Method for producing active peptide through solid state fermentation

Abstract

The invention provides a method for producing active peptide through fermentation. Specifically, radix puerariae is taken as a raw material, a plurality of radix puerariae small-molecule active peptides are obtained by optimizing a solid-state fermentation process, the antioxidant capacity of the obtained radix puerariae small-molecule active peptides is detected, the radix puerariae small-molecule active peptides with high antioxidant capacity are screened to prepare antioxidant drugs, and the radix puerariae small-molecule active peptides have wide application prospects.

Description

Technical Field

[0001] This invention relates to the field of bio-fermentation, specifically to a method for producing small molecule bioactive peptides using kudzu root through solid-state fermentation. Background Technology

[0002] Kudzu root, the dried tuberous root of a legume (Pueraria lobata), is a traditional Chinese medicinal and edible ingredient. Rich in starch, protein, flavonoids, isoflavones, and other nutrients and active ingredients, it has a long history of application and broad development prospects in the food, health product, and pharmaceutical industries. The small-molecule polypeptides formed after the degradation of kudzu root protein not only retain the natural nutritional characteristics of kudzu root but also possess various biological activities such as easy absorption by the human body, antioxidant properties, immune enhancement, metabolic regulation, and liver cell protection. Compared to crude kudzu root protein, its bioavailability is higher and its functions are more clearly defined, further expanding the application boundaries of kudzu root.

[0003] Solid-state fermentation is a technology that relies on the growth and metabolism of microorganisms on a solid culture medium to transform macromolecules in raw materials into small-molecule active products. It has significant advantages such as low energy consumption, low pollution, simple equipment, high product activity, and full utilization of the inherent nutrients in the raw materials. It is highly compatible with the characteristics of kudzu root raw materials—it can fully utilize the carbohydrates such as starch and cellulose in kudzu root as carbon sources for microbial metabolism, promote the secretion of degrading enzymes such as proteases by microorganisms, efficiently transform kudzu root protein into peptides, and effectively retain synergistic active ingredients such as kudzu root flavonoids, thereby enhancing the comprehensive value of the product.

[0004] Despite significant progress in the research of preparing small molecule bioactive peptides from kudzu root through solid-state fermentation, several challenges remain that limit its industrial-scale application. For example, traditional solid-state fermentation methods suffer from low efficiency, resulting in poor product type, yield, purity, and activity. Therefore, there is an urgent need to innovate a method for producing bioactive peptides through solid-state fermentation of kudzu root to improve the type, yield, purity, and activity of the peptides. Summary of the Invention

[0005] To address the above problems, this invention provides a method for producing small molecule bioactive peptides using kudzu root as raw material through solid-state fermentation. The specific steps are as follows: 1. Raw material pretreatment: (1) Select kudzu root varieties with high protein content, select fresh kudzu roots without mold, remove the skin, impurities and rotten parts, put them into a washing machine for washing, and cut them into 5cm pieces; (2) Use a pulverizer to pulverize the kudzu root from step (1), pass it through a 30-mesh sieve, and collect the sieve powder for later use; (3) Mix the powder from step (2) with distilled water at a ratio of 1:3 to 1:5 (g:mL) at room temperature and stir. Soak for 30-60 minutes to allow the powder to fully absorb water and expand, which will facilitate subsequent steam explosion. (4) Evenly load the moistened kudzu root material into the steam explosion kettle to ensure that the material is loose and avoids clumping (clumping will cause uneven steam distribution and inconsistent explosion effect). Do not separate the solid and liquid and directly carry out the explosion treatment (explosion parameters: temperature 180-240℃, steam pressure 1.8-2.5MPa, pressure holding 30-90s). (5) After the blasting is completed, quickly open the discharge port to take out the kudzu material, spread it out and cool it to room temperature to avoid residual heat causing protein denaturation; (6) Place the cooled material in an 85°C constant temperature water bath and heat for 5-10 minutes to inactivate endogenous enzymes, then cool to 45-55°C for later use. (7) Add 0.05 mol / L citrate-sodium citrate buffer to the kudzu material in step (6), adjust the material-liquid ratio to 1:12-1:20 (g:mL), adjust the pH to 5, add cellulase (0.8%-2.0%) and pectinase (0.5%-1.2%), stir evenly to ensure that the enzymes are fully in contact with the kudzu material, stir at 150-200 rpm at 48-52℃ for 4-7 hours to obtain the enzymatic hydrolysate; (8) Place the enzymatic hydrolysate from step (7) in a 95°C constant temperature water bath and heat for 10 minutes to inactivate the enzyme. Cool to 40-50°C and set aside.

