A recombinant collagenase, a rabbit collagen peptide, and a preparation method and application thereof

Abstract

The application discloses a recombinant collagenase, a rabbit collagen peptide and a preparation method and application thereof, and belongs to the technical field of biology. The recombinant collagenase is obtained by connecting a catalytic domain of ColG collagenase and a FnII type binding domain of human fibronectin through a flexible connecting peptide; and the flexible connecting peptide is (GGGGS) 3. The recombinant collagenase has stronger binding specificity and higher affinity to rabbit skin type I collagen, greatly improves the local relative concentration of the enzyme and the substrate, thereby significantly improving the hydrolysis efficiency and reaction speed, and can more accurately release a peptide segment with biological activity.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a recombinant collagen hydrolase, rabbit collagen peptides, their preparation methods and applications. Background Technology

[0002] China boasts the world's largest rabbit industry, being the world's leading rabbit-raising nation, with rabbit meat production accounting for approximately 50% of global output. According to the "China Animal Husbandry Yearbook" and related industry reports, the annual output of meat rabbits has remained stable at 300-350 million in recent years. However, in this rabbit meat-centric industrial chain, the value of rabbit skin, the largest by-product, is severely underestimated. The valuable collagen resources in rabbit skin are wasted, and potential environmental problems arise.

[0003] Collagen peptides, as a functional food ingredient, have been widely recognized for their efficacy in improving skin health. However, rabbit skin collagen peptides prepared using enzymatic hydrolysis methods in existing technologies often have large molecular weights and low levels of bioactive peptides, failing to achieve the expected effects. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a recombinant collagen hydrolase, rabbit collagen peptides, their preparation methods and applications, to overcome the problem that the content of bioactive peptides in rabbit skin collagen peptides prepared by enzymatic hydrolysis in the prior art is low, resulting in weak efficacy of rabbit skin collagen peptides.

[0005] In a first aspect, the present invention provides a recombinant collagen hydrolase, which is obtained by linking the catalytic domain of ColG collagenase and the FnII type binding domain of human fibronectin via a flexible linker peptide; the flexible linker peptide is... .

[0006] Compared to existing collagen hydrolases, the recombinant collagen hydrolase of this invention exhibits stronger binding specificity and higher affinity for rabbit skin type I collagen. This allows for precise "immobilization" of the enzyme molecule onto the rabbit skin collagen fiber substrate, significantly increasing the local relative concentration of the enzyme and substrate. This results in a substantial improvement in hydrolysis efficiency and reaction rate, and more precise release of bioactive peptides. Furthermore, the specific combination of the catalytic domain of ColG collagenase and the FnII-type binding domain of human fibronectin in this invention, when linked by a flexible peptide, allows for correct folding and maintenance of their respective biological functions in a Pichia pastoris expression system. This prevents functional loss due to misfolding or steric hindrance. The flexible linker peptide... Sufficient spatial distance and degrees of freedom of movement are provided to ensure that the catalytic domain can still efficiently cleave the substrate after the binding domain binds to it.

[0007] Furthermore, the amino acid sequence of the recombinant collagen hydrolase is shown in SEQ ID NO:1.

[0008] Optionally, the gene sequence encoding the recombinant collagen hydrolase is shown in SEQ ID NO:2.

[0009] Secondly, the present invention provides a method for preparing a recombinant collagen hydrolase, which includes the following steps: The gene encoding recombinant collagen hydrolase was introduced into a host strain to obtain a recombinant strain; The recombinant strain was fermented to express recombinant collagen hydrolase.

[0010] Compared with existing technologies, the present invention prepares recombinant collagen hydrolase through bio-fermentation. This method is low in cost, has good reproducibility, and can prepare recombinant collagen hydrolase simply, conveniently and quickly.

[0011] Thirdly, the present invention provides a method for preparing rabbit collagen peptides, comprising the following steps: Rabbit skin was subjected to enzymatic degreasing and enzymatic hair removal in sequence to obtain rabbit skin collagen; Rabbit skin collagen was hydrolyzed with the above-mentioned recombinant collagen hydrolase in an aqueous solution to obtain collagen hydrolysate. The hydrolysis reaction was carried out at a temperature of 40℃~50℃ and a pH value of 7.0~8.0. The collagen hydrolysate was filtered and purified to obtain rabbit collagen peptides.

