CN110229769B - 缓解pfoa毒害作用的多功能发酵乳杆菌ccfm1051、其发酵食品及应用 - Google Patents
缓解pfoa毒害作用的多功能发酵乳杆菌ccfm1051、其发酵食品及应用 Download PDFInfo
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Abstract
本发明公开了缓解PFOA毒害作用的多功能发酵乳杆菌CCFM1051、其发酵食品及应用,本发明筛选出对PFOA具有高吸附能力且不在人体中定植并且具有高抗氧化能力的益生菌,不仅能够抑制PFOA造成的氧化应激,而且能够从根本上清除人体内的PFOA。发酵乳杆菌CCFM1051可用于制备具有缓解PFOA毒性的食品、保健品和药品,具有非常广泛的应用前景。
Description
技术领域
本发明属于微生物技术领域,具体涉及缓解PFOA毒害作用的多功能发酵乳杆菌CCFM1051、其发酵食品及应用。
背景技术
全氟化合物由于其疏水疏油特性,并且具有良好的热稳定性,化学稳定性和生物稳定性,在各行各业受到了广泛的应用。用于成衣(如防水,防污户外服饰)及家居纺织品(如地毯,家具布料等),外卖食品容器,个人护理产品(如牙线)以及灭火泡沫等等。而其中PFOA(PFOA)作为是多种氟类化合物的最终转化产物之一,可以随着食物链富集。在全球各种环境介质,例如水,土壤,大气层,灰尘等,以及动物人体内中均检测到PFOA的存在,并且在人体内的半衰期为2-3年,因此受到了研究学者越来越多的重视。在2013年就作为持久,累积和毒性化学物质被《化学品注册、评估、授权和限制法规》(REACH法规)纳入《高度关注物质候选名单》,在2017年正式列入REACH法规,在欧盟各国实施限制。然而,在部分国家PFOA仍在大量使用,且环境中残留的PFOA还会在未来很长一段时间对整个生态系统造成持久影响。
暴露人群血液中PFOA含量与可能的健康影响之间的关联研究发现,暴露于PFOA可能与血液中总胆固醇浓度的升高,肝酶ALT浓度上升与出生体重降低有较明显的关系。而且已经发现PFOA暴露与疫苗接种反应减少存在关联。这些迹象表明,PFOA可能影响人体的肝脏功能,脂代谢与免疫功能。而在人体中出现的影响在哺乳动物体内已经被明确发现,PFOA具有肝脏毒性,免疫毒性,生殖毒性,发育毒性,神经毒性等多种毒性作用。PFOA可以导致肝肿大,同时可诱发小鼠肝组织氧化应激,使自由基异常增多,可能是造成肝脏损伤的主要原因。PFOA暴露对水生动物和啮齿动物免疫系统的多个免疫器官均产生不同程度的损伤,造成免疫器官脾脏和胸腺的萎缩老化,明显干扰斑马鱼脾脏白介素的表达,淋巴细胞的凋亡与衰退。在对哮喘小鼠的暴露实验中,高剂量PFOA暴露相对哮喘模型组外周血炎症因子IL-4升高,IFN-γ明显较低,即诱导Th2型免疫反应加剧肺部炎症。
目前缓解PFOA毒性的方法多是从具有高抗氧化活性的天然化学物质着手,研究中有缓解作用的如枸杞多糖、桑色素、三羟异黄酮、番茄红素等。但这些天然物质都存在价格昂贵且难以获得,此外由于人体的承受能力,大量的摄入对人体是否存在的潜在危害尚不得知。因此,寻找一种能够有效缓解PFOA毒性,并且对人体不存在其他可能的有害作用的有效方法显然十分有必要。
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,提供一种发酵乳杆菌CCFM1051,保藏编号为GDMCC No:60649。
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,其中:所述发酵食品为使用发酵乳杆菌CCFM1051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。
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、果蔬制品,所述乳制品包括牛奶、酸奶油、干酪;所述果蔬制品包括黄瓜、胡萝卜、甜菜、芹菜、圆白菜制品。
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,提供发酵乳杆菌CCFM1051在制备体内非定植益生菌中的应用。
