CN116987626A - 一种副干酪乳杆菌ap14及其在肠道调节与尿酸代谢的应用 - Google Patents
一种副干酪乳杆菌ap14及其在肠道调节与尿酸代谢的应用 Download PDFInfo
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Abstract
本发明公开了一种副干酪乳杆菌AP14及其在肠道调节与尿酸代谢的应用。本发明所提供的副干酪乳杆菌具体为副干酪乳杆菌AP14(Lactobacillus paracasei AP14),它在中国微生物菌种保藏管理委员会普通微生物中心的保藏编号为CGMCC No.25462。本发明的副干酪乳杆菌AP14(Lactobacillus paracasei AP14)CGMCC No.25462具有良好的降尿酸功能,同时能够抑制肠道中有害细菌的生长,调节肠道菌群,促进肠道中嘌呤和尿酸的分解代谢,调控尿酸代谢。同时,本发明的副干酪乳杆菌可制成菌剂用于预防或治疗高尿酸血症、痛风,是一种绿色、安全的菌剂。
Description
技术领域
本发明属于生物技术领域,尤其涉及一种副干酪乳杆菌AP14及其在肠道调节与尿酸代谢的应用。
背景技术
随着经济的飞速发展,人们的生活方式发生了巨大的改变。其中,愈来愈丰盛的饮食使得人们对高蛋白、高脂肪的摄入不断增加,从而提高了许多疾病发生的几率,如:高尿酸血症。长期的高尿酸会引起痛风、高血压、心血管疾病及糖尿病等相关代谢障碍及疾病。痛风已成为我国仅次于糖尿病的第二大代谢性疾病,严重威胁着人们的健康。市场上常用的治疗高尿酸血症的方法主要是服用合成药物,包括别嘌呤醇、非布司他等,但这类药物长期服用会损伤肠胃、肾脏,引起红斑、光过敏等诸多不良影响,这些药物的副作用越发引起人们的担忧。
人体中有三分之一的尿酸是在肠道中通过细菌降解而排出体外的,有文献报道人体是由10%的体细胞和90%的微生物组成,而在人消化***聚集了绝大多数人体的微生物,而肠道菌群承担了大部分转运至肠腔的尿酸的分解。有研究报道在中国人群中高血酸和痛风患者的肠道微生物相比正常的人菌群结构发生了明显变化,痛风的人群中其肠道菌富含Bacteroides caccae和Bacteroides xylanisolvens这两种菌,但是Faecalibacterium prausnitzii和Bidobacterium pseudocatenulatum显著性缺失。此外的许多研究也都证实了人体肠道菌群的变化与高尿酸的发生有密切关系,因此,肠道菌群是降低尿酸的一股强大力量。肠道菌群是相互作用的,可以通过群体感应调控来调节其结构。另外,细菌的氮代谢也可能是通过群体感应进行调控的。所以,利用益生菌对肠道菌群结构进行调整来调控尿酸代谢从而达到一定的降低血液尿酸含量以及高尿酸炎症是可行的,在未来高尿酸血症及痛风等相关领域将有广阔前景。
发明内容
本发明的目的是提供一种副干酪乳杆菌AP14及应用。我们从南极企鹅肠道中筛选得到的副干酪乳杆菌AP14具有良好的降尿酸功能,同时能够抑制肠道中有害细菌的生长,调节肠道菌群。此外,通过海藻多糖及羧甲基纤维素钠进行菌剂包埋缓释,两种多糖能够选择性地利于有益的肠道微生物的生长,其分解产生的短链脂肪酸也是重要的益生元,菌剂和多糖结合在一起能够产生更多的功能特性,维持人体健康。
根据以上内容,本项目以小鼠作为动物模型,利用具有高效降尿酸和抑菌功能益生菌,利用群体感应调控调节肠道微生物结构和氮代谢,从而促进肠道中嘌呤和尿酸的分解代谢,调控尿酸代谢。
为了实现本发明的上述目的,采用了以下技术方案:
一种副干酪乳杆菌AP14,该副干酪乳杆菌保藏于中国普通微生物菌种保藏管理中心(简称:CGMCC,北京市朝阳区北辰西路1号院3号),其保藏编号为CGMCC No.25462。
上述的副干酪乳杆菌的培养物。
上述的副干酪乳杆菌或副干酪乳杆菌的培养物在制备菌剂中的应用。
进一步地,上述副干酪乳杆菌或其培养物用于调节肠道菌群和/或预防或治疗高尿酸血症、痛风。
进一步地,上述副干酪乳杆菌总活菌数不低于108CFU。
一种菌剂,所述菌剂包括上述副干酪乳杆菌或副干酪乳杆菌的培养物。
进一步地,上述副干酪乳杆菌总活菌数不低于108CFU。
术语“培养物”是指经人工接种和培养后,长有微生物群体的液体或固体产物(培养容器内的所有物质,发酵产物)的统称。即通过将微生物进行生长和/或扩增而获得的产物,其可以是微生物的生物学纯培养物,也可以含有一定量的培养基、代谢物或培养过程中产生的其他成分。
与现有技术相比,本发明的有益效果为:
本发明所提供的副干酪乳杆菌AP14能够显著降低降尿酸水平,显著缓解高尿酸血症,使血尿酸水平基本恢复正常水平,显著减缓高尿酸引发的炎症反应,提高免疫力,缓解高尿酸血症带来的不良影响,同时,还可使肠道微生物菌群中典型益生菌的丰度提高,改善肠道菌群结构,是一种安全、有效的益生菌。
保藏说明
保藏地址: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保藏日期:2022年8月1日
菌种名称:副干酪乳杆菌
拉丁名:Lactobacillus paracasei
菌株编号:AP14
保藏机构:中国微生物菌种保藏管理委员会普通微生物中心
保藏机构简称:CGMCC
保藏中心登记入册编号:CGMCC No.25462
附图说明
下面参照附图结合实施例对本发明作进一步的说明。
图1为本发明副干酪乳杆菌AP14菌株***发育进化树。
图2为企鹅肠道样品处理后涂布于尿酸平板37℃培养后菌落生长情况图。
图3为分离出的副干酪乳杆菌AP14菌株于尿酸平板37℃培养后菌落生长情况图。
图4为副干酪乳杆菌AP14菌株24h内的生长和尿酸代谢曲线。
图5为不同组大鼠尿酸水平随时间变化曲线。
图6为养殖15天内大鼠肌酐浓度变化曲线。
图7为大鼠体重、进食量、饮水量变化曲线。
图8为副干酪乳杆菌AP14菌株血平板图。
图9为0-18h副干酪乳杆菌AP14中试发酵产酸曲线。
具体实施方式
为了更好地理解本发明,下面结合实施例和附图对本发明做进一步的详细说明,但本领域技术人员了解,下述实施例不是对本发明保护范围的限制,任何在本发明基础上做出的改变和变化,都在本发明的保护范围之内。
下述实施例中所使用的实验方法如无特殊说明,均为常规方法。
下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例1降尿酸益生菌的筛选及鉴定。
(一)实验材料
样品
本专利的副干酪乳杆菌(Lactobacillus paracasei)筛选自南极西摩海滩企鹅肠道样品,为产乳酸芽孢杆菌属,保藏于中国微生物菌种保藏中心,保藏号为CGMCCNo.25462。
培养基
尿酸固体培养基:取硫酸镁0.5g/L,氯化钠0.1g/L,三水合磷酸氢二钾0.5g/L,磷酸二氢钾0.5g/L,尿酸2g/L,琼脂20g/L,再利用10mol/L NaOH溶液调节pH到7;121℃高温高压蒸汽灭菌20min,倒板,存于4℃冰箱。
MRS培养基:取蛋白胨10g/L,牛肉膏8g/L,酵母膏4g/L,葡萄糖20g/L,磷酸氢二钾2g/L,柠檬酸三氨2g/L,三水合乙酸钠5g/L,七水合硫酸镁0.2g/L,四水合硫酸锰0.05g/L,吐温80 1g/L,再利用10mol/L NaOH溶液调节pH到6.2;121℃高温高压蒸汽灭菌20min,倒板,存于4℃冰箱中备用。
LB培养基:取酵母膏5g/L,蛋白胨10g/L,氯化钠10g/L,去离子水1000mL,若需固体培养基则再加入20g/L琼脂,再利用10mol/L NaOH溶液调节pH到7;121℃高温高压蒸汽灭菌20min,4℃冰箱中备用。
(二)菌种筛选
取3g样品于50mL塑料离心管中,加入20mL无菌水后,置于震荡仪上充分震荡。震荡完成后,使用高速冷冻离心机,于4℃,8000r/min离心5分钟后,取100μL涂布于尿酸固体培养基中,置于37℃恒温培养箱中培养24h。将长出的单菌落划线于MRS平板,37℃培养,进行多次分离纯化直至长出形态一致的单菌落,命名为AP14。
(三)菌种鉴定
16S rDNA基因序列***进化分析
16S rDNA基因PCR扩增
以基因组DNA为模板PCR扩增16S rDNA基因,扩增引物采用27F(5'-AGAGTTTGATCCTGGCTCAG-3')(如SEQ ID NO:2所示)和1492R(5'-TACGACTTAACCCCAATCGC-3')(如SEQ ID NO:3所示)(均由南京金斯瑞生物科技有限公司合成)。PCR反应体系(20μL):引物各1μL,Prime STAR酶10μL,DNA模板8μL,ddH2O补足至20μL。PCR反应程序为:95℃预变性30s;95℃变性10s,55℃退火15s,72℃延伸90s,32个循环,最后72℃5min,扩增的PCR产物在1%琼脂糖凝胶上进行电泳检测,回收无杂带样品。
(四)序列比对与***进化分析
