阅读说明：本技术 微生物发酵生产氨基葡萄糖硫酸钾盐的方法 (Method for producing glucosamine potassium sulfate salt by microbial fermentation ) 是由 卢健行 韩宁 吴祥舟 张建华 卢建功 卢建智 于 2019-12-02 设计创作，主要内容包括：本发明公开了一种微生物发酵生产氨基葡萄糖硫酸钾盐的方法,将大肠杆菌、枯草芽孢杆菌、葡萄球菌进行种子活化后制得混合发酵剂,将混合发酵剂接种至含有葡萄糖和氮源的发酵培养基中恒温摇床培养,得到含有氨基葡萄糖的发酵液,经分离纯化后进一步制得氨基葡萄糖硫酸钾盐并提纯。本发明通过混合发酵剂提高大肠杆菌的发酵活力,半胱氨酸、丙氨酸的加入可以降低发酵过程中谷氨酸的产生速度,提高氨基葡萄糖合成速率和产品纯度,从而提高最终产品氨基葡萄糖硫酸钾盐的纯度。(The invention discloses a method for producing glucosamine potassium sulfate by microbial fermentation, which comprises the steps of activating seeds of escherichia coli, bacillus subtilis and staphylococcus to prepare a mixed starter culture, inoculating the mixed starter culture into a fermentation medium containing glucose and a nitrogen source, carrying out constant-temperature shaking table culture to obtain a fermentation broth containing glucosamine, separating and purifying, and further preparing and purifying the glucosamine potassium sulfate. The fermentation activity of the escherichia coli is improved by the mixed starter, the production speed of the glutamic acid in the fermentation process can be reduced by adding the cysteine and the alanine, the synthesis rate of the glucosamine is increased, the purity of the product is improved, and the purity of the final product, namely the glucosamine potassium sulfate is improved.)

1. A method for producing glucosamine potassium sulfate salt by microbial fermentation is characterized by comprising the following steps: the method comprises the following steps:

(1) activating the seeds of escherichia coli, bacillus subtilis and staphylococcus to prepare a mixed starter;

(2) inoculating the mixed leaven into a fermentation medium containing glucose and a nitrogen source for constant temperature shaking table culture, controlling the rotation speed of the shaking table to be 230-;

(3) the fermentation process is carried out in a constant-speed feeding manner, the glucose feeding speed is controlled to be 3-5L/h, the pH value in the fermentation process is controlled to be 6.8-7.0 by ammonia water, and the fermentation culture is carried out for 35-48h to obtain fermentation liquor;

(4) centrifuging the fermentation liquor, removing thallus, collecting supernatant, adding hydrochloric acid into the supernatant, and hydrolyzing to obtain hydrolysate;

(5) concentrating the hydrolysate in vacuum at 75-90 deg.c and vacuum degree of-0.05-0.1 MPa;

(6) cooling the concentrated solution to 5-10 deg.C, crystallizing under stirring for 5-7 hr, precipitating for 3-4 hr, and centrifuging to obtain glucosamine hydrochloride crude product;

(7) dissolving the obtained crude product with water, subjecting the water solution to pretreated macroporous adsorbent resin column chromatography, eluting with water until no chloride ion exists, concentrating under reduced pressure, and recrystallizing to obtain glucosamine hydrochloride;

(8) dissolving glucosamine hydrochloride in water, adding potassium sulfate under stirring, stirring at 40-50 deg.C for 30-60min, and freeze drying the reaction product under vacuum to obtain glucosamine potassium sulfate.

2. The method for producing glucosamine potassium sulfate by microbial fermentation according to claim 1, wherein the method comprises the following steps: and (2) performing activation culture on the escherichia coli, the bacillus subtilis and the staphylococcus for 6-8h on a plate culture medium before performing seed activation.

3. The method for producing glucosamine potassium sulfate by microbial fermentation according to claim 2, wherein the method comprises the following steps: the plate culture medium in the step (1) comprises 11.5g/L of fish peptone, 6g/L of yeast extract powder, 5g/L of sodium chloride, 2g/L of ammonium sulfate and 15g/L of agar.

4. The method for producing glucosamine potassium sulfate by microbial fermentation according to claim 1 or 2, wherein: the mixed leaven in the step (1) is prepared according to the volume ratio of 3 (1-2) to 0.8-1) of escherichia coli, bacillus subtilis and staphylococcus.

5. The method for producing glucosamine potassium sulfate by microbial fermentation according to claim 1 or 2, wherein: the seeds of the escherichia coli, the bacillus subtilis and the staphylococcus in the step (1) are activated by inoculating strains in a seed culture medium, controlling the culture temperature to be 36-38 ℃, the shaking table speed to be 220-270rpm, and the culture time to be 12-14 h.

6. The method for producing glucosamine potassium sulfate by microbial fermentation according to claim 5, wherein the method comprises the following steps: the seed culture medium in the step (1) comprises 11.5g/L of fish peptone, 20g/L of yeast extract powder, 5g/L of sodium chloride, 2g/L of ammonium sulfate, 5.5g/L of glycerol and pH 6.8-7.0.

