CN113549567A - 一株具有促排便功能的鼠李糖乳杆菌nsl0401及其应用 - Google Patents
一株具有促排便功能的鼠李糖乳杆菌nsl0401及其应用 Download PDFInfo
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- CN113549567A CN113549567A CN202110622104.6A CN202110622104A CN113549567A CN 113549567 A CN113549567 A CN 113549567A CN 202110622104 A CN202110622104 A CN 202110622104A CN 113549567 A CN113549567 A CN 113549567A
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- nsl0401
- lactobacillus rhamnosus
- defecation
- lactobacillus
- rhamnosus
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Abstract
本发明公开了一株具有促排便功能的鼠李糖乳杆菌NSL0401及其应用。所述鼠李糖乳杆菌NSL0401的分类命名为鼠李糖乳杆菌Lactobacillus rhamnosus，保藏于中国典型培养物保藏中心，保藏编号为CTCC M 2021627，其核苷酸序列如SEQ ID No.1所示。所述鼠李糖乳杆菌NSL0401无致病性，不具有急性毒性，安全可靠，且经实验验证，鼠李糖乳杆菌NSL0401能够有效改善机体的排便参数，增加排便次数和粪便含水量，促进体内益生菌的种类和增殖，并增加多种短链脂肪酸的浓度，进而起到促进排便，缓解或治疗弛缓性便秘的作用，并增强机体的肠道功能和健康情况。
Description
技术领域
本发明属于微生物技术领域，具体涉及一株具有促排便功能的鼠李糖乳杆菌NSL0401及其应用。
背景技术
弛缓性便秘属于功能性便秘，是由于全身因素或各有关排便肌肉衰弱，张力低下，特别是肠管平滑肌张力低下、肠管运动弛缓等，使排便动力不足或缺乏，肠内容物在结肠内运进过于缓慢而形成的便秘。而目前多采用肠道水疗法，但这种疗法存在需要去专业医疗机构治疗，或者家庭使用的水疗设备价格比较昂贵等缺点，且盲目的使用会造成一定的健康危险。
益生菌是可以通过定殖在人体内，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，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、抗肿瘤等方面均具有重要的作用。因此，通过益生菌来达到缓解或治疗弛缓性便秘，非常有利于患者的身心健康。
发明内容
本发明的目的在于提供一株具有促排便功能的鼠李糖乳杆菌NSL0401及其应用。所述鼠李糖乳杆菌NSL0401来源安全，无致病性，具有增加排便次数的作用。
为实现上述发明目的，本发明采用以下技术方案予以实现：
本发明提供了一株具有促排便功能的鼠李糖乳杆菌NSL0401，所述鼠李糖乳杆菌NSL0401的分类命名为鼠李糖乳杆菌Lactobacillus rhamnosus，保藏于中国典型培养物保藏中心，保藏编号为CTCC M 2021627。
进一步的，所述鼠李糖乳杆菌NSL0401的核苷酸序列如SEQ ID No.1所示。
进一步的，所述鼠李糖乳杆菌NSL0401的菌落呈圆形，边缘整齐，表面光滑，中间突出，颜色呈乳白色，质地均匀；菌体短杆状，两头钝圆，表面光滑，不产芽孢，无运动性，链状排列。
本发明还提供了所述的鼠李糖乳杆菌NSL0401在用于制备促进排便的保健品或药品中的应用。
本发明还提供了所述的鼠李糖乳杆菌NSL0401在用于制备缓解或治疗弛缓性便秘的保健品或药品中的应用。
进一步的，所述鼠李糖乳杆菌NSL0401通过增加排便数量和粪便含水量来起到缓解或治疗弛缓性便秘的作用。
本发明还提供了所述的鼠李糖乳杆菌NSL0401在用于制备生产短链脂肪酸的微生物制剂中的应用。
进一步的，所述短链脂肪酸包括丙酸、丁酸和异戊酸。
进一步的，所述保健品或药品或微生物制剂中包含菌含量不少于1×107 CFU/mL的鼠李糖乳杆菌NSL0401发酵液或者菌含量不少于1×107 CFU/g的鼠李糖乳杆菌NSL0401菌粉。
与现有技术相比，本发明具有以下优点和技术效果：
