CN110468070A - 鼠李糖乳杆菌ccfm1060、其发酵食品及菌剂制备方法 - Google Patents
鼠李糖乳杆菌ccfm1060、其发酵食品及菌剂制备方法 Download PDFInfo
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Abstract
本发明公开了鼠李糖乳杆菌CCFM1060、其发酵食品及菌剂制备方法,显著降低高脂高胆固醇饮食诱导的Proteus属的丰度升高,减少尿路堵塞、肠道细菌迁移以及肠炎等疾病发生;显著改善NAFLD患者的脂质代谢紊乱;显著改善NAFLD小鼠的胰岛素抵抗;能够回调血清中谷丙转氨酶和谷草转氨酶的升高;显著降低血清中低密度脂蛋白胆固醇的浓度,减少心血管疾病的风险;能够显著提高NAFLD患者肝脏中的SOD水平;能够显著改善肝脏炎症;能够显著改善NAFLD患者肝脏组织损伤;能够显著改善脂肪肝细胞的Nrf2基因的表达;体外吸附全氟辛酸的能力较强。
Description
技术领域
本发明属于功能性微生物技术领域,具体涉及鼠李糖乳杆菌CCFM1060、其发酵食品及菌剂制备方法。
背景技术
近年来,经济的发展导致我国人民生活方式的改变、活动量减少,肥胖的比例明显增加,糖尿病和代谢综合征(Metabolic Syndrome)得患病率大幅度增高。国际糖尿病联合会(IDF)表示,2017年,全球有4.25亿年龄大于19岁的人患有糖尿病,如果维持这种趋势,约30年后,糖尿病患者人数将高达6.93亿人。因此,控制糖尿病已经成为迫在眉睫的事情。
二型糖尿病是以空腹血糖升高、高密度脂蛋白胆固醇降低和甘油三酯升高、胰岛素抵抗等表现同时存在,以糖代谢、脂代谢和蛋白质代谢异常为病理改变基础的多种危险因素聚集,促发动脉粥样硬化等各种心脑血管疾病发生发展的临床综合征。由于二型糖尿病是多种代谢成分异常聚集的病理状态,其集簇发生与胰岛素抵抗有关,目前已成为心血管疾病和肝脏疾病研究领域共同关注的热点。此外,二型糖尿病均伴随着肠道微生态的紊乱,同时也与抑郁、焦虑等精神性疾病紧密相关。另外有研究表明,职业暴露于PFOA的工人的Ⅱ型糖尿病死亡风险增加,因此日常生活中的PFOA暴露,是人类Ⅱ型糖尿病发病的潜在风险。
目前针对二型糖尿病的药物治疗多为围绕缓解胰岛素分泌不足和胰岛素抵抗两方面,包括减轻胰岛素抵抗的二甲双胍、噻唑烷二酮类;良好控制血糖的磺脲类、罗格列酮等。这些药物均有一定的治疗作用,但随着病情加重,用药量增大、药物间相互作用、药物毒副作用等也显著增加,导致消化道出现不良反应,并表现出一定的肝肾毒性,如长期服用二甲双胍会引起刺激一些患者的胃肠道造成不适并且可能会影响患者对维生素B12的吸收,罗格列酮会引起肝功能损伤及水肿等。
非酒精性脂肪性肝病(non-alcoholic fatty liver disease,NAFLD),为肝细胞中脂肪过度堆积而引起的脂肪变性,是一种肝脏代谢紊乱的症状,未加干预可逐渐演化为非酒精性脂肪型肝炎(NASH)进而恶化为死亡率极高的肝纤维化、肝硬化、肝癌。近年来,随着生活水平的提高,越来越多的高脂肪、高能量饮食摆上餐桌,而人们的运动量却并未随之增加,从而使得以非酒精性脂肪性肝病为代表的代谢综合征的发生率逐年升高。由于其病理机制不完善,并无专门针对NAFLD的药物在市面上出售,治疗方式只能从饮食及生活方式上进行改善,同时辅以降低血脂或血糖的药物以达到更好的效果。有研究表明,职业暴露于PFOA的工人的因脂肪肝死亡风险增加,因此日常生活中的PFOA暴露,是人类肝脏疾病发病的潜在风险。
发明内容
本部分的目的在于概述本发明的实施例的一些方面以及简要介绍一些较佳实施例。在本部分以及本申请的说明书摘要和发明名称中可能会做些简化或省略以避免使本部分、说明书摘要和发明名称的目的模糊,而这种简化或省略不能用于限制本发明的范围。
鉴于上述的技术缺陷,提出了本发明。
因此,作为本发明其中一个方面,本发明克服现有技术中存在的不足,提供鼠李糖乳杆菌CCFM1060,保藏编号为GDMCC No:60705。
作为本发明的另一个方面,本发明克服现有技术中存在的不足,提供制备鼠李糖乳杆菌CCFM1060菌剂的方法。
为解决上述技术问题,本发明提供了如下技术方案:制备鼠李糖乳杆菌CCFM1060菌剂的方法,其包括,
菌株培养基的制备;
菌株保护剂的制备;
接种培养、冻干。
作为本发明所述的制备鼠李糖乳杆菌CCFM1060菌剂的方法的一种优选方案:所述菌株培养基,为改良MRS培养基,其配方为胰蛋白胨10g、牛肉浸膏10g、酵母粉5g、葡萄糖20g、醋酸钠5g、柠檬酸氢二铵2g、磷酸氢二钾2g、七水硫酸镁0.5g、吐温-80 1mL、一水合硫酸锰0.25g、半胱氨酸盐酸盐0.5g,水1000mL;调整其pH为6.5±0.2。
作为本发明所述的制备鼠李糖乳杆菌CCFM1060菌剂的方法的一种优选方案:所述菌株保护剂,其配方为100g/L~150g/L脱脂奶粉、100g/L~150g/L麦芽糊精、140g/L~160g/L海藻糖、余量为水,冻干,得到冻干保护剂。