将上述无杂带样品送至生工生物工程(上海)股份有限公司进行测序。将得到的测序结果的序列拼接,在NCBI数据库(https://blast.ncbi.nlm.nih.gov/Blast.cgi)中进行比对,根据细菌16S rRNA基因序列相似性,利用MEGA 7.0软件进行***进化分析。副干酪乳杆菌AP14的16S rDNA序列(如SEQ ID NO:1所示)如下:
CTACTACATGCAGTCGAACGAGTTCTCGTTGATGATCGGTGCTTGCACCGAGATTCAACATGGAACGAGTGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCTTAAGTGGGGGATAACATTTGGAAACAGATGCTAATACCGCATAGATCCAAGAACCGCATGGTTCTTGGCTGAAAGATGGCGTAAGCTATCGCTTTTGGATGGACCCGCGGCGTATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCGATGATACGTAGCCGAACTGAGAGGTTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTGGAGAAGAATGGTCGGCAGAGTAACTGTTGTCGGCGTGACGGTATCCAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCCTCGGCTTAACCGAGGAAGCGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAATGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCTTTTGATCACCTGAGAGATCAGGTTTCCCCTTCGGGGGCAAAATGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATGACTAGTTGCCAGCATTTAGTTGGGCACTCTAGTAAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTTGCGAGACCGCGAGGTCAAGCTAATCTCTTAAAGCCATTCTCAGTTCGGACTGTAGGCTGCAACTCGCCTACACGAAGTCGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCGAAGCCGGTGGCGTAACCCTTTTAGGGAGCGAGCCGTCTAAGTGACTAT
菌株AP14的测序结果与NCBI数据库中已知标准菌株比对表明,该菌株与副干酪乳杆菌的同源性最高,达100%,AP14菌株***发育进化树见图1。
(五)全基因组测序与功能分析
用50mL MRS液体培养基以37℃、200rpm隔夜培养AP14菌株至OD600nm=1.0,以8000rpm离心10min,去除上清液,将菌体送广东美格基因科技有限公司(美格基因)进行基因组测序及分析。
实施例2体外实验的功效验证
(一)平板对峙实验
企鹅样品涂布于尿酸平板培养后的图片,如图2。如图2所示,企鹅样品中存在可降解尿酸的菌株。从图中可以看到有明显的白色菌落,同时部分菌落旁还形成了尿酸降解圈。将上述平板中的菌落分别挑取到新的尿酸平板上,划线分离纯化得到单菌落。共得到12株可降解尿酸的菌株。图3为部分菌株在尿酸平板上的生长情况。结果显示分离出来的单株菌也存在尿酸降解能力,部分菌株的菌落旁还存在降解圈。
(二)生长代谢曲线与尿酸代谢曲线
1.菌株的活化
从冰箱中找到保存的甘油管,用接种环划线到MRS平板上,置于37℃恒温培养36h至有单菌落生成;挑取单菌落接种到7mL液体MRS培养基中,置于37℃摇床培养20h至平台期。
2.接种
取高压蒸汽灭菌后的50mL离心管,每根离心管中加入14.25mL LB液体培养基,600μl 20mmol/l的尿酸标准样品,150μl隔夜培养的菌液,置于37℃摇床培养,每株菌做3个平行组。
3.菌体浓度的测定
从接种为0h开始,每隔2h取200μl菌液于96孔板中,置于酶标仪测定OD595。减去空白培养基的OD595后,按照OD595从0到1对应的菌体浓度为108CFU到109CFU计算测定时刻的菌体浓度。测定到24h时结束。
4.尿酸含量的测定
