Assessment of the in vitro digestibility of dietary proteins and of the degree of the breakdown of their antigenic structures by using polyenzyme systems

A comparative assessment was made of the digestion of bovine serum albumin (BSA), chicken ovalbumin (OVA), and casein by means of the gastric juice--duodenal contents floccular gel structures (FGS) system and a four-enzymic system including trypsin, chymotrypsin, peptidase, and bacterial protease preparations. Decomposition of the BSA and OVA antigenic structures with the use of the two systems was also studied. Significant differences in BSA and OVA digestion by the gastric juice--FGS system were detected both with respect to amino nitrogen content and to the degree of their antigenic structure decomposition, whereas no such differences were observed when the four-enzymic system was used. The systems most accurately simulating the 'proteolytic conveyer' conditions of the gastrointestinal tract are preferable for the studies. The developed method is recommended for use in comparative assessment of the nutrient protein sensitizing properties.     

Response of intestinal proteinase activities to the feeding of isolated winged bean (Psophocarpus tetragonolobus) and soya bean (Glycine max) trypsin inhibitors in rats

The antinutritional activities of trypsin inhibitors (TIs) were compared between winged beans (Psophocarpus tetragonolobus) and soya beans (Glycine max). The inhibitors of the two beans were isolated by trypsin‐bound Sepharose 4B, and 50 mg of lyophilised powders were intubated intragastrically into 24 h fasted rats. The activities of trypsin and chymotrypsin were compared after 30, 60 and 180 min in the washings of the upper, middle and lower parts of the small intestine. The elution profiles of TI and non‐TI compounds in the affinity chromatography were similar in the two beans, and the antitryptic activities were concentrated 5.5 and 6.2 times (based on specific activity) for winged beans and soya beans respectively. Regardless of the TI fed to rats, trypsin activity in the upper intestine was suppressed to almost undetectable levels at 30 and 60 min after intubation. The activities in the middle and lower intestines were also substantially lowered when rats were fed winged bean TI, and significant differences were detected at 30 and 60 min after intubation when compared with rats fed soya bean TI. However, at 180 min after feeding, no differences were found in the trypsin activity in any gut segments. Similar inhibitory properties of isolated TIs were observed in chymotrypsin activities in the small intestine. The results suggest that winged bean TI may have greater inhibitory activity on the intestinal proteinase compared with soya bean TI. © 2001 Society of Chemical Industry     

Determination of Antigenicity by Radioimmunoassay and of Trypsin Inhibitory Activities in Heat or Enzyme Denatured Ovomucoid

The effects of heat treatment and enzymic digestion on the antigenic reactivity of ovomucoid were studied. This reactivity was mainly examined by a solid phase competitive radioimmunoassay. The trypsin inhibitory activity was also measured to elucidate its relationship to the antigenic reactivity. The antigenic reactivity and trypsin inhibitory activity diminished in parallel with increase of heating time. However, enzymic digestion caused a more rapid decrease in antigenic reactivity than in trypsin inhibitory activity. From these results, it is suggested that the structure of the antigenic determinants is destroyed in a similar manner to that of the trypsin inhibitory active site when ovomucoid is heated, while the former structure is destroyed more easily than the latter by enzymic digestion.     

Effect of soybean trypsin inhibitor and chicken ovomucoid on the immunogenic and sensitizing properties of various dietary proteins

Adult male guinea pigs were sensitized by intragastric administration of bovine serum albumin (BSA) and chick ovalbumin (OA) and their mixtures with soybean Kunitz trypsin inhibitor (SBTI) and chick ovomucoid (OM). Sensitization of the animals was evaluated by the anaphylactic shock reaction and also by the levels of serum specific IgG antibodies against BSA and OA as measured in the solid phase radioimmunoassay. The experiment revealed pronounced desensitizing properties of SBTI combined both with OA and BSA. OM produced no effect on the animal sensitization caused by OA and enhanced the BSA-induced sensitization. The results obtained demonstrate the necessity of differential approach to the evaluation of the action of varying trypsin inhibitors on food sensitization.     

