CN112898410A

CN112898410A - Tachypleus tridentatus hemocyanin and preparation method and application thereof

Info

Publication number: CN112898410A
Application number: CN202110109125.8A
Authority: CN
Inventors: 胡梦红; 赵忠浩; 王有基
Original assignee: Shanghai Ocean University
Current assignee: Shanghai Ocean University
Priority date: 2021-01-27
Filing date: 2021-01-27
Publication date: 2021-06-04
Anticipated expiration: 2041-01-27
Also published as: CN112898410B

Abstract

The invention relates to the field of biotechnology, in particular to Tachypleus tridentatus hemocyanin with antioxidant activity and a preparation method thereof. The preparation method comprises the following steps: (1) removing the precipitate after the low-temperature low-speed primary centrifugation of the blood plasma of the tachypleus tridentatus and reserving the supernatant; (2) mixing the supernatant with a saturated ammonium sulfate solution and standing for 10-24 hours; (3) collecting the precipitate after the low-temperature low-speed secondary centrifugation of the mixed solution, and suspending the precipitate in PBS (phosphate buffer solution) with the pH value of 7.0-7.6 to obtain a horseshoe crab hemocyanin crude extract; (4) filling the crude extract into a dialysis bag, and dialyzing at 0-4 ℃ for 12-48 h. The limulus tridentatus hemocyanin with the antioxidant activity is obtained by the method. The method uses the Tachypleus tridentatus waste plasma remained in the preparation of the limulus reagent as a raw material, reduces the cost, is simple to operate, saves the time, obtains the Tachypleus tridentatus hemocyanin with stronger oxidation resistance, can be used for storing medicines, cosmetics, foods and the like, and has good market prospect and industrial value.

Description

Tachypleus tridentatus hemocyanin and preparation method and application thereof

Technical Field

The invention relates to the field of biotechnology, in particular to a Tachypleus tridentatus hemocyanin with antioxidant activity and a preparation method and application of freeze-dried powder of the Tachypleus tridentatus hemocyanin.

Background

Hemocyanin is a protein widely present in arthropod and mollusk hemolymph, and is called three respiratory proteins in nature together with hemoglobin and earthworm hemoglobin. However, as the research progresses, it has been found that the function of hemocyanin is far from this, and hemocyanin is involved in storage of energy, molting of animals, regulation of immune function, and the like in addition to oxygen transport in arthropods. Chinese horseshoe crab is an arthropod living in seabed. The blood of Tachypleus tridentatus, a member of arthropods, contains a large amount of hemocyanin. The research proves that the hemocyanin has various catalytic effects, and the hemocyanin can denaturalize and express the activities of phenol oxidase, catalase, lipoxygenase and the like under specific conditions.

Cosmetics are highly popular commodities, and their consumption is moving with the social trend, and have a distinct epoch characteristic. With the improvement of the quality of life accompanied by economic development, how to delay the aging process becomes the most popular topic. Nowadays, the age-old population characteristics and the special industrial background of cosmetics provide wide space for the application of natural extracts with physiological activities such as free radical resistance, oxidation resistance and the like. With the intensive research on the theory of free radicals, natural extracts having antioxidant activity are also increasingly used in the pharmaceutical industry and food storage.

CN104788541A discloses a preparation method of small peptide with antioxidant activity, wheat germ albumin is subjected to enzymolysis by trypsin to obtain albumin hydrolysate I; taking the components with the molecular weight less than 10KDa in the hydrolysate I, and separating by adopting gel filtration chromatography to obtain the components with antioxidant activity as albumin extract II; carrying out enzymolysis on the extract II by using alkaline protease to obtain an albumin hydrolysate III; and separating the hydrolysate III by gel filtration chromatography and reversed-phase high performance liquid chromatography in sequence to obtain the small peptide with antioxidant activity.

CN102080118A discloses a preparation method of rice bran antioxidant active protein peptide, wherein fresh rice bran is subjected to low-temperature degreasing treatment, rice bran protein is prepared by an alkaline method, and rice bran antioxidant peptide is prepared by using compound protease.

The existing methods are complex in preparation method, and finished products are not easy to store and transport.

Tachypleus tridentatus, a kind of arthropod, has a large amount of hemocyanin in hemolymph. Because of the need to produce limulus reagents, Tachypleus tridentatus is often taken to collect blood, blood cells are used to produce limulus reagents, and the remaining plasma is discarded completely. In fact, these discarded plasmas still contain large amounts of hemocyanin. These hemocyanins existing in the plasma of horseshoe crab can be utilized.

The inventor finds that the residual blood plasma is used for preparing the hydrolysis peptide with the antioxidant activity, and CN106148465A discloses the limulus plasma protein hydrolysis peptide with the antioxidant activity and a preparation method and application thereof, wherein the limulus plasma is subjected to constant-temperature water bath heat treatment denaturation at the temperature of 70-100 ℃, and then the limulus plasma is subjected to suction filtration and is washed by water; hydrolyzing the obtained limulus plasma by using protease, and then centrifuging the limulus plasma hydrolysate to obtain supernatant; passing the obtained supernatant through Sephadex G-50 Sephadex column, and collecting the component with maximum absorbance at wavelength of 230nm to obtain limulus plasma proteolytic peptide.

The raw material of the method is horseshoe crab plasma, the hydrolysis peptide with antioxidant activity is obtained, the steps of suction filtration, enzymolysis and the like are needed, and the sephadex chromatography method is adopted, so that the sephadex chromatography operation is complex, the experiment consumes long time, and the column loading, the column bed pH adjustment and the repeated balance of the chromatographic column by buffer solution are needed before the experiment, which takes time; chromatography columns are also prone to clogging during the experiment, take more time to process once clogged, and are of narrow use, mainly food and drugs. And has the problems of inconvenient product storage and transportation and the like.

Therefore, the prior art has the disadvantages of complicated operation, long time consumption, narrow application of the prepared hydrolyzed peptide, and difficult preservation of the prepared hydrolyzed peptide.

Based on the hemocyanin and the freeze-dried powder with the antioxidant activity, the hemocyanin and the freeze-dried powder with the antioxidant activity are developed, and the aim is to efficiently and simply obtain the hemocyanin with the antioxidant activity so as to meet the market demand.

Disclosure of Invention

The invention aims to provide limulus tridentatus hemocyanin with antioxidant activity, freeze-dried powder and a preparation method thereof.

A method for preparing horseshoe crab hemocyanin comprises the following steps:

(1) removing the precipitate after the low-temperature low-speed primary centrifugation of the blood plasma of the tachypleus tridentatus and taking the supernatant;

(2) and (2) mixing the supernatant obtained by the primary centrifugation in the step (1) with a saturated ammonium sulfate solution according to the ratio of 1: mixing the materials in a volume ratio of 1.5-3, and standing for 10-24 h;

(3) performing low-temperature low-speed secondary centrifugation on the mixed solution after placement in the step (2), collecting precipitates, and suspending the precipitates in a PBS (phosphate buffer solution) with the pH value of 7.0-7.6 to obtain a horseshoe crab hemocyanin crude extract;

(4) and (3) adding the crude limulus hemocyanin extracting solution obtained in the step (3) into a dialysis bag, dialyzing for 12-48 h at 0-4 ℃ by using PBS (phosphate buffer solution) with the pH of 7.0-7.6, and taking a part with the molecular weight of more than or equal to 3500 Da.

The primary centrifugation in the step (1) and the secondary centrifugation in the step (3) are carried out, wherein the centrifugation temperature is 0-4 ℃, the centrifugation speed is 6000-8000 rpm, and the time is 0.5-2 h. More preferably, the temperature of the primary centrifugation or the secondary centrifugation is 4 ℃, the centrifugation speed is 8000rpm, and the time is 1 h.

Preferably, the pH of the saturated ammonium sulfate solution in step (2) is 7.

Preferably, in the step (2), the volume ratio of the supernatant to the saturated ammonium sulfate solution is 1: 2.

the preparation method of the saturated ammonium sulfate solution in the step (2) comprises the following steps: dissolving ammonium sulfate in water at 55-65 ℃, cooling to room temperature, filtering, and adjusting the pH of the filtrate to 7.0 by using ammonia water.

