CN117511779A - 一株改善睡眠质量的植物乳杆菌nsl0101及其应用 - Google Patents
一株改善睡眠质量的植物乳杆菌nsl0101及其应用 Download PDFInfo
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- CN117511779A CN117511779A CN202311397121.XA CN202311397121A CN117511779A CN 117511779 A CN117511779 A CN 117511779A CN 202311397121 A CN202311397121 A CN 202311397121A CN 117511779 A CN117511779 A CN 117511779A
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Abstract
本发明公开了一株改善睡眠质量的植物乳杆菌NSL0101及其应用。所述植物乳杆菌NSL0101的分类命名为Lactobacillus plantarum,保藏于中国典型培养物保藏中心,保藏编号为CCTCC NO:M 2023175,其核苷酸序列如SEQ ID No.1所示。所述植物乳杆菌NSL0101无致病性,不具有急性毒性,安全可靠,且经实验验证,植物乳杆菌NSL0101能够在肠道内长期定植,其在肠道内生长代谢的过程中会分泌产生γ‑氨基丁酸被机体吸收利用,从而有效改善机体的睡眠质量,且其已获得了较成熟的生产工艺,因此具有很好的应用价值。
Description
技术领域
本发明属于微生物技术领域,具体涉及一株改善睡眠质量的植物乳杆菌NSL0101及其应用。
背景技术
益生菌是可以通过定植在人体内,改变宿主某一部位菌群组成的一类对宿主有益的活性微生物,当摄入充足的数量时,其能够产生有利于机体健康的作用。目前发现,益生菌在增强免疫功能、维持肠道生态平衡、抗肿瘤等方面均具有重要的作用。
γ-氨基丁酸(GABA)是一种重要的中枢神经系统抑制性神经递质,具备改善机体睡眠质量、降血压等生理功效。相关研究证实,作为小分子量非蛋白质氨基酸的GABA具备食用安全性,并可用于饮料等食品的生产。
现代研究表明,多种益生菌具有生产γ-氨基丁酸的能力。通过补充具有生产γ-氨基丁酸能力的益生菌或益生菌代谢产物来达到改善睡眠质量的目的,有利于患者的身心健康,且不具有其他药物普遍具备的副作用,因此,该项研究工作具有远大前景和重要意义。
发明内容
本发明的目的在于提供一株改善睡眠质量的植物乳杆菌NSL0101及其应用。所述植物乳杆菌NSL0101来源安全,无致病性,具有改善睡眠质量的作用。
为实现上述发明目的,本发明采用以下技术方案予以实现:
本发明提供了一株改善睡眠质量的植物乳杆菌NSL0101,所述植物乳杆菌NSL0101的分类命名为Lactobacillus plantarum,保藏于中国典型培养物保藏中心,保藏编号为CCTCC NO:M 2023175。
进一步的,所述植物乳杆菌NSL0101的核苷酸序列如SEQ ID No.1所示。
进一步的,所述植物乳杆菌NSL0101为兼性厌氧菌,厌氧环境下生长良好,革兰氏染色阳性,无芽孢,无荚膜,短杆状,两端钝圆形,无运动性,不规则单一、短链排列或栅栏状排列。
进一步的,所述植物乳杆菌NSL0101在MRS固体培养基上的单菌落呈圆形,直径约1mm,边缘整齐,表面光滑,颜色呈乳白色,质地均匀。
进一步的,所述植物乳杆菌NSL0101通过在肠道内代谢产生γ-氨基丁酸来起到改善睡眠质量的作用。
本发明还提供了一种植物乳杆菌冻干菌粉,其是由所述的植物乳杆菌NSL0101的菌泥和冻干保护剂混合后,经真空冷冻干燥获得的。
所述植物乳杆菌NSL0101的菌泥和冻干保护剂的质量比为1~3:1。
进一步的,所述冻干保护剂的配方及其用量为:海藻糖1-2 g/L,脱脂乳粉1-2 g/L,谷氨酸钠0.1-0.2 g/L,吐温80 g/L,纯水 1L。
