CN115572689A - 一株戊糖片球菌m6及其应用 - Google Patents
一株戊糖片球菌m6及其应用 Download PDFInfo
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- CN115572689A CN115572689A CN202210886892.4A CN202210886892A CN115572689A CN 115572689 A CN115572689 A CN 115572689A CN 202210886892 A CN202210886892 A CN 202210886892A CN 115572689 A CN115572689 A CN 115572689A
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Abstract
本发明公开了一株戊糖片球菌M6及其在预防和治疗动物腹泻、调节宿主免疫功能等方面的应用。保藏编号为CGMCC No.24767。本发明戊糖片球菌M6具有良好的胃肠道定植能力和益生性能,能够显著下调肠道炎症因子,在畜牧生产中有良好的应用前景。
Description
技术领域
本发明涉及微生物技术领域,更具体的说是涉及一株戊糖片球菌M6及其应用。
背景技术
随着养殖业集约化程度和动物生产性能的提高,饲养动物的应激水平不断增加,对各种病原微生物的抵抗能力下降,时有临床或亚临床病理状态出现。过去为了预防疾病和提高经济效益,动物饲养中会向饲料中添加抗生素,但抗生素的使用会导致耐药菌株的出现,不利于养殖业的健康发展。
因此,提供增强饲养动物机体免疫力且无副作用的产品或饲养方法是本领域技术人员亟需解决的问题。
发明内容
有鉴于此,本发明提供了一株戊糖片球菌M6及其在预防和治疗动物腹泻、调节宿主免疫功能等方面的应用。
为了实现上述目的,本发明采用如下技术方案:
一株戊糖片球菌M6,于2022年04月25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.24767,保藏地址北京市朝阳区北辰西路1号院3号。该菌具有良好的胃肠道定植能力和益生性能,能够显著下调肠道炎症因子。
本发明的另一目的是提供上述戊糖片球菌M6的应用,所述应用为以下任意一方面:
A.制备预防和治疗腹泻产品中的应用;
B.制备调节宿主免疫功能产品中的应用;
C.制备以替代抗生素药物的生物制剂产品中的应用;
D.制备提高宿主的生长性能产品中的应用;
E.制备胃肠道应激反应产品中的应用;
F.制备牲畜及宠物日常保健产品中的应用。
优选的,所述产品为药品、功能性食品、保健品或饲料。
更优选的,所述戊糖片球菌M6的应用群体为马属动物。
本发明的再一目的是,提供一种药品,含有上述戊糖片球菌M6。
提供一种功能性食品,含有上述戊糖片球菌M6。
提供一种保健品,含有上述戊糖片球菌M6。
提供一种饲料,含有上述戊糖片球菌M6。
经由上述的技术方案可知,与现有技术相比,本发明公开提供了一株戊糖片球菌M6,该菌株具有良好的胃肠道定植能力和益生性能,能够显著下调肠道炎症因子,在畜牧生产中有良好的应用前景。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据提供的附图获得其他的附图。
图1为本发明戊糖片球菌M6的菌落示意图;
图2为本发明戊糖片球菌M6的革兰氏染色示意图;
图3为本发明戊糖片球菌M6系统进化树示意图;
图4为本发明戊糖片球菌M6的生长曲线示意图;
图5为本发明戊糖片球菌M6耐热性能鉴定结果示意图;
图6为本发明戊糖片球菌M6耐酸性能鉴定结果示意图;
图7为本发明戊糖片球菌M6耐胆盐性能鉴定结果示意图;
图8为本发明戊糖片球菌M6胃液耐受性能鉴定结果示意图;
图9为本发明戊糖片球菌M6肠液耐受性能鉴定结果示意图;
图10为本发明戊糖片球菌M6溶血性能鉴定结果示意图;
图11为本发明动物实验各组小鼠体重变化;
图12为本发明动物试验各组小鼠肠道中IL-1β的含量;*表示与模型组存在显著差异,*表示p<0.05,**表示p<0.01,***表示p<0.001;
