Oceanobacillus possesses idebenone-producing ability and high antioxidant capacity.

Abstract

The present invention provides a bacterial strain that possesses higher free radical scavenging ability and idebenone production ability. [Solution] The Oceanobacillus Y-3 strain of the present invention was obtained by screening from black mud of Yuncheng Salt Lake in Shanxi Province. It possesses high free radical scavenging ability and strong iron ion reduction ability, exhibits high antioxidant activity, and has the ability to produce idebenone. The strain of the present invention has great potential for application in the fields of health foods and cosmetics.

Description

Technical Field

[0001] The present invention relates to Oceanobacillus having the ability to produce idebenone and high antioxidant ability, and belongs to the field of microbial strains.

Background Art

[0002] Free radicals, as atomic or molecular clusters with unpaired electrons generated in the metabolic process of the living body, play an important role in signal transduction under physiological conditions. However, if free radicals accumulate too much in the body, they will attack cell structures, causing a series of oxidative stress reactions such as DNA damage, lipid peroxidation, and protein denaturation, and further causing various diseases. Therefore, exploring a method to effectively remove free radicals in the body is of great significance for maintaining human health.

[0003] Currently, the removal of free radicals is mainly achieved in two ways. One is achieved by the antioxidant enzyme system naturally present in the body, such as superoxide dismutase (SOD), etc., and the other is achieved by externally ingesting substances with antioxidant activity such as vitamins, minerals, and specific natural products. However, with aging and differences in physical constitution, the activity of antioxidant enzymes in the body gradually decreases, and there are also certain restrictions on the absorption and utilization efficiency of conventional antioxidants. Therefore, the development of new and efficient free radical scavengers has become the focus of current research.

[0004] Idebenone, as a highly efficient antioxidant, protects cells from oxidative stress by efficiently scavenging and removing free radicals through its benzoquinone group structure. In the biopharmaceutical field, idebenone is widely used in the treatment of neurological and cardiovascular diseases, as well as in improving skin health. In cosmetics and skincare products, idebenone can significantly improve skin condition by performing actions such as free radical scavenging, lipid peroxidation inhibition, inflammation suppression, and DNA damage inhibition. However, chemically synthesizing idebenone is not only costly but also potentially causes environmental pollution. Therefore, exploring sustainable and environmentally friendly methods for producing idebenone is of particular importance.

[0005] In the field of microbiology, probiotics such as lactic acid bacteria and bifidobacteria are widely applied in areas such as food fermentation, pharmaceuticals, and healthcare due to their excellent biological activity and safety. Recent research has shown that some strains of lactic acid bacteria and bifidobacteria possess strong antioxidant capabilities, removing free radicals in the body and protecting cells from oxidative damage. However, although various probiotics have been demonstrated to have antioxidant activity, there are significant differences in free radical scavenging ability among different strains, and relatively few strains can efficiently remove multiple types of free radicals. Furthermore, conventional techniques for microorganisms to produce idebenone have not been disclosed or reported. Therefore, screening and isolating strains that possess both higher free radical scavenging ability and idebenone production ability is of great significance for the development of novel functional products that can be used in multiple fields such as food, health foods, and cosmetics. [Overview of the project]

[0006] This invention relates to Oceanobacillus Y-3 screened from black mud of Yuncheng Salt Lake in Shanxi Province, in order to solve the above problems. This bacterium efficiently removes free radicals and possesses antioxidant and idebenone production capabilities. This invention also provides a comprehensive identification of this bacterium through physiological and biochemical analysis and phenotypic characteristic analysis, laying a certain foundation for the development of novel and efficient free radical scavengers from a microbiological perspective, and providing ideas and bacterial species resources for development in fields such as pharmaceuticals, cosmetics, and health foods.

[0007] The first object of the present invention is to provide Oceanobacillus sp. Y-3, which has idebenone production ability and high antioxidant capacity. Taxonomically named Oceanobacillus sp. Y-3, it was deposited with the China Typical Culture Depository Center on November 5, 2024, with deposit number CCTCC NO:M 20242434.

[0008] The novel Oceanobacillus sp. CCTCC NO:M 20242434, which possesses the ability to produce idebenone and high antioxidant capacity according to the present invention, has the following characteristics. (1) Colony characteristics: The colonies are orange, circular, opaque, with a central protrusion, and the colony edges are well-defined. The size and shape are stable. (2) Morphological characteristics: It is a Gram-positive bacterium, lacking flagella, and its species cells are short rod-shaped, with a length range of 1.5-1.8 μm and a width range of 0.3-0.4 μm. (3) Growth characteristics: It can tolerate salinity from 0% to 5%, the optimal growth temperature is 30°C, the growth pH range is 6.5 to 9.0, and the optimal growth pH range is 7.0 to 7.2. It can be grown using various sugars such as starch, glucose, sucrose, and maltose as its sole carbon source. (4) Physiological and biochemical characteristics: Positive for contact enzymes, positive for starch hydrolysis, positive for cellulose decomposition, positive for glucose oxidation fermentation, negative for protease, positive for esterase, positive for indole; Belongs to facultative anaerobic bacteria and grows well under both aerobic and anaerobic conditions. (5) Antioxidant properties: a. It can effectively remove DPPH free radicals, ABTS cation free radicals, and hydroxyl radicals, and has strong antioxidant capacity. When the cell lysate concentration of Oceanobacillus CCTCC NO:M 20242434 is higher than 12 mg / mL, the scavenging rate of DPPH free radicals reaches 90% or more. When the cell lysate concentration of Oceanobacillus CCTCC NO:M 20242434 is higher than 10 mg / mL, the scavenging rate of ABTS cation free radicals reaches 90% or more. Preferably, when the cell lysate concentration of the said bacteria is 20 mg / mL, the scavenging rate of ABTS cation free radicals reaches 99% or more. The scavenging rate of anhydrous ethanol intracellular extract with a concentration of 10 mg / mL against hydroxyl radicals reaches 72.23%. b. It has a strong iron ion reducing ability, and when the cell lysate concentration of Oceanobacillus sp. CCTCC NO:M 20242434 is 16 mg / mL, the FRAP value is 4.28 mmol / L. (6) It has the ability to produce idebenone.

