茶多酚对米曲霉α-淀粉酶的回收及其特性的影响

研究绿茶中多酚类对米曲霉来源的α－淀粉酶特性的影响。从绿茶中提取茶多酚（TP），对米曲霉α－淀粉酶进行络合，沉淀及回收；以Bernfeild法测定α－淀粉酶与TP络合后，在不同温度，不同pH值，不同底物浓度的活性变化。结果表明：茶多酚对米曲霉α－淀粉酶无活性抑制作用，两者之间具有起混作用；0．3％的茶多酚浓度，获得最大α－淀粉酶活性回收率（约71％）；α－淀粉酶与TP络合后，阳适反应温度由30－50℃范围变为60－70℃；最适pH值由3．0－8．0变为5．0－6．0；在80℃下，活性变化总趋向与游离的α－淀粉酶；180min后能够保85％的酶活力，但是在前40min，酶活力下降较快；Lineweever-Burk图表明，络合后的α－淀粉酶Km由0．18％变为1．03％（可溶性淀粉底物浓度）。结论：米曲霉α－淀粉酶与TP络合后活性不受抑制并可通过这种络合回收，络合后的α－淀粉酶，最适反应温度及最达pH值变大，变窄，对底物的亲和力下降。     

茶多酚对菠萝蛋白酶的回收及其特性的影响

研究了茶多酚对菠萝蛋白酶特性的影响。采用不同茶多酚浓度、温度、pH、保温时间及冷存时间对菠萝蛋白酶进行络合、沉淀及回收,并研究其对络合效果的影响。结果表明,茶多酚对菠萝蛋白酶无抑制作用,两者具有起混活性;最佳络合条件为:茶多酚浓度0.8%,温浴温度30℃,pH5,温浴时间0.5h,冷存时间0.5h,此时获得最大的菠萝蛋白酶活性回收率(约82.1%);菠萝蛋白酶与TP络合后,最适反应温度及最适pH无明显改变但范围变窄,均为55℃与pH7左右;Lineweaver-Burk图表明,络合后的菠萝蛋白酶Km值由0.06%变为0.14%。     

底物亲和法分离纯化蕲蛇胃蛋白酶

利用酶(胃蛋白酶)与其底物(酪蛋白)的亲和性,从蕲蛇胃中提取得到一种胃蛋白酶.让底物和酶在pH 2.5的乳酸缓冲液中充分结合,然后调pH至4.0(底物的等电点)沉淀底物和酶的结合物,随后让沉淀物再溶解于乳酸缓冲液中,添加低浓度的SDS将底物和酶分离,最后用DEAE离子交换色谱柱进行纯化,经SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)鉴定,分离得到的胃蛋白酶为电泳纯,其分子量为33KD.酶的最适pH值小于3.0,最适温度为50℃.该结果证明胃蛋白酶能利用其与酪蛋白的亲合性及SDS沉淀作用有效地分离,这种胃蛋白酶能在食品生产的加热、强酸处理过程中应用.     

罗非鱼肠道蛋白酶的分离纯化及部分性质研究

目的:以罗非鱼加工下脚料肠道为原料,分离提取其中的蛋白酶,并对其部分性质进行研究,旨在提供一种简便的食品级蛋白酶制备方法.方法:用茶多酚络合沉淀,Sephadex G-50分离纯化蛋白酶,SDS-PAGE检测分离效果及相对分子质量;以酪蛋白溶液为底物,采用Folin-酚法测定蛋白酶活力.结果:经茶多酚络合沉淀,得到一种复合蛋白酶,命名为TP-肠道蛋白酶;经Sephadex G-50凝胶层析除去茶多酚,得到游离肠道蛋白酶.TP-肠道蛋白酶的最适温度50℃,pH7.2～9.6,米氏常数Km=1.125×103mg/L;游离肠道蛋白酶的最适温度50℃,pH7.2～10.0,米氏常数Km=8.5×102mg/L.结论:TP-肠道蛋白酶和游离肠道蛋白酶均为复合蛋白酶,有望开发成食品级蛋白酶制剂.     

胃蛋白酶水解甘薯蛋白制备血管紧张素转化酶抑制肽的研究

采用胃蛋白酶水解甘薯蛋白制备血管紧张素转化酶(ACE)抑制肽,通过四元二次回归正交旋转组合设计,考察底物浓度、酶与底物浓度比、pH值、温度对ACE抑制率的影响,并确定最优酶解工艺参数,最终建立了ACE抑制率与各影响因素的回归模型。在此基础上,确定了胃蛋白酶水解甘薯蛋白的最适条件为:底物浓度2.3%、酶与底物浓度比3.7%、pH2.3、温度37℃、时间8h,采用该优化工艺,得到ACE抑制率最大为78.37%的水解产物。为开发防治高血压的保健食品提供了理论依据。     

大鲵胃蛋白酶分离纯化及其性质的研究

通过研究大鲵胃蛋白酶,可以了解大鲵消化机能,同时为食品加工提供新的材料.经80％硫酸铵沉淀、DEAE-52离子交换层析、Sephadex G-100凝胶过滤层析以及HPLC,得到大鲵胃蛋白酶电泳纯制品.纯化倍数为239.87,回收率为4.4％,经SDS-PAGE测得该胃蛋白酶分子量为31ku,大鲵胃蛋白酶的最适温度是40℃,低于40℃稳定,当温度上升,活性迅速下降;最适pH为2,在pH2～6有很好的稳定性.EDTA、BrAc、NBS使该酶活性下降.以酪蛋白为底物,在pH2.0、40℃时测得米氏常数Km值为7.3×103mg/L,V…是2.674 μg/min.     

猪血红蛋白酶解制备ACE抑制肽的研究

本实验选用碱性蛋白酶、胰蛋白酶、胃蛋白酶、风味蛋白酶、中性蛋白酶和木瓜蛋白酶等六种商业蛋白酶在各自最适反应条件下分别水解猪血红蛋白12h,研究其水解产物对血管紧张素转换酶抑制率和蛋白水解度的影响。结果显示:采用胃蛋白酶酶解获得的产物ACE抑制率最高。胃蛋白酶的酶解条件为底物5%(质量分数),酶与底物浓度比E:S=3%,温度37℃,pH2.0,水解4h后其ACE抑制率为81.10%,水解度为6.64%。     

不同储藏时间马铃薯中多酚氧化酶特性的研究

本文以不同储藏时间的马铃薯为原料,采用分光光度法研究了热稳定性、pH值、温度、底物浓度、酶液浓度、抑制剂对多酚氧化酶活性的影响。结果表明：新旧马铃薯的PPO最适温度为20℃,最适pH值为6．0,最适底物为邻苯二酚;新旧马铃薯最适酶液浓度为0．5mL;新马铃薯最适底物浓度为0．1mol／L,旧马铃薯最适底物浓度为0．15mol／L：新旧马铃薯PPO的强抑制剂为抗坏血酸与柠檬酸。     

不同储藏时间马铃薯中多酚氧化酶特性的研究

本文以不同储藏时间的马铃薯为原料,采用分光光度法研究了热稳定性、pH值、温度、底物浓度、酶液浓度、抑制剂对多酚氧化酶活性的影响。结果表明：新旧马铃薯的PPO最适温度为20℃,最适pH值为6．0,最适底物为邻苯二酚;新旧马铃薯最适酶液浓度为0．5mL;新马铃薯最适底物浓度为0．1mol／L,旧马铃薯最适底物浓度为0．15mol／L：新旧马铃薯PPO的强抑制剂为抗坏血酸与柠檬酸。     

蛋壳膜硫酸软骨素的提取工艺及其优化

以蛋壳膜为原料,采用稀碱与酶解相结合的方法提取硫酸软骨素。以胰蛋白酶、胃蛋白酶分解去除杂蛋白,经酒精沉淀并干燥后得到产品。实验得到的最佳提取工艺为:料液比1:1.5,碱浓度5%,碱提温度40℃,胰蛋白酶、胃蛋白酶的用量分别为1.5%、0.8%,pH值分别为8.2和5.5,酶的各自最适条件作用时间均为2.0h。     

Optimization and Scale‐Up Preparation of Egg White Hydrolysate with Angiotensin I Converting Enzyme Inhibitory Activity

Angiotensin I converting enzyme (ACE) plays an important role in regulation of blood pressure as it converts angiotensin I into angiotensin II (a potent vasoconstrictor). Food protein‐derived ACE inhibitory peptides have been considered as a safer alternative to antihypertensive drugs. In our previous study, three ACE inhibitory peptides were characterized from egg white ovotransferrin and their antihypertensive activity has been validated in spontaneously hypertensive rats. However, it is too costly to prepare these peptides from purified egg white ovotransferrin. The aims of the study were to determine the feasibility of preparing these peptides using egg white and then to optimize the conditions of preparing egg white hydrolysate. Taguchi's method was used to design experiments for optimization, which was established as follows: substrate %, pH of thermoase, time of thermoase digestion, ratio of pepsin to substrate, pH of pepsin, temperature of pepsin, and time of pepsin digestion were 7.5%, pH 8, 90 min, 1%, pH 2.5, 55 °C, and 180 min, respectively. The ACE inhibitory activity (IC₅₀ value) and peptide yield obtained under optimal condition were 30 ± 2 μg/mL and 77.5% ± 0.3%, respectively, which were comparable to the predicted values. Hydrolysate prepared at 150 L reactor showed comparable activity but low peptide yield. Results of this study demonstrated the feasibility of using egg white protein as the starting material to prepare a functional ingredient with potent ACE inhibitory activity.     

