阅读说明：本技术 一种提高乙酰氨基葡萄糖发酵单位的方法 (Method for improving acetylglucosamine fermentation unit ) 是由 谢沛 李静 张兆坤 于 2020-12-31 设计创作，主要内容包括：本发明涉及一种提高乙酰氨基葡萄糖发酵单位的方法,属于发酵工程技术领域,采用大肠杆菌发酵生产乙酰氨基葡萄糖,在发酵培养阶段,对IPTG诱导进行控制,选择在不同阶段分批次添加IPTG,同时在第二次IPTG诱导时将葡萄糖酸钙和碘化钾与IPTG一同加入,发酵周期短、产酸效率高、菌体密度高；底物转化率高；而且工艺简单,环境污染低,降低了乙酰氨基葡萄糖的生产成本,有利于工业化生产的推广和应用。(The invention relates to a method for improving fermentation unit of acetylglucosamine, belonging to the technical field of fermentation engineering, adopting Escherichia coli to ferment and produce the acetylglucosamine, controlling IPTG induction in a fermentation culture stage, selectively adding IPTG in different stages in batches, simultaneously adding calcium gluconate and potassium iodide together with IPTG in the second IPTG induction, and having short fermentation period, high acid production efficiency and high thallus density; the conversion rate of the substrate is high; and the process is simple, the environmental pollution is low, the production cost of the acetylglucosamine is reduced, and the popularization and the application of industrial production are facilitated.)

1. A method for increasing the fermentation unit of acetylglucosamine, which is characterized by comprising the following steps: the method comprises the following steps:

activating an original strain of escherichia coli through a slant, culturing for 20 hours in a biochemical incubator at 37 ℃, performing gradient dilution, coating on a complete culture medium, selecting single bacterial colonies, respectively inoculating the single bacterial colonies on the slant, and culturing for 12 hours in the biochemical incubator at 30-36 ℃ to obtain an activated strain;

step two, seed culture: inoculating the activated strain obtained in the step one into a seed bottle filled with a seed culture medium, wherein the culture conditions are as follows: the temperature is 30-36 ℃, the rotating speed of a shaking table is 200rmp, and the culture time is 6-8 h, so as to obtain a seed solution;

step three, inoculating the seed liquid obtained in the step two into a fermentation bottle filled with a fermentation culture medium, wherein the volume of the fermentation culture medium is x L; inoculation amount: 5-15%, culture conditions: the initial fermentation temperature is 30-36 ℃, the air flow is 5-50L/min, the stirring speed is 200-800r/min, the dissolved oxygen is maintained at 10-40%, and the fermentation pH value is controlled to be 6.0-7.2; when the OD of the bacteria is 10-15, adding x mL of IPTG with the concentration of 0.1 mol/L for induction, continuing to culture until the OD value of the bacteria grows to 50-80, adding x mL of IPTG with the concentration of 0.1 mol/L, 10x mL of calcium gluconate solution with the concentration of 5g/L and x mL of potassium iodide solution with the concentration of 0.5g/L for one time, and continuing to culture for 10-20 hours.

2. The method of claim 1, wherein the fermentation unit of acetylglucosamine is increased by: the seed culture medium of the second step comprises the following components in percentage by weight: glucose 20-30 g/L, K₂HPO₄10~20g/L，MgSO₄·7H₂O 0.5~3.0g/L，KH₂PO₄ 10-20 g/L, 0.5-3.0 g/L citric acid, (NH)₄)₂SO₄5-10.0 g/L and 5-10.0 mL/L of trace elements.

3. The method of claim 1, wherein the fermentation unit of acetylglucosamine is increased by: and step three, the fermentation medium comprises the following components in percentage by weight: glucose 20-30 g/L, K₂HPO₄3.0~10.0g/L, MgSO₄·7H₂0.5-3.0 g/L of O, 1.0-7.0 g/L of citric acid, (NH)₄)₂SO₄5-10.0 g/L and 5-10.0 mL/L of trace elements.

4. The method of claim 1, wherein the fermentation unit of acetylglucosamine is increased by: and step three, the inoculation amount is 7%.

Technical Field

The invention belongs to the technical field of fermentation engineering, and particularly relates to a method for improving an acetylglucosamine fermentation unit.

Background

N-acetylglucosamine is a monomer of chitin, and the chitin is very large in stock in nature, and is second to cellulose. N-acetylglucosamine has important physiological functions in medicine, has the functions of diminishing inflammation, resisting tumor and resisting oxidation, is an effective medicine for treating osteoarthritis and rheumatic arthritis, and has important application in the industries of food, chemical industry and cosmetics.

