CN113943681A - 一株降低炎症反应且具有缓解便秘作用的长双歧杆菌 - Google Patents
一株降低炎症反应且具有缓解便秘作用的长双歧杆菌 Download PDFInfo
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- CN113943681A CN113943681A CN202111340203.1A CN202111340203A CN113943681A CN 113943681 A CN113943681 A CN 113943681A CN 202111340203 A CN202111340203 A CN 202111340203A CN 113943681 A CN113943681 A CN 113943681A
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Abstract
本发明公开了一株降低炎症反应且具有缓解便秘作用的长双歧杆菌,属于微生物领域。本发明提供的长双歧杆菌CCFM1112能够有效降低全身代谢循环以及结肠部位的炎症反应,改善便秘小鼠的首粒黑便时间、肠道推进率、粪便含水量以及结肠组织的病理损伤。对于生理指标,该菌株能够影响兴奋型和抑制型胃肠活性肽的含量,增加四种短链脂肪酸的含量,从根本上缓解便秘的症状。长双歧杆菌不仅在生理和病理两方面具有一定的可观效果,较泻药而言缺少一些副作用,还能提高芦丁在机体内的生物利用度有所提高,从而使芦丁能够更好地发挥抗炎、抗氧化的作用因此,本发明提供的长双歧杆菌菌株可以被广泛的添加到各种食品或药品中,从而得到更好的应用价值。
Description
技术领域
本发明涉及一株降低炎症反应且具有缓解便秘作用的长双歧杆菌,属于微生物领域。
背景技术
便秘是一种世界范围的常见胃肠疾病,其患病率高达15%。总的来说,非白种人的患病率要比白种人高,其次,男女比例大致为1:1.5,但是这种疾病无论是对各个年龄、种族、民族,还是对不同社会经济群体而言,都是一种影响十分广泛的疾病。
便秘的主要症状为排便不频繁,每周次数少于3次,大便紧张,有排不尽的感觉,需要手指辅助排便,腹胀,粪便干、硬、呈块状。便秘的分类有很多,便秘根据结肠运输和肛门直肠的功能评估来看,可分为正常运输型便秘、慢传输型便秘、以及盆底功能障碍三种,而根据有无器质性病变又可分为器质性便秘与功能性便秘。引发便秘的原因有很多,例如饮食的不均衡、微生物紊乱、药物的副作用、糖尿病等代谢性疾病所导致的并发症、遗传疾病、机械性肠梗阻、神经疾病等。正是由于引发便秘的原因有很多,便秘这种疾病才更应该引起大家的关注。
面对如此普遍的疾病,其治疗手段也各不相同。对于便秘患者,常鼓励他们多食用新鲜蔬菜、水果、豆类等膳食纤维含量丰富的食物;还可以使用渗透性或刺激性泻药,如聚乙二醇、乳果糖或蒽醌衍生物等,但是这类泻药常会引起依赖性,甚至恶心、腹痛、腹泻等副作用。因此,通过使用益生菌来缓解便秘的方法将会是一种可采取的重要手段。
引发便秘的一个重要原因就是肠道微生物群的紊乱,便秘患者粪便中的厚壁菌门丰度降低,而拟杆菌门的丰度升高。从属水平来看,一些潜在致病菌(肠球菌、梭杆菌等)丰度升高,而部分有益菌(双歧杆菌属、乳杆菌属等)丰度降低。因此,可以考虑利用双歧杆菌来缓解便秘的症状。
目前,针对双歧杆菌相关的生理功能已有国内外的学者进行研究,而其对便秘缓解作用的应用还应该得到更深刻的探究。无论是将其制成菌粉还是加入到发酵乳中进行食用,都有非常大的应用前景,能够预防便秘的发生甚至缓解便秘的症状。通过对双歧杆菌缓解便秘进行研究,将会对食品科学、微生物学以及预防医学等各方面产生巨大的影响,因此,双歧杆菌缓解便秘的研究是十分必要的。
发明内容
