CN108517343B

CN108517343B - Preparation method of porphyra yezoensis antioxidant protein peptide

Info

Publication number: CN108517343B
Application number: CN201810385544.2A
Authority: CN
Inventors: 黄君阳
Original assignee: Xiamen Yuanzhidao Biotech Co ltd
Current assignee: Xiamen Yuanzhidao Biotech Co ltd
Priority date: 2018-04-26
Filing date: 2018-04-26
Publication date: 2021-02-02
Anticipated expiration: 2038-04-26
Also published as: CN108517343A

Abstract

The invention provides a preparation method of porphyra yezoensis antioxidant protein peptide, which comprises the following steps: carrying out crushing treatment, water extraction and sterilization treatment on porphyra yezoensis serving as a raw material to obtain a leaching liquor; inoculating lactobacillus to the obtained leaching liquor for fermentation, and sterilizing the mixed liquor after fermentation; carrying out enzymolysis treatment on the sterilized mixed solution by adopting papain; after enzymolysis, carrying out inactivation treatment, cooling, carrying out centrifugal separation, and collecting supernatant; and filtering the collected supernatant, and drying to obtain the porphyra yezoensis antioxidant protein peptide. According to the preparation method of the porphyra yezoensis antioxidant protein peptide, provided by the invention, the fermentation of lactic acid bacteria and the enzymolysis method can be combined, so that a peptide product which is free of bitter taste, has a fermentation flavor and is good in palatability can be prepared; the removal capability of the prepared antioxidant protein peptide on OH free radicals and DPPH free radicals can be greatly improved; has positive influence on the increase of the added value of the laver.

Description

Preparation method of porphyra yezoensis antioxidant protein peptide

Technical Field

The invention relates to the technical field of biological application, in particular to a preparation method of porphyra yezoensis antioxidant protein peptide.

Background

Porphyra yezoensis is marine edible algae and widely exists in the field of Jiangsu coastal areas. Laver is nutritious, usually has high protein content, and is several times higher than that of kelp. The laver protein is a high-quality protein, has been widely noticed by people due to its high content and unique biological function, and is a research hotspot in the development and utilization of laver.

The laver protein peptide is used as a main active substance of laver protein, and has various physiological effects of reducing blood pressure, resisting bacteria, tumors and oxidation, participating in the regulation of immunity of organisms and the like. The laver protein peptide can be prepared by enzyme hydrolysis method and fermentation method.

In the prior art, the method takes Jiangsu coastal porphyra yezoensis as a raw material, and prepares laver protein antibacterial peptide and a laver alpha-glucosidase inhibitor by enzymolysis under different conditions; the porphyra haitanensis is also taken as a raw material, the porphyra haitanensis protein is hydrolyzed by trypsin to prepare porphyra haitanensis protein peptide, and the in vitro experiment verifies that the porphyra haitanensis protein peptide obtained by the enzymolysis of the optimal process has good activity of reducing blood pressure, cholesterol and oxidation resistance; and the method also comprises the step of separating and purifying laver enzymolysis products by using an enzymolysis-membrane separation technology by taking porphyra yezoensis as a raw material to prepare the antioxidant protein peptide.

As can be seen from the prior art, the method for preparing the antioxidant protein peptide by enzymolysis is common, the enzymolysis method has the advantages of low factory cost, mild conditions and easily controlled hydrolysis process, but the peptide produced by the enzymolysis method has heavier bitter taste and poor palatability, and the hydroxyl radical and DPPH clearance of the prepared protein peptide are lower.

In the prior art, a method for preparing the antioxidant protein peptide by a fermentation method is adopted, compared with an enzymolysis method, the yield of the peptide prepared by the fermentation method is not high, and the removal rate of the prepared protein peptide on removing OH free radicals and DPPH free radicals is also low.

