Eggshell membrane peptide for enhancing bone mineral density as well as preparation method and application of eggshell membrane peptide

Abstract

The invention discloses an eggshell membrane peptide for enhancing bone mineral density as well as a preparation method and application thereof, belongs to the technical field of egg by-product processing, and particularly relates to a preparation method of the eggshell membrane peptide for enhancing bone mineral density, which comprises the following steps: carrying out vacuum freeze drying and crushing on an eggshell membrane to obtain eggshell membrane powder; mixing the eggshell membrane powder with deionized water to obtain an eggshell membrane solution; adding keratinase, performing enzymolysis, and taking supernate; adding collagenase, carrying out enzymolysis, and centrifuging to take supernate, so as to obtain primary enzymatic hydrolysate; adding alkaline phosphatase, performing enzymolysis and centrifugation, and taking supernate to obtain enzymatic hydrolysate; and performing ultrafiltration and drying to obtain the eggshell membrane peptide. According to the eggshell membrane peptide for enhancing the bone mineral density, keratin cross-linking is destroyed through enzymolysis of keratinase, active peptide capable of stimulating osteoblast proliferation is released through enzymolysis of collagenase, phosphopeptide calcium binding sites are further activated through enzymolysis of alkaline phosphatase, calcium salt deposition is promoted, and the eggshell membrane peptide for enhancing the bone mineral density is provided.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of egg by-product processing, and particularly relates to an eggshell membrane peptide for enhancing bone density and a preparation method and application thereof. BACKGROUND

[0002] With the aggravation of global population aging, osteoporosis has become a major public health problem threatening the health of the middle-aged and elderly population. Global osteoporosis patients have brought heavy burden to the medical system and social economy. Currently, the commonly used anti-osteoporosis drugs such as bisphosphonates, estrogen, RANKL inhibitors, etc. have certain curative effects, but long-term use may be accompanied by problems such as mandibular osteonecrosis, cardiovascular risk and gastrointestinal side effects, and it is urgent to develop safe, efficient and natural alternative therapies.

[0003] Eggshell membrane (ESM) is an important component in the waste of poultry egg processing, and the annual waste amount is in the tens of millions of tons worldwide. Studies have shown that ESM is rich in collagen (types I, V, X), chondroitin sulfate, hyaluronic acid and various functional peptides, and has significant anti-inflammatory, antioxidant and tissue repair activities. In recent years, the application of ESM extract in the field of joint health has been reported, but its potential in bone density regulation has not been fully explored.

[0004] In the prior art, the preparation of eggshell membrane peptides mostly adopts conventional enzymatic hydrolysis method, such as using trypsin or papain, which has many limitations, for example, the target specificity of active peptides is insufficient, the molecular weight distribution of traditional enzymatic hydrolysate is wide (1-50 Da), and the content of specific peptide segments with osteogenic activity is low; the bioavailability is low, and large molecular peptides are difficult to be effectively absorbed by the intestinal tract, affecting the in vivo activity expression; the process efficiency is low, and the peptide yield under single enzymatic hydrolysis condition is usually less than 15%, and the active ingredients are easy to be inactivated in the high-temperature sterilization process.

[0005] Therefore, it is of great significance to develop a high-purity, low-molecular-weight active peptide based on eggshell membrane to improve its ability to promote osteogenic differentiation and calcium deposition by optimizing the preparation process, so as to realize the green prevention and control of osteoporosis and the high-value utilization of eggshell waste. SUMMARY

[0006] To solve the above technical problems, the application provides an eggshell membrane peptide for enhancing bone density and a preparation method and application thereof. The application utilizes keratinase to hydrolyze and destroy keratin cross-linking, collagenase to hydrolyze and release active peptides that can stimulate osteoblast proliferation, and alkaline phosphatase to hydrolyze and activate phosphopeptide calcium binding sites to promote calcium salt deposition, thereby providing an eggshell membrane peptide for enhancing bone density.

