A preparation method of catfish bone collagen peptide-calcium chelate

Abstract

The invention discloses a preparation method of catfish bone collagen peptide-calcium chelate, belonging to the technical field of bioactive peptides. The preparation method is based on high static pressure modification, coordinated glycosylation-enzyme cross-linking composite modification, and specifically includes the following steps: decalcification and defatting pretreatment of catfish bones, preparation of catfish bone collagen with decalcified and defatted catfish bone powder, high static pressure modification of catfish bone collagen, enzymolysis with alkaline protease, glycosylation-enzyme cross-linking composite modification with D-glucosamine and transglutaminase, macromolecular separation and desalination of the modified product, chelation of catfish bone collagen peptide with calcium chloride, separation and drying of precipitate. The benefits of the present invention are: (1) the preparation method can increase the yield of catfish bone collagen peptide-calcium chelate to more than 75%, and the peptide-calcium chelation rate and calcium absorption rate are both increased to more than 85%; (2) water volume and energy consumption are reduced, and wastewater discharge is reduced.

Description

Technical Field

[0001] The invention relates to a method for preparing a chelate, in particular to a method for preparing a catfish bone collagen peptide-calcium chelate, and belongs to the technical field of bioactive peptides. Background Art

[0002] Calcium plays an extremely important role in the growth and development of the human body. It can maintain the normal functions of the human nerves, bones, muscles, etc., promote the development of bones, and prevent short stature, rickets, osteoporosis, etc. Dietary intake is the main way for the body to supplement calcium, but those who have a large calcium demand, calcium loss, and calcium deficiency, such as pregnant women, the elderly, and teenagers, generally need to supplement calcium through oral calcium products.

[0003] Peptide-calcium chelate is a complex with a ring structure formed by connecting calcium ions and peptides through coordination bonds. Calcium ions are embedded between amino acid molecules and will not react with phytic acid, oxalic acid, etc. in food to affect human absorption. Therefore, peptide-calcium chelate can effectively transport calcium ions and be absorbed and utilized by intestinal epithelial cells, increasing the bioavailability of calcium. Compared with other calcium supplements, peptide-calcium chelate has good development prospects due to its high solubility, good bioavailability, and low irritation to the human gastrointestinal tract.

[0004] According to the source of peptides, peptide-calcium chelates can be divided into animal-derived peptides, plant-derived peptides, and aquatic-derived peptides. In recent years, the aquatic processing industry has developed rapidly. While achieving industrial value-added, a large number of by-products such as bones, skins, and scales rich in protein have been produced during the processing process. At present, there have been many related studies on the preparation of peptide-calcium complex chelates using fish bones, fish skins, etc. as raw materials, but the existing technology has the problem that the yield of peptide-calcium chelates, peptide calcium chelation rate, and calcium absorption rate are not high. The development of new peptide-calcium chelate preparation technology, the improvement of the yield of peptide-calcium chelates, peptide calcium chelation rate, and calcium absorption rate are of great significance to promote the development of collagen peptide-calcium chelates using bones, scales, etc. as raw materials, and the realization of efficient preparation of collagen peptide-calcium chelates and the development of new calcium supplements. Summary of the invention

[0005] The object of the present invention is to provide a method for preparing catfish bone collagen peptide-calcium chelate which can simultaneously improve the peptide-calcium chelate yield, peptide calcium chelation rate and calcium absorption rate.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] A method for preparing catfish bone collagen peptide-calcium chelate is based on high static pressure modification and coordinated glycosylation-enzyme cross-linking composite modification, and specifically comprises the following steps:

[0008] (1) performing decalcification and defatting pretreatment on catfish bones to obtain decalcified and defatted catfish bone powder;

[0009] (2) preparing catfish bone collagen using decalcified and defatted catfish bone powder;

[0010] (3) performing high static pressure modification on catfish bone collagen to obtain high static pressure modified catfish bone collagen;

[0011] (4) enzymatically hydrolyzing the catfish bone collagen modified by high static pressure with alkaline protease to obtain a catfish bone collagen enzymatic hydrolysis product;

[0012] (5) performing glycosylation-enzyme cross-linking composite modification on catfish bone collagen enzymatic hydrolysis products using D-glucosamine and transglutaminase to obtain glycosylation-enzyme cross-linking composite modified enzymatic hydrolysis products;

[0013] (6) performing macromolecular separation and concentration and desalting on the enzymatic hydrolysis product of glycosylation-enzyme cross-linking composite modification to obtain catfish bone collagen peptide solution;

[0014] (7) The catfish bone collagen peptide is chelated with calcium chloride, and then the chelate is precipitated with alcohol and separated by centrifugation. The precipitate is freeze-dried to obtain the catfish bone collagen peptide-calcium chelate.

