Preparation method of venison peptide with sarcopenia relieving effect and verification method of sarcopenia relieving effect of venison peptide

Abstract

The invention relates to a preparation method of venison peptide with a sarcopenia relieving effect and a verification method of the sarcopenia relieving effect of the venison peptide, belongs to the technical field of biological medicines, and solves the technical problems of tedious conditions in the preparation process of the venison peptide and verification of the sarcopenia relieving effect of the venison peptide. The preparation method of the venison peptide with the sarcopenia relieving effect comprises the following steps: cutting venison into small pieces, adding protease to carry out enzymolysis on the venison, freezing to obtain venison peptide, and obtaining venison peptide powder with small molecular weight through a dialysis bag. And then a verification experiment for relieving sarcopenia can be performed by constructing a mouse with sarcopenia and performing venison peptide on the mouse with sarcopenia. According to the invention, the extraction process is greatly simplified, the experimental method is optimized, the antioxidation performance test finds that the venison peptide still has strong antioxidation performance, and the venison peptide with antioxidation performance can alleviate oxidative damage of cells, maintain normal metabolism of muscle cells and improve muscle quality, so as to better relieve sarcopenia.

Description

Technical Field

[0001] The preparation method of deer meat peptide with the function of alleviating sarcopenia and the verification method of its function of alleviating sarcopenia belong to the field of biomedical technology. Background Art

[0002] Deer meat generally refers to the meat of sika deer or red deer, which contains a variety of active ingredients similar to those in velvet antler, such as various amino acids, fatty acids, mineral elements, etc. It is a good medicine for enhancing the body's metabolic function and the function of the body's muscle system. As a high-quality protein source, deer meat has attracted much attention in the fields of food and bioactive ingredient research in recent years. Deer meat is rich in protein, with a content between 20% and 25%, which plays an important role in promoting the main functional activities. Deer meat is also rich in essential amino acids required by the human body, with an amino acid content of 55.36 g / 100 g to 73.66 g / 100 g, and the essential amino acids account for 38% - 50% of the total amino acid content. Studies have found that after enzymatic hydrolysis and other treatments of deer meat protein, polypeptides with various biological activities can be produced, and these polypeptides show significant effects in antioxidant, immunity improvement, anti-fatigue and other aspects.

[0003] Sarcopenia, also known as muscle attenuation syndrome, is a comprehensive degenerative disease that appears with age, often manifested as a decrease in the volume and number of skeletal muscle fibers (especially type II muscle fibers, i.e., fast muscle fibers), a decrease in muscle strength, a decrease in muscle mass, a decrease in muscle function, etc. It has a relatively high incidence rate in the elderly population, seriously affecting the quality of life and health status of the elderly. Clinical experimental results show that sarcopenia patients are prone to fractures, falls and disability, and are closely related to an increased risk of various chronic diseases such as osteoporosis, hypertension, diabetes and cardiovascular diseases, greatly increasing the social medical burden.

[0004] And the glutamine contained in deer meat can increase the volume of muscle cells, inhibit protein decomposition, promote the differentiation and protein synthesis of skeletal muscle cells, and reduce skeletal muscle atrophy; leucine has the effect of promoting skeletal muscle protein synthesis and inhibiting protein decomposition; proline can promote the synthesis of skin and muscle proteins, suggesting that deer meat may play a role in alleviating sarcopenia by promoting skeletal muscle protein synthesis and inhibiting skeletal muscle atrophy. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art, in order to solve the technical problems such as the cumbersome conditions in the preparation process of deer meat peptide and the verification of the function of deer meat peptide in alleviating sarcopenia, the present invention now provides a preparation method of deer meat peptide with the function of alleviating sarcopenia and a verification method of its function of alleviating sarcopenia.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] Preparation method of deer meat peptide with the function of alleviating sarcopenia, comprising the following steps:

[0008] S1. Take deer meat, remove the skin and fascia, wash until there is no blood, cut into small pieces, add physiological saline for tissue homogenization to obtain a slurry, where the mass ratio of deer meat to physiological saline is (0.5 - 1.5):(6.0 - 8.0); then add protease to the slurry, where the mass ratio of deer meat to the added protease is (5.0 - 8.0):(0.5 - 2.0); then adjust the pH value of the slurry to 6.0 - 7.5, and then place the slurry in a constant temperature shaker for enzymatic hydrolysis for 5.0 h - 8.0 h, the temperature in the constant temperature shaker is 50.0 °C - 60.0 °C, after the enzymatic hydrolysis is completed, inactivate at 100 °C for 10.0 min, then adjust the pH value of the enzymatically hydrolyzed slurry to 6.0 - 7.5, use a centrifuge to centrifuge at a rate of 3500 r / min - 5000 r / min for 10.0 min - 20.0 min, take the supernatant to obtain an enzymatic hydrolysate;

