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（P＜0.05）。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（P＞0.05）;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。     

地衣芽孢杆菌凝乳酶对切达干酪成熟过程中蛋白水解的影响

利用地衣芽孢杆菌凝乳酶制作切达干酪和切达干酪类似物,分析干酪成熟过程中各蛋白水解指标的变化规律,以揭示地衣芽孢杆菌凝乳酶对切达干酪成熟过程中蛋白水解的影响。结果表明,CDF组（添加地衣芽孢杆菌D3.11凝乳酶所制切达干酪）、CD3组（添加地衣芽孢杆菌D3.11凝乳酶但未添加发酵剂制成的干酪类似物）和CCF组（添加商品凝乳酶所制切达干酪）干酪蛋白含量、pH 4.6-可溶性氮、12%三氯乙酸-可溶性氮、5%磷钨酸-可溶性氮、总游离氨基酸含量均随着成熟时间延长呈显著增加趋势,并且成熟期间CDF组干酪均显著高于CCF组干酪（P＜0.05）;十二烷基硫酸钠-聚丙烯酰氨凝胶电泳分析表明,CDF组干酪α-酪蛋白水解程度较大;pH 4.6-可溶性肽段分析表明,随着干酪的成熟,总肽含量呈先增加后下降趋势,但疏水性肽与亲水性肽的比值呈持续下降趋势,在成熟第6个月时,CDF组、CD3组和CCF组干酪疏水性肽与亲水性肽比值分别为2.668、2.822、3.788。主成分分析表明,3 组干酪的蛋白水解程度与成熟度呈正相关,与疏水性肽和亲水性肽的比值呈负相关。以上结果表明,利用地衣芽孢杆菌凝乳酶制作的干酪蛋白水解度更高,但其疏水性肽比例较小,研究结果可为地衣芽孢杆菌凝乳酶在干酪生产中的应用提供理论依据。     

甲醇芽孢杆菌凝乳酶的重组表达及其结构特性

为进一步了解微生物凝乳酶的结构特性,根据GenBank数据库中甲醇芽孢杆菌凝乳酶（I3EB99）的氨基酸序列和大肠杆菌密码子的偏爱性,设计合成此凝乳酶的全基因序列,构建原核表达载体,通过BL21（DE3）表达其融合蛋白,将获得的融合蛋白进行His标签特异性亲和纯化,并利用生物信息学方法研究凝乳酶的三维空间立体结构。结果表明,重组表达的甲醇芽孢杆菌凝乳酶质量浓度为0.7 mg/mL,凝乳活力为（15 870±1.17）SU/g,蛋白水解活力为（263.81±0.94）U/g,凝乳活力与蛋白水解活力比值为60.16,符合干酪生产加工的要求。结构特性研究表明,经重组表达后的甲醇芽孢杆菌凝乳酶呈现疏水特性,具有跨膜结构和信号肽,二级结构中α-螺旋少于β-折叠,在分离纯化过程中结构不稳定易降解,该凝乳酶与来自甲醇芽孢杆菌的一种未知蛋白酶是同源蛋白,高级结构与PDB蛋白数据库中的模板蛋白2ra1.1.A相似度最高。通过对甲醇芽孢杆菌凝乳酶结构特性的研究,为深入分析该凝乳酶作用机理及其功能性奠定了理论基础。     

玉米醇溶蛋白基马苏里拉干酪的研制

近年来,植物基干酪受到广泛关注。以玉米醇溶蛋白、水和椰子油为原料,干酪的功能特性（拉伸性、熔化性、油脂析出性）为评价指标,经单因素和正交试验确定玉米醇溶蛋白基马苏里拉干酪的最佳配比为玉米醇溶蛋白∶水∶椰子油=3∶3∶2（质量比）。最佳配方所得玉米醇溶蛋白基马苏里拉干酪的拉伸性、熔化性分别为市售天然马苏里拉干酪和市售植物基马苏里拉干酪的1.4、10.8倍和1.8、1.4倍。硬度、黏着性、弹性与市售天然马苏里拉干酪相近。试验所得玉米醇溶蛋白基马苏里拉干酪有良好的功能特性和质构特性,具有广阔的市场应用前景。     

