A process for preparing fish collagen tripeptide and its application

Abstract

The present invention discloses a process method for preparing fish collagen tripeptide and its application, belonging to the technical field of collagen peptide preparation technology. The present invention performs pretreatment and preprocessing on raw fish skin. Subsequently, the preprocessed fish skin is subjected to two enzymatic hydrolysis treatments to achieve efficient hydrolysis of fish skin collagen and obtain a collagen peptide product with a small molecular weight. First, neutral protease and papain are used to preliminarily hydrolyze the fish skin. Subsequently, a microbial ferment and an activity promoter are used for deep fermentation enzymatic hydrolysis treatment, which can effectively further cleave peptide segments, improve the yield of small peptide segments, and enhance biological activity. The method of this application is simple, the process is easy to control, there is no environmental pollution, and the enzymatic hydrolysis time is short, which can improve the enzymatic hydrolysis speed and the extraction rate of collagen peptides. The collagen peptides obtained by this method have a low molecular weight and good biological activity, and are more easily promoted and applied.

Description

Technical Field

[0001] The present invention belongs to the technical field of collagen peptide preparation processes, and particularly relates to a process method for preparing fish collagen tripeptide and its application. Background Art

[0002] Collagen, or collagen, is a biological macromolecule synthesized by animal cells and is widely present in animal bones, tendons, cartilage, skin and other connective tissues, and has the functions of supporting organs and protecting the body. With the development of collagen extraction technology, the in-depth research on its properties, structure, etc., and the improvement of the understanding of the biological functions and other properties of collagen and collagen polypeptides, the research and application of collagen have become a hot topic. However, due to the unique triple helix structure of collagen, its properties are very stable, and general processing temperatures and short-term heating cannot decompose it, resulting in difficult digestion and absorption and being not easily fully utilized by the human body. Hydrolyzing collagen into collagen polypeptides will significantly improve its digestion and absorption, nutrition, functional characteristics, etc.

[0003] For a long time, people have used the skins and bones of pigs and cows to extract collagen. However, due to the outbreaks of diseases such as mad cow disease and foot-and-mouth disease, the application of collagen from terrestrial animals has been greatly restricted. Marine animals are rich in collagen and have good biological safety, with great development potential.

[0004] Chinese Patent Application Publication No.: CN102217699A, which discloses a preparation method of squid skin protein active peptide. The squid skin is washed and cut into pieces, soaked in an NaOH aqueous solution for 2 - 8 days for decolorization, and then washed with water until the pH is neutral; soaked in a Ca(OH)2 solution for 2 - 4 days for color fixation, and then washed with water until the pH is neutral; treated in pH-neutral water at 55 - 75 °C for 0.5 - 1.5 h and filtered to remove insoluble proteins to obtain fish skin protein solution; finally, the solution is treated under high pressure at 110 - 121 °C for 30 - 120 min to obtain squid skin protein active peptide. Its deficiencies are as follows. Firstly, the invention needs to soak the squid skin in an NaOH aqueous solution for 2 - 8 days for decolorization and then soak it in a Ca(OH)2 solution for 2 - 4 days for color fixation, with a long preparation period. Secondly, the invention uses the high-pressure treatment method to treat squid skin protein to prepare bioactive peptides, with strict preparation conditions, large molecular weight of the product, and poor actual application effects.

[0005] Chinese Patent Application Publication No.: CN 101870995A discloses a process for preparing collagen tripeptide, which includes subjecting fish skin to primary alkali pretreatment, primary boiling denaturation treatment, and stepwise compound hydrolysis with alkaline protease, pancreatin, and compound protease. Among them, after boiling denaturation, residues should be filtered off with gauze, and the hydrolyzate obtained by stepwise hydrolysis with three enzymes can be made into collagen tripeptide with high protein content and good functional activity through inactivation, filtration, debittering, decolorization, vacuum concentration, and spray drying.

[0006] However, the problems and disadvantages of the current existing technologies are as follows: The enzymatic hydrolysis efficiency of the enzymes used is low, the enzymatic hydrolysis time is long, the enzyme addition amount is large, it is necessary to adjust the optimal pH value and optimal temperature for enzymatic hydrolysis, the operation is complex, adjusting the optimal enzymatic hydrolysis pH value will introduce a large amount of inorganic ions, and the commercial enzyme has poor enzyme activity, incomplete enzymatic hydrolysis, the product has a large molecular weight, poor activity, and single function, and is not easily absorbed by the skin. Summary of the Invention

[0007] In view of the problems existing in the prior art, the present invention provides a new process method for fish collagen tripeptide, and the prepared tripeptide has good application effects in the field of repairing cell photo-damage.

[0008] To achieve the above technical objectives, the technical solution adopted by the present invention is as follows:

[0009] T A process method for preparing fish collagen tripeptide, including the following preparation steps:

[0010] (1) Pretreatment: Scraping the remaining meat, fin spines, and scales from the fish skin, washing it with deionized water, and drying it at a low temperature of 25 - 30°C; after drying, cutting the fish skin into small pieces of 1 cm × 1 cm, sealing and storing it in a refrigerator at (-20) - (-10)°C for later use;

[0011] (2) Preprocessing: Adding the fish skin obtained in step (1) to a NaOH solution with a mass concentration of 0.5 - 1% and soaking it for 20 - 30 min to remove non-collagen components; then soaking the fish skin in isopropanol for 3 - 6 h to remove fat; then soaking it in an ethylenediaminetetraacetic acid solution for 20 - 30 min to remove mineral ions, and finally washing it with water until the pH is neutral and draining it;

[0012] (3) Primary enzymatic hydrolysis treatment: Mixing the fish skin obtained in step (2) with water at a mass ratio of 1:20, swelling and homogenizing it to obtain a crude slurry; adding compound protease to the crude slurry for primary enzymatic hydrolysis treatment to obtain a primary hydrolyzate;

[0013] (4) Deep fermentation enzymatic hydrolysis treatment: Add a microbial fermenting agent accounting for 1% of the mass of the crude slurry and an activity promoter accounting for 0.1% to the primary enzymatic hydrolysis solution for deep fermentation enzymatic hydrolysis. The fermentation temperature is 25 - 30 °C, and the fermentation time is 24 - 36 h. After the treatment is completed, perform enzyme inactivation treatment under high temperature and high pressure. After the entire reaction system cools down, centrifuge for 15 - 30 min, remove the precipitate, and take the supernatant;

[0014] (5) Filtration and concentration treatment: Ultrafilter the supernatant through an ultrafiltration membrane with a molecular cut-off not greater than 2000 Da, collect the filtrate. The permeate is a collagen tripeptide solution. After desalination and concentration, perform vacuum freeze-drying to obtain the final product of fish collagen tripeptide.

