以合成桔霉素的关键基因为靶标的PCR检测方法与UPLC法检测红曲中桔霉素含量的一致性分析
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摘要
为了解菌株中桔霉素生物合成基因与其发酵产品红曲中桔霉素含量的相关性,本实验以25株不同来源的红曲菌为研究对象,以桔霉素合成的关键基因为标靶,设计6对引物进行PCR扩增;同时采用传统生产工艺制备红曲,采用免疫亲和柱纯化与UPLC结合的方法测定红曲中桔霉素的含量。对比PCR扩增结果与UPLC检测结果表明,PCR方法得到的结果与UPLC检测结果具有高度一致性,即PCR扩增桔霉素合成关键基因为阳性的菌株,其发酵制备的红曲中桔霉素含量超过了QB/T 2847-2007功能性红曲米(粉)的桔霉素限量标准50μg/kg,而PCR结果中桔霉素合成关键基因为阴性的,桔霉素含量均低于仪器检测限15.92μg/kg,远低于QB/T 2847-2007功能性红曲米(粉)的桔霉素限量标准。结果表明,本研究采用的PCR方法可以作为判断传统工艺条件下生产的红曲产品中桔霉素是否超标的依据,也为了解菌株分泌桔霉素的能力提供了基本信息。
The aim of this study was to understand the correlation between the key genes responsible for citrinin biosynthesis in the strain and the citrinin content in Hongqu. In this study,6 pairs of primers targeting the key genes responsible for citrinin biosynthesis were designed to amplify the corresponding region with genomic DNA of 25 different Monascus strains collected from various Hongqu products as templates. Simultaneously,the traditional production process was used to prepare Hongqu. The citrinin of these Hongqu purified by immuno-affinity columns was subjected to UPLC detection. The results showed an obvious consistency between the PCR amplification and UPLC detection. When the PCR amplification was positive targeting key genes responsible for citrinin synthesis,the citrinin content of Hongqu fermented by the described Monascus strains exceeded 50 μg/kg(the limit standard for citrinin in functional Hongqu,stated in QB/T 2847-2007);while the key genes of citrinin synthesis was negative by PCR amplification,the citrinin content was lower than the instrumental detection limit(15.92 μg/kg),which was much lower than the citrinin limit standard stated in QB/T2847-2007. These results suggested that the PCR method in this study not only could be used to judge whether citrinin in Hongqu produced by traditional process exceeded the limit of the standard stated by QB/T 2847-2007,also provided basic information for evaluating the ability of different strains to secret citrinin.
The aim of this study was to understand the correlation between the key genes responsible for citrinin biosynthesis in the strain and the citrinin content in Hongqu. In this study,6 pairs of primers targeting the key genes responsible for citrinin biosynthesis were designed to amplify the corresponding region with genomic DNA of 25 different Monascus strains collected from various Hongqu products as templates. Simultaneously,the traditional production process was used to prepare Hongqu. The citrinin of these Hongqu purified by immuno-affinity columns was subjected to UPLC detection. The results showed an obvious consistency between the PCR amplification and UPLC detection. When the PCR amplification was positive targeting key genes responsible for citrinin synthesis,the citrinin content of Hongqu fermented by the described Monascus strains exceeded 50 μg/kg(the limit standard for citrinin in functional Hongqu,stated in QB/T 2847-2007);while the key genes of citrinin synthesis was negative by PCR amplification,the citrinin content was lower than the instrumental detection limit(15.92 μg/kg),which was much lower than the citrinin limit standard stated in QB/T2847-2007. These results suggested that the PCR method in this study not only could be used to judge whether citrinin in Hongqu produced by traditional process exceeded the limit of the standard stated by QB/T 2847-2007,also provided basic information for evaluating the ability of different strains to secret citrinin.
引文
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[9]Wong H C,Koehler P E.Production and isolation of an antibiotic from Monascus purpureus and its relationship to pigment production[J].Journal of Food Sciences,2010,46(2):589-592.
[10]QB/T 2847-2007 功能性红曲米(粉)[S].
[11]GB 1886.181-2016 食品安全国家标准食品添加剂红曲红[S].
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[13]Wan Y,Zhang J,Han H,et al.Citrinin-producing capacity of Monascus purpureus in response to low-frequency magnetic fields[J].Process Biochemistry,2017,53:25-29.
[14]Xiong X,Zhang X,Wu Z,et al.Optimal selection of agricultural products to inhibit citrinin production during submerged culture of Monascus anka[J].Biotechnology Bioprocess Engineering,2014,19(6):1005-1013.
[15]Yang J,Chen Q,Wang W,et al.Effect of oxygen supply on Monascus pigments and citrinin production in submerged fermentation[J].Journal of Bioscience Bioengineering,2015,119(5):564-569.
