单离香料对黄曲霉的气相熏杀作用研究

第 34 卷第 2 期 2014 年 4 月 林摇 产摇 化摇 学摇 与摇 工摇 业 Chemistry and Industry of Forest Products Vol. 34 No. 2 Apr. 2014 doi:10. 3969/ j. issn. 0253鄄2417. 2014. 02. 021 Fungicidal Effect of Isolates on Aspergillus flavus by Fumigation 摇 摇 收稿日期:2013鄄04鄄22 摇 摇 基金项目:粤港关键领域重点突破项目(2009A020700005);广东省科技计划项目(2012B020316005);广东省教育部产学研结合项目 (2012B091100477) 摇 摇 作者简介:吴克刚(1961),男,贵州松桃人,教授,硕士生导师,研究领域包括食品绿色加工新技术、农产品的精深加工与安全保藏 技术及食品添加剂与食品质量安全控制技术;E鄄mail:wukegang2003163. com。 WU Ke鄄gang摇摇 WU Ke鄄gang1, LIN Ya鄄hui1,2, DUAN Xue鄄juan1, CHAI Xiang鄄hua1, ZHAO Xin鄄xin1 (1. Department of Food Engineering,Faculty of Chemical Engineering and Light Industry, Guangdong University of Technology, Guangzhou 510006, China; 2. Guangzhou Sugarcane Industry Research Institute, Guangzhou 510316, China) 摇 摇 摇 摇 摇 摇 摇 摇 摇 Abstract:The fungicidal effects of seven natural isolates were evaluated by flat plate fumigation and block transfer methods. Results showed the fungicidal activity of isolates was in the order of cinnamaldehyde thymol, salicylaldehyde, citral anethole eugenol. However, vanillin did not showed any fungicidal activity. The compound of salicylaldehyde, thymol, citral and cinnamaldehyde, in a ratio of 5颐 5颐 5颐 1, showed significant synergy effect, with the minimum fungicidal concentration (MFC) of 0. 031 25 mL/ L. SEM analysis showed that the isolate compound made the hypha of Aspergillus flavus become thinner, breakable and wizened, and the spores become shrunken and deformed.The compound affected the normal growth and metabolism of A. flavus, and led to fungistatic or fungicidal activity. The high electron density positions of isolate molecules were the active sites of fungicidal activity. More active sites with higher electron density and smaller steric hindrance resulted in the stronger fungicidal activity. The findings of the present study demonstrated the possible exploration of plants based isolates as safety preservatives against fungal spoilage during food stuffs storage. Key words:Aspergillus flavus;fungicidal activity;fumigation;isolates CLC number:TQ35摇 摇 摇 摇 Document code:A摇 摇 摇 摇 Article ID: 0253鄄2417(2014)02鄄0119鄄07 单离香料对黄曲霉的气相熏杀作用研究 吴克刚1, 林雅慧1,2, 段雪娟1, 柴向华1, 赵欣欣1 (1. 广东工业大学 轻工化工学院食品科学系, 广东 广州 510006;2. 广州甘蔗糖业研究所,广东 广州 510316) 摘摇 要:为了开发潜在的植源性单离香料用于控制食品储藏过程中的霉变,采用平板熏蒸法和菌块转移法研究 7 种单离 香料的杀菌作用。 实验表明,单离香料对黄曲霉的杀菌作用依次为肉桂醛 百里香酚,水杨醛,柠檬醛 茴香脑 丁香 酚,香兰素对黄曲霉基本没有作用。 水杨醛、百里香酚、柠檬醛和肉桂醛的质量比为5颐 5颐 5颐 1 时有显著的协同增效作用, 最小杀菌体积分数为 0. 031 25 mL/ L。 扫描电镜分析表明复合物使黄曲霉菌丝变薄、破裂、干枯,孢子收缩变形。 复配香 料影响黄曲霉的正常生理代谢,进一步导致抑制、杀灭作用。 单离香料分子的活性中心为高电子云密度处,活性中心电 子云密度高和空间位阻小,其杀菌效果更佳。 关键词:黄曲霉;杀菌作用;熏蒸;单离香料 According to Food Agriculture Organization of the United Nations (FAO) statistics, 25% of the grain supply in the world cannot be consumed due to the fungal contamination, among which Aspergillus flavus contamination is the most serious. A. flavus belongs to Aspergillus fungi and spreads by spore production. It 120摇 摇林摇 产摇 化摇 学摇 与摇 工摇 业第 34 卷 infects plants, plant products, food, feed, etc. , and poses a great threat to the production of crops and to the health of human, livestock and poultry1-2. The biggest harm of A. flavus is to produce A. flavus toxin (AFT), which is highly toxic biotoxin and recognized worldwide as one of the three strongest carcinogens3-4. AFT B1, B2, G1 and G2 are the main forms of AFT in grain and oil products. In particular, AFT B1 has the highest yield and the strongest carcinogenic activity, and