CN116836821A - Preparation method and application of deep sea-source aspergillus versicolor SCAU214 and extracellular polysaccharide AVP-214-1 thereof - Google Patents
Preparation method and application of deep sea-source aspergillus versicolor SCAU214 and extracellular polysaccharide AVP-214-1 thereof Download PDFInfo
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Abstract
本发明公开了一种深海来源杂色曲霉SCAU214及其胞外多糖AVP‑214‑1的制备方法和应用。所述杂色曲霉SCAU214已于2023年6月25日保藏于广东省微生物菌种保藏中心，其保藏号为GDMCC No：63583。对其进行发酵培养，提取纯化其代谢产物，获得其胞外多糖AVP‑214‑1，所述多糖AVP‑214‑1在较宽的浓度范围内(12.5～400μg/mL)都可以促进巨噬细胞RAW 264.7的增殖，NO的释放量大于阳性对照组，剂量依赖性地刺激巨噬细胞RAW 264.7分泌的细胞因子IL‑6、TNF‑α，对巨噬细胞有较强的免疫刺激效果，是具有安全、高效的调节免疫活性作用。
The invention discloses a preparation method and application of deep-sea Aspergillus versicolor SCAU214 and its extracellular polysaccharide AVP-214-1. The Aspergillus versicolor SCAU214 has been deposited in the Guangdong Provincial Microbial Culture Collection Center on June 25, 2023, and its deposit number is GDMCC No: 63583. Ferment and culture it, extract and purify its metabolites, and obtain its extracellular polysaccharide AVP-214-1. The polysaccharide AVP-214-1 can promote macrophage in a wide concentration range (12.5 ~ 400 μg/mL) The proliferation of cells RAW 264.7, the release of NO is greater than that of the positive control group, and dose-dependently stimulates the secretion of cytokines IL-6 and TNF-α by macrophages RAW 264.7, which has a strong immune stimulating effect on macrophages. It has a safe and efficient effect in regulating immune activity.
Description
Technical Field
The invention relates to the technical field of marine microorganisms, in particular to a preparation method and application of deep-sea aspergillus versicolor SCAU214 and extracellular polysaccharide AVP-214-1 thereof.
Background
The existing methods in the industries of chemical industry, food, cosmetics and the like generally depend on chemical agents, and can cause irreversible influence on the environment or human body in the use process, and the biological extracellular polysaccharide is biodegradable and usually nontoxic. Polysaccharides are the most abundant organic matter in the world and are used in a number of industrial fields as thickeners, stabilizers and gels in foods and as antitumor, antioxidants and/or prebiotics in pharmacology. Common sources of industrial polysaccharides are plants, animals, fungi, algae and bacteria, which are currently mainly from algae. However, microbial exopolysaccharides have their unique advantages: since the growth conditions of microorganisms can be strictly monitored, fungi and bacteria have the advantage of high structural reproducibility, which is not possible for plant and animal sources, since the structure of animal and plant polysaccharides depends on climate, environment and feed etc. conditions which are not easily controlled.
In addition, the ocean becomes one of the most bio-diverse habitats on earth due to its special environmental conditions, and the deep sea environmental conditions are more extreme: no sunlight, low temperature, anaerobic and high hydrostatic pressure. In order to adapt to the deep sea environment, the deep sea fungi evolve unique physiological structures and metabolic pathways, can generate extracellular polysaccharide with novel structures, and show wide biological activity, and the biological active compounds provide candidate compounds for research and development of marine medicaments while protecting the fungi. Aspergillus deep sea is an important component population of fungi in the deep sea. The metabolite components of Aspergillus include polyketides, alkaloids, lactones and terpenes. Aspergillus versicolor is one of Aspergillus, and can produce various useful active metabolites with various biological activities such as disease resistance, insect resistance, virus resistance, tumor resistance, oxidation resistance, cellular immunity, inflammation resistance, etc.
However, both the environmental conditions of aspergillus growth and the extraction method of its metabolites have a significant impact on the biological activity of the resulting polysaccharide. And the aspergillus polysaccharide disclosed at present still has certain toxicity to cells at a slightly high concentration, has low immunocompetence and still needs to excavate other safe and efficient aspergillus metabolites.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a preparation method and application of deep-sea aspergillus versicolor SCAU214 and extracellular polysaccharide AVP-214-1 thereof.
