CN113265361A - 一种能够缓解非酒精性脂肪肝的复合益生菌制剂、制备方法及其应用 - Google Patents
一种能够缓解非酒精性脂肪肝的复合益生菌制剂、制备方法及其应用 Download PDFInfo
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- CN113265361A CN113265361A CN202110704759.8A CN202110704759A CN113265361A CN 113265361 A CN113265361 A CN 113265361A CN 202110704759 A CN202110704759 A CN 202110704759A CN 113265361 A CN113265361 A CN 113265361A
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- bifidobacterium animalis
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Abstract
本发明公开了一种复合微生物制剂、制备方法及其应用,涉及微生物技术领域。其特征在于:该复合益生菌制剂包括动物双歧杆菌乳亚种Bifidobacterium animalis subsp.lactis BLa80、鼠李糖乳杆菌Lactobacillus rhamnosus LRa05和植物乳杆菌Lactobacillus plantarum Lp90;其制备方法包括以下步骤:分别制备动物双歧杆菌乳亚种Bifidobacterium animalis subsp.lactis BLa80、鼠李糖乳杆菌Lactobacillus rhamnosus LRa05和植物乳杆菌Lactobacillus plantarum Lp90冻干粉;按比例将三种冻干粉混合均匀制备复合益生菌。本发明提供的复合益生菌能够预防和/或治疗非酒精性脂肪肝,在制备预防和/或治疗非酒精性脂肪肝的产品(如固体饮料)中,具有很大的应用前景。
Description
技术领域
本发明涉及微生物技术领域,特别涉及一种能够缓解非酒精性脂肪肝的复合益生菌制剂、制备方法及其应用。
背景技术
非酒精性脂肪肝(nonalcoholic fatty liver disease, NAFLD)是指除外酒精和其他明确的损肝因素,以弥漫性肝细胞大泡性脂肪变为主要特征的临床病理综合征,根据病变是否伴有炎症和纤维化分为单纯性脂肪肝、NAFLD及相关性肝硬化。目前认为NAFLD是代谢综合征的肝脏表现,其发病机制尚未完全阐明,近期研究发现其发生过程与肠道菌群的变化密切相关,肠道菌群紊乱和肠壁通透性增加等通过免疫反应介导,在NAFLD的发生、发展中有重要作用。有研究表明以乳酸菌、双歧杆菌为代表的益生菌与胆固醇代谢、胃肠道感染、细菌移位等有直接关系。目前益生菌制剂在NAFLD的应用上并不多见,且对NAFLD的治疗效果及机制并不清楚。
因此,筛选出可以有效缓解非酒精性脂肪肝的复合益生菌至关重要。这对于益生菌在缓解胃肠道疾病的研究可以提供重要的参考作用。与此同时,这对于开发更高保健价值的益生菌有重要的意义,对于利用膳食策略缓解非酒精性脂肪肝开辟新的路径和解决方案。
发明内容
本发明在于克服背景技术中存在的问题,提供一种能够缓解非酒精性脂肪肝的复合益生菌制剂。该能够缓解非酒精性脂肪肝的复合益生菌制剂,能够有效缓解非酒精性脂肪肝。本发明还提供了一种复合益生菌制剂的制备方法及其应用。
本发明解决其问题可通过如下技术方案来达到:一种能够缓解非酒精性脂肪肝的复合益生菌制剂,该复合益生菌制剂包括含有动物双歧杆菌乳亚种Bifidobacteriumanimalis subsp. lactis BLa80、鼠李糖乳杆菌Lactobacillus rhamnosus LRa05和植物乳杆菌Lactobacillus plantarum Lp90。