[0009] A second object of the present invention is to provide a microbial agent containing Oceanobacillus sp. CCTCC NO:M 20242434.

[0010] In one embodiment of the present invention, the microbial agent mainly consists of Oceanobacillus sp. CCTCC NO:M 20242434.

[0011] In one embodiment of the present invention, the number of viable Oceanobacillus sp. CCTCC NO:M 20242434 cells in the fungal agent is 10 7 -10 9 The concentration is CFU / mL.

[0012] In one embodiment of the present invention, the range of bacterial concentrations of Oceanobacillus sp. CCTCC NO:M 20242434 in the fungal agent is 10 7 -10 9 The concentration is CFU / mL.

[0013] In one embodiment of the present invention, the fungal agent is obtained by preparing a seed solution of Oceanobacillus sp. CCTCC NO:M 20242434 and then culturing it.

[0014] In one embodiment of the present invention, the fungal agent may exist in the form of live cells of the Oceanobacillus sp. CCTCC NO:M 20242434 strain of the present invention, freeze-dried dried cells of the Oceanobacillus sp. CCTCC NO:M 20242434 strain of the present invention, immobilized cells of the Oceanobacillus sp. CCTCC NO:M 20242434 strain of the present invention, a liquid fungal agent of the Oceanobacillus sp. CCTCC NO:M 20242434 strain of the present invention, a solid fungal agent of the Oceanobacillus sp. CCTCC NO:M 20242434 strain of the present invention, or any other form of the Oceanobacillus sp. Includes strain sp.)CCTCC NO:M 20242434.

[0015] In one embodiment of the present invention, the fungal agent further comprises other auxiliary agents.

[0016] In one embodiment of the present invention, the fungal agent is DPPH·,·OH,ABTS + This microbial preparation efficiently eliminates free radicals, possesses high reducing power, and has strong antioxidant capabilities.

[0017] A third object of the present invention is to provide a product composition having an antioxidant effect, the method for producing the product composition comprising adding the cell material and / or metabolites of Oceanobacillus CCTCC NO:M 20242434 to a product substrate to obtain the product composition.

[0018] In one embodiment of the present invention, the antioxidant effect is characterized by free radical scavenging ability or iron ion reduction ability. In one embodiment of the present invention, the free radical is DPPH·,·OH,ABTS + It is at least one of the following:

[0019] A fourth object of the present invention is to provide the use of Oceanobacillus sp. CCTCC NO:M 20242434, or the above-mentioned microbial agent or product composition containing Oceanobacillus sp. CCTCC NO:M 20242434, in the manufacture of a product having free radical scavenging ability or antioxidant ability.

[0020] In one embodiment of the present invention, the product includes, but is not limited to, pharmaceuticals, health foods, cosmetics, or functional foods.

[0021] In one embodiment of the present invention, the pharmaceutical further comprises a pharmaceutically acceptable adjuvant, the adjuvant including, but not limited to, at least one of diluents, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, solubilizers, osmotic pressure modifiers, surfactants, coating materials, colorants, pH adjusters, antioxidants, bacteriostatic agents, or buffers.

[0022] In one embodiment of the present invention, the pharmaceutical further comprises other pharmaceutical ingredients having antioxidant properties.

[0023] In one embodiment of the present invention, the drug includes, but is not limited to, an injection, an oral solution, a tablet, a capsule, a dropper pill, or a spray.

[0024] In one embodiment of the present invention, the administration method includes, but is not limited to, subcutaneous injection, intravenous injection, oral administration, topical application, airway inhalation administration, local administration, and sublingual administration.

[0025] In one embodiment of the present invention, the dosage form of the cosmetic can be prepared in the form of a solution, topical cream, cream foam, nourishing emollient, soft emollient, filler, soft water, cream lotion, cosmetic base, essence, soap, liquid detergent, bath additive, sunscreen cream, sunscreen oil, suspension, emulsion, paste, gel, lotion, powder, soap, surfactant-containing detergent, oil, foundation, emulsion foundation, wax foundation, patch, and spray.

[0026] In one embodiment of the present invention, the cosmetic further comprises at least one carrier that is acceptable as a cosmetic.

[0027] In one embodiment of the present invention, the carrier may be an oil, water, a surfactant, a humectant, a lower alcohol, a thickener, a chelating agent, a pigment, a preservative, or a fragrance.

[0028] A fifth object of the present invention is to provide a method for producing idebenone using Oceanobacillus sp. CCTCC NO:M 20242434 or a fungal agent containing Oceanobacillus sp. CCTCC NO:M 20242434.

[0029] A sixth object of the present invention is to provide a cosmetic product containing a cell lysate of Oceanobacillus sp. CCTCC NO:M 20242434 or the above product composition.

[0030] In one embodiment of the present invention, the cosmetic is a skincare product.

[0031] In one embodiment of the present invention, the dosage form of the cosmetic can be prepared in the form of a solution, topical cream, cream foam, nourishing emollient, soft emollient, filler, soft water, cream lotion, cosmetic base, essence, soap, liquid detergent, bath additive, sunscreen cream, sunscreen oil, suspension, emulsion, paste, gel, lotion, powder, soap, surfactant-containing detergent, oil, foundation, emulsion foundation, wax foundation, patch, and spray.

[0032] In one embodiment of the present invention, the cosmetic further comprises at least one carrier that is acceptable as a cosmetic.