[0007] To achieve the above purpose, the application provides a preparation method of an eggshell membrane peptide for enhancing bone density, comprising the following steps:

[0008] 1) vacuum freeze-drying eggshell membrane, crushing, obtaining eggshell membrane powder;

[0009] 2) mixing the eggshell membrane powder obtained in step 1) with deionized water, obtaining an eggshell membrane solution; adding keratinase to the eggshell membrane solution, first enzymolysis, first enzyme inactivation, first centrifugation to obtain supernatant;

[0010] 3) adding collagenase to the supernatant obtained in step 2), second enzymolysis, second enzyme inactivation, second centrifugation to obtain supernatant, obtaining a preliminary enzymolysis solution;

[0011] 4) adding alkaline phosphatase to the preliminary enzymolysis solution obtained in step 3), third enzymolysis, third enzyme inactivation, third centrifugation to obtain supernatant, obtaining an enzymolysis solution;

[0012] 5) ultrafiltration and drying of the enzymolysis solution obtained in step 4), obtaining eggshell membrane peptide.

[0013] Preferably, the vacuum degree of the vacuum freeze-drying in step 1) is 0.100-0.150 mbar, the temperature of the vacuum freeze-drying is -30 to -50℃, and the time of the vacuum freeze-drying is 22-26 h; the particle size of the eggshell membrane powder in step 1) is 100-200 mesh.

[0014] Preferably, the mass concentration of the eggshell membrane solution in step 2) is 2-8%; and the addition amount of the keratinase in step 2) is calculated according to 2-3% of the mass of the eggshell membrane powder.

[0015] Preferably, the pH of the first enzymolysis in step 2) is 10.5-11.5, the temperature of the first enzymolysis is 50-60℃, and the time of the first enzymolysis is 1-3 h; the temperature of the first enzyme inactivation in step 2) is 85-90℃, and the time of the first enzyme inactivation is 10-15 min; the speed of the first centrifugation in step 2) is 8000-12000 rpm, and the time of the first centrifugation is 15-20 min.

[0016] Preferably, the addition amount of the collagenase in step 3) is calculated according to 1-2% of the mass of the eggshell membrane powder.

[0017] Preferably, the pH of the second enzymolysis in step 3) is 6.5-7.5, the temperature of the second enzymolysis is 38-42℃, and the time of the second enzymolysis is 2-4 h; the temperature of the second enzyme inactivation in step 3) is 95℃, and the time of the second enzyme inactivation is 5 min; the speed of the second centrifugation in step 3) is 8000-12000 rpm, and the time of the second centrifugation is 15-20 min.

[0018] Preferably, the amount of the alkaline phosphatase added in step 4) is calculated according to 0.5-1.0% of the mass of the eggshell membrane powder; the pH of the third enzymolysis in step 4) is 8.5-9.5, the temperature of the third enzymolysis is 35-39℃, and the time of the third enzymolysis is 1-2h; the temperature of the third enzyme inactivation in step 4) is 95℃, and the time of the third enzyme inactivation is 5min; and the speed of the third centrifugation in step 4) is 8000-12000rpm, and the time of the third centrifugation is 15-20min.

[0019] Preferably, the ultrafiltration in step 5) uses an ultrafiltration membrane with a molecular weight of 1000Da; and the drying in step 5) is specifically: pre-freezing for 4h at-40℃, and vacuum freeze-drying for 24h at a temperature of-25℃ under a pressure of 0.1mbar.

[0020] The application also provides the eggshell membrane peptide prepared by the preparation method.

[0021] The application also provides the use of the eggshell membrane peptide prepared by the preparation method in the preparation of a product for enhancing bone density.

[0022] Compared with the prior art, the application has the following advantages and technical effects:

[0023] The application provides an eggshell membrane peptide for enhancing bone density, and a preparation method and use thereof. The eggshell membrane peptide prepared by the application releases the original type I collagen peptide and phosphoprotein peptide in the eggshell membrane, and improves the active peptide segment for increasing bone density, stimulates osteoblast proliferation, inhibits osteoclasts, and promotes calcium salt deposition. The application optimizes the enzymolysis conditions of keratinase, opens the dense keratin network in the eggshell membrane powder, optimizes the enzymolysis conditions of collagenase, uses the specificity of the Pro-X-Gly-Pro site of collagen protein, efficiently releases the Gly-Pro-Hyp bone synthesis peptide, and then uses alkaline phosphatase phosphorylation to activate and improve the function of the prepared eggshell membrane peptide for enhancing bone density. BRIEF DESCRIPTION OF DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings in the following description are only some embodiments of the application, and other drawings can be obtained by those skilled in the art without any creative effort on the basis of these drawings.

