阅读说明：本技术 一种改善羟脯氨酸发酵前期溶菌的方法 (Method for improving bacterial lysis in early fermentation stage of hydroxyproline ) 是由 张孟涛 徐倩 封浪 张磊鹏 刘永超 马辉 于 2019-09-12 设计创作，主要内容包括：本发明涉及一种改善羟脯氨酸发酵前期溶菌的方法,属于发酵技术领域,所述方法首先收集羟脯氨酸发酵过程中污染的噬菌体,将收集到的噬菌体对羟脯氨酸发酵液进行侵染,分离纯化侵染后长出的羟脯氨酸菌株,挑选种子进行上罐验证,筛选出优良菌株进行发酵生产。在本发明的羟脯氨酸菌株经噬菌体侵染后,种子活力明显提升,发酵过程中不易受到噬菌体的侵染,有效改善了羟脯氨酸发酵前期易产生自溶的情况。(The invention relates to a method for improving bacterial lysis in the early fermentation stage of hydroxyproline, belonging to the technical field of fermentation. After the hydroxyproline strain is infected by the bacteriophage, the activity of seeds is obviously improved, the hydroxyproline strain is not easily infected by the bacteriophage in the fermentation process, and the condition that the hydroxyproline is easy to generate autolysis in the early fermentation stage is effectively improved.)

1. A method for improving the lysis of hydroxyproline in the early fermentation stage is characterized by comprising the following steps: firstly, collecting bacteriophage, infecting hydroxyproline fermentation liquor with the bacteriophage, culturing until the fermentation liquor becomes clear, then continuing culturing, making the fermentation liquor become turbid, selecting the turbid fermentation liquor for purification culture, selecting the purified strain for tank verification, screening the strain without bacteria dissolution, and performing hydroxyproline fermentation production by using the screened strain verified by tank verification.

2. The method for improving hydroxyproline pre-fermentation bacteriolysis as claimed in claim 1, wherein: the phage collection is to inoculate the fermentation liquor polluted by the phage to a bacterium-carrying plate for culture, and to obtain the needed phage after the plaque grows out.

3. The method for improving hydroxyproline pre-fermentation bacteriolysis as claimed in claim 1, wherein: the phage is infected with hydroxyproline fermentation liquor, a loop of phage is selected and transferred to hydroxyproline shake flask fermentation liquor of OD5-6 for culture, the fermentation liquor at the early stage of culture is changed from turbid to clear, the thalli are subjected to microscopic examination to generate fragments, the bacterial strain capable of resisting the phage can resist normal growth and reproduction after being continuously cultured for a period of time, the fermentation liquor becomes turbid again, the OD is obviously increased, and the fermentation liquor at the moment is taken to coat with a purified bacterial strain for selection.

4. The method for improving hydroxyproline pre-fermentation bacteriolysis as claimed in claim 1, wherein: the tank loading verification shows that the bacterial lysis phenomenon does not occur in a plurality of batches of continuous culture of the purified bacterial strain, and the growth vigor and the acid production capacity of the bacterial strain are improved.

5. The method for improving hydroxyproline pre-fermentation bacteriolysis as claimed in claim 4, wherein: the tank-loading verification is that the purified strain is continuously cultured for 12 batches.

Technical Field

The invention belongs to the technical field of fermentation engineering, and particularly relates to a method for improving bacterial lysis in the early fermentation stage of hydroxyproline.

Background

L-hydroxyproline is one of imino acids and is widely applied to the industries of medicine, chemical industry, food, beauty treatment and the like. The production method of L-hydroxyproline mainly comprises 3 methods, namely a biological tissue extraction method, a chemical synthesis method and a microbial fermentation method. The former two methods have the disadvantages of high cost, serious pollution and the like, and the fermentation method for producing the amino acid becomes a production method with the greatest prospect with the development of metabolic engineering and the advantages of the microbial method for producing the amino acid in recent years. At present, the strain used for L-hydroxyproline fermentation expansion is escherichia coli obtained through a gene recombination technology, the growth of seeds is normal in the test process, but the situations of a large amount of dissolved bacteria and OD reduction suddenly occur after 70% of batch culture is carried out for 3-6h after the strain is transferred into a fermentation tank, and even if the strain grows again after continuous culture, the acid production is extremely low, so that the fermentation failure is caused, and the serious economic loss is brought to the production.

Disclosure of Invention

In order to solve the defects in the prior art, the invention aims to provide a method for improving the bacteriolysis in the early fermentation stage of hydroxyproline, and the method adopts a bacteriophage dip-dyeing test to screen out a bacterial strain capable of resisting bacteriophage, so that the seed activity is improved, and the bacteriolysis problem in the fermentation process is improved.

In order to achieve the purpose, the invention adopts the specific scheme that:

a method for improving bacterial lysis in the early stage of hydroxyproline fermentation comprises the steps of collecting phage, infecting the hydroxyproline fermentation liquor with the phage, culturing until the fermentation liquor becomes clear, continuing culturing until the fermentation liquor becomes turbid, selecting the turbid fermentation liquor for purification culture, selecting a purified strain for tank loading verification, screening a strain without bacterial lysis, and performing hydroxyproline fermentation production by using the screened strain subjected to tank loading verification.

As a further optimization of the scheme, the phage collection is to inoculate the fermentation liquor polluted by the phage onto a bacterium-carrying plate for culture, and obtain the needed phage after growing the plaques.

As a further optimization of the scheme, the step of infecting the phage with hydroxyproline fermentation liquor is to select a loop of phage to be transferred to OD5-6 hydroxyproline shake flask fermentation liquor for culture, the fermentation liquor turns clear from turbid in the early stage of culture, the bacteria are subjected to microscopic examination to generate fragments, the bacterial strain capable of resisting the phage can normally grow and propagate after being continuously cultured for a period of time, the fermentation liquor turns turbid again, OD is obviously increased, and the fermentation liquor at the moment is coated with a purified bacterial strain for selection.

As a further optimization of the scheme, the tank loading verification shows that the bacterial strain after purification is continuously cultured for a plurality of batches without bacterial lysis, and the growth vigor and the acid production capacity of the bacterial strain are improved.

As a further optimization of the above protocol, the tank-in validation is that the purified strain is continuously cultured for 12 batches.

Has the advantages that:

after the fermentation liquor is infected by the phage, most thalli are autolyzed, but few bacterial strains capable of resisting the phage survive and are subjected to secondary propagation, bacterial liquid is selected from the culture solution subjected to secondary propagation for purification and culture, and the cultured bacterial strains have stronger vitality, can resist the invasion of external phage and other bacteria to the thalli, and further reduce the risk of fermentation and bacteriolysis. Compared with the prior art, the method has the advantages that the growth rate of the re-screened strain after infection is higher, the fermentation is more stable, the yield is increased, the cost is reduced, the discharge of waste liquid is reduced, and the environment is protected.

Detailed Description

A method for improving the lysis of hydroxyproline in the early fermentation stage specifically comprises the following steps:

1) collecting bacteriophage, inoculating fermentation liquor with autolysis of thallus to hydroxyproline-carrying bacterial plate for 8h, forming plaque, and refrigerating;

2) scraping a ring of the collected phage by fermentation infection, inoculating the phage into hydroxyproline shake flask fermentation liquor cultured by OD to 5-6, culturing for a period of time, clarifying the fermentation liquor, performing microscopic examination on thallus to generate fragments, and continuously culturing until the fermentation liquor becomes turbid, namely the thallus begins to reproduce again; 3) performing purification culture, streaking the re-increased thallus fermentation liquid plate to obtain a single colony, selecting the single colony for purification culture, and performing tank loading verification;

the fermentation liquor with thalli autolysis is characterized in that thalli autolysis occurs 3-6 hours after seed liquor is transferred into a fermentation tank, and the results mainly show that the fermentation liquor becomes clear, the OD (turbidity) is reduced, and the number of thalli is reduced by microscopic examination. The hydroxyproline strain is escherichia coli, the seed activity is obviously improved after the escherichia coli is infected by the phage, and the bacterial lysis phenomenon does not occur in 12 batches of continuous culture in the fermentation tank.

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention.