CN113773978B - 青春双歧杆菌及其应用 - Google Patents
青春双歧杆菌及其应用 Download PDFInfo
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- CN113773978B CN113773978B CN202010520225.5A CN202010520225A CN113773978B CN 113773978 B CN113773978 B CN 113773978B CN 202010520225 A CN202010520225 A CN 202010520225A CN 113773978 B CN113773978 B CN 113773978B
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Abstract
本发明提出了一种青春双歧杆菌(Bifidobacterium adolescentic),于2019年4月24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.17623。本发明所提出的青春双歧杆菌具有优异的产γ‑氨基丁酸的能力,并且显著优于现有青春双歧杆菌菌株,所制备的药物、保健品或食品,能够有效降血压、镇静安神、增加脑细胞活力。
Description
技术领域
本发明涉及微生物领域,具体地,本发明涉及青春双歧杆菌及其应用,更具体地,本发明涉及青春双歧杆菌、包含青春双歧杆菌的药物、保健品和食品以及片剂。
背景技术
γ-氨基丁酸(GABA)是一种非蛋白质氨基酸,广泛存在于细菌,植物和脊椎动物中。它在植物和细菌的克雷布斯循环中起代谢作用,在脊椎动物中起着有效的神经信号传递作用。γ-氨基丁酸(GABA)主要由谷氨酸脱羧酶催化L-谷氨酸或其盐的不可逆α-脱羧反应形成,其被认为是中枢神经系统中重要的抑制性神经递质,具有调节血压及激素分泌、保护肾脏功能、增强机体免疫力,特别是改善大脑机能的生理功能。
近年来,益生菌在人体健康方面的研究一直是人们关注的焦点。其公认的益生功能主要包括:调节肠道菌群平衡、调节免疫、缓解乳糖不耐症等。然而有研究发现:乳酸菌中存在谷氨酸脱羧酶。其可通过生物发酵法产生γ-氨基丁酸,而此法产生的γ-氨基丁酸具有天然、安全、环保的特点。因此其可作为制备调节血压及激素分泌、保护肾脏功能、增强机体免疫力的药品、保健品或食品的重要活性成分。
由于益生菌产γ-氨基丁酸的能力具有菌株特异性,因此对不同菌株仍需要进一步深入研究。
发明内容
本申请是基于发明人对以下事实和问题的发现和认识作出的:
目前高产γ-氨基丁酸的功能益生菌菌株的研究及应用多集中于乳杆菌,双歧杆菌的研究及应用相对较少。而本申请的发明人却在青春双歧杆菌中发现了一株高产γ-氨基丁酸的菌株-青春双歧杆菌95,该株青春双歧杆菌95的发现,为相关药物、保健品和食品的开发提供了新的菌种,具有广泛的应用前景。
在本发明的第一方面,本发明提出了一种青春双歧杆菌(Bifidobacterium adolescentic),于2019年4月24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. 17623,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,分类命名为Bifidobacterium adolescentic,为了便于描述,本申请中将该菌株称为青春双歧杆菌95。根据本发明实施例的青春双歧杆菌具有优异的产γ-氨基丁酸的能力,并且显著优于现有青春双歧杆菌菌株,青春双歧杆菌95制备的药物,具有有效降血压、镇静安神、增加脑细胞活力。
在本发明的第二方面,本发明提出了前面所述的青春双歧杆菌(Bifidobacterium adolescentic)在合成γ-氨基丁酸中的用途。根据本发明实施例的青春双歧杆菌具有优异的产γ-氨基丁酸的能力,并且显著优于现有青春双歧杆菌菌株。
在本发明的第三方面,本发明提出了前面所述的青春双歧杆菌(Bifidobacterium adolescentic)在制备药物、保健品或食品中的用途,所述药物用于降血压、镇静安神、增加脑细胞活力。根据本发明实施例的青春双歧杆菌具有优异的产γ-氨基丁酸的能力,并且显著优于现有青春双歧杆菌菌株,而γ-氨基丁酸(GABA)是中枢神经系统中重要的抑制性神经递质,具有调节血压及激素分泌、保护肾脏功能、增强机体免疫力,特别是改善大脑机能的生理功能,因此,利用根据本发明实施例的青春双歧杆菌(Bifidobacterium adolescentic)所制备药物,在降血压、镇静安神、增加脑细胞活力方面效果更加优越。
