Salt-tolerant bacillus, and fermentation product and application thereof

Abstract

The invention provides a salt-tolerant bacillus and application thereof, the strain is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No.29259. A fermentation product obtained by using the strain has remarkable effects of moisturizing, whitening, relieving, resisting oxidation, resisting wrinkles, controlling oil, removing acnes and tightening, and can be applied to cosmetics.

Description

Technical Field

[0001] The present application relates to the field of cosmetic technology, and in particular to a halotolerant Bacillus, a fermentation product thereof and uses thereof. Background Art

[0002] The cosmetics industry has always faced the challenge of raw material innovation. There are three main sources of cosmetic raw materials: chemical synthesis, natural extraction and biological fermentation. Although traditional chemical synthesis methods can produce raw materials on a large scale, they are often accompanied by environmental and safety issues. Naturally extracted cosmetic raw materials usually come from various plants, but there are many difficulties in the research and application of plant ingredients and it is difficult to control quality standards. In contrast, fermentation technology provides a more environmentally friendly and sustainable option. It can not only reduce dependence on chemical reagents, but also create new active ingredients through natural processes. These ingredients are often more friendly to the human body and have fewer side effects.

[0003] Microbial fermentation raw materials utilize the metabolic process of specific microbial cells and a variety of complex reaction systems to obtain direct metabolites or secondary metabolites of the bacteria, which usually have a variety of skin physiological benefits. In addition to the metabolites of specific bacteria, fermentation raw materials also contain free amino acids, organic acids, polysaccharides, small molecule peptides, etc. Combined with the formulation and development of traditional cosmetics, the addition of biological fermentation raw materials improves the skin feel, efficacy, absorption and other aspects of the product.

[0004] With the development of the cosmetics industry and the rapid upgrading and iteration of cosmetic raw materials, people's demand for cosmetics has gradually shifted to safe, efficient, green and natural. Compared with natural extraction and chemical synthesis, microbial fermentation raw materials are increasingly used in cosmetics due to their high efficacy, low toxicity, green and safe characteristics. Fermentation technology has become the application direction and research and development 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 the Procter & Gamble Group, Estee Lauder and La Mer under the Estee Lauder Group, and Lancome and L'Oréal Paris under the L'Oréal Group have all incorporated different fermentation raw materials into their flagship products, such as bifid yeast fermentation product lysate (derived from bifidobacterium fermentation) and yeast extract.

[0005] Currently, the "Catalogue of Used Cosmetic Raw Materials (2021 Edition)" includes a total of 85 fermented raw materials. Compared with traditional chemical synthetic raw materials and plant extract raw materials, fermented raw materials still have huge development, application space and market prospects. Summary of the invention

[0006] Based on this, the present application provides a halotolerant Bacillus, its fermentation product and its use. The fermentation product is rich in multiple active ingredients, has significant moisturizing, whitening, soothing, anti-oxidation, anti-wrinkle, oil control, acne removal and firming effects, and can be widely used in cosmetics.

[0007] The specific technical solutions of this application are as follows:

[0008] 1. A salt-tolerant Bacillus (Bacillus halotolerans SDY412), which is deposited in China General Microbiological Strain Collection Center with a deposit number of CGMCC No.29259.

[0009] 2. Use of the halotolerant Bacillus described in item 1 in fermentation production, preferably use of the fermentation production of a fermentation product having moisturizing, whitening, soothing, anti-oxidation, anti-wrinkle, oil control, acne removal and firming effects.

[0010] 3. A fermentation product obtained by fermenting the halotolerant Bacillus according to item 1.

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

[0012] activating the halotolerant Bacillus strain to obtain a seed solution;

[0013] Fermenting the seed liquid to obtain a crude fermentation product;

[0014] The crude fermentation product is centrifuged to obtain a fermentation product.

[0015] 5. The fermentation product according to item 4, wherein in 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 in the fermentation 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 sodium 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 halotolerant Bacillus described in item 1.

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

[0019] activating the halotolerant Bacillus strain to obtain a seed solution;

[0020] Fermenting the seed liquid to obtain a crude fermentation product;

[0021] The crude fermentation product is centrifuged to obtain a fermentation product.

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

[0023] Preferably, the fermentation medium used in the fermentation culture 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 sodium 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 according to any one of Items 3 to 5 or the fermentation product according to any one of Items 6 to 8 in cosmetics.

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

[0026] 11. A cosmetic comprising the fermentation product described in any one of items 3 to 5 or the fermentation product prepared by the method described in any one of items 6 to 8.

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

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

[0029] 14. The cosmetic according to any one of items 11 to 13, wherein the cosmetic is in the form of a cream, lotion, microemulsion, gel, aqueous solution, essence, sheet mask liquid, smearable mask, spray or gel.

[0030] Effects of the Invention

[0031] The fermentation product obtained by using the screened halotolerant Bacillus subtilis in the present application has significant moisturizing, whitening, soothing, anti-oxidation, anti-wrinkle, oil control, acne removal and firming effects, and can be used in cosmetics. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0033] Strain deposit information

[0034] The halotolerant Bacillus (Bacillus halotolerans SDY412) of the present application is named as halotolerant Bacillus (Bacillus halotolerans SDY412), and was deposited in the General Microbiology Center of China Microorganism Collection Administration on December 7, 2023, with the deposit number CGMCC No.29259, address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101. DETAILED DESCRIPTION

[0035] The present application is described in detail below in conjunction with 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 by the embodiments set forth herein. On the contrary, these embodiments are provided to enable a more thorough understanding of the present application and to enable the scope of the present application to be fully communicated to those skilled in the art.

[0036] It should be noted that certain words are used in the specification and claims to refer to specific components. Those skilled in the art should understand that technicians may use different nouns to refer to the same component. This specification and claims do not use the difference in nouns as a way to distinguish components, but use the functional differences of components as the criterion for distinction. As mentioned throughout the specification and claims, "including" or "comprising" are open-ended terms and should be interpreted as "including but not limited to". The subsequent description of the specification is a preferred embodiment of the present application, but the description is based on the general principles of the specification and is not intended to limit the scope of the present application. The scope of protection of the present application shall be determined by the attached claims.

[0037] The present application provides a halotolerant Bacillus (Bacillus halotolerans SDY412), which is a strain obtained by high-temperature separation, salt tolerance test screening and purification from the silt and lake water of Chaka Salt Lake. The DNA of the strain is extracted for 16S rDNA amplification, and the amplified product is sent to the China General Microbiological Strain Collection Center for sequencing and species identification, and is confirmed to be halotolerant Bacillus, named Bacillus halotolerans SDY412, which is deposited in the China General Microbiological Strain Collection Center with a deposit number of CGMCC No.29259.

[0038] The present application provides the use of the above-mentioned halotolerant Bacillus in fermentation production, preferably in the fermentation production of fermentation products with moisturizing, whitening, soothing, anti-oxidation, anti-wrinkle, oil control, acne removal and firming effects.

[0039] The present application adopts the above-mentioned strain for fermentation, and the obtained fermentation product has excellent effects, such as moisturizing, whitening, soothing, anti-oxidation, anti-wrinkle, oil control, acne removal, firming, etc.

[0040] The present application provides a fermentation product obtained by fermenting the above-mentioned halodurable Bacillus. In some embodiments, the method for fermenting halodurable Bacillus comprises:

[0041] activating the halotolerant Bacillus strain to obtain a seed solution;

[0042] fermenting the seed liquid to obtain a crude fermentation product;

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

[0044] In the present application, the method for activating the strain to obtain the seed liquid is not limited in any way. A person skilled in the art may use conventional methods for activation, such as inoculating the strain into a seed culture medium for culturing to obtain the seed liquid. For example, the strain may be inoculated into a shake flask seed culture medium for culturing to obtain the seed liquid, for example, culturing at 30-37°C and 150-300rpm for 16-22 hours to obtain the seed liquid.

[0045] For example, the temperature may be 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 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 liquid to obtain a crude fermentation product means inoculating the seed liquid into a fermentation medium for fermentation to obtain a crude fermentation product. In some embodiments, the inoculation amount of the seed liquid is 1-10% of the liquid volume.

[0049] In the present application, there is no limitation on the inoculation amount, which has the meaning generally understood by those skilled in the art. For example, the inoculation amount refers to the ratio of the volume of the seed solution transferred in to the volume of the culture solution after inoculation.

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

[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 sodium 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 50g / L, 60g / L, 70g / L, 80g / L, 90g / L, 100g / L, etc.;

[0053] Glucose 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 0.5g / L, 0.6g / L, 0.7g / L, 0.8g / L, 0.9g / L, 1g / L, etc.;

[0056] The yeast extract may be 1g / L, 1.5g / L, 2g / L, 2.5g / L, 3g / L, 3.5g / L, 4g / L, 4.5g / L, 5g / L, etc.;

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

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

[0059] Disodium hydrogen phosphate dodecahydrate may be 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 0.1g / L, 0.2g / L, 0.3g / L, 0.4g / L, 0.5g / L, etc.;

[0061] The concentration of ferrous sulfate heptahydrate may 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 liquid is inoculated into a fermentation medium, and cultured at 30-37° C. and 150-300 rpm for 40-60 hours to obtain a crude fermentation product.

[0063] In the present application, there is no limitation on the centrifugal separation method, and those skilled in the art can make a conventional selection 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 the present application has excellent efficacy and can be used in cosmetics.

[0065] The present application provides a method for preparing a fermentation product, which comprises fermenting the above-mentioned halodurable Bacillus. In some embodiments, the method comprises:

[0066] activating the halotolerant Bacillus strain to obtain a seed solution;

[0067] Fermenting the seed liquid to obtain a crude fermentation product;

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

[0069] Preferably, the fermentation medium used in the fermentation culture 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 sodium hydrogen phosphate dodecahydrate, 0.1-0.5 g / L calcium chloride and 0.01-0.05 g / L ferrous sulfate heptahydrate.

