Bone-strengthening milk protein extract as well as preparation method and application thereof

Abstract

The invention discloses a bone-strengthening lactoprotein extract as well as 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 bone-strengthening active peptide FYPELFR. The preparation method specifically comprises the following steps: (1) pasteurization; (2) separating casein; (3) pepsin hydrolysis; (4) trypsin hydrolysis; (5) purifying and desalting; and (6) concentrating and drying. The bone-strengthening active peptide and the bone-strengthening lactoprotein extract containing the bone-strengthening active peptide can be combined with integrin alpha v beta 1 through hydrophobic interaction and hydrogen bonds, proliferation and differentiation of osteoblasts are promoted by activating a specific signal channel, proliferation of the osteoblasts is promoted, and the bone-strengthening active peptide and the bone-strengthening lactoprotein extract can be used for development of bone-strengthening functional food or drugs and have important significance on deep processing of cow milk.

Description

Technical Field

[0001] The present invention relates to the technical field of deep processing of cow's milk, and more particularly to a bone-strengthening milk protein extract, a preparation method thereof and an application thereof. Background Art

[0002] Whey protein is a protein extracted from milk. It has the characteristics of high nutritional value, easy digestion and absorption, and contains a variety of active ingredients. It is recognized as one of the high-quality protein supplements for the human body. Whey protein is usually derived from the by-products of cheese processing. However, due to the significant differences in eating habits between my country and European and American countries, there are no large-scale industrial cheese factories in operation. As a result, my country's infant formula milk powder requires a large amount of whey powder that relies on imports. The production of whey powder while solving the problem of the application of excess casein is a bottleneck in the dairy deep processing industry and a major problem for domestic dairy companies in the process of expanding the industrialization of dairy deep processing. Hydrolyzing casein into functional peptides and expanding application areas and scenarios are issues that the industry urgently needs to solve.

[0003] Milk-derived bioactive peptides are small polypeptides with specific biological activities that are released from milk proteins (such as casein and whey protein) through enzymatic hydrolysis or microbial fermentation. They are generally low in molecular weight and easily absorbed by the human body. With the discovery of milk-derived bioactive peptides with different functions, the study of 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 previous simplistic evaluation of the nutritional functions of milk proteins and has great prospects for application development.

[0004] Cow's milk contains a variety of bioactive peptides with diverse functions, which play important physiological roles, particularly in promoting growth and development, and preventing and treating diseases. However, the content of bioactive peptides in natural cow's milk is low, failing to meet the needs of people with sub-health conditions. Furthermore, there is a lack of in-depth research on the chemical structure and physiological functions of milk-derived bioactive peptides, and traditional preparation techniques result in low yields and purity, making it difficult to develop high-quality milk-derived bioactive peptides.

[0005] A search of the BIOPEP database reveals 111 active peptides derived from "Casein." While the number of casein bioactive peptides continues to be researched and reported, the true number far exceeds 111. A search of the Web of Science (WOS) core database reveals 6,080 studies related to "Casein peptides" over the past 25 years, and 34 studies related to "Casein peptides bone density." Bone-strengthening functional products / ingredients using casein as a primary ingredient, with a clear mechanism of action and patented technology, hold significant economic and social value for the industrialization of dairy product deep 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 hydrolyzed peptides that promote bone development. The method mainly involves enzymatic hydrolysis of buffalo casein, and separation of the buffalo casein hydrolyzate using membrane separation technology to obtain a permeate with a molecular weight of less than 1 kDa. The main components are EDVPSER, NAVPITPTL, VLPVPQK, and HPPHHLSF. Animal experiments were also used to verify the bone density-enhancing effect of the product. In the mixture obtained by this method, there are no reports on the functional verification of EDVPSER and NAVPITPTL. VLPVPQK, as a universal marker for casein phosphopeptides, is widely used for the quantitative detection of CPP content in infant formula milk powder, and the bone-strengthening effect of casein phosphopeptides has been widely studied and reported. HPPHHLSF, as a buffalo casein hydrolyzate and its role in promoting osteoblast proliferation, was also reported in 2016. Therefore, the comprehensive effect of its hydrolyzed peptides cannot be verified.

[0007] CN202211530284.6 discloses a milk-derived active peptide GITDPLFKGM, GITDPLFKG, and a method for obtaining and applying the same. The method uses bovine milk protein concentrate as raw material, ferments it using 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 active peptide GITDPLFKGM. The milk-derived active peptide prepared by this method has high antioxidant capacity and immunomodulatory activity, but the preparation process is relatively complicated, and there is no research basis or related reports on its bone-strengthening function.

