CN113234643B - 一株可缓解etec致腹泻的约氏乳杆菌及其应用 - Google Patents
一株可缓解etec致腹泻的约氏乳杆菌及其应用 Download PDFInfo
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Abstract
本发明公开了一株可缓解ETEC致腹泻的约氏乳杆菌及其应用,属于微生物技术领域。本发明筛选出了一株约氏乳杆菌CCFM1181,该菌株具有缓解腹泻的作用,具体体现在:显著降低腹泻小鼠的粪便含水量,显著缓解腹泻小鼠空肠的病理损伤,显著降低腹泻小鼠血清中的炎症因子水平,显著降低腹泻小鼠血清中肠毒素ST的含量,显著提高腹泻小鼠粪便中乙酸的含量,在制备预防和/或治疗腹泻的药物或辅助缓解腹泻症状的功能性食品、保健品中,具有巨大的应用前景。
Description
技术领域
本发明涉及一株可缓解ETEC致腹泻的约氏乳杆菌及其应用,属于微生物技术领域。
背景技术
ETEC,肠毒性大肠埃希杆菌,定居于小肠表面,不损坏也不侵入肠黏膜上皮细胞,通过产生肠毒素引起分泌性腹泻,是人类霍乱样病人大便中新发现的一组致腹泻性大肠埃希杆菌,是发达国家“旅游者腹泻”的主要病原之一,是“成人霍乱综合征”的常见原因,也是小儿腹泻的重要病原,其发病率仅次于轮状病毒。
同时,ETEC也是引起家畜特别是幼畜(初生仔猪、犊牛等)腹泻的重要病原菌。初生仔猪感染ETEC后,常因剧烈水样腹泻和迅速脱水而死亡,发病率和死亡率均很高,给养猪业带来巨大负担。
ETEC会产生热不稳定毒素LT和热稳定性毒素ST,继而改变紧密连接的完整性,引发炎症、肠道功能异常,以及破坏离子、溶质和水的胞旁通道,最终导致腹泻。传统的氟喹诺酮类、利福昔明等抗生素对ETEC引起腹泻的预防是有效的,但通常只用于高危人群,如免疫功能严重抑制的患者。同时,这些传统药物也会产生很大的副作用,例如,常常会引发肠胃不适、中枢系统出现异常反应,以及头痛、头晕等症状。而且,长期服用这些传统药物会对肝造成损害。另外,长期服用这些传统药物还存在成本高、ETEC出现抗菌素耐药性等问题。上述缺陷均使得这些传统药物的使用受到限制,而替代方法的有效性还在研究中。
益生菌是通过定殖在人体内,改变宿主肠道菌群组成,进而代谢产生如短链脂肪酸等有益代谢物,以对宿主产生有益影响的一类菌。与普通的药物相比,益生菌具有安全性高、无副作用、不会出现耐药性和成本低等优势。并且,研究表明,少数益生菌确实可对一些特殊的疾病起到预防和/或治疗作用,虽然已有文献报道约氏乳杆菌缓解ETEC致腹泻的作用,但效果的优良性还不确定。
因此,发现一株可缓解ETEC致腹泻的益生菌或者优于已有报道的同种菌对预防和/或治疗腹泻这一疾病十分关键。
发明内容
本发明提供了一株约氏乳杆菌(Lactobacillus johnsonii)CCFM1181,分类命名为Lactobacillus johnsonii,已于2021年04月16日保藏于广东省微生物菌种保藏中心,保藏编号为GDMCC No:61607,保藏地址为广州市先烈中路100号大院59号楼5楼。
所述约氏乳杆菌(Lactobacillus johnsonii)CCFM1181在MRS培养基上的菌落呈现乳白色、表面光滑、圆形凸起。
本发明还提供含有所述约氏乳杆菌CCFM1181的发酵剂。
在一种实施方式中,所述发酵剂中约氏乳杆菌CCFM1181的菌体数量≥1×1010CFU/mL或1×1010CFU/g。
在一种实施方式中,所述发酵剂的制备方法为:将约氏乳杆菌CCFM1181接种到培养基中,于37℃下培养18h,得到培养液;将培养液离心,得到菌体;将菌体用生理盐水或细胞保护剂重悬,得到液态发酵剂。
在一种实施方式中,所述培养基为MRS培养基。
在一种实施方式中,所述菌体在重悬后还经过了冻干处理,获得固态发酵剂。
