A deep-sea-derived aspergillus versicolor scau214, a preparation method and application of exopolysaccharide avp-214-1 thereof

Abstract

The application discloses a deep-sea-derived Aspergillus versicolor SCAU214, and a preparation method and application of exopolysaccharide AVP-214-1 thereof. The Aspergillus versicolor SCAU214 has been preserved in the Guangdong Microbial Culture Collection Center on June 25, 2023, and has a preservation number of GDMCC No: 63583. Fermentation culture is carried out on the Aspergillus versicolor SCAU214, and a metabolite thereof is extracted and purified to obtain the exopolysaccharide AVP-214-1 thereof. The exopolysaccharide AVP-214-1 can promote the proliferation of macrophage RAW 264.7 in a wide concentration range (12.5-400 μg / mL), the NO release amount is greater than that of a positive control group, and the exopolysaccharide AVP-214-1 dose-dependently stimulates the secretion of cytokines IL-6 and TNF-alpha of the macrophage RAW 264.7, has a strong immune stimulation effect on the macrophage, and has a safe and efficient immune activity regulating effect.

Description

Technical Field

[0001] This invention relates to the field of marine microbial technology, specifically to a method for preparing and applying Aspergillus variegata SCAU214 and its extracellular polysaccharide AVP-214-1 from deep sea sources. Background Technology

[0002] Currently, existing methods in industries such as chemicals, food, and cosmetics largely rely on chemical agents, which may cause irreversible damage to the environment or human health during use. In contrast, biological extracellular polysaccharides are biodegradable and generally non-toxic. Polysaccharides are the most abundant organic matter in the world, used in multiple industrial sectors as thickeners, stabilizers, and gelling agents in food, and as antitumor, antioxidant, and / or prebiotics in pharmacology. Common sources of industrial polysaccharides are plants, animals, fungi, algae, and bacteria, with algae currently being the primary source. However, microbial extracellular polysaccharides have unique advantages: because the growth conditions of microorganisms can be strictly monitored, fungi and bacteria have the advantage of high structural reproducibility, which is impossible with plant and animal sources, as the structure of plant and animal polysaccharides depends on uncontrollable conditions such as climate, environment, and feed.

[0003] Furthermore, the ocean, due to its unique environmental conditions, is one of the most biodiverse habitats on Earth, with the deep-sea environment being even more extreme: lack of sunlight, low temperatures, anaerobic conditions, and high hydrostatic pressure. To adapt to this environment, deep-sea fungi have evolved unique physiological structures and metabolic pathways, producing novel extracellular polysaccharides that exhibit a wide range of biological activities. These bioactive compounds not only protect the fungi themselves but also provide candidate compounds for marine drug development. Deep-sea Aspergillus is an important component of deep-sea fungi. The metabolites of Aspergillus include various structural types such as polyketides, alkaloids, lactones, and terpenes. Aspergillus versicolor, as a species of Aspergillus, can produce a variety of useful active metabolites, possessing diverse biological activities including disease resistance, insect resistance, antiviral activity, antitumor activity, antioxidant activity, cellular immunity, and anti-inflammatory activity.

[0004] However, the environmental conditions for Aspergillus growth and the extraction methods of its metabolites significantly affect the bioactivity of the obtained polysaccharides. Furthermore, the currently disclosed Aspergillus polysaccharides still exhibit some toxicity to cells and low immunogenicity at slightly higher concentrations, necessitating the discovery of other safe and efficient Aspergillus metabolites. Summary of the Invention

[0005] The purpose of this invention is to overcome the above-mentioned deficiencies of the prior art and to provide a method for preparing Aspergillus variegata SCAU214 and its extracellular polysaccharide AVP-214-1 from deep sea and its application.

[0006] The first objective of this invention is to provide a *Aspergillus versicolor* SCAU214.

[0007] A second objective of this invention is to provide a polysaccharide.

[0008] A third object of the present invention is to provide the use of the Aspergillus versicolor SCAU214 or the polysaccharide in the preparation of products that modulate immune activity.

