An alkaline collagen peptide and its preparation method

Abstract

The present invention belongs to the technical field of active substance extraction, and particularly relates to an alkaline collagen peptide and a preparation method thereof. After the raw materials of the present invention are soaked in an alkali solution, lipase is used to remove fat by enzymatic hydrolysis, enhancing the impurity removal effect and improving the product purity. The present invention first uses the acid precipitation method to remove acidic and neutral collagen peptides, and then uses the polymer polyelectrolyte precipitation method to selectively precipitate alkaline protein peptides to prepare the alkaline collagen peptide. The present invention uses gel chromatography for separation and purification, improving the product purity. The alkaline collagen peptide prepared by the method of the present invention can be applied to alkaline foods.

Description

Technical Field

[0001] The present invention belongs to the technical field of active substance extraction, and particularly relates to an alkaline collagen peptide and a preparation method thereof. Background Art

[0002] Collagen peptide is a small molecule peptide prepared by acid or enzymatic hydrolysis of collagen, composed of two or more amino acids connected by peptide bonds. Collagen peptide is easily absorbed by the human gastrointestinal tract, skin and blood vessels, and is widely used in many fields such as food, beauty skin care and biomedicine. In addition, with the development of social economy and the improvement of public health awareness, weakly alkaline foods have gradually become a hot topic pursued by people in their lives.

[0003] However, there is currently a lack of alkaline collagen peptides suitable for slightly alkaline application scenarios. Most of the common collagen peptides on the market are suitable for acidic or neutral application scenarios. When these collagen peptides are applied in a weakly alkaline environment, they are prone to flocculation, reducing the product quality.

[0004] The prior art only reports the preparation method of collagen tripeptide rich in basic amino acids. For example, Chinese Patent CN114634549B discloses a collagen tripeptide product, its preparation method and application. This method uses fish skin as the raw material, and through processes such as crude extraction, refining, protease enzymatic hydrolysis, and fermentation enzymatic hydrolysis, prepares collagen tripeptide rich in basic amino acids. The product prepared by this method is only collagen tripeptide rich in basic amino acids, rather than alkaline collagen suitable for alkaline application scenarios. Moreover, this method uses a two-step enzymatic hydrolysis method of protease enzymatic hydrolysis and fermentation enzymatic hydrolysis, and the enzymatic hydrolysis process is complex.

[0005] In view of this, it is necessary to develop a preparation method of alkaline collagen peptide to meet the current market demand. The isoelectric point of alkaline collagen peptide is relatively high, and it has good solubility in a weakly alkaline environment, which can improve the stability of the product. Summary of the Invention

[0006] In order to overcome the defects and deficiencies of the prior art, the present invention provides a collagen peptide applicable to alkaline foods and a preparation method thereof through the combined use of acid precipitation and polymer polyelectrolyte precipitation technology.

[0007] The object of the present invention can be achieved by the following technical solutions:

[0008] In the first aspect, the present invention provides a preparation method of an alkaline collagen peptide, which comprises the following steps:

[0009] (1) Pretreatment: Add the cleaned fish skin to an alkaline solution with a mass 2 - 6 times that of the fish skin, soak for 3 - 5 h, wash with distilled water until neutral, add deionized water with a mass 6 times that of the fish skin, add lipase accounting for 0.2 - 0.5% of the fish skin mass, enzymatically hydrolyze at 40 - 50 °C for 45 - 90 min, and wash to obtain pretreated fish skin;

[0010] (2) Enzymatic hydrolysis: Add deionized water with a mass 2 - 6 times that of the pretreated fish skin prepared in step (1), add protease accounting for 0.05 - 2% of the fish skin mass, adjust the temperature to 50 - 65 °C, and enzymatically hydrolyze for 2 - 6 h to obtain a collagen peptide enzymatic hydrolysate;

[0011] (3) Enzyme inactivation: Heat the enzymatic hydrolysate prepared in step (2) at 85 - 100 °C for 10 - 15 min;

[0012] (4) Decolorization and deodorization: Use an activated carbon fiber membrane to decolorize and deodorize the enzymatic hydrolysate treated by enzyme inactivation in step (3) to obtain a decolorized solution;

[0013] (5) Acid precipitation: Add acetic acid to the decolorized solution prepared in step (4), adjust the pH to 3 - 7, centrifuge, and take the supernatant;

