Process method for preparing fish collagen tripeptide and application of fish collagen tripeptide

Abstract

The invention discloses a process method for preparing fish collagen tripeptide and application of the fish collagen tripeptide, and belongs to the technical field of collagen peptide preparation processes. According to the method, the raw material fish skin is subjected to pretreatment and pretreatment, and then the pretreated fish skin is subjected to two times of enzymolysis treatment, so that efficient hydrolysis of fish skin collagen is realized, and a collagen peptide product with small molecular weight is obtained. Firstly, neutral protease and papain are used for carrying out primary enzymolysis on fish skin, then a microbial fermentation agent and an activity accelerant are used for carrying out deep fermentation enzymolysis treatment, peptide fragments can be effectively further cut, the yield of small molecule peptide fragments is increased, and the biological activity is improved. The method is simple, the process is easy to control, environmental pollution is avoided, the enzymolysis time is short, the enzymolysis speed and the extraction rate of the collagen peptide can be increased, and the collagen peptide obtained through the method is low in molecular weight, good in biological activity and easier to popularize and apply.

Description

Technical Field

[0001] The invention belongs to the technical field of collagen peptide preparation technology, and specifically relates to a process for preparing fish collagen tripeptide and application thereof. Background Art

[0002] Collagen, or collagen, is a biological polymer synthesized by animal cells. It is widely present in animal bones, tendons, cartilage, skin and other connective tissues. It has the function of supporting organs and protecting the body. With the development of collagen extraction technology, the in-depth study of its properties and structure, and the improvement of the understanding of the biological functions of collagen and collagen polypeptides, the research and application of collagen has become a hot spot. However, due to the unique triple-stranded superhelical structure of collagen, its properties are very stable. The general processing temperature and short-term heating cannot decompose it, making it difficult to digest and absorb, and it is not easy to be fully utilized by the human body. The hydrolysis of collagen into collagen polypeptides will significantly improve its digestion and absorption, nutrition, functional characteristics, etc.

[0003] For a long time, people have used the skin and bones of pigs and cattle to extract collagen, but due to the outbreak of diseases such as mad cow disease and foot-and-mouth disease, the application of collagen from terrestrial animals has been greatly limited. Marine animals are rich in collagen and have good biosafety, which has great development potential.

[0004] Chinese patent application publication number: CN102217699A, which discloses a method for preparing squid skin protein active peptides, washing the squid skin and cutting it into blocks, adding NaOH aqueous solution to soak for 2-8 days for decolorization, and then washing with water until pH is neutral; adding Ca(OH)2 solution to soak for 2-4 days for color fixation, and then washing with water until pH is neutral; treating in pH neutral water at 55-75℃ for 0.5-1.5h and filtering to remove insoluble protein, extracting fish skin protein liquid; finally, high pressure treatment of the liquid at 110-121℃ for 30-120min, and squid skin protein active peptides are obtained. The disadvantage is that, first of all, the invention needs to soak the squid skin in NaOH aqueous solution for 2-8 days for decolorization, and then soak in Ca(OH)2 solution for 2-4 days for color fixation, and the preparation cycle is long. Secondly, the invention uses high pressure treatment to treat squid skin protein to prepare bioactive peptides, and the preparation conditions are strict, the product molecular weight is large, and the actual application effect is not good.

[0005] Chinese patent application publication number: CN 101870995A discloses a process for preparing collagen tripeptide, which includes subjecting fish skin to an alkali solution pretreatment, a boiling denaturation treatment, and a step-by-step composite hydrolysis of alkaline protease, pancreatic enzyme, and composite protease. After boiling denaturation, the residue is filtered out with gauze, and the hydrolyzate obtained by the three-enzyme step-by-step hydrolysis is inactivated, filtered, debittered, decolorized, vacuum concentrated, and spray-dried to obtain collagen tripeptide with high protein content and good functional activity.

[0006] However, the existing technology has the following problems and disadvantages: the enzymatic hydrolysis efficiency of the enzymes used is low, the enzymatic hydrolysis time is long, the amount of enzyme added is large, the optimum pH value and the optimum temperature need to be adjusted for enzymatic hydrolysis, the operation is complicated, and the adjustment of the optimum pH value for enzymatic hydrolysis will bring in a large amount of inorganic ions. In addition, the commercial enzymes have poor enzymatic activity, the enzymatic hydrolysis is not thorough, the product has a large molecular weight and poor activity, and has a single function and is not easily absorbed by the skin. Summary of the invention

[0007] The present invention aims at the problems existing in the prior art and provides a novel process for preparing fish collagen tripeptide. The prepared tripeptide has good application effect in the field of repairing cell light damage.