[0009] The fourth objective of this invention is to provide a method for preparing polysaccharides.

[0010] The fifth objective of this invention is to provide a product that modulates immune activity.

[0011] To achieve the above objectives, the present invention is implemented through the following solution:

[0012] A species of Aspergillus versicolor, SCAU214, was deposited at the Guangdong Provincial Center for Microbial Culture Collection on June 25, 2023, with accession number GDMCC No: 63583.

[0013] This invention also claims protection for a polysaccharide, the structural formula of which is shown in Formula I:

[0014]

[0015] Based on this, the present invention seeks protection for the use of Aspergillus versicolor SCAU214 or the polysaccharide in the preparation of products that modulate immune activity.

[0016] The present invention also provides a method for preparing a polysaccharide, which is obtained by fermentation of the aforementioned Aspergillus versicolor SCAU214.

[0017] Further, the fermentation product of Aspergillus versicolor SCAU214 was filtered and concentrated to obtain a filtered and concentrated fermentation product.

[0018] Further, the proteins in the concentrated fermentation product are removed by filtration to obtain crude polysaccharide. The structural formula of the purified polysaccharide is shown in Formula I:

[0019]

[0020] Furthermore, the crude polysaccharide is purified by macroporous resin separation to obtain macroporous resin-purified polysaccharide.

[0021] Furthermore, the polysaccharide purified by macroporous resin is further separated by ion exchange column separation to obtain purified polysaccharide.

[0022] The present invention also provides a product for regulating immune activity, characterized in that the product contains Aspergillus versicolor SCAU214 and / or its fermentation products.

[0023] The present invention also provides a product for regulating immune activity, characterized in that the product contains polysaccharides.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] This invention isolates Aspergillus versicolor SCAU214 from deep-sea sediment samples in the Mariana Trench (141°57'N, 10°51'E), ferments and culturees it, extracts and purifies its metabolites, and obtains its extracellular polysaccharide AVP-214-1. AVP-214-1 promotes the proliferation of RAW 264.7 macrophages over a wide concentration range (12.5–400 μg / mL), with higher NO release than the positive control group. It also dose-dependently stimulates the secretion of cytokines IL-6 and TNF-α by RAW 264.7 macrophages, exhibiting a strong immunostimulatory effect on macrophages. This invention demonstrates a safe and highly effective immunomodulatory activity. Attached Figure Description

[0026] Figure 1 Phylogenetic tree of Aspergillus versicolor SCAU214.

[0027] Figure 2 DEAE-52 elution curve of extracellular polysaccharide AVP-214-1 from Aspergillus versicolor SCAU214.

[0028] Figure 3 The image shows the HPGPC peaks of the extracellular polysaccharide AVP-214-1 from Aspergillus versicolor SCAU214.

[0029] Figure 4 The image shows the ion chromatograms of the extracellular polysaccharide AVP-214-1 from Aspergillus versicolor SCAU214. In the image, A is the ion chromatogram of the mixed monosaccharide standard; B is the ion chromatogram of the polysaccharide AVP-214-1.

[0030] Figure 5 The GCMS chromatogram shows the acetylation product of the extracellular polysaccharide AVP-214-1 of Aspergillus versicolor SCAU214.

[0031] Figure 6 Mass spectrum of residue A of methylated derivative of AVP-214-1, an extracellular polysaccharide of Aspergillus versicolor SCAU214.

[0032] Figure 7 This is the B-residue mass spectrum of a methylated derivative of the extracellular polysaccharide AVP-214-1 from Aspergillus versicolor SCAU214.

[0033] Figure 8 This is the C-residue mass spectrum of a methylated derivative of the extracellular polysaccharide AVP-214-1 from Aspergillus versicolor SCAU214.

[0034] Figure 9 The mass spectrum of residue D of the methylated derivative of the extracellular polysaccharide AVP-214-1 of Aspergillus versicolor SCAU214.

[0035] Figure 10 The E-mass spectrum of the methylated derivative of the extracellular polysaccharide AVP-214-1 of Aspergillus versicolor SCAU214.

[0036] Figure 11 The F-mass spectrum of the residues of the methylated derivative of the extracellular polysaccharide AVP-214-1 of Aspergillus versicolor SCAU214.

[0037] Figure 12 This is the G-residue mass spectrum of a methylated derivative of the extracellular polysaccharide AVP-214-1 from Aspergillus versicolor SCAU214.

[0038] Figure 13 The mass spectrum of residue H of the methylated derivative of the extracellular polysaccharide AVP-214-1 of Aspergillus versicolor SCAU214.

[0039] Figure 14One-dimensional nuclear magnetic resonance imaging of the extracellular polysaccharide AVP-214-1 of Aspergillus versicolor SCAU214 1 H-NMR spectrum.

[0040] Figure 15 One-dimensional nuclear magnetic resonance imaging of the extracellular polysaccharide AVP-214-1 of Aspergillus versicolor SCAU214 13 C-NMR spectrum.

[0041] Figure 16 Two-dimensional extracellular polysaccharide AVP-214-1 of Aspergillus versicolor SCAU214 1 H- 1 H-COSY spectrum.

[0042] Figure 17 Two-dimensional HMBC spectrum of extracellular polysaccharide AVP-214-1 from Aspergillus versicolor SCAU214.

[0043] Figure 18 Two-dimensional HSQC spectrum of extracellular polysaccharide AVP-214-1 from Aspergillus versicolor SCAU214.

[0044] Figure 19 The effect of the extracellular polysaccharide AVP-214-1 from Aspergillus versicolor SCAU214 on the proliferation of RAW264.7 macrophages.

[0045] Figure 20 The effect of the extracellular polysaccharide AVP-214-1 of Aspergillus versicolor SCAU214 on NO secretion by RAW264.7 macrophages.

[0046] Figure 21 The effect of the extracellular polysaccharide AVP-214-1 from Aspergillus versicolor SCAU214 on the secretion of cytokines IL-6 and TNF-α by RAW264.7 was investigated. Where A represents IL-6 and B represents TNF-α; compared with the blank control group, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001. Detailed Implementation

[0047] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0048] Example 1: Isolation, purification and identification of Aspergillus versicolor SCAU214

[0049] I. Experimental Methods

[0050] 1. Sample collection and incubation: Deep-sea sediment samples were collected in the Mariana Trench (141°57'N, 10°51'E) at a depth of 5455 meters. 1.0 g of sediment sample was weighed in a clean bench, and 9 mL of sterile distilled water containing 0.1% agar was added. The mixture was shaken well to form a sample suspension, labeled as a concentration of 10. -1 A sample suspension with a concentration of 10 g / mL. Take 1 mL of this suspension. -1 Add 9 mL of sterile distilled water containing 0.1% agar to the above sample suspension at a concentration of g / mL, mix well, and label as concentration 10. -2 Sample suspension at g / mL.

[0051] 2. Sample Inoculation: Prepare Red Sea PDA medium containing 0.5 g / L penicillin and 0.03 g / L Bengal rose. After autoclaving, allow it to stand until the temperature reaches approximately 40°C, then mix thoroughly and pour into petri dishes. Allow it to solidify to obtain a solid medium. Take 200 μL of sample suspensions at different concentration gradients and spread them evenly on the solid medium, with three plates for each concentration. Incubate at 10°C until the water evaporates and there is no liquid on the surface of the medium, then invert the plates for further incubation.

[0052] 3. Isolation of bacterial strains: Spread the strains from step 2 onto plates and incubate at 4°C for 30 days until the morphology of fungi and bacteria can be distinguished. Then, based on their morphological differences, transfer them to a new culture medium. Incubate the resulting culture dishes at 4°C for purification. The selected purified strains showed rapid colony growth on PDA medium, forming round colonies that were initially white, turning green after 2-3 days, and brown after 4-5 days. The conidial heads had hemispherical vesicles with double-layered pedicels, the first layer being shorter and the second longer, arranged radially, with chain-like spores at the apex.

