CN111647526B - 植物乳杆菌sd-h9、其益生菌菌剂及其应用 - Google Patents
植物乳杆菌sd-h9、其益生菌菌剂及其应用 Download PDFInfo
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Abstract
本发明提出了一种植物乳杆菌SD‑H9、其益生菌菌剂及其应用。本发明所述的植物乳杆菌SD‑H9保藏于中国普通微生物菌种保藏管理中心(CGMCC)，保藏编号为CGMCC NO.16649。使用本发明提供的益生菌剂，能够有效缓解过敏性鼻炎大鼠鼻部症状，降低鼻炎大鼠血清中组胺、OVA特异性IgE和IL‑4含量，提高大鼠血清中IFN‑γ含量，有助于缓解过敏反应，诱导正确的免疫应答，从而改变促进变态反应的Th2细胞偏倚，通过调节宿主免疫功能而发挥对过敏性鼻炎的治疗作用，可以有效改善鼻炎症状。
Description
技术领域
本发明属于微生物技术领域，具体涉及一种植物乳杆菌SD-H9、其益生菌菌剂及其应用。
背景技术
过敏性鼻炎是呼吸系统常见的、多发的一种疾病，其主要临床表现为鼻塞、鼻痒、阵发性喷嚏、大量水样鼻涕及嗅觉功能障碍等，此外，它还可能导致或并发鼻窦炎、鼻 息肉、咽炎、中耳炎、气管和支气管炎、胸痛、胸闷、哮喘和变应性眼结膜炎等疾病， 严重影响患者生活质量，危害了人们身体健康而且容易诱发多种疾病。过敏性鼻炎主要 是患者吸入变应原，炎症介质的释放，导致鼻黏膜发生炎症，同时发病原因也和体质、 遗传、免疫等因素息息相关。患鼻炎的人数正在逐年增加，而且年龄趋向低龄化，对人 体的危害更不容忽视。
目前鼻炎主要通过杀菌药物或阿斯匹林、异丁苯丙酸、扑尔敏等抗生素药物制备成 的鼻腔清洗药物进行治疗，该鼻腔清洗液虽然能对鼻腔起到杀毒灭菌的作用，但忽视了使用抗生素药物对鼻腔进行清洗或者杀菌会增加对人体的副作用和用药危害，同时鼻腔粘膜受到药物长期的强烈刺激后也容易发生病变等不良问题。
过敏性鼻炎是体外环境因素作用于机体由IgE介导的Ⅰ型超敏反应，造成Th1和Th2免疫反应失衡而引发的炎症反应，其主要的免疫病理学特征是组织中大量表达Th2 细胞因子的细胞浸润。Th1细胞的功能主要依赖IFN-γ实现，而Th2细胞功能主要依赖 IL-4等。研究表明，通过诱导Th1型细胞的反应来调节Th2型细胞的细胞因子分泌可以 改善与Th2相关的疾病。因此，Th1/Th2淋巴细胞亚群平衡及自身调节对过敏性疾病的 发生、发展有着极其重要的作用。
发明内容
本发明的目的在于克服上述技术不足，本发明提供一种植物乳杆菌SD-H9、其益生菌菌剂和其应用。本发明提供的植物乳杆菌SD-H9是植物乳酸菌的一种，可以通过激 活宿主多种免疫细胞和诱生多种细胞因子而发挥免疫调节的作用，在过敏性鼻炎的治疗 中显示了巨大的潜力。
为达到上述技术目的，本发明的技术方案包括：
一种植物乳杆菌(Lactobacillus plantarum)SD-H9，保藏在中国普通微生物菌种保 藏管理中心，保藏编号CGMCC NO.16649。
一种植物乳杆菌SD-H9在制备治疗个体过敏性鼻炎益生菌菌剂中的应用。
一种益生菌菌剂，包括所述植物乳杆菌SD-H9的菌粉及赋型剂。
与现有技术相比，本发明的有益效果包括：
本发明提供的益生菌剂由于其含有植物乳杆菌SD-H9，能够改善大鼠鼻部过敏症状，并且降低鼻炎大鼠血清中组胺、OVA特异性IgE和IL-4含量，有助于缓解过敏反 应；提高大鼠血清中IFN-γ含量，诱导正确的免疫应答，从而改变促进变态反应的Th2 细胞偏倚，通过调节宿主免疫功能而发挥对过敏性鼻炎的治疗作用。
附图说明
图1本发明实施例提供的大鼠试验各组SD大鼠症状评分比较图；
图2本发明实施例提供的大鼠试验各组给药对大鼠血清组胺水平的影响图(注，##表示与CK组相比p＜0.05；**与模型组相比标记p＜0.05)；
图3本发明实施例提供的大鼠试验各组给药对大鼠血清OVA特异性IgE水平的影响图(注，##表示与CK组相比p＜0.05；**与模型组相比标记p＜0.05)；
图4本发明实施例提供的大鼠试验各组给药对大鼠血清IL-4水平的影响图；
图5本发明实施例提供的大鼠试验各组给药对大鼠血清IFN-γ水平的影响图(注，##表示与CK组相比p＜0.05；**与模型组相比标记p＜0.05)。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合实施例及附图，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
