Preparation method and application of an antioxidant fish collagen peptide

Abstract

The present invention discloses a preparation method and application of antioxidant fish collagen peptides, belonging to the technical field of fish collagen peptide preparation. The present invention optimizes the treatment process of preparing collagen peptides by enzymolysis of fish scales. First, a weak acid solution composed of citric acid and lactic acid is used for pretreatment, and at the same time, the bioactive agent sophorolipid is added to improve the extraction rate of collagen. Subsequently, preliminary alkaline hydrolysis and homogenization treatment are carried out, which can preliminarily hydrolyze fish scale proteins, break the hydrogen bonds between collagen molecular chains, and obtain peptide segments of different lengths under the action of hydrolysis. Then, highly active composite bioenzymes are used for enzymolysis to obtain smaller molecular peptide segments. Finally, spray drying is carried out to obtain the final product collagen peptides with high antioxidant activity. The entire preparation process avoids the use of strong acids and high-temperature cooking and other means. The process is mild and pollution-free, with high extraction rate and good product functionality, and has broad market application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of fish collagen peptide preparation, and particularly relates to a preparation method and application of antioxidant fish collagen peptide. Background Art

[0002] Free radicals are groups or atoms with unpaired electrons generated during the body's metabolic process and exposure to the exogenous environment, and they have high chemical reactivity. Excessive free radicals will cause oxidative stress, damage cell structures such as deoxyribonucleic acid, proteins, and lipids, and also participate in lipid oxidation, pigment discoloration, etc., thereby causing various diseases such as inflammation, cancer, neurodegenerative diseases, and cardiovascular diseases, and may also cause spoilage of many foods, producing bad odors and potential toxic reactions. Therefore, effectively controlling the body's oxidative stress response is the key to improving food quality and preventing disease occurrence. Searching for and preparing highly active antioxidants has always been one of the research focuses.

[0003] Antioxidant peptides have the advantages of high activity, stability, safety, absorbability, and rich sources. Some collagen peptides have antioxidant effects. Collagen peptide is the hydrolysis product of collagen, and it is widely used in the fields of food, medicine, chemical industry, etc. due to its low molecular weight, easy absorption, and certain biological activity. Currently, the raw materials for producing collagen products are basically the skins and bones of terrestrial animals such as pigs and cows. Due to reasons such as the mad cow disease panic, the use of collagen and its products is restricted. Finding collagen raw materials other than pig and cow skins and bones has become an urgent matter, and aquatic processing wastes, especially fish skins and fish scales, are ideal alternative raw materials. The preparation of collagen peptides from low-value aquatic resources such as fish scales and fish skins through processes such as high-temperature steaming and controlled hydrolysis has become a conventional method for aquatic collagen peptides. However, most of the existing technologies require repeated treatments with acids and alkalis for swelling, deashing, etc. These treatment methods are mostly cumbersome, with a large amount of enzyme preparations used, complicated preparation, high cost, long reaction cycle, low yield of collagen peptide segments, high ash content, and not a high content of components with a molecular weight lower than 3 kDa. The functionality of the obtained peptides is poor, and the market application prospect is not very good.

[0004] For example, the Chinese patent application with the application number 201611076917.5 discloses a process for preparing antioxidant peptides and antifreeze peptides by enzymatic hydrolysis of salmon collagen. The method uses salmon skin as the raw material to extract collagen, and uses the crude extracellular protease solution obtained by fermenting Vibrio sp. SQS2-3 to hydrolyze it into short peptide solutions. After centrifugal ultrafiltration at 4500×g for 45 min through a 3000 Da ultrafiltration tube, peptides with the ability to protect myosin and slow down its Ca 2+Antifreeze peptides with the activity of -ATPase decreasing during repeated freeze-thaw cycles are used to obtain antioxidant peptides after separating the lower ultrafiltration fraction by Sephadex LH-20 gel filtration chromatography. However, such a processing technology is complex and costly, making it difficult to achieve effective practical application and promotion.

[0005] Therefore, how to optimize the extraction process of fish collagen peptides, expand new processing routes for preparing collagen peptides from fish products, promote the efficient development and utilization of marine resources, and increase the added value of fish collagen peptides are technical problems that need to be solved urgently at present. Summary of the Invention

[0006] In view of the problems existing in the prior art, the present invention extracts two highly active biological proteases from microbial fermentation broth, realizes the efficient hydrolysis of collagen in fish scales at room temperature, and the obtained small-molecule peptides have strong antioxidant activity, enabling the waste fish scales to be resourcefully utilized.

[0007] To achieve the above technical objectives, the technical solution adopted by the present invention is as follows:

[0008] A preparation method of antioxidant fish collagen peptides, comprising the following preparation steps:

[0009] (1) Cleaning: Wash the raw fish scales with clean water to remove the viscous substances, fats and other impurities on the surface of the fish scales, and break the fish scales into fragments of 3-5 mm, and dry them for later use;

[0010] (2) Pretreatment: Immerse the fish scale fragments obtained in step (1) in a weak acid solution at a material-liquid ratio of 1 g:(10-20) mL, and simultaneously perform ultrasonic treatment. After the ultrasonic treatment ends, wash the fish scales with clean water until neutral, then add a sodium chloride solution, soak overnight, and wash until neutral, and dry to obtain pretreated fish scales for later use;

[0011] (3) Alkali treatment: Add the pretreated fish scales to a sodium hydroxide solution with a mass concentration of 30-40% at a solid-liquid ratio of 1 g:20 mL, adjust the pH of the system to 8-9, heat to 90-95 °C, and carry out constant-temperature hydrolysis for 1-2 h. After the hydrolysis is completed, perform homogenization treatment to obtain an alkali hydrolysis solution;

[0012] (4) Enzymatic hydrolysis treatment: Adjust the pH of the alkali hydrolysis solution to 6-7, add an active compound protease accounting for 0.1-0.3% of the mass of the alkali hydrolysis solution, and the enzymatic hydrolysis time is 3-6 h. After the enzymatic hydrolysis ends, heat at 80-90 °C for enzyme inactivation treatment. After cooling to 50-60 °C, filter to remove insoluble residues, and perform centrifugal separation to obtain a crude enzymatic hydrolysis solution;

[0013] (5) Refining and drying treatment: Ultrafilter the obtained crude enzymatic hydrolysis solution with an ultrafiltration membrane, take the permeate, and spray dry to obtain collagen peptide powder.

