CN117286063A - 一种高活性饲用罗伊氏乳杆菌剂的制备方法 - Google Patents
一种高活性饲用罗伊氏乳杆菌剂的制备方法 Download PDFInfo
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- CN117286063A CN117286063A CN202311238374.2A CN202311238374A CN117286063A CN 117286063 A CN117286063 A CN 117286063A CN 202311238374 A CN202311238374 A CN 202311238374A CN 117286063 A CN117286063 A CN 117286063A
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- C—CHEMISTRY; METALLURGY
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Abstract
本发明公布了一种高活性饲用罗伊氏乳杆菌剂的制备方法,属于生物技术应用领域。本发明的猪源Lactobacillus reuteri YSJL‑12保藏号CGMCC No.9062,菌株代谢物能抑制大肠杆菌、单增李斯特菌、沙门氏杆菌等肠道致病菌,基因组中含有耐酸、耐胆盐等相关抗应激基因,可利用多种碳源。本发明经工艺优化获得高密度制备Lactobacillus reuteri YSJL‑12培养基为豆粕5‑7%,葡萄糖6%,碳酸钙1%,3×复合无机盐。离心发酵液,收获菌泥,直接冷冻干燥制得罗伊氏乳杆菌剂。该菌剂制备方法简便、冻干成本低、活菌数目高,应用于生猪养殖,有助提升生猪健康状况。
Description
技术领域
本发明涉及一种高活性饲用罗伊氏乳杆菌的筛选鉴定、培养条件研究以及菌剂的制备等内容,属于饲用微生物技术应用领域。
背景技术
由于养殖业已禁用抗生素,但病原微生物导致的生猪腹泻等严重影响生猪养殖业的健康发展。研究表明,生猪肠道菌群平衡与其健康状况密切相关,益生菌能够对生猪肠道的菌群平衡提供有益影响,可改善肠道组织形态、对肠粘膜细胞代谢发挥积极作用,益生菌及其代谢产物已被公认为能够替代抗生素的理想补助剂。罗伊氏乳杆菌是目前存在于几乎所用脊椎动物和哺乳动物肠道中的天然宿主乳酸菌,无致病性。1989年美国FDA和饲料官方协会(AAFCO)将罗伊氏乳杆菌列入可直接喂饲动物的微生物,罗伊氏乳杆菌用于饲料益生菌越来越被重视。
近年饲用罗伊氏乳杆菌研究报道或专利申请均逐年增多。李婷等人从小型巴马猪粪便中分离获得罗伊氏乳杆菌GL001,对分离菌株的生长特性、产酸性能、模拟胃肠道环境下的耐受能力、药物敏感性及动物安全性进行了研究,获得了饲用罗伊氏乳杆菌GL001。而后又对驯化的罗伊氏乳杆菌GL005菌株的培养基成分进行优化,获得优化的增殖培养基为:红糖20.25g/L、葡萄糖10.13g/L、麦芽糖5.06g/L、大豆蛋白胨114.67g/L、酵母浸粉38.22g/L、柠檬酸氢二铵2g/L、乙酸钠7.5g/m L、磷酸氢二钾2g/L、硫酸镁0.098g/L、硫酸锰0.03g/L、吐温80 1g/L,静态培养16h活菌数为9.8×109CFU/m L。从公开的文献报道中发现,关于罗伊氏乳杆菌的研究主要集中在对菌株的筛选鉴定及菌株益生特性的研究,对于罗伊氏乳杆菌的增殖培养多是采用MRS培养基、TPY培养基等合成培养基,且培养基中所用原料成本偏高。
申请号CN202211156178“一株罗伊氏乳杆菌HLRE05及其应用”的专利申请中,介绍了从健康家禽新鲜排泄物中分离到一株罗伊氏乳杆菌Lactobacillus reuteri HLRE05,将菌株用于仔猪腹泻预防和酸奶加工制作,但所用菌株宿主特异性并不明确,可能影响菌株在动物及人体肠道中的定殖和功效。申请号CN202210809447“一种产酸的民猪罗伊氏乳杆菌及其培养方法与应用”中的罗伊氏乳杆菌采用MRS培养基厌氧培养罗伊氏乳杆菌,所用厌氧培养条件增加了制备成本,菌剂培养的液态形式不利于菌体存活率的保持。
可见,饲用罗伊氏乳杆菌的实际应用仍有很大的提升空间,既要寻求宿主特异性强菌株,又要获得培养条件便利、制备成本低廉、菌剂活菌数高的制备方法,对于罗伊氏乳杆菌制剂的实际应用具有重要意义。
发明内容
[技术问题]
益生菌菌剂的宿主特异性、活菌数量高效性、生产原料廉价性均是影响益生菌剂应用效果的主要因素,也是衡量饲料益生菌剂工业化生产是否可行的重要指标。但目前生猪养殖业的益生菌剂种类繁多,质量参差不齐,就罗伊氏乳杆菌而言,存在菌株特异性不强,菌株培养条件要求高,制备成本偏高、菌剂活菌数量偏低等问题。
[技术方案]
基于罗伊氏乳杆菌实际应用中存在的问题,本发明提供一种高活性饲用罗伊氏乳酸菌剂的制备方法。本发明从猪肠道新鲜排泄物中筛选了一株猪源罗伊氏乳杆菌,菌株能够抑制大肠杆菌、沙门氏杆菌、单增李斯特菌等多种肠道致病菌。本发明在非厌氧条件下,以副产物豆粕(或豆饼)为原料,通过发酵工艺条件优化获得高活菌数量,利用发酵代谢物做天然冻干保护剂,制得了宿主特异性强、制备成本低、冻干活菌数量高的罗伊氏乳杆菌剂,应用于生猪养殖中,将助于提升生猪健康状况。
一种制备高活性饲用罗伊氏乳杆菌剂的方法,其特征包括以下步骤:(1)将罗伊氏乳杆菌(Lactobacillus reuteri)种子液1%-5%(v/v)接入发酵培养基,充分搅拌后静置发酵;(2)无菌条件下取一定体积发酵液置于无菌离心管中,4000rpm/min离心5-15min,弃上清液,收集菌泥;(3)将菌泥置于-20℃预冻20-24h,-50℃预冻3-4h,置冻干机中冷冻干燥至物料干裂后终止。
优选的,步骤(1)中的发酵培养基含有豆粕(或豆饼)5-7%,葡萄糖6%,碳酸钙1.0%,氯化钠0.6%,无水乙酸钠0.9%,柠檬酸三铵0.3%,硫酸镁0.6%,磷酸氢二钾0.6%,121℃高压灭菌20-30min后,接入种子液,37℃静置培养48-72h。
进一步的,步骤(1)所述罗伊氏乳杆菌命名为Lactobacillus reuteri YSJL-12,保存编号CGMCC No.9062,保藏日期2014年4月16日,保藏单位为:中国微生物菌种保藏管理委员会普通微生物中心,保藏地址为:北京市朝阳区北辰西路1号院3号。
进一步的,步骤(1)所述的罗伊氏乳杆菌,其特征在于对大肠杆菌、沙门氏杆菌、单增李斯特菌具有抑菌活性。
进一步的,步骤(1)所述的种子液培养基采用MRS培养基,培养温度37℃,培养至OD600nm≥1.0。
进一步的,本发明还包括任意一项前述方法制备的高活性饲用罗伊氏乳杆菌剂。
[有益效果]
本发明的有益效果如下:
本发明罗伊氏乳杆菌菌株能抑制大肠杆菌、沙门氏杆菌、单增李斯特菌等多种肠道致病菌;本发明罗伊氏乳杆菌菌株性能优异,基因组中含有与耐酸、耐胆盐等相关的抗应激基因;本发明罗伊氏乳杆菌菌剂制备成本低廉,冻干菌剂活菌数高,相比同类菌剂,其性价比更高;本发明可开发为促进生猪生长、预防生猪腹泻的益生菌制剂。
附图说明
图1 Lactobacillus reuteri YSJL-12菌落平板。
图2 Lactobacillus reuteri YSJL-12扫描电镜下菌体形态。
图3 Lactobacillus reuteri YSJL-12光学显微镜下菌体形态(革兰氏染色)。
图4 Lactobacillus reuteri YSJL-12基于16SrRNA基因构建最大似然法系统发育树。
图5 Lactobacillus reuteri YSJL-12热处理与存活率的关系。
图6 Lactobacillus reuteri YSJL-12pH稳定性。
图7 Lactobacillus reuteri YSJL-12全基因组圈图。
图8 Lactobacillus reuteri YSJL-12COGs(cluster orthologous groups,同源基因簇)基因功能预测。
具体实施方式
下面所述实施方式是对本发明的进一步详细说明,但需要注意的是,此处所述的具体实施方式仅用于说明和解释本发明的目的、技术方案及优点等,并不对本发明构成任何限制。
具体实施方式一菌株的分离筛选
产酸菌株的分离筛选:
取适量健康母猪新鲜粪便加入无菌容器,立即密封,冰镇条件下带回实验室。无菌条件下,取上述新鲜粪便少许加入带玻璃珠的无菌水三角瓶中,摇匀,取1mL加入9mL无菌水中,混匀,依次梯度稀释至10-6,分别取各梯度样液1mL注入已灭菌的空平皿中,再倾注已灭菌冷却至45℃左右融化的分离培养基15mL~20mL/个平皿,水平摇匀,凝固后倒置恒温培养箱中,37℃2~3天。挑取周围变红的单菌落穿刺接入灭菌半固体MRS培养基中,37℃24~48h,4℃备用。菌株分离培养基:MRS培养基中添加0.1%甲基红指示剂。
抑制致病菌菌株的筛选:采用抑菌圈法测定抑菌活性。
(1)致病菌菌液制备:分别从致病菌鼠沙门氏杆菌(Salmonella typhimurium)、单增李斯特菌(Listeria monocytogenes)、大肠杆菌(Escherichia coli)的斜面上各挑取一菌环于10mL液体CM培养基/100mL三角瓶,37℃180r/min,无菌生理盐水稀释成106CFU/mL。
(2)抑菌上清液制备:分别从已分离筛选的备用菌株半固体培养基中挑取一菌环,接入灭菌液体MRS培养基中,37℃静止培养24h,4000r/min离心10min,收集上清液。
(3)抑菌试验:取90mm培养皿,倒入CM培养基15mL~20mL/个平皿,凝固,倒置空白培养过夜,无菌检查后,涂布上述稀释的致病菌液,静置30min,用打孔器打孔,每孔滴加(2)中的上清液100μl,静止30min,每孔补足上清液,37℃24h,观察测量抑菌圈直径。以不同pH值液体MRS上清液替代样品,测定抑菌圈直径(表1),以消除产酸抑菌作用。通过抑菌圈试验获得抑制肠道致病菌的菌株6株(表2)。
表1不同pH值抑菌试验
表2筛选菌株抑菌实验数据表
(4)产胞外蛋白酶菌株的筛选:将前述筛选的6株菌株分别接入液体MRS培养基,37℃24h,4000r/min离心取上清液,测定总酸及蛋白酶活力(表3)。综合抑菌效果及产酸和蛋白酶活力,最终选取产酸、产蛋白酶相对较高,同时抑制肠道致病菌的菌株,定名为YSJL-12。
表3筛选菌株的产酸及产蛋白酶试验
具体实施方式二菌株形态生理生化鉴定
对分离菌株进行了菌落形态(图1)、菌体显微镜下形态观察(图2),并进行了生理生化试验及碳水化合物产酸API50CH试验,结果分别见表4、表5。菌株革兰氏染色为阳性,见附图3。依据《伯杰氏系统细菌学手册》、《常见细菌系统鉴定手册》以及InternationalJournal of Systematic and Evolutionary Microbiology有关研究论文等相关内容,初步鉴定为Lactobacillus reuteri。
表4菌株Lactobacillus reuteri YSJL-12生理生化试验结果
表5菌株Lactobacillus reuteri YSJL-12碳水化合物发酵产酸(API50CH)结果
具体实施方式三16SrRNA基因序列分析法鉴定菌株
菌株基因组提取:按照TIANamp Bacteria DNA Kit细菌基因组DNA提取试剂盒提取分离菌株基因组,进行1%琼脂糖凝胶电泳检测。
16SrRNA基因PCR扩增:引物1 799F 5’-AACAGGATTAGATACCCTG-3’和引物21492R5’-GGTTACCTTGTTACGACTT-3’。反应体系(25uL):10×PCR buffer 2.5uL,dNTP(2.5mM)(TaKaRa)2uL,799F(10pmol/uL)(invitrogen)0.1uL,1492R(10pmol/uL)(invitrogen)0.1uL,Taq聚合酶(5U/uL)(TaKaRa)0.125uL,Templet(提取的DNA)0.5uL,ddH2O to 25uL。反应条件:预变性94℃5min,变性94℃1min,复性52℃1min 30cycles,延伸72℃1min,最后延伸72℃10min,PCR反应结束后,用1%琼脂糖凝胶电泳对PCR产物进行检测。
16SrRNA基因扩增片段回收测序:按照DNA回收试剂盒说明书回收纯化16SrRNA基因片段,送英滩捷基(上海)贸易有限公司北京实验室测序。测序比对,构建系统发育树(附图4),菌株YSJL-12鉴定Lactobacillus reuteri,于2014年4月16日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CGMCC No.9062。
具体实施方式四菌株耐热性试验
将菌株YSJL-12经液体MRS培养基活化一代,以1%接种量接入MRS液体培养基中,37℃培养24h,分别取1ml置于40℃、45℃、50℃、55℃、60℃、65℃、70℃处理1min,然后测定菌株的存活率(附图5)。
MRS液体培养基的配方:
每1L培养基:蛋白胨10.0g,牛肉粉5.0g,酵母粉4.0g,葡萄糖20.0g,吐温801.0mL,K2HPO4·7H2O 2.0g,醋酸钠·3H2O 5.0g,柠檬酸三铵2.0g,MgSO4·7H2O 0.2g,MnSO4·4H2O 0.05g,pH 6.2蒸馏水定容至1L。
试验结果:该菌株65℃以下温度短时处理仍保持大部分菌体细胞活性。
具体实施方式五菌株耐酸性试验
将菌株YSJL-12经液体MRS培养基活化一代,以1%接种量分别接入pH1.5、2.5、3.5、4.5的MRS液体培养基中,置于37℃条件下,分别于0h、2h取样测定存活率(附图6)。
试验结果:菌株在pH4.5条件下该菌能够生长;在pH3.5条件下2h存活率96.6%,在pH2.5条件下2h存活率86.2%,而在pH1.5条件下2h存活率下降到10.3%,但仍可存活。
具体实施方式六菌株基因组抗逆性特征
采用全基因组鸟枪法(Whole Genome Shotgun WGS)策略,利用Illumina MiSeqPE平台和PacBio(PacBio)RSII单分子实时(SMRT)测序平台的组合,对罗伊氏乳杆菌YSJL-12进行全基因组测序。Illumina MiSeq测序获得5661,016对末端配对reads中的高质量数据有5,561,238对末端配对reads。PacBio测序获得总序列长度639,675,353bp,总序列数目80,640段。第二代高通量测序数据拼装:采用A5-miseq(版本20160825)软件、SPAdesgenome assembler v3.11.1软件对经过Kmer校正的数据进行拼装,得到contigs和scaffolds序列。第三代单分子测序数据拼装:将Pacbio获得的第三代单分子下机数据使用HGAP 4、CANU(version 1.6)软件进行拼装,得到scaffold序列。此外,需要对第二代和第三代的拼装结果进行整合:利用Mummer软件(version 3)对拼接第二代和第三代测序数据得到的contigs进行分析,确认第二代和第三代测序数据之间的拼接结果,填补contigs之间的空白;最后使用Pilon软件(版本1.22)对结果进行校正,得到完整的拼接序列。全基因组图谱如附图7所示。
结果表明,罗伊氏乳杆菌YSJL-12的全基因组由一条2084748bp的环状染色体(GC含量为39.01%)和2个环状质粒组成,其中一个质粒51906bp,GC含量为35.23%,另一个质粒15134bp,GC含量为39.56%。
基因组中出现了与温度、pH、渗透压、氧化应激等相关的抗应激基因,如冷热休克蛋白,噬菌体休克蛋白、碱性休克蛋白、钠质子反转运蛋白、胆酰甘氨酸水解酶、硫氧还蛋白还原酶等的基因(表6)。
表6罗伊氏乳杆菌YSJL-12基因组的抗性蛋白
具体实施方式七菌株碳源利用能力
菌株可利用核糖、半乳糖、葡萄糖、麦芽糖、乳糖、蜜二糖、蔗糖、棉籽糖等多种碳源产酸(表5),罗伊氏乳杆菌基因组上含有半乳糖苷酶、果糖激酶、丙酮酸激酶、氨基果糖激酶、碳水化合物激酶、甘油酸激酶、己糖磷酸异构酶等多种涉及碳水化合物运输和代谢相关的酶(表7和图8)。
表7涉及碳水化合物运输和代谢相关的部分酶
具体实施方式八YSJL-12菌剂基础培养基配方的确定
罗伊氏乳杆菌YSJL-12属于异型乳酸发酵菌群,兼性厌氧菌,虽然在乳酸菌通用培养基MRS中生长良好,但基于培养基成本过高,试验采用原料成本相对廉价的豆粕、葡萄糖、玉米蛋白粉为培养基原料,上述原料也是粮油加工副产物,来源广泛,原料充足,替代MRS中的蛋白胨、牛肉膏、酵母膏更切合实际。同时鉴于YSJL-12发酵产酸,培养基中添加碳酸钙以中和酸度利于菌体增殖。因此,本试验将豆粕、玉米蛋白粉、碳酸钙、葡萄糖作为基础培养基的成分,利用正交试验设计筛选最佳配比,结果见表8。
表8 YSJL-12基础培养基正交试验结果
根据极差分析法的结果得到最优组合为A3B2C1D3,即碳酸钙1%、豆粕2%、玉米蛋白粉0.25%、葡萄糖6%。按此最佳配比进行三次验证试验,均值为5.69×109CFU/mL,由此说明试验获得的最佳基础配方真实可行。
具体实施方式九YSJL-12在豆粕基础培养基中最适发酵温度的确定
以豆粕基础培养基为发酵培养基,接种菌株YSJL-12后分别置于30℃、33℃、35℃、37℃、39℃、41℃,以发酵48h活菌计数为检测指标,结果如表9。
表9豆粕基础培养基中YSJL-12不同发酵温度的比较
表中可以看出,在豆粕基础培养基中,YSJL-12的在37℃~39℃范围生长较好,最适生长温度37℃,48h菌体活菌数为5.6×109CFU/mL。
具体实施方式十复合无机盐对液态培养YSJL-12的影响
分别添加不同倍数的复合无机盐,测定37℃48h YSJL-12的活菌数量,见表10。表中看出,添加复合无机盐对YSJL-12的增殖有明显的促进作用,添加1×无机盐就可以提高活菌数近1个数量级,随着无机盐浓度的增大,活菌数量呈现先增加后缓慢降低的趋势,当无机盐添加量达到3×无机盐量时,活菌数量达到最大值1.38×1011CFU/mL,因此,选择3×无机盐量(氯化钠0.6%,无水乙酸钠0.9%,柠檬酸三铵0.3%,硫酸镁0.6%,磷酸氢二钾0.6%)添加量为宜。
表10复合无机盐添加量对液态培养YSJL-12的影响
具体实施方式十一高浓度发酵培养基对YSJL-12生长的影响
