Salt-tolerant bacillus, fermentation products thereof, and uses thereof

Abstract

The application provides a salt-tolerant bacillus strain and uses thereof. The strain is preserved in the China General Microbiological Culture Collection Center, and has a preservation number of CGMCC No. 29259. A fermentation product obtained using the strain has significant moisturizing, whitening, soothing, antioxidant, anti-wrinkle, oil control, acne-removing, and firming effects, and can be applied to cosmetics.

Description

Technical Field

[0001] This application relates to the field of cosmetic technology, and more particularly to a salt-tolerant Bacillus, its fermentation products, and their uses. Background Technology

[0002] The cosmetics industry has always faced the challenge of raw material innovation. Cosmetic raw materials are mainly sourced from three sources: chemical synthesis, natural extraction, and bio-fermentation. While traditional chemical synthesis methods can produce raw materials on a large scale, they often come with environmental and safety concerns. Naturally extracted cosmetic raw materials typically originate from various plants, but plant-based ingredients present numerous challenges in research and application, and quality standards are difficult to control. In contrast, fermentation technology offers a more environmentally friendly and sustainable option. It not only reduces reliance on chemical reagents but also creates new active ingredients through natural processes. These ingredients are often more compatible with the human body and have fewer side effects.

[0003] Microbial fermentation raw materials utilize the metabolic processes of specific microbial cells and various complex reaction systems to obtain direct or secondary metabolites of the microorganisms. These metabolites typically offer multiple physiological benefits to the skin. In addition to the metabolites of specific microorganisms, fermentation raw materials also contain free amino acids, organic acids, polysaccharides, and small molecule peptides. Combined with traditional cosmetic formulation and development, the addition of bio-fermentation raw materials enhances the product's feel, efficacy, and absorption.

[0004] With the development of the cosmetics industry and the rapid upgrading and iteration of cosmetic raw materials, people's demand for cosmetics is gradually shifting towards safety, efficiency, greenness, and naturalness. Compared with natural extraction and chemical synthesis methods, microbial fermentation raw materials are increasingly being used in cosmetics due to their high efficacy, low toxicity, and green safety characteristics. Fermentation technology has become an application direction and research hotspot in the cosmetics field. More and more brands and companies are beginning to apply fermentation technology to product terminals. Internationally renowned brands such as SK-II under Procter & Gamble, Estée Lauder and La Mer under Estée Lauder Group, and Lancôme and L'Oréal Paris under L'Oréal Group have all incorporated different fermentation raw materials into their main products, such as Bifida Ferment Lysate (derived from Bifidobacterium fermentation) and yeast extract.

[0005] Currently, the "Catalogue of Cosmetic Raw Materials in Use (2021 Edition)" includes 85 fermentation raw materials. Compared with traditional chemically synthesized raw materials and plant-extracted raw materials, fermentation raw materials still have huge development, application space and market prospects. Summary of the Invention

[0006] Based on this, this application provides a salt-tolerant Bacillus, its fermentation product and its use. The fermentation product is rich in a variety of active ingredients and has significant moisturizing, whitening, soothing, antioxidant, anti-wrinkle, oil-controlling, acne-removing and firming effects, and can be widely used in cosmetics.

[0007] The specific technical solution of this application is as follows:

[0008] 1. A salt-tolerant Bacillus halotolerans SDY412, which is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 29259.

[0009] 2. The use of the salt-tolerant Bacillus described in item 1 in fermentation production is preferably in the fermentation production of fermentation products with moisturizing, whitening, soothing, antioxidant, anti-wrinkle, oil-controlling, acne-removing and firming effects.

[0010] 3. A fermentation product obtained by fermentation with the salt-tolerant Bacillus described in item 1.

[0011] 4. The fermentation product according to claim 3, wherein the method of fermenting halophilic Bacillus comprises:

[0012] The salt-tolerant Bacillus strain was activated to obtain seed liquid;

[0013] The seed liquid was fermented to obtain a crude fermentation product.

[0014] The crude fermentation product is separated by centrifugation to obtain the fermentation product.

[0015] 5. The fermentation product according to item 4, wherein in the process of fermenting the seed liquid to obtain the crude fermentation product, the inoculation amount of the seed liquid is 1-10% of the liquid volume;

[0016] Preferably, the fermentation medium used for fermentation contains 50-100 g / L rapeseed oil, 30-100 g / L glucose, 5-10 g / L NaNO3, 0.5-1 g / L magnesium sulfate heptahydrate, 1-5 g / L yeast extract, 0.1-1 g / L potassium chloride, 0.1-1 g / L potassium dihydrogen phosphate, 0.1-1 g / L disodium hydrogen phosphate dodecahydrate, 0.1-0.5 g / L calcium chloride, and 0.01-0.05 g / L ferrous sulfate heptahydrate.

[0017] 6. A method for preparing a fermentation product, comprising fermenting the salt-tolerant Bacillus described in item 1.

[0018] 7. The method according to item 6, wherein the method comprises:

[0019] The salt-tolerant Bacillus strain was activated to obtain seed liquid;

[0020] The seed liquid was fermented to obtain a crude fermentation product.

[0021] The crude fermentation product is separated by centrifugation to obtain the fermentation product.

[0022] 8. The method according to item 7, wherein, in the process of fermenting the seed liquid to obtain the crude fermentation product, the inoculation amount of the seed liquid is 1-10% of the liquid volume;

[0023] Preferably, the fermentation medium used for fermentation culture contains 50-100 g / L rapeseed oil, 30-100 g / L glucose, 5-10 g / L NaNO3, 0.5-1 g / L magnesium sulfate heptahydrate, 1-5 g / L yeast extract, 0.1-1 g / L potassium chloride, 0.1-1 g / L potassium dihydrogen phosphate, 0.1-1 g / L disodium hydrogen phosphate dodecahydrate, 0.1-0.5 g / L calcium chloride, and 0.01-0.05 g / L ferrous sulfate heptahydrate.

[0024] 9. Use of the fermentation product of any one of items 3-5 or any one of items 6-8 in cosmetics.

[0025] 10. The use according to item 9, wherein the dosage form of the cosmetic is a cream, lotion, microemulsion, gel, aqueous solution, serum, sheet mask liquid, cream mask, spray or gel.

[0026] 11. A cosmetic product comprising the fermentation product of any one of claims 3-5 or the fermentation product prepared by any one of claims 6-8.

[0027] 12. The cosmetic product according to item 11, wherein the fermentation product comprises 0.01-99.99% by mass percentage in the cosmetic product.

[0028] 13. The cosmetic product according to item 11 or 12, further comprising cosmetically acceptable excipients and / or active ingredients.

[0029] 14. The cosmetic product according to any one of claims 11-13, wherein the dosage form of the cosmetic product is a cream, lotion, microemulsion, gel, aqueous solution, serum, sheet mask liquid, cream mask, spray or gel.

[0030] The effects of the invention

[0031] The fermentation products obtained using the screened halophilic Bacillus in this application have significant moisturizing, whitening, soothing, antioxidant, anti-wrinkle, oil-controlling, acne-removing, and firming effects, and can be applied in cosmetics. Attached Figure Description

[0032] Figure 1 This is a schematic diagram of tyrosinase inhibition rate.

[0033] Strain Preservation Information

[0034] The halophilic Bacillus halotolerans SDY412 disclosed in this application was deposited on December 7, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 29259. The address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101, China. Detailed Implementation

[0035] The present application will now be described in detail with reference to the described embodiments. Although specific embodiments of the present application are shown, it should be understood that the present application can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this application will be thorough and complete, and will fully convey the scope of the present application to those skilled in the art.

[0036] It should be noted that certain terms are used in the specification and claims to refer to specific components. Those skilled in the art will understand that different terms may be used to refer to the same component. This specification and claims do not distinguish components based on differences in terminology, but rather on differences in function. The terms "comprising" or "including" used throughout the specification and claims are open-ended and should be interpreted as "comprising but not limited to." The following descriptions in the specification are preferred embodiments for carrying out this application; however, these descriptions are for the purpose of understanding the general principles of the specification and are not intended to limit the scope of this application. The scope of protection of this application shall be determined by the appended claims.

[0037] This application provides a salt-tolerant Bacillus halotolerans SDY412 strain, which is obtained by high-temperature isolation, salt tolerance testing and screening, and purification from the silt and water of Chaka Salt Lake. The DNA of this strain is extracted and 16S rDNA is amplified. The amplified product is sent to the China General Microbiological Culture Collection Center for sequencing and species identification, and it is confirmed to be a salt-tolerant Bacillus, named Bacillus halotolerans SDY412. It is deposited at the China General Microbiological Culture Collection Center with accession number CGMCC No. 29259.

[0038] This application provides the use of the above-described salt-tolerant Bacillus in fermentation production, preferably in the fermentation production of fermentation products with moisturizing, whitening, soothing, antioxidant, anti-wrinkle, oil-controlling, acne-removing and firming effects.

[0039] This application uses the above-mentioned strain for fermentation, and the resulting fermentation product has excellent effects, such as moisturizing, whitening, soothing, anti-oxidation, anti-wrinkle, oil control, acne removal, and firming.

[0040] This application provides a fermentation product obtained by fermentation with the aforementioned halophilic Bacillus. In some embodiments, the method for fermenting the halophilic Bacillus includes:

[0041] The salt-tolerant Bacillus strain was activated to obtain seed liquid;

[0042] The seed liquid is fermented to obtain a crude fermentation product;

[0043] The crude fermentation product is then subjected to further fermentation to obtain a fermentation product.

[0044] In this application, the method for activating the strain to obtain seed liquid is not limited in any way. Those skilled in the art can use conventional methods to activate the strain, such as inoculating the strain into a seed culture medium for cultivation to obtain seed liquid, or inoculating the strain into a shake flask seed culture medium for cultivation to obtain seed liquid, or culturing at 30-37℃ and 150-300rpm for 16-22 hours to obtain seed liquid.

[0045] For example, the temperature can be 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, 36℃, 37℃, etc.;

[0046] The speed can be 150rpm, 160rpm, 170rpm, 180rpm, 190rpm, 200rpm, 210rpm, 220rpm, 230rpm, 240rpm, 250rpm, 260rpm, 270rpm, 280rpm, 290rpm, 300rpm, etc.;

[0047] The time can be 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, etc.

[0048] In some embodiments, fermenting the seed culture to obtain the crude fermentation product refers to inoculating the seed culture into a fermentation medium for fermentation to obtain the crude fermentation product. In some embodiments, the inoculation amount of the seed culture is 1-10% of the volume of the culture.

[0049] In this application, no limitation is made on the inoculation amount, which has the meaning commonly understood by those skilled in the art. For example, the inoculation amount refers to the ratio of the volume of seed liquid transferred in to the volume of culture medium after inoculation.

[0050] For example, the inoculation amount of the seed liquid is 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% of the liquid volume.

[0051] In some embodiments, the fermentation medium comprises 50-100 g / L rapeseed oil, 30-100 g / L glucose, 5-10 g / L NaNO3, 0.5-1 g / L magnesium sulfate heptahydrate, 1-5 g / L yeast extract, 0.1-1 g / L potassium chloride, 0.1-1 g / L potassium dihydrogen phosphate, 0.1-1 g / L disodium hydrogen phosphate dodecahydrate, 0.1-0.5 g / L calcium chloride, and 0.01-0.05 g / L ferrous sulfate heptahydrate.

[0052] For example, rapeseed oil can be available in concentrations of 50g / L, 60g / L, 70g / L, 80g / L, 90g / L, 100g / L, etc.

[0053] Glucose concentrations can be 30g / L, 40g / L, 50g / L, 60g / L, 70g / L, 80g / L, 90g / L, 100g / L, etc.

[0054] NaNO3 can be 5g / L, 6g / L, 7g / L, 8g / L, 9g / L, 10g / L, etc.;

[0055] Magnesium sulfate heptahydrate can be 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1 g / L, etc.

[0056] Yeast extract can be available in concentrations of 1g / L, 1.5g / L, 2g / L, 2.5g / L, 3g / L, 3.5g / L, 4g / L, 4.5g / L, and 5g / L.

[0057] Potassium chloride can be in concentrations of 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1 g / L, etc.

[0058] Potassium dihydrogen phosphate can be in concentrations of 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1 g / L, etc.

[0059] Disodium hydrogen phosphate dodecahydrate can be in concentrations of 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1 g / L, etc.

[0060] Calcium chloride can be in concentrations of 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, etc.

[0061] Ferrous sulfate heptahydrate can be 0.01 g / L, 0.02 g / L, 0.03 g / L, 0.04 g / L, 0.05 g / L, etc.

[0062] In some embodiments, the seed culture is inoculated into a fermentation medium and cultured at 30-37°C and 150-300 rpm for 40-60 hours to obtain the crude fermentation product.

[0063] In this application, no restrictions are placed on the centrifugation method. Those skilled in the art can make conventional choices based on their needs, as long as the crude fermentation product can be centrifuged to obtain the fermentation product.

[0064] The fermentation product described in this application has excellent efficacy and can be used in cosmetics.

[0065] This application provides a method for preparing a fermentation product, which includes fermenting the above-described halophilic Bacillus. In some embodiments, the method includes:

[0066] The salt-tolerant Bacillus strain was activated to obtain seed liquid;

[0067] The seed liquid was fermented to obtain a crude fermentation product.

