Moisturizing cream prepared from lysate extract produced by fermentation of bacillus subtilis

Abstract

The invention discloses a moisturizer prepared from a lysate extract produced by fermentation of bacillus subtilis. The hydroxydecyl ubiquinone is identified in Y-3FLE, and the yield of the hydroxydecyl ubiquinone is 0.45 mu g / g after the hydroxydecyl ubiquinone is cultured for 4 days, so that the quinone substances are identified in the Halophilic bacteria in the Yuncheng Salt Lake for the first time. The Y-3FLE is prepared by adopting absolute ethyl alcohol as a solvent, the oxidation resistance of the Y-3FLE is investigated through an in-vitro experiment, and a result shows that the Y-3FLE is relatively high in reducing capacity and relatively high in oxidation resistance. A human body closed patch test result shows that the Y-3FLE has no obvious irritation to human skin. The method is applied to the moisturizer, and human body efficacy evaluation is carried out through a multi-probe skin tester. Results show that the moisturizer added with 1% of Y-3FLE has multiple remarkable effects of moisturizing, repairing, nourishing and the like.

Description

Technical Field

[0001] The present invention relates to a moisturizing cream prepared from an extract of lysate produced by fermentation of Oceanobacillus, belonging to the technical field of cosmetics. Background Art

[0002] Free radicals, as atoms or molecular groups with unpaired electrons generated during the body's metabolic process, play an important role in signal transduction under physiological conditions. However, with age, the body's ability to scavenge free radicals weakens, leading to the accumulation of free radicals in cells. When excessive free radicals accumulate in the body, they will attack cell structures, resulting in a series of oxidative stress reactions such as DNA damage, lipid peroxidation, and protein denaturation, accelerating skin aging. Therefore, preparing a cream that can effectively scavenge free radicals in skin cells is of great significance for delaying skin aging.

[0003] Currently, the scavenging of free radicals is mainly achieved in two ways: one is through the antioxidant enzyme system naturally present in the body, such as superoxide dismutase (SOD), etc.; the other is through exogenous intake of substances with antioxidant activity, such as vitamins, minerals, and certain natural products. However, with age and physical differences, the activity of antioxidant enzymes in the body will gradually decrease, and there are also certain limitations in the absorption and utilization efficiency of traditional antioxidants. Therefore, the development of new and efficient free radical scavengers has become a research hotspot.

[0004] Hydroxydecylubiquinone (also known as idebenone) is a potent antioxidant that can scavenge free radicals and inhibit lipid peroxidation, thereby reducing the damage of oxidative stress to skin cells. Hydroxydecylubiquinone has a wide range of applications in drugs and cosmetics. For example, in drugs, it is used to treat Parkinson's disease cognitive impairment, inhibit the growth and metastasis of liver cancer; in cosmetics, it can be used as a potent antioxidant in anti-aging cosmetics. Currently, the common synthetic method of hydroxydecylubiquinone is chemical synthesis through the free radical alkylation of coenzyme Q10, and there is no article reporting a microbial synthesis pathway.

[0005] According to the "Cosmetic Raw Material Catalog" (2021 edition), Bacillus extract belongs to cosmetic raw materials, but there are few reports on its application research in cosmetics. The genus Oceanobacillus has been isolated from various habitats, including deep-sea sediments and marine solar salterns. Therefore, it is very important to study the biological activity of its extract and its application in cosmetics. Summary of the Invention

[0006] In previous work, the inventor screened a strain of marine Bacillus Oceanobacillus sp. Y-3, taxonomically named Oceanobacillus sp. Y-3, which was deposited at the China Center for Type Culture Collection on November 05, 2024, with the deposit number CCTCC NO: M 20242434. Research has found that it has the ability to produce hydroxydodecylubiquinone and betaine, and can be used in the production research of cosmetics.

[0007] The present invention is achieved through the following technical solutions:

[0008] The first object of the present invention is to provide a moisturizing cream prepared from an ethanol extract of the lysate of the fermentation product of Bacillus oceanicus, which was deposited at the China Center for Type Culture Collection on November 05, 2024, with the deposit number CCTCC NO: M 20242434. By mass percentage, the moisturizing cream is composed of the following components: 0.1-5% ethanol extract of the lysate of the fermentation product of Bacillus oceanicus CCTCC NO: M 20242434, 5-30% oil, 2-10% emulsifier, 5-30% humectant, 0.1-2% thickener, 0.1-2% preservative, and 0-5.5% auxiliary components, with the balance being deionized water, and the total being 100%.

[0009] In one embodiment of the present invention, the ethanol extract of the lysate of the fermentation product of Bacillus oceanicus CCTCC NO: M 20242434 is prepared by the following method:

[0010] Centrifuge the fermentation broth of Bacillus oceanicus CCTCC NO: M 20242434 to obtain the bacterial cells. After washing, add ethanol to the bacterial cells, break them, collect the supernatant, concentrate under reduced pressure, and dry to obtain the ethanol extract of the lysate of the fermentation product of Bacillus oceanicus CCTCC NO: M 20242434.

[0011] In one embodiment of the present invention, the breaking is by ultrasonic breaking method or high-pressure cell breaking method.

[0012] In one embodiment of the present invention, when adding ethanol to the washed bacterial cells by volume percentage, the addition amount of the ethanol is added according to 1%-5% of the fermentation broth.

[0013] In one embodiment of the present invention, the oil is one or more of Limnanthes alba seed oil, olive oil, coconut oil, camellia seed oil, jojoba seed oil, shea butter, grape seed oil, beeswax, white oil, hydrogenated lecithin, squalane, caprylic / capric triglyceride, cetearyl alcohol, and stearic monoglyceride.

[0014] In one embodiment of the present invention, the emulsifier is one or more of fatty acid esters, sucrose fatty acid esters, sorbitan fatty acid esters, polysorbates, polyoxyethylene ethers, polyoxypropylene ethers, alkyl glucosides, glucose esters, stearyl esters, and alkyl ethers.

