CN112481340B - Tea polypeptide, preparation method thereof and preparation method of tea protein - Google Patents
Tea polypeptide, preparation method thereof and preparation method of tea protein Download PDFInfo
- Publication number
- CN112481340B CN112481340B CN202011391875.0A CN202011391875A CN112481340B CN 112481340 B CN112481340 B CN 112481340B CN 202011391875 A CN202011391875 A CN 202011391875A CN 112481340 B CN112481340 B CN 112481340B
- Authority
- CN
- China
- Prior art keywords
- tea
- polypeptide
- protein
- preparation
- extract
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- 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
-
- 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
- C07K1/145—Extraction; Separation; Purification by extraction or solubilisation
Abstract
The application relates to the field of tea processing, in particular to tea polypeptide and a preparation method thereof, and a preparation method of tea protein. The preparation method of the tea polypeptide mainly comprises the following steps: mixing and stirring the tea leaves, the extract and the alkaline solution to obtain an intermediate product; removing the extract and tea residue in the intermediate product, and performing protein decomposition to obtain protein polypeptide; wherein the extract comprises at least one of cross-linked polyvinylpyrrolidone, bentonite and silica gel. The application can obtain tea polypeptide with higher purity. And the tea polypeptide can effectively inhibit the activity of collagenase. The use of organic reagents can be reduced, and the crosslinked polyvinylpyrrolidone, the bentonite and the silica gel can be repeatedly used, so that the cost can be effectively reduced, the sewage discharge can be reduced, and the extraction rate can be effectively improved.
Description
Technical Field
The application relates to the field of tea processing, in particular to tea polypeptide and a preparation method thereof, and a preparation method of tea protein.
Background
The protein in the tea is mostly water-insoluble, only 1% -2% of the protein is water-soluble, and the water solubility of some proteins is further reduced due to the denaturation and solidification of the protein in the tea making process. Resulting in a lower utilization of tea protein.
At present, some methods for preparing polypeptide by tea enzymolysis and the like appear to increase the utilization rate of tea protein; however, the purity of tea protein extracted in the prior art is not high, which results in low utilization rate of tea protein.
Disclosure of Invention
The embodiments of the present application aim to provide a tea polypeptide and a preparation method thereof, and a preparation method of tea protein, which aim to provide a new preparation method of tea polypeptide, and increase the purity and utilization rate thereof.
The application provides a preparation method of tea polypeptide, which mainly comprises the following steps:
mixing and stirring tea leaves, the extract and the alkaline solution to obtain an intermediate product;
removing the extract and the tea residue in the intermediate product, and then performing protein decomposition to obtain protein polypeptide;
wherein the extract comprises at least one of cross-linked polyvinylpyrrolidone, bentonite, and silica gel.
At least one of cross-linked polyvinylpyrrolidone, bentonite and silica gel is adopted to be stirred and mixed with tea leaves, then the mixture is separated and decomposed to obtain tea polypeptide with higher purity, and the cross-linked polyvinylpyrrolidone, the bentonite and the silica gel can enrich protein in the tea leaves, so that more protein is separated from tea residues and enters the filtrate. And the tea polypeptide can effectively inhibit the activity of collagenase. In addition, the tea polypeptide is prepared by adopting the cross-linked polyvinylpyrrolidone, the bentonite and the silica gel, so that the use of organic reagents can be reduced, and meanwhile, the cross-linked polyvinylpyrrolidone, the bentonite and the silica gel can be reused, so that the cost can be effectively reduced, the sewage discharge can be reduced, and the extraction rate can be effectively improved.
In some embodiments of the first aspect of the present application, the removing of the extract and tea leaves from the intermediate product is by centrifugation;
optionally, the rotation speed of centrifugal separation is 8000-10000rpm, and the centrifugation time is 30-50min.
In some embodiments of the first aspect of the present application, the step of mixing and stirring the tea leaves, the extract and the alkaline solution to obtain the intermediate product comprises:
mixing folium Camelliae sinensis, extract and alkaline solution, adjusting pH to 7-14, and stirring at 40-70 deg.C to obtain intermediate product;
optionally, the stirring speed is 400-500rpm, and the temperature is 65-75 ℃.
In some embodiments of the first aspect of the present application, the mass ratio of the extract to the tea leaves is (0.01-0.1): 1;
optionally, the concentration of the extract is 0.05wt% to 0.5wt%.
In some embodiments of the first aspect of the present application, the alkaline solution is selected from at least one of sodium hydroxide, ammonia, potassium hydroxide, and calcium carbonate.