[0008] In order to achieve the above technical objectives, the technical solution adopted by the present invention is: A process for preparing fish collagen tripeptide comprises the following preparation steps: (1) Pretreatment: scrape the remaining meat, fins and scales from the fish skin, wash with deionized water, and dry at 25-30°C; after drying, cut the fish skin into small pieces of 1 cm×1 cm, seal them, and store them in a refrigerator at (-20)-(-10)°C for later use; (2) Pretreatment: the fish skin obtained in step (1) is added to a NaOH solution with a mass concentration of 0.5-1% and soaked for 20-30 minutes to remove non-collagen components; the fish skin is then soaked in isopropanol for 3-6 hours to remove fat; the fish skin is then soaked in an ethylenediaminetetraacetic acid solution for 20-30 minutes to remove mineral ions, and finally washed with water until the pH is neutral and drained; (3) initial enzymatic hydrolysis treatment: the fish skin obtained in step (2) is mixed with water in a mass ratio of 1:20, and after swelling and homogenization, a crude slurry is obtained; a composite protease is added to the crude slurry for initial enzymatic hydrolysis treatment to obtain an initial enzymatic hydrolyzate; (4) deep fermentation and enzymatic hydrolysis: adding 1% of the mass of the crude slurry to the primary enzymatic hydrolyzate, a microbial fermentation agent and 0.1% of the activity promoter to carry out deep fermentation and enzymatic hydrolysis, the fermentation temperature is 25-30°C, the fermentation time is 24-36h, and after the treatment is completed, the enzyme is inactivated by high temperature and high pressure treatment. After the entire reaction system is cooled, centrifuge for 15-30min, remove the precipitate and take the supernatant; (5) Filtration and concentration: The supernatant is ultrafiltered through an ultrafiltration membrane with a retention molecule of no more than 2000Da, and the filtrate is collected. The permeate is the collagen tripeptide solution, which is desalted and concentrated, and then vacuum-freeze-dried to obtain the final product, fish collagen tripeptide.

[0009] Furthermore, the fish skin in step (1) is cod and / or tilapia skin.

[0010] Furthermore, in step (2), the solid-liquid ratio of fish skin to NaOH solution is 1 g: 20-40 mL; the solid-liquid ratio of fish skin to isopropanol is 1 g: 30-50 mL; the solid-liquid ratio of fish skin to ethylenediaminetetraacetic acid solution is 1 g: 20-40 mL, and the concentration of ethylenediaminetetraacetic acid solution is 0.1-0.3 mol / L.

[0011] Furthermore, in step (3), the amount of the composite protease added is 3-5% of the mass of the crude slurry, the enzymatic hydrolysis time is 1-2h, and the enzymatic hydrolysis temperature is 50-60°C; the composite protease is neutral protease and papain, and the mass ratio of the two is 1:0.5.

[0012] Furthermore, the preparation method of the microbial fermentation agent in step (4) is as follows: the Pseudomonas guguanyi with a strain number of CGMCC1.15627 and the Aspergillus polymorpha with a strain number of CCTCC CF 2008800 are activated and inoculated into a corn and soybean powder medium and an LB liquid medium, respectively, with an inoculation amount of 3%, and cultured for 10-16 hours at 25-30°C and a shaking speed of 150-220 rpm. After the culture is completed, the bacteria are collected by centrifugation, and then sterile water is added to adjust the bacterial concentration to 1×10 8 -1×10 9 / mL, to obtain the culture solutions of Pseudomonas guguanyi and Aspergillus syn., and then mix the two culture solutions in equal volumes to obtain the microbial fermentation agent.

[0013] Furthermore, the strain numbered CGMCC 1.15627 Pseudomonas guguanyi was purchased from the General Microbiology Center of the China Microbiological Culture Collection Administration, and the original preservation date was February 25, 2015. The strain can be purchased through the collection center without the need for repeated biological preservation.

[0014] Furthermore, the strain Aspergillus polymorpha numbered CCTCC CF 2008800 was purchased from the China Center for Type Culture Collection, and the original preservation date was November 10, 2004. The strain can also be purchased through the collection center without the need for repeated biological preservation.

[0015] Furthermore, the activation method of Pseudomonas guguanyi and Aspergillus syn. is as follows: the target strain stored at low temperature is cultured on an LB slant medium at 25-30°C for 10-16 hours, and the weight ratio of the components of the LB slant medium is: 8-12g of tryptone, 4-6g of yeast extract, 4-6g of sodium chloride, 12-18g of agar powder, and 1000mL of water to obtain an activated strain.

[0016] Furthermore, the composition of the corn and soybean meal culture medium is: 1.0-3.0% corn meal, 1.0-3.0% soybean meal, 0.1-1.0% NaCl, 0.5-1.0% MnSO4·H2O, and the rest is water.

[0017] Furthermore, the activity promoter in step (4) is a mixture of potassium chloride, vitamin C and trehalose in a mass ratio of 1:0.1:0.5.

[0018] Furthermore, the conditions for the high temperature and high pressure enzyme inactivation treatment in step (5) are: temperature of 110-130° C., pressure of 0.1-0.3 MPa, and time of 10-20 min.

[0019] The collagen tripeptide produced by the method described in the present application has a small molecular weight and high activity, can play a role in repairing photodamage to cells, and can be used in cosmetics.

[0020] The raw materials used in this application are all commercially available.

[0021] Beneficial effects: (1) First, the raw fish skin is pre-treated and pre-treated to effectively remove impurities and improve the purity of the raw material. The NaOH solution soaking treatment effectively removes non-collagen components and other impurities, and the organic solvent isopropanol treatment plays a role in defatting and removing impurities and pigments; and finally, the ethylenediaminetetraacetic acid solution treatment forms a stable, dissolved and mobile complex with metal ions through chelation or coordination reaction, thereby removing metal ions in the fish skin; (2) Subsequently, the pretreated fish skin is subjected to two enzymatic hydrolysis treatments to achieve efficient hydrolysis of fish skin collagen and obtain a small molecular weight collagen peptide product. First, the fish skin is preliminarily hydrolyzed using neutral protease and papain 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 cut the protein chain, and thus produce effectively cut peptide segments; (3) Subsequently, microbial fermentation agents and activity promoters are used for deep fermentation and enzymatic hydrolysis. Pseudomonas guguanis and Aspergillus syngenes are mixed in equal proportions for fermentation, which can produce highly active extracellular proteases, further cut the peptides after preliminary enzymatic hydrolysis, increase the yield of small molecule peptides, and improve biological activity. When adding activity promoters composed of potassium chloride, vitamin C and trehalose, potassium ions can effectively activate and enhance protease activity, while vitamin C and trehalose promote the production of extracellular proteases by regulating sugar metabolism pathways during the fermentation metabolism of Pseudomonas guguanis, thereby increasing 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 resulting peptides have high biological activity and have a high application prospect in repairing cell photodamage. (4) In summary, the method of the present 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 no odor, uniform color, good biological properties, and are easier to be promoted and applied. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The experimental results of the effects of samples from different experimental groups on the COL-I content of HSF cells; Figure 2 The experimental results of the effects of samples from different experimental groups on the ELN content of HSF cells; Figure 3 The experimental results of the effects of samples from different experimental groups on the HA content of HSF cells; Figure 4 The experimental results of the effects of samples from different experimental groups on the total antioxidant capacity of HSF cells; Figure 5 HE staining images of mouse skin in different experimental groups (100X); Figure 6 This is a photo of the product obtained in Example 1. DETAILED DESCRIPTION