作为本发明所述的制备鼠李糖乳杆菌CCFM1060菌剂的方法的一种优选方案:所述菌株保护剂,其配方为120g/L脱脂奶粉、120g/L麦芽糊精、150g/L海藻糖、余量为水,冻干,得到冻干保护剂。
作为本发明所述的制备鼠李糖乳杆菌CCFM1060菌剂的方法的一种优选方案:所述接种培养、冻干,包括,鼠李糖乳杆菌CCFM1060以2%的接种量接种到在119~123℃条件下灭菌15~25min后的所述菌株培养基中,在温度35~39℃厌氧条件下培养24~48h,用pH为6.8~7.2磷酸盐缓冲液清洗2~4次,用所述菌株保护剂重悬,使菌浓达到1010CFU/mL;接着,让该菌株重悬液在温度37℃厌氧条件下预培养50~70min,再在-15~-20℃预冻8~14h,之后真空冷冻干燥。
作为本发明所述的制备鼠李糖乳杆菌CCFM1060菌剂的方法的一种优选方案:鼠李糖乳杆菌CCFM1060以2%的接种量接种到在121℃条件下灭菌20min后的培养基中,在温度37℃厌氧条件下培养24h,用pH为6.8磷酸盐缓冲液清洗2~4次,用所述的保护剂重悬,使菌浓达到1010CFU/mL;接着,让该悬浮液在温度37℃厌氧条件下预培养60min,再在-15℃预冻12h,之后真空冷冻干燥。
作为本发明的另一个方面,本发明克服现有技术中存在的不足,提供一种发酵食品。
为解决上述技术问题,本发明提供了如下技术方案:一种发酵食品,其使用所述的鼠李糖乳杆菌CCFM1060菌剂进行发酵,所述菌剂含有大于106CFU/g的活性鼠李糖乳杆菌CCFM1060。
作为本发明所述的发酵食品的一种优选方案:所述发酵食品包括乳制品、豆制品与果蔬制品。
作为本发明所述的发酵食品的一种优选方案:所述发酵食品,包括黄瓜梨汁乳酸菌饮料,其制备方法为,将梨榨汁得到梨汁,将黄瓜切块,放入90℃的热水中热烫30s,打浆,过滤,得到黄瓜汁;将梨汁、黄瓜汁与脱脂乳粉以5:4:1的质量比混合,得到黄瓜汁梨汁混合乳液,均质后加入5%的木糖醇,分装,115℃灭菌20min,冷却至室温后加入所述鼠李糖乳杆菌CCFM1060菌剂以107CFU/m L接种到所述黄瓜汁梨汁混合乳液,37℃恒温培养箱中发酵16h。
本发明的有益效果:本发明筛选的鼠李糖乳杆菌CCFM1060,对模拟胃肠液具有较好的耐受能力;显著降低高脂高胆固醇饮食诱导的Proteus属的丰度升高,减少尿路堵塞、肠道细菌迁移以及肠炎等疾病发生;显著改善NAFLD患者的脂质代谢紊乱;显著改善NAFLD小鼠的胰岛素抵抗;能够回调血清中谷丙转氨酶(ALT)和谷草转氨酶(AST)的升高;显著降低血清中低密度脂蛋白胆固醇的浓度,减少心血管疾病的风险;能够显著提高NAFLD患者肝脏中的SOD水平;能够显著改善肝脏炎症;能够显著改善NAFLD患者肝脏组织损伤;能够显著改善脂肪肝细胞的Nrf2基因的表达;体外吸附全氟辛酸的能力较强;能够显著改善高糖作用下INS-1细胞的增殖和MafA基因的表达。
附图说明
为了更清楚地说明本发明实施例的技术方案,下面将对实施例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一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动性的前提下,还可以根据这些附图获得其它的附图。其中:
图1是发酵用乳酸菌鼠李糖乳杆菌CCFM1060的菌落形态;
图2是鼠李糖乳杆菌CCFM1060对NAFLD小鼠肠道中Proteus菌属的影响;
图3是鼠李糖乳杆菌CCFM1060对NAFLD小鼠血清胆固醇(TC)的影响;
图4是鼠李糖乳杆菌CCFM1060对NAFLD小鼠血清谷丙转氨酶(ALT)的影响;
图5是鼠李糖乳杆菌CCFM1060对NAFLD小鼠血清谷草转氨酶(AST)水平的影响;
图6是鼠李糖乳杆菌CCFM1060对NAFLD小鼠血清低密度脂蛋白胆固醇(LDL-C)水平的影响;
图7是鼠李糖乳杆菌CCFM1060对NAFLD小鼠空腹血糖的影响;
图8是鼠李糖乳杆菌CCFM1060对NAFLD小鼠的胰岛素抵抗的影响
图9是鼠李糖乳杆菌CCFM1060对NAFLD小鼠肝脏甘油三酯(TG)的影响
图10是鼠李糖乳杆菌CCFM1060对NAFLD小鼠肝脏超氧化物歧化酶(SOD)的影响
图11是鼠李糖乳杆菌CCFM1060对NAFLD小鼠肝脏炎症的影响;
图12是鼠李糖乳杆菌CCFM1060对NAFLD小鼠肝脏组织病理的影响;
图13是鼠李糖乳杆菌CCFM1060对脂肪肝细胞Nrf2基因表达的影响;
图14是鼠李糖乳杆菌CCFM1060体外吸附全氟辛酸的能力;
图15是鼠李糖乳杆菌CCFM1060对二型糖尿病小鼠肠道中Desulfovibrio属丰度的影响;
图16是鼠李糖乳杆菌CCFM1060对高糖作用下INS-1细胞增殖情况的影响;
图17是鼠李糖乳杆菌CCFM1060对高糖作用下INS-1细胞MafA基因表达的影响;
