WO2017215313A1 - Procédé de préparation d'un peptide antioxydant en utilisant des coquilles de noix de ginkgo - Google Patents

Procédé de préparation d'un peptide antioxydant en utilisant des coquilles de noix de ginkgo Download PDF

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Publication number
WO2017215313A1
WO2017215313A1 PCT/CN2017/078555 CN2017078555W WO2017215313A1 WO 2017215313 A1 WO2017215313 A1 WO 2017215313A1 CN 2017078555 W CN2017078555 W CN 2017078555W WO 2017215313 A1 WO2017215313 A1 WO 2017215313A1
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ginkgo
aqueous solution
peel
naoh
crude protein
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PCT/CN2017/078555
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English (en)
Chinese (zh)
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王航
王大平
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如皋福大工程技术研究院有限公司
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Publication of WO2017215313A1 publication Critical patent/WO2017215313A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products

Definitions

  • the invention relates to a method for preparing an antioxidant peptide by an enzymatic method, in particular to a method for preparing an antioxidant peptide by using a ginkgo peel, which belongs to the field of biotechnology processing.
  • Ginkgo peel contains protein, fat, sugar, tannin, trace elements, amino acids, as well as flavonoids and phenolic acids such as ginkgo phenol, ginkgo phenol, ginkgo acid, hydrogenated ginkgo acid, hydrogenated ginkgoic acid, and ginkgol.
  • the acidic substances are shikimic acid, D-glycolic acid, and orange such as acid.
  • Ginkgo biloba contains high protein, which is not only wasted resources but also causes environmental pollution.
  • the concept of plant active peptides and the improvement of separation detection technology have led people to gradually pay more attention to the deep processing of protein-rich agricultural by-products and obtain a variety of active peptides, such as casein phosphopeptides, antihypertensive peptides and digestible peptides.
  • active peptides such as casein phosphopeptides, antihypertensive peptides and digestible peptides.
  • the research and development of plant-derived active peptides plays an important role in improving the added value of deep processing of agricultural products in China.
  • antioxidants have developed rapidly at home and abroad, and their use has become more widespread. Antioxidants are not only used for antioxidants in fatty foods, but also as functional factors for the development of health foods and cosmetics. There are many types of antioxidants. However, chemically synthesized antioxidants such as BHA, BHT, etc., have their own toxic side effects, and governments have imposed mandatory amounts on them. People turned their attention to extracting natural antioxidants from various plant and animal tissues. As an antioxidant of foods and organisms, antioxidant active peptides are considered to be ideal substitutes for synthetic antioxidants due to their low toxicity and high efficiency.
  • the present invention uses a ginkgo peel as a raw material to prepare a polypeptide having antioxidant activity by an enzymatic method, and provides a theoretical basis and technical support for the development of an antioxidant functional food.
  • the purpose of the present application is to provide a method for preparing an antioxidant peptide by using ginkgo peel, which specifically comprises the following steps:
  • the dried and pulverized ginkgo peel is put into an aqueous solution of NaOH to extract crude protein of ginkgo peel;
  • the ginkgo peel crude protein is prepared into an aqueous solution having a mass concentration of 1-10% and a pH of 9-10; then a protease is added to adjust the temperature of the aqueous solution to 50-60 ° C, and the hydrolysis reaction is carried out for 2-5 hours. Then, the aqueous solution is kept boiling at 95-100 ° C for 10-15 min, and the enzyme is deactivated to obtain a hydrolyzate; in this step, the aqueous solution prepared by the crude protein of ginkgo peel is preferably adjusted with NaOH; the pH value after adjustment is the most Good for 9;
  • step (1) the ginkgo peel is put into an aqueous solution of NaOH, and then shaken at 55-65 ° C for 1-5 hours, then centrifuged and taken, and the supernatant is taken; the supernatant is adjusted to pH 2 with hydrochloric acid. 3. Centrifugation again, washing the precipitate with water, and drying to obtain crude ginkgo peel protein; the concentration of the aqueous NaOH solution is 0.05-0.1 mol/L; the mass ratio of ginkgo peel to NaOH aqueous solution is 1:10-1: 100. After the ginkgo peel was put into an aqueous NaOH solution, the optimum temperature at the time of shaking was 60 °C.
  • the temperature at the time of shaking is preferably 60 ° C; and when the supernatant is adjusted with hydrochloric acid, the pH is preferably 2.6.
  • the protein in the ginkgo peel can be extracted to the utmost, and the crude protein of ginkgo peel with higher purity can be obtained.
  • the main function of NaOH is to dissolve the protein in the ginkgo peel to facilitate the extraction.
  • concentration of NaOH reaches a certain concentration, the dissolution rate of the protein has been limited, and the concentration of NaOH is controlled at 0.05-0.1 mol/L.
  • concentration of NaOH is controlled at 0.05-0.1 mol/L.
  • Appropriate solid-liquid ratio can extract the protein in the ginkgo peel and enter the NaOH aqueous solution.
  • the extraction rate of protein in the ginkgo peel is affected, and the NaOH aqueous solution is relative to the ginkgo peel. The amount should not be too small.
  • the protease is an alkaline protease. After screening, alkaline protease is the best protease for protein breakdown.
  • the amount of the protease added is 1-5% of the crude protein amount of the ginkgo peel, and more preferably 2-3%.
  • the amount of protease in this range has been well able to complete the decomposition of the protein without excessive addition.
  • the antioxidant peptide prepared by the invention can be used as an antioxidant, and the scavenging rate of DPPH radical is high.
  • concentration of the antioxidant peptide in the solution reaches 0.4 g/L or more, the DPPH free radical scavenging rate is obtained. Stable at more than 80%, has a higher health care effect on the human body.
  • Ginkgo biloba peel was removed, dried at 40 ° C for 12 hours, ground into powder with a pulverizer, 10 kg was put into 100 kg of NaOH aqueous solution, shaken at 60 ° C for 2 hours, centrifuged to remove the supernatant, and then the supernatant was used. The pH was adjusted to 2.6 with hydrochloric acid, and then the precipitate was centrifuged, and the precipitate was washed and dried to obtain crude ginkgo peel protein; wherein the concentration of the aqueous NaOH solution was 0.08 mol/L.
  • the prepared ginkgo biloba crude protein was made into an aqueous solution having a mass concentration of 1% and a pH of 9, and then a 1% alkaline protease was added to the mass of the ginkgo peel crude protein, and the hydrolysis reaction was carried out at 50 ° C for 2 hours.
  • the aqueous solution was kept boiling at 100 ° C for 10 min, and the enzyme was deactivated to obtain a hydrolyzate.
  • An aqueous solution made of ginkgo peel crude protein was adjusted to pH with NaOH.
  • the hydrolyzate was centrifuged, and the supernatant was subjected to vacuum freeze-drying to obtain an antioxidant peptide A.
  • Ginkgo biloba peel was removed, dried at 50 ° C for 8 hours, ground into powder with a pulverizer, 1 kg was put into 100 kg of NaOH aqueous solution, and shaken at 50 ° C for 5 hours, then centrifuged to take the supernatant, and then the supernatant was used. Hydrochloric acid was adjusted to pH 2, and then the precipitate was centrifuged, and the precipitate was washed and dried to obtain crude ginkgo peel protein; wherein the concentration of the aqueous NaOH solution was 0.1 mol/L.
  • the prepared ginkgo biloba crude protein was made into an aqueous solution having a mass concentration of 10% and a pH of 10, and then an alkaline protease of 2% of the crude protein amount of the ginkgo peel was added, and the hydrolysis reaction was carried out at 60 ° C for 5 hours.
  • the aqueous solution was kept at 95 ° C for 12 min, and the enzyme was deactivated to obtain a hydrolyzate.
  • the hydrolyzate was centrifuged, and the supernatant was subjected to vacuum freeze-drying to obtain an antioxidant peptide B.
  • Ginkgo biloba peel was removed, dried at 50 ° C for 8 hours, ground into powder with a pulverizer, 2 kg was put into 100 kg of NaOH aqueous solution, and shaken at 55 ° C for 3 hours, then centrifuged to take the supernatant, and then the supernatant was used.
  • the pH was adjusted to 3 by hydrochloric acid, and then the precipitate was centrifuged, and the precipitate was washed and dried to obtain crude ginkgo peel protein; wherein the concentration of the aqueous NaOH solution was 0.05 mol/L.
  • the prepared ginkgo biloba crude protein was made into an aqueous solution having a mass concentration of 5% and a pH of 9.5, and then an alkaline protease having a crude protein amount of 4% relative to the ginkgo peel was added, and the hydrolysis reaction was carried out at 50 ° C for 2 hours.
  • the aqueous solution was kept at 98 ° C for 11 min, and the enzyme was deactivated to obtain a hydrolyzate.
  • the hydrolyzate was centrifuged, and the supernatant was subjected to vacuum freeze-drying to obtain an antioxidant peptide C.
  • Ginkgo biloba peel was removed, dried at 40 ° C for 12 hours, ground into powder with a pulverizer, 1 kg was put into 90 kg of NaOH aqueous solution, shaken at 60 ° C for 4 hours, centrifuged to take the supernatant, and then the supernatant was used. The pH was adjusted to 2.6 with hydrochloric acid, and then the precipitate was centrifuged, and the precipitate was washed and dried to obtain crude ginkgo peel protein; wherein the concentration of the aqueous NaOH solution was 0.09 mol/L.
  • the prepared ginkgo biloba crude protein is made into an aqueous solution having a mass concentration of 8% and a pH of 9, and then an alkaline protease of 5% relative to the crude protein amount of the ginkgo peel is added, and the hydrolysis reaction is carried out at 50 ° C for 3 hours.
  • the aqueous solution was kept at 97 ° C for 15 min, and the enzyme was deactivated to obtain a hydrolyzate.
  • the hydrolyzate was centrifuged, and the supernatant was subjected to vacuum freeze-drying to obtain an antioxidant peptide D.
  • the activity detection of the antioxidant peptide obtained in the examples was carried out by using the scavenging effect on DPPH radicals, and the specific operation was as follows:
  • the antioxidant peptides were separately prepared into samples of different concentrations, and then 0.4 mL samples were separately mixed with an equal volume of 40 g/LDPPH absolute ethanol solution, and allowed to stand at room temperature for 30 min in the dark, and the absorbance was measured at a wavelength of 517 nm.
  • the samples were replaced with deionized water and reduced glutathione as blank controls and positive controls, respectively.
  • the DPPH free radical scavenging activity of the sample is calculated according to the following formula (1):
  • a i is the absorbance of the DPPH absolute ethanol solution added to the sample
  • a j is the absorbance of the anhydrous ethanol solution added to the sample
  • a 0 is the absorbance of the DPPH absolute ethanol solution with the deionized water instead of the sample .

