CN113832209B - Method for preparing hazelnut peptide with high antioxidant activity - Google Patents
Method for preparing hazelnut peptide with high antioxidant activity Download PDFInfo
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- MTDHILKWIRSIHB-UHFFFAOYSA-N (5-azaniumyl-3,4,6-trihydroxyoxan-2-yl)methyl sulfate Chemical compound NC1C(O)OC(COS(O)(=O)=O)C(O)C1O MTDHILKWIRSIHB-UHFFFAOYSA-N 0.000 claims description 2
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- NJDNXYGOVLYJHP-UHFFFAOYSA-L disodium;2-(3-oxido-6-oxoxanthen-9-yl)benzoate Chemical compound [Na+].[Na+].[O-]C(=O)C1=CC=CC=C1C1=C2C=CC(=O)C=C2OC2=CC([O-])=CC=C21 NJDNXYGOVLYJHP-UHFFFAOYSA-L 0.000 description 1
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- 230000007760 free radical scavenging Effects 0.000 description 1
- 108091005608 glycosylated proteins Proteins 0.000 description 1
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- 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
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- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
- C07K1/113—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides without change of the primary structure
- C07K1/1136—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides without change of the primary structure by reversible modification of the secondary, tertiary or quarternary structure, e.g. using denaturating or stabilising agents
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
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- 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
- C12P21/005—Glycopeptides, glycoproteins
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Abstract
A method for preparing hazelnut peptide with high antioxidant activity belongs to the technical field of peptide modification. The hazelnut peptide DPPH scavenging capacity obtained by using hazelnut as a raw material and adopting an enzyme-controlled hydrolysis technology to prepare antioxidant peptide, carrying out ultrasonic treatment on the raw material before enzymolysis, and carrying out transglutaminase glycosylation modification on the hazelnut peptide, wherein the hazelnut peptide DPPH scavenging capacity obtained by the treatment is obviously higher than that of the hazelnut peptide without modification. The invention has high reaction efficiency, safety and innocuity, is a green enzymatic glycosylation modification, saves time, reduces production cost, greatly improves the product activity, is a key breakthrough for the applicability of hazelnut kernels in food and pharmaceutical industry, and has great significance for the comprehensive utilization of hazelnut resources. The modified hazelnut peptide with high antioxidant activity is used as a raw material of health care products and foods, has high antioxidant activity, and can be used for preventing related diseases.
Description
Technical Field
The invention belongs to the technical field of peptide modification, and particularly relates to a method for preparing hazelnut peptide with high antioxidant activity.
Background
Free radicals and reactive oxygen species generated by the oxidation reaction promote a degenerative reaction in many cellular components, which leads to the development of many diseases such as cancer, alzheimer's disease, inflammation, cardiovascular disease and diabetes. In addition, free radical oxidation directly affects food quality, such as flavor and texture changes, which can shorten the shelf life of the food. Thus, the addition of antioxidants is an effective means of treating these diseases and preventing oxidative spoilage of foods. The bioactive peptide derived from food protein has higher safety than medicine, can be used as single medicine for treatment in clinical test, and has no side effects. The antioxidant peptide is a peptide with the function of scavenging free radicals, can be directly added into food, prolongs the quality guarantee period of the food and slows down the aging of organisms.
At present, an enzyme hydrolysis method is mainly adopted for producing the antioxidant peptide. The method is still limited in the aspects of low enzymatic utilization rate, low substrate conversion rate, longer enzymolysis time and the like, so that the comprehensive utilization of protein resources is adversely affected, and the crude peptide obtained by enzymolysis cannot completely meet the demands of people on the activity of the crude peptide. Therefore, to further enhance the biological utility of the peptide, it is necessary to further modify its structure. Ultrasound can alter the physical and functional properties of proteins, facilitating proteolytic hydrolysis by affecting exposure of binding sites and cleavage sites, thereby improving enzymatic efficiency, protein conversion, enzyme utilization, and peptide bioactivity. The glycosylation modification of the food protein ensures that the glycosylated protein has the functional characteristics of the protein, the hydrophilic characteristics of carbohydrate substances, and the oxidation resistance and other functional characteristics are improved to different degrees. The transglutaminase pathway glycosylation has the advantages of mild reaction conditions, no byproducts and the like, can endow protein with good functional characteristics, and has good application prospects.
