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Process of separating and extracting hyaluronic acid from microbial fermented liquid

A technology of microbial fermentation liquid and hyaluronic acid, which is applied in the field of preparation of hyaluronic acid, can solve the problems of increasing production costs, HA yield and molecular weight loss rate, many operating steps, molecular weight degradation, etc., and achieve the effect of increasing costs

Inactive Publication Date: 2010-04-14
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, U.S. Patent No. 6489467 describes a method that does not require the use of organic solvents, but by adjusting the pH of HA to 2.5 to change the properties of HA and purify it using ultrafiltration, but the filtration rate is very slow and time-consuming, which is not conducive to large-scale Production, and HA should not be kept in an environment with a pH of 2.5 for a long time, which will easily lead to molecular weight degradation
Chinese patents 200610057913.2 and 200610090586.0 also describe adjusting the pH of HA to 3, and performing ultrafiltration with the help of electric and magnetic fields to increase the filtration rate, while avoiding the use of organic solvents, but the filtration rate is still only 25.8L / (m 2 h), which is not conducive to magnification
The process route is long, complicated, time-consuming and costly; more organic solvents are used, which is easy to cause secondary pollution
As a result, the production cost of hyaluronic acid is still very high, which seriously limits the application range of hyaluronic acid
Various alternative methods, such as using some more expensive and stable adsorption resins or ion exchange resins for impurity removal, or using refrigerated centrifugation, pH control, ultrafiltration and other methods to remove impurities, the resulting products have high purity, but the yield and molecular weight of HA are lost. The rate is insufficient due to many operation steps, and involves complex processing and processing equipment, thus increasing the production cost

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] 1) Take 500ml of fermented broth, containing 2g of HA, with a molecular weight of 2.38MDa. Dilute the fermentation broth ten times with deionized water

[0053] 2) Add CPC in an amount of HA: CPC = 1: 1.5 (g / g), stir and complex, and centrifuge at 3000 rpm for 20 minutes to obtain the complex.

[0054] 3) Add 0.5M sodium chloride to the volume of the original fermentation broth, stir until dissociated

[0055] 4) Add ethanol in the amount of HA solution: ethanol = 1:2 (v / v), and get fibrous HA precipitate after stirring;

[0056] 5) Add 0.1M sodium chloride to twice the volume of the fermentation broth, and stir until the precipitate is fully dissolved.

[0057] 6) Add No. 2 perlite of 3g / L, No. 4 perlite of 3g / L and gac of 2g / L in the dissolving solution, after stirring for two hours, filter with cardboard, obtain filtrate.

[0058] 7) Add ethanol to the filtrate in the amount of HA solution: ethanol = 1:2 (v / v), and get HA precipitate after stirring

[0059] 8) Di...

Embodiment 2

[0062] 1) Take 500ml of fermented liquid, containing 2.4g of HA with a molecular weight of 2.14MDa. The fermentation broth needs to be diluted ten times with deionized water

[0063] 2) Add CPC in an amount of HA: CPC = 1: 1.5 (g / g), stir and complex, and centrifuge at 3000 rpm for 20 minutes to obtain the complex.

[0064] 3) Add 0.5M sodium chloride to twice the volume of the fermentation broth, and stir until the precipitate dissolves;

[0065] 4) Add No. 2 perlite of 3g / L, No. 4 perlite of 3g / L and the gac of 2g / L in the dissolving solution, after stirring for two hours, filter with cardboard, obtain first filtrate.

[0066] 5) Add aluminum oxide to the primary filtrate, the addition amount is: HA: aluminum oxide = 1:5 (g / g), add 1-2 g / L activated carbon, stir for 2 hours, and filter with a cardboard to obtain the filtrate;

[0067] 6) Add ethanol to the filtrate in an amount of HA solution: ethanol = 1:2 (v / v), and get HA precipitate after stirring

[0068] 7) The prec...

Embodiment 3

[0071] 1) Take 500ml of fermented liquid, containing 2.4g of HA with a molecular weight of 2.14MDa. fermented broth undiluted

[0072] 2) Add ethanol to the fermentation broth, the amount added is HA solution: ethanol = 1:2 (v / v), and after stirring, a fibrous HA precipitate is obtained;

[0073] 3) Add 0.1M sodium chloride to twice the volume of the fermentation broth, and stir until the precipitate dissolves;

[0074] 4) Add No. 2 perlite of 3g / L, No. 4 perlite of 3g / L and the gac of 2g / L in the dissolving solution, after stirring for two hours, filter with cardboard, obtain first filtrate.

[0075] 5) Add aluminum oxide to the primary filtrate, the addition amount is: HA: aluminum oxide = 1:5 (g / g), add 1-2 g / L activated carbon, stir for 2 hours, and filter with a cardboard to obtain the filtrate;

[0076] 6) Add ethanol to the filtrate in an amount of HA solution: ethanol = 1:2 (v / v), and get HA precipitate after stirring

[0077] 7) Thereafter, the precipitate can be d...

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Abstract

The process of separating and extracting hyaluronic acid from microbial fermented liquid includes the following steps: 1. complexing fermented liquid containing hyaluronic acid with chloro hexadecyl pyridine or precipitating the fermented liquid with ethanol; 2.collecting the complex or alcohol precipitate, and dissociating or dissolving the complex or alcohol precipitate in water solution of sodium chloride; and 3. collecting the hyaluronic acid precipitate, dissolving in water, and drying to obtain target product. The present invention has simple process, hyaluronic acid yield of about 90 %,molecular weight loss of about 15 % and low cost, and is suitable for industrial production.

Description

technical field [0001] The invention relates to a method for preparing hyaluronic acid, in particular to a method for separating and extracting hyaluronic acid from microbial fermentation broth. Background technique [0002] Hyaluronic acid (Hyaluronic Acid, HA) is a linear polymer mucopolysaccharide formed by repeating units of glucuronic acid and acetylglucosamine through β, 1-3 and β, 1-4 glycosidic bonds, and the number of disaccharide units 300-1100 pairs, molecular weight range 10 5 -10 7 Da. [0003] In 1934, Meyer and Palmer isolated a macromolecular polysaccharide from the bovine vitreous, which they named hyaluronic acid. HA is soluble in water, but insoluble in organic solvents such as alcohol, ketone, and ether. Because its solution has super strong water holding capacity, viscoelasticity and bioabsorbability, it is widely used in the fields of eye surgery, adhesion prevention in abdominal surgery, arthritis and cosmetics. [0004] The production of hyaluron...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C08B37/08C12P19/04
Inventor 谭文松胡怡虹张旭蔡海波
Owner EAST CHINA UNIV OF SCI & TECH
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