A first object of the present invention is to provide a aspergillus versicolor (Aspergillus versicolor) SCAU214.
A second object of the present invention is to provide a polysaccharide.
A third object of the present invention is to provide the use of said aspergillus versicolor (Aspergillus versicolor) SCAU214 or said polysaccharide for the preparation of a product for modulating immune activity.
A fourth object of the present invention is to provide a process for the preparation of polysaccharides.
It is a fifth object of the present invention to provide a product that modulates immune activity.
In order to achieve the above object, the present invention is realized by the following means:
an aspergillus versicolor (Aspergillus versicolor) SCAU214, said aspergillus versicolor SCAU214 having been deposited at the cantonese province microorganism strain collection at 25/6/2023 under the accession number GDMCCNo:63583.
the invention also claims a polysaccharide, wherein the structural formula of the polysaccharide is shown in formula I:
based on this, the invention claims the use of the aspergillus versicolor (Aspergillus versicolor) SCAU214 or the polysaccharide for the preparation of a product for modulating immune activity.
The invention also provides a preparation method of the polysaccharide, which is obtained by fermenting the aspergillus versicolor (Aspergillus versicolor) SCAU214.
Further, the fermentation product of the aspergillus versicolor (Aspergillus versicolor) SCAU214 is filtered and concentrated, and the fermentation product is filtered and concentrated.
Further, protein of the filtered and concentrated fermentation product is removed to obtain crude polysaccharide. The structural formula of the purified polysaccharide is shown in formula I:
further, macroporous resin separation and purification are carried out on the crude polysaccharide, so that macroporous resin separation and purification polysaccharide is obtained.
Further, separating the purified polysaccharide by using macroporous resin, and performing ion exchange column separation to obtain the purified polysaccharide.
The invention also provides a product for regulating the immune activity, which is characterized in that the product contains the aspergillus versicolor (Aspergillus versicolor) SCAU214 and/or a fermentation product thereof.
The invention also provides a product for regulating immune activity, which is characterized in that the polysaccharide is contained in the product.
Compared with the prior art, the invention has the following beneficial effects:
according to the invention, aspergillus versicolor (Aspergillus versicolor) SCAU214 is obtained by separating from a deep sea sediment sample of Ma Liya nanometer sea ditches (141 DEG 57'N,10 DEG 51' E), and is subjected to fermentation culture, and the metabolite is extracted and purified to obtain extracellular polysaccharide AVP-214-1, wherein the polysaccharide AVP-214-1 can promote proliferation of macrophage RAW264.7 in a wide concentration range (12.5-400 mu g/mL), the release amount of NO is larger than that of a positive control group, and cytokines IL-6 and TNF-alpha secreted by macrophage RAW264.7 are stimulated in a dose-dependent manner, so that the method has a strong immune stimulation effect on macrophages, and has a safe and efficient immune activity regulating effect.
Drawings
FIG. 1 is a phylogenetic tree of Aspergillus versicolor (Aspergillus versicolor) SCAU214.
FIG. 2 is a graph showing the elution profile of DEAE-52 from Aspergillus versicolor (Aspergillus versicolor) SCAU214 extracellular polysaccharide AVP-214-1.
FIG. 3 shows the HPGPC peak of Aspergillus versicolor (Aspergillus versicolor) SCAU214 extracellular polysaccharide AVP-214-1.
FIG. 4 is an ion chromatogram of Aspergillus versicolor (Aspergillus versicolor) SCAU214 extracellular polysaccharide AVP-214-1. Wherein A is an ion chromatogram of a mixed monosaccharide standard; b is an ion chromatogram of polysaccharide AVP-214-1.
FIG. 5 is a GCMS chromatogram of an acetylation product of the Aspergillus versicolor (Aspergillus versicolor) SCAU214 extracellular polysaccharide AVP-214-1.
FIG. 6 is a residue A mass spectrum of a methylated derivative of the A.variabilis (Aspergillus versicolor) SCAU214 extracellular polysaccharide AVP-214-1.
