CN112602719A - 一种抑菌组合物及其应用 - Google Patents
一种抑菌组合物及其应用 Download PDFInfo
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Abstract
本发明公开了一种抑菌组合物及其应用，属于抑菌剂技术领域。本发明首先提供了一种抑菌防腐组合物，将虫草素与常用防腐剂进行物理混合，组合物中虫草素质量浓度为2‑2000μg/mL，山梨酸钾质量浓度为2‑8000μg/mL，苯甲酸钠质量浓度为2‑8000μg/mL，对羟基苯甲酸酯类质量浓度为2‑4000μg/mL。本发明还提供了其在抗真菌生物膜中的应用。本发明能够有效抑制真菌生长，并对生物膜形成而导致的耐药性有所降低。同时，也降低了传统防腐剂的用量。
Description
技术领域
本发明涉及一种抑菌组合物及其应用，属于抑菌剂技术领域。
背景技术
食品防腐剂是现代食品加工生产中用于提升食品品质、延长食品保质期的重要方式。按照来历差别，防腐剂可以划分成天然有机或者是化学合成防腐剂。但是长期食用人工合成防腐剂对人体带来的潜在风险不容忽视，随着食品安全意识的提高，人们对绿色、健康食品的追求越来越高。因此，需要天然防腐剂或减少合成防腐剂的用量以保证对人具有高度安全性的抗微生物剂用于防止食品腐败变质。
生物膜是指被物体表面吸附的微生物群落通过分泌胞外基质将其嵌入其中的模式，其中，胞外基质有助于保持生物膜结构并维持细胞-细胞，细胞-表面和细胞-环境之间相互作用的稳定，生物膜的形成可能影响某些菌株的耐药性从而导致食源性疾病等，为医疗和食品安全等领域带来很大风险。有研究表明，真菌生物膜的耐药性是游离菌的1000倍。例如，念珠菌形成的生物膜能对抗宿主的防御，并且对临床常用抗真菌药物的敏感性降低。而且，这些药物会伴随着一些呕吐、腹泻、腹痛和食欲不振等问题，并且也会对肝脏、肾脏造成较强的毒性作用。因此，急需可有效针对生物膜形成的方法策略以降低生物膜危害。
发明内容
本发明的目的在于，提供一种有效的抗真菌的虫草素与常见食品防腐剂的组合物，该组合物为虫草素与防腐剂物理混合，同时能显著破坏生物膜形成能力。
本发明的第一个目的是提供一种抑菌组合物，所述组合物包含虫草素和食品防腐剂，所述食品防腐剂选自山梨酸及其钾盐、苯甲酸及其钠盐和对羟基苯甲酸酯类中的一种或多种。
在本发明的一种实施方式中，所述组合物中虫草素质量浓度为2-2000μg/mL。
在本发明的一种实施方式中，所述组合物中山梨酸钾及其钾盐质量浓度为2-8000μg/mL。
在本发明的一种实施方式中，所述组合物中苯甲酸钠及其钠盐质量浓度为2-8000μg/mL。
在本发明的一种实施方式中，所述组合物中对羟基苯甲酸酯类质量浓度为2-4000μg/mL。
在本发明的一种实施方式中，所述抑菌主要针对条件性致病真菌，包括念珠菌和/或曲霉菌。
在本发明的一种实施方式中，所述抑菌组合物的剂型可为多种剂型，包括液剂、乳剂、悬浮剂、粉剂、颗粒剂、可湿性粉剂或水分散粒剂。
本发明的第二个目的是提供一种上述抑菌组合物在抗真菌生物膜、防治果蔬病害、化妆品、饲料中的应用。
本发明的第三个目的是提供一种抑制真菌生物膜的方法，所述方法是以上述抑菌组合物处理真菌生物膜，以抑制真菌生物膜的活性。
本发明的第四个目的是提供一种防治果蔬病害的方法，所述方法是在植物种子萌发时期，向果蔬喷洒上述抑菌组合物，以促进植物生长。
本发明的第五个目的是提供一种化妆品，所述化妆品含有上述抑菌组合物，以达到护理皮肤、维持化妆品质地的效果。
本发明的第六个目的是提供一种饲料，所述饲料含有上述抑菌组合物，避免饲料的腐败变质。
本发明的有益效果：
