CN113073071A - 一株假小链双歧杆菌及其在代谢综合征中的应用 - Google Patents
一株假小链双歧杆菌及其在代谢综合征中的应用 Download PDFInfo
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- CN113073071A CN113073071A CN202110617293.8A CN202110617293A CN113073071A CN 113073071 A CN113073071 A CN 113073071A CN 202110617293 A CN202110617293 A CN 202110617293A CN 113073071 A CN113073071 A CN 113073071A
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- bifidobacterium pseudocatenulatum
- culture supernatant
- liver
- bifidobacterium
- pseudocatenulatum
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Abstract
本发明提供一株假小链双歧杆菌(Bifidobacterium pseudocatenulatum)及其在代谢综合征中的应用,涉及微生物领域,其保藏编号为CGMCC No.22185于2021年4月14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,保藏地址为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,本发明提供的假小链双歧杆菌(Bifidobacterium pseudocatenulatum)能够显著抑制肝细胞脂质积累,降低体重和血脂水平,进而改善肝脏脂肪变性,对于缓解高脂血症、肥胖和脂肪肝的发生具有重要的应用意义。
Description
技术领域
本发明涉及微生物领域,尤其涉及一种假小链双歧杆菌及其在代谢综合征中的应用。
背景技术
血脂异常是导致心脑血管疾病的重要危险因素,与高血压、糖尿病等多种慢性病危险因素一样,会引发冠心病、心肌梗死、卒中等心脑血管疾病。因此,血脂管理目前是我国国民心脑血管疾病防控的重点。随着人们生活水平的提高、饮食结构改变及生活节奏加快、压力增加,“三高”问题也逐渐严重,仅血脂异常一项,患病率就高达40.4%。据国家心血管病中心统计显示,我国血脂异常人数已经超过4亿人。同时,血脂异常也不仅是老年人群的专属疾病,患者群逐渐年轻化,成为30—50岁年龄段人群的“第一杀手”,并且儿童青少年高胆固醇血症患病率也有明显升高,极大加重了我国血脂异常相关疾病防控的严峻形势。
目前,他汀类药物被认为是目前最为有效的降脂药物,但该类药物仍存在一些明显的副作用和服用限制。当前越来越多的研究专注于寻找具有优良降血脂功效的作用物质,并解析其降脂机理,以期发现安全性较高,降脂效果显著的活性物质。近十年来的科学研究发现栖居于人类胃肠道系统内的肠道微生物在调控宿主脂代谢稳态方面发挥不可忽视的作用,肠道菌群紊乱与代谢综合征的发生密切相关。例如,Akkermansia muciniphila菌株是近几年来发现的一类具有减肥,降脂与延缓衰老作用的新型益生菌。因此,对于肠道微生物菌库进行功能挖掘是当期该领域的研究热点,更具体地是寻找具有高效调节代谢稳态功能的菌株。由于肠道菌群,尤其是益生菌,安全性较高,分离纯化成本低,如果能够明确其降血脂功能,并确定其降血脂机制,那么益生菌作为下一代调血脂产品(如食品或/和药物)的市场前景将十分广阔。
发明内容
