CN116987645A - 一种植物乳植杆菌（Lactiplantibacillus plantarum）HEPRO-185及其应用 - Google Patents
一种植物乳植杆菌（Lactiplantibacillus plantarum）HEPRO-185及其应用 Download PDFInfo
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Abstract
本发明涉及医学技术领域，具体公开了一种植物乳植杆菌(Lactiplantibacillus plantarum)HEPRO‑185及其应用。所述的植物乳植杆菌(Lactiplantibacillus plantarum)HEPRO‑185，其保藏编号为GDMCC 63307。该菌株已于2023年3月29日保藏于广东省微生物菌种保藏中心(地址是广州市先烈中路100号大院59号楼5楼广东省科学院微生物研究所)。研究表明，本发明所述的植物乳植杆菌(Lactiplantibacillus plantarum)HEPRO‑185具有优异的抗氧化以及抗衰老活性；并且其抗氧化以及抗衰老作用要显著优于已知的常用的鼠李糖乳杆菌。因此，将本发明所述的植物乳植杆菌(Lactiplantibacillus plantarum)HEPRO‑185为有效成分用于制备具有抗氧化和/或抗衰老作用的产品具有重要的应用价值。
Description
技术领域
本发明涉及益生菌技术领域，具体涉及一种植物乳植杆菌(Lactiplantibacillusplantarum)HEPRO-185及其应用。
背景技术
衰老是由多种因素引起的，其中氧化反应和氧化自由基的生成诱发、加重衰老过程起到重要作用。氧化自由基与细胞的遗传物质、功能性蛋白和脂质能够发生作用，导致细胞损伤和诱发、加速细胞衰老等。而抗氧化可中和细胞中过量生成的氧化自由基，减少其对细胞的损伤，从而延缓衰老过程。
益生菌是一类能够在人体消化道中存活并对宿主有益的活菌，常见的益生菌有乳酸菌、双歧杆菌和酵母菌等。研究报道，益生菌能够调节肠道菌群平衡，抑制有害菌的生长，并促进有益菌的繁殖。同时，益生菌具有维持肠道黏膜屏障完整性、促进食物消化和营养吸收、增强免疫系统功能以及抑制炎症反应等功能。研究也表明，益生菌具有一定的抗氧化作用，能够通过清除自由基、提高抗氧化酶活性和抑制氧化应激等来减少氧化损伤，从而发挥延缓衰老的功能。但是一种有效的益生菌并不能对所有人都发挥出较好的作用。因此，开发更多种类的益生菌具有重要的应用价值。
发明内容
为了克服现有技术中存在的至少之一的技术问题，本发明首先提供了一种植物乳植杆菌(Lactiplantibacillus plantarum)HEPRO-185。
本发明的技术方案如下：
本发明首先提供了一种植物乳植杆菌(Lactiplantibacillus plantarum)HEPRO-185，其保藏编号为GDMCC 63307。
该菌株已于2023年3月29日保藏于广东省微生物菌种保藏中心(地址是广州市先烈中路100号大院59号楼5楼广东省科学院微生物研究所)。
所述的植物乳植杆菌(Lactiplantibacillus plantarum)HEPRO-185，经分析其16s DNA序列(如SEQ ID NO:1所示)，利用测序结果在NCBI数据库中进行比对，与GenBank中植物乳植杆菌(Lactiplantibacillus plantarum)的同源性为99.95％；最终将该菌株鉴定为植物乳植杆菌(Lactiplantibacillus plantarum)HEPRO-185。
发明人在大量的实验中筛选得到了一种全新的植物乳植杆菌(Lactiplantibacillus plantarum)HEPRO-185，发明人在进一步研究中惊奇的发现，其具有抗氧化以及抗衰老作用；并且其抗氧化以及抗衰老作用要显著优于已知的鼠李糖乳杆菌。
本发明还提供了一种上述植物乳植杆菌(Lactiplantibacillus plantarum)HEPRO-185在制备具有抗衰老作用的产品中的应用。
本发明还提供了一种上述植物乳植杆菌(Lactiplantibacillus plantarum)HEPRO-185在制备具有抗氧化作用的产品中的应用。
本发明还提供了一种上述植物乳植杆菌(Lactiplantibacillus plantarum)HEPRO-185在制备具有减少体内脂褐素积累作用的产品中的应用。
