CN116179443A - 一株缓解便秘的乳酸片球菌nss0302及其应用 - Google Patents
一株缓解便秘的乳酸片球菌nss0302及其应用 Download PDFInfo
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- CN116179443A CN116179443A CN202310234367.9A CN202310234367A CN116179443A CN 116179443 A CN116179443 A CN 116179443A CN 202310234367 A CN202310234367 A CN 202310234367A CN 116179443 A CN116179443 A CN 116179443A
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- pediococcus acidilactici
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- acidilactici
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Abstract
本发明公开了一株缓解便秘的乳酸片球菌NSS0302及其应用。所述乳酸片球菌NSS0302的分类命名为乳酸片球菌Pediococcus acidilactici,保藏编号为CCTCC NO:M 20221643,核苷酸序列如SEQ ID No.1所示。所述乳酸片球菌NSS0302无致病性,不具有急性毒性,安全可靠,且经实验验证,乳酸片球菌NSS0302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,具体涉及一株缓解便秘的乳酸片球菌NSS0302及其应用。
背景技术
罗马标准强调了患者主诉便秘的各种症状,包括排便费力,硬大便,排便不完全感觉,肛门直肠梗阻或阻塞的感觉,手工操作促排便,并且每周排便不足3次。在过去的6个月中,至少有3个月出现这些症状中的任何两个,就可以诊断为功能性便秘(functionalconstipation,FC)。
随着饮食结构的改变及社会因素的影响,功能性便秘的发病率显著上升,对人类健康构成严重威胁,其是生物-心理-社会医学模式下的心身疾病之一,严重影响患者的身体健康、精神心理状况及生活质量。
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功能性便秘,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缓解便秘的乳酸片球菌NSS0302及其应用。所述乳酸片球菌NSS0302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:
本发明提供了一株缓解便秘的乳酸片球菌NSS0302,其分类命名为乳酸片球菌Pediococcus acidilactici,保藏编号为CCTCC NO:M 20221643。
进一步的,所述乳酸片球菌NSS0302的核苷酸序列如SEQ ID No.1所示。
本发明还提供了一种乳酸片球菌菌粉,其是由所述的乳酸片球菌NSS0302制备而成的。
进一步的,所述乳酸片球菌菌粉的制备方法为:将所述乳酸片球菌NSS0302活化后,以3%(v/v)的接种量接种于培养基中,37℃-40℃,培养16h-24h得到的发酵液,经低温离心、清洗后,收集菌泥与冻干保护剂以1:2的质量体积比混匀后,干燥过筛,得到乳酸片球菌菌粉。
本发明还提供了所述的乳酸片球菌NSS0302在抑制病原菌中的应用。
进一步的,所述病原菌为大肠杆菌、沙门氏菌、金黄色葡萄球菌、单增李斯特菌和志贺氏菌。
本发明还提供了所述的乳酸片球菌NSS0302在制备生产短链脂肪酸的微生物制剂中的应用。
进一步的,所述短链脂肪酸为丙酸、丁酸、异丁酸和异戊酸。
本发明还提供了所述的乳酸片球菌NSS0302在制备缓解或治疗功能性便秘的保健品或药品中的应用。
进一步的,所述乳酸片球菌NSS0302通过增加排便数量和粪便含水量来起到缓解或治疗功能性便秘的作用。
进一步的,所述保健品或药品中含有菌含量不少于1×107CFU/mL的乳酸片球菌NSS0302。