本发明的目的是提供一种具有降高血糖功效的假小链双歧杆菌(Bifidobacterium pseudocatenulatum)及其应用;该双歧杆菌能够促进HepG2肝癌细胞葡萄糖消耗,抑制α-淀粉酶活性,并且可以显著降低高脂饮食小鼠的体重、血脂和血糖含量,改善葡萄糖耐量及胰岛素抵抗,并缓解肝脏脂肪变性。
本发明第一方面提供保藏编号为CGMCC No.22185的假小链双歧杆菌(Bifidobacterium pseudocatenulatum)或其培养上清液或其死菌体。
其中,所述假小链双歧杆菌(Bifidobacterium pseudocatenulatum)命名为BPW0,已于2021年4月14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. 22185,保藏地址为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。
本发明第二方面提供假小链双歧杆菌或其培养上清液或其死菌体在制备用于改善和/或预防代谢综合征的药物中的应用。
本发明第三方面提供假小链双歧杆菌或其培养上清液或其死菌体在制备用于改善脂质代谢的药物中的应用。
本发明第四方面提供假小链双歧杆菌或其培养上清液或其死菌体在制备用于改善和/或预防脂质积累的药物中的应用。
本发明第五方面提供假小链双歧杆菌或其培养上清液或其死菌体在制备用于改善和/或预防高血脂的药物中的应用。
本发明第六方面提供假小链双歧杆菌或其培养上清液或其死菌体在制备用于改善和/或预防脂肪肝的药物中的应用。
本发明第七方面提供假小链双歧杆菌或其培养上清液或其死菌体在制备用于改善和/或预防非酒精性脂肪肝的药物中的应用。
本发明第八方面提供假小链双歧杆菌或其培养上清液或其死菌体作为食品或食品成分的应用。
本发明第九方面提供药物组合物,其包括第一方面所述的假小链双歧杆菌或其培养上清液或其死菌体。
本发明第十方面提供食品,其包括第一方面所述的假小链双歧杆菌或其培养上清液或其死菌体。
本发明第十一方面涉及治疗患者代谢综合征的方法,包括以第一方面所述的假小链双歧杆菌或其培养上清液或其死菌体或第九方面所述的药物组合物向所述患者给药。
需要说明的是,本发明所称的代谢综合征为包括但不限于肥胖、血脂异常、高血黏、高脂肪肝发生率。
需要说明的是,本发明所称的改善包括但不限于降低体重,降低血脂含量或/和甘油三酯含量或/和血清总胆固醇含量或/和低密度脂蛋白胆固醇水平或/和肝脏甘油三酯或/和肝脏胆固醇含量,缓解肝脏脂肪变性。
需要说明的是,本发明所称的药物组合物还包括剂型上或药学可接受的载体。例如冻干粉、片剂、胶囊、丸剂、粉剂、颗粒、酏剂、酊剂、悬浮液、糖浆和乳剂或药剂领域普通技术人员熟知的剂型。
其中,当剂型或药学上可接受的载体为固态药物组合物时(如胶囊、片剂和粉剂),可以包含合适的粘合剂、润滑剂、崩解剂、着色剂、调味剂、致流动剂和熔化剂等,其中,合适的粘合剂包括淀粉、明胶、天然糖(如葡萄糖或β-乳糖)、玉米甜味剂、天然和合成树胶,如阿拉伯胶、黄耆胶或藻酸钠、羧甲基纤维素、聚乙二醇、蜡等。这些剂型中所用的润滑剂包括油酸钠、硬脂酸钠、硬脂酸镁、苯甲酸钠、乙酸钠、氯化钠等。崩解剂包括(但不限于)淀粉、甲基纤维素、琼脂、膨润土、黄原胶等。
其中,胶囊(例如明胶胶囊)可包含活性成分和粉状载体,如乳糖、淀粉、纤维素衍生物、硬脂酸镁、硬脂酸等。类似的稀释剂可用来制备压制片。片剂和胶囊均可制成即时释放产品或缓释产品。压制片可以包糖衣或包膜以遮蔽任何令人不快的味道及保护片剂不受大气影响,或包肠衣使其在胃肠道中选择性崩解。
其中,当剂型或药学上可接受的载体为液态药物组合物时,包含在水、药学上可接受的脂肪和油、醇包括酯或其它有机溶剂中的溶液或悬浮液、乳剂、糖浆或酏剂、悬浮液、溶液和/或从非泡腾颗粒重建的悬浮液及从泡腾颗粒重建的泡腾制剂等液体药物组合物。这样的液体剂型可包括例如合适的溶剂、防腐剂、乳化剂、悬浮剂、稀释剂、甜味剂、增稠剂和熔化剂。
