CN102337318A - Gingko antioxidant active peptide and preparation method thereof - Google Patents
Gingko antioxidant active peptide and preparation method thereof Download PDFInfo
- Publication number
- CN102337318A CN102337318A CN2011102101198A CN201110210119A CN102337318A CN 102337318 A CN102337318 A CN 102337318A CN 2011102101198 A CN2011102101198 A CN 2011102101198A CN 201110210119 A CN201110210119 A CN 201110210119A CN 102337318 A CN102337318 A CN 102337318A
- Authority
- CN
- China
- Prior art keywords
- ginkgo
- gingko
- active peptides
- preparation
- lyophilize
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Peptides Or Proteins (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention relates to a gingko antioxidant active peptide and a preparation method thereof. The preparation method of the gingko antioxidant active peptide comprises the following steps of: extracting gingko kernel proteins from gingko kernel powder of which the grease content is not higher than 0.95 percent and which serves as a raw material with an alkaline process; hydrolyzing the gingko kernel proteins stepwise by using alkali protease and pepsin; and purifying to obtain the gingko antioxidant active peptide. The sequence of the gingko antioxidant active peptide is Tyr-Val-Gly-Asp or Leu-Gly-Asn-Thr-Asp-Tyr-Ala-Val-His. In the method, the gingko antioxidant active peptide is prepared from gingko kernels serving as a raw material, so that the obtained product has high antioxidant activity, free radicals can be removed, linoleic acid peroxidation activity is suppressed, and high reducing capability is achieved. The preparation method has a scientific and reasonable technical design, and plays an important role in developing the gingko industry; and comprehensive utilization and increase in added value of gingko kernels can be realized.
Description
Technical field
The present invention relates to a kind of ginkgo antioxidation active peptides and preparation method thereof.
Background technology
China has abundant gingko resource, and its stock number accounts for 90% of world's gingko resource total amount.Along with the development of domestic ginkgo industry in recent years, the output of ginkgo kernel also increases sharply, and causes the price of ginkgo kernel to reduce.This is owing to also contain some sensitization compositions in the ginkgo kernel on the one hand, can not directly eat in a large number; On the other hand, the intensive processing product of ginkgo kernel is less, causes that supply exceed demand on the market, and price descends thereupon.The deep processing problem that how to solve ginkgo kernel becomes the key point of ginkgo industry development.
Ginkgo kernel is nutritious; Has very high edible and pharmaceutical use; Contain protein, starch, flavonoid, terpene, vegeto-alkali, polyose, phenols, amino acid, trace element etc., contain compositions such as ginkgolic acid, Hydroginkgolic acid, bilobalide in addition.Protein contnt is more than 10% in the ginkgo kernel, and amino acid is formed rationally, and has many functions, like anti-oxidant, antibacterial, antitumor, anti-ageing and immunomodulatory etc.Therefore, be raw material with ginkgo albumen, promote the deep processing industry of ginkgo, can bring higher added value of product.
Summary of the invention
The present invention provides a kind of preparation method of ginkgo antioxidation active peptides.
The present invention also provides the ginkgo antioxidation active peptides.
The preparation method of said ginkgo antioxidation active peptides; Being not more than 0.95% ginkgo kernel powder with fat content is raw material; Alkaline process extracts ginkgo kernel protein; Adopt Sumizyme MP and stomach en-that said ginkgo kernel protein is carried out fractional hydrolysis then, purifying obtains the ginkgo antioxidation active peptides.
Preferably, fat content is not more than 0.95% ginkgo kernel powder and is with lyophilize, pulverizes the ginkgo kernel powder supercritical fluid extraction degreasing obtain and obtain.Further preferred, the condition of lyophilize ginkgo kernel powder is:, be not higher than lyophilize 36～48h under-35 ℃ of conditions at vacuum tightness 10～20Pa, the parameter of supercritical fluid extraction degreasing is: extracting pressure 30～35MPa, 50 ℃ of extraction temperature, CO
2Flow 20L/h, extraction time 3～4h.The ginkgo kernel powder is pulverized, is crossed 60 mesh sieves by ginkgo kernel and obtains.
Preferably, said alkaline process method for extracting proteins is: be not more than at fat content that to add mass concentration in 0.95% the ginkgo kernel powder be the NaOH solution of 1.5～2.5g/L, the mass volume ratio 1: 25～1 of ginkgo kernel powder and NaOH solution: 30g/ml; Extract 3～5 ℃ of temperature; Extraction time is 11～13h, and is centrifugal then, gets supernatant; Adjustment pH value separates to obtain precipitating being ginkgo kernel protein to iso-electric point.
A.2709 Sumizyme MP enzymolysis
Ginkgo kernel albumen is mixed with the protein solution that concentration is 18～22g/L, and regulating the pH value is 8.5～9.5, is benchmark with the proteic quality of ginkgo kernel, and 2709 Sumizyme MP additions are 6500～7500Ug
-1, 54～56 ℃ of enzymolysis 4～5h, the enzyme that goes out obtains primary enzymolysis liquid;
B. stomach en-enzymolysis
The pH value of primary enzymolysis liquid is adjusted into 2.5～3.5, is benchmark with the proteic quality of ginkgo kernel, and adding pepsic amount is 8500～9500Ug
-1, 49～51 ℃ of enzymolysis 1.5～2.5h, the enzyme that goes out obtains secondary enzymolysis liquid.
Preferably, the method for purifying after the fractional hydrolysis is: centrifugal secondary enzymolysis liquid obtains supernatant, through lyophilize, obtains having the ginkgo anti-oxidation peptide of anti-oxidant activity.Cryodesiccated method after the said fractional hydrolysis in the purification process is: the pre-freeze temperature is-50 ℃, and vacuum tightness is 10～20Pa, and freeze temperature is lower than-35 ℃, freeze-drying time 36～48h.
Further preferred, the method for purifying after the fractional hydrolysis is: centrifugal secondary enzymolysis liquid obtains supernatant, through lyophilize; Obtain having the ginkgo anti-oxidation peptide of anti-oxidant activity, the ginkgo antioxidation active peptides is mixed with the solution of 45～55mg/mL, last Sephadex G-25 gel column carries out separation and purification; The deionized water wash-out, collecting with the DPPH free radical scavenging activity is the highest polypeptide fraction of anti-oxidant activity that detects index screening, lyophilize; Be mixed with the solution of 45～55mg/mL then; Last SephadexG-10 gel column carries out separation and purification, the deionized water wash-out, and collecting with the DPPH free radical scavenging activity is the highest polypeptide fraction of anti-oxidant activity that detects index screening; Lyophilize; Being mixed with the solution of 18～22mg/mL then, adopting half preparative liquid chromatography to carry out separation and purification, is elutriant with 10% acetonitrile+0.1%TFA; Collection serves as to detect two kinds of the highest components of index screening anti-oxidant activity with the DPPH free radical scavenging activity, obtains having first polypeptide fraction and second polypeptide fraction of anti-oxidant activity through lyophilize.First polypeptide fraction that obtains after adopting liquid chromatography-level Four bar flight time mass spectrum to purifying and two peptide sections of second polypeptide fraction are carried out structure and are identified that the aminoacid sequence of two peptide sections is respectively: Tyr-Val-Gly-Asp (molecular weight is 452.21) and Leu-Gly-Asn-Thr-Asp-Tyr-Ala-Val-His (molecular weight is 988.49).
Per-cent is mass percent described in the present invention.
The present invention is feedstock production ginkgo antioxidation active peptides with the ginkgo kernel, and products obtained therefrom has higher anti-oxidant activity, can remove radical, and it is active to suppress linoleic acid peroxidation, has reducing power preferably.Technical project of the present invention is scientific and reasonable, can realize the comprehensive utilization of ginkgo kernel and improve its added value, and the development of ginkgo industry is had great importance.
Description of drawings
Fig. 1 is the liquid chromatography-quadrupole flight time mass spectrum figure of first polypeptide fraction.
Fig. 2 is the liquid chromatography-quadrupole flight time mass spectrum figure of second polypeptide fraction.
Embodiment
Embodiment 1
At extracting pressure 35MPa, 50 ℃ of extraction temperature, CO
2Flow 20L/h carries out degreasing to ginkgo kernel powder (crossing 60 mesh sieves) under the condition of extraction time 4h, and fat content is 0.95% after the degreasing.Take by weighing the ginkgo kernel powder 100g after the degreasing, adding mass concentration is the NaOH solution 2.6L of 2g/L, places 4 ℃ of refrigerators to extract 12h, whenever stirs once at a distance from 30min.Extracting solution is adjusted supernatant pH value to 4.62 through the centrifugal 15min of 3000r/min, and the centrifugal 20min of 5000r/min must precipitate then.Deposition is mixed with the ginkgo kernel protein solution of 20g/L, and regulating the pH value is 8.5, at first adds 2709 Sumizyme MPs, and addition is 6500U/g, places 55 ℃ of constant-temperature shaking water bath enzymolysis 4h; Enzymolysis solution is placed 90 ℃ of water-baths enzyme 10min that goes out, and regulating the pH value is 2.5, and adding pepsic amount is 8500U/g, places 50 ℃ of constant-temperature shaking water bath 2h, and enzymolysis solution is placed 90 ℃ of water-baths enzyme 10min that goes out.Go out enzymolysis solution behind the enzyme in the centrifugal 10min of 3000r/min, collect supernatant, at-50 ℃ of following pre-freeze 5h, at vacuum tightness 10Pa, lyophilize 36h under-35 ℃ of conditions obtains the ginkgo antioxidation active peptides, and yield is 51.39%.When the concentration of ginkgo antioxidation active peptides was 1.0mg/mL, the DPPH free radical scavenging activity was 46.56%, Fe
2+Chelation percent is 76.38%, and reducing power is 0.509.Ginkgo anti-oxidant activity peptide freeze-dried powder is mixed with the solution of 45mg/mL, and last Sephadex G-25 gel column carries out separation and purification, and applied sample amount is 4mL.With the deionized water is elutriant, and elution flow rate is 0.4mL/min, collects the highest polypeptide fraction of anti-oxidant activity.Sephadex G-25 is separated the highest component of activity obtain repeatedly collect, lyophilize is mixed with the solution of 45mg/mL then, and last SephadexG-10 gel column carries out separation and purification, and applied sample amount is 4mL.With the deionized water is elutriant, and elution flow rate is 0.4mL/min, collects the highest polypeptide fraction of anti-oxidant activity.Sephadex G-10 is separated the highest component of activity obtain repeatedly collect, lyophilize is mixed with the solution of 18mg/mL then, adopts half preparative liquid chromatography to carry out separation and purification, and applied sample amount is 1.8mL.With 10% acetonitrile+0.1%TFA is elutriant, and elution flow rate is 10mL/min, collects the highest polypeptide fraction of anti-oxidant activity.The component 1 that obtains after adopting liquid chromatography-level Four bar flight time mass spectrum to purifying is carried out structure with 2 two peptide sections of component and is identified that the aminoacid sequence of two peptide sections is respectively: Tyr-Val-Gly-Asp (molecular weight is 452.21) and Leu-Gly-Asn-Thr-Asp-Tyr-Ala-Val-His (molecular weight is 988.49).When concentration was 1.0mg/mL, the DPPH free radical scavenging activity of peptide section Tyr-Val-Gly-Asp was 79.29%, Fe
2+Chelation percent is 87.38%, and reducing power is 1.026; The DPPH free radical scavenging activity of peptide section Leu-Gly-Asn-Thr-Asp-Tyr-Ala-Val-His is 74.85%, Fe
2+Chelation percent is 91.73%, and reducing power is 0.837.
Embodiment 2
At extracting pressure 30MPa, 50 ℃ of extraction temperature, CO
2Flow 20L/h carries out degreasing to ginkgo kernel powder (crossing 60 mesh sieves) under the condition of extraction time 3h, and fat content is 0.95% after the degreasing.Take by weighing the ginkgo kernel powder 100g after the degreasing, adding mass concentration is the NaOH solution 2.6L of 2g/L, places 4 ℃ of refrigerators to extract 12h, whenever stirs once at a distance from 30min.Extracting solution is adjusted supernatant pH value to 4.62 through the centrifugal 15min of 3000r/min, and the centrifugal 20min of 5000r/min must precipitate then.Deposition is mixed with 2% ginkgo kernel protein solution, and regulating the pH value is 9.0, at first adds 2709 Sumizyme MPs, and addition is 7000U/g, places 55 ℃ of constant-temperature shaking water bath enzymolysis 5h; Enzymolysis solution is placed 90 ℃ of water-baths enzyme 10min that goes out, and regulating the pH value is 3.0, and adding pepsic amount is 9000U/g, places 50 ℃ of constant-temperature shaking water bath 2h, and enzymolysis solution is placed 90 ℃ of water-baths enzyme 15min that goes out.Go out enzymolysis solution behind the enzyme in the centrifugal 10min of 3000r/min, collect supernatant, at-50 ℃ of following pre-freeze 5h, at vacuum tightness 15Pa, lyophilize 48h under-35 ℃ of conditions obtains the ginkgo antioxidation active peptides, and yield is 53.51%.When the concentration of ginkgo antioxidation active peptides was 1.0mg/mL, the DPPH free radical scavenging activity was 48.56%, Fe
2+Chelation percent is 79.26%, and reducing power is 0.537.Ginkgo anti-oxidant activity peptide freeze-dried powder is mixed with the solution of 50mg/mL, and last Sephadex G-25 gel column carries out separation and purification, and applied sample amount is 5mL.With the deionized water is elutriant, and elution flow rate is 0.5mL/min, collects the highest polypeptide fraction of anti-oxidant activity.Sephadex G-25 is separated the highest component of activity obtain repeatedly collect, lyophilize is mixed with the solution of 50mg/mL then, and last Sephadex G-10 gel column carries out separation and purification, and applied sample amount is 5mL.With the deionized water is elutriant, and elution flow rate is 0.5mL/min, collects the highest polypeptide fraction of anti-oxidant activity.Sephadex G-10 is separated the highest component of activity obtain repeatedly collect, lyophilize is mixed with the solution of 20mg/mL then, adopts half preparative liquid chromatography to carry out separation and purification, and applied sample amount is 2mL.With 10% acetonitrile+0.1%TFA is elutriant, and elution flow rate is 10mL/min, collects the highest polypeptide fraction of anti-oxidant activity.The component 1 that obtains after adopting liquid chromatography-level Four bar flight time mass spectrum to purifying is carried out structure with 2 two peptide sections of component and is identified that the aminoacid sequence of two peptide sections is respectively: Tyr-Val-Gly-Asp (molecular weight is 452.21) and Leu-Gly-Asn-Thr-Asp-Tyr-Ala-Val-His (molecular weight is 988.49).When concentration was 1.0mg/mL, the DPPH free radical scavenging activity of peptide section Tyr-Val-Gly-Asp was 79.05%, Fe
2+Chelation percent is 87.31%, and reducing power is 1.018; The DPPH free radical scavenging activity of peptide section Leu-Gly-Asn-Thr-Asp-Tyr-Ala-Val-His is 74.85%, Fe
2+Chelation percent is 92.14%, and reducing power is 0.826.
Embodiment 3
At extracting pressure 35MPa, 50 ℃ of extraction temperature, CO
2Flow 20L/h carries out degreasing to ginkgo kernel powder (crossing 60 mesh sieves) under the condition of extraction time 3h, and fat content is 0.95% after the degreasing.Take by weighing the ginkgo kernel powder 100g after the degreasing, adding mass concentration is the NaOH solution 2.6L of 2g/L, places 4 ℃ of refrigerators to extract 12h, whenever stirs once at a distance from 30min.Extracting solution is adjusted supernatant pH value to 4.62 through the centrifugal 15min of 3000r/min, and the centrifugal 20min of 5000r/min must precipitate then.Deposition is mixed with the ginkgo kernel protein solution of 2g/L, and regulating the pH value is 9.5, at first adds 2709 Sumizyme MPs, and addition is 7500U/g, places 55 ℃ of constant-temperature shaking water bath enzymolysis 4h; Enzymolysis solution is placed 90 ℃ of water-baths enzyme 10min that goes out, and regulating the pH value is 3.0, and adding pepsic amount is 9500U/g, places 50 ℃ of constant-temperature shaking water bath 2h, and enzymolysis solution is placed 90 ℃ of water-baths enzyme 15min that goes out.Go out enzymolysis solution behind the enzyme in the centrifugal 15min of 3000r/min, collect supernatant, at-50 ℃ of following pre-freeze 5h, at vacuum tightness 20Pa, lyophilize 48h under-40 ℃ of conditions obtains the ginkgo antioxidation active peptides, and yield is 50.79%.When the concentration of ginkgo antioxidation active peptides was 1.0mg/mL, the DPPH free radical scavenging activity was 47.34%, Fe
2+Chelation percent is 76.98%, and reducing power is 0.526.Ginkgo anti-oxidant activity peptide freeze-dried powder is mixed with the solution of 45mg/mL, and last Sephadex G-25 gel column carries out separation and purification, and applied sample amount is 6mL.With the deionized water is elutriant, and elution flow rate is 0.4mL/min, collects the highest polypeptide fraction of anti-oxidant activity.Sephadex G-25 is separated the highest component of activity obtain repeatedly collect, lyophilize is mixed with the solution of 45mg/mL then, and last Sephadex G-10 gel column carries out separation and purification, and applied sample amount is 6mL.With the deionized water is elutriant, and elution flow rate is 0.6mL/min, collects the highest polypeptide fraction of anti-oxidant activity.Sephadex G-10 is separated the highest component of activity obtain repeatedly collect, lyophilize is mixed with the solution of 22mg/mL then, adopts half preparative liquid chromatography to carry out separation and purification, and applied sample amount is 1.8mL.With 10% acetonitrile+0.1%TFA is elutriant, and elution flow rate is 10mL/min, collects the highest polypeptide fraction of anti-oxidant activity.The component 1 that obtains after adopting liquid chromatography-level Four bar flight time mass spectrum to purifying is carried out structure with 2 two peptide sections of component and is identified that the aminoacid sequence of two peptide sections is respectively: Tyr-Val-Gly-Asp (molecular weight is 452.21) and Leu-Gly-Asn-Thr-Asp-Tyr-Ala-Val-His (molecular weight is 988.49).When concentration was 1.0mg/mL, the DPPH free radical scavenging activity of peptide section Tyr-Val-Gly-Asp was 80.04%, Fe
2+Chelation percent is 87.15%, and reducing power is 1.033; The DPPH free radical scavenging activity of peptide section Leu-Gly-Asn-Thr-Asp-Tyr-Ala-Val-His is 74.36%, Fe
2+Chelation percent is 91.57%, and reducing power is 0.828.
Embodiment 4
At extracting pressure 35MPa, 50 ℃ of extraction temperature, CO
2Flow 20L/h carries out degreasing to ginkgo kernel powder (crossing 60 mesh sieves) under the condition of extraction time 4h, and fat content is 0.95% after the degreasing.Take by weighing the ginkgo kernel powder 100g after the degreasing, add mass concentration and be 0.2% NaOH solution 2.6L, place 4 ℃ of refrigerators to extract 12h, whenever stir once at a distance from 30min.Extracting solution is adjusted supernatant pH value to 4.62 through the centrifugal 15min of 3000r/min, and the centrifugal 20min of 5000r/min must precipitate then.Deposition is mixed with the ginkgo kernel protein solution of 2g/L, and regulating the pH value is 9.5, at first adds 2709 Sumizyme MPs, and addition is 6500U/g, places 55 ℃ of constant-temperature shaking water bath enzymolysis 5h; Enzymolysis solution is placed 90 ℃ of water-baths enzyme 10min that goes out, and regulating the pH value is 3.5, and adding pepsic amount is 9000U/g, places 50 ℃ of constant-temperature shaking water bath 2h, and enzymolysis solution is placed 90 ℃ of water-baths enzyme 10min that goes out.Go out enzymolysis solution behind the enzyme in the centrifugal 15min of 3000r/min, collect supernatant, at-50 ℃ of following pre-freeze 5h, at vacuum tightness 15Pa, lyophilize 36h under-40 ℃ of conditions obtains the ginkgo antioxidation active peptides, and yield is 50.24%.When the concentration of ginkgo antioxidation active peptides was 1.0mg/mL, the DPPH free radical scavenging activity was 46.88%, Fe
2+Chelation percent is 77.06%, and reducing power is 0.519.Ginkgo anti-oxidant activity peptide freeze-dried powder is mixed with the solution of 55mg/mL, and last Sephadex G-25 gel column carries out separation and purification, and applied sample amount is 4mL.With the deionized water is elutriant, and elution flow rate is 0.4mL/min, collects the highest polypeptide fraction of anti-oxidant activity.Sephadex G-25 is separated the highest component of activity obtain repeatedly collect, lyophilize is mixed with the solution of 55mg/mL then, and last Sephadex G-10 gel column carries out separation and purification, and applied sample amount is 4mL.With the deionized water is elutriant, and elution flow rate is 0.4mL/min, collects the highest polypeptide fraction of anti-oxidant activity.Sephadex G-10 is separated the highest component of activity obtain repeatedly collect, lyophilize is mixed with the solution of 22mg/mL then, adopts half preparative liquid chromatography to carry out separation and purification, and applied sample amount is 2mL.With 10% acetonitrile+0.1%TFA is elutriant, and elution flow rate is 11mL/min, collects the highest polypeptide fraction of anti-oxidant activity.The component 1 that obtains after adopting liquid chromatography-level Four bar flight time mass spectrum to purifying is carried out structure with 2 two peptide sections of component and is identified that the aminoacid sequence of two peptide sections is respectively: Tyr-Val-Gly-Asp (molecular weight is 452.21) and Leu-Gly-Asn-Thr-Asp-Tyr-Ala-Val-His (molecular weight is 988.49).When concentration was 1.0mg/mL, the DPPH free radical scavenging activity of peptide section Tyr-Val-Gly-Asp was 79.17%, Fe
2+Chelation percent is 87.31%, and reducing power is 1.022; The DPPH free radical scavenging activity of peptide section Leu-Gly-Asn-Thr-Asp-Tyr-Ala-Val-His is 74.97%, Fe
2+Chelation percent is 92.12%, and reducing power is 0.841.
Embodiment 5
At extracting pressure 30MPa, 50 ℃ of extraction temperature, CO
2Flow 20L/h carries out degreasing to ginkgo kernel powder (crossing 60 mesh sieves) under the condition of extraction time 4h, and fat content is 0.95% after the degreasing.Take by weighing the ginkgo kernel powder 100g after the degreasing, adding mass concentration is the NaOH solution 2.6L of 2g/L, places 4 ℃ of refrigerators to extract 12h, whenever stirs once at a distance from 30min.Extracting solution is adjusted supernatant pH value to 4.62 through the centrifugal 15min of 3000r/min, and the centrifugal 20min of 5000r/min must precipitate then.Deposition is mixed with the ginkgo kernel protein solution of 20g/L, and regulating the pH value is 9.0, at first adds 2709 Sumizyme MPs, and addition is 7000U/g, places 55 ℃ of constant-temperature shaking water bath enzymolysis 4h; Enzymolysis solution is placed 90 ℃ of water-baths enzyme 15min that goes out, and regulating the pH value is 3.0, and adding pepsic amount is 9500U/g, places 50 ℃ of constant-temperature shaking water bath 2h, and enzymolysis solution is placed 90 ℃ of water-baths enzyme 15min that goes out.Go out enzymolysis solution behind the enzyme in the centrifugal 10min of 3000r/min, collect supernatant, at-50 ℃ of following pre-freeze 5h, at vacuum tightness 15Pa, lyophilize 36h under-45 ℃ of conditions obtains the ginkgo antioxidation active peptides, and yield is 51.93%.When the concentration of ginkgo antioxidation active peptides was 1.0mg/mL, the DPPH free radical scavenging activity was 47.13%, Fe
