Method for preparing ultraviolet absorbent by utilizing Dermatococcus nishiinomiyaensis and application of method for preparing ultraviolet absorbent by utilizing Dermatococcus nishiinomiyaensis

Abstract

The invention belongs to the field of microbial natural products, and particularly relates to a method for preparing an ultraviolet absorbent by utilizing Dermaccus nishiinomiyaensis and application of the ultraviolet absorbent. The invention relates to a method for separating and preparing an ultraviolet absorbent from pediococcus siuteri, wherein the ultraviolet absorbent is 3-hydroxyacetyl indole, 1-H-indole-3-pyrrole formaldehyde, perindoprine, (E)-3-methylthio acrylic acid, 1-acetyl-beta-carboline and dimethylisorolizine. The invention also relates to a method for preparing the ultraviolet absorbent by separating and preparing the ultraviolet absorbent from the pediococcus siuteri. Compared with the common ultraviolet absorbent benzophenone in the traditional sunscreen cosmetics, the sunscreen composition has stronger ultraviolet absorption capacity, and particularly, the perindoprine, the 1-acetyl-beta-carboline and the dimethylisoxazine have stronger absorption in the wave band of 320-400nm, so that the sunscreen composition can effectively prevent the skin from being tanned. Meanwhile, the ultraviolet absorbents come from coral mucus microorganisms, so that the ultraviolet absorbents have better environmental ecology and biological friendliness compared with the traditionally used chemically synthesized ultraviolet absorbents, and are safer and more environment-friendly to use.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method and application for preparing ultraviolet absorbers using *Dermacoccus nishinomiyaensis*. Background Technology

[0002] Coral reefs are widely distributed in shallow waters between 30 degrees north and south latitude. Exposed to high levels of ultraviolet radiation, coral mucus is rich in UV-resistant substances, thus protecting the coral host from UV damage. A diverse range of microorganisms exist in coral mucus, utilizing the organic nutrients to produce UV-resistant compounds, further protecting the coral host from UV damage.

[0003] Traditional sunscreens use UV absorbers such as benzophenone and cinnamate, which pose risks of skin allergies or cancer. Furthermore, their large-scale use can lead to contamination of rivers, lakes, and oceans, causing adverse effects on the environment and wildlife. In addition, over 98% of sunlight's UVA rays, with wavelengths between 320 and 400 nm, can penetrate the ozone layer and clouds to reach the Earth's surface, reaching the dermis and damaging elastic and collagen fibers, causing sunburn. Less than 2% of UVB rays, with wavelengths between 280 and 320 nm, can also penetrate the ozone layer and reach the Earth's surface, penetrating the stratum corneum and causing skin damage, including erythema, posing a significant health risk. However, currently used traditional UV absorbers have insufficient absorption capacity in the UVA band. Summary of the Invention

[0004] This invention isolates and screens a naturally occurring microorganism with UV-resistant capabilities from coral mucus in the South China Sea: *Dermacoccus nishinomiyaensis* Z-5, an actinomycete, currently deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M 20252318. Six UV absorbers isolated and prepared from this *Dermacoccus nishinomiyaensis* are: 3-hydroxylacetyl-indole, 1-H-indole-3-carboxaldehyde, perlolyrine, (E)-3-(methylthio)acrylic acid, 1-acetyl-β-carboline, and 7,8-dimethylisoalloxazine. Compared to benzophenone, a commonly used UV absorber in traditional sunscreens, these products exhibit stronger UV absorption capabilities. In particular, ligustrazine, 1-acetyl-β-carboline, and dimethylisopyrrolizidine demonstrate very strong UV absorption in the UVA band (320-400nm), where benzophenone absorption is weak. Furthermore, these UV absorbers are derived from coral slime microorganisms, making them more environmentally friendly and biocompatible than traditionally used chemically synthesized UV absorbers, thus ensuring safer use.

[0005] This invention is specifically implemented through the following scheme:

[0006] In a first aspect, the present invention provides a natural strain of Dermacoccus nishinomiyaensis Z-5, which is currently deposited at the China Center for Type Culture Collection, accession number CCTCC M 20252318.

[0007] On the other hand, the present invention provides a method for preparing an ultraviolet absorber, comprising the following steps:

[0008] (1) The *Coccus nidus* was added to YPD medium and cultured to obtain a seed culture;

[0009] (2) Inoculate the seed culture into YPD liquid medium and culture to obtain fermentation broth;

[0010] (3) Add ethyl acetate to the fermentation broth for extraction to obtain an extract. Then, evaporate the extract under reduced pressure to obtain a crude extract.

[0011] (4) The crude extract was separated and purified to obtain 3-hydroxyacetylindole, 1-H-indole-3-pyrrolecarboxaldehyde, ligustrol, (E)-3-methylthioacrylic acid, 1-acetyl-β-carboline, and dimethylisocoridine, which are the ultraviolet absorbers.

