Herein,a novel method for fl uorometric detection of soybean trypsin inhibitor(SBTI)activity based on a water-soluble poly(diphenylacetylene)derivative was reported.Fluorescence quenching of the polymer via p-nitroani...Herein,a novel method for fl uorometric detection of soybean trypsin inhibitor(SBTI)activity based on a water-soluble poly(diphenylacetylene)derivative was reported.Fluorescence quenching of the polymer via p-nitroaniline,produced from the trypsin-catalyzed decomposition of N-benzoyl-DL-arginine-4-nitroanilide hydrochloride(L-BAPA),was well described using the Stern-Volmer equation.SBTI activity was quantitatively assessed based on changes in the fl uorescence intensity of the polymer.This strategy has several advantages,such as high sensitivity and ease of operation.Moreover,its applicability to other biochemical analyses is promising.展开更多
By 30% - 60% (NH4)(2)SO4 fractional precipitation, anion-exchange chromatography on DEAE-Sepharose CL-6B, gel filtration on Sephacryl S-200 and anion-exchange chromatography on Waters AP-1 column (Protein(PM)-Pak DEAE...By 30% - 60% (NH4)(2)SO4 fractional precipitation, anion-exchange chromatography on DEAE-Sepharose CL-6B, gel filtration on Sephacryl S-200 and anion-exchange chromatography on Waters AP-1 column (Protein(PM)-Pak DEAE 15HR), a proteinase which can inactivate soybean trypsin inhibitor (STI) was purified from mung bean ( Vigna rabiata (L.) Wilezek) sprouts. Its molecular weight was estimated to be 29.8 kD by SDS-PAGE, and its K-m and V-max for STI were 769.2 N-alpha -benzoyl-L-arginine ethyl ester BAEE/mL and 115.3 BAEE . mL(-1) . min(-1) respectively. This proteinase was stable at temperatures lower than 50 degreesC and pH 6.5 - 8.5, and 90.91% STI activity of defatted soybean powder was inactivated by this preparation, with proteolytic activity 5 000 BAEE/mL at 50 degreesC and pH 8.0 in 4 h.展开更多
The aim of the present study was to optimize trypsin inhibitor degradation in soybean meal by solid-state fermentation （SSF） with Lactobacillus brevis and Aspergillus oryzae, and to determine the effect of SSF on ph...The aim of the present study was to optimize trypsin inhibitor degradation in soybean meal by solid-state fermentation （SSF） with Lactobacillus brevis and Aspergillus oryzae, and to determine the effect of SSF on phytic acid, crude protein, crude fat, and amino acid profile. Response surface methodology （RSM） with Box-Behnken design was used to optimize SSF. The optimal conditions derived from RSM for L. brevis fermentation were： pH=5. 1; inoculum size=10%; duration=72 h; substrate to water ratio=1.5. The minimum content of trypsin inhibitors was 6.4 mg g^-1 dry matter. The optimal conditions derived from RSM for A. oryzae fermentation were： substrate to water ratio= 0.8 1; inoculum size=4%; duration=120 h. The minimum content of trypsin inhibitors was 1.6 mg g^-1 dry matter. Both L. brevis and A. oryzae decreased trypsin inhibitors dramatically （57.1 and 89.2% respectively）. L. brevis fermentation did not affect phytic acid （0.4%） and crude fat （5.2%） considerably, whereas A. oryzae fermentation degraded phytic acid （34.8%） and crude fat （22.0%） contents to a certain extent. Crude protein content was increased after both fermentation （6.4 and 12.9% for L. brevis and A. oryzae respectively）. Urease activity was reduced greatly （83.3 and 58.3% for L. brevis and A. oryzae respectively）. In conclusion, SSF with A. oryzae and L. brevis reduced trypsin inhibitor content and modified major macronutrients in soybean meal.展开更多
Aberrant functioning of serine proteases in inflammatory and carcinogenic processes within the gastrointestinal tract(GIT)has prompted scientists to investigate the potential of serine protease inhibitors,both natural...Aberrant functioning of serine proteases in inflammatory and carcinogenic processes within the gastrointestinal tract(GIT)has prompted scientists to investigate the potential of serine protease inhibitors,both natural and synthetic,as modulators of their proteolytic activities.Protease inhibitors of the Bowman-Birk type,a major protease inhibitor family in legume seeds,which inhibit potently and specifically trypsin-and chymotrypsin-like proteases,are currently being investigated as colorectal chemopreventive agents.Physiologically relevant amounts of Bowman-Birk inhibitors(BBI)can reach the large intestine in active form due to their extraordinary resistance to extreme conditions within the GIT.Studies in animal models have proven that dietary BBI from several legume sources,including soybean,pea,lentil and chickpea,can prevent or suppress carcinogenic and inflammatory processes within the GIT.Although the therapeutic targets and the action mechanism of BBI have not yet been elucidated,the emerging evidence suggests that BBI exert their preventive properties via protease inhibition;in this sense,serine proteases should be considered as primary targets in early stages of carcinogenesis.The validation of candidate serine proteases as therapeutic targets together with the identification,within the wide array of natural BBI variants,of the most potent and specific protease inhibitors,are necessary to better understand the potential of this protein family as colorectal chemopreventive agents.展开更多