[0009] S2. Add the enzymatic hydrolysate obtained in step S1 to a rotary evaporator for concentration, the temperature during rotary evaporation does not exceed 80 °C, after the concentration is completed, put the concentrated enzymatic hydrolysate into a freeze dryer for freeze drying, the freezing temperature is -75 °C - 85 °C, the freezing time is 20.0 h - 26.0 h, to obtain powdery deer meat peptide;

[0010] S3. Dissolve the powdery deer meat peptide in pure water, the concentration does not exceed 10.0 mg / mL, then use a dialysis bag with a molecular weight cut-off of 5000 Da to dialyze the deer meat peptide solution, after the dialysis is completed, perform a second rotary evaporation and a second freeze drying on the dialyzed deer meat peptide solution, the second rotary evaporation temperature does not exceed 80 °C, the temperature of the second freeze drying is -75 °C - 85 °C, the second freezing time is 20.0 h - 26.0 h, thereby obtaining powdery deer meat peptide with a molecular weight of 1000 Da - 5000 Da.

[0011] Furthermore, in step S1, the mass ratio of deer meat to physiological saline is 1.0:7.0.

[0012] Furthermore, in step S1, the protease is papain, where the mass ratio of deer meat to the added protease is

[0013] (5.0 - 8.0):(0.5 - 2.0), and can be 5.0:0.5, 6.0:0.5, 7.0:0.5, 8.0:0.5, 5.0:1.0, 6.0:1.0, 7.0:1.0, 8.0:1.0, 5.0:1.5, 6.0:1.5, 7.0:1.5, 8.0:1.5, 5.0:2.0, 6.0:2.0, 7.0:2.0, 8.0:2.0.

[0014] More preferably, the mass ratio of deer meat to the added protease is 7.0:1.0.

[0015] Furthermore, in step S1, the pH value of the slurry is adjusted to 7.0; the pH value of the slurry after enzymatic hydrolysis is adjusted to 7.0.

[0016] Furthermore, in step S2, the freezing temperature is -80 °C and the freezing time is 24.0 h.

[0017] Furthermore, in step S3, the temperature of the second freeze-drying is -80 °C and the second freezing time is 24.0 h.

[0018] A method for verifying the effect of deer meat peptides in alleviating sarcopenia, characterized by comprising the following steps:

[0019] (1) Select mice with the same behavioral ability to construct 4 different mouse models. The first group of mouse models is the blank control group (CON group), and the mice are normally fed for seven weeks; the second group of mouse models is the dexamethasone group (DEX group), that is, normal mice are injected with the inducing drug dexamethasone to construct mice suffering from sarcopenia, and the mice are normally fed for seven weeks and injected with the same amount of dexamethasone at the same time every day;

[0020] The third group of mouse models is the low-dose deer meat peptide group (D+VE-L group), that is, normal mice are injected with the inducing drug dexamethasone to construct mice suffering from sarcopenia, and the injection amount is the same as that of the second group. Then, the mice are gavaged with low-dose deer meat peptides, and the content of deer meat peptides is 90 mg / kg to 110 mg / kg. The mice are also normally fed for seven weeks and injected with the same amount of dexamethasone at the same time every day, and then gavaged with the same amount of deer meat peptides at the same interval;

[0021] The fourth group of mouse models is the high-dose deer meat peptide group (D+VE-H group), that is, normal mice are injected with the inducing drug dexamethasone to construct mice suffering from sarcopenia, and the injection amount is the same as that of the second group. Then, the mice are gavaged with high-dose deer meat peptides, and the content of deer meat peptides is 190 mg / kg to 210 mg / kg; the mice are also normally fed for seven weeks and injected with the same amount of dexamethasone at the same time every day, and then gavaged with the same amount of deer meat peptides at the same interval;

[0022] (2) When selecting mice with the same behavioral ability, all mice are tested by a climbing cage experiment and data is collected;

[0023] At the same time, within the 7 weeks of feeding the mice, the four groups of mouse models are tested by a climbing cage experiment and a swimming test every day, and the test data of each day is collected;

[0024] (3)When the feeding of the four groups of mouse models ended at seven weeks, the four groups of mouse models were sacrificed respectively, and the cardiac blood of the mice was collected and the gastrocnemius muscles of the mice were isolated. Then, experimental tests were conducted on the size and morphology of the gastrocnemius muscles of the four groups of mouse models, the effect of deer meat peptide on the gastrocnemius muscle coefficient of gavage mice, the effect of deer meat peptide on the mRNA expression of MyoG factor in mice with sarcopenia, and the effect of deer meat peptide on the mRNA expression of Murf-1 factor and Atrgon-1 factor in mice with sarcopenia.

