CN111533784A - Chicken bone antioxidant peptide and preparation method thereof - Google Patents
Chicken bone antioxidant peptide and preparation method thereof Download PDFInfo
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- CN111533784A CN111533784A CN202010452183.6A CN202010452183A CN111533784A CN 111533784 A CN111533784 A CN 111533784A CN 202010452183 A CN202010452183 A CN 202010452183A CN 111533784 A CN111533784 A CN 111533784A
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Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/10—Tetrapeptides
- C07K5/1002—Tetrapeptides with the first amino acid being neutral
- C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
- C07K5/1008—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
-
- 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
Abstract
The invention provides chicken bone antioxidant peptide and a preparation method thereof, which take chicken bones as raw materials, creatively adopt an ultrasonic-microwave combined technology to assist compound enzymolysis of flavourzyme and papain, establish a preparation method of the chicken bone antioxidant peptide and obtain a chicken bone antioxidant peptide product with higher activity. The antioxidant peptide mixture disclosed by the invention has the characteristics of small molecular mass, strong activity, high safety and the like, and 2 new chicken bone peptides are found to be contained through comparison of a UniProt KB database, and the amino acid sequences of the antioxidant peptide mixture are Ser-Cys-Pro-Pro-Gly-His and Ala-Thr-Phe-Gln respectively. The antioxidant peptide and the preparation method thereof not only realize high-valued utilization of chicken bones, but also can be used as an excellent substitute for the existing synthetic antioxidant, and provide theoretical basis and practical reference for research and development of novel natural additives in the industries of food, medicine and cosmetics.
Description
Technical Field
The invention belongs to the technical field of food biology, and particularly relates to chicken bone antioxidant peptide and a preparation method thereof.
Background
The antioxidant can not only delay the oxidation reaction of food, but also effectively inhibit the negative effects caused by oxidative stress in human bodies, thereby arousing wide attention of people. At present, the used antioxidant is mainly synthesized, and although the effect is relatively good, the synthetic antioxidant has certain health risk after being used (or eaten) for a long time. Therefore, the search for safe and nontoxic natural antioxidants is of great significance to industries such as food, cosmetics, medicines and the like.
The food-derived antioxidant peptide has the characteristics of small molecular weight, strong activity, high safety, capability of inhibiting and delaying oxidative stress and harmful effects thereof and the like, and is more popular with the public than synthetic antioxidants. At present, the natural antioxidant peptide is mainly prepared from plant protein and animal protein serving as raw material sources by protease enzymolysis, microbial fermentation and other methods. The enzyme method has the characteristics of high raw material safety, mild reaction conditions, strong specificity and the like, and is more widely applied.
China is a big country for broiler chicken breeding and chicken consumption, and a large amount of processing by-products, namely chicken bones, are generated every year. The chicken bone is the raw material with the highest protein content in the livestock and poultry bone, is up to 16.30 percent, is rich in 8 amino acids necessary for human bodies, has rich amino acid types and balanced proportion, and is an ideal bioactive peptide source. At present, most of chicken bones are processed into animal feed or directly discarded, the comprehensive utilization rate is extremely low, resources are wasted, and the environment is seriously polluted.
At present, the research on chicken bones is relatively weak and lagged, the preparation of Maillard reaction substrates, chicken essence products, natural seasoning base materials and the like by enzymolysis of the chicken bones is mainly focused, and the research and development of antioxidant peptides by taking the chicken bones as raw materials are rarely reported. Therefore, the abundant chicken bone protein resources in China need to be fully utilized, deep system research is carried out on the chicken bone protein resources, and theoretical basis and technical guidance are provided for realizing high-valued utilization of chicken bones and developing food-borne antioxidants.
Disclosure of Invention
The invention aims to provide chicken bone antioxidant peptide and a preparation method thereof, which take chicken bones as raw materials, creatively utilize ultrasonic-microwave combined technology to assist compound enzymolysis of flavourzyme and papain, establish a preparation process of the chicken bone antioxidant peptide, obtain a chicken bone antioxidant peptide product with higher activity, and can apply the antioxidant peptide to the fields of food, medicine, cosmetics and the like.