本发明还提供所述约氏乳杆菌CCFM1181在制备预防和/或治疗腹泻的产品中的应用。
在一种实施方式中,所述治疗腹泻包括但不限于如下至少一种作用:
(1)提高腹泻个体的体重;
(2)降低腹泻个体的粪便含水量;
(3)缓解腹泻个体空肠的病理损伤;
(4)降低腹泻个体血清中的免疫因子水平;
(5)降低腹泻个体血清中肠毒素ST的含量;
(6)提高腹泻个体血清中水通道蛋白AQP3的含量;
(7)提高腹泻个体粪便中短链脂肪酸的含量。
在一种实施方式中,所述腹泻个体是具有腹泻症状的哺乳动物,所述哺乳动物包括但不限于人。
在一种实施方式中,所述腹泻是由肠毒性大肠埃希杆菌诱发的。
在一种实施方式中,所述产品中约氏乳杆菌CCFM1181的活菌数为不低于1×1010CFU/mL或1×1010CFU/g。
在一种实施方式中,所述产品包括功能性食品或药品。
在一种实施方式中,所述药品含有上述约氏乳杆菌CCFM1181、药物载体和/或药用辅料。
在一种实施方式中,所述食品为含有上述约氏乳杆菌CCFM1181的保健食品。
在一种实施方式中,所述食品是使用含有上述约氏乳杆菌CCFM1181的发酵剂生产得到的乳制品、豆制品、肉制品或果蔬制品。
本发明还提供了一种用于预防和/或治疗腹泻的产品,所述产品含有上述约氏乳杆菌CCFM1181。
在一种实施方式中,所述产品中,所述约氏乳杆菌CCFM1181的活菌数不低于1×1010CFU/mL或1×1010CFU/g。
在一种实施方式中,所述产品包括功能性食品、保健食品或药品。
在一种实施方式中,所述药品含有上述约氏乳杆菌(Lactobacillus johnsonii)CCFM1181、药物载体和/或药用辅料。
在一种实施方式中,所述食品包括含有所述约氏乳杆菌(Lactobacillusjohnsonii)CCFM1181的乳制品、豆制品、肉制品或果蔬制品;或使用所述约氏乳杆菌(Lactobacillus johnsonii)CCFM1181生产得到的乳制品、豆制品、肉制品或果蔬制品。
有益效果:本发明筛选出了一株约氏乳杆菌(Lactobacillus johnsonii)CCFM1181,此约氏乳杆菌CCFM1181具有缓解腹泻的作用,具体体现在:
(1)显著提高腹泻小鼠的体重;
(2)显著降低腹泻小鼠的粪便含水量;
(3)显著缓解腹泻小鼠空肠的病理损伤;
(4)显著降低腹泻小鼠血清中的免疫因子水平;
(5)显著降低腹泻小鼠血清中肠毒素ST的含量;
(6)显著提高腹泻小鼠血清中水通道蛋白AQP3的含量;
(7)显著提高腹泻小鼠粪便中短链脂肪酸的含量;
因此,将约氏乳杆菌CCFM1181用于制备预防和/或治疗腹泻的产品,如功能性食品或药品等,具有巨大的应用前景。
生物材料保藏
一株约氏乳杆菌(Lactobacillus johnsonii)CCFM1181,分类学命名为Lactobacillus johnsonii,已于2021年04月16日保藏于广东省微生物菌种保藏中心,保藏编号为GDMCC No:61607,保藏地址为广州市先烈中路100号大院59号楼5楼。
附图说明
图1:不同组小鼠的体重变化情况。
图2:不同组小鼠粪便含水量的变化情况。
图3:不同组小鼠空肠的病理损伤情况。
图4:不同组小鼠空肠绒毛的高度。
图5:不同组小鼠空肠隐窝的深度。
图6:不同组小鼠血清中IFN-γ的含量。
图7:不同组小鼠血清中TNF-α的含量。
图8:不同组小鼠血清中IL-6的含量。
图9:不同组小鼠血清中Toll样受体TLR4的含量。
图10:不同组小鼠血清中肠毒素ST的含量。
图11:不同组小鼠血清中水通道蛋白AQP3的含量。
图12:不同组小鼠粪便中乙酸的含量。
图13:不同组小鼠粪便中丙酸的含量。
图1~13中:*:P<0.05,**:P<0.01,***:P<0.001。
具体实施方式
下面结合具体实施例和附图对本发明进行进一步的阐述。
