Bacillus amyloliquefaciens D189 with high yield of exopolysaccharides, method for producing exopolysaccharides and application of bacillus amyloliquefaciens D189

Abstract

The invention belongs to the technical field of microorganisms, and discloses bacillus amyloliquefaciens D189 with high exopolysaccharide yield. The bacillus amyloliquefaciens D189 is preserved in the China Center for Type Culture Collection on April 29, 2021, and the preservation number of the bacillus amyloliquefaciens D189 is CCTCC (China Center for Type Culture Collection) NO: M2021484. According to the bacillus amyloliquefaciens D189 high-yield exopolysaccharide, the yield of crude exopolysaccharide EPS-OD obtained through optimization is 131.31 + / -2.13 g / L, the crude EPS-OD is purified through a DEAE ion exchange column to obtain the exopolysaccharide EPS, and the components of the crude EPS-OD and the EPS are measured. The invention also determines that the bacillus amyloliquefaciens D189 extracellular polysaccharide has better alpha-glucolase inhibition capability, pancreatic lipase inhibition capability and capability of combining in-vitro total bile acid and cholesterol micelle, so that the bacillus amyloliquefaciens D189 extracellular polysaccharide has a good application prospect in preparation of extracellular polysaccharide and lipid-lowering and sugar-lowering products.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and more specifically, relates to a Bacillus amyloliquefaciens D189 with high exopolysaccharide production, an exopolysaccharide production method and an application thereof. Background Art

[0002] Bacillus amyloliquefaciens belongs to the genus Bacillus and is an aerobic Gram-positive rod-shaped bacterium. The bacterium has the physiological characteristics of forming endospores, generating spores and biofilms, and producing a variety of antimicrobial compounds. It has good stress resistance and is widely distributed in soil, water sources and plants in nature. According to the Global Microbial Catalog, 273 different strains of Bacillus amyloliquefaciens have been isolated, and Bacillus amyloliquefaciens from different sources have different properties and applications. Bacillus amyloliquefaciens is highly safe and has been certified as a safe strain by the U.S. Food and Drug Administration. The bacterium has broad application prospects in agricultural production, biological control, food processing and other fields.

[0003] As a natural high molecular weight substance with broad application prospects, extracellular polysaccharides have shown important value in the fields of medicine, food, agriculture, environmental protection, etc. Especially in the field of medicine, extracellular polysaccharides have good biocompatibility, immunomodulation, antibacterial and anti-tumor functions, and have become a key component of new drug carriers, immunopotentiators and biofilm materials. Extracellular polysaccharides are one of the main metabolites of Bacillus. There are large differences in the molecular weight and monosaccharide composition of different Bacillus extracellular polysaccharides, resulting in huge differences in polysaccharide structure and biological activity. Secondly, its yield and type are directly related to the culture matrix and fermentation conditions required for microbial growth, such as carbon source, nitrogen source, inorganic salt, fermentation time and other factors. The strains with high yield of extracellular polysaccharides currently provided are still far from enough, and the current production of EPS still faces problems such as high cost, low yield and complex production process. Therefore, a new strain with high yield of extracellular polysaccharides and a preparation and optimization method are urgently needed to meet the needs of practical applications. Summary of the invention

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

[0005] The second object of the present invention is to provide the use of the above-mentioned Bacillus amyloliquefaciens D189 in the preparation of blood sugar and lipid lowering products.

[0006] The third object of the present invention is to provide a method for high-yield exopolysaccharide production using Bacillus amyloliquefaciens D189.

[0007] The fourth objective of the present invention is to identify and analyze the structure of the extracellular polysaccharide produced by the above-mentioned Bacillus amyloliquefaciens D189.

[0008] The fifth objective of the present invention is to use the extracellular polysaccharide of Bacillus amyloliquefaciens D189 in reducing blood sugar and lipids.

[0009] The purpose of the present invention is achieved through the following technical solutions:

[0010] A strain of Bacillus amyloliquefaciens, namely Bacillus amyloliquefaciens D189, was deposited in the China Center for Type Culture Collection on April 29, 2021, with the deposit number CCTCC NO: M2021484.