[0053] 4. Strain identification: Extract the genome of the selected purified strain and amplify its ITSDNA gene by PCR. The PCR amplification conditions are: 98℃ pre-mutation for 10 min; 98℃ mutation for 10 s, 55℃ annealing for 10 s, 72℃ extension for 1 min; and finally 72℃ extension for 1 min.

[0054] II. Experimental Results

[0055] like Figure 1 As shown, the selected purified strain (whose ITS sequence accession number in GenBank is OK271389) has a 99% similarity to the ITS sequence of the standard strain Aspergillus versicolor (MH712291) in GenBank. It was named Aspergillus versicolor SCAU214 and deposited at the Guangdong Provincial Microbial Culture Collection Center on June 25, 2023, with accession number GDMCC No: 63583. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0056] Example 2: Extraction, separation and purification of polysaccharide AVP-214-1

[0057] I. Experimental Methods

[0058] Fermentation broth culture medium was prepared as follows: The medium for wheat isolation consisted of 5 g / L malt extract, 5 g / L Czapek Dox broth, 10 g / L peptone, 5 g / L potato glucose broth, 0.5 g / L magnesium chloride, 0.5 g / L calcium chloride, 0.5 g / L potassium chloride, 0.5 g / L sodium bicarbonate, 30 g / L sea salt, 100,000 units / L benzylpenicillin, and 1 g / L Bengal rose. The pH of the fermentation medium was adjusted to 6.0. After preparation, the fermentation medium was sterilized in an autoclave at 121°C for 20 min.

[0059] 1. Inoculation and culture: Aspergillus versicolor SCAU214 spores from Example 1 were inoculated onto the above fermentation broth culture medium and cultured at 18°C ​​for 7 days to obtain the fermentation broth.

[0060] 2. Filtration and concentration: Filter the fermentation broth using a Buchner funnel to remove insoluble impurities, and concentrate it under reduced pressure at 40°C to one-quarter of the original volume to obtain the filtered and concentrated fermentation broth.

[0061] 3. Protein Removal: Add ammonium sulfate to the concentrated fermentation broth obtained in step 2 until saturated, then add an equal volume of tert-butanol. Stir magnetically for 3–4 hours, centrifuge at 5000 rpm for 10 minutes, and retain the aqueous phase to obtain the polysaccharide aqueous solution. Add the polysaccharide aqueous solution to a dialysis bag with a molecular weight cutoff of 3000 Da, seal the bag, place it in distilled water, and dialyze at 4°C. Change the distilled water every 3 hours until the conductivity of the external dialysate no longer changes and small molecules in the dialysis bag are dialyzed into the external dialysate. Remove the solution from the bag, pre-freeze it at -80°C for 24 hours, and then freeze-dry it to obtain the crude extracellular polysaccharide from marine-derived *Aspergillus versicolor* SCAU214, with a yield of approximately 0.3 g / L.

[0062] 4. Macroporous Resin Separation and Purification: Macroporous resins are high-molecular-weight separation materials with large pore sizes. The adsorption on macroporous resins is the result of intermolecular forces, mainly van der Waals forces and hydrogen bonds. Weigh 1000 mg of the crude polysaccharide from step 3, dissolve it in 5 mL of distilled water to prepare a 200 mg / mL solution, and add it to a macroporous resin column; further elute with ultrapure water at a rate of 2 mL / min, collecting a total of 500 mL of eluent, and then dialyze and freeze-dry according to the method in step 3 to obtain the fractional polysaccharide.

[0063] 5. DEAE-52 ion exchange column separation: The DEAE-52 weak anion exchange column is a method for separating polysaccharides using the principles of adsorption and desorption. The pretreated DEAE-52 packing material is packed into a glass chromatography column (2.6×70cm), and four column volumes are balanced with distilled water. 0.04g of the polysaccharide component from step 4 is weighed, dissolved thoroughly in 2mL of tertiary water, and then added to the DEAE-52 ion exchange column.