下述实施例中涉及的BALB/c小鼠购自浙江维通利华公司;下述实施例中涉及的链霉素购自上海生工生物工程(上海)股份有限公司;下述实施例中涉及的ETEC O78:K80购自中国工业微生物菌种保藏管理中心;下述实施例中涉及的布拉迪酵母CNCM I-745购自法国百科达有限公司;下述实施例中涉及的检测IFN-γ(货号:ML720140-2)、TNF-α(货号:ML720852-2)、IL-6(货号:ML720188-2)和IL-8(货号:ML063162-2)的ELISA试剂盒购自上海酶联生物科技有限公司;下述实施例中涉及的检测肠毒素ST(货号:ML701990-2)和水通道蛋白AQP3(货号:ML001869-2)的ELISA试剂盒购自上海酶联生物科技有限公司。
下述实施例中涉及的培养基如下:
MRS固体培养基:蛋白胨10g/L、牛肉膏10g/L、葡萄糖20g/L、乙酸钠2g/L、酵母粉5g/L、柠檬酸氢二铵2g/L、K2HPO4·3H2O 2.6g/L、MgSO4·7H2O 0.1g/L、MnSO4 0.05g/L、吐温80 1mL/L、琼脂15g/L、半胱氨酸氨酸盐0.5g/L。
MRS液体培养基:蛋白胨10g/L、牛肉膏10g/L、葡萄糖20g/L、乙酸钠2g/L、酵母粉5g/L、柠檬酸氢二铵2g/L、K2HPO4·3H2O 2.6g/L、MgSO4·7H2O 0.1g/L、MnSO4 0.05g/L、吐温80 1mL/L、半胱氨酸氨酸盐0.5g/L。
YPD固体培养基:酵母提取物10g/L、蛋白胨20g/L、葡萄糖20g/L、琼脂粉20g/L。
YPD液体培养基:酵母提取物10g/L、蛋白胨20g/L、葡萄糖20g/L。
实施例1:约氏乳杆菌CCFM1181的筛选及菌种鉴定
1、筛选:以来源于吉林桦甸鸭粪便为样本,取0.5g保存于30%(v/v)甘油中的样本在无菌环境下加入装有4.5mL生理盐水的10mL离心管中,得到10-1稀释液,重复上述稀释步骤,依次得到10-2、10-3、10-4、10-5、10-6稀释液;分别吸取100μL不同梯度的梯度稀释液涂布于MRS固体培养基上,37℃培养72h,得到稀释涂布平板;挑取稀释涂布平板上的典型菌落分别接种至MRS液体培养基中,37℃培养48h,得到菌液。
2、鉴定:将分离纯化的各菌液所对应的各菌株编号后,参照教科书《微生物学》(沈萍,陈向东主编)中所记载的步骤进行菌株鉴定、革兰氏染色、生理生化等实验,选取具有约氏乳杆菌典型特征的菌株,经实验获得两株菌,将两株菌分别命名为CCFM1181和FAHBZ6L5(CCFM1181的生理生化特性见表1);其中,菌株鉴定过程如下:
提取CCFM1181和FAHBZ6L5的基因组,将CCFM1181和FAHBZ6L5的16S rDNA进行扩增和测序(由上海生工生物工程股份有限公司完成),将测序分析得到的SEQ ID NO.1所示的CCFM1181的16S rDNA序列和SEQ ID NO.2所示的FAHBZ6L5的16S rDNA序列在GenBank中进行比对,结果显示两株菌株均为约氏乳杆菌,但由于其16S rDNA序列不一致,分别为不同的菌株,故分别命名为约氏乳杆菌(Lactobacillus johnsonii)CCFM1181和约氏乳杆菌(Lactobacillus johnsonii)FAHBZ6L5。
表1 CCFM1181的生理生化特性
苦杏仁苷 | + | 乳糖 | d | 甘露糖 | + | 水杨素 | + |
纤维二糖 | + | 麦芽糖 | + | 蜜利比糖 | d | 蔗糖 | + |
半乳糖 | + | 甘露醇 | - | 棉籽糖 | d | 海藻糖 | d |
注:+,超过90%的菌株为阳性;-大于或等于90%菌株为阳性;d,11%-89%的菌株为阳性;
3、菌液的制备:
挑取步骤2获得的约氏乳杆菌的单菌落接入MRS液体培养基中,于37℃厌氧培养24h,得到活化液;将活化液按照1%(v/v)的接种量接入MRS液体培养基中,于37℃厌氧培养24h,得到一级种子液;将一级种子液按照1%(v/v)的接种量接入MRS液体培养基中,于37℃厌氧培养24h,得到二级种子液;将二级种子液按照1%(v/v)的接种量接入MRS液体培养基中,于37℃厌氧培养24h,得到菌液;将菌液6000g离心15min,收集沉淀;将沉淀用pH为7.4的PBS缓冲液洗涤两次后,6000g再次离心10min,得到菌体;用含有130g/L脱脂乳、20g/L海藻糖和20g/L蔗糖的保护剂溶液将约氏乳杆菌菌体重悬至细胞浓度为5×109CFU/mL,得到约氏乳杆菌菌液。
布拉迪酵母菌液的制备方法如下:
取一勺布拉迪酵母菌粉接入YPD液体培养基中,于28℃培养24h,得到活化液;将活化液按照1%(v/v)的接种量接入YPD液体培养基中,于28℃培养24h,得到一级种子液;将一级种子液按照1%(v/v)的接种量接入YPD液体培养基中,于28℃培养24h,得到二级种子液;将二级种子液按照1%(v/v)的接种量接入YPD液体培养基中,于28℃培养24h,得到菌液;将菌液6000g离心15min,收集沉淀;将沉淀用pH为7.4的PBS缓冲液洗涤两次后,6000g再次离心10min,得到菌体;用含有130g/L脱脂乳、20g/L海藻糖和20g/L蔗糖的保护剂溶液将布拉迪酵母菌体重悬至细胞浓度为5×109CFU/mL,得到布拉迪酵母菌菌液。
研究证明布拉迪酵母菌株没有病源性和扩散性(限定在消化道中,而不会向身体的任何其他位置扩散),通常生长良好,且具有独特生物活性,适合作为人和动物的益生菌使用。自分离出布拉迪酵母菌株以来,已进行了广泛的研究,共发表400多篇相关研究报告。1962年该菌株开始应用于治疗人类腹泻(作为处方药使用),1993开始用于改善单胃动物营养和健康的饲料添加剂,适用于母猪,仔猪,肉鸡,蛋鸡,犊牛,特种皮毛动物、水产动物等。布拉迪酵母已成为商业化菌株并且已有文献报道布拉迪酵母在缓解ETEC引起的腹泻的有益作用,因此,下述实施例中以布拉迪酵母作为阳性对照。
实施例2~7中,约氏乳杆菌(Lactobacillus johnsonii)菌液和布拉迪酵母菌液均按本实施例的方法制备。
实施例2:约氏乳杆菌CCFM1181对腹泻小鼠体重和粪便含水量的影响
具体步骤如下:
取BALB/c小鼠40只,于饲养室温为22~24℃,湿度为40%~60%,12h/12h昼夜交替,自由进食及饮水的条件下饲养1周后,随机分为5组,每组8只,5组分别为:对照组、造模组、灌胃布拉迪酵母菌液的布拉迪酵母组(CNCMI-745组)、灌胃约氏乳杆菌(Lactobacillusjohnsonii)FAHBZ6L5菌液的FAHBZ6L5组、灌胃约氏乳杆菌(Lactobacillus johnsonii)CCFM1181菌液的CCFM1181组。
实验共3周:动物适应性饲养1周后开始实验。从造模前7天开始,一直持续到实验结束(不包括抗生素处理的那3天),对照组和造模组小鼠每只每天灌胃0.2mL按实施例1的方法制备的含有130g/L脱脂乳、20g/L海藻糖和20g/L蔗糖的保护剂溶液,布拉迪酵母组每只每天灌胃0.2mL按实施例1的方法制备的布拉迪酵母菌液,FAHBZ6L5组每只每天灌胃0.2mL按实施例1的方法制备的约氏乳杆菌(Lactobacillus johnsonii)FAHBZ6L5菌液,CCFM1181组每只每天灌胃0.2mL按实施例1的方法制备的约氏乳杆菌(Lactobacillusjohnsonii)CCFM1181菌液;适应后的第2周为造模期,造模第1~3天,在小鼠饮用水中加入5g/L的链霉素,以消除BALB/c小鼠肠道正常菌群,造模第4~7天,用不含抗生素的无菌水代替含有链霉素的水作为小鼠饮用水,并且,对小鼠禁食18h,18h后,给每组小鼠每只灌胃0.2mL浓度为1.2×1011CFU/mL的产肠毒素大肠埃希氏菌(Escherichia coli ETEC O78:K80)悬液(通过将ETEC O78:K80菌体溶于浓度为8.5g/L的生理盐水而得),连续灌胃4天,1天2次,每次间隔2h。