FIG. 7 is a residue B mass spectrum of a methylated derivative of the A.versicolor (Aspergillus versicolor) SCAU214 extracellular polysaccharide AVP-214-1.
FIG. 8 is a residue C mass spectrum of a methylated derivative of the A.versicolor (Aspergillus versicolor) SCAU214 extracellular polysaccharide AVP-214-1.
FIG. 9 is a residue D mass spectrum of a methylated derivative of the A.variabilis (Aspergillus versicolor) SCAU214 extracellular polysaccharide AVP-214-1.
FIG. 10 is a residue E mass spectrum of a methylated derivative of the A.versicolor (Aspergillus versicolor) SCAU214 extracellular polysaccharide AVP-214-1.
FIG. 11 is a residue F mass spectrum of a methylated derivative of the A.versicolor (Aspergillus versicolor) SCAU214 extracellular polysaccharide AVP-214-1.
FIG. 12 is a residue G mass spectrum of a methylated derivative of the A.versicolor (Aspergillus versicolor) SCAU214 extracellular polysaccharide AVP-214-1.
FIG. 13 is a residue H mass spectrum of a methylated derivative of the A.variabilis (Aspergillus versicolor) SCAU214 extracellular polysaccharide AVP-214-1.
FIG. 14 shows one-dimensional nuclear magnetic resonance of the extracellular polysaccharide AVP-214-1 of Aspergillus versicolor (Aspergillus versicolor) SCAU214 1 H-NMR spectrum.
FIG. 15 shows one-dimensional nuclear magnetic resonance of the extracellular polysaccharide AVP-214-1 of Aspergillus versicolor (Aspergillus versicolor) SCAU214 13 C-NMR spectrum.
FIG. 16 is a two-dimensional illustration of the A.variabilis (Aspergillus versicolor) SCAU214 extracellular polysaccharide AVP-214-1 1 H- 1 H-COSY spectrum.
FIG. 17 is a two-dimensional HMBC pattern of the Aspergillus versicolor (Aspergillus versicolor) SCAU214 extracellular polysaccharide AVP-214-1.
FIG. 18 is a two-dimensional HSQC spectrum of Aspergillus versicolor (Aspergillus versicolor) SCAU214 extracellular polysaccharide AVP-214-1.
FIG. 19 shows the effect of Aspergillus versicolor (Aspergillus versicolor) SCAU214 exopolysaccharide AVP-214-1 on RAW264.7 macrophage proliferation.
FIG. 20 shows the effect of Aspergillus versicolor (Aspergillus versicolor) SCAU214 exopolysaccharide AVP-214-1 on the secretion of NO by RAW264.7 macrophages.
FIG. 21 is a graph showing the effect of Aspergillus versicolor (Aspergillus versicolor) SCAU214 exopolysaccharide AVP-214-1 on RAW264.7 secreting cytokines IL-6 and TNF- α. Wherein A is IL-6; b is TNF-alpha; compared to the blank control group: p <0.01,: p <0.001,: p <0.0001.
Detailed Description
The invention will be further described in detail with reference to the drawings and specific examples, which are given solely for the purpose of illustration and are not intended to limit the scope of the invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.
Example 1 isolation and purification and characterization of Aspergillus versicolor (Aspergillus versicolor) SCAU214
1. Experimental method
1. Samples were collected and cultured: collecting deep sea sediment sample (depth 5455 m) in Marina sea ditch (141 deg.57 'N,10 deg.51' E), weighing 1.0g sediment sample in ultra clean bench, adding 9mL sterile distilled water containing 0.1% agar, shaking to form sample suspension, and marking as concentration 10 -1 g/mL of sample suspension. 1mL of the mixture was taken at a concentration of 10 -1 g/mL of the sample suspension was added with 9mL of sterile distilled water containing 0.1% of agar, shaken well and labeled as a concentration of 10 -2 g/mL of sample suspension.
2. Inoculating a sample: preparing a red sea water PDA culture medium containing 0.5g/L penicillin and 0.03g/L rose Bengal, sterilizing with high pressure steam, standing to about 40deg.C, mixing, pouring into a culture dish, and solidifying to obtain a solid culture medium. 200. Mu.L of sample suspensions of different concentration gradients were pipetted and spread evenly on solid medium, 3 plates for each concentration. Culturing at constant temperature of 10deg.C, and culturing upside down after no liquid is present on the surface of the water volatilizing culture medium.
