阅读说明：本技术 一种海鞘抗氧化多肽及其制备方法 (Sea squirt antioxidant polypeptide and preparation method thereof ) 是由 卜迁 麻蕊 侯青 高鸿 于 2021-05-11 设计创作，主要内容包括：本发明公开了一种海鞘抗氧化多肽及其制备方法,所述抗氧化多肽的氨基酸序列为Ile-Ser-Trp。以海鞘内囊肉块为原料,采用复合蛋白酶对其进行酶解,分离纯化、冷冻干燥得到抗氧化多肽。本发明抗氧化多肽缓解了人工抗氧化剂可能引起的副作用,提供了一种新的天然抗氧化剂,可以取代传统的合成食品抗氧化剂。(The invention discloses ascidian antioxidant polypeptide and a preparation method thereof, wherein the amino acid sequence of the antioxidant polypeptide is Ile-Ser-Trp. The sea squirt inner bag meat blocks are used as raw materials, and are subjected to enzymolysis by adopting compound protease, separation and purification, and freeze drying to obtain the antioxidant polypeptide. The antioxidant polypeptide relieves the possible side effect caused by artificial antioxidant, provides a new natural antioxidant, and can replace the traditional synthetic food antioxidant.)

1. An ascidian antioxidant polypeptide, which has the amino acid sequence Ile-Ser-Trp.

2. The method for preparing ascidian antioxidant polypeptide as claimed in claim 1, wherein the ascidian antioxidant polypeptide is prepared from ascidian endocyst meat pieces by enzymolysis with compound protease, separation, purification, and freeze-drying.

3. The method according to claim 2, wherein the complex protease is a neutral protease or a flavourzyme.

4. The method of claim 3, comprising the steps of:

1) taking the inner bag of the sea squirt, removing viscera and fascia, boiling to inactivate enzyme, and making into meat block of the inner bag of the sea squirt;

2) crushing meat, defatting in petroleum ether, adding ethanol to remove pigment, and making into crude protein of sea squirt;

3) performing enzymolysis on ascidian endocystin by using compound protease, inactivating enzyme in boiling water bath after enzymolysis, cooling to room temperature, centrifuging, and taking supernatant for later use;

4) performing ultrafiltration with a spiral-wound polysulfone ultrafiltration membrane to obtain polypeptide components with different molecular weights;

5) freeze-drying the ultrafiltrate after evaporation and concentration in vacuum to obtain a polypeptide component;

6) determining the activity of DPPH and ABTS free radicals, and selecting the component with the highest activity;

7) separating and purifying by column chromatography, freeze-drying, measuring antioxidant activity of eluate corresponding to each absorption peak, and collecting the component with highest antioxidant activity;

8) determining antioxidant activity peak by using C18 chromatographic column and DPPH elimination method, and collecting antioxidant polypeptide.

5. The preparation method according to claim 4, wherein the enzymolysis conditions in step 3) are as follows: heating in water bath to 50 deg.C, and the ratio of enzyme to substrate is 1: 4.

6. The preparation method according to claim 4, wherein the stationary phase in step 7) is Sephadex G-25, the mobile phase is ultrapure water, and wherein the gel column has a volume of 500ml, a sample volume of 24ml, a sample concentration of 20mg/ml, a flow rate of 1.5ml/min, and a detection wavelength of 280 nm.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to ascidian antioxidant polypeptide and a preparation method thereof.

Background

The human body can generate a large amount of free radicals every day, environmental factors can promote the human body to generate the free radicals, the free radicals have extremely strong oxidation capacity, a proper amount of the free radicals are defensive agents of the human body and can kill bacteria and viruses and decompose poisons, but excessive free radicals can oxidize normal substances of the human body, destroy cell structures, promote cell degeneration and cause damage to cells and tissues of the human body, and the negative effects of the free radicals are related to the aging of the human body and a plurality of health diseases, such as diabetes, cancer, neurodegenerative diseases and the like.