优选的,所述益生菌制剂中动物双歧杆菌乳亚种Bifidobacterium animalissubsp. lactis BLa80、鼠李糖乳杆菌Lactobacillus rhamnosus LRa05和植物乳杆菌Lactobacillus plantarum Lp90按照活菌数比为(1~1.5):(1~1.5):(1~1.5)复配。
优选的,所述动物双歧杆菌乳亚种Bifidobacterium animalis subsp. lactisBLa80、鼠李糖乳杆菌Lactobacillus rhamnosus LRa05和植物乳杆菌Lactobacillusplantarum Lp90的活菌数不低于1×106CFU/mL或1×106CFU/g。
优选地,所述动物双歧杆菌乳亚种Bifidobacterium animalis subsp. lactisBLa80,分类命名为Bifidobacteriumanimalis,已于2018年3月5日保藏于中国微生物菌种保藏委员会普通微生物中心,保藏编号为CGMCC No. 15410;鼠李糖乳杆菌Lactobacillusrhamnosus LRa05,分类命名为Lactobacillus rhamnosus,已于2020年7月20日保藏于中国微生物菌种保藏委员会普通微生物中心,保藏编号为CGMCC No. 1.12734;植物乳杆菌Lactobacillus plantarum Lp90,分类命名为Lactobacillus plantarum,已于2015年1月27日保藏于中国微生物菌种保藏委员会普通微生物中心,保藏编号为CGMCC No. 10453,保藏地址均为:北京市朝阳区北辰西路1号院3号。
本发明还提供一种能够缓解非酒精性脂肪肝的复合益生菌制剂的制备方法,包括以下步骤:
(1)分别制备动物双歧杆菌乳亚种Bifidobacterium animalis subsp. lactisBLa80、鼠李糖乳杆菌Lactobacillus rhamnosus LRa05和植物乳杆菌Lactobacillusplantarum Lp90冻干粉;
(2)按比例将动物双歧杆菌乳亚种Bifidobacterium animalis subsp. lactisBLa80冻干粉、鼠李糖乳杆菌Lactobacillus rhamnosus LRa05冻干粉和植物乳杆菌Lactobacillus plantarum Lp90冻干粉混合均匀制备复合益生菌。
优选的,动物双歧杆菌乳亚种Bifidobacterium animalis subsp. lactisBLa80、鼠李糖乳杆菌Lactobacillus rhamnosus LRa05和植物乳杆菌Lactobacillusplantarum Lp90冻干粉的制备方法分别为:
将动物双歧杆菌乳亚种Bifidobacterium animalis subsp. lactis BLa80、鼠李糖乳杆菌Lactobacillus rhamnosus LRa05和植物乳杆菌Lactobacillus plantarum Lp90冻干粉按照占培养基总质量3%的接种量分别接种到MRS培养基中,于37℃下培养18h,得到培养液;将培养液离心,得到菌体;将菌体用海藻糖浓度为100g/L的海藻糖冻干保护剂重悬(冻干保护剂和菌体的质量比为2:1),得到重悬液;将重悬液采用真空冷冻法进行冻干,分别得到三种菌的冻干粉。
优选的,制备三种菌的冻干粉使用的MRS培养基(g/L)为:蛋白胨10g/L、牛肉膏10g/L、葡萄糖20g/L、乙酸钠2g/L、酵母粉5g/L、柠檬酸氢二铵2g/L、K2PO4·3H2O 2.6g/L、MgSO4·7H2O 0.1g/L、MnSO4 0.05g/L、吐温80 1mL/L、半胱氨酸氨酸盐0.5g/L。
本发明还提供一种复合益生菌制剂在预防和/或治疗缓解非酒精性脂肪肝的产品中的应用。
本发明复合益生菌制剂在制备预防和/或治疗非酒精性脂肪肝的固体饮料的应用。所述固体饮料包括含有动物双歧杆菌乳亚种Bifidobacterium animalis subsp.lactis BLa80、鼠李糖乳杆菌Lactobacillus rhamnosus LRa05和植物乳杆菌Lactobacillus plantarum Lp90 。
