Comparative Assessment of Trypsin Inhibitor vis-à-vis Kunitz Trypsin Inhibitor and Bowman-Birk Inhibitor Activities in Soybean

Trypsin inhibitor activity (TIA) in soybean is attributed to two polypeptides, namely, Kunitz trypsin inhibitor (KTI) and Bowman-Birk inhibitor (BBI). Standard spectrophotometric protocol widely followed for estimation of TIA is cumbersome and does not distinguish KTI from BBI. In the present investigation, extraction conditions for KTI were optimized and different forms of this polypeptide were resolved in 180 soybean genotypes of Indian and exotic origin through native PAGE. This led to the identification of three KTI alleles, namely, Ti^a, Ti^b, and Ti^c, with Ti^a occurring in most of the Indian genotypes. Trypsin-KTI complex assay exhibited binding of Ti^a polypeptide with 2.51 fold concentration of trypsin. Subsequently, seeds of selected genotypes were subjected to estimation of KTI and BBI activity through densitometry and enzyme-linked immunosorbent assay (ELISA), respectively; and total TIA through standard spectrophotometric protocol. Summation of KTI and BBI was significantly (P?<?0.05) lower than that of TIA determined through the spectrophotometric method.     

Kunitz trypsin inhibitor in addition to Bowman-Birk inhibitor influence stability of lunasin against pepsin-pancreatin hydrolysis

Soybean contains several biologically active components and one of this belongs to the bioactive peptide group. The objectives of this study were to produce different lunasin-enriched preparations (LEP) and determine the effect of Bowman-Birk inhibitor (BBI) and Kunitz trypsin inhibitor (KTI) concentrations on the stability of lunasin against pepsin-pancreatin hydrolysis (PPH). In addition, the effect of KTI mutation on lunasin stability against PPH was determined. LEP were produced by calcium and pH precipitation methods of 30% aqueous ethanol extract from defatted soybean flour. LEP, lunasin-enriched commercially available products and KTI control and mutant flours underwent PPH and samples were taken after pepsin and pepsin-pancreatin hydrolysis. The concentrations of BBI, KTI, and lunasin all decreased after hydrolysis, but they had varying results. BBI concentration ranged from 167.5 to 655.8 μg/g pre-hydrolysis and 171.5 to 250.1 μg/g after hydrolysis. KTI concentrations ranged from 0.3 to 122.3 μg/g pre-hydrolysis and 9.0 to 18.7 μg/g after hydrolysis. Lunasin concentrations ranged from 8.5 to 71.0 μg/g pre-hydrolysis and 4.0 to 13.2 μg/g after hydrolysis. In all products tested, lunasin concentration after PPH significantly correlated with BBI and KTI concentrations. Mutation in two KTI isoforms led to a lower concentration of lunasin after PPH. This is the first report on the potential role of KTI in lunasin stability against PPH and must be considered in designing lunasin-enriched products that could potentially survive digestion after oral ingestion.     

Changes in inhibitory activity and secondary conformation of soybean trypsin inhibitors induced by tea polyphenol complexation

BACKGROUND: Tea polyphenol (TP) is a new food additive for antioxidant application, while soybean is an important resource for food and feed processing. It is therefore of rational and practical significance to investigate the influence of TP on soybean trypsin inhibitors (TIs). The aim of this study was to determine the effects of TP on the inhibitory activity of Kunitz (KTI) and Bowman–Birk (BBTI) TIs and to reveal the relationship between the inhibitory activity and conformation of KTI and BBTI by measurement of circular dichroism (CD) spectra. RESULTS: KTI and BBTI were found to be partially deactivated by TP. BBTI exhibited stronger resistance than KTI to TP deactivation. The unchanged K_M value of trypsin for benzoyl‐DL ‐arginine‐p‐nitroanilide hydrolysis indicated that KTI and BBTI inhibited trypsin in a non‐competitive pattern when complexed with TP. As the TP/TI ratio was increased and the inhibitory activity of KTI and BBTI decreased, the conformation of KTI and BBTI showed relevant changes and the major CD negative bands shifted progressively towards the near‐UV region. CONCLUSION: These results show the deactivation effects of TP on KTI and BBTI and reveal preliminarily the relationship between the inhibitory activity and secondary structure of KTI and BBTI. Copyright © 2009 Society of Chemical Industry     

Effects of tea polyphenols on the activities of soybean trypsin inhibitors and trypsin