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

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

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

[0073] For example, in terms of mass percentage in the cosmetic, 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.

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

[0075] In the present application, there is no limitation on the excipients and active ingredients acceptable for cosmetics, and those skilled in the art can make routine selections based on needs. There is no limitation on the dosage of the excipients and active ingredients, and those skilled in the art can select the content of the excipients and active ingredients to be added based on needs.

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

[0077] Example

[0078] The present 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, % means wt%, i.e., weight percentage. The reagents or instruments used without indicating the manufacturer are all conventional reagent products that can be obtained commercially.

[0079] Example 1 Acquisition of strains

[0080] In this embodiment, the strains in the sample are separated, screened and identified.

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

[0082] 1. Isolation of strains

[0083] The heat-resistant property of spores was used to isolate Bacillus. 10 g of sludge and water samples from Chaka Salt Lake were taken and diluted to 10% with sterilized pure water. -1 and 10 -2 The dilution was placed in a boiling water bath for 10-20 min, 200 μL of the dilution was added to a test tube containing LB liquid culture medium and cultured in an incubator at 37°C for 24 h to obtain a bacterial solution.

[0084] 2. Screening of strains

[0085] The above bacterial solution was streaked on LB solid medium containing 20% ​​NaCl and cultured in a constant temperature incubator at 37°C until obvious single colonies grew. A single colony was picked and streaked on LB solid medium and cultured in a constant temperature incubator at 37°C. After the colony grew, the streaking was repeated 3 times to obtain a purified single colony.

[0086] 3. Identification of strains

[0087] The above strain was amplified with 16S rDNA using bacterial universal primers (27F / 1492R) and sent for sequencing. The sequencing results were compared in NICI and preliminarily confirmed to be Bacillus. The strain was further identified by the China General Microbiological Culture Collection and confirmed to be a salt-tolerant Bacillus, named Bacillus halotolerans SDY412, which was deposited in the General Microbiological Center of the China Microbiological Culture Collection on December 7, 2023, with a deposit number of CGMCC No. 29259. Sequencing results:

[0088]

[0089] Example 2 Preparation of fermentation products

[0090] 1. Culture Medium Preparation

[0091] (1) Preparation of streak culture medium

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

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

[0096] The culture medium comprises: 50 g / L rapeseed oil, 50 g / L glucose, 5 g / L NaNO3, 1 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, 0.1 g / L calcium chloride, and 0.01 g / L ferrous sulfate heptahydrate.

[0097] 2. Bacteria culture and fermentation:

[0098] (1) Streak the strain obtained in Example 1 on a streak medium and culture it in a constant temperature incubator at 37° C. for 24 h to obtain a single colony;

[0099] (2) Pick a single colony and inoculate it into a 50 mL seed shake bottle with a volume of 20 mL. Incubate it in a shaker at 37°C and 200 rpm for 20 h as the seed solution;

[0100] (3) Inoculate the fermentation seed liquid into a 500 mL fermentation shake flask at a 5% inoculation rate, with a liquid volume of 50 mL. Incubate in a shaker at 37° C. and 220 rpm for 50 h, and harvest the crude fermentation product after fermentation.

[0101] 3. Filter the precipitation to obtain the fermentation product of halotolerant 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 halodurable 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, add glycerin, propylene glycol, butylene glycol, tea tree oil, and halodurable Bacillus fermentation product to the aqueous solution in this order, stir until it is evenly dissolved, and finally add NaOH to adjust the pH value of the aqueous cosmetics to about 6.8.

[0105] Application Example 2: Facial mask products

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

[0107] Its preparation process is:

[0108] Dispersing the modified grafted starch and xanthan gum in glycerol to uniformly disperse them 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 evenly dissolved to obtain an aqueous solution system;

[0110] Then cool to below 45°C, add butylene glycol, hydroxyethyl urea, and halodurogenic Bacillus fermentation product to the aqueous solution system in the order, then add the mixed aqueous solution to the glycerin dispersion system and stir until it is evenly dissolved. Finally, add ethylhexylglycerin, carbomer 981, and sodium hyaluronate to the system, and add sodium hydroxide to adjust the pH value of the product to about 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% caprylhydroxamic acid, 0.4% p-hydroxyacetophenone, 0.3% of the halodurable Bacillus fermentation product prepared in Example 2, 0.2% modified grafted starch, 0.2% acrylate / C10-30 alkyl acrylate cross-linked polymer, 0.05% disodium EDTA, 0.05% allantoin, 0.05% NaOH and 0.02% dipotassium glycyrrhizinate.

