CN117598950B

CN117598950B - Extract containing kava root, preparation method and application thereof in cosmetic with soothing and repairing effects

Info

Publication number: CN117598950B
Application number: CN202311642989.1A
Authority: CN
Inventors: 刘婉; 覃贻佳; 周广祥
Original assignee: Guangzhou Xinghui Biotechnology Co ltd
Current assignee: Guangzhou Convergence Biotechnology Co ltd
Priority date: 2023-12-01
Filing date: 2023-12-01
Publication date: 2024-05-14
Anticipated expiration: 2043-12-01
Also published as: CN117598950A

Abstract

The application discloses an extract containing kava roots, a preparation method and application thereof in a cosmetic with a soothing and repairing effect. The preparation method of the kava root-containing extract comprises the following steps: (1) Inoculating Pichia pastoris seed solution into a fermentation substrate for fermentation to obtain crude fermentation liquor; (2) Sterilizing, cracking and filtering the crude fermentation broth to obtain an extract containing kava roots; wherein the fermentation substrate comprises kava root and mung bean seed. According to the application, the Pichia pastoris is adopted to simultaneously ferment and extract the kava roots and the mung bean seeds, the kava roots and the mung bean seeds play a role in cooperation in the fermentation process, and the finally obtained extract has good relieving and repairing effects, is safe and non-irritating, and is suitable for being applied to preparing cosmetics.

Description

Extract containing kava root, preparation method and application thereof in cosmetic with soothing and repairing effects

Technical Field

The application belongs to the technical field of plant extraction, and particularly relates to an extract containing kava roots, a preparation method and application of the extract in a cosmetic with a soothing and repairing effect.

Background

With the continuous development of human society, the life rhythm becomes faster, the pressure is increased, and the problem of skin sensitivity of people is more and more frequent. Skin sensitivity problems are increasing year by year between men and women and different ages. Climate factors, lifestyle and other problems are important factors that cause skin sensitivity. On the other hand, with the rise of living standard, consumers begin to pay attention to skin problems of themselves, and some skin care products or medical products with high efficacy and quick effect are used for the problems of the skin of themselves. However, improper use of these products exacerbates the skin sensitivity problem, leading to a series of problems such as the skin barrier becoming weaker and the skin defenses decreasing, causing redness, stinging, etc.

At present, some skin care products with relieving and repairing effects are introduced in the cosmetic market to alleviate the problem of redness and stinging of the skin of consumers. Some skin care products with better effects are usually added with antihistamine drugs, external adrenocortical steroid agents, nonsteroidal anti-inflammatory drugs and other hormones as active substances, so that defects and adverse reactions such as hypoadrenocortical function and rebound phenomenon are easy to cause after long-term use, and how to provide a safe and efficient cosmetic raw material with good relaxing and repairing effects becomes the problem to be solved urgently at present.

Disclosure of Invention

In order to overcome the disadvantages and shortcomings of the prior art, a primary object of the present application is to provide a method for preparing an extract containing kava root.

The preparation method of the kava root-containing extract is realized by the following scheme:

a method for preparing kava root-containing extract comprises:

(1) Inoculating Pichia pastoris seed solution into a fermentation substrate for fermentation to obtain crude fermentation liquor;

(2) Sterilizing, cracking and filtering the crude fermentation broth to obtain an extract containing kava roots; wherein the fermentation substrate comprises kava roots and mung bean seeds.

Further, in the step (1), the inoculation amount of the pichia pastoris seed solution is 3-5% (v/v) of the fermentation substrate.

Further, in the step (1), the preparation method of the pichia pastoris seed solution comprises the following steps:

b1, inoculating Pichia pastoris colonies into a liquid culture medium for primary culture to obtain a suspension;

And B2, inoculating the suspension into the liquid culture medium for secondary culture to obtain the pichia pastoris seed liquid.

Still further, in step B1 or step B2, the liquid medium includes at least one of wort medium, PDB medium, YPD medium, and Bengalia red medium.

Further, in the step B1, the temperature of the primary culture is 28-33℃and the time of the primary culture is 20-28 hours.

Further, in step B2, the suspension is inoculated in an amount of 0.8-2% (v/v) of the liquid medium.

Further, in the step B2, the temperature of the secondary culture is 28-33℃and the time of the secondary culture is 36-60 hours.

Further, in the step (1), the total mass of the kava roots and the mung bean seeds is 0.8-2% of the mass of the fermentation substrate.

Further, in the step (1), the mass ratio of the kava root to the mung bean seed is (3-9): 1.

Further, the kava root is dried kava root powder.

Further, the preparation method of the kava root powder comprises the following steps: pulverizing dried kava root, sieving, and collecting lower layer powder.

Further, the mung bean seeds are dried mung bean seed powder.

Further, the preparation method of the mung bean seed powder comprises the following steps: pulverizing dried semen Phaseoli Radiati, sieving, and collecting lower layer powder.

Further, in step (1), the fermentation substrate further comprises nutrients and water.

Further, the nutrient is added in an amount of 1.5-5% by mass of the fermentation substrate, and water is added in an amount of up to 100% by mass of the total fermentation substrate.

Still further, the nutrients include at least one of brewer's grains, fish meal, corn steep liquor.

Further, in the step (1), the fermentation temperature is 28-33 ℃, and the fermentation time is 50-70h.

