CN116656542A - 一株具有缓解酒精性肝损伤功能的发酵粘液乳杆菌及其应用 - Google Patents
一株具有缓解酒精性肝损伤功能的发酵粘液乳杆菌及其应用 Download PDFInfo
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- CN116656542A CN116656542A CN202310515104.5A CN202310515104A CN116656542A CN 116656542 A CN116656542 A CN 116656542A CN 202310515104 A CN202310515104 A CN 202310515104A CN 116656542 A CN116656542 A CN 116656542A
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- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
本发明涉及微生物领域,提供一株具有缓解酒精性肝损伤功能的发酵粘液乳杆菌及其应用。发酵粘液乳杆菌(Limosilactobacillus fermentum)命名为2438菌株,于2023年3月3日在广东省微生物菌种保藏中心保藏,微生物保藏编号为GDMCC No:63220。该菌株具有良好的肠道定植效果,对胃酸、胆盐具有良好的耐特受性,能显著改善慢性酒精性肝损伤,降低酒精对肝脏的氧化损伤,延缓酒精性脂肪肝的形成,因此可以应用于缓解酒精性肝损伤功能。此外本发明菌株灭活后依旧具备益生功能,因此本菌种的适用范围更广,产品形式更加丰富。
Description
技术领域
本发明涉及微生物领域,尤其是涉及一株具有缓解酒精性肝损伤功能的发酵粘液乳杆菌及其应用。
背景技术
酒精性肝损伤是由于长期大量饮酒所致的肝脏疾病。酒精性肝损伤的致病因子明确,即与大量酗酒有关。经口摄入的乙醇在胃肠道内吸收很快,仅5%~10%从肺、肾脏、皮肤排出,主要在小肠空肠段为中心的消化管内吸收,吸收后约90%左右进入肝脏代谢。乙醇氧化的ADH途径会消耗大量的辅酶I(NAD),使NAD+/NADH比值变小,使三羧酸循环受到抑制,影响细胞能量供给。乙醇还可以损害线粒体的功能,使线粒体脂质过氧化物增加,GSH水平下降线粒体形态异常。乙醇氧化过程中的中间产物乙醛也是高活性物质,当乙醛脱氢酶活性降低时,未被氧化的乙醛释放入血,通过黄嘌呤氧化酶转变为超氧化物,导致脂质过氧化,破坏细胞膜,促进肝损伤。乙醛还可以影响线粒体、微管的功能,可以与各种蛋白质结合形成乙醛复合体,成为一个新的抗原刺激,使肝细胞受损。
目前国内外上已经出现很多解酒护肝药物,例如专利CN101978998B公开了解酒护肝药物组合物及制备方法,采用药食同源的原料:枳椇子,葛根、车前草、菊花制成,可以有效地快速分解酒精毒素。但大部分药物都有不同程度的副反应,长期服用保肝药不仅会增加肝脏的负担,同时一些保肝药长期在体内蓄积,还会对肝脏造成损害。因此,获得临床理想安全且可长期服用的预防药物具有重要意义。
已有研究表明乳酸菌能够降低自由基对肝脏的氧化损伤作用,降低炎症因子的产生,改善肠道菌群平衡,降低肠道粘膜的损伤,最终起到保护肝脏的作用。而乳酸菌正是健康个体肠道菌群的重要组成部分,其在护肝功能方面存在一定的潜力。
发明内容