针对现有技术所面临的具有缓解便秘作用的双歧杆菌生长缓慢,不易活化,功能单一等缺陷,本发明的目的在于提供一株能够明显降低炎症反应并有效缓解便秘的长双歧杆菌,且对提高芦丁的抗氧化活性和生物利用度有一定作用,能够克服现有技术中存在的不足,提供相应益生菌制剂、功能性食品,从而预防便秘,或使便秘患者能逐步摆脱药物治疗的副作用与局限性。
本发明提供了一株从江苏省无锡市85岁男性老人粪便中分离筛选出的长双歧杆菌长亚种(Bifidobacterium longum subsp.longum)CCFM1112,于2019年12月30日保藏于广东省微生物菌种保藏中心,保藏地址为广州市先烈中路100号大院59号楼5楼,保藏编号为GDMCC NO.60939。
所述长双歧杆菌长亚种CCFM1112具有下述生物学特性:
(1)菌体特征:革兰氏阳性,无芽孢,菌体约0.6-1.2μm×1.5-7.6μm,存在多个分枝,具有Y、V型。
(2)菌落特征:直径约0.3-2.4mm,圆形且边缘整齐,凸面或透镜状,微白,不透明,表面湿润光滑。
(3)生长特性:该菌株最适生长温度为36-38℃,32-38℃生长良好,最低生长温度为15℃,也能够在45℃下进行生长;最适初始pH为6-7,pH 5.5或以下生长较少;培养20h后进入稳定期前期,最终培养基的pH为4.0-4.8。
(4)对模拟胃肠液具有较好的耐受能力。
(5)具有粘附性,能够较好的粘附在结肠癌细胞HT-29上。
(6)能显著提高便秘小鼠的粪便含水量、首粒黑便时间以及小肠推进率,降低体内炎症反应,调节血清中胃肠活性肽的含量,其缓解便秘的效果良好。
(7)能够增加芦丁在体外模拟消化液中的生物利用度以及抗氧化生物利用度。
本发明还提供一种发酵食品,所述发酵食品为使用长双歧杆菌长亚种CCFM1112发酵生产制得,所述发酵食品包括固态食品、液态食品、半固态食品。
在一种实施方式中,所述发酵食品包括乳制品、豆制品或果蔬制品。
在一种实施方式中,所述乳制品为发酵乳制品,包括发酵乳、风味发酵乳、发酵乳饮料、奶油、乳酪、含乳饮料或乳粉;所述豆制品包括豆奶、豆乳粉;所述果蔬制品包括以白菜、白萝卜、黄瓜、甜菜、黄桃或杨梅为原料发酵的发酵果蔬饮品或食品。
本发明还所述长双歧杆菌长亚种CCFM1112在制备缓解便秘的产品中的应用。
在一种实施方式中,所述产品为药物,所述药物包含长双歧杆菌长亚种CCFM1112和能够在药学上允许的载体。
在一种实施方式中,所述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,所述药物的剂型包括颗粒剂、胶囊剂、片剂、丸剂或口服液。
在一种实施方式中,所述药物含有长双歧杆菌长亚种CCFM1112和芦丁。
在一种实施方式中,所述药物具有如下至少一种作用:
(a)缓解便秘;
(b)改善结肠组织的病理损伤;
(c)影响兴奋型和抑制型胃肠活性肽的含量;
(d)增加乙酸、丙酸、丁酸、戊酸等短链脂肪酸的含量;
(e)增强抗氧化物质的生物利用度。
在一种实施方式中,所述抗氧化物质包括但不限于芦丁。
在一种实施方式中,所述产品为功能性食品或保健品。
本发明还提供含有所述长双歧杆菌长亚种CCFM1112的菌剂。
在一种实施方式中,所述菌剂中长双歧杆菌长亚种CCFM1112的数量≥108cfu/g或108cfu/mL。
在一种实施方式中,所述菌剂是将含有长双歧杆菌长亚种CCFM1112的菌液干燥得到的菌体数量大于108cfu/g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。
本发明还提供所述长双歧杆菌长亚种CCFM1112在食品或药品方面的应用,并且在缓解便秘方面有明显效果。
本发明有益效果:
本发明的长双歧杆菌CCFM1112生长特性较好,具有一定的耐酸碱性,能显著提高便秘小鼠的粪便含水量、首粒黑便时间以及小肠推进率,调节血清中胃肠活性肽的含量,降低体内炎症反应,增加芦丁的抗氧化活性与生物利用度,效果明显,并且同时避免一些泻药存在的副作用,因此,本发明可以视为一种缓解或治疗便秘的药物,还可以应用于药品或者是一些发酵食品中,从而广泛发挥其作用,具有非常有价值的应用前景。