[0033] In one embodiment of the present invention, the carrier may be an oil, water, a surfactant, a humectant, a lower alcohol, a thickener, a chelating agent, a pigment, a preservative, or a fragrance. Effects of the invention:

[0034] (1) The Oceanobacillus sp. CCTCC NO:M 20242434 strain of the present invention has high free radical scavenging ability and can efficiently scavenge DPPH free radicals, ABTS cation free radicals, and hydroxyl radicals. The DPPH free radical scavenging rate of an anhydrous ethanol intracellular extract of Oceanobacillus sp. CCTCC NO:M 20242434 at a concentration of 24 mg / ml reaches 92.81%, the ABTS cation free radical scavenging rate of an anhydrous ethanol intracellular extract at a concentration of 20 mg / ml reaches 99.81%, and the hydroxyl radical scavenging rate of an anhydrous ethanol intracellular extract at a concentration of 10 mg / ml reaches 72.23%. (2) The Oceanobacillus sp. CCTCC NO:M 20242434 strain of the present invention has a strong iron ion reducing ability, and when the cell lysate concentration of Oceanobacillus sp. CCTCC NO:M 20242434 cells is 16 mg / mL, the FRAP value is 4.28 mmol / L. (3) The Oceanobacillus sp. CCTCC NO:M 20242434 strain of the present invention further possesses the ability to produce idebenone. Therefore, Oceanobacillus sp. CCTCC NO:M 20242434 possesses high free radical scavenging ability and total reducing power, exhibiting high antioxidant activity. Furthermore, the Oceanobacillus sp. CCTCC NO:M 20242434 of the present invention also has idebenone production ability, giving it great potential for application in the fields of health foods and cosmetics.

[0035] Deposit of biological materials: This strain is Oceanobacillus Y-3, taxonomically named Oceanobacillus sp.Y-3. The aforementioned strain was deposited with the China Center for Typical Cultures Depository on November 5, 2024, with deposit number CCTCC NO:M 20242434, and the deposit address is Wuhan University, Wuhan City, China. [Brief explanation of the drawing]

[0036] [Figure 1] This is a morphological diagram of a colony of Oceanobacillus sp. Y-3. [Figure 2] This is a diagram of the cellular morphology of Oceanobacillus sp. Y-3. [Figure 3] This diagram shows the growth status of Oceanobacillus sp. Y-3 at different temperatures. [Figure 4] This is a diagram showing the growth status of Oceanobacillus sp. Y-3 at different salinity levels. [Figure 5] This diagram shows the growth status of Oceanobacillus sp. Y-3 under different carbon sources. [Figure 6] This is the total ion chromatogram of an idebenone standard sample. [Figure 7] These are the primary and secondary mass spectra of an idebenone standard sample. [Figure 8] This is the total ion chromatogram of the sample solution. [Figure 9] These are the primary and secondary mass spectra of the peak substance in the sample solution at 4.62 min. [Modes for carrying out the invention]

[0037] The present invention is not limited to the examples provided, which are merely illustrative and do not limit the scope of protection. Any modification or substitution of the methods, steps, or conditions of the present invention falls within the scope of the invention, as long as it does not depart from the spirit and essence of the invention.

[0038] In the following examples, unless otherwise specified, all solutions mentioned use water as the solvent.

[0039] The coarse salt described below is the large salt from the Yuncheng Salt Pond, originating from the Yuncheng Salt Lake.

[0040] The culture medium formulation described in the following examples is as follows: 1. Coarse salt liquid medium: 50g of coarse salt, 20g of MgSO4·7H2O, 2.0g of KCl, 3.0g of sodium citrate, 0.2g of anhydrous calcium chloride, 2.0g of yeast extract, 10.0g of peptone, and distilled water were added to a total volume of 1L, and the pH was adjusted to 7.0-7.2. 2. Coarse salt solid medium: 50g of coarse salt, 20g of MgSO4·7H2O, 2.0g of KCl, 3.0g of sodium citrate, 0.2g of anhydrous calcium chloride, 2.0g of yeast extract, 10.0g of peptone, and distilled water were added to a total volume of 1L, the pH was adjusted to 7.0-7.2, and 15.0g of agar was added. The medium was sterilized at 1 × 10⁵ Pa and 121°C for 30 min, cooled to 50°C-60°C, placed in culture dishes, approximately 15-20mL of medium was poured into each dish, cooled to solidify, and then stored for use.

[0041] Example 1: Morphological observation of strain Y-3 1. Species origin The strain was isolated from the black mud of Yuncheng Salt Lake in Shanxi Province. The specific isolation method is as follows. In a super clean bench, black mud was added to the crude salt medium, and the addition amount was 5% of the medium. It was shaken well to mix uniformly, placed in a shaker incubator at 30 °C for 48 h of induction culture, and then transferred to an incubator at 30 °C. After the supernatant became turbid, the bacterial solution was diluted with sterile water to concentrations of 10 -1 10 -2 10 -3 10 -4 10 -5 10 -6 etc. 100 μL of diluted solutions of different concentrations were taken on the crude salt solid medium, evenly spread with a disposable spreader, left standing to dry the diluted solution, and then placed in a constant temperature incubator at 30 °C for inverted culture. After obvious colonies grew on the medium, different colonies were transferred for culture, and strains with typical characteristics such as orange round edges, neat edges, and surface luster were selected, and each was streaked and cultured on a crude salt solid medium plate for separation, and the strain according to the present invention, orange Y-3 bacteria, was obtained. The purified strain was stored in 20% glycerin and frozen in a -80 °C refrigerator for use.

[0042] 2. Culture characteristics and morphological characteristics The isolated Y-3 strain was streaked on the crude salt solid medium and cultured at 30 °C. Its colony morphology is shown in Figure 1. The colony appeared as an orange circular colony, opaque with a protrusion in the center, the edge of the colony was neat, and the size and shape were stable. When Gram staining was performed on this smear specimen, the morphology of the bacterial cells was as shown in Figure 2. The bacterium showed Gram-positive, had no flagella, was short rod-shaped, with a length range of 1.5 - 1.8 μm and a width range of 0.3 - 0.4 μm.

