阅读说明：本技术 一种提高组氨酸发酵纯度的方法 (Method for improving histidine fermentation purity ) 是由 刘佳 王海波 谢畅丰 于 2021-10-20 设计创作，主要内容包括：本发明属于生物技术的发酵工程技术领域,具体涉及一种提高组氨酸发酵纯度的方法。它包括A、斜面活化培养,从冰箱取出保藏菌种的甘油管,均匀涂布斜面培养茄瓶进行活化,活化温度28-32℃,培养6-10h,形成菌落；B、种子过程控制,取50ml无菌水于步骤A的茄瓶,刮下菌落形成菌悬液,接入种子培养罐中的种子培养基中,培养温度28-32℃,流加氨水控制pH6.5-7.5,培养至种子液的OD650达到8-12；C、发酵过程控制,将步骤B的种子液接种量5-15％入发酵罐的发酵培养基中,控制温度28-32℃,流加氨水控制pH6.5-7.5,过程中控制溶氧20-50％。与现有组氨酸发酵技术相比,发酵放罐液中的组氨酸纯度提高13％以上,同时组氨酸的重量体积百分比也提高很多。(The invention belongs to the technical field of fermentation engineering of biotechnology, and particularly relates to a method for improving histidine fermentation purity. A, slant activation culture, namely taking a glycerol tube for preserving strains out of a refrigerator, uniformly coating a slant culture eggplant bottle for activation, and culturing for 6-10 hours at the activation temperature of 28-32 ℃ to form colonies; B. b, controlling a seed process, namely taking 50ml of sterile water to put into the eggplant bottle in the step A, scraping bacterial colonies to form bacterial suspension, inoculating the bacterial suspension into a seed culture medium in a seed culture tank, controlling the culture temperature to be 28-32 ℃, adding ammonia water in a flowing manner to control the pH to be 6.5-7.5, and culturing until the OD650 of a seed solution reaches 8-12; C. and C, controlling the fermentation process, namely adding 5-15% of the seed liquid inoculation amount in the step B into a fermentation culture medium in a fermentation tank, controlling the temperature to be 28-32 ℃, adding ammonia water in a flowing manner, controlling the pH to be 6.5-7.5, and controlling the dissolved oxygen to be 20-50% in the process. Compared with the prior histidine fermentation technology, the purity of histidine in the fermentation tank discharging liquid is improved by more than 13 percent, and the weight volume percentage of histidine is also improved greatly.)

1. A method for improving histidine fermentation purity comprises the following steps:

A. performing slant activated culture, taking out the glycerol tube for preserving strains from a refrigerator, uniformly coating a slant culture eggplant bottle for activation, and culturing at the activation temperature of 28-32 ℃ for 6-10h to form colonies;

B. b, controlling a seed process, namely taking 50ml of sterile water to put into the eggplant bottle in the step A, scraping bacterial colonies to form bacterial suspension, inoculating the bacterial suspension into a seed culture medium in a seed culture tank, culturing at the temperature of 28-32 ℃, feeding ammonia water to control the pH to be 6.5-7.5, culturing until the OD650 of a seed solution reaches 8-12, and controlling dissolved oxygen to be more than or equal to 30% in the process;

C. controlling the fermentation process, namely adding 5-15% of the seed liquid inoculation amount in the step B into a fermentation culture medium of a fermentation tank, controlling the temperature to be 28-32 ℃, adding ammonia water in a flowing manner to control the pH to be 6.5-7.5, and controlling the dissolved oxygen to be 20-50% in the process;

in the step C, after reducing sugar is reduced to 2-4% after the fermentation medium is hydrolyzed, sucrose with the concentration of 45-75% is fed, and the flow rate of the sucrose is adjusted to control the dissolved oxygen to be 20-50%.

2. The method for improving the purity of histidine fermentation as claimed in claim 1, characterized in that: the mixture ratio of the seed culture medium in the step B is that 20-40g/L of cane sugar, 3.0-11.5g/L of cane molasses, 12-18g/L of corn steep liquor, 0.5-2.0g/L of yeast powder, 2.0-5.0g/L of ammonium sulfate, 0.5-2.0g/L of potassium dihydrogen phosphate, 0.1-0.5g/L of magnesium sulfate, 0.5-2.0mg/L of manganese sulfate, 2-8mg/L of ferrous sulfate, 0.1-0.5mg/L of biotin, 0.1-0.5mg/L of thiamine and 0.1-0.5mg/L of thiamine are dissolved in water.

3. The method for improving the purity of histidine fermentation as claimed in claim 2, characterized in that: and B, dissolving the mixture ratio of the seed culture medium in water, wherein the mixture ratio of the sucrose is 35g/L, the cane molasses is 7g/L, the corn steep liquor is 18g/L, the yeast powder is 2g/L, the ammonium sulfate is 4g/L, the potassium dihydrogen phosphate is 1g/L, the magnesium sulfate is 0.5g/L, the manganese sulfate is 1mg/L, the ferrous sulfate is 5mg/L, the biotin is 0.1mg/L and the thiamine is 0.1 mg/L.

4. The method for improving the purity of histidine fermentation as claimed in claim 1, characterized in that: the proportion of the fermentation medium in the step C is that 40-120g/L of sucrose, 3.0-11.5g/L of cane molasses, 12-18g/L of corn steep liquor, 0.5-2.0g/L of yeast powder, 2.0-4.0g/L of ammonium sulfate, 0.5-2.0g/L of monopotassium phosphate, 0.1-0.5g/L of magnesium sulfate, 0.5-2.0mg/L of manganese sulfate, 2-8mg/L of ferrous sulfate, 0.1-0.5mg/L of biotin and 0.1-0.5mg/L of thiamine are dissolved in water.

5. Method for increasing the purity of histidine fermentation according to claim 4, characterised in that: and C, dissolving the fermentation medium in water according to the proportion of 80g/L of sucrose, 7g/L of cane molasses, 18g/L of corn steep liquor, 2g/L of yeast powder, 4g/L of ammonium sulfate, 1g/L of monopotassium phosphate, 0.5g/L of magnesium sulfate, 1mg/L of manganese sulfate, 5mg/L of ferrous sulfate, 0.2mg/L of biotin and 0.2mg/L of thiamine.

6. The method for improving the purity of histidine fermentation as claimed in claim 1, characterized in that: and B, performing slant activation culture at the activation temperature of 30 ℃ for 8 h.

7. The method for improving the purity of histidine fermentation as claimed in claim 1, characterized in that: and B, in the step B, in the seed process control, the culture temperature is 30 ℃, ammonia water is fed in to control the pH to be 7.0, and the seed is cultured until the OD650 of the seed solution reaches 10.

8. The method for improving the purity of histidine fermentation as claimed in claim 1, characterized in that: and C, in the fermentation process control of the step C, 10% of seed liquid inoculation amount is fed into a fermentation tank, the temperature is controlled to be 30 ℃, ammonia water is fed in a flow manner to control the pH to be 7.0, dissolved oxygen is controlled to be more than or equal to 30% within 10 hours before fermentation, and then the dissolved oxygen is controlled to be 20-30%.

9. The method for improving the purity of histidine fermentation as claimed in claim 1, characterized in that: and C, when the cane sugar is fed in the step C, the cane sugar with the concentration of 60% is fed in after the reducing sugar is reduced to 3% after hydrolysis is controlled by the process, and the flow rate of the cane sugar is adjusted to control the dissolved oxygen to be 20-30%.

Technical Field

The invention belongs to the technical field of fermentation engineering of biotechnology, and particularly relates to a method for improving histidine fermentation purity.

Background

Histidine is one of 20 amino acids and is widely used in the fields of medicines, food additives, feeds and the like. At present, the preparation method for producing histidine by a domestic fermentation method is relatively popular, and various invention patents apply for genetic engineering modification and fermentation application of related strains, but most of the preparation methods are still in an experimental research and development stage. However, in the field of fermentation engineering for practical industrial production, there are few studies on histidine fermentation engineering processes. In the prior technical scheme, the histidine producing bacteria generally have the problems of low histidine content and low histidine purity in fermentation tank discharging liquid and high difficulty in post-extraction treatment caused by other amino acids in industrial application.

Disclosure of Invention

Aiming at the technical problems, the invention controls the dissolved oxygen level of fermentation to reach the specified range by adjusting various technical parameters of slant activation culture, bacterial colony control in the seed process and fermentation process control and feeding and supplementing sugar, thereby achieving the method for improving the histidine fermentation purity.

