Protein-like modified edible peptide capable of promoting osteogenesis and preparation method of protein-like modified edible peptide

Abstract

The invention provides a protein-like modified edible peptide with an osteogenesis promoting effect and a preparation method thereof. The preparation method comprises the following steps: (1) preparing a salmon skin gelatin solution; (2) preparing salmon skin gelatin hydrolysate by using papain; (3) introducing glycine into the hydrolysate under the catalysis of papain; and (4) carrying out proteoid reaction on the salmon skin gelatin hydrolysate to prepare the proteoid modified peptide. According to the novel protein-like modified edible peptide with the osteogenesis promoting effect and the preparation method of the novel protein-like modified edible peptide, the osteogenesis promoting functional characteristics of salmon skin gelatin hydrolysate are improved, and the salmon skin gelatin protein-like modified peptide provides nutritional and health-care effects for promoting bone growth, bone regeneration, bone health care and osteoporosis prevention; the composition can be used as a potential oral supplement for bone joint tissue recovery. The invention belongs to the technical field of health food processing, and the edible peptide has the advantages of edibility, compatibility and safety, can be easily and fully absorbed by human bodies, and has the advantages of strengthening bodies of specific osteoporosis people and the like.

Description

Technical Field

[0001] The invention relates to the technical field of food processing, and in particular to a protein-modified edible peptide having an osteogenic effect and a preparation method thereof. Background Art

[0002] Osteoporosis is a systemic bone metabolic disease and one of the common diseases that affect human health worldwide. According to statistics, more than 1.5 million people die from osteoporosis every year. Osteoporosis is mainly manifested by deterioration of bone structure, decreased bone density, increased bone brittleness and increased risk of fractures. Some drugs such as vitamin D, estrogen replacement drugs, calcium salts, bisphosphonates and calcitonin are commonly used to treat osteoporosis. Although these drugs have positive effects in improving bone density, reducing bone loss, and reducing the occurrence of fractures, long-term use will increase the risk of endometrial cancer and breast cancer, and will also cause cardiovascular and digestive tract diseases. Therefore, the development of new dietary nutritional supplements (including active peptides) that are beneficial to bone health and have no side effects has become a hot issue in the food field.

[0003] Natural proteins and their derivatives (active peptides) have multiple benefits for bone health, which can maintain bone formation and remodeling, promote bone joint tissue recovery, and prevent osteoporosis. For example, in vitro gastrointestinal digests of cheese can promote intestinal calcium absorption and the formation of extracellular bone matrix in Saos-2 cells. Cod bone collagen peptides have a mitigating effect on osteoporosis in ovariectomized rats. Tilapia and Pacific cod collagen both have good biocompatibility and cytocompatibility, and can promote the proliferation of fibroblasts and osteoblasts by accelerating cell cycle cycles, maintain bone balance, and promote bone regeneration. Cod bone collagen peptides increase the bone density of osteoblasts, effectively prevent bone loss, and alleviate the formation of osteoporosis. It is well known that peptide substances further transform the peptide structure through chemical modifications such as insertion of large hydrophobic side chains, D-amino acid substitution, methylation, acetalization, amidation, cyclization, polymerization, and cross-linking, thereby changing the peptide sequence, nutritional composition, and biological activity. For example, porcine plasma protein hydrolysate is modified by oxidized tannins or chlorogenic acid to have higher antioxidant activity. Hydrolyzates prepared by hydrolyzing salmon myofibrillar protein with gastric and pancreatic enzymes, and peptides prepared by Maillard reaction of seaweed oligosaccharides under the action of sorbitol, can improve the inflammation of RAW264.7 macrophages stimulated by LPS. However, how to further modify the salmon skin gelatin hydrolyzate as a natural food ingredient, adjust the key preparation technology, and improve the osteogenic effect of salmon skin gelatin hydrolyzate while ensuring that it is a nutritional health food has become one of the urgent problems to be solved in the current bone health food processing industry.

[0004] At present, there is no report on the use of a specific method to chemically modify salmon skin gelatin hydrolysate and papain through a protein-like reaction to obtain salmon skin gelatin protein-modified edible peptides with osteogenic effect, especially no report on the use of specific glycine composition and ratio to further synergistically improve the osteogenic effect of protein-modified edible peptides. Therefore, the development and preparation of a protein-modified edible peptide with osteogenic effect is expected to become a potential oral supplement for bone and joint tissue recovery and be used in the field of bone health food processing. Summary of the invention

[0005] In view of this, the purpose of the present invention is to propose a method for preparing salmon skin gelatin protein-modified edible peptides with simple process, easy operation, short cycle, no need for complex equipment, and significantly improved osteogenic effect, which can broaden the utilization rate of salmon skin gelatin hydrolysate and improve the osteogenic activity of the product.