[0025] Figure 1 Serum alkaline phosphatase content determination results of mice in each group;

[0026] Figure 2 Serum TRACP content determination results of mice in each group;

[0027] Figure 3 The serum OPG content and RANKL content of each group of mice were determined, wherein A is the serum OPG content and B is the serum RANKL content;

[0028] Figure 4 The femur bone density of each group of mice was determined. DETAILED DESCRIPTION

[0029] Various exemplary embodiments of the present application will now be described in detail, which should be considered to be illustrative of the application and not as limiting the application. It will be appreciated that certain features of the application, which are, for clarity, described in the context of some embodiments, can also be provided separately or in any appropriate combination.

[0030] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. In addition, where particular ranges of values are given, understand that each intervening value, to the upper or lower limit of the ranges is also specifically included. Each smaller range that falls within the broader ranges is also specifically included. The upper and lower limits of these smaller ranges can independently be included or excluded in the range, and each range is inclusive of its end points.

[0031] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All patents, patent applications, publications, and descriptions mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the patents, patent applications, publications, and descriptions are cited.

[0032] Many modifications and variations of this application can be made in the light of the above teachings without departing from the spirit and scope thereof, and it is to be understood that all such modifications and variations fall within the scope of the application. Further, it is to be understood that the disclosure in connection with the specific embodiments described herein illustrates and does not limit the scope of the present application.

[0033] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean including, but not limited to.

[0034] The eggshell membrane used in the present application was purchased from an egg processing factory; the keratinase used was a keratinase from Bacillus licheniformis (enzyme activity ≥ 15000 U / g) purchased from Novozymes; the collagenase (Collagenase NB4) used was purchased from Novozymes; the alkaline phosphatase used was purchased from Desitin; and the alkaline protease used was purchased from Novozymes.

[0035] Example 1

[0036] 1) Eggshell membrane was vacuum freeze-dried for 24 h at a vacuum degree of 0.133 mbar and a temperature of -40°C, crushed, and passed through a 200-mesh sieve to obtain eggshell membrane powder.

[0037] 2) The eggshell membrane powder was mixed with deionized water to prepare an eggshell membrane solution with a mass concentration of 5%, and keratinase was added to the eggshell membrane solution (the amount of keratinase added was calculated as 2.5% of the mass of the eggshell membrane powder), and the first enzymolysis was performed at a pH of 11 and a temperature of 55°C for 2 h, the first enzyme inactivation was performed at 88°C for 12 min, the first centrifugation was performed at 10,000 rpm for 18 min, and the supernatant was taken.

[0038] 3) Collagenase was added to the supernatant (the amount of collagenase added was calculated as 1.5% of the mass of the eggshell membrane powder), and the second enzymolysis was performed at a pH of 7 and a temperature of 40°C for 3 h, the second enzyme inactivation was performed at 95°C for 5 min, the second centrifugation was performed at 10,000 rpm for 18 min, and the supernatant was taken to obtain a preliminary enzymolysis solution.

[0039] 4) Alkaline phosphatase was added to the preliminary enzymolysis solution (the amount of alkaline phosphatase added was calculated as 0.8% of the mass of the eggshell membrane powder), and the third enzymolysis was performed at a pH of 9 and a temperature of 37°C for 1.5 h, the third enzyme inactivation was performed at 95°C for 5 min, the third centrifugation was performed at 10,000 rpm for 18 min, and the supernatant was taken to obtain an enzymolysis solution.

