阅读说明：本技术 一种蛋白核小球藻抗氧化肽及其制备方法 (Chlorella pyrenoidosa antioxidant peptide and preparation method thereof ) 是由 王际辉 肖珊 王璐 王波 蔡燕雪 于 2021-02-05 设计创作，主要内容包括：本发明公开了一种蛋白核小球藻抗氧化肽及其制备方法,以蛋白核小球藻为原料,通过蛋白核小球藻粉破壁处理,提取小球藻蛋白,制备小球藻多肽,分离纯化小球藻多肽,冷冻干燥,得到蛋白核小球藻抗氧化肽。所述抗氧化肽的氨基酸序列为∶VPADDL。本发明采用酶解法超声辅助提取小球藻蛋白,含盐量低,蛋白提取率高。使用多种酶制备小球藻抗氧化肽,以羟基自由基的清除率作为判别指标,能更精准的用于小球藻抗氧化肽的制备。采用截留分子量3000Da的超滤膜制备藻多肽,为下一步色谱分离获得更纯的藻抗氧化肽提供了方便条件。根据藻多肽分子量大小的不同采用凝胶层析分离方式,所得到的藻多肽纯度更高。(The invention discloses chlorella pyrenoidosa antioxidant peptide and a preparation method thereof. The amino acid sequence of the antioxidant peptide is VPADDL. The method adopts an enzymolysis method to extract chlorella protein with ultrasonic assistance, and has low salt content and high protein extraction rate. The chlorella antioxidant peptide is prepared by using various enzymes, and the clearance rate of hydroxyl free radicals is used as a discrimination index, so that the chlorella antioxidant peptide can be more accurately prepared. The ultrafiltration membrane with the molecular weight cutoff of 3000Da is adopted to prepare the algae polypeptide, thereby providing convenient conditions for obtaining purer algae antioxidant peptide by the next chromatographic separation. The purity of the obtained algae polypeptide is higher by adopting a gel chromatography separation mode according to the difference of molecular weight of the algae polypeptide.)

1. The chlorella pyrenoidosa antioxidant peptide is characterized in that: the amino acid sequence of the peptide is VPADDL.

2. A preparation method of chlorella pyrenoidosa antioxidant peptide is characterized in that: comprises the following steps of (1) breaking the wall of the globuline chlorella powder; (2) extracting chlorella protein; (3) preparing chlorella polypeptide; (4) and (5) separating and purifying chlorella polypeptide.

3. The method for preparing chlorella pyrenoidosa antioxidant peptide as claimed in claim 2, wherein: the specific wall breaking method of the globuline nucleus chlorella powder in the step (1) comprises the following steps of sieving the globuline nucleus chlorella powder with a 60-mesh sieve to remove impurities, wherein the liquid-material ratio of the chlorella powder to the purified water is 10: 1; performing ultrasonic treatment for 30min, processing with cell disruptor for 15min (3/2s), freeze drying to obtain cell wall-broken Chlorella powder, and packaging.

4. The method for preparing chlorella pyrenoidosa antioxidant peptide as claimed in claim 2, wherein: weighing wall-broken chlorella powder, adding pure water according to the mass-volume ratio of 1: 30, fully stirring and swelling, adjusting the pH to 5.0, carrying out enzymolysis at 50 ℃, adding 1% cellulase for enzymolysis for 3h, heating to 100 ℃ after enzymolysis is finished, inactivating enzyme for 10min, carrying out ultrasonic-assisted extraction at the ultrasonic power of 480w for 40 min, homogenizing for 10min, centrifuging for 10min at 8000r/min, taking precipitate, and freeze-drying to obtain chlorella protein.

5. The method for preparing chlorella pyrenoidosa antioxidant peptide as claimed in claim 2, wherein: weighing chlorella protein, adding 20 times of pure water for dissolving, adding 3% of alkaline protease, adjusting the pH to 8.0, the enzymolysis temperature to 60 ℃, carrying out enzymolysis for 6 hours, heating to 100 ℃ after the enzymolysis is finished, inactivating the enzyme for 10 minutes, adjusting the pH to 2.0, carrying out enzymolysis at 37 ℃, adding 2% of pepsin for enzymolysis for 2 hours, adjusting the pH to 7.5, carrying out enzymolysis at 37 ℃, adding 2% of pancreatin for enzymolysis for 2 hours, heating to 100 ℃ after the enzymolysis is finished, inactivating the enzyme for 10 minutes, obtaining chlorella polypeptide hydrolysate, and refrigerating for later use.

