酸枣仁加工过程中黄曲霉毒素及污染真菌调查
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摘要
酸枣仁是中药材中黄曲霉毒素污染比较重的品种,为了揭示酸枣仁黄曲霉毒素污染的主要来源并明确产毒菌,收集了采收、加工、贮藏过程的32份酸枣仁样品,利用免疫亲和柱-HPLC柱后光化学衍生法测定黄曲霉毒素含量,通过稀释平板法分离污染真菌,结合形态学和分子生物学特征对菌株进行鉴定。结果表明,共28份样品有黄曲霉毒素检出,污染样品分布在从采收到贮藏的每个生产环节,其中有3份样品仅黄曲霉毒素B_1(AFB_1)超出《中国药典》限量标准,2份样品的AFB_1及总黄曲霉毒素(AFs)均超标,为水漂后及贮藏的样品。水漂后的样品AFB_1和AFs最高检出量分别达到94. 79,121. 43μg·kg~(-1)。所有样品均有真菌污染,新采收的鲜酸枣枣仁污染真菌总数最低,为2. 20×10~2CFU·g~(-1),随着加工过程的推进逐渐增加,水漂后样品的真菌总数升至1. 16×10~6CFU·g~(-1)。共分离鉴定了321株真菌,分属于17属,酸枣仁上黄曲霉毒素污染来源主要是采收加工环境中的黄曲霉Aspergillus flavus,并证实其中1株可以产AFB_1,AFB_2。酸枣采收后的堆沤环节真菌数量明显增加,脱去内果皮后的枣仁上曲霉属Aspergillus真菌成为优势菌群。堆沤和水漂环节增加了黄曲霉毒素污染风险。
Ziziphi Spinosae Semen is one of the Chinese herbal medicine being susceptible to aflatoxins contamination. To investigate the sources of aflatoxins contamination and toxigenic fungi species on Ziziphi Spinosae Semen,32 samples were collected from multiple steps during the post-harvest processing in this study. Aflatoxins in these samples were determined by immunoaffinity column and HPLC coupled with post-column photochemical derivatization. The dilution-plate method was applied to the fungi isolation. The isolated fungi strains were identified by morphological characterization and molecular approaches. The results showed that aflatoxins were detected in 28 samples from every step during the processing of Ziziphi Spinosae Semen. Three samples were detected with aflatoxin B_1 and 2 samples with both aflatoxin B_1 and total aflatoxin exceeding the limit of Chinese Pharmacopoeia. Especially the samples from the washing step,with the highest detected amounts of AFB_1 and AFs were reached 94. 79,121. 43 μg·kg~(-1),respectively. All 32 samples were contaminated by fungi. The fungal counts on the newly harvested samples were 2. 20 × 10~2 CFU·g~(-1). Moreover,it increased as tphreocessing progresses,and achieved 1. 16×10~6 CFU·g~(-1) after washing. A total of 321 isolates were identified to 17 genera. Aspergillus flavus was the main source of aflatoxins during the processing and storage of Ziziphi Spinosae Semen. One isolate of A. flavus was confirmed producing AFB_1 and AFB_2. The fungal count was significantly increased by composting,and Aspergillus was the predominant genus after shell breaking. The contamination level of aflatoxins was increased by composting and washing.
Ziziphi Spinosae Semen is one of the Chinese herbal medicine being susceptible to aflatoxins contamination. To investigate the sources of aflatoxins contamination and toxigenic fungi species on Ziziphi Spinosae Semen,32 samples were collected from multiple steps during the post-harvest processing in this study. Aflatoxins in these samples were determined by immunoaffinity column and HPLC coupled with post-column photochemical derivatization. The dilution-plate method was applied to the fungi isolation. The isolated fungi strains were identified by morphological characterization and molecular approaches. The results showed that aflatoxins were detected in 28 samples from every step during the processing of Ziziphi Spinosae Semen. Three samples were detected with aflatoxin B_1 and 2 samples with both aflatoxin B_1 and total aflatoxin exceeding the limit of Chinese Pharmacopoeia. Especially the samples from the washing step,with the highest detected amounts of AFB_1 and AFs were reached 94. 79,121. 43 μg·kg~(-1),respectively. All 32 samples were contaminated by fungi. The fungal counts on the newly harvested samples were 2. 20 × 10~2 CFU·g~(-1). Moreover,it increased as tphreocessing progresses,and achieved 1. 16×10~6 CFU·g~(-1) after washing. A total of 321 isolates were identified to 17 genera. Aspergillus flavus was the main source of aflatoxins during the processing and storage of Ziziphi Spinosae Semen. One isolate of A. flavus was confirmed producing AFB_1 and AFB_2. The fungal count was significantly increased by composting,and Aspergillus was the predominant genus after shell breaking. The contamination level of aflatoxins was increased by composting and washing.
引文
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[2]卫生部关于进一步规范保健食品原料管理的通知(卫法监发[2002]51号[EB/OL].(2002-03-11)[2018-10-31]. http://www.nhfpc.gov.cn/zwgkzt/wsbysj/200810/38057.shtml.
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[11] Chen A J,Jiao X L,Hu Y J,et al. Mycobiota and mycotoxins in traditional medicinal seeds from China[J]. Toxins,2015,7(10):3858.
[12]王鹭,杨骅,谢国祥,等.普洱茶、红茶、绿茶中真菌毒素的检测[J].中国中药杂志,2017,42(24):4801.
