Xanthophyll carotenoid-producing Flaviramulus sp. SSW2303 strain and uses thereof

Abstract

The present invention relates to Flaviramulus SSW2303, which produces xanthophyll carotenoids, and its uses. More specifically, it has been confirmed that Flaviramulus SSW2303 produces astaxanthin, zeazanthin, and canthaxanthin and has antioxidant efficacy, so it can be utilized in various ways, such as in cosmetic compositions and health functional food compositions.

Description

Technology Field

[0001] The present invention provides a Flaviramulus SSW2303 strain that produces xanthophyll carotenoids and uses thereof. Background Technology

[0002] Carotenoids are a group of isoprenoid pigments naturally synthesized by animals, plants, and microorganisms, C 30 , C 40 , C 50 Carotenoids are fat-soluble pigments of red, orange, and yellow hues, and are known as natural substances with useful physiological functions such as vitamin A precursors, antioxidant, anti-inflammatory, and anticancer properties. Depending on their chemical structure, carotenoids are divided into the oxygen-free carotene series and the oxygen-containing xanthophyll series. Representative carotene series include lycopene, α-carotene, β-carotene, and β-cryptoxanthin, while representative xanthophyll series include carotenoids such as lutein, zeaxanthin, neoxanthin, violaxanthin, fucoxanthin, α-cryptoxanthin, and β-cryptoxanthin.

[0003] Currently, the carotenoid market is largely dominated by synthetic carotenoids due to their lower production costs; however, interest in natural carotenoids is continuously increasing due to resistance to their use and controversies regarding their safety. Nevertheless, natural carotenoids derived from animals and plants have disadvantages, such as low production yields per biomass, high production costs resulting from expensive extraction, isolation, and purification processes, and difficulties in ensuring a consistent supply due to environmental changes including seasonal, climatic, and regional variations.

[0004] Therefore, to overcome these drawbacks, research on carotenoid production using microorganisms has recently been actively underway. Compared to carotenoid production using animals or plants, microbial production enables economically efficient mass production due to the rapid growth rate of microorganisms and the ability to culture them in large quantities at high concentrations. Furthermore, because it is free from spatial constraints and seasonal changes, it allows for reduced production costs and a continuous supply of carotenoids. Additionally, microorganisms offer the advantage of a relatively simple extraction process and easy isolation and purification. Recently, research on mass production of carotenoids is being conducted by improving strains through metabolic engineering or utilizing microorganisms such as E. coli or yeast, which are easy to genetically modify. Consequently, due to these advantages, carotenoid production using microorganisms can serve as an alternative to existing animal and plant-derived carotenoid production and is expected to become a sustainable supply source capable of meeting the growing demand in the carotenoid market. Prior art literature

[0005] 1. INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY Volume 65, Issue Pt_2 “Flaviramulus aquimarinus sp. nov., isolated from seawater” (published on January 1, 2015) The problem to be solved

[0006] The object of the present invention is the genus Flaviramulus SSW2303 ( deposited at KCTC 15595BP) Flaviramulus It lies in providing the sp. SSW2303 strain.

[0007] Another objective of the present invention is to provide a method for producing carotenoids comprising the step of inoculating a strain of the genus Flaviramulus SSW2303, deposited at KCTC 15595BP, into a culture medium.

[0008] Another objective of the present invention is to provide an antioxidant cosmetic composition comprising, as an active ingredient, the strain of the genus Flaviramulus SSW2303 deposited at KCTC 15595BP, a culture thereof, a culture extract thereof, a lysate thereof, or a mixture thereof.

[0009] Another objective of the present invention is to provide a health functional food composition for antioxidant use comprising, as an active ingredient, the strain of the genus Flaviramulus SSW2303 deposited at KCTC 15595BP, a culture thereof, a culture extract thereof, a lysate thereof, or a mixture thereof.

[0010] Another objective of the present invention is to provide a coloring composition for animal feed comprising as an active ingredient the strain of the genus Flaviramulus SSW2303 deposited at KCTC 15595BP, a culture thereof, a culture extract thereof, a lysate thereof, or a mixture thereof.