[0014] Preferably, the fish scales in step (1) are one or more of tilapia scales, grass carp scales or crucian carp scales.

[0015] Preferably, the composition of the weak acid solution in step (2) is: 50 - 60% citric acid, 10 - 20% lactic acid, 1 - 5% sophorolipid, and the balance is water, calculated based on 100% by mass fraction.

[0016] Preferably, the treatment time of the ultrasonic treatment in step (2) is 10 - 20 min, the power is 60 - 80 W, and the treatment temperature is 30 - 40 °C.

[0017] Preferably, the mass concentration of the sodium chloride solution in step (2) is 3 - 5%.

[0018] Preferably, the preparation method of the active complex protease in step (4) is:

[0019] A. Preparation of cell suspension: The cryopreserved Bacillus licheniformis and Saccharomyces paradoxus were activated and then inoculated into a liquid seed medium respectively. After culturing in a shaker at 28 - 30 °C and 160 - 200 r / min for 12 h, seed solutions were obtained. The seed solutions of the two bacteria were mixed at a volume ratio of 1:1 and then inoculated into a fermentation medium at an inoculation amount of 5%. After culturing in a shaker at 28 - 30 °C for 36 h, a fermentation broth was obtained; the fermentation broth was centrifuged at 4 °C and 10,000 r / min for 10 min, and the cell precipitate was collected; the cell precipitate was resuspended with a phosphate buffer solution with a pH of 6.8 and centrifuged again; finally, the cell precipitate was suspended with the phosphate buffer solution again so that the solid - liquid ratio of the cell precipitate to the buffer solution was 1 g:20 mL to prepare a cell suspension;

[0020] B. Extraction of active complex protease solution: Take 20 mL of the cell suspension obtained in step A, and perform fragmentation treatment in an ice bath under the conditions of an ultrasonic power of 400 W, a time of 5 min, an ultrasonic time of 5 s, and a time interval of 5 s (ultrasonic for 5 s, pause for 5 s, then ultrasonic for 5 s, pause for 5 s, and so on for a total of 5 min). The obtained cell lysate was centrifuged at 4 °C and 10,000 r / min for 10 min, and the obtained supernatant was the active complex protease solution.

[0021] More preferably, the preservation number of the Bacillus licheniformis ( Bacillus licheniformis ), purchased from the China General Microbiological Culture Collection Center, was CGMCC No.1.807, and the original preservation time was March 1, 1972; the preservation number of the Saccharomyces paradoxus ( Saccharomyces paradoxus ), purchased from the China General Microbiological Culture Collection Center, was CGMCC No.2.5692, and the original preservation time was May 12, 2016. For the two strains used in the present invention, both can be purchased through the preservation center and there is no need for repeated biological preservation.

[0022] In step A, the activation methods of Bacillus licheniformis and Saccharomyces paradoxus are as follows: inoculate the cryopreserved strains onto LB solid medium and culture at 28 - 30 °C for 12 h.

[0023] In step A, the composition of the liquid seed medium (g / L) is: glucose 100, peptone 5.0, yeast powder 2.0, urea 2.0, MgSO4 0.5, natural pH, and use after autoclaving at 121 °C.

[0024] In step A, the composition of the fermentation medium (g / L) is: glucose 100, peptone 2, yeast powder 1, KH2PO4 2, vegetable oil 10 mL, Tween 80 5 mL, natural pH, and use after autoclaving at 121 °C.

[0025] Preferably, in step (5), the outlet air temperature of spray drying is 70 - 80 °C and the inlet air temperature is 150 - 170 °C.

[0026] An application of a preparation method of antioxidant fish collagen peptides, and the prepared collagen peptides are applied to cosmetics.

[0027] Currently, the common existing technology for preparing collagen peptides is the enzymatic hydrolysis method. The commonly used enzyme types include alkaline protease, neutral protease, trypsin, pepsin, bromelain, papain, etc. The existing technology usually uses commercially available enzyme products, raises the temperature to accelerate enzymatic hydrolysis, and realizes the hydrolysis of collagen to prepare protein peptides. The current defects are: the enzyme performance of commercially available enzyme products is unstable, the activity is not high, the molecular weight of the collagen peptides obtained by enzymatic hydrolysis is relatively large or the distribution is uneven, the functionality is poor, and the actual application value is not high. At the same time, usually heating is required to activate the enzyme activity and accelerate the enzymatic hydrolysis speed, which increases the production cost of enterprises to a certain extent. Therefore, there is an urgent need to optimize and improve the existing enzymatic hydrolysis peptide technology. It can be seen that the selection of protease is the key to improving product quality.

