Collagen peptide as well as preparation method and application thereof

Abstract

The invention relates to the technical field of collagen peptide preparation, and discloses a collagen peptide as well as a preparation method and application thereof. The preparation method of the collagen peptide comprises the following steps: adding water into Hefang crucian carp skin, regulating the Hefang crucian carp skin into feed liquid with the optimal pH value of protease, then adding the protease into the feed liquid for enzymolysis, inactivating after enzymolysis, and centrifuging to take supernate, so as to obtain the collagen peptide. The protease comprises one or more than two of alkaline protease, animal protease and neutral protease. According to the present invention, the He-prescription crucian carp skin is adopted as the preparation raw material, the protease most suitable for the raw material is adopted to perform enzymolysis to prepare the collagen peptide, the optimal enzymolysis process parameters are determined, and compared with the conventional tilapia skin collagen peptide, Hunan crucian carp skin collagen peptide and collagen tripeptide in the antioxidant function, the antioxidant activity is significantly improved, and the antioxidant activity is significantly improved; therefore, the method for preparing the crucian carp skin collagen peptide with remarkable antioxidant activity is established, and the application range of crucian carp processing byproducts is further expanded.

Description

Technical Field

[0001] The present application relates to the technical field of collagen peptide preparation, and particularly relates to a collagen peptide prepared from hybrid crucian carp, a preparation method thereof, and an application thereof. Background Art

[0002] Collagen peptide is a product obtained by protease hydrolysis of collagen, and has advantages such as good biocompatibility, thermal stability, absorbability, water retention, antioxidant property, etc., and is widely used in multiple fields such as food, cosmetics, and health products.

[0003] Currently, the sources of collagen peptides on the market are relatively diverse, mainly including marine-derived and terrestrial-derived processing by-products. For example, pigskin and cowhide are common extraction sources of collagen peptides, and their yields are rich and the costs are relatively low. However, using terrestrial-derived processing by-products, such as cowhide and pigskin, to prepare collagen peptides often has animal disease risks. Using marine-derived processing by-products, such as fish skin, to prepare collagen peptides has higher safety, and the amino acid sequence of fish skin collagen peptide has a higher similarity with that of humans, does not contain specific antigens that may cause allergies in mammals, and is more suitable for sensitive populations. At the same time, fish skin collagen peptide is more easily hydrolyzed into small peptides, has high bioavailability, is rich in specific functional amino acids, and is convenient for processing.

[0004] Among the prepared collagen peptides, collagen tripeptide has a relatively small relative molecular mass and is formed by three amino acids connected by peptide bonds, so it can be quickly absorbed and utilized by the human body and has a wide range of applications. Tilapia is one of the widely cultured species in China, with high yields and rich in nutrients such as protein. The fish skin peptide prepared from tilapia skin contains a variety of functional amino acids and has certain potential in regulating human physiological functions. The collagen peptide prepared from tilapia skin also has a wide range of applications. However, these two types of collagen peptides have certain deficiencies in antioxidant function. Although collagen tripeptide is absorbed quickly, in the complex human body environment, the persistence of its antioxidant effect is poor and it is difficult to exert its efficacy stably for a long time. The processing and utilization methods of tilapia are single, the utilization of protein resources is insufficient, and the antioxidant activity intensity of tilapia skin peptide still has room for improvement compared with some antioxidants in ideal states. Summary of the Invention

[0005] In view of this, the purpose of the present application is to provide a collagen peptide and a preparation method thereof, so that the prepared collagen peptide can significantly improve its antioxidant performance and has a high polypeptide yield and hydrolysis degree;

[0006] Another purpose of the present application only lies in providing the application of the above-mentioned collagen peptide in the preparation of products with antioxidant performance.

[0007] To solve the above technical problems / achieve the above object or at least partially solve the above technical problems / achieve the above object, as the first aspect of the present application, a method for preparing collagen peptides is provided, including:

[0008] Mix the combined Fangji carp skin with water and adjust it to a liquid material with the optimal pH value of alkaline protease, then add alkaline protease to the said liquid material for enzymatic hydrolysis. After enzymatic hydrolysis, inactivate it, centrifuge to obtain the supernatant, and obtain the said collagen peptides.

[0009] Optionally, the enzyme addition amount of the alkaline protease is 0.6 - 1.0%.

[0010] Optionally, the optimal pH value of the alkaline protease is 8 - 11.

[0011] Optionally, the enzymatic hydrolysis temperature of the alkaline protease is 45 - 55 °C, and the enzymatic hydrolysis time is more than 2.5 h.