本发明从四川传统农家泡菜分离到一株鼠李糖乳杆菌NSL0401，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。所述鼠李糖乳杆菌NSL0401经实验验证，能够有效改善机体的排便参数，增加排便次数和粪便含水量，进而促进体内粪便的排出，进而达到缓解和治疗便秘的效果，尤其是对排便肌肉衰弱而引起的弛缓性便秘，具有很好的缓解功效。另外，鼠李糖乳杆菌NSL0401还能够促进体内益生菌的种类和增殖，促进多种营养物质来产生多种有益的短链脂肪酸，增强机体的肠道功能和健康情况，因此其具有很好的应用价值。
附图说明
图1为鼠李糖乳杆菌NSL0401的平板培养照片。
图2为鼠李糖乳杆菌NSL0401的菌体镜检照片。
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：鼠李糖乳杆菌NSL0401的分离和鉴定
1、分离筛选
以来源于3份四川传统农家泡菜为样品。将3份泡菜汁按1%的体积分别接种到MRS液体培养基中，37℃培养16h，培养液梯度稀释至10-8。选择3个适宜的稀释度分别取1mL，倾注改良MRS法倒平板，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。在筛选得到的多种菌株中进一步筛选得到一株编号为编号NSL0401的菌株。
2、形态学观察
（1）将菌株NSL0401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NSL0401的菌落呈圆形，边缘整齐，表面光滑，中间突出，颜色呈乳白色，质地均匀。
（2）取菌株NSL0401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NSL0401呈革兰氏阳性，菌体短杆状，两头钝圆，表面光滑，不产芽孢，无运动性，链状排列。
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NSL0401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NSL0401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
| 项目 | 过氧化氢酶 | 吲哚 | 水解淀粉 | 硝酸盐还原 | 明胶液化 | 硫化氢产生 | 发酵产气 |
| NSL0401 | - | - | - | - | - | - | - |
表2：糖发酵结果
| 项目 | 七叶灵 | 乳糖 | 半乳糖 | 葡萄糖 | 麦芽糖 | 纤维二糖 |
| NSL0401 | + | + | + | + | + | + |
| 项目 | 蔗糖 | 鼠李糖 | 山梨醇 | 水杨 | 甘露醇 | 木糖 |
| NSL0401 | + | + | + | + | + | - |
4、16S rRNA鉴定
提取菌株NSL0401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NSL0401与Lactobacillus rhamnosus的序列相似性为99.44%，因此结合上述实验结果，判断菌株NSL0401为鼠李糖乳杆菌。
将筛选到的菌株NSL0401进行菌种保藏，所述鼠李糖乳杆菌NSL0401的保藏单位：中国典型培养物保藏中心（CCTCC）；地址：湖北省武汉市武昌区八一路珞珈山；保藏日期：2021年5月31日；鼠李糖乳杆菌Lactobacillus rhamnosus的保藏编号为CTCC M 2021627。
实施例2：鼠李糖乳杆菌NSL0401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。
将活化好的鼠李糖乳杆菌NSL0401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，鼠李糖乳杆菌NSL0401周围无明显溶血圈形成，属于没有毒性的γ-溶血，也说明鼠李糖乳杆菌NSL0401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。将处于生长期的鼠李糖乳杆菌NSL0401用PBS缓冲液将浓度调整至1×108 CFU/mL，空腹灌胃一次，每次灌胃容量为20.0mL/kg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，说明鼠李糖乳杆菌NSL0401对小鼠不具有急性毒性，说明鼠李糖乳杆菌NSL0401安全性好。
实施例3：鼠李糖乳杆菌NSL0401对松缓性便秘小鼠模型的影响
1、小鼠实验
将保存的鼠李糖乳杆菌NSL0401活化后，接种于改良MRS液体培养基，37℃下厌氧培养48h，得到发酵液，菌含量不少于1×107 CFU/mL，用于后续实验。
将购得的30只雄性C57BL/6小鼠随机分为对照组、马来酸替加色罗组及鼠李糖乳杆菌NSL0401组，小鼠均可以自由进食及饮水。3组小鼠中，对照组给予正常肉汤培养基，其余2组分别予以马来酸替加色罗蒸馏水溶液（Tegaserod，简为T）及鼠李糖乳杆菌NSL0401发酵液，均予以灌胃处理。小鼠饲养持续时间为7天，7天后记录小鼠排便参数并进行小肠推进试验，并处死小鼠，解剖小鼠并暴露小鼠腹腔脏器，游离并留取结肠组织冻存于-80℃冰箱，以进行后续实验。
2、小鼠排便参数测定
第8天进行小鼠排便参数测定。对小鼠禁食不禁水24h，并对每只小鼠予印度墨汁(0.2 ml/l0g)灌胃，单独放置。记录首粒黑便排出的时间，接着记录2小时内小鼠排便颗粒数，及时收集排出的粪便，称得粪便湿重。烘干粪便，称重得粪便干重。以湿重及干重为原始数据，按照以下公式计算粪便含水率:粪便含水率=(粪便湿重-粪便干重)/粪便湿重×100%。
结果见表3，与对照组相比，T组和NSL0401组小鼠2h排便颗粒数、粪便的湿重和干重以及含水量都增加了，而T组与NSL0401组比较无显著性差异，表明鼠李糖乳杆菌NSL0401可有效改善健康小鼠的排便参数。
表3：小鼠排便参数测定结果
| 对照组 | T组 | NSL0401组 | |
| 2h排便颗粒数 | 5.47±1.08 | 9.45±2.69 | 8.62±1.87 |
| 粪便湿重/mg | 103.4±27.62 | 214.63±49.27 | 185.2±34 |
| 粪便干重/mg | 43.81±10.53 | 68.26±13.95 | 66.57±5.49 |
| 粪便含水量% | 48.52±7.75 | 67.98±5.54 | 53.33±3.29 |
3、小肠推进实验
第8天，每组各5只小鼠进行小肠推进实验。前一天禁食不禁水24h，每只小鼠依据体重印度墨汁灌胃(0.2 ml/l0g)，25min后处死小鼠，解剖，分离出幽门至回盲部肠管，测量小肠总长度以及幽门至墨汁前沿的肠管长。按以下公式计算小肠墨汁推进率：小肠墨汁推进率=墨汁推进长度/小肠总长度×100%。
结果见表4，3个实验组在小肠推进率方面的差异不大。
表4：小肠推进实验结果
| 对照组 | T组 | NSL0401组 | |
| 小肠推进率/% | 95.84±17.52 | 94.63±9.67 | 95.92±11.34 |
4、16S rRNA高通量测序分析肠道菌群
将小鼠粪便样品收集，采用16S rRNA菌群测序方法检测小鼠粪菌组成总数。
结果见表5，对照组、NSL0401组和T组之间的OTUS总数没有显著性差异，但鼠李糖乳杆菌NSL0401会刺激使得该组的OTUS总数比其他组略有增加。
表5：肠道菌群测序结果
| 对照组 | T组 | NSL0401组 | |
| OTUS总数 | 512 | 519 | 523 |
5、对小鼠粪便短链脂肪酸影响
取适量冻存的小鼠回盲部粪便采用气相色谱法技术检测粪便短链脂肪酸水平。结果见表6，与对照组相比，NSL0401组和T组的乙酸、戊酸浓度降低；而NSL0401组的丙酸、丁酸，尤其是异戊酸相较于对照组和T组均显著增加，而异丁酸浓度稍微增加，但不明显。
表6：短链脂肪酸检测结果
| 对照组 | T组 | NSL0401组 | |
| 乙酸mg/g | 4.91±0.85 | 3.27±0.75 | 4.32±0.92 |
| 丙酸μg/g | 279±72 | 259±52 | 293±51 |
| 丁酸μg/g | 328±52 | 291±63 | 382±41 |
| 戊酸μg/g | 59±11 | 31±6 | 37±5 |
| 异丁酸μg/g | 15±3 | 14±3 | 17±4 |
| 异戊酸μg/g | 311±7 | 288±47 | 475±84 |
短链脂肪酸在维持肠道健康、预防和改善包括癌症在内的多种非传染性疾病方面发挥了重要作用。肠道中的短链脂肪酸主要有乙酸、丙酸和丁酸等，其中乙酸随血液进入全身循环，能被大脑、心脏和外周组织氧化；丙酸被肝脏吸收，参与糖异生并抑制胆固醇合成；丁酸能够调节上皮细胞和免疫细胞的生长和凋亡，并对维持肠黏膜屏障完整性、稳定肠道微环境具有重要作用。
