Bone-strengthening milk protein extract, method for preparing same, and use thereof

Abstract

The application discloses a bone-strengthening milk protein extract and a preparation method and application thereof, and belongs to the technical field of deep processing of cow milk. The bone-strengthening milk protein extract contains a bone-strengthening active peptide FYPELFR, and the preparation method specifically comprises the following steps: (1) pasteurization; (2) casein separation; (3) pepsin hydrolysis; (4) trypsin hydrolysis; (5) purification and desalination; and (6) concentration and drying. The bone-strengthening active peptide and the bone-strengthening milk protein extract containing the same can be combined with integrin alpha v beta 1 through hydrophobic interaction and hydrogen bonding, can promote the proliferation and differentiation of osteoblasts by activating specific signal pathways, can promote the proliferation of osteoblasts, can be used for the development of bone-strengthening functional food or medicine, and has important significance for the deep processing of cow milk.

Description

Technical Field

[0001] This invention relates to the field of deep processing technology of cow's milk, and more specifically to a bone-strengthening milk protein extract, its preparation method, and its application. Background Technology

[0002] Whey protein is a protein extracted from milk, characterized by its high nutritional value, easy digestibility and absorption, and the presence of various bioactive components. It is widely recognized as a high-quality protein supplement for the human body. Whey protein is typically derived from a byproduct of cheese processing. However, due to significant differences in dietary habits between my country and Europe and the United States, there are currently no large-scale industrialized cheese factories operating in China. Consequently, my country relies heavily on imports for whey powder needed for infant formula. Addressing the issue of utilizing excess casein while producing whey powder is a bottleneck in the dairy deep-processing industry and a major challenge for domestic dairy companies expanding the industrialization of dairy product deep processing. Hydrolyzing casein into functional peptides to expand its application areas and scenarios is an urgent problem that the industry needs to solve.

[0003] Milk-derived bioactive peptides are small-molecule polypeptides with specific biological activities released from milk proteins (such as casein and whey protein) through enzymatic hydrolysis or microbial fermentation. They typically have low molecular weights and are easily absorbed by the human body. With the discovery of milk-derived bioactive peptides with different functions, research on milk protein bioactive peptides has become a new research hotspot in the fields of physiology and nutrition. The discovery of milk-derived bioactive peptides has changed the past simple evaluation of the nutritional functions of milk proteins and has great prospects for application and development.

[0004] Milk contains a variety of bioactive peptides with different functions, playing important physiological roles, especially in promoting growth and development and preventing diseases. However, the content of bioactive peptides in natural milk is relatively low, which cannot meet the needs of people with sub-health conditions. In addition, there are problems such as a lack of in-depth research on the chemical structure and physiological functions of milk-derived bioactive peptides, low yield and low purity of traditional preparation techniques, and the inability to develop high-quality milk-derived bioactive peptides.

[0005] A search of the BIOPEP database revealed 111 bioactive peptides derived from "Casein," but the actual number of casein bioactive peptides is far greater than 111, as research and reports on their variety continue to emerge. A search of the Web of Science (WOS) core database showed 6080 studies related to "Caseinpeptides" and 34 studies related to "Caseinpeptides Bone Density" over the past 25 years. Functional products / ingredients for strengthening bones, using casein as the main raw material, with clear mechanisms of action and patented technology, possess significant economic and social value for the industrialization of dairy product processing and for meeting the efficacy needs of specific populations.

[0006] In the prior art, CN202210987021.1 discloses a method for preparing and applying buffalo casein hydrolysate peptides that promote bone development. This method mainly involves hydrolyzing buffalo casein and using membrane separation technology to separate the hydrolysate, obtaining a permeate with a molecular weight less than 1 kDa. The main components are EDVPSER, NAVPITPTL, VLPVPQK, and HPHPHLSF. Animal experiments were used to verify the bone density-enhancing effect of this product. However, there are no reports on the functional verification of EDVPSER and NAVPITPTL in the mixture obtained by this method. VLPVPQK, as a universal marker of casein phosphopeptides, is widely used for the quantitative detection of CPP content in infant formula, and the bone-strengthening effects of casein phosphopeptides have been extensively studied and reported. HPHPHLSF, as a buffalo casein hydrolysate, was also reported in 2016 for its effect on promoting osteoblast proliferation. Therefore, the comprehensive effect of the hydrolysate peptides cannot yet be verified.

