A method for simultaneously extracting chondroitin sulfate and collagen peptides from fish bones

Abstract

The invention discloses a method for simultaneously extracting chondroitin sulfate and collagen peptide from fish bones. The method comprises the steps of washing the fish bones, boiling and removing fat, crushing to obtain fish bone powder, hot-pressing and extracting to obtain an extract, adding lactobacillus rhamnosus, pichia pastoris, lactobacillus paracasei and bacillus subtilis to the extract to ferment to obtain fermentation liquid, inactivating enzymes to obtain enzymolysis liquid, and performing column chromatography through an anion exchange resin column to obtain collagen peptide. The method uses hot-pressing extraction instead of alkali extraction, does not use any acid-base reagent, is green and environmentally friendly, avoids environmental pollution, omits the oxidation and decolorization step in the production process of chondroitin sulfate, and has lower production cost and more stable quality of chondroitin sulfate.

Description

Technical Field

[0001] The invention relates to a method for producing collagen peptides, in particular to a method for co-producing chondroitin sulfate and collagen active peptides by using fish bones as raw materials, belonging to the technical field of health food. Background Art

[0002] Fish is deeply loved by the Chinese people and is an indispensable delicacy on the table. my country's annual consumption of marine fish is astonishing. Usually, the bones of fish are discarded after processing or eating, and are not reused. This not only pollutes the environment, but also causes waste of resources.

[0003] Chondroitin sulfate is a type of glycosaminoglycan that is covalently linked to protein to form proteoglycans. It is widely distributed in the extracellular matrix and cell surface of animal tissues. It is mainly used as a drug for the treatment of rheumatic and rheumatoid diseases. It is also used as the main raw material of functional foods. It also has anti-cancer, anti-thrombotic, and lipid-lowering effects. It is now exported to many countries in Europe and the United States as a raw material drug, and the market demand is strong. The collagen in fish bones has many beneficial effects, such as: (1) It can supplement the nutrients needed by all layers of the skin and enhance the activity of collagen in the skin, thereby moisturizing the skin, delaying aging, beautifying, removing wrinkles, and nourishing hair; (2) It is a binder for hydroxyapatite and together with hydroxyapatite, it constitutes the main body of the bone; (3) The fat content of collagen is much lower than that of collagen from other animals, making it more popular among consumers; (4) Fish bone collagen is more extensible and thermally stable than fish skin collagen, making it a top-quality collagen.

[0004] At present, there are three main methods for producing chondroitin sulfate: salting method, alkaline hydrolysis method and enzymatic hydrolysis method. The most commonly used method is to extract chondroitin sulfate by combining dilute alkali or concentrated alkali with enzymatic hydrolysis. The traditional process is: cartilage cooking → alkali extraction → enzyme hydrolysis → hydrogen peroxide decolorization → alcohol precipitation → refining → chondroitin sulfate finished product. The alkaline enzymatic hydrolysis production process has complicated steps and a long cycle, generally taking 3-5 days, and the product quality and yield are unstable. During the high-temperature cooking, alkali extraction, enzymatic hydrolysis and decolorization process, chondroitin sulfate is severely damaged, the product yield is low, the solution color deepens, the ethanol consumption is large, the environment is damaged and the production cost is high, a large amount of salt is generated, and desalination is required, which prolongs the production time and causes environmental pollution. Therefore, we need to improve the traditional production process, avoid environmental pollution, reduce production costs, reduce resource waste, improve production efficiency, and achieve green, environmentally friendly, economical and convenient production.

[0005] In addition, the price of cartilage in the meat market continues to rise, and it is necessary to find a new source of raw materials for the production of chondroitin sulfate. If a method can be developed to extract chondroitin sulfate and collagen peptides from fish bones, it will not only increase the added economic value of aquatic waste, but also reduce environmental pressure, with significant economic value and social benefits. Summary of the invention

[0006] The purpose of the present invention is to provide a method for simultaneously extracting chondroitin sulfate and active collagen peptides from fish bones using fish bones as raw materials. The method does not use acid-base reagents, has a high yield of chondroitin sulfate, and simultaneously obtains a large amount of active collagen peptides with small molecular weight. The method has excellent and stable effects, low industrialization costs, and good application prospects.

