CN111793666A - Tea seed polypeptide, enzymolysis preparation method thereof and antioxidant activity determination method - Google Patents
Tea seed polypeptide, enzymolysis preparation method thereof and antioxidant activity determination method Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A61P39/06—Free radical scavengers or antioxidants
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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Abstract
The invention relates to a tea seed polypeptide, an enzymolysis preparation method thereof and an antioxidant activity determination method, wherein the enzymolysis preparation method comprises the following steps: a pretreatment step; extracting tea seed protein; a tea seed polypeptide preparation step; and (3) measuring the content of the tea seed polypeptide. The method takes camellia seed meal as a raw material, firstly adopts complex enzyme to assist in extracting protein in the camellia seed meal, secondly adopts protease to hydrolyze tea seed protein to obtain micromolecule tea seed polypeptide, and on the basis of protease screening and single-factor influence determination experiments, takes the polypeptide yield as an index, designs a response surface test to optimize the process for preparing the tea seed polypeptide by hydrolyzing the tea seed protein with trypsin, selects optimal process parameters to ensure that the polypeptide yield reaches 74.99%, and shows that the polypeptide prepared by the enzymatic hydrolysis method has better oxidation resistance than the original tea seed protein through an antioxidant activity test result.
Description
Technical Field
The invention relates to the technical field of polypeptide preparation, and particularly relates to tea seed polypeptide, an enzymolysis preparation method of the tea seed polypeptide and an antioxidant activity determination method of the tea seed polypeptide.
Background
The Camellia seeds are mature seeds of evergreen small trees (Camellia oleifera Abel) in the genus of Camellia in the family of Theaceae, and the yield of the Camellia seeds in an aged tea garden, a sexual propagation tea garden and a group variety tea garden can be 60-100 ten thousand tons per year. The tea seed meal is a residue cake of tea seeds after oil extraction, and is widely used in the aspects of chemical industry, agriculture, food and the like. The main components of the camellia seed meal are polysaccharides, tea saponin, crude protein, starch, tea polyphenol and the like, and the tea seed protein can be used as a tryptophan nutrition enhancer to be applied to food.
Research shows that the polypeptide mixture obtained by hydrolyzing the protein raw material has better food processing performance and nutritive value than the raw material protein and amino acid mixture, and the polypeptide product generally has certain physiological health-care functions, such as oxidation resistance, tumor resistance, immunity enhancement and the like. However, the research on the polypeptide extraction and preparation process by using camellia seeds is still in the primary stage at present, the polypeptide yield is low, and the popularization and application are difficult
Disclosure of Invention
In order to solve the technical problems, the invention provides tea seed polypeptide, an enzymolysis preparation method and an antioxidant activity determination method thereof, and the tea seed polypeptide has the advantage of improving the yield of the polypeptide.
In order to achieve the purpose, the technical scheme of the invention is as follows:
an enzymolysis preparation method of tea seed polypeptide comprises the following steps:
a pretreatment step: degreasing camellia seed meal, drying and crushing the camellia seed meal at the temperature of 60 ℃, and sieving the camellia seed meal with a 60-mesh sieve to obtain camellia seed meal;
tea seed protein extraction: adding water into oil tea seed meal to prepare slurry, adding an enzyme preparation for enzymolysis, inactivating the enzyme for 5min by using boiling water, carrying out centrifugal treatment for 10min at the rotating speed of 8000rpm, adjusting the pH value of a supernatant to an isoelectric point, carrying out centrifugal treatment again, taking a precipitate, freeze-drying to obtain tea seed protein, and determining the content of the tea seed protein;
the tea seed polypeptide preparation step comprises: dissolving tea seed protein with deionized water, adjusting pH, adding protease, performing enzymolysis at constant temperature, inactivating enzyme with boiling water for 5min, centrifuging at 8000rpm for 10min, and collecting supernatant to obtain tea seed polypeptide liquid;
the method comprises the following steps of (1) tea seed polypeptide content determination: taking 5.0mL of tea seed polypeptide liquid, adding 5.0mL of 10% trichloroacetic acid aqueous solution, uniformly mixing, standing for 10min, centrifuging for 15min at the rotating speed of 4000rpm, removing protein and macromolecular long-chain peptide fragments, taking 6.0mL of supernatant, taking Gly-Gly-Tyr-Arg as a standard product, measuring the content of the tea seed polypeptide in the supernatant by adopting a biuret method, and calculating the polypeptide yield according to a standard formula.