注:a,b,c表示不同字母所代表的组别都存在显著性差异(P<0.05)。
具体实施方式
为使本发明的上述目的、特征和优点能够更加明显易懂,下面结合具体实施例对本发明的具体实施方式做详细的说明。
在下面的描述中阐述了很多具体细节以便于充分理解本发明,但是本发明还可以采用其他不同于在此描述的其它方式来实施,本领域技术人员可以在不违背本发明内涵的情况下做类似推广,因此本发明不受下面公开的具体实施例的限制。
其次,此处所称的“一个实施例”或“实施例”是指可包含于本发明至少一个实现方式中的特定特征、结构或特性。在本说明书中不同地方出现的“在一个实施例中”并非均指同一个实施例,也不是单独的或选择性的与其他实施例互相排斥的实施例。
鼠李糖乳杆菌CCFM1060(Lactobacilus rhamnosus),于2019年06月28日保藏于广东省微生物菌种保藏中心,地址为广州市先烈中路100号大院59号楼5楼,保藏编号为GDMCCNo:60705。
鼠李糖乳杆菌CCFM1060具有下述生物学特性:
(1)菌体特征:呈革兰氏染色阳性,不形成孢子,不运动的细菌。
(2)菌落特征:有氧或厌氧培养36小时形成明显的菌落,直径在0.5-2mm之间,正面形态圆形,侧面形态呈突起状,边缘不齐,奶油白色,不透明,表面湿润光滑,不产生色素,参见附图1。
(3)生长特性:在37℃恒温有氧或厌氧的条件下,在mMRS培养基中培养约16小时达到对数末期。
(4)对模拟胃肠液具有较好的耐受能力;
(5)显著降低高脂高胆固醇饮食诱导的Proteus属的丰度升高,减少尿路堵塞、肠道细菌迁移以及肠炎等疾病发生;
(6)显著改善NAFLD小鼠的脂质代谢紊乱;
(7)显著改善NAFLD小鼠的胰岛素抵抗;
(8)能够回调NAFLD小鼠的血清中谷丙转氨酶(ALT)和谷草转氨酶(AST)的升高;
(9)显著降低NAFLD小鼠的血清中低密度脂蛋白胆固醇的浓度,减少心血管疾病的风险;
(10)能够显著提高NAFLD小鼠肝脏中的SOD水平;
(11)能够显著改善NAFLD小鼠的的肝脏炎症;
(12)能够显著改善NAFLD小鼠肝脏组织损伤;
(13)能够显著改善脂肪肝细胞的Nrf2基因的表达。
(14)体外吸附全氟辛酸的能力较强。
(15)能够显著改善高糖作用下INS-1细胞的增殖和MafA基因的表达。
本菌株的获得方法为:
(一)乳酸菌的分离筛选:
(l)取1g来自健康人群的新鲜粪便。将样品在含有山梨醇GM17培养基中35℃富集12h;
(2)将富集样品进行梯度稀释后涂布于添加了0.02%嗅甲酚紫的GM17固体平板上,培养24~48h;
(3)选取变色圈明显,并且符合乳酸菌目基本形态的单菌落进行平板划线纯化,筛选分离出乳酸菌;
(4)将上述单菌落培养于液体GM17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)发酵用乳酸菌的初步鉴定:溶钙圈测定法
(l)将步骤(一)所筛选得到的乳酸菌在液体山梨醇GM17培养液中培养24h,然后取l mL培养物8000rpm离心2min;
(2)用0.05M KH2PO4溶液洗涤两次;
(3)将所得菌泥重悬,划线在山梨醇GM17-0.75%CaCO3的固体培养基上,培养24h;
(4)选取溶钙圈明显,且呈凸面圆形、细密色白、无菌丝体的菌落,革兰氏染色后显微镜观察菌体进行初步判定。
(三)发酵用乳酸菌的分子生物学鉴定:
(l)单菌基因组抽提:
A.将步骤(二)所筛选得到的乳酸菌培养过夜,取培养过夜的菌悬液1mL于1.5mL离心管,10000rpm离心2min,弃上清得菌体;
B.用1mL无菌水吹洗菌体后,10000rpm离心2min,弃上清得菌体;
C.加入200μLSDS裂解液,80℃水浴30min;
D.加入酚-氯仿溶液200μL于菌体裂解液中,其中酚-氯仿溶液的组成成分及体积比为Tris饱和酚:氯仿:异戊醇=25:24:1,颠倒混匀后,12000rpm离心5-10min,取上清200μL;
E.加入400μL冰乙醇或冰异丙醇于200uL上清中,﹣20℃静置1h,12000rpm离心5-10min,弃上清;
F.加入500μL70%(体积百分数)冰乙醇重悬沉淀,12000rpm离心1-3min,弃上清;
G.60℃烘箱烘干,或者自然晾干;
H.50μLddH2O重溶沉淀以备PCR;
(2)16S rDNA PCR
A.细菌16S rDNA 50μLPCR反应体系:
10×Taq buffer,5μL;dNTP,5μL;27F,0.5μL;1492R,0.5μL;Taq酶,0.5μL;模板,0.5μL;ddH2O,38μL。
B.PCR条件:
95℃ 5min;95℃10s;55℃ 30s;72℃ 30s;step2-4 30×;72℃ 5min;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5μL,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,将得到的序列结果使用BLAST在GeneBank中进行搜索和相似性比对,选取测序结果鉴定为属于鼠李糖乳杆菌的一种新发现的菌株,-80℃保藏备用。