其中,口服给药用的液态药物组合物可包含着色剂和调味剂以提高患者的接受度。一般来说,水、合适的油、盐水、含水右旋糖(葡萄糖)和相应的糖溶液和乙二醇(如丙二醇或聚乙二醇)是非经胃肠道溶液的合适载体。例如,在口服用片剂或胶囊剂型中,活性成分可与口服无毒的药学上可接受的惰性载体(如乳糖、明胶、琼脂、淀粉、蔗糖、葡萄糖、甲基纤维素、硬脂酸镁、磷酸二钙、硫酸钙、甘露醇、山梨醇等)混合。
有益效果:
本发明提供的假小链双歧杆菌(Bifidobacterium pseudocatenulatum)BPW0能够显著抑制肝细胞脂质积累,降低体重和血脂水平,进而改善肝脏脂肪变性,对于缓解高脂血症、肥胖和脂肪肝的发生具有重要的应用意义。
附图说明
附图是用来提供对本发明的进一步理解和说明,与具体实施方式一起用于解释本发明,但并不限制本发明。
图1显示本发明中的假小链双歧杆菌BPW0的质谱鉴定图。
图2显示本发明中的假小链双歧杆菌BPW0的细菌培养物上清液对HepG2肝癌细胞油红O染色后358nm处的吸光值的影响,辛伐他汀作为阳性药,n=8, *,p<0.05; **,p<0.01;***,p<0.001。
图3显示本发明中假小链双歧杆菌BPW0的细菌培养物上清液对HepG2肝癌细胞内甘油三酯TG含量的影响,辛伐他汀作为阳性药,n=3, *,p<0.0; **,p<0.01; ***,p<0.001。
图4显示灌胃本发明中的假小链双歧杆菌BPW0后高脂血症小鼠的体重曲线。
图5显示本发明中的假小链双歧杆菌BPW0降低高脂血症小鼠的血清总甘油三酯、血清总胆固醇和血清低密度胆固醇水平,n=6, *,p<0.05; **,p<0.01; ***,p<0.001。
图6显示本发明中的假小链双歧杆菌BPW0降低高脂血症小鼠肝脏甘油三酯和肝脏总胆固醇含量,n=6, *,p<0.05; **,p<0.01; ***,p<0.001。
具体实施方式
下述实施例中所使用的实验方法如无特殊说明,均为常规方法。下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。本领域技术人员应当理解,下面所具体描述的内容是说明性而非限制性,不应以此限制本发明的保护范围。
除非另外定义,本发明中使用的所有术语(包括技术术语和科学术语)均具有如本发明所属领域的普通技术人员通常理解的相同含义。在现有技术与本发明冲突的情况下,应当以本发明为准。
实施例1 假小链双歧杆菌BPW0的分离和鉴定
本发明提供的假小链双歧杆菌BPW0的分离过程如下:
1、样品的采集
采集海南百岁老人的粪便作为分离样本,采集前人群未服用过抗生素类药物,无益生菌服用史,无胃肠病史。
2、菌种的分离
将采集的粪便样品稀释后涂布于YCFA培养基,37℃厌氧培养24-48h,挑取单菌落在新的YCFA培养基平板上划线培养,获得纯化的菌落,然后将单菌落涂布在质谱板上,分别加入裂解液和基质干燥后在MALDI-TOF MS 1000质谱仪(Autobio,郑州安图生物科技有限公司)上进行上机鉴定,鉴定结果如图1所示,根据图1的质谱菌种鉴定结果可以看出,本发明分离的BPW0菌株与假小链双歧杆菌B16AC菌株有着很高的相似性,因为可以判断本发明分离的菌株隶属于假小链双歧杆菌种。
需要说明的是,粪便样品的稀释为本领域常规方法,本发明不做限制,在本发明的一个实施例中,使用清水对粪便样品进行稀释。
实施例2 假小链双歧杆菌BPW0活菌液,代谢产物及灭活菌体的制备
1、菌种的培养
取-80℃冻存菌液涂布于YCFA固体平板,在37℃条件下倒置培养24-48h后,取单菌落接种于液体YCFA培养基中,37℃培养18-24h,得到一代菌液;取一代菌液10%接种至新鲜YCFA液体培养基,37℃培养18-24h得到二代菌液;取二代菌液10%接种于新鲜的YCFA液体培养基中,37℃培养18-24h得到工作菌液。
2、活菌液的获得
活菌液还可以通过本技术领域中的其他方式获得,只要能从培养液中富集菌体即可。例如可以通过离心和/或过滤的方法实现。
在本发明的一个实施例中,是将步骤1获得的工作菌液置于13000 rpm,4℃的条件下离心15 min后,弃去上清,收集沉淀,用生理盐水重悬,获得具有活菌体的活菌液。