图13为本发明动物试验各组小鼠结肠中IL-6的含量;*表示与模型组存在显著差异,*表示p<0.05,**表示p<0.01,***表示p<0.001。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
本发明实施例所涉及的试剂均为市售渠道采购,未提及的方法为常规实验方法,在此不再一一赘述。
实施例1
戊糖片球菌M6的鉴定
该菌株来源于内蒙古萨拉齐马场的健康蒙古马肠道内容物,依据菌落、菌体形态、生理生化特性初步鉴定为戊糖片球菌,后经16S rDNA确定具体为戊糖片球菌。
1、M6菌株于MRS固体培养基中纯化培养24h,该菌落呈圆形、白色,菌落表面湿润有光泽,呈现单个、成双或短链排列的卵圆形球菌,在丰富培养基上菌落大而光滑(参见附图1)。革兰氏染色后,在显微镜在观察到该菌为革兰氏阳性球菌(参见附图2)。
2、生理生化鉴定
将M6菌株在MRS培养基上纯化培养,并挑取清晰的单一菌落,进行生化试验。测定结果根据乳酸菌生化鉴定管使用说明书(SHBG13)进行比对,判断各个指标,所有的指标结果在《伯杰氏细菌学手册》和《常见细菌系统鉴定手册》中对照发现。M6菌株可以发酵纤维二糖、麦芽糖、甘露醇、水杨苷、蔗糖、棉子糖、乳糖,不发酵七叶苷、山梨醇、菊糖、葡萄糖;M6菌株可以使过氧化氢酶产气,可以还原硝酸盐溶液,可以水解淀粉酶,有运动性,可以发生吲哚反应,出现吲哚环。可初步判定M6菌株为戊糖片球菌菌株。
3、16s rDNA鉴定
将M6菌株的PCR产物进行胶回收,送上海生工生物工程股份有限公司测序,测得的DNA片段长度为1436bp,菌株的测序结果在NCBI核酸数据库中,使用BLAST方法比对,最后选用MEGA6软件编辑,绘制进化树(参见附图3)。根据同源性分析结果显示M6菌株与戊糖片球菌的同源性为98.7%。综合菌株M6的形态学、生理生化特性以及16S rDNA分子鉴定结果,最终确定M6菌株为戊糖片球菌的一种,命名为戊糖片球菌M6(Pediococcuspentosaceus M6)。
实施例2
1、M6生长曲线
生长曲线是衡量优质乳酸菌的重要指标,作为微生态制剂的候选菌株,乳酸菌的快速生长可以使其成为定植于肠道的优势菌群,并抑制有害细菌的生长。
以OD值为纵坐标,培养时间为横坐标,绘制了戊糖片球菌M6的生长曲线(参见附图4),可知在2h内OD值变化较小,为M6菌株的生长延滞期;2-16h内OD值几乎呈现直线增长,为M6菌株的对数生长期;培养24h以后M6菌株进入稳定期,30h后未见到M6菌株的OD值有较大的下降,说明戊糖片球菌M6稳定性强。
2、耐热性能鉴定
将M6接种于MRS培养基后,振荡混匀,均分装为六组,每组3mL,分别置于37、42、50、55、60、65℃水浴锅中加热10min,再用PBS倍比稀释,涂板,厌氧培养24h,培养结束后平板计数(参见附图5)。
结果表明戊糖片球菌M6在37℃的存活率为百分之百,在45℃,55℃中处理30min仍有60%以上存活率,65℃,75℃条件下的存活率在20%以上,说明该温度也有活菌存在,说明戊糖片球菌M6能在65℃环境下生长,瞬间高温处理也能有细菌存活。
3、耐酸性能鉴定
挑取M6单菌落接种到MRS肉汤中,37℃培养24h,4000rpm/min,离心10min。用PBS洗涤沉淀,调OD为(0.5±0.05),取上述悬浊液以10%加菌量(150μL/1.5mLMRS不同浓度PH溶液,PH=2、3、4)37℃培养24h,在0、2、4、6、8、12、16、24、38h测OD600。每个浓度都是在培养24h后取150μL涂布,培养38h,以其OD值大小代表菌落数,胆盐浓度为0的MRS肉汤常规培养作对照,记录数据分析。
乳酸菌如果在肠道中成功定植,必须要耐受胃液中的酸性环境。以OD值为纵坐标,培养时间为横坐标,绘制M6菌株在不同pH下的生长曲线(参见附图6)。可知,4-16h菌株生长迅速,16-24h生长缓慢,24h以后菌株基本上不再生长,培养基的pH对菌株的生长有影响,酸性环境会抑制戊糖片球菌M6的生长。在pH值5.8的培养基中戊糖片球菌M6正常生长,在pH值为2.0的培养基里,戊糖片球菌M6停止生长或生长滞停,pH3.0的培养基中生长比较缓慢,在pH值为4.0的培养基中戊糖片球菌M6的生长基本不受影响,正常生长。
