CN110157647B - 一种能够缓解焦虑、改善睡眠的短乳杆菌及其用途 - Google Patents
一种能够缓解焦虑、改善睡眠的短乳杆菌及其用途 Download PDFInfo
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- CN110157647B CN110157647B CN201910463154.7A CN201910463154A CN110157647B CN 110157647 B CN110157647 B CN 110157647B CN 201910463154 A CN201910463154 A CN 201910463154A CN 110157647 B CN110157647 B CN 110157647B
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Abstract
本发明公开了一种能够缓解焦虑、改善睡眠的短乳杆菌及其用途,属于微生物技术领域。本发明的短乳杆菌GDMCC No.60605具有优良的模拟胃肠道存活、细胞粘附和GABA产生能力。在旷场实验中能够显著增加小鼠进入中央区域的频率、延长小鼠在旷场中央区域的时间而不影响小鼠总运动距离;在高架十字迷宫实验中能够显著增加小鼠进入开臂的次数;能够延长戊巴比妥钠诱导的睡眠时间、缩短巴比妥钠诱导的睡眠潜伏期;能够调节肠道菌群,显著增加小鼠肠道中Allobaculum的相对丰度。所述的短乳杆菌GDMCC No.60605用于制备缓解焦虑、改善睡眠的药物组合物与发酵食品,具有非常广泛的应用前景。
Description
技术领域
本发明涉及一种能够缓解焦虑、改善睡眠的短乳杆菌及其用途,属于微生物技术领域。
背景技术
失眠症(insomnia)是以入睡和睡眠维持困难所致的睡眠质量或时间达不到正常生理需求的一种主观体验,是最常见的睡眠障碍性疾病。人们常用一些临床症状来诊断失眠,其中包括:入睡困难、夜间易醒并再次入睡困难、次日早醒、无恢复效果的睡眠等。根据发病率和人口统计学资料显示,失眠症是当今社会人们普遍存在的痛苦之一。我国至少有35%的人失眠,17%的人失眠症状相当严重,而睡眠局限在“浅睡眠”的人的比例,高达77.3%。失眠会导致无法恢复精力,因而躯体乏困、精神萎靡、嗜睡、注意力减退、思考困难、反应迟钝、情绪低落、焦躁,此外可能会对心脏功能、免疫功能和血糖调节产生负面影响。
失眠总和焦虑、抑郁相伴而生,40%的失眠患者有一种或几种精神障碍,其中焦虑障碍占到24%。失眠和焦虑存在中等程度的相关性。其中在失眠与焦虑共病的患者中,焦虑障碍先于失眠的情况占73%,而失眠先于焦虑的占69%。因此说失眠是焦虑发作的常见症状之一,也是焦虑发病的主要因素,反过来,焦虑也可以是慢性失眠的危险因素。
现在临床上常选择起效快、作用强的西药治疗焦虑和失眠病症,比较常见的药物主要包含了巴比妥类、非苯二氮类以及苯二氮类几种类型。这些药物能够有效缓解焦虑,增加睡眠的时间,降低睡眠的潜伏期,对睡眠质量进行改善,可是却会抑制中枢神经***,长时间使用就会让患者产生出依赖性与耐受性,例如一旦停药就会产生戒断症状等各种不良反应。鉴于传统治疗方法存在的多种问题,针对焦虑和失眠寻找一种新的干预或治疗方法显得十分必要。
γ-氨基丁酸(GABA)是一种重要的神经递质,日本厚生省、欧洲食品***(EFSA)和美国食品药品管理局(FDA)承认乳酸菌发酵生产的GABA为天然食品添加剂。2009年,中国***批准此类GABA为新资源食品。研究表明,GABA可以舒缓神经,防止神经过于紧张和兴奋,能较好地缓解精神压力、调节情绪,恢复身心健康,同时能缩短入睡时间和延长深睡眠时间,对失眠有一定的缓解效果。现在一般认为,膳食补充的GABA可能是通过改善肠道微生态和迷走神经通路等途径来调节脑功能,进而缓解焦虑、改善睡眠。
