Method for preparing ultraviolet absorbent by utilizing Penicillium oxalicum and application

Abstract

The invention belongs to the field of microbial natural products, and particularly relates to a method for preparing an ultraviolet absorbent by utilizing Penicillium oxalicum and application of the ultraviolet absorbent. According to the invention, four ultraviolet absorbents, which are separated and prepared from the penicillium oxalicum, are 4-hydroxyl cetaxel, 6, 7-dihydroxyl benzopyran-2-ketone, 4-hydroxyl cinnamamide and 1-methoxy-3-methyl-9H-carbazole. The ultraviolet absorbent comes from coral mucus microorganisms, and is more environmentally friendly than a traditionally used chemically synthesized ultraviolet absorbent. Compared with the traditional ultraviolet absorbent benzophenone, the ultraviolet absorbent has higher ultraviolet absorption capacity and can replace the traditional chemically synthesized ultraviolet absorbent.

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 Penicillium oxalicum. 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 within 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 ultimately leads to their release into oceans and lakes, causing adverse effects on the environment and wildlife. In addition, over 98% of UVA ultraviolet rays with wavelengths of 320–400 nm can penetrate the ozone layer and clouds to reach the Earth's surface, directly penetrating the dermis and damaging elastic and collagen fibers, causing sunburn and posing a significant health hazard. However, currently used traditional UV absorbers have insufficient absorption capacity in the UVA band. Summary of the Invention

[0004] This invention utilizes *Penicillium oxalicum*, a natural microorganism with UV-resistant capabilities, isolated and screened from coral mucus in the South China Sea. Four UV absorbers isolated and prepared from this microorganism are 3S,4S-4-Hydroxyscytalone, 6,7-Dihydroxycoumarin, 4-Hydroxycinnamamide, and 1-Methoxy-3-methyl-9H-carbazole. Compared to benzophenone, a commonly used UV absorber in traditional sunscreens, these absorbers exhibit stronger UV absorption capabilities, especially in the UVA band (320-400nm), thus effectively preventing skin tanning. Furthermore, these UV absorbers, derived from coral mucus microorganisms, possess better environmental and biocompatibility than traditionally used chemically synthesized UV absorbers, making them safer to use.

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

[0006] In a first aspect, the present invention provides a Penicillium oxalicum M2, which is currently deposited at the China Center for Type Culture Collection, accession number CCTCC M 20252352, on October 27, 2025.

[0007] Secondly, the present invention provides a method for preparing an ultraviolet absorber, characterized by comprising the following steps:

[0008] (1) The Penicillium oxalate was added to 2216E medium and cultured to obtain seed culture;

[0009] (2) Inoculate the seed liquid into rice culture medium and culture it 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 4-hydroxyxetanone, 6,7-dihydroxybenzopyran-2-one, 4-hydroxycinnamoamide and 1-methoxy-3-methyl-9H-carbazole, which are ultraviolet absorbers.

[0012] Furthermore, the preparation method of the ultraviolet absorber includes the following separation and purification steps:

[0013] (1) Dissolve the crude extract obtained in step (3) in 50-60 mL of methanol, add 34-40 g of 100-200 mesh silica gel powder and evaporate under reduced pressure to obtain the embedded crude extract;

[0014] (2) A normal silica column was filled with 100-200 mesh silica powder and 300-400 mesh silica powder. The embedded crude extract 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] From 0 to 30 minutes, the eluent 1 was collected by mixing petroleum ether and ethyl acetate at a volume ratio of 0:1 as mobile phase 1.

[0016] For 30-60 minutes, use a mixture of petroleum ether and ethyl acetate at a volume ratio of 1:4 as mobile phase 2, and collect the eluent 2.

[0017] For 60-90 min, 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 90-120 min, use a mixture of petroleum ether and ethyl acetate in a volume ratio of 3:2 as mobile phase 4, and collect the eluent 4.