本发明提供一株具有缓解酒精性肝损伤功能的发酵粘液乳杆菌-2438菌株,该菌株具有良好的耐胃酸、胆盐特性和黏附肠细胞特性,可以顺利到达肠道并黏附于肠细胞上,减缓酒精对肠细胞的损伤;所述菌株有较好的抗氧化特性,能降低酒精对肝脏的氧化损伤,延缓酒精性脂肪肝的形成;同时其菌体灭活后依旧有相应功能,有更丰富的产品形式和应用范围。
为了实现上述目的,本发明采用以下技术方案:
一株具有缓解酒精性肝损伤功能的发酵粘液乳杆菌,分离自新疆奶疙瘩样品,经鉴定其属于发酵粘液乳杆菌(Limosilactobacillus fermentum),命名为2438菌株,于2023年3月3日在广东省微生物菌种保藏中心保藏,微生物保藏编号为GDMCC No:63220。
所述2438菌株的生物学性质如下:
(1)形态学特征:在MRS琼脂培养基生长形态为乳白色菌落、不透明、圆形、表面光滑湿润、边缘整齐、中央凸起。革兰氏染色呈典型阳性,显微镜下观察细胞呈短杆状,有圆形末端,呈单个、成对出现,无鞭毛,不产芽孢,不运动。
(2)培养特征:最适生长温度为37℃,兼性厌氧,生长于MRS培养基中。培养基成分:蛋白胨10g,牛肉膏10g,酵母膏5g,葡萄糖20g,乙酸钠5g,K2HPO4 2g,MgSO4·7H2O0.2g,MnSO4·4H2O 0.2g。
(3)生物学鉴定:鉴定采用16s rDNA PCR扩增。扩增序列测序后所得结果于NCBI的GenBank数据库中进行同源性比对分析,结果显示该菌株为(Limosilactobacillusfermentum)2438。
所述2438菌株具有以下优点:
(1)本发明的发酵粘液乳杆菌(Limosilactobacillus fermentum)2438菌株对DPPH和羟自由基有较高的清除率,表明该菌株有良好的抗氧化特性,且效果显著高于商业菌株。
(2)本发明的发酵粘液乳杆菌(Limosilactobacillus fermentum)2438菌株具有良好的耐酸耐胆盐特性和黏附性。在pH=2.5酸环境作用下4h后,活菌数仅降低0.27个数量级;在0.3%(w/v)胆盐作用8h后,活菌数仅降低0.25个数量级;同时该菌株还具有良好的黏附特性。因此,所述发酵粘液乳杆菌2438能够顺利到达肠道,并与肠道上皮细胞发生黏附,发挥其益生作用。
(3)本发明的发酵粘液乳杆菌(Limosilactobacillus fermentum)2438菌株经体外细胞实验证明,该菌株能减缓酒精对肠细胞的损伤作用,添加发酵粘液乳杆菌2438的肠细胞存活率达52.5%,且具有良好的肠屏障保护功能,存在益生菌的情况下,3h肠屏障损失率降至(16.62±2.61)%。
(4)本发明的发酵粘液乳杆菌(Limosilactobacillus fermentum)2438菌株经大鼠动物实验证明,灌胃该菌株的大鼠血清谷丙转氨酶、谷草转氨酶及肝脏中甘油三酯有所降低,说明该菌株能减缓酒精对肝脏细胞的损伤,降低酒精代谢对肝脏的氧化损伤,减缓酒精性脂肪肝的形成。
(5)本发明的发酵粘液乳杆菌(Limosilactobacillus fermentum)2438菌株灭活后依旧具备与活菌相当的益生功能,灭活菌耐温性强,拓展了该菌株的使用范围,产品形式更加丰富。
(6)本发明使用的发酵粘液乳杆菌(Limosilactobacillus fermentum)2438菌株是被囊括于卫生部2010年颁布的《可用于食品的菌种名单》的食品级微生物。
本发明还提供所述发酵粘液乳杆菌在具有缓解酒精性肝损伤功能的组合物中的应用。
本发明还提供一种菌体培养物,含有权利要求1所述发酵粘液乳杆菌。
作为优选,所述发酵粘液乳杆菌经过灭活处理。