its toxicity is much higher than cyanide, arsenic compounds and organic pesticides5-6. World Health Organization (WHO) prescribes that the maximum AFT concentration allowed in food is 15 滋g/ kg, and Food and Drug Administration (FDA) prescribes that the AFT B1 in human food cannot exceed 20 滋g/ kg. European Union implements the most stringent AFT limitation in the world, prescribing 2 滋g/ kg for AFT B1 and 4 滋g/ kg for total AFT. To prevent AFT contamination in food, it is the most primary and critical measure to kill A. flavus in contaminated grains to avoid further breeding and production of massive AFT. Lots of studies have shown that many plant essential oils have antifungal activity on A. flavus, but few on fumigation fungicidal effects7-11. In this study, we investigated into the fungicidal effects and their synergetic effects of several isolates existing in spices on A. flavus by fumigation, hoping to provide theoretical guidance for their application in preventing grain foods from mildewing by fumigation. 1摇 Experimental 1. 1摇 Materials and reagents Thymol, eugenol, cinnamaldehyde, anethole, salicylaldehyde, vanillin and citral were all in the purity of 99 % and were purchased from Guangdong Moda Food Chemical Co. , Ltd. Other reagents were of analytical grade. 1. 2摇 Test fungus A. flavus was purchased from Guangzhou Institute of Microbiology. The culture of test fungus A. flavus was maintained on Potato Dextrose Agar (PDA) medium (potato 200 g, dextrose 20 g, agar 18 g and distilled water 1 000 mL, pH 5. 6 依0. 2). The fungal suspension was adjusted to 106-107CFU/ mL before the tests. 1. 3摇 Fumigation and fungal block transfer experiments 20 mL sterilized culture medium was cooled to about 46 益 and poured into petri dish of 90 mm in diameter. Then 0. 1 mL of fungal suspension of 106- 107CFU/ mL was daubed evenly on the solidified medium with triangular glass rod. Isolates were dissolved in propylene glycol to a certain concentration. 0. 4 mL of isolate solution was dropped to the middle of each petri dish cover. A. flavus were incubated at 28益 for 5 days under the fumigation of the isolate solution. After incubation, the inhibitory concentrations (no visible culture in dish) were recorded. Blocks in 6 mm diameter from the inhibitory plates were removed to fresh PDA plate with sterile punch, and cultured consecutively at 28 益 for 5 days without the fumigation.During this period, the colony diameter was measured every day. Control set without isolate solution was kept parallel to the treatments. All tests were carried out in triplicate. 1. 4摇 Synergetic effects of fungicidal activity of isolates against A. flavus by fumigation Four isolates of stronger fungicidal ability on A. flavus by fumigation were screened out and assembled together to obtain an isolate compound. The optimal ratio among them was selected by orthogonal experiment designed with orthogonal chart L9(34), and the minimum fungicidal concentration (Cmin) of the isolate compound was determined. The synergetic effects among isolates were evaluated by joint fungicidal effect, which can be evaluated by fractional fungicidal concentration (CF). CFwas calculated by the following formula12: CF= Cmin(A)/ C忆min(A)+ Cmin(B)/ C忆min(B)+ Cmin(C)/ C忆min(C)+ Cmin(D)/ C忆min(D) where:Cminisolate A used jointly, mL/ L; C忆minisolate A used individually, mL/ L, A,B,C,D is thymol, 第 2 期吴克刚,等:单离香料对黄曲霉的气相熏杀作用研究121摇 摇 cinnamaldehyde, salicylaldehede,citral. Fungistatic rate = (1 - area of fungal spots with fumigation / area of fungal spots without fumigation) 伊100% 1. 