CN114058665A

CN114058665A - Preparation method of composite flower peptide and application of composite flower peptide in preparation of cosmetics

Info

Publication number: CN114058665A
Application number: CN202111462543.1A
Authority: CN
Inventors: 张大勇; 刘芸芸; 孔英俊; 陈江
Original assignee: Zhejiang Yige Enterprise Management Group Co ltd
Current assignee: Zhejiang Yige Enterprise Management Group Co ltd
Priority date: 2021-12-02
Filing date: 2021-12-02
Publication date: 2022-02-18

Abstract

The scheme discloses a preparation method of composite flower peptide, which comprises the following steps: using various flowers as raw materials, and respectively preparing each raw material flower protein by an alkali-soluble acid-precipitation method; obtaining enzymolysis liquid of each raw material flower protein under the action of protease corresponding to each raw material flower; fermenting the enzymolysis liquid of each raw material flower protein by bifidobacterium to obtain enzymolysis fermentation liquid of different raw material flower proteins; filtering and drying the enzymolysis fermentation liquor to obtain the flower peptide of each raw material flower; the flower peptide of various raw material flowers is mixed to obtain the composite flower peptide. The method utilizes enzymolysis-fermentation combined technology to directionally extract and prepare the small molecular active peptide, and the product has definite main components, low molecular weight, good safety and remarkable biological activity.

Description

Preparation method of composite flower peptide and application of composite flower peptide in preparation of cosmetics

Technical Field

The invention relates to the field of preparation of plant-derived active peptide flower and application of the plant-derived active peptide flower in cosmetics, in particular to a preparation method of composite peptide flower and application of the composite peptide flower in preparation of cosmetics.

Background

The protein is an important component of plants, and the activity of the peptide prepared by the protein in the plants through a certain process in the aspects of oxidation resistance, antibiosis and the like is proved and has more and more extensive application. Compared with synthetic peptide, the active peptide of plant source has certain advantages in the aspects of safety, toxicity and the like.

The flower is an important component of the plant, the contained protein plays an important role in the growth of the flower and the whole plant, the application of the flower extract in skin care products is very wide, but the peptide component extracted from the flower is fresh and common when being used in the cosmetics.

Disclosure of Invention

One purpose of the scheme is to provide a preparation method of composite flower peptide, which obtains the composite flower peptide containing various flower peptides by an enzymolysis-fermentation combined technology, and the skin care product containing the composite flower peptide has good anti-aging effect.

In order to achieve the purpose, the scheme is as follows:

a preparation method of composite flower peptide comprises the following steps:

using various flowers as raw materials, and respectively preparing each raw material flower protein by an alkali-soluble acid-precipitation method;

obtaining enzymolysis liquid of each raw material flower protein under the action of protease corresponding to each raw material flower;

fermenting the enzymolysis liquid of each raw material flower protein by bifidobacterium to obtain enzymolysis fermentation liquid of different raw material flower proteins; filtering and drying the enzymolysis fermentation liquor to obtain the flower peptide of each raw material flower;

the flower peptide of various raw material flowers is mixed to obtain the composite flower peptide.

Preferably, the method further comprises pulverizing each raw material flower by a pretreatment including heat treatment, ultrasonic treatment or high pressure treatment.

Preferably, the alkali-dissolution acid-precipitation method comprises:

mixing each raw material pollen obtained through pretreatment with water according to the mass ratio of the material liquid of 1: 20-1: 30;

beating the mixed material liquid into uniform slurry;

adding a sodium carbonate solution into the mixture solution, standing, centrifuging, filtering, and adjusting the pH value of the filtrate;

adding glacial acetic acid or hydrochloric acid into the filtrate, adjusting the pH value of the solution to 4-5, standing for 12-17 h at 5 ℃, centrifuging to remove supernatant to obtain precipitate, and precipitating to obtain the raw material, namely the floral protein.

Preferably, when the raw material flowers are lily, the mass ratio of the added sodium carbonate solution to the raw material pollen is 1: 1-2: 1, and the concentration of the sodium carbonate solution is 0.5 mol/L; standing for 1.5-2.5 h, centrifuging at a rotating speed of 5000r/min for 20min, and adjusting the pH value of the filtrate to 9-10 after centrifugal filtration;

when the raw material flowers are peony flowers, the mass ratio of the added sodium carbonate solution to the raw material pollen is 2: 1-3: 1, the standing time is 1.5-2.5 h, the centrifugal rotation speed is 5000r/min, the centrifugal time is 20min, and the pH value of the filtrate is adjusted to 6-7 after centrifugal filtration.