[0113] Its preparation process is:

[0114] Dispersing the modified grafted starch in glycerol to uniformly disperse the modified grafted starch 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 evenly dissolved to obtain an aqueous solution system;

[0116] Then cool to below 45°C, add the fermentation product of Bacillus halodurans, sorbitol, aloe vera water, allantoin, and dipotassium glycyrrhizinate to the aqueous solution system in the order, then add the mixed aqueous solution to the glycerin dispersion system and stir until it is evenly dissolved. Finally, add caprylhydroxamic acid and acrylate / C10-30 alkyl acrylate cross-linked polymer to the system, and add sodium hydroxide to adjust the pH value of the product to about 6.8.

[0117] Application Example 4 Emulsion Products

[0118] The emulsion product comprises 74.71% of pure water, 10% of glycerol, 5% of shea butter, 3% of cetearyl ether-25, 2% of olive oil, 2% of monoglyceryl stearate, 1% of sunflower oil, 1% of cetearyl alcohol, 0.4% of p-hydroxyacetophenone, 0.3% of the halodurous bacillus fermentation product prepared in Example 2, 0.2% of caprylhydroxamic acid, 0.28% of carbomer 980, 0.05% of disodium EDTA, 0.05% of sodium hydroxide and 0.01% of sodium hyaluronate (molecular weight is 1000-1700 kDa).

[0119] The preparation process is as follows: the system is divided into a water phase and an oil phase during preparation.

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

[0121] Oil phase: add glycerin, shea butter, cetearyl ether-25, olive oil, monoglyceryl stearate, sunflower seed oil, and cetearyl alcohol in this order, and heat to about 80-90°C to completely dissolve the oil phase and mix evenly to obtain the oil phase.

[0122] The oil phase was poured into the water phase and homogenized by a homogenizer to prepare a completely dispersed emulsion. Caprylylhydroxamic acid and carbomer 980 were added to the system, and sodium hydroxide was added to adjust the pH value of the product to about 6.8.

[0123] Application Example 5: Cream Products

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

[0125] The preparation process is as follows: the system is divided into a water phase and an oil phase during preparation.

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

[0127] Oil phase: Add glycerol, isopropyl palmitate, polyacrylate-13, polyisobutene, polysorbate 20, caprylic / capric triglyceride, dicaprylyl carbonate, cetearyl alcohol, beeswax, silicone wax, diisostearyl malate, dimethicone and tocopheryl acetate in this order and heat to about 80-90°C to completely dissolve the oil phase and mix evenly to obtain the oil phase.

[0128] The oil phase is poured into the water phase and homogenized by a homogenizer to prepare a completely dispersed emulsion. Caprylylhydroxamic acid, carbomer 981, sodium hyaluronate, and sodium hydroxide are added to the system to adjust the pH value of the product to about 6.8.

[0129] Application Example 6: Essential Products

[0130] The essence product comprises 80.61% pure water, 5% butylene glycol, 5% glycerol, 4% isononyl isononanoate, 1.5% cetearyl olivate, 0.3% sorbitan olivate, 1% diisostearyl malate, 1% polydimethylsiloxane, 0.5% tocopherol acetate, 0.4% p-hydroxyacetophenone, 0.3% of the fermentation product of the halotolerant Bacillus 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), and the preparation method thereof is as follows:

[0131] The system is prepared into a water phase and an oil phase.

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

[0133] Oil phase: add butylene glycol, glycerin, isononyl isononanoate, cetearyl olivate, sorbitan olivate, diisostearyl malate, polydimethylsiloxane and tocopheryl acetate in this order, and heat to about 80-90°C to completely dissolve the oil phase and mix evenly to obtain the oil phase.

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

[0135] Application Example 7: Lotion Products

[0136] The lotion product comprises 34.75% of pure water, 8% of glycerin, 40% of sodium cocoyl glycinate, 7% of sodium methyl cocoyl taurate, 2% of sodium lauroyl glutamate, 5% of cocoyl glutamic acid, 1% of cholesterol hydroxystearate, 1.5% of sodium chloride, 0.5% of the fermentation product of halotolerant Bacillus prepared in Example 2, 0.2% of citric acid and 0.05% of 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 into the system according to the prescribed proportion, and heated to 70-80°C to completely dissolve and mix them evenly; the temperature is lowered to below 50°C, and a fermentation product of halotolerant Bacillus, sodium chloride, and disodium EDTA are added and stirred evenly; finally, citric acid is added to adjust the pH to about 6.5, and stirred until a white and delicate paste is formed.

[0138] Experimental Example 1 Moisturizing Efficacy Test

[0139] The essence product of Application Example 6 was used as the experimental group, and the same formula except for the fermentation product of the halophilic Bacillus was removed was used as the control group.

[0140] Ten volunteers were recruited to test the moisturizing performance of the experimental group and the control group. The test method was as follows: after cleaning their faces, the volunteers used the experimental group samples on the left face and the control group samples on the right face. After 30 minutes of use, the water content of the left and right areas of the face was tested using a CK skin tester. After 5 hours of use, the water content of the same areas of the left and right sides of the face was tested again using a CK skin tester. The lower the water loss degree of the test, the better the moisturizing property.

