CN108690820B - 一种高抗氨苄西林植物乳杆菌、其选育方法及应用 - Google Patents
一种高抗氨苄西林植物乳杆菌、其选育方法及应用 Download PDFInfo
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- CN108690820B CN108690820B CN201810577667.6A CN201810577667A CN108690820B CN 108690820 B CN108690820 B CN 108690820B CN 201810577667 A CN201810577667 A CN 201810577667A CN 108690820 B CN108690820 B CN 108690820B
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/169—Plantarum
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
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Abstract
本发明涉及一种高抗氨苄西林植物乳杆菌(Lactobacillus plantarum)Amp及其选育方法。所述菌株是通过出发菌株活化培养、含氨苄西林培养液培养、无氨苄西林培养液培养、进化菌株耐药性的检测等步骤,通过耐药试验,证实所得菌株植物乳杆菌Amp对氨苄西林具有高耐药性的特征,在连续继代培养8个月后最小抑制浓度达到16μg/mL,这一特征可确保植物乳杆菌Amp制品与氨苄西林同时服用时保持较高的活力。
Description
【技术领域】
本发明属于生物技术领域。更具体地,本发明涉及一种高抗氨苄西林植物乳杆菌(Lactobacillusplantarum),还涉及这株高抗氨苄西林植物乳杆菌的选育方法。
【背景技术】
植物乳杆菌在食品工业中应用十分广泛,可作为人体胃肠道益生菌,具有维持肠道内菌群平衡、提高机体免疫力和促进营养物质吸收等作用。通常,服用抗生素期间不建议食用益生菌制品。有研究表明,益生菌和抗生素同时存在可能会导致益生菌的活力减弱,甚至死亡,影响其发挥特有的生理功效。这对日常生活过程中习惯食用益生菌制品的人群极为不利。但事实上,近年来越来越多的临床研究证据表明,如果能够与某些抗菌药物联合应用,其治疗一些疾病的效果则更为理想。从这个角度讲,理想的微生态制剂应该对经常用的抗菌药物有较高的相容性,同时对经常使用的治疗药物具有较高的抗性。因此,采用行之有效的方法选育具有优良耐药特性的植物乳杆菌已经成为时代发展的客观需求。
氨苄西林属于广谱半合成青霉素类抗生素,常常作为一线药物用于肠球菌、痢疾杆菌、伤寒杆菌、大肠杆菌、李斯特菌和奇异变形杆菌等细菌感染所致的疾病治疗,如泌尿系统、呼吸系统、胆道、肠道感染以及脑膜炎、心内膜炎等。目前在中国的抗生素药物市场中占有很大的份额,广泛应用于相关疾病的治疗。围绕这一抗生素选育具有耐药性的植物乳杆菌具有非常重要的意义。
本发明针对植物乳杆菌的抗氨苄西林特性,依据参考文献自行设计了一种基于自然选择的选育方法。这种方法是利用微生物在特定生长条件下的适应性进化实现菌种选育,因为具有安全显效的特点而备受欢迎。通过耐药实验,我们证实所得植物乳杆菌Amp对氨苄西林有高耐药性的特征,该特征可确保植物乳杆菌Amp制品在与氨苄西林同时服用时维持较高的活力,最终筛选出能够耐受一定浓度抗生素的益生菌菌种,以期能够指导益生菌的微生态制剂和抗生素联合使用。这一方法的建立可为我国传统发酵乳制品及微生态制剂中高抗氨苄西林乳酸菌的研究、开发和利用奠定坚实的基础。
【发明内容】
本发明的目的是基于自然选择的方法对植物杆菌抗氨苄西林特性进行选育,提供一株高抗氨苄西林植物乳杆菌。
进一步地,本发明还提供这种高抗氨苄西林植物菌的选育方法,及其在作为食品或食品添加剂的使用。