[0007] The present invention is achieved through the following technical solutions:

[0008] A method for simultaneously extracting chondroitin sulfate and collagen active peptides from fish bones comprises the following steps:

[0009] (1) Using fish bones as raw materials, the fish bones are washed and boiled to remove fat, then taken out, dried and crushed to obtain fish bone powder;

[0010] (2) mixing the fish bone powder obtained in step (1) with water in a mass ratio of 1:(3-6), and then placing the mixture in an autoclave for hot-pressing extraction to obtain an extract. The conditions for hot-pressing extraction are a pressure of 0.1-0.2 MPa, a temperature of 130-150° C., and an extraction time of 0.5-2 h;

[0011] (3) adding the following microorganisms to the extract obtained in step (2): Lactobacillus rhamnosus, Pichia pastoris, Lactobacillus paracasei and Bacillus subtilis, and fermenting for 48-72 hours at 30-35°C, a rotation speed of 150-200 r / min, and a ventilation ratio of 1:(1-2). After the fermentation is completed, a fermentation liquid is obtained, and the enzyme in the fermentation liquid is inactivated to obtain an enzymatic hydrolyzate;

[0012] (4) ultrafiltration and concentration of the enzymatic hydrolyzate obtained in step (3) to obtain a concentrated solution; column chromatography of the concentrated solution using an anion exchange resin column to collect the effluent; and then eluting with deionized water to obtain an eluate; and mixing the effluent and the eluate uniformly, and then concentrating and drying to obtain collagen peptides;

[0013] (5) The anion exchange resin column after elution with deionized water in step (4) is eluted with a NaCl solution, the eluate is collected, oxidized, desalted and concentrated using a hollow fiber membrane device, precipitated with alcohol, centrifuged, and dried to obtain chondroitin sulfate.

[0014] Furthermore, the boiling time in step (1) is 20-30 minutes.

[0015] Furthermore, in step (2), the mass ratio of fish bone powder to water is 1:(4-5).

[0016] Furthermore, the amounts of the microorganisms added in step (3) are: based on the weight of the extract, 80-100 CFU / g of Lactobacillus rhamnosus, 50-80 CFU / g of Pichia pastoris, 30-50 CFU / g of Lactobacillus paracasei, and 30-50 CFU / g of Bacillus subtilis.

[0017] Furthermore, the temperature of the enzyme inactivation process in step (3) is 90-100° C. and the time is 10-15 min.

[0018] Furthermore, the model of the anion exchange resin column in step (4) is: Rohm and Haas acrylic acid strong base anion exchange resin FPA94CL (food grade), with a particle size of 0.630 to 0.850 mm.

[0019] Furthermore, the alcohol precipitation step in step (5) is as follows: adding anhydrous ethanol to the concentrated solution until the final volume concentration of ethanol is 70-75%, and standing for 4-5 hours to obtain precipitation.

[0020] The ventilation ratio described in the present invention refers to the ratio of the volume of air introduced into the fermentation tank per minute to the total volume of the extract.

[0021] The Lactobacillus rhamnosus, Pichia pastoris, Lactobacillus paracasei and Bacillus subtilis used in the technical solution of the present invention can be selected from products of any manufacturer on the market.

[0022] The method provided by the present invention has the following advantages:

[0023] 1) This method uses hot-pressing extraction instead of alkaline extraction, does not use any acid or alkali reagents, is green and environmentally friendly, avoids environmental pollution, and omits the oxidation and decolorization step in the production process of chondroitin sulfate. The production cost of chondroitin sulfate is lower and the quality is more stable;

[0024] 2) The Lactobacillus rhamnosus, Pichia pastoris, Lactobacillus paracasei and Bacillus subtilis used in the present method can produce a variety of hydrolytic proteases during the fermentation growth process. The combined use of these different types of proteases can increase the enzyme cleavage sites, thereby being able to fully enzymatically hydrolyze various types of collagen in fish bones that are difficult to dissolve and obtain a large number of polypeptides.

[0025] 3) The process of the present invention makes full use of the high-quality protein in fish bones, solves the environmental pollution problem and obtains a large amount of small-molecule collagen active peptides and chondroitin sulfate. The extraction rate and purity of chondroitin sulfate and collagen peptides are high, which effectively improves the comprehensive utilization value of fish bones and is very practical and popularizable. DETAILED DESCRIPTION

[0026] The principles and features of the present invention are described below in conjunction with examples. The examples are only used to explain the present invention and are not used to limit the scope of the present invention.