造模期间以及造模结束后,通过体重计测定每组小鼠体重;实验结束后,收集小鼠粪便,通过Lyobeta 5ps冻干机(西班牙泰事达公司)测定小鼠粪便的含水量,测定结果分别见图1~2。
由图1可知,在整个实验期间,造模组小鼠的体重变化和对照组并没有显著差异,药物组、布拉迪酵母组和约氏乳杆菌CCFM1181干预组均没有显著变化。
由图2可知,对照组的粪便含水量为46.73%,造模组小鼠粪便中的含水量(61.98%)是对照组小鼠的1.33倍(p<0.01),CCFM1181组小鼠粪便中的含水量为46.71%,与造模组小鼠相比,存在显著差异(p<0.01);布拉迪酵母组的粪便含水量为54.01%,同样与造模组具有显著性差异;而FAHBZ6L5组小鼠粪便中的含水量为55.77%,与造模组小鼠相比,没有显著差异。
可见,约氏乳杆菌CCFM1181可有效缓解腹泻小鼠粪便含水量升高的症状,缓解效果比布拉迪酵母要好,而约氏乳杆菌FAHBZ6L5则无此作用。
实施例3:约氏乳杆菌CCFM1181对腹泻小鼠空肠病理损伤情况的影响
动物模型的构建方法同实施例2,实验结束后,处死小鼠,取小鼠空肠,于4%(v/v)多聚甲醛中浸泡24h,得到固定好的空肠组织;将固定好的空肠组织依次进行脱水、透明、浸蜡后,用莱卡石蜡包埋机将组织包埋于蜡块中,得到包埋好空肠组织的蜡块;其中,脱水、透明、浸蜡的具体步骤如下:(1)脱水:将固定好的组织先依次经70%、80%和90%(v/v)梯度乙醇溶液进行脱水,每个梯度各30min,然后放入95%和100%(v/v)酒精溶液各2次,每次20min;(2)透明:将组织先放入酒精和二甲苯等体积比混合液中15min,然后放入二甲苯I和二甲苯II各15min;(3)浸蜡:将组织样放入62℃的石蜡I和石蜡II液体中各30min。
将包埋好空肠组织的蜡块用莱卡手动轮转切片机进行切片,切片厚度为5μm,得到空肠组织切片;将空肠组织切片经展片和捞片、烤片、苏木精染色、分化、漂洗、伊红复染、脱水、透明、封片,得到H&E空肠切片;其中,展片和捞片、烤片、苏木精染色、分化、漂洗、伊红复染、脱水、透明、封片的具体操作如下:(1)展片和捞片:将切片放于42℃恒温水浴中进行展片后用载玻片小心地捞出;(2)烤片:将切片放于60℃烘箱中过夜烤片;(3)苏木精染色:将切片先进行水化(即先将切片置于二甲苯I和二甲苯II各5min,然后依次放入100%、95%、90%、80%和70%(v/v)梯度酒精溶液中各5min,最后放入蒸馏水中3min),然后进行染色(即将切片放入苏木精染色液中约20s),最后进行水洗(即将切片用自来水冲洗约30min);(4)分化:将切片放入1%(v/v)盐酸乙醇溶液中7s,进行褪色;(5)漂洗:用自来水冲洗切片约20min;(6)复染:将切片浸入伊红染色液,立即取出;(7)脱水:将切片先依次放入95%(v/v)乙醇溶液I、95%(v/v)乙醇溶液II、70%(v/v)乙醇溶液,放入后立即取出,然后浸入80%(v/v)乙醇溶液50s,最后浸入100%(v/v)乙醇2min;(8)透明:将切片先浸入乙醇和二甲苯等体积比混合液1min,然后浸入二甲苯I和二甲苯II各2min;(9)封片:将切片用中性树胶封片。
用Pannoramic MIDI数字切片扫描仪扫描制作好的H&E空肠切片,并且,拍照观察小鼠空肠的绒毛长度和隐窝深度,观察结果见图3~5。
由图3~5可知,与对照组小鼠相比,造模组小鼠空肠的绒毛长度明显缩短了22.78%,隐窝深度明显增加了70.40%;与造模组小鼠相比,CCFM1181组小鼠空肠的绒毛长度增加了67.66%,隐窝深度降低了28.07%;FAHBZ6L5组小鼠空肠的绒毛长度增加了17.43%,隐窝深度降低了35.68%;布拉迪酵母组小鼠空肠的绒毛长度增加了20.72%,隐窝深度降低了30.58%。
