Alkaline collagen peptide and preparation method thereof

Abstract

The invention belongs to the technical field of active substance extraction, and particularly relates to alkaline collagen peptide and a preparation method thereof. The raw materials are soaked in alkali liquor and then subjected to lipase enzymolysis to remove fat, the impurity removal effect is enhanced, and the product purity is improved. The preparation method comprises the following steps: firstly, removing acidic and neutral collagen peptides by adopting an acid precipitation method, and then selectively precipitating alkaline collagen peptides by adopting a polymer electrolyte precipitation method to prepare the alkaline collagen peptides. Gel chromatography is adopted for separation and purification, and the product purity is improved. The alkaline collagen peptide prepared by the method can be applied to alkaline food.

Description

Technical Field

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

[0002] Collagen peptides are small molecule peptides prepared by hydrolyzing collagen with acid or enzymes. They are composed of two or more amino acids connected by peptide bonds. Collagen peptides are easily absorbed by the human gastrointestinal tract, skin and blood vessels, and are widely used in food, beauty and skin care, biomedicine and other fields. In addition, with the development of social economy and the improvement of public health awareness, weak alkaline foods have gradually become a hot spot in people's lives.

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

[0004] The prior art only reports the preparation method of collagen tripeptides rich in basic amino acids. For example, Chinese patent CN114634549B discloses a collagen tripeptide product and its preparation method and application. This method uses fish skin as raw material, and prepares collagen tripeptides rich in basic amino acids through processes such as crude extraction, refining, protease hydrolysis, and fermentation hydrolysis. The product prepared by this method is only a collagen tripeptide rich in basic amino acids, but not an alkaline collagen protein suitable for alkaline application scenarios. In addition, this method adopts a two-step enzymatic hydrolysis method of protease hydrolysis and fermentation hydrolysis, and the enzymatic hydrolysis process is complicated.

[0005] In view of this, it is necessary to develop a preparation method for alkaline collagen peptides to meet the current market demand. Alkaline collagen peptides have a high isoelectric point and 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 shortcomings of the prior art, the present invention provides a collagen peptide that can be used in alkaline foods and a preparation method thereof through a combination of acid precipitation and polymer high molecular electrolyte precipitation technology.

[0007] The purpose of the present invention can be achieved through the following technical solutions:

[0008] In a first aspect, the present invention provides a method for preparing a basic collagen peptide, comprising the following steps:

[0009] (1) Pretreatment: the cleaned fish skin is added with 2-6 times the mass of alkaline solution and soaked for 3-5h, washed with distilled water until neutral, added with 6 times the mass of deionized water, added with 0.2-0.5% lipase of the mass of the fish skin, and hydrolyzed at 40-50°C for 45-90min, washed, and the pretreated fish skin is obtained;

[0010] (2) enzymolysis: adding 2-6 times the mass of deionized water to the pretreated fish skin prepared in step (1), adding 0.05-2% of the mass of protease of the fish skin, adjusting the temperature to 50-65° C., and enzymolyzing for 2-6 hours to obtain collagen peptide enzymolysis solution;

[0011] (3) Inactivating the enzyme: heating the enzymatic solution prepared in step (2) at 85-100° C. for 10-15 min;

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

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

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

[0015] (7) Precipitate dissolution: add 10-25 times the mass of the precipitate prepared in step (6) of deionized water, adjust the pH to 10-12 with a 1M 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: separating and purifying the supernatant prepared in step (7) by gel chromatography;

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

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

[0019] As an optional manner, in the above preparation method, in step (1), the base is one of sodium hydroxide, sodium carbonate or sodium bicarbonate.

[0020] As an optional manner, in the above preparation method, in step (1), 0.3-0.4% lipase by weight of fish skin is added.

[0021] As an optional manner, in the above preparation method, in step (2), 0.1-0.8% of protease by weight of fish skin is added.

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

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

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

[0025] As an optional manner, in the above preparation method, in step (6), 0.1-0.2% of PAA by weight of fish skin is added to the supernatant prepared in step (5).

[0026] As an optional manner, 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, centrifuged, and the supernatant is taken.

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

[0028] Preferably, the alkaline collagen peptide is used in alkaline food.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

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

[0031] (2) The raw materials of the present invention are soaked in alkali solution and then enzymatically degreased by lipase to enhance the impurity removal effect and improve the product purity.

[0032] (3) The present invention first uses an acid precipitation method to remove acidic and neutral collagen peptides, and then selectively precipitates basic protein peptides through a polymer high molecular electrolyte precipitation method to prepare basic collagen peptides.

