CN113881610B - 多形拟杆菌bp8e及其应用 - Google Patents
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Abstract
本发明属于微生物及其应用领域,提供了多形拟杆菌(Bacteroides thetaiotaomicron)BP8E,其保藏编号为CGMCC No.22149。本发明还提供了多形拟杆菌(Bacteroides thetaiotaomicron)BP8E在生产GABA和预防和/或治疗抑郁症中的应用。本发明提供的多形拟杆菌BP8E能够在不额外添加谷氨酸的培养条件下高产GABA,并且能够改善小鼠抑郁样行为,可以为抑郁症、失眠等压力情绪障碍提供益生菌治疗的潜在菌株,具有很大的潜在市场价值。
Description
技术领域
本发明属于微生物及其应用领域,特别涉及多形拟杆菌BP8E及其应用。
背景技术
γ-氨基丁酸(γ-AminobutyricAcid,又称GABA)是一种非蛋白质天然氨基酸,广泛存在于哺乳动物的神经***中,也存在与植物中,如番茄、马铃薯、葡萄、南瓜及茶叶等。发酵食品如泡菜、发酵糙米中也富含GABA。
GABA是神经***中重要的抑制性神经递质。研究者发现GABA含量不足会产生焦虑、不安、疲倦和焦虑等情绪。然而,随着年龄的增长,人体内的人GABA含量会呈现不断下降趋势。据悉,每10年人体GABA含量下降约5%。目前GABA被报道具有以下生理作用:(1)缓解焦虑和抑制兴奋,(2)促进代谢,(3)提高记忆力,(4)延缓神经细胞衰老,(5)美容,(6)调节血压作用。适当补充GABA能较好的缓解精神压力、调节情绪、改善睡眠。鉴于GABA的有益生理作用以及扎实的理论依据,GABA已开始进入各国市场,早在2001年,日本批准GABA为新资源食品,随后我国也于2009年批准GABA为新资源食品(***2009年第12号公告)。至此,GABA入食应用正式打开了国门,GABA作为一种新食品原料,被誉为“神经维他命”,被广泛应用于针对高压力人群的舒压助眠类产品以及针对时尚爱美的美容升级产品。
除了直接补充GABA膳食补充剂以外,人们可以从天然食物,如谷物,十字花科植物,西红柿等中获取。微生物发酵是当下研究GABA合成的一个热点。肠道微生物栖居于宿主胃肠道***内,对机体代谢和免疫平衡起着至关重要的作用。乳杆菌和双歧杆菌中包含较多合成GABA的菌株,除此之外,其他菌种是否具有高产GABA的能力仍鲜为人知。由于肠道微生物,尤其是具有益生作用的肠道菌,不仅高产GABA,而且存在较高的安全性,因此,挖掘和开发具有高产GABA能力的肠道菌株具有很好的市场应用前景,同时也为抑郁症、失眠等压力情绪障碍提供益生菌治疗的潜在菌株。
发明内容
为了解决现有技术中存在的上述问题,本发明提供了一种具有高效产GABA能力的多形拟杆菌BP8E。该多形拟杆菌能够在不额外添加谷氨酸的培养条件下高产GABA,两次高效液相色谱HPLC检测其GABA产生量平均值为528.6mg/L,并且动物实验显示BP8E能明显够改善小鼠抑郁样行为。
本发明第一方面提供了多形拟杆菌(Bacteroides thetaiotaomicron)BP8E,其保藏编号为CGMCC No. 22149。
本发明第二方面提供了含有下述至少一种的制剂在生产GABA中的应用:
1)多形拟杆菌(Bacteroides thetaiotaomicron)BP8E;
2)多形拟杆菌(Bacteroides thetaiotaomicron)BP8E菌剂;
3)多形拟杆菌(Bacteroides thetaiotaomicron)BP8E活菌悬液;
4)多形拟杆菌(Bacteroides thetaiotaomicron)BP8E死菌悬液;
5)多形拟杆菌(Bacteroides thetaiotaomicron)BP8E代谢产物;
6)多形拟杆菌(Bacteroides thetaiotaomicron)BP8E提取物;
其中,所述多形拟杆菌(Bacteroides thetaiotaomicron)BP8E为本发明第一方面所述的保藏编号为CGMCC No.22149的多形拟杆菌(Bacteroides thetaiotaomicron)BP8E。
