Method for obtaining salmon-source active peptide through fermentation and application of salmon-source active peptide

Abstract

The invention belongs to the field of salmon processing by-product utilization, and particularly relates to a method for obtaining salmon processing by-product active peptide through fermentation and application of the salmon processing by-product active peptide. According to the invention, five uric acid lowering peptides with relatively high xanthine oxidase inhibitory activity are prepared by combining a microbial fermentation method with an ultrafiltration and gel chromatographic column technology, and the specific sequences are IGAPDMAFPR, FDKPVSPLL, FVDPDNFRV, GEADFMDYGR and GNGPDWAKDL. The raw material adopted by the invention is salmon processing waste (brown meat), the price is low, the utilization rate of resources is improved, and waste and environmental pollution are avoided. The method is simple in process operation, low in production cost and good in application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of salmon fine processing, and in particular to a method for obtaining salmon-derived active peptides by fermentation and application thereof. Background Art

[0002] Hyperuricemia is a metabolic disease caused by purine metabolism disorders in the human body, which is mainly manifested as elevated serum uric acid levels. Xanthine oxidase (XOD) is a key enzyme involved in purine metabolism, catalyzing the conversion of hypoxanthine to xanthine, and converting xanthine to uric acid. Inhibiting the activity of xanthine oxidase can reduce the production of uric acid to a certain extent. However, existing drugs that inhibit uric acid synthesis by inhibiting the activity of xanthine oxidase, such as allopurinol and febuxostat, can produce side effects and harm human health during treatment. Therefore, it is necessary to develop safer and more effective means of lowering uric acid.

[0003] At present, food-derived bioactive peptides have been reported to have xanthine oxidase inhibitory activity in vitro and uric acid-lowering effects in vivo, which may become a new method to effectively control uric acid levels. The main methods for preparing bioactive peptides are microbial fermentation, chemical synthesis, and protease hydrolysis. Among them, peptides prepared by microbial fermentation are rich in variety, low in cost, and may have higher biological activity.

[0004] The salmon processing industry is large, and during the processing, fish bones and scraps are produced as by-products, which are often not processed and not effectively utilized. The discarding of some by-products causes environmental pollution and waste of resources. Salmon brown meat is the insulation layer under the salmon skin, which is rich in collagen. Salmon has always been an important source of bioactive peptides, and the active peptide resources based on salmon brown meat need to be further developed and explored. Summary of the invention

[0005] In view of the deficiencies in the prior art, the purpose of the present invention is to provide a method for obtaining salmon-derived active peptides by fermentation and its application.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions.

[0007] The invention discloses a salmon brown meat peptide with uric acid lowering effect, characterized in that the amino acid sequence of the peptide is: IGAPDMAFPR, FDKPVSPLL, FVDPDNFRV, GEADFMDYGR or GNGPDWAKDL, as shown in SEQ ID NO.1-5.

[0008] Furthermore, the use of one or a combination of the above-mentioned salmon brown meat peptides in the preparation of auxiliary uric acid-lowering drugs.

[0009] The present invention also discloses a method for preparing the above-mentioned salmon brown meat peptide, which is characterized in that the preparation is carried out by the following method: Step 1: Preparation of salmon brown meat fermentation liquid: S1 Pre-treatment of salmon brown meat: Wash the salmon brown meat, cut it into small pieces and break it up using a beating homogenizer; Preparation of S2 fermentation medium: The salmon surimi in S1 is prepared into fish meat suspension with distilled water at a solid-liquid ratio of 1:3-1:5, pasteurized for 1-2 hours, and then added with 1% Bacillus amyloliquefaciens after cooling, and fermented at 37°C for 48 hours. After fermentation, the supernatant is collected by centrifugation to obtain the crude fermentation product; Step 2: Preparation of salmon brown meat peptide: S1: Separate the crude fermentation product obtained in step 1 using an ultrafiltration tube to collect components <3 kDa; S2 further separates and purifies the peptide of less than 3 kDa obtained in S1 using a Sephadex G15 gel chromatography column, collects the active components, and concentrates them by rotary evaporation to obtain salmon brown meat peptide.

