CN114805474B - 一株干酪乳杆菌fn345及其在制备预防辅食期婴儿腹泻菌剂中的应用 - Google Patents
一株干酪乳杆菌fn345及其在制备预防辅食期婴儿腹泻菌剂中的应用 Download PDFInfo
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Abstract
本发明公开了一株干酪乳杆菌FN345及其在制备预防辅食期婴儿腹泻菌剂中的应用,属于益生菌健康食品技术领域。本发明公开的一株干酪乳杆菌FN345,保藏编号为CCTCCNO:M2020822,FN345具有显著地抗腹泻效果并可促进大鼠乳鼠肠道多糖的产生;FN345胞外多糖具有显著的抗腹泻效果,抗腹泻的机制为促进肠道CSF1R依赖性巨噬细胞调控肠道稳态,增强与肠道细胞增殖分化相关的CSF1R‑MAPK信号通路;促进肠道细胞增殖,使小肠绒毛高度显著增加,同时促进肠道潘氏细胞、杯状细胞分化及功能成熟;FN345丰富了有益婴儿胃肠道健康的益生菌库,并能开发成相应益生菌制剂应用于辅食期婴儿腹泻预防。
Description
技术领域
本发明涉及益生菌健康食品技术领域,更具体的说是涉及一株干酪乳杆菌FN345及其在制备预防辅食期婴儿腹泻菌剂中的应用。
背景技术
婴儿喂养是国际社会十分关注的领域,婴儿的喂养方式、辅食添加时间、辅食种类均与婴儿健康状况有关。辅食期的婴儿腹泻是一组多因素引起的消化道综合征,是我国婴幼儿最常见的疾病,是造成小儿营养不良、生长发育障碍的主要原因之一。腹泻是婴儿常见的异常生理反应,对婴儿健康有很大影响,研究表明婴幼儿腹泻可导致其营养不良,若得不到及时治疗,婴儿将出现电解质紊乱、贫血、免疫力低下及生长发育障碍。动物研究表明,断乳时肠道的发育主要受肠道干细胞增殖分化的影响,断奶刺激肠道干细胞快速增殖分化,以适应断奶引起的变化,但细胞进程的加快导致未成熟肠上皮细胞和杯状细胞比例增加,功能成熟的潘氏细胞严重减少,肠道形态呈现明显缺陷,通透性变差,屏障功能降低,易感腹泻。因此促进辅食期婴儿肠道各类细胞的分化和成熟对预防辅食期婴儿腹泻至关重要。
因此,提供一株干酪乳杆菌FN345及其在制备预防辅食期婴儿腹泻菌剂中的应用是本领域技术人员亟需解决的问题。
发明内容
有鉴于此,本发明提供了一株干酪乳杆菌FN345及其在制备预防辅食期婴儿腹泻菌剂中的应用,筛选产胞外多糖促巨噬细胞CSF1-R表达的益生菌,菌体胞外多糖通过调控辅食导致的婴儿肠道稳态异常,从而预防辅食期腹泻。
为了实现上述目的,本发明采用如下技术方案:
一株干酪乳杆菌(Lactobacillus casei)FN345,其保藏编号为CCTCC NO:M2020822,已保藏于中国典型培养物保藏中心,简称CCTCC,地址:中国.武汉.武汉大学,保藏日期为2020年12月02日,分类命名为:干酪乳杆菌FN345,Lactobacillus casei FN345。
进一步,所述的一株干酪乳杆菌FN345在产胞外多糖中的应用。
进一步,所述的一株干酪乳杆菌FN345在制备预防辅食期婴儿腹泻菌剂中的应用。
利用脱脂乳培养FN345菌株制备冻干菌粉,后添加辅料制备活菌颗粒。
进一步,所述的一株干酪乳杆菌FN345产的胞外多糖在制备预防辅食期婴儿腹泻药物中的应用。
集落刺激因子1(CSF1)促进肌层巨噬细胞的生长和分化,集落刺激因子1受体(CSF1-R)依赖性肌层巨噬细胞调控隐窝发育,促进潘氏细胞成熟,而CSF1-R的表达受肠道菌群的影响。因此筛选可促巨噬细胞CSF1-R表达的菌株,通过调控辅食后肠道细胞的分化可预防婴儿腹泻。肌层巨噬细胞位于肠道黏膜肌层,不与菌体直接接触,因此推测菌是通过代谢产物与巨噬细胞进行作用,在菌的代谢产物中,胞外多糖可与巨噬细胞发生作用。
