CN104450831A - Method for extracting chondroitin sulfate from fish cartilage - Google Patents
Method for extracting chondroitin sulfate from fish cartilage Download PDFInfo
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- CN104450831A CN104450831A CN201310427809.8A CN201310427809A CN104450831A CN 104450831 A CN104450831 A CN 104450831A CN 201310427809 A CN201310427809 A CN 201310427809A CN 104450831 A CN104450831 A CN 104450831A
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Abstract
The present invention discloses a method for extracting chondroitin sulfate from fish cartilage. The method is characterized by comprising: (1) primary microorganism screening, (2) secondary microorganism screening, (3) fermentation culture, (4) chondroitin sulfate enzyme extraction, (5) cartilage pre-treatment, (6) enzymolysis ultra-filtration, and (7) chondroitin sulfate preparation. According to the present invention, the fish cartilage is adopted to extract the shark chondroitin sulfate, the chondroitin sulfate enzyme produced through fermentation has the high activity, the small molecule chondroitin sulfate obtained through enzymolysis of the chondroitin sulfate enzyme has characteristics of small viscosity, good solubility, easy absorption, high bioavailability and the like, the hydrolysate adopts the ultra-filtration membrane concentration process so as to improve the product yield and the purity, and the extraction method of the present invention has characteristics of convenient operation, short period, easy operation, and large-scale industrial production achieving.
Description
Technical field
The invention belongs to bioengineering field, be specifically related to a kind of method extracting chondroitin sulfate in fish cartilage.
Background technology
Chondroitin sulfate is the acidic mucopolysaccharide extracting from cartilages such as Mammals tracheaes and obtain.White powder, odorless, tasteless, easy moisture absorption, soluble in water, be insoluble to ethanol and acetone and other organic solvent, namely chance water expand or become and stick slurry, more unstable to heat.Need lucifuge to seal to preserve.The mucopolysaccharide that chrondroitin is made up of D-Glucose aldehydic acid and N-acetyl-D-galactosamine.Chondroitin sulfate is the sulfuric ester of chrondroitin, forms the main component of reticular tissue.It has clarification lipid, improves body function of detoxification, the effect such as diuresis and analgesia.Very effective to Collagen illness, also effective to the dysacousis caused by Streptomycin sulphate.Acidic mucopolysaccharide is one of endemic element in connective tissue matrix in organism, and chondroitin sulfate A (CSA) is the one of acidic mucopolysaccharide.Medically prevent and treat for coronary heart disease.
Chondroitin sulfate A (CSA) (CSA) chondroitin sulfate C (CSC) is mainly divided into according to sulfate position difference on semi-lactosi.Recent study finds, the relative molecular weight of chondroitin sulfate (CS) is larger, its large volume often hinders it to pass through cytolemma smoothly, thus can not well play its multiple pharmacological function in body, and low-molecular-weight chondroitin sulfate (LMWCHS) has that viscosity is little, solvability good, easy absorption and bioavailability high.
In recent years, small molecules chondroitin sulfate prevent and treat coronary heart disease, sacroiliitis and antitumor etc. in just day by day in widespread attention, the preparation of small molecules chondroitin sulfate obtains mainly through chondrosulphatase enzymolysis.Chondrosulphatase is that a class is mainly present in microbe, can by multiple acidic mucopolysaccharide as dermatan sulfate, chondroitin sulfate, hyaluronic acid etc. are degraded to the lyase of oligosaccharides and unsaturated disaccharide.
At present because chondrosulphatase yields poorly, expensive, seriously constrain the development of small molecules chondroitin sulfate industry, how to invent a kind of method that efficient, easy to operate, with short production cycle, applicable Large scale processesization is produced, thus produce micromolecular chondroitin sulfate, be extremely important.
