An eggshell membrane peptide for enhancing bone density and a preparation method and application thereof

Abstract

The application discloses an eggshell membrane peptide for enhancing bone density and a preparation method and application thereof, belongs to the technical field of egg by-product processing, and particularly relates to a preparation method of an eggshell membrane peptide for enhancing bone density, which comprises the following steps: vacuum freeze-drying eggshell membranes, crushing the eggshell membranes 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 supernatant; adding collagenase, performing enzymolysis, centrifuging to take supernatant, and obtaining a primary enzymolysis liquid; adding alkaline phosphatase, performing enzymolysis, centrifuging to take supernatant, and obtaining an enzymolysis liquid; performing ultrafiltration, and drying to obtain the eggshell membrane peptide. The application utilizes keratinase to break the cross-linking of keratin by enzymolysis, utilizes collagenase to release active peptides capable of stimulating the proliferation of osteoblasts by enzymolysis, further utilizes alkaline phosphatase to activate phosphopeptide calcium binding sites by enzymolysis, promotes the deposition of calcium salt, and provides the eggshell membrane peptide for enhancing bone density.

Description

Technical Field

[0001] This invention belongs to the field of egg by-product processing technology, and particularly relates to an eggshell membrane peptide for enhancing bone density, its preparation method and application. Background Technology

[0002] With the increasing aging of the global population, osteoporosis has become a major public health problem threatening the health of middle-aged and elderly people. Osteoporosis patients worldwide place a heavy burden on healthcare systems and socioeconomic systems. Currently used anti-osteoporosis drugs, such as bisphosphonates, estrogen, and RANKL inhibitors, while having some efficacy, may lead to problems such as mandibular osteonecrosis, cardiovascular risks, and gastrointestinal side effects with long-term use. Therefore, there is an urgent need to develop safe, effective, and naturally derived alternative therapies.

[0003] Eggshell membrane (ESM) is a significant component of poultry egg processing waste, with global waste amounting to tens of millions of tons annually. Studies have shown that ESM is rich in collagen (types I, V, and X), chondroitin sulfate, hyaluronic acid, and various functional peptides, exhibiting significant anti-inflammatory, antioxidant, and tissue repair-promoting activities. In recent years, the application of ESM extracts in joint health has been reported, but its potential in bone mineral density regulation has not yet been fully explored.

[0004] In existing technologies, the preparation of eggshell membrane peptides mostly adopts conventional enzymatic hydrolysis methods, such as using trypsin or papain. These methods have several limitations, such as insufficient targeting of active peptides, a wide molecular weight distribution of traditional enzymatic hydrolysis products (1-50 Da), and low content of specific peptides with osteogenic activity; low bioavailability, as large molecular weight peptides are difficult to be effectively absorbed by the intestines, affecting in vivo activity expression; and low process efficiency, with peptide yield usually below 15% under single enzymatic hydrolysis conditions, and active ingredients are easily inactivated during high-temperature sterilization.

[0005] Therefore, developing a high-purity, low-molecular-weight active peptide based on eggshell membrane and improving its ability to promote bone differentiation and calcium deposition through optimized preparation process is of great significance for achieving green prevention and control of osteoporosis and high-value utilization of eggshell waste. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention proposes an eggshell membrane peptide for enhancing bone density, its preparation method, and its application. This invention utilizes keratinase to enzymatically break down keratin crosslinks, and collagenase to enzymatically release active peptides that can stimulate osteoblast proliferation. Furthermore, alkaline phosphatase is used to enzymatically activate phosphopeptide calcium binding sites, promoting calcium salt deposition, thus providing an eggshell membrane peptide for enhancing bone density.

[0007] To achieve the above objectives, the present invention provides a method for preparing eggshell membrane peptides for enhancing bone density, comprising the following steps:

[0008] 1) The eggshell membrane is vacuum freeze-dried and pulverized to obtain eggshell membrane powder;

[0009] 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.

[0010] 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;

[0011] 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;

[0012] 5) The enzymatic hydrolysate obtained in step 4) is ultrafiltered and dried to obtain eggshell membrane peptides.

