培养温度、水分活度对稻谷和大米黄曲霉生长及产毒的影响
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摘要
为了解析不同培养温度和水分活度对稻谷和大米黄曲霉生长和产毒的影响,揭示温度和水分活度(a_w)调控稻米上黄曲霉生长和产毒的分子机制,以湖南黄华占稻谷和黑龙江稻花香大米为原料,分析稻谷和大米的真菌菌相,并采用高效液相色谱法测定稻谷和大米AFB_1含量,利用荧光定量PCR分析稻谷和大米水分活度及温度对黄曲霉毒素生物合成关键基因表达的影响。结果表明,当a_w降低至0.75以下,稻谷和大米上不适合黄曲霉生长繁殖和产毒。在33℃、a_w为0.96条件下大米黄曲霉产毒最多,33℃、a_w为0.90条件下大米黄曲霉的生长最好;稻谷则是在温度37℃,a_w为0.94和0.92条件下分别为最适产毒和生长,可见黄曲霉在稻谷和大米上生长和产毒的最适条件并不统一。荧光定量PCR结果显示,低温25℃可以促进黄曲霉产毒基因表达,但毒素在转录合成过程中受到了阻抑,高温37℃则抑制部分结构基因的表达,但菌的生长受到极显著影响而导致毒素量极低。因此,稻谷和大米储藏期及加工时,保持较低的a_w是有效预防黄曲霉侵染及AFB_1污染的关键。本研究结果为制定储藏及加工期稻谷和大米黄曲霉毒素污染的防控措施提供了一定的理论依据和数据支撑。
In order to analyze the effects of temperature and water activity(a_w)on the growth and toxicity of Aspergillus flavus in rice, we studied the molecular mechanism of temperature and water activity regulating the growth and toxin production of Aspergillus flavus on rice. Hunan paddy and Heilongjiang rice were selected as the raw materials. Analysis of fungi and determination of AFB_1 content in rice was done by high performance liquid chromatography. Finally, the effect of rice water activity and temperature on the expression of key genes of aflatoxin biosynthesis was investigate by fluorescence quantitative PCR. A. flavus grew very slowly when temperature ≤ 25℃ and a_(w )≤ 0.75.For the growth of A. flavus in rice, the maximum amounts of AFB_1 in the rice was produced by A. flavus at 33℃ and a_w was 0.96. The most suitable conditions for the growth of Aspergillus flavus were at 33℃ and a_w was 0.90. As for paddy, the maximum amounts of AFB_1 in the rice was produced by A. flavus at 37℃ and a_(w )was 0.94. The most suitable conditions for the growth of Aspergillus flavus were at 37℃ and a_wwas 0.92.The optimum conditions for the growth and AFB_1 production of Aspergillus flavus on rice were not uniform. The expression of toxin producing genes was promoted at 25℃, but the toxin was inhibited in the process of transcriptional synthesis. The expression of sporulation genes and some structural genes were inhibited, thereby inhibiting the production of toxin. Therefore, it is important to keep low a_w and temperature for preventing the infection of Aspergillus flavus and AFB_1 contamination during storage. The results of this study provide a theoretical basis to develop control methods for the whole course of Aspergillus flavus during storage and processing.
In order to analyze the effects of temperature and water activity(a_w)on the growth and toxicity of Aspergillus flavus in rice, we studied the molecular mechanism of temperature and water activity regulating the growth and toxin production of Aspergillus flavus on rice. Hunan paddy and Heilongjiang rice were selected as the raw materials. Analysis of fungi and determination of AFB_1 content in rice was done by high performance liquid chromatography. Finally, the effect of rice water activity and temperature on the expression of key genes of aflatoxin biosynthesis was investigate by fluorescence quantitative PCR. A. flavus grew very slowly when temperature ≤ 25℃ and a_(w )≤ 0.75.For the growth of A. flavus in rice, the maximum amounts of AFB_1 in the rice was produced by A. flavus at 33℃ and a_w was 0.96. The most suitable conditions for the growth of Aspergillus flavus were at 33℃ and a_w was 0.90. As for paddy, the maximum amounts of AFB_1 in the rice was produced by A. flavus at 37℃ and a_(w )was 0.94. The most suitable conditions for the growth of Aspergillus flavus were at 37℃ and a_wwas 0.92.The optimum conditions for the growth and AFB_1 production of Aspergillus flavus on rice were not uniform. The expression of toxin producing genes was promoted at 25℃, but the toxin was inhibited in the process of transcriptional synthesis. The expression of sporulation genes and some structural genes were inhibited, thereby inhibiting the production of toxin. Therefore, it is important to keep low a_w and temperature for preventing the infection of Aspergillus flavus and AFB_1 contamination during storage. The results of this study provide a theoretical basis to develop control methods for the whole course of Aspergillus flavus during storage and processing.
引文
[1] 寇光涛.东北稻米全产业链增值的创新路径及机制研究[D].北京:中国农业大学,2017
[2] 杜欣谊.东北三省水稻生产概况变动比较分析[J].农民致富之友,2016(7):76-77
[3] Castells M,Ramos A J,Sanchis Ⅴ.Distribution of total aflatoxins in milled fractions of hulled rice[J].Journal of Agricultural and Food Chemistry,2007,55(7):2760-2764
[4] 吴芳,严晓平,杨玉雪.我国农户2016年储藏稻谷黄曲霉毒素B1污染情况调查[J].粮食储藏,2017(6):35-39
[5] 滕南雁,宋宁宁,刘涛.广西地区市售食用植物油和大米中黄曲霉毒素B1的采样调查和分析[J].中国卫生检验杂志,2011,(6):1531-1532
[6] 李可,丘汾,杨梅,梁肇海,周海涛.深圳粮油食品中4种黄曲霉毒素联合污染状况[J].卫生研究,2013,42(4):610-614
[7] 白飞荣,姚粟,凌空,程池.黄曲霉和米曲霉的多相鉴定方法[J].微生物学通报,2018,45(1):215-226
[8] 乔宏兴,姜亚乐,王永芬,史洪涛,边传周.黄曲霉毒素的危害及其脱毒方法研究进展[J].动物医学进展,2017,38(1):89-93
[9] 武瑞霞,李云姣,霍超,王向红,桑亚新.乳及乳制品中黄曲霉毒素M1污染及检测技术研究进展[J].中国食品卫生杂志,2016,28(1):124-128
[10] Horn B W,Dorner J W.Regional differences in production of aflatoxin B1 and cyclopiazonic acid by soil isolates of Aspergillus flavus along a transect within the United States[J].Applied & Environmental Microbiology,1999,65(4):1444-1449
[11] Chawanthayatham S,Valentine C C,Fedeles B I,Slocum S L,Wogan G N.Abstract LB-229:Mutation spectra of aflatoxin B1 in vivo establish biomarkers of exposure for human hepatocellualr carcinoma[J].Cancer Research,2017,77(13 Supp):229
[12] Ayerst G.The effects of moisture and temperature on growth and spore germination in some fungi [J].Journal of Stored Products Research,1969,5(2):127-141
[13] Magan N,Lacey J.The effect of temperature and pH on water relations of field and storage fungi[J].Transactions of the British Mycological Society,1984,82:71-81
[14] Cotty P J,Jaime-Garcia R.Influences of climate on aflatoxin producing fungi and aflatoxin contamination[J].International Journal of Food Microbiology,2007,119(1/2):109-115
[15] 岳晓禹,李自刚,郝修振.主要生态因子对贮藏玉米中黄曲霉生长影响的模拟[J].农业工程学报,2013,29(18):269-276
[16] 蔡静平.储粮微生物活性及其应用的研究[J].中国粮油学报,2004,19(4):76-79
[17] Abdel-Hadi A,Carter D,Magan N.Temporal monitoring of the nor-l(aflD) gene of Aspergillus flavus in relation to aflatoxin B1 production during storage of peanuts under different water activity levels[J].Journal of Applied Microbiology,2010,109(6):1914-1922
[18] 张初署,刘阳,邢福国,魏丹丹,刘阳.七种培养基对黄曲霉分离效果的比较[J].核农学报,2013,27(2):208-212
[19] Cotty D P J.Comparison of four media for the isolation of Aspergillus flavus group fungi[J].Mycopathologia,1994,125(3):157-162
[20] Cui L,Selvaraj J N,Xing F.A minor survey of deoxynivalenol in Fusarium infected wheat from Yangtze-Huaihe river basin region in China[J].Food Control,2013,30(2):469-473
[21] 魏丹丹.黄曲霉菌不产毒的分子机制及其在控制黄曲霉毒素污染方面的研究[D].北京:中国农业科学院,2014
[22] 路子显,伍松陵,孙长坡.黄曲霉毒素合成相关基因表达与环境因素的关系[J].生物技术通报,2010(11):56-61
[23] Abdel-Hadi A,Schmidtheydt M,Parra R.A systems approach to model the relationship between aflatoxin gene cluster expression,environmental factors,growth and toxin production by Aspergillus flavus[J].Journal of the Royal Society Interface,2012,9(69):757-767