Tea polyphenols (TPs) and other materials were extracted from Chinese green tea, and their effects on trypsin inhibitors and trypsin were analysed. TPs were found to have a deactivation effect on both Kunitz trypsin inhibitor (KTI) and Bowman–Birk trypsin inhibitor (BBTI). KTI was more easily deactivated than BBTI by complexing with TPs. The deactivation effect of TPs on KTI and BBTI reached a maximum at a TP/KTI ratio of 25 and a TP/BBTI ratio of 16. However, the deactivation effect of TPs on KTI and BBTI was reduced dramatically when KTI and BBTI were already complexed with trypsin. TPs were also found to inhibit trypsin. The inhibitory activity of TPs, KTI and BBTI on trypsin was found to decrease in the order BBTI > gtTI > gtPs. Complete inhibition of trypsin by TPs could not be achieved. When the TP concentration was increased to about 17 µg ml^?1, the residual activity of trypsin was maintained at 400 TU mg^?1, equivalent to 32% of the initial trypsin activity. In TP inhibition the K_M value for trypsin remained unchanged at 5.88 × 10^?4 mol l^?1 and V_max decreased when benzoyl‐DL ‐arginine‐p‐nitroanilide (BAPNA) was used as substrate. The pattern of trypsin inhibition by TPs is non‐competitive. Copyright © 2004 Society of Chemical Industry     

基于Exo III信号放大的荧光适配体传感器检测Kunitz型大豆胰蛋白酶抑制因子

Kunitz型大豆胰蛋白酶抑制因子（Kunitz Soybean Trypsin Inhibitor，KTI）是一种很关键的抗营养因子，不仅对动物消化系统和生长发育有不良影响，还制约各个行业对大豆的利用率，因此快速有效的检测方法是非常必要的。该研究建立一种基于核酸外切酶III（Exonuclease III，Exo III）和碳纳米颗粒（Carbon Nanoparticles，CNPs）的信号辅助放大荧光传感体系用于KTI的检测。具体体系包括KTI适配体（Aptamer，APT）、互补链（Complementary DNA，cDNA）、信号探针（Signal Probe，SP）、Exo III和CNPs共5个部分。通过可行性分析和CNPs浓度优化试验，测得KTI在100~600 ng/mL范围内呈线性相关，检测限为12.59 ng/mL。以豆浆作为样品，采用加标回收测得回收率为97.42%~102.85%，RSD在0.61%~2.36%之间，该方法可对实际样品中的KTI进行测定。     

The biochemical and functional food properties of the bowman-birk inhibitor

The Bowman-Birk inhibitor (BBI) is a small water-soluble protein present in soybean and almost all monocotyledonous and dicotyledonous seeds. The molecular size of BBI ranges from 1,513 Da to about 20,000 Da. BBI is to seeds what alpha(1)-antitrypsin is to humans. Soy-based food products rich in BBI include soybean grits, soymilk, oilcake, soybean isolate, and soybean protein concentrate. BBI is stable within the pH range encountered in most foods, can withstand boiling water temperature for 10 min, resistant to the pH range and proteolytic enzymes of the gastrointestinal tract, bioavailable, and not allergenic. BBI reduces the proteolytic activities of trypsin, chymotrypsin, elastase, cathepsin G, and chymase, serine protease-dependent matrix metalloproteinases, urokinase protein activator, mitogen activated protein kinase, and PI3 kinase, and upregulates connexin 43 (Cx43) expression. Several studies have demonstrated the efficacy of BBI against tumor cells in vitro, animal models, and human phase IIa clinical trials. FDA considers BBI as a drug. FDA also approves labels claiming that consumption of at least 3 to 4 oz of tofu or 8 oz of soymilk or soy protein may reduce the risk of coronary heart disease and breast cancer. This review highlights the biochemical and functional food properties of the Bowman-Birk inhibitor.     

Comparative Effects of Ohmic,Induction Cooker,and Electric Stove Heating on Soymilk Trypsin Inhibitor Inactivation

