双酶水解四角蛤蜊工艺优化研究

以四角蛤蜊为原料,使用内切-外切两步酶解工艺制备复合氨基酸选用多种蛋白酶对四角蛤蜊肉进行降解,以酶解液中氨基酸态氯含量和总氮回收率为考察指标,进而确定内-外两步水解的的蛋白酶种类,开在单因素实验的基础上.采用正交实验优化酶解工艺条件研究结果表明内-外两步酶解制备四角蛤蜊复合氨基酸的最佳工艺为:四角蛤蜊肉加3倍量水匀浆,匀浆液中加中性蛋白酶1500U/g肉,在酶解温度45℃、初始pH7.5下水解6h,灭酶活后改变条件,调pH7.0、温度为45℃,再加入复合蛋白酶4200U/g肉,在此条件下酶解11h.通过实验验证,中性蛋白酶和复合蛋白酶内-外两步酶解具有较好的水解效果,其氨基氮含量为20.78mg/g,总氮回收率为88.19％.     

脱脂米糠抗氧化肽的制备工艺研究

为了得到脱脂米糠抗氧化肽的最佳制备工艺,研究了胰蛋白酶、胃蛋白酶、中性蛋白酶、碱性蛋白酶、木瓜蛋白酶酶解脱脂米糠蛋白的进程特性以及不同的酶解条件对脱脂米糠抗氧化肽活性的影响。从5种蛋白酶中筛选出最合适的酶,通过单因素实验考察了底物浓度、加酶量、pH、温度以及时间对酶解产物水解度和ABTS自由基清除率的影响,在单因素实验的基础上,以酶解产物的ABTS自由基清除率为响应值,进行Box-Behnken中心组合实验。结果表明:选用碱性蛋白酶制备脱脂米糠抗氧化肽效果最好;最佳酶解工艺条件为加酶量1.8%、温度50℃、时间276 min、pH9.0、底物浓度5%;在最佳酶解工艺条件下,所得脱脂米糠抗氧化肽对ABTS自由基清除率可达71.85%。     

胰蛋白酶水解谷朊粉的酶解产物抗氧化性研究

研究了胰蛋白酶水解谷朊粉后酶解产物的抗氧化活性,在单因素实验基础上,运用响应面(RSM)对水解度、肽含量、还原力三个响应值进行分析,探讨酶解产物抗氧化活性最高的最佳水解条件。结果表明:各因素对提高水解度的重要性依次为pH>酶解时间>酶浓度>反应温度;最佳的实验条件为:谷朊粉浓度5%、酶浓度[E/S]29mg/g、pH10.5、反应温度45℃、反应时间2.7h,此时水解度21.4%,肽含量0.12mg/mL,还原力0.81,并通过实验验证了此结论。这说明小麦面筋蛋白经过胰蛋白酶水解后的酶解产物具有较好的抗氧化能力。     

酶解制备苦荞蛋白抗氧化肽及其分离纯化研究

以苦荞麦粉为原料,提取苦荞蛋白,分别采用碱性蛋白酶、胃蛋白酶、胰蛋白酶对蛋白进行酶解,采用DPPH法比较不同酶解产物的抗氧化活性,从而筛选水解制备苦荞蛋白抗氧化肽的最适酶。以水解度为指标,利用单因素试验和响应面法优化酶解工艺条件。结果表明,不同蛋白酶酶解产物的抗氧化活性大小为:胃蛋白酶胰蛋白酶碱性蛋白酶,其中胃蛋白酶酶解产物的DPPH自由基清除率最高,为68.47%。胃蛋白酶最佳水解工艺条件为:时间2.5 h、温度38℃、pH 2.0,在此条件下苦荞蛋白水解度为32.68%。采用超滤对苦荞蛋白水解物进行分离纯化,结果表明,分子量3 kDa的水解物具有显著的抗氧化活性;经凝胶过滤色谱进一步分离得到3个峰,小分子量峰组分显示出最强的抗氧化活性。     