[0023] The technical solution of the present invention is further described below in conjunction with specific embodiments, but is not limited thereto.

[0024] Example 1 A process for preparing fish collagen tripeptide comprises the following preparation steps: (1) Pretreatment: scrape the remaining meat, fins and scales from the fish skin, wash with deionized water, and dry at 25-30°C; after drying, cut the fish skin into small pieces of 1 cm×1 cm, seal them, and store them in a refrigerator at (-20)-(-10)°C for later use; (2) Pretreatment: The fish skin obtained in step (1) is added to a 0.5% NaOH solution and soaked for 30 min to remove non-collagen components; the fish skin is then soaked in isopropanol for 3 h to remove fat; the fish skin is then soaked in an ethylenediaminetetraacetic acid solution for 20 min to remove mineral ions, and finally washed with water until the pH is neutral and drained; (3) initial enzymatic hydrolysis treatment: the fish skin obtained in step (2) is mixed with water in a mass ratio of 1:20, and after swelling and homogenization, a crude slurry is obtained; a composite protease is added to the crude slurry for initial enzymatic hydrolysis treatment to obtain an initial enzymatic hydrolyzate; (4) Deep fermentation and enzymatic hydrolysis: add 1% of the mass of the crude slurry to the initial enzymatic hydrolyzate. The microbial fermentation agent and 0.1% of the activity promoter are added to the primary enzymatic hydrolyzate for deep fermentation and enzymatic hydrolysis. The fermentation temperature is 25-30°C and the fermentation time is 24 hours. After the treatment is completed, the enzyme is inactivated by high temperature and high pressure treatment. After the entire reaction system is cooled, centrifuge for 15 minutes to remove the precipitate and take the supernatant; (5) Filtration and concentration: The supernatant is ultrafiltered through an ultrafiltration membrane with a retention molecule of no more than 2000Da, and the filtrate is collected. The permeate is the collagen tripeptide solution, which is desalted and concentrated, and then vacuum-freeze-dried to obtain the final product, fish collagen tripeptide.

[0025] The fish skin in step (1) is cod fish skin.

[0026] In step (2), the solid-liquid ratio of fish skin to NaOH solution is 1 g:40 mL; the solid-liquid ratio of fish skin to isopropanol is 1 g:50 mL; the solid-liquid ratio of fish skin to ethylenediaminetetraacetic acid solution is 1 g:20 mL, and the concentration of ethylenediaminetetraacetic acid solution is 0.1 mol / L.

[0027] In step (3), the amount of the composite protease added is 3% of the mass of the crude slurry, the enzymolysis time is 1 hour, and the enzymolysis temperature is 50-60°C; the composite protease is neutral protease and papain, and the mass ratio of the two is 1:0.5.

[0028] The preparation method of the microbial fermentation agent in step (4) is as follows: Pseudomonas guguanyi with a strain number of CGMCC 1.15627 and Aspergillus polydophorus with a strain number of CCTCC CF 2008800 are activated and inoculated into a corn and soybean powder medium and an LB liquid medium, respectively, with an inoculation amount of 3%, and cultured for 10-16 hours at 25-30°C and a shaking speed of 150-220 rpm. After the culture is completed, the bacteria are collected by centrifugation, and then sterile water is added to adjust the bacterial concentration to 1×10 8 -1×10 9 / mL, to obtain the culture solutions of Pseudomonas guguanyi and Aspergillus syn., and then mix the two culture solutions in equal volumes to obtain the microbial fermentation agent.

[0029] The strain numbered CGMCC 1.15627 Pseudomonas guguanyi was purchased from the General Microbiology Center of the China Microbiological Culture Collection Administration, and the original preservation date was February 25, 2015. The strain can be purchased through the collection center without the need for repeated biological preservation.

[0030] The strain number is CCTCC CF 2008800 and is purchased from the China Center for Type Culture Collection. The original preservation date is November 10, 2004. The strain can be purchased through the collection center without repeated biological preservation.

[0031] The activation method of Pseudomonas guguanyi and Aspergillus syn. is as follows: the target strain stored at low temperature is cultured on an LB slant medium at 25-30° C. for 10-16 hours, wherein the weight ratio of the components 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 an activated strain.

[0032] The composition of the corn and soybean meal culture medium is: 1.0-3.0% corn meal, 1.0-3.0% soybean meal, 0.1-1.0% NaCl, 0.5-1.0% MnSO4·H2O, and the rest is water.

[0033] The activity promoter in step (4) is a mixture of potassium chloride, vitamin C and trehalose in a mass ratio of 1:0.1:0.5.