[0019] For 120-150 min, 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] For 150-180 min, use a mixture of petroleum ether and ethyl acetate at a volume ratio of 1:0 as mobile phase 6, and collect the eluent 6.

[0021] For 180-210 min, a mixture of dichloromethane and methanol at a volume ratio of 0:1 was used as the mobile phase 7, and the eluent 7 was collected.

[0022] For 210~240 min, a mixture of dichloromethane and methanol at a volume ratio of 1:3 was used as the mobile phase 8, and the eluent 8 was collected.

[0023] For 240-270 min, a mixture of dichloromethane and methanol at a volume ratio of 1:1 was used as the mobile phase 9, and the eluent 9 was collected.

[0024] (3) Thin-layer chromatography analysis and component combination: eluents 1 and 2, 7 and 8 were combined and evaporated under reduced pressure to obtain a mixture;

[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 an aqueous solution of 0.05% formic acid and an acetonitrile solution containing 0.05% formic acid at a volume ratio of 1:9 was used as mobile phase 10. The eluent collected with a retention time of 25-27 min was denoted as Fr2.

[0028] A mobile phase 11 was prepared by mixing an aqueous solution of 0.05% formic acid and an acetonitrile solution containing 0.05% formic acid at a volume ratio of 1:9. The eluent collected with a retention time of 27-28 min was denoted as Fr3.

[0029] A mobile phase 12 was prepared by mixing an aqueous solution of 0.05% formic acid and an acetonitrile solution containing 0.05% formic acid at a volume ratio of 9:1. The eluent collected with a retention time of 28-30 min was denoted as Fr4.

[0030] (5) Add the eluent Fr2-4 to the column at a flow rate of 4 mL / min and perform the following elution.

[0031] A 0.1% formic acid aqueous solution and acetonitrile were mixed at a volume ratio of 5:1 as mobile phase 13. The eluent was collected and retained for 10-12 min, which was 4-hydroxyxetanone.

[0032] A 0.1% formic acid aqueous solution and acetonitrile were mixed at a volume ratio of 4:1 as mobile phase 14. The eluent with a retention time of 8-10 min was collected, which is 6,7-dihydroxybenzopyran-2-one.

[0033] Using a 0.1% formic acid aqueous solution and acetonitrile at a volume ratio of 6:1 as mobile phase 15, the eluent with a retention time of 10-12 min is collected, which is 4-hydroxycinnamate.

[0034] A 0.1% formic acid aqueous solution and acetonitrile were mixed at a volume ratio of 5:1 as mobile phase 16. The eluent with a retention time of 17.5~18.5 min was collected, which is 1-methoxy-3-methyl-9H-carbazole.

[0035] 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.

[0036] Furthermore, the method for preparing the ultraviolet absorber, wherein the 2216E 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 0.0016 g / L, disodium hydrogen phosphate 0.008 g / L, pH 7.4~7.8.

[0037] Furthermore, in the preparation method of the ultraviolet absorber, the rice culture medium formula is as follows: 80 g of rice is added to each liter of artificial seawater, wherein the artificial seawater formula is: NaCl: 132.6 g, MgCl2·6H2O: 26.14 g, MgSO4: 16.53 g, CaCl2: 5.71 g, KCl: 3.63 g, NaHCO3: 1.01 g, NaBr: 0.42 g, distilled water: 5000 mL, pH 7.0-7.2.

[0038] Thirdly, the present invention provides an ultraviolet absorber, characterized in that it comprises at least one of 4-hydroxyxetine, 6,7-dihydroxybenzopyran-2-one, 4-hydroxycinnamoamide, and 1-methoxy-3-methyl-9H-carbazole.

[0039] Fourthly, the present invention provides an application of the aforementioned Penicillium oxalate in the preparation of UV-resistant products.

[0040] Fifthly, the present invention provides the application of the aforementioned 4-hydroxyxetanone, 6,7-dihydroxybenzopyran-2-one, 4-hydroxycinnamoamide, and 1-methoxy-3-methyl-9H-carbazole as ultraviolet absorbers.