4、耐胆盐性能鉴定
肠道是乳酸菌发挥益生作用的重要场所,乳酸菌能否对机体产生益生作用,主要取决于是否能在肠道内黏附、存活并繁殖。菌株对胆盐耐受力的高低可以反映出戊糖片球菌M6在肠道中存活能力的高低。
挑取MRS平板上单个典型菌落,接种于肉汤培养基,37℃培养24h,4000rpm/min,离心10min。沉淀用PBS洗涤后,调OD为(0.5±0.05),取上述悬浊液以10%加菌量,不同胆盐浓度0.2%、0.3%、0.4%,37℃培养24h,37℃培养24h,在0、2、4、6、8、12、16、24、38h测OD600。每个浓度都是在培养24h后取150μL涂布,培养38h,以其OD值大小代表菌落数,并以空白PH和空白胆盐的MRS肉汤常规培养作对照,记录数据分析。以OD值为纵坐标,培养时间为横坐标,绘制M6菌株在不同浓度胆盐下的生长曲线(参见附图7)。
可知,M6菌株在4-16h菌株生长迅速,16-24h生长缓慢,24h以后菌株基本上不再生长,可见胆盐浓度对戊糖片球菌M6的生长有影响。与空白对照相比,胆盐浓度为0.1%时,戊糖片球菌M6的生长没有受到胆盐的抑制;当胆盐浓度为0.2%和0.3%时,戊糖片球菌M6的生长受到胆盐的抑制,生长缓慢。
5、胃液耐受性能鉴定
将M6菌液吸取1ml置于9ml无菌人工胃液中,37℃、150r/min条件下培养2h。二次接种到菌落平板计数液中,37℃、100r/min培养10h。利用菌落平板计数法分别法分别于0.5h、1h、2h、3h进行活菌计数,计算存活率(参见附图8)。
存活率=测定时间菌落数/0时菌落数*100%
马属动物的的内脏器官pH值一般在1-6之间,有益乳酸菌进入机体后,不仅需要对胃酸有耐受能力,还需要不被胃内的各类蛋白酶所影响,只有少数耐酸又不被胃蛋白酶杀死的乳酸菌才能存活下来进入小肠,这可以防止食物来源的致病菌侵染机体。一般情况下,食物会在胃内停留2-4h,本试验用pH为3的人工胃液模拟胃液消化乳酸菌3h,分别在0.5h、1h、2h、3h后涂板计数戊糖片球菌M6的存活率作为菌株耐受胃液的依据。表8和图13为乳酸菌在模拟胃液中消化3h内的存活率,M6菌株在0h的存活率为百分之百,0.5小时的存活率为9.5%,1小时的存活率为9.2%,2小时的存活率为4%,3小时的存活率为2.6%。
6、肠液耐受性能鉴定
将M6菌液接入9mL无菌的人工模拟肠液中,37℃、150r/min条件下培养2h。二次接种到菌落平板计数液中,37℃、100r/min培养10h。利用菌落平板计数法,分别法分别于0h、1h、2h、3h进行活菌计数,计算存活率(参见附图9)。
存活率=测定时间菌落数/0时菌落数*100%
乳酸菌进入小肠后,小肠中的胆汁酸盐对菌株有一定的抑制作用,正常情况下,肠道中胆汁酸盐和肠道中的胰蛋白酶都会抑制微生物的生长。而肠道是乳酸菌发挥作用的重要场所,所以乳酸菌在肠液中存活率至关重要。M6菌株在模拟肠液液中消化0h、1h、2hh的存活率为100%,3h的存活率为95%。
实施例3
M6益生作用评定
1、溶血性能鉴定
将M6菌株活化培养24h后的分离菌种液200μL均匀涂布于哥伦比亚血琼脂板,37℃恒温培养箱厌氧培养24h,观察是否有溶血现象(参见附图10)。
培养结果可见,戊糖片球菌M6不产生溶血环,说明戊糖片球菌M6是安全的菌株,可以作为潜在益生菌的菌株应用于生产。
2、抑菌性能鉴定
菌株的抑菌性能采用牛津杯琼脂扩散法进行测定。取活化后的M6菌株厌氧培养24h,以10000r/min离心5min,分离上清液,置于4℃冰箱备用。取伤寒沙门氏菌(CMCC50071)、大肠杆菌(ATCC 25922)和金黄色葡萄球菌(CMCC 26003)新鲜菌液各200μL,均匀涂于MH固体培养基中。待菌液完全吸收后,在每个平板上放置3个牛津杯,在每个孔中加入200μL的M6菌株上清液,在37℃培养箱中培养24h,观察是否形成抗菌圈并测量直径大(结果见表1)。
表1
菌株 | 抑菌圈直径(mm) |
大肠杆菌 | 9.4967±0.06877 |
伤寒沙门氏菌 | 16.7100±0.6702 |
金黄色葡萄球菌 | 14.2869±0.6880 |