[0040] 5) The enzymolysis solution was ultrafiltered using an ultrafiltration membrane with a molecular weight of 1000 Da, dried (-40°C pre-freezing for 4 h, vacuum freeze-drying at a temperature of -25°C for 24 h at a vacuum degree of 0.1 mbar), and eggshell membrane peptides were obtained.

[0041] Example 2

[0042] 1) Eggshell membrane was vacuum freeze-dried for 22 h at a vacuum degree of 0.1 mbar and a temperature of -30°C, crushed, and passed through a 100-mesh sieve to obtain eggshell membrane powder.

[0043] 2) The eggshell membrane powder was mixed with deionized water to prepare an eggshell membrane solution with a mass concentration of 2%, and keratinase was added to the eggshell membrane solution (the amount of keratinase added was calculated as 2% of the mass of the eggshell membrane powder), and the first enzymolysis was performed at a pH of 10.5 and a temperature of 50°C for 1 h, the first enzyme inactivation was performed at 85°C for 10 min, the first centrifugation was performed at 8000 rpm for 15 min, and the supernatant was taken.

[0044] 3) Collagenase was added to the supernatant (the amount of collagenase added was calculated as 1% of the mass of the eggshell membrane powder), and the second enzymolysis was performed at a pH of 6.5 and a temperature of 38°C for 2 h, the second enzyme inactivation was performed at 95°C for 5 min, the second centrifugation was performed at 8000 rpm for 15 min, and the supernatant was taken to obtain a preliminary enzymolysis solution.

[0045] 4) The preliminary enzymatic hydrolysate was added with alkaline phosphatase (the amount of alkaline phosphatase was calculated as 0.5% of the mass of eggshell membrane powder), and third enzymatic hydrolysis was carried out at pH 8.5 and 35°C for 1 h, third enzyme inactivation was carried out at 95°C for 5 min, and third centrifugation was carried out at 8000 rpm for 15 min to obtain the supernatant, and an enzymatic hydrolysate was obtained.

[0046] 5) The enzymatic hydrolysate was subjected to ultrafiltration using an ultrafiltration membrane with a molecular weight of 1000 Da, and was dried (pre-frozen at -40°C for 4 h, vacuum freeze-dried at 0.1 mbar and -25°C for 24 h), and an eggshell membrane peptide was obtained.

[0047] Example 3

[0048] 1) The eggshell membrane was vacuum freeze-dried at a vacuum degree of 0.15 mbar and a temperature of -50°C for 26 h, was crushed, and was passed through a 200-mesh sieve to obtain eggshell membrane powder.

[0049] 2) The eggshell membrane powder was mixed with deionized water to prepare an eggshell membrane solution with a mass concentration of 8%, and keratinase was added to the eggshell membrane solution (the amount of keratinase was calculated as 3% of the mass of eggshell membrane powder), and first enzymatic hydrolysis was carried out at pH 11.5 and 60°C for 3 h, first enzyme inactivation was carried out at 90°C for 15 min, and first centrifugation was carried out at 12000 rpm for 20 min to obtain the supernatant.

[0050] 3) Collagenase was added to the supernatant (the amount of collagenase was calculated as 2% of the mass of eggshell membrane powder), and second enzymatic hydrolysis was carried out at pH 7.5 and 42°C for 4 h, second enzyme inactivation was carried out at 95°C for 5 min, and second centrifugation was carried out at 12000 rpm for 20 min to obtain the supernatant, and a preliminary enzymatic hydrolysate was obtained.

[0051] 4) Alkaline phosphatase was added to the preliminary enzymatic hydrolysate (the amount of alkaline phosphatase was calculated as 1% of the mass of eggshell membrane powder), and third enzymatic hydrolysis was carried out at pH 9.5 and 39°C for 2 h, third enzyme inactivation was carried out at 95°C for 5 min, and third centrifugation was carried out at 12000 rpm for 20 min to obtain the supernatant, and an enzymatic hydrolysate was obtained.

[0052] 5) The enzymatic hydrolysate was subjected to ultrafiltration using an ultrafiltration membrane with a molecular weight of 1000 Da, and was dried (pre-frozen at -40°C for 4 h, vacuum freeze-dried at 0.1 mbar and -25°C for 24 h), and an eggshell membrane peptide was obtained.

