Methods for promoting eggshell membrane protein dissolution and preparing chelated calcium through high-pressure crushing

Abstract

This invention discloses a method for promoting the dissolution of eggshell membrane proteins and preparing chelated calcium through high-pressure crushing. The method includes the following steps: separating and drying the eggshell and eggshell membrane, then pulverizing them separately to obtain eggshell powder and eggshell membrane powder; adding water to the eggshell membrane powder and homogenizing under high pressure, followed by centrifugation to obtain an eggshell membrane solution; adding eggshell powder to the eggshell membrane solution, adjusting the pH to 1-3, and adding pepsin to react; after the reaction, adjusting the pH of the eggshell membrane solution to 6.5-7.5, and adding trypsin to react; after the reaction, inactivating the enzymes, centrifuging to collect the supernatant, adding anhydrous ethanol to the supernatant for alcohol precipitation, centrifuging to obtain the precipitate, which is chelated calcium. This invention overcomes the technical difficulty of dissolving eggshell membrane proteins, providing a new approach for the industrial extraction of soluble eggshell membrane proteins.

Description

Technical Field

[0001] This invention relates to the field of egg product processing technology, specifically to a method for high-pressure crushing to promote the dissolution of eggshell membrane proteins and to prepare chelated calcium. Background Technology

[0002] Eggshells are an ideal source of calcium supplementation due to their high calcium content and bioavailability. Primarily composed of calcium carbonate, with an average content of 93%, their calcium content can reach as high as 380 mg / g, far exceeding many commonly used calcium supplements. The eggshell membrane, a thin membrane inside the eggshell, is rich in various proteins, mainly composed of collagen, keratin, lysozyme, and various glycoproteins. It is not only a high-quality protein source but also possesses various biological activities such as antioxidant, anti-inflammatory, antibacterial, bone health, skin health, immune regulation, and digestive health benefits, showing broad application potential in the food, pharmaceutical, and cosmetic fields and possessing significant development value. However, the eggshell membrane is composed of a multi-layered fibrous network structure, mainly 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 structure that is difficult to destroy by conventional solvents in its natural state.

[0003] Chemical hydrolysis and enzymatic hydrolysis are relatively traditional methods for extracting protein and peptide products. However, chemical hydrolysis suffers from problems such as reagent residues affecting product quality. Enzymatic hydrolysis is widely used due to its high substrate specificity, but it is difficult to industrialize due to its high cost and long reaction time. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a method for promoting the dissolution of eggshell membrane proteins and preparing chelated calcium through high-pressure crushing. This invention promotes the dissolution of soluble proteins by subjecting eggshell membrane solutions to high-pressure crushing, overcoming the problems of low efficiency and difficulty in industrialization in traditional protein extraction methods.

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

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

[0007] (1) Separate and dry the eggshell and eggshell membrane of the egg, and then pulverize them separately to obtain eggshell powder and eggshell membrane powder.

[0008] (2) Water was added to the eggshell membrane powder and homogenized under high pressure, and then centrifuged to obtain an eggshell membrane solution;

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

[0010] (4) After the reaction, adjust the pH of the eggshell membrane solution to 6.5-7.5, and add trypsin to carry out the reaction;

[0011] (5) After the reaction, the enzyme is inactivated, 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 step (1), the ultrafine pulverization time is 1 to 8 hours.

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

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

[0015] Furthermore, the conditions for high-pressure homogenization are: 800 Bar, and the number of high-pressure homogenization cycles is 20.

[0016] Furthermore, in step (3), the mass-to-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℃, and the reaction time is 1–3 h.

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

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

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

[0021] The present invention also provides a chelated calcium prepared using the method described above.

[0022] The present invention also provides an application of the chelated calcium described above in the preparation of calcium supplement products.

[0023] The principle of this invention:

[0024] High-pressure homogenization is a common technique used to break down, disperse, or homogenize samples through physical mechanical forces. It is widely applied in food, pharmaceuticals, biotechnology, cosmetics, and other fields. Its core principle is to force the sample through a narrow valve or homogenizing valve under high pressure, utilizing the combined effects of shear force, cavitation effect, turbulence, and collision to achieve particle refinement, cell disruption, or component homogenization.

