CN116590204A - 副干酪乳酪杆菌及其在抗衰老和改善皮肤抗氧化中的应用 - Google Patents
副干酪乳酪杆菌及其在抗衰老和改善皮肤抗氧化中的应用 Download PDFInfo
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Abstract
本发明提供副干酪乳酪杆菌及其在抗衰老和改善皮肤抗氧化中的应用,属于微生物领域。本发明公开了一株副干酪乳酪杆菌(Lacticaseibacillus paracasei),于2023年01月09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.26439。本发明提供的副干酪乳酪杆菌3Q225能够提高D‑半乳糖诱导小鼠的氧化衰老模型中多种抗氧化相关酶的活性,并降低了氧化衰老小鼠模型血清和肝脏中丙二醛的水平,减轻氧化性衰老损伤,效果优于现有菌株和VC,具有很好的应用前景。
Description
技术领域
本发明属于微生物领域,具体涉及副干酪乳酪杆菌及其在抗衰老和改善皮肤抗氧化中的应用。
背景技术
随着人类寿命的延长,衰老是不可避免的,这是压力和劳累、伤害和感染、免疫反应受损、营养不良、代谢紊乱以及过失和滥用药物造成的结果。衰老与许多疾病密切相关,包括高血压、2型糖尿病、动脉粥状硬化和阿尔茨海默病,这些疾病的发病可以通过延迟衰老来减缓。
抗氧化是抗衰老的一个方向,但合成抗氧化剂对于人体肝脏、肺部、脾部都有不利影响。有些人口服抗氧化剂后,存在高铁红细胞贫血。如果服用抗氧化剂量超标,就会出现腹泻、肠蠕动亢进、腹痛以及消化道出血等症状,还会引起高尿酸血症等副作用。开展具有抗氧化抗衰老且安全的产品十分必要,益生菌是当前研究一个方向。
益生菌中较为常用的菌是乳酸菌(LAB)。LAB在酸奶中发挥着重要作用,因为这些细菌产生的有机酸,包括乳酸,可以作为天然防腐剂和增味剂。LAB作为益生菌的接受度越来越高,它有助于刺激宿主的免疫系统,降低癌症风险,提供抗高血压、抗炎、抗糖尿病、抗氧化、免疫调节、抗胆固醇或微生物组调节,同时防止病原微生物的感染。乳酸菌,包括乳杆菌和双歧杆菌,已经被认为可以防止D-半乳糖诱导的氧化应激。
申请号为202211367692 .4的中国专利中公开了一株副干酪乳杆菌及其应用,涉及微生物技术领域。该发明公开的副干酪乳杆菌(Lactobacillus paracasei),该菌株被命名为GforU-31,其保藏编号为CCTCC No:M20221354的副干酪乳杆菌。实验表明,GforU-31具有促进细胞增殖、抗衰老、提升皮肤抗菌能力的功能,可用于制备食品、药品、化妆品等。
申请号为202011038787 .2的中国专利中公开了具有抗氧化功能的副干酪乳杆菌(Lactobacillus Paracasei)ZJUIDS05及应用。保藏号为CGMCC NO .20515,其具有很强的体内和体外抗氧化能力。该副干酪乳杆菌ZJUIDS05的用途是:制备具有抗氧化功能的活菌制剂,制备具有抗氧化功能的各类产品。
但以上现有技术均是针对过氧化氢诱导的氧化损伤,与自然衰老的机制相去较远,其实际抗氧化抗衰老作用并不能达到预期。
发明内容
为了解决上述问题,本发明提供了一种新筛菌株,具体为一株副干酪乳酪杆菌,并通过对小鼠血清和组织的分析,观察到副干酪乳酪杆菌3Q225对氧化衰老的抑制作用,验证了其抗氧化和抗衰老作用,以期扩展抗衰老和抗氧化方向的益生菌种质资源,为进一步深入人体研究和产业发展奠定了理论基础。
一方面,本发明提供了一株副干酪乳酪杆菌(Lacticaseibacillus paracasei)。
所述的副干酪乳酪杆菌保藏编号为CGMCC NO.26439,于2023年01月09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。
所述的副干酪乳酪杆菌生理特征为:革兰氏阳性,菌体呈杆状,无芽胞,菌落圆形,接触酶阴性,兼性厌氧,蛋白水解活力较高。
另一方面,本发明提供了前述的副干酪乳酪杆菌的培养方法。
所述的培养方法中包括将前述副干酪乳酪杆菌接种在培养基中进行培养。
所述的培养基的类型可以是固体培养基、半固体培养基或液体培养基。
所述的培养基包括但不限于:MRS培养基、BHI培养基、M17培养基。
所述的培养基也可以是根据现有技术中的培养基针对上述副干酪乳酪杆菌进行优化的培养基。
所述的培养条件可以是:37℃,厌氧或兼性厌氧培养24小时。
所述的培养条件也可以是根据现有技术针对上述副干酪乳酪杆菌进行优化的条件,包括但不限于温度、时间、补料、接种量、搅拌程度、氧含量、二氧化碳含量等。
本发明同时提供了前述的培养方法制备的产物。
所述的产物中包括副干酪乳酪杆菌。
所述的产物包括但不限于发酵液、发酵液上清、发酵液沉淀、冻干粉、活菌、死菌中的任意一种或多种。
再一方面,本发明提供了前述的副干酪乳酪杆菌或产物在抗衰老或抗皮肤氧化中的应用。
具体地,用于制备抗衰老或抗皮肤氧化的产品。
所述的衰老的表现包括但不限于:皮肤松弛、消化功能减退、记忆力下降、毛发变白、骨关节退变等。
所述的氧化可以是皮肤氧化,皮肤氧化表现包括但不限于:皮肤蜡黄、嘴角暗沉、毛孔粗大、色斑、皮肤干燥粗糙。
本发明同时提供包括前述的副干酪乳酪杆菌或产物的抗衰老或抗氧化的口服产品。
具体地,所述的口服产品可以是药品、食品或保健品。
所述的药品、食品、保健品满足国家质量标准。
优选地,所述的口服产品中可以包括107CFU/g或107CFU/mL以上的副干酪乳酪杆菌。
所述的口服产品中可以包括108-1011CFU/g或108-1011CFU/mL的副干酪乳酪杆菌。优选地,所述的口服产品中可以包括109-1010CFU/g或109-1010CFU/mL的副干酪乳酪杆菌。
事实上,所述的口服产品中的副干酪乳酪杆菌含量可以调整,具体的副干酪乳酪杆菌摄入量可以根据口服产品的服用量进行调整,当口服产品中的副干酪乳酪杆菌含量较低时,可以通过增加口服产品服用量保证足量副干酪乳酪杆菌的摄入。
所述的口服产品中还可以包括其他抗氧化或者抗衰老成分已发挥共同作用。
所述的共同作用可以是相加作用或协同作用。
具体地,如还可以包括大豆异黄酮、番茄红素、虾青素、维生素、辅酶Q10、烟酰胺中的一种或多种。
所述的抗氧化或抗衰老成分还包括选自植物提取物、动物提取物或中药提取物。
所述的口服产品中还可以包括辅料。
所述的辅料可以是药学上可接受的载体或赋形剂,也可以是食品或保健品领域常规的辅料或添加剂。
所述的辅料可以选自:淀粉、低聚糖、蛋白类、膳食纤维、香辛料、调味剂、调香剂、调色剂中的一种或多种。
在一些实施例中,所述的口服产品可以是口服液或口服胶囊。
本发明的有益效果:
本发明提供的副干酪乳酪杆菌3Q225能够提高D-半乳糖诱导小鼠的氧化衰老模型中超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)、过氧化氢酶(CAT)和谷胱甘肽(GSH)的活性,同时降低了氧化衰老小鼠模型血清和肝脏中丙二醛(MDA)的水平。病理观察显示,副干酪乳酪杆菌3Q225减轻了氧化性衰老小鼠的肝脏、脾脏和皮肤的损伤。本发明的副干酪乳酪杆菌3Q225能够比保加利亚乳杆菌(LB)和维生素C更好地保护D-半乳糖诱导的衰老模型免受氧化应激。
保藏说明
菌种名称:副干酪乳酪杆菌3Q225(TMPC 3Q225);
分类命名:副干酪乳酪杆菌Lacticaseibacillus paracasei;
保藏编号:CGMCC NO.26439;
保藏时间:2023年01月09日;
保藏中心:中国微生物菌种保藏管理委员会普通微生物中心;
保藏地址:北京市朝阳区北辰西路1号院3号。
附图说明
图1为小鼠血清中SOD、GSH、GSH-Px、CAT和MDA的水平。
图2为小鼠肝脏中SOD、GSH、GSH-Px、CAT和MDA的水平。
图3为小鼠肝脏的H&E病理观察(组织学显微照片)。
图4为小鼠脾脏的H&E病理观察(组织学显微照片)。
图5为小鼠皮肤的H&E病理观察(组织学显微照片)。
图6为小鼠肝脏组织中SOD1、SOD2、CAT、eNOS、nNOS和iNOS的mRNA表达水平。
图7为小鼠脾脏组织中SOD1、SOD2、CAT、eNOS、nNOS和iNOS的mRNA表达水平。
具体实施方式
下面结合具体实施例,对本发明作进一步详细的阐述,下述实施例不用于限制本发明,仅用于说明本发明。以下实施例中所使用的实验方法如无特殊说明,实施例中未注明具体条件的实验方法,通常按照常规条件,下述实施例中所使用的材料、试剂等,如无特殊说明,均可从商业途径得到。
以下实施例中,所采用的动物验证模型为D-半乳糖诱导的衰老模型,D-半乳糖是一种可以控制衰老的药剂,D-半乳糖诱导的衰老模型与自然衰老相似,并已被广泛用于动物建模。这个模型的机制是,细胞内的半乳糖被还原成半乳糖醇,不能进一步降解,半乳糖醇的积累扰乱了细胞的新陈代谢,并诱导产生活性氧(ROS),导致细胞损伤和衰老。
实施例1
(1)实验室菌种
从中国云南省德宏傣族景颇族自治州芒市的酸黄笋分离出副干酪乳酪杆菌3Q225,鉴定后保存在中国微生物菌种保藏中心,分类命名为Lacticaseibacillus paracasei,保藏编号为CGMCC NO.26439。德氏乳杆菌保加利亚亚种(LB,CGMCC 1.16075)从中国微生物菌种保藏中心获得,作为与副干酪乳酪杆菌3Q225的对比菌株。
(2)氧化诱导衰老的动物模型
60只昆明(KM)小鼠(6周龄,雌性,20-25g)购自重庆医科大学实验动物中心(中国重庆)。给予它们自由的饮食和水,并适应实验室环境。一周后,将所有小鼠随机分为6组(n=10/组):正常组、对照组(模型组)、维生素C(Vc)、德氏乳杆菌保加利亚亚种(LB)、副干酪乳酪杆菌3Q225处理的低剂量组(3Q225-L)、副干酪乳酪杆菌3Q225处理的高剂量组(3Q225-H)。在前三周,正常组和对照组没有接受任何处理,LB组的小鼠每天以2.0×109cfu/kg体重的浓度灌服LB;3Q225-L组和3Q225-H组的小鼠每天分别被给予2.0×108cfu/kg体重和2.0×109cfu/kg体重的副干酪乳酪杆菌3Q225;Vc组的小鼠以每天100mg/kg体重的浓度灌服维生素C(灌服体积与其他组相当)。三周后,除正常组外,所有组的小鼠每天腹腔注射浓度为120mg/kg体重的D-半乳糖,持续六周。最后,所有小鼠禁食24小时,并以颈椎脱位法处死。获得血液、脾脏、皮肤和肝脏用于后续实验。
(3)血清和肝脏中SOD、GSH、GSH-Px、CAT和MDA水平的测定
取小鼠血液,在4℃下以4500r/min离心15min,制备血清,血清样品保存于-80℃备用。血清和肝脏中的超氧化物歧化酶(SOD,货号:A001-1-1)、谷胱甘肽(GSH,货号:A005-1-1)、谷胱甘肽过氧化物酶(GSH-Px,货号:A005-1-1)、过氧化氢酶(CAT,货号:A007-2-1)和丙二醛(MDA,货号:A003-1-1)水平由试剂盒(南京建成生物工程研究所,江苏南京)测定。
(4)皮肤、肝脏和脾脏组织的病理观察
每只小鼠各取0.5 cm2的皮肤、肝脏和脾脏,立即在10%福尔马林溶液中固定48小时。经过脱水、透明处理、浸蜡、包埋和切片后,进行H&E染色,在光学显微镜下观察这些组织的形态变化。
(5)定量PCR(qPCR)检测
根据说明书,用TRIzol试剂(Invitrogen,Waltham, MA, USA)提取肝脏和脾脏组织总RNA。总RNA的纯度和浓度由超微分光光度计(Nano-100,All For Life Science,杭州,中国)测定。总RNA样品被用作模板,通过逆转录生成cDNA;根据转录试剂盒( ThermoFisherScientific)的说明,所有样品中cDNA的浓度被调整到1pg/μL的相同水平。PCR反应的总体积为20μL,其中包括1μL的cDNA,10μL的荧光核酸染料预混液(SYBR Green PCRMaster Mix,购自Thermo Fisher Scientific, Inc., Waltham, MA, USA),7.8μL的ddH2O和0.6μL的每种引物。PCR参数如下:95℃60秒;95℃15秒,55℃30秒,72℃35秒,40个循环;95℃30秒,55℃35秒。以GAPDH为内参,用2-ΔΔCt方法计算目标基因的相对表达量(Zhang等,2018)。
(6)统计分析
实验3次重复,结果为平均数±标准差。用SPSS软件(IBM公司,North Castle, NY,USA)进行统计分析,采用单向方差分析(ANOVA)和LSD检验。P<0.05的值被认为是显著的。所有的测量都至少重复三次,数据以平均值±标准差表示。所有数字均由Origin 8.0软件绘制。
(7)结果
SOD、GSH、GSH-Px、CAT和MDA的水平:
如图1-2所示,与正常组小鼠相比,模型组小鼠血清和肝脏中的MDA含量最高,SOD、GSH、GSH-Px和CAT的含量最低。用LB、副干酪乳酪杆菌3Q225或维生素C处理后,氧化诱导衰老小鼠的MDA含量下降,SOD、GSH、GSH-Px和CAT的含量升高。特别是高剂量副干酪乳酪杆菌3Q225组小鼠的MDA、SOD、GSH、GSH-Px和CAT的指标水平与正常组非常接近,说明副干酪乳酪杆菌3Q225菌对小鼠的衰老指标的延缓作用比LB和维生素C好。
小鼠肝脏、脾脏和皮肤的病理观察:
肝脏的组织学显微照片见图3。正常组的小鼠,肝细胞形态规则,大小和染色均匀,没有炎症的迹象,肝细胞线排列有序,边界清晰,中心静脉呈放射状分布。然而,与正常组的小鼠相比,对照组的小鼠所含的肝细胞排列紊乱,形态不规则,细胞边界消失,中心静脉形状不规则,细胞肿胀,并有广泛的炎症浸润痕迹。与对照组的小鼠相比,用维生素C、LB或副干酪乳酪杆菌3Q225治疗的小鼠显示出这种化学损害的减少。在副干酪乳酪杆菌3Q225治疗组中,肝脏损伤的程度比对照组(仅D-半乳糖治疗)要轻,因此副干酪乳酪杆菌3Q225在预防肝脏萎缩方面的疗效更好。此外,保护肝脏的效果与剂量有关。
脾脏的组织学显微照片见图4。正常组中,脾囊平坦,脾小梁结构正常,红髓和白髓的界限清晰,未见明显的眼病。对照组与正常组相比,脾脏失去了正常的组织形态和细胞组织,伴有红髓窦扩张,大量红细胞,白髓淋巴细胞减少,红髓索变窄,细胞密度降低。与对照组相比,经维生素C、LB或副干酪乳酪杆菌3Q225处理后,脾脏组织形态明显改善,表现为红髓和白髓界限清晰,脾囊扁平,生发中心明显,细胞组织良好。各治疗组显示出与正常组小鼠相似的显微照片,其中副干酪乳酪杆菌3Q22显示出最佳效果。
皮肤的组织学显微照片显示在图5。正常皮肤组织的表皮结构清晰而完整.真皮中的胶原纤维含量丰富。真皮和表皮的边界清晰而完整。真皮层中的胶原纤维含量丰富,脂肪空泡的数量正常。与正常组的小鼠相比,对照组的小鼠胶原纤维含量减少,脂肪空泡的数量增加,真皮内发生内渗,真皮和表皮之间的界限模糊不清。与对照组小鼠相比,用维生素C、LB或副干酪乳酪杆菌3Q225治疗可明显改善皮肤组织脂质空泡、内渗现象和表皮与真皮之间的界限,缓解D-半乳糖诱导的衰老小鼠的皮肤病变。另外,副干酪乳酪杆菌3Q225-H 组的小鼠比LB组和Vc组的小鼠的显微照片更接近于正常小鼠,因此副干酪乳酪杆菌3Q225 在预防皮肤病变方面的疗效更好。
小鼠肝脏氧化相关基因的表达:
小鼠肝脏组织中SOD1、SOD2、CAT、eNOS、nNOS和iNOS的mRNA表达水平见图6。
结果显示,正常组小鼠肝脏中SOD1、SOD2、CAT、eNOS、nNOS的mRNA水平最高,iNOS的mRNA水平最低。对照组的上述基因的mRNA表达水平与正常组的趋势相反。经维生素C、LB或3Q225处理后,衰老小鼠的SOD1、SOD2、CAT、eNOS、nNOS和iNOS的表达得到明显改善(P<0.05),其中高剂量的3Q225效果最好。
小鼠脾脏氧化相关基因的表达
小鼠脾脏组织中SOD1、SOD2、CAT、eNOS、nNOS和iNOS的mRNA表达水平见图7。
脾脏组织的qPCR结果与肝脏的结果相似。对照组小鼠脾脏组织中SOD1、SOD2、CAT、eNOS和nNOS的mRNA表达最弱,但正常组小鼠的这些表达最强。与对照组相比,维生素C、LB或3Q225处理可以增加表达。3Q225处理的小鼠的表达比维生素C和LB处理的小鼠强,并接近于正常小鼠,维生素C处理的小鼠也比LB处理的小鼠表达强。
(8)讨论
乳酸菌(LAB)因其众多的益生特性而被广泛应用于食品行业,成为日常生活中相对不可缺少的健康食品。从发酵食品中分离的LAB菌株可能具有抗氧化活性。因此,本发明重点研究了从传统发酵食品中分离的副干酪乳酪杆菌3Q225菌株的抗氧化活性。通过连续皮下注射D-半乳糖已被证明可以诱发建立小鼠的衰老模型,导致小鼠体内活性氧自由基(ROS)的增加和抗氧化酶活性的降低,自由基的增加可能导致衰老相关机制的退化,最终导致氧化应激的增加,加速衰老过程。
病理观察是观察器官、组织或细胞的形态变化,以确定身体的病理变化。皮肤、脾脏和肝脏可以反映出身体因衰老而发生的特征性变化。根据这项研究,与正常组小鼠相比,衰老组的肝脏和脾脏组织萎缩,皮肤受损。维生素C、LB或副干酪乳酪杆菌3Q225处理后,组织的异常形态得到明显改善,其中副干酪乳酪杆菌3Q225的改善效果最明显。副干酪乳酪杆菌3Q225治疗的小鼠的形态组织最接近于正常组。
在人体的生物氧化过程中,会产生大量的自由基。体内自由基的平衡是由一些抗氧化防御系统完成的,包括GSH-Px、SOD、CAT、还原GSH和维生素C,它们的协同作用将体内多余的自由基转化为氧分子和水分子,从而起到抗氧化的作用。乳酸菌具有抗氧化活性,具有清除自由基的功能,可以协助抗氧化酶完成抗氧化作用。此外,细菌在体内代谢过程中会释放抗氧化酶,如超氧化物歧化酶和谷胱甘肽过氧化物酶,以阻止氧化过程。临床研究表明,在接受抗氧化治疗的患者中,SOD、GSH-Px、GSH、CAT和MDA水平发生变化。SOD保护细胞免受氧化造成的损害,间接反映其抗氧化活性。GSH-Px可以清除活性氧和羟基自由基诱发的脂质过氧化,保护细胞膜结构和功能的完整性。在本发明中,副干酪乳酪杆菌3Q225能显著提高小鼠血清和肝脏组织中的SOD水平和GSH-Px活力。作为非酶抗氧化防御系统之一,GSH负责在谷胱甘肽过氧化物酶(GSH-PX)的催化下还原过氧化氢。与正常组相比,肝脏GSH含量明显下降,表明D-半乳糖造成了严重的细胞氧化应激。此外,CAT水平也明显降低,表明衰老模型小鼠的总抗氧化能力下降。在本发明中,副干酪乳酪杆菌3Q225、保加利亚乳杆菌LB和维生素C被证明可以抑制肝脏和血清GSH、GSH-Px和CAT水平的下降。副干酪乳酪杆菌3Q225的抗氧化作用高于其他实验组。有研究表明,益生菌能明显改善高脂血症小鼠的抗氧化能力,提高SOD、GSH-PX、GPT和GOT等抗氧化酶的活性。因此,进一步证明了乳酸菌具有增强抗氧化酶合成的能力。MDA是脂质过氧化的最终产物之一,会引起蛋白质和核酸等生物大分子的交联聚合,并具有细胞毒性和基因毒性作用。因此,MDA的水平是氧化损伤和衰老的一个重要指标。本发明中,与D-半乳糖模型组相比,副干酪乳酪杆菌3Q225低剂量组小鼠肝组织中的MDA含量显著降低,高剂量实验组小鼠肝组织中的MDA含量接近于正常水平。本发明中,副干酪乳酪杆菌3Q225可以降低血清和肝脏中的MDA含量,进一步证明3Q225可以增强机体的抗氧化防御系统。本发明的研究证实,副干酪乳酪杆菌3Q225能缓解衰老对小鼠的影响,使衰老小鼠模型中SOD、GSH-Px、GSH、CAT和MDA水平恢复至与正常小鼠相似。
Claims (10)
1.一株副干酪乳酪杆菌(Lacticaseibacillus paracasei),其特征在于,保藏编号为CGMCC NO.26439。
2.权利要求1所述的副干酪乳酪杆菌的培养方法,其特征在于,包括将副干酪乳酪杆菌接种在培养基中进行培养。
3.权利要求2所述的培养方法制备的产物,其特征在于,包括副干酪乳酪杆菌,选自发酵液、发酵液上清、发酵液沉淀、冻干粉、活菌、死菌中的任意一种或多种。
4.权利要求1所述的副干酪乳酪杆菌或权利要求3所述的产物在抗衰老或抗氧化中的应用。
5.包括权利要求1所述的副干酪乳酪杆菌或权利要求3所述的产物的抗衰老或抗皮肤氧化的口服产品。
6.根据权利要求5所述的口服产品,其特征在于,所述的口服产品为药品、食品或保健品。
7.根据权利要求6所述的口服产品,其特征在于,包括107CFU/g或107CFU/mL以上的副干酪乳酪杆菌。
8.根据权利要求6所述的口服产品,其特征在于,还包括大豆异黄酮、番茄红素、虾青素、维生素、辅酶Q10、烟酰胺中的一种或多种。
9.根据权利要求6所述的口服产品,其特征在于,还包括植物提取物、动物提取物或中药提取物;还包括辅料,所述的辅料选自:淀粉、低聚糖、蛋白类、膳食纤维、香辛料、调味剂、调香剂、调色剂中的一种或多种。
10.根据权利要求6所述的口服产品,其特征在于,为口服液或口服胶囊。
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