[0027] Example 1

[0028] A method for simultaneously extracting chondroitin sulfate and collagen active peptides from fish bones comprises the following steps:

[0029] (1) Using fish bones as raw materials, wash the fish bones, boil them in water for 20 minutes to remove the fat, remove them, dry and grind them to obtain fish bone powder;

[0030] (2) mixing the fish bone powder obtained in step (1) with water in a mass ratio of 1:3, and then placing the mixture in an autoclave for hot pressing extraction to obtain an extract. The conditions for hot pressing extraction are a pressure of 0.1-0.2 MPa, a temperature of 130° C., and extraction for 2 h;

[0031] (3) adding the following microorganisms to the extract obtained in step (2): Lactobacillus rhamnosus, Pichia pastoris, Lactobacillus paracasei and Bacillus subtilis, wherein the amount of each microorganism added is: 80-100 CFU / g of Lactobacillus rhamnosus, 50-80 CFU / g of Pichia pastoris, 30-50 CFU / g of Lactobacillus paracasei and 30-50 CFU / g of Bacillus subtilis, based on the weight of the extract; and fermenting the extract at 30-35° C., a rotation speed of 150 r / min and a ventilation ratio of 1:1 for 72 h. After the fermentation is completed, a fermentation liquid is obtained, and the fermentation liquid is inactivated to obtain an enzymatic solution;

[0032] (4) ultrafiltration and concentration of the enzymatic hydrolyzate obtained in step (3) to obtain a concentrated solution; column chromatography of the concentrated solution using an anion exchange resin column, collecting the effluent, wherein the model of the anion exchange resin column is: Rohm and Haas acrylic acid strong base anion exchange resin FPA94CL (food grade), with a particle size of 0.630 to 0.850 mm; elution with deionized water to obtain an eluate; the effluent and the eluate are evenly mixed and then concentrated and dried to obtain collagen peptides;

[0033] (5) The anion exchange resin column after deionized water elution in step (4) is eluted with a NaCl solution, the eluate is collected, oxidized, desalted and concentrated using a hollow fiber membrane device, anhydrous ethanol is added to the concentrate to a final volume concentration of ethanol of 70-75%, and the concentrate is allowed to stand for 4-5 hours to obtain a precipitate, which is then centrifuged and dried to obtain chondroitin sulfate.

[0034] Example 2

[0035] A method for simultaneously extracting chondroitin sulfate and collagen active peptides from fish bones comprises the following steps:

[0036] (1) Using fish bones as raw materials, wash the fish bones, boil them in water for 30 minutes to remove the fat, remove them, dry and grind them to obtain fish bone powder;

[0037] (2) mixing the fish bone powder obtained in step (1) with water in a mass ratio of 1:6, and then placing the mixture in an autoclave for hot pressing extraction to obtain an extract, wherein the conditions for hot pressing extraction are a pressure of 0.1-0.2 MPa, a temperature of 150° C., and an extraction time of 0.5 h;

[0038] (3) adding the following microorganisms to the extract obtained in step (2): Lactobacillus rhamnosus, Pichia pastoris, Lactobacillus paracasei and Bacillus subtilis, wherein the amount of each microorganism added is: 80-100 CFU / g of Lactobacillus rhamnosus, 50-80 CFU / g of Pichia pastoris, 30-50 CFU / g of Lactobacillus paracasei and 30-50 CFU / g of Bacillus subtilis, based on the weight of the extract; and fermenting the extract at 30-35° C., a rotation speed of 200 r / min and a ventilation ratio of 1:1 for 60 h. After the fermentation is completed, a fermentation liquid is obtained, and the fermentation liquid is inactivated to obtain an enzymatic solution;

[0039] (4) ultrafiltration and concentration of the enzymatic hydrolyzate obtained in step (3) to obtain a concentrated solution; column chromatography of the concentrated solution using an anion exchange resin column, collecting the effluent, wherein the model of the anion exchange resin column is: Rohm and Haas acrylic acid strong base anion exchange resin FPA94CL (food grade), with a particle size of 0.630 to 0.850 mm; elution with deionized water to obtain an eluate; the effluent and the eluate are evenly mixed and then concentrated and dried to obtain collagen peptides;

[0040] (5) The anion exchange resin column after deionized water elution in step (4) is eluted with a NaCl solution, the eluate is collected, oxidized, desalted and concentrated using a hollow fiber membrane device, anhydrous ethanol is added to the concentrate to a final volume concentration of ethanol of 70-75%, and the concentrate is allowed to stand for 4-5 hours to obtain a precipitate, which is then centrifuged and dried to obtain chondroitin sulfate.

