CN111334543A - Novel method for extracting chondroitin sulfate in fish cartilage - Google Patents
Novel method for extracting chondroitin sulfate in fish cartilage Download PDFInfo
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Abstract
The invention provides a novel method for extracting chondroitin sulfate in fish cartilage, belonging to the field of bioengineering. The invention treats the filtrate in advance, can control the temperature to be kept at the set temperature by using the thermostat, and can simultaneously perform the disinfection function on the packaged chondroitin.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to a novel method for extracting chondroitin sulfate in fish cartilage.
Background
Chondroitin sulfate is an acidic mucopolysaccharide extracted from cartilage of mammal trachea, etc. White powder, odorless, tasteless, easy to absorb moisture, easily soluble in water, insoluble in organic solvents such as ethanol and acetone, and unstable to heat. And (4) sealing and storing in dark. Chondroitin is a mucopolysaccharide composed of D-glucuronic acid and N-acetyl-D-galactosamine. Chondroitin sulfate is a sulfate of chondroitin, and constitutes a main component of connective tissue. It has effects in clarifying lipid, improving organism detoxicating function, promoting urination, and relieving pain. It is effective for treating collagen diseases, and auditory disorder caused by streptomycin. Acid mucopolysaccharide is one of the specific components in connective tissue matrix in vivo, and chondroitin sulfate A is one of acid mucopolysaccharide. Can be used for preventing and treating coronary heart disease.
However, the prior art has the problems of long working time, incapability of constant temperature control and low intelligent degree in the process of extracting chondroitin sulfate in fish cartilage.
Therefore, it is necessary to develop a new method for extracting chondroitin sulfate from fish cartilage.
Disclosure of Invention
In order to solve the technical problems, the invention provides a novel method for extracting chondroitin sulfate in fish cartilage, which solves the problems of long working time, incapability of constant temperature control and low intelligent degree in the process of extracting the chondroitin sulfate in the fish cartilage in the prior art, and specifically comprises the following steps:
the method comprises the following steps: pretreating materials, namely degreasing selected fish cartilage, crushing the degreased fish cartilage by using a crusher, adding a 6-9% hydrogen NaOH solution and a sodium chloride solution, extracting for 2-4 hours and 30 minutes at the temperature of 61-75 ℃, filtering, and adjusting the pH of the filtrate to be = 8-9;
screening and fermenting microorganisms, suspending a soil sample with sterile water to prepare 11-20% soil solution, performing suction filtration by using multilayer filter paper to clarify liquid, then diluting the liquid to 11-15 ℃ in a gradient manner, coating 0.5ml of diluent on a separation medium plate for plate culture, then purifying a single bacterial colony on the separation medium plate for 3 times by using a plate streaking separation method, selecting a purified single bacterial colony to be spotted on a primary screening medium plate, culturing in an incubator at 33-35 ℃ for three days to five days, pouring 8ml of 4mol/L glacial acetic acid into the plate, soaking for 2min, storing a bacterial strain generating a transparent ring on a test tube solid inclined plane medium by using a streaking method for later use at 5 ℃, inoculating the bacterial strain generating the transparent ring in a secondary screening medium, controlling the temperature to be 33 ℃, 145r/min, performing shake, aeration culture for 1 day, diluting the gradient to 11-15 ℃, taking 0.5ml of 11-15 bacterial strain liquid, coating the bacterial strain on the primary screening medium, coating the bacterial strain in the secondary screening medium, controlling the temperature to 35-35 ℃ for 33 ℃, performing shake, culturing for 1 day, inoculating the bacterial colony in a fermentation tank for 2 v, measuring the strain by using a fermentation tank, inoculating the strain for 3-32 hours, measuring the strain, inoculating the strain in a fermentation tank by using a fermentation tank at 35 ℃ for 3 v, and measuring the strain, wherein the strain, and measuring the strain, wherein the strain, and measuring the strain, wherein the strain;
step three: extracting chondroitinase sulfate;
firstly, primary extraction, namely placing the obtained fermentation liquor in a centrifuge, centrifuging for 30min to 35min at 5 ℃ and 4300r/min, removing thallus precipitates, carrying out ice bath, slowly adding ammonium sulfate into the fermentation supernatant to reach the saturation of 92 percent under the stirring of a magnetic stirrer, and standing for 24 hours in a chromatography cabinet at 5 ℃;
secondly, extracting again, placing the obtained fermentation liquor in a centrifuge, centrifuging for 30min to 35min at the temperature of 5 ℃ and at the speed of 4500r/min, removing thallus precipitates, carrying out ice bath, slowly adding ammonium sulfate into the fermentation supernatant to reach the saturation of 92 percent under the stirring of a magnetic stirrer, and standing for 24 hours in a chromatography cabinet at the temperature of 5 ℃;
thirdly, fine extraction, then centrifuging the fermentation liquor again at 5 ℃ and 5000r/min, collecting protein precipitate, dissolving by using triple distilled water, placing into a dialysis bag, dialyzing and desalting in a chromatography cabinet at 5 ℃, and freeze-drying under the condition that (NH4)2SO4 solution detects that the liquid outside the dialysis bag does not contain SO 42-;
step four: carrying out enzymolysis and ultrafiltration on the extracted chondroitin sulfate enzyme;
adding cartilage feed, adding the chondroitinase powder prepared in the third step into the filtrate obtained in the first step, and carrying out enzymolysis for 2 to 4 hours at the pH value of 8 to 10 and the temperature of 55 to 60 ℃;
secondly, stirring and dehydrating, namely stirring the raw materials subjected to enzymolysis for 5 to 10 minutes by using a stirrer, and then performing ultrafiltration, dehydration and concentration to 2/3 in the original volume by using an ultrafiltration membrane with the cut-off molecular weight of 8100 at room temperature;
thirdly, waste treatment, namely temporarily storing liquid materials discharged by ultrafiltration membrane dehydration in a storage tank;
step five: preparing chondroitin sulfate and sterilizing;
firstly, primarily manufacturing, adjusting the pH value of the concentrated solution after ultrafiltration dehydration to 8-9, adding hydrogen peroxide which is 4-6% of the weight of the cartilage feed, oxidizing for 2-4 hours at the temperature of 36-45 ℃, filtering, adjusting the pH value of the filtrate to 5-6, adding 96% ethanol for crystallization, and enabling the ethanol concentration to reach 76-80%;
step two, carrying out centrifugal washing treatment, and centrifuging and washing by using a centrifugal machine after crystallization;
and thirdly, drying, packaging and sterilizing, washing and drying to obtain fish chondroitin sulfate, packaging the chondroitin by using packaging equipment, and irradiating and sterilizing the outside of the package by using ultraviolet ray sterilization equipment.
Preferably, in step one, 7% to 9% of hydrogen NaOH solution and sodium chloride solution are added.
Preferably, in step one, 7% to 8% of hydrogen NaOH solution and sodium chloride solution are added.
Preferably, in step one, the extraction is carried out at a temperature of 65 ℃ to 75 ℃ for 2 hours to 4 hours.
Preferably, in the step one, the extraction is performed at a temperature of 65 ℃ to 70 ℃ for 3 hours to 4 hours.
Preferably, in the second step, the soil sample is suspended by sterile water to prepare a 12% to 20% soil solution.
Preferably, in the second step, the soil sample is suspended by sterile water to prepare a 12% to 15% soil solution.
Preferably, in the second step, the liquid is clarified after the suction filtration by using multiple layers of filter paper, and the multiple layers can be set to be 3 layers or 5 layers.
Preferably, in the second step, the liquid is clarified after suction filtration by using multiple layers of filter paper, and the multiple layers can be set to be 3 layers.
Preferably, in step two, the incubation is carried out in an incubator at 34 ℃ to 35 ℃ for three days to five days.
Preferably, in step two, the incubation is carried out in an incubator at 35 ℃ for three to five days.
Preferably, in the second step, the fermentation is carried out for 40 to 48 hours at 33 to 35 ℃.
Preferably, in the second step, the fermentation is carried out at 34 ℃ for 40 to 45 hours.
Preferably, in step three, the first step is centrifuged at 5 ℃ and 4300r/min for 30 min.
Preferably, in step three, the first step is centrifugation at 5 ℃ and 4300r/min for 35 min.
Preferably, in step three, the second step is centrifuged at 4500r/min for 30min at 5 ℃.
Preferably, in step three, the second step 5 ℃, 4500r/min centrifugation for 35 min.