[0034] The conditions of high temperature and high pressure enzyme inactivation treatment in step (5) are: temperature of 110° C., pressure of 0.1 MPa, and time of 20 min.

[0035] Example 2 A process for preparing fish collagen tripeptide comprises the following preparation steps: (1) Pretreatment: scrape the remaining meat, fins and scales from the fish skin, wash with deionized water, and dry at 25-30°C; after drying, cut the fish skin into small pieces of 1 cm×1 cm, seal them, and store them in a refrigerator at (-20)-(-10)°C for later use; (2) Pretreatment: The fish skin obtained in step (1) is added to a 0.7% NaOH solution and soaked for 30 min to remove non-collagen components; the fish skin is then soaked in isopropanol for 5 h to remove fat; the fish skin is then soaked in an ethylenediaminetetraacetic acid solution for 30 min to remove mineral ions, and finally washed with water until the pH is neutral and drained; (3) initial enzymatic hydrolysis treatment: the fish skin obtained in step (2) is mixed with water in a mass ratio of 1:20, and after swelling and homogenization, a crude slurry is obtained; a composite protease is added to the crude slurry for initial enzymatic hydrolysis treatment to obtain an initial enzymatic hydrolyzate; (4) Deep fermentation and enzymatic hydrolysis: add 1% of the mass of the crude slurry to the initial enzymatic hydrolyzate. The microbial fermentation agent and 0.1% of the activity promoter are added to the primary enzymatic hydrolyzate for deep fermentation and enzymatic hydrolysis. The fermentation temperature is 25-30°C and the fermentation time is 30 hours. After the treatment is completed, the enzyme is inactivated by high temperature and high pressure treatment. After the entire reaction system is cooled, centrifuge for 30 minutes to remove the precipitate and take the supernatant; (5) Filtration and concentration: The supernatant is ultrafiltered through an ultrafiltration membrane with a retention molecule of no more than 2000Da, and the filtrate is collected. The permeate is the collagen tripeptide solution, which is desalted and concentrated, and then vacuum-freeze-dried to obtain the final product, fish collagen tripeptide.

[0036] The fish skin in step (1) is tilapia fish skin.

[0037] In step (2), the solid-liquid ratio of fish skin to NaOH solution is 1 g:30 mL; the solid-liquid ratio of fish skin to isopropanol is 1 g:40 mL; the solid-liquid ratio of fish skin to ethylenediaminetetraacetic acid solution is 1 g:30 mL, and the concentration of ethylenediaminetetraacetic acid solution is 0.2 mol / L.

[0038] In step (3), the amount of the composite protease added is 4% of the mass of the crude slurry, the enzymolysis time is 1 hour, and the enzymolysis temperature is 50-60°C; the composite protease is neutral protease and papain, and the mass ratio of the two is 1:0.5.

[0039] The preparation method of the microbial fermentation agent in step (4) is as follows: Pseudomonas guguanyi with a strain number of CGMCC 1.15627 and Aspergillus polydophorus with a strain number of CCTCC CF 2008800 are activated and inoculated into a corn and soybean powder medium and an LB liquid medium, respectively, with an inoculation amount of 3%, and cultured for 10-16 hours at 25-30°C and a shaking speed of 150-220 rpm. After the culture is completed, the bacteria are collected by centrifugation, and then sterile water is added to adjust the bacterial concentration to 1×10 8 -1×10 9 / mL, to obtain the culture solutions of Pseudomonas guguanyi and Aspergillus syn., and then mix the two culture solutions in equal volumes to obtain the microbial fermentation agent.

[0040] The strain numbered CGMCC 1.15627 Pseudomonas guguanyi was purchased from the General Microbiology Center of the China Microbiological Culture Collection Administration, and the original preservation date was February 25, 2015. The strain can be purchased through the collection center without the need for repeated biological preservation.

[0041] The strain number is CCTCC CF 2008800 and is purchased from the China Center for Type Culture Collection. The original preservation date is November 10, 2004. The strain can be purchased through the collection center without repeated biological preservation.

[0042] The activation method of Pseudomonas guguanyi and Aspergillus syn. is as follows: the target strain stored at low temperature is cultured on an LB slant medium at 25-30° C. for 10-16 hours, wherein the weight ratio of the components 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 an activated strain.

[0043] The composition of the corn and soybean meal culture medium is: 1.0-3.0% corn meal, 1.0-3.0% soybean meal, 0.1-1.0% NaCl, 0.5-1.0% MnSO4·H2O, and the rest is water.

[0044] The activity promoter in step (4) is a mixture of potassium chloride, vitamin C and trehalose in a mass ratio of 1:0.1:0.5.

[0045] The conditions of high temperature and high pressure enzyme inactivation treatment in step (5) are: temperature of 130° C., pressure of 0.3 MPa, and time of 15 min.