[0011] Furthermore, the present application claims to protect the use of the Bacillus amyloliquefaciens D189 in the preparation of blood sugar and lipid lowering products.

[0012] Furthermore, the present application also claims to protect the use of the Bacillus amyloliquefaciens D189 in the preparation of extracellular polysaccharides.

[0013] A method for preparing Bacillus amyloliquefaciens exopolysaccharide comprises the following steps:

[0014] Step S1-strain activation and expansion culture: activating the Bacillus amyloliquefaciens D189, then inoculating the activated single colony into LB solid culture medium and continuing to culture for 24 to 48 hours to obtain a seed fermentation liquid, adjusting the absorbance value of the bacterial liquid to about 1.0 at 600nm, and then inoculating it into a fermentation medium for expansion culture;

[0015] Step S2-crude polysaccharide extraction: centrifuge the fermentation liquid after the expanded culture in step S1, remove the precipitated bacteria, retain the supernatant and add ethanol, let it stand and then centrifuge to collect the precipitate, and redissolve to obtain a crude polysaccharide solution;

[0016] Step S3-deproteinization: adding sevage reagent to the polysaccharide solution of step S2, and collecting the upper aqueous phase;

[0017] Step S4-dialysis: After evaporating the aqueous phase solution in step S3 to remove the organic solvent, the aqueous phase solution is transferred to a dialysis bag for dialysis, and the dialyzed polysaccharide solution is collected and dried to obtain crude extracellular polysaccharide of Bacillus amyloliquefaciens D189.

[0018] Preferably, 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, soy peptone concentration 11.7±0.1 g / L.

[0019] Preferably, the time for inoculating into the fermentation medium for expansion culture in step S1 is 60 to 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 below 2000 Da.

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

[0023] The present invention also obtains EPS by purification, especially purification by DEAE-52 cellulose column chromatography. By measurement, the Mw of the EPS is 5135Da. The EPS is mainly composed of glucose (Glc) and fructose (Fru), with a molar ratio of Glc:Fru=0.139:0.861, of which fructose accounts for a larger proportion, exceeding 85% by molar ratio, followed by glucose, which accounts for about 14%, thereby confirming that the polysaccharide is fructoglucan. Combined with a large number of polysaccharide structure analysis and identification, the main structure of the polysaccharide is obtained as follows:

[0024]

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

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

[0027] The present invention provides a strain of Bacillus amyloliquefaciens D189, and the extracellular polysaccharide obtained after fermentation by the strain has a good in vitro lipid-lowering and blood sugar-lowering effect. At the same time, the strain of Bacillus amyloliquefaciens D189 has the ability to produce high extracellular polysaccharides, and the yield of extracellular polysaccharides obtained after optimization is 131.31±2.13g / L; further, the present invention identifies and analyzes the chemical structure of refined polysaccharide EPS of Bacillus amyloliquefaciens D189 by molecular weight, monosaccharide composition, ultraviolet, infrared, methylation, nuclear magnetic resonance spectroscopy, and scanning electron microscopy. The present invention also determines that the extracellular polysaccharide of Bacillus amyloliquefaciens D189 has good α-glucose enzyme inhibition ability and pancreatic lipase inhibition ability, and has the ability to bind to total bile acid and cholesterol micelles in vitro, so it has good application prospects in the preparation of extracellular polysaccharides and lipid-lowering and blood sugar-lowering products. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The effects of carbon source type and sucrose concentration on extracellular polysaccharide production;

[0029] Figure 2 The effects of nitrogen source and soy peptone concentration on the production of extracellular polysaccharides;

[0030] Figure 3 The effects of inorganic salt types and magnesium sulfate concentration on the production of extracellular polysaccharides;

[0031] Figure 4 The effect of fermentation time on the production of extracellular polysaccharides;

[0032] Figure 5 It is the response surface diagram of each factor on the extracellular polysaccharide production;