本发明与上述背景技术相比较可具有如下有益效果:
本发明提供的一种复合益生菌制剂,其含有动物双歧杆菌乳亚种Bifidobacterium animalis subsp. lactis BLa80、鼠李糖乳杆菌Lactobacillusrhamnosus LRa05和植物乳杆菌Lactobacillus plantarum Lp90,三种组分联合使用后,具有明显的协同增效作用。具体体现在:
(1)显著降低 NAFLD模型组大鼠体重;
(2)显著降低NAFLD模型组大鼠血清谷丙转氨酶(Alaninetransaminase,ALT),谷草转氨酶( Aspartate transaminase,AST),血清总胆固醇( Total cholesterol,TC)和甘油三酯(Triglyceride,TG)水平;
(3)显著降低NAFLD模型组大鼠血清炎症因子肿瘤坏死因子-α(Tumor NecrosisFactor-α,TNF-a),白介素1b(interleukin-1B,IL-1),白介素6(Interleukin- 6,IL-6),白介素23(Interleukin-23,IL-23),趋化因子IP-10(interferon-inducible protein-10)和T细胞活化标记物OX-40水平;
(4)所述复合益生菌能降低肝细胞肿胀程度,减少脂肪空泡数量;
(5)此外,所述复合益生菌还能够提高肠道异丁酸和丁酸的含量。
本发明所述的复合益生菌能够用于制备具有降低体重,降低血清ALT,AST,TC和TG水平,降低血清炎症因子,降低肝细胞肿胀程度,减少脂肪空泡数量,提高肠道异丁酸和丁酸的含量的作用,具有非常广泛的应用前景。
附图说明
附图1为本发明实施例试验期间大鼠的体重变化示意图;
附图2为本发明实施例复合益生菌干预14周后的大鼠血清丙氨酸氨基转移酶变化示意图;*P<0.05,**P<0.01,***P<0.001(vs NAFLD模型组);
附图3为本发明实施例复合益生菌干预14周后,实验大鼠血清天门冬氨酸氨基转移酶变化示意图;*P<0.05,**P<0.01,***P<0.001(vs NAFLD模型组);
附图4为本发明实施例复合益生菌干预14周后,实验大鼠血清TC变化示意图;*P<0.05,**P<0.01,***P<0.001(vs NAFLD模型组);
附图5为本发明实施例复合益生菌干预14周后,实验大鼠血清TG变化示意图;*P<0.05,**P<0.01,***P<0.001(vs NAFLD模型组);
附图6为本发明实施例复合益生菌干预14周后,实验大鼠血清TNFa变化示意图;*P<0.05,**P<0.01,***P<0.001(vs NAFLD模型组);
附图7为本发明实施例复合益生菌干预14周后,实验大鼠血清IL1b变化示意图;*P<0.05,**P<0.01,***P<0.001(vs NAFLD模型组);
附图8为本发明实施例复合益生菌干预14周后,实验大鼠血清IL6变化示意图;*P<0.05,**P<0.01,***P<0.001(vs NAFLD模型组);
附图9为本发明实施例复合益生菌干预14周后,实验大鼠血清IL23变化示意图;*P<0.05,**P<0.01,***P<0.001(vs NAFLD模型组);
附图10为本发明实施例复合益生菌干预14周后,实验大鼠血清IP10变化示意图;*P<0.05,**P<0.01,***P<0.001(vs NAFLD模型组);
附图11为本发明实施例复合益生菌干预14周后,实验大鼠血清OX-40变化示意图;*P<0.05,**P<0.01,***P<0.001(vs NAFLD模型组);
附图12为本发明实施例复合益生菌干预14周后,实验大鼠肝脏组织HE染色后比较图;
附图13为本发明实施例复合益生菌干预14周后,实验大鼠血清NAFLD activityscore变化示意图;*P<0.05,**P<0.01,***P<0.001(vs NAFLD模型组);
附图14为本发明实施例复合益生菌干预14周后,实验大鼠粪便异丁酸和丁酸含量变化示意图;箱线图不同的字母表示组间有显著性差异(P<0.05)。
具体实施方式:
下面将结合本发明附图及实施例,对本发明的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其它实施例,都属于本发明保护的范围。
下述实施例中涉及的蛋白胨、牛肉膏、葡萄糖、乙酸钠与酵母粉、柠檬酸氢二铵、K2PO4·3H2O、MgSO4·7H2O、MnSO4、吐温80和半胱氨酸氨酸盐购自国药集团化学试剂有限公司。SPF级雄性3周龄SD大鼠,高脂及普通饲料购自上海斯莱克公司;测定TNF-α、IL-1β、IL-6、IL-17和IL-23检测用ELISA试剂盒购自上海酶联生物科技有限公司。
下述实施例中涉及的培养基如下:
MRS培养基(g/L):蛋白胨10g/L、牛肉膏10g/L、葡萄糖20g/L、乙酸钠2g/L、酵母粉5g/L、柠檬酸氢二铵2g/L、K2PO4·3H2O 2.6g/L、MgSO4·7H2O 0.1g/L、MnSO4 0.05g/L、吐温80 1mL/L、半胱氨酸氨酸盐0.5g/L。
实施例1:动物双歧杆菌乳亚种Bifidobacterium animalis subsp. lactisBLa80冻干粉的制备
将动物双歧杆菌乳亚种Bifidobacterium animalis subsp. lactis BLa80按照占培养基总质量3%的接种量接种到培养基中,于37℃下培养18h,得到培养液;将培养液离心,得到菌体;将菌体海藻糖浓度为100g/L的海藻糖冻干保护剂重悬(冻干保护剂和菌体的质量比为2:1),得到重悬液;将重悬液采用真空冷冻法进行冻干,得到动物双歧杆菌乳亚种Bifidobacterium animalis subsp. lactis BLa80冻干粉。
实施例2:鼠李糖乳杆菌Lactobacillus rhamnosus LRa05冻干粉的制备
将鼠李糖乳杆菌Lactobacillus rhamnosus LRa05按照占培养基总质量3%的接种量接种到培养基中,于37℃下培养18h,得到培养液;将培养液离心,得到菌体;将菌体用海藻糖浓度为100g/L的海藻糖冻干保护剂重悬(冻干保护剂和菌体的质量比为2:1),得到重悬液;将重悬液采用真空冷冻法进行冻干,得到鼠李糖乳杆菌Lactobacillus rhamnosusLRa05冻干粉。
实施例3:植物乳杆菌Lactobacillus plantarum Lp90冻干粉的制备
将植物乳杆菌Lactobacillus plantarum Lp90按照占培养基总质量3%的接种量接种到培养基中,于37℃下培养18h,得到培养液;将培养液离心,得到菌体;将菌体用海藻糖浓度为100g/L的海藻糖冻干保护剂重悬(冻干保护剂和菌体的质量比为2:1),得到重悬液;将重悬液采用真空冷冻法进行冻干,得到植物乳杆菌Lactobacillus plantarum Lp90冻干粉。
实施例4:复合益生菌制剂的制备
动物双歧杆菌乳亚种Bifidobacterium animalis subsp. lactis BLa80、鼠李糖乳杆菌Lactobacillus rhamnosus LRa05和植物乳杆菌Lactobacillus plantarum Lp90冻干粉混合物制备复合益生菌制剂。
取1×108CFU的动物双歧杆菌乳亚种Bifidobacterium animalis subsp. lactisBLa80冻干粉、1×108CFU的鼠李糖乳杆菌Lactobacillus rhamnosus LRa05和1×108CFU植物乳杆菌Lactobacillus plantarum Lp90冻干粉混合均匀制备复合益生菌制剂,用水复溶后灌胃给NAFLD大鼠,连续4周。
实施例5:动物模型的建立及分组