[0015] Preferably, in step (2), the method for preparing catfish bone collagen from decalcified and defatted catfish bone powder is as follows: adding the decalcified and defatted catfish bone powder to an acetic acid solution with a concentration of 0.5 mol / L at a ratio of 1 kg:3 L, stirring and extracting at 80° C. for 3 h, centrifuging to obtain a precipitate and then repeating the extraction once, centrifuging, combining the two extracts, filtering with absorbent cotton, adjusting the pH of the extract to neutral, and concentrating and desalting through a 200 Da nanofiltration membrane to obtain a solution containing catfish bone collagen.

[0016] Preferably, in step (3), the high static pressure modification method is specifically: maintaining the pressure at 400 MPa for 35 minutes.

[0017] Preferably, in step (4), the enzymatic hydrolysis method is specifically as follows: adjusting the protein concentration to 100 mg / mL and the pH value to 8, adding alkaline protease to the solution so that the concentration of alkaline protease is 4%, performing enzymatic hydrolysis at 45°C for 2h, inactivating the enzyme, cooling, and centrifuging to collect the enzymatic hydrolyzate to obtain catfish bone collagen enzymatic hydrolysis product.

[0018] Preferably, in step (5), the method of glycosylation-enzyme cross-linking composite modification is specifically as follows: adjusting the concentration of crude protein to 50 mg / mL, adding a 50 mg / mL D-glucosamine solution to the enzymatic hydrolyzate at a ratio of 1:1 between amino sugar and crude protein, adjusting the pH value to 7.5, adding transglutaminase, the amount of transglutaminase added is 110 U / g crude protein, reacting at 50°C for 5 hours, and obtaining a glycosylation-enzyme cross-linking composite modified enzymatic hydrolyzate.

[0019] Preferably, in step (6), the method for obtaining the catfish bone collagen peptide solution is specifically as follows: a ceramic membrane and a 3 kDa nanofiltration membrane are used in sequence to separate the macromolecules of the enzymatic hydrolysis product of the glycosylation-enzyme cross-linking composite modification, and then a 200 Da nanofiltration membrane is used for concentration and desalting, and the separated liquid is collected to obtain the catfish bone collagen peptide solution.

[0020] Preferably, in step (7), the method for chelating catfish bone collagen peptide with calcium chloride is as follows: mixing the catfish bone collagen peptide solution and the calcium chloride solution in a mass ratio of 1:5, and chelating at 50° C. for 60 minutes to obtain a chelate.

[0021] The present invention is beneficial in that:

[0022] (1) The present invention uses catfish bone as raw material, based on high static pressure modification and coordinated glycosylation-enzyme cross-linking composite modification, to prepare catfish bone collagen peptide-calcium chelate with a yield of more than 75% (calculated based on the protein content in fish bone), a peptide calcium chelation rate of more than 85%, and a calcium absorption rate of more than 85%. The peptide-calcium chelate yield, peptide calcium chelation rate and calcium absorption rate are all greatly improved, and there is no cytotoxicity in the concentration range of 0.25 mg / mL to 2.0 mg / mL.

[0023] (2) The present invention does not involve any drying process from protein extraction and modification to polypeptide preparation and modification to peptide-calcium chelation, and uses a membrane separation device to purify and concentrate the peptide. At the same time, the modification parameters and chelation parameters are calculated based on the protein content in the solution, which reduces water consumption and energy consumption, reduces wastewater discharge, thereby reducing production costs and improving processing efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is a comparison chart of peptide calcium chelation rates of catfish bone collagen peptide-calcium chelates prepared in various embodiments and comparative examples;

[0025] Figure 2 It is a comparison chart of the yields of catfish bone collagen peptide-calcium chelate prepared in each embodiment and each comparative example;

[0026] Figure 3It is a comparison chart of calcium absorption rate of catfish bone collagen peptide-calcium chelate, calcium citrate and calcium chloride prepared in various embodiments and comparative examples at different transport times;

[0027] Figure 4 It is a comparison chart of the effects of the catfish bone collagen peptide-calcium chelate prepared in various embodiments and comparative examples at different concentrations on the survival rate of Caco-2 cells. DETAILED DESCRIPTION

[0028] The present invention is described in detail below with reference to the accompanying drawings and embodiments.