Further, the sterilization in the step (2) is to keep the crude fermentation broth in the step (1) at 80-95 ℃ for 15-30min.

Further, the cracking in the step (2) is high-pressure homogenization treatment, the pressure of the high-pressure homogenization treatment is 20-50Mpa, and the time of the high-pressure homogenization treatment is 15-50min.

Kava is a perennial upright shrub medicinal plant of Piperaceae, and its roots, stems and leaves can be used as medicine. Kava contains kavalactone, kavaine, kavain, etc. as active ingredients. Modern pharmacological research shows that kava extract has the functions of resisting anxiety, tranquilizing, hypnotizing, local anesthesia, inhibiting bacteria, relaxing muscle, improving mental state, etc.

Mung bean seeds are commonly called mung beans and are also called green beans. The mung beans belong to high-protein, low-fat and medium-starch crops, are rich in nutrition and have high economic utilization value. In addition, mung beans contain a large amount of bioactive substances such as amino acids, flavonoid compounds, phenolic compounds, oligosaccharides and the like, and the mung beans are known to have good effects in anti-inflammatory, antibacterial, lipid-lowering, anti-tumor and detoxification aspects in the current medicine.

The application adopts pichia pastoris as a fermentation strain, and simultaneously carries out fermentation extract on kava roots and mung bean seeds. In the fermentation process, the Pichia pastoris utilizes kava roots, mung bean seeds and other nutrients in a fermentation matrix to complete the growth and propagation of the Pichia pastoris, and the growth and propagation of the Pichia pastoris in the fermentation process promote the cell walls of the kava roots and the mung bean seeds to be broken, so that the effective components in the cells of the kava roots and the mung bean seeds are exposed, and the outflow of active substances is facilitated. In addition, pichia pastoris produces secondary metabolites during growth, so that the final extract has good soothing, repairing, calming and stinging reducing effects.

Another object of the present application is to provide an extract containing kava roots prepared by the above method.

Still another object of the present application is to provide the use of the kava root-containing extract for preparing a cosmetic with soothing and repairing effects.

Further, the addition amount of the kava root-containing extract in the cosmetic with the soothing and repairing effects is 0.01-30% by mass.

Compared with the prior art, the application has the beneficial effects that:

(1) The application uses the Pichia pastoris to ferment and extract the kava roots and the mung bean seeds simultaneously, thereby avoiding the independent extraction of the kava roots and the mung bean seeds, simplifying the fermentation and extraction process and reducing the production cost and the fermentation time.

(2) According to the application, active substances in kava roots and mung bean seeds are extracted simultaneously through a fermentation process, and the pichia pastoris utilizes nutrients to complete self growth and reproduction in a fermentation substrate, so that the effects of various enzymes in the metabolism process of the pichia pastoris are beneficial to cell wall fragmentation of the kava roots and mung bean seeds, and the active substances in the kava roots and mung bean seeds are promoted to be dissolved into a crude fermentation liquid, so that the extraction efficiency of the kava roots and mung bean seeds is improved.

(3) The application carries out a large number of screening on strains, parts of kava plants, types of beans and optimal fermentation states of mung beans, and finally discovers that the extract obtained by fermenting the kava roots and mung bean seeds with proper mass ratio by taking the roots of the kava and mung bean seeds which are not germinated as fermentation raw materials and taking pichia pastoris as fermentation strains has good relieving and repairing effects. In the evaluation of human body efficacy, the extract also has the effects of calming skin and relieving skin itch.

(4) The application does not use organic solvent in the fermentation extraction process, the production process is environment-friendly, the obtained extract is safe and efficient, has good effects of relieving, repairing and calming skin and relieving skin itch, and is suitable for being applied to preparing cosmetics with the effect of relieving and repairing.

Detailed Description

The present application will be described in further detail with reference to examples, but embodiments of the present application are not limited thereto. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

The reagents used in the examples were commercially available unless otherwise specified.

All percentages mentioned in the present application are mass percentages unless otherwise indicated.

The Pichia pastoris used in the application is Pichia pastoris BNCC225716, and the strain is purchased from Beijing North Nachuang biological technology research institute.

The Saccharomyces cerevisiae used in the application is Saccharomyces cerevisiae BNCC337309, and the strains are purchased from Beijing North Nakau biological technology institute.

The aspergillus oryzae used in the application is aspergillus oryzae BNCC336549,336549, and the strain is purchased from Beijing Beidou Nakau biological technology institute.

The Lactobacillus delbrueckii subspecies bulgaricus used in the application is Lactobacillus delbrueckii subspecies bulgaricus BNCC187941, and the strains are purchased from Beijing North Naja biological technology institute.

Wort medium, PDB medium, YPD medium, bengalia medium, MRS medium used in the present application may be obtained commercially, or may be prepared as follows:

The preparation method of the wort medium is (taking 1L of wort medium as an example): adding 4 parts of water into 1 part of malt flour, and preserving heat for 3-4 hours in a water bath kettle at 65 ℃ to ensure that the malt flour is saccharified automatically until saccharification is complete; filtering the saccharified solution with 4-6 layers of gauze, detecting sugar concentration in the saccharified solution with Baume gravimeter, diluting filtrate with water to 10-15 Baume, adjusting pH to 6.4, sterilizing at 115-130deg.C for 20-30min, and storing.