[0053] Comparative Example 1

[0054] 1) The eggshell membrane was vacuum freeze-dried at a vacuum degree of 0.133 mbar and a temperature of -40°C for 24 h, was crushed, and was passed through a 200-mesh sieve to obtain eggshell membrane powder.

[0055] 2) Eggshell membrane powder is mixed with deionized water to prepare an eggshell membrane solution with a mass concentration of 5%, and keratinase is added to the eggshell membrane solution (the amount of keratinase added is calculated according to 8% of the mass of the eggshell membrane powder), and the first enzymolysis is performed at pH 11 and 55°C for 4h, the first enzyme inactivation is performed at 88°C for 12min, the first centrifugation is performed at 10000rpm for 18min, and the supernatant is taken.

[0056] 3) Collagenase is added to the supernatant (the amount of collagenase added is calculated according to 1.5% of the mass of the eggshell membrane powder), and the second enzymolysis is performed at pH 7 and 40°C for 3h, the second enzyme inactivation is performed at 95°C for 5min, the second centrifugation is performed at 10000rpm for 18min, and the supernatant is taken to obtain a preliminary enzymolysis solution.

[0057] 4) Alkaline phosphatase is added to the preliminary enzymolysis solution (the amount of alkaline phosphatase added is calculated according to 0.8% of the mass of the eggshell membrane powder), and the third enzymolysis is performed at pH 9 and 37°C for 1.5h, the third enzyme inactivation is performed at 95°C for 5min, the third centrifugation is performed at 10000rpm for 18min, and the supernatant is taken to obtain an enzymolysis solution.

[0058] 5) The enzymolysis solution is subjected to ultrafiltration using an ultrafiltration membrane with a molecular weight of 1000Da, and is dried (pre-frozen at -40°C for 4h, vacuum freeze-dried at 0.1mbar and -25°C for 24h) to obtain eggshell membrane peptides.

[0059] Comparative Example 2

[0060] 1) Eggshell membrane is vacuum freeze-dried at a vacuum degree of 0.133mbar and a temperature of -40°C for 24h, is crushed, and is passed through a 200-mesh sieve to obtain eggshell membrane powder.

[0061] 2) Eggshell membrane powder is mixed with deionized water to prepare an eggshell membrane solution with a mass concentration of 5%, and keratinase is added to the eggshell membrane solution (the amount of keratinase added is calculated according to 2.5% of the mass of the eggshell membrane powder), and the first enzymolysis is performed at pH 11 and 55°C for 2h, the first enzyme inactivation is performed at 88°C for 12min, the first centrifugation is performed at 10000rpm for 18min, and the supernatant is taken.

[0062] 3) Collagenase is added to the supernatant (the amount of collagenase added is calculated according to 5% of the mass of the eggshell membrane powder), and the second enzymolysis is performed at pH 7 and 40°C for 6h, the second enzyme inactivation is performed at 95°C for 5min, the second centrifugation is performed at 10000rpm for 18min, and the supernatant is taken to obtain a preliminary enzymolysis solution.

[0063] 4) The primary enzymatic hydrolysate was added with alkaline phosphatase (the amount of alkaline phosphatase was calculated as 0.8% of the mass of eggshell membrane powder), and third enzymatic hydrolysis was carried out at pH 9 and 37°C for 1.5h, third enzyme inactivation was carried out at 95°C for 5min, and the supernatant was obtained by third centrifugation at 10000rpm for 18min, to obtain the enzymatic hydrolysate.

[0064] 5) The enzymatic hydrolysate was subjected to ultrafiltration using an ultrafiltration membrane with a molecular weight of 1000Da, and dried (vacuum freeze-drying at 0.1mbar and -25°C for 24h after pre-freezing at -40°C for 4h), to obtain eggshell membrane peptides.

[0065] Comparative Example 3

[0066] 1) The eggshell membrane was vacuum freeze-dried at 0.133mbar and -40°C for 24h, crushed, and sieved through a 200-mesh sieve, to obtain eggshell membrane powder.

