CN107019138A - 一种丁香酚组合液及制备方法和应用 - Google Patents
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Abstract
本发明提供了一种丁香酚组合液及制备方法和应用，所述组合液由以下体积份的原料组成：水99.8555％～99.9711％，吐温80溶液0.01％～0.05％，丁香酚0.0189％～0.0945％，以及制备上述丁香酚组合液的方法。该丁香酚组合液能抑制金黄色葡萄球菌生长，甚至丁香酚浓度更高时可作为杀菌剂使用。丁香酚组合液影响了金黄色葡萄球菌的SDH酶和MDH酶的分子结构，使其发生了变化，从而改变了构像，使酶活性降低。丁香酚组合液作用于金黄色葡萄球菌后，因为起细胞透性的改变，使得与蛋白合成相关的核酸类物质外泄，进而导致蛋白质的合成受到抑制，并最终导致细胞死亡。
Description
技术领域
本发明涉及一种丁香酚组合液及制备方法和应用。
背景技术
随着食品安全的话题不断受到关注，人们对食品的质量有了更高的要求，不但要求营养，还要求绿色健康。为了方便食品运输贮藏，人们往往在食品中添加防腐剂，如苯甲酸、山梨酸及其盐类、对羟基甲酸酯类、丙酸盐、二氧化硫、亚硫酸及其盐类、硝酸盐及亚硝酸盐类等，已经被普遍应用。长期使用化学防腐剂，导致致癌性、致突变性和致畸性等潜在安全问题时有发生，同时会对自然界的生态环境造成不利的影响。因此，寻找广谱、高效、低毒、安全的食品防腐剂成为食品领域研究的热点之一。
金黄色葡萄球菌是人类生活中最常接触的一种细菌之一，它在自然界中可以说无处不在，是引起食品污染以及细菌性食物中毒的重要细菌之一，食品作坊以及食品加工工厂都希望对其避而远之，其所分泌的肠毒素(SES)是引起食物中毒的最主要因素。通过李彦媚等发现，金黄色葡萄球菌对环境具有很强的适应性，其菌体细胞可耐受70℃的温度1h，80℃下30min不能被杀死灭活，在温度极低的环境下不被冻死照样生存，其所分泌的肠毒素在100℃的高温下煮沸30min还能够保持其免疫活性和生物活性，所以，在日常的生活中，我们是比较难做到让金黄色葡萄球菌远离我们的食物，俨然金黄色葡萄球菌已对人类健康与食品安全构成威胁，据了解，近年来金黄色葡萄球菌造成的食品安全事例层出不穷，据美国疾控中心报告，在美国，金黄色葡萄球菌引起的食物中毒居第二位，仅次于大肠埃希菌，占细菌性食物中毒约33％，在加拿大约占45％，在一些欧洲国家如匈牙利、芬兰则占食物中毒事件超过50％，特别是2000年发生的“雪印奶粉”事件，有14000多人受感染，所以我们更加期待一种更安全，天然的，无毒无害能够克制金黄色葡萄球菌的食品添加剂。
发明内容
本发明提供一种能够有效针对金黄色葡萄球菌的丁香酚组合液，可以使其更加广泛地应用到食品加工工艺、果蔬、粮油产品、奶制品、调味品等等领域当中，让其发挥更大作用，降低化学合成防腐剂的使用率，避免食品遭受细菌污染，保障食品安全卫生和人类身体健康。
为了解决上述技术问题，本发明是通过以下技术方案实现的：1.一种丁香酚组合液，由以下体积份的原料组成：水99.8555％～99.9711％，吐温80溶液0.01％～0.05％，丁香酚0.0189％～0.0945％。
制备上述丁香酚组合液的方法，包括步骤：向经过高压灭菌的、浓度为0.1％的吐温80溶液中加入丁香酚，经超声波震荡使丁香酚溶解，随后将溶液通过0.22μL的一次性滤头过滤灭菌，最后加水稀释制得丁香酚组合液。
制得的丁香酚组合液可作为金黄色葡萄球菌的抑菌剂或杀菌剂；作为抑菌剂的丁香酚组合液优选为吐温80溶液含量为0.03％，丁香酚含量为0.0567％；作为杀菌剂的丁香酚组合液优选为吐温80溶液含量为0.035％，丁香酚含量为0.06615％。
本发明的有益效果是：
丁香酚组合液能抑制金黄色葡萄球菌生长，甚至丁香酚浓度更高时可作为杀菌剂使用。丁香酚组合液影响了金黄色葡萄球菌的SDH酶和MDH酶的分子结构，使其发生了变化，从而改变了构像，使酶活性降低。丁香酚组合液作用于金黄色葡萄球菌后，因为起细胞透性的改变，使得与蛋白合成相关的核酸类物质外泄，进而导致蛋白质的合成受到抑制，并最终导致细胞死亡。
附图说明
图1是实施例10中空白对照样与丁香酚组合液测定样的对比图；
图2是实施例11中空白对照样与丁香酚组合液测定样的对比图。
图1中(a)对应未经丁香酚组合液处理的金黄色葡萄球菌；(b)-(f)分别对应丁香酚组合液F、E、D、C、B；图2中(a)对应未经丁香酚处理的金黄色葡萄球菌；(b)-(f)分别为丁香酚组合液F、E、D、C、B。
具体实施方式
下面通过具体实施方式结合附图对本发明作进一步详细说明。但本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1：制备丁香酚组合液A
向50份经过高压灭菌的吐温80溶液中加入94.5份丁香酚，经超声波震荡使丁香酚溶解，随后将溶液通过0.22μL的一次性滤头过滤灭菌，最后加99855.5份水稀释。
实施例2：制备丁香酚组合液B
向45份经过高压灭菌的吐温80溶液中加入85.05份丁香酚，经超声波震荡使丁香酚溶解，随后将溶液通过0.22μL的一次性滤头过滤灭菌，最后加99868.95份水稀释。
实施例3：制备丁香酚组合液C
向40份经过高压灭菌的吐温80溶液中加入75.6份丁香酚，经超声波震荡使丁香酚溶解，随后将溶液通过0.22μL的一次性滤头过滤灭菌，最后加99884.4份水稀释。
实施例4：制备丁香酚组合液D
向35份经过高压灭菌的吐温80溶液中加入66.15份丁香酚，经超声波震荡使丁香酚溶解，随后将溶液通过0.22μL的一次性滤头过滤灭菌，最后加99898.85份水稀释。
实施例5：制备丁香酚组合液E
向30份经过高压灭菌的吐温80溶液中加入56.7份丁香酚，经超声波震荡使丁香酚溶解，随后将溶液通过0.22μL的一次性滤头过滤灭菌，最后加99913.3份水稀释。
实施例6：制备丁香酚组合液F
向25份经过高压灭菌的吐温80溶液中加入47.25份丁香酚，经超声波震荡使丁香酚溶解，随后将溶液通过0.22μL的一次性滤头过滤灭菌，最后加99927.75份水稀释。
实施例7：制备丁香酚组合液G
向20份经过高压灭菌的吐温80溶液中加入37.8份丁香酚，经超声波震荡使丁香酚溶解，随后将溶液通过0.22μL的一次性滤头过滤灭菌，最后加99942.2份水稀释。
实施例8：制备丁香酚组合液H
向15份经过高压灭菌的吐温80溶液中加入30.24份丁香酚，经超声波震荡使丁香酚溶解，随后将溶液通过0.22μL的一次性滤头过滤灭菌，最后加9954.76份水稀释。
实施例9：制备丁香酚组合液I
向10份经过高压灭菌的吐温80溶液中加入18.9份丁香酚，经超声波震荡使丁香酚溶解，随后将溶液通过0.22μL的一次性滤头过滤灭菌，最后加99971.1份水稀释。
实施例10：对金黄色葡萄球菌的MIC测定
采用96孔板微量稀释法和琼脂平板稀释法。取96孔板，第一列为空白对照样，仅加入MH肉汤200μL。第2～10列为丁香酚组合液测定样，首先，每列的第一个孔加入MH肉汤100μL，第2～5个孔加入100μL金黄色葡萄球菌悬液；然后在第2～10列的第1～5个孔分别加入丁香酚组合液,使每个孔最终比例分别符合丁香酚组合液A～I的配比，同时每个孔的菌浓度为5*105CFU/mL。同时设置阳性对照。37℃培养24h，观察结果。以阳性对照中细菌生长呈浑浊和空白对照透明为前提，观察其他孔内浑浊情况。按下列标准比较：0.无可见生长；1.轻微模糊；2.浊度明显减少(约50％)；3.浊度轻微减少；4.浊度无明显改变。本试验药物取1.(轻微模糊)或2.(浊度明显减少)为判定终点。同时，以620nm的波长在酶标仪上读取吸光度，以备计算。
以抑菌率为90％的浓度为MIC，采用琼脂平板稀释法，取10μL菌悬液分别置于含丁香酚组合B、丁香酚组合C、丁香酚组合D，丁香酚组合E，丁香酚组合F的平板中，琼脂未凝固前摇匀，37℃培养24h，观察结果，实验重复3次，以平板未见菌落的最低药物浓度为MIC，实验重复3次。
实施例11：对金黄色葡萄球菌的MBC测定
以抑菌率为99％的浓度为MBC，从96孔板微量稀释法未见细菌生长各孔,接种至无菌琼脂，摇匀，37℃培养24h，未见菌落生长的为MBC，实验重复3次。
实施例12：丁香酚组合液对金黄色葡萄球菌体内MDH和SDH酶活性的影响
将金黄色葡萄球菌接分别接入LB培养基和含丁香酚组合液E的LB培养基，150rpm震荡培养24h，4000r/min离心培养液10min收集菌体，并用pH7.3的PBS缓冲液洗涤菌体3次，每组设置3个平行样。加入与菌体等体积的2mg/mL的溶菌酶溶液，搅拌后置于37℃水浴锅中，当菌体开始发粘时取出，按照1ml菌体加6mLPBS缓冲液，10000r/min低温离心10min,取出上清液备用。按照考马斯亮蓝法测定蛋白含量，以小牛血清蛋白对照。按照试剂盒说明书方法进行检测。
测定MDH：340nm处进行比色，双蒸水调零后，加入50μL上清液，取已预温好的1mL工作液迅速冲入0.5cm的石英比色皿中，于20s时读吸光度OD1值，反应1min后，即80s读取吸光度OD2值，记录ΔOD＝OD1值-OD2值，空白对照取50μL双蒸水，加入1ml工作液，其他操作与测定相同，于20s时读吸光度A1值，反应1min后，即80s读取吸光度A2值，记录记ΔA＝A1值-A2值以备计。
测定SDH：600nm处进行比色，蒸馏水调零后，加入100μL上清液，取已预温好的2.6mL工作液迅速冲入1cm的比色皿中，于5s时读吸光度OD1′值，反应1min后，即65s,读取吸光度OD2′值，记录ΔΟD′＝OD1′值-OD2′值，以备计算。
结果：1)如图1所示，与阳性对照相比，丁香酚组合液B、C、D、E、F对金黄色葡萄球菌具有抑制作用，其中最合适的比例为E，与琼脂平板稀释法一致，同时用96孔板法计算出来的抑菌率为90％；如图2所示，在丁香酚组合B、C、D对金黄色葡萄球菌有杀菌作用，其中最适比例为丁香酚D，同时用96孔板法计算出来的抑菌率为99％。不同成分比例的丁香酚组合液作对金黄色葡萄球菌的抑制作用存在依赖；
2)丁香酚组合液对金葡菌体内MDH酶活力的影响：对照组培养基中金黄色葡萄球菌的MDH酶活力17.151U/mgprot，丁香酚组培养基中金黄色葡萄球菌的MDH为8.214U/mgprot，通过配对样品T检验分析，p＜0.01，说明丁香酚组合对照组的MDH酶活性具有极显著差异；丁香酚组与对照组相比，细菌体内MDH酶活性降低；
3)丁香酚组合液对金葡菌体内SDH酶活力的影响：对照组培养基中金黄色葡萄球菌的SDH酶活力为4.00U/mL，丁香酚组培养基中金黄色葡萄球菌的SDH为2.400U/mL，通过配对样品T检验分析，p<0.05，说明丁香酚组合对照组的SDH酶活性差异显著；丁香酚组与对照组相比，细菌体内SDH酶活性降低。
丁香酚组合液E能抑制金黄色葡萄球菌生长，能对金黄色葡萄球菌具有杀菌作用为丁香酚组合液D。从酶活力的角度分析，可能的作用机理是丁香酚组合液影响了这两个酶的分子结构，使其发生了变化，从而改变了构像，使酶活性降低。最后，从丁香酚组合液对金葡菌蛋白浓度的影响结果表现，丁香酚组合液能够抑制部分蛋白质的合成。从蛋白质合成的角度来分析作用机理，可能的作用机理是丁香酚组合液对蛋白质作用于金黄色葡萄球菌后，因为起细胞透性的改变，使得与蛋白合成相关的核酸类物质外泄，进而导致蛋白质的合成受到抑制，并最终导致细胞死亡。
Claims (6)
1.一种丁香酚组合液，其特征在于由以下体积份的原料组成：水99.8555％～99.9711％，吐温80溶液0.01％～0.05％，丁香酚0.0189％～0.0945％。
2.权利要求1所述的丁香酚组合液的制备方法，包括步骤：向经过高压灭菌的、浓度为0.1％的吐温80溶液中加入丁香酚，经超声波震荡使丁香酚溶解，随后将溶液通过0.22μL的一次性滤头过滤灭菌，最后加水稀释制得丁香酚组合液。
3.应用如权利要求1所述的丁香酚组合液作为金黄色葡萄球菌的抑菌剂。
4.根据权利要求3的应用，其特征在于：所述丁香酚组合液中的吐温80溶液含量为0.03％，丁香酚含量为0.0567％。
5.应用如权利要求1所述的丁香酚组合液作为金黄色葡萄球菌的杀菌剂。
6.根据权利要求5的应用，其特征在于：所述丁香酚组合液中的吐温80溶液含量为0.035％，丁香酚含量为0.06615％。
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