[0015] Further, in step (1), the fish skin is cod fish skin and / or tilapia fish skin.

[0016] Further, in step (2), the solid-liquid ratio of the fish skin to the NaOH solution is 1 g : 20 - 40 mL; the solid-liquid ratio of the fish skin to the isopropanol is 1 g : 30 - 50 mL; the solid-liquid ratio of the fish skin to the ethylenediaminetetraacetic acid solution is 1 g : 20 - 40 mL, and the concentration of the ethylenediaminetetraacetic acid solution is 0.1 - 0.3 mol / L.

[0017] Further, in step (3), the addition amount of the compound protease is 3 - 5% of the mass of the crude slurry, the enzymatic hydrolysis time is 1 - 2 h, and the enzymatic hydrolysis temperature is 50 - 60 °C; the compound protease is neutral protease and papain, and the mass ratio of the two is 1:0.5.

[0018] Further, the preparation method of the microbial fermenting agent in step (4) is as follows: Activate the Pseudomonas guangdongensis with strain number CGMCC 1.15627 and Aspergillus sydowii with strain number CCTCC CF 2008800 respectively, and then inoculate them into the corn soy flour medium and LB liquid medium. The inoculation amount is 3% for both. Cultivate under the conditions of 25 - 30 °C and a shaker speed of 150 - 220 rpm for 10 - 16 hours. After the cultivation ends, centrifuge to collect the bacterial cells, and then add sterile water to adjust the bacterial concentration to 1×10 8 -1×10 9 CFU / mL to obtain the culture solutions of Pseudomonas guangdongensis and Aspergillus sydowii. Then mix the two culture solutions in equal volumes to obtain the microbial fermenting agent.

[0019] Even further, the Pseudomonas guangdongensis with strain number CGMCC 1.15627 is purchased from the China General Microbiological Culture Collection Center. The original preservation date is February 25, 2015. This strain can be purchased openly through the preservation center without the need for repeated biological preservation.

[0020] Furthermore, the strain Aspergillus sydowii CCTCC CF 2008800 was purchased from the China Center for Type Culture Collection. The original preservation date was November 10, 2004. This strain can also be purchased openly from the preservation center without the need for repeated biological preservation.

[0021] Furthermore, the activation methods for Pseudomonas guanacastensis and Aspergillus sydowii are as follows: The target strains stored at low temperature are cultured on an LB slant medium at 25 - 30 °C for 10 - 16 hours. The weight ratio of each component of the LB slant medium is: tryptone 8 - 12 g, yeast extract 4 - 6 g, sodium chloride 4 - 6 g, agar powder 12 - 18 g, and water 1000 mL to obtain the activated strains.

[0022] Furthermore, the composition of the corn - soybean meal medium is: corn flour 1.0 - 3.0%, soybean meal 1.0 - 3.0%, NaCl 0.1 - 1.0%, MnSO4·H2O 0.5 - 1.0%, and the rest is water.

[0023] Further, the activity promoter in step (4) is obtained by mixing potassium chloride, vitamin C, and trehalose in a mass ratio of 1:0.1:0.5.

[0024] Further, the conditions for the high - temperature and high - pressure enzyme inactivation treatment in step (5) are: temperature 110 - 130 °C, pressure 0.1 - 0.3 MPa, and time 10 - 20 min.

[0025] The collagen tripeptide produced by the method of the present application has a small molecular weight and high activity, and can play a role in repairing cell photo - damage and can be used in cosmetics.

[0026] All raw materials used in the present application are commercially available.

[0027] Beneficial effects:

[0028] (1) First, the raw material fish skin is pretreated and pre - processed to effectively remove the impurities of the raw material and improve the purity of the raw material. The treatment with NaOH solution can effectively remove miscellaneous proteins such as non - collagen components. The treatment with the organic solvent isopropanol plays a role in degreasing and removing miscellaneous proteins and pigments. And finally, the treatment with ethylenediaminetetraacetic acid solution forms stable, dissolved and mobile complexes with metal ions through chelation or coordination reactions, thereby removing metal ions in the fish skin.

[0029] (2) Subsequently, the pre-treated fish skin is subjected to two enzymatic hydrolysis treatments to achieve efficient hydrolysis of fish skin collagen and obtain a collagen peptide product with a small molecular weight. First, neutral protease and papain are used to preliminarily hydrolyze the fish skin to obtain fish skin collagen peptides. The simultaneous use of neutral protease and papain can improve the hydrolysis efficiency and the yield of collagen peptides, effectively cleave the protein chain, and thus produce effectively cleaved peptide segments;

[0030] (3) Subsequently, a microbial fermenting agent and an activity promoter are used for deep fermentation and enzymatic hydrolysis treatments. When Pseudomonas guangdongensis and Aspergillus sydowii are mixed in equal proportions and fermented, extracellular proteases with high activity can be produced to further cleave the peptide segments after preliminary enzymatic hydrolysis, enhancing the yield of small molecular peptide segments and the biological activity; while adding an activity promoter composed of potassium chloride, vitamin C, and trehalose, potassium ions can effectively activate and enhance the protease activity, and vitamin C and trehalose promote the production of extracellular proteases by regulating the sugar metabolism pathway during the fermentation metabolism of Pseudomonas guangdongensis, enhancing the yield and activity of extracellular proteases. The two-step enzymatic hydrolysis treatment effectively improves the extraction rate of collagen peptides and the yield of low molecular weight peptides. The obtained peptides have high biological activity and have high application prospects in repairing cell photo-damage;

