Bacillus amyloliquefaciens d189 with high yield of exopolysaccharide, method for producing exopolysaccharide and application thereof

Abstract

The application belongs to the technical field of microorganisms, and discloses a bacillus amyloliquefaciens D189 with high yield of extracellular polysaccharide. The bacillus amyloliquefaciens D189 is preserved in China Center for Type Culture Collection on April 29, 2021, and the preservation number is CCTCC NO: M2021484. The bacillus amyloliquefaciens D189 provided in the application has high yield of extracellular polysaccharide, the yield of the crude extracellular polysaccharide EPS-OD is 131.31+ / -2.13 g / L after optimization, the extracellular polysaccharide EPS is obtained by purifying the crude EPS-OD through a DEAE ion exchange column, and the composition of the crude EPS-OD and the EPS is determined. The extracellular polysaccharide of the bacillus amyloliquefaciens D189 has good alpha-glucosidase inhibitory capacity and pancreatic lipase inhibitory capacity, and good total bile acid and cholesterol micelle binding capacity in vitro, so that the extracellular polysaccharide and the lipid-lowering and glucose-lowering product have good application prospects.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, more particularly, relates to a high-yield exopolysaccharide-producing Bacillus amyloliquefaciens D189, a method for producing exopolysaccharide and application thereof. BACKGROUND

[0002] Bacillus amyloliquefaciens belongs to the genus Bacillus and is an aerobic gram-positive bacillus. The bacteria have physiological characteristics such as endospore formation, spore formation and biofilm production, and produce various antibacterial compounds, have good stress resistance, and are widely distributed in soil, water and plants in nature. According to the global microbial catalog, 273 different Bacillus amyloliquefaciens strains have been isolated, and Bacillus amyloliquefaciens from different sources has different properties and applications. Bacillus amyloliquefaciens has high safety, and has been certified by the US Food and Drug Administration as a safe strain. The bacteria have a wide application prospect in the fields of agricultural production, biological control and food processing.

[0003] Exopolysaccharide is a natural high molecular substance with wide application prospect, and has shown important value in the fields of medicine, food, agriculture, environmental protection and the like. Especially in the field of medicine, exopolysaccharide has good biocompatibility, immunomodulation, antibacterial and antitumor functions, and becomes a key component of new drug carriers, immune enhancers and biomembrane materials. Exopolysaccharide is one of the main metabolic products of Bacillus, and the molecular weight and monosaccharide composition of exopolysaccharide produced by different Bacillus vary greatly, resulting in great differences in polysaccharide structure and biological activity. Secondly, the yield and type are directly related to the culture medium and fermentation conditions required for microbial growth, such as carbon source, nitrogen source, inorganic salt, fermentation time and the like. At present, the strains with high yield of exopolysaccharide are still far from enough, and the production of EPS still faces problems such as high cost, low yield and complex production process. Therefore, a new strain with high yield of exopolysaccharide and a preparation and optimization method are urgently needed to meet the actual application needs. SUMMARY

[0004] The first object of the present application is to provide a new Bacillus amyloliquefaciens D189.

[0005] The second object of the present application is to provide the application of the above-mentioned Bacillus amyloliquefaciens D189 in the preparation of a product for reducing blood sugar and blood fat.

[0006] The third object of the present application is to provide a method for producing exopolysaccharide by using Bacillus amyloliquefaciens D189.

[0007] The fourth object of the present application is to identify and analyze the structure of exopolysaccharide produced by the above-mentioned Bacillus amyloliquefaciens D189.

[0008] A fifth object of the present application is the use of the above-mentioned exopolysaccharide of Bacillus amyloliquefaciens D189 in reducing blood sugar and blood lipid.

[0009] The object of the present application is achieved by the following technical solutions:

[0010] The strain of Bacillus amyloliquefaciens is Bacillus amyloliquefaciens D189, which has been preserved in China Center for Type Culture Collection on April 29, 2021, with the preservation number of CCTCC NO: M2021484.

[0011] Further, the present application claims the use of the Bacillus amyloliquefaciens D189 in preparing a product for reducing blood sugar and blood lipid.

[0012] Further, the present application also claims the use of the Bacillus amyloliquefaciens D189 in preparing an exopolysaccharide.

[0013] A preparation method of an exopolysaccharide of Bacillus amyloliquefaciens, comprising the following steps:

[0014] Step S1: strain activation and expansion culture: the Bacillus amyloliquefaciens D189 is activated, then the activated single colony is inoculated in LB solid culture medium for further culture for 24-48 hours to obtain seed fermentation broth, the optical density of the broth at 600 nm is adjusted to about 1.0, and then the broth is inoculated in fermentation medium for expansion culture;

[0015] Step S2: crude polysaccharide extraction: the fermentation broth after expansion culture in step S1 is centrifuged to remove the precipitated bacterial cells, the supernatant is retained, ethanol is added, and after standing, the precipitate is collected by centrifugation to obtain a crude polysaccharide solution;

[0016] Step S3: protein removal: sevage reagent is added to the polysaccharide solution in step S2, and the upper aqueous phase is collected;

[0017] Step S4: dialysis: after evaporation to remove the organic solvent from the aqueous solution in step S3, the solution is transferred to a dialysis bag for dialysis, the polysaccharide solution after dialysis is collected, and dried to obtain the crude exopolysaccharide of Bacillus amyloliquefaciens D189.

[0018] Preferably, the composition of the fermentation medium in step S1 is as follows: sucrose concentration 451±1g / L, magnesium sulfate concentration 8.8±0.1g / L, and soybean peptone concentration 11.7±0.1g / L.

[0019] Preferably, the expansion culture time in step S1 is 60-72 hours.

[0020] Preferably, the inoculation ratio in step S1 is 1-3%.

[0021] Preferably, the molecular weight cut-off of the dialysis in step S4 is 2000 Da or less.

[0022] A Bacillus amyloliquefaciens D189 crude exopolysaccharide (EPS-OD) prepared by the above preparation method. Further, the Bacillus amyloliquefaciens D189 crude exopolysaccharide is composed of 83.95±3.53% total sugar, 5.64±2.51% uronic acid, and 1.34±0.35% protein. The EPS total sugar accounts for 94.89±2.68%, the uronic acid accounts for 0.82±0.62%, and the protein accounts for 0.78±0.18%.

[0023] The present application also obtains EPS by purification, especially DEAE-52 cellulose column chromatography purification. By determination, the Mw of the EPS is 5135 Da. The EPS is mainly composed of glucose (Glc) and fructose (Fru), and the molar ratio is Glc:Fru=0.139:0.861, wherein, the proportion of fructose is more than 85%, and the proportion of glucose is about 14%, thereby confirming that the polysaccharide is fructo-oligosaccharide. In combination with a large amount of polysaccharide structure analysis and identification, the main structure of the polysaccharide is as follows:

[0024]

[0025] The EPS-OD and the EPS have good α-glucosidase and pancreatic lipase inhibitory activity, and can better bind to total bile acid and cholesterol micelles in vitro, and have good prospects for preparing lipid-lowering and glucose-lowering products.

[0026] Compared with the prior art, the present application has the following beneficial effects:

[0027] The application provides a Bacillus amyloliquefaciens D189, and extracellular polysaccharide obtained through fermentation of the strain has good in-vitro lipid-lowering and glucose-lowering effects. Meanwhile, the Bacillus amyloliquefaciens D189 has the ability of high-yield of extracellular polysaccharide, and the yield of the extracellular polysaccharide can reach 131.31±2.13 g / L after optimization. Further, the application identifies and analyzes the chemical structure of the refined polysaccharide EPS of the Bacillus amyloliquefaciens D189 through molecular weight, monosaccharide composition, ultraviolet, infrared, methylation, nuclear magnetic resonance spectrum and scanning electron microscope. The application also determines that the extracellular polysaccharide of the Bacillus amyloliquefaciens D189 has good alpha-glucosidase inhibiting ability and pancreatic lipase inhibiting ability, and has good total bile acid and cholesterin micelle binding ability in-vitro, and thus has good application prospect in preparation of the extracellular polysaccharide and lipid-lowering and glucose-lowering products. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 Effect of carbon source type and sucrose concentration on the yield of extracellular polysaccharide;

[0029] Figure 2 Effect of nitrogen source type and soybean peptone concentration on the yield of extracellular polysaccharide;

[0030] Figure 3 Effect of inorganic salt type and magnesium sulfate concentration on the yield of extracellular polysaccharide;

[0031] Figure 4 Effect of fermentation time on the yield of extracellular polysaccharide;

[0032] Figure 5 Response surface graph of the yield of extracellular polysaccharide under the influence of various factors;

[0033] Figure 6 Elution curve of DEAE-52 cellulose column chromatography;

[0034] Figure 7 Ultraviolet absorption spectrum of extracellular polysaccharide;

[0035] Figure 8 FT-IR spectrum of extracellular polysaccharide;

[0036] Figure 9 Molecular weight determination of extracellular polysaccharide;

[0037] Figure 10 Ion chromatogram of monosaccharide mixed standard;

[0038] Figure 11 Ion chromatogram of extracellular polysaccharide;

[0039] Figure 12 1H NMR spectrum (A), 13C NMR spectrum (B) and DEPT135 spectrum (C) of the exopolysaccharide;

[0040] Figure 13 COSY spectrum (A), HSQC spectrum (B), HMBC spectrum (C) and NOESY spectrum (D) of the purified polysaccharide component EPS;

[0041] Figure 14 Scanning electron microscope image of the exopolysaccharide. DETAILED DESCRIPTION

[0042] Further description will be given to the specific embodiments of the present application. It should be noted that the description of the embodiments is used to help understand the present application, but does not constitute a limitation on the present application. In addition, the technical features involved in each embodiment of the present application described below can be combined with each other as long as there is no conflict between them.

[0043] The experimental methods used in the following examples and experimental examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are commercially available reagents and materials unless otherwise specified; and the equipment used is conventional experimental equipment unless otherwise specified.

[0044] A Bacillus amyloliquefaciens strain was isolated from the fermentation process of Liupu tea by the research group in the early stage, and it was found that the strain had the ability to produce exopolysaccharide, and was named D189, and the exopolysaccharide produced by it was preliminarily detected to have certain in vitro lipid-lowering activity. By observing its colony morphology and its morphology under scanning electron microscope, the colony morphology of the Bacillus amyloliquefaciens D189 was white, round, flat, translucent, relatively moist, smooth and shiny, and the edge was neat. The microscopic examination result of the Bacillus amyloliquefaciens D189 was that the cell morphology was dispersed rod-shaped, and the gram staining was purple positive bacteria. In order to further verify the species of the strain and study the structure and properties of the exopolysaccharide produced by the strain, 16S rDNA sequence analysis was carried out (upstream primer 27F: AGTTTGATCMTGGCTCAG; downstream primer 1492R: GGTTACCTTGTTACGACTT). The 16S rDNA sequence of the strain is shown as SEQ ID No. 1. Based on the 16S rRNA gene sequence analysis, it was found that the strain D189 belonged to Bacillus amyloliquefaciens.

[0045] Further, strain preservation was carried out, and the preservation information of the Bacillus amyloliquefaciens D189 is as follows:

[0046] Preservation time: April 29, 2021;

[0047] Depositary Name: China General Microbiological Culture Collection Center

[0048] Depositary Number: CCTCC NO: M2021484

[0049] Address of Depositary: Wuhan, China, Wuhan University

[0050] Classification Name: Bacillus amyloliquefaciens D189

[0051] Example 1: A preparation method of an extracellular polysaccharide of Bacillus amyloliquefaciens

[0052] Fermentation liquid scale culture: the Bacillus amyloliquefaciens D189 strain cryopreserved tube at -80 DEG C was thawed, inoculated on ISP2 solid culture medium in a clean bench, and placed in a 37 DEG C constant temperature incubator for 36 h of culture. Single colonies growing on the ISP2 solid culture medium were inoculated in LB liquid culture medium, and cultured in a 30 DEG C, 200 r / min shaking bed for 24 h. The liquid culture medium was adjusted to an absorbance value of about 1.0 at 600 nm, inoculated into fermentation medium at a proportion of 2% (volume fraction), and cultured in a 30 DEG C, 200 r / min shaking bed for 24 h.

[0053] Crude polysaccharide extraction: the fermentation liquid was centrifuged at a speed of 8500 r / min for 10 min, and the supernatant was collected and added with 3 times the volume of anhydrous ethanol, and placed at 4 DEG C overnight. The precipitate was collected by centrifugation at 8000 r / min for 20 min, and redissolved to obtain a crude polysaccharide solution.

[0054] Protein removal: Sevag reagent was prepared by mixing n-butanol and chloroform solution at a proportion of 1:4. 1 / 3 volume of Sevag reagent was added to the crude polysaccharide solution, and stirred by a magnetic stirrer for 20 min, then placed in a separating funnel and stood until the liquid phase was obviously layered, and the upper water phase was collected. The residual organic reagent in the polysaccharide aqueous solution was removed by rotary evaporation at 55 DEG C.

[0055] Dialysis: the polysaccharide aqueous solution obtained by rotary evaporation was transferred into a dialysis bag with a molecular weight cut-off of 2000 Da, and dialyzed in distilled water for 48 h, and changed every 4 h. The polysaccharide solution after dialysis was collected, freeze-dried to obtain a Bacillus amyloliquefaciens D189 crude extracellular polysaccharide sample EPS-OD, and stored in a dry and sealed environment.

[0056] Based on the above method, the application also protects an optimized extraction method of the extracellular polysaccharide produced by the above Bacillus amyloliquefaciens D189, and the specific steps are as follows:

[0057] Step S1: Single factor experiment: Select carbon source (sucrose, glucose, maltose, lactose), nitrogen source (beef extract, peptone, tryptone, ammonium sulfate, soybean peptone), inorganic salt (disodium hydrogen phosphate, dipotassium hydrogen phosphate, calcium carbonate, sodium chloride, magnesium sulfate), fermentation time (9, 18, 27, 36, 45, 54, 63h) 4 variables for single factor experiment, fermentation extraction of exopolysaccharide,

[0058] 1. The effect of carbon source type on exopolysaccharide yield

[0059] Sucrose, glucose, maltose and lactose were selected to study the effect of carbon source type on the yield of exopolysaccharide of strain D189. As Figure 1 The yield of exopolysaccharide was the highest when sucrose was selected as the carbon source, which was 411.26±14.76mg / L. When the mass concentration of sucrose substrate increased to 400g / L, the maximum yield was 62.30±1.30g / L.

[0060] 2. The effect of nitrogen source type on exopolysaccharide yield

[0061] Ammonium sulfate, beef extract, soybean peptone, peptone and tryptone were selected to study the effect of nitrogen source type on the yield of exopolysaccharide. As Figure 2 When soybean peptone was selected as the nitrogen source of the culture medium, the yield of exopolysaccharide was the highest, which was 119.35±7.30mg / mL. When the mass concentration of soybean peptone substrate increased to 12g / L, the maximum value was reached, and the yield was 155.99±3.41mg / L.

[0062] 3. The effect of inorganic salt type on exopolysaccharide yield

[0063] Disodium hydrogen phosphate, dipotassium hydrogen phosphate, calcium carbonate, sodium chloride and magnesium sulfate were selected to study the effect of inorganic salt type on the yield of exopolysaccharide of D189. As Figure 3 When magnesium sulfate was selected as the inorganic salt of the culture medium, the yield of exopolysaccharide was the highest, and the yield was 144.04±4.58mg / L. When the concentration of magnesium sulfate was not more than 8g / L, the yield of exopolysaccharide increased steadily with the increase of concentration, and reached the maximum value at the mass concentration of 8g / L, which was 153.16±6.08mg / L.

[0064] 4. The effect of fermentation time on exopolysaccharide yield

[0065] Different fermentation times 9, 18, 27, 36, 45, 54, 63h were selected to study the effect on the yield of exopolysaccharide of D189. As Figure 4As shown, in this period of 9-36h, with the increase of fermentation time, the microbial EPS yield also increased, and reached the maximum value of 143.92±2.24mg / L at 36h, and the EPS yield after 36h changed little. After the EPS yield reached the maximum value, with the increase of fermentation time, the EPS yield showed horizontal fluctuation, and changed little.

[0066] Step S2: calculating the optimal fermentation condition: according to the results of single factor test, four factors of sucrose concentration (A), magnesium sulfate concentration (B), soybean peptone concentration (C) and fermentation time (D) were selected, and the EPS yield was used as the response value, and a four-factor three-level response surface test was designed according to the Box-Behnken principle, and the response surface analysis test software was used, and according to the variance analysis table and the response surface contour map and three-dimensional model, the optimal extraction condition was determined;

[0067] (1) Fitting of response surface regression equation

[0068] Table 1 Response surface experiment design and results

[0069]

[0070]

[0071] Table 2 Regression model variance analysis results

[0072]

[0073]

[0074] According to Table 2, the model is extremely significant (P<0.01), and the model has no significant difference (P=>0.05), indicating that the model fitting degree is high, and has statistical significance. The determination coefficient R 2 =0.9951 and the adjusted determination coefficient R 2 adj =0.9903 are close, indicating that the model has strong correlation, and the regression equation has practical significance.

[0075] After the modified insignificant items, the quadratic multinomial equation of the multivariate regression fitting model can be obtained as follows:

[0076] Y=133.42+13.87A+6.07B-2.02C+2.28D+0.4475AB+1.38AC+0.95AD+0.4025BC-0.435BD+0.4775CD-18.98A 2 -11.62B 2 -8.28C 2 -8.51D 2 .

[0077] The order of the F-values ​​of each factor reflects their influence on extracellular polysaccharide production. By comparison, we can see that the order of influence of each factor on polysaccharide production is: A > B > D > C. The linear terms A, B, C, and D of the model have highly significant effects, while the quadratic term A... 2 B 2 C 2 D 2 The impact is extremely significant.

[0078] (2) Response surface optimization extraction conditions

[0079] Response surface methodology uses graphical techniques to display functional relationships, facilitating direct observation of the relationships between experimental values ​​and response values ​​of individual variables, as well as the interactions between pairs of variables. The interaction results among four fermentation conditions affecting the production of extracellular polysaccharides by *Bacillus amyloliquefaciens* D189 are shown below. Figure 5 As shown, the steeper slope of the response surface indicates that the response value is more significantly affected by fermentation factors. Therefore, it can be concluded that the four factors—sucrose concentration, magnesium sulfate concentration, soybean peptone concentration, and fermentation time—have a strong interaction effect on the production of extracellular polysaccharides (EPS) by Bacillus amyloliquefaciens D189. Analysis using DesignExpert software revealed the optimal fermentation conditions for Bacillus amyloliquefaciens D189 EPS: sucrose concentration 451.198 g / L, magnesium sulfate concentration 8.786 g / L, soybean peptone concentration 11.667 g / L, and fermentation time 64.789 h, with a theoretical EPS yield of 134.639 g / L.

[0080] Step S3: Confirmation and verification of the optimal combination fermentation method: sucrose concentration 451.198 g / L, magnesium sulfate concentration 8.786 g / L, soybean peptone concentration 11.667 g / L, fermentation time 64.789 h. After three parallel experiments, the EPS yield of Bacillus amyloliquefaciens D189 was 131.31 ± 2.13 g / L, with an error of 2.47% compared to the theoretical value.

[0081] Example 2: Extraction, purification, and content determination of crude polysaccharides from Bacillus amyloliquefaciens.

[0082] 100 mg of extracellular polysaccharide sample EPS-OD was dissolved in 5 mL of distilled water and centrifuged at 8000 rpm for 10 min. The supernatant was filtered through a 0.45 μm aqueous filter membrane. The polysaccharide solution was slowly and evenly added along the inner wall of the chromatography column using a dropper. Elution was performed sequentially with distilled water, 0.1 mol / L NaCl, 0.3 mol / L NaCl, 0.5 mol / L NaCl, and 0.7 mol / L NaCl solutions at a flow rate of 1 mL / min, 10 mL per tube. The polysaccharide content in each tube was determined using the phenol-sulfuric acid method, and a polysaccharide elution curve was plotted. The results are as follows:Figure 6 As shown, mainly one polysaccharide elution component EPS was obtained. According to the elution curve, the polysaccharide elution component was collected and concentrated and freeze-dried to obtain the polysaccharide sample EPS. The recovery rate of the crude polysaccharide purification was calculated to be 88.2%.

[0083] The analysis results of each component of the exopolysaccharide are shown in Table 3. The total sugar content of EPS-OD was 83.95±3.53%, and the total sugar content of EPS after DEAE-52 purification increased to 94.89±2.68%, indicating that the main substance obtained by extraction and purification was polysaccharide. The uronic acid content of EPS-OD was 5.64±2.51%, and the uronic acid content of EPS after DEAE-52 purification decreased to 0.82±0.62%, indicating that the EPS obtained by extraction and purification almost did not contain uronic acid, and it was speculated to be neutral sugar. The protein content of EPS-OD was 1.34±0.35%, and the protein content of EPS after DEAE-52 purification decreased to 0.78±0.18%, indicating that EPS-OD contained a small amount of protein, and the EPS after purification almost did not contain protein.

[0084] Table 3 Analysis of polysaccharide components

[0085]

[0086] Example 3 Structure identification of Bacillus amyloliquefaciens polysaccharide

[0087] 3.1 Ultraviolet spectrum analysis

[0088] The ultraviolet absorption spectrum of the exopolysaccharide was scanned by the ultraviolet spectrophotometer at 200-400 nm wavelength range, and the spectrum is shown in Figure 7 The results show that EPS has a gentle trend at 260 nm without ultraviolet absorption peak, indicating that there is no nucleic acid substance in EPS; and EPS has no obvious characteristic absorption peak at 280 nm, indicating that there is no protein in EPS.

[0089] 3.2 Fourier transform infrared spectrum (FT-IR) analysis

[0090] The infrared spectrum of EPS was determined by FT-IR method. A small amount of polysaccharide sample to be tested was mixed with potassium bromide at a ratio of 1:100. The FT-IR spectrometer was used to record the FT-IR spectrum of the exopolysaccharide at 4000 cm -1 -500 cm -1 wavelength range.

[0091] The infrared scanning spectrum of EPS at 4000-500 cm -1 is shown in Figure 8 . The infrared spectrum of EPS shows obvious polysaccharide absorption peaks. The absorption peaks of EPS at 924.44 cm -1 and 807.3 cm-1 The presence of an absorption peak near the wavelength indicates the presence of β-glycosidic bonds.

[0092] 3.3 Determination of the molecular weight of polysaccharides by gel permeation chromatography

[0093] The molecular weight of EPS was determined by high performance gel permeation chromatography (HPGPC) with a mobile phase of 0.05 mol / L NaCl solution, a flow rate of 0.6 ml / min, a column temperature of 40°C, an injection volume of 20 uL, and a chromatographic column of BRT105-104-102 series gel column (8 x 300 mm). The peak curve was plotted with the elution time as the abscissa and the logarithmic value of the molecular weight of the polysaccharide as the ordinate. Figure 9 The molecular weight of EPS was 5135 Da.

[0094] Table 4 Size of the molecular weight of EPS

[0095]

[0096] 3.4 Determination of monosaccharide composition by ion chromatography

[0097] The sample (5 mg) was hydrolyzed with trifluoroacetic acid (TFA, 3 M, 2 mL) (3 h, 80°C) and blown dry with nitrogen to completely remove TFA. Then deionized water (5 mL) was vortexed and mixed, 100 uL was pipetted, 900 uL of deionized water was added, centrifuged (12000 rpm, 5 min), and 25 uL of the supernatant was injected for IC analysis. The chromatographic method was as follows: mobile phase A: H2O; B: 15 mmol / L NaOH; C: 15 mmol / L NaOH & 100 mmol / L NaOAc; flow rate: 0.3 mL / min; injection volume: 25 uL; chromatographic column: Dionex Carbopac™ PA20 (3*150 mm); column temperature: 30°C; detector: electrochemical detector. Quantitative analysis was performed by the external standard method, and the ion exchange chromatogram of 16 standard monosaccharides is shown in Figure 10 .

[0098] The ion exchange chromatogram of EPS is shown in Figure 11 . No uronic acid monosaccharide was detected in the determination of the monosaccharide composition of EPS, which is consistent with the results of polysaccharide composition determination. EPS is mainly composed of glucose (Glc) and fructose (Fru) with a molar ratio of Glc:Fru = 0.139:0.861. The proportion of fructose is more than 85%, and the proportion of glucose is about 14%, thus confirming that the polysaccharide is a fructopolysaccharide.

[0099] 3.5 Methylation analysis

[0100] 3.5.1 Methylation: The methylation reaction was performed by methylation kit. 3 mg of polysaccharide sample was accurately weighed into a reaction bottle, 1 mL of DMSO was added, and the methylation reagent A solution (anhydrous alkali solution) was quickly added. Under airtight conditions, it was dissolved by ultrasonic, and the methylation reagent B solution (iodomethane solution) was quickly added. Under the condition of 30°C water bath, magnetic stirring for 60 min. Add 2 mL of ultrapure water to the mixed system to terminate the methylation reaction.

[0101] 3.5.2 Acetylation: Take the above reaction liquid, add 1 mL of 2M TFA and hydrolyze for 90 min, and rotary evaporate to dryness. Add 2 ml of ddH2O and 60 mg of NaBH4 in sequence, and react for 8 h. Add glacial acetic acid to neutralize, rotary evaporate, and dry. Add 1 mL of acetic anhydride, and react at 100°C for 1 h, and cool to room temperature. Add 3 mL of toluene, and rotary evaporate to dryness, repeat 4-5 times to remove excess acetic anhydride.

[0102] The acetylated product was dissolved in 3 mL of CH2Cl2, transferred to a separatory funnel, and a suitable amount of ultrapure water was added, shaken and mixed, the upper layer was removed, and the operation was repeated 5-6 times. The organic layer was dried with a suitable amount of anhydrous sodium sulfate, and the volume was adjusted to 10 mL. The sample was determined by Agilent GCMS 6890-5973 gas chromatography-mass spectrometer. Equipped with RXI-5SILMS chromatographic column 30m x 0.25mm x 0.25um; the initial temperature is 120°C, and the temperature is increased to 250°C / min at 3°C / min, and maintained for 5 min; the injection port temperature is 250°C, and the detector temperature is 250°C / min.

[0103] After derivatization of the polysaccharide sample, the linkage information can be obtained by GC-MS determination. The mass spectrum is shown in the appendix, and the linkage mode of EPS (Table 5), in which the four kinds of residual sugars of fructose and glucose have the largest proportion of →1)-Fruf-(2→ residual sugar, which constitutes the main chain of glucan, followed by about 10% of the fructose end sugar. In addition, one kind of monosaccharide glucose has two kinds of residual sugars, which are →3,6)-Glcp-(1→ with branching structure and end sugar residual sugar. Therefore, it can be inferred that EPS is fructan, and the main linkage mode is Fruf-(2→.

[0104] Table 5 EPS methylation sugar alcohol acetyl ester (PMAA) results

[0105]

[0106] 3.6 Nuclear magnetic resonance spectroscopy

[0107] The completely dissolved refined polysaccharide component EPS sample was placed in 0.5 mL of D2O, and its 1 D nuclear magnetic resonance spectroscopy1 H and 13 C) and 2 D Nuclear magnetic resonance spectroscopy (COSY, HSQC, HMBC and NOESY), the purified polysaccharide component EPS-3 sample was dissolved in 0.5 mL D2O, and the NMR spectrum was recorded on a Bruker Avance III 600 MHz NMR spectrometer. 1 H and 13 CNMR spectrum.

[0108] Nuclear magnetic resonance (NMR) reveals accurate structural information of EPS. As Figure 12 (A), in the nuclear magnetic hydrogen spectrum 1 In the anomeric region (4.3-5.5 ppm) of HNMR, a group of weak anomeric hydrogen signals were found with main peak positions at 5.30, 5.27, 5.12, 4.53 ppm, confirming that the polysaccharide is composed of multiple residual sugars. In the 3.0-4.2 ppm region of the hydrogen spectrum, there are typical signals of protons outside the anomeric hydrogen of residual sugar ring, which is consistent with the general characteristics of polysaccharide hydrogen spectrum.

[0109] As Figure 12 (B) can be seen, the nuclear magnetic carbon spectrum 13 In CNMR, no obvious uronic acid carbonyl carbon signal was found in the range of 170-210 ppm, and the polysaccharide was neutral. The anomeric region (90-110 ppm) of the carbon spectrum can identify a group of main anomeric carbon signals, which are 104.15, 104.05, 103.95, 103.74, 103.56, 103.45, 101.74 ppm, respectively. Multiple anomeric carbon signals correspond to multiple anomeric hydrogen signals; there are multiple typical polysaccharide residual sugar carbon signals in addition to anomeric carbon in the range of 55-85 ppm, in which the signals at 60.31 and 63.32 ppm are attributed to the methylene-CH2- secondary carbon of the residual sugar ring, Figure 12 (C) EPS DEPT135 spectrum confirmed the assignment of signals at this position; the multiple signals at 74-82 ppm represent the C2-C4 of fructose residual sugar and the C2-C5 of glucose carbon information.

[0110] The structure of the polysaccharide was further analyzed by two-dimensional nuclear magnetic spectrum. Figure 13COSY, HSQC, HMBC, NOESY spectra of EPS. In the HSQC two-dimensional nuclear magnetic spectrum reflecting the hydrogen-carbon correlation of the connected, a group of weak signals of H1 / C1 correlation in the anomeric region were identified, respectively 5.32 / 92.33, 5.28 / 91.89, 5.13 / 91.89, 4.53 / 95.80 ppm. Combined with the methylation results and the integral intensity of the anomeric hydrogen, it was judged that the carbon spectrum peak of 104.15 ppm was the C2 position signal of β→1)-Fruf-(2→, the carbon spectrum peak of 103.74 ppm was the C2 position signal of β-Fruf-(2→, 5.32 / 92.33 was the H1 / C1 signal of α→3,6)-Glcp-(1→, and 4.53 / 95.80 was the H1 / C1 signal of β→t-Glcp-(1→.