3. Isolating the strain: the coated plates of step 2 were incubated at 4℃for 30 days until the morphology of the fungus and bacteria could be distinguished. And then picking and transferring to a new culture medium according to the morphological difference. The resulting dish was cultured at 4℃and subjected to purification culture. The selected purified strain grows faster on PDA culture medium, is round, starts to be white, turns green after 2-3 days, and turns brown after 4-5 days. The top sac of the conidiophore is hemispherical, the small stalk is double-layered, the first layer is short, the second layer is long, the conidiophore is radially arranged, and chain spores are arranged at the top end.
4. Identification of strains: extracting genome of the selected purified strain, and amplifying ITSDNA genes by PCR under the following conditions: pre-changing at 98 ℃ for 10min; changing the temperature to 98 ℃ for 10s, annealing the temperature to 55 ℃ for 10s, and extending the temperature to 72 ℃ for 1min; finally, the extension is carried out for 1min at 72 ℃.
2. Experimental results
As shown in FIG. 1, the purified strain selected (whose ITS sequence is accession No. OK271389 at GenBank) has a similarity of 99% to the ITS sequence of the standard strain Aspergillus versicolor (MH 712291) at GenBank, and is designated as A.versicolor (Aspergillus versicolor) SCAU214 and deposited at the Canon province microorganism strain deposit at month 25 of 2023 under the accession number GDMCC No:63583, the preservation address is 5 buildings of Guangzhou Md.A. No. 100 college, no. 59.
EXAMPLE 2 extraction and isolation of purified polysaccharide AVP-214-1
1. Experimental method
Preparing a fermentation broth culture medium: the culture medium for wheat separation is malt extract 5g/L, czapek Dox broth 5g/L, peptone 10g/L, potato dextrose broth 5g/L, magnesium chloride 0.5g/L, calcium chloride 0.5g/L, potassium chloride 0.5g/L, sodium bicarbonate 0.5g/L, sea salt 30g/L, benzyl penicillin 10 ten thousand units/L and rose Bengal 1g/L. Adjusting pH of fermentation medium to 6.0, and sterilizing at 121deg.C for 20min in autoclave after fermentation medium preparation.
1. Inoculating and culturing: inoculating Aspergillus versicolor (Aspergillus versicolor) SCAU214 spores of example 1 onto the fermentation broth, and culturing at 18deg.C for 7d to obtain fermentation broth.
2. And (3) filtering and concentrating: the broth was filtered using a buchner funnel to remove insoluble impurities and concentrated to one-fourth of the original volume at 40 ℃ under reduced pressure, and the broth was filtered and concentrated.
3. Removing protein: and (3) adding ammonium sulfate into the filtered concentrated fermentation liquor obtained in the step (2) to be saturated, then adding equal volume of tertiary butanol, magnetically stirring for 3-4 h, centrifuging for 10min at 5000r/min, and reserving a water phase to obtain the polysaccharide water solution. Adding the polysaccharide water solution into a dialysis bag with a cut-off molecular weight of 3000Da, sealing, placing into distilled water, dialyzing in a refrigerator at 4deg.C, and changing distilled water every 3 hr until the conductivity of external dialysate is no longer changed, and dialyzing small molecular substances in the dialysis bag into external dialysate. Taking out the solution in the bag, pre-freezing in an ultralow temperature refrigerator at-80 ℃ for 24 hours, and freeze-drying to obtain the extracellular crude polysaccharide of the marine aspergillus versicolor (Aspergillus versicolor) SCAU214 with the yield of about 0.3g/L.
4. Separating and purifying by macroporous resin: macroporous resin is a high molecular separation material with large aperture, and the adsorption effect on macroporous resin is the result of intermolecular forces mainly comprising Van der Waals force and hydrogen bond. Weighing 1000mg of the crude polysaccharide in the step 3, dissolving in 5mL of distilled water, preparing 200mg/mL solution, and loading the solution into a macroporous resin column; eluting with ultrapure water at an elution rate of 2mL/min, collecting 500mL of eluent, dialyzing and freeze-drying to obtain component polysaccharide.
