CN114958608A - 一种凝结芽孢杆菌的保藏方法 - Google Patents
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Abstract
本发明提供一种凝结芽孢杆菌的保藏方法,包括使用改良MRS培养基,制备成斜面,37℃空培24h备用。用接种环挑取凝结芽孢杆菌甘油菌液在改良MRS斜面上自下而上划线;将划线斜面置于37‑42℃恒温培养箱中培养48‑72h;取出斜面,用灭过菌的竹签刮取一环接入灭过菌的沙土管中,置于4℃或‑20℃冰箱保存。通过本发明方法培养后,菌体可较长时间保存,取用方面且可以反复使用,方便工业化生产。本发明在4℃条件下保存2年,‑20℃保存3‑5年活力不受影响。且在4℃冷藏条件下保存2年仍具有很高的活性,重复使用菌种活力不衰退。
Description
技术领域
本发明涉及微生物技术领域,尤其是一种凝结芽孢杆菌的保藏方法。
背景技术
凝结芽孢杆菌(Bacillus coagulans)是芽孢杆菌属、乳酸菌类益生菌。既具有乳酸菌产酸的特性,又具有芽孢杆菌耐高温、抗逆性强和耐储存的优点。作为一种新型的微生态制剂,受到众多专家学者的关注,并对其进行开发研究。目前已被广泛应用于医疗、保健、食品、畜禽及水产养殖等领域。
凝结芽孢杆菌属于兼性厌氧的革兰氏阳性菌,其孢子具有很高的抗性、耐热性和加工性。使用沙土管保存不需特殊设备而且重复使用不会引起菌种退化,有利于凝结芽孢杆菌工业化大生产。
由于菌株的差异性及生产工艺等方面的影响,导致市面上关于凝结芽孢杆菌的产品质量参差不齐。现有技术主要是使用甘油对凝结芽孢杆菌进行保藏,使用一般菌种保藏方法,存在菌种后期活化困难以及操作繁琐等缺点。
发明内容
针对现有技术的不足,本发明提供一种凝结芽孢杆菌的保藏方法。
本发明的技术方案为:一种凝结芽孢杆菌的保藏方法,包括以下步骤:
S1)、将改良MRS培养基,制备成斜面,37℃空培24h备用;
S2)、将河沙洗净晾干,过60目筛、80目筛,留60-80目筛之间的沙子,然后用吸铁石吸去铁质,并用10%盐酸浸泡24h,并用纯化水泡数次至中性,烘干,并装入灭菌后的试管中,塞好棉塞,121℃灭菌1h,每天1次,重复3天,无菌抽样后备用;
S3)、用接种环挑取一环凝结芽孢杆菌菌液,自下而上接入改良MRS斜面上;
S4)、将接入结芽孢杆菌菌液的改良MRS斜面置于37-42℃的恒温培养箱中培养48-72h;
S5)、镜检斜面上产生大量芽孢时,取出斜面,用无菌竹签刮取一环接入备好的无菌沙土管中,置于冰箱中4℃或-20℃保存。
作为优选的,步骤S1)中,所述的改良MRS的配方为:
葡萄糖12g/L;胰蛋白胨10g/L;酵母浸粉10g/L;牛肉膏0.5g/L;氯化钠1g/L;碳酸钙1g/L;硫酸镁0.5g/L;磷酸氢二钾0.5g/L;磷酸二氢钾1g/L;硫酸锰0.2g/L;琼脂粉18g;PH6.8。
作为优选的,步骤S4)中,所述的恒温培养箱的温度为42℃。
作为优选的,步骤S4)中,斜面上菌有大量芽孢时进行保藏,冰箱的保藏温度为4℃或-20℃。
本发明的有益效果为:
1、本发明提供的保藏方法利用芽孢具有抗逆性强的使凝结芽孢杆菌在斜面上大量产孢后进行保藏,在4℃或-20℃条件下均可以进行保藏;
2、本发明在4℃条件下保存2年,-20℃保存3-5年活力不受影响;现有技术常用的甘油管保藏,存在长期保藏活化困难,反复冻融活力下降,冷冻干燥保藏,需要冷冻干燥机,对人员要求高等不便;
3、本发明不需特殊设备,操作简单,且在4℃冷藏条件下保存2年仍具有很高的活性,重复使用菌种活力不衰退。
附图说明
