CN112831444A - 鼠李糖乳杆菌lr2y及应用 - Google Patents
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Abstract
本发明涉及微生物技术领域,具体涉及鼠李糖乳杆菌LR2Y及应用。所述罗伊氏乳杆菌LR2Y已于2019年11月04号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.18800;本发明提供的鼠李糖乳杆菌LR2Y有较强的胃肠道存活率和肠道定植能力,可调节血清中总胆固醇含量,使其降低至正常水平,能够缓解动脉硬化病症;发酵培养基中以山茱萸多糖为促生因子,促进鼠李糖乳杆菌的增殖,提高鼠李糖乳杆菌制剂的稳定性和对不良环境的耐受能力,更好地发挥功效。
Description
技术领域
本发明涉及微生物技术领域,具体涉及鼠李糖乳杆菌LR2Y及应用。
背景技术
心血管疾病已成为全世界范围内的头号杀手,其中动脉硬化是血管衰老的一种表现,会影响心脏泵血功能,增加高血压、心肌梗塞、中风和心衰等患病的风险,如果人们长期饮食结构不合理,摄入高胆固醇食物过高,体内胆固醇的合成与代谢负荷过重,血液中胆固醇升高,引起包括动脉血管壁胆固醇的沉淀,诱发动脉硬化等心血管等疾病的发生,严重威胁人类健康。乳酸菌是存在于人体内的一类特殊的微生物菌群,它们通常具有多种益生功能,如促进人体对营养物质的吸收、合成多种人体必需维生素、抑菌、降血氨和胆固醇、调节免疫等。生物治疗已被全世界公认为安全绿色的新型治疗方法,寻找可有效缓解动脉硬化的微生物菌种成为解决问题的关键。
发明内容
针对现有技术中存在的问题,本发明提供鼠李糖乳杆菌LR2Y,保藏编号为CGMCCNo.18800,该菌株可定植于人体肠道,耐受性较强,可有效降低胆固醇含量,缓解动脉硬化等症状。
本发明提供一株鼠李糖乳杆菌LR2Y,所述罗伊氏乳杆菌LR2Y已于2019年11月04号保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC),保藏编号为:CGMCCNo.18800;分类命名为鼠李糖乳杆菌Lactobacillus rhamnosus;保藏地址:北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所。
本发明还提供一种所述鼠李糖乳杆菌LR2Y在制备食品中的应用。
一种食品,含有鼠李糖乳杆菌LR2Y菌粉。
所述的食品中含有鼠李糖乳杆菌LR2Y菌粉有效菌数量为1×107~1×109CFU/mL。
所述鼠李糖乳杆菌LR2Y菌粉是将鼠李糖乳杆菌LR2Y发酵得到菌液,菌液经过离心,真空冷冻干燥,得到菌粉。
发酵培养基为PFC/MRS复配增殖培养基,PFC/MRS复配增殖培养基为向MRS液体培养基中加入PFC(山茱萸多糖),加热溶解,配制APS/MRS复配增殖培养基,调整pH为7.0,灭菌备用。
将山茱萸多糖按质量体积比1.0~4.0g/100mL溶于MRS培养基中。不同种类的中药多糖及同一种中药多糖的不同浓度对益生菌的增殖效果是具有差异性的。山茱萸多糖对鼠李糖乳杆菌的增殖作用并非浓度越高越好,高浓度山茱萸多糖反而会产生抑制作用。这可能是由于山茱萸多糖作为一种功能性多糖,在合适的添加范围内对于鼠李糖乳杆菌的代谢调节有着积极的促进作用;而多糖浓度的增加使发酵液渗透压增大,从而抑制了鼠李糖乳杆菌的生长。
MRS培养基包括以下组分:蛋白胨10g、牛肉膏5g、酵母粉4g、K2HPO4·7H2O 2g、柠檬酸三铵2g、醋酸钠·3H2O 4g、葡萄糖20g、吐温80 1g、MgSO4·7H2O 0.2g、MnSO4·4H2O0.05g、蒸馏水1000mL。
发酵工艺为静置培养,温度为25~45℃,时间为12~36h;优选地,37℃培养18h。鼠李糖乳杆菌为厌氧菌,在静置培养条件下,能快速地生长繁殖。当发酵温度低于优选范围值时,菌种代谢缓慢,会导致菌体翻代速率有所降低。
所述食品包括益生菌固体饮料,所述益生菌固体饮料,以质量份数计,还包括以下原料:低聚果糖10~20份、麦芽糊精粉10~20份和水65~80份。
本发明的目的之三在于提供一种所述鼠李糖乳杆菌LR2Y在制备缓解动脉硬化的食品中的应用。
