CN113846031A - 一种抑制多种致病菌的混菌菌粉的制备及其应用 - Google Patents
一种抑制多种致病菌的混菌菌粉的制备及其应用 Download PDFInfo
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Abstract
本发明涉及一种抑制多种致病菌的混菌菌粉的制备方法及其应用,属于微生物技术领域,本发明所述抑制多种致病菌的混菌菌粉,为植物乳杆菌、乳酸片球菌和凝结芽孢杆菌混合发酵后的菌体干燥后得到的固态粉末。本发明所述其特征是包括如下步骤:(1)植物乳杆菌、乳酸片球菌和凝结芽孢杆菌混合发酵后的菌体冻干粉的制备(2)混菌菌粉的发酵浓缩液的制备。该方法具有无毒无害、抑菌性能良好等优点。本发明提供的混菌菌粉,对人和环境安全,易于保存和运输并且可有效抑制青贮中有害微生物的生长。因而本发明为研发优质的青贮饲料添加剂、优化发酵环境、抑制有害微生物的生长、提高青贮饲料营养品质提供了新方向。
Description
技术领域
本发明微生物技术领域,具体涉及一种抑制多种致病菌的混菌菌粉的制备方法和应用。
背景技术
青贮饲料就是将半干的、青绿的饲料放入青贮窖内,维持厌氧条件,依赖乳酸菌发酵产生饲料,可长时间保存。青贮最常用的原料是玉米和紫花苜蓿,它们具有较高的营养价值和高纤维含量,已成为奶牛养殖最为广泛使用的饲粮组成成分,特别是在欧美发达国家,有60%~80%的奶牛使用青贮玉米饲料。有害微生物与有益微生物均同时存在于天然的青绿作物,所存在的有害微生物会造成青贮作物营养物质丢失。青贮材料中的有害微生物包括很多腐败细菌,主要是大肠杆菌、李斯特菌和志贺氏菌等。大肠杆菌属于革兰氏阴性菌,在很长的一段时间内,被认为是非致病菌,直到20世纪中叶,才认识到一些特殊血清型的大肠杆菌对人和动物有病原性,尤其对婴儿和幼畜。反刍动物被认为是产志毒素大肠杆菌的主要携带者,人类通过摄入受污染的食物或水,或通过直接接触受污染的动物或环境而感染,感染大肠杆菌常引起严重腹泻和败血症。在腐败的青贮饲料中通常能够检测出大量大肠杆菌,青贮厌氧时间不足可推迟乳酸发酵,进而延缓pH的降低,增加致病性大肠杆菌的存活。李斯特菌广泛存在于水、牧草、粪便等环境中,在青贮饲料和粪便中广泛存在的李斯特菌,会增加牛奶感染的风险,继而传播给人类中,李斯特菌中毒严重时可引起脑组织和血液感染。在牧草和玉米青贮饲料中发现了单核细胞增生李斯特菌,当pH超过4.5时,会进一步增加李斯特菌存在的风险,全年饲喂青贮饲料的农场比不饲喂青贮饲料的农场,感染单核细胞增生李斯特菌几率高3~7倍。志贺氏菌属是一类革兰阴性短小杆菌,是人类细菌性痢疾最为常见的病原菌,通称痢疾杆菌,耐寒,能在普通琼脂培养基上经过24小时生长,形成直径达2mm大小、半透明的光滑型菌落,同时志贺氏菌也是饲料中通常监测的卫生指标。