[0064] Elution was performed sequentially with NaCl solutions of 0, 0.1, 0.3, 0.6, 0.9, and 1.2 mol / L, at a flow rate of 1 mL / min. 10 mL of eluent was collected from each tube, and the collected eluent was analyzed in parallel tubes to plot the elution curve.

[0065] II. Experimental Results

[0066] like Figure 2 As shown, two symmetrical elution peaks were obtained. The eluent containing 0 mol / L NaCl was collected, dialyzed, and lyophilized to obtain the white flocculent polysaccharide AVP-214-1.

[0067] Example 3: Analysis of the purity and basic components of polysaccharide AVP-214-1

[0068] I. Experimental Methods

[0069] 1. Molecular weight: The polysaccharide AVP-214-1 from Example 2 was prepared into 5 mg / mL polysaccharide solutions with known molecular weight (5000, 116000, 23800, 48600, 80900, 148000, 273000, 409800 and 667800 Da) dextran standards. The solutions were placed in sample vials, mobile phase was added, and the solutions were incubated at room temperature (25℃) for 12 h. The molecular weight of polysaccharide AVP-214-1 was then determined using gel permeation chromatography.

[0070] Chromatographic conditions: column temperature 40℃, column type BRT105-104-102 tandem gel column (8mm×300mm), mobile phase 0.05mol / L NaCl, flow rate 0.6mL / min, injection volume 20μL, and peak time chromatogram of polysaccharide AVP-214-1 recorded using RI-10A differential detector.

[0071] Data analysis and standard curve plotting: A regression equation was established with the logarithm of the relative molecular mass (logMw) of the known dextran standard as the ordinate and retention time as the abscissa. The fitted regression equation is: y = -0.1796x + 11.518, (R² - R²)² = 0.1796x + 11.518. 2 =0.9957), lgMp-RT; y=-0.1914x+12.069, (R 2 =0.9941), lgMw-RT; y=-0.1774x+11.357, (R 2 =0.9921), lgMn-RT; obtain the standard curve, and calculate the relative molecular weight of AVP-214-1 based on the standard curve.

[0072] 2. Monosaccharide Composition: Polysaccharides are polymers formed by the dehydration condensation of several monosaccharides. Analyzing the monosaccharide composition of polysaccharides helps in structural analysis and structure-activity relationship analysis. The monosaccharide composition of AVP-214-1 was determined by ion chromatography. Sixteen dried monosaccharide standards (fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, glucuronic acid, galactosyl hydrochloride, glucosamine hydrochloride, N-acetyl-D-glucosamine, guluronic acid, and mannuronic acid) and the AVP-214-1 sample were accurately weighed to prepare the test solution.

[0073] The preparation method is as follows: Accurately weigh 5 mg of sample into an ampoule, add 2 mL of 3M TFA, and hydrolyze at 120℃ for 3 h to obtain an acid hydrolysis solution. Accurately pipette the acid hydrolysis solution into a tube and blow it dry with nitrogen. Add 5 mL of water and vortex to mix, obtaining a homogenate. Pipe 50 μL of the homogenate into 950 μL of deionized water and centrifuge at 12000 rpm for 5 min. Take the supernatant for analysis.

[0074] Chromatographic conditions: column temperature 30℃, column type Dionex Carbopac™ PA20 (3mm×150mm), flow rate 0.3mL / min, injection volume 5μL, gradient elution.

[0075] Mobile phase A: H2O; Mobile phase B: 15 mmol / L NaOH; Mobile phase C: 15 mmol / L NaOH and 100 mmol / L NaOAC. Elution curves were recorded using an electrochemical detector.

[0076] II. Experimental Results

[0077] like Figure 3 As shown, the HPGPC elution curve of AVP-214-1 shows a single, symmetrical peak shape, consistent with the DEAE-52 elution curve, confirming that AVP-214-1 is a homogeneous polysaccharide. Its peak molecular weight (Mp) is 7402 Da, weight-average molecular weight (Mw) is 8277 Da, and number-average molecular weight (Mn) is 6340 Da.

[0078] like Figure 4 As shown in Figures A and B, the peaks around 2.0 min are both sodium hydroxide peaks, while the peaks around 41 min are both sodium acetate peaks. Comparing the ion chromatograms of the two figures indicates that the monosaccharide composition of polysaccharide AVP-214-1 is glucose, mannose, and galactose, with content ratios of 86.4%, 6.3%, and 7.3%, respectively.

[0079] Example 4: Structural analysis of polysaccharide AVP-214-1

[0080] I. Experimental Methods

[0081] 1. Methylation analysis: Methylation analysis is an important method for polysaccharide structure analysis. By analyzing polysaccharides that have been methylated, completely acid-hydrolyzed, and completely acetylated to produce Aldiol acetate derivatives, important information such as the monosaccharide composition and glycosidic bond type of polysaccharide AVP-214-1 in Example 2 can be obtained from the data.

[0082] 2. Nuclear magnetic resonance analysis: The polysaccharide AVP-214-1 of Example 2 was scanned using a pulsed Fourier transform spectrometer.

[0083] II. Experimental Results

[0084] 1. For example Figures 5-13As shown in Table 1, the content of residue A [Glcp-(1→)] is 8.0%, residue B [Manp-(1→)] is 4.0%, residue C [→3)-Glcp-(1→] is 6.3%, residue D [→4)-Galp-(1→) is 4.6%, residue E [→4)-Glcp-(1→) is 59.0%, residue F [→6)-Glcp-(1→) is 5.6%, residue G [→3,4)-Glcp-(1→) is 3.0%, and residue H [→4,6)-Glcp-(1→) is 9.5%. Based on the molecular weight, the composition ratio of each residue in polysaccharide AVP-214-1 is approximately A:B:C:D:E:F:G:H = 5:2:3:2:30:2:2:5.

[0085] Table 1. Analytical results of methylated AVP-214-1 products

[0086]

[0087] 2. For example Figure 14 As shown 1 One-dimensional H-NMR spectrum, such as Figure 15 As shown 13 C-NMR one-dimensional spectrum, such as Figure 16 The diagram shows a two-dimensional image. 1 H- 1 H-COSY spectrum, such as Figure 17 The image shown is an HMBC spectrum. Figure 18 The image shows the HSQC spectrum. The sequence connection order of eight residues in the polysaccharide AVP-214-1 was analyzed by nuclear magnetic resonance (NMR) analysis, as shown below. Figure 14 and Figure 15 In the table, residue A is [α-D-Glcp-(1→], residue B is [α-D-Manp-(1→], residue C is [→3)-α-D-Glcp-(1→], residue D is [→4)-α-D-Galp-(1→], residue E is [→4)-α-D-Glcp-(1→], residue F is [→6-α-D-Glcp-(1→], residue G is [→3,4)-α-D-Glcp-(1→], and residue H is [→4,6)-α-D-Glcp-(1→]. (This is in conjunction with Table 2.) Figure 14 and Figure 15 , residues 1 The H-NMR chemical shifts were 5.25 ppm, 5.32 ppm, 5.01 ppm, 5.21 ppm, 5.43 ppm, 4.97 ppm, 5.00 ppm, and 5.36 ppm, respectively, for the residues. 13The C NMR chemical shifts were 98.75 ppm, 92.61 ppm, 98.74 ppm, 99.80 ppm, 99.55 ppm, 98.06 ppm, 97.93 ppm, and 99.80 ppm, respectively. Figure 16 of 1 The H-1H-COSY spectrum can distinguish separated spin systems of H-1 / H-2, H-2 / H-3, H-3 / H-4, H-4 / H-5, and H-5 / H-6. Figure 17 The HMBC spectra provided support for the presence of glucose residue A. Furthermore, the further structural resolution of monosaccharide residue A was achieved with the aid of HMBC experiments. In addition, the chemical shifts of residues B, C, D, E, F, G, and H were also confirmed. A cross-peak exists between δH 5.32 (B: H-1) and δC 78.26 (C: C-3), indicating a glucosidic bond between B and C. A cross-peak also exists between δH 5.32 (B: H-1) and δC 75.65 (E: C-4), indicating a glucosidic bond between B and E. A cross-peak exists between δH 5.43 (E: H-1) and δC 71.16 (F: C-6), indicating a glucosidic bond between E and F. Cross-peaks exist between δH 4.97 (F: H-1) and δH 5.36 (H: H-1) and δC 83.52 (G: C-3), indicating glucosidic bonds between F and A and G. There is a cross peak between δH 5.43 (E: H-1) and δC 81.43 (G: C-4), indicating that there is a glucosidic bond between E and G. There is also a cross peak between δH 5.25 (A: H-1), δH 5.01 (C: H-1), and δH 5.21 (D: H-1) and δC 75.07 (H: C-4), indicating that there is a glucosidic bond between A, C, D and E. There is also a cross peak between δH 5.25 (A: H-1), δH 5.00 (G: H-1) and δC 68.27 (H: C-6), indicating that there is a glucosidic bond between A and G.