[0011] Another objective of the present invention is to provide an antioxidant composition comprising the genus Flaviramulus SSW2303 strain deposited at KCTC 15595BP, a culture thereof, a culture extract thereof, a lysate thereof, or a mixture thereof as an active ingredient. means of solving the problem

[0012] To achieve the above objective, the present invention relates to the genus Flaviramulus SSW2303 (deposited as KCTC 15595BP) Flaviramulus Provides the sp. SSW2303 strain.

[0013] In addition, the present invention provides a method for producing carotenoids comprising the step of inoculating a strain of the genus Flaviramulus SSW2303, deposited at KCTC 15595BP, into a culture medium.

[0014] In addition, the present invention provides an antioxidant cosmetic composition comprising, as an active ingredient, the strain of the genus Flaviramulus SSW2303 deposited at KCTC 15595BP, a culture thereof, a culture extract thereof, a lysate thereof, or a mixture thereof.

[0015] In addition, the present invention provides a health functional food composition for antioxidant use comprising, as an active ingredient, the strain of the genus Flaviramulus SSW2303 deposited at KCTC 15595BP, a culture thereof, a culture extract thereof, a lysate thereof, or a mixture thereof.

[0016] In addition, the present invention provides a coloring composition for animal feed comprising as an active ingredient the genus Flaviramulus SSW2303 strain deposited at KCTC 15595BP, a culture thereof, a culture extract thereof, a lysate thereof, or a mixture thereof.

[0017] In addition, the present invention provides an antioxidant composition comprising the genus Flaviramulus SSW2303 strain deposited at KCTC 15595BP, a culture thereof, a culture extract thereof, a lysate thereof, or a mixture thereof as an active ingredient. Effects of the invention

[0018] The present invention relates to the microalgae Flaviramulus SSW2303, which produces xanthophyll carotenoids, and its uses. More specifically, it has been confirmed that Flaviramulus SSW2303 produces astaxanthin, zeazanthin, and canthaxanthin and possesses antioxidant efficacy, allowing it to be utilized in various ways, such as in cosmetic compositions and health functional food compositions. Brief explanation of the drawing

[0019] Figure 1A shows the colony formation of the Flaviramulus SSW2303 strain on a solid agar medium. Figure 1B shows the morphology of microalgae observed with an electron microscope of the Flaviramulus SSW2303 strain. Figure 2 shows a taxonomic phylogenetic tree based on the 16S rRNA base sequence of the Flaviramulus SSW2303 strain. Figure 3A shows the results of analyzing standard substances [neoxanthin (NZ), violaxanthin (VI), astaxanthin (AS), zeaxanthin (ZE), canthaxanthin (CA), lycopene (LY), beta-carotene (β-CA)] dissolved in methanol using high-performance liquid chromatography (HPLC). Figure 3B shows the results of analyzing the methanol extract of the Flaviramulus SSW2303 strain using high-performance liquid chromatography (HPLC). Figure 3C shows the results of analyzing a mixture of the methanol extract of the Flaviramulus SSW2303 strain and standard substances [neoxanthin (NZ), violasanthin (VI), astaxanthin (AS), zeaxanthin (ZE), canthaxanthin (CA), lycopene (LY), beta-carotene (β-CA)] using high-performance liquid chromatography (HPLC). Figure 3D shows the results of UV-chromatogram analysis of standard substances astaxanthin, zeaxanthin, and canthaxanthin using high-performance liquid chromatography (HPLC). Figure 3E shows the results of UV-chromatogram analysis using high-performance liquid chromatography (HPLC) on the methanol extract of the Flaviramulus SSW2303 strain. Figure 4A shows the results of analyzing mass spectrometry data for standard substances astaxanthin and methanol extracts of Flaviramulus SSW2303 strain through liquid chromatography-mass spectrometry (LC-MS). Figure 4B shows the results of analyzing mass spectrometry data for standard substances zeaxanthin and methanol extracts of Flaviramulus SSW2303 through liquid chromatography-mass spectrometry (LC-MS). Figure 4C shows the results of analyzing mass spectrometry data for the standard substance canthaxanthin and the methanol extract of the Flaviramulus SSW2303 strain through liquid chromatography-mass spectrometry (LC-MS). Figure 5A shows a graph representing the astaxanthin, zeaxanthin, and canthaxanthin production (Titer) of the Flaviramulus SSW2303 strain under different temperature conditions. Figure 5B shows a graph representing the production (Titer) of astaxanthin, zeaxanthin, and canthaxanthin of the Flaviramulus SSW2303 strain according to sodium chloride (NaCl) concentration. Figure 5C shows a graph representing the production (Titer) of astaxanthin, zeaxanthin, and canthaxanthin according to pH conditions of the Flaviramulus SSW2303 strain. Figure 6 is a graph showing the ABTS radical scavenging activity of the methanol extract of the Flaviramulus SSW2303 strain. Specific details for implementing the invention

