Deep-sea fungus sub-isolation chambertia exopolysaccharide DSP-1 and application thereof

Abstract

The application discloses a deep-sea fungus sub-isolation of Aspergillus ustus exopolysaccharide DSP-1 and application thereof. Didymella keratinophila The product of SCAU273 fermentation is separated and purified to obtain an exopolysaccharide DSP-1, the molecular weight of the polysaccharide is 4116 Da, the polysaccharide has a double-branch structure and is mainly formed by glucose polymerization. Further research shows that the polysaccharide DSP-1 can improve the activity of macrophage RAW 264.7 and promote cell proliferation in a wide concentration range (25-400 μg / mL), and in a certain concentration, the polysaccharide dose-dependently promotes the secretion of NO, TNF-alpha, IL-6 and IL-1 beta of RAW264.7 macrophages, and exhibits good immunocompetence on the macrophage RAW 264.7, and has a wide prospect in the production and popularization and application of environment-friendly marine biological agents.

Description

Technical Field

[0001] This invention relates to the field of extracellular polysaccharide preparation technology, specifically to an extracellular polysaccharide DSP-1 from the shell of a deep-sea fungus and its applications. Background Technology

[0002] As the largest ecosystem on Earth, the ocean harbors a diverse array of microorganisms with biotechnological potential. Furthermore, the extreme conditions within marine ecosystems (such as temperature, pH, and salinity) not only encourage the development of unique metabolic pathways in microorganisms but also increase the likelihood of isolating polysaccharides with novel properties. However, despite this immense potential, only a handful of microbial polysaccharides have successfully entered the market, and even fewer are derived from the ocean. While polysaccharides can be produced by various organisms (such as bacteria, fungi, algae, crustaceans, and plants), those derived from bacteria and fungi are highly sought after due to their rapid reproduction, ease of cultivation, and high yield. Moreover, microbial polysaccharides are easier to isolate and extract compared to non-microbial polysaccharides.

[0003] In fact, compounds derived from microorganisms, especially polysaccharides from marine species, have attracted considerable attention due to their wide range of applications in medicine, pharmaceuticals, food, and other industries. For example, those derived from coral-associated fungi... Aspergillus versicolor The extracellular polysaccharide AVP of LCJ-5-4 exhibits a strong superoxide radical scavenging ability; from marine fungi Aspergillus medius A novel polysaccharide called ASMP was isolated from SCAU-236 cells. ASMP significantly affected the proliferation, NO production, and immune cytokines (TNF-α, IL-6, IL-1β) of RAW264.7 macrophages, and may induce ferroptosis by regulating the Nrf2 / SLC7A11 / GPX4 axis and activating ACSL4, thus exhibiting immunomodulatory effects. In summary, the field of polysaccharides has made phased progress in chemical structure analysis, mechanism of action exploration, and practical application expansion.

[0004] Therefore, providing a novel polysaccharide from marine microorganisms and exploring its role in immune regulation is of great significance for the development of new immunomodulatory products. Summary of the Invention

[0005] The purpose of this invention is to overcome the aforementioned defects and shortcomings in the prior art and to provide a deep-sea fungus. Didymella keratinophila SCAU273.

[0006] The second objective of this invention is to provide an extracellular polysaccharide DSP-1 from the shell of a deep-sea fungus, Subseptospora.

[0007] A third objective of this invention is to provide the application of the above-mentioned deep-sea fungal subseptate shell extracellular polysaccharide DSP-1 in the preparation of immunomodulatory products.

[0008] A fourth objective of this invention is to provide a drug comprising the above-mentioned deep-sea fungus Subseptospora exopolysaccharide DSP-1.

[0009] The above-mentioned objective of this invention is achieved through the following technical solution:

[0010] This invention provides a deep-sea fungus Didymella keratinophila SCAU273, the Didymella keratinophila SCAU273 was deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on October 31, 2025, with accession number GDMCC No: 67190.

[0011] This invention involves the isolation, cultivation, purification, and identification of a strain of the genus *Subspora* from deep-sea sediment samples in the South China Sea (9°39′ N, 112°59′ E). Didymella sp . Deep-sea fungi Didymella keratinophila The strain SCAU273 was deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on October 31, 2025, with accession number GDMCCNo: 67190, and classified as follows: Didymella keratinophila The address of the collection is No. 100, Xianlie Middle Road, Guangzhou City, Guangdong Province.

[0012] The present invention also provides an extracellular polysaccharide DSP-1 from the deep-sea fungus Subseptospora shell, which is derived from the above-mentioned deep-sea fungus. Didymella keratinophila Obtained by fermentation and extraction of SCAU273 strain;

[0013] The structural formula of the DSP-1 is shown in equation (Ⅰ):

[0014] .

