Effects of soaking soybeans in dilute acids on biologically active components

Soaking soybeans in dilute acids affected activities of lipoxygenase, trypsin inhibitor and urease. Effects of soaking time, acid concentration and soaking temperature were investigated. Lipoxygenase activity was completely eliminated by soaking in 0.3 M HC1 at either 23°C or 40°C for 8 hr. Less than 50% trypsin inhibitor remained and urease was inactivated to an acceptable level (0.04 ΔpH).     

An International Collaborative Study on Trypsin Inhibitor Assay for Legumes,Cereals, and Related Products

For determining trypsin inhibitor activity (TIA) in soy products, the American Oil Chemists' Society (AOCS) Method Ba 12-75 has been used. It measures differences in absorbance at 410 nm of bovine trypsin activity toward a synthetic substrate (Nα-benzoyl-DL-arginine-p-nitroanilide) in the absence and presence of an inhibitor. Recently, a significantly improved method was developed (JAOCS, 2019, 96:635–645), featuring 5 mL of total assay volume, enzyme-last sequence, and single inhibitor level in duplicate. It is proposed as the AOCS Method Ba 12a-2020. As a part of the AOCS method approval process, a collaborative study involving 12 international laboratories was conducted to evaluate the performance of the proposed method. The study involved measuring TIA in 10 selected test samples plus a blind duplicate. They included soybeans, pulses, cereals, and their processed products (flours, concentrates, and isolates). After rigorous statistical treatment of the data, only three outliers were removed from the data of two samples. Repeatability relative standard deviations (RSDr) for the 11 samples ranged from 0.99% to 5.52%. Reproducibility RSD (RSD_R) ranged from 7.07% to 22.92%, with seven samples having RSD_R around 10% or less. The remaining four samples had very low TIA, and their RSD_R values ranged from 13.34% to 22.92%. The study has demonstrated reliable performance of the proposed AOCS method. Several collaborators carried out additional experiments addressing some aspects of the method, leading to further refinements. The proposed method is undergoing evaluation by the AOCS Uniform Methods Committee for adoption as an Official Method for measuring TIA in various legume and grain products.     

胰蛋白酶在合成高分子阴离子交换树脂上的固定化作用

研究了胰蛋白酶在合成高分子聚苯乙烯阴离子交换树脂上的固定化技术,固定化酶的动力学性质以及稳定性特征。阴离子交换树指GM201经预处理除去杂质后与偶联剂成二醛反应,再与胰蛋白酶反应,可把酶固定化在载体上。动力学分析表明:固定化胰蛋白酶的K_m(米氏常数)值高于溶液酶,最适温度及最适pH值均有较大变化。稳定性试验表明:固定化酶的热稳定性低于溶液酶,在微酸性,中性及碱性介质中的稳定性高于溶液酶。用固定化胰蛋白酶水解酪蛋白及提取大豆胰蛋白酶抑制剂的试验结果表明,固定化酶具有良好的操作稳定性。     

苦荞麦浸提液对胰蛋白酶和淀粉酶抑制作用的研究

以酪蛋白为底物测定胰蛋白酶活性,结果发现高浓度的芦丁和苦荞麦浸提液对胰蛋白酶有明显的抑制作用,表明苦荞麦对胰蛋白酶的抑制作用中,芦丁可能起主要作用。对淀粉酶活性的研究还发现,苦荞麦浸提液对α-淀粉酶和β-淀粉酶都有明显的抑制作用,其中对α-淀粉酶起主要抑制作用的物质可能是芦丁,对β-淀粉酶的抑制作用可能是非芦丁物质所致。     

Tid基因对水稻的转化及转基因植株的抗虫性

利用基因枪转化法将大豆Kunitz型胰蛋白酶抑制剂(SKTI)基因Ti^d转入北方推广的水稻(Oryza sativa L)品种丰优301和通887，所获得的潮霉素抗性植株通过GUS组织化学分析、PCR检测、Southern blot分子检测，证实Ti^d基因已经转入水稻基因组中，为转基因植株。用转基因水稻植株叶片进行了室内饲喂水稻二化螟(Chilo suppressalis)实验，抗虫性分析结果表明，与对照比较，部分转基因水稻植株明显地增强了对水稻二化螟虫的抗性。对转基因R1代植株进行PCR和PCR Southern分析，表明外源基因在转基因植株后代今稳定遗传。     

Giant Amazonian fish pirarucu (Arapaima gigas): Its viscera as a source of thermostable trypsin

A trypsin was purified from pyloric caeca of pirarucu (Arapaima gigas). The effect of metal ions and protease inhibitors on its activity and its physicochemical and kinetic properties, as well its N-terminal sequence, were determined. A single band (28.0 kDa) was observed by SDS–PAGE. Optimum pH and temperature were 9.0 and 65 °C, respectively. The enzyme was stable after incubation for 30 min in a wide pH range (6.0–11.5) and at 55 °C. The kinetic parameters K_m, k_cat and k_cat/K_m were 0.47 ± 0.042 mM, 1.33 s⁻¹ and 2.82 s⁻¹ mM⁻¹, respectively, using BApNA as substrate. This activity was shown to be very sensitive to some metal ions, such as Fe²⁺, Hg²⁺, Zn²⁺, Al³⁺, Pb²⁺, and was highly inhibited by trypsin inhibitors. The trypsin N-terminal sequence IVGGYECPRNSVPYQ was found. The features of this alkaline peptidase suggest that it may have potential for industrial applications (e.g. food and detergent industries).     