原辅料对再制马苏里拉干酪品质特性的影响

以天然马苏里拉干酪为目标,以干酪融化后特性(融化性、油脂析出率、拉伸性和褐变性)、感官、微观结构为指标,研究原辅料对再制马苏里拉干酪融化后品质特性的影响。结果表明:选择比例为1∶1(m/m,下同)的成熟度为3个月切达干酪和马苏里拉干酪,配合比例为1∶4色拉油和奶油进行再制马苏里拉干酪的制作,既保证了再制干酪所需的质构,同时也赋予产品特有的风味;添加量为1.3%的乳化盐和2.0%的柠檬酸能提供再制干酪所需的乳化性和合理的p H值,产品的加工特性最佳。     

凝乳酶对低脂干酪微观结构和功能特性的影响

研究了低脂干酪成熟过程中蛋白质水解程度对干酪本身的微观结构和功能特性的影响.以低脂乳为原料,添加不同剂量的凝乳酶制备低脂干酪,测定干酪不同成熟期的熔度及质量分数为12%的TCA-SN并观察干酪的微观结构.结果表明,在低脂干酪中添加双倍的凝乳酶时可以减少干酪的硬度、增加熔度和改善其感官状态;当添加3倍凝乳酶时将会导致产品比添加正常凝乳酶量时更有胶弹性.因此,添加双倍凝乳酶时能有效改善低脂干酪的质构、功能特性及感官状态.     

黄酒麦曲中产凝乳酶菌株的分离鉴定

本研究利用梯度稀释法和划线纯化法对黄酒麦曲中的微生物进行初筛,再利用酪蛋白平板法和液态培养基发酵法进行复筛,最终得到了两株产凝乳酶的优良细菌菌株LB-1和LB-2。通过形态学特征观察、生理生化实验以及16S rDNA序列分析鉴定,这两株菌分别确定为甲醇芽孢杆菌(Bacillus methanolicus)和枯草芽孢杆菌(Bacillus subtilis)。根据凝乳活力曲线发现,这两株菌在发酵24 h时其发酵液的凝乳活力最高,分别为269.66±0.78 SU/m L和187.50±1.4 SU/m L,此时的蛋白水解活力分别为1.476±0.49 U/m L和1.29±1.41 U/m L,凝乳活力与蛋白水解活力比值(C/P)分别为187.50和145.34。通过凝乳效果评价,其发酵液所形成的凝块组织结构、质构参数和风味物质均与商品凝乳酶形成的凝块相当,适用于干酪的加工。     

解淀粉芽孢杆菌GSBa-1凝乳酶对羊奶干酪成熟特性的影响

为探究解淀粉芽孢杆菌GSBa-1凝乳酶制备的羊奶干酪(干酪B)成熟特性的变化,以采用商业凝乳酶和同批次羊奶制作的干酪(干酪A)为对照组,比较两组干酪在60d成熟期主要组分、质构特性、微生物指标及风味物质的变化。结果表明,两组干酪得率相差不大。成熟期间干酪的水分、蛋白质及脂肪含量呈先上升后下降趋势,干酪B始终高于干酪A;干酪游离氨基酸总量在成熟期间呈先下降后上升趋势,且干酪B中苯丙氨酸、天冬氨酸、异亮氨酸、甲硫氨酸、丝氨酸含量高于干酪A。成熟前期干酪B质构特性优于干酪A。干酪A成熟后乳酸乳球菌数量增加了(5.22±0.02)%,干酪B无显著变化(P>0.05)。成熟期内,两组干酪中挥发性风味物质种类和含量均增加,但干酪B中的壬酸、辛醇、2-庚酮、2-壬酮、二甲基砜使羊奶干酪风味独特、浓郁。因此,GSBa-1凝乳酶具备替代商业凝乳酶用于羊奶干酪生产的潜力,可对干酪风味的形成和品质的提升起到一定促进作用。     