[0015] In one embodiment of the present invention, the humectant is one or more of butylene glycol, pentylene glycol, hexylene glycol, 1,3 - propanediol, caprylyl glycol, dipropylene glycol, sodium hyaluronate, hyaluronic acid, hydrolyzed hyaluronic acid, sodium hydrolyzed hyaluronate, sodium hyaluronate cross - polymer, acetylated sodium hyaluronate, ectoine, glycerol polyether - 26, hydrolyzed corn starch, carbohydrate isomers, polyquaternium - 51, sodium pyrrolidone carboxylate, zinc pyrrolidone carboxylate, sorbitol, trehalose, urea, β - glucan, yeast β - glucan, methyl gluceth - 20, dendrobium officinale stem extract, ceramide, dihydroxypropyl arginine hydrochloride, sodium alginate, N - acetylchitooligosaccharide, niacinamide, betaine, allantoin, panthenol, tremella polysaccharide, maltodextrin, algae extract, lysate of saccharomyces cerevisiae fermentation product, yeast polypeptides, yeast fermentation product, yeast lysate, yeast extract, filtrate of pichia pastoris fermentation lysate, bacillus fermentation product, lactobacillus fermentation product, lactobacillus fermentation lysate.

[0016] In one embodiment of the present invention, the thickener is one or more of ammonium acryloyldimethyltaurate / behenyl alcohol polyether - 25 methacrylate cross - polymer, hydroxypropyl starch phosphate, sodium acryloyldimethyltaurate, ammonium acryloyldimethyltaurate / VP copolymer, xanthan gum, carbomer, sodium chloride.

[0017] In one embodiment of the present invention, the preservative is one or more of phenoxyethanol, sodium benzoate, sorbic acid, potassium sorbate, chlorphenesin, methylparaben, ethylparaben, propylparaben.

[0018] In one embodiment of the present invention, the excipient component is active substance and / or essence.

[0019] The second object of the present invention is to provide a preparation method of the moisturizing cream, comprising the following steps:

[0020] (1) Mix the oil and the emulsifier, heat to 80 - 90 °C to fully dissolve it, and obtain phase A;

[0021] (2) Mix the humectant, the thickener and water to make it fully swell and homogeneous, and obtain phase B;

[0022] (3) At 80 - 90 °C, add phase A to phase B under stirring, and emulsify at 5000 - 10000 rpm for 1 - 10 min;

[0023] (4) Stir and cool to 40 - 60 °C, add auxiliary ingredients, stir evenly, and then cool to 20 - 30 °C to obtain the moisturizing cream.

[0024] Biological material preservation:

[0025] Bacillus oceanisediminis Y-3, taxonomically named Oceanobacillus sp. Y-3, was deposited at the China Center for Type Culture Collection on November 05, 2024, with the deposit number CCTCC NO: M 20242434, and the deposit address is Wuhan University, Wuhan, China.

[0026] Advantages of the present invention:

[0027] Using UPLC-Q-TOF / MS technology, ubiquinol decyl ether was identified in the ferment lysate extract (FLE) of Y-3 fermentation products, and its yield was 0.45 μg / g at 4 days of culture. This is the first time to identify quinone substances in halophilic bacteria from Yuncheng Salt Lake. The present invention uses absolute ethanol as a solvent to prepare Y-3 FLE, and its scavenging ability for ABTS + ·, DPPH· and iron reduction ability were investigated through in vitro experiments. The results showed that Y-3 FLE had strong reducing ability and scavenging effects on both ABTS + · and DPPH·, showing strong antioxidant performance. The results of the human closed patch test showed that Y-3 FLE (1%, w / w) had no obvious irritation to human skin. When it was applied to the moisturizing cream, the human efficacy was evaluated by a multi-probe skin tester. The results showed that the moisturizing cream added with 1% (w / w) Y-3 FLE had significant multiple effects such as moisturizing, repairing and nourishing. Description of the drawings

[0028] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0029] Figure 1 Total ion chromatograms of ubiquinol decyl ether standard (a) and Y-3 FLE (b);

[0030] Figure 2 First-level (a) and second-level (b) mass spectra of ubiquinol decyl ether standard;

[0031] Figure 3It is the first-level (a) and second-level (b) mass spectrometry diagrams of the substance eluting at 4.62 min in Y-3FLE;

[0032] Figure 4 It is the standard curve graph of ubiquinol decyl;

[0033] Figure 5 It is the ability of Y-3FLE and vitamin C to scavenge ABTS + ·;

[0034] Figure 6 It is the ability of Y-3FLE and vitamin C to scavenge DPPH·;

[0035] Figure 7 It is the iron-reducing ability of Y-3FLE and vitamin C;

[0036] Figure 8 It is the change in the water content of the stratum corneum of the skin during 4 h after using emollient cream T1 (a) and emollient cream T2 (b);

[0037] Figure 9 It is the change in the transdermal water loss during 4 h after using emollient cream T1 (a) and emollient cream T2 (b);

[0038] Figure 10 It is the change in gloss during 4 h after using emollient cream T1 (a) and emollient cream T2 (b);

[0039] Figure 11 It is the change in skin color during 4 h after using emollient cream T1 (a) and emollient cream T2 (b). Specific embodiments

[0040] The following combines specific examples to further elaborate on this invention patent. These implementation cases are only used to illustrate the present invention and not to limit the scope of the present invention. In addition, after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

[0041] Source of raw materials:

[0042] Experimental materials and reagents:

[0043] Ubiquinol decyl standard product, purchased from Shanghai Yuanye Bio-Technology Co., Ltd.;

[0044] Glycerol monostearate, purchased from Shanghai Macklin Biochemical Co., Ltd.;

[0045] Acrylamide dimethyltaurine / VP copolymer, purchased from Clariant Chemicals (China) Co., Ltd.;

[0046] Stearyl alcohol, beeswax, Tween-80, ethylparaben, were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0047] In the following examples, unless otherwise specified, the solutions mentioned are all water-based solvents.

[0048] The crude salt involved below is the large salt from Yuncheng Salt Lake, sourced from Yuncheng Salt Lake.