本发明第三方面提供了含有下述至少一种的制剂在制备用于预防和/或治疗抑郁症的产品中的应用:
1)多形拟杆菌(Bacteroides thetaiotaomicron)BP8E;
2)多形拟杆菌(Bacteroides thetaiotaomicron)BP8E菌剂;
3)多形拟杆菌(Bacteroides thetaiotaomicron)BP8E活菌悬液;
4)多形拟杆菌(Bacteroides thetaiotaomicron)BP8E死菌悬液;
5)多形拟杆菌(Bacteroides thetaiotaomicron)BP8E代谢产物;
6)多形拟杆菌(Bacteroides thetaiotaomicron)BP8E提取物;
其中,所述多形拟杆菌(Bacteroides thetaiotaomicron)BP8E为本发明第一方面所述的保藏编号为CGMCC No. 22149的多形拟杆菌(Bacteroides thetaiotaomicron)BP8E。
本发明第四方面提供了一种用于预防和/或治疗抑郁症的产品,含有下述至少一种:
1)多形拟杆菌(Bacteroides thetaiotaomicron)BP8E;
2)多形拟杆菌(Bacteroides thetaiotaomicron)BP8E菌剂;
3)多形拟杆菌(Bacteroides thetaiotaomicron)BP8E活菌悬液;
4)多形拟杆菌(Bacteroides thetaiotaomicron)BP8E死菌悬液;
5)多形拟杆菌(Bacteroides thetaiotaomicron)BP8E代谢产物;
6)多形拟杆菌(Bacteroides thetaiotaomicron)BP8E提取物;
其中,所述多形拟杆菌(Bacteroides thetaiotaomicron)BP8E为本发明第一方面所述的保藏编号为CGMCC No. 22149的多形拟杆菌(Bacteroides thetaiotaomicron)BP8E。
保藏说明:
菌种名称:BP8E
拉丁名:Bacteroides thetaiotaomicron
分类命名:多形拟杆菌
菌株编号:22149
保藏机构:中国微生物菌种保藏管理委员会普通微生物中心
保藏机构简称:CGMCC
地址:北京市朝阳区北辰西路1号院3号
保藏日期:2021年4月9日
保藏中心登记入册编号:CGMCC No. 22149
本发明提供的多形拟杆菌(Bacteroides thetaiotaomicron)BP8E具有良好的生产GABA能力,能够明显缓解抑郁症模型小鼠的抑郁样行为,为日后开发有效的抗抑郁益生菌产品提供了很好的科学依据,具有很大的潜在市场价值。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案,下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图是本发明的一些实施方式,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1示出了本发明中的多形拟杆菌BP8E的质谱鉴定图。
图2示出了本发明中多形拟杆菌BP8E对抑郁模型小鼠悬尾实验的影响。n=8,*p<0.05, ***p<0.001。
具体实施方式
为使本发明的技术方案、目的和优点更加清楚,下面通过具体的实施例子对本发明做进一步的详细描述。应当理解的是,此处所描述的具体实施方式仅用于说明和解释本发明,并不用于限制本发明。
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。
下述实施例中所使用的各种试剂、材料等,若无特别说明,均为可以从商业渠道获得的产品;下述实施例中所使用的各种测试、检测方法若无特别说明,均为本领域中的常规测试、检测方法,均可以从教科书、工具书或学术期刊中获得。
实施例1:多形拟杆菌BP8E的分离鉴定