蛋白质酶法修饰的初步探讨──大豆蛋白和芝麻蛋白的合成类蛋白质研究

本文报道了以大豆蛋白和芝麻蛋白的胰蛋白酶水解物为原料，以胃蛋白酶为催化剂，通过合成类蛋白反应制取合成类蛋白质。结果表明，在上述反应中，胃蛋白酶是一种适宜的催化剂。底物水解度为８０％时效果最佳。大部分底物的分子量在１０００以下。底物最佳浓度为３０％，最佳ｐＨ为５．０。合成类蛋白质分子量约为１０，０００。     

Enzymatic Modification of Fish Frame Protein Isolate

Protein isolate was hydrolyzed with pepsin yielding an approximate degree of hydrolysis of 80%. Frame protein hydrolysates were then employed for plastein synthesis. Enzyme induced plastein was optimized at: (1) pH 5 for pepsin and pH 7 for alpha-chymotrypsin; (2) substrate concentration, 40% w/v; (3) time of incubation, 24 hr; (4) enzyme/substrate ratio, 1:100 w/w. Percent plastein yields of 33.8 and 27.3 were found for pepsin and alpha-chymotrypsin, respectively, when trichloracetic acid was used as the precipitating agent. However, when plastein was precipitated by ethanol, the percent yield was found to be 46 and 40.5 for pepsin and alpha-chymotrypsin, respectively.     

FAILURE OF SEPHAROSE-PEPSIN AS AN IMMOBILIZED MILK CLOTTING ENZYME

Porcine pepsinogen A was attached to CNBr-activated Sepharose 4B and self activated to form Sepharose-pepsin A. Immobilized pepsin is active in hydrolyzing hemoglobin at acidic pH but has very low milk clotting activity. The low milk clotting activity of freshly prepared Sepharose-pepsin can be accounted for by leaching of small amounts of free pepsin during contact of the complex with milk substrate. The failure of bound pepsin to catalyze milk clotting appears to be due to part of the micellar casein in milk substrate being inaccessible to the immobilized rennet and may also be influenced by differences in pH optima with casein substrate for pepsin and Sepharose-pepsin. Porcine pepsin A, which is electrophoretically pure, and casein substrate exhibit a distinct bimodal optimum reaction pH at 2 and 6. The reaction at pH6 is not evident when the products are assayed by A_280nm because the specific products formed at this pH exhibit low A_280nm. The finding that a pure isoenzyme of pepsin can exhibit a bimodal pH profile for hydrolysis has probably been overlooked by other investigators because of the frequent assay by A_280nm of products.     

罗非鱼下脚料蛋白合成类蛋白反应的工艺优化

以罗非鱼下脚料为原料,采用Alcalase蛋白酶对其进行控制酶解,制备不同水解度的罗非鱼下脚料酶解蛋白,在此基础上添加胃蛋白酶对其进行合成类蛋白反应的研究。以类蛋白产率为指标,在反应温度恒定(37℃)的条件下,通过单因素实验,确定底物水解度为40%、反应时间24h,选取底物浓度、加酶量和pH值3个因素进行响应面实验,通过响应面分析法优化合成类蛋白反应的最佳工艺组合为:底物浓度44.16%,加酶量4.12%,pH值5.08;在此条件下,类蛋白产率达到(10.08±0.03)%,与响应面二次模型预测值(10.11%)无显著差异。     

Pepsinogen and pepsin from the stomach of smooth hound (Mustelus mustelus): Purification,characterization and amino acid terminal sequences