In the step (2), a saturated ammonium sulfate solution is slowly added dropwise to the supernatant.

In steps (3) and (4), the pH of the PBS solution is preferably 7.0.

In steps (3) and (4), the concentration of the PBS solution is 0.005 to 0.1mol/L, more preferably 0.005 to 0.05 mol/L. In a preferred embodiment of the present invention, the PBS solution has a pH of 7.0 and a concentration of 0.01 mol/L.

In the step (4), the dialysis time is 24h, and the dialysis solution is replaced at the 2h, 8h and 22h of dialysis respectively.

Preferably, the limulus tridentatus plasma of step (1) is limulus tridentatus waste plasma remaining after the limulus reagent preparation.

And (4) carrying out freeze drying, vacuum freeze drying or spray drying on the hemocyanin solution obtained in the step (4) to obtain the limulus hemocyanin freeze-dried powder.

Preferably, the hemocyanin solution of Tachypleus tridentatus obtained in the step (4) is freeze-dried by a vacuum freeze-dryer and ground into powder by a low-temperature grinder. More preferably, the temperature of freeze drying is-70 to-80 ℃, and the time is 48 to 96 hours. More preferably, the temperature of the freeze-drying is-80 ℃ and the time is 72 hours.

A Tachypleus tridentatus hemocyanin is prepared by the above method.

The Tachypleus tridentatus hemocyanin has good antioxidant activity, and can be used for preparing antioxidant. The Chinese horseshoe crab hemocyanin can be used for preparing cosmetics, foods, food preservatives or medicines, and is applied to the fields of functional foods, food storage and preservation, pharmacy and cosmetics.

A pharmaceutical or cosmetic with antioxidant activity contains the above Tachypleus tridentatus hemocyanin. Preferably, the above horseshoe crab hemocyanin is used as an active ingredient.

The limulus tridentatus hemocyanin obtained by the method has strong oxidation resistance, and can effectively eliminate various free radicals and peroxides and inhibit the generation of the free radicals and the peroxides, such as superoxide, superoxide radicals, hydroxyl free radicals, hydrogen peroxide and peroxides generated by lipid.

The invention has the beneficial effects that:

(1) the limulus hemocyanin with the antioxidant activity is obtained by taking the Chinese limulus waste plasma remained in the limulus reagent preparation as a raw material, so that byproducts are fully utilized, and the cost is reduced;

(2) the extraction and preparation method is simple, and the time is saved; the method of ammonium sulfate fractional precipitation and dialysis is adopted, the whole operation is simple, convenient and quick, and the obtained tachypleus tridentatus hemocyanin has good anti-oxidation effect;

freeze-drying and grinding the purified horseshoe crab hemocyanin into powder, which not only facilitates storage and transportation, but also ensures that the quality problems of protein character change and the like of the horseshoe crab hemocyanin in China cannot occur in the subsequent use process;

(3) the obtained limulus hemocyanin product has strong antioxidant activity and wide application, can be widely used in antioxidant markets as an antioxidant, such as the fields of medicines, food storage and preservation, cosmetic preparation and the like, and has good market prospect and industrial value.

Detailed Description

The technical solution of the present invention will be described below with reference to specific examples.

Example 1

A method for preparing Tachypleus tridentatus hemocyanin with antioxidant activity comprises the following steps:

(1) taking out the residual blood plasma of Tachypleus tridentatus from a refrigerator at minus 80 ℃, and sequentially thawing in a refrigerator at minus 20 ℃ and 4 ℃;

(2) and after the plasma is completely thawed, taking 5mL of the plasma in a centrifuge tube, and centrifuging at a low temperature and a low speed under the conditions of 4 ℃ of temperature, 8000rpm/min of rotation speed and 1h of centrifugation time. And (4) discarding the precipitate after the centrifugation is finished, and keeping the supernatant for later use.

(3) Taking the supernatant and a saturated ammonium sulfate solution with the pH value of 7.0, and using a liquid transfer gun to transfer the saturated ammonium sulfate solution into a liquid state according to the weight ratio of 2: 1, dropwise and slowly adding the mixture into the supernatant obtained in the step (2); the resulting suspension was placed in a refrigerator at 4 ℃ overnight.