与此同时,长期科学研究结果表明,肠道微生物通过直接接触、可溶性分子与宿主生物相互作用、自身代谢为宿主提供多种有益功能。胃肠道和中枢神经***(CNS)之间的肠-脑轴双向通信还支持肠道微生物群与宿主的神经回路(包括CNS)之间的通信路径。越来越多的证据表明,肠道微生物群能够调节肠道和大脑功能,包括人类的情绪,认知功能和行为。其中乳酸菌是肠道微生物的重要组成部分,许多种属的乳酸菌也具有产GABA的能力。在这种情况下,充分发挥乳酸菌可以产生GABA的优势,使菌株自身和产生的GABA形成复合效应,共同作用于机体来缓解焦虑、改善睡眠。
但是,产GABA的乳酸菌在体内却不一定能具有缓解焦虑、改善睡眠的效果,因为有很多体外产GABA的乳酸菌在经过胃肠道时无法存活,或细胞粘附能力差,无法在肠道定植发挥其有益作用。而有一些具有较强胃肠道耐受能力、细胞粘附能力强的乳酸菌,却不具备较高的GABA产生能力,难以发挥较好的有益作用。此外,益生菌具有耐受胃肠道的酸和胆盐环境以及在肠道定殖是益生菌在体内发挥功能的必要前提条件。因此,寻求一种具有优良的胃肠道存活能力、细胞粘附能力和GABA产生能力的乳酸菌是亟待解决的问题。
目前,一些专利文献涉及缓解焦虑、改善睡眠保健食品及其制备方法,例如专利CN103141855A公布了一种将γ-氨基丁酸、5-羟色胺、维生素B60和五味子复配的改善睡眠质量的保健品及其制备方法;专利CN103976351A公布了一种乳酸菌进行液体深层发酵生产GABA,制备同时具有增强免疫力和改善睡眠功能的发酵液的方法;CN107427539A公开了一种至少包含两歧双歧杆菌W23且能够控制肠道屏障功能的益生菌组合物,可以治疗和预防涉及脑-肠轴的人类疾病。
但是上述专利并未涉及同时具有优良的胃肠道存活能力、细胞粘附能力和GABA产生能力的乳酸菌,无法真正用于改善睡眠。
发明内容
本发明的目的是提供一种短乳杆菌(Lactobacillus brevis)GDMCC No.60605。
如上所述的本发明涉及一种短乳杆菌,利用形态特征、培养性状和生理生化特征等微生物学特性对该乳酸菌鉴定为短乳杆菌(Lactobacillus brevis)GDMCC No.60605,该菌菌株已在广东微生物菌种保藏中心保藏,其保藏号为GDMCC No.60605。
本发明的短乳杆菌,具有如下特性:
1、形态特征:
革兰氏染色结果为革兰氏阳性菌;镜检呈短杆状,两端圈,单个或成短链,无鞭毛,无孢子。
2、培养性状:
在MRS培养基上培养48h后的菌落一般呈乳白色,粗糙扁平,不产生色素。
3、生理生化特征:
能利用***糖、果糖、葡萄糖、麦芽糖、乳糖、蜜二糖、核糖和蔗糖;不利用七叶树苷、水杨苷、棉子糖,山梨醇、肌醇、卫矛醇、鼠李糖、半乳糖;过氧化氢酶、H2S产生、尿素分解、硝酸盐还原等均为阴性。具有优良的模拟胃肠道存活能力、细胞粘附能力和GABA产生能力;其在MMRS培养基中培养48h发酵上清液中的GABA的含量为5072±280mg/L。
4、灌胃小鼠效果:
(1)在旷场实验中使小鼠进入中央区域的频率增加了55.4%、使小鼠在旷场中央区域的时间延长了121.1%,与空白组小鼠相比有显著性差异而不影响小鼠总运动距离;
(2)在高架十字迷宫实验中使小鼠进入开臂的次数增加了69.4%,与空白组小鼠相比有显著性差异;
(3)能够显著延长戊巴比妥钠诱导的睡眠时间,延长的比例为149.2%,使巴比妥钠诱导的睡眠潜伏期缩短了31.0%;
(4)能够调节肠道菌群,显著增加小鼠肠道中Allobaculum的相对丰度。
本发明的第二个目的是提供含有所述短乳杆菌GDMCC No.60605的发酵剂。
在一种实施方式中,所述发酵剂是利用含有所述短乳杆菌GDMCC No.60605的菌液制备所得到的粉剂,它含有106CFU/g以上的活性短乳杆菌GDMCC No.60605。
在一种实施方式中,所述粉剂是将含有所述短乳杆菌GDMCC No.60605的菌液通过常规冷冻干燥工艺或其它工艺制备所得到的。
在一种实施方式中,所述的发酵剂是通过下述制备步骤得到的:
A、培养基的制备:使用以所述培养基总重量计87.7%的水将10%酶水解脱脂乳、0.5%葡萄糖、1.5%胰蛋白胨与0.3%酵母浸膏溶解,然后调整其pH为6.8,这样得到所述的培养基;
B、保护剂的制备:使用水与保护剂原料混合制备得到含有100g/L脱脂奶粉、30mL/L甘油、100g/L麦芽糊精、150g/L海藻糖、10g/L L-谷氨酸钠的保护剂;
C、将短乳杆菌GDMCC No.60605菌种按照以所述培养基的重量计2~4%接种量接种到在温度110~120℃下灭菌8~12min的所述培养基中,然后在温度37℃的条件下培养18h,用pH7.2磷酸盐缓冲液清洗2~4次,用所述的保护剂重悬达到浓度1010CFU/mL;接着,让该悬浮液在温度37℃的条件下预培养60min,再进行冷冻干燥得到所述的发酵剂。
在一种实施方式中,所述发酵剂还含有可用于食品的微生物。可选地,还含有耐酸乳杆菌、植物乳杆菌、保加利亚乳杆菌中的一种或者两种以上。
本发明的第三个目的是提供所述短乳杆菌GDMCC No.60605的应用。
可选地,所述应用,是用于制备发酵食品。所述发酵食品是使用本发明的短乳杆菌GDMCC No.60605或者含有短乳杆菌GDMCC No.60605菌种的发酵剂生产得到的。
在一种实施方式中,所述发酵食品中含有短乳杆菌GDMCC No.60605活菌。
在一种实施方式中,所述发酵食品为乳制品、豆制品或者果蔬制品。可选地,所述的乳制品是牛奶、酸奶油或干酪。可选地,所述的豆制品是豆奶、豆豉或豆酱。可选地,所述的果蔬制品是黄瓜、胡萝卜、甜菜、芹菜或圆白菜制品。
可选地,所述应用,是用于制备具有改善睡眠功能的药物组合物。
在一种实施方式中,所述药物组合物是由短乳杆菌GDMCC No.60605菌剂与在药学上可接受的载体组成的。
在一种实施方式中,在药学上可接受的载体是一种或多种选自在药学上通常使用的填充剂、粘合剂、润湿剂、崩解剂、润滑剂或矫味剂的载体。可选地,所述的药物组合物是颗粒剂、胶囊剂、片剂、丸剂或口服液剂型。
本发明有益效果和优点如下:
本发明的短乳杆菌GDMCC No.60605,具有优良的模拟胃肠道存活、细胞粘附和GABA产生能力(在MMRS培养基中培养48h发酵上清液中的GABA的含量为5072±280mg/L。)。在旷场实验中能够显著增加小鼠进入中央区域的频率,延长小鼠在旷场中央区域的时间而不影响小鼠总运动距离;在高架十字迷宫实验中能够显著增加小鼠进入开臂的次数;能够延长戊巴比妥钠诱导的睡眠时间、缩短巴比妥钠诱导的睡眠潜伏期;能够调节肠道菌群,显著增加小鼠肠道中Allobaculum的相对丰度。所述的短乳杆菌GDMCC No.60605用于制备缓解焦虑、改善睡眠的药物组合物与发酵食品,具有非常广泛的应用前景。
生物材料保藏
短乳杆菌(Lactobacillus brevis),分类命名为Lactobacillus brevis,已与2019年3月12日保藏于广东省微生物菌种保藏中心,保藏地址为广州市先烈中路100号大院59号楼5楼,保藏编号为GDMCC No.60605。
附图说明
图1是旷场实验中短乳杆菌GDMCC No.60605对小鼠总运动距离的影响;
图2是旷场实验中短乳杆菌GDMCC No.60605对小鼠在中央区域停留时间的影响;
图3是旷场实验中短乳杆菌GDMCC No.60605对小鼠进入中央区域频率的影响;
图4是旷场实验中不同处理组小鼠运动轨迹图对比。
图5是高架十字迷宫实验中短乳杆菌GDMCC No.60605对小鼠在开臂停留时间的影响。
图6是高架十字迷宫实验中短乳杆菌GDMCC No.60605对小鼠进入开臂次数的影响。
图7是短乳杆菌GDMCC No.60605对戊巴比妥钠诱导的睡眠时间实验的影响。
图8是短乳杆菌GDMCC No.60605对巴比妥钠诱导的睡眠潜伏期实验的影响。
图9是短乳杆菌GDMCC No.60605对小鼠肠道菌群β多样性的影响。
图10是短乳杆菌GDMCC No.60605对Allobaculum相对丰度变化的影响。
(注:*p<0.05,**p<0.01)
具体实施方式
下述实施例中所述实验方法,如无特殊说明,均为常规方法;所述试剂和生物材料,如无特殊说明,均可从商业途径获得。
实施例1短乳杆菌GDMCC No.60605的筛选
1实验方法
以新鲜人群粪便和泡菜水为样本进行菌株筛选。
(1)吸取样品0.5mL于5mL的MRS培养基中,37℃培养18~24h,进行富集。
(2)梯度稀释:吸取富集后的样品0.5mL于4.5mL无菌生理盐水中获得10-1稀释液,然后吸取0.5mL 10-1稀释液于4.5mL生理盐水中,得到10-2稀释液,按此操作,依次得到10-3,10-4,10-5,10-6稀释液。
(3)涂板培养:吸取100μL梯度稀释液涂布于MRS平板,10-4,10-5,10-6每个梯度1个板;于37℃培养48h。
(4)划线分离:根据菌落形状、大小、边缘、透明度等选择涂布平板上有典型特征的菌落,用接种环挑取菌落在选择性培养基平板上进行划线,37℃培养48h。
(5)得到纯化的单菌落,挑取一个单菌落接种至相应的5mL液体培养基中,培养18~24h。将各菌株编号,进行菌株鉴定、革兰氏染色、生理生化等实验。
其中,MRS培养基的配方为:在1L蒸馏水中加入10g蛋白胨、10g牛肉膏、20g葡萄糖、5g酵母浸膏、2g无水乙酸钠、0.25g一水硫酸锰、1mL吐温80、2.6g三水合磷酸氢二钾、0.5g七水硫酸镁、2g柠檬酸二铵,pH 6.2~6.5。
2实验结果
对分离得到的菌株进行PCR扩增16S rDNA,PCR产物送至华大基因测序有限公司进行测序,GDMCC No.60605鉴定结果为短乳杆菌(Lactobacillus brevis),其特征为:
(1)、形态特征:
革兰氏染色结果为革兰氏阳性菌;镜检呈短杆状,两端圈,单个或成短链,无鞭毛,无孢子。
(2)、培养性状:
在MRS培养基上培养48h后的菌落一般呈乳白色,粗糙扁平。
(3)、生理生化特征:
能利用***糖、果糖、葡萄糖、麦芽糖、乳糖、蜜二糖、核糖和蔗糖;不利用七叶树苷、水杨苷、棉子糖,山梨醇、肌醇、卫矛醇、鼠李糖、半乳糖;过氧化氢酶、H2S产生、尿素分解、硝酸盐还原等均为阴性。
实施例2短乳杆菌GDMCC No.60605产GABA能力研究
1试验方法
1.1短乳杆菌GDMCC No.60605的活化
将短乳杆菌GDMCC No.60605以2%的接种量接种至MRS培养基中,在37℃下,培养18h,得到活化后的短乳杆菌GDMCC No.60605;
1.2发酵液的制备
从1.1中所述活化两次的菌液中取2%接种到添加1.5%L-谷氨酸钠(L-MSG)的MMRS培养基中37℃培养48h,即得到待测发酵液。
MMRS培养基配方为(g/L):在MRS培养基的基础上添加1.5%的L-谷氨酸钠作为GABA合成的前体物质。
1.3发酵液中GABA的测定
使用HPLC测定发酵上清液中的GABA的含量。分析条件按照国标QBT 4587-2013修改而成。色谱条件为:色谱柱:Hypersil GOLD色谱柱(100mm×2.1mm);柱温30℃;进样量:5μL;流动相A为20mmol/L醋酸钠水溶液,流动相B为V(40mmol/L醋酸钠水溶液):V(乙腈)=1:1;流速0.2mL/min;检测波长338nm。洗脱梯度为:0~6min,B由30%升至50%;6~11min,B由50%升至60%;11~12min,B由60%升至100%并保持3min;15~16min,B由100%降至30%;16~20min,30%B保持4min。
样品预处理:取发酵后的样品1mL,8000r/min离心5min,将上清液稀释一定倍数,经0.22μm微孔滤膜过滤,即可用于衍生反应。
样品衍生化:(1)0.4mol/L硼酸缓冲液的制备:准确称取2.47g硼酸,加水约80mL,用NaOH将pH调至10.2,用水定容至100mL。(2)衍生试剂的制备:称取0.1g邻苯二甲醛(o-phthalaldehyde,OPA),用1mL乙腈溶解,然后加入130μL巯基乙醇,用水定容至100mL。(3)样品柱前衍生:吸取衍生试剂和预处理后样品各10μL,混合均匀后在室温下反应90s后进样。
2实验结果
使用HPLC测定MMRS培养基中培养48h发酵上清液中的GABA的含量后发现,短乳杆菌GDMCC No.60605在体外有较高的GABA产生水平,其发酵上清液中GABA含量为5072±280mg/L。
实施例3短乳杆菌GDMCC No.60605模拟胃肠道存活能力探究
1试验方法
(1)模拟胃液的配置:将胃蛋白酶溶于灭菌的生理盐水(0.9%w/v,盐酸调pH至3.0)中,使终浓度为3g/L。以0.22μm无菌滤膜过滤,现配现用。
(2)模拟肠液的配置:将胰蛋白酶溶于灭菌的生理盐水(0.9%w/v,NaOH调pH至8.0)中,使终浓度为1g/L,并加入胆盐使终浓度为0.3%。以0.22μm无菌滤膜过滤,现配现用。
(3)将短乳杆菌GDMCC No.60605连续活化三代(每次18h)后进行实验。测定连续活化三代的菌株在A600下的OD值,通过计算求出OD 5所需的菌液量。
(4)将OD 5的菌液以8000×g离心10min,弃上清,重悬于1mL模拟胃液中,37℃培养3h后进行平板活菌计数。
(5)取模拟胃液处理后的菌液8000×g离心10min,弃上清,重悬于等体积的模拟肠液中,37℃培养4h后进行平板活菌计数。
其中,耐受胃液后的存活率(%)=(耐受模拟胃液后菌液中的活菌数/菌液中的原始活菌数)×100%。耐受肠液后的存活率(%)=(耐受模拟肠液后菌液中的活菌数/耐受模拟胃液后菌液中的活菌数)×100%。耐受胃肠液后的存活率(%)=(耐受模拟肠液后菌液中的活菌数/菌液中的原始活菌数)×100%。
2实验结果
表1短乳杆菌GDMCC No.60605在模拟胃肠道中的耐受能力
通过活菌计数,发现短乳杆菌GDMCC No.60605在模拟胃液培养3h后的存活率为92.08±3.09%,模拟肠液中培养4h后的存活率为18.30±0.97%,在模拟胃肠道中的总存活率为16.85±1.06%。表明短乳杆菌GDMCC No.60605具有优良的胃肠道耐受能力,可在膳食补充后到达肠道,发挥益生功能。
实施例4短乳杆菌GDMCC No.60605对HT-29细胞的体外粘附能力探究
1试验方法
将正常HT-29培养细胞并传代,短乳杆菌GDMCC No.60605连续活化3代。收集培养好的细胞,以血球计数板计数,并用1640培养基(添加双抗、胎牛血清)重悬至2×105个/mL。在6孔板中加入经酸洗并灭菌的盖玻片。接种细胞悬液2mL,待细胞贴壁于盖玻片后(约12h)无菌PBS洗涤3次。取一定量菌液离心收集菌体,以无菌PBS清洗菌体1次后再次离心收集菌体,以1640培养基(不添加双抗、胎牛血清)重悬至菌浓度为108CFU/mL。6孔板每孔中加入2mL 1640培养基菌悬液,孵育2h。孵育结束后,弃去培养液,无菌PBS洗涤3~6次。清洗结束后,每孔中加入2mL甲醇室温固定1h。固定结束后,弃去甲醇,革兰氏(结晶紫)染色,在显微镜下观察。
2实验结果
经过HT-29细胞的体外粘附试验,显微镜观察发现短乳杆菌GDMCC No.60605的细胞粘附个数为28.67±4.73个/细胞。而短乳杆菌GDMCC No.60605较好的细胞黏附能力有利于其在肠道定植,产生GABA,发挥自身的有益功能。
实施例5短乳杆菌GDMCC No.60605冻干菌粉的制备
将活化后的菌株以2%的接种量接种至MRS培养基中,在37℃下,培养18h后,8000×g离心10min后弃掉上清,用无菌生理盐水洗涤3次后收集菌体,将收集得到的上述菌体用保护剂重悬,冷冻干燥得到冻干菌粉,所述的保护剂可以为脱脂奶粉、海藻糖或蔗糖等;
实施例6短乳杆菌GDMCC No.60605缓解焦虑效果评价
1动物实验设计
取20~25g的6周龄健康雄性ICR小鼠30只,随机分为3组:空白对照组,阳性对照组,短乳杆菌GDMCC No.60605治疗组。空白对照组每日灌胃无菌生理盐水;短乳杆菌GDMCCNo.60605治疗组每日灌喂本说明书实施例5制备的浓度3×109CFU/mL短乳杆菌GDMCCNo.60605脱脂乳悬液,阳性对照组灌胃3mg/kg.bw的***溶液。经口每日一次给予小鼠相应剂量的受试物,小鼠灌胃量为20mL/kg.bw,持续30天。
1.1旷场实验
于末次灌胃30min后进行旷场实验,测试时间为8:00~15:00,测试前将小鼠放入铺有垫料的大盒子里,任其自由探索5min。实验开始时,将小鼠迅速放入旷场中央,用视频跟踪记录小鼠10min内的活动情况。测试结束后用湿布擦拭旷场,清除粪便,用干布擦净后再进行下一只小鼠的测试。以10min内运动总距离、进入中央区域次数、停留时间为指标,旷场实验结果列于附图1~4中。
1.2高架十字迷宫实验
于末次灌胃30min后进行旷场实验,测试时间为8:00~15:00。小鼠高架十字迷宫由两个相对的开放臂、两个相对的封闭臂和一个连接四只臂的中央平台构成。中央平台连接两个开放臂和两个封闭臂。迷宫整体固定于支架上,使迷宫底板距实验室地面50cm处。实验室内以工作灯照明,保持安静。测试前将小鼠放入动物盒中,任其自由活动5min。实验开始时,将小鼠置于中央平台处,小鼠头朝向开臂,任其自由探索。分别记录5min内小鼠进入开臂次数(open arms entries,OE)、闭臂次数(close arms entries,CE)、进入开臂时间(time spent in open arms,OT)、进入闭臂时间(time spent in close arms,CT)。以进入开臂次数与入臂总次数的百分比(OE%)和在开臂内运动时间与总时间的百分比(OT%)作为评价小鼠焦虑状态的指标。每次测试结束后用酒精擦拭迷宫,清除***物,再进行下一次测试。每只小鼠只使用一次,若掉下高架,则结束试验。高架十字迷宫实验结果列于附图5~6中。
2实验结果
2.1旷场实验结果如图1~3所示,和空白组相比,短乳杆菌GDMCC No.60605在旷场实验中使小鼠进入中央区域的频率增加了55.4%、使小鼠在旷场中央区域的时间延长了121.1%,与空白组小鼠相比有显著性差异,但上述差异并不是由于小鼠运动总距离的差异引起的。图4中小鼠运动轨迹图也可以明显看出灌胃短乳杆菌GDMCC No.60605后小鼠探索行为的增加,焦虑症状减轻。
2.2高架十字迷宫实验结果
小鼠由于嗜暗性会倾向于在闭臂中活动,但出于好奇心和探究性又会在开臂中活动,在面对新奇刺激时,动物同时产生探究的冲动与恐惧,这就造成了探究与回避的冲突行为,从而产生焦虑心理。从图5~6我们可以看出,灌胃短乳杆菌GDMCC No.60605后小鼠进入开臂的次数显著增多,增加了69.4%。在开臂停留时间有所增加,但和空白组比无显著性差异,说明短乳杆菌GDMCC No.60605可以有效缓解焦虑。
实施例7短乳杆菌GDMCC No.60605改善睡眠效果评价
1动物实验设计
取20~25g的6周龄健康雄性ICR小鼠30只,随机分为3组:空白对照组,阳性对照组,短乳杆菌GDMCC No.60605治疗组。空白对照组每日灌胃无菌生理盐水;短乳杆菌GDMCCNo.60605治疗组每日灌喂本说明书实施例1制备的浓度3×109CFU/mL短乳杆菌GDMCCNo.60605脱脂乳悬液,阳性对照组灌胃3mg/kg.BW的***溶液。经口每日一次给予小鼠相应剂量的受试物,小鼠灌胃量为20mL/kg.bw,持续30天。改善睡眠实验参考《保健食品检验与评价技术规范(2003版)》中改善睡眠功能评价规范部分进行。
1.1延长戊巴比妥钠睡眠时间实验
做正式实验前先进行预实验,确定使动物100%入睡,但又不使睡眠时间过长的戊巴比妥钠剂量(30~60mg/kg.bw),用此剂量正式实验。动物末次给予样品30min后给各组动物腹腔注射戊巴比妥钠,注射量为0.2mL/20g,以翻正反射消失为指标,观察受试样品能否延长戊巴比妥钠睡眠时间。实验结果列于附图7中。
1.2戊巴比妥钠阈下剂量催眠实验
正式实验前先进行预实验,确定戊巴比妥钠阈下催眠剂量(16~30mg/kg.bw),即80~90%小鼠翻正反射不消失的戊巴比妥钠最大阈下剂量。动物末次给予样品30min后,各组动物腹腔注射戊巴比妥钠最大阈下催眠剂量,记录30min内入睡动物数(翻正反射消失达1分钟以上者)。实验在24~25℃安静环境下进行。
1.3巴比妥钠睡眠潜伏期实验
做正式实验前先进行预实验,确定使动物100%入睡,但又不使睡眠时间过长的巴比妥钠的剂量(200~300mg/kg.bw),用此剂量正式实验。动物末次给于样品30min后,给各组动物腹腔注射巴比妥钠,注射量为0.2mL/20g,以翻正反射消失为指标,观察短乳杆菌GDMCC No.60605对巴比妥钠睡眠潜伏期的影响。实验结果列于附图8中。
2实验结果
2.1延长戊巴比妥钠睡眠时间实验通过预实验,确定了戊巴比妥钠腹腔注射剂量为49mg/kg.bw。如图7所示,与空白组相比,作为阳性对照组的***(3mg/kg.bw)极显著延长了戊巴比妥钠诱导睡眠的持续时间。与空白组相比,灌胃短乳杆菌GDMCC No.60605的小鼠睡眠时间延长了149.2%,和空白组相比有显著性差异,说明短乳杆菌GDMCC No.60605可以有效地增加小鼠睡眠时间,具有改善睡眠的效果。
2.2戊巴比妥钠阈下剂量催眠实验
表2短乳杆菌GDMCC No.60605对戊巴比妥钠诱导小鼠睡眠率的影响
实验结果如表2所示,用戊巴比妥钠(33mg/kg.bw)腹腔注射小鼠后,阳性对照组小鼠睡眠率为50%,与空白组相比有显著性差异。灌胃短乳杆菌GDMCC No.60605的小鼠睡眠率为20%,与空白组相比有所提高但无显著性差异。
2.3巴比妥钠睡眠潜伏期实验
通过预实验确定了巴比妥钠腹腔注射的剂量为320mg/kg.bw。如图8所示,与空白组相比,作为阳性对照组的***(3mg/kg.bw)显著缩短了巴比妥钠诱导睡眠的潜伏期。与空白组相比,灌胃短乳杆菌GDMCC No.60605使小鼠的睡眠潜伏期缩短了31.0%,说明短乳杆菌GDMCC No.60605可以缩短小鼠睡眠潜伏期,使其更快入睡,具有改善睡眠的效果。
综上,根据《保健食品检验与评价技术规范(2003版)》中改善睡眠功能评价规范的要求,延长戊巴比妥钠睡眠时间实验、戊巴比妥钠阈下剂量催眠实验、巴比妥钠睡眠潜伏期实验三项实验中二项阳性,且无明显直接睡眠作用,可判定短乳杆菌GDMCC No.60605具有改善睡眠功能作用。
实施例8短乳杆菌GDMCC No.60605对小鼠肠道菌群的影响
1试验方法
取20~25g的6周龄健康雄性ICR小鼠30只,随机分为3组:空白对照组,阳性对照组,短乳杆菌GDMCC No.60605治疗组。空白对照组每日灌胃无菌生理盐水;短乳杆菌GDMCCNo.60605治疗组每日灌喂本说明书实施例5制备的浓度3×109CFU/mL短乳杆菌GDMCCNo.60605脱脂乳悬液,阳性对照组灌胃3mg/kg.bw的***溶液。经口每日一次给予小鼠相应剂量的受试物,小鼠灌胃量为20mL/kg.bw,持续30天。收集新鲜小鼠粪便样品,用MP粪便试剂盒提取总DNA,然后以得到的细菌基因组为模板,进行粪便样品16S rRNA V4区PCR扩增,PCR产物胶回收,混样、文库构建以及上机测序。
2实验结果
通过数据分析得到的小鼠肠道菌群β多样性分析如图9所示,可以看出,灌胃短乳杆菌GDMCC No.60605后小鼠菌群组成发生了显著性变化,从图10可以看出其中Allobaculum的相对丰度显著增加,而大多数Allobaculum是短链脂肪酸(SCFAs)的重要生产者,SCFAs除了能够缓解炎症及其保护肠屏障外,也是微生物与宿主信号传导最重要功能成分之一。所以短乳杆菌GDMCC No.60605能够改善睡眠,可能也与Allobaculum的相对丰度显著增加,促进了SCFAs的产生有关。
实施例9:短乳杆菌GDMCC No.60605的应用
(1)利用短乳杆菌GDMCC No.60605制造乳杆菌奶饮料
将原料乳脱脂奶在95℃热杀菌20min,然后冷却至4℃,再加入本发明的短乳杆菌GDMCC No.60605工作发酵剂,使其浓度达到106CFU/mL以上,在4℃冷藏保存即得到含短乳杆菌GDMCC No.60605活菌的全菌奶饮料。
(2)利用短乳杆菌GDMCC No.60605制造豆奶
采用软水浸泡大豆,水量为原大豆量三倍体积,在温度80℃下浸泡1~2h,再去除大豆皮。接着,沥去浸泡水,另加沸水磨浆,并在温度高于80℃的条件下保温10~15min。浆体用150目滤膜过滤后接着进行离心,得到的离心液即为粗豆奶,再将它加热到温度140~150℃,然后将热粗豆奶迅速导入真空冷却室进行抽真空,所述粗豆奶中的异味物质随着水蒸汽迅速排出。经过真空脱气后,将其温度降至37℃左右,再接入本发明的短乳杆菌GDMCCNo.60605工作发酵剂,使其浓度达到106CFU/mL以上,在4℃冷藏保存即得到含短乳杆菌GDMCC No.60605活菌的豆奶。
(3)利用短乳杆菌GDMCC No.60605制造果蔬饮料
选用新鲜蔬菜洗净后榨汁,接着进行高温瞬间灭菌,在温度140℃下高温热杀菌2s后,立即降温到37℃左右,再接入本发明的短乳杆菌GDMCC No.60605发酵剂,使其浓度达到106CFU/mL以上,在4℃冷藏保存即得到含短乳杆菌GDMCC No.60605活菌的果蔬饮料。
(4)利用短乳杆菌GDMCC No.60605制胶囊制品
本发明的短乳杆菌GDMCC No.60605在MRS培养基上培养24h,在温度4℃与4000r/min的条件下离心20min,用PBS冲洗两次,再加入以最后得到含短乳杆菌GDMCC No.60605的粉剂重量计4%脱脂奶粉和6%乳糖混合10min,再加入无菌2%氯化钙和3%海藻酸钠,同时以150r/min搅拌10min,再静止固化30min,最后清洗过滤,得到的滤液进行冷冻干燥20h,得到含短乳杆菌GDMCC No.60605的粉剂,把这种粉剂装入目前市场上销售的药用微胶囊,得到所述的胶囊制品。
虽然本发明已以较佳实施例公开如上,但其并非用以限定本发明,任何熟悉此技术的人,在不脱离本发明的精神和范围内,都可做各种的改动与修饰,因此本发明的保护范围应该以权利要求书所界定的为准。
Claims (16)
1.一种短乳杆菌(Lactobacillus brevis)GDMCC No.60605,其特征在于,所述菌株已于2019年3月12日保藏于广东省微生物菌种保藏中心,保藏地址为广州市先烈中路100号大院59号楼5楼,保藏编号为GDMCC No.60605。
2.根据权利要求1所述的短乳杆菌GDMCC No.60605,其特征在于它具有下述性质:
(1)具有优良的模拟胃肠道存活能力、细胞粘附能力和GABA产生能力;
(2)在旷场实验中能够显著增加小鼠进入中央区域的频率、延长小鼠在旷场中央区域的时间而不影响小鼠总运动距离;
(3)在高架十字迷宫实验中能够显著增加小鼠进入开臂的次数;
(4)能够延长戊巴比妥钠诱导的睡眠时间,缩短巴比妥钠诱导的睡眠潜伏期;
(5)能够调节肠道菌群,显著增加小鼠肠道中Allobaculum的相对丰度。
3.一种发酵剂,其特征在于,含有权利要求1所述的短乳杆菌GDMCC No.60605。
4.根据权利要求3所述的发酵剂,其特征在于,所述发酵剂是利用含有所述短乳杆菌GDMCC No.60605的菌液制备所得到的粉剂,它含有106CFU/g以上的活性短乳杆菌GDMCCNo.60605。
5.根据权利要3-4任一所述的发酵剂,其特征在于,所述发酵剂中还含有可用于食品的微生物。
6.根据权利要3或4所述的发酵剂,其特征在于,所述发酵剂中还含有耐酸乳杆菌、植物乳杆菌、保加利亚乳杆菌中的一种或者两种以上。
7.一种发酵食品,其特征在于,所述发酵食品是使用权利要求1所述的短乳杆菌GDMCCNo.60605或者含有短乳杆菌GDMCC No.60605菌种的发酵剂生产得到的。
8.根据权利要求7所述的发酵食品,其特征在于,所述发酵食品为乳制品、豆制品或者果蔬制品。
9.根据权利要求7所述的发酵食品,其特征在于,所述发酵食品中含有短乳杆菌GDMCCNo.60605活菌。
10.根据权利要求8所述的发酵食品,其特征在于,所述的乳制品是牛奶、酸奶油或干酪。
11.根据权利要求8所述的发酵食品,其特征在于,所述的豆制品是豆奶、豆豉或豆酱。
12.根据权利要求8所述的发酵食品,其特征在于,所述的果蔬制品是黄瓜、胡萝卜、甜菜、芹菜或圆白菜制品。
13.权利要求1所述的短乳杆菌GDMCC No.60605在制备具有改善睡眠功能的药物组合物或者发酵食品中的应用。
14.根据权利要求13所述的应用,其特征在于,所述药物组合物是由短乳杆菌GDMCCNo.60605菌剂与在药学上可接受的载体组成的。
15.根据权利要求14所述的应用,其特征在于,在药学上可接受的载体是一种或多种选自在药学上通常使用的填充剂、粘合剂、润湿剂、崩解剂、润滑剂或矫味剂的载体。
16.根据权利要求13-15任一所述的应用,其特征在于,所述的药物组合物是颗粒剂、胶囊剂、片剂、丸剂或口服液剂型。
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