Preparation method and application of milk-derived peptide capable of efficiently promoting calcium absorption and bone formation

Abstract

The invention discloses a preparation method and application of milk-derived peptide capable of efficiently promoting calcium absorption and bone formation, and relates to the field of dairy product processing. Casein in milk is hydrolyzed through papain or protease of similar enzymolysis sites, a primary product is obtained through concentration, and a final product is obtained through subsequent storage or drying and other technological operations. The obtained hydrolysate has outstanding functions of promoting calcium absorption and bone formation, especially in bone formation.

Description

Technical Field

[0001] The present invention relates to the field of dairy processing, and more specifically, to a method for preparing peptides that promote calcium absorption and bone formation. The peptides prepared by this technology have the dual-dimensional functional characteristics of precise calcium absorption and bone formation. Background Art

[0002] Currently, both infants and young children in their growth and development stages and middle-aged and elderly people experiencing severe calcium loss have an urgent need for products that promote calcium absorption and improve bone density. This growing market demand has prompted researchers to delve deeper into the potential bioactive peptides found in milk. Currently, there are numerous calcium supplements available on the market, but the true significance of calcium supplementation lies not in the absorption of calcium into the body but rather in its absorption and deposition into bone cells, promoting bone formation. Therefore, the development of functional peptides with high efficacy in promoting calcium absorption and bone formation is essential. Summary of the Invention

[0003] To address the problems of the prior art, the present invention provides a method for preparing and applying a milk-derived peptide that effectively promotes calcium absorption and bone formation. The method utilizes casein from milk, hydrolyzed with papain or a similar protease, followed by molecular weight cutoff and concentration to obtain a preliminary product. The final product is then stored or dried to obtain the final product. The resulting hydrolyzate exhibits outstanding calcium absorption and bone formation-promoting functionality, particularly in bone formation.

[0004] The purpose of the present invention is achieved through the following technical solutions:

[0005] A method for preparing a milk-derived peptide having high efficiency in promoting calcium absorption and bone formation comprises the following steps:

[0006] (1) dissolving casein or milk-derived casein raw material to obtain a casein solution or a milk-derived casein raw material solution;

[0007] (2) enzymatically hydrolyzing the casein or milk-derived casein raw material obtained in step (1) using an enzyme;

[0008] (3) removing unhydrolyzed casein macromolecular components from the hydrolyzate obtained in step (2) by isoelectric precipitation, and / or performing molecular weight cutoff on the casein hydrolyzate to remove macromolecular peptides;

[0009] (4) concentrating the hydrolyzate obtained in step (3);

[0010] (5) The concentrated solution obtained in step (4) is stored or dried to obtain a solid product.

[0011] According to the above technical solution, in step (1), the casein includes the whole casein itself, α S1 -Casein, α S2-One or more of casein, β-casein, and κ-casein.

[0012] According to the above technical solution, in step (1), the milk-derived casein raw material includes one or more of cow's milk, goat's milk, skim milk, reconstituted milk, and sodium caseinate.

[0013] According to the above technical solution, in step (1), the casein concentration (i.e., protein percentage) in the casein solution is less than 12%, preferably 3-5%; the casein percentage in the milk-derived casein raw material solution is less than 8%, preferably 3-5%.

[0014] According to the above technical solution, in step (1), the casein or milk-derived casein raw material is pretreated to better expose the enzymatic hydrolysis sites of the casein. The pretreatment method can be heat treatment, crushing ultrasonic treatment, and high-pressure homogenization. The heat treatment conditions are: temperature 40-140°C, time 4s-30min. The crushing is ultrafine grinding, and the particle size after crushing is preferably 400 mesh or finer.

[0015] According to the above technical solution, the enzyme selected for the enzymatic hydrolysis can be a protease isolated from microorganisms or an enzyme that has the same enzymatic effect on casein hydrolysis during the microbial fermentation process.

[0016] According to the above technical solution, the enzyme used for the enzymatic hydrolysis is one or more of papain, bromelain, trypsin, and neutral protease.