[0049] The medium formulations in the following examples are as follows:

[0050] 1. Crude salt liquid medium: 50 g of crude salt, 20 g of MgSO4·7H2O, 2.0 g of KCl, 3.0 g of sodium citrate, 0.2 g of anhydrous calcium chloride, 2.0 g of yeast extract, 10.0 g of peptone, add distilled water to make up to 1 L, and adjust the pH to 7.0 - 7.2.

[0051] 2. Crude salt solid medium: 50 g of crude salt, 20 g of MgSO4·7H2O, 2.0 g of KCl, 3.0 g of sodium citrate, 0.2 g of anhydrous calcium chloride, 2.0 g of yeast extract, 10.0 g of peptone, add distilled water to make up to 1 L, adjust the pH to 7.0 - 7.2, and add 15.0 g of agar. Autoclave at 1×10 5 Pa, 121 °C for 30 min. After cooling the medium to 50 - 60 °C, pour it into petri dishes, about 15 - 20 mL of the medium is poured into each petri dish, and store it for later use after cooling and solidifying.

[0052] Experimental instruments and equipment:

[0053] Multi-probe skin test system, CK Company, Germany, product specification MAP 10; Intradermal spectrophotometric imager, Delfin Company, Finland, product specification SIAscope V.

[0054] Testing methods:

[0055] 1. ABTS + · Scavenging ability determination:

[0056] (1) Preparation of solutions

[0057] Potassium persulfate solution: Weigh 6.6 mg of potassium persulfate and dissolve it in 10 mL of distilled water to obtain a potassium persulfate solution with a concentration of 2.45 mmol / L;

[0058] ABTS working solution: Weigh 7.7 mg of ABTS and dissolve it in 2 mL of potassium persulfate solution. After reacting in the dark for 16 h, dilute it to obtain a 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) working solution with an absorbance of 0.7 at 734 nm.

[0059] (2) Prepare ethanol solutions with concentrations of 20 mg / mL, 10 mg / mL, 5.0 mg / mL, 2.5 mg / mL, 1.25 mg / mL, 0.63 mg / mL, and 0.31 mg / mL respectively using absolute ethanol from the ethanol extract of the lysate of the fermentation product of Bacillus oceanisediminis CCTCC NO: M 20242434.

[0060] (3) Pipette 10 μL of the ethanol solutions of the fermentation product lysate with different concentrations obtained in step (2) into a 96-well plate respectively, then add 190 μL of the ABTS working solution obtained in step (1) respectively. After reacting in the dark for 15 min, measure the absorbance A at 734 nm. 样品 。

[0061] Control group: The procedure is the same as above, except that absolute ethanol with an equal volume is used instead of the sample solution (the sample solution refers to the above-mentioned ethanol solution of the bacterial cell lysate); measure the absorbance A at 734 nm. 对照 。

[0062] Blank group: The procedure is the same as above, except that absolute ethanol is used instead of the ABTS working solution. The specific steps are the same as above, and the absorbance A is measured at 734 nm. 空白 。

[0063] Each test group needs to be repeated 3 times, and the results are averaged to calculate the ABTS + · scavenging rate.

[0064] ABTS + The calculation formula for the · scavenging rate is as follows:

[0065] ABTS + · scavenging rate / % = [1 - (A 样品 - A 空白 ) / A 对照 )] × 100%.

[0066] (4) Use vitamin C solutions with concentrations of 4.0 mg / mL, 2.0 mg / mL, 0.80 mg / mL, 0.12 mg / mL, 0.10 mg / mL, 0.08 mg / mL, 0.05 mg / mL, and 0.025 mg / mL as positive controls. Specifically: Pipette 10 μL of vitamin C solutions with different concentrations into a 96-well plate respectively, then add 190 μL of the ABTS working solution obtained in step (1) respectively. After reacting in the dark for 15 min, measure the absorbance A at 734 nm. 样品-1 。

[0067] Control group: The specific method is the same as above, except that ultrapure water with an equal volume is used instead of the vitamin C solution; measure the absorbance A at 734 nm. 对照-1 。

[0068] Blank group: The specific method is the same as above, except that an equal volume of ultrapure water is used to replace the DPPH solution; measure the absorbance A at 734 nm. 空白-1

[0069] ABTS + The calculation formula for the scavenging rate is as follows:

[0070] ABTS + Scavenging rate / % = [1 - (A 样品-1 - A 空白-1 ) / A 对照-1 )] × 100%.

[0071] 2. Determination of the scavenging ability of DPPH·:

[0072] (1) Prepare ethanol solutions with mass concentrations of 24 mg / mL, 12 mg / mL, 6.0 mg / mL, 3.0 mg / mL, 1.5 mg / mL, 0.75 mg / mL, 0.38 mg / mL, and 0.19 mg / mL respectively with anhydrous ethanol using the ethanol extract of the lysate of the fermentation product of Bacillus oceanisediminis CCTCC NO: M 20242434.

[0073] (2) Prepare a 2,2-diphenyl-1-picrylhydrazyl (DPPH) solution with a concentration of 0.3 mmol / L using anhydrous ethanol.

[0074] (3) Set up experimental groups, control groups, and blank groups respectively. The specific groups are as follows:

[0075] Experimental group: Pipette 100 μL of the ethanol solution of the lysate of the fermentation product obtained in step (1) into a 96-well plate respectively, and then add 100 μL of the DPPH-ethanol solution obtained in step (2) respectively. After mixing evenly and reacting in the dark for 30 min, measure the absorbance A of the mixture at 517 nm. 样品

[0076] Control group: The same as the experimental group, except that an equal volume of anhydrous ethanol is used to replace the sample solution (the sample solution refers to the above-mentioned ethanol solution of the cell lysate); the specific steps are the same as above, and the absorbance A is measured at 517 nm. 对照

[0077] Blank group: The same as the experimental group, except that an equal volume of anhydrous ethanol is used to replace the DPPH solution, and the specific steps are the same as above. The absorbance A is measured at 517 nm. 空白

[0078] Each group needs to be repeated 3 times, and the results are averaged to calculate the DPPH radical scavenging rate.