[0028] Therefore, the present invention abandons traditional commercial enzyme products, screens two microbial strains producing biological proteases - Bacillus licheniformis and Saccharomyces paradoxus. After mixing them in equal proportions and fermenting, a variety of proteases with biological activities can be produced. These proteases can adapt to different temperature and pH environments, maintain high enzyme activity, and can achieve efficient hydrolysis of fish scale collagen at room temperature. At the same time, the composite enzyme obtained by mixed fermentation has more cleavage sites in the protein peptide chain, and can achieve deep endonuclease digestion of collagen. The peptide chain is effectively cleaved, and the obtained collagen peptides have a smaller molecular weight and good functionality, especially having good antioxidant activity, and having good scavenging effects on DPPH free radicals, superoxide anion free radicals, hydroxyl free radicals, etc.

[0029] The beneficial effects of the technical solution of the present invention are as follows:

[0030] The present invention optimizes the processing technology for preparing collagen peptides by enzymatic hydrolysis of fish scales. First, a weak acid solution composed of citric acid and lactic acid is used for pretreatment, and at the same time, the bioactive agent sophorolipid is added to effectively promote the dissolution of collagen, thereby improving the extraction rate of collagen. At the same time, under the combined action of the weak acid solution and ultrasound, efficient deashing and decalcification of fish scales can be achieved. Subsequently, preliminary alkaline hydrolysis and homogenization treatment are carried out to preliminarily hydrolyze fish scale proteins, break the hydrogen bonds between collagen molecular chains, and obtain peptide segments of different lengths under hydrolysis. Then, highly active composite bioenzymes are used for enzymatic hydrolysis to obtain smaller molecular peptide segments. Finally, spray drying is carried out to obtain the final product collagen peptides with high antioxidant activity. The entire preparation process avoids the use of strong acids and high-temperature cooking, etc. The process is mild and pollution-free, with a high extraction rate and good product functionality, and has broad market application prospects. Description of the Drawings

[0031] Figure 1 It is a test chart of the total antioxidant activity of the collagen peptides obtained in the examples and comparative examples of the present invention. Detailed Embodiments

[0032] The technical solution of the present invention will be further described below in conjunction with specific embodiments, but not limited thereto.

[0033] Example 1

[0034] A method for preparing antioxidant fish collagen peptides, comprising the following preparation steps:

[0035] (1) Cleaning: The raw material fish scales are washed with clean water to remove the viscous substances, fats and other impurities on the surface of the fish scales, and the fish scales are broken into fragments of 3-5 mm, and dried for later use;

[0036] (2) Pretreatment: The fish scale fragments obtained in step (1) are soaked in a weak acid solution at a material-liquid ratio of 1 g:10 mL, and at the same time, ultrasonic treatment is carried out. After the ultrasonic treatment ends, the fish scales are rinsed with clean water until neutral, then a sodium chloride solution is added, soaked overnight, and then washed until neutral and dried to obtain pretreated fish scales for later use;

[0037] (3) Alkaline treatment: The pretreated fish scales are added to a sodium hydroxide solution with a mass concentration of 30% at a solid-liquid ratio of 1 g:20 mL, the pH of the system is adjusted to 8-9, heated to 90-95 °C, and hydrolyzed at a constant temperature for 1 h. After the hydrolysis is completed, homogenization treatment is carried out to obtain an alkaline hydrolysis solution;

[0038] (4)Enzymatic hydrolysis treatment: Adjust the pH of the alkaline hydrolysis solution to 6 - 7, add active compound protease accounting for 0.1% of the mass of the alkaline hydrolysis solution, and the enzymatic hydrolysis time is 3 h. After the enzymatic hydrolysis is completed, heat at 80 - 90 °C for enzyme inactivation treatment. After cooling to 50 - 60 °C, filter to remove insoluble residues, and perform centrifugal separation to obtain a crude enzymatic hydrolysis solution;

[0039] (5)Refining and drying treatment: Use an ultrafiltration membrane to ultrafilter the obtained crude enzymatic hydrolysis solution, take the permeate, and spray dry to obtain collagen peptide powder.

[0040] The fish scales described in step (1) are grass carp scales.

[0041] The composition of the weak acid solution described in step (2) is: citric acid 50%, lactic acid 10%, sophorolipid 1%, and the balance is water, calculated according to 100% by mass fraction.

[0042] The treatment time of the ultrasonic treatment in step (2) is 10 min, the power is 60 W, and the treatment temperature is 30 - 40 °C.

[0043] The mass concentration of the sodium chloride solution in step (2) is 3%.

[0044] The preparation method of the active compound protease in step (4) is:

[0045] A. Preparation of cell suspension: After activating the cryopreserved Bacillus licheniformis and Saccharomyces paradoxus and inoculating them into the liquid seed medium respectively, cultivate them in a shaker at 28 - 30 °C and 160 r / min for 12 h to obtain seed liquid. After mixing the seed liquids of the two bacteria in a volume ratio of 1:1, inoculate them into the fermentation medium at an inoculation amount of 5% and cultivate them in a shaker at 28 - 30 °C for 36 h to obtain a fermentation broth; Centrifuge the fermentation broth at 4 °C and 10,000 r / min for 10 min, and collect the cell precipitate; Resuspend the cell precipitate with a phosphate buffer solution with a pH of 6.8 and centrifuge again; Finally, resuspend the cell precipitate with the phosphate buffer solution so that the solid-liquid ratio of the cell precipitate to the buffer solution is 1 g:20 mL to prepare a cell suspension;

[0046] B. Extraction of active compound protease solution: Take 20 mL of the cell suspension obtained in step A, perform fragmentation treatment in an ice bath under the conditions of an ultrasonic power of 400 W, a time of 5 min, an ultrasonic time of 5 s, and a time interval of 5 s. The obtained cell fragmentation solution is centrifuged at 4 °C and 10,000 r / min for 10 min, and the obtained supernatant is the active compound protease solution.

