CN113041200A

CN113041200A - Cosmetic containing multiple fermentation liquor of custard apple and preparation method thereof

Info

Publication number: CN113041200A
Application number: CN202110384568.8A
Authority: CN
Inventors: 何秋星; 罗婷婷; 孔祥灵; 黄俊紫; 易小梅; 曹华
Original assignee: Guangdong Pharmaceutical University
Current assignee: Guangdong Pharmaceutical University
Priority date: 2021-04-09
Filing date: 2021-04-09
Publication date: 2021-06-29
Anticipated expiration: 2041-04-09
Also published as: CN113041200B

Abstract

The invention provides a cosmetic containing custard apple multiple fermentation broth and a preparation method thereof, belonging to the technical field of cosmetics. Aiming at the analysis of the components of the fermentation liquor of the custard apple and the exploration of the fermentation process, fermentation liquor in different stages is obtained one by utilizing batch fermentation, the fermentation synergistic effect of fermentation strains is explored so as to improve the effect of the fermentation liquor, and the multiple fermentation filtrate of the custard apple is obtained, and the fermentation liquor belongs to natural plants, is rich in nutrition, is not easy to be allergic, is safe and reliable, has good moisturizing effect, can effectively remove free radicals, inhibits tyrosinase, reduces melanin, and further achieves the purpose of whitening; the strain used in the fermentation process is reliable and easily available in source, is not pathogenic bacteria, is mostly human intestinal probiotics, is simple in preparation method, has no organic reagent residue, has the effects of easy absorption, moisture retention, whitening and skin condition improvement, can be used as an effect raw material additive to be applied to cosmetics, is wide in application and has great market value.

Description

Cosmetic containing multiple fermentation liquor of custard apple and preparation method thereof

Technical Field

The invention belongs to the technical field of cosmetics, and particularly relates to a moisturizing and whitening cosmetic containing multiple fermentation liquor of custard apple and a preparation method thereof.

Background

In recent years, with the change of consumption ideas, cosmetics are more advocated with the concept of nature and being far away from chemical pollution, and particularly, from the development trend of the Japanese cosmetic industry, more than 200 kinds of Chinese herbal medicine plant raw materials are used by various cosmetic companies, and Chinese herbal medicines are widely accepted due to mild natural medicine effects and few adverse effects. At present, cosmetics containing natural Chinese herbal medicines account for over 50% of the entire cosmetic market in Japan. At home, the prepared Chinese herbal medicine cosmetics enterprises have the highest index number. Therefore, the search for the development of natural herbal skin cosmetic agents with high efficacy and no or low side effects on human body has become a more important issue in the field of cosmetics. Therefore, developing a natural plant-derived cosmetic raw material with multiple effects, high safety level and good stability becomes one of the new directions of researchers.

Annona squamosa L, also known as Annona squamosa L, belongs to Annonaceae (Annonaceae). Zhejiang, Taiwan, Fujian, Guangdong, Guangxi, Hainan and Yunnan provinces of China are all cultivated with 15. Is a famous fruit in tropical regions, can be used for treating acute dysentery, mental depression and spinal cord osteopathia; the fruit can be used for treating sore and swelling, tonifying spleen, and is recorded in "Kaihu Kaishangchang Shu" (compilation of plant Mingmi charts), and can be eaten as a cause and has a similar shape to the Buddha of Sakyamuni.

The custard apple contains abundant polysaccharide, polyphenol, flavonoid, other organic acids and other substances, and has a very wide application scene. According to the database data at home and abroad, most scholars treat the custard apple by using hydrophilic organic solvents, and the method has more problems, such as: the valuable components in the custard apple are not effectively extracted at one time by using a single solvent for extraction, and the extracting solution has large active substance molecular weight and excessive impurities, thereby causing great waste; the extraction solvent such as methanol, acetone, ethanol and the like is relatively limited in the application of cosmetics, and is easy to cause fire safety problems and the like, so that the custard apple which is a nutrient-rich raw material is very limited in the use scene of the cosmetics.

Because microorganisms can generate a large amount of cellulase, pectinase and other extracellular enzymes with special activity in the growth and metabolism processes, the microorganisms can decompose the cell walls of plant cells, so that the plant cells are broken, active ingredients are dissolved out, macromolecular impurities are metabolized and converted, and the efficacy of the plant extracting solution is greatly increased. In addition, extracellular polysaccharide substances released by part of microorganisms in the growth and metabolism process have excellent cosmetic effects, and a microbial fermentation method has the advantages of simple and safe production process, low production requirement, capability of enhancing efficiency and reducing toxicity, small molecular weight of products, easiness in skin absorption and the like, and gradually draws attention of domestic and foreign cosmetic raw material suppliers. As the microorganism is utilized to ferment the plants, the product has better effect and more application scenes compared with solvent extraction, and the method is a hot method which is sought by researchers at present.

For example, Chinese patent publication CN110731941A discloses a cosmetic for skin care with beneficial bacteria, which comprises the following raw materials by mass percent: 1-3% of lactobacillus/eriodictyon fermentation product extract, 1-3% of lactobacillus/soybean fermentation product extract, 1-3% of lactobacillus/rice fermentation product, 1-3% of lactobacillus/ailanthus fermentation product, 1-3% of lactobacillus/rye fine powder fermentation product, 1-3% of antioxidant, 0.5-3% of adsorbent, 1-10% of skin conditioner, 1-10% of humectant, 0.01-1% of preservative, 0.01-1% of essence and the balance of water; the invention can increase the metabolism speed of epithelial cells, promote the skin to be renewed, inhibit the activity of melanin enzyme and strengthen the skin barrier.

Chinese patent publication CN109843263A discloses a cosmetic composition of a ginseng fruit fermentation product obtained by double fermentation of ginseng fruit using black yeast (Aureobasidium pullulans CXHB-8) and lactic acid bacteria. The composition has effects in promoting cell proliferation, recovering damaged cells, inhibiting collagen degradation, inhibiting skin aging, and improving wrinkle.

Chinese granted patent CN108245479B discloses a facial mask containing bifidobacterium lactis fermentation active extract, which obtains the anti-allergy facial mask with breakthrough effect superior to hydrocortisone and collagen regeneration stimulation effect by optimizing the fermentation process and combining with scientific proportioning. During the fermentation stage, the brevicoside R is added, and other optimized process conditions are combined, so that the physiological performance of the bifidobacterium lactis is changed, and active substances such as biological polysaccharide and the like with extremely strong activity, anti-allergy and regeneration-promoting biological activity are generated.