[0046] Example 3 A process for preparing fish collagen tripeptide comprises the following preparation steps: (1) Pretreatment: scrape the remaining meat, fins and scales from the fish skin, wash with deionized water, and dry at 25-30°C; after drying, cut the fish skin into small pieces of 1 cm×1 cm, seal them, and store them in a refrigerator at (-20)-(-10)°C for later use; (2) Pretreatment: The fish skin obtained in step (1) is added to a 1% NaOH solution and soaked for 30 min to remove non-collagen components; the fish skin is then soaked in isopropanol for 6 h to remove fat; the fish skin is then soaked in an ethylenediaminetetraacetic acid solution for 30 min to remove mineral ions, and finally washed with water until the pH is neutral and drained; (3) initial enzymatic hydrolysis treatment: the fish skin obtained in step (2) is mixed with water in a mass ratio of 1:20, and after swelling and homogenization, a crude slurry is obtained; a composite protease is added to the crude slurry for initial enzymatic hydrolysis treatment to obtain an initial enzymatic hydrolyzate; (4) Deep fermentation and enzymatic hydrolysis: add 1% of the mass of the crude slurry to the initial enzymatic hydrolyzate. The microbial fermentation agent and 0.1% of the activity promoter are added to the primary enzymatic hydrolyzate for deep fermentation and enzymatic hydrolysis. The fermentation temperature is 25-30°C and the fermentation time is 36 hours. After the treatment is completed, the enzyme is inactivated by high temperature and high pressure treatment. After the entire reaction system is cooled, centrifuge for 30 minutes to remove the precipitate and take the supernatant; (5) Filtration and concentration: The supernatant is ultrafiltered through an ultrafiltration membrane with a retention molecule of no more than 2000Da, and the filtrate is collected. The permeate is the collagen tripeptide solution, which is desalted and concentrated, and then vacuum-freeze-dried to obtain the final product, fish collagen tripeptide.

[0047] The fish skin in step (1) is cod fish skin.

[0048] In step (2), the solid-liquid ratio of fish skin to NaOH solution is 1 g:20 mL; the solid-liquid ratio of fish skin to isopropanol is 1 g:30 mL; the solid-liquid ratio of fish skin to ethylenediaminetetraacetic acid solution is 1 g:40 mL, and the concentration of ethylenediaminetetraacetic acid solution is 0.3 mol / L.

[0049] In step (3), the amount of the composite protease added is 5% of the mass of the crude slurry, the enzymolysis time is 2 hours, and the enzymolysis temperature is 50-60°C; the composite protease is neutral protease and papain, and the mass ratio of the two is 1:0.5.

[0050] The preparation method of the microbial fermentation agent in step (4) is as follows: Pseudomonas guguanyi with a strain number of CGMCC 1.15627 and Aspergillus polydophorus with a strain number of CCTCC CF 2008800 are activated and inoculated into a corn and soybean powder medium and an LB liquid medium, respectively, with an inoculation amount of 3%, and cultured for 10-16 hours at 25-30°C and a shaking speed of 150-220 rpm. After the culture is completed, the bacteria are collected by centrifugation, and then sterile water is added to adjust the bacterial concentration to 1×10 8 -1×10 9 / mL, to obtain the culture solutions of Pseudomonas guguanyi and Aspergillus syn., and then mix the two culture solutions in equal volumes to obtain the microbial fermentation agent.

[0051] The strain numbered CGMCC 1.15627 Pseudomonas guguanyi was purchased from the General Microbiology Center of the China Microbiological Culture Collection Administration, and the original preservation date was February 25, 2015. The strain can be purchased through the collection center without the need for repeated biological preservation.

[0052] The strain number is CCTCC CF 2008800 and is purchased from the China Center for Type Culture Collection. The original preservation date is November 10, 2004. The strain can be purchased through the collection center without repeated biological preservation.

[0053] The activation method of Pseudomonas guguanyi and Aspergillus syn. is as follows: the target strain stored at low temperature is cultured on an LB slant medium at 25-30° C. for 10-16 hours, wherein the weight ratio of the components 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 an activated strain.

[0054] The composition of the corn and soybean meal culture medium is: 1.0-3.0% corn meal, 1.0-3.0% soybean meal, 0.1-1.0% NaCl, 0.5-1.0% MnSO4·H2O, and the rest is water.

[0055] The activity promoter in step (4) is a mixture of potassium chloride, vitamin C and trehalose in a mass ratio of 1:0.1:0.5.

[0056] The conditions of high temperature and high pressure enzyme inactivation treatment in step (5) are: temperature of 130° C., pressure of 0.3 MPa, and time of 20 min.

[0057] Comparative Example 1 A process for preparing fish collagen tripeptide comprises the following preparation steps: (1) Pretreatment: scrape the remaining meat, fins and scales from the fish skin, wash with deionized water, and dry at 25-30°C; after drying, cut the fish skin into small pieces of 1 cm×1 cm, seal them, and store them in a refrigerator at (-20)-(-10)°C for later use; (2) Pretreatment: The fish skin obtained in step (1) is added to a 0.5% NaOH solution and soaked for 30 min to remove non-collagen components; the fish skin is then soaked in isopropanol for 3 h to remove fat; the fish skin is then soaked in an ethylenediaminetetraacetic acid solution for 20 min to remove mineral ions, and finally washed with water until the pH is neutral and drained; (3) initial enzymatic hydrolysis treatment: the fish skin obtained in step (2) is mixed with water in a mass ratio of 1:20, and after swelling and homogenization, a crude slurry is obtained; a composite protease and an activity promoter of 0.1% by mass of the crude slurry are added to the crude slurry for preliminary enzymatic hydrolysis treatment to obtain an initial enzymatic hydrolyzate; after the treatment is completed, the enzyme is inactivated by high temperature and high pressure treatment, and the entire reaction system is cooled and centrifuged for 15 minutes to remove the precipitate and obtain the supernatant; (4) Filtration and concentration: The supernatant is ultrafiltered through an ultrafiltration membrane with a retention molecule of no more than 2000Da, and the filtrate is collected. The permeate is the collagen tripeptide solution, which is desalted and concentrated, and then vacuum-freeze-dried to obtain the final product, fish collagen tripeptide.