[0068] The crude fermentation product is centrifuged to obtain the fermentation product. In some embodiments, in the process of fermenting the seed liquid to obtain the crude fermentation product, the inoculation amount of the seed liquid is 1-10% of the liquid volume;

[0069] Preferably, the fermentation medium used for fermentation culture contains 50-100 g / L rapeseed oil, 30-100 g / L glucose, 5-10 g / L NaNO3, 0.5-1 g / L magnesium sulfate heptahydrate, 1-5 g / L yeast extract, 0.1-1 g / L potassium chloride, 0.1-1 g / L potassium dihydrogen phosphate, 0.1-1 g / L disodium hydrogen phosphate dodecahydrate, 0.1-0.5 g / L calcium chloride, and 0.01-0.05 g / L ferrous sulfate heptahydrate.

[0070] This application provides the use of the above-described fermentation product or the fermentation product prepared by the above-described method in cosmetics. In some embodiments, the dosage form of the cosmetic is a cream, lotion, microemulsion, gel, aqueous solution, serum, sheet mask liquid, spreadable mask, spray, or gel.

[0071] Since the fermentation product has been tested and found to have excellent moisturizing, whitening, soothing, antioxidant, anti-wrinkle, oil-controlling, acne-removing, and firming effects, it can be used in cosmetics.

[0072] This application provides a cosmetic product comprising the fermentation product described above or the fermentation product prepared by the method described above. In some embodiments, the fermentation product comprises 0.01-99.99% by mass percentage in the cosmetic product.

[0073] For example, the fermentation product can be 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 80%, 90%, 95%, 99%, 99.99%, etc., as a percentage by mass in the cosmetic.

[0074] In some embodiments, the cosmetic product further comprises cosmetically acceptable excipients and / or active ingredients.

[0075] In this application, no restrictions are placed on the acceptable excipients and active ingredients for cosmetics. Those skilled in the art can make conventional selections based on their needs. Similarly, no restrictions are placed on the amount of excipients and active ingredients used. Those skilled in the art can select the content of excipients and active ingredients to be added based on their needs.

[0076] In some embodiments, the dosage form of the cosmetic is a cream, lotion, microemulsion, gel, aqueous solution, serum, sheet mask liquid, spreadable mask, spray, or gel.

[0077] Example

[0078] This application provides a general and / or specific description of the materials and test methods used in the experiments. In the following examples, unless otherwise specified, % represents wt%, i.e., weight percentage. Reagents or instruments used, unless otherwise specified, are all commercially available conventional reagent products.

[0079] Example 1: Obtaining the strain

[0080] This embodiment describes the isolation, screening, and identification of bacterial strains in the sample.

[0081] The culture media used for strain isolation and screening were: LB liquid medium (tryptone 10.0 g / L, yeast extract 5.0 g / L, NaCl 10.0 g / L, sterilized at 121℃ for 20 min) and LB solid medium (tryptone 10.0 g / L, yeast extract 5.0 g / L, NaCl 10.0 g / L, agar 15.0 g / L, sterilized at 121℃ for 20 min).

[0082] 1. Isolation of strains

[0083] Bacillus was isolated by utilizing the heat-resistant properties of spores. 10g samples of silt and water from Chaka Salt Lake were diluted separately with sterilized pure water to a concentration of 10 g / L. -1 and 10 -2 The diluted solution was boiled in a water bath for 10-20 minutes. 200 μL of the diluted solution was added to a test tube containing LB liquid medium and incubated at 37°C for 24 hours to obtain the bacterial culture.

[0084] 2. Screening of strains

[0085] The above bacterial culture was streaked onto LB solid medium containing 20% ​​NaCl and incubated at 37°C until distinct single colonies grew. Single colonies were picked and streaked onto LB solid medium and incubated at 37°C. After colonies grew, the streaking was repeated 3 times to obtain purified single colonies.

[0086] 3. Strain identification

[0087] The above-mentioned strain was amplified with 16S rDNA using universal bacterial primers (27F / 1492R) and sequenced. The sequencing results were compared with those in NICI, and it was preliminarily confirmed as belonging to the genus *Bacillus*. Further species identification by the China General Microbiological Culture Collection Center (CGMCC) confirmed the strain as a halophilic *Bacillus halotolerans* SDY412, which was deposited at the CGMCC on December 7, 2023, with accession number CGMCCNo. 29259. Sequencing results:

[0088]

[0089] Example 2 Preparation of fermentation products

[0090] 1. Culture medium preparation

[0091] (1) Preparation of streak plate culture medium

[0092] The culture medium contains: 5 g / L yeast extract, 10 g / L peptone, 10 g / L sodium chloride, and 20 g / L agar powder.

[0093] (2) Preparation of seed culture medium

[0094] The culture medium contains: 5 g / L yeast extract, 10 g / L peptone, and 10 g / L sodium chloride.

[0095] (3) Preparation of shake flask fermentation medium

[0096] The culture medium contains: rapeseed oil 50 g / L, glucose 50 g / L, NaNO3 5 g / L, magnesium sulfate heptahydrate 1 g / L, yeast extract 1 g / L, potassium chloride 1 g / L, potassium dihydrogen phosphate 1 g / L, disodium hydrogen phosphate dodecahydrate 1 g / L, calcium chloride 0.1 g / L, and ferrous sulfate heptahydrate 0.01 g / L.

[0097] 2. Microbial culture and fermentation:

[0098] (1) The strain obtained in Example 1 was streaked on a plate streak medium and cultured in a constant temperature incubator at 37°C for 24 hours to obtain a single colony;

[0099] (2) Pick a single colony and inoculate it into a 50mL seed flask, filling the flask with 20mL of liquid. Incubate at 37℃ and 200rpm on a shaker for 20h to obtain the seed culture;

[0100] (3) Inoculate the fermentation seed liquid into a 500mL fermentation shake flask at a 5% inoculation rate, with a total volume of 50mL. Incubate at 37℃ and 220rpm on a shaker for 50h. Harvest the crude fermentation product after fermentation is complete.

[0101] 3. Sedimentation and filtration were performed to obtain the fermentation product of salt-tolerant Bacillus.

[0102] Application Example 1: Aqueous Products

[0103] The aqueous product comprises: 86.48% pure water, 5% butylene glycol, 5% propylene glycol, 3% glycerol, 0.3% of the salt-tolerant Bacillus fermentation product prepared in Example 2, 0.2% p-hydroxyacetophenone, 0.01% tea tree oil, and 0.01% NaOH.

[0104] The preparation process is as follows: heat pure water to about 80°C, add p-hydroxyacetophenone, and stir until it is evenly dissolved; then cool to below 45°C, and add glycerol, propylene glycol, butylene glycol, tea tree oil, and halophilic Bacillus fermentation products to the aqueous solution in that order, stirring until evenly dissolved. Finally, add NaOH to adjust the pH of the aqueous cosmetic to about 6.8.

[0105] Application Example 2: Facial Mask Products

[0106] The mask product comprises: 74.31% pure water, 8% butylene glycol, 8% glycerin, 8% hydroxyethyl urea, 0.4% p-hydroxyacetophenone, 0.3% halophilic Bacillus fermentation product prepared in Example 2, 0.28% carbomer 981, 0.2% modified grafted starch, 0.2% xanthan gum, 0.2% ethylhexylglycerin, 0.05% disodium EDTA, 0.05% sodium hydroxide, and 0.01% sodium hyaluronate (molecular weight 1000-1700 kDa).

