CN105265995A - 紫苏油作为山梨酸钾抗菌增效剂的应用 - Google Patents

紫苏油作为山梨酸钾抗菌增效剂的应用 Download PDF

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CN105265995A
CN105265995A CN201510723832.0A CN201510723832A CN105265995A CN 105265995 A CN105265995 A CN 105265995A CN 201510723832 A CN201510723832 A CN 201510723832A CN 105265995 A CN105265995 A CN 105265995A
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郭娜
刘宗慧
孟日增
黄延军
赵星辰
史册
陈向荣
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Abstract

本发明公开了紫苏油作为山梨酸钾抗菌增效剂的应用，以金黄色葡萄球菌ATCC？29213，大肠杆菌O157：H7，大肠杆菌25922为对照用质控菌株，8株大肠杆菌食品分离株，9株金黄色葡萄球菌食品分离株，为试验菌，实验结果表明，紫苏油与山梨酸钾联合作用金黄色葡萄球菌，测试菌株的FICI值均小于0.5，说明两者对金黄色葡萄球菌具有协同抗菌作用。当紫苏油与山梨酸钾联合作用时，测试菌株FICI值均小于0.5，两者对大肠杆菌具有协同抗菌作用。

Description

紫苏油作为山梨酸钾抗菌增效剂的应用
技术领域
本发明属食品添加剂领域，具体涉及紫苏油增强山梨酸钾抗菌活性的作用。
背景技术
现如今食品保藏技术飞速发展，但是在全球范围内由食源性致病菌造成的食品污染依然是一个重要的问题。金黄色葡萄球菌是一种革兰氏阳性菌，广泛的分布在自然界中，是最常见的食源性致病菌之一，具有致病力强、感染率高和死亡率高等特点，可引起皮肤感染和败血症等一系列复杂的临床症状，金黄色葡萄球菌引起的食物中毒是世界性的公共卫生问题。大肠埃希氏菌俗称大肠杆菌，是一种常见的革兰氏阴性菌，是一种人和动物肠道内的常居菌，但是某些特殊的血清型治病性强，可以造成严重腹泻和败血症。
今年来抗生素的滥用导致一些细菌产生了耐药性，有细菌感染引起的感染率和死亡率居高不下。在这样的条件下，人们希望找到新的方法来抑制这些食源性致病菌，特别是大肠杆菌和金黄色葡萄球菌。协同作为一种可以提高抗菌剂抗菌性的方法近年来受到了关注。山梨酸钾作为一种不饱和脂肪酸，可在体内参与新陈代谢，最终被分解为二氧化碳和水，并且几乎没有毒性，目前山梨酸钾已经成为一种在全球范围内广泛使用的化学防腐剂。但是随着山梨酸钾的普遍，山梨酸钾也许会像抗生素一样在自然界中产生相应的耐药性菌株，从而影响和限制山梨酸钾的抑菌作用。因此寻找山梨酸钾抑制食源性致病菌的增效剂，为新型食品防腐剂的研究开发奠定基础。
紫苏为唇形科一年生草本植物，是国家卫生部首批颁布的既是食品又是药品的60种中药之一。紫苏油是从从紫苏的干燥成熟果实中提取所得植物油。经气相色谱-质谱联用法检测鉴定，紫苏油主要含有软脂酸、亚油酸、α-亚麻酸、硬脂酸、花生一烯酸和花生酸。其中α-亚麻酸是ω-3系列脂肪酸,有独特的保健作用,抗衰老、降血脂、抗癌、消炎、抗过敏,在体内能转化成二十碳五烯酸(EPA)和二十二碳六烯酸(DHA),已成为功能性食品的重要原料。
本发明采用紫苏油作为一种提高山梨酸钾食源性致病菌作用的增效剂，为新型食品防腐剂的研究开发奠定基础。
发明内容
本发明的目的是为了解决长期使用山梨酸钾防腐剂作为食品防腐剂，产生耐药菌株，山梨酸钾添加量增加的问题，而提供一种山梨酸钾抗菌增效剂紫苏油。
紫苏油作为山梨酸钾抗菌增效剂的应用；
所述的菌为大肠杆菌和金黄色葡萄球菌；
所述的应用为山梨酸钾8-16mg/ml和紫苏油0.25-1mg/ml；
一种防腐剂，它包括：山梨酸钾和紫苏油，山梨酸钾和紫苏油的重量比为：8-16:0.25-1；
所述的重量比为8:0.5。
本发明提供了紫苏油作为山梨酸钾抗致病菌增效剂的应用，以金黄色葡萄球菌ATCC29213，大肠杆菌O157：H7，大肠杆菌25922为对照用质控菌株，8株大肠杆菌食品分离株，9株金黄色葡萄球菌食品分离株，为试验菌，实验结果表明，紫苏油与山梨酸钾联合作用金黄色葡萄球菌，测试菌株的FICI值均小于0.5，说明两者对金黄色葡萄球菌具有协同抗菌作用。当紫苏油与山梨酸钾联合作用时，测试菌株FICI值均小于0.5，两者对大肠杆菌具有协同抗菌作用。
附图说明
图1金黄色葡萄球菌ATCC29213的杀菌曲线；
图2大肠杆菌O157：H7的杀菌曲线。
具体实施方式
实施例1微量稀释法药物敏感性实验
用Muller-Hinton肉汤（MHB）培养基将紫苏油贮存液进行倍比稀释每孔加入100μL。将过夜培养的待测菌液用MHB培养基适当稀释后，使得用分光光度计在600nm波长处测得的金黄色葡萄球菌OD值为0.4，大肠杆菌的OD值为1，并进一步稀释至浓度为106CFU/mL。在96孔板中每孔加入100μL菌悬液，以MHB作阴性对照，不加药物的菌悬液为阳性对照。混合均匀后放37℃温箱静置培养，24h后观察结果。抑制细菌生长的最低浓度为菌株的MIC值，将每孔的细菌吹打均匀后移植到MHA平板，菌种不生长的最低浓度为MBC值，试验重复3次，取中位数为最终值。受试菌包括：8株大肠杆菌食品分离株，9株金黄色葡萄球菌食品分离株，以及3株对照用质控菌株：金黄色葡萄球菌ATCC29213，大肠杆菌O157：H7，大肠杆菌25922。
其结果如表1、表2。
表1结果表明，当两者联合作用时，其对金黄色葡萄球菌的MIC有明显的下降。表2结果表明，当两者联合作用时，其对大肠杆菌MIC值明显小于单药时的MIC值。
实施例2棋盘式微量稀释实验
将联用的两种药物至于96孔微量板上，以二维棋盘的纵向（B-H，山梨酸钾）和横向（2-12，紫苏油）分别像两个方向进行倍比稀释，根据例1所测定的两药单独使用时受试菌株的MIC，将紫苏油配制成4×MIC、2×MIC、1×MIC、1/2×MIC、1/4×MIC、1/8×MIC、1/16×MIC、1/32×MIC、1/64×MIC、1/128×MIC、1/256×MIC的浓度，山梨酸钾配制成4×MIC、2×MIC、1×MIC、1/2×MIC、1/4×MIC、1/8×MIC、1/16×MIC的浓度，在此我们选用菌液（浓度为105CFU/mL）来稀释山梨酸钾每孔100μL按依次加入，考察每一种药物联用时的MIC，并通过计算部分抑菌浓度指数（thefractionalinhibitoryconcentrationindex,FICI）来判断两药物体外相互作用。FICI值由以下公式计算：
FICI=FICA+FICB=CA comb/MICA alone+CB comb/MICB alone
其中，MICA alone和MICB alone分别是化合物A和B单用时的MIC值，CA comb和CB comb为两药联用时达到相同药效（等效位点）时各自的浓度。FICI值都判定标准如下：FICI≤0.5，表明两种化合物之间存在协同作用；0.5＜FICI≤4表明两种化合物之间无相关作用；FICI＞4表明两种化合物之间存在拮抗作用。其结果如表3，表4。
表3结果表明，当紫苏油与山梨酸钾联合作用时，测试菌株的FICI值均小于0.5，两者对金黄色葡萄球菌具有协同抗菌作用。表4结果表明，当紫苏油与山梨酸钾联合作用时，测试菌株FICI值均小于0.5，两者对大肠杆菌具有协同抗菌作用。
实施例3杀菌曲线
根据例1所测定的两药单独使用时受试菌株的MIC，将山梨酸钾和紫苏油分别用菌液稀释（菌液浓度为106CFU/mL），使三组的浓度分别达到山梨酸钾（1/2×MIC)、紫苏油（1/2×MIC)、山梨酸钾（1/2×MIC)+紫苏油（1/2×MIC)。空白对照组不加药物。混合均匀后放37℃温箱静置培养，在第3、6、9、12、24、48小时取菌液稀释到适当的倍数涂到琼脂板上，培养24小时后菌落计数。协同的判定标准为联合药物组的菌落个数对比单药的菌落个数减少≥2log10CFU/ml。从杀菌曲线可以看出，单独加入山梨酸钾金黄色葡萄球菌和大肠杆菌的菌落个数对比空白并没有明显的降低，而紫苏油的加入使得菌落个数明显降低，在第12小时，联合药物组的菌落个数对比单药的菌落个数减少的量已2log10CFU/ml，并且在第48小时联合药物组的菌落个数都降为0。金黄色葡萄球菌ATCC29213的杀菌曲线结果见图1，大肠杆菌O157：H7的杀菌曲线见图2。

Claims (5)

1.紫苏油作为山梨酸钾抗菌增效剂的应用。
2.权利要求1所述的紫苏油作为山梨酸钾抗菌增效剂应用，其特征在于：所述的菌为大肠杆菌和金黄色葡萄球菌。
3.权利要求1或2所述的紫苏油作为山梨酸钾抗菌增效剂应用，其特征在于：所述的应用为山梨酸钾8-16mg/ml和紫苏油0.25-1mg/ml。
4.一种防腐剂，它包括：山梨酸钾和紫苏油，山梨酸钾和紫苏油的重量比为：8-16:0.25-1。
5.权利要求4所述的一种防腐剂，其特征在于：所述的重量比为8:0.5。
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