[0020] The present invention will be described in more detail below.

[0022] The present invention relates to the genus Flaviramulus SSW2303 (deposited as KCTC 15595BP) Flaviramulus Provides the sp. SSW2303 strain.

[0023] The above strain can produce carotenoids.

[0024] The above carotenoids may be one or more selected from the group consisting of astaxanthin, zeazanthin, and canthaxanthin, but are not limited thereto.

[0026] In addition, the present invention provides a method for producing carotenoids comprising the step of inoculating a strain of the genus Flaviramulus SSW2303, deposited at KCTC 15595BP, into a culture medium.

[0027] The method may further include a step of culturing the above culture medium under conditions of 20 to 30°C, pH 7, and 2 to 3% (w / v) of NaCl.

[0029] In addition, the present invention provides an antioxidant cosmetic composition comprising, as an active ingredient, the strain of the genus Flaviramulus SSW2303 deposited at KCTC 15595BP, a culture thereof, a culture extract thereof, a lysate thereof, or a mixture thereof.

[0030] The above composition may have radical scavenging activity.

[0031] The cosmetic composition of the present invention is not particularly limited in its formulation and, for example, can be formulated into cosmetics such as softening lotion, astringent lotion, nourishing lotion, nourishing cream, massage cream, essence, eye cream, eye essence, cleansing cream, cleansing foam, cleansing water, pack, powder, body lotion, body cream, body oil, and body essence, and may be applied in a form applied to the skin or in a form absorbed into the skin using micro-needles, etc.

[0032] In the cosmetic composition of the present invention, carriers acceptable under pharmaceutical or cosmetic standards include, depending on the formulation, hydrocarbons such as petroleum jelly, liquid paraffin, and gelling hydrocarbons (alias: plastibase); animal and vegetable oils such as medium-chain fatty acid triglycerides, lard, hard fat, and cocoa fat; higher fatty acid alcohols and fatty acids and their esters such as cetanol, stearyl alcohol, stearic acid, and isopropyl palmitate; water-soluble bases such as macrogol (polyethylene glycol), 1,3-butylene glycol, glycerol, gelatin, sucrose, and sugar alcohols; emulsifiers such as glycerin fatty acid esters, polyoxychloride stearate, and polyoxyethylene hardened castor oil; adhesives such as acrylic acid esters and sodium alginate; and propellants such as liquefied petroleum gas and carbon dioxide. Examples of preservatives include parahydroxybenzoic acid esters, and the external preparation of the present invention can be manufactured using these according to conventional methods. In addition to these, stabilizers, fragrances, colorants, pH adjusters, diluents, surfactants, preservatives, antioxidants, etc., may also be incorporated as needed. The external preparation of the present invention can be applied to a local wound by conventional methods.

[0034] In addition, the present invention provides a health functional food composition for antioxidant use comprising, as an active ingredient, the strain of the genus Flaviramulus SSW2303 deposited at KCTC 15595BP, a culture thereof, a culture extract thereof, a lysate thereof, or a mixture thereof.