[0107] Its preparation process is as follows:

[0108] Modified grafted starch and xanthan gum were dispersed in glycerol and the dispersion was made uniform to obtain a glycerol dispersion system.

[0109] Heat pure water to about 80°C, add p-hydroxyacetophenone and disodium EDTA, and stir until they are dissolved evenly to obtain an aqueous system.

[0110] The mixture was then cooled to below 45°C. Butylene glycol, hydroxyethyl urea, and the fermentation product of halophilic Bacillus were added to the aqueous solution in that order. The well-mixed aqueous solution was then added to the glycerol dispersion and stirred until homogeneous. Finally, ethylhexylglycerol, carbomer 981, and sodium hyaluronate were added to the system, and sodium hydroxide was added to adjust the pH to approximately 6.8.

[0111] Application Example 3: Gel Products

[0112] The gel product comprises: 85.33% pure water, 5% glycerol, 5% sorbitol, 3% aloe vera water, 0.4% capryloyl hydroxamic acid, 0.4% p-hydroxyacetophenone, 0.3% halophilic Bacillus fermentation product prepared in Example 2, 0.2% modified grafted starch, 0.2% acrylate / C10-30 alkyl acrylate crosspolymer, 0.05% disodium EDTA, 0.05% allantoin, 0.05% NaOH, and 0.02% dipotassium glycyrrhizate.

[0113] Its preparation process is as follows:

[0114] Modified grafted starch was dispersed in glycerol and the dispersion was made uniform to obtain a glycerol dispersion system.

[0115] Heat pure water to about 80°C, add p-hydroxyacetophenone and disodium EDTA, and stir until they are dissolved evenly to obtain an aqueous system.

[0116] The mixture was then cooled to below 45°C. The following ingredients were added to the aqueous solution in the following order: Bacillus halophilus fermentation product, sorbitol, aloe vera water, allantoin, and dipotassium glycyrrhizate. The well-mixed aqueous solution was then added to the glycerol dispersion and stirred until homogeneous. Finally, capryloyl hydroxamic acid and acrylate / C10-30 alkyl acrylate crosspolymer were added to the system, and sodium hydroxide was added to adjust the pH to approximately 6.8.

[0117] Application Example 4: Emulsion Products

[0118] The emulsion product comprises 74.71% purified water, 10% glycerol, 5% shea butter, 3% cetearyl alcohol ether-25, 2% olive fruit oil, 2% monoglyceride stearate, 1% sunflower seed oil, 1% cetearyl alcohol, 0.4% p-hydroxyacetophenone, 0.3% halophilic Bacillus fermentation product prepared in Example 2, 0.2% capryloyl hydroxamic acid, 0.28% carbomer 980, 0.05% disodium EDTA, 0.05% sodium hydroxide, and 0.01% sodium hyaluronate (molecular weight 1000-1700 kDa).

[0119] Its preparation process is as follows: the system is prepared by separating an aqueous phase and an oil phase.

[0120] Aqueous phase: Heat pure water to about 80°C, add p-hydroxyacetophenone and disodium EDTA, stir until they are dissolved evenly, then cool to below 45°C, add the fermentation product of salt-tolerant Bacillus to obtain the aqueous phase;

[0121] Oil phase: Add glycerin, shea butter, cetearyl alcohol ether-25, olive fruit oil, monoglyceride stearate, sunflower seed oil, and cetearyl alcohol in that order, and heat to about 80-90℃ to completely dissolve and mix the oil phase to obtain the oil phase.

[0122] The oil phase was poured into the aqueous phase and homogenized using a homogenizer to prepare a fully dispersed emulsion. Then, octanoyl hydroxamic acid and carbomer 980 were added to the system, and sodium hydroxide was added to adjust the pH of the product to about 6.8.

[0123] Application Example 5: Cream Products

[0124] The cream product contains 67.71% pure water, 12% glycerin, 4% isopropyl palmitate, 2% polyacrylate-13, 1.5% polyisobutylene, 1.5% polysorbate 20, 2% caprylic / capric triglyceride, 2% dioctyl carbonate, 1.5% cetearyl alcohol, 1% beeswax, 1% silicone wax, 1% diisostearyl malate, 1% polydimethylsiloxane, 0.5% tocopheryl acetate, 0.4% p-hydroxyacetophenone, 0.3% halophilic Bacillus fermentation product prepared in Example 2, 0.2% capryloyl hydroxamic acid, 0.28% carbomer 981, 0.05% disodium EDTA, 0.05% sodium hydroxide, and 0.01% sodium hyaluronate (molecular weight 1000-1700 kDa).

[0125] Its preparation process is as follows: the system is prepared by separating an aqueous phase and an oil phase.

[0126] Aqueous phase: Heat pure water to about 80°C, add p-hydroxyacetophenone and disodium EDTA, stir until they are dissolved evenly, then cool to below 45°C, add the fermentation product of salt-tolerant Bacillus to obtain the aqueous phase;

[0127] Oil phase: Glycerin, isopropyl palmitate, polyacrylate-13, polyisobutylene, polysorbate 20, caprylic / capric triglyceride, dioctyl carbonate, cetearyl alcohol, beeswax, silicone wax, diisostearyl malate, polydimethylsiloxane, and tocopheryl acetate are added in that order, and the mixture is heated to about 80-90℃ to completely dissolve and mix evenly to obtain the oil phase.

[0128] The oil phase was poured into the aqueous phase and homogenized using a homogenizer to prepare a fully dispersed emulsion. Then, octanoyl hydroxamic acid, carbomer 981, sodium hyaluronate, and sodium hydroxide were added to the system to adjust the pH of the product to about 6.8.

[0129] Application Example 6: Essence Products

[0130] The essence product comprises 80.61% purified water, 5% butylene glycol, 5% glycerin, 4% isononyl isononanoate, 1.5% cetearyl oleate, 0.3% sorbitan oleate, 1% diisostearyl malate, 1% polydimethylsiloxane, 0.5% tocopheryl acetate, 0.4% p-hydroxyacetophenone, 0.3% halophilic Bacillus fermentation product prepared in Example 2, 0.28% carbomer 980, 0.05% disodium EDTA, 0.05% sodium hydroxide, and 0.01% sodium hyaluronate (molecular weight 1000-1700 kDa). Its preparation method is as follows:

[0131] The system is prepared by separating an aqueous phase and an oil phase.

[0132] Aqueous phase: Heat pure water to about 80°C, add p-hydroxyacetophenone and disodium EDTA, stir until they are dissolved evenly, then cool to below 45°C, add the fermentation product of salt-tolerant Bacillus to obtain the aqueous phase;

[0133] Oil phase: Add butylene glycol, glycerol, isononyl isononate, cetearyl oleate, sorbitan oleate, diisostearyl malate, polydimethylsiloxane, and tocopheryl acetate in that order, and heat to about 80-90℃ to completely dissolve and mix the oil phase.