[0017] According to the above technical solution, the enzymatic hydrolysis conditions are pH = 5-10, temperature is 25-80 ° C, enzyme dosage is 100-10000 U / g, and enzymatic hydrolysis time is 30min-24h. The optimal hydrolysis conditions are the optimal hydrolysis conditions for the selected enzyme, and the enzyme dosage is preferably 4000 U / g.

[0018] According to the above technical solution, the main enzymatic cleavage preferences of the hydrolyzate after papain enzymatic hydrolysis are: EK, EE, QQ, VR, GS, LN, KR, AR, ES, KH, KY, QS, TV, IN, PY, AS, ES, or its enzymatic cleavage sites are concentrated in ES, EK, and QS.

[0019] According to the above technical solution, in step (3), the isoelectric precipitation method is an isoelectric centrifugation method, under the following conditions: pH = 4-5, temperature of 20-40°C, centrifugal speed of 4000-10000 r / min, centrifugation time of 5min-30min, preferably 5000-8000 r / min, 15min; the optimal molecular weight cut-off is less than 3000Da or a narrower range.

[0020] According to the above technical solution, the peptide segments of the hydrolyzate obtained after the enzymatic hydrolysis preferably include amino acid sequences such as glycine-serine-glutamate (GSE), glycine-glutamate-proline (GEP), and glycine-glutamate-isoleucine (GEI), and the peptides formed include NPIGSENSE, EDVPSER, LIEGPPG PAGP, KNEILRL, KLTEEEKNRL, HQPHQPLPPTVMFPPQ, YPFPGPIH, LTL TDVENLHLPLPLL, HPPHHLSF, ESPPEINTVQ, GSESTEDQAMEDIKQMEA, PNPIGSENS, KEVVR, NAVPITPTL, QPKTKVIPYVRYL, WMHQPHQPLPPTVM, NIIRKNYIPNP, VLPVPQK, RELEELNVPGEIVE, SGEPTSTPTT, and VIES One or more of: PPEIN, DIGSESTEDQAMEDIK, YKVPQLEIVPNSAEER, LLAVA, AVPYPQR, FQSEEQQQTEDELQDK, DRITGGKDFR, FFSDKIAK, PEVFGKEKVNELSKD, IGSESTEDQAMEDIKQM, HKEMPFPKYPVEPF, PPKKNQDKTEIPT, in particular: HPHPHLSF, VLPVPQK, LLAVA, KNEILRL, KEVVR, NPI GSENSE, LIEGPPGPAGP, EDVPSER and YPFPGPIH.

[0021] According to the above technical solution, in step (4), the concentration method is to concentrate the protein to a concentration of more than 8%, which can be one or more of ultrafiltration, nanofiltration, evaporation, etc. The best choice is low-temperature vacuum rotary evaporation at a temperature below 10°C, preferably 4°C.

[0022] According to the above technical solution, in step (5), the drying method can be spray drying, freeze drying, vacuum drying, or vacuum freeze drying. In order to ensure that the activity of the functional peptide is not destroyed, vacuum freeze drying is the best choice.

[0023] The present invention also relates to a milk-derived peptide having the function of promoting calcium absorption and bone formation and prepared by the above-mentioned preparation method.

[0024] The use of the above-mentioned milk-derived peptide in the preparation of a product that promotes calcium absorption and bone formation, which is a medicine, food, or health product, provides an effective product for people with bone health needs, especially the elderly and children, to promote bone growth and development and alleviate bone diseases such as osteoporosis.

[0025] The present invention is based on the discovery that peptides obtained by papain hydrolysis have excellent calcium absorption and bone formation promoting effects. Analysis of the peptides revealed that the peptide fragments contained therein have significant calcium absorption and bone formation promoting effects. To increase the types of calcium absorption and bone formation promoting peptides and the relative content of functional peptides, molecular interception is used to remove other large molecular peptides, thereby reducing the content of other calcium absorption and bone formation promoting peptides after enzymatic hydrolysis, thereby achieving the development of highly efficient targeted functional products. The present invention can provide new raw materials for potential calcium absorption and bone formation promoting products and lay a certain foundation for the development of functional foods.

