CN113025530A - 一株缓解泻剂结肠的两歧双歧杆菌及其应用 - Google Patents
一株缓解泻剂结肠的两歧双歧杆菌及其应用 Download PDFInfo
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Abstract
本发明公开了一株缓解泻剂结肠的两歧双歧杆菌及其应用,属于微生物技术领域。本发明提供的两歧双歧杆菌CCFM1163能够缩短首粒黑便时间,提高粪便含水量,提高结肠组织中胶质细胞数量,提高结肠中胶质细胞源性神经营养因子GDNF表达量,提高结肠中乙酰胆碱酯酶AChE含量,提高粪便中丁酸含量,增加结肠中Cajal间质细胞数量,降低结肠中胃肠激素VIP含量,降低结肠中水通道蛋白AQP4和AQP8表达量,改善肠道健康状态。从根本上缓解泻剂结肠。本发明可应用于缓解泻剂结肠的功能食品及药品中,具有广阔的应用前景。
Description
技术领域
本发明涉及一株缓解泻剂结肠的两歧双歧杆菌及其应用,属于微生物技术领域。
背景技术
泻剂结肠是由于长期应用泻剂,损害结肠神经系统,导致肠神经退行性变化,出现胃肠动力障碍,对泻剂反应性下降,使病人对泻剂产生依赖性的一种状况,常见于饱受便秘困扰的中老年人。
在我国,慢性便秘的发病率高达20%,诸如番泻叶、大黄、芦荟等一系列刺激性泻药由于见效快、成本低饱受便秘患者青睐,患者因便秘需长期使用泻剂,随着病程的进展,泻剂的剂量与效果成反比,最终失去导泄作用,形成“泻剂结肠”,严重者甚至可发展为结肠黑变病。但消费者对于这类泻药的副作用了解甚少,且国内目前对于泻剂结肠的报道较少,很少引起消费者尤其是便秘患者的关注,因此,刺激性泻药仍是很多家庭出现便秘症状时的首要选择,长此以往,泻剂结肠患者将越来越多,而目前对于泻剂结肠的治疗并无有效手段,严重患者需进行结肠切除术来缓解症状。
益生菌作为缓解便秘的有效辅助治疗手段,具有良好的通便效果,但由于引发便秘的原因是多样的,益生菌对于不同类型的便秘作用效果不尽相同。而目前的研究中,还未将益生菌纳入治疗泻剂结肠的手段。因此,通过对益生菌进行缓解泻剂结肠的研究,将从根本上缓解泻剂结肠,造福便秘人群。
发明内容
针对当前对于泻剂结肠缺乏有效治疗手段的技术缺陷,本发明提供一株容易活化且生长期短的两歧双歧杆菌CCFM1163,该菌能够显著提高肠道蠕动能力,缩短首粒黑便时间,促进肠神经健康发育,提供相应的益生菌制剂或功能性食品,有效缓解泻剂结肠。
本发明提供了一株从甘肃永昌成年男子粪便中分离筛选出的两歧双歧杆菌(Bifidobacterium bifidum),于2021年1月29日保藏于广东省微生物菌种保藏中心,保藏编号为GDMCC No:61478,保藏地址为广州市先烈中路100号大院59号楼5楼广东省微生物研究所。
本发明还提供了含有所述两歧双歧杆菌CCFM1163细胞的组合物。
在一种实施方式中,所述组合物为发酵食品或膳食补充剂。
在一种实施方式中,所述发酵食品为发酵乳制品、豆制品以及果蔬制品。所述乳制品包括牛奶、羊奶、乳酪、奶油、含乳饮料或乳粉;所述豆制品包括大豆、豆乳、豆腐或豆乳粉;所述果蔬制品以包括白菜、圆白菜、萝卜、黄瓜、甜菜、豆角、苹果或杨梅制品中至少一种为原料制得的果蔬制品。
在一种实施方式中,所述组合物为膳食补充剂。
在一种实施方式中,所述膳食补充剂为包含所述两歧双歧杆菌CCFM1163的粉剂、胶囊、软糖或液体制剂。
本发明还提供所述两歧双歧杆菌在制备缓解慢传输型便秘的食品或药物中的应用