综上所述，鼠李糖乳杆菌NSL0401能够改善机体的排便参数，增加排便次数，进而促进机体粪便的排出，达到缓解和治疗便秘的效果，尤其是对排便肌肉衰弱而引起的弛缓性便秘，具有很好的缓解功效。另外，鼠李糖乳杆菌NSL0401能够促进体内益生菌的增殖，还能够促进多种营养物质来产生多种有益的短链脂肪酸，增强机体肠道功能和健康情况。
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。
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<110> 青岛诺森生物技术有限责任公司
<120> 一株具有促排便功能的鼠李糖乳杆菌NSL0401及其应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1439
<212> DNA
<213> 鼠李糖乳杆菌(Lactobacillus rhamnosus)
<400> 1
cttccgtggg tgctatctgc agtcgaacga gttctgatta ttgaaaggtg cttgcatctt 60
gatttaattt tgaacgagtg gcggacgggt gagtaacacg tgggtaacct gcccttaagt 120
gggggataac atttggaaac agatgctaat accgcataaa tccaagaacc gcatggttct 180
tggctgaaag atggcgtaag ctatcgcttt tggatggacc cgcggcgtat tagctagttg 240
gtgaggtaac ggctcaccaa ggcaatgata cgtagccgaa ctgagaggtt gatcggccac 300
attgggactg agacacggcc caaactccta cgggaggcag cagtagggaa tcttccacaa 360
tggacgcaag tctgatggag caacgccgcg tgagtgaaga aggctttcgg gtcgtaaaac 420
tctgttgttg gagaagaatg gtcggcagag taactgttgt cggcgtgacg gtatccaacc 480
agaaagccac ggctaactac gtgccagcag ccgcggtaat acgtaagtgg caagcgttat 540
ccggatttat tgggcgtaaa gcgagcgcag gcggtttttt aagtctgatg tgaaagccct 600
cggcttaacc gaggaagtgc atcggaaact gggaaacttg agtgcagaag aggacagtgg 660
aactccatgt gtagcggtga aatgcgtaga tatatggaag aacaccagtg gcgaaagcgg 720
ctgtctgggt ctgtaactga cgctgaagct cgaaagcatg ggtaggcgaa caggattaga 780
taccctggta gtccatgccg taaacgatga atgctagggt gttggagggt ttccgccctt 840
cagtgccgca gctaacgcat taagcattcc gcctggggga gtacgaccgc aaggttgaaa 900
ctcaaaggaa ttgacggggg cccgcacaag cggtggagca tgtggtttaa ttcgaagcaa 960
cgcgaagaac cttaccaggt cttgacatct tttgatcacc tgagagatca ggtttcccct 1020
tcgggggcaa aatgacaggt ggtgcatggt tgtcgtcagc tcgtgtcgtg agatgttggg 1080
ttaagtcccg caacgagcgc aacccttatg actagttgcc agcatttagt tgggcactct 1140
agtaagactg ccggtgacaa accggaggaa ggtggggatg acgtcaaatc atcatgcccc 1200
ttatgacctg ggctacacac gtgctacaat ggatggtaca acgagttgcg agaccgcgag 1260
gtcaagctaa tctcttaaag ccattctcag ttcggactgt aggctgcaac tcgcctacac 1320