[0031] (4) In summary, the method of this application is simple, the process is easy to control, there is no environmental pollution, and the enzymatic hydrolysis time is short. It can improve the enzymatic hydrolysis speed and the extraction rate of collagen peptides. The collagen peptides obtained by this method have no peculiar smell, uniform color, and good biological properties, and are more likely to be promoted and applied. Description of the Drawings

[0032] Figure 1 Experimental results of the effects of samples from different experimental groups on the COL-I content of HSF cells;

[0033] Figure 2 Experimental results of the effects of samples from different experimental groups on the ELN content of HSF cells;

[0034] Figure 3 Experimental results of the effects of samples from different experimental groups on the HA content of HSF cells;

[0035] Figure 4 Experimental results of the effects of samples from different experimental groups on the total antioxidant capacity of HSF cells;

[0036] Figure 5 HE staining images of the skin of mice in different experimental groups (100X);

[0037] Figure 6 Physical photo of the product obtained in Example 1. Detailed Description of the Invention

[0038] The technical solution of the present invention will be further described below in conjunction with specific embodiments, but it is not limited thereto.

[0039] Example 1

[0040] A process for preparing fish collagen tripeptide includes the following preparation steps:

[0041] (1) Pretreatment: Scrape off the remaining meat, fin spines and scales from the fish skin, wash it with deionized water, and dry it at a low temperature of 25 - 30 °C; after drying, cut the fish skin into small pieces of 1 cm × 1 cm, seal it and store it in a refrigerator at (-20) - (-10) °C for later use;

[0042] (2) Pre - treatment: Immerse the fish skin obtained in step (1) in a NaOH solution with a mass concentration of 0.5% for 30 min to remove non - collagen components; then immerse the fish skin in isopropanol for 3 h to remove fat; then immerse it in an ethylenediaminetetraacetic acid solution for 20 min to remove mineral ions, and finally wash it with water until the pH is neutral and drain it;

[0043] (3) Primary enzymatic hydrolysis treatment: Mix the fish skin obtained in step (2) with water at a mass ratio of 1:20, and after swelling and homogenization, obtain a crude slurry; add a compound protease to the crude slurry for primary enzymatic hydrolysis treatment to obtain a primary enzymatic hydrolysate;

[0044] (4) Deep - fermentation enzymatic hydrolysis treatment: Add a microbial fermenting agent accounting for 1% of the mass of the crude slurry and an activity promoter of 0.1% to the primary enzymatic hydrolysate for deep - fermentation enzymatic hydrolysis. The fermentation temperature is 25 - 30 °C, and the fermentation time is 24 h. After the treatment is completed, inactivate the enzyme by high - temperature and high - pressure treatment. After the whole reaction system cools down, centrifuge for 15 min, remove the precipitate and take the supernatant;

[0045] (5) Filtration and concentration treatment: Ultrafilter the supernatant through an ultrafiltration membrane with a cut - off molecular weight not greater than 2000 Da, collect the filtrate. The permeate is a collagen tripeptide solution. After desalination and concentration, perform vacuum freeze - drying to obtain the final product, fish collagen tripeptide.

[0046] In step (1), the fish skin is cod fish skin.

[0047] In step (2), the solid - liquid ratio of the fish skin to the NaOH solution is 1 g:40 mL; the solid - liquid ratio of the fish skin to isopropanol is 1 g:50 mL; the solid - liquid ratio of the fish skin to the ethylenediaminetetraacetic acid solution is 1 g:20 mL, and the concentration of the ethylenediaminetetraacetic acid solution is 0.1 mol / L.

[0048] In step (3), the addition amount of the compound protease is 3% of the mass of the crude slurry, the enzymatic hydrolysis time is 1 h, and the enzymatic hydrolysis temperature is 50 - 60 °C; the compound protease is neutral protease and papain, and the mass ratio of the two is 1:0.5.

[0049] The preparation method of the microbial ferment in step (4) is as follows: The Pseudomonas guangdongensis with strain number CGMCC 1.15627 and the Aspergillus sydowii with strain number CCTCC CF 2008800 are respectively activated and then inoculated into a corn soy flour medium and an LB liquid medium, with an inoculation amount of 3% for both. They are cultured for 10 - 16 hours under the conditions of 25 - 30°C and a shaking table speed of 150 - 220 rpm. After the cultivation ends, the cells are collected by centrifugation, and then sterile water is added to adjust the cell concentration to 1×10 8 -1×10 9 cells / mL to obtain the culture solutions of Pseudomonas guangdongensis and Aspergillus sydowii. Then, the two culture solutions are mixed in equal volumes to obtain the microbial ferment.

[0050] The Pseudomonas guangdongensis with strain number CGMCC 1.15627 is purchased from the China General Microbiological Culture Collection Center. The original preservation date is February 25, 2015. This strain can be purchased through the opening of the preservation center and does not require repeated biological preservation.

[0051] The Aspergillus sydowii with strain number CCTCC CF 2008800 is purchased from the China Center for Type Culture Collection. The original preservation date is November 10, 2004. This strain can be purchased through the opening of the preservation center and does not require repeated biological preservation.

[0052] The activation methods of Pseudomonas guangdongensis and Aspergillus sydowii are as follows: The target strains stored at low temperature are cultured on an LB slant medium at 25 - 30°C for 10 - 16 hours. The weight ratio of each component of the LB slant medium is: tryptone 8 - 12 g, yeast extract 4 - 6 g, sodium chloride 4 - 6 g, agar powder 12 - 18 g, and water 1000 mL to obtain the activated strains.

[0053] The composition of the corn soy flour medium is: corn flour 1.0 - 3.0%, soy flour 1.0 - 3.0%, NaCl 0.1 - 1.0%, MnSO4·H2O 0.5 - 1.0%, and the rest is water.

[0054] The activity promoter in step (4) is obtained by mixing potassium chloride, vitamin C, and trehalose in a mass ratio of 1:0.1:0.5.

[0055] The conditions for the high-temperature and high-pressure enzyme inactivation treatment in step (5) are: temperature 110°C, pressure 0.1 MPa, and time 20 min.