[0014] (6) Precipitation with a high - molecular polymer: Add PAA accounting for 0.02 - 0.5% of the fish skin mass to the supernatant prepared in step (5), mix well, let stand for 15 - 30 min, centrifuge, and take the precipitate;

[0015] (7) Dissolution of the precipitate: Add deionized water with a mass 10 - 25 times that of the precipitate prepared in step (6), adjust the pH to 10 - 12 with a 1 M Na2CO3 solution, add CaCl2, the mass ratio of PAA to CaCl2 is 2 - 5:8 - 15, adjust the pH to 6 - 7 with hydrochloric acid, centrifuge, and take the supernatant;

[0016] (8) Separation and purification: Separate and purify the supernatant prepared in step (7) by gel chromatography;

[0017] (9) Freeze - drying: Collect the purified collagen peptide solution in step (8) and conduct freeze - drying.

[0018] As an alternative, in the above - mentioned preparation method, in step (1), the fish skin is tilapia skin or cod skin.

[0019] As an alternative, in the above - mentioned preparation method, in step (1), the alkali is one of sodium hydroxide, sodium carbonate, or sodium bicarbonate.

[0020] As an alternative, in the above - mentioned preparation method, in step (1), add lipase accounting for 0.3 - 0.4% of the fish skin mass.

[0021] As an alternative, in the above preparation method, in step (2), protease accounting for 0.1-0.8% of the mass of fish skin is added.

[0022] As an alternative, in the above preparation method, in step (2), the protease is one or a combination of several of alkaline protease, papain, bromelain, neutral protease, and flavor protease.

[0023] Preferably, the protease is one or a combination of several of alkaline protease, papain, bromelain, and neutral protease.

[0024] As an alternative, in the above preparation method, in step (5), acetic acid is added to the decolorized solution prepared in step (4) to adjust the pH to 3-4.

[0025] As an alternative, in the above preparation method, in step (6), PAA accounting for 0.1-0.2% of the mass of fish skin is added to the supernatant prepared in step (5).

[0026] As an alternative, in the above preparation method, in step (7), deionized water 20 times the mass of the precipitate prepared in step (6) is added, the pH is adjusted to 11 with a 1M Na2CO3 solution, CaCl2 is added, the mass ratio of PAA to CaCl2 is 3:10, the pH is adjusted to 6 with hydrochloric acid, and centrifuged to obtain the supernatant.

[0027] In the second aspect, the present invention provides an alkaline collagen peptide prepared by the preparation method described in the first aspect above.

[0028] Preferably, the alkaline collagen peptide is applied to alkaline foods.

[0029] The present invention has the following beneficial effects compared with the prior art:

[0030] (1) The present invention prepares an alkaline collagen peptide suitable for an alkaline application environment.

[0031] (2) After the raw materials of the present invention are soaked in an alkali solution and then degreased by lipase enzymeolysis, the impurity removal effect is enhanced and the product purity is improved.

[0032] (3) The present invention first uses the acid precipitation method to remove acidic and neutral collagen peptides, and then uses the polymer polyelectrolyte precipitation method to selectively precipitate alkaline protein peptides to prepare alkaline collagen peptides.

[0033] (4) The present invention uses gel chromatography for separation and purification, improving the product purity. Detailed implementation manners

[0034] The present invention will be further described below with reference to specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the scope of the present invention.

[0035] For those technical or conditions not specified in the examples, they shall be carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. For the reagents or instruments whose manufacturers are not indicated, they are all conventional products that can be obtained through regular channels.

[0036] Unless otherwise specified, the experimental methods in the following examples are all conventional methods. Unless otherwise specified, the test materials used in the following examples are all commercially available products.

[0037] As used herein, "basic collagen peptide" refers to a collagen peptide with a relatively high isoelectric point pH value and can be dissolved in a slightly alkaline environment.

[0038] As used herein, "polymer polyelectrolyte" is divided into polycationic precipitants and polyanionic precipitants, which precipitate acidic proteins and basic proteins respectively.

[0039] As used herein, "polyacrylic acid (PAA)" is a kind of polyanionic precipitant.