[0067] 2) The eggshell membrane powder was mixed with deionized water to prepare an eggshell membrane solution with a mass concentration of 5%, and keratinase was added to the eggshell membrane solution (the amount of keratinase was calculated as 2.5% of the mass of eggshell membrane powder), and first enzymatic hydrolysis was carried out at pH 11 and 55°C for 2h, first enzyme inactivation was carried out at 88°C for 12min, and the supernatant was obtained by first centrifugation at 10000rpm for 18min.

[0068] 3) Alkaline protease was added to the supernatant (the amount of alkaline protease was calculated as 1.5% of the mass of eggshell membrane powder), and second enzymatic hydrolysis was carried out at pH 8.0 and 55°C for 3h, second enzyme inactivation was carried out at 95°C for 5min, and the supernatant was obtained by second centrifugation at 10000rpm for 18min, to obtain a primary enzymatic hydrolysate.

[0069] 4) The primary enzymatic hydrolysate was added with alkaline phosphatase (the amount of alkaline phosphatase was calculated as 0.8% of the mass of eggshell membrane powder), and third enzymatic hydrolysis was carried out at pH 9 and 37°C for 1.5h, third enzyme inactivation was carried out at 95°C for 5min, and the supernatant was obtained by third centrifugation at 10000rpm for 18min, to obtain the enzymatic hydrolysate.

[0070] 5) The enzymatic hydrolysate was subjected to ultrafiltration using an ultrafiltration membrane with a molecular weight of 1000Da, and dried (vacuum freeze-drying at 0.1mbar and -25°C for 24h after pre-freezing at -40°C for 4h), to obtain eggshell membrane peptides.

[0071] Experimental Example 1

[0072] 60 SPF level, 21 days old, body weight of 14 ± 2 g female C57 / BL6 mice, free access to calcium-containing feed and water, adaptive feeding for one week, calcium-containing feed is commercially available feed added with 5 g CaCO3 per kilogram of feed. After one week of adaptive feeding, the mice were divided into 6 groups, 10 in each group, divided into control group, model group, example 1 group, comparative example 1 group, comparative example 2 group and comparative example 3 group. The experiment lasted for 8 weeks, at the end of the 8th week, the eyeball blood of each group of mice was taken; centrifuged at 4℃ 3500r / min for 10min, the supernatant was taken for serum related index determination; the mice were sacrificed and the femur was soaked in polyformaldehyde and stored in a-80℃ refrigerator.

[0073] The control group: 1-4 weeks of feeding calcium-containing feed; 5-8 weeks of feeding calcium-containing feed, while 1 mL / 100 g (calculated by weight) of normal saline was given by gavage every day. The model group: 1-4 weeks of feeding commercially available feed; 5-8 weeks of feeding commercially available feed, while 1 mL / 100 g (calculated by weight) of normal saline was given by gavage every day. Example 1 group: 1-4 weeks of feeding commercially available feed; 5-8 weeks of feeding calcium-containing feed, while the aqueous solution of eggshell membrane peptide prepared in example 1 was given by gavage every day, and the feeding amount of eggshell membrane peptide was calculated as 100 mg / kg (body weight). Comparative example 1 group: 1-4 weeks of feeding commercially available feed; 5-8 weeks of feeding calcium-containing feed, while the aqueous solution of eggshell membrane peptide prepared in comparative example 1 was given by gavage every day, and the feeding amount of eggshell membrane peptide was calculated as 100 mg / kg (body weight). Comparative example 2 group: 1-4 weeks of feeding commercially available feed; 5-8 weeks of feeding calcium-containing feed, while the aqueous solution of eggshell membrane peptide prepared in comparative example 2 was given by gavage every day, and the feeding amount of eggshell membrane peptide was calculated as 100 mg / kg (body weight). Comparative example 3 group: 1-4 weeks of feeding commercially available feed; 5-8 weeks of feeding calcium-containing feed, while the aqueous solution of eggshell membrane peptide prepared in comparative example 3 was given by gavage every day, and the feeding amount of eggshell membrane peptide was calculated as 100 mg / kg (body weight).

