阅读说明：本技术 一种n-乙酰氨基葡萄糖的发酵生产方法 (Fermentation production method of N-acetylglucosamine ) 是由 穆晓玲 唐浩 刘干 王舒 吴琼琼 葛严峰 于 2020-12-31 设计创作，主要内容包括：本发明提供了一种N-乙酰氨基葡萄糖的发酵生产方法。所述方法先利用大肠杆菌进行发酵,在发酵过程中接种枯草芽孢杆菌继续进行发酵；所述大肠杆菌为大肠埃希氏菌(Escherichia coli),于2018年5月14日保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC),保藏号为CGMCC 15756。本发明提供的方法通过利用大肠杆菌CGMCC 15756与枯草芽孢杆菌配合,进行混菌发酵,使二者平衡共生,避免了单独采用大肠杆菌CGMCC 15756发酵时,因副产物快速积累导致发酵速度明显减慢甚至提前终止的问题,显著提高了发酵终点N-乙酰氨基葡萄糖的产量和转化率,降低了谷氨酸、乙酸等副产物的含量。(The invention provides a fermentation production method of N-acetylglucosamine. The method comprises the steps of firstly utilizing escherichia coli for fermentation, inoculating bacillus subtilis in the fermentation process, and continuing fermentation; the Escherichia coli is Escherichia coli (Escherichia coli), and is preserved in China general microbiological culture Collection center (CGMCC) in 2018, 5 months and 14 days, with the preservation number of CGMCC 15756. The method provided by the invention utilizes the cooperation of the Escherichia coli CGMCC15756 and the Bacillus subtilis to carry out mixed fermentation, so that the Escherichia coli CGMCC15756 and the Bacillus subtilis are in balanced symbiosis, the problem that the fermentation speed is obviously slowed down or even stopped in advance due to the rapid accumulation of byproducts when the Escherichia coli CGMCC15756 is singly used for fermentation is avoided, the yield and the conversion rate of the N-acetylglucosamine at the fermentation end point are obviously improved, and the content of the byproducts such as glutamic acid, acetic acid and the like is reduced.)

1. A fermentation production method of N-acetylglucosamine is characterized in that the method firstly utilizes escherichia coli for fermentation, and bacillus subtilis is inoculated in the fermentation process for continuous fermentation;

the Escherichia coli is Escherichia coli (Escherichia coli), and is preserved in China general microbiological culture Collection center (CGMCC) in 2018, 5 months and 14 days, with the preservation number of CGMCC 15756.

2. The method according to claim 1, wherein the bacillus subtilis is inoculated when the fermentation broth has an OD value at 660nm of 40 to 50;

more preferably, the amount of inoculation of said Bacillus subtilis is 0.5-1.5%;

preferably, the amount of said Escherichia coli is 5-10%.

3. The method according to claim 1 or 2, characterized in that the method further comprises: adding isopropyl-beta-D-thiogalactoside for induction in the fermentation process;

preferably, when the OD value of the fermentation broth at 660nm reaches 20-35, isopropyl-beta-D-thiogalactoside is added for induction;

more preferably, the addition amount of the isopropyl-beta-D-thiogalactoside is 0.05-0.2 mmol/L.

4. A method according to any one of claims 1-3, characterized in that the method comprises the steps of:

inoculating the activated escherichia coli into a fermentation culture solution for fermentation, adding a glucose solution in the fermentation process, adding isopropyl-beta-D-thiogalactoside for induction when the OD value of the fermentation broth at 660nm reaches 20-35, inoculating the activated bacillus subtilis when the OD value of the fermentation broth at 660nm reaches 40-50, and continuing to ferment.

5. A process according to any one of claims 1 to 4, wherein the fermentation broth used in the process comprises the following components:

5-10g/L glucose, 7-12g/L potassium dihydrogen phosphate, 1-4g/L citric acid, and 10-15g/L, MgSO ammonium sulfate₄ 0.5-1.0g/L、CaCl₂0.05-0.1g/L feather powder or hair powder 15-30g/L, FeSO₄ 0.04-0.08g/L、ZnCl₂0.015-0.03g/L、MnSO₄0.002-0.004g/L、CoCl₂ 0.004-0.008g/L、CuSO₄0.006-0.012g/L, and the solvent is water.