Preferably, in step four, the first step is carried out at a temperature of 56 ℃ to 60 ℃ for 2 hours to 4 hours.
Preferably, in step four, the first step is carried out at a temperature of 56 ℃ to 58 ℃ for 2 hours to 4 hours.
Preferably, in the fourth step, the second step uses a stirrer to stir the raw material after enzymolysis for 6 to 10 minutes.
Preferably, in the fourth step, the second step uses a stirrer to stir the raw material after enzymolysis for 5 to 8 minutes.
Preferably, in the fifth step, hydrogen peroxide accounting for 5 to 6 percent of the weight of the cartilage feed is added in the first step.
Preferably, in step five, the first step is oxidized at a temperature of 40 ℃ to 45 ℃ for 2 hours to 4 hours and filtered.
Preferably, in step five, the first step is oxidized at a temperature of 45 ℃ for 2 hours to 4 hours and filtered.
Compared with the prior art, the invention has the following beneficial effects: the new method for extracting chondroitin sulfate in fish cartilage is widely applied to the field of bioengineering. The invention treats the filtrate in advance, can control the temperature to be kept at the set temperature by using the thermostat, and can simultaneously perform the disinfection function on the packaged chondroitin.
Drawings
FIG. 1 is a flow chart of a novel process for the extraction of chondroitin sulfate in fish cartilage.
FIG. 2 is a flow chart of chondroitinase extraction.
FIG. 3 is a flow chart of enzymatic ultrafiltration of extracted chondroitinase.
FIG. 4 is a flow chart of the process of making chondroitin sulfate and disinfection.
Detailed Description
The invention is further described below with reference to the accompanying drawings:
in the figure:
as shown in figures 1 to 4
A new method for extracting chondroitin sulfate in fish cartilage comprises the following steps:
s101: pretreating materials, namely degreasing selected fish cartilage, crushing the degreased fish cartilage by using a crusher, adding a 6-9% hydrogen NaOH solution and a sodium chloride solution, extracting for 2-4 hours and 30 minutes at the temperature of 61-75 ℃, filtering, and adjusting the pH of the filtrate to be = 8-9;
s102, screening and fermenting microorganisms, suspending a soil sample with sterile water to prepare 11-20% soil solution, performing suction filtration by using multilayer filter paper to clarify liquid, then diluting the liquid to 11-15 ℃ in a gradient manner, coating 0.5ml of diluent on a separation medium plate for plate culture, then purifying a single bacterial colony on the separation medium plate for 3 times by using a plate streaking separation method, selecting a purified single bacterial colony to be spotted on a primary screening medium plate, culturing in an incubator at 33-35 ℃ for three days to five days, pouring 8ml of 4mol/L glacial acetic acid into the plate, soaking for 2min, storing a bacterial strain generating a transparent ring on a test tube solid inclined plane medium by using a streaking method for later use at 5 ℃, inoculating the bacterial strain generating the transparent ring into a secondary screening medium, controlling the temperature to be 33 ℃, 145r/min, performing oscillatory aeration culture for 1 day, diluting the gradient to 11-15 ℃, taking 0.5ml of 11-15 bacterial strain liquid, coating the bacterial strain on the primary screening medium, coating the bacterial strain in the secondary screening medium, controlling the temperature to 35-35 ℃ for 33 ℃, performing oscillatory aeration culture for 1 day, inoculating the bacterial strain into a fermentation tank for 20-35 ℃ and observing the strain, inoculating the bacterial strain to a fermentation tank for 20 ℃ by using a fermentation tank, controlling the strain, inoculating the strain to 32 v, measuring the strain, inoculating the strain to obtain a fermentation strain, inoculating the strain, performing fermentation characteristic strain, measuring the strain, inoculating the strain in the strain, inoculating the strain, fermenting tank for 52 v, and performing fermentation at a fermentation tank, wherein the;
s103: extracting chondroitinase sulfate;
s301, primary extraction, namely placing the obtained fermentation liquor in a centrifuge, centrifuging for 30-35 min at 5 ℃ and 4300r/min, removing thallus precipitates, carrying out ice bath, slowly adding ammonium sulfate into the fermentation supernatant to reach 92% of saturation under the stirring of a magnetic stirrer, and standing for 24 hours in a chromatography cabinet at 5 ℃;