[0079] The calculation formula for the DPPH· scavenging rate is as follows:​​​​

[0080] DPPH· scavenging rate / % = [1 - (A 样品 - A 空白 ) / A 对照 )] × 100%

[0081] (4) Using vitamin C solutions at 0.08 mg / mL, 0.04 mg / mL, 0.02 mg / mL, and 0.01 mg / mL as positive controls. Specifically: Pipette 100 μL of vitamin C solutions at different concentrations into a 96-well plate respectively, then add 100 μL of the DPPH-ethanol solution obtained in step (2) respectively, mix well and react in the dark for 30 min, and then measure the absorbance A of the mixture at 517 nm 样品-1 .

[0082] Control group: The specific method is the same as above, except that an equal volume of ultrapure water is used instead of the vitamin C solution; measure the absorbance A at 517 nm 对照-1 .

[0083] Blank group: The specific method is the same as above, except that an equal volume of ultrapure water is used instead of the DPPH solution and measure the absorbance A at 517 nm 空白-1 .

[0084] The calculation formula for DPPH· scavenging rate is as follows:

[0085] DPPH· scavenging rate / % = [1 - (A 样品-1 - A 空白-1 ) / A 对照-1 )] × 100%.

[0086] 3. Determination of ferric reducing ability (FRAP):

[0087] (1) Preparation of TPTZ working solution: Mix 25 mL of acetate buffer (pH = 3.6), 2.5 mL of 10 mmol / L 2,4,6-tripyridyltriazine (TPTZ) solution, and 2.5 mL of 20 mmol / L ferric chloride solution to prepare the TPTZ working solution

[0088] (2) Prepare ethanol solutions of 16 mg / mL, 8.0 mg / mL, 4.0 mg / mL, 3.0 mg / mL, 2.0 mg / mL, 1.0 mg / mL, 0.50 mg / mL, and 0.25 mg / mL of the ethanol extract of the cell lysate of the fermentation product of Bacillus oceanisediminis CCTCC NO: M 20242434 with absolute ethanol

[0089] (3) Add 10 μL of the lysate ethanol solution of the fermentation products with different concentrations obtained in the above step (2) and 190 μL of the TPTZ working solution obtained in step (1) into a 96-well plate in sequence. Shake for 10 s using a microplate reader, incubate at 37 °C for 10 min, and measure the absorbance at a wavelength of 593 nm, which is A 样品 .

[0090] Control group: The specific steps are the same as above. The difference is that acetic acid buffer solution is used instead of the TPTZ working solution, and the absorbance is measured at a wavelength of 593 nm, which is A 对照 .

[0091] Blank group: The specific steps are the same as above. The difference is that absolute ethanol is used instead of the sample solution (the sample solution refers to the above-mentioned lysate ethanol solution of the bacterial cells). The specific steps are the same as above, and the absorbance A is measured at 593 nm 空白 .

[0092] Each experimental group needs to be repeated 3 times, and the results are averaged.

[0093] Prepare a ferrous sulfate standard solution with a concentration in the range of 0.1 - 1 mmol / L, and measure its absorbance under the same conditions. Using the ferrous sulfate concentration as the abscissa and the absorbance as the ordinate, draw a standard curve, and the regression equation is: y = 0.675x + 0.019 (R 2 = 0.999). Calculate the iron ion reducing ability of the sample according to the regression equation, expressed as the equivalent concentration of ferrous ions (FRAP value), with the unit of mmol / L.

[0094] y = A 样品 - A 对照 - A 空白 , substitute it into the equation to obtain the x value, that is, the FRAP value.

[0095] (4) Use vitamin C solutions with concentrations of 11 mg / mL, 5.5 mg / mL, 1.38 mg / mL, 0.69 mg / mL, 0.34 mg / mL, 0.02 mg / mL, and 0.01 mg / mL as positive controls. Specifically: Add 10 μL of the above vitamin C solution and 190 μL of the TPTZ working solution obtained in step (1) into a 96-well plate in sequence. Shake for 10 s using a microplate reader, incubate at 37 °C for 10 min, and measure the absorbance at a wavelength of 593 nm, which is A 样品-1 .

[0096] Control group: The specific steps are the same as above. The difference is that acetic acid buffer solution is used instead of the TPTZ working solution, and the absorbance is measured at a wavelength of 593 nm, which is A 对照-1 .

[0097] Blank group: The specific steps are the same as above. The difference is that an equal volume of ultrapure water is used to replace the vitamin C solution. The specific steps are the same as above, and the absorbance A is measured at 593 nm. 空白-1 .

[0098] Each experimental group needs to be repeated 3 times, and the results are averaged.

[0099] Prepare ferrous sulfate standard solution with a concentration range of 0.1 - 1 mmol / L, and measure its absorbance under the same conditions. Using the ferrous sulfate concentration as the abscissa and the absorbance as the ordinate, draw a standard curve, and the regression equation is: y = 0.675x + 0.019 (R 2 = 0.999). Calculate the iron ion reducing ability of the sample according to the regression equation, expressed as the equivalent concentration of ferrous ions (FRAP value), with the unit of mmol / L.

[0100] y = A 样品-1 - A 对照-1 - A 空白-1 , substitute it into the equation to get the x value, which is the FRAP value.

[0101] Use UPLC-Q-TOF / MS technology to qualitatively and quantitatively analyze ubiquinol decyl in Y-3FLE:

[0102] Sample pretreatment: Take the fermentation broth of the strain cultured for 4 days under the conditions of salinity 2% (w / v), 30 °C and pH 7.0 respectively, prepare FLE, add 1 mL of methanol and vortex to redissolve, centrifuge at 8500 rpm, and take the supernatant to obtain the sample to be tested.

