柠檬醛和丁香酚对黄曲霉生长的协同抑菌作用研究_艾淑娜.pdf

2023 年 2 月第 44 卷第 1 期河南工业大学学报(自然科学版)Journal of Henan University of Technology(Natural Science Edition)Feb.2023Vol.44 No.1收稿日期:2022-04-26基金项目:郑州市重大科技创新专项(2020CXZX0077)作者简介:艾淑娜(1998),女,河南洛阳人,硕士研究生,研究方向为食品安全控制理论与技术。通信作者:谢岩黎,教授,博导,E-mail:。柠檬醛和丁香酚对黄曲霉生长的协同抑菌作用研究艾淑娜,陈琳元,黄靖文,李倩,谢岩黎河南工业大学 粮油食品学院,河南 郑州 450001摘要:为了降低抑菌剂的用量及成本,同时达到更好的抑菌效果,分别检测麝香草酚、香芹酚、邻香兰素、柠檬醛和丁香酚的最小抑菌浓度(MIC),通过棋盘法筛选到具有协同抑菌作用的复配组合为柠檬醛和丁香酚,进一步研究其抑菌机制。测定柠檬醛和丁香酚协同处理下黄曲霉菌丝干重变化,通过碘化丙啶染色试验测定细胞内容物释放、相对电导率和 pH 值变化,检测其对细胞膜完整性的影响,通过荧光白染色试验研究其对隔膜的影响,最后评估其在花生中的抑菌效果。结果表明:麝香草酚、香芹酚、柠檬醛和丁香酚的 MIC 分别为 135、47、71、43 g/mL,通过联合抑菌法得出当 1/4 MIC柠檬醛+1/4 MIC丁香酚时,具有协同抑菌作用;柠檬醛和丁香酚协同处理能完全抑制菌丝生长、破坏黄曲霉细胞膜的完整性,细胞外 pH 值依次降低,相对电导率依次升高,黄曲霉菌丝内的隔膜被破坏;柠檬醛和丁香酚对黄曲霉的抗真菌活性与细胞膜通透性和细胞壁完整性受损有关;1/4 MIC柠檬醛+1/4 MIC丁香酚处理完全抑制了黄曲霉孢子在花生上的萌发。柠檬醛和丁香酚处理在防控黄曲霉污染中展示了良好的效果,同时协同处理方式降低了天然化合物的用量和成本。关键词:黄曲霉;柠檬醛;丁香酚;协同抑菌作用中图分类号:TS201.3文献标志码:A文章编号:1673-2383(2023)01-0059-09DOI:10.16433/j.1673-2383.2023.01.008Synergistic antifungal effect of citral and eugenol against the growth of Aspergillus flavusAI Shuna,CHEN Linyuan,HUANG Jingwen,LI Qian,XIE YanliCollege of Food Science and Engineering,Henan University of Technology,Zhengzhou 450001,ChinaAbstract:In order to reduce the dosage and cost of antifungal agents,five green and safe small molecular natural compounds thymol,carvacrol,o-vanillin,citral,and eugenol were selected in this study,and their antifungal effects against Aspergillus flavus(A.flavus)were determined by combination method.Firstly,the minimum inhibitory concentration(MIC)of each compound was detected individually.Then,through chessboard method,citral and eugenol showed a synergistic antifungal effect,and its antifungal mechanism was further studied.The changes of mycelial dry weight of A.flavus under the treatment of citral and euge-nol were measured.The effects on cell membrane integrity were detected by propidium iodide(PI)stai-ning,cell content release,relative conductivity and pH value changes.The effect on cell walls was studied by fluorescent white(CW)staining experiment.Finally,the antifungal effect on peanuts was evaluated.The results showed that the MIC of thymol,carvacrol,citral and eugenol were 135,47,71 and 43 g/mL,respectively.Citral and eugenol at the ratio of 1/4+1/4 could inhibit the growth of A.flavus,where the fractional inhibitory concentration index(FIC)was 0.5,indicating that they exerted a synergistic antifungal effect.The dry weight test of mycelia showed that the combination of citral and eugenol(1/41/4)河南工业大学学报(自然科学版)2023 