[0025] Beneficial effects of the present invention:

[0026] 1. In the present invention, deer meat peptide is extracted from deer meat, and there is no need to remove other components in deer meat, such as dry matter, crude fat and crude ash, during the whole extraction process, which greatly simplifies the extraction process and saves costs. Compared with the prior art of adding two or three proteases, in the process of extracting deer meat peptide, only one protease is added to enzymatically hydrolyze deer meat to achieve effective extraction of deer meat peptide, which greatly optimizes the experimental method and obtains deer meat peptide still having biological activity and antioxidant capacity. The antioxidant capacity of the deer meat peptide obtained by enzymatic hydrolysis with papain added in the present invention is relatively strong, and the antioxidant performance of the deer meat peptide extracted in the present invention is verified through DPPH free radical scavenging rate determination, ABTS

[0027] free radical scavenging activity determination, metal chelating ability determination and total antioxidant capacity determination. At the same time, it also further proves that the extracted deer meat peptide still has biological activity and antioxidant performance on the premise of simplifying the extraction process. + 2. In the present invention, a blank control group of mice and a mouse model with sarcopenia are constructed, and then the mouse model with sarcopenia is gavaged with deer meat peptide having antioxidant performance. By testing the climbing cage experiment of the mice to verify the grasping force of the mice and testing the swimming time of the mice, it will be found that the deer meat peptide having antioxidant performance can reduce the oxidative damage of the diseased mice, protect the integrity of the cell membrane, maintain the normal metabolic environment of muscle cells, significantly improve the grasping force and swimming time of the mice, and thus verify that the deer meat peptide having antioxidant performance has a relieving effect on the mice with sarcopenia. In addition, by analyzing the size and morphology of the gastrocnemius muscles of different groups of mouse models and testing the effect of deer meat peptide on the mRNA expression of MyoG factor and the mRNA expression of Murf-1 and Atrgon-1 factors in mice with sarcopenia, the deer meat peptide for the mouse model with sarcopenia can relieve the muscle atrophy process more effectively by inhibiting the decomposition of skeletal muscle cells and promoting the proliferation and differentiation of muscle cells, and can indirectly promote the synthesis of vital proteins and improve muscle quality, thereby better relieving the symptoms of the mice with sarcopenia.

[0028]

[0029] ​3. The deer meat peptides extracted in the present invention are dialyzed through a dialysis bag with a molecular weight cut-off of 5000 Da to obtain deer meat peptides with a molecular weight in the range of 1000 Da to 5000 Da. Since the molecular weight is relatively small, when the deer meat peptides are used for gavage in mice with sarcopenia, the mice will absorb them better, and the stronger the alleviating effect on sarcopenia in mice. In addition, the deer meat peptides with antioxidant properties obtained in the present invention have an alleviating effect on sarcopenia, thus providing a new direction for the deep processing of deer meat. Description of the Drawings

[0030] Figure 1 It is a bar graph showing the ability of the deer meat peptides with the effect of alleviating sarcopenia prepared in the present invention to scavenge DPPH free radical activity at different concentrations, and this figure is used as the abstract drawing.

[0031] Figure 2 It is a bar graph showing the ability of the deer meat peptides with the effect of alleviating sarcopenia prepared in the present invention to scavenge ABTS + free radical activity at different concentrations.

[0032] Figure 3 It is a bar graph showing the metal chelating ability of the deer meat peptides with the effect of alleviating sarcopenia prepared in the present invention at different concentrations.

[0033] Figure 4 It is a bar graph showing the total antioxidant ability of the deer meat peptides with the effect of alleviating sarcopenia prepared in the present invention at different concentrations.

[0034] Figure 5 It is a bar graph showing the cage climbing time of mice in the CON group, DEX group, D+VE-L group and D+VE-H group before the verification experiment in the present invention.

[0035] Figure 6 It is a bar graph showing the average cage climbing time of mice in the CON group, DEX group, D+VE-L group and D+VE-H group within 7 weeks after administration in the verification experiment in the present invention.

[0036] Figure 7 It is a bar graph showing the average swimming time of mice within 7 weeks after administration in the verification experiment in the present invention.

[0037] Figure 8 It is a morphological diagram of the gastrocnemius muscles of mice in the CON group, DEX group, D+VE-L group and D+VE-H group in the verification experiment in the present invention.

[0038] Figure 9 It is a bar graph showing the effect of deer meat peptides on the weight of gastrocnemius muscles of four groups of mice in the verification experiment in the present invention.

[0039] Figure 10 It is a bar graph showing the effect of deer meat peptides on the gastrocnemius muscle coefficient of four groups of mice in the verification experiment in the present invention.

[0040] Figure 11 Gel electrophoresis images of the mRNA expression of MyoG factor in mice of the CON group, DEX group, D+VE-L group, and D+VE-H group by the deer meat peptide with the function of alleviating sarcopenia prepared in the present invention.