[0006] The present invention provides a protein-modified edible peptide with osteogenic effect and a preparation method thereof, which solves the problem that the active peptides prepared from edible bioactive peptides in the field of bone health nutritional health food research at home and abroad have no obvious osteogenic effect, which ultimately leads to the inability to mass-produce nutritional edible peptides with osteogenic effect, limiting the application of bioactive peptides in the functional food processing industry, especially limiting the processing of bone health nutritional health foods.

[0007] The technical solution of the present invention is achieved in this way:

[0008] A method for preparing a protein-modified edible peptide having an osteogenic effect comprises the following steps:

[0009] S1: Weigh salmon skin gelatin, prepare a salmon skin gelatin solution with deionized water, take the salmon skin gelatin solution, add papain, adjust the pH of the solution, perform enzymatic hydrolysis at 55-65°C for 7-9h, inactivate the enzyme, centrifuge, collect the supernatant, and freeze-dry to obtain salmon skin gelatin hydrolysate; the suitable enzymatic hydrolysis temperature of papain is 55-65°C, and the hydrolysis effect is poor under other temperature conditions. Compared with other enzymes, papain is used to hydrolyze salmon skin to obtain salmon skin gelatin hydrolysate for subsequent protein-like reaction, and the efficiency is improved.

[0010] S2: preparing a high-concentration hydrolyzate of salmon skin gelatin, adding glycine methyl ester hydrochloride, stirring, adding specific activity papain, adjusting the pH of the solution, performing an enzymatic hydrolysis reaction at 35-40° C. for 5-7 hours, inactivating the enzyme, and terminating the reaction to obtain a protein-modified peptide solution; the glycine methyl ester hydrochloride is added in an amount such that the ratio of the free amino group content to the total free amino group content is at least 0.4 mol / mol;

[0011] S3: freeze-drying the protein-modified peptide solution, and then air-drying it to a constant weight to obtain the protein-modified edible peptide having an osteogenesis-promoting effect.

[0012] The protein-like reaction between salmon skin gelatin, glycine and papain of the present invention requires a four-step process: the first step is a hydrolysis reaction. Under the optimum pH value, papain catalyzes the breakage of low-concentration salmon skin gelatin peptide chains at different locations and hydrolyzes them into small-molecule peptide segments. When papain combines with salmon skin gelatin, an acylase intermediate is formed. When the intermediate reacts with water, the salmon skin gelatin is hydrolyzed into small-molecule peptide segments, and the average relative molecular mass of the obtained hydrolyzate decreases, the free end group content increases accordingly, and the free amino group content increases. At the same time, under the action of papain, the hydrolysis reaction exposes the hydrophobic groups inside the hydrolyzed salmon skin gelatin. The second step is a condensation reaction. The high salmon skin gelatin hydrolyzate concentration forms an acylase intermediate with papain, which promotes the condensation reaction between the amino acids of the hydrolyzed peptides to form new peptide bonds, introduces new groups, and realizes the modification of proteins. Among them, the presence of papain is the material basis of the protein-like reaction, because only the formation of an acylase intermediate can promote the generation of new peptide bonds. The third step is transpeptidation. When the acylase intermediate reacts with high-concentration salmon skin gelatin hydrolysate and glycine methyl ester hydrochloride, these nucleophilic reagents, the reaction will proceed in the direction of protein synthesis, and its free end group content will not change. During the process of protease transpeptidation, the small peptides of salmon skin gelatin hydrolysate can be converted into high molecular weight peptides, the peptide chain grows, and a high-molecular-weight protein-like substance is formed. At the same time, it also promotes the transfer of amide groups of glycine methyl ester hydrochloride, and introduces glycine into the salmon skin gelatin hydrolyzed peptide segment, thereby transforming the hydrolyzed peptide structure and affecting its physicochemical properties, biological activity and functional properties. The fourth step is hydrophobic interaction (physical polymerization). Salmon skin gelatin is a collagen derivative with a unique amino acid composition, namely, rich in glycine (molar ratio of about 1 / 3), proline (molar ratio close to 10%), and hydroxyproline (non-collagen protein content is extremely low). Although salmon skin gelatin is a hydrophilic colloid, as the protein-like reaction proceeds, under the action of papain, the hydrophobic surface inside the hydrolyzed polypeptide chain of salmon skin gelatin is exposed, so that hydrophobic interactions occur between the hydrolyzed polypeptide chains to form polymers. At the same time, glycine is a non-polar, hydrophobic amino acid. The presence of glycine methyl ester hydrochloride with strong hydrophobicity induces more hydrophobic amino acids to be exposed, so that hydrophobic glycine amino acids can be enriched on the peptide chain matrix, forming hydrophobic interactions, causing the hydrophobic index of the protein-like substance to increase, which is more conducive to the occurrence of protein-like reactions. Therefore, hydrophobic interaction is the main force for the formation of protein-like substances. The formation of protein-like substances involves a process of molecular rearrangement, which is physically aggregated into a gel-like substance and precipitated through non-covalent bonds. The formation of protein-like substances also promotes the polypeptide to proceed in the positive reaction direction of polymerization, thereby maintaining the structure of the protein-like reaction product.