Effects of limited enzymatic hydrolysis with trypsin on the functional properties of hemp (Cannabis sativa L.) protein isolate

Effects of limited enzymatic hydrolysis induced by trypsin on the physicochemical and functional properties of hemp (Cannabis sativa L.) protein isolate (HPI) were investigated. The enzymatic hydrolysis was confirmed by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and size exclusion chromatography (SEC). SEC and differential scanning calorimetry (DSC) analyses confirmed the presence of aggregates in the corresponding hydrolysates (with the degree of hydrolysis of 2.3–6.7%). Functional properties, including protein solubility (PS), thermal properties, emulsifying and foaming properties, and water holding and fat adsorption capacities (WHC and FAC) were evaluated. The PS was remarkably improved by the limited enzymatic hydrolysis at all tested pH values. However, the enzymatic hydrolysis led to the marked decreases in emulsifying activity index, foaming capacity and foam stability, WHC and FAC. These decreases were to a great extent related to the presence of aggregates in the hydrolysates.     

Effective Diffusivity and Kinetics of Urease Inactivation and Color Change During Processing of Soybeans with Superheated-Steam Fluidized Bed

Abstract

Influences of temperature and moisture content on diffusional transport property and kinetics of both urease inactivation and color change during soybean treatment with superheated steam in the temperatures of 120–150°C were investigated. The experimental results have shown that the effective diffusivity and apparent rates of such reactions are related to temperature in a way that the rates of water transport, urease inactivation, and brown pigment formation are higher with higher temperature. In addition, these rates are also enhanced by increasing moisture content, except for the browning reaction that is inhibited by this factor due to the dilution effect. The reduction in urease activity is reasonably described by modified first-order reaction and the color change of soybeans is characterized by three Hunter parameters in which the changes of L-, a- and b-values are followed according to zero-order, modified Monod and first-order reactions, respectively. The kinetics of inactivation and color change, along with the experimental data of protein solubility and lysine content, have been suggested that the soybean should be treated with superheated steam at the temperature ranging from 120 to 135°C. The superheated-steam fluidized-bed technique can simultaneously be applied for both drying and inactivating such components in soybeans by a single operation when the initial moisture content falls within a suitable range, approximately lower than 20%d.b. At elevated initial moisture contents, two-stage drying technique is recommended in order to avoid excessive cooked soybeans.     

Soybean Trypsin Inhibitor Assay: Further Improvement of the Standard Method Approved and Reapproved by American Oil Chemists’ Society and American Association of Cereal Chemists International

For measuring trypsin inhibitor (TI) activities in soybean products, the current standard method, approved and reapproved by American Oil Chemists Society (Method Ba 12-75) and American Association of Cereal Chemists International (Method 22-40.01), features mixing trypsin with a series of inhibitor levels and then adding a substrate to start the colorimetric reaction. Yet, previous studies have shown flaws with the method, particularly with using several inhibitor levels and the sequence of adding the substrate last. The present study showed that with varying levels of dilution and volumes of a dilute sample extract, the pH of the premix (the mixture of a dilute sample extract and trypsin solution) ranged 3.30–3.60 for raw soy flour, and 3.20–6.70 for toasted soy. Within these premix pH ranges, the standard method of adding substrate last would give TI values equal to or less than those measured by the same method except for adding the enzyme last. The standard method was subsequently improved by using a single sample extract level and the enzyme-last sequence. Other modifications included making stock solutions for reagents, adding Ca²⁺ to the trypsin solution, diluting sample extracts to a level that causes 30–70% of inhibition, and running both reference and sample blanks for better controls. Alternatively, the full volume assay (10 mL total, as in the standard method) was further modified by using half the volume of each reagent with the same concentration. Compared to the standard method, the improved methods gave more consistent results when assaying 11 selected soy products. The half volume (5 mL) and full volume methods gave the same results, but the former could increase assay sensitivity and reduce amounts of reagents used.     

Trypsins from the pyloric ceca of jacopever (Sebastes schlegelii) and elkhorn sculpin (Alcichthys alcicornis): Isolation and characterization

Trypsins from the pyloric ceca of jacopever (Sebastes schlegelii), TR-J, and elkhorn sculpin (Alcichthys alcicornis), TR-E, were purified by gel filtration on Sephacryl S-200 and Sephadex G-50. The molecular weights of TR-J and TR-E were estimated to be 24,000 Da by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. TR-J and TR-E revealed optimum temperatures of 60 and 50 °C, respectively, and showed the same optimum pH (pH 8.0) for hydrolysis of N-p-tosyl-l-arginine methyl ester. TR-J and TR-E were unstable at above 50 and 40 °C, respectively, and were more stable at alkaline pH than at acidic pH. Thermal stabilities of TR-J and TR-E were highly calcium dependent. These purified trypsin enzymes were inhibited by serine protease inhibitors, such as TLCK and soybean trypsin inhibitor. The N-terminal amino acid sequences of TR-J and TR-E were also investigated. The N-terminal amino acid sequences of TR-J, IVGGYECKPYSQPHQVSLNS, and TR-E, IVGGYECTPHSQAHQVSLNS, were found, and these sequences showed highly homology to other fish trypsins.