产细菌素乳酸菌的分离鉴定及培养条件的研究

【作者】 邹鹏；

【导师】 葛菁萍；

【作者基本信息】 黑龙江大学 ， 生物化学与分子生物学， 2006， 硕士

【摘要】 本研究的主要目的是筛选和鉴定产细菌素的乳酸菌菌株、优化发酵条件以提高细菌素的活性并探讨粗细菌素的生物学特性。 本试验从乳酸酸菜发酵液中筛选到8株产细菌素的乳酸杆菌菌株HDRS1～HDRS8，根据形态特征观察，常规生理生化鉴定，API 50 CH试剂条鉴定和16S rDNA序列分析，确定HDRS1～HDRS7为植物乳杆菌(Lactobacillus plantarum)，HDRS8为短乳杆菌(Lactobacillus brevis)。 在排除过氧化氢和有机酸对指示菌的抑制作用后，HDRS1和HDRS8菌株的发酵液对指示菌均有强烈的抑制作用。而经胰蛋白酶和蛋白酶K处理后，发酵液抑菌活性明显下降，证明HDRS1和HDRS8的发酵液中含有蛋白质性质的抑菌物质，即细菌素。 本研究探讨了营养要素(包括培养基组成和不同的起始pH值)和环境要素(包括培养温度、装液量和接种量)对细菌素产生的影响，以求得产生细菌素的最佳发酵条件。正交试验得出HDRS1产细菌素的最佳营养要素和环境要素为：蔗糖2％，牛肉膏2.5％，吐温80 0.1％，pH6.5，30℃培养，80mL／100mL装液量；HDRS8产细菌素的最佳营养要素和环境要素为，葡萄糖2％，牛肉膏2.5％，吐温80 0.1％，pH6.5，37℃培养，40mL／100mL装液量。 分别取HDRS1和HDRS8在对数期和稳定期的发酵液进行抑菌试验，确定对数期的细菌素产量较高。比较了四种不同的菌种保藏方法，结果发现用螺口试管穿刺法保藏HDRS1和HDRS8菌种效果最好，有效保藏时间在5个月以上。 粗细菌素的生物学特性研究表明，HDRS1和HDRSD8产生的细菌素在微酸性条件下抑菌活性较高，在pH为5.0时活性最高，显示活性更多还原

【Abstract】 The aim of this project was to screen lactobacillus strains of producing bacteriocin. Fermentation conditions of bacteriocin were studied in order to enhance the titer of bacteriocin and the biological features of coarse bacteriocin were also determined.Eight bacteriocin-producing lactic acid bacteria strains were screened from Chinese sauerkraut juice, which named as HDRS1-HDRS8. According to the morphological features, physiological and biochemical features, API identification kit and 16S rDNA sequence analyse of the strains, strains HDRS1-HDRS7 were identified as Lactobacillus plantarum and HDRS8 was identified as Lactobacillus brevis.The supernatant of HDRS1 and HDRS8 could strongly inhibit indicator strains after excluded inhibitive effect of H₂O₂ and organic acid, but the inhibitive activity decreased sharply after treatment with trypsin and proteinase K. The results confirmed that the anti-bacteria product produced by HDRSl and HDRS8 were both protein, which could be classed as bacteriocin.Different factors were investigated such as the ingredients of medium, temperature, pH, inoculating amount and medium capacity on production of bacteriocin in order to optimize the fermentation condition. The optimum carbon source of HDRS1 and HDRS8 were sucrose and glucose, respectively, and the optimal concentration were both 2%; the optimum nitrogen source and growth factor of them were beef extract and Tween80, respectively, and the optimal concentration was 2.5% and 0.1%, by orthogonal experiment. The optimum initial pH for HDRS1 and HDRS8 was 6.5 and they produced the highest amount of bacteriocin at 30℃ and 37℃, respectively. The optimum amount in one flask of HDRS1 and HDRS8 were 80mL/100mL and更多还原