CN114507621B - 一株植物乳杆菌及在降尿酸、减重、抗炎症方面的应用 - Google Patents
一株植物乳杆菌及在降尿酸、减重、抗炎症方面的应用 Download PDFInfo
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Abstract
本发明公开了一株植物乳杆菌及在降尿酸、减重、抗炎症方面的应用。该植物乳杆菌名称为植物乳杆菌(Lactobacillus plantarum)FS4722,保藏编号为CGMCC No.22750,该菌株于2021年6月21日保藏于北京市朝阳区北辰西路1号院3号的中国微生物菌种保藏管理委员会普通微生物中心。本发明中通过实验发现:本发明筛选获得的植物乳杆菌FS4722具有降尿酸、抗痛风、减重、抗炎症的作用,还可以改善肠道菌群多样性,提高肠道乙酸和丙酸的含量,因此,可将其用于开发具有降尿酸、抗痛风、减重、抗炎症等作用的发酵食品、保健品和药品。
Description
技术领域
本发明属于活性微生物基功能性食品技术领域,特别涉及一株植物乳杆菌及在降尿酸、减重、抗炎症方面的应用。
背景技术
相关研究表明,高尿酸血症的全球患病率在7~36%之间,而痛风的全球患病率在0.5~10.3%之间。其中,美国和澳大利亚痛风和高尿酸血症的患病率已到达平稳期,而其他大多数国家(包括发达国家和发展中国家)的患病率仍然在持续上升。而且,男性高尿酸血症和痛风的患病率明显高于女性,为女性的1~21倍。显然,高尿酸血症和痛风已成为常见的炎症性疾病,属于医疗健康的全球公共问题。
高尿酸血症是一种由于嘌呤代谢障碍引起的血尿酸水平异常升高的代谢性疾病,国内诊断标准是空腹血尿酸水平男性超过7.0mg/dL(416.0μmol/L),女性超过6.0mg/dL(357.0μmol/L)。人体嘌呤代谢紊乱或摄入过多嘌呤核苷酸类食物都会引起血清尿酸浓度异常升高。尿酸在血液中主要以尿酸盐的形式存在,其在生理温度和pH下(37℃,pH 7.4)的饱和点为392μmol/L(6.6mg/dL),浓度达到或超过饱和点时会以晶体形式析出并沉积在关节处,由此形成痛风。痛风病的发生常伴有四肢及身体不适的症状,并可诱发肾功能不全、高血压、糖尿病及冠心病等综合性病症,重症者可引起关节炎症反复发作,疼痛难忍,并形成痛风石造成关节畸形,给患者带来巨大痛苦,直接影响生活质量,甚至有可能危及生命。
由于高尿酸血症和痛风是一种病程慢长的退行性疾病,受饮食、生活习惯、环境等因素影响,具有糖尿病、肥胖等诱因复杂的特点,以防为主、防治结合、综合调理是痛风疾病临床的共识。目前主要通过长期服用降尿酸药物,达到血尿酸控制标准,但现有药物的使用往往会对人体带来不同程度的毒副作用。开发毒性低、副作用小、能够长期服用的安全新型药物是当前的研究热点。
益生菌是一种通过改善肠道微生物平衡从而对宿主施加有益影响的微生物添加物。目前,已有研究表明高尿酸血症和痛风患者的肠道菌群与健康人群相比存在显著差异。通过益生菌调节肠道菌群和血代谢指标成为缓解高尿酸血症和痛风的一种新的治疗手段。目前,植物乳杆菌UA149(CN110055199A-一株植物乳杆菌UA149株及其应用)和短乳杆菌(Lactobacillus brevis)DM9218(邓英,何春阳,唐艳,等.短乳杆菌DM9218对高果糖饮食诱导的小鼠高尿酸血症的缓解作用及机制研究[J].中国微生态学杂志,2017,29(12):4.)的降血尿酸作用已被证实,但其需连续口服分别15和56天后才发挥降尿酸效果,前者下降尿酸的幅度最多61.2umol/L,后者最多50umol/L,效果较差。因此,提供一种降尿酸作用更好的益生菌,在开发降尿酸、抗痛风的食品、保健品和药品中具有更大的应用潜力。
发明内容
本发明的第一个目的在于克服现有技术的缺点与不足,提供一株植物乳杆菌。
本发明的第二个目的在于提供一种培养所述植物乳杆菌的方法。
本发明的第三个目的在于提供一种植物乳杆菌胞内酶。
本发明的第四个目的在于提供所述的植物乳杆菌在制备发酵食品中的应用。
本发明的第五个目的在于提供一种含植物乳杆菌的发酵制品。
本发明的第六个目的在于提供所述的植物乳杆菌、植物乳杆菌胞内酶及含植物乳杆菌的发酵制品在制备防治高尿酸血症(降尿酸)、抗痛风、减重,抗炎症、改善肠道菌群多样性、提高肠道乙酸和/或丙酸的含量的产品中的应用。
本发明的目的通过下述技术方案实现:
一株植物乳杆菌,名称为植物乳杆菌(Lactobacillus plantarum)FS4722,保藏编号为CGMCC No.22750,该菌株于2021年6月21日保藏于北京市朝阳区北辰西路1号院3号的中国微生物菌种保藏管理委员会普通微生物中心(简称中国微生物菌种保藏中心)。
一种培养所述植物乳杆菌的方法,具体步骤为:将所述的植物乳杆菌接种于培养基中,于28℃~37℃条件下进行培养。
所述的培养基为MRS肉汤培养基。
所述的培养的pH值优选为6.5。
所述的培养的时间优选为12~20小时。
一种植物乳杆菌胞内酶,通过如下方法制备得到:将上述植物乳杆菌接种到培养基中,于28℃~37℃条件下培养12~20小时,离心收集菌体,生理盐水清洗后,加水稀释细胞,再超声破碎细胞,最后在4℃条件下离心收集细胞内容物,过滤,得到植物乳杆菌胞内酶。
所述的培养的条件优选为:37℃培养20小时。
所述的离心收集菌体的条件为:8000r离心5min。
所述的加水稀释后的细胞浓度优选为106CFU/mL。
所述的超声破碎细胞的时间优选为:20min。
所述的离心收集细胞内容物的条件为:12000rpm离心20min。
所述的过滤为采用0.22um的滤膜进行过滤。
所述的植物乳杆菌在制备发酵食品中的应用。
所述的发酵食品使用上述植物乳杆菌发酵生产制得,包括固态、液态和半液态发酵食品。
所述的发酵食品包括果蔬品、乳制品和豆制品。
所述的乳制品包括牛奶、酸奶、黄油、奶酪等。
所述果蔬品包括苹果、雪梨、萝卜、黄瓜、卷心菜、大白菜、大蒜、姜片、洋葱等。
一种含植物乳杆菌的发酵制品,通过使用上述植物乳杆菌发酵生产制得。
所述的含植物乳杆菌的发酵制品在口服期间能持续降低血尿酸水平;所述含植物乳杆菌的发酵制品能降低肝细胞中甘油三酯含量,具有减重作用;所述含植物乳杆菌的发酵制品能降低血清中IL-1β、IL-6和TNF-α水平,具有抗炎作用;所述含植物乳杆菌的发酵制品能提高肠道菌群多样性,修复肠道菌群结构,提高肠道乙酸和丙酸含量,上调肠道紧密连接蛋白的转录水平,由此提高肠道的免疫屏障;所述含植物乳杆菌的发酵制品服用15天即可改善肠道菌群多样性,提高肠道乙酸和丙酸的含量,降低肥胖、高尿酸血症、痛风和炎症的患病风险。
所述的植物乳杆菌、植物乳杆菌胞内酶以及含植物乳杆菌的发酵制品中的至少一种在制备防治高尿酸血症(降尿酸)、抗痛风、减重(降低肥胖)和/或抗炎症的产品中的应用;
所述的产品包括功能性食品、保健品或药物。
所述的植物乳杆菌通过抑制嘌呤吸收达到降低血尿酸水平的目的。
所述的植物乳杆菌通过抑制嘌呤吸收以降低血尿酸水平,进而达到抗痛风的目的。
所述的减重通过降低肝脏细胞内的胆固醇和甘油三酯水平,进而实现减重的目的;具体通过如下方式实现:所述植物乳杆菌能够降解胆固醇,由此降低小肠对胆固醇的吸收,降低肝脏细胞内甘油三酯含量,达到减重的目的;或所述的植物乳杆菌胞内酶能降低肝脏细胞内的胆固醇和甘油三酯水平,进而达到减重的目的。
所述的抗炎症的产品包括能够清除肝脏组织中的炎性细胞的产品,或能够降低血清中促炎因子IL-1β、IL-6和TNF-α表达的产品。
所述的植物乳杆菌通过清除肝脏组织中的炎性细胞达到抗炎症的目的,和/或植物乳杆菌通过降低血清中的促炎因子IL-1β、IL-6和TNF-α的浓度,达到缓解炎症的目的。
所述的植物乳杆菌、植物乳杆菌胞内酶以及含植物乳杆菌的发酵制品中的至少一种在制备改善肠道菌群多样性、提高肠道乙酸和/或丙酸的含量的产品中的应用,所述的植物乳杆菌能够提高小肠紧密连接蛋白Occludin的转录水平,修复肠道粘膜屏障;所述的植物乳杆菌能够修复高尿酸血症的肠道菌群失衡,上调肠道菌群的α-多样性;能够提高产乙酸和丙酸的Alistipes属、Bacteroides属丰度,进而上调乙酸和丙酸的含量。
本发明相对于现有技术具有如下的优点及效果:
1、本发明的植物乳杆菌(Lactobacillus plantarum)FS4722株系从广州菜市场售的酸菜中筛选而来的有益活性微生物菌剂,具有以下生物学特征:(1)菌体特征:革兰氏阳性菌,不形成孢子的细菌;乳白色、圆形、边缘整齐、不透明、表面光滑;(2)生长特征:最适生长温度37℃,最适生长pH6.5,摇床培养20h后进入稳定期;(3)在高尿酸小鼠模型中能够显著降低血尿酸水平:植物乳杆菌FS4722能够抑制嘌呤吸收,3天即可显著降低小鼠的血尿酸水平;(4)在高尿酸小鼠模型中能显著降低血清中炎症因子IL-1β、IL-6和TNF-α水平,缓解炎症;(5)全细胞可降解胆固醇,胞内酶能降低肝脏细胞内的胆固醇和甘油三酯水平:植物乳杆菌FS4722能够降解胆固醇,由此降低小肠对胆固醇的吸收,降低肝脏细胞内甘油三酯含量,能降低小鼠体重,具有减重效果;(6)在高尿酸小鼠模型中能清除高尿酸血症或痛风小鼠肝脏细胞内的炎性细胞;(7)植物乳杆菌FS4722能提高小肠紧密连接蛋白Occludin的转录水平,修复肠道粘膜屏障,在高尿酸小鼠模型中能够修复高尿酸血症小鼠的肠道菌群失衡,提高肠道菌群α-多样性,逆转肠道菌群结构;提高肠道乙酸和丙酸的Alistipes、Bacteroides属丰度,以此提高肠道乙酸和丙酸的含量,降低机体炎症、高尿酸血症、痛风等患病风险。