In a preferred embodiment of the present invention, the enzyme preparation in the tea seed protein extraction step is alpha-amylase with a total enzyme addition amount of 300U/g: the laccase is 3: 2, the ratio of material to liquid is 1:25g/mL, the pH value is 5.0, the temperature in the enzymolysis treatment process is 35 ℃, and the enzymolysis treatment time is 60 min.
As a preferable scheme of the present invention, the determining the content of the tea seed protein in the tea seed protein extraction step specifically includes:
the method comprises the steps of accurately weighing dried tea seed protein, dissolving the dried tea seed protein with deionized water, transferring the tea seed protein into a 250mL volumetric flask, adding water to a constant volume, and measuring the concentration of the tea seed protein by adopting a Coomassie brilliant blue method and taking bovine serum albumin as a standard product.
As a preferable scheme of the invention, the standard formula in the tea seed polypeptide content determination step is as follows: the yield of the polypeptide is equal to the amount of the tea seed polypeptide in the hydrolysate per the mass of the tea seed protein multiplied by 100 percent.
In a preferred embodiment of the present invention, the method further comprises a single factor influence measurement step, wherein the single factor influence measurement step comprises: the method comprises the steps of protease screening, enzyme adding quantity comparison and measurement, reaction temperature comparison and measurement, pH comparison and measurement and reaction duration comparison and measurement.
As a preferred embodiment of the present invention, the protease screening step specifically includes: according to the operation mode in the tea seed polypeptide preparation step, carrying out enzymolysis treatment at the temperature of 40 ℃ by taking trypsin, papain, neutral protease, alkaline protease and acid protease as proteases in sequence, and determining the enzymolysis treatment effect of each protease by determining the polypeptide yield;
the enzyme adding amount comparison and determination step specifically comprises the following steps: and (3) fixing the ratio of material to liquid of 1:25, sequentially adding enzyme amounts of 100U/g, 300U/g, 500U/g and 900U/g under the conditions that the temperature is 60 ℃ and the pH value is 8.0, carrying out enzymolysis treatment for 3 hours, and determining the enzymolysis treatment effect of each enzyme amount by measuring the polypeptide yield;
the reaction temperature comparison determination step specifically comprises: and (3) fixing the ratio of material to liquid of 1:25, sequentially carrying out enzymolysis treatment for 3 hours at the conditions of 30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃ and 80 ℃ under the conditions of 700U/g of enzyme adding amount and 8.0 pH, and determining the enzymolysis treatment effect under each temperature condition by measuring the polypeptide yield;
the pH comparison determination step specifically comprises: and (3) fixing the ratio of material to liquid of 1:25, sequentially carrying out enzymolysis treatment for 3 hours under the conditions of the enzyme adding amount of 700U/g and the temperature of 60 ℃ and the conditions of pH values of 5, 6, 7, 8, 9 and 10, and determining the enzymolysis treatment effect of each pH value by measuring the polypeptide yield;
the step of measuring the reaction duration by contrast specifically comprises the following steps: and (3) fixing the ratio of material to liquid of 1:25, carrying out continuous 6h enzymolysis treatment under the conditions of 700U/g enzyme adding amount, 60 ℃ temperature and 8.0 pH value, sampling once every 1h, and determining the enzymolysis treatment effect in each reaction time length by measuring the polypeptide yield.
As a preferable embodiment of the present invention, the single factor influence determining step further comprises a four-factor three-level response surface test, and the four-factor three-level response surface test specifically comprises: and (3) carrying out a response surface test by taking the temperature, the reaction duration, the pH and the total enzyme addition amount as condition variables, and constructing a mathematical model between the condition variables and the response values and carrying out variance analysis by taking the polypeptide yield as a response value according to the test result.
As a preferred embodiment of the present invention, the four-factor three-level response surface test further comprises: and establishing a response surface graph and a contour graph between the response value and any two condition variables according to the test result.
On the other hand, the invention also provides a method for measuring the antioxidant activity of the tea seed polypeptide, which comprises the step of respectively carrying out DPPH free radical scavenging activity experiment, hydroxyl free radical scavenging activity experiment and ABTS free radical scavenging activity experiment on the tea seed polypeptide prepared by the preparation method of any one technical scheme.
On the other hand, the invention also provides a tea seed polypeptide prepared by the preparation method of any one of the technical schemes.
In summary, compared with the prior art, the invention has the following beneficial effects:
the embodiment of the invention provides a tea seed polypeptide, an enzymolysis preparation method thereof and an antioxidant activity determination method, wherein camellia seed meal is used as a raw material, complex enzyme is adopted to assist in extracting protein in the tea seed polypeptide, protease is adopted to hydrolyze tea seed protein to obtain micromolecule tea seed polypeptide, a response surface test is designed to optimize a process for preparing the tea seed polypeptide by performing enzymolysis on the tea seed protein through trypsin by taking the polypeptide yield as an index on the basis of a protease screening and single-factor influence determination experiment, optimal process parameters are selected to enable the polypeptide yield to reach 74.99%, and an antioxidant activity test result shows that the polypeptide prepared by the enzymatic hydrolysis method has better antioxidant property than original tea seed protein.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a graph showing the effect of different proteases on the proteolysis of tea seeds in a first embodiment of the present invention.
FIG. 2 is a graph showing the effect of different enzyme dosages on the yield of polypeptide in the first embodiment of the present invention.
FIG. 3 is a graph showing the effect of different enzymolysis temperatures on the yield of polypeptide in the first embodiment of the present invention.
FIG. 4 is a graph showing the effect of different pH on the yield of polypeptide in the first embodiment of the present invention.
FIG. 5 is a graph showing the effect of different enzymolysis durations on the yield of polypeptide in the first embodiment of the present invention.
FIG. 6 is a graph illustrating temperature and PH response surface analysis in accordance with an embodiment of the present invention.
FIG. 7 is an analysis chart of the response surface of temperature and enzyme dosage in the first embodiment of the present invention.
FIG. 8 is a graph of time versus pH response surface analysis in accordance with one embodiment of the present invention.
FIG. 9 is a graph showing the analysis of the response of enzyme addition amount to time in the first embodiment of the present invention.
FIG. 10 is a graph showing the analysis of the amount of enzyme added and the pH response surface in the first embodiment of the present invention.
Fig. 11 is an analysis graph of temperature and time response surface according to a first embodiment of the present invention.
FIG. 12 is a graph comparing the DPPH free radical clearance of tea seed proteins and tea seed polypeptides in one embodiment of the invention.
FIG. 13 is a graph comparing the hydroxyl radical scavenging rate of tea seed protein and tea seed polypeptide in the first embodiment of the present invention.
FIG. 14 is a graph comparing the ABTS free radical clearance rates of tea seed proteins and tea seed polypeptides in one embodiment of the invention.
Detailed Description
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 only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example one
An enzymolysis preparation method of tea seed polypeptide comprises the following steps:
s100, a preprocessing step: degreasing the camellia seed meal to enable the content of residual oil to be less than 1%, drying and crushing the camellia seed meal at the temperature of 60 ℃, and sieving the camellia seed meal with a 60-mesh sieve to obtain camellia seed meal for later use.
S200, tea seed protein extraction: adding water into oil tea seed meal to prepare slurry, adding an enzyme preparation for enzymolysis, inactivating enzyme with boiling water for 5min, centrifuging at the rotation speed of 8000rpm for 10min, adjusting the pH of the supernatant to the isoelectric point, centrifuging at the rotation speed of 8000rpm for 10min, taking out the precipitate, freeze-drying to obtain tea seed protein, and determining the content of the tea seed protein.
Wherein the enzyme preparation is alpha-amylase with the total enzyme adding amount of 300U/g: the laccase is 3: 2, the ratio of material to liquid is 1:25g/mL, the pH is 5.0, the temperature in the enzymolysis treatment process is 35 ℃, the enzymolysis treatment time is 60min, in the embodiment, the alpha-amylase is 2 ten thousand U/g, and the laccase is 2 ten thousand U/g.
The method for determining the content of the tea seed protein specifically comprises the following steps: the method comprises the steps of accurately weighing dried tea seed protein, dissolving the dried tea seed protein with deionized water, transferring the tea seed protein into a 250mL volumetric flask, adding water to a constant volume, and measuring the concentration of the tea seed protein by using bovine serum albumin as a standard substance by a Coomassie brilliant blue method, wherein the content of the obtained tea seed protein in the embodiment is 23.50%.
S300, tea seed polypeptide preparation: dissolving tea seed protein with deionized water, adjusting pH, adding protease, performing enzymolysis at constant temperature, inactivating enzyme with boiling water for 5min, centrifuging at 8000rpm for 10min, and collecting supernatant to obtain tea seed polypeptide liquid, wherein the pH value and the temperature of the enzymolysis at constant temperature are determined according to the selected protease, and the protease can be selected from neutral protease (2 ten thousand U/g), papain (10 ten thousand U/g), alkaline protease (10 ten thousand U/g), acidic protease (10 ten thousand U/g), trypsin (4000U/g), etc.
S400, tea seed polypeptide content determination: taking 5.0mL of tea seed polypeptide liquid, adding 5.0mL of 10% (w/v) trichloroacetic acid aqueous solution (TCA), uniformly mixing, standing for 10min, centrifuging for 15min at the rotating speed of 4000rpm, removing protein and macromolecular long-chain peptide fragments, taking 6.0mL of supernatant, placing the supernatant in another test tube, taking Gly-Gly-Tyr-Arg as a standard substance, measuring the content of the tea seed polypeptide in the supernatant by adopting a biuret method, and calculating the polypeptide yield according to a standard formula, wherein the standard formula is as follows: the yield of the polypeptide is equal to the amount of the tea seed polypeptide in the hydrolysate/the mass of the tea seed protein x 100%, and in this example, the content of the polypeptide is calculated by acid-soluble polypeptide.
S500, a single factor influence determination step, comprising: the method comprises the steps of protease screening, enzyme adding quantity comparison and measurement, reaction temperature comparison and measurement, pH comparison and measurement and reaction duration comparison and measurement.
The S501 protease screening step specifically comprises the following steps: according to the operation mode in the tea seed polypeptide preparation step, Trypsin (Trypsin), Papain (Papain), Neutral Protease (Neutral Protease), Alkaline Protease (alkali Protease) and Acid Protease (Acid Protease) are sequentially used as proteases to carry out enzymolysis treatment at the temperature of 40 ℃, and the pH value is adjusted according to the optimal conditions of each Protease during the enzymolysis treatment, wherein the optimal pH value of the Neutral Protease is 7.0, the optimal pH value of the Alkaline Protease is 10.5, the optimal pH value of the Trypsin is 7.5, the optimal pH value of the Acid Protease is 3.0, the optimal pH value of the Papain is 7.0, and the enzymolysis treatment effect of each Protease is determined by measuring the polypeptide yield after the enzymolysis treatment.
As shown in FIG. 1, FIG. 1 shows a comparative graph of the proteolytic effect of different proteases on tea seeds, wherein the different lower case letters marked in the table indicate significant differences (P < 0.05) between the test results; as can be seen from fig. 1, the hydrolysis effect of trypsin on tea seed protein is relatively best, the polypeptide yield of the hydrolysate is significantly higher than that of other proteases, which indicates that the peptide bond selectivity of trypsin has better adaptability to the composition and distribution of the peptide bonds of tea seed protein, so trypsin is selected as the protease for enzymolysis treatment in this embodiment.
S502 enzyme adding quantity comparison and determination steps specifically comprise: and (3) fixing the ratio of material to liquid of 1:25, sequentially adding enzyme amounts of 100U/g, 300U/g, 500U/g and 900U/g under the conditions that the temperature is 60 ℃ and the pH value is 8.0, carrying out enzymolysis treatment for 3 hours, and determining the enzymolysis treatment effect of each enzyme amount by measuring the polypeptide yield.
As shown in FIG. 2, FIG. 2 shows a graph of the effect of different enzyme dosages on the polypeptide yield, and the results of FIG. 2 show that the enzyme dosages significantly affect the polypeptide yield after other conditions are fixed. When the enzyme adding amount is between 100U/g and 700U/g, the polypeptide yield is continuously improved along with the increase of the enzyme adding amount; when the enzyme adding amount exceeds 700U/g, the polypeptide yield is basically kept constant. This is probably because with increasing enzyme dosage, the number of active centers capable of binding to the substrate increases, the rate of the enzyme-catalyzed reaction increases accordingly, and the yield of the polypeptide increases; when the enzyme amount reaches 700U/g, the enzyme-substrate ratio reaches a saturation state, and the polypeptide yield does not increase. Therefore, in this example, the amount of enzyme to be added for the enzymatic hydrolysis was determined to be 700U/g.
The step of S503 reaction temperature comparison determination specifically comprises the following steps: and (3) fixing the ratio of material to liquid of 1:25, sequentially carrying out enzymolysis treatment for 3 hours at 30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃ and 80 ℃ under the conditions that the enzyme adding amount is 700U/g and the pH value is 8.0, and determining the enzymolysis treatment effect under each temperature condition by measuring the polypeptide yield.
As shown in FIG. 3, FIG. 3 shows a graph of the influence of different enzymolysis temperatures on the polypeptide yield, and FIG. 2 shows that the polypeptide yield increases with the increase of the temperature within the temperature range of 30-60 ℃, and the polypeptide yield begins to decrease after the temperature exceeds 60 ℃. Within a certain range, the temperature rise is beneficial to the improvement of the catalytic performance of enzyme molecules, and when the enzymolysis temperature is too high, the conformation of the enzyme is changed and gradually denatured and inactivated, so that the enzymolysis efficiency is reduced. Therefore, in this example, 60 ℃ is determined as the suitable enzymolysis temperature.
The step of S504 pH comparison determination specifically comprises: and (3) fixing the ratio of material to liquid of 1:25, sequentially carrying out enzymolysis treatment for 3 hours under the conditions of 700U/g enzyme adding amount and 60 ℃ and pH values of 5, 6, 7, 8, 9 and 10, and determining the enzymolysis treatment effect of each pH value by measuring the polypeptide yield.
As shown in FIG. 4, FIG. 4 shows a graph of the effect of different pHs on the polypeptide yield, and the results in FIG. 4 show that the polypeptide yield increases gradually with increasing pH, reaches a maximum at pH8.0, and starts to decrease as pH is increased. The pH is one of important factors influencing the enzyme activity, and can influence the combination of the enzyme and the substrate by changing the active center of the enzyme or the dissociation state of the enzyme and the substrate, and finally influence the polypeptide yield. Thus, in this example, a suitable pH for enzymatic digestion was determined to be 8.0.
The step of S505 reaction duration comparison determination specifically comprises: and (3) fixing the ratio of material to liquid of 1:25, carrying out continuous 6h enzymolysis treatment under the conditions of 700U/g enzyme adding amount, 60 ℃ temperature and 8.0 pH value, sampling once every 1h, and determining the enzymolysis treatment effect in each reaction time length by measuring the polypeptide yield.
As shown in fig. 5, fig. 5 shows a graph of the influence of different enzymolysis durations on the polypeptide yield, and the results of fig. 5 show that in the initial stage of enzymolysis, the polypeptide yield rises along with the extension of the reaction time, and the hydrolysis time is about 3.5h, so that the polypeptide yield reaches the maximum value; with the further extension of the enzymolysis time, the increase of the polypeptide yield tends to be flat. Too long enzymolysis time may cause further hydrolysis of part of the polypeptide product to generate amino acids, and may also cause decrease in polypeptide yield. Therefore, in this example, the suitable time for enzymolysis was determined to be 3.5 hours.
S600, a four-factor three-horizontal response surface test specifically comprises the following steps: according to the experimental result of single-factor influence measurement, after the optimal value level of each condition variable is determined, response surface tests are carried out by taking the polypeptide yield as an index and taking the temperature, the reaction duration, the pH and the total enzyme addition amount as the condition variables, and a mathematical model between the condition variables and the response values is constructed and variance analysis is carried out by taking the polypeptide yield as the response values according to the experimental result.
The condition variables and horizontal design in this example were as per table 1:
table 1 design table of condition variable levels
The experimental arrangement and results in this example are shown in table 2:
TABLE 2 response surface test design and results
According to the test results in Table 2, curve fitting is performed with the polypeptide yield as the response value, the temperature (A), the reaction time (B), the pH (C) and the total enzyme addition (D) as variables, and a mathematical model between the variables and the response value can be constructed:
Y＝71.60+1.87A+2.64B+4.83C+4.55D+0.90AB-0.84AC-1.35AD+0.49BC+0.63BD+1.20CD-8.03A2-11.24B2-8.78C2-4.72D2。
then, variance analysis was performed on the mathematical model and the data in table 2 by using Design Expert 10.0.4 statistical analysis software, and the results are shown in table 3:
TABLE 3 regression model ANOVA
As can be seen from table 3, the model is very significant (P < 0.01), the correlation coefficient R2 of the model is 0.9670, and the mismatch term of the model is not significant (P0.1880 > 0.1), which indicates that the above model has very high fitting degree to the test result, and the model can be used for predicting and optimizing the test result within the value range of each factor. And (3) performing significance analysis on each condition variable item, wherein the result shows that the pH (C) and the total enzyme adding amount (D) have extremely significant influence on the camellia seed meal hydrolysis process.
S700, drawing a response surface 3D graph and a contour graph, which specifically comprises the following steps: and establishing a response surface graph and a contour graph between the response value and any two condition variables according to the test result.
The response surface analysis diagram is a three-dimensional curved surface diagram formed by a response value and two condition variables in a test under the condition that other condition variables are horizontally fixed, and the influence of the interaction of each condition variable on the response value can be intuitively reflected. The influence of the 4 condition variables and their interactions on the degree of hydrolysis can be known by a multiple regression equation, and can be represented by a response surface diagram and a contour diagram, as shown in fig. 6 to 11.
According to the established response surface model, the optimal process model for obtaining the tea seed polypeptide by enzymolysis is as follows: the enzymolysis temperature is 63.6 ℃, the enzymolysis time is 3.99h, the pH is 10.48, the total enzyme addition amount is 1064U/g, and under the condition, the yield of the tea seed polypeptide is 73.79%. In order to verify the optimal process conditions of the experiment, the enzymolysis temperature is 63 ℃, the enzymolysis time is 4h, the pH is 10.5, and the total enzyme addition amount is 1064U/g, under the conditions, the average polypeptide yield measured by three experiments is 74.99%, the experiment result is close to the predicted value, the process for extracting the tea seed polypeptide by optimizing the response surface method is feasible, and the fitting model is suitable for optimizing the extraction process.
According to the embodiment of the invention, camellia seed meal is used as a raw material, firstly, complex enzyme is adopted to assist in extracting protein in the camellia seed meal, secondly, protease is adopted to hydrolyze tea seed protein to obtain small molecular tea seed polypeptide, and on the basis of protease screening and single factor influence determination experiments, the response surface test is designed to optimize the process for preparing the tea seed polypeptide by performing enzymolysis on the tea seed protein by using trypsin, and the optimal process parameters are selected to ensure that the polypeptide yield reaches 74.99%.
Example two
A method for measuring the antioxidant activity of tea seed polypeptide comprises the step of carrying out a DPPH free radical scavenging activity experiment, a hydroxyl free radical scavenging activity experiment and an ABTS free radical scavenging activity experiment on the tea seed polypeptide prepared by the preparation method in the embodiment I respectively.
The experimental steps of the DPPH free radical scavenging activity experiment, the hydroxyl free radical scavenging activity experiment and the ABTS free radical scavenging activity experiment are the conventional steps, and are not described in detail herein.
As shown in fig. 12, fig. 12 shows a comparison of DPPH radical scavenging rates of tea seed protein and tea seed polypeptide, and it can be seen from fig. 12 that DPPH radical scavenging ability of tea seed polypeptide exhibits concentration dependence, and increases with the increase in concentration. And the DPPH free radical scavenging capacity of the tea seed polypeptide is obviously higher than that of the tea seed protein which is not hydrolyzed under the same concentration. When the concentration of the tea seed polypeptide is 100mg/L, the clearance rate of DPPH free radicals is 22.39%, which is 2.3 times of that of tea seed protein with the same concentration. The DPPH clearance rate of the tea seed polypeptide obtained after the hydrolysis of the tea seed protein is greatly improved.
As shown in fig. 13, fig. 13 is a graph comparing the hydroxyl radical scavenging rates of tea seed protein and tea seed polypeptide, and it can be seen from fig. 13 that the hydroxyl radical scavenging capacity of tea seed polypeptide is overall stronger than that of tea seed protein, and gradually improves with the increase of mass concentration. When the concentration of the tea seed polypeptide is 1.0g/L, the clearance rate of hydroxyl free radicals is 70.26%, and is improved by 36.7% compared with the tea seed protein with the same concentration. The clearance rate of hydroxyl free radicals after the hydrolysis of the tea seed protein is obviously improved.
As shown in fig. 14, fig. 14 is a graph showing a comparison of the ABTS free radical scavenging rates of tea seed protein and tea seed polypeptide, and it is understood from fig. 14 that the ABTS free radical scavenging ability of tea seed polypeptide is stronger than that of tea seed protein as a whole, and the ABTS free radical scavenging ability is slowly improved as the mass concentration increases. The ABTS free radical scavenging capacity of the tea seed polypeptide is 1.2-1.8 times higher than that of tea seed protein under the same concentration.
The antioxidant activity test result shows that the polypeptide prepared by the enzymatic hydrolysis method has better antioxidant property than the original tea seed protein.
EXAMPLE III
A tea seed polypeptide is prepared by the preparation method of the embodiment I.
Tea seed polypeptide is prepared by taking tea seed meal as a raw material and adopting a trypsin hydrolysis method. The results of single-factor experiments and response surface experiments show that the influence of pH and enzyme addition on the yield of tea seed polypeptide is the most obvious, and the influence is the enzymolysis time and the enzymolysis temperature. The optimal technological parameters for preparing the polypeptide are that the pH is 10.5, the feed-liquid ratio is 1:25(w/v), the enzyme adding amount is 1064U/g, the enzymolysis time is 4.0h, the enzymolysis temperature is 63 ℃, and under the optimal condition, the polypeptide yield reaches 74.99%. Along with the increase of the concentration of the tea seed polypeptide, the DPPH free radical clearance rate, hydroxyl free radical clearance rate and ABTS free radical clearance rate are improved, wherein the hydroxyl free radical clearance capacity is most obvious. And the free radical scavenging capacity of the tea seed polypeptide is obviously higher than that of unhydrolyzed tea seed protein on the whole, and the tea seed polypeptide has excellent antioxidant activity.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (10)
1. An enzymolysis preparation method of tea seed polypeptide is characterized by comprising the following steps:
a pretreatment step: degreasing camellia seed meal, drying and crushing the camellia seed meal at the temperature of 60 ℃, and sieving the camellia seed meal with a 60-mesh sieve to obtain camellia seed meal;
tea seed protein extraction: adding water into oil tea seed meal to prepare slurry, adding an enzyme preparation for enzymolysis, inactivating the enzyme for 5min by using boiling water, carrying out centrifugal treatment for 10min at the rotating speed of 8000rpm, adjusting the pH value of a supernatant to an isoelectric point, carrying out centrifugal treatment again, taking a precipitate, freeze-drying to obtain tea seed protein, and determining the content of the tea seed protein;
the tea seed polypeptide preparation step comprises: dissolving tea seed protein with deionized water, adjusting pH, adding protease, performing enzymolysis at constant temperature, inactivating enzyme with boiling water for 5min, centrifuging at 8000rpm for 10min, and collecting supernatant to obtain tea seed polypeptide liquid;
the method comprises the following steps of (1) tea seed polypeptide content determination: taking 5.0mL of tea seed polypeptide liquid, adding 5.0mL of 10% trichloroacetic acid aqueous solution, uniformly mixing, standing for 10min, centrifuging for 15min at the rotating speed of 4000rpm, removing protein and macromolecular long-chain peptide fragments, taking 6.0mL of supernatant, taking Gly-Gly-Tyr-Arg as a standard product, measuring the content of the tea seed polypeptide in the supernatant by adopting a biuret method, and calculating the polypeptide yield according to a standard formula.
2. The enzymatic hydrolysis preparation method of tea seed polypeptide according to claim 1, wherein the enzyme preparation in the tea seed protein extraction step is alpha-amylase with a total enzyme addition amount of 300U/g: the laccase is 3: 2, the ratio of material to liquid is 1:25g/mL, the pH value is 5.0, the temperature in the enzymolysis treatment process is 35 ℃, and the enzymolysis treatment time is 60 min.