Therefore, the invention organically combines the two methods, carries out molecular modification on the bioactive peptide and synergistically improves the antioxidant activity of the bioactive peptide. The content of protein in hazelnut is about 15-30%, hazelnut protein is a main byproduct in hazelnut oil processing industry, most of the hazelnut protein is discarded or used as animal feed, and the content of protein in grease processing byproduct defatted meal is up to more than 40%. The peptide prepared from hazelnut kernel has the functions of resisting oxidation, reducing blood pressure, reducing blood fat, resisting fatigue and the like, and can be widely applied to foods and medicines. The development of various active peptides by taking the hazelnut as a raw material has great significance on the comprehensive utilization of hazelnut resources.
Disclosure of Invention
The invention aims to provide a method for preparing hazelnut peptide with high antioxidant activity, which comprises the following steps:
(1) Hazelnut peptide preparation
Adding distilled water into hazelnut meal to prepare a solution with the mass concentration of 2% -35% of the hazelnut meal, carrying out ultrasonic pretreatment for 10-60 min at the power of 100-2400 w and the temperature of 20-60 ℃, and carrying out water bath for 10-20 min at the temperature of 90-96 ℃ to denature protein; cooling to room temperature, regulating pH value to 7-10 with 0.5mol/L NaOH, adding alkaline protease Alcalase 2.4L, adding enzyme amount to 4000-15000U/g hazelnut meal, carrying out enzymolysis for 2-5 h at 40-65 ℃, continuously adding NaOH in the enzymolysis process to keep the pH value unchanged, heating at 85-95 ℃ rapidly for 10-20 min after the reaction is finished to inactivate enzyme, cooling to room temperature, regulating pH value to be neutral with 0.5mol/L HCl, centrifuging for 10-20 min at 5000-8000 r/min, freeze-drying supernatant fluid (-50-30 ℃ for 20-30 h), and obtaining hazelnut peptide, and storing at 4 ℃;
(2) Hazelnut peptide modification
Adding distilled water into the hazelnut peptide obtained after freeze drying in the step (1) to prepare a solution with the mass concentration of Cheng Zhenren percent and 2-30 percent, regulating the pH value to 6-10 by using 0.5mol/L NaOH, then adding transglutaminase with the enzyme quantity of 1-15U/g hazelnut peptide, then adding sugar containing amino with the mass of 0.2-2.0 times of the hazelnut peptide, reacting for 2-5 h in a water bath with the temperature of 30-50 ℃, heating for 10-20 min at 70-80 ℃ rapidly after the reaction is finished to inactivate the enzyme, freeze-drying at (-50-30 ℃ for 20-30 h) to obtain the hazelnut peptide with high antioxidant activity, and preserving at the temperature of 4 ℃.
The hazelnut peptide may be prepared from Sichuan hazelnut needle, hazel, corylus corylifolia, corylus corylifolia a big-fruit hazel, corylus, or turkish hazel.
The amino group-containing sugar may be glucosamine, glucosamine hydrochloride, glucosamine sulfate or chitosan.
Compared with the prior art, the invention has the beneficial effects that:
The invention discloses a method for preparing hazelnut peptide with high antioxidant activity for the first time, which takes hazelnuts as raw materials, adopts an enzyme-controlled hydrolysis technology to prepare the hazelnut peptide, is assisted with ultrasonic treatment, and is added with transglutaminase to carry out enzymatic glycosylation modification, so that the antioxidant activity of the hazelnut peptide is obviously improved after the two treatments, and the method has great significance for comprehensive utilization of hazelnut resources.
The method disclosed by the invention is to combine ultrasonic technology to remove substrate restriction during enzymolysis, then combine the two methods organically through transglutaminase modification, carry out molecular modification on the bioactive peptide, synergistically improve the antioxidation activity of the bioactive peptide, and provide a feasible alternative method for modifying the bioactive peptide by traditional glycosylation, the traditional glycosylation reaction time is longer, the nutritive value of protein is reduced, meanwhile, the food safety problem exists, the transglutaminase modification reaction speed is faster, the energy is saved, the generation of byproducts is greatly reduced, the defects of the prior art are overcome, and the hazelnut has great development prospect in the food and medicine field. The modified hazelnut peptide with high antioxidant activity is used as a raw material of health care products and foods, has high antioxidant activity, and can be used for preventing related diseases.
Drawings
The drawings in the following description are only examples of embodiments of the present invention and other drawings may be made from these drawings by those of ordinary skill in the art without inventive faculty.
Fig. 1: DPPH clearance contrast plot;
Fig. 2: ORAC value versus graph;
1. ORAC detection
Oxygen radical adsorption capacity (Oxigen Radical Absorbance Capacity, ORAC) is a novel tube antioxidant assay based on terminating free radical chain reactions by inhibiting hydrogen proton transfer. Trolox was diluted with PBS buffer to 4 standard solutions of different concentrations (6.25, 12.5, 25 and 50. Mu.M). Two control groups were simultaneously set, one group with free radical (+AAPH) added to the 96-well blackboard and one group with no free radical (-AAPH) added to the 96-well blackboard. To each well were added 100. Mu.L of sodium Fluorescein (FL) at a final concentration of 63mmol/L, 50. Mu.L of Trolox at different concentrations, and 10. Mu.L of glutathione, the product of step (1) of example 1, or the aqueous solution of the product of step (2) of example 1, each at a concentration of 1 mg/mL. Subsequently, the 96-well plate was thoroughly shaken for 2min and incubated in a temperature-controlled incubator at 37℃for 10min, and, except for (-AAPH) wells, 50. Mu.L of AAPH was added to each well at a final concentration of 6mmol/L, and after thoroughly mixing, the measurement was immediately carried out with an excitation wavelength of 485nm and an emission wavelength of 528nm and the values were recorded, and the fluorescence value (denoted as fn) was measured every 2min during the reaction. The standard curve is plotted with fluorescence half decay time on the ordinate and Trolox concentration on the abscissa.
2. DPPH radical scavenging rate detection
2.0ML of glutathione, the product of the step (1) of the example 1 or the aqueous solution of the product of the step (2) of the example 1 (with the concentration of 1 mg/mL) are respectively added into test tubes, then 2.0mL of 0.2mmol/L of DPPH absolute ethanol solution is added into each test tube, and after mixing, the test tubes are placed at room temperature and protected from light for reaction for 30min. When the reaction was terminated, the absorbance of the reaction solution at 517nm was measured for each sample, and 3 replicates were performed for each sample in the experiment, with absolute ethanol as a reference control. The discrimination of the DPPH free radical scavenging capacity of the sample is based on the absorbance difference.
As shown in fig. 1 and 2, the product of step (1) of example 1 was not modified, and the modified product of step (2) of example 1 was obtained. The hazelnut peptide with high antioxidant activity prepared by the invention has good antioxidant capacity, and compared with unmodified active peptide, the antioxidant activity of the modified peptide is remarkably improved.
Detailed Description
The present invention is further illustrated by the following examples, which are not intended to limit the scope of the invention in any way.
Example 1
(1) Hazelnut peptide preparation
Taking 8g of hazelnut meal, adding 100mL of distilled water, and stirring to prepare a solution with the mass concentration of 8% of the hazelnut meal; carrying out ultrasonic treatment at a power of 600w and a temperature of 34 ℃ for 26min, and then carrying out water bath at a temperature of 95 ℃ for 15min to denature protein; cooling to room temperature, regulating pH to 8.9 with 0.5mol/L NaOH, adding Alcalase 2.4L (one of alkaline proteases), adding enzyme 8710U/g hazelnut meal, performing enzymolysis at 52.3deg.C for 3 hr, continuously adding NaOH during enzymolysis to maintain pH unchanged, rapidly heating at 90deg.C for 15min to inactivate enzyme after reaction, cooling to room temperature, regulating pH to neutrality with 0.5mol/L HCl, centrifuging at 6000r/min for 15min, lyophilizing supernatant at-40deg.C for 24 hr to obtain hazelnut peptide, and storing at 4deg.C;
(2) Hazelnut peptide modification
Adding 100mL of distilled water into 6g of freeze-dried hazelnut peptide, adding Cheng Zhenren mass percent solution of the peptide, adjusting the pH value to 8.1 by using 0.5mol/L NaOH, adding transglutaminase (with the enzyme amount of 7U/g hazelnut peptide), adding glucosamine (4.8 g) with the weight of 0.8 times that of the hazelnut peptide, reacting for 2h in a water bath at 43 ℃, heating for 15min at 75 ℃ rapidly after the reaction is finished, inactivating enzymes, freeze-drying at 40 ℃ for 24h to obtain the hazelnut peptide with high antioxidant activity, and preserving at 4 ℃.