[0141] The test data of three volunteers were randomly selected and shown in Table 1.

[0142] Table 1

[0143]

[0144] As shown in Table 1, the essence products containing the fermentation products of Bacillus halophilus have lower water loss on the face after use compared with the cosmetics without the fermentation products of Bacillus halophilus, that is, they have better moisturizing effect.

[0145] Experimental Example 2 Whitening Efficacy Test

[0146] In this experimental example, the tyrosinase inhibition rate of the fermentation product of the halotolerant Bacillus in Example 2 was tested to evaluate the whitening effect of the fermentation product of the halotolerant Bacillus.

[0147] Experimental principle: In the biosynthesis of melanin in the skin, tyrosinase is a key enzyme that acts on DOPA to form DOPA quinone, which spontaneously undergoes a series of reactions to form melanin. In a phosphoric acid solution with a pH of 6.8, tyrosinase can catalyze the conversion of DOPA into DOPA quinone, and the absorbance value can be measured at 475nm on a spectrophotometer. Cosmetics that inhibit tyrosinase activity can reduce the conversion of DOPA into DOPA quinone, thereby reducing the absorbance value. Based on the change in absorbance value, the inhibitory effect of cosmetics on tyrosinase activity can be evaluated.

[0148] Instruments and reagents: analytical balance (accurate to 0.0001g); pipette (5mL, 100μL); constant temperature water bath, GENESYS180 UV spectrophotometer; disodium hydrogen phosphate dodecahydrate (McLean, AR, 99%), levodopa (McLean, 99%), kojic acid (McLean, 99%), tyrosinase (Yuanye, 25KU), citric acid monohydrate (Aladdin, AR, 99.5%).

[0149] Experimental methods:

[0150] 1. Solution preparation

[0151] (1) Sodium hydrogen phosphate-citric acid buffer solution at pH 6.8

[0152] Preparation of 0.2mol / L Na2HPO4·12H2O: Dissolve 14.33g of Na2HPO4·12H2O in 200mL 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 with 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 at pH=6.8 to obtain 100u / mL tyrosinase solution, which is prepared 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 sodium hydrogen phosphate-citrate buffer with a pH of 6.8 to obtain a 1 mg / mL levodopa solution.

[0159] (4) Test substance 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 respectively using sodium hydrogen phosphate-citrate buffer at pH 6.8;

[0162] Test substance: Fermentation product of halotolerant Bacillus

[0163] The halodurable Bacillus fermentation product described in Example 2 was diluted to 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 using a pH 6.8 sodium hydrogen phosphate-citrate buffer.

[0164] 2. Experimental methods

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

[0166] 0.5 mL of tyrosinase solution was added to each of the sample tube (T) and the enzyme reaction tube (C). The sample background (T0) and the solvent background (C0) were replaced with 0.5 mL of disodium hydrogen phosphate-citrate buffer. The sample and tyrosinase were fully mixed and placed in a 37°C water bath for reaction for 10 minutes. 2 mL of L-DOPA solution was added to each tube in turn. The reaction time of each tube was controlled to 5 minutes. The reaction solution of each tube was immediately transferred to a cuvette, and the absorbance was measured at 475 nm.

[0167] Validation: The IC50 (the sample test concentration corresponding to 50% inhibition effect) of the positive control kojic acid should be between 0.05 mg / mL and 0.15 mg / mL, and the test system is considered to be effective.

[0168] Result calculation

[0169] The tyrosinase inhibition rate was calculated according to the absorbance values ​​measured in each test tube.

[0170] Tyrosinase inhibition rate (%) = (1-(T-T0) / (C-C0))*100%

[0171] In the formula,

[0172] T- the average value of the absorbance of the sample tube for 3 times, that is, the absorbance of the solution after the sample reacts with tyrosinase;

[0173] T0-sample background absorbance;

[0174] C- average of two absorbance values ​​of the enzyme reaction tube, i.e., the absorbance value of the reaction between tyrosinase and DOPA when no sample is added;

[0175] C0-solvent background absorbance.

[0176] Validation: Under the experimental conditions, the IC50 of kojic acid is in the range of 0.05mg / mL-0.15mg / mL, which proves that the experimental data is valid. The experimental results are shown in Table 2 and Figure 1 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 Tyrosinase inhibition results of Bacillus fermentation products

[0181]

[0182] From the above data, it can be seen that the fermentation product of Bacillus halodurans has a good inhibitory effect on tyrosinase, with an IC50 of about 0.04 mg / mL, which is less than the IC50 of the positive control kojic acid, and the whitening effect is better than kojic acid.

[0183] Experimental Example 3 Soothing Efficacy Test

[0184] The aqueous product of Application Example 1 was used as the experimental group, and the same formula without the fermentation product of halophilic Bacillus was used as the blank control group. The soothing efficacy was evaluated by the zebrafish larvae neutrophil migration detection method.