[0058] In step (3), the amount of the composite protease added is 4% of the mass of the crude slurry, the enzymolysis time is 25 hours, and the enzymolysis temperature is 50-60°C; the composite protease is neutral protease and papain, and the mass ratio of the two is 1:0.5.

[0059] Compared with Example 1, this comparative example uses the same raw materials and process steps as Example 1 except that deep fermentation and enzymolysis treatment are not performed.

[0060] Comparative Example 2 A process for preparing fish collagen tripeptide comprises the following preparation steps: (1) Pretreatment: scrape the remaining meat, fins and scales from the fish skin, wash with deionized water, and dry at 25-30°C; after drying, cut the fish skin into small pieces of 1 cm×1 cm, seal them, and store them in a refrigerator at (-20)-(-10)°C for later use; (2) Pretreatment: The fish skin obtained in step (1) is added to a 0.5% NaOH solution and soaked for 30 min to remove non-collagen components; the fish skin is then soaked in isopropanol for 3 h to remove fat; the fish skin is then soaked in an ethylenediaminetetraacetic acid solution for 20 min to remove mineral ions, and finally washed with water until the pH is neutral and drained; (3) enzymatic hydrolysis: the fish skin obtained in step (2) is mixed with water in a mass ratio of 1:20, and after swelling and homogenization, a crude slurry is obtained. 4% by mass of a microbial fermentation agent and 0.1% of an activity promoter are added to the crude slurry for deep fermentation at a fermentation temperature of 25-30° C. for 25 h. After the treatment is completed, the enzyme is inactivated by high temperature and high pressure treatment. After the entire reaction system is cooled, the reaction system is centrifuged for 15 min, and the precipitate is removed to obtain a supernatant; (4) Filtration and concentration: The supernatant is ultrafiltered through an ultrafiltration membrane with a retention molecule of no more than 2000Da, and the filtrate is collected. The permeate is the collagen tripeptide solution, which is desalted and concentrated, and then vacuum-freeze-dried to obtain the final product, fish collagen tripeptide.

[0061] Compared with Example 1, this comparative example has the same raw materials and process steps as Example 1 except that the preliminary enzymatic hydrolysis is not performed.

[0062] Comparative Example 3 A process for preparing fish collagen tripeptide comprises the following preparation steps: (1) Pretreatment: scrape the remaining meat, fins and scales from the fish skin, wash with deionized water, and dry at 25-30°C; after drying, cut the fish skin into small pieces of 1 cm×1 cm, seal them, and store them in a refrigerator at (-20)-(-10)°C for later use; (2) Pretreatment: The fish skin obtained in step (1) is added to a 0.5% NaOH solution and soaked for 30 min to remove non-collagen components; the fish skin is then soaked in isopropanol for 3 h to remove fat; the fish skin is then soaked in an ethylenediaminetetraacetic acid solution for 20 min to remove mineral ions, and finally washed with water until the pH is neutral and drained; (3) initial enzymatic hydrolysis treatment: the fish skin obtained in step (2) is mixed with water in a mass ratio of 1:20, and after swelling and homogenization, a crude slurry is obtained; a composite protease is added to the crude slurry for initial enzymatic hydrolysis treatment to obtain an initial enzymatic hydrolyzate; (4) Deep fermentation enzymolysis treatment: add 1% of the mass of the crude slurry to the primary enzymolysis liquid. The microbial fermentation agent and 0.1% of the activity promoter are added to the primary enzymolysis liquid for deep fermentation. The fermentation temperature is 25-30°C and the fermentation time is 24 hours. After the treatment is completed, the enzyme is inactivated by high temperature and high pressure treatment. After the entire reaction system is cooled, centrifuge for 15 minutes to remove the precipitate and take the supernatant; Filtration and concentration treatment: The supernatant is ultrafiltered through an ultrafiltration membrane with a retention molecule of no more than 2000Da, and the filtrate is collected. The permeate is the collagen tripeptide solution, which is desalted and concentrated, and then vacuum-freeze-dried to obtain the final product, fish collagen tripeptide.

[0063] The preparation method of the microbial fermentation agent in step (4) is as follows: the activated Pseudomonas guguanyi strain numbered CGMCC 1.15627 is inoculated into a corn and soybean powder culture medium at an inoculation amount of 3%, and cultured for 10-16 hours at 25-30°C and a shaking speed of 150-220 rpm. After the culture is completed, the bacteria are collected by centrifugation, and then sterile water is added to adjust the bacterial concentration to 1×10 8 -1×10 9 / mL to obtain a microbial fermentation agent.

[0064] Compared with Example 1, this comparative example is the same as Example 1 except that in step (4), Aspergillus polydorum with strain number CCTCC CF2008800 is not used to prepare the microbial fermentation agent. The other raw materials and process steps are the same as those in Example 1.

[0065] Comparative Example 4 Compared with Example 1, this comparative example uses the same raw materials and process steps as Example 1, except that in step (4), the Pseudomonas guguanyi strain numbered CGMCC 1.15627 is not used to prepare the microbial fermentation agent.

[0066] Comparative Example 5 Compared with Example 1, this comparative example is the same as Example 1 except that the volume ratio of Pseudomonas guguanyi with strain number CGMCC 1.15627 and Aspergillus polymorpha with strain number CCTCC CF 2008800 is changed in the preparation of the microbial fermentation agent in step (4). The other raw materials and process steps are the same as those in Example 1.