In order to solve the technical problems, the technical scheme provided by the invention is as follows: a method for improving histidine fermentation purity comprises the following steps:

A. performing slant activated culture, taking out the glycerol tube for preserving strains from a refrigerator, uniformly coating a slant culture eggplant bottle for activation, and culturing at the activation temperature of 28-32 ℃ for 6-10h to form colonies;

B. b, controlling a seed process, namely taking 50ml of sterile water to put into the eggplant bottle in the step A, scraping bacterial colonies to form bacterial suspension, inoculating the bacterial suspension into a seed culture medium in a seed culture tank, controlling the culture temperature to be 28-32 ℃, adding ammonia water in a flowing manner to control the pH to be 6.5-7.5, and culturing until the OD650 of a seed solution reaches 8-12; OD650 is an absorbance value at a wavelength of 650nm of a spectrophotometer, wherein the OD value is the absorbance of the seed solution measured by the spectrophotometer, the OD value represents the number of thalli in the seed solution, and the larger the OD value is, the more thalli are represented; controlling the dissolved oxygen to be more than or equal to 30 percent in the process;

C. controlling the fermentation process, namely adding 5-15% of the seed liquid inoculation amount in the step B into a fermentation culture medium of a fermentation tank, controlling the temperature to be 28-32 ℃, adding ammonia water in a flowing manner to control the pH to be 6.5-7.5, and controlling the dissolved oxygen to be 20-50% in the process;

in the step C, after reducing sugar is reduced to 2-4% after the fermentation medium is hydrolyzed, sucrose with the concentration of 45-75% is fed, and the flow rate of the sucrose is adjusted to control the dissolved oxygen to be 20-50%. Namely, the fermentation medium is consumed along with the rapid growth of the thalli, and the concentration of reducing sugar is detected at any time by a film reagent method. In the present invention, the percentage concentrations are the raw material weight g/total culture medium volume L100%, i.e. weight volume percentage.

Further: in the method for improving the histidine fermentation purity, in the step B, the mixture ratio of the seed culture medium is that 20-40g/L of sucrose, 3.0-11.5g/L of cane molasses, 12-18g/L of corn steep liquor, 0.5-2.0g/L of yeast powder, 2.0-5.0g/L of ammonium sulfate, 0.5-2.0g/L of monopotassium phosphate, 0.1-0.5g/L of magnesium sulfate, 0.5-2.0mg/L of manganese sulfate, 2-8mg/L of ferrous sulfate, 0.1-0.5mg/L of biotin and 0.1-0.5mg/L of thiamine are dissolved in water.

And B, dissolving the mixture ratio of the seed culture medium in water, wherein the mixture ratio of the sucrose is 35g/L, the cane molasses is 7g/L, the corn steep liquor is 18g/L, the yeast powder is 2g/L, the ammonium sulfate is 4g/L, the potassium dihydrogen phosphate is 1g/L, the magnesium sulfate is 0.5g/L, the manganese sulfate is 1mg/L, the ferrous sulfate is 5mg/L, the biotin is 0.1mg/L and the thiamine is 0.1 mg/L.

The proportion of the fermentation medium in the step C is that 40-120g/L of sucrose, 3.0-11.5g/L of cane molasses, 12-18g/L of corn steep liquor, 0.5-2.0g/L of yeast powder, 2.0-4.0g/L of ammonium sulfate, 0.5-2.0g/L of monopotassium phosphate, 0.1-0.5g/L of magnesium sulfate, 0.5-2.0mg/L of manganese sulfate, 2-8mg/L of ferrous sulfate, 0.1-0.5mg/L of biotin and 0.1-0.5mg/L of thiamine are dissolved in water.

And C, dissolving the fermentation medium in water according to the proportion of 80g/L of sucrose, 7g/L of cane molasses, 18g/L of corn steep liquor, 2g/L of yeast powder, 4g/L of ammonium sulfate, 1g/L of monopotassium phosphate, 0.5g/L of magnesium sulfate, 1mg/L of manganese sulfate, 5mg/L of ferrous sulfate, 0.2mg/L of biotin and 0.2mg/L of thiamine.