[16]李琦,高健信,陈福生,等.不产桔霉素高产红曲色素的基因工程红曲菌株构建[J].中国酿造,2018(6):30-35.
[17]Sambrook J,Russel D W. Molecular cloning:a laboratory manual[R].New York:Cold Spring Harbor Laboratory Press,2001.
[18]Shao Y,Xu L,Chen F.Genetic diversity analysis of Monascus strains using SRAP and ISSR markers[J].Mycoscience,2011,52(4):224-233.
[19]Li Y P,Xu Y,Huang Z B.Isolation and characterization of the citrinin biosynthetic gene cluster from Monascus aurantiacus[J].Biotechnology Letters,2012,34(1):131-136.
[20]Feng Y,Chen W,Chen F.A Monascus pilosus MS-1 strain with high-yield monacolin K but no citrinin[J].Food Science and Biotechnology,2016,25(4):1115-1122.
[21]何坤. 不同红曲菌菌株代谢产物初步分析[D]. 武汉:华中农业大学,2017.
[22]贾离离,祁志红,彭立军,等.超高效液相色谱仪器检出限计算方法的比较分析[J].现代食品科技,2018(2):212-217.
[2]颜延宁.红曲色素的研究进展[J].轻工科技,2007,23(2):17.
[3]Demeyer D,Honikel K,Smet S.The world cancer research fund report 2007:A challenge for the meat processing industry[J].Meat Science,2008,80(4):953-959.
[4]吴衍庸.红曲酯化酶新技术及在中国白酒上的应用[J].酿酒科技,2004,25(6):249-250.
[5]Hong M Y,Seeram N P,Zhang Y,et al.Anti-cancer effects of Chinese red yeast rice beyond Monacolin K alone in colon cancer cells[J].Journal of Nutritional Biochemistry,2008,19(7):448-458.
[6]Blanc P J,Laussac J P,Le B J,et al.Characterization of monascidin A from Monascus as citrinin[J].International Journal of Food Microbiology,1995,27(2-3):201.
[7]Blanc P J,Loret M O,Goma G. Production of citrinin by various species of Monascus[J].Biotechnology Letters,1995,17(3):291-294.
[8]Ciegler A,Bennett J W. Mycotoxins and mycotoxicoses[J].Bioscience,1980,30(8):512-515.
[9]Wong H C,Koehler P E.Production and isolation of an antibiotic from Monascus purpureus and its relationship to pigment production[J].Journal of Food Sciences,2010,46(2):589-592.
[10]QB/T 2847-2007 功能性红曲米(粉)[S].
[11]GB 1886.181-2016 食品安全国家标准食品添加剂红曲红[S].
[12]许赣荣,陈蕴,顾玉梅,等.低产桔霉素红曲霉菌种筛选[J].无锡轻工大学学报,2000,19(4):358-360.
[13]Wan Y,Zhang J,Han H,et al.Citrinin-producing capacity of Monascus purpureus in response to low-frequency magnetic fields[J].Process Biochemistry,2017,53:25-29.
[14]Xiong X,Zhang X,Wu Z,et al.Optimal selection of agricultural products to inhibit citrinin production during submerged culture of Monascus anka[J].Biotechnology Bioprocess Engineering,2014,19(6):1005-1013.
[15]Yang J,Chen Q,Wang W,et al.Effect of oxygen supply on Monascus pigments and citrinin production in submerged fermentation[J].Journal of Bioscience Bioengineering,2015,119(5):564-569.
[16]李琦,高健信,陈福生,等.不产桔霉素高产红曲色素的基因工程红曲菌株构建[J].中国酿造,2018(6):30-35.
[17]Sambrook J,Russel D W. Molecular cloning:a laboratory manual[R].New York:Cold Spring Harbor Laboratory Press,2001.
[18]Shao Y,Xu L,Chen F.Genetic diversity analysis of Monascus strains using SRAP and ISSR markers[J].Mycoscience,2011,52(4):224-233.
[19]Li Y P,Xu Y,Huang Z B.Isolation and characterization of the citrinin biosynthetic gene cluster from Monascus aurantiacus[J].Biotechnology Letters,2012,34(1):131-136.
[20]Feng Y,Chen W,Chen F.A Monascus pilosus MS-1 strain with high-yield monacolin K but no citrinin[J].Food Science and Biotechnology,2016,25(4):1115-1122.
[21]何坤. 不同红曲菌菌株代谢产物初步分析[D]. 武汉:华中农业大学,2017.
[22]贾离离,祁志红,彭立军,等.超高效液相色谱仪器检出限计算方法的比较分析[J].现代食品科技,2018(2):212-217.