In some embodiments of the first aspect of the present application, the mixing of the tea leaves, the extract and the alkaline solution further comprises pulverizing the tea leaves into 60 mesh powder;
optionally, the protein is decomposed to obtain protein polypeptide; filtering the decomposed product to obtain filtrate.
In some embodiments of the first aspect of the present application, the step of performing proteolytic cleavage to obtain a protein polypeptide comprises;
adjusting the pH value of the filtrate obtained by removing the extract and the tea residue in the intermediate product to 3-5 to precipitate the filtrate, and performing ultrasonic decomposition on the precipitate;
optionally, ultrasonic decomposing for 10-20min with ultrasonic power of 500-800w.
In some embodiments of the first aspect of the present application, the step of performing proteolytic cleavage to obtain a protein polypeptide comprises;
adjusting pH of the filtrate to 3-5 to precipitate, and decomposing precipitate with protease;
optionally, the protease is selected from at least one of neutral protease, alkaline protease, complex protease, trypsin and papain.
In a second aspect, the present application provides a tea polypeptide prepared by the above-described process for preparing a tea polypeptide; the tea polypeptide contains 45.0% -65.0% of components with molecular weight of (5 +/-0.2) KD calculated by the total weight of the polypeptide.
The tea polypeptide with the molecular weight of about 5KD has better effect on inhibiting the activity of collagenase. The tea polypeptide has the component content of 45.0% -65.0% in (5 +/-0.2) KD of molecular weight, is relatively large in ratio, and has a good effect of inhibiting the activity of collagenase. Can effectively slow down the generation of wrinkles.
The third aspect of the application provides a preparation method of tea protein, which mainly comprises the following steps:
mixing and stirring the tea leaves, the extract and the alkaline solution to obtain an intermediate product;
removing the extract and the tea residue in the intermediate product, and taking filtrate;
wherein the extract comprises at least one of cross-linked polyvinylpyrrolidone, bentonite and silica gel.
The cross-linked polyvinylpyrrolidone, bentonite and silica gel are adopted as extracts to be fully mixed with tea leaves, and then centrifugal separation is carried out, so that the purity of protein in tea protein can be improved to a great extent.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are required to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present application and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained from the drawings without inventive effort.
FIG. 1 is a graph showing the collagenase inhibitory effects of the tea polypeptides of examples 1 to 8 and comparative example 1.
FIG. 2 shows the molecular weight distributions of the tea polypeptides provided in example 1 and comparative example 1.
Figure 3 shows the molecular weight distribution of the tea polypeptide of example 1.
FIG. 4 shows the levels of collagen secretion by the tea polypeptide of example 1 on 3T3 cell I.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions of the embodiments of the present application will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The tea polypeptides and the methods of making the same of the examples of the present application are described in detail below.
A preparation method of tea polypeptide mainly comprises the following steps:
mixing and stirring the tea leaves, the extract and the alkaline solution to obtain an intermediate product;
removing the extract and the tea residue in the intermediate product, and then performing protein decomposition to obtain protein polypeptide;
wherein the extract comprises at least one of cross-linked polyvinylpyrrolidone, bentonite and silica gel.
Cross-linked polyvinylpyrrolidone, CTFA name: crosslinked Polyvinylpyrrolidone, PVPP for short, has very strong expansion performance and complexing ability with various substances.
The inventors have surprisingly found that mixing tea leaves, extract and alkaline solution and stirring to obtain an intermediate product; can separate more protein in tea leaves from tea leaves into water phase or combine with extract. And then removing the extract and the tea residue in the intermediate product to obtain the protein with higher purity and higher content and improve the extraction rate of the protein. The above proteins are decomposed to obtain tea polypeptide with high content and molecular weight of about 5 KD.
In some embodiments of the present application, the mixing the tea leaves, the extract and the alkaline solution further comprises pulverizing the tea leaves into a powder of 60 mesh. It will be appreciated that in other embodiments of the present application, the tea leaves may not be comminuted, or the tea leaves may be comminuted to a powder of smaller or larger particle size, irrespective of yield and the like.
The alkaline solution may be at least one of ammonia, potassium hydroxide, sodium carbonate, and sodium hydroxide, for example, in the present embodiment, an aqueous sodium hydroxide solution is used as the alkaline solution. It should be noted that in other embodiments of the present application, other alkaline solutions may be used to mix with the tea leaves and adjust the pH.
The extract comprises at least one of cross-linked polyvinylpyrrolidone, bentonite and silica gel.