2、本发明的植物乳杆菌FS4722口服3天即可达到降尿酸目的,下降幅度接近94umol/L,尤其第12天的效果突出,下降幅度高达154umol/L,在开发降尿酸、抗痛风的食品、保健品和药品中具有更大的应用潜力。
3、本发明所述的植物乳杆菌FS4722能够用于制备具有减重、抗炎症等功能的食品、保健品和药品,还可以用于发酵制备发酵食品,包括固态、液态和半液态食品,具有非常广泛的应用前景。
附图说明
图1是小鼠第3、6和12天的血尿酸水平统计图(图中,Δ尿酸代表高尿酸模型组与FS4722干预组小鼠血尿酸值平均值之差;****表示P<0.0001,ns表示P>0.05);其中,A为小鼠第3天的血尿酸水平;B为小鼠第6天的血尿酸水平;C为小鼠第12天的血尿酸水平。
图2是FS4722减重潜力评价结果图(图中,****表示P<0.0001,***表示P<0.001,**表示P<0.01,*表示P<0.05,ns表示P>0.05);其中,A为胆固醇降解能力;B为HepG2细胞内甘油三酯含量变化;C为小鼠体重变化。
图3是FS4722对小鼠炎症的影响图(图中,***表示P<0.001,**表示P<0.01,*表示<0.05);其中,A为小鼠肝脏组织病理切片;B为血清中炎症因子IL-1β水平;C为血清中IL-6水平;D为血清中TNF-α水平。
图4是FS4722对小鼠肠道菌群的影响图(图中,****表示P<0.0001,**表示P<0.01,*表示P<0.05,ns表示P>0.05);其中,A为Shanon指数;B为Chao指数;C为NMDS非度量多维尺度分析图。
图5是FS4722对肠道代谢产物及相关菌群丰度的影响图(图中,****表示P<0.0001,**表示P<0.01,*表示P<0.05,ns表示P>0.05);其中,A为粪便中乙酸含量;B为粪便中丙酸含量;C为Alistipes属丰度;D为Bacteroides属丰度;E为occludin转录水平。
具体实施方式
下面结合实施例对本发明作进一步详细的描述,但本发明的实施方式不限于此。除非特别说明,本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。下列实施例中未注明具体实验条件的试验方法,通常按照常规实验条件。除非特别说明,本发明所用试剂和原材料均可通过市售获得。
实施例1植物乳杆菌FS4722的筛选、分离纯化以及鉴定
(一)菌株FS4722的筛选方法,步骤包括:
(1)取100uL天然发酵的泡菜汁(来自广州菜市场售的酸菜),加入900uL 0.85%无菌生理盐水,摇匀后作为母液。在1.5mL离心管内加入100uL母液,加入900uL上述无菌生理盐水,使母液稀释10倍。依此类推,当母液稀释至106时,取100uL悬浮液于固体MRS平板(MRS肉汤培养基购于广东环凯微生物科技有限公司,其配方如下:每升含酪蛋白酶消化物10g,牛肉膏粉10g,酵母膏粉4g,柠檬酸三铵2g,乙酸钠5g,七水硫酸镁0.2g,四水硫酸锰0.05g,磷酸氢二钾2g,葡萄糖20g,吐温-80 1.08g,pH 5.7±0.2。使用方法:取54g,加入1L去离子水,完全溶解后,121℃高压灭菌15min。MRS固体培养基是在MRS肉汤培养基中加入琼脂粉而得,每100mL加入0.4g琼脂粉)(含0.001%溴甲酚紫指示剂)上,用涂布棒顺时针方向推开,直至固体MRS平板表面的悬浮液不可流动为止,整个过程在超净台进行。将涂布后的平板用封口膜封口,在37℃培养箱倒置培养24小时。
(2)乳酸菌在新陈代谢过程中会伴随乳酸等酸性有机物的产生,平板上溴甲酚紫指示剂会伴随乳酸菌有机酸的产生而变色,由紫色变成黄色。挑取黄色圈内的单菌落于MRS固体培养基(含溴甲酚紫)进行划线纯化,封口膜封口,在37℃培养箱培养24h,重复纯化2次后,将黄色的单菌落接种在含10mL MRS肉汤培养基的玻璃试管内,封口膜封口后37℃,200rpm摇床培养12小时,以富集纯化的乳酸菌。将其命名为菌株FS4722。