[0041] Example 3

[0042] A method for simultaneously extracting chondroitin sulfate and collagen active peptides from fish bones comprises the following steps:

[0043] (1) Using fish bones as raw materials, wash the fish bones, boil them in water for 30 minutes to remove the fat, remove them, dry and grind them to obtain fish bone powder;

[0044] (2) mixing the bone powder obtained in step (1) with water in a mass ratio of 1:4, and then placing the mixture in an autoclave for hot-pressing extraction to obtain an extract, wherein the conditions for hot-pressing extraction are a pressure of 0.1-0.2 MPa, a temperature of 140° C., and an extraction time of 1.5 h;

[0045] (3) adding the following microorganisms to the extract obtained in step (2): Lactobacillus rhamnosus, Pichia pastoris, Lactobacillus paracasei and Bacillus subtilis, wherein the amount of each microorganism added is: 80-100 CFU / g of Lactobacillus rhamnosus, 50-80 CFU / g of Pichia pastoris, 30-50 CFU / g of Lactobacillus paracasei and 30-50 CFU / g of Bacillus subtilis, based on the weight of the extract; and fermenting the extract at 30-35° C., a rotation speed of 180 r / min and a ventilation ratio of 1:2 for 48 h. After the fermentation is completed, a fermentation liquid is obtained, and the enzyme in the fermentation liquid is inactivated to obtain an enzymatic solution;

[0046] (4) ultrafiltration and concentration of the enzymatic hydrolyzate obtained in step (3) to obtain a concentrated solution; column chromatography of the concentrated solution using an anion exchange resin column, collecting the effluent, wherein the model of the anion exchange resin column is: Rohm and Haas acrylic acid strong base anion exchange resin FPA94CL (food grade), with a particle size of 0.630 to 0.850 mm; elution with deionized water to obtain an eluate; the effluent and the eluate are evenly mixed and then concentrated and dried to obtain collagen peptides;

[0047] (5) The anion exchange resin column after deionized water elution in step (4) is eluted with a NaCl solution, the eluate is collected, oxidized, desalted and concentrated using a hollow fiber membrane device, anhydrous ethanol is added to the concentrate to a final volume concentration of ethanol of 70-75%, and the concentrate is allowed to stand for 4-5 hours to obtain a precipitate, which is then centrifuged and dried to obtain chondroitin sulfate.

[0048] Comparative Example 1:

[0049] A method for preparing chondroitin sulfate and co-producing collagen peptides from fish bones comprises the following steps:

[0050] (1) Steaming

[0051] Fresh marine fish bones are selected as raw materials, sent to a continuous cooking tank for cooking for 30 minutes, and separated with high-pressure air to obtain fish bone protein and fish bones;

[0052] (2) Crushing and fermentation

[0053] The fish bones obtained in step (1) are washed and crushed, and the average particle size is controlled to be 0.3 mm to obtain fish bone powder; the obtained fish bone powder is added with an equal amount of purified water and stirred evenly, and then 2% of the weight of the fish bone powder is inoculated with lactic acid bacteria, the pH value is adjusted to 5.5, and the fermentation is carried out at a constant temperature, the temperature is controlled at 37° C., and the fermentation is carried out for 18 hours to obtain a fermentation liquid;

[0054] (3) Separation

[0055] The fermentation liquid obtained in step (2) is centrifuged to obtain a clear liquid and a precipitate;

[0056] (4) Enzymatic hydrolysis and enzyme inactivation of precipitate

[0057] The precipitate obtained in step (3) is washed clean, purified water is added and stirred evenly, and then 0.7% of the weight of the precipitate is added with a neutral protease for enzymolysis at a temperature of 50° C. for 8 h to obtain an enzymolysis solution; the enzymolysis solution is heated to 90° C. and the enzyme is inactivated for 40 min to obtain an enzyme-inactivated enzymolysis solution;

[0058] (5) Preparation of collagen peptides

[0059] The enzyme-killed enzymatic hydrolysate obtained in step (4) is ultrafiltered and concentrated to obtain a concentrated solution; the concentrated solution is subjected to column chromatography using an anion exchange resin column, and the effluent is collected; the effluent is eluted with deionized water to obtain an eluate; the effluent and the eluate are evenly mixed, and spray-dried to obtain collagen peptides;

[0060] (6) Preparation of Chondroitin Sulfate

[0061] The anion exchange resin column after elution with deionized water in step (5) is eluted with NaCl solution, the eluate is subjected to ultrafiltration desalination, and then anhydrous ethanol is added to a final volume concentration of ethanol of 70%, and the mixture is allowed to stand for 4 hours to obtain a precipitate; the precipitate is decolorized and oxidized with hydrogen peroxide, the mass ratio of the precipitate to hydrogen peroxide being 1:3, and then dried under reduced pressure to obtain chondroitin sulfate.