水牛αS1-酪蛋白多态性对马苏里拉干酪品质的影响

目的分析水牛α_(S1)-酪蛋白多态性对马苏里拉干酪品质的影响。方法以α_(S1)-酪蛋白AB型、α_(S1)-酪蛋白BB型和混合组水牛乳样为原料分别制成全脂马苏里拉干酪,比较3组水牛乳样和干酪在组成、功能特性和微观结构等方面的差异。结果α_(S1)-酪蛋白AB型水牛乳在脂肪、蛋白质和总固形物含量上显著高于BB型。制成马苏里拉干酪后,AB型干酪的蛋白质含量显著高于BB型和混合组干酪。BB型和AB型干酪在硬度、咀嚼性和胶黏性上显著高于混合组。结论不同基因型α_(S1)-酪蛋白水牛乳的乳成分存在差异,α_(S1)-酪蛋白多态性与水牛乳马苏里拉干酪的组成、质构和融化特性等存在显著关联。     

凝乳酶的研究进展

凝乳酶是一种最早在未断奶的小牛胃中发现的天门冬氨酸蛋白酶,可专一地切割乳中κ-酪蛋白的Phe105-Met106之间的肽键,破坏酪蛋白胶束使牛奶凝结,凝乳酶的凝乳能力及蛋白水解能力使其成为干酪生产中形成质构和特殊风味的关键性酶,被广泛地应用于奶酪和酸奶的制作。本文以牛凝乳酶为例介绍了凝乳酶的结构、理化特性和凝乳机理,综述了凝乳酶主要来源以及不同来源凝乳酶之间酶性质差异,旨在为凝乳酶研究提供些许参考。     

Characteristics of Miniature Cheddar‐Type Cheese Made by Microbial Rennet from Bacillus amyloliquefaciens: A Comparison with Commercial Calf Rennet

Miniature Cheddar‐type cheeses were produced using microbial rennet from Bacillus amyloliquefaciens (milk‐clotting enzyme [MCE]) or calf rennet (CAR). With the exception of pH, there were no significant differences in gross composition between MCE‐cheese (MCE‐C) and CAR‐cheese (CAR‐C). The pH value of CAR‐C was significantly higher than that of MCE‐C at 40 and 60 d of ripening. The total nitrogen content of the pH 4.6‐soluble fraction obtained from MCE‐C was higher than that obtained from CAR‐C. However, nitrogen content of the 12% TCA‐soluble fraction was similar between CAR‐C and MCE‐C. The extent of α_s1‐casein and β‐casein hydrolysis, measured by urea‐PAGE, was similar in both cheese samples. The hydrolysis of β‐casein was lower than that of α_s1‐casein. Different reverse phase‐high‐performance liquid chromatography peptide profiles of ethanol‐soluble and ethanol‐insoluble fractions were obtained from CAR‐C and MCE‐C. The peptide content in the 2 cheese samples increased throughout ripening; the ratio of hydrophobic to hydrophilic peptides was lower in MCE‐C than in CAR‐C. Compared with CAR‐C, MCE‐C was softer as a result of higher protein hydrolysis. Microbial rennet from B. amyloliquefaciens contributed to higher proteolytic rates, which reduced ripening time.     

Vatless manufacturing of low-moisture part-skim mozzarella cheese from highly concentrated skim milk microfiltration retentates

Low-moisture, part-skim (LMPS) Mozzarella cheeses were made from concentration factor (CF) 6, 7, 8, and 9, pH 6.0 skim milk microfiltration (MF) retentates using a vatless cheese-making process. The compositional and proteolytic effects of cheese made from 4 CF retentates were evaluated as well as their functional properties (meltability and stretchability). Pasteurized skim milk was microfiltered using a 0.1-microm ceramic membrane at 50 degrees C to a retentate CF of 6, 7, 8, and 9. An appropriate amount of cream was added to achieve a constant casein:fat ratio in the 4 cheesemilks. The ratio of rennet to casein was also kept constant in the 4 cheesemilks. The compositional characteristics of the cheeses made from MF retentates did not vary with retentate CF and were within the legal range for LMPS Mozzarella cheese. The observed reduction in whey drained was greater than 90% in the cheese making from the 4 CF retentates studied. The development of proteolytic and functional characteristics was slower in the MF cheeses than in the commercial samples that were used for comparison due to the absence of starter culture, the lower level of rennet used, and the inhibition of cheese proteolysis due to the inhibitory effect of residual whey proteins retained in the MF retentates, particularly high molecular weight fractions.     