6. The method for preparing chlorella pyrenoidosa antioxidant peptide as claimed in claim 2, wherein: the specific method for separating and purifying the chlorella polypeptide in the step (4) is that an ultrafiltration membrane with the cut-off molecular weight of 3000Da is used for preparing chlorella polypeptide solution with the molecular weight of less than 3000 Da; vacuum concentrating the polypeptide solution at 60 deg.C, and freeze drying to obtain Chlorella polypeptide with molecular weight less than 3000 Da; separating chlorella polypeptide with molecular weight less than 3000Da by SephadexG-15 gel chromatography to obtain the chlorella antioxidant peptide.

Technical Field

The invention relates to a chlorella pyrenoidosa antioxidant peptide and a preparation method thereof.

Background

The traditional synthetic antioxidant may cause some unexpected side effects, the concern of consumers about the safety of the artificial antioxidant in food is large, green health food is increasingly concerned by consumers, and chlorella pyrenoidosa (chlorella pyrenoidosa) is single-cell green microalgae and widely distributed in fresh water and seawater. Chlorella is of great interest because it is rich in proteins, dietary fibers, lipids, vitamins, chlorophyll and carotenoids, as well as active metabolites. In addition, chlorella has excellent immunoregulatory effect, antioxidant ability and hypoglycemic and hypolipidemic effects, and is evaluated as a "green health food" by Food and Agricultural Organizations (FAO) of the united nations. The chlorella pyrenoidosa antioxidant peptide is a natural antioxidant, at present, the utilization rate of the chlorella pyrenoidosa protein in China is low, the preparation method is complex, the extracted chlorella pyrenoidosa protein is high in salt content, and the protein extraction rate and the purity are low.

Disclosure of Invention

In order to solve the defects of the prior art, the invention provides the chlorella pyrenoidosa antioxidant peptide and the preparation method thereof, and the antioxidant peptide has good antioxidant capacity. Has important significance for the development of science and technology, economy and food industry.

The technical scheme adopted by the invention is that the protein chlorella pyrenoidosa antioxidant peptide has an amino acid sequence of VPADDL.

A preparation method of chlorella pyrenoidosa antioxidant peptide comprises the following steps of (1) breaking the wall of chlorella pyrenoidosa powder; (2) extracting chlorella protein; (3) preparing chlorella polypeptide; (4) and (5) separating and purifying chlorella polypeptide.

Further, the specific wall breaking method of the globuline chlorella powder in the step (1) is that the globuline chlorella powder is sieved by a 60-mesh sieve to remove impurities, and the liquid-material ratio of the chlorella powder to the purified water is 10: 1; performing ultrasonic treatment for 30min, processing with cell disruptor for 15min (3/2s), freeze drying to obtain cell wall-broken Chlorella powder, and packaging.

Further, the specific method for extracting chlorella protein in the step (2) is that wall-broken chlorella powder is weighed, pure water is added according to the mass-to-volume ratio of 1: 30, the mixture is fully stirred and swelled, the pH value is adjusted to 5.0, the enzymolysis temperature is 50 ℃, 1% cellulase is added for enzymolysis for 3 hours, after the enzymolysis is finished, the temperature is raised to 100 ℃ for enzyme deactivation for 10 minutes, then ultrasonic assisted extraction is carried out for 40 minutes under the ultrasonic power of 480w, homogenization is carried out for 10 minutes, centrifugation is carried out for 10 minutes at 8000r/min, and the precipitate is taken for freeze drying, so that the chlorella protein is obtained.

Further, the preparation method of the chlorella polypeptide in the step (3) comprises the specific steps of weighing chlorella protein, adding 20 times of pure water for dissolving, adding 3% of alkaline protease, adjusting the pH to 8.0, adjusting the enzymolysis temperature to 60 ℃, carrying out enzymolysis for 6 hours, heating to 100 ℃ after the enzymolysis is finished, inactivating enzyme for 10 minutes, adjusting the pH to 2.0, adjusting the enzymolysis temperature to 37 ℃, adding 2% of pepsin for enzymolysis for 2 hours, adjusting the pH to 7.5, carrying out enzymolysis at 37 ℃, adding 2% of pancreatin for enzymolysis for 2 hours, heating to 100 ℃ after the enzymolysis is finished, inactivating enzyme for 10 minutes, obtaining chlorella polypeptide enzymolysis liquid, and refrigerating for later use.