为了实现上述目的,本发明提供一种高抗氨苄西林植物乳杆菌(Lactobacillusplantarum)Amp,该菌株已于2018年04月25日在北京市朝阳区北辰西路1号院3号中国科学院微生物研究所中国微生物菌种保藏管理委员会普通微生物中心保藏,其保藏号为CGMCC No.15741。
在本发明中,所述高抗氨苄西林植物乳杆菌Amp的最小抑菌浓度(Minimuminhibitory concentration,MIC)为16μg/mL。
进一步地,本发明还提供一株高抗氨苄西林植物乳杆菌的选育方法,所述选育方法包括下述步骤:
A、菌株活化培养
取分离自内蒙古地区传统自然发酵酸粥样品的植物乳杆菌(Lactobacillusplantarum)LC,在液体MRS培养基中在活化,传代培养3代,然后将得到的培养液在固体MRS平板上划线,在37℃培养72h,随机选取三个单菌落作为出发菌株,在MRS培养基中活化传代培养3代,然后冻干保藏,它们分别命名为植物乳杆菌LC-1、植物乳杆菌LC-2和植物乳杆菌LC-3;
植物乳杆菌(Lactobacillusplantarum)LC于2018年04月25日在北京市朝阳区北辰西路1号院3号中国科学院微生物研究所中国微生物菌种保藏管理委员会普通微生物中心保藏,其保藏号为CGMCCNo.15742;
B、含氨苄西林培养液培养
取步骤A中活化培养的3代植物乳杆菌LC-1、植物乳杆菌LC-2和植物乳杆菌LC-3菌液,按照LSM培养基体积的1%接种量分别加入含有终浓度为0.0625、0.125、0.25、0.5、1μg/mL氨苄西林的LSM培养基中,在37℃恒温培养24h,得到一组试验发酵液;
C、无氨苄西林培养液培养
采用与步骤B操作方式相同的方式,按1%的接种量让植物乳杆菌LC-1、植物乳杆菌LC-2和植物乳杆菌LC-3在不添加氨苄西林的LSM培养基中进行空白对照发酵,得到一组空白对照发酵液;
D、继代与突变
在步骤B与步骤C结束后,分别测定所述试验发酵液与空白对照发酵液的OD值,确定能够抑制50%植物乳杆菌LC生长的氨苄西林浓度,即测定氨苄西林IC50值;
接着,以LSM培养基体积计1%的接种量将植物乳杆菌LC-1、植物乳杆菌LC-2和植物乳杆菌LC-3转接至含有上述氨苄西林浓度的LSM培养基中培养,得到第一次发酵液,次日将第一次发酵液再按照以LSM体积计1%接种量再次接入含有上述氨苄西林浓度的LSM培养基中,进行第二次发酵,每日重复操作进行继代培养,使菌株发生适应性进化,产生突变;
F、菌株耐药性的检测
将出发菌株和传代过程中的植物乳杆菌LC用MRS培养基活化3代后,划线于固体MRS培养基中,在37℃下培养24h,挑取单菌落置于6mL 0.85重量%的NaCl溶液中,混匀,测定在波长600nm处的OD值,用该NaCl溶液将其OD值调整至0.16-0.2,其活菌数为3×108CFU/mL,再用MRS培养基稀释500倍后作为种子液待用,所述种子液的活菌数为6×105CFU/mL;
吸取2mL所述种子液,转接于含有0.032-64μg/mL氨苄西林的LSM培养基中,则氨苄西林浓度和菌液活菌数分别减半,每支试管中的活菌数为3×105CFU/mL;以4mL未添加氨苄西林的含有活菌数3×105CFU的LSM与4mL添加氨苄西林又未接种的LSM分别作为阳性对照与阴性对照,混匀后在恒温培养箱中在温度37℃下培养48h;
测定三株植物乳杆菌的最小抑制浓度,在连续继代培养1个月后,耐药性开始发生变化,8个月后达到16μg/mL,将这种突变株命名为植物乳杆菌Amp。
在本发明中,植物乳杆菌(Lactobacillusplantarum)LC保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCC No.15742。
在本发明中,所述LSM培养基由以体积计90%IST培养基与10%MRS培养基组成。
本发明还提供上述高抗氨苄西林植物乳杆菌Amp在食品或食品添加剂中的应用。