[0056] Example 2

[0057] A process for preparing fish collagen tripeptides includes the following preparation steps:

[0058] (1) Pretreatment: Scrape off the remaining meat, fin spines, and scales from the fish skin, wash it with deionized water, and dry it at a low temperature of 25 - 30°C; after drying, cut the fish skin into small pieces of 1 cm × 1 cm, seal it, and store it in a refrigerator at (-20) - (-10)°C for later use;

[0059] (2) Preconditioning: Immerse the fish skin obtained in step (1) in a NaOH solution with a mass concentration of 0.7% for 30 min to remove non - collagen components; then soak the fish skin in isopropanol for 5 h to remove fat; then soak it in an ethylenediaminetetraacetic acid solution for 30 min to remove mineral ions, and finally wash it with water until the pH is neutral and drain it;

[0060] (3) Primary enzymatic hydrolysis treatment: Mix the fish skin obtained in step (2) with water at a mass ratio of 1:20, and after swelling and homogenization, obtain a crude slurry; add a complex protease to the crude slurry for primary enzymatic hydrolysis treatment to obtain a primary enzymatic hydrolysate;

[0061] (4) Deep - fermentation enzymatic hydrolysis treatment: Add a microbial fermenting agent accounting for 1% of the mass of the crude slurry and an activity promoter of 0.1% to the primary enzymatic hydrolysate for deep - fermentation enzymatic hydrolysis. The fermentation temperature is 25 - 30°C, and the fermentation time is 30 h. After the treatment is completed, perform high - temperature and high - pressure enzyme inactivation treatment. After the entire reaction system cools down, centrifuge for 30 min, remove the precipitate, and take the supernatant;

[0062] (5) Filtration and concentration treatment: Ultrafilter the supernatant through an ultrafiltration membrane with a molecular cut - off not greater than 2000 Da, collect the filtrate. The permeate is a collagen tripeptide solution. After desalination and concentration, perform vacuum freeze - drying to obtain the final product, fish collagen tripeptide.

[0063] The fish skin in step (1) is tilapia fish skin.

[0064] In step (2), the solid - to - liquid ratio of the fish skin to the NaOH solution is 1 g:30 mL; the solid - to - liquid ratio of the fish skin to isopropanol is 1 g:40 mL; the solid - to - liquid ratio of the fish skin to the ethylenediaminetetraacetic acid solution is 1 g:30 mL, and the concentration of the ethylenediaminetetraacetic acid solution is 0.2 mol / L.

[0065] In step (3), the addition amount of the complex protease is 4% of the mass of the crude slurry, the enzymatic hydrolysis time is 1 h, and the enzymatic hydrolysis temperature is 50 - 60°C; the complex protease is neutral protease and papain, and the mass ratio of the two is 1:0.5.

[0066] The preparation method of the microbial ferment in step (4) is as follows: The Pseudomonas guangaii with strain number CGMCC 1.15627 and the Aspergillus sydowii with strain number CCTCC CF 2008800 are respectively activated and then inoculated into a corn soybean meal medium and an LB liquid medium, with an inoculation amount of 3% for both. They are cultured for 10 - 16 hours under the conditions of 25 - 30 °C and a shaker speed of 150 - 220 rpm. After the cultivation ends, the cells are collected by centrifugation, and then sterile water is added to adjust the cell concentration to 1×10 8 -1×10 9 CFU / mL to obtain the culture solutions of Pseudomonas guangaii and Aspergillus sydowii. Then, the two culture solutions are mixed in equal volume to obtain the microbial ferment.

[0067] The Pseudomonas guangaii with strain number CGMCC 1.15627 is purchased from the China General Microbiological Culture Collection Center. The original preservation date is February 25, 2015. This strain can be purchased openly through the preservation center without the need for repeated biological preservation.

[0068] The Aspergillus sydowii with strain number CCTCC CF 2008800 is purchased from the China Center for Type Culture Collection. The original preservation date is November 10, 2004. This strain can be purchased openly through the preservation center without the need for repeated biological preservation.

[0069] The activation methods of Pseudomonas guangaii and Aspergillus sydowii are as follows: The target strains stored at low temperature are cultured on an LB slant medium at 25 - 30 °C for 10 - 16 hours. The weight ratio of each component of the LB slant medium is: tryptone 8 - 12 g, yeast extract 4 - 6 g, sodium chloride 4 - 6 g, agar powder 12 - 18 g, and water 1000 mL to obtain the activated strains.

[0070] The composition of the corn soybean meal medium is: corn flour 1.0 - 3.0%, soybean meal 1.0 - 3.0%, NaCl 0.1 - 1.0%, MnSO4·H2O 0.5 - 1.0%, and the rest is water.

[0071] The activity promoter in step (4) is obtained by mixing potassium chloride, vitamin C, and trehalose in a mass ratio of 1:0.1:0.5.

[0072] The conditions for the high-temperature and high-pressure enzyme inactivation treatment in step (5) are: temperature 130 °C, pressure 0.3 MPa, and time 15 min.

[0073] Example 3

[0074] A process for preparing fish collagen tripeptide includes the following preparation steps:

[0075] (1) Pretreatment: Scrape off the remaining meat, fin spines and scales from the fish skin, wash it with deionized water, and dry it at a low temperature of 25 - 30°C; after drying, cut the fish skin into small pieces of 1 cm × 1 cm, seal it and store it in a refrigerator at (-20) - (-10)°C for later use;

[0076] (2) Preprocessing: Immerse the fish skin obtained in step (1) in a NaOH solution with a mass concentration of 1% for 30 min to remove non - collagen components; then soak the fish skin in isopropanol for 6 h to remove fat; then soak it in an ethylenediaminetetraacetic acid solution for 30 min to remove mineral ions, and finally wash it with water until the pH is neutral and drain it;

[0077] (3) Primary enzymatic hydrolysis treatment: Mix the fish skin obtained in step (2) with water at a mass ratio of 1:20, and after swelling and homogenization, obtain a crude slurry; add a compound protease to the crude slurry for primary enzymatic hydrolysis treatment to obtain a primary enzymatic hydrolysate;

[0078] (4) Deep fermentation enzymatic hydrolysis treatment: Add a microbial fermenting agent accounting for 1% of the mass of the crude slurry and a 0.1% activity promoter to the primary enzymatic hydrolysate for deep fermentation enzymatic hydrolysis. The fermentation temperature is 25 - 30°C, and the fermentation time is 36 h. After the treatment is completed, perform high - temperature and high - pressure enzyme inactivation treatment. After the whole reaction system cools down, centrifuge for 30 min, remove the precipitate and take the supernatant;

[0079] (5) Filtration and concentration treatment: Ultrafilter the supernatant through an ultrafiltration membrane with a molecular cut - off not greater than 2000 Da, collect the filtrate. The permeate is a collagen tripeptide solution. After desalination and concentration, perform vacuum freeze - drying to obtain the final product of fish collagen tripeptide.

