一株产凝乳酶解淀粉芽孢杆菌的筛选、鉴定及酶学性质

采用酪蛋白培养基,从甘南牦牛放牧区分离筛选产凝乳酶细菌。通过形态学、生理生化特征和16SrDNA序列同源分析对菌株进行鉴定,并对该菌株所产凝乳酶的特性进行了研究。从牧区采集的56个样品中共筛选得到6株产凝乳酶细菌,复筛得到一株凝乳活力高、蛋白水解力低的菌株GN4.1,经鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens),在麸皮培养基中发酵48h,凝乳活力可达1011.56SU/mL,蛋白水解力为14.61U/mL。凝乳酶的最适作用温度为60℃,65℃加热10min后凝乳活力丧失;最适作用pH为5.5,在pH3.5～8.5内稳定性较好。预期该菌株所产凝乳酶有用于乳品加工业的潜力。     

酒曲中产凝乳酶微生物菌株的分离筛选及鉴定

针对酒曲中的微生物进行分离纯化,得到11株细菌和2株真菌,并采用酪蛋白平板法和Arima时间法筛选出了1株产凝乳酶的细菌菌株编号为LB-51。通过形态学观察、生理生化实验和16S rDNA序列分析鉴定该菌株为解淀粉芽孢杆菌,将该产凝乳酶菌株命名为解淀粉芽孢杆菌GSBa-1。该菌株在液体LB培养基中发酵72 h产凝乳酶的凝乳活力为(431.53±15.89)SU/mL,蛋白水解活力为(5.05±0.59)U/mL,所产凝乳酶凝乳活力高而蛋白水解活力低,凝乳酶粗酶单位酶活力为1.54×10~5SU/g。解淀粉芽孢杆菌GSBa-1是分离筛选自酒曲中的一株高产凝乳酶细菌,因此其来源安全,可作为工业化候选菌株进一步研究开发。     

产凝乳酶解淀粉芽孢杆菌的诱变选育

为了获得高产凝乳酶菌株,以产凝乳酶解淀粉芽孢杆菌为出发菌株,采用紫外线和硫酸二乙酯进行诱变。诱变后筛选得了一个突变株L-6,凝乳活力为1419.7 SU/m L,比原始菌株提高了40.2%;水解活力降低了22.39%,凝乳活力与蛋白水解活力的比值为125.64,比出发菌株提高了81.32%。传代实验表明,该菌株具有良好的遗传稳定性。     

江米酒凝乳酶酶学特性的研究

江米酒中的凝乳酶是引起我国传统乳制品米酒奶凝乳的原因.本实验对从江米酒中纯化得到的凝乳酶的酶学特性进行了研究.纯化凝乳酶的最适反应温度为45℃,酶活力在45～55℃之间比较稳定.纯化凝乳酶的最适反应pH为5.5,酶活力在pH值3.0～7.0之间比较稳定.Na 、K 、Ca2 、Mg2 、Zn2 、Mn2 、Fe2 均对凝乳酶的凝乳活力有促进作用,其中Ca2 对凝乳酶的凝乳活力有着显著地促进作用,Cu2 对凝乳活力有抑制作用.凝乳酶特异性的水解酪蛋白,而对乳白蛋白和乳球蛋白不产生水解作用.凝乳酶的酶切主要位点在α-酪蛋白第95位M的N-端,同时还存在其他酶切位点.     

酒曲发酵产凝乳酶条件优化及其凝乳特性研究

采用响应面分析法对酒曲发酵产凝乳酶的条件进行优化,确定其最佳产酶条件是:酒曲接种量5.7%,发酵时间72 h,摇床转速120 r/min,在此条件下凝乳酶凝乳活力为51.05 Su/m L,与理论预测值(52.29 Su/m L)接近。与商业凝乳酶作对照,探究不同因素对酒曲凝乳酶凝乳特性的影响,结果表明:在温度30~35℃,pH 5.5~6.1范围时,酒曲凝乳酶的凝乳效果较好。随着CaCl_2和凝乳酶添加量的增加,酒曲凝乳酶的凝乳效果增强。流变学特性表明,与商业凝乳酶相比,酒曲凝乳酶的凝乳时间较短,凝乳的粘性和弹性较高。微观结构观测结果:酒曲凝乳酶可使凝乳形成连续的、不规则且较为致密的网状结构,有利于其在干酪中的应用。     