实施例1:鼠李糖乳杆菌CCFM1060对模拟胃肠液具有良好的耐受性
将冷冻保存的鼠李糖乳杆菌CCFM1060接种于mMRS培养基(MRS培养基+0.05%半胱氨酸盐酸盐)中,在温度37℃厌氧培养48h,再经mMRS培养液传代培养2~3次后,取1mL鼠李糖乳杆菌CCFM1060的培养液,与9.0mL pH 2.5人工模拟胃液(含1%胃蛋白酶、pH=2.5的mMRS培养基)混合,并在37℃下厌氧培养,分别在0h、0.5h、1h和2h时取样,用mMRS琼脂培养基浇注培养进行平板菌落计数,测定活菌数并计算其存活率。
存活率是在该培养液中在取样时的活菌数对数值与在第0h时活菌数对数值之比,以%表示。取取1mL鼠李糖乳杆菌CCFM1060的培养液加入9mL人工模拟肠液(含0.3%胆盐、1%胰蛋白酶、pH=8.0的mMRS培养基)中,在37℃下厌氧培养,分别在0h、0.5h、1h、2h、3h和4h时取样,用mMRS琼脂培养基浇注培养进行平板菌落计数,测定活菌数并计算其存活率。存活率是在该培养液中在取样时的活菌数对数值与在第0h时活菌数对数值之比,以%表示。实验结果如表1和表2所示。结果表明,鼠李糖乳杆菌CCFM1060对人工胃肠液具有较好的耐受性。
表1鼠李糖乳杆菌CCFM1060在人工模拟胃液中的耐受性
表2鼠李糖乳杆菌CCFM1060在人工模拟肠液中的耐受性
实施例2:鼠李糖乳杆菌CCFM1060对NAFLD小鼠肠道菌群的调节作用
取体重16-20g的健康雄性C57BL/6J小鼠48只,适应环境1周,随机分为6组:空白对照组(NC)、模型对照组(M)、罗格列酮对照组(RC)、辛伐他汀对照组(SC)、鼠李糖乳杆菌CCFM1060干预组(CCFM1060)、鼠李糖乳杆菌L10干预组(LC),每组含小鼠8只,灌胃菌株由3%的蔗糖溶液重悬。实验动物分组及处理方法见表3:
表3实验动物分组
试验末期收集小鼠新鲜粪便冻存于-80℃,提取粪便中的宏基因组,并利用二代测序仪对肠道菌群结构进行分析。试验结束时,小鼠禁食不禁水12h,腹腔注射0.5mL/10g 1%的戊巴比妥钠溶液麻醉后,心脏采血,辅以颈椎脱臼法处死。血液样本3000×g、4℃条件下离心15min,取上清,-80℃冻存用于测定相关血清指标。部分肝脏收集后迅速置于预冷的生理盐水中漂洗去血,放入4%中性多聚甲醛溶液中固定,剩余部分肝脏于液氮中速冻并转移至-80℃冻存,后续制成肝匀浆以用于测定相关指标,具体制备方法如下:称取一定量肝脏组织,按1:9比例加入生理盐水进行组织研磨,3000r离心10min,取上清冻存于-80℃备用。
菌群分析实验结果如图2所示,显著降低高脂高胆固醇饮食诱导的Proteus属的丰度升高,减少炎症和尿道感染风险,减少肥胖及免疫性疾病的发生
NAFLD小鼠粪便中Proteus属显著上升,鼠李糖乳杆菌CCFM1060的摄入可将Proteus属丰度回调,而Proteus属与尿路堵塞、肠道细菌迁移以及肠炎有关,表明本发明具有减少尿路堵塞、肠道细菌迁移以及肠炎等疾病发生的功能。
实施例3:鼠李糖乳杆菌CCFM1060显著降低NAFLD小鼠血清总胆固醇(TC)的水平
C57BL/6J小鼠分组、造模及处理方法同实施例2。
实验结果如图3所示。模型组小鼠血清总胆固醇含量明显升高,灌胃鼠李糖乳杆菌CCFM1060明显降低了模型小鼠的TC水平并接近于空白对照组。其降低小鼠血清TC的能力与辛伐他汀药物组相似。
实施例4:鼠李糖乳杆菌CCFM1060降低NAFLD小鼠血清谷丙转氨酶(ALT)水平
C57BL/6J小鼠分组、造模及处理方法同实施例2。按照ALT试剂盒的检测方法测定血液中谷丙转氨酶(ALT)的含量。
实验结果如图4所示。模型组小鼠空腹ALT显著升高,鼠李糖CCFM1060的干预明显降低了NAFLD小鼠的ALT水平,其降低小鼠空腹血糖水平的能力与辛伐他汀相似,而鼠李糖乳杆菌L10的摄入并未对ALT的升高有所逆转,值得注意的罗格列酮组的ALT水平显著高于模型组,提示长期服用罗格列酮会对肝脏造成药物损伤。
实施例5:鼠李糖乳杆菌CCFM1060降低NAFLD小鼠血清谷草转氨酶(AST)的水平
C57BL/6J小鼠分组、造模及处理方法同实施例2。按照试剂盒的检测方法测定血液中谷草转氨酶(AST)的含量。
实验结果如图5所示。由图5可以看出,模型组小鼠血清AST含量明显升高,灌胃鼠李糖乳杆菌CCFM1060显著降低血清AST的含量,且其趋势和ALT趋势一致,提示鼠李糖乳杆菌CCFM1060能够缓解肝脏损伤。
实施例6:鼠李糖乳杆菌CCFM1060降低NAFLD小鼠血清低密度脂蛋白胆固醇(LDL-C)的水平
C57BL/6J小鼠分组、造模及处理方法同实施例2。按照试剂盒的检测方法测定低密度脂蛋白胆固醇(LDL-C)的含量。
