CN114874954A - 一种能够促进肠道中短链脂肪酸产生的弯曲乳杆菌及其应用 - Google Patents
一种能够促进肠道中短链脂肪酸产生的弯曲乳杆菌及其应用 Download PDFInfo
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- CN114874954A CN114874954A CN202210691849.2A CN202210691849A CN114874954A CN 114874954 A CN114874954 A CN 114874954A CN 202210691849 A CN202210691849 A CN 202210691849A CN 114874954 A CN114874954 A CN 114874954A
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Abstract
本发明公开了一种能够促进肠道中短链脂肪酸产生的弯曲乳杆菌及其应用,属于微生物技术领域以及医药技术领域。本发明提供的弯曲乳杆菌CCFM1268能够促进肠道健康,降低结肠炎哺乳动物疾病活动指数,减轻结肠的缩短,降低结肠组织的损伤;提高结肠炎哺乳动物结肠组织紧密连接蛋白的含量;(3)降低结肠炎哺乳动物结肠组织中促炎因子IL‑6、IL‑17、IL‑1β和TNF‑α的含量;(4)促进结肠炎哺乳动物肠道中短链脂肪酸含量;(5)调节结肠炎哺乳动物肠道菌群结构。因此,弯曲乳杆菌CCFM1268在制备保护肠道健康的产品(如食品或药品等)中,具有巨大的应用前景。
Description
技术领域
本发明涉及一种能够促进肠道中短链脂肪酸产生的弯曲乳杆菌及其应用,属于微生物技术领域以及医药技术领域。
背景技术
炎症性肠病(Inflammatory bowel disease,IBD),主要包括克罗恩病(Crohn’sdisease,CD)和溃疡性结肠炎(Ulcerative colitis,UC),是一种威胁人类健康的胃肠道炎症,且其发病率在世界范围内逐年上升。CD主要是引发肠道的跨壁炎症,且对胃肠道的任意部分都可能产生影响。在临床上多表现为慢性反复起病,体重下降。而UC主要表现为粘膜炎症,并仅局限于结肠,临床特征为慢性或急性起病并伴有体重下降及腹泻。
有研究表明,炎症性肠病的发生发展可能与结肠中短链脂肪酸的代谢缺陷有关。结肠炎患者肠道内一些短链脂肪酸产生菌如柔嫩梭菌相对丰度降低,导致了肠道内短链脂肪酸含量的降低,进而限制了结肠上皮细胞的能量供应以及对粘膜炎症反应的局部控制(公开于Nemoto H,Kataoka K,Ishikawa H,et al.Reduced diversity and imbalance offecal microbiota in patients with ulcerative colitis[J].Digestive Diseasesand Sciences,2012,57(11):2955-2964.论文中)。研究表明,短链脂肪酸能够有效维持机体肠道和免疫稳态,这些酸主要通过抑制肠上皮促炎细胞因子活性并抑制巨噬细胞中NF-κB信号通路的激活来发挥抗炎功效(公开于Markowiak-Kopec P,Slizewska K.The Effectof Probiotics on the Production of Short-Chain Fatty Acids by HumanIntestinal Microbiome[J].Nutrients,2020,12(4).论文中)。此外,短链脂肪酸能够抑制肠道内大肠杆菌、沙门氏菌等致病性微生物的生长,与其竞争定殖位点(公开于HavenaarR.Intestinal health functions of colonic microbial metabolites:a review[J].Beneficial Microbes,2011,2(2):103-114.论文中)。Shan等人研究发现,短链脂肪酸还能够修复受损的肠粘膜,因为其具有刺激杯状细胞分泌粘液的能力(公开于Shan M,Gentile M,Yeiser J R,et al.Mucus Enhances Gut Homeostasis and Oral Toleranceby Delivering Immunoregulatory Signals[J].Science,2013,342(6157):447-453.论文中)。
弯曲乳杆菌是一种广泛应用于食品中的益生菌。目前有研究表明,弯曲乳杆菌能够通过NF-κB和细胞外调节蛋白激酶(ERK)信号通路介导树突状细胞IL-10的产生从而有效改善DSS诱导的小鼠结肠炎。但现阶段,尚未有研究者关注弯曲乳杆菌对结肠炎小鼠肠道短链脂肪酸含量的影响。因此,有必要探究弯曲乳杆菌对结肠炎小鼠肠道短链脂肪酸含量的促进效果,并开发出能够有效提高结肠炎小鼠肠道内短链脂肪酸含量的弯曲乳杆菌。
发明内容
为了提供一株能够预防和/或治疗验证并且安全无副作用的弯曲乳杆菌(Latilactobacillus curvatus),本发明提供了一株弯曲乳杆菌(Latilactobacilluscurvatus)CCFM1268,已于2022年04月26日保藏于广东省微生物菌种保藏中心,保藏编号为GDMCC No:62425。