[0041] Figure 12 Bar graph of the mRNA expression of MyoG factor in four groups of mice by the deer meat peptide with the function of alleviating sarcopenia prepared in the present invention.

[0042] Figure 13 Gel electrophoresis images of the mRNA expression of Murf-1 factor and Atrgon-1 factor in mice of the CON group, DEX group, D+VE-L group, and D+VE-H group by the deer meat peptide with the function of alleviating sarcopenia prepared in the present invention.

[0043] Figure 14 Bar graph of the mRNA expression of Murf-1 factor in four groups of mice by the deer meat peptide with the function of alleviating sarcopenia prepared in the present invention.

[0044] Figure 15 Bar graph of the mRNA expression of Atrgon-1 factor in four groups of mice by the deer meat peptide with the function of alleviating sarcopenia prepared in the present invention. Detailed implementation manners

[0045] The technical solutions in the embodiments of the present application will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present application without creative efforts shall fall within the scope of protection of the present application.

[0046] Example 1

[0047] S1. Take deer meat, remove the skin and fascia, wash until there is no blood, cut into small pieces, and add 7.0 times the amount of physiological saline of the deer meat for tissue homogenization to obtain a slurry; then add papain to the slurry, and the weight of the added papain is 1 / 7 of the weight of the deer meat; then adjust the pH value of the slurry to 7.0, and then place the slurry in a constant temperature shaker for enzymatic hydrolysis for 7.0 h, and the temperature in the constant temperature shaker is 55.0 °C. After the enzymatic hydrolysis is completed, inactivate it at 100 °C for 10.0 min, and then adjust the pH value of the enzymatically hydrolyzed slurry to 7.0, and use a centrifuge to centrifuge at a rate of 5000 r / min for 20.0 min, and take the supernatant to obtain an enzymatic hydrolysate;

[0048] S2. Add the enzymolysis solution obtained in step S1 to a rotary evaporator for concentration. The temperature during rotary evaporation is 70 °C. After the concentration is completed, put the concentrated enzymolysis solution into a freeze dryer for freeze-drying. The freezing temperature is -80 °C and the freezing time is 24.0 h to obtain powdery deer meat peptides, and the yield of the deer meat peptides is 0.43%.

[0049] S3. Dissolve the powdery deer meat peptides in pure water at a concentration of 5 mg / mL. Then dialyze the deer meat peptide solution using a dialysis bag with a molecular weight cut-off of 5000 Da. After dialysis, perform a second rotary evaporation and a second freeze-drying on the dialyzed deer meat peptide solution. The temperature of the second rotary evaporation is 70 °C, the temperature of the second freeze-drying is -80 °C, and the second freezing time is 24.0 h to obtain powdery deer meat peptides with a molecular weight between 1000 Da and 5000 Da. Then take 0.5 mg of the powdery deer meat peptides and dissolve them in 1.0 mL of pure water to prepare a 0.5 mg / mL deer meat peptide solution for standby.

[0050] Example 2

[0051] The difference between this example and Example 1 is only in step S3. Take 1.0 mg of the powdery deer meat peptides with a molecular weight between 1000 Da and 5000 Da and dissolve them in 1.0 mL of pure water to prepare a 1.0 mg / mL deer meat peptide solution for standby. Other conditions and parameters are exactly the same as those in Example 1.

[0052] Example 3

[0053] The difference between this example and Example 1 is only in step S3. Take 1.5 mg of the powdery deer meat peptides with a molecular weight between 1000 Da and 5000 Da and dissolve them in 1.0 mL of pure water to prepare a 1.5 mg / mL deer meat peptide solution for standby. Other conditions and parameters are exactly the same as those in Example 1.

[0054] Example 4

[0055] The difference between this example and Example 1 is only in step S3. Take 2.0 mg of the powdery deer meat peptides with a molecular weight between 1000 Da and 5000 Da and dissolve them in 1.0 mL of pure water to prepare a 2.0 mg / mL deer meat peptide solution for standby. Other conditions and parameters are exactly the same as those in Example 1.

[0056] Test Example

[0057] 1. Determine the in vitro antioxidant capacity of DPPH radical scavenging activity

[0058] Take a certain amount of the 4 different concentrations of deer meat peptide solutions prepared in Examples 1 to 4 above respectively and determine the in vitro antioxidant capacity of DPPH radical scavenging activity.

[0059] Conclusion: As Figure 1 shown, when the concentration of venison peptide solution is between 0.5 mg / mL and 2 mg / mL, the TEAC values of Examples 1 to 4 are 13.10 ± 0.87 µM TE / mg, 22.43 ± 0.46 µM TE / mg, 29.80 ± 1.58 µM TE / mg, and 36.62 ± 1.20 µM TE / mg respectively. The higher the TEAC value, the stronger the antioxidant capacity of the solution, the higher the scavenging rate of free radicals, and there is a certain dose-effect relationship between the scavenging rate and the concentration of venison peptide.