6. The method according to any one of claims 1 to 5, wherein the temperature of the fermentation is 34 to 38 ℃;

and/or the fermentation period is 60-70 h.

7. The method according to any one of claims 1 to 6, wherein the pH of the fermentation broth is maintained between 6.5 and 7.0 during the fermentation; preferably, the pH is adjusted by adding ammonia water during the fermentation process; preferably, the concentration of the aqueous ammonia is 15 to 25 wt%.

8. The process according to any one of claims 1 to 7, wherein the initial sterile air aeration ratio for the fermentation is 0.15 to 0.3vvm, the initial pressure is 0.015 to 0.03MPa and the initial stirring speed is 150-300 rpm;

and/or in the fermentation process, after the dissolved oxygen of the fermentation liquor is reduced to 20-40% of the initial dissolved oxygen, maintaining the dissolved oxygen to be 20-40% of the initial dissolved oxygen;

and/or the method for maintaining the dissolved oxygen in the fermentation process is to adjust the aeration ratio and the stirring speed.

9. The method according to any one of claims 1 to 8, wherein during the fermentation, when the glucose content in the fermentation broth is reduced to below 0.5g/L, feeding of the glucose solution is started to maintain the glucose content in the fermentation broth at 0.1-0.5 g/L;

preferably, the concentration of the glucose solution is 50-70 wt%.

10. A method according to any of claims 1-9, characterized in that the method comprises the steps of:

adding a sterilized fermentation culture solution into a fermentation tank, wherein the fermentation culture solution comprises the following components: 5-10g/L glucose, 7-12g/L potassium dihydrogen phosphate, 1-4g/L citric acid, and 10-15g/L, MgSO ammonium sulfate₄ 0.5-1.0g/L、CaCl₂0.05-0.1g/L feather powder or hair powder 15-30g/L, FeSO₄ 0.04-0.08g/L、ZnCl₂ 0.015-0.03g/L、MnSO₄0.002-0.004g/L、CoCl₂ 0.004-0.008g/L、CuSO₄0.006-0.012g/L, solvent is water;

adjusting the initial sterile air ventilation ratio to 0.15-0.3vvm, the initial pressure to 0.15-0.03Mpa and the initial stirring rotation speed to 150-;

in the fermentation process, the temperature is maintained at 34-38 ℃, and ammonia water with the concentration of 15-25 wt% is added at the same time, so that the pH value is maintained at 6.5-7.0; when the dissolved oxygen of the fermentation liquor is reduced to be below 40 percent of the initial dissolved oxygen, adjusting the aeration ratio and the stirring speed to maintain the dissolved oxygen to be 20 to 40 percent of the initial dissolved oxygen; when the glucose content in the fermentation liquor is reduced to be below 0.5g/L, feeding a glucose solution with the concentration of 50-70 wt% to ensure that the glucose content in the fermentation liquor is maintained to be 0.1-0.5 g/L; when the OD value of the fermentation liquor at 660nm reaches 20-35, adding isopropyl-beta-D-thiogalactoside for induction at one time, wherein the addition amount is 0.05-0.2 mmol/L; when the OD value of the fermentation liquor at 660nm reaches 40-50, inoculating the activated bacillus subtilis according to the inoculation amount of 0.5-1.5%, and continuing to ferment for 60-70 h.

Technical Field

The invention belongs to the technical field of fermentation engineering, and particularly relates to a fermentation production method of N-acetylglucosamine.

Background

Glucosamine (GlcN, glucosamine for short) and its derivatives have wide application in medicine, food, cosmetics, etc. Particularly, from the aspect of medical application, glucosamine is also a substance that can be synthesized in the human body, is an important nutrient for forming chondrocytes, and is a natural tissue component of healthy articular cartilage. With age, glucosamine deficiency becomes more severe, leading to the progressive degeneration and wear of articular cartilage, resulting in a variety of bone diseases. Glucosamine, in turn, can help repair and maintain cartilage and stimulate the growth of chondrocytes. Has obvious therapeutic effect on arthritis, hyperosteogeny, meniscus injury, cervical spondylosis, etc. Glucosamine is widely used as a food ingredient in japan and the united states because it is not toxic to the human body.

