CN115581272B - 甜菊酚酸和益生菌在制备抑制幽门螺杆菌的产品中的应用 - Google Patents

甜菊酚酸和益生菌在制备抑制幽门螺杆菌的产品中的应用 Download PDF

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CN115581272B
CN115581272B CN202211034230.0A CN202211034230A CN115581272B CN 115581272 B CN115581272 B CN 115581272B CN 202211034230 A CN202211034230 A CN 202211034230A CN 115581272 B CN115581272 B CN 115581272B
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helicobacter pylori
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陈育如
毛丹丹
傅咏南
卞雪莲
冯志营
甘甜
陈睿思
黄芬芬
庄佳俊
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Shanghai Likang Precision Medical Technology Co ltd
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Abstract

本申请提供了甜菊酚酸和益生菌在制备抑制幽门螺杆菌的药物中的应用，所述益生菌为肠膜明串珠菌，所述肠膜明串珠菌在中国普通微生物菌种保藏管理中心的保藏编号为CGMCC No.24366；其中甜菊酚酸的浓度为10、12或14mg/mL；所述的甜菊酚酸是指甜叶菊提取物中的咖啡酰喹尼酸和黄酮成分；咖啡酰喹尼酸包括单咖啡酰喹尼酸、双咖啡酰喹尼酸和叁咖啡酰喹尼酸。本申请发现甜菊酚酸与益生菌组合的抑菌比其单组份使用具有更佳的效果，在幽门螺旋杆菌的防治方面具有重要的应用价值。

Description

甜菊酚酸和益生菌在制备抑制幽门螺杆菌的产品中的应用
发明领域：
本申请属于微生物以及幽门螺杆菌治疗领域。具体地，本申请提供了甜菊酚酸成分和益生菌在制备抑制幽门螺杆菌产品中的应用，以及用于相应的产品。
背景技术：
幽门螺旋杆菌(Helicobacter pylori，简称Hp)是胃癌、慢性胃炎、消化性溃疡、胃黏膜相关淋巴组织淋巴瘤等疾病的主要病因之一。菌体形态呈螺旋或S形，是能够定植于胃黏膜的微厌氧的革兰阴性杆菌(彭超，谢川，吕农华.幽门螺旋杆菌在胃内定植的相关影响因素[J].中国微生态学杂志，2017，29(3)：363-366)。1982年，澳大利亚西澳大学的临床微生物教授Barry J.Marshall发现了幽门螺旋杆菌，获得2005年诺贝尔医学奖(BarryJ.Marshall，1991)。一般认为幽门螺旋杆菌仅寄居于人类，人是唯一的传染源，幽门螺旋杆菌感染在家庭内有明显的聚集现象，自愈率几乎为零。1994年，世界卫生组织(WHO)国际癌症研究机构将幽门螺旋杆菌定为Ⅰ类致癌原。全世界有超过50％的人有Hp感染。流行病学调查表明，我国成人中Hp感染率达到40％-60％，平均为55％(毛丹丹,傅咏南,卞雪莲,王校.一种抑制幽门螺旋杆菌的复合益生菌组合物及其应用[P].CN111632084A)。
随着幽门螺旋杆菌治疗的广泛开展，幽门螺旋杆菌对克拉霉素、甲硝唑和左氧氟沙星(氟喹诺酮类)的耐药率呈上升趋势(Zhang YX，Zhou LY，Song ZQ，et al.Primaryantibiotic resistance of Helicobacter pylori strains isolated from patientswith dyspeptic symptoms in Beijing:a prospective serial study[J].World JGastroenterol，2015，21(9):2786-2792)，近年来，益生菌、黏膜保护剂、中药等非抗生素药物在幽门螺旋杆菌防治中发挥了重要作用，益生菌在提高幽门螺旋杆菌根除率及减少药物不良反应方面具有显著优势(Ables A Z，Simon I，Melton E R Update on Helicobacterpylori treatment[J].Am Fam Physician，2007，75:351-358；Yoon H，Kim N，Kim JY，etal.Effects of multistrain probiotic containing yogurt on second-line tripletherapy for Helicobacter pyloriinfection[J]J.Gastroenterol.Hepatol.2011，26:44-48)。
益生菌是一类对宿主有益的活性微生物。是目前临床使用最为广泛的微生态制剂。乳酸菌是国内外公认的对人类健康有益的安全的(generally recognized as safe，GRAS)食品级微生物，被广泛应用于食品、医药、农业等领域中，它不仅能够调节胃肠道菌群平衡，提高人体免疫力，改善机体营养状况，还具有降低胆固醇、血脂等多种生理功能。很多乳酸菌还能够产生对致病微生物具有拮抗作用的物质，如有机酸类、乙醇、过氧化氢、罗伊氏素、细菌素类等，这些具有抑菌效果的代谢产物能够抑制食源性致病菌和腐败菌的生长，在食品以及医药等行业具有潜在的应用价值，是乳酸菌研究热点之一(王彦伟.产细菌素乳酸菌的筛选及其所产细菌素leucocin B-K7的研究[D].江南大学,2016)。
罗伊氏乳杆菌(Lactobacillus reuteri)：罗伊氏乳杆菌是普遍存在于人体肠道的有益乳酸菌，其重要特性是产生独特的抑菌素reuterin。reuterin是一种低分子量的非蛋白、中性、可溶性的细菌素，具有很宽的抑菌谱带，可以有效抑制多种有害菌的生长，原理是罗氏乳杆菌与Hp具有相同的糖脂结合特性，通过竞争性地结合共同受体达到抑制Hp的目的(Mukai T,Asasaka T,Sato E,etal.Inhibition of binding of Helicobacter pylorito the glycolipid receptors by probiotic Lactobacillus reuteri[J].FEMSImmunol Med Microbiol,2002,32(2):105-110)。罗伊氏乳杆菌具有抗菌，抗过敏，降低胆固醇的作用，其分泌的罗氏菌素能够下调如空泡毒素A基因(Va-cA)、鞭毛蛋白A基因(flaA)等毒力基因的表达水平，抑制Hp的生长，显著减少Hp菌落数量(Urrutia-Baca VH，2018Urrutia-Baca VH,Escamilla-Garcia E,de la Garza-Ramos MA,et al.In VitroAntimicrobial Activity and Downregulation of Virulence Gene Expression onHelicobacter pylori by Reuterin[J].Probiotics Antimicrob Proteins,2018,10(2):168-175)。罗伊氏乳杆菌添加到酸牛奶中，饮用后可以进入人体发挥作用。
鼠李糖乳杆菌(Lactobacillus rhamnosus)：乳杆菌抑制Hp生长主要是酸或酸依赖性成分的作用(heng P X,Fang H Y,Yang H B,et al.Lactobacillus pentosusstrainLPS16 produces lactic acid,inhibiting multidrug-resistant Helicobacter pylori[J].Journal of Microbiology Immunology and Infection,2016,49(2):168-174)，鼠李糖乳杆菌处理后，Hp对人胃腺细胞的黏附率显著下降。鼠李糖乳杆菌能够降低患者体内Hp的定殖量，有效缓解Hp感染和其导致的胃肠症状，但对免疫调节的作用效果并不显著(张美怡,翟齐啸,赵建新,张灏,陈卫.鼠李糖乳杆菌抗幽门螺杆菌感染的临床效果评价[J].食品与发酵工业,2020,46(23):29-34)。