植物乳杆菌SD-H9的分离及鉴定
取10g健康人肠道排泄物用无菌生理盐水进行10倍梯度稀释，将样品稀释106倍后涂布于MRS琼脂培养基上，培养24h，观察菌落形态，从中挑取疑似乳酸菌的单菌落 划线分离至新的MRS琼脂培养基上，培养24h后再次进行划线分离纯化。采用无菌牙 签从第二次划线分离的培养基上挑取若干个单菌落，分别进行编号，选取其中20个单 菌落进行16S rDNA测序。
本实施例16SrDNA扩增测序结果显示，编号为SD-H9的菌株，其16SrDNA 测序结果为SEQ ID NO.1所示序列。用BLAST工具在NCBI数据库中进行比对， 结果显示本发明涉及的菌株SD-H9与植物乳杆菌亲缘关系最近，相似性达到99％ 以上。因此，鉴定为植物乳杆菌，命名为植物乳杆菌(Lactobacillus plantarum) SD-H9，于2018年10月29日保藏于中国普通微生物菌种保藏管理中心(CGMCC)， 保藏编号为CGMCC NO.16649，地址为北京市朝阳区北辰西路1号院3号。
植物乳杆菌SD-H9的益生菌菌剂
本发明还涉及一种益生菌菌剂，包括植物乳杆菌SD-H9的菌粉及赋型剂。其中植物乳杆菌SD-H9菌粉的有效活菌数不低于1011cfu/g。赋型剂为益生菌的乳化剂、保护 剂和稳定剂，如低聚麦芽糖、水溶性壳聚糖、乳糖、脱脂奶粉、海藻酸钠、谷氨酸钠、 抗坏血酸钠；赋型剂于益生菌菌剂中质量百分比为5-8％。
植物乳杆菌SD-H9对OVA致敏鼻炎的动物实验验证
1、材料
(1)受试品植物乳杆菌SD-H9混悬溶液的配制：取本发明提供的益生菌菌剂(其 中有效活菌数不低于1011cfu/g)，置于相应供试品管中，加入氯化钠注射液搅拌混悬， 即得植物乳杆菌SD-H9混悬溶液(混悬悬液中有效活菌数不低于109cfu/ml)。
(2)其他试剂及配制
鸡卵白蛋白(98％)，sigma公司；氢氧化铝佐剂，Thermo scientific公司；乌来糖，国药集团化学试剂有限公司；组胺、OVA特异性IgE、IL-4、IFN-γELISA试剂盒(美 国R&D公司进口分装)。
0.8mg/mL OVA溶液的配制：称取0.0496g鸡卵白蛋白(OVA)，加38mL氯化钠 注射液溶解，再加24mL氢氧化铝佐剂，磁力搅拌器搅拌约30min，即可。
60mg/mLOVA溶液的配制：称取0.24g鸡卵白蛋白(OVA)，加4mL氯化钠注射 液溶解，得60mg/mL OVA溶液即可。
20％乌来糖溶液的配制：称取20g乌来糖，加入氯化钠注射液至100mL，摇匀。
2、动物饲养及试验
SPF级SD大鼠来源和动物合格证号：广东省医学实验动物中心提供，实验动物生产许可证号：SCXK(粤)2013-0002，动物合格证号：44007200054944。
检疫观察：对购入的45只SD大鼠检疫3d。期间每日检查动物一次，如发现不健 康的动物立即剔除，选用健康SD大鼠进行实验。
动物饲养条件：群养，饲养温度与湿度：20～26℃，40～70％，采用12h:12h昼夜 间断照明；饲养室条件始终保持稳定，以保证试验结果的可靠性。试验期间动物均按实 验要求喂以相应颗粒饲料，动物自由进食饮水。
试验分组：最终选用36只健康的体重为139.3～168.8g雄性SD大鼠检疫观察和适应性饲养3天，每天观察1次。动物检疫合格后，将健康大鼠随机分为三组，正常对照 组(如图1-5中记为CK)、模型对照组(如图1-5中记为MOL)、益生菌治疗组(如图 1-5中记为SD-H9)，每组12只。
致敏：将各试验组分别于饲养第1天、第3天、第5天、第7天、第9天、第11 天、第13天腹腔注射0.8mg/mL OVA溶液，1mL/只大鼠，共7次致敏，正常对照组同 法注射等体积的氯化钠注射液，即完成致敏。
给药与激发：第14天(末次致敏次日)进行被动皮肤过敏试验，确认造模成功后，第16天开始给药。益生菌治疗组灌胃植物乳杆菌SD-H9混悬溶液，2mL/只大鼠，1次/ 日，连续给药28天；正常对照组和模型对照组均灌胃给予氯化钠注射液，2mL/只大鼠， 1次/日，连续给药28天。益生菌治疗组和模型对照组于第24天、第26天、第28天、 第30天、第32天、第34天、第36天、第43天，用60mg/mL的OVA溶液双侧鼻腔 滴鼻激发，每侧滴鼻30μL进行双侧滴鼻激发；正常对照组同样于第24天、第26天、 第28天、第30天、第32天、第34天、第36天和第43天双侧鼻腔滴鼻氯化钠注射液， 每侧滴鼻30μL。
3、检测及分析方法
(1)鼻部症状观察：按表1标准，于第43天观察滴鼻激发后30min内动物的鼻 部症状，并给予评分。