Pepsinogen from the stomach of smooth hound (Mustelus mustelus) was purified to homogeneity by 20–70% ammonium sulphate precipitation, Sephadex G-100 gel filtration and DEAE-cellulose anion exchange chromatography with a 9.4-fold increase in specific activity and 38.36% recovery. Upon activation at pH 2.0, M. mustelus pepsinogen was converted to active form in one-step pathway. Molecular weights of the purified pepsinogen and the active pepsin were estimated to be 40,000 and 35,000 Da using SDS-PAGE and gel filtration, respectively. The optimum pH and temperature for the pepsin activity were pH 2.0 and 40 °C, respectively, using haemoglobin as a substrate. Activity was completely inhibited by Pepstatin A but not by phenylmethylsulphonyl fluoride, a serine-protease inhibitor and ethylenediaminetetraacetic acid, a metalloenzyme inhibitor. The N-terminal amino acid sequences of the first 15 amino acids of the activation segment of the pepsinogen and the first 20 amino acids of the active pepsin were LLRVPLRKGKSTLDV and ATEPLSNYLDSSYFGDISIG, respectively. M. mustelus pepsinogen, which showed high homology to rat C pepsinogen, had Thr-Leu-Asp sequence at amino acid positions 12–14 not found in all pepsinogen sequences. A remarkable substitution was found in the activation segment of M. mustelus pepsinogen: the Arg-13 conserved in all gastric proteinases, whose sequences are known, is replaced by Leu-13.     

湛江对虾副产物酶解制备抗氧化肽

以湛江对虾加工副产物虾头为研究对象,研究虾头的酶解条件和酶解产物的抗氧化活性,选用木瓜蛋白酶和胃蛋白酶分别对虾头酶解,制备高活性的抗氧化肽。在单因素的基础上,以清除DPPH自由基为指标,研究时间、温度、加酶量、pH对酶解产物抗氧化活性的影响。结果表明,与木瓜蛋白酶相比,胃蛋白酶酶解产物的抗氧化效果更好;通过正交试验,确定胃蛋白酶酶解产物的最佳条件为温度32℃、酶解时间1 h、pH 1、加酶量为1.33×10-5kat/g(以底物计),在此条件下酶解产物的抗氧化活性最高,对DPPH自由基清除率为89.12%。     

螺旋藻蛋白水解产物对ACE抑制活性的研究

采用胃蛋白酶和胰蛋白酶水解螺旋藻藻胆蛋白制备ACE 抑制肽,通过体外实验测定其ACE 抑制率,以ACE 抑制率为指标确定两种蛋白酶的水解条件。结果表明：胃蛋白酶水解条件为：水解温度37℃,酶与底物比1:50,pH3.0,底物质量分数5%,水解产物的ACE 抑制率为82.16%,其IC50 值为0.104mg/ml;胰蛋白酶水解条件为：水解温度42℃,酶与底物1:50,pH8.0,底物质量分数6%,水解产物的ACE 抑制率为93.54%,其IC50值为0.017mg/ml。此外,胃蛋白酶水解产物再用胰蛋白酶水解,其产物的IC50 值为0.087mg/ml。     

响应面试验优化酶解法制备海洋微藻微拟球藻抗氧化肽工艺

以海洋微藻饵料微拟球藻蛋白为原料,采用胃蛋白酶酶解制备微拟球藻抗氧化肽。以酶解产物的水解度和1,1-二苯基-2-三硝基苯肼自由基清除活性为评价指标,研究酶底比（质量百分比）、底物质量浓度、酶解温度和pH值对酶解产物的影响。在单因素试验的基础上,利用响应面法优化制备高抗氧化活性抗氧化肽的工艺条件,确定最佳酶解工艺条件为：底物质量浓度5.0?mg/mL、酶解温度32?℃、pH?1.36、酶底比6%。在最佳酶解条件下实际测得微拟球藻抗氧化肽的DPPH自由基清除率为51.85%。     

The effects of homogenisation method and freezing on the determination of quality parameters in red grape berries of Vitis vinifera

The effect of sample processing with three different blenders, sample freezing and storage time on the determination of berry quality parameters – total anthocyanins, total soluble solids (TSS), total phenolics (TP) and pH – in homogenates of red grape berries has been examined. Samples of Cabernet Sauvignon, Merlot and Shiraz from four regions of South Australia were homogenised using Ultra-Turrax, Waring or Retsch commercial blenders applied to fresh samples, and to samples held in frozen storage overnight or for 1, 3, 6 and 12 months. All samples were analysed immediately after homogenisation. The results showed that neither homogeniser type nor freezing had any significant effect on total anthocyanins or TSS. However, freezing had an effect on total anthocyanins concentrations in samples with higher colour. The homogeniser type had a significant effect on TP and pH for all varieties analysed together. Freezing had no significant effect on TP, but significant effect on pH, the value being greater in samples at the lower end of the pH range. Frozen storage time of up to 3 months had no significant effect on total anthocyanins, TSS or T P. Statistically significant differences were observed in the concentration of total anthocyanins in samples after 3 months of frozen storage.