在一种实施方式中,所述慢传输型便秘包括但不限于泻剂结肠。
在一种实施方式中,所述泻剂结肠是由番泻叶或其提取物诱导引起的。
在一种实施方式中,所述缓解慢传输型便秘包括如下至少一种功能:
1)缩短泻剂结肠小鼠的首粒黑便时间,提高粪便含水量;
2)提高结肠组织中神经胶质细胞数量;
3)提高结肠中胶质细胞源性神经营养因子GDNF表达量;
4)提高结肠中乙酰胆碱酯酶AChE含量;
5)提高粪便中丁酸含量;
6)增加结肠组织中Cajal间质细胞数量;
7)降低结肠中胃肠激素VIP含量;
8)降低结肠中水通道蛋白AQP4和AQP8表达量。
在一种实施方式中,所述药物还含有药学上可接受的载体,包括医学上通常使用的填充剂、粘合剂、润湿剂、崩解剂、润滑剂、矫味剂中的一种或多种。
在一种实施方式中,所述药物的剂型是颗粒剂、胶囊剂、片剂、丸剂或口服液
本发明还提供一种含有所述两歧双歧杆菌CCFM1163的菌剂,所述菌剂为将含有两歧双歧杆菌CCFM1163的菌液干燥得到的粉剂。
在一种实施方式中,所述干燥是指真空冷冻干燥或其它菌液干燥工艺。
在一种实施方式中,所述菌剂中两歧双歧杆菌的细胞数量≥1×108CFU/g。
本发明有益效果
本发明的两歧双歧杆菌CCFM1163生长特性优良,能够显著缓解泻剂结肠小鼠的症状:能够缩短首粒黑便时间,提高粪便含水量,提高结肠组织中胶质细胞数量,提高结肠中胶质细胞源性神经营养因子GDNF表达量,提高结肠中乙酰胆碱酯酶AChE含量,提高粪便中丁酸含量,增加结肠中Cajal间质细胞数量,降低结肠中胃肠激素VIP含量,降低结肠中水通道蛋白AQP4和AQP8表达量,改善肠道健康状态。因此,本发明可以视为缓解或治疗泻剂结肠的成分,应用于缓解泻剂结肠的药物、保健品或发酵食品中,或作为现有药物(例如莫沙必利)的药物替代物,从而发挥其作用,具有广阔的应用前景。
生物材料保藏
一株两歧双歧杆菌(Bifidobacterium bifidum)CCFM1163,分类命名为Bifidobacterium bifidum,于2021年1月29日保藏于广东省微生物菌种保藏中心,保藏编号为GDMCC No:61478,保藏地址为广州市先烈中路100号大院59号楼5楼广东省微生物研究所。
附图说明
图1为不同组别的泻剂结肠小鼠症状(首粒黑便时间、粪便含水量、小肠推进率)的示意图
图2为不同组别的泻剂结肠小鼠结肠组织中胶质细胞特征蛋白S100 beta变化示意图;
图3为不同组别的泻剂结肠小鼠结肠组织中胶质细胞源性神经营养因子GDNF表达变化示意图;
图4为不同组别的泻剂结肠小鼠结肠组织中乙酰胆碱酯酶AChE变化示意图;
图5为不同组别的泻剂结肠小鼠粪便中丁酸含量示意图;
图6为不同组别的泻剂结肠小鼠结肠组织中干细胞因子受体c-kit基因表达量变化示意图;
图7为不同组别的泻剂结肠小鼠结肠中胃肠激素VIP变化示意图;
图8为不同组别的泻剂结肠小鼠结肠组织中水通道蛋白AQP4和AQP8转录水平变化示意图;
注:柱形图上方符号表示数据显著性水平,*表示p<0.05,**表示p<0.01,***表示p<0.001,****表示p<0.0001(与model组比较),#表示p<0.05,##表示p<0.01,###表示p<0.001,####表示p<0.0001(与normal组比较)。
具体实施方式
实施例1两歧双歧杆菌CCFM1163的筛选、鉴定和培养
(一)菌株的分离筛选:
(1)使用一次性无菌取便器采集甘肃永昌成年男子的粪便,将粪便样品涂布在MRS+质量百分数0.08%半胱氨酸的培养基中,于厌氧培养箱(N2:CO2:H2=80:10:10)中富集12h;
(2)将粪便样品用无菌生理盐水进行梯度稀释后涂布于添加了无菌的100μg/mL莫匹罗星、50U/mL制霉菌素的MRS+质量百分数0.08%L-半胱氨酸盐酸盐的固体平板上,培养24-48h;
(3)选取符合双歧杆菌基本形态的单菌落进行平板划线纯化,筛选分离出所选菌株;
(4)将上述单菌落培养于液体MRS+质量百分数0.08%半胱氨酸培养液中培养24h后进行革兰氏染色,选取革兰氏阳性菌进行后续试验。
(二)双歧杆菌的初步鉴定:果糖-6-磷酸盐磷酸酮酶测定法
(1)将步骤(一)所筛选得到的乳酸菌在液体MRS+质量百分数0.08%半胱氨酸培养液中培养24h,然后取1mL培养物8000rpm离心2min;
(2)用含0.08%(质量百分数)半胱氨酸的pH6.5的0.05M KH2PO4溶液洗涤两次;
(3)重悬于200μL添加了0.25%(质量百分数)Triton X-100的上述磷酸盐缓冲液;
(4)添加50μL浓度为6mg/mL氟化钠和10mg/mL碘乙酸钠的混合液以及50μL浓度为80mg/mL的果糖-6-磷酸,37℃孵育1h;
(5)添加300μL浓度为0.139g/mL、pH 6.5的盐酸轻胺,并于室温放置10min;
(6)分别添加200μL 15%(质量百分数)的三氯乙酸和4M HCl;
(7)添加200μL含有5%(质量百分数)三氯化铁的0.1M HCl,若体系迅速变为红色,即为F6PPK阳性,可初步断定其为双歧杆菌。
(三)双歧杆菌的分子生物学鉴定
(1)取步骤(二)筛选出并且活化3代的菌体(培养12-48h)1mL用于菌种鉴定,6000r/min离心3min,弃上清得菌体。
(2)加入1mL无菌水吹打洗菌体后,10000r/min离心1min,弃上清得菌体,加入500μL无菌水重悬,作为菌液模板。
(3)16S rDNA PCR体系:
A.细菌16S rDNA,20μLPCR反应体系:
27F,0.5μL;1492R,0.5μL;Taq酶,1μL;模板,1μL;ddH20,8μL。
B.PCR条件:
94℃ 5min;94℃ 30s;55℃ 30s;72℃ 2min;72℃ 10min;step2-4 30×;12℃2min。
(3)制备1%琼脂糖凝胶,之后将PCR产物与10000×Loading buffer混合,上样量2μL,120V跑30min,然后进行凝胶成像;
(4)将16S rDNA的PCR产物进行测序分析,并将得到的序列结果使用BLAST在GenBank中进行搜索和相似性比对,选取测序结果鉴定为两歧双歧杆菌(Bifidobacteriumbifidum)的菌株,-80℃保藏备用。
所述两歧双歧杆菌具有以下生物学特性:
(1)菌体特征:革兰氏阳性,细胞球状,直径0.8~1.0μm,无鞭毛,无芽孢;
(2)菌落特征:菌落乳白色,边缘整齐,球状,凸起,不透明,表面湿润光滑;
(3)生长特性:该菌株的最低生长温度为15℃,最高生长温度为45℃,在温度35-37℃下生长最佳,最适生长pH为6.5,培养16h后进入对数后期或稳定期前期;
(四)两歧双歧杆菌菌悬液的制备
