CN117448404A

CN117448404A - Enzymolysis method and application of haematococcus pluvialis extract

Info

Publication number: CN117448404A
Application number: CN202310992328.5A
Authority: CN
Inventors: 王凡; 洪奇
Original assignee: Meishang Guangzhou Cosmetics Co ltd
Current assignee: Meishang Guangzhou Cosmetics Co ltd
Priority date: 2023-08-08
Filing date: 2023-08-08
Publication date: 2024-01-26

Abstract

The invention relates to an enzymolysis method of haematococcus pluvialis extract and application of an enzymolysis product obtained by the enzymolysis method. The enzymolysis method comprises the steps of adding protease into an algae protein solution prepared by haematococcus pluvialis, optimizing a process by adopting a Box-Behnken response surface method, taking the total in-vitro antioxidant capacity as a response value, selecting four response variables of enzyme addition quantity, enzymolysis pH, enzymolysis temperature and enzymolysis time, taking the optimal point of a single factor experiment as a center, respectively taking a horizontal value around the center as a response surface level, and performing multiple regression analysis on data of each response variable to obtain a regression equation and obtain optimal enzymolysis conditions.

Description

Enzymolysis method and application of haematococcus pluvialis extract

Technical Field

The invention relates to the field of biotechnology, in particular to an enzymolysis method of haematococcus pluvialis extract, and application of an enzymolysis product obtained by using the enzymolysis method, wherein the enzymolysis product of haematococcus pluvialis extract has excellent antioxidant capacity.

Background

This section provides background information related to the present application, which does not necessarily constitute prior art.

Haematococcus pluvialis (Haematococcus Pluvialis) is a single-cell, spherical, green dinoflagellate oily cell with a diameter of about 5-50 μm. Depending on their life cycle, morphology and physiology, haematococcus pluvialis can exist as a vegetative green cell, being able to swim due to all of their two flagella, which is closely related to green phase and biomass accumulation; it may also exist as red cyst cells that accumulate astaxanthin under pressure. Ultrastructural changes occur when haematococcus pluvialis cells transition from green to red. The chemical composition of cellular material is also evolving. According to Dry Biomass (DBW), lutein is present in up to 1% in the green phase, with a total lipid content varying between 20% and 25%, while in the red phase lipids are present in 32% to 37% and are deposited with 1% to 5% astaxanthin. Most haematococcus pluvialis cells have high protein content in the green stage, which accounts for 29% -45% of dry weight, and the protein content in palmar cells is reduced to 36%; in the red phase, the protein represents about 21% -23% of the dry cell weight. The haematococcus pluvialis protein has various compositions, mainly comprises aspartic acid, glutamic acid and leucine, wherein the content of essential amino acids reaches 38.76% of the total amino acid content, is close to the EAA/TAA ratio 40% proposed by the United nations grain and agricultural organization (FAO) and the World Health Organization (WHO), and is a good potential source of high-quality protein. Among the microalgae of great commercial value, haematococcus pluvialis is the most abundant source of known natural astaxanthin.

Currently, existing technical solutions for preparing haematococcus pluvialis extract (i.e. phycin solution) include supercritical carbon dioxide (CO ₂ ) Extraction, ultrasonic extraction, microwave extraction, high-pressure homogenization orGrinding, extracting with solvent, and performing enzymolysis. For haematococcus pluvialis, the cell wall is composed of glycoprotein molecules and a small amount of cellulose and chitin, so enzymes used in the process of preparing haematococcus pluvialis extract (i.e. phycocyanin solution) by enzymolysis extraction mainly comprise cellulase, hemicellulase, pectinase, chitinase, protease, crashing enzyme, eductase and the like. Tjahjono in 1993 proposed the preparation of Haematococcus pluvialis extract with proteinase K. CN201610218349.1 (culture method of haematococcus pluvialis zoocells and preparation method of protoplast) discloses that haematococcus pluvialis zoocells are resuspended in a buffer containing collagenase for enzymolysis at a certain initial density, wherein the collagenase is selected from collagenase I, II, III or IV type. CN2004100075358 (technology for preparing and regenerating haematococcus pluvialis protoplast) discloses that haematococcus pluvialis cells are treated by a pretreatment agent prepared from an acidic buffer solution, EDTA and dithiothreitol, and then the protoplast is separated by a compound hypertonic enzyme solution composed of cellulase, helicase, pectase and the acidic buffer solution, wherein the preparation rate can reach 80%.

Although development of haematococcus pluvialis has been carried out for decades, research has focused mostly on astaxanthin and polysaccharides, and the use of haematococcus pluvialis derived polypeptides has often been neglected. According to previous studies on other microalgae derived polypeptides, they have various effects of antibacterial, anticancer, anti-inflammatory and antioxidant. Bioactive peptides are defined as peptide sequences in proteins, mostly containing 2-20 amino acids, usually rich in hydrophobic amino acids, which have a beneficial effect on body function or a positive effect on human health, beyond their known nutritional value. In the last few years there has been an increasing interest in finding different bioactive peptide sequences which can reduce or prevent the risk of chronic diseases and provide immune protection. They regulate important bodily functions through their various activities, including antihypertensive, antibacterial, antithrombotic, immunomodulating, opioid, antioxidant and mineral binding functions. Currently, bioactive peptides are prepared mainly by two methods, namely enzymatic hydrolysis and fermentation production by using a starter. In addition, bioactive peptides can also be chemically synthesized because the content of these peptides found in nature is very low and commercial value in producing synthetic bioactive peptides is increasing. After the protein is processed and hydrolyzed to form the bioactive peptide, it is often subjected to further isolation and purification. Most scholars employ an ultrafiltration step to fractionate the compounds of the hydrolysate according to molecular weight; other purification techniques may be used, including chromatographic techniques, primarily reverse phase high performance liquid chromatography (RP-HPLC) and ultra-high performance liquid chromatography (UPLC), with the particular choice of different techniques depending on the desired level of purity of the final product.