[0088] Based on the above evidence, the structural formula of polysaccharide AVP-214-1 in Example 2 is shown in Formula I and Formula II.

[0089]

[0090]

[0091] Table 2. Sugar residues of polysaccharide AVP-214-1 13 C 1 Chemical shift of H

[0092]

[0093] Example 5: Effect of polysaccharide AVP-214-1 on the proliferation of RAW 264.7 cells

[0094] I. Experimental Methods

[0095] The effect of AVP-214-1 on the proliferation of RAW 264.7 macrophages was detected using the CCK-8 assay.

[0096] 1. Discard the old culture medium from the cell culture dish, wash twice with 2 mL of PBS buffer, add 2 mL of new complete culture medium, gently pipette the RAW264.7 cells to form a cell suspension, and count the cells using a cell counting chamber.

[0097] 2. Transfer the cell suspension from step 1 to a blank cell culture dish, add fresh complete culture medium, and adjust the cell density to 1.0 × 10⁶ cells / mL. 4 A fresh cell suspension was obtained by measuring cells per mL. 100 μL of the fresh cell suspension was transferred to a 96-well plate and incubated in a cell culture incubator (37°C, 5% CO2) for 24 h. The supernatant was discarded, and different concentrations of the polysaccharide AVP-214-1 sample from Example 2 (0, 12.5, 25, 100, 200, and 400 μg / mL) were added, with 6 replicates per group. The plates were then incubated in a cell culture incubator (37°C, 5% CO2) for another 24 h.

[0098] 3. After the culture in step 2 is completed, add 10 μL of CCK-8 reagent to each well, incubate at 37℃ in the dark for 2 h, and measure the absorbance at 450 nm to analyze the effect of polysaccharide AVP-214-1 on the proliferation of RAW264.7 cells.

[0099] II. Experimental Results

[0100] like Figure 19 As shown, the polysaccharide AVP-214-1 (12.5–400 μg / mL) promoted the proliferation of RAW 264.7 macrophages, and the cell viability of the treated groups was higher than that of the control group. Within the concentration range of 12.5–400 μg / mL, AVP-214-1 showed no cytotoxicity to RAW264.7 macrophages. When the AVP-214-1 concentration was 12.5–100 μg / mL, cell viability was significantly different from that of the control group; when the AVP-214-1 concentration was 200–400 μg / mL, cell viability was not significantly different from that of the control group. This may be because high sugar concentrations promote cell proliferation followed by the secretion of growth-inhibiting metabolites.