The antioxidant peptide mixture disclosed by the invention is found to contain 2 new chicken bone peptides through the comparison of a UniProt KB database, and the amino acid sequences of the antioxidant peptide mixture are Ser-Cys-Pro-Pro-Gly-His and Ala-Thr-Phe-Gln respectively.
A chicken bone antioxidant peptide and a preparation method thereof are characterized in that: preparing by using an ultrasonic-microwave combined technology assisted with a flavourzyme and papain complex enzyme method; the method specifically comprises the following steps:
(1) preparing a chicken bone protein sample solution: soaking the chicken bone powder in distilled water, wherein the concentration of substrate protein is 6-10%, placing the chicken bone powder in an ultrasonic and microwave combined reaction system, and the corresponding ultrasonic and microwave treatment conditions comprise ultrasonic power of 400-600W, ultrasonic time of 25-35 min, microwave power of 400-600W and microwave time of 6.0-10.0 min;
(2) preparing zymolyte: the chicken bone protein is subjected to enzymolysis through flavourzyme and papain, wherein the enzymolysis condition is that the enzyme adding amount is 7000-10000U/g, the enzyme activity ratio of the papain to the flavourzyme is 1:4, the pH value is 6.0-8.0, the enzymolysis temperature is 40-50 ℃, and the enzymolysis time is 3.5-4.5 h;
(3) separation and purification: performing ultrafiltration separation on the obtained zymolyte by adopting an ultrafiltration membrane with the molecular weight cutoff of 3kDa, setting the treatment temperature to be 20-25 ℃, and the operation pressure to be 0.10-0.15 MPa, collecting components with the molecular weight less than 3kDa, and further separating and purifying; and (2) performing separation by Sephadex G-25 gel chromatography, taking deionized water as an eluent, controlling proper sample concentration and elution speed, measuring the light absorption value of the elution component at the wavelength of 220nm, measuring the antioxidant activity of the elution component corresponding to each absorption peak, collecting the component with higher activity, and performing freeze drying to obtain the antioxidant peptide.
The invention has the following beneficial effects:
(1) the protein content in the chicken bone powder is 62.98 +/-0.23%, which is beneficial to preparing enzymolysis liquid;
(2) the invention firstly utilizes the ultrasonic-microwave combined technology to assist the flavor protease and papain complex enzyme method to prepare the antioxidant peptide, thereby not only promoting the high-efficiency extraction of the chicken bone protein, but also fully combining the action characteristics of the exonuclease and the endonuclease and obviously improving the enzymolysis effect. Compared with the common complex enzymolysis method, the products prepared by the ultramicro auxiliary complex enzyme method have the removal rates of superoxide anion free radicals, DPPH free radicals and hydroxyl free radicals respectively improved by 50.80%, 41.88% and 8.57%, the chelating rate of ferrous ions is improved by 16.24%, and the reducing power is improved by 41.37%. The comprehensive antioxidant capacity of an enzymolysis product can be obviously improved by adopting an ultramicro coupling technology to assist the compound enzymolysis, and a new way is provided for the preparation of antioxidant peptides;
(3) the reducing power of the antioxidant peptide prepared by the invention is 0.496, the DPPH free radical scavenging activity is up to 96.99%, the antioxidant peptide has a strong antioxidant function, a new utilization approach is provided for livestock and poultry byproducts, the resource waste and pollution are reduced, and the commercial value and the utilization rate of chicken bones are improved;
(4) the antioxidant peptide mixture provided by the invention is derived from food and can be applied to the fields of food, health care, cosmetics and the like.
Drawings
FIG. 1: antioxidant activity of ultrafiltration components of different molecular weights. Wherein CBH-I: MW > 10 kDa; CBH-II: MW 6-10 kDa; CBH-III: MW 3-6 kDa; CBH-IV: MW < 3 kDa;
FIG. 2: sephedex G-25 gel chromatography separation of component CBH-IV;
FIG. 3: nanoliter liquid chromatogram of component CBH-IV-2;
FIG. 4: the antioxidant activity of the elution fraction is obtained by gel chromatography.