[0029] 1. Preparation of Catfish Bone Collagen Peptide-Calcium Chelate Example 1

[0030] The preparation method of catfish bone collagen peptide-calcium chelate provided in this embodiment is based on high static pressure modification and coordinated glycosylation-enzyme cross-linking composite modification, and specifically comprises the following steps:

[0031] 1. Catfish bone decalcification and defatting pretreatment

[0032] In order to improve the yield of catfish bone collagen, the catfish bone is firstly subjected to decalcification and defatting pretreatment, specifically:

[0033] The catfish bones were immersed in a 0.1 mol / L sodium hydroxide solution to remove non-collagenous substances, repeatedly rinsed with tap water until neutral, and then immersed in a 6% mass concentration hydrochloric acid for decalcification. After washing with tap water three times to remove the hydrochloric acid, they were immersed in a 15% volume concentration isopropanol solution for degreasing. After washing with tap water three times to remove the isopropanol, the catfish bones were naturally dried and crushed, and passed through a 40-mesh sieve to obtain decalcified and defatted catfish bone powder (particle size below 0.4 mm).

[0034] 2. Preparation of Catfish Bone Collagen

[0035] The decalcified and defatted catfish bone powder was added to an acetic acid solution with a concentration of 0.5 mol / L at a ratio of 1 kg: 3 L, and the mixture was stirred and extracted at 80°C for 3 h. The precipitate was taken out by centrifugation and the extraction was repeated once. The two extracts were combined and filtered with absorbent cotton. The pH of the extract was adjusted to neutral. The extract was concentrated and desalted by passing through a 200 Da nanofiltration membrane to obtain a solution containing catfish bone collagen.

[0036] 3. High static pressure modification of catfish bone collagen

[0037] In order to improve the enzymatic hydrolysis efficiency of catfish bone collagen, catfish bone collagen was first subjected to high static pressure modification treatment before enzymatic hydrolysis. Specifically:

[0038] The solution containing catfish bone collagen is added into the high static pressure reaction chamber, and the pressure is maintained at 400 MPa for 35 minutes to obtain high static pressure modified catfish bone collagen.

[0039] 4. Enzymatic hydrolysis of catfish collagen modified by high static pressure

[0040] The concentration of high static pressure modified catfish bone collagen in the solution was detected and the protein concentration was adjusted to 100 mg / mL. At the same time, the pH value of the solution was adjusted to 8. Alkaline protease was added to the solution to make the concentration of alkaline protease 4%. Enzyme hydrolysis was carried out at 45°C for 2h. After the enzyme was inactivated and cooled, the enzymatic hydrolyzate (containing catfish bone collagen enzymatic hydrolysis products) was collected by centrifugation.

[0041] 5. Glycosylation-enzyme cross-linking composite modification of catfish bone collagen enzymatic hydrolysis products

[0042] In order to improve the yield of peptide-calcium chelate and calcium absorption rate, the catfish bone collagen enzymatic hydrolysate was subjected to glycosylation-transglutaminase cross-linking composite modification treatment, specifically:

[0043] The crude protein concentration in the enzymatic hydrolysate was detected and adjusted to 50 mg / mL. A D-glucosamine solution with a concentration of 50 mg / mL was added to the enzymatic hydrolysate at a mass ratio of 1:1 between amino sugar and crude protein. The pH value was adjusted to 7.5, and transglutaminase was added in an amount of 110 U / g crude protein. The reaction was carried out at 50°C for 5 h to perform a glycosylation-enzyme cross-linking composite modification treatment to obtain a glycosylation-enzyme cross-linking composite modified enzymatic hydrolysate.

[0044] 6. Membrane separation of catfish bone collagen peptides

[0045] The ceramic membrane and 3kDa nanofiltration membrane were used successively to separate the macromolecules of the enzymatic hydrolysate modified by glycosylation-enzyme cross-linking composite, and then a 200Da nanofiltration membrane was used for concentration and desalination. The separated liquid was collected to obtain the catfish bone collagen peptide solution.