作为优选,所述菌体培养物为菌液或菌剂。
作为优选,所述菌液的浓度为0.5~2×109CFU/mL。
本发明还提供所述菌体培养物在具有缓解酒精性肝损伤功能的组合物中的应用。
附图说明
图1为本发明分离得到的发酵粘液乳杆菌新菌株2438的菌株外观特性。其中,菌株菌落特征如图1A所示,革兰氏染色显微镜观察特征如图1B所示。
图2为本发明中小鼠的肝体比(HIS)。其中,CK为对照组、Model为模型组、L2438为菌处理组(低)、M2438为菌处理组(中)、H2438为菌处理组(高)、D2438为菌处理组(死),详见实施例6的表7,下同。
图3为本发明中小鼠血清的谷草转氨酶酶活(GOT)。
图4为本发明中小鼠血清的谷丙转氨酶酶活(GPT)。
图5为本发明中小鼠肝脏的甘油三酯(TG)含量。
图6为本发明中小鼠肝脏的还原型谷胱甘肽(GSH)含量。
图7为本发明中小鼠肝脏的HE染色切片。
具体实施方式
下面通过具体实施例,对本发明的技术方案做进一步说明。
本发明中,若非特指,所采用的原料和设备等均可从市场购得或是本领域常用的,实施例中的方法,如无特别说明,均为本领域的常规方法。
本发明提供一株具有缓解酒精性肝损伤功能的发酵粘液乳杆菌,分离自新疆奶疙瘩样品,经鉴定其属于发酵粘液乳杆菌(Limosilactobacillus fermentum),命名为2438菌株,于2023年3月3日在广东省微生物菌种保藏中心保藏,微生物保藏编号为GDMCC No:63220。
所述2438菌株的生物学性质如下:
(1)形态学特征:在MRS琼脂培养基生长形态为乳白色菌落、不透明、圆形、表面光滑湿润、边缘整齐、中央凸起。革兰氏染色呈典型阳性,显微镜下观察细胞呈短杆状,有圆形末端,呈单个、成对出现,无鞭毛,不产芽孢,不运动。如图1所示,其中,菌株菌落特征如图1A所示,革兰氏染色显微镜观察特征如图1B所示。
(2)培养特征:最适生长温度为37℃,兼性厌氧,生长于MRS培养基中。培养基成分:蛋白胨10g,牛肉膏10g,酵母膏5g,葡萄糖20g,乙酸钠5g,K2HPO4 2g,MgSO4·7H2O 0.2g,MnSO4·4H2O 0.2g。
(3)生物学鉴定:鉴定采用16s rDNA PCR扩增。扩增序列测序后所得结果于NCBI的GenBank数据库中进行同源性比对分析,结果显示该菌株为(Limosilactobacillusfermentum)2438。
实施例1发酵粘液乳杆菌2438的抗氧化特性
1.清除DPPH自由基的能力
本发明菌株发酵粘液乳杆菌2438和对照商业菌株鼠李糖乳杆菌GG(LGG)经二代活化后,取对数生长末期菌液,4000rpm离心10min,弃上清液,获得菌泥,PBS(pH=7.4)洗涤2次,菌悬液OD600调至0.5±0.1。在反应体系中加入待测益生菌的菌悬液1m L,再加入1mL0.1mmol/L DPPH的无水乙醇溶液,充分混匀后,室温下避光振荡反应30min,然后经过6000rpm离心10min,取上清液,测定517nm处的吸光度(OD值)。以等体积的生理盐水代替样品溶液作为对照组,并用等体积的生理盐水和无水乙醇的混合液作为空白调零。按下式计算DPPH自由基的清除率:
DPPH自由基清除率=(A对照-A样品)/A对照×100%
DPPH是一种很稳定的人工合成的以氮为中心的自由基,DPPH法是一种极为常见的用于筛选和评价抗氧化效果的有效方法。结果如表1所示,本发明菌株发酵粘液乳杆菌2438菌体相比鼠李糖乳杆菌GG对DPPH自由基有更高的清除效果。