[0010] Furthermore, the Bacillus amyloliquefaciens described in S2 in step 1 is stored in the General Microbiological Center of China National Microbiological Culture Collection Administration, with the storage number being CGMCC No.27124.

[0011] Furthermore, the active peptide obtained by the above-mentioned method for preparing salmon brown meat peptide is used in the preparation of auxiliary uric acid-lowering drugs.

[0012] The present invention also discloses a pharmaceutical composition, characterized in that it contains one or more of the above-mentioned salmon brown meat peptides.

[0013] Furthermore, the pharmaceutical composition also comprises a pharmaceutically acceptable carrier.

[0014] Furthermore, the above-mentioned pharmaceutical composition is used in the preparation of auxiliary uric acid-lowering drugs.

[0015] The present invention also discloses a strain of Bacillus amyloliquefaciens, characterized in that the strain is classified and named Bacillus amyloliquefaciens Y11 (Bacillus amyloliquefaciens), and is preserved in the General Microbiological Center of China Microorganism Culture Collection Administration, located in Beijing, China, with a preservation number of CGMCC No.27124 and a preservation date of April 17, 2023.

[0016] Furthermore, the Bacillus amyloliquefaciens is used in the preparation of salmon brown meat peptide with uric acid lowering effect.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0018] The present invention adopts microbial fermentation method in combination with ultrafiltration and gel chromatography column technology to prepare uricosuric acid-lowering peptides with high xanthine oxidase inhibitory activity. The raw materials used in the present invention are salmon processing waste, which is low in price, improves the reuse rate of resources, and avoids waste and environmental pollution. The present invention has simple process operation, low production cost, and good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Graph showing XOD inhibition activity of different ultrafiltration fractions of the present invention.

[0020] Figure 2 This is a molecular weight distribution diagram of the ultrafiltration component <3 kDa of the present invention.

[0021] Figure 3 This is a Sephadex G15 gel chromatography separation diagram of the present invention and a XOD inhibitory activity determination diagram of the separated components.

[0022] Figure 4 It is the mass spectrum of the mixed component of G1+G5 of the present invention.

[0023] Figure 5 This is a graph showing the in vitro XOD inhibitory activity of the uricosuric acid-lowering peptide of the present invention.

[0024] Figure 6 This is a diagram showing the cytotoxicity of the uricosuric acid-lowering peptide of the present invention after acting on HK-2 cells.

[0025] Figure 7 The uric acid lowering activity of FDKPVSPLL and allopurinol of the present invention in a hyperuric acid cell model. DETAILED DESCRIPTION

[0026] The present invention is further described in detail below with specific embodiments, but it should not be understood that the scope of the above subject matter of the present invention is limited to the following embodiments, and all technologies realized based on the content of the present invention belong to the scope of the present invention.

[0027] Unless otherwise specified, all reagents and materials used in the present invention are commercially available.

[0028] Example 1 Preparation of salmon brown meat fermentation liquid by microbial fermentation method.

[0029] (1) Pretreatment of salmon brown meat: Clean the salmon brown meat, cut it into small pieces and break it up using a beater homogenizer.

[0030] (2) Preparation of fermentation medium: The salmon surimi prepared in step 1 is prepared into a fish meat suspension with a solid-liquid ratio of 1:3 to 1:5 using distilled water, and pasteurized for 1 to 2 hours. After cooling, 1% of Bacillus amyloliquefaciens Y11 is added thereto by volume, and fermented at 37°C for 48 hours. After fermentation, the supernatant is collected by centrifugation to obtain the crude fermentation product.

[0031] The strain Bacillus amyloliquefaciens Y11 is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration with the deposit number CGMCC No.27124.

[0032] The culture conditions of the strain Bacillus amyloliquefaciens Y11 were as follows: cultured in broth medium at 37°C and 150 rpm for 24 h.

[0033] Example 2 Colorimetric determination of xanthine oxidase activity.

[0034] (1) Solution preparation: Accurately weigh 6.4 mg of xanthine, dissolve it in 1 mL of 1 M NaOH, and then add 100 mL of distilled water to prepare a xanthine solution with a final concentration of 0.48 mM; weigh 0.2 mg of xanthine oxidase and add 50 mL of distilled water to prepare a final concentration of 0.02 U / mL; weigh 10 mg of allopurinol and add 10 mL of distilled water to prepare a 1 mg / mL solution.

[0035] (2) XOD inhibitory activity determination: The following examples all use a colorimetric method to determine the XOD inhibitory activity. The specific operation is to prepare the different component samples purified in Example 3 and Example 4 into a 1 mg / mL solution, add 50 μL of the sample to be tested and 50 μL of a xanthine oxidase solution with a concentration of 0.02 U / mL to each well of a 96-well plate, mix well, react at 25°C for 5 minutes, then add 150 μL of a 0.48 mM xanthine solution, mix well, react at 25°C for 25 minutes, and add 80 μL of 1 M hydrochloric acid to terminate the reaction after the reaction. Determine the absorbance at 290 nm. The formula for calculating the xanthine oxidase inhibitory activity is: (1) Where: A1 is the absorbance of the sample solution with enzyme added; A2 is the absorbance of the sample solution without enzyme added; A3 is the absorbance of the blank group with distilled water replacing the sample solution.

[0036] Example 3 Separation and purification of fermentation broth products.

[0037] (1) The crude fermentation product obtained in Example 1 was filtered through a 0.22 μm sterile filter membrane and then through ultrafiltration membranes with molecular weights of 10 kDa and 3 kDa, thereby obtaining three types of crude salmon brown meat fermentation broth with molecular weights of <3 kDa, 3-5 kDa, and >5 kDa, and the XOD inhibitory activity of different components was determined.

[0038] The experimental results are as follows Figure 1 As shown in the figure, it can be seen that the three salmon fermentation broths with different molecular weights can effectively inhibit the activity of XOD, among which the XOD inhibitory activity of the F3 (<3kDa) component is significantly higher than that of the other two components, and the XOD inhibitory activity of F3 is 87.71±1.61%.

[0039] (2) Size exclusion chromatography (SEC) was used to determine the molecular weight distribution of <3 kDa. The sample was dissolved and centrifuged to obtain the supernatant. The sample was analyzed using a BioCore SEC-120 5 μm, 7.8 × 300 mm column with a sample volume of 10 μL and a mobile phase of 150 mmol phosphate buffer solution at a flow rate of 0.7 mL / min. The detection was performed at 214 nm.

[0040] The experimental results are shown in Table 1. The molecular weight of F3 is mainly concentrated below 1 kDa, of which 500 Da-1 kDa is 20.78%, 200-500 Da is 44.48%, and <200 Da is 18.43%. Small molecule peptides may have higher XOD inhibitory activity.

[0041] Table 1 Molecular weight distribution of F3 .

[0042] (3) The ultrafiltration fraction <3 kDa was further separated and purified using a Sephadex G15 gel chromatography column with ultrapure water as the eluent, a flow rate of 0.5 mL / min, and a detection wavelength of 280 nm.

[0043] The results are as follows Figure 2 As shown, five different components were purified and named G1 to G5.

[0044] The XOD inhibitory activity of each component of G1 to G5 is as follows Figure 3 As shown, the XOD inhibitory activities of the two components G1 and G5 were the highest at 93.96±0.99% and 93.67±2.35%, respectively, and there was no significant difference between the two. The mixed component of the two was used for subsequent mass spectrometry identification.

[0045] Example 4 Screening and identification of XOD inhibitory peptides.

[0046] After the G1+G5 mixed components were dissolved, they were desalted using a Waters SEP-PAK C18 solid phase extraction cartridge, freeze-dried and reconstituted with formic acid water, and the supernatant was taken for detection. The chromatographic separation conditions were that the A phase was a 0.1% formic acid aqueous solution, and the B phase was a 0.1% formic acid acetonitrile aqueous solution (acetonitrile was 80%). The liquid chromatography column (50μm×150mm, AcclaimPepMapTM RSLC, thermo scientific Technology Inc.) was equilibrated with 92% A solution, and the injection volume was 1μL for separation by the chromatographic column. The relevant liquid phase gradient was set as follows: 0min~98min, the linear gradient of B solution was from 8% to 28%; 98min~113min, the linear gradient of B solution was from 28% to 37%; 113min~117min, the linear gradient of B solution was from 37% to 100%; 117min~120min, B solution was maintained at 100%. After capillary HPLC separation, mass spectrometry analysis was performed using a Thermo QE HF mass spectrometer (ThermoFisher), and the analysis time was 120 min. Detection method: positive ion. The mass-to-charge ratio of the peptide and its fragments was collected according to the following method: 20 fragment spectra (MS2 scan) were collected after each full scan (full scan). The scanning range was 400-1800, the primary resolution was 60000, the secondary resolution was 15000, and the collision energy was CE28eV.

[0047] The mass spectrometry results are as follows Figure 4 , a total of 4122 peptides and 526 oligopeptides were identified. The biological activity, toxicity, sensitization and water solubility of oligopeptides were predicted by Pepideranker, Toxinpred, AllTOP Server Page and Innovagen. The results are shown in Table 2. A total of 30 bioactive scores greater than 0.5 and 5 bioactive scores greater than 0.7 were screened out. Then, the XOD inhibitory activity of the 5 bioactive peptides with scores greater than 0.7 was measured. The results are shown in Table 2. Figure 5 As shown, five peptides, IGAPDMAFPR, FDKPVSPLL, FVDPDNFRV, GEADFMDYGR, and GNGPDWAKDL, have high XOD inhibitory activity, and the XOD inhibitory activity is between 88.49 and 92.01.

[0048] Table 2 Prediction of biological activity of peptides .

[0049] Example 5 XOD inhibits the cytotoxicity of peptides.

[0050] HK-2 cells were cultured in MEM complete culture medium supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin at 37°C and 5% CO 2 The culture medium was changed every 2 days, and the cells were passaged after the cell growth density reached 80%.

[0051] Cytotoxicity: Take 100 μL of cell suspension (5×10 3 cells / mL) were inoculated in a 96-well plate and incubated for 24 hours to allow it to adhere to the wall. Then the culture medium was discarded. 100 μL of basal culture medium was added to the blank control group; 100 μL of five XOD inhibitory peptides at 0.025, 0.05, 0.1, 0.25, 0.5, and 1.0 mg / mL were added to the peptide group and cultured for 24 hours. Then the culture medium was discarded and 100 μL of 10% CCK8 solution diluted with basal culture medium was added to each well. The cells were cultured for 1-4 hours in the dark and their absorbance at 450 nm was measured. The formula for calculating cell viability is: (2) Where: A 1 A is the absorbance after culturing cells with samples of different concentrations; 2 A is the absorbance of CCK-8 without cells; 3 It is the absorbance of cells cultured in medium without adding sample.

[0052] The results are as follows Figure 6 As shown, the survival rate of HK-2 cells with peptide FDKPVSPLL was above 80% at different concentrations, the survival rate of FVDPDNFRV, GEADFMDYGR and IGAPDMAFPR was higher than 80% when the concentration was lower than 0.5 mg / mL, and the survival rate of GNGPDWAKDL was higher than 80% at 0.1 mg / mL, which showed certain toxicity at high concentrations.

[0053] The uric acid-lowering effect of the sample in Example 6 in the hyperuricemia cell model.

[0054] HK-2 cells were cultured at a rate of 2×10 5 cell / mL was inoculated in a 6-well plate and cultured for 24 h. After stable adhesion, 1 mg / mL of XOD inhibitory peptide and allopurinol were added to the wells. Complete culture medium was added to the blank group and the control group, and cultured for 24 h. The culture medium was discarded, and the cells were washed three times with PBS. A 1 mmol / L adenosine solution prepared with basal culture medium was added and cultured for 24 h. Then 0.05 U / mL XO was added to the wells and cultured for 12 h. The supernatant was taken to determine the uric acid content.

[0055] The results are as follows Figure 7As shown in the figure, the uric acid content in the model group was significantly higher than that in the control group, indicating that the hyperuric acid model was successfully established. The XOD inhibitory peptide FDKPVSPLL can significantly reduce the uric acid level in the hyperuric acid model and has a certain uric acid-lowering effect.

[0056] In summary, the enzymatic hydrolysate of salmon brown meat fermented by microorganisms has a strong in vitro uric acid-lowering activity, and the components obtained after a series of purifications also have a high XOD inhibitory activity. The peptide prepared by the present invention has application prospects.

[0057] The above description is only a preferred embodiment of the present invention and is not intended to limit the scope of the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A salmon brown meat peptide having uric acid lowering effect, characterized in that: The amino acid sequence of the peptide is: IGAPDMAFPR, FDKPVSPLL, FVDPDNFRV, GEADFMDYGR or GNGPDWAKDL, as shown in SEQ ID NO.1-5.

2. Use of one or a combination of the salmon brown meat peptides described in claim 1 in the preparation of auxiliary uric acid-lowering drugs.

3. A method for preparing salmon brown meat peptide as claimed in claim 1, characterized in that: Prepared by the following method: Step 1: Preparation of salmon brown meat fermentation liquid: S1 Pre-treatment of salmon brown meat: Wash the salmon brown meat, cut it into small pieces and break it up using a beating homogenizer; Preparation of S2 fermentation medium: The salmon surimi in S1 is prepared into fish meat suspension with distilled water at a solid-liquid ratio of 1:3-1:5, pasteurized for 1-2 hours, and then added with 1% Bacillus amyloliquefaciens after cooling, and fermented at 37°C for 48 hours. After fermentation, the supernatant is collected by centrifugation to obtain the crude fermentation product; Step 2: Preparation of salmon brown meat peptide: S1: Separate the crude fermentation product obtained in step 1 using an ultrafiltration tube to collect components <3 kDa; S2 further separates and purifies the peptide of less than 3 kDa obtained in S1 using a Sephadex G15 gel chromatography column, collects the active components, and concentrates them by rotary evaporation to obtain salmon brown meat peptide.

4. The preparation method according to claim 3, characterized in that: The Bacillus amyloliquefaciens described in S2 in step 1 is stored in the General Microbiological Center of China Microorganism Culture Collection Administration, with the storage number of CGMCC No.27124.

5. Use of the active peptide obtained by the method for preparing salmon brown meat peptide according to claims 3 to 4 in the preparation of auxiliary uric acid-lowering drugs.

6. A pharmaceutical composition, characterized in that Contains one or more salmon brown meat peptides as described in claim 1.

7. The pharmaceutical composition according to claim 6, characterized in that The pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

8. Use of the pharmaceutical composition according to claims 6 to 7 in the preparation of auxiliary uric acid-lowering drugs.

9. A strain of Bacillus amyloliquefaciens, characterized in that The strain is classified and named as Bacillus amyloliquefaciens Y11, and is preserved in the General Microbiological Center of China Microbiological Culture Collection Administration with a preservation number of CGMCC No.27124.

10. Use of the Bacillus amyloliquefaciens according to claim 9 in the preparation of salmon brown meat peptide with uric acid lowering effect.

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