所述弯曲乳杆菌(Latilactobacillus curvatus)CCFM1268来源于新疆省女性粪便样本,该菌株经测序分析,其16S rDNA序列如SEQ ID NO.1所示,将测序得到的序列在GenBank中进行核酸序列比对,结果显示菌株为弯曲乳杆菌,命名为弯曲乳杆菌(Latilactobacillus curvatus)CCFM1268。
所述弯曲乳杆菌(Latilactobacillus curvatus)CCFM1268在MRS固体培养基上的菌落呈乳白色,半圆形凸起,表面光滑、湿润,边缘整齐。
本发明还提供了含所述弯曲乳杆菌CCFM1268的产品。
在一种实施方式中,所述产品为食品、药品或保健品。
在一种实施方式中,所述产品中,弯曲乳杆菌的活菌数为不低于5×109CFU/mL或5×109CFU/g。
在一种实施方式中,所述食品包括但不限于酸奶、奶粉、乳酪、乳酸菌饮料、冰激凌及植物基发酵制品;所述植物基发酵制品为发酵豆奶或发酵果汁。
在一种实施方式中,所述药品含有上述弯曲乳杆菌(Latilactobacilluscurvatus)CCFM1268、药物载体和/或药用辅料。
在一种实施方式中,所述食品为保健食品。
在一种实施方式中,所述食品为使用含有上述弯曲乳杆菌(Latilactobacilluscurvatus)CCFM1268的发酵剂生产得到的乳制品、豆制品或果蔬制品;或所述食品为含有上述弯曲乳杆菌(Latilactobacillus curvatus)CCFM1268的饮料或零食。
在一种实施方式中,所述发酵剂的制备方法为将上述弯曲乳杆菌按照占培养基总质量2~4%(v/v)的接种量接种到培养基中,于37℃下培养18h,得到培养液;将培养液离心,得到菌体;将菌体用生理盐水清洗3次后用冻干保护剂重悬,得到重悬液;将重悬液采用真空冷冻法进行冻干,得到发酵剂。
在一种实施方式中,所述冻干保护剂和菌体的质量比为2:1。
在一种实施方式中,所述冻干保护剂包含130g/L的脱脂奶粉。
在一种实施方式中,所述培养基包含占培养基总质量87.7%的水、占培养基总质量10%的脱脂乳、占培养基总质量0.5%的葡萄糖、占培养基总质量1.5%的胰蛋白胨以及占培养基总质量0.3%的酵母浸膏。
在一种实施方式中,所述培养基的pH为6.8。
本发明还提供了所述弯曲乳杆菌CCFM1268在制备预防和/或治疗炎症性肠病的药物中的应用。
在一种实施方式中,所述预防和/或治疗炎症性肠病包括如下至少一种作用:
(1)降低结肠炎哺乳动物的疾病症状,减轻结肠缩短,缓解结肠组织损伤;
(2)提高结肠炎哺乳动物结肠组织Z0-1、Occludin、Claudin-1和Claudin-3等紧密连接蛋白的含量;
(3)降低结肠炎哺乳动物结肠组织中促炎因子IL-6、IL-17、IL-1β和TNF-α的含量;
(4)促进结肠炎哺乳动物肠道中短链脂肪酸的浓度;所述短链脂肪酸包括但不限于乙酸、丙酸、丁酸;
(5)调节结肠炎哺乳动物的肠道菌群结构。
在一种实施方式中,所述药物含有所述弯曲乳杆菌CCFM1268、药物载体和/或药用辅料。
在一种实施方式中,所述药物载体包含微囊、微球、纳米粒和/或脂质体。
在一种实施方式中,所述药用辅料包含赋形剂和/或附加剂。
在一种实施方式中,所述赋形剂包含黏合剂、填充剂、崩解剂和/或润滑剂。
在一种实施方式中,所述附加剂包含增溶剂、助溶剂、潜溶剂和/或防腐剂。
在一种实施方式中,所述药品的剂型为粉剂、颗粒剂、胶囊剂、片剂、丸剂或口服液。
在一种实施方式中,所述食品为保健食品;或所述食品为使用含有上述弯曲乳杆菌CCFM1268的发酵剂生产得到的乳制品、豆制品或果蔬制品;或所述食品为含有上述弯曲乳杆菌CCFM1268的饮料或零食。
在一种实施方式中,所述发酵剂的制备方法为将上述弯曲乳杆菌CCFM1268按照占培养基总质量2~4%(v/v)的接种量接种到培养基中,于37℃下培养18h,得到培养液;将培养液离心,得到菌体;将菌体用生理盐水清洗3次后用冻干保护剂重悬,得到重悬液;将重悬液采用真空冷冻法进行冻干,得到发酵剂。
在一种实施方式中,所述冻干保护剂和菌体的质量比为2:1。
在一种实施方式中,所述冻干保护剂包含130g/L的脱脂奶粉。
在一种实施方式中,所述培养基含有脱脂乳、葡萄糖、胰蛋白胨和酵母浸膏。
在一种实施方式中,所述培养基的pH为6.8。
本发明还提供了一种含有上述弯曲乳杆菌(Latilactobacillus curvatus)CCFM1268的微生物制剂。
在一种实施方式中,所述微生物制剂中,弯曲乳杆菌(Latilactobacilluscurvatus)CCFM1268的活菌数为不低于5×109CFU/mL或5×109CFU/g。
本发明还提供了应用所述弯曲乳杆菌CCFM1268制备的发酵制品。
在一种实施方式中,所述发酵制品包括发酵食品、发酵乳、发酵果蔬饮料、后生元或合生元制剂。
在一种实施方式中,所述发酵制品是将所述弯曲乳杆菌CCFM1268培养,再收集所需的发酵产物。
有益效果
1、本发明筛选出了一株弯曲乳杆菌(Latilactobacillus curvatus)CCFM1268,此弯曲乳杆菌CCFM1268能够改善肠道健康,具体体现在:
(1)显著降低DSS诱导结肠炎期间小鼠的疾病活动指数,减轻结肠的缩短,降低结肠组织的损伤。
