A collagen tripeptide, and a preparation method and application thereof

Abstract

The application discloses collagen tripeptide and a preparation method and application thereof, and belongs to the technical field of bioengineering. The preparation method comprises the following steps: obtaining bacillus velezensis with high collagenase yield through separation and identification, the bacillus velezensis belongs to probiotics, and high-activity collagenase is obtained through fermentation culture and purification; taking shark cartilage or blue fish skin as raw materials, and obtaining collagen tripeptide through the following steps of combined enzymolysis of alkaline protease and collagenase after pretreatment, inactivation, centrifugal separation and freeze-drying. The collagen tripeptide prepared by the application has a molecular weight of less than 500 Dalton, contains active components such as GHK tripeptide, is easy to absorb and has high safety, and can be used for preparing antioxidant products, anti-inflammatory products, memory-enhancing products or immunity-enhancing products. The application solves the problems of unsafe collagenase source and low collagen tripeptide preparation efficiency, and has a good industrial application prospect.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a collagen tripeptide, its preparation method, and its application. Background Technology

[0002] Collagen is a veteran in the field of oral beauty supplements, and it has become a trend for more and more young people to combat early signs of aging by supplementing with collagen. As an important connective tissue protein in the skin, collagen is known for its triple helix structure and stable molecular properties. Among them, type I collagen is the most widely distributed, accounting for 80% to 90% of the total. It is synthesized by fibroblasts and supports the skin structure, maintaining elasticity and moisture. Studies have shown that collagen is in dynamic balance in the skin, but with age, the rate of loss exceeds the rate of regeneration, leading to dry, rough skin, increased wrinkles, and a gradual loss of elasticity and radiance. Therefore, collagen supplementation has become an important direction in the field of oral beauty and anti-aging.

[0003] However, the molecular weight of ordinary collagen products is usually between 10,000 and 300,000 Daltons, making them difficult for the human body to absorb and utilize directly, resulting in extremely low bioavailability. In contrast, collagen tripeptide (CTP), as the smallest bioactive unit of collagen composed of three amino acids, has a molecular weight of less than 500 Daltons. It can pass through the epithelial cells of the small intestine villi and directly enter the bloodstream, allowing for "point-to-point" absorption and synthesis into the collagen needed for the skin. It also boasts advantages such as high purity, non-antigenicity, and low allergenicity. Among these, GHK tripeptide (glycyl-L-histidyl-L-lysine, GHK) and its GHK-Cu complex with copper also possess functions such as improving blood circulation, promoting the synthesis of nerve and neurotrophic factors, anti-oxidation, anti-inflammation, and potential anti-tumor effects.

[0004] In existing technologies, most mainstream collagen tripeptide products at home and abroad (such as Japanese NPPI collagen tripeptide and Korean Ameco collagen tripeptide) are derived from the enzymatic hydrolysis products of type I collagen from tilapia skin or fish scales, which are suitable for skin repair. Type II collagen is mainly found in cartilage, and its enzymatic hydrolysis products are more suitable for cartilage repair and joint health. However, most products made from shark cartilage (rich in type II collagen, accounting for 90% to 95% of the total cartilage protein) (such as shark cartilage powder capsules) have not undergone enzymatic hydrolysis, resulting in difficulties in digestion and absorption and insufficient function.

[0005] Collagenase (collagen hydrolase) is a key tool for degrading collagen to generate collagen tripeptides. Based on its source, it can be divided into three categories: animal-derived, plant-derived, and microbial-derived. Among these, microbial collagenases have become the preferred choice for industrial applications due to their broad substrate range, numerous cleavage sites, and low production costs. However, existing microbial collagenases have the following drawbacks: 1) Most are derived from pathogenic bacteria, posing safety risks; 2) Few collagenase strains from non-pathogenic bacteria have been developed, and those that exist generally suffer from low yield and low enzyme activity, making it difficult to meet the needs of large-scale production; 3) Collagenase purification processes are complex, and enzymatic hydrolysis efficiency is unstable, resulting in a low proportion of active components in the collagen tripeptide products.

[0006] Therefore, developing a method for efficiently preparing highly active collagen tripeptides (especially collagen tripeptides derived from type II collagen) based on safe probiotic-derived collagenase, and expanding the application scenarios of these collagen tripeptides, has become a pressing technical problem to be solved in this field. Summary of the Invention

[0007] To address the aforementioned issues, this invention provides a collagen tripeptide, its preparation method, and its applications. This method involves screening a safe, high-yield collagenase strain (Bacillus belyssus), optimizing the collagenase fermentation and purification process, and the collagen tripeptide enzymatic hydrolysis preparation process to obtain a highly active and easily absorbed collagen tripeptide, and expanding its applications in multiple fields.

[0008] The present invention adopts the following technical solution: In a first aspect, the present invention provides a method for preparing collagen tripeptides, comprising the following steps: S1. Isolation and Identification of High-Producing Collagenase-Producing Bacteria: Soil samples were collected from near the sewage outlet of a seafood market. After dilution with sterile phosphate buffer, the resuspension was spread onto gelatin plates and incubated overnight. Colonies with a clear ring around the periphery were picked, spread onto new gelatin plates, and incubated overnight. Single colonies were inoculated onto LB agar and incubated overnight. The supernatant was added to solidified gelatin tubes for incubation. Colonies exhibiting significant gelatin liquefaction were screened to obtain collagenase-producing bacteria. The bacterial species was identified using 16S rDNA, gyrA, and gyrB genes. The identified collagenase-producing bacteria were *Bacillus belesiensis*, a member of the *Bacillus* genus. *Bacillus belesiensis* is listed in the European Food Safety Authority's Qualified Presumption of Safety (QPS) list and is deemed generally safe by the US Food and Drug Administration. Furthermore, *Bacillus belesiensis* is also listed in the food ingredient list of the Korean Ministry of Food and Drug Safety. These standards indicate that *Bacillus belesiensis* is safe for use in the food industry. S2, Collagenase fermentation culture: The collagenase bacteria identified in step S1 were inoculated into LB medium and cultured in a shaker at 37°C until the logarithmic phase. They were then inoculated into fermentation medium at an inoculation ratio of 1%-8% and fermented at a constant temperature of 28°C-37°C for 24-48 hours. S3. Collagenase purification: The fermentation medium from step S2 is purified to obtain purified collagenase. S4. Preparation of collagen tripeptides: Wash cartilage or fish skin, then rinse with NaOH solution and distilled water in sequence, dry and pulverize; mix the pulverized cartilage powder or fish skin powder with water to swell, add protease containing purified collagenase, and enzymatically hydrolyze at pH 7-8 and 37℃-50℃ for 2-48 hours to obtain a reaction solution; heat the reaction solution to 80℃ and let it stand for 30 minutes to inactivate the enzyme, centrifuge at 4℃ and 12000rpm / min for 30 minutes, collect the supernatant; freeze-dry the supernatant to obtain collagen tripeptides.

[0009] Preferably, in step S1, the gelatin plate is selected from one or more of fish gelatin plates, bovine gelatin plates, and chicken gelatin plates; the incubation temperature in the incubator is 37°C, and the incubation time is 24 hours.

[0010] Preferably, in steps S1 and S2, the LB culture medium includes a nitrogen source, a carbon source, and water, with a pH of 7-8; the nitrogen source is selected from one or more of beef extract, peptone, yeast powder, and gelatin; and the carbon source is selected from one or more of glucose, sucrose, and glycerol.

[0011] Preferably, in step S2, the inoculation ratio is 3%, the constant temperature fermentation temperature is 31°C, and the constant temperature fermentation time is 36 hours.

[0012] Preferably, the specific process of step S3 is as follows: S31. Collect the fermentation medium from step S2 and centrifuge at 3000g for 30 minutes at 4℃. S32. Take the supernatant, add ammonium sulfate powder while stirring until the ammonium sulfate concentration is 10%-60%, and let it settle on ice for 1 hour; Centrifuge at 33°C, 4°C, and 3000g for 30 minutes. Take the supernatant and pass it sequentially through a cation exchange chromatography column and an anion exchange chromatography column. The eluted fraction is concentrated by a 10kDa ultrafiltration tube to obtain purified collagenase.