[0007] CN202211530284.6 discloses a milk-derived bioactive peptide GITDPLFKGM, GITDPLFKG, its preparation method, and its application. The method uses bovine milk protein concentrate as raw material, ferments it with Lactobacillus paracasei, and then filters it using an ultrafiltration membrane to obtain a polypeptide mixture. The polypeptide mixture is analyzed and screened to obtain the milk-derived bioactive peptide GITDPLFKGM. The milk-derived bioactive peptide prepared by this method has high antioxidant capacity and immunomodulatory activity, but the preparation process is relatively complex, and there is no research basis or related reports on its bone health function.

[0008] Therefore, how to prepare and screen bone-strengthening milk protein extract using industrial methods is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0009] In view of this, the purpose of the present invention is to provide a bone-strengthening milk protein extract, its preparation method and application, so as to overcome the shortcomings of the prior art.

[0010] To achieve the above objectives, the present invention adopts the following technical solution:

[0011] A bone-strengthening active peptide with the amino acid sequence FYPELFR, specifically as shown in SEQ ID No. 1: Phe-Tyr-Pro-Glu-Leu-Phe-Arg (N-terminus-C-terminus).

[0012] A method for preparing the above-mentioned bone-strengthening active peptide specifically includes the following steps:

[0013] (1) Pasteurization

[0014] Skim milk is pasteurized to obtain sterilized skim milk;

[0015] (2) Casein isolation

[0016] Sterilized skim milk is concentrated through a microfiltration membrane, washed, and filtered to obtain a casein solution;

[0017] (3) Pepsin hydrolysis

[0018] Adjust the pH of the casein solution to 1.5-3.0, add pepsin to hydrolyze it, and obtain pepsin hydrolysate;

[0019] (4) Trypsin hydrolysis

[0020] Adjust the pH of the pepsin hydrolysate to 5.5-7.0, add trypsin to hydrolyze, and obtain trypsin hydrolysate;

[0021] (5) Purification and desalting

[0022] The trypsin hydrolysate was purified by membrane filtration, desalted by nanofiltration, and the retentate was collected to obtain a polypeptide mixture.

[0023] (6) Analysis and screening

[0024] The peptide mixture was analyzed and screened to obtain the bone-strengthening active peptide with the amino acid sequence FYPELFR.

[0025] Furthermore, in step (1) above, the pasteurization temperature is 75-89℃, preferably 83-87℃; the pasteurization time is 10-60s, preferably 13-17s.

[0026] Furthermore, in step (2) above, the microfiltration membrane is a ceramic membrane or an organic membrane, preferably a ceramic membrane; the pore size of the microfiltration membrane is 0.1μm, 0.14μm or 0.2μm, preferably 0.14μm; it is concentrated to 20%-40% of the original volume, preferably 30%; and the washing is performed by adding 2-5 times the volume of pure water, preferably 3 times.

[0027] Furthermore, in step (3) above, the amount of pepsin added is 1000-5000 U / g protein, preferably 2000 U / g protein; the hydrolysis temperature is 35-45℃, preferably 36-38℃; and the hydrolysis time is 1-3h, preferably 2h.

[0028] Furthermore, in step (4) above, the amount of trypsin added is 1000-5000 U / g protein, preferably 2000 U / g protein; the hydrolysis temperature is 35-45℃, preferably 36-38℃; and the hydrolysis time is 1-4h, preferably 2.5h.

[0029] Furthermore, in step (5) above, the membrane pore size for filtration purification is 500-5000 Da, preferably 500-2000 Da; the membrane pore size for nanofiltration desalination is 1-100 nm, preferably 1-10 nm.

[0030] Furthermore, in step (6) above, the analysis and screening are as follows: First, the peptide mixture is detected using an LC-ESI-Q-TOF high-resolution liquid chromatography-mass spectrometry system, and the amino acid sequence of the peptides is analyzed in conjunction with peptide omics identification; then, the target receptor protein αvβ1 (PDB ID: 3VI4) is obtained using the Protein Data Bank (http: / / www.rcsb.org / pdb) database, and molecular docking and affinity scoring are performed using AutoDockVina software to select peptides with high affinity and exclude publicly available peptides.

[0031] A bone-strengthening milk protein extract containing the aforementioned bone-strengthening active peptides.

[0032] A method for preparing the above-mentioned bone-strengthening milk protein extract specifically includes the following steps:

[0033] (1) Pasteurization

[0034] Skim milk is pasteurized to obtain sterilized skim milk;

[0035] (2) Casein isolation

[0036] Sterilized skim milk is concentrated through a microfiltration membrane, washed, and filtered to obtain a casein solution;

[0037] (3) Pepsin hydrolysis

[0038] Adjust the pH of the casein solution to 1.5-3.0, add pepsin to hydrolyze it, and obtain pepsin hydrolysate;

[0039] (4) Trypsin hydrolysis

[0040] Adjust the pH of the pepsin hydrolysate to 5.5-7.0, add trypsin to hydrolyze, and obtain trypsin hydrolysate;

[0041] (5) Purification and desalting

[0042] The trypsin hydrolysate was purified by membrane filtration, desalted by nanofiltration, and the retentate was collected to obtain a polypeptide mixture.

[0043] (6) Concentration and drying

[0044] The polypeptide mixture was concentrated and freeze-dried to obtain the bone-strengthening milk protein extract.

[0045] By preparing the bone-strengthening milk protein extract using the method described above, a polypeptide mixture with a high content of the characteristic polypeptide FYPELFR can be obtained, thereby enhancing the bone-strengthening efficacy of the product. Simultaneously, the bone-strengthening active peptide in the extract can be a single peptide fragment, a polypeptide mixture, or a composition. For a single peptide fragment, it can be prepared by enzymatic hydrolysis and purification of casein solution, by chemical synthesis, or by biosynthesis, preferably by enzymatic hydrolysis and purification of casein solution or by biosynthesis, more preferably by enzymatic hydrolysis and purification of casein solution. For a polypeptide mixture, it can be prepared by a combination of enzymatic purification and biosynthesis, preferably by enzymatic hydrolysis and purification of casein solution. The characteristic polypeptide composition includes the bone-strengthening active peptide FYPELFR and its derivatives.

[0046] Furthermore, in step (1) above, the pasteurization temperature is 75-89℃, preferably 83-87℃; the pasteurization time is 10-60s, preferably 13-17s.

[0047] Furthermore, in step (2) above, the microfiltration membrane is a ceramic membrane or an organic membrane, preferably a ceramic membrane; the pore size of the microfiltration membrane is 0.1μm, 0.14μm or 0.2μm, preferably 0.14μm; it is concentrated to 20%-40% of the original volume, preferably 30%; and the washing is performed by adding 2-5 times the volume of pure water, preferably 3 times.

[0048] Furthermore, in step (3) above, the amount of pepsin added is 1000-5000 U / g protein, preferably 2000 U / g protein; the hydrolysis temperature is 35-45℃, preferably 36-38℃; and the hydrolysis time is 1-3h, preferably 2h.

[0049] Furthermore, in step (4) above, the amount of trypsin added is 1000-5000 U / g protein, preferably 2000 U / g protein; the hydrolysis temperature is 35-45℃, preferably 36-38℃; and the hydrolysis time is 1-4h, preferably 2.5h.

[0050] Furthermore, in step (5) above, the membrane pore size for filtration purification is 500-5000 Da, preferably 500-2000 Da; the membrane pore size for nanofiltration desalination is 1-100 nm, preferably 1-10 nm.

[0051] Furthermore, in step (6) above, the concentration method is RO concentration, the transmembrane pressure difference is 1.0-2.5MPa, and the temperature is 10-25℃; the freeze-drying temperature is -50~-30℃, the pressure is 0.03MPa, and the time is 24-48h.

[0052] The present invention also claims protection for a derivative of the above-mentioned bone-strengthening active peptide or the bone-strengthening active peptide prepared by the above-mentioned preparation method, including a polypeptide derivative obtained by hydroxylation, carboxylation, carbonylation, methylation, acetylation, phosphorylation, esterification or glycosylation modification on the amino acid side chain group, amino terminus or carboxyl terminus of the bone-strengthening active peptide FYPELFR.

[0053] The present invention also claims protection for the use of the above-mentioned bone-strengthening active peptide, the above-mentioned bone-strengthening milk protein extract containing the above-mentioned bone-strengthening active peptide, or the derivative of the above-mentioned bone-strengthening active peptide in the preparation of food (especially including health products, with a mass percentage of 1.0%-2%) or medicine with bone-strengthening effects, which can obtain better bone-strengthening activity.