(2)显著提高DSS造模后小鼠结肠组织Z0-1、Occludin、Claudin-1和Claudin-3等紧密连接蛋白的含量。
(3)显著降低结肠炎小鼠结肠组织中促炎因子IL-6、IL-17、IL-1β和TNF-α的含量。
(4)显著促进DSS造模后小鼠肠道中短链脂肪酸如乙酸、丙酸、丁酸的浓度。
(5)显著调节结肠炎小鼠肠道菌群结构。
2、本发明的弯曲乳杆菌CCFM1268及其有效成分具有良好的安全性,并具备治疗用途,可用于开发益生菌产品。
生物材料保藏
弯曲乳杆菌(Latilactobacillus curvatus)CCFM1268,分类学命名为Latilactobacillus curvatus,已于2022年04月26日保藏于广东省微生物菌种保藏中心,保藏编号为GDMCC No:62425,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:弯曲乳杆菌CCFM1268菌落形态图。
图2:不同组别小鼠结肠长度。
图3:不同组别小鼠体重变化。
图4:不同组别小鼠DAI疾病活动指数变化。
图5:不同组别小鼠结肠H&E染色。
图6:不同组别小鼠结肠组织中紧密连接蛋白含量;其中:A为不同组别小鼠结肠组织中ZO-1的含量;B为不同组别小鼠结肠组织中Occludin的含量;C为不同组别小鼠结肠组织中Claudin-1的含量;D为不同组别小鼠结肠组织中Claudin-3的含量。
图7:不同组别小鼠结肠组织中细胞因子含量;其中:A为不同组别小鼠结肠组织中TNF-α的含量;B为不同组别小鼠结肠组织中IL-17的含量;C为不同组别小鼠结肠组织中IL-6的含量;D为不同组别小鼠结肠组织中IL-1β的含量。
图8:不同组别小鼠粪便中短链脂肪酸含量;A为不同组别小鼠粪便中总短链脂肪酸含量;B为不同组别小鼠粪便中乙酸含量;C为不同组别小鼠粪便中丙酸含量;D为不同组别小鼠粪便中异丁酸含量;E为不同组别小鼠粪便中丁酸含量;F为不同组别小鼠粪便中戊酸含量。
图9:不同组别小鼠肠道菌群多样性,其中:A为不同组别小鼠肠道菌群α多样性;B为不同组别小鼠肠道菌群β多样性。
图10:不同组别小鼠肠道菌群门水平堆积图。
图11:不同组别小鼠肠道菌群差异属相对丰度图,其中:A为不同组别小鼠肠道菌群乳杆菌属相对丰度;B为不同组别小鼠肠道菌群另枝菌属相对丰度;C为不同组别小鼠肠道菌群拟杆菌属相对丰度;D为不同组别小鼠肠道菌群蓝绿藻菌属相对丰度。
具体实施方式
技术术语:
本发明所用术语“微生物制剂”是指经过分离纯化、菌种改良、工业化生产扩繁或上述任一种工序的微生物加工制成的活菌制剂,通常以简单有机质作为载体制备成可吸附微生物菌液,或加入其它辅助成分制备成固体制剂。
本发明所用术语“药物载体”是指能够改变药物进入体内的方式和在体内的分布,控制药物的释放速度,或将药物输送到靶器官的物质。
本发明所用术语“后生元”是指益生菌产出的有益物质,包括但不限于益生菌经加工处理后产生的对人体健康有益的菌体和/或代谢产物。
本发明所用术语“合生元制剂”是指益生菌和益生元按一定比例结合的生物制剂,能够同时发挥二者的共同作用。
本发明所用术语“菌株”是指具有共同特征的一种特定物种的微生物。除非指示相反含义,否则术语“菌株”与“细胞”在本文中可互换使用。
本发明所用术语“平板”是指平板培养基,是被用于获得微生物纯培养的最常用的固体培养基形式,它是冷却凝固后的固体培养基在无菌培养皿中形成的培养基固体平面,常被简称为培养平板,或平板。
本发明所用术语“培养基”是指用于微生物培养的基质或营养液,它包括对于所述微生物的生长所需的营养物质和水分,足够的能源和适宜的pH环境等。
本发明所用术语“培养”是指对所述微生物持续一段时期的培养直到达到一种希望的目标为止。
本发明所用术语“预防和/或治疗炎症性肠病”是指预防以及降低肠炎的频率和/或发生率和/或严重程度和/或缩短其持续时间;发生率是指任何肠炎发生的数量;频率是指发生相同肠炎的数量;此预防涵盖降低该肠炎在以后生活中的频率和/或严重程度。
本发明所用术语“菌悬液”是指含有微生物细胞的悬浮液。
下述实施例中涉及的材料:
下述实施例中涉及的胰蛋白胨、酵母粉等化学品购自国药集团;下述实施例中涉及的小鼠为6-8周龄的雄性SPF(Specific pathogen free,无特定病原体)级C57BL/6N小鼠购自北京维通利华实验动物技术有限公司;下述实施例中涉及的ELISA试剂盒购自南京森贝伽生物科技有限公司;下述实施例中涉及的葡聚糖硫酸钠(DSS)购自MP Biomedicals公司;下述实施例中涉及的Fast DNA Spin Kit for Feces试剂盒购自MP Biomedicals公司;下述实施例中涉及的检测细胞因子的ELISA试剂盒购自Sigma-Aldrich公司;下述实施例中涉及的多聚甲醛购自武汉塞维尔生物科技有限公司。
下述实施例中涉及的培养基:
LBS固体培养基(g/L):胰蛋白胨10g/L、酵母提取物5g/L、葡萄糖20g/L、K2PO4·3H2O 6g/L、柠檬酸铵2g/L、无水乙酸钠17g/L、MgSO4·7H2O 1.18g/L、MnSO4·H2O 0.134g/L、FeSO4·H2O 0.036g/L、吐温80 1mL/L、琼脂20g/L
LBS液体培养基(g/L):胰蛋白胨10g/L、酵母提取物5g/L、葡萄糖20g/L、K2PO4·3H2O 6g/L、柠檬酸铵2g/L、无水乙酸钠17g/L、MgSO4·7H2O 1.18g/L、MnSO4·H2O 0.134g/L、FeSO4·H2O 0.036g/L、吐温80 1mL/L