The preparation method of the PDB culture medium is (taking 1L of PDB culture medium as an example) as follows: peeling potato, cutting into small pieces (200 g), adding water, boiling for 30ml, filtering with double-layer gauze, collecting filtrate, adding 20g glucose, supplementing 1L with water, sterilizing at 115-130deg.C for 20-30min, and storing.

The YPD medium was prepared by the following method (1L of YPD medium was prepared as an example): dissolving 10g of yeast extract, 20g of peptone and 20g of glucose in 1L of water, uniformly mixing, sterilizing at 115-130 ℃ for 20-30min, and storing for later use.

The preparation method of the Bengalia culture medium is (taking Bengalia culture medium for preparing 1L as an example): 5-10g of peptone, 10-20g of glucose, 0.5-3g of KH ₂PO₄ 1-3g、MgSO₄·7H₂ O, 0.1-0.5g of chloramphenicol, 100-200mL of 1/3000 manalar red solution, adding distilled water to 1L, sterilizing at 115-130 ℃ for 20-30min, and storing for later use.

The preparation method of the MRS culture medium is (taking the MRS culture medium of 1L as an example) that: 10g of peptone, 8g of beef extract powder, 4g of yeast powder, 20g of glucose, 0.2g of magnesium sulfate, 5g of sodium acetate, 2g of diammonium citrate, 2g of dipotassium hydrogen phosphate, 0.04g of manganese sulfate, 1g of tween 80 and 1g of distilled water, and sterilizing at 115-130 ℃ for 20-30min and storing for later use.

The preparation methods of the fermentation substrates in the examples and comparative examples of the present application are: mixing plant powder (at least one of kava root powder, semen Phaseoli Radiati seed powder, kava leaf powder, kava stem powder, semen glycines powder, and semen Phaseoli powder according to the requirements of each example or comparative example), nutrients and water, sterilizing at 115-130deg.C for 20-30min, and storing.

Example 1

An extract containing kava root, which is prepared by the following steps:

(1) Inoculating Pichia pastoris seed solution into a fermentation substrate according to an inoculum size of 4% (v/v), and fermenting at 30 ℃ for 60 hours to obtain a crude fermentation broth;

(2) Preserving heat of the crude fermentation broth prepared in the step (1) at 90 ℃ for 20min to obtain sterilized crude fermentation broth; homogenizing the sterilized crude fermentation broth under high pressure of 35Mpa for 30min to obtain cracked crude fermentation broth, and filtering the cracked crude fermentation broth to obtain filtrate as extract containing kava root.

The preparation method of the pichia pastoris seed liquid comprises the following steps:

b1, picking pichia pastoris colonies by an inoculating loop, inoculating the pichia pastoris colonies into a YPD culture medium, and culturing the pichia pastoris colonies at 30 ℃ for 24 hours at one time to obtain a suspension;

b2, inoculating the suspension in the step B1 into YPD medium with an inoculum size of 1.3% (v/v), and culturing for 48 hours at 30 ℃ for the second time to obtain Pichia pastoris seed solution.

The fermentation substrate comprises, by mass, 1.0% of kava root powder, 0.2% of mung bean seed powder, 3% of brewer's grains and 95.8% of deionized water.

In this example, the total mass percent of kava root powder and mung bean seed powder is 1.2%, and the mass ratio of kava root to mung bean seed is 5:1.

Example 2

An extract containing kava root, which is prepared by the following steps:

(1) Inoculating Pichia pastoris seed solution into a fermentation substrate with an inoculum size of 3% (v/v), and fermenting at 28 ℃ for 50h to obtain a crude fermentation broth;

(2) Preserving the temperature of the crude fermentation broth prepared in the step (1) at 80 ℃ for 30min to obtain sterilized crude fermentation broth; homogenizing the sterilized crude fermentation broth under high pressure of 50Mpa for 15min to obtain cracked crude fermentation broth, and filtering the cracked crude fermentation broth to obtain filtrate as extract containing kava root.

b1, picking pichia pastoris colonies by an inoculating loop, inoculating the pichia pastoris colonies into a PDB culture medium, and culturing the pichia pastoris colonies for 28 hours at the temperature of 28 ℃ for one time to obtain a suspension;

B2, inoculating the suspension in the step B1 into a PDB culture medium with an inoculum size of 0.8% (v/v), and culturing for 60 hours at 33 ℃ for the second time to obtain a Pichia pastoris seed solution.

The fermentation substrate comprises, by mass, 1.75% of kava root powder, 0.25% of mung bean seed powder, 3.5% of corn steep liquor and 94.5% of deionized water.

In this example, the total mass percent of kava root powder and mung bean seed powder was 2.0%, and the mass ratio of kava root to mung bean seed was 7:1.