[13] Varga J,Frisvad J C,KocsubéS,et al. New and revisited species in Aspergillus section Nigri[J]. Stud Mycol,2011,69(1):1.
[14] Visagie C M,Houbraken J,Frisvad J C,et al. Identification and nomenclature of the genus Penicillium[J]. Stud Mycol,2014,78(3):343.
[15] Chen A J,Tang D,Zhou Y Q,et al. Identification of ochratoxin A producing fungi associated with fresh and dry liquorice[J].PLoS ONE,2013,8(10):e78285.
[16]于莉,吕国忠.东北地区茎点霉(Phoma)和叶点霉(Phyllosticta)两属真菌分类研究[J].沈阳农业大学学报,1994,25(2):153.
[17]宋美芳,陈娟,李学兰,等.云南地区三七和草果上真菌污染的初步分析[J].中国中药杂志,2012,37(12):1734.
[18] Su C Y,Hu Y J,Gao D,et al. Occurrence of toxigenic fungi and mycotoxins on root herbs from Chinese markets[J]. J Food Protect,2018,81(5):754.
[19] Culliao A G,Barcelo J M. Fungal and mycotoxin contamination of coffee beans in Benguet province,Philippines[J]. Food Addit Contam A,2015,32(2):250.
[20] Ezekiel C N,Sulyok M,Frisvad J C,et al. Fungal and mycotoxin assessment of dried edible mushroom in Nigeria[J]. Int J Food Microbiol,2013,162(3):231.
[21] Greco M,Pardo A,Pose G. Mycotoxigenic fungi and natural cooccurrence of mycotoxins in rainbow trout(Oncorhynchus mykiss)feeds[J]. Toxins,2015,7(11):4595.
[2]卫生部关于进一步规范保健食品原料管理的通知(卫法监发[2002]51号[EB/OL].(2002-03-11)[2018-10-31]. http://www.nhfpc.gov.cn/zwgkzt/wsbysj/200810/38057.shtml.
[3] The International Agency for Research on Cancer. Some naturally occurring substances:food items and constituents,heterocyclic aromatic amines,and mycotoxins,IARC monographs on the evaluation of carcinogenic risks to humans[M]. Lyon:World Health Organization,1993:245.
[4]郑荣,毛丹,王柯,等.柱后衍生-高效液相色谱法测定酸枣仁中黄曲霉毒素G2,G1,B2,B1[J].中国卫生检验杂志,2010,20(1):36.
[5]王少敏,张甦,陈洁,等. UHPLC-MS/MS测定中药材中4种黄曲霉毒素[J].中国卫生检验杂志,2014,24(2):190.
[6]杨文武,熊凌云,王瑞芳,等.免疫亲和柱净化HPLC挂后光化学衍生法测定34批中药材中黄曲霉毒素G2,G1,B2,B1[J].现代中药研究与实践,2013,27(1):43.
[7]刘丽娜,金红宇,孙磊,等.中药材及部分制剂中黄曲霉毒素残留筛查报告及初步风险评估[J].中国药学杂志,2015,50(17):1541.
[8]林方芬,郑志勇,黄剑,等.中药饮片中黄曲霉毒素B1含量的快速测定研究[J].江苏中医药,2016,48(12):65.
[9] Smedsgaard J. Micro-scale extraction procedure for standardized screening of fungal metabolite production in cultures[J]. J Chromatogr A,1997,760(2):264.
[10] Samson R A,Peterson S W,Frisvad J C,et al. New species in Aspergillus section Terrei[J]. Stud Mycol,2011,69(1):39.
[11] Chen A J,Jiao X L,Hu Y J,et al. Mycobiota and mycotoxins in traditional medicinal seeds from China[J]. Toxins,2015,7(10):3858.
[12]王鹭,杨骅,谢国祥,等.普洱茶、红茶、绿茶中真菌毒素的检测[J].中国中药杂志,2017,42(24):4801.
[13] Varga J,Frisvad J C,KocsubéS,et al. New and revisited species in Aspergillus section Nigri[J]. Stud Mycol,2011,69(1):1.
[14] Visagie C M,Houbraken J,Frisvad J C,et al. Identification and nomenclature of the genus Penicillium[J]. Stud Mycol,2014,78(3):343.
[15] Chen A J,Tang D,Zhou Y Q,et al. Identification of ochratoxin A producing fungi associated with fresh and dry liquorice[J].PLoS ONE,2013,8(10):e78285.
[16]于莉,吕国忠.东北地区茎点霉(Phoma)和叶点霉(Phyllosticta)两属真菌分类研究[J].沈阳农业大学学报,1994,25(2):153.
[17]宋美芳,陈娟,李学兰,等.云南地区三七和草果上真菌污染的初步分析[J].中国中药杂志,2012,37(12):1734.
[18] Su C Y,Hu Y J,Gao D,et al. Occurrence of toxigenic fungi and mycotoxins on root herbs from Chinese markets[J]. J Food Protect,2018,81(5):754.
[19] Culliao A G,Barcelo J M. Fungal and mycotoxin contamination of coffee beans in Benguet province,Philippines[J]. Food Addit Contam A,2015,32(2):250.
[20] Ezekiel C N,Sulyok M,Frisvad J C,et al. Fungal and mycotoxin assessment of dried edible mushroom in Nigeria[J]. Int J Food Microbiol,2013,162(3):231.
[21] Greco M,Pardo A,Pose G. Mycotoxigenic fungi and natural cooccurrence of mycotoxins in rainbow trout(Oncorhynchus mykiss)feeds[J]. Toxins,2015,7(11):4595.