CN114317326B - 一种用于缓解过敏反应的清酒乳杆菌及制备方法 - Google Patents
一种用于缓解过敏反应的清酒乳杆菌及制备方法 Download PDFInfo
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Abstract
本发明公开了一种用于缓解过敏反应的清酒乳杆菌及制备方法，一种用于缓解小麦依赖‑运动诱发过敏反应的清酒乳杆菌，所述清酒乳杆菌的分类命名为米酒乳杆菌Lactobacillus sakei；所述清酒乳杆菌保藏于中国典型培养物保藏中心，保藏编号为CCTCC DB 20082413，保藏时间为2003年04月12日。本发明有益效果：本发明清酒乳杆菌可以通过降低小麦依赖‑运动激发过敏反应患者血清中特异性IgE和组胺含量，从而调节机体免疫功能预防/或治疗小麦依赖‑运动诱发性过敏反应。
Description
技术领域
本发明涉及的微生物技术领域，尤其涉及一种用于缓解小麦依赖-运动诱发过敏反应的清酒乳杆菌、制备方法、微生态制剂及应用。
背景技术
麸质相关疾病是一种新出现的世界性疾病，在普通人群中，其全球流行率约为5％。麸质相关疾病包括乳糜泻、非乳糜泻、面筋过敏和小麦过敏。其中包含一种特殊的过敏反应，即小麦依赖-运动诱发性过敏反应(Wheat-dependent,exercise-inducedanaphylax，WDEIA)，其具体发生在摄入小麦后进行体育锻炼。它的临床特征是在摄入小麦并进行体育运动后1-4小时出现过敏反应，通常症状为从局部或者全身性荨麻诊发展为呼吸道、胃肠或心血管疾病，包括血管性水肿、呼吸困难、虚脱和休克。阿司匹林、酒精的摄入，机体存在感染，压力或者女性激素刺激可以替代或者协同运动，在摄入小麦后将即可引起超敏反应。
目前缓解和控制这种小麦依赖-运动诱发性过敏反应的普遍措施是避免摄入包含小麦制品的食物，包括各类面食、饼干、面包等；进食小麦制品后，6小时内不进行剧烈运动。现今也存在去除麸质的小麦制品，然而这些制品失去了部分营养，同时对于生产加工产生影响，且不能从根本上缓解或控制小麦依赖-运动诱发性过敏反应。
发明内容
为解决上述技术问题，本发明根据不同益生菌菌种，包括是嗜酸乳杆菌、鼠李糖乳杆菌、干酪乳杆菌、副干酪乳杆菌、罗伊乳杆菌、植物乳杆菌等，通过致敏原小麦蛋白与免疫佐剂CT混合灌胃方式构建小麦过敏小鼠模型，筛选有益于缓解食物过敏，尤其是缓解小麦依赖-运动诱发过敏反应的益生菌菌种，最终筛选得到一株清酒乳杆菌，可用于缓解小麦依赖-运动诱发过敏反应。因此，本发明目的在于提供一种用于缓解小麦依赖-运动诱发过敏反应的清酒乳杆菌。
为达上述目的，本发明提供如下技术方案：一种用于缓解小麦依赖-运动诱发过敏反应的清酒乳杆菌，所述清酒乳杆菌的分类命名为米酒乳杆菌Lactobacillus sakei；所述清酒乳杆菌保藏于中国典型培养物保藏中心，保藏编号为CCTCC DB 20082413，保藏时间为2003年04月12日。
优选的，所述清酒乳杆菌能够降低小麦依赖-运动激发过敏反应患者血清中特异性IgE和组胺含量。
优选的，所述清酒乳杆菌以微胶囊、片剂或冻干粉的形式，用以食品辅料或药用载体。
一种清酒乳杆菌的制备方法，包括上述所述清酒乳杆菌，还包括以下步骤，将传统发酵乳样品处理培养，根据菌落特征，分离纯化不同特征的菌落的菌株，通过G氏染色，过氧化酶实验初步鉴定乳酸菌；对分离菌株进行菌落形态和显微镜观察，确定目标菌株；挑选所述目标菌株用MRS液体培养基进行活化，利用细菌基因组DNA提取试剂盒进行DNA提取，进行16srRNA扩增；PCR产物经1.0％琼脂糖凝胶电泳检验后，进行测序分析。
