CN112410251B - 一种产酸快、产酸量高的植物乳杆菌及其应用 - Google Patents
一种产酸快、产酸量高的植物乳杆菌及其应用 Download PDFInfo
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Abstract
本发明提供了一种产酸快、产酸量高的植物乳杆菌及其应用,其为植物乳杆菌(Lactobacillus plantarum)WG03,保藏单位为中国普通微生物菌种保藏管理中心,保藏号为CGMCC 20876,保藏日期为2020年10月12日。本发明提供了一种包含上述植物乳杆菌的微生物菌剂。可将上述的植物乳杆菌或菌剂应用于青贮饲料发酵中。本发明通过原生质体融合技术结合传统诱变的方法获得了一株产酸速度快且产酸量高的植物乳杆菌,使用该菌株作为青贮添加剂,能够很好的抑制腐败菌的生长,快速启动厌氧发酵过程,改善青贮饲料的品质。
Description
技术领域
本发明涉及微生物技术领域,具体地说是涉及一种产酸快、产酸量高的植物乳杆菌及其应用。
背景技术
乳酸菌是一大类发酵糖产生大量乳酸的细菌的统称,主要包括乳杆菌、双歧杆菌、乳球菌、链球菌、肠球菌、明串珠菌、片球菌、芽孢杆菌等属。乳酸菌在医药、工业、农业等与人类生活密切相关的重要领域有很高的应用价值,如乳杆菌、双歧杆菌、湿热链球菌发酵的酸奶,应用于保健食品;乳杆菌、双歧杆菌等应用于临床医疗;植物乳杆菌应用于青贮饲料。青贮发酵过程中原料附着的乳酸菌等微生物利用碳水化合物形成乳酸等有机酸,并产生厌氧酸性环境,从而抑制酵母和霉菌等腐败菌生长,进而有限保存原料营养成分。由于发酵原料附着的乳酸菌一般较少,所以添加能够快速降低pH值的同型发酵乳酸菌将有利于青贮发酵的快速启动。植物乳杆菌是最常见的乳酸菌添加剂。但目前市面售卖的乳酸菌添加剂产酸速率和产酸量达不到理想效果,容易造成青贮物质腐败,影响青贮品质。
例如在专利文献CN202010612259.7中公开了一株植物乳杆菌及应用该菌株生产发酵饲料的方法,其植物乳杆菌的产酸量为12.3g/L,相对较低。目前加快产酸速率和提高产酸量的方法主要集中在自然选育和诱变育种的研究,但突变的方向难以掌握,突变体难以集中多个理想状态,为克服这些局限性,本发明采用原生质体融合技术将多个优良性状集中到一株菌株上,加快产酸速率,提高产酸量。
发明内容
本发明的目的是提供一种产酸快、产酸量高的植物乳杆菌及其应用,以解决现有植物乳杆菌产酸速率和产酸量不理想的问题。
本发明采用的技术方案是:一种产酸快、产酸量高的植物乳杆菌,其为植物乳杆菌(Lactobacillus plantarum)WG03,保藏单位为中国普通微生物菌种保藏管理中心,保藏地址为北京市朝阳区北辰西路1号院3号,保藏号为CGMCC 20876,保藏日期为2020年10月12日。
一种包含上述植物乳杆菌的微生物菌剂。
所述菌剂中植物乳杆菌的活菌数不低于109CFU/g。
上述的植物乳杆菌在青贮饲料发酵中的应用。
上述的微生物菌剂在青贮饲料发酵中的应用。
本发明运用原生质体融合技术提高植物乳杆菌的产酸速率和产酸量,与传统的育种技术相比,具有快速、高效及正突变率高等优点。原生质体融合技术将发生正突变的菌株进行多次递推式融合使菌株间优势相互叠加,也更容易获得目标菌株,原生质体融合技术通常结合传统诱变的方法来进行目标菌株的筛选。通过此技术获得的植物乳杆菌产酸速度快且产酸量高,使用该菌株作为青贮添加剂,能够很好的抑制腐败菌的生长,快速启动厌氧发酵过程,改善青贮饲料的品质。
附图说明
图1为本发明植物乳杆菌MRS培养基平板菌落图。
图2为第一组发酵袋苜蓿自然青贮发酵产品效果图。
图3为第二组发酵袋苜蓿添加植物乳杆菌HBU03青贮发酵产品效果图。
图4为第三组发酵袋苜蓿添加植物乳杆菌CGMCC 20876青贮发酵产品效果图。
具体实施方式
以下结合具体实施例,对本发明进行详细说明,实施例中未提及的试剂和操作均按本领域的常规操作实施。