实验结果如附图6所示。由实验结果可以看出,与正常对照组相比,模型组小鼠血清低密度脂蛋白胆固醇含量显著升高,灌胃鼠李糖乳杆菌CCFM1060可降低血清低密度脂蛋白胆固醇的含量,且鼠李糖乳杆菌CCFM1060对血清低密度脂蛋白胆固醇水平的回调能力明显优于鼠李糖乳杆菌L10。
实施例7:鼠李糖乳杆菌CCFM1060降低NAFLD小鼠的空腹血糖水平C57BL/6J小鼠分组、造模及处理方法同实施例2。
实验结果如图7所示。模型组小鼠空腹血糖显著升高,鼠李糖乳杆菌CCFM1060的干预明显降低了NAFLD小鼠的空腹血糖水平,其空腹血糖控制能力显著强于鼠李糖乳杆菌L10的干预,且其降低小鼠空腹血糖水平的能力与罗格列酮相似。
实施例8:鼠李糖乳杆菌CCFM1060缓解NAFLD小鼠的胰岛素抵抗C57BL/6J小鼠分组、造模及处理方法同实施例2。按照试剂盒的检测方法测定胰岛素(INS)的含量,并结合空腹血糖结果计算胰岛素抵抗指数。
实验结果如图8所示。与空白组相比,高脂高胆固醇饮食24周后,模型组小鼠胰岛素抵抗指数显著升高,鼠李糖乳杆菌L10干预后NAFLD小鼠胰岛素抵抗指数有所降低,但其效果不如鼠李糖乳杆菌CCFM1060,提示鼠李糖乳杆菌CCFM1060能够提高NAFLD小鼠的胰岛素敏感性,可能对二型糖尿病有一定缓解效果。
实施例9:鼠李糖乳杆菌CCFM1060降低NAFLD小鼠的肝脏中甘油三酯(TG)的水平
C57BL/6J小鼠分组、造模及处理方法同实施例2。按照试剂盒的检测方法测定甘油三酯(TG)的含量,以肝脏蛋白浓度进行校正。
实验结果如图9所示。由实验结果可以看出,与正常对照组相比,模型组小鼠肝脏TG显著升高,灌胃鼠李糖乳杆菌CCFM1060降低NAFLD小鼠肝脏中TG的水平,且鼠李糖乳杆菌CCFM1060对肝脏TG的调节能力与辛伐他汀相当。
实施例10:鼠李糖乳杆菌CCFM1060提高了NAFLD小鼠的肝脏中超氧化物歧化酶(SOD)的水平,以肝脏蛋白浓度进行校正。
C57BL/6J小鼠分组、造模及处理方法同实施例2。按照超氧化物歧化酶(SOD)试剂盒的说明书测定肝脏中SOD的含量。
实验结果如图10所示。由实验结果可以看出,空白对照组的SOD水平高于模型组,但无显著差异,灌胃鼠李糖乳杆菌CCFM1060显著提高NAFLD小鼠肝脏中SOD的水平而鼠李糖乳杆菌L10干预后并未呈现出相似的结果。
实施例11:鼠李糖乳杆菌CCFM1060减轻NAFLD小鼠肝脏中的炎症水平C57BL/6J小鼠分组、造模及处理方法同实施例2。按照肿瘤坏死因子-α(TNF-α)试剂盒的说明书测定肝脏中TNF-α的含量,以肝脏蛋白浓度进行校正。
实验结果如图11所示。由实验结果可以看出,高脂高胆固醇饮食24周后,模型组的TNF-α水平显著升高,灌胃鼠李糖乳杆菌CCFM1060显著降低NAFLD小鼠肝脏中TNF-α水平,且其炎症缓解效果强于辛伐他汀和罗格列酮,而鼠李糖乳杆菌L10干预后NAFLD小鼠炎症缓解效果一般。
实施例12:鼠李糖乳杆菌CCFM1060缓解NAFLD小鼠的肝脏组织损伤C57BL/6J小鼠分组、造模及处理方法同实施例2。取部分经4%中性多聚甲醛固定的肝脏制作石蜡切片,经HE染色后光镜下观察组织形态并拍照,进行病理学评价。具体步骤如下:
(1)固定:组织样品用生理盐水洗一下,立即投入4%中性多聚甲醛固定液中固定,一般固定时间在72h之内。
(2)洗涤:流水冲洗或浸泡数小时或过夜。
(3)脱水:样品依次经70%、80%、90%各级乙醇溶液脱水,各30min,再放入95%1次20min、100%2次每次10min。
(4)透明:1/2纯酒精+1/2二甲苯混合液10min、二甲苯Ⅰ10min、Ⅱ10min(至透明为止)。
(5)浸蜡:将样品放入石蜡(62℃)透蜡2h。
(6)包埋:以最大的面在底层,使切出的面组织面所占最大。
(7)切片:用手动切片机,将蜡块切成5μm厚度的片段。
(8)展片和粘片(捞片):打开水浴锅,使水温维持在42℃,使切片平整铺展在水面上。
(9)烤片:将载玻片连同载玻片架放入55℃的干燥箱,约2h至蜡熔化。
(10)水化:石蜡切片经二甲苯Ⅰ、Ⅱ脱蜡各10min,然后放入100%、95%、90%、80%、70%各级酒精溶液中各5min,再放入蒸馏水中3min。
(11)初染:切片放入苏木精中染色约20s。
(12)水洗:用自来水流水冲洗约15min。使切片颜色变蓝,但要注意流水不能过大,以防切片脱落。
(13)分化:将切片放入1%盐酸乙醇溶液中褪色,7s。见切片变红,颜色较浅即可。
(14)漂洗:切片再放入自来水流水中冲洗15-20min使其恢复蓝色。
(15)复染:浸入伊红染液,立即取出进行脱水。
(16)脱水:将切片依次过95%乙醇Ⅰ、95%乙醇Ⅱ、70%乙醇,再放入80%乙醇50s、无水乙醇2min。
(17)透明:切片放入1/2无水乙醇,1/2二甲苯中1min,二甲苯Ⅰ、Ⅱ中各2min。
(18)封片:切片经二甲苯透明后,使用中性树胶作为封藏剂,树胶可用二甲苯稀释至合适的稠度。