[0040] Preparation Example:

[0041] Example 1

[0042] A preparation method of basic collagen peptide, which comprises the following steps:

[0043] (1) Pretreatment: Add the cleaned tilapia skin to an alkali solution with 6 times the mass and soak for 3 h, wash with distilled water until neutral, add 6 times the mass of deionized water, add lipase accounting for 0.3% of the fish skin mass, and enzymolyze at 40 °C for 50 min, then wash to obtain pretreated fish skin, wherein the alkali solution is sodium hydroxide with a mass concentration of 0.05%;

[0044] (2) Enzymolysis: Add 2 times the mass of deionized water to the pretreated fish skin prepared in step (1), add alkaline protease accounting for 0.2% of the fish skin mass, adjust the temperature to 55 °C, and enzymolyze for 3.5 h to obtain a collagen peptide enzymolysis solution;

[0045] (3) Inactivating enzyme: Heat the enzymolysis solution prepared in step (2) at 85 °C for 15 min;

[0046] (4) Decolorization and deodorization: Use an activated carbon fiber membrane to decolorize and deodorize the enzymolysis solution treated by inactivating enzyme in step (3) to obtain a decolorized solution;

[0047] (5) Acid precipitation: Add acetic acid to the decolorized solution prepared in step (4), adjust the pH to 3, centrifuge, and take the supernatant;

[0048] (6) Polymer precipitation: Add PAA accounting for 0.1% of the fish skin mass to the supernatant prepared in step (5), mix well, let stand for 30 min, centrifuge, and take the precipitate.

[0049] (7) Precipitate dissolution: Add deionized water 20 times the mass of the precipitate prepared in step (6), adjust the pH to 11 with a 1 M Na2CO3 solution, add CaCl2, with the mass ratio of PAA to CaCl2 being 3:10, adjust the pH to 6 with hydrochloric acid, centrifuge, and take the supernatant.

[0050] (8) Separation and purification: Separate and purify the supernatant prepared in step (7) by gel chromatography.

[0051] (9) Freeze-drying: Collect the purified collagen peptide solution in step (8) and conduct freeze-drying.

[0052] Example 2

[0053] A method for preparing alkaline collagen peptides, which comprises the following steps:

[0054] (1) Pretreatment: Add the cleaned tilapia skin to an alkaline solution 6 times its mass and soak for 3 h, wash with distilled water until neutral, add 6 times the mass of deionized water, add lipase accounting for 0.4% of the fish skin mass, enzymatically hydrolyze at 40 °C for 50 min, wash, to obtain pretreated fish skin, wherein the alkaline solution is sodium hydroxide with a mass concentration of 0.05%.

[0055] (2) Enzymatic hydrolysis: Add 2 times the mass of deionized water to the pretreated fish skin prepared in step (1), add papain accounting for 0.1% of the fish skin mass, adjust the temperature to 55 °C, and enzymatically hydrolyze for 3.5 h to obtain a collagen peptide enzymatic hydrolysate.

[0056] (3) Enzyme inactivation: Heat the enzymatic hydrolysate prepared in step (2) at 85 °C for 15 min.

[0057] (4) Decolorization and deodorization: Use an activated carbon fiber membrane to decolorize and deodorize the enzymatic hydrolysate treated by enzyme inactivation in step (3) to obtain a decolorized solution.

[0058] (5) Acid precipitation: Add acetic acid to the decolorized solution prepared in step (4) to adjust the pH to 3, centrifuge, and take the supernatant.

[0059] (6) Polymer precipitation: Add PAA accounting for 0.1% of the fish skin mass to the supernatant prepared in step (5), mix well, let stand for 30 min, centrifuge, and take the precipitate.

[0060] (7)Precipitate dissolution: Add deionized water 20 times the mass of the precipitate prepared in step (6), adjust the pH to 11 with 1M Na2CO3 solution, add CaCl2, with the mass ratio of PAA to CaCl2 being 3:10, adjust the pH to 6 with hydrochloric acid, centrifuge, and take the supernatant;

[0061] (8)Separation and purification: Separate and purify the supernatant prepared in step (7) by gel chromatography;

[0062] (9)Freeze-drying: Collect the purified collagen peptide solution in step (8) and conduct freeze-drying.

