【作者】 杜新永；

【导师】 何国庆；

【作者基本信息】 浙江大学 ， 食品科学， 2014， 博士

【摘要】 本课题以中国传统发酵食品为材料,筛选产α-半乳糖苷酶的乳酸菌,并且进行了酶活力及菌种特性研究。经相关性分析,确立了菌体生物量与α-半乳糖苷酶活力之间的正相关性,再结合培养基优化、生物膜诱导,实现了乳酸菌的高密度培养,获得了酶活力稳定、抗逆性强的全细胞唾液乳杆菌XH4B的α-半乳糖苷酶制剂。通过PY-SE半选择性培养基的培养,结合X-α-Gal显色及pNPG酶活测定,获得8株具有较高α-半乳糖苷酶活性的乳酸菌。经生理生化鉴定的结果,生长健旺、酶活力高的三株菌分别为：Lactobacillus salivarius XAIR (JX125455), L. salivarius XH4B (JX125456), Pediococcus acidilactici XS1B (JQ927329)。充分考虑到菌种本身的生长势、酶活力,以及我国关于食品中食用菌菌种的规定,最终确定唾液乳杆菌XH4B用于后续的研究。对于α-半乳糖苷酶诱导效果最好的碳源是水苏糖,其次为棉子糖、α-乳糖。采用溶菌酶法提取α-半乳糖苷酶,可以获得48.2%的酶活；而冻融法、超声破碎法、蛋白酶法可以获得25.5-36.1%的酶活力。相对于超声波法提取的粗酶,超滤纯化效率为149.80%,Sephadex G200柱层析纯化效率为391.91%。最佳酶促反应条件为：柠檬酸缓冲液pH值5.5,离子强度0.15mol/L,反应温度52。C。该酶以pNPG为底物时的Km值为0.817,以棉子糖为底物时Km值为6.672。仅有K+和Na+对酶活力有正向的激活作用。通过结晶紫染色法及电子扫描显微镜观察,当培养基中存在豆粕浸液(SE)蛋白沉淀时,其中的唾液乳杆菌XH4B倾向于形成悬浮式生物膜,而不是吸附式生物膜。生物膜的形成提高了唾液乳杆菌XH4B对不利环境的抗性,并且改变了α-半乳糖苷酶的积累趋势,生物量与酶活力之间的相关性系数为0.936,建立了生物量与α-半乳糖苷酶活力之间的正相关性,为高密度培养奠定了基础。以生物量为考察指标时,最好的氮源为酵母粉(4.38g/L),碳源则是乳糖(5.01g/L)、葡萄糖(4.91g/L)、蔗糖(4.89g/L)。乙酸钠、柠檬酸钠及磷酸氢二钠具有较好的pH值缓冲作用,同时也不会对唾液乳杆菌XH4B生长造成影响。30-50%的PY-SE培养中碳、氮源较为平衡,又有足量的蛋白质沉淀可以诱导产生悬浮式生物膜,是最优培养基。采用1-2-3补料模式,并且弃去发酵液组的生物量最高,达到17.89g/L。保留发酵废液进行补料时,两种补料模式(1-2-3)和(3-2-1)并没有显著性差异,但是1-2-3的补料模式可以充分稀释前期发酵积累的代谢废物,因此,生物量会稍微高一些。在发酵豆乳中添加唾液乳杆菌益生菌制剂,水苏糖和棉子糖的降解效率可以达到90.63%和69.89%,显著高于(P<0.05)单纯使用唾液乳杆菌XH4B单一发酵剂的效果。本课题创新性成果包括筛选到a-半乳糖苷酶活力高的唾液乳杆菌XH4B,并且,通过SE对悬浮生物膜的诱导,为乳酸菌发酵剂高密度培养提供了全新的思路,在发酵工业中具有广阔的应用前景。更多还原

【Abstract】 In this study, we took Chinese traditional foods as material to isolate lactic acid bacteria (LAB) with a-galactosidase activity. Both catalyst activity and LAB strain properties were tested and a correlationship between catalyst activity and biomass were established. After culture medium optimization as well as biofilm inducement, LAB high cell density fermentation was attained which contributed to the stable, high a-galactosidase activity whole cell Lactobacillus salivarius XH4B probiotic.8LAB strains with higher a-galactosidase activity were obtained through incubation by semi-selective culture medium PY-SE, staining by X-a-Gal and catalyst activity measurement by pNPG. After16S rDNA identification,3out of8strains were named as:Lactobacillus salivarius XA1R (JX125455), L. salivarius XH4B (JX125456), Pediococcus acidilactici XS1B (JQ927329). According to the growth viability and a-galactosidase activity of the strains, as well as the Chinese edible fungi regulation in foods, we eventually chose the strain of L. salivarius XH4B for the following researches.The best carbohydrate for a-galactosidase activity inducement was stachyose, followed by raffinose, a-lactose. As to extraction, the lysozyme method could attain48.2%activity, while the methods of freeze-thawing, supersonic extraction and protease only attained25.5-36.1%activity compared with whole-cell a-galactosidase activity. The purifying ratios of hyper-filtration and Sephadex G200column chromatography were149.80%and391.91%respectively compared with crude enzyme extracted by supersonic. The best catalyst activity conditions were:citric buffer pH value5.5, ion strength0.15mol/L and temperature52℃. The Km values of the a-galactosidase were0.817to pNPG and6.672to raffinose. Only K+and Na+activated the catalyst activity positively.When SE (Soybean meal extract) added into the culture medium, there was floating biofilm instead of attached biofilm formation which observed by crystal violet staining as well as electronic scanning microscope. The biofilm formation enhanced the LAB cells resistance to circumstantial stresses, and changed the α-galactosidase accumulation trends. There was a positive correlation between L. salivarius XH4B biomass and a-galactosidase activity with an index of0.936, which promised the high cell density fermentation for higher a-galactosidase activity.The best hydrogen for biomass yield was yeast extract (4.38g/L), and carbohydrates were lactose (5.01g/L), glucose (4.91g/L) and sucrose (4.89g/L). Sodium acetate, sodium citrate and disodium hydrogen phosphate could buffer the culture media pH value and did not inhibit the L. salivariu.s XH4B growth. The culture media of30～50%PY-SE balanced nitro-and carbon-hydrates and adequate protein coagulation to induce floating biofilm which was the best ones for high cell density fermentation. With1-2-3fed-batch method when fermented iluid was discarded, the biomass could reach17.89g/L. When the fermented iluid remained in batches, two fed-batches (1-2-3and3-2-1) were not different significantly, however, the1-2-3fed-batch could thoroughly dilute the former metabolic waste and the biomass was higher than3-2-1.With the additive of α-galactosidase or freeze dried L. salivasrius XH4B could hydrate stachyose and raffinose90.63%and69.89%respectively, which significantly higher than the starter only.This study isolated L. salivarius XH4B with high α-galactosidase activity and broadened the method to LAB high cell density fermentation with SE inducement to floating biofilm. This would benefit the fermentation industry in yoghurt or probiotic manufacturing.更多还原