不同来源乳酸菌的耐药性分析及药敏性乳酸菌的应用

【作者】 张爱民；

【导师】 顾瑞霞；

【作者基本信息】 扬州大学 ， 微生物学， 2008， 硕士

【摘要】 乳酸菌在自然界分布广泛,且广泛应用于食品工业、饲料业和医药业等相关的领域。近年来研究表明,抗生素在治疗疾病的同时,也使得环境中耐药菌株的种类及数目增加。乳酸菌,作为在食品工业中广泛应用的发酵剂,携带有可转移耐药因子的乳酸菌应用于生产,将严重影响到食品的安全性。本文对新疆牧区酸马奶、酸牛奶、骆驼奶、奶疙瘩,云南大理乳饼、本地市售乳制品及扬州本地健康婴儿粪便中分离鉴定的乳酸菌的耐药性情况进行了研究,对消除耐药性的乳酸菌的耐药性回复情况、生长及发酵性能、储藏过程中的存活率以及耐药性消除菌株在豆乳中的应用进行了研究。1、从不同来源的样品中共分离到45株乳酸菌,其中从新疆的传统乳制品中分离到了16株乳酸菌,包括米酒乳杆菌5株,德氏乳杆菌保加利亚亚种2株,植物乳杆菌2株,食品乳杆菌、干酪乳杆菌、短乳杆菌各1株,乳酸乳球菌乳脂亚种1株,屎肠球菌2株、牛链球菌各1株;从云南乳饼中分离到11株乳酸菌,包括德氏乳杆菌保加利亚亚种3株,瑞士乳杆菌、干酪乳杆菌、绿色乳杆菌各2株,短乳杆菌、屎肠球菌1株;从本地乳品中分离到6株德氏乳杆菌保加利亚亚种,5株嗜热链球菌;从婴儿粪便中分离到6株鼠李糖乳杆菌。2、乳酸菌耐药性的研究结果显示,分离株对青霉素G的耐药率为35.6%（16/45）;对万古霉素的耐药率为15.6%（7/45）;对红霉素的耐药率为73.3%（33/45）;对四环素耐药率为48.9%（22/45）;对阿莫西林的耐药率为20%（9/45）;对头孢曲松的耐药率为17.8%（8/45）;对庆大霉素的耐药率为68.9%（31/45）;对诺氟沙星的耐药率为64.4%（29/45）;对氯霉素的耐药率为24.2%（11/45）。其中,新疆来源的乳酸菌表现出对红霉素、庆大霉素、诺氟沙星的高度耐药,具有多重耐药的菌株有13株,云南来源的乳酸菌表现出对红霉素的高度耐药,具有多重耐药的菌株7株,市售乳品来源的乳酸菌表现出对庆大霉素的高度耐药,具有多重耐药的菌株有9株,婴儿肠道来源的乳酸菌表现出对庆大霉素的高度耐药,具有多重耐药的菌株有5株。3、耐药性消除实验表明:SDS结合47℃培养交替处理96h后,乳酸菌的耐药性就可以全部被消除,盲传50代后耐药性虽有所回复,但并没有超出药敏范围。与耐药性消除前相比,耐药性消除后,XL1（L.Lactic subsp.cremoris）、XN₅3（S.bovis）在不同温度下的生长速度都有所降低;菌株G4（L.casei）、90-M2X（E.faecium）的产酸能力分别降低了13%和28%;4℃放置2周后菌株XN₅3（S.bovis）、YC-380（E.faecium）和XL1（L.Lactic subsp.cremoris）的存活率,分别是耐药性消除前的0.3%、0.7%和1.5%。4、用逐步提高培养液中豆乳浓度的方法,对消除耐药性以后的菌株进行驯化,驯化后乳酸菌的凝乳时间缩短0.5-2h,产酸能力提高5-15°T,显示乳酸菌有较强的适应能力。应用驯化后的耐药性消除菌株进行酸豆乳发酵试验,结果表明:酸豆乳生产的最佳工艺是:大豆∶水按1∶8（W/W）磨浆,蔗糖和奶油的最佳添加量分别为8%和1.0%,发酵剂的最佳球杆组合是CP7与BG按1∶1混合,发酵剂的接种量为3-4%,42℃发酵8h。发酵结束后生产出的豆乳有较高的酸度和黏度、有特殊的香气。更多还原

【Abstract】 Lactic acid bacteria（LAB） exists widely in the natural world and has high value of application in the food industry,agriculture,medicine and other fields which have closely relationship to the human.Antibiotics have been widely used in bacteria caused infections disease treament,however,they also brings hostile effects such as antibiotic resistance,the number and types of antibiotics resistant bacteria,including varieties of LAB,increased rapidly.The LAB carrying transferable antibiotics resistant factors will bring harmful consequences to consumers when they are used as starter.LAB were isolated from different samples of:koumiss,yogurt,acid camel milk, acid milk clot collected in the pastoral areas,in Xinjiang province,China;dairy cake,collected in Yunnan province,China;healthy infant faeces,collected from Yangzhou,Jiangsu province,China,and dairy products,bought from local market.The isolates were identified with physiological and biochemical methods.Antibiotics resistance of the isolated LAB was tested.SDS combined with 47℃training was used to eliminate the antibiotics resistance factors.The effects of elimination on the growth,the fermentation performance and the stability during storage of the antibiotics sensitance eliminated strains were studied.These strains were used to produce soymilk and the results were descripted as follows:1.A total of 45 strains of LAB were isolated and identified from varieties of samples of different sources of which16 strains of LAB isolated from Xinjiang include five L.sake,two L.delbrueckii subsp.Bulgaricus,two L.plantarum,one L.alimentarius,one L.casei,one L.brevic,two E.faecium,one L.Lactic subsp. Cremoris,one S.bovis;eleven strains of LAB isolated from Yunnan dairy cake,there L.delbrueckii subsp.bulgaricus,two L.helveticus,two L.casei subsp tolerans,two L.viridescens,one E.faecium;eleven strains of LAB isolated from the local dairy include six L.delbrueckii subsp.bulgaricus,five Str.thermopilu;six strains of LAB were isolated from the healthy infant faeces,they were all L.rhamnosus.2.The result of antibiotics resistance testing showed that 35.6%（16/45）of the strain was resistant to penicillin G,15.6%（7/45）was resistant to vancomycin;73.3%（33/45）was resistant to erythromycin,48.9%（22/45）was resistant to tetracycline,20%（9/45）was resistant to amoxicillin,17.8%（8/45）was resistant to Ceftriaxone,68.9%（31/45）was resistant to gentamicin,64.4%（29/45）was resistant to norfloxacin,24.2%（11/45）was resistant to chloramphenicol.Among them,the strains which isolatied from Xinjiang province showd high level resistant to erythromycin,gentamicin,norfloxacin,13 strains were multi-drug resistant;the strains which isotatied from Yunnan province showd high level resistant to erythromycin,7 strains were multi-drug resistant;the strains which isolatied from bought dairy showd high level resistant to gentamicin,9 strains were multi-drug resistant;the strains which isolatied from the infant intestinal showd high level resistant to gentamicin,5 strains were multi-drug resistant.3.The result of antibiotics resistant elimination testing showed that 1/8 MIC concentration of SDS combined with 47℃incubation were used to eliminate the antibiotics resistance of LAB,and the result showed that treated 96h under above mentioned conditions,the antibiotics resistance of the LAB could be the totally eliminated,fifty continuous generations later the antibiotics sensitive was found to be stable.However after the elimination of antibiotics resistance,the growth rate of XL1,XN₅3 had been decreased,the acid production capacity of strain G4 and 90-M2X had been reduced by 13%and 28%,respectively;after storaged at 4℃for two weeks, the survival rates of XN₅3,YC-380 and XL1 were 0.3%,0.7%and 1.5%of their parents, respectively.4.Taming can shorten the Antibiotic-sensitive LAB’s curd time by 0.5 to 2 hour,and increase the LAB’s acid production by 5 to 15°T.The proportion of soybean and water,streptococcus and bacilli,and the quantity of sugar,butter and starter were studied by the soymilk fermentation test.The result showed that the best ration of soybean and water is 1:8（W/W）,the best conment of sugar and butter is 8.0%and 1.0 %respectively.There percent starter,mixed by CP7 and BG at ration of 1:1,incubated for 8h at 42℃,could produce fine sensory quality.更多还原