Collagen peptides capable of promoting the expression of type XVII collagen, preparation method thereof, and applications thereof

Abstract

The present invention provides a collagen peptide capable of promoting the expression of type XVII collagen. Among them, the collagen peptide is prepared by the following steps: 1) Add water to the crude collagen extract, adjust the pH value of the crude collagen extract solution to 7-9, and add a compound protease accounting for 0.5-3% of the weight of the crude collagen extract; under the condition of a temperature of 40-80 °C, enzymatically hydrolyze for 2-8 hours, and after inactivating the enzyme, obtain an enzymatic hydrolysate; wherein, the compound protease is composed of alkaline protease, aminopeptidase and ginger protease; 2) Adjust the pH value of the enzymatic hydrolysate to 6-7.5, add a fermenting agent accounting for 1-10% of the weight of the crude collagen extract, and ferment at a temperature of 30-50 °C for 40-100 hours, and obtain a fermentation broth after sterilization; the fermenting agent is composed of Bacillus subtilis, Bacillus coagulans and Bacillus cereus; 3) After separating the solid and liquid of the fermentation broth, collect the filtrate. The present invention also provides a preparation method and application of the collagen peptide.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a collagen peptide capable of promoting the expression of type XVII collagen, and a preparation method and application thereof. Background Art

[0002] Type XVII collagen is a transmembrane non-fibroblast collagen, which is a homogeneous trimer composed of three identical α1(XVII) chains, and is divided into three major structural domains: intracellular, transmembrane, and extracellular. The hemidesmosomes formed by type XVII collagen connect epidermal stem cells to the basement membrane, and at the same time regulate the actin and keratin filament networks, coordinating the migration and renewal of epidermal stem cells and self-cleaning epidermal cells. Therefore, type XVII collagen plays a very important regulatory role in regulating skin renewal and self-cleaning. Insufficient content of type XVII collagen will present skin atrophy and fragility, pigmentation and hair loss. Research shows that in addition to decreasing with age, the increasing life and work pressure, environmental pollution, electronic radiation, staying up late and the intake of high-calorie foods also accelerate the loss of type XVII collagen. The content of type XVII collagen is extremely scarce in various animals, and the extraction difficulty is also very high.

[0003] Therefore, there is an urgent need in the art for a new and effective method to delay the loss rate of type XVII collagen. Summary of the Invention

[0004] To solve the above problems, the present invention provides a collagen peptide capable of inducing the expression of type XVII collagen in vivo to increase the content of type XVII collagen in vivo.

[0005] On the one hand, the present invention provides a collagen peptide capable of promoting the expression of type XVII collagen, which is characterized in that the collagen peptide is prepared by a method comprising the following steps:

[0006] 1) Add water to the crude collagen extract, adjust the pH value of the crude collagen extract solution to 7-9, and add a composite protease accounting for 0.5-3% of the weight of the crude collagen extract; under the condition of a temperature of 40-80°C, enzymatically hydrolyze for 2-8 hours, and after inactivating the enzyme, obtain an enzymatic hydrolysate; wherein, the composite protease is composed of alkaline protease, aminopeptidase and ginger protease;

[0007] 2) Adjust the pH value of the enzymatic hydrolysate to 6-7.5, add a fermenting agent accounting for 1-10% of the weight of the crude collagen extract, and ferment at a temperature of 30-50°C for 40-100 hours, and after sterilization, obtain a fermentation broth; the fermenting agent is composed of Bacillus subtilis, Bacillus coagulans and Bacillus cereus;

[0008] 3) After solid-liquid separation of the fermentation broth, collect the filtrate.

[0009] On the other hand, the present invention also provides the application of the collagen peptide in the preparation of cosmetics and functional foods.

[0010] On the other hand, the present invention also provides a method for preparing a collagen peptide, which is characterized in that the preparation method comprises the following steps:

[0011] 1) Add water to the crude collagen extract, adjust the pH value of the crude collagen extract solution to 7 - 9, and add a compound protease accounting for 0.5 - 3% of the weight of the crude collagen extract; under the condition of a temperature of 40 - 80°C, carry out enzymatic hydrolysis for 2 - 8 hours to obtain an enzymatic hydrolysate; wherein, the compound protease is composed of alkaline protease, aminopeptidase and ginger protease;

[0012] 2) Adjust the pH value of the enzymatic hydrolysate to 6 - 7.5, add a fermenting agent accounting for 1 - 10% of the weight of the crude collagen extract, and ferment at a temperature of 30 - 50°C for 40 - 100 hours, and obtain a fermentation broth after sterilization; the fermenting agent is composed of Bacillus subtilis, Bacillus coagulans and Bacillus cereus;

[0013] 3) After solid - liquid separation of the fermentation broth, collect the filtrate.