[0026] Beneficial effects of the present invention:

[0027] Compared with the trypsin hydrolysis product, the papain enzymatic hydrolysis product of the present invention has a relatively high purity of functional peptides prepared under uniform experimental conditions, wherein the molecular weight is less than 3000Da and the content is relatively high, and the peptides are easily absorbed; at the same time, the casein peptide developed by this technology has outstanding performance in bone mineralization function, especially: compared with the trypsin hydrolysis product preparation process, the papain hydrolysis product has the effect of increasing bone mineral density by about 15%, increasing trabecular thickness by about 16%, and increasing bone volume / tissue volume by about 2.5%. The development of this peptide product helps to improve the overall quality and stability of bones. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is the casein phosphopeptide content of casein hydrolyzed products by different enzymes.

[0029] Figure 2 The relative molecular weights of casein hydrolyzed by different enzymes are shown in Figure 1. (A) shows the relative molecular weight distribution of bromelain hydrolyzate, (B) shows the relative molecular weight distribution of papain hydrolyzate, (C) shows the relative molecular weight distribution of neutral protease hydrolyzate, and (D) shows the relative molecular weight distribution of trypsin hydrolyzate. Different lowercase letters indicate significant differences (P < 0.05) between the molecular weight intervals in the figure.

[0030] Figure 3 These are CT images of the femur and trabecular microstructure of low calcium model mice fed with peptides treated in Example 1-4.

[0031] Figure 4 Figure 1 shows the physicochemical parameters of the femurs of mice fed a calcium-deficient model treated with peptides from Examples 1-4. Data represent mean ± SD (n = 8). Different lowercase letters indicate significant differences (P < 0.05). (A) is the femur radius, (B) is the femur length, and (C) is the femur weight.

[0032] Figure 5The following are analysis diagrams of femoral microstructural indicators in mice fed a calcium-deficient model treated with peptides from Examples 1-4. Different lowercase letters indicate significant differences (P<0.05): (A) is bone density, (B) is trabecular thickness, (C) is trabecular number, and (D) is bone volume / tissue volume. DETAILED DESCRIPTION

[0033] The technical scheme of the present invention will be described in detail below. It should be understood that the embodiments proposed here are merely implementation cases for illustrative purposes and are not intended to limit the scope of the present invention. Unless otherwise specified, the reagents and instruments used in the following examples are all commercially available products.

[0034] Example 1

[0035] The method for preparing casein calcium absorption-bone formation promoting peptide by enzymatic hydrolysis of casein with papain, wherein the sample preparation is completed by the following steps:

[0036] The casein solid was crushed with a grinder and passed through a 400-mesh sieve. 1.0 g was weighed and a 1% casein solution was prepared with deionized water. The solution was heated in a water bath at 60° C. until the casein solid was dissolved. The pH was adjusted to 5.0 with 0.1 mol / L hydrochloric acid. The solution was hydrolyzed with papain at an enzyme addition amount of 4000 U / g and heated in a water bath at 60° C. for 120 min to obtain solution I. The pH was adjusted to 4.6 with 0.1 mol / L hydrochloric acid. After 30 min, the solution was centrifuged at 4° C. and 5000 r / min for 30 minutes to collect solution II. The solution II was redissolved three times with deionized water and then centrifuged for enrichment. The solution was concentrated by low-temperature (4° C.) vacuum rotary evaporation at 100-150 r / min to reduce the volume of the solution to less than 1 / 3 of the original volume. The solution was then vacuum freeze-dried to obtain a casein calcium absorption-bone formation peptide casein product (papain hydrolyzed casein product).

[0037] Example 2

[0038] The method for preparing calcium absorption-bone formation-promoting peptides by enzymatic hydrolysis of sodium caseinate by bromelain is to prepare the sample in the following steps:

[0039] Weigh 1.0g sodium caseinate solid, adopt deionized water to configure 1% casein solution, and heat in a water bath at 50℃ until the sodium caseinate solid dissolves, use 0.01mol / L hydrochloric acid and sodium hydroxide solution, adjust pH to 7.0, and under 50℃ of water bath, adopt bromelain to hydrolyze, enzyme addition is 4000U / g, 50℃ water bath 120min, obtain solution I, use 0.1mol / L hydrochloric acid to adjust pH=4.6, after 30min, 4℃, 5000r / min, centrifuge for 30 minutes, collect solution II, with deionized water by solution II through centrifugal enrichment after 3 times of redissolving, carry out 100-150r / min and carry out low temperature (4℃) vacuum rotary evaporation and concentration, make the solution volume be less than original 1 / 3, carry out vacuum freeze-drying afterwards, obtain casein calcium absorption-bone formation peptide casein product (bromelain hydrolyzed casein product).