7. The method for producing glucosamine potassium sulfate by microbial fermentation according to claim 1, wherein the method comprises the following steps: the fermentation medium in the step (2) comprises: 25g/L glucose, 20g/L yeast extract powder, 10g/L alanine, 10g/L cysteine, 3g/L sodium nitrate, 1.05g/L dipotassium phosphate, 0.45g/L potassium dihydrogen phosphate, 0.1g/L sodium chloride, 0.5g/L magnesium sulfate, 0.03g/L ferrous sulfate, 3.2g/L lactose and 5.5g/L glycerol.

8. The method for producing glucosamine potassium sulfate by microbial fermentation according to claim 1, wherein the method comprises the following steps: the macroporous adsorption resin column in the step (7) is a 732 cation exchange column; the flow rate of the aqueous solution passing through the column bed is 3-5ml cm^-2·min^-1(ii) a The eluted solution is 3-5% AgNO₃The solution checks the eluate for chloride ions.

9. The method for producing glucosamine potassium sulfate by microbial fermentation according to claim 1, wherein the method comprises the following steps: and (4) recrystallizing in 85-95% ethanol solution in the step (7).

10. The method for producing glucosamine potassium sulfate by microbial fermentation according to claim 1, wherein the method comprises the following steps: in the step (8), the mass ratio of the potassium sulfate to the glucosamine hydrochloride to the water is 1 (2-4) to 10-15.

Technical Field

The invention belongs to the technical field of biological fermentation, and particularly relates to a method for producing glucosamine potassium sulfate salt by microbial fermentation.

Background

Glucosamine (GleN) is an important hexosamine formed by substituting one hydroxyl group of glucose with an amino group, and there are two main types of glucosamine on the market today, one is glucosamine hydrochloride and the other is glucosamine sulfate. D-Glucosamine Hydrochloride (D-Glucosamine Hydrochloride), molecular formula C₆H₁₃NO₅HCl, a white crystal, odorless, slightly sweet, easily soluble in water, slightly soluble in methanol, insoluble in organic solvents such as ethanol, has important physiological functions for human body, participates in liver and kidney detoxification, plays a role in anti-inflammation and liver protection, has good curative effect on rheumatic arthritis and gastric ulcer, and is a main raw material for synthesizing antibiotics and anticancer drugs; can also be used in food, cosmetic and feed additive. Glucosamine hydrochloride is extracted from natural chitin, is a marine biological agent, and is the main component of chondroitin sulfate. It can promote the synthesis of mucopolysaccharide, raise the viscosity of joint synovial fluid, improve the metabolism of joint cartilage, promote the repair of joint cartilage and has obvious antiphlogistic and analgesic effects. It has the effect of promoting the injection efficiency of antibiotics, and can be used as nutritional supplement for diabetic patients.

The current methods for producing glucosamine mainly comprise an acid hydrolysis method, an enzymolysis method and a microbial fermentation method. The production raw materials of the acid hydrolysis method and the enzymolysis method are from exoskeletons of fishes, shrimps, crabs and the like, and glucosamine is obtained by extracting chitin and chitosan and then carrying out acidolysis or enzymolysis; however, a large amount of concentrated hydrochloric acid is needed in the acid hydrolysis process, which can cause serious industrial pollution; the enzymolysis method is to degrade the exoskeleton of fishes, shrimps and crabs by using chitosan, and has low process efficiency and higher production cost, so that the search for a proper microbial fermentation method to realize the industrial production of glucosamine is the current environmental and market demand. Escherichia coli is a common glucosamine synthetase producing strain. The research shows that: in Escherichia coli, glucosamine is produced from glutamine as an amino donor and fructose-6-phosphate under the catalytic action of glucosamine synthetase (GlmS). However, in the prior art, the production amount of glucosamine is low, the industrial high-efficiency production is difficult to realize, and the product purity can not reach the eyeball of a consumer.

Disclosure of Invention

In order to make up the defects of the prior art, the invention provides the method for producing glucosamine potassium sulfate by microbial fermentation, which has the advantages of high production efficiency, low cost and simple operation.

The invention is realized by the following technical scheme:

a method for producing glucosamine potassium sulfate by microbial fermentation is characterized in that: the method comprises the following steps:

(1) activating the seeds of escherichia coli, bacillus subtilis and staphylococcus to prepare a mixed starter;

(2) inoculating the mixed leaven into a fermentation medium containing glucose and a nitrogen source for constant temperature shaking table culture, controlling the rotation speed of the shaking table to be 230-;

(3) the fermentation process is carried out in a constant-speed feeding manner, the glucose feeding speed is controlled to be 3-5L/h, the pH value in the fermentation process is controlled to be 6.8-7.0 by ammonia water, and the fermentation culture is carried out for 35-48h to obtain fermentation liquor;

(4) centrifuging the fermentation liquor, removing thallus, collecting supernatant, adding hydrochloric acid into the supernatant, and hydrolyzing to obtain hydrolysate;

