WO2017215313A1

WO2017215313A1 - Method for the preparation of antioxidant peptide using gingko nut shells

Info

Publication number: WO2017215313A1
Application number: PCT/CN2017/078555
Authority: WO
Inventors: 王航; 王大平
Original assignee: 如皋福大工程技术研究院有限公司
Priority date: 2016-06-16
Filing date: 2017-03-29
Publication date: 2017-12-21
Also published as: CN106086132A

Abstract

Provided is a method for preparing an antioxidant peptide using gingko nut shells, comprising the following steps: (1) placing dried, crushed gingko nut shells in an aqueous solution of NaOH so as to extract a crude protein of the gingko nut shells; (2) preparing the crude protein of the gingko nut shells at a mass density of 1 to 10% in an aqueous solution of pH 9-10, then adding proteinase, adjusting the temperature of the aqueous solution to 50-60°C and conducting a hydrolysis reaction for 2 to 5 hours, then maintaining the aqueous solution at a rolling boil at 95-100°C for 10-15 minutes, inactivating the enzyme and obtaining a hydrolyzed solution; (3) precipitating the described hydrolyzed solution by centrifuging, extracting the supernatant and vacuum-lyophilizing same to prepare an antioxidant peptide; When the density of the antioxidant peptide in the solution reaches 0.4g/L. or more, the removal rate thereof for DPPH free radicals stabilizes at above 80%.

Description

Method for preparing antioxidant peptide by using ginkgo peel

Technical field

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.

Background technique

At present, the annual output of ginkgo in China exceeds 12,000 tons, and the output of ginkgo leaves reaches 15,000 tons. The yield of ginkgo peel is relatively large, reaching 1.5-2.0 million tons. 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.

At present, the research on ginkgo at home and abroad mainly focuses on ginkgo leaves, while the outer skin of ginkgo biloba is mainly thrown away in the form of waste, which is not effectively utilized, which wastes a lot of resources and pollutes the environment. Therefore, the comprehensive utilization of ginkgo peels is extremely important. At present, the comprehensive utilization of ginkgo peel at home and abroad is still in a blank stage, and there is no practical application.

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. 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.

In recent years, 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.

Therefore, 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.

Summary of the invention

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:

(1), the dried and pulverized ginkgo peel is put into an aqueous solution of NaOH to extract crude protein of ginkgo peel;

(2) 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;

(3), the above hydrolyzate is centrifuged, and the supernatant is vacuum-dried to obtain an antioxidant peptide.

Further, in 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. In the above step (1), after the ginkgo peel is put into the NaOH aqueous solution, 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.

By extracting the crude protein of ginkgo peel by this method, 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. When the 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. Under the premise of ensuring that the protein is dissolved, the consumption of NaOH is reduced. Appropriate solid-liquid ratio can extract the protein in the ginkgo peel and enter the NaOH aqueous solution. In order to avoid the excessive concentration of protein in 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. Of course, after exceeding a certain ratio, too much NaOH aqueous solution can not extract more protein, and the mass ratio of ginkgo peel and NaOH solution can be controlled between 1:10-1:100. Ensure that the protein in the ginkgo peel can be Extract it as much as possible.

Further, the protease is an alkaline protease. After screening, alkaline protease is the best protease for protein breakdown.

Further, 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. When the 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.

detailed description

Example 1

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.

Example 2

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.

Example 3

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.

Example 4

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):

Where 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 .

The test results are shown in Table 1.

Table 1 Effect of antioxidant peptides on DPPH free radical scavenging

Claims

A method for preparing an antioxidant peptide by using a ginkgo peel, which comprises the following steps:

(1), the dried and pulverized ginkgo peel is put into an aqueous solution of NaOH to extract crude protein of ginkgo peel;

(2) 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. , the aqueous solution is kept boiling at 95-100 ° C for 10-15 min, and the enzyme is deactivated to obtain a hydrolyzate;

(3), the above hydrolyzate is centrifuged, and the supernatant is vacuum-dried to obtain an antioxidant peptide.
The method of claim 1 wherein

After the ginkgo peel is put into the NaOH aqueous solution in the step (1), the mixture is shaken at 55-65 ° C for 1-5 hours, then the precipitate is centrifuged, and the supernatant is taken; the supernatant is adjusted to pH 2-3 with hydrochloric acid. The precipitate was centrifuged again, and the precipitate was washed with water and dried to obtain crude protein of ginkgo peel;

The concentration of the aqueous NaOH solution is 0.05-0.1 mol/L; the mass ratio of the ginkgo peel to the aqueous NaOH solution is 1:10-1:100.
The method according to claim 2, wherein in the step (1), after the ginkgo peel is put into the aqueous NaOH solution, the temperature at the time of shaking is 60 °C.
The method according to claim 2, wherein in the step (1), the supernatant is adjusted with hydrochloric acid to have a pH of 2.6.
The method of claim 1 wherein the protease is an alkaline protease.
The method according to claim 1, wherein the protease is added in an amount of from 1 to 5% by weight of the crude protein of the ginkgo peel.
The method according to claim 1, wherein in the step (2), the aqueous solution prepared by the ginkgo rind crude protein is adjusted to pH with NaOH.
The method according to claim 1, wherein in the step (2), the aqueous solution of the ginkgo rind crude protein is formulated to have a pH of 9.