Hebei Province is one of the main distribution areas growing wild soybean( Glycine soja ) in China. In this study, 461 seed samples,collected from 18 natural populations in this province, were used to electrophoretica...Hebei Province is one of the main distribution areas growing wild soybean( Glycine soja ) in China. In this study, 461 seed samples,collected from 18 natural populations in this province, were used to electrophoretically observe the change in forms and their frequencies of the Kunitz trypsin inhibitor protein (KTI)in individual populations and geographical areas. Allelic frequencies accounted for 85% for Tia and 15% for Tib in the total samples. Twelve populations examined were polymorphic at the KTI locus, accounting for over 50% in the populations investigated. Four populations, 22% of all the populations, were found to have natural cross-pollination with varied heterozygote rates of 3% -5.5%, and the average was 1% in the total sampies. Geographically, the mean Tib frequency in the north areas was higher than in the south, and higher in the mountainous area than in the plain areas. The populations in a lake ecological environment (Baiyangdian Lake) were almost monomorphic. No obvious relationship between the frequency and the geographical distance was observed. In addition, we first found a mutation for the absence of the KTI in wild soybean.展开更多
A significant reduction of trypsin inhibitory activity by selected bacterial proteolytic enzymes was demonstrated in vitro.Two trials were conducted to examine the capacity of the tested enzymes to inactivate soybean ...A significant reduction of trypsin inhibitory activity by selected bacterial proteolytic enzymes was demonstrated in vitro.Two trials were conducted to examine the capacity of the tested enzymes to inactivate soybean ANFs in vivo.In trial I,twenty four piglets weaned at four weeks of age were assigned in replicate groups of 4 piglets per pen to one of three dietary treatments:(1)control;(2)Enzyme 1 supplemented(E 1);(3)Enzyme 2 supplemented (E 2).In trial II,twenty piglets weaned at five weeks of age were alloted to five treatment diets:(1)contro,l:(2)0.1% P4 supplemented;(3)0.5% P4 supplemented;(4)0.1% P7 supplemented;(5)0.5% P7 supplemented.The optimum pH for hydrolysis was 8 for E ,9 11 for E 2,8.5 for P4 and nuctral for P7.After 17 days of the trial,daily gain of piglets on enzymes E 1 and E 2 was 36% and 18% more than that in the control group,although the difference was not significant.The animals on the treated groups had a tendency to have lighter heart(7.8 and 5.9%),spleen(11.1 and 7.4%) and pancreas(16.7 and 12.5% for E 1 and E 2 respectively)in relation to empty body weight than those in the control.The small intestine of pigs on the treated groups was significantly lighter(18.9 for E 1 and 7.7% for E 2) than that in the control(P<0.05).The stomach (26.4 and 24%,p=0.198) and cecum(21.9 and 9.4%,p=0.114) also showed the same pattern.The growth depression was attributed to reduced feed intake caused by antinutritional factors in soybeans.It is concluded that supplements of proteolytic enzymes E 1 or E 2 had a positive effect on growth and efficiency and caused much less reaction in the gut as manifested by the weight of the tract and of its accessory organs.Dietary saupplements of P4 or P7 had no significant effect on growth,but reduced reaction of soybean antinutritional factors in the gut,especialy P4 in dose of 0.5%.The growth depression was attributed to low feed intake caused by antinutritional factors in soybeans.展开更多
Trypsin inhibitors have been found in various animals, plants and microorganisms.There were two types of trypsin inhibitors in soybean including Bowman-Birk protease inhibitors(BBI) and Kunitz in-hibitors(KTI).The dif...Trypsin inhibitors have been found in various animals, plants and microorganisms.There were two types of trypsin inhibitors in soybean including Bowman-Birk protease inhibitors(BBI) and Kunitz in-hibitors(KTI).The different BBI genes from wild soybean(G.soja) and cultivated soybean(G.max) formed a multigene family.We constructed a cDNA library of cultivar 'SuiNong 14' seed at the R7 growth stage using the SMART Kit.Seventeen contigs or singletons were highly homologous to soy-bean protease inhibitors.Contigs of 5, 35, 8 and 9 were highly homologous to BBI family members BBI-A1, BBI-A2, BBI-C and BBI-D, respectively.Sequence analyses showed there were novel allelic varia-tions among the 4 BBI members in SuiNong 14.Based on the comparison of soybean seed cDNA li-braries from different developmental stages, it was apparent that the expression of trypsin inhibitors increased during seed development in soybean.Phylogenetic analysis of BBI gene sequences among dicotyledonous and monocotyledonous plants demonstrated that these genes shared a common pro-genitor.展开更多
基金The authors appreciate the support from the Zhe-jiang Province Lingyan Key R&D Project(No.2022C01177)the Zhejiang Administration for Market Regulation Eyas Program Cultiva-tion Project(No.CY2022355).