(5) concentrating the hydrolysate in vacuum at 75-90 deg.c and vacuum degree of-0.05-0.1 MPa, and recovering hydrochloric acid;

(6) cooling the concentrated solution to 5-10 ℃, crystallizing for 5-7h under stirring, precipitating for 3-4h, centrifuging to obtain glucosamine hydrochloride crude product, and adding concentrated hydrochloric acid into the mother solution obtained by centrifuging for hydrolyzing the concentrated solution of airplane after staying up;

(7) dissolving the obtained crude product with water, subjecting the water solution to pretreated macroporous adsorbent resin column chromatography, eluting with water until no chloride ion exists, concentrating under reduced pressure, and recrystallizing to obtain glucosamine hydrochloride;

(8) dissolving glucosamine hydrochloride in water, adding potassium sulfate under stirring, stirring at 40-50 deg.C for 30-60min, and freeze drying the reaction product under vacuum to obtain glucosamine potassium sulfate.

Preferably, the Escherichia coli, the Bacillus subtilis and the staphylococcus in the step (1) are activated and cultured on a plate culture medium for 6-8h before being subjected to seed activation.

Further, the plate culture medium in the step (1) comprises 11.5g/L of fish peptone, 6g/L of yeast extract, 5g/L of sodium chloride, 2g/L of ammonium sulfate and 15g/L of agar.

Further, the mixed starter in the step (1) is prepared according to the volume ratio of 3 (1-2) to 0.8-1 of escherichia coli, bacillus subtilis and staphylococcus.

Further, the seeds of the escherichia coli, the bacillus subtilis and the staphylococcus are activated in the step (1) by inoculating strains in a seed culture medium, controlling the culture temperature to be 36-38 ℃, the shaking table speed to be 220-270rpm, and the culture time to be 12-14 h.

Further, the seed culture medium in the step (1) comprises 11.5g/L of fish peptone, 20g/L of yeast extract powder, 5g/L of sodium chloride, 2g/L of ammonium sulfate, 5.5g/L of glycerol and 6.8-7.0 of pH.

Preferably, the fermentation medium in step (2) comprises: 25g/L glucose, 20g/L yeast extract powder, 10g/L alanine, 10g/L cysteine, 3g/L sodium nitrate, 1.05g/L dipotassium phosphate, 0.45g/L potassium dihydrogen phosphate, 0.1g/L sodium chloride, 0.5g/L magnesium sulfate, 0.03g/L ferrous sulfate, 3.2g/L lactose and 5.5g/L glycerol.

Further, the mixed starter in the step (2) is inoculated on the fermentation medium according to the inoculation amount of 10%.

Preferably, the hydrochloric acid in the step (4) is 30% concentrated hydrochloric acid.

Preferably, the stirring speed of the stirring crystallization in the step (6) is 50 rpm.

Preferably, the macroporous adsorption resin column in the step (7) is 732 cation exchange columns; the flow rate of the aqueous solution passing through the column bed is 3-5ml cm^-2·min^-1(ii) a The eluted solution is 3-5% AgNO₃The solution checks the eluate for chloride ions.

Further, in the step (7), recrystallization is carried out by using an 85-95% ethanol solution.

Preferably, the mass ratio of the potassium sulfate, the glucosamine hydrochloride and the water in the step (8) is 1 (2-4) to (10-15).

The invention has the beneficial effects that: according to the invention, the fermentation activity of escherichia coli is improved by the mixed leaven, the glucosamine is efficiently produced by fermentation, the production speed of glutamic acid in the fermentation process can be reduced by adding cysteine and alanine, the synthesis rate of the glucosamine is improved, the purity of the product is improved by acidolysis, concentration, crystallization and decoloration, the reaction cost is reduced, and the yield of the product is improved.

Detailed Description

The present invention will be described in further detail with reference to specific embodiments thereof to assist those skilled in the art in providing a more complete, accurate and thorough understanding of the inventive concept and aspects thereof, and the scope of the present invention includes, but is not limited to, the following examples, and any modifications in the details and form of the technical aspects thereof that fall within the spirit and scope of the present application are intended to be included therein.

The following examples all follow the following medium composition:

the plate culture medium comprises: 11.5g/L of fish peptone, 6g/L of yeast extract powder, 5g/L of sodium chloride, 2g/L of ammonium sulfate and 15g/L of agar.

The seed culture medium comprises: 11.5g/L of fish peptone, 20g/L of yeast extract powder, 5g/L of sodium chloride, 2g/L of ammonium sulfate and 5.5g/L of glycerol;

the fermentation medium comprises: 25g/L glucose, 20g/L yeast extract powder, 10g/L alanine, 10g/L cysteine, 3g/L sodium nitrate, 1.05g/L dipotassium phosphate, 0.45g/L potassium dihydrogen phosphate, 0.1g/L sodium chloride, 0.5g/L magnesium sulfate, 0.03g/L ferrous sulfate, 3.2g/L lactose and 5.5g/L glycerol.