[0008] Therefore, how to prepare and screen bone-strengthening milk protein extracts using industrial methods is an urgent problem that those skilled in the art need to solve. Summary of the Invention

[0009] In view of this, the object of the present invention is to provide a bone-strengthening milk protein extract and a preparation method and application thereof, so as to solve the deficiencies in the prior art.

[0010] In order to achieve the above object, the present invention adopts the following technical solutions:

[0011] A bone-strengthening active peptide, the amino acid sequence of which is 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 comprises the following steps:

[0013] (1) Pasteurization

[0014] pasteurizing the skim milk to obtain sterilized skim milk;

[0015] (2) Casein Isolate

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

[0017] (3) Pepsin hydrolysis

[0018] The pH value of the casein solution is adjusted to 1.5-3.0, and pepsin is added for hydrolysis to obtain a pepsin hydrolyzate;

[0019] (4) Trypsin hydrolysis

[0020] The pH value of the pepsin hydrolyzate is adjusted to 5.5-7.0, and trypsin is added for hydrolysis to obtain a trypsin hydrolyzate;

[0021] (5) Purification and desalting

[0022] The trypsin hydrolyzate is filtered and purified through a membrane, desalted by nanofiltration, and the retentate is collected to obtain a polypeptide mixture;

[0023] (6) Analysis and screening

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

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

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

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

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

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

[0030] Furthermore, in the above step (6), the analysis and screening are specifically as follows: first, the peptide mixture is detected using LC-ESI-Q-TOF high-resolution liquid chromatography-mass spectrometry, and the peptide amino acid sequence is analyzed in combination with peptidomics identification; then, the target receptor protein αvβ1 (PDB ID: 3VI4) is obtained using the Protein Data Bank (http: / / www.rcsb.org / pdb) database, and the AutoDockVina software is used for molecular docking and affinity scoring, and high-affinity peptides are selected, while publicly available peptides are excluded.

[0031] A bone-strengthening milk protein extract contains the above-mentioned bone-strengthening active peptide.

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

[0033] (1) Pasteurization

[0034] pasteurizing the skim milk to obtain sterilized skim milk;

[0035] (2) Casein Isolate

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

[0037] (3) Pepsin hydrolysis

[0038] The pH value of the casein solution is adjusted to 1.5-3.0, and pepsin is added for hydrolysis to obtain a pepsin hydrolyzate;

[0039] (4) Trypsin hydrolysis

[0040] The pH value of the pepsin hydrolyzate is adjusted to 5.5-7.0, and trypsin is added for hydrolysis to obtain a trypsin hydrolyzate;

[0041] (5) Purification and desalting

[0042] The trypsin hydrolyzate is filtered and purified through a membrane, desalted by nanofiltration, and the retentate is collected to obtain a polypeptide mixture;

[0043] (6) Concentration and drying

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

[0045] By preparing the bone-strengthening milk protein extract using the above-mentioned method of the present invention, a polypeptide mixture with a high content of the characteristic polypeptide FYPELFR can be obtained, thereby improving the bone-strengthening effect of the product. At the same time, the bone-strengthening active peptide in the extract can be a single peptide segment, or it can be a polypeptide mixture or composition. For a single peptide segment, it can be prepared by a casein solution through an enzymatic separation and purification method, by a chemical synthesis method, or by a biosynthesis method. It is preferably prepared by a casein solution through an enzymatic separation and purification method or by a biosynthesis method, and more preferably prepared by a casein solution through an enzymatic separation and purification method. For a polypeptide mixture, it can be prepared by an enzymatic purification method and a biosynthesis mixed method, and is preferably prepared by a casein solution through an enzymatic separation and purification method. The characteristic polypeptide composition includes the bone-strengthening active peptide FYPELFR and its derivatives.

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

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

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

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

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

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

[0052] The present invention also seeks to protect a derivative of the above-mentioned bone-strengthening active peptide or the bone-strengthening active peptide obtained by the above-mentioned preparation method, including polypeptide derivatives 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 seeks to protect the use of the above-mentioned bone-strengthening active peptide, the above-mentioned bone-strengthening milk protein extract containing the bone-strengthening active peptide, or the above-mentioned bone-strengthening active peptide derivative in the preparation of foods (especially including health products, with a mass percentage of 1.0%-2%) or medicines with bone-strengthening efficacy, which can obtain better bone-strengthening activity.

