CN114652753B - 两歧双歧杆菌b11在制备抑制幽门螺旋杆菌及修复胃黏膜屏障产品中的应用 - Google Patents
两歧双歧杆菌b11在制备抑制幽门螺旋杆菌及修复胃黏膜屏障产品中的应用 Download PDFInfo
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Abstract
本发明涉及微生物技术领域,具体涉及保藏编号为CGMCC No.24381的两歧双歧杆菌B11在制备抑制幽门螺杆菌及修复胃黏膜屏障产品中的应用。本发明提供的两歧双歧杆菌B11对幽门螺杆菌有显著的抑制作用,与胃癌细胞AGS存在强粘附作用,为两歧双歧杆菌B11能够粘附在胃上皮细胞及其周围黏液层提供可能,使两歧双歧杆菌B11在胃部有定植的潜力,可以显著抑制幽门螺杆菌在胃上皮细胞的粘附,同时,两歧双歧杆菌B11可抑制促炎因子IL‑8的转录水平,抑制幽门螺杆菌导致的炎症,对胃黏膜起到保护作用。同时,该菌株的活菌株还能够促进黏膜保护因子TFF1、iNOS和COX‑2的转录,对胃黏膜屏障和细胞修复有积极作用。
Description
技术领域
本发明涉及微生物技术领域,具体涉及两歧双歧杆菌B11在制备抑制幽门螺杆菌及修复胃黏膜屏障产品中的应用。
背景技术
幽门螺杆菌又称幽门螺旋菌,拉丁属名Helicobacterpylori,简称Hp,是革兰氏阴性、微需氧的细菌,生存于胃部及十二指肠的各区域内。它会引起胃黏膜轻微的慢性发炎,甚或导致胃及十二指肠溃疡与胃癌,被世界卫生组织确定为第Ι类致癌因子,因此开展预防和治疗幽门螺杆菌的研究具有极大的应用价值。
随着菌株变异、继发性耐药、交叉耐药以及不同菌株交叉感染等诸多因素的增加,幽门螺杆菌的根除率逐年下降。与此同时,在以抗生素治疗幽门螺杆菌感染时,不规范的抗生素滥用会对胃肠微生态***产生不良影响,破坏胃肠道微生态平衡及微生物屏障,使肠道中抗菌药物敏感菌株逐步减少,耐药菌株增加,并且抗生素还会导致部分患者出现诸多不良反应,如腹痛、恶心、呕吐、腹泻等,且发生呈现增高趋势。研究表明,益生菌具有抑制幽门螺杆菌的作用,但目前市面上的益生菌对于幽门螺杆菌的抑制效果不佳,因此,开发一种能有效抑制幽门螺杆菌、改善胃肠道不适,提高幽门螺杆菌感染临床治疗效果的益生菌很有必要。
发明内容
针对现有技术中的缺陷,本发明提供两歧双歧杆菌B11在抑制幽门螺杆菌、修复胃黏膜屏障中的应用,该菌株的活菌株或代谢产物能够抑制幽门螺旋杆菌的生长、粘附胃癌细胞、抑制幽门螺杆菌粘附胃癌细胞以及消除幽门螺杆菌引起的炎症,同时该菌株的活菌株还能够修复胃黏膜屏障,促进黏膜保护因子转录。
为达到上述发明目的,本发明采用了如下的技术方案:
保藏编号为CGMCC No.24381的两歧双歧杆菌B11在制备抑制幽门螺杆菌及修复胃黏膜屏障产品中的应用。该菌株已于2022年01月21日保藏在中国微生物菌种保藏管理委员会普通微生物菌种保藏中心,保藏地址为北京市朝阳区北辰西路1号院3号,中科院微生物研究所。
本发明提供的两歧双歧杆菌B11的代谢产物中的有机酸、细菌素等物质,对幽门螺杆菌有显著的抑制作用,其活菌株与胃癌细胞AGS存在强粘附作用,存在黏附于胃上皮细胞进而定植胃部的潜力,为两歧双歧杆菌B11通过竞争抑制来发挥抑制幽门螺杆菌的作用提供可能。经试验证明,两歧双歧杆菌B11 对于抑制幽门螺杆菌在胃上皮细胞的粘附的抑制率可达17.8%。同时,两歧双歧杆菌B11还能抑制促炎因子IL-8的转录水平,抑制幽门螺杆菌导致的炎症,从而对胃黏膜起到保护作用。该菌株的活菌株还能够促进黏膜保护因子三叶肽 (TFF1)、iNOS和COX-2的转录,对胃黏膜屏障和细胞修复有积极作用。