The existing preparation method of N-acetylglucosamine comprises a sodium methoxide method, a direct chitin enzymolysis method and the like. The process of the sodium methoxide method comprises the following steps: adding sodium methoxide into glucosamine raw materials for replacement reaction, removing chloride ions in the glucosamine, adding acetic anhydride for acetylation, removing harmful substances such as bacteria, rust, colloid and the like in the glucosamine raw materials through ultrafiltration, then carrying out fractional precipitation and filtration, then carrying out re-dissolution crystallization and solid-liquid separation, and obtaining the finished product after vacuum drying. The direct enzymolysis method means that chitin can be directly produced by enzymolysis of chitin with chitinase in the production of N-acetylglucosamine, and the enzymolysis process of the chitin is longer due to the particularity of the chemical properties of the chitin, the hydrolysis is incomplete, the hydrolysis rate is below 75%, the efficiency is lower, the actual large-scale industrial production cost is higher, and the benefit is poor.

The construction of recombinant Escherichia coli by metabolic engineering is an effective way for producing food safety-grade acetylglucosamine. However, the fermentation level is not ideal especially for the fermentation yield of acetylglucosamine by adopting the method, and how to further optimize the fermentation process so as to effectively improve the fermentation unit is a problem to be researched by researchers in the field.

Disclosure of Invention

In view of the above problems, an object of the present invention is to provide a method for increasing the fermentation unit of acetylglucosamine. The method controls the IPTG induction in the fermentation culture stage, selects to add IPTG in different stages in batches, simultaneously adds calcium gluconate and potassium iodide together with the IPTG in the second IPTG induction, effectively improves the fermentation yield,

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

a method for increasing acetylglucosamine fermentation units, comprising the steps of:

activating an original strain of escherichia coli through a slant, culturing for 20 hours in a biochemical incubator at 37 ℃, performing gradient dilution, coating on a complete culture medium, selecting single bacterial colonies, respectively inoculating the single bacterial colonies on the slant, and culturing for 12 hours in the biochemical incubator at 30-36 ℃ to obtain an activated strain;

step two, seed culture: inoculating the activated strain obtained in the step one into a seed bottle filled with a seed culture medium, wherein the culture conditions are as follows: the temperature is 30-36 ℃, the rotating speed of a shaking table is 200rmp, and the culture time is 6-8 h, so as to obtain a seed solution;

step three, inoculating the seed liquid obtained in the step two into a fermentation bottle filled with a fermentation culture medium, wherein the volume of the fermentation culture medium is x L; inoculation amount: 5-15%, culture conditions: the initial fermentation temperature is 30-36 ℃, the air flow is 5-50L/min, the stirring speed is 200-800r/min, the dissolved oxygen is maintained at 10-40%, and the fermentation pH value is controlled to be 6.0-7.2; when the OD of the bacteria is 10-15, adding x mL of IPTG with the concentration of 0.1 mol/L for induction, continuing to culture until the OD value of the bacteria grows to 50-80, adding x mL of IPTG with the concentration of 0.1 mol/L, 10x mL of calcium gluconate solution with the concentration of 5g/L and x mL of potassium iodide solution with the concentration of 0.5g/L for one time, and continuing to culture for 10-20 hours.

As a further optimization of the above scheme, the seed culture medium in the second step comprises the following components in percentage by weight: glucose 20-30 g/L, K₂HPO₄10~20g/L，MgSO₄·7H₂O 0.5~3.0g/L，KH₂PO₄ 10-20 g/L, 0.5-3.0 g/L citric acid, (NH)₄)₂SO₄5-10.0 g/L and 5-10.0 mL/L of trace elements.

As a further optimization of the above scheme, the components and their contents contained in the fermentation medium in step three are respectively: glucose 20-30 g/L, K₂HPO₄3.0~10.0g/L, MgSO₄·7H₂0.5-3.0 g/L of O, 1.0-7.0 g/L of citric acid, (NH)₄)₂SO₄5-10.0 g/L and 5-10.0 mL/L of trace elements.

As a further optimization of the above scheme, the inoculation amount in the third step is 7%.

Has the advantages that:

the method adopts the escherichia coli for fermentation production of the acetylglucosamine, the strain fermentation is aerobic fermentation, the thallus grows faster, IPTG induction is controlled in the fermentation culture stage, IPTG is added in different stages in batches, and calcium gluconate and potassium iodide are added together with the IPTG in the second IPTG induction, so that the fermentation yield is effectively improved, the process is simple, the control is easy, and the method is suitable for industrial production.

Detailed Description

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.

Example 1

A method for improving acetylglucosamine fermentation units comprises the following specific steps:

inoculating an original strain (Escherichia coli CGMCC NO. 13924) to a slant, culturing for 20h at 37 ℃ in a biochemical incubator, then performing gradient dilution, coating on a complete culture medium (LB), selecting single colonies, respectively inoculating to the slant, and culturing for about 12h at 30-36 ℃ in the biochemical incubator. Inoculated into a 500mL seed flask, culture conditions: the temperature is 30-36 ℃, the rotating speed of a shaking table is 200rmp, and the culture time is 6-8 h; when the OD value is about 1.0-2.0, inoculating the strain into a 5L fermentation bottle, wherein the inoculation amount is as follows: about 7%, culture conditions: when the temperature is 30-36 ℃, the rotating speed of a shaking table is 200rmp, and the OD of the thallus is about 10-15, 5ml of 0.1 mol/L IPTG induction is added once, when the OD value of the strain is continuously cultured to about 50-80, 5ml of 0.1 mol/L IPTG induction, 50ml of 5g/L calcium gluconate and 5ml of 0.5g/L potassium iodide are added once again, the culture is continuously carried out for 10-20 h, and the content of the acetylglucosamine is detected to be 70.25g/L after the fermentation is finished.