Preferably, the enzymatic hydrolysate for obtaining different raw material flower proteins under the action of different proteases comprises:

adding each raw material flower protein, protease suitable for the flower and water into a reaction container, fully dispersing reactants, adjusting the pH value of the solution, and heating in a water bath; centrifuging, and collecting supernatant to obtain enzymolysis solution of the material, i.e. the floral protein.

Preferably, when the raw material flower is lily, the protease is Alcalase alkaline protease, the enzyme activity is more than 600000U/g, and the enzyme-substrate ratio is more than 5000U/g;

when the raw material flower is peony, the protease is Neutrase protease, the enzyme activity is more than 300000U/g, and the enzyme substrate ratio is more than 5000U/g.

Preferably, when the raw material flowers are lily, the pH value of the solution is adjusted to 8-8.5 before water bath heating; when the raw material flowers are peony flowers, adjusting the pH value of the solution to 6-7 before heating in a water bath;

the water bath heating comprises two continuous heating stages at different temperatures, wherein the water bath heating temperature in the first stage is 50-55 ℃, and the heating time is 3-4 hours; the temperature of the second stage of water bath heating is 90-95 ℃, and the heating time is 15-20 min.

Preferably, the enzymolysis liquid of each raw material flower protein is fermented by bifidobacterium to obtain enzymolysis fermentation liquid of different raw material flower proteins, and the enzymolysis fermentation liquid is filtered and dried to obtain the flower peptide of each raw material flower, which comprises:

centrifuging and filtering the enzymolysis fermentation liquor at normal temperature, performing ultrafiltration and classification on the obtained filtrate to obtain a filtrate, and performing freeze drying on the filtrate with the relative molecular mass of more than 80% of components in the filtrate being below 200Da through molecular weight test to obtain the raw material floridin.

Preferably, the step of mixing the flower peptides of the plurality of raw material flowers to obtain the composite flower peptide comprises the step of mixing the first raw material flower peptide and the second raw material flower peptide according to the mass ratio of 1: 1-10: 1 to obtain the composite flower peptide.

In a second aspect, the use of a complex peptide prepared according to any one of the above-described methods for preparation of a cosmetic.

The scheme has the following beneficial effects:

the method utilizes enzymolysis-fermentation combined technology to directionally extract the active peptide, has clear main components of the product, greatly reduces the safety risk, has obvious biological activity, fully utilizes resources of peony and lily and improves the cost performance of the product; and through screening different pretreatment modes, the yield of the protein in the alkali extraction and acid precipitation process is improved, and the full utilization of resources is facilitated; different proteases are screened to obtain a product with lower molecular weight and stronger oxidation resistance, so that the cost performance of the product is improved; compared with the screening and drying process, the antioxidant and anti-aging effects of the product are improved.

Drawings

In order to illustrate the implementation of the solution more clearly, the drawings that are needed in the description of the embodiments will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the solution, and that other drawings may be derived from these drawings by a person skilled in the art without inventive effort.

FIG. 1 is a schematic representation of the crude protein content of each example;

FIG. 2 is a graph showing ORAC values of antioxidant ability of each example;

FIG. 3 is a graph showing the inhibition of beta-galactosidase activity in each example;

FIG. 4A is a schematic diagram of the synergistic effect of the combination of lily peptide and peony peptide;

FIG. 4B is a schematic diagram of the synergistic effect of the combination of lily peptide and peony peptide;

FIG. 5 shows the chorioallantoic membrane blood vessel experiment of peptide chicken embryo by enzymolysis;

FIG. 6 shows the allantoic membrane blood vessel experiment of peptide chick embryo by enzymolysis-fermentation method.

Detailed Description

Embodiments of the present solution will be described in further detail below with reference to the accompanying drawings. It is clear that the described embodiments are only a part of the embodiments of the present solution, and not an exhaustive list of all embodiments. It should be noted that, in the present embodiment, features of the embodiment and the embodiment may be combined with each other without conflict.

The terms "first," "second," and the like in the description and in the claims, and in the drawings described above, if any, are used for distinguishing between similar elements and not necessarily for describing a particular sequential or chronological order. It will be appreciated that the data so used may be interchanged under appropriate circumstances such that the embodiments described herein may be practiced otherwise than as specifically illustrated or described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed, but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.

It should be understood that the term "and/or" as used herein is merely one type of association that describes an associated object, meaning that three relationships may exist, e.g., a and/or B may mean: a exists alone, A and B exist simultaneously, and B exists alone. In addition, the character "/" herein generally indicates that the former and latter related objects are in an "or" relationship.

The preparation of the active peptide from the plant source has multiple ways, such as hydrolysis method, enzymolysis method and the like, and the preparation of the active peptide by the enzymolysis method has the advantages of environmental protection, low cost and high activity. In the enzymolysis method, the pretreatment mode, the protease type, the material-liquid ratio, the leaching temperature, the separation mode and other process links have certain influence on the molecular weight and the activity of the product. The inventor researches to find that if the enzymolysis method is used, the protein in the target plant needs to be extracted first, and the product obtained by the currently common enzymolysis method has certain safety risk and high proportion of large molecular weight in the molecular weight distribution. Accordingly, the inventors of the present application provide a method for preparing a composite flower peptide, comprising the steps of:

1. pretreatment of

Respectively crushing the peony and the lily, and pretreating the peony and the lily by using microwaves, wherein the microwave power is 300W, and the microwave time is 3-7 minutes. The pretreatment process is not limited to microwave treatment, and includes heat treatment, ultrasonic treatment, high-pressure treatment, and the like.

2. Protein production

The peony protein and lily protein are prepared by an alkali-soluble acid-precipitation method.

Component 1: lily peptide protein

Alkali dissolution: mixing the pretreated lily powder (the mass of the pollen is M1) according to a material-liquid ratio of 1:20 to 1:30, adding the mixture into water, and beating the feed liquid into uniform slurry by using a wall breaking machine. Adding a sodium carbonate solution with the concentration of 0.5mol/L (the mass of the sodium carbonate solution is M) (M: M1 is 1: 1-2: 1), standing for 2 hours, centrifuging and filtering, wherein the centrifugation speed is 5000r/min, centrifuging for 20min, and the pH value of the filtrate is 9-10;

acid precipitation: adding glacial acetic acid or hydrochloric acid into the filtrate, adjusting pH to 4.6, standing in a refrigerator at 5 deg.C for 15 hr, centrifuging at a rate of 5000r/min for 20min, and removing supernatant to obtain precipitate, i.e. lily peptide.

And (2) component: peony protein

Alkali dissolution: and (3) mixing the pretreated peony powder (the mass of the pollen is M2) according to a material-liquid ratio of 1: 26, adding the mixture into water, and beating the feed liquid into uniform slurry by using a wall breaking machine. Adding a sodium carbonate solution with the concentration of 0.5mol/L (the mass of the sodium carbonate solution is M) (M: M2 is 2: 1-3: 1), standing for 2 hours, centrifuging and filtering, wherein the centrifugation speed is 5000r/min, centrifuging for 20min, and the pH value of the filtrate is 6-7;

acid precipitation: adding glacial acetic acid or hydrochloric acid into the filtrate, adjusting pH to 4.6, standing in a refrigerator at 5 deg.C for 15 hr, centrifuging at a speed of 5000r/min for 20min, and removing supernatant to obtain precipitate, i.e. peony protein.

3. Enzymolysis

And (3) component: lily enzymatic hydrolysate

Adding water, lily protein (component 1) accounting for 5% of the total mass of the enzymatic hydrolysate, Alcalase alkaline protease (enzyme activity is more than 60 ten thousand U/g, enzyme substrate ratio is more than 5000U/g) and other materials into a reaction container, fully dispersing the materials by using a water bath oscillator, adjusting the pH value to 8-8.5, heating in a water bath at 55 ℃ for 3-4 hours, inactivating the protease by using a water bath at 90-95 ℃ for 15-20 min, refrigerating at 0-5 ℃ for over night, centrifugally removing impurities, and taking supernatant to obtain lily enzymatic hydrolysate; in this step, the selected enzyme is not limited to Alcalase alkaline protease, and may include animal protease, plant protease, microbial protease, etc., and the relevant technological parameters, pH value, temperature and reaction time, are adjusted correspondingly according to the kind of enzyme.

And (4) component: peony enzymolysis liquid

Adding water, peony protein (component 2) accounting for 5% of the total mass of the enzymolysis liquid, Neutrase protease (enzyme activity is more than 30 ten thousand U/g, enzyme bottom is more than 5000U/g) and other materials into a reaction container, fully dispersing the materials by using a water bath oscillator, adjusting the pH value to 6-7, heating in a water bath at 50 ℃ for 3-4 hours, using a water bath at 90-95 ℃ for 15-20 minutes to inactivate the protease, refrigerating in a refrigerator at 0-5 ℃ overnight, centrifuging to remove impurities, and taking supernatant to obtain the peony enzymolysis liquid.

In the step, the selected enzyme is not limited to Alcalase alkaline protease, and can comprise animal protease, plant protease, microbial protease and the like, and relevant process parameters such as pH value, temperature and reaction time are correspondingly adjusted according to the enzyme type;

4. fermentation of

And (5) component: lily enzymolysis fermentation liquor

(1) Preparing a fermentation medium

Mixing 0.1-1.0% of agar, 1-3% of glucose, 1-0.3% of tween-80, 0.1-0.5% of citric acid and 89.7-97.8% of lily enzymatic hydrolysate according to the mass percentage of all substances in the total mass of the culture medium to prepare a fermentation culture medium, and performing high-temperature sterilization and cooling at room temperature for later use;

(2) preparation of lily enzymolysis fermentation liquor

Inoculating bifidobacterium into a sterilized fermentation medium, and performing anaerobic fermentation culture; during inoculation, the mass ratio of the bifidobacterium seed liquid to the fermentation medium is 1-3: 50; the anaerobic fermentation condition is that the fermentation is carried out for 20-24 h at 36-38 ℃;

and (4) component 6: peony enzymolysis fermentation liquor

(1) Preparing a fermentation medium

Mixing 0.1-1.0% of agar, 1-3% of glucose, 1-0.3% of tween-80 and 89.7-97.8% of peony enzymatic hydrolysate according to the mass percentage of the substances in the total mass of the culture medium to prepare a fermentation culture medium, and performing high-temperature sterilization and cooling at room temperature for later use;

(2) preparation of peony enzymolysis fermentation liquor

after screening, each raw material flower is subjected to alkali dissolution and acid precipitation enzymolysis by using protease corresponding to the flower, and then probiotic fermentation is carried out, so that the safety can be improved, and the product can be applied to cosmetics;

5. separation of

And (4) component 7: lily peptide

Centrifuging the component 5 at the rotating speed of 2500-3500 rmp/min for 20-30 min, sieving with a 300-mesh sieve, filtering, performing ultrafiltration grading on the filtrate, testing the molecular weight at normal temperature under the operating pressure of 0.35Mpa, selecting an ultrafiltration component with the relative molecular mass of less than 200Da accounting for more than 80%, and freeze-drying to obtain the lily peptide. In this step, the drying mode may further include spray drying, hot air drying, and the like;

and (4) component 8: peony peptide

Centrifuging the component 6 at the rotating speed of 2500-3500 rmp/min for 20-30 min, filtering through a 300-mesh sieve, carrying out ultrafiltration grading on the filtrate, testing the molecular weight at normal temperature under the operating pressure of 0.35Mpa, selecting an ultrafiltration component with the relative molecular mass of less than 200Da accounting for more than 80%, and carrying out freeze drying to obtain the lily peptide. In this step, the drying method may further include spray drying, hot air drying, and the like;

6. preparation of composite flower peptide

Mixing the component 7 and the component 8 according to the mass ratio of 1:1 to 10:1, testing DPPH free radical clearance and anti-aging efficacy evaluation, and selecting a composition with good efficacy evaluation effect, namely the composite flower peptide.

The prepared composite flower peptide is used in cosmetics and has obvious anti-aging effect.

The present invention will be further described with reference to the following specific examples.

Example 1

1. Respectively crushing the peony and the lily, and pretreating the peony and the lily by using microwaves, wherein the microwave power is 300W, and the microwave time is 5 minutes;

2. adding 120g of pretreated lily powder into 2500g of water, beating feed liquid into uniform slurry by using a wall breaking machine, adding 225g of sodium carbonate solution with the concentration of 0.5mol/L, adjusting the pH value of the feed liquid to 10.2-10.3, standing for 2 hours, carrying out centrifugal filtration at the centrifugal rate of 5000r/min for 20min, and adjusting the pH value of filtrate to 9.56; adding hydrochloric acid into the filtrate, adjusting pH to 4.6, standing in a refrigerator at 5 deg.C for 15 hr, centrifuging at a rate of 5000r/min, centrifuging for 20min to remove supernatant to obtain precipitate 125 g, i.e. lily peptide, and measuring lily protein content;

adding 120g of pretreated peony powder into 3000g of water, and beating the feed liquid into uniform slurry by using a wall breaking machine. Adding 350g of sodium carbonate solution with the concentration of 0.5mol/L, adjusting the pH value of the feed liquid to 10-10.2, standing for 2 hours, carrying out centrifugal filtration at the centrifugal rate of 5000r/min for 20min, and adjusting the pH value of the filtrate to 6-7;

adding hydrochloric acid and the like into the filtrate, adjusting the pH value to be 4.6, standing in a refrigerator at 5 ℃ for 15 hours, centrifuging and filtering at the centrifugation speed of 5000r/min, centrifuging for 20min, removing supernatant to obtain 660 g of precipitate, namely peony protein, and measuring the content of the lily protein;