Most fermentation liquor for cosmetics in the market is subjected to single-strain fermentation treatment, or is limited by fermentation conditions or selection of fermentation strains, and fermentation substrates cannot be maximally utilized; thus, various bacterial fermentation processes have emerged; besides extremely high requirements on the skill level of operators, the compound strain fermentation has relatively uncontrollable competitive action among various strains, harsh fermentation conditions, different batch differences of product quality and adverse effects on cosmetic production enterprises; for the one-time fermentation of the composite strain, aiming at the analysis of the components of the fermentation liquor of the custard apple and the exploration of the fermentation process, the fermentation liquor of different stages is obtained one by batch fermentation, and the fermentation synergistic effect of the fermentation strains is explored so as to improve the effect of the fermentation liquor, thereby obtaining the multiple fermentation filtrate of the custard apple with wide application scenes.

Disclosure of Invention

In order to solve the technical problems, the invention provides a method for preparing a multiple fermentation liquid of custard apple by performing multiple fermentation on the custard apple by using a strain combination, and application of the multiple fermentation liquid of custard apple in cosmetics.

The invention adopts the following technical scheme:

a moisturizing and whitening cosmetic containing multiple fermentation liquor of custard apple comprises the following components in percentage by weight: 1-5% of propylene glycol, 1-6% of glycerol, 0.01-0.05% of disodium ethylene diamine tetraacetate, 2-10% of isopropyl myristate, 0.05-0.1% of phenoxyethanol, 0.1-0.15% of ethylhexyl glycerol, 4-15% of custard apple fermentation filtrate, 0.05-0.25% of hydrogenated castor oil and the balance of water.

Preferably, the moisturizing and whitening cosmetic comprises the following components in percentage by weight: 1.5-3% of propylene glycol, 2-4% of glycerol, 0.02-0.04% of disodium ethylene diamine tetraacetate, 4-6% of isopropyl myristate, 0.05-0.08% of phenoxyethanol, 0.12-0.15% of ethylhexyl glycerol, 6-8% of custard apple fermentation filtrate, 0.08-0.15% of hydrogenated castor oil and the balance of water.

Preferably, the moisturizing and whitening cosmetic comprises the following components in percentage by weight: 2% of propylene glycol, 3.2% of glycerol, 0.03% of disodium ethylene diamine tetraacetate, 5% of isopropyl myristate, 0.05% of phenoxyethanol, 0.15% of ethylhexyl glycerol, 7.5% of custard apple fermentation filtrate, 0.1% of hydrogenated castor oil and the balance of water.

Wherein the mass ratio of the custard apple fermentation filtrate to the hydrogenated castor oil is 50-100: 1; preferably 75: 1.

The mass ratio of the phenoxyethanol to the ethylhexyl glycerin is 1: 1-3; preferably 1: 3.

The cosmetic is cream, emulsion, toner, essence, gel or facial mask.

The custard apple fermentation filtrate is custard apple multiple fermentation liquid, and the fermentation strains comprise Bacillus subtilis, Lactobacillus iners, Bifidobacterium and Saccharomyces cerevisiae.

The invention also discloses a preparation method of the custard apple multiple fermentation liquid, which comprises the following steps: the method specifically comprises the following steps:

(1) rejuvenating the strains;

(2) purifying strains;

(3) seed liquid amplification culture:

(3.1) purifying and culturing the Bacillus subtilis: adding 0.1g of strain lyophilized powder into 1ml of sterile physiological saline, mixing, sucking to 2 TSA solid plates on average, shaking, and culturing at 30 deg.C for 50 h; selecting a colony which grows vigorously, inoculating the colony to a TSA slant culture medium, culturing for 24-48h at 25-38 ℃, and washing with 5-10mL of sterile physiological saline to obtain a bacterial suspension A;

(3.2) Lactobacillus (Lactobacillus iners) purification culture: adding 0.1g of strain lyophilized powder into 1ml of sterile physiological saline, mixing, sucking on average to 2 blood plates, shaking, and culturing at 37 deg.C for 50 h; selecting a colony which grows vigorously, inoculating the colony to a slant blood culture medium, culturing for 24-48h at 25-38 ℃, and washing with 5-10mL of sterile physiological saline to obtain a bacterial suspension B;

(3.3) Bifidobacterium (Bifidobacterium adolescent) purification culture: adding 0.1g of strain lyophilized powder into 1ml of sterile physiological saline, mixing, sucking to 2 special culture media on average, shaking, and performing anaerobic culture at 37 deg.C for 50 h; selecting a colony which grows vigorously, inoculating the colony into a slant culture medium to culture for 24-48h at 25-38 ℃, and washing with 5-10mL of sterile physiological saline to obtain a bacterial suspension C;

(3.4) Saccharomyces cerevisiae (Saccharomyces cerevisiae Hansen) purification culture: adding 0.1g of strain lyophilized powder into 1ml of sterile physiological saline, mixing, sucking on 2 TSA plates on average, shaking, and culturing at 37 deg.C for 50 h; selecting a colony which grows vigorously, inoculating the colony into a slant culture medium to be cultured for 24-48h at the temperature of 25-38 ℃, and taking 5-10mL of sterile physiological saline to wash to obtain a bacterial suspension D;

(4) obtaining of custard apple fermentation product:

(4.1) selecting and cleaning mature custard apple, crushing the whole custard apple into pulp, adding sterile water, shaking uniformly and sterilizing to obtain sterilized custard apple pulp, namely fermentation substrate 1;

(4.2) inoculating the fermentation substrate 1 obtained in the step (4.1) into the bacterial suspension A, shaking up, sealing, introducing sterile atmosphere, performing shake culture for 5-15 days under the conditions that the temperature is 25-38 ℃ and the pH is 5-8, sterilizing after fermentation is finished, adjusting the pH to 5-8, performing ultrasonic treatment for 5-10min, and shaking up to obtain a fermentation product 2;

(4.3) inoculating the fermented product 2 obtained in the step (4.2) into the bacterial suspension B, shaking up, sealing, introducing sterile carbon dioxide, performing shake culture for 20-30h at the temperature of 25-38 ℃ and under the condition that the pH value is 5-8, sterilizing after fermentation is finished, adjusting the pH value to 5-8, performing ultrasonic treatment for 5-10min, and shaking up to obtain a fermented product 3;

(4.4) inoculating the fermented product 3 obtained in the step (4.3) into the bacterial suspension C, shaking up, sealing, introducing sterile carbon dioxide, performing shake culture for 20-30h at the temperature of 25-38 ℃ and under the condition that the pH value is 5-8, sterilizing after fermentation is finished, adjusting the pH value to 5-8, performing ultrasonic treatment for 5-10min, and shaking up to obtain a fermented product 4;

(4.5) inoculating the fermentation product 4 obtained in the step (4.4) into the bacterial suspension D, shaking up, sealing, introducing sterile carbon dioxide, shake-culturing for 1-5 days at 25-38 ℃ and pH 5-8, sterilizing after fermentation, breaking the wall by ultrasonic waves for 2min, and centrifuging at 8000rpm for 20min to obtain a fermentation product 5, namely the multi-fermentation liquid of the custard apple.

Preferably, the culture medium of the TSA solid plate in the step (3.1) is tryptone 15g/L, soytone 5g/L, sodium chloride 4g/L, yeast extract 2g/L, agar 15g/L, MgSO₄·7H₂O 0.5g/L，K₂HPO₄ 1g/L KH₂PO₄1g/L and 2g/L glucose;

the culture medium of the blood plate in the step (3.2) is tryptone 4g/L, soytone 5g/L, sodium chloride 4g/L and NaH₂PO₄1g/L, 10g/L of beef extract powder, 5% of defatted fiber sheep blood and MgSO₄·7H₂O 0.5g/L，K₂HPO₄ 1g/L KH₂PO₄1g/L of agar and 15g/L of agar;

the special culture medium in the step (3.3) is soy peptone 4g/L, sodium chloride 10g/L and NaH₂PO₄1g/L, yeast extract 10g/L, tryptone 5g/L, glucose 8g/L, MgSO₄·7H₂O 0.5g/L，K₂HPO₄ 1g/L KH₂PO₄1g/L, 15g/L agar and 0.5 g/L-cysteine;

the culture medium of the plate in the step (3.4) is tryptone 15g/L, soytone 5g/L, yeast extract 2g/L, sodium chloride 4g/L, glucose 2g/L, K₂HPO₄ 1g/L KH₂PO₄1g/L and 15g/L agar.

Preferably, the bacterial suspensions A-D prepared in the steps (3.1) - (3.4) are subjected to turbidimetry by a bacterial turbidimeter, and the turbidity is 1-5 MCF.

The whole fruit is smashed into pulp in the step (4.1), the temperature is 20-30 ℃, and the mixture ratio of custard apple pulp to sterile water is 10 g: 50 g.

Preferably, the mass-to-volume ratio of the fermentation substrate 1 to the bacterial suspension A in the step (4.2) is 50-100: 1-5;

the mass-to-volume ratio of the fermentation substrate 2 to the bacterial suspension B in the step (4.3) is 50-100: 1-5;

the mass-to-volume ratio of the fermentation substrate 3 to the bacterial suspension C in the step (4.4) is 50-100: 1-5;

the mass-to-volume ratio of the fermentation substrate 4 to the bacterial suspension D in the step (4.5) is 50-100: 1-5.

According to the invention, the mass-volume ratio of the fermentation substrate to the bacterial suspension is controlled in the implementation process, so that the fermentation substrate can be well fermented, and the obtained fermentation product has high contents of polysaccharide, polyphenol, flavone and protein, and has better whitening and moisturizing effects.

The custard apple multiple fermentation liquid is pre-frozen in a freeze dryer at-20 to-30 ℃ for 5 to 10 hours to obtain freeze-dried powder.

The invention also discloses application of the custard apple multiple fermentation liquid in preparation of moisturizing and whitening cosmetics.

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

(1) the multiple fermentation liquor of custard apple has good moisturizing effect, can effectively remove free radicals, inhibit tyrosinase, reduce melanin, and further achieve the purpose of whitening;

(2) the invention uses natural plants, has rich nutrition, is not easy to be allergic, is safe and reliable, has no side effect, and is suitable for various crowds to use for a long time;

(3) the multiple fermentation liquid of the custard apple obtained by the invention has smaller active matter molecular weight, and polysaccharide and polypeptide in the filtrate of the product obtained by microbial fermentation have good effect of promoting and repairing damaged skin;

(4) the batch multiple fermentation used in the invention has low requirement on the operation skill level of personnel and good reproducibility for one-time composite fermentation;

(5) according to the invention, the multiple fermentation liquor of the custard apple is fermented for multiple times, enzymes, extracellular polysaccharides and hypha metabolites generated by microorganisms can be accumulated in the fermentation product, the previous step of fermentation is used for preparing for the next step of fermentation, and the contents of polysaccharides, proteins, flavones and the like in the obtained multiple fermentation liquor are obviously improved;

(6) the strain used by the invention has reliable and easily obtained sources, is not pathogenic bacteria, is mostly human intestinal probiotics, has simple preparation method, no organic reagent residue, has the effects of easy absorption, moisture retention, whitening and skin condition improvement, can be used as an effect raw material additive to be applied to cosmetics, has wide application and great market value.

Drawings

FIG. 1 shows the effect of 0.8mg/mL custard fruit ferment 2-5 on HSF and HaCat cells, respectively;

FIG. 2 is the in vitro evaluation of the moisture retention performance of 1.0g/mL custard apple multiple fermentate.

Detailed Description

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.

Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention.

When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The source of the raw materials used in the present invention is not limited, and the raw materials used in the present invention are not particularly limited

Are all common commercial products in the technical field. Wherein Bacillus subtilis (Bacillus subtilis) is ATCC 6633, Lactobacillus iners (Lactobacillus iners) is ATCC 55195, Bifidobacterium (Bifidobacterium adolescent) is ATCC 15703, Saccharomyces cerevisiae Hansen (Saccharomyces cerevisiae Hansen) is ATCC 9763, and tyrosinase (25ku), L-DOPA, HSF, HaCat cells and the like are purchased from Shanghai Baomann Biotech Co., Ltd.

Example 1 preparation of the Annona squamosa fermented extract:

(1) rejuvenation of strains:

and (3) selecting a bacillus subtilis bacterial colony, placing the bacillus subtilis bacterial colony in a liquid culture medium, and activating in a shaking table to obtain activated bacillus subtilis.

And (3) selecting lactic acid rod bacterial colonies, looping the lactic acid rod bacterial colonies in a liquid culture medium, and putting the lactic acid rod bacterial colonies into a shaking table for activation to obtain activated bacillus subtilis.

And (3) selecting a colony of the bifidobacteria rod, namely looping the colony in a liquid culture medium, and putting the colony into a shaking table for activation to obtain the activated bacillus subtilis.