Digestive function of the small intestine and pancreas in rats on artificial feeding

Full-value diets of similar composition were given to male rats weighing 207-230 g, by intravenous (group 1) or intragastric (group 2) routes. The proportion of amino acids, fats and carbohydrates was 9.9:15.7:74.4 (with regard to their calorific value). The diet calorific value comprised 60.6 kcal/rat/day. An average mass increase in group 1 was 2.44 +/- 0.14 g/day, in group 2 - 1.75 +/- 0.11 g/day. The protein content and activities of alpha- and gamma-amylase, invertase, maltase, and glycil-L-leucine dipeptidase were assayed in the intestinal mucosa of the proximal portion of the small intestine in group 1 rats, while a decreased alpha-amylase activity in the distal portion of the small intestine was recorded in the animals of group 2. The mass of the pancreas in the rats of group 1 and 2 was authentically lower than in the control rats which received oral feeding with natural foods. The lowest mass of the pancreas was observed in the rats of group 1. Specific activity of trypsin, lipase and RNase in the pancreatic tissues of rats in groups 1 and 2 was similar. The results of the study have evidenced a lowered function of the digestive system under conditions of artificial feeding, especially in case of intravenous nutrition.     

Preservation of the enzyme activity of horseradish peroxidase administered into the gastrointestinal tract of adult dogs and rats

Enzymatic activity of horse radish peroxidase administered to the gastrointestinal tract of adult dogs and rats was assessed. It was discovered that in adult dogs, 30% of the protein reached the distal parts of the small intestine in a non-splitted form. As early as two hours after administering horse radish peroxidase over 15% of active enzyme was detectable in the gastrointestinal tract of adult rats. 0.5% of non-splitted enzyme was found in the rat large intestine.     

Dietary protein as a modulator of the activity of enzymes metabolizing xenobiotics. UDP glucuronosyltransferase and glutathione transferase

Activity of microsomal UDP-glucuronosyl transferase and cytosol glutathione transferase was studied in the liver and small intestine mucosa of rats which received rations with 5.18 and 33% of protein, during 2 months. Activity of UDP-glucuronosyl transferase GT1-isoform both in native and in detergent-activated microsomes of the liver increased with protein deficiency in the ration, while GT2-isoform activity and that of three glutathione transferase forms decreased. Excess of protein in the ration led to a growth of conjugation enzyme activity in the liver. The food protein level influenced the degree of GT1 and GT2 induction by benzyl but it did not change the capacity for induction of glutathione transferase. Activity of conjugation enzymes in the small intestine mucosa of rats to a lesser degree depended on the protein level in the ration.     

Effect of dietary sub-lethal doses of lima bean lectin on relative organ weights, pancreatic and intestinal trypsin (EC 3.4.21.4) and chymotrypsin (EC 3.4.21.1) in the rat

The dietary implications of feeding sub-lethal doses of extracted and purified lectin from lima bean were assessed in weanling rats using changes in relative organ weights, pancreatic and intestinal trypsin and chymotrypsin activities as the response indices. Liver weights decreased significantly (p less than 0.05) while the heart showed a slight but non-significant increase in response to dietary lectin levels. The kidneys, pancreas and spleen were not significantly affected by dietary lectin. Although the activities of the pancreatic enzymes tended, for the most part, to decrease with increasing dietary lectin, such decreases were not significant when compared with the control. Intestinal trypsin and chymotrypsin activities were significantly (p less than 0.05) decreased in the small intestine while the activity values in both the large intestine and caecum were relatively unaffected. Activities of both enzymes showed significant (p less than 0.05) negative quadratic relationship with dietary lectin levels in the small intestine as judged by the magnitude of the R2, coefficients of multiple determination, of 0.77 and 0.76 for trypsin and chymotrypsin respectively.     

Comparison of the resistance during proteolysis of the antigenic structure of bovine serum albumin, chicken ovalbumin and their carbohydrate-modified forms

A study was made of the effect of Maillard reaction between epsilon-amino groups of proteins and sugar carbonyl groups on the resistance of protein antigens (AG) to proteolysis in model systems and in the course of digestion and absorption in the gastrointestinal tract of adult rats. It was found that in vitro pepsin and trypsin inactivate antigenic determinants of bovine serum albumin (BSA) modified by carbohydrate less readily than those of basic protein. At the same time in the course of studying the cleavage and absorption of 3H-proteins and their glucose derivatives in the rat body, lack of a significant stabilization of modified BSA and ovalbumin as compared with non-modified ones was discovered. The discrepancy of the data derived in vitro and in vivo is likely to be related to the inadequacy of the available enzymatic models with regard to real conditions of digestion in the gastrointestinal tract.     