The traditional glucosamine production method is prepared by hydrolyzing crustacean raw materials by using high-concentration hydrochloric acid, and has the defects of limited raw material sources, easy environmental pollution and anaphylactic reaction, difficult realization of large-scale industrial production and the like.

Currently, a commonly used method for producing glucosamine is to prepare N-acetylglucosamine by fermentation, and then perform extraction treatment after acidolysis and the like to obtain glucosamine and derivatives thereof, such as glucosamine hydrochloride, glucosamine sulfate and the like.

The common strain for preparing the N-acetylglucosamine by fermentation is escherichia coli, and the method generally has the problems of low yield and conversion rate of the N-acetylglucosamine and high content of byproducts such as glutamic acid, acetic acid and the like. CN 104988196A, CN 106191169A improves the existing fermentation method, improves the content and conversion rate of N-acetylglucosamine at the fermentation end point, but still has the problem of high content of by-products.

Therefore, the content and the conversion rate of the N-acetylglucosamine prepared by the fermentation method are further improved, the content of byproducts is reduced, and the method has important significance for the glucosamine production industry.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide a fermentation production method of N-acetylglucosamine. Compared with a method for fermenting by adopting escherichia coli alone, the method provided by the invention can obviously improve the yield and the conversion rate of the N-acetylglucosamine.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the invention provides a fermentation production method of N-acetylglucosamine, which comprises the steps of firstly fermenting by using escherichia coli, inoculating bacillus subtilis in the fermentation process and continuing to ferment;

the Escherichia coli is Escherichia coli (Escherichia coli), and is preserved in China general microbiological culture Collection center (CGMCC) in 2018, 5 months and 14 days, with the preservation number of CGMCC 15756.

In the invention, the Escherichia coli CGMCC15756 is preserved in the international microorganism preservation unit specified by the Budapest treaty in 2018, 5 months and 14 days: china General Microbiological Culture Collection Center (China General Microbiological Culture Collection Center), Address: beijing, West Lu No. 1 Hospital No. 3 of the national academy of sciences, facing the Yangtze region.

The inventor of the invention discovers through research that due to gene modification, inducer and product toxicity and other reasons, the growth and metabolism capacity of the escherichia coli CGMCC15756 is weak when the escherichia coli CGMCC15756 is independently cultured, when the escherichia coli CGMCC15756 is independently adopted for fermentation, byproducts glutamic acid and acetic acid are accumulated in the early stage of fermentation, and qualitative change is generated when the accumulation reaches a certain amount (especially when the acetic acid concentration reaches more than 3 g/L), namely the fermentation speed is obviously slowed down and even is stopped in advance, so that the yield and the conversion rate of the N-acetylglucosamine are greatly influenced.

The invention selects a proper amount of bacillus subtilis strains to be inoculated in the early specific time of fermentation. Firstly, the bacillus subtilis generation can consume byproducts such as glutamic acid, acetic acid and the like and redundant residual sugar accumulated in the early stage of fermentation of escherichia coli as a carbon source of the bacillus subtilis generation, so that the influence of excessive accumulation on subsequent fermentation is avoided; secondly, the bacillus subtilis gradually forms spores after inoculation, enters a dormant state and does not consume nutrient substances, so that the nutrient loss of a carbon source and the like is reduced, and the cost is saved; in addition, partial extracellular secretion of Bacillus subtilis may promote the growth and metabolism of Escherichia coli CGMCC15756 (similar to the promotion of associated bacteria in gulonic acid fermentation).

The problem of accumulation of the byproducts is effectively solved and the yield and the conversion rate of the N-acetylglucosamine are remarkably improved by matching the Escherichia coli CGMCC15756 with the Bacillus subtilis for mixed fermentation.

In some embodiments of the invention, the bacillus subtilis is a standard strain purchased from the chinese medical bacterial collection management center (CMCC) under code number CMCC 63501.