[0025] The beneficial effects of this invention are:

[0026] This invention uses eggshells as raw material, aiming to utilize the calcium and soluble proteins within the eggshell under the premise of human gastrointestinal digestion. Simultaneously, a high-pressure homogenization process is used to promote the dissolution of soluble proteins from the eggshell membrane. This maximizes the formation of soluble protein-chelated calcium complexes between eggshell membrane proteins and eggshell calcium under the action of complex digestive enzymes in the gastrointestinal tract, thereby promoting the absorption of calcium from the eggshell by the human body. This invention opens up an innovative technological path for the industrial utilization of egg by-products and provides a complete solution for the food industry to develop natural and healthy products that meet modern consumer demands. Attached Figure Description

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

[0028] In the figure, ESM8h is the eggshell membrane solution that has not been homogenized under high pressure, ESM8h HP10 is eggshell membrane solution 2, ESM8hHP20 is eggshell membrane solution 3, ESM8h HP30 is eggshell membrane solution 4, and ESM8h HP40 is eggshell membrane solution 6.

[0029] Figure 2 The graph shows the results of the amount of soluble protein dissolved in the eggshell membrane solution after different high-pressure homogenization cycles.

[0030] In the figure, CK is the eggshell membrane solution that has not been homogenized under high pressure, 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 different high-pressure homogenization cycles.

[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 A graph showing the results of calcium uptake in Caco-2 cells;

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

[0035] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can understand it.

[0036] Example 1

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

[0038] (1) Using eggshells as raw materials, the eggshells and eggshell membranes are separated and dried, and then subjected to ultrafine grinding for 1 hour to obtain eggshell powder and eggshell membrane powder.

[0039] (2) Dissolve 30g of eggshell membrane powder in 600mL of deionized water, homogenize under high pressure 5 times, centrifuge and collect the supernatant to obtain a homogeneous eggshell membrane solution 1. The parameters for high pressure homogenization are: 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℃ for 1 h.

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

[0042] (5) Inactivate enzymes at high temperature (100℃), centrifuge and take supernatant 1. Take 10 μL of supernatant 1 for total calcium determination and set aside. Add anhydrous ethanol to the remaining supernatant 1 and precipitate overnight (10-12 h). Centrifuge and take precipitate 1. Precipitate 1 is chelated calcium 1.

[0043] Example 2

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

[0045] (1) Using eggshells as raw materials, the eggshells and eggshell membranes are separated and dried, and then subjected to ultra-fine pulverization for 3 hours to obtain eggshell powder and eggshell membrane powder.

[0046] (2) Dissolve 50g of eggshell membrane powder in 1000mL of deionized water, homogenize under high pressure 10 times, centrifuge and collect the supernatant to obtain a homogeneous eggshell membrane solution 2. The parameters for high pressure homogenization are: 500 Bar.

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

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

[0050] Example 3

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

[0052] (1) Using eggshells as raw materials, the eggshells and eggshell membranes are separated and dried, and then subjected to ultra-fine pulverization for 5 hours to obtain eggshell powder and eggshell membrane powder.

[0053] (2) Dissolve 50g of eggshell membrane powder in 1000mL of deionized water, homogenize under high pressure 20 times, centrifuge and collect the supernatant to obtain a homogeneous eggshell membrane solution 3. The parameters for high pressure homogenization are: 800 Bar.

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

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

[0056] (5) Inactivate enzymes at high temperature (100℃), centrifuge and take supernatant 3. Take 10μL of supernatant 3 for total calcium determination and set aside. Add anhydrous ethanol to the remaining supernatant 3 and precipitate overnight (10-12h). Centrifuge and take precipitate 3. Precipitate 3 is chelated calcium 3.

[0057] Example 4

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

[0059] (1) Using eggshells as raw materials, the eggshells and eggshell membranes are separated and dried, and then subjected to ultrafine grinding for 8 hours to obtain eggshell powder and eggshell membrane powder.

[0060] (2) Dissolve 50g of eggshell membrane powder in 1000mL of deionized water, homogenize under high pressure 30 times, centrifuge and collect the supernatant to obtain a homogeneous eggshell membrane solution 4. The parameters for high pressure homogenization are: 600 Bar.

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

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

[0064] Example 5

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

[0066] (1) Using eggshells as raw materials, the eggshells and eggshell membranes are separated and dried, and then subjected to ultrafine grinding for 8 hours to obtain eggshell powder and eggshell membrane powder.

[0067] (2) Dissolve 50g of eggshell membrane powder in 1000mL of deionized water, homogenize under high pressure 35 times, centrifuge and collect the supernatant to obtain a homogeneous eggshell membrane solution 5. The parameters for high pressure homogenization are: 700 Bar.

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

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

[0070] (5) Inactivate enzymes at high temperature (100℃), centrifuge and take supernatant 5. Take 10μL of supernatant 5 for total calcium determination and set aside. Add anhydrous ethanol to the remaining supernatant 5 and precipitate overnight (10-12h). Centrifuge and take precipitate 5. Precipitate 5 is chelated calcium 5.

[0071] Example 6

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

[0073] (1) Using eggshells as raw materials, the eggshells and eggshell membranes are separated and dried, and then subjected to ultrafine grinding for 8 hours to obtain eggshell powder and eggshell membrane powder.

[0074] (2) Dissolve 50g of eggshell membrane powder in 1000mL of deionized water, homogenize under high pressure for 40 times, centrifuge and collect the supernatant to obtain a homogeneous eggshell membrane solution 6. The parameters for high pressure homogenization are: 800 Bar.

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

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

[0077] (5) Inactivate enzymes at high temperature (100℃), centrifuge and take supernatant 6. Take 10 μL of supernatant 6 for total calcium determination and set aside. Add anhydrous ethanol to the remaining supernatant 6 and precipitate overnight (10-12 h). Centrifuge and take precipitate 6. Precipitate 6 is chelated calcium 6.

[0078] I. Testing of Eggshell Membrane Solution After High-Pressure Homogenization

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

[0080] Eggshell membrane solutions 1-6 from Examples 1-6 were diluted 5-15 times and then tested using a laser particle size analyzer.

[0081] The results are shown in Table 1: After different cycles of high-pressure homogenization, the particle size decreased significantly and tended to be uniformly distributed.

[0082] Table 1 Particle size of eggshell membrane solution

[0083]

[0084]

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

[0086] Eggshell membrane solutions 1-6 from Examples 1-6 were dropped onto aluminum foil and dried. The solutions were then sputtered with gold using a particle sputtering apparatus, and the microstructure of the samples was observed using a scanning electron microscope at 10kV.

[0087] The results are as follows Figure 1 As shown: After 20 high-pressure homogenization cycles, the size of the eggshell membrane particles tends to stabilize and the distribution becomes more uniform. Therefore, 20 high-pressure homogenization cycles are 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 8000-12000 rpm, and the supernatant was collected to obtain soluble protein solutions 1-6. The soluble protein content was determined using a BCA reagent kit. Protein standard solution was added to the BCA reagent and dissolved thoroughly to prepare protein standard solutions. An appropriate amount of protein standard solution was diluted to prepare standards. The standards were added to 96-well plates in a gradient, and ultrapure water was added to make up the volume to create a standard curve.

[0090] The soluble protein solutions were diluted 1–6 times and added to 96-well plates, then incubated at 25–60°C for 10–60 min. The absorbance at 562 nm was measured using a microplate reader. The protein concentration of the samples was calculated based on the standard curve and the sample volume used.

[0091] The results are as follows Figure 2 As shown: After 0 cycles of high-pressure homogenization, the amount of soluble protein dissolved reached a maximum of 256.46 mg and tended to stabilize. Therefore, 20 cycles of high-pressure homogenization are preferred.