5. DEAE-52 ion exchange column separation: the DEAE-52 weak anion exchange column is a method for separating polysaccharide by utilizing adsorption and desorption principles, pretreated DEAE-52 packing is filled into a glass chromatographic column (2.6X70 cm), 4 column volumes are balanced by distilled water, 0.04g of component polysaccharide in the step 4 is weighed, and after being fully dissolved by 2mL of tertiary water, the component polysaccharide is added into the DEAE-52 ion exchange column.
Eluting with 0, 0.1, 0.3, 0.6, 0.9 and 1.2mol/L NaCl solution in sequence, collecting 10mL of eluent from each tube, detecting by separating tube, and drawing an elution curve.
2. Experimental results
As shown in FIG. 2, 2 symmetrical elution peaks are obtained, and eluent of 0mol/L NaCl is collected for dialysis and freeze-drying to obtain white flocculent component polysaccharide AVP-214-1.
EXAMPLE 3 analysis of purity and essential Components of polysaccharide AVP-214-1
1. Experimental method
1. Molecular weight: polysaccharide AVP-214-1 of example 2 and dextran standards of known molecular weights (5000, 116000, 23800, 48600, 80900, 148000, 273000, 409800 and 667800 Da) were prepared as 5mg/mL polysaccharide solutions, placed in sample bottles, mobile phases were added, and placed at room temperature (25 ℃) for 12 hours, and the molecular weight of polysaccharide AVP-214-1 was detected by gel chromatography.
Chromatographic conditions: the column temperature is 40 ℃, the chromatographic column is BRT105-104-102 series gel column (8 mm multiplied by 300 mm), the mobile phase is 0.05mol/L NaCl, the flow rate is 0.6mL/min, the sample injection volume is 20 mu L, and the peak time spectrum of polysaccharide AVP-214-1 is recorded by an RI-10A differential detector.
Data analysis and standard curve plotting: establishing a regression equation by taking the logarithm (logMw) of the relative molecular weight of a known glucan standard substance as an ordinate and the retention time as an abscissa, wherein the fitted regression equation is as follows: y= -0.1796x+11.518, (R) 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; a standard curve was obtained and the relative molecular weight of AVP-214-1 was calculated from the standard curve.
2. Monosaccharide composition: the polysaccharide is a polymer formed by dehydrating and condensing a plurality of monosaccharides, and analysis of monosaccharide composition of the polysaccharide is helpful for structural analysis and analysis of structure-activity relationship. The monosaccharide composition of the AVP-214-1 fraction was determined by ion chromatography. Accurately weighing dried 16 monosaccharide standard substances (fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, glucuronic acid, galactosamine hydrochloride, glucosamine hydrochloride, N-acetyl-D glucosamine, guluronic acid and mannuronic acid) and AVP-214-1 samples to prepare a solution to be measured.
The preparation method comprises the following steps: 5mg of the sample is precisely weighed and placed in an ampoule bottle, 3M TFA2mL is added, and the mixture is hydrolyzed for 3h at 120 ℃ to obtain an acid hydrolysis solution. Accurately sucking the acid hydrolysis solution, transferring to a pipe, blowing with nitrogen, drying, adding 5mL of water, and mixing by vortex to obtain a uniform mixed solution; mu.L of deionized water was added by pipetting 50. Mu.L of the mixture, and the mixture was centrifuged at 12000rpm for 5min. The supernatant was taken and analyzed.
Chromatographic conditions: the column temperature is 30 ℃, the chromatographic column is DionexCarbopacTM PA (3 mm multiplied by 150 mm), the flow rate is 0.3mL/min, the sample injection amount is 5 mu L, and the gradient elution is carried out.
Mobile phase a: h 2 O; mobile phase B:15mmol/L NaOH; mobile phase C: elution profiles were recorded with an electrochemical detector with 15mmol/L NaOH and 100mmol/L NaOAC.
2. Experimental results
As shown in FIG. 3, the HPGPC of AVP-214-1 showed a single symmetry of peak pattern, which is consistent with the DEAE-52 elution profile, demonstrating that AVP-214-1 is a homogeneous polysaccharide. The peak molecular weight (Mp) was 7402Da, the weight average molecular weight (Mw) was 8277Da, and the number average molecular weight (Mn) was 6340Da.