[0134] The oil phase is poured into the aqueous phase and homogenized using a homogenizer to prepare a fully dispersed emulsion. Then, Carbomer 980, sodium hyaluronate, and sodium hydroxide are added to the system to adjust the pH value of the product to about 6.8 to obtain the essence product.

[0135] Application Example 7: Detergent Products

[0136] The detergent product contains 34.75% pure water, 8% glycerin, 40% sodium cocoyl glycinate, 7% sodium methyl cocoyl taurate, 2% sodium lauroyl glutamate, 5% cocoyl glutamate, 1% cholesterol hydroxystearate, 1.5% sodium chloride, 0.5% of the halophilic Bacillus fermentation product prepared in Example 2, 0.2% citric acid, and 0.05% disodium EDTA.

[0137] The preparation process is as follows: pure water, glycerin, sodium cocoyl glycinate, sodium methyl cocoyl taurate, sodium lauroyl glutamate, cocoyl glutamic acid, and cholesterol hydroxystearate are weighed and added to the system according to the specified ratio. The mixture is heated to 70-80℃ to completely dissolve and mix evenly. The temperature is then lowered to below 50℃, and halophilic Bacillus fermentation product, sodium chloride, and disodium EDTA are added and stirred evenly. Finally, citric acid is added to adjust the pH to about 6.5, and the mixture is stirred until a white and delicate paste is formed.

[0138] Experiment Example 1: Moisturizing Efficacy Test

[0139] The essence product of Example 6 was used as the experimental group, while the same formula with the halophilic Bacillus fermentation products removed was used as the control group.

[0140] Ten volunteers were recruited to test the moisturizing performance of the experimental and control groups. The testing method was as follows: after cleansing their faces, volunteers applied the experimental group sample to the left cheek and the control group sample to the right cheek. After 30 minutes of use, the moisture content of the left and right facial areas was measured using a CK skin tester. After 5 hours of use, the moisture content of the same area on both sides of the face was measured again using a CK skin tester. The lower the water loss, the better the moisturizing performance.

[0141] The test data of 3 randomly selected volunteers are shown in Table 1.

[0142] Table 1

[0143]

[0144] As shown in Table 1, serums containing Bacillus halophilus fermentation products have lower facial moisture loss after use compared to cosmetics without Bacillus halophilus fermentation products, meaning they have better moisturizing effects.

[0145] Experiment Example 2: Whitening Efficacy Test

[0146] This experiment tested the tyrosinase inhibition rate of the halophilic Bacillus fermentation product in Example 2 to evaluate its whitening effect.

[0147] Experimental Principle: In the biosynthesis of melanin in the skin, tyrosinase is a key enzyme that acts on dopa to form dopaquinone, which then spontaneously undergoes a series of reactions to ultimately form melanin. Tyrosinase catalyzes the conversion of dopa to dopaquinone in a phosphoric acid solution at pH 6.8, and the absorbance can be measured at 475 nm using a spectrophotometer. Cosmetics that inhibit tyrosinase activity can reduce the conversion of dopa to dopaquinone, thereby lowering the absorbance. The inhibitory effect of cosmetics on tyrosinase activity is evaluated based on changes in absorbance.

[0148] Instruments and reagents: analytical balance (accurate to 0.0001g); pipettes (5mL, 100μL); constant temperature water bath; GENESYS 180 UV spectrophotometer; disodium hydrogen phosphate dodecahydrate (Maclean, AR, 99%), levodopa (Maclean, 99%), kojic acid (Maclean, 99%), tyrosinase (Yuanye, 25KU), citric acid monohydrate (Aladdin, AR, 99.5%).

[0149] Experimental methods:

[0150] 1. Solution preparation

[0151] (1) Disodium hydrogen phosphate-citric acid buffer solution with pH = 6.8

[0152] Preparation of 0.2 mol / L Na2HPO4·12H2O: Dissolve 14.33 g of Na2HPO4·12H2O in 200 mL of water and stir with a glass rod until dissolved;

[0153] Preparation of 0.1 mol / L citric acid monohydrate: Dissolve 2.1 g of citric acid monohydrate in 100 mL of water and stir with a glass rod until dissolved;

[0154] Preparation of disodium hydrogen phosphate-citric acid buffer solution with pH=6.8: 154.5 mL of 0.2 mol / L Na2HPO4·12H2O aqueous solution was added to 45.5 mL of 0.1 mol / L citric acid monohydrate to prepare 200 mL of disodium hydrogen phosphate-citric acid buffer solution.

[0155] (2) Tyrosinase solution

[0156] Preparation of 100u / mL tyrosinase solution: Add 25KU of tyrosinase to 250mL of disodium hydrogen phosphate-citrate buffer solution with pH=6.8 to obtain a 100u / mL tyrosinase solution. Prepare immediately before use.

[0157] (3) Levodopa solution

[0158] Preparation of levodopa solution: Accurately weigh 0.5 g of levodopa (accurate to 0.0001 g) and add it to 500 mL of disodium hydrogen phosphate-citrate buffer solution with pH = 6.8 to obtain a 1 mg / mL levodopa solution.

[0159] (4) Test solution

[0160] Positive control: kojic acid

[0161] Kojic acid was diluted to 0.008 mg / mL, 0.04 mg / mL, 0.2 mg / mL, 1 mg / mL, and 3 mg / mL using a pH 6.8 disodium hydrogen phosphate-citrate buffer solution.

[0162] Test substance: Fermentation product of halophilic Bacillus

[0163] The salt-tolerant Bacillus fermentation product described in Example 2 was diluted with disodium hydrogen phosphate-citrate buffer at pH 6.8 to concentrations of 0.008 mg / mL, 0.04 mg / mL, 0.2 mg / mL, 1 mg / mL, 3 mg / mL, 8 mg / mL, and 10 mg / mL, respectively.

[0164] 2. Experimental Methods

[0165] Set up sample tubes (T), sample background (T0), enzyme reaction tubes (C), and solvent background (C0) using 15 mL centrifuge tubes. For each sample and each test concentration, set up three parallel tubes for the sample tube (T) and two parallel tubes for the enzyme reaction tube. Add 1 mL of the same concentration of the test substance solution to each of the sample tubes (T) and sample background (T0). Add 1 mL of disodium hydrogen phosphate-citrate buffer to each of the enzyme reaction tubes (C) and solvent background (C0).

[0166] Add 0.5 mL of tyrosinase solution to each of the sample tube (T) and enzyme reaction tube (C). Replace the sample background (T0) and solvent background (C0) with 0.5 mL of disodium hydrogen phosphate-citric acid buffer. Mix the sample and tyrosinase thoroughly and react in a 37°C water bath for 10 minutes. Add 2 mL of levodopa solution to each tube sequentially, controlling the reaction time of each tube to 5 minutes. Immediately transfer the reaction solution from each tube to a cuvette and measure the absorbance at 475 nm.

[0167] Validity verification: The IC50 of the positive control kojic acid (the sample concentration at which 50% inhibition is achieved) should be between 0.05 mg / mL and 0.15 mg / mL for the test system to be considered effective.

[0168] Result Calculation

[0169] The tyrosinase inhibition rate was calculated using the following formula based on the absorbance values ​​measured in each test tube.

[0170] Tyrosinase inhibition rate (%) = (1 - (T - T0) / (C - CO)) * 100%

[0171] In the formula,

[0172] The T-sample tube absorbance is the average of three absorbance values, which is the absorbance of the solution after the sample reacts with tyrosinase.

[0173] T0 - Sample background absorbance;

[0174] The average of two absorbance values ​​of the C-enzyme reaction tube is the absorbance value of the reaction between tyrosinase and dopa when no sample is added.

[0175] C0 - Solvent background absorbance.

[0176] Validity verification: Under the experimental conditions, the IC50 of kojic acid was in the range of 0.05 mg / mL to 0.15 mg / mL, proving the validity of the experimental data. The experimental results are shown in Table 2 and... Figure 1 As shown.

[0177] Table 2 Results of tyrosinase inhibition rate of kojic acid

[0178]

[0179] The experimental results are shown in Table 3.

[0180] Table 3 Results of tyrosinase inhibition rate of Bacillus fermentation products

[0181]

[0182] The data above show that the fermentation product of halophilic Bacillus has a good inhibitory effect on tyrosinase, with an IC50 of about 0.04 mg / mL, which is lower than the IC50 of the positive control kojic acid, and the whitening effect is better than that of kojic acid.

[0183] Experiment Example 3: Soothing Efficacy Test

[0184] The aqueous product from Example 1 was used as the experimental group, while the same formula without the fermentation products of halophilic Bacillus was used as the blank control group. The soothing efficacy was evaluated using the zebrafish juvenile neutrophil migration assay.

[0185] Experimental principle: Hyaluronidase is a specific lysing enzyme of hyaluronic acid and a participant in allergic reactions, strongly correlated with the release of histamine from mast cells. The soothing effect of a test sample can be determined using the hyaluronidase inhibition rate; a higher inhibition rate indicates a stronger soothing effect, and vice versa.

[0186] This experiment references the laboratory method (HMC-WI-029 hyaluronidase inhibition rate). The results of the hyaluronidase inhibition rate test of the test sample and the negative control were compared. If the inhibition rate of the test sample was higher than that of the negative control and the difference was statistically significant (P<0.05), the test sample could be considered to have a certain soothing effect.

[0187] Experimental instruments: analytical balance; ELISA reader

[0188] Preparation of experimental reagents:

[0189] 1. Preparation of acetate buffer: Solution A: Dilute 1155 μL of glacial acetic acid reagent to a 100 mL volumetric flask with purified water; Solution B: Dilute 1.64 g of anhydrous sodium acetate to a 100 mL volumetric flask with purified water. Take 4.8 mL of solution A and 45.2 mL of solution B from the two solutions prepared above.

[0190] Mix solutions A and B, and dilute to 100 mL in a volumetric flask with pure water to obtain the acetate buffer solution.

[0191] 2. Sample dilution solution: Dilute the aqueous product of Application Example 1 with pure water to a sample concentration of 2%.

[0192] 3. Positive control: Dipotassium glycyrrhizate (purity ≥98%): Dilute with water to a positive control concentration of 3%.

[0193] 4. Negative control: pure water.

[0194] Experimental steps:

[0195] Take four centrifuge tubes: tubes 1 and 2 (0.5 mL sample solution and 0.5 mL 600 U / mL hyaluronidase), and tubes 3 and 4 (0.5 mL distilled water and 0.5 mL acetate buffer). Incubate at 37°C for 20 min. Add 0.1 mL (2.5 mol / L) calcium chloride solution and incubate at 37°C for 20 min. Add 0.5 mL 0.5 mg / mL sodium hyaluronate to tubes 1 and 3, and 0.5 mL acetate buffer to tubes 2 and 4. Incubate at 37°C for 40 min. Let stand at room temperature for 10 min, then add 0.5 mL acetylacetone solution (3.5 mL acetylacetone dissolved in 50 mL 0.1 mL / L sodium carbonate solution) and 0.1 mL... Mix 5 mol / L NaOH solution and 0.5 mL distilled water, place in a boiling water bath for 15 min; cool for 5 min, then place in an ice water bath for 10 min; let stand at room temperature for 10 min, then add 1 mL of P-DAB colorimetric reagent (P-DAB solution is prepared by mixing 0.8 g p-dimethylaminobenzaldehyde, 15 mL concentrated hydrochloric acid, and 15 mL anhydrous ethanol solution); after thorough shaking, add 3.5 mL of anhydrous ethanol, let stand at room temperature for 30 min, and develop color. Take 150 μL and place it in a 96-well plate, then place it in a microplate reader and measure the absorbance A1, A2, A3, and A4 of the four test tubes (1, 2, 3, and 4) at 530 nm.

[0196] The inhibition rate of hyaluronidase is calculated using the following formula: Hyaluronidase inhibition rate = [1 - (A1 - A2) / (A3 - A4)] × 100%.

[0197] Statistical analysis was performed using SPSS software. Independent samples t-tests were used to compare the hyaluronidase inhibition rates of the experimental samples, positive controls, and negative controls. All statistical analyses were two-tailed tests, with a significance level of α = 0.05. P > 0.05 indicated no significant difference between the two groups; P < 0.05 indicated a significant difference between the two groups. The experimental results are shown in Table 4.

[0198] Table 4

[0199]

[0200]

[0201] As shown in Table 4, the positive control showed an inhibition rate of >50% for hyaluronidase, indicating that the reaction system was effective. When the sample concentration was 2%, the average inhibition rate of hyaluronidase was 39.618%, which was significantly different from that of the negative control (P<0.05), indicating that the sample had a certain soothing effect.

[0202] Experiment Example 4 Antioxidant Efficacy Test

[0203] The emulsion product of Example 4 was used as the experimental group, while the same formula with the halophilic Bacillus fermentation product removed was used as the blank control group.

[0204] Experimental Principle: 1,1-Diphenyl-2-trinitrophenylhydrazine (DPPH) is a stable, long-lived free radical. In the presence of a free radical scavenger, the light absorption of the DPPH ethanol solution is reduced due to its single electron pairing with DPPH. The degree of DPPH ethanol solution decolorization is linearly related to the number of electrons it accepts, thus allowing evaluation of the test sample's ability to scavenge free radicals, i.e., its antioxidant activity.

[0205] Instruments and reagents: analytical balance (accuracy 0.0001g), 10mL test tubes, adjustable pipette (1.00mL), UV-Vis spectrophotometer, 95% ethanol, 1,1-diphenyl-2-trinitrophenylhydrazine (≥95%), ascorbic acid (AR, ≥99%).