实验结果如图12所示。由实验结果可以看出,模型组小鼠肝细胞排列稀疏,肝脂滴数量多且大小不一,脂滴之间相互粘连,肝小叶有炎性细胞浸润,少量肝细胞发生气球样病变,而灌胃鼠李糖乳杆菌CCFM1060可明显改善上述病变,且效果明显好于鼠李糖乳杆菌L10。
实施例13:鼠李糖乳杆菌CCFM1060提高脂肪肝细胞中Nrf2的水平
将L02细胞在含有10%的FBS连续稳定传代三次后,接种于6孔板上,于37℃、5%CO2环境中培养24h,待细胞贴壁后,给予2mg/mL棕榈酸孵育24h后再分别单独接入1mL的鼠李糖乳杆菌CCFM1060和鼠李糖乳杆菌L10(接入PBS作为空白对照)孵育24h。所有孵育均在37℃、5%CO2环境中进行。鼠李糖乳杆菌CCFM1060刺激实验组、鼠李糖乳杆菌L10刺激实验组和PBS对照组各三个孔,且重复三次。
弃去培养液,先用PBS缓冲液清洗每个孔,每次1mL,清洗3次后,加TRIZOL裂解,进行细胞RNA提取。将提取的RNA反转录为cDNA后进行qPCR测定BA3与脂肪肝细胞共孵育后Nrf2基因的表达水平。Nrf2引物信息如表5所示,结果以GAPDH作为内参,表示为2-△△CT。
表4引物信息
引物名称 | 正向 | 反向 |
NRF2 | CAACCCTTGTCACCATCTCA | GTGTTCTCACATTGGGCATC |
GAPDH | AGGTCGGTGTGAACGGATTTG | TGTAGACCATGTAGTTGAGGTCA |
实验结果如图13所示。由实验结果可以看出,鼠李糖乳杆菌CCFM1060刺激显著提高了脂肪肝细胞的Nrf2基因的表达水平,鼠李糖乳杆菌L10刺激组的Nrf2基因的表达水平也有升高但明显不及鼠李糖乳杆菌CCFM1060刺激组,表明鼠李糖乳杆菌CCFM1060可能具有一定抗氧化能力。
实施例14:鼠李糖乳杆菌CCFM1060对PFOA具有良好的吸附能力
对鼠李糖乳杆菌CCFM1060进行纯化和活化培养,按1%(v/v)接种量接种于MRS液体培养基中,37℃厌氧培养24h。然后在8000r/min离心5min收集菌体,取沉淀用生理盐水清理后继续在8000r/min离心5min,去沉淀得到活菌体细胞,即湿菌体。将湿菌体重悬于50mg/LPFOA溶液中,并使最终菌体浓度达到1g干菌体/L(将湿菌体重悬于不含PFOA的超纯水中作为空白对照)。使用0.1M的NaOH或HCl溶液将含菌液的PFOA溶液的pH迅速调整至3.0,添加少量的NaOH或HCl(少于0.5ml)其离子强度对PFOA吸附的影响可以忽略。随后将装有100ml样液的250ml锥形瓶置于37℃、150rpm摇床培养,6h后取样测定,2次平行试验取平均值。
PFOA吸附量的测定:吸附实验后,样液在8000r/min离心5min,并用0.22μm的水膜过滤,PFOA浓度用具有Waters SYNAPT MS***的UPLC-MS测定,采用Acquity UPLC BEH c18柱(2.1×100mm,1.7μm,Waters Co.),柱温35℃,进样量1μL。用100%(v/v)的乙腈溶液(溶液A)和0.1%(v/v)甲酸水溶液(溶液B)作为洗脱液,进行梯度清洗,流速是0.3mL/min,梯度清洗条件如表5所示。
表5梯度洗脱条件
t/min | 0-0.5 | 0.5-5.0 | 5.0-7.0 | 7.0-7.5 |
溶剂A比例 | 70% | 70-100% | 100% | 100-70% |
质谱条件:电离源为ESI源;MRM检测;MS+检测;Capillary(毛细管);3.0kV;Conc(椎体):40.00V;Source Temperature(放射源温度):120℃;Desolvation(去溶剂化)温度:400℃;Conc Gas Flow:50L/h;Desolvation Gas Flow:700L/h.气体流速为0.1ml/min;质子比扫描范围:100-2000;扫面时间1s,间隔0.061s。结果用MassLynxV4.1(Waters公司)分析;根据吸附前后PFOA的浓度差异计算乳酸菌对PFOA的吸附量。测定结果如图14所示,鼠李糖乳杆菌CCFM1060对50mg/L PFOA的吸附率为70.00%±2.88%,显著高于其他菌株。
实施例15:
鼠李糖乳杆菌CCFM1060菌剂的制备方法为:
①菌株培养基的制备:鼠李糖乳杆菌的培养基为改良MRS培养基(mMRS),配方为胰蛋白胨10g、牛肉浸膏10g、酵母粉5g、葡萄糖20g、醋酸钠5g、柠檬酸氢二铵2g、磷酸氢二钾2g、七水硫酸镁0.5g、吐温-80 1mL、一水合硫酸锰0.25g、半胱氨酸盐酸盐0.5g,水1000mL;调整其pH为6.5±0.2。
②菌株保护剂的制备:保护剂配方为:120g/L脱脂奶粉、120g/L麦芽糊精、150g/L海藻糖、余量为水,冻干,得到冻干保护剂;