As shown in FIG. 4A and B, about 2.0min is the peak of sodium hydroxide, and about 41min is the peak of sodium acetate. Ion chromatography comparing the two figures shows that the monosaccharide composition in polysaccharide AVP-214-1 is glucose, mannose and galactose, and the content ratio is 86.4%, 6.3% and 7.3%, respectively.
EXAMPLE 4 structural resolution of polysaccharide AVP-214-1
1. Experimental method
1. Methylation analysis: methylation analysis is an important means for polysaccharide structure analysis, and analysis of polysaccharide which is prepared into an Aldi alcohol acetate derivative through methylation, complete acid hydrolysis and complete acetylation can obtain important information of monosaccharide composition and glycosidic bond type of the polysaccharide AVP-214-1 of example 2 from data.
2. Nuclear magnetic resonance analysis: the polysaccharide AVP-214-1 of example 2 was scanned using a pulse Fourier transform spectrometer.
2. Experimental results
1. As shown in fig. 5 to 13, and incorporating the relevant residue information in table 1, the content of the a residue [ Glcp- (1→ ] is 8.0%, the content of the B residue [ Manp- (1→ ] is 4.0%, the content of the C residue [ →3) -Glcp- (1→ ] is 6.3%, the content of the D residue [ →4) -Galp- (1→ ] is 4.6%, the content of the E residue [ →4) -Glcp- (1→ ] is 59.0%, the content of the F residue [ →6) -Glcp- (1→ ] is 5.6%, the content of the G residue [ →3, 4) -Glcp- (1→ ] is 3.0%, the content of the H residue [ →4, 6) -Glcp- (1→ ] is 9.5%, and the respective residue composition ratio of the polysaccharide AVP-214-1 is about a: B: C: D: F: G: h=5:2:3:2:2:2:5:5.
TABLE 1 analysis of methylated AVP-214-1 products
2. As shown in FIG. 14 1 The H-NMR one-dimensional spectrum is shown in FIG. 15 13 The C-NMR one-dimensional spectrum is shown in FIG. 16 as two-dimensional 1 H- 1 An H-COSY spectrum, shown in FIG. 17, an HMBC spectrum, shown in FIG. 18HSQC spectra are shown. The eight sequence residues of polysaccharide AVP-214-1 were analyzed by nuclear magnetic resonance analysis for their ligation sequence, as shown in FIGS. 14 and 15, residue A was [ alpha-D-Glcp- (1 → ]]Residue B [ alpha-D-Manp- (1 →)]Residue C [. Fwdarw.3) -alpha-D-Glcp- (1 → beta.)]Residues D [. Fwdarw.4) -alpha-D-Galp- (1. Fwdarw.)]Residue E is [. Fwdarw.4) -alpha-D-Glcp- (1. Fwdarw.)]Residue F [. Fwdarw.6-. Alpha. -D-Glcp- (1 →)]Residue G [. Fwdarw.3, 4) - α -D-Glcp- (1. Fwdarw]Residues H [. Fwdarw.4, 6) -alpha-D-Glcp- (1 →)]. Combining Table 2 with FIGS. 14 and 15, residues 1 H-NMR chemical shifts were 5.25ppm, 5.32ppm, 5.01ppm, 5.21ppm, 5.43ppm, 4.97ppm, 5.00ppm, 5.36ppm, respectively, of residues 13 The C NMR chemical shifts were 98.75ppm, 92.61ppm, 98.74ppm, 99.80ppm, 99.55ppm, 98.06ppm, 97.93ppm, 99.80ppm, respectively, FIG. 16 1 The H-1H-COSY spectra can distinguish between the separate spin systems of H-1/H-2, H-2/H-3, H-3/H-4, H-4/H-5 and H-5/H-6, which data support the presence of glucose residue A in conjunction with the HMBC spectra of FIG. 17. Likewise, further structural resolution of monosaccharide residue a was accomplished with the aid of HMBC experiments. Furthermore, chemical shifts of residues B, C, D, E, F, G and H were also confirmed. A cross peak between δH25.32 (B: H-1) and δC78.26 (C: C-3) indicates that there is a glycosidic bond between B and C, a cross peak between δ0H25.32 (B: H-1) and δ1C75.65 (E: C-4) also indicates that there is a glycosidic bond between B and E, a cross peak between δ2H25.43 (E: H-1) and δ3C71.16 (F: C-6) indicates that there is a glycosidic bond between E and F, a cross peak between δ4H2.97 (F: H-1) and δ5H2.36 (H: H-1) both have a glycosidic bond between F and A and G, a cross peak between δ7H2.43 (E: H-1) and δC 81.43 (G: C-4) indicates that there is a glycosidic bond between E and G, a cross peak between δ4H2.35 (F: H-1) and δ5H2.36 (H: H-1) both have a glycosidic bond between δ6C 83.52 (G: C-3) and a cross peak between δ7H2.43 (E: H-1) and δC 81.43 (G: C-4) indicates that there is a glycosidic bond between E and a cross peak between δ7H2.43 (E: H-1) and a cross peak between δ7H.37.16 (F: C-6) and a cross peak between δ4H.35.20.36 (E and a.20.20.20.20.20 and a).