[0012] Furthermore, the separation and purification method for the ultraviolet absorber, as described above, includes the following steps:

[0013] (1) Dissolve the crude extract obtained in step (3) in 30-40 ml of methanol, add 40-50 g of 100-200 mesh silica gel powder and evaporate under reduced pressure to obtain the embedded crude extract powder;

[0014] (2) A normal silica column was filled with 100-200 mesh silica powder. The embedded coarse extract powder was loaded onto the column and the following gradient elution was performed using a mixture of petroleum ether and ethyl acetate, and a mixture of dichloromethane and methanol as the mobile phase.

[0015] For 0-10 min, a mixture of petroleum ether and ethyl acetate at a volume ratio of 0:1 was used as mobile phase 1, and the eluent 1 was collected.

[0016] After 10-20 minutes, the eluent 2 was collected by mixing petroleum ether and ethyl acetate at a volume ratio of 1:4 as mobile phase 2.

[0017] For 20-30 minutes, use a mixture of petroleum ether and ethyl acetate in a volume ratio of 2:3 as mobile phase 3, and collect the eluent 3.

[0018] For 30-40 minutes, use a mixture of petroleum ether and ethyl acetate at a volume ratio of 3:2 as mobile phase 4, and collect the eluent 4.

[0019] For 40-50 minutes, use a mixture of petroleum ether and ethyl acetate at a volume ratio of 4:1 as mobile phase 5, and collect the eluent 5.

[0020] After 50-60 minutes, the eluent 6 was collected by mixing petroleum ether and ethyl acetate at a volume ratio of 1:0 as the mobile phase 6.

[0021] After 60-70 minutes, a mixture of dichloromethane and methanol at a volume ratio of 1:0 was used as the mobile phase 7, and the eluent 7 was collected.

[0022] After 70-80 minutes, a mixture of dichloromethane and methanol at a volume ratio of 1:1 was used as the mobile phase 8, and the eluent 8 was collected.

[0023] After 80-90 minutes, a mixture of dichloromethane and methanol at a volume ratio of 0:1 was used as the mobile phase 9, and the eluent 9 was collected.

[0024] (3) Thin-layer chromatography analysis and component combination: eluents 4 and 5 were combined, evaporated under reduced pressure, and the mixture was obtained after evaporation under reduced pressure.

[0025] (4) Add the mixture to ethanol to obtain a mixture solution;

[0026] The mixture solution was added to the chromatographic column at a flow rate of 4 mL / min, and the following elution was performed:

[0027] A mixture of 0.1% formic acid aqueous solution and acetonitrile at a volume ratio of 17:3 was used as mobile phase 10, and the eluent with a retention time of 2.5~3.5 min was collected and denoted as Fr2.

[0028] The eluent with a retention time of 5.5 to 6 min was collected and denoted as Fr3, using a mixture of 0.1% formic acid aqueous solution and acetonitrile at a volume ratio of 8:2 as mobile phase 11.

[0029] The eluent with a retention time of 6-6.5 min was collected and denoted as Fr4, using a mixture of 0.1% formic acid aqueous solution and acetonitrile at a volume ratio of 3:1 as mobile phase 12.

[0030] (5) After distilling the eluent Fr2-4 under reduced pressure, add 4 mL of methanol to dissolve it, centrifuge and collect the supernatant, add it to the chromatographic column at a flow rate of 4 mL / min, and perform the following elution.

[0031] Using a 0.1% formic acid aqueous solution and acetonitrile at a volume ratio of 4:1 as mobile phase 13, the eluent with a retention time of 10-12 min is collected, which is chuanxiong indole;

[0032] A 0.1% formic acid aqueous solution and acetonitrile were mixed at a volume ratio of 17:3 as mobile phase 14. The eluent with a retention time of 11-13 min was collected, which is 1-acetyl-β-carbamoline.

[0033] A mobile phase 15 was prepared by mixing 0.1% formic acid aqueous solution and acetonitrile at a volume ratio of 3:2. The eluent with a retention time of 10-12 min was collected, which is dimethylisopyridine.

[0034] Using a 0.1% formic acid aqueous solution and acetonitrile at a volume ratio of 5:1 as mobile phase 16, the eluent with a retention time of 10-12 min is collected, which is 3-hydroxyacetylindole;

[0035] A mobile phase 17 was prepared by mixing 0.1% formic acid aqueous solution and acetonitrile at a volume ratio of 5:1. The eluent with a retention time of 11-13 min was collected, which is 1-H-indole-3-pyrrolecarboxaldehyde.

[0036] A mobile phase 18 was prepared by mixing 0.1% formic acid aqueous solution and acetonitrile at a volume ratio of 4:1. The eluent with a retention time of 9-11 min was collected, which is (E)-3-methylthioacrylic acid.

[0037] Furthermore, the method for preparing the ultraviolet absorber is characterized in that the chromatographic column is an Agient EclipseXDB-CI8 with dimensions of 9.4×250 mm and a particle size of 5 μm.