蛤蜊酶解条件的优化及其酶解物抗疲劳活性的研究

以水解度为评价指标,比较了复合蛋白酶、木瓜蛋白酶、水产蛋白酶、中性蛋白酶、风味蛋白酶、胰蛋白酶和碱性蛋白酶对蛤蜊的酶解效果,结果表明,水产蛋白酶对蛤蜊的酶解效果优于其它蛋白酶;选用水产蛋白酶,利用单因素试验,比较了pH、温度、时间、料液比和加酶量对蛤蜊酶解效果的影响,并采用响应面试验设计对酶解条件进行了优化,结果表明,该酶的最优酶解条件为:酶的添加量2.0%,温度50.5℃,水解时间3 h,pH 6.0;利用小鼠负重游泳试验对蛤蜊酶解产物进行抗疲劳活性研究,结果表明,蛤蜊酶解物具有显著的抗疲劳活性。     

响应面法优化坛紫菜抗氧化肽酶法制备工艺

以坛紫菜为原料,通过酸性蛋白酶、中性蛋白酶、碱性蛋白酶、胰蛋白酶和纤维素酶酶解制备活性肽,以DPPH自由基清除率和多肽得率为评价指标,研究坛紫菜水解肽的抗氧化能力。结果表明:5种酶的酶解产物都具有抗氧化能力,中性蛋白酶酶解产物DPPH自由基清除率最高,选择它为最佳工具酶。通过单因素和响应面试验优化酶解工艺,得到最佳酶解工艺:酶解时间3.6 h、酶解温度47℃、酶用量16362 U/g、底物浓度3.0%、pH7.0。此条件下制备得到的水解肽具有较强抗氧化能力,DPPH自由基清除率可达(91.83±0.81)%。     

响应面试验优化四角蛤蜊调味料风味前体物质酶解制备工艺

以四角蛤蜊肉为原料,利用酶解技术获得四角蛤蜊调味料风味前体物质。以水解度和感官评价为指标,确定酶的种类与比例。在单因素试验的基础上,使用中性蛋白酶和复合风味蛋白酶,通过Box-Behnken响应面分析法,研究双酶酶解四角蛤蜊的工艺条件。研究结果：当中性蛋白酶和复合风味蛋白酶质量比为2∶1时,其酶解液的水解度和鲜度都最佳;双酶酶解最佳条件为酶添加量2.4‰、时间5.5 h、温度52 ℃、料液比1∶4（g/g）、自然pH值,此时酶解液的水解度较大（44.32%）,风味较好。     

鮟鱇鱼肝抗氧化肽的酶法制备及对羟自由基的清除作用

以脱脂鮟鱇鱼肝为原料,以水解度及对羟自由基的清除活性为考察指标,用木瓜蛋白酶、碱性蛋白酶、中性蛋白酶、胃蛋白酶及胰蛋白酶进行单酶解筛选试验。以水解度及对羟自由基的清除作用为指标,应用二次正交旋转组合设计试验研究加酶量、酶解时间、pH值及温度对制备鮟鱇鱼肝抗氧化肽工艺的影响。综合考虑水解度和对羟自由基的清除活性因素,最终确定碱性蛋白酶酶解鮟鱇鱼肝制备抗氧化肽的最佳工艺条件是:加酶量3000U/g,酶解时间6h,pH8.5,酶解温度55℃。该条件下制备的鮟鱇鱼肝抗氧化肽产物水解度和对羟自由基的清除分别为69.52%、76.74%。     

双酶法制备豌豆肽及其抗氧化活性

研究豌豆蛋白双酶水解的最佳工艺条件及产物的抗氧化活性。以豌豆蛋白粉为原料,通过单因素试验和正交试验优化出双酶分段水解豌豆蛋白的工艺条件,并初步研究豌豆肽的抗氧化活性。结果表明,双酶法制备豌豆肽的最佳工艺条件为:底物浓度10%,复合蛋白酶加酶量3.0%,pH 9.0,温度55℃,酶解3.5 h;用碱性蛋白酶酶解,加酶量3.0%,pH 9.5,温度50℃,酶解4.0 h。由此酶解得到水解物的水解度为39.61%。水解液蛋白浓度0.125 mg/mL时,其对Fe2+螯合能力为83.22%。试验表明和单酶水解相比,双酶水解工艺可提高豌豆蛋白的水解度和抗氧化活性。     

Box-Benhnken响应面优化巴旦杏抗氧化肽的制备工艺

本文研究了以巴旦杏粕蛋白为实验原料,通过Box-Benhnken响应面优化巴旦杏粕蛋白抗氧化肽的酶法制备工艺。以酶解产物的水解度及DPPH?清除率为评价标准从碱性蛋白酶、中性蛋白酶、胰蛋白酶、木瓜蛋白酶、复合蛋白酶中挑选最优水解酶,考察酶的添加量、pH值、酶解时间及酶解温度对酶解产物DPPH?清除率的影响。在单因素试验基础上,采用四因素三水平响应面法确定巴旦杏抗氧化肽酶法制备工艺。结果表明：碱性蛋白酶较适合制备巴旦杏抗氧化肽,其最佳酶解工艺条件为：酶解时pH为9.1,酶添加量为10000 U/g,酶解温度为58 ℃,酶解时间为4 h,此时酶解物的DPPH?清除率为74.45%。该条件适于制备的巴旦杏抗氧化肽,通过对巴旦杏抗氧化肽制备工艺的优化可为抗氧化肽的开发与应用提供理论借鉴。     

三种蛋白酶酶解核桃饼粕的抗氧化活性研究

该研究以核桃饼粕（WC）、石油醚脱脂核桃饼粕（PEWC）、石油醚及丙酮脱脂核桃饼粕（PEAWC）、核桃饼粕分离蛋白（WCP）、石油醚脱脂核桃饼粕分离蛋白（PEWCP）、石油醚及丙酮脱脂核桃饼粕分离蛋白（PEAWCP）为研究对象,分别采用碱性蛋白酶、胃蛋白酶、胰蛋白酶超声水解,以抗氧化活性为评价指标,筛选最佳的蛋白酶及原料处理方法。结果表明,碱性蛋白酶水解PEWC对OH·、O2-·和DPPH·清除率最高,分别为18.74%、28.44%和79.96%;胰蛋白酶酶解PEWC的总还原力最大;胰蛋白酶水解PEWCP的OH·和DPPH·清除率最高,分别为21.44%和80.22%,但O2-·清除率较低;胃蛋白酶水解PEWCP的O2-·清除率最高,达30.23%;碱性蛋白酶酶解PEWCP的总还原力最大;核桃饼粕经石油醚脱脂后的酶解产物抗氧化活性最高,且以碱性蛋白酶为最佳水解酶。     

Determination of the extracellular and cell-associated hydrolase profiles of Pseudomonas fluorescens sp. using the Analytab API ZYM system

The extracellular and cell-associated hydrolase profiles of a number of Pseudomonas fluorescens strains were examined with the Analytab API ZYM system. Esterase/lipase was the only strong extracellular enzyme activity detected (mean 3.33): weak esterase, lipase, and leucine aminopeptidase activities were found with some strains (mean activities of 1.08, 1.53, and 1.40, respectively). Very strong leucine aminopeptidase activity (4.5) was associated with the cells. Cell-associated trypsin, esterase/lipase, acid phosphatase, and phosphoamidase were also found. Neither extracellular nor cell-associated hydrolase profiles changed significantly when cells were grown in skim milk or mineral salts medium at either 5 or 20 degrees C. Similarly, added calcium did not seem required for synthesis of any of the enzymes. The extracellular enzyme profiles differed considerably from those of the cell-associated enzymes for all strains tested. An extracellular proteinase-deficient mutant of strain 32A (RM14) failed to produce significant quantities of extracellular esterase/lipase activity. Production of cell-associated enzymes was unaffected by the mutation. These results suggest that the Analytab API ZYM system may be useful in identifying psychrotrophs isolated from milk.     