优选地,所述产品为抑制幽门螺杆菌的产品时,所述两歧双歧杆菌B11 为活菌株或其代谢产物。
优选地,所述产品为用于抑制幽门螺旋杆菌生长的产品。
优选地,所述产品为用于粘附胃癌细胞的产品。
优选地,所述产品为用于抑制幽门螺杆菌粘附胃癌细胞的产品。
优选地,所述产品为用于消除幽门螺杆菌引起的炎症的产品。
优选地,所述产品为修复胃黏膜屏障产品时,所述两歧双歧杆菌B11为活菌株。
优选地,所述产品为用于促进黏膜保护因子转录的产品。
优选地,所述黏膜保护因子包括TFF1、iNOS和COX-2中的至少一种。
以上抑制幽门螺旋杆菌及修复胃黏膜屏障的产品包括药品、保健食品或食品,药品、保健食品的剂型包括但不限于粉剂、片剂、胶囊剂、颗粒剂或溶液剂等常规剂型,食品的形式包括但不限于饮料、冷制糕点、乳制品等。
附图说明
图1为实施例1提供的两歧双歧杆菌B11对幽门螺杆菌的抑制作用;
图2为实施例2提供的两歧双歧杆菌B11对胃癌细胞AGS的粘附作用;
图3为实施例3提供的两歧双歧杆菌B11抑制幽门螺杆菌对胃癌细胞的粘附情况;
图4为实施例4提供的两歧双歧杆菌B11抑制幽门螺杆菌Hp介导IL-8的分泌的情况;
图5为实施例5提供的两歧双歧杆菌B11对黏膜保护因子TFF1转录的影响;
图6为实施例6提供的两歧双歧杆菌B11对促进黏膜保护因子一氧化氮合酶iNOS和环氧合酶COX-2的转录的影响。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合实施例,对本发明进行进一步详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。
目前市面上的益生菌对于幽门螺杆菌的抑制效果不佳,本发明提供的保藏编号为CGMCC No.24381的两歧双歧杆菌B11,具有抑制幽门螺杆菌、修复胃黏膜屏障中的作用。第一方面,两歧双歧杆菌B11对幽门螺杆菌的生长有显著的抑制作用,且与胃癌细胞AGS存在强粘附作用,具备在胃部定植的潜力,能够通过竞争抑制来抑制幽门螺杆菌在胃上皮细胞的粘附,同时两歧双歧杆菌B11可抑制促炎因子IL-8的转录水平,抑制幽门螺杆菌导致的炎症,对胃黏膜起到保护作用。第二方面,两歧双歧杆菌B11的活菌株还能够促进黏膜保护因子TFF1、iNOS和COX-2的转录,对胃黏膜屏障和细胞修复有积极作用。
以下实施例中使用的改良MRS培养基的配方为:葡萄糖20g,蛋白胨10g,牛肉粉6.5g,酵母浸粉5g,乙酸钠5g,柠檬酸氢二铵2g,磷酸氢二钾2g, MgSO4·7H2O 0.58g,MnSO4·H2O 0.25g,半胱氨酸盐酸盐0.5g,吐温80 1ml,蒸馏水1000mL。
实施例1
本实施例提供了两歧双歧杆菌B11对幽门螺杆菌的抑制作用。
将两歧双歧杆菌B11活化3代,接种于改良MRS培养基中,37℃厌氧培养18h后,调整菌液密度为1.0×108CFU/mL左右,4℃、5000r/min离心5min,收集培养上清液,用0.22μm的滤膜过滤除菌,备用。
通过牛津杯法进行抑菌试验,将固体培养基倒入平板中,凝固之后将活化三代稳定传代的幽门螺杆菌用无菌PBS悬浮,菌浓度约(1×108),加1ml到平板上,晃匀,静置3分钟,晾干。将牛津杯放在平板上,在孔中加入100μ L上述过滤的两歧双歧杆菌B11培养上清液,在厌有氧环境下于37℃培养18h,观察并测量抑菌圈直径大小,改良MRS培养基作为阴性对照。
