Method for promoting dissolution of eggshell membrane protein and preparing chelated calcium by high-pressure crushing

Abstract

The invention discloses a method for promoting dissolution of eggshell membrane protein and preparing chelated calcium through high-pressure crushing. Comprising the following steps: separating and drying eggshells and eggshell membranes of egg shells, and respectively carrying out superfine grinding to obtain eggshell powder and eggshell membrane powder; adding water into the eggshell membrane powder, carrying out high-pressure homogenization, and centrifuging to obtain an eggshell membrane solution; adding eggshell powder into the eggshell membrane solution, adjusting the pH value to 1-3, and adding pepsase for reaction; after the reaction, adjusting the pH value of the eggshell membrane solution to 6.5-7.5, and adding trypsin for reaction; and after the reaction, inactivating the enzyme, centrifuging to take a supernatant, adding absolute ethyl alcohol into the supernatant for alcohol precipitation, and centrifuging to obtain a precipitate, namely the chelated calcium. The technical problem that the eggshell membrane protein is difficult to dissolve out is solved, and a new way is provided for industrial extraction of the eggshell membrane soluble protein.

Description

Technical Field

[0001] The invention relates to the technical field of egg product processing, and in particular to a method for promoting the dissolution of eggshell membrane protein by high-pressure crushing and preparing chelated calcium. Background Art

[0002] Eggshell is an ideal source of calcium supplements, boasting a high calcium content and high bioavailability. It is primarily composed of calcium carbonate, averaging 93% calcium carbonate, and can contain up to 380 mg / g of calcium, a value far exceeding that of many commonly used calcium supplements. The eggshell membrane, a thin layer within the eggshell, is rich in various proteins, primarily collagen, keratin, lysozyme, and various glycoproteins. Not only is it a high-quality protein source, it also possesses numerous bioactivities, including antioxidant, anti-inflammatory, and antimicrobial properties, as well as benefits for bone health, skin health, immune regulation, and digestive health. It holds great potential for development in food, pharmaceuticals, and cosmetics, offering broad application potential. However, the eggshell membrane is composed of a multilayered fibrous network, primarily composed of fibrous proteins such as collagen and keratin. These proteins are tightly cross-linked through numerous disulfide bonds, hydrogen bonds, and hydrophobic interactions, forming a highly stable three-dimensional network that is resistant to disruption by conventional solvents in its native state.

[0003] Chemical hydrolysis and enzymatic hydrolysis are traditional methods for extracting proteins and peptides. However, chemical hydrolysis can pose challenges such as residual reagents that can affect product quality. Enzymatic hydrolysis, while widely used due to its high substrate specificity, is limited by high costs and long reaction times, making it difficult to commercialize. Summary of the Invention

[0004] The present invention aims to overcome the shortcomings of existing technologies and provides a method for promoting the dissolution of eggshell membrane protein and preparing chelated calcium by high-pressure crushing. The present invention promotes the dissolution of soluble protein by high-pressure crushing of the eggshell membrane solution, overcoming the low efficiency and difficulty in industrialization of traditional protein extraction.

[0005] To achieve the above purpose, the technical solution designed by the present invention is as follows:

[0006] The present invention provides a method for promoting the dissolution of eggshell membrane protein by high-pressure crushing and preparing chelated calcium, comprising the following steps:

[0007] (1) separating and drying the eggshell and eggshell membrane of an eggshell, and then ultrafine grinding the eggshell and eggshell membrane to obtain eggshell powder and eggshell membrane powder;

[0008] (2) adding water to the eggshell membrane powder, homogenizing under high pressure, and centrifuging to obtain an eggshell membrane solution;

[0009] (3) adding eggshell powder to the eggshell membrane solution, adjusting the pH to 1-3, and adding pepsin to react;

[0010] (4) After the reaction, the pH of the eggshell membrane solution is adjusted to 6.5-7.5, and trypsin is added to react;

[0011] (5) After the reaction, the enzyme is inactivated, and the supernatant is collected by centrifugation. Anhydrous ethanol is added to the supernatant for alcohol precipitation, and the precipitate is obtained by centrifugation. The precipitate is chelated calcium.

[0012] Furthermore, in the step (1), the ultrafine grinding time is 1 to 8 hours.

[0013] Furthermore, in step (2), the mass volume ratio of eggshell membrane powder to water is 1:20 g / mL;

[0014] The conditions of the high-pressure homogenization are: 500-1000 Bar; the number of high-pressure homogenization is 1-40 times.

[0015] Furthermore, the high-pressure homogenization condition is: 800 Bar, and the number of high-pressure homogenization times is 20 times.

[0016] Furthermore, in step (3), the mass volume ratio of eggshell powder to eggshell membrane solution is 0.1-0.6:10 g / mL;

[0017] The amount of pepsin added is 500-2000 U / mL, the reaction temperature is 25-40° C., and the reaction time is 1-3 hours.

[0018] Furthermore, the mass volume ratio of the eggshell powder and the eggshell membrane solution is 0.3:10 g / mL.

[0019] Furthermore, in step (4), the amount of trypsin added is 10 to 300 U / mL, the reaction temperature is 25 to 40° C., and the reaction time is 1 to 3 hours.

[0020] Furthermore, in the step (5), the enzyme inactivation condition is 100-110° C., and the alcohol precipitation time is 10-12 h.

[0021] The present invention also provides chelated calcium prepared by the method.

[0022] The present invention also provides a use of the chelated calcium in preparing calcium supplement products.

[0023] Principle of the present invention:

[0024] High-pressure homogenization is a common technique for breaking up, dispersing, or homogenizing samples through physical mechanical forces. It is widely used in the food, pharmaceutical, biotechnology, cosmetics, and other fields. Its core principle is to use high pressure to force the sample through a narrow valve gap or homogenizing valve, utilizing the combined effects of shear force, cavitation, turbulence, and collision to achieve particle refinement, cell disruption, or component homogenization.

[0025] Beneficial effects of the present invention:

[0026] This invention uses eggshells as raw materials, aiming to utilize the calcium and soluble protein in eggshells for digestion in the human gastrointestinal tract. High-pressure homogenization promotes the dissolution of soluble protein from the eggshell membranes. This maximizes the formation of a soluble protein-chelated calcium complex between eggshell membrane protein and eggshell calcium under the action of complex digestive enzymes in the gastrointestinal tract, thereby promoting the absorption of eggshell calcium by the human body. This opens up an innovative technological path for the industrial utilization of egg byproducts and provides a complete solution for the food industry to develop natural and healthy products that meet modern consumer needs. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Electron micrographs of eggshell membrane solutions after high-pressure homogenization for different times;

[0028] In the figure, ESM8h is an eggshell membrane solution that has not been subjected to high-pressure homogenization, ESM8h HP10 is eggshell membrane solution 2, ESM8h HP20 is eggshell membrane solution 3, ESM8h HP30 is eggshell membrane solution 4, and ESM8h HP40 is eggshell membrane solution 6;

[0029] Figure 2 This is a graph showing the amount of soluble protein released from eggshell membrane solution after high-pressure homogenization for different times;

[0030] In the figure, CK is an eggshell membrane solution that has not been subjected to high-pressure homogenization, HP10 is eggshell membrane solution 2, HP20 is eggshell membrane solution 3, and HP40 is eggshell membrane solution 6;

[0031] Figure 3 The graph shows the results of eggshell calcium chelation rate in the supernatant after high-pressure homogenization for different times;

[0032] In the figure, CK is the supernatant without high-pressure homogenization, HP10 is supernatant 2, HP20 is supernatant 3, HP30 is supernatant 4, and HP40 is supernatant 6;

[0033] Figure 4 This is the result graph of Caco-2 cell calcium uptake;

[0034] In the figure, CaCl2 is an inorganic calcium chloride solution, CK is a chelated calcium solution without high-pressure homogenization treatment, and Highpressure is a chelated calcium solution. DETAILED DESCRIPTION

[0035] The present invention is further described in detail below with reference to specific embodiments so that those skilled in the art can understand.

[0036] Example 1

[0037] A method for promoting the dissolution of eggshell membrane protein by high-pressure crushing and preparing chelated calcium comprises the following steps:

[0038] (1) Eggshells were used as raw materials, and the eggshells and eggshell membranes were separated, dried, and ultrafinely ground for 1 hour to obtain eggshell powder and eggshell membrane powder.

[0039] (2) Dissolve 30 g of eggshell membrane powder in 600 mL of deionized water, perform high-pressure homogenization five times, and centrifuge to obtain a homogeneous eggshell membrane solution 1. The high-pressure homogenization parameter is 500 bar.

[0040] (3) Take 10 mL of high-pressure homogenized eggshell membrane solution 1, add 0.1 g of eggshell powder, adjust the pH to 1, add 1000 U / mL of pepsin, and react at 25°C for 1 h.

[0041] (4) Adjust the pH of eggshell membrane solution 1 to 6.5, add 100 U / mL trypsin, and react at 25°C for 1 h.

[0042] (5) Inactivate the enzyme at high temperature (100°C), centrifuge and obtain supernatant 1, take 10 μL of supernatant 1 for total calcium determination and set aside; add anhydrous ethanol to the remaining supernatant 1, precipitate overnight (10-12 hours), centrifuge and obtain precipitate 1, which is chelated calcium 1.

[0043] Example 2

[0044] A method for promoting the dissolution of eggshell membrane protein by high-pressure crushing and preparing chelated calcium comprises the following steps:

[0045] (1) Eggshells were used as raw materials, and the eggshells and eggshell membranes were separated, dried, and ultrafinely ground for 3 hours to obtain eggshell powder and eggshell membrane powder.

[0046] (2) Dissolve 50 g of eggshell membrane powder in 1000 mL of deionized water, perform high-pressure homogenization 10 times, and centrifuge to obtain a homogeneous eggshell membrane solution 2. The high-pressure homogenization parameter is 500 bar.

[0047] (3) Take 10 mL of eggshell membrane solution 2 after high-pressure homogenization, add 0.2 g of eggshell powder, adjust the pH to 2, add 1000 U / mL of pepsin, and react at 33°C for 1 h.

[0048] (4) Adjust the pH of eggshell membrane solution 2 to 7, add 100 U / mL of trypsin, and react at 33°C for 1 h.

[0049] (5) Inactivate the enzyme at high temperature (100°C), centrifuge and obtain supernatant 2. Take 10 μL of supernatant 2 for total calcium determination and set aside. Add anhydrous ethanol to the remaining supernatant 2 and precipitate overnight (10 to 12 hours). Centrifuge and obtain precipitate 2. Precipitate 2 is chelated calcium 2.

[0050] Example 3

[0051] A method for promoting the dissolution of eggshell membrane protein by high-pressure crushing and preparing chelated calcium comprises the following steps:

[0052] (1) Eggshells were used as raw materials, and the eggshells and eggshell membranes were separated, dried, and ultrafinely ground for 5 hours to obtain eggshell powder and eggshell membrane powder.

[0053] (2) Dissolve 50 g of eggshell membrane powder in 1000 mL of deionized water, perform high-pressure homogenization 20 times, and centrifuge to obtain a homogeneous eggshell membrane solution 3. The high-pressure homogenization parameter is 800 bar.

[0054] (3) Take 10 mL of eggshell membrane solution 3 after high-pressure homogenization, add 0.3 g of eggshell powder, adjust the pH to 3, add 1000 U / mL of pepsin, and react at 37°C for 1.5 h.

[0055] (4) Adjust the pH of eggshell membrane solution 3 to 7.5, add 100 U / mL trypsin, and react at 37°C for 1.5 h.

[0056] (5) Inactivate the enzyme at high temperature (100°C), centrifuge and obtain supernatant 3, take 10 μL of supernatant 3 for total calcium determination and set aside; add anhydrous ethanol to the remaining supernatant 3, precipitate overnight (10-12 hours), centrifuge and obtain precipitate 3, which is chelated calcium 3.

[0057] Example 4

[0058] A method for promoting the dissolution of eggshell membrane protein by high-pressure crushing and preparing chelated calcium comprises the following steps:

[0059] (1) Eggshells were used as raw materials, and the eggshells and eggshell membranes were separated, dried, and ultrafinely ground for 8 hours to obtain eggshell powder and eggshell membrane powder.

[0060] (2) Dissolve 50 g of eggshell membrane powder in 1000 mL of deionized water, perform high-pressure homogenization 30 times, and centrifuge to obtain a uniform eggshell membrane solution 4. The high-pressure homogenization parameter is 600 bar.

[0061] (3) Take 10 mL of eggshell membrane solution 4 after high-pressure homogenization, add 0.4 g of eggshell powder, adjust the pH to 2, add 1000 U / mL of pepsin, and react at 40°C for 1.5 h.

[0062] (4) Adjust the pH of eggshell membrane solution 4 to 7, add 100 U / mL of trypsin, and react at 40°C for 2 h.

[0063] (5) Inactivate the enzyme at high temperature (100°C), centrifuge and obtain supernatant 4. Take 10 μL of supernatant 4 for total calcium determination and set aside. Add anhydrous ethanol to the remaining supernatant 4 and precipitate overnight (10 to 12 hours). Centrifuge and obtain precipitate 4. Precipitate 4 is chelated calcium 4.

[0064] Example 5

[0065] A method for promoting the dissolution of eggshell membrane protein by high-pressure crushing and preparing chelated calcium comprises the following steps:

[0066] (1) Eggshells were used as raw materials, and the eggshells and eggshell membranes were separated, dried, and ultrafinely ground for 8 hours to obtain eggshell powder and eggshell membrane powder.

[0067] (2) Dissolve 50 g of eggshell membrane powder in 1000 mL of deionized water, perform high-pressure homogenization 35 times, and centrifuge to obtain a uniform eggshell membrane solution 5. The high-pressure homogenization parameters are: 700 Bar.

[0068] (3) Take 10 mL of the eggshell membrane solution 5 after high-pressure homogenization, add 0.5 g of eggshell powder, adjust the pH to 2, add 1000 U / mL of pepsin, and react at 37°C for 2 h.

[0069] (4) Adjust the pH of the eggshell membrane solution 5 to 7, add 100 U / mL of trypsin, and react at 37°C for 2 h.

[0070] (5) Inactivate the enzyme at high temperature (100°C), centrifuge and obtain supernatant 5, take 10 μL of supernatant 5 for total calcium determination and set aside; add anhydrous ethanol to the remaining supernatant 5, precipitate overnight (10-12 hours), centrifuge and obtain precipitate 5, which is chelated calcium 5.

[0071] Example 6

[0072] A method for promoting the dissolution of eggshell membrane protein by high-pressure crushing and preparing chelated calcium comprises the following steps:

[0073] (1) Eggshells were used as raw materials, and the eggshells and eggshell membranes were separated, dried, and ultrafinely ground for 8 hours to obtain eggshell powder and eggshell membrane powder.

[0074] (2) Dissolve 50 g of eggshell membrane powder in 1000 mL of deionized water, perform high-pressure homogenization 40 times, and centrifuge to obtain a uniform eggshell membrane solution 6. The high-pressure homogenization parameter is 800 bar.

[0075] (3) Take 10 mL of eggshell membrane solution 6 after high-pressure homogenization, add 0.6 g of eggshell powder, adjust the pH to 3, add 1000 U / mL of pepsin, and react at 40°C for 2 h.

[0076] (4) Adjust the pH of the eggshell membrane solution 6 to 7.5, add 100 U / mL of trypsin, and react at 40°C for 2 h.

[0077] (5) Inactivate the enzyme at high temperature (100°C), centrifuge and obtain supernatant 6, take 10 μL of supernatant 6 for total calcium determination and set aside; add anhydrous ethanol to the remaining supernatant 6, precipitate overnight (10-12 hours), centrifuge and obtain precipitate 6, which is chelated calcium 6.

[0078] 1. Testing of eggshell membrane solution after high-pressure homogenization

[0079] 1. Particle size measurement of eggshell membrane solution after high-pressure homogenization

[0080] The eggshell membrane solutions 1 to 6 in Examples 1 to 6 were respectively diluted 5 to 15 times and tested using a laser particle size distribution analyzer.

[0081] The results are shown in Table 1: After high-pressure homogenization for different times, the particle size is significantly reduced and tends to be evenly distributed.

[0082] Table 1 Particle size of eggshell membrane solution

[0083]

[0084]

[0085] 2. SEM analysis of eggshell membrane solution after high-pressure homogenization

[0086] The eggshell membrane solutions 1 to 6 in Examples 1 to 6 were respectively added dropwise onto aluminum foil and dried. The samples were then gold-sputtered using a particle sputtering apparatus, and the microscopic morphology of the samples was observed using a scanning electron microscope at 10 kV.

[0087] The results are as follows Figure 1 As shown in the figure: after 20 times of high-pressure homogenization, the eggshell membrane particle size tends to be stable and the distribution is more uniform, so 20 times of high-pressure homogenization is preferred.

[0088] 3. Test of the amount of soluble protein dissolved in eggshell membrane solution after high-pressure homogenization

[0089] Eggshell membrane solutions 1-6 from Examples 1-6 were centrifuged at 8,000-12,000 rpm, and the supernatants were collected to obtain soluble protein solutions 1-6, respectively. Soluble protein content was determined using a BCA assay kit. The protein standard solution was added to the BCA reagent and fully dissolved to prepare a protein standard solution. An appropriate amount of the protein standard solution was diluted to prepare a standard. The standard solution was added to a 96-well plate in a gradient and topped up with ultrapure water to generate a standard curve.

[0090] Dilutions of soluble protein solution 1 to 6 were added to a 96-well plate and incubated at 25–60°C for 10–60 min. The absorbance at A562 nm was measured using a microplate reader. The protein concentration of the sample was calculated based on the standard curve and the sample volume used.

[0091] The results are as follows Figure 2 As shown in the figure: after 0 times of high-pressure homogenization, the amount of soluble protein dissolved reached a maximum of 256.46 mg and tended to be stable, so 20 times of high-pressure homogenization was preferred.

[0092] From the above, it can be seen that the eggshell membrane solution generated by high-pressure homogenization 20 times in Example 3 has the best effect.

[0093] 2. Test of chelation rate in supernatant

[0094] 1. Total Calcium Determination

[0095] 10 μL of each of the supernatants 1 to 6 in Examples 1 to 6 was taken for total calcium determination.

[0096] 2. Chelated Calcium Assay

[0097] The chelated calcium 1 to 6 of Examples 1 to 6 were added to deionized water to dissolve the precipitate, diluted, and the chelated calcium was determined by atomic absorption.

[0098] 3. Calculate the chelation rate using the values ​​of total calcium and chelated calcium.

[0099] The results are as follows Figure 3 As shown, the chelation rate reaches a maximum of 12.49% after 20 high-pressure homogenizations and tends to be stable. Therefore, the eggshell membrane solution generated by 20 high-pressure homogenizations in Example 3 has a high chelation rate and the best chelated calcium yield.

[0100] 3. Calcium uptake test of Caco-2 cells

[0101] 1. A chelated calcium precipitate without high-pressure homogenization treatment was prepared by the method of Example 3, except that the number of high-pressure homogenizations was 0.

[0102] 2. The precipitate 3 obtained in Example 3 and the chelated calcium precipitate not subjected to high-pressure homogenization were re-dissolved and freeze-dried to obtain chelated calcium powder and chelated calcium powder not subjected to high-pressure homogenization, respectively. Complete culture medium was used as a solvent to prepare 100 μg / mL chelated calcium solution and 100 μg / mL chelated calcium solution not subjected to high-pressure homogenization, respectively, and the mixture was heated in a 37°C water bath.

[0103] 3. Caco-2 cells were seeded in a Transwell plate and cultured in an incubator for 21 days. The upper and lower chambers of the 12-well TransWell™ plate were rinsed once with HBSS buffer and incubated in an incubator for 40 minutes. After two more rinses, 1 mL of preheated HBSS buffer was added to the lower chamber. 0.5 mL of a chelated calcium solution (100 μg of calcium), an inorganic calcium chloride solution, and a chelated calcium solution without high-pressure homogenization were added to the upper chamber, respectively. After a 3-hour incubation, the solution in the lower chamber was removed and the calcium content was determined by atomic absorption spectrometry.

[0104] 4. The results are as follows Figure 4 As shown in the figure, after 3 hours of incubation, chelated calcium treated by high-pressure homogenization can significantly increase the amount of calcium transported by cells, reaching 5.21 μg.

[0105] Although the above embodiments have been described in detail, they are only a part of the embodiments of the present invention, not all of them. People can also obtain other embodiments based on this embodiment without inventiveness, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A method for promoting the dissolution of eggshell membrane protein by high-pressure crushing and preparing chelated calcium, characterized in that: The following steps are involved: (1) separating and drying the eggshell and eggshell membrane of an eggshell, and then ultrafine grinding the eggshell and eggshell membrane to obtain eggshell powder and eggshell membrane powder; (2) adding water to the eggshell membrane powder, homogenizing under high pressure, and centrifuging to obtain an eggshell membrane solution; (3) adding eggshell powder to the eggshell membrane solution, adjusting the pH to 1-3, and adding pepsin to react; (4) After the reaction, the pH of the eggshell membrane solution is adjusted to 6.5-7.5, and trypsin is added to react; (5) After the reaction, the enzyme is inactivated, and the supernatant is collected by centrifugation. Anhydrous ethanol is added to the supernatant for alcohol precipitation, and the precipitate is obtained by centrifugation. The precipitate is chelated calcium.

2. The method according to claim 1, wherein: In the step (1), the ultrafine grinding time is 1 to 8 hours.

3. The method according to claim 1, wherein: In the step (2), the mass volume ratio of eggshell membrane powder to water is 1:15-30 g / mL; The conditions of the high-pressure homogenization are: 500-1000 Bar; the number of high-pressure homogenization is 1-40 times.

4. The method according to claim 3, wherein: The high-pressure homogenization condition is: 800 Bar, and the number of high-pressure homogenization is 20 times.

5. The method according to claim 1, wherein: In the step (3), the mass volume ratio of eggshell powder to eggshell membrane solution is 0.1-0.6:10 g / mL; The amount of pepsin added is 500-2000 U / mL, the reaction temperature is 25-40° C., and the reaction time is 1-3 hours.

6. The method according to claim 5, characterized in that: The mass volume ratio of the eggshell powder and the eggshell membrane solution is 0.3:10 g / mL.

7. The method according to claim 1, wherein: In the step (4), the amount of trypsin added is 10 to 300 U / mL, the reaction temperature is 25 to 40° C., and the reaction time is 1 to 3 hours.

8. The method according to claim 1, wherein: In the step (5), the enzyme inactivation condition is 100-110° C., and the alcohol precipitation time is 10-12 h.

9. Chelated calcium prepared by the method according to any one of claims 1 to 8.

10. Use of the chelated calcium according to claim 9 in preparing calcium supplement products.

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