[0060] 2. Determination of in vitro antioxidant capacity of ABTS ＋ radical scavenging activity

[0061] Take a certain amount of the four different concentrations of venison peptide solutions prepared in Examples 1 to 4 above respectively, and conduct the determination of in vitro antioxidant capacity of ABTS ＋ radical scavenging activity.

[0062] Conclusion: As Figure 2 shown, venison peptide has the ability to scavenge ABTS + radicals. When the concentration of venison peptide solution is between 0.5 mg / mL and 2 mg / mL, the TEAC values of Examples 1 to 4 are 460.62 ± 9.37 µM TE / mg, 668.79 ± 1.62 µM TE / mg, 874.74 ± 12.76 µM TE / mg, and 958.71 ± 8.76 µM TE / mg respectively. The higher the concentration of venison peptide, the stronger the ability to scavenge ABTS + radicals, and the absorbance at 734 nm decreases, and the ability to scavenge ABTS + radicals increases accordingly, that is, there is a certain dose-effect relationship between the scavenging ability and the concentration of venison peptide.

[0063] 3. Determination of metal chelating ability

[0064] Take a certain amount of the four different concentrations of venison peptide solutions prepared in Examples 1 to 4 above respectively, and conduct the determination of metal chelating ability.

[0065] Conclusion: As Figure 3 shown, venison peptide can chelate with Fe 2+ . When the concentration of venison peptide solution is between 0.5 mg / mL and 2 mg / mL, the TEAC values of Examples 1 to 4 are 10.55 ± 0.70%, 38.84 ± 0.72%, 56.46 ± 0.72%, and 64.59 ± 2.86% respectively. The higher the concentration of venison peptide, the more it chelates with Fe2+ The stronger the chelating ability is, so the venison peptide has a strong metal chelating ability.

[0066] 4. Determine the total antioxidant capacity

[0067] Take a certain amount of the 4 different concentrations of venison peptide solutions prepared in the above Examples 1 to 4 respectively to determine the total antioxidant capacity.

[0068] Conclusion: As Figure 4 shown, when the concentration of the venison peptide solution is between 0.5 mg / mL and 2 mg / mL, the TEAC values of Examples 1 to 4 are 43.24 ± 3.82 µM TE / mg, 90.24 ± 6.33 µM TE / mg, 135.38 ± 8.75 µM TE / mg, 179.76 ± 5.74 µM TE / mg respectively. The venison peptide has the ability to reduce Fe 3+ . As the concentration of the venison peptide solution increases, the solution color becomes lighter, and the ability to reduce Fe 3+ increases accordingly, and there is a certain dose-effect relationship between the reduction rate and the venison peptide concentration.

[0069] The above comprehensive tests show that the venison peptide has a strong antioxidant ability.

[0070] Verify Example 1

[0071] A verification method for the effect of venison peptide in alleviating sarcopenia, characterized by including the following steps:

[0072] (1) Select mice with the same behavioral ability to construct 4 different mouse models. The first group of mouse models is the blank control group (CON group), and the mice are normally fed for seven weeks;

[0073] The second group of mouse models is the dexamethasone group (DEX group), that is, inject the induction drug dexamethasone into normal mice to construct mice with sarcopenia, and normally feed the mice for seven weeks and inject the same amount of dexamethasone at the same time every day;

[0074] The third group of mouse models is the low-dose venison peptide group (D+VE-L group), that is, inject the induction drug dexamethasone into normal mice to construct mice with sarcopenia, and the injection amount is the same as that of the second group. Then, perform low-dose gavage of venison peptide on the mice, and the content of venison peptide is 90 mg / kg to 110 mg / kg. Also, normally feed the mice for seven weeks and inject the same amount of dexamethasone at the same time every day, and then gavage the same amount of venison peptide to the mice at the same interval;

[0075] The fourth group of mouse models was the high-dose venison peptide group (D+VE-H group). That is, normal mice were injected with the inducing drug dexamethasone to construct mice with sarcopenia. The injection amount was the same as that of the second group. Then, the mice were gavaged with high-dose venison peptide, and the content of venison peptide was 190 mg / kg - 210 mg / kg. The mice were also normally fed for seven weeks and injected with the same amount of dexamethasone at the same time every day, and then gavaged with the same amount of venison peptide at the same interval.

[0076] (2) When selecting mice with the same behavioral ability, all mice were tested by the climbing cage experiment and data were collected. At the same time, during the seven weeks of feeding the mice, the four groups of mouse models were tested by the climbing cage experiment and swimming test every day, and the test data of each day were collected.

[0077] (3) When the four groups of mouse models were fed until the end of seven weeks, the four groups of mouse models were sacrificed respectively, and the cardiac blood of the mice was collected and the gastrocnemius muscles of the mice were isolated. Then, the size and morphology of the gastrocnemius muscles of the four groups of mouse models, the effect of venison peptide on the gastrocnemius muscle coefficient of the gavaged mice, the effect of venison peptide on the mRNA expression of MyoG factor in mice with sarcopenia, and the effect of venison peptide on the mRNA expression of Murf-1 factor and Atrgon-1 factor in mice with sarcopenia were tested by experiments.

[0078] Verification conclusion:

[0079] 1. As Figure 5 and Figure 6 shown, through Figure 5 it can be seen that before starting the experiment on the mice, the climbing cage times of the mice in the CON group, DEX group, D+VE-L group and D+VE-H group were basically the same, indicating that the grasping forces of the four groups of mice selected before the experiment were the same, proving that the abilities of the selected mice were basically the same. Through Figure 6 it can be seen that after the mice were normally fed, injected with dexamethasone, and gavaged with low- and high-dose venison peptide for 7 weeks, comparing the average climbing cage time data of the mice collected in 7 weeks: compared with the CON group and the DEX group, the grasping force of the mice in the CON group was significantly higher than that of the mice in the DEX group (**P < 0.01), indicating that the construction of the mouse model with sarcopenia was successfully constructed. Comparing the D+VE-L group and the D+VE-H group, the grasping force of the mice in the D+VE-H group was significantly higher than that of the mice in the D+VE-H group (**P < 0.01). By gavaging high-dose venison peptide to the mice with sarcopenia, the grasping force of the mice can be significantly improved, indicating that gavaging the antioxidant venison peptide to the mice can effectively relieve the muscle atrophy process of the mice with sarcopenia, improve the muscle mass, and thus better relieve the sarcopenia of the mice.

[0080] 2. As Figure 7As shown in the figure, after normal feeding of mice for 7 weeks, injection of dexamethasone, and intragastric administration of low and high doses of venison peptide, the average swimming time data of mice collected at 7 weeks were compared: By comparing the CON group and the DEX group, the swimming time of mice in the CON group was significantly higher than that of mice in the DEX group (**P < 0.01), indicating that the construction of a mouse model with sarcopenia was successful and the experimental operation was correct; In addition, by comparing the swimming time of mice in the DEX group, the D+VE-L group, and the D+VE-H group, it was found that the swimming time of mice in the DEX group was significantly lower than that of mice in the D+VE-L group and the D+VE-H group, and the swimming time of mice in the D+VE-H group was the longest. Because the concentration of venison peptide administered intragastrically was the highest, it could indirectly promote the synthesis of myogenic proteins, improve the muscle mass of sarcopenic mice, significantly enhance the muscle endurance of sarcopenic mice, and there was a certain quantitative relationship between muscle endurance and the concentration of venison peptide, which further proved that intragastric administration of venison peptide could better relieve sarcopenia in mice.

[0081] 3. A decrease in muscle mass is a clinical manifestation of sarcopenia. The gastrocnemius muscle weight coefficient can be used to more accurately judge whether the construction of a sarcopenic mouse model is successful. As Figures 8 to 10 shown, through Figure 8 it can be seen that on the left are the left leg muscle diagrams of four groups of mice, and on the right are the right leg muscle diagrams of four groups of mice. By comparison, it can be seen that the leg muscles of mice in the D+VE-L group and the D+VE-H group are more firm, indicating that intragastric administration of venison peptide can inhibit the decomposition of dexamethasone on mouse muscle mass; Through Figure 9 it can be seen that compared with the gastrocnemius muscle weight of mice in the CON group, the gastrocnemius muscle weights of the DEX group, the D+VE-L group, and the D+VE-H group are relatively lower (**P < 0.01), and there is muscle atrophy in all cases, proving that the construction of a sarcopenic mouse model is successful and the experimental operation is correct. Compared with the gastrocnemius muscle weight of mice in the DEX group, the gastrocnemius muscle weights of the D+VE-L group and the D+VE-H group have both increased, indicating that venison peptide with antioxidant properties can relieve muscle atrophy caused by dexamethasone. Through Figure 10 it can be seen that compared with the CON group, the gastrocnemius muscle coefficient of the DEX group is relatively lower (**P < 0.01), the gastrocnemius muscle coefficients of the D+VE-L group and the D+VE-H group are significantly higher than that of the DEX group, and the gastrocnemius muscle coefficient of the D+VE-H group is the highest. By intragastric administration of high-concentration venison peptide to mice, the muscle strength and muscle endurance of mice are improved, which helps the nutritional supply of muscles and the clearance of metabolic wastes, thereby maintaining the normal function of muscles, and then increasing the gastrocnemius muscle coefficient of mice, significantly relieving the symptoms of muscle atrophy in mice, and then relieving sarcopenia in mice.

[0082] 4. As Figure 11 andFigure 12 As shown by Figure 11 it can be seen that the gel electrophoresis of the mRNA of MyoG factor in the gastrocnemius cells of mice. The band of the D+VE-H group mouse model is the brightest. The MyoG factor promotes the differentiation of muscle fiber cells by activating the transcription of muscle generation-related factors. During the growth of animals, MyoG plays a regulatory role in the generation of muscle cells centered on muscle fiber cells, directly controlling the muscle cells of the mesoderm, the normal growth of animals, and the generation of muscle cells. By Figure 12 it can be seen that compared with the CON group, the mRNA expression level of the MyoG factor in the DEX group mice was significantly decreased (**P < 0.01), which proved that the construction of the mouse model with sarcopenia was successfully constructed and the experimental operation was correct. By comparing the mRNA expression levels of the MyoG factor in the DEX group with those in the D+VE-L group and D+VE-H group mice, the mRNA expression levels of the MyoG factor in the D+VE-L group and D+VE-H group mice were significantly higher than those in the DEX group mice, which proved that venison peptides could significantly increase the mRNA expression level of the MyoG factor in the sarcopenia mice. Furthermore, by inhibiting the decomposition of skeletal muscle cells, it promoted the proliferation and differentiation of skeletal muscle cells in the sarcopenia mice, was more helpful to alleviate the muscle atrophy process, and could indirectly promote the synthesis of vital proteins, improve muscle quality, and thus better alleviate the symptoms of the sarcopenia mice.

[0083] 5. As Figures 13 to 15 shown by Figure 13 it can be seen from the gel electrophoresis of the mRNA of the MuRF-1 factor and Atrogin-1 factor in Figure 14 that Figure 15From the mRNA expression levels of MuRF1 and Atrogin-1 factors in the gastrocnemius cells of the four groups of mice, it can be seen that compared with the DEX group of mouse models, the mRNA expression levels of MuRF1 and Atrogin-1 factors in the D+VE-L group and D+VE-H group of mice were significantly decreased. Moreover, by gavage with a high concentration of venison peptide, the mRNA expression levels of MuRF1 and Atrogin-1 factors in the D+VE-H group of mice were the lowest, almost not expressed in the mice, indicating that the contents of MuRF1 and Atrogin-1 factors in the mice were the lowest. And MuRF1 and Atrogin-1 factors are related genes controlling skeletal muscle atrophy, belonging to the ubiquitin ligase E3 system, which control the muscle decomposition rate through MuRF1 and Atrogin-1 factors. The low mRNA expression levels of MuRF1 and Atrogin-1 factors indicate a decrease in the muscle decomposition rate, suggesting that gavage with venison peptide with antioxidant properties can effectively reduce the mRNA expression levels of MuRF1 and Atrogin-1 factors in sarcopenia mice, reduce the muscle decomposition rate in sarcopenia mice, and thus effectively relieve sarcopenia in mice.

[0084] In summary, using dexamethasone as an inducing drug to construct a mouse model with sarcopenia and administering venison peptide to the sarcopenia model mice, venison peptide can significantly increase the cage climbing time and swimming time of mice, and the gastrocnemius muscle weights of both the low-dose venison peptide group and the high-dose venison peptide group have increased, indicating that venison peptide can relieve muscle atrophy caused by dexamethasone; the mRNA expression levels of MuRF1 and Atrogin-1 factors in the low-dose venison peptide group and the high-dose venison peptide group were significantly decreased. MuRF1 and Atrogin-1 factors are related genes controlling skeletal muscle atrophy, promoting muscle fiber cell differentiation and relieving muscle atrophy; the mRNA expression levels of MyoG factors in the low-dose venison peptide group and the high-dose venison peptide group were also significantly increased. MyoG factor promotes the differentiation of muscle fiber cells by activating the transcription of muscle generation-related factors, and thus significantly relieves sarcopenia.