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,提供发酵乳杆菌CCFM1051在制备缓解PFOA的毒性作用、预防和治疗便秘、抗肝病、抗高血压、抗肥胖的药物和保健品中的应用。
作为本发明所述的应用的一种优选方案:所述发酵乳杆菌CCFM1051能够吸附PFOA、清除二苯基三硝基苯肼自由基(DPPH)、清除羟自由基、抗氧化、改善因PFOA暴露造成的脾脏萎缩、降低PFOA暴露后血清中IL-4含量、升高PFOA暴露后血清中谷丙转氨酶(ALT)、谷草转氨酶(AST)、γ-谷氨酰转肽酶(γ-GT)的含量、改善PFOA暴露后的肠道菌群紊乱、降低肠道中S24-7科、乳酸菌属(Lactobacilus)的丰度、升高拟杆菌属(Bacteroides)和Eubacteriaceae科的丰度,减少肝病、高血压和肥胖的发生、提高便秘患者粪便含水量和首粒黑便排出时间、改善便秘情况。
所述发酵乳杆菌CCFM1051还能够提高高糖作用下INS-1细胞的增殖和MafA基因的表达、缓解PFOA相关糖尿病。
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,提供所述的发酵食品在制备缓解PFOA毒性、抗肝病、抗高血压、抗肥胖的功能性食品中的应用。
本发明的有益效果:本发明筛选出对PFOA具有高吸附能力且不在人体中定植并且具有高抗氧化能力的益生菌,不仅能够抑制PFOA造成的氧化应激,而且能够从根本上清除人体内的PFOA。发酵乳杆菌CCFM1051可用于制备具有缓解PFOA毒性的食品、保健品和药品,具有非常广泛的应用前景。
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为本发明菌株在体外重悬于浓度为10mg/L PFOA中经过37℃,150rpm摇床6h后,过0.22μm水系滤膜进入超高效液相色谱质谱联用仪中,吸附前后PFOA浓度变化示意图。
图2为本发明菌株在体外清除二苯基三硝基苯肼自由基(DPPH)能力(图a),清除羟自由基能力(图b)、还原能力(图c)。
图3为本发明菌株干预10天后小鼠暴露于PFOA,小鼠脾脏比的变化图。其中*P<0.05(vs模型组)。
图4为本发明菌株干预10天后小鼠暴露于PFOA,小鼠血清中白介素4示意图。其中*P<0.05,**P<0.01(vs模型组)。
图5为本发明菌株干预10天后小鼠暴露于PFOA,小鼠血清中ALT(图a)、AST(图b)、γ-GT(图c)水平示意图。其中*P<0.05,**P<0.01,***P<0.001,****P<0.0001(vs模型组)。
图6为本发明菌株干预10天后小鼠暴露于PFOA,小鼠肠道菌群α多样性变化示意图;其中*P<0.05,**P<0.01,***P<0.001(vs模型组)。
图7为本发明菌株干预10天后小鼠暴露于PFOA,小鼠肠道中S24-7科、乳酸菌属(Lactobacilus)、拟杆菌属(Bacteroides)和Eubacteriaceae科丰度的变化示意图;其中*P<0.05,**P<0.01(vs模型组)。
图8为本发明菌株干预后便秘小鼠粪便含水量的改善情况;其中*P<0.05,**P<0.01,***P<0.001(vs模型组)。
图9为本发明菌株干预后便秘小鼠首粒黑便排出时间的降低情况;其中*P<0.05,**P<0.01,***P<0.001(vs模型组)。
图10是本发明菌株对高糖作用下INS-1细胞增值情况的影响。
图11是本发明菌株对高糖作用下INS-1细胞MafA基因表达的影响。
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。
本发明的发酵乳杆菌CCFM1051,保藏于广东省微生物菌种保藏中心,保藏编号为GDMCC No:60649。
所述发酵乳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18h后进入稳定期;
(4)体外具有良好的PFOA吸附能力;