植物乳杆菌、乳酸片球菌和凝结芽孢杆菌三者均为同型发酵乳酸菌。在发酵过程中的代谢产物如有机酸和细菌素等物质均对杂菌有一定的抑制作用,乳酸菌是动物消化道内的有益微生物,代谢产生抑菌物质,具有抗菌谱广、不易产生耐药性等优点。凝结芽孢杆菌具有抗胃酸、抗干燥、耐高温高压、易贮存等独特的生物特性。目前,国内外对于某些致病菌的处理方法有热处理,添加乳酸、苯甲酸等添加剂的化学方法等,但是伴随着应用,这些方法的弊端也逐渐显现出来。范寰公开了一种复合发酵物,克服了现有缺乏一种有效抑制黄曲霉的制剂的缺陷,提供了一种抑制黄曲霉的复合发酵物及其制备方法,但是液态菌剂保存条件复杂且运输困难。研究表明,部分菌种对大肠杆菌、李斯特菌的生长有一定的抑制作用,但是尚没有成熟的、适用于青贮作物的抑制致病菌的复合菌剂的应用。
发明内容
本发明的目的在于提供一种新型的环保的抑制多种致病菌的植物乳杆菌、乳酸片球菌、凝结芽孢杆菌混菌菌粉。该复合菌粉易于保存,对人和环境安全,运输便利,并且可以有效抑制青贮饲料中部分有害微生物的生长,是一种安全有效的青贮饲料添加剂。
本发明所采用的技术方案得以实现:
步骤1:菌种的活化
从甘油管中取出植物乳杆菌、乳酸片球菌和凝结芽孢杆菌,分别于MRS固体斜面上划线,置37℃培养箱中培养至斜面上出现菌落,重复操作2次;
步骤2:接种植物乳杆菌、乳酸片球菌和凝结芽孢杆菌
分别挑起三种菌的单菌落于MRS培养基中培养过夜;
步骤3:固态菌粉的制备
将步骤2中培养好的发酵液,离心弃上清,加入保护剂进行喷雾干燥;
步骤4:固态菌粉的发酵
将步骤3中得到的混菌菌粉接种于MRS培养基中,置于35℃摇床震荡培养24-48h;
步骤5:发酵液的浓缩
将步骤3中的发酵液离心,弃沉淀取上清,并将发酵上清液旋蒸浓缩6-10倍;
步骤6:大肠杆菌、李斯特菌和志贺氏菌的接种
分别挑起三种的单菌落于LB液体培养基中,振荡培养过夜;
步骤7:混菌菌粉中活菌数的测定
将上述得到的混菌菌粉与PBS缓冲溶液混合,吸取100μL于装有900μL的1.5mL离心管中,依次进行稀释涂布至10-11倍;
步骤8:抑菌活性的测定
抑菌实验采用琼脂扩散法进行测定,取指示菌悬液与经融化后保持在45℃左右的半固体培养基混匀后倒入平板上,凝固后用打孔器打孔,将混菌菌粉的发酵浓缩液注入孔中,并于4℃水平放置预扩散后,培养12h测量其抑菌圈直径,每组试验3个重复。
上述步骤2的培养条件:培养温度为30℃,湿度为50-65%,转速为100-180rpm;
上述步骤3的离心参数为转速10000rpm,15-20min,4℃;
上述步骤4的植物乳杆菌、乳酸片球菌和凝结芽孢杆菌接种量为1%;
上述步骤5的旋蒸温度为50-60℃,时间为20-60min;
上述步骤6中大肠杆菌、李斯特菌和志贺氏菌的培养温度为37℃,150-180rpm,培养过夜;
上述步骤8中打孔器的直径为8mm,半固体培养基中指示菌悬液的添加量为1%,发酵浓缩液的预扩散时间为24-30h,抑菌圈的培养时间为12-24h。
本发明的有益效果在于:青贮制作和使用过程中,不免携带和产生一些不良和有害的微生物,从而危害青贮作物的长期保存和动物的生产,进而影响动物和人类的健康。本发明制备的植物乳杆菌、乳酸片球菌、凝结芽孢杆菌混菌菌粉对青贮中有害微生物例如大肠杆菌、李斯特菌和志贺氏菌均具有良好的抑菌效果。与传统的混菌菌剂相比,本发明提供了一种广谱的更稳定,更便于运输和保存的青贮中有害微生物的抑制方法,也为达到青贮饲料长期保存这一目的提供了新思路。
