CN113564216A

CN113564216A - Method for obtaining tridecapeptide through yeast fermentation and application thereof

Info

Publication number: CN113564216A
Application number: CN202110819459.4A
Authority: CN
Inventors: 曹原; 眭春; 魏明明; 孙玮哲
Original assignee: Lanke Yimei Science And Technology Jilin Co ltd
Current assignee: Lanke Yimei Science And Technology Jilin Co ltd
Priority date: 2021-07-20
Filing date: 2021-07-20
Publication date: 2021-10-29
Anticipated expiration: 2041-07-20
Also published as: CN113564216B

Abstract

The invention discloses a method for obtaining tridecapeptide by yeast fermentation and application thereof, which comprises the following steps of S1 and preparation of a culture medium: s11, preparing a YPD culture medium; s12, preparation of BSM medium: mixing glycerol, phosphoric acid, trace elements and biotin, adding deionized water, stirring uniformly, adding nutrients, and adjusting pH with ammonia water to obtain a BSM culture medium; s2, fermentation expression; s3, processing fermentation liquor: separating the obtained product, separating by using an ultrafiltration membrane to obtain the tridecapeptide, and recovering the waste liquid. The tridecapeptide is a commonly used raw material in the field of cosmetics, is a polypeptide consisting of a plurality of amino acids, can promote the generation of new cells when being used in the field of cosmetics, can repair scars on the surface of human skin and promote the growth of cells, has more stable and safer performance compared with forbidden EGF, and is an ideal substitute for EGF.

Description

Method for obtaining tridecapeptide through yeast fermentation and application thereof

Technical Field

The invention relates to the technical field of biological fermentation, in particular to a method for obtaining tridecapeptide through yeast fermentation and application thereof.

Background

Epidermal growth factor, also known as human oligopeptide-1, is an active polypeptide consisting of a plurality of amino acids, and can make skin elastic and ruddy when used in cosmetics, but the epidermal growth factor has large side effects and can cause the phenomenon of skin overgrowth, so that the epidermal growth factor is regulated by the national drug administration and is prohibited from being used as a cosmetic raw material.

At present, the tridecapeptide is an ideal substitute of a high-activity epidermal growth factor, can be used as a cosmetic raw material, can promote the growth of cells, reduce the generation of wrinkles, and has the effects of brightening the skin and lightening scars, so the tridecapeptide has wide application in the field of cosmetics.

At present, the product obtained by the preparation method of the tridecapeptide contains too many impurities, which is not beneficial to large-scale industrial continuous production, so the invention of the method for obtaining the tridecapeptide by yeast fermentation and the application thereof are particularly important.

Disclosure of Invention

The present invention aims to provide a method for obtaining tridecapeptide by yeast fermentation and the application thereof, so as to solve the problems in the background art.

In order to solve the technical problems, the invention provides the following technical scheme: a method for obtaining tridecapeptide by yeast fermentation comprises the following steps,

s1, preparation of medium:

s11, preparation of YPD Medium:

(1) dissolving yeast extract and 10-15 parts of peptone, and sterilizing to obtain solution A;

(2) dissolving glucose, and sterilizing to obtain solution B;

(3) mixing the solution A and the solution B, and uniformly stirring to obtain a YPD culture medium;

s12, preparation of BSM medium:

mixing glycerol, phosphoric acid, trace elements and biotin, adding deionized water, stirring uniformly, adding nutrients, and adjusting pH with ammonia water to obtain a BSM culture medium;

s2, fermentation expression:

(1) inoculating active bacteria into YPD culture medium, and culturing until bacterial colony grows out;

(2) putting the YPD culture medium into the cultured bacterial colony, and culturing in a shaking table to obtain a seed solution;

(3) adding YPD culture medium and defoaming agent into the fermentation tank, and sterilizing;

(4) inoculating the seed liquid into a fermentation tank by using a flame inoculation method for fermentation;

(5) adding a BSM culture medium and a defoaming agent into the induced expression fermentation tank, sterilizing, and then performing high-pressure steam sterilization;

(6) putting the seed liquid in the fermentation tank into an inducible expression fermentation tank, adding methanol, starting fermentation, and obtaining fermentation liquid after fermentation;

s3, processing fermentation liquor:

separating the obtained product, separating by using an ultrafiltration membrane to obtain the tridecapeptide, and recovering the waste liquid.

