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Method for Separating and Extracting Hyaluronic Acid from Chinese Rana Skin by Membrane Filtration

A hyaluronic acid and membrane filtration technology, which is applied in the field of separation and extraction of hyaluronic acid, can solve the problems of reagent residue, inability to effectively remove protein, and reduced safety, and achieve high yields

Active Publication Date: 2016-09-14
吉林省东北亚生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Nylon filter cloth with a pore size of 0.088-0.074 mm cannot effectively remove protein and retain hyaluronic acid; organic solvents in the solvent will also cause reagent residue problems and reduce safety
Nowadays, people have gradually realized that the preparation of HA by modern biological separation means and methods such as membrane filtration can effectively solve the above problems, but no specific methods and steps have been proposed so far.

Method used

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  • Method for Separating and Extracting Hyaluronic Acid from Chinese Rana Skin by Membrane Filtration

Examples

Experimental program
Comparison scheme
Effect test

Embodiment l

[0030] Embodiment 1, select the Chinese wood frog skin after getting the oil, the Chinese wood frog skin is lower in fat content and easily minced, therefore, directly add the Chinese wood frog skin 8 times of water and soak for 20 hours. Homogenize at 10,000 r / min for 2 minutes. After homogenization, it becomes a paste. Stir and heat to 85°C for 5 minutes. Cool to 45°C. Wash the residue twice.

[0031] HCl adjusted to pH 1.5, 500nm pore size ceramic membrane filtration, 9 times the amount of water was added for 3 washes, retentate, alkaline conditions (pH 7.5), pepsin (amount: 45mg / g dry tissue) enzymatic hydrolysis at 50 ° C for 12 After 1 hour, the enzymatic hydrolyzed solution was centrifuged to get the clear liquid, HCl was adjusted to pH 1.5, 500nm pore size ceramic membrane was filtered, 30 times the amount of water was added for 10 washings, NaCl was added to make the concentration of NaCl in the solution 0.1mol / L, and HCl was adjusted to pH 6.5 , add 3 times the volu...

Embodiment 2

[0033] Example 2. Select the Rana chinensis skin after oil extraction. The Rana chinensis skin has low fat content and is easy to be minced. Therefore, directly add 10 times the amount of water for 24 hours. Homogenize at 12,000 rpm for 3 minutes. After homogenization, it becomes a paste. Stir and heat to 90 °C, keep warm for 10 min, cool to 50 °C, autolyse for 12 hours, centrifuge at 4,000 rpm, 4 times the volume of water, and divide Wash the residue twice.

[0034]HCl adjusted to pH 2.0, 500nm pore size ceramic membrane filtration, 9 times the amount of water was added for 3 washings, retentate, alkaline conditions (pH 8.5), pepsin (amount: 40 mg / g dry tissue) enzymatic hydrolysis at 50 ° C for 12 After 1 hour, the enzymatic hydrolyzed solution was centrifuged to get the clear liquid, HCl was adjusted to pH 2.0, 500nm pore size ceramic membrane was filtered, 30 times of water was added for 10 times to wash, and NaCl was added to make the concentration of NaCl in the solution...

Embodiment 3

[0036] Example 3. Select the Rana chinensis skin after oil extraction. The Rana chinensis rind has a low fat content and is easy to be minced. Therefore, directly add 12 times the amount of water to soak for 28 hours. Homogenize at 14,000 rpm for 4 minutes. After homogenization, it becomes a paste. Stir and heat to 95°C, keep warm for 15 minutes, cool to 55°C, autolyse for 12 hours, centrifuge at 4,000 rpm, and 4 times the volume of water. Wash the residue twice.

[0037] HCl adjusted to pH 2.5, 500nm pore size ceramic membrane filtration, 9 times the amount of water was added for 3 washings, retentate, alkaline conditions (pH 8.0), pepsin (amount: 50mg / g dry tissue) enzymatic hydrolysis at 50 ° C for 12 hours, the enzymatic hydrolyzate was centrifuged to get the clear liquid, HCl was adjusted to pH 2.5, filtered with a 500nm pore size ceramic membrane, 30 times the amount of water was added for 10 washings, and NaCl was added to make the concentration of NaCl in the solution ...

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Abstract

The invention discloses a method for separating and extracting hyaluronic acid (HA) from rana chensinensis skins by using a membrane filtration process, and belongs to the field of methods for separating and extracting HA. According to the method disclosed by the invention, modern biological separation measures and methods such as membrane filtration are adopted, a ceramic membrane of which the membrane pore is 500nm is selected, the molecular weight cut off of the ceramic membrane is 1,000,000 Daltons, and thus protein can be effectively removed and HA can be intercepted. According to the method for extracting HA from the rana chensinensis skins, disclosed by the invention, 2.8g of high-quality HA can be extracted from per kilogram of the rana chensinensis skins, and by virtue of inspection, the glucuronic acid content of a product is 36%, and the protein content is 0.58%, so that the product can reach a cosmetic grade. According to the method, protein is removed by using the membrane filtration process, HA is desalted, graded and concentrated, high yield can be achieved, very little damage to the biological activity of polysaccharides can be caused, and the problem of reagent residues existing in a conventional organic solvent method also cannot be caused, so that the method is an economic, simple, convenient and effective extraction process and is suitable for industrial production of HA.

Description

technical field [0001] The invention belongs to the field of methods for separating and extracting hyaluronic acid, in particular to a method for separating and extracting hyaluronic acid from Rana chinensis skin by using a membrane filtration method. Background technique [0002] Hyaluronic acid (HA) is an internationally recognized biological macromolecule moisturizing agent, which is composed of (1-3)-2-acetamido-2-deoxy-D-glucose (1-4)-D-D. - A linear mucopolysaccharide composed of repeating units of glucuronic acid disaccharide. After years of research, people have a clear understanding of the structure, physicochemical properties and physiological functions of HA, and it is clear that it has many advantages such as high viscoelasticity, plasticity, permeability and good biocompatibility. At present, HA is mainly used in ophthalmic microsurgery, arthritis treatment, advanced cosmetics, obstetrics and gynecology and other fields. Natural hyaluronic acid with a molecula...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C08B37/08
Inventor 张辉吴楠高晓晨刘冬孙佳明杜延佳刘侗
Owner 吉林省东北亚生物科技有限公司
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