuming beef extended with soy protein was evaluated by measuring serum ferritin and clinical parameters of iron status during a six-month study. Fifty-two families (245 participants) were randomly assigned to consume, for 180 days, 1 of 7 beef products: all beef, beef extended with either soy isolate, soy concentrate or soy flour (20% reconstituted soy product, 80% beef), or beef extended with each of the three soy products fortified with 60 mg Fe and 25 mg Zn/100 g protein. The beef product was consumed by the subjects as their principal source of protein for 1 meal a day (children 1–18 yr) or 1–2 meals a day (11 per week; adult men and women). A control group consumed their usual self-selected diets. No evidence was found that consumption of beef extended with soy protein deleteriously affected the iron status of men, women or children. Consumption of beef extended with soy protein, at the levels used in this study, by military men and women and by school lunch participants would not appear to impose a risk in these population groups.     

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.     

The degradation of lectins,phaseolin and trypsin inhibitors during germination of white kidney beans,Phaseolus vulgaris L.

White kidney beans (Phaseolus vulgaris), cv Processor, contain a relatively high content of phaseolin (storage protein), lectins and a special group of glycoproteins as well as a considerable amount of protein-type trypsin inhibitors. Protein digestion of raw Processor beans in monogastrics, for example pigs, is disturbed by poorly digested, phaseolin lectins, which can bind to carbohydrates in brush border membranes of the small intestinal epithelium, and trypsin inhibitors. The effect of the germination of white kidney beans on lectins, phaseolin and trypsin inhibitors was studied in order to achieve a degradation of lectins, phaseolin and trypsin inhibitors and an increase ofin vitro enzymatic hydrolysis of the protein of bean flour. Therefore, whole bean extracts were examined throughout a germination period of up to seven days for their lectin and phaseolin pattern, lectin content, binding capacities of functional lectins towards brush border membranes and trypsin inhibitor content. In addition thein vitro enzymatic hydrolysis by pepsin and pancreatin of the protein from flours of (un)germinated white kidney beans was studied. SDS-PAGE demonstrated a degradation of E-lectins and a disappearance of L-lectins and phaseolin during germination. Results indicated a decrease of the lectin content by 85%, a loss of binding capacities of functional lectins towards brush border membranes by 91%, and a decrease of trypsin inhibitors by 76%, in bean flour after germination for seven days. A maximum inin vitro enzymatic hydrolysis of protein from bean flour was already established after germination for half a day.     

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.     

Proximate composition and some functional properties of extrusion cooked soybean and sweet potato blends.

Mixtures of sweet potato flour and soy flour were made in a pilot mixer. They were moisturized with 18, 25, and 30% water and extruded in a single screw extruder at 80 rpm, using a die of 6mm. Extrusion temperature was maintained at 100 +/- 3 degrees C. Effects of adding soy flour into sweet potato flour, as well as variation in feed moisture on the composition and some functional properties of the extrudates were investigated. Increase in sweet potato content increased carbohydrate values. Protein increased with increase in soy flour. Feed moisture did not significantly (p < or = 0.05) affect extrudate composition. Increase in sweet potato content and feed moisture increased expansion ratio. Bulk density decreased with decrease in feed moisture, but increased with increase in soy flour. Starch content increased as sweet potato content increased. Degree of gelatinization increased with sweet potato content. Lower feed moisture enhanced gelatinization. Water absorption index (WAI) increased as sweet potato content increased. Feed moisture had a slight effect on WAI and water solubility index (WSI). Amylose increased with increase in sweet potato content. Increase in soy flour led to an increase in yellowness (b*) of extrudates.