MRS固体培养基(g/L):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、K2PO4·3H2O 2.6g/L、MgSO4·7H2O 0.1g/L、MnSO4 0.05g/L、吐温80 1mL/L、琼脂20g/L。
MRS液体培养基(g/L):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、K2PO4·3H2O 2.6g/L、MgSO4·7H2O 0.1g/L、MnSO4 0.05g/L、吐温80 1mL/L。
下述实施例中涉及的弯曲乳杆菌菌悬液的制备方法:
将弯曲乳杆菌划线于MRS固体培养基上,37℃条件下培养48h,得到单菌落;挑取单菌落接种于MRS液体培养基中,37℃条件下培养18h进行活化,连续活化两代,得到活化液;将活化液按2%(v/v)的接种量接种于MRS液体培养基中,37℃条件下培养18h,得到菌液;将菌液经8000g离心10min,得到弯曲乳杆菌菌体;将弯曲乳杆菌菌体经生理盐水洗涤后用0.9%生理盐水重悬,获得菌浓为1×1010CFU/mL的菌悬液。需保藏菌种时,将弯曲乳杆菌菌体重悬于浓度为200g/L的甘油溶液,保存于于-80℃下待用。
清酒乳杆菌的菌悬液按照上述相同方法制备,区别在于,将弯曲乳杆菌替换为清酒乳杆菌。
实施例1:弯曲乳杆菌CCFM1268的筛选、菌种鉴定及培养
1、筛选
以来源于四川省的发酵泡菜水为样本,将样本经预处理后,在30%左右甘油中保存于-80℃冰箱,取出解冻后,混匀样本吸取0.2mL样本加到5mL LBS培养基中富集8h,将富集液用0.9%生理盐水进行梯度稀释,选择合适的梯度稀释液涂布在LBS固体培养基上,于37℃培养48h,挑取典型菌落至MRS平板上划线纯化,挑取单菌落转接至液体MRS液体培养基增菌,30%甘油保藏,得到菌株CCFM1268。
2、鉴定
提取所筛选弯曲乳杆菌的基因组,将其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),经测序分析,该菌株的16S rDNA序列如SEQ ID NO.1所示,将该序列在GenBank中进行比对,与弯曲乳杆菌的同源度为:98.46%;结果显示菌株均为弯曲乳杆菌,命名为弯曲乳杆菌CCFM1268。
同期筛选得到清酒乳杆菌QJSNT1L10。
3、培养
将弯曲乳杆菌CCFM1268接入MRS固体培养基上于37℃培养48h后,观察其菌落。图1为弯曲乳杆菌CCFM1268在MRS平板固体培养基上培养48小时后的菌落形态,发现其菌落呈呈乳白色,半圆形凸起,表面光滑、湿润,边缘整齐。
实施例2:弯曲乳杆菌CCFM1268对结肠炎小鼠结肠长度、体重变化疾病活动指数及结肠组织的影响
取6-8周龄健康雌性C57小鼠32只,随机分为4组,每组8只,4组分别为:空白对照组、造模组和分别灌胃弯曲乳杆菌CCFM1268、清酒乳杆菌QJSNT1L10菌悬液的CCFM1268、QJSNT1L10干预组。其中,空白组和造模组每只老鼠按照0.2mL的剂量每天灌胃一次生理盐水,其余各组每只老鼠按0.2mL的剂量灌胃1×109CFU/mL的菌悬液。实验过程中,空白组给予正常饮水,其余各组给予25g/L的DSS饮水。实验结束后,对每只小鼠进行异氟烷麻醉并处死。
结肠长度的检测方法:小鼠处死后,取整段结肠(盲肠末端至肛门),测量长度,并对结肠外观进行观察。
由图2可知,空白组小鼠结肠长度为5.87cm,DSS造模后小鼠结肠长度缩短至4.56cm。弯曲乳杆菌CCFM1268干预后,小鼠结肠长度恢复至5.26cm;而清酒乳杆菌QJSNT1L10干预后对小鼠结肠长度无明显影响,结肠长度为4.48cm。
体重变化测定方法:在实验过程中每天记录小鼠体重,并以造模前一天小鼠体重为基线,观察小鼠体重变化情况。
由图3可知,与空白组相比,造模组小鼠体重明显下降,造模期间体重降低14%。弯曲乳杆菌CCFM1268干预后减轻了小鼠体重的下降程度,体重降低10%左右。而清酒乳杆菌QJSNT1L10干预后对小鼠体重降低无明显影响。
疾病活动指数(Disease activity index,DAI)的检测方法:DAI评分参照Murthy的评分系统,包括体重变化,便血情况和大便性状三个方面(具体评分标准如表1所示)。造模期间,每天测量小鼠体重,检测小鼠便血情况和大便性状,根据表1进行评分。
表1:疾病活动指数评分标准
体重下降(%) | 大便稠度 | 便血 | 分数 |
0 | 正常 | 无血便 | 0 |
1~5 | 大便稀溏 | 无血便 | 1 |
6~10 | 水样腹泻 | 有出血但是不多 | 2 |
11~15 | 黏糊糊的腹泻,少量血 | 有出血但是不多 | 3 |
>15 | 带血的严重水样泻 | 大出血 | 4 |
由图4可知,从造模第四天开始,造模组小鼠的疾病活动指数开始增加,到第七天达到最大值,为3.05。弯曲乳杆菌CCFM1268和清酒乳杆菌QJSNT1L10干预后均能够有效降低结肠炎小鼠的疾病活动指数。其中,弯曲乳杆菌CCFM1268的缓解效果最好,造模第七天结肠炎小鼠的疾病活动指数为2.22。