[0006] 2. Culture medium preparation: Add the following to every 100 mL of kudzu enzymatic hydrolysate: 25 g wheat bran, 0.3% urea, and 0.2% KH2PO4. Adjust the pH to 6.5 and the moisture content of the material to 50%-60%. Transfer the prepared culture medium to a fermenter and sterilize it with high-pressure steam at 121℃ and 101 kPa for 20 min. Cool it to 37℃.

[0007] 3. Fermentation: Add the enzymatic hydrolysate from step 1 (8) to the culture medium in step 2, and dynamically adjust the pH to 6.5 throughout the process; (1) Early stage (0-24h): The fermentation temperature is 30-32℃. Aseptically inoculate Aspergillus niger with an inoculation amount of 10%. After culturing for 12h, aseptically inoculate Bacillus subtilis with an inoculation amount of 10%. Introduce air at 0.1-0.5vvm through the ventilation equipment. (2) Mid-term (24-72h): Adjust the fermentation temperature to 35-37℃ and introduce air at a rate of 0.5-1.5vvm through the aeration equipment; (3) Later stage (72-96h): Adjust the fermentation temperature to 32-35℃ and introduce air at 1.5-2.0vvm through the ventilation equipment; During fermentation, the material is turned over every 12 hours to ensure uniform aeration of the system.

[0008] 4. Extraction of small molecule active peptides from kudzu root: After fermentation, the fermented material was transferred to a beaker, and 50℃ distilled water was added at a material-to-liquid ratio of 1:8-1:12 (mass ratio). The mixture was stirred evenly and placed in a constant temperature water bath for extraction at 50℃ for 1-2 hours, with stirring every 20 minutes during the extraction. After extraction, the mixture was filtered through gauze, and the filtrate was collected. The remaining filter residue was then extracted again with distilled water at the same material-to-liquid ratio. The two filtrates were combined. The combined filtrate was transferred to a high-speed refrigerated centrifuge and centrifuged at 8000 rpm and 4℃ for 15 minutes to remove the precipitate. The supernatant was collected to obtain crude liquid of kudzu root small molecule active peptides.

[0009] 5. Separation and purification: (1) The crude extract was ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 2 kDa, and the permeate was collected; (2) The permeate from step (1) is transferred into a nanofiltration concentrator for concentration, and concentrated to 1 / 5-1 / 3 of the original volume to obtain a concentrated solution of small molecule active peptides from kudzu root. (3) Add 0.5%-1% activated carbon to the concentrate, stir at 50℃ for 30 min to decolorize, centrifuge at 8000 rpm for 15 min to remove activated carbon, and obtain clear kudzu small molecule active peptide solution. (4) Transfer the kudzu root small molecule active peptide liquid from step (3) into a spray dryer, set the inlet air temperature to 180-200℃, the outlet air temperature to 80-90℃, and the feed rate to 10-15mL / min, and spray dry to obtain light yellow kudzu root small molecule active peptide powder, and seal and store for later use.

[0010] 6. Testing: The amino acid sequence of the obtained kudzu root small molecule active peptide powder was detected, and a total of 12 small molecule active peptides were detected, as follows: Table 1 SEQ ID No. sequence SEQ ID No. sequence 1 LLVGADYPS 7 VVLYDWPAW 2 VVYDAVLGP 8 VVPYWADSRG 3 LLVGADYPSRVW 9 DSAPWYYLPG 4 WVVDAVPGRS 10 VGGYDWVLYA 5 YLVDAVLLDDA 11 PGDLVVYYPA 6 LVGAPWDSGAV 12 VYLDWGAYG Another aspect of the present invention is to provide a small molecule active peptide from kudzu root with strong antioxidant and free radical scavenging capabilities, the amino acid sequence of which is shown in SEQ ID No. 1-12.

[0011] Another aspect of the present invention is to provide a composition, specifically, comprising a small molecule bioactive peptide of kudzu root with an amino acid sequence as shown in SEQ ID No. 1-12.

[0012] Another aspect of the invention is an application, specifically, of using compositions comprising small molecule active peptides of kudzu root with amino acid sequences as shown in SEQ ID No. 1-12 in the preparation of antioxidant and free radical scavenging drugs.

[0013] Beneficial effects: (1) This invention uses kudzu root as raw material. The mechanical force generated by the instantaneous depressurization of steam explosion destroys the cell wall structure of kudzu root, exposing the nutrients such as protein and starch encapsulated within. At the same time, it reduces the crystallinity of cellulose and lignin, creating conditions for subsequent enzymatic hydrolysis and fermentation. Combined with compound cellulase and pectinase, it further degrades the cellulose and pectin remaining in the cell wall, breaking down the structural barrier of the raw material. Kudzu root is fermented by mixed fermentation of Aspergillus niger and Bacillus subtilis. The cellulase secreted by Aspergillus niger further destroys the cell wall of kudzu root, promoting protein release. The protease secreted by Bacillus subtilis efficiently degrades the protein to generate polypeptides. In the fermentation process, segmented temperature-controlled fermentation is adopted. In the early stage, it promotes the adaptation and proliferation of the strain. In the middle stage, it accelerates the secretion of protease and the degradation of kudzu root protein. In the later stage, it reduces polypeptide degradation and enhances product activity.

[0014] (2) The prepared kudzu small molecule active peptides not only have high yield and high purity, but also have many types and strong free radical scavenging and antioxidant capabilities. Detailed Implementation

[0015] The specific implementation methods provided by the present invention will be described in detail below with reference to the embodiments.

[0016] Example 1: Preparation of small molecule bioactive peptides from kudzu root 1. Raw material pretreatment: (1) Select kudzu root varieties with high protein content, select fresh kudzu roots without mold, remove the skin, impurities and rotten parts, put them into a washing machine for washing, and cut them into 5cm pieces; (2) Use a pulverizer to pulverize the kudzu root from step (1), pass it through a 30-mesh sieve, and collect the sieve powder for later use; (3) Mix the powder from step (2) with distilled water at a ratio of 1:3 (g:mL) at room temperature and stir. Soak for 30 minutes to allow the powder to fully absorb water and expand, which will facilitate subsequent steam explosion. (4) Evenly load the moistened kudzu root material into the steam explosion kettle to ensure that the material is loose and avoids clumping (clumping will cause uneven steam distribution and inconsistent explosion effect). Do not separate the solid and liquid and directly carry out the explosion treatment (explosion parameters: temperature 180℃, steam pressure 1.8MPa, pressure holding 30s). (5) After the blasting is completed, quickly open the discharge port to take out the kudzu material, spread it out and cool it to room temperature to avoid residual heat causing protein denaturation; (6) Place the cooled material in an 85°C constant temperature water bath and heat for 5 minutes to inactivate endogenous enzymes, then cool to 45°C for later use. (7) Add 0.05 mol / L citrate-sodium citrate buffer to the kudzu material in step (6), adjust the material-liquid ratio to 1:12 (g:mL), adjust the pH to 5, add 0.8% cellulase and 0.5% pectinase, stir evenly to ensure that the enzymes are fully in contact with the kudzu material, stir at 150 rpm at 48℃ for 4 hours to obtain the enzymatic hydrolysate; (8) Place the enzymatic hydrolysate from step (7) in a 95°C constant temperature water bath and heat for 10 minutes to inactivate the enzyme. Cool to 40°C and set aside.

[0017] 2. Culture medium preparation: Add the following to every 100 mL of kudzu root enzymatic hydrolysate: 25 g wheat bran, 0.3% urea, and 0.2% KH2PO4. Adjust the pH to 6.5 and the moisture content of the material to 50%. Transfer the prepared culture medium to a fermenter and sterilize it with high-pressure steam at 121℃ and 101 kPa for 20 min. Cool it to 37℃.

[0018] 3. Fermentation: Add the enzymatic hydrolysate from step 1 (8) to the culture medium in step 2, and dynamically adjust the pH to 6.5 throughout the process; (1) Early stage (0-24h): The fermentation temperature is 30℃. Aseptically inoculate Aspergillus niger with an inoculation amount of 10%. After culturing for 12h, aseptically inoculate Bacillus subtilis with an inoculation amount of 10%. Introduce 0.1vvm of air through the ventilation equipment. (2) Mid-term (24-72h): Adjust the fermentation temperature to 35℃ and introduce 0.5vvm of air through the ventilation equipment; (3) Later stage (72-96h): Adjust the fermentation temperature to 32℃ and introduce 1.5vvm of air through the ventilation equipment; During fermentation, the material is turned over every 12 hours to ensure uniform aeration of the system.

[0019] 4. Extraction of small molecule active peptides from kudzu root: After fermentation, the fermented material was transferred to a beaker, and 50°C distilled water was added at a material-to-liquid ratio of 1:8 (mass ratio). The mixture was stirred evenly and placed in a constant temperature water bath for extraction at 50°C for 1 hour, with stirring every 20 minutes during the extraction. After extraction, the mixture was filtered through gauze, and the filtrate was collected. The remaining filter residue was then extracted again with distilled water at the same material-to-liquid ratio, and the two filtrates were combined. The combined filtrate was transferred to a high-speed refrigerated centrifuge and centrifuged at 8000 rpm and 4°C for 15 minutes to remove the precipitate. The supernatant was collected to obtain crude liquid of kudzu root small molecule active peptides.

[0020] 5. Separation and purification: (1) The crude extract was ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 2 kDa, and the permeate was collected; (2) The permeate from step (1) is transferred into a nanofiltration concentrator for concentration until it is reduced to 1 / 5 of its original volume, thus obtaining a concentrated solution of small molecule active peptides from kudzu root. (3) Add 0.5% activated carbon to the concentrate, stir at 50℃ for 30 min to decolorize, centrifuge at 8000 rpm for 15 min to remove activated carbon, and obtain clear kudzu small molecule active peptide solution. (4) Transfer the kudzu root small molecule active peptide liquid from step (3) into a spray dryer, set the inlet air temperature to 180℃, the outlet air temperature to 80℃, and the feed rate to 10mL / min, and spray dry to obtain light yellow kudzu root small molecule active peptide powder, and seal and store for later use.

[0021] The yield (mass of kudzu small molecule active peptide powder / mass of kudzu raw material) and purity (reversed-phase high-performance liquid chromatography) of the obtained kudzu small molecule active peptides were determined.

[0022] Example 2: 1. Raw material pretreatment: (1) Select kudzu root varieties with high protein content, select fresh kudzu roots without mold, remove the skin, impurities and rotten parts, put them into a washing machine for washing, and cut them into 5cm pieces; (2) Use a pulverizer to pulverize the kudzu root from step (1), pass it through a 30-mesh sieve, and collect the sieve powder for later use; (3) Mix the powder from step (2) with distilled water at a ratio of 1:5 (g:mL) at room temperature and stir. Soak for 60 minutes to allow the powder to fully absorb water and expand, which will facilitate subsequent steam explosion. (4) Evenly load the moistened kudzu root material into the steam explosion kettle to ensure that the material is loose and avoids clumping (clumping will cause uneven steam distribution and inconsistent explosion effect). Do not separate the solid and liquid and directly carry out the explosion treatment (explosion parameters: temperature 240℃, steam pressure 2.5MPa, pressure holding 90s). (5) After the blasting is completed, quickly open the discharge port to take out the kudzu material, spread it out and cool it to room temperature to avoid residual heat causing protein denaturation; (6) Place the cooled material in an 85°C constant temperature water bath and heat for 10 minutes to inactivate endogenous enzymes, then cool to 55°C for later use. (7) Add 0.05 mol / L citrate-sodium citrate buffer to the kudzu material in step (6), adjust the material-liquid ratio to 1:20 (g:mL), adjust the pH to 5, add 2% cellulase and 1.2% pectinase, stir evenly to ensure that the enzyme and kudzu material are fully in contact, stir at 200 rpm at 52℃ for 7 hours to obtain the enzymatic hydrolysate; (8) Place the enzymatic hydrolysate from step (7) in a 95°C constant temperature water bath and heat for 10 minutes to inactivate the enzyme. Cool to 50°C and set aside.

[0023] 2. Culture medium preparation: Add the following to every 100 mL of kudzu enzymatic hydrolysate: 25 g wheat bran, 0.3% urea, and 0.2% KH2PO4. Adjust the pH to 6.5 and the moisture content of the material to 60%. Transfer the prepared culture medium to a fermenter and sterilize it with high-pressure steam at 121℃ and 101 kPa for 20 min. Cool it to 37℃.

[0024] 3. Fermentation: Add the enzymatic hydrolysate from step 1 (8) to the culture medium in step 2, and dynamically adjust the pH to 6.5 throughout the process; (1) Early stage (0-24h): The fermentation temperature is 32℃. Aseptically inoculate Aspergillus niger with an inoculation amount of 10%. After culturing for 12h, aseptically inoculate Bacillus subtilis with an inoculation amount of 10%. Introduce 0.5vvm of air through the ventilation equipment. (2) Mid-term (24-72h): Adjust the fermentation temperature to 37℃ and introduce 1.5vvm of air through the ventilation equipment; (3) Later stage (72-96h): Adjust the fermentation temperature to 35℃ and introduce 2.0vvm of air through the ventilation equipment; During fermentation, the material is turned over every 12 hours to ensure uniform aeration of the system.

[0025] 4. Extraction of small molecule active peptides from kudzu root: After fermentation, the fermented material was transferred to a beaker, and 50°C distilled water was added at a material-to-liquid ratio of 1:12 (mass ratio). The mixture was stirred evenly and placed in a constant temperature water bath for extraction at 50°C for 2 hours, with stirring every 20 minutes during the extraction. After extraction, the mixture was filtered through gauze, and the filtrate was collected. The remaining filter residue was then extracted again with distilled water at the same material-to-liquid ratio, and the two filtrates were combined. The combined filtrate was transferred to a high-speed refrigerated centrifuge and centrifuged at 8000 rpm and 4°C for 15 minutes to remove the precipitate. The supernatant was collected to obtain crude liquid of kudzu root small molecule active peptides.

[0026] 5. Separation and purification: (1) The crude extract was ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 2 kDa, and the permeate was collected; (2) The permeate from step (1) is transferred into a nanofiltration concentrator for concentration until it is reduced to 1 / 3 of its original volume, thus obtaining a concentrated solution of small molecule active peptides from kudzu root. (3) Add 1% activated carbon to the concentrate, stir at 50℃ for 30 min to decolorize, centrifuge at 8000 rpm for 15 min to remove activated carbon, and obtain clear kudzu small molecule active peptide solution. (4) Transfer the kudzu root small molecule active peptide liquid from step (3) into a spray dryer, set the inlet air temperature to 200℃, the outlet air temperature to 90℃, and the feed rate to 15mL / min, and spray dry to obtain light yellow kudzu root small molecule active peptide powder, and seal and store for later use.

[0027] The yield (mass of kudzu small molecule active peptide powder / mass of kudzu raw material) and purity (reversed-phase high-performance liquid chromatography) of the obtained kudzu small molecule active peptides were determined.

[0028] Comparative Example 1 The experimental steps are the same as in Example 2, the only difference being that only steam explosion treatment is performed during the pretreatment process.

[0029] Comparative Example 2 The experimental steps are the same as in Example 2, the only difference being that only the compound enzyme is treated during the pretreatment process.

[0030] Comparative Example 3 The experimental steps were the same as in Example 2, the only difference being that only Aspergillus niger was used for single-strain fermentation during the fermentation process.

[0031] Comparative Example 4 The experimental steps were the same as in Example 2, the only difference being that only Bacillus subtilis was used for single-strain fermentation during the fermentation process.

[0032] Comparative Example 5 The experimental steps were the same as in Example 2, the only difference being that the temperature was controlled at 35°C throughout the fermentation process.

[0033] The yield and purity data of small molecule active peptides from kudzu root in each group are as follows: Table 2: Group Yield purity Example 1 10.6% 95% Example 2 11.5% 96% Comparative Example 1 6.9% 90%% Comparative Example 2 5.4% 88% Comparative Example 3 7.2% 91% Comparative Example 4 7.1% 90% Comparative Example 5 9.3% 92% Therefore, pretreatment with steam explosion in conjunction with cellulase and pectinase can effectively disrupt the cell wall structure of kudzu, exposing the encapsulated nutrients such as protein and starch, while reducing the crystallinity of cellulose and lignin, further degrading residual cellulose and pectin in the cell wall, and breaking down the structural barriers of the raw materials. The co-fermentation of kudzu with Aspergillus niger and Bacillus subtilis allows the cellulase secreted by Aspergillus niger to further disrupt the kudzu cell wall and promote protein release, while the protease secreted by Bacillus subtilis efficiently degrades the protein to generate peptides. The fermentation process employs segmented temperature-controlled fermentation, promoting strain adaptation and proliferation in the early stage, accelerating protease secretion and kudzu protein degradation in the middle stage, and reducing peptide degradation in the later stage.

[0034] Example of results: Detection of the free radical scavenging ability of small molecule active peptides from kudzu root. The amino acid sequence of the kudzu root small molecule active peptide powder of Example 2 was identified, and 12 kudzu root small molecule active peptides were obtained (Table 1). The free radical scavenging ability of the above 12 kudzu root small molecule active peptides was then tested.

[0035] (1) Determination of DPPH free radical scavenging ability: The synthesized 12 small molecule bioactive peptides from kudzu root were prepared into 2 mL peptide samples with a concentration of 2 mg / mL (three replicates for each group), and labeled as groups 1-12 according to their sequence numbers. Each of the 12 peptide samples was mixed with 2 mL of 0.1 mmol / L DPPH free radical solution, shaken thoroughly, and incubated in the dark for 30 min. The absorbance (A) of the mixture was measured at 517 nm. i The absorbance value A was measured using 2 mL of distilled water instead of the polypeptide sample solution in the control group (three replicates per group). 对 The absorbance value A was measured using 2 mL of anhydrous ethanol instead of DPPH solution in the blank control group (three replicates per group). 空 The DPPH free radical scavenging rate is calculated using the following formula: DPPH free radical scavenging rate (%) = [1 - (A i- A 空 ) / A 对 】×100 (2) Determination of ABTS free radical scavenging ability: Prepare an ABTS reagent solution containing 7 mmol / L ABTS and 2.45 mmol / L potassium persulfate, and incubate at room temperature for 12-16 hours. Dilute with anhydrous ethanol at 734 nm to obtain an absorbance of 0.700 ± 0.005 to obtain the ABTS radical cation working solution. Mix 3.9 mL of the ABTS radical cation working solution with 0.1 mL of a polypeptide sample solution (three replicates per group) containing 12 synthesized kudzu small molecule active peptides at a concentration of 2 mg / mL, shake thoroughly, and incubate in the dark for 6 min. Measure the absorbance at 734 nm. i Anhydrous ethanol was used as a blank control instead of the peptide sample solution (three replicates for each group), and the absorbance value A was measured. 空 The ABTS free radical scavenging rate is calculated using the following formula: ABTS free radical scavenging rate (%) = (A 空- A i ) / A 空 ×100 (3) Determination of hydroxyl radical scavenging ability: The 12 synthesized small molecule active peptides from kudzu root were prepared into 2 mL peptide sample solutions with a concentration of 2 mg / mL (three replicates per group) and placed in 10 mL stoppered test tubes. Then, 2 mL of hydrogen peroxide solution (6 mmol / L) and 2 mL of ferrous sulfate solution (6 mmol / L) were added. After thorough shaking, the solutions were allowed to stand in the dark for 30 min. Next, 2 mL of freshly prepared salicylic acid solution (6 mmol / L) was added to the tubes, and after shaking to mix, the solutions were allowed to stand for 10 min. The absorbance A of the reaction mixture was measured at 510 nm. i The absorbance value A was measured using distilled water instead of the peptide sample solution in the blank control (three replicates in each group). 空 The absorbance value was measured using distilled water instead of salicylic acid solution (three replicates per group). 对 The hydroxyl radical scavenging rate is calculated using the following formula: Hydroxyl radical scavenging rate (%) = (A 空 +A 对- A i ) / A 空 ×100 The results are as follows: Table 3

