• KOREAN JOURNAL OF CROP SCIENCE
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• v.57 no.1
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• pp.78-82
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• 2012

Soybean [$Glycine$ $max$ (L.) Merr.] protein is a high quality source for food and feed. But, antinutritional factors in the raw mature soybean are exist. Kunitz trypsin inhibitor (KTI) protein is a main antinutritional factor in soybean seed. Also, P34 protein, referred as $Gly$ $m$ Bd 30K, has been identified as a predominant immunodominant allergen. Genetic relationship between KTI protein and P34 protein could be useful in soybean breeding program for the genetic elimination or reduction of these factors. The objective of this study was to determine the independent inheritance or linkage between KTI protein and P34 protein in soybean seed. A total of 479 $F_2$ seeds were obtained from the cross of 07B1 and PI567476 parents. KTI protein and relative amount of P34 protein were analysed from $F_2$ seeds harvested from the F1 plants by using SDS-PAGE and Western blot analysis. The segregation ratios of 3 : 1 for KTI protein (353 KTI protein present : 126 KTI protein absent) and relative amount of P34 protein (363 normal amount of P34 protein : 116 low amount of P34 protein). The segregation ratio of 3 : 1 suggested that KTI protein and relative amount of P34 protein in mature soybean seed were controlled by a single major gene. The segregation ratios of 9 : 3 : 3 : 1 (266 KTI protein present, normal amount of P34 protein: 88 KTI protein present, low amount of P34 protein: 102 KTI protein absent, normal amount of P34 protein: 23 KTI protein absent, low amount of P34 protein) and Chi-square value (${\chi}^2$=3.31, P=0.346) were observed in $F_2$ seeds. This data showed that KTI protein was inherited independently with relative amount of P34 protein in soybean. These results will be helpful in breeding program for selecting the line with lacking KTI protein and reduced amount of P34 protein in soybean.

• Journal of Applied Biological Chemistry
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• v.57 no.2
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• pp.183-188
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• 2014

In order to increase the nutritional quality of soybean meal (SBM) as an animal feed, Bacillus subtilis TP6, a previously isolated strain from an Indonesian traditional fermented soybean food, Tempeh, was used as a starter organism for solid-state fermentation. In the pre-treated SBM with water content of 60% (v/w), B. subtilis TP6 was grown to a maximum viable cell number of $3.5{\times}10^9CFU/g$. Compared to control, crude protein in Bacillus fermented SBM was increased by 16%, while raffinose, stachyose, and trypsin inhibitors were reduced by 31, 37, and 90%, respectively. The Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that proteins in the fermented SBM were remarkably hydrolyzed into smaller molecular masses, resulting in a decrease in large sized proteins. Our data suggested that B. subtilis fermentation could increase the nutritive value of SBM through reduction of anti-nutritive factors and improvement of protein quality by hydrolysis of soy protein. In addition, B. subtilis TP6 produced a functional ingredient, poly-${\gamma}$-glutamic acid which has various health benefits.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.24 no.5
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• pp.273-288
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• 1991

To elucidate the physiological and biochemical differences between chondrichthyes and osteichthyes, the properties of the specific digestive enzymes in cat-shark, Cephaloscyllium umbratile, and mackerel, Scomber japonicus, were studied. Homogenous trypsin proved through the disc-electrophoresis, SDS-PAG electrophoresis and gel filtration was obtained from the pancreas of cat-shark by $50-70\%$ saturated ammonium sulphate fractionation, DEAE-Sephadex A-50 column chromatography, benzamidine-Sepharose 6B affinity chromatography and Sephadex G-75-120 gel filtration. Two types of trypsins were also obtained from the pyloric caeca of mackerel by $30-70\%$ saturated ammonium sulphate fractionation and the slightly modified procedure from the method adopted in the purification of cat-shark trypsin. The two trypsins, designated trypsin A and B, were proved their homogeneity by disc- and SDS-PAG electrophoresis and gel filtration. The molecular weights of the trypsins were estimated to be 31,700 for cat-shark trypsin, 30,000 for mackerel trypsin A and 29,000 for mackerel trypsin B by SDS-PAG electrophoresis, but those were estimated to be 21,500 for cat-shark trypsin, 23,700 for mackerel trypsin A and 21,500 for mackerel trypsin B by gel filtration. The trypsins exhibited their optimum conditions at pH 9.0 and on temperature ranged from $45^{\circ}C\;to\;50^{\circ}C$ for cat-shark, and at pH 8.0 and a temperature of $50^{\circ}C$ for mackerel trypsin A and B, respectively. The cat-shark trypsin was stable at pH 10.0 and the temperature below $10^{\circ}C$, whereas the mackerel trypsin A and B, were stable in the range over pH 7.0 to pH 9.0 below $10^{\circ}C$ and at pH 8.0 below $35^{\circ}C$, respectively. The mackerel trypsins were severely inhibited by some heavy metal ions such as $Ag^{2+},\;Cu^{2+}\;and\;Hg^{2+}$ compared to cat-shark trypsin. All of the enzymes were also inhibited by antipain, leupeptin, TLCK(tosyllysine chloromethyl ketone) and SBTI(soybean trypsin inhibitor) remarkably. The inhibitory effects of PMSF(phenylmethane sulphonylfluoride), DFP(diisopropyl fluorophosphate) and benzamidine were indicated that these enzymes belong to serine-proteases.