结肠组织病理学特征的检测方法:取1cm远端结肠(距肛门1cm)于4%多聚甲醛溶液中4℃下浸泡24h后,送至武汉塞维尔生物科技有限公司完成结肠组织H&E染色并制备切片,用Pannoramic MIDI数字切片扫描仪扫描制作好的H&E结肠切片,进行拍照,观察结肠组织损伤情况。
由图5可知,DSS造模后,结肠组织大面积粘膜层坏死,固有层中肠腺坏死消失,同时伴有中等量的中性粒细胞浸润;黏膜下层小面积水肿并伴有少量的炎性细胞浸润。清酒乳杆菌QJSNT1L10干预后一定程度上加重了结肠炎小鼠的结肠损伤,结肠组织广泛粘膜溃疡,毛细血管扩张且轻微出血,粘膜下层水中,且存在多量中性粒细胞和淋巴细胞浸润。相较于清酒乳杆菌QJSNT1L10,弯曲乳杆菌CCFM1268对结肠损伤表现出一定的保护作用。
上述结果表明,弯曲乳杆菌CCFM1268能够有效缓解DSS造成的结肠损伤,减少体重变化,恢复结肠长度。
实施例3:弯曲乳杆菌CCFM1268对结肠炎小鼠结肠紧密连接蛋白表达的影响
取6-8周龄健康雌性C57小鼠32只,随机分为4组,每组8只,4组分别为:空白对照组、造模组和分别灌胃弯曲乳杆菌CCFM1268、清酒乳杆菌QJSNT1L10菌悬液的CCFM1268、QJSNT1L10干预组。其中,空白组和造模组每只老鼠按照0.2mL的剂量每天灌胃一次生理盐水,其余各组每只老鼠按0.2mL的剂量灌胃1×109CFU/mL的菌悬液。实验过程中,空白组给予正常饮水,其余各组给予2.5%的DSS饮水。实验结束后,对每只小鼠进行异氟烷麻醉并处死。
将各组小鼠处死后,取出结肠组织,利用高通量破碎仪破碎结肠组织得到匀浆,然后12000g、4℃离心15min,收集上清,得到结肠组织上清液。使用BCA试剂盒测定上清液的总蛋白含量后,利用森贝伽的ELISA试剂盒,测定结肠组织中ZO-1、Occludin、Claudin-1和Claudin-3的含量。
由图6可知,弯曲乳杆菌CCFM1268干预后能够显著增加小鼠结肠组织中ZO-1、Occludin、Claudin-1和Claudin-3四类紧密连接蛋白的含量,将结肠组织中ZO-1的含量从3.16pg/mg上调至6.82pg/mg;Occludin的含量从3.39pg/mg上调至5.78pg/mg;Claudin-1的含量从4.48pg/mg上调至7.80pg/mg;Claudin-3的含量从2.88pg/mg上调至5.33pg/mg。而清酒乳杆菌QJSNT1L10干预后对上述四类紧密连接蛋白的含量无明显影响。
实施例4:弯曲乳杆菌CCFM1268对结肠炎小鼠结肠细胞因子表达水平的影响
取6-8周龄健康雌性C57小鼠32只,随机分为4组,每组8只,4组分别为:空白对照组、造模组和分别灌胃弯曲乳杆菌CCFM1268、清酒乳杆菌QJSNT1L10菌悬液的CCFM1268、QJSNT1L10干预组。其中,空白组和造模组每只老鼠按照0.2mL的剂量每天灌胃一次生理盐水,其余各组每只老鼠按0.2mL的剂量灌胃1×109CFU/mL的菌悬液。实验过程中,空白组给予正常饮水,其余各组给予2.5%的DSS饮水。实验结束后,对每只小鼠进行异氟烷麻醉并处死。
将各组小鼠处死后,取出结肠组织,利用高通量破碎仪破碎结肠组织得到匀浆,然后12000g、4℃离心15min,收集上清,得到结肠组织上清液。使用BCA试剂盒测定上清液的总蛋白含量后,按照结肠组织生化指标测定方法检测结肠组织上清液中细胞因子TNF-ɑ、IL-17、IL-6、IL-1β的含量。
由图7可知,DSS造模后,小鼠结肠组织中TNF-ɑ、IL-17、IL-6、IL-1β四类促炎细胞因子的含量较空白组小鼠均显著上调,分别为67.22pg/mg、195.63pg/mg、267.64pg/mg和284.64pg/mg。弯曲乳杆菌CCFM1268干预后能够显著下调小鼠结肠组织中TNF-ɑ和IL-17的含量,将TNF-ɑ和IL-17的含量分别降低至30.84pg/mg和69.7pg/mg。而清酒乳杆菌QJSNT1L10虽然能够下调小鼠结肠组织中TNF-ɑ和IL-17的含量,但与造模组相比,在统计学上无显著性差异。
实施例5:弯曲乳杆菌CCFM1268对结肠炎小鼠短链脂肪酸含量的影响
取6-8周龄健康雌性C57小鼠32只,随机分为4组,每组8只,4组分别为:空白对照组、造模组和分别灌胃弯曲乳杆菌CCFM1268、清酒乳杆菌QJSNT1L10菌悬液的CCFM1268、QJSNT1L10干预组。其中,空白组和造模组每只老鼠按照0.2mL的剂量每天灌胃一次生理盐水,其余各组每只老鼠按0.2mL的剂量灌胃1×109CFU/mL的菌悬液。实验过程中,空白组给予正常饮水,其余各组给予2.5%的DSS饮水。实验结束后,对每只小鼠进行异氟烷麻醉并处死。
将收集得到的小鼠粪便置于液氮中,后转移至-80℃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0.8mL乙醚溶液,震荡30s后离心15min(8000g,4℃),移取上清液至含有0.3g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GC-MS检测短链脂肪酸含量,检测结果见图。