2+Chelation percent is 77.57%, and reducing power is 0.528.Ginkgo anti-oxidant activity peptide freeze-dried powder is mixed with the solution of 50mg/mL, and last Sephadex G-25 gel column carries out separation and purification, and applied sample amount is 6mL.With the deionized water is elutriant, and elution flow rate is 0.4mL/min, collects the highest polypeptide fraction of anti-oxidant activity.Sephadex G-25 is separated the highest component of activity obtain repeatedly collect, lyophilize is mixed with the solution of 45mg/mL then, and last SephadexG-10 gel column carries out separation and purification, and applied sample amount is 6mL.With the deionized water is elutriant, and elution flow rate is 0.4mL/min, collects the highest polypeptide fraction of anti-oxidant activity.Sephadex G-10 is separated the highest component of activity obtain repeatedly collect, lyophilize is mixed with the solution of 20mg/mL then, adopts half preparative liquid chromatography to carry out separation and purification, and applied sample amount is 2.2mL.With 10% acetonitrile+0.1%TFA is elutriant, and elution flow rate is 11mL/min, collects the highest polypeptide fraction of anti-oxidant activity.The component 1 that obtains after adopting liquid chromatography-level Four bar flight time mass spectrum to purifying is carried out structure with 2 two peptide sections of component and is identified that the aminoacid sequence of two peptide sections is respectively: Tyr-Val-Gly-Asp (molecular weight is 452.21) and Leu-Gly-Asn-Thr-Asp-Tyr-Ala-Val-His (molecular weight is 988.49).When concentration was 1.0mg/mL, the DPPH free radical scavenging activity of peptide section Tyr-Val-Gly-Asp was 78.65%, Fe
2+Chelation percent is 86.87%, and reducing power is 1.033; The DPPH free radical scavenging activity of peptide section Leu-Gly-Asn-Thr-Asp-Tyr-Ala-Val-His is 74.27%, Fe
2+Chelation percent is 91.46%, and reducing power is 0.829.
Polypeptide 1:Tyr-Val-Gly-Asp
Polypeptide 2:Leu-Gly-Asn-Thr-Asp-Tyr-Ala-Val-His
Claims (10)
1. the preparation method of a ginkgo antioxidation active peptides; It is characterized in that; Being not more than 0.95% ginkgo kernel powder with fat content is raw material; Alkaline process extracts ginkgo kernel protein, adopts Sumizyme MP and stomach en-that said ginkgo kernel protein is carried out fractional hydrolysis then, purifies to obtain the ginkgo antioxidation active peptides.
2. the preparation method of ginkgo antioxidation active peptides as claimed in claim 1 is characterized in that, fat content is not more than 0.95% ginkgo kernel powder and is with lyophilize, pulverizes the ginkgo kernel powder supercritical fluid extraction degreasing that obtains and obtain.
3. the preparation method of ginkgo antioxidation active peptides as claimed in claim 2; It is characterized in that; The condition of lyophilize ginkgo kernel powder is:, be not higher than lyophilize 36～48h under-35 ℃ of conditions at vacuum tightness 10～20Pa, the parameter of supercritical fluid extraction degreasing is: extracting pressure 30～35MPa; 50 ℃ of extraction temperature, CO
2Flow 20L/h, extraction time 3～4h.
4. the preparation method of ginkgo antioxidation active peptides as claimed in claim 1; It is characterized in that said alkaline process method for extracting proteins is: be not more than at fat content that to add mass concentration in 0.95% the ginkgo kernel powder be the NaOH solution of 1.5～2.5g/L, the mass volume ratio 1: 25～1 of ginkgo kernel powder and NaOH solution: 30g/ml; Extract 3～5 ℃ of temperature; Extraction time is 11～13h, and is centrifugal then, gets supernatant; Adjustment pH value separates to obtain precipitating being ginkgo kernel protein to iso-electric point.
5. the preparation method of ginkgo antioxidation active peptides as claimed in claim 1 is characterized in that, said Sumizyme MP is 2709 Sumizyme MPs, and the method for said ginkgo kernel protein being carried out fractional hydrolysis is:
A.2709 Sumizyme MP enzymolysis
Ginkgo kernel albumen is mixed with the protein solution that concentration is 18～22g/L, and regulating the pH value is 8.5～9.5, is benchmark with the proteic quality of ginkgo kernel, and 2709 Sumizyme MP additions are 6500～7500Ug
-1, 54～56 ℃ of enzymolysis 4～5h, the enzyme that goes out obtains primary enzymolysis liquid;
B. stomach en-enzymolysis
The pH value of primary enzymolysis liquid is adjusted into 2.5～3.5, is benchmark with the proteic quality of ginkgo kernel, and adding pepsic amount is 8500～9500Ug
-1, 49～51 ℃ of enzymolysis 1.5～2.5h, the enzyme that goes out obtains secondary enzymolysis liquid.
6. the preparation method of ginkgo antioxidation active peptides as claimed in claim 1 is characterized in that, the method for purifying after the fractional hydrolysis is: centrifugal secondary enzymolysis liquid obtains supernatant, through lyophilize, obtains having the ginkgo anti-oxidation peptide of anti-oxidant activity.
7. the preparation method of ginkgo antioxidation active peptides as claimed in claim 6; It is characterized in that the cryodesiccated method after the said fractional hydrolysis in the purification process is: the pre-freeze temperature is-50 ℃, vacuum tightness is 10～20Pa; Freeze temperature is lower than-35 ℃, freeze-drying time 36～48h.
8. like the preparation method of claim 6 or 7 described ginkgo antioxidation active peptides, it is characterized in that the method for purifying after the fractional hydrolysis is: centrifugal secondary enzymolysis liquid obtains supernatant; Through lyophilize, obtain having the ginkgo anti-oxidation peptide of anti-oxidant activity, the ginkgo antioxidation active peptides is mixed with the solution of 45～55mg/mL; Last Sephadex G-25 gel column carries out separation and purification, the deionized water wash-out, and collecting with the DPPH free radical scavenging activity is the highest polypeptide fraction of anti-oxidant activity that detects index screening; Lyophilize is mixed with the solution of 45～55mg/mL then, and last Sephadex G-10 gel column carries out separation and purification; The deionized water wash-out; Collection serves as the highest polypeptide fraction of anti-oxidant activity that detects index screening with the DPPH free radical scavenging activity, and lyophilize is mixed with the solution of 18～22mg/mL then; Adopt half preparative liquid chromatography to carry out separation and purification; With 10% acetonitrile+0.1%TFA is elutriant, and collecting with the DPPH free radical scavenging activity serves as to detect two kinds of the highest components of index screening anti-oxidant activity, obtains having first polypeptide fraction and second polypeptide fraction of anti-oxidant activity through lyophilize.
9. ginkgo antioxidation active peptides, its sequence is Tyr-Val-Gly-Asp.
10. ginkgo antioxidation active peptides, its sequence is Leu-Gly-Asn-Thr-Asp-Tyr-Ala-Val-His.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011102101198A CN102337318A (en) | 2011-07-26 | 2011-07-26 | Gingko antioxidant active peptide and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011102101198A CN102337318A (en) | 2011-07-26 | 2011-07-26 | Gingko antioxidant active peptide and preparation method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102337318A true CN102337318A (en) | 2012-02-01 |
Family
ID=45513268
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2011102101198A Pending CN102337318A (en) | 2011-07-26 | 2011-07-26 | Gingko antioxidant active peptide and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102337318A (en) |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103819541A (en) * | 2014-03-06 | 2014-05-28 | 福州大学 | Microalgae oxidation prevention polypeptide |
| CN103911412A (en) * | 2014-03-14 | 2014-07-09 | 昌吉市新祥林食品有限责任公司 | Preparation method of melon seed polypeptide |
| CN104313099A (en) * | 2014-10-28 | 2015-01-28 | 扬州大学 | Method for preparing egg albumin peptide with high antioxidant activity |
| CN104543328A (en) * | 2014-12-29 | 2015-04-29 | 徐州绿之野生物食品有限公司 | Preparation method and application for fermented gingko cell protein concentrated solution |
| CN105475487A (en) * | 2015-12-09 | 2016-04-13 | 中国农业大学 | Preparation method of ginkgo protein peptide fermented milk and ginkgo protein peptide fermented milk beverage |
| CN106636277A (en) * | 2016-12-29 | 2017-05-10 | 沧州医学高等专科学校 | Preparation technology of antioxidant polypeptide of cherry pits |
| WO2017215312A1 (en) * | 2016-06-16 | 2017-12-21 | 如皋福大工程技术研究院有限公司 | Method for the preparation of calcium-chelating peptide using gingko nut shells |
| WO2017215313A1 (en) * | 2016-06-16 | 2017-12-21 | 如皋福大工程技术研究院有限公司 | Method for the preparation of antioxidant peptide using gingko nut shells |
| CN112913572A (en) * | 2019-12-06 | 2021-06-08 | 宏润生物科技股份有限公司 | Method for culturing cordyceps militaris, culture product and pharmaceutical composition |
| CN116491631A (en) * | 2023-05-26 | 2023-07-28 | 青岛农业大学 | Braised pork with prefabricated dish and preparation method thereof |
-
2011
- 2011-07-26 CN CN2011102101198A patent/CN102337318A/en active Pending
Non-Patent Citations (5)
| Title |
|---|
| 孟如杰和田亚平: "银杏种仁中一种抗氧化活性蛋白的纯化及性质", 《天然产物研究与开发》 * |
| 牛卫宁和郭蔼光: "银杏种仁中抗菌蛋白的纯化及性质", 《西北植物学报》 * |
| 贾韶千等: "均匀设计优化碱法提取银杏蛋白工艺", 《食品科学》 * |
| 贾韶千等: "银杏抗氧化肽的分离纯化及活性鉴定", 《农业机械学报》 * |
| 黄小炯等: "银杏抗菌蛋白的提取及其抗菌性研究", 《食品工业科技》 * |
Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103819541A (en) * | 2014-03-06 | 2014-05-28 | 福州大学 | Microalgae oxidation prevention polypeptide |