解淀粉芽孢杆菌GSBa-1凝乳酶的生产及其酶学性质研究

采用正交试验设计优化了解淀粉芽孢杆菌GSBa-1发酵产凝乳酶的工艺条件:发酵温度35℃,装液量40%,摇床转速180 r/min,发酵时间84 h。在此优化条件下,获得的凝乳酶凝乳活力为558.14 Su/m L。进一步研究了该酶的酶学性质,凝乳酶最适反应温度为55℃,酶活力在25~45℃比较稳定,60℃保持50 min完全失活。在p H5.5时凝乳酶活力最高,在pH 5.5~7.0范围内,随着pH增大,凝乳酶活力逐渐下降,p H 6.5时,凝乳酶活力稳定性最高。Ca~(2+)、Mg~(2+)、Fe~(2+)、Zn~(2+)以及Al~(3+)均对凝乳酶的凝乳活力有促进作用,其中Ca~(2+)对凝乳活力的促进作用最为显著,且Ca~(2+)浓度为0.020 mol/L时凝乳酶的凝乳活力达到最大值,而Na~+、K~+和Cu~(2+)对凝乳活力均有抑制作用;凝乳酶Km为2.35 g/L,Vmax=1.18 U/m L。     

发酵法生产凝乳酶的概述

凝乳酶是干酪生产过程中的关键性酶,其主要作用是凝聚牛奶中的酪蛋白.本文简要介绍了凝乳酶的起源和它的凝乳机制.并着重阐述了微生物源凝乳酶的特点及研究者们在提高微生物凝乳酶活性降低其蛋白分解活力上所做的研究.     

红曲米中凝乳酶产生菌的筛选及液态发酵条件的优化

红曲米是我国古代流传下来的一种传统添加剂,红曲米中微生物所产凝乳酶具有凝乳作用.采用酪蛋白平板法和Arima时间法筛选出了1株产生凝乳酶能力强的菌株M5.通过正交试验,对该霉菌产凝乳酶发酵条件进行了初步优化, 结果表明,PDA 培养基是该霉菌产凝乳酶的最佳培养基,最佳培养条件为温度30℃、转速为160 r/min、振荡培养72 h,凝乳活力可达到150.6 SU.     

青海牧区产凝乳酶细菌多样性分析

为发掘性能优良的产凝乳酶细菌菌株,开发新型细菌凝乳酶。本文采用酪蛋白培养基从采自青海牧区土壤样品分离筛选产凝乳酶细菌,利用菌株16S r DNA基因序列对菌株进行鉴定并分析其多样性。结果表明,36个样品中分离得到21株产凝乳酶细菌,沉淀圈与菌落直径比值为1.41~5.5;分离菌株在不同培养基上凝乳酶和蛋白水解活性有明显差异,麸皮汁培养基中凝乳酶活性为11.3~1215.6 SU/m L,蛋白水解活力为14.6~59.5U/m L;21株菌中有14株菌属芽孢杆菌属(Bacillus),是产凝乳酶细菌的优势属;多样性指数分析表明,该地区产凝乳酶细菌具有丰富多样性。     

解淀粉芽孢杆菌GSBa-1凝乳酶在马苏里拉干酪中的应用

对分离自酒曲的1株解淀粉芽孢杆菌GSBa-1发酵所产凝乳酶进行研究,该酶凝乳活力高而蛋白水解活力低,纯酶凝乳活力可达1.46×106 SU/g;使用该凝乳酶和商品凝乳酶制作马苏里拉干酪,并对干酪理化成分、成熟过程中pH值和微生物指标及干酪成熟前后质构特性、游离脂肪酸、可溶性蛋白、风味和干酪性能等指标进行对比分析。结果显示,理化成分上菌株凝乳酶与商品凝乳酶制作的干酪相接近(P0.05)。干酪在成熟过程中,发酵剂存活数先增加后减少,但其差异不大;菌株凝乳酶制作的干酪pH 4.6可溶性蛋白含量较多,干酪的游离氨基酸总量(76 mg/100 g)也高于商品凝乳酶制作的干酪游离氨基酸总量(55.3 mg/100 g);菌株凝乳酶制作的干酪质构特性优于商品凝乳酶制作的干酪;电镜结果显示,菌株凝乳酶制作的干酪内部网状结构更充实;菌株凝乳酶具有稍强的蛋白水解活力,导致其制作的干酪风味物质种类多于商品凝乳酶制作的干酪,风味物质更加丰富。干酪样品的保形性和拉丝性实验测定结果显示,2种凝乳酶制作的干酪性能差异不大(P0.05);对2种凝乳酶制作的干酪进行感官评定,其总评分相接近。以上结果表明,解淀粉芽孢杆菌GSBa-1凝乳酶在一定程度上可代替小牛凝乳酶应用于马苏里拉干酪的生产。     