因玉米蛋白粉溶解性差,酸度高,玉米蛋白粉的增加,增大了体系酸度,因此试验采用增加初始豆粕代替玉米蛋白粉氮源的培养基(豆粕5-7%,葡萄糖6%,碳酸钙1%,3×复合无机盐)。与前述优化的豆粕培养基进行对比,发酵72小时检测活菌数量,结果如表11,随着初始豆粕浓度的增加,72小时发酵体系中的活菌数量明显提高,豆粕7%的发酵培养基中活菌数可达到8.56×1012CFU/mL。但若豆粕浓度过高会增加制备成本,因此选择适当增加豆粕浓度5-7%为宜。
表11高浓度豆粕发酵培养基对YSJL-12生长的影响
具体实施方式十二罗伊氏乳杆菌YSJL-12冻干菌粉的制备
7%豆粕发酵培养基中接入1%罗伊氏乳杆菌YSJL-12种子液,37℃静置培养72h,发酵液4000rpm/min离心,取菌泥,-20℃预冻20-24h,-50℃预冻3-4h,置于冻干机中,真空度26pa,冷阱温度-55℃,层板温度-50℃,120min;-40℃,120min;-30℃,120min;-20℃,120min;-10℃,至物料干裂后结束冻干。冻干菌粉活菌计数3.06×1013CFU/g(绝干)。
具体实施方式十三罗伊氏乳杆菌YSJL-12冻干菌粉的制备
6%豆粕发酵培养基中接入3%罗伊氏乳杆菌YSJL-12种子液,37℃静置培养72h,发酵液4000rpm/min离心,取菌泥,-20℃预冻20-24h,-50℃预冻3-4h,置于冻干机中,真空度26pa,冷阱温度-55℃,层板温度-50℃,120min;-40℃,120min;-30℃,120min;-20℃,120min;-10℃,至物料干裂后结束冻干。冻干菌粉活菌计数1.76×1013CFU/g(绝干)。
具体实施方式十四罗伊氏乳杆菌YSJL-12冻干菌粉的制备
5%豆粕发酵培养基中接入5%罗伊氏乳杆菌YSJL-12种子液,37℃静置培养72h,发酵液4000rpm/min离心,取菌泥,-20℃预冻20-24h,-50℃预冻3-4h,置于冻干机中,真空度26pa,冷阱温度-55℃,层板温度-50℃,120min;-40℃,120min;-30℃,120min;-20℃,120min;-10℃,至物料干裂后结束冻干。冻干菌粉活菌计数1.08×1013CFU/g(绝干)。
Claims (6)
1.一种制备高活性饲用罗伊氏乳杆菌剂的方法,其特征包括以下步骤:
(1)将罗伊氏乳杆菌(Lactobacillus reuteri)种子液1%-5%(v/v)接入发酵培养基,充分搅拌后静置发酵;
(2)无菌条件下取一定体积发酵液置于无菌离心管中,4000rpm/min离心5-15min,弃上清液,收集菌泥;
(3)将菌泥置于-20℃预冻20-24h,-50℃预冻3-4h,置冻干机中冷冻干燥至物料干裂后终止。
2.根据权利要求1所述的一种制备高活性饲用罗伊氏乳杆菌剂的方法,其特征在于:发酵培养基含有豆粕(或豆饼)5-7%,葡萄糖6%,碳酸钙1.0%,氯化钠0.6%,无水乙酸钠0.9%,柠檬酸三铵0.3%,硫酸镁0.6%,磷酸氢二钾0.6%,121℃高压灭菌20-30min后,接入种子液,37℃静置培养48-72h。
3.根据权利要求1所述的罗伊氏乳杆菌,其特征在于所述罗伊氏乳杆菌命名为YSJL-12,保存编号CGMCCNo.9062,保藏日期2014年4月16日,保藏单位为:中国微生物菌种保藏管理委员会普通微生物中心,保藏地址为:北京市朝阳区北辰西路1号院3号。
4.根据权利要求1所述的罗伊氏乳杆菌,其特征在于对大肠杆菌、沙门氏杆菌、单增李斯特菌具有抑菌活性。
5.根据权利要求1所述的一种制备高活性饲用罗伊氏乳杆菌剂的方法,其特征在于:种子液培养基采用MRS培养基,培养温度37℃,培养至OD600nm≥1.0。
6.权利要求1-5任意一项所述方法制备的高活性饲用罗伊氏乳杆菌剂。
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