[0040] Example 3

[0041] The method for preparing casein calcium absorption-promoting bone formation peptide by trypsin hydrolysis of casein is to complete the sample preparation according to the following steps:

[0042] Weigh 2.0g casein solid, prepare 2% casein solution with deionized water, heat in a water bath at 50℃ until the casein solid dissolves, then cool to room temperature, adjust pH to 7.0 with 0.1mol / L hydrochloric acid, hydrolyze with trypsin, add 4000U / g enzyme, and incubate in a 37℃ water bath for 120min to obtain solution I, adjust pH to 4.6 with 0.1mol / L hydrochloric acid, centrifuge at 4℃ and 5000r / min for 30min after 30min, collect solution II, redissolve solution II three times with deionized water and then centrifuge for enrichment, carry out low-temperature (4℃) vacuum rotary evaporation and concentration at 100-150r / min to make the solution volume less than 1 / 3 of the original, and then carry out vacuum freeze-drying to obtain casein calcium absorption-bone formation peptide casein product (trypsin hydrolyzed casein product).

[0043] Example 4

[0044] The method for preparing casein calcium absorption-promoting bone formation peptide by enzymatic hydrolysis of sodium caseinate with neutral protease is to complete sample preparation according to the following steps:

[0045] Weigh 3g of sodium caseinate solid, use deionized water to configure 3% sodium caseinate solution, heat in a water bath at 50℃ until the casein solid dissolves, use 0.01mol / L hydrochloric acid and sodium hydroxide solution to adjust the pH to 7.0, heat in a water bath at 50℃, use neutral protease to hydrolyze, the enzyme addition amount is 4000U / g, and the solution is in a 50℃ water bath for 120min to obtain solution I, adjust the pH=4.6, after 30min, centrifuge at 4℃, 5000r / min for 30 minutes, collect solution II, redissolve solution II three times with deionized water and then centrifuge and enrich, carry out low-temperature (4℃) vacuum rotary evaporation and concentration at 100-150r / min to make the solution volume less than 1 / 3 of the original, and carry out vacuum freeze-drying to obtain a casein calcium absorption-bone formation peptide casein product (neutral protease hydrolyzed casein product).

[0046] Example 5

[0047] In order to better illustrate the product effect of casein phosphopeptide, the product in Example 1-4 was used to measure casein phosphopeptide. Figure 1 It can be found that the casein phosphopeptide content in the trypsin product is the highest, while the casein phosphopeptide content in papain is significantly lower than that in the trypsin hydrolyzate. This also shows that the effect of the casein phosphopeptide product is not as good as the products in Examples 1, 2 and 4 of the present invention.

[0048] Example 6

[0049] The relative molecular weights of the casein hydrolyzed products by different enzymes in Examples 1-4 were measured. Figure 2 It can be found that comparing the molecular weights of casein hydrolyzates prepared by different enzyme hydrolysis can well explain why the commercially available casein phosphopeptides prepared by trypsin have a larger molecular weight, while the casein calcium absorption and bone formation peptides developed by this technology have a smaller molecular weight and are easily absorbed.