[0046] 7. Preparation of Catfish Bone Collagen Peptide-Calcium Chelate

[0047] The concentration of protein peptides in the catfish bone collagen peptide solution was detected. The catfish bone collagen peptide solution and a calcium chloride solution with a concentration of 500 mg / mL were mixed in a mass ratio of 1:5, and chelated at 50°C for 60 min. Then, 8 times the volume of anhydrous ethanol was added to carry out alcohol precipitation separation on the chelate at 4°C for 6 h. After centrifugation at 9000 r / min for 10 min, the precipitate was collected and freeze-dried to obtain the catfish bone collagen peptide-calcium chelate, which was recorded as S1. Example 2

[0048] The preparation method of catfish bone collagen peptide-calcium chelate provided in this embodiment is basically the same as that in Example 1, the only difference is that when the catfish bone collagen enzymatic hydrolysis product is subjected to glycosylation-enzyme cross-linking composite modification (step 5), the amount of transglutaminase added is different, specifically, the amount of transglutaminase added is 90 U / g.

[0049] The finally obtained catfish bone collagen peptide-calcium chelate was recorded as S2. Example 3

[0050] The preparation method of catfish bone collagen peptide-calcium chelate provided in this embodiment is basically the same as that in Example 1, except that:

[0051] (1) When catfish bone collagen is modified under high static pressure (step 3), the pressure and holding time of the high static pressure reaction chamber are different. Specifically, the pressure is held at 450 MPa for 30 min.

[0052] (2) When enzymatically hydrolyzing catfish bone collagen modified by high static pressure (step 4), the protein concentration and enzymatic hydrolysis conditions were different. Specifically, the protein concentration was adjusted to 80 mg / mL and the enzymatic hydrolysis was carried out at 40°C for 2 h.

[0053] (3) When the catfish bone collagen enzymatic hydrolysis product was subjected to glycosylation-enzyme cross-linking composite modification (step 5), the enzyme cross-linking conditions were different. Specifically, the pH value was adjusted to 7.0 and the reaction was carried out at 55°C for 4.5 hours.

[0054] The finally obtained catfish bone collagen peptide-calcium chelate was recorded as S3.

[0055] Comparison Example 1

[0056] The preparation method of catfish bone collagen peptide-calcium chelate provided in this comparative example is based on glycosylation-enzyme cross-linking composite modification, and the difference from Example 1 is that after the catfish bone collagen is prepared, the catfish bone collagen is not subjected to high static pressure modification, but is directly subjected to enzymolysis.

[0057] The finally obtained catfish bone collagen peptide-calcium chelate was recorded as D1.

[0058] Comparison Example 2

[0059] The preparation method of catfish bone collagen peptide-calcium chelate provided in this comparative example is based on high static pressure modification and coordinated glycosylation modification, and the difference from Example 1 is:

[0060] (1) When catfish bone collagen is modified under high static pressure, the pressure and holding time of the high static pressure reaction chamber are different. Specifically, the pressure is maintained at 450 MPa for 30 min.

[0061] (2) The enzymatic hydrolysis products of catfish bone collagen were only subjected to glycosylation modification, but not to transglutaminase cross-linking modification.

[0062] The finally obtained catfish bone collagen peptide-calcium chelate was recorded as D2.

[0063] Comparison Example 3

[0064] The preparation method of catfish bone collagen peptide-calcium chelate provided in this comparative example is based on high static pressure modification and coordinated enzyme cross-linking modification, and the difference from Example 1 is:

[0065] (1) When catfish bone collagen is modified under high static pressure, the pressure and holding time of the high static pressure reaction chamber are different. Specifically, the pressure is maintained at 450 MPa for 30 min.

[0066] (2) The enzymatic hydrolysis product of catfish bone collagen was only subjected to transglutaminase cross-linking modification, but not glycosylation modification.

[0067] The finally obtained catfish bone collagen peptide-calcium chelate was recorded as D3.

[0068] 2. Detection of calcium chelation ability of catfish bone collagen peptide-calcium chelate

[0069] The ratio of the calcium content in the peptide-calcium chelate to the calcium content in the reaction system is the peptide-calcium chelation rate. The peptide-calcium chelation rate can directly reflect the calcium binding ability of collagen peptides. The calculation formula of the peptide-calcium chelation rate is as follows:

[0070]

[0071] in, C 1 is the calcium content added to the reaction system, C 2 is the calcium content in the supernatant after alcohol precipitation and centrifugation after the chelation reaction is completed (determined by a calcium content colorimetric detection kit and detected at 575 nm on an enzyme reader).

[0072] The calculation results of peptide-calcium chelation rate of catfish bone collagen peptide-calcium chelate prepared in each embodiment and each comparative example are shown in Figure 1 .

[0073] Depend on Figure 1 It can be seen that the peptide-calcium chelation rate of the catfish bone collagen peptide-calcium chelate prepared in each embodiment of the present invention is 88.20% to 92.08%, which is much higher than the peptide-calcium chelation rate of the catfish bone collagen peptide-calcium chelate prepared in each comparative example.