[0074] The alkaline phosphatase kit (Nanjing Jiancheng Biological Co., Ltd.) was used to determine the alkaline phosphatase (ALP) activity. ALP can decompose phenylphosphonate disodium to produce free phenol and phosphoric acid. Phenol reacts with 4-aminoantipyrine in an alkaline solution and generates a red quinone derivative under the oxidation of potassium ferricyanide. The absorbance was measured at 520 nm. The absorbance was measured by an enzyme marker.

[0075] The bone protection factor ELISA kit (Wuhan Enzyme-free Biotechnology Co., Ltd.) was used to detect the content of serum bone protection factor (OPG), the receptor activator of nuclear factor-κβ ligand kit (Wuhan Enzyme-free Biotechnology Co., Ltd.) was used to detect the content of receptor activator of nuclear factor-κβ ligand (RANKL), and the anti-tartaric acid acid phosphatase ELISA kit (Wuhan Enzyme-free Biotechnology Co., Ltd.) was used to detect the activity of anti-tartaric acid acid phosphatase (TRACP). The method steps were carried out according to the kit instructions, and the absorbance was measured at 450 nm.

[0076] Micro-CT was used to analyze the bone mineral density (BMD) of the femur, and the scanning system was Skyscan-1276-CT system with a voxel size of 6 μm and an aluminum filter of 0.25 mm.

[0077] As shown in Figure 1 , compared with the control group, the activity of ALP in the model group was significantly increased (P<0.05), indicating that the model group of mice had the phenomenon of insufficient bone calcium deposition. After feeding the eggshell membrane peptides prepared in Example 1, the eggshell membrane peptides prepared in Comparative Example 1, the eggshell membrane peptides prepared in Comparative Example 2 or the eggshell membrane peptides prepared in Comparative Example 3, the activity of ALP was lower than that of the model group. The activity of ALP in Example 1 group was significantly lower than that in the model group (P<0.05), indicating that the intake of calcium supplements could alleviate the calcium deficiency of the body.

[0078] As shown in Figure 2 , TRACP is synthesized and released by osteoclasts after differentiation, and its serum content can indicate the activity of osteoclasts. The serum TRACP content of the model group was significantly higher than that of the control group (P<0.05), indicating that the activity of osteoclasts in the model group was increased. After feeding the eggshell membrane peptides prepared in Example 1, the eggshell membrane peptides prepared in Comparative Example 1, the eggshell membrane peptides prepared in Comparative Example 2 or the eggshell membrane peptides prepared in Comparative Example 3, the content of TRACP was reduced, among which the eggshell membrane peptides prepared in Example 1 could significantly reduce the content of TRACP, indicating that the eggshell membrane peptides prepared in Example 1 could significantly alleviate the calcium deficiency of the body (P<0.05).

[0079] As shown in Figure 3 A and Figure 3 B, compared with the control group, the OPG content of the model group was significantly decreased, and the serum free RANKL content was significantly increased, indicating that the probability of activation of osteoclasts in the model group was higher. The OPG content of the mice in Example 1 group was significantly increased, and the RANKL content was significantly reduced (P<0.05), which also indicated that the OPG / RANKL ratio was further increased, and the differentiation process of osteoclasts was further inhibited, indicating that the eggshell membrane peptides prepared in Example 1 had the effect of promoting calcium supplementation and could reduce the function of osteoclasts and alleviate bone loss.

[0080] like Figure 4 As shown, bone mineral density (BMD) of the femur represents the change in mass per unit area of ​​bone at a selected site. The BMD of the model group mice was significantly lower than that of the control group, indicating that bone loss occurred in the model group mice. After feeding the eggshell membrane peptide prepared in Example 1, the eggshell membrane peptide prepared in Comparative Example 1, the eggshell membrane peptide prepared in Comparative Example 2, or the eggshell membrane peptide prepared in Comparative Example 3, the BMD of the mice in each group increased. In particular, the mice fed the eggshell membrane peptide prepared in Example 1 showed a significant increase in bone mineral density, proving that the eggshell membrane peptide prepared in this invention can significantly improve bone microstructure.

[0081] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing eggshell membrane peptides for enhancing bone density, characterized in that, Includes the following steps: 1) The eggshell membrane is vacuum freeze-dried and pulverized to obtain eggshell membrane powder; 2) The eggshell membrane powder obtained in step 1) is mixed with deionized water to obtain an eggshell membrane solution; keratinase is added to the eggshell membrane solution, followed by a first enzymatic hydrolysis, a first enzyme inactivation, and a first centrifugation to collect the supernatant. 3) Add collagenase to the supernatant obtained in step 2), perform a second enzymatic hydrolysis, a second enzyme inactivation, and a second centrifugation to collect the supernatant to obtain the preliminary enzymatic hydrolysate; 4) Add alkaline phosphatase to the preliminary enzymatic hydrolysate obtained in step 3), perform a third enzymatic hydrolysis, a third enzyme inactivation, and a third centrifugation to collect the supernatant to obtain the enzymatic hydrolysate; 5) The enzymatic hydrolysate obtained in step 4) is ultrafiltered and dried to obtain eggshell membrane peptides.

2. The preparation method according to claim 1, characterized in that, The vacuum degree of the vacuum freeze-drying in step 1) is 0.100 to 0.150 mbar, the temperature of the vacuum freeze-drying is -30 to -50℃, and the time of the vacuum freeze-drying is 22 to 26 hours; the particle size of the eggshell membrane powder in step 1) is 100 to 200 mesh.

3. The preparation method according to claim 1, characterized in that, The mass concentration of the eggshell membrane solution in step 2) is 2-8%; the amount of keratinase added in step 2) is calculated as 2-3% of the mass of the eggshell membrane powder.

4. The preparation method according to claim 1, characterized in that, In step 2), the pH of the first enzymatic hydrolysis is 10.5–11.5, the temperature of the first enzymatic hydrolysis is 50–60°C, and the time of the first enzymatic hydrolysis is 1–3 h; in step 2), the temperature of the first enzyme inactivation is 85–90°C, and the time of the first enzyme inactivation is 10–15 min; in step 2), the speed of the first centrifugation is 8000–12000 rpm, and the time of the first centrifugation is 15–20 min.

5. The preparation method according to claim 1, characterized in that, The amount of collagenase added in step 3) is calculated as 1-2% of the mass of eggshell membrane powder.

6. The preparation method according to claim 1, characterized in that, In step 3), the pH of the second enzymatic hydrolysis is 6.5–7.5, the temperature of the second enzymatic hydrolysis is 38–42°C, and the time of the second enzymatic hydrolysis is 2–4 h; in step 3), the temperature of the second enzyme inactivation is 95°C, and the time of the second enzyme inactivation is 5 min; in step 3), the speed of the second centrifugation is 8000–12000 rpm, and the time of the second centrifugation is 15–20 min.

7. The preparation method according to claim 1, characterized in that, The amount of alkaline phosphatase added in step 4) is calculated as 0.5-1.0% of the mass of eggshell membrane powder; the pH of the third enzymatic hydrolysis in step 4) is 8.5-9.5, the temperature of the third enzymatic hydrolysis is 35-39℃, and the time of the third enzymatic hydrolysis is 1-2 hours; the temperature of the third enzyme inactivation in step 4) is 95℃, and the time of the third enzyme inactivation is 5 minutes; the speed of the third centrifugation in step 4) is 8000-12000 rpm, and the time of the third centrifugation is 15-20 minutes.

8. The preparation method according to claim 1, characterized in that, The ultrafiltration described in step 5) utilizes an ultrafiltration membrane with a molecular weight of 1000 Da; the drying described in step 5) specifically involves: pre-freezing at -40℃ for 4 hours, followed by vacuum freeze-drying at 0.1 mbar and a temperature of -25℃ for 24 hours.

9. Eggshell membrane peptides prepared by the preparation method according to any one of claims 1 to 8.

10. The use of the eggshell membrane peptide prepared by the preparation method according to any one of claims 1 to 8 in the preparation of products for enhancing bone density.

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