[0080] The fish skin in step (1) is cod fish skin.

[0081] In step (2), the solid - to - liquid ratio of the fish skin to the NaOH solution is 1 g:20 mL; the solid - to - liquid ratio of the fish skin to isopropanol is 1 g:30 mL; the solid - to - liquid ratio of the fish skin to the ethylenediaminetetraacetic acid solution is 1 g:40 mL, and the concentration of the ethylenediaminetetraacetic acid solution is 0.3 mol / L.

[0082] In step (3), the addition amount of the compound protease is 5% of the mass of the crude slurry, the enzymatic hydrolysis time is 2 h, and the enzymatic hydrolysis temperature is 50 - 60°C; the compound protease is neutral protease and papain, and the mass ratio of the two is 1:0.5.

[0083] The preparation method of the microbial ferment in step (4) is as follows: The Pseudomonas gunnii with strain number CGMCC 1.15627 and the Aspergillus sydowii with strain number CCTCC CF 2008800 are respectively activated and then inoculated into a corn soy flour medium and an LB liquid medium, with an inoculation amount of 3% for both. They are cultured for 10 - 16 hours under the conditions of 25 - 30°C and a shaker speed of 150 - 220 rpm. After the cultivation ends, the cells are collected by centrifugation, and then sterile water is added to adjust the cell concentration to 1×10 8 -1×10 9 CFU / mL to obtain the culture solutions of Pseudomonas gunnii and Aspergillus sydowii. Then, the two culture solutions are mixed in equal volumes to obtain the microbial ferment.

[0084] The Pseudomonas gunnii with strain number CGMCC 1.15627 was purchased from the China General Microbiological Culture Collection Center. The original preservation date was February 25, 2015. This strain can be purchased openly through the preservation center and does not require repeated biological preservation.

[0085] The Aspergillus sydowii with strain number CCTCC CF 2008800 was purchased from the China Center for Type Culture Collection. The original preservation date was November 10, 2004. This strain can be purchased openly through the preservation center and does not require repeated biological preservation.

[0086] The activation methods for Pseudomonas gunnii and Aspergillus sydowii are as follows: The target strains stored at low temperature are cultured on an LB slant medium at 25 - 30°C for 10 - 16 hours. The weight ratio of each component of the LB slant medium is: 8 - 12 g of tryptone, 4 - 6 g of yeast extract, 4 - 6 g of sodium chloride, 12 - 18 g of agar powder, and 1000 mL of water to obtain the activated strains.

[0087] The composition of the corn soy flour medium is: 1.0 - 3.0% corn flour, 1.0 - 3.0% soy flour, 0.1 - 1.0% NaCl, 0.5 - 1.0% MnSO4·H2O, and the rest is water.

[0088] The activity promoter in step (4) is obtained by mixing potassium chloride, vitamin C, and trehalose in a mass ratio of 1:0.1:0.5.

[0089] The conditions for the high-temperature and high-pressure enzyme inactivation treatment in step (5) are: temperature 130°C, pressure 0.3 MPa, and time 20 min.

[0090] Comparative Example 1

[0091] A process for preparing fish collagen tripeptide includes the following preparation steps:

[0092] (1) Pretreatment: Scrape off the remaining meat, fin spines and scales from the fish skin, wash it with deionized water, and dry it at a low temperature of 25 - 30 °C; after drying, cut the fish skin into small pieces of 1 cm × 1 cm, seal it and store it in a refrigerator at (-20) - (-10) °C for later use;

[0093] (2) Preliminary treatment: Immerse the fish skin obtained in step (1) in a NaOH solution with a mass concentration of 0.5% for 30 min to remove non-collagen components; then immerse the fish skin in isopropanol for 3 h to remove fat; then immerse it in an ethylenediaminetetraacetic acid solution for 20 min to remove mineral ions, and finally wash it with water until the pH is neutral and drain it;

[0094] (3) Primary enzymatic hydrolysis treatment: Mix the fish skin obtained in step (2) with water at a mass ratio of 1:20, and after swelling and homogenization, obtain a crude slurry; add a compound protease and an activity promoter accounting for 0.1% of the mass of the crude slurry to the crude slurry for primary enzymatic hydrolysis treatment to obtain a primary enzymatic hydrolysate; after the treatment is completed, perform enzyme inactivation treatment under high temperature and high pressure. After the entire reaction system cools down, centrifuge for 15 min, remove the precipitate and take the supernatant;

[0095] (4) Filtration and concentration treatment: Ultrafilter the supernatant through an ultrafiltration membrane with a molecular cut-off not greater than 2000 Da, collect the filtrate, the permeate is a collagen tripeptide solution, after desalting and concentration, perform vacuum freeze-drying to obtain the final product of fish collagen tripeptide.

[0096] In step (3), the addition amount of the compound protease is 4% of the mass of the crude slurry, the enzymatic hydrolysis time is 25 h, and the enzymatic hydrolysis temperature is 50 - 60 °C; the compound protease is neutral protease and papain, and the mass ratio of the two is 1:0.5.

[0097] This comparative example is compared with Example 1. Except for not performing deep fermentation enzymatic hydrolysis treatment, the other raw materials and process steps are the same as those in Example 1.