将30只6周龄、体重170-190 g的无特定病原体(Specific Pathogen Free,SPF)雄性Sprague Dawley大鼠饲养在严格控制的温度22±2℃下,每笼4只,12h光照/黑暗周期,自由进食和饮水。适应养殖2周后,将大鼠随机分配至正常对照(Control,CTL)组(n = 10)和HFD(high-fat diet)组(n = 20)。CTL组动物定期饲喂常规大鼠普通饲料,HFD组饲喂高脂肪饲料。喂食10周后,将HFD组20只大鼠进一步平均随机分配至HFD组、益生菌干预组。为消除生物节律影响,均于每日上午9:00灌胃,连续灌胃干预4周。
实施例6:益生菌干预对NAFLD大鼠体重的影响
实验持续14周,每两周记录每只大鼠的体重。试验期间大鼠的体重变化如图1所示。
大鼠一般情况:如图1所示,整个实验过程中,所有大鼠均正常生长。CTL组大鼠毛色光亮浓密,活动正常,精力充沛,反应较敏捷。HFD组大鼠毛色暗黄,较毛糙,活动量减少,反应比较迟钝,进食量和饮水量均增多,体重增加迅速,大便较软,有恶臭。BLa80+LRa05+Lp90益生菌组合物干预后,大鼠一般情况较HFD组明显改善,体重在用药第2周以后增长速度较HFD组逐渐减缓并出现体重下降的趋势。实验第10周,CTL组和HFD组大鼠体重均有增加,其中HFD组大鼠体重明显高于CTL组,差异具有统计学意义(P<0.05)。实验第14周,HFD组大鼠体重较CTL组显著增加,差异具有统计学意义(P<0.05);BLa80+LRa05+Lp90益生菌组合物干预组较HFD组大鼠体重显著降低(P<0.05)。
实施例7:益生菌干预对NAFLD大鼠血清生化指标的影响
实验第14周,末次灌胃给药后24h,所有大鼠禁食不禁水。称体重完毕后,采用10%水合氯醛腹腔注射麻醉大鼠,随后采集血液和肝脏组织标本。
血液采集:剖开胸腔,暴露心脏,用静脉采血针刺破心尖,连接EDTA真空静脉采血管取血,以3000r/min在离心机上离心10min,留取血浆,置于-80 ℃储存待检。
肝脏组织采集:剖开大鼠腹腔,充分暴露其肝脏,剪取整个肝脏组织,用0.9%氯化钠溶液冲洗干净后,记录肝脏组织的一般情况,使用电子天平准确测量肝脏的重量并记录。切取同一部位肝脏组织约1cm3置于4%多聚甲醛溶液中固定,储存于4℃冰箱中备用检测。
血清ALT、AST、TC、TG的测定:取出预留的血清,融化后采用自动生化分析仪按照机器和说明书对大鼠血清ALT、AST、TC、TG进行测定,分析ALT、天AST、TC、TG水平。
如图2-5所示,与CTL组比较,HFD组大鼠血清ALT、AST、TC、TG水平明显升高,差异有统计学意义(P<0.05)。与HFD组比较,BLa80+LRa05+Lp90益生菌组合物干预组大鼠血清ALT、AST、TC、TG水平均降低,差异具有统计学意义(P< 0.05)。
实施例8:益生菌干预对NAFLD大鼠血清炎症因子的影响
血液采集方法同实施例6。
酶联免疫吸附(ELISA)法检测炎症因子:根据生产商(上海酶联生物科技公司)说明书,使用ELISA测定试剂盒检测炎症因子包括TNF-α,IL-1β、IL-6、IL-23、IP-10和OX-40的浓度。
如图6-11所示,与CTL组比较,HFD组大鼠血清ALT、AST、TC、TG水平明显升高,差异有统计学意义(P<0.05)。与HFD组相比,经BLa80+LRa05+Lp90益生菌组合物干预后大鼠血清TNF-α、IL-1β、IL-6、IL-23、IP-10、OX-40的含量均降低,差异有显著统计学意义(P<0.05)。
实施例9:益生菌干预对NAFLD大鼠肝脏病理的影响
肝脏组织HE染色:每只大鼠相同部位的肝组织经4%多聚甲醛固定,乙醇脱水,二甲苯脱醇,石蜡包埋,切成3-4 μm切片,苏木精伊红(H&E)染色,在光镜下观察。然后根据NAFLD诊断和治疗指南,通过新NAFLD活动度评分系统对染色的肝脏进行组织学分析,对肝组织的脂肪变性、气球样变和炎症进行分级。实验大鼠肝脏组织HE染色后比较见图12。
如图12所示,CTL组肝小叶结构完整、清晰,肝索分布呈放射状,排列较整齐,肝窦结构清晰,肝细胞大小较一致,细胞核结构清晰可见,细胞内几乎无脂质沉积。HFD组大鼠肝细胞肿胀明显、可见气球样变,部分可见小叶内有坏死,汇管区可见大量炎性细胞。经BLa80+LRa05+Lp90益生菌组合物干预后大鼠也出现不同程度的肝细胞脂肪变性,但脂肪变性的程度和炎性细胞浸润程度较HFD组明显减轻。
进一步通过NAFLD活动度评分分析,如图13所示,与CTL组比较,HFD组大鼠NAFLD活动度评分明显升高,差异有统计学意义(P<0.05)。与HFD组相比,经BLa80+LRa05+Lp90益生菌组合物干预后大鼠NAFLD活动度评分降低,差异有显著统计学意义(P<0.05)。
实施例10:益生菌干预对NAFLD大鼠粪便丁酸和异丁酸含量的影响
分别于0、10和14周收集粪便标本,冷冻于液氮中,并在-80℃保存以便进一步分析。
样品处理,取适量样本,加50 μL15%磷酸,再加125 μg/mL的内标(异己酸)溶液100μL和乙醚400 μL匀浆1min,于4 ℃12000 rpm离心10min,取上清进行GC-MS检测。GC-MS检测方法,色谱条件:色谱柱Agilent HP-INNOWAX毛细管柱(30 m*0.25 mm ID*0.25 μm);分流进样,进样量1 μL,分流比10:1。进样口温度250 ℃;离子源温度230 ℃;传输线温度250℃,四极杆温度150 ℃。程序升温起始温度90 ℃;然后以10 ℃/min 升温至120 ℃;再以5℃/min 升温至150 ℃;最后以25 ℃/min升温至250 ℃维持2 min。载气为氦气,载气流速1.0 mL/min。MS 条件:电子轰击电离(EI)源,SIM扫描方式,电子能量70 eV。
如图14所示,与CTL组比较,HFD组大鼠丁酸和异丁酸含量明显降低,差异有统计学意义(P<0.05)。与HFD组相比,经BLa80+LRa05+Lp90益生菌组合物干预后大鼠丁酸和异丁酸含量显著升高,差异有显著统计学意义(P<0.05)。
实施例11:动物双歧杆菌乳亚种Bifidobacterium animalis subsp. lactisBLa80、鼠李糖乳杆菌Lactobacillus rhamnosus LRa05和植物乳杆菌Lactobacillusplantarum Lp90冻干粉制备的复合益生菌用于固体饮料
本发明的一种复合益生菌固体饮料,该产品由复合益生菌2.0克加工而成;可制成小袋产品用于冲饮或即食。按每袋2.0克计,该产品每袋内含有20亿CFU益生菌。所述20亿CFU益生菌包括10亿CFU的动物双歧杆菌乳亚种Bifidobacterium animalis subsp.lactis BLa80,5亿CFU的鼠李糖乳杆菌Lactobacillus rhamnosus LRa05和5亿CFU的植物乳杆菌Lactobacillus plantarum Lp90,填充剂为麦芽糊精。制备的复合益生菌固体饮料能够有利于预防及缓解非酒精性脂肪肝。
虽然本发明已以较佳实施例公开如上,但其并非用以限定本发明,任何熟悉此技术的人,在不脱离本发明的精神和范围内,都可做各种的改动与修饰,因此本发明的保护范围应该以权利要求书所界定的为准。
Claims (9)
1.一种能够缓解非酒精性脂肪肝的复合益生菌制剂,其特征在于,该复合益生菌制剂包括含有动物双歧杆菌乳亚种Bifidobacterium animalis subsp. lactis BLa80、鼠李糖乳杆菌Lactobacillus rhamnosus LRa05和植物乳杆菌Lactobacillus plantarum Lp90。
2.根据权利要求1所述的一种能够缓解非酒精性脂肪肝的复合益生菌制剂,其特征在于,所述益生菌制剂中动物双歧杆菌乳亚种Bifidobacterium animalis subsp. lactisBLa80、鼠李糖乳杆菌Lactobacillus rhamnosus LRa05和植物乳杆菌Lactobacillusplantarum Lp90按照活菌数比为(1~1.5):(1~1.5):(1~1.5)复配。