辅食引起的婴儿腹泻是一个原因复杂的病理过程,可能与辅食的喂养不合理,如辅食不卫生、过早辅食补充、辅食添加量太多,辅食刺激导致婴儿肠道出现异常生理反应等均有关。本发明针对的腹泻诱因是添加辅食后婴儿肠道绒毛高度及隐窝深度降低,杯状细胞增多出现炎症反应,肠道稳态被破坏导致的腹泻,益生菌胞外多糖能调节婴儿肠道稳态,抑制辅食期婴儿腹泻。对于辅食的喂养不合理导致的腹泻不具备针对性。
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干酪乳杆菌FN345及其在制备预防辅食期婴儿腹泻菌剂中的应用,具有以下有益效果:
(1)筛选得到一株显著降低辅食引起腹泻的益生菌—干酪乳杆菌FN345(FN-345),其主要通过产胞外多糖起到抗腹泻作用;
(2)干酪乳杆菌FN345具有显著地抗腹泻效果并可促进大鼠乳鼠肠道多糖的产生;
(3)FN345胞外多糖具有显著的抗腹泻效果,抗腹泻的机制为促进肠道CSF1R依赖性巨噬细胞调控肠道稳态,增强与肠道细胞增殖分化相关的CSF1R-MAPK信号通路;促进肠道细胞增殖,使小肠绒毛高度显著增加,同时促进肠道潘氏细胞、杯状细胞分化及功能成熟;
(4)干酪乳杆菌FN345丰富了有益婴儿胃肠道健康的益生菌库,并能开发成相应益生菌制剂应用于辅食期婴儿腹泻预防。
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附图为本发明不同菌株发酵液促进RAW264.7细胞中CSF1R表达的能力;
图2附图为本发明菌株16S rRNA鉴定得到的系统发育树;
图3附图为本发明不同组大鼠乳鼠(施用菌悬液)的粪便评分;
图4附图为本发明不同组大鼠乳鼠(施用菌悬液)的粪便含水量;
图5附图为本发明不同组大鼠乳鼠(施用菌悬液)血液中FITC-葡聚糖浓度;
图6附图为本发明不同组大鼠乳鼠(施用菌悬液)结肠内容物和粪便中的多糖含量;
图7附图为本发明不同组大鼠乳鼠(施用粗胞外多糖)的粪便评分;
图8附图为本发明不同组大鼠乳鼠(施用粗胞外多糖)的粪便含水量;
图9附图为本发明不同组大鼠乳鼠(施用粗胞外多糖)血液中FITC-葡聚糖浓度;
图10附图为本发明抗腹泻菌株粗胞外多糖对大鼠乳鼠肠道组织结构的影响;
图11附图为本发明免疫荧光检测大鼠乳鼠肠道增殖的细胞和潘氏细胞;
图12附图为本发明RT-PCR检测CSF1R的表达量;
图13附图为本发明RT-PCR检测SRF的表达量;
图14附图为本发明RT-PCR检测CreB的表达量;
Jejunum,空肠;Ileum,回肠;Colon,结肠;Rectum,直肠。
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。
实施例1筛选促CSF1-R表达的产胞外多糖菌株