The bacillus subtilis CMCC63501 has strong metabolic capability and can fully consume byproducts accumulated in the early stage of fermentation of escherichia coli CGMCC 15756; and the time from inoculation to spore formation is 20-30 hours, which is matched with the time of 60-70 hours of the whole fermentation period, so that the loss of nutrients such as carbon source can be reduced to the maximum extent, and the cost is saved. The bacillus subtilis CMCC63501 and the escherichia coli CGMCC15756 are adopted for mixed fermentation, so that the yield and the conversion rate of the N-acetylglucosamine can be further improved.

In some embodiments of the invention, the bacillus subtilis is inoculated when the fermentation broth has an OD (absorbance) value at 660nm of 40-50 (which may be, for example, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50).

In some embodiments of the invention, the bacillus subtilis is inoculated in an amount of 0.5 to 1.5%; for example, it may be 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, or 1.5%.

In the invention, by selecting a proper inoculation time and optimizing the inoculation amount, the two bacteria basically reach a balanced symbiotic relationship, and excessive competition is avoided, so that the yield and the conversion rate of the N-acetylglucosamine are further improved.

In some embodiments of the invention, the Escherichia coli is inoculated in an amount of 5-10%; for example, it may be 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, etc.

In some embodiments of the invention, the method further comprises: isopropyl-beta-D-thiogalactoside is added in the fermentation process for induction.

Preferably, when the fermentation broth OD at 660nm reaches 20-35 (e.g. can be 20, 22, 23, 25, 26, 28, 30, 32, 33 or 35 etc.), isopropyl- β -D-thiogalactoside (IPTG) is added for induction.

In some embodiments of the invention, the isopropyl-beta-D-thiogalactoside is added in an amount of 0.05-0.2 mmol/L; for example, it may be 0.05mmol/L, 0.06mmol/L, 0.08mmol/L, 0.1mmol/L, 0.12mmol/L, 0.13mmol/L, 0.15mmol/L, 0.16mmol/L, 0.18mmol/L or 0.2 mmol/L.

In some embodiments of the invention, the method comprises the steps of:

inoculating the activated escherichia coli into a fermentation culture solution for fermentation, adding a glucose solution in the fermentation process, adding isopropyl-beta-D-thiogalactoside for induction when the OD value of the fermentation broth at 660nm reaches 20-35, inoculating the activated bacillus subtilis when the OD value of the fermentation broth at 660nm reaches 40-50, and continuing to ferment.

In some embodiments of the invention, the fermentation broth used in the method comprises the following components:

5-10g/L glucose, 7-12g/L potassium dihydrogen phosphate, 1-4g/L citric acid, and 10-15g/L, MgSO ammonium sulfate₄0.5-1.0g/L、CaCl₂0.05-0.1g/L feather powder or hair powder 15-30g/L, FeSO₄ 0.04-0.08g/L、ZnCl₂0.015-0.03g/L、MnSO₄ 0.002-0.004g/L、CoCl₂ 0.004-0.008g/L、CuSO₄0.006-0.012g/L, and the solvent is water.

The hair powder used in the culture medium is generally pig hair and the like, the feather powder is chicken hair, duck hair and the like, the raw materials are rich in keratin and low in price, and the bacillus subtilis has strong catabolism capability on the keratin and high utilization rate. After the hair powder or feather powder is decomposed and metabolized into small molecular amino acid by the bacillus subtilis, the further use of the escherichia coli in the invention is facilitated. Therefore, hair powder or feather powder is selected as the optimal fermentation nitrogen source in the present invention.

In some embodiments of the invention, the temperature of the fermentation is 34-38 ℃; for example, the temperature may be 34 ℃, 34.5 ℃, 35 ℃, 35.5 ℃, 36 ℃, 36.5 ℃, 37 ℃, 37.5 ℃ or 38 ℃.

In the invention, when the content of the product N-acetylglucosamine in the fermentation liquor is not increased obviously any more, the fermentation is finished, and the fermentation time is the fermentation period. In the present invention, the fermentation period is generally 60 to 70 hours.

In some embodiments of the invention, the pH of the fermentation broth is maintained between 6.5 and 7.0 during the fermentation process; for example, it may be 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, etc.

Preferably, the pH is adjusted during the fermentation by adding ammonia.

Preferably, the concentration of the ammonia water is 15-25 wt%; for example, it may be 15 wt%, 16 wt%, 18 wt%, 20 wt%, 22 wt%, 23 wt%, 25 wt%, or the like.