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

[0055] 1. The bone-strengthening active peptide of the present invention and the bone-strengthening milk protein extract containing the same can bind to integrin αvβ1 through hydrophobic interaction and hydrogen bonding, promote the proliferation and differentiation of osteoblasts by activating specific signaling pathways, promote osteoblast proliferation, and can be used for the development of bone-strengthening functional foods or drugs, which is of great significance to the deep processing of cow's milk.

[0056] 2. The present invention first uses skim milk as raw material, sterilizes and separates casein to obtain a casein solution, then hydrolyzes it with pepsin, hydrolyzes it with trypsin, purifies it with two-step membrane filtration, and purifies it with one-step desalination 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. The present invention uses specific raw materials and enzymatic hydrolysis methods to hydrolyze bovine casein, and then analyzes and screens to obtain a new polypeptide with ideal bone-strengthening efficacy, enriching the types of milk-derived bioactive peptides and providing new raw materials for the preparation of bone-strengthening efficacy products. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 The process flow chart of the preparation method of Example 1 and Example 2;

[0059] Figure 2 This is the analysis result of the polypeptide mixture in Example 1. DETAILED DESCRIPTION

[0060] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0061] Example 1

[0062] The preparation method of bone strengthening active peptide FYPELFR is as follows: Figure 1 As shown, the specific steps include:

[0063] (1) Pasteurization

[0064] The skim milk was pasteurized at 85°C for 15s to obtain sterilized skim milk;

[0065] (2) Casein Isolate

[0066] The sterilized skim milk was first concentrated to 30% of the original volume through a 0.14 μm ceramic microfiltration membrane, and then washed and filtered 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 value 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 h to obtain a pepsin hydrolyzate.

[0069] (4) Trypsin hydrolysis

[0070] The pH value of the pepsin hydrolyzate was adjusted to 6.8, 2000 U / g trypsin was added, and the mixture was hydrolyzed at 37°C for 2.5 h to obtain a trypsin hydrolyzate;

[0071] (5) Purification and desalting

[0072] The trypsin hydrolyzate was first filtered and purified through a 2000Da membrane, and the permeate was collected; the permeate was then concentrated and diafiltered using a 500Da membrane, and the concentrate was collected; finally, the concentrate was nanofiltered and desalted using a 10nm membrane, and the retentate was collected to obtain a polypeptide mixture;

[0073] (6) Analysis and screening

[0074] First, the peptide mixture was detected using an LC-ESI-Q-TOF high-resolution liquid chromatography-mass spectrometer, and the peptide amino acid sequence was analyzed in combination with peptidomics identification; then, the target receptor protein αvβ1 was obtained using the Protein DataBank database, and molecular docking and affinity scoring were performed using AutoDock Vina software. The high-affinity peptides HPHHLSF, FYPELFR, FFSDK, and YLGY were selected, and the publicly available peptides HPHHLSF, FFSDK, and YLGY were excluded to obtain the bone-strengthening active peptide FYPELFR.

[0075] Example 2

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

[0077] (1) Pasteurization

[0078] The skim milk was pasteurized at 85°C for 15s to obtain sterilized skim milk;

[0079] (2) Casein Isolate

[0080] The sterilized skim milk was first concentrated to 30% of the original volume through a 0.14 μm ceramic microfiltration membrane, and then washed and filtered 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 value 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 h to obtain a pepsin hydrolyzate.

[0083] (4) Trypsin hydrolysis

[0084] The pH value of the pepsin hydrolyzate was adjusted to 6.8, 2000 U / g trypsin was added, and the mixture was hydrolyzed at 37°C for 2.5 h to obtain a trypsin hydrolyzate;

[0085] (5) Purification and desalting

[0086] The trypsin hydrolyzate was first filtered and purified through a 2000Da membrane, and the permeate was collected; the permeate was then concentrated and diafiltered using a 500Da membrane, and the concentrate was collected; finally, the concentrate was nanofiltered and desalted using a 10nm membrane, and the retentate was collected to obtain a polypeptide mixture;

[0087] (6) Concentration and drying

[0088] The polypeptide mixture was concentrated by RO with 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 hours 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 is different from that of Example 2 only in that the step of "casein powder dilution" is added, and specifically comprises the following steps:

[0091] (1) Pasteurization

[0092] The skim milk was pasteurized at 85°C for 15s to obtain sterilized skim milk;

[0093] (2) Casein Isolate

[0094] The sterilized skim milk was first concentrated to 30% of the original volume through a 0.14 μm ceramic microfiltration membrane, and then washed and filtered 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 value 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 h to obtain a pepsin hydrolyzate.