CHARACTERIZATION OF PROTEINASE CLASSES IN LANGOSTILLA (PLEURONCODES PLANIPES) AND CRAYFISH (PACIFASTACUS ASTACUS) EXTRACTS

Proteinases (EC 3.4.21-24) in langostilla extract and crayfish hepatopancreas were classified using site specific effectors or chelators for either serine, cysteine, aspartic or metallo classes. Azocasein hydrolysis by langostilla and crayfish preparations was inhibited 50% and 40%, respectively, when assayed in the presence of serine proteinase inhibitor, phenylmethylsulfonyl fluoride. A similar degree of inhibition was observed with a trypsin inhibitor. However, no inhibition occurred with a chymotrypsin inhibitor. Less inhibition of azocasein hydrolysis, i.e., 20% and 14%, respectively, resulted with 1,10 phenanthroline. Inhibitors for cysteine and aspartic proteinases did not reduce the azocasein hydrolysis activities significantly. The amidase activity, with N-benzoyl-DL-Arg-p-nitroanilide as substrate was more sensitive, in both extracts, to serine proteinase inhibitors than the azocasein hydrolase activity. Results showed the presence of serine proteinases, i.e., trypsin but not chymotrypsin, as the major component and some minor activity of metallo dependent proteinases in the decapods extracts. Zymograms obtained after SDS-PAGE showed a dozen proteinases in each extract. Some of them were inhibited by a serine proteinase inhibitor and two to three were inhibited by 1,10 phenanthroline, supporting the results of the test tube proteinase activity assays. Furthermore, the reported technique for zymograms allowed direct comparison between extracts and provided information about their composition and the molecular weight of the targeted enzymes.     

Debittering and Hydrolysis of a Tryptic Hydrolysate of β-casein with Purified General and Proline Specific Aminopeptidases from Lactococcus lactis ssp. cremoris AM2

ABSTRACT: In this study, purified β-casein was hydrolysed with trypsin to produce a bitter substrate. The role of 3 aminopeptidases, a general aminopeptidase lysyl- para -nitroanilide hydrolase (KpNA-H), X-prolyl dipeptidyl aminopeptidase (Pep X) and aminopeptidase P (Pep P) each purified from Lactococcus lactis ssp. cremoris AM2, in the hydrolysis and debittering of the tryptic hydrolysate of β-casein, was then studied. The hydrolysates were analyzed for percentage degree of hydrolysis (DH%) and bitterness score. Results indicate that the hydrolysis and debittering potential of the general aminopeptidase (KpNA-H) is limited in the absence of proline specific aminopeptidases. Statistically significant (p < 0.001) reductions in bitterness were obtained following incubation of the tryptic digest of β-casein with specific combinations of the above aminopeptidases.     

Measurement of bile salt hydrolase activity from Lactobacillus acidophilus based on disappearance of conjugated bile salts

Bile salt hydrolase activity of Lactobacillus acidophilus was measured based on the disappearance of sodium glycocholate and sodium taurocholate from the reaction mixture using HPLC. The amount of sodium glycocholate and sodium taurocholate that disappeared was proportional to the amount of sodium cholate that appeared in the mixture as detected by HPLC. Sodium glycocholate did not precipitate at the enzyme reaction conditions (37 degrees C and pH 5.4) for determining bile salt hydrolase activity. The bile salt hydrolase assay was insensitive to low oxidation-reduction potential when measuring bile salt hydrolase from L. acidophilus, an intestinal microorganism. However, EDTA and freezing temperatures were necessary to maintain stability of the partially purified enzyme during storage.     

Detection and localization of a peptidoglycan hydrolase in Lactobacillus delbrueckii subsp. bulgaricus

Peptidoglycan hydrolase activities in Lactobacillus delbrueckii subsp. bulgaricus were detected by analysis of bacterial extracts on denaturing polyacrylamide gel electrophoresis containing lyophilized Micrococcus lysodeikticus cells as substrate. A hydrolase with an estimated molecular mass of 80 kDa was found to cross-react on Western blot with monoclonal antibodies raised against muramidase-2 of Enterococcus hirae. These antibodies were also used to demonstrate that the method of cell sample preparation affected protein detection. Slot and Western blots indicate that the peptidoglycan hydrolase from L. bulgaricus is bound to the cell wall. Immuno-labeling followed by optical and electron microscopic observations suggest that this hydrolase is intracellular and restricted mainly to the space between the membrane and the cell wall.     