与现有技术相比,本发明具有以下优点和技术效果:
本发明从四川三台县传统农家发酵豆豉分离到一株全新的乳酸片球菌NSS0302,其安全性强,无致病性,不具有急性毒性。所述乳酸片球菌NSS0302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,促进粪便的排出,进而达到缓解和治疗便秘的效果,尤其是对由结肠平滑肌或神经支配功能障碍引起的,导致神经结肠运动异常引起的功能性便秘,具有很好的治疗效果。另外,乳酸片球菌NSS0302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为乳酸片球菌NSS0302的平板培养照片。
图2为乳酸片球菌NSS0302的菌体镜检照片。
图3为乳酸片球菌NSS0302的抑菌能力结果图。
图4为乳酸片球菌NSS0302的溶血性检测结果图。
图5为安全性试验的肠道组织切片结果图。
图6为小鼠2小时排便颗粒数。
图7为小鼠粪便湿重、干重及含水量情况。
图8为小肠推进实验结果。
图9为粪便不同脂肪酸含量结果。
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:乳酸片球菌NSS0302的分离和鉴定
1、分离筛选
以来源于3份四川三台县传统农家发酵豆豉为样品。将多份豆豉按5%的体积分别接种到MRS液体培养基中,37℃培养16h,无菌试管中加入1.0mL菌液和9.0mL 0.85%无菌生理盐水,稀释成浓度为10-1的菌液,重复上述操作依次将菌液稀释成浓度为10-2、10-3、10-4、10-5四个梯度,用无菌吸管各吸取0.1mL的10-3、10-4、10-5三个浓度梯度的菌液涂于MRS固体培养基上,每个浓度梯度做3个平行试验,37℃静置培养24h。进行菌落形态观察,挑选不同形态的单菌落再次于MRS固体培养基上划线分离,37℃下培养24h,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NSS0302的菌株。
2、形态学观察
(1)将菌株NSS0302接种于MRS固体平板上,37℃恒温培养24h,观察菌落形态特征。结果见图1,菌株NSS0302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NSS0302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NSS0302呈革兰氏阳性,菌体呈现圆球状,对生或四联,少见单生,不成链状排列,革兰氏阳性,兼性厌氧,不产芽孢,无运动性。
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。具体的生理生化特性见表1、2。
表1:生理生化检测结果
表2:糖发酵结果
特征 | 果糖 | 乳糖 | 葡萄糖 | 水杨苷 | 半乳糖 | 麦芽糖 |
结果 | + | + | + | + | + | - |
特征 | 海藻糖 | 松三糖 | 蜜二糖 | 阿拉伯糖 | 纤维二糖 | 木糖 |
结果 | + | - | - | + | + | + |
特征 | 蔗糖 | 菊糖 | 淀粉 | 鼠李糖 | 棉籽糖 | 甘露醇 |
结果 | + | + | + | + | + | - |
特征 | 甘露糖 | 核糖 | 七叶灵 | 苦杏仁苷 | 葡萄糖酸钠 | |
结果 | + | + | + | + | - |
注:+为阳性结果,-为阴性结果。
4、16S rRNA鉴定
提取菌株NSS0302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NSS0302与Pediococcus acidilactici的序列相似性为99.72%,因此结合上述实验结果,判断菌株NSS0302为乳酸片球菌。
将筛选到的菌株NSS0302进行菌种保藏,所述乳酸片球菌NSS0302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:2022年10月24日;乳酸片球菌Pediococcus acidilactici的保藏编号为CCTCC NO:M 20221643。
实施例2:乳酸片球菌NSS0302的安全性评价
1、抑菌性试验