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  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicines Containing Plant Substances (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne un procédé utilisant des coques de noix de ginkgo pour préparer un peptide antioxydant, ledit procédé comprenant les étapes suivantes consistant à : (1) placer les coques de noix de ginkgo broyées et séchées dans une solution aqueuse de NaOH pour extraire une protéine brute des coques des noix de ginkgo ; (2) préparer la protéine brute des coques des noix de ginkgo à une densité de masse de 1 à 10 % dans une solution aqueuse de pH 9 à 10, puis ajouter une protéinase, ajuster la température de la solution aqueuse à 50 à 60 °C et effectuer une réaction d'hydrolyse pendant 2 à 5 heures, puis maintenir la solution à ébullition vive à 95 à 100 °C pendant 10 à 15 minutes, inactiver l'enzyme et obtenir une solution hydrolysée ; (3) précipiter la solution hydrolysée par centrifugation, extraction du surnageant et lyophilisation sous vide de celui-ci pour préparer un peptide antioxydant. Lorsque la densité du peptide antioxydant en solution atteint au moins 0,4 g/l, son pouvoir antiradicalaire par le test DPPH se stabilise à plus de 80 %.
PCT/CN2017/078555 2016-06-16 2017-03-29 Procédé de préparation d'un peptide antioxydant en utilisant des coquilles de noix de ginkgo WO2017215313A1 (fr)

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CN201610420906.8A CN106086132A (zh) 2016-06-16 2016-06-16 一种利用银杏果皮制备抗氧化肽的方法
CN201610420906.8 2016-06-16

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Cited By (2)

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CN111616362A (zh) * 2020-06-29 2020-09-04 山东省经济林管理站 一种核桃雄花抗氧化剂及其制备方法和应用
CN114774499A (zh) * 2022-04-24 2022-07-22 山东省农业科学院 一种珍珠油杏抗氧化肽APHs-1-c的提取方法及其应用

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106086132A (zh) * 2016-06-16 2016-11-09 如皋福大工程技术研究院有限公司 一种利用银杏果皮制备抗氧化肽的方法

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CN106119324A (zh) * 2016-06-16 2016-11-16 如皋福大工程技术研究院有限公司 一种利用银杏果皮制备钙螯合肽的方法

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CN106086132A (zh) * 2016-06-16 2016-11-09 如皋福大工程技术研究院有限公司 一种利用银杏果皮制备抗氧化肽的方法
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111616362A (zh) * 2020-06-29 2020-09-04 山东省经济林管理站 一种核桃雄花抗氧化剂及其制备方法和应用
CN111616362B (zh) * 2020-06-29 2022-03-08 山东省林业保护和发展服务中心 一种核桃雄花抗氧化剂及其制备方法和应用
CN114774499A (zh) * 2022-04-24 2022-07-22 山东省农业科学院 一种珍珠油杏抗氧化肽APHs-1-c的提取方法及其应用

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