Anti-osteoporosis milk-derived peptide YPSYG and application thereof

Abstract

The invention provides an anti-osteoporosis milk-derived peptide YPSYG and application thereof, and a preparation method of the anti-osteoporosis milk-derived peptide YPSYG comprises the following steps: taking milk protein, firstly adding papain for enzymolysis, and then adding alkaline protease for enzymolysis to obtain the milk-derived peptide YPSYG. The milk-derived peptide with bone synthesis promoting activity can significantly improve bone biomechanical characteristics and bone microstructure, and can be used for preparing anti-osteoporosis drugs for treating osteoporosis. The milk-derived peptide YPSYG disclosed by the invention is convenient to synthesize, can be industrially produced, and has a good application prospect in the fields of medicines, cosmetics, health-care products and the like.

Description

Technical Field

[0001] The invention belongs to the field of bioactive peptides, and specifically relates to an anti-osteoporosis milk-derived peptide YPSYG and an application thereof. Background Art

[0002] Osteoporosis is a systemic, metabolic bone degenerative disease characterized by decreased bone mass, bone micronodule damage, decreased bone strength, and increased risk of fractures, which is common in the elderly. Osteoporosis can be divided into the following three types according to the cause: primary, secondary, and idiopathic osteoporosis. Primary osteoporosis is a physiological degenerative disease that occurs with aging and is common in the elderly. The cause of osteoporosis can be explained based on the law of bone metabolism. Normal bone metabolism includes two parts. One part is the acidic substances produced by osteoclasts to decompose the minerals in the bone, and the proteases to decompose the bone matrix, thereby forming bone resorption pits; the other part is the migration of osteoblasts to the bone resorption area, secreting bone matrix, and mineralizing it into new bones. The balance of bone metabolism depends on the number and work efficiency of osteoclasts and osteoblasts. When the balance of bone metabolism tends to excessive bone resorption, osteoporosis will occur. Studies have shown that osteoporosis caused by imbalanced bone metabolism may be related to oxidative stress. The level of inflammatory factors in the body will increase due to the occurrence of oxidative stress, thereby stimulating the activity of osteoclasts, leading to enhanced bone resorption efficiency, imbalanced bone metabolism, and reduced bone mass during bone metabolism, increasing the incidence of osteoporosis. The anti-osteoporosis drugs currently available on the market can be divided into bone resorption inhibitors and bone formation promoters according to different treatment methods. Bone resorption inhibitors include bisphosphonates, estrogen replacement therapy, vitamin D and its metabolites, and calcitonin. Bone formation promoters include fluoride, parathyroid hormone, androgen therapy, and calcium supplements. Although the above drugs have achieved significant clinical efficacy, they still have some limitations and side effects. For example, alendronate can severely irritate the esophagus when taken orally, causing esophagitis or digestive tract reactions; estrogen replacement therapy can cause adverse reactions such as endometrial hyperplasia and breast cancer; fluorine preparations can accelerate bone formation, but excessive accumulation of fluorine preparations will produce excessive osteocalcin, thereby reducing the effect of bone mineralization; in testosterone treatment, the combined use of calcium and vitamin D can stop the further development of osteoporosis and increase bone density, but it is accompanied by changes in blood lipids, cardiovascular complications, and the risk of prostate cancer; calcium intake can only accelerate the speed at which the human body reaches peak bone mass, but cannot improve its final peak bone mass. Therefore, there is an urgent need to find safe, effective and side-effect-free methods for the prevention of osteoporosis. Food-derived natural bioactive peptides are highly active, easy to prepare, widely available, and naturally safe. They can be used as a new, potential active factor to improve or prevent osteoporosis. Milk protein contains a variety of bioactive peptides that have a positive impact on human metabolism and physiological activities. It has the advantages of a comprehensive amino acid composition and physiological functional activity. It can be used as an important raw material for the preparation of bioactive peptides. Enzymatic hydrolysis of milk protein can greatly reduce the problems of indigestion and allergic reactions caused by its macromolecular structure, and release active peptides with physiological functions. Therefore, the development of enzymatic preparation of milk-derived anti-osteoporosis bioactive peptides is a very promising direction. Summary of the invention

[0003] Technical problem to be solved: The purpose of the present invention is to provide a new use of a bioactive peptide in anti-osteoporosis.