[0013] Preferably, in step S4, the cartilage is selected from one or more of shark cartilage, sturgeon cartilage, ray cartilage, beef cartilage, and chicken cartilage; the fish skin is selected from one or more of grass carp, tilapia, shark, and sturgeon; the concentration of NaOH in the NaOH solution is 0.1 mol / L, and the washing conditions of the NaOH solution are stirring at 25°C for 2 hours.

[0014] Preferably, in step S4, the ratio of cartilage powder or fish skin powder to water is 1:4-1:20; the protease is a combination of alkaline protease and purified collagenase; the pH of the enzymatic hydrolysis is 7.5, and the temperature of the enzymatic hydrolysis is 45°C; the amount of alkaline protease is 20U / g-400U / g, and the hydrolysis time is 2 hours; the amount of collagenase is 20U / g-400U / g, and the hydrolysis time is 48 hours; the freeze-drying conditions are -60°C freeze-drying for 3 hours.

[0015] Secondly, the present invention provides a collagen tripeptide, which is prepared by the method for preparing the collagen tripeptide.

[0016] Thirdly, the present invention provides an application of collagen tripeptide in the preparation of antioxidant products, anti-inflammatory products, memory-enhancing products, or immunity-enhancing products.

[0017] Preferably, the antioxidant product is composed of one or more of the collagen tripeptide, elastin peptide, nicotinamide, and sodium hyaluronate; the anti-inflammatory product, memory-enhancing product, or immunity-enhancing product is composed of one or more of the collagen tripeptide, spearmint extract, rosemary extract, DHA, γ-aminobutyric acid, curcumin, red beet, and probiotics.

[0018] By adopting the above technical solution, the present invention has the following advantages compared with the prior art: 1. The Bacillus berberis strain obtained by screening in this invention is a probiotic. The strain is safe and efficient and can be safely applied in the food industry. This strain has a strong ability to produce collagenase and the fermentation process is stable, which solves the safety hazards of collagenase from traditional pathogenic bacteria and the problem of low yield of non-pathogenic strains.

[0019] 2. The collagenase of this invention has high purity and strong enzyme activity. Through the purification process of "salting out + double chromatography + ultrafiltration", a collagenase with high purity and stable enzyme activity is obtained, which ensures efficient enzymatic hydrolysis.

[0020] 3. The collagen tripeptide prepared by this invention has a molecular weight of less than 500 Daltons and can be directly absorbed by the human body. Furthermore, by optimizing the enzymatic hydrolysis process (alkaline protease + collagenase combination), the proportion of active components (such as GHK tripeptide) in the product is significantly increased. The collagen tripeptide exhibits high activity, and experiments have shown that it inhibits UVB-induced photoaging, delays cartilage degeneration, and significantly inhibits CD4+. + T-cell activation has a good anti-inflammatory effect.

[0021] 4. This invention uses aquatic processing by-products such as shark cartilage and fish skin as raw materials to realize the resource utilization of waste, with high raw material utilization rate and reduced production costs; at the same time, enzymatic hydrolysis solves the problem of poor absorption of traditional shark cartilage powder and gives full play to the function of its type II collagen.

[0022] 5. The collagen tripeptide prepared by this invention can be used in various products such as anti-oxidation, anti-inflammation, memory enhancement, and immunity enhancement, meeting different consumer needs, with a wide range of applications and broad market prospects. Attached Figure Description

[0023] Figure 1 This is a diagram showing the phylogenetic analysis results of collagenase bacteria strains in this invention; Figure 2 This is a graph showing the SDS-PAGE detection results of collagenase in this invention. Figure 3 The figure shows the results of the enzymatic hydrolysis of shark cartilage powder by different enzyme combinations using the ninhydrin method of this invention. Figure 4 This is a diagram showing the results of the present invention regarding the inhibition of T cell activity by shark cartilage collagen protein hydrolysate, etc. Figure 5 The image shows the results of treating arthritis in mice with shark cartilage collagen protein hydrolysate and other products of the present invention. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0025] See Figures 1 to 5 Table 1.

[0026] Example 1: Isolation and Identification of High Collagenase-Producing Bacteria a) Take a certain amount of soil from near the sewage outlet of the seafood market, dilute it with 20 ml of sterile phosphate buffer, take 200 μl of the resuspension and spread it on a gelatin plate, and incubate it overnight at 37°C. b) Pick out colonies with a clear ring around them and spread them on a plate containing 1% fish gelatin. Incubate overnight at 37°C. c) Pick 100 different monoclonal colonies and inoculate them into LB medium. Incubate overnight at 37°C. Take 100 μL of the culture supernatant and add it to solidified gelatin medium. Incubate at 37°C for 24 hours and observe the liquefaction of the gelatin. Store the completely liquefied colonies. The LB medium includes a nitrogen source, a carbon source, and water, with a pH of 7.5. The nitrogen source is beef extract, and the carbon source is glucose. d) 16S rDNA, gyrA and gyrB were amplified using PCR, and the genes were sequenced and a phylogenetic tree was constructed, such as... Figure 1 As shown, strain CN24055 was identified as *Bacillus belyssae*, a member of the *Bacillus* genus and a probiotic. *Bacillus belyssae* is listed in the European Food Safety Authority's Qualified Presumption of Safety (QPS) list and is deemed generally safe by the US Food and Drug Administration. Furthermore, *Bacillus belyssae* is also listed in the food ingredient list of the Korean Ministry of Food and Drug Safety. These standards indicate that *Bacillus belyssae* is safe for use in the food industry. Meanwhile, *Bacillus belyssae* (… Bacillus velezensis The strain is a well-known and legally available strain, and is not subject to additional patent preservation here.

[0027] Example 2: Preparation method of collagenase Collagenase (Bacillus belye) was inoculated into LB medium and cultured on a shaker at 37°C until the OD600 reached 2.5. It was then inoculated at a 3% inoculation ratio into beef extract fermentation medium and fermented at 31°C for 36 hours. The fermentation broth was collected, centrifuged at 3000g at 4°C for 30 min, and the supernatant was collected. The supernatant was placed on ice, and ammonium sulfate powder was added while stirring until the final concentration reached 30%, and allowed to stand for 1 h. The supernatant was then collected by centrifugation at 3000g at 4°C for 30 min. The supernatant was incubated with cation exchange medium MMC for 1 h and eluted with a gradient of Tris buffer containing 10-100% NaCl. 10-20% and 30-80% fractions were collected separately and incubated with anion exchange medium MMA for 1 h, eluted with a gradient of Tris buffer containing 10-100% NaCl, and the flow-through was collected and concentrated using a 10 kDa ultrafiltration tube. The buffer was then replaced with PBS. Prepare a protein sample using 40 μL of enzyme solution and perform SDS-PAGE, as shown below. Figure 2 As shown, the purified collagenase band is approximately 40 kDa.

[0028] Example 3: Preparation method of shark cartilage collagen tripeptide a) Wash the shark cartilage, then wash it with 0.1 mol / L NaOH solution at 25℃ for 2 hours with stirring, and wash it 5 times with distilled water with stirring. After drying, crush it. b) Mix the pulverized cartilage powder with water at a ratio of 1:5, allow it to swell fully, adjust the pH to 7.5, add 100 U / g of alkaline protease, papain or bromelain respectively, and stir at 45°C for 2 h for enzymatic hydrolysis; then add 200 U / g of collagenase prepared in Example 2, and stir at 45°C for 48 h for enzymatic hydrolysis.

[0029] c) After the enzymatic hydrolysis is complete, heat the reaction solution to 80°C, let it stand for 30 minutes to fully inactivate the enzyme activity, and then centrifuge at 4°C, 12000 rpm / min for 30 minutes to collect the supernatant.

[0030] d) The supernatant from step c was freeze-dried at -60°C for 3 hours to obtain shark cartilage collagen tripeptide.

[0031] like Figure 3 As shown, different combinations of proteases yield different amounts of collagen tripeptides, with the combination of alkaline protease and collagenase yielding the highest amount of collagen tripeptides.

[0032] The products of collagenase hydrolysis of shark type II collagen were analyzed by LC-MS. The proportion of peptides with a molecular weight ≤500kDa was 42.96%, and the proportion of tripeptides was 10.11%, as shown in Table 1.

[0033] Table 1: LC-MS analysis of products from collagenase hydrolysis of shark type II collagen Example 4: Shark cartilage collagen tripeptide inhibits T cell activation Shark type II collagen was enzymatically hydrolyzed into collagen tripeptides using the collagenase prepared above, and the product content was detected using the ninhydrin method. PBMC cells were isolated from the blood of healthy individuals and stimulated with 1 μg / ml anti-human CD3 and CD28 antibodies. Simultaneously, 10 μg / ml of shark type II collagen enzymatic hydrolysate (SII hydrolysate), shark type II collagen, and 50 μg / ml chondroitin sulfate sodium were added, with a saline control included. After 6 h of culture, cells were collected and washed twice with 1% BSA+PBS, resuspended in 100 μl of 1% BSA+PBS, and APC-CD4 and PE-CD69 flow cytometry antibodies were added. The cells were incubated on ice in the dark for 30 min. After washing twice with 1% BSA+PBS, the cells were resuspended in 300 μl of 1% BSA+PBS and analyzed by flow cytometry. Figure 4 As shown, shark type II collagenase hydrolysates affect CD4. + The inhibitory effect on T cell activity was most significant.

[0034] Example 5: Shark cartilage enzymatic hydrolysates alleviate inflammatory markers in a mouse model of rheumatoid arthritis. Shark cartilage and shark type II collagen were enzymatically hydrolyzed using the collagenase prepared above. A Babl / c mouse model of arthritis was induced by injection of complete Freund's adjuvant into the paw. After 14 days of continuous observation and recording, mice with an inflammation score higher than 3 were divided into four groups: Group A (arthritis model group, fed with saline); Group B (fed with shark cartilage hydrolysate); Group C (fed with shark type II collagen hydrolysate); Group D (fed with undenatured shark type II collagen); and Group E (uninduced group). Feeding was performed once daily for 35 days at a dose of 1 mg / kg, and inflammatory symptoms were recorded. Finally, the mice were euthanized, and mRNA was extracted from the paw. The levels of inflammatory factors IL-6, IL-7A, IL-1β, and TNF-α were analyzed using qPCR. Figure 5 As shown, in the arthritis model group, all of the above inflammatory factors were significantly elevated, while in the BD group, the inflammation score and inflammatory factor mRNA levels were decreased to varying degrees. Among them, the group fed shark cartilage enzymatic hydrolysate showed the most significant decrease, even to the level of normal. There was no significant difference between the group fed shark type II collagen enzymatic hydrolysate and the group fed non-denatured shark type II collagen.

[0035] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for preparing collagen tripeptide, characterized in that, Includes the following steps: S1. Isolation and identification of high collagenase-producing bacteria: Soil was collected from near the sewage outlet of the seafood market, diluted with sterile phosphate buffer, and the resuspension was spread on gelatin plates and incubated overnight. Colonies with a clear zone were picked and spread on new gelatin plates and incubated overnight. Single colonies were picked and inoculated on LB medium and incubated overnight. The culture supernatant was added to solidified gelatin tubes and incubated. Colonies that significantly liquefy gelatin were screened. The bacterial species were identified using 16S rDNA, gyrA, and gyrB genes to obtain collagenase-producing bacteria. S2, Collagenase fermentation culture: The collagenase bacteria identified in step S1 were inoculated into LB medium and cultured in a shaker at 37°C until the logarithmic phase. They were then inoculated into fermentation medium at an inoculation ratio of 1%-8% and fermented at a constant temperature of 28°C-37°C for 24-48 hours. S3. Collagenase purification: The fermentation medium from step S2 is purified to obtain purified collagenase. S4. Preparation of collagen tripeptides: Wash cartilage or fish skin, then rinse with NaOH solution and distilled water in sequence, dry and pulverize; mix the pulverized cartilage powder or fish skin powder with water to swell, add protease containing purified collagenase, and enzymatically hydrolyze at pH 7-8 and 37℃-50℃ for 2-48 hours to obtain a reaction solution; heat the reaction solution to 80℃ and let it stand for 30 minutes to inactivate the enzyme, centrifuge at 4℃ and 12000rpm / min for 30 minutes, collect the supernatant; freeze-dry the supernatant to obtain collagen tripeptides.