In some embodiments of the invention, the fermentation has an initial sterile air aeration ratio of 0.15 to 0.3vvm (e.g., can be 0.15vvm, 0.18vvm, 0.2vvm, 0.22vvm, 0.25vvm, 0.28vvm, 0.3vvm, etc.), an initial pressure of 0.015 to 0.03Mpa (e.g., can be 0.015Mpa, 0.0.018 Mpa, 0.018Mpa, 0.022Mpa, 0.025Mpa, 0.028Mpa, 0.03Mpa, etc.), and an initial agitation speed of 150 to 300rpm (e.g., can be 150rpm, 180rpm, 200rpm, 220rpm, 250rpm, 280rpm, 300rpm, etc.).

In some embodiments of the present invention, the dissolved oxygen is maintained at 20-40% (e.g., 20%, 22%, 25%, 28%, 30%, 32%, 35%, 38%, 40%, etc.) of the initial dissolved oxygen after the dissolved oxygen in the fermentation broth is reduced to 20-40% of the initial dissolved oxygen during the fermentation process.

In some embodiments of the invention, the method for maintaining dissolved oxygen during fermentation is to adjust aeration ratio and stirring speed.

In some embodiments of the invention, during the fermentation process, when the glucose content in the fermentation liquor is reduced to below 0.5g/L, the glucose solution is fed in, so that the glucose content in the fermentation liquor is maintained to be 0.1-0.5 g/L; for example, it may be 0.1g/L, 0.15g/L, 0.2g/L, 0.25g/L, 0.3g/L, 0.35g/L, 0.4g/L, 0.45g/L, or 0.5 g/L.

Preferably, the concentration of the glucose solution is 50-70 wt%; for example, it may be 50 wt%, 52 wt%, 53 wt%, 55 wt%, 56 wt%, 58 wt%, 60 wt%, 62 wt%, 65 wt%, 68 wt%, 70 wt%, or the like.

In some embodiments of the invention, the method comprises the steps of:

adding a sterilized fermentation culture solution into a fermentation tank, wherein the fermentation culture solution comprises the following components: 5-10g/L glucose, 7-12g/L potassium dihydrogen phosphate, 1-4g/L citric acid, and 10-15g/L, MgSO ammonium sulfate₄ 0.5-1.0g/L、CaCl₂0.05-0.1g/L feather powder or hair powder 15-30g/L, FeSO₄ 0.04-0.08g/L、ZnCl₂ 0.015-0.03g/L、MnSO₄0.002-0.004g/L、CoCl₂ 0.004-0.008g/L、CuSO₄0.006-0.012g/L, and the solvent is water.

Adjusting the initial sterile air ventilation ratio to 0.15-0.3vvm, the initial pressure to 0.015-0.03Mpa and the initial stirring rotation speed to 150-;

in the fermentation process, the temperature is maintained at 34-38 ℃, and ammonia water with the concentration of 15-25 wt% is added at the same time, so that the pH value is maintained at 6.5-7.0; when the dissolved oxygen of the fermentation liquor is reduced to be below 40 percent of the initial dissolved oxygen, adjusting the aeration ratio and the stirring speed to maintain the dissolved oxygen to be 20 to 40 percent of the initial dissolved oxygen; when the glucose content in the fermentation liquor is reduced to be below 0.5g/L, feeding a glucose solution with the concentration of 50-70 wt% to ensure that the glucose content in the fermentation liquor is maintained to be 0.1-0.5 g/L; when the OD value of the fermentation liquor at 660nm reaches 20-35, adding isopropyl-beta-D-thiogalactoside for induction at one time, wherein the addition amount is 0.05-0.2 mmol/L; when the OD value of the fermentation liquor at 660nm reaches 40-50, inoculating the activated bacillus subtilis according to the inoculation amount of 0.5-1.5%, and continuing to ferment for 60-70 h.