采集长期生活在北京城区的健康成年人的粪便样品,稀释后涂布于YCFA培养基,37℃厌氧培养24h,分离获得平板上不同的单菌落。用灭菌后的接种环挑取不同单菌落在新的YCFA固体培养基平板上划线纯化,37℃厌氧培养24h,得到纯化单菌落。将每个纯化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)上进行质谱上机鉴定。鉴定完成后将各菌株于-80℃冻存在本公司的菌种资源库中,以待后用。其中,本发明所涉及的多形拟杆菌BP8E菌株鉴定结果如图1所示。根据图1结果可见,BP8E菌株在分类上与多形拟杆菌存在极高的相似性,据此,将其命名为多形拟杆菌BP8E菌株。
实施例2:多形拟杆菌BP8E的制备
取-80℃冻存的BP8E菌液涂布于YCFA固体平板,在37℃条件下倒置培养24h后,取单菌落接种于液体YCFA培养基中,37℃培养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24h得到工作菌液。将工作菌液置于13000 rpm,4℃的条件下离心15 min后,收集上清即为BP8E菌株代谢物,后续实验用,其中菌体沉淀用生理盐水重悬,即可获得具有活菌体的活菌液。
实施例3:GABA产生量检测
本实施例通过纸层析法和高效液相色谱法对多形拟杆菌BP8E菌株产生的GABA进行定量检测。
(1)纸层析法
取多形拟杆菌BP8E上清液、YCFA培养基和GABA标准溶液(0.2,0.4,0.8,1.2,1.6和2.0 g/L)各4 μL在层析纸上点样,然后放在展开剂(V(正丁醇)∶V(冰醋酸)∶V(水)=5∶3∶2,茚三酮用量为1.2%(w/v))中展开。层析结束后,立即将层析纸放入90℃烘箱中干燥显色30min。将与标准品位置一致的斑点剪下,用5 mL洗脱液(V75%乙醇:V0.6%硫酸铜=38:2)在40℃、50rpm条件下洗脱。取混匀后的洗脱液200 μL用酶标仪检测510 nm处的吸光值。根据GABA标准品绘制的标准曲线可计算样本中所含GABA的浓度值(单位为mg/L)。
通过三次纸层析实验,多形拟杆菌BP8E在YCFA液体培养基中(无外源L-谷氨酸)培养24小时后产生GABA的量如表1所示:
(2)HPLC法
准确称量GABA标准品,配制质量浓度为0.04,0.08,0.12,0.16和0.20 g/L的GABA标准溶液,取各浓度的GABA标准溶液100μL,向其中分别加入100μL邻苯二甲醛,充分混匀后室温条件下孵育至少90s。反应后溶液各取20 μL进样,于338 nm处测吸收峰值。以峰面积建立GABA浓度检测标准曲线。然后,将稀释后的BP8E上清液中加入邻苯二甲醛,孵育混匀后进样,于338 nm处测吸收峰值。
为了得到较可靠的GABA产生量的数据,共进行了两次高效液相色谱HPLC检测,多形拟杆菌BP8E产生GABA的量如下表2所示。
表2显示两次HPLC实验检测到的GABA浓度分别为564.6mg/L和492.7mg/L,平均值为528.6mg/L。文献报道的许多菌株需要在培养体系中添加外源谷氨酸,用以刺激菌株合成GABA,而本发明中的BP8E菌株能够在没有外源刺激的条件下产生高于文献报道的GABA量(232.8mg/L, 朱宗涛等,高产氨基丁酸的双歧杆菌的筛选及对抑郁细胞模型的改善作用,《食品工业科技》,2020),表明本发明的多形拟杆菌BP8E菌株具有优异的高产GABA的能力。
实施例4:多形拟杆菌BP8E对慢性应激抑郁模型小鼠悬尾实验的影响
通过慢性应激刺激构建的抑郁症模型小鼠是筛选抑郁症治疗药物研究中最常用的动物模型。因此,在以下实施例中采用通过慢性应激刺激构建的抑郁症模型小鼠作为受试对象。
(1)分组与小鼠模型建立
采购雄性昆明种小鼠32只,体重20g左右,适应性喂养一周后随机分为4组:正常组,模型组,BP8E活菌组和盐酸氟西汀组,每组8只小鼠。除正常对照组外,其他组小鼠按照慢性不可预知应激(CUMS)方法,每周以随机的次序给予以下应激刺激,持续4周:禁食12h,束缚1h,潮湿垫料12h,禁水12h,昼夜颠倒24h,震荡1分钟,电击足底。BP8E活菌组小鼠每日每只灌胃109CFU/mL多形拟杆菌BP8E活菌悬液,持续28天。正常组和模型组小鼠则给予灌胃对应的YCFA培养基,阳性组小鼠腹腔注射盐酸氟西汀(10mg/kg)。
(2)小鼠悬尾实验