(4) Taking the suspension, centrifuging at low temperature and low speed under the conditions of 4 ℃, 8000rpm/min of rotation speed and 1h of centrifugation time. And (3) observing blue precipitates of the Chinese horseshoe crab hemocyanin at the bottom after the centrifugation is finished, discarding supernate, and suspending the precipitates in PBS (phosphate buffer solution) with the pH value of 7.0 to obtain the crude extract of the horseshoe crab hemocyanin.

(5) Adding the crude extractive solution of Limulus hemocyanin into dialysis bag of MD3500, standing at 4 deg.C for 24 hr, and respectively replacing dialysis solution at 2 hr, 8 hr, and 22 hr after dialysis. The dialyzed solution was 0.01mol/L PBS solution at pH 7.0. Taking the part with molecular weight more than or equal to 3500 Da.

(6) Freezing and crystallizing the dialyzed limulus hemocyanin solution at-80 deg.C for 72 hr, grinding into powder with low temperature grinder, and storing at 4 deg.C.

Example 2 determination of the antioxidant Activity of Limulus tridentatus hemocyanin

(1) Determination of superoxide radical scavenging Capacity

Superoxide radical is one of the most abundant oxygen radicals produced in the body. Pyrogallol is subjected to oxidative decomposition under the condition of alkaline (pH8.2) buffer solvent to generate superoxide anion and yellow intermediate product, and the intermediate product has maximum absorption peak at the wavelength of 320 nm. When antioxidant substances are added into the reaction system, the oxidation reaction of the pyrogallol is inhibited, the intermediate product is reduced, and the light absorption value of the sample solution at 320nm is weakened. The specific steps for measuring the scavenging capacity of the superoxide radical are as follows:

taking 0.5mL of Tachypleus tridentatus hemocyanin solution into a sample tube, adding 2.3mL of 50mmol/L Tris-HCl buffer solution with the pH value of 8.2, adding 20 mu L of 10mmol/L pyrogallol, quickly and uniformly mixing for reaction, measuring the absorbance at 320nm after 1min, and recording the absorbance value every 30s until the reaction is carried out for 6 min. According to the formula [ A_{Blank space}-(A_{Sample (I)}-A_Control)]/A_{Blank space}X 100 calculation of clearance, where A_{Blank space}I.e. absorbance, A, of the treatment groups without addition of hemocyanin_{Sample (I)}I.e. absorbance, A, of the treatment group to which hemocyanin was added_ControlI.e. the absorbance of only hemocyanin.

The results show that the Chinese horseshoe crab hemocyanin eliminates the IC of superoxide radical₅₀The value was 0.42 mg/mL.

(2) Determination of Hydrogen peroxide scavenging Capacity

The capacity of hydrogen peroxide scavenging is determined by reference to the method of Gulcin, and the specific steps are as follows: taking 3.4mL of hemocyanin solution to a sample tube, adding 0.6mL of 40mmol/L H₂O₂And (3) solution. Standing for 200min, zeroing with distilled water, and measuring absorbance at 230 nm. According to the formula [ A_{Blank space}-(A_{Sample (I)}-A_Control)]/A_{Blank space}X 100 calculation of clearance, where A_{Blank space}I.e. absorbance, A, of the treatment groups without addition of hemocyanin_{Sample (I)}I.e. absorbance, A, of the treatment group to which hemocyanin was added_ControlI.e. the absorbance of only hemocyanin.

IC for eliminating hydrogen peroxide from Chinese horseshoe crab hemocyanin₅₀The value was 0.89 mg/mL.

(3) Determination of anti-lipid peroxidation Capacity

Referring to the method of Zain, 200g of lecithin was dissolved in 200mL of 0.1mol/L phosphate buffer solution pH7.4 and dissolved with shaking in ice bath. Taking 4mL of hemocyanin solution, adding 3.6mL of lecithin solution, 0.4mL of 10mmol/L ferrous dichloride solution and 0.4mL of 10mmol/L VC solution into a sample tube. And carrying out constant temperature water bath at 37 ℃ in dark for 60 min. Cooled to room temperature, 1mL of 0.8% TBA solution and 1mL of 20% TCA solution were added, respectively, to the mixture in a boiling water bath for 15min, cooled to room temperature and centrifuged for 10min, and the absorbance at 535nm was measured.