Preparation of trypsin‐immobilised chitosan beads and their application to the purification of soybean trypsin inhibitor

BACKGROUND: Trypsin inhibitors are among the most important antinutritional factors in legumes. Recent research has shown that soybean trypsin inhibitor (SBTI) exhibits multiple bioactivities, but very few studies on the purification of SBTI are available. Enzymes are commonly used as biospecific ligands in affinity purification of their substrates or inhibitors. The aim of the present study was to prepare trypsin (EC 3.4.21.4)‐immobilised chitosan beads and use them to purify trypsin inhibitor from soybean whey. RESULTS: Compared with free trypsin, the immobilised trypsin had higher thermal and pH stability. The adsorption ratio of SBTI from crude SBTI aqueous solution by trypsin‐immobilised chitosan beads was 33.3%. The purified SBTI obtained by affinity chromatography was characterised by sodium dodecyl sulfate polyacrylamide gel electrophoresis as a single polypeptide band with an M_r of 8.3 kDa belonging to the Bowman–Birk family. CONCLUSION: Trypsin‐immobilised chitosan beads were effectively used in the affinity separation of trypsin inhibitor from soybean seeds, thus indicating that immobilised trypsin may have practical application in the soybean‐processing industry. The results of this study provide a background for further investigation of potential applications of soybean bioactive constituents in the areas of agriculture and food. Copyright © 2008 Society of Chemical Industry     

Breakdown of the soluble soybean antigen in the digestive tract of adult rats

Adult male Wistar rats received intragastrically a protein mixture containing 3H-labeled reserve protein of soy beans (PSB), the soy protein isolate 500-E of Ralston Purina Co (USA) being chosen as a protein source. Immunoreactive and TCA-precipitable 3H-PSB was determined in the stomach and small bowel content, 20 and 60 minutes after the administration. The experiment showed more complete inactivation of soy antigen as compared to chick ovalbumin (the authors' data), bovine serum albumin, chick ovomucoid and Kunitz trypsin inhibitor (the literature data). Basing on the results obtained, it is suggested that the soy reserve protein possesses low allergenic properties.     

Influence of an inhibitor of monoaminooxidase (nialamide) on the absorption and retention of nitrogen in adult rats

A study was made on adult rats (170 g) of the effect of nialamide (20 mg/100 g of diet), an inhibitor of monoaminooxidase, administered for 15 or 30 days (short or long-term, respectively), with food intake controlled (pair fed), on the absorption and retention of nitrogen, and on the nitrogen content in the liver, the gastronemio and longissimus dorsi muscles and in the small intestine. Nialamide administered for a time which can be considered prolonged (30 days) negatively affects absorption of alimentary protein, while metabolic utilization is reduced when the drug is administered both in the short and the long term. Proteic catabolism is less marked in male rats, due to the hormonal anabolizant situation in this sex. Nitrogen content under the effect of nialamide showed no significant variation in the gastronemio muscle or in the small intestine. There is a clear reduction in the longissimus dorsi muscle and an increase in the liver of those animals of both sexes treated with the drug, with the exception of male rats given nialamide in the long term.     

Protease inhibitors and lectins in soya beans and effects of aqueous heat-treatment

Protease inhibitor and lectin concentrations and N and lipid levels were evaluated in 27 soya bean samples. All contained trypsin and chymotrypsin inhibiting activity and lectin. Shinano midori, Shinano wasediro, Shinano-oomame and Tsuronoku had high protein contents and average lipid levels, and their lectin and trypsin and chymotrypsin inhibitory activities were not unduly high. These lines, in particular, merit further nutritional evaluation. Food intake, feed conversion efficiency and growth by rats was reduced when soya bean was included in their diet, but the extent to which these parameters were affected did not appear to depend only on the protease inhibitor or lectin content. In contrast, pancreatic and small intestinal growth did seem to reflect the levels of these factors in the diet. Chymotrypsin inhibitor activity in soya bean was more readily abolished by heat-treatment than was trypsin inhibitory activity. Lectin activity was also relatively heat-resistant. However, all of these activities could be abolished by aqueous heat-treatment of fully imbibed seeds at 100°C for 10 min. The effects of a soya diet on pancreas and small intestine weights were also abolished by this pre-treatment, and food intake and nutrient utilisation by rats was greatly improved. © 1998 Society of Chemical Industry     

Organ distribution of bovine serum albumin administered intragastrically in the rat

Radioimmunoassay was employed to examine distribution of antigenic structures of bovine serum albumin (BSA), absorbed by the gastrointestinal tract, in the blood serum and organs of intact rats. It was shown that 3 h after administering 3H-BSA an appreciable amount of its antigenic structures could be identified in the blood serum, liver, spleen, and carcass of the animals. The total amount of antigenic determinants of BSA which got into the internal environment of the body from the intestine amounted to about 0.2% of the dose administered. The highest specific content of antigenic structures of BSA supplied via the intestinal barrier was detected in the spleen.     