Disclosure of Invention

In order to solve the problems in the background art, the invention provides a preparation method of porphyra yezoensis antioxidant protein peptide, which comprises the following steps:

s100, carrying out crushing treatment, water extraction and sterilization treatment on porphyra yezoensis serving as a raw material to obtain a leaching solution;

s200, inoculating lactic acid bacteria to the obtained leaching liquor for fermentation, wherein the inoculation amount of the lactic acid bacteria is 1% -3%, the fermentation temperature is 35-40 ℃, the fermentation time is 20-36 hours, and after fermentation, sterilizing the mixed liquor;

s300, carrying out enzymolysis treatment on the sterilized mixed solution by using papain, wherein the addition amount of the papain is 0.5-2% of the mass of the porphyra yezoensis, the enzymolysis temperature is 55-65 ℃, and the enzymolysis reaction time is 2-4 hours; after enzymolysis, carrying out inactivation treatment, cooling, carrying out centrifugal separation, and collecting supernatant;

s400, filtering the collected supernatant, and drying to obtain the porphyra yezoensis antioxidant protein peptide.

Further, in S100, the pulverization treatment, the water extraction, and the sterilization include: crushing porphyra yezoensis, mixing the crushed porphyra yezoensis with water according to the material-water ratio of 1: 20-1: 50, and leaching the porphyra yezoensis for 20-30 min after stirring treatment; the unit of the material-water ratio is g/mL;

treating the obtained leaching liquor at 115-125 ℃ for 10-30 min to kill pathogenic bacteria, and cooling to 30-40 ℃ for later use.

Further, in step S200, the lactic acid bacteria include at least one of lactobacillus bulgaricus, lactobacillus fermentum, lactobacillus helveticus, and lactobacillus plantarum.

Further, in step S200, before inoculating the leaching liquor, lactobacillus is inoculated in MRS medium and activated for 10-12 hours at 37 ℃ with the rotation speed of the shaker being 200 rpm.

Further, the MRS culture medium comprises the following components: 10g/L of casein peptone, 5g/L of beef extract, 5g/L of yeast extract, 5g/L of glucose, 5g/L of sodium acetate, 2g/L of diamine citrate, 801g/L of tween, 2g/L of dipotassium phosphate, 0.2g/L of magnesium sulfate heptahydrate, 0.05g/L of manganese sulfate heptahydrate and 20g/L of calcium carbonate;

the pH value of the MRS culture medium is 6.8, and the MRS culture medium is sterilized at 115 ℃ for 30-40 min.

Further, after enzymolysis is carried out for 2-4 hours, controlling the temperature at 0-5 ℃, flushing nitrogen into the reaction chamber, adjusting the air inflow by a pressure reducing valve for 2 liters/hour, inflating for 2 hours, and controlling the pressure of the reaction chamber at 1.5 bar; ultrasonic treatment is adopted at the same time, and ultraviolet light is used for 12-15 min; after the ultraviolet light treatment is finished, continuously introducing nitrogen gas, controlling the pressure of the reaction chamber to be 1.0bar, raising the temperature to be below 100 ℃, and inactivating for 15 min; after natural cooling, centrifuging for 15 minutes at 5000r/min, and collecting supernatant.

Further, in S400, the method for filtering the supernatant comprises: loading into a chromatographic column of phi 1.0 × 150cm, filtering with macroporous adsorbent resin HPD400A, removing impurities with ultrafiltration membrane with cut-off molecular weight of 10Ka, and collecting filtrate.

Further, the drying method in S400 includes: drying the concentrated solution into powder by adopting a vacuum drying oven; the concentration and drying temperature is 55-60 ℃, and the drying time is 10-15 h.

According to the technical scheme provided by the invention, the fermentation and enzymolysis method of the lactic acid bacteria can be combined, the steps of separating and purifying the enzyme are omitted by one step of producing the enzyme and synthesizing the enzyme by enzymolysis of the lactic acid bacteria, and the terminal peptidase generated by the metabolism of the lactic acid bacteria can modify and recombine the tail end of the small peptide to rearrange and transfer the amino acid, so that a peptide product without bitter taste and with fermentation flavor can be prepared, and the palatability is good; the removal capability of the prepared antioxidant protein peptide on OH free radicals and DPPH free radicals can be greatly improved; the invention adopts edible lactobacillus fermentation combined with an enzymolysis method to produce the laver antioxidant protein peptide product, and has positive influence on developing laver health food and improving the added value of the laver.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed to be used in the description of the embodiments or the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.

FIG. 1 is a graph showing the relationship between hydroxyl radical scavenging rate and concentration of Porphyra yezoensis protein peptide prepared by the method of the present invention;

FIG. 2 is a graph showing the relationship between DPPH.radical scavenging rate and concentration of Porphyra yezoensis protein peptide prepared by the method of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention. 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.

The invention provides a preparation method of porphyra yezoensis antioxidant protein peptide, which comprises the following steps:

s300, carrying out enzymolysis treatment on the sterilized mixed solution by adopting papain, wherein the addition amount of the papain is 0.5-2% of the mass of the porphyra yezoensis, the enzymolysis temperature is 55-65 ℃, and the enzymolysis reaction time is 2-4 hours; after enzymolysis, carrying out inactivation treatment, cooling, carrying out centrifugal separation, and collecting supernatant;

wherein the enzyme activity of the papain is 2 multiplied by 105U/g; the temperature of inactivation treatment is 100-105 ℃, and the treatment time is 15-20 min;

Wherein, in S100, the pulverization treatment, the water extraction and the sterilization comprise the following steps: crushing porphyra yezoensis, sieving by using a 40-mesh sieve, and mixing the porphyra yezoensis with water according to a material-water ratio of 1: 20-1: 50, wherein the unit is g/mL; leaching after stirring for 20-30 min; after leaching, the obtained leaching liquor is treated for 10min to 30min at the temperature of 115 ℃ to 125 ℃ to kill pathogenic bacteria, and then is cooled to 30 ℃ to 40 ℃ for standby.

In step S200, the Lactobacillus may be Lactobacillus strains listed in "list of strains available for food" (No. 2010) 65 issued by ministry of health), including but not limited to at least one or more combinations of Lactobacillus bulgaricus (Lactobacillus bulgaricus), Lactobacillus fermentum (Lactobacillus), and Lactobacillus helveticus (Lactobacillus helveticus).

Inoculating the lactic acid bacteria to a fresh MRS culture medium before inoculating the leaching liquor, and activating for 10-12 hours at the culture temperature of 37 ℃ and the rotating speed of a shaking table of 200 revolutions per minute.

Wherein, the MRS culture medium comprises the following components: 10g/L of casein peptone, 5g/L of beef extract, 5g/L of yeast extract, 5g/L of glucose, 5g/L of sodium acetate, 2g/L of diamine citrate, 801g/L of tween, 2g/L of dipotassium phosphate, 0.2g/L of magnesium sulfate heptahydrate, 0.05g/L of manganese sulfate heptahydrate and 20g/L of calcium carbonate;

In S300, preferably, after 2-4 hours of enzymolysis, controlling the temperature at 0-5 ℃, flushing nitrogen into the reaction chamber, adjusting the air inflow by using a pressure reducing valve for 2 liters/hour, inflating for 2 hours, and controlling the pressure of the reaction chamber to be 1.5 bar; ultrasonic treatment is adopted at the same time, and ultraviolet light is used for 12-15 min; after the ultraviolet light treatment is finished, continuously introducing nitrogen gas, controlling the pressure of the reaction chamber to be 1.0bar, raising the temperature to be below 100 ℃, and inactivating for 15 min; after natural cooling, centrifuging for 15 minutes at 5000r/min, and collecting supernatant.

According to the preferred scheme, ultraviolet light is adopted for processing in the enzymolysis process, so that cell walls of the laver can be better destroyed, proteins are further dissolved, and the preparation efficiency is improved; on the other hand, when papain is adopted for enzymolysis reaction, ultraviolet light can provide energy to promote the enzymolysis reaction of the papain to be more complete, so that the side chain of amino acid is more fully exposed; however, the use of uv light has many problems, and improper use thereof causes hydrolysis of peptide chains, thereby reducing the yield and quality of the product. In the step, nitrogen is firstly used as inert protection under the condition of low temperature to prevent a peptide chain from being oxidized, ultrasonic treatment is adopted to enable the nitrogen to be better attached to the periphery of protein, and then ultraviolet light is used for irradiation; in the process, the temperature, the nitrogen consumption and the ultraviolet light treatment time are controlled, so that the ultraviolet light improves the enzymolysis reaction of the papain, the product yield and the removal capacity of OH free radicals and DPPH free radicals, and simultaneously, the ultraviolet light is ensured not to generate adverse effects.

In S400, the method of filtering the supernatant includes: loading into a chromatographic column of phi 1.0 × 150cm, filtering with macroporous adsorbent resin HPD400A, removing impurities with ultrafiltration membrane with cut-off molecular weight of 10Ka, and collecting filtrate.

In S400, the drying method includes: drying the concentrated solution into powder by adopting a vacuum drying oven, wherein the model of the vacuum drying oven is DZG-6020, the concentration drying temperature is 55-60 ℃, and the drying time is 10-15 h, preferably 12 h.

The present invention provides the following examples:

example one

S100, taking 20g of porphyra yezoensis, crushing, sieving by using a 40-mesh sieve, and adding 500mL of distilled water for uniformly mixing; leaching under stirring for 20 min; after leaching, treating the leaching liquor at 121 ℃ for 10min to kill pathogenic bacteria, and then cooling to 40 ℃ for later use;

s200, inoculating lactobacillus bulgaricus to a fresh MRS culture medium, activating for 12 hours, and controlling the culture temperature to be 37 ℃ and the rotating speed of a shaking table to be 200 r/min;

inoculating the activated culture solution into the leaching liquor prepared by S100; wherein the inoculation amount of the lactobacillus bulgaricus is 3 percent, the fermentation temperature is 37 ℃, and the fermentation time is 36 hours; the mixed solution after fermentation is subjected to heat treatment at 90 ℃ for 10 minutes.

S300, adding 0.3g of papain (2 multiplied by 105U/g), wherein the enzymolysis temperature is 60 ℃, and the enzymolysis reaction is 4 hours; inactivating at 100 deg.C for 15min after enzymolysis; naturally cooling, centrifuging at 5000r/min for 15min, and collecting supernatant;

s400, filling a chromatographic column with the specification of phi 1.0 multiplied by 150cm into treated macroporous adsorption resin HPD400A, carrying out suction filtration on the clear liquid, removing impurities from the clear liquid by using an ultrafiltration membrane with the molecular weight cutoff of 10Ka, and collecting filtrate; drying the concentrated solution at 55 deg.C for 12h in a DZG-6020 vacuum drying oven, controlling the water content of the final product to 3%, taking out the final product, pulverizing, and sieving with 80 mesh sieve to obtain Porphyra yezoensis protein peptide powder.

Example two

s200, inoculating lactobacillus fermentum to a fresh MRS culture medium, activating for 12 hours, and controlling the culture temperature to be 37 ℃ and the rotating speed of a shaking table to be 200 r/min;

inoculating the activated culture solution into the leaching liquor prepared by S100; wherein the inoculation amount of the lactobacillus fermentum is 3 percent, the fermentation temperature is 37 ℃, and the fermentation time is 36 hours; the mixed solution after fermentation is subjected to heat treatment at 90 ℃ for 10 minutes.

EXAMPLE III

S300, adding 0.3g of papain (2 multiplied by 105U/g), wherein the enzymolysis temperature is 60 ℃, and the enzymolysis reaction is 4 hours; controlling the temperature at 0 ℃, flushing nitrogen into the reaction chamber, adjusting the air inflow by a pressure reducing valve for 2 liters/hour, inflating for 2 hours, and controlling the pressure of the reaction chamber to be 1.5 bar; simultaneously, ultrasonic treatment is adopted, and ultraviolet light is used for 12 min;

after the ultraviolet treatment is finished, continuously introducing nitrogen into the reaction chamber, controlling the pressure of the reaction chamber to be 1.0bar, raising the temperature to be below 100 ℃, and inactivating for 15 min; naturally cooling, centrifuging at 5000r/min for 15min, and collecting supernatant;

The first example is that lactobacillus bulgaricus is adopted as the fermented lactic acid bacteria, the second example is that lactobacillus fermentum is adopted as the fermented lactic acid bacteria, the third example is a further optimization scheme on the basis of the second example, and the porphyra yezoensis protein peptides prepared in the first, second and third examples are subjected to the following physicochemical property and oxidation resistance analysis.

(1) Determination and analysis of protein peptide yield

The yield Y (%) of the protein peptide is calculated by taking the mass of the protein peptide powder finally obtained by vacuum drying and the mass percentage of the dried laver powder as a calculation method, and the yield of the laver antioxidant protein peptide is tested by taking the porphyra yezoensis protein peptide prepared by adopting a fermentation method in the prior art as a comparative example I, and the result is shown in table 1.

TABLE 1

Name (R)	Example one	Example two	EXAMPLE III	Comparative example 1
					Yield of laver protein peptide(％)	14.28	15.21	19.82	11.01

As can be seen from Table 1, the yield of the laver antioxidant protein peptide prepared by the enzymolysis method by combining lactic acid bacteria and papain is obviously improved compared with the antioxidant protein peptide prepared by the fermentation method in the prior art; by comparing the first and second examples, it can be seen that the yield of protein peptide produced by lactobacillus fermentum is higher than the yield of protein peptide produced by lactobacillus bulgaricus; in the third example, the yield is obviously improved by improving the process.

(2) Measurement and analysis of amino acid composition of Porphyra yezoensis protein peptide

Amino acid composition and content were measured according to GB/T5009.124-2003 "determination of amino acids in food", Porphyra yezoensis protein peptide was treated by hydrochloric acid hydrolysis method and then measured by an amino acid autoanalyzer, and the measurement results are shown in Table 2:

TABLE 2

Note: the symbol indicates essential amino acids,. DELTA.indicates taste-imparting amino acids, "-" indicates no detection

As can be seen from Table 2, in the porphyra yezoensis protein, the total amount of amino acids is 28.46%, the contents of umami amino acids such as glutamic acid, aspartic acid and the like are higher, and the total amount of 8 flavor development amino acids such as aspartic acid, serine, glutamic acid, proline, glycine, alanine, phenylalanine, tyrosine and the like is 15.85%, which accounts for 55.69% of the total amount of the amino acids, so that the porphyra is delicious in taste, and the contents of the flavor development amino acids are increased through a fermentation enzymolysis process;

wherein 8 flavor amino acids in the lactobacillus bulgaricus fermented zymolyte account for 60.75 percent of the total amount of the amino acids, and 8 flavor amino acids in the lactobacillus fermentum fermented zymolyte account for 59.33 percent of the total amount of the amino acids, and the content is improved to a certain extent, which indicates that the taste of the laver is further improved by the lactobacillus fermentation enzymolysis method.

It can also be seen from table 2 that, although the content of amino acids such as aspartic acid, glycine and glutamic acid is increased after the fermentation and enzymolysis of lactic acid bacteria, the content of amino acids such as leucine, histidine and arginine is reduced to a certain extent, which indicates that in the fermentation and enzymolysis process of lactic acid bacteria, lactic acid bacteria degrade and utilize the nitrogen source in part of amino acids, secrete a large amount of enzyme to destroy the cell wall of laver, dissolve intracellular proteins, and degrade the intracellular proteins into laver protein peptides under the synergistic action of papain. As can be seen from Table 2, the total amount of amino acids increased after fermentation, indicating that the lactic acid bacteria produce enzymes during decomposition and fermentation of Porphyra yezoensis, and the enzymes can effectively degrade macromolecular proteins, saccharides and the like and convert into amino acids and other substances, thereby improving the nutritive value.

(3) Measurement of hydroxyl radical (. OH) scavenging ability of Porphyra yezoensis protein peptide

Preparing the porphyra yezoensis protein peptide prepared in the step 4 into a solution to be detected with the concentration gradients of 0.05, 0.1, 0.15, 0.2 and 0.5mg/mL, taking 1mL of the solution to be detected, and sequentially adding 1mL of FeSO with the concentration of 9mM₄Solution and 6mM H₂O₂The solution was mixed with a shaker for 10 minutes, followed by addition of 1mL of 6mM salicylic acid solution, mixing with a shaker, standing at room temperature for 30 minutes, and then measuring the absorbance at 510nm, and recording the value.

The clearance effect of the protein peptide on OH free radicals is expressed by clearance rate, and the clearance rate is calculated by the following formula:

clearance ═ 1- (sample absorbance/blank absorbance) ] × 100%;

the results are shown in FIG. 1, which is based on the different graphs of the removal rate of hydroxyl radical by porphyra yezoensis protein peptide solution with different concentrations.

As can be seen from FIG. 1, the Porphyra yezoensis protein peptide prepared by the method has the capability of scavenging hydroxyl radicals.

Within the mass concentration range of the researched enzymolysis products (namely the porphyra yezoensis antioxidant protein peptide), the clearance rate of the peptide prepared by combining lactobacillus bulgaricus and lactobacillus fermentum with the enzymolysis method is increased along with the increase of the mass concentration of the enzymolysis products, and the clearance rate is in positive correlation. When the mass concentration of the enzymolysis product is 0.15mg/mL, the clearance rates of hydroxyl radicals of the porphyra yezoensis protein peptide prepared by the fermentation enzymolysis method of lactobacillus bulgaricus and lactobacillus fermentum are 45.89% and 54.36% respectively. When the mass concentration of the enzymolysis product is 0.5mg/mL, the removal rate of hydroxyl radicals of porphyra yezoensis protein peptide prepared by the fermentation enzymolysis method of lactobacillus fermentum is 93.25%.

The patent with application number 201110362654.5 discloses a method for purifying, analyzing and identifying antioxidant active peptide after controllable enzymolysis of laver, laver antioxidant protein peptide prepared by the method is used as a comparative example II, antioxidant protein peptide prepared by the patent with application number 201410533079.4 is used as a comparative example III, the clearance rate of hydroxyl radical is tested, and the results are shown in the following table:

TABLE 3

Concentration of	Example one	Example two	EXAMPLE III	Comparative example No. two	Comparative example No. three
						0.15mg/mL	45.89％	54.36％	60.13％	/	/
0.5mg/mL	89.12％	93.25％	96.57％	/	/
						1.0mg/mL	/	/	/	/	20.26％
100mg/mL	/	/	/	47.12％	/

As can be seen from table 3, the examples provided by the present invention have a significantly improved effect compared to the comparative examples and comparative examples, in that the examples use lower concentrations but have higher hydroxyl radical scavenging rates at appropriate pH and temperature.

In addition, for accurate quantitative comparison of two porphyra yezoensis proteinsThe activity of the peptide scavenging hydroxyl radical is obtained by fitting a curve of the drawn clearance rate to the concentration of the substrate, and the mass concentration of the protein peptide solution, namely IC, when the clearance rate is 50 percent is calculated by a regression equation₅₀Value, IC₅₀The smaller the value, the stronger the capability of the enzymolysis liquid to remove hydroxyl radicals and the stronger the antioxidant activity.

The dose-effect relationship of porphyra yezoensis protein peptide prepared by lactobacillus bulgaricus and lactobacillus fermentum fermentation and enzymolysis method to hydroxyl free radical is respectively as follows:

Y_{lactobacillus bulgaricus}＝32.023ln(X)+109.55,R²0.9771, thus IC₅₀＝0.1557mg/mL；

Y_{Lactobacillus fermentum}＝33.796ln(X)+118.48,R²0.9938, thus IC₅₀＝0.1326mg/mL。

Lactobacillus bulgaricus IC₅₀Value 0.1557mg/mL, Lactobacillus fermentum IC₅₀The value was 0.1326mg/mL, from which it was found that the Porphyra yezoensis protein peptide produced by Lactobacillus fermentum has a higher ability to scavenge hydroxyl radicals than Lactobacillus bulgaricus, since the two lactic acid bacteria have different abilities to hydrolyze laver proteins, and the laver peptide produced by Lactobacillus fermentum has a higher ability to scavenge the reactive amino acid side chains.

(4) Measurement of DPPH-removing ability of Porphyra yezoensis protein peptide

Taking the porphyra yezoensis protein peptide prepared in the step 4, preparing a solution to be detected with the concentration gradient of 0.1, 0.3, 0.5, 0.8 and 1.0mg/mL, taking 1mL of the solution to be detected, adding 1mL0.04mg/mL of DPPH solution, oscillating and mixing uniformly, reacting for 30min, centrifuging for 10min at 3000r/mim, and taking supernatant; measuring the light absorption value at the wavelength of 517nm, and recording the value; the removal effect of the porphyra yezoensis protein peptide on DPPH free radical is expressed by the removal rate; the clearance calculation formula is as follows:

clearance ═ 1- (sample absorbance/blank absorbance) ] × 100%;

the results are shown in FIG. 2, which is a graph plotting the difference in DPPH.removing rate of Porphyra yezoensis protein peptide solutions of different concentrations.

As can be seen from FIG. 2, the prepared porphyra yezoensis protein peptide has scavenging ability to DPPH, and both of the scavenging ability of the two products are weaker than that of vitamin C in the experimental concentration range of 0.1mg/mL to 1.0 mg/mL. In the mass concentration range of the enzymatic hydrolysate, the DPPH clearance is gradually increased along with the increase of the mass concentration of the enzymatic hydrolysate. When the mass concentration of the enzymolysis product is 0.8mg/mL, the DPPH clearance of the porphyra yezoensis protein peptide prepared by the fermentation enzymolysis method of lactobacillus bulgaricus and lactobacillus fermentum is 70.05% and 81.33% respectively. The DPPH.multidot.protein peptide produced by fermentation and enzymolysis of Lactobacillus fermentum had a DPPH.multidot.clearance of 84.11% when the mass concentration of the enzymatic hydrolysate was 1.0 mg/mL.

The laver antioxidant protein peptide prepared in the patent application No. 201110362654.5 was used as a comparative example II to test the DPPH.radical scavenging rate, and the test results are shown in the following table:

TABLE 4

Concentration of	Example one	Example two	EXAMPLE III	Comparative example No. two
					0.8mg/mL	70.05％	81.33％	89.52％	/
1.0mg/mL	75.12％	84.11％	92.12％	/
					100mg/mL	/	/	/	47.04％

As can be seen from table 4, in comparison with the comparative example, the examples provided by the present invention have lower concentrations, but can achieve higher clearance of hydroxyl radicals, and have significant progress, under the condition that the respective pH and temperature are kept appropriate.

In order to accurately quantitatively compare the DPPH removal and activity of two porphyra yezoensis protein peptides, a curve of the drawn clearance rate and the concentration of a substrate is fitted, and the mass concentration of the protein peptide solution, namely IC when the clearance rate is 50 percent is calculated by a regression equation₅₀Value, IC₅₀The smaller the value, the stronger the DPPH.removing ability of the enzymolysis liquid and the stronger the antioxidant activity. The dose-effect relationship of porphyra yezoensis protein peptide prepared by the lactobacillus bulgaricus and lactobacillus fermentum fermentation and enzymolysis method to DPPH is as follows:

Y_{lactobacillus bulgaricus}＝20.708ln(X)+73.791,R²0.9852, thus IC₅₀＝0.3142mg/mL；

Y_{Lactobacillus fermentum}＝23.534ln(X)+85.679,R²0.9976, thus IC₅₀＝0.2196mg/mL。

Lactobacillus bulgaricus IC₅₀Value 0.3142mg/mL, Lactobacillus fermentum IC₅₀The value was 0.2196mg/mL, and it was found that DPPH.was more efficiently removed by the laver protein peptide produced by Lactobacillus fermentum than by Lactobacillus bulgaricus.

The combination of the experimental data and the conclusions shows that the laver protein peptides prepared by the two lactic acid bacteria have oxidation resistance, but the laver protein peptides prepared by the lactobacillus fermentum have stronger scavenging capability in scavenging OH free radicals and DPPH free radicals. The laver protein peptide prepared by fermentation has good inoxidizability, can be used as a bioactive substance to be applied to health food and food industry, and has wide application value.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A preparation method of porphyra yezoensis antioxidant protein peptide is characterized by comprising the following steps:

s400, filtering the collected supernatant, and drying to obtain porphyra yezoensis antioxidant protein peptide;

in S300: after 2-4 hours of enzymolysis, controlling the temperature at 0-5 ℃, flushing nitrogen into the reaction chamber, adjusting the air inflow by a pressure reducing valve for 2 liters/hour, inflating for 2 hours, and controlling the pressure of the reaction chamber at 1.5 bar; ultrasonic treatment is adopted at the same time, and ultraviolet light is used for 12-15 min; after the ultraviolet light treatment is finished, continuously introducing nitrogen gas, controlling the pressure of the reaction chamber to be 1.0bar, raising the temperature to be below 100 ℃, and inactivating for 15 min; naturally cooling, centrifuging at 5000r/min for 15min, and collecting supernatant;

in S400: the method for filtering the supernatant comprises the following steps: loading into adsorption resin HPD400A with chromatographic column of phi 1.0 × 150cm, vacuum filtering, removing impurities with ultrafiltration membrane with cut-off molecular weight of 10Ka, and collecting filtrate.

2. The method for preparing porphyra yezoensis antioxidant protein peptide according to claim 1, wherein in S100, the pulverization treatment, the water extraction and the sterilization comprise: crushing porphyra yezoensis, mixing the crushed porphyra yezoensis with water according to the material-water ratio of 1: 20-1: 50, and leaching the porphyra yezoensis for 20-30 min after stirring treatment; the unit of the material-water ratio is g/mL;

3. The method for preparing porphyra yezoensis antioxidant protein peptide according to claim 1, wherein the peptide comprises the following steps: in step S200, the lactic acid bacteria include at least one of lactobacillus bulgaricus, lactobacillus fermentum, lactobacillus helveticus, and lactobacillus plantarum.

4. The method for preparing porphyra yezoensis antioxidant protein peptide according to claim 1, wherein the peptide comprises the following steps: in step S200, before inoculating the leaching liquor, the lactobacillus is inoculated in an MRS culture medium and activated for 10-12 hours at the culture temperature of 37 ℃ and the rotation speed of a shaking table of 200 revolutions per minute.

5. The method for preparing porphyra yezoensis antioxidant protein peptide according to claim 4, wherein MRS medium comprises the following components: 10g/L of casein peptone, 5g/L of beef extract, 5g/L of yeast extract, 5g/L of glucose, 5g/L of sodium acetate, 2g/L of diamine citrate, 801g/L of tween, 2g/L of dipotassium phosphate, 0.2g/L of magnesium sulfate heptahydrate, 0.05g/L of manganese sulfate heptahydrate and 20g/L of calcium carbonate;

6. The method for preparing porphyra yezoensis antioxidant protein peptide according to claim 1, wherein the drying method in S400 comprises: drying the concentrated solution into powder by adopting a vacuum drying oven; the concentration and drying temperature is 55-60 ℃, and the drying time is 10-15 h.