结果如图1所示,阴性对照的抑菌直径为10.5±1.2mm,两歧双歧杆菌 B11的抑菌直径为14.8±1.7mm。两歧双歧杆菌B11的代谢产物中的有机酸、细菌素等物质,对幽门螺杆菌有显著的抑制作用。有机酸乳酸能够改变革兰氏阴性菌细胞膜的渗透性,进而提升抑菌效果。由于代谢产物中的乳酸含量与其对幽门螺杆菌的抑制程度正相关,从图中的抑菌直径的大小可知,两歧双歧杆菌B11的培养上清液对幽门螺杆菌生长有显著的抑制作用。
实施例2
本实施例提供了两歧双歧杆菌B11对胃癌细胞AGS的粘附作用。
将连续三代稳定培养的贴壁胃癌细胞AGS用胰酶消化后终止反应,离心洗涤后用无抗性的F12(胎牛血清10%)培养液悬浮处理,调整细胞液浓度达到5×104个/mL),进行爬片处理,静置过夜培养。待细胞铺满玻片,用PBS 缓冲液清洗载玻片3次;将两歧双歧杆菌B11活化3代,接种于改良MRS培养基中,37℃厌氧培养18h后,调整菌液密度为1.0×108CFU/mL左右,4℃、 5000r/min离心收集菌体,用无抗性的F12(胎牛血清10%)培养基调整菌液浓度至1×108个/mL,将调整后的菌液浸没上述铺满胃癌细胞AGS的玻片培养3小时,再用PBS洗涤三次,利用甲醇固定液固定2h,革兰氏染色后镜检。
结果如图2所示,两歧双歧杆菌B11与胃癌细胞AGS存在强粘附作用,证明两歧双歧杆菌B11在胃部有定植的潜力。幽门螺杆菌主要定植于胃上皮细胞表面及其***黏液层,两歧双歧杆菌B11在胃部定植就能够通过竞争抑制而发挥对幽门螺杆菌的抑制作用。
实施例3
本实施例提供了两歧双歧杆菌B11在抑制幽门螺杆菌对胃癌细胞粘附的应用。
将连续三代稳定培养的贴壁胃癌细胞AGS,用胰酶消化后终止反应,离心洗涤后用无抗性的F12(胎牛血清10%)培养液悬浮处理,调整细胞液浓度达到5×104个/mL),每孔100μL接种于96孔板,37℃静置过夜培养。待细胞铺满孔板的底部,用PBS缓冲液清洗3次,设置下列不同组与胃癌细胞 AGS共孵育。
对照组:将活化三代的幽门螺杆菌,用无抗性的F12(胎牛血清10%)培养基调整菌液浓度1×108个/mL,加入100μL后再加入100μL改良MRS培养基作为对照组;
试验组:将两歧双歧杆菌B11活化3代,接种于改良MRS培养基中,37℃厌氧培养18h后,调整菌液密度为1.0×108CFU/mL左右,4℃、5000r/min离心收集菌体,用F12(胎牛血清10%)培养基调整菌液浓度1×108个/mL,将两歧双歧杆菌B11与幽门螺杆菌悬液按1:1的体积比混合,共加入200μL作为试验组;
将对照组和试验组样品置于细胞培养箱中,在37℃下培养3h,再用PBS 洗涤三次,加入50μL尿素酶测试液,振荡后用550nm的酶标仪测定吸光值。结果如图3所示,两歧双歧杆菌B11能够通过与胃癌细胞AGS的粘附作用定植于胃部,抑制幽门螺杆菌在胃上皮细胞的粘附,抑制率可达17.8%,可见,两歧双歧杆菌B11能够与幽门螺杆菌竞争细胞上的粘附位点抑制幽门螺杆菌的数量。
实施例4
本实施例提供了两歧双歧杆菌B11在抑制幽门螺杆菌Hp介导IL-8的分泌的应用。
将连续三代稳定培养得到的胃癌细胞AGS接种于24孔板(接种浓度为5 ×104个/mL,培养体积200μL/孔),37℃培养24h,用无抗性的F12(胎牛血清10%)培养基洗去死亡的AGS细胞,设置下列不同组与胃癌细胞AGS共孵育。
空白对照组(AGS):加入200μL/孔的无抗性的F12(胎牛血清10%) 培养基;
AGS+Hp组:加入180μL的无抗性的F12(胎牛血清10%)培养基和 20μL的幽门螺杆菌菌悬液(菌体终浓度1×108个/mL);