[0103] Liquid chromatography conditions: The chromatographic column is ACQUITY UPLC BEH C 18 Column (2.1 mm × 100 mm, 1.7 μm); Mobile phase: A is 0.1% formic acid - aqueous solution, B is acetonitrile; The gradient elution program is shown in Table 1, the flow rate is 0.3 mL / min; Diode array (PDA) detector; Column temperature 45 °C; Injection volume 5.0 μL.

[0104] Table 1 Gradient elution program

[0105] Time (min) Mobile phase A (%) Mobile phase B (%) 0.00 100 0 7.00 70 30 8.00 20 80 9.00 0 100 10.00 100 0

[0106] Mass spectrometry conditions: Positive ion mode, electrospray ionization source, multiple reaction monitoring mode, ion source temperature 100 °C, capillary voltage 3.5 kV, cone voltage 20 V, desolvation gas temperature 400 °C, IMS gas flow rate 24 mL / min, collision energy 6 / 20 eV.

[0107] Drawing of the standard curve: Using anhydrous methanol as the solvent, prepare a 1.0 mg / mL ubiquinone decyl standard solution. Dilute this standard solution successively into standard working solutions with concentrations of 12.5, 25.0, 50.0, 62.5, and 125 μg / L, and perform injection analysis respectively. Using the ubiquinone decyl concentration as the abscissa and the peak area as the ordinate, draw the standard curve to obtain the regression equation.

[0108] The following will elaborate on the technical solution of the present invention in combination with specific embodiments. In the following embodiments, unless otherwise specified, the reagents, materials, and equipment used can be obtained through commercial channels, or prepared by conventional methods, or commonly used in this industry.

[0109] Example 1:

[0110] Fresh culture medium: 20 g of coarse salt, 20 g of MgSO4·7H2O, 2.0 g of KCl, 3.0 g of sodium citrate, 0.2 g of anhydrous calcium chloride, 2.0 g of yeast extract, 10.0 g of peptone. Add distilled water to make up to 1 L and adjust the pH to 7.2.

[0111] (1) Take samples from the cryopreservation tube of the obtained Oceanobacillus sp. Y-3 strain, inoculate it into the coarse salt liquid medium, and place it in a shaking incubator at 30 °C for 3 days. When the culture medium becomes a turbid and opaque red liquid, pick single colonies and continue to inoculate them on the coarse salt solid medium plate for 4 days to activate the strain. Pick single colonies and inoculate them into a conical flask containing 100 mL of coarse salt liquid medium, and culture them in a shaking incubator at 30 °C for 3 days to detect OD 600 up to 0.5, which can be used as the coarse salt seed liquid. Add the cultured coarse salt seed liquid to a sterile conical flask containing 300 mL of the above fresh culture medium at a ratio of 10% by volume, and culture it in a shaking incubator at 30 °C for more than 4 days until OD 600 is 0.4 - 0.5 to obtain the cultured fermentation broth.

[0112] (2) Centrifuge the fermentation broth obtained in step (1) at 4 °C and 8000 rpm for 10 min, remove the supernatant, wash the cell precipitate with PBS, centrifuge again to remove the liquid phase, wash it twice with PBS, and then resuspend the cell precipitate with 1% anhydrous ethanol by the volume of the liquid culture medium to obtain the cell liquid. Break the cell wall of the cell liquid in an ultrasonic disruptor for 3 cycles, centrifuge and collect the supernatant to obtain the ethanol extract of the cell lysate, which is Y-3FLE.

[0113] Qualitative analysis of ubiquinone decyl: Use UPLC-Q-TOF / MS technology to analyze the ubiquinone decyl standard (5 ppm) and the Y-3FLE sample, and the total ion chromatograms obtained are respectively as Figure 1 shown in a and b in the figure. FromFigure 1 As can be seen from a, the hydroxydodecylubiquinone standard showed a peak at 4.62 min. Its first-order mass spectrum (TOF MS) and second-order mass spectrum (TOF MS / MS) are as Figure 2 shown. The maximum number of protons was m / z 339, which was [M+H] + ion peak, and the fragment peaks were +321, +307, +289, +197. The first-order mass spectrum (TOF MS) and second-order mass spectrum (TOF MS / MS) of the substance with a peak at 4.62 min in the Y-3FLE sample are as Figure 3 shown. The maximum number of protons was m / z 339, which was [M+H] + ion peak, and the fragment peaks were +321, +307, +289, +197. By comparing Figure 1 , 2 with Figure 3 the control, it can be seen that the substance with a peak at 4.62 min in the sample had the same retention time as the hydroxydodecylubiquinone standard, and the [M+H] + ion peak was the same as that of the hydroxydodecylubiquinone standard. Therefore, it was determined that the substance with a peak at 4.62 min was hydroxydodecylubiquinone, and Oceanobacillus sp. Y-3FLE contained hydroxydodecylubiquinone.

[0114] Quantitative analysis of hydroxydodecylubiquinone: The standard curve of hydroxydodecylubiquinone is as Figure 4 shown. In the range of 12.5 - 125 μg / L, the peak area had a good linear relationship with the mass concentration: Y = 4.517X - 4.258 (R 2 = 0.999). The hydroxydodecylubiquinone in strain Y-3FLE was determined by UPLC-Q-TOF / MS. When the strain was cultured for 4 days, the content of hydroxydodecylubiquinone was 81.4 μg / L. Since the bacterial liquid used was 250 mL, the converted content was 0.33 μg / L. Since the mass of the freeze-dried Y-3 cells was 0.1809 g, the production capacity of hydroxydodecylubiquinone by Oceanobacillus sp. Y-3 strain was calculated to be 0.45 μg / g.

[0115] Example 2: Antioxidant performance test of Y-3FLE

[0116] (1) ABTS + · scavenging rate determination

[0117] The scavenging effect of Y-3FLE on ABTS + · is as Figure 5 shown. It can be seen from the figure that both Y-3FLE and vitamin C had good scavenging effects on ABTS + ·, and with the increase of concentration, the scavenging effect on ABTS +The scavenging effect of · also gradually increases. Both increase with the increase in concentration. The scavenging rate of Y-3FLE reaches the maximum at 20 mg / mL, which is 99.81%. Its IC 50 value is shown in Table 2. The IC50 value of Y-3FLE is 4.442 mg / mL, and the scavenging effect is lower than that of vitamin C.

[0118] Table 2 IC + values of Y-3FLE and vitamin C for scavenging ABTS 50 ·

[0119]

[0120] (2) Determination of DPPH· scavenging rate

[0121] The results of the scavenging effect of Y-3FLE on DPPH· are shown as follows Figure 6 . As can be seen from the figure, both Y-3FLE and vitamin C have a scavenging effect on DPPH·, and the scavenging rate increases with the increase in the sample concentration. The scavenging rate of Y-3FLE reaches the maximum (91.26%) at 12.00 mg / mL. As can be seen from Table 3, the IC 50 value of Y-3FLE is 2.64 mg / mL, and the scavenging effect is lower than that of vitamin C.

[0122] Table 3 IC 50 values of Y-3FLE and vitamin C for scavenging DPPH·

[0123]

[0124] (3) Determination of reducing power

[0125] The results of the determination of the reducing power of Y-3FLE are shown as follows Figure 7 . It can be seen that both Y-3FLE and vitamin C have good reducing power, and with the increase in concentration, the reducing power gradually increases. When the concentration of Y-3FLE is 16.25 mg / mL, the FRAP value is 4.29 mmol / L, which is close to the highest FRAP (4.56 mmol / L) value of vitamin C.

[0126] Generally speaking, the FLE of Oceanobacillus sp. Y-3 has strong abilities to scavenge ABTS + ·, DPPH· and has strong iron-reducing ability. It has strong antioxidant ability and antioxidant potential.

[0127] Example 3: Safety evaluation of Y-3FLE

[0128] Using white petrolatum as a solvent, prepare a 1% (w / w) Y-3FLE solution. Refer to the "Technical Specifications for Cosmetics Safety" (2015 Edition) to conduct a human closed patch test on the sample. Select 30 volunteers who meet the subject selection criteria, weigh 0.020 g - 0.025 g of the sample into the small chamber of the patch tester, and use white petrolatum as the control hole.

[0129] The results of the human skin patch test of Y-3FLE (1%, w / w) are shown in Table 4. The results show that none of the 30 subjects had symptoms such as erythema, edematous erythema, significant swelling or papules, indicating that Y-3FLE (1%, w / w) has no obvious irritation to human skin.

[0130] Table 4 Results of the human skin patch test of Y-3FLE (1%, w / w)

[0131]

[0132] Example 4: Preparation of moisturizing cream

[0133] Prepare the W / O type moisturizing cream T2 according to the formula in Table 5. Preparation method: Accurately weigh the oil phase (octadecanol, beeswax, white oil, glycerol monostearate, Tween-80, ethylparaben, Y-3FLE) and the water phase (glycerol, acrylamidodimethyltaurine / VP copolymer and water). Place them in separate dry beakers, set the water bath temperature to 80 °C, heat the oil phase and the water phase in the water bath, and stir until dissolved and homogeneous. When the water bath temperature rises to the set temperature, add the oil phase to the water phase under homogenization conditions, emulsify at 8000 rpm for 5 min, and cool to room temperature and bottle. Prepare another blank moisturizing cream T1 without Y-3FLE as a blank control according to the above steps.

[0134] Table 5 Formulation for the preparation of moisturizing cream

[0135]

[0136] Refer to the "T / CAB 0152-2022 Test Methods for Cosmetic Efficacy" to conduct a human efficacy evaluation on the moisturizing cream T1 (blank group) and T2 (sample group) using a multi-probe skin tester. The specific test method is as follows:

[0137] (1) Product usage method

[0138] Weigh 0.4 g of the moisturizing cream T1 and the moisturizing cream T2 respectively, and apply them evenly on the left and right faces.

[0139] (2) Test environment

[0140] The test environment is at a temperature of 22.0°C ± 1.0°C and a humidity of 50% ± 10%. Seventeen volunteers were selected according to the subject inclusion criteria. Before the test, the volunteers needed to sit quietly in the test environment for 30 minutes.

[0141] (3) Test items

[0142] ① Water content of the stratum corneum

[0143] Since there are significant differences in the dielectric constants between water and other substances, the water content test probe can reflect the water content by measuring the change in the capacitance of the skin stratum corneum. In this study, a moisture tester was used to measure the water content of the stratum corneum on the right cheek, and the average value was taken after measuring 3 times. A before-and-after comparison was adopted to evaluate the moisturizing effect of the product.

[0144] ② Trans-epidermal water loss

[0145] Based on Fick's diffusion law, a trans-epidermal water loss instrument for the skin reflects the trans-epidermal water loss by calculating the amount of water diffused per unit time and per unit area. In this study, the trans-epidermal water loss on the right face was measured, and the average value was taken after measuring 20 times. A before-and-after comparison was adopted to evaluate the repair effect of the product.

[0146] ③ Glossiness

[0147] The glossiness can be reflected by measuring the reflection of light irradiated on the skin surface. In this study, a skin glossiness test probe was used to measure the glossiness of the left and right faces, and the average value was taken after measuring 3 times. A before-and-after comparison was adopted to evaluate the nourishing effect (enhancing the skin glossiness) of the product.

[0148] ④ Skin color

[0149] The main factors determining skin color include the content and distribution of melanin, the state of hemoglobin, the deposition of carotenoids, and the characteristics of the stratum corneum, etc. The tristimulus values refer to the amounts of stimulation of the three theoretical primary colors (red, green, and blue) required to achieve visual equivalence with the test light in a color matching experiment. In this study, a skin color test probe was used to measure the color (ITA° value) of the left and right faces, and the average value was taken after measuring 3 times for each part to evaluate the nourishing and certain whitening (enhancing the skin ITA° value) effects of the product.

[0150] (4) Test period

[0151] The test period is short-term, 4 hours. Data of the volunteers before use and at 5 minutes, 2 hours, and 4 hours after use were collected. The specific test item arrangements are shown in Table 6.

[0152] Table 6 Arrangements of moisturizing cream test items

[0153]

[0154] Note: "○" indicates implementation.

[0155] The human efficacy evaluation test results of skin cream T1 (blank group) and T2 (sample group) are as follows:

[0156] (1) Stratum corneum water content

[0157] The changes in the stratum corneum water content during the 4 hours after applying skin cream T1 and T2 are as Figure 8 shown. After applying T1, compared with the initial value, the stratum corneum water content of the skin slightly increased at 5 minutes, but slightly decreased at 2 hours and 4 hours, and there were no significant differences in the data, indicating that skin cream T1 has no moisturizing effect; while after applying T2 at 5 minutes, 2 hours and 4 hours, the water content of the stratum corneum in the test area was significantly increased compared with the initial value, and the improvement rates were 15.14%, 11.43% and 10.87% respectively. Among them, the data at 5 minutes had a significant difference (*p<0.05), indicating that skin cream T2 has a significant moisturizing effect.

[0158] (2) Transdermal water loss

[0159] The changes in transdermal water loss during the 4 hours after applying skin cream T1 and T2 are as Figure 9 shown. After applying T1, at 5 minutes, the transdermal water loss value increased significantly compared with the initial value, and showed a downward trend at 2 hours and 4 hours, but there were no significant differences in the data, indicating that skin cream T1 has no repair effect. On the contrary, it has a certain damage to the skin stratum corneum barrier function at 5 minutes of use; while after applying T2 at 5 minutes, 2 hours and 4 hours, the transdermal water loss value in the test area decreased compared with the initial value, and the improvement rates were -6.08%, -6.19% and -18.06% respectively. Among them, the data at 4 hours had a significant difference (*p<0.05), indicating that skin cream T2 has a significant repair effect.

[0160] (3) Glossiness

[0161] The changes in glossiness during the 4 hours after applying skin cream T1 and T2 are as Figure 10 shown. After applying T1 at 5 minutes, 2 hours and 4 hours, the skin glossiness in the test area was significantly increased compared with the initial value, and the data at 5 minutes and 2 hours both had significant differences (**p<0.01, *p<0.05), indicating that skin cream T1 has a significant effect on improving glossiness; while after applying T2 at 5 minutes, 2 hours and 4 hours, the glossiness in the test area increased rapidly compared with the initial value, and the improvement rates were 32.83%, 33.97% and 24.98% respectively, and the data all had significant differences (***p<0.001, ***p<0.001, ***p<0.001), indicating that skin cream T2 can significantly improve skin glossiness and has a nourishing effect.

[0162] Although the emollient cream T1 itself has a certain effect of brightening the skin, which may be caused by the oil-phase components in the formula, the emollient cream T2 has a more significant brightening effect than emollient cream T1 at any time in the measurement range, and the brightening effect has a certain persistence, indicating that Y-3FLE is playing a role. Since the emollient cream T2 has significant moisturizing and repair effects, it may cause the water-rich stratum corneum to form regular reflections, thereby producing a bright luster and effectively improving the skin gloss.

[0163] (4) Skin color

[0164] The changes in skin color (ITA° value) during the 4 h after using emollient creams T1 and T2 are as Figure 11 shown. At 5 min and 2 h after using T1, the ITA° values of the skin in the test area decreased slightly compared with the initial values, and increased slightly at 4 h, but the data had no significant differences, indicating that emollient cream T1 had no effect on increasing the ITA° value; while at 5 min, 2 h, and 4 h after using T2, the skin color in the test area was significantly improved compared with the initial value, and the improvement rates were 4.55%, 7.75%, and 9.46% respectively. Among them, the data at 2 h and 4 h had significant differences (*p < 0.05, *p < 0.05), indicating that emollient cream T2 could significantly increase the ITA° value of the skin, lighten the skin color, and had nourishing and certain whitening effects.

[0165] In summary, emollient cream T2 can significantly increase the water content of the skin stratum corneum, significantly reduce transepidermal water loss, significantly improve skin gloss, and significantly increase the ITA° value of the skin. Therefore, it has multiple effects such as moisturizing, repairing, and nourishing.

[0166] Comparative Example 1: Extraction of Y-3 extracellular crude polysaccharide and determination of its free radical scavenging ability

[0167] (1) Extraction method of Y-3 extracellular crude polysaccharide

[0168] Take 1 mL of the fermentation broth of the strain cultured under the conditions of 2% (w / v) salinity, 30 °C, and pH 7.0 for 4 d, dilute it 10 times with distilled water, centrifuge it at 4 °C and 8000 rpm for 10 min, discard the thallus, and obtain the supernatant. Take 1 mL of the supernatant, add 3 mL of absolute ethanol, mix well, let it stand for 10 h, then centrifuge it at 4 °C and 8000 rpm for 10 min, discard the supernatant, wash the precipitate with 75% ethanol until the effluent shows a negative reaction in the reducing sugar reaction, and then vacuum dry it (40 °C) to obtain the extracellular crude polysaccharide.

[0169] (2) Determination of the ABTS free radical scavenging ability of Y-3 extracellular crude polysaccharide

[0170] Prepare a 7 mg / mL extracellular crude polysaccharide extract and measure its ABTS+ · The clearance rate was 10.77%.

[0171] Comparative Example 2: Preparation of Y-3FLE (aqueous extract) and determination of its free radical scavenging ability

[0172] (1) Preparation method of Y-3FLE (aqueous extract)

[0173] Take 100 mL of the fermentation broth of the strain cultured under the conditions of 2% (w / v) salinity, 30 °C and pH 7.0 for 4 days, centrifuge at 4 °C and 8000 rpm for 10 min, pour off the upper culture broth, wash the cells twice with PBS, after centrifuging and pouring off the supernatant, resuspend the cells with 1 mL of PBS, perform three cycles of ultrasonic disruption (650 W, 10 min), centrifuge again to collect the upper clear liquid, and freeze-dry the filtrate to obtain FLE (aqueous extract).

