地衣芽孢杆菌D3.11凝乳酶及其作用机制研究

Rennet is the key enzyme in the production of cheese. Bacteria is one of the most important strain producing milk-clotting enzyme. Attention has been focused on the milk-clotting enzymes from bacteria because of its extensive sources, lower cost, and easy extraction. Bacillus licheniformis D3.11, an efficient rennet producing strain, was isolated from the pastoral area in Tibetan Plateau of China. However,.the mechanism has not been demonstrated clearly. In this program, we intent to isolated and purified milk-clotting enzyme from Bacillus licheniformis. Based on the analysis of the structure, the enzyme property was studied, and the most suitable substrates and the hydrolysis site of the milk-clotting enzymes were determined by Urea SDS-PAGE, RP-HPLC and MALDI-TOF/MS to reveal the specificity of.the enzymes. The change laws of micelle diameters, rheological property and the three dimensional microarchitecture were studied by laser particle sizeteste, rheometer and confocal laser scanning microscope. On this condition, the coagulation mechanism of milk-clotting enzyme from Bacillus licheniformis was studied. Changes and relationships of residual rennet, protein degradation, textural rheological properties, microstructure and flavor during ripening period of the cheese were studied by TPA, rheometer, scanning electron microscopy and GC/MS to clarify the action mechanism of milk-clotting enzyme from Bacillus licheniformis D3.11.

凝乳酶是干酪生产的关键酶。细菌是重要的产凝乳酶微生物，细菌凝乳酶以其来源广泛、成本低和提取方便等优点成为研究热点。地衣芽孢杆菌D3.11是我们前期从青藏高原牧区筛选得到的一株产凝乳酶的优良菌株，但目前对其作用机制尚不明确。本项目拟分离纯化地衣芽孢杆菌凝乳酶，在结构分析的基础上，研究其基本酶学特性，同时采用Urea SDS-PAGE、RP-HPLC和MALDI-TOF/MS等方法确定其最适作用底物和作用位点，揭示地衣芽孢杆菌凝乳酶的特异性；利用激光粒径分析仪、流变仪和激光共聚焦扫描电镜等分析乳凝胶形成过程中的胶粒直径、流变性和微观三维结构的变化规律，揭示地衣芽孢杆菌凝乳酶的凝乳机理；采用质构仪、流变仪、扫描电镜和气质联用仪等研究干酪成熟过程中残留凝乳酶活力与蛋白水解程度、流变性、质构特征、微观结构的变化规律及相关性，阐明地衣芽孢杆菌D3.11凝乳酶对干酪成熟的影响机制。

本项目研究了地衣芽孢杆菌凝乳酶的酶学特性和凝乳机理，主要研究成果如下：.1、经 SDS-PAGE分析，地衣芽孢杆菌凝乳酶的分子量为56.1 kda，抑制剂实验表明该酶是一种天冬氨酸蛋白酶，该酶在 pH5.5-9.5范围内具有活性，在60℃加热20min 后完全失活。在60℃、pH5.5时酶活力最高。作用位点分析表明该酶凝乳是由于水解-酪蛋白Thr121-Ile122，破坏了酪蛋白微粒的稳定性，从而引起酪蛋白微粒凝结。.2、对不同pH条件下地衣芽孢杆菌D3.11凝乳酶的凝乳特性进行研究。浊度分析表明，在pH5.2~6.7范围内，凝乳pH值越低浊度增长率越高；粒径结果显示，降低凝乳pH值可促进蛋白粒径值增加；粘度分析表明，凝乳pH越低，凝乳前10 min内脱脂乳粘度增长越快；流变分析表明，凝乳pH越低凝乳时间越短，在凝乳pH5.8时所形成的凝胶强度最大，地衣芽孢杆菌凝乳酶和商品凝乳酶最大储能模量值分别达到101.34Pa和158.38Pa。.3、理化指标分析表明，随着成熟期的增加，CDF组、CD3组和CCF组干酪水分含量和水分活度显著下降（p<0.05），蛋白含量显著上升（p<0.05），pH值呈现先下降后上升的趋势；蛋白水解分析发现，三组干酪pH4.6-SN、12%TCA-SN、5%PTA-SN、总游离氨基酸含量均随着干酪的成熟呈显著增加趋势（p<0.05）；SDS-PAGE电泳图分析表明，CDF组干酪α-CN水解程度较大；pH4.6可溶性肽段分析表明，随着干酪的成熟，总肽含量呈现先增加后下降的趋势，但疏水性肽与亲水性肽的比值呈现持续下降的趋势。.4、TPA分析表明，三组干酪的硬度和胶着性随着干酪的成熟而显著增加（p<0.05），而弹性、咀嚼性、粘聚性和回复性则呈现下降趋势；扫描电镜结果表明，蛋白结构的致密性随成熟度的增加而减弱，CDF组干酪结构相比于CCF组干酪较为松散。流变性能结果显示，在6个月的成熟期内，储能模量和损耗模量随着成熟时间和扫描温度的增加而降低，Tanδ值在成熟期内下降，表明干酪流动性随着成熟时间的增加而减弱。