[0043] 3. Growth characteristics of Oceanobacillus sp. Y-3 The Y-3 strain has a growth temperature range of 30-37°C (Figure 3), can tolerate a chloride concentration of 0-5% (Figure 4), has a growth pH range of 6.5-9.0, and an optimal growth pH range of 7.0-7.2. It belongs to the category of facultative anaerobic bacteria, grows well under both anaerobic and aerobic conditions, and can grow using multiple sugars such as starch, glucose, sucrose, and maltose as its sole carbon source (Figure 5).

[0044] Example 2: 16S rRNA classification and physiological and biochemical characteristics of the Y-3 strain. Molecular biological identification was performed on the Y-3 strain of Example 1, its 16S rRNA gene was amplified by PCR, and after passing electrophoresis, it was sequenced. The primers used for PCR were 27F primer sequence (5'-3'): AGAGTTTGATCCTGCTCAG (SEQ ID NO. 1) and 1492R primer sequence (5'-3'): GGTTACCTTGTTACGACTT (SEQ ID NO. 2).

[0045] The sequencing results were as shown in SEQ ID NO.3. The sequence was subjected to Blast sequence comparison analysis in the NCBI database, and finally, it was confirmed that the Y-3 strain is Oceanobacillus sp., with a homology score of 99.73%.

[0046] The Oceanobacillus Y-3 of the present invention was deposited with the China Center for Typical Cultures Depositary on November 5, 2024, with depositary number CCTCC NO:M 20242434.

[0047] The 16s rDNA sequence of Oceanobacillus sp.Y-3 is shown below (SEQ ID NO.3). The physiological and biochemical characteristics of Oceanobacillus sp.Y-3 were tested using the "General Manual for Systematic Identification of Bacteria <Common Bacterial Systematic Identification Handbook>" as a reference. The results showed that Oceanobacillus sp.Y-3 was positive for contact enzymes, starch hydrolysis, cellulose degradation, glucose oxidation fermentation, protease, esterase, and indole.

[0048] Example 3: Preparation of Oceanobacillus sp. Y-3 cell lysate Fresh culture medium: 20g of coarse salt, 20g of MgSO4·7H2O, 2.0g of KCl, 3.0g of sodium citrate, 0.2g of anhydrous calcium chloride, 2.0g of yeast extract, 10.0g of peptone, and distilled water were added to a final volume of 1L, and the pH was adjusted to 7.2. (1) The obtained Oceanobacillus sp. Y-3 strain was sampled from a frozen storage tube, inoculated into a coarse salt liquid medium, and cultured for 3 days in a shaking incubator at 30°C (rotation speed: 200 rpm). When the culture medium became a cloudy, opaque red liquid, single colonies were selected from each and inoculated into coarse salt solid medium plates. These were then incubated statically in a constant temperature and humidity incubator for 4 days (30°C, 70% humidity) to activate the strains.

[0049] Single colonies were selected and inoculated into Erlenmeyer flasks containing 100 mL of coarse salt liquid medium. They were then cultured for 3 days at 30°C in a shaking incubator (rotation speed: 200 rpm). At this time, the OD of the test bacterial suspension was measured. 600 = 0.5, and it can be used as a coarse salt seed solution.

[0050] Add the cultured coarse salt seed solution to a sterile Erlenmeyer flask containing 300 mL of the above fresh culture solution at a volume ratio of 10% (v / v), and incubate in a 30°C shaking incubator (rotation speed: 200 rpm) for at least 4 days, i.e., OD 600 The culture was continued until the concentration reached 0.4-0.5, and the cultured fermentation liquid was obtained.

[0051] (2) Fermentation liquid obtained in step (1) (OD600 (0.4) was centrifuged for 10 minutes at 4°C and 8000 rpm, the supernatant was removed, the bacterial cell precipitate was washed with PBS, centrifuged again to remove the liquid phase, washed twice with PBS, and then the precipitate was obtained. The obtained precipitate was resuspended in an equal volume of anhydrous ethanol to obtain a cell suspension. The cell suspension was broken up for 3 cycles using an ultrasonic disruptor (1200W, power: 80%, time: 30 min), centrifuged for 10 min at 4°C and 8500 rpm, and the supernatant was collected to obtain an ethanol extract of cell lysates.

[0052] (3) Add an equal volume of anhydrous ethanol to the bacterial cell precipitate after centrifugation in step (2), and repeat the extraction step of step (2) once to obtain bacterial cell lysate ethanol extract-1. Combine the above bacterial cell lysate ethanol extract-1 with the bacterial cell lysate ethanol extract obtained in step (2), concentrate under reduced pressure, and dry to obtain Oceanobacillus sp.Y-3 bacterial cell lysate, which was stored in a refrigerator at -80°C for use.

[0053] Example 4: Measurement of antioxidant activity of Oceanobacillus sp. Y-3 bacterial cell lysates 1. Measurement of DPPH free radical (DPPH·) scavenging ability (1) Prepare ethanol solutions of Oceanobacillus sp.Y-3 cell lysates obtained in Example 3 with anhydrous ethanol at 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. (2) A 0.3 mmol / L solution of 2,2-diphenyl-1-picrylhydrazyl (DPPH) was prepared using anhydrous ethanol. (3) An experimental group, a control group, and a blank group were established. The specific composition of each group is as follows: Experimental group: Transfer 100 μL of the bacterial cell lysate ethanol solution obtained in step (1) to a 96-well plate, then add 100 μL of the DPPH-ethanol solution obtained in step (2) to each plate, mix uniformly, and then react for 30 minutes in the dark. The absorbance A of the mixture at 517 nm was then measured. 試料 We measured it.