Example 3

An extract containing kava root, which is prepared by the following steps:

(1) Inoculating Pichia pastoris seed solution into a fermentation substrate with an inoculum size of 5% (v/v), and fermenting at 33 ℃ for 70 hours to obtain a crude fermentation broth;

(2) Preserving heat of the crude fermentation broth prepared in the step (1) for 15min at 95 ℃ to obtain sterilized crude fermentation broth; homogenizing the sterilized crude fermentation broth under high pressure of 30Mpa for 50min to obtain cracked crude fermentation broth, and filtering the cracked crude fermentation broth to obtain filtrate as extract containing kava root.

b1, picking pichia pastoris colonies by an inoculating loop, inoculating the pichia pastoris colonies into a Bengalia red culture medium, and culturing the pichia pastoris colonies for 20 hours at 33 ℃ for one time to obtain a suspension;

B2, inoculating the suspension in the step B1 into a Bengalia red culture medium with an inoculum size of 2% (v/v), and culturing for a second time at 28 ℃ for 36 hours to obtain a Pichia pastoris seed solution.

The fermentation substrate comprises, by mass, 0.64% of kava root powder, 0.16% of mung bean seed powder, 4% of fish meal and 95.2% of deionized water.

In this example, the total mass percent of kava root powder and mung bean seed powder was 0.8%, and the mass ratio of kava root to mung bean seed was 4:1.

Example 4

The preparation method of the kava root-containing extract of this example is different from that of example 1 in that: the fermentation substrate comprised 0.9% kava root powder and 0.3% mung bean seed powder, the remaining steps and parameters were the same as in example 1.

In this example, the total mass percent of kava root powder and mung bean seed powder was 1.2%, and the mass ratio of kava root to mung bean seed was 3:1.

Example 5

The preparation method of the kava root-containing extract of this example is different from that of example 1 in that: the fermentation substrate comprised 1.08% kava root powder and 0.12% mung bean seed powder, the remaining steps and parameters were the same as in example 1.

In this example, the total mass percent of kava root powder and mung bean seed powder was 1.2%, and the mass ratio of kava root to mung bean seed was 9:1.

Example 6

The preparation method of the kava root-containing extract of this example is different from that of example 1 in that: the 3% beer in the fermentation substrate was changed to 3% fish meal, the rest of the steps and parameters were the same as in example 1.

Example 7

The preparation method of the kava root-containing extract of this example is different from that of example 1 in that: the 3% beer in the fermentation substrate was changed to 3% corn steep liquor, and the remaining steps and parameters were the same as in example 1.

Example 8

The preparation method of the kava root-containing extract of this example is different from that of example 1 in that: the 3% spent grain in the fermentation substrate was changed to 1.5% spent grain, and the amount of ionized water was adjusted to 100% of the total mass of the fermentation substrate, and the remaining steps and parameters were the same as in example 1.

Example 9

The preparation method of the kava root-containing extract of this example is different from that of example 1 in that: the 3% spent grain in the fermentation substrate was changed to 5% spent grain, and the amount of ionized water was adjusted to 100% of the total mass of the fermentation substrate, and the remaining steps and parameters were the same as in example 1.

Example 10

The preparation method of the kava root-containing extract of this example is different from that of example 1 in that: the pressure of the high-pressure homogenization treatment in the step (2) was changed to 20Mpa, and the rest of the steps and parameters were the same as in example 1.

Comparative example 1

The preparation method of the extract of this comparative example is different from that of example 1 in that: in this comparative example, kava roots and mung bean seeds were fermented and extracted using Saccharomyces cerevisiae, and the remaining steps and parameters were the same as in example 1.

The preparation method of the saccharomyces cerevisiae seed liquid comprises the following steps:

b1, inoculating Saccharomyces cerevisiae colonies extracted by an inoculating loop into YPD culture medium, and culturing at 30 ℃ for 24 hours once to obtain suspension;

b2, inoculating the suspension in the step B1 into YPD culture medium with an inoculum size of 1.3% (v/v), and culturing for 48 hours at 30 ℃ to obtain saccharomyces cerevisiae seed liquid.

Comparative example 2

The preparation method of the extract of this comparative example is different from that of example 1 in that: in this comparative example, kava roots and mung bean seeds were fermented and extracted using Aspergillus oryzae, and the remaining steps and parameters were the same as in example 1.

The preparation method of the aspergillus oryzae seed liquid comprises the following steps:

B1, inoculating Aspergillus oryzae colonies extracted by an inoculating loop into a PDB culture medium, and culturing at 30 ℃ for 24 hours at one time to obtain a suspension;

B2, inoculating the suspension in the step B1 into a PDB culture medium with an inoculum size of 1.3% (v/v), and culturing for 48 hours at 30 ℃ for the second time to obtain aspergillus oryzae seed liquid.

Comparative example 3

The preparation method of the extract of this comparative example is different from that of example 1 in that: the comparative example adopts Lactobacillus delbrueckii subspecies bulgaricus to ferment and extract kava roots and mung bean seeds, and the preparation method comprises the following steps:

(1) Inoculating lactobacillus delbrueckii subspecies bulgaricus seed solution into a fermentation substrate in an inoculum size of 4% (v/v), and fermenting at 37 ℃ for 60 hours to obtain a crude fermentation broth;

(2) Preserving heat of the crude fermentation broth prepared in the step (1) at 90 ℃ for 20min to obtain sterilized crude fermentation broth; homogenizing the sterilized crude fermentation broth under high pressure of 35Mpa for 30min to obtain cracked crude fermentation broth, and filtering the cracked crude fermentation broth to obtain filtrate as extract.