[0033] (4) The present invention adopts gel chromatography for separation and purification, thereby improving the purity of the product. DETAILED DESCRIPTION

[0034] The present invention will be further described 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 intended to limit the scope of the present invention.

[0035] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0036] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0037] As used herein, "alkaline collagen peptide" refers to a collagen peptide having a relatively high isoelectric point pH value and being soluble in a slightly alkaline environment.

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

[0039] As used herein, "polyacrylic acid (PAA)" is a polyanionic precipitation agent.

[0040] Preparation Example:

[0041] Example 1

[0042] A method for preparing alkaline collagen peptide comprises the following steps:

[0043] (1) Pretreatment: adding 6 times the mass of alkaline solution to the cleaned tilapia skin and soaking it for 3 hours, washing it with distilled water until it is neutral, adding 6 times the mass of deionized water, adding 0.3% of the mass of lipase to the skin, enzymolysis at 40° C. for 50 minutes, and washing to obtain the pretreated skin, wherein the alkaline 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 0.2% of the mass of alkaline protease of the fish skin, adjust the temperature to 55° C., and perform enzymolysis for 3.5 h to obtain collagen peptide enzymolysis solution;

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

[0046] (4) Decolorization and deodorization: using an activated carbon fiber membrane to decolorize and deodorize the enzymatic hydrolyzate treated with enzyme inactivation in step (3) to obtain a decolorized solution;

[0047] (5) Acid precipitation: adding acetic acid to the decolorizing solution prepared in step (4), adjusting the pH to 3, centrifuging, and collecting the supernatant;

[0048] (6) polymer precipitation: add 0.1% of the weight of fish skin PAA to the supernatant prepared in step (5), mix well, let stand for 30 min, centrifuge, and collect the precipitate;

[0049] (7) Precipitation dissolution: add 20 times the mass of the precipitate prepared in step (6) of deionized water, adjust the pH to 11 with a 1 M 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;

[0050] (8) separation and purification: separating and purifying the supernatant prepared in step (7) by gel chromatography;

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

[0052] Example 2

[0053] A method for preparing alkaline collagen peptide comprises the following steps:

[0054] (1) Pretreatment: adding 6 times the mass of alkaline solution to the cleaned tilapia skin and soaking it for 3 hours, washing it with distilled water until it is neutral, adding 6 times the mass of deionized water, adding 0.4% of the mass of lipase to the skin, enzymolyzing it at 40° C. for 50 minutes, and washing it to obtain the pretreated skin, wherein the alkaline solution is sodium hydroxide with a mass concentration of 0.05%;

[0055] (2) Enzymolysis: Add 2 times the mass of deionized water to the pretreated fish skin prepared in step (1), add 0.1% of papain by mass of the fish skin, adjust the temperature to 55° C., and perform enzymolysis for 3.5 h to obtain a collagen peptide enzymolysis solution;

[0056] (3) Inactivating the enzyme: heating the enzymatic solution prepared in step (2) at 85° C. for 15 min;

[0057] (4) Decolorization and deodorization: using an activated carbon fiber membrane to decolorize and deodorize the enzymatic hydrolyzate treated with enzyme inactivation in step (3) to obtain a decolorized solution;

[0058] (5) Acid precipitation: adding acetic acid to the decolorizing solution prepared in step (4), adjusting the pH to 3, centrifuging, and collecting the supernatant;

[0059] (6) polymer precipitation: add 0.1% of the weight of fish skin PAA to the supernatant prepared in step (5), mix well, let stand for 30 min, centrifuge, and collect the precipitate;

[0060] (7) Precipitation dissolution: add 20 times the mass of the precipitate prepared in step (6) of deionized water, adjust the pH to 11 with a 1 M 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;

[0061] (8) separation and purification: separating and purifying the supernatant prepared in step (7) by gel chromatography;

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

[0063] Example 3

[0064] A method for preparing alkaline collagen peptide comprises the following steps:

[0065] (1) Pretreatment: adding 6 times the mass of alkaline solution to the cleaned cod skin and soaking it for 3 hours, washing it with distilled water until it is neutral, adding 6 times the mass of deionized water, adding 0.3% of the mass of fish skin lipase, enzymolysis at 40° C. for 50 minutes, washing, and obtaining pretreated fish skin, wherein the alkaline solution is sodium hydroxide with a mass concentration of 0.05%;