[0054] Control group: Except for replacing the sample solution (the sample solution refers to the above-mentioned bacterial cell lysate ethanol solution) with an equal volume of anhydrous ethanol, the procedure was the same as the experimental group, and the specific steps were the same as above, with absorbance A at 517 nm. 対照 We measured it.

[0055] Blank group: Except for replacing the DPPH solution with an equal volume of anhydrous ethanol, the blank group was identical to the experimental group, and the specific steps were the same as above, with absorbance A at 517 nm. ブランク We measured it.

[0056] Each group was tested three times, the average of the results was taken, and the DPPH free radical scavenging rate was calculated.

[0057] The formula for calculating the DPPH free radical scavenging rate is shown below, and the results are shown in Table 1.

[0058] DPPH free radical scavenging rate / % = [1 - (A 試料 -A ブランク ) / A 対照 )] × 100% (4) As positive controls, ascorbic acid solutions of 0.08 mg / mL, 0.04 mg / mL, 0.02 mg / mL, and 0.01 mg / mL were used. Specifically, 100 μL of each of the different concentrations of ascorbic acid solutions was transferred to a 96-well plate, then 100 μL of the DPPH-ethanol solution obtained in step (2) was added to each, and after homogeneous mixing, the mixture was reacted for 30 minutes in the dark, and the absorbance A of the mixture at 517 nm was measured. 試料-1 We measured it.

[0059] Control group: The specific method was the same as above, except that the ascorbic acid solution was replaced with an equal volume of ultrapure water, and absorbance A was measured at 517 nm. 対照-1 We measured it. Blank group: The specific method was the same as above, except that the DPPH solution was replaced with an equal volume of ultrapure water, and absorbance A was observed at 517 nm. ブランク-1 We measured it. Each of them follows the above method, A 試料-1 , A 対照-1 and A ブランク-1 The DPPH free radical scavenging rate was calculated based on the obtained data. The formula for calculating the DPPH free radical scavenging rate is shown below, and the results are shown in Table 1. DPPH free radical scavenging rate / % = [1 - (A 試料-1 -A ブランク-1 ) / A 対照-1 )] × 100%.

[0060] JPEG2026104772000002.jpg90170

[0061] 2. ABTS cation free radical (ABTS + • Measurement of erasure ability (1) Preparation of the solution 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. ABTS working solution: 7.7 mg of ABTS was weighed and dissolved in 2 mL of potassium persulfate solution. After reacting in the dark for 16 hours, it was diluted with a 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)diammonium salt (ABTS) working solution having an absorbance of 0.7 at 734 nm.

[0062] (2) The Oceanobacillus sp. Y-3 cell lysates obtained in Example 3 were prepared as ethanol solutions of Oceanobacillus sp. Y-3 cell lysates at 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 anhydrous ethanol.

[0063] (3) Transfer 10 μL each of the different concentrations of bacterial cell lysate ethanol solutions obtained in step (2) to a 96-well plate, then add 190 μL of the ABTS working solution obtained in step (1) to each, and allow to react for 15 minutes in the dark, then measure the absorbance A at 734 nm. 試料 We measured it.

[0064] Control group: The control group was the same as above, except that the sample solution (the sample solution refers to the above-mentioned bacterial cell lysate ethanol solution) was replaced with an equal volume of anhydrous ethanol, and its absorbance at 734 nm was A. 対照 We measured it. Blank group: Except for replacing the ABTS working solution with anhydrous ethanol, the specific steps are the same as above, and the absorbance at 734 nm is A ブランク We measured it. Each test group was repeated three times, and the average result was taken to calculate the ABTS cation free radical scavenging rate. The formula for calculating the ABTS cation free radical scavenging rate is as follows, and the results obtained are shown in Table 2. ABTS cation free radical scavenging rate / % = [1 - (A 試料 -A ブランク ) / A 対照 )] × 100%.

[0065] (4) As positive controls, ascorbic acid solutions 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 were used. Specifically, 10 μL of each different concentration of ascorbic acid solution was transferred to a 96-well plate, and then 190 μL of the ABTS working solution obtained in step (1) was added to each. The reaction was then allowed to proceed for 15 minutes in the dark, and the absorbance A at 734 nm was measured. 試料 We measured it.

[0066] Control group: The specific method was the same as above, except that the ascorbic acid solution was replaced with an equal volume of ultrapure water, and the absorbance A at 734 nm was measured. 対照-1 We measured it. Blank group: The specific method was the same as above, except that the DPPH solution was replaced with an equal volume of ultrapure water, and the absorbance A at 734 nm was measured. ブランク-1 We measured it. Each of them follows the above method, A 試料 , A 対照 and A ブランク The ABTS cation free radical scavenging rate was calculated based on the obtained results. The formula for calculating the ABTS cation free radical scavenging rate is as follows, and the results obtained are shown in Table 2. ABTS cation free radical scavenging rate / % = [1 - (A 試料-1 -A ブランク-1 ) / A 対照-1 )] × 100%.

[0067] JPEG2026104772000003.jpg109170

[0068] 3. Measurement of hydroxyl radical (·OH) scavenging ability (1) The Oceanobacillus sp. Y-3 cell lysates obtained in Example 3 were prepared as ethanol solutions of Oceanobacillus sp. Y-3 cell lysates at concentrations of 0.63 mg / mL, 1.25 mg / mL, 2.5 mg / mL, 5.0 mg / mL, and 10 mg / mL, respectively, using anhydrous ethanol. (2) Take 50 μL each of the solutions of each concentration gradient obtained in step (1) above, add 50 μL each of 9 mmol / L H2O2 solution, 9 mmol / L FeSO4 solution, and 9 mmol / L salicylic acid-anhydrous ethanol solution, vortex to homogenize, and react in a 37°C water bath for 35 min. Measure the absorbance at 510 nm, A 試料 That's what I decided.