[0062] The collagen peptides and chondroitin sulfate obtained in Examples 1-3 and Comparative Example 1 were tested as follows:

[0063] 1. Extraction rate and purity test

[0064] Chondroitin sulfate testing standard: GB / T 20365-2006 "Determination of chondroitin sulfate and glucosamine hydrochloride content by liquid chromatography";

[0065] Detection standard for collagen peptides: YY / T 1805.3-2022 "Collagen for tissue engineering medical device products Part 3: Detection of collagen content based on characteristic peptide determination-liquid chromatography-mass spectrometry".

[0066] The extraction results of chondroitin sulfate and collagen peptides of Examples 1-3 and Comparative Example 1 are shown in Table 1.

[0067] Table 1 Test results

[0068]

[0069] It can be seen from the data in Table 1 that, compared with Comparative Example 1, the extraction rate and purity of chondroitin sulfate obtained by the method of the present invention are both more than 2 percentage points higher, and the extraction rate and purity of collagen peptides are also slightly higher.

[0070] 2. Molecular weight distribution test of collagen peptide

[0071] The molecular weight distribution of the collagen peptide obtained in Example 2 was measured by size exclusion chromatography. The results are shown in Table 2.

[0072] Table 2 Molecular weight distribution of collagen peptides

[0073]

[0074] As can be seen from Table 2, 100% of the collagen peptides obtained in Example 2 were below 2.0 kDa, and more than 80% were below 0.5 kDa. Therefore, the collagen peptides obtained by the method of the present invention were all oligopeptides.

[0075] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for simultaneously extracting chondroitin sulfate and collagen peptides from fish bones, characterized in that: The steps include: (1) Using fish bones as raw materials, the fish bones are washed and boiled to remove fat, then taken out, dried and crushed to obtain fish bone powder; (2) mixing the fish bone powder obtained in step (1) with water in a mass ratio of 1:(3-6), and then placing the mixture in an autoclave for hot-pressing extraction to obtain an extract. The conditions for hot-pressing extraction are a pressure of 0.1-0.2 MPa, a temperature of 130-150° C., and an extraction time of 0.5-2 h; (3) adding the following microorganisms to the extract obtained in step (2): Lactobacillus rhamnosus, Pichia pastoris, Lactobacillus paracasei and Bacillus subtilis, and fermenting for 48-72 hours at 30-35°C, a rotation speed of 150-200 r / min, and a ventilation ratio of 1:(1-2). After the fermentation is completed, a fermentation liquid is obtained, and the enzyme in the fermentation liquid is inactivated to obtain an enzymatic hydrolyzate; (4) ultrafiltration and concentration of the enzymatic hydrolyzate obtained in step (3) to obtain a concentrated solution; column chromatography of the concentrated solution using an anion exchange resin column to collect the effluent; and then eluting with deionized water to obtain an eluate; and mixing the effluent and the eluate uniformly, and then concentrating and drying to obtain collagen peptides; (5) The anion exchange resin column after elution with deionized water in step (4) is eluted with a NaCl solution, the eluate is collected, oxidized, desalted and concentrated using a hollow fiber membrane device, precipitated with alcohol, centrifuged, and dried to obtain chondroitin sulfate.

2. The method according to claim 1, characterized in that: The amounts of the microorganisms added in step (3) are: based on the weight of the extract, 80-100 CFU / g of Lactobacillus rhamnosus, 50-80 CFU / g of Pichia pastoris, 30-50 CFU / g of Lactobacillus paracasei, and 30-50 CFU / g of Bacillus subtilis.

3. The method according to claim 1 or 2, characterized in that: The boiling time in step (1) is 20-30 minutes.

4. The method according to claim 1 or 2, characterized in that: The mass ratio of fish bone powder to water in step (2) is 1:(4-5).

5. The method according to claim 1 or 2, characterized in that: The temperature of the enzyme inactivation process in step (3) is 90-100°C and the time is 10-15 minutes.

6. The method according to claim 1 or 2, characterized in that: The model of the anion exchange resin column in step (4) is: food grade Rohm and Haas acrylic acid strong base anion exchange resin FPA94CL, with a particle size of 0.630-0.850 mm.

7. The method according to claim 1 or 2, characterized in that: The alcohol precipitation step in step (5) is as follows: adding anhydrous ethanol to the concentrated solution until the final volume concentration of ethanol is 70-75%, and standing for 4-5 hours to obtain precipitation.

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