进一步的,所述植物乳杆菌冻干菌粉的活菌数不小于4×1011CFU/g。
本发明还提供了所述的植物乳杆菌NSL0101或者所述的植物乳杆菌冻干菌粉在制备改善睡眠质量的食品、保健品或药物中的应用。
进一步的,所述食品、保健品或药物中含有活菌数不少于1×108CFU/g的植物乳杆菌NSL0101。
本发明还提供了所述的植物乳杆菌NSL0101或者所述的植物乳杆菌冻干菌粉在制备γ-氨基丁酸的微生态制剂中的应用。
进一步的,所述微生态制剂包括即食型益生菌粉、益生菌活性饮料和后生元益生菌饮品。
与现有技术相比,本发明具有以下优点和技术效果:
本发明从广西搜集到的农家老泡菜汤汁中分离到一株植物乳杆菌NSL0101,其安全性强,无致病性,不具有急性毒性,且能够发酵多种营养物质供能生长。所述植物乳杆菌NSL0101经实验验证,能够在肠道内长期定植,其在肠道内生长代谢的过程中会分泌产生γ-氨基丁酸被机体吸收利用,从而有效改善机体的睡眠质量。另外,植物乳杆菌NSL0101具有优秀的发酵特性,发酵水平高,适合大规模产业化生产。通过大量实验研究,植物乳杆菌NSL0101已获得了较成熟的生产工艺,因此具有很好的应用价值。
附图说明
图1为植物乳杆菌NSL0101的平板培养照片。
图2为植物乳杆菌NSL0101的菌体镜检照片。
图3为实验小鼠伺喂期间体重变化曲线。
图4为植物乳杆菌NSL0101对实验小鼠睡眠潜伏期的影响实验结果。
图5为植物乳杆菌NSL0101对小鼠睡眠时长的影响实验结果。
具体实施方式
结合以下具体实例对本发明的技术方案作进一步详细的说明。
下述实施例中,如无特殊说明,所使用的实验方法均为常规方法,所用材料、试剂等均可从生物或化学试剂公司购买。
实施例1:植物乳杆菌NSL0101的分离和鉴定
1、分离筛选
以来源于3份广西壮族自治区农家老泡菜汤汁为样品。将3份泡菜汤汁按1%的体积分别接种到MRS液体培养基中,37℃培养24h,培养液梯度稀释至10-8。选择3个适宜的稀释度分别取1mL,接种至MRS固体平板,37℃下厌氧培养48h,进行菌落形态观察,挑选不同形态的单菌落再次于改良MRS固体培养基上划线分离,37℃下厌氧培养48h,重复划线分离3代,得到纯化后菌株,进行革兰氏染色并显微镜观测,挑选出革兰氏染色阳性,显微镜观测呈球状、杆状、多形状的菌株。在筛选得到的多株菌株中进一步筛选得到一株编号为NSL0101的菌株。
2、形态学观察
(1)将菌株NSL0101接种于改良MRS固体平板上,37℃恒温培养48 h,观察菌落形态特征。结果见图1,菌株NSL0101在MRS固体培养基上的单菌落呈圆形,直径约1mm,边缘整齐,表面光滑,颜色呈乳白色,质地均匀。
(2)取菌株NSL0101的典型菌落涂片,用革兰氏染色法进行染色,显微镜下观察菌体形态。结果见图2,菌株NSL0101革兰氏染色阳性,无芽孢,无荚膜,短杆状,两端钝圆形,无运动性,不规则单一、短链排列或栅栏状排列。
3、生理生化特征
生理生化特征观察参照《常见细菌系统鉴定手册》和《乳酸细菌分类鉴定及实验方法》方法进行。
结果见表1和表2,菌株NSL0101的过氧化氢酶实验、吲哚实验、水解淀粉实验、硝酸盐还原实验、明胶液化实验、硫化氢产生实验、发酵产气实验均为阴性,符合乳杆菌属的特征,因此初步判定其为乳杆菌属。菌株NSL0101能发酵七叶灵、乳糖、半乳糖、葡萄糖、麦芽糖、纤维二糖、蔗糖、鼠李糖、山梨醇、水杨酸、甘露醇,不发酵木糖,可发酵多种营养物质供能。
表1:生理生化检测结果
表2:糖发酵结果
4、16S rRNA鉴定
提取菌株NSL0101的DNA作为模板,利用细菌16S rRNA通用引物进行扩增,扩增片段送至生物公司进行序列测定,测序结果如SEQ ID No.1所示。BLAST比对结果表明,菌株NSL0101与Lactobacillus plantarum的序列相似性为99.93%,因此结合上述实验结果,判断菌株NSL0101为植物乳杆菌。