[0063] Example 3

[0064] A preparation method of alkaline collagen peptide, which comprises the following steps:

[0065] (1)Pretreatment: Add the cleaned cod skin to an alkaline solution 6 times its mass and soak for 3 h, wash with distilled water until neutral, add 6 times its mass of deionized water, add lipase accounting for 0.3% of the fish skin mass, enzymatically hydrolyze at 40 °C for 50 min, and wash to obtain pretreated fish skin. Among them, the alkaline solution is sodium hydroxide with a mass concentration of 0.05%;

[0066] (2)Enzymatic hydrolysis: Add 2 times the mass of deionized water to the pretreated fish skin prepared in step (1), add neutral protease accounting for 0.8% of the fish skin mass, adjust the temperature to 55 °C, and enzymatically hydrolyze for 3.5 h to obtain a collagen peptide enzymatic hydrolysate;

[0067] (3)Enzyme inactivation: Heat the enzymatic hydrolysate prepared in step (2) at 85 °C for 15 min;

[0068] (4)Decolorization and deodorization: Use an activated carbon fiber membrane to decolorize and deodorize the enzymatic hydrolysate treated by enzyme inactivation in step (3) to obtain a decolorized solution;

[0069] (5)Acid precipitation: Add acetic acid to the decolorized solution prepared in step (4), adjust the pH to 4, centrifuge, and take the supernatant;

[0070] (6)Precipitation with high molecular polymer: Add PAA accounting for 0.1% of the fish skin mass to the supernatant prepared in step (5), mix well, let stand for 30 min, centrifuge, and take the precipitate;

[0071] (7)Precipitate dissolution: Add deionized water 20 times the mass of the precipitate prepared in step (6), adjust the pH to 11 with 1M Na2CO3 solution, add CaCl2, with the mass ratio of PAA to CaCl2 being 3:10, adjust the pH to 6 with hydrochloric acid, centrifuge, and take the supernatant;

[0072] (8) Separation and purification: The supernatant prepared in step (7) is separated and purified by gel chromatography;

[0073] (9) Freeze-drying: The purified collagen peptide solution in step (8) is collected and freeze-dried.

[0074] Example 4

[0075] A method for preparing basic collagen peptides, comprising the following steps:

[0076] (1) Pretreatment: The cleaned cod skin is added to an alkali solution with 6 times its mass and soaked for 3 h, washed with distilled water until neutral, added with 6 times its mass of deionized water, 0.3% of lipase based on the mass of the fish skin is added, and enzymolysis is carried out at 40 °C for 50 min, then washed to obtain pretreated fish skin, wherein the alkali solution is sodium carbonate with a mass concentration of 0.05%;

[0077] (2) Enzymolysis: 2 times the mass of deionized water is added to the pretreated fish skin prepared in step (1), 0.1% of alkaline protease and 0.05% of bromelain based on the mass of the fish skin are added, the temperature is adjusted to 55 °C, and enzymolysis is carried out for 3.5 h to obtain a collagen peptide enzymolysis solution;

[0078] (3) Enzyme inactivation: The enzymolysis solution prepared in step (2) is heated at 85 °C for 15 min;

[0079] (4) Decolorization and deodorization: The enzymolysis solution treated by enzyme inactivation in step (3) is decolorized and deodorized by an activated carbon fiber membrane to obtain a decolorized solution;

[0080] (5) Acid precipitation: Acetic acid is added to the decolorized solution prepared in step (4) to adjust the pH to 3, centrifuged, and the supernatant is taken;

[0081] (6) High molecular polymer precipitation: 0.2% of PAA based on the mass of the fish skin is added to the supernatant prepared in step (5), mixed well, allowed to stand for 30 min, centrifuged, and the precipitate is taken;

[0082] (7) Precipitate dissolution: 20 times the mass of deionized water based on the mass of the precipitate prepared in step (6) is added, the pH is adjusted to 11 with a 1 M Na2CO3 solution, CaCl2 is added, the mass ratio of PAA to CaCl2 is 3:10, the pH is adjusted to 6 with hydrochloric acid, centrifuged, and the supernatant is taken;

[0083] (8) Separation and purification: The supernatant prepared in step (7) is separated and purified by gel chromatography;

[0084] (9) Freeze-drying: The purified collagen peptide solution in step (8) is collected and freeze-dried.

[0085] Comparative Example 1

[0086] Delete the treatment process of lipase in step (1) of Example 1. The fish skin is only treated with alkaline soaking, and other conditions are the same as those in Example 1.