[0174] (2) Determination of the free radical scavenging ability of Y-3FLE (aqueous extract)

[0175] Prepare FLE (aqueous extract) at a concentration of 10 mg / mL and determine its ABTS + · The clearance rate was 98.84%, and its clearance rate for DPPH· was 22.46%.

[0176] Table 7 lists the comparison of the free radical scavenging abilities of different extracts of Oceanobacillus sp. Y-3. It can be seen from Table 7 that the extracellular crude polysaccharide of Y-3 has a certain scavenging effect on ABTS free radicals; Y-3FLE (aqueous extract) has a relatively high scavenging effect on ABTS free radicals, but a relatively low scavenging effect on DPPH free radicals; while Y-3FLE (ethanol extract) has a relatively high scavenging effect on both ABTS and DPPH free radicals.

[0177] Table 7 Comparison of the free radical scavenging abilities of extracts of Oceanobacillus sp. Y-3 strain

[0178] Extract of strain Y-3 ABTS radical scavenging rate / % DPPH radical scavenging rate / % Crude extracellular polysaccharide of Y-3 7.0 mg / mL, 10.77% / Y-3FLE (water extract) 10 mg / mL, 98.84% 10 mg / mL, 22.46% Y-3FLE (ethanol extract) 10 mg / mL, 91.54% 6.0 mg / mL, 89.88%

[0179] The examples provided above are not intended to limit the scope covered by the present invention, nor are the described steps intended to limit the order of their execution. Obvious improvements made by those skilled in the art in combination with the existing common general knowledge also fall within the protection scope defined by the claims of the present invention.

Claims

1. A skin cream prepared from an ethanol extract of the lysate of the fermentation product of Bacillus oceanisediminis, characterized in that, The Bacillus oceanisediminis was deposited at the China Center for Type Culture Collection on November 05, 2024, with the deposit number CCTCC NO: M 20242434; calculated by mass percentage, the moisturizing cream is composed of the following components: 0.1-5% ethanol extract of Bacillus oceanisediminis fermentation product lysate, 5-30% oil, 2-10% emulsifier, 5-30% humectant, 0.1-2% thickener, 0.1-2% preservative, and 0-5.5% auxiliary components, with the balance being water, and the total being 100%.

2. The moisturizing cream according to claim 1, characterized in that, The ethanol extract of Bacillus oceanisediminis fermentation product lysate is prepared by the following method: Centrifuge the Bacillus oceanisediminis fermentation broth to obtain thalli, add ethanol to the washed thalli, break them, collect the supernatant, concentrate under reduced pressure, and dry to obtain the ethanol extract of Bacillus oceanisediminis fermentation product lysate.

3. The moisturizing cream according to claim 2, characterized in that, The breaking is by ultrasonic breaking method or high-pressure cell breaking method.

4. The moisturizing cream according to claim 1, characterized in that, The oil is one or more of Limnanthes alba seed oil, olive oil, coconut oil, Camellia oleifera seed oil, Simmondsia chinensis seed oil, shea butter, grape seed oil, beeswax, white oil, hydrogenated lecithin, squalane, caprylic / capric triglyceride, cetearyl alcohol, and stearic monoglyceride.

5. The moisturizing cream according to claim 1, characterized in that, The emulsifier is one or several of fatty acid esters, sucrose fatty acid esters, sorbitan fatty acid esters, polysorbates, polyoxyethylene ethers, polyoxypropylene ethers, alkyl glucosides, glucose esters, stearyl esters, and alkyl ethers.

6. The moisturizing cream according to claim 1, characterized in that, The humectant is one or several of butanediol, pentanediol, hexanediol, 1,3-propanediol, octyldecanol, dipropylene glycol, sodium hyaluronate, hyaluronic acid, hydrolyzed hyaluronic acid, sodium hydrolyzed hyaluronate, sodium hyaluronate cross-linked polymer, acetylated sodium hyaluronate, ectoine, glycerol polyether-26, hydrolyzed corn starch, carbohydrate isomers, polyquaternium-51, sodium pyrrolidone carboxylate, zinc pyrrolidone carboxylate, sorbitol, trehalose, urea, β-glucan, yeast β-glucan, methyl glucitol polyether-20, Dendrobium nobile stem extract, ceramide, dihydroxypropyl arginine hydrochloride, sodium alginate, chitosan oligosaccharide, niacinamide, betaine, allantoin, panthenol, Tremella fuciformis polysaccharide, maltodextrin, algal extract, lysate of Saccharomyces cerevisiae fermentation product, yeast polypeptides, yeast fermentation product, yeast lysate, yeast extract, filtrate of Pichia pastoris fermentation lysate, Bacillus fermentation product, Lactobacillus fermentation product, and Lactobacillus fermentation lysate.

7. The moisturizing cream according to claim 1, characterized in that, The thickener is one or more of ammonium acryloyldimethyltaurate / behenyl alcohol polyether-25 methacrylate cross-linked polymer, hydroxypropyl starch phosphate, sodium acryloyldimethyltaurate, ammonium acryloyldimethyltaurate / VP copolymer, xanthan gum, carbomer, and sodium chloride.

8. The moisturizing cream according to claim 1, characterized in that, The preservative is one or more of phenoxyethanol, sodium benzoate, sorbic acid, potassium sorbate, chlorphenesin, methylparaben, ethylparaben, and propylparaben.

9. The moisturizing cream according to claim 1, characterized in that, The auxiliary component is an active substance and / or a fragrance.

10. A method for preparing the moisturizing cream according to any one of claims 1 to 9, characterized in that, It includes the following steps: (1) Mix the oil and the emulsifier, heat to 80-90°C to fully dissolve them to obtain Phase A; (2) Mix the humectant, thickener and water to make them fully swell and homogenize to obtain Phase B; (3) At 80 - 90 °C, add Phase A to Phase B under stirring, and emulsify at 5000 - 10000 rpm for 1 - 10 min; (4) Stir and cool to 40 - 60 °C, add the auxiliary ingredients, stir evenly, and cool to 20 - 30 °C to obtain the said moisturizing cream.

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