The existence of some natural antioxidants in human body can eliminate the harm of free radicals to human body, such as vitamin C and vitamin E, and some synthetic antioxidants can also play a role in eliminating free radicals. However, research shows that the artificially synthesized antioxidant has certain toxic and side effects on human bodies. For example, 2-tert-butylhydroquinone (TBHQ) causes DNA damage and may cause mutation in animals, 2, 6-di-tert-butyl-p-methylphenol (BHT) causes carcinogenesis, and Propyl Gallate (PG) causes damage to the kidney as well. To alleviate the concerns of the safety of synthetic antioxidants, the development of natural antioxidants is receiving increasing attention from the food industry and the scientific community. The protein is hydrolyzed by enzyme to obtain bioactive polypeptide, and the amino acid residue in the polypeptide endows the protein with certain bioactivity, such as antioxidant activity, antitumor activity, anti-inflammatory activity, immunoregulation, etc. The antioxidant peptide is a bioactive peptide with the capacity of eliminating matrix free radicals, inhibiting and delaying lipid peroxidation, chelating metal ions and the like.

The ocean is the most huge ecosystem of the earth, contains extremely abundant biological resources, and marine organisms generate a large amount of functional substances different from terrestrial organisms in order to adapt to the complex marine environment. Many polypeptides with good antioxidant activity have been isolated from marine organisms. The sea squirt is commonly called as the sea pineapple, about 1500 kinds of the sea squirt are usually attached to the bottom of a ship and a reef on the sea bottom. Halocynthia Roretzi is an economical marine product in which cultivation is widely performed. The content of protein in Halocynthia Roretzi is 34%, which is the most abundant material except water, but research on antioxidant active peptide in Halocynthia Roretzi is less. How to obtain the high-efficiency antioxidant polypeptide with a specific amino acid sequence from edible sea squirts becomes an urgent research direction.

Disclosure of Invention

The invention aims to provide a method for preparing antioxidant polypeptide from the inner capsule of Halocynthia Roretzi, which replaces synthetic antioxidant with natural antioxidant, and lays the foundation for developing antioxidant polypeptide based on food source and exploring its wide application in food and medicine.

The invention is realized by the following technical scheme:

the invention provides an ascidian antioxidant polypeptide, the amino acid sequence of which is Ile-Ser-Trp.

On the other hand, the invention provides a preparation method of the ascidian antioxidant polypeptide, which takes ascidian inner capsule meat blocks as raw materials, adopts compound protease of neutral protease and flavourzyme to carry out enzymolysis, separation and purification, and freeze drying to obtain the antioxidant polypeptide.

The invention adopts the compound protease as an incision enzyme, acts on peptide bonds in protein molecules, does not cut tail ends to generate free amino acids, and ensures that polypeptide chains are generated. Meanwhile, the most obvious side effect in the preparation of the active peptide by the enzyme method is that in the enzyme cutting process, a missed cutting site and a non-specific enzyme cutting site appear, and finally, the protein cannot be absolutely cut into a conventional peptide fragment, so that a plurality of unnecessary products are generated. The composite protease contains various single enzymes and various enzyme cutting sites, so that the missed cutting sites are prevented. The flavourzyme plays an optimizing role in the bitter taste and the flavour of the hydrolysis, and is widely applied to the hydrolysis of animal protein.

The preparation method of the ascidian antioxidant polypeptide specifically comprises the following steps:

1) taking the inner bag of the sea squirt, removing viscera and fascia, boiling to inactivate enzyme, and making into meat block of the inner bag of the sea squirt;

2) crushing meat, defatting in petroleum ether, adding ethanol to remove pigment, and making into crude protein of sea squirt;

3) performing enzymolysis on ascidian endocystin by using compound protease, inactivating enzyme in boiling water bath after enzymolysis, cooling to room temperature, centrifuging, and taking supernatant for later use;

4) performing ultrafiltration with a spiral-wound polysulfone ultrafiltration membrane to obtain polypeptide components with different molecular weights;

5) freeze-drying the ultrafiltrate after evaporation and concentration in vacuum to obtain a polypeptide component;

6) determining the activity of DPPH and ABTS free radicals, and selecting the component with the highest activity;

7) separating and purifying by column chromatography, freeze-drying, measuring antioxidant activity of eluate corresponding to each absorption peak, and collecting the component with highest antioxidant activity;

8) determining antioxidant activity peak by using C18 chromatographic column and DPPH elimination method, and collecting antioxidant polypeptide.