可见,约氏乳杆菌CCFM1181可有效缓解腹泻小鼠空肠的病理损伤情况,且在绒毛长度恢复方面的效果优于布拉迪酵母,而约氏乳杆菌FAHBZ6L5的缓解效果要差一些。在隐窝深度降低方面各菌株缓解效果相当。
实施例4:约氏乳杆菌CCFM1181对腹泻小鼠免疫因子水平的影响
动物模型的构建方法同实施例2,实验结束后,取血并处死小鼠,取小鼠血清,通过ELISA试剂盒测定每组小鼠血清中IFN-γ、TNF-α、IL-6、IL-8和TLR4的含量,检测结果见图6~10。
如图6所示,造模组小鼠血清中IFN-γ的含量为162.13pg/mL,比对照组(91.27pg/mL)显著升高;相比于造模组小鼠,CCFM1181组小鼠血清中IFN-γ的含量显著降低,降低了50.28%;FAHBZ6L5组小鼠降低了24.74%,没有显著差异;布拉迪酵母组小鼠降低了13.63%。
如图7所示,造模组小鼠血清中TNF-α的含量为236.13pg/mL,与对照组(157.42pg/mL)相比显著升高;相比于造模组小鼠,CCFM1181组小鼠血清中TNF-α的含量显著降低,降低了25.90%;FAHBZ6L5组小鼠降低了21.43%,没有显著差异;布拉迪酵母组小鼠降低了14.53%。
如图8所示,造模组小鼠血清中IL-6的含量为14.38pg/mL,比对照组(7.94pg/mL)显著升高;相比于造模组小鼠,CCFM1181组小鼠血清中IL-6的含量显著降低,降低了32.95%;FAHBZ6L5组小鼠降低了15.86%,没有显著差异;布拉迪酵母组小鼠降低了24.24%。
如图9所示,造模组小鼠血清中TLR4的含量为5.23pg/mL,比对照组(3.30pg/mL)显著降低;相比于造模组小鼠,CCFM1181组小鼠血清中TLR4的含量显著降低,降低了45.29%;FAHBZ6L5组小鼠降低了44.59%,具有显著差异;布拉迪酵母组小鼠降低了19.99%。
可见,约氏乳杆菌CCFM1181可显著降低腹泻小鼠的免疫因子水平且效果优于布拉迪酵母;而约氏乳杆菌FAHBZ6L5并无抗炎作用。
实施例5:约氏乳杆菌CCFM1181对腹泻小鼠血清中肠毒素ST和水通道蛋白AQP3含量的影响
动物模型的构建方法同实施例2,实验结束后,取血并处死小鼠,取小鼠血清,通过ELISA试剂盒测定每组小鼠血清中肠毒素ST和水通道蛋白AQP3的含量,检测结果见图10~11。
如图10所示,造模组小鼠血清中肠毒素ST的含量为970.41pg/mL,比对照组(623.44pg/mL)显著升高;相比于造模组小鼠,CCFM1181组小鼠血清中肠毒素ST的含量显著降低,降低了28.98%;FAHBZ6L5组小鼠血清中肠毒素ST的含量降低了18.76%;布拉迪酵母组小鼠血清中肠毒素ST的含量降低了28.99%。
如图11所示,造模组小鼠血清中水通道蛋白AQP3的含量为163.67pg/mL,比对照组(185.95pg/mL)显著降低;相比于造模组小鼠,CCFM1181组小鼠血清中AQP3的含量显著升高,升高了20.57%,FAHBZ6L5组小鼠血清中AQP3的含量升高了1.77%,两组均无显著性,布拉迪酵母组小鼠血清中AQP3的含量升高了31.31%,与造模组存在显著性差异。
可见,约氏乳杆菌CCFM1181可显著降低腹泻小鼠血清中肠毒素ST的含量,效果和布拉迪酵母相当,而约氏乳杆菌FAHBZ6L5并不明显;约氏乳杆菌CCFM1181提高腹泻小鼠血清中水通道蛋白AQP3的效果没有布拉迪酵母的好,但比FAHBZ6L5效果好。
实施例6:约氏乳杆菌CCFM1181对腹泻小鼠粪便中短链脂肪酸含量的影响
