【作者】 朱洁；

【导师】 徐同； 陈卫良；

【作者基本信息】 浙江大学 ， 植物病理， 2009， 博士

【摘要】 乳酸链球菌素Nisin是由部分Lactococcus lactis(乳酸链球菌)菌株产生的一种细菌素，可抑制多种革兰氏阳性细菌，对人体安全无毒，在食品行业已经应用了50多年，目前尚未见应用中的耐药菌株报道。随着Nisin应用的快速增加和普及，Nisin也许也会象抗生素一样在自然界中产生相应的耐药性菌株，从而影响和限制Nisin的使用。本研究从浙江杭州自然界及应用Nisin为防腐剂的食品样品中尝试分离Nisin的耐药性菌株，分析在自然界或实际应用环境中Nisin耐药菌株产生的原因，并对耐药菌株的耐药机理进行探讨。主要研究结果如下：1．乳酸链球菌素耐药菌株的分离筛选和鉴定从杭州十个不同市场采集的土壤、植物叶片及食品样品中，分离共获得102个革兰氏阳性细菌菌株，以不同浓度的Nisin筛选，50 IU Nisin抑制其中47个菌株，占所有菌株的49.0％；100 IU Nisin抑制61个菌株(59.8％)；200 IUNisin抑制84个菌株(82.4％)；500 IU Nisin抑制97个菌株(95.1％)；剩下5个菌株(NIS06、NIS13、NIS17、NIS41、NIS98)为Nisin高度耐药菌株，500 IU Nisin不能抑制其生长。最小抑菌浓度(MIC)测定发现NIS41菌株对Nisin的MIC达2560IU，NIS13、NIS17、NIS98 3个菌株的MIC为1280IU，NIS06菌株的MIC为640IU。通过生理生化方法及16S rDNA序列分析5个细菌均鉴定为葡萄球菌属，其中NIS41为表皮葡萄球菌(Staphylococcusepidermidis)，NIS06，NIS13，NIS98为松鼠葡萄球菌(Staphylococcus sciuri)，NIS17为木糖葡萄球菌(Staphylococcus xylosus)2．5个Nisin耐药菌株的耐药性分析分离自自然界及市场样品的5个Nisin耐药菌株S．epidermidis NIS 41、S．sciuri NIS06、S．sciuri NIS13、S．sciuri NIS98、S．xylosus NIS17在无Nisin的培养基中连续转接30个世代后，对Nisin的耐药性没有发生显著改变，说明这5个Nisin耐药菌株的耐药性具有较好的遗传稳定性。用碱裂解法提取，5个菌株均没有发现质粒；以这5个菌株的基因组DNA为模板，以NisI／NisFEG部分序列为引物的聚合酶链式反应法(PCR)未能扩增到NisI／NisFEG疑似基因，说明上述菌株不存在类似乳酸链球菌的相关免疫基因。以LB液体培养基、黄色微球菌的去菌液为对照，5个耐药菌株的培养去菌液37℃分别处理Nisin8h，与对照相比，Nisin活性明显降低，表明这5个耐药菌株的去菌液中存在着Nisin失活物质，这可能是其主要的耐药机制。3．耐药菌株S．epidermidis NIS 41 Nisin失活物质(NIS)研究Nisin失活物质(NIS)在Nisin耐药菌株Staphylococcus epidermidis NIS 41的生长稳定期开始大量产生。培养液中的NIS在80％饱和度硫酸铵浓度下无法沉淀。37℃、60℃处理20分钟对NIS活力无明显影响，80℃以上处理活力下降。NIS在pH4～10内具有活性，最适pH值在8.0左右。NIS 4℃下保存8天以内活力基本稳定，8天以后活力开始下降，15天左右活力完全丧失。二氯甲烷、三氯甲烷、乙酸乙酯、正己烷、无水乙醚无法从水中萃取NIS，乙醇、丙酮、甲醇无法沉淀NIS。30℃真空条件下NIS可挥发。NIS对Nisin具有抑制作用，但对地衣芽孢杆菌Bacillus licheniformis ZJU12产生的细菌素无抑制。Tricine-SDS-PAGE检测发现NIS对Nisin具有降解作用，但用NIS处理BSA没有观察到降解现象。NIS的初提液经过冻融分管浓缩，高效液相色谱HPLC分离后，电喷雾质谱(ESI-MS)分析得到NIS的分子量174.94．Nisin的商业产品的纯化及对其抑菌作用的影响Nisin的商业产品经过CM-32离子交换色谱，1Kda的透析袋透析脱盐浓缩，并以Tricine-SDS-PAGE检测纯度，获得Nisin纯品。检测Nisin粗品和Nisin纯品对黄色微球菌M．flavus NCIB 8166，表皮葡萄球菌S．epidermidis NIS 41，大肠杆菌Escherichia coli BL21，乳酸链球菌Lactococcus lactis LY3的最小抑菌浓度，Nisin粗品和纯品对M．flavus NCIB 8166的MIC为50IU；对Nisin耐药菌株S．epidermidis NIS 41的MIC都是3200IU；25600IU的Nisin纯品与Nisin粗品都无法抑制E．coli BL21和L．lactis LY3，说明Nisin纯品与Nisin粗品对这四个菌株的抑菌活性没有显著差异，纯化作用对Nisin的抑菌并没有影响。平板计数琼脂法检测NaCl对Nisin抑菌作用的影响，发现0mM，50mM，100mM NaCl浓度条件下，Nisin粗品与Nisin纯品对4个菌株的抑菌曲线均保持相对平稳，没有明显的变化，说明在小于100mM NaCl浓度条件下NaCl对Nisin的抑菌作用没有明显影响。更多还原

