甲醇芽孢杆菌凝乳酶对马苏里拉干酪加工特性的影响

为了探究甲醇芽孢杆菌（Bacillus methanolicus）凝乳酶在马苏里拉干酪加工中的应用,分别以使用甲醇芽孢杆菌凝乳酶、混合酶制剂（含质量分数10%甲醇芽孢杆菌凝乳酶和90%商品凝乳酶）制作的马苏里拉干酪作为实验组,以商品凝乳酶干酪作为对照组,测定不同组别干酪成熟期间的蛋白水解特性、质构、风味和微观结构变化,研究甲醇芽孢杆菌凝乳酶对马苏里拉干酪加工特性的影响。结果表明,实验组干酪在成熟过程中pH值（4.6～5.3）、微生物数量（8.80～9.68（lg（CFU/g）））与对照组无显著差异（P＞0.05）;实验组干酪水分质量分数（混合酶干酪为（43.21±1.17）%、甲醇芽孢杆菌凝乳酶干酪为（46.15±0.94）%）均显著高于对照组（（41.08±1.04）%）,得率（混合酶干酪为（9.27±0.17）%、甲醇芽孢杆菌凝乳酶干酪为（9.46±0.16）%）也显著高于对照组（（8.98±0.13）%）（P＜0.05）;且实验组干酪的蛋白水解特性（pH 4.6时可溶性蛋白、酪蛋白水解程度和游离氨基酸质量分数）以及风味物质种类和相对含量等指标也优于对照组干酪。但是实验组中甲醇芽孢杆菌凝乳酶干酪保形性相对欠佳,感官评定得分偏低,混合酶干酪与对照组质构及感官基本得分一致,因此甲醇芽孢杆菌凝乳酶可以作为商品酶的部分代替品应用于干酪的生产中。     

契达干酪浆中使用热激瑞士乳杆菌的研究

在食品加工中添加酶解干酪比添加天然干酪更加营养、经济、有效。为了改善酶解干酪的风味,研究经优化热激(67℃/20s,68℃/15s,69℃/10s)条件处理与未处理的瑞士乳杆菌对干酪浆蛋白水解的影响。研究结果表明,经过5d成熟时间,蛋白水解产生肽段的数量增加,疏水性肽对亲水性肽的比率降低。感官评价结果显示,在适当条件下热激组的干酪浆滋气味显著增加,酸味降低,硬度减小。     

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。     

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。     

干酪中含氮物的快速测定方法

目前，人们已经可以利用蛋白质、肽以及氨基酸的结构特点定量测定出复杂食品体系中的含氮物含量。这些方法中，主要是光谱法。但是由于浊度的干扰，该分析法仅限于测定可溶性氮含量。在干酪制品中，如在切达干酪成熟过程中，可溶性氮的形成可以表示出蛋白水解程度，而且干酪中风味物质及其前体物质的形成，以及干酪的质地都与蛋白水解有关。因此，快速准确地测定干酪蛋白质、多     

天祝放牧牦牛生活环境土壤中一株产凝乳酶细菌的分离与鉴定

筛选高产凝乳酶细菌,并对其进行鉴定和产凝乳酶活力测定。利用酪蛋白培养基从天祝放牧牦牛生活环境土壤中筛选高产凝乳酶细菌,利用形态观察、生理生化检测、16S rDNA 序列分析对其进行鉴定,并采用Arima法和福林法分别测定凝乳酶活力和蛋白水解活力。获得18 株产凝乳酶细菌,其中菌株D3.11 产凝乳酶活力相对最高,鉴定为地衣芽孢杆菌(Bacillus licheniformis),其在麸皮培养基中发酵72 h 产凝乳酶活力高达89.56 SU/ml,蛋白水解力为21.27U/ml。菌株D3.11 是一株高产凝乳酶细菌,可作为候选菌株进行进一步研究。     

