米黑毛霉产凝乳酶固体发酵培养基优化

对米黑毛霉产凝乳酶固体发酵培养基进行优化,以提高凝乳酶的产量,为米黑毛霉凝乳酶的工业化生产提供技术依据。首先通过单因素实验,得到最佳碳源为葡萄糖,最佳氮源为硝酸铵,最佳产酶诱导物为乳清粉。在此基础上通过正交实验进一步优化,得到培养基外加成分的最优组合为葡萄糖浓度2.5%,硝酸铵浓度1.0%,乳清粉浓度2.0%。培养基优化后米黑毛霉的凝乳活力达到(3649.52±3.62)SU/mL,比优化前提高了54.5%。     

一种新型凝乳酶生产干酪工艺的研究

采用蓝色刺孢霉所产的新型凝乳酶生产干酪。研究探讨了发酵剂添加量、CaCl2添加量、凝乳酶质量浓度和pH对其凝乳特性的影响。研究确定用新型凝乳酶生产干酪的最佳工艺条件为:发酵剂添加量0.2 g/L,CaCl2添加量为0.02%,pH 5.9,新型凝乳酶添加量为2%。试验中选用的这种新型凝乳酶生产的干酪风味柔和,组织状态细腻,产品质量好。     

霉菌固态发酵产凝乳酶培养基成分优化

本实验在单因素实验基础上,利用Plackett-burman方法筛选得到影响霉菌产凝乳酶的主要因素:葡萄糖、Na2SO4、吐温-80,通过Box-Behnken实验设计对培养基中葡萄糖、Na2SO4、吐温-80的浓度进行了优化,所得培养基的最优成分为液固比为0.9∶1、CaSO40.04%、K2SO4 0.04%、葡萄糖2.8%、Na2SO4 0.031%、吐温-80 0.34%,优化后霉菌产凝乳酶活力平均值为868.3U/g曲,比基础培养基提高了2.25倍。     

重组毕赤酵母产凝乳酶发酵参数优化的研究

以重组毕赤酵母GS115PJ5(含微小毛霉凝乳酶基因)为研究对象,通过单因素实验确定其最佳生长条件:培养温度30℃、培养基pH值5.2、摇床转速280r/min、培养基装瓶量5%(12.5mL/250mL)。在单因素实验的基础上通过Plackett-Burman实验筛选出培养基装瓶量、甲醇添加量、培养基pH值等3个影响重组毕赤酵母产凝乳酶的主要因素,并通过响应面实验确定三者有利于产酶的最佳值分别为13.06%、1.52%、6.15,在此条件下,凝乳酶活力达到最大值即491.7SU/mL,验证实验证明模型预测值准确、可靠。     

软质干酪凝乳特性的研究

研究探讨了软质干酪生产过程中原料乳的比重、CaCl2的添加量、凝乳酶质量浓度以及pH值对其凝乳特性的影响,结果表明:当原料乳比重为1.029g/mL、CaCl2的添加量为0.02%、凝乳酶质量浓度为2.5g/L牛乳、pH值为5.8时可以制作出风味柔和、组织状态细腻的软质干酪。     

Paenibacillus spp.BD3526发酵小麦麸皮生产凝乳酶

对Paenibacillus spp.BD3526凝乳酶的发酵条件和凝乳性能进行了研究,探讨了不同培养基、发酵时间、碳源、氮源和装液量对凝乳酶活力和活菌数的影响,并与商业化的小牛皱胃酶、重组凝乳酶、Rhizomucor miehei来源凝乳酶进行凝乳性能比较。实验结果表明:发酵时间、碳源、氮源和装液量对BD3526凝乳酶活力(milkclotting activity,MCA)和蛋白水解活力(proteolytic activity,PA)均有不同程度的影响。选用小麦麸皮培养基进行发酵至24 h时,MCA达到最大至3 279.76±67.11 SU/m L,凝乳性能强,PA值在整个发酵期间变化不大;添加碳源或氮源会不同程度地降低MCA值;装液量为30 m L时,发酵上清液中菌体浓度最高、MCA达到6 000 SU/m L。与商业化的凝乳酶相比,BD3256凝乳酶在凝乳过程中未使凝乳块产生过度水解,也无乳清析出现象,具有应用到干酪生产中的潜力。     

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%.     