Summary of the invention
The present invention is in order to overcome the above-mentioned shortcoming existed in prior art, and provide a kind of method extracting chondroitin sulfate in fish cartilage, it is characterized in that, described method comprises the steps:
(1) microorganism primary dcreening operation: with sterilized water, soil sample suspension is made the soil solution of 10%, obtain clarified liq after two layers of filter paper suction filtration, liquid gradient is diluted to 10
-5get diluent 0.3ml and be coated with isolation medium slat chain conveyor, by single bacterium colony plate streaking separation method purifying 2 times on isolation medium flat board, single bacterium colony dibbling after picking purifying is in primary dcreening operation culture medium flat plate, after cultivating 6-7 days in 32 DEG C of incubators, in flat board, pouring the Glacial acetic acid of 6ml 3mol/L into, soak 3min, utilizing method of scoring to be stored on test tube solid slant culture base and stand-by in 4 DEG C of preservations by producing the bacterial strain of transparent circle;
(2) microorganism is sieved again: be inoculated in multiple sieve substratum by the thalline producing transparent circle, 32 DEG C, 135r/min oscillatory ventilation cultivates 2 days, by bacterium liquid gradient dilution to 10
-9, get 0.3ml 10
-7, 10
-8, 10
-9bacterium liquid, coat in primary dcreening operation substratum, cultivate 6-7 days in 32 DEG C of incubators after, the transparent circle bacterial strain larger relative to bacterium colony is selected by measuring, be inoculated in multiple sieve substratum, 32 DEG C, 135r/min oscillatory ventilation cultivation 24h, obtaining cell density through plate count is 2 × 10
7-3 × 10
7individual/ml as kind of a daughter bacteria liquid, the morphological specificity of visual inspection bacterium colony;
(3) fermentation culture: fermented in kind of daughter bacteria liquid access 5L fermentor tank by fermention medium by 2-2.5% v/v inoculum size, 28-32 DEG C, fermentation 55-65h;
(4) chondrosulphatase is extracted: gained fermented liquid is placed in whizzer, 4 DEG C, the centrifugal 35min of 4200r/min, discard bacterial sediment, ice bath, under the stirring of magnetic stirring apparatus, in fermented supernatant fluid, slowly add the saturation ratio of ammonium sulfate to 90%, leave standstill 2 days in 4 DEG C of chromatography cabinets, then 4 DEG C, 4200r/min recentrifuge fermented liquid, collect albumen precipitation, utilize tri-distilled water to dissolve and be placed in dialysis tubing, dialysis desalting in 4 DEG C of chromatography cabinets, at (NH
4)
2sO
4solution detects the outer liquid of dialysis tubing not containing SO
4 2-when, lyophilize;
(5) cartilage pre-treatment: pulverized after selecting the degreasing of fish cartilage, the hydrogen NaOH solution and the sodium chloride solution that add 3-5% extract 5-6h under temperature is the condition of 50-60 DEG C, adjust filtrate pH=8-9 after filtering;
(6) enzymolysis ultrafiltration: the chondrosulphatase powder that the step (4) adding cartilage charged material weight 3-5% to above-mentioned filtrate makes, be 6-7 in pH value, temperature is enzymolysis 5-6h under the condition of 40-50 DEG C, is at room temperature concentrated into 2/3 of original volume with the ultra-filtration membrane ultrafiltration that molecular weight cut-off is 8000;
(7) Sulphuric acid chrondroitin: adjust pH value to be 8-9 the concentrated solution after ultrafiltration, be incorporated as the hydrogen peroxide of cartilage charged material weight 2-3%, be be oxidized 5-6h under the condition of 25-35 DEG C to filter in temperature, filtrate adjusts pH value to be 5-6, add 95% alcohol crystal, make alcohol concn reach 65-75%, centrifugal after crystallization, washing, obtain fish chondroitin sulfate after drying.
A kind of above-mentioned method extracting chondroitin sulfate in fish cartilage, is characterized in that, the substratum used in described step (1) comprises chondroitin sulfate 0.5%, (NH
4)
2sO
40.5%, K
3pO
40.05%, NaCl 0.6%, pH=7.0.