本发明是基于自然选择方法对植物乳杆菌抗氨苄西林特性进行选育,通过耐药试验,证实选育所得的植物乳杆菌Amp对氨苄西林具有高耐药性的特征,MIC达到16μg/mL,这一特征可确保植物乳杆菌Amp及其制品与氨苄西林同时服用时保持较高的活力。
本发明的植物乳杆菌(Lactobacillusplantarum)Amp于2018年04月25日保藏于北京市朝阳区北辰西路1号院3号中国科学院微生物研究所中国微生物菌种保藏管理委员会普通微生物中心保藏,其保藏号为CGMCC No.15741。
【具体实施方式】
通过下述实施例将能够更好地理解本发明。
在本发明中如无特殊说明,表示浓度的“%”均为重量百分比,“:”均为重量比,“份”均为重量份。
本发明涉及以下培养基:
固体MRS培养基:由10克蛋白胨、10克牛肉膏、5克酵母膏、2克柠檬酸氢二铵、20克葡萄糖、1.0ml吐温80、5克三水合乙酸钠、2克三水合磷酸氢二钾、0.58克七水合硫酸镁、0.25克一水合硫酸锰、18克琼脂与1000ml蒸馏水组成,其pH 6.2-6.6。
液体MRS培养基:由10克蛋白胨、10克牛肉膏、5克酵母膏、2克柠檬酸氢二铵、20克葡萄糖、1.0ml吐温80、5克三水合乙酸钠、2克三水合磷酸氢二钾、0.58克七水合硫酸镁、0.25克一水合硫酸锰与1000ml蒸馏水组成,其pH 6.2-6.6。
IST培养基:11g水解酪蛋白、3g蛋白胨、3g葡萄糖、3g氯化钠、1g可溶淀粉、2g磷酸氢二钠、1g乙酸钠、0.2g甘油磷酸镁、0.1g葡萄糖酸钙、0.001g硫酸钴、0.001g硫酸铜、0.001g硫酸锌、0.001g硫酸亚铁、0.002g氯化锰、0.001g甲萘醌、0.001g氰钴胺、0.02g L-半胱氨酸盐酸盐、0.02g L-色氨酸、0.003g维生素B6、0.003g泛酸、0.003g烟酰胺、0.0003g生物素、0.0004g硫胺、0.01g腺嘌呤、0.01g鸟嘌呤、0.01g黄嘌呤、0.01g尿嘧啶与1000ml蒸馏水。
实施例1:高抗氨苄西林植物乳杆菌的突变选育
该实施例的实施步骤如下:
A、菌株活化培养
取植物乳杆菌(Lactobacillusplantarum)LC(CGMCC No.15742),在液体MRS培养基中在活化,传代培养3代,然后将得到的培养液在固体MRS平板上划线,在37℃培养72h,随机选取三个单菌落作为出发菌株,在MRS培养基中活化传代培养3代,然后冻干保藏,它们分别命名为植物乳杆菌LC-1、植物乳杆菌LC-2和植物乳杆菌LC-3;
B、含氨苄西林培养液培养
取步骤A中活化培养的3代植物乳杆菌LC-1、植物乳杆菌LC-2和植物乳杆菌LC-3菌液,按照LSM培养基体积的1%接种量分别加入含有终浓度为0.0625、0.125、0.25、0.5、1μg/mL氨苄西林的LSM培养基中,在37℃恒温培养24h,得到一组试验发酵液;
C、无氨苄西林培养液培养
采用与步骤B操作方式相同的方式,按1%的接种量让植物乳杆菌LC-1、植物乳杆菌LC-2和植物乳杆菌LC-3在不添加氨苄西林的LSM培养基中进行空白对照发酵,得到一组空白对照发酵液;
D、继代与突变
在步骤B与步骤C结束后,用紫外可见光分光光度计在波长为600nm条件下分别测定所述试验发酵液与空白对照发酵液的OD值,确定能够抑制50%植物乳杆菌LC生长的氨苄西林浓度,即测定氨苄西林IC50值;
接着,以LSM培养基体积计1%的接种量将植物乳杆菌LC-1、植物乳杆菌LC-2和植物乳杆菌LC-3转接至含有上述氨苄西林浓度的LSM培养基中培养,得到第一次发酵液,次日将第一次发酵液再按照以LSM体积计1%接种量再次接入含有上述氨苄西林浓度的LSM培养基中,进行第二次发酵,每日重复操作进行继代培养,使菌株发生适应性进化,产生突变。
实施例2菌株耐药性的检测
将出发菌株和传代过程中的植物乳杆菌用MRS培养基活化3代后,划线于固体MRS培养基中,在37℃下培养24h,挑取单菌落置于6mL 0.85重量%的NaCl溶液中,混匀,测定在波长600nm处的OD值,用该NaCl溶液将其OD值调整至0.16-0.2,其活菌数为3×108CFU/mL,再用MRS培养基稀释500倍后作为种子液待用,所述种子液的活菌数为6×105CFU/mL;