[0015] This invention relates to the above-mentioned deep-sea fungi Didymella keratinophilaFermentation was performed on SCAU273. After fermentation, the fermentation broth underwent a series of treatments (filtration (filtering mycelia), protein removal, dialysis, and lyophilization) to obtain crude extracellular polysaccharides. The crude polysaccharides were further separated and purified (column chromatography, lyophilization) to obtain a homogeneous fraction, polysaccharide DSP-1. Further molecular weight and purity determination (HPGPC) were performed. The weight-average molecular weight (Mw) of polysaccharide DSP-1 was 4116 Da; the number-average molecular weight (Mn) was 4127 Da; and the peak molecular weight (Mp) was 4289 Da. The monosaccharide composition of polysaccharide DSP-1 was glucose and mannose, with a content ratio of 92% and 8%, respectively. Subseptate shell polysaccharide DSP-1 is a novel polysaccharide with unique structural features. DSP-1 has a bibranched structure, a structural feature extremely rare in marine fungal extracellular polysaccharides. These unique structural properties make DSP-1 a structurally unique marine fungal polysaccharide, and it is expected to provide a new perspective for the study of the structure-function relationship of fungal extracellular polysaccharides.

[0016] Furthermore, the extraction method involves filtering the mycelia and proteins from the fermentation broth, dialysis, and freeze-drying to obtain crude extracellular polysaccharides; the crude extracellular polysaccharides are then separated and purified to obtain the final product.

[0017] Furthermore, the mycelium is filtered by using a Buchner funnel to separate the fermentation broth from the mycelium.

[0018] Furthermore, the molecular weight cutoff during dialysis is 2800–3500 Da.

[0019] Preferably, the molecular weight cutoff of the dialysis bag used for dialysis is 3000 Da.

[0020] Furthermore, the separation is achieved by sequentially eluting with macroporous resin and ion exchange column.

[0021] Preferably, the ion exchange column is a DEAE-52 ion exchange column.

[0022] Preferably, the macroporous resin is of type NKA-9.

[0023] Furthermore, the purification is performed using dextran gel column chromatography.

[0024] Preferably, the dextran gel column is a G-75 gel chromatography column.

[0025] Furthermore, the fermentation medium contains 16–24 g / L mannitol, 6–14 g / L glucose, 16–24 g / L maltose, 1–8 g / L yeast extract, 0.6–1.4 g / L corn steep liquor, 0.4–1.2 g / L magnesium sulfate, 0.1–0.8 g / L potassium dihydrogen phosphate, 0.1–0.8 g / L L-cysteine, 26–34 g / L sea salt, and a pH of 5.3–6.7.

[0026] Preferably, the fermentation medium contains 20 g / L mannitol, 10 g / L glucose, 10 g / L maltose, 3 g / L yeast extract, 1 g / L corn steep liquor, 0.5 g / L magnesium sulfate, 0.5 g / L potassium dihydrogen phosphate, 0.5 g / L L-cysteine, 30 g / L sea salt, and pH=6.0.

[0027] The preparation method of this invention is simple, efficient, easy to operate, and environmentally friendly. Further investigation was conducted into the effects of the extracellular polysaccharide DSP-1 from the deep-sea fungus *Subspinipes* shell prepared in this invention on the viability, NO production, and cytokine secretion of RAW 264.7 cells. The results showed that DSP-1 could enhance the viability of RAW 264.7 cells, promote cell proliferation, and dose-dependently promote the secretion of NO, TNF-α, IL-6, and IL-1β by RAW 264.7 cells. It also exhibited a strong immunostimulatory effect on RAW 264.7 macrophages, significantly influencing the immune response and demonstrating immunomodulatory activity without cytotoxicity. Therefore, the extracellular polysaccharide DSP-1 from the deep-sea fungus *Subspinipes* shell prepared in this invention can regulate macrophage immune activity and has broad prospects for the production and widespread application of environmentally friendly marine biological agents.

[0028] Therefore, this invention also protects the use of the above-mentioned deep-sea fungal subseptospora shell extracellular polysaccharide DSP-1 in the preparation of immunomodulatory products.

[0029] Furthermore, the immune regulation involves enhancing macrophage activity and proliferation, increasing macrophage NO release, and / or promoting macrophage secretion of anti-inflammatory cytokines.

[0030] Furthermore, the macrophages are RAW 264.7 macrophages.

[0031] Furthermore, the anti-inflammatory cytokines are IL-6, IL-1β, and TNF-α.

[0032] The present invention also provides a drug comprising the above-mentioned deep-sea fungus Subseptospora exopolysaccharide DSP-1.

[0033] Furthermore, the concentration of the extracellular polysaccharide DSP-1 from the deep-sea fungus Subseptospora shell in the drug is 25–400 μg / mL.

[0034] Furthermore, the concentration of the deep-sea fungus Subseptospora exopolysaccharide DSP-1 in the drug is 100–200 μg / mL.