| CN103911412A (en) * | 2014-03-14 | 2014-07-09 | 昌吉市新祥林食品有限责任公司 | Preparation method of melon seed polypeptide |
| CN104313099B (en) * | 2014-10-28 | 2017-08-25 | 扬州大学 | A kind of preparation method of the active egg white powder peptide of high anti-oxidation |
| CN104313099A (en) * | 2014-10-28 | 2015-01-28 | 扬州大学 | Method for preparing egg albumin peptide with high antioxidant activity |
| CN104543328A (en) * | 2014-12-29 | 2015-04-29 | 徐州绿之野生物食品有限公司 | Preparation method and application for fermented gingko cell protein concentrated solution |
| CN105475487A (en) * | 2015-12-09 | 2016-04-13 | 中国农业大学 | Preparation method of ginkgo protein peptide fermented milk and ginkgo protein peptide fermented milk beverage |
| WO2017215312A1 (en) * | 2016-06-16 | 2017-12-21 | 如皋福大工程技术研究院有限公司 | Method for the preparation of calcium-chelating peptide using gingko nut shells |
| WO2017215313A1 (en) * | 2016-06-16 | 2017-12-21 | 如皋福大工程技术研究院有限公司 | Method for the preparation of antioxidant peptide using gingko nut shells |
| CN106636277A (en) * | 2016-12-29 | 2017-05-10 | 沧州医学高等专科学校 | Preparation technology of antioxidant polypeptide of cherry pits |
| CN112913572A (en) * | 2019-12-06 | 2021-06-08 | 宏润生物科技股份有限公司 | Method for culturing cordyceps militaris, culture product and pharmaceutical composition |
| CN112913572B (en) * | 2019-12-06 | 2022-08-02 | 陈衫润 | Method for culturing cordyceps militaris, culture product and pharmaceutical composition |
| CN116491631A (en) * | 2023-05-26 | 2023-07-28 | 青岛农业大学 | Braised pork with prefabricated dish and preparation method thereof |
| CN116491631B (en) * | 2023-05-26 | 2024-06-25 | 青岛农业大学 | Braised pork with prefabricated dish and preparation method thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102337318A (en) | Gingko antioxidant active peptide and preparation method thereof | |
| CN101617787B (en) | Process for continuously extracting a plurality of products from highland barley | |
| CN102657332B (en) | Preparation method for successively producing yak bone marrow oil and ultramicro polypeptide bone calcium powder once | |
| CN103255186A (en) | Combined production method for abalone polysaccharide, lipid and protein peptide | |
| CN101182357A (en) | Globefish skin active collagen and method for preparing peptide thereof | |
| CN107557421A (en) | A kind of method of the effective component extracting from ganoderma lucidum | |
| CN103445159A (en) | Enzymatic preparation method of all-component ginseng juice | |
| CN105079492A (en) | Dendrobium officinale and solomon's seal capsule and preparation method thereof | |
| CN105924495B (en) | High-efficiency preparation method of high-purity flaxseed protein | |
| CN103202419A (en) | A compound fish scale peptide and calcium compound chewable tablet for antioxidation and calcium supplementing and a preparation method thereof | |
| CN104187539A (en) | Method for preparing chocolate essence from cocoa bean husk | |
| CN103436581A (en) | Method for extracting rapeseed peptides through wet milling combined with enzymic method | |
| CN110699412A (en) | Method for extracting selenium polypeptide from selenium-rich passion fruit seeds | |
| CN103262998A (en) | Wild lycium ruthenicum tablet and preparation method | |
| CN100526471C (en) | Method for preparing avenin ACE inhibiting peptide | |
| CN106801079A (en) | The method that a kind of pair of enzyme stepwise discretization Carapax Eriocheir sinensis prepare antioxidation active peptides | |
| CN104531814A (en) | Preparation method of oil-tea meal enzymatically-modified protein | |
| CN105146311A (en) | Highland barley dietary fiber instant powder and production method thereof | |
| CN111073933A (en) | Preparation method of protein peptide selenium chelate suitable for ruminants | |
| CN102246994A (en) | Fructus cannabis and sesame vegetable protein beverage and preparation method thereof | |
| CN102160563B (en) | Preparation method of anti-fatigue oligopeptides biscuits | |
| CN104357215B (en) | A kind of selenium-enriched tea leaf tobacco aromaticss and preparation method thereof and the application in cigarette preparation | |
| CN108949876A (en) | A kind of preparation method of the peony seeds polypeptide oral liquor with antioxidant activity | |
| CN103284273B (en) | Gynostemma pentaphylla and lotus root tea powder and preparation method thereof | |
| CN105053858A (en) | Moringa oleifera leaf extract containing instant noodles |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20120201 |