文摘Herein,a novel method for fl uorometric detection of soybean trypsin inhibitor(SBTI)activity based on a water-soluble poly(diphenylacetylene)derivative was reported.Fluorescence quenching of the polymer via p-nitroaniline,produced from the trypsin-catalyzed decomposition of N-benzoyl-DL-arginine-4-nitroanilide hydrochloride(L-BAPA),was well described using the Stern-Volmer equation.SBTI activity was quantitatively assessed based on changes in the fl uorescence intensity of the polymer.This strategy has several advantages,such as high sensitivity and ease of operation.Moreover,its applicability to other biochemical analyses is promising.
文摘By 30% - 60% (NH4)(2)SO4 fractional precipitation, anion-exchange chromatography on DEAE-Sepharose CL-6B, gel filtration on Sephacryl S-200 and anion-exchange chromatography on Waters AP-1 column (Protein(PM)-Pak DEAE 15HR), a proteinase which can inactivate soybean trypsin inhibitor (STI) was purified from mung bean ( Vigna rabiata (L.) Wilezek) sprouts. Its molecular weight was estimated to be 29.8 kD by SDS-PAGE, and its K-m and V-max for STI were 769.2 N-alpha -benzoyl-L-arginine ethyl ester BAEE/mL and 115.3 BAEE . mL(-1) . min(-1) respectively. This proteinase was stable at temperatures lower than 50 degreesC and pH 6.5 - 8.5, and 90.91% STI activity of defatted soybean powder was inactivated by this preparation, with proteolytic activity 5 000 BAEE/mL at 50 degreesC and pH 8.0 in 4 h.
基金supported by a research project of the Science and Technology Key Group in Zhejiang Provincethe research projects from the Science and Technology Department of Zhejiang Province,China (2009C12068)
文摘The aim of the present study was to optimize trypsin inhibitor degradation in soybean meal by solid-state fermentation （SSF） with Lactobacillus brevis and Aspergillus oryzae, and to determine the effect of SSF on phytic acid, crude protein, crude fat, and amino acid profile. Response surface methodology （RSM） with Box-Behnken design was used to optimize SSF. The optimal conditions derived from RSM for L. brevis fermentation were： pH=5. 1; inoculum size=10%; duration=72 h; substrate to water ratio=1.5. The minimum content of trypsin inhibitors was 6.4 mg g^-1 dry matter. The optimal conditions derived from RSM for A. oryzae fermentation were： substrate to water ratio= 0.8 1; inoculum size=4%; duration=120 h. The minimum content of trypsin inhibitors was 1.6 mg g^-1 dry matter. Both L. brevis and A. oryzae decreased trypsin inhibitors dramatically （57.1 and 89.2% respectively）. L. brevis fermentation did not affect phytic acid （0.4%） and crude fat （5.2%） considerably, whereas A. oryzae fermentation degraded phytic acid （34.8%） and crude fat （22.0%） contents to a certain extent. Crude protein content was increased after both fermentation （6.4 and 12.9% for L. brevis and A. oryzae respectively）. Urease activity was reduced greatly （83.3 and 58.3% for L. brevis and A. oryzae respectively）. In conclusion, SSF with A. oryzae and L. brevis reduced trypsin inhibitor content and modified major macronutrients in soybean meal.
基金Supported by ERDF-co-financed grant from the Spanish CICYT,No.AGL2011-26353 to Clemente AClemente A involved in COST Action FA1005 INFOGEST on Food Digestion
文摘Aberrant functioning of serine proteases in inflammatory and carcinogenic processes within the gastrointestinal tract(GIT)has prompted scientists to investigate the potential of serine protease inhibitors,both natural and synthetic,as modulators of their proteolytic activities.Protease inhibitors of the Bowman-Birk type,a major protease inhibitor family in legume seeds,which inhibit potently and specifically trypsin-and chymotrypsin-like proteases,are currently being investigated as colorectal chemopreventive agents.Physiologically relevant amounts of Bowman-Birk inhibitors(BBI)can reach the large intestine in active form due to their extraordinary resistance to extreme conditions within the GIT.Studies in animal models have proven that dietary BBI from several legume sources,including soybean,pea,lentil and chickpea,can prevent or suppress carcinogenic and inflammatory processes within the GIT.Although the therapeutic targets and the action mechanism of BBI have not yet been elucidated,the emerging evidence suggests that BBI exert their preventive properties via protease inhibition;in this sense,serine proteases should be considered as primary targets in early stages of carcinogenesis.The validation of candidate serine proteases as therapeutic targets together with the identification,within the wide array of natural BBI variants,of the most potent and specific protease inhibitors,are necessary to better understand the potential of this protein family as colorectal chemopreventive agents.