[0004] Technical solution: An anti-osteoporosis milk-derived peptide YPSYG, wherein the amino acid sequence of the milk-derived peptide YPSYG is Tyr-Pro-Ser-Tyr-Gly (wherein Tyr is tyrosine, Pro is proline, Ser is serine, and Gly is glycine). Furthermore, the method comprises the following steps: taking milk protein, first adding papain for enzymolysis, and then adding alkaline protease for enzymolysis to obtain milk-derived peptide YPSYG. Furthermore, the milk protein concentration is 1-5%. Furthermore, the amount of papain added is 1-5%, and the enzymolysis time is 1-3 hours; the amount of alkaline protease added is 1-5%, and the enzymolysis time is 1-3 hours. Furthermore, an anti-osteoporosis milk-derived peptide YPSYG is used in the preparation of drugs and health products for preventing osteoporosis. Furthermore, the drug also contains a pharmaceutically acceptable carrier. Furthermore, the application includes improving the biomechanical properties of bones. Furthermore, the application includes improving bone microstructure. Furthermore, the health care product includes a functional fermented dairy product. The application of the anti-osteoporosis milk-derived peptide YPSYG in the preparation of materials promoting bone synthesis and calcium absorption. Beneficial effects: 1. The milk-derived peptide with osteosynthesis-promoting activity provided by the present invention can significantly improve the biomechanical properties and bone microstructure of bones, and can also participate in the regulation of multiple hormone synthesis and oxidative stress response by up-regulating the expression levels of glutathione transferase and CYP aromatase or related genes, thereby improving osteoporosis. Therefore, it can be used to prepare anti-osteoporosis drugs and be used for the treatment of osteoporosis; 2. The milk-derived peptide with bone anabolic activity provided by the present invention has good bone anabolic activity, can significantly improve the bone biomechanical properties of osteoporotic rats, and effectively reduce the number and activity of osteoclasts observed by TRAP staining, alleviate the structural destruction and quantitative loss of trabecular bone induced by retinoic acid observed by HE staining, and can improve the bone microstructure of osteoporotic rats. 3. The YPSYG of the present invention is easy to synthesize and can be industrially produced, and has good application prospects in the fields of medicines, cosmetics and health products. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 The virtual enzymolysis results of four enzymes are shown; Figure 2 The effects of different enzymatic hydrolysis sequences, substrate concentrations, papain addition and enzymatic hydrolysis time, as well as alkaline protease addition and enzymatic hydrolysis time on the DPPH free radical scavenging rate and soluble calcium binding capacity of the hydrolyzate, where A is different enzymatic hydrolysis sequences; B is substrate concentration; C is papain concentration; D is papain enzymatic hydrolysis time; E is alkaline protease concentration; and F is alkaline protease enzymatic hydrolysis time. Figure 3 This is the total ion current of milk-derived peptides; Figure 4 It is the primary mass spectrum with a retention time of 18.12min; Figure 5 The secondary mass spectrum of m / z=674.4 ion; Figure 6 Different concentrations of milk peptide + 4mM CaCl 2 Effects on the proliferation activity of MC3T3-E1 cells, wherein A is KEDVPSER; B is HKEMPFPK; C is YPSYG; D is EDVPSE; E is VPQLE; F is IPAVF; Figure 7 is the maximum load diagram of the rat's bones; Figure 8 HE-stained microscope images of the cross-section of the femoral epiphysis (magnification ×200), where A is the normal group; B is the model group; C is the pure peptide group; D is the milk-derived peptide group; and E is the positive control group. DETAILED DESCRIPTION The present invention is further described below in conjunction with the accompanying drawings and embodiments. The following embodiments are explanations of the present invention and the present invention is not limited to the following embodiments: Example 1 Artificial synthesis of milk-derived bioactive peptide YPSYG (1) Computer virtual enzymatic hydrolysis First, the sequences of five milk proteins were searched in the UniProt database, including α-S1-casein, α-S2-casein, β-lactoglobulin, β-casein, and κ-casein. Four common commercial enzymes, trypsin (Try), pepsin (Pep), papain (Pap), and alkaline protease (Alk), were selected, and the BIOPEP active peptide database was used to simulate enzymatic hydrolysis of the selected five protein amino acid sequences, and the virtual enzymatic hydrolysis results of different combinations were recorded. The statistical results are shown in Figure 2. Figure 1 As shown, the two enzymes most suitable for hydrolyzing milk protein are papain and alkaline protease (Pap+Alk). (2) Screening of the order of adding two