CN115232761B - 一种具有改善afb1引起的肝损伤及肠菌失衡的植物乳杆菌 - Google Patents
一种具有改善afb1引起的肝损伤及肠菌失衡的植物乳杆菌 Download PDFInfo
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Abstract
本发明提供了一种具有改善AFB1引起的肝损伤及肠菌失衡的植物乳杆菌,属于微生物技术领域。本发明提供的植物乳杆菌CGMCCNo.24527热灭活后制备生物制剂,并将其应用于改善AFB1引起的肝损伤和肠菌失衡的产品中。能够调节肠道菌群,显著减少了Muribaculaceae和Alloprevotella的丰度,增加了乳酸杆菌属(Lactobacillus)的丰度;减少AFB1的吸收,促使AFB1的排出;并且能够有效改善AFB1引起的肝损伤以及肠道菌群失调。
Description
技术领域
本发明涉及微生物技术领域,具体涉及一种具有改善AFB1引起的肝损伤及肠菌失衡的植物乳杆菌。
背景技术
在目前已知的黄曲霉毒素中,黄曲霉毒素B1(AFB1)是毒性最强、危害最大、分布最广的一种生物毒素,在多种食物及食品原料中均有检出。AFB1在人体中具有极高的蓄积性,一旦摄入几乎很难排出体外;同时,AFB1表现出明显的肝毒性、免疫毒性、致突变性、致癌性和致畸性,是肝癌的重要致病因子,还会引发机体生长抑制、炎症反应和营养不良等健康问题。因此,国际癌症研究机构(International Agency for Research on Cancer, IARC)已将AFB1列为第一类致癌物。
预防食物霉变和加强源头检测仍然是目前控制AFB1染污的主要技术手段,但由于AFB1极易在食品中存在,即使食物中含量很低,但长期食用,仍然会在体内造成较高的蓄积量,进而引起不可逆转的严重后果。对于高温高湿地区的人而言,由于食物极易霉变,暴露于AFB1的风险会更高。
因此,如何有效降低AFB1在机体内的残留,并减轻其毒性作用成为本领域亟待解决的技术问题。虽然有活性的微生物可以有效吸附AFB1,但其贮存条件要求苛刻,而灭活的微生物则可以避免活性微生物不易贮存的缺点,具有更广泛的应用潜力。
发明内容
要解决的技术问题:本发明的目的是提供一种具有改善AFB1引起的肝损伤及肠菌失衡的植物乳杆菌,能够降低AFB1在体内的残留量,从而减少机体的吸收,有效改善AFB1引起的肝损伤以及肠道菌群失调。
技术方案:一种植物乳杆菌(Lactobacillus plantarum T3),所述植物乳杆菌已于2022年04月22日保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCC No. 24527。
本发明还提供了含有上述植物乳杆菌的微生物制剂,所述植物乳杆菌为热灭活的。
在本发明的一种实施方式中,所述的微生物制剂,其特征在于,所述微生物制剂中,热灭活的植物乳杆菌的菌数不低于1×106CFU/mL或1×106CFU/g。
本发明还提供了上述微生物制剂在制备改善AFB1引起的肝损伤的产品中的应用。
本发明还提供了上述微生物制剂在制备改善AFB1引起的肠菌失衡的产品中的应用。
在本发明的一种实施方式中,所述的改善AFB1引起的肝损伤的产品中,热灭活的植物乳杆菌的菌数不低于1×106CFU/mL或1×106CFU/g。
在本发明的一种实施方式中,所述的改善AFB1引起的肠菌失衡的产品中,热灭活的植物乳杆菌的菌数不低于1×106CFU/mL或1×106CFU/g。
在本发明的一种实施方式中,所述的改善AFB1引起的肝损伤的产品包括药物。
在本发明的一种实施方式中,所述的改善AFB1引起的肠菌失衡的产品包括药物。
在本发明的一种实施方式中,所述的改善AFB1引起的肠菌失衡的产品,产品效果在于调节肠道菌群,包括调节厚壁菌门和拟杆菌门的丰度。
有益效果:
本发明中热灭活的植物乳杆菌T3(iT3)能够调节肠道菌群,显著减少了Muribaculaceae和Alloprevotella的丰度,增加了乳酸杆菌属(Lactobacillus)的丰度。一些研究表明,Muribaculaceae与肠道生态失调有关,它能够编码降解粘蛋白聚糖的酶,参与小鼠结肠粘蛋白的降解,破坏肠道屏障,增加肠粘膜的通透性,从而使 AFB1更容易穿透肠道粘膜屏障进入血液循环,引起中毒;而Alloprevotella易引起肝脏损伤。