[0047] The Bacillus licheniformis ( Bacillus licheniformis ), the preservation number is CGMCC No. 1.807, purchased from the China General Microbiological Culture Collection Center, and the original preservation time is March 1, 1972; The Saccharomyces paradoxus ( Saccharomyces paradoxusThe preservation number of () is CGMCC No. 2.5692. It was purchased from the China General Microbiological Culture Collection Center, and the original preservation time was May 12, 2016. Both of the two strains used in the present invention can be purchased openly through the preservation center, and there is no need for repeated biological preservation.

[0048] In step A, the activation method of Bacillus licheniformis and Saccharomyces paradoxus is as follows: The cryopreserved strains are inoculated on an LB solid medium and cultured at 28 - 30 °C for 12 h.

[0049] In step A, the composition of the liquid seed medium is (g / L): glucose 100, peptone 5.0, yeast powder 2.0, urea 2.0, MgSO4 0.5, natural pH, and it is used after sterilization at 121 °C.

[0050] In step A, the composition of the fermentation medium is (g / L): glucose 100, peptone 2, yeast powder 1, KH2PO4 2, vegetable oil 10 mL, Tween 80 5 mL, natural pH, and it is used after sterilization at 121 °C.

[0051] In step (5), the outlet air temperature of spray drying is 70 - 80 °C, and the inlet air temperature is 150 - 170 °C.

[0052] Example 2

[0053] A preparation method of antioxidant fish collagen peptide comprises the following preparation steps:

[0054] (1) Cleaning: The raw material fish scales are cleaned with clear water to remove the viscous substances, fats and other impurities on the surface of the fish scales, and the fish scales are broken into fragments of 3 - 5 mm, and then dried for later use;

[0055] (2) Pretreatment: The fish scale fragments obtained in step (1) are soaked in a weak acid solution at a material-liquid ratio of 1 g:15 mL, and ultrasonic treatment is carried out simultaneously. After the ultrasonic treatment ends, the fish scales are rinsed with clear water until neutral, then sodium chloride solution is added, soaked overnight, and then rinsed until neutral and dried to obtain pretreated fish scales for later use;

[0056] (3) Alkali treatment: The pretreated fish scales are added to a sodium hydroxide solution with a mass concentration of 35% at a solid-liquid ratio of 1 g:20 mL, the pH of the system is adjusted to 8 - 9, heated to 90 - 95 °C, and hydrolyzed at a constant temperature for 1.5 h. After the hydrolysis is completed, homogenization treatment is carried out to obtain an alkali hydrolysis solution;

[0057] (4) Enzymatic hydrolysis treatment: The pH of the alkali hydrolysis solution is adjusted to 6 - 7, active compound protease accounting for 0.2% of the mass of the alkali hydrolysis solution is added, the enzymatic hydrolysis time is 5 h. After the enzymatic hydrolysis ends, heat treatment is carried out at 80 - 90 °C to inactivate the enzyme, cooled to 50 - 60 °C, and then filtered to remove insoluble residues, and centrifuged to obtain a crude enzymatic hydrolysis solution;

[0058] (5) Refining and drying treatment: The obtained crude enzymatic hydrolysate is ultrafiltered using an ultrafiltration membrane, the permeate is taken, and spray drying is performed to obtain collagen peptide powder.

[0059] The fish scales described in step (1) are carp scales.

[0060] The composition of the weak acid solution described in step (2) is: 50% citric acid, 15% lactic acid, 3% sophorolipid, and the balance is water, calculated as 100% by mass fraction.

[0061] The treatment time of the ultrasonic treatment in step (2) is 15 min, the power is 70 W, and the treatment temperature is 30 - 40 °C.

[0062] The mass concentration of the sodium chloride solution in step (2) is 4%.

[0063] The preparation method of the active complex protease in step (4) is as follows:

[0064] A. Preparation of cell suspension: The cryopreserved Bacillus licheniformis and Saccharomyces paradoxus are activated and then respectively inoculated into a liquid seed medium, cultured in a shaker at 28 - 30 °C and 200 r / min for 12 h to obtain seed solutions. The seed solutions of the two strains are mixed at a volume ratio of 1:1, and then inoculated into a fermentation medium at an inoculation amount of 5%, and cultured in a shaker at 28 - 30 °C for 36 h to obtain a fermentation broth; the fermentation broth is centrifuged at 4 °C and 10,000 r / min for 10 min, and the cell precipitate is collected; the cell pellet is resuspended with a phosphate buffer solution with a pH of 6.8 and centrifuged again; finally, the cell pellet is resuspended with the phosphate buffer solution to make the solid-liquid ratio of the cell pellet to the buffer solution 1 g:20 mL to prepare a cell suspension;

[0065] B. Extraction of active complex protease solution: Take 20 mL of the cell suspension obtained in step A, and perform fragmentation treatment in an ice bath under the conditions of an ultrasonic power of 400 W, a time of 5 min, an ultrasonic time of 5 s, and a time interval of 5 s. The obtained cell lysate is centrifuged at 4 °C and 10,000 r / min for 10 min, and the obtained supernatant is the active complex protease solution.

[0066] The Bacillus licheniformis ( Bacillus licheniformis ) has a preservation number of CGMCC No. 1.807, is purchased from the China General Microbiological Culture Collection Center, and the original preservation time is March 1, 1972; the Saccharomyces paradoxus ( Saccharomyces paradoxus ) has a preservation number of CGMCC No. 2.5692, is purchased from the China General Microbiological Culture Collection Center, and the original preservation time is May 12, 2016. The two strains used in the present invention can be openly purchased through the preservation center, and no repeated biological preservation is required.

