CN116790404A - 一株高效降解玉米赤霉烯酮的贝莱斯芽孢杆菌 - Google Patents
一株高效降解玉米赤霉烯酮的贝莱斯芽孢杆菌 Download PDFInfo
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- CN116790404A CN116790404A CN202310139402.9A CN202310139402A CN116790404A CN 116790404 A CN116790404 A CN 116790404A CN 202310139402 A CN202310139402 A CN 202310139402A CN 116790404 A CN116790404 A CN 116790404A
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- bacillus
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- zearalenone
- velezensis
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Abstract
本发明公开一株玉米赤霉烯酮降解菌贝莱斯芽孢杆菌Bacillus velezensis PA26‑7菌株。该菌株已于2022年10月27日保藏于广东省微生物菌种保藏中心，保藏编号：GDMCC.No 62926。该菌分离自微生态制剂产品，是一株非致病性芽孢杆菌，可高效降解玉米赤霉烯酮，可应用于制备玉米赤霉烯酮脱毒菌剂，在农产品加工及饲料生产等领域具有良好的应用前景。
Description
技术领域
本发明属于微生物筛选及霉菌毒素降解技术领域。更具体地，涉及一株高效降解玉米赤霉烯酮的贝莱斯芽孢杆菌Bacillus velezensis PA26-7菌株。
背景技术
玉米赤霉烯酮(Zearalenone，ZEN)是由镰刀菌属真菌产生的一种非甾体雌激素类真菌毒素，广泛存在于霉变的玉米、小麦、高粱、大麦和燕麦等谷物中。ZEN可溶于碱性溶液和各种有机溶剂，其熔点达164℃-165℃。从农作物的种植到收获、运输、储存、碾磨加工，甚至高温烹饪等过程中，ZEN均可保持稳定，因此农产品及饲料中的ZEN难以被完全清除。此外，温度、湿度、大雾天气或虫害等多种环境因素，以及一些未遵循良好农业规范的操作都可能加剧ZEN污染。ZEN具有肝毒性、高雌激素性、免疫毒性、遗传毒性、致癌致畸性等，其存在不仅导致受污染的农产品质量下降，造成巨大的经济损失，同时这些受污染的谷物产品的国际贸易还进一步促成了ZEN的全球传播，危害公众健康。因此，采用合理高效的方法解决饲料中ZEN的污染问题尤为重要。
迄今为止，许多策略已被开发用于谷物和饲料中ZEN的清除，包括物理法、化学法和生物法。生物脱毒法是通过微生物细胞壁对ZEN的吸附作用或微生物分泌的酶对ZEN的降解作用以实现清除ZEN的目的。相比其他脱毒方法，生物法成本更低、效率更高、特异性更强，并且不会造成饲料和环境的二次污染，具有更高的安全性和实用性，已被证明是ZEN脱毒的最佳方法。
目前已发现多种可以代谢ZEN的微生物，包括粉红黏帚霉(Gliocladium roseum)、热带假丝酵母(Candida tropicalis)、鲁氏酵母(Zygosaccharomyces rouxii)、毕赤酵母(Pichia sp.)等在内的真菌和芽孢杆菌(Bacillus sp.)、假单胞菌(Psesudomonas sp.)鲁氏不动杆菌(Acinetobacter lwoffii)等在内的细菌。目前国内外ZEN降解菌虽有较多报道，但是多数降解菌存在降解效率不高、产物雌激素活性未知、菌株本身安全性未知等问题，因此筛选安全的高效降解菌株具有重要意义。
发明内容
本发明要解决的技术问题是克服现有的玉米赤霉烯酮降解技术的缺陷与不足，提供一株高效降解玉米赤霉烯酮(ZEN)的贝莱斯芽孢杆菌Bacillus velezensis PA26-7，该菌株不存在抗生素耐药性或其他潜在毒性，且其代谢产物的雌激素活性和毒性减弱，可应用于饲料和食品原料的脱毒。
