Use of hermanella atlantica lp-1 for the co-production of exopolysaccharide and indole-3-acetic acid

Abstract

This invention relates to the application of *H. atlantis lanceolata* LP-1 in the co-production of extracellular polysaccharides and indoleacetic acid (IAA), belonging to the field of microbial technology. *H. atlantis lanceolata* LP-1 was inoculated into a fermentation medium and cultured aerobically to obtain indoleacetic acid and extracellular polysaccharides. *H. atlantis lanceolata* LP-1 was deposited on March 17, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 33858. The highest accumulated extracellular polysaccharide concentration of this *H. atlantis lanceolata* LP-1 in the fermentation medium was 10.05 g / L, and the highest IAA concentration was 45.37 mg / L.

Description

Application of Hermann's Atlanta bacillus LP-1 in the co-production of extracellular polysaccharides and indoleacetic acid Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to the application of Hermann's Atlanta bacillus LP-1 in the co-production of extracellular polysaccharides and indoleacetic acid. 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, the potential biological activities of microbial polysaccharides, such as antioxidant activity, cholesterol-lowering, immunomodulatory, hypoglycemic, and anticancer abilities, are rapidly making them an important emerging biomaterial. However, the current synthesis of most microbial polysaccharides uses expensive food raw materials as substrates, resulting in high production costs and limiting their application.

[0003] Indoleacetic acid (IAA) is a plant growth regulator that plays a crucial role in plant cell division, elongation, differentiation, seed germination, root development, and vegetative growth. Applying IAA can promote crop growth and increase crop yield, reducing the use of pesticides and chemical fertilizers, and is therefore widely used in agricultural production. Currently, there are two main industrial processes for IAA production: the chemical method and the microbial method. The chemical method utilizes raw materials such as indole and chloroacetic acid through multiple reactions to obtain IAA. Although the process is relatively mature, it suffers from high raw material costs and environmental pollution. The microbial method, on the other hand, offers advantages such as mild conditions, low energy consumption, no use of toxic or harmful substances, and minimal environmental pollution, making it a promising candidate for IAA production. Existing technologies all utilize microbial strains to produce microbial polysaccharides or IAA alone, resulting in low raw material utilization and high costs. Therefore, developing a strain capable of efficiently co-producing microbial polysaccharides and IAA has significant practical importance and application value. Summary of the Invention

[0004] The purpose of this invention is to provide an application of Hermann's Atlanta bacillus LP-1 in the co-production of extracellular polysaccharides and indoleacetic acid.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] The application of Hermann's Atlanta LP-1 in the co-production of extracellular polysaccharides and indoleacetic acid includes the following steps:

[0007] I. Strain Activation: Hermann's Atlanta LP-1 was inoculated onto modified TB medium and incubated statically at 28-32 °C for 18-22 h. Single colonies were picked and streaked onto modified TB medium containing 20 g / L sucrose and incubated statically at 28-32 °C for 18-22 h to obtain the activated strain. The modified TB medium consisted 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. Hermann's Atlanta 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.

[0008] II. Seed Culture Preparation: Under aseptic conditions, inoculate 4 loops of the activated bacterial strain into a shake flask containing seed culture medium, then place it on a shaker at 200 rpm and incubate at 28-32 ℃ for 16 h to obtain the fermentation seed culture. Seed culture medium composition: sucrose 20 g / L, disodium hydrogen phosphate 6.5 g / L, potassium nitrate 3 g / L, magnesium sulfate 0.5 g / L, ferrous sulfate 0.018 g / L, calcium chloride 0.013 g / L, tryptone 2 g / L, solvent: water, pH 7.0-8.0.

[0009] III. Fermentation Culture: Under aseptic conditions, the fermentation seed liquid is inoculated into the fermentation medium at a volume ratio of 1-8%, and placed on a shaker at 200 rpm. The initial pH is 6.5-7.5, and the culture is carried out at 28-32 ℃ for 24-48 h. When the concentrations of polysaccharides and IAA in the fermentation broth no longer increase, the fermentation is stopped to obtain Hermann's Atlanta bacillus LP-1 fermentation broth.

[0010] Alternatively, the fermentation seed culture 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 6.5-7.5, and a culture time of 24-48 h. When the concentrations of polysaccharides and IAA in the fermentation broth no longer increase, fermentation is stopped to obtain Hermann's Atlanta bacillus LP-1 fermentation broth.

[0011] The fermentation medium comprises: 0-50 g / L carbon source, 0-5 g / L organic nitrogen source, 0-10 g / L inorganic nitrogen source, 0-500 mg / L L-tryptophan, 1.5-4 g / L metal salt, and water as the solvent; the pH of the fermentation medium is 6.5-7.5; the carbon source is one or more of maltose, fructose, lactose, glucose, and sucrose; the organic nitrogen source is one or more of beef extract, yeast extract, tryptone, yeast extract powder, and soybean peptone; the inorganic nitrogen source is one or more of potassium nitrate, sodium nitrate, ammonium nitrate, ammonium sulfate, and diammonium hydrogen phosphate; and the metal salt is a mixed metal salt of magnesium sulfate, ferrous sulfate, and disodium hydrogen phosphate.

[0012] Furthermore, the fermentation medium consists of: 30 g / L sucrose, 2 g / L beef extract, 6 g / L potassium nitrate, 300 mg / L L-tryptophan, 10 mg / L ferrous sulfate, 3 g / L disodium hydrogen phosphate, and 300 mg / L magnesium sulfate.

[0013] IV. Isolation of extracellular polysaccharides and IAA co-produced by Hermann's Atlanta bacillus LP-1:

[0014] S1: Cell removal: The fermentation broth of Hermann's Atlanta bacillus LP-1 was heated to 80 °C and kept in a constant temperature water bath for 20 min to denature some of the proteins; then it was centrifuged in a high-speed centrifuge at 9000 rpm and 4 °C for 40 min to remove the cells and obtain the supernatant.

[0015] S2: Separation: Concentrate the supernatant under reduced pressure to 1 / 5 of its original volume, add 3 times the volume of anhydrous ethanol, and let stand at 4 °C overnight to allow precipitation to complete; then centrifuge at 9000 rpm and 4 °C for 10 min, and collect the precipitate containing crude polysaccharide and the supernatant containing IAA and other small molecules respectively.

[0016] S3: Preparation of crude polysaccharide: The precipitate containing crude polysaccharide was washed and dehydrated successively with anhydrous ethanol, acetone and diethyl ether, and then vacuum dried to obtain crude polysaccharide powder;

[0017] S4: Preparation of crude IAA extract: The supernatant containing IAA and other small molecules was concentrated under reduced pressure at <40 °C, and most of the ethanol was recovered to obtain a concentrated aqueous IAA mixture. The pH was adjusted to 2.5-3.0 with 1M HCl, and an equal volume or twice the volume of ethyl acetate was added. The mixture was shaken vigorously and allowed to stand for separation. Anhydrous sodium sulfate was added to the organic phase for dehydration and drying, and the mixture was filtered. The organic solvent was recovered by rotary evaporation under reduced pressure at <40 °C to obtain the crude IAA extract.

[0018] V. Purification of extracellular polysaccharides:

[0019] S1: Decolorization: The crude polysaccharide powder was dissolved in distilled water to obtain a crude polysaccharide solution with a concentration of 5 mg / mL. The pH of the crude polysaccharide solution was adjusted to 4.5-5.5, and 5‰ activated carbon powder was added. The mixture was stirred continuously in a constant temperature water bath at 60 ℃ for 1 h to obtain a polysaccharide-activated carbon mixture. After stirring in the water bath, the mixture was filtered while hot using a Büchner funnel. A filter cake was first prepared using diatomaceous earth, and then the polysaccharide-activated carbon mixture was filtered to obtain a clear and transparent polysaccharide solution. The pH of the decolorized polysaccharide solution was adjusted to 8.

[0020] S2: Protein Removal: The polysaccharide solution obtained in S1 was concentrated to 1 / 20 of its original volume at 55 °C to obtain a concentrated solution. Trypsin (0.5-1‰) and neutral protease (1‰) 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, 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.

[0021] S3: Dialysis: Dialyze the liquid in S2 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 the extracellular crude polysaccharide of Hermann's Atlanta bacillus LP-1.

[0022] S4: Adsorption: After reconstituted the extracellular crude polysaccharide of Hermann's Atlanta bacillus LP-1 from S3, 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;

[0023] S5: Elution: Elution was performed with 0.5 mol / L sodium chloride solution at a flow rate of 1 mL / min to obtain the eluent. The eluent was subjected to a delay treatment and then freeze-dried to obtain the extracellular polysaccharide of Hermann's Atlanta bacillus LP-1.

[0024] 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.

[0025] 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.

[0026] VI. Purification of indoleacetic acid:

[0027] S1: Solid-phase extraction purification: The activated C18 solid-phase extraction column is equilibrated sequentially with methanol and water. The sample dissolved in a small amount of acidic aqueous phase (pH 3.0) is loaded onto the column. Strongly polar impurities are washed away with an acidic aqueous solution containing 20% ​​methanol (pH 3.0), and then IAA is eluted with 70-80% methanol to obtain the eluent.

[0028] S2: High performance liquid chromatography determination of content: The eluent is dried under nitrogen, redissolved in 0.1% formic acid aqueous solution containing 20% ​​methanol, filtered through a 0.22 μm microporous membrane, and placed into an HPLC sample vial for analysis;

[0029] S3: Chromatographic conditions: Agilent ZORBAX SB-C18 column (250 mm × 4.6 mm, 3-5 μm), flow rate 0.8 mL / min, column temperature 35 °C, injection volume 20 μL, fluorescence detector (excitation wavelength 280 nm, emission wavelength 350 nm), and mobile phase as shown in Table 1.

[0030] Table 1. Mobile phase for IAA content determination

[0031]

[0032] S4: Standard Curve Construction: IAA was dissolved in methanol to prepare a 1 mg / mL IAA solution as a stock solution. Then, it was diluted with an acidic aqueous solution containing 20% ​​methanol (pH 3.0) to prepare a series of standard solutions with concentrations of 0, 0.01, 0.05, 0.1, 0.5, 1.0, and 2.0 μg / mL. These standard solutions were then injected sequentially for analysis. A standard curve was constructed with the IAA peak area and concentration as the ordinate.

[0033] The beneficial effects of this invention are:

[0034] The *Hermannia Atlantaobacter* LP-1 strain of this invention can co-produce polysaccharides and IAA, and has potential application value. The *Hermannia Atlantaobacter* LP-1 strain accumulated up to 10.05 g / L of extracellular polysaccharides and 45.37 mg / L of IAA in the fermentation medium. Compared with traditional chemical synthesis, the co-production of extracellular polysaccharides and IAA by *Hermannia Atlantaobacter* LP-1 reduces production costs and has the advantages of simple operation, high safety, and environmental friendliness, making it highly promising for industrial application. Attached Figure Description

[0035] Figure 1 shows the observation of Hermann's Atlanta bacillus LP-1 in Example 1, where A is a microscopic image, B is a colony morphology image on TB plate, and C is a colony morphology image on seed plate.

[0036] Figure 2 shows the agarose gel electrophoresis diagram of 16S rDNA purification by PCR of Hermann's Atlanta bacillus LP-1 in Example 1;

[0037] Figure 3 is a phylogenetic tree diagram of Hermann's Atlanta bacillus LP-1 in Example 1;

[0038] Figure 4 shows the high-performance liquid chromatogram of the monosaccharide composition of extracellular polysaccharides of Hermann's Atlanta bacterium LP-1;

[0039] Figure 5 shows the nuclear magnetic resonance (NMR) image of the extracellular polysaccharide of Hermann's Atlanta bacillus LP-1. 1 H-map;

[0040] Figure 6 shows the nuclear magnetic resonance (NMR) image of the extracellular polysaccharide of Hermann's Atlanta bacillus LP-1. 13 C-map;

[0041] Figure 7 shows the high-performance liquid chromatogram of the molecular weight of extracellular polysaccharides from Hermann's Atlanta LP-1;

[0042] Figure 8 shows the inhibition of α-glucosidase activity by the extracellular polysaccharide of Hermann's Atlanta bacillus LP-1;

[0043] Figure 9 shows the effect of extracellular polysaccharides from Hermann's Atlanta bacillus LP-1 on glycogen content in HepG2 cells;

[0044] Figure 10 shows the effect of extracellular polysaccharides from Hermann's Atlanta bacillus LP-1 on glucose consumption in HepG2 cells;

[0045] Figure 11 shows the effect of carbon source type in fermentation medium on the co-production of extracellular polysaccharide and IAA by Hermann's Atlanta bacterium LP-1.

[0046] Figure 12 shows the effect of carbon source concentration in the fermentation medium on the co-production of extracellular polysaccharides and IAA by Hermann's Atlanta LP-1.

[0047] Figure 13 shows the effect of the type of organic nitrogen source in the fermentation medium on the co-production of extracellular polysaccharide and IAA by Hermann's Atlanta LP-1.

[0048] Figure 14 shows the effect of organic nitrogen source concentration in fermentation medium on the co-production of extracellular polysaccharides and IAA by Hermann's Atlanta LP-1.

[0049] Figure 15 shows the effect of the type of inorganic nitrogen source in the fermentation medium on the co-production of extracellular polysaccharide and IAA by Hermann's Atlanta bacterium LP-1.

[0050] Figure 16 shows the effect of inorganic nitrogen source concentration in fermentation medium on the co-production of extracellular polysaccharides and IAA by Hermann's Atlanta bacterium LP-1.

[0051] Figure 17 shows the effect of L-tryptophan concentration in the fermentation medium on the co-production of extracellular polysaccharides and IAA by Hermann's Atlanta LP-1.

[0052] Figure 18 shows the effect of FeSO4 concentration in the fermentation medium on the co-production of extracellular polysaccharides and IAA by Hermann's Atlanta LP-1.

[0053] Figure 19 shows the effect of Na2HPO4 concentration in the fermentation medium on the co-production of extracellular polysaccharides and IAA by Hermann's Atlanta bacterium LP-1.

[0054] Figure 20 shows the effect of MgSO4 concentration in the fermentation medium on the co-production of extracellular polysaccharides and IAA by Hermann's Atlanta LP-1.

[0055] Figure 21 shows the progress curve of Hermann's Atlanta bacillus LP-1 in batch fermentation in a 5 L reactor for the co-production of extracellular polysaccharides and IAA. Detailed Implementation

[0056] The present invention will be further described below with reference to the embodiments and accompanying drawings.

[0057] In the diagram, EPS represents extracellular polysaccharide, and IAA represents indoleacetic acid. *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, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, and is classified as *Atlantibacter hermannii*.

[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: sucrose 20 g / L, beef extract 3 g / L, sodium chloride 1.645 g / L, magnesium sulfate 0.61 g / L, calcium chloride 0.07 g / L, ferrous sulfate 0.027 g / L, manganese sulfate 0.003 g / L, zinc chloride 0.0075 g / L, solvent: water, 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: sucrose 30 g / L, organic nitrogen source 2 g / L, potassium nitrate 6 g / L, L-tryptophan 300 mg / L, ferrous sulfate 10 mg / L, disodium hydrogen phosphate 3 g / L, magnesium sulfate 300 mg / L, solvent: water, pH 6.5-7.5.

[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 -9Two 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 glycerol-free 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 yields of polysaccharides and indoleacetic acid (IAA) of the initially screened strains were measured to obtain the strain capable of co-producing polysaccharides and IAA with the highest yield.

[0067] Genomic DNA was extracted from the strain with the highest yield using a bacterial genomic DNA extraction kit. PCR amplification was performed using upstream primer 27F and downstream primer 1492R. The 16S rDNA sequence is shown in Figure 1. The PCR product was purified by gel extraction and sent to Suzhou Genewiz Biotechnology Co., Ltd. for sequencing. The nucleotide sequence length of the 16S rDNA gene of the sequenced strain was 1367 bp, 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, and named *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 2.

[0072] Table 2. Colony morphology and physiological and biochemical characteristics of Hermann's Atlanta bacillus LP-1

[0073]

[0074] Example 2

[0075] Application of extracellular polysaccharides and indoleacetic acid co-produced by Hermann's Atlanta bacillus LP-1

[0076] Hermann's Atlanta bacillus LP-1 was inoculated into modified TB medium and cultured at 30 °C for 16 h. Single colonies were then streaked onto modified TB medium containing 20 g / L sucrose and cultured at 30 °C for 16 h to obtain activated inoculum. The activated inoculum was aseptically inoculated into a shake flask containing 4 loops of seed liquid medium and cultured on a shaker at 200 rpm at 30 °C for 16 h to obtain fermentation seed liquid. The fermentation seed liquid was 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, stirring speed of 220 rpm, and aeration rate of 1.2 VVM. The initial pH of fermentation was 7.0. During fermentation, an automatic pH control device was activated, and the pH of the fermentation broth was maintained at around 7.0 using ammonia or hydrochloric acid. The fermentation time was 48 hours. The concentration of microbial polysaccharides in the fermentation broth was measured every 3 hours. At 48 h, the extracellular polysaccharide and IAA yields of *Hermannia telsonii* LP-1 were 10.05 g / L and 45.37 mg / L, respectively (Figure 21). The fermentation medium consisted of: 30 g / L sucrose, 2 g / L beef extract, 6 g / L potassium nitrate, 300 mg / L L-tryptophan, 10 mg / L ferrous sulfate, 3 g / L disodium hydrogen phosphate, and 300 mg / L magnesium sulfate.

[0077] Isolation of extracellular polysaccharides and IAA co-produced by Hermann's Atlanta bacillus LP-1:

[0078] S1: Cell removal: The fermentation broth of Hermann's Atlanta bacillus LP-1 was heated to 80 °C and kept in a constant temperature water bath for 20 min to denature some of the proteins; then it was centrifuged in a high-speed centrifuge at 9000 rpm and 4 °C for 40 min to remove the cells and obtain the supernatant.

[0079] S2: Separation: Concentrate the supernatant under reduced pressure to 1 / 5 of its original volume, add 3 times the volume of anhydrous ethanol, and let stand at 4 °C overnight to allow precipitation to complete; then centrifuge at 9000 rpm and 4 °C for 10 min, and collect the precipitate containing crude polysaccharide and the supernatant containing IAA and other small molecules respectively.

[0080] S3: Preparation of crude polysaccharide: The precipitate containing crude polysaccharide was washed and dehydrated successively with anhydrous ethanol, acetone and diethyl ether, and then vacuum dried to obtain crude polysaccharide powder;

[0081] S4: Preparation of crude IAA extract: The supernatant containing IAA and other small molecules was concentrated under reduced pressure at <40 °C, and most of the ethanol was recovered to obtain a concentrated aqueous IAA mixture. The pH was adjusted to 2.5-3.0 with 1M HCl, an equal volume of ethyl acetate was added, the mixture was vigorously shaken, and allowed to stand for separation. Anhydrous sodium sulfate was added to the organic phase for dehydration and drying, and the mixture was filtered. The organic solvent was recovered by rotary evaporation under reduced pressure at <40 °C to obtain the crude IAA extract.

[0082] Purification of extracellular polysaccharides:

[0083] S1: Decolorization: The crude polysaccharide powder was dissolved in distilled water to obtain a crude polysaccharide solution with a concentration of 5 mg / mL. The pH of the crude polysaccharide solution was adjusted to 4.5-5.5, and 5‰ activated carbon powder was added. The mixture was stirred continuously in a constant temperature water bath at 60 ℃ for 1 h to obtain a polysaccharide-activated carbon mixture. After stirring in the water bath, the mixture was filtered while hot using a Büchner funnel. A filter cake was first prepared using diatomaceous earth, and then the polysaccharide-activated carbon mixture was filtered to obtain a clear and transparent polysaccharide solution. The pH of the decolorized polysaccharide solution was adjusted to 8.

[0084] S2: Protein Removal: The polysaccharide solution obtained in S1 was concentrated to 1 / 20 of its original volume at 55 °C to obtain a concentrated solution. Trypsin (0.5-1‰) and neutral protease (1‰) 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, 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.

[0085] S3: Dialysis: Dialyze the liquid in S2 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 the extracellular crude polysaccharide of Hermann's Atlanta bacillus LP-1.

[0086] S4: Adsorption: After reconstituted the extracellular crude polysaccharide of Hermann's Atlanta bacillus LP-1 from S3, 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;

[0087] S5: Elution: Elution was performed with 0.5 mol / L sodium chloride solution at a flow rate of 1 mL / min to obtain the eluent. The eluent was subjected to a delay treatment and then freeze-dried to obtain the extracellular polysaccharide of Hermann's Atlanta bacillus LP-1.

[0088] Determination of extracellular polysaccharide content:

[0089] 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.

[0090] Identification of microbial polysaccharides:

[0091] High-performance liquid chromatography, infrared spectroscopy, and nuclear magnetic resonance were used to identify the fermentation products of Hermann's Atlanta bacillus LP-1.

[0092] 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).

[0093] 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 Hermann's Atlanta bacillus LP-1 is composed of four monosaccharides: glucuronic acid, glucose, galactose, and fucose, with a molar ratio of 1:1.1:2.1:2.3.

[0094] 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-detection. The results are shown in Figures 5 and 6.

[0095] 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 glycosyl complex research library of the University of Georgia to determine the glycosidic bond linkage mode in each polysaccharide structure. The results are shown in Table 3.

[0096] 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.

[0097] Table 3 Polysaccharide methylation data

[0098]

[0099] Purification of indoleacetic acid:

[0100] S1: Solid-phase extraction purification: The activated C18 solid-phase extraction column is equilibrated sequentially with methanol and water. The sample dissolved in a small amount of acidic aqueous phase (pH 3.0) is loaded onto the column. Strongly polar impurities are washed away with an acidic aqueous solution containing 20% ​​methanol (pH 3.0), and then IAA is eluted with 70-80% methanol to obtain the eluent.

[0101] S2: High performance liquid chromatography determination of content: The eluent is dried under nitrogen, redissolved in 0.1% formic acid aqueous solution containing 20% ​​methanol, filtered through a 0.22 μm microporous membrane, and placed into an HPLC sample vial for analysis;

[0102] S3: Chromatographic conditions: Agilent ZORBAX SB-C18 column (250 mm × 4.6 mm, 3-5 μm), flow rate 0.8 mL / min, column temperature 35 °C, injection volume 20 μL, fluorescence detector (excitation wavelength 280 nm, emission wavelength 350 nm). The mobile phase for IAA content determination is shown in Table 1.

[0103] S4: Standard Curve Construction: Accurately weigh IAA and dissolve it in methanol to prepare a 1 mg / mL IAA solution as a stock solution. Then, dilute it with an acidic aqueous solution containing 20% ​​methanol (pH 3.0) to prepare a series of standard solutions with concentrations of 0, 0.01, 0.05, 0.1, 0.5, 1.0, and 2.0 μg / mL, and inject them sequentially for analysis. Plot a standard curve with IAA peak area on the ordinate and concentration on the ordinate.

[0104] Example 3

[0105] Hypoglycemic ability of Hermann's Atlanta bacillus LP-1 extracellular polysaccharide

[0106] I. α-Glucosidase Inhibitory Activity

[0107] Take 100 μL of Hermann's Atlanta LP-1 extracellular 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, terminate the reaction by adding 1 mL of Na2CO3 solution (1 M). 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 measured using distilled water as a control group to replace α-glucosidase. The results, shown in Figure 8, indicate that the inhibitory effect of Hermann's Atlanta LP-1 extracellular polysaccharide on α-glucosidase increased with increasing concentration, reaching 69.68% at a concentration of 160 μg / mL. This demonstrates that Hermann's Atlanta LP-1 extracellular polysaccharide exhibits good hypoglycemic activity.

[0108] II. Glucose Inhibition in HepG2 Cells

[0109] HepG2 cells were placed in DMEM high-glucose medium and cultured in a CO2 cell incubator at 37 ℃ with 5% CO2 (v / v) until 80% confluence. The cell number was then adjusted to 2 × 10⁶ cells / year. 5 Cells were seeded at a density of 100 μL / mL into 96-well plates for culture. Four control groups (blank, model, positive control, and experimental groups) were established. After cell adhesion, the medium was replaced with 100 μL of serum-free medium for starvation culture. Except for the blank control group, the model, positive control, and experimental groups were treated with 10 μL of a 1×10⁻⁶ mcg / mL serum-free medium. -7 mol / L insulin. Two hours later, 10 μL of serum-free culture medium was added to the blank group and the model group, 10 μL of serum-free culture medium containing metformin at a mass concentration of 1000 mg / L was added to the positive control group, and 10 μL of serum-free culture medium containing Hermann's Atlanta bacillus LP-1 extracellular polysaccharide solution at mass concentrations of 5, 10, 20, and 40 mg / L were added to the experimental groups, respectively. After 48 h, cells were collected, centrifuged, and the supernatant was discarded. The cells were resuspended in 1 mL of phosphate-buffered saline (PBS, pH 7.4) and divided into two parts. One part was sonicated in an ice-water bath at 60 W for 3–5 seconds each time, with a 30-second interval between sonication sessions, for a total of 3–5 sonication sessions, following the instructions of the glucose kit. The other part was incubated with 0.225 mL of NaOH solution in a boiling water bath for 20 min, followed by 0.225 mL of double-distilled water. After mixing, the mixture was incubated in a boiling water bath for 5 min, cooled, and the absorbance was measured at 620 nm to evaluate the effect of Hermann's Atlanta bacillus LP-1 extracellular polysaccharide on glycogen synthesis and glucose consumption in HepG2 cells.

[0110] As shown in Figures 9 and 10, in the range of 50-400 μg / mL, the extracellular polysaccharide of Hermann's Atlanta bacterium significantly promoted glucose consumption and increased glycogen synthesis, effectively improved insulin resistance, promoted glucose metabolism, and showed a good hypoglycemic effect.

[0111] Example 4

[0112] Effects of carbon source type in fermentation medium on the yield of extracellular polysaccharides and IAA

[0113] Hermann's Atlanta bacillus LP-1 was inoculated into TB medium and cultured at 30 ℃ for 18-22 h. Single colonies were picked and streaked onto TB medium containing 20 g / L sucrose and cultured at 30 ℃ for 18-22 h to obtain activated strains. The activated strains were aseptically inoculated into shake flasks containing seed medium and placed on a shaker at 200 rpm for 16 h to obtain fermentation seed liquid. The fermentation seed liquid was inoculated at a volume ratio of 4% into fermentation medium without carbon source (CK) and with maltose, fructose, lactose, glucose, and sucrose at a final concentration of 20 g / L. The initial pH was 7.0. The fermentation was carried out at 30 ℃ with shaking at 200 rpm in 500 mL Erlenmeyer flasks with 80 mL of fermentation medium and fermented for 48 h. The contents of extracellular polysaccharides and IAA were calculated from the fermentation broths with different carbon sources. As shown in Figure 11, when sucrose is used as the carbon source for fermentation, the yields of polysaccharides and IAA are the highest, at 6.12 g / L and 30.11 mg / L, respectively. Therefore, sucrose is chosen for subsequent fermentation.

[0114] Example 5

[0115] Effect of carbon source concentration in fermentation medium on the yield of extracellular polysaccharides and IAA

[0116] The fermentation seed culture 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 30 °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 contents of microbial polysaccharides and IAA were calculated from the fermentation broth. As shown in Figure 12, considering the yield of polysaccharides and IAA and the substrate conversion rate, the fermentation effect was optimal at a sucrose concentration of 30 g / L, with polysaccharide and IAA yields of 6.12 g / L and 34.87 mg / L, respectively. Therefore, a sucrose concentration of 30 g / L was selected for subsequent fermentation.