[0067] In step A, the activation method of Bacillus licheniformis and Saccharomyces paradoxus is as follows: inoculate the cryopreserved strains on an LB solid medium and culture at 28 - 30 °C for 12 h.

[0068] In step A, the composition of the liquid seed medium (g / L) is: glucose 100, peptone 5.0, yeast extract 2.0, urea 2.0, MgSO4 0.5, natural pH, sterilize at 121 °C before use.

[0069] In step A, the composition of the fermentation medium (g / L) is: glucose 100, peptone 2, yeast extract 1, KH2PO4 2, vegetable oil 10 mL, Tween 80 5 mL, natural pH, sterilize at 121 °C before use.

[0070] In step (5), the outlet temperature of spray drying is 70 - 80 °C and the inlet temperature is 150 - 170 °C.

[0071] Example 3

[0072] A preparation method of antioxidant fish collagen peptide, comprising the following preparation steps:

[0073] (1) Cleaning: Wash the raw material fish scales with clean water to remove the viscous substances, fats and other impurities on the surface of the fish scales, and break the fish scales into fragments of 3 - 5 mm, dry and reserve for later use;

[0074] (2) Pretreatment: Immerse the fish scale fragments obtained in step (1) in a weak acid solution at a material - liquid ratio of 1 g:20 mL, and simultaneously perform ultrasonic treatment. After the ultrasonic treatment ends, rinse the fish scales with clean water until neutral, then add a sodium chloride solution, soak overnight, and wash until neutral, dry to obtain pretreated fish scales for later use;

[0075] (3) Alkali treatment: Add the pretreated fish scales to a sodium hydroxide solution with a mass concentration of 40% at a solid - liquid ratio of 1 g:20 mL, adjust the pH of the system to 8 - 9, heat to 90 - 95 °C, carry out constant - temperature hydrolysis for 2 h, and homogenize the hydrolyzed product after hydrolysis to obtain an alkali - hydrolyzed solution;

[0076] (4) Enzymatic hydrolysis treatment: Adjust the pH of the alkali - hydrolyzed solution to 6 - 7, add an active compound protease accounting for 0.3% of the mass of the alkali - hydrolyzed solution, carry out enzymatic hydrolysis for 6 h. After the enzymatic hydrolysis ends, heat at 80 - 90 °C for enzyme inactivation treatment, cool to 50 - 60 °C, filter to remove insoluble residues, and carry out centrifugal separation to obtain a crude enzymatic hydrolysis solution;

[0077] (5) Refining and drying treatment: Ultrafilter the obtained crude enzymatic hydrolysis solution with an ultrafiltration membrane, take the permeate, and spray - dry to obtain collagen peptide powder.

[0078] The fish scales mentioned in step (1) are tilapia fish scales.

[0079] The composition of the weak acid solution described in step (2) is: 60% citric acid, 20% lactic acid, 5% sophorolipid, and the balance is water, calculated as 100% by mass fraction.

[0080] In step (2), the treatment time of ultrasonic treatment is 20 min, the power is 80 W, and the treatment temperature is 30 - 40 °C.

[0081] In step (2), the mass concentration of the sodium chloride solution is 5%.

[0082] The preparation method of the active compound protease in step (4) is as follows:

[0083] A. Preparation of cell suspension: The cryopreserved Bacillus licheniformis and Saccharomyces paradoxus were activated and then inoculated into a liquid seed medium respectively. After culturing in a shaker at 28 - 30 °C and 200 r / min for 12 h, seed solutions were obtained. The seed solutions of the two bacteria were mixed at a volume ratio of 1:1 and then inoculated into a fermentation medium at an inoculation amount of 5%. After culturing in a shaker at 28 - 30 °C for 36 h, a fermentation broth was obtained; the fermentation broth was centrifuged at 4 °C and 10,000 r / min for 10 min to collect the cell precipitate; the cell precipitate was resuspended with a phosphate buffer solution with a pH of 6.8 and centrifuged again; finally, the cell precipitate was suspended with the phosphate buffer solution to make the solid-liquid ratio of the cell precipitate to the buffer solution 1 g:20 mL to obtain the cell suspension.

[0084] B. Extraction of active compound protease solution: Take 20 mL of the cell suspension obtained in step A, and perform fragmentation treatment in an ice bath under the conditions of an ultrasonic power of 400 W, a time of 5 min, an ultrasonic time of 5 s, and a time interval of 5 s. The obtained cell lysate was centrifuged at 4 °C and 10,000 r / min for 10 min, and the obtained supernatant is the active compound protease solution.

[0085] The preservation number of the Bacillus licheniformis ( Bacillus licheniformis ) is CGMCC No. 1.807, purchased from the China General Microbiological Culture Collection Center, and the original preservation time is March 1, 1972; the preservation number of the Saccharomyces paradoxus ( Saccharomyces paradoxus ) is CGMCC No. 2.5692, purchased from the China General Microbiological Culture Collection Center, and the original preservation time is May 12, 2016. For the two strains used in the present invention, both can be purchased openly through the preservation center, and there is no need for repeated biological preservation.

[0086] In step A, the activation method of Bacillus licheniformis and Saccharomyces paradoxus is: inoculate the cryopreserved strain on an LB solid medium and culture at 28 - 30 °C for 12 h.

[0087] In step A, the liquid seed medium is composed of (g / L): glucose 100, peptone 5.0, yeast powder 2.0, urea 2.0, MgSO4 0.5, with natural pH, and it is used after sterilization at 121°C.

[0088] In step A, the fermentation medium is composed of (g / L): glucose 100, peptone 2, yeast powder 1, KH2PO4 2, vegetable oil 10 mL, Tween 80 5 mL, with natural pH, and it is used after sterilization at 121°C.

[0089] In step (5), the outlet air temperature for spray drying is 70 - 80°C and the inlet air temperature is 110 - 170°C.

[0090] Comparative Example 1

[0091] The method for preparing collagen peptide in this comparative example is the same as that in Example 3 in terms of the remaining raw materials and preparation methods, except that sophorolipid is not added in the weak acid solution. That is:

[0092] The weak acid solution described in step (2) is composed of: citric acid 60%, lactic acid 20%, and the balance is water, calculated as 100% by mass fraction.

[0093] Comparative Example 2

[0094] The method for preparing collagen peptide in this comparative example is the same as that in Example 3 in terms of the remaining raw materials and preparation methods, except that only Bacillus licheniformis is used in the preparation method of the active compound protease.

[0095] The preparation method of the active compound protease in step (4) is as follows:

[0096] A. Prepare cell suspension: Activate the cryopreserved Bacillus licheniformis and inoculate it into the liquid seed medium. Incubate it in a shaker at 28 - 30°C and 200 r / min for 12 h to obtain the seed liquid. Inoculate the seed liquid into the fermentation medium at an inoculation amount of 5% and incubate it in a shaker at 28 - 30°C for 36 h to obtain the fermentation broth; Centrifuge the fermentation broth at 4°C and 10000 r / min for 10 min to collect the cell precipitate; Resuspend the cell precipitate with phosphate buffer solution with a pH of 6.8 and centrifuge again; Finally, resuspend the cell precipitate with phosphate buffer solution to make the solid-liquid ratio of the cell precipitate to the buffer solution 1 g:20 mL to obtain the cell suspension;

[0097] B. Extract the active compound protease solution: Take 20 mL of the cell suspension obtained in step A and perform fragmentation treatment in an ice bath under the conditions of ultrasonic power of 400 W, time of 5 min, ultrasonic time of 5 s, and time interval of 5 s. Centrifuge the obtained cell fragmentation liquid at 4°C and 10000 r / min for 10 min, and the obtained supernatant is the active compound protease solution.

[0098] Comparative Example 3

[0099] The method for preparing collagen peptides in this comparative example is the same as that in Example 3 for the rest of the raw materials and preparation methods, except that in the preparation method of the active compound protease, only Saccharomyces paradoxus is used.

[0100] The preparation method of the active compound protease in step (4) is as follows:

[0101] A. Preparation of cell suspension: The cryopreserved Saccharomyces paradoxus is activated and inoculated into a liquid seed medium, and cultured in a shaker at 28 - 30 °C and 200 r / min for 12 h to obtain a seed solution. The seed solution is inoculated into a fermentation medium at an inoculation amount of 5% and cultured in a shaker at 28 - 30 °C for 36 h to obtain a fermentation broth. The fermentation broth is centrifuged at 4 °C and 10,000 r / min for 10 min to collect the cell precipitate. The cell precipitate is resuspended with a phosphate buffer solution with a pH of 6.8 and centrifuged again. Finally, the cell precipitate is resuspended with the phosphate buffer solution to make the solid-liquid ratio of the cell precipitate to the buffer solution 1 g:20 mL to obtain the cell suspension.

[0102] B. Extraction of active compound protease solution: Take 20 mL of the cell suspension obtained in step A, and perform fragmentation treatment in an ice bath under the conditions of an ultrasonic power of 400 W, a time of 5 min, an ultrasonic time of 5 s, and a time interval of 5 s. The obtained cell fragmentation solution is centrifuged at 4 °C and 10,000 r / min for 10 min, and the obtained supernatant is the active compound protease solution.

[0103] Comparative Example 4

[0104] The method for preparing collagen peptides in this comparative example is the same as that in Example 3 for the rest of the raw materials and preparation methods, except that in the preparation method of the active compound protease, the dosage ratio of Bacillus licheniformis and Saccharomyces paradoxus is changed.

[0105] The preparation method of the active compound protease in step (4) is as follows:

[0106] A. Preparation of cell suspension: The cryopreserved Bacillus licheniformis and Saccharomyces paradoxus are activated and inoculated into a liquid seed medium respectively, and cultured in a shaker at 28 - 30 °C and 200 r / min for 12 h to obtain seed solutions. The seed solutions of the two bacteria are mixed at a volume ratio of 2:1 and then inoculated into a fermentation medium at an inoculation amount of 5% and cultured in a shaker at 28 - 30 °C for 36 h to obtain a fermentation broth. The fermentation broth is centrifuged at 4 °C and 10,000 r / min for 10 min to collect the cell precipitate. The cell precipitate is resuspended with a phosphate buffer solution with a pH of 6.8 and centrifuged again. Finally, the cell precipitate is resuspended with the phosphate buffer solution to make the solid-liquid ratio of the cell precipitate to the buffer solution 1 g:20 mL to obtain the cell suspension.

[0107] B. Extraction of active complex protease solution: Take 20 mL of the cell suspension obtained in step A, and perform fragmentation treatment in an ice bath under the conditions of an ultrasonic power of 400 W, a time of 5 min, an ultrasonic time of 5 s, and a time interval of 5 s. The obtained cell lysate is centrifuged at 4°C and 10,000 r / min for 10 min, and the supernatant is the active complex protease solution.

