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.     

原核表达重组牛凝乳酶原及重组牛凝乳酶酶学特性

凝乳酶能够专一性裂解κ-酪蛋白,是制造干酪的关键酶。运用大肠杆菌表达系统对牛凝乳酶原进行了原核表达和初步纯化,活化重组凝乳酶原及测定凝乳活性,对重组凝乳酶的酶学特性进行分析。结果表明:大肠杆菌表达的重组蛋白约占菌体总蛋白的66.3%,每升培养液可纯化约200 mg的重组凝乳酶原,活化后的凝乳酶活力可达600 000 SU/g。经测定凝乳酶最适作用温度为57⁶2℃,并在pH 262℃,并在pH 2⁷、低于40℃的温度范围内稳定。金属离子中Al3+,Fe3+和Cu2+能显著增强酶活;胃蛋白酶抑制剂pepstatin A对酶有明显的抑制作用。     

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。     

江米酒乳凝固机理研究

对江米酒的乳凝固机理进行研究,结果表明：江米酒的凝乳作用是由酒药中的霉菌在江米酒发酵过程中产生的酸性蛋白酶引发的,属于酶凝固,利用江米酒进行凝乳是我国传统乳制品奶酪制作的独特工序。     

牛凝乳酶原基因在大肠杆菌中的高效表达及活性检测

以实验室保存的携带凝乳酶原前体基因的重组载体pMD 19-T/bPPC为模板克隆凝乳酶原基因,经双酶切后与载体pET-30a连接得到重组载体pET-30a/bPC,转化大肠杆菌BL21(DE3),经IPTG诱导后,采用SDS-PAGE检测目的蛋白表达情况。重组蛋白经变性/复性、DEAE-Sepharose Fast Flow纯化和自催化后检测凝乳活性。结果表明,重组凝乳酶原基因在大肠杆菌中高效表达,表达量占菌体总蛋白的68%,采用Arima K方法检测,其凝乳活力达到80 SU/mL。因此,通过大肠杆菌表达系统大量制备具有生物活性的重组牛凝乳酶原的策略是可行的,研究结果为弥补国内天然牛凝乳酶的短缺提供一种途径。     

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。     

原核表达重组牛凝乳酶的纯化

原核表达获得的重组牛凝乳酶与商品重组牛凝乳酶有相似的酶学特性,具商业潜力,故进一步研究其纯化方法。使用离心与饱和硫酸铵沉淀,成功纯化出有活性的重组牛凝乳酶。蛋白最终回收率为1.82%,总活力比纯化前提高了73.3%,比活力提高了95.3倍。同时,讨论了如何提高包涵体复性率与产物回收率,为今后该酶的工业化生产提供技术参考。     

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。     

牛凝乳酶原基因在毕赤酵母中的表达及其酶学特性的研究

凝乳酶是制造干酪的关键酶。为获得高活性的牛凝乳酶(chymosin,B-chy),用电脉冲法将线性化的pGAPZαA-B-pchy重组表达载体转化到毕赤酵母GS115中。该菌株在以葡萄糖为碳源的YPD培养基中可分泌表达牛凝乳酶原。SDS-PAGE分析表明所表达的牛凝乳酶,分子量约为37ku,符合预期大小。发酵培养96h后,酶活力达到96SU/mL。酶学特性分析表明,其最适凝乳温度为60℃,在pH2~6、小于50℃的温度范围内稳定。Ca2+浓度为40mmol/L时,钙促酶反应活性达到最高,此后随着Ca2+浓度的增加酶活力逐渐降低。金属离子Al3+、Mn2+、Fe2+、Mg2+和K+对酶活力具有显著的促进作用,而Co2+、Zn2+、Cu2+、Ni2+对酶活力具有显著的抑制作用。本研究优化了凝乳酶的表达条件,为凝乳酶工业化生产提供了理论基础。     

辣木叶凝乳酶提取条件优化及凝乳特性研究

鲜辣木叶为原料,在单因素试验的基础上,利用二次通用旋转试验设计,优化辣木叶凝乳酶的提取条件,并对其凝乳特性进行研究。结果表明,辣木叶中存在可凝乳蛋白酶。凝乳蛋白酶的提取方法是将新鲜辣木叶切碎、加入5倍体积0.15 mol·L~(-1) NaCl溶液45℃浸泡90 min、浸泡液10倍真空浓缩后硫酸铵盐析(饱和度60%)、沉淀加水透析和真空冷冻干燥得辣木凝乳酶。凝乳酶特性:最适凝乳温度60℃、凝乳活力19.99 SU·mL~(-1),70℃凝乳活力丧失,在pH 4.0~8.0之间活力稳定;酪蛋白水解活力温度50℃最高,pH 4.0~10.0之间稳定。辣木凝乳酶的研究为新型植物源凝乳酶和云南特色生物资源的开发提供了新的线索。     

Manufacture and Characterization of Gua-Nai: A New Dairy Food Produced with an Oriental-Type Culture

Gua-nai is a sweet-set gel produced from pasteurized whole or skim milk. The milk-clotting enzymes were elaborated from Chinese wine cake culture raised on steamed glutinous rice (sweet rice). The organisms involved were isolated and identified as Amylomyces rouxii, Rhizopus oryzae, Aspergillus oryzae, and a single yeast, Endomycopsis burtonii. These organisms were employed in the reconstitution of the wine cake culture using sweet rice flour as the medium. During the rice fermentation, a clear, effervescent, pale yellow liquid phase appeared exhibiting both proteolytic and milk-clotting activities and containing ethanol. Taxonomic studies of the mycoflora of the fermenting rice also revealed a gradual disappearance of the filamentous molds, leaving only the yeast in the medium at completion of fermentation. A consumer taste panel did not indicate significant differences between gua-nai made with commercial wine cake culture and that made with the culture reconstituted from isolated microorganisms.     