根据本发明的具体实施例,所述药物用于治疗或预防抑郁症。
在本发明的第四方面,本发明提出了一种药物。根据本发明的实施例,所述药物包括:前面所述的青春双歧杆菌作为活性成份。根据本发明实施例的药物在降血压、镇静安神、增加脑细胞活力方面效果更优。
根据本发明的实施例,所述药物进一步包括药学上可接受的辅剂。
在本发明的第五方面,本发明提出了一种保健品。根据本发明的实施例,所述保健品包括所述的青春双歧杆菌(Bifidobacterium adolescentic)。
在本发明的第六方面,本发明提出了一种食品。根据本发明的实施例,所述食品包括:所述的青春双歧杆菌(Bifidobacterium adolescentic)。
根据本发明的实施例,所述药物或保健品或食品是粉剂、片剂、饮料或胶囊。
根据本发明的实施例,基于所述药物或保健品或食品的总质量,所述青春双歧杆菌的含量为0.8~1.2×109CFU/g ,优选,1.0×109CFU/g。发明人发现,所述青春双歧杆菌的含量在0.8~1.2×109CFU/g范围内,所述药物在降血压、镇静安神、增加脑细胞活力方面效果更优。
在本发明的第七方面,本发明提出了一种片剂。根据本发明的实施例,所述片剂包括: 质量分数为15~25%的所述的青春双歧杆菌(Bifidobacterium adolescentic)、质量分数为50~60%的调味剂、质量分数为15~25%的益生元以及质量分数为2.5~10%的植物油。根据本发明具体实施例的片剂在降血压、镇静安神、增加脑细胞活力方面效果优越。
根据本发明的实施例,所述益生元包括选自低聚果糖、低聚木糖、低聚半乳糖、低聚异麦芽、螺旋藻、节旋藻、蔬菜提取物、中草药提取物、野生植物提取物、蛋白质水解物、和多元醇的至少之一。
根据本发明的实施例,所述益生元为低聚麦芽糖。
根据本发明的实施例,所述调味剂为柑橘粉。
根据本发明的实施例,所述植物油为棕榈油。
根据本发明的实施例,所述片剂包括: 质量分数为20%的所述的青春双歧杆菌、质量分数为55%的柑橘粉、质量分数为20%的低聚麦芽糖以及质量分数为5%的棕榈油。
根据本发明的实施例,所述青春双歧杆菌是以冻干粉的形式提供的。
附图说明
图1是根据本发明实施例的青春双歧杆菌产γ-氨基丁酸的结果图;
图2是根据本发明实施例的青春双歧杆菌耐酸耐胆盐的结果图;以及
图3是根据本发明实施例的青春双歧杆菌粘附能力的结果图。
具体实施方式
下面详细描述本发明的实施例,所述实施例的示例在附图中示出。下面通过参考附图描述的实施例是示例性的,旨在用于解释本发明,而不能理解为对本发明的限制。
本发明提供了一种新的青春双歧杆菌菌株-青春双歧杆菌95及其用途。
本申请公开了一种药物或保健品或食品,该药物或保健品或食品中含有青春双歧杆菌95。优选的,本申请的药物为用于降血压、镇静安神、增加脑细胞活力的活菌制剂。需要说明的是,本申请的药物或保健品或食品,可以是单独的活性青春双歧杆菌95的各种制剂,也可以与其它活性成份配合使用,只要相互之间不影响活性即可;进一步地,所述药物的最好情况是各活性成分之间能够具有功能互补或具有促进作用,例如可以将青春双歧杆菌95与其他益生菌组合制成复合益生菌片,达到更好或更多的活性功能,除具降血压、镇静安神、增加脑细胞活力作用以外,还可以起到缓解其他精神类疾病的功能,具体的,可以根据复合益生菌片的活性和组分而定,在此不做限定。任选地,所述药物进一步包括药物开发上可接受的载体或辅料。需要说明的是,在制活菌制剂时,通常都需要添加载体或辅料,只要添加的载体或辅料与青春双歧杆菌95之间没有相互抑制或不良副作用即可。药物或保健品或食品可以是粉剂、片剂、饮料、胶囊。
本申请的一种优选方案中,青春双歧杆菌片剂料中包括青春双歧杆菌95的冻干粉20 %、柑橘粉55%、低聚麦芽糖20 %、棕榈油5%,以上组分均为重量份,将其混合均匀后,压片制成片剂。
本申请的有益效果在于:
本申请的青春双歧杆菌95,具有降血压、镇静安神、增加脑细胞活力作用,可以广泛应用于各种益生菌类药物、保健品或食品的制备。因此该菌株的应用潜力巨大。
下面通过具体实施例进一步详细描述本发明。
实施例1 青春双歧杆菌95的分离和鉴定
1)本实施例以健康人粪便为样品,用无菌生理盐水,10倍梯度稀释样品至103倍,将其涂布于MRS-Cys固体培养基中,置于37℃培养箱中48h,观察菌落形态,并挑取双歧杆菌的疑似菌落,分别转接到厌氧的MRS培养基中进行纯培养。
2)对菌株进行16s rDNA菌种鉴定:
抽提细菌DNA,进行16s rDNA扩增,引物(F:5’-AGAGTTTGATGGCTCAG-3’(SEQ IDNO:1); R: 5’-TAGGGTTACCTTGTTACGACTT-3’(SEQ ID NO:2);).利用上述引物进行PCR扩增和琼脂糖凝胶电泳,然后切胶回收,测序。引物序列如下:
GTGGGGGGTCTTATACTGGCAAGCCGACCATGGAACCCACATTGCTACTGGGTGAGAGTGGAGAGGAACTAAGTAACGCGTGACCGACCTGCTACATACACCGGAATAGCTCCTGGAAACGGGTGGTAATGCCGGATGCTCCAGTTGGATGCATGTCCTTCTGGGAAAGATTCATCGGTATGGGATGGGGTCGCGTCCTATCAGCTTGATGGCGGGGTAACGGCCCACCATGGCTTCGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCACATTGGGACTGAGATACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGCGTGCGGGATGACGGCCTTCGGGTTGTAAACCGCTTTTGACTGGGAGCAAGCCCTTCGGGGTGAGTGTACCTTTCGAATAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCAAGCGTTATCCGGAATTATTGGGCGTAAAGGGCTCGTAAGCGGTTCGTCGCGTCCGGTGTGAAAGTCCATCGCTTAACTGTTGATCCGCGCCGGGTACTTGCGGGCTTTAATGGCGGAAGGGAGCTTGGAATTCCCGGCGCCAACGGGGGACTGTGTATATCCCGAAAAAACACCCATCCGCAGATGACGGTCTGTAGACGACGTATCTCATGATGGTCGACCCCGGGGGTATCGATCAGCATCAGACACGAGGACCACACCCCTGTATTCGGTGTTGTTGTACTGGAGGATATCTTCCACGGCCTCGTGCAGGAGTAATTCTATCTCCATCGCCCTAGGGCAGTCCGCCGTACGGTGAGACTACAGAATTGACGGTGTCTTCCAGCGGAGACTAGCTGATTATTCATCTTCGCGAATCTACTGCCTGACTGCTCTATGCCGAGTTGTCTCTACGGCTGTCTAAGTAGGCATGCCTCCCAG(SEQ ID NO:3).
测序结果利用BLAST工具在NCBI数据库中进行比对,分离获得的菌株16s rDNA序列与青春双歧杆菌显示出99%的同源性,表明分离获得的菌株为青春双歧杆菌。将该菌株命名为青春双歧杆菌95。该菌株于2019年4月保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏号为CGMCC No. 17623
实施例2
(一)青春双歧杆菌95、对照菌株青春双歧杆菌150培养及制备
1、青春双歧杆菌150购自中国微生物菌种保藏管理委员会普通微生物中心,菌株编号150。
2、BS培养基:由蛋白胨10g/L,肝浸粉5g/L,牛肉浸粉3g/L,酵母浸粉5g/L,胰酪蛋白胨8g/L,可溶性淀粉0.5g/L,氯化钠1g/L,,磷酸氢二钾1g/L,磷酸二氢钾1g/L,葡萄糖10g/L,吐温80 1ml/L,七水硫酸亚铁0.01g/L,硫酸锰0.005g/L,调整pH至7.2±0.1,116℃灭菌30min制得。
3、将保存的青春双歧杆菌95和青春双歧杆菌150取出活化后,分别接种至BS液体培养基于37℃培养15h,活化3代备用。
(二)菌株产γ-氨基丁酸能力测定
青春双歧杆菌95和青春双歧杆菌150在含有3% 的L-谷氨酸钠的MRS培养基中,37℃培养36h 后,12000r/min离心15min,取上清液100 μL,加入20 μL衍生试剂、100 μL硼酸缓冲液混合均匀,室温反应5 min。分别以浓度为0 g/L、0.05 g/L、0.1 g/L、0.15 g/L、0.2g/L、0.25 g/L、0.3 g/L的γ-氨基丁酸标准品制作标准曲线。进样量:20 μL,流速:0.8 mL/min,柱温:30℃,360 nm处检测吸收峰。
流动相A为20 mmol/L醋酸钠缓冲液(pH 7.3),流动相B为乙腈,其比例为4∶1。衍生试剂:邻苯二甲醛(OPA) 20 mg、β-巯基乙醇20 μL及5 mL乙腈,混匀。硼酸缓冲液:硼酸24.7g,加超纯水定容至1 L,pH值为10.4。
实验结果如图1所示,青春双歧杆菌95和青春双歧杆菌150都可产γ-氨基丁酸,但青春双歧杆菌95产γ-氨基丁酸的能力显著高于青春双歧杆菌150。
(三)耐酸、耐胆盐能力测定
1、耐酸能力测定