[0035] Preferably, the concentration of the deep-sea fungus Subseptospora exopolysaccharide DSP-1 in the drug is 200 μg / mL.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] This invention discloses an extracellular polysaccharide DSP-1 from the shell of a deep-sea fungus, *Subseptospora*, and its applications. Didymella keratinophila The fermentation product of SCAU273 was isolated and purified to obtain an extracellular polysaccharide DSP-1. This polysaccharide has a molecular weight of 4116 Da, a bibranched structure, and is mainly composed of glucose polymers. It is a novel polysaccharide with unique structural features, extremely rare among marine fungal extracellular polysaccharides. Further studies showed that DSP-1 can enhance the activity and promote the proliferation of RAW 264.7 macrophages over a wide concentration range (25–400 μg / mL). Furthermore, at certain concentrations, it dose-dependently promoted the secretion of NO, TNF-α, IL-6, and IL-1β by RAW 264.7 macrophages, exhibiting good immunomodulatory activity against RAW 264.7 macrophages. Therefore, the polysaccharide DSP-1 prepared in this invention has broad prospects for the production and widespread application of environmentally friendly marine biological agents. Attached Figure Description

[0038] Figure 1 The DEAE-52 elution curve of crude polysaccharide DSP is shown.

[0039] Figure 2 This is the HPGPC calibration curve for the standard sample. Figure 2 In this context, a represents lgMp-RT; b represents lgMw-RT; and c represents lgMn-RT.

[0040] Figure 3 The image shows the HPGPC chromatogram of component polysaccharide DSP-1.

[0041] Figure 4 The ion chromatography of the polysaccharide component DSP-1 was performed. Figure 4 In the table, a is a 5 ppm mixed monosaccharide standard; b is DSP-1.

[0042] Figure 5 The infrared scanning spectrum of component polysaccharide DSP-1 is shown.

[0043] Figure 6 The GC-MS chromatogram of the methylated product of the polysaccharide DSP-1 component is shown.

[0044] Figure 7 One-dimensional nuclear magnetic resonance of component polysaccharide DSP-1 1 H NMR spectrum.

[0045] Figure 8 One-dimensional nuclear magnetic resonance of component polysaccharide DSP-1 13 C10 NMR spectrum.

[0046] Figure 9 Two-dimensional polysaccharide DSP-1 1 H- 1 H-COSY spectrum.

[0047] Figure 10 The image shows the two-dimensional HSQC spectrum of the component polysaccharide DSP-1.

[0048] Figure 11 The image shows the two-dimensional HMBC spectrum of the component polysaccharide DSP-1.

[0049] Figure 12 The predicted structure of the component polysaccharide DSP-1.

[0050] Figure 13 The effect of polysaccharide DSP-1 on the viability of RAW 264.7 cells.

[0051] Figure 14 The effect of component polysaccharide DSP-1 on NO release from RAW 264.7.

[0052] Figure 15 The effect of the polysaccharide DSP-1 on the secretion of cytokines by RAW 264.7 was investigated. Figure 15 In the table, a represents IL-6; b represents TNF-α; and c represents IL-1β. Detailed Implementation

[0053] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0054] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0055] The fermentation broth culture medium consisted of: mannitol 20 g / L, glucose 10 g / L, maltose 10 g / L, yeast extract 3 g / L, corn steep liquor 1 g / L, magnesium sulfate 0.5 g / L, potassium dihydrogen phosphate 0.5 g / L, L-cysteine ​​0.5 g / L, sea salt 30 g / L, and pH 6.0.

[0056] Example 1: Extraction, isolation, and purification of DSP-1, an extracellular polysaccharide from the shell of the deep-sea fungus Subseptospora.

[0057] I. Experimental Methods

[0058] 1. Isolation, purification and identification of deep-sea fungi

[0059] Take 1 g of deep-sea sediment sample (sampling depth > 1000 m) collected in the South China Sea (9°39′ N, 112°59′ E), place it in 9 mL of sterilized artificial seawater, vortex for 10 min, and prepare 1×10⁻⁶ ppm. -1 Gradient dilution buffer. Then take 1 mL of 1×10⁻⁶ [diluted buffer -1 The diluent was transferred to 9 mL of sterile artificial seawater and vortexed thoroughly to obtain 1×10⁻⁶ solution. -2 Serial dilution buffer. Using a sterile pipette, aspirate the original sample solution, 1×10⁻⁶, and... -1 and 1×10 -2 200 μL of each serial dilution solution was evenly spread onto the surface of seawater potato dextrose agar (PDA) plates. The seawater PDA medium was prepared according to the instructions, with an additional 30 g / L of sea salt added to simulate the marine environment. Four plates were spread at each concentration and statically incubated at 26°C for 7 days. Colony growth was observed and recorded daily until the morphological differences between different fungi were clearly distinguishable.

[0060] Fungal colonies with diverse phenotypes were screened based on their morphological characteristics (including color, size, edge morphology, degree of elevation, and texture). In a sterile laminar flow hood, target colonies were picked using a sterile inoculation loop and streaked for purification. The streaked plates were then placed in a 26°C incubator for 7 days. After incubation, the plates were examined under a microscope to check for the formation of single colonies. If no pure colonies were obtained, the streaking purification process was repeated, picking suspected single colonies from the first purification and streaking them again until a homogeneous pure culture was obtained.