[0206] Test substance:

[0207] Experimental group: Emulsion products containing halophilic Bacillus fermentation products, with a test concentration of 100 mg / mL.

[0208] Blank control group: The same formulation with the fermentation product of halophilic Bacillus removed, tested at a concentration of 100 mg / mL.

[0209] Positive control group: ascorbic acid, tested concentration 0.04 mg / mL

[0210] Test method:

[0211] Preparation of DPPH solution: Dissolve 12 mg of DPPH in 100 mL of 95% ethanol and sonicate for 5 min to obtain a 0.12 mg / mL DPPH ethanol solution.

[0212] Emulsion solution preparation: Take 2.0g of the experimental group emulsion and the blank control group emulsion respectively, and add 20mL of deionized water to obtain an emulsion solution of 100mg / mL.

[0213] Preparation of ascorbic acid solution: Take 4 mg of ascorbic acid and add 100 mL of deionized water to obtain an ascorbic acid solution with a concentration of 0.04 mg / mL.

[0214] Add the solution according to Table 5:

[0215] Table 5

[0216] T-sample tube <![CDATA[T0-Sample Background]]> C-DPPH pipe <![CDATA[C0 - solvent background]]> Test solution (mL) 1 1 0 0 Water or 95% ethanol (mL) 2 2 3 3 DPPH ethanol solution (mL) 1 0 1 0 95% ethanol (mL) 0 1 0 1 Parallel number 3 1 3 1

[0217] Note: The third row of the first column in the table, "water or 95% ethanol solution", means that water is added to T-sample tubes, T0-sample background, and C0 solvent background, and 95% ethanol is added to C-DPPH tubes.

[0218] After each tube was allowed to stand at room temperature in the dark for 30 minutes, the absorbance at 517 nm was measured, and the free radical scavenging ability of the sample was calculated using the following formula:

[0219] Free radical scavenging rate, % = (1 - (T - T0) / (C - C0)) × 100%

[0220] In the formula: T is the absorbance of the sample tube, that is, the absorbance of the solution after the test substance reacts with DPPH; T0 is the background absorbance of the sample (test substance); C-DPPH tube absorbance is the average of three measurements, that is, the DPPH absorbance without the test substance; C0 is the background absorbance of the solvent. The statistical difference P value is calculated. If there is a significant difference (P < 0.05), the test sample can be considered to have a certain antioxidant effect. The results are shown in Table 6.

[0221] Table 6

[0222] Test Project Blank control group experimental group Positive control group Free radical scavenging rate, % -0.10 89.76 75.49 p-value / ＜0.05 ＜0.05

[0223] As shown in Table 6, the free radical scavenging rate of the experimental group was 89.76%, and the free radical scavenging rate of the experimental sample was higher than that of the blank control group, with a statistical difference of P < 0.05. The experimental group sample has antioxidant effect.

[0224] Example 5 Anti-wrinkle test

[0225] The essence product from Example 6 was used as the experimental group, while the same formula, with the fermentation products of halophilic Bacillus removed, was used as the control group.

[0226] The test method is as follows:

[0227] Volunteer requirements: 100 volunteers aged 30-50, gender not limited, no history of skin diseases or skin allergies, good health, good compliance, able to use samples as required by staff, and must not use any cosmetics other than the test samples during the trial.

[0228] Volunteers were randomly divided into two groups of 50 each. The experimental group received samples from one group, and the control group received samples from the other for 30 days, twice daily (morning and evening). On days 0 and 30, the VISIA-7 was used to assess facial wrinkles. Wrinkle counts were used to represent wrinkle characteristics; higher counts indicated more wrinkles. The results are shown in Table 7, where the wrinkle count represents the number of corresponding skin problems, i.e., the number of wrinkles detected.

[0229] Table 7

[0230]

[0231] As can be seen from Table 7, the wrinkle feature count was greatly reduced when using the essence product containing the fermentation product of halophilic Bacillus, indicating that the fermentation product of halophilic Bacillus has an anti-wrinkle effect.

[0232] Example 6: Oil Control Efficacy Test

[0233] The essence product from Example 6 was used as the experimental group, while the same formula with the halophilic Bacillus fermentation product removed was used as the experimental control group, and finasteride was used as the positive control group.

[0234] 1. Experimental Principle

[0235] The oil-controlling efficacy of the sample was verified by testing its inhibition rate against 5α-reductase. 5α-reductase, under the catalysis of reduced coenzyme II, converts testosterone into dihydrotestosterone (DHT). Increased DHT levels lead to increased sebum secretion, i.e., increased oil production. Therefore, the best way to control oil production is to address the root cause by directly inhibiting 5α-reductase to block the conversion of testosterone into DHT and reduce oil production.

[0236] The experiment used the absorbance of testosterone at 242 nm to test the inhibition rate of 5α-reductase. The higher the absorbance, the higher the testosterone content, which indicates a higher inhibition rate of 5α-reductase.

[0237] 2. Experimental steps:

[0238] Blank group: 50 μL of testosterone, 25 μL of 5α-reductase, and 50 μL of reduced coenzyme II were added to the reaction tube in sequence. After mixing, the mixture was incubated at 37℃ for 60 min. The absorbance of the mixture at 242 nm was measured. The blank background value was subtracted to obtain the absorbance value of the blank group (A0).

[0239] Assay: Add 50 μL of testosterone, 25 μL of 5α-reductase, 50 μL of reduced coenzyme II, and 10 μL of experimental sample, experimental control group, or finasteride to the reaction tube in sequence. After mixing, incubate at 37℃ for 60 min. Measure the absorbance of the mixture at 242 nm and subtract the background value to obtain the absorbance (An).

[0240] The formula for calculating the absorbance ratio (I) is: I% = An - A0 / A0 × 100%.

[0241] The absorbance ratios of each group of samples are shown in Table 8 below.

[0242] Table 8

[0243] sample absorbance ratio Finasteride 80.11％ experimental group 91.83％ Experimental control group 20.52％ Blank control group 15.69％

[0244] As shown in Table 8, the absorbance ratio of the serum containing Bacillus halophilus fermentation products was higher than that of the experimental control group without Bacillus halophilus fermentation products. This indicates that the serum containing Bacillus halophilus fermentation products has a better inhibition rate of 5α-reductase and has a significant oil-control effect.

[0245] Experiment Example 7: Acne Treatment Efficacy Test

[0246] The cream product from Example 5 was used as the test group, while a sample of the same formula without the fermentation products of halophilic Bacillus was used as the control group. The efficacy was evaluated by using the Propionibacterium acnes inhibition test according to T / CNMIA 0010—2020 "Safety / Efficacy Evaluation Standard for Acne-Removing Efficacy Skin Care Products".

