CN109022313B - 一株植物乳杆菌 - Google Patents
一株植物乳杆菌 Download PDFInfo
- Publication number
- CN109022313B CN109022313B CN201810870915.6A CN201810870915A CN109022313B CN 109022313 B CN109022313 B CN 109022313B CN 201810870915 A CN201810870915 A CN 201810870915A CN 109022313 B CN109022313 B CN 109022313B
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- fbl
- lactobacillus plantarum
- strain
- lactobacillus
- bacteria
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- C—CHEMISTRY; METALLURGY
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
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- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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Abstract
本发明公开一株能防治牛羊腹泻病的菌株。本发明的植物乳杆菌,于2018年4月2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,保藏编号为CGMCC NO:15546。本发明的植物乳杆菌可在制备食草动物的饲料添加剂中的应用,还可在制备食草动物的饲料中的应用。本发明的植物乳杆菌菌株,具有很强的黏附及抑菌能力,有很好的耐酸、耐胃肠道以及耐胆盐能力,能有效的防治牛羊腹泻病。
Description
技术领域
本发明涉及一株能防治牛羊腹泻病的菌株。
背景技术
益生菌被定义为活的微生物,当摄入足够的数量时,能给予宿主健康作用。这些能对健康有益处的特定属的益生菌菌属有:乳杆菌属,双歧杆菌,酵母属,肠球菌属,小球菌属,明串珠菌属,芽孢杆菌属等。目前研究和应用最为广泛的益生菌是乳杆菌(Lactobacillus)以及双歧杆菌(Bifidobacterium)。其中存在于机体肠道中并在食品中广泛添加的乳杆菌种主要有:卷曲乳杆菌(Lactobacillus crispatus)、格氏乳杆菌(Lactobacillus gassed)和植物乳杆菌(Lactobacillus plantarum)。根据国家《食品安全法》及其实施条例的有关规定,国家***卫办监督发〔2010〕65号文件规定了《可用于食品的菌种名单》,其中乳杆菌属有14种,植物乳杆菌在列。乳酸菌在发酵过程中,不仅可使机体中的乳糖、蛋白质等物质转化成乳酸、半乳糖、氨基酸及小分子肽链等易被人体消化和吸收的小分子物质,还可代谢产生机体需要的多种B族维生素等物质为机体提供营养。因此,在饲料中添加乳酸菌可很好的满足机体对各类营养物质的需求,大大提高机体对营养物质例如钙类物质的吸收利用效率,饲料中摄入的乳酸菌可作用于机体肠道内壁形成有益菌群及生物屏障,并通过产生大量的短链脂肪酸(乳酸、乙酸等)、过氧化氢、细菌素或多肽等物质,从而发挥生物学屏障作用,阻止病原微生物的侵袭,形成良好的机体肠道微生态平衡***。另外,乳酸菌菌体抗原及代谢物通过刺激肠粘膜***,激活免疫活性细胞,从而产生特异性抗体与致敏淋巴细胞,从而控制炎症性肠病、缓解过敏反应、增强天然免疫。食用添加益生菌的饲料还对可解决肉牛、牦牛以及绵羊的腹胀、腹泻等问题。
随着抗生素在畜产品中的残留对人类健康的危害、过量***对环境的污染、长期使用导致耐药菌株的产生等问题日益被人们所重视,目前很多国家已经禁止或限制了抗生素在饲料中的使用,无抗、减抗畜禽产品将成为发展主流,而当前益生菌制剂作为安全、绿色的饲料添加剂引起了极大的关注。开发具有强抑菌性、黏附性,强耐酸、耐胃肠道及耐胆盐能力,并能增强动物免疫力的益生菌菌株极具应用价值,而益生性能与其发酵的乳酸菌种类密切相关,因此获得优质的有特异功能的乳酸菌菌种是作为益生菌饲料添加剂最核心的技术。