将实验室现有分离自母乳和婴儿粪便中的菌株接种于MRS-S(5%蔗糖)琼脂上,37℃培养48h,然后检测粘稠/粘液菌落。选择具有“粘稠”表型(当菌落接触接种环时形成牢不可破的链)和“粘液非粘稠”表型(闪亮、光滑和粘稠的外观,但不出现细丝)的菌株作为胞外多糖生产菌株。将产胞外多糖菌株在MRS-S培养基(5%蔗糖)中厌氧培养24小时,离心得到不含菌体的发酵液。将500μL发酵液与1500μL RPMI培养基混合,加入RAW264.7细胞单层的6孔板中,并在5%(v/v)CO2与95%空气的气体环境下于37℃培养24h。将1mL RIPA和10μLPMSF混合,使PMSF终浓度为1mM,获得混合缓冲液。取150μL混合缓冲液添加到每个孔中,然后将六孔板置于冰上30分钟。离心(12,000rpm,5min)收集上清液。以未加发酵液的细胞所表达的CSF1R浓度为空白对照(Control),使用小鼠CSF1R酶联免疫吸附测定(ELISA)KIT确定细胞中CSF1R的浓度。计算菌株发酵液促进RAW264.7细胞中CSF1R表达的能力,结果见图1。
图1结果显示,共17株产胞外多糖菌株发酵液CSF1R表达量高于对照组,其中FN-345可显著促进CSF1-R的表达(P<0.05)。
菌株鉴定结果见图2,FN-345为Lactobacillus caseiFN-345。
实施例2菌株抗腹泻效果筛选
根据哈尔滨工业大学动物护理和使用伦理委员会(IACUC-2019023)的指南对所有使用的动物进行饲养。30只14天大的Sprague Dawley(SD)大鼠乳鼠和它们的母亲被安置在有软木颗粒地板的聚碳酸酯笼中。乳鼠基础饮食是母乳喂养,Medilac-Vita(妈咪爱,枯草杆菌二联活菌)被用作阳性对照。菌株在MRS肉汤中37℃培养24h,5000g离心5min收集菌体,以磷酸盐缓冲液(PBS)冲洗菌体三次后重悬,制备菌悬液(含108CFU/mL菌体)。在实验中,大鼠乳鼠被随机分为3组(每组N=10):菌株FN-345组(FN-345),对照组(PBS;Control),阳性对照组(妈咪爱;Positive control)。根据大鼠乳鼠食物摄入量与体重的比值,每天向每只乳鼠喂1克饲料,持续7天。根据先前实验中益生菌的补充剂量,在FN-345组中,每天通过管饲法向每只大鼠乳鼠施用100μL的菌悬液(含108CFU/mL菌体)。在对照组中,向大鼠乳鼠施用100μL PBS。在阳性组中,每天向每只大鼠乳鼠施用100μL妈咪爱(枯草杆菌二联活菌,含108CFU/mL菌体)溶液。
每天早晨8:00进行腹部按摩以收集新鲜的大鼠乳鼠粪便,通过对粪便稠度的视觉评估进行粪便评分,粪便稠度评分为0=固体,1=半固体,2=半液体和3=液体。腹泻的发生被定义为等级为2或3的粪便的产生。根据每组所有大鼠的粪便评分,进行差异统计学分析,确定菌株对粪便状态的影响。
用热重法测定粪便中的水分含量。
将FITC-葡聚糖(4000Da)灌胃于21天SD大鼠乳鼠(50mg/100g体重)。2h后,将大鼠乳鼠麻醉取血。FITC-葡聚糖在PBS中连续稀释,并添加到不含FITC-右旋糖酐的大鼠乳鼠血浆中,作为标准曲线。使用发光光谱仪在490nm的激发波长和530nm的发射波长下读取样品。
结果如图3-5所示,在5株菌中,菌株FN-345组大鼠乳鼠的粪便评分,粪便含水量及血液中FITC-葡聚糖浓度显著低于对照组(P<0.05),被认为可以具有明显的抗腹泻效果。
实施例3抗腹泻菌株对大鼠乳鼠肠道多糖含量的影响
采样苯酚硫酸法检测FN-345及对照组大鼠乳鼠肠道内容物的多糖含量。结果如图6所示,菌株FN-345可以使结肠内容物中多糖含量显著增高(P<0.05),使粪便(stool)中多糖含量显著增高(P<0.05)。
实施例4抗腹泻菌株粗胞外多糖提取及多糖抗腹泻效果分析