[0038] Furthermore, in the preparation method of the ultraviolet absorber, the YPD culture medium formula is as follows: 20 g yeast extract, 20 g glucose, and 10 g peptone are added per liter of pure water, with a pH of 6.4-6.6.

[0039] Thirdly, the present invention provides an ultraviolet absorber, characterized in that it comprises at least one of ligustrazine, 1-acetyl-β-carbazoline, dimethylisocoridine, 3-hydroxyacetylindole, 1-H-indole-3-pyrrolecarboxaldehyde, and (E)-3-methylthioacrylic acid.

[0040] Fourthly, the present invention provides an application of the aforementioned *Penicillium nidulans* in the preparation of anti-ultraviolet products.

[0041] Fifthly, the present invention provides an application of the aforementioned 3-hydroxyacetylindole, 1-H-indole-3-pyrrolecarboxaldehyde, ligustrol, (E)-3-methylthioacrylic acid, 1-acetyl-β-carboline, and dimethylisopyrrolizine as ultraviolet absorbers.

[0042] Furthermore, the application is characterized in that ligustrazine, 1-acetyl-β-carbazoline, dimethylisocoridine, 3-hydroxyacetylindole, 1-H-indole-3-pyrrolecarboxaldehyde, and (E)-3-methylthioacrylic acid absorb ultraviolet light with wavelengths of 280 nm to 400 nm. Attached Figure Description

[0043] Figure 1 UV absorption diagram at 254 nm of natural *Staphylococcus niger* metabolites isolated from coral mucus

[0044] Figure 2 UV absorption at 300 nm of metabolites of *Staphylococcus niger* isolated from coral mucus

[0045] Figure 3 UV absorption spectrum at 365 nm of natural *Staphylococcus niger* metabolites isolated from coral mucus

[0046] Figure 4 DAD detection spectrum of the peak containing perlolyrine

[0047] Figure 5 The peak containing 3-hydroxylacetyl-indole is shown in the DAD detection spectrum.

[0048] Figure 6The peak containing 1-H-indole-3-carboxaldehyde is shown in the DAD detection chromatogram.

[0049] Figure 7 The peak of (E)-3-methylthio)acrylic acid is detected by DAD.

[0050] Figure 8 The peak containing 1-acetyl-β-carboline is shown in the DAD detection spectrum.

[0051] Figure 9 The peak containing 7,8-dimethylisoalloxazine is detected by DAD.

[0052] Figure 10 It is 3-hydroxylacetyl-indole. 13 C NMR spectrum

[0053] Figure 11 It is 3-hydroxylacetyl-indole. 1 H NMR image

[0054] Figure 12 It is 1-H-indole-3-carboxaldehyde. 13 C NMR spectrum

[0055] Figure 13 It is 1-H-indole-3-carboxaldehyde. 1 H NMR image

[0056] Figure 14 It is perlolyrine. 13 C NMR spectrum

[0057] Figure 15 It is perlolyrine. 1 H NMR image

[0058] Figure 16 It is (E)-3-methylthio)acrylic acid. 13 C NMR spectrum

[0059] Figure 17It is (E)-3-methylthio)acrylic acid. 1 H NMR image

[0060] Figure 18 It is 1-acetyl-β-carboline. 13 C NMR spectrum

[0061] Figure 19 It is 1-acetyl-β-carboline. 1 H NMR image

[0062] Figure 20 It is 7,8-dimethylisoalloxazine. 13 C NMR spectrum

[0063] Figure 21 It is 7,8-dimethylisoalloxazine. 1 H NMR image

[0064] Figure 22 The structural formulas of six compounds with ultraviolet absorption capabilities are as follows: (A: 3-hydroxylacetyl-indole, B: 1H-indole-3-carboxaldehyde, C: perlolyrine, D: (E)-3-(methylthio)acrylic acid, E: 1-acetyl-β-carboline, F: 7,8-dimethylisoalloxazine)

[0065] Figure 23 UV absorption spectrum of benzophenone (control)

[0066] Figure 24 UV absorption spectrum of 3-hydroxylacetyl-indole

[0067] Figure 25 UV absorption spectrum of 1-H-indole-3-carboxaldehyde

[0068] Figure 26The ultraviolet absorption spectrum of perlolyrine.

[0069] Figure 27 The UV absorption spectrum of (E)-3-methylthio)acrylic acid.

[0070] Figure 28 UV absorption spectrum of 1-acetyl-β-carboline

[0071] Figure 29 UV absorption spectrum of 7,8-dimethylisoalloxazine Detailed Implementation

[0072] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application. Reagents not specifically described in detail herein are all conventional reagents and are commercially available; methods not specifically described in detail are all conventional experimental methods and can be learned from the prior art.

[0073] It should be noted that the terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion, for example, a process, method, system, product, or device that includes a series of steps or units is not necessarily limited to those steps or units that are explicitly listed, but may include other steps or units that are not explicitly listed or that are inherent to such process, method, product, or device.