[0247] Experimental steps

[0248] The 24-hour slant culture of Propionibacterium acnes (purchased from the China Center for Type Culture Collection) was washed down with PBS phosphate buffer (PBS buffer prepared by mixing 1 / 15 mol / L KH2PO4 and 1 / 15 mol / L Na2HPO4·2H2O at a volume ratio of 18.2:81.8) to prepare a bacterial suspension (the required concentration is: when 100 μL is dropped onto the sample, the recovered bacterial count is 1 × 10⁻⁶). 4 ~9×10 4 CFU / tablet or ml).

[0249] Using the smear method, samples from the experimental group and the control group were smeared onto the sample slides to obtain the test sample slides and control samples, respectively. Four test sample slides (2.0cm×3.0cm) and four control sample slides (of the same material and size as the test sample, but without antibacterial material and sterilized) were placed in sterilized petri dishes.

[0250] Take the above bacterial suspension and add 100 μL to each sample and control sample. Spread evenly and start timing. After 2, 5, 10 and 20 min, use sterile forceps to put the sample into a test tube containing 5 mL PBS and mix thoroughly. Dilute to 10%, 20% and 50% dilutions, respectively. Then, take 0.5 mL of each and place it in two petri dishes. Pour 15 mL of Sabouraud agar (yeast) at 40-45℃ into the petri dishes, rotate the petri dishes to mix thoroughly, and after the agar solidifies, turn the plates over and incubate at 35℃±2℃ for 72 h (yeast). Count the viable colonies.

[0251] The experiment was repeated 3 times, and the antibacterial rate was calculated using the following formula:

[0252] X = (AB) / A × 100%

[0253] Where: X - antibacterial rate, %;

[0254] A - Average colony count of control sample;

[0255] B - Average colony count of the tested sample.

[0256] When the antibacterial rate is ≥50% to 90%, the product has antibacterial effect; when the antibacterial rate is ≥90%, the product has strong antibacterial effect.

[0257] Experimental data

[0258] This antibacterial detection method was used to test samples from the experimental group and the control group. The antibacterial rate of the control group was 22.64% against Propionibacterium acnes, while the antibacterial rate of the experimental group against Propionibacterium acnes reached 76.43%. The tested samples showed obvious antibacterial effects, that is, they have acne-removing efficacy.

[0259] Experiment Example 8: Firming Efficacy Test

[0260] Application Example 5 was used as the test sample; a sample made with the same formula but without halophilic Bacillus was used as the control sample for the firming efficacy test.

[0261] Ten volunteers were recruited to test skin elasticity using the CK skin analyzer. The testing method was as follows: After cleansing their faces daily, volunteers applied a control cream to their left cheek and a similar amount of the test sample to their right cheek. Using the elasticity measurement probe of the CK skin analyzer, skin elasticity values ​​were measured on the left and right cheeks at 0, 3, 5, 7, and 14 days. The skin's rebound rate was recorded, and the numerical changes were observed. If the skin elasticity value of the right cheek (with the test sample applied) increased compared to the left cheek (with the regular cream applied), it indicated that the test sample could restore skin elasticity, i.e., it had a firming effect. The results of the experiment from five randomly selected volunteers are shown in Table 9.

[0262] Table 9

[0263]

[0264] As shown in Table 7, among the test values ​​of 5 randomly selected volunteers, the skin elasticity test value of the right cheek that was treated with the test sample increased compared to the left cheek that was treated with ordinary cream. This indicates that the test sample can restore skin elasticity and has a firming effect.

[0265] The above description is merely a preferred embodiment of this application and is not intended to limit the application in any other way. Any person skilled in the art may make changes or modifications to the disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of this application without departing from the scope of the technical solution of this application shall still fall within the protection scope of this application.

Claims

1. A salt-tolerant Bacillus, deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 29259.

2. The use of the salt-tolerant Bacillus of claim 1 in fermentation production.

3. The use according to claim 2, wherein the use is in the fermentation production of fermented products having moisturizing, whitening, soothing, antioxidant, anti-wrinkle, oil-controlling, acne-removing and firming effects.

4. A fermentation product obtained by fermentation with the salt-tolerant Bacillus of claim 1.

5. The fermentation product according to claim 4, wherein the method of fermenting halophilic Bacillus comprises: The salt-tolerant Bacillus strain was activated to obtain seed liquid; The seed liquid was fermented to obtain a crude fermentation product. The crude fermentation product is separated by centrifugation to obtain the fermentation product.

6. The fermentation product according to claim 5, wherein in the process of fermenting the seed liquid to obtain the crude fermentation product, the inoculation amount of the seed liquid is 1-10% of the liquid volume.

7. The fermentation product according to claim 5, wherein, The fermentation medium used for fermentation contains 50-100 g / L rapeseed oil, 30-100 g / L glucose, 5-10 g / L NaNO3, 0.5-1 g / L magnesium sulfate heptahydrate, 1-5 g / L yeast extract, 0.1-1 g / L potassium chloride, 0.1-1 g / L potassium dihydrogen phosphate, 0.1-1 g / L disodium hydrogen phosphate dodecahydrate, 0.1-0.5 g / L calcium chloride, and 0.01-0.05 g / L ferrous sulfate heptahydrate.

8. A method for preparing a fermentation product, comprising fermenting the salt-tolerant Bacillus of claim 1.

9. The method of claim 8, wherein the method comprises: The salt-tolerant Bacillus strain was activated to obtain seed liquid; The seed liquid was fermented to obtain a crude fermentation product. The crude fermentation product is separated by centrifugation to obtain the fermentation product.

10. The method according to claim 9, wherein, In the process of fermenting the seed liquid to obtain the crude fermentation product, the inoculation amount of the seed liquid is 1-10% of the liquid volume.

11. The method according to claim 9, wherein, The fermentation medium used for fermentation culture contains 50-100 g / L rapeseed oil, 30-100 g / L glucose, 5-10 g / L NaNO3, 0.5-1 g / L magnesium sulfate heptahydrate, 1-5 g / L yeast extract, 0.1-1 g / L potassium chloride, 0.1-1 g / L potassium dihydrogen phosphate, 0.1-1 g / L disodium hydrogen phosphate dodecahydrate, 0.1-0.5 g / L calcium chloride, and 0.01-0.05 g / L ferrous sulfate heptahydrate.

12. Use of the fermentation product according to any one of claims 4-7 or the fermentation product prepared by the method according to any one of claims 8-11 in the preparation of cosmetics.

13. The use according to claim 12, wherein, The dosage forms of the cosmetics are creams, lotions, microemulsions, gels, liquids, serums, sheet masks, smear masks, or sprays.

14. A cosmetic product comprising the fermentation product of any one of claims 4-7 or the fermentation product prepared by any one of claims 8-11.

15. The cosmetic product according to claim 14, wherein, The fermentation product is 0.01-99.99% by mass percentage in cosmetics.

16. The cosmetic product according to claim 14, wherein, It also contains cosmetic-acceptable excipients and / or active ingredients.

17. The cosmetic product according to any one of claims 14-16, wherein, The dosage forms of the cosmetics are creams, lotions, microemulsions, gels, liquids, serums, sheet masks, smear masks, or sprays.

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