Extraction of Carotenoprotein from Shrimp Process Wastes with the Aid of Trypsin from Atlantic Cod

Atlantic cod trypsin or bovine trypsin were used to aid the extraction of carotenoprotein from shrimp wastes at 4°C. When 25 mg% cod trypsin was added to extraction medium containing 0.5N ethylene diaminetetraacetic acid (EDTA) 64% of the astaxanthin and 81% of the protein of shrimp waste was recovered as carotenoprotein in 24 hr. With 25 mg% bovine trypsin, under otherwise identical conditions, the carotenoprotein recovered represented 49% of the astaxanthin and 65% of the protein of the waste. Semi-purified cod trypsin was not as effective as pure trypsin in facilitating recovery of carotenoprotein from shrimp waste. The recovery of carotenoprotein from shrimp waste, during extraction at 4°C with or without trypsin, was facilitated by EDTA.     

固定化胰蛋白酶水解乌鸡肉制备功能肽的研究

以海藻酸钠为载体,戊二醛为交联剂固定化胰蛋白酶,考察胰蛋白酶的固定化工艺,固定化酶水解乌鸡肉的工艺条件及固定化酶的稳定性。结果表明：固定化胰蛋白酶的最佳条件为：海藻酸钠浓度4%、加酶量10%、pH7.5、温度70℃,酶活力回收率为38.84%;水解乌鸡肉的最佳条件为：固液比1:3(m/V)、pH7.5、加酶量10%、温度60℃,氨基氮含量最高为2.18mg/ml,固定化酶重复使用8 次,酶活力仍保持50% 以上。     

塔拉单宁水解酶性质的研究

黑曲霉塔拉单宁水解酶经丙酮初提、葡聚糖凝胶G-150柱层析及PEG-6000浓缩,纯度为纯化前的8.9倍,比活力为6.77 nkat/g。SDS-PAGE电泳分析表明,塔拉单宁水解酶由2个亚基组成,其分子量分别为93.1 ku和39.8 ku。酶学性质显示,其作用的最适pH为4.0,稳定pH为3.5～5.5;最适作用温度为40℃,稳定温度小于50℃;以单宁酸为底物时,单宁酶的米氏常数Km值为1.276 mmol/L。研究结果可为其在实际生产中的应用提供一定的参考。     

一种胰蛋白酶亲和材料的制备及应用

基于抑制剂与酶的特异亲和作用,将具有良好稳定性的荞麦胰蛋白酶抑制剂(Buckwheat trypsin inhibitor,BTI)固定化,制备成一种胰蛋白酶的亲和吸附剂,实现胰蛋白酶的高效纯化。通过原核表达、Ni2+-NTA亲和层析和Superdex G-25凝胶过滤技术得到电泳纯的BTI,以溴化氰活化琼脂糖凝胶(CNBr-Sepharose CL-4B)作为亲和层析载体,制备亲和吸附剂BTI-Sepharose,检测其对胰蛋白酶的特异性吸附作用,并进一步研究BTI-Sepharose对胰蛋白酶吸附及解吸附的条件。制备的BTI-Sepharose对胰蛋白酶具有特异吸附性,可用于胰蛋白酶的亲和纯化。缓冲液p H对BTI-Sepharose与胰蛋白酶的吸附及解吸附均有重要影响,二者吸附作用的最适pH为7.2,在pH为3.5时两者能够完全分离,且不影响胰蛋白酶的生物活性。试验制备的BTI-Sepharose可实现一步层析制备高纯度胰蛋白酶,为胰蛋白酶的高效纯化及新型胰蛋白酶的研究开发提供理论依据。