[0074] To better understand the invention and not to limit its scope, all figures indicating amounts, percentages, and other numerical values ​​used in this application should, in all cases, be understood to be modified by the word "approximately." Therefore, unless specifically stated otherwise, the numerical parameters listed in the specification and appended claims are approximate values ​​and may vary depending on the desired properties being sought. Each numerical parameter should at least be considered as obtained based on reported significant figures and through conventional rounding methods.

[0075] Example 1: Isolation and Identification of Microorganisms in Coral Slime

[0076] Coral was collected from the coral reef of Xidao Island in Sanya. The coral was washed with sterile artificial seawater, and the mucus was extracted using a sterile pipette and stored in a centrifuge tube. Sterile artificial seawater (formula shown in Table 1) was added and mixed to dilute the coral mucus.

[0077] Add 20 mL of culture medium to a 50 mL centrifuge tube. The culture medium formula is as follows: peptone 5.0 g / L, yeast extract 1.0 g / L, ferric citrate 0.1 g / L, sodium chloride 19.45 g / L, magnesium chloride 5.98 g / L, sodium sulfate 3.24 g / L, calcium chloride 1.8 g / L, potassium chloride 0.55 g / L, sodium carbonate 0.16 g / L, potassium bromide 0.08 g / L, strontium chloride 0.034 g / L, boric acid 0.022 g / L, sodium silicate 0.004 g / L, sodium fluoride 0.0024 g / L, sodium nitrate (or ammonium nitrate) 0.0016 g / L, disodium hydrogen phosphate 0.008 g / L, pH 7.6 ± 0.2 (25℃). Add 2 μL of coral mucus to the culture medium and incubate in a constant temperature shaker at 28℃ and 220 rpm.

[0078] Table 1. Artificial Seawater Formulation

[0079]

[0080] Prepare solid plates for the corresponding culture medium by adding 1.8% agar. Spread 45 μL of the enriched culture solution onto the corresponding solid medium, with three replicates for each medium. Incubate the plates upside down in a 28 ℃ incubator for 7-14 days. Once colonies have grown on the plates, select single colonies based on morphological characteristics for streak plating for further purification.

[0081] In a clean bench, use a sterile pipette tip to pick up a single colony from the purification plate and inoculate it into a 50 mL centrifuge tube containing 20 mL of the liquid culture medium corresponding to the purification plate. Incubate the tube in a constant temperature shaker at 28 ℃ and 220 rpm for 3-4 days to obtain the bacterial suspension.

[0082] In a clean bench, add 1 μL of bacterial culture to a PCR tube. Following the PCR reaction system in Table 2, use universal primers to perform PCR amplification of 16S rDNA and ITS sequences. Perform electrophoresis with 3 μL of the PCR solution. Observe the DNA bands under UV light. Cut the bands and place them in a centrifuge tube for sequencing. Compare the results with the NCBI database to determine the strain species information and complete the strain identification.

[0083] Table 2.16S rDNA PCR System

[0084]

[0085] Example 2: Screening of *Staphylococcus niger* strains with UV resistance

[0086] Colonies were picked from the agar plate and inoculated into 50 mL centrifuge tubes, each containing 20 mL of sterile culture medium with the following formula: peptone 5.0 g / L, yeast extract 1.0 g / L, ferric citrate 0.1 g / L, sodium chloride 19.45 g / L, magnesium chloride 5.98 g / L, sodium sulfate 3.24 g / L, calcium chloride 1.8 g / L, potassium chloride 0.55 g / L, sodium carbonate 0.16 g / L, potassium bromide 0.08 g / L, strontium chloride 0.034 g / L, boric acid 0.022 g / L, sodium silicate 0.004 g / L, sodium fluoride 0.0024 g / L, sodium nitrate 0.0016 g / L, disodium hydrogen phosphate 0.008 g / L, pH 7.4–7.8. The centrifuge tubes were incubated in a constant temperature shaker at 28°C and 220 rpm for 48 h to obtain the seed culture.

[0087] The seed culture was inoculated into 250 mL Erlenmeyer flasks at a 1% (v / v) inoculation rate. Each flask contained 200 mL of YPD medium, formulated with 20 g yeast extract, 20 g glucose, and 10 g peptone per liter of purified water, at pH 6.4-6.6. The culture was incubated for 7 days in a constant-temperature shaker at 30°C and 220 rpm.