附图说明
图1为本发明制备的植物乳杆菌、乳酸片球菌、凝结芽孢杆菌混菌菌粉。
图2为本发明植物乳杆菌、乳酸片球菌、凝结芽孢杆菌混菌菌粉对大肠杆菌、李斯特菌和酵母菌的抑菌圈实验效果图。(其中混菌菌粉发酵浓缩液、PBS缓冲液和空白MRS培养基的添加量均为100μL)
具体实施方式
下面结合优选实施例进行详细阐述,以使发明的优点和特征能更易于被本领域技术人员理解,从而对本发明的保护范围做出更为清楚明确的界定。
实施例1:
本发明的一种抑制多种致病菌的植物乳杆菌、乳酸片球菌、凝结芽孢杆菌混菌菌粉混菌菌粉,该菌粉由植物乳杆菌、乳酸片球菌、凝结芽孢杆菌发酵而来,其菌粉发酵浓缩液具有抑菌活性,对于青贮中部分有害微生物具有良好的抑菌活性。
本优选实施例中,一种抑制多种致病菌的植物乳杆菌、乳酸片球菌、凝结芽孢杆菌混菌菌粉混菌菌粉的制备方法包括如下步骤:
(1)菌种的活化
用无菌接种环从冻存管中取出植物乳杆菌、乳酸片球菌和凝结芽孢杆菌,分别于MRS固体斜面上划线,置35℃培养箱中培养至出现菌落,重复操作活化2次后,用接种环挑起单菌落接于MRS液体培养基中35℃,120rpm培养过夜,备用。
(2)液态混菌菌剂的制备
接种凝结芽抱杆菌、乳酸片球菌和植物乳杆菌,至同一培养基中混合培养,即得到液态混菌菌剂。培养温度为30-38℃,湿度45-65%,转速100-160rpm,接种量为10%。
(3)固态混菌菌粉的制备
将上述得到的液态混菌菌剂在4℃、10 000r/min下离心15min,弃上清液,保存菌体沉淀。喷雾干燥采用离心式喷雾干燥设备,条件设置为进风温度110-150℃,出风温度80-100℃,加入保护剂,该条件下得到的干粉即为混菌菌粉。
(4)发酵上清浓缩液的制备
将上述干燥后的植物乳杆菌、乳酸片球菌、凝结芽孢杆菌混菌菌粉接种到MRS液体培养基中,将1g混菌菌粉接种于100mL MRS培养基中,于35℃、120r/min摇床中培养24h后,在4℃、10 000r/min下离心15min,取上清液,然后将上清液旋蒸浓缩7倍。
(5)混菌菌粉中活菌数的测定
将上述得到的混菌菌粉与PBS缓冲溶液混合均匀,吸取100μL于装有900μL PBS缓冲液的1.5mL离心管中,依次进行稀释涂布至10-11倍。
(6)抑菌活性的测定
抑菌实验采用琼脂扩散法进行测定,取指示菌悬液100μL与经融化后保持在45℃左右的半固体培养基(每个平板中约加入20ml培养基)混匀后倒入平板上,凝固后用内径8mm打孔器打孔,分别将100μL植物乳杆菌、乳酸片球菌、凝结芽孢杆菌混菌菌粉的上清浓缩液注入孔中,于4℃水平放置预扩散24h后,30℃培养12h,测量其抑菌圈直径,每组平行试验3个。
上述MRS培养基的配制:蛋白胨10.0g/L、葡萄糖20g/L、乙酸钠5g/L、硫酸镁0.2g/L、硫酸锰0.05g/L、牛肉粉5g/L、酵母粉4g/L、磷酸氢二钾2.0g/L、柠檬酸三铵2g/L、吐温801ml,加蒸馏水定容至1L。