Example 2
(1) Hazelnut peptide preparation
Adding 100mL distilled water into 10g Mao Zhenzi pieces of meal, and stirring to prepare a solution with the mass concentration of 10% of hazelnut meal; carrying out ultrasonic treatment at a power of 800w and a temperature of 40 ℃ for 30min, and then carrying out water bath at a temperature of 95 ℃ for 15min to denature protein; cooling to room temperature, regulating pH to 8.9 with 0.5mol/L NaOH, adding Alcalase 2.4L (one of alkaline proteases), adding 8900U/g hazelnut meal, performing enzymolysis at 50.3deg.C for 2.5 hr, continuously adding NaOH during enzymolysis to maintain pH unchanged, rapidly heating at 90deg.C for 15min to inactivate enzyme after reaction, cooling to room temperature, regulating pH to neutrality with 0.5mol/L HCl, centrifuging at 6000r/min for 15min, lyophilizing supernatant at-40deg.C for 24 hr to obtain hazelnut peptide, and storing at 4deg.C;
(2) Hazelnut peptide modification
Adding 100mL of distilled water into 4% of solution with the mass concentration of Cheng Zhenren peptide, adjusting the pH value to 7.9 with 0.5mol/L NaOH, adding transglutaminase (with the enzyme amount of 5U/g hazelnut peptide), adding glucosamine (3.2 g) with the mass of 0.8 times that of the hazelnut peptide, reacting for 2.5h in a water bath at 42 ℃, heating for 15min at 75 ℃ to inactivate enzyme after the reaction is finished, freeze-drying at-40 ℃ for 24h to obtain hazelnut peptide with high antioxidant activity, and preserving at 4 ℃.
The foregoing is merely a preferred embodiment of the invention and it should be noted that numerous changes and modifications could be made to the polypeptide without departing from the principles of the invention and it is intended to cover the invention as set forth in the claims.
Claims (1)
1. A method for preparing hazelnut peptide with high antioxidant activity comprises the following steps:
(1) Hazelnut peptide preparation
Adding distilled water into hazelnut meal to prepare a solution with the mass concentration of 2% -35% of the hazelnut meal; carrying out ultrasonic pretreatment for 10-60 min at the power of 100-2400 w and the temperature of 20-60 ℃, and carrying out water bath for 10-20 min at the temperature of 90-96 ℃ to denature protein; cooling to room temperature, regulating the pH value to 7-10 by using 0.5 mol/L NaOH, adding 2.4L of alkaline protease Alcalase, adding 4000-15000U/g hazelnut meal, carrying out enzymolysis for 2-5 h at 40-65 ℃, continuously adding NaOH in the enzymolysis process to keep the pH value unchanged, heating at 85-95 ℃ rapidly for 10-20 min after the reaction is finished to inactivate enzymes, cooling to room temperature, regulating the pH value to be neutral by using 0.5 mol/L HCl, centrifuging for 10-20 min at 5000-8000 r/min, and freeze-drying supernatant to obtain hazelnut peptide and storing at 4 ℃;
(2) Hazelnut peptide modification
Adding distilled water into the hazelnut peptide obtained after freeze drying in the step (1), preparing a solution with the mass concentration of Cheng Zhenren peptide being 2% -30%, adjusting the pH value to be 6-10 by using 0.5 mol/L NaOH, then adding transglutaminase with the enzyme quantity being 1-15U/g hazelnut peptide, adding sugar containing amino with the mass being 0.2-2.0 times that of the hazelnut peptide, reacting for 2-5 hours in a water bath with the temperature being 30-50 ℃, heating for 10-20 minutes at the temperature being 70-80 ℃ after the reaction is finished, inactivating the enzyme, obtaining the hazelnut peptide with high antioxidant activity after freeze drying, and preserving at the temperature of 4 ℃;
Wherein, the hazelnut peptide is prepared from Sichuan hazelnut, needle, hazelnut, corylus avellana a big-fruit hazel, corylus corm, corylus corm, corylus americana or turkish hazel; the sugar containing amino is glucosamine, glucosamine hydrochloride, glucosamine sulfate or chitosan; the freeze drying temperature is-50 to-30 ℃, and the freeze drying time is 20-30 hours.
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