[0061] A small amount of fungal hyphae purified and grown on PDA medium for one week was transferred to a 1.5 mL sterile EP tube. Fungal DNA was extracted using the Jebes kit according to the kit instructions. The extracted DNA was then used as a template for PCR amplification. Universal primers were used: ITS1: 5'-TCCGTAGGTGAACCTGCGG-3' (SEQ ID No. 1), ITS4: 5'-TCCTCCGCTTATTGATATGC-3' (SEQ ID No. 2). The amplification program was 30 cycles of 94℃ pre-denaturation for 4 min, 94℃ denaturation for 45 s, 55℃ annealing for 45 s, and 72℃ extension for 1 min, followed by a final extension at 72℃ for 10 min.

[0062] Agarose gel electrophoresis was used to verify the quality of PCR products. Agarose gel with a volume ratio of 1.2% and a thickness of 1 mm was prepared; 5 μL of PCR product was added to each well, using a 1000 bp DNA molecular weight standard (DNAladder) as a reference. Electrophoresis was performed in 1×TAE buffer at a constant voltage of 150 V for 20 min. After electrophoresis, the results were observed and recorded using a gel imaging system. PCR products that passed electrophoresis verification were sent to a professional sequencing company for bidirectional sequencing. The obtained ITS gene sequence was submitted to the National Center for Biotechnology Information (NCBI) database, and sequence homology analysis was performed using BLAST to confirm that the strain belonged to the genus *Subspinipes*. Didymella sp.)- Didymella keratinophila Therefore, the strain was named Didymella keratinophila SCAU273, NCBI accession number PX720622, was deposited on October 31, 2025, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), with accession number GDMCC No: 67190, and classified as follows: Didymella keratinophila The address of the collection is No. 100, Xianlie Middle Road, Guangzhou City, Guangdong Province.

[0063] 2. Extraction of extracellular polysaccharides from the shell of *Pseudomonas subsp.*

[0064] (1) Fermentation: The mycelium (subseptate shell) identified in Method 1 was fermented. Didymella keratinophila SCAU273 was aseptically inoculated into 1000 mL shake flasks containing 300–400 mL of fermentation broth. The flasks were incubated at 26°C and 140 r / min for 7 days. A total of 20 L of the culture was fermented.

[0065] (2) Extraction: The fermentation broth and mycelium were separated by filtration using a Buchner funnel, and the obtained fermentation broth was concentrated by rotary vacuum at 60°C and 60 rpm / min.

[0066] (3) Protein removal: Take the crude polysaccharide extract prepared in the previous step, add pre-cooled Sevage reagent at a volume ratio of crude polysaccharide solution to Sevage reagent of 3:1, and stir at room temperature for 20 min on a magnetic stirrer to promote full interaction between the protein and Sevage reagent and selective adsorption to the organic phase. Then place the mixture in a high-speed refrigerated centrifuge and centrifuge at 12000 r / min for 10 min, carefully collect the upper aqueous phase component; repeat the above deproteinization operation until the protein is completely removed. Combine the aqueous phase solutions after multiple deproteinizations to obtain the pre-purified polysaccharide aqueous solution. Slowly transfer the solution into a dialysis bag with a molecular weight cutoff of 3000 Da, and seal the bag opening with a special dialysis bag sealing clamp to prevent leakage. Then immerse the sealed dialysis bag in sufficient distilled water and perform static dialysis at 4℃ for 3 days, changing the fresh distilled water every 8 h to maintain the concentration gradient of the dialysis system and improve the efficiency of impurity removal. After dialysis, the polysaccharide solution was removed from the dialysis bag and transferred to sterile cryovials. The solution was pre-frozen at -80°C for 48 hours until completely frozen, then freeze-dried to constant weight to obtain the deep-sea fungus. Didymella keratinophila SCAU273 extracellular crude polysaccharide DSP.

[0067] (4) Separation: Deep-sea fungi were isolated using macroporous resin (model NKA-9, 150g). Didymella keratinophila Extracellular crude polysaccharide DSP was isolated from SCAU273 cells. After treating the macroporous resin according to the manufacturer's instructions, the crude polysaccharide DSP was prepared into a 500 mg / mL solution with tertiary water, and 5 mL of the solution was slowly loaded onto the resin wall. After equilibration and standing for 5–10 min, the solution was eluted with tertiary water, the eluent was collected, dialyzed, and lyophilized to obtain a homogeneous polysaccharide fraction.