Detailed Description
The present embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation manner and process are given, but the scope of the present invention is not limited to the following implementation examples.
Example 1
The preparation method of the antioxidant peptide comprises the following steps:
chicken bone pretreatment: cleaning chicken bone, removing meat, cutting into pieces, drying in a 50 deg.C constant temperature drying oven for 48h, pulverizing, defatting with petroleum ether, and sieving with 40 mesh sieve to obtain chicken bone powder material.
Extraction of chicken bone protein: soaking the chicken bone powder in distilled water, placing the chicken bone powder in an ultrasonic and microwave combined reaction system under the corresponding ultrasonic and microwave treatment conditions of 500W of ultrasonic power, 30min of ultrasonic time, 520W of microwave power and 7.9min of microwave time, wherein the concentration of substrate protein is 8% (W/v).
Enzymolysis of chicken bone protein: the chicken bone protein is subjected to compound enzymolysis by adopting papain and flavourzyme according to the enzyme activity ratio of 1:4, the enzyme adding amount is 9000U/g, the pH value is 7.0, the enzymolysis temperature is 45 ℃, and the enzymolysis time is 4 hours. Inactivating enzyme in boiling water bath for 10min after enzymolysis, rapidly cooling to room temperature, centrifuging at 4000rpm for 20min, and collecting supernatant.
And (3) ultrafiltration separation: and (3) primarily separating the supernatant through ultrafiltration membranes with the molecular weight cut-off of l0kDa, 6kD and 3kDa, setting the treatment temperature to be 25 ℃ and the operation pressure to be 0.12 MPa. The membrane underflow was collected to obtain four ultrafiltration fractions of different molecular weights, and their reducing power and DPPH radical scavenging activity were compared (FIG. 1). Collecting high-activity components CBH-IV (molecular weight is less than 3kDa) for further separation and purification.
Sephadex G-25 gel chromatography separation: dissolving the ultrafiltration component CBH-IV in deionized water, centrifuging at 10000rpm for 10min, and keeping the supernatant. Sephadex G-25 gel column (. PHI.1.5 cm. times.60 cm) was equilibrated with deionized water, and the supernatant was applied to the column. Eluting with deionized water, with sample concentration of 25mg/mL and elution flow rate of 0.7mL/min, detecting the absorbance of the eluate at 220nm wavelength, and drawing elution curve, as shown in FIG. 2. And collecting the elution peak CBH-IV-2, and freeze-drying to obtain the antioxidant peptide.
The collected antioxidant peptide is prepared into 0.4 mu g/mu L solution, and the amino acid sequence is determined by adopting a Nano-liter liquid chromatography-mass spectrometry combined method (Nano-LC-ESI-MS/MS), and the liquid chromatogram of the amino acid sequence is shown in figure 3. The results of the alignment of the UniProt KB database show that the sequence contains 2 new amino acid sequences, and are shown in Table 1.
TABLE 1 peptide fragments identified in component CBH-IV-2
Example 2
The activity of the natural antioxidant peptide obtained in example 1 was investigated:
reducing power: weighing the freeze-dried sample to prepare a sample solution of 5 mg/mL. 1.2mL of the sample solution was subjected to the reduction force measurement, 3mL of 0.2mol/L phosphate buffer solution and 3mL of 1% potassium ferricyanide solution were added, and the mixture was subjected to a water bath at 50 ℃ for 20 min. And taking out after the water bath is finished, adding 3mL of 10% trichloroacetic acid when the temperature is returned to the room temperature, and centrifuging for 10min at the rotating speed of 3000 r/min. 2mL of the supernatant was mixed with 2mL of distilled water, 0.5mL of a 0.1% ferric chloride solution was added, the mixture was allowed to stand for 10min, and the absorbance was measured at a wavelength of 700 nm. Meanwhile, 1.2mL of distilled water was used as a blank control instead of the enzymatic hydrolysate.