[0012] Optionally, the preparation method further includes; ultrafiltering the collagen peptides and selecting the collagen peptide component with a molecular weight cut-off of < 3 kDa.

[0013] As the second aspect of the present application, collagen peptides prepared by the preparation method described in the present application are provided.

[0014] As the third aspect of the present application, the application of the collagen peptides described in the present application in the preparation of products with antioxidant functions is provided.

[0015] Optionally, the products include cosmetics, foods, health products, additives, and medicines.

[0016] As the fourth aspect of the present application, a product with antioxidant function is provided, including the collagen peptides described in the present application.

[0017] Optionally, it further includes other functional active ingredients that do not react with the collagen peptides described in the present application, and / or non-functional auxiliary components.

[0018] The present application uses the combined Fangji carp skin as the preparation raw material, adopts the protease most suitable for this raw material for enzymatic hydrolysis to prepare collagen peptides, and at the same time determines the optimal enzymatic hydrolysis process parameters. Compared with conventional tilapia skin collagen peptides, Xiangji carp skin collagen peptides, and collagen tripeptides in terms of antioxidant function, the antioxidant activity is significantly improved. Thus, the present application establishes a method for preparing combined Fangji carp skin collagen peptides with significant antioxidant activity, further expanding the application scope of combined Fangji carp processing by-products. Description of the Drawings

[0019] The accompanying drawings of the specification, which form a part of this application, are used to provide a further understanding of this application. The schematic embodiments of this application and their descriptions are used to explain this application and do not constitute an improper limitation to this application;

[0020] Figure 1 The shown are the results of DPPH, ABTS, and superoxide radical scavenging activities of collagen peptides with different molecular weights (W1 - W3) from hybrid common carp and collagen peptides with different molecular weights (J1 - J3) from Xiang carp; different lowercase letters indicate significant differences between polypeptide solutions at the same concentration (P < 0.05);

[0021] Figure 2 The shown is the comparison of antioxidant activities of four collagen peptides at the same concentration; different lowercase letters indicate significant differences between polypeptide solutions at the same concentration (P < 0.05);

[0022] Figure 3 The shown is the comparison of polypeptide yields and degrees of hydrolysis of different enzymolysis solutions from hybrid common carp skin; different lowercase letters indicate significant differences between polypeptide solutions at the same concentration (P < 0.05);

[0023] Figure 4 The shown is the effect of enzyme dosage on polypeptide yield and degree of hydrolysis; different lowercase letters indicate significant differences between polypeptide solutions at the same concentration (P < 0.05);

[0024] Figure 5 The shown is the effect of enzymolysis temperature on polypeptide yield and degree of hydrolysis; different lowercase letters indicate significant differences between polypeptide solutions at the same concentration (P < 0.05);

[0025] Figure 6 The shown is the effect of enzymolysis time on polypeptide yield and degree of hydrolysis; different lowercase letters indicate significant differences between polypeptide solutions at the same concentration (P < 0.05);

[0026] Figure 7 The shown is the effect of enzymolysis pH on polypeptide yield and degree of hydrolysis; different lowercase letters indicate significant differences between polypeptide solutions at the same concentration (P < 0.05). Detailed implementation manners

[0027] The present application discloses a collagen peptide, its preparation method and application. Those skilled in the art can draw on the content of this article and appropriately modify the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in this application. The products, processes and applications described in this application have been described through preferred embodiments. Relevant personnel can obviously make changes or appropriate changes and combinations to the methods described in this article without departing from the content, spirit and scope of this application to implement and apply the technology of this application. Obviously, the described embodiments are part of the embodiments of this application, rather than all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without creative work belong to the scope of protection of this application.

[0028] It should be noted that in this article, if relational terms such as "first" and "second", "step 1" and "step 2", and "(1)" and "(2)" appear, they are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variant thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements not only includes those elements, but also includes other elements not expressly listed, or also includes elements inherent to such process, method, article or device. Without further limitation, an element defined by the statement "including one..." does not exclude the existence of additional identical elements in the process, method, article or device including the said element. At the same time, without conflict, the embodiments in this application and the features in the embodiments can be combined with each other.

[0029] Hybrid crucian carp is a new high-quality crucian carp variety cultivated by Hunan Normal University through distant hybridization. It uses Japanese white crucian carp (♀) as the female parent and red crucian carp (♂) as the male parent, and is cultivated through distant hybridization technology. It has the characteristics of fast growth, strong stress resistance and excellent meat quality, and is a new excellent variety of freshwater fish in the field of aquaculture. Through research, the present application has found that using the skin of hybrid crucian carp as a preparation raw material can obtain collagen peptides with higher antioxidant activity, which can solve the problem of insufficient antioxidant activity of conventional tilapia skin collagen peptides and collagen tripeptides. At present, there is no report on the preparation of collagen peptides using the skin of hybrid crucian carp:

[0030] Therefore, in the first aspect of this application, a preparation method of a collagen peptide is provided, including:

[0031] Add the skin of hybrid crucian carp to water and adjust it to a feed liquid with the optimal pH value for protease. Then, add protease to the feed liquid for enzymatic hydrolysis. After enzymatic hydrolysis, inactivate the enzyme, centrifuge to obtain the supernatant, and obtain the collagen peptide. The protease includes one or more of alkaline protease, animal protease, and neutral protease.

[0032] In some embodiments of the present application, compared with animal protease, neutral protease, flavor protease, and papain, the collagen peptide prepared by enzymatic hydrolysis with alkaline protease has the highest yield and also has a better degree of hydrolysis. Therefore, in the examples of the present application, alkaline protease is used to verify the antioxidant activity and establish the optimal enzymatic hydrolysis process. The alkaline protease is purchased from Nanning Pangbo Bioengineering Co., Ltd., with an enzyme activity of 200,000 u / g, and the components are bacillus licheniformis protease and glucose. The enzyme addition amount is 0.6 - 1.0%, such as 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, etc. Within this enzyme addition amount range, the prepared collagen peptide has both a high yield and a degree of hydrolysis.

[0033] In some other embodiments of the present application, the optimal pH value of the alkaline protease is 8 - 11, such as 8, 9, 10, 11, etc. Within this pH value range, the prepared collagen peptide has both a high yield and a degree of hydrolysis.

[0034] In some other embodiments of the present application, the temperature of the enzymatic hydrolysis is 45 - 55°C, such as 45°C, 50°C, 55°C, etc.; the time of the enzymatic hydrolysis is more than 2.5 h, and further is 2.5 - 3.5 h, such as 2.5 h, 3 h, 3.5 h, etc.; within this enzymatic hydrolysis temperature range and / or enzymatic hydrolysis time range, the prepared collagen peptide has both an extremely high yield and a degree of hydrolysis.

[0035] In some embodiments of the present application, the preparation method further includes; ultrafiltration of the collagen peptide, and selecting the collagen peptide component with a molecular weight cut-off of <3 kDa; through the comparison of four components with a molecular weight cut-off of >10 kDa, a molecular weight cut-off of 5 - 10 kDa, a molecular weight cut-off of 3 - 5 kDa, and a molecular weight cut-off of <3 kDa for ultrafiltration, the collagen peptide component with a molecular weight cut-off of <3 kDa has significantly higher antioxidant activities in DPPH radical scavenging activity, ABTS radical scavenging activity, and superoxide anion radical scavenging activity than other molecular weight cut-off components.

[0036] In some embodiments of the present application, the hybrid crucian carp skin also includes pre-treatment before enzymatic hydrolysis to prepare collagen peptide. The pre-treatment includes: drying and crushing the fresh fish skin after removing the fish meat, and then, after treatment for removing impurity proteins and fat, drying and crushing it as the raw material for enzymatic hydrolysis to prepare collagen peptide.

[0037] In some other embodiments of the present application, the impurity proteins are removed by soaking fish skin with sodium hydroxide solution. For example, 0.1 mol / L sodium hydroxide is used, the material-liquid ratio can be selected as 1:30 (w / v), soaked for 12 h, and the sodium hydroxide solution is replaced every 6 h.

[0038] In some other embodiments of the present application, the degreasing is carried out by soaking fish skin with isopropanol. For example, 10% isopropanol is used, the material-liquid ratio can be selected as 1:30 (w / v), soaked for 24 h, and the isopropanol is replaced every 12 h.

[0039] In the second aspect of the present application, a collagen peptide prepared by the preparation method described in the present application is provided. In some embodiments of the present application, the molecular weight of the collagen peptide < 3 kDa.

[0040] In the third aspect of the present application, the present application compared the antioxidant activities of collagen peptides prepared from hybrid crucian carp skin, collagen peptides prepared from Hunan crucian carp skin, and purchased tilapia skin collagen peptides and collagen tripeptides. The results showed that the collagen peptide obtained by enzymatic hydrolysis of hybrid crucian carp skin in the present application was significantly higher than the antioxidant activities of other fish skin collagen peptides in terms of DPPH radical scavenging activity, ABTS radical scavenging activity, and superoxide anion radical scavenging activity. Based on this excellent technical effect, the present application provides the application of the collagen peptide in the preparation of products with antioxidant functions. Among them, the products include but are not limited to cosmetics, foods, health products, additives, and pharmaceuticals, and the form of the products can be determined according to the excipients used, including but not limited to freeze-dried powder, gel, solution, granule, paste, capsule, tablet, and so on.

[0041] In the fourth aspect of the present application, a product with antioxidant function is provided, including the collagen peptide described in the present application, and the amount of the collagen peptide in the product can be adjusted according to actual needs.

[0042] In some embodiments of the present application, the product can also select to add other functional active ingredients that do not react with the collagen peptide described in the present application according to the additional effects required or the product performance expected to be achieved, such as functional active ingredients for improving blood lipid, functional active ingredients for improving blood sugar, anti-inflammatory functional active ingredients, moisturizing functional active ingredients, whitening functional active ingredients, anti-wrinkle functional active ingredients, and so on; at the same time, the product can also include or not include non-functional excipient components, such as thickeners, preservatives, stabilizers, pH regulators, wetting agents, and so on.

[0043] In each group of comparative experiments provided in this application, unless otherwise specified, except for the differences pointed out in each group, other experimental conditions, materials, etc. are kept consistent to ensure comparability. In addition, the materials used in this application can all be obtained through commercial channels.

[0044] The following further describes a collagen peptide provided in this application, its preparation method and applications.

[0045] Example 1:

[0046] 1. Take fish skin

[0047] Hybrid common carp: Purchased from the local aquatic product market in Changsha, Hunan; take fresh fish skin, dry it at 60 °C after removing the fish meat, cut it into pieces and set aside.

[0048] Remove miscellaneous proteins: Add 0.1 mol / L NaOH at a material-liquid ratio of 1:30 (w / v), soak for 12 h, and change the liquid every 6 h.

[0049] Degrease: Add 10% isopropanol at a material-liquid ratio of 1:30 (w / v), soak for 24 h, and change the liquid every 12 h. The treated fish skin turns white and swells, dry it at 60 °C and break it into pieces for later use.

[0050] 2. Enzymatic hydrolysis

[0051] Weigh a certain amount of hybrid common carp skin powder and place it in a conical flask. Add ultrapure water at a material-liquid ratio of 1:15, adjust the pH value to 9 - 11, add 0.6 - 1.0% alkaline protease (Nanning Pangbo Bioengineering Co., Ltd., 200,000 u / g, the components are bacillus licheniformis protease and glucose), carry out enzymatic hydrolysis at 45 - 55 °C for at least 2.5 h. After the reaction is completed, inactivate at 95 °C for 15 min, cool and centrifuge to obtain the supernatant, and obtain the collagen peptide.

[0052] Example 2:

[0053] 1. Grouping

[0054] Hybrid common carp: Purchased from the local aquatic product market in Changsha, Hunan, and prepare collagen peptide according to the method of Example 1; use alkaline protease to carry out enzymatic hydrolysis on hybrid common carp skin, and the enzymatic hydrolysis conditions are enzyme addition amount of 0.8%, enzymatic hydrolysis time of 3 h, pH 9, and enzymatic hydrolysis temperature of 50 °C.

[0055] Xiang carp: Purchased from Zhumatang Farmers' Market, and prepare collagen peptide by the same method as in Example 1 for hybrid common carp;

[0056] Tilapia skin collagen peptide is purchased from Shandong Baolijia Biotechnology Co., Ltd.;

[0057] Collagen tripeptide is purchased from Hunan Filler Biotechnology Co., Ltd.

[0058] 2. Detection of antioxidant activity

[0059] (1) DPPH free radical scavenging activity

[0060] Weigh 0.0789 g of DPPH (1,1-diphenyl-2-picrylhydrazyl) powder, dissolve it with absolute ethanol and transfer it to a 100 ml volumetric flask, make up the volume to 100 ml to prepare a 0.2 mol / L DPPH stock solution. Dilute the DPPH stock solution 10 times with absolute ethanol and dilute the sample by an appropriate multiple. Use a pipette to aspirate 1 mL of the sample solution and 1 mL of DPPH into a 2 mL Eppendorf tube for the experimental group, aspirate 1 mL of the sample solution and 1 mL of absolute ethanol for the control group, and aspirate 1 mL of absolute ethanol and 1 mL of DPPH for the CK group, and react in the dark for 30 min. After the reaction, centrifuge at 7500 r / min for 5 min, aspirate 200 μL of the supernatant into an enzyme-linked immunosorbent assay (ELISA) plate, repeat the spotting 3 times for each group and take the average value, measure the OD value at a wavelength of 517 nm, and substitute it into the standard curve to obtain the DPPH free radical scavenging rate in the sample.

[0061] R 1 = 1 - (A - A 0 ) / A CK

[0062] In the formula: R 1 : DPPH free radical scavenging rate; A: absorbance of the experimental group; A 0 : absorbance of the control group; A CK : absorbance of the blank group.

[0063] (2) ABTS free radical scavenging activity

[0064] Weigh 3 mg of ABTS, add 0.735 mL of water to obtain an ABTS stock solution; weigh 1 mg of K 2 S 2 O 8 , add 1.43 mL of water to obtain a K 2 S 2 O 8 stock solution, mix the two solutions in a ratio of 1:1 and dilute 40 - 60 times, and measure the OD value to be around 0.7. For the experimental group, take 0.8 mL of ABTS and 0.2 mL of the sample, mix well, let stand for 6 min, spot the sample to obtain A 0 ; for the control group, take 0.8 mL of ABTS and 0.2 mL of 95% ethanol, mix well, let stand for 6 min, spot the sample to obtain A. Calculate:

[0065]

[0066] In the formula: R 2 : ABTS free radical scavenging rate; A: absorbance of the experimental group; A 0 : absorbance of the control group

[0067] (3) Superoxide anion radical scavenging activity

[0068] Take 1 mL of the sample to be tested and mix it evenly with 4.5 mL of 0.05 M Tris-HCl (pH = 8.2) solution. React at a constant temperature of 25 °C in a water bath for 20 min. Add 0.3 mL of 0.05 M pyrogallol and react at a constant temperature of 25 °C in a water bath for 4 min. Add 1 mL of 8 mmol / L HCl to terminate the reaction, and measure the absorbance at a wavelength of 325 nm. Calculate the scavenging rate according to the following formula:

[0069]

[0070] In the formula: R 3 : Superoxide anion radical scavenging rate; A: Absorbance of the experimental group; B: Absorbance of the control group (pure water replaces pyrogallol); C: Absorbance of the blank group (pure water replaces the sample).

[0071] 3. Ultrafiltration retention of different molecular weights

[0072] Select ultrafiltration membranes with a molecular weight cut-off of 10 kDa, 5 kDa, and 3 kDa to separate the collagen peptides of hybrid crucian carp. Use the permeate as the initial feed solution for the next stage of ultrafiltration to obtain peptide components with different molecular weight ranges. Separate the hydrolyzed crude peptides successively through ultrafiltration membranes with molecular weight cut-offs of 10 kDa, 5 kDa, and 3 kDa,

[0073] The collagen peptides from hybrid crucian carp skin are divided into three components: W1 (molecular weight < 3 kDa), W2 (molecular weight 3 - 5 kDa), and W3 (molecular weight 5 - 10 kDa);

[0074] The collagen peptides from Xiang crucian carp skin are divided into three components: J1 (molecular weight < 3 kDa), J2 (molecular weight 3 - 5 kDa), and J3 (molecular weight 5 - 10 kDa).

[0075] Collect the permeates and retentates of each component for antioxidant activity detection.

[0076] 4. Results

[0077] (1) Antioxidant activity detection results of ultrafiltration retention of different molecular weights

[0078] Table 1 DPPH radical scavenging rate of collagen peptides with different molecular weights at 3 mg / ml

[0079]

[0080] Table 2 ABTS radical scavenging rate of collagen peptides with different molecular weights at 1 mg / ml

[0081]

[0082] Table 3 Superoxide anion radical scavenging rate of 1 mg / ml collagen peptides with different molecular weights

[0083]

[0084] As shown in the above Tables 1-3 and the corresponding Figure 1 It can be seen that the antioxidant activities of the collagen peptides W1 and J1 of hybrid common carp and Xiang carp are relatively high in their respective groups; the collagen peptides of different molecular weights of hybrid common carp are generally better than those of Xiang carp, especially the antioxidant property of the <3Kda molecular weight is the most obvious. It is speculated in this application that peptides with lower molecular weights may be more likely to approach free radicals to provide protons or electrons, and the free radical scavenging efficiency is higher. Generally speaking, the collagen peptides W1 and J1 have the highest antioxidant activity, and the collagen peptide W1 of hybrid common carp is the most prominent.

[0085] (2) Detection results of antioxidant properties of collagen peptides from different sources

[0086] Table 4 DPPH radical scavenging rate of different peptides at 15 mg / ml

[0087]

[0088] Table 5 ABTS radical scavenging rate of different peptides at 1 mg / ml

[0089]

[0090] Table 6 Superoxide radical scavenging rate of different peptides at 1 mg / ml

[0091]

[0092] The antioxidant activities of skin peptides of Xiang carp (molecular weight < 3KDa), skin peptides of hybrid common carp (molecular weight < 3KDa), tilapia skin peptides and collagen tripeptides were detected at the same concentration. According to Tables 4-6 and their corresponding columnar Figure 2 It can be clearly seen that the DPPH radical scavenging rate and ABTS radical scavenging rate of the skin peptides of hybrid common carp are significantly higher than those of the other three skin peptides (P<0.05), and the superoxide anion scavenging rate is significantly higher than that of the skin peptides of Xiang carp (<3Kda) and tilapia skin peptides (P<0.05). Among them, the scavenging ability of ABTS radicals is the strongest, and the scavenging rate at 1 mg / ml is 82.40%, indicating that the skin peptides of hybrid common carp prepared by the enzymatic hydrolysis process in this application have strong antioxidant activity.

[0093] Example 3:

[0094] (1) Comparison of enzymatic hydrolysis effects of different proteases

[0095] Weigh a certain amount of powdered Hefang crucian carp skin and place it in a conical flask. Add ultrapure water according to a solid-liquid ratio of 1:15. Adjust the reaction temperature and pH according to Table 7 below. Add a certain amount of protease and enzymatically hydrolyze for 3 h. After the reaction is completed, inactivate at 95 °C for 15 min. After cooling, centrifuge and take the supernatant. Using the degree of hydrolysis and the polypeptide yield as evaluation indicators, compare the enzymatic hydrolysis effects of different proteases.

[0096] Table 7 Hydrolysis conditions of five proteases

[0097]

[0098] (2) Determination of polypeptide yield

[0099] Preparation of Folin-phenol solution A:

[0100] Before use, reagent A is prepared by mixing A and B at a volume ratio of 50:1 and is limited to use on the same day.

[0101] Reagent A: Weigh 10 g of sodium carbonate, 2 g of sodium hydroxide, and 0.25 g of potassium sodium tartrate. Dissolve with distilled water and make up to 500 mL in a volumetric flask.

[0102] Reagent B: Weigh 0.5 g of copper sulfate pentahydrate, dissolve with water, and make up to 100 mL in a volumetric flask.

[0103] Preparation of Folin-phenol standard curve:

[0104] Table 8 Folin-phenol reagent table

[0105]

[0106] Weigh a certain amount of bovine serum albumin (BSA) and prepare a standard solution with a concentration of 250 μg / mL.

[0107] Add reagents according to Table 5, measure the absorbance at 640 nm, and make a standard curve.

[0108]

[0109] (3) Detection of degree of hydrolysis

[0110] OPA reagent (prepared immediately before use): First, completely dissolve 7.62 g of sodium tetraborate and 200 mg of SDS in 150 mL of distilled water. Then dissolve 160 mg of OPA in 4 mL of absolute ethanol. After mixing, add 176 mg of DTT to solution 1. After mixing the above solutions, make up to 200 mL with distilled water to obtain the OPA reagent.

[0111] Serine standard solution (1 mM / L): Weigh 105 mg of serine, dissolve with deionized water, and make up to 1000 mL.

[0112] Drawing of the standard curve: Take 0, 100, 200, 300, 400, 500 μL of serine standard solution into 1 mL centrifuge tubes, supplement the volume to 500 μL with deionized water. Take 400 μL of the standard solution into 5 mL test tubes, add 3 mL of OPA reagent, mix well, react at room temperature for 2 min, and measure the absorbance at 340 nm (using deionized water as the reference). Use the measured absorbance and serine concentration to make a standard curve.

[0113] Determination of the degree of hydrolysis of the sample: Take 1 mL of the hydrolyzate and dilute it 150 times with deionized water (the measured value of the absorbance should fall within the range of the standard curve, and the specific dilution factor can be adjusted according to the absorbance value). Take 400 μL of the diluted hydrolyzate, add 3 mL of OPA reagent, mix well, react at room temperature for 2 min, and measure the absorbance at 340 nm. Each treatment is repeated 5 times. According to the average value of the measured absorbance, find Cserine-NH 2 (mM / L) from the standard curve, and calculate the degree of hydrolysis according to the following formula.

[0114]

[0115] Note: SerineNH 2 : Content of SerineNH 2 in each gram of protein (mmol / g); N: Dilution factor; V: Volume of the supernatant (L); X: Mass of the sample (g); P: Protein content in the sample (%); The constants α and β are taken as 1 and 0.4 respectively.

[0116] (3) Results

[0117] Polypeptide yield of different composite crucian carp skin hydrolysates

[0118] Table 9 Polypeptide yield of different composite crucian carp skin hydrolysates

[0119]

[0120] Table 10 Detection of the degree of hydrolysis of different hydrolysates

[0121]

[0122] Combined with Table 9 and Table 10 and their corresponding column analysis charts Figure 3 The results show that the overall trend of the polypeptide yield is alkaline protease > animal protease > neutral protease > flavor protease > papain, and the overall trend of the degree of hydrolysis is flavor protease > animal protease > alkaline protease > neutral protease > papain. Taking the polypeptide yield in the hydrolysate as the main consideration, the polypeptide yield of the alkaline protease hydrolysate is the highest, and its hydrolysis ability is moderate. The flavor protease has the strongest hydrolysis ability, but its polypeptide yield is low, and often the higher the degree of hydrolysis, the heavier the fishy and bitter taste of the hydrolysate. Considering the polypeptide yield and the degree of hydrolysis comprehensively, alkaline protease is selected as the optimal one.

[0123] Example 4:

[0124] Alkaline protease was selected as the optimal enzyme. Taking the polypeptide yield and hydrolysis degree as evaluation indexes, the results of enzymatic hydrolysis of Hybrid crucian carp skin were compared under different enzyme dosages, different enzymatic hydrolysis temperatures, different enzymatic hydrolysis times and different enzymatic hydrolysis pH values.

[0125] Weigh a certain amount of Hybrid crucian carp skin powder and place it in a conical flask. Add ultrapure water according to the solid-liquid ratio of 1:15, and preheat it for 10 min at different temperatures of alkaline protease. Use NaOH or HCl solution to adjust to different pH values of the enzyme. Then add a certain amount of hydrolytic enzyme to start the reaction and carry out constant-temperature enzymatic hydrolysis at different temperatures of the enzyme. After the reaction is completed, immediately take out the hydrolysis mixture from the water bath and inactivate it at 95 °C for 15 min. After cooling to room temperature, centrifuge at 8000 r / min for 15 min, and the supernatant is the collagen peptide enzymatic hydrolysate.

[0126] The conditions of single-factor experiment are as follows:

[0127] Enzyme dosage: Under the conditions of temperature 50 °C, pH 9 and enzymatic hydrolysis time 3 h, the enzyme dosages are 0.2%, 0.4%, 0.6%, 0.8% and 1.0% respectively.

[0128] Temperature: Under the conditions of enzyme dosage 0.6%, enzymatic hydrolysis time 3 h and pH 9, the enzymatic hydrolysis temperatures are 30, 40, 50, 60 and 70 °C respectively.

[0129] Time: Under the conditions of enzyme dosage 0.6%, temperature 50 °C and pH 9, enzymatic hydrolysis is carried out for 1.5 h, 2.0 h, 2.5 h, 3.0 h and 3.5 h respectively.

[0130] pH: Under the conditions of enzyme dosage 0.6%, temperature 50 °C and enzymatic hydrolysis time 3 h, the pH values are 7, 8, 9, 10 and 11 respectively.

[0131] The detection methods of polypeptide yield and hydrolysis degree are the same as those in Example 3.

[0132] (1) Comparison results of enzyme dosage

[0133] Table 11 Polypeptide yields of enzymatic hydrolysates of Hybrid crucian carp skin with different enzyme dosages

[0134]

[0135] Table 12 Hydrolysis degrees of enzymatic hydrolysates of Hybrid crucian carp skin with different enzyme dosages

[0136]

[0137] Combined with Table 11, Table 12 and their corresponding line analysis charts Figure 4It can be seen from the results that the degree of hydrolysis of the enzymatic hydrolysate increases with the increase of the enzyme dosage, while the polypeptide yield tends to remain unchanged with the increase of the enzyme dosage, reaching the highest value at 0.8% in both cases. When the enzyme dosage is in the range of 0.6 - 1.0%, both the polypeptide yield and the degree of hydrolysis can be ensured to be relatively high, with 0.8% being the best. Enzyme dosages in other ranges cannot guarantee a high level.

[0138] (2) Results of comparison of enzymatic hydrolysis temperature

[0139] Table 13 Polypeptide yields of enzymatic hydrolysates of Hefang crucian carp skin at different temperatures

[0140]

[0141] Table 14 Degrees of hydrolysis of enzymatic hydrolysates of Hefang crucian carp skin at different temperatures

[0142]

[0143] Combined with Table 13 and Table 14 and their corresponding line analysis diagrams Figure 5 It can be seen from the results that the polypeptide yield and the degree of hydrolysis of the enzymatic hydrolysate first increase and then decrease with the increase of the enzymatic hydrolysis temperature, and are at a relatively high level in the range of 45 - 55°C, reaching the maximum value at 50°C.

[0144] (3) Results of comparison of enzymatic hydrolysis time

[0145] Table 15 Polypeptide yields of enzymatic hydrolysates of Hefang crucian carp skin at different times

[0146]

[0147] Table 16 Degrees of hydrolysis of enzymatic hydrolysates of Hefang crucian carp skin at different times

[0148]

[0149]

[0150] Combined with Table 15 and Table 16 and their corresponding line analysis diagrams Figure 6 It can be seen from the results that the degree of hydrolysis of the enzymatic hydrolysate first increases and then tends to be stable with the increase of the enzymatic hydrolysis time, reaching the maximum value at 3.5 h, and the polypeptide yield changes little with the increase of the enzymatic hydrolysis time, reaching the maximum value at 3 h; overall, an enzymatic hydrolysis time of more than 2.5 h can ensure a relatively high level. Considering efficiency, an enzymatic hydrolysis time of 2.5 - 3.5 h or 3 - 3.5 h is relatively high.

[0151] (4) Results of comparison of enzymatic hydrolysis pH

[0152] Table 17 Polypeptide yields of enzymatic hydrolysates of Hefang crucian carp skin at different pH values

[0153]

[0154] Table 18 Degree of hydrolysis of enzymatically hydrolyzed solution of crucian carp skin at different pH values

[0155]

[0156] Combined with Table 17 and Table 18 and their corresponding line analysis diagrams Figure 7 The results show that the polypeptide yield and degree of hydrolysis of the enzymatically hydrolyzed solution first increase and then decrease with the increase of the enzymatic hydrolysis pH. They are both at a relatively high level in the pH range of 8 - 11 or 9 - 11, and reach the maximum value at pH 9. Therefore, pH 9 is selected as the optimal enzymatic hydrolysis pH.

[0157] The above are only specific embodiments of the present application, enabling those skilled in the art to understand or implement the present application. Various modifications to these embodiments will be obvious to those skilled in the art. The general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to these embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A method for preparing collagen peptide, characterized in that: include: The crucian carp skin is added with water and adjusted to a feed liquid with an optimal pH value for protease, and then protease is added to the feed liquid for enzymolysis, which is inactivated after enzymolysis, and the supernatant is collected by centrifugation to obtain the collagen peptide; the protease includes one or more of alkaline protease, animal protease, and neutral protease.

2. The preparation method according to claim 1, characterized in that: The amount of alkaline protease added is 0.6-1.0%.

3. The preparation method according to claim 1, characterized in that: The optimum pH value of the alkaline protease is 8-11.

4. The preparation method according to claim 1, characterized in that: The enzymatic hydrolysis temperature of the alkaline protease is 45-55° C., and the enzymatic hydrolysis time is more than 2.5 hours.

5. The preparation method according to any one of claims 1 to 4, characterized in that: The method also includes: ultrafiltration of the collagen peptide to select collagen peptide components with a molecular weight cutoff of <3 kDa.

6. Collagen peptide prepared by the preparation method according to any one of claims 1 to 5.

7. Use of the collagen peptide according to claim 6 in the preparation of products with antioxidant function.

8. The use according to claim 7, characterized in that: The products include cosmetics, food, health products, additives, excipients and medicines.

9. A product with antioxidant function, characterized in that: Comprising the collagen peptide according to claim 6.

10. The product according to claim 9, characterized in that It also includes other functional active ingredients that do not react with the collagen peptide described in claim 6, and / or non-functional auxiliary components.

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