[0185] Experimental principle: Hyaluronidase is a specific cleavage enzyme of hyaluronic acid, a participant in allergic reactions, and has a strong correlation with the release of histamine by mast cells. Whether the test sample has a soothing effect can be judged by the hyaluronidase inhibition rate. The higher the hyaluronidase inhibition rate, the stronger the soothing effect of the substance, and vice versa.

[0186] This test refers to the laboratory method (HMC-WI-029 hyaluronidase inhibition rate) and compares the hyaluronidase inhibition rate test results of the test sample with that of the negative control. If the inhibition rate of the test sample is higher than that of the negative control and there is a significant difference (P < 0.05), it can be considered that the test sample has a certain soothing effect.

[0187] Experimental instruments: analytical balance; microplate reader

[0188] Experimental reagent configuration:

[0189] 1. Preparation of acetate buffer: Solution A: Use purified water to make up 1155 μL of glacial acetic acid reagent in a 100mL volumetric flask; Solution B: Use pure water to make up 1.64g of anhydrous sodium acetate in a 100mL volumetric flask, take 4.8mL of the above two solutions A and 45.2mL of solution B.

[0190] Mix solutions A and B and dilute to 100 mL with pure water to obtain acetate buffer.

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

[0192] 3. Positive control: dipotassium glycyrrhizinate (purity ≥ 98%): dilute with water to a positive control concentration of 3%.

[0193] 4. Negative control: pure water.

[0194] Experimental steps:

[0195] Take 4 centrifuge tubes, namely test tubes 1 and 2 (0.5 mL sample solution and 0.5 mL hyaluronidase with a concentration of 600 U / mL), test tubes 3 and 4 (0.5 mL distilled water and 0.5 mL acetate buffer), keep warm at 37°C for 20 min; add 0.1 mL (2.5 mol / L) calcium chloride solution, keep warm at 37°C for 20 min; add 0.5 mL 0.5 mg / mL sodium hyaluronate to test tubes 1 and 3, add 0.5 mL acetate buffer to test tubes 2 and 4, keep warm at 37°C for 40 min; let stand at room temperature for 10 min, add 0.5 mL acetylacetone solution (3.5 mL acetylacetone dissolved in 50 mL 0.1 ml / L sodium carbonate solution), 0.1 mL NaOH solution (5 mol / L) and 0.5 mL of distilled water were placed in a boiling water bath for 15 min; cooled for 5 min, and placed in an ice water bath for 10 min; placed at room temperature for 10 min, and 1 mL of P-DAB color developer was added (P-DAB solution was prepared by mixing 0.8 g of p-dimethylaminobenzaldehyde, 15 mL of concentrated hydrochloric acid, and 15 mL of anhydrous ethanol solution); after sufficient shaking, 3.5 mL of anhydrous ethanol was added, and the mixture was placed at room temperature for 30 min for color development. 150 μL was taken and placed in a 96-well plate and placed in an ELISA reader to measure the absorbances A1, A2, A3, and A4 of four test tubes No. 1, 2, 3, and 4 at 530 nm.

[0196] The inhibition rate of hyaluronidase was calculated according to the following formula: Hyaluronidase inhibition rate = [1-(A1-A2) / (A3-A4)] x 100%.

[0197] The statistical analysis software was SPSS, and the comparison of hyaluronidase inhibition rates between the test samples, positive controls, and negative controls was performed using an independent sample t-test. The above statistical analyses were all two-tailed tests, with a significance level of α = 0.05. P>0.05 indicated that there was no significant difference between the two groups; P<0.05 indicated that there was a significant difference between the two groups. The experimental results are shown in Table 4

[0198] Table 4

[0199]

[0200]

[0201] As can be seen from Table 4, the hyaluronidase inhibition rate of the positive control is >50%, and the reaction system is effective. When the sample concentration is 2%, the average hyaluronidase inhibition rate is 39.618%, which is significantly different from the negative control (P<0.05), indicating that the sample has a certain soothing effect.

[0202] Experimental Example 4 Antioxidant Efficacy Test

[0203] The emulsion product of Application Example 4 was used as the experimental group, and the same formula except for the fermentation product of the halophilic Bacillus was used as the blank control group.

[0204] Experimental principle: 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) is a stable long-lived free radical. When a free radical scavenger is present, the light absorption of DPPH ethanol solution is weakened due to pairing with its single electron. The degree of fading of DPPH ethanol solution is linearly related to the number of electrons it accepts, which can be used to evaluate the ability of the test sample to scavenge free radicals, that is, the size of the antioxidant activity.

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

[0206] Test substance:

[0207] Experimental group: emulsion containing fermentation product of halotolerant Bacillus, tested at a concentration of 100 mg / mL

[0208] Blank control group: the same formula without the fermentation product of Bacillus halophilus, the test concentration was 100 mg / mL

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

[0210] Test method:

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

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

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

[0214] Add liquid according to Table 5:

[0215] Table 5

[0216] T-sample tube <![CDATA[T0 - Sample background]]> C-DPPH tube <![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 times 3 1 3 1

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

[0218] After each tube was left to stand at room temperature and protected from light 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] Wherein: 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 value of 3 measurements, that is, the DPPH absorbance without adding the test substance; C0 is the background absorbance of the solvent and the statistical difference P value is calculated. If there is a significant difference (P < 0.05), it can be considered that the test sample has a certain antioxidant effect. The results are shown in Table 6.