年could completely inhibit the growth of mycelia.PI staining experiment showed that citral and eugenol combination treatment destroyed the integrity of A.flavus cell membranes.Compared with the control group,the release of cell content(OD260)in 1/16+1/16,1/8+1/8 and 1/4+1/4 groups increased by about 1.1,1.2,and 1.5 times respectively.Compared with the control group,at 180 minutes,the extracellular pH value of 1/16+1/16,1/8+1/8,and 1/4+1/4 decreased by 0.29,0.32,and 0.49 respec-tively.Compared with the control group,the relative conductivity of 1/16+1/16,1/8+1/8 and 1/8+1/8 increased by 5.02%,18.38%and 33.01%respectively.CW staining experiment showed that the number of septa decreased with the increase of citral and eugenol concentration.When the concentrations of citral and eugenol were 1/16+1/16 and 1/8+1/8,there were two septa per picture.When the concentrations of citral and eugenol were 1/8+1/8,the number of septa decreased to one.These results suggested that the synergistic antifungal activity of citral and eugenol against A.flavus was related to the impairment of cell membrane permeability and cell wall integrity.In addition,1/4+1/4 treatment completely inhibited the germination of A.flavus spores on peanuts.Therefore,citral and eugenol treatment showed a promis-ing efficacy in the prevention and control of A.flavus pathogenicity.Meanwhile,such combination treat-ment method reduced the dosage and cost of natural compounds,and provided a research approach for ensuring food safety.Key words:Aspergillus flavus;citral;eugenol;synergistic antifungal activity黄曲霉是一种空气传播的腐生真菌1,常污染收获前后的农作物,如花生和玉米等。黄曲霉具有 55 个次级代谢基因簇,能调控产生多种次级代谢物,如黄曲霉毒素、环匹阿尼酸等真菌毒素2。黄曲霉毒素是由曲霉产生的有害次级代谢产物,尤其是黄曲霉、寄生曲霉和诺氏曲霉3,是天然存在的剧毒物质,具有致突变性、致畸性、强致癌性,已被世界卫生组织的癌症研究机构列为 I 类致癌物质4。针对黄曲霉及黄曲霉毒素的防治,常见的方法及其优缺点如下:辐照法5、吸附法6、热处理7等物理方法,虽然操作简单、方便、高效,但会造成粮食的品质和营养价值下降;唑类、有机磷等化学杀菌剂,能有效控制粮食储藏期间的霉变,但是化学剂长期使用会导致菌体产生耐药性,而且会在粮食中残留,危害人体健康8;筛选抗黄曲霉的微生物等生物方法特异性强、污染小,能够保证食品营养安全,但该方法目前尚处于实验室阶段9。植物源天然化合物因高安全性和高生物利用度等优点成为近年来抑菌领域的一个研究热点。然而,低剂量的植物源天然化合物通常不能达到满意的抑菌效果,高剂量则会增加成本,限制其推广应用10。因此,解决上述问题的有效方式之一是联合使用不同的化合物,如香芹酚与百里香酚、百里香酚和柠檬醛、香芹酚和紫苏醛联合使用分别对茄镰刀菌11、沙门氏菌12和罗克福青霉13具有协同抑菌作用。这些化合物的抑菌机制多与破坏细胞膜的完整性有关,由于它们是疏水性化合物,能够和细胞膜的磷脂双分子层结合14。截至目前,发挥协同抑菌作用的植物源天然化合物的种类和数量仍然极为有限,筛选新型的协同组在食品的储藏和保鲜中具有重要的意义。作者首先检测了麝香草酚、香芹酚、邻香兰素、柠檬醛和丁香酚的最小抑菌浓度(minimum inhibitory concentration,MIC),然后通过联合抑菌法评价 5 种化合物两两复配的抑菌功效,确定了柠檬醛和丁香酚具有协同抑菌作用。在此基础上进行菌丝称重、细胞膜染色、细胞内容物释放、相对电导率、pH 值和细胞壁染色等试验,探索该协同组的抑菌机制,通过在花生上的抑菌试验评估其在短期储藏中的抑菌效果。该研究为植物源天然化合物在粮食抑菌领域的应用提供理论指导。1材料与方法1.1材料与试剂花生、