[0069] Blank group: The specific steps are the same as above, except that the sample solution (the sample solution refers to the above bacterial cell lysate ethanol solution) is replaced with distilled water, and the absorbance is measured at 510 nm. ブランク year, Control group: The specific steps were the same as above, except that the H2O2 solution was replaced with distilled water, and the absorbance was measured at 510 nm. 対照 The hydroxyl radical scavenging rate was then calculated. Each group's experiment was conducted three times in parallel, and the average value was taken. The formula for calculating the hydroxyl radical scavenging rate is as follows, and the results obtained are shown in Table 3. Hydroxyl radical scavenging rate / % = [A ブランク -(A 試料 -A 対照 )] / A ブランク ×100%.

[0070] (3) As positive controls, ascorbic acid solutions with concentrations of 0.84 mg / mL, 0.42 mg / mL, 0.21 mg / mL, 0.10 mg / mL, and 0.05 mg / mL were used. Specifically, 50 μL each of the above ascorbic acid solutions was taken, and 50 μL each of 9 mmol / L H2O2 solution, 9 mmol / L FeSO4 solution, and 9 mmol / L salicylic acid-anhydrous ethanol solution were added, and the mixture was vortexed to make it homogeneous. The reaction was then carried out in a 37°C water bath for 35 minutes. The absorbance was measured at 510 nm, and A 試料-1 That's what I decided.

[0071] Blank group: The specific steps are the same as above, except that the ascorbic acid solution was replaced with an equal volume of ultrapure water, and the absorbance was measured at 510 nm. ブランク-1 year, Control group: The specific steps were the same as above, except that the H2O2 solution was replaced with an equal volume of ultrapure water, and the absorbance was measured at 510 nm. 対照-1 The hydroxyl radical scavenging rate was then calculated. Each of them follows the above method, A 試料-1 , A 対照-1 and A ブランク-1 The hydroxyl radical scavenging rate was calculated based on the obtained results. Each group's experiment was conducted three times in parallel, and the average value was taken. The formula for calculating the hydroxyl radical scavenging rate is as follows, and the results obtained are shown in Table 3. Hydroxyl radical scavenging rate / % = [A ブランク-1 -(A 試料-1 -A 対照-1 )] / A ブランク-1 ×100%.

[0072] JPEG2026104772000004.jpg77170

[0073] As can be seen from Tables 1, 2, and 3, an ethanol solution of Oceanobacillus sp. Y-3 bacterial cell lysate has a high scavenging effect against DPPH free radicals, ABTS cationic free radicals, and hydroxyl radicals.

[0074] 4. Measurement of iron ion reduction capacity (FRAP) (1) Preparation of TPTZ working solution A TPTZ working solution was prepared by mixing 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.

[0075] (2) The Oceanobacillus sp.Y-3 cell lysates obtained in Example 3 were prepared as ethanol solutions of Oceanobacillus sp.Y-3 cell lysates at concentrations 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, respectively.

[0076] (3) Add 10 μL of ethanol solution of bacterial cell lysate of different concentrations obtained in step (2) above, and 190 μL of TPTZ working solution obtained in step (1) to each of the 96-well plates in order, 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. 試料 That's what I decided.

[0077] Control group: The specific steps were the same as above, except that the TPTZ working solution was replaced with acetate buffer, and the absorbance was measured at a wavelength of 593 nm. 対照 That's what I decided. Blank group: The specific steps are the same as above, except that the sample solution (the sample solution refers to the above bacterial cell lysate ethanol solution) is replaced with anhydrous ethanol, and the absorbance at 593 nm is A ブランク We measured it. Each test group was repeated three times, and the average results were taken.

[0078] Ferrous sulfate standard solutions with concentrations in the range of 0.1–1 mmol / L were prepared, and their absorbance was measured under the same conditions. A standard curve was created with ferrous sulfate concentration on the x-axis and absorbance on the y-axis. The resulting regression equation was y = 0.675x + 0.019(R 2 The regression equation is 0.999). The iron ion reducing capacity of the sample was calculated using this regression equation and expressed as the equivalent concentration of ferrous ions (FRAP value). The unit is mmol / L. y=A 試料 -A 対照 -A ブランク Substituting this into the formula, we obtained the x-value, i.e., the FRAP value.

[0079] (4) As positive controls, ascorbic acid 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 were used. Specifically, 10 μL of the above ascorbic acid solution and 190 μL of the TPTZ working solution obtained in step (1) were added sequentially to a 96-well plate, shaken for 10 seconds with a microplate reader, incubated at 37°C for 10 minutes, and the absorbance was measured at a wavelength of 593 nm. 試料-1 That's what I decided.

[0080] Control group: The specific steps were the same as above, except that the TPTZ working solution was replaced with acetate buffer, and the absorbance was measured at a wavelength of 593 nm. 対照-1 That's what I decided. Blank group: The specific steps are the same as above, except that the ascorbic acid solution was replaced with an equal volume of ultrapure water, and the absorbance at 593 nm is A ブランク-1 We measured it. Each of them follows the above method, A 試料-1 , A 対照-1 and A ブランク-1 I obtained it. Each test group was repeated three times, and the average results were taken.

[0081] Ferrous sulfate standard solutions with concentrations in the range of 0.1–1 mmol / L were prepared, and their absorbance was measured under the same conditions. A standard curve was created with ferrous sulfate concentration on the x-axis and absorbance on the y-axis. The resulting regression equation was y = 0.675x + 0.019(R 2 The regression equation is 0.999). The iron ion reducing capacity of the sample was calculated using this regression equation and expressed as the equivalent concentration of ferrous ions (FRAP value). The unit is mmol / L. y=A 試料 -A 対照 -A ブランク Substituting this into the formula, we obtained the x-value, i.e., the FRAP value. The results obtained are shown in Table 4.