1株高产凝乳酶菌株的鉴定及N~+注入诱变选育

为获得高产凝乳酶的菌株,以市售干酪为原料,从中分离得到1株具有高凝乳活力的菌株XC-1。通过生理生化实验、16S rDNA同源性序列分析,鉴定该菌株为枯草芽孢杆菌(Bacillus subtilis)。采用N+注入法对筛选出的菌株进行诱变,确定最佳诱变条件为能量20 keV、剂量2.08×1015ions/cm2,获得1株特性优良的诱变菌株XCYB-6,其凝乳活力达到2 181.82 SU/mL,比出发菌株提高65.63%,蛋白水解活力为2.53 U/mL,凝乳活力与蛋白水解活力的比值高达862.38。传代(6代)实验表明,诱变菌株具有良好的遗传稳定性。     

Production and characterization of milk-clotting enzyme from <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> JNU002 by submerged fermentation

Milk-clotting enzymes produced by microorganisms have been developed to replace calf rennet, yet the enzymatic level and the ratio of milk-clotting activity to proteolytic activity still need further improvement. This work described a strain Bacillus amyloliquefaciens JNU002 that screened from wheat bran that has a promising characterization. After optimization, B. amyloliquefaciens JNU002 showed a high milk-clotting activity (4969 SU/mL) and low proteolytic activity (4.02 U/mL) at 48 h with an inoculum size of 0.2% (v/v) at initial pH 6.0 in 15-L bioreactor. After purification, the purified enzyme gave a single protein band on SDS–PAGE, corresponding to 28 kDa. The purified enzyme showed a high ratio (2,575) of milk-clotting activity to proteolytic activity at 35 °C, and the ratio would be even higher (22,992) at 70 °C. The milk-clotting reaction and proteolytic reaction were prevented at 75 °C. This enzyme was stable at pH 4–6 and below 40 °C, and this was convenient for storage and transportation.     

Novel milk-clotting enzyme produced by Coprinus lagopides basidial mushroom

Submerged cultured higher basidiomycetes were screened for milk-clotting activity. The native liquid of Coprinus lagopides demonstrated high milk-clotting activity, while having relatively low general proteolytic activity. Optimization of growth media composition and ultrafiltration of the native liquid allows increasing the ratio of milk-clotting and proteolytic activities. The Enzyme was purified and characterized.     

江米酒凝乳酶的纯化及凝乳机制初探

江米酒凝乳酶是扣碗酪生产中起关键作用的凝乳因子。文中采用纤维素离子交换柱从江米酒中得到分子质量为36·4ku的单一蛋白酶,其凝乳活力与蛋白酶水解活力的比值从纯化前的3·1提高到16·0。对纯化酶的基本特性研究表明,该酶在30~55℃,pH值3·0~7·0保持比较稳定的酶活性,而与胃蛋白酶、米黑毛霉凝乳酶及牛凝乳酶凝乳过程中的组织状态变化比较,初步认为江米酒凝乳酶的动力学或凝乳机制可能与牛凝乳酶等其他天冬氨酸蛋白酶不完全相同。     