(5)体外具有良好的清除二苯基三硝基苯肼自由基(DPPH)能力,清除羟自由基能力和还原能力。
(6)发酵乳杆菌CCFM1051能显著改善PFOA暴露小鼠脾脏萎缩;
(7)发酵乳杆菌CCFM1051能够显著降低PFOA暴露小鼠血清中IL-4的含量;
(8)发酵乳杆菌CCFM1051能显著降低PFOA暴露小鼠血清中谷丙转氨酶(ALT)、谷草转氨酶(AST)和γ-谷氨酰转肽酶(γ-GT)的水平;
(9)发酵乳杆菌CCFM1051能显著降低PFOA暴露小鼠肠道中S24-7科和乳酸菌属(Lactobacilus)的丰度,提高拟杆菌属(Bacteroides)和Eubacteriaceae科丰度,改善PFOA暴露造成的肠道紊乱,减少肝病、高血压和肥胖的发生。
(10)发酵乳杆菌CCFM1051能够显著提高便秘小鼠粪便含水量和首粒黑便排出时间,明显改善小鼠的便秘情况。
(11)发酵乳杆菌CCFM1051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,缓解PFOA相关糖尿病。
所述发酵乳杆菌CCFM1051的提取方法为:
(一)乳酸菌的分离筛选:
(l)取1g健康成年人的新鲜粪便。将样品在含有山梨醇MRS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MRS固体平板上,培养24-48h;
(3)选取变色圈明显,并且符合乳酸菌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MRS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)乳杆菌的初步鉴定: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MRS培养液中培养24h,然后取lmL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MRS-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为杆状即初步判定为乳杆菌。
(三)发酵乳杆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10×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发酵乳杆菌的一种新发现的菌株,-80℃保藏备用。
实施例1:体外具有良好的PFOA吸附能力;
菌体吸附对发酵乳杆菌CCFM1051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℃培养18h。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10mg/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系统的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。
表1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。测定结果列于图1,CCFM1051对10mg/L的PFOA的吸附量为57.5%±1.5%。
实施例2:体外具有良好的清除二苯基三硝基苯肼自由基(DPPH)能力,清除羟自由基能力和还原能力;
将1mL乳酸菌完整细胞悬液与1mL新鲜配制的DPPH无水乙醇溶液(0.2mmol/L)充分混匀后,37℃条件下避光反应30min。将DPPH与PBS(pH7.2)混合作为对照样品,同样条件培养。7000×g离心10min后,在517nm测定吸光度,并按照以下公式计算乳酸菌清除DPPH自由基的能力:
DPPH自由基清除率(%)=[1-A517(样品)/A517(对照)]×100%。