用MRS-S(5%蔗糖)培养基发酵菌株FN-345,37℃培养48h,然后在4℃下10000r/min离心10min除去沉淀菌体,上清液除蛋白后浓缩,加入95%乙醇醇沉,在4℃下10000r/min离心10min收集沉淀,将沉淀用适量超纯水溶解后透析、冷冻干燥,即得粗胞外多糖。在实验中,大鼠乳鼠被随机分为2组(每组N=10):FN-345粗胞外多糖组(c-eps345),对照组(磷酸盐缓冲液,PBS;Control)。根据大鼠乳鼠食物摄入量与体重的比值,每天向每只乳鼠喂1克饲料,持续7天。根据先前实验中益生菌的补充剂量,在FN-345组中,每天通过管饲法按80mg多糖/kg体重的比例向每只大鼠乳鼠补充粗胞外多糖溶液100μL。在对照组中,向大鼠乳鼠施用100μL PBS。
每天早晨8:00进行腹部按摩以收集新鲜的大鼠乳鼠粪便。腹泻的发生通过对粪便稠度的视觉评估确定,以粪便评分判定粪便稠度,粪便稠度评分为0=固体,1=半固体,2=半液体和3=液体。腹泻的发生被定义为等级为2或3的粪便的产生。根据每组所有大鼠的粪便评分,进行差异统计学分析,确定多糖对粪便状态的影响。
用热重法测定粪便中的水分含量。
将FITC-葡聚糖(4000Da)灌胃于21天SD大鼠乳鼠(50mg/100g体重)。2h后,将大鼠乳鼠麻醉取血。FITC-葡聚糖在PBS中连续稀释,并添加到不含FITC-右旋糖酐的大鼠乳鼠血浆中,作为标准曲线。使用发光光谱仪在490nm的激发波长和530nm的发射波长下读取样品。
结果如图7-9所示,FN-345粗胞外多糖组大鼠乳鼠的粪便评分,粪便含水量及血液中FITC-葡聚糖浓度显著低于对照组(P<0.05),被认为具有明显的抗腹泻效果。
实施例5FN-345粗胞外多糖作用后大鼠乳鼠肠道病理学分析
将FN-345粗胞外多糖灌胃后大鼠乳鼠的小肠和大肠固定在聚甲醛中以进行形态测定。按照标准程序将样品脱水并包埋。切成5μm的切片后,用苏木精和曙红(HE)染色。在常规BX-51显微镜下检查标本。使用Vistron系统(Puchheim)收集小肠(空肠、回肠)和大肠(结肠、直肠)形态的代表性照片。结果如图10所示,与对照组相比,FN-345胞外多糖组大鼠乳鼠回肠绒毛明显提高,回肠和结肠段杯状细胞数量增加且分布更均匀。
实施例6FN-345粗胞外多糖抗腹泻机制研究
利用免疫荧光检测大鼠乳鼠肠道增殖的细胞和潘氏细胞,利用RT-PCR检测与肠道细胞增殖分化相关的MAPK信号通路CSF1R、CreB和SRF的转录。结果如图11-14所示,与对照组相比,FN-345胞外多糖组大鼠乳鼠肠道ki-67标记的增殖细胞及Lysozyme标记的潘氏细胞表达量明显增加;在MAPK信号通路中,CSF1R和SRF在FN-345胞外多糖组不同肠段表达量均显著高于对照组(P<0.05),CreB在FN-345胞外多糖组结肠和直肠中的表达量显著高于对照组(P<0.05)。
机制研究表明,菌株FN-345粗胞外多糖通过MAPK通路,促进CSF1R表达,进而通过上调ERK蛋白,促进SRF和CreB表达增加,从而调控肠道细胞增殖及分化过程。
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 (4)
1.一株干酪乳杆菌(Lactobacillus casei)FN345,其特征在于,其保藏编号为CCTCCNO:M 2020822。
2.权利要求1所述的一株干酪乳杆菌FN345在产胞外多糖中的应用。
3.权利要求1所述的一株干酪乳杆菌FN345在制备预防辅食期婴儿腹泻菌剂中的应用。
4.权利要求1所述的一株干酪乳杆菌FN345产的胞外多糖在制备预防辅食期婴儿腹泻药物中的应用。
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