[0068] The crude polysaccharide, after treatment with macroporous resin, was further separated using a DEAE-52 ion exchange column. The packing material was prepared and the column packed according to the instructions, and after standing, three volumes of equilibration solution were formed. Elution was performed using a gradient of different concentrations of NaCl solution (0, 0.1, 0.3, 0.6, 0.9, and 1.2 mol / L), with the peristaltic pump speed controlled at 1 mL / min and the automatic receiver time set to 10 min per tube, i.e., collecting 10 mL per tube. 200 μL from each tube was used to determine the sugar content (phenol-sulfuric acid method), and a elution curve was plotted. The eluent in pure water was collected based on the corresponding elution peaks in the elution curve. The eluent to be retained was injected into a prepared 3000 Da dialysis bag. The eluent was collected and analyzed in parallel tubes, and an elution curve was plotted to obtain a symmetrical single elution peak.

[0069] (5) Purification: The neutral polysaccharide from step (4) was further purified using a G-75 gel chromatography column, eluted with ultrapure water, and finally named the obtained polysaccharide DSP-1.

[0070] II. Experimental Results

[0071] Step (4) Plot the elution curve as follows Figure 1 As shown, a symmetrical single elution peak was obtained.

[0072] Example 2: Purity and basic component analysis of extracellular polysaccharides from the shell of deep-sea fungus Subseptospora.

[0073] I. Experimental Methods

[0074] 1. The molecular weight and purity of the extracellular polysaccharide DSP-1 of the deep-sea fungus Subseptospora shell obtained in Example 1 were determined by high performance gel permeation chromatography (HPGPC).

[0075] Accurately weigh the deep-sea fungus *Subspinoptera* exopolysaccharide DSP-1 sample and dextran standard, and dissolve them separately in 0.05 mol / L NaCl to prepare 5 mg / mL *Subspinoptera* exopolysaccharide DSP solutions and standard solutions of different concentrations. Centrifuge at 12000 rpm for 10 min, filter the supernatant through a 0.22 μm microporous membrane, and place them into sample vials. Use a BRT105-104-102 tandem gel column (8 × 300 mm) and an LC-10A high-performance liquid chromatography system (Shimadzu Corporation, Japan) at 40℃, with 0.05 mol / L NaCl as the mobile phase, a flow rate of 0.6 mL / min, and inject 20 μL. Record the peak time chromatogram using an RI-10A differential detector. A regression equation was established with retention time as the x-axis, yielding calibration curves for lgMp-RT (peak molecular weight), lgMw-RT (weight-average molecular weight), and lgMn-RT (number-average molecular weight). Figure 2 ).

[0076] Based on the standard curve, a calculation formula was derived to calculate the molecular weight of the extracellular polysaccharide DSP-1 of the deep-sea fungus Subseptospora shell.

[0077] 2. Monosaccharide composition: The monosaccharide composition of component polysaccharide DSP-1 was determined by high performance anion exchange chromatography-pulse amperometry (HPAEC-PAD).

[0078] The Thermo ICS 5000+ ion chromatography system was used, and an electrochemical detector was employed to analyze and detect monosaccharide components. Thirteen monosaccharide standards were prepared into 10 mg / mL standard stock solutions. Appropriate amounts of these stock solutions were then mixed to prepare standard mixtures with maximum index concentrations of 60 μg / mL, 50 μg / mL, or 40 μg / mL. The mass of different monosaccharides was determined using an absolute quantification method, and the molar ratio was calculated based on the molar mass of the monosaccharides. A clean chromatographic bottle was used, and an appropriate amount of polysaccharide sample was weighed. 1 mL of 2M TFA acid solution was added, and the mixture was heated at 121℃ for 2 hours. Nitrogen gas was purged, and the sample was dried. The sample was then washed with 99.99% methanol, dried again, and the methanol washing was repeated 2-3 times. The sample was dissolved in sterile water and transferred to a chromatographic bottle for analysis. A Dionex standard was used. TM CarboPac TM PA20 (150*3.0 mm, 10 μm) liquid chromatography column; injection volume: 5 μL. Mobile phase A (H2O), mobile phase B (0.1 M NaOH), mobile phase C (0.1 M NaOH, 0.2 M NaAc), flow rate: 0.5 mL / min; column temperature: 30℃.

[0079] Data analysis: The ion chromatograms of DSP-1 polysaccharide samples and mixed polysaccharide standards were compared to analyze the monosaccharide composition of DSP-1 polysaccharide samples.

[0080] II. Experimental Results

[0081] A regression equation was established with retention time as the x-axis, and the HPGPC calibration curves for the standards were obtained as follows: lgMp-RT (peak molecular weight, a), lgMw-RT (weight-average molecular weight, b), and lgMn-RT (number-average molecular weight, c). Figure 2 As shown.

[0082] Figure 3 The image shows the HPGPC chromatogram of polysaccharide component DSP-1. This result confirms that DSP-1 is a homogeneous component. By comparing with the dextran standard curve and calculating the retention time, the weight-average molecular weight (Mw) of DSP-1 was further determined to be 4116 Da; the number-average molecular weight (Mn) was 4127 Da; and the peak molecular weight (Mp) was 4289 Da.