[0013] The formation of new peptide bonds in protein-like reactions has a transpeptidation mechanism. The hydrolysis and synthesis reaction mechanism between salmon skin gelatin, papain and glycine methyl ester hydrochloride is as follows:

[0014]

[0015] At the same time, the present invention creatively uses glycine to mediate the protein-like reaction of salmon skin gelatin hydrolysate in the presence of papain. The ratio of the free amino group content of glycine methyl ester hydrochloride to the total free amino group content is 0.4 mol / mol, 0.6 mol / mol and 0.8 mol / mol, respectively. The three different proportions of glycine-mediated protein-like modified peptides achieve unexpected technical effects. Introducing different proportions of glycine during the protein-like reaction causes the salmon skin gelatin hydrolysate to undergo different numbers of transpeptidation and transpeptidation sites under the catalysis of papain, resulting in different sequences, arrangements and spatial structures of the peptide chains, forming protein-like modified peptides with different physical and chemical properties and biological activities.

[0016] Preferably, the glycine methyl ester hydrochloride is added according to the ratio of free amino group content to total free amino group content of 0.6 to 0.8 mol / mol.

[0017] Preferably, in step S1, the mass concentration of the salmon skin gelatin solution is 3-7 g / 100 mL; in step S2, the mass concentration of the high-concentration hydrolyzate is 30-50 g / 100 mL.

[0018] Preferably, in steps S1 and S2, the enzyme inactivation temperature is 85-95° C., and the enzyme inactivation time is 10-20 min.

[0019] Preferably, in step S1, the activity of the added papain is 3.5 kU / g protein, the pH value of the system is adjusted to 7.0, the water bath temperature is 60° C., and the hydrolysis time is 8 h.

[0020] Preferably, in step S2, the amount of papain added is 3.5 kU / g protein, the pH value of the system is adjusted to 7.0, the shaking table temperature is 37° C., and the reaction time is 6 h.

[0021] Preferably, in steps S1 and S3, the freeze drying includes vacuum freeze drying, the vacuum degree of the vacuum freeze drying is 0.01 to 0.5 mbar, the cold trap temperature of the vacuum freeze drying is -72°C to -45°C, and the vacuum freeze drying time is 48 hours.

[0022] On the one hand, the present invention provides a protein-modified edible peptide prepared according to the above preparation method.

[0023] On the other hand, the present invention provides the use of the above-mentioned protein-modified edible peptide in the preparation of health foods for restoring bone and joint tissues. The protein-modified edible peptide with osteogenic effect obtained by the present invention can be directly soluble in water, has osteogenic effect, can be used as a potential oral supplement for bone and joint tissue restoration, and is particularly suitable for the field of bone health nutritional health food processing; in addition, this salmon skin gelatin protein-modified peptide has the advantages shared by protein and glycine, has good biocompatibility, can provide the body with the required amino acids, and improves the nutritional value of bioactive peptide health foods, thereby achieving the purpose of promoting osteogenic activity to a greater extent.

[0024] Preferably, the protein-modified edible peptide is used in the preparation of health food for promoting osteoblast proliferation. The osteoblast proliferation activity assay of the present invention shows that after the protein-modified edible peptide acts on osteoblasts for 24 to 48 hours, the osteoblast proliferation activity increases to 115.0% to 138.8%.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] (1) The present invention creatively reacts salmon skin gelatin hydrolysate with papain to undergo a protein-like reaction for structural modification; further, a specific component dosage and specific amino acids are selected to obtain a glycine-mediated protein-like modified peptide of salmon skin gelatin hydrolysate. After the specific peptide acts on osteoblasts for 24 to 48 hours, multiple comparisons are made with salmon skin gelatin hydrolysate, a protein-like modified peptide without the introduction of glycine, and a mixture of salmon skin gelatin hydrolysate, inactivated papain, and glycine. The salmon skin gelatin protein-like modified peptide introduced with glycine has a more obvious osteogenic effect. After the specific peptide acts on osteoblasts for 24 to 48 hours, the osteoblast proliferation activity increases to 115.0% to 138.8%, which proves that the salmon skin gelatin protein-like modified peptide introduced with glycine promotes the proliferation activity of osteoblasts to a greater extent, has better osteogenic activity, and is of great significance to bone health.