[0041] Furthermore, the application is characterized in that 4-hydroxyxetanone, 6,7-dihydroxybenzopyran-2-one, 4-hydroxycinnamoamide, and 1-methoxy-3-methyl-9H-carbazole absorb ultraviolet light with a wavelength of 200~400 nm. Attached Figure Description

[0042] Figure 1 The UV absorption spectrum at 254 nm for the metabolites of natural Penicillium oxalate isolated from coral mucus.

[0043] Figure 2 The UV absorption spectrum at 310 nm for natural Penicillium oxalate metabolites isolated from coral mucus.

[0044] Figure 3 The UV absorption spectrum at 340 nm for natural Penicillium oxalate metabolites isolated from coral mucus.

[0045] Figure 4 DAD detection chromatogram of 4-hydroxyxetanone (3S,4S-4-Hydroxyscytalone)

[0046] Figure 5 DAD detection chromatogram for 6,7-dihydroxybenzopyran-2-one (6,7-Dihydroxycoumarin)

[0047] Figure 6 DAD detection chromatogram of 4-hydroxycinnamamide

[0048] Figure 7 DAD detection chromatogram of 1-methoxy-3-methyl-9H-carbazole

[0049] Figure 8 It is 4-hydroxyxetanone (3S,4S-4-Hydroxyscytalone) 13 C NMR spectrum

[0050] Figure 9 It is 4-hydroxyxetanone (3S,4S-4-Hydroxyscytalone) 1 H NMR image

[0051] Figure 10 It is 6,7-dihydroxybenzopyran-2-one. 13 C NMR spectrum

[0052] Figure 11 It is 6,7-dihydroxybenzopyran-2-one. 1 H NMR image

[0053] Figure 12 It is 4-hydroxycinnamamide. 13 C NMR spectrum

[0054] Figure 13 It is 4-hydroxycinnamamide. 1 H NMR image

[0055] Figure 14 It is 1-methoxy-3-methyl-9H-carbazole. 13 C NMR spectrum

[0056] Figure 15 It is 1-methoxy-3-methyl-9H-carbazole. 1 H NMR image

[0057] Figure 16 The structural formulas of four compounds with ultraviolet absorption capabilities are shown: (A: 3S,4S-4-Hydroxyscytalone, B: 6,7-Dihydroxycoumarin, C: 4-Hydroxycinnamamide, D: 1-Methoxy-3-methyl-9H-carbazole)

[0058] Figure 17 UV absorption spectrum of 4-hydroxyxetanone (3S,4S-4-Hydroxyscytalone)

[0059] Figure 18 UV absorption spectrum of 6,7-dihydroxybenzopyran-2-one (6,7-Dihydroxycoumarin)

[0060] Figure 19UV absorption spectrum of 4-hydroxycinnamamide

[0061] Figure 20 UV absorption spectrum of 1-methoxy-3-methyl-9H-carbazole

[0062] Figure 21 UV absorption spectrum of benzophenone (control) Detailed Implementation

[0063] 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.

[0064] 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.

[0065] 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.

[0066] Example 1: Isolation and Identification of Microorganisms from Coral Slime

[0067] 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.

[0068] 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.

[0069] 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.

[0070] Table 1. Artificial Seawater Formulation

[0071]

[0072] 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.

[0073] Add 1 μL of bacterial culture to a PCR tube and perform PCR amplification of 18S rDNA and ITS sequences using universal primers according to the PCR reaction systems in Tables 2 and 3. Perform electrophoresis with 3 μL of PCR buffer, observe the DNA bands under UV light, cut the bands, place them in a centrifuge tube for sequencing, and compare with the NCBI database to determine the strain species information, thus completing the strain identification.

