CN113215013A - 一株降解和抑制黄曲霉毒素的双功能深海海迪茨氏菌 - Google Patents
一株降解和抑制黄曲霉毒素的双功能深海海迪茨氏菌 Download PDFInfo
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Abstract
本发明提供一株不仅能降解黄曲霉毒素B1、也能抑制黄曲霉毒素合成的深海放线菌菌株，为海迪茨氏菌M2PC‑M2Mix1‑11菌株（Dietzia maris M2PC‑M2Mix1‑11），分离自在西太平洋牛郎平顶海山的山顶1617m水深处原位定向富集培养1年的培养物，已保藏于广东省微生物菌种保藏中心，保藏编号为GDMCC No.61241。将M2PC‑M2Mix1‑11菌株在M2培养基中发酵2‑5天获得的发酵无细胞离心上清液，在37度下1天对黄曲霉毒素B1的降解率达90.89±0.24%，对黄曲霉毒素合成的抑制率达100%。本发明的海迪茨氏菌M2PC‑M2Mix1‑11菌株及其发酵液不仅可用于田间作物栽培、粮食储藏、食品与饮料加工、饲料生产等的抑制黄曲霉毒素的产生，而且也可用于相关粮食、饮品、饲料等已经污染黄曲霉毒素的去除脱毒。
Description
技术领域：
本发明涉及深海微生物技术与真菌毒素生物防控技术领域，具体的说是一株降解和抑制黄曲霉毒素的双功能深海海迪茨氏菌。
背景技术：
黄曲霉毒素是自然界中已发现的毒性最强的真菌毒素，不仅其急性毒性强，而且具有致癌、致畸和免疫抑制性，被国际癌症机构列为I类致癌物质。自然界中的黄曲霉毒素主要包括B1、B2、G1、G2等，其中B1的毒性最强，其半数致死量为0.36毫克/公斤体重，为氰化钾的10倍，黄曲霉毒素中毒主要危害肝脏，出现肝炎、坏死、肝硬化等，黄曲霉毒素还可诱发多种癌症，如肝癌、胃癌、肾癌、直肠癌、乳腺癌等，其中以肝癌为主，其致癌力是剧毒农药“六六六”的1万倍。黄曲霉毒素可广泛污染粮食和饲料，导致食用者的身心健康受到严重危害，因此，如何抑制黄曲霉毒素的产生、以及如何降解已经产生的黄曲霉毒素，对保障粮食和饲料安全，防止或减少对人类和动物健康的危害，极为重要。
发明内容：
本发明所要解决的技术问题是克服上述现有技术的不足，本发明解决上述技术问题采用的技术方案是：一株降解和抑制黄曲霉毒素的双功能深海海迪茨氏菌，其特征包括如下步骤：
（1）本发明所说的深海海迪茨氏菌为海迪茨氏菌M2PC-M2Mix1-11菌株（DietziamarisM2PC-M2Mix1-11），并已于2020年10月26日保藏于广东省微生物菌种保藏中心，保藏单位地址为广州市先烈中路100号大院59号楼5楼，保藏编号为GDMCC No.61241；
（2）本发明所说的深海海迪茨氏菌M2PC-M2Mix1-11菌株分离自西太平洋牛郎平顶海山的山顶1617m水深处原位定向富集培养1年的培养物，通过以下方法分离获得：深海原位富集培养物用无菌水稀释10倍后，涂布到含有香豆素为唯一碳源的M2培养基，20度下恒温培养，及时挑取单菌落，并经进一步划线纯化，获得纯菌种M2PC-M2Mix1-11菌株；
（3）本发明所说的M2PC-M2Mix1-11菌株经16S rRNA基因分子鉴定为海迪茨氏菌（Dietzia maris )的一个新菌株；
（4）将本发明的深海海迪茨氏菌M2PC-M2Mix1-11菌株接种于M2培养基中，在18-28℃温度下，振荡培养2-5天，即可得到含有高效降解黄曲霉毒素和抑制黄曲霉毒素产生的活性物质的发酵液；
（5）本发明的发酵液无细胞离心上清液与黄曲霉毒素B1混合后，在37度培养1天，对黄曲霉毒素B1的降解率可达90.89±0.24%；
（6）本发明的发酵液无细胞离心上清液接入黄曲霉毒素产生菌，在28度培养6天后，可100%抑制黄曲霉毒素产生菌合成黄曲霉毒素；
（7）本发明的海迪茨氏菌M2PC-M2Mix1-11菌株及其发酵液不仅可用于田间作物栽培、粮食储藏、食品与饮料加工、饲料生产等的抑制黄曲霉毒素的产生，而且也可用于相关粮食、饮品、饲料等已经污染黄曲霉毒素的去除脱毒。
具体实施方式
下面结合实施例对本发明做进一步说明，但并不因此将本发明限制在所述的实施例范围之中。
