Preparation method and verification method of deer skin collagen peptide with skin repairing and immunoregulation effects

Abstract

The invention relates to a preparation method and a verification method of deer skin collagen peptide with skin repairing and immunoregulation effects, and belongs to the technical field of biological medicines. The technical problems that the technology for extracting the deer skin collagen peptide from the deer skin of the sika deer is tedious, and whether the prepared deer skin collagen peptide has skin repairing and immunoregulation effects or not are solved. The preparation method of the deer skin collagen peptide with the skin repairing and immunoregulation effects comprises the following steps: cutting deer skin into small pieces, and preparing the deer skin collagen peptide with the molecular weight of less than 10kDa by adopting a double-step enzymolysis method. And a verification experiment of an immunosuppressive mouse is constructed through cyclophosphamide induction. According to the invention, the extraction process is greatly simplified, and a double-step enzymolysis method is adopted, so that the deer skin collagen peptide subjected to enzymolysis is small-molecule peptide, has strong antioxidant capacity, can repair light-damaged cells and promote skin cell growth, and is good in safety; by injecting the deer skin collagen peptide, the immune function of the mouse is improved, and damage caused by mouse cells is relieved.

Description

Technical Field

[0001] The invention discloses a preparation method of deerskin collagen peptide with skin repair and immune regulation effects and a verification method thereof, belonging to the field of biomedicine technology. Background Art

[0002] As the largest organ in the human body, the skin is not only the first line of defense against the invasion of external pathogens, but also plays an important role in maintaining the stability of the body's internal environment. However, factors such as environmental pollution, ultraviolet radiation, unhealthy living habits, and aging in modern life often lead to various skin problems such as dryness, wrinkles, inflammation, and slow wound healing. These problems not only affect the beauty of the skin, but may also pose a potential threat to physical health. At the same time, the normal functioning of the immune system is crucial to skin health, and immune imbalance may cause or aggravate a variety of skin diseases. Therefore, finding a substance that can both effectively repair skin damage and regulate immune function is of great significance for improving skin health.

[0003] Collagen, the primary structural protein of the skin, plays an indispensable role in key physiological processes such as cellular metabolism, maintaining skin elasticity, and wound repair. Collagen peptides, due to their small molecular weight, high bioactivity, and ease of absorption by the human body, have gradually become a research hotspot in the fields of skin repair and beauty. Existing Chinese patent CN115177535A (filing date: May 27, 2022) discloses a deerskin collagen peptide liposome hydrogel, a preparation method, and its application. This invention, by preparing deerskin collagen peptides into a liposome hydrogel, improves the stability and skin permeability of the collagen peptides, effectively delaying skin aging. However, the collagen peptide liposome hydrogel prepared in this invention has a single function, namely skin repair.

[0004] Immune function also plays a crucial role in skin health, with immune cells and factors involved in skin defense, repair, and inflammatory responses. When the skin is damaged or infected, the immune system is activated, initiating an immune response to eliminate pathogens and repair damaged tissue. However, excessive or abnormal immune responses can lead to the occurrence and exacerbation of skin inflammation. Therefore, regulating the immune system to maintain a balanced state is important for the prevention and treatment of skin diseases. Currently, the main industrial sources of collagen are pigs, cattle, and marine organisms. However, these traditional sources present numerous challenges. For example, obtaining collagen from cattle carries the risk of pathogens such as mad cow disease. Furthermore, due to religious beliefs and other factors, the use of porcine collagen is strictly restricted in some regions. Furthermore, while traditional collagen sources (such as pigs and cattle) are widely used in skin repair, research on their immunomodulatory properties is relatively scarce.

[0005] As a traditional tonic, sika deer hide is a valuable ingredient in traditional Chinese medicine. Rich in essential amino acids such as Glu, Gly, Ala, and Pro, it is also known for its medicinal properties in traditional Chinese medicine, such as replenishing Qi and blood, and combating fatigue. As a result, it is gradually emerging as a promising new collagen source. However, the acid-based or ultrasound-assisted methods used in early collagen extraction techniques have numerous drawbacks. These methods are energy-intensive and require extremely harsh extraction conditions, typically requiring high temperatures and strong acids. This can easily damage the active structure of collagen, resulting in poor quality. Summary of the Invention

[0006] In view of the shortcomings of the existing technology, in order to solve the technical problems such as the complicated process of extracting deerskin collagen peptides from sika deer deerskin and whether the obtained deerskin collagen peptides have skin repair and immune regulation effects, a preparation method of deerskin collagen peptides with skin repair and immune regulation effects and a verification method thereof are proposed.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] The preparation method of deerskin collagen peptide having skin repair and immunomodulatory effects comprises the following steps:

[0009] S1. Deer hide was removed, hair removed, fat and fascia removed, minced into 0.5 cm × 0.5 cm pieces, and homogenized with distilled water to prepare a slurry for later use.

[0010] S2, pepsin was added to the above-mentioned slurry, wherein the amount of pepsin added was 3000 U / g~4500 U / g, and the amount of pepsin added to every 1.0mL of slurry was 10.0mg~14.0mg, the pH value of the slurry was adjusted to 1.5~3.0, and the slurry was subsequently placed in a constant temperature oscillator for enzymolysis for 2.0h~5.0h, and the temperature in the constant temperature oscillator was 30.0℃~50.0℃. After the enzymolysis was completed, it was inactivated at 100℃ for 8.0min, and then the pH value of the slurry after enzymolysis was adjusted to 1.5~3.0. Centrifuge was used to centrifuge at a rate of 2500r / min~4000r / min for 20.0min~40.0min, and the supernatant was taken to obtain an enzymolyzed solution;