[0050] Example 7

[0051] In order to better evaluate the effects of the calcium absorption-bone formation peptides prepared in Examples 1-4, the following animal experiments were performed: This study used a hypocalcemic mouse model. The mouse experiments were approved by the Liaoning Provincial Experimental Animal Management Committee. All procedures used followed the care and animal guidelines of the National Institute of Health. The experiments were approved by the Animal Ethics Committee of Dalian Polytechnic University (Ethics Number: DLPU2024004). A total of 60 SPF male KM mice (4 weeks old) were purchased from Liaoning Changsheng Experimental Animal Research Center (Dalian, Liaoning, China) and used in this study. Mice were raised under laboratory conditions (temperature of (22±2)℃ and relative humidity of (60±5)%. Casein hydrolyzed products of papain, bromelain, trypsin and neutral protease were divided into 4 experimental groups. There were also three comparative experimental groups, namely CaCl2, low calcium control group (i.e., model group in the figure) and normal control group (i.e., control group or blank group in the figure), with 8 mice in each group. The normal control group was fed with AIN-93 standard animal feed (calcium content of 0.5%) until the end of the experiment (8 weeks), and the low calcium control group continued to be fed with the same low calcium diet for 8 weeks (calcium content of 0.05%) until the end of the experiment. The other groups were fed with a low calcium diet (calcium content of 0.05%) for the first four weeks and a low calcium diet (calcium content of 0.05%) for the last four weeks. While maintaining a low-calcium diet for 8 weeks, the animals were supplemented with a solution of trypsin-hydrolyzed casein, bromelain-hydrolyzed casein, neutral protease-hydrolyzed casein, papain-hydrolyzed casein, and CaCl2 via oral gavage once daily. The oral calcium intake for each experimental group was set at 20.0 mg / kg body weight per day; the peptide (enzymatic casein hydrolyzed product) and CaCl2 intake were set at 19.95 × 8 mg / kg body weight per day. The pancreas, bromelain-hydrolyzed casein, neutral protease-hydrolyzed casein, and papain-hydrolyzed casein, as well as CaCl2, were dissolved in distilled water and fed separately from the diet via gavage. After 8 weeks, the animals were euthanized, and blood and femur samples were collected for analysis.

[0052] Measurement of mouse femur weight, length, and diameter: The femurs of mice in each group were separated, and the adhesion tissue was removed. The femurs were then dried in a drying oven at 105°C to constant weight, and the weights were recorded. The length and diameter of the femurs were measured with a vernier caliper and recorded for analysis.

[0053] Mouse bone CT imaging analysis:

[0054] (1) CT scan

[0055] All specimens were fixed and then scanned with Micro-CT. Scanning parameters were: tube current 200 μA, voltage 70 kV, full-body scan, resolution 6.534165 μm, exposure time 350 ms, and scan angle 180 degrees. A phantom (Phantom; provided by the equipment manufacturer) was scanned under the same conditions for calibration. Raw images were obtained after scanning.

[0056] from Figure 3 It can be seen that the products of Examples 1-4 all have good effects in promoting calcium absorption and bone formation, among which the effect of Example 3 is weaker. The trabecular structures in Examples 1, 3 and 4 are relatively compact and thick.

[0057] (2) CT reconstruction

[0058] Reconstruct selected regions of the original image using the 3D reconstruction software NRecon (version V1.7.4.2, Bruker, Germany). Preview the reconstructed image before reconstruction. To minimize image artifacts and achieve optimal reconstruction results, adjust the reconstruction parameters to the following: Smoothing = 5 (smoothing), Beam-hardening = 8 (beam intensity), and Ring artifacts = 25%. Once the parameters are set, edit and select the designated folder to begin image reconstruction.

[0059] from Figure 4 It can be seen that there is no significant difference in the femoral radius, length and weight of the products in Examples 1-4, but the femoral length and weight of Example 1 (i.e., the papain group) are slightly higher than those of the other groups.

[0060] (3) CT analysis

[0061] CT Analyser (software version 1.20.3.0, Bruker, Germany) was used for analysis. Uniform parameters were set, and the software calculated parameters such as total tissue volume (TV), bone volume (BV), volume ratio (BV / TV), bone area (BS), trabecular number (Tb.N), and trabecular thickness (Tb.th).

[0062] from Figure 5 It can be clearly seen that the values of bone density, trabecular thickness, number, and bone volume / tissue volume in Example 1 group are higher than those in other treatment groups, indicating that the preparation method of the present invention can indeed obtain an efficient product that promotes calcium absorption and bone formation.