本发明的虫草素与常见防腐剂的组合物对真菌的具有较强的抗菌活性，当达到相同效果时，与单独作用相比，其虫草素使用量下降了5-20倍，传统防腐剂使用量下降了2.5-20倍。同时，组合物也对真菌生物膜具有显著的抑制作用，其抗生物膜活性远远大于单独使用时，如使用6.25μg/mL虫草素和1000μg/mL山梨酸钾的组合物处理的生物膜活性仅有45.50％，而当单独作用时，生物膜活性分别为87.22％和78.27％。因此，本发明不仅仅能够降低传统防腐剂的使用量，而且还能有效抑制生物膜的形成，降低真菌的耐药性。
附图说明
图1为实施例1中抑菌组合物对白色念珠菌抗生物膜作用效果。
图2为实施例2中抑菌组合物对黑曲霉菌抗生物膜作用效果。
图3为实施例3中抑菌组合物对白色念珠菌抗生物膜作用效果。
图4为实施例4中抑菌组合物对黑曲霉菌抗生物膜作用效果。
图5为实施例5中抑菌组合物对白色念珠菌抗生物膜作用效果。
图6为实施例6中抑菌组合物对黑曲霉菌抗生物膜作用效果。
具体实施方式
以下对本发明的优选实施例进行说明，应当理解实施例是为了更好地解释本发明，不用于限制本发明。
1、生物材料：
念珠菌生物膜培养：所用的白色念珠菌(白假丝酵母，Candida albicans)来自北纳创联生物技术公司，编号为BNCC 186382。白色念珠菌在PDB液体培养基中活化后，使用RPMI-1640培养基进行稀释，取100μL的菌液(1×106cells)加入到96孔板中35℃静态培养24h形成成熟生物膜。
黑曲霉菌生物膜的培养：黑曲霉菌(Aspergillus niger van Tieghem)来自广东微生物研究所保藏中心，GIM编号为3.422。黑曲霉在固体SDA培养基上35℃培养48h并收集孢子，用RPMI-1640培养基重悬孢子，调节孢子液浓度为1×106孢子/mL。将100μL孢子悬液接种于96孔板中35℃静态培养24h以形成成熟生物膜。
2、最小抑菌浓度(MIC)测定：
根据临床和实验室标准协会(CLSI)标准，采用标准肉汤稀释法对用于本研究的虫草素、山梨酸钾以及虫草素与山梨酸钾混合物等对其MIC进行分析。
3、生物膜活性测试：
在培养前分别加入不同比例的虫草素与一种或多种防腐剂组合物，以未加混合物处理的为对照组，并采用XTT法检测生物膜活性。
4、协同作用评价方法：
以FICI法测定联合用药效果，即利用LA理论评价药物联合应用的相互作用。表述如下：∑FIC＝FICA+FICB＝CA/MICA+CB/MICB，MICA和MICB分别是A、B单用药时最小抑菌浓度，CA和CB分别是两药联合用药时达到等效位点的浓度。FICI>4时为拮抗作用，FICI在0.5与4之间为相加或无关作用，FICI≤0.5为协同作用。
其中，根据临床和实验室标准协会(CLSI)标准，采用标准肉汤稀释法对用于本研究的虫草素、山梨酸钾以及虫草素与山梨酸钾混合物等对其MIC进行分析。
实施例1：一种抑菌组合物
一种抑菌组合物，所述组合物包含6.25μg/mL虫草素和1000μg/mL山梨酸钾。
实施例2：一种抑菌组合物
一种抑菌组合物，所述组合物包含12.5μg/mL虫草素和375μg/mL山梨酸钾。
以单独虫草素和单独山梨酸钾作为对照组，将虫草素和山梨酸钾单独加入到含有真菌及新鲜培养基的96孔板中，培养24h，观察真菌的生长情况，其MIC结果见表1。虫草素与山梨酸钾复配后对真菌抗真菌活性具有协同作用，而且虫草素与山梨酸钾使用量减少明显。
以单独虫草素和山梨酸钾作为对照组，检测其对白色念珠菌和黑曲霉菌生物膜作用，结果见表2和图1、2。从表2和图1、2中看出，虫草素与山梨酸钾组合物对生物膜的形成有抑制作用；且与虫草素或者山梨酸钾单独使用相比，组合物的抑制作用增强非常明显。
表1虫草素与山梨酸钾对念珠菌、曲霉菌的联合作用
表2虫草素与山梨酸钾对生物膜活性的影响
实施例3：一种抑菌组合物
一种抑菌组合物，所述组合物包含12.5μg/mL虫草素和500μg/mL苯甲酸钠。
实施例4：一种抑菌组合物
一种抑菌组合物，所述组合物包含50μg/mL虫草素和93.75μg/mL苯甲酸钠。
将虫草素和苯甲酸钠单独加入到含有真菌及新鲜培养基的96孔板中，培养24h，观察真菌的生长情况，其MIC结果见表3。虫草素与苯甲酸钠物理混合后对白色念珠菌和黑曲霉菌抗真菌作用为协同作用。
以单独虫草素和单独苯甲酸钠作为对照组，检测其对白色念珠菌和黑曲霉菌生物膜形成抑制作用，结果见表4和图3、4。结果显示，与单独作用相比，组合物对白色念珠菌和黑曲霉菌的生物膜形成的抑制作用更强。
表3虫草素与苯甲酸钠对念珠菌、曲霉菌的联合作用
表4虫草素与苯甲酸钠对生物膜活性的影响
实施例5：一种抑菌组合物
一种抑菌组合物，所述组合物包含12.5μg/mL虫草素和250μg/mL对羟基苯甲酸正丁酯。
实施例6：一种抑菌组合物
一种抑菌组合物，所述组合物包含25μg/mL虫草素和12.5μg/mL对羟基苯甲酸正丁酯。
将虫草素和对羟基苯甲酸正丁酯单独加入到含有真菌及新鲜培养基的96孔板中，培养24h，观察真菌的生长情况，其MIC结果见表5。虫草素与对羟基苯甲酸正丁酯物理混合后对念珠菌和曲霉菌抗真菌作用满足协同作用。
以单独虫草素和单独对羟基苯甲酸正丁酯作为对照组，检测其对白色念珠菌和黑曲霉菌生物膜形成的抑制作用，结果见表6和图5、6。虫草素与对羟基苯甲酸正丁酯复配后对白色念珠菌和黑曲霉菌生物膜活性也有影响，生物膜形成抑制作用增强非常明显。
表5虫草素与对羟基苯甲酸正丁酯对念珠菌、曲霉菌的联合作用
表6虫草素与对羟基苯甲酸正丁酯对生物膜活性的影响
实施例7：一种防治果蔬病害的方法
一种防治果蔬病害的方法，所述方法是在植物种子萌发时期，向果蔬喷洒实施例1中的抑菌组合物，以促进植物生长。
实施例8：一种化妆品
一种化妆品，向现有的化妆品中添加实施例3中的抑菌组合物，以达到护理皮肤、维持化妆品质地的效果。
实施例9：一种饲料
一种饲料，向现有的饲料中添加实施例5中的抑菌组合物，避免饲料的腐败变质。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。
Claims (10)
1.一种抑菌组合物，其特征在于，所述组合物包含虫草素和食品防腐剂，所述食品防腐剂选自山梨酸及其钾盐、苯甲酸及其钠盐和对羟基苯甲酸酯类中的一种或多种。
2.根据权利要求1所述的抑菌组合物，其特征在于，所述组合物中虫草素质量浓度为2-2000μg/mL；和/或，山梨酸钾及其钾盐质量浓度为2-8000μg/mL；和/或，苯甲酸钠及其钠盐质量浓度为2-8000μg/mL；和/或，对羟基苯甲酸酯类质量浓度为2-4000μg/mL。
3.根据权利要求1或2所述的抑菌组合物，其特征在于，所述抑菌主要针对条件性致病真菌，包括念珠菌和/或曲霉菌。
4.根据权利要求1-3任一项所述的抑菌组合物，其特征在于，所述抑菌组合物的剂型可为多种剂型，包括液剂、乳剂、悬浮剂、粉剂、颗粒剂、可湿性粉剂或水分散粒剂。
5.权利要求1-4任一项所述的抑菌组合物在抗真菌生物膜、防治果蔬病害、化妆品、饲料中的应用。
6.一种抑制真菌生物膜的方法，其特征在于，所述方法是以权利要求1-4任一项所述的抑菌组合物处理真菌生物膜。
7.一种食品或医疗设备的抑菌方法，其特征在于，所述方法是以权利要求1-4任一项所述的抑菌组合物冲洗或浸泡食品或医疗设备。
8.一种防治果蔬病害的方法，其特征在于，所述方法是在植物种子萌发时期，向果蔬喷洒权利要求1-4任一项所述的抑菌组合物，以促进植物生长。
9.一种化妆品，其特征在于，所述化妆品含有权利要求1-4任一项所述的抑菌组合物。
10.一种饲料，其特征在于，所述饲料含有权利要求1-4任一项所述的抑菌组合物。
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