肠膜明串珠菌(Leuconostoc mesenteroides)：是一类重要的乳酸菌，能发酵糖类产生乳酸，具有高产酸、抗氧化和拮抗致病菌等能力。肠膜明串珠菌可以产生对多种病原菌和腐败菌具有抑制作用的细菌素，并且该细菌素具有很好的热稳定性和酸稳定性，并且对蛋白酶的敏感，抑菌谱广等优点(谈重芳,焦浈,杨飞飞,庞会利,蔡义民,秦广雍,王雁萍,李宗伟,常胜合.一株肠膜明串珠菌及其应用[P].CN101974450B)。可以应用为具有广阔应用前景的天然食品防腐剂和饲料添加剂；同时能发酵产生乳酸及双乙酰类物质(李祎,吴晓敏,杜航,景如贤,杨刚刚.一株肠膜明串珠菌的分离鉴定及其抑菌特性[J].微生物学通报,2021,48(12):4776-4788.DOI:10.13344/j.microbiol.china.210350)
嗜酸乳杆菌(Lactobacillus acidophilus)：具有调节肠道菌群、增强免疫力与促消化的作用，具有一定的耐酸，但对胃酸的抵抗能力较弱，通常采用微胶囊包被使嗜酸乳杆菌顺利到达肠道发挥作用。
甜叶菊(Stevia rebaudiana(Bertoni)Hemsl)：菊科甜叶菊属的多年生草本植物。其中的甜菊酚酸具有抗氧化、抗病毒、降压、降脂等功效(陈育如,杨凤平,杨帆,宋婷婷,束成杰,尹慧慧.甜叶菊及甜菊糖的多效功能与保健应用[J].南京师大学报(自然科学版),2016,39(02):56-60)。郎青云以甜叶菊为原料，采用超声辅助纤维素酶法提取甜菊糖，以甜菊糖提取率为指标，在单因素试验的基础上，通过响应面法分析得出最佳工艺条件；再用滤纸片法测定甜菊糖的抑菌活性，用液体培养法测定甜菊糖的最小抑菌浓度(MIC)。甜菊糖对大肠杆菌、枯草芽孢杆菌、毛霉、根霉4种供试菌的最小抑菌浓度分别为20、40、10、80mg/mL(郎青云,李慧,祝谢民,周艳,史保国.超声辅助纤维素酶提取甜菊糖及其抑菌活性研究[J].安徽农学通报,2019,25(21):30-35)。与其他使用甜菊提取物(主要是甜菊糖)不同，本申请使用甜菊酚酸(又名咖啡酰喹尼酸，发明人获得授权的发明专利，已转让企业生产，至此甜菊产业从以往的以提取甜菊糖为目的变成甜菊糖和甜菊酚酸可同时生产)与益生菌协同抑制Hp菌。
对甜叶菊提取物进行细菌敏感性测试，利用二倍稀释法测定最低抑菌浓度，结果表明：甜叶菊提取物具有很强的抑大肠杆菌、金黄色葡萄球菌和枯草芽孢杆菌的作用，明显强于山梨酸钾，对大肠杆菌、金黄色葡萄球菌和枯草芽孢杆菌的最低抑菌浓度分别为8.4、8.4、4.2mg/mL(任晓静,奚印慈,洪怡蓝,陈洁,徐文斌.甜叶菊提取物抑菌作用的研究[J].食品工业科技,2014,35(14):174-177)。
发明内容
在上述研究的基础上，本申请研究了甜叶菊提取物特别是甜菊酚酸对幽门螺杆菌的抑制效果，甜菊酚酸(15mg/ml)以下浓度，单独抑Hp菌效果并不明显，而在与肠膜明串珠菌CGMCC No.24366合用时，抑菌效果大为增强。
一方面，本申请提供了甜菊酚酸和益生菌在制备抑制幽门螺杆菌的产品中的应用。
另一方面，本申请提供了抑制幽门螺杆菌的产品，其中包含甜菊酚酸和益生菌成分。
进一步地，所述益生菌可选肠膜明串珠菌(Leuconostoc mesenteroides)，罗伊氏乳杆菌(Lactobacillus reuteri)，鼠李糖乳杆菌(Lactobacillus rhamnosus)，约氏乳杆菌(Lactobacillus johnsonii)，嗜酸乳杆菌(Lactobacillus acidophilus)中的一种或多种。
进一步地，所述益生菌为肠膜明串珠菌或者嗜酸乳杆菌。
进一步地，所述产品中还包含小球藻、甘油、乳粉中的一种或多种成分。
进一步地，所述产品为药品或药用辅料。
进一步地，所述益生菌为肠膜明串珠菌CGMCC No.24366或者嗜酸乳杆菌。
本申请中所述的甜菊酚酸是指甜叶菊提取物中的酚酸类和黄酮成分，酚酸类也称为咖啡酰喹尼酸(包括单咖啡酰喹尼酸、双咖啡酰喹尼酸和叁咖啡酰喹尼酸)、黄酮类可以是芹菜素葡萄糖苷、槲皮素葡萄糖苷，山萘酚鼠李糖苷，咖啡酰喹尼酸和黄酮其鉴别和提取方法可以依据本领域常规做法和现有技术的记载进行(如本申请中提及中国专利ZL201210055626.3或其他同类文献中的方法)，提取的甜菊酚酸中酚酸含量或黄酮含量可以在5％-95％之间。
本申请中的益生菌不局限于实施例中使用的具体菌株，其他同种菌株经过适当的验证和选择也可以选用。益生菌菌粉、菌泥等培养物的制备方法可以使用本领域已知的方法，如冷冻干燥、喷雾干燥制备。
本申请中的产品制备可以采用本领域中已知的各种制备方法，包括但不限于混合、过筛、压片、灌装、发酵等。
附图说明
图1为加甜菊酚酸(15mg/ml)前后对益生菌的抑菌效果比较。
具体实施方式
下面结合具体实施例详述本发明，但本发明的保护范围不局限于以下实施例。
以下实施例中的甜菊酚酸来自本发明的第一发明人已转让企业的授权发明专利—一种以甜叶菊为原料同时制备总咖啡酰奎尼酸和甜菊糖的方法ZL 201210055626.3中的制备方法(使用AB-8或D201树脂)，此专利中的“以甜叶菊为原料制备总咖啡酰奎尼酸”即是甜菊酚酸.提取过程中还可同时分离出黄酮类成份。
以下实施例中肠膜明串珠菌(Leuconostoc mesenteroides)使用申请人分离保藏的CGMCC No.24366，罗伊氏乳杆菌(Lactobacillus reuteri)、鼠李糖乳杆菌(Lactobacillus rhamnosus)、约氏乳杆菌(Lactobacillus johnsonii)、嗜酸乳杆菌(Lactobacillus acidophilus)为本申请人所在实验室保藏。
实施例1
本申请的实验工作中，采用8至14mg/mL甜菊酚酸对Hp菌进行抑制时，作用不明显，相关结果见表1。
表1甜菊酚酸单独使用和与益生菌合用后抑菌圈的情况(CM)
从表1可见，甜菊酚酸单独使用时，在实验浓度范围内抑菌圈的大小变化并不明显，明串珠菌单独使用时，效果与剂量有一定的关联，而在甜菊酚酸与明串珠菌合用时，则有明显的增效结果。
进一步验证了甜菊酚酸对其他几种益生菌的增效作用
表2益生菌加甜菊酚酸前后各抑菌圈的情况(CM)
罗伊氏乳杆菌 鼠李糖乳杆菌 嗜酸乳杆菌
加酚酸前 1.3 1.3 1.1
加酚酸后 1.5 1.7 1.8
表2中实验的具体方法：表中益生菌通过常规的液态培养条件，待菌量达到10亿以上活菌数(cfu/ml)后，用发酵菌液单独使用或与甜菊酚酸(>15mg/ml)联合使用,、采用牛津杯抑菌法测定得到表2中的抑菌圈具体数据。结果表明，甜菊酚酸对其他三种益生菌抗Hp也有增效作用。

