Filtering method of N-acetylglucosamine fermentation liquor

Abstract

The invention discloses a filtering method of N-acetylglucosamine fermentation liquor, and belongs to the technical field of bioengineering. The method comprises the following steps: fermenting escherichia coli to obtain fermentation liquor of N-acetylglucosamine, carrying out inactivation, acidification and flocculation pretreatment on the fermentation liquor, and separating by using a horizontal screw centrifuge. Compared with a common separation mode at present, the method has the advantages that solid-liquid separation is carried out on the fermentation liquor by using the horizontal screw centrifuge, due to the absence of a filtering medium, the method is not restricted by the area and the filtering speed, the solid-liquid separation speed is high, the clear liquid yield is high, and the water content of filter residues is low. The process is simple to operate, short in working time and low in labor intensity, and is a simple and efficient filtering method for the N-acetylglucosamine fermentation liquor.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to a method for filtering N-acetylglucosamine fermentation liquid. Background Art

[0002] N-acetylglucosamine (GlcNAc) is a derivative of glucose and is the basic unit of many important polysaccharides in organisms. Studies have shown that N-acetylglucosamine has anti-inflammatory, anti-tumor and antioxidant effects; it can be used as a therapeutic drug for rheumatic and rheumatoid arthritis in clinical practice, as an infant food additive, antioxidant and sweetener for diabetic patients; it can also treat gastrointestinal inflammation, functional disorders and gastric ulcers and other gastrointestinal diseases, and is widely used in nutritional supplements, pharmaceuticals, cosmetics and food.

[0003] At present, the domestic production method is still the chitin extraction method, but with the enhancement of people's environmental awareness, this method will also face elimination. More and more manufacturers have turned their attention to the microbial fermentation method, which is a relatively advantageous method in microorganisms when constructing genetic engineering bacteria fermentation. This method directly uses glucose fermentation to produce N-acetylglucosamine through engineering bacteria, and the product concentration can reach 140g / L, which greatly meets the future industrial production needs. The present invention is the first step of the microbial fermentation process for producing N-acetylglucosamine-solid-liquid separation, which lays a solid foundation for the preparation and purification of N-acetylglucosamine.

[0004] Since N-acetylglucosamine fermentation broth has a high bacterial count and high viscosity, a corresponding GlcNAc fermentation broth filtration method needs to be developed to achieve solid-liquid separation. Therefore, the current filtration methods for GlcNAc fermentation broth often have the disadvantages of low separation efficiency, high energy consumption, and high environmental pollution.

[0005] For example, Chinese patents CN106831894A (patent name: "A method for separating D-glucosamine hydrochloride by deacetylation coupling adsorption") and CN105543312A (patent name: "Preparation method of N-acetyl-D-glucosamine based on microbial fermentation") both use ceramic membrane filtration to remove microbial cells, thereby achieving solid-liquid separation. However, this process has the disadvantages of long filtration cycle, dilution of target product concentration during microfiltration membrane removal, large amount of filter residue and wastewater, easy clogging of membrane components, high equipment investment and maintenance costs.

[0006] For another example, Chinese patent CN113354698A (patent name: "A method for preparing N-acetylglucosamine fermentation clear liquid using a composite flocculant"), although this method achieves solid-liquid separation of bacteria through flocculation and plate and frame filter pressing, it still has some defects, such as the plate and frame filter pressing requires intermittent operation, high labor intensity, long working hours, a large number of people, a poor working environment, a large amount of solid waste (waste filter aids), etc., especially when dealing with low filtration rates and abnormal tank batches, it is basically impossible to filter, resulting in losses. Summary of the invention

[0007] In view of the above-mentioned deficiencies in the prior art, the present invention provides a method for filtering N-acetylglucosamine fermentation broth which has simple process operation, low energy consumption and is environmentally friendly.