江米酒凝乳机理的初步研究

研究了江米酒制作米酒奶的凝乳机理。试验结果证实,江米酒中的凝乳活性因子,即江米酒发酵过程中微生物产生的蛋白酶是米酒奶凝乳的主要原因。     

江米酒脂肪酶对米酒奶脂解风味的影响

利用传统江米酒制备凝乳剂来制作米酒奶、其中江米酒脂肪酶活力为1.0～1.5LU/mL。通过酸度值(ADV)分析发现,脂肪酸的积累主要发生在凝乳期间。采用气相色谱- 质谱联用仪(GC-MS)检测各游离脂肪酸的含量,棕榈酸、油酸、豆蔻酸和硬脂酸占有较高的比例。     

Influence of rennet milk-clotting activity on the proteolytic and sensory characteristics of an ovine cheese

Roncal cheese (regulated by an Apellation of Origin) is a traditional hard cheese manufactured from raw ewe's milk in the region of Navarre in Spain. Roncal cheeses, manufactured using two lamb rennets with different milk-clotting activity levels, were evaluated to compare their chemical, proteolytic, and sensory characteristics. A preliminary study of samples of lamb rennets indicated that a large proportion of such rennets did not fulfil current microbiological requirements and likewise revealed considerable variation in the milk-clotting activity of the samples examined. Trends in the overall physicochemical parameter values (pH, dry matter, fat, and protein) were similar in both cheese batches. Proteolysis of the nitrogen fractions was observed to take place at a faster rate in the cheeses made using the rennet with the higher milk-clotting activity (soluble nitrogen, non-protein nitrogen, and amino acid nitrogen values around 13–20% higher than in the cheeses made using the rennet with the lower milk-clotting activity after 180 days of ripening). Urea-PAGE electrophoretic analysis of the caseins from the cheeses manufactured using both types of rennet showed that the β-caseins were less susceptible to proteolysis than the α_s-caseins. The effect of the different milk-clotting activity levels was most pronounced on the α_s-caseins, in which the rennet with the higher milk-clotting activity gave higher breakdown. Nevertheless, the differences in the proteolysis rates did not yield any appreciable sensory differences.     

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).     

用Y85—8512毛霉凝乳酶生产鲜奶酪饮料初探

采用本实验室研制的Y85—8512毛霉凝乳酶、以鲜牛乳等为原料、利用消毒奶设备、进行鲜奶酪饮料生产的初步探索。本文对影响凝乳效果的两个重要因素、鲜奶酪饮料的配方及保存期进行了试验,经试生产,初步确定了工艺流程。用凝乳酶来生产鲜奶酪饮料尚属首次。     

Characterization of wine rennet and its kinetics by gel electrophoresis

The rennet of glutinous rice wine (wine rennet) is an exclusive clotting agent for Chinese Royal cheese production. Some characterizations are reported herein in an attempt to provide evidence about the use of the protease as either a rennet substitute or an accelerator in cheese making and ripening. The results showed that wine rennet was a monomeric and unglycosylated protease. The N-sequencing indicated a high degree of similarity to other fungal rennets. The cleavage sites of wine rennet on oxidized insulin B chain identified by HPLC-mass spectrometry included Gln⁴-His⁵, Ala¹⁴-Leu¹⁵, Leu¹⁵-Tyr¹⁶, Tyr¹⁶-Leu¹⁷, and Phe²⁴-Phe²⁵ at pH 6.5, which were similar to those observed for Mucor rennet, but different from calf chymosin except for Leu¹⁵-Tyr¹⁶. A comparison study of the kinetic properties of wine rennet on bovine caseins with that of rennets from calf and Mucor miehei by gel electrophoresis showed that these rennets had similar coagulation efficiency but different reaction rates. Wine rennet exhibited a higher degree of degradation than the calf and Mucor enzymes at pH 6.5 and 40°C. Therefore, wine rennet would be an adjunct for calf rennet or an accelerator in cheese making.     

羊奶牛奶混合干酪加工工艺研究

为改进羊奶牛奶混合干酪加工工艺,本实验以羊奶粉和鲜牛奶为原料,研究了牛奶添加比例、不同凝乳酶、杀菌条件、酸化条件和氯化钙添加量对混合干酪凝乳效果、出品率和感官品质的影响。结果表明,添加35% 的鲜牛奶,选用羔羊皱胃酶为凝乳酶,巴氏杀菌(63℃,30min),酸化pH6.20,氯化钙添加量为0.02% 时混
合干酪的品质较好。     

Diffuse Reflectance Profiles of Eight Milk-Clotting Enzyme Preparations

Eight milk-clotting enzyme prepartations were standardized to equal clot time and used to coagulate pasteurized whole milk. Diffuse reflectance profiles were monitored for 60-min using a fiber optic sensor sensitive to infrared light at 950 nm. Modified M. miehei and M. pusillus protease, recombinant chymosin and calf rennet produced similar profiles. Rates of increase in diffuse reflectance were E. parasitica recombinant chymosin > calf rennet > modified M. miehei, M. pusillus var. Lindt > 50:50 blend of calf rennet and bovine pepsin > unmodified M. miehei > pepsin. Monitoring milk coagulation as described may be useful during cheese making and allow setting optimal conditions for milk-clotting enzyme preparations.