将菌株NSL0101进行菌种保藏,所述植物乳杆菌NSL0101的保藏单位:中国典型培养物保藏中心(CCTCC);地址:中国.武汉.武汉大学;保藏日期:2023年02月22日;植物乳杆菌Lactobacillus plantarum的保藏编号为CCTCC NO:M 2023175。
实施例2:植物乳杆菌NSL0101的安全性评价
1、溶血性检测
菌株的溶血活性检测溶血是指在溶血毒素及其他理化因素的作用下,红细胞破裂,血红蛋白逸出。菌株代谢产物有无溶血活性被认为是菌株安全性评价的重要指标。
将活化好的植物乳杆菌NSL0101以及质控菌株大肠杆菌用灭菌的接菌环划线接种于哥伦比亚血琼脂平板中,于37℃培养48 h,同时做空白试验,观察菌落周围有无溶血圈出现。
结果显示,植物乳杆菌NSL0101周围无明显溶血圈形成,属于没有毒性的γ-溶血,也说明植物乳杆菌NSL0101无致病性,安全性好。
2、动物经口急性毒理试验
选用18-22 g的健康成年SPF级KM小鼠20只,雌雄各半。将处于生长期的植物乳杆菌NSL0101用PBS缓冲液将浓度调整至1×108CFU/mL,空腹灌胃一次,每次灌胃容量为0.2mL/10g体重。灌胃后立即观察其活动表现、体重以及身体状况变化,观察期为14d。
结果显示,在观察期内小鼠的活动、食欲正常,体重增加;试验结束后,对存活的小鼠进行解剖,肉眼未见心肝脾肺肾肠等内脏器官异常,说明植物乳杆菌NSL0101对小鼠不具有急性毒性,说明植物乳杆菌NSL0101安全性好。
实施例3:植物乳杆菌NSL0101对小鼠睡眠的影响
1、小鼠实验
将活化的植物乳杆菌NSL0101划线于MRS固体培养基上,37℃培养48h,挑取得到的单菌落接种于MRS液体培养基中,37℃培养16h,连续活化三代后,按3%(v/v)的接种量接种于MRS液体培养基中,37℃下培养12h,经12000rpm,4℃离心,得到菌泥,超纯水清洗后将菌泥与已灭菌处理的冻干保护剂(配方:16g海藻糖,12g脱脂乳粉,1g谷氨酸钠,1g吐温80,100g纯水)按照质量比2:1充分混合,真空冷冻干燥后得到冻干菌粉,粉碎后过80目筛,最终得到植物乳杆菌NSL0101冻干菌粉。植物乳杆菌NSL0101冻干菌粉的活菌数不小于4×1011CFU/g。菌粉真空包装后保存在-18℃的冰箱中,以备后续实验使用。
将购得的90只雄性C57BL/6小鼠随机分为甲、乙、丙三批,每批30只,分别进行肠道成分、入睡率和睡眠时长实验。每批小鼠随机分为阴性对照组、植物乳杆菌NSL0101组和阳性对照组,每组10只,小鼠均可以自由进食及饮水。3组小鼠中,阴性对照组给予生理盐水,植物乳杆菌NSL0101组给予上述制得的植物乳杆菌NSL0101冻干菌粉10 mg/kg(用生理盐水溶解),阳性对照组给予地西泮2 mg/kg(用生理盐水溶解),均予以每天灌胃一次处理,灌胃体积0.2mL/10g体重。小鼠饲养持续时间为30天,期间每隔6天记录一次小鼠体重,30天后停止灌胃,准备开展后续实验。
体重结果如图3所示,三组小鼠的体重变化均无显著差异,说明植物乳杆菌NSL0101对小鼠体重没有影响,安全性好。
2、小鼠肠道内乳酸菌及γ-氨基丁酸含量检测
甲批小鼠末次灌胃24h后脱颈处死,及时解剖分离获得各组小鼠新鲜肠道组织,将肠道组织放入研钵并迅速加入液氮冷冻,研磨均匀后制成肠道组织样品。采用实时荧光定量PCR技术,应用SYBR GreenⅡ试剂,利用比较Ct法分析上述小鼠肠道组织样品内乳酸菌的相对含量;采用ELISA法测定上述小鼠肠道组织样品内γ-氨基丁酸含量。
结果见表3,与对照组相比,NSL0101组小鼠肠道内乳酸菌相对含量极显著增高(P<0.01),表明植物乳杆菌NSL0101可以在小鼠肠道内定植。与对照组相比,NSL0101组小鼠肠道内γ-氨基丁酸显著升高,说明成功定植于小鼠肠道的植物乳杆菌NSL0101在肠道内分泌产生了γ-氨基丁酸,γ-氨基丁酸是一种重要的中枢神经系统抑制性神经递质,具备改善机体睡眠质量、降血压等生理功效。
表3:小鼠肠道组织样品内乳酸菌及γ-氨基丁酸含量检测结果(`x±SD)
与阴性对照组相比,*表示差异显著(P<0.05)。
3、小鼠入睡率实验
乙批小鼠开展戊巴比妥钠阈下实验,末次灌胃1h后对各组小鼠给予腹腔注射戊巴比妥钠35mg/kg,注射体积0.1mL/10g体重,以小鼠翻正反射消失作为其入睡判断标准,从腹腔注射完毕到翻正反射消失的时间作为睡眠潜伏期,统计各组小鼠的睡眠情况,计算得出入睡率。
结果见表4和图4,与阴性对照组相比,NSL0101组睡眠潜伏期无显著变化(P>0.05),阳性对照组睡眠潜伏期显著缩短(P<0.05);与阴性对照组相比,NSL0101组入睡率明显升高,结合表3实验结果,说明植物乳杆菌NSL0101在肠道内定植并分泌γ-氨基丁酸,因此具有一定的助眠作用。
表4:戊巴比妥钠阈下实验结果
与阴性对照组相比,*表示差异显著(P<0.05)。
4、小鼠睡眠时长实验
丙批小鼠开展戊巴比妥钠阈下实验,末次灌胃1h后对各组小鼠给予腹腔注射戊巴比妥钠50 mg/kg,注射体积0.1mL/10g体重,以小鼠翻正反射消失作为其入睡判断标准,从翻正反射消失到翻正反射恢复的时间作为睡眠时长,观察记录各组小鼠的睡眠时长情况,因此具有一定的助眠作用。
结果见表5和图5,与阴性对照组相比,NSL0101组睡眠时长明显增加(P<0.05),阳性对照组睡眠时长显著增加(P<0.05),说明植物乳杆菌NSL0101能够起到延长睡眠的效果。
表5:戊巴比妥钠睡眠延长实验结果
与阴性对照组相比,*表示差异显著(P<0.05),**表示差异极显著。
综上所述,植物乳杆菌NSL0101能够在肠道内定植并分泌功能性物质γ-氨基丁酸,γ-氨基丁酸可作为神经递质对机体的神经系统进行调节,起到缓解压力延长睡眠时长的作用,进而达到安神助眠的效果。
植物乳杆菌NSL0101具有优秀的发酵特性,发酵水平高,适合大规模产业化生产。通过大量实验研究,已获得了较成熟的生产工艺,因此具有很好的应用价值。
以上实施例仅用于说明本发明的技术方案,而非对其进行限制;尽管参照前述实施例对本发明进行了详细的说明,对于本领域的普通技术人员来说,依然可以对前述实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换;而这些修改或替换,并不使相应技术方案的本质脱离本发明所要求保护的技术方案的精神和范围。
Claims (10)
1.一株改善睡眠质量的植物乳杆菌(Lactobacillus plantarum)NSL0101,其特征在于,所述植物乳杆菌NSL0101保藏于中国典型培养物保藏中心,保藏编号为CCTCC NO:M2023175。
2.根据权利要求1所述的植物乳杆菌NSL0101,其特征在于,所述植物乳杆菌NSL0101的核苷酸序列如SEQ ID No.1所示。
3.一种植物乳杆菌冻干菌粉,其特征在于,所述植物乳杆菌冻干菌粉是由权利要求1所述的植物乳杆菌NSL0101的菌泥和冻干保护剂混合后,经真空冷冻干燥获得的。
4.根据权利要求3所述的植物乳杆菌冻干菌粉,其特征在于,所述植物乳杆菌NSL0101的菌泥和冻干保护剂的质量比为1~3:1。
5.根据权利要求3所述的植物乳杆菌冻干菌粉,其特征在于,所述冻干保护剂的配方及其用量为:海藻糖1-2 g/L,脱脂乳粉1-2 g/L,谷氨酸钠0.1-0.2 g/L,吐温80 g/L,纯水1L。
6.根据权利要求3所述的植物乳杆菌冻干菌粉,其特征在于,所述植物乳杆菌冻干菌粉的活菌数不小于4×1011 CFU/g。
7.权利要求1所述的植物乳杆菌NSL0101或者权利要求3所述的植物乳杆菌冻干菌粉在制备改善睡眠质量的食品、保健品或药物中的应用。
8.根据权利要求7所述的应用,其特征在于,所述食品、保健品或药物中含有活菌数不少于1×108 CFU/g的植物乳杆菌NSL0101。
9.权利要求1所述的植物乳杆菌NSL0101或者权利要求3所述的植物乳杆菌冻干菌粉在制备γ-氨基丁酸的微生态制剂中的应用。
10.根据权利要求9所述的应用,其特征在于,所述微生态制剂包括即食型益生菌粉、益生菌活性饮料和后生元益生菌饮品。
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