Disclosure of Invention

The application provides an enzymolysis method of haematococcus pluvialis extract, which is based on the technology of preparing bioactive peptide by enzymolysis, wherein the antioxidation capability of an enzymolysis product is measured by using an FRAP method, and on the basis of a single factor experiment, the optimal conditions of 4 factors of time used for enzymolysis, enzyme concentration in an enzymolysis system, pH of a solution in the enzymolysis process and reaction temperature in the enzymolysis system in the enzymolysis reaction are optimized by using a response surface method, so that the enzymolysis product with the optimal total antioxidation capability is obtained.

In order to achieve the above purpose, the invention adopts the following technical scheme: the enzymolysis method of haematococcus pluvialis extract comprises the steps of adding protease into an algae protein solution prepared by haematococcus pluvialis, selecting four response variables of enzyme addition amount, enzymolysis pH, enzymolysis temperature and enzymolysis time based on a Box-Behnken response surface method by taking the total in-vitro antioxidant capacity as a response value, taking the optimal point of a single factor experiment as a center, taking a horizontal value around the center as a response surface level, and performing multiple regression analysis on data of each response variable to obtain a regression equation:

Y＝163.86-3.27A+3.46B+2.34C+1.91D-1.55AB-0.7175AC-0.8475AD+3.81BC-1.78BD-1.70CD-9.36A2-3.70B2-6.77C2-3.03D2；

wherein A is enzymolysis time, B is enzyme addition amount, C is enzymolysis pH, D is enzymolysis temperature, and Y is total in-vitro antioxidation capability.

In one or more embodiments, the method of preparing haematococcus pluvialis extract (i.e., phycin solution) includes the steps of:

preparing an algae solution with the mass concentration of 10wt% by using haematococcus pluvialis dry powder and deionized water;

adding cellulase and pectase into the algae liquid for enzymolysis, then using an ultrasonic cell grinder to carry out ultrasonic crushing on the solution, centrifuging for 15min after crushing, and collecting the supernatant to obtain the algae protein solution.

In one or more embodiments, the protease is selected from neutral protease or trypsin.

In one or more embodiments, the enzymatic hydrolysis method of haematococcus pluvialis extract includes the steps of: adding protease into the algae protein solution for enzymolysis, wherein the addition amount of the protease is 2.7%, the enzymolysis pH is 8.6, the enzymolysis temperature is 40 ℃, the enzymolysis time is 53min, inactivating the algae protein solution for 10min under the water bath condition of 90 ℃ after the enzymolysis is finished, cooling the algae protein solution to room temperature, centrifuging the cooled algae protein solution, taking the supernatant, and filtering the obtained algae protein solution to obtain an enzymolysis product.

The enzymolysis method of haematococcus pluvialis extract comprises adding protease into the algae protein solution prepared from haematococcus pluvialis, wherein the addition amount of the protease is 0.1%, the enzymolysis temperature is 50 ℃, the enzymolysis time is 4 hours, and preferably, the enzymolysis pH is 6.5, 7.0, 7.5, 8.0, 8.5 or 9.0.

The enzymolysis method of haematococcus pluvialis extract comprises adding protease into the algae protein solution prepared from haematococcus pluvialis, wherein the addition amount of the protease is 0.1%, the enzymolysis pH is 7.0, the enzymolysis time is 4h, and the enzymolysis temperature is preferably 30 ℃, 40 ℃, 50 ℃ or 60 ℃.

The enzymolysis method of haematococcus pluvialis extract comprises adding protease into the algae protein solution prepared by haematococcus pluvialis, wherein the addition amount of the protease is 0.1%, the enzymolysis pH is 7.0, the enzymolysis temperature is 50 ℃, and the enzymolysis time is preferably 0.5h, 1h, 1.5h, 2h, 3h, 4h or 5h.

The enzymolysis method of haematococcus pluvialis extract comprises adding protease into the algae protein solution prepared from haematococcus pluvialis, wherein the enzymolysis pH is 7.0, the enzymolysis temperature is 50 ℃, the enzymolysis time is 4 hours, and preferably, the addition amount of the protease is 0.1%, 0.4%, 1.0%, 2.0%, 3.0% or 4.0%.

The invention optimizes the enzymolysis process of haematococcus pluvialis extract by using a Box-Behnken response surface method on the basis of single factor test investigation of enzyme addition amount, enzymolysis pH, enzymolysis temperature and enzymolysis time. Through fitting of a plurality of equations, the surface fitting equation is obvious, and the determined optimal enzymolysis process condition of the haematococcus pluvialis extract is as follows: the algae protein solution is subjected to enzymolysis by protease, the enzyme addition amount is 2.709%, the enzymolysis pH is 8.61, the enzymolysis temperature is 40.357 ℃, the enzymolysis time is 0.876h, and under the enzymolysis condition, the predicted value of the oxidation capability of the enzymolysis product is 165.978. In the verification process, the process conditions are adjusted according to actual operation: the enzymolysis time is 53min, the enzyme addition amount in the enzymolysis system is 2.7%, the pH of the solution in the enzymolysis process is 8.6, and the reaction temperature in the enzymolysis system is 40 ℃.

In another aspect, the present application also relates to an enzymatic hydrolysate obtained by the enzymatic hydrolysis method described above.

In another aspect, the application also relates to the application of the enzymolysis product in preparing skin care products or cosmetics with effects of relieving, anti-inflammatory, brightening skin, barrier repairing and aging delaying, wherein the action parts of the skin care products or cosmetics comprise: head, hair, eyes, lips, torso, face.

On the other hand, the application also relates to application of the enzymolysis product in preparing medicines with anti-inflammatory, whitening, skin barrier repairing and anti-aging effects.

In another aspect, the present application also relates to the use of the enzymatic hydrolysate described above for the preparation of an antioxidant or anti-aging agent.

The following description is made with reference to specific embodiments.

Drawings

The invention is further illustrated by the accompanying drawings, which are not to be construed as limiting the invention in any way.

FIG. 1 shows the results of the enzymatic protease screening (Yusheng-alkali: yusheng red algae alkaline protease enzymatic hydrolysate; yusheng-acid: yusheng red algae alginic acid protease enzymatic hydrolysate; yusheng-Zhonger: yusheng red algae neutral protease enzymatic hydrolysate; yusheng-pancreas: yusheng red algae trypsin enzymatic hydrolysate; yusheng-stomach: yusheng red algae pepsin enzymatic hydrolysate).

FIG. 2 is a graph showing the effect of pH on the antioxidant capacity of Haematococcus pluvialis polypeptides.

