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Preparation method of microbial fermentation-based N-acetyle-D-glucosamine

A technology of glucosamine and microbial fermentation, applied in the field of preparation of N-acetyl-D-glucosamine, can solve the problems of reduced product yield, high price of chitinase, product oxidative denaturation, etc., and achieves reduction of the probability of other impurities, The effect of protecting from oxidation discoloration and reducing impurities

Inactive Publication Date: 2016-05-04
浙江澳兴生物科技有限公司
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  • Abstract
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  • Claims
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Problems solved by technology

[0006] At present, there are two main processes for the production of N-acetyl-D-glucosamine. One process is that shrimp and crab shells are processed into chitin by acid decalcification, alkali degreasing and protein, and then the chitin is hydrolyzed by hydrochloric acid into D-amino Glucose hydrochloride is then prepared by the acetylation reaction of D-glucosamine hydrochloride and acetic anhydride, but there is a precursor chemical acetic anhydride in the process, which has the risk of drug diffusion. The raw material of chitin is shrimp and crab Shells, whose supply is susceptible to seasonal changes
Another process is to prepare N-acetyl-D-glucosamine from chitin through the action of chitinase, but chitinase is expensive, making the production cost too high to realize industrialization, and the existing process is concentrated in the purification process When the temperature is high, it is easy to partially oxidize and denature the product and reduce the yield and content of the product

Method used

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  • Preparation method of microbial fermentation-based N-acetyle-D-glucosamine
  • Preparation method of microbial fermentation-based N-acetyle-D-glucosamine
  • Preparation method of microbial fermentation-based N-acetyle-D-glucosamine

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preparation example Construction

[0051] The preparation method that the embodiment of the present invention provides comprises the following steps:

[0052] S101, melting the glycerol tube for preserving the engineered bacteria at room temperature to form a mixed solution;

[0053] Specifically, the engineering strain is N-acetyl-D-glucosamine-producing Brevibacterium metabolizing.

[0054] S102, use an inoculation loop to dip the mixed solution onto a plate or slope containing streptomycin and apramycin, and incubate at 37°C for 18-24 hours. In a preferred embodiment, the incubation time is 19 to 22 hours;

[0055] Of course, before the activation of the strain, the strain needs to be preserved, and the preservation can be carried out at -80°C or -20°C.

[0056] Preservation methods specifically include: picking single colonies of engineering bacteria grown on resistant plates, adding them to resistant LB medium to form bacterial liquid, and culturing at 37°C for 8 to 12 hours; the culture time is preferab...

example 1

[0101] 1) Take the glycerol tube with bacteria stored at -20°C, melt it at room temperature, then use an inoculation loop to dip some into a plate or slope containing streptomycin and apramycin, and incubate at 37°C for 20 hours;

[0102] 2) Pick a single colony and place it in a 100mL Erlenmeyer flask with a liquid volume of 10ml LB (including streptomycin 50mg / L and apramycin 50mg / L), and shake and culture at 37°C for 8 hours;

[0103] 3) Inoculate 10ml of primary seeds in a 5L shake flask with 1L of secondary seed medium (comprising streptomycin 50mg / L and apramycin 50mg / L), culture with shaking at 34°C for 18 hours, OD600= 3, and 5 bottles are put into a 3000L seed tank, and the formula is shown in Table 1. Only streptomycin was added, not apramycin. Stirring speed 220rpm, ventilation ratio 1:0.8;

[0104] basic salt medium

Content (g / L)

Remark

K H 2 PO 4

14

CaCl 2

0.02

K H 2 PO 4 .3H 2 o

21

...

example 2

[0130] 1) Take the glycerol tube with strains preserved at -20°C, melt at room temperature, then use an inoculation loop to dip some into a plate or slope containing streptomycin and apramycin, and incubate at 37°C for 21 hours;

[0131] 2) Pick a single colony and place it in a 100 mL Erlenmeyer flask with a liquid volume of 10 ml LB (containing 50 mg / L streptomycin and 50 mg / L apramycin), and culture it with shaking at 37° C. for 8 hours.

[0132] 3) Inoculate 10 ml of primary seeds in a 5L shake flask with 1L of secondary seed medium (containing 50 mg / L streptomycin and 50 mg / L apramycin), and culture with shaking at 34°C for 17 hours, OD600= 4, and 4 bottles are inserted into a 3000L seed tank, and the formula is shown in Table 4. Only streptomycin was added, not apramycin. The stirring speed is 220rpm, and the ventilation ratio is 1:0.8.

[0133]

[0134] Sterilization: 121°C 20min

[0135] Table 4

[0136] 4) Prepare the fermentation medium, add various ingredient...

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Abstract

The embodiment of the invention relates to a preparation method of microbial fermentation-based N-acetyle-D-glucosamine. The preparation method comprises the steps of firstly carrying out two-stage cultivation on engineering strains (metabolism brevibacterium producing N-acetyle-D-glucosamine) subjected to special preservation, then transferring a fermentation medium into a fermentation tank of 30000L, at the same time adding sterilized glucose, then inoculating seeds cultivated in two stages into the fermentation tank of 30000L, strictly controlling fermentation parameters until the fermentation is sufficient, then sterilizing fermentation liquor, removing the strains by filtration through a ceramic membrane or a sheet frame after sterilization, adding chitosan into filtrate for flocculation to remove proteins, adding activated carbon for decoloration, filtering, performing ion exchange and bed mixing, adding a certain amount of ethyl alcohol into feed liquor for low-temperature concentration and crystallization, adding ethyl alcohol of the concentration over 95 percent into a crystalized material for rinsing, and performing centrifugal drying to obtain the high-purity N-acetyle-D-glucosamine.

Description

technical field [0001] The invention relates to the technical field of pharmaceutical processing technology, in particular to a preparation method of N-acetyl-D-glucosamine based on microbial fermentation. Background technique [0002] N-acetyl-D-glucosamine, English name N-Acetylglucosamine, molecular structure formula is: [0003] [0004] Chemical name: 2-(acetylamino)-2-deoxy-D-glucose. [0005] N-acetyl-D-glucosamine is a kind of reducing monosaccharide with high sweetness. It has many important physiological functions in organisms. It is clinically used as a drug for treating rheumatism and rheumatoid arthritis. Oxidants, food additives for infants and sweeteners for diabetics and cosmetics are of great significance in the fields of life sciences, medicine and fine chemicals. [0006] At present, there are two main processes for the production of N-acetyl-D-glucosamine. One process is that shrimp and crab shells are processed into chitin by acid decalcification, a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/26C12P19/02C12N1/21C12R1/13
CPCC12P19/26C12N1/20C12P19/02
Inventor 詹小远陈进利张福明
Owner 浙江澳兴生物科技有限公司
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