图1为本发明保藏方法中凝结芽孢杆菌在改良MRS斜面上培养48h时,进行染色,凝结芽孢杆菌镜检情况图。
图2为凝结芽孢杆菌在MRS斜面上培养48h时,进行染色,凝结芽孢杆菌镜检情况;
图3为凝结芽孢杆菌在PCA斜面上培养48h时,进行染色,凝结芽孢杆菌镜检情况;
图4为本发明保藏方法保藏的沙土管图片;
具体实施方式
下面结合附图对本发明的具体实施方式作进一步说明:
实施例1
本实施例提供一种凝结芽孢杆菌的保藏方法,包括以下步骤:
S1)、配置改良MRS固体培养基,用电炉加热至琼脂完全溶解后,倒入试管,用棉塞封口,牛皮纸包扎。按照115℃,30min的条件进行高压灭菌,灭菌后取出摆成斜面,待培养基凝固后,置于37℃培养24h备用;
其中,改良MRS的配方为:葡萄糖12g/L;胰蛋白胨10g/L;酵母浸粉10g/L;牛肉膏0.5g/L;氯化钠1g/L;碳酸钙1g/L;硫酸镁0.5g/L;磷酸氢二钾0.5g/L;磷酸二氢钾1g/L;硫酸锰0.2g/L;琼脂粉18g;PH 6.8。
S2)、将河沙洗净晾干,过60目筛、80目筛,留60-80目筛之间的沙子,然后用吸铁石吸去铁质,并用10%盐酸浸泡24h,并用纯化水泡数次至中性,烘干,并装入灭菌后的试管中,塞好棉塞,121℃灭菌1h,每天1次,重复3天,无菌抽样后备用;
S3)、用接种环挑取一环凝结芽孢杆菌菌液,自下而上接入改良MRS斜面上;
S4)、将接入结芽孢杆菌菌液的改良MRS斜面置于42℃的恒温培养箱中培养48h;
S5)、镜检斜面上产生大量芽孢时,取出斜面,用无菌竹签刮取一环接入备好的无菌沙土管中,置于冰箱中4℃或-20℃保存。将沙土管分别置于常温、4℃、-20℃条件下保藏半年,1年、2年、3年检测沙土中活菌数与芽孢数,检测结果如下表:
根据上表检测结果可知:本发明保藏方法4℃保存2年的存活率为90.0%,-20℃保藏3年的存活率为91.1%,说明该方法长时间保藏稳定性好。
实施例2
本实施例提供一种凝结芽孢杆菌的保藏方法,包括以下步骤:
S1)、配置改良MRS固体培养基,用电炉加热至琼脂完全溶解后,倒入试管,用棉塞封口,牛皮纸包扎。按照115℃,30min的条件进行高压灭菌,灭菌后取出摆成斜面,待培养基凝固后,置于37℃培养24h备用;
其中,改良MRS的配方为:葡萄糖12g/L;胰蛋白胨10g/L;酵母浸粉10g/L;牛肉膏0.5g/L;氯化钠1g/L;碳酸钙1g/L;硫酸镁0.5g/L;磷酸氢二钾0.5g/L;磷酸二氢钾1g/L;硫酸锰0.2g/L;琼脂粉18g;PH 6.8。
S2)、将河沙洗净晾干,过60目筛、80目筛,留60-80目筛之间的沙子,然后用吸铁石吸去铁质,并用10%盐酸浸泡24h,并用纯化水泡数次至中性,烘干,并装入灭菌后的试管中,塞好棉塞,121℃灭菌1h,每天1次,重复3天,无菌抽样后备用;
S3)、用接种环挑取一环凝结芽孢杆菌菌液,自下而上接入改良MRS斜面上;
S4)、将接入结芽孢杆菌菌液的改良MRS斜面置于37℃的恒温培养箱中培养72h;
S5)、镜检斜面上产生大量芽孢时,取出斜面,用无菌竹签刮取一环接入备好的无菌沙土管中,置于冰箱中4℃或-20℃保存。
实施例3
本实施例提供一种凝结芽孢杆菌的保藏方法,包括以下步骤:
S1)、配置改良MRS固体培养基,用电炉加热至琼脂完全溶解后,倒入试管,用棉塞封口,牛皮纸包扎。按照115℃,30min的条件进行高压灭菌,灭菌后取出摆成斜面,待培养基凝固后,置于37℃培养24h备用;
其中,改良MRS的配方为:葡萄糖12g/L;胰蛋白胨10g/L;酵母浸粉10g/L;牛肉膏0.5g/L;氯化钠1g/L;碳酸钙1g/L;硫酸镁0.5g/L;磷酸氢二钾0.5g/L;磷酸二氢钾1g/L;硫酸锰0.2g/L;琼脂粉18g;PH 6.8。