[0008] The technical solution adopted to achieve the above-mentioned invention object is: (1) inactivating the N-acetylglucosamine fermentation broth produced by fermentation; (2) The inactivated N-acetylglucosamine fermentation broth is acidified and the pH is adjusted to 4.0-5.0; (3) first adding an inorganic flocculant to the acidified N-acetylglucosamine fermentation broth and stirring evenly, and then adding an organic flocculant for flocculation; (4) The solution after flocculation in step (3) is subjected to horizontal centrifugation to obtain N-acetylglucosamine fermentation clear liquid and filter residue.

[0009] In step (1), the N-acetylglucosamine fermentation broth is inactivated by heating, the heating temperature is 70-100° C., and the inactivation time is 30-60 min.

[0010] In step (2), the acidification adopts one of hydrochloric acid, sulfuric acid or phosphoric acid.

[0011] In step (3), the inorganic flocculant is one of polyferric sulfate, polyaluminium chloride, polyaluminium sulfate, ferric chloride and aluminium chloride.

[0012] In step (3), the amount of the inorganic flocculant is 0.005-0.01 of the volume of the N-acetylglucosamine fermentation broth.

[0013] In step (3), the organic flocculant is a polyacrylamide solution, the concentration of polyacrylamide in the polyacrylamide solution is 0.3-1%; the amount of the polyacrylamide solution used is 0.005-0.01 of the volume of the N-acetylglucosamine fermentation broth.

[0014] In step (4), the operation method of the horizontal screw centrifuge is: start the horizontal screw centrifuge, adjust the speed to 800-1500rpm, use a pump to pump the N-acetylglucosamine flocculant into the horizontal screw centrifuge, and maintain the speed at 800-1500rmp. After the centrifugation of the flocculant, wash the filter residue once with purified water with 10%-20% volume of the N-acetylglucosamine flocculant, and then perform a second centrifugation separation, and combine the separated liquids.

[0015] The present invention uses a horizontal screw centrifuge to separate the fermentation liquid into solid and liquid. Because there is no filter medium, it is not restricted by the area agent filtration rate, so it can process some liquids that cannot be processed by a plate and frame filter press. Its advantages are mainly reflected in less equipment, small footprint, short working time, low labor intensity, and environmental friendliness. The degree of mechanization and automation is significantly better than the plate and frame filter press filtration process. DETAILED DESCRIPTION

[0016] The present invention is further illustrated by the following examples, which are not intended to limit the present invention.

[0017] Example 1 Take 30L of N-acetylglucosamine fermentation broth with a bacterial concentration of OD=100, heat it to 75℃ for inactivation, and keep it warm for 40min; cool it to room temperature, adjust the pH to 4.0 with hydrochloric acid, add 150g of polyferric sulfate while stirring, stir for 20min, then slowly add 150mL of 0.3% polyacrylamide solution, stir evenly, and let it stand for flocculation for 30min; then use horizontal screw centrifugation (speed: 1000rmp); after the fermentation broth is completely centrifuged, wash the filter residue with 10% purified water, perform secondary centrifugation, combine the two separated liquids, and finally obtain 32L of N-acetylglucosamine fermentation clear liquid (OD=0.2), the filter residue weighs 4.5kg, and the water content is 75%.

[0018] Example 2 Take 300L of N-acetylglucosamine fermentation broth with a fermentation broth bacterial concentration of OD=140, heat it to 85℃ for inactivation, and keep it warm for 60min; cool it to room temperature, adjust the pH to 4.5 with hydrochloric acid, add 2.1kg of polyaluminium chloride while stirring, stir for 20min, then slowly add 2100mL of 0.5% polyacrylamide solution, stir evenly, let it stand for flocculation for 30min; then use horizontal screw centrifuge (speed: 1500rmp); after the fermentation broth is completely centrifuged, wash the filter residue with 10% purified water, perform secondary centrifugation, combine the two separated liquids, and finally obtain 320L of N-acetylglucosamine fermentation clear liquid (OD=0.12), the filter residue weighs 41kg, and the water content is 65%.