Further, in the step 1), ultrapure water is adopted for boiling for 15min at 100 ℃ to inactivate enzyme.

Further, the enzymolysis conditions in the step 3) are as follows: heating in water bath to 50 deg.C, and the ratio of enzyme to substrate is 1: 4.

Further, the cut-off amounts of the scroll type polysulfone ultrafiltration membrane in the step 4) comprise 30kDa, 5kDa, 1kDa and 500 Da; the obtained polypeptide components comprise polypeptides with molecular weight of 30kDa-5kDa, 5kDa-1kDa, 1kDa-500Da and <500 Da.

Further, the evaporation concentration conditions in the step 5) are as follows: 55 deg.C and 0.1 MPa.

Further, in the step 7), the stationary phase is sephadex G-25, the mobile phase is ultrapure water, wherein the volume of the gel column is 500ml, the sample injection amount is 24ml, the sample injection concentration is 20mg/ml, the flow rate is 1.5ml/min, and the detection wavelength is 280 nm.

The invention has the beneficial effects that:

the invention prepares a high-efficiency antioxidant polypeptide with an amino acid sequence of Ile-Ser-Trp from edible sea squirt inner capsules, provides a new natural antioxidant, can replace a synthetic antioxidant, eliminates the worry of people about the safety problem of the artificially synthesized antioxidant, and simultaneously meets the market demand of the antioxidant. Meanwhile, a preparation and separation and purification method aiming at the echinocystis euonymus antioxidant peptide is developed, the additional value of echinocystis euonymus processing is improved, and the research on marine natural products is widened.

Drawings

FIG. 1 is a graph showing the ABTS free radical scavenging activity of the individual components after ultrafiltration in example 1 of the present invention;

FIG. 2 is a graph showing DPPH radical scavenging activity of E fraction (<500Da) after ultrafiltration in example 1 of the present invention;

FIG. 3 is a Sephadex G-25 separation and purification map of example 1 of the present invention;

FIG. 4 shows the radical scavenging activity of each fraction after Sephadex G-25 separation and purification in example 1 of the present invention;

FIG. 5 shows the determination of peaks of antioxidant activity by DPPH elimination in example 1 of the present invention;

FIG. 6 shows the radical scavenging ability of the synthetic polypeptide Ile-Ser-Trp.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to specific embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

The preparation of ascidian antioxidant polypeptide comprises the following steps:

1) treating Halocynthia Roretzi: thawing Halocynthia Roretzi, removing shell, collecting inner capsule, removing viscera and fascia, adding ultrapure water, decocting at 100 deg.C for 15min, and inactivating enzyme to obtain Halocynthia Roretzi inner capsule meat block.

2) Removing impurities: crushing the meat, degreasing in petroleum ether, adding ethanol to remove pigment, and making into crude sea squirt protein.

3) Enzymolysis of sea squirt inner capsule protein: performing enzymolysis on ascidian endocystin by using compound protease, wherein the pH is 7.0, heating in water bath at 50 deg.C, the ratio of enzyme to substrate is 1:4, performing enzymolysis for 7h, inactivating enzyme in boiling water bath for 15min, rapidly cooling to room temperature, 12000rpm/min, centrifuging for 15min, and collecting supernatant;

the compound protease is purchased from Jiangsu Ruiyang biotechnology (China. Wuxi).