S2)、将河沙洗净晾干,过60目筛、80目筛,留60-80目筛之间的沙子,然后用吸铁石吸去铁质,并用10%盐酸浸泡24h,并用纯化水泡数次至中性,烘干,并装入灭菌后的试管中,塞好棉塞,121℃灭菌1h,每天1次,重复3天,无菌抽样后备用;
S3)、用接种环挑取一环凝结芽孢杆菌菌液,自下而上接入改良MRS斜面上;
S4)、将接入结芽孢杆菌菌液的改良MRS斜面置于40℃的恒温培养箱中培养60h;
S5)、镜检斜面上产生大量芽孢时,取出斜面,用无菌竹签刮取一环接入备好的无菌沙土管中,置于冰箱中4℃或-20℃保存。
对比例1
将菌种在液体培养基中扩培18-24h后与50%甘油1:1混合,置于-20℃或--80℃条件下保藏
凝结芽孢杆菌在不同培养基制成的斜面上生长情况如图1-4所示,从镜检结果明显可以看出,凝结芽孢杆菌在本发明所用的改良MRS培养基上芽孢率高,芽孢成熟度高。
上述实施例和说明书中描述的只是说明本发明的原理和最佳实施例,在不脱离本发明精神和范围的前提下,本发明还会有各种变化和改进,这些变化和改进都落入要求保护的本发明范围内。
Claims (4)
1.一种凝结芽孢杆菌的保藏方法,其特征在于,包括以下步骤:
S1)、将改良MRS培养基,制备成斜面,37℃空培24h备用;
S2)、将河沙洗净晾干,过60目筛、80目筛,留60-80目筛之间的沙子,然后用吸铁石吸去铁质,并用10%盐酸浸泡24h,并用纯化水泡数次至中性,烘干,并装入灭菌后的试管中,塞好棉塞,121℃灭菌1h,每天1次,重复3天,无菌抽样后备用;
S3)、用接种环挑取一环凝结芽孢杆菌菌液,自下而上接入改良MRS斜面上;
S4)、将接入结芽孢杆菌菌液的改良MRS斜面置于37-42℃的恒温培养箱中培养48-72h;
S5)、镜检斜面上产生大量芽孢时,取出斜面,用无菌竹签刮取一环接入备好的无菌沙土管中,置于冰箱中4℃或-20℃保存。
2.根据权利要求1所述的一种凝结芽孢杆菌的保藏方法,其特征在于:步骤S1)中,所述的改良MRS的配方为:
葡萄糖12g/L;
胰蛋白胨10g/L;
酵母浸粉10g/L;
牛肉膏0.5g/L;
氯化钠1g/L;
碳酸钙1g/L;
硫酸镁0.5g/L;
磷酸氢二钾0.5g/L;
磷酸二氢钾1g/L;
硫酸锰0.2g/L;
琼脂粉18g;
PH 6.8。
3.根据权利要求1所述的一种凝结芽孢杆菌的保藏方法,其特征在于:步骤S4)中,所述的恒温培养箱的温度为42℃。
4.根据权利要求1所述的一种凝结芽孢杆菌的保藏方法,其特征在于:步骤S5)中,斜面上菌有大量芽孢时进行保藏,冰箱的保藏温度为4℃或-20℃。
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CN108102967A (zh) * | 2018-01-11 | 2018-06-01 | 天津生机集团股份有限公司 | 一种鸡源凝结芽孢杆菌菌株及其产孢方法 |
CN113736689A (zh) * | 2021-08-02 | 2021-12-03 | 昆明三正生物科技(集团)有限公司 | 一种凝结芽孢杆菌培养基及培育方法 |
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CN113736689A (zh) * | 2021-08-02 | 2021-12-03 | 昆明三正生物科技(集团)有限公司 | 一种凝结芽孢杆菌培养基及培育方法 |
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