青春双歧杆菌95和青春双歧杆菌150分别以3%接种量接种于BS液体培养基中,菌株培养至稳定期后4℃下6000 r/min离心10 min收集菌体,磷酸盐缓冲液(pH 7.2)洗涤2次,重悬于pH 2和pH 3的无菌磷酸盐缓冲液(PBS)中,并将细菌浓度调至1×109CFU/mL。37℃培养2 h后,取样涂板计数,并计算存活率。
2、耐胆盐能力测定
青春双歧杆菌95和青春双歧杆菌150分别以3%接种量接种于BS液体培养基中,37℃条件下培养到稳定期后4℃下6000 r/min离心10 min收集菌体,重悬于含有0.3%胆盐的无菌磷酸盐缓冲液(PBS)中,分别在3 h和6 h取样涂板计数,并计算存活率。
存活率=(平板菌落数量×稀释倍数)/初始细菌数量
实验结果如图2所示:青春双歧杆菌95和青春双歧杆菌150在pH2、pH3的酸性条件下都有一定的耐受能力,但青春双歧杆菌95无论是在pH2还是pH3条件下,其存活能力都强于青春双歧杆菌150。在胆盐方面,青春双歧杆菌95在培养3h及6h时的存活能力也都优于青春双歧杆菌150。
(四)抗生素敏感性测定
将四环素、红霉素、克林霉素、氯霉素、氨苄西林、甲氧苄啶、环丙沙星、利福平、万古霉素等9种抗生素分别进行一系列的2倍比稀释,分别得到浓度为64μg/mL、16μg/mL、8μg/mL、4μg/mL、2μg/mL、1μg/mL、0.5μg/mL、0.25μg/mL、0.125μg/mL、0.0625μg/mL、0.03125μg/mL的抗生素稀释液。
将抗生素稀释液分别加入到对应的96孔板中,随后加入青春双歧杆菌95的BS培养液,混匀后,37℃培养48h,观察实验结果。以每个孔的抗菌药物最小抑菌浓度(缩写MIC)来表征青春双歧杆菌95的抗生素敏感性。
结果显示,青春双歧杆菌95对四环素、红霉素、克林霉素、氯霉素、氨苄西林、甲氧苄啶、环丙沙星、利福平、万古霉素等抗生素的敏感性符合欧洲食品监督局(EFSA)规定,且小于EFSA规定的安全值。因此,青春双歧杆菌95对这9种抗生素敏感性较好(如表1所示)。
表1:
(五)粘附能力测定
将HT-29细胞(人结肠癌细胞)以每孔1×105的接种量接种于12孔板中,每孔含2mL PMI 1640完全培养基(10% 胎牛血清)。收集培养至稳定期的青春双歧杆菌95和青春双歧杆菌150(4℃,6000 r/min,10 min),并用磷酸盐缓冲溶液洗涤3次,然后用细胞培养液将其重悬至5×108CFU/mL。待细胞长至单层,去除细胞培养液,并用无菌磷酸盐缓冲溶液冲洗细胞2-3次,然后加入1 mL双歧杆菌悬浮液(每株菌3个重复)。37℃ 、5% CO2-95%空气培养箱中培养2 h后,弃除细菌悬浮液,磷酸盐缓冲溶液冲洗3-5次,胰蛋白酶消化3 min,加入适量含有10% FBS的1640培养液终止反应。然后,加入1 mL 0.05% TritonX-100,反复吹打使细胞完全裂解,收集菌体并进行系列稀释,涂板计数。另选取长至单层的培养孔,进行细胞计数并计算其粘附能力。
粘附能力=每个培养皿内粘附的细菌总数/每个培养皿的总细胞数
实验结果如图3所示,青春双歧杆菌95和青春双歧杆菌150都可粘附于人结肠癌细胞(HT-29细胞),但青春双歧杆菌95的粘附能力显著高于青春双歧杆菌150。
实施例3药品、保健品或食品的制备
本实施例将青春双歧杆菌95制成常用片剂。 109CFU/g青春双歧杆菌片剂的制备方法如下:
青春双歧杆菌95的冻干粉20%、柑橘粉55%、低聚麦芽糖20%、棕榈油5%,以上组分均为重量份,将其混合均匀后,压片制成片剂。其中,青春双歧杆菌95的冻干粉为采用冻干机将其菌悬液在无菌环境下冷冻抽真空干燥而成。
此外,术语“第一”、“第二”仅用于描述目的,而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此,限定有“第一”、“第二”的特征可以明示或者隐含地包括至少一个该特征。在本发明的描述中,“多个”的含义是至少两个,例如两个,三个等,除非另有明确具体的限定。
在本说明书的描述中,参考术语“一个实施例”、“一些实施例”、 “示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中,对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且,描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外,在不相互矛盾的情况下,本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例,可以理解的是,上述实施例是示例性的,不能理解为对本发明的限制,本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。