(3)取1mL 106CFU/mL乳酸菌(OD600nm≈0.45),5000rpm离心1min,收集菌体。并用无菌生理盐水清洗3次。加入750uL含1.8mM肌苷和1.3mM鸟苷的0.1M中性磷酸钾缓冲液,充分混匀,37℃、180rpm反应1h后,5000rpm离心1min,收集270uL上清液,加入30uL 5%(v/v)三氟乙酸以终止反应,用0.22um滤膜过膜后,液相色谱HPLC对肌苷和鸟苷进行定性定量。其中,液相色谱条件如下:
流动相:含5%甲醇的25mM KH2PO4。具体配置如下,3.4g KH2PO4用950mL去离子水溶解后,与50mL甲醇混匀,用0.22um有机滤膜过滤2次以除去杂质,超声30min除去气泡后,4℃冰箱保存备用。色谱柱:ODS-3(4.6×250mm,5um),购自岛津(上海)实验器材有限公司。Waters 1525高效液相色谱仪、流速1mL/min、光二极管检测器、254nm、35℃,保留时间20min。
①降解速率计算公式:
c:肌苷(鸟苷)的初始浓度,单位g·L-1;
x:反应60min后,肌苷(鸟苷)的剩余浓度,单位g·L-1;
v:肌苷(鸟苷)的反应速率,单位g·L-1·min-1;
②降解率计算公式:
a:肌苷(鸟苷)的降解率。
最后,菌株FS4722株在60min内对肌苷和鸟苷的降解率均超过98%,用50%(v/v)甘油等体积混合后,-80℃保存。
(二)菌株FS4722的菌落形态特征观察
菌株FS4722为革兰氏阳性菌,不形成孢子的细菌,最适生长温度37℃,最适生长pH6.5,摇床培养20h后进入稳定期。在MRS固体平板上菌斑呈乳白色、圆形、边缘整齐、不透明、表面光滑。
(三)植物乳杆菌FS4722的分子生物学鉴定,步骤包括:
(1)16S rDNA测序:挑取MRS固体平板上的FS4722菌落,加入30uL裂解液,85℃水浴15min后,12000r/min离心5min,取上清液1uL作为DNA模板。采用通用引物27F/1492R进行PCR扩增。其中,
PCR体系组分如表1所示:
表1
引物序列如下:
27F序列:5’-AGAGTTTGATCCTGGCTCAG-3’;
1492R序列:5’-TACGGYTACCTTGTTACGACTT-3’。
PCR程序如表2所示:
表2
(2)PCR扩增产物的鉴定:本发明采用琼脂糖电泳检测DNA分子量。具体地,0.3g琼脂粉与30mL 1×Bis-Tris缓冲溶液混合,微波炉加热至琼脂粉完全溶解,加入3uLgoldview核酸染料摇匀后,倒入模具定型。PCR扩增产物与核酸染料Gel-Green等体积混合。将琼脂胶放入Bio-Rad水平电泳仪,每孔依次加入2uL Marker(100~3000bp)和PCR扩增产物,在125V电压下运行30min后,用Bio-Rad凝胶成像***观察DNA条带。最终得知,目标片段长度1504bp。
(3)16s rDNA测序:将上述验证后的PCR产物送至生工生物工程(上海)股份有限公司进行测序,获得的16s rDNA序列在NCBI上BLAST,与已知菌株的测序信息进行同源性比对,以鉴定菌株。使用MEGA 5.0对本发明筛选的菌株序列与NCBI数据库中调取的参考菌株序列进行多序列匹配分析。本发明筛选的菌株被鉴定为Lactiplantibacillus(中文名:乳杆菌),推测为Lactiplantibacillus plantarum(中文名:植物乳杆菌)。最终,本发明将该菌株命名为植物乳杆菌(Lactobacillus plantarum)FS4722。
菌株的16S rDNA基因序列如下(SEQ ID NO.1):
GGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAACGAACTCTGGTATTGATTGGTGCTTGCATCATGATTTACATTTGAGTGAGTGGCGAACTGGTGAGTAACACGTGGGAAACCTGCCCAGAAGCGGGGGATAACACCTGGAAACAGATGCTAATACCGCATAACAACTTGGACCGCATGGTCCGAGTTTGAAAGATGGCTTCGGCTATCACTTTTGGATGGTCCCGCGGCGTATTAGCTAGATGGTGGGGTAACGGCTCACCATGGCAATGATACGTAGCCGACCTGAGAGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAACTCTGTTGTTAAAGAAGAACATATCTGAGAGTAACTGTTCAGGTATTGACGGTATTTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCTTCGGCTCAACCGAAGAAGTGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGTATGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCATACCGTAAACGATGAATGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATACTATGCAAATCTAAGAGATTAGACGTTCCCTTCGGGGACATGGATACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTATCAGTTGCCAGCATTAAGTTGGGCACTCTGGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTTGCGAACTCGCGAGAGTAAGCTAATCTCTTAAAGCCATTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGTCGGTGGGGTAACCTTTTAGGAACCAGCCGCCTAAGGTGGGACAGATGATTAGGGTGAAGTCGTA。
(4)全基因组测序:将提取的全基因组送至生工生物工程(上海)股份有限公司进行二代测序,将得到的序列进行数据库比对,发现能所述植物乳杆菌FS4722胞内具有3个能降解肌苷和鸟苷的同工酶。
根据菌株FS4722的菌落形态特征和分子生物学鉴定结果,将植物乳杆菌(Lactobacillus plantarum)FS4722保藏于中国微生物菌种保藏中心,保藏编号为CGMCCNo.22750,保藏日期为2021年6月21日。
实施例2植物乳杆菌FS4722对小鼠的降尿酸效果评价
所述植物乳杆菌FS4722对小鼠的降尿酸效果评价,具体步骤如下:
将24只3周龄(体重16~24g)的昆明小鼠(购于北京维通利华实验动物技术有限公司)随机分成4组:正常对照组、高尿酸模型组、FS4722干预组和药物对照组,每组各6只。高尿酸模型组每天灌胃造模剂(250mg/kg氧嗪酸钾盐、400mg/kg肌苷和400mg/kg鸟苷,0.5%(w/v)羧甲基纤维素钠为分散剂)同时灌胃100uL生理盐水;FS4722干预组和药物对照组每天灌胃造模剂的同时分别灌胃109CFU植物乳杆菌FS4722和100uL 10mg/kg的别嘌醇;正常对照组只灌胃100uL的生理盐水,在连续喂养的第3、6和12天收集小鼠血清,采用尿酸检测试剂盒(购于南京建成)对血清中的尿酸定量,三次重复。
结果表明,无论在第几天FS4722干预组小鼠的血尿酸水平显著低于高尿酸模型组,与正常对照组和药物治疗组相比无显著性差异,说明所述植物乳杆菌FS4722株具有显著的降尿酸作用,喂养第3天即可起效(图1)。
实施例3植物乳杆菌FS4722的减重效果评价
所述植物乳杆菌FS4722的减重效果评价,具体步骤如下:
(1)将二次活化的植物乳杆菌FS4722、植物乳杆菌1(植物乳杆菌1为植物乳杆菌ZJUF T17(Lactobacillus plantarum ZJUF T17),来源于酸面团,根据中国专利获得:申请号为201810724365.7,发明名称为“植物乳杆菌ZJUF T17及其应用”)和干酪乳杆菌(干酪乳杆菌(Lactobacillus casi)已在文献中公开:Zhong,S R,Li M F,Zhang Z H,Zong M H,WuX L,Lou W Y.Novel Antioxidative Wall Materials for Lactobacillus caseiMicroencapsulation via the Maillard Reaction between the Soy Protein Isolateand Prebiotic Oligosaccharides[J].J.Agric.Food Chem.2021,69(46),13744–13753.其中该干酪乳杆菌来源于广州益力多),按1%(v/v)接种量接种至含100ug/mL胆固醇的MRS肉汤培养基中,37℃培养12h后,采用总胆固醇检测试剂盒(购于南京建成)检测胆固醇含量,胆固醇降解能力(%)=(胆固醇投入量–胆固醇含量)/胆固醇投入量×100。三次重复。结果表明,所述植物乳杆菌FS4722的胆固醇降解能力与某减肥代餐粉筛选出来的植物乳杆菌1相比无显著性差异,显著高于某酸奶筛选出来的干酪乳杆菌2(图2A)。