将活化3代后的菌液以2%的接种量接种至1L液体MRS培养基中,振荡混匀后于厌氧培养箱中37℃培养24h。在8000g/min,4℃的条件下离心15min,去上清后,用含0.05%-0.1%L-半胱氨酸盐酸盐的无菌生理盐水清洗2次,同样以相同条件进行离心,去上清后,用10%脱脂乳进行保存,-80℃冰箱冻存一周。在进行动物实验前,将菌液复温,震荡均匀后用平板倾注法测定初始和冻存一周后的活菌数量。MRS培养基的配方为:牛肉膏10g;胰蛋白胨10g;酵母粉5g;葡萄糖20g;无水乙酸钠5g;MgSO4·7H2O 0.1g;MnSO4·H2O 0.05g;柠檬酸氢二铵2g;K2HPO4·3H2O 2.6g;吐温80 1mL;另添加L-半胱氨酸盐酸盐0.8g;调节pH为6.8±0.2;定容至1L。高压灭菌115℃20min。
冻存一周后数量级并没有明显变化,说明将菌液冻存后的菌体活力变化微小,不会对实验产生明显影响,可用于动物实验。
可选地,还可将菌液冻干,制备获得干粉状菌剂。
实施例2:两歧双歧杆菌CCFM1163对番泻叶致泻剂结肠小鼠的缓解效果
将两歧双歧杆菌CCFM1163菌种于-80℃冰箱取出后,划线于MRS平板中,37℃培养24h,挑取单菌落于MRS液体管中,37℃培养20h,以2%的体积量接种于新的MRS液体培养基中,于37℃培养16h,按照同样的方式再次培养一代,然后将两歧双歧杆菌悬液在6000r/min、4℃条件下离心5min,然后用10%(m/v)的脱脂乳(将10g脱脂乳粉溶解于100mL水中配制而成)进行重悬,制得菌浓为5×109CFU/mL的菌悬液,用于动物实验。
取7周龄的健康雄性C57BL/6J小鼠24只,适应环境饲养1周,随机分为4组:对照组(normal)、模型组(model)、莫沙必利组(mosapride)和两歧双歧杆菌CCFM1163干预组(CCFM1163),每组含小鼠6只,每天早上9点开始灌胃,每次0.2mL。实验动物分组及处理方法见表1。
表1实验动物分组及处理方法
第16周灌胃结束后,将小鼠单只放入垫有吸水纸的笼盒中,收集粪便,称重即为湿重,冻干后,即为干重,按照如下公式计算粪便含水量。
粪便含水量=(粪便湿重-粪便干重)/粪便湿重×100%
分别向每只小鼠灌胃0.2mL墨汁,从灌胃开始,记录每只小鼠排首粒黑便时间。
小鼠处死之前,每只小鼠灌胃0.2mL墨汁,30min后处死解剖,剪取上端自幽门下端至盲肠,测量小肠全长为“小肠总长度”,从幽门到墨汁前沿为“墨汁推进长度”,按照以下公式计算小肠推进率。
小肠推进率=(墨汁推进长度(cm))/(小肠总长度(cm))×100%
首粒黑便时间、粪便含水量、小肠推进率结果如图1所示,由图可知,相比于对照组,造模后model组小鼠首粒黑便时间延长至normal组的1.83倍(p<0.001),粪便含水量由52.88%下降至45.5%(p<0.01),而小肠推进率无显著变化,即模型小鼠存在结肠转运障碍,泻剂结肠造模成功。相较于泻剂结肠模型组,灌胃两歧双歧杆菌CCFM1163处理可将泻剂结肠小鼠的首粒黑便时间缩短至model组的58.18%(p<0.001),粪便含水量提高了19.93%(p<0.001),增强泻剂结肠小鼠的结肠蠕动能力,且效果优于药物莫沙必利mosapride组(p<0.01)。说明灌胃两歧双歧杆菌CCFM1163可以显著增强泻剂结肠小鼠的结肠转运能力,缓解泻剂结肠小鼠的症状。
实施例3:两歧双歧杆菌CCFM1163提高泻剂结肠小鼠结肠组织中胶质细胞数量
C57BL/6J小鼠分组、造模及处理方法同实施例2。采用免疫荧光标记胶质细胞特征性蛋白S100 beta来定量肠神经中胶质细胞的数量。具体方法如下:
小鼠处死时去新鲜结肠组织用生理盐水冲洗干净,立即投入中性缓冲多聚甲醛固定液中固定24h,流水冲洗过夜,样品依次经70%、80%、90%各级乙醇溶液脱水,各30min,再放入95%、100%各2次,每次20min。样品一次经过1/2纯酒精,1/2二甲苯等量混合液15min,二甲苯Ⅰ15min、Ⅱ15min至透明,放入二甲苯和石蜡各半的混合液15min,再放入石蜡Ⅰ、石蜡Ⅱ透蜡各50-60分钟。以结肠横截面在底层,使用徕卡手轮式切片机切片。将切片连同切片架放入60℃的干燥箱,30~60min至蜡熔化,将切片用二甲苯浸泡10min,更换二甲苯后再浸泡10min脱蜡,样品依次经过100%,95%,85%,75%乙醇,ddH2O水化,投入柠檬酸钠缓冲液(10mM柠檬酸钠,0.05%Tween 20,pH 6.0)煮沸20min抗原修复,TBS洗涤3次,每张切片滴加3%BSA-PBST 50μl,湿盒中室温封闭30分钟封闭,弃去封闭液,每张切片滴加50μl一抗(Anti-S100 beta抗体,Abcam),4℃湿盒中孵育过夜,次日取出切片,室温复温60分钟。TBS浸泡5分钟,清洗4次最后除去组织上的液体,每张切片滴加荧光标记的二抗50μl避光孵育45-60min,TBS浸泡5分钟,清洗4次,每张切片滴加50μl DAPI复染细胞核,抗荧光淬灭封片液封片,指甲油密封盖玻片。使用数字切片扫描仪扫片,软件Image J分析,结果如图2所示。
黏膜的胶质细胞主要参与上皮屏障功能,神经节内的胶质细胞主要作用于神经修复,与神经元的密切互动,支持这些细胞和胶质细胞的分化、参与神经的发生和形成。泻剂结肠区别于普通便秘最明显的特征即神经受损严重,胶质细胞的数量可以很好地反应肠神经的健康状况。由图2可知,泻剂结肠小鼠S100 beta阳性面积减少了46.81%,说明肠胶质细胞明显减少(p<0.01),肠神经受损,灌胃CCFM1163后,S100 beta阳性表达面积较模型组提高了76.50%,说明胶质细胞数量明显增多(p<0.01),肠神经重建。可知灌胃CCFM1163可恢复肠神经健康,从而促进结肠蠕动,提高结肠动力,缓解泻剂结肠。
实施例4:两歧双歧杆菌CCFM1163提高泻剂结肠小鼠结肠组织中GDNF水平
C57BL/6J小鼠分组、造模及处理方法同实施例2。采用ELISA方法对结肠组织中GDNF表达量进行定量。具体方法如下:
用预冷的PBS冲洗结肠组织,去除残留血液,剔除周围脂肪组织,称重后将其剪碎,将剪碎的组织与PBS溶液按照1:9的重量体积比在高通量组织破碎仪上进行破碎,匀浆,再于5000×g离心5-10分钟,取上清检测,参照相应试剂盒说明书进行实验,根据标准曲线计算中组织中GDNF。结果如图3所示。
GDNF是一种多肽类的神经营养因子,对损伤的神经细胞有营养和保护作用,在退变性神经元中表达降低,提示神经元损伤。由图3可知,泻剂结肠小鼠GDNF水平较normal组降低了39.82%(p<0.001),提示存在肠神经受损,灌胃CCFM1163后GDNF水平显著提高至model组的1.86倍至4600pg/mL左右,与药物对照莫沙必利mosapride组相比多提高了31.81%(p<0.001),说明CCFM1163处理可以滋养肠神经,恢复正常的肠神经功能,从而缓解泻剂结肠。
实施例5:两歧双歧杆菌CCFM1163提高泻剂结肠小鼠结肠组织中AChE水平
C57BL/6J小鼠分组、造模及处理方法同实施例2。采用ELISA方法对结肠组织中乙酰胆碱酯酶AChE表达量进行定量。具体方法同实施例4,结果如图4所示。