将1mL1,10-邻二氮菲1mLPBS(pH7.2),1mL乳酸菌完整细胞悬液或以及1mLFeSO4混合均匀(称为“混合物1”)。向“混合物1”中加入1mLH2O2,37℃条件下水浴1.5h,在536nm测定吸光度,表示为A536(样品)。将“混合物1”中的完整细胞悬液换成同体积的蒸馏水,以同样条件培养并检测,表示为A536(空白)。将向“混合物1”加入的H2O2换成同体积的蒸馏水,以同样条件培养并检测,表示为A536(对照)。按照以下公式计算乳酸菌清除羟自由基的能力:
羟自由基清除率(%)=[A536(样品)-A536(空白)]/[A536(对照)-A536(空白)]×100%
将0.5mL乳酸菌完整细胞悬液与同体积的铁氰化钾(1%)及PBS缓冲液(pH6.6)混合,振荡使体系均匀。将蒸馏水与铁氰化钾及PBS混合作为空白对照。混合体系在50℃条件下培养20min,快速冷却,并加入0.5mL10%的三氯乙酸。在2000×g离心5min后,取1mL上清与1mL0.1%的氯化铁混合并反应10min。随后在700nm波长下测定吸光度,并使用半胱氨酸(Cysteine)作为表征还原力的标准。实验结果见图2。
实施例3:发酵乳杆菌CCFM1051能显著改善PFOA暴露小鼠脾脏萎缩;
6周龄雄性C57BL/6J小鼠50只,适应环境一周后,根据体重随机分为五组:对照组、模型组、槲皮素干预组、发酵乳杆菌CCFM1051干预组、LGG干预组,每组含10只小鼠,动物分组及处理方法见表2
表2动物实验分组及处理方法
实施例3中的小鼠于第13天称体重,随后安乐处死,取出脾脏称湿重以计算脏器系数,按以下公式计算小鼠脾脏的脏器系数:
脾脏脏器系数=脾脏湿重/安乐死前小鼠体重
实验结果如图3所示,结果表明服用发酵乳杆菌CCFM1051能够显著逆转由于PFOA染毒造成的小鼠脾脏萎缩。
实施例4:发酵乳杆菌CCFM1051能够显著降低PFOA暴露小鼠血清中IL-4的含量;
实施例4中的小鼠于第13天安乐处死。收集血清,3000g离心15min获得血清,用ELISA试剂盒检测血清中IL-4的含量。实验结果表明服用发酵乳杆菌CCFM1051能显著改善PFOA染毒造成的小鼠免疫损伤(图4)。
实施例5:发酵乳杆菌CCFM1051能显著降低PFOA暴露小鼠血清中谷丙转氨酶(ALT)、谷草转氨酶(AST)和γ-谷氨酰转肽酶(γ-GT)的水平;
取实施例3中的血清,采用全自动生化分析仪检测血清中谷丙转氨酶(ALT)、谷草转氨酶(AST)和γ-谷氨酰转肽酶(γ-GT)的含量。ALT主要存在于肝细胞原浆的可溶部分,ALT活性升高提示肝细胞遭到破坏,细胞膜通透性增强。AST主要存在于肝细胞线粒体中,AST活性提高提示线粒体损伤。实验结果表明(图5),服用发酵乳杆菌CCFM1051能够显著降低PFOA暴露小鼠血清中谷丙转氨酶(ALT)、谷草转氨酶(AST)和γ-谷氨酰转肽酶(γ-GT)的含量。表明服用发酵乳杆菌CCFM1051能够显著缓解PFOA造成的小鼠肝细胞膜结构和功能的损伤。
实施例6:发酵乳杆菌CCFM1051能显著降低PFOA暴露小鼠肠道中S24-7和Lactobacillus的丰度,提高Bacteroides、Eubacteriaceae和Eubacteriaceae丰度,改善PFOA暴露造成的肠道紊乱,减少肝病、高血压和肥胖的发生。
取实施例3中小鼠第12天的新鲜粪便,采用MP的粪便试剂盒提取小鼠粪便样品中总DNA。具体操作步骤如下主要参照试剂盒说明书进行。以小鼠粪便基因组为模板,以上游引物520F(5′-AYTGGGYDTAAAGNG-3′)、下游引物802R(5′-TACNVGGGTATCTAATCC-3′)为引物扩增16S rDNA的V3-V4区片段,目的片段长度为247bp左右。PCR反应结束,将观察到目的条带的所有PCR样品再次进行电泳,配制2.0%琼脂糖凝胶,于120V条件下电泳40min,跑胶结束后,在紫外灯下快速进行目的条带的切割。按照QIAquickGel Extraction Kit胶回收试剂盒说明书进行目的条带胶回收。根据QubitDNA3.0试剂盒检测样品DNA浓度,随后根据TurSeq DNA LT SamplePreparation Kit及其说明构建文库,最后根据MiSeq Regent Kit及其说明经过Illumina Miseq测序仪上机测定。