s302, extracting again, placing the obtained fermentation liquor in a centrifuge, centrifuging for 30min to 35min at 5 ℃ and 4500r/min, removing thallus precipitates, carrying out ice bath, slowly adding ammonium sulfate into the fermentation supernatant to reach 92% of saturation degree under the stirring of a magnetic stirrer, and standing for 24 hours in a chromatography cabinet at 5 ℃;
s303, fine extraction, then centrifuging the fermentation liquor again at 5 ℃ and 5000r/min, collecting protein precipitate, dissolving by using triple-distilled water, placing into a dialysis bag, dialyzing and desalting in a chromatography cabinet at 5 ℃, and freeze-drying under the condition that (NH4)2SO4 solution detects that the liquid outside the dialysis bag does not contain SO 42-;
s104: carrying out enzymolysis and ultrafiltration on the extracted chondroitin sulfate enzyme;
s401, adding cartilage feeding materials, adding the chondroitinase powder prepared in the third step into the filtrate obtained in the first step, and carrying out enzymolysis for 2 to 4 hours at the pH value of 8 to 10 and the temperature of 55 to 60 ℃;
s402, stirring and dehydrating, namely stirring the raw materials subjected to enzymolysis for 5 to 10 minutes by using a stirrer, and then performing ultrafiltration, dehydration and concentration to 2/3 in the original volume by using an ultrafiltration membrane with the cut-off molecular weight of 8100 at room temperature;
s403, treating the waste, namely temporarily storing the liquid material dehydrated and discharged by the ultrafiltration membrane in a storage tank;
s105: preparing chondroitin sulfate and sterilizing;
s501, primarily making, namely adjusting the pH value of the concentrated solution after ultrafiltration dehydration to 8-9, adding hydrogen peroxide which accounts for 4-6% of the weight of the cartilage feed, oxidizing the mixture at the temperature of 36-45 ℃ for 2-4 hours, filtering the mixture, adjusting the pH value of the filtrate to 5-6, adding 96% ethanol for crystallization, and enabling the ethanol concentration to reach 76-80%;
s502, carrying out centrifugal washing treatment, and centrifuging and washing by using a centrifugal machine after crystallization;
s503, drying, packaging and sterilizing, washing and drying to obtain fish chondroitin sulfate, packaging the chondroitin by using packaging equipment, and irradiating and sterilizing the outside of the package by using ultraviolet ray sterilizing equipment.
Preferably, in S101, 7% to 9% hydrogen NaOH solution and sodium chloride solution are added.
Preferably, in S101, 7% to 8% hydrogen NaOH solution and sodium chloride solution are added.
Preferably, in S101, the extraction is performed at a temperature of 65 ℃ to 75 ℃ for 2 hours to 4 hours.
Preferably, in S101, the extraction is performed at a temperature of 65 ℃ to 70 ℃ for 3 hours to 4 hours.
Preferably, in S102, the soil sample is suspended with sterile water to form a 12% to 20% soil solution.
Preferably, in S102, the soil sample is suspended with sterile water to form a 12% to 15% soil solution.
Preferably, in S102, the liquid is clarified after suction filtration by using multiple layers of filter paper, and the multiple layers may be set to be 3 layers or 5 layers.
Preferably, in S102, the liquid is clarified after suction filtration by using multiple layers of filter paper, and the multiple layers may be set to be 3 layers.
Preferably, in S102, the incubation is performed in an incubator at 34 ℃ to 35 ℃ for three days to five days.
Preferably, in S102, the incubation is performed in an incubator at 35 ℃ for three to five days.
Preferably, in S102, the fermentation is carried out at 33 ℃ to 35 ℃ for 40 hours to 48 hours.
Preferably, in S102, the fermentation is carried out at 34 ℃ for 40 to 45 hours.
Preferably, in S103, the mixture is centrifuged at 4300r/min for 30min at S3015 ℃.
Preferably, in S103, the S3015 ℃ and 4300r/min are centrifuged for 35 min.
Preferably, in S103, the second step is centrifuged at 4500r/min for 30min at 5 ℃.
Preferably, in S103, the centrifugation is carried out at 4500r/min at S3025 ℃ for 35 min.
Preferably, in S104, the S401 temperature is 56 ℃ to 60 ℃ for 2 hours to 4 hours.