[0101] Example 6: Effect of polysaccharide AVP-214-1 on NO release from RAW 264.7 cells

[0102] I. Experimental Methods

[0103] In immune responses, NO release is a key indicator of immunomodulatory activity and plays a vital role in immunity. NO plays an important role in various immunomodulatory diseases, such as inhibiting cancer, preventing cardiovascular disease, improving reproductive performance, enhancing antioxidant capacity, and promoting cell proliferation. This study measured NO release from RAW 264.7 macrophages treated with the marine-derived Aspergillus versicolor polysaccharide AVP-214-1 to determine the stimulatory effect of AVP-214-1 on macrophages. An NO kit was used to determine the effect of the polysaccharide AVP-214-1 from Example 2 on NO release from RAW 264.7 cells.

[0104] 1. The method for preparing the cell suspension is the same as step 1 in Example 5.

[0105] 2. The cell density of the new cell suspension was 1.0 × 10⁻⁶. 5 Cells / mL, 500 μL of fresh cell suspension was transferred to a 24-well plate, and the concentration of polysaccharide AVP-214-1 sample was set to 0, 12.5, 25, 50 and 100 μg / mL. A 1 μg / mL lipopolysaccharide LPS solution was set as a positive control group. Other culture methods were the same as step 2 of Example 5.

[0106] 3. Centrifuge each well of the solution after the culture in step 2 at 1000×g for 3 min, collect the supernatant, and follow the instructions of the NO kit to analyze the effect of polysaccharide AVP-214-1 on NO release in RAW264.7 cells.

[0107] II. Experimental Results

[0108] like Figure 20 As shown, compared with the blank control group, the 12.5, 25, 50, and 100 μg / mL AVP-214-1 polysaccharide treatment groups dose-dependently promoted NO release, and the NO release in the 100 μg / mL polysaccharide AVP-214-1 treatment group was greater than that in the positive control group treated with 1 μg / mL LPS. The increased NO release indicates that polysaccharide AVP-214-1 has an immunostimulatory effect on macrophages.

[0109] Example 7: Effect of polysaccharide AVP-214-1 on cytokine secretion by RAW 264.7 cells

[0110] I. Experimental Methods

[0111] Cytokines are a class of small protein molecules with broad biological activity, playing an important role in regulating the body's immune response and inflammatory response. IL-6 and TNF-α are common cytokines. This study measured the secretion of IL-6 and TNF-α by AVP-214-1-stimulated RAW 264.7 macrophages.

[0112] The supernatant collected in the NO experiment was tested using Example 6. The TNF-α and IL-6 secretion was detected by following the instructions of the TNF-α and IL-6 kits. The effect of polysaccharide AVP-214-1 on the secretion of cytokines in RAW264.7 cells was analyzed.

[0113] II. Experimental Results

[0114] like Figure 21 As shown in Figures A and B, compared with the blank control group, the secretion of cytokines IL-6 and TNF-α increased in a dose-dependent manner under treatment with polysaccharide AVP-214-1 at concentrations of 12.5, 25, 50, and 100 μg / mL.

[0115] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description and ideas, and it is neither necessary nor possible to exhaustively describe all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A type of Aspergillus variegated ( Aspergillus versicolor SCAU214, characterized in that, The Aspergillus variegata SCAU214 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on June 25, 2023, with accession number GDMCC No: 63583.

2. A polysaccharide, characterized in that, The structural formula of the polysaccharide is shown in Formula I: 。 3. The use of the polysaccharide of claim 2 in promoting the proliferation of macrophages.

4. A method for preparing a polysaccharide with the structural formula shown in Formula I, characterized in that, The Aspergillus variegata as described in claim 1 ( Aspergillus versicolor The fermentation product of SCAU214 was filtered and concentrated to obtain the filtered and concentrated fermentation product. The protein in the concentrated fermentation product was removed by filtration to obtain crude polysaccharide; the crude polysaccharide was then purified by macroporous resin separation to obtain macroporous resin purified polysaccharide. The polysaccharide was purified by ion exchange column separation after separation using macroporous resin. 。 5. A product that promotes the proliferation of macrophages, characterized in that, The product contains a polysaccharide with the structural formula shown in Formula I. 。

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