③鼠李糖乳杆菌CCFM1060菌种按照以上所述培养基的质量计2%的接种量接种到在121℃条件下灭菌20min后的培养基中,在温度37℃厌氧条件下培养24h,用pH为6.8磷酸盐缓冲液清洗2~4次,用所述的保护剂重悬,使菌浓达到1010CFU/mL;接着,让该悬浮液在温度37℃厌氧条件下预培养60min,再在-15℃预冻12h,最后进行真空冷冻干燥得到所述的鼠李糖乳杆菌CCFM1060菌剂。
实施例16:
将鸭梨去皮后切块,在含有0.5%的抗坏血酸溶液中浸泡30s,捞出,榨汁,得到梨汁,将黄瓜切块,放入90℃的热水中热烫30s,按照料液比1:4打浆,过滤,得到黄瓜汁;将梨汁、黄瓜汁与脱脂乳粉以5:4:1的质量比混合,得到黄瓜汁梨汁混合乳液,均质后加入5%的木糖醇,分装到锥形瓶中于115℃灭菌20min,待其冷却至室温后利用无菌操作技术将实施例15制备的鼠李糖乳杆菌CCFM1060菌剂以107CFU/m L接种到上述混合物内,37℃恒温培养箱中发酵16h,冰浴终止发酵得到黄瓜梨汁乳酸菌饮料,于4℃保藏。
实施例17:鼠李糖乳杆菌CCFM1060对二型糖尿病小鼠的肠道Desulfovibrio属的影响
取体重16-20g的健康雄性C57BL/6J小鼠40只,适应环境1周,随机分为5组:空白对照组(NC)、模型对照组(M)、罗格列酮对照组(RH)、鼠李糖乳杆菌CCFM1060干预组(CCFM1060)、鼠李糖乳杆菌4-1对照组(4-1)每组含小鼠8只,灌胃菌悬液的剂量为3.0×109CFU/mL,重悬于3%的蔗糖溶液中。实验动物分组及处理方法见表6:
表6实验动物分组
第2-7周:正常组小鼠喂食普通饲料,其余小鼠喂食高脂饲料。
在第11周的第1d,所有小鼠禁食不禁水12h,正常组注射50mmol/L柠檬酸-柠檬酸钠缓冲液(pH 4.5),其余组注射按照100mg/(kg体重)的剂量注射50mmol/L STZ(冰上避光,现配现用),其中STZ的配制是用50mmol/L柠檬酸-柠檬酸钠缓冲液溶解。
试验末期收集小鼠新鲜粪便冻存于-80℃,提取粪便中的宏基因组,并利用二代测序仪对肠道菌群结构进行分析。试验结束时,小鼠禁食不禁水12h,腹腔注射0.5mL/10g 1%的戊巴比妥钠溶液麻醉后,心脏采血,辅以颈椎脱臼法处死。血液样本3000×g、4℃条件下离心15min,取上清,-80℃冻存用于测定相关血清指标。部分肝脏收集后迅速置于预冷的生理盐水中漂洗去血,放入多聚甲醛中固定,剩余部分肝脏于液氮中速冻并转移至-80℃冻存,后续制成肝匀浆以用于测定相关指标,具体制备方法如下:称取一定量肝脏组织,按1:9比例加入生理盐水进行组织研磨,3000r离心10min,取上清冻存于-80℃备用。
菌群分析实验结果如图15所示,二型糖尿病小鼠粪便中Desulfovibrio属的肠道微生物显著上升,而鼠李糖乳杆菌CCFM1060的摄入可将Desulfovibrio属的丰度回调,这表明本发明筛选的鼠李糖乳杆菌CCFM1060具有减少帕金森等疾病发生的功能。
实施例18:鼠李糖乳杆菌CCFM1060可促进高糖诱导的INS-1细胞的增殖及Maf AmRNA的表达
实验分成5组:正常组(含11.1mmol/L葡萄糖的普通培养液),高糖组(含22.2mmol/L葡萄糖的高糖培养液),罗格列酮组(高糖培养液+80μmol/L的罗格列酮),CCFM1060组(高糖培养液+含1*109CFU/mL CCFM1060菌液)4-1组(高糖培养液+含1*109CFU/mL 4-1菌液)。
将INS-1细胞(编号:BH-AC0530)培养于RPMI-1640培养液(含11.1mmol/L葡萄糖,10%FBS,50μmol/L 2-巯基乙醇,1mmol/L丙酮酸,10mmol/L HEPES)中,并放入37℃,5%CO2的培养箱中。
CCK-8法检测细胞增殖:将状态良好的细胞消化离心并接种于96孔板上,各孔约5×103个细胞,板的周边孔不接种细胞,为防止边缘效应同时向其中加入PBS溶液。待细胞贴壁,各孔中加入含0.5%胎牛血清的RPMI-1640培养基,同步化处理24h。同步化结束,依据分组向各孔加入相应培养基培养48h,每组设三个复孔,同时设置调零孔。药物干预结束,吸去旧培养基,PBS清洗2次,加入180μL无血清培养基和20μL CCK-8溶液,孵育3-4h。孵育结束,使用酶标仪于450nm下测定各孔吸光度值。
Maf A mRNA表达的测定:Trizol法提取RNA,吸弃6孔板中原培养液,同时用预冷的PBS清洗2次,各孔中分别加入1.0mL Trizol裂解细胞并将含细胞的裂解液转至无酶EP管,移液枪吹打至无明显沉淀静置5min。向各EP管加入0.2mL三氯甲烷,剧烈震荡15s,室温放置2-3min。4℃,12000rpm离心15min,吸取上清0.4m L左右,转入到另一无酶EP管中,加入0.5mL的异丙醇,颠倒混匀,室温静置10min。4℃,12000rpm离心10min,小心弃去上清,加入1.0mL 75%乙醇并颠倒混匀。4℃,12000rpm离心5min,弃上清,室温干燥2-5分钟。加入20μLDEPC处理水溶解,保存于80℃待用。测定RNA的浓度和质量,并按照反转录试剂盒说明书进行反转录。反转录得到的cDNA进行q RT-PCR检测,其中MafA特异性引物:F:5'-atcactctgcccaccatcac-3',R:5'-atgacctcctccttgctgaa-3'。PCR体系为:F(10μM),0.50μL;R(10μM),0.50μL;c DNA Template,1.00μL;dd H2O,3.00μL;mix,5.00μL。PCR程序:95℃,2min;(95℃,30sec;60℃,30sec;72℃,20sec)*35;72℃,5min;目的基因经过Real-timePCR检测后,采用2-△△CT法进行相对基因表达分析。先用CFX Manager软件分析各组大鼠INS-1细胞目标基因的表达量,再以正常组表达量为1,其他各组与之相比较,计算各组基因表达水平。
CCK-8法检测结果如图16所示,与正常组相比,高糖作用组细胞生长明显降低(P<0.05),罗格列酮对照组细胞增殖较高糖组有明显增加(P<0.05),CCFM1060组与高糖组相比细胞增殖状况也明显增加(P<0.05)。
Maf A mRNA表达情况如图17显示,高糖作用组细胞的MafA mRNA的表达量明显低于正常组(P<0.05),而罗格列酮阳性对照组和CCFM1060组的Maf A mRNA表达量较高糖作用组明显上升(P<0.05)。
应说明的是,以上实施例仅用以说明本发明的技术方案而非限制,尽管参照较佳实施例对本发明进行了详细说明,本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的精神和范围,其均应涵盖在本发明的权利要求范围当中。
Claims (10)
1.鼠李糖乳杆菌CCFM1060,保藏编号为GDMCC No:60705。
2.制备鼠李糖乳杆菌CCFM1060菌剂的方法,其特征在于:包括,
菌株培养基的制备;
菌株保护剂的制备;
接种培养、冻干。
3.如权利要求2所述的制备鼠李糖乳杆菌CCFM1060菌剂的方法,其特征在于:所述菌株培养基,为改良MRS培养基,其配方为胰蛋白胨10g、牛肉浸膏10g、酵母粉5g、葡萄糖20g、醋酸钠5g、柠檬酸氢二铵2g、磷酸氢二钾2g、七水硫酸镁0.5g、吐温-80 1mL、一水合硫酸锰0.25g、半胱氨酸盐酸盐0.5g,水1000mL;调整其pH为6.5±0.2。
4.如权利要求2或3所述的制备鼠李糖乳杆菌CCFM1060菌剂的方法,其特征在于:所述菌株保护剂,其配方为100g/L~150g/L脱脂奶粉、100g/L~150g/L麦芽糊精、140g/L~160g/L海藻糖、余量为水,冻干,得到冻干保护剂。
5.如权利要求4所述的制备鼠李糖乳杆菌CCFM1060菌剂的方法,其特征在于:所述菌株保护剂,其配方为120g/L脱脂奶粉、120g/L麦芽糊精、150g/L海藻糖、余量为水,冻干,得到冻干保护剂。
6.如权利要求2所述的制备鼠李糖乳杆菌CCFM1060菌剂的方法,其特征在于:所述接种培养、冻干,包括,鼠李糖乳杆菌CCFM1060以2%的接种量接种到在119~123℃条件下灭菌15~25min后的所述菌株培养基中,在温度35~39℃厌氧条件下培养24~48h,用pH为6.8~7.2磷酸盐缓冲液清洗2~4次,用所述菌株保护剂重悬,使菌浓达到1010CFU/mL;接着,让该菌株重悬液在温度37℃厌氧条件下预培养50~70min,再在-15~-20℃预冻8~14h,之后真空冷冻干燥。
7.如权利要求6所述的制备鼠李糖乳杆菌CCFM1060菌剂的方法,其特征在于:鼠李糖乳杆菌CCFM1060以2%的接种量接种到在121℃条件下灭菌20min后的培养基中,在温度37℃厌氧条件下培养24h,用pH为6.8磷酸盐缓冲液清洗2~4次,用所述的保护剂重悬,使菌浓达到1010CFU/mL;接着,让该悬浮液在温度37℃厌氧条件下预培养60min,再在-15℃预冻12h,之后真空冷冻干燥。
8.一种发酵食品,其特征在于:使用权利要求2所述的鼠李糖乳杆菌CCFM1060菌剂进行发酵,所述菌剂含有大于106CFU/g的活性鼠李糖乳杆菌CCFM1060。
9.如权利要求8所述的发酵食品,其特征在于:所述发酵食品包括乳制品、豆制品与果蔬制品。
10.如权利要求9所述的发酵食品,其特征在于:所述发酵食品,包括黄瓜梨汁乳酸菌饮料,其制备方法为,将梨榨汁得到梨汁,将黄瓜切块,放入90℃的热水中热烫30s,打浆,过滤,得到黄瓜汁;将梨汁、黄瓜汁与脱脂乳粉以5:4:1的质量比混合,得到黄瓜汁梨汁混合乳液,均质后加入5%的木糖醇,分装,115℃灭菌20min,冷却至室温后加入所述鼠李糖乳杆菌CCFM1060菌剂以107CFU/mL接种到所述黄瓜汁梨汁混合乳液,37℃恒温培养箱中发酵16h。
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