[0011] S3. Add a composite enzyme of papain and trypsin to the above enzymatic solution, wherein the mass ratio of papain and trypsin is (1~3): (1~3); the amount of the composite enzyme of papain and trypsin added is 4000 U / g~6000 U / g, and the amount of the composite enzyme of papain and trypsin added to each 1.0 mL of enzymatic solution is 4.0 mg~6.0 mg, the pH value of the enzymatic solution is adjusted to 3.0~5.0, and then the enzymatic solution is placed in a constant temperature oscillator for enzymolysis for 2.0h~5.0h, and the temperature in the constant temperature oscillator is 35.0℃~50.0℃. After the enzymatic hydrolysis is completed, it is inactivated at 100℃ for 8.0min, and then the pH value of the enzymatic hydrolysis slurry is adjusted to 3.0~5.0, and a centrifuge is used to centrifuge at a rate of 2500r / min~4000r / min for 20.0min~40.0min, and the supernatant is taken to obtain a secondary enzymatic hydrolysis enzymatic solution;

[0012] S4. The enzymatic hydrolysis solution obtained in step S3 is added to a rotary evaporator for concentration. The temperature during rotary evaporation is 50°C to 70°C. After concentration, the concentrated enzymatic hydrolysis solution is placed in a freeze dryer for freeze drying. The freezing temperature is -45°C to -55°C and the freezing time is 40h to 50h, thereby obtaining deerskin collagen peptide.

[0013] Furthermore, in step S1, the volume of distilled water added to 1.0 g of deerskin is 18 mL to 25 mL.

[0014] The method for verifying deerskin collagen peptides having skin repair and immune regulation effects comprises the following steps:

[0015] (1) Select female mice with the same behavioral abilities to construct five different mouse models.

[0016] Six female mice were selected as the blank control group (CON group) in the first group. From the first to the third day of the experiment, the same amount of normal saline was injected into the abdominal cavity of each mouse at a frequency of once a day, with the amount of normal saline injected ranging from 5.0 mL / kg to 15.0 mL / kg. Starting from the fourth day, each mouse was gavaged with distilled water at a frequency of 5.0 mL / kg to 15.0 mL / kg once a day for 11 consecutive days, and each mouse was fed for a total of 14 days.

[0017] Six female mice were selected as the model control group in the second group. From the first to the third day of the experiment, each mouse was intraperitoneally injected with the same amount of 80 mg / kg of cyclophosphamide, with the injection frequency being once a day, and the amount of 80 mg / kg of cyclophosphamide injected was 5.0 mL / kg-15.0 mL / kg. Starting from the fourth day, each mouse was gavaged with distilled water, with the amount of distilled water gavage being 5.0 mL / kg-15.0 mL / kg, once a day, for 14 consecutive days, and each mouse was fed for a total of 14 days.

[0018] The third group selected 6 female mice as the low-dose collagen peptide group. From the first to the third day of the experiment, each mouse was intraperitoneally injected with the same amount of 80 mg / kg of cyclophosphamide, with the injection frequency being once a day, and the amount of 80 mg / kg of cyclophosphamide injected was 5.0 mL / kg~15.0 mL / kg; starting from the fourth day, each mouse was gavaged with a low dose of deerskin collagen peptide, with the gavage being once a day, and the amount of low-dose deerskin collagen peptide injected was 5.0 mL / kg~15.0 mL / kg, for 14 consecutive days, and each mouse was fed for a total of 14 days;

[0019] The fourth group selected 6 female mice as the medium-dose collagen peptide group. From the first to the third day of the experiment, each mouse was intraperitoneally injected with the same amount of 80 mg / kg cyclophosphamide, with an injection frequency of once a day, and the amount of 80 mg / kg cyclophosphamide injected was 5.0 mL / kg~15.0 mL / kg; starting from the fourth day, each mouse was gavaged with a medium-dose deerskin collagen peptide, with an oral gavage of 5.0 mL / kg~15.0 mL / kg once a day, for 14 consecutive days, and each mouse was fed for a total of 14 days;

[0020] The fifth group selected 6 female mice as the high-dose collagen peptide group. From the first to the third day of the experiment, each mouse was intraperitoneally injected with the same amount of 80 mg / kg cyclophosphamide, with the injection frequency being once a day, and the amount of 80 mg / kg cyclophosphamide injected was 5.0 mL / kg~15.0 mL / kg; starting from the fourth day, each mouse was gavaged with a high dose of deerskin collagen peptide, with the gavage being once a day, and the amount of high-dose deerskin collagen peptide being gavaged was 5.0 mL / kg~15.0 mL / kg, for 14 consecutive days, and each mouse was fed for a total of 14 days;

[0021] (2) During the 14 days of feeding, the mental state, fur glossiness, balance, and reaction ability of the mice were observed in real time, and the weight changes of each group of mice were recorded in real time and the data were collected;

[0022] (3) After the five groups of mice were fed for 14 days, they were anesthetized and blood was collected from the heart. The mice were then killed by cervical dislocation and dissected. The number of peripheral blood leukocytes in each group of mice was detected, the organ index of the mice was weighed, the organ lesions were checked, and the spleen immune-related indicators were detected.

[0023] The beneficial effects of the present invention are as follows: the present invention extracts deerskin collagen peptides from deerskin, and no impurity removal operation is performed during the entire extraction process, thereby greatly simplifying the extraction process and saving costs. Furthermore, under the premise of simplifying the process, deerskin collagen peptides with biological activity can still be prepared. Compared with the prior art, the present invention adopts a two-step enzymatic hydrolysis method when extracting deerskin collagen peptides, so that the deerskin collagen peptides after enzymatic hydrolysis are small molecule peptides. Furthermore, through various experiments such as antioxidant capacity determination, cytotoxicity detection, in vitro hemolysis test, light damage test and cell scratch test, it is fully proved that the deerskin collagen small molecule peptides have strong antioxidant capacity, can repair light-damaged cells, promote skin cell growth, and have good safety, further illustrating that the deerskin collagen peptides of the present invention have strong skin repair ability.

[0024] The present invention uses cyclophosphamide to induce the construction of immunosuppressive mice, and then injects different doses of deerskin collagen peptide into the immunosuppressive mice. By testing the mRNA expression levels of NF-κB, TNF-α, IL-5 and IL-10 in the mice, it is found that the mice injected with deerskin collagen peptide, whether high or low doses, can promote the expression levels of immunosuppressive mice, enhance the immune function of the mice, and reduce the damage to mouse cells caused by cyclophosphamide. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The figure is the SDS-PAGE electrophoresis diagram of the deerskin collagen peptide prepared in the present invention.

[0026] Figure 2 The figure is a bar graph showing the DPPH free radical scavenging ability of the deerskin collagen peptide prepared in the present invention at different concentrations.

[0027] Figure 3 The deerskin collagen peptide prepared in the present invention reacts with ABTS at different concentrations. + Histogram of free radical scavenging activity.

[0028] Figure 4 The figure is a bar graph showing the total antioxidant capacity of the deerskin collagen peptide prepared in the present invention at different concentrations.

[0029] Figure 5This is a bar graph showing the cytotoxicity test of the deerskin collagen peptide prepared in the present invention at different concentrations.

[0030] Figure 6 The figure is a bar graph showing the in vitro hemolysis experiment of the deerskin collagen peptide prepared in the present invention at different concentrations.

[0031] Figure 7 This is a bar graph showing the effect of the deerskin collagen peptide prepared in the present invention on repairing photodamaged skin at different concentrations.

[0032] Figure 8 This is a diagram showing the repair of cell scratches by the deerskin collagen peptide prepared in the present invention at different concentrations.

[0033] Figure 9 The figure is a bar graph showing the cell growth promoting ability test of the deerskin collagen peptide prepared in the present invention at different concentrations.

[0034] Figure 10 The figure is a line graph showing the weight changes of mice in the verification experiment of the present invention, and is also used as an abstract figure.

[0035] Figure 11 This is a bar graph showing the effect of different concentrations of deerskin collagen peptides on the peripheral blood leukocyte counts in the validation experiment of the present invention when mice were injected with the deerskin collagen peptides.

[0036] Figure 12 This is a bar graph showing the effects of injecting different concentrations of deerskin collagen peptides into mice on mouse thymocytes in the verification experiment of the present invention.

[0037] Figure 13 This is a bar graph showing the effects of injecting different concentrations of deerskin collagen peptides into mice on mouse spleen cells in the verification experiment of the present invention.

[0038] Figure 14 This is a bar graph showing the effects of injecting different concentrations of deerskin collagen peptides into mice on mouse liver cells in the verification experiment of the present invention.

[0039] Figure 15 These are pathological sections of the spleen of mice injected with different concentrations of deerskin collagen peptides in the verification experiment of the present invention.

[0040] Figure 16 These are pathological sections of the thymus of mice injected with different concentrations of deerskin collagen peptides in the verification experiment of the present invention.

[0041] Figure 17 This is a bar graph showing the effect of different concentrations of deerskin collagen peptides on the mRNA expression of NF-κB in the spleen of mice injected with the deerskin collagen peptides in the verification experiment of the present invention.

[0042] Figure 18This is a bar graph showing the effect of different concentrations of deerskin collagen peptides on the mRNA expression of TNF-α in the spleen of mice injected with the deerskin collagen peptides in the verification experiment of the present invention.

[0043] Figure 19 This is a bar graph showing the effect of different concentrations of deerskin collagen peptides on the mRNA expression of IL-5 in the spleen of mice injected with the deerskin collagen peptides in the verification experiment of the present invention.

[0044] Figure 20 This is a bar graph showing the effect of different concentrations of deerskin collagen peptides on the mRNA expression of IL-10 in the spleen of mice injected with the deerskin collagen peptides in the verification experiment of the present invention. DETAILED DESCRIPTION

[0045] The following is a clear and complete description of the technical solutions in the embodiments of this application. Obviously, the embodiments described are only part of the embodiments of this application, not all of them. Based on the embodiments of this application, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of this application.

[0046] The amount of pepsin added to each 1.0 mL of the slurry is 10.0 mg to 14.0 mg, and the pH value of the slurry is adjusted to 1.5 to 3.0. The slurry is then placed in a constant temperature shaker for enzymatic hydrolysis for 2.0 h to 5.0 h, and the temperature of the constant temperature shaker is 30.0° C. to 50.0° C. After the enzymatic hydrolysis is completed, it is inactivated at 100° C. for 8.0 min, and then the pH value of the enzymatically hydrolyzed slurry is adjusted to 1.5 to 3.0, and a centrifuge is used to centrifuge at a rate of 2500 r / min to 4000 r / min for 20.0 min to 40.0 min, and the supernatant is taken to obtain an enzymatic hydrolyzate.

[0047] S3. Add a composite enzyme of papain and trypsin to the above enzymatic solution, wherein the mass ratio of papain and trypsin is (1~3): (1~3); the amount of the composite enzyme of papain and trypsin added is 4000 U / g~6000 U / g, and the amount of the composite enzyme of papain and trypsin added to each 1.0 mL of enzymatic solution is 4.0 mg~6.0 mg, the pH value of the enzymatic solution is adjusted to 3.0~5.0, and then the enzymatic solution is placed in a constant temperature oscillator for enzymolysis for 2.0h~5.0h, and the temperature in the constant temperature oscillator is 35.0℃~50.0℃. After the enzymatic hydrolysis is completed, it is inactivated at 100℃ for 8.0min, and then the pH value of the enzymatic hydrolysis slurry is adjusted to 3.0~5.0, and a centrifuge is used to centrifuge at a rate of 2500r / min~4000r / min for 20.0min~40.0min, and the supernatant is taken to obtain a secondary enzymatic hydrolysis enzymatic solution;

[0048] S4. The enzymatic hydrolysis solution obtained in step S3 is added to a rotary evaporator for concentration. The temperature during rotary evaporation is 50°C to 70°C. After concentration, the concentrated enzymatic hydrolysis solution is placed in a freeze dryer for freeze drying. The freezing temperature is -45°C to -55°C and the freezing time is 40h to 50h, thereby obtaining deerskin collagen peptide.

[0049] Furthermore, in step S1, the volume of distilled water added to 1.0 g of deerskin is 18 mL to 25 mL.

[0050] Example 1

[0051] S1. Deer hide was removed, hair removed, fat and fascia removed, minced into 0.5 cm × 0.5 cm pieces, and homogenized with distilled water to prepare a slurry for later use.

[0052] S2, pepsin was added to the above-mentioned slurry, wherein the amount of pepsin added was 4000 U / g, and the amount of pepsin added to every 1.0mL of slurry was 12.5mg, the pH value of the slurry was adjusted to 2.0, and the slurry was placed in a constant temperature oscillator for enzymolysis for 3.0h, and the temperature in the constant temperature oscillator was 37.0°C. After the enzymolysis was completed, it was inactivated at 100°C for 8.0min, and then the pH value of the slurry after enzymolysis was adjusted to 2.0, and a centrifuge was used to centrifuge at a speed of 3000r / min for 35.0min. The supernatant was taken to obtain an enzymolyzed solution;

[0053] S3. Add a composite enzyme of papain and trypsin to the above enzymatic hydrolysate, wherein the mass ratio of papain and trypsin is 1:1; the amount of pepsin added is 5000 U / g, and the amount of papain and trypsin composite enzyme added to each 1.0 mL of enzymatic hydrolysate is 5.0 mg, the pH value of the enzymatic hydrolysate is adjusted to 4.0, and the enzymatic hydrolysate is placed in a constant temperature oscillator for enzymolysis for 3.0 h, the temperature in the constant temperature oscillator is 45.0 ° C. After the enzymatic hydrolysis is completed, it is inactivated at 100 ° C for 8.0 min, and then the pH value of the enzymatic hydrolyzed slurry is adjusted to 4.0, and a centrifuge is used to centrifuge at a rate of 3000 r / min for 35.0 min, and the supernatant is taken to obtain a secondary enzymatic hydrolysate;

[0054] S4. The enzymatic hydrolysis solution obtained in step S3 is added to a rotary evaporator for concentration at a temperature of 60° C. After concentration, the concentrated enzymatic hydrolysis solution is placed in a freeze dryer for freeze drying at a freezing temperature of −50° C. for 48 h, thereby obtaining deerskin collagen peptides. The yield of deerskin collagen peptides is 6%.

[0055] Weigh 0.05 mg, 0.5 mg, 1.0 mg, 2.0 mg, 2.5 mg, 5.0 mg, and 10 mg of deerskin collagen peptide respectively, and dissolve them in 1.0 mL of pure water to prepare 0.05 mg / mL, 0.5 mg / mL, 1.0 mg / mL, 2.0 mg / mL, 2.5 mg / mL, 5.0 mg / mL, and 10 mg / mL deerskin collagen peptide solutions for use. Depending on the actual concentration of deerskin collagen peptide used, different weights of deerskin collagen peptide can be weighed to prepare solutions of different concentrations.

[0056] Comparative Example 1

[0057] The only difference between this comparative example and Example 1 is that trypsin is added in step S3 to replace the composite enzyme of papain and trypsin in Example 1 to obtain deerskin collagen peptides.

[0058] Comparative Example 2

[0059] The only difference between this comparative example and Example 1 is that papain is added in step S3 to replace the papain and trypsin complex enzyme in Example 1 to obtain deerskin collagen peptides.

[0060] Comparative Example 3

[0061] The only difference between this comparative example and Example 1 is that pepsin is added in step S3 to replace the papain and trypsin complex enzyme in Example 1 to obtain deerskin collagen peptides.

[0062] Test Examples

[0063] 1. Determination of the molecular weight of deerskin collagen peptide

[0064] Conclusion: If Figure 1 As shown in the SDS-PAGE electrophoresis diagram, it can be seen that through the above Example 1 and Comparative Examples 1 to 3, it can be found that in step S3, the method of using papain and trypsin in combination in the second enzymatic hydrolysis is the best, and deerskin collagen peptides with a molecular weight of less than 10 kDa are prepared, which are small molecule peptides, indicating that the deerskin collagen peptides are better absorbed when used, so that the deerskin collagen peptides have a stronger repair ability for damaged skin cells.

[0065] 2. DPPH free radical scavenging activity and ABTS ＋ In vitro antioxidant capacity assay for free radical scavenging activity and total antioxidant capacity assay

[0066] Conclusion: If Figure 2As shown, taking the deerskin collagen peptide solution with a concentration of 1.0 mg / mL and 2.0 mg / mL as an example, with the TEAC value as an indicator, the higher the TEAC value of the deerskin collagen peptide solution with a concentration of 2.0 mg / mL, the higher the free radical scavenging rate of the deerskin collagen peptide. The scavenging rate and the venison peptide concentration show a certain dose-effect relationship, and the free radical scavenging rate is much higher than that of the deerskin collagen peptide solution with a concentration of 1.0 mg / mL.

[0067] 3. Conduct ABTS ＋ In vitro antioxidant capacity assay for free radical scavenging activity

[0068] Conclusion: If Figure 3 As shown, taking the deerskin collagen peptide solution concentrations of 0.5 mg / mL and 1.0 mg / mL as an example, with the TEAC value as an indicator, the higher the TEAC value of the deerskin collagen peptide solution with a concentration of 1.0 mg / mL, the stronger the free radical scavenging ability of the deerskin collagen peptide, and the scavenging ability and the concentration of the deerskin collagen peptide solution show a certain dose-effect relationship.

[0069] 4. Determination of total antioxidant capacity

[0070] Conclusion: If Figure 4 As shown in the figure, taking the deerskin collagen peptide solution with a concentration of 0.5 mg / mL and 1.0 mg / mL as an example, with the TEAC value as an indicator, the deerskin collagen peptide solution with a concentration of 1.0 mg / mL has a higher TEAC value, indicating that the deerskin collagen peptide has a strong reducing ability. With the increase of the deerskin collagen peptide concentration, the reducing ability is enhanced accordingly, and the reducing ability and the deerskin collagen peptide solution show a certain dose-effect relationship.

[0071] The above tests show that the deerskin collagen peptide of the present invention is a small molecule peptide and has good in vitro antioxidant activity, which can effectively repair photodamaged skin cells and promote the growth of skin cells.

[0072] 5. Conduct security testing

[0073] The CCK-8 kit was used to detect the cytotoxicity of deerskin collagen peptide on skin cells.

[0074] Conclusion: If Figure 5 As shown, the concentrations of deerskin collagen peptide solution were 0.05 mg / mL, 0.5 mg / mL and 5.0 mg / mL.

[0075] For example, according to GB / T16886.5-2003 (ISO10993-5:1999), samples with a cell viability exceeding 75% are generally considered non-cytotoxic. Using a CCK-8 test kit, different concentrations of deerskin collagen peptide solutions were tested, and the cell viability was found to be over 95%, fully demonstrating that the deerskin collagen peptide of the present invention is safe and non-toxic.

[0076] 6. Perform in vitro hemolysis performance test

[0077] The in vitro hemolysis of deerskin collagen peptide was detected using a colorimetric method. First, a 2% mouse erythrocyte suspension was prepared. 500 μL of the mouse erythrocyte suspension was mixed with deerskin collagen peptide solutions at concentrations of 0.1 mg / mL, 1.0 mg / mL, and 5.0 mg / mL, respectively, and incubated at 37°C for 1 h. After centrifugation, the absorbance of the supernatant at 540 nm was measured. 500 μL of the mouse erythrocyte suspension was mixed with distilled water as a positive control, and 500 μL of the mouse erythrocyte suspension was mixed with physiological saline as a negative control. After centrifugation, the absorbance of the supernatant at 540 nm was measured.

[0078] Conclusion: If Figure 6 As shown, the supernatants of the deerskin collagen peptide solution groups were all colorless and transparent, while the supernatant of the positive control group was bright red. Quantitative results showed that the hemolysis rate of the deerskin collagen peptides was less than 1.5%, indicating that they did not cause in vitro hemolysis when used, further demonstrating the safety of the deerskin collagen peptides of the present invention.

[0079] 7. Conduct skin repair test

[0080] In order to study the protective and restorative effects of deerskin collagen peptide on the viability of cells irradiated by ultraviolet light, HaCaT cells were cultured.

[0081] The cells were irradiated with an optimal dose of UV light in the presence of different concentrations of deerskin collagen peptide solution. After irradiation, they were added to fresh DNEN medium containing 10% FBS and incubated for another 24 hours. The control group (CON) was incubated in fresh DNEN medium containing 10% FBS for another 24 hours under the same UV light irradiation condition. The cell viability was then analyzed.

[0082] Conclusion: If Figure 7 As shown in the figure, *** compared with the light damage group, there was a very significant difference (P < 0.001); **** compared with the light damage group

[0083] There was a highly significant difference between the two groups (P < 0.0001). Cell activity was highest when the deerskin collagen peptide content was 156.25 μg / mL, indicating that these cells had the strongest ability to maintain normal physiological functions, including cell metabolism, growth, and division. Experiments have shown that deerskin collagen small molecule peptides can have a certain ability to repair skin cells damaged by light.

[0084] 8. Test the ability to promote cell growth

[0085] The ability of deerskin collagen peptide to promote the growth of HaCaT cells was explored, and the cell scratch test was used to detect the

[0086] Proliferation capacity.

[0087] Conclusion: If Figure 8 and Figure 9 As shown in the table, **there is a significant difference compared with the blank group (P < 0.01). When the deerskin collagen peptide concentration is 0.1 mg / mL, the longest cell migration distance reaches 75 μm, indicating that deerskin collagen peptide can significantly promote cell proliferation and improve cell growth ability.

[0088] Verification Example 1

[0089] The method for verifying deerskin collagen peptides having skin repair and immune regulation effects comprises the following steps:

[0090] (1) Five different mouse models were constructed using female mice with similar behavioral abilities. Female mice (N = 36, 8 weeks old, weighing 23 g to 26 g) were purchased from the Animal Research Center of Jilin University. Housing conditions included: temperature: (22 ± 1)°C; relative humidity: 30%-50%; 12 / 12 h light / dark cycle; free access to food and water. The mice were acclimated to the laboratory environment for 7 days before the experiment.

[0091] Six female mice were selected as the blank control group (CON group) in the first group. From the first to the third day of the experiment, the same amount of normal saline was injected into the abdominal cavity of each mouse at a frequency of once a day, and the amount of normal saline injected was 10.0 mL / kg. Starting from the fourth day, each mouse was gavaged with distilled water at a volume of 10.0 mL / kg once a day for 13 consecutive days, and each mouse was fed for a total of 16 days.

[0092] Six female mice were selected as the model control group in the second group. From the first to the third day of the experiment, each mouse was intraperitoneally injected with the same amount of 80 mg / kg of cyclophosphamide, with the injection frequency being once a day, and the injection volume of 80 mg / kg of cyclophosphamide was 10.0 mL / kg. Starting from the fourth day, each mouse was gavaged with distilled water, with the gavage volume of distilled water being 10.0 mL / kg, once a day, for 13 consecutive days, for a total of 16 days for each mouse.

[0093] The third group selected 6 female mice as the low-dose collagen peptide group. From the first to the third day of the experiment, each mouse was intraperitoneally injected with the same amount of 80 mg / kg cyclophosphamide, with the injection frequency being once a day, and the amount of 80 mg / kg cyclophosphamide injected was 10.0 mL / kg; starting from the fourth day, each mouse was gavaged with a low dose of deerskin collagen peptide, with the gavage being once a day, and the amount of low-dose deerskin collagen peptide injected was 10.0 mL / kg, for 13 consecutive days, and each mouse was fed for a total of 16 days;

[0094] The fourth group selected 6 female mice as the medium-dose collagen peptide group. From the first to the third day of the experiment, each mouse was intraperitoneally injected with the same amount of 80 mg / kg cyclophosphamide, with an injection frequency of once a day and an injection volume of 80 mg / kg cyclophosphamide of 10.0 mL / kg. Starting from the fourth day, each mouse was gavaged with a medium-dose deerskin collagen peptide, with an oral gavage of 10.0 mL / kg once a day, for 13 consecutive days. Each mouse was fed for a total of 16 days.

[0095] The fifth group selected 6 female mice as the high-dose collagen peptide group. From the first to the third day of the experiment, each mouse was intraperitoneally injected with the same amount of 80 mg / kg cyclophosphamide, with the injection frequency being once a day, and the amount of 80 mg / kg cyclophosphamide injected was 10.0 mL / kg; starting from the fourth day, each mouse was gavaged with a high dose of deerskin collagen peptide, with the gavage being once a day, and the amount of high-dose deerskin collagen peptide gavage was 10.0 mL / kg, for 13 consecutive days, and each mouse was fed for a total of 16 days;

[0096] (2) During the 16 days of feeding, the mental state, fur glossiness, balance, and reaction ability of the mice were observed in real time, and the weight changes of each group of mice were recorded in real time and the data were collected;

[0097] (3) After the five groups of mice were fed for 16 days, they were anesthetized and blood was collected from the heart. The mice were then killed by cervical dislocation and dissected. The number of peripheral blood leukocytes in each group of mice was detected, the organ index of the mice was weighed, the organ lesions were checked, and the spleen immune-related indicators were tested.

[0098] Verification conclusion:

[0099] 1. If Figure 10 As shown in the figure, after three consecutive days of intraperitoneal injection of cyclophosphamide into mice, we observed that the model mice had reduced food intake and showed symptoms of lethargy, indicating that the cyclophosphamide-induced immunosuppression model in mice was successful. Among them, * the high-dose deerskin collagen peptide group was significantly different from the model control group (P < 0.05), ** the medium-dose deerskin collagen peptide group was significantly different from the model control group (P < 0.01), and *** the low-dose deerskin collagen peptide group was extremely significantly different from the model control group (P < 0.001). Figure 10 It can be seen that after 3 consecutive days of injection of cyclophosphamide, the body weight of mice in each group decreased significantly compared with the CON group. Afterwards, except for the model control group, the mice in the deerskin collagen peptide low-dose group, deerskin collagen peptide medium-dose group and deerskin collagen peptide high-dose group were treated by gavage. From the statistical results, it can be seen that the body weight of mice in the model control group continued to decrease one day after the termination of cyclophosphamide injection. From the 7th day, the weight of mice in each group began to recover and the degree of recovery was different. Among them, the weight recovery of mice in the deerskin collagen peptide low-dose group and deerskin collagen peptide medium-dose group was significantly better than that of mice in the deerskin collagen peptide high-dose group; compared with the model control group, the weight of mice treated by gavage with deerskin collagen peptide recovered significantly faster.

[0100] 2. After anesthetizing the mice fed for 16 days, blood was collected from the heart, and the mice were killed by cervical dislocation and dissected. The number of peripheral blood white blood cells was detected, the organ index of the mice was weighed, the organ lesions were checked, and the spleen immune-related indicators were tested. Figure 11 As shown, the detection of peripheral blood leukocyte counts found that after the mice were injected with cyclophosphamide, the peripheral blood leukocyte counts of the model control group, the deerskin collagen peptide low-dose group, the deerskin collagen peptide medium-dose group and the deerskin collagen peptide high-dose group were significantly decreased compared with the blank control group, with **significant differences (P < 0.01), ***extremely significant differences (P < 0.001), and ****extremely significant differences (P < 0.0001). However, compared with the model control group, the peripheral blood leukocyte counts of the deerskin collagen peptide low-dose group, the deerskin collagen peptide medium-dose group and the deerskin collagen peptide high-dose group were significantly increased, indicating that deerskin collagen peptide can significantly improve the immunosuppression of cyclophosphamide.

[0101] 3. If Figures 12 to 14 As shown in the figure, the thymus, spleen and liver indexes of the five groups of mice were detected. The model control group was significantly lower than the blank control group, indicating that the immunosuppression model was successfully established. * had significant differences (P<0.05), ** had significant differences (P<0.01), *** had extremely significant differences (P<0.001) and **** had extremely significant differences (P<0.0001). It can be seen that the thymus, spleen and liver indexes of the deerskin collagen peptide low-dose group and the deerskin collagen peptide medium-dose group were higher than those of the other groups of mice.

[0102] 4. If Figure 15 and Figure 16 As shown in the figure, spleen and thymus pathological sections of five groups of mice are shown: A is the blank control group; B is the model control group; C is the low-dose deerskin collagen peptide group; D is the medium-dose deerskin collagen peptide group; and E is the high-dose deerskin collagen peptide group. The red and white medulla of the spleen tissue of the blank control mice showed clear boundaries, and the cells were tightly and neatly arranged. Compared with the blank control group, the red and white medulla of the spleen tissue of the model control mice showed unclear boundaries and chaotic cell arrangement, indicating that the immunosuppressive model was successfully established. As the concentration of injected deerskin collagen peptide increased, the red and white medulla of the spleen tissue gradually became clearer, the white matter was intact, and the cells were densely arranged, indicating that deerskin collagen peptide can alleviate spleen cell damage. Thymus cells of the blank control mice were neatly arranged, without cell damage, and the red and white pulp cortex boundaries were clear. Compared with the blank control group. The thymocytes of mice in the model control group showed deformation, nuclear condensation and fragmentation, and were separated from the surrounding tissue in the cortex in a multifocal manner. However, as the concentration of deerskin collagen peptide injected increased, the thymocyte damage in the mice in the low-dose, medium-dose and high-dose deerskin collagen peptide groups was significantly reduced, as shown in the sections. The boundaries between the red and white medulla gradually became clear, and the cells were densely arranged, indicating that deerskin collagen peptide can reduce the damage to the thymus and greatly improve the immunosuppression of mice induced by cyclophosphamide.

[0103] 5. If Figures 17 to 20 As shown in Figure 2, the mRNA expression levels of NF-κB in the five groups of mice showed that Figure 17As shown in the figure, compared with the blank control group mice, the cyclophosphamide-induced immunosuppression in the model control group significantly reduced the mRNA expression of NF-κB in the spleen of the mice, and was lower than that in the CON group (P < 0.001), indicating that the immunosuppression model was successfully established. After the mice were injected with different doses of deerskin collagen peptide intervention, it was found that the mRNA expression of NF-κB in the high-dose deerskin collagen peptide group was significantly higher than that in the other four groups (P < 0.0001), indicating that high-dose deerskin collagen peptide can effectively upregulate the mRNA expression level of NF-κB in the spleen of immunosuppressive model mice. It can be seen from the mRNA expression of TNF-α in the five groups of mice that Figure 18 As shown in the figure, compared with the blank control group, the model control group mice had significantly lower TNF-α mRNA expression in the spleen of the mice induced by cyclophosphamide-induced immunosuppression, and the expression was lower than that of the CON group (P < 0.01, P < 0.0001), indicating that the immunosuppression model was successfully established. After the mice were injected with different doses of deerskin collagen peptides for intervention, it was found that the mRNA expression of TNF-α in the low-dose deerskin collagen peptide group was significantly higher than that in the other four groups (P < 0.05), indicating that the injection of low-dose deerskin collagen peptides has a certain promoting effect on the mRNA expression of TNF-α in the spleen of the immunosuppressive model mice. It can be seen from the mRNA expression of IL-5 in the five groups of mice that Figure 19 As shown, compared with the blank control group mice, the cyclophosphamide-induced immunosuppression in the model control group mice significantly reduced the mRNA expression of IL-5 in the spleen of the mice, and was lower than that in the CON group (P < 0.05), indicating that the immunosuppression model was successfully established. After the mice were injected with different doses of deerskin collagen peptide intervention, it was found that the mRNA expression of IL-5 in the low-dose deerskin collagen peptide group was significantly higher than that in the other four groups (P < 0.05), and the increase in the medium-dose deerskin collagen peptide group was more significant (P < 0.01), indicating that the low-dose deerskin collagen peptide group and the medium-dose deerskin collagen peptide group can significantly promote the mRNA expression of IL-5 in the spleen of immunosuppressive model mice. It can be seen from the mRNA expression of IL-10 in the five groups of mice, as shown in the figure below. Figure 20As shown, compared with the blank control group, cyclophosphamide-induced immunosuppression in the model control group significantly reduced IL-10 mRNA expression in the spleen of the mice, and the expression was lower than that in the CON group (P < 0.0001), indicating that the immunosuppression model was successfully established. After the mice were injected with different doses of deerskin collagen peptide, it was found that the IL-10 mRNA expression in the spleen of the mice in the low-dose deerskin collagen peptide group, the medium-dose deerskin collagen peptide group, and the high-dose deerskin collagen peptide group was significantly higher than that in the other groups (P < 0.0001), indicating that the injection of different doses of deerskin collagen peptide can significantly and effectively promote the expression of IL-10 mRNA in the spleen of the immunosuppressed model mice. In summary, the injection of different doses of deerskin collagen peptide into cyclophosphamide-induced immunosuppressed mice can effectively enhance the immunity of the mice and enable them to live a normal life.

[0104] The above embodiments are merely examples for clarity of explanation and are not intended to limit the embodiments. Those skilled in the art will appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all embodiments here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A method for preparing deerskin collagen peptide with skin repair and immunomodulatory effects, characterized in that: The steps include: S1. Deer hide was removed, hair removed, fat and fascia removed, minced into 0.5 cm × 0.5 cm pieces, and homogenized with distilled water to prepare a slurry for later use. S2, pepsin was added to the above-mentioned slurry, wherein the amount of pepsin added was 3000 U / g~4500 U / g, and the amount of pepsin added to every 1.0mL of slurry was 10.0mg~14.0mg, the pH value of the slurry was adjusted to 1.5~3.0, and the slurry was subsequently placed in a constant temperature oscillator for enzymolysis for 2.0h~5.0h, and the temperature in the constant temperature oscillator was 30.0℃~50.0℃. After the enzymolysis was completed, it was inactivated at 100℃ for 8.0min, and then the pH value of the slurry after enzymolysis was adjusted to 1.5~3.

0. Centrifuge was used to centrifuge at a rate of 2500r / min~4000r / min for 20.0min~40.0min, and the supernatant was taken to obtain an enzymolyzed solution; S3. Add a composite enzyme of papain and trypsin to the above enzymatic hydrolysate, wherein the mass ratio of papain and trypsin is (1-3): (1-3); the amount of the composite enzyme of papain and trypsin added is 4000 U / g-6000 U / g, and the amount of the composite enzyme of papain and trypsin added to each 1.0 mL of enzymatic hydrolysate is 4.0 mg-6.0 mg, the pH value of the enzymatic hydrolysate is adjusted to 3.0-5.0, and then the enzymatic hydrolysate is placed in a constant temperature oscillator for enzymolysis for 2.0 h-5.0 h, the temperature in the constant temperature oscillator is 35.0°C-50.0°C, after the enzymatic hydrolysis is completed, it is inactivated at 100°C for 8.0 min, and then the pH value of the enzymatic hydrolysate slurry is adjusted to 3.0-5.0, and a centrifuge is used to centrifuge at a rate of 2500 r / min-4000 r / min for 20.0 min-40.0 min, and the supernatant is taken to obtain a secondary enzymatic hydrolysate; S4. The enzymatic hydrolysis solution obtained in step S3 is added to a rotary evaporator for concentration. The temperature during rotary evaporation is 50°C to 70°C. After concentration, the concentrated enzymatic hydrolysis solution is placed in a freeze dryer for freeze drying. The freezing temperature is -45°C to -55°C and the freezing time is 40h to 50h, thereby obtaining deerskin collagen peptide.

2. The method for preparing deerskin collagen peptide with skin repair and immunomodulatory effects according to claim 1, characterized in that: In step S1, 18 mL to 25 mL of distilled water is added to 1.0 g of deerskin.

3. A method for verifying deerskin collagen peptides with skin repair and immune regulation effects, characterized in that: The steps include: (1) Select female mice with the same behavioral abilities to construct five different mouse models. Six female mice were selected as the blank control group (CON group) in the first group. From the first to the third day of the experiment, the same amount of normal saline was injected into the abdominal cavity of each mouse at a frequency of once a day, with the amount of normal saline injected ranging from 5.0 mL / kg to 15.0 mL / kg. Starting from the fourth day, each mouse was gavaged with distilled water at a frequency of 5.0 mL / kg to 15.0 mL / kg once a day for 11 consecutive days, and each mouse was fed for a total of 14 days. Six female mice were selected as the model control group in the second group. From the first to the third day of the experiment, each mouse was intraperitoneally injected with the same amount of 80 mg / kg of cyclophosphamide, with the injection frequency being once a day, and the amount of 80 mg / kg of cyclophosphamide injected was 5.0 mL / kg-15.0 mL / kg. Starting from the fourth day, each mouse was gavaged with distilled water, with the amount of distilled water gavage being 5.0 mL / kg-15.0 mL / kg, once a day, for 14 consecutive days, and each mouse was fed for a total of 14 days. The third group selected 6 female mice as the low-dose deerskin collagen peptide group. From the first to the third day of the experiment, each mouse was intraperitoneally injected with the same amount of 80 mg / kg of cyclophosphamide, and the injection frequency was once a day. The amount of 80 mg / kg of cyclophosphamide injected was 5.0mL / kg~15.0mL / kg; starting from the fourth day, each mouse was gavaged with a low dose of deerskin collagen peptide, and the gavage was once a day. The amount of low-dose deerskin collagen peptide was 5.0mL / kg~15.0mL / kg. This was continued for 14 days, and each mouse was fed for a total of 14 days. The fourth group selected 6 female mice as the deerskin collagen peptide medium-dose group. From the first to the third day of the experiment, each mouse was intraperitoneally injected with the same amount of 80 mg / kg of cyclophosphamide. The injection frequency was once a day, and the amount of 80 mg / kg of cyclophosphamide injected was 5.0 mL / kg~15.0 mL / kg. Starting from the fourth day, each mouse was gavaged with a medium-dose deerskin collagen peptide once a day, and the amount of the medium-dose deerskin collagen peptide was 5.0 mL / kg~15.0 mL / kg. This was continued for 14 days, and each mouse was fed for a total of 14 days. The fifth group selected 6 female mice as the high-dose deerskin collagen peptide group. From the first to the third day of the experiment, each mouse was intraperitoneally injected with the same amount of 80 mg / kg of cyclophosphamide. The injection frequency was once a day, and the amount of 80 mg / kg of cyclophosphamide injected was 5.0 mL / kg~15.0 mL / kg. Starting from the fourth day, each mouse was gavaged with a high dose of deerskin collagen peptide once a day, and the amount of high-dose deerskin collagen peptide was 5.0 mL / kg~15.0 mL / kg. This was continued for 14 days, and each mouse was fed for a total of 14 days. (2) During the 14 days of feeding, the mental state, fur glossiness, balance, and reaction ability of the mice were observed in real time, and the weight changes of each group of mice were recorded in real time and the data were collected; (3) After the five groups of mice were fed for 14 days, they were anesthetized and blood was collected from the heart. The mice were then killed by cervical dislocation and dissected. The number of peripheral blood leukocytes in each group of mice was detected, the organ index of the mice was weighed, the organ lesions were checked, and the spleen immune-related indicators were detected.

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