The preparation method of the lactobacillus delbrueckii subspecies bulgaricus seed solution comprises the following steps:

B1, inoculating lactobacillus delbrueckii subspecies bulgaricus colonies into an MRS culture medium by using an inoculating loop, and culturing at 37 ℃ for 24 hours at one time to obtain a suspension;

B2, inoculating the suspension in the step B1 into MRS culture medium with an inoculum size of 1.3% (v/v), and culturing for 48 hours at 37 ℃ for the second time to obtain lactobacillus delbrueckii subspecies bulgaricus seed solution.

The fermentation substrate of comparative example 3 was the same as in example 1.

Comparative example 4

The preparation method of the extract of this comparative example is different from that of example 1 in that: this comparative example changed from 1.0% kava root powder and 0.2% mung bean seed powder to 1.2% kava root powder in the fermentation substrate, and the remaining steps and parameters were the same as in example 1.

Comparative example 5

The preparation method of the extract of this comparative example is different from that of example 1 in that: in this comparative example, 1.0% kava root powder and 0.2% mung bean seed powder in the fermentation substrate were changed to 1.2% mung bean seed powder, and the remaining steps and parameters were the same as in example 1.

Comparative example 6

The preparation method of the extract of this comparative example is different from that of example 1 in that: this comparative example changed from 1.0% kava root powder and 0.2% mung bean seed powder to 0.6% kava root powder and 0.6% mung bean seed powder in the fermentation substrate, and the remaining steps and parameters were the same as in example 1.

Comparative example 7

The preparation method of the extract of this comparative example is different from that of example 1 in that: in this comparative example, 1.0% of kava root powder in the fermentation substrate was changed to 1.0% of kava leaf powder, and the rest of the steps and parameters were the same as in example 1.

Comparative example 8

The preparation method of the extract of this comparative example is different from that of example 1 in that: in this comparative example, 1.0% kava root powder in the fermentation substrate was changed to 1.0% kava stem powder, and the remaining steps and parameters were the same as in example 1.

Comparative example 9

The preparation method of the extract of this comparative example is different from that of example 1 in that: in this comparative example, 0.2% of mung bean seed powder in the fermentation substrate was changed to 0.2% of soybean powder, and the rest of the steps and parameters were the same as in example 1.

Comparative example 10

The preparation method of the extract of this comparative example is different from that of example 1 in that: in this comparative example, 0.2% of mung bean seed powder in the fermentation substrate was changed to 0.2% of red bean powder, and the rest of the steps and parameters were the same as in example 1.

Comparative example 11

The preparation method of the extract of this comparative example is different from that of example 1 in that: in this comparative example, 0.2% of mung bean seeds in the fermentation substrate were germinated and then added to the fermentation substrate for fermentation (the mass of deionized water in the fermentation substrate may be adjusted so that the total mass of the fermentation substrate is 100%).

The fermentation method of the mung bean seeds comprises the following steps: spreading a wet towel on a tray as a bottom, spreading semen Phaseoli Radiati on the towel, adding water 3 times the weight of semen Phaseoli Radiati into the tray to soak semen Phaseoli Radiati, covering the surface of semen Phaseoli Radiati with the wet towel, and germinating at 40deg.C with relative humidity of 90% to obtain germinated semen Phaseoli Radiati. Pulverizing germinated semen Phaseoli Radiati, and adding into fermentation substrate.

Comparative example 12

The preparation method of the extract of this comparative example is different from that of example 1 in that: in the comparative example, after the crude fermentation broth is sterilized, the sterilized crude fermentation broth is directly subjected to filtration treatment without cracking, and the obtained filtrate is the extract of the comparative example, and the specific preparation method is as follows:

(2) Preserving heat of the crude fermentation broth prepared in the step (1) at 90 ℃ for 20min to obtain sterilized crude fermentation broth; filtering the sterilized crude fermentation broth to obtain filtrate which is the extract of the comparative example.

The preparation method and fermentation substrate of the Pichia pastoris seed solution are the same as in example 1.

Comparative example 13

The preparation method of the extract of this comparative example is different from that of example 1 in that: this comparative example changed 3% brewer's grains in the fermentation substrate to 3% skim milk powder, and the remaining steps and parameters were the same as in example 1.

Comparative example 14

An extract, which is prepared by the following steps:

Mixing 1.0% of kava root powder, 0.2% of mung bean seed powder and the balance of water uniformly, performing ultrasonic extraction at 60 ℃ and 30KHz frequency for 3h, and filtering to obtain filtrate as extract.

The extract of this comparative example differs from that of example 1 in that: the comparative example adopts an ultrasonic extraction method to extract kava root powder and mung bean seed powder.

Test example 1: relaxation performance test

Test sample: the extracts of examples 1-10 and comparative examples 1-14 were prepared as sample concentrates for use; matrix essence.

The formula of the sample essence is as follows: the deionized water is filled up to 100%;3% of the extract of examples 1 to 10 or comparative examples 1 to 14; 3% glycerol; 0.2% allantoin; 0.5% betaine; 5% butanediol; 1% tocopheryl acetate; 0.03% carbomer; 0.2% triethanolamine; 0.5% phenoxyethanol.

The preparation method of the sample essence comprises the following steps: adding deionized water, the extracts of examples 1-10 or comparative examples 1-14, glycerol, allantoin, betaine, butanediol and tocopheryl acetate into a preparation container, heating to 70deg.C, stirring, and maintaining for 30min to obtain a mixture; and (3) when the temperature of the mixture is reduced to 45 ℃, carbomer, triethanolamine and phenoxyethanol are added, the mixture is stirred uniformly, and the sample essence is obtained after the temperature is reduced to room temperature.