FIG. 3 is a graph showing the effect of temperature on the antioxidant capacity of Haematococcus pluvialis polypeptides.

FIG. 4 shows the effect of enzymolysis time on the antioxidant capacity of Haematococcus pluvialis polypeptides.

FIG. 5 shows the effect of enzyme loading on the antioxidant capacity of Haematococcus pluvialis polypeptides.

FIG. 6 is a graph of response surface and contour plot of enzymatic hydrolysis conditions for total antioxidant capacity of Haematococcus pluvialis polypeptides in vitro.

FIG. 7 shows the effect of HPE on LPS damage and UVA damage (FIG. 7A shows LPS-induced damage modification experiments and FIG. 7B shows UVA treatment damage repair experiments).

Detailed Description

It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.

It should be noted that, some reagents involved in the embodiments of the present invention are shown in table 1, and apparatuses and devices involved in the embodiments of the present invention are shown in table 2.

Table 1: reagent(s)

Table 2: apparatus and device

Example 1: preparation of algae protein solution

The embodiment provides a preparation method of an algae protein solution, which adopts a pectinase and cellulase combined enzymolysis combined ultrasonic method to crush cell walls of haematococcus pluvialis and extract haematococcus pluvialis protein, and specifically comprises the following steps:

dissolving haematococcus pluvialis dry powder into 10% concentration algae solution (W/V) by deionized water;

adding cellulase and pectase into the solution for enzymolysis, adding 4000U of cellulase and 5000U of pectase according to the amount of dry powder of haematococcus pluvialis per gram, carrying out enzymolysis for 3 hours at the pH of the solution of 4.5 and 45 ℃ in the enzymolysis process, then carrying out ultrasonic crushing on the solution for 30 minutes (power of 60%, ultrasonic on-off of 2.0 and ultrasonic off of 2.0) by using a BILON-1000E ultrasonic cell grinder, repeating for two times, centrifuging for 15 minutes by using a high-speed centrifuge at 4000rpm after ultrasonic finish, and collecting supernatant to obtain the phycocyanin solution.

Example 2: enzymolysis protease screening experiment

The type I collagen is one of the main components of the extracellular matrix of dermis, and is synthesized in the dermis by the dermis fibroblast, secreted to the outside of the dermis, separated by terminal peptide under the action of terminal procollagenase, and polymerized to form collagen fiber. The human dermal fibroblast can be used as a cell model for researching the improvement of the collagen I content of cosmetics, and whether the test substance has efficacy in promoting collagen synthesis is evaluated by measuring the up-regulation rate of the collagen I content of a blank control and the test substance after the test substance is dosed. The content of the type I collagen is determined by adopting an enzyme-linked immunosorbent assay (ELISA) method, and the specific principle is as follows: the type I collagen is combined with the collagen antibody coated on the ELISA plate after being specifically combined with the anti-type I collagen antibody with a substrate mark, and a colored product is generated after the substrate is catalyzed by enzyme, wherein the content of the type I collagen is positively correlated with the color depth of the colored product. The optical density (OD value) was measured at a wavelength of 450nm by using an enzyme-labeled instrument, and the type I collagen content was calculated.

1. Preparation of reagents or sample solutions

Preparing HFF-1 cell culture medium: 90% DMEM (high sugar) +10% FBS (Gibco fetal bovine serum) +1% green-streptomycin diabody;

TGF-beta 1 reagent preparation: TGF-. Beta.1 stock solution was 100. Mu.g/ml and diluted to 100ng/ml with complete medium as required by the experiment.

2. Preparation of sample solution to be tested

The sample is determined to be soluble before the test, and products with unspecified test concentration need to be subjected to cytotoxicity test in advance (the maximum concentration without toxicity is selected for subsequent tests); the specified concentration of the product or cosmetic raw material is formulated with the corresponding solvent to the corresponding concentration.

3. Cell culture

(1) Cell resuscitation: taking out the cell cryopreservation tube, rapidly placing in a 37 ℃ water bath kettle, shaking to be dissolved, transferring into a centrifuge tube (complete culture medium containing DMEM) under an ultra-clean bench environment, placing the centrifuge tube into a centrifuge, balancing, and setting the procedures as follows: the rotation speed is 800rpm; the time is 3min. After the operation is finished, the supernatant is discarded, a proper amount of culture medium (containing serum) is added, the mixture is lightly blown by a gun head, the mixture is added into a 10ml T75 culture flask containing 10% of serum culture medium, and the mixture is placed into a cell culture box for static culture after uniform mixing.

(2) Cell passage: after cells are resuscitated for 2 days, taking out a T75 culture flask, observing under a microscope, when the cell density is about 90%, discarding the supernatant in an ultra clean bench, adding 3ml of pancreatin after PBS is washed once, standing and digesting for 30-60s in an incubator, adding 3ml of DMEM complete medium to stop digestion, blowing the cells at the bottom of a culture dish, collecting the cells into a centrifuge tube, centrifuging the cells by using a centrifuge (the same procedure as before), discarding the supernatant, adding 2ml of complete medium to suspend the cells, uniformly blowing the cells by a gun head, absorbing 750 μl of the cells into the T75 culture flask containing 10ml of DMEM medium, mixing the cells uniformly, and then placing the cells into the incubator for standing and culturing.

(3) Cell cryopreservation: taking out a T75 culture bottle, discarding the culture medium, washing with PBS once, adding 3ml of pancreatin, standing in an incubator for digestion for 60 seconds, adding 3ml of DMEM culture medium to stop digestion, blowing off cells at the bottom of a culture dish, collecting the cells into a centrifuge tube, centrifuging the cells by using a centrifuge (the same procedure as before), discarding the supernatant, adding 3ml of cell cryopreservation solution (serum: DMSO: culture medium=2:1:7), suspending the cells, and then adding 1ml of cell cryopreservation solution into each tube of cryopreservation tube. The frozen storage tube is put into a program cooling box and is transferred into a refrigerator with the temperature of minus 80 ℃ for preservation.

4. Concentration of test substance

(1) And (3) paving: taking out T75 flask, discarding culture medium, washing with PBS once, adding 3ml pancreatin, standing in incubator for digestion for 60s, adding 3ml DMEM culture medium to stop digestion, blowing cell at bottom of culture dish, collecting into centrifuge tube, and centrifuging (procedureThe same as before), the supernatant was discarded, 2ml of DMEM medium was added, 180. Mu.l of the cells were removed after resuspension, 20. Mu.l of trypan blue dye was added, the number of cells was counted by using a countstar cell counter, and finally the medium containing the cells was added to a 24-well plate, and the final cell density was 6X 10 ⁴ And/or holes. After the plating is finished, the cells are placed into a cell culture box for culturing for 24 hours.

(2) Sample adding: when the cells are cultured to about 70% of fusion degree, the culture medium is discarded, fresh culture medium is added, UVA induction is performed with a certain amount, and the total irradiation amount is controlled to be 15J/cm < 2 >. The supernatant was discarded and complete medium containing a concentration of the test substance or yang ginseng was added. Incubation was carried out for 24h.

Test object group setting: 15J UVA ultraviolet treated cells + complete medium + test object 1-5 (1. Yusheng-alkali: yusheng red algae alkaline protease enzymolysis product; 2. Yusheng-acid: yusheng red algae alginic acid protease enzymolysis product; 3. Yusheng-middle: yusheng red algae neutral protease enzymolysis product; 4. Yusheng-Yi: yusheng red algae trypsin enzymolysis product; 5. Yusheng-Wei: yusheng red algae pepsin enzymolysis product).

Positive group settings: 15J UVA UV treated cells+complete medium+TGF-. Beta.1;

blank set setting: complete medium;

negative control group settings: 15J UVA UV treated cells+complete medium.

5. Sample collection

After 24h incubation in incubator, the cell cultures were collected and stored in a-20℃refrigerator based on 1.5mL centrifuge tubes.

6. I type collagen content detection

According to the I-type collagen detection instruction, the concentration of the I-type collagen is detected, and two compound holes are formed in each sample.

On the ordinate, the concentration of the standard substance is OD ₄₅₀ The value is the abscissa, a standard curve is manufactured, a regression equation of the standard curve is calculated, and the OD of the sample is calculated ₄₅₀ The values are substituted into the equation and the type I collagen content of the sample is calculated. Type I collagen up-regulation rate calculation formula:

up-regulation (%) = (T/C-1) ×100%, where T is the average of the type i collagen content of the test object and C is the average of the type i collagen content of the blank/solvent control.

The experimental results are shown in figure 1, and the concentration of type i collagen can be significantly increased by enzymatic hydrolysis using neutral protease relative to control and other proteases. Based on the consideration of improving the antioxidant capacity of the proteolytic products, a related experiment will be performed with neutral protease.

Example 3: single factor experiment to confirm the optimal enzymolysis condition

10mL of algae protein solution is measured, neutral proteinase is added, enzymolysis is carried out under certain pH, temperature, time and enzyme proportion, and after the enzymolysis is finished, the algae protein solution is inactivated for 10min under the water bath condition of 90 ℃. After the sample was cooled to room temperature, the supernatant was centrifuged at 8000rpm for 5min using a high-speed centrifuge and filtered with a Mibo 0.45 μm aqueous needle filter to obtain the final sample.

1. Determination of optimal parameters

(1) Determination of optimal pH for enzymolysis

The enzymolysis condition is that the concentration of enzyme in the enzymolysis system is 0.1% (W/V), the reaction temperature in the enzymolysis system is 50 ℃, and the time for the enzymolysis reaction is 4 hours. The pH of the solution in the enzymolysis process is set to 6.5, 7.0, 7.5, 8.0, 8.5 and 9.0.

(2) Determination of optimal temperature for enzymolysis

The enzymolysis condition is that the concentration of enzyme in an enzymolysis system is 0.1% (W/V), the pH of a solution in the enzymolysis process is 7.0, and the time for enzymolysis reaction is 4 hours. The reaction temperature in the enzymolysis system is set to be 30, 40, 50 and 60 ℃.

(3) Determination of optimal time for enzymolysis

The enzymolysis condition is that the concentration of enzyme in the enzymolysis system is 0.1% (W/V), the pH of the solution in the enzymolysis process is 7.0, and the reaction temperature in the enzymolysis system is 50 ℃. The time for the enzymolysis reaction is set to 0.5, 1, 1.5, 2, 3, 4 and 5 hours.

(4) Determination of optimal proportion of enzyme concentration in neutral protease enzymolysis system

The enzymolysis condition is that the pH value of the solution in the enzymolysis process is 7.0, the reaction temperature in the enzymolysis system is 50 ℃, and the time for the enzymolysis reaction is 4 hours. The enzyme concentration in the enzymolysis system is set to 0.1%, 0.4%, 1.0%, 2.0%, 3.0% and 4.0%.

2. Response surface method optimized experimental design

The experiment adopts Design-Expert 12.0 software to Design a 4-factor 3-level Box-Behnken response surface experiment by taking 4 factors of the time used for enzymolysis reaction, the enzyme concentration in an enzymolysis system, the pH of a solution in the enzymolysis process and the reaction temperature in the enzymolysis system as experimental factors and taking the total in-vitro antioxidant capacity as a response value according to the result of the single-factor experiment. The factor levels are shown in Table 3.

Table 3: response surface analysis factor level table

Wherein the detection steps of the total antioxidant capacity in vitro are as follows:

s1, preparing a solution

2.04g of anhydrous sodium acetate is weighed, 8mL of glacial acetic acid is added, distilled water is added to constant volume to 100mL, and finally an acetic acid buffer solution with pH of 3.6 can be obtained. 15.6mg of TPTZ powder is weighed again, poured into 5mL of deionized water, 2-3 drops of concentrated hydrochloric acid are added dropwise, and stirring and shaking are carried out to fully mix the liquid phase and the solid phase until the liquid phase and the solid phase are completely dissolved, so that a TPTZ solution with the concentration of 10mM is obtained. Then, 0.54g of ferric trichloride hexahydrate was weighed and poured into 100mL of distilled water with stirring and shaking to thoroughly mix the liquid phase and the solid phase until complete dissolution, thereby obtaining a ferric trichloride solution having a concentration of 20 mM.

The three reagents are mixed according to the ratio of acetic acid buffer solution to 10mM TPTZ to 20mM ferric chloride=10:1:1 to prepare TPTZ working solution for standby.

S2.FeSO ₄ Drawing of a Standard Curve

Accurately weigh 40mg FeSO ₄ Dissolving in 100mL distilled water to obtain standard FeSO ₄ Solution (c=400 μg/mL), 14 centrifuge tubes were divided into two groups and added with 0, 0.0125, 0.125, 0.625, 1.25, 2.5, 5mL standard FeSO respectively ₄ The solution was made up to 5ml with deionized water and mixed well. Taking 20 mu L of each spot into a 96-well plate, adding 180 mu L of TPTZ working solution into the well, standing at normal temperature for 5min, measuring the absorbance at 596nm, recording as A standard, and recording as 2The 00 μl deionized water blank was designated as a blank, and the difference between the a standard and the a blank was calculated. Taking the average value of the two groups of measured differences, and recording FeSO ₄ And (3) drawing a standard curve by taking the concentration as an FRAP value and taking the FRAP value as an abscissa and the absorbance as an ordinate, and calculating the total in-vitro antioxidant capacity of the sample according to the standard curve.

S3, measuring total antioxidant capacity in vitro

Taking 20 mu L of sample solution to be spotted into a 96-well plate, adding 180 mu L of TPTZ working solution into the well, standing for 5min at normal temperature, measuring the absorbance at 596nm, marking the absorbance as a sample A, and marking 200 mu L of deionized water as a blank control as a blank A. And calculating the difference between the sample A and the blank A, and substituting the difference into a standard curve to calculate the total in vitro antioxidant capacity of the sample. The 3 replicates were repeated.

3. Analysis of experimental results

Determination of the experimental curve: drawing a standard curve as shown in fig. 1 with FRAP value as abscissa and absorbance as ordinate to obtain regression equation of y=0.0048x+0.0495, r ² The standard curve has extremely high reliability and can be used for in vitro total antioxidant capacity measurement.

Single factor effect on total antioxidant capacity in vitro:

(1) Determination of optimal pH for enzymolysis

As shown in fig. 2, under the condition that the reaction temperature, the reaction time, the enzyme concentration and other conditions in the enzymolysis system are the same, the oxidation resistance of the enzymolysis liquid is gradually increased in the process of increasing the pH of the solution from 6.5 to 8.5, and the oxidation resistance of the enzymolysis liquid starts to be reduced as the pH continues to be increased. The reason for this may be that when the pH is raised to 8.5, the enzyme activity is also gradually raised to the highest level; when the pH is more than 8.5, the protease activity is inhibited, resulting in a decrease in the enzymolysis efficiency, a decrease in the content of the antioxidant peptide obtained by enzymolysis, and a decrease in the total antioxidant capacity. Therefore, the experiment determines that the optimal pH for enzymolysis is 8.5.

(2) Determination of optimal temperature for enzymolysis

As shown in FIG. 3, under the condition that the pH, time and enzyme concentration in an enzymolysis system are the same in the enzymolysis process, the activity of protease is enhanced along with the temperature rise, so that the total in-vitro antioxidant capacity of the enzymolysis liquid is increased, and the total in-vitro antioxidant capacity of the enzymolysis product is highest when the reaction temperature is 40 ℃. With the continuous rising of the temperature, the activity of protease is inhibited, so that the enzymolysis efficiency is reduced, the content of the antioxidant peptide obtained by enzymolysis is reduced, and the total antioxidant capacity of the enzymolysis liquid is reduced. Therefore, the reaction temperature in the enzymolysis system is optimally selected to be 40 ℃.

(3) Determination of optimal time for enzymolysis

As shown in FIG. 4, under the condition that the pH, the temperature and the enzyme concentration in the enzymolysis system are the same in the enzymolysis process, the total in-vitro antioxidant capacity of the haematococcus pluvialis extract reaches the highest when the time for the enzymolysis reaction reaches 1h. And the oxidation resistance of the material tends to decrease and tends to be balanced with the time. This is probably because the antioxidant peptide in the enzymatic hydrolysate is further hydrolyzed into free amino acids with the time taken for the enzymatic hydrolysis reaction being prolonged, most of the amino acids are not reduced, and the total antioxidant capacity in vitro tends to decrease. The time for the enzymatic hydrolysis reaction was thus determined to be 1h.

(4) Determination of optimal ratio of neutral protease to enzyme

As shown in fig. 5, under the same conditions of controlling the pH, temperature, time for the enzymolysis reaction, etc. of the solution in the enzymolysis process, the total antioxidant capacity in vitro shows an ascending trend along with the increase of the protease usage amount, and reaches the highest enzyme concentration when the enzyme concentration in the enzymolysis system is 2%, and the antioxidant capacity continuously increases and decreases to a gentle trend. The reason is probably that in the enzymolysis reaction system, when the substrate concentration is enough, the enzyme concentration is in direct proportion to the speed of the enzymatic reaction, and when the enzyme concentration is 2%, the content of the antioxidant peptide obtained by enzymolysis is the highest, and the total antioxidant capacity in vitro is the highest. The enzyme concentration continues to increase, the protease possibly performs enzymolysis with each other, so that the inhibition effect on the enzymolysis efficiency is generated, the antioxidant peptide obtained by enzymolysis is reduced, and the total antioxidant capacity in vitro is reduced. Therefore, the most suitable enzyme concentration in the enzymolysis system is 2% in the experiment enzymolysis system.

Example 4: design of response surface experiment and results

Box-Behnken experimental design and experimental results

The Box-Behnken experiment design in the response surface method is adopted, the time used for enzymolysis reaction, the enzyme concentration in an enzymolysis system, the pH of a solution in the enzymolysis process and the reaction temperature in the enzymolysis system are taken as independent variables, and the total in-vitro antioxidant capacity of haematococcus pluvialis polypeptide is taken as a response value to carry out a Box-Behnken response surface optimization experiment with 4 factors and 3 levels. The experimental design and treatment results for each group are shown in table 4.

2. Regression equation and analysis of variance

Performing multiple regression fitting on the data in Table 2 by adopting Design-Expert 12.0 software to obtain a multiple quadratic regression equation taking the total antioxidant capacity of haematococcus pluvialis polypeptides in vitro as an objective function:

Y＝163.86-3.27A+3.46B+2.34C+1.91D-1.55AB-0.7175AC-0.8475AD+3.81BC-1.78BD-1.70CD-9.36A ² -3.70B ² -6.77C ² -3.03D ² . The regression model was analyzed by the method, and the meaning of the relevant parameters, regression equation and analysis of variance are shown in Table 5.

Table 4: box-Behnken experimental design and experimental result

Table 5: regression equation and analysis of variance

And (3) injection: ^* p is less than 0.05, which indicates that the difference is significant, ^** p <0.01 indicates that the difference is extremely significant.

From Table 5, it can be seen that the regression equation fits better (R ² ＝88.55％，R ² Adj= 77.10), the mismatch value P > 0.05 is not significant). Equation P<0.01 shows that the regression model of the total in vitro antioxidant capacity of haematococcus pluvialis polypeptide is very remarkable, and experimental data CV=2.18% shows that the experimental result is reliable. The primary term A, B in the equation affects very significantly (P<0.01 C has a significant effect on response values (P<0.05 D) influence of notIs remarkable; interaction item A ² 、C ² Has extremely remarkable effect (P<0.01)，BC、B ² 、D ² Has remarkable effect (P)<0.05 AB, AC, AD, BD, CD effect was insignificant (P > 0.05). This means that only the enzymolysis of haematococcus pluvialis has a significant interaction between the pH of the solution and the concentration of the enzyme in the enzymolysis system in the enzymolysis process, and the interaction has a significant influence on the total in vitro antioxidant capacity of haematococcus pluvialis polypeptides. The influence of each factor on the total in vitro antioxidant capacity of the haematococcus pluvialis polypeptide can be obtained by the P value and the F value in table 3, and the influence is as follows: the enzyme concentration in the enzymolysis system is more than the time of enzymolysis reaction is more than the pH value of the solution in the enzymolysis process is more than the reaction temperature in the enzymolysis system.

3. Interaction analysis

And using Design-Expert 12.0 software as a response curve diagram to analyze the influence of pairwise interaction among 4 factors of the time used for enzymolysis reaction, the enzyme concentration in an enzymolysis system, the pH of a solution in the enzymolysis process and the reaction temperature in the enzymolysis system on a response value. As shown in FIG. 6A, the interaction between the time used for the enzymolysis reaction and the enzyme concentration in the enzymolysis system has no significant effect on the response value, and the total in-vitro antioxidant capacity of the haematococcus pluvialis polypeptide also increases with the prolongation of the time used for the enzymolysis reaction and the increase of the enzyme concentration in the enzymolysis system. When the time for the enzymolysis and the enzyme concentration in the enzymolysis system reach a certain value, the total in-vitro antioxidant capacity of the haematococcus pluvialis polypeptide reaches a peak value, and at the moment, the time for the enzymolysis and the enzyme concentration in the enzymolysis system are continuously increased, so that the antioxidant capacity of the haematococcus pluvialis polypeptide is reduced. And when the enzyme concentration in the enzymolysis system is about 2%, the response curved surface reaches the highest point, namely the highest oxidation resistance. As shown in fig. 6B, the interaction between the time taken for the enzymatic hydrolysis reaction and the pH of the solution during the enzymatic hydrolysis process did not significantly affect the response value. Similar to the interaction relationship between the time used in the enzymolysis reaction and the enzyme concentration in the enzymolysis system, the influence of the interaction between the time used in the enzymolysis reaction and the pH of the solution in the enzymolysis process on the response value is also parabolic, namely, the relationship has a maximum point, and the change trend is shown to be increased and then decreased. When the time for the enzymolysis reaction is about 1h and the pH value of the solution in the enzymolysis process is about 8.5, the response surface reaches the highest point. As shown in FIG. 6C, the interaction between the time used for the enzymolysis reaction and the reaction temperature in the enzymolysis system has no significant effect on the response value, and the response surface reaches the highest point when the time used for the enzymolysis reaction is about 1h and the reaction temperature in the enzymolysis system is about 40 ℃. As shown in fig. 6D, the interaction between the enzyme concentration in the enzymolysis system and the pH of the solution during enzymolysis has a significant influence on the response value, the gradient of the response surface is steep, and the contour line is densely distributed. Along with the increase of the enzyme concentration in the enzymolysis system and the increase of the pH value of the solution in the enzymolysis process, the total in-vitro antioxidant capacity of the haematococcus pluvialis polypeptide is also increased. When the enzyme concentration in the enzymolysis system and the pH value of the solution in the enzymolysis process reach a certain value, the total in-vitro antioxidant capacity of the algae polypeptide reaches a peak value, and at the moment, the enzyme concentration in the enzymolysis system and the pH value of the solution in the enzymolysis process continuously rise, so that the antioxidant capacity of the algae polypeptide is reduced. When the enzyme concentration in the enzymolysis system is about 2% and the pH of the solution in the enzymolysis process is about 8.5, the response surface reaches the highest point. As shown in fig. 6E, the interaction between the enzyme concentration in the enzymatic hydrolysis system and the reaction temperature in the enzymatic hydrolysis system has no significant effect on the response value, and the response curve reaches the highest point when the enzyme concentration in the enzymatic hydrolysis system is about 2% and the reaction temperature in the enzymatic hydrolysis system is about 40 ℃. As shown in fig. 6F, the interaction between the pH of the solution during the enzymolysis and the reaction temperature in the enzymolysis system has no significant effect on the response value, and the response surface reaches the highest point when the pH of the solution during the enzymolysis is about 8.5 and the reaction temperature in the enzymolysis system is about 40 ℃.

Finally, analysis and simulation are carried out through Design-Expert 12.0 software to obtain the best enzymolysis conditions of haematococcus pluvialis as follows: the time for the enzymolysis reaction is 0.876h, the enzyme concentration in the enzymolysis system is 2.709%, the pH value of the solution in the enzymolysis process is 8.61, the reaction temperature in the enzymolysis system is 40.357 ℃, and the predicted value of the antioxidant capacity of the haematococcus pluvialis polypeptide is 165.978. In the verification process, the experimental conditions are adjusted according to actual operation: the time for the enzymolysis reaction is 53min, the enzyme concentration in the enzymolysis system is 2.7%, the pH of the solution in the enzymolysis process is 8.6, and the reaction temperature in the enzymolysis system is 40 ℃. Experiments are carried out by adopting the optimized haematococcus pluvialis enzymolysis technology, and the average value after 3 times of parallel experiments is taken to measure that the value of the total antioxidant capacity of the haematococcus pluvialis polypeptide in vitro is 171.65 and is close to the predicted value. Therefore, the curved surface response method is used for optimizing the enzymolysis and extraction of the haematococcus pluvialis antioxidant peptide, and the haematococcus pluvialis proteolytic enzyme product with optimal antioxidant capacity is obtained.

Example 5: anti-inflammatory and anti-aging experiments on enzymatic products of haematococcus pluvialis extract

The enzymatic Hydrolysate (HPE) of haematococcus pluvialis extract obtained using the optimal production conditions of example 4 was tested by the following steps:

1. high sugar DMEM medium containing 10% FBS (Gibico high quality fetal bovine serum) and 2% Green streptomycin was used at 5% CO ₂ Culturing Raw 264.7 cells and HFF cells in a 37 ℃ environment;

2. taking mouse macrophages in log phase (Raw 264.7 cells) and adjusting the density to 2 x 10 ⁵ Inoculating 1mL of each well into a 12-well plate, discarding the supernatant after culturing for 24 hours (the cell fusion degree is 60% -75%), adding 1mL of culture solution into a blank control group, adding 1mL of culture solution containing 1 mug/mL of Lipopolysaccharide (LPS) into a negative control group, adding 1mL of culture solution containing 100 mug/mL of dexamethasone into a positive control group, adding 100 mug of culture solution containing 0.5 volume percent and 1% of HPE into a sample group, continuously culturing for 24 hours, taking the supernatant, setting 2 compound wells in each group, and detecting the NO content by using an ELISA kit (Beyotime, china);

3. taking human foreskin fibroblasts in logarithmic growth phase (HFF cells) and adjusting the density to 1 x 10 ⁵ Inoculating 500 mu L of each well into a 24-well plate, discarding supernatant after culturing for 24 hours (cell fusion degree 60% -75%), adding 100 mu L of culture solution containing 0.1 mu g/mL TGF-beta 1 into a positive control group, adding 100 mu L of culture solution containing 0.5% HPE by volume fraction into a sample group, adding 100 mu L of culture solution into the rest groups, placing into an ultraviolet therapeutic instrument, and irradiating at a dose of 2-5J/cm ² Irradiating for 15min. After the irradiation was completed, the supernatant was discarded, the cells were washed with PBS 1 to 2 times, the sample addition was repeated, and after further culturing for 24 hours, the supernatant was taken and 2 wells were multiplexed into each group, and the type I Collagen (Collagen I, CO I) content was detected using ELISA kit (Lianke, china).

The results show that: as shown in fig. 7A, after induction by LPS, the expression of inflammatory factor NO in Raw 264.7 cells was reduced (p < 0.05) by dexamethasone (positive control), and HPE, indicating that 0.5% volume fraction of HPE significantly (p < 0.05) reduced the expression of NO inflammatory factor in Raw 264.7 cells, and the HPE obtained in the present application had significant soothing and barrier repairing effects; as shown in fig. 7B, after damage by UVA irradiation, the content of CO i in HFF cells was reduced, and after TGF- β (positive control) and HPE action, the content of CO i was increased (p < 0.05), indicating that 5% volume fraction of HPE was able to significantly (p < 0.01) promote CO i secretion in HFF cells, repair cell damage caused by UVA irradiation, indicating that HPE obtained in the present application has significant anti-inflammatory and anti-aging effects.

Example 6: application of enzymolysis product of haematococcus pluvialis extract

The enzymolysis product (HPE) of haematococcus pluvialis extract obtained under the optimal production conditions of the embodiment 4 is added into cosmetics or skin care products to obtain the beneficial effects of relieving, anti-inflammatory, brightening skin, repairing barrier, delaying aging and the like. The cosmetic or skin care product may further comprise: one or more of moisturizer, emulsifier, thickener, preservative or ingredient with antiseptic and antibacterial effects, solvent, skin conditioner, solubilizer, film forming agent, emollient, colorant, essence, chelating agent and antioxidant.

The humectant includes, but is not limited to, at least one of butylene glycol, glycerin, 1, 3-propanediol, pentylene glycol, isopentylene glycol, diglycerol, triglycerol, polyglycerol, sorbitol, polyethylene glycol, polypropylene glycol, ethylene glycol, or diethylene glycol.

The emulsifier includes, but is not limited to, at least one of stearic acid, cetostearyl alcohol, cetostearyl glucose, cetostearyl alcohol ethyl hexanoate, stearyl polyether-20, potassium cetyl phosphate, lanolin wax, or coconut oil PEG-10 esters.

The thickener includes, but is not limited to, one or a combination of several of C14-C22 alcohol, C12-C20 alkyl glucoside, carbomer, xanthan gum, gellan gum, gum arabic, sclerotium gum, hydroxymethyl cellulose, hydroxyethyl cellulose, acrylic acid (ester)/C10-30 alkanol acrylate cross-linked polymer, polyacrylate-13, ammonium acrylate and acrylamide copolymer, hydroxyethyl acrylate, sodium acryloyldimethyl taurate copolymer, sodium polyacrylate, ammonium acryloyldimethyl taurate/VP copolymer, or ammonium acryloyldimethyl taurate/behenate-25 methacrylate cross-linked polymer.

The preservative comprises at least one of methyl hydroxybenzoate, ethyl hydroxybenzoate, propyl hydroxybenzoate, phenoxyethanol, chlorpheniramine, potassium sorbate, sodium benzoate, benzoic acid or imidazolidinyl urea.

The antioxidants include, but are not limited to, rutin (flavone), quercetin (flavone), hesperidin (flavone), dioscin (flavone), mangiferin (flavone), mangosteen (flavone), anthocyanin (carotenoid), astaxanthin (carotenoid), lutein (carotenoid), lycopene (carotenoid), carotene (carotenoid), resveratrol (polyphenol), tetrahydrocurcumin (polyphenol), rosmarinic acid (polyphenol), ellagic acid (polyphenol), hypericin (polyphenol), chlorogenic acid (polyphenol), oleuropein (polyphenol), lipoic acid (disulfide), glutathione oxidation (disulfide), cystine (disulfide), N-acetylcystine (disulfide), reduced glutathione (mercapto), cysteine (mercapto) and N-acetylcysteine (mercapto).