[0098] Comparative Example 2

[0099] A process for preparing fish collagen tripeptide includes the following preparation steps:

[0100] (1) Pretreatment: Scrape off the remaining meat, fin spines and scales from the fish skin, wash it with deionized water, and dry it at a low temperature of 25 - 30 °C; after drying, cut the fish skin into small pieces of 1 cm × 1 cm, seal it and store it in a refrigerator at (-20) - (-10) °C for later use;

[0101] (2) Preliminary treatment: Immerse the fish skin obtained in step (1) in a NaOH solution with a mass concentration of 0.5% for 30 min to remove non-collagen components; then immerse the fish skin in isopropanol for 3 h to remove fat; then immerse it in an ethylenediaminetetraacetic acid solution for 20 min to remove mineral ions, and finally wash it with water until the pH is neutral and drain it;

[0102] (3) Enzymatic hydrolysis treatment: Mix the fish skin obtained in step (2) with water at a mass ratio of 1:20. After swelling and homogenization, a crude slurry is obtained. Add a microbial fermenting agent accounting for 4% of its mass and an activity promoter of 0.1% to the crude slurry for deep fermentation. The fermentation temperature is 25 - 30 °C, and the fermentation time is 25 h. After the treatment is completed, perform enzyme inactivation treatment under high temperature and high pressure. After the entire reaction system cools down, centrifuge for 15 min, remove the precipitate, and take the supernatant;

[0103] (4) Filtration and concentration treatment: Ultrafilter the supernatant through an ultrafiltration membrane with a molecular cut-off not greater than 2000 Da, collect the filtrate. The permeate is a collagen tripeptide solution. After desalting and concentration, perform vacuum freeze-drying to obtain the final product, fish collagen tripeptide.

[0104] This comparative example is compared with Example 1. Except for not performing preliminary enzymatic hydrolysis treatment, the other raw materials and process steps are the same as those in Example 1.

[0105] Comparative Example 3

[0106] A process method for preparing fish collagen tripeptide includes the following preparation steps:

[0107] (1) Pretreatment: Scrape off the remaining meat, fin spines, and scales from the fish skin, wash it with deionized water, and dry it at a low temperature of 25 - 30 °C; after drying, cut the fish skin into small pieces of 1 cm × 1 cm, seal it, and store it in a refrigerator at (-20) - (-10) °C for later use;

[0108] (2) Pretreatment: Immerse the fish skin obtained in step (1) in a NaOH solution with a mass concentration of 0.5% for 30 min to remove non-collagen components; then immerse the fish skin in isopropanol for 3 h to remove fat; then immerse it in an ethylenediaminetetraacetic acid solution for 20 min to remove mineral ions, and finally wash it with water until the pH is neutral and drain it;

[0109] (3) Preliminary enzymatic hydrolysis treatment: Mix the fish skin obtained in step (2) with water at a mass ratio of 1:20. After swelling and homogenization, a crude slurry is obtained; add compound protease to the crude slurry for preliminary enzymatic hydrolysis treatment to obtain a preliminary enzymatic hydrolysate;

[0110] (4) Deep fermentation enzymatic hydrolysis treatment: Add a microbial fermenting agent accounting for 1% of the mass of the crude slurry and an activity promoter of 0.1% to the preliminary enzymatic hydrolysate for deep fermentation. The fermentation temperature is 25 - 30 °C, and the fermentation time is 24 h. After the treatment is completed, perform enzyme inactivation treatment under high temperature and high pressure. After the entire reaction system cools down, centrifuge for 15 min, remove the precipitate, and take the supernatant;

[0111] Filtration and concentration treatment: The supernatant is ultrafiltered through an ultrafiltration membrane with a molecular cut-off not greater than 2000 Da, and the filtrate is collected. The permeate is a collagen tripeptide solution, which is desalted and concentrated, and then vacuum freeze-dried to obtain the final product of fish collagen tripeptide.

[0112] The preparation method of the microbial ferment used in step (4) is as follows: The Pseudomonas gunnii with the strain number CGMCC 1.15627 is activated and then inoculated into the corn soy flour medium, and the inoculation amount is 3% in both cases. It is cultured for 10 - 16 hours under the conditions of 25 - 30 °C and a shaking speed of 150 - 220 rpm. After the cultivation is completed, the cells are collected by centrifugation, and then sterile water is added to adjust the cell concentration to 1×10 8 -1×10 9 cells / mL to obtain the microbial ferment.

[0113] This comparative example is compared with Example 1. Except that in step (4), the microbial ferment is not prepared using Aspergillus sydowii with the strain number CCTCC CF2008800, the remaining raw materials and process steps are the same as those in Example 1.

[0114] Comparative Example 4

[0115] This comparative example is compared with Example 1. Except that in step (4), the microbial ferment is not prepared using Pseudomonas gunnii with the strain number CGMCC 1.15627, the remaining raw materials and process steps are the same as those in Example 1.

[0116] Comparative Example 5

[0117] This comparative example is compared with Example 1. Except that in the preparation of the microbial ferment in step (4), the volume ratio of Pseudomonas gunnii with the strain number CGMCC 1.15627 and Aspergillus sydowii with the strain number CCTCC CF 2008800 is changed, the remaining raw materials and process steps are the same as those in Example 1.

[0118] That is: The preparation method of the microbial ferment described in step (4) is as follows: The Pseudomonas gunnii with the strain number CGMCC 1.15627 and the Aspergillus sydowii with the strain number CCTCC CF 2008800 are respectively activated and then inoculated into the corn soy flour medium and the LB liquid medium, and the inoculation amount is 3% in both cases. It is cultured for 10 - 16 hours under the conditions of 25 - 30 °C and a shaking speed of 150 - 220 rpm. After the cultivation is completed, the cells are collected by centrifugation, and then sterile water is added to adjust the cell concentration to 1×10 8 -1×10 9 cells / mL to obtain the culture solutions of Pseudomonas gunnii and Aspergillus sydowii. Then, the two culture solutions are mixed according to a volume ratio of 1:2 to obtain the microbial ferment.

[0119] Comparative Example 6

[0120] In this comparative example, compared with Example 1, except that in the preparation of the microbial fermenting agent in step (4), the volume ratio of Pseudomonas guangaii with strain number CGMCC 1.15627 and Aspergillus sydowii with strain number CCTCC CF 2008800 was changed, the other raw materials and process steps were the same as those in Example 1.

