马铃薯胰蛋白酶抑制剂的抑菌活性

本研究探讨了马铃薯胰蛋白酶抑制剂（PTI）抑菌活性及其抑菌机理。采用滤纸片扩散法测定抑菌圈直径,微量二倍稀释法测定最低抑菌浓度（MIC）、最低杀菌浓度（MBC）,以此评价PTI的抑菌性。通过扫描电镜观察并测定胞膜通透性、胞膜完整性、胞内活性氧含量及对细菌蛋白酶的抑制率等实验,探讨了PTI的抑菌机理。结果表明：PTI对枯草芽孢杆菌、蜡样芽孢杆菌和铜绿假单胞菌有较明显的抑制作用,其抑菌圈直径分别为12.56 mm、12.35 mm和11.76 mm,MIC为1.88~3.75 mg/mL,MBC均为3.75 mg/mL。PTI破坏了菌体细胞的形态,导致菌悬液中相对电导率升高和核酸的泄漏,影响了细菌的膜通透性;通过荧光显微镜观察,1 MIC的PTI使菌体胞膜的完整性遭到破坏并促进胞内ROS的产生,造成了菌体氧化损伤;PTI对菌体蛋白酶的活性表现出一定的抑制作用。通过实验得出PTI可能通过这种多靶点协同增效作用达到其抑菌效果。     

Trypsin Inhibitor from 3 Legume Seeds: Fractionation and Proteolytic Inhibition Study

ABSTRACT: The trypsin inhibitor from navy beans (Phaseoulus vulgaris), red kidney beans (Phaseoulus vulgaris L.), and adzuki beans (Vigna angularis) provided by the Royal Project Foundation in Thailand was isolated by heat and ammonium sulfate (AS) precipitation. Incubation at 70 °C for 10 min produced the highest trypsin inhibitor recovery for all legumes. The AS precipitation with 60% to 80% saturation (precipitate IV) resulted in 41-, 88-, and 34-fold of the purity and (−)26%, 126%, and (−)47% of percentage of activity increase for navy beans, red kidney beans, and adzuki beans, respectively. The trypsin inhibitors had a molecular weight of 132 kDa for navy beans, 118 kDa for red kidney beans, and 13 kDa for adzuki beans under nonreducing conditions. The obtained precipitate IV fraction from each legume effectively prevented the degradation of the tilapia muscle with concentration dependent. The myosin heavy chain increased as the concentration of the inhibitor fraction increased, especially at the highest level of addition. The result indicated that the precipitate IV from these legumes have potential for use as a protease inhibitor in fishery related products.     

豆浆豆腐中胰蛋白酶抑制因子活性的研究

豆制品中含有丰富的营养物质,但其中的抗营养因子阻碍人体对营养物质的吸收。分别采用6种不同制浆工艺将5种原料豆进行豆浆、豆腐的制备,测定大豆和豆浆中蛋白含量以及豆浆、豆腐的产量,通过分析不同大豆品种及制浆工艺与豆制品产量及蛋白含量之间的关系,以产量为指标,研究不同大豆品种及其加工工艺对豆制品产量及蛋白含量的影响,选取高产量、高蛋白的豆制品。并采用紫外分光光度计法分别对原料豆、豆浆、豆腐等进行大豆胰蛋白酶抑制因子活性的检测,得出低大豆胰蛋白酶抑制因子活性的品种及加工豆制品的制浆工艺。结果表明,豆制品产量与蛋白含量成正比,不同大豆品种采用不同制浆工艺所制得的豆浆、豆腐中胰蛋白酶抑制因子活性具有显著性差异,为不同大豆品种适用性加工及工业化生产提供理论依据。     

转基因食品胰蛋白酶抑制剂活性的测定

目的通过测定转基因食品的胰蛋白酶抑制剂活性,为建立转基因食品营养评价标准提供数据。方法首先通过探讨胰蛋白酶-底物-胰蛋白酶抑制剂的剂量反应关系,建立转基因食品胰蛋白酶抑制剂活性测定的最佳反应体系,并以此为基础分析了部分转基因玉米、大豆的胰蛋白酶抑制剂活性及与亲本食品的差别。结果所检转基因食品胰蛋白酶抑制剂活性大多与亲本食品差别不大,尽管个别产品出现胰蛋白酶抑制剂活性增高或降低现象,但都在可接受范围。结论在规范检测技术前提下加强对非转基因食品基础数据建设对制定转基因食品营养评价标准十分必要。     