[0097] (4) Trypsin hydrolysis

[0098] The pH value of the pepsin hydrolyzate was adjusted to 6.8, 2000 U / g trypsin was added, and the mixture was hydrolyzed at 37°C for 2.5 h to obtain a trypsin hydrolyzate;

[0099] (5) Purification and desalting

[0100] The trypsin hydrolyzate was first filtered and purified through a 2000Da membrane, and the permeate was collected; the permeate was then concentrated and diafiltered using a 500Da membrane, and the concentrate was collected; finally, the concentrate was nanofiltered and desalted using a 10nm membrane, and the retentate was collected to obtain a polypeptide mixture;

[0101] (6) Concentration and drying

[0102] The polypeptide mixture was concentrated by RO with 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 hours to obtain a bone-strengthening milk protein extract containing the bone-strengthening active peptide FYPELFR;

[0103] (7) Casein powder dilution

[0104] The content of the bone-strengthening active peptide FYPELFR in the bone-strengthening milk protein extract containing the bone-strengthening active peptide FYPELFR is diluted to 1.5% by using casein powder to obtain 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 is different from that of Example 2 only in the source of the "casein solution", and specifically comprises the following steps:

[0107] (1) Pasteurization

[0108] Commercially available casein powder was dissolved in 40°C warm water with a protein content of 8% under stirring, 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 value 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 h to obtain a pepsin hydrolyzate.

[0111] (3) Trypsin hydrolysis

[0112] The pH value of the pepsin hydrolyzate was adjusted to 6.8, 2000 U / g trypsin was added, and the mixture was hydrolyzed at 37°C for 2.5 h to obtain a trypsin hydrolyzate;

[0113] (4) Purification and desalting

[0114] The trypsin hydrolyzate was first filtered and purified through a 2000Da membrane, and the permeate was collected; the permeate was then concentrated and diafiltered using a 500Da membrane, and the concentrate was collected; finally, the concentrate was nanofiltered and desalted using a 10nm membrane, and the retentate was collected to obtain a polypeptide mixture;

[0115] (5) Concentration and drying

[0116] The polypeptide mixture was concentrated by RO with 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 hours 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 is different from that of Example 2 only in that the step of "trypsin hydrolysis" is not included. Specifically, the following steps are included:

[0119] (1) Pasteurization

[0120] The skim milk was pasteurized at 85°C for 15s to obtain sterilized skim milk;

[0121] (2) Casein Isolate

[0122] The sterilized skim milk was first concentrated to 30% of the original volume through a 0.14 μm ceramic microfiltration membrane, and then washed and filtered 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 value 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 h to obtain a pepsin hydrolyzate.

[0125] (4) Purification and desalting

[0126] The pepsin hydrolyzate was first filtered and purified through a 2000Da membrane, and the permeate was collected; the permeate was then concentrated and diafiltered using a 500Da membrane, and the concentrate was collected; finally, the concentrate was nanofiltered and desalted using a 10nm membrane, and the retentate was collected to obtain a polypeptide mixture;

[0127] (5) Concentration and drying

[0128] The polypeptide mixture was concentrated by RO with 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 hours 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 milk protein extract containing the bone-strengthening active peptide FYPELFR is different from that of Example 2 only in that the step of "pepsin hydrolysis" is not included. Specifically, the method comprises the following steps:

[0131] (1) Pasteurization

[0132] The skim milk was pasteurized at 85°C for 15s to obtain sterilized skim milk;

[0133] (2) Casein Isolate

[0134] The sterilized skim milk was first concentrated to 30% of the original volume through a 0.14 μm ceramic microfiltration membrane, and then washed and filtered 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 value 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 a trypsin hydrolyzate.