由图8可知,与空白组相比,DSS造模后结肠炎小鼠粪便内乙酸、丙酸、异丁酸、丁酸、戊酸及总短链脂肪酸含量降低。弯曲乳杆菌CCFM1268干预后能够显著上调乙酸、丙酸、异丁酸、丁酸及总短链脂肪酸含量,将粪便内乙酸含量从175.52μmol/g上调至429.87μmol/g;丙酸含量从63.37μmol/g上调至147.48μmol/g;异丁酸含量从10.28μmol/g上调至17.74μmol/g;丁酸含量从43.10μmol/g上调至114.28μmol/g。而清酒乳杆菌QJSNT1L10干预后仅能够显著上调粪便中乙酸的含量,对丙酸、异丁酸、丁酸、戊酸及总短链脂肪酸含量无显著影响。
实施例6:弯曲乳杆菌CCFM1268对结肠炎小鼠肠道菌群多样性的影响
取6-8周龄健康雌性C57小鼠32只,随机分为4组,每组8只,4组分别为:空白对照组、造模组和分别灌胃弯曲乳杆菌CCFM1268、清酒乳杆菌QJSNT1L10菌悬液的CCFM1268、QJSNT1L10干预组。其中,空白组和造模组每只老鼠按照0.2mL的剂量每天灌胃一次生理盐水,其余各组每只老鼠按0.2mL的剂量灌胃1×109CFU/mL的菌悬液。实验过程中,空白组给予正常饮水,其余各组给予2.5%的DSS饮水。实验结束后,对每只小鼠进行异氟烷麻醉并处死。
采用Fast DNA Spin Kit for Feces试剂盒提取每组小鼠粪便细菌宏基因组后,对16s V3-V4区序列进行PCR扩增后,通过二代测序仪粪便样品中肠道菌群多样性。
由图9可知,清酒乳杆菌QJSNT1L10干预后显著降低了结肠炎小鼠肠道菌群α多样性,而弯曲乳杆菌CCFM1268干预后对结肠炎小鼠肠道菌群α多样性无明显影响。弯曲乳杆菌CCFM1268干预组与造模组存在离散,这表明弯曲乳杆菌CCFM1268干预组与造模组肠道菌群结构存在差异。
实施例7:弯曲乳杆菌CCFM1268对结肠炎小鼠肠道菌群组成的影响
具体步骤如下:
取6-8周龄健康雌性C57小鼠32只,随机分为4组,每组8只,4组分别为:空白对照组、造模组和分别灌胃弯曲乳杆菌CCFM1268、清酒乳杆菌QJSNT1L10菌悬液的CCFM1268、QJSNT1L10干预组。其中,空白组和造模组每只老鼠按照0.2mL的剂量每天灌胃一次生理盐水,其余各组每只老鼠按0.2mL的剂量灌胃1×109CFU/mL的菌悬液。实验过程中,空白组给予正常饮水,其余各组给予2.5%的DSS饮水。实验结束后,对每只小鼠进行异氟烷麻醉并处死。
采用Fast DNA Spin Kit for Feces试剂盒提取每组小鼠粪便细菌宏基因组后,对16s V3-V4区序列进行PCR扩增后,通过二代测序仪粪便样品中肠道菌群组成。结果如图10和图11所示。
由图10可知,弯曲乳杆菌CCFM1268干预后,结肠炎小鼠肠道菌群中厚壁菌门的相对丰度从30.19%显著上调至71.69%;而拟杆菌门的相对丰度则从37.93%显著降低至14.50%。与造模组相比,清酒乳杆菌QJSNT1L10则上调了变形菌门的相对丰度,从19.37%上调至22.03%。
由图11可知,弯曲乳杆菌CCFM1268干预后能够显著增加乳杆菌属的相对丰度,同时降低另枝菌属的相对丰度。此外,弯曲乳杆菌CCFM1268干预后还能够降低拟杆菌属相对丰度并增加蓝绿藻菌属相对丰度,但是与造模组相比,在统计学上无显著性差异。这表明弯曲乳杆菌CCFM1268干预后能够有效改善结肠炎小鼠肠道菌群结构。
实施例8:弯曲乳杆菌CCFM1268用于制备菌粉
弯曲乳杆菌CCFM1268可用于制备菌粉,菌粉的具体制备过程如下:
弯曲乳杆菌CCFM1268划线于MRS固体培养基上,37℃条件下培养48h,得到单菌落;挑取单菌落接种于MRS液体培养基中,37℃条件下培养18h进行活化,连续活化两代,得到活化液;将活化液按2%(v/v)的接种量接种于培养基中,37℃条件下培养18h,得到菌液;将菌液经8000g离心10min,得到菌泥;将菌泥用生理盐水清洗3次后用保护剂重悬至浓度为1×1010CFU/mL,得到菌悬液;将菌悬液在温度37℃下预培养60min后冻干,得到弯曲乳杆菌CCFM1268菌粉;
其中,培养基的制备方法为:使用以培养基总重量计87.7%的水将10%脱脂乳、0.5%葡萄糖、1.5%胰蛋白胨与0.3%酵母浸膏溶解,然后调整其pH为6.8,得到培养基;
保护剂的成分包含:130g/L脱脂奶粉。
实施例9:弯曲乳杆菌CCFM1268制剂的制备
乳杆菌制剂的剂型为片剂、散剂或胶囊。
乳杆菌制剂的制备方法,包括:发酵收集乳杆菌菌泥,经冻干后与预胶化淀粉混合再加入硬脂酸镁,制得乳杆菌制剂。
实施例10:弯曲乳杆菌CCFM1268用于制备活菌制剂
将弯曲乳杆菌CCFM1268进行大规模培养,将培养物进行冷冻干燥。
实施例11:弯曲乳杆菌CCFM1268用于制备药物
弯曲乳杆菌CCFM1268可用于制备胶囊制品,胶囊制品的具体制备过程如下:
弯曲乳杆菌CCFM1268划线于MRS固体培养基上,37℃条件下培养48h,得到单菌落;挑取单菌落接种于MRS液体培养基中,37℃条件下培养18h进行活化,连续活化两代,得到活化液;将活化液按2%(v/v)的接种量接种于培养基中,37℃条件下培养18h,得到菌液;将菌液经6000r/min离心10min,得到菌泥;将菌泥用生理盐水清洗3次后用保护剂重悬至浓度1×1010CFU/mL,得到菌悬液;菌悬液添加至浓度为30g/L的海藻酸钠溶液中至浓度为2×109CFU/mL后,充分搅拌,使得弯曲乳杆菌CCFM1268的细胞均匀地分散于海藻酸钠溶液中,得到混合液;将混合液挤压到浓度为20g/L的氯化钙溶液中形成胶粒;待形成的胶粒静止固化30min后,过滤收集胶粒;将收集得到的胶粒进行冷冻干燥48h,得到粉剂;将粉剂装入到药用胶囊中,得到胶囊制品;