测序完成后,剔除掉序列长度<200bp、引物序列、不能拼接的单序列,按照重叠碱基>10bp且无错配的标准拼接序列。将相似度大于97%序列定义为一个分类单元(Operational Taxonomic Unit,OTU),通过RibosomalDatabase Project(RDP)Bayesclassifier来确定物种。计算样品的α-多样性、β-多样性,用来评估样品的菌群多样性。其中α-多样性用chao1和observed species指数来表征,结果(图6)显示模型组小鼠的肠道菌群α多样性升高,说明PFOA暴露会伴随着一定程度的肠道紊乱。服用发酵乳杆菌CCFM1051能明显降低肠道菌群的α多样性,改善肠道紊乱情况。
此外,S24-7科和乳酸杆菌(Lactobacilus)丰度在PFOA染毒小鼠中显著提高,而服用发酵乳杆菌CCFM1051能够显著的逆转这一情况;S24-7高度定位于恒温动物的胃肠道,革兰氏阴性非运动型厌氧型微生物,能够发酵多种碳水化合物,和非酒精性脂肪肝和高血压的发生发展有关。乳酸杆菌(Lactobacilus)是正常胃肠道和泌尿生殖器的一部分,是常见的益生菌,在乳酸菌预防实验中PFOA模型组和染毒模型出现丰度上升的情况,乳酸菌可能在PFOA暴露后出现了反馈调节的现象。服用发酵乳杆菌CCFM1051还能显著提高PFOA染毒小鼠中拟杆菌属(Bacteroides)和Eubacteriaceae科的丰度(图7)。拟杆菌属(Bacteroides)又称类杆菌属,是拟杆菌科的一属,为革兰氏染色阴性、无芽孢、专性厌氧的小杆菌。拟杆菌属正常寄居于人和动物的肠道、口腔、上呼吸道和生殖道。拟杆菌是人和动物体内大量存在的正常菌群,约占成年个体肠道菌群的1/4以上。是肠道细菌的营养来源;能够调节多种宿主基因的表达,包括那些涉及营养吸收,黏膜屏障强化和血管因子生成的基因;激活T细胞依赖性免疫应答;影响潘氏细胞蛋白的表达;限制病原体在胃肠道的定植。而Eubacteriaceae与肝性脑病,即肝硬化中功能失调的肠-肝-脑轴的恢复有关,在严重肥胖患者的胆肠道旁路术后其丰度显著减少。以上结果表明发酵乳杆菌CCFM1051在缓解PFOA毒性的基础上,还同时具备调节肠道菌群、调节免疫及肠道屏障、减少肝病、高血压和肥胖的发生。
实施例7:发酵乳杆菌CCFM1051对小鼠便秘的缓解作用
SPF级雄性BALB/c小鼠40只(20-25g),随机分为5组:空白对照组、便秘模型对照组、发酵乳杆菌CCFM1051干预组、植物乳杆菌对照组和酚酞治疗组,每组含小鼠10只。
将发酵乳杆菌CCFM1051冻干菌粉重悬于脱脂乳粉中,制成浓度为4.0×109CFU/mL的菌悬液。实验前14天每天给干预组小鼠灌喂0.25mL的发酵乳杆菌CCFM1051脱脂乳悬液(4.0×109CFU/mL),植物乳杆菌组灌胃等量的L.plantarum ST-III,其余3组灌喂等量的不含菌的脱脂乳。试验第15-17天,阴性对照组灌胃0.25mL生理盐水,其余四组灌胃0.25mL,1mg/mL的洛哌丁胺溶液,确保小鼠洛哌丁胺的灌胃量为10mg/kgBW。
灌胃结束1h后,阴性对照组和便秘模型对照组灌胃脱脂乳,发酵乳杆菌CCFM1051干预组小鼠灌喂0.25mL的发酵乳杆菌CCFM1051(4.0×109CFU/mL),酚酞治疗对照组灌胃0.25mL的7mg/mL的酚酞溶液,确保小鼠酚酞灌胃量为70mg/kgBW。植物乳杆菌组灌胃0.25mL的L.plantarum ST-III(4.0×109CFU/mL)。
实验期间的每天收集小鼠粪便,用于小鼠粪便含水量的计算,按下式计算粪便含水量:
粪便含水量(%)=(粪便湿重-粪便干重)/粪便湿重x100。
第17天上午除空白对照组灌胃生理盐水,其余组均灌胃洛哌丁胺,灌胃1h后,对所有小鼠灌胃活性炭阿拉伯树胶水溶液0.25mL,然后将每只小鼠单独放置在一个干净的铺有吸水纸的不锈钢笼中,记录从灌胃活性炭开始到第一粒黑便排出的时间(min),作为首粒排黑便时间,用于评价发酵乳杆菌CCFM1051对小鼠便秘的缓解作用,期间小鼠自由进食和饮水。实验结果见图8和图9。