[0087] Comparative Example 2

[0088] Delete steps (6) and (7) of Example 1, and other conditions are the same as those in Example 1.

[0089] Comparative Example 3

[0090] Change the addition amount of PAA in step (6) of Example 1 to 1% of the mass of the fish skin, and other conditions are the same as those in Example 1.

[0091] Comparative Example 4

[0092] Change PAA in step (6) of Example 1 to sodium carboxymethylcellulose, and other conditions are the same as those in Example 1.

[0093] Effect Example:

[0094] Effect Example 1

[0095] Calculate the yield of basic collagen peptide in each example and comparative example. Detect the isoelectric point by isoelectric focusing electrophoresis. The yields and isoelectric points of basic collagen peptide in each example and comparative example are shown in Table 1.

[0096] Table 1: Isoelectric points and yields of each example and comparative example

[0097] Example Yield Isoelectric point Example 1 2.5 10 Example 2 2.3 9.5 Example 3 2.5 9.2 Example 4 2.4 9.2 Comparative Example 1 1.5 9.1 Comparative Example 2 0.8 8.7 Comparative Example 3 0.9 8.3 Comparative Example 4 2.2 8.6

[0098] As can be seen from Table 1, the isoelectric points of Examples 1-4 are all higher than 9, and the yields are all higher than 2. Compared with Example 1, the yield of Comparative Example 1 decreased significantly, indicating that impurities may affect the precipitation effect of PAA on basic collagen peptide; the yields and isoelectric points of the samples prepared in Comparative Example 2 and Comparative Example 3 both decreased, indicating that PAA significantly precipitates basic collagen peptide, and the precipitation effect is highly correlated with its addition amount. Too high or too low addition amount will affect the precipitation effect, thus affecting the yield and isoelectric point of the sample; the isoelectric point of Comparative Example 4 decreased significantly, indicating that the precipitation effect of PAA on basic collagen peptide is significantly better than that of sodium carboxymethylcellulose.

[0099] Effect Example 2: Determination of antioxidant activity

[0100] Refer to the instructions of the ABTS free radical scavenging ability kit, superoxide anion free radical scavenging ability kit and hydroxyl free radical scavenging ability kit to determine the antioxidant activity of basic collagen peptide in each example and comparative example. The test results are shown in Table 2.

[0101] Table 2: Antioxidant activity of basic collagen peptide

[0102]

[0103] The results show that, compared with Comparative Examples 1-4, the alkaline collagen peptides in Examples 1-4 have stronger antioxidant ability, with the ABTS radical scavenging rate higher than 75%, and the superoxide anion radical scavenging rate and hydroxyl radical scavenging rate both higher than 65%.

[0104] Effect Example 3: Evaluation of anti-glycation ability

[0105] For the alkaline collagen peptides obtained in each example and comparative example, experiments were carried out according to the anti-glycation ability evaluation method. The specific evaluation method is as follows: Mix 1 mL of fructose solution (0.5 mol / L) with 1 mL of alkaline collagen peptide, and then add 1 mL of BSA solution (5 mg / mL). All reactants are dissolved in 0.2 mol / L phosphate buffer (pH = 7.4, containing 0.02% sodium azide). Use the reaction solution added with aminoguanidine (with the same concentration as the alkaline collagen peptide) as the positive control group, and the reaction solution of BSA and fructose as the blank group. After culturing in a constant temperature incubator at 37°C for 7 days, take 1.5 mL of the reaction solution and measure the fluorescence intensity under the conditions of excitation wavelength 370 nm and emission wavelength 440 nm. Calculate the inhibition rate of the alkaline collagen peptide on fluorescent AGE according to the following formula:

[0106]

[0107] In the formula: F0: fluorescence intensity of the blank group, F1: fluorescence intensity of the experimental group.

[0108] The results of the anti-glycation experiment are shown in Table 3.

[0109] Table 3: Inhibitory activity of alkaline collagen peptide on AGE

[0110]

[0111]

[0112] The results show that the AGE inhibition rates of the alkaline collagen peptides in Examples 1-4 all reach 40%, showing good anti-glycation effects.

[0113] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these changes and modifications.