4) And (3) ultrafiltration: ultrafiltering with roll-type polysulfone ultrafiltration membranes with cut-off amounts of 30kDa, 5kDa, 1kDa and 500Da respectively to obtain 4-component ultrafiltrates (A, B, C, D, E) with molecular weights of 30kDa-5kDa, 5kDa-1kDa, 1kDa-500Da and <500Da respectively, and having ABTS free radical scavenging activity shown in FIG. 1. The DPPH free radical scavenging activity of polypeptide fractions with molecular weight <500Da is shown in FIG. 2.

Wherein the molecular interception of the roll type polysulfone ultrafiltration membrane can be replaced by 20k-800 Da.

5) And (3) evaporation and concentration: setting the temperature of a rotary evaporator at 55 ℃ and the pressure at 0.1Mpa, and respectively evaporating and concentrating the ultrafiltrates of 4 components.

6) And (3) freeze drying: freeze drying the rotary evaporated ultrafiltrate at-80 deg.C under vacuum to obtain 4-component polypeptide powder.

7) And (3) measuring antioxidant activity: and (4) determining the activity of DPPH and ABTS free radicals, and selecting the component with the highest activity.

8) Gel column chromatography: and separating and purifying the component E with the optimal antioxidant activity by column chromatography, wherein a stationary phase is sephadex G-25, a mobile phase is ultrapure water, measuring the antioxidant activity of eluent corresponding to each absorption peak after freeze drying, and collecting the component with the highest antioxidant activity.

Wherein the gel column volume is 500ml, the sample volume is 24ml, the sample concentration is 20mg/ml, the flow rate is 1.5ml/min, the detection wavelength is 280nm, and F is obtained₁、F₂Two absorption peaks (fig. 3). DPPH radical scavenging ability and ABTS radical scavenging ability are shown in FIG. 4.

In addition, other chromatographic methods, such as ion chromatography and hydrophobic chromatography, can be selected and used in the invention.

9) Reversed phase high performance liquid chromatography RP-HPLC: selecting the optimal antioxidant active component F₂The peak of antioxidant activity was determined by DPPH digestion using a C18 column (fig. 5).

10) Mass spectrum identification: the molecular weight is 405Da through LC-MS/MS identification, and the amino acid sequence is Ile-Ser-Trp.

Example 2 antioxidant Activity assay

1) DPPH radical scavenging ability

Preparing 0.1mM DPPH free radical solution by 95% ethanol, mixing samples with different concentrations and DPPH according to the ratio of 1:1, and measuring the light absorption value at 517nm after the room temperature dark reaction is carried out for 30 min. The blank group was replaced with an equal volume of 95% ethanol and the DPPH radical scavenging capacity of the sample was calculated as follows:

in the formula, A_controlAbsorbance of blank set, A_sampleThe absorbance values of the experimental groups are shown.

2) ABTS free radical scavenging ability

Preparing ABTS solution with concentration of 7mM and potassium persulfate solution with concentration of 2.45mM by using ultrapure water, mixing according to the proportion of 1:1, and incubating at room temperature in a dark place for 12-16h to obtain ABTS free radical mother liquor. Diluting with phosphate buffer solution (5mM, pH7.4) to give ABTS free radical working solution with absorbance of 0.70 + -0.02 at 734nm, and making into preparation. The experimental group is 0.1ml sample and 3.9ml ABTS working solution, and the light absorption value is measured at 734nm after reacting for 6min at room temperature. The blank group was replaced with the same volume of ultrapure water, and the ABTS radical scavenging ability of the sample was calculated as follows:

in the formula, A_controlAbsorbance of blank set, A_sampleThe absorbance values of the experimental groups are shown.

3) The complete sequence of the amino acid of the antioxidant polypeptide obtained in example 1 is identified to be Trp-Leu-Pro by using UPLC/Q-TOF-MS, and the molecular weight is 405 Da. The antioxidant activity is shown in figure 6, wherein DPPH free radical clearance rate is 58%, ABTS free radical clearance rate is 64%, and the antioxidant activity is close to that of positive control Glutathione (GSH).

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Sequence listing

<110> Sichuan university

<120> ascidian antioxidant polypeptide and preparation method thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 3

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Ile Ser Trp

1