Method for extracting hyaluronic acid from rana chensinensis skin
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A technology of hyaluronic acid and frog skin, applied in the biological field, can solve the problems of complicated steps, low HA extraction rate and low HA extraction rate, and achieve the effects of improving extraction rate, high purity and good activity
Active Publication Date: 2018-01-26
ACADEMY OF MILITARY MEDICAL SCI +1
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This method is a traditional method for preparing HA. The disadvantages are: the steps are cumbersome, resulting in a low extraction rate of HA; at the same time, a variety of toxic substances such as chloroform and quaternary ammonium salts are used, which is not friendly to humans and the environment.
Zhang Hui et al. separated and extracted HA from wood frog skin by ceramic membrane filtration method. After hydrolysis with a single enzyme pepsin, a ceramic membrane with a certain pore size was selected to filter out impurities, and the extraction rate of HA was not high.
[0007] Therefore, the technology for extracting hyaluronic acid from Rana skin needs to be further improved at present
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Embodiment 1
[0098] (1) Obtain freshly stripped Rana skin, remove subcutaneous tissue and adhesions, wash, dry at room temperature, and pulverize to obtain Rana skin powder;
[0099] (2) Dissolution: Weigh 10 g of Rana skin powder and dissolve it in 100 ml of distilled water, stir with a magnetic stirrer for 6 hours, and put it in a refrigerator at 4°C overnight to fully dissolve it.
[0100] (3) Ultrasonic treatment: the dissolved Rana skin was made into a solution, and treated with ultrasonic waves, specifically at 20KHz-35KHz, 100W-300W, for 20min-30min.
[0101] (4) Ultra-high pressure treatment: put the solution after ultrasonic treatment in an ultra-high pressure container, start the high-pressure oil pump, keep it at 200-400Mpa for 2-5min at this pressure, then quickly open the valve controlling the high-pressure oil circuit, The pressure is released within 2s, and the sample after ultra-high pressure treatment is ready for use.
[0110] (1) Obtain freshly stripped Rana skin, remove subcutaneous tissue and adhesions, wash, dry at room temperature, and pulverize to obtain Rana skin powder;
[0111] (2) Dissolution: Weigh 10g of Rana skin powder and dissolve it in 150ml of distilled water, stir it with a magnetic stirrer for 8 hours, and put it in a refrigerator at 4°C overnight to fully dissolve it.
[0112] (3) Ultrasonic treatment: the dissolved Rana skin was made into a solution, and treated with ultrasonic waves, specifically at 20KHz-35KHz, 100W-300W, for 20min-30min.
[0113] (4) Ultra-high pressure treatment: put the solution after ultrasonic treatment in an ultra-high pressure container, start the high-pressure oil pump, keep it at 200-400Mpa for 2-5min at this pressure, then quickly open the valve controlling the high-pressure oil circuit, The pressure is released within 2s, and the sample after ultra-high pressure treatment is ready for use.
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Abstract
The invention provides a method for extracting hyaluronic acid from rana chensinensis skin, which comprises the following steps of: (1) obtaining rana chensinensis skin powder; (2) dissolving the ranachensinensis skin powder by using distilled water; (3) carrying out super-high pressure treatment on the rana chensinensis skin powder dissolving liquid; (4) carrying out enzymolysis treatment on thesuper-high pressure leaching liquid by using trypsin and alkaline protease; (5) adding ethanol to the enzymolysis liquid to generate precipitate, carrying out centrifugation, collecting the precipitate, and carrying out drying treatment; (6) dissolving the dried precipitate by using sodium chloride; (7) adding hydrogen peroxide to the precipitate dissolving liquid, and carrying out decoloration;(8) adjusting the pH value to 4.5-5.0, adding the mixed solution of chloroform and n-butanol, carrying out centrifugation to separate protein, and collecting the water phase; (9) dialyzing the water phase, and collecting the dialysate; and (10) carrying out alcohol precipitation and drying treatment on the dialysate to obtain the hyaluronic acid fine product. According to the present invention, the method has advantages of simple process and high hyaluronic acid extraction rate, and the extracted hyaluronic acid has high purity and good activity.
Description
technical field [0001] The invention relates to the field of biotechnology, in particular to a method for extracting hyaluronic acid from wood frog skin. Background technique [0002] Hyaluronic acid (HA for short) is a disaccharide unit formed by condensation of β-D-N-acetylglucose and β-D-glucuronic acid as structural units with β-1,4-glucosidic bonds. The sugar units are then repeatedly and alternately connected by β-1,3-glycosidic bonds to form HA chains. The HA chain length can reach up to 25,000 disaccharide units, and the relative molecular mass is 10 4 -10 7 between Dalton. [0003] HA has a wide range of applications in the fields of medicine, food and cosmetics, and has unique moisturizing properties. It can heal wounds, promote postoperative adhesion, and can be used as a raw material to synthesize new drugs with anti-inflammatory and analgesic effects. Due to the large number of carboxyl and hydroxyl groups contained in HA molecules, it can form hydrogen bon...
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