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[0083] As can be seen from Table 4, when the concentration of the Oceanobacillus sp. cell lysate ethanol solution is 16 mg / mL, its FRAP value is 4.28 mmol / L, which is close to the highest FRAP value of vitamin C (4.68 mmol / L), proving that Oceanobacillus sp. cell lysates have high reducing ability.

[0084] Example 5: Qualitative analysis of idebenone production ability of Oceanobacillus sp. Y-3 Idebenone in the cell lysates of Oceanobacillus sp. Y-3 was analyzed qualitatively and quantitatively using ultrafast liquid chromatography-tandem quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF / MS). Liquid chromatography conditions: Chromatography column is ACQUITY UPLC BEH C 18 The column was 2.1 mm × 100 mm, 1.7 μm thick. Mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was acetonitrile. The gradient elution program is as shown in Table 5. The flow rate was 0.3 mL / min. A diode array (PDA) detector was used, the column temperature was 45°C, and the sample injection volume was 5.0 μL.

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[0086] Mass spectrometry conditions: positive ion mode, electrospray ion source, multi-reaction monitoring mode, ion source temperature 100°C, capillary voltage 3.5kV, cone voltage 20V, desolvation gas temperature 400°C, IMS gas flow rate 24mL / min, collision energy 6 / 20eV.

[0087] The total ion chromatogram of the idebenone standard solution is shown in Figure 6. The peak-emitting substance at 4.62 min is idebenone. Its primary mass (TOF MS) and secondary mass (TOF MS / MS) spectra are shown in Figure 7. The maximum proton number m / z 339.2154 is [M+H] + This is an ion peak.

[0088] Standard working solutions of 12.5, 25, 50, 62.5, and 125 μg / L were prepared from idebenone standard solutions, and samples were added to each solution for analysis. A regression equation was created with idebenone concentration on the x-axis and peak area on the y-axis. The results of the analysis under the above chromatographic conditions showed a good linear relationship between peak area and mass concentration within the range of 12.5–125 μg / L: Y = 4.5168X - 4.2582(R 2 This indicates that the value is 0.9986. Subsequently, the idebenone content in the sample was calculated using the calibration curve described above.

[0089] Following step (1) of Example 3, 500 mL of cultured Oceanobacillus sp.Y-3 fermentation broth was obtained, and further processed according to steps (2)-(3) of Example 3 to finally obtain 65.6 mg of Oceanobacillus sp.Y-3 bacterial cell lysate. 1 mL of methanol was added and the lysate was redissolved by sonication. After centrifugation at 8000-9000 rpm, the supernatant was taken to obtain the measurement sample. Using the above chromatography and mass spectrometry conditions, the measurement sample was injected into a chromatograph for chromatographic separation, and the chromatographic effluent was directly placed into a mass spectrometer for detection. Idebenone was qualitatively and quantitatively analyzed by mass spectrometry.

[0090] Figure 8 shows the total ion chromatogram of the cell lysate solution of Oceanobacillus sp. Y-3, and Figure 9 shows the primary mass spectrometry (TOF MS) and secondary mass spectrometry (TOF MS / MS) spectra of the peak-emitted substance at 4.62 min. The maximum proton number m / z 339.2187 is [M+H] + This is an ion peak. As can be seen by comparing Figures 8 and 9 with Figures 6 and 7, the peak emission time of the substance in the sample at 4.62 min matches that of the idebenone standard sample, and [M+H] +Since the ion peak matches that of the idebenone standard sample, the peak-emitting substance at 4.62 min is benone, confirming that the Oceanobacillus sp. Y-3 bacterial cell lysate solution contains idebenone.

[0091] Example 6: Quantitative analysis of idebenone production capacity of Oceanobacillus sp. Y-3 Fresh culture medium: 20g of coarse salt, 20g of MgSO4·7H2O, 2.0g of KCl, 3.0g of sodium citrate, 0.2g of anhydrous calcium chloride, 2.0g of yeast extract, 10.0g of peptone, and distilled water were added to a total volume of 1L, and the pH was adjusted to 7.2.

[0092] (1) Preparation of seed juice: The obtained Oceanobacillus sp. Y-3 strain was sampled from a cryopreserved tube, inoculated into a coarse salt liquid medium, and incubated in a 30°C shaking incubator (rotation speed: 200 rpm) for 3 days. When the culture medium became a cloudy, opaque red liquid, single colonies were selected from each and inoculated onto a coarse salt solid medium plate. These were then incubated statically in a constant temperature and humidity incubator for 4 days (30°C, 70% humidity) to activate the strains. Single colonies were selected and inoculated into Erlenmeyer flasks containing 100 mL of coarse salt liquid medium. They were then cultured for 3 days at 30°C in a shaking incubator (rotation speed: 200 rpm). At this time, the OD of the test bacterial suspension was measured. 600 = 0.5, and it can be used as a coarse salt seed solution. (2) Add the prepared coarse salt seed solution to a sterile Erlenmeyer flask containing 300 mL of the above fresh medium (salt concentration 2%, pH 7.2) at a volume ratio of 10% (v / v), and incubate in a 30°C shaking incubator (rotation speed: 200 rpm) for 5 days, OD 600 A culture fermentation solution with a concentration of 0.7-0.8 was obtained. (3) The fermented liquid obtained (OD 600 The sample (0.7) was centrifuged for 10 minutes at 4°C and 8000 rpm, the supernatant was removed, the bacterial cell precipitate was washed with PBS, the liquid phase was removed by centrifugation again, and the precipitate was obtained after washing twice with PBS. The obtained precipitate was resuspended in an equal volume of anhydrous ethanol to obtain a bacterial suspension. The bacterial suspension was disrupted for 3 cycles using an ultrasonic disruptor (1200W, power: 80%, time: 30 min), centrifuged for 10 min at 4°C and 8500 rpm, and the supernatant was collected to obtain an ethanol extract of bacterial cell lysates. (4) Add an equal volume of anhydrous ethanol to the bacterial cell precipitate after centrifugation in step (3), and repeat the extraction step of step (3) once to obtain bacterial cell lysate ethanol extract-1. Combine the above bacterial cell lysate ethanol extract-1 and the bacterial cell lysate ethanol extract obtained in step (3), concentrate under reduced pressure, and then add methanol to a final volume of 1 mL. (5) The idebenone content in the ethanol extract of the Oceanobacillus sp. Y-3 bacterial cell lysate obtained in step (4) was measured using the measurement method and calibration curve of Example 5, and was found to be 81.4 μg / L. Since the freeze-dried mass of Oceanobacillus sp.Y-3 cells was 0.1809 g, the idebenone production capacity of the Oceanobacillus sp.Y-3 strain was calculated to be 0.45 μg / g.

