CN108728381A - 一种具有对克林霉素稳定性的植物乳杆菌、其评价方法及应用 - Google Patents
一种具有对克林霉素稳定性的植物乳杆菌、其评价方法及应用 Download PDFInfo
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Abstract
本发明涉及一种具有对克林霉素稳定性的植物乳杆菌(Lactobacillus plantarum)LC及其评价方法。该菌株通过菌株活化培养、含克林霉素培养液培养、无克林霉素培养液培养、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LC在连续继代培养10个月后,耐药性维持在0.032μg/mL,即对克林霉素具有高耐药性的特征,这一特征可确保植物乳杆菌LC制品与克林霉素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,本发明涉及一种具有对克林霉素稳定性的植物乳杆菌(Lactobacillus plantarum),还涉及这株具有对克林霉素稳定性的植物乳杆菌的评价方法。
【背景技术】
植物乳杆菌作为人体胃肠道益生菌被广泛应用于食品和药品领域中。克林霉素的主要成份为克林霉素磷酸酯,其抗菌活性较强,适用于革兰阳性菌引起的呼吸道、关节与软组织、骨组织、胆道等感染以及厌氧菌引起的腹腔和盆腔等部位感染,其在中国的抗生素药物市场中占有很大的份额,并被广泛应用于相关疾病的治疗。
通常,服用抗生素期间不建议食用益生菌制品。但是目前,随着滥用现象的加剧,对于益生菌而言,抗生素已经成为一种的新的应激环境。近年来一些研究表明,这种应激环境会抑制益生菌的蛋白质合成、扰乱细菌分裂繁殖的能力,从而影响菌株的生长繁殖。益生菌在应激环境中若想发挥其原有的益生功效,必需具有良好的稳定性。因此,评价益生特性的植物乳杆菌在克林霉素环境下是否具有良好的稳定特性,对于在服用抗生素情况下能否食用益生菌具有非常重要的意义。
本发明针对植物乳杆菌在克林霉素环境下具有稳定性的特征,依据参考文献自行设计了一种基于适应性进化的实验方法。这种方法一般是利用微生物在特定生长条件下的适应性进化实现菌种评价,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LC对克林霉素具有良好的稳定特征。该特征可确保植物乳杆菌LC制品在与克林霉素同时服用时具有良好的稳定性。这一方法的建立可为克林霉素环境下可稳定使用的益生菌制品的研究、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提供一株具有对克林霉素稳定性的植物乳杆菌。
进一步地,本发明还提供这株植物乳杆菌对克林霉素稳定性的评价方法,及其在作为食品或食品添加剂的使用。
为了实现上述目的,本发明提供一种具有对克林霉素稳定性的植物乳杆菌(Lactobacillus plantarum)LC,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CGMCCNo.15742。
在本发明中,所述具有对克林霉素稳定性的植物乳杆菌LC的最小抑菌浓度(Minimum inhibitory concentration,MIC)为0.032μg/mL。
进一步地,本发明还提供一株具有对克林霉素稳定性的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
取植物乳杆菌,然后在液体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代得到菌液,所述菌液中的菌株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、含克林霉素培养液培养
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,分别加入到含有终浓度为0.0625、0.125、0.25、0.5、1μg/mL克林霉素的LSM培养基中,在37℃恒温培养24h,得到一组试验发酵液;
C、无克林霉素培养液培养
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在不添加克林霉素的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
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生长的克林霉素浓度,即测定克林霉素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转接至含有上述克林霉素浓度的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克林霉素浓度的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;
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0.032-16μg/mL克林霉素的LSM培养基中,则克林霉素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未添加克林霉素的含有活菌数3×105CFU的LSM与4mL添加克林霉素又未接种的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10个月后,耐药性仍然稳定在0.032μg/mL,将这株植物乳杆菌命名为植物乳杆菌LC。
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。
本发明还提供上述植物乳杆菌LC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克林霉素特性进行评价,通过耐药试验,证实所得的植物乳杆菌LC对克林霉素具有高度稳定性,连续传代10个月MIC仍维持在0.032μg/mL。
本发明的植物乳杆菌(Lactobacillus plantarum)LC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.15742。
【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:具有对克林霉素稳定性的植物乳杆菌
该实施例的实施步骤如下:
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,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代得到菌液,所得菌液中的菌株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、含克林霉素培养液培养
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,分别加入到含有终浓度为0.0625、0.125、0.25、0.5、1μg/mL克林霉素的LSM培养基中,在37℃恒温培养24h,得到一组试验发酵液;
C、无克林霉素培养液培养
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在不添加克林霉素的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
在步骤B与步骤C结束后,通过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生长的克林霉素浓度,即测定克林霉素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转接至含有上述克林霉素浓度的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克林霉素浓度的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。
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0.032-16μg/mL克林霉素的LSM培养基中,则克林霉素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未添加克林霉素的含有活菌数3×105CFU的LSM与4mL添加克林霉素又未接种的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传代过程中不同时期的最小抑制浓度,结果如表1所示
表1.适应性进化不同时间段对应菌株的MIC值(μg/mL)
在连续继代培养10个月后,耐药性维持在0.032μg/mL,将这株菌命名为植物乳杆菌LC。
上述实验证实本发明的植物乳杆菌LC在克林霉素环境下具有良好的稳定性,因此可以预期该植物乳杆菌LC及其制品在与克林霉素同时服用时能够具有良好的稳定性。该菌株对克林霉素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 (5)
1.一株具有对克林霉素稳定性的植物乳杆菌(Lactobacillusplantarum)LC,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CGMCCNo.15742。
2.根据权利要求1所述的具有对克林霉素稳定性的植物乳杆菌LC,其特征在于它的最小抑菌浓度为0.032μg/mL。
3.植物乳杆菌对克林霉素稳定性的评价方法,其特征在于该方法包括下述步骤:
A、菌株活化培养
取植物乳杆菌,然后在液体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,所述出发菌株在MRS培养基中活化传代培养3代得到菌液,所述菌液中的菌株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、含克林霉素培养液培养
取A步骤的植物乳杆菌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%接种量分别加入到含有终浓度为0.002、0.004、0.008、0.016、0.032μg/mL克林霉素的LSM培养基中,37℃培养24h,得到一组试验发酵液;
C、无克林霉素培养液培养
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在不添加克林霉素的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
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生长的克林霉素浓度,即测定克林霉素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转接至含有上述克林霉素浓度的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克林霉素浓度的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;
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,使其活菌数为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0.032-16μg/mL克林霉素的LSM培养基中,则克林霉素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未添加克林霉素的含有活菌数3×105CFU的LSM与4mL添加克林霉素又未接种的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10个月后,耐药性保持在0.032μg/mL,将这种稳定性菌株命名为植物乳杆菌LC。
4.根据权利要求4所述的评价方法,其特征在于所述的LSM培养基由以体积计90%IST培养基与10%MRS培养基组成。
5.权利要求1或2所述的具有对克林霉素稳定性的植物乳杆菌LC在食品或食品添加剂中的应用。
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Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
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CN104140937A (zh) * | 2014-05-24 | 2014-11-12 | 东北农业大学 | 植物乳杆菌klds1001及其在抑制口腔变异链球菌中的用途 |
-
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Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
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Non-Patent Citations (1)
Title |
---|
靳妲等: "一株植物乳杆菌体外抗氧化和耐药性的研究", 《食品工业科技》 * |
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