[0027] (2) The present invention creatively uses glycine to mediate the protein-like reaction of salmon skin gelatin hydrolysate. The ratio of the free amino content of glycine methyl ester hydrochloride to the free amino content of salmon skin gelatin hydrolysate is 0.4 mol / mol, 0.6 mol / mol and 0.8 mol / mol, respectively. The protein-like modified peptides mediated by glycine in different proportions achieve unexpected technical effects. Introducing glycine in different proportions during the protein-like reaction makes the salmon skin gelatin hydrolysate undergo different numbers of transpeptidation and transpeptidation sites under the catalysis of papain, resulting in different sequences, arrangements and spatial structures of the peptide chains, forming protein-like modified peptides with different physical and chemical properties and biological activities.

[0028] (3) The salmon skin gelatin, glycine and papain involved in the present invention are common food raw materials. The glycine-mediated salmon skin gelatin protein-modified peptide prepared by a protein-like reaction between the three has good biocompatibility with cells, can be absorbed and utilized by the human body, and has the characteristics of edibility and safety.

[0029] (4) The protein-modified edible peptides involved in the present invention improve the osteogenic activity of salmon skin gelatin, increase the biological efficacy and nutritional value of salmon skin gelatin, and creatively provide new raw materials for the production of functional foods or health products with the effect of preventing osteoporosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The invention discloses a preparation flow chart of a protein-modified edible peptide with osteogenic effect and a preparation method thereof. DETAILED DESCRIPTION

[0031] In order to better understand the technical content of the present invention, specific embodiments are provided below to further illustrate the present invention.

[0032] Unless otherwise specified, the experimental methods used in the embodiments of the present invention are all conventional methods.

[0033] Unless otherwise specified, the materials, reagents, etc. used in the embodiments of the present invention can be obtained from commercial sources.

[0034] Example 1

[0035] A method for preparing a protein-modified edible peptide with an osteogenic effect, comprising the following steps:

[0036] (1) Weigh constant weight of salmon skin gelatin and prepare a solution with a mass concentration of 5 g / 100 mL using deionized water;

[0037] (2) adding papain with an activity of 3.5 kU / g protein to the salmon skin gelatin solution, adjusting the solution pH to 7.0, the water bath temperature to 60°C, and the hydrolysis time to 8 h;

[0038] (3) inactivating the enzyme in the solution at 90° C. for 15 min, cooling to room temperature, centrifuging at 5000 r / min for 20 min, collecting the supernatant for vacuum freeze drying, the vacuum degree being 0.01 to 0.5 mbar, the cold trap temperature being -72° C. to -45° C., and the freeze drying time being 48 h to obtain salmon skin gelatin hydrolyzate;

[0039] (4) Weighing salmon skin gelatin hydrolysate, and preparing a hydrolyzate with deionized water to have a substrate concentration of 40 g / 100 mL;

[0040] (5) adding glycine methyl ester hydrochloride having a free amino group content ratio of 0.4 mol / mol to the total free amino group content to the high concentration salmon skin gelatin hydrolyzate;

[0041] (6) adding papain with an activity of 3.5 kU / g protein, adjusting the system pH to 7.0, the shaker temperature to 37°C, and the reaction time to 6 h;

[0042] (7) After the reaction is completed, the solution is inactivated at 90° C. for 15 min to terminate the reaction, thereby obtaining a protein-modified peptide solution; the solution is cooled and freeze-dried in a vacuum at a vacuum degree of 0.01 to 0.5 mbar, a cold trap temperature of -72° C. to -45° C., and a freeze-drying time of 48 h to obtain a protein-modified edible peptide having an osteogenic effect.

[0043] (8) Blast drying for 6 to 8 h until the protein-modified edible peptide has a constant weight, and the sample is recorded as PGH-Gly-0.4.