Claims

1. A preparation method of an alkaline collagen peptide with antioxidant and anti-glycation effects, characterized in that: The steps of the preparation method are as follows: (1) Pretreatment: Add the cleaned tilapia skin to an alkaline solution with a mass 6 times that of the skin, soak for 3 h, wash with distilled water until neutral, add deionized water with a mass 6 times that of the skin, add lipase at 0.3% of the mass of the fish skin, enzymatically hydrolyze at 40 °C for 50 min, and wash to obtain pretreated fish skin. Among them, the alkaline solution is sodium hydroxide with a mass concentration of 0.05%; (2) Enzymatic hydrolysis: Add deionized water with a mass 2 times that of the pretreated fish skin prepared in step (1), add alkaline protease at 0.2% of the mass of the fish skin, adjust the temperature to 55 °C, and enzymatically hydrolyze for 3.5 h to obtain a collagen peptide enzymatic hydrolysate; (3) Enzyme inactivation: Heat the enzymatic hydrolysate prepared in step (2) at 85 °C for 15 min; (4) Decolorization and deodorization: Use an activated carbon fiber membrane to decolorize and deodorize the enzymatic hydrolysate treated by enzyme inactivation in step (3) to obtain a decolorized solution; (5) Acid precipitation: Add acetic acid to the decolorized solution prepared in step (4) to adjust the pH to 3, centrifuge, and take the supernatant; (6) High molecular polymer precipitation: Add PAA at 0.1% of the mass of the fish skin to the supernatant prepared in step (5), mix well, let stand for 30 min, centrifuge, and take the precipitate; (7) Precipitate dissolution: Add deionized water with a mass 20 times that of the precipitate prepared in step (6), adjust the pH to 11 with a 1 M Na2CO3 solution, add CaCl2, and the mass ratio of PAA to CaCl2 is 3:

10. Adjust the pH to 6 with hydrochloric acid, centrifuge, and take the supernatant; (8) Separation and purification: Separate and purify the supernatant prepared in step (7) by gel chromatography; (9) Freeze-drying: Collect the purified collagen peptide solution in step (8) and perform freeze-drying, The isoelectric point of the alkaline collagen peptide is 10.

2. A preparation method of an alkaline collagen peptide with antioxidant and anti-glycation effects, characterized in that: The steps of the preparation method are as follows: (1) Pretreatment: Add the cleaned tilapia skin to an alkaline solution with a mass 6 times that of the skin, soak for 3 h, wash with distilled water until neutral, add deionized water with a mass 6 times that of the skin, add lipase at 0.4% of the mass of the fish skin, enzymatically hydrolyze at 40 °C for 50 min, and wash to obtain pretreated fish skin. Among them, the alkaline solution is sodium hydroxide with a mass concentration of 0.05%; (2) Enzymatic hydrolysis: Add deionized water with a mass 2 times that of the pretreated fish skin prepared in step (1), add papain at 0.1% of the mass of the fish skin, adjust the temperature to 55 °C, and enzymatically hydrolyze for 3.5 h to obtain a collagen peptide enzymatic hydrolysate; (3) Enzyme inactivation: Heat the enzymatic hydrolysate prepared in step (2) at 85 °C for 15 min; (4) Decolorization and deodorization: Use an activated carbon fiber membrane to decolorize and deodorize the enzymatic hydrolysate treated by enzyme inactivation in step (3) to obtain a decolorized solution; (5) Acid precipitation: Add acetic acid to the decolorized solution prepared in step (4) to adjust the pH to 3, centrifuge, and take the supernatant; (6) High molecular polymer precipitation: Add PAA at 0.1% of the mass of the fish skin to the supernatant prepared in step (5), mix well, let stand for 30 min, centrifuge, and take the precipitate; (7)Precipitate dissolution: Add deionized water 20 times the mass of the precipitate prepared in step (6), adjust the pH to 11 with 1 M Na2CO3 solution, add CaCl2, with the mass ratio of PAA to CaCl2 being 3:10, adjust the pH to 6 with hydrochloric acid, centrifuge, and take the supernatant; (8)Separation and purification: Separate and purify the supernatant prepared in step (7) by gel chromatography; (9)Freeze-drying: Collect the purified collagen peptide solution in step (8) and perform freeze-drying, The isoelectric point of the basic collagen peptide is 9.

5.