乙酰胆碱酯酶AChE是生物神经传导中的一种关键性酶,存在于胆碱能突触间,该酶能降解乙酰胆碱,终止神经递质对突触后膜的兴奋作用,保证神经信号在生物体内的正常传递。此外,乙酰胆碱酯酶参与细胞的发育和成熟,能促进神经元发育和神经再生。由图4可知,泻剂结肠小鼠结肠中乙酰胆碱酯酶AChE水平较normal组明显下降了24%(p<0.01),无法及时分解神经递质,干扰神经信号传递,破坏肠道正常的蠕动机制,灌胃CCFM1163后,AChE水平较model组提高了48%(p<0.001),比药物作用效果提高9%左右,促进受损肠神经的发育和再生,恢复正常的肠神经功能,从而缓解泻剂结肠。
实施例6:两歧双歧杆菌CCFM1163提高泻剂结肠小鼠粪便中丁酸含量
C57BL/6J小鼠分组、造模及处理方法同实施例2。将实验结束前所收集的测定粪便含水量的小鼠粪便冻存于-80℃。具体方法如下,称取20mg冻干后粪便,用500μL饱和NaCl溶液重悬,加入20μL 10%H2SO4溶液;加入1000μL无水乙醚,震荡均匀,提取脂肪酸,然后12000rpm 4℃离心15min;取上层乙醚相,加入0.25g无水Na2SO4进行干燥;静置30min后12000rpm 4℃离心5min取上层乙醚相,利用GC-MS测定小鼠冻干粪便中的短链脂肪酸含量。使用Rtx-Wax柱(柱长30m,内径25μm);载气为He,流速为2mL/min;进样体积1μL,按7.5℃/min升温至140℃,然后按60℃/min升温至200℃保持3min,离子化温度为20℃;分析采用全扫描模式,为通过外标法测得标准曲线,从而计算出丁酸的浓度。结果如图6所示。
丁酸是维持结肠上皮的重要底物,可诱导健康的结肠细胞增殖,促进黏蛋白分泌和紧密连接蛋白重新分布,维持正常的结肠屏障功能,且丁酸可以作为结肠的能量物质为结肠提供动力。由图5可知,泻剂结肠模型小鼠粪便中丁酸含量较normal组降低至原来的19%左右(p<0.01),灌胃CCFM1163后,粪便中丁酸含量显著提高至model组的12.89倍(p<0.0001),而药物对照莫沙必利仅仅提高了36%,因此,CCFM1163干预后可显著提高结肠能量物质丁酸的水平,为结肠提供动力,增强结肠蠕动能力,从而缓解泻剂结肠。
实施例7:两歧双歧杆菌CCFM1163提高泻剂结肠小鼠结肠组织中Cajal间质细胞数量
C57BL/6J小鼠分组、造模及处理方法同实施例2。采用实时荧光定量聚合酶链反应(RT-qPCR)来测定MUC-2基因的表达量,首先去要从新鲜结肠组织中提取RNA,具体方法如下:
将小鼠解剖后取出的新鲜结肠组织0.2g在加有液氮的研钵(180℃,4h高温灭酶)中反复研磨,再向研钵中加入1mL Trizol试剂,继续研磨,待液体基本澄清后,收集至1.5mL无酶离心管中,室温静置15min,向离心管中加入200μL三氯甲烷溶液,轻摇15s,室温静置10min,4℃、12000r/min离心15min,取600μL上层无色水相至另一只无酶离心管中,加入500μL异丙醇。上下颠倒混匀,室温下静置10min,静置结束后,4℃、12000r/min离心10min,弃去上清,留下RNA在离心管底部形成的白色沉淀,加入1mL用DEPC水配制的75%的乙醇溶液,漩涡震荡重悬,4℃、7500r/min离心5min,弃去上清,室温自然挥发干燥。向干燥的RNA中加入30μL RNase free water,待RNA溶解后,以Nanodrop测定RNA浓度及纯度,并通过琼脂糖凝胶电泳检测RNA的质量。以提取的总RNA为模板,根据诺唯赞生物科技股份有限公司的HiScript II Q Select RT SuperMix for qPCR反转录试剂盒说明书操作步骤逆转录合成cDNA,-20℃下保存。