[0035] The above health functional food may contain various nutritional supplements, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc.

[0036] In addition, it may contain fruit pulp for the production of natural fruit juice, synthetic fruit juice, and vegetable drinks. These ingredients may be used independently or in combination. Furthermore, the health functional food composition may be in the form of any one of meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, chewing gum, ice cream, soup, beverage, tea, functional water, drinkables, alcohol, and vitamin complexes.

[0037] In addition, the above-mentioned health functional food may further include food additives, and unless otherwise stipulated, suitability as a "food additive" shall be determined in accordance with the specifications and standards for the relevant item, based on the general provisions and general test methods of the Food Additives Codex approved by the Ministry of Food and Drug Safety.

[0038] Examples of items listed in the above "Food Additives Codex" include chemically synthesized products such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamon acid; natural additives such as persimmon dye, licorice extract, crystalline cellulose, kohlrabi dye, and guar gum; L-sodium glutamate preparations; alkaline agents added to noodles; preservative preparations; and mixed preparations such as tar dye preparations.

[0039] At this time, the content of the active ingredient added to the food during the process of manufacturing the health functional food can be appropriately increased or decreased as needed, and preferably, it can be added in an amount of 1 to 90 parts by weight per 100 parts by weight of the food.

[0041] In addition, the present invention provides a coloring composition for animal feed comprising as an active ingredient the genus Flaviramulus SSW2303 strain deposited at KCTC 15595BP, a culture thereof, a culture extract thereof, a lysate thereof, or a mixture thereof.

[0043] In addition, the present invention provides an antioxidant composition comprising the genus Flaviramulus SSW2303 strain deposited at KCTC 15595BP, a culture thereof, a culture extract thereof, a lysate thereof, or a mixture thereof as an active ingredient.

[0045] Hereinafter, to aid in understanding the present invention, examples and the like will be described in detail. However, the following examples and the like are merely illustrative of the content of the present invention and the scope of the present invention is not limited to the following examples and the like. The examples and the like of the present invention are provided to more completely explain the present invention to those with average knowledge in the art.

[0047] <Example 1> Isolation of Strains

[0048] Seawater from the coast of Sinan-gun, Jeollanam-do was collected and diluted stepwise (10-fold, 100-fold, 1,000-fold, etc.) with sterile seawater. 80 μL of the diluted solution was then plated onto MA medium (marine agar 2216, BD, USA) plates and cultured at 25°C for 3 to 5 days. After 3 to 5 days of culture, red-colored colonies were isolated and subcultured at least three times in the same medium until pure isolation was achieved. As shown in Figure 1A, the purely isolated strain with red colonies was provisionally named SSW2303. To observe the cell morphology and flagella of the SSW2303 strain, the liquid culture medium was stained with 3% uranyl acetate (EMS, USA) reagent, and imaging analysis was performed using a transmission electron microscope (LIBRA120, Carl-Zeiss, Germany). As a result, as shown in Fig. 1B, it was observed that it was a rod-shaped bacterium with a width of 0.4–0.5 μm and a length of 1.5–2 μm, without flagella.

[0050] <Example 2> Identification of Strains

[0051] The identification of the strain selected in Example 1 was performed using 16S rRNA gene sequencing. Chromosomal DNA of the isolated bacterial strain was extracted using the DNeasy Blood & Tissue kit (Quiagen, Germany). The extracted chromosomal DNA was used as a template to amplify the 16S rRNA gene using the polymerase chain reaction (PCR) method, and the primers used were 27f (AGA GTT TGA TCC TGG CTC AG) and 1492r (TAC GGY TAC CTT GTT ACG AC). After performing PCR, the amplified 16S rRNA gene was purified using a PCR Purification kit (Bioneer, Korea) to identify a fragment of approximately 1.5 kb, and the gene sequence of 1,422 base pairs was analyzed using an ABI 3730XL automatic sequencer (ABI, USA) with 27f, 518r (ATT ACC GCG GCT GCT GG), 785f (GGA TTA GAT ACC CTG GTA) and 1492r universal primers.