[0121] That is, the preparation method of the microbial fermenting agent described in step (4) is as follows: Pseudomonas guangaii with strain number CGMCC 1.15627 and Aspergillus sydowii with strain number CCTCC CF 2008800 were separately activated and then inoculated into a corn soya bean powder medium and an LB liquid medium, and the inoculation amount was 3% for both. They were cultured for 10 - 16 hours under the conditions of 25 - 30 °C and a shaking speed of 150 - 220 rpm. After the cultivation ended, the bacterial cells were collected by centrifugation, and then sterile water was added to adjust the bacterial concentration to 1×10 8 -1×10 9 CFU / mL to obtain the culture broths of Pseudomonas guangaii and Aspergillus sydowii. Then, the two culture broths were mixed according to a volume ratio of 2:1 to obtain the microbial fermenting agent.

[0122] Performance Test

[0123] According to the methods of the example and the comparative example, the preparation of fish collagen tripeptide was carried out, and the performance of the obtained products was detected. The detection methods are as follows:

[0124] For the detection of molecular weight distribution, the method in Appendix A of "National Standard of the People's Republic of China GB / T22729 - 2008" was used; for the detection method of hydroxyproline, it was carried out with reference to "National Standard of the People's Republic of China GB / T9695.23 - 2008", for the total nitrogen detection method, it was carried out with reference to "National Standard of the People's Republic of China GB / T5009.5 - 2016", and for the ash detection method, it was carried out with reference to "National Standard of the People's Republic of China GB / T5009.4 - 2016". All experimental results were repeated 5 times, and the results were averaged. The results are shown in Table 1 below.

[0125] Table 1 Performance Test Results

[0126]

[0127] Table 2 Performance Test Results

[0128]

[0129] From the data in Table 1-2, we can see that the final product obtained in the embodiments of the present invention has a low molecular weight and a high proportion of small molecular weights. This is because in the two-step enzymatic hydrolysis of the present invention, the first-step enzymatic hydrolysis realizes the first cleavage of collagen, and subsequently, the active protease produced by microbial fermentation is used to realize the deep cleavage of the peptides obtained from the primary enzymatic hydrolysis, promoting the complete enzymatic hydrolysis of proteins and significantly reducing the proportion of small molecular weight peptides. In Comparative Examples 1-2 where the enzymatic hydrolysis process was changed, a single enzymatic hydrolysis method was difficult to achieve effective cleavage of peptides, and the enzymatic hydrolysis was incomplete, resulting in an increase in the proportion of high molecular weight peptides. Subsequently, the biological activity of the product was further tested. The test method is as follows: The ability of the collagen peptide samples obtained in Examples 1-3 and Comparative Examples 1-6 to repair photo-damage was tested.

[0130] Cell culture:

[0131] HSF cells were cultured in DMEM medium containing 10% fetal bovine serum, 1% penicillin (1×10 5 U / L) and streptomycin (100 mg / L), and cultured in an incubator at an environmental temperature of 37 °C, a carbon dioxide content of 5%, and saturated humidity. When the cell confluence reached 80%, the medium was replaced, and the cells were digested with 0.25% trypsin (diluted with EDTA) for subculture.

[0132] Establishment of UVA-induced photo-damage model:

[0133] 1×10 4 cells in the logarithmic growth phase were seeded in a 96-well plate and cultured for 24 h. When the cell confluence reached 80%, the medium was replaced with PBS (pH 7.4, 0.01 mol / L), and the cells were stimulated with UVA at a dose of 20 J / cm 2 Then, the medium was replaced with the basal medium, and the cell viability was measured after 24 h of culture. IC 50 was selected to establish the UVA damage model.

[0134] Determination of the contents of HA, COL-I, ELN and total antioxidant capacity:

[0135] The contents of HA, ELN, and COL-I in the cells were detected by enzyme-linked immunosorbent assay (ELISA), and their total antioxidant capacity was determined by the ABTS method. HSF cells in the logarithmic growth phase were taken, and 2×10 5Cells were seeded at 100 μg / well in 6-well cell culture plates. When cell confluence reached 80%, the medium was replaced with PBS. After UVA irradiation, the PBS was discarded and treated with 2 mL of serum-free DMEM, 3% aqueous collagen peptide solution for the experimental and control groups, and 50 μg / mL VC solution. These served as the model, experimental, and VC-positive control groups, respectively. A blank control group was also established. Cell samples were collected according to the instructions for HA, ELN, and COL-I enzyme-linked immunosorbent assay kits and the total antioxidant capacity assay kit. HA, ELN, and COL-I content, as well as total antioxidant capacity, were determined in each group. After cell collection, protein content was determined in each group. The measured content of each indicator was then divided by the corresponding protein content to normalize the cell weight and eliminate the influence of sample-induced cell proliferation on the indicators. All experiments were repeated five times, and the results were averaged.

[0136] Table 3 Verification of light damage repair capability

[0137]

[0138] From the data in Tables 1-3, we can see that: compared with the blank control group, the content of each indicator in the model group was extremely significantly reduced, showing the characteristic indicators of light-damaged cells. Compared with the model group, the COL-I, ELN, HA content and TAOC in the VC positive control group were extremely significantly improved, indicating that the model was successfully established. From the results in Table 2, it can be seen that compared with the model group, the embodiment can extremely significantly improve the relevant indicators. At the same time, compared with VC, although the total antioxidant capacity of the embodiment group failed to exceed the VC positive control group, it was very close to the antioxidant level of VC. It shows the excellent performance of the fish collagen peptide of the present invention in repairing cell light damage.

[0139] Taking Example 1 as an example, the performance of the protein peptides in Example 1 and Comparative Examples 1-6 in repairing cell light damage was tested. The test method was the same as above. The experimental results are as follows: Figures 1-4 As shown, from Figures 1-4 We can see that the product of Example 1 of the present invention significantly increases the content of HA, COL-I and ELN in the cells in the model, and plays a positive role in repairing UVA-induced skin light damage. At the same time, the TAOC of the model cells is significantly improved, which may be attributed to the effective relief of intracellular oxidative stress by small molecule collagen peptides, thereby protecting the cells from oxidative damage. However, the contents of various indicators of Comparative Examples 1-6 are not as good as those of Example 1. This is because the enzymatic hydrolysis effect of Comparative Examples 1-2, which changed the enzymatic hydrolysis process, was weakened, while the synergistic effect of the two bacteria decreased in Comparative Examples 3-6, which changed the composition of the microbial fermentation agent, and the deep enzymatic hydrolysis effect was weakened, resulting in a decrease in the content of small molecule peptides and biological activity of the final product. In order to further verify the effectiveness of the example products, mouse experiments were conducted again.