In some embodiments, the mass ratio of the extract to the tea leaves is (0.01-0.1): 1. for example, the mass ratio of extract to tea leaf may be 0.01: 1. 0.02: 1. 0.05: 1. 0.07: 1. 0.09: 1. 0.1:1, etc.
In the examples of the application, the extract, the tea leaves and the alkaline solution may be mixed first with the two and then with the remaining one, for example, the tea leaves and the alkaline solution are mixed first and then with the extract. Or mixing the three components at the same time.
For example, in some embodiments, a 60 mesh powder is taken, water is added in an amount of 20-60 times the weight of the powder, and sodium hydroxide is used to adjust the pH to 7-14. For example, the pH may be adjusted to 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 12, 12.5, 13, 13.5, or 14, and so forth.
In some embodiments of the present application, the step of mixing and stirring the tea leaves, the extract and the alkaline solution to obtain the intermediate product comprises:
mixing folium Camelliae sinensis, extract and alkaline solution, and stirring at 40-70 deg.C to obtain intermediate product; for example, the temperature may be 40 ℃, 42 ℃, 45 ℃, 50 ℃, 55 ℃,60 ℃, 62 ℃, 65 ℃, 68 ℃ or 70 ℃ or the like.
Illustratively, in the step of stirring at 40-70 ℃ to obtain the intermediate product, the stirring speed is 400-500rpm, and the temperature is 65-75 ℃; for example, the rotation speed of the stirring may be 400rpm, 410rpm, 420rpm, 450rpm, 470rpm, 490rpm, 500rpm, or the like, and the temperature during the stirring may be, for example, 65 ℃, 68 ℃, 70 ℃, 72 ℃, 75 ℃, or 78 ℃, or the like.
After stirring at 40-70 deg.C to obtain intermediate product, removing extract and tea residue from the intermediate product, in the examples of the application, centrifuging to separate intermediate product, and collecting filtrate after centrifugation.
Centrifugation facilitates separation of the proteins attached to the extract from the extract into the filtrate. It should be noted that in other embodiments of the present application, the intermediate product may be isolated in other manners.
Illustratively, the step of centrifuging the intermediate product has a rotation speed of 8000-10000rpm and a centrifugation time of 30-50min. For example, in the centrifugal separation process, the rotation speed may be 8000rpm, 8200rpm, 8600rpm, 9000rpm, 9200rpm, 9500rpm, 10000rpm, or the like, and the time of centrifugation may be 30min, 32min, 35min, 40min, 42min, 46min, or 50min, or the like.
Centrifuging, collecting filtrate, adjusting pH to 3-5 to precipitate, and performing protein decomposition on the precipitate to obtain protein polypeptide. For example, the filtrate is precipitated by adjusting the pH of the filtrate to 3, 3.5, 4, 4.5 or 5. Hydrochloric acid may be used for adjusting the pH.
Adjusting pH value to precipitate the filtrate, and performing protein decomposition on the precipitate to obtain protein polypeptide.
The protein decomposition of the precipitate can be carried out by means of enzymatic decomposition or by means of ultrasonic decomposition. The protein is decomposed by adopting ultrasound, so that the decomposition efficiency is improved, and impurities are not easily introduced in the ultrasonic decomposition.
For example, the precipitate is mixed with water and then is subjected to ultrasonic decomposition for 10-20min, and the ultrasonic power is 500-800w. The ultrasonic power may be 500w, 550w, 580w, 600w, 650w, 700w, 750w, 800w, or the like. The ultrasound time can be 10min, 12min, 13min, 15min, 18min or 20min, etc.
Or, decomposing the precipitate with a protease; in some embodiments, the protease is selected from at least one of a neutral protease, an alkaline protease, a complex protease, trypsin, and papain. Mixing the precipitate and water according to a mass ratio of 1:10 to obtain an aqueous solution of the precipitate, and adding an enzyme, wherein the ratio of the enzyme activity to the aqueous solution of the precipitate may be 1000u/mL.
In some embodiments of the present application, the decomposing of the protein further comprises filtering the decomposition product with a 5kD ceramic membrane to obtain a filtrate. And filtering the 5kD ceramic membrane to intercept the polypeptide with the molecular weight of about 5 kD.
In some embodiments, for convenience of storage, drying the filtrate is further included, for example, freeze drying, vacuum drying, spray drying, etc. may be used.
The preparation method provided by the embodiment of the application has at least the following advantages:
the tea polypeptide with high purity can be obtained by adopting at least one of cross-linked polyvinylpyrrolidone, bentonite and silica gel to stir and mix with tea leaves, and then separating and decomposing the mixture. And the tea polypeptide can effectively inhibit the activity of collagenase. In addition, the tea polypeptide is prepared by adopting the cross-linked polyvinylpyrrolidone, the bentonite and the silica gel, so that the use of organic reagents can be reduced, and meanwhile, the cross-linked polyvinylpyrrolidone, the bentonite and the silica gel can be reused, so that the cost can be effectively reduced, the sewage discharge can be reduced, and the extraction rate can be effectively improved.
The application also provides a tea polypeptide, which is prepared by the preparation method of the tea polypeptide;
the tea polypeptide contains 45.0% -65.0% of components with molecular weight of (5 +/-0.2) KD calculated by the total weight of the polypeptide.
In other words, the content of components having a molecular weight of (5. + -. 0.2) KD in the polypeptide produced by the above-mentioned method for producing tea polypeptide is 45.0wt% to 65.0wt%.
Collagenase is an enzyme that hydrolyzes protein, and causes loss of moisture and elasticity of the skin, and wrinkle formation. Therefore, the activity of collagenase is inhibited, and the generation of wrinkles can be effectively slowed down. The tea polypeptide with molecular weight of about 5KD has better effect on inhibiting activity of collagenase. Therefore, the tea polypeptide provided by the embodiment of the application has a better contribution to inhibiting the activity of collagenase.
In addition, the content of protein in the tea polypeptide obtained by the method provided by the embodiment of the application is higher.
The application also provides a preparation method of the tea protein, which mainly comprises the following steps:
mixing folium Camelliae sinensis, extract and alkaline solution, adjusting pH to 7-14, and stirring at 40-70 deg.C to obtain intermediate product; and centrifuging the intermediate product, and taking the centrifuged filtrate.
Wherein the extract comprises at least one of cross-linked polyvinylpyrrolidone, bentonite and silica gel.
Mixing the tea leaves, the extract and the alkaline solution, adjusting the pH value to 7-14, and stirring at 40-70 ℃ to obtain an intermediate product; and centrifuging the intermediate product, and taking the centrifuged filtrate. Corresponding process parameters can be referred to above, and are not described herein again.
After the research of the inventor, the inventor finds that in the process of preparing tea polypeptide, the purity of protein in tea protein can be greatly improved by adopting crosslinked polyvinylpyrrolidone, bentonite and silica gel as extracts to be fully mixed with tea leaves and then carrying out centrifugal separation. And the inhibition effect of the obtained protein molecules on collagenase is obviously improved after the decomposition.
The features and properties of the present application are described in further detail below with reference to examples.
Example 1
The embodiment provides a tea polypeptide, which is mainly prepared by the following preparation method:
(1) Dried tea leaves are taken, crushed and sieved by a 60-mesh sieve to obtain 1.0kg of tea powder.
(2) Taking 1.0kg of tea powder obtained in the step (1), adding water with the weight being 40 times of the weight of the powder, uniformly stirring, adjusting the pH value to 11 by using sodium hydroxide, adding 0.2wt% of PVPP, wherein the mass ratio of the PVPP to the tea powder is 0.1; stirring for 1.0h. The stirring speed was 500rpm and the temperature was maintained at 60 ℃.
(3) Centrifuging at 8000rpm for 30min, adjusting pH of the supernatant to 4.0 with hydrochloric acid, centrifuging, collecting precipitate, adding water 10 times the weight of the precipitate, performing ultrasonic treatment at 600w power for 10min, and filtering to obtain filtrate.
(4) And (5) carrying out freeze drying to obtain the tea polypeptide.
Example 2
The embodiment provides a tea polypeptide, which is mainly prepared by the following preparation method:
(1) Taking dry tea leaves, crushing, and sieving by a 60-mesh sieve to obtain 1.0kg of tea powder.
(2) Taking 1.0kg of tea powder obtained in the step (1), adding water which is 50 times of the weight of the powder, uniformly stirring, adjusting the pH value to 13 by using sodium hydroxide, adding 0.5wt% of PVPP, wherein the mass ratio of the PVPP to the tea powder is 0.1; stirring for 2.0h. The stirring speed was 500rpm and the temperature was maintained at 70 ℃.
(3) Centrifuging at 8000rpm for 30min, adjusting pH of the supernatant to 4.0 with hydrochloric acid, centrifuging, collecting precipitate, and adding 10 times of water; carrying out ultrasonic treatment at 600w for 10min, and filtering to obtain filtrate.
(4) And (5) carrying out freeze drying to obtain the tea polypeptide.
Example 3
The embodiment provides a tea polypeptide which is mainly prepared by the following preparation method:
(1) Taking dry tea leaves, crushing, and sieving by a 60-mesh sieve to obtain 1.0kg of tea powder.
(2) Taking 1.0kg of tea powder obtained in the step (1), adding water which is 40 times of the weight of the powder, uniformly stirring, adjusting the pH value to 12 by using sodium hydroxide, adding 0.2wt% of bentonite, wherein the mass ratio of the bentonite to the tea powder is 0.1; stirring for 1.0h. The stirring speed was 500rpm and the temperature was maintained at 60 ℃.
(3) Centrifuging at 8000rpm for 30min, adjusting pH of the supernatant to 4.0 with hydrochloric acid, centrifuging, collecting precipitate, and adding 10 times of water; carrying out ultrasonic treatment at 600w for 10min, and filtering to obtain filtrate.
(4) And (5) carrying out freeze drying to obtain the tea polypeptide.
Example 4
The embodiment provides a tea polypeptide, which is mainly prepared by the following preparation method:
(1) Taking dry tea leaves, crushing, and sieving by a 60-mesh sieve to obtain 1.0kg of tea powder.
(2) Taking 1.0kg of tea powder obtained in the step (1), adding water 40 times of the weight of the powder, uniformly stirring, adjusting the pH value to 11 by using sodium hydroxide, adding 0.2wt% of PVPP, wherein the mass ratio of the PVPP to the tea powder is 0.05; stirring for 1.0h. The stirring speed was 500rpm and the temperature was maintained at 60 ℃.
(3) Centrifuging at 8000rpm for 30min, adjusting pH of the supernatant to 4.0 with hydrochloric acid, centrifuging, collecting precipitate, and adding water 10 times the weight of the precipitate; adding 1000u/mL (enzyme activity/volume of extractive solution) of alkaline protease, performing enzymolysis at 45 deg.C for 5.0h, and filtering to obtain filtrate.
(4) And (4) carrying out freeze drying to obtain the tea polypeptide.
Example 5
The embodiment provides a tea polypeptide which is mainly prepared by the following preparation method:
(1) Taking dry tea leaves, crushing, and sieving by a 60-mesh sieve to obtain 1.0kg of tea powder.
(2) Taking 1.0kg of tea powder obtained in the step (1), adding water which is 40 times of the weight of the powder, uniformly stirring, adjusting the pH value to 7 by using sodium hydroxide, adding 0.2wt% of silica gel, wherein the mass ratio of the silica gel to the tea powder is 0.05; stirring evenly for 1.0h. The stirring speed was 500rpm and the temperature was maintained at 60 ℃.
(3) Centrifuging at 8000rpm for 30min, adjusting pH of the supernatant to 4.0 with hydrochloric acid, centrifuging, collecting precipitate, and adding 10 times of water; carrying out ultrasonic treatment at 600w for 10min, and filtering to obtain filtrate.
(4) And (4) carrying out freeze drying to obtain the tea polypeptide.
Example 6
The embodiment provides a tea polypeptide, which is mainly prepared by the following preparation method:
(1) Dried tea leaves are taken, crushed and sieved by a 60-mesh sieve to obtain 1.0kg of tea powder.
(2) Taking 1.0kg of tea powder obtained in the step (1), adding water 40 times of the weight of the powder, uniformly stirring, adjusting the pH value to 11 by using sodium hydroxide, adding 0.2wt% of PVPP, wherein the mass ratio of the PVPP to the tea powder is 0.05; stirring for 1.0h. The stirring speed was 500rpm and the temperature was maintained at 60 ℃.
(3) Centrifuging at 8000rpm for 30min, adjusting pH of the supernatant to 4.0 with hydrochloric acid, centrifuging, collecting precipitate, and adding 10 times of water; adding 1000u/mL (enzyme activity/volume of extractive solution) neutral protease, performing enzymolysis at 65 deg.C for 5.0h, and filtering to obtain filtrate.
(4) And (4) carrying out freeze drying to obtain the tea polypeptide.
Example 7
The embodiment provides a tea polypeptide, which is mainly prepared by the following preparation method:
(1) Dried tea leaves are taken, crushed and sieved by a 60-mesh sieve to obtain 1.0kg of tea powder.
(2) Taking 1.0kg of the tea powder obtained in the step (1), adding water which is 40 times of the weight of the powder, uniformly stirring, adjusting the pH value to 11 by using potassium hydroxide, adding 0.2wt% of PPVP, wherein the mass ratio of the PPVP to the tea powder is 0.1; stirring for 1.0h. The stirring speed was 500rpm and the temperature was maintained at 60 ℃.
(3) Centrifuging at 8000rpm for 30min, adjusting pH of the supernatant to 4.0 with hydrochloric acid, centrifuging, collecting precipitate, and adding 10 times of water; carrying out ultrasonic treatment at 600w for 10min, and filtering to obtain filtrate.
(4) And (5) carrying out freeze drying to obtain the tea polypeptide.
Example 8
The embodiment provides a tea polypeptide, which is mainly prepared by the following preparation method:
(1) Taking dry tea leaves, crushing, and sieving by a 60-mesh sieve to obtain 1.0kg of tea powder.
(2) Taking 1.0kg of the tea powder obtained in the step (1), adding water which is 40 times of the weight of the powder, uniformly stirring, adjusting the pH value to 11 by using ammonia water, adding 0.2wt% of PPVP, wherein the mass ratio of the PPVP to the tea powder is 0.1; stirring for 1.0h. The stirring speed was 500rpm and the temperature was maintained at 60 ℃.
(3) Centrifuging at 8000rpm for 30min, adjusting pH of the supernatant to 4.0 with hydrochloric acid, centrifuging, collecting precipitate, and adding 10 times of water; carrying out ultrasonic treatment at 600w for 10min, and filtering to obtain filtrate.
(4) And (5) carrying out freeze drying to obtain the tea polypeptide.
Comparative example 1
The comparative example provides a tea polypeptide, prepared mainly by the following preparation method:
(1) Taking dry tea leaves, crushing, and sieving by a 60-mesh sieve to obtain 1.0kg of tea powder.
(2) Taking 1.0kg of tea powder obtained in the step (1), adding water 40 times of the weight of the powder, uniformly stirring, adjusting the pH value to 11 by using sodium hydroxide, and uniformly stirring for 1.0h. The stirring speed was 500rpm and the temperature was maintained at 60 ℃.
(3) Centrifuging at 8000rpm for 30min, adjusting pH of the supernatant to 4.0 with hydrochloric acid, centrifuging, collecting precipitate, adding water 10 times the weight of the precipitate, performing ultrasonic treatment at 600w power for 10min, and filtering to obtain filtrate.
(4) And (5) carrying out freeze drying to obtain the tea polypeptide.
Test example 1
The protein content of the tea polypeptides obtained in examples 1-8 and comparative example 1 was tested.
The content of the protein was measured by BCA (bicinchoninic acid) protein assay kit method.
The protein reduces the divalent copper ions into monovalent copper ions under alkaline conditions, and then reacts with the BCA reagent to generate a purple compound, and the absorbance at 562nm is detected.
Protein content (%) = determination of sample protein mass (g)/sample lyophilized powder mass (g);
5KD molecular weight distribution (%) = 5KD protein mass (g)/sample freeze-dried powder mass (g) are obtained through separation;
the test results are shown in Table 1.
TABLE 1
As can be seen from Table 1, the tea polypeptides obtained in examples 1-8 have a much higher protein content than comparative example 1. The tea polypeptides obtained in examples 1-6 have higher protein content than in examples 8 and 7. The tea polypeptide obtained in example 1 has the highest protein content, and the protein extraction rate is also high.
The protein content in the tea polypeptide can be improved, the impurity content in the tea extract can be reduced, and the purity of the tea polypeptide can be improved by adding the cross-linked polyvinylpyrrolidone, the bentonite and the silica gel.
In the embodiment 7 and the embodiment 8, potassium hydroxide and ammonia water are respectively adopted to adjust the pH value, the protein purity is lower, the ratio of the molecular weight of about 5KD is smaller, and the protein dissolution is less. The alkaline solution is beneficial to leaching tea protein by adopting sodium hydroxide, and the extraction rate of the tea protein is improved, so that the subsequent steps are facilitated. The ultrasonic decomposition of tea protein has short time and does not attract other substances.
Test example 2 collagenase inhibition test
Collagenase action on FALGPA (C) 23 H 32 N 4 O 7 ) The resulting material had an absorption at 335nm and the collagenase activity was examined by monitoring the absorbance at 335 nm. The collagenase inhibitory effect of individual samples was determined spectrophotometrically using FALGPA as a substrate.
The tea polypeptides of example 1-example 8 and comparative example 1 were each dissolved in water to a concentration of 0.5mg/mL (lyophilized powder mass mg/solvent volume mL) and tested for collagenase. The test results are shown in fig. 1.
As can be seen from fig. 1, the tea polypeptides of examples 1 to 8 have a superior collagenase inhibitory effect, compared to comparative example 1, and collagen, which is a main component of extracellular matrix, provides skin with stretchability and supplements skin nutrients required for each layer of skin. Collagenase is a protein hydrolyzing enzyme, and causes loss of moisture and elasticity of the skin, and wrinkle formation. Therefore, the activity of collagenase is inhibited, and the generation of wrinkles can be effectively slowed down.
Test example 3
Tea polypeptides with different molecular weights are separated by adopting 1KD, 3KD, 5KD, 8KD and 10KD ceramic membranes, the tea polypeptides provided by the embodiment 1 and the comparative example 1 are respectively prepared into aqueous solutions with the concentration of 10mg/mL, and the ceramic membrane separation is carried out. Freeze-drying the filtrate and carrying out content measurement. The content determination method is carried out according to the following formula, and the mass percentages of different molecular weights in the extract are calculated according to the following formula;
content (%) = A/B100%
In the formula:
a: the total protein mass of the filtrate freeze-dried powder is g;
b: corresponding to the mass of the freeze-dried powder before filtration, g.
The results are shown in FIG. 2. It can be seen from FIG. 2 that the method provided in example 1 improves the distribution of 5kD in the extract.
Tea polypeptides with different molecular weights are separated by adopting 1KD, 3KD, 5KD, 8KD and 10KD ceramic membranes, the preparation concentration of the tea polypeptide prepared in the embodiment 1 is 10mg/mL, and ceramic membrane separation is carried out. Then, the filtrates were lyophilized to a concentration of 1mg/mL, respectively, for efficacy measurement. The test results are shown in FIG. 3.
As can be seen from FIG. 3, the 5KD tea polypeptide is the main functional component and is the main target product, which shows that the method provided by the application can effectively improve the leaching of the target product.
Test example 4
Effect on collagen secretion from 3T3 cells I
By Western blotting, 3T3 cells were used at a density of 1.7 x 10^6 cells/well and for a duration of 48h. After 24h plating, the tea polypeptide provided in example 1 containing 0.2mg/ml and 0.4mg/ml mass fraction of drug was added to each of the different experimental groups, after 48h of culture, the culture medium supernatant was aspirated, washed twice with PBS solution, cells were lysed with 700 μ L of RAPI lysate, the lysate was collected, centrifuged at 13200rpm and 4 ℃ for 30min, the supernatant was aspirated to remove the pellet, and the protein concentration in the cell culture supernatant was determined. And (3) carrying out SDS-PAGE protein gel electrophoresis, ensuring that the total amount of protein of each sample is the same when loading, and carrying out immunoblotting detection. As shown in FIG. 4, it is understood from FIG. 4 that example 1 promotes the secretion of collagen I from 3T3 cells and effectively inhibits collagenase activity.
In conclusion, the product obtained by the preparation method of tea polypeptide provided by the embodiment of the application has higher polypeptide content, and the tea polypeptide accounts for a higher ratio of about 5 KD. Has excellent effect on inhibiting the activity of collagenase.
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
Claims (1)
1. A tea polypeptide with collagenase inhibitory activity is characterized in that the content of components with molecular weight of (5 +/-0.2) KD is 45.0% -65.0% calculated by the total weight of the tea polypeptide; the preparation method of the tea polypeptide mainly comprises the following steps:
(1) Taking dry tea leaves, crushing, and sieving by a 60-mesh sieve to obtain 1.0kg of tea powder;
(2) Taking 1.0kg of tea powder obtained in the step (1), adding water 40 times of the weight of the powder, uniformly stirring, adjusting the pH value to 11 by using sodium hydroxide, adding 0.2wt% of PVPP, wherein the mass ratio of the PVPP to the tea powder is 0.1; uniformly stirring for 1.0h at the stirring speed of 500rpm and the temperature of 60 ℃;
(3) Centrifuging at 8000rpm for 30min, adjusting pH of the supernatant to 4.0 with hydrochloric acid, centrifuging, collecting precipitate, adding water 10 times the weight of the precipitate, performing ultrasonic treatment at 600w power for 10min, and filtering to obtain filtrate;