[0221] Table 6

[0222] Test items 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 can be seen from Table 6, the free radical scavenging rate of the experimental group is 89.76%, and the free radical scavenging rate of the experimental samples is higher than that of the blank control group, and the statistical difference P value is <0.05, indicating that the samples in the experimental group have antioxidant effects.

[0224] Example 5 Anti-wrinkle test

[0225] The essence product of Application Example 6 was used as the experimental group, and the same formula except for the fermentation product of halotolerant Bacillus was used as the control group.

[0226] The test method is as follows:

[0227] Volunteer requirements: 100 volunteers aged 30-50, regardless of gender, with no history of skin diseases or skin allergies, good health, good compliance, able to use samples as required by staff, and no cosmetics other than test samples may be used during the trial.

[0228] The volunteers were randomly divided into 2 groups, each with 50 people. The experimental group samples and the blank control group samples were used for 30 days, once in the morning and evening. VISIA-7 was used to test the facial wrinkles of the volunteers on the 0th day and the 30th day. The wrinkle condition was represented by the characteristic count of wrinkles. The higher the characteristic count, the more wrinkles. The results are shown in Table 7, wherein the characteristic count of wrinkles represents the number of corresponding skin problems, that is, the number of wrinkles detected.

[0229] Table 7

[0230]

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

[0232] Example 6 Oil Control Efficacy Test

[0233] The essence product of Application Example 6 was used as the experimental group, while the same formula without the fermentation product of halophilic Bacillus was used as the experimental control group, and finasteride was used as the positive control group.

[0234] 1. Experimental Principle

[0235] The oil control effect of the sample is verified by testing the inhibition rate of the sample on 5α-reductase. 5α-reductase can convert testosterone into dihydrotestosterone DHT under the catalysis of reduced coenzyme II. The increase of DHT will lead to increased sebum secretion, that is, increased oil production. Therefore, the best way to control oil is to start from the root and directly inhibit 5α-reductase to block the conversion of testosterone into DHT and reduce oil production.

[0236] The experiment uses the absorbance value of testosterone at 242nm to test the inhibition rate of 5α-reductase, wherein the higher the absorbance, that is, the higher the testosterone content, means the higher the 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, mixed and incubated at 37°C for 60 min, and the absorbance of the mixture at 242 nm was measured, and the blank background value was deducted to obtain the absorbance value of the blank group (A0).

[0239] Test group: 50 μL of testosterone, 25 μL of 5α-reductase, 50 μL of reduced coenzyme II, and 10 μL of experimental group sample or experimental control group or finasteride were added to the reaction tube in sequence, mixed and incubated at 37°C for 60 min, and the absorbance of the mixture at 242 nm was measured, and the background value was deducted to obtain the absorbance (An).

[0240] The calculation formula of 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] It can be seen from Table 8 that the essence product containing the fermentation product of Bacillus halophilus has a larger absorbance ratio than the experimental control group without the fermentation product of Bacillus halophilus, indicating that the essence product containing the fermentation product of Bacillus halophilus has a better inhibition rate on 5α-reductase and has a significant oil control effect.

[0245] Experimental Example 7: Acne-Removal Efficacy Test

[0246] The cream product of Application Example 5 was used as the test experimental group, and the same formula sample without the fermentation product of halophilic Bacillus was used as the control experimental group. According to T / CNMIA 0010-2020 "Safety / Efficacy Evaluation Standards for Anti-Acne Functional Skin Care Products", the Propionibacterium acnes inhibition test was used to conduct efficacy evaluation.

[0247] Experimental procedures

[0248] The 24-hour slant culture of Propionibacterium acnes (purchased from China Center for Type Culture Collection) was washed with PBS phosphate buffer (1 / 15 mol / L KH2PO4 and 1 / 15 mol / L Na2HPO4·2H2O were mixed in a volume ratio of 18.2:81.8 to obtain PBS buffer) and prepared into a bacterial suspension (the required concentration was: 100 μL was dropped on the sample and the number of recovered bacteria was 1×10 4 ~9×10 4 cfu / piece or ml).

[0249] Using the smearing method, smear the samples of the test group and the control group on the sample pieces to obtain the test pieces and the control samples respectively, take 4 test pieces (2.0cm×3.0cm) and 4 control pieces (the same material and size as the sample, but without antibacterial material and sterilized) (placed in a sterilized plate).