[0111] By combining multiple two-dimensional nuclear magnetic spectra, the carbon and hydrogen signal positions and structural corresponding information of various residual sugars in the polysaccharide structure were sequentially assigned. For example, it was determined that the anomeric carbon 104.15 ppm belonged to the quaternary carbon C2 in the residual sugar →1)-Fruf-(2→. By identifying the HMBC spectrum of hydrogen-carbon long-range correlation, it was found that the strongly correlated signals were 3.56 and 3.64 ppm, confirming that the positions of H1a and H1b were 3.56 and 3.64 ppm; combined with the HSQC spectrum, it was confirmed that the signal of C1 was 59.80; according to the HMBC information, the related signal of H1a and H1b was found to be 76.06, confirming that the hydrogen spectrum position of C3 was 76.06 ppm; combined with HSQC, 4.08 / 76.06 was determined, and the H3 carbon spectrum position was 4.08 ppm; by the same method and combined with the COSY spectrum, it was determined that the H4 / H3 related signal was 4.08 / 3.98, and H4 was 3.98 ppm; further, the H5 / C5 and H6 / C6 position information was determined to be 3.85 / 80.18, 3.46, 3.80 / 63.05. According to this method, other main position residual sugars were found and the same signal position assignment was performed. The analysis results are shown in Table 6.

[0112] Table 6 Analysis results of main residual sugar assignment of EPS

[0113]

[0114] HMBC is more suitable for determining the hydrogen-carbon correlation of 2-3 chemical bonds apart, and it is useful for determining the linkage of glycosidic bond in polysaccharide analysis. In the β-1)-Fruf-(2→ residual sugar with a relatively high content, the anomeric carbon correlation signals were found in the HMBC spectrum: 3.56 / 104.04, 3.64 / 104.04, 4.08 / 104.04. Through the above attribution, it was confirmed that the anomeric hydrogen of the residual sugar was correlated with H3 in the same ring and H1 of the same residual sugar, and the relatively strong signals 3.56 / 104.04 and 3.64 / 104.04 confirmed the existence of 1→2 glycosidic bond of the same residual sugar 1→2, and constituted most of the glycosidic bonds in the fructo-inulin; the correlation signals 3.56 / 103.74 and 3.64 / 103.74 confirmed the existence of the end sugar β-t-Fruf-(2→ and β-1)-Fruf-(2→. In addition, the correlation signal 5.27 / 104.04 suggested that there was a 1→2 glycosidic bond between α-3,6)-Glcp-(1→ and β-1)-Fruf-(2→, and the signal 3.81 / 104.15 suggested that there was a 2→3 glycosidic bond between β-1)-Fruf-(2→ and α-3,6)-Glcp-(1→; for the end sugar β-t-Glcp-(1→, due to the weak signal, the signal 3.67 / 95.80 was found, which was guessed to have a 1→6 glycosidic bond with α-3,6)-Glcp-(1→. The correlation signal 3.66 / 103.74 suggested that there was a 2→6 branched chain between the end sugar t-β-Fruf-(2→ and α-3,6)-Glcp-(1→.

[0115] Therefore, it is speculated that the main structure of the polysaccharide is as shown below:

[0116]

[0117] 3.7 Scanning electron microscopy analysis

[0118] The polysaccharide powder was adhered to the sample stage by conductive glue, and the un-fixed powder was blown away by nitrogen. After gold spraying, nitrogen blowing was performed again, and finally the sample was placed under a scanning electron microscope for observation. The images were magnified by 500x, 1000x and 2000x for observation.

[0119] The apparent morphology of the refined polysaccharide is as shown in Figure 14 The EPS appears as regular spherical or ellipsoidal, with a smooth surface, and the average pore size is about 30-50 microns. The spherical conformation of EPS may be due to the high branching of the polysaccharide molecules, with the branches stretching in all directions, thereby forming a dense spherical structure.

[0120] Example 4 In vitro hypoglycemic and hypolipidemic activity of Bacillus amyloliquefaciens polysaccharide

[0121] 4.1 Inhibition of pancreatic lipase and α-glucosidase by exopolysaccharide

[0122] 4.1.1 Inhibition of pancreatic lipase

[0123] Take 20 uL of polysaccharide solution and add 30 uL of Tris-HCl buffer, 50 uL of pancreatic lipase solution or Tris-HCl buffer, and react at 37°C for 10 min. Add 100 uL of pNPP substrate solution to the reaction system, and react at 37°C for 10 min. Measure the absorbance at 405 nm. Use orlistat solution as a positive control, and blank buffer as a negative control.

[0124] 4.1.2 Inhibition of α-glucosidase

[0125] Take 50 uL of polysaccharide solution and mix with 100 uL of 0.1 U / mL α-glucosidase solution, and incubate at 37°C for 10 min. Add 100 uL of 1.5 mmol / L pNPG solution prepared with phosphate buffer (0.1 M, pH = 6.9), and incubate at 37°C for 20 min. Add 1 mL of 1 mol / L sodium carbonate solution to terminate the reaction. Use acarbose as a positive control, and ultrapure water as a negative control. Measure the OD value of the reaction at 400 nm.

[0126] Experimental results: EPS-OD and EPS have certain inhibitory activity on α-glucosidase and pancreatic lipase. As shown in Table 7, the inhibition rates of EPS-OD on α-glucosidase and pancreatic lipase are 29.12 ± 0.67% and 32.15 ± 1.86%, respectively, and the inhibition rates of EPS on α-glucosidase and pancreatic lipase are 29.42 ± 0.47% and 36.48 ± 4.34%, respectively. Compared with EPS-OD, the α-glucosidase inhibitory activity of EPS does not change significantly, but the pancreatic lipase inhibitory activity increases. It is speculated that the change in the pancreatic lipase inhibitory activity of EPS polysaccharide is related to the purity of the polysaccharide.