[0093] Example 7: Use of Oceanobacillus CCTCC NO:M 20242434 in emulsion preparation This embodiment provides an emulsion composition with the following formulation. 1. Aqueous phase: Mix water, glycerin, hydrolyzed sodium hyaluronate, xanthan gum, carbomer, and methylparaben, then heat to 80°C and stir until uniformly dissolved. 2. Oil phase: Cetyl alcohol, glyceryl stearate / PEG-100 glyceryl stearate, caprylic / capric triglyceride, glyceryl triethylhexanoate, shea butter. Heat the above ingredients to 80°C and stir to dissolve uniformly. 3. The aqueous phase and oil phase were mixed and then homogenized for 5 minutes. After the emulsification was complete, the Oceanobacillus sp.Y-3 bacterial cell lysate prepared in Example 3 was added and the mixture was uniformly stirred.

[0094] Although the present invention is disclosed using preferred embodiments as described above, this does not limit the invention, and any person familiar with the art can make various modifications and alterations without departing from the spirit and scope of the invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. Oceanobacillus sp. Y-3, The aforementioned Oceanobacillus is characterized by being deposited with the China Typical Culture Depository Center on November 5, 2024, with deposit number CCTCC NO: M 20242434.

2. A microbial agent characterized by containing Oceanobacillus cCTCC NO:M 20242434 as described in claim 1.

3. The microbial agent according to claim 2, characterized in that the microbial agent mainly consists of Oceanobacillus cCTCC NO:M 20242434.

4. The microbial agent according to claim 2, characterized in that the microbial agent includes live cells of the Oceanobacillus CCTCC NO:M 20242434 strain, freeze-dried cells of the Oceanobacillus CCTCC NO:M 20242434 strain, immobilized cells of the Oceanobacillus CCTCC NO:M 20242434 strain, liquid microbial agent of the Oceanobacillus CCTCC NO:M 20242434 strain, solid microbial agent of the Oceanobacillus CCTCC NO:M 20242434 strain, or the Oceanobacillus CCTCC NO:M 20242434 strain existing in any other form.

5. Product compositions having antioxidant effects, The product composition is characterized in that it is manufactured by adding the cell material and / or metabolites of Oceanobacillus cCTCC NO:M 20242434 described in claim 1 to a product substrate to obtain the product composition.

6. The product composition according to claim 5, characterized in that the antioxidant effect has the ability to scavenge free radicals or reduce iron ions.

7. The aforementioned free radicals are DPPH•,•OH,ABTS ＋ The product composition according to claim 6, characterized in that it is at least one of the following.

8. Use of Oceanobacillus CCTCC NO:M 20242434 as described in claim 1, a fungicide containing Oceanobacillus CCTCC NO:M 20242434 as described in claim 1, or a product composition as described in claim 4 in the manufacture of a product having free radical scavenging ability or antioxidant function.

9. The use according to claim 8, characterized in that the aforementioned product includes, but is not limited to, pharmaceuticals, health foods, cosmetics, or functional foods.

10. The use according to claim 9, characterized in that the product further contains an adjuvant that is acceptable in pharmaceuticals, food, or health foods.

11. The use according to claim 9, wherein the pharmaceutical product further comprises pharmaceutically acceptable adjuvants, the adjuvants comprising, but not limited to, at least one of diluents, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH adjusters, antioxidants, bacteriostatic agents, or buffers.

12. The use according to claim 9, characterized in that the pharmaceutical product includes, but is not limited to, injections, oral solutions, tablets, capsules, drops, and sprays.

13. The use according to claim 9, characterized in that the method of administration of the pharmaceutical product includes, but is not limited to, subcutaneous injection, intravenous injection, oral administration, topical application, airway inhalation, local administration, and sublingual administration.

14. A method for producing idebenone by biological law, A method characterized by producing and obtaining idebenone by fermentation of Oceanobacillus CCTCC NO:M 20242434 as described in claim 1 or a fungal agent containing Oceanobacillus CCTCC NO:M 20242434.

15. A cosmetic product characterized by comprising a cell lysate of Oceanobacillus cCTCC NO:M 20242434 as described in claim 1, or the product composition as described in claim 4.

16. The cosmetic according to claim 15, characterized in that the cosmetic is a skincare product.

17. The cosmetic according to claim 16, characterized in that the dosage form of the cosmetic is prepared in the form of a solution, topical cream, cream foam, nourishing emollient, soft emollient, filler, soft water, cream lotion, cosmetic base, essence, soap, liquid cleanser, bath additive, sunscreen cream, sunscreen oil, suspension, emulsion, paste, gel, lotion, powder, soap, surfactant-containing cleanser, oil, foundation, emulsion foundation, wax foundation, patch, and spray.

18. The cosmetic according to claim 17, characterized in that the cosmetic further comprises at least one carrier that is acceptable as a cosmetic.

19. The cosmetic according to claim 18, characterized in that the carrier is oil, water, surfactant, humectant, lower alcohol, thickener, chelating agent, pigment, preservative, and fragrance.

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