CN109499039B - Luria-Bertani培养基在降解棒曲霉素中的应用 - Google Patents

Luria-Bertani培养基在降解棒曲霉素中的应用 Download PDF

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CN109499039B
CN109499039B CN201811390665.2A CN201811390665A CN109499039B CN 109499039 B CN109499039 B CN 109499039B CN 201811390665 A CN201811390665 A CN 201811390665A CN 109499039 B CN109499039 B CN 109499039B
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patulin
culture medium
bertani
tryptone
degradation
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田世平
李博强
张占全
徐勇
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Abstract

本发明公开了Luria‑Bertani培养基在降解棒曲霉素中的应用。本发明提供了Luria‑Bertani培养基或胰蛋白胨在降解棒曲霉素中的应用。还提供了Luria‑Bertani培养基或胰蛋白胨在制备降解棒曲霉素产品中的应用。本发明用Luria‑Bertani培养基脱除棒曲霉素不仅操作简单而且脱除效果能达到80％以上，具有广阔的应用前景。

Description

Luria-Bertani培养基在降解棒曲霉素中的应用
技术领域
本发明属于生物技术领域，尤其涉及一种Luria-Bertani培养基在降解棒曲霉素中的应用。
背景技术
棒曲霉素(patulin)是由青霉属、曲霉属等真菌产生的一种真菌毒素，具有潜在的致癌、致畸、致突变性。棒曲霉素污染引起的果蔬及其加工制品的食品安全问题给消费者的健康带来巨大的威胁，亟需研发有效的毒素脱除剂。目前，已有的棒曲霉素脱除剂绝大部分属于化学试剂，如各种化学氧化剂、巯基类化合物等，不但其脱除效果一般并且极易产生二次污染。还有一些脱除剂因成本高、安全性低，在实际应用中也未得到很好的推广。
发明内容
本发明的一个目的是提供Luria-Bertani培养基或胰蛋白胨的用途。
本发明提供了Luria-Bertani培养基或胰蛋白胨在降解棒曲霉素中的应用。
或，本发明提供了Luria-Bertani培养基或胰蛋白胨在制备降解棒曲霉素产品中的应用。
或，本发明提供了Luria-Bertani培养基或胰蛋白胨在使棒曲霉素降解为E-ascladiol中的应用。
或，本发明提供了Luria-Bertani培养基或胰蛋白胨在制备使棒曲霉素降解为E-ascladiol产品中的应用。
上述胰蛋白胨均为Luria-Bertani培养基中的胰蛋白胨。
上述应用中，所述Luria-Bertani培养基包括胰蛋白胨、酵母提取物和氯化钠。
本发明另一个目的是提供一种降解棒曲霉素的方法。
本发明提供的方法，包括如下步骤：将棒曲霉素在LB培养基中，培养，实现降解棒曲霉素。
本发明第3个目的是提供一种制备E-ascladiol的方法。
本发明提供的方法，包括如下步骤：将棒曲霉素在LB培养基中，培养，实现制备E-ascladiol。
上述方法中，所述棒曲霉素在LB培养基中的浓度为50mg/L。
上述方法中，所述培养时间为大于等于12小时。
Luria-Bertani培养基是实验室培养大多数细菌的常用培养基，其基本成分包括胰蛋白胨、酵母提取物以及氯化钠，生产成本低且无安全隐患，本发明用Luria-Bertani培养基脱除棒曲霉素不仅操作简单而且脱除效果能达到80％以上，具有广阔的应用前景。
附图说明
图1为Luria-Bertani培养基降解棒曲霉素检测结果。
图2为Luria-Bertani培养基中各组分对降解棒曲霉素的影响研究。
图3为Luria-Bertani培养基对棒曲霉素的降解作用与培养时间的关系。
图4为Luria-Bertani培养基对棒曲霉素的降解作用与培养时间的关系的HPLC检测结果。
图5为棒曲霉素和其降解产物E-ascladiol的紫外吸收光谱表征和HPLC检测结果。
具体实施方式
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
棒曲霉素(Macklin，P816488)。
