【作者】 宋达峰；

【导师】 朱睦元； 顾青；

【作者基本信息】 浙江大学 ， 遗传学， 2013， 博士

【摘要】 防腐剂是食品工业中最重要的添加剂之一,使用防腐剂是防止食品变质的有效方法。目前我国大量使用的还是化学防腐剂,存在食品安全隐患。Nisin是世界公认安全的天然食品防腐剂；但Nisin抗菌谱较窄,只能抑制革兰氏阳性菌,对革兰氏阴性细菌、酵母菌、霉菌及病毒无抑制作用。植物乳杆菌素(Plantaricin)是由植物乳杆菌(Lactobacillus plantarum)产生的一种耐高、低温、耐酸、在人体中可降解、安全无毒,且具有较强抑菌活性的抗菌短肽。本实验室己分离到一株对典型革兰氏阳性和革兰氏阴性食品腐败菌均具有抑制作用的植物乳杆菌,本研究确定发挥抗菌作用的为肽类物质PZJ5,并进一步研究了PZJ5的分离纯化、理化特性、基因克隆及生物学信息、合成条件优化等因素。获得主要试验结果如下：(1)在酸性条件(pH≤5)下,细菌素PZJ5对革兰氏阳性菌和革兰氏阴性菌都有明显的抑菌作用。PZJ5具有较好的热稳定性。该细菌素对胃蛋白酶非常敏感,胰蛋白酶、蛋白酶K均可使PZJ5抑菌活性降低。(2)发酵上清液经硫酸铵梯度沉淀、离子交换色谱、疏水层析和凝胶色谱纯化后,PZJ5得到了较好的分离,出现了单独活力峰,表明该细菌素样品已经达到了单一组分。经质谱测定PZJ5的分子量为2573.8Da,采用Edman法进行N端测序,获得了PZJ5的氨基酸序列：KTKQQFLIKAQTQLFKVFGYTLKK。(3)通过对细菌素作用后的指示菌恢复生长情况观察,发现细菌素PZJ5的作用模式为杀菌。通过透射电镜和扫描电镜观察,发现细菌素PZJ5通过导致细胞壁成孔或破裂,使指示菌细胞内容物泄漏,从而细胞代谢无法正常进行,最终导致细菌死亡。通过测定指示菌细胞内K+和核物质的泄露,发现乳酸菌素PZJ5可在极短的时间使指示菌菌体破裂,细菌内容物大量外泄,导致菌体死亡。(4)经基因克隆和测序分析,发现PZJ5的成熟肽氨基酸全序列为22个氨基酸残基：KTKQQFLIKAQTQLFKVFGYTL,在其N端还有一段信号肽序列：MSLVKENKTLVDELIAQITFGG。通过Blast比对,发现PZJ5是一种新的乳酸菌细菌素。经生物信息学分析,PZJ5成熟肽带有4个正电荷氨基酸,并且在第3位到第18位残基的位置上组成了一个两亲a-螺旋结构,符合膜作用原理。(5)通过对不同培养条件、培养基成分等因素的研究,采用PB试验和响应面法优化发酵条件。得到最佳产PZJ5条件为葡萄糖2.00%,酵母提取物为1.02%,接种量为2.06%。经过优化后,植物乳杆菌ZJ5所产细菌素的效价从未优化的474IU/ml增加到872IU/ml.更多还原

【Abstract】 The preservatives is one of the most important additives in the food industry. The preservatives is an effective way to prevent food spoilage. At present, China is extensive use of chemical preservatives, which may cause food safety risks. Nisinis used widely as one of a natural preservative, but it can inhibit G+bacteria only and cannot inhibit G-bacteria, yeast and fungus. Plantaricin, produced by Lactobacillus plantarum, has a high resistance of low temperature and acid. Plantaricin, which can be biodegradable in body, was considered safe and has a strong antibacterial activity. Our group screened a Lactobacillus plantarum, which has an antibacterial effect, and separated a novel bacteriocin named plantaricin PZJ5. Therefore, we study on purification, cloning and biological information, physic-chemical characteristic and production condition optimization of bacteriocin were developed. The results are as follows:(1) Under acidic conditions (pH≤5), bacteriocins PZJ5have significant inhibitory effect on Gram-positive bacteria and gram-negative bacteria. PZJ5performance good thermal stability, and very sensitive to protease, which the antibacterial activity can be decreased by pepsin and trypsin.(2) After the gradient ammonium sulfate precipitation, ion exchange chromatography, hydrophobic interaction chromatography, gel permeation chromatography and reverse-phase HPLC purified, the PZJ5was separation and collected by a single vitality peak. This indicates that the PZJ5has reached a single component. The mass spectrometry of PZJ5showed a molecular weight of2573.8Da. Edman method for sequencing of the N-terminal amino acid sequence of PZJ5, the sequence is KTKQQFLIKAQTQLFKVFGYTLKK.(3) Through the restore growth methods by observed indicator strain, we found the mode of action of bacteriocins PZJ5is sterilization. By transmission electron microscopy and scanning electron microscopy, we found the bacteriocins PZJ5could leading to cell contents leak of the indicator bacteria by causing cell walls break down, and the cell metabolism can not be continue, that eventually leading to the death of the indicator bacteria. By measuring the indicator bacteria intracellular K+and leakage of nuclear substances, the lactobacillus PZJ5can bacterial indicator bacteria in a very short time.(4) Cloning and sequencing analysis found that the mature peptide of PZJ5has22amino acid residues:KTKQQFLIKAQTQLFKVFGYTL, and there is a signal peptide in the N-terminal:MSLVKENKTLVDELIAQITFGG. Blast alignment found PZJ5is a new bacteriocins. By the bioinformatics analysis, the mature peptide of PZJ5with four positively charged amino acids had an amphiphilic alpha-helix structure at the position of3to18residues. These comply with the principle of membrane-bound.(5) To optimize the fermentation conditions by the different culture conditions, medium composition, the use of PB test and response surface methodology, the best conditions is glucose2.00%, yeast extract1.02%and inoculum2.06%. After the optimization, the titer of plantaricin ZJ5increased from474IU/ml to872IU/ml.更多还原