[0036] Analysis showed that among the 12 kudzu root small molecule active peptides obtained in this invention, the ability to scavenge free radicals is closely related to the number of amino acids in the peptides. Among them, peptide 9 has the highest scavenging ability, and SEQ ID No. 7 has the highest activity, followed by SEQ ID No. 1, 2, and 12.

[0037] It can be seen that all 12 kudzu small molecule active peptides mentioned above have certain free radical scavenging capabilities, and the kudzu small molecule active peptide with the amino acid sequence shown in SEQ ID No. 7 has the strongest activity and high antioxidant capacity.

[0038] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.

Claims

1. A method for producing bioactive peptides by fermentation, characterized in that, The method includes the following steps: a. Raw material pretreatment: (1) Select kudzu root varieties with high protein content, select fresh kudzu roots without mold, remove the skin, impurities and rotten parts, put them into a washing machine for washing, and cut them into 5cm pieces; (2) Use a pulverizer to pulverize the kudzu root from step (1), pass it through a 30-mesh sieve, and collect the sieve powder for later use; (3) Mix the powder from step (2) with distilled water at a ratio of 1:3 to 1:5 (g:mL) at room temperature and stir. Soak for 30-60 minutes to allow the powder to fully absorb water and expand, which will facilitate subsequent steam explosion. (4) The kudzu root material from step (3) is evenly loaded into the steam explosion reactor to ensure that the material is loose, avoids clumping, and does not separate from the solid and liquid, and is directly subjected to explosion treatment; the explosion parameters are temperature 180-240℃, steam pressure 1.8-2.5MPa, and pressure holding for 30-90s. (5) After the blasting is completed, quickly open the discharge port to take out the kudzu material, spread it out and cool it to room temperature to avoid residual heat causing protein denaturation; (6) Place the cooled material in an 85°C constant temperature water bath and heat for 5-10 minutes to inactivate endogenous enzymes, then cool to 45-55°C for later use. (7) Add 0.05mol / L citrate-sodium citrate buffer to the kudzu material in step (6), adjust the material-liquid ratio to 1:12-1:20 (g:mL), adjust the pH to 5, add 0.8%-2.0% cellulase and 0.5%-1.2% pectinase, stir evenly to ensure that the enzyme and kudzu material are fully in contact, stir at 150-200rpm at 48-52℃ for 4-7h to obtain the enzymatic hydrolysate; (8) Place the enzymatic hydrolysate from step (7) in a 95°C constant temperature water bath and heat for 10 min to inactivate the enzyme, then cool to 40-50°C for later use. b. Culture medium preparation: Add the following to every 100 mL of kudzu enzymatic hydrolysate: 25 g wheat bran, 0.3% urea, and 0.2% KH2PO4. Adjust the pH to 6.5 and the moisture content of the material to 50%-60%. Transfer the prepared culture medium to a fermenter and sterilize it with high-pressure steam at 121℃ and 101 kPa for 20 min. Then cool it to 37℃. c. Fermentation: Add the enzymatic hydrolysate from step a (8) to the culture medium in step b, and dynamically adjust the pH to 6.5 throughout the process; (1) Early stage (0-24h): The fermentation temperature is 30-32℃. Aseptically inoculate Aspergillus niger with an inoculation amount of 10%. After culturing for 12h, aseptically inoculate Bacillus subtilis with an inoculation amount of 10%. Introduce