病原指示菌:大肠杆菌(BNCC336902),沙门氏菌(BNCC103134),金黄色葡萄球菌(BNCC186335)和单增李斯特菌(BNCC336877),志贺氏菌(CCTCC AB 91106)。
在LB琼脂板上活化3代,挑取单菌落接种至LB肉汤,于37℃恒温摇床200r/min扩增培养12h,采用琼脂扩散法进行抑菌试验。
抑菌试验结果如图3所示,菌株NSS0302对大肠杆菌、沙门氏菌、金黄色葡萄球菌、单增李斯特菌、志贺氏菌等5种常见的致病菌都表现出很好的抑菌能力,抑菌圈直径为15.69㎜-20.01㎜。
2、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。
普通培养基高压灭菌,冷却至40-50℃,无菌加入新鲜绵羊血,轻晃混匀倾倒成5%脱脂绵羊血的血平板。采用三区划线法,用接种环将菌株NSS0302接种至血平板,倒置血平板于37℃恒温培养箱培养24h,观察有无溶血环产生并判断溶血类型。菌落周围出现的草绿色环被评估为α-溶血;菌落周围形成透明区域被认为是β-溶血,也称为完全溶血;菌落没有发生变化被认为是γ-溶血,即不溶血。
结果如图4显示,乳酸片球菌NSS0302周围无明显溶血圈形成,属于没有毒性的γ-溶血,也说明乳酸片球菌NSS0302无致病性,安全性好。
3、动物经口急性毒理试验
试验采用单因素完全随机设计。40只体重为25±2g的雄性雌性各半小鼠随机分为2组,每组20只,一组为空白对照组,另一组为NSS0302组。适应环境3d后开始试验。空白对照组小鼠每天灌胃无菌生理盐水0.2mL,NSS0302组小鼠每天灌胃乳酸片球菌NSS0302(109CFU/mL,0.2mL),连续灌胃21d,脱颈处死小鼠。
在整个试验期间,每天观察小鼠精神状况,采食与饮水,粪便形态与颜色、体重、脏器指数及肠道组织病理学观察。
结果显示,整个试验期间,所有小鼠精神良好,采食饮水无异常表现,无腹泻及其它疾病表现,各组小鼠的粪便形态和颜色无明显差别,生命体征良好,无死亡现象。NSS0302组小鼠体重在整个试验期间与空白对照组小鼠基本无差别。
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,脏器指数NSS0302组与空白对照组相比无显著差别。小鼠解剖后,腹腔无溃疡、粘连等病理变化,小鼠脏器也未发现淤血、肿大等异常,肠道结构完整(图5),无明显的损伤状况。
整体上看,乳酸片球菌NSS0302对小鼠不具有急性毒性,说明乳酸片球菌NSS0302是安全无害的。
实施例3:乳酸片球菌NSS0302菌粉的制备
将活化的乳酸片球菌NSS0302划线于MRS固体培养基上,37℃培养16h,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9h,经12000rpm,4℃离心,得到菌泥,超纯水清洗后将菌泥加入无菌的冻干保护剂(配方:30g海藻糖,10g脱脂乳粉,0.2g甘氨酸,1g吐温80,1000mL纯水)中,其中保护剂与菌泥的体积质量比为2:1,充分混合,真空冷冻干燥,得到冻干菌粉,再经80目筛网进行摇摆式粉碎,最终得到乳酸片球菌NSS0302冻干菌粉。
乳酸片球菌NSS0302冻干菌粉的活菌数≥6×1011CFU/g。菌粉真空保存在-18℃的冰箱中以备后用。
实施例4:乳酸片球菌NSS0302对功能性便秘小鼠模型的影响
1、小鼠实验模型建立
将40只小鼠按照随机数字表分为空白对照组10只,模型对照组10只,NSS0302组10只,药物阳性组10只。小鼠均可以自由进食及饮水。每逢周一、周三,周五称取小鼠体重一次,作为给药的依据(与成人的药物用量相同,给予生理盐水或复方地芬诺酯混悬液的量=[体重(g)/100]ml),一共给药20天,具体给药方案见表3。其中,将实施例3制备的乳酸片球菌NSS0302菌粉使用生理盐水稀释成菌含量不少于1×107CFU/mL的菌液。
表3:给药方案
2、粪便含水量检测及含水率计算
造模第20天收集小鼠粪便,将新鲜的粪便收集到10ml的离心管中,并记录2小时内的排出颗粒数。使用电子天平称量离心管及其内粪便的总质量。粪便湿重=离心管与粪便的总质量-离心管质量。
收集的粪便置于恒温风干机中,设置通风,温度为80℃。持续烘烤粪便12小时后,把装有烘干后粪便的烘干后10ml离心管置于电子天平上称量,得出烘干后粪便与10ml离心管的总质量。粪便干重=离心管与烘干后粪便的总质量-离心管质量。