[0108] Comparative Example 5

[0109] In the method for preparing collagen peptides in this comparative example, except for changing the dosage ratio of Bacillus licheniformis and Saccharomyces paradoxus in the preparation method of the active complex protease, the other raw materials and preparation methods are the same as those in Example 3.

[0110] The preparation method of the active complex protease in step (4) is as follows:

[0111] A. Preparation of cell suspension: Activate the cryopreserved Bacillus licheniformis and Saccharomyces paradoxus and inoculate them into the liquid seed medium respectively. After culturing in a shaker at 28 - 30°C and 200 r / min for 12 h, seed solutions are obtained. After mixing the seed solutions of the two bacteria in a volume ratio of 1:2, inoculate them into the fermentation medium at an inoculation amount of 5% and culture in a shaker at 28 - 30°C for 36 h to obtain a fermentation broth; centrifuge the fermentation broth at 4°C and 10,000 r / min for 10 min to collect the cell precipitate; resuspend the cell precipitate with a phosphate buffer solution with a pH of 6.8 and centrifuge again; finally, suspend the cell precipitate with the phosphate buffer solution to make the solid-liquid ratio of the cell precipitate to the buffer solution 1 g:20 mL to obtain the cell suspension.

[0112] B. Extraction of active complex protease solution: Take 20 mL of the cell suspension obtained in step A, and perform fragmentation treatment in an ice bath under the conditions of an ultrasonic power of 400 W, a time of 5 min, an ultrasonic time of 5 s, and a time interval of 5 s. The obtained cell lysate is centrifuged at 4°C and 10,000 r / min for 10 min, and the supernatant is the active complex protease solution.

[0113] Performance test

[0114] The test method is as follows:

[0115] The molecular weight distribution is carried out according to the method in Appendix A of the "National Standard of the People's Republic of China GB / T 22729-2008", the detection method of hydroxyproline is carried out according to the "National Standard of the People's Republic of China GB / T 9695.23-2008", the detection methods of ash content, etc. are carried out according to the "National Standard of the People's Republic of China GB / T 5009.4-2016", and the determination method of total nitrogen content refers to GB 5009.5—2016 "Determination of Protein in Foods".

[0116] Determination of degree of protein hydrolysis

[0117] Degree of hydrolysis of protein (%) = (Content of free amino acids in 1 g of hydrolyzed fish scale solution / Total nitrogen content in 1 g of fish scales) × 100%

[0118] The determination method of free amino nitrogen content refers to the ninhydrin method.

[0119] Table 1 Performance test results

[0120]

[0121] Determination of total antioxidant capacity:

[0122] Reference method: Lu Xin, Hu Dongbin, Jia Cong, et al., Analysis of antioxidant properties of polypeptides produced by in vitro simulated digestion of sesame protein, China Oils and Fats, 2020, 45(05): 63-66+81.

[0123] Take the collagen peptide products obtained in Examples 1-3 and Comparative Examples 1-5, prepare them into a 2 mg / mL solution, take 50 μL of the hydrolyzed solution and add it to a 96-well plate, add 150 μL of FRAP working solution (a mixture of 0.3 mol / L acetate buffer at pH 3.6, 10 mmol / L TPTZ, and 20 mmol / L FeCl3 solution in a volume ratio of 10:1:1), shake for 30 s, react in the dark at 37 °C for 30 min, and measure the absorbance at a wavelength of 593 nm. Another standard curve was made with a 0-2 mmol / L FeSO4 standard solution instead of the sample. The total antioxidant capacity of the sample is expressed as the concentration of FeSO4 with the same absorbance value, and the unit is mmol / g. The test results are as Figure 1 shown.

[0124] Determination of hydroxyl radical scavenging ability:

[0125] Take the collagen peptide products obtained in Examples 1-3 and Comparative Examples 1-5, prepare them into a solution, and detect according to the instructions of the DPPH free radical kit (Nanjing Jiancheng Bioengineering Institute), with glutathione as the positive control. The DPPH free radical scavenging rate is calculated according to the formula:

[0126] DPPH free radical scavenging rate / % = [1 - × 100%;

[0127] wherein, A 空 is the absorbance of the white tube, A 样 is the absorbance of the measurement tube, and A 对 is the absorbance of the control tube.

[0128] O 2‒ · scavenging activity determination: Take the collagen peptide products obtained in Examples 1-3 and Comparative Examples 1-5, prepare them into a solution, and according to O 2‒· Test according to the kit instructions. Use glutathione as the positive control, O 2‒ · The clearance rate is calculated according to the formula:

[0129] O 2‒ · Clearance rate / % = × 100%

[0130] Wherein, A 标 is the absorbance of the standard tube, A 样 is the absorbance of the measurement tube, and A 对 is the absorbance of the control tube.

[0131] · OH scavenging activity determination: Take the collagen peptide products obtained in Examples 1-3 and Comparative Examples 1-5, prepare them into solutions, and determine according to the · OH kit instructions. Use glutathione as the positive control. The · OH clearance rate is calculated according to the formula:

[0132] · OH clearance rate / % = × 100%

[0133] Wherein, A 标 is the absorbance of the standard tube, A 样 is the absorbance of the measurement tube, A 对 is the absorbance of the control tube, and A 空 is the absorbance of the white tube.