And (3) selecting saccharomyces cerevisiae bacterial colonies, placing the saccharomyces cerevisiae bacterial colonies in a liquid culture medium, and activating the saccharomyces cerevisiae bacterial colonies in a shaking table to obtain activated bacillus subtilis.

(2) And (3) strain purification:

and (2) respectively diluting the activated bacteria liquid obtained in the step (1), and uniformly coating the diluted bacteria liquid on the culture plate so as to detect the condition of the bacterial strain and obtain four purified probiotics.

(3) Seed liquid expanded culture

(3.1) purifying and culturing the Bacillus subtilis: adding 0.1g strain lyophilized powder into 1ml sterile physiological saline, mixing, sucking to 2 TSA solid plates with culture medium of tryptone 15g/L, soybean peptone 5g/L, sodium chloride 4g/L, yeast extract 2g/L, and MgSO₄·7H₂O 0.5g/L，K₂HPO₄ 1g/L KH₂PO₄1g/L, 2g/L glucose and 15g/L agar, shaking up, and culturing at 30 ℃ for 50 h; selecting a colony which grows vigorously, inoculating the colony to a TSA slant culture medium, culturing for 48 hours at 28 ℃, and washing with 10mL of sterile physiological saline to obtain a bacterial suspension A, wherein the Mach turbidity value of the bacterial suspension A is 2.10 MCF;

(3.2) Lactobacillus (Lactobacillus iners) purification culture: adding 0.1g of lyophilized strain powder into 1ml of sterile physiological saline, mixing, sucking on average to 2 blood plates, and culturing in the medium of tryptone 4g/L, soybean peptone 5g/L, sodium chloride 4g/L, NaH₂PO₄1g/L, 10g/L of beef extract powder, 5% of defatted fiber sheep blood and MgSO₄·7H₂O 0.5g/L，K₂HPO₄ 1g/L KH₂PO₄1g/L of agar and 15g/L of agar, shaking up, and culturing for 50h at 37 ℃; selecting a colony which grows vigorously, inoculating the colony to a slant blood culture medium, culturing for 48h at 36.5 ℃, and washing with 10mL of sterile physiological saline to obtain a bacterial suspension B, wherein the Maifanshi turbidity value of the bacterial suspension B is 2.50 MCF;

(3.3) Bifidobacterium (Bifidobacterium adolescent) purification culture: adding 0.1g of strain lyophilized powder into 1ml of sterile physiological saline, mixing, and sucking to 2 special culture media (soybean peptone 4g/L, sodium chloride 10g/L, NaH)₂PO₄1g/L, yeast extract 10g/L, tryptone 5g/L, glucose 8g/L, MgSO₄·7H₂O 0.5g/L，K₂HPO₄ 1g/L KH₂PO₄1g/L of agar, 15g/L of agar and 0.5g/L of L-cysteine, shaking up, and carrying out anaerobic culture at 37 ℃ for 50 hours; selecting a colony which grows vigorously, inoculating the colony into a slant culture medium to be cultured for 48h at 36.5 ℃, and washing 10mL of sterile physiological saline to obtain a bacterial suspension C, wherein the Mach turbidity value of the bacterial suspension C is 1.10 MCF;

(3.4) Saccharomyces cerevisiae (Saccharomyces cerevisiae Hansen) purification culture: adding 0.1g strain lyophilized powder into 1ml sterile physiological saline, mixing, sucking on 2 plates with medium of casein peptone 15g/L, soybean peptone 5g/L, yeast extract 2g/L, sodium chloride 4g/L, glucose 2g/L, K₂HPO₄ 1g/L KH₂PO₄1g/L of agar and 15g/L of agar, shaking up, and culturing for 50h at 37 ℃; and selecting a colony which grows vigorously, inoculating the colony into a slant culture medium, culturing for 48h at 28 ℃, and washing with 10mL of sterile physiological saline to obtain a bacterial suspension D, wherein the Maifand turbidity value of the bacterial suspension D is 1.50 MCF.

(4) Obtaining of custard apple fermentation product:

(4.1) selecting and cleaning mature custard apple, crushing the whole fruit into pulp, adding 80g of sterile water into 20g of custard apple pulp, shaking up and sterilizing to obtain sterilized custard apple pulp, namely a fermentation substrate 1;

(4.2) inoculating the fermentation substrate 1 obtained in the step (4.1) into the bacterial suspension A according to the mass-to-volume ratio of 100:5, shaking up, sealing, introducing sterile atmosphere, performing shake culture for 180h under the conditions that the temperature is 32 ℃ and the pH is 6.8, sterilizing for 15min under the condition of 121 ℃ after fermentation is finished, adjusting the pH to 7.2, performing ultrasonic treatment for 5min, and shaking up to obtain a fermentation product 2;

(4.3) inoculating the fermented product 2 obtained in the step (4.2) into the bacterial suspension B according to the mass-to-volume ratio of 100:5, shaking up, sealing, introducing sterile carbon dioxide, performing shake culture for 24h under the conditions that the temperature is 36.5 ℃ and the pH is 7.2, sterilizing for 30min under the condition of 80 ℃ after fermentation is finished, adjusting the pH to 6.8, performing ultrasonic treatment for 5min, and shaking up to obtain a fermented product 3;

(4.4) inoculating the fermented product 3 obtained in the step (4.3) into the bacterial suspension C according to the mass-to-volume ratio of 100:5, shaking up, sealing, introducing sterile carbon dioxide, performing shake culture for 20h at the temperature of 36.5 ℃ and under the condition that the pH value is 6.8, sterilizing for 30min at the temperature of 80 ℃ after fermentation is finished, adjusting the pH value to 7.2, performing ultrasonic treatment for 5min, and shaking up to obtain a fermented product 4;

(4.5) inoculating the fermentation product 4 obtained in the step (4.4) into the bacterial suspension D according to the mass-volume ratio of 100:5, shaking up, sealing, introducing sterile carbon dioxide, performing shake culture for 50h at the temperature of 30 ℃ and the pH value of 7.2, sterilizing at the temperature of 121 ℃ for 15min after fermentation is finished, performing ultrasonic wall breaking for 2min, and centrifuging at 8000rpm for 20min to obtain the fermentation product 5, namely the multiple fermentation liquid of the custard apple.

Meanwhile, the fermented products 2, 3 and 4 are respectively prepared for standby by the same batch and method.

Comparative example 1 preparation of aqueous extract of Annona squamosa

Taking the same amount of custard apple pulp as the fermentation substrate 1 in example 1, heating the pulp and deionized water at a mass ratio of 1:4 to 80 ℃, and extracting under reflux for 2.0h to obtain the water extract of custard apple.

Comparative example 2 preparation of 50% ethanol extract of custard apple

Taking the custard apple pulp with the same amount as the fermentation substrate 1 in the example 1, adding 50% ethanol by volume fraction according to the material-liquid ratio of 1:4 (mass ratio), heating to 90 ℃, and performing reflux extraction for 2.0h to obtain the custard apple 50% ethanol extract.

Comparative example 3 preparation of Sakyamuni fruit fermentation broth a

Taking the custard apple pulp with the same amount as the fermentation substrate 1 in the example 1, inoculating the bacteria suspension A, the bacteria suspension B, the bacteria suspension C and the bacteria suspension D according to the mass-volume ratio of 100:5, introducing sterile carbon dioxide, performing shake culture for 50h at the temperature of 30 ℃ and the pH value of 7.2, sterilizing for 15min at the temperature of 121 ℃ after the fermentation is finished, performing ultrasonic wall breaking for 2min, and centrifuging at 8000rpm for 20min to obtain custard apple fermentation liquid a.

Comparative example 4 preparation of multiple fermentation broth of Sakyamuni fruit

The difference from example 1 is that: the adding sequence of each bacterial suspension is different, and the specific steps are as follows:

(4.2) inoculating the fermented product 2 obtained in the step (4.1) into the bacterial suspension B according to the mass-to-volume ratio of 100:5, shaking up, sealing, introducing sterile carbon dioxide, performing shake culture for 24h under the conditions that the temperature is 36.5 ℃ and the pH is 7.2, sterilizing for 30min under the condition of 80 ℃ after fermentation is finished, adjusting the pH to 6.8, performing ultrasonic treatment for 5min, and shaking up to obtain a fermented product II;

(4.3) inoculating the fermentation substrate II obtained in the step (4.2) into the bacterial suspension A according to the mass-to-volume ratio of 100:5, shaking up, sealing, introducing sterile atmosphere, performing shake culture for 180h under the conditions that the temperature is 32 ℃ and the pH is 6.8, sterilizing for 15min under the condition of 121 ℃ after fermentation is finished, adjusting the pH to 7.2, performing ultrasonic treatment for 5min, and shaking up to obtain a fermentation product III;

(4.4) inoculating the fermentation product III obtained in the step (4.3) into the bacterial suspension D according to the mass-to-volume ratio of 100:5, shaking up, sealing, introducing sterile carbon dioxide, performing shake culture for 50h at the temperature of 30 ℃ and under the condition that the pH value is 7.2, sterilizing for 15min at the temperature of 121 ℃ after fermentation is finished, performing ultrasonic wall breaking for 2min, and centrifuging for 20min at 8000rpm to obtain a fermentation product IV;

(4.5) inoculating the fermentation product IV obtained in the step (4.4) into the bacterial suspension C according to the mass-to-volume ratio of 100:5, shaking up, sealing, introducing sterile carbon dioxide, performing shake culture for 20h at the temperature of 36.5 ℃ and under the condition of pH6.8, sterilizing for 30min at the temperature of 80 ℃ after fermentation is finished, adjusting the pH to 7.2, performing ultrasonic treatment for 5min, and shaking up to obtain the fermentation product V.

The custard apple fermentation broth extract prepared in example 1 is viscous liquid in appearance, foams by shaking, and has a color ranging from yellow to brownish yellow and a certain flower fragrance. The pH is 4.0-7.0, the plate colony count is less than 50CFU/mL, no pathogenic bacteria is detected, and the cosmetic hygiene standard GB7916-87 is met.

Experimental example 1 analysis of the composition of the custard apple fermentation broth

Protein detection methods refer to GB 5009.5-2010; the polysaccharide is detected by adopting a phenol-sulfuric acid method; the measurement methods of total flavonoids, polyphenols and the like refer to the industrial methods, and are specifically shown in table 1 below.

TABLE 1

Test example 2 biological Performance measurement of fermentation broth of Sakyamuni fruit at various stages

The MTT method is adopted to examine the influence of the fermentation liquor of the custard apple (multiple fermentation liquor of custard apple, fermentation product 2, fermentation product 3 and fermentation product 4) in different fermentation periods on the activity of Human Skin Fibroblasts (HSF) and human immortalized keratinocyte (HaCat) cells under the same mass concentration, wherein the HSF cells and the HaCat cells are respectively important components of the dermis layer and the epidermis layer of the human skin. The biological performance of the multiple fermentation liquids in the first experimental example is verified through a cell level experiment, and the effect of the multiple fermentation liquids in improving the skin condition is verified. HSF and HaCat at concentrations were seeded into 96-well plates and after 24h incubation, the solution was removed from the wells. Adding 0.8mg/mL solution prepared by taking DMEM as a solution, placing 100 mu L of solution in each well, and culturing for 24h in an incubator by taking culture solution without samples as a control. The wells were removed, washed 2-3 times with PBS, and their OD at 490nm was measured using a microplate reader, and cell viability was calculated as follows.

In the formula A_S-absorbance of the sample set; a. the₀-blank set absorbance values; a. the_i-control absorbance values.

After the fermentation liquid of the custard apple acts on HSF and HaCat cells for 24h, the cell activity is respectively shown in figure 1, after 2-5 of the custard apple fermentation product of 0.8mg/mL acts on the HSF and HaCat cells respectively, and the multiple fermentation liquid (namely fermentation liquid 5) of the custard apple acts on the cells for 24h, the proliferation rate of the custard apple on the HSF cells reaches 121.1 percent and the proliferation rate of the HaCat cells reaches 118.3 percent through the MTT method. Meanwhile, the experimental data can also show that the fermentation liquid of the custard apple at the other stages obtained in the experimental example 1 has different proliferation effects on HSF and HaCat for a blank control group and a water extraction group, but the effect is slightly worse than that of the multiple fermentation liquid of the custard apple obtained in the experimental example 1, which indicates that the fermentation method of the experimental example 1 can obtain the fermentation liquid with better proliferation effects on human skin fibroblasts and human immortalized keratinocytes.

Test example 3 safety testing of the fermented extract of custard apple:

1. the experimental method comprises the following steps:

the chick embryo allantoic membrane test is mainly classified into chick embryo chorioallantoic membrane test (CAMVA) and chick embryo chorioallantoic membrane test (HET-CAM). The chick embryo allantoic membrane has rich blood vessel networks and is similar to the structure of an eye conjunctiva, and the chick embryo allantoic membrane test is a stimulation experiment in-vitro replacement method with high sensitivity. The experiment operation is strictly executed according to the export-import inspection and quarantine industry standard SN/T2329-2009 of the people's republic of China, and the details are shown in the standard.

2. The experimental results are as follows:

and judging and selecting a reaction time method according to an experimental result.

Concentrating the multiple fermentation extraction filtrate of the custard apple to different gradients, wherein the maximum gradient IS 2g/mL (the ratio of raw materials to the filtrate), dripping 0.3mL of fermentation filtrate on the surface of a chorioallantoic membrane, the range IS about 1.5cm in diameter, lightly washing with normal saline after contacting 30S, observing the change of CAM blood vessels within 5min, recording the initial time of bleeding, hemolysis and blood coagulation, calculating the stimulation score (IS) according to the observation result and the application formula (1), and keeping two digits after the decimal point:

in the formula:

sec H (bleeding time) -the average time to onset of bleeding observed on CAM membranes in seconds(s);

sec L (vascular melting time) -the average time to onset of vascular melting observed on the CAM membrane in seconds(s);

sec C (clotting time) -the average time to onset of clotting observed on the CAM membrane in seconds(s).

ps: bleeding: blood flows from the CAM membrane into the blood vessel and out of the blood vessel, and may be manifested in various forms such as punctate hemorrhage or flocculent diffuse hemorrhage.

Blood coagulation: denaturation of intravascular and extravascular proteins is manifested by slow intravascular blood flow or thrombosis, a brownish black appearance of the blood vessels, and turbidity and opacity outside the blood vessels.

Vessel melting: the small vessel wall on the CAM membrane is ruptured, and the small vessel is dissolved and disappears.

The ocular irritation of the test subjects was classified according to the calculated IS values as shown in table 2 below.

TABLE 2 evaluation of stimulation scoring results

Stimulation scoring	Irritation classification
		IS＜1	Has no irritation
1≤IS＜5	Light irritation
		5≤IS＜9	Moderate irritation
IS≥10	Strong irritation/corrosion

The experimental results show that IS IS less than 1 at each concentration, and that the custard apple fermentation extract obtained in the first embodiment has no irritation, which shows that the custard apple fermentation extract provided by the invention has safety and does not bring adverse reactions to human bodies.

Test example 4 measurement of antioxidant Properties of Annona squamosa fermentation extract filtrate

Dissolving the multiple fermentation extract of the custard apple obtained in the first embodiment in distilled water to obtain a series of custard apple fermentation filtrates with concentrations (0.10g/mL, 0.25g/mL, 0.5g/mL, 0.75g/mL, 1.0g/mL), and performing antioxidant property detection.

4.1 determination of ABTS + clearing Capacity

Diluting the ABTS + solution with phosphate buffer solution (10mmol/L, pH7.4) to make its absorbance at 734nm wavelength be 0.700 + -0.020 to obtain ABTS + working solution. Respectively taking 1mL of 2mg/mL samples of the custard apple fermentation extraction filtrate with the antioxidant function into a test tube, adding 3mL of ABTS + working solution, fully and uniformly mixing, storing for 10min in a dark place at room temperature, measuring absorbance at 734nm by taking phosphate buffer as a blank control), making 3 parallel samples for each sample, and calculating the clearance rate according to the following formula:

wherein Ai is the absorbance value of 1mL of sample solution +3mL of ABTS + solution; aj is the absorbance value of 1mL of sample solution plus 3mL of phosphate buffer; ac was the absorbance value of 1mL phosphate buffer +3mL LABTS + solution.

4.2 determination of hydroxyl radical scavenging Capacity

To 0.40mL of 6mmol/L ferrous sulfate solution, 1mL of 8.8mmol/L hydrogen peroxide solution was added, and then 1mL of 9 mmol/L salicylic acid solution and 1.60mL of distilled water were added and mixed well, heated in a water bath at 37 ℃ for 15min (distilled water was a blank control), and the absorbance value Ac was measured at a wavelength of 510 nm. Ai is the absorbance value measured by replacing 1.60mL of distilled water with 2.0mg/mL of sample of the custard apple fermentation extraction filtrate with the antioxidant function, and the absorbance value is Aj measured by replacing hydrogen peroxide solution with 1mL of distilled water. And taking the Vc solution as a positive control, and calculating the clearance rate of the sample on the hydroxyl radical.

4.3 determination of superoxide anion radical scavenging ability

1mL of distilled water was added to 1.40mL of a Tris-HCl buffer solution (0.05mol/L, pH 8.2), 0.2mL of 5mmol/L pyrogallol solution was added thereto, the mixture was mixed well, and the absorbance value Ac was measured at a wavelength of 320nm after standing for 5 min. Ai is the absorbance value measured by replacing 1mL of distilled water with 2mg/mL of sample of the custard apple fermentation extraction filtrate with the antioxidant function, and 0.2mL of 10mmol/L hydrochloric acid solution is used for replacing pyrogallol solution to measure the absorbance value Aj. 3 replicates of each sample were taken and the clearance of superoxide anion radicals from the samples was calculated.

The results are shown in Table 3.

TABLE 3 measurement result of antioxidant property of filtrate obtained by fermenting and extracting custard apple

From the above data, it can be seen that the custard apple fermentation extract filtrate with antioxidant function of the first embodiment of the present invention has better cleaning capability for ABTS +, hydroxyl radical and superoxide anion radical, wherein when the concentration of the custard apple fermentation extract is increased, the antioxidant capacity of the fermentation liquid is correspondingly increased, and in 1.0g/mL of the fermentation liquid, the ABTS + clearance rate is 93.6%, the hydroxyl radical clearance rate is 90.1%, and the superoxide anion radical clearance rate is 93.3%. The ABTS + clearance rate of the extracting filtrate of the non-fermented custard apple is 61.1%, the hydroxyl radical clearance rate is 42.9%, and the superoxide anion radical clearance rate is 36.4%, which shows that the radical clearance rate of the custard apple fermentation extracting solution with each concentration is greatly increased after the treatment by the method.

Test example 5 study on in vitro moisture retention of Annona squamosa fruit fermentation extract

Different humectant molecules have different acting forces on water molecules, different capacities of absorbing water and keeping water, large acting force, strong binding force on the water molecules and larger water absorbing and keeping amount.

The experimental method comprises the following steps:

1. and preparing supersaturated solutions of magnesium chloride or calcium chloride, sodium chloride and disodium hydrogen phosphate or potassium chloride 12h before the experiment is carried out, and preparing constant humidity environments with relative humidity of 30%, 75% and 98%.

2. The weighing bottle is marked, placed in a drying oven at 105 ℃ to be dried to constant weight, and weighed after being cooled to room temperature, and the weight is m 1. 0.0200g of dextran (control group) and the lyophilized powder of the custard apple fermentation extract are weighed respectively until the powder or liquid level just exceeds the bottom of the bottle, and the mark is m2 (weighing bottle + sample). The weighing flask containing the sample was placed in a hygrostat (supersaturated solution of disodium hydrogen phosphate) having a humidity of about 98% and left for 3 hours.

3. Marking the weighing bottle, placing the bottle in a drying oven at 105 ℃ for drying to constant weight, and weighing after cooling to room temperature. Is designated m 1. The appropriate amount of 10% dextran and 1.0g/mL custard apple fermentation extract were weighed respectively so that the liquid level just fell over the bottom of the bottle as m2 (weigh bottle + sample). The weighing flask with the sample was placed in a humidistat (supersaturated solution of calcium chloride) having a humidity of about 30% and left for 3 hours.

4. After standing for 3h, the sample was quickly taken out and weighed as m3 (weigh bottle + sample).

5. And calculating the moisture absorption rate and the water loss rate or the moisture retention rate of the sample.

Moisture absorption rate (m3-m2)/(m2-m 1). times.100%

The water loss rate was (m2-m3)/(m2-m 1). times.100%

The experimental result is shown in figure 2, and the figure 2 shows that the custard apple fermentation extract prepared by the invention has better moisturizing effect.

In conclusion, the custard apple fermentation extract filtrate prepared by the invention can effectively remove free radicals, and data show that the custard apple fermentation extract filtrate has the effects of moisturizing and the like, can be used as an effect raw material additive to be applied to cosmetics, has wide application and has great market value.

Test example 6 tyrosinase activity inhibition Effect test

Adding PBS phosphate buffer solution with negative control pH6.8, sample solution, and 100U/mL tyrosinase into each test tube, keeping the temperature in 37 deg.C water bath for 10min, adding 1.5mmol/L L-DOPA solution, reacting at 37 deg.C for 5min, immediately measuring absorbance at 475nm, averaging for 3 times, and averaging with PBS phosphate buffer solution with pH6.8 as negative control.

The experimental system design is shown in Table 4, A1 is blank zero adjustment, A2 is blank control, A3 is sample zero adjustment, and A4 is sample tube sample, which is the fermentation liquid of Sakyamur fruit in example 1. The results of the tyrosinase activity inhibition assay for each sample are shown in Table 5.

TABLE 4

Group of	sample/mL	PBS/mL	L-DOPA/mL	tyrosinase/mL
					A1
	0	2.0	1	0
					A2	0	1.5	1	0.5
A3	0.5	1.5	1	0
					A4	0.5	1.0	1	0.5

TABLE 5

The data in the table show that the tyrosinase inhibition rates of different concentrations of the multiple fermentation extraction filtrate of custard apple with the whitening function, prepared by the method, are 56.2-84.2%, and the whitening effect is good. From the experiments of groups 1-6, the tyrosinase inhibition rate is higher when the concentration is higher, and the tyrosinase inhibition rate reaches 84.2% under the concentration of 1.0g/mL in the experiments listed above.

Example 2 Annona squamosa fermented filtrate skin care essence

The paint comprises the following components in percentage by mass:

the preparation method comprises the following steps: stirring phase A to 90 deg.C, cooling to 50 deg.C, adding phase B, stirring for 3 min, adding phase C, and stirring to obtain multiple fermentation filtrate essence of Annona squamosa, numbered essence A.

Example 3 Annona squamosa fermented filtrate skin care essence

The preparation method comprises the following steps: stirring phase A to 90 deg.C, cooling to 50 deg.C, adding phase B, stirring for 3 min, adding phase C, and stirring to obtain multiple fermentation filtrate essence of Annona squamosa, numbered essence B.

Comparative example 5

The difference from example 3 is that: the fermented product 5 was replaced with the fermented product 2, and the other components and contents were the same as those in example 3, and the number was designated as essence C.

Comparative example 6

The difference from example 3 is that: the fermented product 5 was replaced with the fermented product 3, and the other components and contents were the same as in example 3, and the product was numbered as essence D.

Comparative example 7

The difference from example 3 is that: the fermented product 5 was replaced with fermented product 4, and the other components and contents were the same as in example 3, and the product was numbered as essence E.

Comparative example 8

The difference from example 3 is that: the fermentation product 5 was replaced with the custard apple fermentation broth a, and the other components and contents were the same as those in example 3, and numbered as essence F.

Comparative example 9

The difference from example 3 is that: the fermentation product 5 is replaced by the multiple fermentation liquid V of the custard apple, other components and contents are the same as those in the example 3, and the number is the essence M.

Comparative example 10

The difference from example 3 is that: the fermented product 5 was replaced with ordinary deionized water, and the other components and contents were the same as in example 3, and the number was designated as a control group.

And (3) trial detection effect:

selecting 40 subjects, half of all the subjects, with the age of 18-40 years, and living habits of all staying up for a long time, and the problems of dryness, scales, pigmentation and the like of facial skin in different degrees, randomly dividing the subjects into 4 groups, wherein each group comprises 10 subjects, cleaning the face with clear water, and then applying the product on the face until the product is absorbed without cleaning; the product is used for 1 time per day for 4 weeks, and no other product is used during the use period; before testing, the test subjects cleaned their faces with clear water, wiped clean with facial tissue, and started to measure after sitting still in a constant temperature and humidity laboratory (temperature 20. + -. 1 ℃ C., humidity 55. + -. 3) for 30 minutes. An area of 4 x 4cm in the lower middle of the right face of each human subject was selected and tested for skin condition using an Antera 3D imaging system developed by Miravex corporation.

Antera 3D has multiple detection functions, including skin wrinkles, texture, melanin, hemoglobin levels, etc., and can extract relevant data from images associated with three-dimensional patterns of skin to determine treatment efficacy and monitor changes in treatment over time.

The evaluation method comprises the following steps: this example analyzes its experimental results according to wrinkle depth, texture, melanin, hemoglobin levels in Antera 3D.

The wrinkle change was scored by using the above essence on the basis of the number of fine lines in the lower right part of the right face of the subject analyzed with a small size (1mm filter).