Preferably, the activation temperature of the slant activation culture in the step A is 30 ℃, and the slant activation culture is cultured for 8 hours. And B, in the step B, in the seed process control, the culture temperature is 30 ℃, ammonia water is fed in to control the pH to be 7.0, and the seed is cultured until the OD650 of the seed solution reaches 10. And C, in the fermentation process control of the step C, 10% of seed liquid inoculation amount is fed into a fermentation tank, the temperature is controlled to be 30 ℃, ammonia water is fed in a flow mode, the pH value is controlled to be 7.0, the former stage of dissolved oxygen is controlled to be more than or equal to 30% within 10 hours before fermentation, and then the dissolved oxygen is controlled to be 20-30%. And C, when the sucrose with the concentration is fed in the step C, the sucrose with the concentration of 60% is fed in after reducing sugar is reduced to 3% after hydrolysis is controlled by the process, and the flow rate of the sucrose is adjusted to control dissolved oxygen to be 20-30%.

Compared with the prior art, the method for improving the histidine fermentation purity comprises the following steps: A. performing slant activated culture, taking out the glycerol tube for preserving strains from a refrigerator, uniformly coating a slant culture eggplant bottle for activation, and culturing at the activation temperature of 28-32 ℃ for 6-10h to form colonies; B. b, controlling a seed process, namely taking 50ml of sterile water to put into the eggplant bottle in the step A, scraping bacterial colonies to form bacterial suspension, inoculating the bacterial suspension into a seed culture medium in a seed culture tank, controlling the culture temperature to be 28-32 ℃, adding ammonia water in a flowing manner to control the pH to be 6.5-7.5, and culturing until the OD650 of a seed solution reaches 8-12; OD650 is an absorbance value at a wavelength of 650nm of a spectrophotometer, wherein the OD value is the absorbance of the seed solution measured by the spectrophotometer, the OD value represents the number of thalli in the seed solution, and the larger the OD value is, the more thalli are represented; controlling the dissolved oxygen to be more than or equal to 30 percent in the process; C. controlling the fermentation process, namely adding 5-15% of the seed liquid inoculation amount in the step B into a fermentation culture medium of a fermentation tank, controlling the temperature to be 28-32 ℃, adding ammonia water in a flowing manner to control the pH to be 6.5-7.5, and controlling the dissolved oxygen to be 20-50% in the process; particularly, in the step C, after reducing sugar is reduced to 2-4% after the fermentation medium is hydrolyzed, sucrose with the concentration of 45-75% (weight volume percentage) is fed, and the flow rate of the sucrose is adjusted to control the dissolved oxygen to be 20-50%. The invention is the key control point for controlling the thallus to be in the basic metabolism growth state, the flow rate of the cane sugar is adjusted to control the dissolved oxygen to be 20-50%, the data parameters are obtained by repeated adjustment through repeated tests of the inventor, the technical effect is obvious, compared with the prior art for controlling the residual sugar to be in the range of 2-3%, the purity of the histidine in the fermentation tank-discharging liquid is improved by more than 13%, and meanwhile, the weight volume percentage of the histidine is also greatly improved.

Detailed Description

The present invention will be further described with reference to the following examples, which are not intended to limit the scope of the present invention, but are merely illustrative, and the selection of the raw materials in the process can be made according to the circumstances without substantially affecting the result.

Comparative example 1

Seed culture medium proportioning: 35g/L of sucrose, 7g/L of cane molasses, 18g/L of corn steep liquor, 2g/L of yeast powder, 4g/L of ammonium sulfate, 1g/L of monopotassium phosphate, 0.5g/L of magnesium sulfate, 1mg/L of manganese sulfate, 5mg/L of ferrous sulfate, 0.1mg/L of biotin and 0.1mg/L of thiamine, and is dissolved in water. The fermentation medium proportion is as follows: 80g/L of sucrose, 7g/L of cane molasses, 18g/L of corn steep liquor, 2g/L of yeast powder, 4g/L of ammonium sulfate, 1g/L of monopotassium phosphate, 0.5g/L of magnesium sulfate, 1mg/L of manganese sulfate, 5mg/L of ferrous sulfate, 0.2mg/L of biotin and 0.2mg/L of thiamine, and is dissolved in water.

Slant activation culture: taking out the glycerol tube for preserving strains from the ultra-low temperature refrigerator, uniformly coating a slant culture eggplant bottle for activation, and culturing for 8h at 30 ℃.

