CN112553115A - 植物乳杆菌zdy2013在制备缓解肾损伤产品中应用 - Google Patents
植物乳杆菌zdy2013在制备缓解肾损伤产品中应用 Download PDFInfo
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Abstract
植物乳杆菌ZDY2013在制备缓解肾损伤产品中应用,本发明分别提出了一种制备缓解肾损伤药物或保健品或食品中的应用、一种缓解高盐饮食诱导肾损伤的保健品或食品、一种用于缓解高盐诱导肾损伤的益生菌菌剂及制备方法。所述的植物乳杆菌ZDY2013极耐酸耐胆盐,能在胃肠液中保持较高的存活率,并能抑制食源性致病菌的增长。此外,还能因为能调节肠道菌群多样性,达到改善宿主胃肠道健康。具有缓解肾脏纤维化,改善炎症表观症状,调节氧化应激,调控炎症因子的表达,达到有效防治高盐饮食诱导的肾损伤功效。
Description
技术领域
本发明属微生物领域,具体涉及的植物乳杆菌ZDY2013的新应用。
背景技术
高盐饮食(HSD)导致的各类疾病越来越得到人们的重视,然而高盐饮食与高血压、肾组织损伤、自身免疫性疾病、糖尿病及骨质疏松症等疾病有关。有研究表明高盐饮食组的小鼠粪便经过移植后可引起受体小鼠肠道渗漏和肾脏损伤,此外,高盐的摄入和氧化应激有很强的相关性,它可导致肾小管多巴胺氧化,进而发生溶酶体和线粒体的功能障碍和肾脏的炎症反应。目前用于治疗肾损伤的药物主要有肝素、科素亚、利妥昔单抗、阿德福伟、替诺福韦等, 药价高,且副作用不可控。
植物乳杆菌作为肠道革兰氏阳性益生菌,可调节肠道菌群平衡,抑制有害菌的生长,在肠道免疫作用中占有重要地位。
本发明所述的植物乳杆菌ZDY2013,2015年4月28日保存于武汉市中国典型培养物保藏中心(China Center for Type Culture Collection,简称CCTCC M,地址:湖北省武汉市武昌区八一路299号武汉大学校内,邮编:430072),保藏编号为CCTCC M2014170。该菌株已在中国专利“高耐酸植物乳杆菌ZDY2013”(专利号ZL201510552202.1)中公开,任何个人只要不是商业用途都可以从中国典型培养物保藏中心获取。
发明内容
本发明的目的之一是提出一种植物乳杆菌ZDY2013在制备缓解肾损伤药物或保健品或食品中的应用。所述植物乳杆菌ZDY2013的保藏号为CCTCC M2014170。
优选地,所述肾损伤是高盐饮食所导致的。
优选地,所述肾损伤是肾脏组织炎症和/或纤维化。
本发明的第二个目的是提出一种缓解高盐饮食诱导肾损伤的保健品或食品。其活性成分包括有植物乳杆菌ZDY2013,所述植物乳杆菌ZDY2013的保藏号为CCTCC M2014170。
优选的,所述食品为饮料、酸奶、果冻等多种乳杆菌可生存的食品,只要植物乳杆菌可生存的食品中都可以,具体根据实际需要。
优选的,本发明所用的缓解高盐饮食诱导肾损伤的植物乳杆菌菌剂剂量为1×108cfu/kg,根据具体情况计算使用,即每kg动物、人体所用菌剂中含植物乳杆菌ZDY2013达108cfu。
本发明第三个目的是提出一种用于缓解高盐诱导肾损伤的益生菌菌剂,由植物乳杆菌ZDY2013制备而成,以一种处于稳定期的新鲜活菌菌体形式存在。
本发明所述的一种用于缓解肾损伤的植物乳杆菌菌剂的制备方法,包括如下步骤。
(1)植物乳杆菌ZDY2013 MRS液体培养基37±1℃厌氧培养24±2 h,到达稳定期的菌体,所述植物乳杆菌ZDY2013的保藏号为CCTCC M2014170。
(2)离心收集步骤(1)中菌体,用无菌1×PBS洗涤2-4次。
(3)用无菌1×PBS调整步骤(2)所得菌体浓度到1-2×108 cfu/mL。
(4)向上述所得菌体中加入适宜辅料制成的药物或食物或保健品。
本发明所述的植物乳杆菌ZDY2013极耐酸耐胆盐,能在胃肠液中保持较高的存活率,并能抑制食源性致病菌的增长。此外,还能因为能调节肠道菌群多样性,达到改善宿主胃肠道健康。具有缓解肾脏纤维化,改善炎症表观症状,调节氧化应激,调控炎症因子的表达,达到有效防治高盐饮食诱导的肾损伤功效。
附图说明
图1为不同组小鼠体重变化。
图2为不同组小鼠肾脏组织HE染色染色切片。其中,相比于NSD空白组,HSD组短箭头所指位置粘膜下层边缘模糊,长箭头所指位置出现明显的炎症细胞浸润,益生菌缓解后,HHL,HHZ,HLZ组均得到缓解,其中结肠肌层平滑,炎症现象明显减少。
图3为不同组小鼠肾脏组织Masson染色切片。观察组织纤维化程度,其中,相比于NSD空白组,HSD组箭头所指位置肾脏组织纤维化明显,益生菌缓解后,HHL,HHZ,HLZ组肾脏组织纤维化均得到缓解。
图4为不同组小鼠肾脏组织纤维化量化图。
图5 不同组小鼠结肠组织炎症因子及抗氧化因子表达差异。
图6不同组小鼠肾脏组织炎症因子及抗氧化因子表达差异。
具体实施方式
为了便于理解本发明,下面将对本发明进行更全面的描述。本发明可以以许多不同的形式来实现,并不限于本文所描述的实施例。相反地,提供这些实施例的目的是使对本发明公开内容的理解更加透彻全面。
下列实施例中未注明具体条件的实验方法,通常按照常规条件,例如Sambrook等人,分子克隆:实验室手册(New York: Cold Spring Harbor Laboratory Press, 1989)中所述的条件,或按照制造厂商所建议的条件。实施例中所用到的各种常用化学试剂,均为市售产品。
除非另有定义,本发明所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本发明的说明书中所使用的术语只是为了描述具体的实施例的目的,不用于限制本发明。本发明所使用的术语“和/或”包括一个或多个相关的所列项目的任意的和所有的组合。
实施例1。
菌体的活化:将保存于甘油中的植物乳杆菌ZDY2013旋涡混匀后吸取10μL涂布于MRS固体培养基中,37℃厌氧培养24h,挑取单菌落接种于4mL液体MRS培养基中,37℃厌氧培养24h,按1%接种量接种于10mL MRS液体培养基中,37℃厌氧培养24h,达到稳定期。
MRS 培养基配制 :蛋白胨 5g/L,牛肉膏 5g/L,胰蛋白胨 10g/L,酵母粉 5g/L,葡萄糖 20g/L,无水乙酸钠 5g/L,柠檬酸氢二胺 2g/L,磷酸氢二钾 2g/L,硫酸镁 0.58g/L,硫酸锰0.25g/L,吐温-80 1m L /L,pH6.3±0.1,121℃、15 分钟灭菌。
实施例2。
植物乳杆菌菌剂的制备:将活化好的处于稳定期的植物乳杆菌ZDY2013 离心,收集新鲜菌体,用无菌1×PBS洗涤3次,用无菌1×PBS调菌体浓度到108 cfu/mL,制成实验所需菌剂,备用。
实施例3。
高盐饮食小鼠模型的构建及实验设计。
C57BL/6J小鼠在无菌鼠房内适应一周后,NSD组(n=10)小鼠给予正常日粮和正常饮水,实验组小鼠给予4%Nacl高盐日粮和正常饮水,持续4周后将实验组小鼠分为高盐组,HSD组(n=7),即每日灌胃200µL 1xPBS作为对照,HHZ组(n=8),即每日灌胃200µL1x1010 CFU/mL植物乳杆菌ZDY2013,HLZ组(n=8),即每日灌胃200µL 1x108 CFU/mL植物乳杆菌ZDY2013。HHL组(n=8),每日灌胃200µL 1x1010CFU/mL鼠李糖乳杆菌(GG)。(鼠李糖乳杆菌(GG)作为国际生公认功能和作用开发较完全的益生菌株,与植物乳杆菌ZDY2013起对照的作用。)实验过程中两天一次对小鼠体重进行测量,观察小鼠体重变化,灌胃2w后,对小鼠进行***安乐死,在无菌环境下取小鼠的血清、结肠、以及肾脏组织备用。另取结肠、肾脏组织,生理盐水迅速冲洗干净,标本于10%***溶液浸泡,做HE染色。实验结果。