enzymes The enzyme was hydrolyzed by double enzyme segmented hydrolysis process, and the double enzyme addition method was set as follows: first add papain and then add alkaline protease, or first add alkaline protease and then add papain. The enzyme amount was 5%, and the DPPH free radical scavenging rate and soluble calcium binding capacity of the enzymatic solution were determined after 2 hours of enzymatic hydrolysis at the respective optimal enzymatic hydrolysis temperature and pH value. Figure 2 A. The appropriate enzymatic hydrolysis order can be selected as adding papain first and then alkaline protease. Among them, the determination method of DPPH free radical scavenging rate is as follows: ① Use anhydrous ethanol as solvent to prepare 1×10 -4 mol / L DPPH solution, store the prepared solvent away from light; ② Add 2 mL of enzymatic solution and 2 mL of DPPH solution into the same test tube and shake well. After mixing the solutions, react in the dark for 30 minutes to obtain a mixed solution. ③Measure the absorbance A of the mixed solution at 517nm i , and at the same time measure the absorbance A after mixing 2mL DPPH solution with 2mL anhydrous ethanol 0 , and the absorbance Aj after 2mL of enzymatic solution is mixed with 2mL of anhydrous ethanol. The calculation formula is as follows; The soluble calcium binding capacity was determined as follows: ① First, mix 20mM pH 7.8 phosphate buffer and 5mM CaCl 2 After mixing, 0.6 mM milk-derived peptide was added; ② Place the above solution in a 37℃ water bath for 2h, centrifuge (10000r / min, 15min) and take the supernatant; ③ Determine the soluble calcium binding amount in the supernatant using the o-cresolphthalein complexone colorimetric method. (3) Single factor test of double enzyme hydrolysis of milk protein 1. Substrate concentration The papain addition amount was fixed at 3%, the papain enzymolysis time was 2h, the alkaline protease addition amount was 3%, and the alkaline protease enzymolysis time was 2h. The lactoenzyme enzymolysis reaction was carried out at different substrate concentrations (1%, 2%, 3%, 4%, 5%) at the optimal enzymolysis temperature and pH value of the two enzymes to investigate the effects of different substrate concentrations on the DPPH free radical scavenging rate and soluble calcium binding capacity of milk-derived peptides. Figure 2 B can be obtained, and the optimal substrate concentration is 2%. 2. Amount of papain added The substrate concentration was fixed at 2%, the papain enzymolysis time was 2h, the alkaline protease addition amount was 3%, and the alkaline protease enzymolysis time was unchanged at 2h. At the optimal enzymolysis temperature and pH value of the two enzymes, different papain addition amounts (1%, 2%, 3%, 4%, 5%) were used to perform lactolysis reaction to investigate the effects of different papain addition amounts on the DPPH free radical scavenging rate and soluble calcium binding capacity of milk-derived peptides. Figure 2 C shows that the optimal addition amount of papain is 1%. 3. Papain enzymatic hydrolysis time The substrate concentration was fixed at 2%, the amount of papain added was 1%, the amount of alkaline protease added was 3%, and the alkaline protease enzymolysis time was kept constant at 2h. The lactic acid enzymolysis reaction was carried out at different papain enzymolysis times (1h, 1.5h, 2h, 2.5h, 3h) at the optimal enzymolysis temperature and pH value of the two enzymes to investigate the effects of different papain enzymolysis times on the DPPH free radical scavenging rate and soluble calcium binding capacity of milk-derived peptides. Figure 2 D shows that the optimal enzymatic hydrolysis time of papain is 2h. 4. Amount of alkaline protease added The substrate concentration was fixed at 2%, the amount of papain added was 1%, the papain hydrolysis time was 2h, and the alkaline protease hydrolysis time was kept constant at 2h. At the optimal hydrolysis temperature and pH value of the two enzymes, different alkaline protease addition amounts (1%, 2%, 3%, 4%, 5%) were used to perform lactolysis reactions to investigate the effects of different alkaline protease addition amounts on the DPPH free radical scavenging rate and soluble calcium binding capacity of milk-derived peptides. Figure 2 E shows that the optimal addition amount of alkaline protease is 1%. 