5摇 Microscopic morphology of A. flavus fumigated by the isolate compound 0. 1mL of A. flavus spore suspension was inoculated on a PDA plate which was inserted into a sterilized glass slide at 45 益, and incubated at 28 益 for 5 days. 0. 4 mL of the isolate compound solution was then dropped to the petri dish cover. After being incubated for 24 h at 28 益 under the fumigation, the agar block and glass slide were cut to make electron microscopy samples by the following steps:fix samples in 25% glutaraldehyde solution overnight at 4 益, rinse samples for 40 min with 0. 1 mol/ L phosphate buffer (pH 7郾 0) for three times after discard fixative, fix samples in 1% osmium tetroxide solution for 2. 5 -3 h, rinse samples for 5 min with 0. 1 mol/ L phosphate buffer (pH 7. 0) for three times after discarding fixative, dehydrate samples for 10 min with different concentrations of ethanol (30%, 50%, 70 %, 80% and 90%), respectively, treat samples for 10 min with pure ethanol and tert鄄butanol for three times respectively, coat samples with gold in an ion coater after frozen鄄dry. Samples were observed by scanning electron microscopy JSM鄄6360LV (JEOL Ltd). 2摇 Results and Discussion 2. 1摇 Fungicidal activity of isolates against A. flavus by fumigation Antifungal activity of isolates against A. flavus by fumigation was shown in Table 1. Except for vanillin, fumigation of the other six isolates showed various degree of antifungal effects on A. flavus. Salicylaldehyde exhibited the strongest activity and inhibited completely the growth of A. flavus at 0. 031 25 mL/ L, followed by cinnamaldehyde at 0. 062 5 mL/ L, thymol, citral and anethole at 0. 125 mL/ L, eugenol at 0. 25 mL/ L. Table 1摇 Antifungal activity of isolates on A. flavus by fumigation1) concentration/ (mL L-1) blank control propylen glycol eugenolcitralsalicylaldehydeanetholethymolvanillincinnamaldehyde 0. 00780+ + + + + + + + + + 0. 01560+ + + + + + + + 0. 03125+ + + +-+ + 0. 06250+ + +-+ +- 0. 12500+ + +-+ +- 0. 25000+ + +-+ +- 0. 50000+ + +-+ +- 1) + + :good growth, + :bad growth, - : without growth;same as in the following tables To determine which antifungal activity of isolates against A. flavus was fungistatic or fungicidal, fungal block transfer experiments were performed for flat plates without fungal growth.Results were shown in Table 2. If the colony diameter of fungal block increases gradually with incubation time, isolates should show fungistatic effect on A. flavus at this concentration. If the colony diameter does not increase, isolates should show fungicidal effect at this concentration. As shown by the results, cinnamaldehyde, salicylaldehyde, thymol, citral, anethole and eugenol show fungicidal effect on A. flavus at the Cminof 0. 062 5, 0. 125, 0. 125, 0. 125, 0. 25 and 0. 5 mL/ L, respectively. The lower Cmin, the stronger fungicidal effect. Thus, cinnamaldehyde had the strongest fungicidal activity against A. flavus among the six isolates, and followed by salicylaldehyde, thymol, citral, anethole and eugenol. 2. 2摇 Synergetic effects of fungicidal activity of isolates against A. flavus by fumigation Salicylaldehyde, thymol, citral and cinnamaldehyde were chosen for further experiment according to orthogonal table L9(34). The total concentration of isolates was 0. 