The extracts of examples 1-10 or comparative examples 1-14 were not added to the base concentrate, and the same mass of deionized water was used instead of the base concentrate, and the base concentrate was prepared in the same manner as the sample concentrate.

The testing method comprises the following steps: selecting 250 volunteers with sensitive muscle skin and facial skin in a reddish state and a percutaneous water loss value (TEWL value) of the facial skin of more than 20 (the volunteers are randomly divided into 25 groups, each 10 people use one essence, and the test results are averaged.) and using the sample essence or the matrix essence by the volunteers for 2 times a day in the morning and in the evening. During the test period, volunteers were not allowed to use skin care products of similar efficacy. The skin transdermal moisture loss value (TEWL value) of the volunteer was tested before using the sample essence or the matrix essence or after using for 5 days, and the rate of change of the skin transdermal moisture loss value of the volunteer was calculated.

Rate of change in skin transdermal moisture loss value (%) = (T _5d-T_0d)/T_0d x 100%

T _5d refers to the skin transdermal moisture loss value of the volunteer's face after 5 days of use;

t _0d refers to the percutaneous water loss value of the facial skin of the pre-volunteer using.

Test instrument: trans-epidermal moisture loss meter vaponter.

The percutaneous moisture loss value of skin refers to the evaporation value of skin moisture (unit: g/m ² h), and proper skin moisture evaporation is the normal skin metabolism. An excessive skin moisture loss value indicates an impaired skin barrier in the test subject. After the test sample is used, the skin barrier of the tester is restored, the skin state is relaxed, and the evaporation value of the skin moisture is reduced when the skin percutaneous moisture loss value is reduced. The greater the absolute value of the rate of change of the skin transdermal moisture loss value, the better the skin barrier recovery, the better the soothing effect of the test sample.

The test results are shown in Table 1.

TABLE 1

The test results show that the absolute value of the change rate of the skin percutaneous water loss value of the essence containing the extracts of the examples and the comparative examples is larger than that of the matrix essence, and the extracts of the examples and the comparative examples can reduce the water evaporation on the skin surface to a certain extent and have a certain relieving effect. Wherein the absolute value of the rate of change of the percutaneous water loss values of the extracts of examples 1 to 10 was greater than that of the extracts of comparative examples 1 to 14, indicating that the soothing effect of examples 1 to 10 was superior to that of comparative examples 1 to 14. Further, examples 1-10 exhibited better soothing effects than comparative examples 1-3, demonstrating that the use of Pichia pastoris for fermentation extraction of kava roots and mung bean seeds gave extracts with better soothing effects than the use of Saccharomyces cerevisiae, aspergillus oryzae, and Lactobacillus fermentum for fermentation extraction, while the use of Lactobacillus fermentum gave extracts with the worst soothing effects. Examples 1-10 have better soothing effects than comparative examples 4 and 5, which shows that there is a synergistic effect between kava roots and mung bean seeds during fermentation, and the extract obtained by co-fermentation of the two has the best soothing effect, and the obtained extract has poor soothing effect by only fermenting kava roots or mung bean seeds. Further, the soothing effect of examples 1, 4, 5 is superior to comparative example 6, demonstrating that the mass ratio of kava roots to mung bean seeds has an effect on the soothing effect of the final extract during fermentation, and that the soothing effect is best when the mass ratio of kava roots to mung bean seeds is 5:1 under certain conditions, and that the soothing effect of the extract is poor when the mass ratio of kava roots to mung bean seeds is 1:1. In the application, the mass ratio of kava root to mung bean seeds is controlled to be (3-9): 1. Examples 1 to 10 have a soothing effect superior to that of comparative examples 7 to 11, showing that the effect of co-fermenting kava roots and mung bean seeds is superior, and that the effect of soothing the extract obtained by replacing mung bean seeds with soybeans in comparative example 9 is even worse than that obtained by fermenting kava roots alone by replacing kava leaves, kava stems or mung bean seeds with soybeans or red beans, or by germination of mung bean seeds in advance. Examples 1-10 have a better soothing effect than comparative example 12, demonstrating that the crude fermentation broth is subjected to lysis in the resulting fermentation broth, resulting in a better soothing effect of the resulting extract; if fermentation is finished, the step of cracking is not performed, the crude fermentation broth is directly sterilized and filtered, the obtained culture broth is only pichia pastoris extracellular culture broth, active substances in pichia pastoris cells cannot be effectively dissolved into the fermentation broth, active components of the fermentation broth are greatly reduced, and the relief effect of a final product is poor. The results of comparative examples 1, 6 to 10 and comparative example 13 show that the kind and content of the nutrients in the fermentation medium and the pressure of the high-pressure homogenization treatment during the preparation of the extract have an effect on the soothing effect of the finally obtained extract, and therefore, the addition amount of the nutrients in the present application is 1.5 to 5%, the nutrient is at least one selected from the group consisting of brewer's grains, fish meal and corn steep liquor, and the pressure of the high-pressure homogenization treatment should be set to 20 to 50Mpa.

