Method for separating and extracting hyaluronic acid (HA) from rana chensinensis skins by using membrane filtration process

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: 2015-07-01
吉林省东北亚生物科技有限公司
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  • 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 (HA) from rana chensinensis skins by using membrane filtration process

Examples

Experimental program
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Effect test

Embodiment l

[0030] Embodiment 1, select the Chinese forest frog skin after getting the oil, the Chinese forest frog skin has a lower fat content, and is easily crushed, therefore, directly add 8 times the amount of water of the Chinese forest frog skin and soak for 20 hours. Homogenize at a speed of 10,000 rpm for 2 minutes, form a paste after homogenization, stir and heat to 85°C, keep warm for 5 minutes, cool to 45°C, autolyze for 12 hours, centrifuge at 4,000 rpm, add 4 times the amount of water, and divide Wash residue twice.

[0031] Adjust the pH to 1.5 with HCl, filter with a 500nm pore ceramic membrane, add 9 times the amount of water to wash with water 3 times, retentate, alkaline condition (pH7.5), pepsin (dosage: 45mg / g dry tissue) at 50°C for 12 hours, centrifuge the enzymolysis solution to get the clear liquid, adjust the pH to 1.5 with HCl, filter through a ceramic membrane with a pore size of 500nm, add 30 times the amount of water to wash with water 10 times, add NaCl to m...

Embodiment 2

[0033] Embodiment 2, select the Rana sinensis skin after oil extraction, the Rana sinensis skin has a low fat content and is easily crushed, therefore, directly add 10 times the amount of water of the Rana sinensis skin and soak for 24 hours. Homogenize at a speed of 12,000 rpm for 3 minutes, form a paste after homogenization, stir and heat to 90°C, keep warm for 10 minutes, cool to 50°C, autolyze for 12 hours, centrifuge at 4,000 rpm, add 4 times the amount of water, and separate Wash residue twice.

[0034]Adjust the pH to 2.0 with HCl, filter with a 500nm pore ceramic membrane, add 9 times the amount of water to wash with water 3 times, retentate, alkaline condition (pH8.5), pepsin (dosage: 40mg / g dry tissue) at 50°C for 12 hours, centrifuge the enzymatic solution to get the clear liquid, adjust the pH to 2.0 with HCl, filter with a 500nm pore size ceramic membrane, add 30 times the amount of water to wash with water 10 times, add NaCl to make the concentration of NaCl in t...

Embodiment 3

[0036] Embodiment 3, select the Rana sinensis skin after oil extraction, the Rana sinensis skin has a low fat content and is easy to grind, therefore, directly add 12 times the amount of Rana sinensis skin and soak in water for 28 hours. Homogenize at a speed of 14,000 rpm for 4 minutes, form a paste after homogenization, stir and heat to 95°C, keep warm for 15 minutes, cool to 55°C, autolyze for 12 hours, centrifuge at 4,000 rpm, add 4 times the amount of water, and separate Wash residue twice.

[0037] Adjust the pH to 2.5 with HCl, filter with a 500nm pore ceramic membrane, add 9 times the amount of water to wash with water 3 times, retentate, alkaline condition (pH8.0), pepsin (dosage: 50mg / g dry tissue) at 50°C for 12 hours, the enzymolysis solution was centrifuged to get the clear liquid, HCl was adjusted to PH2.5, 500nm pore size ceramic membrane was filtered, 30 times the amount of water was added to wash 10 times, NaCl was added to make the concentration of NaCl in th...

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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 Chinese forest frog skin by using a membrane filtration method. Background technique [0002] Hyaluronic acid (Hyaluronic acid referred to as HA) is an internationally recognized biological macromolecular moisturizer, which is composed of (1-3)-2-acetylamino-2-deoxy-D-glucose (1-4)-D-D - Linear mucopolysaccharide composed of repeating units of glucuronic acid disaccharides. After years of research, people have a clear understanding of the structure, physical and chemical 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. Nat...

Claims

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Application Information

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Patent Type & Authority Applications(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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