From the above evidence, the structural formula of polysaccharide AVP-214-1 of example 2 was determined to be shown as formula I and formula II.
TABLE 2 polysaccharide AVP-214-1 sugar residues 13 C、 1 Chemical shift of H
EXAMPLE 5 Effect of polysaccharide AVP-214-1 on RAW264.7 cell proliferation
1. Experimental method
The effect of AVP-214-1 on RAW264.7 macrophage proliferation was examined using CCK-8.
1. After the old medium in the cell culture dish was aspirated, washed 2 times with 2mL of PBS buffer, 2mL of new complete medium was added, RAW264.7 cells were gently blown down to form a cell suspension, and counted with a cell counting plate.
2. Sucking the cell suspension in step 1, transferring to a blank cell culture dish, adding new complete culture solution, and adjusting cell density to 1.0X10 4 New cell suspensions were obtained per mL. 100. Mu.L of the new cell suspension was pipetted into a 96-well plate and placed in a cell incubator (37 ℃ C., 5% CO) 2 ) Is cultured for 24 hours. The supernatant was discarded, samples of the polysaccharide AVP-214-1 of example 2 (0, 12.5, 25, 100, 200 and 400. Mu.g/mL) were added at different concentrations, 6 wells were set up in each group, and the mixture was placed in a cell incubator (37 ℃, 5% CO) 2 ) Is cultured for 24 hours.
3. And (2) adding 10 mu L of CCK-8 reagent into each hole after the culture in the step (2), incubating for 2 hours at 37 ℃ in a dark place, measuring the absorbance at 450nm, and analyzing the influence of polysaccharide AVP-214-1 on RAW264.7 cell proliferation.
2. Experimental results
As shown in FIG. 19, polysaccharide AVP-214-1 (12.5-400 μg/mL) promotes proliferation of macrophage RAW264.7, cell viability of the treated group is higher than that of the control group, and polysaccharide AVP-214-1 has no cytotoxicity to RAW264.7 macrophages in the concentration range of 12.5-400 μg/mL. When the concentration of AVP-214-1 is 12.5-100 mug/mL, the cell activity has obvious statistical difference compared with the control group; at AVP-214-1 concentrations of 200-400 μg/mL, there was no significant statistical difference in cell viability compared to the control, probably due to the high sugar concentration promoting secretion of growth-inhibiting metabolites after cell proliferation.
Example 6 Effect of polysaccharide AVP-214-1 on NO Release by RAW264.7 cells
1. Experimental method
In immune response, NO release is a marker for determining immunomodulatory activity, and plays an important role in immunization. NO plays an important role in various immunomodulatory diseases, such as inhibiting cancer, preventing cardiovascular diseases, improving reproductive performance, enhancing antioxidant capacity in vivo, promoting cell proliferation, etc. Determining the condition of the RAW264.7 macrophage treated by the marine Aspergillus versicolor component polysaccharide AVP-214-1 to release NO, thereby judging the stimulation of the AVP-214-1 on the macrophage. The effect of polysaccharide AVP-214-1 of example 2 on the release of NO from RAW264.7 cells was determined experimentally using the NO kit.