5. Alkaline protease enzymatic hydrolysis time The substrate concentration was fixed at 2%, the amount of papain added was 1%, the papain enzymatic hydrolysis time was 2h, and the amount of alkaline protease added was unchanged at 1%. The lactic acid enzymatic hydrolysis reaction was carried out at different alkaline protease enzymatic hydrolysis times (1h, 1.5h, 2h, 2.5h, 3h) at the optimal enzymatic hydrolysis temperature and pH value of the two enzymes to investigate the effects of different alkaline protease enzymatic hydrolysis times on the DPPH free radical scavenging rate and soluble calcium binding capacity of milk-derived peptides. Figure 2It can be seen from F that the optimal enzymatic hydrolysis time of alkaline protease is 3h. In summary, the results are as follows Figure 2 As shown, the enzymatic substrate concentration of the double-enzyme hydrolysis of milk protein is 2%, the amount of papain added is 1%, the enzymatic hydrolysis time is 2h, and the amount of alkaline protease added is 1%, and the enzymatic hydrolysis time is 3h, so as to obtain milk-derived peptides. Structural identification and mass spectrometry analysis of milk-derived peptides Preparation of test and reference solutions: Take the lyophilized milk peptide powder and pass it through a 24-mesh sieve, weigh 1g of milk peptide powder and reference respectively and add ultrapure water to dissolve, filter through a 0.45μm filter membrane, and use liquid chromatography-mass spectrometry to identify the structure of the milk peptide. According to the total ion current of milk-derived peptides ( Figure 3 ), the retention time of milk-derived peptides is mainly concentrated in the first 41 minutes. According to the molecular ion peaks and fragment ion peaks in the corresponding secondary mass spectrum, the peptide sequence corresponding to the secondary mass spectrum is deduced in combination with the amino acid molecular weight. Taking a quasi-molecular ion (m / z=674.4) with a retention time of 18.12 minutes as an example, the structure of the milk-derived peptide is analyzed; according to Figure 4 , Figure 5 , after analysis by proteome discover2.4 software, combined with the molecular weight of amino acids, the amino acid sequence of the peptide corresponding to the secondary mass spectrum can be calculated. The UPLC-Q-Extractive Orbitrap MS results were analyzed and a total of 34 peptides were obtained, which are listed in Table 1 below: Table 1 Sequences of milk-derived peptides identified by UPLC-Q-Extractive Orbitrap MS The amino acid sequences of most of the milk-derived peptides in Table 1 are consistent with the structural characteristics of peptides with antioxidant and calcium absorption promoting activities, confirming that bioinformatics virtual enzymatic hydrolysis technology can effectively screen bioactive peptides. Molecular docking screening and activity verification Autodock4.2 software was used to complete the docking of the milk-derived peptides in Table 1 with DPPH·. The minimum binding energy was used as the evaluation index, and the peptide segment with the minimum binding energy was selected to screen the anti-osteoporosis milk-derived peptides. Table 2 Binding free energy and docking model scores of milk peptides and DPPH· / EGFR SYBYL2.0 software was used to screen potential milk-derived peptides with osteogenic activity. EGFR was selected as the receptor, 34 milk-derived peptides were selected as ligands, and the binding pocket was set to receptor surface intelligent screening. The affinity between the receptor and the peptide was judged according to the model Total score. The larger the total score, the better the binding model effect of the receptor and the peptide, which means that the peptide has a stronger osteogenic activity. Table 2 lists the binding free energy of the milk peptides after docking with DPPH·, as well as the Total score of the docking model of the milk peptides with EGFR molecules. The sum of the Total score and the absolute value of Binding energy was used as the evaluation index to screen milk peptides with anti-osteoporosis activity. The larger the total score, the stronger the affinity between the receptor protein and the peptide segment, which means the better the enrichment effect on the active molecules. According to the total score in Table 2, the four milk peptides with the highest scores, KEDVPSER, HKEMPFPK, YPSYG, and EDVPSE, were screened. Considering the possibility of false negatives in the Total Score value, the two milk peptides with the lowest total scores, VPQLE and IPAVF, were selected as the negative control group for subsequent verification experiments. Example 2 Effect of milk-derived peptides on MC3T3-E1 cell proliferation MC3T3-E1 cells in the logarithmic growth phase were selected for digestion and prepared into a cell suspension. After counting with a hemocytometer, the suspension was diluted to 1.5 × 10 5 cells / mL, inoculated into 96-well plates, 100 μL per well. Incubate at 37°C, 5% CO 2 Incubate in the incubator for 24 hours, discard the old culture medium, add culture medium to the blank zero group, add cells without drug to the control group, and add different concentrations of KEDVPSER, YPSYG, HKEMPFPK, EDVPSE, IPAVF, and VPQLE solutions (0.02-0.8mM) to the experimental group. Each group has 6 replicate wells, and select a milk-derived peptide with the highest proliferation rate of MC3T3-E1 cells. After 24 hours of culture in the incubator, discard the old culture medium, add 200μMTT (0.5mg / mL) to each well, continue