响应面法优化红曲米中凝乳酶高产菌株的发酵条件

对从红曲米中分离得到的产凝乳酶能力强的菌株M5传代菌株的液态发酵培养基及产酶条件进行优化。首先进行单因素实验得到适宜的氮源为(NH4)2SO4、酪蛋白,无机盐为FeSO4、KH2PO4,适宜的接种量为1%,培养温度为30℃,培养时间为5d,发酵培养基初始pH为6.0。在此基础上通过Plackett-Burman实验筛选出对酶活影响显著的三个因素:(NH4)2SO4含量、培养时间和培养温度。再用Box-Behnken响应曲面实验对三个显著因素进行优化。结果表明,产酶的最佳培养基组分:(NH4)2SO40.53%、FeSO40.05‰、KH2PO40.05%、干酪素0.5%、葡萄糖2%、接种量1%。最佳发酵条件为:培养温度31.3℃、摇床转速为180r/min、培养时间113h、pH6.0。基于响应曲面优化的产凝乳酶培养基组成与发酵条件效果显著,供试菌株M5传代菌株所产凝乳酶活性由45.34SU/mL提高到190.68SU/mL。     

MULTIVARIATE ANALYSIS OF STRUCTURE-RELATED DATA TO EXPLAIN MILK CLOTTING ACTIVITY OF PROTEOLYTIC ENZYMES

Multivariate analysis was used for investigating the relationships between milk-clotting activity and physical/chemical properties, such as CD spectra, hydrophobicity and zeta potential, of ten proteolytic enzymes measured at six different pH values. Cluster analysis of CD data and zeta potential values classified the enzymes into five groups, i.e., three active enzyme groups and two inactive enzyme groups with respect to milk clotting activity. Classification of the enzymes into three groups, i.e., enzymes with high, medium and low milk-clotting activity, was achieved by discriminant analysis after adding the ratio of secondary structure parameters to the predictor variables. Results indicated that β-sheet, β-turn and random structure features were important for milk-clotting activity.     

Comparison of the milk-clotting properties of three plant extracts

Several proteases from plant sources have been proposed as milk coagulants, however, limited research has been done on their milk-clotting properties. The effect of temperature on the milk-clotting activity of kiwi fruit, melon and ginger extracts was evaluated, as well as the effects of the different extracts on curd properties. Melon extracts showed high milk-clotting activity over a broad temperature range (45–75 °C) while kiwi fruit and ginger extracts showed high activity over a narrower temperature range, with a maximum at 40 and 63 °C, respectively. Curds produced using kiwi extracts had textural properties comparable with those obtained using commercial rennet, while melon extracts produced a fragile gel and low curd yield. The milk-clotting behavior of the three plant extracts was related to the protease specificity present in these extracts. The kiwi proteases displayed chymosin-like properties and thus hold the best potential for use as a milk coagulant in cheese production.     

Production and characterization of a milk-clotting protease in the crude enzymatic extract from the newly isolated Thermomucor indicae-seudaticae N31: (Milk-clotting protease from the newly isolated Thermomucor indicae-seudaticae N31)

Microbial rennet-like milk-clotting enzymes are aspartic proteinases that catalyze milk coagulation, substituting calf rennet. Crude enzymatic extract produced by the thermophilic fungus, Thermomucor indicae-seudaticae N31, on solid state fermentation (SSF) using wheat bran, exhibited high milk-clotting activity and low proteolytic activity after 24 h of fermentation. Highest milk-clotting activity (MCA) was at pH 5.7, at 70 °C and in 0.04 M CaCl₂; it was stable in the pH range 3.5–4.5 for 24 h and up to 45 °C for 1 h. MCA was highly inhibited by pepstatin A. Hydrolytic activity profile of the crude enzymatic extract on whole bovine casein, analyzed by gel electrophoresis (Urea–PAGE) and RP-HPLC revealed low proteolytic action towards casein fractions and a peptide profile similar to the one obtained with commercial Rhizomucor miehei protease (Hannilase).     

凝乳酶高产菌株的超声波-紫外复合诱变育种及其发酵条件的优化

以编号为Jl-1的黑曲霉为出发菌株,通过超声波和紫外诱变处理,在酪蛋白培养基上以凝乳圈直径为指标进行初筛,在基础固态发酵培养基上进行复筛,选育得到一株具有良好遗传稳定性的突变菌株.其经固态发酵凝乳酶产量为凝乳活力600U/mL,较出发菌株Jl-1提高57%.菌株经8次传代培养,凝乳酶产量仅降低7.1%.在此基础上应用单因素试验和正交设计对菌株产凝乳酶的固态发酵条件进行了系统研究和优化,得到最佳的试验组合为发酵温度28℃.发酵时间为6d,加水量为20mL,加样量为4份.采用此条件,凝乳酶产量达828U/mL,比Jl-3优化前提岛38%.