高产乳链菌肽菌种选育及发酵培养基优化-制药工程专业论文.docxVIP

肉制品、罐头产品、饮品等行业 肉制品、罐头产品、饮品等行业 安全、无毒、易被人体内消化酶 菌肽效价低、生产成本高等因素 链菌肽的效价，人们对乳链菌肽 株Lactococcus lactis subsp．1actis W28进行紫外线诱变处理，通过乳链菌肽耐受平板法、葡萄糖耐受平板法以及 双重抗性筛选法获得乳链菌肽高产突变菌株，并通过摇瓶发酵实验进行复筛，最 终选育得到四株遗传稳定性好、乳链菌肽效价高的突变菌株，为GNK．1、GNK-．4、 SNK．5、SNK．10，四株菌中耐受nisin的最大浓度为7000IU／mL，葡萄糖为15％， 其乳链菌肽效价分别为2200IU／mL、2105IU／mL、2274IU／mL、21 78IU／mL，较 原始菌株分别提高了1．90、1．81、1．96、1．88倍。 以紫外诱变获得的乳链茵肽高产菌株GNK．1、GNK-4、SNK一5、SNK．10作 为融合亲本，进行Genome Shuffling，提高乳酸链球菌对乳链菌肽的耐受水平。 通过高浓度乳链菌肽(8000IU／mL)和高浓度葡萄糖(15％)对融合细胞进行初筛， 以摇瓶发酵实验进行复筛，得到乳链菌肽效价高的融合子F2、F6，乳链菌肽效 价能够达到2426IU／mL、2525IU／mL，较原始菌株提高了2．09倍和2．17倍。 为了降低生产成本，采用菌糠处理液作为乳链菌肽生产菌的培养基质。实验 首先对菌糠进行稀硫酸预处理，获得实验原料菌糠处理液。本研究主要考察了处 理液的离子浓度、不同碱液回调处理液pH值对乳酸链球菌生长以及乳链菌肽效 价的影响，发现当采用Ca(OH)2调节pH值的处理液原液培养乳酸链球菌时，菌 q 体生长最好，菌体密度可达2．9×1011cfu／mL，只是乳链菌肽效价变化不明显。 本研究对以菌糠处理液生产乳链菌肽培养基进行了优化，确定了最适补加碳 气·， 源、氮源、磷酸盐分别为蔗糖、酵母膏、玉米浆、KH2P04，其最适补加浓度分 别为39／L、7edt,、39／L、19／L，此时乳链菌肽效价为335IU／mL，较仅以菌糠处 理液为培养基质提高了23％。为乳链菌肽的生产开发了一种新的廉价原料，同时 使菌糠得到了有效利用。 关键词：乳链菌肽紫外诱变菌种选育培养基优化Genome Shuffling ABSTRACTNisin ABSTRACT Nisin is applied as a natural preservative in various food products，such as dairy, meat，call，beverage and SO on．It shows an obvious antimicrobial activity against a wide range of Gram positive bacteria,SO it can prevent food decay effectively． Compared with chemical preservative，nisin exhibits some advantages：effective，safe， nontoxic．Also it can be degraded by enzymes in human’S digestive system，SO there will be no cumulation of nisin．Nevertheless，the low yield of nisin and high cost of nisin production lead to a high price，which restricts its widespread use．Currently, people do a lot of research about nisin medium optimization and fermentation process optimization for improving the yield of nisin． In our research，Lactococcus lactis subsp．1actis W28 saved in our laboratory WaS mutanted by two rounds of UV irradiation．The mutants were screened by nisin resistance，glucose tolerance and both of them as selection marker,and then with a second shake flask experiments to obtain four mutants with gene