[0085] The above embodiments are merely examples for clear illustration and not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to enumerate all the implementation manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. A method for preparing a venison peptide having the effect of alleviating sarcopenia, characterized in that: The steps include: S1. Take venison, remove the skin and fascia, wash until there is no blood, cut into small pieces, add physiological saline for tissue homogenization, and prepare slurry, wherein the mass ratio of venison to physiological saline is (0.5-1.5): (6.0-8.0); then add protease to the slurry, wherein the mass ratio of venison to the added protease is (5.0-8.0): (0.5-2.0); then adjust the pH value of the slurry to 6.0-7.5, and then place the slurry in a constant temperature oscillator for enzymolysis for 5.0h-8.0h, the temperature in the constant temperature oscillator is 50.0℃-60.0℃, after the enzymolysis is completed, inactivate at 100℃ for 10.0min, and then adjust the pH value of the slurry after enzymolysis to 6.0-7.5, use a centrifuge at a rate of 3500r / min-5000r / min for 10.0min-20.0min, take the supernatant, and obtain an enzymolysis solution; S2, adding the enzymatic hydrolysate obtained in step S1 to a rotary evaporator for concentration, wherein the temperature during rotary evaporation is not higher than 80°C, and after the concentration is completed, placing the concentrated enzymatic hydrolysate in a freeze dryer for freeze drying, wherein the freezing temperature is -75°C to -85°C, and the freezing time is 20.0h to 26.0h, to obtain powdered venison peptide; S3. Dissolve the powdered venison peptide in pure water at a concentration not exceeding 10.0 mg / mL, and then dialyze the venison peptide solution using a 5000Da dialysis bag. After the dialysis is completed, perform a second rotary evaporation and a second freeze-drying on the dialyzed venison peptide solution. The second rotary evaporation temperature is not higher than 80°C, the second freeze-drying temperature is -75°C~-85°C, and the second freezing time is 20.0h~26.0h, thereby obtaining a powdered venison peptide with a molecular weight of 1000Da~5000Da.

2. The method for preparing venison peptide having the effect of alleviating sarcopenia according to claim 1, characterized in that: In step S1, the mass ratio of venison to physiological saline is 1.0:7.

0.

3. The method for preparing venison peptide having the effect of alleviating sarcopenia according to claim 1, characterized in that: In step S1, the protease is papain.

4. The method for preparing venison peptide having the effect of alleviating sarcopenia according to claim 1, characterized in that: The mass ratio of venison to added protease is 7.0:1.

0.

5. The method for preparing venison peptide having the effect of alleviating sarcopenia according to claim 1, characterized in that: In step S1, the pH value of the slurry is adjusted to 7.0; the pH value of the slurry after enzymatic hydrolysis is adjusted to 7.

0.

6. The method for preparing venison peptide having the effect of alleviating sarcopenia according to claim 1, characterized in that: In step S2, the freezing temperature is -80°C and the freezing time is 24.0 hours.

7. The method for preparing venison peptide having the effect of alleviating sarcopenia according to claim 1, characterized in that: In step S3, the temperature of the second freeze-drying is -80°C, and the second freezing time is 24.0 h.

8. A method for verifying the effect of venison peptide in alleviating sarcopenia, characterized in that: The steps include: (1) Mice with the same behavioral abilities were selected to construct four different mouse models. The first group of mouse models was the blank control group (CON group), and the mice were fed normally for seven weeks; The second group of mouse models was the dexamethasone group (DEX group), which was to inject the inducing drug dexamethasone into normal mice to create mice with sarcopenia. The mice were fed normally for seven weeks and injected with the same amount of dexamethasone at the same time every day. The third group of mouse models was the low-dose venison peptide group (D+VE-L group), that is, normal mice were injected with the inducing drug dexamethasone to construct mice with sarcopenia. The injection amount was the same as that of the second group, and then the mice were gavaged with a low dose of venison peptide, the content of which was 90mg / kg~110mg / kg. The mice were also fed normally for seven weeks and injected with the same amount of dexamethasone at the same time every day, and then the mice were gavaged with the same amount of venison peptide at the same interval; The fourth group of mouse models was the high-dose venison peptide group (D+VE-H group), that is, normal mice were injected with the inducing drug dexamethasone to construct mice with sarcopenia, and the injection amount was the same as that of the second group, and then the mice were gavaged with a high dose of venison peptide, and the content of venison peptide was 190mg / kg~210mg / kg; the mice were also fed normally for seven weeks and injected with the same amount of dexamethasone at the same time every day, and then the mice were gavaged with the same amount of venison peptide at the same interval; (2) When selecting mice with the same behavioral ability, all mice were subjected to cage climbing test and data were collected; at the same time, during the 7 weeks of feeding the mice, the four groups of mouse models were subjected to cage climbing test and swimming test every day, and the test data were collected every day; (3) After the four groups of mouse models were fed for seven weeks, they were killed, and their cardiac blood was collected and their gastrocnemius muscles were isolated. Then, experimental tests were conducted on the size and morphology of the gastrocnemius muscles of the four groups of mouse models, the effect of venison peptide on the gastrocnemius muscle coefficient of gavaged mice, the effect of venison peptide on the mRNA expression of MyoG factor in mice with sarcopenia, and the effect of venison peptide on the mRNA expression of Murf-1 factor and Atrgon-1 factor in mice with sarcopenia.

Images