During thermal treatment of soymilk, a rapid incorporation of Kunitz trypsin inhibitor (KTI) into protein aggregates by covalent (disulfide bond, SS) and/or noncovalent interactions with other proteins is responsible for its fast inactivation of trypsin inhibitor activity (TIA). In contrast, the slow cleavage of a single Bowman–Birk inhibitor (BBI) peptide bond is responsible for its slow inactivation of TIA and chymotrypsin inhibitor activity (CIA). In this study, the effects of Ohmic heating (220 V, 50 Hz) on soymilk TIA and CIA inactivation were examined and compared to induction cooker and electric stove heating with similar thermal histories. It was found that: (1) TIA and CIA inactivation was slower from 0 to 3 min, and faster after 3 min as compared to induction cooker and electric stove. (2) The thiol (SH) loss rate was slower from 0 to 3 min, and similar to induction cooker and electric stove after 3 min. (3) Ohmic heating slightly increased protein aggregate formation. (4) In addition to the cleavage of one BBI peptide bond, an additional reaction might occur to enhance BBI inactivation. (5) Ohmic heating was more energy‐efficient for TIA and CIA inactivation. (6) TIA and CIA inactivation was accelerated with increasing electric voltage (110, 165, and 220 V) of Ohmic heating. It is likely that the enhanced inactivation of TIA by Ohmic heating is due to its combined electrochemical and thermal effects.     

大豆胰蛋白酶抑制剂的制备及性质

采用硫酸钠盐析法从大豆乳清废水中选择性回收大豆胰蛋白酶抑制剂(soybean trypsin inhibitor,STI),且以商品化的Kunitz型胰蛋白酶抑制剂(soybean Kunitz trypsin inhibitor,KTI)为对照表征STI的理化性质和界面性质。结果表明,STI提取优化条件为:乳清溶液固形物含量13%、pH 4、加盐量9 g/100 m L,此条件下STI的得率为20.54%;十二烷基硫酸钠-聚丙烯酰胺凝胶电泳图谱显示,其主要成分为KTI,以苯甲酰-DL-精氨酸-p-硝基酰替苯胺盐酸盐为底物的胰蛋白酶抑制活力为2 135.00 TIU/mg,且具有良好的温度和pH值稳定性(80℃加热30 min后仍保持73.19%的抑制活力,在pH 2~11范围内抑制活力无明显变化);傅里叶变换红外光谱和圆二色性结果显示,其与KTI(Sigma T9218)的结构类似,二级结构主要是β-折叠和无规卷曲;界面性质数据表明,STI分子能很快吸附到气水界面形成高弹性界面,从而使其具有良好的起泡性和泡沫稳定性。因此,简单的硫酸钠盐析法是大规模制备高纯度且功能性质良好的STI的有效方法,所获得的STI在医药及功能性食品领域有潜在的应用价值。     

Lunasin and Bowman-Birk protease inhibitor (BBI) in US commercial soy foods

The inverse association between the intake of soybean foods and cancer incidence and mortality rates supported by published literature has led to studies on identifying bioactive components. The cancer preventive properties of the soybean peptides lunasin and Bowman-Birk protease inhibitor (BBI) have been demonstrated by in vitro and in vivo assays. Since there is no comprehensive information on the concentrations of these two peptides, US commercially available soy foods, including soy milk, soy-based infant formula, tofu, bean curd, soybean cake, tempeh, natto, miso and su-jae samples, were analyzed for lunasin and BBI. Both peptides were present in most of the products, in varying concentrations, depending mainly on the soybean variety and the manufacturing process. Lunasin and BBI were absent in the fermentation products natto and miso, suggesting that fermentation destroys both peptides. To study the bioavailability of lunasin and BBI, three soy milk samples with different concentrations of these peptides were subjected to an enzymatic hydrolysis process simulating physiological digestion. The results confirm the important role BBI plays in the protection of lunasin from digestion by pepsin and pancreatin.     

The cytotoxic effect of Bowman–Birk isoinhibitors,IBB1 and IBBD2, from soybean (Glycine max) on HT29 human colorectal cancer cells is related to their intrinsic ability to inhibit serine proteases

Bowman–Birk inhibitors (BBI) from soybean and related proteins are naturally occurring protease inhibitors with potential health‐promoting properties within the gastrointestinal tract. In this work, we have investigated the effects of soybean BBI proteins on HT29 colon adenocarcinoma cells, compared with non‐malignant colonic fibroblast CCD‐18Co cells. Two major soybean isoinhibitors, IBB1 and IBBD2, showing considerable amino acid sequence divergence within their inhibitory domains, were purified in order to examine their functional properties, including their individual effects on the proliferation of HT29 colon cancer cells. IBB1 inhibited both trypsin and chymotrypsin whereas IBBD2 inhibited trypsin only. Despite showing significant differences in their enzyme inhibitory properties, the median inhibitory concentration values determined for IBB1 and IBBD2 on HT29 cell growth were not significantly different (39.9±2.3 and 48.3±3.5 μM, respectively). The cell cycle distribution pattern of HT29 colon cancer cells was affected by BBI treatment in a dose‐dependent manner, with cells becoming blocked in the G0–G1 phase. Chemically inactive soybean BBI had a weak but non‐significant effect on the proliferation of HT29 cells. The anti‐proliferative properties of BBI isoinhibitors from soybean reveal that both trypsin‐ and chymotrypsin‐like proteases involved in carcinogenesis should be considered as potential targets of BBI‐like proteins.     