表1 OVA诱发模型动物鼻超敏反应鼻部症状评分标准
(2)细胞免疫因子的检测：动物末次给药当天晚上禁食不禁水，末次给药次日按0.6mL/100g体重腹腔注射20％乌来糖溶液麻醉后，腹主动脉采血，3000rpm离心15min， 取血清，采用ELISA法检测各组大鼠组胺、OVA特异性IgE、IL-4及IFN-γ的含量。
(3)数据统计方法：所有数据采用
表示，应用SPSS 21.0软件进行统计 分析。超敏反应鼻部症状评分采用卡方检验，组胺采用秩和检验，OVA特异性IgE含量 采用方差分析，IL-4含量采用cos转换后进行方差分析，IFN-γ含量采用log转换后进行 方差分析。检验水平α＝0.05。
4、结果
如图1，鼻部症状观察比较结果如下，模型对照组中SD大鼠出现鼻痒、喷嚏和流 清涕症状，评分集中于2分和3分，说明鼻炎大鼠模型建立成功，与正常组比较，鼻部 症状评分分布比较差异有统计学意义(P<0.05)。与模型对照组相比，益生菌治疗组评 分为0分的无过敏症状小鼠数量增加，益生菌治疗组大鼠鼻部过敏症状有较好改善。
如图2，与正常对照组比较，模型对照组大鼠血清组胺水平显著升高(P<0.05)； 与模型对照组比较，益生菌治疗组大鼠血清组胺水平显著降低(P<0.05)。如图3，模 型对照组大鼠血清OVA特异性IgE含量增加，高于正常对照组，差异有统计学意义 (P<0.05)；与模型对照组比较，益生菌治疗组大鼠血清OVA特异性IgE含量降低， 差异有统计学意义(P<0.05)。如图4、图5，模型对照组大鼠血清IL-4水平增加，益 生菌治疗组血清IL-4含量有所下降但对IL-4浓度无显著影响(P>0.05)，然而益生菌 治疗组显著增加IFN-γ的浓度(P<0.05)。
因此，植物乳杆菌SD-H9能缓解过敏性鼻炎大鼠鼻部症状，降低鼻炎大鼠血清中组胺、IgE和IL-4含量，提高IFN-γ含量，改变促进变态反应的Th2细胞偏倚，通过调 节宿主免疫功能而发挥对过敏性鼻炎的治疗作用。
以上所述本发明的具体实施方式，并不构成对本发明保护范围的限定。任何根据本 发明的技术构思所做出的各种其他相应的改变与变形，均应包含在本发明权利要求的保 护范围内。
序列表
<110> 深圳市华大农业应用研究院
<120> 一种植物乳杆菌SD-H9、其益生菌菌剂及其应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 503
<212> DNA
<213> SD-H9(Artificial Sequence)
<400> 1
agagtttgaa cctggctcag gacgaacgct ggcggcgtgc ctaatacatg caagtcgaac 60
gaactctggt attgattggt gcttgcatca tgatttacat ttgagtgagt ggcgaactgg 120
tgagtaacac gtgggaaacc tgcccagaag cggggataac acctggaaac agatgctaat 180
accgcataac aacttggacc gcatggtccg agtttgaaag atggcttcgg ctatcacttt 240
tggatggtcc cgcggcgtat tagctagatg gtggggtaac ggctcaccat ggcaatgata 300
cgtagccgac ctgagagggt aatcggccac attgggactg agacacggcc caaactccta 360
cgggaggcag cagtagggaa tcttccacaa tggacgaaag tctgatggag caacgccgcg 420
tgagtgaaga agggtttcgg ctcgtaaaac tctgttgtta aagaagaaca tatctgagag 480
taactgttca ggtatggcag tat 503
Claims (3)
1.一种植物乳杆菌SD-H9在制备治疗个体过敏性鼻炎的益生菌菌剂中的应用；所述治疗个体过敏性鼻炎指降低过敏性鼻炎个体血清中组胺的含量；所述植物乳杆菌SD-H9保藏在中国普通微生物菌种保藏管理中心，保藏编号为CGMCC NO.16649。
2.如权利要求1所述的应用，其特征在于，所述过敏性鼻炎为OVA致敏性鼻炎。
3.如权利要求1所述的应用，其特征在于，所述植物乳杆菌SD-H9的有效活菌数不低于1011cfu/g。
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