[0088] After fermentation, the fermentation broth was obtained by vacuum filtration and extracted with ethyl acetate. The extract was then subjected to rotary evaporation under reduced pressure to remove the solvent ethyl acetate, yielding a crude extract. Methanol was added to the crude extract in small, repeated additions of 4 mL to dissolve the extract. The mixture was centrifuged at 12000 rpm for 5 min to remove solid impurities. 50 μL of the supernatant was transferred to a LC-MS vial with an inner tube for high-performance liquid chromatography (HPLC, Agilent). UV absorption: HPLC: 1260 Infinity II; Column: Agilent Eclipse XDB-C18, 4.6 × 150 mm, 5 μm particle size. Detector: Diode array detector (DAD), detection wavelengths 254 nm (UVC), 290 nm (UVB), and 340 nm (UVA). Injection volume: 10 μL; Column temperature: 28℃. Mobile phase consisted of an aqueous solution containing 0.1% formic acid (phase A) and acetonitrile (phase B), with a flow rate of 1.0 mL / min. Injection volume: 10 μL. Mobile phase gradient: 5% A phase (v / v), 0 min; 5% A phase (v / v), 2 min; 0% A phase (v / v), 22 min; 0% A phase (v / v), 27 min; 5% A phase (v / v), 28 min; 5% A phase (v / v), 30 min; Column temperature: 30℃; Detection wavelength: 254 nm (UVC), 290 nm (UVB), and 340 nm (UVA). Strains with UV-absorbing metabolites were screened based on the number of UV absorption peaks and the total UV absorption peak area. The metabolites of the isolated coral slime actinomycete *Dermacoccus nishinomiyaensis* fermented in YPD medium showed strong UV absorption at 254 nm (UVC), 290 nm (UVB), and 340 nm (UVA). Figure 1-3 (As shown), this strain was therefore prepared as a UV absorber. The strain was deposited at the China Center for Type Culture Collection on October 29, 2025, at Wuhan University, Wuhan, China.

[0089] Example 3: Isolation and purification of ultraviolet-absorbing compounds from the fermentation broth of *Coral Slime Actinomycetes*

[0090] (1) *Coccus nidus* was inoculated into 20 L of YPD medium and cultured in a constant temperature shaker at 30 ℃ and 220 rpm for 7 days. Metabolites in the culture medium were extracted by sonication with 2 volumes of ethyl acetate (2v / v) for 2 h. The extract was then evaporated under reduced pressure to remove the solvent ethyl acetate and obtain a crude extract. The crude extract was dissolved in 30 mL of methanol, and 40 g of 100-200 mesh silica gel powder was added. The mixture was then evaporated under reduced pressure to embed the crude extract components.

[0091] (2) Dissolve the crude extract obtained in step (3) of claim 2 in 30 mL of methanol, add 40 g of 100-200 mesh silica gel powder and evaporate under reduced pressure to obtain the embedded crude extract powder.

[0092] (3) A positive silica column was filled with 100-200 mesh silica powder. The embedded coarse extract powder was loaded onto the column and gradient elution was performed using a mixture of petroleum ether and ethyl acetate, and a mixture of dichloromethane and methanol as the mobile phase.

[0093] A mixture of petroleum ether and ethyl acetate at a volume ratio of 0:1 was used as the mobile phase. The eluent was collected from 0 to 10 min and recorded as eluent 1.

[0094] A mixture of petroleum ether and ethyl acetate in a volume ratio of 1:4 was used as the mobile phase. The eluent was collected for 10-20 min and recorded as eluent 2.

[0095] A mixture of petroleum ether and ethyl acetate in a volume ratio of 2:3 was used as the mobile phase. The eluent was collected after 20-30 min and recorded as eluent 3.

[0096] A mixture of petroleum ether and ethyl acetate in a volume ratio of 3:2 was used as the mobile phase. The eluent was collected after 30-40 minutes and recorded as eluent 4.

[0097] A mixture of petroleum ether and ethyl acetate in a volume ratio of 4:1 was used as the mobile phase. The eluent was collected after 40-50 minutes and recorded as eluent 5.

[0098] Petroleum ether and ethyl acetate were mixed at a volume ratio of 1:0 as the mobile phase, and the eluent was collected after 50-60 min and recorded as eluent 6.

[0099] Dichloromethane and methanol were mixed in a volume ratio of 1:0 as the mobile phase, and the eluent was collected for 60-70 minutes and recorded as eluent 7.

[0100] Dichloromethane and methanol were mixed in a volume ratio of 1:1 as the mobile phase, and the eluent was collected after 70-80 minutes and recorded as eluent 8.

[0101] Dichloromethane and methanol were mixed in a volume ratio of 0:1 as the mobile phase, and the eluent was collected for 80-90 minutes and recorded as eluent 9.

[0102] (4) Spot each eluent onto a thin-layer chromatography silica gel plate for thin-layer chromatography analysis. Combine eluents 4 and 5, evaporate to dryness under reduced pressure, and add the mixture to ethanol to obtain a mixture solution.

[0103] (5) Dissolve the mixture obtained in step (3) in 4 ml of methanol, centrifuge and collect the supernatant. Inject 50 µL of the supernatant into an Agilent 1260 Infinity II series HPLC semi-preparative system at 28 °C. Use an Agilent Eclipse XDB-C18 column (9.4 × 250 mm, 5 μm particle size) and a diode array detector to detect 300 nm UV light. Collect the following peaks using an automatic fraction collector:

[0104] The retention time was 2.5–3.5 min. Elution was performed using a mixture of 0.1% formic acid aqueous solution and acetonitrile at a volume ratio of 17:3 as the mobile phase. The flow rate was 4 mL / min, and the collected eluent was denoted as Fr2.

[0105] The retention time was 5.5-6 min. The eluent was prepared by using a mixture of 0.1% formic acid aqueous solution and acetonitrile at a volume ratio of 8:2 as the mobile phase, the flow rate was 4 mL / min, and the collected eluent was recorded as Fr3.

[0106] The retention time was 6-6.5 min. The eluent was prepared by using a mixture of 0.1% formic acid aqueous solution and acetonitrile at a volume ratio of 3:1 as the mobile phase, the flow rate was 4 mL / min, and the collected eluent was recorded as Fr4.

[0107] (5) After distilling the eluent Fr2~4 in step (4) under reduced pressure, add 4 mL of methanol to dissolve it, centrifuge and take the supernatant. With an injection volume of 50 µL, in an Agilent 1260 Infinity II series HPLC semi-preparative system, use an Agilent Eclipse XDB-C18 column (9.4 × 250 mm, particle size 5 μm) and a diode array detector to detect the UV absorption peaks of the components at a wavelength of 300 nm. Collect each peak.

[0108] In this mixture, phase A was a 0.1% formic acid aqueous solution mixed with phase B acetonitrile at a volume ratio of 4:1, with a retention time of 10-12 min, denoted as peak 4-2, which is ligustrazine (e.g., ligustrazine). Figure 4 (as shown)

[0109] In this mixture, phase A was a 0.1% formic acid aqueous solution mixed with phase B acetonitrile at a volume ratio of 17:3, with a retention time of 11–13 min, denoted as peaks 2–5, representing 1-acetyl-β-carbazoline (e.g., Figure 5 (as shown)

[0110] In this mixture, phase A is a 0.1% formic acid aqueous solution mixed with phase B acetonitrile at a volume ratio of 3:2, with a retention time of 10-12 min, and is designated as peaks 4-5, representing dimethylisopyrrolizine (e.g., Figure 6 (as shown)

[0111] In this mixture, phase A was a 0.1% formic acid aqueous solution mixed with phase B acetonitrile at a volume ratio of 5:1, with a retention time of 10-12 min, denoted as peaks 3-4, representing 3-hydroxyacetylindole (e.g., Figure 7 (as shown)

[0112] In this phase, phase A is a 0.1% formic acid aqueous solution mixed with phase B acetonitrile at a volume ratio of 5:1, with a retention time of 11-13 min, denoted as peaks 3-5, representing 1-H-indole-3-pyrrolecarboxaldehyde (e.g., Figure 8 (as shown)

[0113] In this phase, phase A is a 0.1% formic acid aqueous solution mixed with phase B acetonitrile at a volume ratio of 4:1, with a retention time of 9–11 min, denoted as peak 3-3, which represents (E)-3-methylthioacrylic acid (e.g., Figure 9 (As shown).

[0114] Example 4: Identification of the structure of ultraviolet-absorbing compounds from *Staphylococcus nigricans*

[0115] The prepared pure compound component was dissolved in methanol to prepare a 0.1 mg / mL solution. 500 μL of the methanol solution of the compound was pipetted into a 1.5 mL centrifuge tube and centrifuged at 12000 rpm for 5 min to remove solid impurities.

[0116] 50 μL of supernatant was pipetted into a LC-MS vial with an inner tube and analyzed by LC-MS with an injection volume of 1 μL. The Vanquish UPLC / Q Exactive plus mass spectrometer from Thermo Fisher Scientific was used. LC-MS / MS analysis was performed on a Q Exactive Plus UHPLC system equipped with an ESI ion source. The chromatographic column was an Angilent EC-C18, 2.1 × 50 mm, with a particle size of 1.9 μm. The mobile phase consisted of an aqueous solution of 0.05% formic acid (A) and acetonitrile (B), with an injection volume of 1 μL. The column temperature was 30 °C. The ESI ion source parameters were set as follows: capillary temperature 320 °C, nozzle voltage 3.2 kV, lens RF voltage 50 V, positive ion mode. Mass spectrometric data of the compounds were acquired using a Thermo Fisher Xcalibur 3.0 spectrometer to obtain the molecular weights of the compounds. After acquiring the mass spectrometry data, the raw data was converted to .mzXML format using MSConvertGUI 3.0 software, connected to the GNPs molecular network (https: / / gnps.ucsd.edu / ), and the MS / MS results were compared with the molecular network database to observe whether there were any compounds with high matching degree.

[0117] The structure was then further identified using nuclear magnetic resonance (NMR) spectroscopy (NMR). Deuterated methanol was used as the solvent for the more polar compounds, while deuterated chloroform was used for the less polar compounds. Small, repeated applications of 500 μL of deuterated solvent were added to the sample vial, and the dissolved sample was transferred to the NMR tube until the liquid level reached 3-4 cm. The compounds were then analyzed using a Bruker Avance 600 MHz III NMR spectrometer. 1 H NMR and 13 C NMR analysis (results are as follows) Figure 10-21 (As shown), the spectra were analyzed using MestReNova software. Finally, dimethylisopyrrolizidine was isolated from fraction Fr 2, 3-hydroxyacetylindole and 1-H-indole-3-pyrrolecarboxaldehyde were isolated from fraction Fr 3, and ligustrol, (E)-3-methylthioacrylic acid, and 1-acetyl-β-carboline were isolated from fraction Fr 4. The structural formulas of the compounds are shown below. Figure 22 As shown.

[0118] Example 5: Evaluation of the UV absorption capacity of the compound

[0119] The compound was completely dissolved in methanol to achieve a concentration of 1 mM. 0.364 g of benzophenone was weighed and added to 2 ml of methanol to prepare a 1 mM solution as a positive control, while a pure methanol solution was used as a negative control.

[0120] Absorb 500 Each compound was placed in a methanol solution in a centrifuge tube and centrifuged at 12,000 rpm for 5 min to remove solid impurities. 50 μL of the solution was then collected. The supernatant was transferred to a LC-MS vial with an inner tube and diluted with 10 mL of water. The UV absorption of the compounds was analyzed by HPLC across the entire wavelength range (230-400 nm) using the same conditions as described above. The maximum absorption wavelength of each compound was determined by analyzing its UV absorption spectrum. The absorbance of each compound and benzophenone in the UVA and UVB bands was then compared using a microplate reader. 280 μL of sample was injected. Add 1 mM of the test solution to each well of a 96-well plate, select a wavelength of 280-400 nm and a bandwidth of 2 nm, and read the values ​​three times for each well.

[0121] The UV absorption spectra of the six compounds prepared by isolation are as follows: Figure 24-29 As shown (where Figure 24-27 29. The vertical axis, mAU, represents milliampere, and the horizontal axis represents wavelength. Figure 28 The vertical axis represents AU (Absorbance unit), and the horizontal axis represents wavelength, indicating strong ultraviolet absorption. Compared with the traditional ultraviolet absorber benzophenone, all of them have stronger ultraviolet absorption capabilities, especially perlolyrine, 1-acetyl-β-carbazoline, and dimethylisocoroxazine, which have very strong ultraviolet absorption in the UVA band (320-400 nm) where benzophenone absorption is weak (Table 3).

[0122] Table 3. UV absorbance of the four compounds compared to benzophenone (control).

[0123]

[0124] The above description is only a preferred embodiment of this application, but the scope of protection of this application is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology provided in this application should be included within the scope of protection of this application.

Claims

1. A naturally occurring strain of Dermacoccus nishinomiyaensis Z-5, which is deposited in China Center for Type Culture Collection (CCTCC) and has the accession number CCTCC M 20252318. 2.A method for preparing an ultraviolet absorbent, comprising the following steps: (1) adding the Dermacoccus nishinomiyaensis of claim 1 into YPD culture medium to obtain a seed liquid; (2) inoculating the seed liquid into YPD liquid culture medium to obtain a fermentation liquid; (3) adding ethyl acetate into the fermentation liquid to obtain an extraction liquid, and then performing rotary evaporation under reduced pressure to obtain a crude extract; (4) separating and purifying the crude extract to obtain 3-hydroxyacetylindole, 1-H-indole-3-pyrrole carboxaldehyde, ligustilide, (E)-3-methylthiopropenoic acid, 1-acetyl-β-carboline and dimethyl isoazoline, which are the ultraviolet absorbent. 3.A method for separating and purifying the ultraviolet absorbent of claim 2, comprising the following steps: (1) dissolving the crude extract obtained in step (3) of claim 2 with 30-40 ml of methanol, adding 40-50 g of 100-200 mesh silica gel powder, and then performing rotary evaporation under reduced pressure to obtain embedded crude extract powder; (2) filling a forward silica gel column with 100-200 mesh silica gel powder, and then loading the embedded crude extract powder thereon, and using a mixture of petroleum ether and ethyl acetate, a mixture of dichloromethane and methanol as the mobile phase, and performing the following gradient elution: 0-10 min, using a mixture of petroleum ether and ethyl acetate with a volume ratio of 0:1 as the mobile phase 1 to obtain eluate 1; 10-20 min, using a mixture of petroleum ether and ethyl acetate with a volume ratio of 1:4 as the mobile phase 2 to obtain eluate 2; 20-30 min, using a mixture of petroleum ether and ethyl acetate with a volume ratio of 2:3 as the mobile phase 3 to obtain eluate 3; 30-40 min, using a mixture of petroleum ether and ethyl acetate with a volume ratio of 3:2 as the mobile phase 4 to obtain eluate 4; 40-50 min, using a mixture of petroleum ether and ethyl acetate with a volume ratio of 4:1 as the mobile phase 5 to obtain eluate 5; 50-60 min, using a mixture of petroleum ether and ethyl acetate with a volume ratio of 1:0 as the mobile phase 6 to obtain eluate 6; 60-70 min, using a mixture of dichloromethane and methanol with a volume ratio of 1:0 as the mobile phase 7 to obtain eluate 7; 70-80 min, using a mixture of dichloromethane and methanol with a volume ratio of 1:1 as the mobile phase 8 to obtain eluate 8; 80-90 min, using a mixture of dichloromethane and methanol with a volume ratio of 0:1 as the mobile phase 9 to obtain eluate 9; (3) thin layer chromatography analysis and component combination: combining eluate 4 and 5, and then performing rotary evaporation under reduced pressure to obtain a mixture; (4) adding the mixture into ethanol to obtain a mixture solution; adding the mixture solution into a chromatographic column at a flow rate of 4 mL / min, and performing the following elution: The mixture prepared by mixing 0.1% formic acid aqueous solution and acetonitrile in a volume ratio of 17:3 as mobile phase 10, and collecting the eluate with a retention time of 2.5-3.5 min as Fr2; The mixture prepared by mixing 0.1% formic acid aqueous solution and acetonitrile in a volume ratio of 8:2 as mobile phase 11, and collecting the eluate with a retention time of 5.5-6 min as Fr3; The mixture prepared by mixing 0.1% formic acid aqueous solution and acetonitrile in a volume ratio of 3:1 as mobile phase 12, and collecting the eluate with a retention time of 6-6.5 min as Fr4; (5) After the eluate Fr2-4 is distilled under reduced pressure, 4 mL of methanol is added for dissolution, and the supernatant is obtained by centrifugation and added to the chromatographic column, with a flow rate of 4 mL / min, for the following elution, The mixture prepared by mixing 0.1% formic acid aqueous solution and acetonitrile in a volume ratio of 4:1 as mobile phase 13, and collecting the eluate with a retention time of 10-12 min as ligustilide; The mixture prepared by mixing 0.1% formic acid aqueous solution and acetonitrile in a volume ratio of 17:3 as mobile phase 14, and collecting the eluate with a retention time of 11-13 min as 1-acetyl-β-carboline; The mixture prepared by mixing 0.1% formic acid aqueous solution and acetonitrile in a volume ratio of 3:2 as mobile phase 15, and collecting the eluate with a retention time of 10-12 min as dimethylisoazepine; The mixture prepared by mixing 0.1% formic acid aqueous solution and acetonitrile in a volume ratio of 5:1 as mobile phase 16, and collecting the eluate with a retention time of 10-12 min as 3-hydroxyacetylindole; The mixture prepared by mixing 0.1% formic acid aqueous solution and acetonitrile in a volume ratio of 5:1 as mobile phase 17, and collecting the eluate with a retention time of 11-13 min as 1-H-indole-3-pyrrole aldehyde; The mixture prepared by mixing 0.1% formic acid aqueous solution and acetonitrile in a volume ratio of 4:1 as mobile phase 18, and collecting the eluate with a retention time of 9-11 min as (E)-3-methylthioacrylic acid.

4. The method for preparing the ultraviolet absorber as described in claim 3, characterized in that, The chromatographic column is Agient EclipseXDB-CI8, with a specification of 9.4x250 mm and a particle size of 5 μm.

5. The preparation method of the ultraviolet absorber of claim 2, wherein the YPD culture medium formula is: adding 20 g of yeast extract, 20 g of glucose, and 10 g of peptone per liter of pure water, with a pH of 6.4-6.

6.

6. An ultraviolet absorber characterized by comprising at least one of ligustilide, 1-acetyl-β-carboline, dimethylisoazepine, 3-hydroxyacetylindole, 1-H-indole-3-pyrrole aldehyde, and (E)-3-methylthioacrylic acid.

7. The use of the Myroides pedrosoi of claim 1 in the preparation of anti-ultraviolet products.

8. The use of 3-hydroxyacetylindole, 1-H-indole-3-pyrrole aldehyde, ligustilide, (E)-3-methylthioacrylic acid, 1-acetyl-β-carboline, and dimethylisoazepine as ultraviolet absorbers according to claim 6.

9. Use according to claim 8, characterized in that, Ligustilide, 1-acetyl-beta-carboline, dimethylisoazepine, 3-hydroxyacetylindole, 1-H-indole-3-pyrrole carboxaldehyde, (E)-3-methylthiopropenoic acid absorb UV light at 280 nm-400 nm. Ligustilide, 1-acetyl-beta-carboline, dimethylisoazepine, 3-hydroxyacetylindole, 1-H-indole-3-pyrrole carboxaldehyde, (E)-3-methylthiopropenoic acid absorb UV light at 280 nm-400 nm.

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