小麦肽对大鼠氮代谢以及胃肠黏膜结构和功能的影响

目的：观察灌胃小麦肽对于大鼠氮代谢、胃肠形态和小肠黏膜酶活的影响。方法：SD大鼠50只,随机分为空白对照组及低、中、高剂量小麦肽组(灌胃剂量分别为20、100、500mg/(kg·d))和小麦蛋白组20mg/(kg·d)),每天灌胃1次,连续灌胃30d。分别于第10、20、30天用代谢笼采集大鼠24h粪、尿样品。灌胃30d后断颈处死大鼠,取血清、胃和小肠黏膜,观察氮代谢指标、血清生化指标、胃肠内表面扫描电镜、小肠黏膜酶活等的变化。结果：灌胃30d后,低剂量小麦肽能显著提高大鼠蛋白质消化率、氮沉积、净蛋白质利用率(P＜0.05);低、中剂量小麦肽均能显著增加大鼠蛋白质生物学价值(P＜0.05);低、中、高剂量小麦肽均能显著增加大鼠血清总蛋白含量(P＜0.05);低、中剂量小麦肽大鼠胃肠道上皮细胞较空白对照组饱满、连接紧密,排列整齐有序;低、中、高剂量小麦肽均能显著上调大鼠小肠黏膜氨基肽酶活力(P＜0.05);中、高剂量小麦肽均能上调大鼠小肠黏膜Na+-K+-ATP酶活力(P＜0.05)。结论：一定剂量的小麦肽能提高大鼠对于蛋白质的吸收与利用,其可能的途径是：促进胃肠道上皮细胞的生长;小肠黏膜氨基肽酶、Na+-K+-ATP酶活力的上调。     

Lactose--a potential dietary fiber. The regulation of its microecological effect in the intestinal tract. 2. The nutrient effect of lactose]

In the small intestine lactose is subjected to the hydrolytic impact of beta-galactosidase originating mainly from the mucosa. In rats about two thirds of the enzyme activity are located in the first part of the small intestine, and one third in the second one. A part of the mucosal enzyme does not remain in the mucosa. It becomes detached and can be determined in the chymus. The ratio of the transient to the resident proportion amounts to 1.8: 1 in germfree and 0.23: 1 in conventional rats. Bacterial settlement causes an increase in the mucosal mass resulting in higher total activity whereas the specific activity of the mucosal enzyme remains unchanged. Microorganisms occurring close to the small intestine mucosa take part in lactose degradation. Lactose-containing diet leads to an increase in both the bacterial and the mucosal activity, the latter one to varying degrees. Lactose concentration in the ileal chymus rises with increasing intake of lactose and decreasing protein and phosphate intake. Following a saturation kinetics the velocity of lactose hydrolysis is correlated with the lactose concentration of the diet. alpha-lactose is hydrolysed more rapidly in the small intestine of both human sucklings and rats than beta-lactose. As the results of a mathematical model show lactose mutarotation does not effect on the degree of lactose degradation in the small intestine. Depending on the intake of lactose and the food composition the rate of lactose hydrolysis amounts to: --max. 50% after small intestine perfusion in human sucklings, --max. 80% after small intestine perfusion in rats, --max. 60% in rats with ileostomata.     

Soybean trypsin inhibitor neutralization of its inhibitory effect upon the casein precipitating reaction of trypsin by rabbit antiserum

Summary Antiserum produced against Soybean Trypsin Inhibitor (SBTI) was shown to neutralize the SBTI inhibition of the Casein precipitating activity of trypsin thus indicating that the precipitating interactions observed in agar gel double diffusion analysis were at least partly due to reaction between SBTI and specific antibody.On this basis the use of antiserum for the detection of SBTI in various soy proteins is discussed.