苦瓜中胰蛋白酶抑制剂活性的测定方法研究

目的用一种改进的测定方法,测定从苦瓜籽中提取分离的胰蛋白酶抑制剂的抑制活性。方法通过稳定性、重复性和专属性实验,分析和考察此测定方法。结果苦瓜籽中的胰蛋白酶抑制剂的相对分子质量1×10~3,比活1 153 U/mg;配制的抑制剂在2 h内测得的活性变化小于2%;RSD 2．5%;0．05 mg/mL牛血清白蛋白对测定体系没有干扰。结论此方法适用于苦瓜胰蛋白酶抑制剂活性的测定。     

热处理对鲜豆奶中胰蛋白酶抑制子活性的影响

分别以95℃、120℃、140℃三种温度处理鲜豆奶,研究热处理对胰蛋白酶抑制子活性的影响。测定结果表明要达到胰蛋白酶抑制子90％的失活率,95℃时需要35min;120℃时需要7min;而140℃时只需要60s左右。胰蛋白酶抑制子的热致死时间曲线表明,加热温度增加30℃,钝化胰蛋白酶抑制子90％的热处理时间可缩短10倍。     

商品大豆饮料胰蛋白酶抑制剂活性的研究

本研究详细测定了未经热处理的生豆奶、各种经现代技术加工的大豆饮料产品、东方传统大豆食品豆腐等样品的胰蛋白酶抑制剂活性（ＴＩＡ）。其中生豆奶的ＴＩＡ（以每克蛋白质所抑制的纯胰蛋白酶毫克数表示）是６６．４ｍｇ／ｇ蛋白质，经巴氏杀菌的商品大豆饮料ＴＩＡ是２３．７ｍｇ／ｇ蛋白质。检验了经超高温灭菌处理的７种不同商标大豆饮品，结果表明这些饮品的ＴＩＡ在１３．３～３１．６ｍｇ／ｇ蛋白质之间。另有两种已知是经灭菌的大豆饮料，其ＴＩＡ最低，分别为４．１ｍｇ／ｇ蛋白质和７．７ｍｇ／ｇ蛋白质。又分析了一种采用东方传统加工方法生产的豆腐，其ＴＩＡ仅为６．４ｍｇ／ｇ蛋白质。上述这些分析结果反映了某些经现代加工方法生产的商品大豆饮料，在消除抗营养因子的处理上是不够充分的。     

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.     

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。     

枯草杆菌蛋白酶对大豆胰蛋白酶抑制剂的水解钝化研究

研究了枯草杆菌蛋白酶(Subtilisin)对大豆胰蛋白酶抑制剂(STI)酶解活性的影响。实验在50℃,pH7.5条件下反应1h,并以BAPNA为底物,采用改进的方法测定枯草杆菌蛋白酶对大豆胰蛋白酶抑制剂的水解钝化程度,用SDS-PAGE方法和分子排阻色谱法研究其蛋白酶钝化敏感性。结果证明,枯草杆菌蛋白酶可以水解大部分的抑制剂,并通过SDS-PAGE进一步证实了比色法得出的结论。而由分子排阻色谱图可以分析得出抑制剂经枯草杆菌蛋白酶酶解后,活性降低。分析认为,可能是由于抑制剂中二硫键被打断使得其结构发生改变,同时生成大量复杂的小分子多肽物质。因此,可以推断大豆胰蛋白酶抑制剂的稳定性与二硫键的存在有关。     

A Chondroitin Sulfate Chain of Urinary Trypsin Inhibitor Enhances Protease Inhibitory Activity of the Core Protein

Human urinary trypsin inhibitor (UTI) is a proteoglycan composed of one core protein covalently linked to one glycosaminoglycan, which is a low sulfated chondroitin 4-sulfate. It is used as an anti-inflammatory medicine based on the protease inhibitory activity of the core protein. However, functions of the chondroitin sulfate have not been clarified. Recently, we succeeded in remodeling the UTI chondroitin sulfate to hyaluronan to create hyaluronan hybrid UTI, without changing the core protein. Here, we investigated the effect of the remodeled chondroitin sulfate on the activities of serine proteases. Native UTI showed stronger protease inhibitory activity than hyaluronan hybrid UTI or hydrolyzed glycosaminoglycan UTI. Chondroitin 4-sulfate chains with a small peptide derived from the native UTI did not show any protease inhibitory activity. These results suggest that the chondroitin sulfate chain linked covalently to core protein enhances protease inhibitor activity of UTI although the chondroitin sulfate chain itself does not.     