[0013] Preferably, 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°C, 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.

[0014] Preferably, 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.

[0015] Preferably, 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.

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

[0017] Preferably, 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.

[0018] Preferably, 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.

[0019] Preferably, the ultrafiltration in step 5) utilizes an ultrafiltration membrane with a molecular weight of 1000 Da; the drying in step 5) specifically involves: pre-freezing at -40°C for 4 hours, followed by vacuum freeze-drying at 0.1 mbar and a temperature of -25°C for 24 hours.

[0020] The present invention also provides eggshell membrane peptides prepared by the aforementioned preparation method.

[0021] The present invention also provides the application of the eggshell membrane peptides prepared by the above preparation method in the preparation of products for enhancing bone density.

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

[0023] This invention provides an eggshell membrane peptide for enhancing bone density, its preparation method, and its applications. The eggshell membrane peptide prepared by this invention releases the type I collagen peptides and phosphoprotein peptides originally present in the eggshell membrane, while simultaneously enhancing the active peptides that increase bone density, stimulating osteoblast proliferation, inhibiting osteoclasts, and promoting calcium salt deposition. This invention optimizes the enzymatic hydrolysis conditions of keratinase to open the dense keratin network in eggshell membrane powder, utilizes collagenase to optimize its hydrolysis conditions, and leverages its specific cleavage of the Pro-X-Gly-Pro sites of collagen to efficiently release Gly-Pro-Hyp osteogenic peptides. Then, alkaline phosphatase phosphorylation is used for activation, enhancing the bone density-enhancing function of the prepared eggshell membrane peptides. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 The results of serum alkaline phosphatase levels in mice of each group are shown.

[0026] Figure 2 The results of serum TRACP content measurement in mice of each group;

[0027] Figure 3 The results of serum OPG and RANKL content measurements in mice of each group are shown. In this figure, A represents serum OPG content and B represents serum RANKL content.

[0028] Figure 4 Results of femoral bone mineral density measurement in each group of mice. Detailed Implementation

[0029] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0030] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0031] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0032] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0033] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0034] The eggshell membrane used in this invention was purchased from an egg processing plant; the keratinase used was keratinase derived from Bacillus licheniformis (enzyme activity ≥15000U / g) purchased from Novozymes; the collagenase (Collagenase NB4) used was purchased from Novozymes; the alkaline phosphatase used was purchased from DSM; and the alkaline protease used was purchased from Novozymes.

[0035] Example 1

[0036] 1) Eggshell membrane was freeze-dried under vacuum conditions of 0.133 mbar and -40℃ for 24 hours, then pulverized and passed through a 200-mesh sieve to obtain eggshell membrane powder.

[0037] 2) Eggshell membrane powder is mixed with deionized water to prepare an eggshell membrane solution with a mass concentration of 5%. Keratinase is added to the eggshell membrane solution (the amount of keratinase added is calculated as 2.5% of the mass of eggshell membrane powder). The first enzymatic hydrolysis is carried out at pH 11 and temperature of 55℃ for 2 hours, followed by the first enzyme inactivation at 88℃ for 12 minutes, and the first centrifugation at 10000rpm for 18 minutes. The supernatant is then collected.

[0038] 3) Add collagenase to the supernatant (the amount of collagenase added is calculated as 1.5% of the mass of eggshell membrane powder), and perform a second enzymatic hydrolysis for 3 hours at pH 7 and temperature of 40℃, followed by a second enzyme inactivation at 95℃ for 5 minutes, and a second centrifugation at 10000rpm for 18 minutes to obtain the supernatant, thus obtaining the preliminary enzymatic hydrolysate.

[0039] 4) Add alkaline phosphatase to the initial enzymatic hydrolysate (the amount of alkaline phosphatase added is calculated as 0.8% of the mass of eggshell membrane powder). Under the conditions of pH 9 and temperature 37℃, perform the third enzymatic hydrolysis for 1.5h, the third enzyme inactivation at 95℃ for 5min, and the third centrifugation at 10000rpm for 18min to collect the supernatant and obtain the enzymatic hydrolysate.

[0040] 5) The enzymatic hydrolysate was ultrafiltered using an ultrafiltration membrane with a molecular weight of 1000 Da and dried (pre-frozen at -40℃ for 4 hours, then freeze-dried under vacuum at 0.1 mbar and -25℃ for 24 hours) to obtain eggshell membrane peptides.

[0041] Example 2

[0042] 1) Eggshell membrane was freeze-dried under vacuum conditions of 0.1 mbar and -30℃ for 22 h, pulverized, and passed through a 100-mesh sieve to obtain eggshell membrane powder.

[0043] 2) Eggshell membrane powder is mixed with deionized water to prepare an eggshell membrane solution with a mass concentration of 2%. Keratinase is added to the eggshell membrane solution (the amount of keratinase added is calculated as 2% of the mass of eggshell membrane powder). The first enzymatic hydrolysis is carried out at pH 10.5 and temperature 50℃ for 1 hour, followed by the first enzyme inactivation at 85℃ for 10 minutes, and the first centrifugation at 8000rpm for 15 minutes. The supernatant is then collected.

[0044] 3) Add collagenase to the supernatant (the amount of collagenase added is calculated as 1% of the mass of eggshell membrane powder), and perform a second enzymatic hydrolysis for 2 hours at pH 6.5 and temperature 38℃, followed by a second enzyme inactivation at 95℃ for 5 minutes, and a second centrifugation at 8000rpm for 15 minutes to obtain the supernatant, thus obtaining the preliminary enzymatic hydrolysate.

[0045] 4) Add alkaline phosphatase to the initial enzymatic hydrolysate (the amount of alkaline phosphatase added is calculated as 0.5% of the mass of eggshell membrane powder). Under the conditions of pH 8.5 and temperature 35℃, perform the third enzymatic hydrolysis for 1 hour, the third enzyme inactivation at 95℃ for 5 minutes, and the third centrifugation at 8000 rpm for 15 minutes to collect the supernatant and obtain the enzymatic hydrolysate.

[0046] 5) The enzymatic hydrolysate was ultrafiltered using an ultrafiltration membrane with a molecular weight of 1000 Da and dried (pre-frozen at -40℃ for 4 hours, then freeze-dried under vacuum at 0.1 mbar and -25℃ for 24 hours) to obtain eggshell membrane peptides.

[0047] Example 3

[0048] 1) Eggshell membrane was freeze-dried under vacuum conditions of 0.15 mbar and -50℃ for 26 h, pulverized, and passed through a 200-mesh sieve to obtain eggshell membrane powder.

[0049] 2) Eggshell membrane powder is mixed with deionized water to prepare an eggshell membrane solution with a mass concentration of 8%. Keratinase is added to the eggshell membrane solution (the amount of keratinase added is calculated as 3% of the mass of eggshell membrane powder). The solution is subjected to first enzymatic hydrolysis for 3 hours at pH 11.5 and temperature of 60℃, first enzyme inactivation at 90℃ for 15 minutes, and first centrifugation at 12000rpm for 20 minutes. The supernatant is then collected.

[0050] 3) Add collagenase to the supernatant (the amount of collagenase added is calculated as 2% of the mass of eggshell membrane powder), and perform a second enzymatic hydrolysis for 4 hours at pH 7.5 and temperature 42℃, followed by a second enzyme inactivation at 95℃ for 5 minutes, and a second centrifugation at 12000rpm for 20 minutes to collect the supernatant and obtain the preliminary enzymatic hydrolysate.

[0051] 4) Add alkaline phosphatase to the initial enzymatic hydrolysate (the amount of alkaline phosphatase added is calculated as 1% of the mass of eggshell membrane powder). Under the conditions of pH 9.5 and temperature 39℃, perform the third enzymatic hydrolysis for 2 hours, the third enzyme inactivation at 95℃ for 5 minutes, and the third centrifugation at 12000rpm for 20 minutes to collect the supernatant and obtain the enzymatic hydrolysate.

[0052] 5) The enzymatic hydrolysate was ultrafiltered using an ultrafiltration membrane with a molecular weight of 1000 Da and dried (pre-frozen at -40℃ for 4 hours, then freeze-dried under vacuum at 0.1 mbar and -25℃ for 24 hours) to obtain eggshell membrane peptides.

[0053] Comparative Example 1

[0054] 1) Eggshell membrane was freeze-dried under vacuum conditions of 0.133 mbar and -40℃ for 24 hours, then pulverized and 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%. Keratinase is added to the eggshell membrane solution (the amount of keratinase added is calculated according to 8% of the mass of eggshell membrane powder). The first enzymatic hydrolysis is carried out at pH 11 and temperature of 55℃ for 4 hours, followed by the first enzyme inactivation at 88℃ for 12 minutes, and the first centrifugation at 10000rpm for 18 minutes. The supernatant is then collected.

[0056] 3) Add collagenase to the supernatant (the amount of collagenase added is calculated as 1.5% of the mass of eggshell membrane powder), and perform a second enzymatic hydrolysis for 3 hours at pH 7 and temperature of 40℃, followed by a second enzyme inactivation at 95℃ for 5 minutes, and a second centrifugation at 10000rpm for 18 minutes to obtain the supernatant, thus obtaining the preliminary enzymatic hydrolysate.

[0057] 4) Add alkaline phosphatase to the initial enzymatic hydrolysate (the amount of alkaline phosphatase added is calculated as 0.8% of the mass of eggshell membrane powder). Under the conditions of pH 9 and temperature 37℃, perform the third enzymatic hydrolysis for 1.5h, the third enzyme inactivation at 95℃ for 5min, and the third centrifugation at 10000rpm for 18min to collect the supernatant and obtain the enzymatic hydrolysate.

[0058] 5) The enzymatic hydrolysate was ultrafiltered using an ultrafiltration membrane with a molecular weight of 1000 Da and dried (pre-frozen at -40℃ for 4 hours, then freeze-dried under vacuum at 0.1 mbar and -25℃ for 24 hours) to obtain eggshell membrane peptides.

[0059] Comparative Example 2

[0060] 1) Eggshell membrane was freeze-dried under vacuum conditions of 0.133 mbar and -40℃ for 24 hours, then pulverized and 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%. Keratinase is added to the eggshell membrane solution (the amount of keratinase added is calculated as 2.5% of the mass of eggshell membrane powder). The first enzymatic hydrolysis is carried out at pH 11 and temperature of 55℃ for 2 hours, followed by the first enzyme inactivation at 88℃ for 12 minutes, and the first centrifugation at 10000rpm for 18 minutes. The supernatant is then collected.

[0062] 3) Add collagenase to the supernatant (the amount of collagenase added is calculated as 5% of the mass of eggshell membrane powder), and perform a second enzymatic hydrolysis for 6 hours at pH 7 and temperature of 40℃, followed by a second enzyme inactivation at 95℃ for 5 minutes, and a second centrifugation at 10000rpm for 18 minutes to obtain the supernatant, thus obtaining the preliminary enzymatic hydrolysate.

[0063] 4) Add alkaline phosphatase to the initial enzymatic hydrolysate (the amount of alkaline phosphatase added is calculated as 0.8% of the mass of eggshell membrane powder). Under the conditions of pH 9 and temperature 37℃, perform the third enzymatic hydrolysis for 1.5h, the third enzyme inactivation at 95℃ for 5min, and the third centrifugation at 10000rpm for 18min to collect the supernatant and obtain the enzymatic hydrolysate.

[0064] 5) The enzymatic hydrolysate was ultrafiltered using an ultrafiltration membrane with a molecular weight of 1000 Da and dried (pre-frozen at -40℃ for 4 hours, then freeze-dried under vacuum at 0.1 mbar and -25℃ for 24 hours) to obtain eggshell membrane peptides.

[0065] Comparative Example 3

[0066] 1) Eggshell membrane was freeze-dried under vacuum conditions of 0.133 mbar and -40℃ for 24 hours, then pulverized and passed through a 200-mesh sieve to obtain eggshell membrane powder.

[0067] 2) Eggshell membrane powder is mixed with deionized water to prepare an eggshell membrane solution with a mass concentration of 5%. Keratinase is added to the eggshell membrane solution (the amount of keratinase added is calculated as 2.5% of the mass of eggshell membrane powder). The first enzymatic hydrolysis is carried out at pH 11 and temperature of 55℃ for 2 hours, followed by the first enzyme inactivation at 88℃ for 12 minutes, and the first centrifugation at 10000rpm for 18 minutes. The supernatant is then collected.

[0068] 3) Add alkaline protease to the supernatant (the amount of alkaline protease added is calculated as 1.5% of the mass of eggshell membrane powder). Under the conditions of pH 8.0 and temperature 55℃, perform the second enzymatic hydrolysis for 3 hours, the second enzyme inactivation at 95℃ for 5 minutes, and the second centrifugation at 10000rpm for 18 minutes to collect the supernatant and obtain the preliminary enzymatic hydrolysate.

[0069] 4) Add alkaline phosphatase to the initial enzymatic hydrolysate (the amount of alkaline phosphatase added is calculated as 0.8% of the mass of eggshell membrane powder). Under the conditions of pH 9 and temperature 37℃, perform the third enzymatic hydrolysis for 1.5h, the third enzyme inactivation at 95℃ for 5min, and the third centrifugation at 10000rpm for 18min to collect the supernatant and obtain the enzymatic hydrolysate.

[0070] 5) The enzymatic hydrolysate was ultrafiltered using an ultrafiltration membrane with a molecular weight of 1000 Da and dried (pre-frozen at -40℃ for 4 hours, then freeze-dried under vacuum at 0.1 mbar and -25℃ for 24 hours) to obtain eggshell membrane peptides.

[0071] Experimental Example 1

[0072] Sixty SPF-grade, 21-day-old female C57 / BL6 mice, weighing 14±2g, were given free access to calcium-containing feed and water for one week of acclimatization. The calcium-containing feed consisted of commercially available feed supplemented with 5g CaCO3 per kilogram of feed. After one week of acclimatization, the mice were divided into 6 groups of 10 mice each: 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, blood samples were collected from the eyeballs of mice in each group; the samples were centrifuged at 3500 rpm for 10 min at 4℃, and the supernatant was used for serum-related index determination; the mice were sacrificed, and the femurs were collected, soaked in paraformaldehyde, and stored at -80℃.

[0073] Control group: fed calcium-containing feed for 1–4 weeks; fed calcium-containing feed for 5–8 weeks, and administered 1 mL / 100g of physiological saline daily by gavage (calculated based on body weight). Model group: fed commercially available feed for 1–4 weeks; fed commercially available feed for 5–8 weeks, and administered 1 mL / 100g of physiological saline daily by gavage (calculated based on body weight). Example 1 group: fed commercially available feed for 1–4 weeks; fed calcium-containing feed for 5–8 weeks, and administered an aqueous solution of eggshell membrane peptides prepared in Example 1 daily by gavage, with the dosage of eggshell membrane peptides calculated at 100 mg / kg (body weight). Comparative example 1 group: fed commercially available feed for 1–4 weeks; fed calcium-containing feed for 5–8 weeks, and administered an aqueous solution of eggshell membrane peptides prepared in Comparative example 1 daily by gavage, with the dosage of eggshell membrane peptides calculated at 100 mg / kg (body weight). Comparative Example 2: Feeded with commercially available feed for weeks 1-4; fed with calcium-containing feed for weeks 5-8, while simultaneously administering an aqueous solution of eggshell membrane peptides prepared in Comparative Example 2 via gavage daily. The dosage of eggshell membrane peptides was calculated at 100 mg / kg (body weight). Comparative Example 3: Feeded with commercially available feed for weeks 1-4; fed with calcium-containing feed for weeks 5-8, while simultaneously administering an aqueous solution of eggshell membrane peptides prepared in Comparative Example 3 via gavage daily. The dosage of eggshell membrane peptides was calculated at 100 mg / kg (body weight).

[0074] Alkaline phosphatase (ALP) activity was determined using an alkaline phosphatase kit (Nanjing Jiancheng Biotechnology Co., Ltd.). ALP decomposes disodium phenyl phosphate, producing free phenol and phosphate. The phenol reacts with 4-aminoantipyridine in alkaline solution and, upon oxidation by potassium ferricyanide, generates a red quinone derivative, the absorbance of which was measured at 520 nm. All absorbance measurements were performed using a microplate reader.

[0075] The corresponding enzyme-linked immunosorbent assay (ELISA) kits were used for detection. The osteoprotegerin ELISA kit (Wuhan Wumei Biotechnology Co., Ltd.) was used to detect serum osteoprotegerin (OPG) levels, the nuclear factor-κB ligand receptor activator (RANKL) kit (Wuhan Wumei Biotechnology Co., Ltd.) was used to detect RANKL levels, and the tartrate-resistant acid phosphatase (TRACP) ELISA kit (Wuhan Wumei Biotechnology Co., Ltd.) was used to detect TRACP activity. The procedures were performed according to the kit instructions, and absorbance was measured at 450 nm.

[0076] Bone mineral density (BMD) of the femur was analyzed using micro-computed tomography (Micro-CT). The scanning system was a Skyscan-1276-CT system with a voxel size of 6 μm and an aluminum filter of 0.25 mm.

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

[0078] like Figure 2 As shown, TRACP is synthesized and released by osteoclasts after differentiation, and its serum content can indicate osteoclast activity. The serum TRACP content in the model group was significantly higher than that in the control group (P<0.05), indicating that osteoclast activity was increased in the model group. Feeding with the eggshell membrane peptide prepared in Example 1, Comparative Example 1, Comparative Example 2, or Comparative Example 3 all reduced the TRACP content. Among them, feeding with the eggshell membrane peptide prepared in Example 1 significantly reduced the TRACP content, indicating that the eggshell membrane peptide prepared in Example 1 can significantly alleviate calcium deficiency in the body (P<0.05).

[0079] like Figure 3 China A and Figure 3 As shown in Figure B, compared with the control group, the OPG content in the model group was significantly decreased, and the serum free RANKL content was significantly increased, indicating that the osteoclasts in the model group mice were more likely to be activated. In the Example 1 group, the OPG content was significantly increased, and the RANKL content was significantly decreased (P<0.05), indicating that the OPG / RANKL ratio further increased and the osteoclast differentiation process was further inhibited. This suggests that the eggshell membrane peptide prepared in Example 1 has a calcium-promoting effect and can reduce osteoclast function, thus alleviating 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 freeze-dried under vacuum and then 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; The amount of keratinase added in step 2) is calculated as 2-3% of the mass of the eggshell membrane powder; 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℃, and the time of the first enzymatic hydrolysis is 1~3h; in step 2), the temperature of the first enzyme inactivation is 85~90℃, and the time of the first enzyme inactivation is 10~15min; in step 2), the speed of the first centrifugation is 8000~12000rpm, and the time of the first centrifugation is 15~20min. The amount of collagenase added in step 3) is calculated as 1-2% of the mass of eggshell membrane powder; 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℃, and the time of the second enzymatic hydrolysis is 2-4h; in step 3), the temperature of the second enzyme inactivation is 95℃, and the time of the second enzyme inactivation is 5min; in step 3), the speed of the second centrifugation is 8000-12000rpm, and the time of the second centrifugation is 15-20min. 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 1~2h; the temperature of the third enzyme inactivation in step 4) is 95℃, and the time of the third enzyme inactivation is 5min; the speed of the third centrifugation in step 4) is 8000~12000rpm, and the time of the third centrifugation is 15~20min.

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

3. The preparation method according to claim 1, characterized in that, The mass concentration of the eggshell membrane solution mentioned in step 2) is 2-8%.

4. 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.

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