其中,培养基的制备方法为:使用以培养基总重量计87.7%的水将10%酶水解脱脂乳、0.5%葡萄糖、1.5%胰蛋白胨与0.3%酵母浸膏溶解,然后调整其pH为6.8,得到培养基。
实施例12:弯曲乳杆菌CCFM1268的用于制备发酵乳
弯曲乳杆菌CCFM1268可用于制备发酵乳,发酵乳的具体制备过程如下:
弯曲乳杆菌CCFM1268划线于MRS固体培养基上,37℃条件下培养48h,得到单菌落;挑取单菌落接种于MRS液体培养基中,37℃条件下培养18h进行活化,连续活化两代,得到活化液;将活化液按2%(v/v)的接种量接种于培养基中,37℃条件下培养18h,得到菌液;将菌液经8000g离心10min,得到菌泥;将菌泥用生理盐水清洗3次后用保护剂重悬至浓度为1×1010CFU/mL,得到菌悬液;将菌悬液在温度37℃下预培养60min后冻干,得到弯曲乳杆菌CCFM1268菌粉;
其中,培养基的制备方法为:使用以培养基总重量计87.7%的水将10%酶水解脱脂乳、0.5%葡萄糖、1.5%胰蛋白胨与0.3%酵母浸膏溶解,然后调整其pH为6.8,得到培养基;
保护剂的成分包含:130g/L脱脂奶粉。
将弯曲乳杆菌CCFM1268与商业干粉发酵剂保加利亚乳杆菌(保加利亚乳杆菌数量级1×109CUF/g菌粉)和商业干粉发酵剂嗜热链球菌(嗜热链球菌数量1×109CUF/g菌粉)按照菌粉重量比1:1:1的比例混合,得到发酵剂;将糖添加至鲜奶中至浓度为50g/L,得到混合液;将混合液在65℃、20MPa的条件下进行均质后在95℃下保温杀菌5min,得到发酵原料;将发酵原料降温至35℃后以0.03%(v/v)的接种量将发酵剂接种至发酵原料中,于35℃下保温发酵16h,得到发酵乳;将发酵乳于42℃下放置4h进行凝乳后,在4℃下冷藏24h进行后熟,得到发酵乳成品。
实施例13:弯曲乳杆菌CCFM1268用于制备豆奶
弯曲乳杆菌CCFM1268可用于制备豆奶,豆奶的具体制备过程如下:
弯曲乳杆菌CCFM1268划线于MRS固体培养基上,37℃条件下培养48h,得到单菌落;挑取单菌落接种于MRS液体培养基中,37℃条件下培养18h进行活化,连续活化两代,得到活化液;将活化液按2%(v/v)的接种量接种于培养基中,37℃条件下培养18h,得到菌液;将菌液经8000g离心10min,得到菌泥;将菌泥用生理盐水清洗3次后用保护剂重悬至浓度为1×1010CFU/mL,得到菌悬液;将菌悬液在温度37℃下预培养60min后冻干,得到弯曲乳杆菌CCFM1268菌粉;
其中,培养基的制备方法为:使用以培养基总重量计87.7%的水将10%酶水解脱脂乳、0.5%葡萄糖、1.5%胰蛋白胨与0.3%酵母浸膏溶解,然后调整其pH为6.8,得到培养基;
保护剂的成分包含:130g/L脱脂奶粉。
将大豆在温度80℃下浸泡2h后去除大豆皮,得到去皮大豆;将去皮大豆沥去浸泡水后加沸水磨浆,得到豆浆;将豆浆在高于80℃的温度条件下保温12min,得到熟豆浆;将熟豆浆用150目筛网过滤后离心分离,得到粗豆奶;将粗豆奶加热到温度140~150℃后迅速导入真空冷却室进行抽真空,使得粗豆奶中的异味物质随着水蒸汽迅速排出,得到熟豆奶;将熟豆奶降温至约37℃后在熟豆奶中添加弯曲乳杆菌CCFM1268菌粉至浓度为不低于1×106CFU/mL,得到含弯曲乳杆菌CCFM1268的豆奶(豆奶需在4℃下冷藏保存)。
实施例14:弯曲乳杆菌CCFM1268用于制备果蔬饮料
弯曲乳杆菌CCFM1268可用于制备果蔬饮料,果蔬饮料的具体制备过程如下:
弯曲乳杆菌CCFM1268划线于MRS固体培养基上,37℃条件下培养48h,得到单菌落;挑取单菌落接种于MRS液体培养基中,37℃条件下培养18h进行活化,连续活化两代,得到活化液;将活化液按2%(v/v)的接种量接种于培养基中,37℃条件下培养18h,得到菌液;将菌液经8000g离心10min,得到菌泥;将菌泥用生理盐水清洗3次后用保护剂重悬至浓度为1×1010CFU/mL,得到菌悬液;将菌悬液在温度37℃下预培养60min后冻干,得到弯曲乳杆菌CCFM1268菌粉;
其中,培养基的制备方法为:使用以培养基总重量计87.7%的水将10%酶水解脱脂乳、0.5%葡萄糖、1.5%胰蛋白胨与0.3%酵母浸膏溶解,然后调整其pH为6.8,得到培养基;
保护剂的成分包含:130g/L脱脂奶粉。
将新鲜水果和蔬菜洗净后榨汁,得到果蔬汁;将果蔬汁在温度140℃下高温热杀菌2秒,得到杀菌后的果蔬汁;将杀菌后的果蔬汁降温至约37℃后在杀菌后的果蔬汁中添加弯曲乳杆菌CCFM1268菌粉至浓度为不低于1×106CFU/mL,得到果蔬饮料(果蔬饮料需在4℃下冷藏保存)。
实施例15:弯曲乳杆菌CCFM1268用于制备乳饮料
弯曲乳杆菌CCFM1268可用于制备乳饮品,乳饮品的具体制备过程如下:
弯曲乳杆菌CCFM1268划线于MRS固体培养基上,37℃条件下培养48h,得到单菌落;挑取单菌落接种于MRS液体培养基中,37℃条件下培养18h进行活化,连续活化两代,得到活化液;将活化液按2%(v/v)的接种量接种于培养基中,37℃条件下培养18h,得到菌液;将菌液经8000g离心10min,得到菌泥;将菌泥用生理盐水清洗3次后用保护剂重悬至浓度为1×1010CFU/mL,得到菌悬液;将菌悬液在温度37℃下预培养60min后冻干,得到弯曲乳杆菌CCFM1268菌粉;
