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1 8 5 Vol. 8 No Journal of Food Safety and Quality May., 2017 魏 1, 超 2* 1,, 代晓航 2 1,, 郭灵安 2, 雷欣宇 1 (1., ; 2. (), ) 摘要 : 目的, 方法, ITS ; A(ochratoxin A, OTA) 结果 4 1, OTA, OAT 结论,,, 关键词 : ; ; A Identification and toxin-producing capability of causing-spoilage fungi in strawberry WEI Chao 1, 2*, DAI Xiao-Hang 1, 2, GUO Ling-An 1, 2, LEI Xin-Yu 1 (1. Analysis and Testing Center of Sichuan Academy of Agricultural Sciences, Chengdu , China; 2. Security Risk Assessment Laboratory of Ministry of Agriculture (Chengdu), Chengdu , China) ABSTRACT: Objective To investigate the main causing-spoilage fungi on the surface of strawberries and analyze whether it generates toxins. Methods The fungi were isolated from strawberries. Taxonomic status of fungi was conducted by colony morphology observation, reproduction microscopic observation and ITS gene sequence phylogenetic analysis. High performance liquid chromatography (HCLP) was used to detect the content of ochratoxin A (OTA) in spoilage strawberries and fungal fermentation broth. Results Four kinds of Mucor and one Penicillium could cause strawberry decay, which were identified as Mucor fragilis, Mucor circinelloides, Mucor racemosus, Rhizomucor variabilis and Penicillium sp. at molecular level. OTA could not be detected form spoilage strawberries and Mucor fermentation broth, but OAT toxins were detected in Penicillium fermentation broth. Conclusion Fungi are the main cause of strawberry decay, and Mucor spp. play a dominated role in the decay process, but Penicillium which can produce toxins coexist with it. Risk monitoring of strawberry and its products should be strengthened. KEY WORDS: strawberry; fungi; ochratoxin A 1 引言,, [1], 基金项目 : (GJFP ) Fund: Supported by the National Major Project for Risk of Assessment of Agricultural Products Quality and Safety (GJFP ) * 通讯作者 :,,,. cc123qqq@163.com *Corresponding author: WEI Chao, Master, Research Assistant, Analysis and Testing Center of Sichuan Academy of Agricultural Sciences; Security Risk Assessment Laboratory of Ministry of Agriculture (Chengdu), Chengdu , China. cc123qqq@163.com

2 1722 8, [2,3],,,, [4-6], [7,8] (Botrytinia spp.) (Penicillium spp.)(alternaria spp.) [9], [10,11] 5, ITS(internal transcribed spacer), A(ochratoxin A, OTA), 2 材料与方法 2.1 材料 20, , 20,, 3 kg 2.2 仪器与试剂 : PD ( ); YM-75-Z (); MJX-500 () : (1TS1): 5 -TCCGTAG GTGAACCTGCGG-3 ; (ITS4):5 -TCCTCCGCTTA TTGATATGC-3 ; (); DNA ( DP336 ) : (); 0.22 μm (); (, Fisher Scientific); OTA (SUPELCO ); Agilent 1200 ( ) 2.3 试验方法霉菌的分离培养 g, 25 g 10, 10 1 ~10 5, 1 ml 28 72~120 h, PDA 2~3, 72~90 h, PDA, 30 72~90 h PD h 霉菌的鉴定 (1), 28 1~2 d,, 1 (2) DNA PCR 25 μl : MasterMix 12.5 μl; ITS1 ITS4 1 μl DNA1 μl ddh 2 O 25 μl PCR : 95, 3 min; 94, 30 s 55, 30 s 72, 30, 35 ; 72, 7 min PCR, NCBI, PRIMER PREMIER, MAGA6.0 (maximum parsimony) OTA 的检测 3 5 OTA 20, 2~5 d, PD h, 4000 r/min 2 min : 20 g, 4 g NaCl(AR), 70% 100 ml, 1.5 h,, 5 ml 15 ml ddh 2 O, 4000 r/min 10 min, OTA,, 20 ml ddh 2 O, 1 ml ( ), 1 ml ddh 2 O : C 18 ; E x =330, E m =477; H 2 O: : =50:49:1(V:V:V); 1.0 ml/min; 7 min; 40 ; 20.0 μl 3 结果与分析 3.1 霉菌的分离与显微鉴定 1, 1,,,,, [9] 5 1, F-3547 F-3550 F-3766, F-3583, F 霉菌的 ITS 鉴定与归类分析 ITS GENE BANK, 2,,, F-3547 (Mucor fragilis), F-3550 (Mucor circinelloides), F-3766 (Mucor racemosus), F-3583 (Rhizomucor variabilis), F , 3

fungi F-3547,,,,,,,,,,,, F-3550,,,,,,,,,,

3 5, : 1723 Fig. 1 1 Photographs of spoilage strawberry infected by fungi Table 1 表 1 真菌的菌落形态及显微观察 Colony morphology and microscopic identification of fungi F-3547,,,,,,,,,,,, F-3550,,,,,,,,,, F-3766,,,,,,,,, F-3583,,,,,,,,,, F-3522,,,,,,,, 2 Fig. 2 Phylogenetic tree of Mucor spp. from strawberries

