【作者】 章检明；

【导师】 易华西；

【作者基本信息】 哈尔滨工业大学 ， 食品科学， 2015， 硕士

【摘要】 乳酸菌细菌素是乳酸菌在代谢过程中由核糖体产生的一类具有抑菌活性的多肽或蛋白类物质。它可被人体降解,具有无毒、无残留、高效、稳定性好、无抗药性等优点,因此成为当前天然食品防腐剂研究与开发的热点。但是,目前只有Nisin实现了工业化生产,其它的细菌素都还不能进行大规模的生产或还处于研究阶段。细菌素产量低,是限制其工业化生产最大的瓶颈。因此,如何提高细菌素产量一直是国内外研究的热点。本课题以课题组前期筛选得到的植物乳杆菌B23作为细菌素生产菌,该菌能在MRS培养基中稳定的产生具有抗单增李斯特菌活性的细菌素Lac-B23,但产量较低。本文首先利用单因素试验对MRS培养基成分进行优化和筛选,在此基础上运用PB试验、最陡爬坡试验和CCD试验得到了最佳产细菌素Lac-B23的培养基配方:葡萄糖20 g/L、酵母浸粉10 g/L、磷酸氢二钾5 g/L、硫酸锰0.1 g/L、吐温-80 1.88 m L/L、乙酸钠9.9 g/L。并在此条件下发酵,细菌素Lac-B23产量比未优化前提高了8倍。研究了初始p H、发酵温度、初始接菌浓度、培养时间对细菌素Lac-B23合成的影响。结果表明当初始p H=7,发酵温度为37℃、初始接菌浓度为109 CFU/m L、培养时间在12-16 h时最有利于细菌素Lac-B23合成。研究了外源添加物对细菌素Lac-B23合成的影响,发现酪氨酸、精氨酸、苯丙氨酸、谷氨酰胺和亮氨酸对细菌素Lac-B23的合成具有正向刺激作用。然而,甘油和丙酮酸对细菌素产量没有明显诱导作用。研究自我诱导对细菌素Lac-B23合成的影响,发现当初始接菌浓度≥106 CFU/m L时,发酵上清的抑菌活性随着初始接菌浓度的增加而增加,低于这个值时发酵上清液中检测不到抑菌活性。说明细菌素Lac-B23的合成依赖于菌体密度。研究信号分子对细菌素合成的影响,发现当信号分子添加量≤10%时能促进细菌素的合成。此外,蛋白酶E和蛋白酶K能降解发酵液中的信号分子,这说明信号分子是一类肽或蛋白物质。研究了微生物共培养对细菌素Lac-B23合成的影响。首先对诱导菌进行筛选,得出单增李斯特菌和金黄色葡萄球菌与植物乳杆菌B23共培养后能增加发酵上清液中的抑菌活性。并且,当单增李斯特的初始接菌浓度低于103 CFU/m L或金黄色葡萄球菌的初始接菌浓度在104-105 CFU/m L之间时才具有诱导作用。此外,诱导分子并没有释放到诱导菌的培养基中,且死诱导菌体也不能诱导细菌素的合成,植物乳杆菌B23只有与活的诱导菌共培养时才能增加细菌素的产量。更多还原

【Abstract】 At present, bacteriocins of lactic acid bacteria are the most popular natural food preservative in the food industry. They are ribosomally synthesized bioactive peptides or proteins with antimicrobial activity against many Gram-positive species, which have lots of advantages including non-toxic, no residue, non-resistance, high stability, etc. Therefore bacteriocins of lactic acid bacteria have became very important in the research and development of the field of microbial food preservative. However, the commercial application of bacteriocins can be hampered by low production. Thus, the development of low-cost culture medium and the research approach of increasing of bacteriocins production by lactic acid bacteria is always the focus in the lactic acid bacteria bacteriocins research field.In our laboratory, a bacterial strain producing bacteriocin Lac-B23 had been isolated from fermented milk and the strain had been named Lactobacillus plantarum B23 after bacterial identification. But bacteriocin Lac-B23 production was low. So one variable at a time(OVAT) approach, Plackett-Burman, Steepest ascent and Central Composite Design were used to obtain a best culture media for bacteriocins production. Results of the experiment indicated that we got a novel modify MRS(m MRS): glucose 20 g/L, yeast extract 10 g/L, dipotassium phosphate 5 g/L, manganese sulfate 0.1 g/L, Tween 80 1.88 m L/L, Sodium acetate anhydrous 9.9 g/L. On this condition, bacteriocin Lac-B23 production increased from 280 AU/m L to 2560 AU/m L, and the cost drastically reduced from 12. 26 RMB of MRS broth to 5.16 RMB of m MRS. After that, we researched the effect of initial p H, temperature and initial inoculum size on bacteriocin production. Results showed that bacteriocin production reached the maximum when initial p H=7, 37℃and 109 CFU/m L.We researched the effect of exogenous inducer on bacteriocin production. We found that tyrosine, arginine, phenylalanine, glutamine and leucine were favored for the synthesis of bacteriocin; glutamate, aspartate, cysteine, glycine, aspartyl and methionine inhibited bacteriocin production. In addition, glycerinum and pyruvic acid had no effect on bacteriocin production. We also found that we could not detect bacteriocin activity when initial inoculum size < 106 CFU/m L, which illustrated synthesis of bacteriocin depended on initial cell concentration. After that, signal molecule(autoinducing peptide) was added to m MRS, results showed that adding 5% and 10% signal molecule could increase bacteriocin production. Moreover, protease E and protease K could degrade signal molecule, which indicated signal molecule was a kind of polypeptide or protein.At last, we researched influence of co-culture on bacteriocin production. We found bacteriocin production could be increased when lactobacillus plantarum was cocultivated with Listeria monocytogenes and Staphylococcus aureus, and initial induction bacteria concentration strongly affected bacteriocin production. In addition, we proved that inducers were no released into medium, and only live induction bacteria could improve bancteriocin production.更多还原