本发明的热灭活植物乳杆菌T3(iT3)能够高效吸附黄曲霉毒素B1(AFB1),减少AFB1的吸收;并且能够有效改善AFB1引起的氧化应激损伤以及肠道菌群失衡。
本发明的热灭活植物乳杆菌T3(iT3)所具有的吸附效应以及增加免疫球蛋白(IgG、IgM)的生成可促进粪便中AFB1的排泄。
本发明的热灭活植物乳杆菌T3(iT3)能够保护肝脏正常形态,维护肝脏的解毒功能。
本发明的热灭活植物乳杆菌T3(iT3)能够起到免疫调节作用,增强机体对AFB1的免疫应答作用。
AFB1可以破坏小鼠的食欲,产生厌食行为,从而严重影响小鼠生长性能,而热灭活植物乳杆菌T3(iT3)能有效改善AFB1对小鼠生长发育的不利影响,使小鼠体重稳定增加。
附图说明
图1为对照组、植物乳杆菌T3和热灭活植物乳杆菌T3(iT3)对AFB1的吸附率;
图2为喂养30天后各组小鼠体重变化;
图3为实施例2中小鼠粪便中AFB1含量;
图4为实施例2中正常对照组(Control)小鼠肝脏切片HE染色;
图5为实施例2中AFB1组小鼠肝脏切片HE染色;
图6为实施例2中AFB1+iT3组小鼠肝脏切片HE染色;
图7为实施例2中小鼠肝脏器官指数;
图8为实施例2中小鼠肝脏中GSH含量;
图9为实施例2中小鼠肝脏中MDA含量;
图10为实施例2中小鼠血清中ALT含量;
图11为实施例2中小鼠血清中AST含量;
图12为实施例2中小鼠血清中Cr含量;
图13为实施例2中小鼠血清中TNF-α含量;
图14为实施例2中小鼠血清中IL-4含量;
图15为实施例2中小鼠血清中IgG含量;
图16为实施例2中小鼠血清中IgM含量;
图17为实施例2中小鼠粪便菌群微生物属水平上的PLS-DA物种分析;
图18为实施例2中小鼠粪便菌群微生物门水平上的物种组成;
图19为实施例2中小鼠粪便菌群微生物属水平上的物种组成;
图20为实施例2中小鼠粪便菌群微生物韦恩图;
图21为实施例2中AFB1组特有粪便菌群微生物组成图;
图22为实施例2中小鼠显著差异的粪便菌群微生物(丰度>1%)。
具体实施方式
本发明提出了一种具有改善AFB1引起的肝损伤及肠菌失衡的植物乳杆菌,为使本发明的目的、技术方案及效果更加清楚、明确,以下将配合实施例来对本发明进一步详细说明。应当理解,此处所描述的具体实施例仅用以解释本发明,并不用于限定本发明。
植物乳杆菌T3菌株的分离、鉴定及其热灭活形式的制备
于黑龙江地区从粘面子中分离得到植物乳杆菌T3菌株,经16S rRNA鉴定,T3菌株为植物乳杆菌(Lactobacillus plantarun)。将在液体培养基中培养的植物乳杆菌T3在100℃灭活30 min得到其热灭活的菌剂—热灭活植物乳杆菌T3(iT3),此时的菌株已无生命活动,不能生长繁殖。
实施例1
热灭活植物乳杆菌T3(iT3)对黄曲霉毒素B1(AFB1)的吸附率
将菌体浓度为1010 CFU/mL的活的和热灭活的植物乳杆菌T3离心(3000 r/min, 4℃,10 min),收集菌体沉淀,重悬于无菌PBS缓冲液中。向990 μL PBS菌悬液中加入10 μLAFB1工作液(100 μg/mL),混匀,37 ℃共培养30 min,离心。向收集的上清液中加入2倍体积的二氯甲烷,剧烈震荡10 min,静置分层,收集下层液,重复该过程2次。将2次收集到的下层液合并后真空干燥,复溶于等体积的35%甲醇,用ELISA试剂盒(上海酶联生物科技有限公司)检测上清液中AFB1的残留量,通过下列公式计算出植物乳杆菌T3和热灭活植物乳杆菌T3(iT3)对AFB1的吸附率。
式中:A1为添加活的或热灭活的植物乳杆菌T3时,上清液中AFB1含量;A0为不添加任何形式的植物乳杆菌T3时,上清液中AFB1含量(即对照组)。
实验数据用平均值和标准误差(平均值±标准误差)表示,图中不同的小写字母表示不同处理组之间存在显著性差异,即P<0.05。采用Excel软件进行试验数据的处理,应用SPSS 25软件进行试验的差异性分析。
如图1所示,当向AFB1溶液中加入热灭活植物乳杆菌T3(iT3)共同培养30 min后,iT3对AFB1的吸附率达到84.0%,显著高于其T3的吸附率(68.5%),极大地降低了原溶液中毒素的残留量。
可见,iT3具有减少AFB1在体内蓄积,进而减轻其毒性的潜力。
实施例2
1、动物饲养实验设计
选用18-22 g的ICR雄性小鼠(5周龄,辽宁长生生物技术股份有限公司,合格证编号:No.210726210100917863),在25 ℃、12 h光照/黑暗循环、相对湿度50%±5%的标准条件下饲养。涉及小鼠的程序严格按照国际动物护理标准进行,并经吉林农业大学动物保护机构和使用委员会批准。
将小鼠随机分为3组,每组7只:
(1)对照组(Control):正常饮食(饲料来自北京科澳协力饲料有限公司);
(2)AFB1组:正常饮食+AFB1(300 μg /kg bw);
(3)AFB1+iT3组:正常饮食+AFB1(300 μg /kg bw)+热灭活植物乳杆菌T3(4.0×1010 CFU)
实验期间均自由进食和饮水,连续喂养30天,对照组每天灌胃等量的生理盐水。每天记录小鼠体重、健康状态和进食情况。在喂养第1天,在小鼠灌胃AFB1或植物乳杆菌T3后的12小时收集粪便,检测其中AFB1残留量。其它实验均在喂养30天后,禁食12h,采集血液、肝脏和粪便,用于后续分析。
2、结果分析:
2.1、体重变化
每天记录小鼠的体重。
如图2所示,动物实验结束时,正常对照组的小鼠体重与第1天相比增加了76.1%,显著高于其他两组(P<0.05)。AFB1组小鼠的平均体重最低,与第1天相比,仅增长了51.4%。而AFB1+iT3组的小鼠平均体重增长了68.5%。
可见,热灭活植物乳杆菌T3能够有效改善AFB1对小鼠生长发育的不利影响。
2.2、 AFB1的排泄量检测
在动物实验的第1天,小鼠灌胃AFB1或AFB1+iT3后的12小时,收集粪便,经液氮冷冻后研磨成粉,用5倍体积的苯-乙腈将其溶解,离心15 min(2000 r/min),上清液经0.22 μm微孔滤膜过滤后冻干,重新溶于35%的甲醇溶液中,使用ELISA试剂盒(上海酶联生物科技有限公司)检测粪便中AFB1的含量。
如图3所示,AFB1组小鼠粪便中仅含有0.9 μg/g的AFB1。而AFB1+iT3组的小鼠粪便中AFB1的含量分别为1.8 μg/g,排泄量是AFB1组的2倍(P<0.05)。
可见,热灭活植物乳杆菌T3促进了AFB1的排泄,减少了毒素在体内的蓄积,具有应用于制备AFB1吸附剂产品中的潜力。
2.3、肝脏组织病理学与氧化损伤分析
动物实验第30天时,将小鼠禁食12 h,迅速取肝脏,用生理盐水冲洗干净,吸干水分后称重。将肝脏在10%福尔马林固定液中处理48 h后,包埋于石蜡中,切成厚度约5 μm的切片进行HE染色,并通过下列公式计算出小鼠的肝脏指数。
式中:B1为肝脏重量,B0为小鼠体重。
准确称取小鼠肝脏组织,放入冷生理盐水中,经过组织研磨得到10%的肝脏组织匀浆,离心15 min(4000 r/min, 4 ℃),收集上清液,使用谷胱甘肽(GSH)和丙二醛(MDA)试剂盒(南京建成生物工程研究所)检测其含量。
如图4-6所示为小鼠肝脏组织病理学分析:对照组小鼠肝脏组织结构完好,肝细胞排列规整(图4)。AFB1组小鼠的肝脏出现玻璃样变区,肝细胞的排列不规则,肝板结构遭到破坏(图5)。AFB1+iT3组的肝脏未出现明显病变(图6)。
如图7所示,与对照组相比,AFB1组小鼠的肝脏指数显著升高(P<0.05),而AFB1+iT3组有效缓解了AFB1导致的肝脏指数升高(P<0.05)。
如图8-9所示为肝脏氧化应激的水平:AFB1组小鼠肝脏中具有抗氧化功能的GSH含量明显低于对照组(P<0.05),而AFB1+iT3组的GSH含量显著高于AFB1组(P<0.05,图8)。与对照组相比,AFB1组小鼠肝脏中的氧化物MDA含量显著升高(P<0.05),而AFB1+iT3组的MDA含量显著低于AFB1组(P<0.05,图9)。
可见,热灭活植物乳杆菌T3具有应用于制备预防、改善或治疗AFB1引起的肝损伤的产品中的潜力。
2.4、小鼠血清中生化指标的检测
将获取的血液样品离心(3000 r/min, 4 ℃, 15 min),收集血清,使用丙氨酸转氨酶(ALT)、冬氨酸转氨酶(AST)以及肌酐(Cr)试剂盒(南京建成生物工程研究所)检测其含量;使用白细胞介素4(IL-4)、肿瘤坏死因子α(TNF-α)、免疫球蛋白G(IgG)和免疫球蛋白M(IgM)试剂盒(上海酶联生物科技有限公司)检测其含量。
如图10-12所示为小鼠血清中转氨酶活性和肌酐的浓度:与对照组相比,AFB1组小鼠血清中ALT和AST的活性显著升高(<0.05),而AFB1+iT3组中ALT和AST的活性均显著低于AFB1组(P<0.05)。相比于对照组,AFB1组小鼠血清中Cr含量明显升高(P<0.05),而AFB1+iT3组中Cr含量显著低于AFB1组(P<0.05)。
如图13-16所示为小鼠血清中的免疫指标:与对照组相比,AFB1导致了引发炎症效应的TNF-α水平显著升高(P<0.05,图13),同时显著抑制了具有抗炎作用的IL-4水平(P<0.05,图14),而AFB1+iT3组的TNF-α水平显著低于AFB1组(P<0.05,图13),并且IL-4水平显著高于AFB1组(P<0.05,图14)。与对照组相比,AFB1显著降低了血清中两种增强免疫功能的IgG与IgM的水平(P<0.05),而AFB1+iT3组中IgG与IgM的水平均显著高于AFB1组(P<0.05,图15-16)。
2.5、小鼠肠道微生物群16S rRNA基因测序分析
动物实验结束前12h在无菌环境下收集各组小鼠的粪便,置于无菌冻存管中,液氮速冻后,转移至-80℃保存备用。
根据E.Z.N.A.®soil DNA kit(Omega Bio-tek, Norcross, GA, U.S.)说明书进行微生物群落总DNA抽提。使用338F和806R引物对16SrRNA基因V3-V4可变区进行PCR扩增。将同一样本的PCR产物混合后使用2%琼脂糖凝胶回收PCR产物,利用AxyPrep DNA GelExtraction Kit(Axygen Biosciences, Union City, CA, USA)进行回收产物纯化,2%琼脂糖凝胶电泳检测,并用Quantus™ Fluorometer(Promega, USA)对回收产物进行检测定量。利用Illumina公司的Miseq PE300/NovaSeq PE250平台进行测序(Shanghai MajorbioBio-pharm Technology Co., Ltd)。
使用fastp软件(https://github.com/OpenGene/fastp, version 0.20.0)对原始测序序列进行质控;使用FLASH软件(http://www.cbcb.umd.edu/software/flash,version 1.2.7)进行拼接;使用UPARSE软件(http://drive5.com/uparse/, version7.1),根据97%的相似度对序列进行OTU聚类并剔除嵌合体。数据在美吉生信云计算平台上进行分析。
如图17所示,三组小鼠的肠道微生物组成在属水平上明显被分离,表明各组的菌群结构显著不同,这说明热灭活植物乳杆菌T3可改善AFB1诱导的异常肠道菌群结构。
如图18-19所示为在门水平和属水平上小鼠肠道微生物的组成:在门水平上,与对照组相比,AFB1减少了小鼠肠道厚壁菌门,增加了拟杆菌门,厚壁菌门/拟杆菌门的比例(Firmicutes/Bacteroidetes ratio, F/B ratio)降低(9:10),而AFB1+T3组的F/B ratio高于AFB1组(22:10,图18)。在属水平上,与对照组相比,AFB1显著增加了Muribaculaceae的丰度(36.3%,对照组为14.7%),减少了有益菌Lactobacillus的丰度(1.6%,对照组为12.6%),而AFB1+iT3组的Muribaculaceae的丰度下降至22.7%,有益菌Lactobacillus的丰度上升至4.8%(图19)。
如图20-21所示,虽然不同处理组的肠道微生物组成大部分(68.97%)是相同的,但AFB1组仍有28个特有的物种(图20),其中85.08%是由Muribaculaceae组成(OTU701,图21),而在AFB1+iT3组未检测出这一物种。Muribaculaceae是一类与肠道生态失调有关的微生物,它能够编码降解粘蛋白聚糖的酶,参与小鼠结肠粘蛋白的降解,破坏肠道屏障,增加肠粘膜的通透性。因此,热灭活植物乳杆菌T3减少Muribaculaceae的丰度表明了其对肠道粘膜的保护作用。
如图22所示,对丰度超过1%的物种进行差异分析,发现各组之间有5个显著不同的物种,其中Muribaculaceae和Alloprevotella是AFB1组中显著富集的两个物种,而其他3个物种(Roseburia, Lactobacillus, Candidatus_Saccharimonas)在正常对照组和AFB1+iT3组中显着富集,尤其是有益菌Lactobacillus(乳酸杆菌)的丰度最高。因此,可考虑使用热灭活植物乳杆菌T3预防、改善或治疗AFB1引起的肠道菌群失调。
对比例1
热灭活植物乳杆菌T3(iT3)对AFB1的清除作用显著高于其它60株乳酸菌
这60株乳酸菌分别来自:青酒乳酸杆菌(Lactobacillus sakei)、发酵乳杆菌(Lactobacillus fermentum)、副干酪乳杆菌(Lactobacillus paracasei)、肠膜明串珠菌亚种(Leuconostoc mesenteroides subsp. Mesenteroides)、类肠膜魏斯氏菌(Weissella paramesenteroides)以及植物乳杆菌(Lactobacillus plantarun)。将60株乳酸菌的菌体浓度调整到1010 CFU/mL,按实施例1中的处理和计算方法计算每株菌对AFB1的清除率。
如表1所示,虽然这60株乳酸菌对AFB1具有一定的清除能力,但它们的清除率为16.0%-68.5%,显著低于热灭活植物乳杆菌T3(iT3)的清除率(84%),说明iT3在对AFB1的毒性削减作用方面具有更好的应用价值。
表1 其它60株乳酸菌对AFB1的清除作用
本说明书中各个实施例采用递进的方式描述,每个实施例重点说明的都是与其他实施例的不同之处,各个实施例之间相同相似部分互相参见即可。
对所公开的实施例的上述说明,使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的,本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下,在其它实施例中实现。因此,本发明将不会被限制于本文所示的这些实施例,而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。
Claims (10)
1.一种植物乳杆菌(Lactobacillus plantarum T3),其特征在于,所述植物乳杆菌已于2022年03月14日保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCC No. 24527。
2.含有权利要求1所述的植物乳杆菌的微生物制剂,其特征在于,所述植物乳杆菌为热灭活的。
3.如权利要求2所述的微生物制剂,其特征在于,所述微生物制剂中,热灭活的植物乳杆菌的菌数不低于1×106CFU/mL或1×106CFU/g。
4.如权利要求2所述的微生物制剂在制备改善AFB1引起的肝损伤的产品中的应用。
5.如权利要求2所述的微生物制剂在制备改善AFB1引起的肠菌失衡的产品中的应用。
6.如权利要求4所述的应用,其特征在于,所述产品中,热灭活的植物乳杆菌的菌数不低于1×106CFU/mL或1×106CFU/g。
7.如权利要求5所述的应用,其特征在于,所述产品中,热灭活的植物乳杆菌的菌数不低于1×106CFU/mL或1×106CFU/g。
8.如权利要求4所述的应用,所述产品包括药物。
9.如权利要求5所述的应用,所述产品包括药物。
10.如权利要求5所述的应用,其特征在于,所述产品效果在于调节肠道菌群,包括调节厚壁菌门和拟杆菌门的丰度。
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