[0067] That is, the preparation method of the microbial fermentation agent in step (4) is as follows: Pseudomonas guguanyi with a strain number of CGMCC 1.15627 and Aspergillus polydophorus with a strain number of CCTCC CF 2008800 are activated and inoculated into corn and soybean powder medium and LB liquid medium respectively, with an inoculation amount of 3%, and cultured for 10-16 hours at 25-30°C and a shaking speed of 150-220 rpm. After the culture is completed, the bacteria are collected by centrifugation, and then sterile water is added to adjust the bacterial concentration to 1×10 8 -1×109 / mL, to obtain the culture solutions of Pseudomonas guguanyi and Aspergillus syn., and then mix the two culture solutions in a volume ratio of 1:2 to obtain a microbial fermentation agent.

[0068] Comparative Example 6 Compared with Example 1, this comparative example is the same as Example 1 except that the volume ratio of Pseudomonas guguanyi with strain number CGMCC 1.15627 and Aspergillus polymorpha with strain number CCTCC CF 2008800 is changed in the preparation of the microbial fermentation agent in step (4). The other raw materials and process steps are the same as those in Example 1.

[0069] That is, the preparation method of the microbial fermentation agent in step (4) is as follows: Pseudomonas guguanyi with a strain number of CGMCC 1.15627 and Aspergillus polydophorus with a strain number of CCTCC CF 2008800 are activated and inoculated into corn and soybean powder medium and LB liquid medium respectively, with an inoculation amount of 3%, and cultured for 10-16 hours at 25-30°C and a shaking speed of 150-220 rpm. After the culture is completed, the bacteria are collected by centrifugation, and then sterile water is added to adjust the bacterial concentration to 1×10 8 -1×10 9 / mL, to obtain the culture solutions of Pseudomonas guguanyi and Aspergillus syn., and then mix the two culture solutions in a volume ratio of 2:1 to obtain a microbial fermentation agent.

[0070] Performance Testing The fish collagen tripeptide was prepared according to the example and comparative example methods, and the obtained product was tested for performance. The testing method is as follows: The molecular weight distribution test was carried out using the method in Appendix A of the National Standard of the People's Republic of China GB / T22729-2008; the hydroxyproline test method was carried out in accordance with the National Standard of the People's Republic of China GB / T9695.23-2008, the total nitrogen test method was carried out in accordance with the National Standard of the People's Republic of China GB / T5009.5-2016, and the ash test method was carried out in accordance with the 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.

[0071] Table 1 Performance test results

[0072] Table 2 Performance test results

[0073] From the data in Table 1-2, we can see that the final product obtained in the embodiment of the present invention has a low molecular weight and a high proportion of small molecular weight. This is because the present invention has two-step enzymolysis, and the initial enzymolysis realizes the first step of cutting of collagen, and the active protease produced by microbial fermentation is subsequently used to achieve deep cutting of the initial enzymolysis peptide segment, promote thorough enzymolysis of the protein, and greatly reduce the proportion of small molecular weight peptide segments. However, in Comparative Example 1-2, which changed the enzymolysis process, it is difficult to achieve effective cutting of peptide segments with a single enzymolysis method, and the enzymolysis is not thorough, which leads to an increase in the proportion of high molecular weight peptide segments. The biological activity of the product was further tested later, and the testing method was as follows: the collagen peptide samples obtained in Example 1-3 and Comparative Example 1-6 were tested for their ability to repair light damage.

[0074] Cell culture: HSF cells were cultured in a medium containing 10% fetal bovine serum and 1% penicillin (1×10 5 The cells were cultured in DMEM medium containing 100 mg / L (100 μg / L) and streptomycin (100 mg / L) at 37°C in an incubator with 5% carbon dioxide 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.

[0075] Establishment of UVA-induced photodamage model: 1×10 per well 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 incubated at 20 J / cm 2 The cells were stimulated with UVA of 1000 μg / mL and then replaced with basal medium. Cell viability was measured after 24 h of culture. IC 50 Establishment of UVA damage model.

[0076] HA, COL-I, ELN content and total antioxidant capacity determination: The contents of HA, ELN and COL-I in cells were detected by enzyme-linked immunosorbent assay (ELISA), and the total antioxidant capacity was determined by ABTS method. HSF cells in logarithmic growth phase were taken and 2×10 5The cells were inoculated in 6-well cell culture plates. When the cell confluence reached 80%, the culture medium was replaced with PBS. After UVA irradiation, the PBS was discarded, and 2 mL of serum-free DMEM, experimental group and comparative group collagen peptide aqueous solution (mass concentration 3%), and 50 μg / mL VC solution were added for treatment, corresponding to the model group, experimental group and VC positive control group, respectively, and a blank control group was set up. According to the instructions of HA, ELN, COL-I enzyme-linked immunosorbent assay kit and total antioxidant capacity assay kit, cell samples were collected, and the HA, ELN and COL-I content and total antioxidant capacity of each group were determined. After collecting the cells, the protein content of each group needs to be determined. After measuring the content of the above indicators, it is divided by the corresponding protein content to normalize the cell amount and eliminate the influence of the differences in various indicators caused by the proliferation of the samples on the cells. All experimental results were repeated 5 times, and the results were averaged.

[0077] Table 3 Verification of the ability to repair light damage

[0078] From the data in Tables 1-3, we can see that: compared with the blank control group, the content of each index in the model group was 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 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 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 positive control group of VC, 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.

[0079] 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: Figure 1-4 As shown, from Figure 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, plays a positive role in repairing UVA-induced skin light damage, and 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 cells from oxidative damage. The contents of various indicators in Comparative Examples 1-6 are not as good as those in 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 in Comparative Examples 3-6, which changed the composition of the microbial fermentation agent, decreased, 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.

[0080] The experimental method for mice is as follows: 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 gavaged with an equal amount of physiological saline, and the gavage time was 30 min before each irradiation.