Further, the concrete steps are as follows,

s1, preparation of medium:

s11, preparation of YPD Medium:

(1) dissolving 5-7 parts of yeast extract and 10-15 parts of peptone, and sterilizing at 120-125 ℃ for 18-22min to obtain a solution A;

(2) dissolving 10-15 parts of glucose, and sterilizing at 110-115 ℃ for 13-16min to obtain a solution B;

s12, preparation of BSM medium:

mixing 50-70 parts of glycerol, 20-30 parts of phosphoric acid, 1-2 parts of trace elements and 0.5-1 part of biotin, adding deionized water, stirring uniformly, adding nutrients, and adjusting the pH to 5 by using ammonia water to obtain a BSM culture medium;

s2, fermentation expression:

(1) inoculating active bacteria into YPD culture medium, and culturing at 25-30 deg.C until colony grows out;

(2) putting the YPD culture medium into the cultured colony, and culturing in a shaking table at 28-30 deg.C and 200r/min to obtain seed solution;

(3) adding YPD culture medium and defoaming agent into fermentation tank, and sterilizing at 120 deg.C for 30 min;

(4) inoculating the seed liquid into a fermentation tank by using a flame inoculation method for fermentation, wherein the fermentation time is 10-12 h;

(5) adding BSM culture medium and defoaming agent into the induced expression fermentation tank, sterilizing at 120 deg.C for 30min, and then sterilizing with high pressure steam for 15 min;

(6) putting the seed liquid in the fermentation tank into an inducible expression fermentation tank, adding methanol, starting fermentation, and fermenting for 72h to obtain fermentation liquid;

s3, processing fermentation liquor:

Further, the preparation steps of the nutrient are as follows,

(1) adding ether into the obtained waste liquid, extracting, heating at 35 deg.C, centrifuging, filtering, collecting the upper layer product, and recovering the lower layer product;

(2) adding pyrrolidine and deionized water into the upper layer product, controlling the pH value to be 2.5-3.5, and carrying out contact glow discharge point electrolytic reaction to obtain a product A;

(3) and mixing the product A and the lower-layer product to obtain the nutrient.

Further, the absorbance (OD) of the seed solution obtained needs to be measured_600nm) Control of OD_600nmAt 4-6.

Further, the mass ratio of the amount of methanol added to the seed liquid in step S2(6) is 5-8: 1.

Further, the active bacteria are yeast and methanol-assimilating bacteria, and the ratio of the yeast to the methanol-assimilating bacteria is 5-6: 1.

Further, the yeast is pichia pastoris, and the methanol-assimilating bacterium is a methylophilus bacterium.

Further, the application of the tridecapeptide obtained by the method for obtaining the tridecapeptide through yeast fermentation in the dressing.

Further, mixing the obtained tridecapeptide with anthocyanin, natural tea tree oil and honeysuckle extract, adding glycerol, carbomer and deionized water, stirring uniformly, adding triethanolamine, and stirring to obtain the dressing.

Furthermore, the mass ratio of the tridecapeptide to the anthocyanin to the honeysuckle extract is 2:1: 1.

Compared with the prior art, the invention has the following beneficial effects: the tridecapeptide is a commonly used raw material in the field of cosmetics, is a polypeptide consisting of a plurality of amino acids, can promote the generation of new cells, repair scars on the skin surface of a human body and promote the growth of the cells when being used in the field of cosmetics, has more stable and safer performance compared with forbidden EGF, and is an ideal substitute for the EGF.

The tridecapeptide is produced mainly by chemical synthesis and microbial preparation, and the tridecapeptide is prepared by using a yeast fermentation method. The yeast used in the application is pichia pastoris, and the pichia pastoris is selected because the pichia pastoris is a facultative anaerobe, can survive in aerobic and anaerobic environments, has a high growth speed, is a methanol nutritional yeast, and can be regulated and controlled by adding methanol to the gene expression of the pichia pastoris, so that the pichia pastoris is selected when strains are selected, and the methanol is added to regulate and control.