Claims (4)

1.甜菊酚酸和益生菌在制备抑制幽门螺杆菌的药物中的应用，所述益生菌为肠膜明串珠菌，所述肠膜明串珠菌在中国普通微生物菌种保藏管理中心的保藏编号为CGMCCNo.24366；其中甜菊酚酸的浓度为10、12或14mg/mL；所述的甜菊酚酸是指甜叶菊提取物中的咖啡酰喹尼酸和黄酮成分；咖啡酰喹尼酸包括单咖啡酰喹尼酸、双咖啡酰喹尼酸和叁咖啡酰喹尼酸。
2.根据权利要求1所述的应用，其中所述药物中还包含小球藻、甘油、乳粉中的一种或多种成分。
3.抑制幽门螺杆菌的药物，其中包含甜菊酚酸和益生菌成分，所述益生菌为肠膜明串珠菌，所述肠膜明串珠菌在中国普通微生物菌种保藏管理中心的保藏编号为CGMCCNo.24366；其中甜菊酚酸的浓度为10、12或14mg/mL；所述的甜菊酚酸是指甜叶菊提取物中的咖啡酰喹尼酸和黄酮成分；咖啡酰喹尼酸包括单咖啡酰喹尼酸、双咖啡酰喹尼酸和叁咖啡酰喹尼酸。
4.根据权利要求3所述的药物，其中所述药物中还包含小球藻、甘油、乳粉中的一种或多种成分。
CN202211034230.0A 2022-08-26 2022-08-26 甜菊酚酸和益生菌在制备抑制幽门螺杆菌的产品中的应用 Active CN115581272B (zh)

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