生物材料保藏
一株长双歧杆菌长亚种(Bifidobacterium longum subsp.longum)CCFM1112,其分类学命名为(Bifidobacterium longum subsp.longum),已于2019年12月30日保藏于广东省微生物菌种保藏中心,保藏编号为GDMCC NO.60939,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为不同组小鼠缓解便秘相关指标(排首粒黑便时间、粪便含水量、小肠推进率)的示意图;
图2为不同组小鼠血清中生长抑素含量变化示意图;
图3为不同组小鼠血清中P物质含量变化示意图;
图4为不同组小鼠血清与结肠组织中白介素1β含量变化示意图;
图5为不同组小鼠结肠组织中肿瘤坏死因子α含量变化示意图。
图6为不同组小鼠粪便中短链脂肪酸含量变化示意图。
图7为不同组小鼠结肠组织切片病理变化示意图。
具体实施方式
所述长双歧杆菌CCFM1112的提取方法为:
(一)菌株的分离筛选:
(1)使用一次性无菌取便器采集江苏省无锡市85岁男性的粪便,将粪便样品在含有低聚果糖MRS+质量百分数(0.05%~0.1%)半胱氨酸培养基中,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(0.05%~0.1%)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(0.05%~0.1%)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(0.05%~0.1%)半胱氨酸培养液中培养24h,然后取1mL培养物8000rpm离心2min;
(2)用含质量百分数0.05%半胱氨酸的pH6.5的0.05M KH2PO4溶液洗涤两次;
(3)重悬于200μL添加了质量百分数0.25%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质量百分数15%的三氯乙酸和4M HCL;
(7)添加200μL含有质量百分数5%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代的培养12-48h的菌液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:
20μLPCR反应体系:
27F,0.5μL;1492R,0.5μL;Taq酶,1μL;基因组DNA,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,其序列结果为TTTGCAGAGCGGAGCGGGTCACCTTGACCGGGTCGGTCACACCGGCGGCCAGCAGGTCTTCGTAGGTGTCGGTGGCGGCGTTGAAGCCTTCGCCATCAGGCAGGGAGCGGACGGTGTTGATGACCACGTCACCGGACACGCCGGCGTTCTCGGCGATCTGCTTGATCGGGGCCTCGATGGCGCGGAACACGATGGCGGCACCGGTAGCCTCTTCGCCGGTCAGGGAGGTGACGGCCTCGGTCTTCTCGGCCTTGGCAGCAGCCTGAACGAGGGCCACGCCACCGCCAGGCAGCAGGCCTTCCTCGATGGCGGCCTTGGCGTTACGCACGGCATCTTCGATGCGGTGCTTGCGCTCCTTGGCCTCGACCTCGGTGGCAGCGCCGACCTTGATGACAGCCACGCCGCCAGCCAGCTTGGCCAGACGCTCCTGCAGCTTCTCACGACG(SEQ ID NO.1),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长双歧杆菌的菌株,-80℃保藏备用;