Trypsin Inhibitor Activity and Tannin Content Do Not Affect Calcium Bioavailability of Three Commonly Consumed Legumes

Calcium bioavailability from legumes containing a range in trypsin inhibitor activity (TIA) and tannin concentration was studied. Three cultivars (Phaseolus vulgaris) were hydroponically grown and intrinsically labeled with ^4SCa. Raw and cooked legumes were fed to six groups of 6-wk old male Sprague-Dawley rats. Another group received an intraperitoneal (IP) injection of ⁴⁵Ca. An eighth group was fed a casein metal extrinsically labeled with ⁴⁵Ca. The absorption of calcium from legumes by rats averaged 47.1 ± 7.5% of IP dose. Average phytate content of the legumes was 1.7% and oxalate 0.37%. Raw legumes had an average of 15000 TIA units/g of whole bean, which were completely removed by cooking. Calcium absorption was unaffected by TIA or tannin content. Reduced availability of bean calcium was likely due to phytate and/or oxalate present in legumes.     

Alcalase蛋白酶降解大豆胰蛋白酶抑制剂的研究

研究了不同酶解条件下 (pH值、温度、时间、加酶量和添加巯基还原剂 ) ,碱性内切蛋白酶Alcalase对大豆蛋白和大豆胰蛋白酶抑制剂的降解作用。研究结果表明 ,Alcalase可同时降解大豆蛋白和胰蛋白酶抑制剂。该酶解反应的最适条件为 :pH 8 0、温度 6 0℃、最适加酶量 10 μL/g蛋白 (约 0 0 2 832AU/ g蛋白 ) ,添加Na2 SO3为ω(Na2 SO3) =0 3% ,水解时间 4h。在此条件下 ,残留胰蛋白酶抑制活性为对照的 2 0 % ,可溶性蛋白含量可达 2 7mg/mL ,游离氨基酸含量为 7 1mg/mL ,大豆蛋白的水解度为 8 9%。还讨论了Alcalase蛋白酶降解大豆蛋白生成小肽的最佳反应条件     

Biochemical Characterization of a Low Trypsin Inhibitor Soybean

A low trypsin inhibitor soybean (LTI) was characterized using electrophoresis, enzyme activity measurements, and gel exclusion chromatography. The protein profiles were similar to a control soybean. Gel exclusion chromatography resulted in two peaks of trypsin inhibitor activity in the control. The first peak, absent in LTI, proved to be Kunitz trypsin inhibitor and was electrophoretically isomorphic. The second inhibitor consisted of at least five isotypes and co-eluted with Bowman-Birk trypsin inhibitor. Shorter heating times were required to inactivate both trypsin and chymotrypsin inhibitor activity in LTI compared to control soybeans. The use of LTI may increase the economic viability of soybeans as protein supplements for humans.     

An Enzymatic Method for Methanol Quantification in Methanol/Ethanol Mixtures with a Microtiter Plate Fluorometer

In response to the need for a rapid, high-throughput screening of methanol contamination in spirits, a new microplate-based assay was developed. In this assay, alcohol oxidase first oxidizes methanol to formaldehyde, which is further oxidized to formate by formaldehyde dehydrogenase while reducing NAD⁺ to NADH. The latter product then reacts with resazurin under catalysis by FerB, a diaphorase-type enzyme, to give the highly fluorescent resorufin. These reactions are run simultaneously in 200 μL final volume in a 96-well plate and quantified using a plate reader and fluorescence detector. It is shown that the rate of fluorescence change is related to methanol and ethanol concentrations according to the rate law for two competing substrates. Quantification of methanol in real samples is carried out by applying the standard additions technique with four different spiking concentrations of the methanol standard; methanol content in the sample is calculated from the x-intercept of the fitted line. The high activity of FerB with resazurin and low rate of further conversion of resorufin to non-fluorescent dihydroresorufin indicate that FerB may be advantageous over commercially available diaphorases for use in fluorescence enzyme assays.     

测量中的不确定度及其数学处理方法

论述了国际上新提出的不确定度概念和它的数学处理方法 ,以及与误差概念的区别 ,并说明了为什么要用不确定度的概念取代过去长期使用的误差概念来对测量结果的质量作出评定 .     

Trypsin Inhibitor Activity In Vitro Digestibility and Sensory Quality of Meat-Like Yuba Products as Affected by Processing

ABSTRACT: :
Soybeans ( Glycine max ) were soaked and ground to obtain soymilk. The soymilk was cooked in an open tank and held at 85 to 90 deg;C. Yuba films were picked up in 20 min intervals and dried for 20 min. Yuba films were soaked in chicken-flavor solutions (25% and 35%), and baking soda (BS) solutions (0%, 1%, 2%, and 3% BS), and cooked at 100 °C for 30 min, 60 min, and 90 min. TIA decreased (p < 0.05) with the increase of heating time and BS concentration. In vitro protein digestibility (IVPD) decreased with heating time and BS concentration (p < 0.05). Sensory characteristics were affected by flavor concentration. By using 0% BS, 25% of the chicken flavor concentration, and a short heating time method, meat-like products with low TIA, high IVPD, and good sensory characteristics were obtained.