The selection of the medium is important because it directly affects the growth, expression and gene stability of the yeast. According to the method, glucose is selected as a main carbon source during preparation of the culture medium, and nutrients such as inorganic salt and peptone are added to ensure normal growth of yeast.

The method uses pichia pastoris as a main body, controls gene expression of the pichia pastoris by adding methanol, can increase the yield of products, but needs to control the adding amount of the methanol, limits the mass ratio of the adding amount of the methanol to seed liquid to be optimal at 5-8:1, and can control the seed liquid according to OD (optical density) in the seed liquid_600nmThe value is judged and needs to be controlled between 4 and 6. The normal induction expression of the pichia pastoris can be ensured by controlling the amount of the added methanol, and the yield of the product can be obviously increased.

This application is by OD_600nmThe value can be used for judging the growth condition of Pichia pastoris, and OD is used_600nmThe amount of methanol added is controlled, but in order to realize large-scale continuous industrial production, methanol is continuously added, and the large amount of methanol addition and accumulation bring safety risks, so that the methanol assimilation bacteria are added on the basis of the original pichia pastoris.

The ratio of pichia pastoris to the methylophilus bacterium is controlled to be 5-6:1, so that most of methanol can be ensured to be used by the pichia pastoris, and the fermentation condition can be judged according to the growth condition of the methylophilus bacterium in the production process.

After the production is finished, fermentation liquor is obtained, the added Methylophilus bacteria can produce protein and formic acid, wherein the formic acid has certain corrosivity and can corrode skin to produce redness and swelling, therefore, the method can obtain a tridecapeptide product, a large biomolecule and formic acid by processing the obtained fermentation liquor for multiple times, sequentially separate the tridecapeptide product, the large biomolecule and the formic acid, and reprocess the formic acid, because the boiling point of the formic acid is higher, the method can obtain an amino acid compound by adding tetrahydropyrrole, adding acid liquor to control the pH to be 2.5-3.5, and adopting a contact glow discharge point electrolytic reaction, and then mix the amino acid compound with a lower-layer product, wherein the lower-layer product contains a large amount of large biomolecules, contains more protein, obtains nutrient, and contains glycine and proline, wherein the glycine has a certain inhibition effect on escherichia coli, the produced proline can regulate the osmotic balance of yeast and Methylophilus bacteria, and further can ensure the normal growth and gene expression of the yeast and Methylophilus bacteria.

The tridecapeptide prepared by the application is widely used in the field of preparation of dressings, and in the preparation process of the dressings, anthocyanin, natural tea tree oil and honeysuckle extract are added, wherein the anthocyanin is also called anthocyanidin, is a bioflavonoid substance and has a certain antioxidant capacity, the added natural tea tree oil has wide antibacterial, bacteriostatic and anti-inflammatory effects, and is a natural antibacterial and anti-inflammatory product which is effective in the market at present.

Detailed Description

The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all 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 1

A method for obtaining tridecapeptide by yeast fermentation comprises the following steps,

s1, preparation of medium:

s11, preparation of YPD Medium:

(1) dissolving 5 parts of yeast extract and 10 parts of peptone, and sterilizing at 120 ℃ for 18min to obtain a solution A;

(2) dissolving 10 parts of glucose, and sterilizing at 110 ℃ for 13min to obtain a solution B;

s12, preparation of BSM medium:

mixing 50 parts of glycerol, 20 parts of phosphoric acid, 1 part of trace elements and 0.5 part of biotin, adding deionized water, stirring uniformly, adding nutrients, and adjusting the pH to 5 by using ammonia water to obtain a BSM culture medium;

s2, fermentation expression:

(1) inoculating active bacteria into a YPD culture medium, and culturing at 25 ℃ until colonies grow, wherein the active bacteria are yeast and methanol assimilating bacteria, the ratio of the yeast to the methanol assimilating bacteria is 5:1, the yeast is pichia pastoris, and the methanol assimilating bacteria are bacteria of the genus Methylophilus;

(2) placing YPD culture medium into the cultured colony, culturing in shaking table at 28 deg.C and 200r/min to obtain seed solution, and testing absorbance (OD)_600nm) Control of OD_600nmAt 4;

(4) inoculating the seed liquid into a fermentation tank by using a flame inoculation method for fermentation, wherein the fermentation time is 10 h;

(6) putting the seed liquid in the fermentation tank into an inducible expression fermentation tank, adding methanol, wherein the mass ratio of the added methanol to the seed liquid is 5:1, starting fermentation, and fermenting for 72 hours to obtain fermentation liquid;

s3, processing fermentation liquor:

S4, the preparation steps of the nutrient are as follows,

(2) adding pyrrolidine and deionized water into the upper-layer product, controlling the pH value to be 2.5, and carrying out contact glow discharge point electrolytic reaction to obtain a product A;

Mixing the obtained tridecapeptide with anthocyanin, natural tea tree oil and honeysuckle extract, wherein the mass ratio of the tridecapeptide to the anthocyanin to the honeysuckle extract is 2:1:1, adding glycerol, carbomer and deionized water, stirring uniformly, adding triethanolamine, and stirring to obtain the dressing.

Example 2

s1, preparation of medium:

s11, preparation of YPD Medium:

(1) dissolving 6 parts of yeast extract and 13 parts of peptone, and sterilizing at 123 ℃ for 20min to obtain a solution A;

(2) dissolving 13 parts of glucose, and sterilizing at 113 ℃ for 15min to obtain a solution B;

s12, preparation of BSM medium:

mixing 60 parts of glycerol, 25 parts of phosphoric acid, 1.5 parts of trace elements and 0.8 part of biotin, adding deionized water, stirring uniformly, adding nutrients, and adjusting the pH to 5 by using ammonia water to obtain a BSM culture medium;

s2, fermentation expression:

(1) inoculating active bacteria into a YPD culture medium, and culturing at 27 ℃ until colonies grow, wherein the active bacteria are yeast and methanol assimilating bacteria, the ratio of the yeast to the methanol assimilating bacteria is 5.5:1, the yeast is pichia pastoris, and the methanol assimilating bacteria are bacteria of the genus Methylophilus;

(2) placing YPD culture medium into the cultured colony, culturing in shaker at 29 deg.C and 200r/min to obtain seed solution, and testing absorbance (OD)_600nm) Control of OD_600nmAt 5;

(4) inoculating the seed liquid into a fermentation tank by using a flame inoculation method for fermentation, wherein the fermentation time is 11 h;

(6) putting the seed liquid in the fermentation tank into an inducible expression fermentation tank, adding methanol, wherein the mass ratio of the added methanol to the seed liquid is 6:1, starting fermentation, and fermenting for 72 hours to obtain fermentation liquid;

s3, processing fermentation liquor:

S4, the preparation steps of the nutrient are as follows,

(2) adding pyrrolidine and deionized water into the upper-layer product, controlling the pH value to be 3.0, and carrying out contact glow discharge point electrolytic reaction to obtain a product A;

Example 3

s1, preparation of medium:

s11, preparation of YPD Medium:

(1) dissolving 7 parts of yeast extract and 15 parts of peptone, and sterilizing at 125 ℃ for 22min to obtain a solution A;

(2) dissolving 15 parts of glucose, and sterilizing at 115 ℃ for 16min to obtain a solution B;

s12, preparation of BSM medium:

mixing 70 parts of glycerol, 30 parts of phosphoric acid, 2 parts of trace elements and 1 part of biotin, adding deionized water, stirring uniformly, adding nutrients, and adjusting the pH to 5 by using ammonia water to obtain a BSM culture medium;

s2, fermentation expression:

(1) inoculating active bacteria into a YPD culture medium, and culturing at 30 ℃ until colonies grow, wherein the active bacteria are yeast and methanol assimilating bacteria, the ratio of the yeast to the methanol assimilating bacteria is 6:1, the yeast is pichia pastoris, and the methanol assimilating bacteria are bacteria of the genus Methylophilus;

(2) placing YPD culture medium into the cultured colony, culturing in shaking table at 30 deg.C and 200r/min to obtain seed solution, and testing absorbance (OD)_600nm) Control of OD_600nmAt 6;

(4) inoculating the seed liquid into a fermentation tank by using a flame inoculation method for fermentation, wherein the fermentation time is 12 h;

(6) putting the seed liquid in the fermentation tank into an inducible expression fermentation tank, adding methanol, wherein the mass ratio of the added methanol to the seed liquid is 8:1, starting fermentation, and fermenting for 72 hours to obtain fermentation liquid;

s3, processing fermentation liquor:

S4, the preparation steps of the nutrient are as follows,

(2) adding pyrrolidine and deionized water into the upper-layer product, controlling the pH value to be 3.5, and carrying out contact glow discharge point electrolytic reaction to obtain a product A;

Comparative example 1

s1, preparation of medium:

s11, preparation of YPD Medium:

s12, preparation of BSM medium:

mixing 70 parts of glycerol, 30 parts of phosphoric acid, 2 parts of trace elements and 1 part of biotin, adding deionized water, uniformly stirring, and adjusting the pH to 5 by using ammonia water to obtain a BSM culture medium;

s2, fermentation expression:

(1) inoculating active bacteria into a YPD culture medium, and culturing at 30 ℃ until colonies grow out, wherein the active bacteria are yeasts, and the yeasts are pichia pastoris;

s3, processing fermentation liquor:

and separating the obtained product by using an ultrafiltration membrane to obtain the tridecapeptide.

Comparative example 2

s1, preparation of medium:

s11, preparation of YPD Medium:

s12, preparation of BSM medium:

s2, fermentation expression:

s3, processing fermentation liquor:

Comparative example 3

s1, preparation of medium:

s11, preparation of YPD Medium:

s12, preparation of BSM medium:

s2, fermentation expression:

(1) inoculating active bacteria in YPD medium, and culturing at 30 deg.C until colonies grow, wherein the active bacteria are yeast and methanol-assimilating bacteria at a ratio of 6:1, and the methanol-assimilating bacteria are bacteria of genus Methylophilus;

s3, processing fermentation liquor:

S4, the preparation steps of the nutrient are as follows,

Comparative example 4

s1, preparation of medium:

s11, preparation of YPD Medium:

s12, preparation of BSM medium:

s2, fermentation expression:

(6) putting the seed liquid in the fermentation tank into an inducible expression fermentation tank, starting fermentation, and fermenting for 72h to obtain fermentation liquid;

s3, processing fermentation liquor:

S4, the preparation steps of the nutrient are as follows,

Experiment of

The comparative examples 1, 2 and 3 were set up by using example 3 as a control, wherein the strains of example 1, 2, 3, 1, 2 and 3 were inoculated in the same amount, and a control experiment was conducted in which no methanol-assimilating bacterium was added in comparative example 1, formic acid was not treated in comparative example 2, a common yeast was used in comparative example 3, and no methanol was added in comparative example 4.

The fermentation liquids obtained in example 1, example 2, example 3, comparative example 1, comparative example 2, comparative example 3 and comparative example 4 were analyzed, and as a result,

experimental group	Peptides	Protein	Formic acid
				Example 1	Detect out	Detect out	Detect out
Example 2	Detect out	Detect out	Detect out
				Example 3	Detect out	Detect out	Detect out
Comparative example 1	Detect out	Detect out	Not detected out
				Comparative example 2	Detect out	Detect out	Detect out
Comparative example 3	Detect out	Detect out	Detect out
				Comparative example 4	Detect out	Detect out	Detect out

Watch 1

The dressings obtained in example 1, example 2, example 3, comparative example 1, comparative example 2, comparative example 3, and comparative example 4 were analyzed for formic acid content, and as a result,

experimental group

Example 1

Example 2

Example 3

Comparative example 1

Comparative example 2

Comparative example 3

Comparative example 4

Formic acid

Not detected out

Detect out

Not detected out

Watch two

The nutrients of example 1, example 2, example 3, comparative example 1, comparative example 2, comparative example 3, and comparative example 4 were analyzed, and the results were as follows,

experimental group	Formic acid	Tetrahydropyrroles	Glycine	Proline
					Example 1	Not detected out	Not detected out	Detect out	Detect out
Example 2	Not detected out	Not detected out	Detect out	Detect out
					Example 3	Not detected out	Not detected out	Detect out	Detect out
Comparative example 1	---	---	---	---
					Comparative example 2	---	---	---	---
Comparative example 3	Not detected out	Not detected out	Detect out	Detect out
					Comparative example 4	Not detected out	Not detected out	Detect out	Detect out

Watch III

The dressings obtained in example 1, example 2, example 3, comparative example 1, comparative example 2, comparative example 3 and comparative example 4 were subjected to the escherichia coli bacteriostatic performance test, and the results were as follows,

experimental group	Example 1	Example 2	Example 3	Comparative example 1	Comparative example 2	Comparative example 3	Comparative example 4
								The antibacterial rate is%	94	95	94	91	94	93	90

Watch four

As can be seen from the Table I, the fermentation liquids of examples 1, 2, 3, 4 contain a certain amount of formic acid because formic acid is generated with methanol as a main carbon source with the added methanol-assimilating bacteria, and thus the formic acid contained in the product may cause damage to human skin if the fermentation liquids are not treated.

As can be seen from table two, the dressings of examples 1, 2, 3, 1, 3, and 4 do not contain formic acid, and formic acid can be detected in comparative example 2, which further illustrates that the dressings prepared in examples 1, 2, and 3 have high safety.

In the third table, it can be seen that formic acid and tetrahydropyrrole are not detected in the nutrients of example 1, example 2, example 3, comparative example 3 and comparative example 4, glycine and proline can be detected, and can promote the growth of microorganisms, so as to achieve the purpose of improving the yield.

As can be seen in Table IV, the bacteriostatic ratios of example 1, example 2, example 3, comparative example 1, comparative example 2, comparative example 3 and comparative example 4 to Escherichia coli are all above 90%, which indicates that the dressing prepared by the application has excellent antibacterial performance, but the bacteriostatic ratio of comparative example 4 is lower than that of example 1, example 2 and example 3, but methanol is not added in comparative example 4, but a slight content can be detected in the detection of glycine and proline, which indicates that the growth rate of the methanol-assimilating bacteria is lowest in an environment without methanol, i.e., the methanol-assimilating bacteria grows slowly or even does not grow in a condition with only a glucose carbon source, and indicates that the application selects the correctness of adding the methanol-assimilating bacteria.

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for obtaining tridecapeptide by yeast fermentation, characterized in that: the steps are as follows,

s1, preparation of medium:

s11, preparation of YPD Medium:

(1) dissolving yeast extract and peptone, and sterilizing to obtain solution A;

(2) dissolving glucose, and sterilizing to obtain solution B;

s12, preparation of BSM medium:

s2, fermentation expression:

s3, processing fermentation liquor:

2. Method for obtaining tridecapeptide by yeast fermentation according to claim 1, characterized in that: the specific steps are as follows,

s1, preparation of medium:

s11, preparation of YPD Medium:

s12, preparation of BSM medium:

s2, fermentation expression:

s3, processing fermentation liquor:

3. Method for obtaining tridecapeptide by yeast fermentation according to claim 2, characterized in that: the preparation steps of the nutrient are as follows,

4. Method for obtaining tridecapeptide by yeast fermentation according to claim 2, characterized in that: after obtaining the seed liquid, the absorbance (OD) of the seed liquid needs to be tested_600nm) Control of OD_600nmAt 4-6.

5. Method for obtaining tridecapeptide by yeast fermentation according to claim 2, characterized in that: the mass ratio of the amount of methanol added to the seed liquid in the step S2(6) is 5-8: 1.

6. Method for obtaining tridecapeptide by yeast fermentation according to claim 2, characterized in that: the active bacteria are yeast and methanol assimilating bacteria, and the ratio of the yeast to the methanol assimilating bacteria is 5-6: 1.

7. A method for obtaining tridecapeptide by yeast fermentation according to claim 6, characterized in that: the methanol-assimilating bacterium is a Methylophilus bacterium.

8. Use of tridecapeptide obtained by fermentation with yeast according to one of claims 1 to 7 in dressings.

9. Use of tridecapeptide obtained by fermentation with yeast according to one of claims 1 to 7, characterized in that: mixing the obtained tridecapeptide with anthocyanin, natural tea tree oil and honeysuckle extract, adding glycerol, carbomer and deionized water, stirring uniformly, adding triethanolamine, and stirring to obtain the dressing.

10. Use according to claim 8, characterized in that: the mass ratio of the tridecapeptide to the anthocyanin to the honeysuckle extract is 2:1: 1.