吸取2mL所述种子液,转接于含有0.032-64μg/mL氨苄西林的LSM培养基中,则氨苄西林浓度和菌液活菌数分别减半,每支试管中的活菌数为3×105CFU/mL;以4mL未添加氨苄西林的含有活菌数3×105CFU的LSM与4mL添加氨苄西林又未接种的LSM分别作为阳性对照与阴性对照,混匀后在恒温培养箱中在温度37℃下培养48h;
测定三株植物乳杆菌在传代过程中不同时期的的最小抑制浓度,结果如表1所示
表1.适应性进化不同时间段对应突变菌株的MIC值(μg/mL)
在连续继代培养1个月后,耐药性开始发生变化,8个月后达到16μg/mL,将这种突变株命名为植物乳杆菌Amp。
上述实验证实本发明的植物乳杆菌Amp在氨苄西林环境下具有良好的稳定性,因此可以预期该植物乳杆菌Amp及其制品在与氨苄西林同时服用时能够具有良好的稳定性。该菌株对氨苄西林环境下可稳定使用的益生菌制品的研究、开发和利用奠定了基础。
Claims (2)
1.一种高抗氨苄西林植物乳杆菌(Lactobacillus plantarum)Amp,该菌株已于2018年04月25日在北京市朝阳区北辰西路1号院3号中国科学院微生物研究所中国微生物菌种保藏管理委员会普通微生物中心保藏,其保藏号为CGMCC No. 15741。
2.权利要求1所述的高抗氨苄西林植物乳杆菌Amp在食品或食品添加剂中的应用。
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Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR20060113123A (ko) * | 2005-04-29 | 2006-11-02 | (주)엠지인투바이오 | 신균주 락토바실러스 플랜타럼 ib 213(kctc 18106p),락토바실러스 퍼멘텀 ib 261(kctc 18107p) 및 이를 이용한양돈용 프로바이오틱스 |
CN105002125B (zh) * | 2015-08-19 | 2017-11-21 | 内蒙古农业大学 | 一种高抗庆大霉素干酪乳杆菌及其选育方法 |
CN107446841A (zh) * | 2017-07-13 | 2017-12-08 | 华南理工大学 | 一种直投式植物乳杆菌高密度培养方法及制得的冻干粉和应用 |
-
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Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR20060113123A (ko) * | 2005-04-29 | 2006-11-02 | (주)엠지인투바이오 | 신균주 락토바실러스 플랜타럼 ib 213(kctc 18106p),락토바실러스 퍼멘텀 ib 261(kctc 18107p) 및 이를 이용한양돈용 프로바이오틱스 |
CN105002125B (zh) * | 2015-08-19 | 2017-11-21 | 内蒙古农业大学 | 一种高抗庆大霉素干酪乳杆菌及其选育方法 |
CN107446841A (zh) * | 2017-07-13 | 2017-12-08 | 华南理工大学 | 一种直投式植物乳杆菌高密度培养方法及制得的冻干粉和应用 |
Non-Patent Citations (2)
Title |
---|
两株植物乳杆菌作为潜在益生菌的体外评价;孟祥晨等;《东北农业大学学报》;20150923;第46卷(第9期);第44-51页 * |
细菌抗生素耐药机制研究进展;马瑞芬等;《中国微生态学杂志》;20140730;第26卷(第7期);第854-857页 * |
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