gaagtcggaa tcgctagtaa tcgcggatca gcacgccgcg gtgaatacgt tcccgggcct 1380
tgtacacacc gcccgtcaca ccatgagagt ttgtaacacc cgaagccggt ggcgtaacc 1439
Claims (9)
1.一株具有促排便功能的鼠李糖乳杆菌NSL0401，其特征在于，所述鼠李糖乳杆菌NSL0401的分类命名为鼠李糖乳杆菌Lactobacillus rhamnosus，保藏于中国典型培养物保藏中心，保藏编号为CTCC M 2021627。
2.根据权利要求1所述的鼠李糖乳杆菌NSL0401，其特征在于，所述鼠李糖乳杆菌NSL0401的核苷酸序列如SEQ ID No.1所示。
3.根据权利要求1所述的鼠李糖乳杆菌NSL0401，其特征在于，所述鼠李糖乳杆菌NSL0401的菌落呈圆形，边缘整齐，表面光滑，中间突出，颜色呈乳白色，质地均匀；菌体短杆状，两头钝圆，表面光滑，不产芽孢，无运动性，链状排列。
4.权利要求1所述的鼠李糖乳杆菌NSL0401在用于制备促进排便的保健品或药品中的应用。
5.权利要求1所述的鼠李糖乳杆菌NSL0401在用于制备缓解或治疗弛缓性便秘的保健品或药品中的应用。
6.根据权利要求5所述的应用，其特征在于，所述鼠李糖乳杆菌NSL0401通过增加排便数量和粪便含水量来起到缓解或治疗弛缓性便秘的作用。
7.权利要求1所述的鼠李糖乳杆菌NSL0401在用于制备生产短链脂肪酸的微生物制剂中的应用。
8.根据权利要求7所述的应用，其特征在于，所述短链脂肪酸包括丙酸、丁酸和异戊酸。
9.根据权利要求4所述的应用、或者权利要求5所述的应用，或者权利要求7所述的应用，其特征在于，所述保健品或药品或微生物制剂中包含菌含量不少于1×107 CFU/mL的鼠李糖乳杆菌NSL0401发酵液或者菌含量不少于1×107 CFU/g的鼠李糖乳杆菌NSL0401菌粉。
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114774313A (zh) * | 2022-04-13 | 2022-07-22 | 微康益生菌(苏州)股份有限公司 | 鼠李糖乳杆菌LRa05在制备缓解便秘制品或调解肠道菌群制品方面的用途 |
| CN116925984A (zh) * | 2023-09-15 | 2023-10-24 | 山东益昊生物科技有限公司 | 一株解淀粉芽孢杆菌yhj32及其复合益生菌制剂和应用 |
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| CN109136131A (zh) * | 2018-08-27 | 2019-01-04 | 南昌大学 | 一株具有缓解结肠炎功效的鼠李糖乳杆菌及其应用 |
| CN109182166A (zh) * | 2018-08-27 | 2019-01-11 | 南昌大学 | 一株具有缓解便秘功效的鼠李糖乳杆菌及其应用 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109136131A (zh) * | 2018-08-27 | 2019-01-04 | 南昌大学 | 一株具有缓解结肠炎功效的鼠李糖乳杆菌及其应用 |
| CN109182166A (zh) * | 2018-08-27 | 2019-01-11 | 南昌大学 | 一株具有缓解便秘功效的鼠李糖乳杆菌及其应用 |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114774313A (zh) * | 2022-04-13 | 2022-07-22 | 微康益生菌(苏州)股份有限公司 | 鼠李糖乳杆菌LRa05在制备缓解便秘制品或调解肠道菌群制品方面的用途 |
| CN114774313B (zh) * | 2022-04-13 | 2024-01-26 | 微康益生菌(苏州)股份有限公司 | 鼠李糖乳杆菌LRa05在制备缓解便秘制品或调解肠道菌群制品方面的用途 |
| CN116925984A (zh) * | 2023-09-15 | 2023-10-24 | 山东益昊生物科技有限公司 | 一株解淀粉芽孢杆菌yhj32及其复合益生菌制剂和应用 |
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