3、代谢产物的获得
菌体代谢产物一般存在菌体的培养液中,因此,可以通过将菌体的培养液进行固液分离,获得上清液的方式来获得代谢产物。当然,细菌培养物上清液的制备也可在厌氧环境下进行。在本发明的一个实施例中,具体是将工作菌液于13000 rpm,4℃离心15 min后留取上清液转移至无菌离心管后即可获得细菌培养物上清液,4℃存放备用,获得代谢产物。
4、死菌液的获得
死菌体可以通过本领域常规的手段进行制备,例如,加热,辐射等。在本发明的一个实施例中,通过将活菌体在温度为65-85℃条件下,加热0.5-1.5h致死获得死菌体的死菌液。
实施例3 HepG2细胞实验
本发明使用的人源肝癌HepG2细胞购自于国家生物医学实验细胞资源库,培养方式为本领域常规使用的培养方法,在本发明的一个实施例中,培养方式为:将人源肝癌HepG2细胞在37°C和5% CO2条件下用含10%胎牛血清(FBS)的高糖DMEM培养基培养,培养基中按1:100的比例添加双抗(100ug/ml青霉素和100ug/ml链霉素),每1-2d更换一次新鲜的培养液。
本申请将本发明提供的菌株作用于HepG2细胞构建的脂质积累模型中,观察本发明提供的菌株对脂类代谢的影响,具体如下:
1、油红O染色试验
取生长状态良好的对数生长期HepG2细胞,以适宜浓度接种于96孔板中,每孔加入100µl DMEM培养基培养24h至细胞完全贴壁。待细胞生长汇合至70-80%时,弃去原培养基,加入含有油酸的新鲜培养基,油酸用以进行脂质积累模型的构建,终浓度为100uM,并添加30%(v/v)实施例2制备的代谢产物。过夜培养22-24h后,弃掉培养基,用PBS洗涤3次后,使用4%多聚甲醛室温固定30min,然后用PBS清洗一遍,弃去PBS后每孔加入按产品说明书配制的油红O染液室温染色20min。染色完毕后,PBS漂洗三次,最后一次弃掉PBS后每孔加入100µl二甲亚砜DMSO,置于摇床上轻轻摇晃5min使油红充分溶解,然后用酶标仪读取358nm处吸光值,该数值用以评估细胞内脂质积累的含量。每个样品设置4个附孔,以仅加入同等体积含量的假小链双歧杆菌BPW0培养基YCFA(30%,v/v)为阴性对照,以50uM辛伐他汀为阳性对照,结果如图2所示。
根据图2所示,对照组中油酸刺激导致HepG2细胞内脂质蓄积显著增加,而经过30%体积浓度的上清液(即代谢产物)处理后,油红O染色后在358nm下的吸光值已经降至0.2以下,明显降低;这与加入50µM辛伐他汀处理组的数值相当,可见,使用30%假小链双歧杆菌BPW0的代谢产物的抑制脂质蓄积能力已经接近使用50µM的辛伐他汀的阳性药能力。
2、细胞内甘油三酯TG含量的检测
取生长状态良好的对数生长期HepG2细胞,以适宜浓度接种于12孔板中,每孔加入1ml DMEM培养基培养24h至细胞完全贴壁。待细胞生长汇合至70-80%时,弃去原培养基,加入含有油酸的新鲜培养基,油酸用以进行脂质积累模型的构建,终浓度为100µM,并添加30%(v/v)如上所述的假小链双歧杆菌BPW0细菌培养物上清液代谢物。过夜培养22-24h后,弃掉培养基,用PBS洗涤3次后,每孔加入预冷的0.1% TritonX-100裂解液裂解细胞,并用细胞刮收集细胞至1.5ml Eppendorf离心管内,超声破碎裂解细胞,随后,用微量TG测定试剂盒按照生产厂商提供的操作流程检测细胞内TG的含量,同时用BCA蛋白浓度试剂盒检测细胞样品的蛋白质浓度,用mmol/g为单位表征细胞细胞内甘油三酯(TG)的含量。以仅加入同等体积含量的假小链双歧杆菌培养基YCFA(30%,v/v)为阴性对照,以50µM辛伐他汀为阳性对照。
根据图3可以明显看出,经油酸处理后的肝HepG2细胞内脂质蓄积显著增加,用体积百分比30%的假小链双歧杆菌BPW0培养上清液以及50µM辛伐他汀处理后,HepG2细胞内甘油三酯含量都将至0.2mmol/mg蛋白以下,且用本申请提供的菌株培养上清液处理的效果与用50µM辛伐他汀处理的效果相当,可见,使用本申请的菌株代谢产物对脂质蓄积能力的抑制效果接近50µM的辛伐他汀的阳性药力,对脂类代谢的影响效果显著。
