Hermannia atlantica strain lp-1, methods for synthesis of exopolysaccharide and uses thereof

Abstract

The present application relates to a kind of helman atlantic bacillus LP-1 and the method and application of the exocellular polysaccharide synthesized by it, belong to the field of microbial technology.The helman atlantic bacillus LP-1 is preserved in China Microbial Culture Collection Center on March 17, 2025, and the preservation number is CGMCC No.33858.The helman atlantic bacillus LP-1 of the present application can ferment and synthesize exocellular polysaccharide with sucrose as carbon source and potassium nitrate as inorganic nitrogen source, and the yield of exocellular polysaccharide can reach 10.14 g / L, the production rate is 0.21 g / L, and the substrate conversion rate is as high as 50.70%, which greatly reduces the production cost.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of Hermann's Atlanta bacillus LP-1 and its method for synthesizing extracellular polysaccharides and its applications. Background Technology

[0002] Microbial polysaccharides are high-molecular polymers synthesized by microorganisms (such as bacteria and fungi) and possess unique physiological activities and broad application prospects. Studies have shown that microbial polysaccharides, due to their unique physicochemical properties, rheological characteristics, and superior biological activity and nutritional value, have applications in the pharmaceutical, food, and chemical industries. Furthermore, microbial polysaccharides possess potential biological activities, such as antioxidant activity, cholesterol-lowering, immunomodulatory, hypoglycemic, and anticancer abilities. However, currently, the synthesis of most microbial polysaccharides uses grain raw materials as substrates, resulting in high production costs and limiting their application. Summary of the Invention

[0003] The first objective of this invention is to provide a strain of Hermann's Atlanta bacillus LP-1, which produces an extracellular polysaccharide yield of 10.14 g / L by fermentation using sucrose as a carbon source, potassium nitrate as a nitrogen source, and ferrous sulfate, disodium hydrogen phosphate, magnesium sulfate, and calcium chloride as metal salts.

[0004] The second objective of this invention is to provide a method for synthesizing extracellular polysaccharides from Hermann's Atlanta bacillus LP-1, wherein the extracellular polysaccharides from Hermann's Atlanta bacillus LP-1 have significant antioxidant, hypoglycemic, and HPV inhibitory effects.

[0005] A third objective of this invention is to provide an application of the extracellular polysaccharide of Hermann's Atlanta bacillus LP-1.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] Hermann's Atlanta bacillus LP-1 was deposited at the China General Microbiological Culture Collection Center on March 17, 2025, with accession number CGMCC No. 33858.

[0008] A method for synthesizing extracellular polysaccharides using Hermann's Atlanta bacillus LP-1 includes the following steps:

[0009] I. Activation of the strain: Hermann's Atlanta bacillus LP-1 was inoculated into TB medium and incubated statically at 26-38 °C for 18-22 h. A single colony was picked and streaked onto TB medium containing 20 g / L sucrose and incubated statically at 26-38 °C for 18-22 h to obtain the activated strain.

[0010] II. Preparation of seed culture: Under aseptic conditions, 4 loops of the activated strain were inoculated into a shake flask containing seed culture medium and placed on a shaker at 200 rpm for 16 h at 26-38 ℃ to obtain the fermentation seed culture.

[0011] III. Fermentation Culture: Under aseptic conditions, the fermentation seed liquid is inoculated into the fermentation medium at an inoculation rate of 1-8% by volume, and placed on a shaker at 200 rpm. The initial pH is 7.0-8.0, and the culture is carried out at 28-32 ℃ for 12-24 h. When the polysaccharide concentration in the fermentation broth no longer increases, the fermentation is stopped to obtain Hermann's Atlanta bacillus LP-1 fermentation broth.

[0012] Alternatively, the fermentation seed liquid can be inoculated into a fermenter at a volume ratio of 1-15% under aseptic conditions, with a liquid volume of 3 L or 5 L, a rotation speed of 200-500 rpm, an aeration ratio of 1.0-1.2 VVM, a culture temperature of 28-32 ℃, an initial pH of 7.0-8.0, and a culture time of 24-48 h. When the polysaccharide concentration in the fermentation broth no longer increases, fermentation is stopped to obtain Hermann's Atlanta bacillus LP-1 fermentation broth.

[0013] IV. Extraction of extracellular polysaccharides from Hermann's Atlanta bacillus LP-1:

[0014] S1: Cell removal: The Hermann's Atlanta bacillus LP-1 fermentation broth was diluted with water at a volume ratio of 1:4, and centrifuged in a high-speed centrifuge at 9000 rpm and 4 ℃ for 40 min to remove the bacterial cells and obtain the supernatant.

[0015] S2: Decolorization: Adjust the pH of the supernatant from S1 to 4.5-5.5, add 5‰ activated carbon powder, and incubate in a constant temperature water bath at 60 ℃ with continuous stirring for 1 h to obtain a polysaccharide-activated carbon mixture. After stirring in the water bath, filter while hot using a Büchner funnel. First, prepare a filter cake using diatomaceous earth, then filter the polysaccharide-activated carbon mixture to obtain a clear and transparent polysaccharide solution. Adjust the pH of the decolorized polysaccharide solution to 8.

[0016] S3: Protein Removal: The polysaccharide solution obtained in S2 was concentrated to 1 / 20 of its original volume at 55 °C to obtain a concentrated solution. 0.5-1‰ trypsin and 1‰ neutral protease were added to the concentrated solution, and the reaction was carried out at 50 °C for 4 h. The reaction was then terminated by boiling for 10 min. The pH was adjusted to neutral, and 1 / 3 volume of Sevag reagent (chloroform: n-butanol = 4:1) was added. The solution was stirred overnight at 4 °C. Subsequently, the solution was centrifuged at 9000 rpm at 4 °C for 30 min. The supernatant was collected, and Sevag reagent was added again, repeating the process until no protein layer appeared.

[0017] S4: Dialysis: Dialyze the liquid in S3 with double-distilled water for 1-2 days, evaporate and concentrate, add anhydrous ethanol for precipitation overnight, centrifuge for 9000 min to collect the precipitate, freeze dry to obtain crude extracellular polysaccharide of Hermann's Atlanta bacillus LP-1;

[0018] S5: Adsorption: After reconstitution of the crude extracellular polysaccharide of Hermann's Atlanta bacillus LP-1 from S4, the insoluble matter was removed by filtration through a 0.22 μm filter membrane, and then pumped into DEAE Sepharose. ® An anion exchange column, after adsorption treatment, yields a chromatography column adsorbed with microbial polysaccharides;

[0019] S6: Elution: Elution was performed with 0.5 mol / L sodium chloride solution at a flow rate of 1 mL / min. The resulting eluent was subjected to a delay treatment and then freeze-dried to obtain the extracellular polysaccharide of Hermann's Atlanta bacillus LP-1.