为解决上述问题,寻找一种安全绿色的饲料添加剂是业内工作人员的一个工作方向。
发明内容
本发明提供一株植物乳杆菌。
本发明的一株植物乳杆菌,于2018年4月2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,保藏编号为CGMCC NO:15546。
本发明的的植物乳杆菌可在食草动物饲养中的应用,也可在制备食草动物的饲料添加剂中的应用,还可在制备食草动物的饲料中的应用。
本发明的植物乳杆菌菌株,具有很强的黏附及抑菌能力,有很好的耐酸、耐胃肠道以及耐胆盐能力,能有效的防治牛羊腹泻病。
附图说明
图1FBL-3a的菌落形态图。
图2FBL-3a的革兰氏染色图。
图3FBL-3a的PCR扩增片段图谱。
图4FBL-3a的生长以及发酵pH曲线图。
图5温度对菌FBL-3a生长的影响。
图6菌株FBL-3a黏附性。
图7植物乳杆菌标准株CICC6009黏附性。
具体实施方式
以下是结合附图详细阐述本发明的方案。
1.菌株的分离
本发明植物乳杆菌FBL-3a(Lactobacillusplantarum FBL-3a)是从新疆肉牛粪便分离获得的。本发明的菌株分离纯化方法是:从新疆牦牛粪便稀释液中吸取1mL,分别稀释10-4,10-5,10-6次方,各取0.1mL涂布于含钙的MRS平板上,待稀释液被平板吸收后,37℃厌氧培养24h,挑选出具有溶钙圈疑似乳酸菌的单菌落12个,分别在MRS固体平板上划线纯化培养3次,各挑一单菌落于MRS液体培养基中37℃静置培养24h,取发酵菌液进行抗逆性以及体外益生特性试验,最终挑选出1株黏附性强及抑菌性好,有很好的耐酸、耐胃肠道及耐胆盐能力的菌株保存并命名为FBL-3a。于2018年4月2日提交并保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCC NO:15546。
本发明前述使用的培养基的配方如下:
(1)MRS培养基(1L):蛋白胨10.0g;牛肉浸粉10.0g;酵母浸粉5.0g;葡萄糖20.0g;吐温801.0mL;K2HPO4·3H2O 2.0g;无水乙酸钠5.0g;柠檬酸三铵2.0g;MgSO4·7H2O0.29g;MnSO4·H2O 0.058g。(将以上成分加入蒸馏水中,定容到1000mL,加热溶解,调节pH为6.3,分装后121℃高压灭菌20min。(注:固体培养基在此基础上加1.5%琼脂粉)
(2)LB培养基(1L):胰蛋白胨10g;氯化钠10g;酵母浸粉5g,琼脂20g,PH调节为7.5。
2.菌种的鉴定
2.1菌种的形态观察
对菌FBL-3a进行菌落形态观察、参见图1,对菌株进行革兰氏染色,其电子显微镜观察菌体形态参见图2。菌FBL-3a的主要形态特征如下:菌落圆,光滑,在含钙的MRS培养基上呈乳黄色,不透明,菌落周围具有透明溶钙圈。革兰氏染色显示阳性,菌体杆状,宽:0.5-0.6μm,长:1.2-3.0μm。
生理生化特征的鉴定:
菌FBL-3a生理生化特征见表1,具体为:明胶实验和吲哚实验显示阴性,奈试产氨实验为阳性,VP、MR、淀粉水解、过氧化氢实验显示阴性,说明该菌能分解利用大分子含氮物质,不能将葡萄糖分解产生丙酮酸,不能产生淀粉酶将淀粉水解为糖类物质,不能利用明胶。同时菌FBL-3a能发酵利用α-乳糖、D-果糖、麦芽糖、葡萄糖、蔗糖产酸,不产气。
表1菌FBL-3a的生理生化特征
注:生理学特征:(“+”表示阳性;“-”表示阴性);糖类发酵实验结果记录表:(“+,+”表示产酸,产气;“-,-”表示不产酸,不产气。)
2.2菌FBL-3a的分子生物学鉴定
①细菌基因组DNA的提取采用Takara公司的细菌基因组DNA提取试剂盒提取,提取后将DNA分装于EP管冻存于-20℃。②16S rDNA序列的PCR扩增,得到目的片段长约为1500bp(图3)。将含有目标片段的产物委托南京金斯瑞生物科技有限公司完成测序,得到的实际有效核苷酸序列为1425bp,在NCBI数据中对各16S rRNA序列进行Blast比对分析,得到序列与Lactobacillus plantarum的序列同源性大于99%。将FBL-3a基因序列提交NCBI数据库中,进行细菌登入号的申请,申请得到的登录号为MH517566。
菌FBL-3a的16SrDNA基因序列:植物乳杆菌FBL-3a的16SrDNA基因序列为SEQ IDNO.6,结合菌FBL-3a的形态观察和生理生化实验,以及***发育树中的同源性分析,确定菌FBL-3a为植物乳杆菌(LactobacilluspLantarum)菌种。
3.植物乳杆菌FBL-3a的微生物学特性研究
3.1植物乳杆菌FBL-3a母液的制备
取100μL 30%甘油保存的植物乳杆菌FBL-3a接种于10mL的MRS液体培养基中,37℃静置培养24h,制成菌种母液备用。
3.2植物乳杆菌FBL-3a生长以及发酵PH曲线测定,取3.1中植物乳杆菌FBL-3a母液(下同)各100μL分别接种于57管装有10mL MRS液体培养基的试管中,37℃静置发酵培养,以未接菌的液体培养基为对照,分别在发酵后0-96(h)的不同时间段内取3管测定菌体OD(600nm)值以及PH值,结果表明(图4),菌FBL-3a发酵到10h时进入对数生长期,发酵到18h时进入稳定期,此时菌体OD(600nm)值为3.09±0.02。菌发酵到稳定期时pH已降低到4.17,且稳定期pH稳定在3.66左右。
3.3植物乳杆菌FBL-3a最适发酵温度的测定
取植物乳杆菌FBL-3a母液各100μL分别接种于装有10mL MRS液体培养基的试管中,20、25、30、35、40、45、50(℃)静置发酵培养24h,测定发酵液OD600nm)值,结果表明(图5),菌FBL-3a最适生长温度为35℃,菌体OD(600nm)值为3.66±0.02;菌在40℃时,OD(600nm)值仍然可到达3.02±0.02,发酵到50℃时菌的OD(600nm)值降到0.80±0.01处于基本不生长状态,说明该菌适合生长温度20-40℃。
3.4植物乳杆菌FBL-3a最适发酵pH值的测定
取植物乳杆菌FBL-3a母液各100μL分别接种于装有10mL的不同pH值的MRS液体培养基的试管中37℃静置发酵培养24h,测定发酵液OD600nm值,结果见表2,植物乳杆菌FBL-3a最适发酵pH值为6.5,此pH值下培养24h菌体OD(600nm)值为3.81±0.02,当pH值小于1.0或大于9.0时,菌的OD(600nm)值均低于0.1,处于不生长状态,说明菌适合生长的pH值范围为3.0-8.0之间。pH值范围在4.0-7.5的MRS培养基经植物乳杆菌FBL-3a发酵后,菌液pH值下降到3.2-3.8之间。说明植物乳杆菌FBL-3a具有很强的产酸及耐酸能力。
表2菌FBL-3a最适发酵pH值的测定
4.植物乳杆菌FBL-3a的益生特性研究
4.1FBL-3a对胃肠道耐受性研究
收集过夜培养的菌体,用无菌的PBS重悬,将其浓度调整为1x109CFU/mL,并将其放入模拟胃液(pH3.0)中37℃培养1,2,3h,分别取样。3h后,再放入到模拟肠液中,1mL培养物加9mL模拟肠液(pH8.0),37℃培养2,4,8h后,分别取样并采用平板计数法测定活菌总数,结果见表3,经过模拟胃液处理3h以及模拟肠液处理8h过后,菌FBL-3a的活率仍在90%以上,可知该菌能很好地耐受胃肠道环境。
4.2菌FBL-3a菌株的耐胆盐试验
取FBL-3a菌液以2%接种量接种于含有不同胆盐(含量梯度为0、0.1%、0.2%、0.3%、0.6%)的MRS液体培养基(PH6.3),置于37℃下分别培养0、2、4h,每个处理3个重复。各取1mL培养物于9mL生理盐水中混匀,制备10-4、10-5、10-6个稀释度溶液,取0.1mL稀释液于MRS固体平板上涂布,于37℃二氧化碳培养箱中厌氧培养24h(每个稀释度做3个平行)记录计算平板上的菌落数。结果见表4,在胆盐浓度为0.3%处理4h后菌FBL-3a的生长量依然达到(3.13±0.28)×107(CFU/mL),可知菌FBL-3a有很好的耐胆碱能力。
4.3菌FBL-3a菌株的耐酸试验
取菌FBL-3a母液按100μL接种菌种于不同pH值(pH梯度为1.0、1.5、2.0、2.5、3.0)的10mL MRS液体培养基,置于37℃下分别培养0、2、4h,每一处理3个重复。各取1mL样品菌液于9mL PBS中混匀,制备10-4、10-5、10-6个稀释度溶液,取0.1mL稀释液于MRS固体平板上涂布,于37℃二氧化碳培养箱中倒置培养24h(每个稀释度做3个平行)记录计算平板上的菌落数。结果见表5,菌FBL-3a在pH值为1.0以及1.5时基本不生长,但在2.0及以上时,培养2h及4h后均有生长,在pH为2,培养4h后,菌落可达到(3.77±0.25)×107(CFU/mL),生长良好,说明该菌具有很强的耐酸能力,在2.0-3.0之间,培养4h的菌落少于2h,说明在这一pH范围内,随着处理时间的延长,菌的生长受到抑制。
4.4菌FBL-3a的表面性质测定
过夜培养植物乳杆菌FBL-3a(MRS液体培养基)、金黄色葡萄球菌ATCC6538(LB液体培养基)18h得到菌液,分别置于5000r/min、4℃下离心10min,收集菌体,分别用pH 7.2的无菌磷酸盐缓冲液(PBS)洗涤2次(即在菌体中加入PBS,吹打混合均匀后,置5000r/min、4℃下离心10min,收集菌体)。自凝集率(%):用无菌的PBS将植物乳杆菌FBL-3a制成在波长600nm处的吸光值为0.5±0.02(A0)的菌悬液,静置24h后测定吸光值A24,自凝集率(%)公式为(A0-A24)/A0×100%;他凝集率(%):用无菌的PBS将FBL-3a和金黄色葡萄球菌ATCC 6538的悬菌液调节成在波长600nm处的吸光值为0.5±0.02(A0)的混合悬浮菌液。静置24h后测定吸光值A24,他凝集率(%)公式为(A0-A24)/A0×100%;疏水性(%):用无菌的PBS将菌FBL-3a制成在波长600nm处的吸光值为0.5±0.02(A0)的菌悬液,将二甲苯加入到菌悬液中,在室温条件下预培养10min后将该两相体系涡旋振荡120s,37℃静置共培养1h,然后小心去除二甲苯相,于600nm处测定水相的吸光值A1。疏水性(%):公式为(A0-A1)/A0×100%。测定结果见表6,可知FBL-3a的自凝集率为77.43%,他凝集率为25.24%,疏水性为48.28%,说明植物乳杆菌FBL-3a可与致病菌(金黄色葡萄球菌)聚集,且疏水性能良好,可为黏附实验增加试验依据。
表6菌株FBL-3a表面性质表
凝集试验 | A<sub>0</sub>±s | A<sub>24</sub>±s | 凝集率(%) |
自凝试验 | 0.506±0.027 | 0.115±0.042 | 77.43% |
他凝试验 | 0.511±0.014 | 0.382±0.035 | 25.24% |
疏水性试验 | A<sub>0</sub>±s | A<sub>1</sub>±s | 疏水性(%) |
0.495±0.028 | 0.256±0.036 | 48.28% |
4.5菌FBL-3a的黏附能力测定
将活化好的植物乳杆菌FBL-3a以2%的接种量接种于MRS液体培养基中,37℃培养18h,离心收集菌体(8000r/min,4℃,10min)然后用无菌PBS(pH7.2)洗涤2次,将菌体重悬于Caco-2细胞培养液中,用培养液将菌体浓度调整至1×108CFU/mL。将已长成单层的Caco-2细胞用无菌PBS(p H 7.2)漂洗2次,每孔加入200μL含菌体的细胞培养液中。置37℃、5%CO2、相对湿度95%的二氧化碳培养箱中孵育2h,用无菌PBS(p H 7.2)漂洗细胞3次,以除去未黏附的细菌。向24孔板中加入无水甲醇固定10min,姬姆萨染色,油镜镜检。每孔随机计数30个视野,通过计数黏附在单个细胞上的乳杆菌数,来判断黏附能力的大小,以植物乳杆菌标准株CICC6009为对照。黏附结果见表7,植物乳杆菌FBL-3a与结肠癌细胞Caco-2黏附率达到2375±112.4CFU/30cell,而对照植物乳杆菌标准株CICC6009与结肠癌细胞Caco-2黏附率仅达到359±45.2CFU/30cell,则说明自分离植物乳杆菌FBL-3a有很强的黏附能力。
表7菌株FBL-3a黏附能力表
5菌FBL-3a在牛羊腹泻病的应用研究
将植物乳杆菌FBL-3a与实验室自分离的另三株益生菌株按一定比例混合,制成微生态制剂。对张掖、苏南不同程度腹泻病例共计40只羊80头牛进行饲喂试验。结果表明,饲喂第二天观察病畜,发现腹泻症状有明显好转,连续饲喂三天后,症状较轻的病畜基本痊愈,症状较严重的病畜在饲喂一周后也基本痊愈。而有严重症状的1头牛,饲喂前已经多日便血,已不进食,病情过于严重,未能治愈。但总体来说,治愈效果十分显著,经过一周饲喂,40只羊的腹泻症状均好转,治愈率达到97.5%,且后续观察并未出现病情反复等现象,而80头牛的腹泻症状也好转,但有两头病重牛未能治愈,但治愈率仍达到98.75%。
Claims (3)
1.一株植物乳杆菌(Lactobacillus plantarum)FBL-3a,于2018年4月2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,保藏编号为CGMCC NO:15546。
2.权利要求1所述的植物乳杆菌FBL-3a 在制备食草动物的饲料添加剂中的应用。
3.权利要求1所述的植物乳杆菌FBL-3a 在制备食草动物的饲料中的应用。
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