Purification and Quantification of Kunitz Trypsin Inhibitor in Soybean Using Two-Dimensional Liquid Chromatography

Kunitz trypsin inhibitor (KTI) is one of the major antinutritional factors in soybean and results in inhibition of digestion of dietary protein. In this study, we developed a novel strategy to purify and quantify KTI from soybean using two-dimensional liquid chromatography. Lipids from ground soybean were removed using hexane after which the ground soybean was extracted with protein extraction buffer. The crude extract was first purified by weak anion exchange chromatography, and then the fraction containing KTI was further separated by size exclusion chromatography. The fraction containing KTI was collected and analyzed by SDS-PAGE and electrospray ionization mass spectrometry. Results indicated that purified KTI has a molecular mass of 20 kDa and a purity of ~98% with inhibitory activity of 2425 TIU/mg protein. This assay was used for the quantification of KTI in soybean samples. The assay showed concentrations with a range between 7.81 and 500.00 μg/mL and a limit of detection of 0.12 mg/g. The recoveries of KTI in spiked soybean samples were between 82.19% and 86.65%, and intra- and interday precisions (% CV) were less than 7.35% and 8.42%. The developed method was used to analyze soybean samples from different sources and soybean products derived from different processing techniques, which demonstrated that the developed procedure provided an accurate and sensitive tool for separation and quantification of intact KTI in soybean.     

STI的抑制作用及茶多酚作用下STI的失活探讨

大豆中的两种主要的胰蛋白酶抑制剂对胰蛋白酶的活性有很强的抑制作用,随着胰蛋白酶抑制剂的增加,胰蛋白酶活性几科能完全被抑制;茶多酚能有效地和胰蛋白酶抑制剂络合而使抑制剂对胰蛋白酶的抑制作用减弱,同时研究表明茶多酚络合失活胰蛋白酶抑制剂还受温度的影响。     

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     

ELISA-Based Detection of Soybean Proteins: A Comparative Study Using Antibodies Against Modified and Native Proteins

Soybean, the world’s primary provider of protein and oil, is widely used in foodstuffs which might pose a serious threat to allergic consumers due to the presence of some allergenic proteins. Enzyme-linked immunosorbent assay (ELISA) is the preferred method for the determination of allergen contamination in foodstuffs. Due to food processing, the antibody–antigen interaction in these routinely used methods are disrupted, therefore leading to erroneous results. A comparison between an ELISA using antibodies against modified soybean proteins and against the Kunitz trypsin inhibitor (KTI) is described. Limits of detection and quantification of 115.6 ng soybean protein/mL and 346.8 ng/mL were obtained for soybean-ELISA and 117.3 ng KTI/mL and 351.9 ng/mL for KTI-ELISA, respectively. Minimal cross-reactivity with other legumes and food ingredients were obtained. The applicability of these ELISAs was evaluated to detect the presence of soybean proteins in cookies. Both matrix interferences and the baking process affected analytical recovery. However, the recoveries were found to be much higher using the soybean-ELISA. The low recovery obtained using the KTI-ELISA might be due to the inability of the antibodies used to recognize the modified proteins. These results indicate that using antibodies developed towards allergens modified through food processing simulating reactions is a better approach to be used in food allergen detection.     

Soybean Protease Inhibitors in Foods

The major types of soybean protease inhibitors are the Kunitz inhibitor (KSTI) and the Bowman-Birk inhibitor (BBI). Enzymatic analysis and rocket Immunoelectrophoresis were used to specifically quantitate the amounts of KSTI and BBI in various soy foods. Many soy products such as soy flours, soy concentrates and soy isolates contained significant amounts of KSTI and BBI. Some soy foods contained small but still detectable levels of KSTI and BBI.     