【Abstract】 Nisin, a lanthionine-containing peptide produced by certain strains ofLactococcus lactis, is widely used in the food industry as a safe and naturalpreservative because of its antimicrobial activity against a broad range ofgram-positive bacteria. However, the mutant study on some gram-positive bacteriashowed that some strains become resistance to nisin after inducing by graduatelyincreasement of nisin concentration These results indicated the efficacy of nisin asa food preservative could be compromised by the emergence of nisin-resistantmutants. But no nisin resistant bacterial strains from nature were reported before.The present work attempted to isolate and screen nisin resistant bacterial strainsfrom market samples in Hangzhou, China, and to explore the nisin resistantmechanism.The main results was obtained as followings:1. Isolation, screen and identification of nisin resistant bacterial strains102 bacterial strains were isolated from the samples collected at ten different markets inHangzhou, Zhejiang province, China, and screened with different nisin concentration. It wasfound that 50IU nisin inhibited 47 strains (49.0%); 100IU nisin inhibited 61 strains(59.8%);200IU nisin inhibited 84 strains (82.4%); 500IU nisin inhibited 97 strains (95.1%); theremaining five strains(NIS06、NIS13、NIS17、NIS41、N1S98)were high nisin resistant bacteria.MIC test showed that the MIC of nisin to NIS41, NIS13, NIS17, NIS98 and NIS06 was2560IU; 1280IU, 1280IU, 1280IU and 640IU, respectively. Analysis of physiological-chemicalcharacteristics and 16S rDNA gene sequences showed that the five strains were all belong toStaphylococcus, NIS41 was identified as Staphylococcus epidermidis; NIS06, NIS13 andNIS98 were all identified as Staphylococcus sciuri, N1S17 was identified as Staphylococcusxylosus.2. Observation on the mechanism of nisin resistant strains.S. epidermidis NIS 41、S. sciuri NIS06、S. sciuri NIS13、S. sciuri NIS98、S. xylosus NIS17, remained their resistance or tolerance to nisin even after 30transfers in a nisin-free medium, indicatingt the 5 nisin-resistant strains with highgenetic stability. No plasmid was extraced from the 5 bacterial strains, and no PCRproducts were detected in PCR using genomic DNA of the strains as template andpartial NisI/NisFEG sequence as primers. Nisin activity were significantly reducedafter treated with supernatant of the 5 nisin-resistant strains, indicating that theproduce of Nisin inactive substance (NIS) would be the main mechanism for theirresistance to Nisin.3. Nisin inactive substance (NIS) from Staphylococcus epidermidis NIS 41.NIS was detected in stationary growth phase of Staphylococcus epidermidisNIS 41. The NIS was not precipitated with 80% saturation of ammonium sulfateadded to cell-free supernatants. The activity of the NIS was stable duringtemperature exposure to 37 and 60℃for 20 min, but lost obviously up to 80℃.The NIS was shown to retain the activity at 4℃within 8 days, but lost completelyin 15th day. The NIS was shown to retain the activity within the pH range of 2-9,and the optimum pH for the activity was about 8.0. The NIS was not extracted byDichloromethane, chloroform, ethyl acetate, n-hexane, and anhydrous ether fromwater, also it was not precipited by Ethanol, acetone and methanol. Cell-freesupematant of S. epidermidis NIS 41 was distilled at 30℃vacuum condition, andthe NIS was found in the volatile liquid. The NIS led to suppression of nisinactivity but did no effect on the bacteriocin produced by Bacillus licheniformisZJU12. Tricine-SDS-PAGE showed that nisin was degradated by the NIS. The NISwas purified by freeze-thaw treatment and HPLC, and the molecular masswas174.9 based on ESI-MS analysis.4. Nisin purification and its effect on inhibition of tested bacterial strains.Purified Nisin was obtained from commercial Nisin by ion-exchangechromatography (CM-32) and dialysis with 1Kda cut. Then Tricine-SDS-PAGEwere applied to judge the purification. The results from MIC test for M. flavusNCIB 8166, S.epidermidis NIS 41, Escherichia.coli BL21 and Lactococcus lactisLY3 showed no obvious difference on antibacterial activity between the purified and the unpurified nisin. The purified and unpurified nisin both showed a MIC of50IU for M. flavus NCIB 8166 and a MIC of 3200IU for S. epidermidis NIS 41;both 25600IU purified nisin and unpurifed nisin failed to show antibacterialactivity against E.coli BL21 and L. lactis LY3. It is suggested that purification hadno effect on antibacterial activity of nisin. By agar plate count method, inhibitioncurve of purified and unpurifed nisin against the 4 strains remained stable in thepresence of NaCl at concentration of 0mM, 50mM and 100mM NaCl, indicatingNaCl below 100mM concentration hadno effect on antibacterial activity of nisin.更多还原