文摘Hebei Province is one of the main distribution areas growing wild soybean( Glycine soja ) in China. In this study, 461 seed samples,collected from 18 natural populations in this province, were used to electrophoretically observe the change in forms and their frequencies of the Kunitz trypsin inhibitor protein (KTI)in individual populations and geographical areas. Allelic frequencies accounted for 85% for Tia and 15% for Tib in the total samples. Twelve populations examined were polymorphic at the KTI locus, accounting for over 50% in the populations investigated. Four populations, 22% of all the populations, were found to have natural cross-pollination with varied heterozygote rates of 3% -5.5%, and the average was 1% in the total sampies. Geographically, the mean Tib frequency in the north areas was higher than in the south, and higher in the mountainous area than in the plain areas. The populations in a lake ecological environment (Baiyangdian Lake) were almost monomorphic. No obvious relationship between the frequency and the geographical distance was observed. In addition, we first found a mutation for the absence of the KTI in wild soybean.
文摘A significant reduction of trypsin inhibitory activity by selected bacterial proteolytic enzymes was demonstrated in vitro.Two trials were conducted to examine the capacity of the tested enzymes to inactivate soybean ANFs in vivo.In trial I,twenty four piglets weaned at four weeks of age were assigned in replicate groups of 4 piglets per pen to one of three dietary treatments:(1)control;(2)Enzyme 1 supplemented(E 1);(3)Enzyme 2 supplemented (E 2).In trial II,twenty piglets weaned at five weeks of age were alloted to five treatment diets:(1)contro,l:(2)0.1% P4 supplemented;(3)0.5% P4 supplemented;(4)0.1% P7 supplemented;(5)0.5% P7 supplemented.The optimum pH for hydrolysis was 8 for E ,9 11 for E 2,8.5 for P4 and nuctral for P7.After 17 days of the trial,daily gain of piglets on enzymes E 1 and E 2 was 36% and 18% more than that in the control group,although the difference was not significant.The animals on the treated groups had a tendency to have lighter heart(7.8 and 5.9%),spleen(11.1 and 7.4%) and pancreas(16.7 and 12.5% for E 1 and E 2 respectively)in relation to empty body weight than those in the control.The small intestine of pigs on the treated groups was significantly lighter(18.9 for E 1 and 7.7% for E 2) than that in the control(P<0.05).The stomach (26.4 and 24%,p=0.198) and cecum(21.9 and 9.4%,p=0.114) also showed the same pattern.The growth depression was attributed to reduced feed intake caused by antinutritional factors in soybeans.It is concluded that supplements of proteolytic enzymes E 1 or E 2 had a positive effect on growth and efficiency and caused much less reaction in the gut as manifested by the weight of the tract and of its accessory organs.Dietary saupplements of P4 or P7 had no significant effect on growth,but reduced reaction of soybean antinutritional factors in the gut,especialy P4 in dose of 0.5%.The growth depression was attributed to low feed intake caused by antinutritional factors in soybeans.
基金the "863" Project of National High Technology Research and Devel-opment Program of China (Grant No. 2006AA100104 and 2006AA10A110)Na-tional Natural Science Foundation of China (Grant No. 30490251)+1 种基金National Key Technologies R&D Program in the 11th Five-Year Plan (Grant No. 2006BAD13B05)Basic Research Funding of the Institute of Crop Science
文摘Trypsin inhibitors have been found in various animals, plants and microorganisms.There were two types of trypsin inhibitors in soybean including Bowman-Birk protease inhibitors(BBI) and Kunitz in-hibitors(KTI).The different BBI genes from wild soybean(G.soja) and cultivated soybean(G.max) formed a multigene family.We constructed a cDNA library of cultivar 'SuiNong 14' seed at the R7 growth stage using the SMART Kit.Seventeen contigs or singletons were highly homologous to soy-bean protease inhibitors.Contigs of 5, 35, 8 and 9 were highly homologous to BBI family members BBI-A1, BBI-A2, BBI-C and BBI-D, respectively.Sequence analyses showed there were novel allelic varia-tions among the 4 BBI members in SuiNong 14.Based on the comparison of soybean seed cDNA li-braries from different developmental stages, it was apparent that the expression of trypsin inhibitors increased during seed development in soybean.Phylogenetic analysis of BBI gene sequences among dicotyledonous and monocotyledonous plants demonstrated that these genes shared a common pro-genitor.