to culture for 4 hours, discard MTT, and add 150μL dimethyl sulfoxide. Oscillate at room temperature in the dark for 5 minutes, mix evenly, and measure the absorbance A570 of each well at a wavelength of 570nm. Repeat 3 times and take the average value. Calculate according to the following formula: The MTT method was used to detect the effects of different concentrations of milk peptides on the proliferation activity of MC3T3-E1 cells cultured for 24 h. Figure 5 It can be seen that KEDVPSER, HKEMPFPK, YPSYG, and EDVPSE have stronger proliferation activity in promoting MC3T3-E1 cells, while VPQLE and IPAVF have no effect on the proliferation activity of MC3T3-E1 cells or even have an inhibitory effect. MTT method was used to detect different concentrations of milk peptide + 4mM CaCl 2 Effects on the proliferation activity of MC3T3-E1 cells (such as Figure 6 ), different concentrations of milk peptides + 4mM CaCl 2 YPSYG+4mM CaCl 2 The promoting effect is better than adding other milk-derived peptides. Example 3 Study on the intervention effect of milk-derived peptides on osteoporosis in elderly rats (1) Animal grouping: 60 female SD rats (about 12 weeks old) were housed under controlled environmental conditions (12-h light-dark cycle). After acclimation for one week, they were randomly divided into 6 groups according to body weight, with 8-10 rats in each group. (2) Establishment of osteoporosis rat model: The blank group was gavaged with 0.9% saline (0.9 g sodium chloride plus 100 mL distilled water) once a day, and the remaining groups were gavaged with retinoic acid (75 mg retinoic acid / kg bw) once a day for 2 weeks to complete the modeling; (3) Drug administration: Starting from the third week, oral administration was performed once a day according to the group for 6 weeks: Normal group (CK): intragastrically administered with 0.9% saline; Model group (MG): intragastric administration of 0.9% saline; Positive control group (PG): intragastric administration of alendronate sodium (5 mg alendronate sodium / kg bw) Milk peptide group (MP): The laboratory pre-experiment set the intragastric concentration to be 400 mg milk peptide / kg bw, 800 mg milk peptide / kg bw, and 1000 mg milk peptide / kg bw. According to the pre-experiment results, the intragastric concentration of milk peptide was determined to be (800 mg milk peptide / kg bw) Pure peptide group (PP): intragastric administration of milk-derived peptide YPSYG (100 mMYPSYG / kgbw); (4) Bone biomechanical indicators: After removing the excess tissue of the right femur and tibia of the rat, the length and diameter were measured using an electronic vernier caliper and the data were recorded. A three-point bending test was performed on the right femur and tibia using a texture analyzer. The test temperature was 23°C, the load measurement accuracy of the testing machine was 0.001N, the support span was 20mm, the pressure head diameter was 1.5mm, the range of 1000N was selected, and the pressure was applied at a loading speed of 10mm / min until a fracture occurred. The maximum load of the right femur and tibia of each group was recorded. according to Figure 7The results showed that compared with the blank group, the maximum load of the femur and tibia in the model group was significantly reduced, indicating that the modeling was successful. Compared with the model group, the maximum load of the femur and tibia in the milk peptide group, pure peptide group and positive control group was significantly increased, indicating that milk peptide can significantly improve the bone biomechanical indicators of osteoporotic rats and has a good effect on improving bone quality and cortical bone strength. (5) Bone histomorphological parameters: Hematoxylin-eosin staining. After decalcification, embedding, and sectioning, the cross-section of the left femoral epiphysis and the longitudinal section of the left femoral shaft of the rat were stained with HE, and the histopathological characteristics of the bones were observed under an optical electron microscope. Figure 8 The results of the observation of the cross section of the femoral epiphysis under an optical electron microscope are shown. The HE staining results show that after treatment with milk-derived peptides, the bone microstructure and bone quality of osteoporotic rats were effectively improved, which played a positive role in improving osteoporosis. The above is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any technician familiar with the art can make many possible changes and modifications to the technical solution of the present invention by using the above disclosed methods and technical contents without departing from the spirit and technical solution of the present invention, or modify it into an equivalent embodiment of equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still falls within the scope of protection of the technical solution of the present invention.