表1不同菌株对DPPH自由基清除的效果
菌株 | 对照商业菌LGG | 发酵粘液乳杆菌2438 |
清除率/% | 24±1.52 | 52±2.55 |
2.清除羟自由基的能力
本发明菌株发酵粘液乳杆菌2438和对照商业菌株鼠李糖乳杆菌GG(LGG)经二代活化后,取对数生长末期菌液,4000rpm离心10min,弃上清液,获得菌泥,PBS(pH=7.4)洗涤2次,菌悬液OD600调至0.5±0.1。在反应体系中加入OD值为5的发酵粘液乳杆菌WHH3906菌悬液1mL,再加入1mL生理盐水和1mL FeSO4(3mmol/L)。混匀后加入1mL H2O2(3mmol/L),室温静置10min后加入1mL水杨酸(3mmol/L,乙醇溶解),混匀,37℃水浴20min,离心取上清测定510nm处的吸光值。以等体积的生理盐水代替样品溶液作为对照组,并用等体积的生理盐水和无水乙醇的混合液作为空白调零。
按照下式计算羟自由基的清除率:羟自由基清除率=(As-Ap)/As×100%
式中,Ap:菌液的OD510值,As:菌悬液换成0.9%的生理盐水的OD510值。
羟自由基是活泼性最强、氧化性最大的自由基,对DNA、蛋白质和脂类有较强的结合能力,是引起体内氧化损伤的主要因素,所得结果如表2所示,本发明菌株2438的羟自由基清除率为55%,显著优于商业菌株LGG,说明发酵粘液乳杆菌2438在抗氧化能力方面有更优秀的性能。
表2不同菌株对羟自由基的清除效果
菌株 | 对照商业菌LGG | 发酵粘液乳杆菌2438 |
清除率/% | 37±3.52 | 55±4.21 |
实施例2发酵粘液乳杆菌2438的耐受特性
将本发明菌株与市售商业菌株均培养至对数生长末期后,获取菌悬液,将菌悬液分成2组,分别进行耐酸特性与耐胆盐特性的测定,操作如下:
(1)取一定量菌液,4000g/10min离心后弃上清。加入相同体积pH=2.5的MRS溶液,吹打混匀后,在37℃环境下孵育,用稀释涂布计数法测定0点及孵育2h、4h后菌数的变化。(2)取4000g菌液,10min离心后弃上清。加入相同体积的含0.3%(w/v)胆盐的MRS溶液,吹打混匀后,在37℃环境下孵育,用稀释涂布计数法测定0点及孵育4h、8h后菌数的变化。
菌株存活率计算公式:菌株存活率(%)=N1/N0*100%。
式中,N1为菌株处理后活菌数,N0为菌株初始活菌数。
结果如表3所示,发酵粘液乳杆菌2438表现出良好的耐酸、耐胆盐特性。在酸环境下孵育4h后,菌数降低0.27数量级。在胆盐环境下孵育8h后,菌数降低2.27数量级。说明该菌株不管在酸环境下还是胆盐环境下,耐受性均优于对照的商业菌株。
表3不同菌株耐酸性、耐胆盐测试结果
实施例3发酵粘液乳杆菌2438的肠道黏附能力
本发明菌株与对照商业菌株培养至对数末期后收集菌液。4000g,10min离心后,用含10%(v/v)胎牛血清的DMEM完全培养基重悬,并调整菌液菌数为2×108CFU/mL。HT29细胞培养后,采用胰酶进行消化,用含10%(v/v)胎牛血清的DMEM培养基(含青霉素100U/mL,链霉素100mg/mL)进行重悬。经细胞计数后,将细胞悬液浓度调整至1×106cells/mL,接种于已放置细胞爬片的12孔细胞培养板中,37℃,5%(v/v)CO2培养24h。加入受试菌菌悬液1mL,37℃条件下于CO2培养箱中孵育2h。孵育结束后,经PBS洗涤3次、甲醇固定后,取出细胞爬片。经革兰氏染色后,用中性树脂封片,光学显微镜下进行观察。
结果如表4所示,发酵粘液乳杆菌2438的黏附率略优于对照商业菌。