[0014] Collagen peptide is a peptide substance with collagen as the raw material source, and is a class of small - molecule amino acid sequences with various biological functions. Different raw material sources, different pretreatment processes and different preparation methods will result in collagen peptides with different average molecular weights and different characteristic peptide segments.

[0015] The present invention provides a collagen peptide that can promote the expression of type XVII collagen, has a relatively low average molecular weight, can be easily absorbed through digestion or through the skin, and can significantly promote the expression of type XVII collagen. Compared with the control, using the collagen peptide provided in the present application can increase the expression of type XVII collagen by more than one - fold.

[0016] In addition, microorganisms synthesize and secrete various enzyme systems during growth, and use microbial fermentation to degrade collagen, thereby generating specific small - molecule active peptides.

[0017] The present invention provides a method for preparing a collagen peptide. By adopting specific enzymatic hydrolysis steps and specific bacterial fermentation steps, a collagen peptide that can promote the expression of type XVII collagen is obtained. Description of the Drawings

[0018] Figure 1 It is the cumulative permeation amount per unit area within 24 hours.

[0019] Figure 2To detect the expression of type XVII collagen gene (COL17A1) in human skin fibroblasts by RT-qPCR method.

[0020] Figure 3 To detect the change of CoXVII protein level in human skin fibroblasts by ELISA. Specific implementation mode

[0021] The present invention provides a collagen peptide capable of promoting the expression of type XVII collagen, which is characterized in that the collagen peptide is prepared by a method comprising the following steps:

[0022] 1) Add water to the crude collagen extract, adjust the pH value of the crude collagen extract solution to 7-9, preferably 7-8, and add 0.5-3%, preferably 0.8-2% of the composite protease based on the weight of the crude collagen extract; enzymatically hydrolyze at a temperature of 40-80°C, preferably 50-70°C, more preferably 55-65°C for 2-8 hours, preferably 3-6 hours; after inactivating the enzyme, obtain the enzymolysis solution; wherein, the composite protease is composed of alkaline protease, aminopeptidase and ginger protease;

[0023] 2) Adjust the pH value of the enzymolysis solution to 6-7.5, and add 1-10%, preferably 2-8%, more preferably 3-6% of the fermenting agent based on the weight of the crude collagen extract; ferment at a temperature of 30-50°C, preferably 35-45°C for 40-100 hours, preferably 45-80 hours, more preferably 48-60 hours; after sterilization, obtain the fermentation broth; the fermenting agent is composed of Bacillus subtilis, Bacillus coagulans and Bacillus cereus;

[0024] 3) After solid-liquid separation of the fermentation broth, collect the filtrate.

[0025] The crude collagen extract can be a commercially available product or can be prepared according to the prior art. For example, the raw material containing collagen is washed to remove impurities, degreased, extracted with water and / or acid, etc. to obtain a crude collagen product.

[0026] The raw material containing collagen can come from the skin, scales or bones of animals; for example, it can be common livestock, poultry and aquatic products such as cattle, pigs, sheep, chickens, ducks, fish, etc., as long as the skin, bones and scales of the animals can be obtained. The fish can be tilapia fish skin or cod fish skin.

[0027] The collected filtrate can be used directly or used directly after simple processing, such as filtration, becoming solid, etc.

[0028] The conditions for enzyme inactivation and sterilization can be conventional conditions in the art. Preferably, it is carried out at 85-95°C for 8-60 minutes, preferably 20-50 minutes.

[0029] In a preferred embodiment, the inoculation amounts of the Bacillus subtilis, the Bacillus coagulans and the Bacillus cereus are 1 - 9×10 8 cfu / g.

[0030] In a preferred embodiment, the content of the collagen peptide in the collected filtrate is 70 - 90% by weight, and it also contains 2 - 12% by weight of free amino acids.