[0134] Table 2 Free radical scavenging rate

[0135]

[0136] From the data in Table 1-2, we can see that the collagen peptides obtained in Examples 1-3 of the present invention have higher quality, a higher proportion of small molecular weight peptides, and also show better DPPH free radicals, O 2‒ ·, · OH scavenging rates, and have good antioxidant activity. For Comparative Example 1 without sophorolipid, the pretreatment effect of fish skin decreased, resulting in the weakening of the physical and chemical properties of the obtained product. For Comparative Examples 2-5 with different strain compositions, the balance of synergistic effects between the two was broken, the types and activities of enzymes produced by fermentation changed, resulting in a decrease in the enzymatic hydrolysis effect on fish skin. Therefore, using two strains to prepare an active composite enzyme to enzymatically hydrolyze fish skin to prepare high-activity collagen peptide is a key technical means and is indispensable.

[0137] It should be noted that the above-mentioned embodiments are only some embodiments of the preferred implementation modes of the present invention, not all embodiments. Obviously, based on the above embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

Claims

1. A preparation method of antioxidant fish collagen peptide, characterized in that It includes the following preparation steps: (1) Cleaning: The raw material fish scales are cleaned with clear water to remove the viscous substances, fats and other impurities on the surface of the fish scales, and the fish scales are broken into fragments of 3-5 mm, and dried for later use; (2) Pretreatment: The fish scale fragments obtained in step (1) are soaked in a weak acid solution at a material-liquid ratio of 1 g:(10-20) mL, and ultrasonic treatment is carried out at the same time. After the ultrasonic treatment ends, the fish scales are rinsed with clear water until neutral, then sodium chloride solution is added, soaked overnight, washed until neutral, and dried to obtain pretreated fish scales for later use; (3) Alkali treatment: The pretreated fish scales are added to a sodium hydroxide solution with a mass concentration of 30-40% according to a solid-liquid ratio of 1 g:20 mL, the pH of the system is adjusted to 8-9, heated to 90-95 °C, and hydrolyzed at a constant temperature for 1-2 h. After the hydrolysis is completed, homogenization treatment is carried out to obtain an alkali hydrolysis solution; (4) Enzymatic hydrolysis treatment: The pH of the alkali hydrolysis solution is adjusted to 6-7, and an active compound protease accounting for 0.1-0.3% of the mass of the alkali hydrolysis solution is added. The enzymatic hydrolysis time is 3-6 h. After the enzymatic hydrolysis ends, heat treatment is carried out at 80-90 °C to inactivate the enzyme. After cooling to 50-60 °C, insoluble residues are removed by filtration, and centrifugal separation is carried out to obtain a crude enzymatic hydrolysis solution; (5) Refining and drying treatment: The obtained crude enzymatic hydrolysis solution is ultrafiltered by an ultrafiltration membrane, the permeate is taken, and spray drying is carried out to obtain collagen peptide powder; The composition of the weak acid solution described in step (2) is: citric acid 50-60%, lactic acid 10-20%, sophorolipid 1-5%, and the balance is water, calculated according to 100% by mass fraction; The preparation method of the active compound protease in step (4) is: A. Preparation of cell suspension: The cryopreserved Bacillus licheniformis and Saccharomyces paradoxus are activated and then respectively inoculated into a liquid seed culture medium, and cultured in a shaker at 28-30 °C and 160-200 r / min for 12 h to obtain seed liquid. After mixing the seed liquids of the two bacteria according to a volume ratio of 1:1, inoculate them into the fermentation culture medium according to an inoculation amount of 5%, and culture in a shaker at 28-30 °C for 36 h to obtain a fermentation broth; The fermentation broth is centrifuged at 4 °C and 10,000 r / min for 10 min, and the cell precipitate is collected; The cell precipitate is resuspended with a phosphate buffer solution with a pH of 6.8, and centrifuged again; Finally, the cell precipitate is suspended with a phosphate buffer solution again so that the solid-liquid ratio of the cell precipitate to the buffer solution is 1 g:20 mL to prepare a cell suspension; B. Extraction of compound protease solution: Take 20 mL of the cell suspension obtained in step A, and carry out fragmentation treatment in an ice bath under the conditions of an ultrasonic power of 400 W, a time of 5 min, an ultrasonic time of 5 s, and a time interval of 5 s. The obtained cell fragmentation solution is centrifuged at 4 °C and 10,000 r / min for 10 min, and the supernatant obtained is the compound protease solution; The Bacillus licheniformis ( Bacillus licheniformis ) has a deposit number of CGMCC No. 1.807 and is purchased from the China General Microbiological Culture Collection Center; the Saccharomyces paradoxus ( Saccharomyces paradoxus ) has a deposit number of CGMCC No. 2.5692 and is purchased from the China General Microbiological Culture Collection Center.

2. The preparation method of the antioxidant fish collagen peptide according to claim 1, characterized in that, The fish scales described in step (1) are one or more of tilapia scales, grass carp scales or crucian carp scales.

3. The preparation method of the antioxidant fish collagen peptide according to claim 1, characterized in that, The preparation method of the antioxidant fish collagen peptide according to claim 1 is characterized in that, in step (2), the treatment time of ultrasonic treatment is 10 - 20 min, the power is 60 - 80 W, and the treatment temperature is 30 - 40 °C.

4. The preparation method of the antioxidant fish collagen peptide according to claim 1, characterized in that In step (2), the mass concentration of the sodium chloride solution is 3 - 5%.

5. The preparation method of the antioxidant fish collagen peptide according to claim 1, characterized in that The preparation method of the antioxidant fish collagen peptide according to claim 1 is characterized in that, in step (5), the outlet air temperature of spray drying is 70 - 80 °C, and the inlet air temperature is 150 - 170 °C.

6. Use of the method for preparing the antioxidant fish collagen peptide according to any one of claims 1-5, characterized in that, The prepared collagen peptide is applied to cosmetics.

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