[0054] As can be seen from the above technical solution, compared with the prior art, the beneficial effects of the present invention are as follows:

[0055] 1. The bone-strengthening active peptides and bone-strengthening milk protein extracts containing them of the present invention can bind to integrin αvβ1 through hydrophobic interactions and hydrogen bonds, and promote osteoblast proliferation and differentiation by activating specific signaling pathways. They can be used for the development of bone-strengthening functional foods or drugs, and are of great significance for the deep processing of milk.

[0056] 2. This invention first uses skim milk as raw material, and obtains a casein solution through sterilization and casein separation. Then, it undergoes pepsin hydrolysis, trypsin hydrolysis, two-step membrane filtration purification, and one-step purification and desalting to obtain a polypeptide mixture. Finally, the polypeptide mixture is analyzed and screened to obtain the bone-strengthening active peptide FYPELFR with ideal efficacy. This preparation method is simple, efficient, economical, environmentally friendly, and has a wide range of applications.

[0057] 3. This invention hydrolyzes bovine milk casein using specific raw materials and enzymatic methods, and then analyzes and screens to obtain a new polypeptide with ideal bone-strengthening effects, enriching the types of milk-derived bioactive peptides and providing new raw materials for the preparation of bone-strengthening products. Attached Figure Description

[0058] Figure 1 The process flow diagrams are for the preparation methods of Examples 1 and 2;

[0059] Figure 2 The results are the analytical results of the polypeptide mixture in Example 1. Detailed Implementation

[0060] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0061] Example 1

[0062] Preparation method of bone-strengthening active peptide FYPELFR, such as Figure 1 As shown, the specific steps include:

[0063] (1) Pasteurization

[0064] Skim milk is pasteurized at 85°C for 15 seconds to obtain sterilized skim milk.

[0065] (2) Casein isolation

[0066] The sterilized skim milk was first concentrated to 30% of its original volume through a 0.14μm ceramic microfiltration membrane, and then washed with 3 times the volume of purified water to obtain a casein solution with a protein content of 8%.

[0067] (3) Pepsin hydrolysis

[0068] The pH of the casein solution was adjusted to 2.5, 2000 U / g pepsin was added, and the solution was hydrolyzed at 37°C for 2 hours to obtain pepsin hydrolysate.

[0069] (4) Trypsin hydrolysis

[0070] The pH of the pepsin hydrolysate was adjusted to 6.8, 2000 U / g trypsin was added, and hydrolysis was carried out at 37℃ for 2.5 h to obtain the trypsin hydrolysate.

[0071] (5) Purification and desalting

[0072] The trypsin hydrolysate was first purified by filtration through a 2000 Da membrane, and the permeate was collected. Then, the permeate was concentrated and washed using a 500 Da membrane, and the concentrate was collected. Finally, the concentrate was desalted by nanofiltration using a 10 nm membrane, and the retentate was collected to obtain a peptide mixture.

[0073] (6) Analysis and screening

[0074] First, the peptide mixture was detected using LC-ESI-Q-TOF high-resolution liquid chromatography-mass spectrometry, and the amino acid sequences of the peptides were analyzed using peptidomics identification. Then, the target receptor protein αvβ1 was obtained from the Protein DataBank database, and molecular docking and affinity scoring were performed using AutoDock Vina software. The high-affinity peptides HPHPHLSF, FYPELFR, FFSDK, and YLGY were selected, while the publicly available peptides HPHPHLSF, FFSDK, and YLGY were excluded, thus obtaining the bone-strengthening active peptide FYPELFR.

[0075] Example 2

[0076] The preparation method of the bone-strengthening active peptide FYPELFR-containing bone-strengthening milk protein extract differs from Example 1 only in that the "analytical screening" step is replaced with a "concentration and drying" step, such as... Figure 2 As shown, the specific steps include:

[0077] (1) Pasteurization

[0078] Skim milk is pasteurized at 85°C for 15 seconds to obtain sterilized skim milk.

[0079] (2) Casein isolation

[0080] The sterilized skim milk was first concentrated to 30% of its original volume through a 0.14μm ceramic microfiltration membrane, and then washed with 3 times the volume of purified water to obtain a casein solution with a protein content of 8%.

[0081] (3) Pepsin hydrolysis

[0082] The pH of the casein solution was adjusted to 2.5, 2000 U / g pepsin was added, and the solution was hydrolyzed at 37°C for 2 hours to obtain pepsin hydrolysate.

[0083] (4) Trypsin hydrolysis

[0084] The pH of the pepsin hydrolysate was adjusted to 6.8, 2000 U / g trypsin was added, and hydrolysis was carried out at 37℃ for 2.5 h to obtain the trypsin hydrolysate.

[0085] (5) Purification and desalting

[0086] The trypsin hydrolysate was first purified by filtration through a 2000 Da membrane, and the permeate was collected. Then, the permeate was concentrated and washed using a 500 Da membrane, and the concentrate was collected. Finally, the concentrate was desalted by nanofiltration using a 10 nm membrane, and the retentate was collected to obtain a peptide mixture.

[0087] (6) Concentration and drying

[0088] The peptide mixture was concentrated by RO at a transmembrane pressure difference of 2.5 MPa and a temperature of 25 °C, and the RO concentrate was collected. Finally, the RO concentrate was freeze-dried at -50 °C and 0.03 MPa for 48 h to obtain a bone-strengthening milk protein extract containing the bone-strengthening active peptide FYPELFR.

[0089] Example 3

[0090] The preparation method of the product containing the bone-strengthening active peptide FYPELFR differs from Example 2 only in that it adds a "casein powder dilution" step, which specifically includes the following steps:

[0091] (1) Pasteurization

[0092] Skim milk is pasteurized at 85°C for 15 seconds to obtain sterilized skim milk.

[0093] (2) Casein isolation

[0094] The sterilized skim milk was first concentrated to 30% of its original volume through a 0.14μm ceramic microfiltration membrane, and then washed with 3 times the volume of purified water to obtain a casein solution with a protein content of 8%.

[0095] (3) Pepsin hydrolysis

[0096] The pH of the casein solution was adjusted to 2.5, 2000 U / g pepsin was added, and the solution was hydrolyzed at 37°C for 2 hours to obtain pepsin hydrolysate.

[0097] (4) Trypsin hydrolysis

[0098] The pH of the pepsin hydrolysate was adjusted to 6.8, 2000 U / g trypsin was added, and hydrolysis was carried out at 37℃ for 2.5 h to obtain the trypsin hydrolysate.

[0099] (5) Purification and desalting

[0100] The trypsin hydrolysate was first purified by filtration through a 2000 Da membrane, and the permeate was collected. Then, the permeate was concentrated and washed using a 500 Da membrane, and the concentrate was collected. Finally, the concentrate was desalted by nanofiltration using a 10 nm membrane, and the retentate was collected to obtain a peptide mixture.

[0101] (6) Concentration and drying

[0102] The peptide mixture was concentrated by RO at a transmembrane pressure difference of 2.5 MPa and a temperature of 25 °C, and the RO concentrate was collected. Finally, the RO concentrate was freeze-dried at -50 °C and 0.03 MPa for 48 h to obtain a bone-strengthening milk protein extract containing the bone-strengthening active peptide FYPELFR.

[0103] (7) Casein powder dilution

[0104] The content of FYPELFR in the bone-strengthening active peptide FYPELFR in the bone-strengthening milk protein extract was diluted to 1.5% by using casein powder, thus obtaining a product containing the bone-strengthening active peptide FYPELFR.

[0105] Comparative Example 1

[0106] The preparation method of the bone-strengthening milk protein extract containing the bone-strengthening active peptide FYPELFR differs from Example 2 only in the source of the "casein solution," and specifically includes the following steps:

[0107] (1) Pasteurization

[0108] Under stirring, commercially available casein powder with a protein content of 8% was dissolved in warm water at 40°C, hydrated for 30 minutes, heated to 70°C and homogenized at 200 bar, and finally pasteurized at 85°C for 15 seconds to obtain a casein solution with a protein content of 8%.

[0109] (2) Pepsin hydrolysis

[0110] The pH of the casein solution was adjusted to 2.5, 2000 U / g pepsin was added, and the solution was hydrolyzed at 37°C for 2 hours to obtain pepsin hydrolysate.

[0111] (3) Trypsin hydrolysis

[0112] The pH of the pepsin hydrolysate was adjusted to 6.8, 2000 U / g trypsin was added, and hydrolysis was carried out at 37℃ for 2.5 h to obtain the trypsin hydrolysate.

[0113] (4) Purification and desalting

[0114] The trypsin hydrolysate was first purified by filtration through a 2000 Da membrane, and the permeate was collected. Then, the permeate was concentrated and washed using a 500 Da membrane, and the concentrate was collected. Finally, the concentrate was desalted by nanofiltration using a 10 nm membrane, and the retentate was collected to obtain a peptide mixture.

[0115] (5) Concentration and drying

[0116] The peptide mixture was concentrated by RO at a transmembrane pressure difference of 2.5 MPa and a temperature of 25 °C, and the RO concentrate was collected. Finally, the RO concentrate was freeze-dried at -50 °C and 0.03 MPa for 48 h to obtain a bone-strengthening milk protein extract containing the bone-strengthening active peptide FYPELFR.

[0117] Comparative Example 2

[0118] The preparation method of the bone-strengthening milk protein extract containing the bone-strengthening active peptide FYPELFR differs from Example 2 only in that it does not include the "trypsin hydrolysis" step. Specifically, it includes the following steps:

[0119] (1) Pasteurization

[0120] Skim milk is pasteurized at 85°C for 15 seconds to obtain sterilized skim milk.

[0121] (2) Casein isolation

[0122] The sterilized skim milk was first concentrated to 30% of its original volume through a 0.14μm ceramic microfiltration membrane, and then washed with 3 times the volume of purified water to obtain a casein solution with a protein content of 8%.

[0123] (3) Pepsin hydrolysis

[0124] The pH of the casein solution was adjusted to 2.5, 2000 U / g pepsin was added, and the solution was hydrolyzed at 37°C for 2 hours to obtain pepsin hydrolysate.

[0125] (4) Purification and desalting

[0126] The pepsin hydrolysate was first purified by filtration through a 2000 Da membrane, and the permeate was collected. Then, the permeate was concentrated and washed using a 500 Da membrane, and the concentrate was collected. Finally, the concentrate was desalted by nanofiltration using a 10 nm membrane, and the retentate was collected to obtain a peptide mixture.

[0127] (5) Concentration and drying

[0128] The peptide mixture was concentrated by RO at a transmembrane pressure difference of 2.5 MPa and a temperature of 25 °C, and the RO concentrate was collected. Finally, the RO concentrate was freeze-dried at -50 °C and 0.03 MPa for 48 h to obtain a bone-strengthening milk protein extract containing the bone-strengthening active peptide FYPELFR.

[0129] Comparative Example 3

[0130] The preparation method of the bone-strengthening active peptide FYPELFR-containing bone-strengthening milk protein extract differs from Example 2 only in that it does not include the "pepsin hydrolysis" step, and specifically includes the following steps:

[0131] (1) Pasteurization

[0132] Skim milk is pasteurized at 85°C for 15 seconds to obtain sterilized skim milk.

[0133] (2) Casein isolation

[0134] The sterilized skim milk was first concentrated to 30% of its original volume through a 0.14μm ceramic microfiltration membrane, and then washed with 3 times the volume of purified water to obtain a casein solution with a protein content of 8%.

[0135] (3) Trypsin hydrolysis

[0136] The pH of the casein solution was adjusted to 6.8, 2000 U / g trypsin was added, and the solution was hydrolyzed at 37°C for 2.5 h to obtain trypsin hydrolysate.

[0137] (4) Purification and desalting

[0138] The trypsin hydrolysate was first purified by filtration through a 2000 Da membrane, and the permeate was collected. Then, the permeate was concentrated and washed using a 500 Da membrane, and the concentrate was collected. Finally, the concentrate was desalted by nanofiltration using a 10 nm membrane, and the retentate was collected to obtain a peptide mixture.

[0139] (5) Concentration and drying

[0140] The peptide mixture was concentrated by RO at a transmembrane pressure difference of 2.5 MPa and a temperature of 25 °C, and the RO concentrate was collected. Finally, the RO concentrate was freeze-dried at -50 °C and 0.03 MPa for 48 h to obtain a bone-strengthening milk protein extract containing the bone-strengthening active peptide FYPELFR.

[0141] Performance testing

[0142] 1. Obtain effect verification

[0143] The polypeptide mixture prepared in step (5) of Example 1 was detected using an LC-ESI-Q-TOF high-resolution liquid chromatography-mass spectrometry system. A total of 36 effective peptides were detected (see...). Figure 2 Of the ligands, 3 were dipeptides, 7 were tripeptides, 5 were tetrapeptides, 7 were pentapeptides, 8 were hexapeptides, 3 were heptapeptides, 2 were octapeptides, and 1 was decapeptide. Molecular docking and affinity analysis revealed that 4 peptides exhibited stronger affinity for the target receptor protein αvβ1 (PDB ID: 3VI4) compared to the original ligand RDG (see Table 1).

[0144] Table 1 Molecular docking affinity

[0145] HPHPHLSF -9.1 FYPELFR -8.5 FFSDK -8.3 YLGY -8.3 Original ligand RDG -6.1

[0146] Meanwhile, literature and patent searches revealed relevant reports on the octapeptide HPHPHLSF, pentapeptide FFSDK, and tetrapeptide YLGY, with reports indicating that the octapeptide HPHPHLSF promotes osteoblast proliferation. However, no reports were found on the heptapeptide "FYPELFR," making it a newly discovered bone-strengthening functional peptide sequence in this invention.

[0147] 2. Validation of the preparation scheme

[0148] The bone-strengthening active peptide prepared in Example 1, the bone-strengthening milk protein extract prepared in Examples 2 and Comparative Examples 1-3, and the product containing the bone-strengthening active peptide prepared in Example 3 were taken as samples, and their protein content and the content of the target peptide "FYPELFR" were detected respectively. The results are shown in Table 2.

[0149] Table 2 shows the protein and target peptide content in samples from Examples 1-3 and Comparative Examples 1-3.

[0150] Example 1 99.67 98.10 Example 2 95.27 5.34 Example 3 87.68 1.50 Comparative Example 1 93.66 2.53 Comparative Example 2 94.03 Not detected Comparative Example 3 93.89 0.36

[0151] As shown in Table 2:

[0152] ① Example 1 of the present invention can obtain bone-strengthening active peptide FYPELFR with high purity;

[0153] ② In the bone-strengthening milk protein extract prepared in Example 2 of this invention, the content of bone-strengthening active peptide FYPELFR can reach more than 5%, which is much higher than that of the preparation scheme using casein powder in Comparative Example 1 (2.53%) or the preparation scheme using other enzymatic hydrolysis methods in Comparative Examples 2-3 (0.36%).

[0154] 3. Verification of bone-strengthening effects

[0155] The MTT assay was used to determine the proliferation of MC3T3-E1 osteoblasts in the samples.

[0156] The osteoblast-derived active peptides prepared in Example 1, the osteoblast-derived milk protein extracts prepared in Examples 2 and Comparative Examples 1-3, and the product containing osteoblast-derived active peptides prepared in Example 3 were used as samples. Each sample was cultured in 10% FBS α-MEM medium, with a blank control group included. Cells were cultured at 37°C for 24 h, then treated with tetramethylazoazolium blue MTT solution (5 mg / mL, dissolved in 10 mM PBS) for 4 h. Afterward, 150 μL of DMSO was added to dissolve purple dinoflagellates, and the plates were gently shaken for 10 min. The absorbance of the solution in each well was measured at 490 nm using a microplate reader to determine the cell proliferation rate. The calculation formula was: relative osteoblast proliferation rate (%) = (OD0.05)2 样本组 / OD 培养基组 ()×100%. The sample from Example 1 was diluted to three protein concentrations. The results are shown in Table 3.

[0157] Table 3. Relative osteoblast proliferation rates of samples from Examples 1-3 and Comparative Examples 1-3

[0158]

[0159] As shown in Table 3:

[0160] ① The samples in Examples 1-3 of this invention have a good proliferative effect on osteoblasts.

[0161] ② The samples in Examples 1-3 of this invention effectively promoted osteoblast proliferation under certain concentration conditions. As the FYPELFR content increased, the relative proliferation rate of osteoblasts showed a trend of first increasing and then decreasing, and was related to the FYPELFR content in the sample.

[0162] ③Comparative Examples 2-3 had significantly lower FYPELFR content, resulting in a lower bone-strengthening effect compared to Examples 1-3.

[0163] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The application of a bone-strengthening active peptide or a bone-strengthening milk protein extract containing a bone-strengthening active peptide in the preparation of food with bone-strengthening effects, characterized in that, The amino acid sequence of the bone-strengthening active peptide is FYPELFR.

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