[0031] In a preferred embodiment, in step 1), the mass ratio of the alkaline protease, the aminopeptidase and the ginger protease is 2.5 - 4:0.5 - 1.5:1; preferably, the addition amount of the compound protease is 0.5 - 2% of the crude collagen extract, the enzymatic hydrolysis temperature is 55 - 75°C, and the enzymatic hydrolysis time is 3 - 6 hours.

[0032] In a preferred embodiment, in step 2), the mass ratio of the Bacillus subtilis, the Bacillus coagulans and the Bacillus cereus is 1 - 3:1 - 3:1; preferably, the mass ratio of the Bacillus subtilis, the Bacillus coagulans and the Bacillus cereus is 1 - 2:1 - 2:1, and they are fermented at a temperature of 35 - 42°C for 50 - 80 hours.

[0033] Preferably, the collagen peptide provided by the present invention has a relatively small average molecular weight, mainly distributed between 500 - 2000 Da, preferably between 1000 - 2000 Da. Therefore, the collagen peptide provided by the present invention is not only easily digested and absorbed by oral administration, but also can be transdermally absorbed through the skin.

[0034] The collagen peptide provided by the present invention can be used in foods or cosmetics, and is absorbed through the digestive tract or the skin to promote the expression of type XVII collagen.

[0035] Therefore, the present invention also provides the application of the collagen peptide in the preparation of cosmetics and functional foods.

[0036] The present invention also provides a method for preparing a collagen peptide, which is characterized in that the preparation method comprises the following steps:

[0037] 1) Add water to the crude collagen extract, adjust the pH value of the crude collagen extract solution to 7 - 9, and add 0.5 - 3% of the compound protease based on the weight of the crude collagen extract; under the condition of a temperature of 40 - 80°C, perform enzymatic hydrolysis for 2 - 8 hours, and after inactivating the enzyme, obtain an enzymatic hydrolysate; wherein, the compound protease is composed of alkaline protease, aminopeptidase and ginger protease;

[0038] 2) Adjust the pH value of the enzymatic hydrolysate to 6 - 7.5, and add a starter accounting for 1 - 10% by weight of the crude collagen extract, preferably 3 - 6% of the starter; ferment at a temperature of 30 - 50 °C for 40 - 100 hours, and obtain a fermentation broth after sterilization; the starter is composed of Bacillus subtilis, Bacillus coagulans, and Bacillus cereus;

[0039] 3) After solid - liquid separation of the fermentation broth, collect the filtrate.

[0040] The raw material containing collagen can be derived from the skin, scales, or bones of animals; for example, it can be common livestock, poultry, and aquatic products such as cows, pigs, sheep, chickens, ducks, and fish, as long as the skin, bones, and scales of the animals can be obtained. The fish part can be tilapia fish skin or cod fish skin.

[0041] The collected filtrate can be used directly or used directly after simple processing, such as filtration, becoming solid, etc.

[0042] Both the enzymes and bacteria mentioned above can be obtained through commercial purchase.

[0043] The conditions for enzyme inactivation and sterilization can be conventional conditions in the art. Preferably, it is carried out at 85 - 95 °C for 8 - 60 minutes.

[0044] In a preferred embodiment, the inoculation amounts of Bacillus subtilis, Bacillus coagulans, and Bacillus cereus are 1 - 9×10 8 cfu / g.

[0045] In a preferred embodiment, in step 1), the mass ratio of the alkaline protease, aminopeptidase, and zingiber protease is 2.5 - 4:0.5 - 1.5:1; preferably, the addition amount of the compound protease is 0.5 - 2% of the crude collagen extract, the temperature for enzymatic hydrolysis is 55 - 75 °C, and the time for enzymatic hydrolysis is 3 - 6 hours.

[0046] In a preferred embodiment, in step 2), the mass ratio of Bacillus subtilis, Bacillus coagulans, and Bacillus cereus is 1 - 3:1 - 3:1, and ferment at a temperature of 35 - 42 °C for 50 - 80 hours.

[0047] In a preferred embodiment, the crude collagen extract is obtained according to the following steps: subject the raw material containing collagen to degreasing, water extraction, and acid extraction in sequence.

[0048] In a preferred embodiment, the degreasing is carried out as follows: immerse the raw material containing collagen in a NaOH solution for a period of time, take it out and wash it with water until neutral; repeat 1 - 3 times. Preferably, the mass concentration of the NaOH solution is 0.1 - 5%, and the soaking time is 12 - 24 hours.