1. The cell suspension preparation method was the same as in example 5, step 1.
2. The cell density of the novel cell suspension was 1.0X10 5 mu.L of the new cell suspension was pipetted into 24 well plates at polysaccharide AVP-214-1 sample concentrations of 0, 12.5, 25, 50 and 100. Mu.g/mL, and 1. Mu.g/mL LPS solution was set as positive control, otherwise the culture was the same as in example 5, step 2.
3. And (3) centrifuging the solution of each hole after the culture in the step (2), wherein the centrifugation condition is 1000 Xg, centrifuging for 3min, collecting supernatant, operating according to the instruction of the NO kit, and analyzing the influence of polysaccharide AVP-214-1 on the NO release amount of RAW264.7 cells.
2. Experimental results
As shown in FIG. 20, 12.5, 25, 50 and 100. Mu.g/mL of AVP-214-1 polysaccharide treated groups dose-dependently promoted the release of NO, and 100. Mu.g/mL of polysaccharide AVP-214-1 treated groups released more NO than the positive control group 1. Mu.g/mL of LPS treated group, as compared to the blank control group. The increased NO release indicates that polysaccharide AVP-214-1 has an immunostimulatory effect on macrophages.
Example 7 Effect of polysaccharide AVP-214-1 on RAW264.7 cell secretion of cytokines
1. Experimental method
Cytokines are a class of small molecule proteins with a broad range of biological activities that play an important role in regulating the immune and inflammatory responses of the body, with IL-6 and TNF- α being common cytokines. The cytokine IL-6 and TNF-alpha secreted by AVP-214-1 stimulated macrophage RAW264.7 is measured,
the supernatant collected in the NO assay of example 6 was used to examine the effects of polysaccharide AVP-214-1 on secreted cytokines in RAW264.7 cells by following the procedure of TNF-alpha kit and IL-6 kit instructions, and detecting the secretion of TNF-alpha and IL-6.
2. Experimental results
As shown in FIGS. 21A and B, the secretion amounts of cytokines IL-6 and TNF- α increased in a dose-dependent manner under treatment with polysaccharide AVP-214-1 of 12.5, 25, 50 and 100. Mu.g/mL, as compared to the control group.
It should be noted that the above embodiments are merely for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and that other various changes and modifications can be made by one skilled in the art based on the above description and the idea, and it is not necessary or exhaustive to all embodiments. Any modification, equivalent replacement, improvement, etc. which come within the spirit and principles of the invention are desired to be protected by the following claims.
Claims (10)
1. An aspergillus versicolor (Aspergillus versicolor) SCAU214, wherein the aspergillus versicolor SCAU214 has been deposited at the collection of microorganism strains in the cantonese province at 25/2023 under the accession number GDMCC No:63583.
2. the polysaccharide is characterized in that the structural formula of the polysaccharide is shown as a formula I:
3. use of aspergillus versicolor (Aspergillus versicolor) SCAU214 as claimed in claim 1 or of a polysaccharide as claimed in claim 2 for the preparation of a product for modulating immune activity.
4. A method for preparing polysaccharide, characterized in that the polysaccharide is obtained by fermenting Aspergillus versicolor (Aspergillus versicolor) SCAU214 according to claim 1.
5. The method according to claim 4, wherein the fermentation product of Aspergillus versicolor (Aspergillus versicolor) SCAU214 according to claim 1 is concentrated by filtration, and the fermentation product is concentrated by filtration.
6. The process according to claim 5, wherein the protein of the fermentation product is removed by filtration and concentrated to obtain a crude polysaccharide.
7. The method according to claim 6, wherein the crude polysaccharide is subjected to macroporous resin separation and purification to obtain macroporous resin separation and purification polysaccharide.
8. The method according to claim 7, wherein the purified polysaccharide is obtained by ion exchange column separation of the macroporous resin-separated purified polysaccharide.
9. A product for modulating immune activity, comprising aspergillus versicolor (Aspergillus versicolor) SCAU214 and/or a fermentation product thereof according to claim 1.
10. A product for modulating immune activity, comprising the polysaccharide of claim 2.
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