(2)将二次活化的植物乳杆菌FS4722按1%(v/v)接种量接种于MRS肉汤培养基,37℃培养20h后,8000r离心5min,收集菌体,用0.85%生理盐水清洗3次后,稀释至106CFU/mL,超声20min以破碎细胞,4℃、12000rpm离心20min收集细胞内容物,用0.22um滤膜过滤备用。然后将实验分为4组:对照组、模型组、FS4722组和药物组;其中将复苏后的肝脏细胞HepG2(购于中国科学院典型培养物保藏委员会细胞库,目录号:TCHu 72)接种至6孔板,加入高胆固醇诱导液(200mg/mL胆固醇和20mg/mL 25-羟基胆固醇(孵育48h以建立高胆固醇模型(模型组);FS4722组在高胆固醇模型的基础上再加入植物乳杆菌FS4722的细胞内容物(5%,(v/v))继续孵育24h;药物组在高胆固醇模型的基础上再加入0.2ug/mL辛伐他汀作为药物对照组;以不加入任何药物作为空白对照(对照组),三次重复,采用普利莱的甘油三酯TG酶法检测试剂盒进行检测。结果表明,FS4722能显著降低HepG2细胞内的甘油三酯含量,且与药物对照组无显著性差异(图2B)。
(3)选取18只3周龄的雄性昆明小鼠(购于北京维通利华实验动物技术有限公司),随机平均分成3组,分别为正常对照组(简称正常组)、高脂组和FS4722组。高脂组每天喂养60%高脂模型饲料(60%脂肪供能高脂饲料,购于协同生物,产品货号:XTHF60),FS4722组在喂养高脂模型饲料的同时灌胃1010CFU FS4722菌剂,正常对照组喂养普通饲料(购于协同生物,产品货号:SWS9102),49天后对其称重,三次重复。结果表明,服用FS4722的小鼠体重明显低于高脂组,且与正常组无显著性差异(图2C)。
实施例4植物乳杆菌FS4722的抗炎症效果评价
所述植物乳杆菌FS4722的抗炎症效果评价,具体步骤如下:
将实施例2中实验后的小鼠养至15天时安乐处死,取新鲜肝脏组织(>50mg),4%多聚甲醛浸没后放4℃固定。石蜡包埋后冷冻切片,H&E染色后用正置白光拍照显微镜观察基本病理。血清中IL-1β、IL-6和TNF-α水平选用ELISA试剂盒检测。
结果表明,高尿酸模型组小鼠较多肝细胞胞浆轻度疏松淡染(黑色箭头),少量静脉周围见炎性细胞小灶性浸润(红色箭头),而FS4722干预组肝索结构清晰,肝细胞排列紧密,除了少量肝细胞胞浆轻度疏松淡染外(黑色箭头),未见明显坏死或炎症反应(图3A)。高尿酸模型组小鼠血清中IL-1β、IL-6和TNF-α水平明显升高(图3B、3C、3D),说明高尿酸血症伴随炎症发生,且服用FS4722具有抗炎效果。
实施例5植物乳杆菌FS4722对高尿酸血症小鼠肠道菌群及其代谢物的调节作用
所述植物乳杆菌FS4722对高尿酸血症小鼠肠道菌群及其代谢物的调节作用,具体步骤如下:
取实施例1中小鼠养至15天时的新鲜粪便,干冰运输,送至生工生物工程(上海)股份有限公司进行为生物分类测序,以细菌V3-V4为扩增区域,上游引物为341F(5’-CCTACGGGNGGCWGCAG-3’),下游引物为805R(5’-GACTACHVGGGTATCTAATCC-3’),测序平台为Miseq。α-多样性和β-多样性用于评估样本菌群的多样性和差异性。其中Shanon指数和Chao指数反映样本的α-多样性。结果表明,高尿酸血症小鼠的Shanon和Chao指数与正常对照组相比显著下降,α-多样性降低,但是FS4722能显著上调小鼠的α-多样性(图4A、4B)。NMDS非度量多维尺度分析图反映样本菌群的β-多样性,即差异性,两样本点越接近说明两样本物种组成越相似,图中表明,药物对照组与正常对照组的距离最远,而FS4722干预组与正常对照组的95%置信圈几乎重叠(图4C),说明服用FS4722能修复肠道菌群结构。
另取50mg新鲜粪便采用气质联用仪(色谱柱:VF-MAXms石英毛细管柱(30m×0.25mm×0.25μm),进样口温度280℃,EI离子源温度230℃,四级杆温度150℃,高纯度氦气(纯度大于99.999%)作为载气,进样量3uL,不分流进样;柱流量1mL/min,20PSI恒压模式;升温程序:初始温度70℃保持1min,先以10℃/min升温至200℃,然后以30℃/min升温至240℃并保持3min;采用全自动扫描模式进行质谱检测,质谱检测范围为30-150(m/z))对游离脂肪酸进行定性定量分析,结果表明服用FS4722能明显上调粪便中乙酸和丙酸的含量(图5A、5B),Alistipes属和Bacteroides属是宿主体内一类重要的乙酸、丙酸产生菌,而FS4722的服用均能显著上调这两物种的丰度(图5C、5D)。乙酸、丙酸等可增加肠上皮细胞紧密连接蛋白occludin的转录水平(图5E),从而加强细胞紧密连接,抑制肠道通透性,增强肠道免疫屏障。
实施例6植物乳杆菌FS4722的发酵食品
所述植物乳杆菌FS4722的发酵食品,步骤如下:
在含糖和不含糖的无菌纯牛奶(本实验采用市售燕塘甜牛奶和燕塘纯牛奶)中加入植物乳杆菌FS4722作为发酵剂,最终浓度>108CFU/mL,37℃孵育0h、6h和12h即在4℃冷藏保存,得到含本发明植物乳杆菌FS4722活菌的乳制品。同样,在洗净及高温瞬时灭菌(130℃灭菌8s)后的新鲜蔬菜及蔬菜汁(卷心菜、大白菜)中加入植物乳杆菌FS4722作为发酵剂,37℃发酵或者直接4℃冷藏保存即得含植物乳杆菌FS4722活菌的果蔬制品。
利用本发明植物乳杆菌FS4722所发酵生产的发酵食品,包括固态、液态和半固态食品。所述发酵食品包括果蔬品、乳制品和豆制品。所述乳制品包括牛奶、酸奶、黄油、奶酪等;所述果蔬品包括苹果、雪梨、萝卜、黄瓜、卷心菜、大白菜、大蒜、姜片、洋葱等。
所述发酵制品能够降低小鼠血尿酸水平,减重,抗炎症等效果。此外,所述发酵制品能够改善肠道菌群多样性,修复肠道紊乱,提高肠道乙酸和丙酸含量,提高肠道免疫屏障,减少高尿酸血症、痛风、肥胖、炎症的患病风险。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
序列表
<110> 华南理工大学
<120> 一株植物乳杆菌及在降尿酸、减重、抗炎症方面的应用
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1504
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 菌株的16S rDNA基因序列
<400> 1
ggctcaggac gaacgctggc ggcgtgccta atacatgcaa gtcgaacgaa ctctggtatt 60
gattggtgct tgcatcatga tttacatttg agtgagtggc gaactggtga gtaacacgtg 120
ggaaacctgc ccagaagcgg gggataacac ctggaaacag atgctaatac cgcataacaa 180
cttggaccgc atggtccgag tttgaaagat ggcttcggct atcacttttg gatggtcccg 240
cggcgtatta gctagatggt ggggtaacgg ctcaccatgg caatgatacg tagccgacct 300
gagagggtaa tcggccacat tgggactgag acacggccca aactcctacg ggaggcagca 360
gtagggaatc ttccacaatg gacgaaagtc tgatggagca acgccgcgtg agtgaagaag 420
ggtttcggct cgtaaaactc tgttgttaaa gaagaacata tctgagagta actgttcagg 480
tattgacggt atttaaccag aaagccacgg ctaactacgt gccagcagcc gcggtaatac 540
gtaggtggca agcgttgtcc ggatttattg ggcgtaaagc gagcgcaggc ggttttttaa 600
gtctgatgtg aaagccttcg gctcaaccga agaagtgcat cggaaactgg gaaacttgag 660
tgcagaagag gacagtggaa ctccatgtgt agcggtgaaa tgcgtagata tatggaagaa 720
caccagtggc gaaggcggct gtctggtctg taactgacgc tgaggctcga aagtatgggt 780
agcaaacagg attagatacc ctggtagtcc ataccgtaaa cgatgaatgc taagtgttgg 840
agggtttccg cccttcagtg ctgcagctaa cgcattaagc attccgcctg gggagtacgg 900