[0066] (2) Enzymolysis: Add 2 times the mass of deionized water to the pretreated fish skin prepared in step (1), add 0.8% of the mass of fish skin neutral protease, adjust the temperature to 55° C., and perform enzymolysis for 3.5 h to obtain collagen peptide enzymolysis solution;

[0067] (3) Inactivating the enzyme: heating the enzymatic solution prepared in step (2) at 85° C. for 15 min;

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

[0069] (5) Acid precipitation: adding acetic acid to the decolorizing solution prepared in step (4), adjusting the pH to 4, centrifuging, and collecting the supernatant;

[0070] (6) polymer precipitation: add 0.1% of the weight of fish skin PAA to the supernatant prepared in step (5), mix well, let stand for 30 min, centrifuge, and collect the precipitate;

[0071] (7) Precipitation dissolution: add 20 times the mass of the precipitate prepared in step (6) of deionized water, adjust the pH to 11 with a 1 M 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;

[0072] (8) separation and purification: separating and purifying the supernatant prepared in step (7) by gel chromatography;

[0073] (9) Freeze-drying: Collect the collagen peptide solution purified in step (8) and freeze-dry it.

[0074] Example 4

[0075] A method for preparing alkaline collagen peptide comprises the following steps:

[0076] (1) Pretreatment: adding 6 times the mass of alkaline solution to the cleaned cod skin and soaking it for 3 hours, washing it with distilled water until it is neutral, adding 6 times the mass of deionized water, adding 0.3% of the mass of fish skin lipase, enzymolysis at 40° C. for 50 minutes, washing, and obtaining pretreated fish skin, wherein the alkaline solution is sodium carbonate with a mass concentration of 0.05%;

[0077] (2) Enzymolysis: Add 2 times the mass of deionized water to the pretreated fish skin prepared in step (1), add 0.1% alkaline protease and 0.05% bromelain by mass of the fish skin, adjust the temperature to 55° C., and perform enzymolysis for 3.5 h to obtain a collagen peptide enzymolysis solution;

[0078] (3) Inactivating the enzyme: heating the enzymatic solution prepared in step (2) at 85° C. for 15 min;

[0079] (4) Decolorization and deodorization: using an activated carbon fiber membrane to decolorize and deodorize the enzymatic hydrolyzate treated with enzyme inactivation in step (3) to obtain a decolorized solution;

[0080] (5) Acid precipitation: adding acetic acid to the decolorizing solution prepared in step (4), adjusting the pH to 3, centrifuging, and collecting the supernatant;

[0081] (6) polymer precipitation: add 0.2% of the weight of fish skin PAA to the supernatant prepared in step (5), mix well, let stand for 30 min, centrifuge, and collect the precipitate;

[0082] (7) Precipitation dissolution: add 20 times the mass of the precipitate prepared in step (6) of deionized water, adjust the pH to 11 with a 1 M 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;

[0083] (8) separation and purification: separating and purifying the supernatant prepared in step (7) by gel chromatography;

[0084] (9) Freeze-drying: Collect the collagen peptide solution purified in step (8) and freeze-dry it.

[0085] Comparative Example 1

[0086] The treatment process of lipase in step (1) of Example 1 was deleted, and the fish skin was only subjected to alkaline bubble treatment, and other conditions were the same as in Example 1.

[0087] Comparative Example 2

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

[0089] Comparative Example 3

[0090] The amount of PAA added in step (6) of Example 1 was changed to 1% of the fish skin mass, and the other conditions were the same as in Example 1.

[0091] Comparative Example 4

[0092] The PAA in step (6) of Example 1 was replaced by sodium carboxymethyl cellulose, and the other conditions were the same as those of Example 1.

[0093] Effect embodiment:

[0094] Effect Example 1

[0095] The yield of the alkaline collagen peptide of each example and comparative example was calculated. The isoelectric point was detected by isoelectric focusing electrophoresis. The yield and isoelectric point of the alkaline collagen peptide of each example and comparative example are shown in Table 1.

[0096] Table 1: Isoelectric point and yield of each embodiment 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 shown in 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 is significantly reduced, indicating that impurities may affect the precipitation effect of PAA on alkaline collagen peptides; the yields and isoelectric points of the samples prepared in Comparative Examples 2 and 3 are both reduced, indicating that PAA significantly precipitates alkaline collagen peptides, and the precipitation effect is highly related to its addition amount. Too high or too low an addition amount will affect the precipitation effect, thereby affecting the yield and isoelectric point of the sample; the isoelectric point of Comparative Example 4 is significantly reduced, indicating that the precipitation effect of PAA on alkaline collagen peptides is significantly better than that of sodium carboxymethyl cellulose.

[0099] Effect Example 2: Antioxidant Activity Determination

[0100] The antioxidant activity of the alkaline collagen peptides in each example and comparative example was measured with reference to the instructions of the ABTS free radical scavenging ability kit, the superoxide anion free radical scavenging ability kit and the hydroxyl free radical scavenging ability kit. The test results are shown in Table 2.

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

[0102]

[0103] The results showed that compared with comparative examples 1-4, the alkaline collagen peptides in examples 1-4 had stronger antioxidant capacity, with ABTS free radical scavenging rates higher than 75%, and superoxide anion free radical scavenging rates and hydroxyl free radical scavenging rates both higher than 65%.

[0104] Effect Example 3: Evaluation of Anti-glycation Ability

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

[0106]

[0107] Where: F0: fluorescence intensity of blank group, F1: fluorescence intensity of experimental group.

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

[0109] Table 3: Inhibitory activity of basic collagen peptides against AGE

[0110]

[0111]

[0112] The results show that the AGE inhibition rate of the basic collagen peptides in Examples 1-4 all reached 40%, and had a good anti-glycation effect.

[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 equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for preparing alkaline collagen peptide, characterized in that: The following steps are involved: (1) Pre-treatment: The cleaned fish skin is added to an alkaline solution with a mass of 2-6 times and soaked for 3-5h, washed with distilled water until neutral, added with 6 times the mass of deionized water, added with 0.2-0.5% lipase of the fish skin mass, and hydrolyzed at 40-50°C for 45-90min, and washed to obtain the pre-treated fish skin; (2) enzymolysis: adding 2-6 times the mass of deionized water to the pretreated fish skin prepared in step (1), adding 0.05-2% of the mass of protease of the fish skin, adjusting the temperature to 50-65° C., and enzymolyzing for 2-6 hours to obtain a collagen peptide enzymolysis solution; (3) Inactivating the enzyme: heating the enzymatic solution prepared in step (2) at 85-100° C. for 10-15 min; (4) Decolorization and deodorization: using an activated carbon fiber membrane to decolorize and deodorize the enzymatic hydrolyzate treated in step (3) to obtain a decolorized solution; (5) Acid precipitation: Add acetic acid to the decolorizing solution prepared in step (4), adjust the pH to 3-7, centrifuge, and collect the supernatant; (6) polymer precipitation: adding 0.02-0.5% of the weight of fish skin PAA to the supernatant prepared in step (5), mixing, standing for 15-30 minutes, centrifuging, and collecting the precipitate; (7) Precipitate dissolution: Add 10-25 times the mass of the precipitate prepared in step (6) to deionized water, adjust the pH to 10-12 with a 1M Na2CO3 solution, add CaCl2, with a mass ratio of PAA to CaCl2 of 2-5:8-15, adjust the pH to 6-7 with hydrochloric acid, centrifuge, and collect the supernatant; (8) Separation and purification: The supernatant prepared in step (7) is separated and purified by gel chromatography; (9) Freeze-drying: The collagen peptide solution purified in step (8) is collected and freeze-dried.

2. The preparation method according to claim 1, wherein: In step (1), the fish skin is tilapia skin or cod skin.

3. The preparation method according to claim 1, wherein: In step (1), the base is one of sodium hydroxide, sodium carbonate or sodium bicarbonate.

4. The preparation method according to claim 1, wherein: In step (1), 0.3-0.4% lipase by weight of fish skin is added.

5. The preparation method according to claim 1, wherein: In step (2), 0.1-0.8% of the weight of fish skin protease is added.

6. The preparation method according to claim 1, wherein: In step (2), the protease is one or a combination of alkaline protease, papain, bromelain, neutral protease, and flavor protease.

7. The preparation method according to claim 1, wherein: In step (5), acetic acid is added to the decolorizing solution prepared in step (4) to adjust the pH to 3-4.

8. The preparation method according to claim 1, wherein: In step (6), 0.1-0.2% of the weight of fish skin PAA is added to the supernatant prepared in step (5).

9. The preparation method according to claim 1, wherein: In step (7), 20 times the mass of deionized water as the precipitate prepared in step (6) was added, the pH was adjusted to 11 with a 1M Na2CO3 solution, CaCl2 was added, the mass ratio of PAA to CaCl2 was 3:10, the pH was adjusted to 6 with hydrochloric acid, the mixture was centrifuged, and the supernatant was collected.

10. Basic collagen peptide prepared by the preparation method according to any one of claims 1 to 9.

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