SEQUENCE LISTING
<110> 深圳华大生命科学研究院
深圳市华大农业应用研究院
<120> 青春双歧杆菌及其应用
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tgggatgggg tcgcgtccta tcagcttgat ggcggggtaa cggcccacca tggcttcgac 240
gggtagccgg cctgagaggg cgaccggcca cattgggact gagatacggc ccagactcct 300
acgggaggca gcagtgggga atattgcaca atgggcgcaa gcctgatgca gcgacgccgc 360
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gggtgcaagc gttatccgga attattgggc gtaaagggct cgtaagcggt tcgtcgcgtc 540
cggtgtgaaa gtccatcgct taactgttga tccgcgccgg gtacttgcgg gctttaatgg 600
cggaagggag cttggaattc ccggcgccaa cgggggactg tgtatatccc gaaaaaacac 660
ccatccgcag atgacggtct gtagacgacg tatctcatga tggtcgaccc cgggggtatc 720
gatcagcatc agacacgagg accacacccc tgtattcggt gttgttgtac tggaggatat 780
cttccacggc ctcgtgcagg agtaattcta tctccatcgc cctagggcag tccgccgtac 840
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ctcccag 967
Claims (18)
1.一种青春双歧杆菌(Bifidobacterium adolescentic),于2019年4月24日保藏于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. 17623。
2.权利要求1所述的青春双歧杆菌在合成γ-氨基丁酸中的用途。
3.权利要求1所述的青春双歧杆菌在制备药物中的用途,所述药物用于降血压、镇静安神。
4.权利要求1所述的青春双歧杆菌在制备药物中的用途,所述药物用于治疗抑郁症。
5.一种用于降血压、镇静安神、治疗抑郁症的药物,其特征在于,包括:权利要求1所述的青春双歧杆菌作为活性成分。
6.根据权利要求5所述的药物,其特征在于,进一步包括药学上可接受的辅剂。
7.一种保健品,其特征在于,包括:权利要求1所述的青春双歧杆菌。
8.一种食品,其特征在于,包括:权利要求1所述的青春双歧杆菌。
9.根据权利要求5~8任一项所述的药物或保健品或食品,其特征在于,所述药物或保健品或食品是粉剂、片剂、饮料或胶囊。
10.根据权利要求9所述的药物或保健品或食品,其特征在于,所述药物或保健品或食品是片剂。
11.根据权利要求10所述的药物或保健品或食品,其特征在于,包括: 质量分数为15~25%的权利要求1所述的青春双歧杆菌、质量分数为50~60%的调味剂、质量分数为15~25%的益生元以及质量分数为2.5~10%的植物油。
12.根据权利要求11所述的药物或保健品或食品,其特征在于,所述益生元为低聚麦芽糖。
13.根据权利要求11所述的药物或保健品或食品,其特征在于,所述调味剂为柑橘粉。
14.根据权利要求11所述的药物或保健品或食品,其特征在于,所述植物油为棕榈油。
15.根据权利要求10所述的药物或保健品或食品,其特征在于,包括: 质量分数为20%的权利要求1所述的青春双歧杆菌、质量分数为55%的柑橘粉、质量分数为20%的低聚麦芽糖以及质量分数为5%的棕榈油。
16.根据权利要求5~15任一项所述的药物或保健品或食品,其特征在于,所述青春双歧杆菌是以冻干粉的形式提供的。
17.根据权利要求5~16任一项所述的药物或保健品或食品,其特征在于,基于所述药物或保健品或食品的总质量,所述青春双歧杆菌的含量为0.8~1.2×109 CFU/g。
18.根据权利要求5~17任一项所述的药物或保健品或食品,其特征在于,基于所述药物或保健品或食品的总质量,所述青春双歧杆菌的含量为1.0×109 CFU/g。
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