表4不同菌株的肠道黏附能力
菌株 | 发酵粘液乳杆菌2438 | 对照菌 |
黏附率(菌数/细胞数) | 7.01±0.65 | 3.75±0.30 |
实施例4发酵粘液乳杆菌2438抑制酒精损伤肠细胞能力
HT29细胞的培养:从-80℃低温冰箱中取出HT-29细胞进行复苏,复苏后的HT-29细胞传至第三代,采用0.25%(w/v)的胰酶(含EDTA)消化后1000rpm离心5min,加入5mL含10%(v/v)胎牛血清的DMEM培养基(含青霉素100U/mL,链霉素100mg/mL)进行重悬(直至细胞呈单细胞悬液),取适量细胞用血球细胞计数板计数,根据计数结果,用含10%(v/v)胎牛血清的DMEM培养基(含青霉素100U/mL,链霉素100mg/mL)将细胞悬液浓度稀释到1×105cells/mL,取1mL接种于已放置细胞爬片的96孔细胞培养板中,于37℃,5%(v/v)CO2培养1d。
益生菌对抑制乙醇损伤肠细胞能力方法:将活化后的益生菌菌液进行清洗,浓度为1OD/mL,以DMEM培养基为对照,以含10%(v/v)酒精DMEM培养基为处理组,于37℃、含5%(v/v)CO2浓度的培养箱中孵育30min后清洗一次,之后用MTT法测定活性。
结果如表5所示,添加发酵粘液乳杆菌2438的肠细胞存活率远高于没有添加益生菌的肠细胞,达52.5%,也高于对照商业菌LGG,说明发酵粘液乳杆菌2438有较好的抑制乙醇对肠细胞的损伤。
表5不同菌株抑制酒精损伤肠细胞的能力
菌株 | 发酵粘液乳杆菌2438 | 对照商业菌LGG | 模型/不加菌 |
细胞存活率/% | 52.50±3.65 | 31.53±4.15 | 18.46±0.61 |
实施例5发酵粘液乳杆菌2438的肠屏障保护功能
IPEC单层细胞的培养:从-80℃低温冰箱中取出IPEC细胞进行复苏,复苏后的IPEC细胞传至第三代,采用0.25%(w/v)的胰酶(含EDTA)消化后1000rpm离心5min,加入5mL含10%(v/v)胎牛血清的DMEM培养基(含青霉素100U/mL,链霉素100mg/mL)进行重悬(直至细胞呈单细胞悬液),取适量细胞用血球细胞计数板计数,根据计数结果,用含10%(v/v)胎牛血清的DMEM培养基(含青霉素100U/mL,链霉素100mg/mL)将细胞悬浮,取0.2mL接种于Transwell小室中,于37℃,5%(v/v)CO2培养7d以上。
益生菌菌株的活化:分别以1%的接种量接种于液体MRS培养基中,在37℃恒温培养箱中,经活化培养后,收集菌液。每株菌取出3mL菌液,测定菌液OD600,用空白培养基进行适当稀释,至OD600=0.90±0.10。剩余菌液取5mL在4200g室温下离心10min收集菌体,用5mL含10%(v/v)胎牛血清的DMEM完全培养基中培养(不加双抗)重悬,并根据之前的稀释倍数,将菌液OD统一稀释至0.900。
益生菌保护肠屏障实验:将活化后的益生菌菌液进行清洗,浓度为0.9OD/mL,以DMEM培养基为空白,以含2.5%(v/v)酒精和益生菌的DMEM培养基为处理组,仅含2.5%(v/v)酒精DMEM培养基为对照组于37℃,含5%(v/v)CO2浓度的培养箱中孵育30min、1h、2h、3h,用电阻仪测定细胞屏障通透性。
结果如表6所示,2.5%(v/v)酒精会破坏肠细胞的屏障功能,而存在益生菌的情况下,肠屏障损失率有所减少,说明发酵粘液乳杆菌2438有较好的保护肠屏障的功能,且优于对照商业菌LGG。