3. A preparation method of an alkaline collagen peptide with antioxidant and anti-glycation effects, characterized in that: The steps of the preparation method are as follows: (1)Pretreatment: Add the cleaned cod skin to an alkaline solution 6 times its mass and soak for 3 h, wash with distilled water until neutral, add 6 times its mass of deionized water, add lipase at 0.3% of the fish skin mass, enzymatically hydrolyze at 40 °C for 50 min, and wash to obtain pretreated fish skin. Among them, the alkaline solution is sodium hydroxide with a mass concentration of 0.05%; (2)Enzymatic hydrolysis: Add 2 times the mass of deionized water to the pretreated fish skin prepared in step (1), add neutral protease at 0.8% of the fish skin mass, adjust the temperature to 55 °C, and enzymatically hydrolyze for 3.5 h to obtain a collagen peptide enzymatic hydrolysate; (3)Enzyme inactivation: Heat the enzymatic hydrolysate prepared in step (2) at 85 °C for 15 min; (4)Decolorization and deodorization: Use an activated carbon fiber membrane to decolorize and deodorize the enzymatic hydrolysate treated by enzyme inactivation in step (3) to obtain a decolorized solution; (5)Acid precipitation: Add acetic acid to the decolorized solution prepared in step (4), adjust the pH to 4, centrifuge, and take the supernatant; (6)High molecular polymer precipitation: Add PAA at 0.1% of the fish skin mass to the supernatant prepared in step (5), mix well, let stand for 30 min, centrifuge, and take the precipitate; (7)Precipitate dissolution: Add deionized water 20 times the mass of the precipitate prepared in step (6), adjust the pH to 11 with 1 M Na2CO3 solution, add CaCl2, with the mass ratio of PAA to CaCl2 being 3:10, adjust the pH to 6 with hydrochloric acid, centrifuge, and take the supernatant; (8)Separation and purification: Separate and purify the supernatant prepared in step (7) by gel chromatography; (9)Freeze-drying: Collect the purified collagen peptide solution in step (8) and perform freeze-drying, The isoelectric point of the basic collagen peptide is 9.

2.

4. A method for preparing an alkaline collagen peptide with antioxidant and anti-glycation effects, characterized in that: The steps of the preparation method are as follows: (1)Pretreatment: Add the cleaned cod skin to an alkaline solution 6 times its mass and soak for 3 h, wash with distilled water until neutral, add 6 times its mass of deionized water, add lipase at 0.3% of the fish skin mass, enzymatically hydrolyze at 40 °C for 50 min, and wash to obtain pretreated fish skin. Among them, the alkaline solution is sodium carbonate with a mass concentration of 0.05%; (2)Enzymatic hydrolysis: Add 2 times the mass of deionized water to the pretreated fish skin prepared in step (1), add alkaline protease at 0.1% of the fish skin mass and bromelain at 0.05% of the fish skin mass, adjust the temperature to 55 °C, and enzymatically hydrolyze for 3.5 h to obtain a collagen peptide enzymatic hydrolysate; (3)Enzyme inactivation: Heat the enzymatic hydrolysate prepared in step (2) at 85 °C for 15 min; (4) Decolorization and deodorization: Use activated carbon fiber membrane to decolorize and deodorize the enzymolysis solution treated by enzyme inactivation in step (3) to obtain a decolorized solution; (5) Acid precipitation: Add acetic acid to the decolorized solution prepared in step (4), adjust the pH to 3, centrifuge, and take the supernatant; (6) High molecular polymer precipitation: Add PAA accounting for 0.2% of the mass of fish skin to the supernatant prepared in step (5), mix well, let stand for 30 min, centrifuge, and take the precipitate; (7) Precipitate dissolution: Add deionized water 20 times the mass of the precipitate prepared in step (6), adjust the pH to 11 with 1M Na2CO3 solution, add CaCl2, the mass ratio of PAA to CaCl2 is 3:10, adjust the pH to 6 with hydrochloric acid, centrifuge, and take the supernatant; (8) Separation and purification: Separate and purify the supernatant prepared in step (7) by gel chromatography; (9) Freeze drying: Collect the purified collagen peptide solution in step (8) for freeze drying, The isoelectric point of the basic collagen peptide is 9.

2.

5. The basic collagen peptide with antioxidant and anti-glycation effects prepared by the preparation method according to any one of claims 1 to 4.

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