air at 0.1-0.5vvm through the ventilation equipment. (2) Mid-term (24-72h): Adjust the fermentation temperature to 35-37℃ and introduce air at a rate of 0.5-1.5vvm through the aeration equipment; (3) Later stage (72-96h): Adjust the fermentation temperature to 32-35℃ and introduce air at 1.5-2.0vvm through the ventilation equipment; During fermentation, the material is turned over every 12 hours to ensure uniform aeration of the system; d. Extraction of small molecule active peptides from kudzu root: After fermentation, the fermented material was transferred to a beaker, and 50℃ distilled water was added at a material-to-liquid ratio of 1:8-1:12 (mass ratio). The mixture was stirred evenly and placed in a constant temperature water bath for extraction at 50℃ for 1-2 hours, with stirring every 20 minutes during the extraction. After extraction, the mixture was filtered through gauze, and the filtrate was collected. The remaining filter residue was then extracted again with distilled water at the same material-to-liquid ratio. The two filtrates were combined. The combined filtrate was transferred to a high-speed refrigerated centrifuge and centrifuged at 8000 rpm and 4℃ for 15 minutes to remove the precipitate. The supernatant was collected to obtain crude kudzu root small molecule active peptide solution. e. Separation and purification: (1) The crude extract was ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 2 kDa, and the permeate was collected; (2) The permeate from step (1) is transferred into a nanofiltration concentrator for concentration, and concentrated to 1 / 5-1 / 3 of the original volume to obtain a concentrated solution of small molecule active peptides from kudzu root. (3) Add 0.5%-1% activated carbon to the concentrate, stir at 50℃ for 30 min to decolorize, centrifuge at 8000 rpm for 15 min to remove activated carbon, and obtain clear kudzu small molecule active peptide solution. (4) Transfer the kudzu root small molecule active peptide liquid from step (3) into a spray dryer, set the inlet air temperature to 180-200℃, the outlet air temperature to 80-90℃, and the feed rate to 10-15mL / min, and spray dry to obtain light yellow kudzu root small molecule active peptide powder, and seal and store for later use.

2. The method according to claim 1, characterized in that, The active peptide is a small molecule active peptide from kudzu root, and its amino acid sequence is shown in SEQ ID No. 1-12.

3. An antioxidant active peptide, characterized in that, The antioxidant active peptide is a small molecule active peptide from kudzu root, and its amino acid sequence is shown in SEQ ID No. 1-12.

4. The antioxidant active peptide according to claim 3, characterized in that, The antioxidant active peptide is a small molecule active peptide from kudzu root, and its amino acid sequence is shown in SEQ ID No.

7.

5. An antioxidant composition, characterized in that, The composition comprises one or more of the small molecule active peptides of kudzu root with amino acid sequences as shown in SEQ ID No. 1-12.

6. The antioxidant composition according to claim 5, characterized in that, The composition comprises a small molecule active peptide from kudzu root with an amino acid sequence as shown in SEQ ID No.

7.

7. The use of the antioxidant active peptides according to claims 3-4 and / or the antioxidant compositions according to claims 5-6 in the preparation of antioxidant drugs.

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