Compared with the prior art, the invention has the following beneficial effects:

according to the invention, bacillus subtilis is introduced in the fermentation process of escherichia coli CGMCC15756 for mixed fermentation, so that the bacillus subtilis and the escherichia coli CGMCC15756 are in balanced symbiosis, the problem that the fermentation speed is obviously slowed down or even stopped in advance due to rapid accumulation of byproducts when the escherichia coli CGMCC15756 is singly adopted for fermentation is avoided, the yield and the conversion rate of the N-acetylglucosamine at the fermentation end are improved, and the content of the byproducts such as glutamic acid and acetic acid is reduced.

The preservation information of the bacteria employed in the present invention is as follows:

escherichia coli (Escherichia coli) deposited in the International depositary organization for microorganisms designated by the Budapest treaty on 5/14.2018: china General Microbiological Culture Collection Center (CGMCC), Address: the preservation number of the microorganism culture is CGMCC15756 in the institute of microbiology of national institute of sciences 3, Xilu No. 1, Beijing, Chaoyang, and Beiyang.

Detailed Description

The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the specific embodiments are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.

In the embodiment of the invention, the test method of each item is as follows:

OD value: measuring the light absorption value by adopting a 72306 visible spectrophotometer under the visible light with the wavelength of 660 nm;

glucose content: measured according to the method of GB/T5009.7-2008;

n-acetylglucosamine content: high performance liquid chromatography;

acetic acid content: high performance liquid chromatography;

content of glutamic acid: taking supernatant after the fermentation liquor is centrifuged, diluting until the concentration of the residual glutamic acid is within L g/L, and measuring by using a biosensor analyzer;

conversion rate N-acetylglucosamine/total glucose consumption × 100%.

The strains adopted in the embodiment of the invention are activated according to the following method:

activating the seeds of escherichia coli CGMCC 15756: inoculating glycerol tube (original strain) to test tube slant (LB culture medium), culturing at 37 deg.C for 48 hr, inoculating test tube slant strain to liquid seed culture medium (LB culture medium, 100ml/500ml conical flask), and culturing at 37 deg.C and 200r/min for 18 hr in a shaking table.

B, B.subtilis CMCC63501 seed activation: inoculating glycerol tube (original strain) to test tube slant (LB culture medium), culturing at 37 deg.C for 56 hr, inoculating test tube slant strain to liquid seed culture medium (LB culture medium, 100ml/500ml conical flask), and culturing at 37 deg.C and 250r/min for 24 hr in a shaking table.

Example 1

The embodiment provides a fermentation production method of N-acetylglucosamine, which comprises the following specific steps:

adding 15L of fermentation culture solution into a 50L fermentation tank, and sterilizing; the fermentation culture solution comprises the following components: 5g/L glucose, 12g/L potassium dihydrogen phosphate, 1g/L citric acid, 15g/L, MgSO ammonium sulfate₄ 0.5g/L、CaCl₂0.1g/L of pig hair powder and 15g/L, FeSO of pig hair powder₄ 0.08g/L、ZnCl₂ 0.015g/L、MnSO₄ 0.004g/L、CoCl₂ 0.004g/L、CuSO₄0.012g/L, solvent is water;

adjusting the initial air ventilation of sterile air to 4.5L/min (0.3vvm), the initial pressure to 0.03Mpa, the initial stirring speed to 300rpm and the initial dissolved oxygen to 100%, inoculating the activated Escherichia coli CGMCC15756 according to the inoculum size of 10% (v/v), and fermenting;

in the fermentation process, the temperature is maintained at 37 ℃, and meanwhile, the pH is adjusted by ammonia water with the concentration of 20 wt% to maintain the pH at 7.0; the dissolved oxygen of the fermentation liquor begins to decrease along with the progress of fermentation, and when the dissolved oxygen is reduced to 40 percent of the initial dissolved oxygen, the aeration ratio and the stirring speed are adjusted to maintain the dissolved oxygen to be 20 to 30 percent of the initial dissolved oxygen; when the glucose content in the fermentation liquor is reduced to be below 0.5g/L, feeding a 56 wt% glucose solution to maintain the glucose content in the fermentation liquor at 0.1-0.5 g/L; when the OD value of the fermentation liquor at 660nm reaches 27, adding isopropyl-beta-D-thiogalactoside for induction at one time, wherein the addition amount is 0.1 mmol/L; when the OD value of the fermentation liquor at 660nm reaches 45, inoculating the activated bacillus subtilis CMCC63501 according to the inoculation amount of 0.5% (v/v), and continuing to perform fermentation for 65 h.