[0019] Example 3 Take 1000L of N-acetylglucosamine fermentation liquid with a fermentation liquid bacterial concentration of OD=110, heat it to 95℃ for inactivation, and keep it warm for 60min; cool it to room temperature, adjust the pH to 5.0 with hydrochloric acid, add 9kg of aluminum chloride while stirring, stir for 20min, slowly add 9L of 0.8% polyacrylamide solution, stir evenly, let it stand for flocculation for 30min; then use horizontal screw centrifuge (speed: 1500rmp); after the fermentation liquid is completely centrifuged, wash the filter residue with 10% purified water, perform secondary centrifugation, combine the two separated liquids, and finally obtain 1120L of N-acetylglucosamine fermentation clear liquid (OD=0.18), the filter residue weighs 128kg, and the water content is 68%.

[0020] Example 4 Take 4000L of N-acetylglucosamine fermentation liquid with a fermentation liquid bacterial concentration of OD=130, heat it to 95℃ for inactivation, and keep it warm for 60min; cool it to room temperature, adjust the pH to 4.5 with hydrochloric acid, add 28kg of polyaluminium chloride while stirring, stir for 20min, slowly add 28L of 0.5% polyacrylamide solution, stir evenly, let it stand for flocculation for 30min; then use horizontal screw centrifuge (speed: 1500rmp); after the fermentation liquid is completely centrifuged, wash the filter residue with 20% purified water, perform secondary centrifugation, combine the two separated liquids, and finally obtain 4380L of N-acetylglucosamine fermentation clear liquid (OD=0.13), the filter residue weighs 530kg, and the water content is 66%.

[0021] The above is a detailed introduction to a method for filtering N-acetylglucosamine fermentation broth provided by the present invention. Specific examples are used herein to illustrate the implementation methods and preferred conditions of the invention, and the description of the above examples is intended to help understand the method and core concept of the present invention.

Claims

1. A method for filtering N-acetylglucosamine fermentation broth, characterized in that: The specific steps are as follows: (1) inactivating the N-acetylglucosamine fermentation broth produced by fermentation; (2) The inactivated N-acetylglucosamine fermentation broth is acidified and the pH is adjusted to 4.0-5.0; (3) first adding an inorganic flocculant to the acidified N-acetylglucosamine fermentation broth and stirring evenly, and then adding an organic flocculant for flocculation; (4) The solution after flocculation in step (3) is subjected to horizontal centrifugation to obtain N-acetylglucosamine fermentation clear liquid and filter residue.

2. The method for filtering N-acetylglucosamine fermentation broth according to claim 1, characterized in that: In step (1), the N-acetylglucosamine fermentation broth is inactivated by heating, the heating temperature is 70-100° C., and the inactivation time is 30-60 min.

3. The method for filtering N-acetylglucosamine fermentation broth according to claim 1, characterized in that: In step (2), the acidification adopts one of hydrochloric acid, sulfuric acid or phosphoric acid.

4. The method for filtering N-acetylglucosamine fermentation broth according to claim 1, characterized in that: In step (3), the inorganic flocculant is one of polyferric sulfate, polyaluminium chloride, polyaluminium sulfate, ferric chloride and aluminium chloride.

5. The method for filtering N-acetylglucosamine fermentation broth according to claim 1, characterized in that: In step (3), the amount of the inorganic flocculant is 0.005-0.01 of the volume of the N-acetylglucosamine fermentation broth.

6. The method for filtering N-acetylglucosamine fermentation broth according to claim 1, characterized in that: In step (3), the organic flocculant is a polyacrylamide solution, the concentration of polyacrylamide in the polyacrylamide solution is 0.1-1%; the amount of the polyacrylamide solution is 0.005-0.01 of the volume of the N-acetylglucosamine fermentation broth.

7. The method for filtering N-acetylglucosamine fermentation broth according to claim 1, characterized in that: In step (4), the operation method of the horizontal screw centrifuge is: start the horizontal screw centrifuge, adjust the speed to 800-1500rpm, use a pump to pump the N-acetylglucosamine flocculant into the horizontal screw centrifuge, and maintain the speed at 800-1500rmp. After the centrifugation of the flocculant, wash the filter residue once with purified water with 10%-20% volume of the N-acetylglucosamine flocculant, and then perform a second centrifugation separation, and combine the separated liquids.