不同温度条件下地衣芽孢杆菌凝乳酶的凝乳性能

为研究温度对地衣芽孢杆菌D3.11凝乳酶凝乳性能的影响,以脱脂乳为底物,并以商品凝乳酶为对照,分析凝乳过程中黏度、浊度、持水力、OD值及流变学特性的变化规律,观察凝块的微观结构。结果表明,随着凝乳温度的升高,2种酶体系的黏度值逐渐增大,温度较高时,长时间凝乳会导致体系黏度值下降;在30～45℃时2种酶体系的浊度分别随温度升高显著增大(P<0.05);流变学特性表明,商品酶体系所能达到的储能模量(G′)的峰值为269.01 Pa,大于地衣芽孢杆菌D3.11凝乳酶体系的峰值232.91 Pa,且随着凝乳温度的上升,2种酶体系的G′不断下降,商品酶体系比地衣芽孢杆菌体系的G′下降更快;2种酶体系持水力均随温度的上升呈先上升后下降的趋势,在35℃时2种酶体系的持水力均达到最大;2种酶体系的乳清OD值随温度升高显著增大(P<0.05),地衣芽孢杆菌凝乳酶体系的乳清OD值均大于商品酶体系;利用激光扫描共聚焦显微镜观察微观结构,发现40℃条件下2种酶体系的凝乳效果最好。该研究发现温度对地衣芽孢杆菌D3.11凝乳酶的凝乳性能影响显著,实验结果可为地衣芽孢杆菌凝乳酶的应用提供理论依据。     

添加蛋白酶微胶囊干酪的反相高效液相色谱分析

利用阿拉伯胶和明胶为壁材,以毛霉蛋白酶为芯材,采用冷冻干燥技术制备蛋白酶微胶囊,用于干酪制备,促进其成熟。在干酪成熟过程中采用反相高效液相色谱技术分析添加蛋白酶微胶囊的干酪中水溶性氮提取液,并对色谱峰的分布情况进行主成分分析。试验结果表明:在干酪成熟期间,随着成熟时间的延长,亲水性小分子质量肽含量逐渐增大;亲水性中分子质量肽的含量先增加后降低;大分子质量疏水性肽的含量无太大变化。     

乳酸菌自溶对切达干酪成熟中蛋白质分解的影响

选取自溶度不同的乳杆菌作为附属发酵剂混合商业发酵剂制作切达干酪,研究了乳酸菌自溶对干酪成熟的影响.测定成熟期间各干酪中乳酸菌活菌数、pH值、pH4.6可溶氮质量分数和12%TCA可溶氮质量分数.并结合SDS-PAGE分析干酪蛋白质的水解情况.结果表明,各实验组干酪的12%TCA可溶性氮含量随着成熟时间延长逐渐增加.在1个月后不同组别之间差异显著(P<0.05).与对照组干酪相比,加入高自溶度菌株的实验组干酪蛋白分解程度较高,非蛋白氮的质量分数为8.00%.同时,SDS-PAGE结果显示,各干酪的蛋白质都有-定程度的分解.产物略有不同.乳酸菌自溶可以加速蛋白质分解.     

一种富含天然γ-氨基丁酸干酪的制备及对干酪的评价

以具有合成γ-氨基丁酸能力的嗜热链球菌(QYW-LYS-1)及植物乳杆菌(QYW-L-11)为干酪附属发酵剂制作切达干酪,检测添加这两种附属发酵剂的干酪在成熟过程中γ-氨基丁酸的含量及乳酸菌总数的变化,并研究这两种附属发酵剂在干酪成熟过程中对其蛋白水解程度、感官、组成成分等方面的影响。结果表明:在干酪中添加产γ-氨基丁酸的菌株对干酪的蛋白水解、感官评价及组成成分无不良影响。在4℃贮存条件下,经过90d的成熟,添加QYW-LYS-1的干酪中γ-氨基丁酸的含量最多,达到0.43mg/g。本研究为制备一种富含天然γ-氨基丁酸的功能性干酪提供理论基础。     