本发明有益效果：本发明清酒乳杆菌可以通过降低小麦依赖-运动激发过敏反应患者血清中特异性IgE和组胺含量，从而调节机体免疫功能预防/或治疗小麦依赖-运动诱发性过敏反应。
附图说明
图1为本发明所述清酒乳杆菌在光学显微镜下的结构图；
图2为本发明所述清酒乳杆菌对WDEIA患者血清中特异性IgE的影响；
图3为本发明所述清酒乳杆菌对WDEIA患者血清中组胺含量的影响。
具体实施方式
下面结合附图，对本发明的具体实施方式进行详细描述，但应当理解本发明的保护范围并不受具体实施方式的限制。应当说明的是，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，均可从商业途径可购得。
实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。为使本发明实施例的目的、技术方案和优点更加清楚，下面将对本发明实施例中的技术方案进行清楚、完整地描述。
实施例1
本发明提供一种清酒乳杆菌(Lactobacilus sakei)，其用于缓解小麦依赖-运动诱发性过敏反应，该清酒乳杆菌于2003年4月12日保藏于中国典型培养物保藏中心，保藏编号为CCTCC DB 20082413
分类命名为：米酒乳杆菌Lactobacilus sakei，保藏地址为：中国.武汉.武汉大学。
本实施例中该清酒乳杆菌为圆端直杆菌，通常0.9-1微米长，单个、成对或成短链。通常缺乏鞭毛，但能运动。厌氧，表面菌落约微米宽，凸起，圆，光滑，细密，白色，偶尔浅黄或深黄色。培养液中的生长物甚至很浑浊。
进一步的，该清酒乳杆菌以微胶囊、片剂或冻干粉的形式食用，包含剂量为1×108～1010cfu/kg的菌量，应用于制备药物、保健品、或食品方面，清酒乳杆菌摄入方式为每天1×108～1×1010cfu。
研究发现清酒乳杆菌可以通过降低小麦依赖-运动激发过敏反应患者血清中特异性IgE和组胺含量，从而调节机体免疫功能预防/或治疗小麦依赖-运动诱发性过敏反应。
需要说明的是，小麦依赖-运动诱发性过敏反应，其主要过敏原为ω-5醇溶蛋白(Tria 19)。在利用WDEIA患者血清和外周血单个核细胞开展的研究中发现，WDEIA患者具有醇溶蛋白特异性IgE抗体，并鉴定出ω-5醇溶蛋白(Tria19)为主要过敏原。运动时血液重新分布导致胃肠道粘膜缺血，未消化完全的小麦蛋白(主要是醇溶蛋白)通过消化道进入血液，与特异性IgE结合，激活嗜碱性粒细胞和肥大细胞，引起其脱颗粒，释放炎症介质，从而引发过敏反应。在WDEIA患者中，其嗜碱性粒细胞释放出高水平的组胺，ω-5醇溶蛋白特异性IgA抗体水平显著升高，IL-10降低。本发明的清酒乳杆菌(Lactobacillus sakei)能够抑制Th0向Th2分化，显著调节升高的IgE和IL-4，增强肠系膜淋巴结中Treg分化和IL-10分泌，减少噬碱性粒细胞组胺分泌，来减少小麦依赖-运动诱发性过敏反应，为小麦依赖-运动诱发性过敏反应的预防和治疗提供可行的途径。
实施例2
本实施例为上述实施例提出一种用于缓解过敏反应的清酒乳杆菌的制备方法，包括菌株的分离与鉴定，具体如下：
一、菌株的分离
(1)将传统发酵乳样品用无菌生理盐水进行适当稀释，取100μL涂布于MRS(蛋白胨10g，酵母膏5g，K2HPO4 2g(含结晶水2.62g)，柠檬酸三胺2g，葡萄糖20g，MnSO4 0.05(MnSO4.H2O 0.198g)，MgSO4 0.28g，肉浸膏8g，琼脂15-20g，NaAc(无水乙酸钠)5g，(吐温-801mL或1g),蒸馏水1L，Ph7.2，琼脂培养基平板，在37℃培养48h后，计数，根据菌落特征，分离纯化不同特征的菌落的菌株，通过G氏染色，过氧化酶实验初步鉴定乳酸菌。
(2)对分离菌株进行菌落形态和显微镜观察，选取G+，触酶阴性的杆菌，按照Harrigan&McCance的方法做进一步鉴定。乳酸构型用D-乳酸和L-乳酸脱氢酶试剂盒进行确定。乳酸菌菌株的碳水化合物发酵模式是用API试剂盒进行确定。目标清酒乳杆菌生理生化特性及其表型鉴定如下表1所示。
表1：清酒乳杆菌生理生化特性及其表型鉴定。
注：结果中“+”表示阴性、“-”表示阴性。
(3)挑选目标菌株用MRS液体培养基进行活化，液体培养条件为37℃，培养12h-16h。利用细菌基因组DNA提取试剂盒进行DNA提取，而后进行16srRNA扩增。16srRNA扩增釆用通用引物，其上游引物pA(5’-AGA GTT TGA TCC TGG CTC AG-3’)，下游引物pH(5’GGCTAC CTT GTT ACG ACT 3’)；PCR体系为The 50μL，PCR反应体系中带MgCl2 1.5mmol/L的缓冲液，dNTP 0.2mmol/L，每一个引物0.5μmol/L，DNA模板1μg/mL，1U of Taq DNApolymerase。PCR条件为：95℃，4min；35×[94℃45s，55℃45s，72℃1min]；72℃，5min。
(4)PCR产物经1.0％琼脂糖凝胶电泳检验后，进行测序分析。
光学显微镜下观察革兰氏染色后的清酒乳杆菌，结果显示，清酒乳杆菌呈杆状、形态一致，具体光学显微镜照片参见图1的示意。
实施例3
清酒乳杆菌缓解小麦依赖-运动诱发过敏反应
(1)以10名WDEIA患者为研究对象，将10名患者随机分为对照组(3人)、安慰剂组(3人)和益生菌组(4人)。受试期间，益生菌组受试者每日午餐后1小时内服用独立包装的清酒乳杆菌(2g，活菌数为0.5×1010CFU/g)，安慰剂组受试者每日午餐后1小时内服用安慰剂粉末(烘干多孔糊精2g)，连续服用28天。所有人群在试验前和试验期间所食用的食物均保持一致。服用益生菌或安慰剂第28天采集受试者的血液样品，并对其进行高速离心获取血清进行免疫指标检测。
(2)血清中特异性IgE测定：利用酶联免疫吸附法(ELISA法)进行测定。小麦醇溶蛋白用PBS稀释至15μg/mL，然后将其固定在96孔板中4℃过夜，用200μL PBS洗涤3次，在25℃下，用200μL 5％BSA封闭2h。之后，将平板洗涤3次并与100μL 10位受试者的血清(1:30稀释)，并在37℃下孵育1.5小时，并使用非过敏血清作为阴性对照。洗涤3次后，每孔加入100μL HRP偶联山羊抗人IgE(1:5000)，37℃孵育1.5小时。然后每孔加入100μL四甲基联苯胺溶液，37℃孵育15分钟。50μL 2M H2SO4溶液终止显色反应。使用酶标仪(Molecular Devices，USA)在450nm处测量每个孔的光学密度。
结果如图2的示意，与对照组相比，安慰剂组WDEIA人群血清中IgE水平无显著变化，清酒乳杆菌干预28天益生菌组WDEIA人群血清中IgE水平显著低于对照组与安慰剂组。说明清酒乳杆菌干预对WDEIA患者具有调节作用，能缓解小麦依赖-运动激发过敏反应。
实施例3
清酒乳杆菌缓解小麦依赖-运动诱发过敏反应。
(1)以10名WDEIA患者为研究对象，将10名患者随机分为对照组(3人)、安慰剂组(3人)和益生菌组(4人)。受试期间，益生菌组受试者每日午餐后1小时内服用独立包装的清酒乳杆菌(4g，活菌数为1×1010CFU/g)，安慰剂组受试者每日午餐后1小时内服用安慰剂粉末(烘干多孔糊精2g)，连续服用28天。所有人群在试验前和试验期间所食用的食物均保持一致。服用益生菌或安慰剂第28天采集受试者的血液样品，并对其进行高速离心获取血清进行免疫指标检测。
(2)血清中组胺测定：血清中组胺的测定采用人组胺ELISA试剂盒(购于ThermoFisher Scientific)，按照其说明书进行测定，在450nm处测定各孔的吸光度OD值。
结果如图3的示意，与对照组相比，安慰剂组WDEIA人群血清中组胺水平无显著变化，清酒乳杆菌干预28天益生菌组WDEIA人群血清中组胺水平显著低于对照组与安慰剂组。说明清酒乳杆菌干预对WDEIA患者具有调节作用，能缓解小麦依赖-运动激发过敏反应。
综上，本发明的清酒乳杆菌能够显著降低小麦依赖-运动激发患者过敏反应特异性抗体IgE和血清中组胺含量，从而缓解过敏反应。因此，该清酒乳杆菌可用于缓解小麦依赖-运动激发过敏反应。
应当理解的是，本发明通过实施方式加以描述，实施例仅为针对本发明权利要求所提出技术方案能够实现所给出清楚完整的说明，即对权利要求的解释说明，因此当评判本发明说明书记载的技术方案是否公开充分时，应当予以充分考虑权利要求所限定方案的旨在核心要义，而在说明书中必然存在与本实施例所提出解决核心技术问题相无关的其他技术问题，其对应的技术特征、技术方案均不属于本实施例要义所指，属于非必要技术特征，故可参照隐含公开，本领域技术人员完全可以结合现有技术和公知常识进行实现，因此无任何必要做详述。
应说明的是，以上实施例仅用以说明本发明的技术方案而非限制，尽管参照较佳实施例对本发明进行了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的精神和范围，其均应涵盖在本发明的权利要求范围当中。
Claims (3)
1. 一种清酒乳杆菌用于制备缓解小麦依赖-运动诱发过敏反应药物中的应用，其特征在于：所述清酒乳杆菌的分类命名为米酒乳杆菌Lactobacillus sakei；所述清酒乳杆菌保藏于中国典型培养物保藏中心，保藏编号为CCTCC DB 20082413，保藏时间为2003年04月12日。
2.如权利要求1所述的一种清酒乳杆菌用于制备缓解小麦依赖-运动诱发过敏反应药物中的应用，其特征在于：所述清酒乳杆菌能够降低小麦依赖-运动激发过敏反应患者血清中特异性IgE和组胺含量。
3.如权利要求1所述的一种清酒乳杆菌用于制备缓解小麦依赖-运动诱发过敏反应药物中的应用，其特征在于：所述清酒乳杆菌以微胶囊、片剂或冻干粉的形式，用以食品辅料或药用载体。
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| WO2010013143A2 (en) * | 2008-08-01 | 2010-02-04 | Institut Pasteur | Methods for inhibiting mast cell activation and treating mast cell-dependent inflammatory diseases and disorders using lactobacillus |
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| CN101575582A (zh) * | 2008-05-08 | 2009-11-11 | 景岳生物科技股份有限公司 | 具有抗炎活性的乳杆菌分离株及其用途 |
| EP2332557A1 (en) * | 2009-12-08 | 2011-06-15 | Campina Nederland Holding B.V. | Probiotic lactic acid bacteria |
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