[0020] Furthermore, the extracellular polysaccharide of Hermann's Atlanta bacillus LP-1 includes glucuronic acid, glucose, galactose, and fucose, wherein the molar ratio of glucuronic acid, glucose, galactose, and fucose is 1:1.1:2.1:2.3.

[0021] Furthermore, the extracellular polysaccharide structure of the Hermann's Atlanta bacterium LP-1 contains nine linkage modes, namely T-Fucp-(1→), T-Glcp-(1→), T-GlcAp-(1→), T-Galp-(1→,→3)-Glcp-(1→,→4)-GlcAp-(1→,→6)-GlcAp-(1→,→6)-Galp-(1→,→3,4)-Glcp-(1→), with a molar ratio of 8.8:1.2:1.7:5.4:2.6:2.8:1.1:2.6:1.5.

[0022] Furthermore, the TB culture medium consists of: yeast extract 24 g / L, tryptone 12 g / L, potassium dihydrogen phosphate 2.31 g / L, dipotassium hydrogen phosphate 12.54 g / L, and agar 20 g / L;

[0023] The seed culture medium consists of: 20 g / L sucrose, 6.5 g / L disodium hydrogen phosphate, 3 g / L potassium nitrate, 0.5 g / L magnesium sulfate, 0.018 g / L ferrous sulfate, 0.013 g / L calcium chloride, 2 g / L tryptone, and water as the solvent, with a pH of 7.0-8.0.

[0024] The fermentation medium comprises: 0-50 g / L carbon source, 0-5 g / L nitrogen source, 0-7 g / L metal salt, water as solvent, and pH value of 7.0-8.0, preferably 8.0; the carbon source is one or more of sucrose, lactose, arabinose, glucose, and fructose; the nitrogen source is one or more of potassium nitrate, ammonium nitrate, sodium nitrate, diammonium hydrogen phosphate, and calcium nitrate; and the metal salt is one or more of magnesium sulfate, ferrous sulfate, zinc chloride, disodium hydrogen phosphate, manganese sulfate, and calcium chloride.

[0025] Furthermore, the fermentation medium comprises: 20 g / L sucrose, 3 g / L potassium nitrate, 9.7 mg / L ferrous sulfate, 6.0 g / L disodium hydrogen phosphate, 0.3 g / L magnesium sulfate, and 0.14 g / L calcium chloride, and the pH of the fermentation medium is 8.0.

[0026] The extracellular polysaccharide of Hermann's Atlanta bacillus LP-1 was prepared using the above-described method for synthesizing extracellular polysaccharides from Hermann's Atlanta bacillus LP-1.

[0027] The use of extracellular polysaccharides from Hermann's Atlanta bacterium LP-1 in the preparation of drugs for the prevention and / or treatment and / or synergistic treatment of diabetes.

[0028] The use of extracellular polysaccharides from Hermann's Atlanta bacterium LP-1 in the preparation of drugs for the prevention and / or treatment and / or synergistic treatment of HPV.

[0029] Application of extracellular polysaccharides of Hermann's Atlanta bacillus LP-1 in scavenging DPPH free radicals and inhibiting linoleic acid peroxidation.

[0030] The beneficial effects of this invention are:

[0031] The Hermann's Atlanta bacterium LP-1 of the present invention can synthesize extracellular polysaccharides by fermentation using sucrose as a carbon source and potassium nitrate as an inorganic nitrogen source. The yield of extracellular polysaccharides can reach 10.14 g / L, the production rate is 0.21 g / L, and the substrate conversion rate is as high as 50.70%, which greatly reduces the production cost.

[0032] The extracellular polysaccharide synthesized from Hermann's Atlanta bacillus LP-1 of this invention exhibits low toxicity, significant antioxidant, hypoglycemic, and human papillomavirus (HPV) inhibitory effects. The highly efficient and low-toxicity anti-HPV activity of Hermann's Atlanta bacillus LP-1 extracellular polysaccharide offers significant advantages in the development of anti-HPV drugs as an active lead compound, expanding its novel pharmaceutical functions and demonstrating promising application prospects.

[0033] The extracellular polysaccharide of Hermann's Atlanta bacillus LP-1 of this invention exhibits excellent free radical scavenging and lipid peroxidation resistance. At a concentration of 10 mg / mL, the scavenging rate of DPPH free radicals reached 52.29%, and at a concentration of 5 mg / mL, the inhibition rate of linoleic acid peroxidation reached 68.57%.

[0034] The extracellular polysaccharide of Hermann's Atlanta bacillus LP-1 of this invention has a good hypoglycemic effect. At a concentration of 160 μg / mL, the inhibitory rate on α-glucosidase reaches 69.68%, and the inhibitory rate on α-amylase reaches 16.73%.

[0035] The extracellular polysaccharide of *Hermannia Atlantai* LP-1 of this invention exhibits excellent anti-HPV properties. Within a concentration range of 25-200 μg / mL, it shows strong inhibitory effects on the infection process of both HPV16 and HPV18 pseudovirus particles, and the inhibitory effect is dose-dependent. Even at a relatively low concentration of 25 μg / mL, the inhibition rates for HPV16 and HPV18 are still as high as 68.7% and 52.5%, respectively. Attached Figure Description

[0036] Figure 1 Agarose gel electrophoresis image of 16S rDNA purified by PCR from Hermann's Atlanta bacillus LP-1;

[0037] Figure 2 Phylogenetic tree diagram of Hermann's Atlanta bacillus LP-1;

[0038] Figure 3 High-performance liquid chromatography (HPLC) chromatogram of the monosaccharide composition of extracellular polysaccharides from Hermann's Atlanta bacterium LP-1;

[0039] Figure 4 Nuclear magnetic resonance (NMR) image of the extracellular polysaccharide of Hermann's Atlanta bacillus LP-1. 1 H-map;

[0040] Figure 5 Nuclear magnetic resonance (NMR) image of the extracellular polysaccharide of Hermann's Atlanta bacillus LP-1. 13 C-map;

[0041] Figure 6 The graph shows the effect of the type of carbon source in the fermentation medium on the extracellular polysaccharides of Hermann's Atlanta LP-1.

[0042] Figure 7 The graph shows the effect of carbon source concentration in the fermentation medium on the extracellular polysaccharides of Hermann's Atlanta LP-1.

[0043] Figure 8 The effect of the type of organic nitrogen source in the fermentation medium on the extracellular polysaccharides of Hermann's Atlanta LP-1 is shown in the figure.

[0044] Figure 9 The figure shows the effect of organic nitrogen source concentration in the fermentation medium on the extracellular polysaccharides of Hermann's Atlanta LP-1.

[0045] Figure 10 The effect of the type of inorganic nitrogen source in the fermentation medium on the extracellular polysaccharides of Hermann's Atlanta bacillus LP-1 is shown in the figure.

[0046] Figure 11 The figure shows the effect of inorganic nitrogen source concentration in the fermentation medium on the extracellular polysaccharides of Hermann's Atlanta LP-1.

[0047] Figure 12 The graphs show the effects of different metal salts and their concentrations in the fermentation medium on the extracellular polysaccharides of *H. tarmaniae* LP-1. A represents the effect of ferrous sulfate concentration on the extracellular polysaccharides of *H. tarmaniae* LP-1, B represents the effect of disodium hydrogen phosphate concentration on the extracellular polysaccharides of *H. tarmaniae* LP-1, C represents the effect of magnesium sulfate concentration on the extracellular polysaccharides of *H. tarmaniae* LP-1, and D represents the effect of calcium chloride concentration on the extracellular polysaccharides of *H. tarmaniae* LP-1.

[0048] Figure 13 The effect of fermentation medium pH on the extracellular polysaccharides of Hermann's Atlanta LP-1 is shown in the figure.

[0049] Figure 14 The figure shows the effect of fermentation medium temperature on the extracellular polysaccharides of Hermann's Atlanta LP-1.

[0050] Figure 15 A graph showing the process of batch synthesis of extracellular polysaccharides from Hermann's Atlanta bacillus LP-1 in a 10 L fermenter;

[0051] Figure 16 The molecular weight of the extracellular polysaccharide of Hermann's Atlanta bacillus LP-1 is shown in the high-performance liquid chromatogram.

[0052] Figure 17The graph shows the antioxidant capacity of extracellular polysaccharides from Hermann's Atlanta bacterium LP-1, where A represents the DPPH free radical scavenging rate and B represents the anti-linoleic acid peroxidation activity.

[0053] Figure 18 The graph shows the hypoglycemic ability of extracellular polysaccharides from Hermann's Atlanta bacterium LP-1, where A represents the inhibition of α-glucosidase activity and B represents the inhibition of α-amylase activity.

[0054] Figure 19 Figure showing the effect of extracellular polysaccharides from Hermann's Atlanta bacillus LP-1 on the proliferation ability of HeLa cells;

[0055] Figure 20 The graph shows the inhibitory effect of extracellular polysaccharides of Hermann's Atlanta bacillus LP-1 on HPV16 and HPV18 pseudovirus infection, where A represents HPV16 and B represents HPV18. Detailed Implementation

[0056] The present invention will be further described below with reference to embodiments and accompanying drawings. Hermann's Atlanta bacillus Atlantibacter hermannii LP-1 is deposited at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 33858, on March 17, 2025. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. It is classified and named as follows: Atlantibacter hermannii .

[0057] In the diagram, EPS represents extracellular polysaccharide, and DCW represents cell mass.

[0058] Example 1

[0059] Isolation and identification of Hermann's Atlanta bacillus LP-1

[0060] I. Composition of the culture medium used in Example 1

[0061] Enrichment liquid culture medium: 20 g / L sucrose, 3 g / L beef extract, 1.645 g / L sodium chloride, 0.61 g / L magnesium sulfate, 0.07 g / L calcium chloride, 0.027 g / L ferrous sulfate, 0.003 g / L manganese sulfate, 0.0075 g / L zinc chloride, water as solvent, pH 7.0-8.0.

[0062] Solid screening medium: 20 g / L sucrose, 3 g / L beef extract, 1.645 g / L sodium chloride, 0.61 g / L magnesium sulfate, 0.07 g / L calcium chloride, 0.027 g / L ferrous sulfate, 0.003 g / L manganese sulfate, 0.0075 g / L zinc chloride, 20 g / L agar powder, water as solvent, pH 7.0-8.0.

[0063] Seed culture medium: 20 g / L sucrose, 3 g / L potassium nitrate, 2 g / L tryptone, 0.018 g / L ferrous sulfate, 6.5 g / L disodium hydrogen phosphate, 0.5 g / L magnesium sulfate, 0.013 g / L calcium chloride, water as solvent, pH 7.0-8.0.

[0064] Fermentation medium: 20 g / L sucrose, 3 g / L potassium nitrate, 0.018 g / L ferrous sulfate, 6.5 g / L disodium hydrogen phosphate, 0.5 g / L magnesium sulfate, 0.013 g / L calcium chloride, solvent: water, pH 7.0-8.0.

[0065] II. Separation and Identification

[0066] Two g samples were taken from each of 36 soil samples and inoculated into Erlenmeyer flasks containing enrichment liquid culture medium. The samples were enriched and cultured at 30 °C and 200 rpm for 48 h to obtain the culture medium. Two mL of this culture medium was transferred to the aforementioned enrichment liquid culture medium and enriched and cultured at 32 °C and 200 rpm for 24 h, repeated twice. The third enrichment culture medium was diluted to 10 mL. -8 10 -9 Two 200 μL samples were spread onto solid screening medium and incubated at 30 °C for 24 h to obtain colonies. Positive strains were screened based on the wetness and viscosity of the colony surface and then isolated and purified to obtain purified bacterial strains. The purified strains were then inoculated onto TB medium and then into seed culture medium, incubated at 30 °C and 200 rpm for 24 h to obtain seed culture. The seed culture was inoculated into fermentation medium at a rate of 4% (v / v) and incubated at 30 °C and 200 rpm for 48 h. The polysaccharide yield of the initially screened strains was measured, and the strain with the highest yield was obtained.

[0067] Genomic DNA was extracted from the bacterial genomic DNA extraction kit of the strain with the highest yield. PCR amplification was performed using upstream primer 27F and downstream primer 1492R. The 16S rDNA sequence is shown below. Figure 1 As shown, the PCR amplification product was purified by gel extraction and sent to Suzhou Genewise Biotechnology Co., Ltd. for sequencing. The nucleotide sequence of the 16S rDNA gene of the obtained strain was 1400 bp in length, and its gene sequence is shown in SEQ ID No. 1. The primer sequence of upstream primer 27F was 5′-GAGAGTTTGATCCTGGCTCAG-3′, and the primer sequence of downstream primer 1492R was 5′-ACGGCTACCTTGTTACGACTT-3′.

[0068] SEQ ID No. 1:

[0069]

[0070] The sequencing results were compared with known 16S rDNA sequences in the GeneBank database using BLAST, and homology comparisons were performed using the BLAST program to construct a phylogenetic tree based on the complete 16S rDNA sequence. The results showed that this strain achieved 100% homology with *Atlantibacter hermannii*. Based on morphological observation and physiological and biochemical analysis, the selected strain was identified as *Atlantibacter hermannii* LP-1. *Atlantibacter hermannii* LP-1 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33858, deposited on March 17, 2025, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0071] The colony morphology and physiological and biochemical characteristics of Hermann's Atlanta bacillus LP-1 are shown in Table 1.

[0072] Table 1. Colony morphology and physiological and biochemical characteristics of LP-1

[0073]

[0074] Example 2

[0075] Preparation of extracellular polysaccharides from Hermann's Atlanta bacillus LP-1

[0076] Hermann's Atlanta bacillus LP-1 was inoculated into TB medium and cultured at 30 °C for 18-22 h. Single colonies were then streaked onto TB medium containing 20 g / L sucrose and cultured at 30 °C for 18-22 h to obtain activated inoculum. The activated inoculum was aseptically inoculated into a shake flask containing seed medium and cultured at 30 °C for 16 h on a shaker at 200 rpm to obtain the fermentation seed solution. The fermentation seed solution was then inoculated into sterile fermentation medium at a volume ratio of 4%, with a total fermentation tank volume of 3 L. Fermentation was carried out at 30 °C, with a stirring speed of 220 rpm and an aeration rate of 1.2 VVM to obtain the Hermann's Atlanta bacillus LP-1 fermentation broth. The initial pH of fermentation was 8. During fermentation, an automatic pH control device was used to maintain the pH of the fermentation broth at 8.0 using ammonia or hydrochloric acid. The fermentation time was 48 hours. Samples were taken every 3 hours to determine the concentration of microbial polysaccharides in the fermentation broth. Analysis: The above fermentation broth was centrifuged at 9000 rpm for 10 minutes. The supernatant was diluted and the content of microbial polysaccharides and cell count in the fermentation broth were measured. It was found that the optimal extracellular polysaccharide yield was 10.14 g / L at 48 h, the production rate was 0.21 g / L / h, and the total sugar conversion rate was 50.7%. The fermentation medium consisted of: 20 g / L sucrose, 3 g / L potassium nitrate, 0.064 mmol / L ferrous sulfate, 0.42 mmol / L disodium hydrogen phosphate, 2.48 mmol / L magnesium sulfate, and 1.28 mmol / L calcium chloride. The initial pH of the fermentation broth was adjusted to 8.0 using ammonia.

[0077] Purification of extracellular polysaccharides from Hermann's Atlanta LP-1:

[0078] S1: Cell removal: The Hermann's Atlanta bacillus LP-1 fermentation broth was diluted with water at a volume ratio of 1:4, and centrifuged in a high-speed centrifuge at 9000 rpm and 4 ℃ for 40 min to remove the bacterial cells and obtain the supernatant.

[0079] S2: Decolorization: Adjust the pH of the supernatant from S1 to 4.5-5.5 with 1 mol / L hydrochloric acid, add 5‰ activated carbon powder, and incubate in a constant temperature water bath at 60 ℃ with continuous stirring for 1 h to obtain a polysaccharide-activated carbon mixture. After stirring in the water bath, filter while hot using a Büchner funnel. First, prepare a filter cake using diatomaceous earth, then filter the polysaccharide-activated carbon mixture to obtain a clear and transparent polysaccharide solution. Adjust the pH of the decolorized polysaccharide solution to 8 with 1 mol / L sodium hydroxide solution.

[0080] S3: Protein Removal: The liquid obtained in S2 was concentrated to 1 / 20 of its original volume at 55 °C to obtain a concentrated solution. 0.5-1‰ trypsin and 1‰ neutral protease were added to the concentrated solution, and the reaction was carried out at 50 °C for 4 h. The reaction was then stopped by boiling for 10 min. The pH was adjusted to neutral with 1 mol / L hydrochloric acid solution, and 1 / 3 volume of Sevag reagent (chloroform: n-butanol = 4:1) was added. The solution was stirred overnight at 4 °C, then centrifuged at 9000 rpm for 30 min at 4 °C. The supernatant was collected, and Sevag reagent was added again, repeating the process until no protein layer appeared.

[0081] S4: Dialysis: Dialyze the liquid in S3 with double-distilled water for 1-2 days, evaporate and concentrate, add anhydrous ethanol for precipitation overnight, centrifuge for 9000 min to collect the precipitate, freeze dry to obtain crude extracellular polysaccharide of Hermann's Atlanta bacillus LP-1;

[0082] S5: Adsorption: After reconstitution of the crude Hermann's Atlanta bacillus LP-1 extracellular polysaccharide from S4, the insoluble matter was removed by filtration through a 0.22 μm filter membrane, and then pumped into DEAE Sepharose. ® An anion exchange column, after adsorption treatment, yields a chromatography column adsorbed with microbial polysaccharides;

[0083] S6: Elution: Elution was performed with 0.5 mol / L sodium chloride solution at a flow rate of 1 mL / min. The resulting eluent was subjected to a delay treatment and then freeze-dried to obtain the high-quality extracellular polysaccharide of Hermann's Atlanta bacillus LP-1.

[0084] Determination of extracellular polysaccharide content:

[0085] The polysaccharide content was determined using the phenol-sulfuric acid method: Dry, constant-weight glucose was weighed and prepared into standard solutions of 0.00, 0.01, 0.02, 0.04, 0.08, and 0.10 mg / mL, respectively. Simultaneously, a 0.1 mg / mL extracellular polysaccharide solution was prepared. 1 mL of each standard solution and extracellular polysaccharide solution was accurately pipetted into test tubes, and 1 mL of 6% phenol reagent and 5 mL of concentrated sulfuric acid were added to each. After mixing, the tubes were heated at 40 °C for 30 min, cooled to room temperature, and the absorbance was measured at 490 nm. Using the 0 mg / mL standard solution as a blank control, a standard curve was plotted with absorbance on the ordinate and concentration on the abscissa. The regression equation was obtained, and the extracellular polysaccharide content was calculated.

[0086] Identification of microbial polysaccharides:

[0087] High-performance liquid chromatography, infrared spectroscopy, and nuclear magnetic resonance were used to identify fermentation products of A. hermannii LP-1.

[0088] ① Molecular weight determination: The extracellular polysaccharide was prepared into an aqueous solution of 2 mg / mL and filtered through a 0.22 μm microporous membrane. Dextran with different molecular weights (180-135350 Da) was used as standards. The molecular weight was determined using an Ultrahydrogel™ Linear (7.8 mm × 300 mm) gel chromatography column and an Empower 3 workstation with a high performance liquid chromatograph (1525, Waters).

[0089] ② Monosaccharide component study: 0.1 g of extracellular polysaccharide was placed in a hydrolysis flask, and 5 mL of 1 mol / L sulfuric acid was added. Hydrolysis was carried out at 100 ℃ for 12 h. The pH of the hydrolyzed product was then adjusted to neutral using NaOH solution. The hydrolyzed extracellular polysaccharide and each standard monosaccharide were then injected to analyze the structural composition of the monosaccharides. All injected samples were filtered through a 0.22 μm filter membrane. The chromatographic conditions were: HPLC column (organic acid column); column temperature: 50 ℃; mobile phase: 0.2 mol / L NaOH solution; flow rate: 0.3 mL / min; injection volume: 20 µL. The monosaccharide composition in the polysaccharide was determined based on the retention time of the standard sugars, and the molar ratio of each monosaccharide component was calculated based on the peak area. As can be seen from the high performance liquid chromatogram, the fermentation product of A. hermannii LP-1 is composed of four monosaccharides: glucuronic acid, glucose, galactose, and fucose, in a molar ratio of 1:1.1:2.1:2.3.

[0090] ③ Infrared spectroscopy and nuclear magnetic resonance studies: 1 mg of dried extracellular polysaccharide and 99 mg of dried KBr were thoroughly ground, compressed into tablets, and then the infrared spectra of the samples were measured using an infrared spectrometer with a scanning range of 4000 cm⁻¹. -1 -400 cm -1 Infrared spectra of microbial polysaccharides were obtained. 50 mg of extracellular polysaccharide sample was loaded into an NMR tube and completely dissolved in 0.5 mL of D₂O. To ensure the polysaccharide sample dissolved in high-purity D₂O, it was freeze-dried and exchanged three times. Finally, NMR analysis of the microbial polysaccharides was performed at 25 °C. 1 H and NMR 13 C-test. Results are as follows: Figure 4 and Figure 5 As shown.

[0091] ④ Methylation analysis: The methylation of extracellular polysaccharides was analyzed using the modified Hakomori method. The gas chromatography-mass spectrometry peaks were analyzed according to the glycosylation library of the Glycocomplex Research Center at the University of Georgia to determine the glycosidic bond linkage mode in each polysaccharide structure. The results are shown in Table 2.

[0092] The extracellular polysaccharide obtained in this invention is a homogeneous polysaccharide with a molecular weight of 1159.93 kDa. It mainly contains nine linkage modes, namely T-Fucp-(1→), T-Glcp-(1→), T-GlcAp-(1→), T-Galp-(1→, →3)-Glcp-(1→, →4)-GlcAp-(1→, →6)-GlcAp-(1→, →6)-Galp-(1→, →3,4)-Glcp-(1→), with a molar ratio of 8.8:1.2:1.7:5.4:2.6:2.8:1.1:2.6:1.5.

[0093] Table 2 Polysaccharide methylation data

[0094]

[0095] Example 3

[0096] The fermentation seed culture obtained in Example 2 was inoculated at a rate of 4% (v / v) into fermentation media containing no carbon source (C) and those containing sucrose (SUC), lactose (LAC), arabinose (ARA), fructose (FRU), and glucose (GLU) at a final concentration of 20 g / L. The initial pH was 7.0, and the cultures were incubated at 32 °C with shaking at 200 rpm in 500 mL Erlenmeyer flasks containing 80 mL of fermentation medium. Fermentation was carried out for 48 h. Fermentation broths with different carbon sources were collected, and the cell count was measured at 600 nm. The extracellular polysaccharide content was calculated using the method described in Example 2. Figure 6 It can be seen that fermentation using sucrose as the carbon source resulted in the highest cell mass and the highest production of extracellular polysaccharides; therefore, sucrose was selected as the optimal carbon source. The microbial polysaccharide yield reached 5.65 g / L, the total sugar conversion rate was 28.25%, and the production rate was 0.12 g / L / h.

[0097] Example 4

[0098] The fermentation seed culture obtained in Example 2 was inoculated at a rate of 4% (v / v) into fermentation media with sucrose concentrations of 0 g / L, 10 g / L, 20 g / L, 30 g / L, 40 g / L, and 50 g / L, respectively. The initial pH was 7.0. Fermentation was carried out at 32 °C with shaking at 200 rpm in 500 mL Erlenmeyer flasks containing 80 mL of fermentation medium. Fermentation was carried out for 48 h. Cell counts were measured at 600 nm using the fermentation broth, and the content of microbial polysaccharides was calculated using the procedures described in Example 2. Figure 7 It can be seen that when the carbon source concentration is 20 g / L, the extracellular polysaccharide yield and cell mass are the highest, the total sugar conversion rate is 31.20%, and the production rate is 0.13 g / L / h. Therefore, a sucrose concentration of 20 g / L was selected for subsequent fermentation.

[0099] Example 5

[0100] The fermentation seed culture obtained in Example 2 was inoculated at a 4% (v / v) inoculation rate into fermentation media containing no organic nitrogen source (C), beef extract (BE) at a final concentration of 3 g / L, yeast extract (YEJ), tryptone (TRY), (domestic) yeast extract powder (YEP), and (imported) yeast powder (YEP) (M). The initial pH was 7.0, and the culture was carried out at 32 °C with shaking at 200 rpm in 500 mL Erlenmeyer flasks containing 80 mL of fermentation medium. Fermentation was carried out for 48 h, and the cell count was measured at 600 nm using the fermentation broth. The content of microbial polysaccharides was calculated using the procedure in Example 2. Figure 8 It can be seen that when YEP(M) is used as the organic nitrogen source, the extracellular polysaccharide yield reaches the highest level of 7.31 g / L, the total sugar conversion rate is 36.55%, and the production rate is 0.15 g / L / h. Therefore, YEP(M) is selected for subsequent fermentation.

[0101] Example 6

[0102] The fermentation seed culture obtained in Example 2 was inoculated at a rate of 4% (v / v) into fermentation media with YEP(M) concentrations of 0 g / L, 1 g / L, 2 g / L, 3 g / L, 4 g / L, and 5 g / L, respectively. The initial pH was 7.0. Fermentation was carried out at 32 °C with shaking at 200 rpm in 500 mL Erlenmeyer flasks containing 80 mL of fermentation medium. Fermentation was carried out for 48 h. Cell count was measured at 600 nm using the fermentation broth. The extracellular polysaccharide content was calculated using the procedure described in Example 2. The results are as follows: Figure 9 As shown, imported yeast powder has no significant effect on cell growth and the amount of extracellular polysaccharides produced, and it is expensive. Therefore, considering all factors, no organic nitrogen source is added to the fermentation medium.

[0103] Example 7

[0104] The fermentation seed culture obtained in Example 2 was inoculated at a rate of 4% (v / v) into fermentation media containing either an inorganic nitrogen source (Con) or ammonium nitrate (A), calcium nitrate (B), potassium nitrate (C), sodium nitrate (D), or diammonium hydrogen phosphate (E) at a final concentration of 3 g / L. The initial pH was 7.0. Fermentation was carried out at 32 °C with shaking at 200 rpm in 500 mL Erlenmeyer flasks containing 80 mL of fermentation medium. Fermentation was carried out for 48 h. Cell count was measured at 600 nm using the fermentation broth, and the extracellular polysaccharide content was calculated using the procedure described in Example 2. Figure 10 It can be seen that when potassium nitrate is used as the inorganic nitrogen source, the extracellular polysaccharide yield reaches 6.95 g / L, the total sugar conversion rate is 34.75%, and the production rate is 0.14 g / L / h. Therefore, potassium nitrate was selected as the optimal inorganic nitrogen source for subsequent studies.

[0105] Example 8

[0106] The fermentation seed culture obtained in Example 2 was inoculated at a rate of 4% (v / v) into fermentation media with potassium nitrate concentrations of 0 g / L, 1 g / L, 2 g / L, 3 g / L, 4 g / L, and 5 g / L, respectively. The initial pH was 7.0. Fermentation was carried out at 32 °C with shaking at 200 rpm in 500 mL Erlenmeyer flasks containing 80 mL of fermentation medium. Fermentation was carried out for 48 h. Cell count was measured at 600 nm using the fermentation broth, and the extracellular polysaccharide content was calculated using the procedure described in Example 2. Figure 11 It can be seen that when the potassium nitrate concentration is 3 g / L, the extracellular polysaccharide yield reaches 6.88 g / L, and the production rate is 0.14 g / L / h. Therefore, 3 g / L was selected as the optimal inorganic nitrogen source concentration for subsequent studies.

[0107] Example 9

[0108] The fermentation seed culture obtained in Example 2 was inoculated at an inoculation rate of 4% (v / v) into fermentation media containing the following metal salt concentrations, and the single-factor variable experiments were conducted as follows:

[0109] Ferrous sulfate: 0.032, 0.064, 0.128, 0.256, 0.384 mmol / L and control group; the optimal concentration of ferrous sulfate was 0.064 mmol / L.

[0110] The concentration of ferrous sulfate was controlled at 0.064 mmol / L. The concentrations of disodium hydrogen phosphate were 0.014, 0.028, 0.042, 0.056, and 0.084 mol / L, and the control group was also tested. The optimal concentration of disodium hydrogen phosphate was 0.042 mol / L.

[0111] The concentrations of ferrous sulfate (0.064 mmol / L), disodium hydrogen phosphate (0.042 mol / L), and magnesium sulfate (1.22, 2.48, 3.66, 6.1, and 9.76 mmol / L) were controlled, along with a control group. The optimal concentration of magnesium sulfate was 2.48 mmol / L.

[0112] The concentrations of ferrous sulfate (0.064 mmol / L), disodium hydrogen phosphate (0.042 mol / L), magnesium sulfate (2.48 mmol / L), and calcium chloride (0.64, 0.96, 1.28, 1.6, 1.92 mmol / L) were controlled, along with a control group. The optimal concentration of calcium chloride was 1.28 mmol / L.

[0113] The initial pH was 7.0. Fermentation was carried out at 32 °C with shaking at 200 rpm in 500 mL Erlenmeyer flasks containing 80 mL of fermentation medium. Fermentation was carried out for 48 h. Cell counts were measured at 600 nm using samples of the fermentation broth. The content of microbial polysaccharides was calculated using the procedure described in Example 2. Figure 12 It can be seen that when ferrous sulfate is 0.064 mmol / L (9.7 mg / L), disodium hydrogen phosphate is 0.042 mol / L (6.0 g / L), magnesium sulfate is 2.48 mmol / L (0.3 g / L), and calcium chloride is 1.28 mmol / L (0.14 g / L), the strain produces 8.68 g / L of extracellular polysaccharides, with a total sugar conversion rate of 43.4% and a production rate of 0.18 g / L / h. The control group consists of blank controls with an addition amount of 0 mmol / L.

[0114] Example 10

[0115] The fermentation seed culture obtained in Example 2 was inoculated at a rate of 4% (v / v) into fermentation media at pH 6.0, 7.0, 8.0, 9.0, 10.0, and the natural control (C), respectively. The cultures were incubated at 32 °C with shaking at 200 rpm in 500 mL Erlenmeyer flasks containing 80 mL of fermentation medium. Fermentation was carried out for 48 h. Cell counts were determined at 600 nm using the fermentation broth, and the extracellular polysaccharide content was calculated using the procedures described in Example 2. Figure 13 It can be seen that when pH=8, the extracellular polysaccharide yield reaches 8.60 g / L, the total sugar conversion rate is 43.0%, and the production rate is 0.18 g / L / h. Therefore, pH=8 was selected as the optimal pH for subsequent studies.

[0116] Example 11

[0117] The fermentation seed culture obtained in Example 2 was inoculated into the same fermentation medium at an inoculum size of 4% (v / v), with an initial pH of 7.0. The culture was then placed in 500 mL Erlenmeyer flasks containing 80 mL of fermentation medium at different temperatures (22 °C, 24 °C, 28 °C, 30 °C, 32 °C, and 36 °C) with shaking at 200 rpm for 48 h. Cell count was determined at 600 nm using the fermentation broth, and the extracellular polysaccharide content was calculated using the procedure described in Example 2. Figure 14 It can be seen that when the fermentation temperature is 30 °C, the extracellular polysaccharide yield reaches 9.21 g / L, the total sugar conversion rate is 46.05%, and the production rate is 0.19 g / L / h. Therefore, 30 °C was selected as the optimal temperature for subsequent fermentation.

[0118] Example 12

[0119] Antioxidant capacity of Hermann's Atlanta bacillus LP-1 extracellular polysaccharide

[0120] I. Free radical scavenging ability of 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH)

[0121] An equal volume of LP-1 polysaccharide solution was mixed with DPPH-ethanol solution (0.1 mmol / L) and reacted at room temperature in the dark for 30 min. The absorbance at 517 nm was measured (A1). The DPPH scavenging rate (%) was calculated as [1-(A1-A2) / A0]×100% to represent the antioxidant activity (where A1 refers to the absorbance of LP-1 polysaccharide, A2 refers to the absorbance of the control group with anhydrous ethanol instead of DPPH solution, and A0 refers to the absorbance of the blank group with distilled water instead of the sample). The results are as follows. Figure 17 As shown in Figure A, the scavenging effect of LP-1 polysaccharide on DPPH free radicals increased with increasing concentration, reaching 52.29% at a concentration of 10 mg / mL, indicating that LP-1 polysaccharide has certain antioxidant activity. Ascorbic acid (VC) served as a positive control.

[0122] II. Linoleic acid peroxidation inhibition ability

[0123] The ability of polysaccharides to inhibit linoleic acid peroxidation was evaluated by determining the malondialdehyde (MDA) content in the linoleic acid system. 4.1 mL of linoleic acid-ethanol solution (2.5%, v / v), 10 mL of phosphate buffer (0.2 M, pH 7.4), 4.9 mL of distilled water, and 1 mL of 4 mM ferrous sulfate heptahydrate were mixed. Then, 4 mL of LP-1 polysaccharide solution of different concentrations was added, and the mixture was thoroughly mixed and incubated at 40 °C in the dark for 24 hours. After incubation, 1 mL of the reaction solution was taken, and 1 mL of 25% trichloroacetic acid and 2 mL of 0.67% thiobarbituric acid were added sequentially. The mixture was heated in a boiling water bath for 15 minutes, cooled, and then 4 mL of n-butanol was added. Deionized water was used as a blank control, and VC was used as a positive control. The absorbance was measured at 532 nm. The linoleic acid peroxidation inhibition rate (%) was calculated as (1-A) / (25%). S / A0)×100% Reactive antioxidant activity (A S A represents the absorbance containing polysaccharides, while A0 represents the absorbance when deionized water is used instead of polysaccharides. Results are as follows: Figure 17 As shown in B, the inhibitory effect of LP-1 polysaccharide on linoleic acid peroxidation increases with increasing mass concentration. At a concentration of 5 mg / mL, the inhibition rate reaches 68.57%, which means that LP-1 polysaccharide has a good ability to resist lipid peroxidation.

[0124] Example 13

[0125] Hypoglycemic ability of Hermann's Atlanta bacillus LP-1 extracellular polysaccharide

[0126] I. α-Glucosidase Inhibitory Activity

[0127] Take 100 μL of LP-1 polysaccharide solution, add 100 μL of α-glucosidase (57 U / 100 mL), and incubate at 37 ℃ for 10 min. Then add 100 μL of p-nitrophenol-β-D-galactopyranoside (PNPG) (5 mM), and incubate at 37 ℃ for 20 min. Finally, add 1 mL of Na2CO3 solution (1 M) to terminate the reaction. Use distilled water as a blank control and acarbose as a positive control. Measure the absorbance at 405 nm. α-glucosidase inhibition rate = (1-(A x -A x0 ) / A0)×100 %(A x A0 is the absorbance of the sample; A0 is the absorbance of the blank group using distilled water instead of the sample; A0 is the absorbance of the blank group. x0 (The absorbance was used as a control group with distilled water instead of α-glucosidase). Results are as follows: Figure 18 As shown in Figure A, the inhibitory effect of LP-1 polysaccharide on α-glucosidase increases with increasing mass concentration, reaching 69.68% at a concentration of 160 μg / mL. This indicates that LP-1 polysaccharide has good hypoglycemic activity.

[0128] II. α-Amylase Inhibitory Activity

[0129] Take 500 μL of LP-1 polysaccharide solution, add 20 U / mL of α-amylase (250 μL), and incubate at 25 ℃ for 10 min. Add 1% of soluble starch (500 μL), and incubate at 25 ℃ for 10 min. Then add 1 mL of 3,5-dinitrosalicylic acid (DNS) to stop the reaction, boil for 5 min, cool to room temperature, and add 10 mL of distilled water. Use distilled water as a blank control and acarbose as a positive control. Measure the absorbance at 540 nm. α-Amylase inhibition rate = (1-(A x -A x0 ) / A0)×100%(A x A0 is the absorbance of the sample; A0 is the absorbance of the blank group using distilled water instead of the sample solution; A0 is the absorbance of the blank group. x0 (The absorbance was used as a control group with distilled water instead of α-amylase). Results are as follows: Figure 18 As shown in B, the inhibitory effect of LP-1 polysaccharide on α-amylase increases with increasing mass concentration, reaching an inhibition rate of 16.73% at a concentration of 160 μg / mL.

[0130] Example 14

[0131] I. Effect of LP-1 polysaccharide on HeLa cell proliferation by CCK8 assay

[0132] HeLa cells were fed at a rate of 1×10 5 Cells were seeded at a concentration of 0.1 mL / well in 96-well cells and cultured completely in DMEM containing 10% serum. The cells were then cultured adherently in a standard incubator (37 °C, 5% CO2) for 12 h, followed by co-incubation with different concentrations of LP-1 polysaccharide for 48 h. The culture medium was discarded, and fresh culture medium was mixed with CCK8 reagent at a volume ratio of 9:1 to prepare the assay reagent. 100 μL was added to each well, and the cells were returned to the incubator for 2 h. The absorbance at 450 nm was then measured.

[0133] Cell viability = (Average optical density of drug delivery wells / Average optical density of control group) × 100%

[0134] The results are as follows Figure 19 As shown, within the concentration range of 50-1000 μg / mL, LP-1 polysaccharide did not exhibit a significant inhibitory effect on the cell viability of HeLa cells, indicating that LP-1 polysaccharide has no cytotoxic effect on HeLa cells.

[0135] II. Plasmid transfection and acquisition of HPV pseudovirus particles

[0136] Pre-lay 7×10 6 293T / 17 cells at 75 cm 2 In culture flasks, cells were transfected when they reached approximately 40% confluency. First, 38 μg of HPV capsid protein and GFP reporter plasmid were mixed in 2 mL of OptiMEM, and 85 μL of Lipofectamine 2000 liposomes were also mixed in 2 mL of OptiMEM. Both were incubated at room temperature for 10 min. The corresponding DNA and liposomes were then mixed and incubated at room temperature for at least 20 min. The DNA-liposome mixture was then added directly to the cells and incubated overnight at 37 °C. The next morning, the medium was replaced with fresh DMEM, and the cells were cultured at 37 °C for another 30 h. 48 h after transfection, the cells were harvested, and 1 / 20 volume of 10% Triton X-100 (final volume 0.5%), 0.1% Benzonase, and 0.1% Plasmid Safe reagent were added to the solution. The cell lysate was then matured at 37 °C for 24 h, aliquoted, and stored at -80 °C for later use.

[0137] III. Dose-response relationship study of the inhibitory effect of LP-1 polysaccharide on HPV pseudovirus infection

[0138] HeLa cells were fed at a rate of 1×10 5Cells were seeded at a concentration of 0.1 mL / mL into 96-well cell culture plates and incubated at 37°C in a 5% CO2 incubator for 24 h. 50 μL of a 100-fold diluted HPV pseudovirus particle mixture was added, followed by five 2-fold diluted LP-1 polysaccharide solutions (200, 100, 50, 25, 12.5, and 0 μg / mL) below the non-virulent concentration. After 48 h of incubation, the cells were washed twice with PBS, and the fluorescence intensity of each group was observed using an inverted fluorescence microscope. The number of positive cells (N) and the average optical density (AOI) were analyzed using ImageJ software, and the relative infection total (RA = N × AOI) was obtained by multiplying the two. The infection inhibition rate was calculated as (RA = N × AOI). 空白组 -RA 样品组 ) / RA 空白组 .

[0139] The results are as follows Figure 20 As shown, within the concentration range of 25-200 μg / mL, LP-1 polysaccharide exhibited strong inhibitory effects on the infection process of HPV16 and HPV18 pseudovirus particles, and the inhibitory effect was dose-dependent. At a lower concentration (25 μg / mL), its inhibition rates were still as high as 68.7% and 52.5%, respectively. The highly effective and low-toxicity anti-HPV activity of LP-1 polysaccharide has significant advantages in its development as an active lead compound for anti-HPV drugs.

[0140] Other embodiments of this disclosure will readily occur to those skilled in the art upon consideration of the specification and practice of the disclosure herein. This disclosure is intended to cover any variations, uses, or adaptations of this disclosure that follow the general principles of this disclosure and include common knowledge or customary techniques in the art not disclosed herein. The specification and examples are to be considered exemplary only, and the true scope and spirit of this disclosure are indicated by the claims.

[0141] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. The scope of patent protection of the present invention shall be determined by the claims. Similarly, any equivalent structural changes made based on the content of the present invention's specification shall also be included within the scope of protection of the present invention.

Claims

1. Hermann's Atlanta bacillus LP-1, characterized in that, Hermann's Atlanta bacillus LP-1 was deposited at the China General Microbiological Culture Collection Center on March 17, 2025, with accession number CGMCC No. 33858.

2. A method for synthesizing extracellular polysaccharides using Hermann's Atlanta bacillus LP-1, characterized in that, The method includes the following steps: inoculating the Hermann's Atlanta bacillus LP-1 of claim 1 into a fermentation medium for aerobic culture to obtain the extracellular polysaccharide of Hermann's Atlanta bacillus LP-1.

3. The method for synthesizing extracellular polysaccharides from Hermann's Atlanta bacillus LP-1 according to claim 2, characterized in that, The extracellular polysaccharide of Hermann's Atlanta bacillus LP-1 includes glucuronic acid, glucose, galactose, and fucose, wherein the molar ratio of glucuronic acid, glucose, galactose, and fucose is 1:1.1:2.1:2.

3.

4. The method for synthesizing extracellular polysaccharides from Hermann's Atlanta LP-1 according to claim 2, characterized in that, The extracellular polysaccharide structure of *Hermann's Atlanta bacillus* LP-1 contains nine linkage modes, namely T-Fuc p -(1→、T-Glc p -(1→、T-GlcA p -(1→、T-Gal p -(1→、→3)-Glc p -(1→、→4)-GlcA p -(1→、→6)-GlcA p -(1→、→6)-Gal p -(1→、→3,4)-Glc p -(1→, its molar ratio is 8.8:1.2:1.7:5.4:2.6:2.8:1.1:2.6:1.

5.

5. The method for synthesizing extracellular polysaccharides from Hermann's Atlanta bacillus LP-1 according to claim 2, characterized in that, The aerobic culture is either a shake flask culture or a fermenter culture. The culture conditions for the shake flask culture are: pH 7.0-8.0, culture temperature 28-32 ℃, inoculum size 1-8%, and culture time 12-24 h; The fermentation conditions in the fermenter are as follows: pH 7.0-8.0, culture temperature 28-32 ℃, inoculum size 1-15%, culture time 24-48 h, aeration ratio 1.0-1.2 VVM, and batch fermentation.

6. The method for synthesizing extracellular polysaccharides from Hermann's Atlanta LP-1 according to claim 2, characterized in that, The fermentation medium comprises: 10-50 g / L sucrose, 1-5 g / L potassium nitrate, 0.032-0.584 mmol / L ferrous sulfate, 0.014-0.084 mol / L disodium hydrogen phosphate, 1.22-9.76 mmol / L magnesium sulfate, and 0.64-1.28 mmol / L calcium chloride; the pH of the fermentation medium is 7.0-8.

0.

7. The extracellular polysaccharide of Hermann's Atlanta bacillus LP-1, characterized in that, The extracellular polysaccharide was prepared using the method for synthesizing extracellular polysaccharides from *H. tarda* LP-1 according to any one of claims 2-6. The extracellular polysaccharide of *H. tarda* LP-1 comprises glucuronic acid, glucose, galactose, and fucose, wherein the molar ratio of glucuronic acid, glucose, galactose, and fucose is 1:1.1:2.1:2.

3. The extracellular polysaccharide structure of *H. tarda* LP-1 includes nine linkage schemes, namely T-Fuc... p -(1→、T-Glc p -(1→、T-GlcA p -(1→、T-Gal p -(1→、→3)-Glc p -(1→、→4)-GlcA p -(1→、→6)-GlcA p -(1→、→6)-Gal p -(1→、→3,4)-Glc p -(1→, the molar ratio of which is 8.8:1.2:1.7:5.4:2.6:2.8:1.1:2.6:1.5; the molecular weight of the extracellular polysaccharide of Hermann's Atlanta bacillus LP-1 is 1159.93 kDa.

8. The use of the extracellular polysaccharide of Hermann's Atlanta bacillus LP-1 as described in claim 7 in the preparation of medicaments for the prevention and / or treatment and / or synergistic treatment of diabetes.

9. The use of the extracellular polysaccharide of Hermann's Atlanta bacillus LP-1 as described in claim 7 in the preparation of drugs for the prevention and / or treatment and / or synergistic treatment of HPV.

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