降解大豆胰蛋白酶抑制剂的短小芽孢杆菌菌株及其胞外蛋白的鉴定

本研究以大豆胰蛋白酶抑制剂为唯一碳源,从豆类内部筛选获得3株具有降解大豆胰蛋白酶抑制剂的细菌。通过对菌株的16Sr DNA基因的分析,3株细菌分别被鉴定为枯草芽孢杆菌LZ013-1、短小芽孢杆菌LZ013-2和类芽孢杆菌LZ013-3。进一步研究发现LZ013-2菌株的上清液具有高效降解大豆胰蛋白酶抑制剂的活性,2h可将胰蛋白酶抑制剂抑制率降低73.60%。将LZ013-1和LZ013-2菌株应用于豆粕发酵中,两株芽孢杆菌均能高效降解胰蛋白酶抑制剂。原始豆粕的胰蛋白酶抑制剂活性为22.26 mg/g,经过LZ013-1和LZ013-2菌株的液态发酵后分别降低为0.50 mg/g和0.63 mg/g,经过固态发酵后分别降低为1.06 mg/g和1.03 mg/g。通过硫酸铵分级沉淀和凝胶柱层析,分离LZ013-2菌株发酵上清液中具有降解活性的蛋白质,并采用质谱鉴定分离得到的蛋白质,筛选并鉴定出2种活性蛋白,分别为肽酶S8(Uniprot ID:A0A2T0DB16)和肽酶M84(Uniprot ID:A8FIH7)。     

Trypsin inhibitor activity of soybean as affected by genotype and fertilisation

The presence of protease inhibitors in soybean prohibits the utilisation of the raw beans for food and feed. However, little information is available about environmental influences and the effects of nitrogen and sulphur supply on the antinutritional constituents of soybean. As these factors may influence protease inhibitors, soybean genotypes segregated according to the presence or absence of the Kunitz trypsin inhibitor have been evaluated for trypsin inhibitor activity (TIA) in field trials. TIA was affected significantly by environment (geographical location), fertilisation treatment and genotype. Environmental means of TIA were between 69.5 and 104.8 mg g^?1. Nitrogen application, which caused an increase in seed protein content, resulted in a reduction in TIA by about 15% as compared with the control. Remarkably, simultaneous application of nitrogen and sulphur in the form of ammonium sulphate had a similar reductive effect on TIA to that of nitrogen application alone, although soybean protease inhibitors are rich in sulphur amino acids. Significant genetic variation in TIA was found both within the genotype class with the Kunitz inhibitor present as well as within the class lacking this inhibitor. The results suggest that TIA of soybean may be modified considerably by genetic improvement and appropriate agronomic management. Copyright © 2003 Society of Chemical Industry     

大豆胰蛋白酶抑制剂失活方法研究进展

胰蛋白酶抑制剂是大豆食品与饲料的主要抗营养因子,大豆胰蛋白酶抑制剂的失活能明显提高大豆食品与饲料的营养价值和食用安全性。大豆胰蛋白酶抑制剂的钝化方法有物理、化学、生物还原、酶解、发酵以及天然化合物络合法等,介绍了研究概况,大豆胰蛋白酶抑制剂失活诸方面与技术,并对其发展前景作了初步探讨。     

Thermal inactivation of trypsin inhibitors of soybean preparations added to meat

The activity of trypsin inhibitors was investigated in minced meat in which 30% of meat protein was replaced by soybean preparations (flakes, concentrate, isolate, textured flour). Trypsin inhibitors were determined in 20% suspensions of these soybean preparations. The activity of trypsin inhibitors in soybean preparations added to meat, or suspended in water, appeared to be higher than that of dry products. The heat treatment (70, 80, 90 and 100°C for 15 min) reduced the inhibitor activity in all soybean preparations, both in model meat systems (MMS) and in suspensions. Trypsin inhibitor activity was higher in MMS than in water suspensions of soybean preparations, suggesting that meat components protect trypsin inhibitor proteins against heat treatment. Trypsin inhibitor activity in heated meat model systems was too low to produce unfavourable nutritional effects.     

Effects of heating on protein quality of soybean flour devoid of Kunitz inhibitor and lectin

The effects of autoclaving on protein quality of soybean flours prepared from a conventional soybean (CSB) and an isoline lacking Kunitz inhibitor and lectin (KFLF) were studied. The heating was efficient in the urease, trypsin inhibitors and lectin inactivation, being 15 min sufficient to reduce more than 90% of these compounds and provide protein solubility over 80%. The results of PER, NPR and weight gain showed that heating equally improved the nutritional quality of both soybean flours, although higher autoclaving time was required for KFLF. No significant improvement was observed on NPU and in vivo digestibility of the diets containing KFLF at any heating time. As these later results were similar to those obtained with diets containing CSB, they show the importance of the heating to improve the nutritional value and show that other components rather than trypsin inhibitors and lectins impair the nutritive value of raw soybean.