CN116410868B - 一种产生杂色曲霉素的曲霉菌及其应用 - Google Patents
一种产生杂色曲霉素的曲霉菌及其应用 Download PDFInfo
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Abstract
本发明属于曲霉菌的技术领域，具体涉及一种产生杂色曲霉素的曲霉菌及其应用。所述曲霉菌经鉴定为詹森曲霉菌(Aspergillus jensenii)，所述詹森曲霉菌的保藏编号为GDMCC No:62668，其生物材料名称为Aspergillus jensenii DM‑33，其核酸序列如序列表SEQ IDNO.1所示，所述詹森曲霉菌及其产生的杂色曲霉素的可用于食品中杂色曲霉素风险评估、污染控制及其标准品制备等相关研究；以及所筛选的曲霉菌在大米培养基中，能够在较短的时间内(7天)产生杂色曲霉素，STC的产量为2.96μg/kg，在适合28℃的环境条件培养28d，STC的产量最大。
Description
技术领域
本发明属于曲霉菌的技术领域，具体涉及一种产生杂色曲霉素的曲霉菌及其应用。
背景技术
杂色曲霉素(Sterigmatocystin，STC)是1954年由初田勇一和久山真平首次从杂色曲霉(Aspergillus versicolor)培养物中分离出来，是含呋喃环的氧杂蒽酮类化合物。STC是黄曲霉毒素B1的合成前体，具有潜在的致癌性，被IARC划分为2B级致癌物。由于真菌可侵染农产品收获前后各个阶段，因此STC经常在农产品及其加工品中被检出，给相关行业造成经济损失的同时严重地威胁着人类健康。此外，由于数据的缺乏，世界各国还未对食品中的STC进行限量规定。鉴于此，对STC污染情况、生成机制、迁移及降解规律以及早期预警的研究已成为当今的研究热点。
目前对于STC的产生机制已经有了一定的进展，是由某些真菌代谢产生的多肽类物质，包括黄曲霉A.flavus，寄生曲霉(A.parasiticus)，构巢曲霉(A.nidulans)和杂色曲霉(A.versicolor)，其中A.versicolor是代谢STC的主要菌种。近期一项对潜在STC产生真菌的研究表明，包括翘孢霉属(Emericella)、曲霉属(Aspergillus)、毛壳菌属(Chaetomium)，枝葡萄孢属(Botryotrichum)和腐质霉属(Humicola)在内的55种真菌均可产生STC。
发明内容
针对上述问题，本发明的目的在于提供一种产生杂色曲霉素的曲霉菌及其应用。
本发明的技术内容如下：
本发明提供了一种产生杂色曲霉素的曲霉菌，所述曲霉菌经鉴定为詹森曲霉菌(Aspergillus jensenii)，所述詹森曲霉菌的保藏编号为GDMCC No:62668，其生物材料名称为Aspergillus jensenii DM-33，已于2022年7月29日保存到广东省科学院微生物研究所，地址为广州市先烈中路100号大院59号楼5楼；
所述詹森曲霉菌Aspergillus jensenii DM-33的核酸序列如序列表SEQ ID NO.1所示。
所述曲霉菌的培养方法为，置于大米培养基中，20～30℃培养并通过筛选得到；
所述大米培养基的组分为大米和水；
所述培养温度为28℃、静置培养28天时，所述曲霉菌产生杂色曲霉素的量达到最大；
当温度超过30℃，所述曲霉菌不产生杂色曲霉素。
本发明还提供了一种詹森曲霉菌的应用，所述詹森曲霉菌Aspergillus jenseniiDM-33用于生产杂色曲霉素；
所述詹森曲霉菌Aspergillus jensenii DM-33还包括用于食品中杂色曲霉素风险评估、污染控制及其标准品制备的研究。
本发明的有益效果如下：
本发明的产生杂色曲霉素的曲霉菌，所述曲霉菌命名为詹森曲霉菌(Aspergillusjensenii)，其保藏编号为GDMCC No:62668，所述詹森曲霉菌及其产生的杂色曲霉素的可用于食品中杂色曲霉素风险评估、污染控制及其标准品制备等相关研究；以及所筛选的曲霉菌在大米培养基中，能够在较短的时间内(7天)产生杂色曲霉素STC，其产量为2.96μg/kg，在28℃的环境条件培养28d，此时产量最大，而超过30℃之后，几乎不产生杂色曲霉素，为杂色曲霉素的产生环境提供了参考价值。
附图说明
图1为鉴定菌株在CA培养基上的生长形态；
图2为鉴定菌株在CYA培养基上的生长形态；
图3为鉴定菌株在PDA培养基上的生长形态；
图4为本发明的菌株DM-33菌丝体电子显微镜照片(X400)；
图5为本发明的菌株DM-33的孢子电镜照片(X1000)；
图6为本发明的菌株DM-33产杂色曲霉素的最适时间图；
图7为本发明的菌株DM-33产杂色曲霉素的最适温度图。
具体实施方式
以下通过具体的实施案例以及附图说明对本发明作进一步详细的描述，应理解这些实施例仅用于说明本发明而不用于限制本发明的保护范围，在阅读了本发明之后，本领域技术人员对本发明的各种等价形式的修改均落于本申请所附权利要求所限定。
若无特殊说明，本发明的所有原料和试剂均为常规市场的原料、试剂。
实施例
一种产生杂色曲霉素的曲霉菌的筛选以及鉴定
1)菌株的分离：取10.0g稻米样品，装入盛有250mL无菌水并带有玻璃珠的三角瓶中，充分振荡后依次稀释到浓度为10-5、10-6、10-7、10-8，分别取100μL的稀释液于PDA培养基中涂布，每个梯度3个平行，静置5min后放入恒温培养箱中，28℃，倒置培养7天；
所述PDA培养基的组分为：马铃薯(去皮)200.0g；葡萄糖20.0g；琼脂14.0g；最终pH5.6；
观察不同菌株的形态，待长出小菌落后，挑取形态不同的菌落接种于PDA培养基上纯化，反复进行平板划线，最终获得纯化菌株接种于上述PDA斜面上保存，随后对纯化得到的菌株进行测序以及产杂色曲霉素能力鉴定，对于不同形态的菌株进行初步鉴定，所获菌株及其产杂色曲霉素结果见表1。
表1分离菌种产杂色曲霉素能力的鉴定
由表1可见，根据形态不同挑取的菌落中，产生杂色曲霉素的仅有詹森曲霉菌，所筛选的詹森曲霉菌的核酸序列如序列表SEQ ID NO.1所示。
2)菌株的鉴定
将分离得到的詹森曲霉菌菌株分别接种到PDA、CA和CYA培养基，28℃培养8天，具体接种操作为：将接种针先在火焰上烧红灭菌并在平板培养基的边缘冷却且蘸湿，用针尖挑取一个孢子，以垂直法把接种针上的孢子点到预先标记好的部位，切勿刺破培养基；
2.1)根据菌落形态及显微特征鉴定，所筛选以及鉴定得的詹森曲霉菌菌株在CA培养基上菌落边缘不整齐，最外面有一圈白色菌丝，中间孢子颜色呈黄绿色，背面白色，表面干燥，无皱褶(图1)；在CYA培养基上边缘整齐，正面有褶皱，且从中间向外沿呈放射状，孢子整体颜色为棕黄色，菌落背面有深沟，颜色为棕色(图2)；在PDA培养基上菌落边缘整齐，最外面有一圈白色菌丝，孢子整体颜色为绿色，背面为白色，经标准对比鉴定为詹森曲霉菌(图3)；
如图4所示，光学显微镜下，PDA培养基上的菌株的分生孢子梗细长，顶端膨大呈顶囊，球形，梨形至匙形，分生孢子球形至近球形，少数椭圆形至梨形；如图5所示，为PDA培养基上的菌株分生孢子的电镜图；
结合菌株的菌落形态以及以上微观形态、ITS DNA同源性分析等方面的区别特征，进一步鉴定所筛选的菌株为詹森曲霉(Aspergillus jensenii)，于2022年7月29日保存到广东省科学院微生物研究所，保藏编号为GDMCC No:62668，其生物材料名称为Aspergillusjensenii DM-33。
3)菌株产杂色曲霉素能力测定
孢子悬液的制备：用吐温水洗下平板上新鲜、生长旺盛的孢子，然后移入装有玻璃珠的三角瓶中，在振荡器中充分震荡，使孢子均匀分散。
吸取孢子悬液1mL接入到大米培养基(120g大米，60mL水)中，培养温度为28℃，培养28天。
4)HPLC-MS/MS检测与鉴定方法
称取灭菌后的大米5.00g，加入25mL 90％的乙腈，充分震荡30min，超声3min，加入1.00g无水硫酸镁和1.00g NaCl，振荡1min，4500rpm离心5min。吸取5mL上清液至装有0.05gPSA中，振荡1min，4500rpm离心5min。吸取1mL上清液，过0.22μm滤膜后上机检测STC含量；
色谱柱：XR-ODSⅢC18色谱柱(75mm×2.0mm，1.6μm)；流动相A：含0.1％甲酸水；流动相B：乙腈；梯度洗脱程序：0min 30％流动相B；3.0min 50％流动相B；5.0min 80％流动相B；5.1min 30％流动相B；7.0min 30％流动相B；流速：0.3mL/min；柱温：40℃；进样体积：1μL。
离子源：电喷雾离子源；检测方式：多反应监测；离子源接口温度：300℃；脱溶剂温度：250℃；加热块温度：400℃；雾化气流速：3L/min；加热气流速：10L/min；干燥气流速：10L/min。
如图6和图7所示，可见本发明的杂色曲霉素的产生菌所产杂色曲霉素最适温度为28℃，发酵28天产量达到最大，以及其在30℃以上虽然菌体生长良好但不会产生杂色曲霉素。
本发明涉及的菌株在大米培养基中，在较短的时间内(7天)即可产生杂色曲霉素，产量为2.96μg/kg。
Claims (3)
1.一种产生杂色曲霉素的曲霉菌，其特征在于，所述曲霉菌为詹森曲霉菌Aspergillus jensenii，所述詹森曲霉菌的保藏编号为GDMCC No:62668，其生物材料名称为Aspergillus jensenii DM-33。
2.一种权利要求1所述的曲霉菌的应用，其特征在于，所述詹森曲霉菌Aspergillus jensenii DM-33用于生产杂色曲霉素。
3.根据权利要求2所述的曲霉菌的应用，其特征在于，所述詹森曲霉菌Aspergillus jensenii DM-33还包括用于食品中杂色曲霉素风险评估、污染控制研究及其标准品研制的研究和制备。
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