从接种为0h开始,每隔2h取100μl菌液,采用尿酸含量检测试剂盒测定菌液中的尿酸含量。测定到24h时结束,结果如图4所示。菌株AP14前0到8h处于延迟期,尿酸含量先升后降,8h时降低了接近100μmol/L;8到20h属于生长期,尿酸含量回升后又稍微下降;20到24h处于稳定期,尿酸含量在此阶段下降很快,从20h的700μmol/L降至550μmol/L左右,24h内总体降解比例达到了31%。
实施例3大鼠动物实验
选用40只雄性SD大鼠,共4组,每组10只,编号为A、B、C、D组,分别对应对照组、模型组、益生菌组、治疗组,具体处理如下:
表1小鼠养殖分组及处理表
(一)大鼠尿酸水平变化情况
于给药第0、3、6、9、12、15天,中途取血制血清,用尿酸含量检测试剂盒(Solarbio)测定不同处理组大鼠血清中尿酸浓度,对比不同时间不同组间大鼠血清中尿酸浓度差异变化,结果如图5所示。结果显示,高尿酸模型组的尿酸水平显著高于对照组,说明高尿酸模型建立成功。而治疗组尿酸水平处于对照组和模型组之间,说明使用AP14能够显著缓解高尿酸血症。
(二)大鼠肌酐水平变化情况
于给药第0、3、6、9、12、15天,中途取血制血清,每组5只,用检测试剂盒(南京建成,中国)测定小鼠血清中肌酐(CRE)含量测定,结果如图6所示。由图6可知,对照组和益生菌组的肌酐含量相对稳定,无太大波动,说明益生菌对大鼠肾小球的滤过作用没有产生不良影响。而高尿酸模型组和AP14治疗组的肌酐波动较大,且含量较高,说明高尿酸使大鼠的肾小管滤过作用受到较大影响,大鼠肾功能受损,而在此情况下添加AP14益生菌也难以修复高尿酸对肾脏的损伤。
(三)大鼠体重、进食量、饮水量变化情况
将实验大鼠进行分组,每3天检查各组大鼠的体重、平均饮水和食物摄入量。验证菌株AP14对大鼠体重、进食和饮水方面的影响,结果如图7所示。由图7分析可见,0-15天内,高尿酸模型大鼠的体重、进食量和饮水量均远远低于对照组,且为四组中最低;单独使用AP14菌株的大鼠体重、进食量和饮水量与对照组基本一致,说明AP14菌株不会对大鼠本身产生不良影响;而同时进行药饮和AP14灌胃的治疗组大鼠的体重、进食量和饮水量都比高尿酸模型组有一定程度的提高,说明在有高尿酸血症的情况下,食用AP14菌株可以在一定程度上缓解高尿酸血症给大鼠带来的不良影响,表面AP14菌株具有较好的治疗效果。
实施例4血平板溶血实验
配制血平板:15%琼脂,0.85%的氯化钠;121℃灭菌20min后冷却到50℃,无菌地加入已灭菌的血细胞溶液(每10ml琼脂加0.5ml)。用手旋转或翻转试管使其混合,倒入培养皿中,冷却凝固。
在血平板上划线副干酪乳杆菌AP14,于37℃恒温培养箱培养24h,取出,观察溶血情况。
结果如图8所示,菌落周围未出现明显环带,说明AP14菌株不会造成溶血。
实施例5中试发酵产酸实验
在200L中试发酵罐中按60%装液量装入MRS液体培养基,调节pH至6.8,高温灭菌后,按2%(v/v)接入活化的AP14菌株种子液。取样测定发酵0h、3h、6h、9h、12h、15h和18h的OD600nm和发酵液pH,用Origin软件绘制0-18h AP14中试发酵产酸曲线(图9)。结果显示,随着发酵的进行,菌液PH值逐渐降低并稳定在5.7的弱酸范围,说明副干酪乳杆菌的发酵代谢产酸特性,可在肠道工作下形成弱酸环境,抑制病原菌的致病性。
虽然以上描述了本发明的具体实施方式,但是熟悉本技术领域的技术人员应当理解,我们所描述的具体的实施例只是说明性的,而不是用于对本发明的范围的限定,熟悉本领域的技术人员在依照本发明的精神所作的等效的修饰以及变化,都应当涵盖在本发明的权利要求所保护的范围内。
Claims (7)
1.一种副干酪乳杆菌,其特征在于,所述副干酪乳杆菌为副干酪乳杆菌AP14(Lactobacillus paracasei AP14),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.25462。
2.如权利要求1所述的副干酪乳杆菌的培养物。
3.如权利要求1所述的副干酪乳杆菌或权利要求2所述的副干酪乳杆菌的培养物在制备菌剂中的应用。
4.如权利要求3所述的应用,其特征在于,所述副干酪乳杆菌或其培养物用于调节肠道菌群和/或预防或治疗高尿酸血症、痛风。
5.如权利要求4所述的应用,其特征在于,所述副干酪乳杆菌总活菌数不低于108CFU。
6.一种菌剂,其特征在于,所述菌剂包括权利要求1所述的副干酪乳杆菌或权利要求2所述的副干酪乳杆菌的培养物。
7.如权利要求6所述的菌剂,其特征在于,所述副干酪乳杆菌总活菌数不低于108CFU。
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