AGS+Hp+B11组:加入160μL的无抗性的F12(胎牛血清10%)培养基、 20μL的幽门螺杆菌菌悬液(菌体终浓度1×108个/mL)和20μL的两歧双歧杆菌B11菌悬液(菌体终浓度1×108个/mL);
AGS+B11组:加入体积180μL的无抗性的F12(胎牛血清10%)培养基、 20μL的两歧双歧杆菌B11(菌体终浓度1×108个/mL);
静置于培养箱37℃共孵育4h。收集菌液,将菌液经8000g离心10min,收集沉淀,提取RNA反转cDNA进行检测,根据2-△△Ct值计算IL-8的转录水平。
结果如图4所示,幽门螺杆菌感染人体后可刺激胃粘膜上皮细胞生成种类多样的细胞因子,会引起IL-8产量增加,进而诱发和促进一系列炎症反应。两歧双歧杆菌B11可抑制促炎因子IL-8的转录水平,抑制幽门螺杆菌导致的炎症,对胃黏膜起到保护作用。
实施例5
本实施例提供了两歧双歧杆菌B11在促进黏膜保护因子三叶肽(TFF1) 转录的应用
1、制备细胞悬液:
酶解:用胰蛋白酶消化单层培养胃癌细胞AGS,收集悬浮培养细胞,制成单个细胞悬液,细胞密度为104个/mL。
加样:吸取上述细胞悬液40μL与40μL的0.4%台盼蓝溶液,吹吸混匀染色2min,将盖玻片盖在计数板两槽中间,用吸管轻轻吹打细胞悬液,吸取 20μL细胞悬液,在计数板上盖玻片一侧加细胞悬液,加样量不要溢出盖玻片。
计数:在显微镜下,用10×物镜观察计数板四角大方格中的细胞数,细胞压中线时,只计左侧和上方者,不计右侧和下方者。
计算:将计算结果代入下式,得出细胞密度。
细胞数/毫升原液=(4大格细胞数之和/4)×104×稀释倍数
2、细胞增殖试验
根据合适的铺板细胞数,取纯化的细胞接种到96孔板中,密度为5×104个/mL,每孔约100μL细胞悬液,每组可设置5个重复孔,在37℃培养箱中培养。
细胞接种后贴壁培养16h左右,镜检观察每孔的细胞生长状态,生长密度接近90%时,实验孔每孔加入10μL密度为1×108个/mL的两歧双歧杆菌B11 菌悬液,对照孔每孔加入等量的无抗性的F12(胎牛血清10%)培养基,37℃培养箱中培养4h。另取未接种AGS细胞的96孔板,实验孔每孔加入10μL密度为1×108个/mL的两歧双歧杆菌B11菌悬液,空白孔每孔加入等量的无抗性的F12(胎牛血清10%)培养基,37℃培养箱中培养4h。
每组去除原有培养基更换新培养基,然后每孔加入10μL的CCK-8溶液,终浓度10wt%,轻轻敲击培养板以帮助混匀。放于细胞培养箱内培养1h,用双波长进行测定检测结果,检测波长450nm,参比波长600nm。最终用检测波长数值减去参比波长数值,参考计算公式计算AGS细胞存活率。计算公式:
细胞存活率=[(As-Ab1)/(Ac-Ab)]×100%;
式中,As表示实验孔(含有AGS细胞的培养基、CCK-8、两歧双歧杆菌 B11)的吸光度;
Ac表示对照孔(含有AGS细胞的培养基、CCK-8)的吸光度;
Ab表示未接种AGS细胞的空白孔(空白培养基、CCK-8)的吸光度;
Ab1表示未接种AGS细胞的实验孔(空白培养基、CCK-8、两歧双歧杆菌B11)的吸光度。
3、TFF1试验:
将连续三代稳定培养得到的胃癌细胞AGS接种于24孔板(接种浓度为5 ×104个/mL,培养体积200μL)培养24h,用无抗性的F12(胎牛血清10%) 培养基洗去死亡的AGS细胞,设置下列不同组与细胞共孵育,每组3个重复。
空白对照组(AGS):加入200μL的无抗性的F12(胎牛血清10%)培养基;
试验组(AGS+B11):加入180μL的无抗性的F12(胎牛血清10%)培养基和20μL的两歧双歧杆菌B11(菌体终浓度1×108个/mL);