夸克干酪加工工艺的优化

通过凝乳酶和排乳清条件单因素,以产率、校正产率和感官评价为指标的正交实验,确定了夸克干酪生产以产率为指标,3个因素的较优工艺条件为离心时间10 s,凝乳酶添加量为0.004％,发酵剂添加量为5％(均为体积分数,下同).以校正产率为指标,3个因素较优工艺条件为离心时间10 s,凝乳酶添加量为0.004％,发酵剂添加量为3％.以感官评价为指标,3个因素的较优工艺条件为,发酵剂添加量为5％,凝乳酶添加量为0.005％,离心时间30 s.     

响应面法优化“曲拉”凝乳酶干酪素凝乳工艺

以"曲拉"为原料,采用筛选的微小毛霉凝乳酶为凝乳剂,在酶添加量、凝乳温度、凝乳pH值、CaCl2添加量4个单因素试验基础上,利用响应面试验设计法进行试验设计,取得曲拉凝乳酶干酪素出品率与各单因素的函数关系,并建立曲拉凝乳酶干酪素凝乳工艺模型。回归方程和响应曲面结果表明:4个因素对曲拉凝乳酶出品率影响大小依次为:凝乳pH值>凝乳酶添加量>CaCl2添加量>凝乳温度;最佳凝乳工艺为凝乳pH6.2、凝乳酶添加量0.05g/kg、CaCl2添加量2.5%、凝乳温度42℃。在此条件下,曲拉凝乳酶干酪素的理论出品率为75.23%,实验验证出品率为73.54%。     

微小毛霉发酵制取凝乳酶的培养基优化研究

利用响应面分析法优化微小毛霉的发酵培养基,提高微小毛霉凝乳酶的凝乳活力。在单因素实验的基础上,用Design-Expert软件对实验数据进行多元回归分析,建立了3种因素与凝乳酶活力之间的函数关系,得出在基础发酵培养基中加入的氯化钙、乳清粉和葡萄糖的最佳浓度分别为0.58%、0.64%和1.13%,此时,微小毛霉凝乳酶的凝乳活力的理论值为1150.81SU/mL,验证平均值为1109.7SU/mL,与预测值基本一致。     

均匀设计法优化廉价型牛凝乳酶工程菌发酵培养基

为了获得生产用廉价型牛凝乳酶工程菌发酵培养基,通过单因素试验考察发酵培养基中各组分对产酶的影响。结果显示：葡萄糖、玉米浆、酵母提取物、尿素质量浓度对产酶影响显著。以上述因素作为随机因子,进行均匀设计试验,采用逐步回归方法对试验结果进行分析。结果表明：在葡萄糖45g/L、玉米浆17g/L、酵母提取物6g/L、尿素12g/L的条件下,凝乳酶活性达342.86SU/mL,比优化前提高了1.22倍。所得培养基为重组牛凝乳酶的高效低成本生产提供了参考。     

乳糖替代凝乳酶制作大豆干酪技术初探

对加酶发酵制作干酪的传统方法进行了改进,用添加乳糖的方法来代替添加酶进行发酵.试验综合考察了影响混合豆乳(豆乳和牛乳)干酪凝乳的几个因素,即豆乳添加量、发酵剂添加量、乳糖添加量和乳酸钙添加量,并确定混合豆乳干酪的最佳凝乳工艺参数.试验结果表明,最佳工艺参数为:80%豆乳 3%发酵剂 3%乳糖 0.1%乳酸钙.     

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。     

印度毛霉MJ229固态发酵产凝乳酶的研究

由于从小牛皱胃中提取的皱胃酶已无法满足干酪生产的需要,因而不得不寻找其他凝乳酶来替代小牛皱胃酶.本研究在前期小试实验基础上,研究毛霉MJ229固态发酵产凝乳酶的中试制备条件.确定其固态发酵培养基配方为:大米、麸皮和稻草的质量比为5:4:1,吐温-80为0.3％(w/w);加入自来水使液固比为1:1,最佳载曲量为每盘3kg.分别在自然状态与调控水分和温度状态下进行曲盘固态发酵试验,结果表明,毛霉MJ229在调节状态下发酵48h后获得凝乳酶活力可达5333Su/g,与自然发酵条件相比提高了25％.     