表6不同菌株的肠屏障保护功能
实施例6发酵粘液乳杆菌2438缓解酒精性肝损伤功能
本实验采取随机分组设计,将健康的雄性C57BL/6小鼠(8周龄,23~24g),72只,食用普通饲料适应7天后,随机分为10组,对照组12只,模型组12只,各益生菌处理组12只。动物饲养保持环境温度为21±2℃,湿度为30-70%,12h光照交替,自由饮水,摄入饲料需人为控制。每三天换一次垫料。液体饲料购自江苏南通特洛菲饲料科技有限公司,酒精液体饲料的酒精浓度为4%(w/v)。
分组及处理方式如表7所示。
表7菌株缓解酒精性肝损伤功能实验的分组及处理方式
整个实验周期为7周,第一周为适应期,酒精浓度(w/v)从0、1.6%、2.4%、3.2%上升到4%,之后持续6周后,过夜禁食后进行眼眶取血获取血液样本,取肝脏并称重,血液样本取出后,静置30min,4℃,4000rpm离心15min,取血清。小鼠血清谷草转氨酶(GOT)、谷丙转氨酶(GPT)、肝脏甘油三酯(TG)、谷胱甘肽(GSH)含量的测定,严格按照相应试剂盒(购自北京普利莱基因技术有限公司)说明书的具体操作步骤进行。
图2-6的结果显示,模型小鼠在血清指标、肝脏指标均与对照组有显著的差异(p<0.05),说明模型小鼠的酒精性肝损伤症状明显,模型成立。图2结果显示,高剂量组和死菌组小鼠的肝体比与模型组有显著性差异(p<0.05)。图3、图4结果显示,高剂量组和死菌组的谷丙转氨酶和谷草转氨酶有明显的下降,接近对照组水平,相比模型组均有显著性的差异。所述死菌组即灭活菌组。从图5、图6发现,高剂量组和死菌组的肝脏指标甘油三脂和还原型谷胱氨肽有显著的下降,说明高剂量组和死菌组有较好的缓解酒精性肝损伤功能。图7的肝脏HE染色切片中也可验证,高剂量组和死菌组的小鼠肝脏中的脂肪颗粒明显比模型小鼠的少。发明人经过实验发现,所述菌悬液或者死菌悬液的浓度为0.5~2×109CFU/mL(用量0.2mL)时,具有“高剂量组”的优势,即具有相对较好的缓解酒精性肝损伤功能。
以上所述,仅是本发明的较佳实施例而已,并非对本发明作任何形式上的限制,虽然本发明已以较佳实施例揭露如上,然而并非用以限定本发明,任何熟悉本专业的技术人员,在不脱离本发明技术方案范围内,当可利用上述揭示的技术内容作出些许更动或修饰为等同变化的等效实施例,但凡是未脱离本发明技术方案内容,依据本发明的技术实质对以上实施例所作的任何简单修改、等同变化与修饰,均仍属于本发明技术方案的范围内。
Claims (8)
1.一株具有缓解酒精性肝损伤功能的发酵粘液乳杆菌,其特征在于,属于发酵粘液乳杆菌 (Limosilactobacillus fermentum),命名为2438菌株,于2023年3月3日在广东省微生物菌种保藏中心保藏,微生物保藏编号为GDMCC No:63220。
2.权利要求1所述发酵粘液乳杆菌在具有缓解酒精性肝损伤功能的组合物中的应用。
3.一种菌体培养物,含有权利要求1所述发酵粘液乳杆菌。
4.根据权利要求3所述的菌体培养物,其特征在于,所述发酵粘液乳杆菌经过灭活处理。
5.根据权利要求3所述的菌体培养物,其特征在于,所述菌体培养物为菌液。
6.根据权利要求5所述的菌体培养物,其特征在于,所述菌液的浓度为0.5~2×109CFU/mL。
7.根据权利要求3所述的菌体培养物,其特征在于,所述菌体培养物为菌剂。
8.权利要求3所述菌体培养物在具有缓解酒精性肝损伤功能的组合物中的应用。
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