[0033] Figure 6 This is the elution curve of DEAE-52 cellulose column chromatography;

[0034] Figure 7 is the UV absorption spectrum of extracellular polysaccharide;

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

[0036] Fig. 9 For the determination of molecular weight of extracellular polysaccharides;

[0037] Fig.10 This is the ion chromatogram of the monosaccharide standard mixture;

[0038] Fig.11 is the ion chromatogram of extracellular polysaccharide;

[0039] Fig.12 1HNMR spectrum (A), 13CNMR spectrum (B) and DEPT135 spectrum (C) of extracellular polysaccharide;

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

[0041] Fig.14 This is a scanning electron micrograph of extracellular polysaccharide. DETAILED DESCRIPTION

[0042] The specific embodiments of the present invention are further described below. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in each embodiment of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0043] Unless otherwise specified, the test methods used in the following embodiments and experimental examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, are reagents and materials that can be obtained from commercial channels; the equipment used, unless otherwise specified, are all conventional experimental equipment.

[0044] In the early stage, our research group isolated a strain of Bacillus amyloliquefaciens from the fermentation process of Liubao tea, and found that the strain has the ability to produce extracellular polysaccharides, and named it D189. Preliminary detection showed that its extracellular polysaccharide has certain in vitro lipid-lowering activity. By observing its colony morphology and its morphology under a scanning electron microscope, the colony morphology of Bacillus amyloliquefaciens D189 is white, round, flat, translucent, relatively moist, smooth and shiny, and has neat edges; the microscopic examination results of Bacillus amyloliquefaciens D189 are: the cell morphology is dispersed rod-shaped, and Gram staining is purple positive bacteria. In order to further verify the species of the strain and then study the structure and properties of the extracellular polysaccharides produced by the strain, 16S rDNA sequence analysis was performed (upstream primer 27F: AGTTTGATCMTGGCTCAG; downstream primer 1492R: GGTTACCTTGTTACGACTT). The 16S rDNA sequence of the strain is shown in SEQ ID No.1. Based on 16S rRNA gene sequence analysis, strain D189 was found to belong to Bacillus amyloliquefaciens.

[0045] Furthermore, the strain was preserved, and the deposit information of Bacillus amyloliquefaciens D189 is as follows:

[0046] Deposit date: April 29, 2021;

[0047] Name of depository: China Center for Type Culture Collection;

[0048] Deposit number: CCTCC NO: M2021484;

[0049] Address of depository: Wuhan University, Wuhan, China;

[0050] Classification and nomenclature: Bacillus amyloliquefaciens D189.

[0051] Example 1 A method for preparing Bacillus amyloliquefaciens exopolysaccharide:

[0052] Fermentation broth expansion culture: Take a frozen tube of Bacillus amyloliquefaciens D189 strain stored at -80℃, thaw it, inoculate it on ISP2 solid culture medium in a clean bench, and culture it in a 37℃ constant temperature incubator for 36h. Pick a single colony grown on ISP2 solid culture medium and inoculate it into LB liquid culture medium, and culture it in a shaking table at 30℃ and 200r / min for 24h. Adjust the absorbance of the liquid culture medium to about 1.0 at 600nm, inoculate it into the fermentation medium at an inoculation ratio of 2% (volume fraction), and culture it in a shaking table at 30℃ and 200r / min for 24 hours.

[0053] Extraction of crude polysaccharides: The fermentation broth was centrifuged at 8500 r / min for 10 min, the supernatant was collected and 3 volumes of anhydrous ethanol were added, and the mixture was allowed to stand overnight at 4°C. The precipitate was collected by centrifugation at 8000 r / min for 20 min, and the crude polysaccharide solution was obtained by re-dissolving.

[0054] Protein removal: Take n-butanol and chloroform solution, mix them in a ratio of 1:4 and let them stand to prepare Sevag reagent. Add 1 / 3 volume of Sevag reagent to the crude polysaccharide solution, stir with a magnetic stirrer for 20 minutes, place it in a separatory funnel and let it stand until the liquid phase is clearly separated, and collect the upper aqueous phase. Rotary evaporate at 55°C to remove the residual organic reagent in the polysaccharide aqueous solution.