Through determination, the content of N-acetylglucosamine in the fermentation liquor is 120.3g/L, the content of glutamic acid is 0.9g/L, the content of acetic acid is 0.6g/L, and the mass conversion rate of glucose to N-acetylglucosamine is 40.1%.

Comparative example 1

The method for producing N-acetylglucosamine by fermentation only differs from example 1 in the following specific steps: and (3) not inoculating the bacillus subtilis CMCC63501 in the fermentation process, stopping the fermentation due to acetic acid accumulation after 51 hours of fermentation, and continuing to 65 hours.

Through determination, the content of N-acetylglucosamine in the fermentation liquor is 52g/L, the content of glutamic acid is 7.4g/L, the content of acetic acid is 5.9g/L, and the mass conversion rate from glucose to N-acetylglucosamine is 25%.

Comparative example 2

The method for producing N-acetylglucosamine by fermentation only differs from example 1 in the following specific steps: inoculating bacillus subtilis CMCC63501 instead of Escherichia coli CGMCC15756 initially, wherein the inoculation amount is 0.5%, no bacillus subtilis CMCC63501 is inoculated in the fermentation process, and the fermentation is stopped after the total fermentation time reaches 65 h.

Through determination, the content of N-acetylglucosamine in the fermentation liquor is 0.04g/L, the content of glutamic acid is 0.05g/L, the content of acetic acid is 0.2g/L, and the mass conversion rate of glucose to N-acetylglucosamine is about 0.

Example 2

The embodiment provides a fermentation production method of N-acetylglucosamine, which comprises the following specific steps:

adding 300L of fermentation culture solution into 1m³Sterilizing in a fermentation tank; the fermentation culture solution comprises the following components: 10g/L glucose, 7g/L potassium dihydrogen phosphate, 4g/L citric acid, 10g/L, MgSO ammonium sulfate₄ 1.0g/L、CaCl₂0.05g/L of chicken feather powder and 30g/L, FeSO of chicken feather powder₄ 0.04g/L、ZnCl₂ 0.03g/L、MnSO₄ 0.002g/L、CoCl₂ 0.008g/L、CuSO₄0.006g/L, and the solvent is water;

adjusting the initial air ventilation rate of sterile air to 60L/min (0.2vvm), the initial pressure to 0.03Mpa, the initial stirring speed to 150rpm and the initial dissolved oxygen amount to 100%, inoculating the activated escherichia coli CGMCC15756 according to the inoculation amount of 8% (v/v), and fermenting;

in the fermentation process, the temperature is maintained at 37 ℃, and meanwhile, the pH is adjusted by ammonia water with the concentration of 20 wt% to maintain the pH at 7.0; the dissolved oxygen of the fermentation liquor begins to decrease along with the progress of fermentation, and when the dissolved oxygen is reduced to 40 percent of the initial dissolved oxygen, the aeration ratio and the stirring speed are adjusted to maintain the dissolved oxygen to be 20 to 30 percent of the initial dissolved oxygen; when the glucose content in the fermentation liquor is reduced to be below 0.5g/L, a glucose solution with the concentration of 58 wt% is fed in to ensure that the glucose content in the fermentation liquor is maintained to be 0.1-0.5 g/L; when the OD value of the fermentation liquor at 660nm reaches 25, adding isopropyl-beta-D-thiogalactoside for induction at one time, wherein the addition amount is 0.1 mmol/L; when the OD value of the fermentation liquor at 660nm reaches 45, inoculating the activated bacillus subtilis CMCC63501 according to the inoculation amount of 0.5% (v/v), and continuing to perform fermentation for 71 h.

Through determination, the content of N-acetylglucosamine in the fermentation liquor is 118.5g/L, the content of glutamic acid is 1.1g/L, the content of acetic acid is 0.7g/L, and the mass conversion rate of glucose to N-acetylglucosamine is 38.6%.

Example 3

The embodiment provides a fermentation production method of N-acetylglucosamine, which comprises the following specific steps:

adding 15L of fermentation culture solution into a 50L fermentation tank, and sterilizing; the fermentation culture solution comprises the following components: glucose 8g/L, potassium dihydrogen phosphate 10g/L, citric acid 2g/L, ammonium sulfate 12g/L, MgSO₄ 0.8g/L、CaCl₂0.08g/L of duck feather powder and 20g/L, FeSO of duck feather powder₄0.06g/L、ZnCl₂ 0.02g/L、MnSO₄ 0.003g/L、CoCl₂ 0.006g/L、CuSO₄0.01g/L, and the solvent is water;

adjusting the initial air ventilation of sterile air to 4.5L/min (0.3vvm), the initial pressure to 0.03Mpa, the initial stirring speed to 300rpm and the initial dissolved oxygen to 100%, inoculating the activated Escherichia coli CGMCC15756 according to the inoculation amount of 5% (v/v), and fermenting;

in the fermentation process, the temperature is maintained at 35 ℃, and the pH is adjusted by ammonia water with the concentration of 20 wt% to maintain the pH at 6.5; the dissolved oxygen of the fermentation liquor begins to decrease along with the progress of fermentation, and when the dissolved oxygen is reduced to 40 percent of the initial dissolved oxygen, the aeration ratio and the stirring speed are adjusted to maintain the dissolved oxygen to be 20 to 30 percent of the initial dissolved oxygen; when the glucose content in the fermentation liquor is reduced to be below 0.5g/L, feeding a 56 wt% glucose solution to maintain the glucose content in the fermentation liquor at 0.1-0.5 g/L; when the OD value of the fermentation liquor at 660nm reaches 35, adding isopropyl-beta-D-thiogalactoside for induction at one time, wherein the addition amount is 0.05 mmol/L; when the OD value of the fermentation liquor at 660nm reaches 50, inoculating the activated bacillus subtilis CMCC63501 according to the inoculation amount of 1.5% (v/v), and continuing to perform fermentation for 62 h.

Through determination, the content of N-acetylglucosamine in the fermentation liquor is 122.5g/L, the content of glutamic acid is 0.5g/L, the content of acetic acid is 0.5g/L, and the mass conversion rate of glucose to N-acetylglucosamine is 41.2%.

Example 4

This example provides a method for producing N-acetylglucosamine by fermentation, which only differs from example 1 in the following steps: when the OD value of the fermentation liquor at 660nm reaches 35, inoculating activated bacillus subtilis CMCC63501, and the total fermentation time is 60 h.

Through determination, the content of N-acetylglucosamine in the fermentation liquor is 112.7g/L, the content of glutamic acid is 0.4g/L, the content of acetic acid is 0.4g/L, and the mass conversion rate of glucose to N-acetylglucosamine is 36.5%.

Example 5

This example provides a method for producing N-acetylglucosamine by fermentation, which only differs from example 1 in the following steps: when the OD value of the fermentation liquor at 660nm reaches 55, inoculating activated bacillus subtilis CMCC63501, and the total fermentation time is 79 h.

Through determination, the content of N-acetylglucosamine in the fermentation liquor is 85g/L, the content of glutamic acid is 3.9g/L, the content of acetic acid is 3.2g/L, and the mass conversion rate from glucose to N-acetylglucosamine is 28%.

As can be seen from the above examples and comparative examples, compared with the method of fermenting with Escherichia coli CGMCC15756 alone (comparative example 1) or the method of fermenting with Bacillus subtilis CMCC63501 alone (comparative example 2), the method provided by the invention has the advantages that the final N-acetylglucosamine content and the conversion rate are remarkably improved, the content of the byproducts such as glutamic acid and acetic acid is remarkably reduced, and the reduction of the byproducts is beneficial to the post-purification work. The method provided by the invention is simple and effective, and has no obvious increase on large-scale production cost, so that the method is suitable for large-scale industrial fermentation production of the N-acetylglucosamine.

In example 4, the inoculation time of the bacillus subtilis CMCC63501 is earlier, so that the N-acetylglucosamine content and the conversion rate in the tank are reduced; in example 5, the inoculation time of the bacillus subtilis CMCC63501 is relatively late, so that the N-acetylglucosamine content and the conversion rate in the tank are greatly reduced, and the fermentation period and the contents of acetic acid and glutamic acid as byproducts are obviously increased.

Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.