CN114621990A - 一种利用铜绿假单胞菌发酵生产鼠李糖脂的培养基 - Google Patents
一种利用铜绿假单胞菌发酵生产鼠李糖脂的培养基 Download PDFInfo
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Abstract
本发明涉及发酵产鼠李糖脂的方法，特别是涉及一种利用铜绿假单胞菌以及经过基因改造的铜绿假单胞菌工程菌发酵生产鼠李糖脂的培养基。按重量百分比计，碳源4％‑5％，氮源0.25％‑0.4％，磷酸盐1％‑2％，氯化钾0.09％‑0.15％，氯化镁0.06％‑0.08％，氯化钙0.01％‑0.03％，硫酸锰0.015％‑0.018％，硫酸锌0.02％‑0.03％，钼酸钠0.001％‑0.002％，余量为水，PH6.8‑7.0。本发明提供的培养基配方，经验证能够有效地促进菌株合成次级代谢产物鼠李糖脂，提高鼠李糖脂产量，并且使用廉价的碳源有利于降低鼠李糖脂发酵成本更适合于工业发酵。
Description
技术领域
本发明涉及发酵产鼠李糖脂的方法，特别是涉及一种利用铜绿假单胞菌以及经过基因改造的铜绿假单胞菌工程菌发酵生产鼠李糖脂的培养基。
背景技术
表面活性剂被广泛应用于生产生活中，目前，大部分表面活性剂是具有石化来源的化学合成表面活性剂，其大量使用会对环境和生态系统造成很大的风险。
鼠李糖脂作为生物来源的表面活性剂，具有优异的表/界面活性，易降解，环境友好，具有替代化学合成表面活性剂的潜力，然而，在实际的生产中鼠李糖脂产量普遍偏低，导致鼠李糖脂市场价格偏高，无法与价格低廉的化学合成表面活性剂相竞争,从而限制了鼠李糖脂的规模化市场化的应用，因此，提高发酵产量是鼠李糖脂应用领域需要解决的问题，培养基配方优化是提高鼠李糖脂产量的有效手段之一。
碳源和氮源是培养基最主要的成分，碳源包括亲水性和疏水性的碳源，亲水性碳源具有良好的水溶性更容易被菌株利用，而单位重量的疏水碳源相比其他结构的碳源来说含有更高的碳含量，即产同样重量鼠李糖脂所需的疏水碳源量更少，因此，少量亲水碳源复配疏水碳源，可以减少碳源使用量，还有利于前期鼠李糖脂的快速合成以及疏水碳源被鼠李糖脂乳化后更易被菌株利用，另外，使用廉价的碳源将进一步降低发酵成本，有机速效氮源虽然不利于鼠李糖脂的合成，却有利于前期菌体的快速繁殖，因此，发酵培养基添加少量的速效有机氮源复配无机氮源，使菌株生物量快速增加以合成鼠李糖脂，有利于缩短发酵周期。
发明内容
为了提高鼠李糖脂产量和工业化发酵鼠李糖脂，本发明提供一种利用铜绿假单胞菌以及经过基因改造的铜绿假单胞菌工程菌发酵生产鼠李糖脂的培养基。
为实现上述目的，本发明所采用的技术方案为：
一种利用铜绿假单胞菌发酵生产鼠李糖脂的培养基，按重量百分比计，碳源4％-5％，氮源0.25％-0.4％，磷酸盐1％-2％，氯化钾0.09％-0.15％，氯化镁0.06％-0.08％，氯化钙0.01％-0.03％，硫酸锰0.015％-0.018％，硫酸锌0.02％-0.03％，钼酸钠0.001％-0.002％，余量为水，PH6.8-7.0。
所述碳源为疏水碳源和亲水碳源；氮源为有机氮源和无机氮源；无机氮源为硝酸钾和/或硝酸钠；有机氮源为蛋白胨、酵母浸出物、牛肉膏中的一种或几种；磷酸盐为磷酸氢二钠和磷酸二氢钠
优选，按重量百分比计，亲水碳源1％，疏水碳源3％，无机氮源0.25％，有机氮源0.07％，磷酸氢二钠0.7％，磷酸二氢钠0.5％，氯化钾0.12％，氯化镁0.07％，氯化钙0.015％，硫酸锰0.016％，硫酸锌0.025％，钼酸钠0.001％，余量为水，PH6.8-7.0。
所述培养基用于利用铜绿假单胞菌或经过基因改造后的铜绿假单胞菌经好氧条件下发酵生产鼠李糖脂。
所述培养基用于好氧发酵生产鼠李糖脂过程中，培养温度为37℃，接种量为3％，转速为180rpm，溶解氧浓度范围是：1.0mg/L～15mg/L。
本发明具有以下优点：
本发明提供的培养基配方能够使铜绿假单胞菌的鼠李糖脂产量得到显著的提高，廉价的原料使鼠李糖脂发酵成本更低，使菌种更适合于工业发酵，有利于促进鼠李糖脂市场化和规模化的应用。
附图说明
图1为本发明实施例1，实施例2，实施例3所用的排油圈法测定鼠李糖脂产量的标准曲线。
具体实施方式
以下结合实例对本发明的具体实施方式做进一步说明，应当指出的是，此处所描述的具体实施方式只是为了说明和解释本发明，并不局限于本发明。
实施例1：
原始发酵培养基为：按重量百分比计，甘油10％(纯度≥99.7％)，硝酸钠0.6％，磷酸氢二钠0.1％，磷酸二氢钠0.1％，氯化钾0.08％，氯化钙0.007％，硫酸锌0.03％，氯化镁0.08％，余量为水，PH6.8-7.0。
培养基的制备方法：按重量百分比计，将发酵培养基除去甘油外的组分逐一与水混合均匀，最后加入甘油，并在115℃条件下蒸汽灭菌30min。
菌株为Pseudomonas aeruginosa SG(Pseudomonas aeruginosa SG相关记载参见Zhao F et al.2015Production of biosurfactant by a Pseudomonas aeruginosaisolate and its applicability to in situ microbial enhanced oil recoveryunder anoxic conditions)。
种子液培养方法：将铜绿假单胞菌SG在含LB的琼脂平板上划线活化后接种于斜面试管固体LB琼脂培养基上备用，试管中的菌种接种于含有50mL种子液的摇瓶中，于37℃，转速为180rpm，培养时间为16小时。种子液配方为：每100mL水中，含有酵母粉1g，蛋白胨1g，氯化钠1g，PH调节到7.0。
利用上述培养基通过铜绿假单胞菌发酵产鼠李糖脂的方法：按照3wt％的接种量将上述获得铜绿假单胞菌的种子液接种到上述发酵培养基中，培养温度为37℃，180rpm，培养时间为7天，在摇瓶水平进行3次重复的平行发酵实验，排油圈法测定鼠李糖脂产量，最终初始培养基发酵得到的鼠李糖脂产量为11.26g/L，
实施例2：铜绿假单胞菌培养基配方的优化
根据上述原始发酵培养基进行获得最适铜绿假单胞菌培养产鼠李糖脂的优化培养基：
(1)碳源的优化：氮源为硝酸钠用量为0.6％，其他培养基组分和含量与初始配方一致，单因素法考察不同碳源包括亲水碳源工业甘油(纯度≥80％)、蔗糖、葡萄糖、可溶性淀粉，疏水碳源工业废油、餐厨废油；以及疏水碳源与亲水碳源不同比例复配等对铜绿假单胞菌鼠李糖脂产量的影响，碳源用量为10％，按照3wt％的接种量将铜绿假单胞菌的种子液接种到发酵培养基中，培养温度为37℃，180rpm，培养时间为5天，结果表明工业甘油与疏水碳源复配时(质量比1：3)，有最大的鼠李糖脂产量(13.66g/L)，因此选为最佳碳源，其中疏水碳源为餐厨废油。
(2)氮源的优化：固定碳源为工业甘油和疏水碳源(质量比1：3)，碳源用量为10％，其他培养基组分和含量不变，单因素法考察了不同种类无机氮源包括硝酸钠、硝酸钾、硫酸铵、氯化铵，有机氮源包括：酵母浸出物、蛋白胨、牛肉膏等及无机氮源和有机氮源不同比例的复配，氮源用量为0.6％，按照3wt％的接种量将铜绿假单胞菌的种子液接种到发酵培养基中，培养温度为37℃，180rpm，培养时间为5天。实验结果显示硝酸盐和有机氮源复配(质量比3：1)，有最大的鼠李糖脂产量(14.07g/L)为最佳氮源。
(3)磷酸盐用量的优化：固定碳源为工业甘油和疏水碳源(质量比1：3)，碳源用量为10％，氮源硝酸盐和有机氮源复配(质量比3：1)，氮源用量为0.6％,磷源为磷酸氢二钠和磷酸二氢钠，其它培养基组分和用量维持和初始培养基一致，磷源用量梯度变化(0～3％)，按照3wt％的接种量将铜绿假单胞菌的种子液接种到发酵培养基中，培养温度为37℃，180rpm，培养5天,实验结果显示磷酸盐用量为1.2％时有最大的鼠李糖脂产量(14.89g/L)。
(4)微量元素：碳源为工业甘油和餐厨废油(质量比1：3)，用量10％，碳源为硝酸盐和有机氮源(3：1)，用量为0.6％，磷酸盐为磷酸氢二钠和磷酸二氢钠，用量为1.2％，添加硫酸锰0.016％，钼酸钠0.001％和硫酸锌0.025％，按照3wt％的接种量将上述获得铜绿假单胞菌的种子液接种到发酵培养基中，培养温度为37℃，180rpm，培养时间为5天，结果表明，鼠李糖脂产量为15.98g/L，相较未添加之前鼠李糖脂产量得到提高，微量元素的添加有利于合成鼠李糖脂。
(5)培养基各组分最优浓度用量：进行两水平的Plackett-Burman设计，每组实验设置三个平行，实验结果进行分析得到对鼠李糖脂产量有显著影响作用的培养基组分为：碳源，氮源和磷源。通过响应面法Box-Behnken实验设计拟合方程得到的最佳培养基配方用量为：亲水碳源1％，疏水碳源3％，无机氮源0.25％，有机氮源0.07％，磷酸氢二钠0.7％，磷酸二氢钠0.5％，氯化钾0.12％，氯化镁0.07％，氯化钙0.015％，硫酸锰0.016％，硫酸锌0.025％，钼酸钠0.001％，其中碳源包括疏水碳源和亲水碳源，亲水碳源为工业甘油，疏水碳源为工业废油和餐厨废油中的至少一种，氮源包括有机氮源和无机氮源，无机氮源为硝酸钾和硝酸钠中的至少一种，有机氮源为蛋白胨、酵母浸出物和牛肉膏中的至少一种，水补足剩余体积，PH6.8-7.0。
实施例3
优化后培养基为：按重量百分比计，亲水碳源1％，疏水碳源3％，无机氮源0.25％，有机氮源0.07％，磷酸氢二钠0.7％，磷酸二氢钠0.5％，氯化钾0.12％，氯化镁0.07％，氯化钙0.015％，硫酸锰0.016％，硫酸锌0.025％，钼酸钠0.001％，余量为水，PH6.8-7.0。
所述亲水碳源为工业甘油，疏水碳源为3％餐厨废油，无机氮源为0.25％硝酸钾，有机氮源为0.07％酵母浸出物。
培养基的制备方法：按重量百分比计，将发酵培养基除去疏水碳源外的组分逐一与水混合均匀，最后加入疏水碳源，并在115℃条件下蒸汽灭菌30min。
种子液培养的方法：铜绿假单胞菌在含LB的琼脂平板上划线活化后接种于斜面试管固体LB琼脂培养基上备用，试管中的菌种接种于含有100mL种子液的摇瓶中，于37℃，转速为180rpm，培养时间为16小时。种子液配方为：每100mL水中，含有酵母粉1g，蛋白胨1g，氯化钠1g，PH调节到7.0。
利用上述培养基通过铜绿假单胞菌发酵产鼠李糖脂的方法：按照3wt％的接种量将上述获得铜绿假单胞菌的种子液接种到发酵培养基中好氧条件下培养，培养温度为37℃，180rpm，溶解氧浓度7.5mg/L，培养时间为5天。
优化后培养基配方的验证：将铜绿假单胞菌以3wt％的接种量分别接种到原始发酵培养基和优化后的培养基中，培养温度为37℃，180rpm，培养时间为5天，在摇瓶水平进行3次重复的平行发酵实验，最终，优化后的培养基中获得摇瓶发酵鼠李糖脂平均产量为17.51g/L，较原始发酵培养基的11.26g/L提高了55.5％。因此，最终优化后培养基配中碳源和氮源用量都大幅度降低，并且使用了大量廉价的碳源，不仅有效提高了鼠李糖脂产量，并且有利于降低鼠李糖脂的发酵成本。
Claims (5)
1.一种利用铜绿假单胞菌发酵生产鼠李糖脂的培养基，其特征在于：按重量百分比计，碳源4％-5％，氮源0.25％-0.4％，磷酸盐1％-2％，氯化钾0.09％-0.15％，氯化镁0.06％-0.08％，氯化钙0.01％-0.03％，硫酸锰0.015％-0.018％，硫酸锌0.02％-0.03％，钼酸钠0.001％-0.002％，余量为水，PH6.8-7.0。
2.按权利要求1所述的利用铜绿假单胞菌发酵生产鼠李糖脂的培养基，其特征在于：所述碳源为疏水碳源和亲水碳源；氮源为有机氮源和无机氮源；无机氮源为硝酸钾和/或硝酸钠；有机氮源为蛋白胨、酵母浸出物、牛肉膏中的一种或几种；磷酸盐为磷酸氢二钠和磷酸二氢钠。
3.按权利要求2所述的利用铜绿假单胞菌发酵生产鼠李糖脂的培养基，其特征在于：按重量百分比计，亲水碳源1％，疏水碳源3％，无机氮源0.25％，有机氮源0.07％，磷酸氢二钠0.7％，磷酸二氢钠0.5％，氯化钾0.12％，氯化镁0.07％，氯化钙0.015％，硫酸锰0.016％，硫酸锌0.025％，钼酸钠0.001％，余量为水，PH6.8-7.0。
4.按权利要求1或2所述的利用铜绿假单胞菌发酵生产鼠李糖脂的培养基，其特征在于：所述培养基用于利用铜绿假单胞菌或经过基因改造后的铜绿假单胞菌经好氧条件下发酵生产鼠李糖脂。
5.按权利要求4所述的利用铜绿假单胞菌发酵生产鼠李糖脂的培养基，其特征在于：所述培养基用于好氧发酵生产鼠李糖脂过程中，培养温度为37℃，接种量为3％，转速为180rpm，溶解氧浓度范围是：1.0mg/L～15mg/L。
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