The solubilizing agents include, but are not limited to, glycol ethers, benzalkonium chloride, benzethonium chloride, benzyl alcohol, benzyl benzoate, cetylpyridinium chloride, cyclodextrin, glyceryl monostearate, hypromellose, inulin, lecithin, meglumine, nonionic emulsifying waxes, phospholipids, poloxamers, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, polyoxylglycerides, povidone, pyrrolidone, sodium bicarbonate, sorbitan esters, starch, stearic acid, sulfobutyl ether-cyclodextrin, glyceryl tricaprylate, glyceryl trioleate, vitamin E polyethylene glycol succinate, or mixtures thereof.

The skin conditioning agents include, but are not limited to, polymethacrylates, polymethylvinyl esters, polyamides, modified celluloses, starches.

Such colorants include, but are not limited to, inorganic colorants (e.g., titanium dioxide, iron oxide, titanated mica, iron oxide coated mica, ultramarine, chromium oxide, chromium hydroxide, manganese violet, bismuth oxychloride, guanine and aluminum) organic colorants (e.g., ferric ammonium ferrocyanide). The colorant may also include one or more pigments; these pigments may be white or colored, as well as inorganic or organic; examples of the inorganic pigment include titanium dioxide, zirconium oxide and cerium oxide, which are optionally surface-treated, and iron oxide and chromium oxide, manganese violet, ultramarine blue, chromium hydrate and ferric blue, and metallic pigments such as aluminum and bronze; examples of organic pigments include carbon black, D & C type pigments, and lakes based on coix seed pigment, barium, strontium, calcium, aluminum, and guanine, etc.

The enzymatic Hydrolysate (HPE) of haematococcus pluvialis extract obtained under the optimal production conditions of example 4 may be added to a cosmetic or skin care product while being compounded with an organic solvent and/or an inorganic solvent, wherein the organic solvent may be selected from hydrophilic organic solvents, lipophilic organic solvents, and amphiphilic solvents, and mixtures thereof.

The hydrophilic organic solvents include, but are not limited to, straight or branched chain lower monohydric alcohols containing 1 to 8 carbon atoms, such as ethanol, propanol, butanol, isopropanol, isobutanol; acetone; polyethylene glycol containing 6-80 alkenyloxy units, polyhydric alcohols such as propylene glycol, butylene glycol, glycerol or sorbitol; esters such as ethyl acetate and methyl acetate, wherein the alkyl group contains 1 to 5 carbon atoms of-or dialkyl isosorbides, for example dimethyl isosorbide, for example diethylene glycol monomethyl ether or monoethyl ether and propylene glycol ethers such as dipropylene glycol methyl ether. The lipophilic organic solvents include, but are not limited to, hydrocarbons such as: hexane, heptane and octane; fatty acid esters such as diisopropyl adipate and dioctyl adipate; alkyl benzoate and dioctyl malate. Other organic solvents include, but are not limited to, polyols, such as: polypropylene Glycol (PPG) derivatives, such as: polypropylene glycol esters of fatty acids, and PPG ethers of fatty alcohols, such as: PPG-36 oleate and PPG-23 oleyl ether.

The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.

The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims

1. An enzymolysis method of haematococcus pluvialis extract, which is characterized by comprising the following steps:

adding protease into the algae protein solution prepared by haematococcus pluvialis;

based on a Box-Behnken response surface method, taking the total in-vitro antioxidant capacity as a response value, selecting four response variables of enzyme addition amount, enzymolysis pH, enzymolysis temperature and enzymolysis time, taking the optimal point of a single factor experiment as a center, taking a horizontal value around the center up and down as a response surface level, and performing multiple regression analysis on the response variable data to obtain a regression equation:

2. The enzymatic hydrolysis method according to claim 1, wherein the protease is selected from neutral protease and trypsin.

3. The enzymolysis method according to claim 1, wherein the preparation method of haematococcus pluvialis extract comprises the following steps:

4. The method according to claim 1, wherein the enzyme addition amount is 0.1%, the enzyme hydrolysis temperature is 50 ℃, the enzyme hydrolysis time is 4 hours, and the enzyme hydrolysis pH is 6.5, 7.0, 7.5, 8.0, 8.5 or 9.0.

5. The method according to claim 1, wherein the enzyme addition amount is 0.1%, the enzyme hydrolysis pH is 7.0, the enzyme hydrolysis time is 4 hours, and the enzyme hydrolysis temperature is 30 ℃, 40 ℃, 50 ℃ or 60 ℃.

6. The method according to claim 1, wherein the enzyme addition amount is 0.1%, the enzyme hydrolysis pH is 7.0, the enzyme hydrolysis temperature is 50 ℃, and the enzyme hydrolysis time is 0.5h, 1h, 1.5h, 2h, 3h, 4h or 5h.

7. The method according to claim 1, wherein the enzymatic hydrolysis pH is 7.0, the enzymatic hydrolysis temperature is 50 ℃, the enzymatic hydrolysis time is 4 hours, and the enzyme addition amount is 0.1%, 0.4%, 1.0%, 2.0%, 3.0% or 4.0%.

8. The method according to claim 1, wherein the protease is added to the algae protein solution for enzymolysis, the enzyme addition amount is 2.7%, the enzymolysis pH is 8.6, the enzymolysis temperature is 40 ℃, the enzymolysis time is 53min, after the completion, the algae protein solution is inactivated for 10min under the water bath condition of 90 ℃, and the algae protein solution is cooled to room temperature, centrifuged to obtain a supernatant, and an enzymolysis product is obtained by filtration.

9. Use of the enzymatic hydrolysate obtained by the enzymatic hydrolysis method according to any of claims 1-8 for the preparation of skin care products or cosmetics.

10. Use of the enzymatic hydrolysate obtained by the enzymatic hydrolysis process of any of claims 1-8 for the preparation of an antioxidant or anti-aging agent.