A control was set using the protocol described above, which differs from the protocol described above in that: when the fermentation culture is carried out until the OD of the thalli is about 10-15, 10ml of IPTG with the same concentration is added for induction at one time, and then the culture is continued until the fermentation is finished. And after the fermentation is finished, detecting that the content of the acetylglucosamine is 49.69 g/L.

The yield of acetylglucosamine in example 1 was increased by 41.37% compared to the control.

Example 2

Picking a ring of escherichia coli (escherichia coli CGMCC NO. 13924) from a fresh slant, inoculating the Escherichia coli into a 500mL seed bottle, and culturing under the following conditions: the temperature is 30-36 ℃, the rotating speed of a shaking table is 200rmp, and the culture time is 6-8 h; when the OD value is about 1.0-2.0, inoculating the shake flask seeds into a 10L full-automatic fermentation tank filled with 6L fermentation medium in an inoculation amount of 10% for secondary seed culture. The culture temperature is 30-36 ℃, the tank pressure is 0.01-0.05 MPa, the air flow is 1-8L/min, the stirring speed is 200-800r/min, the dissolved oxygen is maintained at 10-50%, the pH value is controlled at 7.0-7.2, and the culture time is 5-8 h. The second-level seeds are respectively inoculated into a 50L full-automatic fermentation tank filled with 18L fermentation medium according to the inoculation amount of 10 percent for fermentation. The fermentation temperature is 30-36 ℃, the tank pressure is 0.01-0.15 MPa, the air flow is 5-50L/min, the stirring speed is 200-800r/min, the dissolved oxygen is maintained at 10-40%, the fermentation pH value is controlled to be 6.2-7.2, 18ml of 0.1 mol/L IPTG induction is added once when the thallus OD is about 10-15, the culture is continued until the thallus OD value is about 50-80, 18ml of 0.1 mol/L IPTG induction, 180ml of 5g/L calcium gluconate and 18ml of 0.5g/L potassium iodide are added once again, the culture is continued for 10-20 h, and the content of the acetylglucosamine is detected to be 220.76g/L after the fermentation is finished.

A control was set using the protocol described above, which differs from the protocol described above in that: when the fermentation culture is carried out until the OD of the thalli is about 10-15, 36ml of IPTG with the same concentration is added for induction at one time, and then the culture is continued until the fermentation is finished. And after the fermentation is finished, detecting that the content of the acetylglucosamine is 170.74 g/L.

The yield of acetylglucosamine in example 2 was increased by 29.30% compared to the control.

Example 3

Picking a ring of escherichia coli (escherichia coli CGMCC NO. 13924) from a fresh slant, inoculating the Escherichia coli into a 500mL seed bottle, and culturing under the following conditions: the temperature is 30-36 ℃, the rotating speed of a shaking table is 200rmp, and the culture time is 6-8 h; when the OD value is about 1.0-2.0, inoculating the shake flask seeds with 0.6m³2m of fermentation Medium³And performing secondary seed culture in a fermentation tank. The culture temperature is 30-36 ℃, the tank pressure is 0.01-0.05 MPa, and the air flow is 0.1-0.5 m³The dissolved oxygen is maintained at 10-50% at a stirring speed of 100-300 r/min, the pH value is controlled at 7.0-7.2, and the culture time is 5-8 h. Inoculating the second-level seeds with 10% inoculum size to a container with 4m³10m of fermentation Medium³Fermenting in a fermentation tank. The fermentation temperature is 30-36 ℃, the tank pressure is 0.01-0.15 MPa, the air flow is 5-50L/min and the stirring speed is 200-Controlling the culture medium to be between 7.0 and 7.2, adding 4L of 0.1 mol/L IPTG induction once when the OD of the bacteria is about 10 to 15, continuing to culture until the OD value of the bacteria grows to about 50 to 80, adding 4L of 0.1 mol/L IPTG induction and 40L of 5g/L calcium gluconate once again and 4L of 0.5g/L potassium iodide once again, continuing to culture for 10 to 20 hours, and detecting the content of the acetylglucosamine to be 198.76g/L after the fermentation is finished.

A control was set using the protocol described above, which differs from the protocol described above in that: when the fermentation culture is carried out until the OD of the thalli is about 10-15, 8L of IPTG with the same concentration is added for induction at one time, and then the culture is continued until the fermentation is finished. And after the fermentation is finished, detecting that the content of the acetylglucosamine is 150.74 g/L.

The yield of acetylglucosamine in example 3 was increased by 31.86% compared to the control.

It should be noted that the above-mentioned embodiments illustrate rather than limit the scope of the invention, which is defined by the appended claims. It will be apparent to those skilled in the art that certain insubstantial modifications and adaptations of the present invention can be made without departing from the spirit and scope of the invention.