一种毛霉表面成熟干酪的蛋白质水解作用评价

从毛豆腐中分离出一株毛霉,并应用于表面成熟干酪,以研究干酪成熟过程中所发生的蛋白质水解作用。在90d 的成熟过程中,干酪的pH 值增加;蛋白质水解作用的评价指标,如干酪外层的水溶性氮- 总氮比、pH4.6水溶性氮- 总氮比、12g/100mL 三氯乙酸可溶性氮- 总氮比,在成熟90d 后分别增加至(23.68 ± 1.07)%、(19.38 ± 1.32)%和(8.61 ± 0.85)%,并且高于干酪的内部相应指标。SDS-PAGE 和毛细管电泳分析干酪的pH4.6 不溶性组分,结果表明酪蛋白在干酪成熟过程中被降解。对干酪成熟过程中分离出的水溶性组分进行RP-HPLC 分析,结果显示成熟过程中蛋白质被水解以及形成一些新肽分子。     

Proteolysis during the ripening of goats’ milk cheese made with plant coagulant or calf rennet

Powdered plant coagulant (PPC) obtained from the cardoon (Cynara cardunculus) was compared with calf rennet (CR) for the manufacture of goats’ milk cheese, by determining difference in the proteolysis throughout ripening. There were no substantial differences between the compositions of cheeses made using the two types of coagulants. However, cheeses manufactured with PPC exhibited higher levels of pH 4.6-SN than cheese made using CR. The extent of breakdown of α_s-casein, as measured by urea-PAGE, was greater in cheese made using PPC than cheese made using CR. The formation of hydrophobic peptides and the ratio of hydrophobic/hydrophilic peptides throughout the ripening were higher in cheeses made with PPC than in cheeses made with CR. Principal component analysis (PCA) of peak heights of RP-HPLC peptide profiles of the ethanol-soluble and ethanol-insoluble fractions distributed the samples according to the coagulant used in their manufacture. Quantitative differences in several peptides were evident between the two types of cheese.     

干酪介电特性与成熟期及成熟度相关性分析

以半硬质契达干酪为研究对象,对同一加工样品在9℃贮存成熟,分别对成熟期0、15、30、45、60、90、120、150、180 d的干酪样品进行了介电特性和成熟度指标的测定,包括干酪样品总氮(total nitrogen,TN)含量、p H 4.6可溶性氮(soluble nitrogen,SN)含量及三氯乙酸(trichloroacetic acid,TCA)溶液中SN含量的测定。测试频率选定为500、915、1 500、2 000 MHz,对测试结果进行了统计分析,建立了干酪介电特性与成熟度指标之间的相关性。结果表明:随着成熟期的延长,干酪水分含量略有减少、水分活度明显降低、p H值先降低后升高、成熟度指数p H 4.6-SN/TN和12%TCA-SN/TN均随成熟期延长而增大;介电常数ε’在所选的测试频率下与成熟期和成熟度指数的变化均呈线性相关关系;介电损耗因数ε’’在所选测试频率范围内,随成熟期的延长和成熟度的增大呈现总体下降趋势,与成熟期和成熟度在500 MHz和915 MHz频率条件下线性相关性极显著(P0.01),而在高频1 500 MHz和2 000 MHz条件下线性相关性不显著;损耗角正切值变化不明显,表明在测试频率范围介电常数ε’和介电损耗因数ε’’的变化方向一致,同时变化幅度相近。     

Proteolysis and microstructure of Piacentinu Ennese cheese made using different farm technologies

The aim of this study was to provide the biochemical and structural characterization of Piacentinu Ennese cheese and to evaluate the impact of different farm technologies on cheese proteolysis and microstructure. Fifteen cheeses were manufactured according to traditional technology, i.e., from raw milk and farmhouse rennet in the absence of starter culture. Pasteurized milk, commercial rennet, and starter were used for production of 20 nontraditional cheeses. Proteolysis in Piacentinu Ennese cheese was monitored during a 2- to 10-mo ripening time. Low rates of overall proteolysis were observed in cheese, as percentages of total N soluble at pH 4.6 and in 12% trichloroacetic acid were about 11.40 and 8.10%, respectively, after 10 mo of age. Patterns of primary proteolysis by urea-PAGE showed that alpha(s)-caseins were degraded to a larger extent than were beta-caseins, although a considerable amount of both caseins was still intact after 10 mo. Reversed phase-HPLC analysis of the cheese peptide fractions showed a slow decrease in the levels of hydrophobic peptides coupled to increasing levels of hydrophilic compounds as the cheese aged. The structural characteristics of Piacentinu Ennese cheese were evaluated by scanning electron microscopy after 2, 4, and 6 mo of age. The micrographs showed a sponge-like structural network with a well-distributed system of empty spaces, originally occupied by whey and fat. The microstructure changed during cheese ripening to become more compact with cavities of smaller size. Farm technology significantly affected cheese proteolysis and microstructure. Nontraditional cheeses had higher levels of pH 4.6-soluble N and showed a larger hydrolysis of alpha(s)-casein fractions by urea-PAGE analysis than did traditional cheeses. Large differences between cheese-types also concerned the patterns of secondary proteolysis. Nontraditional cheeses had higher levels of 12% trichloroacetic acid-soluble N and showed larger proportions of free amino acids and hydrophilic peptides in the HPLC profiles of the corresponding 70% ethanol-soluble N fraction than traditional cheeses. Nontraditional cheeses also had a more open structure with a coarser and less continuous appearance than did traditional cheeses. A large amount of variability in cheese proteolysis and structure within nontraditional treatment reflected farm-dependent changes in manufacturing conditions related to the use of various types of rennet and starter.     

发酵剂对牦牛乳硬质干酪蛋白降解和苦味关系的影响

用甘肃天祝新鲜牦牛乳为原料分别添加嗜温、嗜热和混合发酵剂制作硬质干酪,以pH 4.6-可溶性氮（soluble nitrogen,SN）、12%三氯乙酸氮（trichloroacrtic acid-N,TCA-N）、游离氨基酸（free amino acid,FAA）含量和疏水性肽/亲水性肽（S/Q）为蛋白水解度指标,研究3 种牦牛乳硬质干酪在6 个月内成熟过程中苦味和蛋白质降解之间的关系。结果表明：3 种干酪在成熟过程中pH 4.6-SN、12% TCA-N和FAA含量均呈上升趋势,苦味值与pH 4.6-SN、12% TCA-N和FAA含量成正相关,相关系数分别为0.400、0.412和0.458。3 种干酪成熟过程中S/Q的变化趋势和程度不同,嗜温发酵剂干酪中S/Q呈现降低趋势;嗜热和混合发酵剂干酪中S/Q均呈现先降低后增大的趋势,但在这两种干酪中S/Q的变化程度不同,嗜热发酵剂干酪在1～3 个月S/Q略有降低,在3～6 个月S/Q快速增大,而混合发酵剂干酪正好相反。S/Q与苦味值成极显著正相关（r=0.895）,S/Q可很好地反映干酪中苦味的强弱。而干酪中苦味强弱与蛋白质降解强弱密切相关,对蛋白降解程度越大的发酵剂制作的干酪越容易产生苦味,其中,嗜热发酵剂对干酪蛋白降解程度最大,混合发酵剂次之,嗜温发酵剂最小。     

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.     

Impact of Autolytic and Proteolytic Lactobacilli and Nisin-Producing Culture on Proteolysis and Sensory Characteristics in Cheddar Cheese

ABSTRACT: Cheddar cheeses were made using a nisin-tolerant starter culture with either Lactobacillus delbrueckii subsp. bulgaricus UL12 (autolytic strain), Lactobacillus casei subsp. casei L2A (proteolytic strain), Lactococcus lactis subsp. lactis biovar. diacetylactis UL719 (nisin producer), or of Lb. bulgaricus UL12 and Lc. diacetylactis UL719. Lb. bulgaricus UL12 produced more trichloroacetic acid-soluble nitrogen than did Lb. casei L2A, which produced more phosphotungstic acid-soluble nitrogen than did Lc. diacetylactis UL719. High-performance liquid chromatography analyses showed that either lactobacilli or Lc. diacetylactis UL719 increased the hydrophilic and hydrophobic peptide contents. Cheeses containing both Lb. bulgaricus UL12 and Lc. diacetylactis UL719 had the most intense old Cheddar cheese flavor after 6 mo of ripening.     

Thermal inactivation of chymosin during cheese manufacture

The aspartic proteinase, chymosin (EC 3.4.23.4) is the principal milk clotting enzyme used in cheese production and is one of the principal proteolytic agents involved in cheese ripening. Varietal differences in chymosin activity, due to factors such as cheese cooking temperature, fundamentally influence cheese characteristics. Furthermore, much chymosin is lost in whey, and further processing of this by-product may require efficient inactivation of this enzyme, with minimal effects on whey proteins. In the first part of this study, the thermal inactivation kinetics of Maxiren 15 (a recombinant chymosin preparation) were studied in skim milk ultrafiltration permeate, whole milk whey and skim milk whey. Inactivation of chymosin in these systems (at pH 6.64) followed first order kinetics with a D45.5 value of 100 +/- 21 min and a z-value of 5.9 +/- 0.3 degrees C. D-Values increased linearly with decreasing pH from 6.64 to 6.2, while z-values decreased as pH decreased from 6.64 to 6.4, but were similar at pH 6.4 and 6.2. Subsequent determination of chymosin activity during manufacture of Cheddar and Swiss-type cheese showed good correlations between predicted and experimental values for thermal inactivation of chymosin in whey. However, both types of cheese curd exhibited relatively constant residual chymosin activity throughout manufacture, despite the higher cooking temperature applied in the manufacture of Swiss cheese. Electrophoretic analysis of slurries made from Cheddar and Swiss cheese indicated decreased proteolysis due to chymosin activity during storage of the Swiss cheese slurry, but hydrolysis of sodium caseinate by coagulant extracted from both cheese types indicated similar levels of residual chymosin activity. This may suggest that some form of conformational change other than irreversible thermal denaturation of chymisin takes place in cheese curd during cooking, or that some other physico-chemical difference between Swiss and Cheddar cheese controls the activity of chymosin during ripening.     

快速成熟契达干酪成熟期间的理化特性变化

对天然契达(Cheddar)干酪快速成熟期间理化特性进行了研究,结果表明,在21 d内,pH值呈下降趋势(P<0.05),在21 d到35 d的成熟中,pH值缓慢增加(P>0.05);Cheddar干酪快速成熟过程中由于蛋白酶和脂肪酶的作用使蛋白发生水解,pH值为4.6醋酸溶液—水溶性氮质量分数、质量分数为12%的三氯乙酸—可溶性氮和游离氨基酸(FAA)的质量分数都呈明显增加趋势(P<0.05);成熟过程中TPA各项指标均有不同程度的变化,硬度和凝聚性呈先下降后增加的趋势,弹性和咀嚼性呈先上升后下降的趋势。     

Application of exopolysaccharide-producing cultures in reduced-fat Cheddar cheese: composition and proteolysis

Proteolysis during ripening of reduced fat Cheddar cheeses made with different exopolysaccharide (EPS)-producing and nonproducing cultures was studied. A ropy strain of Lactococcus lactis ssp. cremoris (JFR1) and capsule-forming nonropy and moderately ropy strains of Streptococcus thermophilus were used in making reduced-fat Cheddar cheese. Commercial Cheddar starter was used in making full-fat cheese. Results showed that the actual yield of cheese made with JFR1 was higher than that of all other reduced-fat cheeses. Cheese made with JFR1 contained higher moisture, moisture in the nonfat substance, and residual coagulant activity than all other reduced-fat cheeses. Proteolysis, as determined by PAGE and the level of water-soluble nitrogen, was also higher in cheese made with JFR1 than in all other cheeses. The HPLC analysis showed a significant increase in hydrophobic peptides (causing bitterness) during storage of cheese made with JFR1. Cheese made with the capsule-forming nonropy adjunct of S. thermophilus, which contained lower moisture and moisture in the nonfat substance levels and lower chymosin activity than did cheese made with JFR1, accumulated less hydrophobic peptides. In conclusion, some EPS-producing cultures produced reduced-fat Cheddar cheese with moisture in the nonfat substance similar to that in its full-fat counterpart without the need for modifying the standard cheese-making protocol. Such cultures might accumulate hydrophobic (bitter) peptides if they do not contain the system able to hydrolyze them. For making high quality reduced-fat Cheddar cheese, EPS-producing cultures should be used in conjunction with debittering strains.