CN111690574B - 一株类肠膜魏斯氏菌及其应用 - Google Patents
一株类肠膜魏斯氏菌及其应用 Download PDFInfo
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Abstract
本发明公开了一株类肠膜魏斯氏菌及其应用。本发明的的一株类肠膜魏斯氏菌,该菌株为类肠膜魏斯氏菌Weissella paramesenteroides;保藏名称为类肠膜魏斯氏菌LL2018353;保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC),保藏地址是北京市朝阳区北辰西路1号院3号;保藏日期:2019年10月21日;保藏编号:CGMCC NO.18710。所述的类肠膜魏斯氏菌的16S rDNA基因序列为SEQ ID No.1所示的核苷酸序列。本发明抑菌和产酸性强,能低温(4摄氏度)长期保存,并能够降低猪的腹泻率和便秘率的类肠膜魏斯氏菌。
Description
技术领域
本发明涉及微生物技术领域,尤其涉及一株类肠膜魏斯氏菌及其应用。
背景技术
乳酸菌是近年来推广的饲用微生物添加剂之一。其能利用碳水化合物发酵产生有机酸等物质。研究资料表明,乳酸菌能促进动物生长,调节肠道pH,维持肠道微生态平衡,从而改善胃肠道功能、提高消化率和生物效价、抑制肠道内腐败菌生长、提高机体免疫力等。目前市场上的乳酸菌多数为厌氧生长,且品种单一,不能满足实际市场的需求。类肠膜魏斯氏菌是今年来发现的一种乳酸菌,主要存在于泡菜中,应用于泡菜等的发酵,而未见于对大肠杆菌、金黄色葡萄球菌和鼠伤寒沙门氏菌的抑菌报道。
当我国畜禽养殖过程中,抗生素、激素等药物在促进动物生长,提高饲料报酬,降低发病率以及提高生产性能等方面具有明显的优势,继而被广泛应用于动物饲养中。但是抗生素的大量使用,易造成动物肠道菌群失衡,抑制免疫反应,造成生物产品中抗生素的残留,动物排泄物污染环境等问题,土壤环境恶化等一系列反应;类肠膜魏斯氏菌具有良好的产酸和抑菌特性,具有抑制病原菌促进生长的特性,可作为新型饲料添加剂替代部分抗生素。
目前,缺乏一株抑菌和产酸性强的类肠膜魏斯氏菌及其应用。
发明内容
本发明所要解决的技术问题是提供了一株抑菌和产酸性强的类肠膜魏斯氏菌及其应用。
为了实现上述目的,本发明通过如下技术方案实现:本发明的一株类肠膜魏斯氏菌,该菌株为类肠膜魏斯氏菌Weissellaparamesenteroides;保藏名称为类肠膜魏斯氏菌LL2018353;保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC),保藏地址是北京市朝阳区北辰西路1号院3号;保藏日期:2019年10月21日;保藏编号:CGMCC NO.18710。
本发明所述的类肠膜魏斯氏菌制备的类肠膜魏斯氏菌菌剂。
本发明所述的类肠膜魏斯氏菌菌剂,其活性成分为如下(a)(b)(c)中的至少一种:
(a)所述的类肠膜魏斯氏菌的发酵培养物;
(b)所得类肠膜魏斯氏菌细胞的超声裂解上清;
(c)所得类肠膜魏斯氏菌细胞的超声裂解沉淀。
本发明所述的类肠膜魏斯氏菌菌剂的制备方法,包括如下步骤:
(1)菌种制备:MRS液体培养基中接种,35~38℃培养16~24h,5000~8000rpm离心获得菌体。
(2)将菌体进行冷冻干燥。
进一步地,在步骤(1)中,所述MRS培养基培养液为:蛋白胨10.0g;牛肉膏5.0g;酵母粉4.0g;葡萄糖20.0g;磷酸氢二钾2.0g;柠檬酸三铵2.0g;醋酸钠5.0g;硫酸镁0.2g;硫酸锰0.05g;吐温80 1.0mL;水1L。
进一步地,所述微生物制剂中菌体的有效活菌数为1010~1011CFU/g。
本发明所述的类肠膜魏斯氏菌或类肠膜魏斯氏菌菌剂在饲料和水中的应用。
有益效果:本发明抑菌和产酸性强,能低温(4摄氏度)长期保存,并能够降低猪的腹泻率和便秘率的类肠膜魏斯氏菌。
与现有技术相比,本发明具有如下优点:
(1)本发明的类肠膜魏斯氏菌有显著的抑菌效果,能抑制金黄色葡萄球菌、鼠伤寒沙门氏菌和大肠杆菌K88的生长。本发明提供的类肠膜魏斯氏菌能快速生长,降低pH,耐受低温,耐酸,并可长期液体保存。所述的类肠膜魏斯氏菌可耐低温,耐酸,4℃液体保存,pH为3.4,保存75d,活菌数为105~106CFU/mL。
本发明提供的类肠膜魏斯氏菌(Weissellaparamesenteroides)从健康迪庆藏猪体内分离的类肠膜魏斯氏菌,通过菌落形态观察,革兰氏染色、生理生化鉴定和分子生物学等鉴定其为类肠膜魏斯氏菌(Weissella paramesenteroides),该菌生长速度快,产酸效率高,对大肠杆菌K88、金黄色葡萄球菌和鼠伤寒沙门氏菌均具有良好的抑制作用。
(2)本发明体外实验表明,其对大肠杆菌K88、金黄色葡萄球菌和鼠伤寒沙门氏菌均有抑制作用。
(3)本发明动物实验表明,采用0.1%添加量到迪庆藏猪中,能降低粪便中粗蛋白和铵态氮的含量,降低猪的腹泻率和便秘率,微生物多样性分析结果表明,添加类肠膜魏斯氏菌后,能显著提高与肠道炎症等疾病负相关的菌群(理研氏菌科RC9肠群、克里斯滕森菌科R-7群、瘤胃菌科UCG-005、Lachnospiraceae_XPB1014_group和unclassified_f__Lachnospiraceae)的相对丰度。
(4)本发明动物实验表明,采用0.1%添加量到迪庆藏猪中,可在猪肠道内提高2-羟基鲸乙酸、3-氧-十八烷酸、3-羟基十四烯二酸、羟基十六碳烯酸和10,20-二羟基二十碳饱和脂肪酸等脂肪酸的含量,从而促进脂类代谢,提高维生素B2前体物质Furanofukinin含量,促进VB2的合成,提高与乙醇代谢相关的生物标记物乙基葡萄糖醛酸苷的含量,显示了机体内有显著的乙醇代谢的过程。
附图说明
图1为本发明所述的800×显微镜检的图;
图2为本发明的353的菌落形态图;
图3为本发明的基于OTU水平的韦恩图分析图;
图4为本发明的基于种水平的韦恩图分析图。
具体实施方式
本发明结合附图和具体实施例作进一步说明。应该理解,这些实施例仅用于说明目的,而不用于限制本发明范围。
实施例1
本发明的一株类肠膜魏斯氏菌,该菌株为类肠膜魏斯氏菌Weissellaparamesenteroides;保藏名称为类肠膜魏斯氏菌LL2018353;保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC),保藏地址是北京市朝阳区北辰西路1号院3号;保藏日期:2019年10月21日;保藏编号:CGMCC NO.18710。
所述的类肠膜魏斯氏菌的16S rDNA基因序列为SEQ ID No.1所示的核苷酸序列。
在MRS琼脂培养基中,经过35℃,72h有氧培养后,菌落呈乳白色、圆形,表面光滑、边缘整齐,中间凸起,无色素沉着,容易挑起,无丝,有乳酸的香味,无刺鼻酸味;革兰氏染色为阳性,菌体为杆状,单个或者两两呈现,兼性厌氧,最适生长温度38℃。
本发明所述的类肠膜魏斯氏菌制备的类肠膜魏斯氏菌菌剂。
其活性成分为如下(a)(b)(c)中的至少一种:
(a)所述的类肠膜魏斯氏菌的发酵培养物;
(b)所得类肠膜魏斯氏菌细胞的超声裂解上清;
(c)所得类肠膜魏斯氏菌细胞的超声裂解沉淀。
本发明所述的类肠膜魏斯氏菌菌剂的制备方法,包括如下步骤:
(1)菌种制备:MRS液体培养基中接种,38℃培养16h,5000rpm离心获得菌体。
(2)将菌体进行冷冻干燥。
在步骤(1)中,所述MRS培养基为:蛋白胨10.0g;牛肉膏5.0g;酵母粉4.0g;葡萄糖20.0g;磷酸氢二钾2.0g;柠檬酸三铵2.0g;醋酸钠5.0g;硫酸镁0.2g;硫酸锰0.05g;吐温801.0mL;水1L。
所述微生物制剂中菌体的有效活菌数为6×1010CFU/g。
本发明所述的类肠膜魏斯氏菌或类肠膜魏斯氏菌菌剂在饲料和水中的应用。
实施例2
实施例2与实施例1的区别在于:
在MRS琼脂培养基中,经过30℃,72h有氧培养后,菌落呈乳白色、圆形,表面光滑、边缘整齐,中间凸起,无色素沉着,容易挑起,无丝,有乳酸的香味,无刺鼻酸味;革兰氏染色为阳性,菌体为杆状,单个或者两两呈现,兼性厌氧,生长温度30℃。
本发明所述的类肠膜魏斯氏菌菌剂的制备方法,包括如下步骤:
(1)菌种制备:MRS液体培养基中接种,30℃培养24h,8000rpm离心获得菌体。
(2)将菌体进行冷冻干燥。
所述微生物制剂中菌体的有效活菌数为1010CFU/g。
实施例3
实施例3与实施例1的区别在于:
在MRS琼脂培养基中,经过35℃,72h有氧培养后,菌落呈乳白色、圆形,表面光滑、边缘整齐,中间凸起,无色素沉着,容易挑起,无丝,有乳酸的香味,无刺鼻酸味;革兰氏染色为阳性,菌体为杆状,单个或者两两呈现,兼性厌氧,生长温度35℃。
本发明所述的类肠膜魏斯氏菌菌剂的制备方法,包括如下步骤:
(1)菌种制备:MRS液体培养基中接种,35℃培养20h,6000rpm离心获得菌体。
(2)将菌体进行冷冻干燥。
所述微生物制剂中菌体的有效活菌数为1011CFU/g。
试验例1
本发明提供的类肠膜魏斯氏菌具有以下微生物特征:在MRS琼脂培养基中,经过35℃,72h有氧培养后,菌落呈乳白色、圆形,表面光滑、边缘整齐,中间凸起,无色素沉着,容易挑起,无丝,有乳酸的香味,无刺鼻酸味。革兰氏染色为阳性,菌体为杆状,单个或者两两呈现,兼性厌氧,最适生长温度30~38℃,类肠膜魏斯氏菌生理生化鉴定表如表1所示:
表1
注:“+”为阳性反应,“-”为阴性反应
抑菌性能测定:
将大肠杆菌K88、鼠伤寒沙门氏菌和金黄色葡萄球菌作为指示菌,用0.9%的盐水制得混悬菌液并调节浓度,制备约含有指示菌105CFU/mL的琼脂平板,待冷却后,用凝胶打孔法在平板上打直径为4mm的孔径加入待测菌液60uL,35℃培养17h测定抑菌圈直径,结果如表2。353对三种病原菌的体外拮抗效果如表2所示:
表2
本发明的类肠膜魏斯氏菌可耐低温,耐酸,4℃液体保存,pH 3.4,保存75d,活菌数为105~106CFU/mL。
表2低温条件下存活率比较
试验在云南省畜牧兽医科学院进行,选择迪庆藏猪四元杂交猪,平均体重为95kg左右的健康育肥猪20头,体重差异不显著。随机分成2组(1个试验组和1个对照组),每组2个重复,每个重复5头。试验期为2019年11月19日-12月11日。对照组日粮为基础日粮,试验组日粮,饮水中添加实施例1中制得的添加剂,自由采食和饮水,常规免疫和驱虫,试验期内每天观察并记录猪的发病情况,试验期共21天,试验结束计算平均日增重、日采食量、料重比和成活率。添加353对育肥猪生长性能的影响如表3所示:
表3
添加353对粪便常规成分指教的影响如表4所示:
表4
表3~4数据显示,饲粮中添加类肠膜魏斯氏菌后,能降低粪便中粗蛋白和铵氮的含量,具有潜在提高饲粮利用率和降低铵氮排放的效果。由表格中试验数据可以看出,本发明的添加剂能有效促进生长,防治育肥猪腹泻的效果。实施例腹泻率仅为0.9%,与对照组相比腹泻率降低了50%,便秘率为1.8%,与对照组相比较降低了57.5%,抗病效果非常明显。同时本发明产品对提高仔猪生长性能也具有一定的帮助,料重比降低了2.3%,平均日采食量增加了9.37%,平均日增重增加了11.78%。
试验例2
试验分析
OTU(Operational Taxonomic Units)是在系统发生学或群体遗传学研究中,为了便于进行分析,人为给某一个分类单元(品系,属,种、分组等)设置的统一标志。对实验组和对照组的猪粪便采用高通量测序技术分析,结果如下,基于OUT水平的图3的韦恩图分析结果显示,未添加本试验菌株353的实验猪肠道内OTU数为888,其中独有的OTU数为104,添加了干菌粉的试验组共有OTU数为979,独有OTU数为195,而图4显示对照组细菌种类较试验组少24种,由此可看本发明的干菌粉添加剂可以提高猪肠道内细菌物种数量。添加353后对育肥猪属水平的肠道菌群结构影响如表5所示:
表5
根据属水平上的分类结果分析,发现在育肥猪的饲粮中添加类肠膜魏斯氏菌后,能显著或极显著提高与肠道炎症负相关的理研氏菌科RC9肠群、克里斯滕森菌科R-7群、瘤胃菌科UCG-005、毛螺菌科_XPB1014群和未分类的毛螺菌科的相对丰度。其中毛螺菌科与宿主粘膜完整性、胆汁酸代谢和多糖分解相关。结合生长试验发现,证明添加353后具有调节肠道菌群结构,促进生长的效果。
Alpha多样性分析如表6所示:
表6
Alpha多样性是反映样品物种多样性的指标之一,Shannon农指数、Ace指数和Chao1指数越高,Simpson指数越低,则物种的多样性则越高。从表中可知试验组D的的Shannon指数、Ace指数和Chao1指数均高于对照组C,Simpson指数则低于对照组,则说明试验组的物种多样性较对照组高,但差异不显著说明添加353后,具有提高菌群Alpha多样性的趋势。
由表7可知,对粪便中代谢产物分析,寻找其差异的代谢产物后发现:2-羟基肉豆蔻酸、磷酸脱氧腺苷、Furanofukinin、乙基葡萄糖醛酸苷、10,20-二羟基二十碳饱和脂肪酸、N-(1-去氧-1-果糖基)甘氨酸、DTMP、2-羟基鲸乙酸、Scillipheosidin 3-[glucosyl-(1->2)-rhamnoside]、羟基十六碳烯酸和3-氧-十八烷酸均显著或极显著上升,3-羟基十三酰肉碱和3-羟基十四烯二酸则显著下降。2-羟基鲸乙酸、3-氧-十八烷酸、3-羟基十四烯二酸、羟基十六碳烯酸和10,20-二羟基二十碳饱和脂肪酸与脂类代谢相关,3-羟基十三酰肉碱与代谢疾病呈反比,故而添加353后,促进代谢;Furanofukinin是维生素B2前体物质,是由微生物合成维生素B2的中间体之一,添加353后,具有促进肠道内合成VB2的作用,磷酸脱氧腺苷和DTMP与遗传物质相关,乙基葡萄糖醛酸苷作为临床上一种与乙醇代谢相关的生物标记物,显示了机体内有显著的乙醇代谢的过程。说明添加353后,肠道内有乙醇代谢的发生。结合生长试验发现,353具有调节肠道菌群结构,促进生长的效果。差异代谢产物分析如表7所示:
表7
注:FC(E/C)为该代谢物在两组间的差异表达倍数(fold change)。
C:该代谢物在对照组X中的表达量,
E:该代谢物在实验组Y中的表达量,
C为对照;
以上显示和描述了本发明的基本原理、主要特征和本发明的优点。本行业的技术人员应该了解,本发明不受上述实施例的限制,上述实施例和说明书中描述的只是说明本发明的原理,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,本发明要求保护范围由所附的权利要求书、说明书及其等效物界定。
序列表
<110> 云南省畜牧兽医科学院
<120> 一株类肠膜魏斯氏菌及其应用
<130> 2020
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1485
<212> DNA
<213> 人工序列(类肠膜魏斯氏菌353的16S rDNA基因序列)
<400> 1
ggggtggggg tgtataatgc agtcgaacgc tttgtcttta attgatctga cgagcttgct 60
ctgatttgat tttatctgac aaagagtggc gaacgggtga gtaacacgtg ggtaacctac 120
ctcttagcag gggataacat ttggaaacaa gtgctaatac cgtataatac caacaaccgc 180
atggttgttg gttgaaagat ggttctgcta tcactaagag atggacccgc ggtgcattag 240
ctagttggta aggtaacggc ttaccaaggc aatgatgcat agccgagttg agagactgat 300
cggccacaat gggactgaga cacggcccat actcctacgg gaggcagcag tagggaatct 360
tccacaatgg gcgcaagcct gatggagcaa cgccgcgtgt gggaagaagg gtttcggctc 420
gtaaaacact gttataagag aagaaccgca ctgagagtaa ctgttcagtg tgtgacggta 480
tcttaccaga aaggaacggc taaatacgtg ccagcagccg cggtaatacg tatgttccaa 540
gcgttatccg gatttattgg gcgtaaagcg agcgcagacg gttatttaag tctgaagtga 600
aagccctcag ctcaactgag gaatggcttt gggaaactgg gatgacttga gtgcagtaga 660
ggaaagtgga actccatgtg tagcggtgaa atgcgtagat atatggaaga acaccagtgg 720
cgaaggcggc ttttctggac tgtaactgac gttgaggctc gaaagtggtg ggtagcaaac 780
aggattagaa taccctggca gtccacaccg taaaccgatg agtgcctaga tgatcgaagg 840
ctttccggcc catgagtggt cgcaactaac gcattaagca ctccgcctgg ggagtacgac 900
cgcaaggttg aaactcaaag gaattgacgg ggacccgcac aagcggtgga gcatgtggtt 960
taattcgaag caacgcgaag aaccttacca ggtcttgaca tcccttgcta atcctagaaa 1020
taggacgttc ccttcgggga caaggtgaca ggtggtgcat ggttgtcgtc agctcgtgtc 1080
gtgagatgtt gggttaagtc ccgcaacgag cgcaaccctt attattagtt gccagcattc 1140
agttgggcac tctagtgaga ctgccggtga caaaccggag gaaggtgggg atgacgtcaa 1200
atcatcatgc cccttatgac ctgggctaca cacgtgctac aatggcatat acaacgagtc 1260
gccaacccgc gagggtgcgc taatctctta aagtatgtct cagttcggat tgtaggctgc 1320
aactcgccta catgaagtcg gaatcgctag taatcgcgga tcagcacgcc gcggtgaata 1380
cgttcccggg tcttgtacac accgcccgtc acaccatgag agtttgtaac acccaaagcc 1440
ggtggggtaa ccttttagga gccagccgtc taaggtaccc aagat 1485
Claims (6)
1.一株类肠膜魏斯氏菌(Weissella paramesenteroides)LL2018353,其特征在于:保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC),保藏编号:CGMCCNO.18710。
2.根据权利要求1所述的类肠膜魏斯氏菌制备的类肠膜魏斯氏菌菌剂。
3.根据权利要求2所述的类肠膜魏斯氏菌菌剂的制备方法,其特征在于包括如下步骤:
(1) 菌种制备:MRS液体培养基中接种,35~38℃培养16~24h,5000~8000rpm离心获得菌体;
(2)将菌体进行冷冻干燥。
4.根据权利要求3所述的类肠膜魏斯氏菌菌剂的制备方法,其特征在于:所述MRS液体培养基为:蛋白胨10.0g;牛肉膏5.0g;酵母粉4.0g;葡萄糖20.0g;磷酸氢二钾2.0g;柠檬酸三铵2.0g;醋酸钠5.0g;硫酸镁0.2g;硫酸锰0.05g;吐温80 1.0mL; 水1L。
5.根据权利要求3所述的类肠膜魏斯氏菌菌剂的制备方法,其特征在于:所述菌剂中菌体的有效活菌数为1010~1011CFU/g。
6.根据权利要求2所述的类肠膜魏斯氏菌菌剂在添加于猪用饲料以及猪饮用水的应用。
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