[0052] The homology of the 16S rRNA gene information of the above strain was confirmed by performing a BLAST search on the 16S rRNA gene information of standard strains registered in NCBI (National Center for Biotechnology Information) and EzBioCloud (https: / / www.ezbiocloud.net / ). As a result of comparing the similarity between the above strain and the standard strain (type strain) through the BLAST search, the 16S rRNA gene information of the strain was identical to the nucleotide sequence described in SEQ ID NO. 1, and Flaviramulus Standard strains reported in the genus, Flaviramulus aestuariivivens OITF-51 (98.7%) and Flaviramulus aquimarinus It showed the highest similarity to KYW499 (95.6%).

[0053] Phylogenetic tree analysis to elucidate the evolutionary relationships between the taxa of this strain was performed using the ClustalW program to perform multiple alignment between the 16S rRNA sequences of this strain and standard strains obtained through BLAST search, followed by the use of the MEGA X program with Neighbor-joining (NJ), Maximum-parsimony (MP), and Maximum-likelihood (ML) algorithms. The bootstrap value of the phylogenetic tree was expressed as a percentage of the proportion of matches with the phylogenetic trees calculated from the NJ, MP, and ML algorithms by randomly selecting (resampling) alignment data 1,000 times. A closed circle (●) was used to indicate a clade that matched all three algorithms, while a circle (○) was used to indicate a clade that matched two of the algorithms.

[0054] As shown in Fig. 2, based on the 16S rRNA gene sequence, the NJ, MP, and ML phylogenetic tree analysis results show that the SSW2303 strain is Flaviramulus Showing a very high taxonomic relationship with standard strains reported in the genus, Flaviramulus aestuariivivens It formed a phylogenetic group that is evolutionarily closest to the OITF-51 standard strain and distinct from the standard strains of other species. These results indicate that the SSW2303 strain Flaviramulus Based on indications that it belongs to the genus, this isolated strain was identified and named Flaviramulus SSW2303.

[0056] <Example 3> Preparation of Extract and Analysis of High-Addition Pigments Using High-Performance Liquid Chromatography (HPLC)

[0057] To extract pigments contained in the Flaviramulus SSW2303 strain, the cells cultured in MB liquid medium were centrifuged at 6,000 xg for 5 minutes to separate them from the culture medium. They were then washed twice with an equal volume of culture medium using PBS buffer (Sigma, USA) and freeze-dried for 24 hours using a freeze-dryer. The freeze-dried cells were finely ground in a mortar and pestle and mixed thoroughly. Subsequently, 10 mg of the cells were weighed, and 10 mL of 99.9% methanol (HPLC grade) was added to a concentration of 1 mg / mL. After mixing thoroughly with a Vortex for 1 minute, pigment extraction was performed using an ultrasonic cell disruptor.

[0058] Pigment extract analysis was performed using high-performance liquid chromatography (HPLC-DAD system, 1260 Infinity, Agilent, Germany). For HPLC analysis, a C18 reversed-phase column (Spherisorb S5 ODS1 4.6 x 250 mm, Waters, USA) was used, and two mobile phase solvents, A (14% 0.1 M Tris-HCl (pH 8.0), 84% acetonitrile, and 2% methanol) and B (68% methanol, 32% ethyl acetate), were used under gradient conditions. The flow rate was set to 1.2 mL / min, and absorbance was detected at UV 445 nm. For the analysis, representative high-value carotenoid pigments such as neoxanthine (NZ), violaxanthine (VI), astaxanthin (AS), zeaxanthin (ZE), canthaxanthin (CA), lycopene (LY), and beta-carotene (β-CA) were used as standard substances.