[0074] 3. Detection of the Yield of Catfish Bone Collagen Peptide-Calcium Chelate

[0075] The yield of catfish bone collagen peptide-calcium chelate was calculated based on the protein content in fish bones. The calculation formula is:

[0076]

[0077] in, M 1 is the yield of catfish bone collagen peptide-calcium chelate, M For the quality of raw fish bones, C It is the protein content in fish bones (detected using the Kjeldahl method in the national standard GB50095-2010 Determination of protein content in food).

[0078] The calculation results of the yield of catfish bone collagen peptide-calcium chelate prepared in each embodiment and each comparative example are shown in Figure 2 .

[0079] Depend on Figure 2 It can be seen that the yield of catfish bone collagen peptide-calcium chelate prepared in each embodiment of the present invention is 76% to 80%, which is much higher than the yield of catfish bone collagen peptide-calcium chelate prepared in each comparative example.

[0080] 4. Detection of calcium absorption rate of catfish bone collagen peptide-calcium chelate

[0081] Select monolayers with good density and permeability (cells are in good condition under microscope and the resistance value is 600Ωcm 2 The Caco-2 cells (above) were used for calcium absorption and transport assay.

[0082] After the Caco-2 cell culture was completed, the culture medium was discarded, and the Caco-2 cells were gently washed twice with HBSS buffer without calcium and magnesium, and then the Caco-2 cells were transferred to a new Transwell culture plate containing 1.5 mL of HBSS buffer on the basal side, and cultured for 30 minutes at 37°C and 5% CO2. Then, 0.5 mL of 1.0 mg / mL catfish bone collagen peptide-calcium chelate solution, calcium citrate solution or calcium chloride solution prepared in each embodiment or comparative example was added to the basal side of the culture plate, and 1.0 mL of solution was drawn from the top side of the culture plate at 30 minutes, 60 minutes, 120 minutes, and 180 minutes to determine the calcium content, and 1.0 mL of HBSS buffer was added to maintain the constant volume of the basal side of the culture plate. The calcium content was determined by atomic absorption spectrometry.

[0083] The calcium absorption rate of Caco-2 cells at different transport times was calculated according to the following formula:

[0084]

[0085] in,C 1 is the calcium content in the system at the initial moment of transport, C 2 is the calcium content in the system when the transport reaches time t.

[0086] The calculation results of calcium absorption rates of catfish collagen peptide-calcium chelate, calcium citrate and calcium chloride prepared in each embodiment and each comparative example at different transport times are shown in Figure 3 .

[0087] Depend on Figure 3 It can be seen that:

[0088] (1) Under the same transit time, the calcium absorption rate of the catfish bone collagen peptide-calcium chelate prepared in each embodiment of the present invention is much higher than the calcium absorption rate of the catfish bone collagen peptide-calcium chelate prepared by calcium citrate, calcium chloride and each comparative example;

[0089] (2) After 180 minutes of transport, the calcium absorption rate of the catfish bone collagen peptide-calcium chelate prepared in each embodiment of the present invention reached 85% to 88%, which was much higher than the calcium absorption rate of the catfish bone collagen peptide-calcium chelate prepared by calcium citrate, calcium chloride and each comparative example, and increased by nearly 1 times.

[0090] 5. Detection of cytotoxicity of catfish bone collagen peptide-calcium chelate

[0091] The CCK-8 method was used to determine the effects of the catfish bone collagen peptide-calcium chelates prepared in the examples and comparative examples at different concentrations (0.25 mg / mL, 0.5 mg / mL, 1.0 mg / mL and 2.0 mg / mL) on the survival rate of Caco-2 cells.

[0092] The effects of different concentrations of catfish collagen peptide-calcium chelate on the survival rate of Caco-2 cells were tested. Figure 4 .

[0093] When the cell survival rate is above 80%, the sample to be tested is considered to have no cytotoxicity at the corresponding concentration. Figure 4 It can be seen that when the concentration of catfish bone collagen peptide-calcium chelate is 0.5 mg / mL to 1.5 mg / mL, the survival rate of Caco-2 cells is higher than 100%, indicating that it can promote the proliferation of Caco-2 cells. When the concentration of catfish bone collagen peptide-calcium chelate is 2.0 mg / mL, the survival rate of Caco-2 cells is higher than 89%, indicating that the catfish bone collagen peptide-calcium chelate prepared in each embodiment and each comparative example has no cytotoxicity within the concentration range of 0.25 mg / mL to 2.0 mg / mL.

[0094] In summary, the catfish bone collagen peptide-calcium chelate prepared in each embodiment of the present invention has a high yield, peptide-calcium chelation rate and calcium absorption rate, and has no cytotoxicity in the concentration range of 0.25 mg / mL to 2.0 mg / mL.

[0095] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is impossible to list all the embodiments here. Any obvious changes or modifications derived from the technical solution of the present invention are still within the scope of protection of the present invention.

Claims

1. A method for preparing catfish bone collagen peptide-calcium chelate, characterized in that: Based on high static pressure modification, synergistic glycosylation-enzyme cross-linking composite modification, specifically including the following steps: (1) performing decalcification and defatting pretreatment on catfish bones to obtain decalcified and defatted catfish bone powder; (2) preparing catfish bone collagen using decalcified and defatted catfish bone powder; (3) performing high static pressure modification on catfish bone collagen to obtain high static pressure modified catfish bone collagen; (4) enzymatically hydrolyzing the catfish bone collagen modified by high static pressure with alkaline protease to obtain a catfish bone collagen enzymatic hydrolysis product; (5) The catfish bone collagen enzymatic hydrolysate was subjected to glycosylation-enzyme cross-linking composite modification using D-glucosamine and transglutaminase. Specifically, the crude protein concentration was adjusted to 50 mg / mL, and a 50 mg / mL D-glucosamine solution was added to the enzymatic hydrolysate at a ratio of 1:1 of amino sugar to crude protein. The pH value was adjusted to 7.5, and transglutaminase was added in an amount of 110 U / g crude protein. The reaction was carried out at 50° C. for 5 h to obtain a glycosylation-enzyme cross-linking composite modified enzymatic hydrolysate. (6) performing macromolecular separation and concentration and desalting on the enzymatic hydrolysis product of glycosylation-enzyme cross-linking composite modification to obtain catfish bone collagen peptide solution; (7) The catfish bone collagen peptide is chelated with calcium chloride, and then the chelate is precipitated with alcohol and separated by centrifugation. The precipitate is freeze-dried to obtain the catfish bone collagen peptide-calcium chelate.

2. The method for preparing catfish bone collagen peptide-calcium chelate according to claim 1, characterized in that: In step (2), the method for preparing catfish bone collagen using decalcified and defatted catfish bone powder is specifically as follows: The decalcified and defatted catfish bone powder was added to an acetic acid solution with a concentration of 0.5 mol / L at a ratio of 1 kg: 3 L, and the mixture was stirred and extracted at 80°C for 3 h. The precipitate was taken out by centrifugation and the extraction was repeated once. The two extracts were combined and filtered with absorbent cotton. The pH of the extract was adjusted to neutral. The extract was concentrated and desalted by passing through a 200 Da nanofiltration membrane to obtain a solution containing catfish bone collagen.

3. The method for preparing catfish bone collagen peptide-calcium chelate according to claim 1, characterized in that: In step (3), the high static pressure modification method is specifically as follows: Maintain pressure at 400 MPa for 35 minutes.

4. The method for preparing catfish bone collagen peptide-calcium chelate according to claim 1, characterized in that: In step (4), the enzymatic hydrolysis method is specifically as follows: The protein concentration was adjusted to 100 mg / mL and the pH value was adjusted to 8. Alkaline protease was added to the solution to make the concentration of alkaline protease 4%. The solution was enzymolyzed at 45°C for 2 hours. After the enzyme was inactivated and cooled, the enzymolysis solution was collected by centrifugation to obtain the catfish bone collagen enzymolysis product.

5. The method for preparing catfish bone collagen peptide-calcium chelate according to claim 1, characterized in that: In step (6), the method for obtaining catfish bone collagen peptide solution is specifically as follows: The ceramic membrane and 3kDa nanofiltration membrane were used successively to separate the macromolecules of the enzymatic hydrolysate modified by glycosylation-enzyme cross-linking composite, and then a 200Da nanofiltration membrane was used for concentration and desalination. The separated liquid was collected to obtain the catfish bone collagen peptide solution.

6. The method for preparing catfish bone collagen peptide-calcium chelate according to claim 1, characterized in that: In step (7), the method of chelating catfish bone collagen peptide with calcium chloride is specifically as follows: The catfish bone collagen peptide solution and the calcium chloride solution were mixed in a mass ratio of 1:5, and chelated at 50° C. for 60 minutes to obtain a chelate.

Images