Further, the specific method for separating and purifying the chlorella polypeptide in the step (4) is that an ultrafiltration membrane with the cut-off molecular weight of 3000Da is used for preparing a chlorella polypeptide solution with the molecular weight of less than 3000 Da; vacuum concentrating the polypeptide solution at 60 deg.C, and freeze drying to obtain Chlorella polypeptide with molecular weight less than 3000 Da; separating chlorella polypeptide with molecular weight less than 3000Da by SephadexG-15 gel chromatography to obtain the chlorella antioxidant peptide.

The invention has the beneficial effects that: the method adopts an enzymolysis method to extract chlorella protein with ultrasonic assistance, and has low salt content and high protein extraction rate. The chlorella antioxidant peptide is prepared by using various enzymes, and the clearance rate of hydroxyl free radicals is used as a discrimination index, so that the chlorella antioxidant peptide can be more accurately prepared. The ultrafiltration membrane with the molecular weight cutoff of 3000Da is adopted to prepare the algae polypeptide, thereby providing convenient conditions for obtaining purer algae antioxidant peptide by the next chromatographic separation. The purity of the obtained algae polypeptide is higher by adopting a gel chromatography separation mode according to the difference of molecular weight of the algae polypeptide.

Drawings

FIG. 1 is a diagram of Sephadex G-15 gel chromatography separation and purification.

FIG. 2 is a mass spectrum of Chlorella antioxidant peptide.

FIG. 3 is a graph comparing the hydroxyl radical scavenging capacity of purified antioxidant polypeptides with that of glutathione.

FIG. 4 is a graph comparing iron ion chelation capacity of purified antioxidant polypeptides with glutathione.

FIG. 5 is a graph comparing superoxide anion radical scavenging capacity of purified antioxidant polypeptides and glutathione.

Detailed Description

The technical solution of the present invention will be described in detail with reference to specific embodiments. The following examples are merely intended to more clearly illustrate the technical solutions of the present invention and are therefore only given as examples and are not intended to limit the present invention in any way.

First, example 1

Step 1, sieving 10g of chlorella powder by a 60-mesh sieve to remove impurities, and adding 100ml of purified water; performing ultrasonic treatment for 30min, processing with cell disruptor for 15min (3/2s), freeze drying to obtain cell wall-broken Chlorella powder, and packaging.

Step 2, weighing 10g of wall-broken chlorella powder, adding 300ml of pure water, fully stirring and swelling, adjusting the pH to 5.0, carrying out enzymolysis at 50 ℃, adding 0.1g of cellulase for enzymolysis for 3h, heating to 100 ℃ after the enzymolysis is finished, carrying out enzyme deactivation for 10min, carrying out ultrasonic-assisted extraction at an ultrasonic power of 480w for 40 min, homogenizing for 10min, centrifuging for 10min at 8000r/min, removing supernatant, precipitating, and freeze-drying to obtain chlorella protein.

Step 3, weighing 5g of chlorella protein, adding 100ml of pure water for dissolving, adding 0.15g of alkaline protease, adjusting the pH to 8.0, carrying out enzymolysis at 60 ℃ for 6h, heating to 100 ℃ after enzymolysis is finished, inactivating enzyme for 10min, adjusting the pH to 2.0, carrying out enzymolysis at 37 ℃, adding 0.1g of pepsin for enzymolysis for 2h, adjusting the pH to 7.5, carrying out enzymolysis at 37 ℃, adding 0.1g of pancreatin for enzymolysis for 2h, heating to 100 ℃ after enzymolysis is finished, inactivating enzyme for 10min, obtaining chlorella polypeptide enzyme hydrolysate, and refrigerating for later use.