紫外线和硫酸二乙酯诱变高产凝乳酶地衣芽孢杆菌的研究

以产凝乳酶地衣芽孢杆菌为出发菌株,通过紫外线诱变和硫酸二乙酯诱变,提高菌株的凝乳活力。经反复诱变筛选获得一株凝乳活力较高且水解活力较低的突变株DES-6,其凝乳活力为184.65SU/mL,比原菌株增加了15.41%,水解活力为23.35U/mL,比原菌株降低了64.80%。传代实验表明,突变株DES-6具有稳定的遗传性。     

枯草芽孢杆菌克隆与表达微小毛霉凝乳酶基因

从自制的酒酿利用酪蛋白培养基分离纯化到了一株产凝乳酶的微小毛霉菌株（ZZMZ-19）,从ZZM-19菌株的cDNA文库筛选到了两个凝乳酶基因chl和ch2并实现了凝乳酶基因ch1和ch2在枯草芽孢杆菌菌株（ZZMZ-01）中的的克隆与表达。chl和曲2阳性克隆菌株在酪蛋白培养基r1]发酵30h左右的时间凝乳酶酶活达到最人,分别为48．27SU／mL和41．02SU／mL,相比出发菌株发酵时间缩短了15h。用交联葡聚糖凝胶柱G100分离纯化其发酵卜清液后,用十二烷基硫酸钠聚丙烯酰胺凝胶电泳方法测得CHII和cHIII分子草分别为32ku和30ku。     

Structure and shelf stability of milk protein gels created by pressure-assisted enzymatic gelation

In this work, pressure-assisted enzymatic gelation was applied to milk proteins, with the goal of enhancing the structure and stability of pressure-created milk protein gels. High-pressure processing (HPP) at 600 MPa, 3 min, and 5°C was applied to milk protein concentrate (MPC) samples of 12.5% protein concentration, both in the absence and in the presence of calf chymosin [up to 60 IMCU (international milk-clotting units)/kg of milk] or camel chymosin (up to 45 IMCU/kg of milk). Gel hardness, water-holding capacity, and degree of proteolysis were used to assess network strength and shelf stability. The processing trials and all measurements were conducted in triplicate. Statistical analyses of the data were performed by ANOVA, at a 95% confidence level. After HPP treatment, we observed significant structural changes for all samples. Pressurization of MPC, with or without chymosin addition, led to extensive protein aggregation and network formation. The strength of HPP-created milk protein gels without chymosin addition, as measured by the elastic modulus (G′), had a value of 2,242 Pa. The value of G′ increased with increasing chymosin concentration, reaching as high as 4,800 Pa for samples with 45 IMCU/kg of camel chymosin. During 4 wk of refrigerated storage, the HPP and chymosin MPC gels maintained higher gel hardness and better structural stability compared with HPP only (no chymosin) MPC gels. The water-holding capacity of the gels without chymosin remained at 100% during 28 d of refrigerated storage. The HPP and chymosin MPC gels had a lower water-holding capacity (91–94%) than the HPP-only counterparts, but their water-holding capacity did not decrease during storage. Overall, these findings demonstrate that controlled, fast structural modification of high-concentration protein systems can be obtained by HPP-assisted enzymatic treatment, and the created gels have a strong, stable network. This study provides insights into the possibility of using HPP for the development of milk-protein-based products with novel structures and textures and long refrigerated shelf life, along with the built-in safety imparted by the HPP treatment.     

凝乳酶蛋白质工程的研究

由于微生物凝乳酶过多的非专一性水解和热稳定性高导致干酪产量降低和成熟中出现苦味。应用重组技术和蛋白质工程技术，使酶的一级结构发生可选择的和系统的变化，得到的蛋白质便具有所期望的功能特性。应用蛋白质工程对酶改性涉及到改变亚基结构和更换残基，改变酶的动力学参数和热稳定性，改变底物特异性以及最适pH等。     

A novel electrochemical assay for chymosin determination using a label-free peptide as a substrate

Chymosin is a predominant enzyme in rennet and is used in cheese production because of its excellent milk-clotting activity. Herein, we proposed a facile and label-free electrochemical method for determining chymosin activity based on a peptide-based enzyme substrate. The synthesized substrate peptide for chymosin was assembled onto the surface of the Au-deposited grassy carbon electrode. The current was proportional to chymosin activity, and thus chymosin activity could be determined. The detection ranges of chymosin activity were 2.5 to 25 U mL^?1. The detection limit of chymosin activity was 0.8 U mL^?1. The sensing platform was used to quantify chymosin activity in commercial rennet with high selectivity, excellent stability, and satisfactory reproducibility. We developed a facile, fast, and effective electrochemical assay for detecting chymosin activity, which has potential applications in cheesemaking.