3. adding water, lily protein accounting for 5% of the total mass of the enzymatic hydrolysate, Alcalase alkaline protease (enzyme activity is more than 60 ten thousand U/g, and enzyme-substrate ratio is more than 5000U/g) and other materials into a reaction container, fully dispersing the materials by using a water bath oscillator, adjusting the pH value to 8-8.5, heating in a water bath at the temperature of 55 ℃ for 3 hours, using a water bath at the temperature of 95 ℃ for 15min to inactivate the protease, refrigerating at the temperature of 0-5 ℃ overnight in a refrigerator, centrifuging to remove impurities, and taking supernatant to obtain lily enzymatic hydrolysate;

adding water, peony protein accounting for 5% of the total mass of the enzymolysis liquid, Neutrase neutral protease (the enzyme activity is more than 30 ten thousand U/g, and the enzyme bottom is more than 5000U/g) and other materials into a reaction container, fully dispersing the materials by using a water bath oscillator, adjusting the pH value to 8-8.5, heating in a water bath at 37 ℃ for 4 hours, using a water bath at 95 ℃ for 15min to inactivate the protease, refrigerating at 0-5 ℃ overnight in a refrigerator, centrifuging to remove impurities, and taking supernatant to obtain peony enzymolysis liquid;

4. preparation of Lily enzymolysis fermentation broth

(1) Preparing a fermentation medium

(2) preparation of lily enzymolysis fermentation liquor

Inoculating bifidobacterium into a sterilized fermentation medium, and performing anaerobic fermentation culture; during inoculation, the mass ratio of the bifidobacterium seed liquid to the fermentation medium is 1: 50; fermenting for 20-24 h at 36-38 ℃ under the anaerobic fermentation condition to obtain lily enzymolysis fermentation liquor;

5. obtaining lily peptide

Centrifuging the lily enzymolysis fermentation liquor, wherein the centrifugation rotation speed is 2500-3500 rmp/min, the centrifugation time is 20-30 min, sieving the lily enzymolysis fermentation liquor by a 300-mesh sieve to obtain filtrate, carrying out ultrafiltration grading on the filtrate, carrying out ultrafiltration grading at the operation pressure of 0.35Mpa, testing the molecular weight at normal temperature, selecting an ultrafiltration component with the relative molecular mass of less than 200Da and accounting for more than 80%, and carrying out spray drying to obtain lily peptide;

6. preparation of peony enzymolysis fermentation liquor

(1) Preparing a fermentation medium

(2) preparation of peony enzymolysis fermentation liquor

Inoculating bifidobacterium into a sterilized fermentation medium, and performing anaerobic fermentation culture; during inoculation, the mass ratio of the bifidobacterium seed liquid to the fermentation medium is 1: 50; carrying out anaerobic fermentation for 20-24 h at the temperature of 36-38 ℃ to obtain peony enzymolysis fermentation liquor;

7. obtaining peony flower peptide

Centrifuging the peony enzymolysis fermentation liquor, wherein the centrifugation rotation speed is 2500-3500 rmp/min, the centrifugation time is 20-30 min, filtering through a 300-mesh sieve to obtain filtrate, carrying out ultrafiltration grading on the filtrate, carrying out operation pressure of 0.35Mpa, testing the molecular weight at normal temperature, selecting an ultrafiltration component with the relative molecular weight of less than 200Da accounting for more than 80%, and carrying out spray drying to obtain peony peptide;

8. mixing lily peptide and peony peptide according to the weight ratio of 10:1, testing the DPPH free radical clearance and the anti-aging efficacy evaluation to obtain the composite flower peptide.

Example 2

2. adding 120g of the pretreated lily powder into 2500g of water, and stirring the feed liquid into uniform slurry by using a wall breaking machine. Adding 225g of sodium carbonate solution with the concentration of 0.5mol/L, adjusting the pH value of the feed liquid to 10.2-10.3, standing for 2 hours, carrying out centrifugal filtration at the centrifugal rate of 5000r/min for 20min, and adjusting the pH value of the filtrate to 9.56;

adding glacial acetic acid into the filtrate, adjusting pH to 4.6, standing in a refrigerator at 5 deg.C for 15 hr, centrifuging at a rate of 5000r/min, centrifuging for 20min to remove supernatant to obtain precipitate 125 g, i.e. lily peptide, and measuring lily protein content.

adding glacial acetic acid and the like into the filtrate, adjusting the pH value to be 4.6, standing in a refrigerator at 5 ℃ for 15 hours, centrifuging and filtering at the centrifugation speed of 5000r/min, centrifuging for 20min, removing supernatant to obtain 660 g of precipitate, namely peony protein, and measuring the content of the lily protein;

3. adding water, lily protein accounting for 5% of the total mass of the enzymatic hydrolysate, Alcalase alkaline protease (enzyme activity is more than 60 ten thousand U/g, enzyme-substrate ratio is more than 5000U/g) and other materials into a reaction container, fully dispersing the materials by using a water bath oscillator, adjusting the pH value to 8-8.5, heating in a water bath at the temperature of 55 ℃ for 3 hours, using a water bath at the temperature of 95 ℃ for 15 minutes, inactivating the protease, refrigerating at the temperature of 0-5 ℃ overnight in a refrigerator, centrifuging to remove impurities, and taking supernatant fluid to obtain the lily enzymatic hydrolysate.

4. preparation of Lily enzymolysis fermentation broth

(1) Preparing a fermentation medium

(2) preparation of lily enzymolysis fermentation liquor

5. obtaining lily peptide

6. preparation of peony enzymolysis fermentation liquor

(1) Preparing a fermentation medium

(2) preparation of peony enzymolysis fermentation liquor

7. obtaining peony flower peptide

Example 3

1. Respectively crushing the peony and the lily, and pretreating the peony and the lily by adopting a high pressure method and a heat treatment method under the pressure of 50kPa at the temperature of 110 ℃ for 5 minutes;

adding glacial acetic acid into the filtrate, adjusting pH to 4.6, standing in a refrigerator at 5 deg.C for 15 hr, centrifuging at a rate of 5000r/min for 20min, removing supernatant to obtain precipitate 125 g, i.e. lily peptide, and measuring lily protein content;

adding 120g of pretreated peony powder into 3000g of water, and beating the feed liquid into uniform slurry by using a wall breaking machine. Adding 350g of sodium carbonate solution with the concentration of 0.5mol/L, adjusting the pH value of the feed liquid to 10-10.2, standing for 2 hours, carrying out centrifugal filtration at the centrifugal rate of 5000r/min, adjusting the centrifugation for 20min, and adjusting the pH value of the filtrate to 6-7;

4. preparation of Lily enzymolysis fermentation broth

(1) Preparing a fermentation medium

(2) preparation of lily enzymolysis fermentation liquor

5. obtaining lily peptide

6. preparation of peony enzymolysis fermentation liquor

(1) Preparing a fermentation medium

(2) preparation of peony enzymolysis fermentation liquor

7. obtaining peony flower peptide

Example 4

adding water, peony protein accounting for 5% of the total mass of the enzymolysis liquid, Neutrase neutral protease (the enzyme activity is more than 30 ten thousand U/g, and the enzyme bottom is more than 5000U/g) and other materials into a reaction container, fully dispersing the materials by using a water bath oscillator, adjusting the pH value to 6-7, heating in a water bath at the temperature of 50 ℃ for 3 hours, inactivating the protease by using a water bath at the temperature of 95 ℃ for 15 minutes, refrigerating at the temperature of 0-5 ℃ for over night in a refrigerator, centrifuging to remove impurities, and taking supernatant to obtain peony enzymolysis liquid;

4. preparation of Lily enzymolysis fermentation broth

(1) Preparing a fermentation medium

(2) preparation of lily enzymolysis fermentation liquor

5. obtaining lily peptide

6. preparation of peony enzymolysis fermentation liquor

(1) Preparing a fermentation medium

(2) preparation of peony enzymolysis fermentation liquor

7. obtaining peony flower peptide

Example 5

4. preparation of Lily enzymolysis fermentation broth

(1) Preparing a fermentation medium

(2) preparation of lily enzymolysis fermentation liquor

5. obtaining lily peptide

6. preparation of peony enzymolysis fermentation liquor

(1) Preparing a fermentation medium

(2) preparation of peony enzymolysis fermentation liquor

7. obtaining peony flower peptide

Example 6

adding the pretreated peony flower powder 120 into 3000g of water, and beating the feed liquid into uniform slurry by using a wall breaking machine. Adding 350g of sodium carbonate solution with the concentration of 0.5mol/L, adjusting the pH value of the feed liquid to 10-10.2, standing for 2 hours, carrying out centrifugal filtration at the centrifugal rate of 5000r/min for 20min, and adjusting the pH value of the filtrate to 6-7;

4. preparation of Lily enzymolysis fermentation broth

(1) Preparing a fermentation medium

(2) preparation of lily enzymolysis fermentation liquor

5. obtaining lily peptide

6. preparation of peony enzymolysis fermentation liquor

(1) Preparing a fermentation medium

(2) preparation of peony enzymolysis fermentation liquor

7. obtaining peony flower peptide

The composite flower peptide prepared in each example is detected, and the detection method and the detection result are as follows:

the detection and evaluation method comprises the following steps:

1. crude protein content test

The protein in the sample is decomposed under catalytic heating, and the resulting ammonia is combined with sulfuric acid to produce ammonium sulfate. Alkalifying and distilling to free ammonia, absorbing with boric acid, titrating with sulfuric acid or hydrochloric acid standard titration solution, calculating nitrogen content according to acid consumption, and multiplying by conversion coefficient to obtain protein content.

2. Evaluation of Oxidation resistance index ORAC value

Accurately sucking 1g of sample into a 15ml centrifuge tube, diluting the sample with 10ml of ultrapure water to obtain a sample solution of 100mg/ml, and evaluating the appropriate concentration by pre-experiment groping DPPH to finally obtain the concentration series of the sample:

based on the ORAC reaction in 75mmol potassium phosphate buffer (pH 7.4) environment, at 37 degrees C after a preset 5min, using a multi-channel pipette rapidly in each hole to add AAPH 140 u l start the reaction, and the plate placed in the fluorescence analyzer at 37 degrees C at excitation wavelength 485nm, emission wavelength 538nm for continuous measurement, every 2min to determine the fluorescence intensity of each hole, the determination time is generally set to the fluorescence decay after baseline.

3. Evaluation of safety

According to the following: the technical specification of cosmetic safety (2015 edition) is based on clinical medicine as a theoretical basis, and a test population is formed by specific experimental populations, and spot-sticking tests are carried out on specified parts of a test subject, so that the potential possibility that the cosmetics have adverse reactions on human skin is determined. Selecting qualified spot test materials. The test substance is put into a spot tester, and the dosage is about 0.020 g-0.025 g (solid or semisolid) or 0.020 mL-0.025 mL (liquid can be dripped on a filter paper sheet attached to the spot tester and placed in the spot tester).

When the test substance is the original product of the final cosmetic product, the control hole is a blank control (without any substance), and when the test substance is the diluted cosmetic product, the diluent of the cosmetic product is used in the control hole.

The spot test device with the tested object is applied to the back or the curved side of the forearm of the tested object by using a non-irritating adhesive tape, and is lightly pressed by the palm to be uniformly applied to the skin for 24 hours.

And (5) removing the tested substance spot tester for 30min, and observing skin reaction after the indentation disappears. If the result is negative, the test is observed once more at 24h and 48h after the patch test. The results were recorded as in Table 1 (Standard Table for grading adverse skin reactions).

TABLE 1

If the skin closed patch test result shows that the test object has adverse reactions to the human body, for example, 30 subjects have more than 5 people with grade 1 adverse skin reactions, or more than 2 people with grade 2 adverse skin reactions, or any 1 example, 3 grades or more than 3 grades adverse skin reactions.

The prepared composite flower peptide is used for skin care products, and the anti-aging effect of the skin care products is detected.

Evaluation of anti-aging efficacy

Research shows that the senescent cells have remarkable characteristics, the size of the senescent cells is usually increased, and the activity of senescence-associated beta-galactosidase (SA-beta-Gal) is up-regulated in a pH 6.0 environment, so that the anti-senescence of the active substances is approved and widely applied by the beta-galactosidase activity inhibition experiment, and the anti-senescence efficacy evaluation test results of examples 1 to 6 are shown in Table 2.

TABLE 2

From the detection, evaluation and analysis of the composite peptide in different embodiments of table 2, it can be seen that the pretreatment method microwave method is superior to the high pressure method, and glacial acetic acid has better safety than hydrochloric acid in the acid precipitation process. As shown in FIG. 1, the product using trypsin has a smaller molecular weight and a slightly higher crude protein content than the other proteases. As shown in figure 3, the 1:1 compounding ratio of the lily peptide and the peony peptide has a higher beta-galactosidase activity inhibition rate than the 1:10 compounding ratio, but the oxidation resistance index ORAC value of the flower-derived peptide (1:1) is slightly lower, as shown in figure 2. Freeze drying is superior to spray drying in terms of maintaining the antioxidant and anti-aging activities of the product, but adds significant cost. After the lily peptide and the peony peptide are compounded, the oxidation resistance index ORAC value and the beta-galactosidase activity inhibition rate of the anti-aging evaluation have synergistic effect, and the comprehensive efficacy is improved, as shown in fig. 4A and 4B.

As can be seen from the above tests, examples 4, 5 and 6 have a good anti-aging effect.

Through the embodiment, the yield of the alkali extraction and acid precipitation process protein is improved by screening different pretreatment modes, and the full utilization of resources is facilitated; different proteases are screened to obtain a product with lower molecular weight and stronger oxidation resistance, so that the cost performance of the product is improved; compared with the screening and drying process, the antioxidant and anti-aging effects of the product are improved.

Comparative example 1: the comparative example aims at researching the influence of the fermentation process on the enzymolysis liquid, the lily enzymolysis liquid and the peony enzymolysis liquid are respectively prepared by the enzymolysis process according to the steps 1-3 in the embodiment 4, then the enzymolysis liquid is ultrafiltered and graded, the operation pressure is 0.35Mpa, the temperature and the normal temperature are realized, the molecular weight is tested, the ultrafiltration component accounting for more than 80 percent of the relative molecular weight of less than 200Da is selected, and spray drying is carried out, so that the lily peptide and the peony peptide are obtained. The lily peptide and the peony peptide are prepared according to the following steps of 10: mixing the components in a mass ratio of 1 to obtain the composite flower peptide.

The composite peptide flower prepared in the comparative example 1 and the example 4 is respectively subjected to a blood vessel experiment of a chick embryo chorioallantoic membrane, and the experiment results prove that enzymolysis liquid can cause hemolysis of the chick embryo chorioallantoic membrane blood vessel to form blood spots as shown in figure 5, and the hemolysis phenomenon does not occur after fermentation as shown in figure 6, which shows that the safety risk of the composite peptide flower is reduced by the fermentation process.

Compared with the existing extraction technology of peony and lily, the method has the advantages that the enzymolysis technology is utilized to directionally extract the active peptide, the main components of the product are clear, the safety is good, the biological activity is obvious, the resources of peony and lily are fully utilized, and the cost performance of the product is improved.

It should be understood that the above-mentioned embodiments of the present invention are only examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention, and it will be obvious to those skilled in the art that other variations or modifications may be made on the basis of the above description, and all embodiments may not be exhaustive, and all obvious variations or modifications may be included within the scope of the present invention.

Claims

1. A preparation method of composite flower peptide is characterized by comprising the following steps:

2. The method of claim 1, further comprising pulverizing each raw flower by a pretreatment comprising heat treatment, ultrasonic treatment or high pressure treatment.

3. The method for preparing composite flower peptide according to claim 2, wherein the alkali-soluble acid-precipitation method comprises:

beating the mixed material liquid into uniform slurry;

4. The method for preparing composite flower peptide according to claim 3, wherein when the raw material flower is lily, the mass ratio of the added sodium carbonate solution to the raw material pollen is 1: 1-2: 1, and the concentration of the sodium carbonate solution is 0.5 mol/L; standing for 1.5-2.5 h, centrifuging at a rotating speed of 5000r/min for 20min, and adjusting the pH value of the filtrate to 9-10 after centrifugal filtration;

5. The method for preparing composite flower peptide according to claim 1, wherein the enzymatic hydrolysate for obtaining different raw material flower proteins under the action of different proteases comprises:

6. The method for preparing composite flower peptide according to claim 5, wherein when the raw material flower is lily, the protease is Alcalase alkaline protease, the enzyme activity is more than 600000U/g, and the enzyme base ratio is more than 5000U/g;

7. The method for preparing composite flower peptide according to claim 5, wherein when the raw material flower is lily, the pH value of the solution is adjusted to 8-8.5 before water bath heating; when the raw material flowers are peony flowers, adjusting the pH value of the solution to 6-7 before heating in a water bath;

8. The method for preparing composite flower peptide according to claim 1, wherein the step of fermenting the enzymolysis liquid of each raw material flower protein by bifidobacterium to obtain enzymolysis fermentation liquid of different raw material flower proteins, and the step of filtering and drying the enzymolysis fermentation liquid to obtain the flower peptide of each raw material flower comprises the following steps:

9. The method for preparing composite flower peptide according to claim 1, wherein the step of mixing flower peptides of multiple raw material flowers to obtain the composite flower peptide comprises the step of mixing a first raw material flower peptide and a second raw material flower peptide according to a mass ratio of 1: 1-10: 1 to obtain the composite flower peptide.

10. Use of the complex peptide flower prepared by the preparation method according to any one of claims 1 to 9 in the preparation of cosmetics.