实施例1：深海海迪茨氏菌M2PC-M2Mix1-11菌株的发酵
将过夜培养的海迪茨氏菌M2PC-M2Mix1-11菌株的液体种子，按4%接种量接种到含有乙酸钠 0.5%，蛋白胨 0.05%，酵母浸粉0.05%，牛肉膏0.05%，葡萄糖 0.05%，蔗糖 0.05%，可溶性淀粉 0.05%，柠檬酸三钠 0.005%，苹果酸 0.005%，酒石酸钾钠 0.005%，硝酸铵0.1%，氯化铵 0.02%、水100ml的液体培养基中，在18-28度、150-250转/分钟下培养2-5天，结束发酵，对发酵液进行离心，获得的无细胞上清液即是含有高效降解黄曲霉毒素和抑制黄曲霉毒素产生的活性物质的发酵液。
实施例2：深海海迪茨氏菌M2PC-M2Mix1-11菌株的发酵无细胞上清液降解黄曲霉毒素的活性
向实施例1中发酵2天获得的无细胞发酵上清液中，加入AFB1，使AFB1的终浓度为4微克/毫升，以相应的培养基加入AFB1使AFB1的终浓度为4微克/毫升为对照，在37度静止培养1天后，向对照和处理中加入氯仿提取AFB1，用色谱甲醇定容提取的AFB1，HPLC测定AFB1，监测波长为203nm，记录AFB1的峰面积，计算AFB1的降解率，降解率(%)=(对照AFB1的峰面积-无细胞发酵上清液AFB1的峰面积)/对照AFB1的峰面积x 100，经测定和计算，海迪茨氏菌M2PC-M2Mix1-11菌株的发酵无细胞上清液对黄曲霉毒素的降解率达90.89±0.24%。
实施例3：深海海迪茨氏菌M2PC-M2Mix1-11菌株的发酵无细胞上清液抑制黄曲霉毒素产生的活性
向实施例1中发酵5天获得的无细胞发酵上清液中，加入2%葡萄糖和0.5%酵母提取物，作为培养黄曲霉毒素产生菌-寄生曲霉的培养液，对照培养液为实施例1中的液体培养基中加入2%葡萄糖和0.5%酵母提取物，向上述培养基中分别接种1%的寄生曲霉的孢子悬液后，在28度培养6天，离心收集菌丝体，用甲醇-氢氧化钠溶液提取菌丝体中的黄曲霉毒素生物合成途径的中间产物，在560nm处测量光密度值（OD560），计算抑毒率，抑毒率(%)=(对照培养液的OD560-无细胞发酵上清液的OD560)/对照培养液的OD560 x 100，经测定和计算，海迪茨氏菌M2PC-M2Mix1-11菌株的发酵无细胞上清液对黄曲霉毒素合成的抑制率达100%，同时对寄生曲霉菌丝生长的抑制率为31.8%。
Claims (1)
1.一株分离自在西太平洋牛郎平顶海山的山顶1617m水深处原位定向富集培养1年的培养物的深海放线菌菌株，为海迪茨氏菌M2PC-M2Mix1-11菌株（Dietzia maris M2PC-M2Mix1-11），已于2020年10月26日保藏于 广东省微生物菌种保藏中心，保藏编号为GDMCCNo.61241。
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| CN117603818A (zh) * | 2023-11-14 | 2024-02-27 | 山东省农业科学院 | 分泌锰过氧化物酶的茶树菇菌株及其降解真菌毒素的应用 |
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| EA201800010A1 (ru) * | 2017-08-16 | 2019-02-28 | Товарищество С Ограниченной Ответственностью "Экостандарт.Kz" | Способ получения биологического препарата для очистки почвы от нефти и нефтепродуктов |
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| CN117603818A (zh) * | 2023-11-14 | 2024-02-27 | 山东省农业科学院 | 分泌锰过氧化物酶的茶树菇菌株及其降解真菌毒素的应用 |
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