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

[0093] II. Test of chelation rate in supernatant

[0094] 1. Total calcium determination

[0095] Take 10 μL of the supernatant from Examples 1 to 6 respectively for the determination of total calcium.

[0096] 2. Chelated calcium determination

[0097] The chelated calcium 1-6 from Examples 1-6 were dissolved and precipitated in deionized water and diluted. The chelated calcium was then determined by atomic absorption spectrometry.

[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 reached a maximum of 12.49% after 20 high-pressure homogenizations and then tended to stabilize. Therefore, the eggshell membrane solution generated by 20 high-pressure homogenizations in Example 3 had a high chelation rate and the best chelated calcium yield.

[0100] III. Test of calcium uptake in Caco-2 cells

[0101] 1. Chelated calcium precipitate without high-pressure homogenization was prepared by the method in Example 3, except that the number of high-pressure homogenization cycles was 0.

[0102] 2. The precipitate 3 obtained in Example 3 and the chelated calcium precipitate without high pressure homogenization were reconstituted and freeze-dried to obtain chelated calcium powder and chelated calcium powder without high pressure homogenization, respectively. Using complete culture medium as solvent, chelated calcium solutions of 100 μg / mL and chelated calcium solutions without high pressure homogenization were prepared and placed in a water bath at 37°C.

[0103] 3. Caco-2 cells were seeded into Transwell plates and cultured for 21 days. The upper and lower chambers of the 12-well TransWell™ plates were washed once with HBSS buffer and incubated for 40 minutes. After washing twice more, 1 mL of preheated HBSS buffer was added to the lower chamber. 0.5 mL of chelated calcium solution (100 μg of calcium), inorganic calcium chloride solution, and un-homogenized chelated calcium solution were added sequentially to the upper chamber. After incubation for 3 hours, the solution in the lower chamber was collected, and its calcium content was determined by atomic absorption spectrometry.

[0104] 4. Results are as follows Figure 4 As shown, after 3 hours of incubation, the chelated calcium treated with high pressure homogenization significantly increased the amount of calcium transported by cells, reaching 5.21 μg.

[0105] All other parts not described in detail are existing technologies. Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for promoting the dissolution of eggshell membrane proteins by high-pressure crushing and preparing chelated calcium, characterized in that: Includes the following steps: (1) Separate and dry the eggshell and eggshell membrane of the eggshell, and then pulverize them separately to obtain eggshell powder and eggshell membrane powder; (2) Water was added to the eggshell membrane powder and homogenized under high pressure, and then centrifuged to obtain an eggshell membrane solution; (3) Add eggshell powder to the eggshell membrane solution, adjust the pH to 1-3, and add pepsin to react; (4) After the reaction, adjust the pH of the eggshell membrane solution to 6.5-7.5 and add trypsin to carry out the reaction; (5) After the reaction, the enzyme is inactivated, 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. In step (2), the mass-to-volume ratio of eggshell membrane powder to water is 1:15~30 g / mL; the conditions for high-pressure homogenization are: 800 Bar; and the number of high-pressure homogenization cycles is 20.

2. The method according to claim 1, characterized in that: In step (1), the ultrafine pulverization time is 1~8 h.

3. The method according to claim 1, characterized in that: In step (3), the mass-to-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℃, and the reaction time is 1~3 h.

4. The method according to claim 3, characterized in that: The mass-to-volume ratio of the eggshell powder to the eggshell membrane solution is 0.3:10 g / mL.

5. The method according to claim 1, characterized in that: In step (4), the amount of trypsin added is 10~300 U / mL, the reaction temperature is 25~40℃, and the reaction time is 1~3 h.

6. The method according to claim 1, characterized in that: In step (5), the enzyme inactivation conditions are 100~110℃ and the alcohol precipitation time is 10~12 h.

7. A chelated calcium prepared by the method according to any one of claims 1 to 6.

8. The use of the chelated calcium according to claim 7 in the preparation of calcium supplement products.

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