2. The method for preparing collagen tripeptide as described in claim 1, characterized in that: In step S1, the gelatin plate is selected from one or more of fish gelatin plates, bovine gelatin plates, and chicken gelatin plates; the incubation temperature in the incubator is 37°C, and the incubation time is 24 hours; the collagenase bacteria species identified is Bacillus belesiensis of the Bacillus genus.

3. The method for preparing collagen tripeptide as described in claim 1, characterized in that: In steps S1 and S2, the LB medium includes a nitrogen source, a carbon source, and water, with a pH of 7-8; the nitrogen source is selected from one or more of beef extract, peptone, yeast powder, and gelatin; the carbon source is selected from one or more of glucose, sucrose, and glycerol.

4. The method for preparing collagen tripeptide as described in claim 1, characterized in that: In step S2, the inoculation ratio is 3%, the constant temperature fermentation temperature is 31℃, and the constant temperature fermentation time is 36 hours.

5. The method for preparing collagen tripeptide as described in claim 1, characterized in that, The specific process of step S3 is as follows: S31. Collect the fermentation medium from step S2 and centrifuge at 3000g for 30 minutes at 4℃. S32. Take the supernatant, add ammonium sulfate powder while stirring until the ammonium sulfate concentration is 10%-60%, and let it settle on ice for 1 hour; Centrifuge at 33°C, 4°C, and 3000g for 30 minutes. Take the supernatant and pass it sequentially through a cation exchange chromatography column and an anion exchange chromatography column. The eluted fraction is concentrated by a 10kDa ultrafiltration tube to obtain purified collagenase.

6. The method for preparing collagen tripeptide as described in claim 1, characterized in that: In step S4, the cartilage is selected from one or more of shark cartilage, sturgeon cartilage, ray cartilage, beef cartilage, and chicken cartilage; the fish skin is selected from one or more of grass carp, tilapia, shark, and sturgeon; the concentration of NaOH in the NaOH solution is 0.1 mol / L, and the washing conditions of the NaOH solution are stirring at 25°C for 2 hours.

7. The method for preparing collagen tripeptide as described in claim 1, characterized in that: In step S4, the ratio of cartilage powder or fish skin powder to water is 1:4-1:20; the protease is a combination of alkaline protease and purified collagenase; the pH of the enzymatic hydrolysis is 7.5, and the temperature of the enzymatic hydrolysis is 45℃; the enzyme amount of the alkaline protease is 20U / g-400U / g, and the enzymatic hydrolysis time is 2 hours; the enzyme amount of the collagenase is 20U / g-400U / g, and the enzymatic hydrolysis time is 48 hours; the freeze-drying conditions are -60℃ freeze-drying for 3 hours.

8. A collagen tripeptide, characterized in that: It is prepared by the method of any one of claims 1-7.

9. An application of the collagen tripeptide as described in claim 8, characterized in that: The collagen tripeptide is used in the preparation of antioxidant products, anti-inflammatory products, memory-enhancing products, or immunity-enhancing products.

10. The application of the collagen tripeptide as described in claim 9, characterized in that: The antioxidant product is composed of one or more of the collagen tripeptide, elastin peptide, nicotinamide, and sodium hyaluronate; the anti-inflammatory product, memory-enhancing product, or immunity-enhancing product is composed of one or more of the collagen tripeptide, spearmint extract, rosemary extract, DHA, γ-aminobutyric acid, curcumin, red beet, and probiotics.

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