3. The enzymatic hydrolysis preparation method of tea seed polypeptide according to claim 1 or 2, wherein the determination of the content of tea seed protein in the tea seed protein extraction step specifically includes:
the method comprises the steps of accurately weighing dried tea seed protein, dissolving the dried tea seed protein with deionized water, transferring the tea seed protein into a 250mL volumetric flask, adding water to a constant volume, and measuring the concentration of the tea seed protein by adopting a Coomassie brilliant blue method and taking bovine serum albumin as a standard product.
4. The enzymatic hydrolysis preparation method of tea seed polypeptide according to claim 1, wherein the standard formula in the step of measuring the content of tea seed polypeptide is as follows: the yield of the polypeptide is equal to the amount of the tea seed polypeptide in the hydrolysate per the mass of the tea seed protein multiplied by 100 percent.
5. The enzymatic hydrolysis preparation method of tea seed polypeptide according to claim 1, further comprising a single factor influence determination step, wherein the single factor influence determination step comprises: the method comprises the steps of protease screening, enzyme adding quantity comparison and measurement, reaction temperature comparison and measurement, pH comparison and measurement and reaction duration comparison and measurement.
6. The enzymatic hydrolysis preparation method of tea seed polypeptide according to claim 5, wherein the protease screening step specifically comprises: according to the operation mode in the tea seed polypeptide preparation step, carrying out enzymolysis treatment at the temperature of 40 ℃ by taking trypsin, papain, neutral protease, alkaline protease and acid protease as proteases in sequence, and determining the enzymolysis treatment effect of each protease by determining the polypeptide yield;
the enzyme adding amount comparison and determination step specifically comprises the following steps: and (3) fixing the ratio of material to liquid of 1:25, sequentially adding enzyme amounts of 100U/g, 300U/g, 500U/g and 900U/g under the conditions that the temperature is 60 ℃ and the pH value is 8.0, carrying out enzymolysis treatment for 3 hours, and determining the enzymolysis treatment effect of each enzyme amount by measuring the polypeptide yield;
the reaction temperature comparison determination step specifically comprises: and (3) fixing the ratio of material to liquid of 1:25, sequentially carrying out enzymolysis treatment for 3 hours at the conditions of 30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃ and 80 ℃ under the conditions of 700U/g of enzyme adding amount and 8.0 pH, and determining the enzymolysis treatment effect under each temperature condition by measuring the polypeptide yield;
the pH comparison determination step specifically comprises: and (3) fixing the ratio of material to liquid of 1:25, sequentially carrying out enzymolysis treatment for 3 hours under the conditions of the enzyme adding amount of 700U/g and the temperature of 60 ℃ and the conditions of pH values of 5, 6, 7, 8, 9 and 10, and determining the enzymolysis treatment effect of each pH value by measuring the polypeptide yield;
the step of measuring the reaction duration by contrast specifically comprises the following steps: and (3) fixing the ratio of material to liquid of 1:25, carrying out continuous 6h enzymolysis treatment under the conditions of 700U/g enzyme adding amount, 60 ℃ temperature and 8.0 pH value, sampling once every 1h, and determining the enzymolysis treatment effect in each reaction time length by measuring the polypeptide yield.
7. The enzymatic hydrolysis preparation method of tea seed polypeptide according to claim 5 or 6, wherein the single factor influence determination step is followed by a four-factor three-level response surface test, specifically comprising: and (3) carrying out a response surface test by taking the temperature, the reaction duration, the pH and the total enzyme addition amount as condition variables, and constructing a mathematical model between the condition variables and the response values and carrying out variance analysis by taking the polypeptide yield as a response value according to the test result.
8. The enzymatic hydrolysis preparation method of tea seed polypeptide according to claim 7, further comprising, after the four-factor three-level response surface test: and establishing a response surface graph and a contour graph between the response value and any two condition variables according to the test result.
9. A method for measuring antioxidant activity of tea seed polypeptide is characterized by comprising the following steps: a DPPH free radical scavenging activity test, a hydroxyl free radical scavenging activity test and an ABTS free radical scavenging activity test are respectively carried out on the tea seed polypeptide prepared by the preparation method of any one of claims 1 to 8.
10. A tea seed polypeptide produced by the production method according to any one of claims 1 to 8.
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