[0117] Example 6

[0118] Effects of organic nitrogen source types in fermentation medium on the yield of extracellular polysaccharides and IAA

[0119] The fermentation seed culture was inoculated at a 4% (v / v) incubation rate into fermentation media without organic nitrogen sources (CK) and containing beef extract (BE), yeast extract (YEJ), tryptone (TRY), yeast extract powder (YEP), and soybean peptone (PFS) at a final concentration of 3 g / L. The initial pH was 7.0, and the cultures were incubated at 30 °C with shaking at 200 rpm in 500 mL Erlenmeyer flasks with 80 mL of fermentation medium. Fermentation was carried out for 48 h, and the contents of microbial polysaccharides and IAA were calculated. As shown in Figure 13, these organic nitrogen sources did not significantly increase polysaccharide production and even had an inhibitory effect, but they significantly promoted IAA production. In comparison, the fermentation effect was best when BE was used as the organic nitrogen source, with polysaccharide and IAA yields of 5.25 g / L and 31.10 mg / L, respectively. Therefore, BE was selected for subsequent fermentation.

[0120] Example 7

[0121] Effects of organic nitrogen source concentration in fermentation medium on the yield of extracellular polysaccharides and IAA

[0122] The fermentation seed culture was inoculated at a rate of 4% (v / v) into fermentation media with beef extract 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 30 °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 contents of microbial polysaccharides and IAA were calculated from the fermentation broth. As shown in Figure 14, with increasing beef extract concentration, polysaccharide yield did not change significantly, while IAA yield gradually increased. A sucrose concentration of 2 g / L maintained a high IAA yield and substrate conversion rate, resulting in the best fermentation effect. At this concentration, the polysaccharide and IAA yields were 6.41 g / L and 33.28 mg / L, respectively. Therefore, a beef extract concentration of 2 g / L was selected for subsequent fermentation.

[0123] Example 8

[0124] Effects of Inorganic Nitrogen Sources in Fermentation Medium on the Production of Extracellular Polysaccharides and IAA

[0125] The fermentation seed culture was inoculated at a rate of 4% (v / v) into fermentation media without an inorganic nitrogen source (CK) and containing potassium nitrate A, sodium nitrate B, ammonium nitrate C, ammonium sulfate D, and diammonium hydrogen phosphate E at a final concentration of 3 g / L. The initial pH was 7.0, and the cultures were incubated at 30 °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 contents of extracellular polysaccharides and IAA were calculated from the fermentation broth. As shown in Figure 15, the yields of polysaccharides and IAA were the highest when potassium nitrate was used as the inorganic nitrogen source, reaching 9.65 g / L and 40.23 mg / L, respectively. Therefore, potassium nitrate was chosen for subsequent fermentation.

[0126] Example 9

[0127] Effects of inorganic nitrogen source concentration in fermentation medium on the yield of extracellular polysaccharides and IAA

[0128] The fermentation seed culture was inoculated at a rate of 4% (v / v) into fermentation media with potassium nitrate concentrations of 0 g / L, 2 g / L, 4 g / L, 6 g / L, 8 g / L, and 10 g / L, respectively. The initial pH was 7.0. Fermentation was carried out at 30 °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 contents of extracellular polysaccharides (IAA) and intracellular nitrogen (IAA) were calculated. As shown in Figure 16, the highest yields of polysaccharides and IAA were observed at a potassium nitrate concentration of 6 g / L, reaching 10.09 g / L and 45.82 mg / L, respectively. Therefore, 6 g / L was selected as the optimal inorganic nitrogen source concentration for subsequent studies.

[0129] Example 10

[0130] Effect of L-tryptophan concentration in fermentation medium on the yield of extracellular polysaccharides and IAA

[0131] The fermentation seed culture was inoculated at a rate of 4% (v / v) into fermentation media with L-tryptophan concentrations of 0 g / L, 100 mg / L, 200 mg / L, 300 mg / L, 400 mg / L, and 500 mg / L, respectively. The initial pH was 7.0. Fermentation was carried out at 30 °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 contents of extracellular polysaccharides and IAA were calculated from the fermentation broth. As shown in Figure 17, the highest yields of polysaccharides and IAA were observed at an L-tryptophan concentration of 300 mg / L, reaching 9.73 g / L and 48.89 mg / L, respectively. Therefore, a L-tryptophan concentration of 300 mg / L was selected for subsequent fermentation.

[0132] Example 11

[0133] Effects of Metal Ion Types and Concentrations in Fermentation Medium on the Yield of Extracellular Polysaccharides and IAA

[0134] The fermentation seed culture was inoculated at a rate of 4% (v / v) into the fermentation media of each metal salt listed in Table 4. The initial pH was 7.0. Fermentation was carried out at 30 °C with shaking at 200 rpm in 500 mL Erlenmeyer flasks containing 80 mL of culture medium. Fermentation was carried out for 48 h, and the contents of extracellular polysaccharides (e.g., MgSO4, CaCl2, FeSO4, and Na2HPO4) were calculated. As shown in Table 4, MgSO4, CaCl2, FeSO4, and Na2HPO4 promoted the production of e.g., e.g., e.g., MgSO4, CaCl2, FeSO4, and Na2HPO4. FeSO4 showed the most significant promoting effect. The presence of MnSO4 and ZnCl2 inhibited the production of e.g., e.g., MgSO4, Na2HPO4, and FeSO4. Therefore, FeSO4, Na2HPO4, and MgSO4 were selected for optimal combination. The complete culture medium in Table 4 is A+B+C+Fe. 2+ +Na + +Ca 2+ +Mg 2+ +Mn 2+ +Zn 2 The concentrations of each metal salt are: MgSO4 200 mg / L, FeSO4 10 mg / L, ZnCl2 5 mg / L, CaCl2 50 mg / L, MnSO4 2 mg / L, and Na2HPO4 2 g / L.

[0135] Table 4. Effects of single or mixed inorganic salts on the production of extracellular polysaccharides and IAA.

[0136]

[0137] The optimal concentrations of FeSO4, Na2HPO4, and MgSO4 were determined by inoculating the fermentation seed liquid at a rate of 4% (v / v) into fermentation media containing the following metal salts using a single-factor variable experiment.

[0138] After a single-factor variable experiment:

[0139] The fermentation seed liquid was inoculated into fermentation medium with ferrous sulfate concentrations of 0, 5, 10, 20, 30, and 40 mg / L for fermentation culture. The results are shown in Figure 18. When the ferrous sulfate concentration was 10 mg / L, the yield of extracellular polysaccharide and IAA in the fermentation broth was the highest.

[0140] The ferrous sulfate concentration in the fermentation medium was controlled at 10 mg / L. The fermentation seed liquid was inoculated into fermentation medium with disodium hydrogen phosphate concentrations of 1.5, 3.0, 4.5, 6.0, and 7.5 g / L, respectively, and fermented. The results are shown in Figure 19. When the ferrous sulfate concentration was 10 mg / L and the disodium hydrogen phosphate concentration was 3 g / L, the yield of extracellular polysaccharide and IAA in the fermentation broth was the highest.

[0141] The ferrous sulfate concentration in the fermentation medium was controlled at 10 mg / L and the disodium hydrogen phosphate concentration at 3 g / L. The fermentation seed liquid was inoculated into fermentation medium with magnesium sulfate concentrations of 150, 300, 450, 600, and 750 mg / L, respectively, for fermentation culture. The results are shown in Figure 20. When the ferrous sulfate concentration was 10 mg / L, the disodium hydrogen phosphate concentration was 3 g / L, and the magnesium sulfate concentration was 300 mg / L, the yields of extracellular polysaccharides and IAA in the fermentation broth were the highest, with yields of 9.13 g / L and 45.66 mg / L, respectively.

[0142] 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.

[0143] 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. The application of Hermann's Atlanta bacillus LP-1 in the co-production of extracellular polysaccharides and indoleacetic acid, characterized in that, Hermann's Atlanta LP-1 was inoculated into a fermentation medium and cultured aerobically to obtain indoleacetic acid and extracellular polysaccharides of Hermann's Atlanta LP-1. The fermentation medium consisted of 30 g / L sucrose, 2 g / L beef extract, 6 g / L potassium nitrate, 300 mg / L L-tryptophan, 10 mg / L ferrous sulfate, 3 g / L disodium hydrogen phosphate, and 300 mg / L magnesium sulfate. Hermann's Atlanta LP-1 was deposited on March 17, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33858, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

2. The application of Hermann's Atlanta bacillus LP-1 according to claim 1 in the co-production of extracellular polysaccharides and indoleacetic acid, characterized in that, The extracellular polysaccharide of Hermann's Atlanta bacillus LP-1 is a heteropolysaccharide composed of glucuronic acid, glucose, galactose, and fucose in a molar ratio of 1:1.1:2.1:2.

3.

3. The application of Hermann's Atlanta bacillus LP-1 according to claim 1 in the co-production of extracellular polysaccharides and indoleacetic acid, characterized in that, The extracellular polysaccharide structure of *Hermann's Atlanta bacillus* LP-1 contains nine linkages: 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.

4. The application of Hermann's Atlanta bacillus LP-1 according to claim 1 in the co-production of extracellular polysaccharides and indoleacetic acid, 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 6.5-7.5, culture temperature 28-32 ℃, inoculum size 1-8%, and culture time 24-48 h; the culture conditions for the fermenter culture are: pH 6.5-7.5, culture temperature 28-32 ℃, inoculum size 1-15%, culture time 24-48 h, aeration ratio 1.0-1.2 VVM, and the fermentation method is batch fermentation.

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