[0127] 4.2 Binding capacity of exopolysaccharide to total bile acid and cholesterolum in vitro

[0128] 4.2.1 Binding capacity of exopolysaccharide to total bile acid in vitro

[0129] Weigh cholesterolum, oleic acid, and lecithin in 5 ml of methanol solution, and ultrasonically treat to dissolve completely. Dry with nitrogen gas. Add sodium chloride, sodium cholate, and phosphate buffer solution (pH = 7.4), and treat at 37°C for 12 h to prepare a cholesterolum micelle solution (the cholesterolum micelle solution contains 10 mM sodium taurocholate, 2 mM cholesterolum, 5 mM oleic acid, 5 mM lecithin, 132 mM sodium chloride, and 15 mM phosphate buffer).

[0130] In the test tube, 1.0 mL of micelle solution was added, and the sample to be tested was mixed and incubated at 37°C for 2 h. The supernatant was taken for testing after centrifugation. Cholestyramine was used as a positive control, and no sample was used as a negative control. The cholesterol concentration in the supernatant was determined by a total cholesterol kit.

[0131] 4.2.2 Cholesterol micelle binding capacity in vitro

[0132] Cholesterol, oleic acid, and lecithin were weighed into 5 mL of methanol solution and ultrasonically treated. After being fully dissolved, the solution was dried with nitrogen. Sodium chloride, sodium cholate, and phosphate buffer solution (pH = 7.4) were added, and the solution was treated at 37°C for 12 h to prepare a cholesterol micelle solution (containing 10 mM sodium taurocholate, 2 mM cholesterol, 5 mM oleic acid, 5 mM lecithin, 132 mM sodium chloride, and 15 mM phosphate buffer).

[0133] In the test tube, 1.0 mL of micelle solution was added, and the sample to be tested was mixed and incubated at 37°C for 2 h. The supernatant was taken for testing after centrifugation. Cholestyramine was used as a positive control, and no sample was used as a negative control. The cholesterol concentration in the supernatant was determined by a total cholesterol kit.

[0134] Test results: EPS-OD and EPS have good in vitro bile acid and cholesterol binding capacity. 10 mg of cholestyramine was used as a positive control, and the in vitro bile acid and cholesterol micelle binding capacity of 10 mg of EPS-OD and EPS was determined. As shown in Table 7, the binding capacity of EPS-OD to bile acid and cholesterol micelles was 34.35 ± 3.60% and 42.47 ± 1.21%, respectively, and the binding capacity of EPS to bile acid and cholesterol micelles was 33.35 ± 2.54% and 37.23 ± 0.65%, respectively. Compared with EPS-OD, the bile acid binding capacity of EPS changed little, but the cholesterol micelle binding capacity decreased. After purification, the spatial configuration of EPS changed, and the exposed binding sites decreased, resulting in a decrease in the binding capacity of polysaccharide to cholesterol micelles.

[0135] Table 7 Biological activity of EPS-OD and EPS

[0136]

[0137]

[0138] The embodiments of the present application are described in detail above, but the present application is not limited to the described embodiments. Various changes, modifications, replacements, and variations of the embodiments can be made without departing from the principles and spirit of the present application, and still fall within the scope of the present application.

Claims

1. A method for producing exopolysaccharide by Bacillus amyloliquefaciens D 189, characterized by, Comprising the following steps: Step S1 - strain activation and expansion culture: the Bacillus amyloliquefaciens D189 is activated, and then the activated single colony is inoculated into LB solid culture medium for further culture for 24-48 hours to obtain seed fermentation liquor, the absorbance of which at 600 nm is adjusted to about 1.0, and then inoculated into fermentation medium for expansion culture; Step S2 - crude polysaccharide extraction: the fermentation liquor after expansion culture in step S1 is centrifuged to remove the precipitated bacterial cells, and the supernatant is retained, ethanol is added, and after standing, the precipitate is collected by centrifugation to obtain a crude polysaccharide solution; Step S3 - protein removal: sevage reagent is added to the polysaccharide solution of step S2, and the upper aqueous phase is collected; Step S4 - dialysis: after the organic solvent in the aqueous phase solution of step S3 is removed by evaporation, it is transferred to a dialysis bag for dialysis, and the polysaccharide solution after dialysis is collected, dried to obtain crude extracellular polysaccharide of Bacillus amyloliquefaciens D189; The composition of the fermentation medium in step S1 is as follows: sucrose concentration 451±1 g / L, magnesium sulfate concentration 8.8±0.1 g / L, soybean peptone concentration 11.7±0.1 g / L; the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) D189 has been preserved in the China Center for Type Culture Collection on April 29, 2021, and the preservation number is CCTCC NO: M2021484. Bacillus amyloliquefaciens )D189, has been preserved in the China Center for Type Culture Collection on April 29, 2021, and the preservation number is CCTCC NO: M2021484. The structure of the exopolysaccharide is as follows: .

2. The production method according to claim 1, characterized by, The expansion culture time in step S1 is 60-72 hours.

3. The preparation method according to claim 1, characterized in that, The inoculation ratio in step S1 is 1-3%.

4. The production method according to claim 1, characterized by, The molecular weight cut-off for dialysis in step S4 is 2000 Da or less.

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