Claims

1. An anti-osteoporosis milk-derived peptide YPSYG, characterized in that: The amino acid sequence of the milk-derived peptide YPSYG is Tyr-Pro-Ser-Tyr-Gly.

2. The method for preparing the anti-osteoporosis milk-derived peptide YPSYG according to claim 1, characterized in that: The following steps are involved: Take milk protein, first add papain for enzymolysis, then add alkaline protease for enzymolysis, so as to obtain milk-derived peptide YPSYG.

3. The method for preparing the anti-osteoporosis milk-derived peptide YPSYG according to claim 2, characterized in that: The milk protein concentration is 1-5%.

4. The method for preparing the anti-osteoporosis milk-derived peptide YPSYG according to claim 2, characterized in that: The amount of papain added is 1-5%, and the enzymolysis time is 1-3 hours; the amount of alkaline protease added is 1-5%, and the enzymolysis time is 1-3 hours.

5. Use of the anti-osteoporosis milk-derived peptide YPSYG according to claim 1 in the preparation of medicines and health products for preventing osteoporosis.

6. The use according to claim 5, characterized in that: The drug also contains a pharmaceutically acceptable carrier.

7. The use according to claim 5 or 6, characterized in that: Such applications include improving bone biomechanical properties.

8. The use according to claim 5 or 6, characterized in that: Such uses include improving bone microarchitecture.

9. The use according to claim 5, characterized in that: The health care products include functional fermented dairy products.

10. Use of the anti-osteoporosis milk-derived peptide YPSYG according to claim 1 in the preparation of materials promoting bone synthesis and calcium absorption.

Images