[0074] Table 2. 18S rDNA PCR system

[0075]

[0076] Table 3. ITS PCR System

[0077]

[0078] Example 2: Screening of natural Penicillium oxalate molds with UV resistance

[0079] Colonies were picked from the agar plates and inoculated into 50 mL centrifuge tubes, each containing 20 mL of 2216E liquid medium with the following formulation: 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. Seed culture was obtained by placing centrifuge tubes in a constant temperature shaker at 28 ℃ and 220 rpm for 48 h.

[0080] The seed culture was inoculated into 250 mL Erlenmeyer flasks at a 1% (v / v) inoculation rate. Each flask contained 100 mL of one of six fungal fermentation media (No. 1: 80 g rice per liter of artificial seawater; No. 2: 5 g yeast extract, 10 g glucose, and 5 g soybean peptone per liter of artificial seawater; No. 3: 20 g soybean meal, 20 g glucose, and 10 g malt extract per liter of artificial seawater; No. 4: 20 g wheat bran, 5 g sucrose, and 10 g peptone per liter of artificial seawater; No. 5: 20 g cottonseed meal, 10 g glucose, and 5 g soybean peptone per liter of artificial seawater; No. 6: 30 g malt extract and 5 g peptone per liter of artificial seawater). The No. 1 rice medium was incubated statically at 28 °C for 30 days. The other five media were incubated in a constant temperature shaker at 28 °C and 220 rpm for 7 days.

[0081] 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. 4 mL of methanol was added to the crude extract in small, repeated additions 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 was analyzed using: 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), 310 nm (UVB), and 340 nm (UVA). The mobile phase consisted of an aqueous solution (A) containing 0.05% formic acid (v / v) and an acetonitrile solution (B) containing 0.05% formic acid (v / v). The injection volume was 10 μL, and the column temperature was 30 °C. Strains exhibiting UV-absorbing metabolites were screened based on the number of UV absorption peaks and the total peak area of ​​UV absorption. The metabolites of the isolated coral slime fungus *Penicillium oxalicum*, fermented in No. 1 rice medium, all showed strong UV absorption at 254 nm (UVC), 310 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 27, 2025, at Wuhan University, Wuhan, China.

[0082] Example 3: Isolation and purification of UV-absorbing compounds from coral slime fungi

[0083] (1) The coral slime fungus *Penicillium oxalicum* was inoculated into a rice culture medium prepared with 20 L of artificial seawater (the formula was: 80 g of rice per liter of artificial seawater). The medium was incubated at room temperature (28 ℃) for 30 days. The metabolites in the culture medium were extracted with three times the volume of ethyl acetate. The extract was then evaporated under reduced pressure to remove the solvent ethyl acetate, yielding a crude extract. The crude extract was dissolved in 50 mL of methanol, and an appropriate amount of 34 g of 100-200 mesh silica gel powder was added. The mixture was then evaporated under reduced pressure to encapsulate the crude extract components.

[0084] (2) Dissolve the crude extract obtained in step (1) in 50 mL of methanol, add 34 g of 100-200 mesh silica powder and evaporate under reduced pressure to obtain the embedded crude extract;

[0085] (3) A positive silica column was filled with 100-200 mesh silica powder and 300-400 mesh silica powder. The embedded crude extract was loaded onto the column and the following gradient elution was performed by sequentially using a mixture of petroleum ether and ethyl acetate, and a mixture of dichloromethane and methanol as the mobile phase.

[0086] 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 30 min and recorded as eluent 1.

[0087] 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 after 30-60 minutes and recorded as eluent 2.

[0088] 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 60-90 min and recorded as eluent 3.

[0089] 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 90-120 min and recorded as eluent 4.

[0090] 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 120-150 min and recorded as eluent 5.

[0091] A mixture of petroleum ether and ethyl acetate at a volume ratio of 1:0 was used as the mobile phase. The eluent was collected after 150-180 min and recorded as eluent 6.

[0092] Dichloromethane and methanol were mixed in a volume ratio of 0:1 as the mobile phase, and the eluent was collected for 180-210 min and recorded as eluent 7.

[0093] Dichloromethane and methanol were mixed in a volume ratio of 1:3 as the mobile phase, and the eluent was collected for 210-240 min and recorded as eluent 8.

[0094] Dichloromethane and methanol were mixed in a volume ratio of 1:1 as the mobile phase, and the eluent was collected after 240-270 min and recorded as eluent 9.

[0095] (4) Spot eluents 1 to 9 onto a thin-layer chromatography silica gel plate for thin-layer chromatography analysis. Combine eluents 1 and 2, 7 and 8, and evaporate to dryness under reduced pressure to obtain a mixture. Add the mixture to ethanol to obtain a mixture solution.

[0096] (5) HPLC was used to trace compounds with ultraviolet absorption: The HPLC instrument was an Agilent Technologies (China) Co., Ltd. 1260 Infinity II series, the column was an Agient Eclipse XDB-CI8, 9.4×250 mm, particle size 5 μm, and the detector was a diode array detector, detecting short-wave ultraviolet light at 254 nm, medium-wave ultraviolet light at 310 nm, and long-wave ultraviolet light at 340 nm. The mobile phase consisted of an aqueous solution containing 0.05% formic acid and an acetonitrile solution containing 0.05% formic acid. The injection volume was 10 µL, and the column temperature was 30 ℃. The mixture was added to the column, eluted, and the following eluent was collected:

[0097] The retention time was 25–27 min. Elution was performed using a mixture of an aqueous solution of 0.05% formic acid and an acetonitrile solution containing 0.05% formic acid at a volume ratio of 1:9, at a flow rate of 4 mL / min. The collected eluent was denoted as Fr2.

[0098] The retention time was 27–28 min. Elution was performed using a mixture of an aqueous solution of 0.05% formic acid and an acetonitrile solution containing 0.05% formic acid at a volume ratio of 1:9 as the mobile phase. The flow rate was 4 mL / min, and the collected eluent was denoted as Fr3.

[0099] The retention time was 28–30 min. Elution was performed using a mixture of an aqueous solution of 0.05% formic acid and an acetonitrile solution containing 0.05% formic acid at a volume ratio of 9:1 as the mobile phase. The flow rate was 4 mL / min, and the collected eluent was denoted as Fr4.

[0100] (6) The eluent Fr2-4 obtained in step (5) was dissolved in 4 mL of methanol after vacuum distillation, and the supernatant was collected by centrifugation. The supernatant was injected at a volume of 50 µL into an Agilent 1260 Infinity II series HPLC semi-preparative system. The chromatographic column was Agilent Eclipse XDB-C18, with a size of 9.4 × 250 mm and a particle size of 5 μm. The detector was a diode array detector with a detection wavelength of 300 nm for medium-wave ultraviolet light. Each peak was collected by an automatic fraction collector.

[0101] In this process, 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 10-12 min, which is 4-hydroxyxetanone (e.g., ...). Figure 4 (as shown)

[0102] In this mixture, 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 8-10 min, which is 6,7-dihydroxybenzopyran-2-one (e.g. Figure 5 (as shown)

[0103] In this mixture, phase A is a 0.1% formic acid aqueous solution mixed with phase B acetonitrile at a volume ratio of 6:1, with a retention time of 10-12 min, which is 4-hydroxycinnamate (e.g., ...). Figure 6 (as shown)

[0104] In this process, phase A (0.1% formic acid aqueous solution) and phase B (acetonitrile) are mixed at a volume ratio of 5:1, with a retention time of 17.5–18.5 min, to produce 1-methoxy-3-methyl-9H-carbazole (e.g. Figure 7 (As shown).

[0105] Example 4: Structural Identification of Ultraviolet Absorbing Compounds from Coral Slime Fungi

[0106] The pure compound component with ultraviolet absorption prepared above 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.

[0107] 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.

[0108] 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 8-15 (As shown), the spectra were analyzed using MestReNova software to determine the structural formulas of the compounds. The structural formulas of the four compounds are as follows: Figure 16 As shown.

[0109] Example 5 Evaluation of the ultraviolet absorption capacity of compounds

[0110] 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, which served as a positive control. Pure methanol solution was used as a negative control.

[0111] Pipette 500 μL of each compound solution into a centrifuge tube and centrifuge at 12000 rpm for 5 min. Transfer 50 μL of the supernatant to a LC-MS vial with an inner tube. Perform HPLC full-wavelength (190-400 nm) UV absorption analysis of the compounds using a 10 μL injection volume (under the same conditions as above). Determine the maximum absorption wavelength of each compound by analyzing its UV absorption spectrum. Compare the absorbance of each compound with that of benzophenone in the UVA and UVB bands using a microplate reader. Add 280 μL of each 1 mM test solution to a 96-well plate, select a wavelength of 280-400 nm and a bandwidth of 2 nm, and read the data three times per well, taking the average value.

[0112] The UV absorption spectra of the four compounds prepared by separation are as follows: Figure 17-20 As shown in the figure (where the vertical axis represents the response value, with an upper limit of 100 indicating an ultraviolet absorption intensity of 3000 mAU at the corresponding wavelength, and the horizontal axis represents the wavelength), it can be seen that it has a strong ultraviolet absorption capacity. Compared with the traditional ultraviolet absorber benzophenone, it has a stronger ultraviolet absorption capacity (e.g., Figure 21 As shown in Table 4, 6,7-dihydroxybenzopyran-2-one and 4-hydroxyxetanone exhibit very strong UV absorption, especially in the UVA band (320-400 nm) where benzophenone absorption is weak (Table 4).

[0113] Table 4. UV absorbance of the four compounds compared to benzophenone (control).

[0114]

[0115] 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 strain of Penicillium oxalicum M2, currently deposited at the China Center for Type Culture Collection, accession number CCTCC M 20252352.

2. A method for preparing an ultraviolet absorber, comprising the following steps: (1) The Penicillium oxalate strain described in claim 1 is added to 2216E medium to obtain a seed culture; (2) The seed liquid was inoculated into rice culture medium and cultured to obtain fermentation broth; (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. (4) The crude extract was separated and purified to obtain 4-hydroxyxetanone, 6,7-dihydroxybenzopyran-2-one, 4-hydroxycinnamoamide and 1-methoxy-3-methyl-9H-carbazole, which are ultraviolet absorbers.

3. The preparation method of the ultraviolet absorber as described in claim 2, wherein the separation and purification steps are as follows: (1) Dissolve the crude extract obtained in step (3) of claim 2 in 50-60 mL of methanol, add 34-40 g of 100-200 mesh silica gel powder and evaporate under reduced pressure to obtain the embedded crude extract; (2) A normal silica column was filled with 100-200 mesh silica powder and 300-400 mesh silica powder. The embedded crude extract 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. From 0 to 30 minutes, the eluent 1 was collected by mixing petroleum ether and ethyl acetate at a volume ratio of 0:1 as mobile phase 1. For 30-60 minutes, use a mixture of petroleum ether and ethyl acetate at a volume ratio of 1:4 as mobile phase 2, and collect the eluent 2. For 60-90 min, 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. For 90-120 min, use a mixture of petroleum ether and ethyl acetate in a volume ratio of 3:2 as mobile phase 4, and collect the eluent 4. For 120-150 min, 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. For 150-180 min, use a mixture of petroleum ether and ethyl acetate at a volume ratio of 1:0 as mobile phase 6, and collect the eluent 6. The eluent 7 was collected after 180-210 min of a mixture of dichloromethane and methanol at a volume ratio of 0:

1. For 210~240 min, a mixture of dichloromethane and methanol at a volume ratio of 1:3 was used as the mobile phase 8, and the eluent 8 was collected. For 240~270 min, a mixture of dichloromethane and methanol at a volume ratio of 1:1 was used as the mobile phase 9, and the eluent 9 was collected. (3) Thin-layer chromatography analysis and component combination: eluents 1 and 2, 7 and 8 were combined and evaporated under reduced pressure to obtain a mixture; (4) Add the mixture to ethanol to obtain a mixture solution; The mixture solution was added to the chromatographic column at a flow rate of 4 mL / min, and the following elution was performed: A mixture of an aqueous solution of 0.05% formic acid and an acetonitrile solution containing 0.05% formic acid at a volume ratio of 1:9 was used as mobile phase 10. The eluent collected with a retention time of 25-27 min was denoted as Fr2. A mobile phase 11 was prepared by mixing an aqueous solution of 0.05% formic acid and an acetonitrile solution containing 0.05% formic acid at a volume ratio of 1:

9. The eluent collected with a retention time of 27-28 min was denoted as Fr3. A mobile phase 12 was prepared by mixing an aqueous solution of 0.05% formic acid and an acetonitrile solution containing 0.05% formic acid at a volume ratio of 9:

1. The eluent collected with a retention time of 28-30 min was denoted as Fr4. (5) After distilling the eluent Fr2-4 under reduced pressure, add 4 mL of methanol to dissolve it, centrifuge to collect the supernatant, add it to the chromatographic column, and perform the following elution at a flow rate of 4 mL / min. A 0.1% formic acid aqueous solution and acetonitrile were mixed at a volume ratio of 5:1 as mobile phase 13. The eluent was collected and retained for 10-12 min, which was 4-hydroxyxetanone. A 0.1% formic acid aqueous solution and acetonitrile were mixed at a volume ratio of 4:1 as mobile phase 14. The eluent with a retention time of 8-10 min was collected, which is 6,7-dihydroxybenzopyran-2-one. Using a 0.1% formic acid aqueous solution and acetonitrile at a volume ratio of 6:1 as mobile phase 15, the eluent with a retention time of 10-12 min is collected, which is 4-hydroxycinnamate. A 0.1% formic acid aqueous solution and acetonitrile were mixed at a volume ratio of 5:1 as mobile phase 16. The eluent with a retention time of 17.5~18.5 min was collected, which is 1-methoxy-3-methyl-9H-carbazole.

4. The method for preparing the ultraviolet absorber as described in claim 3, characterized in that, The chromatographic column was an Agient EclipseXDB-CI8, with dimensions of 9.4 x 250 mm and a particle size of 5 μm.

5. The method for preparing the ultraviolet absorber according to claim 2, wherein the 2216E 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 0.0016 g / L, disodium hydrogen phosphate 0.008 g / L, pH 7.4~7.

8.

6. The method for preparing the ultraviolet absorber according to claim 2, wherein the rice culture medium formula is as follows: 80 g of rice is added to each liter of artificial seawater, wherein the artificial seawater formula is: NaCl: 132.6 g, MgCl2·6H2O: 26.14 g, MgSO4: 16.53 g, CaCl2: 5.71 g, KCl: 3.63 g, NaHCO3: 1.01 g, NaBr: 0.42 g, distilled water: 5000 mL, pH 7.0-7.

2.

7. An ultraviolet absorber, characterized in that, It includes at least one of 4-hydroxyxetanone, 6,7-dihydroxybenzopyran-2-one, 4-hydroxycinnamoamide, and 1-methoxy-3-methyl-9H-carbazole.

8. The use of Penicillium oxalate of claim 1 in the preparation of UV-resistant products.

9. The use of 4-hydroxyxetanone, 6,7-dihydroxybenzopyran-2-one, 4-hydroxycinnamoamide, and 1-methoxy-3-methyl-9H-carbazole as ultraviolet absorbers as described in claim 7.

10. The application as described in claim 9, characterized in that, 4-Hydroxyxetine, 6,7-dihydroxybenzopyran-2-one, 4-hydroxycinnamoamide, and 1-methoxy-3-methyl-9H-carbazole absorb ultraviolet light with wavelengths of 200-400 nm.

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