实施例四小鼠试验
本申请将菌株施用于小鼠,观察菌株降血脂和缓解脂肪肝的功效,具体操作如下:
动物实验所用小鼠为8周龄雄性C57BL/6小鼠36只,随机分4组,分别为正常组,高脂组,高脂+BPW0活菌液组,高脂+BPW0死菌体组,每组8只动物。
正常组给予标准小鼠饲料,其余各组给予60%脂肪功能的高脂饲料D12492喂养。其中高脂+BPW0活菌组每天给予1x109CFU相应的BPW0活菌;高脂+BPW0死菌体组每天给药灭活菌体重悬液,高脂组则给予等体积的生理盐水。连续给菌4周,每周称量小鼠体重后记录体重数据。
4周后,用水合氯醛麻醉,眼眶取血,4°C离心后取上清作为血清样品,用于测量血清甘油三酯TG,总胆固醇TC和低密度脂蛋白胆固醇LDL-c的含量,同时处死动物;收取肝脏组织,-80°C低温保存,用于测定肝脏总胆固醇TC和甘油三酯TG的含量,其中,解剖前小鼠体重减去灌胃前小鼠体重为体重增加;最终检测结果如图4、5所示。
根据图4可以看出,每天只喂养高脂饲料D12492导致小鼠体重显著增加,显示出高脂饮食能够明显的促进肥胖发生;图5显示高脂组动物的甘油三酯TG,血清总胆固醇TC和低密度脂蛋白胆固醇LDL-c水平明显增加,表现出严重的高脂血症;图6显示高脂组动物中肝脏TG和TC含量也显著增加,具有典型的脂肪肝特征。
而根据图4-6中还可以看出,喂养本申请的假小链双歧杆菌BPW0活菌或者灭活菌体的小鼠,其体重、甘油三酯TG、血清总胆固醇TC、低密度脂蛋白胆固醇LDL-c水平以及肝脏TG、TC含量明显低于高脂组的小鼠。
可见,向小鼠喂食本申请的假小链双歧杆菌BPW0活菌或者灭活菌体就能显著抑制由于高脂饮食导致的体重增加,降低动物的血清TG,TC和LDL-c水平和肝脏内的TG和TC含量,显示出较好的减肥效果、良好的降脂活性,有效防止肝脏脂肪积累活性。
根据上述实验结果,可以确定本申请提供的假小链双歧杆菌BPW0菌株具有优良的调血脂功效,在制备减肥、降脂和预防非酒精性脂肪肝发生的产品方面具有广阔的应用前景。
以上详细描述了本发明的优选实施方式,但是,本发明并不限于上述实施方式中的具体细节,在本发明的技术构思范围内,可以对本发明的技术方案进行多种简单变型,这些变型均属于本发明的保护范围。
另外需要说明的是,在上述具体实施方式中所描述的各个具体技术特征,再不矛盾的情况下,可以通过任何合适的方式进行组合,为了避免不要的重复,本发明对各种可能的组合方式不再另行说明。
Claims (10)
1.保藏编号为CGMCC No. 22185假小链双歧杆菌(Bifidobacterium pseudocatenulatum)或其培养上清液或其死菌体。
2.权利要求1所述的假小链双歧杆菌或其培养上清液或其死菌体在制备用于改善和/或预防代谢综合征的药物中的应用。
3.权利要求1所述的假小链双歧杆菌或其培养上清液或其死菌体在制备用于改善脂质代谢的药物中的应用。
4.权利要求1所述的假小链双歧杆菌或其培养上清液或其死菌体在制备用于改善和/或预防脂质积累的药物中的应用。
5.权利要求1所述的假小链双歧杆菌或其培养上清液或其死菌体在制备用于改善和/或预防高血脂的药物中的应用。
6.权利要求1所述的假小链双歧杆菌或其培养上清液或其死菌体在制备用于改善和/或预防脂肪肝的药物中的应用。
7.权利要求1所述的假小链双歧杆菌或其培养上清液或其死菌体在制备用于改善和/或预防非酒精性脂肪肝的药物中的应用。
8.权利要求1所述的假小链双歧杆菌或其培养上清液或其死菌体作为食品或食品成分的应用。
9.药物组合物,其包括权利要求1所述的假小链双歧杆菌或其培养上清液或其死菌体。
10.食品,其包括权利要求1所述的假小链双歧杆菌或其培养上清液或其死菌体。
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