[0083] like Figure 4 As shown, Figure 4 In Figure 'a', the ion chromatogram of the mixed monosaccharide standard is shown. Figure 4 Figure b shows the ion chromatogram of polysaccharide DSP-1. Analysis and comparison of the ion chromatograms of the two figures indicate that the monosaccharide composition of polysaccharide DSP-1 is glucose and mannose, with contents of 92% and 8%, respectively. This suggests that the extracellular polysaccharide DSP-1 of *Septoria subsp.* is mainly composed of glucose polymers.

[0084] Example 3: Structural analysis of the extracellular polysaccharide DSP-1 from the subseptospora shell

[0085] I. Experimental Methods

[0086] 1. Infrared Spectroscopy: Dry DSP-1 (2 mg) was thoroughly ground and mixed with dry potassium bromide (100 mg). The mixture was then compressed into transparent tablets using a tablet press. The resulting tablets were placed on a Fourier transform infrared spectrometer (Vertex 70, Bruker GmbH, Germany) at 4000–400 cm⁻¹. -1 The frequency range is used to record infrared spectral data.

[0087] 2. Methylation Analysis: Take a small amount of DSP-1 sample (3 mg), dissolve it in 500 µL DMSO, add 1 mg sodium hydroxide and incubate the mixture for 30 minutes. Then add 50 µL iodomethane and allow the reaction to proceed for 1 hour. Add 1 mL water and 2 mL dichloromethane, vortex thoroughly, and centrifuge the mixture. Discard the aqueous phase and repeat the water washing step three times. Collect the lower dichloromethane phase and evaporate it to dryness with a nitrogen stream. Add 100 µL 2M TFA and hydrolyze at 121 °C for 90 minutes, then evaporate the sample at 30 °C. Add 50 µL 2 M ammonia and 50 µL 1 M NaBD4, mix thoroughly, and react at room temperature for 2.5 hours. Add 1 mL water and let stand for 10 minutes. Add 500 μL dichloromethane, vortex, centrifuge, discard the aqueous phase, and repeat the water washing step three times. Finally, the lower dichloromethane phase was collected, and the methylated product sample was determined using an Agilent GCMS 6890A-5977B gas chromatograph-mass spectrometer. The column was a BPX70 (30 m × 0.25 mm × 0.25 µm, SGE, Australia). The injection volume was 1 μL, the split ratio was 10:1, the carrier gas was high-purity helium, and the flow rate was 1.5 mL / min. The column oven was initially set at 140 °C and held for 2.0 min, then programmed to increase to 230 °C at a rate of 3 °C / min and held for 3 min. Data were collected, and the glycosidic bond linkage type of DSP-1 was analyzed.

[0088] 3. Nuclear Magnetic Resonance Spectroscopy Analysis: DSP-1 (40 mg) was dissolved in D2O (550 μL), and then heated in a water bath until completely dissolved. After centrifugation, the supernatant was collected and transferred to an NMR tube. Recording was performed using an AVANCE NEO 600 NMR spectrometer. 1 H-NMR, 13 C-NMR and two-dimensional (2D) NMR spectroscopy: Analysis includes 1 H- 1H-correlation spectroscopy (COSY), heteronuclear single quantum correlation (HSQC), and heteronuclear multibond correlation (HMBC) spectroscopy.

[0089] II. Experimental Results

[0090] The infrared scanning spectrum of polysaccharide DSP-1 is as follows: Figure 5 As shown, 3387 cm -1 The significant absorption peak at [value missing] indicates the presence of typical OH stretching vibration. The stretching and bending vibrations of CH [value missing] are at approximately 2924 cm⁻¹. -1 and 1414 cm -1 An absorption peak is generated at 1359.37 cm⁻¹. The extracyclic CO stretching vibration of the polysaccharide occurs at this point. -1 Found at [location]. 1152 cm -1 and 1021 cm -1 The absorption peak at 925 cm⁻¹ corresponds to the CO bond bending vibration of pyranose, indicating that DSP-1 contains a pyranose structure. -1 and 856 cm -1 The nearby characteristic absorption peaks correspond to α-glycosidic bonds in glucan, indicating that DSP-1 is an α-glucan. Furthermore, the 1700–1735 cm⁻¹ peaks... -1 No absorption peaks were observed within the range, indicating that DSP-1 does not contain uronic acid.

[0091] The GC-MS chromatogram of the methylated product of polysaccharide DSP-1 after methylation treatment is shown in the figure. Figure 6 As shown in Table 1, the sugar residues and their proportions were analyzed by GC-MS. The results showed that the polysaccharide DSP-1 contained four sugar residue linkages: Glcp-(1→(A), →6)-Manp-(1→(B), →4)-Glcp-(1→(C), →4,6)-Glcp-(1→(D), with contents of 16.79%, 8.01%, 58.1%, and 17.1%, respectively. The methylation results were consistent with the monosaccharide composition analysis, indicating that DSP-1 is a dextran containing a small amount of mannose. Based on molecular weight and molar ratio, the composition ratio of each residue in polysaccharide DSP-1 was approximately A∶B∶C∶D = 14∶4∶2∶4.