(1) 本发明明显改善小鼠体重。
经过灌胃植物乳杆菌ZDY2013菌剂处理5天后,处理组小鼠较高盐饮食组小鼠体重有明显的改善,如图1所示,灌胃处理结束后,益生菌菌剂处理组小鼠可明显恢复体重向正常饮食组体重靠近。说明本发明可有效缓解高盐饮食导致的体重下降情况,改善小鼠的体重。
(2) 本发明显著改善小鼠病理学情况。
将不同组小鼠组肾脏组织制成病理学切片,高盐饮食组小鼠肾脏组织与自然对照组进行对比,从图2可以看出,高盐饮食组肾小球出现明显的萎缩、纤维化,肾小管腔扩张。间质出现炎症现象。本发明组(植物乳杆菌菌剂处理组)肾小球部分纤维化,无萎缩现象,炎症细胞部分浸润。说明了本发明菌剂可有效缓解高盐饮食小鼠的肾脏组织炎症和纤维化情况,具有修复高盐诱导的肾脏损伤的功效。
(3) 本发明菌剂显著调节高盐诱导的结肠组织炎症因子的表达。
将结肠组织进行RNA提取,反转录操作后,检测炎症因子TGF-β1,IFN-γ,FN-1的表达水平。
由图3可以看出,本发明中植物乳杆菌组小鼠较高盐饮食组小鼠炎症因子表达水平得到显著调节,下调了促炎症因子IFN-γ表达水平,并上调了抑炎症因子TGF-β1的表达水平。说明了本发明菌剂可通过调节细胞因子表达水平,从而达到保护机体抗炎症的目的。
(4) 本发明菌剂显著调节高盐诱导的肾脏相关因子的表达。
将肾脏组织进行RNA提取,反转录操作后,检测炎症因子TGF-β1,CCL4的表达水平,检测抗氧化因子SOD1, SOD2的表达水平。
由图4可以看出,本发明组(植物乳杆菌组)小鼠较高盐饮食组小鼠炎症因子表达水平得到显著调节,下调了抑炎因子TGF-β1,同时下调了氧化应激反应因子SOD1、SOD2和趋化因子CCL4。说明本发明菌剂可通过调节细胞因子、氧化应激表达水平,从而保护高盐诱导的肾脏损伤的目的。
以上仅为本发明的具体实施例,并不以此限定本发明的保护范围;在不违反本发明构思的基础上所作的任何替换与改进,均属本发明的保护范围。
Claims (8)
1.植物乳杆菌ZDY2013在制备缓解肾损伤药物、保健品或食品中的应用,所述植物乳杆菌ZDY2013的保藏号为CCTCC M2014170。
2.根据权利要求1所述的一种植物乳杆菌ZDY2013在制备缓解肾损伤药物或保健品或食品中的应用,其特征是所述肾损伤是高盐饮食所导致的。
3.根据权利要求1所述的一种植物乳杆菌ZDY2013在制备缓解肾损伤药物或保健品或食品中的应用,其特征是所述肾损伤是肾脏组织炎症和/或纤维化。
4.一种缓解高盐饮食诱导肾损伤的保健品或食品,其特征是其活性成分包括有植物乳杆菌ZDY2013,所述植物乳杆菌ZDY2013的保藏号为CCTCC M2014170。
5.根据权利要求4所述的一种缓解高盐饮食诱导肾损伤的保健品或食品,其特征是所述食品为饮料、酸奶或果冻。
6.根据权利要求4所述的一种缓解高盐饮食诱导肾损伤的保健品或食品,其特征是所述的植物乳杆菌菌剂剂量为1×108 cfu/kg。
7.一种用于缓解高盐诱导肾损伤的益生菌菌剂,其特征是由植物乳杆菌ZDY2013制备而成,并以新鲜活菌菌体形式存在。
8.权利要求7所述的一种用于缓解肾损伤的益生菌菌剂的制备方法,其特征是包括如下步骤:
(1)植物乳杆菌ZDY2013 MRS液体培养基37±1℃厌氧培养24±2 h,到达稳定期的菌体;
(2)离心收集步骤(1)中菌体,用无菌1×PBS洗涤2-4次;
(3)用无菌1×PBS调整步骤(2)所得菌体浓度到1-2×108 cfu/mL;
(4)向上述所得菌体中加入适宜辅料制成的药物、食物或保健品。
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