[24] Gallo A,Solfrizzo M,Epifani F.Effect of temperature and water activity on gene expression and aflatoxin biosynthesis in Aspergillus flavus on almond medium[J].International Journal of Food Microbiology,2015,217(1):162-169
[25] Schmidt-Heydt M,Abdel-Hadi A,Magan N,Geisena R.Complex regulation of the aflatoxin biosynthesis gene cluster of A.flavus in relation to various combinations of water activity and temperature[J].International Journal of Food Microbiology,2009,135(3):231-237
[26] Liu X,Guan X,Xing F,Lv C.Effect of water activity and temperature on the growth of Aspergillus flavus,the expression of aflatoxin biosynthetic genes and aflatoxin production in shelled peanuts[J].Food Control,2017,82:325-332
[27] Georgianna D R,Payne G A.Genetic regulation of aflatoxin biosynthesis:from gene to genome[J].Fungal Genetics & Biology,2009,46(2):113-125
[28] Br?se S,Encinas A,Keck J,Nising C F.Chemistry and biology of mycotoxins and related fungal metabolites[J].Chemical Reviews,2009,109(9):3903-3990
[29] Unnikrishnan Ⅴ,Nath B S.Hazardous chemicals in foods[J].Indian Journal of Dairy & Biosciences,2000,11:155-158
[30] O′Brian G R,Georgianna D R,Wilkinson J R.The effect of elevated temperature on gene transcription and aflatoxin biosynthesis[J].Mycologia,2007,99(2):232-239
[31] Yu J,Fedorova N D,Montalbano B G.Tight control of mycotoxin biosynthesis gene expression in Aspergillus flavus by temperature as revealed by RNA-Seq[J].Fems Microbiology Letters,2011,322(2):145-149
[32] 刘肖.花生储藏过程中水活度、温度对黄曲霉生长和产毒的影响[D].北京:中国农业科学院,2016
[2] 杜欣谊.东北三省水稻生产概况变动比较分析[J].农民致富之友,2016(7):76-77
[3] Castells M,Ramos A J,Sanchis Ⅴ.Distribution of total aflatoxins in milled fractions of hulled rice[J].Journal of Agricultural and Food Chemistry,2007,55(7):2760-2764
[4] 吴芳,严晓平,杨玉雪.我国农户2016年储藏稻谷黄曲霉毒素B1污染情况调查[J].粮食储藏,2017(6):35-39
[5] 滕南雁,宋宁宁,刘涛.广西地区市售食用植物油和大米中黄曲霉毒素B1的采样调查和分析[J].中国卫生检验杂志,2011,(6):1531-1532
[6] 李可,丘汾,杨梅,梁肇海,周海涛.深圳粮油食品中4种黄曲霉毒素联合污染状况[J].卫生研究,2013,42(4):610-614
[7] 白飞荣,姚粟,凌空,程池.黄曲霉和米曲霉的多相鉴定方法[J].微生物学通报,2018,45(1):215-226
[8] 乔宏兴,姜亚乐,王永芬,史洪涛,边传周.黄曲霉毒素的危害及其脱毒方法研究进展[J].动物医学进展,2017,38(1):89-93
[9] 武瑞霞,李云姣,霍超,王向红,桑亚新.乳及乳制品中黄曲霉毒素M1污染及检测技术研究进展[J].中国食品卫生杂志,2016,28(1):124-128
[10] Horn B W,Dorner J W.Regional differences in production of aflatoxin B1 and cyclopiazonic acid by soil isolates of Aspergillus flavus along a transect within the United States[J].Applied & Environmental Microbiology,1999,65(4):1444-1449
[11] Chawanthayatham S,Valentine C C,Fedeles B I,Slocum S L,Wogan G N.Abstract LB-229:Mutation spectra of aflatoxin B1 in vivo establish biomarkers of exposure for human hepatocellualr carcinoma[J].Cancer Research,2017,77(13 Supp):229
[12] Ayerst G.The effects of moisture and temperature on growth and spore germination in some fungi [J].Journal of Stored Products Research,1969,5(2):127-141
[13] Magan N,Lacey J.The effect of temperature and pH on water relations of field and storage fungi[J].Transactions of the British Mycological Society,1984,82:71-81
[14] Cotty P J,Jaime-Garcia R.Influences of climate on aflatoxin producing fungi and aflatoxin contamination[J].International Journal of Food Microbiology,2007,119(1/2):109-115
[15] 岳晓禹,李自刚,郝修振.主要生态因子对贮藏玉米中黄曲霉生长影响的模拟[J].农业工程学报,2013,29(18):269-276
[16] 蔡静平.储粮微生物活性及其应用的研究[J].中国粮油学报,2004,19(4):76-79
[17] Abdel-Hadi A,Carter D,Magan N.Temporal monitoring of the nor-l(aflD) gene of Aspergillus flavus in relation to aflatoxin B1 production during storage of peanuts under different water activity levels[J].Journal of Applied Microbiology,2010,109(6):1914-1922
[18] 张初署,刘阳,邢福国,魏丹丹,刘阳.七种培养基对黄曲霉分离效果的比较[J].核农学报,2013,27(2):208-212
[19] Cotty D P J.Comparison of four media for the isolation of Aspergillus flavus group fungi[J].Mycopathologia,1994,125(3):157-162
[20] Cui L,Selvaraj J N,Xing F.A minor survey of deoxynivalenol in Fusarium infected wheat from Yangtze-Huaihe river basin region in China[J].Food Control,2013,30(2):469-473
[21] 魏丹丹.黄曲霉菌不产毒的分子机制及其在控制黄曲霉毒素污染方面的研究[D].北京:中国农业科学院,2014
[22] 路子显,伍松陵,孙长坡.黄曲霉毒素合成相关基因表达与环境因素的关系[J].生物技术通报,2010(11):56-61
[23] Abdel-Hadi A,Schmidtheydt M,Parra R.A systems approach to model the relationship between aflatoxin gene cluster expression,environmental factors,growth and toxin production by Aspergillus flavus[J].Journal of the Royal Society Interface,2012,9(69):757-767
[24] Gallo A,Solfrizzo M,Epifani F.Effect of temperature and water activity on gene expression and aflatoxin biosynthesis in Aspergillus flavus on almond medium[J].International Journal of Food Microbiology,2015,217(1):162-169
[25] Schmidt-Heydt M,Abdel-Hadi A,Magan N,Geisena R.Complex regulation of the aflatoxin biosynthesis gene cluster of A.flavus in relation to various combinations of water activity and temperature[J].International Journal of Food Microbiology,2009,135(3):231-237
[26] Liu X,Guan X,Xing F,Lv C.Effect of water activity and temperature on the growth of Aspergillus flavus,the expression of aflatoxin biosynthetic genes and aflatoxin production in shelled peanuts[J].Food Control,2017,82:325-332
[27] Georgianna D R,Payne G A.Genetic regulation of aflatoxin biosynthesis:from gene to genome[J].Fungal Genetics & Biology,2009,46(2):113-125
[28] Br?se S,Encinas A,Keck J,Nising C F.Chemistry and biology of mycotoxins and related fungal metabolites[J].Chemical Reviews,2009,109(9):3903-3990
[29] Unnikrishnan Ⅴ,Nath B S.Hazardous chemicals in foods[J].Indian Journal of Dairy & Biosciences,2000,11:155-158
[30] O′Brian G R,Georgianna D R,Wilkinson J R.The effect of elevated temperature on gene transcription and aflatoxin biosynthesis[J].Mycologia,2007,99(2):232-239
[31] Yu J,Fedorova N D,Montalbano B G.Tight control of mycotoxin biosynthesis gene expression in Aspergillus flavus by temperature as revealed by RNA-Seq[J].Fems Microbiology Letters,2011,322(2):145-149
[32] 刘肖.花生储藏过程中水活度、温度对黄曲霉生长和产毒的影响[D].北京:中国农业科学院,2016