CN116784446B - 一种协同抑制铜绿假单胞菌的组合物及其应用 - Google Patents

一种协同抑制铜绿假单胞菌的组合物及其应用 Download PDF

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CN116784446B
CN116784446B CN202310728312.3A CN202310728312A CN116784446B CN 116784446 B CN116784446 B CN 116784446B CN 202310728312 A CN202310728312 A CN 202310728312A CN 116784446 B CN116784446 B CN 116784446B
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张昊
刘宇纾
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China Agricultural University
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Abstract

本发明涉及一种协同抑制铜绿假单胞菌的组合物及其应用。组合物包括绿原酸和阿魏酸的组合，两种多酚类物质共同作用可有效抑制铜绿假单胞菌的生长。绿原酸‑阿魏酸这一组合对铜绿假单胞菌具有协同作用的抑菌效果，能够应用于天然抑菌剂的制备，降低单一抑菌物质的用量和使用成本，以及其对食品各性状的不良影响。绿原酸‑阿魏酸的组合物可为开发成针对食源性致病菌的天然抑菌剂提供参考。

Description

一种协同抑制铜绿假单胞菌的组合物及其应用
技术领域
本发明涉及食品以及医药领域，具体涉及一种协同抑制铜绿假单胞菌的组合物及其应用。
背景技术
食品质量安全问题关系人们的身体健康和生命安全，是一项重要的民生工程。近十年来食源性疾病的发病率在全球范围内呈现逐年递增的趋势，发达国家平均每年有30％的人患有食源性疾病，发展中国家虽未有详细数据统计，但这个数据应该会更高。据世界卫生组织统计的数据来看，全球每年都有上亿的腹泻案例，超过70％是由食源性疾病导致的，而食源性致病菌污染是导致食源性疾病的主要原因，因此食源性致病菌的危害和预防也成为食品相关行业的重点关注问题。食品中的食源性致病菌在预包装食品和散装即食食品中较为常见，多为大肠杆菌、金黄色葡萄球菌、沙门氏菌、单增李斯特菌、铜绿假单胞菌等。
假单胞菌是一种能在低温下生长的食品腐败菌，极易污染肉制品、乳制品和蔬菜等食品并引起腐败变质。因此，该菌是引起食品腐败变质的重要微生物。铜绿假单胞菌是假单胞菌中最常见的一个种，又称绿脓杆菌，广泛分布于水、空气、土壤以及人体的呼吸道、皮肤和肠道等处，尤其在潮湿的自然环境中更易生长繁殖，所以其传播途径广泛，该菌是一种条件性致病菌，可引起人类急性肠道炎、皮肤炎、败血病和脑膜炎等疾病，其产生的内、外毒素具有致病性，内毒素能引起系统炎症反应综合征，对于老人、小孩等抵抗力较弱的群体危害较大；外毒素侵入敏感细胞后，可能引起哺乳动物蛋白合成受阻，临床上常引起继发性感染。
预防微生物导致的食品变质和食物中毒通常是通过使用化学防腐剂来实现的，但这些防腐剂的不当应用会对人体健康产生危害，还会造成食物链中化学残留，以及过度使用从而导致微生物产生耐药性等不良后果。