[0092] Table 1. GC-MS analysis determines the connection type of DSP-1.

[0093]

[0094] DSP-1 1 H and 13 C NMR spectra as follows Figure 7 and Figure 8 As shown, 1Chemical shifts in the 3.2–5.5 ppm signal range in the 1H NMR spectrum revealed polysaccharide characteristics. Except for 4.7 ppm, the anodic signal between 4.3 and 5.5 ppm confirmed that DSP-1 primarily contains the α-configuration of glycan residues. No signal was detected in the 6.5–9.5 ppm concentration range, indicating the absence of aromatic compounds. The proton chemical shifts from 4.1 to 3.2 ppm were attributed to the H2–H6 (C-2–C-6) glycosidic ring. Furthermore, the proton peak at 5.41 ppm was designated as the H-1 chemical shift of the 1,4-linked α-D-Glcp residue, indicating that the configuration of DSP-1 is α-glucan. 13 In the CNMR spectrum, no signal was observed in the 82-88 ppm region indicating the presence of the pyranose ring form of the sugar residues. Further consolidation... 1 HNMR spectroscopy, 13 C NMR spectroscopy, 1 H- 1 H COSY spectrum ( Figure 9 ), HSQC spectroscopy ( Figure 10 The GC-MS results allowed for the differentiation of chemical shifts corresponding to residues A, B, C, and D. Signals at δ 4.97 / 98.65, 3.63 / 72.68, 3.98 / 74.5, 3.68 / 70.32, 3.73 / 73.75, and 3.84 / 60.45 ppm were assigned to the H1 / C1–H6 / C6 of α-D-Glcp-(1→) (residue A). Similarly, the chemical shifts in the NMR spectra were further measured and combined with the GC-MS analysis results to determine that residues B, C, and D were →6)-α-D-Manp-(1→, →4)-α-D-Glcp-(1→, →4,6)-α-D-Glcp-(1→). The specific chemical shifts of residues A and D are determined and recorded in Table 2.

[0095] Table 2. Sugar residues of the component polysaccharide DSP-1 13 C 1 H chemical shift

[0096]

[0097] The key correlation of DSP-1 sugar residues lies in HMBC spectroscopy ( Figure 11 ),according to 1 H- 13Based on the C HMBC spectrum and GC-MS analysis, cross-peaks were observed between δH 5.41 ppm (C:H-1) and δC 74.6 ppm (D:C-4) / δC 69.3 ppm (D:C-6), indicating a correlation between C and D. The structural fragment is →4)-α-D-Glcp-(1→4,6)-α-D-Glcp-(1→). Similarly, the cross-peak between δH 5.41 ppm (C:H-1) and δC 76.66 ppm (C:C-4) indicates a correlation between residue C, with the structural fragment being →4)-α-D-Glcp-(1→4)-α-D-Glcp-(1→). δH 5.37 ppm (D:H-1) and δC 76.66 ppm... The cross-peak between ppm (C:C-4) indicates a correlation between residues D and C, with the structural fragment being →4,6)-α-D-Glcp-(1→4)-α-D-Glcp-(1→). The cross-peak between δH 5.37 ppm (D:H-1) and δC 73.18 ppm (B:C-6) indicates a correlation between residues D and B, with the structural fragment being →4,6)-α-D-Glcp-(1→6)-Manp-(1→). The cross-peak between δH 5.24 ppm (B:H-1) and δC 73.18 ppm (B:C-6) indicates a correlation between residues B, with the structural fragment being →6)-α-D-Manp-(1→6)-α-D-Manp-(1→). The cross-peak between δH 4.97 ppm (A:H-1) and δC 74.6 ppm (D:C-4) / δC 69.3 Cross peaks were observed between ppm (D:C-6), indicating a correlation between A and D. The structural fragment is α-D-Glcp-(1→4,6)-α-D-Glcp-(1→4,6)-α-D-Glcp-(1→4). Cross peaks between δ H 3.64 ppm (C:H-4) and δ C 100.05 ppm (B:C-1) indicate a correlation between residues C and B. The structural fragment is α-D-Glcp-(1→6)-α-D-Manp-(1→4).

[0098] Based on all the above results and combined with the methylation results, the structure of DSP-1 is finally obtained as follows: Figure 12 As shown, DSP-1 has a bibranched structure. These unique structural properties make DSP-1 a structurally unique marine fungal polysaccharide, which is expected to provide a new perspective for the study of the structure-function relationship of fungal extracellular polysaccharides.