Claims

1. A method for preparing a milk-derived peptide that promotes calcium absorption and bone formation, characterized in that: The steps include: (1) dissolving casein or milk-derived casein raw material to obtain a casein solution or a milk-derived casein raw material solution; (2) enzymatically hydrolyzing the casein or milk-derived casein raw material obtained in step (1) using an enzyme; (3) removing unhydrolyzed casein macromolecular components from the hydrolyzate obtained in step (2) by isoelectric precipitation, and / or performing molecular weight cutoff on the casein hydrolyzate to remove macromolecular peptides; (4) concentrating the hydrolyzate obtained in step (3); (5) The concentrated solution obtained in step (4) is stored or dried to obtain a solid product.

2. The preparation method according to claim 1, characterized in that The casein includes casein itself, α S1 -Casein, α S2 -One or more of casein, β-casein, and κ-casein; the milk-derived casein raw materials include one or more of cow's milk, goat's milk, skim milk, reconstituted milk, and sodium caseinate.

3. The preparation method according to claim 1, characterized in that In step (1), the casein mass percentage in the casein solution is less than 12%, and the casein mass percentage in the milk-derived casein raw material solution is less than 8%; The casein or milk-derived casein raw material is pretreated, and the pretreatment method is one or more of heat treatment, crushing, ultrasonic treatment, and high-pressure homogenization.

4. The preparation method according to claim 1, characterized in that In step (2), the enzyme for enzymatic hydrolysis includes proteases isolated from microorganisms and enzymes that have the same enzymatic effect on casein during microbial fermentation, preferably one or more of papain, bromelain, trypsin, and neutral protease; The enzymatic hydrolysis conditions are: pH=5-10, temperature is 25-80°C, enzyme dosage is 100-10000 U / g, and enzymatic hydrolysis time is 30 min-24 h.

5. The preparation method according to claim 4, characterized in that The main enzymatic cleavage preferences of papain hydrolysates are: EK, EE, QQ, VR, GS, LN, KR, AR, ES, KH, KY, QS, TV, IN, PY, AS, ES, or their cleavage sites are concentrated in ES, EK, and QS.

6. The preparation method according to claim 1, characterized in that In step (3), the isoelectric precipitation method is isoelectric centrifugation, and the conditions are: pH = 4-5, temperature is 20-40°C; and the molecular weight cut-off is less than 3000Da.

7. The preparation method according to claim 1, characterized in that The peptide segments of the hydrolyzate obtained after enzymatic hydrolysis preferably include one or more of the amino acid sequences of glycine-serine-glutamate (GSE), glycine-glutamate-proline (GEP), and glycine-glutamate-isoleucine (GEI), and the peptides formed include NPIGSENSE, EDVPSER, LIEGPPGPAGP, KNEILRL, KLTEEEKNRL, HQ PHQPLPPTVMFPPQ, YPFPGPIH, LTLTDVENLHLPLPLL, HPPHHLSF, ESPPEINTVQ, GSESTEDQAMEDIKQMEA, PNPIGSENS, KEVVR, NAVPITPTL, QPKTKVIPYVRYL, WMHQPHQPLPPTVM, NIIRKNYIPNP, VLPVPQK, RE LEELNVPGEIVE, SGEPTSTPTT, VIESPPEIN, DIGSESTEDQAMEDIK, and YKV One or more of PQLEIVPNSAEER, LLAVA, AVPYPQR, FQSEEQQQTEDELQDK, DRITGGKDFR, FFSDKIAK, PEVFGKEKVNELSKD, IGSESTEDQAMEDIKQM, HKEM PFPKYPVEPF, PPKKNQDKTEIPT.

8. The preparation method according to claim 1, characterized in that In step (4), the concentration method is one or more of ultrafiltration, nanofiltration, and evaporation; In step (5), the drying method is spray drying, freeze drying, vacuum drying or vacuum freeze drying.

9. A milk-derived peptide having the effect of promoting calcium absorption and bone formation, prepared by the method according to any one of claims 1 to 8.

10. Use of the milk-derived peptide according to claim 9 in the preparation of a product promoting calcium absorption and bone formation, characterized in that: The product is a medicine, food or health product.

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