其中，铜绿假单胞菌的耐药现象格外严重，同时单一的天然成分抗菌效果相对有限，使得药物和天然成分联合使用成为了抑制铜绿假单胞菌的研究热点，以此达到优势互补作用。徐海瑛等发现双黄连口服液联用庆大霉素对金黄色葡萄球菌和铜绿假单胞菌药敏结果均呈相加作用；王淋荆等的研究发现黄芩、金银花和夏枯草可以通过促进外排泵基因表达而使环丙沙星对铜绿假单胞菌有拮抗作用，穿心莲和五倍子也能促使庆大霉素对铜绿假单胞菌表现出拮抗作用；胡芳等将双黄连粉针与青霉素等多种抗生素联用作用于非耐药菌，使双黄连对细菌的MIC下降，这说明中西药联合应用对铜绿假单胞菌有明显的协同抑菌作用。
绿原酸作为广泛存在于植物中的酚类物质，其协同抑菌功能也受了研究人员的关注。绿原酸与III型细菌素Helveticin-M复配可显著破坏荧光假单胞菌形态，增强细胞膜渗透性，加剧胞内物质外泄，最终加速荧光假单胞菌死亡；李光辉等人研究证明安石榴苷和绿原酸对金黄色葡萄球就具有协同抑制作用，为开发天然食品防腐剂或食源性致病菌的治疗预防提供理论基础。
阿魏酸是一种植物富含的天然抗菌剂，具有优良的抗氧化和抗菌性能，例如对致病菌大肠杆菌0157：H7和单核细胞增生李斯特菌。因此，还被用作天然多糖基质的抗菌包装材料的有效成分。用阿魏酸接枝的复合细菌纤维素-壳聚糖膜可用作延长食品保质期的包装材料和缓慢愈合伤口的敷料。此外，Liu等人使用阿魏酸制备创新的复合薄膜，用作保存虾的包装材料。
绿原酸作为一种重要的植物次生代谢产物，存在于各类蔬果和其他植物中。已有大量研究证明，绿原酸具有抗氧化、抗病毒、抑菌、保肝利胆、降低血脂等生理功能。其中，绿原酸作为一种天然抑菌剂，对大肠杆菌、肺炎克雷伯菌、李斯特菌、金黄葡萄球菌等常见病原体都具有广谱抑菌作用，也因其绿色、健康的特性而受到广泛关注。目前许多研究工作都聚焦于单一植物源抑菌物质的开发利用，但在实际应用过程中，单一抑菌剂使用时可能会因用量较大而造成成本升高、或产生不良感官影响等问题，因此探究天然抑菌物质复配使用时的抑菌作用意义重大。
发明内容
本发明的目的是提供一种抑菌组合物，进一步组合物是由绿原酸和阿魏酸复配组合而成，同时提供一种该抑菌组合物作为抑菌剂的应用，应用可选自食品、保健品、涂层、包装中的应用。
本发明是通过以下技术方案得到的：
本发明提供一种抑菌组合物，该组合物是由绿原酸和阿魏酸复配组合而成；所述绿原酸和阿魏酸的复配重量比例为(2-6)：(0.4-1.5)。优选的，绿原酸含量范围2-6mg/ml、阿魏酸含量范围0.5-2mg/ml；更优选的，绿原酸含量范围3-5mg/ml、阿魏酸含量范围0.8-1.5mg/ml；更优选的，绿原酸含量范围3.5-4.5mg/ml、阿魏酸含量范围0.9-1.2mg/ml。
本发明提供上述抑菌组合物在抑制铜绿假单胞菌、阪崎肠杆菌、变形链球菌、藤黄微球菌的生长中的应用。特别的，对于抑制铜绿假单胞菌的生长中的应用。
本发明提供的抑菌组合物在作为防腐抑菌剂制备产品中的应用。优选，产品为饲料、食品或保健品。
当产品为食品，抑菌组合物在食品中的添加量不高于10、8或6g/100g。
本发明提供一种食品，该食品中添加了前述抑菌组合物作为防腐抑菌剂。
抑菌组合物符合国家食品安全标准，在食品中的添加量不高于8g/100g。本发明使用的绿原酸、阿魏酸为食品级，使用浓度远低于影响人体健康标准。同时本发明的抑菌组合物能够协同抑制致病菌的生长，致病菌包括铜绿假单胞菌、阪崎肠杆菌、变形链球菌、藤黄微球菌。
抑菌组合物在食品中的添加量为1、2、3、4、5、6g/100g。
综上所述，本发明提供了一种由绿原酸-阿魏酸组成的对于铜绿假单胞菌具有协同作用的抑菌组合，该组合物具有良好的抑菌作用，特别是在抑制铜绿假单胞菌方面具有协同作用。本发明的抑菌组合物可应用于食品、化妆品、保健品中，减少原有防腐剂的添加量，同时使产品兼具抗菌作用。筛选出绿原酸-阿魏酸这一对铜绿假单胞菌具有协同作用的抑菌组合，能够应用于天然抑菌剂的制备，降低单一抑菌物质的用量和使用成本，以及其对食品各性状的不良影响。
有益效果：
1、本发明验证了绿原酸-阿魏酸针对铜绿假单胞菌具有协同的抑菌作用。
2、在绿原酸、没食子酸、原儿茶酸、咖啡酸、对香豆酸、阿魏酸等多酚类物质进行筛选，发现绿原酸阿魏酸的组合针对特定的铜绿假单胞菌具有协同的抑制效果。
附图说明
图1最小抑菌浓度判定示意图。
图2不同处理对铜绿假单胞菌的生长曲线的影响。
具体实施方式
以下通过实施例进一步说明本发明，其仅用于说明本发明而不限制本发明的范围。
本发明实施例中涉及的培养基配方：
营养肉汤液体培养基：每1升培养基中，蛋白胨10.0g，牛肉粉3.0g，氯化钠5.0g，葡萄糖1.0g，pH7.5。
脑心浸液培养基：每1升培养基中，牛脑浸出粉10.0，牛心浸液9.0g，蛋白胨10.0g，氯化钠5.0g，葡萄糖2.0g，磷酸氢二钠2.5g，pH7.4。
实施例1细菌活化培养及菌悬液的制备
(1)细菌活化培养
将铜绿假单胞菌和阪崎肠杆菌(实验室保藏菌种)从-80℃冰箱中取出化冻，吸取200μL至装有营养肉汤(NB)培养基的试管中，于37℃恒温恒湿培养箱中培养24h。
变形链球菌、藤黄微球菌保存于冻干管中，先用75％酒精脱脂棉进行表面消毒，在酒精灯外焰加热冻干管，随后立即滴加无菌水使管壁受冷破裂。向管内加入0.5mL相应液体培养基，摇匀并使其中菌粉溶解，再将溶解后的菌悬液全部转移至相应的液体培养基试管中，于菌种最适生长温度下培养24h，各细菌培养条件见下表1。
表1细菌培养条件
(2)菌悬液制备
将活化后的细菌用平板划线的方式获取单菌落，挑取单菌落接种到营养肉汤液体培养基中，在摇床中震荡培养6-8小时，获得指数生长周期的细菌。离心机6000rpm离心5min后获得菌体沉淀，用无菌PBS洗涤后重悬于营养肉汤培养基中，37℃培养过夜。
实施例2抑菌溶液配制
为提高抑菌物质浓度，溶剂采用DMSO和去离子水的混合溶液进行溶解。测定铜绿假单胞菌、阪崎肠杆菌、变形链球菌的最小抑菌浓度时，绿原酸、没食子酸、原儿茶酸储备液浓度为16mg/mL，溶剂为6％DMSO溶液；咖啡酸、对香豆酸、阿魏酸储备液浓度为8mg/mL，溶剂为10％DMSO溶液。测定藤黄微球菌的最小抑菌浓度时，绿原酸、没食子酸储备液浓度为12mg/mL，溶剂为6％DMSO溶液；原儿茶酸储备液浓度为12mg/mL，溶剂为4％DMSO溶液；咖啡酸、对香豆酸、阿魏酸储备液浓度为7mg/mL，溶剂为10％DMSO溶液。配置好的抑菌剂储备液用0.22μm滤膜过滤除菌备用。
实施例3最小抑菌浓度(MIC值)测定
(1)96孔板制备
在无菌的96孔培养板中各孔中都加入100μL无菌营养肉汤培养基。之后向首列加入100μL菌悬液，作为阴性对照列；向第二列中加入抑菌物质储备液，作为阳性对照列；向第三列中加入100μL抑菌物质储备液，移液枪吹打混匀后，吸取100μL混合液加入第四列孔中混匀，依此类推逐列进行梯度二倍稀释。最后向稀释后的各孔中加入100μL菌悬液吹打混匀。
(2)培养及结果判读
处理好的96孔培养板置于37℃恒温恒湿培养箱中培养24h，然后通过阳性对照、阴性对照和各抑菌处理孔的浊度进行观察比较，判断可以抑制生长的最小浓度，此孔中抑菌物质的浓度即为最小抑菌浓度(MIC值)，如图1所示。
(3)MIC值结果
表2六种抑菌物质对铜绿假单胞菌、阪崎肠杆菌、变形链球菌、藤黄微球菌的MIC值
实施例4分级抑菌浓度指数(FICI值)测定
(1)抑菌溶液配制
根据最小抑菌浓度测定的实验结果，配制六种抑菌物质的储备液，储备液浓度为各物质最小抑菌浓度的4倍。在离心管中用无菌营养肉汤培养基对各物质储备液进行梯度二倍稀释，得到浓度分别为2×MIC、1×MIC、1/2×MIC、1/4×MIC、1/8×MIC、1/16×MIC的抑菌溶液。
(2)96孔板制备
绿原酸分别与其他五种抑菌物质组合进行实验。将50μL不同浓度绿原酸溶液从左至右依次加入96孔培养板，每列孔中绿原酸浓度相同。之后将50μL另一种抑菌物质溶液从上至下依次加入96孔培养板，每行孔中抑菌物质溶液浓度相同。将各孔中两种物质溶液吹打混匀后，向每孔中加入100μL菌悬液，再次吹打混匀后将培养板置于相应温度的恒温恒湿培养箱中培养24h。培养结束后观察标记无细菌生长的孔，根据该孔中绿原酸和另一种抑菌物质的浓度，计算对应分级抑菌浓度指数，用于判断该组合的联合抑菌效应。
(3)抑菌效应判定原则
FICI——分级抑菌浓度指数；
FIC——分级抑菌浓度；
A、B——代表组合使用时的两种抑菌物质。
FICI≤0.5时，两种抑菌物质互为协同效应；0.5＜FICI≤1时，两种抑菌物质互为相加效应；当1＜FICI≤4时，两种抑菌物质之间为无关效应；当FICI＞4时，两种抑菌物质互为拮抗效应。
(4)FICI值及抑菌效应
表3抑菌物质组合对铜绿假单胞菌、阪崎肠杆菌、变形链球菌、藤黄微球菌的抑菌效应
如表3所示，通过测定FICI值，可得绿原酸和阿魏酸联合作用于铜绿假单胞菌时FICI＝0.5。即绿原酸和阿魏酸联合作用对铜绿假单胞菌具有协同抑制作用。
实施例6抑菌处理后的细菌生长曲线
不同浓度抑菌物质处理对铜绿假单胞菌生长曲线的影响如图2所示，空白对照组中，铜绿假单胞菌在4h内迅速生长，在10h后逐步进入稳定生长期。低浓度单独抑菌组仅能延缓细菌进入对数生长期，但不能起到抑制细菌生长的作用，可见2mg/mL的绿原酸和0.5mg/mL的阿魏酸不能有效抑制铜绿假单胞菌；而高浓度单独处理组和2mg/mL绿原酸+0.5mg/mL阿魏酸的协同抑菌组的铜绿假单胞菌几乎不生长，OD600维持在0.1-0.2之间，表明此浓度能有效抑制铜绿假单胞菌的生长繁殖，证明了协同组合抑菌效果显著，即抑菌效果：
0.25MIC绿原酸+0.25MIC阿魏酸≈1MIC绿原酸≈1MIC阿魏酸。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。

Claims (7)

1.一种抑制铜绿假单胞菌生长的抑菌组合物，该组合物是由绿原酸和阿魏酸复配组合而成；绿原酸含量范围为4-4.5mg/ml、阿魏酸含量范围为1-1.2mg/ml。
2.如权利要求1所述的抑菌组合物在制备抑制铜绿假单胞菌生长的产品中的应用。
3.如权利要求1所述的抑菌组合物在作为抑制铜绿假单胞菌的防腐抑菌剂制备产品中的应用。
4.根据权利要求3中所述的应用，其特征在于，产品为饲料、食品或保健品。
5.根据权利要求4中所述的应用，其特征在于，产品为食品，抑菌组合物在食品中的添加量不高于6g/100g。
6.一种食品，该食品中添加了权利要求1所述抑菌组合物作为防腐抑菌剂。
7.根据权利要求6所述的食品，其中，
抑菌组合物在食品中的添加量为1-6g/100g。
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