[0137] (4) Purification and desalting

[0138] The trypsin hydrolyzate was first filtered and purified through a 2000Da membrane, and the permeate was collected; the permeate was then concentrated and diafiltered using a 500Da membrane, and the concentrate was collected; finally, the concentrate was nanofiltered and desalted using a 10nm membrane, and the retentate was collected to obtain a polypeptide mixture;

[0139] (5) Concentration and drying

[0140] The polypeptide mixture was concentrated by RO with 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 hours 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 peptide mixture obtained in step (5) of Example 1 was detected using LC-ESI-Q-TOF high-resolution liquid chromatography-mass spectrometry, and a total of 36 effective peptides were detected (see Figure 2 ), including 3 dipeptides, 7 tripeptides, 5 tetrapeptides, 7 pentapeptides, 8 hexapeptides, 3 heptapeptides, 2 octapeptides, and 1 decapeptide. Molecular docking and affinity analysis revealed that 4 peptides exhibited stronger affinity for the target receptor protein αvβ1 (PDB ID: 3VI4) than the original ligand RDG (see Table 1).

[0144] Table 1 Molecular docking affinity

[0145] Peptide sequence Affinity energy (kcal / mol) HPHLSF -9.1 FYPELFR -8.5 FFSDK -8.3 YLGY -8.3 Original ligand RDG -6.1

[0146] Meanwhile, literature and patent searches revealed that the octapeptide HPHHLSF, the pentapeptide FFSDK, and the tetrapeptide YLGY all have relevant literature reports, and the octapeptide HPHHLSF has been reported to promote osteoblast proliferation. However, the heptapeptide "FYPELFR" has not been reported and is a newly discovered bone-strengthening functional peptide sequence.

[0147] 2. Preparation plan verification

[0148] The bone-strengthening active peptide prepared in Example 1, the bone-strengthening milk protein extracts prepared in Example 2 and Comparative Examples 1-3, and the product containing the bone-strengthening active peptide prepared in Example 3 were used as samples and tested for protein content and the target peptide "FYPELFR" content. The results are shown in Table 2.

[0149] Table 2 Contents of proteins and target peptides in samples of Examples 1-3 and Comparative Examples 1-3

[0150] sample Protein content / % FYPELFR content / % 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] From Table 2 we can see that:

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

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

[0154] 3. Verification of bone strengthening efficacy

[0155] The MTT method was used to determine the effect of the samples on the proliferation of MC3T3-E1 osteoblasts.

[0156] Take the bone-strengthening active peptide prepared in Example 1, the bone-strengthening milk protein extract prepared in Example 2 and Comparative Examples 1-3, and the product containing the bone-strengthening active peptide prepared in Example 3 as samples, respectively use α-MEM culture medium with 10% FBS for each sample, and set up a blank control group. Culture the cells at 37°C for 24 hours, then use MTT solution (concentration of 5 mg / mL, dissolved in 10 mM PBS) to stand for 4 hours; then, add 150 μL DMSO to dissolve the purple dinoflagellates, and gently shake the plate for 10 minutes. Use a microplate reader to measure the absorbance of the solution in each well at 490 nm to determine the cell proliferation rate. The calculation formula is: Relative proliferation rate of osteoblasts (%) = (OD 样本组 / OD 培养基组 )×100%. The sample of Example 1 was diluted to three protein concentrations. The results are shown in Table 3.

[0157] Table 3 Relative proliferation rate of osteoblasts in samples of Examples 1-3 and Comparative Examples 1-3

[0158]

[0159] From Table 3 we can see that:

[0160] ① The samples of Examples 1-3 of the present invention have a good proliferation effect on osteoblasts.

[0161] ② The samples of Examples 1-3 of the present invention effectively promoted the proliferation of osteoblasts 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 samples.

[0162] ③ The samples of Comparative Examples 2-3 have a lower FYPELFR content, and their bone strengthening effect is significantly lower than that of the samples of Examples 1-3.

[0163] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one 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 present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A bone-strengthening active peptide, characterized in that: The amino acid sequence is FYPELFR.

2. A method for preparing the bone-strengthening active peptide according to claim 1, characterized in that: The specific steps include: (1) Pasteurization pasteurizing the skim milk to obtain sterilized skim milk; (2) Casein Isolate The sterilized skim milk is concentrated through a microfiltration membrane and washed and filtered to obtain a casein solution; (3) Pepsin hydrolysis The pH value of the casein solution is adjusted to 1.5-3.0, and pepsin is added for hydrolysis to obtain a pepsin hydrolyzate; (4) Trypsin hydrolysis The pH value of the pepsin hydrolyzate is adjusted to 5.5-7.0, and trypsin is added for hydrolysis to obtain a trypsin hydrolyzate; (5) Purification and desalting The trypsin hydrolyzate is filtered and purified through a membrane, desalted by nanofiltration, and the retentate is collected to obtain a polypeptide mixture; (6) Analysis and screening The polypeptide mixture is analyzed and screened to obtain the bone-strengthening active peptide.