4 Fig. 3 Phylogenetic tree of Penicillium spp. from strawberries Table 2 表 2 霉菌的 ITS 鉴定结果 Identification results of fungi by ITS (%) F-3547 Mucor fragilis isolate IA4I2F2 18S ribosomal RNA gene F-3550 Mucor circinelloides genomic DNA containing 18S rrna gene F-3766 Mucor racemosus strain xsd S ribosomal RNA gene F-3583 Rhizomucor variabilis strain CBS S ribosomal RNA gene F-3522 Penicillium sp. genomic DNA sequence contains ITS Table 3 表 3 草莓样品及真菌发酵液的 OTA 检测 OTA detection of strawberries and fungal fermentation broth F~3547 F~3550 F~3766 F~3583 F~3522 OTA (ng/ml) ND ND ND ND ND ND ND 2.22 : ND 3.3 OTA 的检测 3 5 OTA, 3, 4, OTA, 10 ng/ml, 1 ng/ml, 2.3.3, 6, RSD<1.0%, [13], OTA, F-3522 OTA 2.22 ng/ml, OTA, 4 OTA 4 结论,,,,,,,, OTA,, PH,, [12-15],,

5 5, : OTA Fig. 4 HPLC chromatograms of OTA from strawberries and fungal fermentation broth : a: (standard); b:(rotten strawberries); c: F~3522 (fungi fermentation) 参考文献 [1],,,. [J]., 2016, 57(3): Zhang N, Hao HY, Fang XJ, et al. Advances in strawberry postharvest decay fungi disease control [J]. J Zhejiang Agric Sci, 2016, 57(3): [2],,,. [J]., 2014, 27(6): Wei C, Guo LA, Hu L, et al. Pesticide residues detection of strawberries surface and its effects on microflora [J]. Southwest China J Agric Sci, 2014, 27(6): [3],,,. 16SrDNA (I)[J]., 2016, 29(12): Wei C, Guo LA, Dai XH, et al. Research on the microorganism identification, homology analysis of 16SrDNA and adhesion of typical microorganisms on the surface of strawberry(i) [J]. Southwest China J Agric Sci, 2016, 29(12): [4] Antonio B, Barbara S, Daniela C, et al. Inhibition of spoiling yeasts of fruit juices through citrus extracts [J]. J Food Protect, 2013, 76(10): [5] Rosa M. Raybaudi-M, Jonathan MM, et al. Control of pathogenic and spoilage microorganisms in fresh-cut fruits and fruit juices by traditional and alternative natural antimicrobials [J]. Compreh Rev Food Sci Food Saf,

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6 , 8(3): [6] Nasu H, Hatamoto. Control of fruit mold of peach [J]. J Agric Food Chem, 2013,61(25): [7] Mantle PG. Risk assessment and the importance of ochratoxins [J]. Int Biodeterior Biodegrad, 2002, (50): [8] Moss MO. Risk assessment for aflatoxins in foodstuffs [J]. Int Biodeterior Biodegrad, 2002, (50): [9],. [J]., 2016, 9(7): Li JY, Gao XY. Research progress on the detection of key pathogenic microorganisms of fruit spoilage [J]. J Food Saf Qual, 2016, 9(7): [10],,,. A [J]., 2008, 6: 4 7. Yang JL, Yue TL, Gao ZP, et al. Research advance on the determination methods of ochratoxin A [J]. Acad Period Farm Prod Process, 2008, 6: 4 7. [11] Hamid B, Pierrette LB, Joseph LB. Thermo stability of ochratoxin A in wheat under two moisture conditions [J]. Appl Environ Microbiol, 1995, (61): [12]. [M]. :, Wei JC. Fungal identification manual [M]. Shanghai: Shanghai Science and Technology Press, [13] GB A [S]. GB National Food Safety Standard Determination of ochratoxin A in feeds [S]. [14] Stephen JF. Microbiological risk assessment of food [M]. Oxford: Blackwell Publishing Ltd, 2002 [15] FAO. Risk characterization of microbiological hazards in food: Guidelines [M]. Rome: Food & Agriculture Organization of the United Nations (FAO), 作者简介 ( 责任编辑 : 姚菲 ) 魏超, 硕士, 助理研究员, 主要研究方法为微生物检测及相关风险评估 cc123qqq@163.com

Microsoft Word 聂雪梅.doc

Microsoft Word 聂雪梅.doc 4 4 Vol. 4 No. 4 2013 8 Journal of Food Safety and Quality Aug., 2013 聂雪梅 *, 李立, 孙利, 高飞 (, 100123) 摘要 : 2011 12,, 2007~2011,,,, 关键词 : ; ; ; ; Trade and notification situation on food import and export