[0012] Compared with existing technologies, this application uses recombinant collagen hydrolase to enzymatically hydrolyze rabbit skin collagen, enabling more complete and thorough hydrolysis. The recombinant collagen can more effectively further degrade intermediate products (large and medium molecular weight peptides), thereby significantly increasing the relative content and absolute yield of the small-molecule functional dipeptides Pro-Hyp and Hyp-Gly in the final product. Furthermore, due to the more thorough hydrolysis, the molecular weight distribution of the final rabbit collagen peptide product is more concentrated (500 Da~2000 Da), with higher content of key active ingredients and smaller batch-to-batch variations, greatly improving the stability of the rabbit collagen peptide product quality.

[0013] Furthermore, the ratio of recombinant collagen hydrolase to rabbit skin collagen in the aqueous solution is (4000U~6000U):1g.

[0014] Further, enzymatic defatting includes: using lipase to defatt the rabbit skin at a temperature of 30℃~45℃ and a pH of 7.0~9.0; after the reaction, allowing it to stand and separate into layers, separating the upper light phase rich in fatty acids and the lower aqueous phase; filtering the aqueous phase to obtain the defatted rabbit skin as the solid; and / or, The ratio of lipase to rabbit skin is (50g~200g):100kg.

[0015] The above technical solution further defines the reaction conditions, steps, and ratio of lipase to rabbit skin for enzymatic degreasing. Under the above reaction conditions and addition amount, lipase can more effectively remove the oil from rabbit skin.

[0016] Further, enzymatic hair removal includes: using keratinase to perform a hair removal reaction on degreased rabbit skin obtained after enzymatic defatting at a temperature of 40℃~55℃ and a pH value of 8.0~10.0; after the reaction, separating the shed hair from the solid phase of rabbit skin collagen by mechanical separation to obtain hair and rabbit skin collagen respectively; and / or, The ratio of keratinase to defatted rabbit skin is (100g~300g): 100kg.

[0017] The above technical solution further defines the reaction conditions, steps, and ratio of keratinase to defatted rabbit skin for enzymatic hair removal. Under the above reaction conditions and addition amount, keratinase can more effectively hydrolyze hair keratin, causing the hair to fall off the surface of the rabbit skin.

[0018] Furthermore, the rabbit skin is subjected to enzymatic degreasing and enzymatic hair removal in sequence to obtain rabbit skin collagen, which also includes: The upper light phase, rich in fatty acids, obtained after separation, is subjected to a saponification reaction, and the fatty acid salts are recovered after the reaction is completed; and / or, The separated hair was subjected to alkaline hydrolysis to prepare liquid fertilizer.

[0019] The above technical solution further recycles and utilizes the byproducts generated after enzymatic degreasing and hair removal of rabbit skin, reducing resource waste, achieving efficient resource utilization, and improving economic benefits.

[0020] Fourthly, the present invention provides a rabbit collagen peptide, which is prepared by the above preparation method. The total content of Pro-Hyp and Hyp-Gly in the rabbit collagen peptide is 1.5% to 1.7% by mass percentage.

[0021] Compared with existing rabbit collagen peptides, the rabbit collagen peptides of the present invention have a further increased content of small molecule functional dipeptides Pro-Hyp and Hyp-Gly, thereby enabling the rabbit collagen peptides of the present invention to effectively stimulate the proliferation of human dermal fibroblasts and upregulate the gene expression of type I collagen (COL1A1) and elastin (ELN).

[0022] Fifthly, the present invention provides the application of the above-mentioned rabbit collagen peptides in the preparation of products that enhance skin elasticity and promote collagen synthesis.

[0023] Compared with the prior art, the rabbit collagen peptide of the present invention has good intestinal transmembrane absorption capacity, effectively promotes the expression of type I collagen (COL1A1) and elastin (ELN) genes in human dermal fibroblasts, thereby further promoting the synthesis of type I collagen and elastin and improving skin elasticity. Detailed Implementation

[0024] To make the technical problems to be solved, the technical solutions, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are only for explaining the present invention and are not intended to limit the present invention.

[0025] It should be understood that, unless otherwise specified, all raw materials used in the following examples are commercially available.

[0026] Example 1 Construction, expression and purification of recombinant collagen hydrolase Based on publicly available databases, the amino acid sequences of the catalytic domain of ColG collagenase from *Clostridium histolyticum* and the FnII-type binding domain of human fibronectin were selected. Specifically, the amino acid sequence of the catalytic domain of ColG collagenase corresponds to amino acid residues 207-750 of NCBI accession number WP_021687132.1; the FnII-type binding domain of human fibronectin is specifically the FnII1 domain, whose amino acid sequence corresponds to amino acid residues 275-317 of UniProt accession number P02751. The amino acid sequences of these two domains were then linked by a flexible linker peptide. The amino acid sequence of the recombinant collagen hydrolase is obtained by connecting the amino acids.

[0027] The catalytic domain of ColG collagenase corresponds to the amino acid residues 207-750. The inventors discovered that the N-terminal leader peptide of the wild-type ColG collagenase catalytic domain (85-750 segment) has strong hydrophobicity. If it is directly fused to the Pichia pastoris α-factor secretion signal peptide, it will cause steric hindrance, which will interfere with the accurate cleavage of the signal peptidase and result in low secretion efficiency. Therefore, this invention removes it to ensure the efficient secretion of recombinant collagen hydrolase in the host strain.

[0028] The amino acid sequence of the above-mentioned recombinant collagen hydrolase was reverse-translated into a DNA sequence, and optimized according to the codon preference of Pichia pastoris to obtain the target gene sequence, which was then sent to a commercial company for synthesis.

[0029] Using the synthesized target gene sequence as a template, and F1 and R1 as primers, the target gene was amplified by PCR, and the target gene obtained by PCR was double-digested with restriction endonucleases EcoRI and XbaI.

[0030] Forward primer F1 (containing EcoRI recognition site): 5'-CCGGAATTCATGAAAATCGCTTTCAATTCCTAT-3' (SEQ ID NO: 3); Reverse primer R1 (containing the Xba I recognition site): 5'- GCT TCTAGA TGAAGCTCTTGAGATGCAGT-3' (SEQ ID NO: 4).

[0031] The expression vector pPICZα A was double-digested with restriction endonucleases EcoR I and Xba I to obtain the linearized vector pPICZα A. The target gene after digestion was then ligated with the linearized vector pPICZα A to obtain the recombinant plasmid pPICZα-rRC-EP-S1. The recombinant plasmid... After extraction, the enzyme was digested with SacI restriction endonuclease to linearize it in the AOX1 promoter region, which facilitates subsequent homologous recombination and integration.

[0032] The expression vector pPICZαA contains the gene sequence of the secretion signal peptide of Pichia pastoris α-factor and the gene sequence of 6xHis-tag.

[0033] The linearized recombinant plasmid pPICZα-rRC-EP-S1 was transformed into the Pichia pastoris protease A-deficient strain SMD1168H (pep4) by electroporation, and positive transformants were screened out.

[0034] The positive transformants obtained from the above screening were inoculated into a 5L fermenter for high-density fermentation to achieve efficient expression of the target protein. The specific steps are as follows: 1. Preparation of seed culture: The above positive transformants were inoculated into 50 mL of BMGY medium (initial pH 6.0) and cultured with shaking at 30℃ and 250 rpm for 36 hours until... Once the value reaches 5, seed liquid is obtained.

[0035] 2. Fermentation tank culture (glycerol batch culture): Inoculate the seed culture into a 5L fermenter at a 10% (v / v) inoculation rate. The fermenter should contain 4L of basal salt medium (BSM). Maintain the temperature at 30℃, pH at 5.0 (automatically controlled by adding 25% (v / v) ammonia), and DO (dissolved oxygen) above 30% (adjusted by adjusting the turbine speed (500-800 rpm) and aeration rate). Culture for 24 hours until the glycerol is depleted. Reached 100.

[0036] The formulation of the above-mentioned basal salt medium (BSM) is as follows: 26.7 mL / L of 85% phosphate, 0.93 g / L of calcium sulfate, 18.2 g / L of potassium sulfate, and magnesium sulfate. 14.9 g / L, potassium hydroxide 4.13 g / L, glycerol 40.0 g / L.

[0037] 3. Glycerin Feeding Stage: Begin by adding a 50% (w / v) glycerin feed solution (i.e., 50g glycerin per 100mL of feed solution, with the remainder being water). This feed solution also contains 1.2% (v / v) PTM1 trace element. The flow rate should be controlled to maintain a DO of approximately 30%. This feeding stage lasts for 6 hours. Reached 200.

[0038] 4. Methanol Induction Phase: Stop adding glycerol feed solution and start adding methanol containing 1.2% (v / v) PTM1 trace element to induce expression. The initial flow rate was 3.5 mL / h / L, gradually increased to 15 mL / h / L, while maintaining DO above 30% throughout. During methanol induction, the temperature was lowered to 28℃, and induction continued for 96 hours.

[0039] After fermentation, the fermentation broth was centrifuged at 10000xg and 4℃ for 20 minutes, and the fermentation supernatant was collected. The recombinant collagen hydrolase in the supernatant was purified by Ni-NTA affinity chromatography. The purification results are shown in Table 1.

[0040] The specific steps for purifying recombinant collagen hydrolase in the supernatant by Ni-NTA affinity chromatography are as follows: Equilibration: Mix the fermentation supernatant (4.5 L) with an equal volume of binding buffer A (containing 40 mM sodium phosphate, 1 M NaCl and 40 mM imidazole, pH 7.4).

[0041] Sample loading: Flow the mixed sample (total volume) through a Ni-NTA affinity chromatography column (GE HisTrap FF 5mL column) pre-equilibrated with binding buffer A, with a loading flow rate controlled at 2.5 mL / min.

[0042] Elution: Wash away contaminating proteins using 10 column volumes (i.e., 50 mL) of buffer B containing 100 mM imidazole (containing 20 mM sodium phosphate, 500 mM NaCl and 100 mM imidazole, pH 7.4) at a flow rate of 5.0 mL / min.

[0043] Collection: Finally, the target protein was eluted using elution buffer C containing 500 mM imidazole (containing 20 mM sodium phosphate, 500 mM NaCl and 500 mM imidazole, pH 7.4) at a flow rate of 2.0 mL / min, and the elution peak was collected.

[0044] Desalting: The collected protein solution was placed into a dialysis bag with a pore size of 3 nm and dialyzed overnight at 4 °C with 20 mM sodium phosphate buffer (pH 7.4) to remove imidazole and high concentration of salt, resulting in purified recombinant collagen hydrolase solution.

[0045] Table 1 The amino acid sequence of the recombinant collagen hydrolase of the present invention is shown in SEQ ID NO:1, and the nucleotide sequence of its gene is shown in SEQ ID NO:2.

[0046] It should be understood that the last 7 amino acids at the C-terminus of the recombinant collagen hydrolase shown in SEQ ID NO:1 are due to the use of a reverse primer containing an Xba I recognition site during PCR amplification of the target gene, resulting in the presence of these 7 amino acids at the end of the recombinant collagen hydrolase after translational expression. This does not affect the folding and activity of the recombinant collagen hydrolase protein.

[0047] Example 2 Preparation of rabbit skin collagen Raw material receiving and cleaning: Collect 100kg of fresh rabbit skins from a large rabbit slaughterhouse, rinse with running water for 30 minutes to remove blood, feces and other impurities from the surface of the rabbit skins.

[0048] Enzymatic degreasing: The cleaned rabbit skin was placed in a 500L reactor, and 300L of deionized water was added. The pH was adjusted to 8.0 using 1M NaOH solution. 100g of lipase (derived from Aspergillus oryzae, enzyme activity ≥100,000 U / g) was added to the reactor, and stirring was started at 37℃ and 60 rpm for 4 hours. After the reaction, the mixture was allowed to stand and separate into layers. The upper light phase was rich in fatty acids, and the degreased rabbit skin precipitated in the lower aqueous phase. The upper fatty acid-rich liquid light phase was transferred to a saponification reactor using a peristaltic pump. The lower aqueous phase containing the degreased rabbit skin was then filtered to obtain the degreased rabbit skin.

[0049] It should be understood that when using a peristaltic pump to transfer the upper layer of fatty acid-rich liquid light phase, conventional operating procedures in the art can be followed.

[0050] In some embodiments, the fatty acids rich in the upper light phase can also be recycled. Specifically, the upper liquid light phase rich in fatty acids is transferred to a saponification reactor using a pump. A 5M NaOH solution is added to the reactor to adjust the pH to 11. The reactor is heated to 70°C and maintained for 2 hours for saponification. After the reaction, solid NaCl particles are added for salting out. The mixture is stirred until NaCl is saturated and allowed to stand to allow complete precipitation of the soap particles (sodium fatty acid salts). The mixture is cooled, filtered, and the solid soap particles are collected. After drying in a vacuum oven at 60°C for 12 hours, approximately 3.5 kg of cosmetic-grade soap base raw material is obtained.

[0051] Enzymatic hair removal: The defatted rabbit skin obtained after filtration was transferred to another 500L reaction vessel, and 300L of deionized water was added. The pH was adjusted to 9.5 using 1M NaOH solution. 200g of keratinase (derived from Bacillus licheniformis, enzyme activity ≥600 U / mg) was added to the reaction vessel, and the mixture was stirred at 80 rpm at 50℃ for 6 hours until the rabbit hair on the defatted rabbit skin was completely separated from the skin plate. After the reaction, the reaction mixture was passed through a 1mm pore size rotary drum sieve for solid-liquid separation. The detached hair was retained on the sieve, while the solid phase of rabbit skin collagen and aqueous solution passed through the sieve. The rabbit skin collagen and aqueous solution that passed through the sieve were further filtered through a 100-mesh filter cloth, and the filter residue retained on the filter cloth was collected to obtain clean rabbit skin collagen that was basically free of fat and hair. The rabbit skin collagen was then repeatedly washed with water until the pH of the effluent was neutral. After draining, it was weighed for subsequent enzymatic hydrolysis.

[0052] In some embodiments, the shed hair can also be recycled. The specific method is as follows: after the reaction is completed, the hair trapped on the sieve is collected to obtain 12g of wet hair, which is placed in 50L of 10% (w / v) NaOH solution and hydrolyzed at 80°C for 4 hours. After cooling, the reaction mixture is filtered through a 100-mesh filter cloth to remove unhydrolyzed residues. The filtrate is collected to obtain 50L of liquid organic fertilizer rich in amino acids.

[0053] Example 3 The recombinant collagen hydrolase solution purified in Example 1 was used to precisely quantify its protein concentration using the BCA method. Rabbit skin collagen prepared in Example 2 was used as a substrate and suspended in 50 mM Tris-HCl buffer (pH 7.5). The recombinant collagen hydrolase solution was added to bring the final concentration of the recombinant collagen hydrolase in the reaction system to 5 nM. Enzymatic degradation was carried out under optimal reaction conditions of 37°C and pH 7.5. A series of substrate concentrations (0.1 mg / mL, 0.5 mg / mL, 1.0 mg / mL, 2.0 mg / mL, 3.0 mg / mL, 4.0 mg / mL, 5.0 mg / mL) were set. Under each substrate concentration condition, samples were taken at multiple time points (0 min, 5 min, 10 min, 15 min, 30 min) after the start of the reaction. The initial reaction rate at different substrate concentrations was calculated by detecting the amount of amino groups generated in the reaction product. The amino groups of the reaction product were detected using the OPA method, with absorbance measured at 340 nm.

[0054] Data Analysis: Based on the initial reaction rates at different substrate concentrations, the Michaelis constant (Km) was calculated using the Lineweaver-Burk double reciprocal plotting method. m ), maximum reaction rate (V max And according to formula k cat =V max / [E] Calculate the transformation number (k) cat ), where [E] is the total enzyme concentration. Catalytic efficiency is determined by k. cat / K m express.

[0055] The Michaelis constant (Ki) of commercially available, natural Clostridium histolytica ColG collagenase was calculated using the same method described above. m ), maximum reaction rate (V max ), transformation number (k) cat ) and catalytic efficiency (k cat / K m The results of comparing the recombinant collagen hydrolase of the present invention with those of the present invention are shown in Table 2.

[0056] Table 2 From the above results, we can conclude that: Compared to the natural Clostridium histolyticum ColG collagenase, the recombinant collagen hydrolase of this invention has a higher K content. m The significantly decreased value indicates that the recombinant collagen hydrolase has a higher affinity for the substrate rabbit skin collagen; the recombinant collagen hydrolase's... An increase in the value indicates a further increase in the maximum reaction rate of recombinant collagen hydrolase; the recombinant collagen hydrolase... An increased value indicates that the recombinant collagen hydrolase catalyzes a greater number of substrates per unit time per enzyme molecule; the recombinant collagen hydrolase's... The significantly increased value indicates that the catalytic efficiency of the recombinant collagen hydrolase has been significantly improved, by about 3.4 times, which means that it can hydrolyze the substrate rabbit skin collagen more efficiently in the same amount of time.

[0057] Example 4 Preparation of rabbit collagen peptides Preparation of concentrated fermentation enzyme solution: The Pichia pastoris positive transformant constructed in Example 1 was fermented at high density according to the fermentation method in Example 1. After fermentation, the fermentation broth was centrifuged at 8000 xg for 15 minutes to remove the precipitated Pichia pastoris cells. The supernatant obtained was the crude enzyme solution.

[0058] The crude enzyme solution was concentrated to 1 / 10 of its original volume using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa. Small molecule impurities and some culture medium components were removed to obtain the concentrated fermentation enzyme solution. The activity of recombinant collagen hydrolase in the concentrated fermentation enzyme solution was determined to be 50,000 U / mL.

[0059] Enzyme activity unit definition: The amount of enzyme required to release 1 μmol of glycine from collagen substrate per minute under conditions of 37°C and pH 7.5 is defined as 1 activity unit (U).

[0060] Enzymatic hydrolysis: The rabbit skin collagen prepared in Example 2 above was added to deionized water to obtain a 10% (w / v, g / 100mL) collagen suspension. The temperature was raised to 45℃, and the pH was adjusted to 7.5 with 1M NaOH solution. The prepared fermentation concentrate enzyme solution was added to the collagen suspension at a ratio of 50,000 U recombinant collagen hydrolase per gram of rabbit skin collagen. Hydrolysis was carried out at 45℃ and stirred at 120 rpm for 4 hours.

[0061] Enzyme inactivation and product purification: After hydrolysis, the reaction solution was rapidly heated to 90°C and maintained for 15 minutes to completely inactivate the recombinant collagen hydrolytic enzymes in the reaction solution. After cooling, the solution was filtered through a plate and frame filter (10 µm pore size) to remove a small amount of insoluble matter, obtaining a clear rabbit collagen peptide hydrolysate. The rabbit collagen peptide hydrolysate was then passed through activated carbon for decolorization and nanofiltration membrane (500 Da molecular weight cutoff) to remove inorganic salts and very small molecule amino acids. Finally, the retentate was spray-dried to obtain rabbit collagen peptide powder.

[0062] Molecular weight distribution analysis: The above-mentioned rabbit collagen peptide powder was analyzed by gel permeation chromatography (GPC). The results showed that more than 92% (mass percentage) of the peptides in the rabbit collagen peptide powder had a molecular weight of less than 3000 Da, of which 78% (mass percentage) of the peptides were concentrated between 500 Da and 2000 Da.

[0063] LC-MS / MS peptide spectrometry analysis: Mass spectrometry analysis of the above-mentioned rabbit collagen peptide powder identified a variety of dipeptides and tripeptides containing Pro-Hyp and Hyp-Gly. Quantitative analysis confirmed that the total content of Pro-Hyp and Hyp-Gly reached 1.62% (w / w), which is higher than that of conventional enzymatic hydrolysis products.

[0064] Example 5 Testing the intestinal absorption capacity of rabbit collagen peptides The three identical models were established, and the specific steps were as follows: Caco-2 cells were used... Cells / insert were seeded at a density of [number] cells / insert on Transwell chambers coated with type I collagen and incubated at 37°C with 5% [temperature]. A tightly connected monolayer cell model was formed by culturing cells in an incubator for 21 days. Transmembrane resistance was measured. Confirm the integrity of the model.

[0065] Osmosis experiment: A solution (10 mg / mL, 0.5 mL) of rabbit collagen peptide powder prepared in Example 4 dissolved in water, a solution (10 mg / mL, 0.5 mL) of rabbit collagen peptide powder prepared using natural Clostridium histolyticum ColG collagenase according to the method of Example 4 dissolved in water, and a solution (10 mg / mL, 0.5 mL) of commercially available rabbit collagen peptide (purchased from Anhui Shengmeinuo Biotechnology Co., Ltd., model R-CP90) dissolved in water were added to the apical (AP) side of the cells in the three models, respectively. Samples were taken from the basal (BL) side of the three models at different time points (30 min, 60 min, 120 min, and 180 min) to detect the hydroxyproline concentration and calculate the osmosis volume.

[0066] Results: The apparent permeability coefficient (Papp) of the rabbit collagen peptides of this invention was calculated to be: The apparent permeability (Papp) of rabbit collagen peptides prepared using natural Clostridium histolyticum ColG collagenase according to the method of Example 4 was [value missing]. The apparent permeability (Papp) of commercially available rabbit collagen peptide solutions is: .

[0067] As can be seen from the above results, the rabbit collagen peptide of the present invention has a higher apparent permeability coefficient and better potential for transmembrane absorption in the intestine.

[0068] Example 6 In vitro efficacy verification Experimental Design: Human dermal fibroblasts (HDFs) were used in the experiment. The cells were divided into groups A, B, C, D and E. The cells were seeded in 6-well plates and cultured in DMEM medium containing 10% (v / v) FBS until the cell confluence in the wells reached 80%. Then the medium was replaced with serum-free DMEM medium for starvation treatment for 12 hours. The medium was then replaced with serum-free DMEM medium containing different test samples for further treatment.

[0069] Group A: Blank control group: Serum-free DMEM culture medium containing no additional components.

[0070] Group B: Positive control group: Commercially synthesized Pro-Hyp was added to serum-free DMEM medium to a final concentration of 50 µM.

[0071] Group C: Rabbit collagen peptides prepared in Example 4 were added to serum-free DMEM medium to achieve a final concentration of 100 µg / mL.

[0072] Group D: Rabbit collagen peptides prepared using natural Clostridium histolytica ColG collagenase according to the method in Example 4 were added to serum-free DMEM medium to achieve a final concentration of 100 µg / mL.

[0073] Group E: Commercially available rabbit collagen peptides (purchased from Anhui Shengmeinuo Biotechnology Co., Ltd., model R-CP90) were added to serum-free DMEM culture medium to achieve a final concentration of 100 µg / mL.

[0074] 1. Cell proliferation experiment: After 48 hours of cell treatment, 10 µL of CCK-8 solution was added to each well, and incubation continued for 2 hours. The absorbance was measured at 450 nm using a microplate reader, and the results are shown in Table 3.

[0075] 2. Gene expression experiment: After 24 hours of cell treatment, total RNA was extracted from each group of cells and expressed separately. The technology was used to detect the mRNA expression levels of type I collagen (COL1A1) and elastin (ELN) genes. The primers used in the detection are shown in Table 4, and the detection results are shown in Table 5.

[0076] Table 3 Compared to Group A , Compared to Group A .

[0077] From the above results, we can conclude that:

[0078] The cell proliferation rate (OD value) of group C was significantly higher than that of groups A, D, and E, and close to that of the positive control group B, indicating that the rabbit collagen peptide of the present invention can effectively promote the proliferation of HDFs cells.

[0079] Table 4 primers Table 5 Gene Expression Levels Note: Compared with the blank control group .

[0080] From the above results, we can conclude that: Compared to rabbit collagen peptides prepared using natural Clostridium histolyticum ColG collagenase according to the method of Example 4 and commercially available rabbit collagen peptides, the rabbit collagen peptides prepared in Example 4 of this invention can effectively upregulate the gene expression of type I collagen (COL1A1) and elastin (ELN) in human dermal fibroblasts.

[0081] Example 7 Human clinical efficacy verification Recruiting 75 healthy female volunteers aged 35-55, using the German company CK. A skin elasticity tester was used to measure the biomechanical parameters of the skin on the inner forearm of each volunteer, and the volunteers were randomly divided into five groups: Group 1: Volunteers took 5g of placebo orally daily. The placebo was maltodextrin with added citric acid and steviol glycosides to mimic the appearance and flavor of the product. Group Two: Volunteers orally administered 5g of commercially available fish collagen peptides daily. These fish collagen peptides were purchased from Rousselot and their brand name is [Brand Name Missing]. ; Group 3: Volunteers orally administered 5g of the rabbit collagen peptide prepared in Example 4 daily; Group 4: Volunteers orally administered 5g of rabbit collagen peptides prepared using natural Clostridium histolyticum ColG collagenase according to the method in Example 4 daily; Group 5: Volunteers took 5g of commercially available rabbit collagen peptides (purchased from Anhui Shengmeinuo Biotechnology Co., Ltd., model R-CP90) orally every day.

[0082] Eight weeks later, the German company CK was used again. The biomechanical parameters of the skin on the inner forearm of each group of volunteers were measured using a skin elasticity tester and compared with the data before the experiment. The results are shown in Table 6.

[0083] Table 6. Changes in key parameters of skin elasticity after 8 weeks Indicates a comparison with the placebo group (Group 1) .

[0084] The results above show that after oral administration of the rabbit collagen peptides prepared in this invention for 8 weeks, it can significantly improve various elasticity indicators of the skin, and the effect is better than that of commercially available conventional fish collagen peptides, rabbit collagen peptides prepared using natural Clostridium histolyticum ColG collagenase, and commercially available rabbit collagen peptides.

[0085] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A recombinant collagen hydrolase, characterized in that, The recombinant collagen hydrolase is obtained by linking the catalytic domain of ColG collagenase and the FnII type binding domain of human fibronectin through a flexible linker peptide; the flexible linker peptide is (GGGGS)3.

2. The recombinant collagen hydrolase according to claim 1, characterized in that, The amino acid sequence of the recombinant collagen hydrolase is shown in SEQ ID NO:

1.

3. A method for preparing a recombinant collagen hydrolase, used to prepare the recombinant collagen hydrolase according to claim 1 or 2, characterized in that, Includes the following steps: The gene encoding the recombinant collagen hydrolase was introduced into a host strain to obtain a recombinant strain; The recombinant strain was fermented to express the recombinant collagen hydrolase.

4. A method for preparing rabbit collagen peptides, characterized in that, Includes the following steps: Rabbit skin was subjected to enzymatic degreasing and enzymatic hair removal in sequence to obtain rabbit skin collagen; The rabbit skin collagen is hydrolyzed in an aqueous solution with the recombinant collagen hydrolase described in claim 1 or 2 to obtain a collagen hydrolysate. The hydrolysis reaction is carried out at a temperature of 40°C to 50°C and a pH value of 7.0 to 8.

0. The collagen hydrolysate was filtered and purified to obtain rabbit collagen peptides.

5. The preparation method according to claim 4, characterized in that, The ratio of recombinant collagen hydrolase to rabbit skin collagen in the aqueous solution is (4000U~6000U):1g.

6. The preparation method according to claim 4, characterized in that, The enzymatic degreasing method includes: using lipase to degrease the rabbit skin at a temperature of 30℃~45℃ and a pH of 7.0~9.0; after the reaction, allowing the skin to stand and separate into layers, obtaining an upper light phase rich in fatty acids and a lower aqueous phase; filtering the aqueous phase to obtain a solid, which is degreased rabbit skin; and / or, The ratio of the lipase to the rabbit skin is (50g~200g):100kg.

7. The preparation method according to claim 4, characterized in that, The enzymatic hair removal method includes: using keratinase to perform a hair removal reaction on the degreased rabbit skin obtained after enzymatic degreasing at a temperature of 40℃~55℃ and a pH value of 8.0~10.0; after the reaction, the detached hair is separated from the solid phase of rabbit skin collagen by mechanical separation to obtain hair and rabbit skin collagen respectively; and / or, The ratio of the keratinase to the defatted rabbit skin is (100g~300g):100kg.

8. The preparation method according to any one of claims 4 to 7, characterized in that, The process of sequentially performing enzymatic degreasing and enzymatic hair removal on rabbit skin to obtain rabbit skin collagen also includes: The upper light phase, rich in fatty acids, obtained after separation, is subjected to a saponification reaction, and the fatty acid salts are recovered after the reaction is completed; and / or, The separated hair was subjected to alkaline hydrolysis to prepare liquid fertilizer.

9. A rabbit collagen peptide, prepared by the preparation method according to any one of claims 4 to 8, characterized in that, Based on the mass percentage of the rabbit collagen peptides, the total content of Pro-Hyp and Hyp-Gly in the rabbit collagen peptides is 1.5% to 1.7%.

10. The use of the rabbit collagen peptide according to claim 9 in the preparation of products that enhance skin elasticity and promote collagen synthesis.

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