05 mL/ L, fungistatic rate was determined after the culturing at 28 益 for 5 days. The experimental design and results were shown in Table 3. 122摇 摇林摇 产摇 化摇 学摇 与摇 工摇 业第 34 卷 Table 2摇 Fungal block transfer experiments isolates concentration/ (mL L-1) colony diameter under different incubation time/ mm 1 day2 days3 days4 days5 days eugenol 0.2506661112 0.5066666 citral 0.12566666 0.25066666 0.5066666 salicylaldehyde 0.03125611143652 0.062568194159 0.12566666 0.25066666 0.5066666 anethole 0.125612131618 0.25066666 0.5066666 thymol 0.12566666 0.25066666 0.5066666 cinnamaldehyde 0.062566666 0.12566666 0.25066666 0.5066666 Table 3摇 Orthogonal experiment for synergetic effects of antifungal activity No. A thymol/ (mg L-1) B cinnamaldehyde/ (mL L-1) C salicylaldehyde/ (mL L-1) D citral/ (mL L-1) fungistatic rate/ % 10.020.020.020. 0215 20.020.040.060. 0650 30.020.060.100. 10100 40.060.020.060. 1030 50.060.040.100. 0240 60.060.060.020. 0615 70.100.020.100. 0695 80.100.040.020. 1055 90.100.060.060. 0255 k1 55472837 k228484553 k368577862 R40105025 The antifungal effects of the concentration of the optimized isolate compound on A. flavus was further Table 4摇 Effects of concentration of the isolate compound on fungistatic activity concentration/ (mL L-1)growth of A. flavus colony diameter after transfer 5 days/ mm Blank control+ +80 Propylene glycol+ +80 0. 0078+ +80 0. 0156+45 0. 03125-6 0. 0625-6 0. 125-6 investigated and shown in Table 4. The combined fungicidal effects of isolates in compound were shown in Table 5. Range analysis showed that the impacts of factors were in the order of salicylaldehyde thymol citral cinnamaldehyde.The lowest level of cinnamaldehyde was selected due to its minimum impact. Thus, the optimal combination was:salicylaldehyde鄄thymol鄄citral鄄cinnamaldehyde 5 颐 5 颐 5 颐 1.Fungal block transfer experiments showed that the MFC of the isolate compound against A. flavus by fumigation was 0.031 25 mL/ L. Interrelation among isolates is synergetic effect when CF臆0. 5, additive effect when 0. 5 OCH3 CH3 CH(CH3)2, and the electrophilic strength of substituent groups is in the order of CHO 詤CHCH2 詤CHCHCH3. Since various substituents have different electronic effects, the position, number, electron density and steric effects of active sites (shown as Fig. 2) are different. As shown in Fig. 2,cinnamaldehyde contained three active sites and showed the strongest fungicidal activity. Salicylaldehyde, thymol and citral contained two active sites and possessed higher fungicidal activity. Eugenol contained only one active site and had the lowest fungicidal activity. Vanillin also contained an active site, but it was connected to a strong electrophilic group and the electron density of active site was too low. So it didn忆t show any fungicidal activity. Although anethole had two active sites, the electrophobic effect of OCH3was weaker than OH, and the electrophilic effect of 詤CHCHCH3led to lower electron cloud density at active site and plused the steric hindrance. Thus its fungicidal activity was weaker than salicylaldehyde, thymol and citral, but stronger than eugenol. Fig. 2摇 Chemical structure of isolates(for high electron density region) 第 2 期吴克刚,等:单离香料对黄曲霉的气相熏杀作用研究125摇 摇 3摇 Conclusion Except for vanillin, all other six isolates had fungicidal activity on A. flavus. Cinnamaldehyde had the strongest fungicidal activity, followed by thymol, salicylaldehyde, citral an