实施例中采用的植物乳杆菌乳酸含量的测定方法为:向发酵液中加入过量CaCO3,待反应完全,取10mL发酵液,经8000r/min离心10min,再用移液枪吸取上清液2mL,加入25mL水,用10%的NaOH溶液调节pH值至13,最后向三角瓶中加入200mg钙黄绿素(指示剂),待完全溶解,使用0.05mol/L的EDTA标准溶液进行滴定,背光观察,当溶液得颜色由黄绿荧光色变为橙色且颜色不再发生变化,即为终点。
计算公式:W(乳酸)=90VC(g/L)
V:滴定消耗EDTA的量(mL)
C:EDTA浓度(mol/L)
实验材料与试剂
MRS培养基:牛肉膏10.0g,蛋白胨10.0g,酵母浸出汁5.0g,葡萄糖20.0g,磷酸氢二钾2.0g,柠檬酸铵2.0g,醋酸钠5.0g,硫酸镁0.1g,硫酸锰0.05g,吐温-80 1.0mL,加蒸馏水至1.0L,调节pH6.2±0.2,115℃灭菌20min。
再生培养基:在MRS液体培养基的基础上不加吐温-80,加明胶2.5%,25mmol/L氯化钙,25mmol/L六水合氯化镁,0.5mol/L蔗糖,1.6-1.8%琼脂粉,115℃灭菌20min。
原生质体缓冲溶液(LPB):Tris1.21g,蔗糖171.14g,六水合氯化镁4.07g,盐酸调节pH6.8,115℃灭菌20min。
溶菌酶溶液(100mg/mL):1g溶菌酶溶于10mL灭菌LPB中,用0.22μm的水相滤膜过滤除菌,-20℃保存。
0.85%生理盐水:称取8.5g固体NaCl,加蒸馏水定容至1L,于115℃高温灭菌15min后备用。
实施例1 复合诱变
1、DES诱变
以植物乳杆菌HBU03为出发菌株,该菌株分离自雄安新区白洋淀流域底泥中,保藏于河北省微生物多样性研究与应用实验室。取9.4mL该菌株的菌悬液至无菌三角瓶中,加入10mL pH7.0的无菌磷酸缓冲液并加入0.6mL DES溶液,震荡均匀,放置在37℃恒温摇床中振荡,从DES溶液加入开始计时,处理35min,加入10mL25%Na2S2O3来中止反应。取诱变后的菌液稀释至合适梯度,涂布于溴甲酚紫培养基上,将培养皿倒置,并置于深色纸箱中,放置于39℃培养箱培养,4~6天以后取出,用直尺测量菌落及变色圈的直径,计算出后者与前者的比值,即HC值,对HC值大的菌落加以挑选并保藏于斜面上,置于4℃冰箱中。活化试验保藏的平板初筛菌株,并发酵培养,72h后检测产酸速率和产酸量。其中一株突变株,命名为HBU03D,产酸量54.26g/L,比出发菌株HBU03的产酸量(51.65g/L)提高了5.05%,到达最高产酸量的时间与出发菌株基本一致。
2、紫外诱变
以突变株HBU03D为出发菌株,将已稀释至合适稀释度的菌液用移液枪吸取10mL至无菌培养皿,并放置在磁力搅拌器上,紫外灯下20cm处。打开磁力搅拌器的电源,让菌液混合均匀,并开启器皿盖,开启紫外灯,用秒表计时,照射时间为40s。照射完毕,放置于无菌室静置1h,然后吸取菌液200µL于溴甲酚紫培养基,将培养皿倒置,并置于深色纸箱中,放置于39℃培养箱培养,4~6天以后取出并观察,平板上长出的菌落成橘红色,且菌落周围有大小不等的柠檬黄色的变色圈,用直尺测量菌落及变色圈的直径,计算出后者与前者的比值,即HC值,对HC值大的菌落加以挑选并保藏于斜面上,置于 4℃冰箱中。活化试验保藏的紫外诱变平板初筛菌株,并发酵培养,72h后检测产酸速率和产酸量。其中一株突变株,命名为HBU03DV产酸量为68.24g/L,比出发菌株HBU03的产酸量(51.65g/L)提高了32.12%,到达最高产酸量的时间提前了约1h。
3、紫外-氯化锂诱变
活化紫外诱变得到的突变株HBU03DV,接种至种子培养基中,并稀释得到菌悬液,以最佳诱变时间,紫外灯照射,处理菌悬液。将紫外照射试验得到的菌悬液,用移液枪吸取200µL于氯化锂诱变培养基,培养皿倒置,并置于深色纸箱中,放置于39℃培养箱培养,4~6天以后取出,用直尺测量菌落及变色圈的直径,计算出后者与前者的比值,即HC值,对HC值大的菌落加以挑选并保藏于斜面上,置于4℃冰箱中。活化试验保藏的平板初筛菌株,并发酵培养,72h后检测产酸速率和产酸量。其中一株突变株,命名为HBU03DV15,产酸量为67.72g/L,比出发菌株HBU03的产酸量(51.65g/L)提高了31.11%,到达最高产酸量的时间提前了1.67h。