[0049] In a preferred embodiment, the step of water extraction includes extracting in boiling water for 3 - 6 hours.

[0050] In another preferred embodiment, the acid extraction includes extracting in an acidic solution with a pH of 2 - 5 at 80 - 95°C for 1 - 3 hours.

[0051] In a preferred embodiment, the acid extraction product is rapidly frozen and thawed 3 - 5 times, and the freezing temperature is preferably less than or equal to -20°C.

[0052] In the present application, filtration can be carried out by conventional filtration methods in the art, preferably plate - frame filtration. For example, the filtration pressure is 0.2 - 0.5 Mpa, and the flow rate is 1.5 - 4 tons per hour.

[0053] In a preferred embodiment, the method further includes a step of purifying the filtrate after the step 3); preferably, the purification includes ultrafiltration and nanofiltration.

[0054] In the present application, membrane filtration methods are used for separation and purification. Ultrafiltration and nanofiltration are used to remove inorganic salts and water - soluble free amino acids, etc. The operations are all carried out at room temperature, without phase changes, highly energy - efficient, will not affect the activity of small - molecule peptides, and no pollution is generated during the production process.

[0055] In a preferred embodiment, the molecular weight cut - off of the ultrafiltration membrane used for ultrafiltration is 1 - 10 kDa; the ultrafiltration membrane is preferably a polysulfone - type or polyamide - type spiral wound membrane.

[0056] In a preferred embodiment, the molecular weight cut - off of the nanofiltration membrane is 500 - 2000 Da; the nanofiltration membrane is preferably any one of polyamide, sulfonated polyethersulfone, and sulfonated polysulfone hollow fiber membranes.

[0057] In a preferred embodiment, the solution obtained by nanofiltration is spray - dried, and the inlet air temperature of the spray - drying is set at 160 - 220°C, and the outlet air temperature is set at 90 - 120°C.

[0058] In a preferred embodiment, the collagen peptide obtained by the method provided in the present application has a relatively small average molecular weight, mainly distributed between 500 - 2000 Da, preferably distributed between 1000 - 2000 Da.

[0059] Examples

[0060] The present invention will be further described in detail below through examples. However, the following examples are for illustrative purposes only and should not be construed as limiting the scope of the present invention.

[0061] Example 1

[0062] Weigh 1 kg of sheepskin and soak it in 0.1% NaOH solution at room temperature for 8 hours for degreasing. Wash it three times with water until neutral. After washing, add 10 L of water and boil it for 3 hours to obtain a crude extract of total protein. Adjust the pH of the solution to 3.5 with 0.1 mol / l dilute hydrochloric acid, stir it at 90 °C for 2 hours to extract collagen, and obtain an acid extract. Rapidly freeze-thaw the acid extract three times at -20 °C, then freeze-dry the filtered solution and weigh it to obtain 146 g of crude collagen extract.

[0063] Weigh 100 g of the crude collagen extract, add 900 mL of water, and add a compound protease of alkaline protease, aminopeptidase (purchased from Ningxia Xiasheng) and ginger protease (purchased from Shanghai Yimiao Chemical Technology Co., Ltd.) accounting for 1% of the weight of the crude collagen extract. The weight ratio of the alkaline protease, aminopeptidase and ginger protease is 3:1:1. The enzymatic hydrolysis temperature is 55 °C, the enzymatic hydrolysis time is 3 hours, and the pH value is controlled at 7.5 during the enzymatic hydrolysis process.

[0064] Inactivate the enzyme at 90 °C for 30 minutes to obtain an enzymatic hydrolysate.

[0065] Inoculate Bacillus subtilis, Bacillus coagulans and Bacillus cereus in the enzymatic hydrolysate. All three kinds of bacilli are purchased from Ningbo Taisituo Biotechnology Co., Ltd. Based on the crude collagen extract, add a fermenting agent accounting for 5% of the weight of the crude collagen extract. Among them, the inoculation amounts of Bacillus subtilis, Bacillus coagulans and Bacillus cereus are all 2×10 8 cfu / g; the fermentation temperature is 35 °C and the fermentation time is 48 hours.

[0066] Sterilize at 90 °C for 60 minutes to obtain a fermentation broth.

[0067] Separate the solid and liquid of the fermentation broth, then pass the supernatant through a polysulfone ultrafiltration membrane with a molecular weight of 2 KD, and then through a 100 Da sulfonated polysulfone hollow fiber nanofiltration membrane. Spray-dry the obtained filtrate, with an inlet air temperature of 180 °C and an outlet air temperature of 90 °C, to obtain collagen peptide powder.

[0068] Detect the molecular weight of small molecule collagen peptides, and the measured results are shown in Table 1; the average molecular weight of the collagen peptide powder is 1623; the peptide content is 87.3%, the free amino acid is 2.4%, the moisture is 6.02%, and the ash content is 1.65%.

[0069] Table 1 Molecular weight distribution

[0070]

[0071] Control example

[0072] Weigh 1 kg of sheepskin and soak it in 0.1% NaOH solution at room temperature for 8 hours for degreasing. Wash it three times with water until neutral. After cleaning, add 10 L of water and boil it for 3 hours to obtain a crude extract of total protein. Adjust the pH of the solution to 3.5 with 0.1 mol / L dilute hydrochloric acid and stir it at 90 °C for 2 hours to extract collagen. Freeze-dry the solution collected by filtration and weigh it to obtain 146 g of crude collagen extract.

[0073] Weigh 100 g of the crude collagen extract, add 900 mL of water, and add alkaline protease at 1% of the weight of the crude collagen extract. The enzymatic hydrolysis temperature is 55 °C, the enzymatic hydrolysis time is 3 hours, and the pH value is controlled at 7.5 during the enzymatic hydrolysis process.

[0074] Inactivate the enzyme at 90 °C for 30 minutes to obtain the enzymatic hydrolysis solution.

[0075] Perform solid-liquid separation on the enzymatic hydrolysis solution, then pass the supernatant through a polysulfone ultrafiltration membrane with a molecular weight cut-off of 2000 Da, and then through a sulfonated polysulfone hollow fiber nanofiltration membrane with a molecular weight cut-off of 100 Da. Spray-dry the obtained filtrate at an inlet air temperature of 180 °C and an outlet air temperature of 90 °C to obtain collagen peptide powder.

[0076] Detect that the molecular weight of the small molecule collagen peptide is 1871 Da.

[0077] Transdermal absorption experiment

[0078] Use a TP-6 type transdermal diffusion instrument (manufacturer: Tianjin Tianhe Analytical Instrument Co., Ltd.) to conduct a transdermal absorption experiment. First, A special skin membrane (manufacturer: Merck Millipore, USA) is directly fixed between the sample cell and the receiving cell. The sample cell contains a 10 wt% collagen peptide physiological saline solution obtained in Example 1, and physiological saline is used as a blank control. The receiving cell is filled with physiological saline to a volume of 17 mL, and it is kept under constant-speed stirring and constant temperature. The water bath temperature is (37 °C ± 0.5 °C) and the rotation speed is 300 rpm. Take 1 mL of samples at 2 h, 4 h, 6 h, 8 h, and 24 h respectively, and add an equal amount of physiological saline at the same time. Then take a certain amount of the test solution from the receiving solution and place it in a volumetric flask. Measure the content of collagen peptide in the taken-out solution, and calculate the cumulative permeation amount per unit area (Qn) and the transdermal rate constant (J) according to the following formula:

[0079]

[0080] In the formula, Cn is the Hyp concentration measured at the nth sampling point, with the unit of μg / mL; Ci is the Hyp concentration measured at the ith sampling point, with the unit of μg / mL; 10 is the conversion coefficient between hyp and collagen peptide; V is the volume of the receiving cell, which is 17 mL; V0 is the volume taken at each time point, which is 1 mL; A is the permeation area, which is 1.54 cm 2 .

[0081]

[0082] The results of the transdermal absorption experiment are as Figure 1 shown.