[0044] Example 2

[0045] A method for preparing a protein-modified edible peptide with an osteogenic effect, comprising the following steps:

[0046] (1) Weigh constant weight of salmon skin gelatin and prepare a solution with a mass concentration of 5 g / 100 mL using deionized water;

[0047] (2) adding papain with an activity of 3.5 kU / g protein to the salmon skin gelatin solution, adjusting the solution pH to 7.0, the water bath temperature to 60°C, and the hydrolysis time to 8 h;

[0048] (3) inactivating the enzyme in the solution at 90° C. for 15 min, cooling to room temperature, centrifuging at 5000 r / min for 20 min, collecting the supernatant for vacuum freeze drying, the vacuum degree being 0.01 to 0.5 mbar, the cold trap temperature being -72° C. to -45° C., and the freeze drying time being 48 h to obtain salmon skin gelatin hydrolyzate;

[0049] (4) Weighing salmon skin gelatin hydrolysate, and preparing a hydrolyzate with deionized water to have a substrate concentration of 40 g / 100 mL;

[0050] (5) adding glycine methyl ester hydrochloride having a free amino group content to total free amino group content ratio of 0.6 mol / mol to a high concentration of salmon skin gelatin hydrolyzate;

[0051] (6) adding papain with an activity of 3.5 kU / g protein, adjusting the system pH to 7.0, the shaker temperature to 37°C, and the reaction time to 6 h;

[0052] (7) After the reaction is completed, the solution is inactivated at 90° C. for 15 min to terminate the reaction, thereby obtaining a protein-modified peptide solution; the solution is cooled and freeze-dried in a vacuum at a vacuum degree of 0.01 to 0.5 mbar, a cold trap temperature of -72° C. to -45° C., and a freeze-drying time of 48 h to obtain a protein-modified edible peptide having an osteogenic effect.

[0053] (8) Blast drying for 6 to 8 h until the protein-modified edible peptide has a constant weight, and the sample is recorded as PGH-Gly-0.6.

[0054] Example 3

[0055] A method for preparing a protein-modified edible peptide with an osteogenic effect, comprising the following steps:

[0056] (1) Weigh constant weight of salmon skin gelatin and prepare a solution with a mass concentration of 5 g / 100 mL using deionized water;

[0057] (2) adding papain with an activity of 3.5 kU / g protein to the salmon skin gelatin solution, adjusting the solution pH to 7.0, the water bath temperature to 60°C, and the hydrolysis time to 8 h;

[0058] (3) inactivating the enzyme in the solution at 90° C. for 15 min, cooling to room temperature, centrifuging at 5000 r / min for 20 min, collecting the supernatant for vacuum freeze drying, the vacuum degree being 0.01 to 0.5 mbar, the cold trap temperature being -72° C. to -45° C., and the freeze drying time being 48 h to obtain salmon skin gelatin hydrolyzate;

[0059] (4) Weighing salmon skin gelatin hydrolysate, and preparing a hydrolyzate with deionized water to have a substrate concentration of 40 g / 100 mL;

[0060] (5) adding glycine methyl ester hydrochloride having a free amino group content to total free amino group content ratio of 0.8 mol / mol to the high concentration salmon skin gelatin hydrolyzate;

[0061] (6) Add 3.5 kU / g protein of papain, adjust the system pH to 7.0, the shaker temperature to 37 °C, and the reaction time to 6 h;

[0062] (7) After the reaction is completed, the solution is inactivated at 90° C. for 15 min to terminate the reaction, thereby obtaining a protein-modified peptide solution; the solution is cooled and freeze-dried in a vacuum at a vacuum degree of 0.01 to 0.5 mbar, a cold trap temperature of -72° C. to -45° C., and a freeze-drying time of 48 h to obtain a protein-modified edible peptide having an osteogenic effect.

[0063] (8) Blast drying for 6 to 8 h until the protein-modified edible peptide has a constant weight, and the sample is recorded as PGH-Gly-0.8.

[0064] Comparative Example 1

[0065] A method for preparing salmon skin gelatin hydrolysate, comprising the following steps:

[0066] (1) Weigh constant weight of salmon skin gelatin and prepare a solution with a mass concentration of 5 g / 100 mL using deionized water;

[0067] (2) adding papain with an activity of 3.5 kU / g protein to the salmon skin gelatin solution, adjusting the solution pH to 7.0, the water bath temperature to 60°C, and the hydrolysis time to 8 h;

[0068] (3) inactivating the enzyme in the solution at 90° C. for 15 min, cooling to room temperature, centrifuging at 5000 r / min for 20 min, collecting the supernatant for vacuum freeze drying, the vacuum degree being 0.01 to 0.5 mbar, the cold trap temperature being -72° C. to -45° C., and the freeze drying time being 48 h to obtain salmon skin gelatin hydrolyzate;

[0069] (4) Air dry for 6 to 8 h until the salmon skin gelatin hydrolysate has a constant weight and record the sample as GH.

[0070] Comparative Example 2

[0071] A method for preparing an edible peptide, which is different from Example 1 in that glycine methyl ester hydrochloride is not introduced to perform protein-like reaction modification of salmon skin gelatin hydrolysate, and the specific steps are as follows:

[0072] (1) Weigh constant weight of salmon skin gelatin and prepare a solution with a mass concentration of 5 g / 100 mL using deionized water;

[0073] (2) adding papain with an activity of 3.5 kU / g protein to the salmon skin gelatin solution, adjusting the solution pH to 7.0, the water bath temperature to 60°C, and the hydrolysis time to 8 h;

[0074] (3) inactivating the enzyme in the solution at 90° C. for 15 min, cooling to room temperature, centrifuging at 5000 r / min for 20 min, collecting the supernatant for vacuum freeze drying, the vacuum degree being 0.01 to 0.5 mbar, the cold trap temperature being -72° C. to -45° C., and the freeze drying time being 48 h to obtain salmon skin gelatin hydrolyzate;

[0075] (4) Weighing salmon skin gelatin hydrolysate, and preparing a hydrolyzate with deionized water to have a substrate concentration of 40 g / 100 mL;

[0076] (5) adding 3.5 kU / g protein of papain to the high concentration of salmon skin gelatin hydrolysate, adjusting the system pH to 7.0, the shaking temperature to 37°C, and the reaction time to 6 h;

[0077] (6) After the reaction is completed, the solution is inactivated at 90°C for 15 minutes to terminate the reaction and obtain a protein-modified peptide solution; the solution is cooled and freeze-dried in a vacuum at a vacuum degree of 0.01 to 0.5 mbar, a cold trap temperature of -72°C to -45°C, and a freeze-drying time of 48 hours to obtain an edible peptide.

[0078] (7) Air dry for 6 to 8 h until the edible peptide has a constant weight and record the sample as PGH.

[0079] Experimental Test - Osteoblast Activity Assay

[0080] hFOB 1.19 human SV40 transfected osteoblasts were purchased from the cell bank of Shanghai Institutes for Biological Sciences. The culture medium used for hFOB1.19 cells contains 10% (v / v) fetal bovine serum, 0.3 mg / mL G418, DMEM and Ham's F-12 (1:1) culture medium (containing 1.2 g / L NaHCO3). hFOB1.19 cells were routinely cultured in a cell culture incubator at 33.5°C and 5% CO2. In the differentiation experiment, the osteoblasts were first routinely cultured for 3 days to reach 80% confluence, and then differentiated at 39.5°C for 7 days, with the differentiation medium changed every 2 days.

[0081] The cell density was adjusted with complete culture medium, and 100 μL of hFOB 1.19 osteoblasts were inoculated into 96-well plates (5×10 3 / well) and incubate for 24 hours. After adhesion, change to serum-free culture medium for 12 hours to make the cells in G0 phase. Discard the supernatant, add 25-100 μg / mL of the culture medium of each embodiment and comparative sample to each well, and incubate for 24-48 hours; discard the supernatant, add 110 μL CCK-8 diluent to each well, incubate for another 2 hours, and analyze the proliferation activity at a wavelength of 450nm using an enzyme marker. The treatment with only culture medium is the blank group, i.e., the zero adjustment group, and the treatment with only cells and culture medium is the negative control group. The formula for calculating cell activity is as follows:

[0082]

[0083] Each sample was replaced with culture medium, and the activity of osteoblasts was determined to be 100%; β-estradiol (E2) was selected as a positive control, and each sample was replaced with 1×10 -8 mol / L 17β-estradiol, when applied to osteoblasts for 24h and 48h, the cell proliferation activity increased to 122.5% and 132.6%, respectively.

[0084] The experimental data of the osteogenic effect of each embodiment and comparative example on osteoblasts are as follows:

[0085] Table 1 Effects of samples of various embodiments and comparative examples on the proliferation of osteoblasts

[0086]

[0087]

[0088] Note: Each experiment was repeated 3 times, and 5 parallel wells were set for each sample. The value of each sample was the average of five parallel data.

[0089] As can be seen from Table 1 above, the protein-modified edible peptide with a concentration of 50 μg / mL and 0.4 mol / mol glycine introduced in Example 1 has the best osteogenic activity after acting on hFOB 1.19 osteoblasts for 48 hours, and the osteoblast proliferation activity is increased to 125.8% compared with the negative control group; the protein-modified edible peptide with a concentration of 50 μg / mL and 0.6 mol / mol glycine introduced in Example 2 has the best osteogenic activity after acting on hFOB 1.19 osteoblasts for 48 hours, and the osteoblast proliferation activity is increased to 137.6% compared with the negative control group; the protein-modified edible peptide with a concentration of 50 μg / mL and 0.8 mol / mol glycine introduced in Example 3 has the best osteogenic activity after acting on hFOB 1.19 osteoblasts for 48 hours, and the osteoblast proliferation activity is increased to 138.8% compared with the negative control group.

[0090] After the salmon skin gelatin hydrolyzate of comparative example 1 acted on hFOB 1.19 osteoblasts for 48 hours, the osteoblast proliferation activity increased to 113.0% compared with the negative control group. After the protein-modified edible peptide of comparative example 2 without the introduction of glycine methyl ester hydrochloride acted on hFOB 1.19 osteoblasts for 48 hours, the osteoblast proliferation activity increased to 122.7% compared with the negative control group. By comparing Examples 1 to 3 with Comparative Examples 1 to 2, it can be seen that the protein-modified edible peptide introduced with a concentration of 50 μg / mL and 0.6 to 0.8 mol / mol of glycine prepared by the present invention has a more significant osteogenic effect, is more beneficial to human bone health, and can provide new raw materials for the development of functional foods or health products with the risk of preventing osteoporosis or fractures.

[0091] Comparative Example 3

[0092] The difference between this comparative example and Example 3 is that the reaction parameters are different, and the specific steps are as follows:

[0093] (1) weighing a constant weight of salmon skin gelatin, and preparing a salmon skin gelatin solution with a mass concentration of 5 g / 100 mL with deionized water;

[0094] (2) adding papain with an activity of 2.5 kU / g protein to the salmon skin gelatin solution, adjusting the solution pH to 7.0, the water bath temperature to 55°C, and the hydrolysis time to 8 h;

[0095] (3) inactivating the enzyme in the solution at 90° C. for 15 min, cooling to room temperature, centrifuging at 5000 r / min for 20 min, collecting the supernatant for vacuum freeze drying, the vacuum degree being 0.01 to 0.5 mbar, the cold trap temperature being -72° C. to -45° C., and the freeze drying time being 48 h to obtain salmon skin gelatin hydrolyzate;

[0096] (4) Weighing salmon skin gelatin hydrolysate, and preparing a hydrolyzate with deionized water to have a substrate concentration of 30 g / 100 mL;

[0097] (5) adding glycine methyl ester hydrochloride having a free amino group content to total free amino group content ratio of 0.8 mol / mol to the high concentration salmon skin gelatin hydrolyzate;

[0098] (6) adding papain with an activity of 2.5 kU / g protein, adjusting the system pH to 7.0, the shaker temperature to 37°C, and the reaction time to 6 h;

[0099] (7) After the reaction is completed, the solution is inactivated at 90° C. for 15 min to terminate the reaction, thereby obtaining a protein-modified peptide solution; the solution is cooled and freeze-dried in a vacuum at a vacuum degree of 0.01 to 0.5 mbar, a cold trap temperature of -72° C. to -45° C., and a freeze-drying time of 48 h to obtain a protein-modified edible peptide having an osteogenic effect.

[0100] (8) Blast drying for 6 to 8 hours until the protein-modified edible peptide has a constant weight.

[0101] After testing, the protein-modified edible peptide obtained in Comparative Example 3, with a concentration of 50 μg / mL of the protein-modified edible peptide acting on osteoblasts for 24 hours and 48 hours, increased the cell proliferation activity by 121.6% and 123.7%, respectively.

[0102] Comparative Example 4

[0103] The difference between this comparative example and Example 3 is that papain is not added to carry out protein-like reaction after adding glycine methyl ester hydrochloride. The specific steps are as follows:

[0104] (1) Weigh constant weight of salmon skin gelatin and prepare a solution with a mass concentration of 5 g / 100 mL using deionized water;

[0105] (2) adding papain with an activity of 3.5 kU / g protein to the salmon skin gelatin solution, adjusting the solution pH to 7.0, the water bath temperature to 60°C, and the hydrolysis time to 8 h;