Step 4, carrying out ultrafiltration separation on the enzymolysis product by using an ultrafiltration membrane with the molecular weight cut-off range of 3000Da on the obtained polypeptide enzymolysis liquid, and dividing the polypeptide enzymolysis liquid into 2 components with different molecular weight ranges, wherein the components are respectively a component with the molecular weight more than 3000Da and a component with the molecular weight less than 3000 Da; collecting the components with the best antioxidant activity, separating by Sephadex G-15 gel chromatography column (length 60cm, diameter 1.6cm), eluting with pure water at flow rate of 1 ml/min; and collecting the peak with the optimal antioxidant activity to obtain the antioxidant polypeptide.

Secondly, determining the amino acid sequence of the antioxidant polypeptide,

chromatography apparatus: easy-nano 1200; mass spectrometer: q active Plus (Thermo, USA). The column used was Pico Tip EMITTER (C18, 1.9 μm particle size, 120A pore size), the loading was 3ul, and the eluent was A: 0.1% aqueous formic acid solution, B: 80% acetonitrile, 0.1% formic acid, flow rate: 200 nL/min. The data acquisition software is Xcalibur (Thermo, USA); . Mass spectrum conditions: MS scan range (m/z) 355-: high energy collision (HCD), fragmentation energy (NCE): 27; exclusion of ions at valency 1 and above 7, dynamic exclusion time 60s, data dependent acquisition mode (DDA), and one MS1 spectrum selected the 10 strongest parent ions for cascade scanning.

The antioxidant polypeptide of the invention is determined by liquid chromatography-mass spectrometry to be composed of 6 amino acids, and the total sequence of the amino acids of the polypeptide is VPADDL.

Testing of antioxidant activity of algal peptides

1) Hydroxyl radical scavenging ability, as shown in figure 3,

taking 2mL of polypeptide sample with the concentration of 1mg/mL, and respectively adding 0.5mL of 6mm/L ferrous sulfate solution, 2mL of 1.5mmol/L salicylic acid-ethanol solution and 0.5mL of 0.1% H₂O₂Shaking the solution, placing in 37 deg.C water bath for reaction for 15min, and measuring absorbance A at 510nm_i(ii) a Equal amount of distilled water is used to replace H₂O₂Measuring the absorbance A_j(ii) a The sample was replaced with an equal amount of distilled water, and the absorbance was measuredDegree A₀。

Hydroxyl radical clearance rate ═ 1- (A)_i-A_j)/A₀]×100％

2) The iron ion chelating ability was measured, as shown in FIG. 4,

1mL of a 1mg/mL sample of the polypeptide was taken and 2.7mL of deionized water and 0.1mL of Fe Cl were added₂The solution (2mmol/L) was mixed well, 0.2mL of Ferrozine (5mmol/L) was added, and the mixture was allowed to stand for 10 min. Measuring the absorbance A1 at a wavelength of 562 nm; a. the₀The absorbance of the sample was replaced by 1mL of double distilled water as a blank control.

Iron ion chelation rate ═ 1-A/A₀)×100％

3) Superoxide anion radical scavenging ability, as shown in figure 5,

the superoxide anion free radical scavenging ability was measured by pyrogallol autooxidation. Taking 2.5mL of 50mmol/L Tris-HCl buffer solution (pH 8.2), preserving the heat in a water bath at 25 ℃ for 20min, respectively adding 1mL of polypeptide sample solution with the concentration of 1g/mL and 0.6mL of 25mmol/L pyrogallol solution, uniformly mixing, reacting in the water bath at 25 ℃ for 5min, adding 0.5mL of concentrated hydrochloric acid to terminate the reaction, and measuring the absorbance A at 299nm₁Measuring the light absorption value A by using deionized water instead of pyrogallol₂(ii) a Measuring light absorption value A by using deionized water to replace sample solution₀。

Superoxide anion radical scavenging ratio [% ], [ (1- (A1-A2)/A0) × 100%

The purified antioxidant polypeptide has strong antioxidant capacity, and as can be seen from figures 3, 4 and 5, compared with glutathione standard, the algae antioxidant peptide has strong hydroxyl free radical scavenging capacity, iron ion chelating capacity and superoxide anion free radical scavenging capacity.

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> Dongguan school of technology and technology

<120> a protein chlorella pyrenoidosa antioxidant peptide and a preparation method thereof

<141> 2021-02-05

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 6

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Val Pro Ala Asp Asp Leu

1 5