实施例2 原生质体的制备
选取产酸量高的突变株HBU03DV和到达最高产酸量时间最短的突变株HBU03DV15,作为两个亲本菌株进行原生质体融合,以期在同一突变株中实现产酸量大、产酸时间短的性状。
1、取培养8h(对数中后期)的突变菌株HBU03DV和HBU03DV15,离心(4000r/min,5min)收集菌体,LPB缓冲液洗涤2次,重悬于LPB中,菌体浓度约为107-108CFU/mL,加入终浓度为100mg/mL溶菌酶,37℃水浴30min。
2、原生质体灭活
紫外灭活:将上述制备好的诱变菌株HBU03DV原生质体液倾倒在直径为6cm的玻璃培养皿中,平皿中放入磁力针做转子并放置于磁力搅拌器上,将磁力搅拌器置于超净台中,将照射距离调整为19.5cm,开启功率为18W的紫外灯管计时,照射35min进行灭活。
热灭活:将上述制备好的诱变菌株HBU03DV15原生质体液移入离心管中,置于60℃水浴中40min进行灭活。
实施例3 原生质体融合及筛选
1、原生质体融合:各取500μL两种方式灭活后的两亲本原生质体液混合,3000r/min低速离心20min,弃上清液。重悬于500μLLPB中,加9倍体积的40%的PEG6000,室温作用20min,作用时间结束后,立即加5mL LPB稀释,去除PEG6000的作用,3000r/min,10min低速离心两次,重悬于100μL的缓冲液中。融合后涂布于再生平板,37℃培养箱中培养48h。同时涂布未经灭活的原生质体液,作为对照。
2、融合菌株的筛选:待菌落长出,用灭过菌的牙签挑取单个融合菌株到MRS液体培养基中,经过37℃静置发酵42-48h后,以突变菌株HBU03DV、HBU03DV15和出发菌株HBU03作为对照,检测产酸速率和产酸量,获得了一株较理想的融合菌株,该菌株的产酸量达到了89.63g/L,比出发菌株HBU03提高了71.38%,到达最高产酸量的时间提前了2.5h。将该融合菌株进行保藏,保藏号为CGMCC 20876,保藏单位为中国普通微生物菌种保藏管理中心。
将上述保藏的植物乳杆菌进行活化,扩大培养,经液态发酵、喷雾干燥制成约1010CFU/g的菌粉。可将该菌粉应用于青贮饲料发酵中。
实施例4 本发明植物乳杆菌在青贮饲料发酵中的应用
1、青贮苜蓿制备
将晾晒至所需水分含量的紫花苜蓿用铡刀切割成1cm左右的小段,装入密封袋中,设置3个处理组,接入对数生长期的植物乳杆菌,第一组不添加菌剂,自然发酵;第二组添加植物乳杆菌HBU03,含量为 1×109CFU/g,添加量为1g/kg;第三组添加实施例3获得的融合菌株,含量为 1×109CFU/g,添加量为1g/kg。对密封袋充分压实抽真空封口,厌氧发酵30天。青贮饲料的感官评定分析在30天后进行。
2、感官评定分析
取出发酵完成后的青贮样品,参考《德国DLG青贮饲料感官评分标准》的评分方法根据嗅觉、结构和色泽进行青贮质量评定(具体分值如表1所示)。根据评分可将其分为优(20~16分)、良(15~10分)、中(9~5分)、差(4~0分)四个等级。经品质评分,本实验所制作青贮苜蓿品质感官评分及青贮品质评定结果等级见表2。应用本实验获得的植物乳杆菌发酵的青贮品质评定等级为优良等级。
Claims (5)
1.一种产酸快、产酸量高的植物乳杆菌,其特征是,其为植物乳杆菌( Lactobacillus plantarum)WG03,保藏单位为中国普通微生物菌种保藏管理中心,保藏号为CGMCC 20876,保藏日期为2020年10月12日。
2.一种包含权利要求1所述植物乳杆菌的微生物菌剂。
3.根据权利要求2所述的菌剂,其特征是,所述菌剂中植物乳杆菌的活菌数不低于109CFU/g。
4.权利要求1所述的植物乳杆菌在青贮苜蓿发酵中的应用。
5.权利要求2所述的微生物菌剂在青贮苜蓿发酵中的应用。
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