[0099] Example 4: Regulation of the immunomodulatory activity of the extracellular polysaccharide DSP-1 in the subseptospora shell

[0100] I. Experimental Methods

[0101] 1. Effect of DSP-1 on RAW 264.7 cell viability

[0102] RAW 264.7 cell viability was assessed using the Cell Counting Kit-8 (CCK-8, Abcam, USA) assay. Aliquots (100 μL) of the cell suspension were transferred to blank culture dishes, and the cell density was adjusted to 1.0 × 10⁻⁶ cells / mL by adding fresh complete culture medium. 4 Cells / mL. Cells were incubated for 24 hours. After discarding the supernatant, cells were treated with DSP-1 at a concentration of 25-400 μg / mL for 24 hours. The blank control group consisted of cells and complete culture medium, while the positive control group received an equal volume of 1 μg / mL LPS. Six replicates were set up for each group, and all plates were incubated at 37°C and 5% CO2 for 24 hours. Subsequently, 10 μL of CCK-8 reagent was added to each well, and cells were incubated at 37°C in the dark for 2 hours. Absorbance was measured at 450 nm, data were recorded, and the effect of DSP-1 on macrophage viability was analyzed.

[0103] 2. The effect of DSP-1 on NO release in RAW 264.7

[0104] The levels of nitric oxide (NO) and cytokines produced in RAW 264.7 cells were assessed using a commercial assay kit. Cells were loaded at a concentration of 1.0 × 10⁶ cells / year. 5 Cells were seeded at a density of 0.5 mL in 6-well plates and incubated at 37°C with 5% CO2 for 24 hours. After removing the culture medium, cells were treated with DSP-1 at concentrations of 0, 25, 50, 100, 200, and 400 μg / mL, with the positive control group treated with 1 μg / mL DSP. After 24 hours of incubation, NO production was measured using Griess reagent according to the kit instructions.

[0105] 3. Effects of DSP-1 on cytokine secretion by RAW 264.7 cells

[0106] The supernatant collected in the above NO detection experiment was used to detect the secretion of interleukin-6 (IL-6), interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) using an enzyme-linked immunosorbent assay (ELISA) kit, and the effect of component polysaccharide DSP-1 on the secretion of cytokines in RAW264.7 cells was analyzed.

[0107] II. Experimental Results

[0108] like Figure 13As shown, cell viability in all treatment groups was higher than that in the control group. DSP-1 was non-toxic to RAW 264.7 cells, and within the concentration range of 25-400 μg / mL, DSP-1 significantly promoted the proliferation of RAW 264.7 macrophages. Particularly at a concentration of 200 μg / mL, DSP-1 achieved the highest cell viability-promoting ability, although it was not higher than the positive control group. However, this result indicates that DSP-1 has a strong ability to promote cell viability.

[0109] The NO release level of RAW 264.7 macrophages after 24 h of DSP-1 treatment was measured using a NO kit. The results are as follows: Figure 14 As shown in the figure. Compared with the blank control, the polysaccharide DSP-1 significantly promoted the release of NO. At a concentration of 200 μg / mL, the NO level induced by DSP-1 reached its highest level. Although it was still lower than that of the positive control group (LPS), this result shows that DSP-1 has a strong immunostimulatory ability.

[0110] like Figure 15 As shown, DSP-1 at concentrations of 25, 50, and 100 μg / mL significantly increased the secretion of IL-6, IL-1β, and TNF-α in the experimental groups. The secretion of IL-6 and TNF-α reached its highest value at a concentration of 100 μg / mL. Under DSP-1 stimulation, the concentration of IL-1β also increased significantly, with its secretion reaching levels comparable to the LPS-treated group at concentrations of 50 and 100 μg / mL, demonstrating a strong pro-inflammatory effect. These results indicate that DSP-1 effectively promotes the secretion of cytokines (IL-6, TNF-α, and IL-1β) in RAW 264.7 macrophages, exhibiting a strong immunomodulatory effect. These results demonstrate that DSP-1 initiates immune activation of mouse RAW 264.7 macrophages by significantly promoting the production of NO, TNF-α, IL-6, and IL-1β, exhibiting a strong immune activation capacity.

Claims

1. A deep-sea fungus Didymella keratinophila SCAU273 is characterized by, The Didymella keratinophila SCAU273 was deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on October 31, 2025, with accession number GDMCC No: 67190.

2. A method for preparing the extracellular polysaccharide DSP-1 from the shell of a deep-sea fungus, characterized in that, The deep-sea fungus described in claim 1 Didymella keratinophila Obtained by fermentation and extraction of SCAU273; The structural formula of the DSP-1 is shown in equation (Ⅰ): 。 3. The method for preparing the extracellular polysaccharide DSP-1 of the deep-sea fungus subseptate shell according to claim 2, characterized in that, The extraction method involves filtering mycelia and proteins from the fermentation broth, dialysis, and freeze-drying to obtain crude extracellular polysaccharides; the crude extracellular polysaccharides are then separated and purified to obtain the final product.

4. The method for preparing the extracellular polysaccharide DSP-1 of the deep-sea fungus subseptate shell according to claim 3, characterized in that, The molecular weight cutoff during dialysis is 2800–3500 Da.

5. The method for preparing the extracellular polysaccharide DSP-1 of the deep-sea fungus subseptate shell according to claim 3, characterized in that, The separation process involves elution using a macroporous resin and an ion exchange column in sequence.

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