实施例1、Luria-Bertani培养基降解棒曲霉素
一、Luria-Bertani培养基降解棒曲霉素的发现
LB(Luria-Bertani)培养基，其配方为胰蛋白胨10g/L、酵母提取物5g/L、氯化钠10g/L。
实验组：25μg棒曲霉素添加至500μl的LB培养基中，构成LB-棒曲霉素的反应体系，该体系于200rpm、28℃摇床中培养72h(胰蛋白胨：酵母提取物：氯化钠：棒曲霉素的质量比为＝200:100:200:1)；
MES对照组：25μg棒曲霉素添加至500μl的pH＝6的50mM MES缓冲液(9.76g MES溶于1L蒸馏水中，用NaOH调节pH)中，构成MES-棒曲霉素反应体系，该体系于200rpm、28℃摇床中培养72h；
反应结束后，收集上述两组反应液，用0.22μm孔径的滤膜过滤，取200μl于液相小瓶中，用高效液相色谱检测棒曲霉素残留量。色谱仪为美国Waters公司高效液相色谱仪，配备有自动进样器(Waters 2498)，双向HPLC泵(Waters 1525)，紫外检测器(Waters 2487)；所用色谱柱为岛津ODS反相柱(C18柱，5μm，250×4.6mm)；检测时，流动相为乙腈∶水＝10∶90(v/v)，流速1.0mL/min，所用柱温、检测波长、进样量分别为25℃、276nm、10μL。
结果如图1所示，实验组培养72h后棒曲霉素含量与MES对照组相比降低了80％以上；表明，LB培养基对棒曲霉素(棒曲霉素)具有一定的清除作用。
二、Luria-Bertani培养基中各组分对降解棒曲霉素的影响研究
Luria-Bertani培养基主要成分包括酵母提取物、胰蛋白胨以及氯化钠，通过对各成分缺失以后LB对棒曲霉素的清除能力来研究降解棒曲霉素的主要成分。
酵母提取物缺失组：25μg棒曲霉素添加至500μl的酵母提取物缺失的LB培养基中(胰蛋白胨10g/L、氯化钠10g/L、棒曲霉素50mg/L)中，200rpm、28℃培养72h；
胰蛋白胨缺失组：25μg棒曲霉素添加至500μl的胰蛋白胨缺失的LB培养基中(酵母提取物5g/L、氯化钠10g/L、棒曲霉素50mg/L)中，200rpm、28℃培养72h；
氯化钠缺失组：25μg棒曲霉素添加至500μl的氯化钠缺失的LB培养基中(配方：胰蛋白胨10g/L、酵母提取物5g/L、棒曲霉素50mg/L)中，200rpm、28℃培养72h；
MES对照组：25μg棒曲霉素添加至500μl的pH＝6的50mM MES缓冲液(9.76g MES溶于1L蒸馏水中，用NaOH调节pH)中(棒曲霉素浓度为50mg/L)，200rpm、28℃培养72h；
反应结束后，收集上述两组反应液，用0.22μm孔径的滤膜过滤，取200μl于液相小瓶中，用高效液相色谱检测棒曲霉素残留量。(检测方法同前)。
结果如图2所示，1-5依次为MES对照组、LB培养基组、氯化钠缺失组、胰蛋白胨缺失组、酵母提取物缺失组，可以看出，起棒曲霉素清除作用的主要成分为培养基中的胰蛋白胨。
三、Luria-Bertani培养基对棒曲霉素的降解作用与培养时间的关系
实验组：25μg棒曲霉素添加至500μl的LB培养基中，构成LB-棒曲霉素的反应体系(胰蛋白胨10g/L、酵母提取物5g/L、氯化钠10g/L、棒曲霉素50mg/L)，该体系于200rpm、28℃摇床中培养72h；
在不同培养时间取样，用HPLC检测棒曲霉素含量(检测方法同第一部分)。
结果如图3和图4所示，可以看出，跟踪观察0-72h的反应过程发现，随着时间延长，棒曲霉素含量逐渐减少，高效液相色谱检测发现，检测物除了在10min左右出现棒曲霉素特征峰以外，5.8min左右还会出现一个比较稳定的峰，且随着时间延长，该峰面积逐渐增加。
通过对5.8min左右的特征峰进行紫外吸收光谱表征(图5)，发现其最大吸收波长在268nm左右，与棒曲霉素降解产物E-ascladiol的最大紫外吸收波长一致，说明LB培养基在清除棒曲霉素的过程中可将棒曲霉素转化为E-ascladiol。

Claims (5)

1. Luria-Bertani培养基或胰蛋白胨在降解棒曲霉素中的非疾病诊断治疗方法的应用。
2.Luria-Bertani培养基或胰蛋白胨在制备降解棒曲霉素产品中的应用。
3.一种降解棒曲霉素的方法，包括如下步骤：将棒曲霉素在LB培养基中，培养，实现降解棒曲霉素。
4.根据权利要求3所述的方法，其特征在于：所述棒曲霉素在LB培养基中的浓度为50mg/L。
5.根据权利要求3所述的方法，其特征在于：所述培养时间为大于等于12小时。
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