3. The method for preparing a bone-strengthening active peptide according to claim 2, characterized in that: In step (1), the pasteurization temperature is 75-89° C. and the time is 10-60 seconds; In step (2), the microfiltration membrane is a ceramic membrane or an organic membrane with a pore size of 0.1 μm, 0.14 μm or 0.2 μm; the concentration is to 20%-40% of the original volume; the diafiltration is performed by adding 2-5 times the volume of pure water; In step (3), the amount of pepsin added is 1000-5000 U / g protein; the hydrolysis temperature is 35-45° C., and the time is 1-3 h; In step (4), the amount of trypsin added is 1000-5000 U / g protein; the hydrolysis temperature is 35-45° C., and the time is 1-4 h; In step (5), the pore size of the membrane for filtration purification is 500-5000Da; the pore size of the membrane for nanofiltration desalination is 1-100nm.

4. The method for preparing a bone-strengthening active peptide according to claim 2, characterized in that: In step (6), the analysis and screening are specifically as follows: first, the peptide mixture is detected using an LC-ESI-Q-TOF high-resolution liquid chromatography-mass spectrometer, and the peptide amino acid sequence is analyzed in combination with peptide omics identification; then, the target receptor protein αvβ1 is obtained using the Protein Data Bank database, and molecular docking and affinity scoring are performed using AutoDock Vina software, and high-affinity peptides are selected, while publicly available peptides are excluded.

5. A bone-strengthening milk protein extract, characterized in that Contains the bone-strengthening active peptide according to claim 1.

6. A method for preparing the bone-strengthening milk protein extract according to claim 5, characterized in that: The specific steps include: (1) Pasteurization pasteurizing the skim milk to obtain sterilized skim milk; (2) Casein Isolate The sterilized skim milk is concentrated through a microfiltration membrane and washed and filtered to obtain a casein solution; (3) Pepsin hydrolysis The pH value of the casein solution is adjusted to 1.5-3.0, and pepsin is added for hydrolysis to obtain a pepsin hydrolyzate; (4) Trypsin hydrolysis The pH value of the pepsin hydrolyzate is adjusted to 5.5-7.0, and trypsin is added for hydrolysis to obtain a trypsin hydrolyzate; (5) Purification and desalting The trypsin hydrolyzate is filtered and purified through a membrane, desalted by nanofiltration, and the retentate is collected to obtain a polypeptide mixture; (6) Concentration and drying The polypeptide mixture is concentrated and freeze-dried to obtain the bone-strengthening milk protein extract.

7. The method for preparing a bone-strengthening milk protein extract according to claim 5, characterized in that: In step (1), the pasteurization temperature is 75-89° C. and the time is 10-60 seconds; In step (2), the microfiltration membrane is a ceramic membrane or an organic membrane with a pore size of 0.1 μm, 0.14 μm or 0.2 μm; the concentration is to 20%-40% of the original volume; the diafiltration is performed by adding 2-5 times the volume of pure water; In step (3), the amount of pepsin added is 1000-5000 U / g protein; the hydrolysis temperature is 35-45° C., and the time is 1-3 h; In step (4), the amount of trypsin added is 1000-5000 U / g protein; the hydrolysis temperature is 35-45° C., and the time is 1-4 h; In step (5), the pore size of the membrane for filtration purification is 500-5000Da; the pore size of the membrane for nanofiltration desalination is 1-100nm.

8. The method for preparing a bone-strengthening milk protein extract according to claim 5, characterized in that: In step (6), the concentration method is RO concentration, the transmembrane pressure difference is 1.0-2.5 MPa, and the temperature is 10-25°C; the freeze-drying temperature is -50 to -30°C, the pressure is 0.03 MPa, and the time is 24-48 hours.

9. A bone-strengthening active peptide according to claim 1 or a derivative of the bone-strengthening active peptide prepared by the preparation method according to any one of claims 2 to 4.

10. Use of the bone-strengthening active peptide according to claim 1, the bone-strengthening milk protein extract containing the bone-strengthening active peptide according to claim 5, or the derivative of the bone-strengthening active peptide according to claim 9 in the preparation of food or medicine with bone-strengthening efficacy.

Images