[0083] Cell experiment - Transcription level of type XVII collagen mRNA

[0084] (1) Cell culture

[0085] Human skin fibroblasts (HSF) were cultured in a 10 cm cell culture dish using complete medium (10% FBS + RPMI - 1640 + double antibiotics (100 units / mL penicillin + 100 μg / mL streptomycin)). When the cell density increased to 90%, sub - culture was carried out; the medium was aspirated using a pipette, and 2 mL of DPBS was added for washing once; then 1 mL of 0.25% trypsin was added and digested at room temperature for 3 min. After observing that the cells became round under the microscope, an equal volume of complete medium was added to neutralize the trypsin; the cells were blown down by pipetting dozens of times and collected into a 15 mL centrifuge tube; the centrifuge tube was centrifuged at 1500 rpm at room temperature for 5 min, and the supernatant was discarded; 2 mL of fresh medium was added to resuspend the cell pellet, and 10 μL was aspirated for cell counting; the counted cells were inoculated into a new cell culture dish at a density of 2×10 5 and the medium was supplemented to 10 mL, then placed in an incubator at 37°C and 5% CO2 for continued culture.

[0086] (2) Treatment of cells with collagen peptides

[0087] After the cells covered the plate, the cells were collected into a 10 mL centrifuge tube, centrifuged at 1200 rpm for 3 min, the supernatant was removed, and new cell culture medium was added, then blown evenly and counted using a hemocytometer. 2.5×10 5 cells were seeded in each well of a 12 - well plate and cultured overnight. The cultured cells were divided into 6 groups, with 2 parallels in each group: control group (without adding any collagen peptides); group A (collagen peptides obtained from the comparative example, final concentration 200 μg / mL); group B (commercially available collagen peptides, average molecular weight 2000 Da, Beijing Shengmeinuo Biotechnology Co., Ltd., final concentration 200 μg / mL); the treatment groups of the collagen peptides obtained in Example 1 were divided into three groups: final concentrations of 200, 400, and 800 μg / mL respectively. The cells were treated with the above - mentioned collagen peptides for 12 h. Then, 400 μmol / L hydrogen peroxide was added to each well and treated for 6 h.

[0088] Detection of the expression of type XVII collagen gene (COL17A1)

[0089] Extract cellular RNA according to the steps in the kit instruction manual (purchased from Takara Bio Inc. (Dalian)), reverse transcribe to synthesize cDNA, and prepare reagents for RT-qPCR experiments. The results are as Figure 2 shown.

[0090] It can be seen from Figure 2 that: in the control group, Group A (the collagen peptide obtained in the comparative example, final concentration 200 μg / mL); Group B (the commercially available collagen peptide, average molecular weight 2000 Da, Beijing Shengmeinuo Biotechnology Co., Ltd., final concentration 200 μg / mL), the transcriptional levels of type XVII collagen mRNA were similar; while in the treatment group with the collagen peptide obtained in Example 1 at 200 μg / mL, the transcriptional level of type XVII collagen mRNA was about 1.5 times that of the control group; while in the treatment group with the collagen peptide obtained in Example 1 at 400 μg / mL, the transcriptional level of type XVII collagen mRNA was about 2 times that of the control group; while in the treatment group with the collagen peptide obtained in Example 1 at 800 μg / mL, the transcriptional level of type XVII collagen mRNA was about 2.7 times that of the control group;

[0091] Cell experiment - Expression level of type XVII collagen

[0092] (1) Cell culture

[0093] Culture human skin fibroblasts (HSF) in a 10 cm cell culture dish using complete medium (10% FBS + RPMI-1640 + double antibiotics (100 units / mL penicillin + 100 μg / mL streptomycin)). Passage the cells when the cell density proliferates to 90%; use a pipette to aspirate the medium, add 2 mL DPBS to wash once; then add 1 mL of 0.25% trypsin to digest at room temperature for 3 min. After observing that the cells become round under the microscope, add an equal volume of complete medium to neutralize the trypsin; pipette the cells dozens of times to detach them and collect them into a 15 mL centrifuge tube; centrifuge the centrifuge tube at 1500 rpm at room temperature for 5 min, discard the supernatant; add 2 mL of fresh medium to resuspend the cell pellet, aspirate 10 μL for cell counting; seed the counted cells at a density of 2×10 5 into a new cell culture dish, make up the medium to 10 mL, and then place it in an incubator at 37 °C and 5% CO2 for continued culture.