小鼠c-kit基因和内参基因mGAPDH基因引物如表2所示,
表2小鼠c-kit蛋白基因和mGAPDH基因引物序列
qRT-PCR反应体系为:
qRT-PCR反应条件为:95℃ 30s;95℃ 10s,60℃ 30s,共40个循环。
以mGAPDH基因作为内参基因,通过CFX96Manager软件分析结果。实验结果如图6所示。
Cajal间质细胞(ICC)是肠道的起搏细胞,ICC通过与受体c-kit结合发挥作用,开启信号通路,维持ICC的生长与发育,其数量可以用c-kit的表达量来表示。由图6可知,造模后,泻剂结肠小鼠c-kit表达量显著降低至normal组的47.34%(p<0.01),表明泻剂结肠小鼠胃肠动力不足,灌胃CCFM1163后,泻剂结肠小鼠c-kit蛋白表达量较model组显著提高了85.28%(p<0.05),效果优于药物莫沙必利(仅提高36.83%)。因此,两歧双歧杆菌CCFM1163可以显著提高泻剂结肠小鼠结肠中c-kit表达量,增加Cajal间质细胞数量,从而提高结肠动力,缓解泻剂结肠。
实施例8:两歧双歧杆菌CCFM1163降低泻剂结肠小鼠结肠组织中神经肽VIP水平
C57BL/6J小鼠分组、造模及处理方法同实施例2。采用ELISA方法对结肠组织中血管活性肠肽VIP和胃泌素Gas表达量进行定量。具体方法同实施例4,结果如图7所示。
血管活性肠肽VIP在消化系统的主要作用是舒张平滑肌,维持正常的肠道节律性蠕动,并促进水分吸收;由图7可知,泻剂结肠小鼠结肠中VIP水平较normal组明显升高了14%(p<0.01),提示小鼠肠道蠕动紊乱,水分吸收过高,肠道正常功能受损。灌胃CCFM1163后,VIP水平较model组明显降低11%(p<0.01),说明灌胃CCFM1163可降低泻剂结肠小鼠结肠中VIP水平,维持正常的肠道蠕动,并减少水分过度吸收,提高粪便含水量,从而缓解泻剂结肠。
实施例9:两歧双歧杆菌CCFM1163降低泻剂结肠小鼠结肠组织中水通道蛋白AQP4和AQP8水平
C57BL/6J小鼠分组、造模及处理方法同实施例2。采用实时荧光定量聚合酶链反应(qRT-PCR)来测定AQP4和AQP8基因的表达量,具体方法同实施例7。
小鼠AQP4、AQP8基因和内参基因mGAPDH基因引物如表3所示
表3小鼠AQP4、AQP8蛋白基因和mGAPDH基因引物序列
以mGAPDH基因作为内参基因,通过CFX96Manager软件分析结果。实验结果如图8所示。
水通道蛋白是调节肠道水液平衡的关键蛋白,AQP4和AQP8在结肠的表达与水分吸收相关。由图8可知,造模后,泻剂结肠小鼠AQP4和AQP8表达量显著提高至1.42和1.50(p<0.0001,p<0.01),表明泻剂结肠小鼠存在水液调节失衡,肠腔过度失水导致粪便干结,从而影响肠道蠕动,灌胃CCFM1163后,泻剂结肠小鼠AQP4和AQP8蛋白表达量显著降低model组的60.38%和56.30%(p<0.001,p<0.001)。因此,两歧双歧杆菌CCFM1163可以显著降低泻剂结肠小鼠结肠中AQP4表达量,恢复结肠水液平衡,提高粪便含水量,使粪便更容易通过结肠排出,从而缓解泻剂结肠。
实施例10:利用本发明两歧双歧杆菌CCFM1163制造含该菌的发酵食品