Preferably, in S104, the S401 temperature is 56 ℃ to 58 ℃ for 2 hours to 4 hours.
Preferably, in S104, the raw material after the enzymolysis is stirred by the stirrer in S402 for 6 to 10 minutes.
Preferably, in S104, the raw material after the enzymolysis is stirred by the stirrer for 5 to 8 minutes in S402.
Preferably, in S105, hydrogen peroxide in an amount of 5% to 6% by weight of the cartilage charge is added to S501.
Preferably, in S105, the S501 is oxidized at a temperature of 40 ℃ to 45 ℃ for 2 hours to 4 hours and filtered.
Preferably, in S105, the S501 is oxidized at 45 ℃ for 2 to 4 hours and filtered.
Detailed description of the preferred embodiment
Example 1:
defatting fish cartilage, pulverizing with pulverizer, adding 6% to 8% NaOH solution and sodium chloride solution, extracting at 68 deg.C to 75 deg.C for 2 hr to 4 hr for 30min, filtering, adjusting pH =8-9, suspending with sterile water to obtain 11% to 20% soil solution, filtering with multi-layer filter paper, clarifying, diluting the liquid, diluting to 11-15%, coating with 0.5ml diluent, culturing with separation medium plate, purifying the bacterial colony with plate streaking method, inoculating the purified bacterial colony to primary sieve medium plate, culturing at 33 deg.C to 35 deg.C for three days, adding 8ml4mol/L glacial acetic acid, soaking for 2min, marking the bacterial colony to solid medium, storing at 365 deg.C, inoculating to re-sieve medium, inoculating to transparent bacterial colony, culturing at 35 deg.C and 60 deg.C, culturing at 35 deg.C, culturing temperature, culturing at 35 deg.C for 35 deg.C, culturing at 35 deg.C for 35 deg.C, culturing at 35 deg.C, culturing temperature, filtering, culturing at 35min, filtering, culturing at 35 deg.C for 60 min, filtering, culturing at 35 deg.C, culturing at 5min, filtering, culturing at 35 deg.C, culturing at 35min, filtering, culturing at 5min, filtering, culturing at 35 deg.C, filtering, culturing at 35 deg.C, culturing at 5min, filtering, culturing at 35% pH of pH, filtering, culturing at 5min, collecting bacterial colony concentration, culturing at 35min, culturing at 5min, collecting bacterial colony concentration, culturing at 35-35 deg.C, culturing at 5min, filtering, collecting bacterial colony concentration, filtering, culturing at 5min, collecting bacterial colony concentration, culturing at 35 deg.C, culturing at 5min, culturing at 35min, culturing at 5min, centrifuging at 5min, collecting bacterial colony concentration, centrifuging at 5min, culturing at 5min, culturing, centrifuging at 35 deg.C, collecting bacterial colony concentration, centrifuging at 35 deg.C, centrifuging at 35min, centrifuging at 35min, collecting bacterial colony concentration, centrifuging at 35min, centrifuging at 35 deg.C, centrifuging at 35min, collecting bacterial colony concentration, centrifuging at 35 min.
Example 2:
selecting fish cartilage, defatting, pulverizing with pulverizer, adding 6% to 9% hydrogen NaOH solution and sodium chloride solution, extracting at 65 deg.C to 75 deg.C for 2 hr to 4 hr, filtering, adjusting pH =8-9, suspending with sterile water to obtain 12% to 20% soil solution, filtering with 3 layers of filter paper, clarifying, diluting the liquid, diluting to 11-15%, collecting 0.5ml of diluent, coating with separation medium, culturing, purifying the bacterial colony with plate streaking method, inoculating the purified bacterial colony to a primary sieve medium plate, culturing in 34 deg.C to 35 deg.C incubator for three days to five days, adding 8ml of 4mol/L glacial acetic acid, soaking for 2min, placing the bacterial colony on a slant culture medium, storing at 365 deg.C, culturing at 35 deg.C and 34 deg.C, inoculating to 35 deg.C, culturing at 35 deg.C, culturing temperature of 35 deg.C, culturing at 35 deg.C, culturing temperature of 60 deg.C, culturing at 35 deg.C, culturing, filtering, culturing at 35 deg.C, culturing temperature of 60 deg.C, culturing at 5 deg.C, filtering, culturing at 5 deg.C, collecting bacterial colony concentration, culturing at 5min, culturing, filtering, culturing at 5 deg.C, culturing at 5min, filtering, culturing at 5min, collecting bacterial colony concentration, culturing at 5min, culturing at 5min, collecting bacterial colony concentration, filtering, collecting bacterial colony concentration, culturing at 5 deg.C, culturing at 5min, collecting bacterial colony concentration, culturing at 5min, culturing at 5 deg.C, culturing at 5min, collecting bacterial colony concentration, culturing at 5 deg.C, culturing at 5 deg.C, culturing at 5min, culturing at 5min, culturing at 5min, culturing at 35-35 deg.C, culturing at 5min, culturing at 5 deg.C, culturing at 5min, culturing at 5min, culturing at 5min, culturing at 5min, culturing.