小鼠悬尾实验是一种药效学上常用的行为绝望模型,用以评价抗抑郁药物的药效,通过观察小鼠在实验中悬尾的不动时间,来判断小鼠的抑郁样行为的改变。实验时,将小鼠用胶带在尾端1cm处黏贴胶带,使其倒置悬挂鼻部离地面20-25cm高,使用动物行为视频分析***记录6min内小鼠的绝望行为,分析后4min内的累计不动时间(s)。实验中,小鼠被分开测试以避免彼此的视觉和声音的干扰,其中,小鼠悬尾不动时间的判断标准为小鼠停止挣扎,呈倒悬垂状态,静止不动。
统计结果如图2所示,在悬尾实验中,模型组与正常组相比,小鼠悬尾的不动时间明显增多(p<0.001),BP8E组与模型组相比,灌胃BP8E活菌悬液能够显著减少受试小鼠的绝对不动时间(p<0.05),平均不动时间由~120s降至~94s,效果仅稍弱于药物盐酸氟西汀对小鼠绝对不动时间的改善效果(平均不动时间~85s),这些表明本发明中的多形拟杆菌BP8E菌株能够明显缓解抑郁症模型小鼠的在悬尾实验中的抑郁样行为。
以上所述仅为本发明的优选实施例而已,并不用于限制本发明,对于本领域的技术人员来说,本发明可以有各种更改和变化。凡在本发明的精神和原则之内所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (4)
1.多形拟杆菌(Bacteroides thetaiotaomicron)BP8E,其保藏编号为CGMCC No.22149。
2.含有下述至少一种的制剂在生产γ-氨基丁酸中的应用:
1)多形拟杆菌(Bacteroides thetaiotaomicron)BP8E;
2)多形拟杆菌(Bacteroides thetaiotaomicron)BP8E菌剂;
3)多形拟杆菌(Bacteroides thetaiotaomicron)BP8E活菌悬液;
4)多形拟杆菌(Bacteroides thetaiotaomicron)BP8E死菌悬液;
5)多形拟杆菌(Bacteroides thetaiotaomicron)BP8E代谢产物;
6)多形拟杆菌(Bacteroides thetaiotaomicron)BP8E提取物;
其中,所述多形拟杆菌(Bacteroides thetaiotaomicron)BP8E为权利要求1所述的保藏编号为CGMCC No. 22149的多形拟杆菌(Bacteroides thetaiotaomicron)BP8E。
3.含有下述至少一种的制剂在制备用于预防和/或治疗抑郁症的产品中的应用:
1)多形拟杆菌(Bacteroides thetaiotaomicron)BP8E;
2)多形拟杆菌(Bacteroides thetaiotaomicron)BP8E菌剂;
3)多形拟杆菌(Bacteroides thetaiotaomicron)BP8E活菌悬液;
4)多形拟杆菌(Bacteroides thetaiotaomicron)BP8E死菌悬液;
5)多形拟杆菌(Bacteroides thetaiotaomicron)BP8E代谢产物;
其中,所述多形拟杆菌(Bacteroides thetaiotaomicron)BP8E为权利要求1所述的保藏编号为CGMCC No. 22149的多形拟杆菌(Bacteroides thetaiotaomicron)BP8E。
4.一种用于预防和/或治疗抑郁症的产品,含有下述至少一种:
1)多形拟杆菌(Bacteroides thetaiotaomicron)BP8E;
2)多形拟杆菌(Bacteroides thetaiotaomicron)BP8E菌剂;
3)多形拟杆菌(Bacteroides thetaiotaomicron)BP8E活菌悬液;
4)多形拟杆菌(Bacteroides thetaiotaomicron)BP8E死菌悬液;
5)多形拟杆菌(Bacteroides thetaiotaomicron)BP8E代谢产物;
其中,所述多形拟杆菌(Bacteroides thetaiotaomicron)BP8E为权利要求1所述的保藏编号为CGMCC No. 22149的多形拟杆菌(Bacteroides thetaiotaomicron)BP8E。
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