Controlling the seed process: 50ml of sterile water is put into an eggplant bottle, and bacterial colonies are gently scraped off to form bacterial suspension. Inoculating into a seed culture tank, culturing at 30 deg.C, feeding ammonia water to control pH7.0, air volume of 0h 300L/h, rotating speed of 150rpm, increasing air volume and rotating speed according to OD growth condition, adjusting dissolved oxygen to a certain extent before transplanting, and controlling dissolved oxygen to be more than or equal to 30% in the process. The OD growth was monitored during the process and the culture was continued until the OD650 of the seed liquid reached 10. Controlling the fermentation process: the inoculation amount of the seed liquid is 10 percent, the temperature is controlled to be 30 ℃, the pH value is controlled to be 7.0 by feeding ammonia water, the air volume is 800L/h after 0h, the rotating speed is 300rpm, the air volume and the rotating speed are properly increased according to the OD increasing condition, the dissolved oxygen is controlled to be more than or equal to 30 percent at the early stage (the early stage is generally 10 hours before fermentation) in the process, and then the dissolved oxygen is controlled to be 20-30 percent. Controlling the material supplementing process: after the reducing sugar is reduced to 3 percent after hydrolysis, 60 percent of sugar supplement is added, and the residual sugar is controlled to be 2 to 3 percent. Fermenting with 5L fermenter for 40-50h, and determining by high performance liquid chromatography to obtain average histidine content of 17.2g/L and average histidine purity of 83.5%.

Example 1

Seed culture medium proportioning: 35g/L of sucrose, 7g/L of cane molasses, 18g/L of corn steep liquor, 2g/L of yeast powder, 4g/L of ammonium sulfate, 1g/L of monopotassium phosphate, 0.5g/L of magnesium sulfate, 1mg/L of manganese sulfate, 5mg/L of ferrous sulfate, 0.1mg/L of biotin and 0.1mg/L of thiamine, and is dissolved in water. The fermentation medium proportion is as follows: 80g/L of sucrose, 7g/L of cane molasses, 18g/L of corn steep liquor, 2g/L of yeast powder, 4g/L of ammonium sulfate, 1g/L of monopotassium phosphate, 0.5g/L of magnesium sulfate, 1mg/L of manganese sulfate, 5mg/L of ferrous sulfate, 0.2mg/L of biotin and 0.2mg/L of thiamine, and is dissolved in water.

Slant activation culture: taking out the glycerol tube for preserving strains from the ultra-low temperature refrigerator, uniformly coating a slant culture eggplant bottle for activation, and culturing for 8h at 30 ℃.

Controlling the seed process: 50ml of sterile water is put into an eggplant bottle, and bacterial colonies are gently scraped off to form bacterial suspension. Inoculating into a seed culture tank, culturing at 30 deg.C, feeding ammonia water to control pH7.0, air volume of 0h 300L/h, rotating speed of 150rpm, increasing air volume and rotating speed according to OD growth condition, adjusting dissolved oxygen to a certain extent before transplanting, and controlling dissolved oxygen to be more than or equal to 30% in the process. The OD growth was monitored during the process and the culture was continued until the OD650 of the seed liquid reached 10.

Controlling the fermentation process: and (2) putting 10% of seed liquid inoculation amount into a fermentation tank, controlling the temperature to be 30 ℃, controlling the pH value to be 7.0 by feeding ammonia water, controlling the air volume to be 800L/h and the rotating speed to be 300rpm for 0h, properly increasing the air volume and the rotating speed according to the OD increase condition, fermenting for 0-10 h, controlling the dissolved oxygen to be more than or equal to 30% at the early stage and then controlling the dissolved oxygen to be 20-30%.

Controlling the material supplementing process: after the reducing sugar is reduced to 3 percent after hydrolysis, 60 percent of sugar supplement is fed, and the flow rate of the sugar supplement is adjusted to control the dissolved oxygen to be 20 to 30 percent.

Fermenting for 50-60 hr in 5L fermenter, and determining by high performance liquid chromatography to obtain histidine content of 19.2g/L and histidine purity of 94.6%. Compared with the comparative example 1, the improvement is 11.6 percent and 13.3 percent respectively.

The foregoing is only a preferred embodiment of this invention, and any obvious combination and substitution is within the scope of the invention without departing from the spirit of this invention. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the scope of the claims of the present invention.