The technical solutions of the present invention or similar technical solutions designed by those skilled in the art based on the teachings of the technical solutions of the present invention are all within the scope of the present invention.
Claims (10)
1. A novel method for extracting chondroitin sulfate in fish cartilage is characterized by comprising the following steps:
the method comprises the following steps: pretreating materials, namely degreasing selected fish cartilage, crushing the degreased fish cartilage by using a crusher, adding a 6-9% hydrogen NaOH solution and a sodium chloride solution, extracting for 2-4 hours and 30 minutes at the temperature of 61-75 ℃, filtering, and adjusting the pH of the filtrate to be = 8-9;
screening and fermenting microorganisms, suspending a soil sample with sterile water to prepare 11-20% soil solution, performing suction filtration by using multilayer filter paper to clarify liquid, then diluting the liquid to 11-15 ℃ in a gradient manner, coating 0.5ml of diluent on a separation medium plate for plate culture, then purifying a single bacterial colony on the separation medium plate for 3 times by using a plate streaking separation method, selecting a purified single bacterial colony to be spotted on a primary screening medium plate, culturing in an incubator at 33-35 ℃ for three days to five days, pouring 8ml of 4mol/L glacial acetic acid into the plate, soaking for 2min, storing a bacterial strain generating a transparent ring on a test tube solid inclined plane medium by using a streaking method for later use at 5 ℃, inoculating the bacterial strain generating the transparent ring in a secondary screening medium, controlling the temperature to be 33 ℃, 145r/min, performing shake, aeration culture for 1 day, diluting the gradient to 11-15 ℃, taking 0.5ml of 11-15 bacterial strain liquid, coating the bacterial strain on the primary screening medium, coating the bacterial strain in the secondary screening medium, controlling the temperature to 35-35 ℃ for 33 ℃, performing shake, culturing for 1 day, inoculating the bacterial colony in a fermentation tank for 2 v, measuring the strain by using a fermentation tank, inoculating the strain for 3-32 hours, measuring the strain, inoculating the strain in a fermentation tank by using a fermentation tank at 35 ℃ for 3 v, and measuring the strain, wherein the strain, and measuring the strain, wherein the strain, and measuring the strain, wherein the strain;
step three: extracting chondroitinase sulfate;
firstly, primary extraction, namely placing the obtained fermentation liquor in a centrifuge, centrifuging for 30min to 35min at 5 ℃ and 4300r/min, removing thallus precipitates, carrying out ice bath, slowly adding ammonium sulfate into the fermentation supernatant to reach the saturation of 92 percent under the stirring of a magnetic stirrer, and standing for 24 hours in a chromatography cabinet at 5 ℃;
secondly, extracting again, placing the obtained fermentation liquor in a centrifuge, centrifuging for 30min to 35min at the temperature of 5 ℃ and at the speed of 4500r/min, removing thallus precipitates, carrying out ice bath, slowly adding ammonium sulfate into the fermentation supernatant to reach the saturation of 92 percent under the stirring of a magnetic stirrer, and standing for 24 hours in a chromatography cabinet at the temperature of 5 ℃;
thirdly, fine extraction, then centrifuging the fermentation liquor again at 5 ℃ and 5000r/min, collecting protein precipitate, dissolving by using triple distilled water, placing into a dialysis bag, dialyzing and desalting in a chromatography cabinet at 5 ℃, and freeze-drying under the condition that (NH4)2SO4 solution detects that the liquid outside the dialysis bag does not contain SO 42-;
step four: carrying out enzymolysis and ultrafiltration on the extracted chondroitin sulfate enzyme;
adding cartilage feed, adding the chondroitinase powder prepared in the third step into the filtrate obtained in the first step, and carrying out enzymolysis for 2 to 4 hours at the pH value of 8 to 10 and the temperature of 55 to 60 ℃;