Test example 2: repair Performance test

Test sample: the extracts of examples 1-10 and comparative examples 1-14 were prepared with deionized water to form a 2% by mass aqueous solution; the blank control group was deionized water.

The testing steps are as follows: 125 volunteers with intact arms, shielding and smooth skin are selected, the inner sides of the left and right arms of each volunteer are used as test parts, and each volunteer has 2 test parts. Square areas of 2cm x 2cm were marked on the inside of the arm of the volunteer as test areas, 10 test areas were randomly arranged for each test sample, and the test results were averaged. In the test stage, stimulating the test area by using 50% capsaicin aqueous solution with the stimulation time of 30min, and stimulating and collecting the skin heme value of the test area; and coating a test sample on the test area, collecting skin heme values of the skin again after waiting for 20min, and calculating the change rate of the skin heme values before and after coating the test sample.

Skin heme value change rate (%) = (T _20min-T_0min)/T_0min ×100%

T _20min refers to the skin heme value after 20min of use;

t _0min refers to the skin heme value before use (i.e., the skin heme value after 50% capsaicin in water stimulation).

Test instrument: skin color tester CL400.

The skin heme value reflects the redness state of the skin, after the skin is stimulated by the capsaicin aqueous solution, the skin of the test area is rapidly reddened, the skin heme value is increased, and if the skin heme value of the test area is reduced more, the absolute value of the change rate of the skin heme value is larger, the better the stimulation repairing effect and the better the redness eliminating effect of the test sample are indicated.

The test results are shown in Table 2.

TABLE 2

Test sample	Skin heme value change rate (%)
		Blank control	-8.6
Example 1	-35.2
		Example 2	-35.0
Example 3	-27.3
		Example 4	-32.5
Example 5	-33.8
		Example 6	-32.0
Example 7	-29.7
		Example 8	-28.5
Example 9	-34.8
		Example 10	-31.1
Comparative example 1	-15.4
		Comparative example 2	-10.5
Comparative example 3	-12.3
		Comparative example 4	-21.8
Comparative example 5	-14.2
		Comparative example 6	-25.2
Comparative example 7	-16.9
		Comparative example 8	-20.6
Comparative example 9	-18.3
		Comparative example 10	-22.1
Comparative example 11	-20.8
		Comparative example 12	-15.4
Comparative example 13	-21.0
		Comparative example 14	-13.6

The test results show that the skin heme values are significantly reduced after stimulation using the extracts of the examples or comparative examples compared to the blank. The absolute value of the change rate of the skin heme values of the extracts of the examples and the comparative examples is larger than that of the blank control group, which shows that the extracts of the examples and the comparative examples have certain stimulation repairing effects. Among them, the extracts of examples 1 to 10 of the present application were superior in the repairing effect to comparative examples 1 to 14. Examples 1-10 showed better repair effect than comparative examples 1-3, demonstrating that the best repair effect of the extract obtained by fermenting and extracting kava roots and mung bean seeds with pichia pastoris, the second less fermentation and extraction effect of Saccharomyces cerevisiae, and the worst fermentation and extraction effect of Aspergillus oryzae. The repairing effect of examples 1-10 is superior to that of comparative examples 4, 5 and 7-11, which shows that there is a synergistic effect between kava roots and mung bean seeds during fermentation, and the effect of fermenting both is superior to that of fermenting kava roots or mung bean seeds alone, and in addition, the repairing effect of the extract obtained by replacing kava roots with leaves and stems of kava, replacing mung bean seeds with red beans or soybeans, or fermenting mung bean seeds after germination is deteriorated. The repairing effect of the extracts of examples 1-10 was superior to that of comparative example 12, indicating that the crude fermentation broth was subjected to cleavage in the resulting broth, and the resulting extract contained more active material and had better repairing effect. The repairing effect of example 1 is superior to that of examples 4-10 and comparative examples 6 and 13, showing that the mass ratio of kava root to mung bean seed in the fermentation medium, the kind and content of nutrients in the fermentation medium, and the pressure of high-pressure homogenization treatment have an effect on the repairing effect of the finally obtained extract in the preparation of the extract, and therefore, the mass ratio of kava root to mung bean seed in the present application should be set to (3-9): 1, the addition amount of nutrients to be 1.5-5%, the nutrient selected from at least one of brewer grains, fish meal, corn steep liquor, and the pressure of high-pressure homogenization treatment should be set to 20-50Mpa.

Test example 3: questionnaire feedback

Test sample: the same as in test example 1.

The testing method comprises the following steps: selecting 250 volunteers with sensitive muscle skin and facial skin in a reddish state and a percutaneous water loss value (TEWL value) of the facial skin of more than 20 (the volunteers are randomly divided into 25 groups, each 10 people use one essence, and the test results are averaged.) and using the sample essence or the matrix essence by the volunteers for 2 times a day in the morning and in the evening. During the test period, volunteers were not allowed to use skin care products of similar efficacy. After using the sample for 5 days, the volunteers are investigated in terms of skin pricking improvement condition, sedation effect, relaxation effect and repair effect of the test sample, the volunteers evaluate the test sample in a scoring mode, the test sample is fully divided into 10 points, the test result is subjected to the removal of the highest point and the lowest point, the rest result is averaged, and the higher the score is, the better the effect of the test sample is indicated.

The test results are shown in Table 3.

TABLE 3 Table 3

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Test results show that compared with the matrix essence or the essence of the comparative example, the essence containing the extract of the embodiment of the application has better effects of improving skin stinging, relieving skin redness and swelling, calming skin and repairing skin. Examples 1-10 had better effects of soothing, repairing, improving stinging and calming the skin than comparative examples 1-3, showing that in the present application, the effect of the obtained extract on soothing, repairing, improving stinging and calming the skin was best by fermenting kava roots and mung bean seeds with pichia pastoris, and the effect of the obtained extract on soothing, repairing, improving stinging and calming the skin was poor by fermenting and extracting with other strains. The effects of soothing, repairing, improving stinging and calming the skin of examples 1 to 10 are superior to those of comparative examples 4, 5 and 7 to 11, which show that there is a synergistic effect between kava roots and mung bean seeds during fermentation, and the effect of co-fermenting the two is superior to that of single kava roots or single mung bean seeds, and in addition, the effect of replacing kava roots with leaves and stems of kava, replacing mung bean seeds with red beans or soybeans, or fermenting mung bean seeds after germination, is poor. The extracts of examples 1-10 had better effects on soothing, repairing, improving stinging and calming the skin than comparative example 12, indicating that the crude fermentation broth was split in the resulting broth, and the resulting extract contained more active substance and had better effects on soothing, repairing, improving stinging and calming the skin. The effects of relieving, repairing, improving stinging and calming the skin of example 1 are superior to those of examples 4 to 10 and comparative examples 6 and 13, and it is demonstrated that the mass ratio of kava root to mung bean seed in the fermentation substrate, the kind and content of nutrients in the fermentation substrate and the pressure of the high-pressure homogenization treatment have an effect on the relieving, repairing, improving stinging and calming the skin of the finally obtained extract in the preparation of the extract, and therefore, the mass ratio of kava root to mung bean seed in the present application should be set to (3-9): 1, the addition amount of nutrients should be 1.5-5%, the nutrient should be selected from at least one of brewer's grains, fish meal and corn steep liquor, and the pressure of the high-pressure homogenization treatment should be set to 20-50Mpa.

Test example 4: safety performance test

Safety performance tests were performed according to the skin irritation/corrosiveness test of chapter six of cosmetic safety Specification (2015 edition).

Experimental animals: a rabbit; days of experiment: 14 days; skin irritation intensity of rabbits was judged according to skin irritation/corrosiveness test table 2 of cosmetic safety Specification (2015).

Test sample: the extracts prepared in examples 1 to 10 and comparative examples 1 to 14.

The test results are shown in Table 4.

TABLE 4 Table 4

The test result shows that the extract of the embodiment of the application is safe and can not generate irritation to skin when directly applied to skin. However, the extracts of comparative examples 2, 7 and 10 showed a certain irritation to the skin, indicating that the fermented species, the selection of the fermented plant parts, the variety of beans had a certain influence on the safety of the final extract.

The application adopts the Pichia pastoris to ferment and extract the kava stems and the mung bean seeds, and the finally obtained extract containing the kava roots is safe and non-irritating, and is suitable for being applied to the preparation of cosmetics with the effects of relieving and repairing.

The above embodiments are preferred embodiments of the present application, but the embodiments of the present application are not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present application should be made in the equivalent manner, and the embodiments are included in the protection scope of the present application.

Claims

1. A method for preparing an extract comprising kava roots, comprising:

(2) Sterilizing, cracking and filtering the crude fermentation broth to obtain an extract containing kava roots; wherein the fermentation substrate comprises kava roots and mung bean seeds;

the total mass of the kava roots and the mung bean seeds is 0.8-2% of the mass of the fermentation substrate; the mass ratio of the kava root to the mung bean seed is (3-9) 1;

The fermentation substrate further comprises nutrients and water; the addition amount of the nutrient is 1.5-5% of the mass of the fermentation substrate, and the addition amount of the water is 100% of the total mass of the fermentation substrate;

The nutrient comprises at least one of brewer's grains, fish meal and corn steep liquor;

In the step (1), the fermentation temperature is 28-33 ℃, and the fermentation time is 50-70h.

2. The method of claim 1, wherein the sterilization in step (2) is performed by incubating the crude fermentation broth at 80-95 ℃ for 15-30min;

The cracking in the step (2) is high-pressure homogenization treatment, the pressure of the high-pressure homogenization treatment is 20-50Mpa, and the time of the high-pressure homogenization treatment is 15-50min.

3. The method of claim 1, wherein the pichia pastoris seed solution in step (1) is inoculated in an amount of 3-5% (v/v) of the fermentation substrate;

the preparation method of the pichia pastoris seed solution comprises the following steps:

4. The method according to claim 3, wherein in step B1 or step B2, the liquid medium comprises at least one of wort medium, PDB medium, YPD medium, and Bengalia medium;

in the step B1, the temperature of the primary culture is 28-33 ℃, and the time of the primary culture is 20-28h;

In step B2, the inoculation amount of the suspension is 0.8-2% (v/v) of the liquid culture medium;

the temperature of the secondary culture is 28-33 ℃, and the time of the secondary culture is 36-60h.

5. An extract comprising kava root, which is prepared by the method of claim 1.

6. The use of the kava root-containing extract of claim 5 for the preparation of a cosmetic having soothing and repairing effects.