(四)长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,用无菌生理盐水(含0.05%-0.1%的L-半胱氨酸盐酸盐)进行清洗2次,同样以相同条件进行离心,去上清后,用30%的甘油进行保存,-80℃冰箱冻存一周。在进行动物实验前,将菌液以6000r/min离心5min,再用无菌生理盐水清洗两遍,用10%脱脂乳重悬菌液,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;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。
实验结果:初始活菌数为,1周后活菌数为,数量级并没有产生变化,说明将菌液冻存后不会对实验产生影响,可用于动物实验。
实施例1:长双歧杆菌CCFM1112对洛哌丁胺诱导产生便秘相关症状的缓解
将长双歧杆菌CCFM1112、两歧双歧杆菌CGMCC NO.13632(该菌株公开于公开号为CN106834187B的专利文件中)与长双歧杆菌CCFM642(该菌株公开于DOI:10.3389/fmicb.2019.01721的论文中)菌种于-80℃冰箱取出后,划线于MRS平板中,37℃培养48h,挑取单菌落于MRS液体管中,37℃培养24h,以2%的体积量接种于新的MRS液体培养基中,于37℃培养24h,按照同样的方式再次培养一代,然后将双歧杆菌菌悬液在6000r/min、4℃条件下离心5min,然后用10%的脱脂乳进行重悬,制得菌悬液,用于动物实验。
取6周龄的健康雄性Balb/c小鼠30只,适应环境1周,随机分为5组:对照组、模型组、长双歧杆菌CCFM642组、两歧双歧杆菌CGMCC NO.13632组、长双歧杆菌CCFM1112组,每组含小鼠6只,灌胃菌悬液的剂量为5×109CFU/mL,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
在第37天,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;
在第38天,空白对照组给予0.2mL生理盐水、模型组和灌菌组均给予0.2mL盐酸洛哌丁胺溶液(10mg/kg b.w),1h后,分别向每组灌胃墨汁,从灌胃墨汁开始,记录每一只小鼠排首粒黑便的时间。
第38天,各组小鼠禁食不禁水过夜。第39天上午9点空白对照组给予0.2mL生理盐水,模型组和灌菌组均给予0.2mL盐酸洛哌丁胺溶液(10mg/kg b.w),30min后,各组分别灌胃墨汁,30min后处死小鼠,打开腹腔,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
粪便含水量、排首粒黑便时间、小肠推进率实验结果如图1所示,由图可知,长双歧杆菌CCFM1112组相对于便秘模型组,灌胃长双歧杆菌CCFM1112能显著提高小肠推进率、提高粪便含水量,缩短排首粒黑便时间,且整体能力要优于两歧双歧杆菌CGMCC NO.13632,而长双歧杆菌CCFM642在这三个方面均无明显的改善作用。通过比较发现长双歧杆菌CCFM1112对小肠推进率的影响更为显著,它更大程度上会对便秘小鼠的小肠部分产生作用。综上所述,长双歧杆菌CCFM1112从表观指标上凸显出良好的缓解便秘的作用。
实施例2:长双歧杆菌CCFM1112可降低便秘小鼠血清中生长抑素(SS)的含量
Balb/c小鼠分组、造模及处理方法同实施例1。第39天处死小鼠后,将收集到的小鼠血液静置2h,3000g离心15min后获得血清,采用生长抑素(SS)检测试剂盒,根据说明书进行实验,由标准曲线计算出血清中SS的浓度。
有研究表明生长抑素对胃肠运动与消化道激素的分泌均有一定的抑制作用。实验结果如图2所示,由图2可知,长双歧杆菌CCFM1112组相比于模型组,能显著下调便秘小鼠血清SS的含量,而长双歧杆菌CCFM642处理的小鼠血清中SS的含量与模型组相比无显著差异。由实验结果可知,长双歧杆菌CCFM1112能通过下调血清中SS的含量加速胃肠道动力,刺激消化道激素的分泌,从而缓解肠道蠕动减慢的症状。
实施例3:长双歧杆菌CCFM1112可提高便秘小鼠血清中P物质(SP)的含量
SP作为一种神经肽,可以刺激肠道蠕动,使胆囊收缩,促进唾液分泌,同时对免疫功能有促进作用。
Balb/c小鼠分组、造模及处理方法同实施例1。第39天处死小鼠后,将收集到的小鼠血液静置2h,3000×g离心15min后获得血清,采用P物质(SP)检测试剂盒,根据说明书操作,由标准曲线计算出血清中SP的浓度。结果如图3所示,便秘模型小鼠与空白对照组相比,其血清中SP出现了显著降低的现象,由图3可知长双歧杆菌CCFM1112能显著提升便秘小鼠结肠组织中SP的含量至正常水平,且其效果要明显优于长双歧杆菌CCFM642及两歧双歧杆菌CGMCC NO.13632。这说明长双歧杆菌CCFM1112可以通过提高血清中SP的含量提高结肠动力。
实施例4:长双歧杆菌CCFM1112可降低便秘小鼠血清与结肠组织中白介素Iβ(IL-1β)含量Balb/c小鼠分组、造模及处理方法同实施例1。第39天处死小鼠后,将收集到的小鼠血液静置2h,3000×g离心15min后获得血清,采用白介素Iβ(IL-1β)试剂盒,根据说明书进行实验,由标准曲线计算出血清中IL-1β的浓度。将小鼠解剖后取出的新鲜结肠组织0.3g,按1:9(组织重量:PBS)的比例加入预冷的PBS,进行组织研磨,于4℃、12000r/min离心15min,取出上清液。采用白介素Iβ(IL-1β)试剂盒,根据说明书进行实验,由标准曲线计算出结肠组织中IL-1β的浓度。
研究发现,IL-1β能够与抗原协同作用,促进B细胞生长和分化,促进单核-巨噬细胞等APC的抗原递呈能力,引起炎症介质释放。结果如图4所示,灌胃洛哌丁胺后,便秘模型小鼠的血清与结肠组织中IL-1β的含量比空白对照组明显增加,血清中便秘小鼠IL-1β的含量增加了8.81pg/ml,结肠组织中便秘小鼠IL-1β的含量增加了4.64pg/ml,但灌胃长双歧杆菌CCFM1112的小鼠,其血清与结肠组织中IL-1β的含量明显降低,血清中含量降低了14.8pg/ml,结肠组织中降低了4.36pg/ml,两歧双歧杆菌CGMCC NO.13632也使血清中的IL-1β的含量显著降低,但长双歧杆菌CCFM1112的效果强于两歧双歧杆菌CGMCC NO.13632,与空白对照组的含量接近,且明显强于长双歧杆菌CCFM642。对结肠组织中IL-1β的含量,长双歧杆菌CCFM642和两歧双歧杆菌CGMCC NO.13632均无显著降低效果。因此,长双歧杆菌CCFM1112可以明显缓解便秘小鼠中整体代谢循环和结肠组织的炎症反应。
实施例5:长双歧杆菌CCFM1112可降低便秘小鼠结肠组织中肿瘤坏死因子α含量变化示意图Balb/c小鼠分组、造模及处理方法同实施例1。将小鼠解剖后取出的新鲜结肠组织0.3g,按1:9(组织重量:PBS)的比例加入预冷的PBS,进行组织研磨,于4℃、12000r/min离心15min,取出上清液。采用肿瘤坏死因子α(TNF-α)试剂盒,根据说明书进行实验,由标准曲线计算出结肠组织中TNF-α的浓度。
TNF-α主要由活化的单核或巨噬细胞产生,是一种能杀伤和抑制肿瘤细胞的细胞因子。促进中性粒细胞吞噬,抗感染,促进细胞增殖和分化,是重要的炎症介质,并参与某些自身免疫病的病理损伤。如图5所示,灌胃洛哌丁胺后,便秘模型小鼠结肠组织中TNF-α的含量比空白对照组存在升高趋势,灌胃长双歧杆菌CCFM1112后,小鼠结肠组织中TNF-α的含量降低了30.48pg/ml,且较空白对照组更低。虽然两歧双歧杆菌CGMCC NO.13632也显著降低了模型小鼠结肠组织中TNF-α的含量,但长双歧杆菌CCFM1112效果要明显优于两歧双歧杆菌CGMCC NO.13632。相反长双歧杆菌CCFM642在降低模型小鼠结肠组织中TNF-α的含量方面无明显效果。因此,长双歧杆菌CCFM1112可以明显缓解便秘小鼠结肠组织中的炎症反应。
实施例6:长双歧杆菌CCFM1112可提高便秘小鼠粪便中短链脂肪酸含量
Balb/c小鼠分组、造模及处理方法同实施例1。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粪便,用500μL饱和NaCl溶液重悬,加入20μL10%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,从而计算出各种短链脂肪酸的浓度。实验结果如图6所示,用洛哌丁胺造模后,便秘模型组小鼠乙酸、丙酸、丁酸、戊酸含量显著低于空白对照组,而灌胃长双歧杆菌CCFM1112后,粪便中乙酸、丙酸、丁酸、戊酸的含量显著上升,且含量分别比长双歧杆菌CCFM642组高58.7%、45.9%、48.7%、33.4%,比两歧双歧杆菌CGMCC NO.13632高10.4%、48.1%、19.4%、8.01%,并向空白对照组的含量靠拢。结果表明长双歧杆菌CCFM1112对粪便中乙酸、丙酸、丁酸、戊酸这四种短链脂肪酸的含量产生明显变化,能够分别使乙酸、丙酸、丁酸、戊酸相比于模型组提高了17.43μmol/g、3.054μmol/g、1.102μmol/g、0.549μmol/g。乙酸、丁酸等短链脂肪酸能够降低肠道pH值,促进肠道中钙镁离子的吸收,抑制有害菌的侵染,刺激肠道蠕动。因此,长双歧杆菌CCFM1112能够通过增加乙酸、丙酸、丁酸、戊酸这四种短链脂肪酸的含量来缓解便秘。
实施例7:长双歧杆菌CCFM1112可缓解便秘小鼠结肠组织切片中病理变化
Balb/c小鼠分组、造模及处理方法同实施例1。小鼠解剖后取新鲜组织放入多聚甲醛固定液,浸泡后,过夜冲洗。将样品依次经70%、80%、90%各级乙醇溶液脱水,各30min,再放入95%、100%各2次,每次20min进行脱水。放入1/2纯酒精,1/2二甲苯等量混合液15min,二甲苯前洗液15min、二甲苯后洗液15min至透明为止。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分钟来除去透明剂。将处理好的样品进行包埋切片、展片、烤片、H&E染色与封片。
观察小鼠结肠组织的病理切片,如图7所示,便秘组与空白对照组相比,结肠组织的黏液层变薄,隐窝缩短、杯状细胞减少,并且存在炎症浸润的现象。但是长双歧杆菌CCFM1112组能够明显改善这些症状,结肠黏液层变厚,隐窝和杯状细胞界限分明,平滑肌完整,效果强于两歧双歧杆菌CGMCC NO.13632。相反长双歧杆菌CCFM642的改善效果不显著。结果表明,长双歧杆菌CCFM1112能够明显改善便秘小鼠结肠组织的病理损伤。
实施例8:长双歧杆菌CCFM1112可提高芦丁在体外消化的抗氧化活性与生物利用度
芦丁(Rutin)是从槐米等豆科植物花蕾及果实中提取而来,是一种黄酮类化合物,能够抗自由基、抗脂质过氧化、拮抗血小板活化因子以及抗炎等。由于芦丁广泛的药理应用,使得它需要研制出能够在生物体内更好地发挥抗氧化活性的方法,提高其生物利用度。
准确称取25mg芦丁与50mL蒸馏水震荡混匀,取上清液10mL,置于-20℃条件下保存备用,作为未消化样品。称取定量的胃蛋白酶溶解于2/3所需体积的生理盐水(0.5%w/v),用浓盐酸调节pH至3.0±0.05,用0.22μm无菌滤膜过滤后定容到所需体积,使终浓度达到3g/L,此为模拟胃液。称取定量的胰蛋白酶,胆盐溶解于2/3所需体积的生理盐水(0.5%w/v),用0.1mol/L的NaOH调节pH至8.0±0.05,用0.22μm无菌滤膜过滤后定容到所需体积,使终浓度分别达到1g/L和0.3%,此为模拟肠液。以长双歧杆菌GD74为对照,长双歧杆菌GD74是与CCFM1112筛选自同一健康人同一份粪便样本的另一株长双歧杆菌。将活化18h后的CCFM1112与对照组的长双歧杆菌GD74的菌液放置于冰上冷却,取3ml冷却后的菌液于7ml未消化样品中(Rutin组则在7ml未消化样品中加入3mL MRS培养液),混匀后依次经过10ml模拟胃液与10ml模拟肠液,4℃,10000r/min离心10min,取上清液10mL,置于-20℃条件下保存备用,作为胃、肠消化样品。取20mL经过模拟胃、肠消化样品放入烧杯中,再放入装有10mL0.05mol/L KH2PO4(pH 7.0)的透析袋,在37℃条件下分别振荡3与4h,透析袋里的成分为可吸收样品(in),透析袋外样品为不可吸收样品(out),迅速离心4℃,10000r/min,10min,从in取上清液10mL,从out中取上清液7mL,放置于-20℃条件下保存备用。通过高效液相色谱法测定并比较透析袋中芦丁的质量浓度与消化液中芦丁的质量浓度,来判断其生物利用度。采用DPPH乙醇法与铁氰化钾法来测定芦丁对自由基的清除能力和对亚铁离子的还原能力。测定芦丁经消化后保留的抗氧化活性与透析袋中的抗氧化活性,可以得到抗氧化性生物利用度。
实验结果如表2所示:
表2模拟消化液中芦丁的生物利用度与抗氧化性生物利用度
经过长双歧杆菌CCFM1112的孵育后,发现模拟消化液中芦丁的生物利用度与抗氧化性生物利用度有一定程度的增加,这说明长双歧杆菌CCFM1112能够使芦丁在胃肠道的消化过程中更加稳定地发挥抗氧化作用,且优于对照的长双歧杆菌。
实施例9:利用本发明长双歧杆菌CCFM1112制造含该菌的发酵食品
选用新鲜蔬菜或水果洗净后榨汁,包括白菜、白萝卜、黄瓜、甜菜、黄桃、杨梅制品中的一种或上述多种的混合物为原料。
对榨汁后的原料进行高温瞬间灭菌,在温度140℃下高温热杀菌2秒后,立即降温至37℃,再接入本发明制备的长双歧杆菌CCFM1112或含有该菌株的菌剂发酵剂,使CCFM1112的细胞浓度达到108CFU/mL以上,在温度4℃下冷藏保存,于是得到含有本发明长双歧杆菌CCFM1112活菌的果蔬饮料。
实施例10:利用本发明长双歧杆菌CCFM1112制造含该菌的发酵食品
利用长双歧杆菌CCFM1112作为发酵微生物发酵生产食品,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,所述乳制品包括发酵乳制品(发酵乳、风味发酵乳、发酵乳饮料等)、奶油、乳酪、含乳饮料以及乳粉;所述豆制品包括豆奶、豆乳粉;所述果蔬制品包括白菜、白萝卜、黄瓜、甜菜、黄桃、杨梅制品。
实施例9、实施例10制备的发酵食品能够提高粪便含水量与小肠推进率,同时缩短排首粒黑便时间、下调血清中生长抑素(SS)的含量、上调血清中P物质(SP)水平、下调血清与结肠组织中白介素1β(IL-1β)的含量、下调结肠组织中肿瘤坏死因子α(TNF-α)、缓解结肠组织的病理变化、增加粪便中乙酸、丙酸、丁酸和戊酸这几种短链脂肪酸的含量以及提高芦丁在体外消化过程中的抗氧化活性与生物利用度。
本发明所述的长双歧杆菌长亚种CCFM1112可用于含有芦丁的食品中,不仅能够对缓解便秘产生作用,同时对芦丁在模拟人体消化过程中的抗氧化活性与生物利用度产生有益影响,具有非常广泛的应用前景。
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> 一株降低炎症反应且具有缓解便秘作用的长双歧杆菌
<130> BAA211529A
<150> 202011272758.2
<151> 2020-11-12
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 445