In terms of skin texture, in an ideal form, quantitative measurements are made by the vertical deviation of the skin surface-the larger the deviation, the rougher the surface; the smaller the deviation, the flatter the surface. The average roughness of the skin, Sa, was measured using an asperity conditioning option using the andrera software to determine the texture of the skin.

The function of concentration change of melanin and hemoglobin can be measured by using the function of multispectral analysis in Antera 3D. And selecting the same area of the middle lower part of the right face to measure the change conditions of melanin and heme, thus obtaining the corresponding skin color change.

The scoring result is calculated by weight percent, 90-100 scores indicate that the skin improvement condition is very obvious, 70-90 scores indicate that the skin improvement is obvious, 50-70 scores indicate that the skin condition is improved, and less than 40 scores indicate that the skin improvement is not obvious. Skin score was 10 before testing.

The results are shown in Table 7 as average values.

TABLE 7

According to the detection data in the table, the essence prepared in the embodiment and the comparative example has the effect of improving the skin state, which is embodied in the aspects of skin texture and skin whitening degree, and meanwhile, the essence prepared from the custard apple fermentation filtrate prepared in the embodiment 3 has a more obvious skin care effect.

In conclusion, the custard apple fermentation liquor essence prepared by the invention has obvious effect of improving skin conditions after daily skin care for 28 days, and is characterized by improving skin textures, reducing fine lines, reducing melanin level and obviously improving glossiness. Meanwhile, the cell level experiment in the second experimental example shows that the cell activity of the custard apple fermentation liquid obtained by the invention is correspondingly increased after the custard apple fermentation liquid acts on HSF and HaCat cells for 24 hours, which shows that the custard apple fermentation liquid has the function of promoting proliferation on the HSF and HaCat cells,

this effect is also exhibited in the fifth experimental example, specifically, the roughness and glossiness of the skin are exhibited. Therefore, the custard apple fermentation filtrate obtained by the invention can be used as an effect raw material additive to be applied to cosmetics, and has wide application and great market value.

The present invention has been further described with reference to specific embodiments, which are only exemplary and do not limit the scope of the present invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.

Claims

1. A moisturizing and whitening cosmetic containing multiple fermentation liquor of custard apple is characterized in that: the paint comprises the following components in percentage by weight: 1-5% of propylene glycol, 1-6% of glycerol, 0.01-0.05% of disodium ethylene diamine tetraacetate, 2-10% of isopropyl myristate, 0.05-0.1% of phenoxyethanol, 0.1-0.15% of ethylhexyl glycerol, 4-15% of custard apple fermentation filtrate, 0.05-0.25% of hydrogenated castor oil and the balance of water;

the custard apple fermentation filtrate is custard apple multiple fermentation liquid.

2. The moisturizing and whitening cosmetic according to claim 1, characterized in that: the paint comprises the following components in percentage by weight: 2% of propylene glycol, 3.2% of glycerol, 0.03% of disodium ethylene diamine tetraacetate, 5% of isopropyl myristate, 0.05% of phenoxyethanol, 0.15% of ethylhexyl glycerol, 7.5% of custard apple fermentation filtrate, 0.1% of hydrogenated castor oil and the balance of water;

3. The moisturizing and whitening cosmetic composition according to claim 2, wherein: the mass ratio of the custard apple fermentation filtrate to the hydrogenated castor oil is 50-100: 1; preferably 75: 1.

4. A custard apple fermentation filtrate is characterized in that: the Annona squamosa fruit fermentation filtrate is the Annona squamosa fruit multiple fermentation liquid in claim 2, and the fermentation strains of the Annona squamosa fruit multiple fermentation liquid comprise Bacillus subtilis, Lactobacillus iners, Bifidobacterium adolescent and Saccharomyces cerevisiae Hansen.

5. The method for preparing the custard apple fermentation filtrate as claimed in claim 4, wherein the method comprises the following steps: the preparation method comprises the following steps:

(1) rejuvenating the strains;

(2) purifying strains;

(3) seed liquid amplification culture:

(3.4) Saccharomyces cerevisiae (Saccharomyces cerevisiae Hansen) purification culture: taking 0.1g of strain freeze-dried powder, adding 1ml of sterile physiological saline, mixing, averagely sucking to 2 TSA plates, shaking up, and culturing at 37 ℃ for 50 h; selecting a colony which grows vigorously, inoculating the colony into a slant culture medium to be cultured for 24-48h at the temperature of 25-38 ℃, and taking 5-10mL of sterile physiological saline to wash to obtain a bacterial suspension D;

(4) obtaining of custard apple fermentation product:

6. The method of claim 5, wherein: the culture medium of the TSA solid plate in the step (3.1) is peptone 4g/L, sodium chloride 10g/L and NaH₂PO₄1g/L, agar 15g/L, MgSO₄·7H₂O 0.5g/L，K₂HPO₄1g/L KH₂PO₄ 1g/L；

The culture medium of the blood plate in the step (3.2) is peptone 4g/L, sodium chloride 10g/L and NaH₂PO₄1g/L, 10g/L of beef extract powder, 5% of defatted fiber sheep blood and MgSO₄·7H₂O 0.5g/L，K₂HPO₄ 1g/LKH₂PO₄1g/L of agar and 15g/L of agar;

the special culture medium in the step (3.3) is peptone 4g/L, sodium chloride 10g/L and NaH₂PO₄1g/L, yeast extract 10g/L, tryptone 5g/L, glucose 8g/L, MgSO₄·7H₂O 0.5g/L，K₂HPO₄ 1g/LKH₂PO₄1g/L of agar and 15g/L of agar;

the culture medium of the plate in the step (3.4) is as follows: 15g/L of tryptone, 5g/L of soytone, 2g/L of yeast extract, 4g/L of sodium chloride, 2g/L of glucose, 1-3g of dipotassium phosphate and 15g/L of agar.

7. The method of claim 5, wherein: the bacterial suspensions A-D prepared in the steps (3.1) - (3.4) are turbidified by a bacterial turbidimeter, and the turbidity is 1-5 MCF.

8. The method of claim 5, wherein: the mass-to-volume ratio of the fermentation substrate 1 to the bacterial suspension A in the step (4.2) is 50-100: 1-5;

9. The use of the custard apple fermentation liquid of claim 4 and the custard apple fermentation liquid prepared by the preparation method of claims 5-8 in the preparation of moisturizing and whitening cosmetics.

10. Use according to claim 9, characterized in that: the cosmetic is cream, emulsion, toner, essence, gel or facial mask.