本发明的有益效果在于,
(1)本发明提供的鼠李糖乳杆菌LR2Y有较强的胃肠道存活率和肠道定植能力,可调节血清中总胆固醇含量,使其降低至正常水平,能够缓解动脉硬化病症;
(2)发酵培养基中以山茱萸多糖为促生因子,促进鼠李糖乳杆菌的增殖,提高鼠李糖乳杆菌制剂的稳定性和对不良环境的耐受能力,更好地发挥功效;
(3)利用所述鼠李糖乳杆菌LR2Y显著的功能特性使其在食品中,尤其是益生菌固体饮料中具有广泛的应用价值。
具体实施方式
为了使本技术领域的人员更好地理解本发明中的技术方案,下面将对本发明实施例中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做出创造性劳动前提下所获得的所有其他实施例,都应当属于本发明保护的范围。
生物材料保藏说明
生物材料的分类命名:鼠李糖乳杆菌Lactobacillus rhamnosus
生物材料的菌株编号:LR2Y
生物材料的保藏单位名称:中国微生物菌种保藏管理委员会普通微生物中心
生物材料的保藏单位简称:CGMCC
生物材料的保藏单位地址:北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所,邮政编码:100101
生物材料的保藏日期:2019年11月04号
生物材料的保藏中心登记入册编号:CGMCC No.18800
山茱萸果肉中含糖类、有机酸类及其酯类、鞣质类等,是治疗糖尿病、冠心病和高血压的主要药材,其中山茱萸多糖是山茱萸生物学活性物质的重要成分,本发明人经大量实验后发现,山茱萸多糖可作为促生因子,促进鼠李糖乳杆菌增殖,提高其在胃肠道中转运时的耐受性。本发明所述山茱萸多糖的提取可采用现有技术中常规方法,例如可采用申请号为CN200810120569.6的专利所提供的酶解提取山茱萸多糖的方法:
(1)干燥粉碎:取烘干的山茱萸干燥果肉,研磨粉碎成100目的山茱萸干粉。
(2)酶处理:取10.0g山茱萸干粉,加入蒸馏水300g混合,再加入果胶酶(酶活30000U/g,上海麦克林生化科技有限公司)0.3g、木瓜蛋白酶(酶活200U/mg,上海麦克林生化科技有限公司)0.3g、纤维素酶(酶活15000U/g,无锡市雪梅酶制剂科技有限公司)0.3g、淀粉水解酶(酶活2000U/g,北京东华强盛生物技术有限公司)0.3g,用1.0mol/LNaOH和1.0mol/L HCl调节溶液的pH值为7.0,在温度为60℃下酶解提取0.5h;
(3)高温灭酶:酶解完成后升温至90℃灭酶1.5h;
(4)醇沉:提取液离心后上清液浓缩至原体积的1/4,向浓缩液中加入4倍体积的90%乙醇,搅拌均匀后静止12h后离心,沉淀物经冷冻干燥后得到山茱萸多糖。或因试剂购自厂家不同以及操作上的差异,最终山茱萸多糖含量仅为47.5%。
实施例1不同添加量山茱萸多糖对鼠李糖乳杆菌活菌数的影响
将活化3代的菌种按1%(V/V)接种量(约1×106CFU/mL)接种于含有不同量山茱萸多糖的发酵培养基中,平行3组,同时以不加山茱萸多糖的MRS液体培养基为对照组,37℃培养16h后,测定培养基中的活菌数。活菌计数采用稀释平板计数法。结果见表1。
表1不同量山茱萸多糖对鼠李糖乳杆菌活菌数的影响
通过表1可知,添加山茱萸多糖的发酵液中活菌数高于对照组,说明山茱萸多糖可以作为促生因子,促进鼠李糖乳杆菌的增殖。随着山茱萸多糖含量的增加,鼠李糖乳杆菌的生长随之增加,在3.0g/100mL时达到最大,继续增大山茱萸多糖的含量,增值效果反而减小。
实施例2鼠李糖乳杆菌LR2Y模拟胃肠消化液的存活率
模拟胃液的配制:模拟胃液的配制:取9.5%盐酸15mL,加蒸馏水稀释,使pH值达到2.5,按照每100mL加入1.0g胃蛋白酶,混匀,0.22μm无菌滤膜过滤,现配现用。取鼠李糖乳杆菌LR2Y菌粉1g,转移到预热的装有模拟胃液试管中,37℃下80r/min处理0.5h、1h、2h。对样品溶液进行梯度稀释,倾注法活菌计数,测定活菌数并计算其存活率。存活率是在该培养液中取样时的活菌数与在第0h活菌数之比,以%表示。