本发明的目的是提供一株具备高效降解玉米赤霉烯酮(Zearalenone，ZEN)的贝莱斯芽孢杆菌Bacillus velezensis PA26-7。
本发明上述目的通过以下技术方案实现：
本发明研究从微生态制剂产品中筛选分离得到了一株高效降解ZEN的贝莱斯芽孢杆菌PA26-7菌株，可将ZEN代谢为雌激素活性和毒性减弱的产物。对该菌株的安全性评价结果表明该菌具备作为饲料添加剂的必要条件。因此，所述贝莱斯芽孢杆菌PA26-7菌株应在本发明的保护范围之内，该菌株已于2022年10月27日保藏于广东省微生物菌种保藏中心，保藏编号为GDMCC.No 62926。
与现有的技术相比，本发明具有以下有益效果：
本发明研究筛选分离得到了一株高效降解玉米赤霉烯酮的贝莱斯芽孢杆菌Bacillus velezensis PA26-7，可高效将ZEN代谢为毒性和雌激素活性减弱的物质，且该菌株是一株非致病性的芽孢杆菌，可应用于制备玉米赤霉烯酮的脱毒菌剂，在农产品加工、饲料生产等领域具有良好的应用前景。
附图说明
图1为产品PA26对ZEN的降解能力(A为ZEN的空白对照组，B为PA26产品悬液降解ZEN的效果图)。
图2为Bacillus velezensis PA26-7对ZEN的降解能力(A为ZEN的空白对照组，B为PA26-7降解ZEN的效果图)。
图3为Bacillus velezensis PA26-7对ZEN的降解产物细胞毒性。
图4为Bacillus velezensis PA26-7对ZEN的降解产物的雌激素活性。
图5为Bacillus velezensis PA26-7在5％羊血琼脂平板上的溶血毒性检测。
具体实施方式
以下结合说明书附图和具体实施例来进一步说明本发明，但实施例并不对本发明做任何形式的限定。除非特别说明，本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
实施例1微生态制剂的ZEN的降解活性
1、方法
(1)取1g市售微生态制剂，以10倍体积的无菌PBS溶液重悬后，按1％接种量接种至含有ZEN浓度为10μg/mL的LB培养基中，30℃震荡培养48h。
(2)取100μL发酵液，加入等体积甲醇充分震荡混匀，12000rpm离心5min，取上清液经0.22μm微孔滤膜过滤至上样瓶。
(3)使用高效液相色谱HPLC对ZEN残留量进行检测。
(4)ZEN降解率(％)＝(1-ZEN样品峰面积/ZEN对照峰面积)×100％
2、结果如附图1所示，微生态制剂产品对ZEN具有降解能力。
实施例2高效ZEN降解菌的分离鉴定
1、方法
(1)将PA26产品悬液按1％接种量接种含10μg/mL ZEN的MSM培养基中，30℃震荡培养，24h后转接至新的含ZEN的MSM培养基中，重复三到五次后涂布于LB琼脂培养基，培养24h后挑选多个单克隆经HPLC验证其降解ZEN的效果。
(2)经过上述分离筛选过程，最终得到一株降解能力最强的菌株，将其命名为PA26-7。其在含ZEN的LB培养基中培养48h后，培养基中ZEN浓度较少83.2％，证明其具有高效降解玉米赤霉烯酮的能力。
(3)使用DNA抽提试剂盒提取细菌基因组，以16S通用引物对该菌株16S rRNA目的基因进行扩增并测序，测序结果在NCBI上Blast比对鉴定该菌株种属。
2、结果如附图2所示，PA26-7对可降解培养基中83.2％的ZEN，经鉴定PA26-7是一株贝莱斯芽孢杆菌。
实施例3贝莱斯芽孢杆菌Bacillus velezensis PA26-7降解ZEN的产物细胞毒性检测
1、方法
(1)将PA26-7接种至ZEN终浓度为10、20、30μg/mL的LB培养基中，37℃，150rpm震荡培养48h，得到PA26-7降解ZEN产物溶液，同时设置不接种PA26-7的培养基为阴性对照。
(2)人肝癌细胞HepG2细胞复苏后传代培养至细胞生长状态良好，用0.25％胰酶-EDTA消化成单个细胞。用含10％BI血清的DMEM培养液调整细胞浓度为1×105，96孔板每孔接种100μL细胞悬液，96孔板边缘用无菌PBS缓冲液填充。
(3)5％CO2，37℃恒温培养24h至细胞贴壁。
(4)更换96孔板中的细胞培养液为各浓度ZEN及对应浓度下脱毒菌代谢ZEN 48h的产物。每个浓度设置5个平行孔，同时设置细胞培养液为空白对照。
(5)5％CO2，37℃恒温培养24h，更换细胞培养液后每孔加入10μL CCK-8溶液，37℃反应1h后测定每孔在450nm处的吸光度。
(6)计算细胞增殖率：细胞增殖率(％)＝(OD样品-OD阴性)/(OD阴性-OD空白)×100％
2、结果如附图3所示，贝莱斯芽孢杆菌Bacillus velezensis PA26-7降解各浓度ZEN产物的细胞毒性均显著降低。
实施例4贝莱斯芽孢杆菌Bacillus velezensis PA26-7降解ZEN的产物雌激素活性检测
1、方法
(1)按实施例3所述步骤制备PA26-7降解ZEN产物溶液。
(2)人乳腺癌细胞MCF-7细胞复苏后传代培养至细胞生长状态良好，用0.25％胰酶-EDTA消化成单个细胞。用无酚红高糖DMEM培养液调整细胞浓度为5×104，96孔板每孔接种100μL细胞悬液，96孔板边缘用无菌PBS缓冲液填充。
(3)5％CO2，37℃恒温培养24h至细胞贴壁。
(4)更换96孔板中的细胞培养液为各浓度ZEN及对应浓度下脱毒菌代谢ZEN 48h的产物。每个浓度设置5个平行孔，同时设置细胞培养液为空白对照。
(5)5％CO2，37℃恒温培养24h，更换细胞培养液后每孔加入10μL CCK-8溶液，37℃反应1h后测定每孔在490nm处的吸光度。
(6)计算细胞增殖率：细胞增殖率(％)＝(OD样品-OD空白)/-OD空白×100％
2、结果如附图4所示，贝莱斯芽孢杆菌Bacillus velezensis PA26-7降解各浓度ZEN产物的雌激素活性均显著降低，即对ZEN进行了有效脱毒。
实施例5贝莱斯芽孢杆菌Bacillus velezensis PA26-7溶血毒性检测
1、方法
(1)PA26-7接种至LB培养基中，37℃，150rpm震荡培养48h，取发酵液1000rpm离心5min，取上清。
(2)用牛津杯在5％羊血琼脂平板打孔，孔中加入100μL PA26-7发酵上清液，30℃培养24-48h，观察羊血平板上是否有溶血现象。
2、结果如附图5所示，贝莱斯芽孢杆菌Bacillus velezensis PA26-7在5％羊血琼脂平板上没有透明溶血圈形成，该菌株不具有溶血毒性。
实施例6贝莱斯芽孢杆菌Bacillus velezensis PA26-7耐药性检测
采用美国临床实验室标准化委员会(CLSI)推荐的微量肉汤稀释法，以ATCC 29213为质控菌株，测定PA26-7对红霉素、万古霉素、氨苄西林、利奈唑胺、替加环素、庆大霉素、链霉素、利福平、氟苯尼考、泰妙菌素的最低抑菌浓度，判断PA26-7药物敏感性。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都在本发明的保护范围内。
Claims (5)
1.一株高效降解玉米赤霉烯酮的贝莱斯芽孢杆菌Bacillus velezensis PA26-7，其特征在于，该菌株已于2022年10月27日保藏于广东省微生物菌种保藏中心，其保藏编号为GDMCC.No 62926。
2.根据权利要求1所述的贝莱斯芽孢杆菌Bacillus velezensis PA26-7，其特征在于，该菌是筛选、分离自市售微生态制剂产品。
3.根据权利要求1所述的贝莱斯芽孢杆菌Bacillus velezensis PA26-7，其特征在于，该菌株降解玉米赤霉烯酮的产物细胞毒性减弱。
4.根据权利要求1所述的贝莱斯芽孢杆菌Bacillus velezensis PA26-7，其特征在于，该菌株降解玉米赤霉烯酮的产物雌激素活性减弱。
5.根据权利要求1所述的贝莱斯芽孢杆菌Bacillus velezensis PA26-7，其特征在于，该菌株是一株非致病性的芽孢杆菌。
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