[0059] As a result, as shown in Fig. 3A, peaks of the carotenoid standard, NE (6.4 min), VI (7.6 min), AS (8.4 min), ZE (10.6 min), CA (11.7 min), LY (16.7 min), and β-CA (18 min) were identified at each retention time. As shown in Fig. 3B, among the peaks detected in the methanol extract of the Flaviramulus SSW2303 strain, peaks were observed at retention times (within ±0.1) similar to those of the astaxanthin, zeaxanthin, and canthaxanthin standard. As shown in Fig. 3C, when the standard and the Flaviramulus SSW2303 strain pigment methanol extract were mixed and analyzed, peaks were identified at the same retention times as those of the astaxanthin, zeaxanthin, and canthaxanthin standard. In addition, as shown in Fig. 3D, when the UV-chromatograms of the peaks detected in the standard substances of astaxanthin, zeaxanthin, and canthaxanthin and the pigment methanol extract of the SSW2303 strain were examined, peaks were formed in the same wavelength range as the standard substances of astaxanthin (480 nm), zeaxanthin (453 nm and 478 nm), and canthaxanthin (479 nm). Therefore, through these results, it was found that the Flaviramulus SSW2303 strain produces astaxanthin, zeaxanthin, and canthaxanthin.

[0061] <Example 4> Verification of Astaxanthin, Zeaxanthin, and Canthaxanthin Production via Liquid Chromatography-Mass Spectrometry (LC-MS)

[0062] Liquid Chromatography Mass Spectrometry (LC-MS) was performed to precisely confirm whether the peaks in the pigment extract of the Flaviramulus SSW2303 strain, identified by HPLC analysis, were astaxanthin, zeaxanthin, or canthaxanthin. First, for the LC-MS analysis, pigment extract samples were prepared using the previously mentioned extract preparation method, and then the mass patterns of the peaks appearing in the zeaxanthin standard and the cell extract were compared and analyzed using an Exion AD LC and X500R QTOF (SCIEX, USA) system. The scan mode was analyzed in the range of 100–650 m / z, and the analysis was performed in an ESI-positive state. The drying gas was set to 12.0 L / min, the nebulizer pressure to 50 psi, and the drying gas temperature to 350°C, and the operation was conducted at a capillary voltage of 4,000 V. As a result, as shown in Figure 4A, astaxanthin (C 40 H 52 04, Mw=596.84 g / mol) As a result of the ion spectrum analysis of the chromatogram peak of the standard substance, [M] = 597.3917 m / z and [M+H] + = was confirmed at 568.3971 m / z, and as a result of ion spectrum analysis of the chromatogram peak of the pigment extract of Flaviramulus SSW2303 strain, [M] = 597.3925 m / z and [M+H] + = 568.3972 m / z, confirming that it is the same pigment as astaxanthin. As shown in Fig. 4B, zeaxanthin (C 40 H 56 As a result of the ion spectrum analysis of the chromatogram peak of the standard O2 (Mw=568.88 g / mol), [M] = 598.4246 m / z and [M+H] + = was confirmed at 569.4309 m / z, and ion spectrum analysis of the chromatogram peak of the pigment extract of Flaviramulus SSW2303 strain showed [M] = 598.4272 m / z and [M+H] += 569.4333 m / z, confirming that it is the same pigment as astaxanthin. As shown in Fig. 4C, canthaxanthin (C 40 H 52 ion spectrum analysis results of the chromatogram peak of the standard substance (O2, Mw=564.82 g / mol), [M] + = 565.4024 m / z and [M+H] + = confirmed at 566.4072 m / z, and as a result of ion spectrum analysis of the pigment extract chromatogram peak of Flaviramulus SSW2303 strain, [M] + = 565.4033 m / z and [M+H] + = 566.4080 m / z, it was confirmed that it is the same pigment as canthaxanthin. Through this, it was possible to verify that the pigment extract of the Flaviramulus SSW2303 strain contains astaxanthin, zeaxanthin, and canthaxanthin pigments.

[0064] <Example 5> Investigation of Astaxanthin, Zeaxanthin, and Canthaxanthin Production Capacity According to Culture Characteristics of Strains

[0065] The production capacity of astaxanthin, zeaxanthin, and canthaxanthin was investigated according to temperature, pH, and NaCl concentration conditions affecting the growth of the strain according to the present invention. To investigate the effect of growth temperature, the strain was inoculated at 2% (v / v) in MB (marine broth 2216, BD, USA) medium at temperatures of 10℃, 15℃, 20℃, 25℃, 30℃, 35℃, 40℃, 45℃, 50℃, and 55℃, and cultured for 48 hours with stirring at 200 rpm. To investigate the effect of NaCl concentration, MB media composed of 0%, 1%, 2%, 3%, 4%, 5%, 6%, and 7% NaCl (w / v) were used in an MB medium from which NaCl had been removed. To investigate the effect of pH, 10 mM MES buffer (2-(N-morpholino)ethanesulfonic acid) was added to the MB medium under pH 5 and pH 6 conditions, and 10 mM Tris buffer was added under pH 7, pH 8, pH 9, and pH 10 conditions. After adjusting the pH using 1 M HCl and 1 M NaOH, strains were inoculated and cultured using the MB media composed for each pH condition in the same manner as for the temperature conditions. After culturing for each culture condition, pigment extracts were obtained in the same manner as in Example 3, and the production capacity of astaxanthin, zeaxanthin, and canthaxanthin was analyzed using high-performance liquid chromatography (HPLC). To quantify the amount of extracted pigments, a calibration curve was constructed using astaxanthin, zeaxanthin, and canthaxanthin standard substances, and the amount (mg) of astaxanthin, zeaxanthin, and canthaxanthin produced in a unit culture medium (ℓ) was calculated using this. The average experimental values ​​for three experimental groups under different culture conditions are shown in Figure 5.

[0066] As shown in Figure 5A, the production capacity of astaxanthin, zeaxanthin, and canthaxanthin was observed in the culture temperature range of 20–30℃, and the production of astaxanthin (mg) per liter of culture medium was analyzed to be 0.1–1 mg / ℓ, zeaxanthin production 0.7–3 mg / ℓ, and canthaxanthin production 0.1–0.31 mg / ℓ. Except for zeaxanthin, astaxanthin and canthaxanthin showed higher production capacity at 30℃ than under other culture temperature conditions, so subsequent experiments at different concentrations of NaCl (w / v) and pH were conducted at a culture temperature of 30℃.

[0067] As shown in Figure 5B, the production capacity of astaxanthin, zeaxanthin, and canthaxanthin was confirmed in the range of 1–4% NaCl (w / v), and the production of astaxanthin (mg) per culture medium (ℓ) was analyzed to be 0.1–0.7 mg / ℓ, zeaxanthin production 0.7–2.6 mg / ℓ, and canthaxanthin 0.02–0.16 mg / ℓ. It was confirmed that the production capacity of astaxanthin, astaxanthin, and canthaxanthin was highest under 2% NaCl (w / v) conditions compared to other NaCl (w / v) conditions.

[0068] As shown in Figure 5C, astaxanthin, zeaxanthin, and canthaxanthin production was observed in the pH range of 6 to 8, and astaxanthin (mg) production per culture medium (ℓ) was analyzed to be 0.45–1.28 mg / ℓ, zeaxanthin production 2.51–5.56 mg / ℓ, and canthaxanthin 0.13–0.41 mg / ℓ. It was confirmed that astaxanthin, astaxanthin, and canthaxanthin production showed the highest production capacity under pH 7 conditions compared to other pH conditions.

[0069] Based on these results, it was determined that culture conditions of 30°C, 2% NaCl (w / v), and pH 7 are optimal for the production of astaxanthin, zeaxanthin, and canthaxanthin by the Flaviramulus SSW2303 strain.