• Journal of Plant Biotechnology
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• v.47 no.2
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• pp.118-123
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• 2020

The black seed coat of soybeans contain anthocyanins which promote health. However, mature soybean seeds contain anti-nutritional factors like lipoxygenase, lectin and Kunitz Trypsin Inhibitor (KTI) proteins. Furthermore, these seeds can be used only after the genetic elimination of these proteins. Therefore, the objective of this study was to develop novel soybean genotypes with black seed coat and triple recessive alleles (lx1lx1lx2lx2lx3lx3, titilele) for lipoxygenase, lectin, and KTI proteins. From a cross of parent1 (lx1lx2lx3/lx1lx2lx3, ti/ti, Le/Le) and parent2 (lx1lx2lx3/lx1lx2lx3, Ti/Ti, le/le), 132 F₂ seeds were obtained. A 3:1 segregation ratio was observed during F₂ seed generation for the inheritance of lectin and KTI proteins. Between a cross of the Le and Ti genes, the observed independent inheritance ratio in the F₂ seed generation was 9: 3 : 3 : 1 (69 Le_Ti_: 32 leleTi_: 22 Le_titi: 9 leletiti) (χ²=2.87, P=0.5 - 0.1). From nine F₂ seeds with triple recessive alleles (lx1lx1lx2lx2lx3lx3, titilele genotype), one novel strain posessing black seed coat, and free of lipoxygenase, lectin and KTI proteins, was selected. The seed coat color of the new strain was black and the cotyledon color of the mature seed was green. The weight of 100 seeds belonging to the new strain was 35.4 g. This black soybean strain with lx1lx1lx2lx2lx3lx3, titilele genotype is a novel strain free of lipoxygenase, lectin, and KTI proteins.

• The Plant Pathology Journal
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• v.28 no.2
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• pp.197-201
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• 2012

Potyviruses express their RNA genomes through the production of polyproteins that are processed in host cells by three virus-encoded proteases. Soybean plants produce large amounts of protease inhibitors during seed development and in response to wounding that could affect the activities of these proteases. The in vitro activities of two of the proteases of Soybean mosaic virus (SMV) and Tobacco vein mottling virus (TVMV) were compared in the rabbit reticulocyte lysate in vitro translation system using synthetic RNA transcripts. Transcripts produced from SMV and TVMV cDNAs that included the P1 and helper component-protease (HC-Pro) coding regions directed synthesis of protein products that were only partially processed. Unprocessed poly-proteins were not detected from transcripts that included all of the P1, HC-Pro, P3 and portions of the cylindrical inclusion protein coding regions of either virus. Addition of soybean trypsin inhibitor to in vitro translation reactions increased the accumulation of the unprocessed polyprotein from TVMV transcripts, but did not alter the patterns of proteins produced from SMV. These experiments suggest that SMV-and TVMV-encoded proteases are differentially sensitive to protease inhibitors.

• Journal of Life Science
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• v.29 no.2
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• pp.147-151
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• 2019

Soybean [Glycine max (L.) Merr.] is grown worldwide for its high protein and oil content. Anthocyanins from black soybean seed coats are known to have many pharmaceutical effects. Soybean cultivars with large seed sizes and black seed coats are needed by soybean farmers. However, antinutritional factors, like protein, stachyose, and Kunitz trypsin inhibitor (KTI) exist in raw mature soybeans. Genetic elimination or reduction of these components is needed in soybean breeding. The objective of this research was to develop new a soybean strain with black seed coats and green cotyledons that was KTI protein free and low in stachyose. Six parents were used. The presence or absence of KTI protein was detected using the Western blot technique. The content of stachyose in mature seeds was detected using HPLC. One new strain was selected from 11 $F_2$ plants with black seed coats and green cotyledons that lacked KTI protein. The new strain had black seed coats and green cotyledons and was KTI protein free and low in stachyose. The plant height of the new strain was 66 cm, and its 100-seed weight was 28.4 g. The stachyose content of the new strain was 2.59 g/kg. The new strain developed in this research will be used to develop new cultivars that are KTI protein free and low in stachyose.

