High-F-value oligopeptide as well as preparation method and application thereof

Abstract

The invention provides a high-F-value oligopeptide as well as a preparation method and application thereof, and belongs to the technical field of active peptides. The invention provides a preparation method of high-F-value oligopeptide, which comprises the following steps: enriching protein components in a protein raw material, and then carrying out enzyme digestion treatment and enzyme digestion aromatic amino acid treatment. After the high-F-value oligopeptide obtained by the preparation method disclosed by the invention is freeze-dried, the F value of the high-F-value oligopeptide can be up to 23.45. The high-F-value oligopeptide obtained by the invention shows similar DPPH removing activity to vitamin C, and zebra fish experiments prove that the high-F-value oligopeptide has a good liver injury repairing effect.

Description

Technical Field

[0001] The invention belongs to the technical field of active peptides, and specifically relates to a high F value oligopeptide and a preparation method and application thereof. Background Art

[0002] The liver is an important organ in the human body, with multiple functions such as metabolism, detoxification, and immunity. However, the liver is susceptible to damage from various factors, including drugs, alcohol, viral infections, and metabolic diseases. These factors may lead to acute or chronic liver damage, further developing into liver fibrosis, cirrhosis, and even liver cancer. At present, the drugs and treatments used clinically for liver protection and repair are limited, and are often accompanied by certain side effects. Therefore, the development of safe and efficient liver damage repair agents has important clinical significance and market demand.

[0003] In recent years, with the development of biotechnology, bioactive peptides have gradually attracted widespread attention due to their unique functions in anti-oxidation, anti-inflammation, immunomodulation and cell repair. In particular, oligopeptides, as a type of bioactive peptide, have a small molecular weight, are easily absorbed, can quickly reach the target organs, and exert biological effects. Studies have shown that oligopeptides of specific sources and specific sequences have shown significant protective effects in the repair of liver damage, such as reducing liver cell damage, inhibiting inflammatory responses, and promoting liver cell regeneration.

[0004] F value is one of the important parameters to measure the quality of oligopeptides, which is usually related to the molecular weight distribution, purity and biological activity of oligopeptides. Oligopeptides with high F values ​​have higher biological activity, stronger antioxidant capacity and cell protection. By optimizing the preparation process, oligopeptides with high F values ​​can be obtained, thereby further improving their efficacy in liver repair. However, how to efficiently prepare and apply such high F value oligopeptides currently lacks systematic research and mature technology.

[0005] Although there are some oligopeptide-based liver protection products on the market, the oligopeptide F values ​​of most products are low and the biological activity is not ideal. In addition, the existing preparation methods have certain limitations in purification efficiency, production cost and product stability, which are difficult to meet the needs of large-scale production and practical application. Therefore, it is urgent to develop a new method that can efficiently prepare high F value oligopeptides and verify their practical application effect in repairing liver damage. Summary of the invention

[0006] The present invention provides a high F value oligopeptide and a preparation method and application thereof. The high F value oligopeptide has the effect of repairing liver damage.

[0007] The invention provides a method for preparing a high F value oligopeptide, comprising the following steps: enriching protein in a protein raw material, sequentially treating it with alkaline protease, papain and trypsin, and then removing aromatic amino acids to obtain a high F value oligopeptide.

[0008] In a specific embodiment of the present invention, the enrichment comprises treating the protein raw material by an alkali dissolution and acid precipitation method, and collecting the precipitate.

[0009] In a specific embodiment of the present invention, the protein raw material includes concentrated whey protein.

[0010] In a specific embodiment of the present invention, when the alkaline protease is used for treatment, the temperature is 50-55° C., the pH value is 9.0-9.5, and the enzymolysis time is 2h-2.5h.

[0011] In a specific embodiment of the present invention, when the papain is used for treatment, the pH value is 7.0-7.5 and the enzymolysis time is 3h-3.5h.

[0012] In a specific embodiment of the present invention, when the trypsin is used for treatment, the pH value is 7.0-7.5 and the enzymatic hydrolysis is performed for 2h-2.5h.

[0013] In a specific embodiment of the present invention, the removal of aromatic amino acids comprises treating with a mixture of flavor protease and aminopeptidase, inactivating the enzymes and centrifuging, and treating the centrifuge with LK1100 resin and LK1500 resin.

[0014] The present invention also provides a high F value oligopeptide prepared by the above preparation method.

[0015] The present invention also provides the use of the above-mentioned high F value oligopeptide in the preparation of biologically active materials.

[0016] The present invention also provides a biomaterial with liver damage repairing function, wherein the active ingredients include high F value oligopeptides, and the high F value oligopeptides are prepared by using concentrated whey protein as a raw material using the above-mentioned preparation method.

[0017] Beneficial effects: The present invention provides a method for preparing a high F value oligopeptide, which is prepared by enriching the protein components in the protein raw material, and then performing enzymatic cleavage treatment and enzymatic cleavage aromatic amino acid treatment, and then removing the aromatic amino acids by inactivating the enzyme, centrifugal filtration and resin adsorption. The high F value oligopeptide obtained by the preparation method of the present invention was freeze-dried and tested to find that the leucine content was 2.71%, the isoleucine content was 1.03%, and the valine content was 3.98%; the phenylalanine content was 0.44%, the tyrosine content was 0, the tryptophan content was 0, and the F value was 23.45. Using the method of the present invention, compared with the F value (4.92) without resin treatment and the F value (6.97) without dearomatic amino acid treatment, the present invention can obtain an oligopeptide with a high F value.

[0018] The high F value oligopeptide obtained by the present invention exhibits DPPH scavenging activity close to that of vitamin C, and zebrafish experiments confirm that the high F value oligopeptide has a good effect on repairing liver damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a typical image of zebrafish after sample processing. The blue area is the liver and the red area is the yolk sac. DETAILED DESCRIPTION

[0020] The invention provides a method for preparing a high F value oligopeptide, comprising the following steps: enriching protein in a protein raw material, sequentially treating it with alkaline protease, papain and trypsin, and then removing aromatic amino acids to obtain a high F value oligopeptide.

[0021] The protein raw material of the present invention can be a protein-rich raw material, such as the concentrated whey protein used in the embodiment. In the embodiment, the concentrated whey protein is used as a raw material and the principle of alkali dissolution and acid precipitation is adopted to prepare the whey protein with high protein content, wherein the concentrated whey protein is purchased from Shanghai Fuxin Industrial Co., Ltd. The present invention does not specifically limit the alkali dissolution and acid precipitation method, and the operation can be performed using conventional methods in the art.

[0022] The present invention performs alkaline protease pretreatment on the whey protein with high protein content, and performs preliminary enzymolysis on the whey protein; the process includes mixing and enzymolysis of the whey protein with an alkaline protease aqueous solution under alkaline conditions, such as stirring and enzymolysis at a speed of 65 rpm for 2 hours in an environment with a pH value of 9.5 in the embodiment, to obtain an alkaline protease pretreated whey protein hydrolysate. The mass ratio of the alkaline protease to the whey protein in the present invention can be 10 g:1 kg.

[0023] The present invention performs deep enzymatic hydrolysis on the whey protein hydrolysate pretreated with alkaline protease, i.e., enzymatic hydrolysis is performed once with papain and trypsin, and the pH value is kept at 7.0-7.5 during the two enzymatic hydrolysis processes. The enzymatic hydrolysis time of papain is 3 hours, and the enzymatic hydrolysis time of trypsin is 2 hours. ...

[0024] The invention performs enzymatic cleavage of aromatic amino acids on the enzymatic hydrolyzate after deep enzymatic hydrolysis, that is, adding a mixed enzyme of flavor protease and aminopeptidase to the enzymatic hydrolyzate for 2 hours; after enzymatic hydrolysis, activated carbon can also be added for treatment. The mass ratio of the flavor protease and aminopeptidase in the invention is 2:1; and the time of activated carbon treatment is 45 minutes. The mixed enzyme exo-cuts aromatic amino acids, and the activated carbon adsorbs free amino acids.

[0025] The present invention performs enzyme inactivation treatment on the liquid treated with activated carbon, that is, heating the liquid to 100° C. for enzyme inactivation treatment, and then centrifugation filtration is performed, using a centrifuge with a rotation speed of 10,000 rpm and using filter paper for filtration treatment.

[0026] The present invention uses LK1100 resin and LK1500 resin to remove aromatic amino acids in the filtrate. LK1100 resin can adsorb free amino acids and small molecule polypeptides with a molecular weight of less than 500 Daltons, and LK1500 resin can specifically adsorb amino acids with benzene rings (aromatic amino acids all have benzene rings). When the present invention uses the above two resins for treatment, the two resins are first pretreated, and then 100g of LK1100 resin and LK1500 resin are weighed respectively, and the two columns are treated in series, and the enzymatic hydrolyzate after centrifugal filtration is slowly added to the ion exchange column, and the flow rate is controlled at 15ml / min by a constant flow pump, and the permeate after resin adsorption is collected.

[0027] The present invention freeze-dries the liquid adsorbed by the two resins, and then detects the properties of the freeze-dried material, especially the F value. The F value referred to in the present invention is calculated by the molar ratio of the content of branched-chain amino acids (BCAA: Val, Ile, Leu) and aromatic amino acids (AAA: Trp, Tyr, Phe).

[0028] The present invention also detects the obtained high F value oligopeptide, such as using GB / T 22492-2008 to detect amino acid distribution and using GB 5009.294-2023 to detect tryptophan content.

[0029] The present invention also provides a high F value oligopeptide prepared by the above preparation method.

[0030] In one embodiment of the present invention, the oligopeptide obtained by the above preparation method using concentrated whey protein as raw material has an F value as high as 23.45.

[0031] The present invention also provides the use of the above-mentioned high F value oligopeptide in the preparation of biologically active materials.

[0032] In one embodiment of the present invention, the above-mentioned oligopeptide with a high F value of 23.45 was functionally verified, and it was found that the IC value of the oligopeptide with a high F value in clearing DPPH was 50 The value is close to that of vitamin C, showing good DPPH scavenging activity; and it has a repairing effect on liver damage.

[0033] The present invention also provides a biomaterial with liver damage repairing function, wherein the active ingredients include high F value oligopeptides, and the high F value oligopeptides are prepared by using concentrated whey protein as a raw material using the above-mentioned preparation method.

[0034] In order to further illustrate the present invention, a high F value oligopeptide provided by the present invention and its preparation method and application are described in detail below in conjunction with examples, but they should not be construed as limiting the scope of protection of the present invention.

[0035] Example 1

[0036] 1. Preparation of whey protein

[0037] The method is to use concentrated whey protein as raw material and adopt the principle of alkali dissolution and acid precipitation to prepare whey protein with high protein content. The specific operation process is as follows: 500 kg of concentrated whey protein is weighed, 6000 liters of deionized water are added and stirred fully, 1M NaOH is used to adjust its pH to about 10.5, the temperature is raised to about 35° C. at the same time, and stirred for 30 minutes. A centrifuge is used to centrifuge the whey protein liquid, the supernatant is collected and further 1M HCl is used to adjust the pH to below 4, and the static collection precipitation is the target, whey protein (435 kg). The protein content of the whey protein powder is 93.5%, the water content is 3.6%, the ash content is 0.6%, the fat content is 1.6%, and the carbohydrate content is 0.7%.

[0038] 2. Preparation of high F value oligopeptides

[0039] (1) Alkaline protease pretreatment: 1 L of deionized water was heated to 50-55° C., the pH was adjusted to about 9.5, 10 g of alkaline protease (2709 Toyoko Huadao) was added, stirred and dissolved evenly, and then 1 kg of the prepared whey protein was added, and the enzymatic hydrolysis was performed at 65 rpm for 2 h before further treatment;

[0040] (2) Deep enzymatic hydrolysis: The pH of the pretreated whey protein hydrolysate was adjusted to 7.0-7.5, and 20 g of papain (Toyoko Huadao) was added to continue enzymatic hydrolysis for 3 h, and then 50 g of trypsin (Toyoko Huadao) was added to continue enzymatic hydrolysis for 2 h. The pH was maintained at 7.0-7.5 during the enzymatic hydrolysis.

[0041] (3) Enzymatic cleavage of aromatic amino acids: After the enzymatic hydrolyzate treated in step 2 was further hydrolyzed for 2 h by adding 20 g of flavor protease (Toyoko Huadao) and 10 g of aminopeptidase (Toyoko Huadao), then 20 g of activated carbon (Shanghai Quanhu Activated Carbon Co., Ltd.) was added and treated for 45 min;

[0042] (4) Inactivating enzymes: Inactivate the enzymes in the enzymatic hydrolysis liquid of step 3 by heating the liquid to 100° C.;

[0043] (5) Centrifugal filtration: centrifuge at a speed of 10,000 rpm and filter paper for filtration;

[0044] (6) LK1100 resin and LK1500 resin are used to remove aromatic amino acids. LK1100 resin can adsorb free amino acids and small molecular peptides with a molecular weight of less than 500 Daltons, and LK1500 resin can specifically adsorb amino acids with benzene rings (aromatic amino acids all have benzene rings). The specific operation steps are as follows:

[0045] 6.1 Pretreatment of LK1100 resin and LK1500 resin

[0046] Resin pretreatment: The resin should be thoroughly cleaned before the first use, and ethanol 10 to 20 cm above the resin layer is added to soak for 1 to 1 hour, the soaking liquid is released, and ethanol is continued to be washed until the dripping liquid is diluted with water in a test tube and is not turbid, and water is washed until there is no alcohol smell at the outlet. The amount of ethanol and water is about 2 to 5 times the volume of the resin; 1M sodium hydroxide 3 to 5 times the amount of the resin is added to wash out the residual solvent and impurity ions in the resin, and deionized water is used to wash until the pH of the dripping liquid is less than 9; 1M hydrochloric acid 3 to 5 times the amount of the resin is added to wash out the metal ions, and deionized water is used to wash until the pH of the dripping liquid is greater than 4, and the pretreatment is completed.

[0047] 6.2 Resin impurity removal

[0048] Weigh 100 g of LK1100 resin and LK1500 resin respectively, connect the two columns in series, slowly add the enzymatic hydrolyzate after centrifugal filtration into the ion exchange column, control the flow rate at 15 ml / min using a constant flow pump, and collect the permeate after resin adsorption.

[0049] (7) Freeze drying and material property testing: The purified enzymatic hydrolysate is freeze dried, and the physical and chemical properties of the high F value corn peptide are tested with reference to relevant national standards. The specific test items are: amino acid distribution test (GB / T 22492-2008) and tryptophan content test (GB 5009.294-2023).

[0050] Test results: the leucine content was 2.71%, the isoleucine content was 1.03%, the valine content was 3.98%; the phenylalanine content was 0.44%, the tyrosine content was 0, the tryptophan content was 0, and the F value of the prepared high F value corn peptide was calculated to be 23.45.

[0051] Comparative Example 1

[0052] Compared with Example 1, this comparative example does not undergo resin treatment:

[0053] (1) Alkaline protease pretreatment: Measure 1L of deionized water and heat it to 50-55°C, adjust the pH to about 9.5, add 10g of alkaline protease (2709 Toyoko Huadao), stir well to dissolve, then add 1kg of whey protein, stir well at 65 rpm for 2h, and then further treat;

[0054] (2) Deep enzymatic hydrolysis: The pH of the pretreated whey protein hydrolysate was adjusted to 7.0-7.5, and 20 g of papain (Toyoko Huadao) was added to continue enzymatic hydrolysis for 3 h, and then 50 g of trypsin (Toyoko Huadao) was added to continue enzymatic hydrolysis for 2 h. The pH was maintained at 7.0-7.5 during the enzymatic hydrolysis.

[0055] (3) Enzymatic cleavage of aromatic amino acids: After the enzymatic hydrolyzate treated in step 2 was further hydrolyzed for 2 h by adding 20 g of flavor protease (Toyoko Huadao) and 10 g of aminopeptidase (Toyoko Huadao), then 20 g of activated carbon (Shanghai Quanhu Activated Carbon Co., Ltd.) was added and treated for 45 min;

[0056] (4) Inactivating enzymes: Inactivate the enzymes in the enzymatic hydrolysis liquid of step 3 by heating the liquid to 100° C.;

[0057] (5) Centrifugal filtration: centrifuge at a speed of 10,000 rpm and filter paper for filtration;

[0058] Test results: leucine content is 9.36%, isoleucine content is 5.88%, valine content is 5.11%; phenylalanine content is 2.58%, tyrosine content is 2.44%, tryptophan content is 0.69%, and the F value of the sample prepared in Comparative Example 1 is calculated to be 4.92.

[0059] Comparative Example 2

[0060] Compared with Example 1, this comparative example is not subjected to dearomatization:

[0061] (1) Alkaline protease pretreatment: Measure 1L of deionized water and heat it to 50-55°C, adjust the pH to about 9.5, add 10g of alkaline protease (2709 Toyoko Huadao), stir well to dissolve, then add 1kg of whey protein, stir well at 65 rpm for 2h, and then further treat;

[0062] (2) Deep enzymatic hydrolysis: The pH of the pretreated whey protein hydrolysate was adjusted to 7.0-7.5, and 20 g of papain (Toyoko Huadao) was added to continue enzymatic hydrolysis for 3 h, and then 50 g of trypsin (Toyoko Huadao) was added to continue enzymatic hydrolysis for 2 h. During the enzymatic hydrolysis, the pH was maintained at 7.0-7.5, and 20 g of activated carbon (Shanghai Quanhu Activated Carbon Co., Ltd.) was added to treat for 45 min;

[0063] (3) Inactivating enzymes: Inactivate the enzymes in the enzymatic hydrolysis liquid of step 3 by heating the liquid to 100° C.;

[0064] (4) Centrifugal filtration: centrifuge at a speed of 10,000 rpm and filter paper for filtration;

[0065] (5) LK1100 resin and LK1500 resin are used to remove aromatic amino acids. LK1100 resin can adsorb free amino acids and small molecular peptides with a molecular weight of less than 500 Daltons, and LK1500 resin can specifically adsorb amino acids with benzene rings (aromatic amino acids all have benzene rings). The specific operation steps are as follows:

[0066] 6.1 Pretreatment of LK1100 resin and LK1500 resin

[0067] Resin pretreatment: The resin should be thoroughly cleaned before the first use, and ethanol 10 to 20 cm above the resin layer is added to soak for 1 to 2 hours, the soaking liquid is released, and ethanol is continued to be washed until the dripping liquid is not turbid when diluted with water in a test tube, and the outlet is washed with water until there is no alcohol smell. The amount of ethanol and water is about 2 to 5 times the volume of the resin; 1M sodium hydroxide 3 to 5 times the amount of the resin is added to wash out the residual solvent and impurity ions in the resin, and deionized water is used to wash until the pH of the dripping liquid is less than 9; 1M hydrochloric acid 3 to 5 times the amount of the resin is added to wash out the metal ions, and deionized water is used to wash until the pH of the dripping liquid is greater than 4, and the pretreatment is completed.

[0068] 6.2 Resin impurity removal

[0069] Weigh 100 g of LK1100 resin and LK1500 resin respectively, connect the two columns in series, slowly add the enzymatic hydrolyzate after centrifugal filtration into the ion exchange column, control the flow rate at 15 ml / min using a constant flow pump, and collect the permeate after resin adsorption.

[0070] (7) Freeze drying and material property testing: The purified enzymatic hydrolysate is freeze dried, and the physical and chemical properties of the high F value corn peptide are tested with reference to relevant national standards. The specific test items are: amino acid distribution test (GB / T 22492-2008) and tryptophan content test (GB 5009.294-2023).

[0071] Test results: the leucine content was 7.52%, the isoleucine content was 5.01%, the valine content was 4.69%; the phenylalanine content was 1.89%, the tyrosine content was 1.65, and the tryptophan content was 0.23. The F value of the oligopeptide prepared in Comparative Example 2 was calculated to be 6.97.

[0072] Example 2 Study on the Antioxidant Properties of the High F-Value Oligopeptides Described in Example 1

[0073] Determination of DPPH free radical scavenging ability

[0074] (1) The experiment was divided into blank group, negative control group, test sample group, and positive control group;

[0075] (2) Preparation of DPPH free radical working solution: Prepare 1.00 g·L DPPH free radical working solution using anhydrous ethanol as solvent. -1 DPPH free radical stock solution was stored at 4℃ away from light for future use; 1mL DPPH free radical stock solution was mixed with 9mL anhydrous ethanol to obtain a concentration of 100mg·L - 1 DPPH free radical working solution.

[0076] Drawing of DPPH free radical standard curve: DPPH free radical working solution was diluted with anhydrous ethanol to concentrations (mg·L -1 ) were 100, 80, 60, 40, 20, 10, and 5 sample solutions, and 6 replicates were set for each concentration. The absorbance at 517 nm was measured. A standard curve was drawn with the DPPH free radical concentration as the abscissa and the absorbance as the ordinate, and the linear regression equation was fitted: y = 0.0079x + 0.039 (R 2 =0.9999). This indicates that the DPPH free radical concentration is between 5 and 100 mg·L -1 The absorbance has a good linear relationship within this range.

[0077] Determination of DPPH free radical scavenging rate: Take an appropriate amount of high F value oligopeptide and dilute it with anhydrous ethanol to a concentration (g·L -1 ) were 0.13, 0.25, 0.5, 1, 2, 4, and 8 respectively, and 100 mg·L -1The DPPH free radical working solution was mixed evenly at a volume ratio of 1:1, reacted at 37°C in the dark for 30 minutes, and the absorbance at 517 nm was measured; 3 replicates were set for each concentration, and VC of the same concentration was used as a positive control. The DPPH free radical scavenging rate was calculated according to formula (1):

[0078]

[0079] Wherein: A0 is the absorbance of anhydrous ethanol + DPPH free radical working solution; A1 is the absorbance of sample solution + DPPH free radical working solution; A2 is the absorbance of sample solution + anhydrous ethanol.

[0080] Table 1 Antioxidant activity of oligopeptides with high F value

[0081]

[0082] From Table 1, we can see that in the range of 0.13 to 8 mg·mL -1 The ability of high F value oligopeptides to scavenge DPPH free radicals was dose-dependent within the dosage range. When the mass concentration of high F value oligopeptides was 0.13 mg·mL -1 The scavenging capacity of DPPH free radicals was 20.12% when the high F value oligopeptide was 8 mg·mL -1 The ability to scavenge DPPH free radicals was 94.37%, and the IC 50 The value is 0.82 mg·mL -1 ; When the vitamin C concentration is 0.13 mg·mL -1 The ability to scavenge DPPH free radicals was 29.06% when the vitamin C content was 8 mg·mL -1 The ability to scavenge DPPH free radicals was 98.79%, and the IC 50 The value is 0.82 mg·mL -1 ; From the results, it can be seen that the IC of high F value oligopeptides to remove DPPH 50 The value was close to that of the vitamin C group, showing better DPPH scavenging activity.

[0083] Example 3 Repairing effect of high F value oligopeptides on liver damage (zebrafish test)

[0084] 3.1. Sample preparation information

[0085] High F value oligopeptides were prepared into 2.00 mg / mL stock solution with standard dilution water and used immediately after preparation.

[0086] Positive control: Metadoxine, white powder, batch number 201101, Zhejiang Zhenyuan Pharmaceutical Co., Ltd., stored in a cool and dry place. Prepared into 149 mg / mL stock solution with DMSO, used immediately after preparation.

[0087] 3.2 Experimental animals

[0088] Zebrafish were raised in fish farming water at 28°C (water quality: 200 mg of instant sea salt was added to every 1 L of reverse osmosis water, the conductivity was 450-550 μS / cm; pH was 6.5-8.5; hardness was 50-100 mg / L CaCO3), bred and provided by the fish farming center, the experimental animal use license number is: SYXK (Zhejiang) 2022-0004, and the breeding management complies with the requirements of the international AAALAC certification (certification number: 001458).

[0089] Wild-type AB strain zebrafish were bred in natural pairs. Zebrafish aged 3 days post fertilization (3dpf) were used for the determination of the maximum detection concentration (MTC) of the sample to improve alcoholic liver damage and its efficacy evaluation.

[0090] 3.3. Instruments, consumables and reagents

[0091] Dissecting microscope (SZX7, OLYMPUS, Japan); CCD camera (VertA1, Shanghai Tusen Vision Technology Co., Ltd., China); precision electronic balance (CP214, OHAUS, USA); 6-well plate (NestBiotech, China).

[0092] Dimethyl sulfoxide (DMSO, batch number 20171016, Sinopharm Chemical Reagent Co., Ltd., China); methyl cellulose (batch number C2004046, Shanghai Aladdin Biochemical Technology Co., Ltd., China); anhydrous ethanol (batch number 20210901, Sinopharm Chemical Reagent Co., Ltd., China).

[0093] 3.4.MTC

[0094] 3dpf wild-type AB strain zebrafish were randomly selected in a 6-well plate, with 15 fish in each well (experimental group). The samples were dissolved in water (concentrations shown in Table 2), and a normal control group and a model control group were set up at the same time, with a capacity of 3mL per well. Except for the normal control group, the rest of the experimental groups were dissolved in water and given anhydrous ethanol to establish a zebrafish alcoholic liver injury model. The samples were treated at 28℃ until 5dpf, and the MTC of the samples on the improvement of alcoholic liver injury in the model zebrafish was determined.

[0095] Table 2 Results of the concentration experiment to explore the improvement effect of alcoholic liver injury on samples (n=15)

[0096]

[0097] Under the experimental conditions, the maximum detection concentration (MTC) of albumin peptide, whey protein peptide, high F value oligopeptide and spirulina peptide for improving alcoholic liver injury was 250μg / mL. The MTC of corn peptide for improving alcoholic liver injury was 125μg / mL. The MTC of compound peptide for improving alcoholic liver injury was 1000μg / mL.

[0098] 3.5. Evaluation of efficacy in improving alcoholic liver damage

[0099] 3dpf wild-type AB strain zebrafish were randomly selected in a 6-well plate, with 15 in each well (experimental group). The samples were dissolved in water (concentrations shown in Table 3), the positive control was 149μg / mL of metadoxine, and the normal control group and model control group were set up at the same time, with a capacity of 3mL per well. Except for the normal control group, the rest of the experimental groups were dissolved in water and given anhydrous ethanol to establish the zebrafish alcoholic liver injury model. The zebrafish were treated at 28℃ until 5dpf, and 8 zebrafish were randomly selected from each experimental group and photographed under a dissecting microscope. The data were analyzed and collected using NIS-Elements D 3.20 advanced image processing software to analyze 1) liver area; 2) liver brightness average; 3) yolk sac absorption delay area, and the statistical analysis results of these indicators were used to evaluate the improvement effect of the sample on alcoholic liver injury. The statistical analysis results were expressed as mean±SE. SPSS26.0 software was used for statistical analysis, and p<0.05 indicated that the difference was statistically significant.

[0100] Table 3 Results of the experimental evaluation of the improvement effect of alcoholic liver damage on samples (n=8)

[0101]

[0102] Under the experimental conditions, the results are shown in Table 3 and Figure 1 As shown, whey protein peptides and high F value oligopeptides have the effect of improving alcoholic liver damage, which is specifically manifested by a decrease in liver area and an increase in the average liver brightness.

[0103] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing a high F value oligopeptide, characterized in that: The following steps are involved: The protein in the protein raw material is enriched, treated with alkaline protease, papain and trypsin in sequence, and then the aromatic amino acids are removed to obtain high F value oligopeptides.

2. The preparation method according to claim 1, characterized in that: The enrichment comprises treating the protein raw material by alkaline dissolution and acid precipitation, and collecting the precipitate.

3. The preparation method according to claim 1 or 2, characterized in that: The protein raw material includes whey protein concentrate.

4. The preparation method according to claim 1, characterized in that: When the alkaline protease is used for treatment, the temperature is 50-55° C., the pH value is 9.0-9.5, and the enzymolysis time is 2h-2.5h.

5. The preparation method according to claim 1, characterized in that: When the papain is used for treatment, the pH value is 7.0-7.5 and the enzymolysis time is 3h-3.5h.

6. The preparation method according to claim 1, characterized in that: When the trypsin is used for treatment, the pH value is 7.0 to 7.5, and the enzymolysis is performed for 2 hours to 2.5 hours.

7. The preparation method according to claim 1, characterized in that: The method for removing aromatic amino acids comprises treating with a mixed enzyme of flavor protease and aminopeptidase, inactivating the enzymes and centrifuging, and treating the centrifuge liquid with LK1100 resin and LK1500 resin.

8. An oligopeptide with a high F value prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the high F value oligopeptide according to claim 8 in the preparation of biologically active materials.

10. A biomaterial having liver damage repairing effect, characterized in that: The active ingredients include high F value oligopeptides, which are prepared using concentrated whey protein as a raw material using the preparation method described in any one of claims 1 to 7.

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