A kind of above-mentioned method extracting chondroitin sulfate in fish cartilage, is characterized in that, the substratum used in described step (2) comprises chondroitin sulfate 0.5%, CaSO47H
20 0.6%, KH
2pO
40.05%, NaCl 0.6%, Glacial acetic acid 1%, yeast leaching powder 0.8%, pH=7.0.
A kind of above-mentioned method extracting chondroitin sulfate in fish cartilage, is characterized in that, the fermention medium selected in described step (3) comprises chondroitin sulfate 0.7%, CaSO47H
20 0.7%, yeast leaching powder 0.7%, KH
2pO
40.05%, initial pH=6.0.
Beneficial effect of the present invention:
The present invention adopts fish cartilage to extract shark chondroitine, chondrosulphatase enzymolysis is adopted to replace traditional resolvase enzymolysis process, the chondroitin sulfate enzyme activity that fermentation produces is high, by the small molecules chondroitin sulfate obtained with chondrosulphatase enzymolysis, have that viscosity is little, solvability good, easy absorption and a bioavailability high.Enzymolysis solution uses ultra-filtration membrane concentration technology, greatly reduces the consumption of alcohol and the energy consumption of alcohol recycle, and improves yield and the purity of product.Extracting method of the present invention is easy to operate, the cycle is short, and easy handling can realize large-scale industrial production.
Embodiment
Below in conjunction with specific embodiment, the invention will be further described, to help understanding content of the present invention.
embodiment 1
(1) microorganism primary dcreening operation: with sterilized water, soil sample suspension is made the soil solution of 10%, obtain clarified liq after two layers of filter paper suction filtration, liquid gradient is diluted to 10
-5, get diluent 0.3ml and be coated with isolation medium slat chain conveyor, by single bacterium colony plate streaking separation method purifying 2 times on isolation medium flat board, the single bacterium colony dibbling after picking purifying is in by chondroitin sulfate 0.5%, (NH
4)
2sO
40.5%, K
3pO
40.05%, the primary dcreening operation culture medium flat plate of NaCl 0.6% composition, control pH=7.0, cultivate 6-7 days in 32 DEG C of incubators after, the Glacial acetic acid of 6ml 3mol/L is poured in flat board, soaking 3min, utilizing method of scoring to be stored on test tube solid slant culture base and stand-by in 4 DEG C of preservations by producing the bacterial strain of transparent circle;
(2) microorganism is sieved again: be inoculated in multiple sieve substratum by the thalline producing transparent circle, 32 DEG C, 135r/min oscillatory ventilation cultivates 2 days, by bacterium liquid gradient dilution to 10
-9, get 0.3ml 10
-7bacterium liquid, coat in primary dcreening operation substratum, cultivate after 6 days in 32 DEG C of incubators, selecting the transparent circle bacterial strain larger relative to bacterium colony by measuring, being inoculated in by chondroitin sulfate 0.5%, CaSO47H
20 0.6%, KH
2pO
40.05%, in the multiple sieve substratum that NaCl 0.6%, Glacial acetic acid 1%, yeast leaching powder 0.8% form, control pH=7.0, at 32 DEG C, 135r/min oscillatory ventilation cultivation 24h, obtaining cell density through plate count is 2 × 10
7-3 × 10
7individual/ml as kind of a daughter bacteria liquid, the morphological specificity of visual inspection bacterium colony;
(3) fermentation culture: will plant in daughter bacteria liquid access 5L fermentor tank by 2% v/v inoculum size and pass through by chondroitin sulfate 0.7%, CaSO47H
20 0.7%, yeast leaching powder 0.7%, KH
2pO
4the fermention medium fermentation of 0.05% composition, controls initial pH=6.0, at 28 DEG C, and fermentation 65h;
(4) chondrosulphatase is extracted: gained fermented liquid is placed in whizzer, 4 DEG C, the centrifugal 35min of 4200r/min, discard bacterial sediment, ice bath, under the stirring of magnetic stirring apparatus, in fermented supernatant fluid, slowly add the saturation ratio of ammonium sulfate to 90%, leave standstill 2 days in 4 DEG C of chromatography cabinets, then 4 DEG C, 4200r/min recentrifuge fermented liquid, collect albumen precipitation, utilize tri-distilled water to dissolve and be placed in dialysis tubing, dialysis desalting in 4 DEG C of chromatography cabinets, at (NH
4)
2sO
4solution detects the outer liquid of dialysis tubing not containing SO
4 2-when, lyophilize;
(5) cartilage pre-treatment: pulverized after selecting the degreasing of fish cartilage, the hydrogen NaOH solution and the sodium chloride solution that add 3-5% extract 6h under temperature is the condition of 50 DEG C, adjust filtrate pH=8-9 after filtering;
(6) enzymolysis ultrafiltration: the chondrosulphatase powder that the step (4) adding cartilage charged material weight 3% to above-mentioned filtrate makes, be 6-7 in pH value, temperature is enzymolysis 6h under the condition of 40 DEG C, is at room temperature concentrated into 2/3 of original volume with the ultra-filtration membrane ultrafiltration that molecular weight cut-off is 8000;
(7) Sulphuric acid chrondroitin: adjust pH value to be 8-9 the concentrated solution after ultrafiltration, be incorporated as the hydrogen peroxide of cartilage charged material weight 2%, be be oxidized 5h under the condition of 25 DEG C to filter in temperature, filtrate adjusts pH value to be 5-6, add 95% alcohol crystal, make alcohol concn reach 65%, centrifugal after crystallization, washing, obtain fish chondroitin sulfate after drying.
embodiment 2
(1) microorganism primary dcreening operation: with sterilized water, soil sample suspension is made the soil solution of 10%, obtain clarified liq after two layers of filter paper suction filtration, liquid gradient is diluted to 10
-5, get diluent 0.3ml and be coated with isolation medium slat chain conveyor, by single bacterium colony plate streaking separation method purifying 2 times on isolation medium flat board, the single bacterium colony dibbling after picking purifying is in by chondroitin sulfate 0.5%, (NH
4)
2sO
40.5%, K
3pO
40.05%, the primary dcreening operation culture medium flat plate of NaCl 0.6% composition, control pH=7.0, cultivate 6-7 days in 32 DEG C of incubators after, the Glacial acetic acid of 6ml 3mol/L is poured in flat board, soaking 3min, utilizing method of scoring to be stored on test tube solid slant culture base and stand-by in 4 DEG C of preservations by producing the bacterial strain of transparent circle;
(2) microorganism is sieved again: be inoculated in multiple sieve substratum by the thalline producing transparent circle, 32 DEG C, 135r/min oscillatory ventilation cultivates 2 days, by bacterium liquid gradient dilution to 10
-9, get 0.3ml 10
-8bacterium liquid, coat in primary dcreening operation substratum, cultivate 6-7 days in 32 DEG C of incubators after, by measure select the transparent circle bacterial strain larger relative to bacterium colony, be inoculated in by chondroitin sulfate 0.5%, CaSO47H
20 0.6%, KH
2pO
40.05%, in the multiple sieve substratum that NaCl 0.6%, Glacial acetic acid 1%, yeast leaching powder 0.8% form, control pH=7.0, at 32 DEG C, 135r/min oscillatory ventilation cultivation 24h, obtaining cell density through plate count is 2 × 10
7-3 × 10
7individual/ml as kind of a daughter bacteria liquid, the morphological specificity of visual inspection bacterium colony;
(3) fermentation culture: will plant in daughter bacteria liquid access 5L fermentor tank by 2.5% v/v inoculum size and pass through by chondroitin sulfate 0.7%, CaSO47H
20 0.7%, yeast leaching powder 0.7%, KH
2pO
4the fermention medium fermentation of 0.05% composition, controls initial pH=6.0, at 32 DEG C, and fermentation 55h;
(4) chondrosulphatase is extracted: gained fermented liquid is placed in whizzer, 4 DEG C, the centrifugal 35min of 4200r/min, discard bacterial sediment, ice bath, under the stirring of magnetic stirring apparatus, in fermented supernatant fluid, slowly add the saturation ratio of ammonium sulfate to 90%, leave standstill 2 days in 4 DEG C of chromatography cabinets, then 4 DEG C, 4200r/min recentrifuge fermented liquid, collect albumen precipitation, utilize tri-distilled water to dissolve and be placed in dialysis tubing, dialysis desalting in 4 DEG C of chromatography cabinets, at (NH
4)
2sO
4solution detects the outer liquid of dialysis tubing not containing SO
4 2-when, lyophilize;
(5) cartilage pre-treatment: pulverized after selecting the degreasing of fish cartilage, the hydrogen NaOH solution and the sodium chloride solution that add 5% extract 5h under temperature is the condition of 60 DEG C, adjust filtrate pH=8-9 after filtering;
(6) enzymolysis ultrafiltration: the chondrosulphatase powder that the step (4) adding cartilage charged material weight 3-5% to above-mentioned filtrate makes, be 6-7 in pH value, temperature is enzymolysis 5h under the condition of 50 DEG C, is at room temperature concentrated into 2/3 of original volume with the ultra-filtration membrane ultrafiltration that molecular weight cut-off is 8000;
(7) Sulphuric acid chrondroitin: adjust pH value to be 8-9 the concentrated solution after ultrafiltration, be incorporated as the hydrogen peroxide of cartilage charged material weight 3%, be be oxidized 5h under the condition of 35 DEG C to filter in temperature, filtrate adjusts pH value to be 5-6, add 95% alcohol crystal, make alcohol concn reach 75%, centrifugal after crystallization, washing, obtain fish chondroitin sulfate after drying.
embodiment 3
(1) microorganism primary dcreening operation: with sterilized water, soil sample suspension is made the soil solution of 10%, obtain clarified liq after two layers of filter paper suction filtration, liquid gradient is diluted to 10
-5, get diluent 0.3ml and be coated with isolation medium slat chain conveyor, by single bacterium colony plate streaking separation method purifying 2 times on isolation medium flat board, the single bacterium colony dibbling after picking purifying is in by chondroitin sulfate 0.5%, (NH
4)
2sO
40.5%, K
3pO
40.05%, the primary dcreening operation culture medium flat plate of NaCl 0.6% composition, control pH=7.0, cultivate 6-7 days in 32 DEG C of incubators after, the Glacial acetic acid of 6ml 3mol/L is poured in flat board, soaking 3min, utilizing method of scoring to be stored on test tube solid slant culture base and stand-by in 4 DEG C of preservations by producing the bacterial strain of transparent circle;
(2) microorganism is sieved again: be inoculated in multiple sieve substratum by the thalline producing transparent circle, 32 DEG C, 135r/min oscillatory ventilation cultivates 2 days, by bacterium liquid gradient dilution to 10
-9, get 0.3ml 10
-9bacterium liquid, coat in primary dcreening operation substratum, cultivate 6-7 days in 32 DEG C of incubators after, by measure select the transparent circle bacterial strain larger relative to bacterium colony, be inoculated in by chondroitin sulfate 0.5%, CaSO47H
20 0.6%, KH
2pO
40.05%, in the multiple sieve substratum that NaCl 0.6%, Glacial acetic acid 1%, yeast leaching powder 0.8% form, control pH=7.0, at 32 DEG C, 135r/min oscillatory ventilation cultivation 24h, obtaining cell density through plate count is 2 × 10
7-3 × 10
7individual/ml as kind of a daughter bacteria liquid, the morphological specificity of visual inspection bacterium colony;
(3) fermentation culture: will plant in daughter bacteria liquid access 5L fermentor tank by 2.5% v/v inoculum size and pass through by chondroitin sulfate 0.7%, CaSO47H
20 0.7%, yeast leaching powder 0.7%, KH
2pO
4the fermention medium fermentation of 0.05% composition, controls initial pH=6.0, at 32 DEG C, and fermentation 55h;
(4) chondrosulphatase is extracted: gained fermented liquid is placed in whizzer, 4 DEG C, the centrifugal 35min of 4200r/min, discard bacterial sediment, ice bath, under the stirring of magnetic stirring apparatus, in fermented supernatant fluid, slowly add the saturation ratio of ammonium sulfate to 90%, leave standstill 2 days in 4 DEG C of chromatography cabinets, then 4 DEG C, 4200r/min recentrifuge fermented liquid, collect albumen precipitation, utilize tri-distilled water to dissolve and be placed in dialysis tubing, dialysis desalting in 4 DEG C of chromatography cabinets, at (NH
4)
2sO
4solution detects the outer liquid of dialysis tubing not containing SO
4 2-when, lyophilize;
(5) cartilage pre-treatment: pulverized after selecting the degreasing of fish cartilage, the hydrogen NaOH solution and the sodium chloride solution that add 4% extract 5.5h under temperature is the condition of 55 DEG C, adjust filtrate pH=8-9 after filtering;
(6) enzymolysis ultrafiltration: the chondrosulphatase powder that the step (4) adding cartilage charged material weight 4% to above-mentioned filtrate makes, be 6-7 in pH value, temperature is enzymolysis 5.5h under the condition of 45 DEG C, is at room temperature concentrated into 2/3 of original volume with the ultra-filtration membrane ultrafiltration that molecular weight cut-off is 8000;
(7) Sulphuric acid chrondroitin: adjust pH value to be 8-9 the concentrated solution after ultrafiltration, be incorporated as the hydrogen peroxide of cartilage charged material weight 3%, be be oxidized 6h under the condition of 30 DEG C to filter in temperature, filtrate adjusts pH value to be 5-6, add 95% alcohol crystal, make alcohol concn reach 70%, centrifugal after crystallization, washing, obtain fish chondroitin sulfate after drying.
The above, be only the specific embodiment of the present invention, is not limited thereto, and is anyly familiar with those skilled in the art in the technical scope that this patent discloses, and can expect change easily or replace, all should be encompassed within protection scope of the present invention.
Claims (4)
1. extract a method for chondroitin sulfate in fish cartilage, it is characterized in that, described method comprises the steps:
(1) microorganism primary dcreening operation: with sterilized water, soil sample suspension is made the soil solution of 10%, obtain clarified liq after two layers of filter paper suction filtration, liquid gradient is diluted to 10
-5get diluent 0.3ml and be coated with isolation medium slat chain conveyor, by single bacterium colony plate streaking separation method purifying 2 times on isolation medium flat board, single bacterium colony dibbling after picking purifying is in primary dcreening operation culture medium flat plate, after cultivating 6-7 days in 32 DEG C of incubators, in flat board, pouring the Glacial acetic acid of 6ml 3mol/L into, soak 3min, utilizing method of scoring to be stored on test tube solid slant culture base and stand-by in 4 DEG C of preservations by producing the bacterial strain of transparent circle;
(2) microorganism is sieved again: be inoculated in multiple sieve substratum by the thalline producing transparent circle, 32 DEG C, 135r/min oscillatory ventilation cultivates 2 days, by bacterium liquid gradient dilution to 10
-9, get 0.3ml 10
-7, 10
-8, 10
-9bacterium liquid, coat in primary dcreening operation substratum, cultivate 6-7 days in 32 DEG C of incubators after, the transparent circle bacterial strain larger relative to bacterium colony is selected by measuring, be inoculated in multiple sieve substratum, 32 DEG C, 135r/min oscillatory ventilation cultivation 24h, obtaining cell density through plate count is 2 × 10
7-3 × 10
7individual/ml as kind of a daughter bacteria liquid, the morphological specificity of visual inspection bacterium colony;
(3) fermentation culture: fermented in kind of daughter bacteria liquid access 5L fermentor tank by fermention medium by 2-2.5% v/v inoculum size, 28-32 DEG C, fermentation 55-65h;