• Asian-Australasian Journal of Animal Sciences
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• v.30 no.9
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• pp.1303-1313
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• 2017

Objective: This study evaluated the change and function of the pancreas, and small intestine in relation to growth performance of broilers on diets supplemented with raw soybean meal (RSBM) and protease. Samples of test ingredients and diets, after mixing and prior to being used were also assessed on contents of anti-nutritional factors. Methods: A $3{\times}3$ factorial study was used, with three levels of RSBM (commercial soybean meal [SBM] was replaced by RSBM at 0, 10%, or 20%) and protease (0.1, 0.2, or 0.3 g/kg). Each treatment was replicated six times with nine birds per replicate. Birds were housed in cages, in climate-controlled room and fed starter, grower and finisher diets. Results: Levels of trypsin inhibitors in the diets, containing varying levels of RSBM ranged between 1,730.5 and 9,913.2 trypsin inhibitor units/g DM. Neither RSBM nor protease supplementation in diets significantly affected (p>0.05) the body weight of broilers in the entire periods (0 to 35-d). Increasing the level of RSBM in diets increased the weight of the pancreas at d 10 (p<0.000), d 24 (p<0.001), and d 35 (p<0.05). Increasing levels of RSBM in the diets reduced the apparent ileal digestibility of crude protein (CP), and amino acid (AA) at d 24. Increasing level of RSBM in the diets decreased (p<0.01) pancreatic protein content, but this was increased (p<0.05) when protease was added to the diets (0 to 10-d). Increasing the level of protease improved the pancreatic digestive enzymes, including trypsin (p<0.05), chymotrypsin (p<0.01), and general proteolytic enzymes (p<0.05). Conclusion: The commercial SBM could be replaced at up to 20% by RSBM for broilers. Although protease supplementation slightly improved the digestive enzymes, and the ileal digestibilities of CP and AA, the CP and AA were negatively affected by increasing RSBM.

• Food Science and Biotechnology
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• v.14 no.6
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• pp.732-737
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• 2005

The combinational effect of gamma irradiation and moist heating on the trypsin inhibitor activity (TIA) in soaked and dried soybeans was evaluated by measuring the inhibition using N-benzoyl-DL-arginine-p-nitroanilide (BAPNA) as substrate. Gamma irradiation significantly decreased the TIA level in soybean at doses above 5 kGy, and the $ID_{50}$ (the gamma irradiation dose required to reach 50% inhibition) value for TIA was 13.53 kGy. Soaking prior to gamma irradiation significantly lowered the $ID_{50}$ to 8.44 kGy, and the soaking process enhanced the efficiency to inactivate TIA by as much as 48%. When soaking prior to gamma irradiation was followed by subsequent mild heating ($60^{\circ}C$) process, the $IT_{50}$ (heating time required to reach the 50% inhibition of TIA) value at even 1 kGy (5.28 min) was greatly reduced by over 50% compared to the level for the no-soaking process. In addition, the activation energy of soaking prior to gamma irradiation at 1 kGy was 2.45 kcal/mole, which was also about 50% lower than the 5.10 kcal/mole of dried soybean gamma-irradiated. Based on these results, soaking prior to gamma irradiation is an effective method for TIA inhibition. Furthermore, a combination of two or more processing methods such as soaking, heating and gamma irradiation is much more effective than any single processing method.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.29 no.6
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• pp.797-804
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• 1996

Two trypsins were purified from shrimp hepatopancreas through ammonium sulfate fractionation, Q-Sepharose ionic exchange, benzamidine Sepharose-6B affinity, and Sephacryl S-300 gel chromatography. Both enzymes had a single polypeptide chain with a molecular weight (M.W.) of 32 kDa by sodium dodecylsulfate polyacrylamide gel electrophoresis (SOS-PAGE), although trypsin A and B were estimated to be a molecular weight of 27.2 and 22.8 kDa, respectively, using Sephacryl S-300 gel filtration. Both trypsins had similar amino acid compositions and rich in glycine, valine, alanine, aspartic acid, and glutamic acid, but low in methionine and basic amino acids. Both enzymes were completely inactivated by soybean trypsin inhibitor (SBTI), phenylmethylsulfonyl fluoride (PMSF), tosyl-L-lysine chloromethyl ketone (TLCK), benzamidine, leupeptin, however, not affected by tosyl-L-phenylalanine chloromethyl ketone (TPCK) and pepstatin.

• Journal of the Korean Society of Food Culture
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• v.14 no.5
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• pp.477-485
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• 1999

This study was conducted to improve the functional properties of soybean protein isolate by dimethylglutarylation and acetylation. Amino acid composition and solubility of modified soybean protein by dimethylglutarylation were not changed, but lysine and trypsin inhibitor activity was decreased an isoelectric point was moved from pH5 to pH4 as a result of modification. Emulsification capacity and stability, foaming capacity and thermal stability were increased by the modification. In that 91% dimethylglutarylated protein did not coagulate when heating at $100^{\circ}C$ for 20 min. while its foaming stability was decreased. Whereas specific gravity was decreased by the modification of the soybean protein, relative viscosity and whiteness were improved. Generally, dimethylglutarylation produced more conformational changes in protein system than did in acetylation.