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CN-122012664-A - Method for producing active peptide by solid state fermentation

CN122012664ACN 122012664 ACN122012664 ACN 122012664ACN-122012664-A

Abstract

The invention provides a method for producing active peptide by fermentation. Specifically, the kudzuvine root is taken as a raw material, a plurality of kudzuvine root small molecular active peptides are obtained by optimizing a solid state fermentation process, the obtained kudzuvine root small molecular active peptides are detected for the antioxidant capacity, and the kudzuvine root small molecular active peptides with strong antioxidant capacity are screened to prepare antioxidant drugs, so that the kudzuvine root small molecular active peptides have wide application prospect.

Inventors

  • LIU LU
  • LIU TIANJIAO

Assignees

  • 广州牛邦生物技术有限公司

Dates

Publication Date
20260512
Application Date
20260402

Claims (7)

  1. 1. A method for the fermentative production of an active peptide, said method comprising the steps of: a. pretreatment of raw materials: (1) Selecting a variety of kudzuvine root with high protein content, selecting fresh kudzuvine root without mildew, removing epidermis, impurities and rotting parts, putting into a cleaning machine for cleaning, and cutting into small pieces of 5 cm; (2) Crushing the kudzuvine root obtained in the step (1) by adopting a crusher, sieving the crushed kudzuvine root by a 30-mesh sieve, and collecting undersize powder for later use; (3) Mixing and stirring the powder in the step (2) and distilled water according to the feed liquid ratio of 1:3-1:5 (g: mL) at room temperature, and soaking for 30-60min to ensure that the powder fully absorbs water and swells, thereby facilitating the subsequent steam explosion; (4) Uniformly filling the kudzu vine root material in the step (3) into a steam explosion kettle, ensuring that the material is loose, avoiding caking, preventing solid and liquid from separating, and directly carrying out explosion treatment, wherein the explosion parameters are that the temperature is 180-240 ℃, the steam pressure is 1.8-2.5MPa, and the pressure is maintained for 30-90s; (5) After blasting is finished, a discharging hole is opened rapidly to take out the kudzuvine root material, and the kudzuvine root material is flattened and cooled to room temperature, so that protein denaturation caused by waste heat is avoided; (6) Heating the cooled material in a constant-temperature water bath kettle at 85 ℃ for 5-10min, inactivating endogenous enzymes, and cooling to 45-55 ℃ for later use; (7) Adding 0.05mol/L citric acid-sodium citrate buffer solution into the kudzuvine root material in the step (6), adjusting the feed-liquid ratio to 1:12-1:20 (g: mL), adjusting the pH to 5, adding 0.8% -2.0% of cellulase and 0.5% -1.2% of pectase, uniformly stirring, ensuring that the enzyme is fully contacted with the kudzuvine root material, and stirring at a low speed of 150-200rpm for 4-7h at 48-52 ℃ to obtain an enzymolysis product; (8) Heating the zymolyte in the step (7) in a constant-temperature water bath kettle at 95 ℃ for 10min to inactivate enzyme, and cooling to 40-50 ℃ for standby; b. Preparation of a culture medium: 25g of bran, 0.3% of urea and 0.2% of KH 2 PO 4 are added into every 100mL of pueraria enzymolysis product, the pH is regulated to 6.5, the water content of the material is regulated to 50% -60%, the prepared culture medium is transferred into a fermentation tank, and the fermentation tank is sterilized by high-pressure steam for 20min at 121 ℃ and 101kPa and cooled to 37 ℃; c. fermentation: adding the zymolyte in the step (8) into the culture medium in the step b, and dynamically regulating the pH to 6.5 in the whole process; (1) At the early stage (0-24 h), inoculating Aspergillus niger aseptically at 30-32deg.C with 10% of inoculum size, culturing for 12h, inoculating Bacillus subtilis aseptically with 10% of inoculum size, and introducing air of 0.1-0.5vvm via ventilation equipment; (2) Medium term (24-72 h), regulating fermentation temperature to 35-37deg.C, and introducing air of 0.5-1.5vvm via ventilation equipment; (3) Later stage (72-96 h) of adjusting the fermentation temperature to 32-35 ℃ and introducing 1.5-2.0vvm of air through ventilation equipment; in the fermentation process, the material is turned once every 12 hours, so that the ventilation uniformity of the system is ensured; d. extracting kudzuvine root small molecule active peptide: After fermentation, transferring the fermentation material into a beaker, adding distilled water at 50 ℃ according to the feed liquid ratio of 1:8-1:12 (mass ratio), stirring uniformly, placing into a constant-temperature water bath kettle, leaching at the constant temperature of 50 ℃ for 1-2h, stirring once every 20min, filtering with gauze after leaching, collecting filtrate, adding distilled water into the residual filter residue according to the feed liquid ratio, repeatedly leaching once, combining the two filtrates, transferring the combined filtrate into a high-speed refrigerated centrifuge, centrifuging at 8000rpm and 4 ℃ for 15min, removing precipitate, and collecting supernatant to obtain the crude body fluid of the small molecular active peptide of the radix puerariae; e. and (3) separating and purifying: (1) Ultrafiltering the crude extract with ultrafiltration membrane with molecular cutoff of 2Kda, and collecting permeate; (2) Transferring the permeate from the step (1) into nanofiltration concentration equipment for concentration to 1/5-1/3 of the original volume, so as to obtain a kudzuvine root small molecule active peptide concentrated solution; (3) Adding 0.5% -1% of active carbon into the concentrated solution, stirring at constant temperature of 50 ℃ for 30min for decolorization, centrifuging at 8000rpm for 15min, and removing the active carbon to obtain clarified kudzuvine root small molecule active peptide liquid; (4) Transferring the radix Puerariae small molecule active peptide liquid obtained in step (3) into a spray dryer, setting air inlet temperature at 180-200deg.C, air outlet temperature at 80-90deg.C, and feeding speed at 10-15mL/min, spray drying to obtain light yellow radix Puerariae small molecule active peptide powder, and sealing and storing for use.
  2. 2. The method of claim 1, wherein the active peptide is a kudzuvine root small molecule active peptide, and the amino acid sequence of the kudzuvine root small molecule active peptide is shown in SEQ ID No. 1-12.
  3. 3. The antioxidant active peptide is characterized in that the antioxidant active peptide is a kudzuvine root small molecule active peptide, and the amino acid sequence of the antioxidant active peptide is shown as SEQ ID No. 1-12.
  4. 4. The antioxidant active peptide according to claim 3, wherein the antioxidant active peptide is a kudzuvine root small molecule active peptide, and the amino acid sequence of the antioxidant active peptide is shown as SEQ ID No. 7.
  5. 5. An antioxidant composition is characterized by comprising one or more of kudzuvine root small molecule active peptides with amino acid sequences shown in SEQ ID No. 1-12.
  6. 6. The antioxidant composition of claim 5, wherein the composition comprises a kudzuvine root small molecule active peptide with an amino acid sequence shown as SEQ ID No. 7.
  7. 7. Use of an antioxidant active peptide according to claims 3-4 and/or an antioxidant composition according to claims 5-6 for the preparation of an antioxidant medicament.