其中,培养基的制备方法为:使用以培养基总重量计87.7%的水将10%酶水解脱脂乳、0.5%葡萄糖、1.5%胰蛋白胨与0.3%酵母浸膏溶解,然后调整其pH为6.8,得到培养基;
保护剂的成分包含:130g/L脱脂奶粉。
将脱脂奶在95℃热杀菌20min后冷却至4℃,得到原料;在原料中添加弯曲乳杆菌CCFM1268菌粉至浓度为不低于1×106CFU/mL,得到乳饮品(乳饮品需在4℃下冷藏保存)。
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> 一种能够促进肠道中短链脂肪酸产生的弯曲乳杆菌及其应用
<130> GBAA220743B
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 918
<212> DNA
<213> Latilactobacillus curvatus
<400> 1
tgcagtcgaa cgcactctcg ttagattgaa gaagcttgct tctgattgat aacatttgag 60
tgagtggcgg acgggtgagt aacacgtggg taacctgccc taaagtgggg gataacattt 120
ggaaacagat gctaataccg cataaaacct agcaccgcat ggtgcaaggt tgaaagatgg 180
tttcggctat cactttagga tggacccgcg gtgcattagt tagttggtga ggtaaaggct 240
caccaagacc gtgatgcata gccgacctga gagggtaatc ggccacactg ggactgagac 300
acggcccaga ctcctacggg aggcagcagt agggaatctt ccacaatgga cgaaagtctg 360
atggagcaac gccgcgtgag tgaagaaggt tttcggatcg taaaactctg ttgttggaga 420
agaacgtatt tgatagtaac tgatcaggta gtgacggtat ccaaccagaa agccacggct 480
aactacgtgc cagcagccgc ggtaatacgt aggtggcaag cgttgtccgg atttattggg 540
cgtaaagcga gcgcaggcgg tttcttaagt ctgatgtgaa agccttcggc tcaaccgaag 600
aagtgcatcg gaaactggga aacttgagtg cagaagagga cagtggaact ccatgtgtag 660
cggtgaaatg cgtagatata tggaagaaca ccagtggcga aggcggctgt ctggtctgta 720
actgacgctg aggctcgaaa gcatgggtag caaacaggat tagataccct ggtagtccat 780
gccgtaaacg atgagtgcta ggtgttggag ggtttccgcc cttcagtgcc gcagctaacg 840
cattaagcac tccgcctggg gagtacgacc gcaaggttga aactcaaagg aattgacggg 900
ggcccgcaca agcggtgg 918
Claims (10)
1.弯曲乳杆菌(Latilactobacillus curvatus)CCFM1268,已于2022年04月26日保藏于广东省微生物菌种保藏中心,保藏编号为GDMCC No:62425。
2.含有权利要求1所述弯曲乳杆菌CCFM1268的产品。
3.根据权利要求2所述的产品,其特征在于,所述产品为食品、保健品或药物。
4.根据权利要求3所述的应用,其特征在于,所述产品中,弯曲乳杆菌CCFM1268的数量不低于5×109CFU/mL或5×109CFU/g。
5.含有权利要求1所述的弯曲乳杆菌CCFM1268的微生物制剂。
6.权利要求1所述的弯曲乳杆菌CCFM1268在制备预防和/或治疗炎症性肠病的药物中的应用。
7.根据权利要求6所述的应用,其特征在于,所述药物含有权利要求1所述弯曲乳杆菌CCFM1268、药物载体和/或药用辅料。
8.根据权利要求6或7所述的应用,其特征在于,所述药物中弯曲乳杆菌CCFM1268的数量不低于5×109CFU/mL或5×109CFU/g。
9.发酵制品,其特征在于,将发酵微生物培养,收集发酵产物;所述发酵微生物含有权利要求1所述的弯曲乳杆菌CCFM1268。
10.根据权利要求9所述的发酵制品,其特征在于,所述发酵制品包括发酵食品、发酵饮品、后生元或合生元制剂。
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郭慧玲;邵玉宇;孟和毕力格;张和平;: "肠道菌群与疾病关系的研究进展", 微生物学通报, no. 02 * |
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