粪便含水率=(粪便湿重-粪便干重)/粪便湿重
结果见图6和图7,与模型组相比,药物组和NSS0302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,而药物组与NSS0302组比较无显著性差异,表明乳酸片球菌NSS0302可有效改善健康小鼠的排便参数。
3、墨汁推进试验
小鼠造模第20天9:00开始禁食。在造模第21天,每组在给造模药物30分钟后,分别以灌胃的方式给予碳粉液。碳粉液用量为10mL/kg。从灌胃碳粉液开始计算时间,计算小鼠排出第一粒黑便所需时间。
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%。
结果见图8,与模型组相比,药物组和NSS0302组小鼠小肠推进率都较大,而药物组与NSS0302组比较无显著性差异,表明乳酸片球菌NSS0302和药物均能恢复受到复方地芬诺酯抑制的肠蠕动功能。
4、16S rRNA高通量测序分析肠道菌群
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。
小鼠结果见表4,对照组、NSS0302组和空白组之间的OTUS总数没有显著性差异,但乳酸片球菌NSS0302会刺激使得该组的OTUS总数比其他组略有增加。
表4:肠道菌群测序结果
空白对照组 | 模型组 | S0302组 | 药物对照组 | |
OTUS总数 | 512 | 519 | 523 | 517 |
5、粪便短链脂肪酸影响
采用Agilent 7890-5977GC-MS系统进行气相色谱-质谱分析。为定量检测短链脂肪酸,构建了0.1-100ug/ml浓度范围的校准曲线。IS用于校正样品之间的注射变异和仪器响应的微小变化。
结果见图9,与模型组相比,其余3组的脂肪酸含量明显增加,NSS0302组的丙酸、丁酸,尤其是异戊酸相较于对照组和药物组均显著增加,而异丁酸浓度稍微增加,但不明显。
综上所述,乳酸片球菌NSS0302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。
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)
1.一株缓解便秘的乳酸片球菌NSS0302,其特征在于,所述乳酸片球菌NSS0302的分类命名为乳酸片球菌Pediococcus acidilactici,保藏编号为CCTCCNO:M 20221643。
2.根据权利要求1所述的乳酸片球菌NSS0302,其特征在于,所述乳酸片球菌NSS0302的核苷酸序列如SEQ ID No.1所示。
3.一种乳酸片球菌菌粉,其特征在于,所述乳酸片球菌菌粉是由权利要求1所述的乳酸片球菌NSS0302制备而成的。
4.根据权利要求3所述的乳酸片球菌菌粉,其特征在于,所述乳酸片球菌菌粉的制备方法为:将所述乳酸片球菌NSS0302活化后,以3%v/v的接种量接种于培养基中,37℃-40℃,培养16h-24h得到的发酵液,经低温离心、清洗后,收集菌泥与冻干保护剂以1:2的质量体积比混匀后,干燥过筛,得到乳酸片球菌菌粉。
5.权利要求1或2所述的乳酸片球菌NSS0302在抑制病原菌中的应用。
6.根据权利要求5所述的乳酸片球菌NSS0302在制备抑制病原菌中的应用,其特征在于,所述病原菌为大肠杆菌、沙门氏菌、金黄色葡萄球菌、单增李斯特菌和志贺氏菌。
7.权利要求1或2所述的乳酸片球菌NSS0302在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所述的乳酸片球菌NSS0302在制备生产短链脂肪酸的微生物制剂中的应用,其特征在于,所述短链脂肪酸为丙酸、丁酸、异丁酸和异戊酸。
9.权利要求1或2所述的乳酸片球菌NSS0302在制备缓解或治疗功能性便秘的保健品或药品中的应用。
10.根据权利要求9所述的乳酸片球菌NSS0302在制备缓解或治疗功能性便秘的保健品或药品中的应用,其特征在于,所述保健品或药品中含有菌含量不少于1×107CFU/mL的乳酸片球菌NSS0302。
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