模拟肠液的配制:取KH2PO4 6.8g,加入500mL蒸馏水溶解,用4g/L NaOH溶液调pH至6.8,蒸馏水定容至1L,加胆盐0.3%,按照100mL加1g胰蛋白酶,混匀,0.22μm无菌滤膜过滤,现配现用。取LR2Y菌粉1g,转移到装有模拟肠液试管中,37℃条件下80r/min处理0.5h、1h、2h。对样品溶液进行梯度稀释,倾注法活菌计数,测定活菌数并计算其存活率。存活率是在该培养液中取样时的活菌数与在第0h活菌数之比,以%表示。
设置对照组,对照组菌粉将鼠李糖乳杆菌LR2Y于MRS培养基中发酵得到菌液,菌液经过离心,真空冷冻干燥后所得,即对照组中的发酵培养基不含有山茱萸多糖。
表2鼠李糖乳杆菌LR2Y通过模拟胃消化液的存活率
表3鼠李糖乳杆菌LR2Y通过模拟肠消化液的存活率
通过表2和表3可知,鼠李糖乳杆菌LR2Y对人工胃肠液具有较好的耐受性,同时相对于对照组,在添加山茱萸多糖的发酵液中,鼠李糖乳杆菌LR2Y的存活率更高。山茱萸多糖能够促进鼠李糖乳杆菌增殖,提高其在胃肠道中转运时的耐受性。
实施例3菌株发酵液去除胆固醇评价实验
胆固醇降解率的测定采用铁矾显色法,具体操作详见文献《酸菜汁中乳酸菌的分离鉴定及其体外降胆固醇能力研究》。
其中,
鼠李糖乳杆菌LR2Y菌株的胆固醇脱除率为86.55%,具有较高的胆固醇降解能力,从而能够缓解动脉硬化病症。
实施例4
一种含有所述鼠李糖乳杆菌LR2Y菌粉的益生菌固体饮料,以质量份数计,还包括以下原料:低聚果糖20、和麦芽糊精粉15份和水75份,所述益生菌固体饮料中有效菌数量为1×109CFU/mL。
将鼠李糖乳杆菌LR2Y接种到PFC/MRS复配增殖培养基平板中,接种量为2%,在37℃下培养18h后,对菌液进行离心、真空冷冻干燥处理,获得菌粉,将菌粉、低聚果糖粉、麦芽糊精粉按配比混合后即得所述益生菌固体饮料。
所述MRS培养基包括以下组分:蛋白胨10g、牛肉膏5g、酵母粉4g、K2HPO4·7H2O 2g、柠檬酸三铵2g、醋酸钠·3H2O 4g、葡萄糖20g、吐温80 1g、MgSO4·7H2O 0.2g、MnSO4·4H2O0.05g、蒸馏水1000mL,将山茱萸多糖按质量体积比3.0g/100mL溶于MRS培养基中得PFC/MRS复配增殖培养基。
通过优选实施例的方式对本发明进行了详细描述,但本发明并不限于此。在不脱离本发明的精神和实质的前提下,本领域普通技术人员可以对本发明的实施例进行各种等效的修改或替换,而这些修改或替换都应在本发明的涵盖范围内/任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,可轻易想到变化或替换,都应涵盖在本发明的保护范围之内。因此,本发明的保护范围应以权利要求所述的保护范围为准。
Claims (9)
1.一株鼠李糖乳杆菌LR2Y,其特征在于,所述罗伊氏乳杆菌LR2Y已于2019年11月04号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.18800。
2.如权利要求1所述的所述鼠李糖乳杆菌LR2Y在制备食品中的应用。
3.一种食品,其特征在于,所述食品含有鼠李糖乳杆菌LR2Y菌粉。
4.如权利要求3所述的一种食品,其特征在于,所述的食品中含有鼠李糖乳杆菌LR2Y菌粉有效菌数量为1×107~1×109CFU/mL。
5.如权利要求3所述的一种食品,其特征在于,所述鼠李糖乳杆菌LR2Y菌粉是将鼠李糖乳杆菌LR2Y发酵得到菌液,菌液经过离心,真空冷冻干燥,得到菌粉。
6.如权利要求5所述的一种食品,其特征在于,发酵培养基为向MRS液体培养基中加入山茱萸多糖,加热溶解,配制复配增殖培养基,调整pH为7.0,灭菌备用。
7.如权利要求6所述的一种食品,其特征在于,将山茱萸多糖按质量体积比1.0~4.0g/100mL溶于MRS培养基中。
8.如权利要求5所述的一种食品,其特征在于,发酵工艺为静置培养,温度为25~45℃,时间为12~36h。
9.如权利要求3所述的一种食品,其特征在于,所述食品包括益生菌固体饮料,所述益生菌固体饮料,以质量份数计,还包括以下原料:低聚果糖10~20份、麦芽糊精粉10~20份和水65~80份。
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