(4) chondrosulphatase is extracted: gained fermented liquid is placed in whizzer, 4 DEG C, the centrifugal 35min of 4200r/min, discard bacterial sediment, ice bath, under the stirring of magnetic stirring apparatus, in fermented supernatant fluid, slowly add the saturation ratio of ammonium sulfate to 90%, leave standstill 2 days in 4 DEG C of chromatography cabinets, then 4 DEG C, 4200r/min recentrifuge fermented liquid, collect albumen precipitation, utilize tri-distilled water to dissolve and be placed in dialysis tubing, dialysis desalting in 4 DEG C of chromatography cabinets, at (NH
4)
2sO
4solution detects the outer liquid of dialysis tubing not containing SO
4 2-when, lyophilize;
(5) cartilage pre-treatment: pulverized after selecting the degreasing of fish cartilage, the hydrogen NaOH solution and the sodium chloride solution that add 3-5% extract 5-6h under temperature is the condition of 50-60 DEG C, adjust filtrate pH=8-9 after filtering;
(6) enzymolysis ultrafiltration: the chondrosulphatase powder that the step (4) adding cartilage charged material weight 3-5% to above-mentioned filtrate makes, be 6-7 in pH value, temperature is enzymolysis 5-6h under the condition of 40-50 DEG C, is at room temperature concentrated into 2/3 of original volume with the ultra-filtration membrane ultrafiltration that molecular weight cut-off is 8000;
(7) Sulphuric acid chrondroitin: adjust pH value to be 8-9 the concentrated solution after ultrafiltration, be incorporated as the hydrogen peroxide of cartilage charged material weight 2-3%, be be oxidized 5-6h under the condition of 25-35 DEG C to filter in temperature, filtrate adjusts pH value to be 5-6, add 95% alcohol crystal, make alcohol concn reach 65-75%, centrifugal after crystallization, washing, obtain fish chondroitin sulfate after drying.
2. a kind of method extracting chondroitin sulfate in fish cartilage according to claim 1, is characterized in that, the substratum used in described step (1) comprises chondroitin sulfate 0.5%, (NH
4)
2sO
40.5%, K
3pO
40.05%, NaCl 0.6%, pH=7.0.
3. a kind of method extracting chondroitin sulfate in fish cartilage according to claim 1, is characterized in that, the substratum used in described step (2) comprises chondroitin sulfate 0.5%, CaSO47H
20 0.6%, KH
2pO
40.05%, NaCl 0.6%, Glacial acetic acid 1%, yeast leaching powder 0.8%, pH=7.0.
4. a kind of method extracting chondroitin sulfate in fish cartilage according to claim 1, is characterized in that, the fermention medium selected in described step (3) comprises chondroitin sulfate 0.7%, CaSO47H
20 0.7%, yeast leaching powder 0.7%, KH
2pO
40.05%, initial pH=6.0.
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|---|---|---|---|
| CN201310427809.8A CN104450831A (en) | 2013-09-21 | 2013-09-21 | Method for extracting chondroitin sulfate from fish cartilage |
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|---|---|---|---|
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111334543A (en) * | 2020-04-28 | 2020-06-26 | 山东冰文生物技术有限公司 | Novel method for extracting chondroitin sulfate in fish cartilage |
| CN112481338A (en) * | 2020-12-22 | 2021-03-12 | 荣成万盈水产科技有限公司 | Preparation method of squid chondroitin sulfate glycoprotein |
| CN112625147A (en) * | 2020-12-22 | 2021-04-09 | 荣成万盈水产科技有限公司 | Method for preparing chondroitin sulfate from squid cartilage |
-
2013
- 2013-09-21 CN CN201310427809.8A patent/CN104450831A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111334543A (en) * | 2020-04-28 | 2020-06-26 | 山东冰文生物技术有限公司 | Novel method for extracting chondroitin sulfate in fish cartilage |
| CN112481338A (en) * | 2020-12-22 | 2021-03-12 | 荣成万盈水产科技有限公司 | Preparation method of squid chondroitin sulfate glycoprotein |
| CN112625147A (en) * | 2020-12-22 | 2021-04-09 | 荣成万盈水产科技有限公司 | Method for preparing chondroitin sulfate from squid cartilage |
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Application publication date: 20150325 |