(4) And (5) carrying out freeze drying to obtain the tea polypeptide.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011391875.0A CN112481340B (en) | 2020-12-01 | 2020-12-01 | Tea polypeptide, preparation method thereof and preparation method of tea protein |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011391875.0A CN112481340B (en) | 2020-12-01 | 2020-12-01 | Tea polypeptide, preparation method thereof and preparation method of tea protein |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112481340A CN112481340A (en) | 2021-03-12 |
| CN112481340B true CN112481340B (en) | 2022-12-23 |
Family
ID=74939116
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011391875.0A Active CN112481340B (en) | 2020-12-01 | 2020-12-01 | Tea polypeptide, preparation method thereof and preparation method of tea protein |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112481340B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102875691A (en) * | 2012-10-19 | 2013-01-16 | 黄冈师范学院 | Method for synchronously preparing active substances from tea dregs |
| CN111109410A (en) * | 2020-03-04 | 2020-05-08 | 山东先天元食品有限公司 | Polypeptide tea decoction pieces and preparation method thereof |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101744093B (en) * | 2008-12-17 | 2012-07-25 | 福建福大百特生物科技有限公司 | Tea leaf protein polypeptide and preparation method and application thereof |
| CN105982329A (en) * | 2016-01-07 | 2016-10-05 | 安徽省华银茶油有限公司 | Fruit-vegetable nutritional tea leaf seed polypeptide lozenge and preparation method thereof |
| CN108434443B (en) * | 2018-05-28 | 2021-04-09 | 广东康侨药业有限公司 | Whitening antioxidant composition and application thereof |
-
2020
- 2020-12-01 CN CN202011391875.0A patent/CN112481340B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102875691A (en) * | 2012-10-19 | 2013-01-16 | 黄冈师范学院 | Method for synchronously preparing active substances from tea dregs |
| CN111109410A (en) * | 2020-03-04 | 2020-05-08 | 山东先天元食品有限公司 | Polypeptide tea decoction pieces and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| PVPP吸附茶多酚特性及其在绿茶饮料中的应用;徐悦;《茶叶》;20190615;第45卷(第2期);第62页左栏第2段 * |
| 富硒碱性茶蛋白ACE抑制肽的制备、分离纯化及结构鉴定;杜梦珂;《中国优秀硕士学位论文全文数据库 工程科技I辑》;20181115(第11期);第I-II页摘要、第4页1.2.1.3、第15页2.2实验方法、第35页3.2实验方法、第55-56页4.4 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112481340A (en) | 2021-03-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105010720B (en) | A kind of method that high anti-oxidation active peptide is prepared using ultra high pressure treatment rice residue protein | |
| WO2013073404A1 (en) | Rice-protein composition and method for manufacturing same | |
| KR100904631B1 (en) | Preparation of functional hydrolysates from oyster using transglutaminase | |
| JP2013544522A5 (en) | ||
| CN102115690A (en) | Method for comprehensively utilizing rice bran | |
| CN104250286B (en) | Navodon septentrionalis fish-skin antioxidant collagen peptide and preparation method and use thereof | |
| CA1088443A (en) | Method of producing polysaccharides | |
| CN112481340B (en) | Tea polypeptide, preparation method thereof and preparation method of tea protein | |
| CN111471732A (en) | Novel selenium-rich tea source ACE inhibitory peptide and preparation method thereof | |
| CN113603799A (en) | High-oxidation-resistance rice bran polysaccharide-peptide compound and preparation method thereof | |
| CN109091409B (en) | Preparation method of passion flower anthocyanin and egg albumin peptide mask dry powder | |
| CN112175098A (en) | Method for efficiently preparing polysaccharide from sparassis crispa stipe | |
| JPH0430401B2 (en) | ||
| CN115368486A (en) | Ternary eutectic solvent and application thereof in extraction of procambarus clarkia shell chitin | |
| CN112898447B (en) | Method for extracting polysaccharide from radix cynanchi bungei | |
| WO2007041950A1 (en) | Extraction method of patinopecten yessoensis polysaccharide | |
| CN114107415A (en) | Preparation method of sargassum fusiforme extract containing small molecular active peptide | |
| JP3282036B2 (en) | Method for producing acidic polysaccharide | |
| CN111995655B (en) | Walnut protein isolate and preparation method thereof, phosphorylated walnut protein isolate and preparation method thereof, and food | |
| JPS6225167B2 (en) | ||
| CN114835828B (en) | Preparation method of black fungus crude polysaccharide | |
| RU2200424C2 (en) | Method for obtaining pectin-containing product out of sea grasses of zosteraceae species | |
| US4284552A (en) | Process for the preparation of CoA-SPC from Bakers' yeast | |
| CN114752644B (en) | Preparation method of red bean polypeptide with antioxidant effect | |
| US4755618A (en) | Recovery of active tannin from schoene sludge |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |