CN112813003B - 一株植物乳杆菌及其在制备缓解高血脂症引发的疾病的药物或食品中的应用 - Google Patents
一株植物乳杆菌及其在制备缓解高血脂症引发的疾病的药物或食品中的应用 Download PDFInfo
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Abstract
本发明公开了一株植物乳杆菌及其在制备缓解高血脂症引发的疾病的药物或食品中的应用，属于微生物应用技术领域。本发明的植物乳杆菌为植物乳杆菌(lactobacillus plantarum)HH19058，于2020年12月24日保藏于中国典型培养物保藏中心，保藏编号为CCTCC NO：M 2020966，地址为：湖北省武汉市武昌区八一路299号武汉大学校内。本发明的植物乳杆菌HH19058从泡菜中分离得到，具有降低人体血脂含量、减少心血管病发病率的作用，可用于制备缓解高血脂症的食品和药品。
Description
技术领域
本发明属于微生物应用技术领域，具体涉及一株植物乳杆菌及其在制备缓解高血脂症引发的疾病的药物中的应用。
背景技术
随着人们生活水平的提高，高血脂症患者日益增多且日趋年轻化，常表现有肥胖、高血压、糖尿病及血脂异常等代谢性疾病，这些疾病又进一步引起冠心病、脑血管意外等心血管疾病，严重威胁人们的健康。因此，减肥降脂成为预防和治疗上述疾病的重要手段，也越来越受到人们的重视。药物治疗是当前治疗高血脂症的主要方式，但是药物治疗成本较高，并且具有较大的副作用。新型的减肥药物和减肥保健品或者刺激脂肪代谢及外周脂肪燃烧，或者减少机体对食物的摄取，增加能量消耗来实现降脂减肥目的，长期使用可造成油便、排便失禁、胃肠胀气等不良反应，有些产品则通过腹泻和抑制食欲造成脱水或营养不良，导致肌肉、内脏、骨骼、体内水分等活性组织减少。乳酸菌作为人体肠道中重要的益生菌，已被证实某些益生乳酸菌能够降低人体的血清胆固醇含量，改善血脂水平，减少心血管疾病发生。
发明内容
针对现有技术中存在的问题，本发明的目的在于提供一株植物乳杆菌，该菌株具有较强的降胆固醇和甘油三酯功能；为预防和治疗缓解高血脂症的药物的研发奠定基础。
为了达到上述目的，本发明采用如下技术方案：
植物乳杆菌，所述植物乳杆菌为植物乳杆菌(Lactobacillus plantarum)HH19058；该菌于2020年12月24日保藏于中国典型培养物保藏中心，保藏编号为CCTCC NO:M2020966，地址为：湖北省武汉市武昌区八一路299号武汉大学校内。
上述的保藏编号为CCTCC NO:M 2020966的植物乳杆菌在制备缓解高血脂症引发的疾病的药物中的应用。
在上述方案的基础上，所述高血脂症引发的疾病为心血管疾病、糖尿病或肥胖。
一种缓解高血脂症引发的疾病的药物，含有上述的保藏编号为CCTCC NO:M2020966的植物乳杆菌。
在上述方案的基础上，所述高血脂症引发的疾病为脂肪肝、高血压、高血脂、糖尿病或肥胖。
一种缓解高血脂症引发的疾病的药物，含有如权利要求1所述的保藏编号为CCTCCNO:M 2020966的植物乳杆菌。
在上述方案的基础上，所述缓解高血脂症引发的疾病的药物是用于维持血脂指标、降低肝功能损伤的药物。
在上述方案的基础上，所述维持血脂指标为降低血清中TC、TG、LDL-C的含量，升高HDL-C的含量，维持血脂正常水平。
在上述方案的基础上，所述降低肝功能损伤为改善由高脂膳食诱导的肝组织脂肪变性，降低肝细胞脂肪含量；维持正常的肝细胞形态；使血清中的ALT、AST维持在正常水平。
上述的保藏编号为CCTCC NO:M 2020966的植物乳杆菌在制备润肠通便的药物中的应用。
本发明技术方案的优点：
本发明的植物乳杆菌HH19058具有降低人体血脂含量、减少心血管病发病率的作用。
脂膳食诱导肥胖模型小鼠结果显示：本发明的植物乳杆菌HH19058能够降低机体肝指数、脾指数、附睾脂肪指数，改善由高脂膳食诱导的肝组织脂肪变性，使血管周围的脂肪泡变小变少，并能够降低血清中TC、TG、LDL-C的含量，升高HDL-C的含量，使血脂接近正常水平；能够降低血清中ALT、AST的含量，使肝功能得到改善。
附图说明
图1为分离菌株HH19058菌落形态；
图2为菌株HH19058的革兰氏染结果；
图3为小鼠肝指数、脾指数及附睾脂肪指数；
图4为小鼠肝脏组织病理学观察；
图5为植物乳杆菌HH19058对小鼠血清中TC、TG、LDL-C、HDL-C、ALT、AST含量的影响；
图6为植物乳杆菌HH19058对小鼠粪便中TC、TG、TBA及水分的影响。
具体实施方式
在本发明中所使用的术语，除非有另外说明，一般具有本领域普通技术人员通常理解的含义。
下面结合具体实施例，并参照数据进一步详细的描述本发明。以下实施例只是为了举例说明本发明，而非以任何方式限制本发明的范围。
实施例1植物乳杆菌HH19058的分离和鉴定
1、分离纯化和形态鉴定
泡菜汁：于2018年9月采集山东青岛地区农家的卷心菜泡菜，将菜叶和汁水一起均质得到的泡菜汁进行10倍梯度稀释。
用MRS固体培养基、LBS固体培养基和NPNL固体培养基分别分离上述泡菜汁中的乳酸菌，将分离的乳酸菌于厌氧工作站中37℃培养48h，挑取平板上的典型菌落划线分离得到纯菌落，通过革兰氏染色进行形态学观察，并于MRS液体培养基中37℃厌氧培养24h，4℃冰箱冷藏备用。
结果如图1和图2所示，菌株HH19058纯化后在MRS培养基中形成单菌落，菌落形态几乎一致，大多数呈圆形，白色，表面光滑湿润。在15℃-45℃环境下均能生长，接触酶、运动性、硝酸盐还原、吲哚、产H2S及明胶液化试验均为阴性，革兰氏染色后在显微镜下观察到紫色杆状细胞形态，判定为乳酸菌。
2、API鉴定
将1中分离的纯菌株HH19058在37℃培养20h，于3000r/min离心15min，收集菌体，用无菌生理盐水洗涤菌体后重悬为菌悬液，根据API试剂盒说明书进行操作。
利用API50 CHL系列鉴定试纸条对菌株进行鉴定，鉴定结果如表1所示。经API labplus系统最终鉴定，实验菌株为植物乳杆菌(Lactobacillus plantarum)，其ID值为99.0％，T值为0.45，达到鉴定要求。
表1植物乳杆菌HH19058的API 50 CHL系统鉴定结果
3、16S rDNA鉴定
采用细菌基因组DNA提取试剂盒提取纯化菌株的DNA，将提取的乳酸菌基因组DNA作为16S rDNA-PCR模板进行扩增，
引物序列为：
27F：5’-AGAGTTTGATCCTGGCTCAG-3’(SEQ ID No.2)，
1492R：5’-GGTTACCTTGTTACGACTT-3’(SEQ ID No.3)。
采用25uL反应体系进行扩增：2.5uL 10×buffer、1.5uL MgCl2(25.0mM)、上下游引物(12.5pm)各1.0uL、1.0uL dNTPs(10.0mM)、0.15uL rTaq DNA聚合酶(5.0U/uL)、样品DNA模板5uL，加无菌去离子水至25uL。
扩增程序：94℃预变性5min；35cycles(94℃60s，64℃45s，72℃60s)；72℃5min。
扩增产物送至上海生工生物工程股份有限公司进行测序，将测序结果通过BLAST程序与GenBank基因库进行同源性比对。16S rDNA基因扩增产物的序列如SEQ ID No.1所示。
SEQ ID No.1
16S rDNA同源性分析结果显示，菌株与Gene Bank数据库中已知植物乳杆菌(Lactobacillus plantarum)的同源性达99.0％。
结合形态学鉴定、API鉴定和16S rDNA同源性分析，将分离的乳酸菌鉴定为植物乳杆菌，编号为HH19058。
对植物乳杆菌HH19058进行生物保藏，保藏信息如下：
保藏时间：2020年12月24日
保藏单位名称：中国典型培养物保藏中心
保藏编号：CCTCC NO:M 2020966
保藏单位地址：湖北省武汉市武昌区八一路299号武汉大学校内(武汉大学第一附小对面)，武汉大学保藏中心
分类命名：植物乳杆菌HH19058 Lactobacillus plantarum HH19058
实施例2植物乳杆菌HH19058降胆固醇和甘油三酯的研究
将植物乳杆菌HH19058按3％接种量接种至胆固醇培养基(MRS-TC)中37℃培养24h后，取培养液0.2mL，加入4.8mL无水乙醇振荡混匀1min，静置5min后振荡混匀，4000r/min离心10min，取2.0mL上清液用于胆固醇的测定，以未接菌的胆固醇培养基作为对照。
植物乳杆菌HH19058按3％接种量接种至甘油三酯培养基(MRS-TG)中37℃培养24h后，4000r/min离心10min，取2.0mL上清液用于甘油三酯的测定，以未接菌的甘油三酯培养基作为对照。
胆固醇培养基(MRS-TC)配制：胆固醇0.1g、Tween-20 1.0mL、蔗糖酯0.1g、冰乙酸5mL，超声破碎混悬，加入到MRS液体培养基中，使培养基中胆固醇终浓度为0.1mg/mL，添加0.2％牛胆盐和0.2％的巯基乙酸钠，调pH至6.5±0.2，121℃灭菌15min，冷却备用。
甘油三酯培养基(MRS-TG)配制：吐温20与植物油按1:3比例混合，边搅拌边加热至充分溶解，经乳化机充分乳化后作为甘油三酯来源，按6％的比例将植物油乳化液加入到MRS液体培养基中，边加边搅拌，添加0.2％的巯基乙酸钠，调pH至6.5±0.2，121℃灭菌15min，冷却备用。
试验结果发现，本发明分离得到的植物乳杆菌HH19058降解胆固醇和甘油三酯能力较强，降解率分别为62.3％和42.1％。
实施例3植物乳杆菌HH19058对高血脂小鼠的影响
挑选合格的C57成年小鼠108只，13周龄，雌雄各半，同性别体重差异应在平均体重的20％之内，用基础饲料适应性饲喂1周后，按各组间平均体重无显著差异分为3组(表2)。第1组正常饲喂基础饲料，第2、3组饲喂高脂饲料，通过饲喂4周高脂饲料建立高血脂小鼠模型。
表2动物建模分组
模型建立后，每天上午固定时间按照表2中的处理方法对小鼠进行灌胃，连续4周，按0.1mL/10g体重灌胃。实验期间每7天称量一次体重，调整灌胃体积。
植物乳杆菌HH19058菌液配制，植物乳杆菌HH19058以3％接种量接种MRS肉汤培养基，37℃静置培养20h，4000r/min 4℃离心10min，收集菌体，用无菌生理盐水重悬，计数并调整浓度至3×1011CFU/mL。
灌胃后第4周末，收集各组小鼠粪便，所有小鼠禁食不禁水18h，摘眼球取血，室温静置2-4小时，4℃3000r/min离心10min，取上清；取血后脱颈椎处死，解剖取出肝组织、脾组织、附睾组织并迅速称重；剪取适量大小肝组织立即放入10％福尔马林溶液中固定48h，经脱水、透明、浸蜡、包埋、切片后进行HE染色，最后在光学显微镜下观察组织形态变化。
结果发现，灌胃4周后，植物乳杆菌HH19058试验组较高脂模型组肝指数、脾指数、附睾脂肪指数有明显下降(图3)；对照组小鼠肝细胞无脂肪变性，肝组织结构清晰、完整，肝小叶结构正常，细胞界限清楚，细胞核位于中央，高脂模型组小鼠肝组织小泡性脂肪变性，脂肪含量增多，有大量脂肪空泡，细胞内容物流出，细胞肿胀，细胞壁完整性破坏，经过植物乳杆菌HH19058干预后，肝组织脂肪变性程度显著减轻，肝细胞脂肪空泡更小更少，形态与正常对照组小鼠相似(图4)。
按照生化试剂盒说明书对血清中TC(胆固醇)、TG(甘油三酯)、LDL-C(低密度脂蛋白胆固醇)、HDL-C(高密度脂蛋白胆固醇)、AST(谷草转氨酶)、ALT(谷丙转氨酶)含量进行测定。对照组及试验组小鼠血清中的TC、TG、LDL-C、HDL-C、AST、ALT含量显著低于模型组(P＜0.05)，说明植物乳杆菌HH19058可降低血脂水平和肝功能损伤(图5)。
将试验最后3日收集的各组小鼠粪便，烘干后各称取0.5g，研钵中磨细，加入5.0mL氯仿：甲醇(2:1)混合液，振荡混匀，45℃水浴提取1h，4℃10000r/min离心20min，弃上清液，收集氯仿层液体到新的EP管中，加入1mL无菌生理盐水混悬，4℃10000r/min离心10min，收集氯仿层液体，置通风橱吹干，加入80uL异丙醇：Triton-100(9:1)混合液复溶，涡旋震荡2min，加入120uL蒸馏水，涡旋震荡2min，充分溶解，按照生化试剂盒说明书测定TG(甘油三酯)、TC(胆固醇)、TBA(总胆汁酸)含量。
结果发现，饲喂高脂饲料的小鼠粪便中胆固醇及甘油三酯的增加量大于对照组，灌胃4周后，试验组和药物组小鼠粪便中胆固醇、甘油三酯和胆汁酸的含量显著高于模型组(P＜0.05)，而粪便中水分含量显著高于模型组(P＜0.05)(图6)。分析原因，是乳酸菌促进肠腔中的菌群将胆汁酸分解为游离胆汁酸，并沉淀在粪便中被排出体外，由于胆固醇是合成胆汁酸的原料，为补偿由粪便排出的胆汁酸，更多的胆固醇需要转化为胆汁酸，从而降低了体内胆固醇的含量；同时，植物乳杆菌HH19058将胆固醇吸附至其细胞膜或细胞壁上，通过粪便排出体外，从而降低体内血脂水平；粪便的水分含量作为排便通畅的一个重要指标，植物乳杆菌HH19058同时具有潜在的通便能力，加速了被吸收的胆固醇在肠道中运输的速度，从而又是乳酸菌体内降低胆固醇含量的另外一个途径。
以上所述，仅是本发明的较佳实施例而已，并非是对本发明作其它形式的限制，任何熟悉本专业的技术人员可能利用上述揭示的技术内容加以变更或改型为等同变化的等效实施例。但是凡是未脱离本发明技术方案内容，依据本发明的技术实质对以上实施例所作的任何简单修改、等同变化与改型，仍属于本发明技术方案的保护范围。
序列表
<110> 青岛海华莱康生物医药技术有限公司
青岛海华生物医药技术有限公司
<120> 一株植物乳杆菌及其在制备缓解高血脂症引发的疾病的药物或食品中的应用
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1977
<212> DNA
<213> 植物乳杆菌(Lactobacillus plantarum)
<400> 1
tcctatacat gcagtcgaac gaactctggt attgattggt gcttgcatca tgatttacat 60
ttgagtgagt ggcgaactgg tgagtaacac gtgggaaacc tgcccagaag cgggggataa 120
cacctggaaa cagatgctaa taccgcataa caacttggac cgcatggtcc gagtttgaaa 180
gatggcttcg gctatcactt ttggatggtc ccgcggcgta ttagctagat ggtggggtaa 240
cggctcacca tggcaatgat acgtagccga cctgagaggg taatcggcca cattgggact 300
gagacacggc ccaaactcct acgggaggca gcagtaggga atcttccaca atggacgaaa 360
gtctgatgga gcaacgccgc gtgagtgaag aagggtttcg gctcgtaaaa ctctgttgtt 420
aaagaagaac atatctgaga gtaactgttc aggtattgac ggtatttaac cagaaagcca 480
cggctaacta cgtgccagca gccgcggtaa tacgtaggtg gcaagcgttg tccggattta 540
ttgggcgtaa agcgagcgca ggcggttttt taagtctgat gtgaaagcct tcggctcaac 600
cgaagaagtg catcggaaac tgggaaactt gagtgcagaa gaggacagtg gaactccatg 660
tgtagcggtg aaatgcgtag atatatggaa gaacaccagt ggcgaaggcg gctgtctggt 720
ctgtaactga cgctgaggct cgaaagtatg ggtagcaaac aggattagat accctggtag 780
tccataccgt aaacgatgaa tgctaagtgt tggagggttt ccgcccttca gtgctgcagc 840
taacgcatta agcattccgc ctggggagta cggccgcaag gctgaaactc aaaggaattg 900
acgggggccc gcacaagcgg tggagcatgt ggtttaattc gaaggaatct gtcacttagg 960
cggctggttc ctaaaaggtt accccaccga ctttgggtgt tacaaactct catggtgtga 1020
cgggcggtgt gtacaaggcc cgggaacgta ttcaccgcgg catgctgatc cgcgattact 1080
agcgattccg acttcatgta ggcgagttgc agcctacaat ccgaactgag aatggcttta 1140
agagattagc ttactctcgc gagttcgcaa ctcgttgtac catccattgt agcacgtgtg 1200
tagcccaggt cataaggggc atgatgattt gacgtcatcc ccaccttcct ccggtttgtc 1260
accggcagtc tcaccagagt gcccaactta atgctggcaa ctgataataa gggttgcgct 1320
cgttgcggga cttaacccaa catctcacga cacgagctga cgacaaccat gcaccacctg 1380
tatccatgtc cccgaaggga acgtctaatc tcttagattt gcatagtatg tcaagacctg 1440
gtaaggttct tcgcgtagct tcgaattaaa ccacatgctc caccgcttgt gcgggccccc 1500
gtcaattcct ttgagtttca gccttgcggc cgtactcccc aggcggaatg cttaatgcgt 1560
tagctgcagc actgaagggc ggaaaccctc caacacttag cattcatcgt ttacggtatg 1620
gactaccagg gtatctaatc ctgtttgcta cccatacttt cgagcctcag cgtcagttac 1680
agaccagaca gccgccttcg ccactggtgt tcttccatat atctacgcat ttcaccgcta 1740
cacatggagt tccactgtcc tcttctgcac tcaagtttcc cagtttccga tgcacttctt 1800
cggttgagcc gaaggctttc acatcagact taaaaaaccg cctgcgctcg ctttacgccc 1860
aataaatccg gacaacgctt gccacctacg tattaccgcg gctgctggca cgtagttagc 1920
cgtggctttc tgggttaaat accgtcaata cctgaacagt tactctcaga tatgtct 1977
<210> 2
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
agagtttgat cctggctcag 20
<210> 3
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
ggttaccttg ttacgactt 19
Claims (7)
1.植物乳杆菌，其特征在于，所述植物乳杆菌为植物乳杆菌(Lactobacillusplantarum)HH19058；该菌于2020年12月24日保藏于中国典型培养物保藏中心，保藏编号为CCTCC NO:M 2020966，地址为：湖北省武汉市武昌区八一路299号武汉大学校内。
2.权利要求1所述的保藏编号为CCTCC NO:M 2020966的植物乳杆菌在制备缓解高血脂症引发的疾病的药物中的应用。
3.根据权利要求2所述的应用，其特征在于，所述高血脂症引发的疾病为脂肪肝、高血脂。
4.一种缓解高血脂症引发的疾病的药物，其特征在于，含有如权利要求1所述的保藏编号为CCTCC NO:M 2020966的植物乳杆菌。
5.权利要求4所述缓解高血脂症引发的疾病的药物，其特征在于，是用于维持血脂指标、降低肝功能损伤的药物。
6.根据权利要求5所述缓解高血脂症引发的疾病的药物，其特征在于，所述维持血脂指标为降低血清中TC、TG、LDL-C的含量，升高HDL-C的含量，维持血脂正常水平。
7.根据权利要求5所述缓解高血脂症引发的疾病的药物，其特征在于，所述降低肝功能损伤为改善由高脂膳食诱导的肝组织脂肪变性，降低肝细胞脂肪含量；维持正常的肝细胞形态；使血清中的ALT、AST维持在正常水平。
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