Description

Method for producing active peptide by solid state fermentation Technical Field The invention relates to the field of biological fermentation, in particular to a method for producing small molecule active peptide by solid state fermentation of kudzuvine root. Background The kudzu root serving as the dried root tuber of the leguminous pueraria plant is a traditional medicine and food homologous raw material in China, is rich in various nutrition and active ingredients such as starch, protein, flavonoid, isoflavone and the like, and has long application history and wide development prospect in the fields of food, health care products, medicines and the like. The small molecular polypeptide formed by degrading the pueraria protein not only maintains the natural nutrition characteristic of the pueraria, but also has various biological activities of being easily absorbed by human bodies, resisting oxidation, enhancing immunity, regulating metabolism, protecting liver cells and the like, and compared with the pueraria crude protein, the pueraria protein has higher bioavailability and more definite functions, and further expands the application boundary of the pueraria. The solid state fermentation is a technology for converting macromolecular substances in raw materials into micromolecular active products by depending on the growth metabolism of microorganisms on a solid state culture medium, has the remarkable advantages of low energy consumption, small pollution, simple equipment, high product activity, capability of fully utilizing inherent nutritional ingredients of the raw materials and the like, is highly compatible with the characteristics of the raw materials of the radix puerariae, can fully utilize carbohydrates such as starch, cellulose and the like in the radix puerariae as carbon sources for microbial metabolism, promotes degrading enzymes such as protease and the like by microorganisms, efficiently converts the radix puerariae protein into polypeptides, simultaneously can effectively retain synergistic active ingredients such as radix puerariae flavone and the like, and improves the comprehensive value of the products. Although research on preparing small molecule active polypeptide by radix puerariae solid state fermentation has been progressed, some problems still exist at present, and the industrial large-scale application of the small molecule active polypeptide is limited. For example, in terms of fermentation processes, traditional solid state fermentation modes have lower fermentation efficiency, resulting in poor product type, yield, purity and activity. Therefore, there is a need to create a method for producing active peptide by solid state fermentation of radix Puerariae, so as to improve the type, yield, purity and activity of polypeptide. Disclosure of Invention In order to solve the problems, the invention provides a method for producing small molecule active peptide by using kudzuvine root as a raw material through solid state fermentation, which comprises the following specific steps: 1. pretreatment of raw materials: (1) Selecting a variety of kudzuvine root with high protein content, selecting fresh kudzuvine root without mildew, removing epidermis, impurities and rotting parts, putting into a cleaning machine for cleaning, and cutting into small pieces of 5 cm; (2) Crushing the kudzuvine root obtained in the step (1) by adopting a crusher, sieving the crushed kudzuvine root by a 30-mesh sieve, and collecting undersize powder for later use; (3) Mixing and stirring the powder in the step (2) and distilled water according to the feed liquid ratio of 1:3-1:5 (g: mL) at room temperature, and soaking for 30-60min to ensure that the powder fully absorbs water and swells, thereby facilitating the subsequent steam explosion; (4) Uniformly filling the kudzuvine root material which is well moistened with water into a steam explosion kettle, ensuring the loosening of the material, avoiding caking (the caking can lead to uneven steam distribution and inconsistent explosion effect), and directly carrying out explosion treatment (explosion parameters: 180-240 ℃ in temperature, 1.8-2.5MPa in steam pressure and 30-90s in pressure maintaining); (5) After blasting is finished, a discharging hole is opened rapidly to take out the kudzuvine root material, and the kudzuvine root material is flattened and cooled to room temperature, so that protein denaturation caused by waste heat is avoided; (6) Heating the cooled material in a constant-temperature water bath kettle at 85 ℃ for 5-10min, inactivating endogenous enzymes, and cooling to 45-55 ℃ for later use; (7) Adding 0.05mol/L citric acid-sodium citrate buffer solution into the kudzuvine root material in the step (6), adjusting the feed-liquid ratio to 1:12-1:20 (g: mL), adjusting the pH to 5, adding cellulase (0.8% -2.0%) and pectase (0.5% -1.2%), uniformly stirring, ensuring that the enzyme is fully contacted with the kudzuvine root material, and st