实施例8:发酵乳杆菌CCFM1051可促进高糖诱导的INS-1细胞的增殖及Maf A mRNA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),CCFM1051组(高糖培养液+含1*109CFU/mL CCFM1051菌液)LGG组(高糖培养液+含1*109CFU/mL LGG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/LHEPES)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(图11)。
CCK-8法检测结果如图10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),CCFM1051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11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CCFM1051组的Maf AmRNA表达量较高糖作用组明显上升(P<0.05)。
实施例9:利用本发明发酵乳杆菌CCFM1051制造含该菌的发酵食品
选用新鲜蔬菜洗净后榨汁,接着进行高温瞬间灭菌,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发酵乳杆菌CCFM1051菌剂发酵剂,使其浓度达到106CFU/mL以上,在温度4℃下冷藏保存,于是得到含有本发明短双歧杆菌CCFM1051活菌的果蔬饮料。
利用本发明能够使用发酵乳杆菌CCFM1051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、酸奶油、干酪;所述果蔬制品包括黄瓜、胡萝卜、甜菜、芹菜、圆白菜制品。
在PFOA模型小鼠实验中,服用CCFM1051能够显著改善因PFOA暴露造成的小鼠脾脏萎缩;服用CCFM1051能够显著降低PFOA暴露小鼠血清中白介素4(IL-4)含量;服用CCFM1051显著升高PFOA暴露小鼠血清中ALT、AST、γ-GT的含量;服用能够改善PFOA暴露小鼠的肠道菌群紊乱,降低肠道中S24-7和Lactobacillus的丰度,升高、Bacteroides和Eubacteriaceae丰度,并使肠道菌群趋于正常化,减少肝病、高血压和肥胖的发生。服用发酵乳杆菌CCFM1051能够显著提高便秘小鼠粪便含水量和首粒黑便排出时间,明显改善小鼠的便秘情况。细胞实验表明,发酵乳杆菌CCFM1051能够显著提高高糖作用下INS-1细胞的增殖情况和MafA基因的表达,能够缓解PFOA相关糖尿病。体外实验表明,发酵乳杆菌CCFM1051能够良好的吸附PFOA,能够有效地清除二苯基三硝基苯肼自由基(DPPH)、清除羟自由基、表现出良好的还原能力。
本发明筛选出对PFOA具有高吸附能力且不在人体中定植并且具有高抗氧化能力的益生菌,不仅能够抑制PFOA造成的氧化应激,而且能够从根本上清除人体内的PFOA。发酵乳杆菌CCFM1051可用于制备具有缓解PFOA毒性的食品、保健品和药品,具有非常广泛的应用前景。
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 (4)
1.发酵乳杆菌(Lactobacillus fermentum)CCFM1051,保藏编号为GDMCC No:60649。
2.一种发酵食品,其特征在于:所述发酵食品为使用权利要求1所述发酵乳杆菌(Lactobacillus fermentum)CCFM1051发酵生产制得,所述发酵食品为固态食品、液态食品或半固态食品。
3.权利要求1所述的发酵乳杆菌(Lactobacillus fermentum)CCFM1051在制备体内非定植益生菌中的应用。
4.权利要求1所述的发酵乳杆菌(Lactobacillus fermentum)CCFM1051在制备具有缓解PFOA的毒性的食品、药物或保健品中的应用。
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