上述PBS缓冲液的配置:氯化钠8g/L、氯化钾0.2g/L、十二水磷酸氢二钠3.58g/L、磷酸二氢钾0.27g/L,加蒸馏水定容至1L。
上述大肠杆菌为大肠杆菌ATCC25922;李斯特菌为李斯特菌ATCC195115;志贺氏菌为志贺氏菌ATCC25931。
经测定乳酸片球菌、植物乳杆菌、凝结芽孢杆菌混菌菌粉中活菌数约为3.25×109cfu/g,在抑菌试验中发现乳酸片球菌、植物乳杆菌、凝结芽孢杆菌混菌菌粉发酵后得到的上清浓缩液均对大肠杆菌、李斯特菌和志贺氏菌的生长有抑制作用;乳酸片球菌、植物乳杆菌、凝结芽孢杆菌复合混菌菌粉发酵后得到的上清浓缩液对大肠杆菌、李斯特菌和志贺氏菌的抑菌圈直径分别为15.52mm、14.13mm和15.17mm;其中混菌菌粉发酵上清浓缩液对大肠杆菌的抑菌直径最大。
Claims (9)
1.一种抑制多种致病菌的混菌菌粉的制备方法,其特征在于,包括植物乳杆菌、乳酸片球菌和凝结芽孢杆菌活化、混合发酵、菌粉的制备、菌粉的发酵,发酵液旋蒸浓缩等步骤。
2.根据权利要求1所述的一种抑制多种致病菌的混菌菌粉的制备方法,其特征在于,具体步骤为:
(1)菌体活化:从冻存管中取出植物乳杆菌、乳酸片球菌和凝结芽孢杆菌,分别于MRS固体斜面上划线,置培养箱中培养至出现菌落,重复操作活化2次;
(2)接种植物乳杆菌、乳酸片球菌和凝结芽孢杆菌:分别挑起三种菌的单菌落于MRS培养基中培养过夜;
(3)固态菌粉的制备:将上述培养好的菌液,离心弃上清,加入保护剂进行喷雾干燥;
(4)固态菌粉的发酵:称取一定量的混菌菌粉于灭好的MRS培养基中,在35℃摇床中培养24-48h;
(5)浓缩发酵液:将上述发酵好的液体离心,留上清液弃沉淀,将发酵液旋蒸浓缩6-10倍;
(6)混菌菌粉中活菌数的测定
将上述得到的混菌菌粉与PBS缓冲溶液混合均匀,吸取100μL菌液依次进行稀释涂布至10-11倍;
(7)抑菌性能的测定:抑菌实验采用琼脂扩散法进行测定,取指示菌悬液与经融化的半固体培养基混匀后倒入平板上,凝固后打孔器打孔,将植物乳杆菌、乳酸片球菌、凝结芽孢杆菌混合培养的发酵浓缩液注入孔中,于4℃水平放置预扩散后培养12h测量其抑菌圈直径,每组试验3个重复。
3.按权利要求1所述的方法,其特征在于所述植物乳杆菌为植物乳杆菌ACCC11016;所述乳酸片球菌为乳酸片球菌CGMCC1.4;所述凝结芽孢杆菌为凝结芽孢杆菌ACCC10229。
4.按权利要求1所述的方法,其特征在于,混菌菌粉活菌数约为3.25×109cfu/g。
5.按权利要求1所述的方法,其特征在于,菌粉的接种量为发酵培养基体积的1%。
6.按权利要求1所述的方法,其特征在于,所述发酵培养基选自MRS培养基。
7.按权利要求1所述的方法,其特征在于,所述发酵时还伴随搅拌,所述搅拌的速率为150~180rpm。
8.按权利要求1所述的方法,其特征在于,所述混菌菌粉发酵的发酵时间为24-48h。
9.按权利要求1所述的方法,其特征在于,所述抑菌实验中,发酵浓缩液的添加量为100μL。
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