<212> DNA
<213> Bifidobacterium longum subsp. longum
<400> 1
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Claims (10)
1.一种长双歧杆菌长亚种(Bifidobacterium longum subsp.longum)CCFM1112,于2019年12月30日保藏于广东省微生物菌种保藏中心,保藏编号为GDMCC NO.60939。
2.一种发酵食品,其特征在于,应用权利要求1所述的长双歧杆菌长亚种CCFM1112发酵制得,所述发酵食品包括固态食品、液态食品、半固态食品。
3.根据权利要求2所述的发酵食品,其特征在于,包括乳制品、豆制品或果蔬制品。
4.权利要求1所述的长双歧杆菌长亚种CCFM1112在制备具有以下至少一种功能的产品中的应用:
(a)缓解便秘;
(b)改善结肠组织的病理损伤;
(c)影响兴奋型和抑制型胃肠活性肽的含量;
(d)增加短链脂肪酸的含量;所述短链脂肪酸包括但不限于乙酸、丙酸、丁酸或戊酸中的至少一种;
(e)增强抗氧化物质的生物利用度。
5.根据权利要求4所述的应用,其特征在于,所述产品为药品、功能性食品或保健品。
6.含有权利要求1所述长双歧杆菌长亚种CCFM1112的菌剂,其特征在于,所述菌剂中长双歧杆菌CCFM1112的数量≥1×108cfu/g或1×108cfu/mL。
7.根据权利要求6所述的菌剂,其特征在于,是将含有权利要求1所述的长双歧杆菌长亚种CCFM1112的细胞液干燥得到的。
8.含有权利要求1所述长双歧杆菌长亚种CCFM1112的药物,其特征在于,所述药物包含长双歧杆菌长亚种CCFM1112和能够在药学上允许的载体。
9.根据权利要求8所述的药物,其特征在于,所述药物还含有芦丁。
10.根据权利要求8或9所述的药物,其特征在于,所述药物的剂型包括颗粒剂、胶囊剂、片剂、丸剂或口服液。
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CN114958662A (zh) * | 2022-05-13 | 2022-08-30 | 江南大学 | 一株能缓解便秘同时上调il-10缓解炎症的长双歧杆菌长亚种及其应用 |
CN116083327A (zh) * | 2023-04-07 | 2023-05-09 | 天赋能(天津)功能食品研究发展有限公司 | 长双歧杆菌婴儿亚种及在缓解便秘、抗结肠组织炎症和改善肠道菌群方面的应用 |
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CN114958662A (zh) * | 2022-05-13 | 2022-08-30 | 江南大学 | 一株能缓解便秘同时上调il-10缓解炎症的长双歧杆菌长亚种及其应用 |
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CN116555076A (zh) * | 2023-03-13 | 2023-08-08 | 广东悦创生物科技有限公司 | 一株长双歧杆菌长亚种my1及其在制备通便和护肠食品药品中的应用 |
CN116555076B (zh) * | 2023-03-13 | 2023-11-28 | 广东悦创生物科技有限公司 | 一株长双歧杆菌长亚种my1及其在制备通便和护肠食品药品中的应用 |
CN116083327A (zh) * | 2023-04-07 | 2023-05-09 | 天赋能(天津)功能食品研究发展有限公司 | 长双歧杆菌婴儿亚种及在缓解便秘、抗结肠组织炎症和改善肠道菌群方面的应用 |
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