注:菌株的抑菌能力大小与抑菌圈的大小相同,抑菌圈直径在0~10mm表示抑菌作用良好,10~20mm表示抑菌作用优良。
实施例4
对小鼠肠道炎症的调控作用
实验动物:7周龄雄性C57BL/6J小鼠,购自斯贝福(北京)生物技术有限公司。
试验分组:空白组、模型组、低剂量组和高剂量组,随机分组,每组5只。
实验菌株:伤寒沙门氏菌CMCC(B)50071购自广东环凯微生物科技有限公司。
实验方法:
将C57BL/6J小鼠适应性喂养一周,开始为期14天的实验,空白组正常饮食饮水,第1-7天每天灌胃0.2ml生理盐水,第8-14天每天灌胃0.4ml生理盐水;模型组第1-14天每天灌胃0.2ml生理盐水,第8-14天每天灌胃0.2ml浓度为1×1011CFU/mL的伤寒沙门氏菌悬液(根据便血情况、粪便形状等表观指标初步确定造模成功);低剂量组第1-14天每天灌胃0.2ml浓度为1×108CFU/mL的M6菌悬液,第8-14天每天灌胃0.2ml浓度为1×1011CFU/mL的伤寒沙门氏菌悬液;高剂量组第1-14天每天灌胃0.2ml浓度为1×1010CFU/mL的M6菌悬液,第8-14天每天灌胃0.2ml浓度为1×1011CFU/mL的伤寒沙门氏菌悬液。
试验第15天,小鼠颈椎脱位处死,正常解剖后,取出消化系统中十二指肠、空肠、回肠、盲肠和结肠等器官立即放置于液氮,-80℃保存。同时,切除消化器官组织1cm,立即置于4%多聚甲醛溶液中进行固定。
观察指标:造模前后称量小鼠体重(参见附图11),造模期间观察小鼠粪便性状及其粪便隐血情况,并通过ELISA试剂盒检测组织上清中细胞因子IL-1β(参见附图12)、IL-6(参见附图13)(南京建成生物工程研究所)。
附图11可见,造模第七天,模型组小鼠体重降低14.98%,而戊糖片球菌M6组干预显著抑制了体重的降低,其中高剂量组小鼠体重减轻0.74%。可知,抑制模型组小鼠体重降低是乳酸菌缓解肠道炎症的重要体现。
附图12和13可知,模型组显著增加了促炎细胞因子IL-1β和IL-6在肠道各个部位的浓度,这表明伤寒沙门氏菌引起了小鼠严重的肠道炎症反应,尤其在小肠部聚集,免疫反应更强烈。与模型组小鼠相比,戊糖片球菌M6两个组能显著下调IL-1β和IL-6两种促炎因子,尤其是高剂量组的效果更加明显。以上结果表明,戊糖片球菌M6可调节肠炎小鼠的免疫反应失调,从而降低肠道炎症损伤。
本说明书中各个实施例采用递进的方式描述,每个实施例重点说明的都是与其他实施例的不同之处,各个实施例之间相同相似部分互相参见即可。
对所公开的实施例的上述说明,使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的,本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下,在其它实施例中实现。因此,本发明将不会被限制于本文所示的这些实施例,而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。
Claims (8)
1.一株戊糖片球菌M6,其特征在于,保藏编号为CGMCC No.24767。
2.如权利要求1所述一株戊糖片球菌M6的应用,其特征在于,所述应用为以下任意一方面:
A.制备预防和治疗腹泻产品中的应用;
B.制备调节宿主免疫功能产品中的应用;
C.制备以替代抗生素药物的生物制剂产品中的应用;
D.制备提高宿主的生长性能产品中的应用;
E.制备胃肠道应激反应产品中的应用;
F.制备牲畜及宠物日常保健产品中的应用。
3.如权利要求2所述的应用,其特征在于,所述产品为药品、功能性食品、保健品或饲料。
4.如权利要求2或3所述的应用,其特征在于,所述戊糖片球菌M6的应用群体为马属动物。
5.一种药品,其特征在于,含有权利要求1所述戊糖片球菌M6。
6.一种功能性食品,其特征在于,含有权利要求1所述戊糖片球菌M6。
7.一种保健品,其特征在于,含有权利要求1所述戊糖片球菌M6。
8.一种饲料,其特征在于,含有权利要求1所述戊糖片球菌M6。
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