[0094] (2) Treat cells with collagen peptide

[0095] After the cells cover the plate, collect the cells into a 10 mL centrifuge tube, centrifuge at 1200 rpm for 3 min, discard the supernatant, add fresh cell culture medium and pipette evenly, and count with a hemocytometer. In each well of a 12-well plate, seed 2.5×10 5Cells were cultured overnight. The cultured cells were divided into 6 groups, with 2 parallels set in each group: control group (without adding any collagen peptides); group A (the collagen peptides obtained in the comparative example, with a final concentration of 200 μg / mL); group B (commercially available collagen peptides, with an average molecular weight of 2000 Da, Beijing Shengmeinuo Biotechnology Co., Ltd., with a final concentration of 200 μg / mL); the treatment groups with the collagen peptides obtained in Example 1 were divided into three groups: with final concentrations of 200, 400, and 800 μg / mL respectively. The cells were treated with the above-mentioned collagen peptides for 12 h. Then, 400 μmol / L hydrogen peroxide was added to each well and treated for 6 h.

[0096] (3) Detect the content of type XVII collagen (ColXVII)

[0097] ① Extract proteins according to the instructions of the type XVII collagen (Col-XVII) ELISA kit (purchased from Wuhan Aimeijie Technology Co., Ltd.).

[0098] ② Prepare the standard curve: Dilute the standard, prepare 5 EP tubes, and then add 150 μL of standard dilution solution to each EP tube, numbered 1, 2, 3, 4, 5 respectively. Add 150 μL of standard product to tube 1, pipette repeatedly with a pipette tip, vortex for about 5 s, then change the pipette tip, take 150 μL from tube 1 and add it to tube 2, and so on for the subsequent process. The concentration decreases from high to low;

[0099] ③ Add samples: For the standard addition, 2 wells are set for each concentration point (parallel), and 50 μL is added to each well; 50 μL is added to each well in the sample wells (first add 40 μL of sample dilution solution, then add 10 μL of sample); 50 μL of standard dilution solution or sample dilution solution is added to the blank well. The standard addition and sample addition should be completed within 15 min. After adding the samples, cover with a sealing plate membrane and incubate at 37 °C for 30 min;

[0100] ④ Wash the plate: Add 300 μL of washing solution to each well (do not overflow the plate wells), place the plate on a shaker for about 5 s, then let it stand for 30 s, and centrifuge dry within 1 s, then tap the plate. A layer of absorbent paper or filter paper should be placed on the table during the tapping process, and the plate wells should be tapped down a few times. The main criterion for determining the completion of tapping is that there are no obvious water stains on the absorbent paper and filter paper;

[0101] ⑤ Incubate: Add 50 μL of enzyme-labeled reagent to each well. Do not add it to the blank well here. Incubate for 30 min and then wash the plate; Develop color: First add 50 μL of color-developing solution A to each well, and then add 50 μL of color-developing solution B to each well. Operate in the dark and develop color at 37 °C for 15 min;

[0102] ⑥ Read the values: Add 50 μL of stop solution to each well, and read the values within 15 min after adding the stop solution. Reading after the specified time is invalid. Reading within the specified time is valid.

[0103] The results are as Figure 3 shown.

[0104] It can be seen from Figure 3 that: in the control group, Group A (collagen peptide obtained from the comparative example, final concentration 200 μg / mL); Group B (commercially available collagen peptide, average molecular weight 2000 Da, Beijing Shengmeinuo Biotechnology Co., Ltd., final concentration 200 μg / mL), the expression levels of type XVII collagen were similar, approximately 90 pg / mL, 95 pg / mL, and 100 pg / mL respectively; while in the treatment group with the collagen peptide obtained in Example 1 at 200 μg / mL, the expression level of type XVII collagen was approximately 110 pg / mL; while in the treatment group with the collagen peptide obtained in Example 1 at 400 μg / mL, the expression level of type XVII collagen was approximately 120 pg / mL; while in the treatment group with the collagen peptide obtained in Example 1 at 800 μg / mL, the expression level of type XVII collagen was approximately 170 pg / mL, and the up-regulation of the expression level of type XVII collagen was very significant.

Claims

1. A collagen peptide capable of promoting the expression of type XVII collagen, characterized in that: The collagen peptide is prepared by a method comprising the following steps: 1) adding water to the crude collagen extract, adjusting the pH value of the crude collagen extract solution to 7-9, and adding 1% composite protease by weight of the crude collagen extract; performing enzymolysis for 3 hours at a temperature of 55° C. and a pH value of 7.5, and obtaining an enzymolysis solution after inactivating the enzyme; wherein the composite protease is composed of alkaline protease, aminopeptidase and ginger protease, and the weight ratio of the alkaline protease, aminopeptidase and ginger protease is 3:1:1; 2) adding a starter culture of 5% by weight of the crude collagen extract, fermenting at a temperature of 35° C. for 48 hours, and sterilizing to obtain a fermentation liquid; the starter culture is composed of Bacillus subtilis, Bacillus coagulans and Bacillus cereus, and the inoculation amount of Bacillus subtilis, Bacillus coagulans and Bacillus cereus is 2×10 8 cfu / g; 3) After the solid-liquid separation of the fermentation broth, the filtrate is collected and passed through a polysulfone ultrafiltration membrane with a molecular weight of 2KD, and then through a 100Da sulfonated polysulfone hollow fiber nanofiltration membrane.

2. The collagen peptide according to claim 1, characterized in that In the step 1), the crude collagen extract is obtained according to the following steps: defatting, water extraction and acid extraction of the raw material containing collagen are performed in sequence.

3. The collagen peptide according to claim 2, characterized in that The water extraction step comprises extracting in boiling water for 3-6 hours.

4. The collagen peptide according to claim 2, characterized in that The acid extraction comprises extracting in an acidic solution of pH 2-5 at 80-95°C for 1-3 hours.

5. The collagen peptide according to claim 2, characterized in that The acid extraction includes extraction in an acidic solution at pH 3.5 at 90°C for 2 hours.

6. The collagen peptide according to claim 2, characterized in that The degreasing comprises soaking in NaOH with a mass concentration of 0.1% at room temperature for 8 hours.

7. The collagen peptide according to claim 2, characterized in that The step of preparing the crude collagen extract further comprises rapidly freezing and thawing the acid-extracted product at -20°C for three times.

8. The collagen peptide according to claim 2, characterized in that The raw material containing collagen is sheepskin.

9. Use of the collagen peptide according to any one of claims 1 to 8 in the preparation of cosmetics and functional foods.

10. A method for preparing collagen peptide, characterized in that: The preparation method comprises the following steps: 1) adding water to the crude collagen extract, adjusting the pH value of the crude collagen extract solution to 7-9, and adding 1% composite protease by weight of the crude collagen extract; performing enzymolysis for 3 hours at a temperature of 55° C. and a pH value of 7.5, and obtaining an enzymolysis solution after inactivating the enzyme; wherein the composite protease is composed of alkaline protease, aminopeptidase and ginger protease, and the weight ratio of the alkaline protease, aminopeptidase and ginger protease is 3:1:1; 2) adding a starter culture of 5% by weight of the crude collagen extract, fermenting at a temperature of 35° C. for 48 hours, and sterilizing to obtain a fermentation liquid; the starter culture is composed of Bacillus subtilis, Bacillus coagulans and Bacillus cereus, and the inoculation amount of Bacillus subtilis, Bacillus coagulans and Bacillus cereus is 2×10 8 cfu / g; 3) After the solid-liquid separation of the fermentation broth, the filtrate is collected and passed through a polysulfone ultrafiltration membrane with a molecular weight of 2KD, and then through a 100Da sulfonated polysulfone hollow fiber nanofiltration membrane.

11. The method for preparing collagen peptide according to claim 10, characterized in that: The crude collagen extract is obtained according to the following steps: defatting, water extraction and acid extraction are performed on the raw materials containing collagen in sequence.

12. The method for preparing collagen peptide according to claim 11, characterized in that: The water extraction step comprises extracting in boiling water for 3-6 hours.

13. The method for preparing collagen peptide according to claim 11, characterized in that: The acid extraction comprises extracting in an acidic solution of pH 2-5 at 80-95°C for 1-3 hours.

14. The method for preparing collagen peptide according to claim 11, characterized in that: The acid extraction includes extraction in an acidic solution at pH 3.5 at 90°C for 2 hours.

15. The method for preparing collagen peptide according to claim 11, characterized in that: The degreasing comprises soaking in NaOH with a mass concentration of 0.1% at room temperature for 8 hours.

16. The method for preparing collagen peptide according to claim 11, characterized in that: The raw material containing collagen is sheepskin.

17. The method for preparing collagen peptide according to claim 11, characterized in that: The step of preparing the crude collagen extract further comprises rapidly freezing and thawing the acid-extracted product at -20°C for three times.

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