[0081] Table 4 Animal gavage conditions

[0082] Modeling method of model group: Arrange 4 UVA tubes and 2 UVB tubes in parallel as ultraviolet irradiation light sources. Depilate the mice (before each irradiation, the mice need to be depilated). Except for the normal group, the back skin of the mice was irradiated with UV (UVA+UVB) 3 times a week from the 1st week to the 6th week, with an irradiation height of 30cm. The intensity was measured with a UVR irradiator to determine the minimum erythema (MED) dose. The irradiation dose was 0.5MED / time in the first week, 1.0MED / time in the second week, and increased by 0.5MED / time per week until 3.0MED / time in the 6th week.

[0083] Skin tissue section observation: After the mice were killed by dislocating the cervical vertebrae, appropriate mouse skin tissue was taken and fixed in 4% paraformaldehyde, then embedded in paraffin and sliced. The slices were then stained with HE and the stained skin tissue was observed under a microscope at an appropriate magnification. The results are as follows Figure 5 As shown. Figure 5 It can be seen that compared with the normal group, the epidermis of the mice in the model group (MG group) was obviously abnormally thickened, irregularly arranged, and keratinized. At the same time, the basal cells in the MG group showed vacuolar changes and inflammatory infiltration. The epidermal thickness of the mouse skin in the Example 1 group was significantly reduced compared with the MG group, and the epidermal structure of the mouse skin was closer to the normal group.

[0084] It should be noted that the above embodiments are only partial embodiments of the preferred methods of implementing the present invention, rather than all embodiments. Obviously, based on the above embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

Claims

1. A process for preparing fish collagen tripeptide, characterized in that: The method comprises the following preparation steps: (1) Pretreatment: scrape the remaining meat, fins and scales from the fish skin, wash with deionized water, and dry at 25-30°C; after drying, cut the fish skin into small pieces of 1 cm×1 cm, seal them, and store them in a refrigerator at (-20)-(-10)°C for later use; (2) Pretreatment: the fish skin obtained in step (1) is added to a NaOH solution with a mass concentration of 0.5-1% and soaked for 20-30 min; the fish skin is then soaked in isopropanol for 3-6 h; the fish skin is then soaked in an ethylenediaminetetraacetic acid solution for 20-30 min, and finally washed with water until the pH is neutral and drained; (3) initial enzymatic hydrolysis treatment: the fish skin obtained in step (2) is mixed with water in a mass ratio of 1:20, and after swelling and homogenization, a crude slurry is obtained; a composite protease is added to the crude slurry for initial enzymatic hydrolysis treatment to obtain an initial enzymatic hydrolyzate; (4) deep fermentation and enzymatic hydrolysis: adding 1% of the mass of the crude slurry to the primary enzymatic hydrolyzate, a microbial fermentation agent and 0.1% of the activity promoter to carry out deep fermentation and enzymatic hydrolysis, the fermentation temperature is 25-30°C, the fermentation time is 24-36h, and after the treatment is completed, the enzyme is inactivated by high temperature and high pressure treatment. After the entire reaction system is cooled, centrifuge for 15-30min, remove the precipitate and take the supernatant; (5) Filtration and concentration: The supernatant is ultrafiltered through an ultrafiltration membrane with a retention molecule of no more than 2000Da, and the filtrate is collected. The permeate is the collagen tripeptide solution, which is desalted and concentrated, and then vacuum-freeze-dried to obtain the final product, fish collagen tripeptide; The preparation method of the microbial fermentation agent in step (4) is as follows: Pseudomonas gibberellus with a strain number of CGMCC 1.15627 and Aspergillus polydoctorius with a strain number of CCTCC CF 2008800 are activated and inoculated into a corn and soybean powder culture medium and an LB liquid culture medium, respectively, with an inoculation amount of 3%, and cultured for 10-16 hours at 25-30° C. and a shaking speed of 150-220 rpm. After the culture is completed, the bacteria are collected by centrifugation, and then sterile water is added to adjust the bacterial concentration to 1×108-1×109 cells / mL to obtain a culture solution of Pseudomonas gibberellus and Aspergillus polydoctorius, and then the two culture solutions are mixed in equal volumes to obtain a microbial fermentation agent; The activity promoter in step (4) is a mixture of potassium chloride, vitamin C and trehalose in a mass ratio of 1:0.1:0.

5.

2. The process for preparing fish collagen tripeptide according to claim 1, characterized in that: The fish skin in step (1) is cod skin and / or tilapia skin.

3. The process for preparing fish collagen tripeptide according to claim 1, characterized in that: In step (2), the solid-liquid ratio of fish skin to NaOH solution is 1 g: 20-40 mL; the solid-liquid ratio of fish skin to isopropanol is 1 g: 30-50 mL; the solid-liquid ratio of fish skin to ethylenediaminetetraacetic acid solution is 1 g: 20-40 mL, and the concentration of 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: In step (3), the amount of the composite protease added is 3-5% of the mass of the crude slurry, the enzymolysis time is 1-2 hours, and the enzymolysis temperature is 50-60°C; the composite protease is neutral protease and papain, and the mass ratio of the two is 1:0.

5.

5. The process for preparing fish collagen tripeptide according to claim 1, characterized in that: The conditions of the high temperature and high pressure enzyme inactivation treatment in step (5) are: temperature of 110-130° C., pressure of 0.1-0.3 MPa, and time of 10-20 min.

6. A collagen tripeptide produced by the process described in any one of claims 1 to 5.

7. Use of the collagen tripeptide produced by the process described in any one of claims 1 to 5 in repairing cell light damage.

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