3.根据权利要求1所述的一种能够缓解非酒精性脂肪肝的复合益生菌制剂其特征在于,所述动物双歧杆菌乳亚种Bifidobacterium animalis subsp. lactis BLa80、鼠李糖乳杆菌Lactobacillus rhamnosus LRa05和植物乳杆菌Lactobacillus plantarum Lp90的活菌数不低于1×106CFU/mL或1×106CFU/g。
4.根据权利要求1所述的一种能够缓解非酒精性脂肪肝的复合益生菌制剂,其特征在于:所述动物双歧杆菌乳亚种Bifidobacterium animalis subsp. lactis BLa80,分类命名为Bifidobacterium animalis,已于2018年3月5日保藏于中国微生物菌种保藏委员会普通微生物中心,保藏编号为CGMCC 15410;鼠李糖乳杆菌Lactobacillus rhamnosus LRa05,分类命名为Lactobacillus rhamnosus,已于2020年7月20日保藏于中国微生物菌种保藏委员会普通微生物中心,保藏编号为CGMCC 1.12734;植物乳杆菌Lactobacillus plantarumLp90,分类命名为Lactobacillus plantarum,已于2015年1月27日保藏于中国微生物菌种保藏委员会普通微生物中心,保藏编号为CGMCC 10453。
5.一种权利要求1所述的能够缓解非酒精性脂肪肝的复合益生菌制剂的制备方法,其特征在于:包括以下步骤:
(1)分别制备动物双歧杆菌乳亚种Bifidobacterium animalis subsp. lactisBLa80、鼠李糖乳杆菌Lactobacillus rhamnosus LRa05和植物乳杆菌Lactobacillusplantarum Lp90冻干粉;
(2)按比例将动物双歧杆菌乳亚种Bifidobacterium animalis subsp. lactis BLa80冻干粉、鼠李糖乳杆菌Lactobacillus rhamnosus LRa05冻干粉和植物乳杆菌Lactobacillus plantarum Lp90冻干粉混合均匀制备复合益生菌。
6.根据权利要求5所述的制备方法,其特征在于:动物双歧杆菌乳亚种Bifidobacterium animalis subsp. lactis BLa80、鼠李糖乳杆菌Lactobacillusrhamnosus LRa05和植物乳杆菌Lactobacillus plantarum Lp90冻干粉的制备方法分别为:
将动物双歧杆菌乳亚种Bifidobacterium animalis subsp. lactis BLa80、鼠李糖乳杆菌Lactobacillus rhamnosus LRa05和植物乳杆菌Lactobacillus plantarum Lp90冻干粉按照占培养基总质量3%的接种量分别接种到MRS培养基中,于37℃下培养18h,得到培养液;将培养液离心,得到菌体;将菌体用海藻糖浓度为100g/L的海藻糖冻干保护剂重悬(冻干保护剂和菌体的质量比为2:1),得到重悬液;将重悬液采用真空冷冻法进行冻干,分别得到三种菌的冻干粉。
7.根据权利要求6所述的制备方法,其特征在于:制备三种菌的冻干粉使用的MRS培养基(g/L)为:蛋白胨10g/L、牛肉膏10g/L、葡萄糖20g/L、乙酸钠2g/L、酵母粉5g/L、柠檬酸氢二铵2g/L、K2PO4·3H2O 2.6g/L、MgSO4·7H2O 0.1g/L、MnSO4 0.05g/L、吐温80 1mL/L、半胱氨酸氨酸盐0.5g/L。
8.一种权利要求1所述的复合益生菌制剂在预防和/或治疗非酒精性脂肪肝的产品中的应用。
9.一种权利要求1所述的复合益生菌制剂在制备预防和/或治疗非酒精性脂肪肝的固体饮料的应用;所述固体饮料包括含有动物双歧杆菌乳亚种Bifidobacterium animalissubsp. lactis BLa80、鼠李糖乳杆菌Lactobacillus rhamnosus LRa05和植物乳杆菌Lactobacillus plantarum Lp90,填充剂为麦芽糊精。
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