ccgcaaggct gaaactcaaa ggaattgacg ggggcccgca caagcggtgg agcatgtggt 960
ttaattcgaa gctacgcgaa gaaccttacc aggtcttgac atactatgca aatctaagag 1020
attagacgtt cccttcgggg acatggatac aggtggtgca tggttgtcgt cagctcgtgt 1080
cgtgagatgt tgggttaagt cccgcaacga gcgcaaccct tattatcagt tgccagcatt 1140
aagttgggca ctctggtgag actgccggtg acaaaccgga ggaaggtggg gatgacgtca 1200
aatcatcatg ccccttatga cctgggctac acacgtgcta caatggatgg tacaacgagt 1260
tgcgaactcg cgagagtaag ctaatctctt aaagccattc tcagttcgga ttgtaggctg 1320
caactcgcct acatgaagtc ggaatcgcta gtaatcgcgg atcagcatgc cgcggtgaat 1380
acgttcccgg gccttgtaca caccgcccgt cacaccatga gagtttgtaa cacccaaagt 1440
cggtggggta accttttagg aaccagccgc ctaaggtggg acagatgatt agggtgaagt 1500
cgta 1504
<210> 2
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 27F序列
<400> 2
agagtttgat cctggctcag 20
<210> 3
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 1492R序列
<400> 3
tacggytacc ttgttacgac tt 22
Claims (10)
1.一株植物乳杆菌,其特征在于:名称为植物乳杆菌(Lactobacillus plantarum)FS4722,保藏编号为CGMCC No.22750,该菌株于2021年6月21日保藏于北京市朝阳区北辰西路1号院3号的中国微生物菌种保藏管理委员会普通微生物中心。
2.一种培养权利要求1所述植物乳杆菌的方法,其特征在于,具体步骤为:将植物乳杆菌接种于培养基中,于28℃~37℃条件下进行培养。
3.根据权利要求2所述的方法,其特征在于:
所述的培养基为MRS肉汤培养基;
所述的培养的时间为12~20小时。
4.一种植物乳杆菌胞内酶,其特征在于,通过如下方法制备得到:将权利要求1所述的植物乳杆菌接种到培养基中,于28℃~37℃条件下培养12~20小时,离心收集菌体,生理盐水清洗后,加水稀释细胞,再超声破碎细胞,最后在4℃条件下离心收集细胞内容物,过滤,得到植物乳杆菌胞内酶。
5.权利要求1所述的植物乳杆菌在制备发酵食品中的应用。
6.一种含植物乳杆菌的发酵制品,其特征在于:通过使用权利要求1所述的植物乳杆菌发酵生产制得。
7.权利要求1所述的植物乳杆菌以及权利要求6所述的含植物乳杆菌的发酵制品中的至少一种在制备防治高尿酸血症、抗痛风、减重和/或抗炎症的药物中的应用。
8.根据权利要求7所述的应用,其特征在于:
所述的植物乳杆菌通过抑制嘌呤吸收达到降低血尿酸水平的目的;
所述的植物乳杆菌通过抑制嘌呤吸收以降低血尿酸水平,进而达到抗痛风的目的;
所述的植物乳杆菌通过降低肝脏细胞内的胆固醇和甘油三酯水平,进而实现减重的目的;
和/或所述的植物乳杆菌通过清除肝脏组织中的炎性细胞达到抗炎症的目的,和/或通过降低血清中的促炎因子IL-1β、IL-6和TNF-α的浓度,达到缓解炎症的目的。
9.权利要求1所述的植物乳杆菌以及权利要求6所述的含植物乳杆菌的发酵制品中的至少一种在制备改善肠道菌群多样性、提高肠道乙酸和/或丙酸的含量的药物中的应用。
10.根据权利要求9所述的应用,其特征在于:
所述的植物乳杆菌通过修复高尿酸血症的肠道菌群失衡以达到上调肠道菌群的α-多样性的目的、通过提高产乙酸和丙酸的另枝菌属(Alistipes)、拟杆菌属(Bacteroides)丰度以达到上调乙酸和丙酸含量的目的。
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