[0071] [Example 6] Confirmation of Antioxidant Activity of Flaviramulus SSW2303 Strain

[0072] ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging activity measurement is ABTS + ABTS was used to verify the antioxidant capacity of carotenoids produced from the Flaviramulus SSW2303 strain as a method to measure the reaction in which radicals are removed by antioxidants in the sample. + Radicals were generated by mixing 7.4 mM ABTS(Sigma) and 2.6 mM potassium persulphate in a 1:1 ratio and reacting the mixture in a dark room for 16 hours to form ABTS+ radicals. The formed ABTS + The radical solution was adjusted by diluting with ethanol until an absorbance of 0.700 ± 0.100 was obtained at 734 nm. ABTS + Radical scavenging ability is ABTS + 100 µl of the solution was mixed with 100 µl of methanol extract solutions of Flaviramulus SSW2303 strain diluted in DMSO (Dimethyl Sufoxide, Sigma) solution to concentrations of 25, 50, 100, 200, and 400 µg / ml. After reacting for 20 minutes in a dark room at room temperature, the absorbance was measured at 734 nm. The negative control was ABTS without the methanol extract solution of Flaviramulus SSW2303 strain. + A solution containing only and DMSO was used, and the positive control was prepared by dissolving ascorbic acid (Sigma) in DMSO to a concentration of 20 µg / ml and measured using the same method as the experimental group. ABTS + Radical scavenging was calculated according to the formula below and expressed as a percentage.

[0073] ABTS + Radical scavenging efficacy (%) = 1 - (Absorbance of extract-treated group / Absorbance of negative control group) × 100

[0074] As a result, as shown in Figure 6, it was confirmed that the antioxidant activity of the methanol extract of the Flaviramulus SSW2303 strain increased in a concentration-dependent manner at 5, 50, 100, 200, and 400 μg / ml, and it exhibited an antioxidant activity of 86.5% at 100 μg / ml.

[0076] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will understand that other specific forms can be easily modified without altering the technical spirit or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

[0077] The scope of the present invention is defined by the claims set forth below, and all modifications or variations derived from the meaning and scope of the claims and equivalent concepts thereof should be interpreted as being included within the scope of the present invention.

[0078] Depository Name: Korea Research Institute of Biotechnology and Bioengineering Biological Resource Center (KCTC) Trustee Number: KCTC15595BP Date of Deposit: 2023-09-12

Claims

Claim 1 Flaviramulus SSW2303 deposited as KCTC 15595BP ( Flaviramulus A strain of the genus Flaviramulus SSW2303, wherein the strain is characterized by producing one or more carotenoids selected from the group consisting of astaxanthin, zeazanthin, and canthaxanthin. Claim 2 delete Claim 3 delete Claim 4 A method for producing carotenoids comprising the step of inoculating a strain of the genus Flaviramulus SSW2303, deposited as KCTC 15595BP, into a culture medium under conditions of 30℃, pH 7, and NaCl 2% (w / v). Claim 5 delete Claim 6 An antioxidant cosmetic composition comprising, as an active ingredient, a strain of the genus Flaviramulus SSW2303 deposited at KCTC 15595BP, a culture thereof, a culture extract thereof, a lysate thereof, or a mixture thereof, wherein the strain is characterized by producing one or more carotenoids selected from the group consisting of astaxanthin, zeazanthin, and canthaxanthin. Claim 7 An antioxidant cosmetic composition according to claim 6, characterized in that the composition has radical scavenging activity. Claim 8 An antioxidant health functional food composition comprising, as an active ingredient, a strain of the genus Flaviramulus SSW2303 deposited at KCTC 15595BP, a culture thereof, a culture extract thereof, a lysate thereof, or a mixture thereof, wherein the strain is characterized by producing one or more carotenoids selected from the group consisting of astaxanthin, zeazanthin, and canthaxanthin. Claim 9 A coloring composition for animal feed comprising, as an active ingredient, a strain of the genus Flaviramulus SSW2303 deposited at KCTC 15595BP, a culture thereof, a culture extract thereof, a lysate thereof, or a mixture thereof, wherein the strain is characterized by producing one or more carotenoids selected from the group consisting of astaxanthin, zeazanthin, and canthaxanthin. Claim 10 delete

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