将牛乳高温瞬时灭菌,立即降温至37℃,再接入两歧双歧杆菌CCFM1163菌剂作为发酵剂进行发酵,使添加菌剂后的牛乳中两歧双歧杆菌CCFM1163浓度达到106CFU/mL以上,在温度4℃下冷藏保存,于是得到含有本发明两歧双歧杆菌CCFM1163活菌的发酵乳。
实施例11:利用本发明两歧双歧杆菌CCFM1163制造含该菌的发酵食品
利用本发明能够使用两歧双歧杆菌CCFM1163发酵生产制备其他发酵食品,所述发酵食品包括固态食品、液态食品、半固态食品。所述发酵食品包括乳制品、豆制品、果蔬制品,所述乳制品包括牛奶、羊奶、乳酪、奶油、含乳饮料或乳粉;所述豆制品包括大豆、豆乳、豆腐、豆乳粉;所述果蔬制品包括以白菜、圆白菜、萝卜、黄瓜、甜菜、豆角、苹果或杨梅制品中至少一种为原料制得的果蔬制品。
将实施例10、实施例11制备的发酵食品用于泻剂结肠小鼠,结果显示,含两歧双歧杆菌CCFM1163的发酵食品也具备能够缩短首粒黑便时间,提高粪便含水量,提高结肠组织中胶质细胞数量,提高结肠中胶质细胞源性神经营养因子GDNF表达量,提高结肠中乙酰胆碱酯酶AChE含量,提高粪便中丁酸含量,增加结肠中Cajal间质细胞数量,降低结肠中胃肠激素VIP含量,降低结肠中水通道蛋白AQP4和AQP8表达量的作用,可改善泻剂结肠小鼠肠道健康状态。
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> 一株缓解泻剂结肠的两歧双歧杆菌及其应用
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Claims (10)
1.一株两歧双歧杆菌(Bifidobacterium bifidum)CCFM1163,其特征在于,于2021年1月29日保藏于广东省科学院微生物研究所,保藏编号为GDMCC No:61478。
2.含有权利要求1所述两歧双歧杆菌CCFM1163的组合物。
3.根据权利要求2所述的组合物,其特征在于,所述两歧双歧杆菌CCFM1163的细胞数量≥1×108CFU/g或1×108CFU/mL。
4.根据权利要求2所述的组合物,其特征在于,是药物、发酵食品或膳食补充剂。
5.根据权利要求4所述的组合物,其特征在于,所述膳食补充剂是包含两歧双歧杆菌CCFM1163的粉剂、胶囊、软糖或液体制剂。
6.权利要求1所述的两歧双歧杆菌CCFM1163在制备缓解慢传输型便秘的食品或药物中的应用。
7.根据权利要求6所述的应用,其特征在于,所述慢传输型便秘包括但不限于泻剂结肠。
8.根据权利要求7所述的应用,其特征在于,所述缓解慢传输型便秘包括如下至少一种功能:
1)缩短泻剂结肠小鼠的首粒黑便时间,提高粪便含水量;
2)提高结肠组织中神经胶质细胞数量;
3)提高结肠中胶质细胞源性神经营养因子GDNF表达量;
4)提高结肠中乙酰胆碱酯酶AChE含量;
5)提高粪便中丁酸含量;
6)增加结肠组织中Cajal间质细胞数量;
7)降低结肠中胃肠激素VIP含量;
8)降低结肠中水通道蛋白AQP4和AQP8表达量。
9.根据权利要求6所述的应用,其特征在于,所述药物还含有药学上可接受的载体,包括医学上通常使用的填充剂、粘合剂、润湿剂、崩解剂、润滑剂、矫味剂中的一种或多种。
10.含有权利要求1所述两歧双歧杆菌CCFM1163的微生物菌剂。
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