优选地，所述的产品为食品、膳食补充剂、功能性食品、保健品及药物。
优选地，所述产品的剂型为丸剂、胶囊剂、片剂、颗粒剂或粉剂。
有益效果：本发明首先提供了一种全新的植物乳植杆菌(Lactiplantibacillusplantarum)HEPRO-185，研究表明，其具有优异的抗氧化以及抗衰老活性；并且其抗氧化以及抗衰老作用要显著优于已知的常用的鼠李糖乳杆菌。
此外，发明人在研究中还发现，本发明所述的植物乳植杆菌(Lactiplantibacillus plantarum)HEPRO-185，还具有优异的减少体内脂褐素积累的作用；并且其减少体内脂褐素积累作用要显著优于已知的常用的鼠李糖乳杆菌。
由于所述的植物乳植杆菌(Lactiplantibacillus plantarum)HEPRO-185具有抗氧化以及抗衰老作用；因此，将本发明所述的植物乳植杆菌(Lactiplantibacillusplantarum)HEPRO-185为有效成分用于制备具有抗氧化和/或抗衰老作用的产品具有重要的应用价值。
具体实施方式
下面将结合实施例对本发明技术方案进行清楚、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他实施例，都属于本发明保护的范围。
实施例1植物乳植杆菌(Lactiplantibacillus plantarum)HEPRO-185的分离与鉴定
(1)富集培养：取来自健康儿童的粪便处理液加入液体培养基(蛋白胨8.0g/L、酵母粉8.0g/L、谷氨酸8.0g/L、葡萄糖9.0g/L、吐温80 1.0mL/L、醋酸钠5.0g/L，磷酸氢二钾2.0g/L、硫酸镁0.5g/L、硫酸锰0.5g/L)中，在33℃静止培养4h。
(2)对步骤(1)的培养液进行镜检，判断细菌种类之后稀释培养液并涂布含菌培养液于涂布平板培养，挑取不同菌落接种不同培养基，扩大培养。益生菌固体培养基(蛋白胨8.0g/L，牛肉浸粉5.0g/L，酵母浸粉8.0g/L，葡萄糖10.0g/L，磷酸氢二钾2.0g/L，柠檬酸三铵2.0g/L，醋酸钠5.0g/L，硫酸镁0.5g/L，硫酸锰0.5g/L，吐温-80 1.0g/L，琼脂16.0g/L)，33℃培养4h。
(3)具有抗氧化、抗衰老活性益生菌分离：基于构建的秀丽隐杆线虫氧化衰老模型筛选具有抗氧化、抗衰老活性的益生菌；筛选出具有优秀抗氧化、抗衰老活性的菌株之一；经分析其16s DNA序列(如SEQ ID NO:1所示)，利用测序结果在NCBI数据库中进行比对，与GenBank中植物乳植杆菌(Lactiplantibacillus plantarum)的同源性为99.95％；最终将该菌株鉴定为植物乳植杆菌(Lactiplantibacillus plantarum)HEPRO-185。
实施例2植物乳植杆菌(Lactiplantibacillus plantarum)HEPRO-185冻干粉的制备
(1)植物乳植杆菌(Lactiplantibacillus plantarum)HEPRO-185种子液制备：将HCLP01菌种接入培养基(培养基成分为：蛋白胨11.0g/L，酵母粉11.0g/L，葡萄糖11.0g/L，吐温80 1.2mL/L，乙酸钠6.0g/L，磷酸氢二钾2.5g/L，硫酸镁0.7g/L，硫酸锰0.5g/L，谷氨酸11.0g/L)，摇瓶发酵12h，获得OD 600＞1.0的植物乳植杆菌(Lactiplantibacillusplantarum)HEPRO-185种子液。
(2)扩大培养：将步骤(1)所得的植物乳植杆菌(Lactiplantibacillusplantarum)HEPRO-185种子液接入10L发酵罐中的液体培养基(10L，培养基成分为：蛋白胨11.0g/L，酵母粉11.0g/L，葡萄糖11.0g/L，吐温80 1.2mL/L，乙酸钠6.0g/L，磷酸氢二钾2.5g/L，硫酸镁0.7g/L，硫酸锰0.5g/L，谷氨酸11.0g/L)；搅拌培养24h，结束发酵得发酵液；
(3)将步骤(2)所得发酵液离心收集菌体，然后添加水以及冻干保护剂脱脂乳粉混合均匀后经冷冻干燥后制得活菌数为10亿cfu/g的植物乳植杆菌(Lactiplantibacillusplantarum)HEPRO-185冻干粉。
效果实施例1
测试样品为：按实施例2所述方法制备得到的植物乳植杆菌(Lactiplantibacillus plantarum)HEPRO-185冻干粉(活菌数为10亿cfu/g)以及市售鼠李糖乳杆菌LGG冻干粉(活菌数为100亿cfu/g)。
将年龄同步的L1期秀丽隐杆线虫培养至L4期。然后将L4秀丽隐杆线虫(n＝100)转移到含有1mg/mL活性物的(植物乳植杆菌(Lactiplantibacillus plantarum)HEPRO-185冻干粉、市售鼠李糖乳杆菌LGG冻干粉)和50μM 5-氟-2-脱氧尿苷(FUDR，防止产卵)的新鲜NGM平板中，并在20℃下培养。每隔一天将秀丽隐杆线虫转移到含有活性物的新鲜NGM平板中。每天在显微镜下对秀丽隐杆线虫的存活进行计数，直到所有秀丽隐杆虫死亡，实验独立地重复3次。测试结果见表1。
表1.抗衰老实验结果
从表1实验数据中可以看出，本发明活菌数为10亿cfu/g的植物乳植杆菌(Lactiplantibacillus plantarum)HEPRO-185冻干粉，其对线虫的平均寿命要显著高于活菌数为100亿cfu/g的市售鼠李糖乳杆菌LGG冻干粉。这说明：本发明所述的植物乳植杆菌(Lactiplantibacillus plantarum)HEPRO-185具有优异的抗抗衰老活性；并且其抗衰老作用要显著优于已知的常用的鼠李糖乳杆菌。
效果实施例2
测试样品为：按实施例2所述方法制备得到的植物乳植杆菌(Lactiplantibacillus plantarum)HEPRO-185冻干粉(活菌数为10亿cfu/g)以及市售鼠李糖乳杆菌LGG冻干粉(活菌数为100亿cfu/g)。
将年龄同步的L1期秀丽隐杆线虫培养至L4期。然后(n＝100)将L4秀丽隐杆线虫转移到新鲜NGM平板中，10mM百草枯共同培养12h之后。收集秀丽隐杆线虫并将其转移到含有1mg/mL活性物(植物乳植杆菌(Lactiplantibacillus plantarum)HEPRO-185冻干粉、市售鼠李糖乳杆菌LGG冻干粉)和50μM 5-氟-2-脱氧尿苷(FUDR，防止产卵)的新鲜NGM中，并在20℃下培养24小时之后，收集秀丽隐杆线虫，ELISA试剂盒检测SOD、CAT的活性以及ROS的含量结果见表2；采用荧光显微镜检测脂褐素含量，结果见表3。
表2抗氧化实验结果
从表2实验结果可以看出，本发明活菌数为10亿cfu/g的植物乳植杆菌(Lactiplantibacillus plantarum)HEPRO-185冻干粉，其对线虫的体内ROS含量要小于活菌数为100亿cfu/g的市售鼠李糖乳杆菌LGG冻干粉，其对线虫的体内SOD活力以及Catalase活力要高于市售鼠李糖乳杆菌LGG冻干粉。这说明：本发明所述的植物乳植杆菌(Lactiplantibacillus plantarum)HEPRO-185其抗衰老作用要显著优于已知的常用的鼠李糖乳杆菌。
表3.线虫体内脂褐素积累的活力
从表3实验结果可以看出，本发明活菌数为10亿cfu/g的植物乳植杆菌(Lactiplantibacillus plantarum)HEPRO-185冻干粉，其对线虫的体内脂褐素积累要显著小于活菌数为100亿cfu/g的市售鼠李糖乳杆菌LGG冻干粉。这说明：本发明所述的植物乳植杆菌(Lactiplantibacillus plantarum)HEPRO-185其减少体内脂褐素积累作用要显著优于已知的常用的鼠李糖乳杆菌。
以上所揭露的仅为本发明较佳实施例而已，当然不能以此来限定本发明之权利范围，本领域普通技术人员可以理解实现上述实施例的全部或部分流程，并依本发明权利要求所作的等同变化，仍属于发明所涵盖的范围。
Claims (6)
1.一种植物乳植杆菌(Lactiplantibacillus plantarum)HEPRO-185，其保藏编号为GDMCC 63307。
2.权利要求1所述的植物乳植杆菌(Lactiplantibacillus plantarum)HEPRO-185在制备具有抗衰老作用的产品中的应用。
3.权利要求1所述的植物乳植杆菌(Lactiplantibacillus plantarum)HEPRO-185在制备具有抗氧化作用的产品中的应用。
4.权利要求1所述的植物乳植杆菌(Lactiplantibacillus plantarum)HEPRO-185在制备具有减少体内脂褐素积累作用的产品中的应用。
5.根据权利要求2-4任一项所述的应用，其特征在于，所述的产品为食品、膳食补充剂、功能性食品、保健品及药物。
6.根据权利要求2-4任一项所述的应用，其特征在于，所述产品的剂型为丸剂、胶囊剂、片剂、颗粒剂或粉剂。
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