Effect of artisanal kid rennet paste on lipolysis in semi-hard goat cheese

This study examined the use of hygienised kid rennet pastes in model cheese systems and also in goat milk semi-hard cheeses to promote lipolysis. The results obtained indicated that the use of rennet paste caused greater lipolysis and increased, mostly, the short-chain free fatty acid (FFA) content. The model systems made with whole goat’s milk using rennet paste and commercial rennet mixture exhibited a higher FFA content than did the rennet paste-free controls (31,600 vs. 25,600 μmol/kg cheese). For the pilot cheeses made with bovine rennet and rennet paste mixture, the increase in FFA level after 45 days of ripening compared with the cheeses prepared only with commercial rennet was as much as 6600 (μmol/kg cheese) and the increase in the butyric acid content was also 1650 (μmol/kg cheese). Moreover, after 15 days of ripening, industrially prepared cheeses made with rennet paste exhibited greater levels of FFA than did the cheeses made with commercial rennet (11,500 μmol/kg at 45 days of ripening). Their flavour was stronger and the organoleptic characteristics were better accepted, which implies less ripening time for commercial cheese manufacture.     

Effect of the use of three different lamb paste rennets on lipolysis of the PDO Pecorino Romano Cheese

Two rennet pastes were prepared from lambs on different diets, and then, together with a commercial lamb rennet paste, were used to produce protected designation of origin (PDO) Pecorino Romano cheeses. Chymosin activity and lipolytic activity were higher in the prepared rennets than in the commercial rennet. Lipolysis was greatest in cheese produced with one of the prepared rennets. Both quantitative and qualitative differences in free fatty acids were found in the cheeses. The taste of cheeses made with the prepared rennets was preferred by an expert sensory panel and also considered more piquant than that of cheese made with commercial rennet. The results indicate that both the diet of the lambs and slaughtering conditions should be regulated to produce rennet that preserves the traditional characteristics of PDO Pecorino Romano cheese. Both lipolytic and proteolytic properties of rennet pastes should be standardised for the preparation of such cheeses.     

乳清添加对马苏里拉奶酪成熟过程中风味物质及其品质的影响

该研究以未添加乳清的马苏里拉奶酪为对照,考察乳清对马苏里拉奶酪成熟期内（0 d、30 d、60 d、90 d）乳酸菌活菌数、油脂析出性、挥发性风味物质及氨基酸含量的影响,并对挥发性风味物质与氨基酸进行相关性分析。结果表明,在奶酪成熟期内,两种奶酪的活菌数均呈先增加后减少的趋势,油脂析出性均增加,且乳清奶酪均高于对照奶酪;两种奶酪的挥发性风味物质存在差异,从2种奶酪中共检测到61种挥发性风味物质,其中对照奶酪中共检出43种,乳清奶酪中共检出55种。乳清奶酪中的醇、酸、醛、酮、酯类物质的含量及种类均高于对照。两种奶酪成熟期间均分别检测到15种游离氨基酸,乳清奶酪成熟90 d时的氨基酸含量（848.92 mg/kg）高于对照奶酪（663.44 mg/kg）。除精氨酸、丝氨酸、酪氨酸、天冬氨酸外,其余氨基酸对20种挥发性风味物质具有积极作用。综上,乳清添加可提升马苏里拉奶酪的风味。     

Improvement of sensory quality of lowfat kefalograviera-type cheese by using commercial special starter cultures

Two commercially available special starter culture systems, Alp DIP and a mixture of Alp DIP D and Joghurt V1, were compared with one commercial regular starter culture, CH-1, for their effects on the compositional, sensory and textural characteristics of lowfat (9.5%) high moisture (49.6%) Kefalograviera-type cheese during aging. A full-fat control Kefalograviera cheese (30.8% fat, 37.8% moisture) was also made with the regular starter culture. The results indicated that the type of starter did not affect the composition (moisture, fat, protein, salt and pH) of the lowfat cheese. Sensory analysis showed that the lowfat cheeses made with the special cultures received greater body and texture scores and significantly higher flavor scores than the lowfat control cheese after aging for 90 and 180 d. Moreover, the former cheeses received body and texture and flavor scores not significantly different from those of the full-fat cheese. Texture profile analysis by Instron showed that there were no significant differences in the textural characteristics (force and compression to fracture, cohesiveness, springiness, gumminess and chewiness) between lowfat cheeses made with the special cultures and that made with the regular starter, except for hardness which was significantly lower in the former cheeses.