[0140] The experimental method for mice is as follows:

[0141] The experimental animals were female Kunming mice (4 weeks old). All animals were randomly divided into 3 groups and kept in a quiet environment with appropriate temperature and humidity. The specific gavage conditions are shown in Table 4. The normal group and the model group were respectively gavaged with an equal amount of normal saline, and the gavage time was 30 minutes before each irradiation.

[0142] Table 4 Animal gavage conditions

[0143]

[0144] Model establishment method for the model group:

[0145] Four UVA tubes and two UVB tubes were arranged side by side as the ultraviolet irradiation light source. The mice were depilated (the mice needed to be depilated before each irradiation). Except for the normal group, from the 1st week to the 6th week, the dorsal skin of the mice was irradiated with UV (UVA + UVB) 3 times a week, and the irradiation height was 30 cm. The intensity was measured with a UVR irradiometer to determine the minimum erythema (MED) dose. The irradiation dose was 0.5 MED / time in the 1st week, 1.0 MED / time in the 2nd week, and increased by 0.5 MED / time per week until 3.0 MED / time in the 6th week.

[0146] Observation of skin tissue sections:

[0147] After the mice were sacrificed by cervical dislocation, appropriate skin tissues of the mice were immersed in 4% paraformaldehyde for fixation, and then embedded in paraffin for section preparation. Then the sections were stained with HE respectively, and the stained skin tissues were observed under a microscope at an appropriate magnification. The results are as Figure 5 shown. It can be seen from Figure 5 that compared with the normal group, the epidermis of the mice in the model group (MG group) was significantly abnormally thickened, and the arrangement was irregular, and the keratinization phenomenon was obvious. At the same time, the basal layer cells in the MG group showed vacuolar degeneration and inflammatory infiltration. And compared with the MG group, the epidermis thickness of the mice in Example 1 group decreased significantly, and the epidermal structure of the mice was closer to the normal group.

[0148] It should be noted that the above-mentioned embodiments are only some embodiments of the preferred ways to implement the present invention, rather than 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 process for preparing fish collagen tripeptide, characterized in that, It includes the following preparation steps: (1) Pretreatment: Scrape off the remaining meat, fin spines and scales from the fish skin, wash it with deionized water, and dry it at a low temperature of 25 - 30°C; after drying, cut the fish skin into small pieces of 1 cm × 1 cm, seal and store it in a refrigerator at (-20) - (-10)°C for later use; (2) Preconditioning: Immerse the fish skin obtained in step (1) in a NaOH solution with a mass concentration of 0.5 - 1% for 20 - 30 min; then immerse the fish skin in isopropanol for 3 - 6 h; then immerse it in an ethylenediaminetetraacetic acid solution for 20 - 30 min, and finally wash it with water until the pH is neutral and drain it; (3) Primary enzymatic hydrolysis treatment: Mix the fish skin obtained in step (2) with water at a mass ratio of 1:20, and after swelling and homogenization, obtain a crude slurry; add a compound protease to the crude slurry for primary enzymatic hydrolysis treatment to obtain a primary enzymatic hydrolysate; (4) Deep fermentation enzymatic hydrolysis treatment: Add a microbial fermenting agent accounting for 1% of the mass of the crude slurry and an activity promoter of 0.1% to the primary enzymatic hydrolysate for deep fermentation enzymatic hydrolysis. The fermentation temperature is 25 - 30°C, and the fermentation time is 24 - 36 h. After the treatment is completed, perform high-temperature and high-pressure enzyme inactivation treatment. After the entire reaction system cools down, centrifuge for 15 - 30 min, remove the precipitate and take the supernatant; (5) Filtration and concentration treatment: Ultrafilter the supernatant through an ultrafiltration membrane with a molecular cut-off not greater than 2000 Da, collect the filtrate. The permeate is a collagen tripeptide solution. After desalination and concentration, perform vacuum freeze-drying to obtain the final product, fish collagen tripeptide; In step (3), the addition amount of the compound protease is 3-5% of the mass of the crude slurry, the enzymolysis time is 1-2 h, and the enzymolysis temperature is 50-60 °C; the compound protease is neutral protease and papain, and the mass ratio of the two is 1:0.5; the preparation method of the microbial fermenting agent in step (4) is as follows: The Pseudomonas guangguensis with the strain number CGMCC 1.15627 and the Aspergillus sydowii with the strain number CCTCC CF 2008800 are respectively activated and then inoculated into a corn soybean meal medium and an LB liquid medium, and the inoculation amount is 3% for both. They are respectively cultured for 10-16 hours under the conditions of 25-30 °C and a shaking speed of 150-220 rpm. After the culture is ended, the thalli are collected by centrifugation, and then sterile water is added to adjust the cell concentration to 1×10 8 -1×10 9 cells / mL to obtain the culture solutions of Pseudomonas guangguensis and Aspergillus sydowii. Then, the two culture solutions are mixed in equal volume to obtain the microbial fermenting agent; the activity promoter described in step (4) is obtained by mixing potassium chloride, vitamin C and trehalose according to a mass ratio of 1:0.1:0.

5.

2. The process for preparing fish collagen tripeptide according to claim 1, wherein In step (1), the fish skin is cod fish skin and / or tilapia fish skin.

3. The process for preparing fish collagen tripeptide according to claim 1, characterized in that, In step (2), the solid-liquid ratio of the fish skin to the NaOH solution is 1 g:20 - 40 mL; the solid-liquid ratio of the fish skin to isopropanol is 1 g:30 - 50 mL; the solid-liquid ratio of the fish skin to the ethylenediaminetetraacetic acid solution is 1 g:20 - 40 mL, and the concentration of the ethylenediaminetetraacetic acid solution is 0.1 - 0.3 mol / L.

4. The process for preparing fish collagen tripeptide according to claim 1, characterized in that, The conditions for the high-temperature and high-pressure enzyme inactivation treatment in step (4) are: temperature is 110 - 130°C, pressure is 0.1 - 0.3 MPa, and time is 10 - 20 min.

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