动物模型的构建方法同实施例2,实验结束后,收集小鼠粪便置于液氮中,后转移至-80℃冰箱,在进行短链脂肪酸含量的检测前取出,进行真空冷冻干燥,准确称取0.05g冻干后的粪便样品溶解于0.5mL饱和氯化钠溶液中,浸泡30min,组织匀浆机匀浆,加入0.02mL浓度为10%的硫酸,震荡30s,在通风橱内向粪便溶液中准确加入1mL乙醚溶液,震荡30s后离心15min(8000g、4℃),移取上清液至含有0.25g无水硫酸钠的离心管中,震荡均匀,离心15min(8000g、4℃),取上清至气质容量瓶中,通过GCMS检测短链脂肪酸含量,检测结果见图12~13。
如图12~13所示,造模组小鼠粪便中乙酸、丙酸的含量和对照组几乎无明显差别;CCFM1181组小鼠粪便中乙酸的含量与造模组相比显著上调,是造模组的2.07倍,丙酸的含量明显上升但没有显著差异,是造模组的1.54倍;FAHBZ6L5组小鼠粪便中乙酸、丙酸的含量分别是造模组的1.19倍,1.21倍,均没有显著性差异;布拉迪酵母组小鼠粪便中乙酸、丙酸的含量分别是造模组的1.26倍,0.84倍,没有显著性差异。
可见,约氏乳杆菌CCFM1181可提高腹泻小鼠粪便中乙酸和丙酸的含量,且效果优于布拉迪酵母和约氏乳杆菌FAHBZ6L5。
实施例7:约氏乳杆菌CCFM1181用于制备药物
约氏乳杆菌CCFM1181可用于制备片剂,片剂的具体制备过程如下:
挑取实施例1获得的约氏乳杆菌(Lactobacillus johnsonii)CCFM1181的单菌落接入MRS液体培养基中,于37℃培养16h,得到菌浓数量级达1×107CFU/mL的活化液;将活化液按照1%(v/v)的接种量接入MRS液体培养基中,于37℃培养16h,得到一级种子液;将一级种子液按照1%(v/v)的接种量接入MRS液体培养基中,于37℃培养16h,得到二级种子液;将二级种子液按照1%(v/v)的接种量接入MRS液体培养基中,于37℃培养16h,得到菌液;将菌液6000g离心15min,收集沉淀;将沉淀用pH为7.4的PBS缓冲液洗涤两次后,6000g再次离心10min,得到菌体;用含有130g/L脱脂乳、20g/L海藻糖和20g/L蔗糖的保护剂溶液将约氏乳杆菌菌体重悬至细胞浓度为1×1010CFU/mL,得到约氏乳杆菌菌液;将约氏乳杆菌菌液冷冻干燥,得到约氏乳杆菌菌粉;在约氏乳杆菌菌粉中加入占约氏乳杆菌菌粉总重计2%的硬脂酸作润滑剂、3%的羧甲基纤维素钠(CMC-Na)作粘合剂后进行压片,得到片剂。
取上述方法制备的片剂以1g/只的剂量每天灌胃ETEC致腹泻小鼠,连续两周,可有效缓解小鼠腹泻的症状,在预防和/或治疗腹泻上有极好的效果。
实施例8:约氏乳杆菌CCFM1181用于制备菌粉
约氏乳杆菌CCFM1181可用于制备菌粉,菌粉的具体制备过程如下:
挑取实施例1获得的约氏乳杆菌(Lactobacillus johnsonii)CCFM1181的单菌落接入MRS液体培养基中,于37℃培养16h,得到菌浓数量级达1×107CFU/mL的活化液;将活化液按照1%(v/v)的接种量接入MRS液体培养基中,于37℃培养16h,得到一级种子液;将一级种子液按照1%(v/v)的接种量接入MRS液体培养基中,于37℃培养16h,得到二级种子液;将二级种子液按照1%(v/v)的接种量接入MRS液体培养基中,于37℃培养16h,得到菌液;将菌液6000g离心15min,收集沉淀;将沉淀用pH为7.4的PBS缓冲液洗涤两次后,6000g再次离心10min,得到菌体;用含有130g/L脱脂乳、20g/L海藻糖和20g/L蔗糖的保护剂溶液将约氏乳杆菌菌体重悬至细胞浓度为1×1010CFU/mL,得到约氏乳杆菌菌液;将约氏乳杆菌菌液冷冻干燥,得到菌粉。
可选地,还可将其它类型的益生菌与约氏乳杆菌混合,制备复配菌粉。
取上述方法制备菌粉以1g/只的剂量每天灌胃ETEC致腹泻小鼠,连续两周,可有效缓解小鼠腹泻的症状,在预防和/或治疗腹泻上有极好的效果。
实施例9:约氏乳杆菌CCFM1181用于制备发酵食品