[0250] Take the above bacterial suspension and drop 100 μL on each sample piece and control sample piece respectively. Start timing after evenly coating. Act for 2, 5, 10, and 20 minutes. Use sterile tweezers to put the sample pieces into test tubes containing 5 mL PBS and mix them thoroughly. Dilute them to 10%, 20%, and 50% dilutions. Then take 0.5 mL of each and place them on two plates. Pour 15 mL of Sabouraud agar medium (yeast) at 40-45°C, rotate the plates to make it fully uniform. After the agar solidifies, turn the plates over and culture them at 35°C±2°C for 72 hours (yeast) to count the live bacterial colonies.

[0251] The test was repeated 3 times, and the inhibition rate was calculated as follows:

[0252] X=(AB) / A×100%

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

[0254] A-average colony count of control samples;

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

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

[0257] Experimental data

[0258] This antibacterial detection method was used to detect samples from the test group and the control group. The antibacterial rate of the control group's samples against Propionibacterium acnes was 22.64%, while the antibacterial rate of the test group against Propionibacterium acnes could reach 76.43%. The tested samples had obvious antibacterial effects, that is, they had acne-removing effects.

[0259] Experimental Example 8 Firming Effect Test

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

[0261] 10 volunteers were recruited and the skin elasticity value was tested using a CK skin tester. The test method is: after cleaning the facial skin every day, the volunteers applied a control cream on the left face and applied a substantially equal amount of the test sample on the right face. The elasticity measuring probe of the CK skin tester was used to test the skin elasticity values ​​of the left and right faces on 0 days, 3 days, 5 days, 7 days, and 14 days, respectively, and the skin rebound ratio was read and the numerical changes were observed. If the skin elasticity test value of the right face with the test sample applied increases compared to the left face with the ordinary cream applied, it indicates that the test sample can restore the elasticity of the skin, that is, it has a firming effect. The experimental results of 5 volunteers randomly selected are shown in Table 9.

[0262] Table 9

[0263]

[0264] It can be seen from Table 7 that in the test values ​​of 5 randomly selected volunteers, the skin elasticity test values ​​of the right face applied with the test sample increased compared with the left face applied with ordinary cream, which means that the test sample can restore skin elasticity and has a firming effect.

[0265] The above is only the preferred embodiment of the present application, and does not limit the present application in other forms. Any technician familiar with the profession may use the above disclosed technical content to change or modify it into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiment based on the technical essence of the present application without departing from the technical solution of the present application still falls within the protection scope of the technical solution of the present application.

Claims

1. A salt-tolerant Bacillus (Bacillus halotolerans SDY412), which is deposited in China General Microbiological Strain Collection Center with a deposit number of CGMCC No.29259.

2. Use of the halotolerant Bacillus according to claim 1 in fermentation production, preferably in fermentation production of fermentation products having moisturizing, whitening, soothing, anti-oxidation, anti-wrinkle, oil control, acne removal and firming effects.

3. A fermentation product obtained by fermenting the halotolerant Bacillus according to claim 1.

4. The fermentation product according to claim 3, wherein the method for fermenting the halodurogenic Bacillus comprises: activating the halotolerant Bacillus strain to obtain a seed solution; Fermenting the seed liquid to obtain a crude fermentation product; The crude fermentation product is centrifuged to obtain a fermentation product.

5. The fermentation product according to claim 4, wherein in fermenting the seed liquid to obtain the crude fermentation product, the inoculation amount of the seed liquid is 1-10% of the liquid volume; Preferably, the fermentation medium used in the fermentation 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 sodium hydrogen phosphate dodecahydrate, 0.1-0.5 g / L calcium chloride and 0.01-0.05 g / L ferrous sulfate heptahydrate.

6. A method for preparing a fermentation product, comprising fermenting the halotolerant Bacillus according to claim 1.

7. The method according to claim 6, wherein the method comprises: activating the halotolerant Bacillus strain to obtain a seed solution; Fermenting the seed liquid to obtain a crude fermentation product; The crude fermentation product is centrifuged to obtain a fermentation product.

8. The method according to claim 7, wherein: In fermenting the seed liquid to obtain a crude fermentation product, the inoculation amount of the seed liquid is 1-10% of the liquid volume; Preferably, the fermentation medium used in the fermentation culture 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 sodium hydrogen phosphate dodecahydrate, 0.1-0.5 g / L calcium chloride and 0.01-0.05 g / L ferrous sulfate heptahydrate.

9. Use of the fermentation product according to any one of claims 3 to 5 or any one of claims 6 to 8 in cosmetics; Preferably, the dosage form of the cosmetic is a cream, lotion, microemulsion, gel, aqueous solution, essence, sheet mask liquid, smearable mask, spray or gel.

10. A cosmetic comprising the fermentation product according to any one of claims 3 to 5 or the fermentation product prepared by the method according to any one of claims 6 to 8; Preferably, the fermentation product is 0.01-99.99% by mass in the cosmetic; Preferably, it further comprises cosmetically acceptable excipients and / or active ingredients; Preferably, the dosage form of the cosmetic is a cream, lotion, microemulsion, gel, aqueous solution, essence, sheet mask liquid, smearable mask, spray or gel.

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