secondly, stirring and dehydrating, namely stirring the raw materials subjected to enzymolysis for 5 to 10 minutes by using a stirrer, and then performing ultrafiltration, dehydration and concentration to 2/3 in the original volume by using an ultrafiltration membrane with the cut-off molecular weight of 8100 at room temperature;
thirdly, waste treatment, namely temporarily storing liquid materials discharged by ultrafiltration membrane dehydration in a storage tank;
step five: preparing chondroitin sulfate and sterilizing;
firstly, primarily manufacturing, adjusting the pH value of the concentrated solution after ultrafiltration dehydration to 8-9, adding hydrogen peroxide which is 4-6% of the weight of the cartilage feed, oxidizing for 2-4 hours at the temperature of 36-45 ℃, filtering, adjusting the pH value of the filtrate to 5-6, adding 96% ethanol for crystallization, and enabling the ethanol concentration to reach 76-80%;
step two, carrying out centrifugal washing treatment, and centrifuging and washing by using a centrifugal machine after crystallization;
and thirdly, drying, packaging and sterilizing, washing and drying to obtain fish chondroitin sulfate, packaging the chondroitin by using packaging equipment, and irradiating and sterilizing the outside of the package by using ultraviolet ray sterilization equipment.
2. The novel process for the extraction of chondroitin sulfate in fish cartilage as claimed in claim 1, wherein in step one, 7% to 9% NaOH solution and NaCl solution are added.
3. The novel process for the extraction of chondroitin sulfate in fish cartilage as claimed in claim 1, wherein the extraction is carried out at a temperature of 65 ℃ to 75 ℃ for 2 hours to 4 hours.
4. The novel process for the extraction of chondroitin sulfate in fish cartilage as claimed in claim 1, wherein the suspension of the soil sample in sterile water is made up to a 12% to 20% soil solution.
5. The novel process for the extraction of chondroitin sulfate in fish cartilage as claimed in claim 1, wherein in step two, the liquid is clarified after suction filtration using multiple layers of filter paper, wherein the multiple layers may be set to 3 or 5 layers.
6. The novel process for the extraction of chondroitin sulfate in fish cartilage as claimed in claim 1, wherein, in the second step, the incubation is carried out in an incubator at 34 ℃ to 35 ℃ for three days to five days.
7. The novel process for the extraction of chondroitin sulfate in fish cartilage as claimed in claim 1, wherein in the second step, the fermentation is carried out at 33 ℃ to 35 ℃ for 40 hours to 48 hours.
8. The novel process for the extraction of chondroitin sulfate in fish cartilage as claimed in claim 1, wherein in step three, the first step is centrifugation at 5 ℃ and 4300r/min for 30 min.
9. The novel process for the extraction of chondroitin sulfate in fish cartilage as claimed in claim 1, wherein in step three, the second step is centrifuged at 4500 rpm/5 ℃ for 30 min.
10. The novel process for the extraction of chondroitin sulfate in fish cartilage as claimed in claim 1, wherein the first step is an oxidation at a temperature of 40 ℃ to 45 ℃ for 2 hours to 4 hours and a filtration.
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| CN202010348626.7A CN111334543A (en) | 2020-04-28 | 2020-04-28 | Novel method for extracting chondroitin sulfate in fish cartilage |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| 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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| CN103305496A (en) * | 2013-06-27 | 2013-09-18 | 青岛贝尔特生物科技有限公司 | Method for extracting chondrosulphatase through microbe fermentation |
| CN104450831A (en) * | 2013-09-21 | 2015-03-25 | 青岛蓝农谷农产品研究开发有限公司 | Method for extracting chondroitin sulfate from fish cartilage |
| CN106674380A (en) * | 2017-02-09 | 2017-05-17 | 高焕顺 | Preparation method of new chondroitin sulfate |
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| CN103305496A (en) * | 2013-06-27 | 2013-09-18 | 青岛贝尔特生物科技有限公司 | Method for extracting chondrosulphatase through microbe fermentation |
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| 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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