According to the formula [ A_{Blank space}-(A_{Sample (I)}-A_Control)]/A_{Blank space}X 100 calculation of clearance, where A_{Blank space}I.e. absorbance, A, of the treatment groups without addition of hemocyanin_{Sample (I)}I.e. absorbance, A, of the treatment group to which hemocyanin was added_ControlI.e. the absorbance of only hemocyanin.

IC for resisting lipid peroxidation capability of Chinese horseshoe crab hemocyanin₅₀The value was 0.05 mg/mL.

(4) Measurement of hydroxyl radical scavenging ability

Sodium salicylate is free from salicylate in hydrogen peroxide solution, which can react with hydroxyl radical to produce 2, 3-dihydroxybenzoic acid and 2, 5-dihydroxybenzoic acid with color. The colored species has a maximum absorption peak at 510 nm. The absorbance of the reaction can be reduced by adding a substance capable of scavenging hydroxyl radicals to the reaction. The specific steps for determining the scavenging capacity of the hydroxyl free radical are as follows:

taking 0.2mL of hemocyanin solution to a sample tube, and adding 1mL of 2mmol/L FeSO₄Solution, 0.4mL of 2mmol/L sodium salicylate solution and 1mL of 0.1% H₂O₂And (3) solution. The mixture was incubated at 37 ℃ for 1h and, after cooling, the absorbance was measured at 510 nm. According to the formula [ A_{Blank space}-(A_{Sample (I)}-A_Control)]/A_{Blank space}X 100 calculation of clearance, where A_{Blank space}I.e. absorbance, A, of the treatment groups without addition of hemocyanin_{Sample (I)}I.e. absorbance, A, of the treatment group to which hemocyanin was added_ControlI.e. the absorbance of only hemocyanin.

IC for eliminating hydroxyl free radical of Chinese horseshoe crab hemocyanin₅₀The value was 1.63 mg/mL.

The tachypleus tridentatus hemocyanin prepared by the method has good capacity of eliminating free radicals and resisting lipid peroxidation, and can be used in the fields of medicine preparation, food storage, cosmetic preparation and the like.

Claims

1. A method for preparing horseshoe crab hemocyanin is characterized by comprising the following steps:

(1) removing the precipitate after the low-temperature low-speed primary centrifugation of the blood plasma of the tachypleus tridentatus and reserving the supernatant;

2. The method for producing horseshoe crab hemocyanin according to claim 1, wherein the primary centrifugation in step (1) and the secondary centrifugation in step (3) are performed at 0 to 4 ℃ and at 6000 to 8000rpm for 0.5 to 2 hours.

3. The method for producing horseshoe crab hemocyanin according to claim 1, wherein the concentration of the PBS solution in steps (3) and (4) is 0.005 to 0.1 mol/L.

4. The method for producing horseshoe crab hemocyanin having antioxidative activity according to claim 1 or 3, wherein the concentration of PBS solution is 0.01 mol/L.

5. The method for producing horseshoe crab hemocyanin according to claim 1, wherein said horseshoe crab plasma of step (1) is horseshoe crab waste plasma remaining after production of a horseshoe crab reagent.

6. The method for producing horseshoe crab hemocyanin according to claim 1, wherein the dialyzed protein solution of step (4) is freeze-dried or spray-dried.

7. A horseshoe crab hemocyanin produced by the method according to any one of claims 1 to 6.

8. Use of horseshoe crab hemocyanin according to claim 7 for the preparation of an antioxidant.

9. Use of horseshoe crab hemocyanin according to claim 7 for the preparation of cosmetics, foods, food preservatives or pharmaceuticals.

10. A cosmetic or pharmaceutical preparation having an antioxidant activity, which comprises the horseshoe crab hemocyanin of claim 7.