静置于37℃培养箱共孵育4h。收集菌液,将菌液经8000g离心10min,收集沉淀,提取RNA反转cDNA进行检测,根据2-△△Ct值计算的转录水平。
胃黏膜部保护因子对胃黏膜保护或受损后修复至关重要。三叶草因子家族(trefoil factor family,TFF),包括TFF1、TFF2、TFF3三个成员,主要分泌于人体胃肠道组织粘液分泌细胞,其表达与粘蛋白MUC分泌细胞有关。TFFs的分泌可促进胃黏膜上皮细胞的移动,快速修复,防止炎症的发生。以上实验的结果如图5所示,两歧双歧杆菌B11可维持细胞活性,促进TFF1因子的转录,进而对胃黏膜细胞的修复有积极作用。
实施例6
本实施例提供了两歧双歧杆菌B11在促进黏膜保护因子一氧化氮合酶 iNOS和环氧合酶COX-2的转录的应用
将连续3代培养得到的胃癌细胞AGS接种于24孔板(接种浓度为5×104个/mL,培养体积200μL)培养24h,用无抗性的F12(胎牛血清10%)培养基洗去死亡的AGS细胞,设置下列不同组与细胞共孵育。
空白对照组(AGS):加入200μL的无抗性的F12(胎牛血清10%)培养基;
试验组(AGS+B11):加入体积180μL的无抗性的F12(胎牛血清10%) 培养基和20μL的两歧双歧杆菌B11菌悬液(菌体终浓度1×108个/mL);
静置于培养箱共孵育4h。收集菌液,将菌液经8000g离心10min,收集沉淀,提取RNA反转cDNA进行检测,根据2-△△Ct值,计算iNOS和COX-2的转录水平。
NO是由L-精氨酸(L-arg)在一氧化氮合酶(NOS)的催化下产生的。 NOS分为内皮型(eNOS)、神经型(nNOS)和诱导型(iNOS),eNOS主要存在于血管内皮细胞,其合成的NO主要用于调节和维持GMBF,并在抵御损伤因子、促进胃黏膜修复中起重要作用。
COX-2通过合成内源性PGE2起到修复胃黏膜、加快溃疡愈合的作用。 PGE2含量最多,生物活性强,起主要保护作用。研究证实,在大鼠的胃黏膜屏障受到破坏后,PGE2提供了胃细胞保护作用来对抗胃酸、乙醇、吲哚美辛或酸逆弥散引起的损害。
以上实验的结果如图6所示,两歧双歧杆菌B11可以促进黏膜保护因子 iNOS和COX-2的转录,表明两歧双歧杆菌B11能够对胃黏膜屏障和细胞修复有积极作用。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内所作的任何修改、等同替换或改进等,均应包含在本发明的保护范围之内。
Claims (9)
1.保藏编号为CGMCC No.24381的两歧双歧杆菌B11在制备抑制幽门螺杆菌及修复胃黏膜屏障产品中的应用。
2.如权利要求1所述的应用,其特征在于:所述产品为抑制幽门螺杆菌的产品时,所述两歧双歧杆菌B11为活菌株或其培养上清液。
3.如权利要求2所述的应用,其特征在于:所述产品为用于抑制幽门螺旋杆菌生长的产品。
4.如权利要求2所述的应用,其特征在于:所述产品为用于粘附胃癌细胞的产品。
5.如权利要求2所述的应用,其特征在于:所述产品为用于抑制幽门螺杆菌粘附胃癌细胞的产品。
6.如权利要求2所述的应用,其特征在于:所述产品为用于消除幽门螺杆菌引起的炎症的产品。
7.如权利要求1所述的应用,其特征在于:所述产品为修复胃黏膜屏障产品时,所述两歧双歧杆菌B11为活菌株。
8.如权利要求7所述的应用,其特征在于:所述产品为用于促进黏膜保护因子转录的产品。
9.如权利要求8所述的应用,其特征在于:所述黏膜保护因子包括TFF1、iNOS和COX-2中的至少一种。
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