以将约氏乳杆菌CCFM1181或实施例8制备的含约氏乳杆菌CCFM1181的菌粉用于制备发酵食品或发酵饮品。
用于制备发酵饮品的原料可选自:动物乳、植物乳、果蔬汁、谷物汁中的一种或多种的混合。
用于制备发酵食品的原料可选自:动物乳、植物乳、果蔬原料,谷物粉或谷物混合物,肉制品原料。
虽然本发明已以较佳实施例公开如上,但其并非用以限定本发明,任何熟悉此技术的人,在不脱离本发明的精神和范围内,都可做各种的改动与修饰,因此本发明的保护范围应该以权利要求书所界定的为准。
SEQUENCE LISTING
<110> 江南大学
<120> 一株可缓解ETEC致腹泻的约氏乳杆菌及其应用
<130> BAA210616A
<160> 2
<170> PatentIn version 3.3
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<212> DNA
<213> Lactobacillus johnsonii
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aagttaccaa cttcagttga agctgatgaa acagaagcaa cttgagcaat ttcgtcctta 360
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Claims (12)
1.一株约氏乳杆菌(Lactobacillus johnsonii)CCFM1181,其特征在于,已于2021年04月16日保藏于广东省微生物菌种保藏中心,保藏编号为GDMCC No:61607。
2.含有权利要求1所述约氏乳杆菌CCFM1181的发酵剂。
3.根据权利要求2所述的发酵剂,其特征在于,所述发酵剂中约氏乳杆菌CCFM1181的菌体数量≥1×1010 CFU/mL或1×1010 CFU/g。
4.根据权利要求2或3所述的发酵剂,其特征在于,所述发酵剂的制备方法为:将约氏乳杆菌CCFM1181接种到培养基中,于30~37°C下培养,得到培养液;再将培养液离心,收集,用生理盐水或细胞保护剂重悬菌体,得到液态发酵剂。
5.根据权利要求2或3所述的发酵剂,其特征在于,所述发酵剂的制备方法为:将约氏乳杆菌CCFM1181接种到培养基中,于30~37°C下培养,得到培养液;再将培养液离心,收集,用生理盐水或细胞保护剂重悬菌体,得到液态发酵剂,再将液态发酵剂冻干,获得固态发酵剂。
6.权利要求1所述的约氏乳杆菌CCFM1181在制备预防和/或治疗腹泻的药物中的应用。
7.根据权利要求6所述的应用,其特征在于,所述治疗腹泻具有如下至少一种作用:
(1)提高腹泻个体的体重;
(2)降低腹泻个体的粪便含水量;
(3)缓解腹泻个体空肠的病理损伤;
(4)降低腹泻个体血清中的免疫因子水平;
(5)降低腹泻个体血清中肠毒素ST的含量;
(6)提高腹泻个体血清中水通道蛋白AQP3的含量;
(7)提高腹泻个体粪便中短链脂肪酸的含量。
8.根据权利要求7所述的应用,其特征在于,所述腹泻个体是具有腹泻症状的哺乳动物。
9.一种预防和/或治疗腹泻的药物,其特征在于,含有权利要求1所述的约氏乳杆菌CCFM1181和药物载体,或含有权利要求1所述的约氏乳杆菌CCFM1181和药用辅料。
10.权利要求1所述的约氏乳杆菌CCFM1181或权利要求2~5任一所述发酵剂在制备食品中的应用。
11.根据权利要求10所述的应用,其特征在于,所述食品为含有所述约氏乳杆菌(Lactobacillus johnsonii)CCFM1181的乳制品、豆制品、肉制品或果蔬制品。
12.权利要求1所述的约氏乳杆菌CCFM1181或权利要求2~5任一所述发酵剂在制备饮品中的应用。
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