[0106] (3) inactivating the enzyme in the solution at 90° C. for 15 min, cooling to room temperature, centrifuging at 5000 r / min for 20 min, collecting the supernatant for vacuum freeze drying, the vacuum degree being 0.01 to 0.5 mbar, the cold trap temperature being -72° C. to -45° C., and the freeze drying time being 48 h to obtain salmon skin gelatin hydrolyzate;

[0107] (4) Weighing salmon skin gelatin hydrolysate, and preparing a hydrolyzate with deionized water to have a substrate concentration of 40 g / 100 mL;

[0108] (5) adding glycine methyl ester hydrochloride having a free amino group content ratio of 0.8 mol / mol to the total free amino group content to the high concentration salmon skin gelatin hydrolyzate, adjusting the system pH to 7.0, the shaking table temperature to 37° C., and the reaction time to 6 h;

[0109] (6) After the reaction is completed, the mixture is cooled and freeze-dried in a vacuum at a vacuum degree of 0.01 to 0.5 mbar, a cold trap temperature of -72°C to -45°C, and a freeze-drying time of 48 h to obtain an edible peptide.

[0110] (7) Dry with forced air for 6 to 8 hours until the edible peptide has a constant weight.

[0111] After testing, the edible peptide obtained in Comparative Example 4, with a concentration of 50 μg / mL, acted on osteoblasts for 24 hours and 48 hours, and the cell proliferation activity increased by 111.2% and 114.0%, respectively.

[0112] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a protein-modified edible peptide having an osteogenic effect, characterized in that: The steps include: S1: Weigh salmon skin gelatin, prepare a salmon skin gelatin solution with deionized water, add papain to the salmon skin gelatin solution, adjust the pH of the solution, perform enzymatic hydrolysis at 55-65° C. for 7-9 hours, inactivate the enzyme, centrifuge, collect the supernatant, and freeze-dry to obtain salmon skin gelatin hydrolyzate; S2: preparing a high-concentration hydrolyzate of salmon skin gelatin, adding glycine methyl ester hydrochloride, stirring, adding specific activity papain, adjusting the pH of the solution, performing an enzymatic hydrolysis reaction at 35-40° C. for 5-7 hours, inactivating the enzyme, and terminating the reaction to obtain a protein-like modified peptide solution; the glycine methyl ester hydrochloride is added in an amount such that the ratio of the free amino group content to the total free amino group content is at least 0.4 mol / mol; S3: freeze-drying the protein-modified peptide solution, and then air-drying it to a constant weight to obtain the protein-modified edible peptide having an osteogenesis-promoting effect.

2. A method for preparing a protein-modified edible peptide having an osteogenic effect according to claim 1, characterized in that: The glycine methyl ester hydrochloride is added in an amount such that the ratio of the free amino group content to the total free amino group content is 0.6 to 0.8 mol / mol.

3. A method for preparing a protein-modified edible peptide having an osteogenic effect according to claim 1, characterized in that: In step S1, the mass concentration of the salmon skin gelatin solution is 3-7 g / 100 mL; in step S2, the mass concentration of the high-concentration hydrolyzate is 30-50 g / 100 mL.

4. A method for preparing a protein-modified edible peptide having an osteogenic effect according to claim 1, characterized in that: In steps S1 and S2, the enzyme inactivation temperature is 85-95°C and the enzyme inactivation time is 10-20 minutes.

5. The protein-modified edible peptide with osteogenic effect and the preparation method thereof according to claim 1, characterized in that: In step S1, the activity of papain added is 3.5 kU / g protein, the pH value of the system is adjusted to 7.0, the water bath temperature is 60° C., and the hydrolysis time is 8 h.

6. The protein-modified edible peptide with osteogenic effect and the preparation method thereof according to claim 1, characterized in that: In step S2, the amount of papain added was 3.5 kU / g protein, the pH value of the system was adjusted to 7.0, the shaking table temperature was 37° C., and the reaction time was 6 h.

7. A method for preparing a protein-modified edible peptide having an osteogenic effect according to claim 1, characterized in that: In steps S1 and S3, the freeze drying includes vacuum freeze drying, the vacuum degree of the vacuum freeze drying is 0.01 to 0.5 mbar, the cold trap temperature of the vacuum freeze drying is -72°C to -45°C, and the vacuum freeze drying time is 48 hours.

8. A protein-modified edible peptide prepared according to the method for preparing a protein-modified edible peptide with osteogenic effect according to any one of claims 1 to 7.

9. A protein-modified edible peptide according to claim 8, characterized in that: The protein-modified edible peptide can be directly soluble in water, has a bone health care effect, can be used as a potential oral supplement for bone and joint tissue recovery, and is applied in the field of health food processing.

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