DPPH radical clearance: 3.94mg of DPPH is accurately weighed, dissolved by 95% ethanol, and prepared into 0.1mmol/L DPPH solution after the volume is up to 100 mL. Weighing the freeze-dried sample to prepare a sample solution of 5 mg/mL. Mixing 2.5mL sample solution and 2.5mL LDPPH solution, standing at room temperature in dark place for 30min, and measuring light absorption value at 517 nm; 2.5mL of 95% ethanol solution instead of DPPH solution was used as a control; 2.5mL of distilled water was used as a blank instead of the sample. The DPPH radical clearance of the samples was calculated using the following formula:
in the formula: a. the1-absorbance of the sample set; a. the2-control absorbance values;A3-blank control absorbance value
The determination of the embodiment shows that the chicken bone antioxidant peptide has strong activity of removing DPPH free radicals and strong reducing power which are respectively 96.99 percent and 0.496 (shown in figure 4), and the antioxidant activity is good.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
Sequence listing
<110> Jilin university
<120> chicken bone antioxidant peptide and preparation method thereof
<160>2
<170>SIPOSequenceListing 1.0
<210>1
<211>6
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<213> Chicken (Gallus Gallus)
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Ser Cys Pro Pro Gly His
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<210>2
<211>4
<212>PRT
<213> Chicken (Gallus Gallus)
<400>2
Ala Thr Phe Gln
1
Claims (2)
1. A preparation method of chicken bone antioxidant peptide is characterized in that ultrasonic-microwave combined technology is utilized to assist the preparation of flavourzyme and papain complex enzyme method;
the method specifically comprises the following steps:
(1) preparing a chicken bone protein sample solution: soaking the chicken bone powder in distilled water, wherein the concentration of substrate protein is 6-10%, placing the chicken bone powder in an ultrasonic and microwave combined reaction system, and the corresponding ultrasonic and microwave treatment conditions comprise ultrasonic power of 400-600W, ultrasonic time of 25-35 min, microwave power of 400-600W and microwave time of 6.0-10.0 min;
(2) preparing zymolyte: the chicken bone protein is subjected to enzymolysis through flavourzyme and papain, wherein the enzymolysis condition is that the enzyme adding amount is 7000-10000U/g, the enzyme activity ratio of the papain to the flavourzyme is 1:4, the pH value is 6.0-8.0, the enzymolysis temperature is 40-50 ℃, and the enzymolysis time is 3.5-4.5 h;
(3) separation and purification: performing ultrafiltration separation on the obtained zymolyte by adopting an ultrafiltration membrane with the molecular weight cutoff of 3kDa, setting the treatment temperature to be 20-25 ℃, and the operation pressure to be 0.10-0.15 MPa, collecting components with the molecular weight less than 3kDa, and further separating and purifying; and (2) performing separation by Sephadex G-25 gel chromatography, taking deionized water as an eluent, controlling proper sample concentration and elution speed, measuring the light absorption value of the elution component at the wavelength of 220nm, measuring the antioxidant activity of the elution component corresponding to each absorption peak, collecting the component with higher activity, and performing freeze drying to obtain the antioxidant peptide.
2. The chicken bone antioxidant peptide mixture prepared by the method of claim 1, wherein the comparison of UniProt KB database shows that the mixture contains 2 new peptides, and the amino acid sequences of the peptides are respectively: Ser-Cys-Pro-Pro-Gly-His, Ala-Thr-Phe-Gln.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112568320A (en) * | 2020-12-21 | 2021-03-30 | 四川品品食品有限公司 | Application method for chicken breast enzymolysis to produce small molecular peptide |
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| CN102649974A (en) * | 2012-05-14 | 2012-08-29 | 上海海洋大学 | Method for preparing antioxidant peptide through ultrasonic-microwave synergetic enzymatic hydrolysis |
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