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Zusammenfassung Es konnte gezeigt werden, daß Antiserum, welches gegen die Soja-Trypsin-Hemmer hergestellt worden war, die SBTI Hemmung der caseinfällenden Trypsinaktivität neutralisieren kann; daraus wird gefolgert, daß die bei der Ausfällung mit dem Agar-Gel-Diffusions-test beobachteten Reaktionen zumindest teilweise auf eine Reaktion zwischen SBTI und den spezifischen Antikörpern beruhen.Aufgrund dieser Ergebnisse wird die Anwendung von Antiserum zur Bestimmung von SBTI in verschiedenen Soja-Proteinen beschrieben.

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This work was supported by a grant from the Agricultural Research Council of Norway.     

Influence of medium chain triglycerides and vitamin D3 on digestive and metabolic utilization of protein in rats with intestinal resection

The effects of different types of diet and resecting 50% of the distal small intestine on the digestive and metabolic utilization of protein were studied in resected rats and sham-operated controls. Nutritional parameters were analyzed at 1 and 3 months postsurgery. Intestinal resection led to a deterioration in digestive utilization of protein, which improved with time (3 months postsurgery). A qualitative change in the dietary fat source from 100% olive oil to equal parts of medium chain triglycerides, sunflower seed oil and olive oil and this same diet supplemented with vitamin D3 favoured digestive utilization of protein as recorded 1 month after surgery. However, the beneficial effects were maintained at 3 months only in resected rats given dietary fat in the form of 100% olive oil.     

Digestion and absorption of protein along ovine gastrointestinal tract

Digestion and absorption of protein were determined in ovine gastrointestinal tract with cerium-141 as an unabsorbed reference substance. Nitrogen flows changed little in rumen and reticulum, but in the proximal small intestine flows increased because of secretion of .9 g nitrogen per day per kg body weight. This secretion included trypsin, chymotrypsin, elastase, and carboxypeptidases A and B; maximal activity was in proximal segments of the small intestine and decreased with distance from the pylorus. Activity of chymotrypsin decreased more rapidly than that of trypsin. Amino acid flows reflected the influx of protein in the duodenum; absorption was approximately 55% in the terminal ileum. No major changes of proportions of individual amino acids were observed. Overall nitrogen absorption was 72.6% of which 6% was in the large intestine. The major soluble protein fraction in the gastrointestinal tract consisted of peptides with molecular weight 7,000 to 14,000 daltons. Soluble high molecular weight protein was observed only in rumen and duodenum. Low molecular weight peptides and amino acids accumulated only in the proximal small intestine. Solubilization of protein and breakdown of peptides of 7,000 to 14,000 molecular weight appear to be rate limiting for protein absorption in sheep.     

Qualitative and quantitative determination of proteolysis in mastitic milks

Proteolytic activity in mastitic skim-milk was often 5-10 fold higher than in normal milk, its level being related to somatic cell count but not precisely correlated with it. In milks with the highest levels of activity plasmin accounted for about one third of the total proteinase. A further third was sedimented with the micellar fraction together with the plasmin, but unlike plasmin, was not inhibited by addition of soyabean trypsin inhibitor (SBTI). The final third remained in the serum phase. Polyacrylamide gel electrophoresis (PAGE) showed that alpha-sl- and beta-caseins were degraded at about the same overall rate. The plasmin produced the usual readily identified fragments from beta-casein, but incubation of mastitic milk also produced changes in patterns in the gamma-casein region differing from plasmin-induced changes, which were also apparent when the micellar fraction was incubated. As they were inhibited by SBTI, a second trypsin-like enzyme in addition to plasmin may also have been present. Other proteinase(s) not inhibited by SBTI was also associated with casein micelles and produced at least 3 characteristic protein fragments seen on PAGE. The serum phase proteinase(s) was likewise not inhibited by SBTI, and did not produce any well-defined electrophoretic bands, suggesting a rather non-specific breakdown of caseins. After separation of mastitic whole milk, a considerable proportion of the proteolytic activity was found in the cream phase. The proportion was enhanced by freezing and thawing, and the enzyme appeared to be identical to the SBTI-resistant micellar proteinase. Because of the considerable proteolysis likely to occur under the time and temperature conditions involved, our results may provide some explanation for the problems encountered in cheesemaking with mastitic milks (e.g. yield losses, poor curd strength and off-flavour development).