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

[0056] Based on the above method, the present invention also protects the above-mentioned optimized extraction method of exopolysaccharide produced by 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, soy 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 extracellular polysaccharides,

[0058] 1. Effect of carbon source type on extracellular polysaccharide production

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

[0060] 2. Effect of nitrogen source type on extracellular polysaccharide production

[0061] Ammonium sulfate, beef extract, soy peptone, peptone and tryptone were selected to study the effect of nitrogen source type on extracellular polysaccharide production. Figure 2 When soy peptone was used as the nitrogen source of the culture medium, the maximum yield of extracellular polysaccharide was 119.35±7.30 mg / mL. When the mass concentration of the substrate soy peptone increased to 12 g / L, the maximum yield was 155.99±3.41 mg / L.

[0062] 3. Effect of inorganic salt types on extracellular polysaccharide production

[0063] Sodium hydrogen phosphate, potassium hydrogen phosphate, calcium carbonate, sodium chloride and magnesium sulfate were selected to study the effect of inorganic salts on the extracellular polysaccharide production of D189. Figure 3 When magnesium sulfate was used as the inorganic salt of the culture medium, the yield of extracellular polysaccharide was the highest, at which point the yield was 144.04±4.58mg / L. When the magnesium sulfate concentration was no more than 8g / L, the yield of extracellular polysaccharide increased steadily with the increase of concentration, reaching the maximum value of 153.16±6.08mg / L at a mass concentration of 8g / L.

[0064] 4. Effect of fermentation time on extracellular polysaccharide production

[0065] Effects of different fermentation times of 9, 18, 27, 36, 45, 54, and 63 h on the production of extracellular polysaccharides in D189 Figure 4As shown in the figure, during the period of 9-36h, as the fermentation time increased, the microbial extracellular polysaccharide production also increased, reaching a maximum value of 143.92±2.24mg / L at 36h, and the polysaccharide production after 36h did not change much. After the EPS production reached the highest value, as the fermentation time increased, the EPS production fluctuated horizontally and did not change much.

[0066] Step S2: Calculate the optimal fermentation conditions: According to the results of the single factor test, select four factors, namely, sucrose concentration (A), magnesium sulfate concentration (B), soy peptone concentration (C) and fermentation time (D), and take the extracellular polysaccharide yield as the response value. Use the Box-Behnken principle to design a four-factor three-level response surface experiment, substitute it into the response surface analysis test software, and determine the optimal extraction conditions according to the variance analysis table, response surface contour map and three-dimensional model;

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

[0068] Table 1 Response surface experimental design and results

[0069]

[0070]

[0071] Table 2 Results of variance analysis of regression model

[0072]

[0073]

[0074] According to Table 2, the model is extremely significant (P < 0.01), and there is no significant difference in the lack of fit of the model (P = > 0.05), indicating that the model has a high degree of fit and is statistically significant. 2 = 0.9951 and adjusted coefficient of determination R 2 adj =0.9903 is relatively close, which shows that the model has strong correlation and the regression equation has practical significance.

[0075] After correcting the insignificant terms, the quadratic polynomial 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 the magnitude of its influence on the production of extracellular polysaccharides. By comparison, we can know that the order of influence of each factor on polysaccharide production is: A>B>D>C. The first-order terms A, B, C, and D of the model have extremely significant effects, and the second-order term A 2 , B 2 , C 2 , D 2 The impact is extremely significant.

[0078] (2) Response surface optimization of extraction conditions

[0079] The response surface diagram uses graphical technology to display the functional relationship, which is convenient for directly observing the relationship between the test value of each variable and the response value, as well as the interaction between two variables. The interaction results between the four fermentation conditions that affect the production of extracellular polysaccharides by Bacillus amyloliquefaciens D189 are shown in Figure 2. Figure 5 As shown. The slope of the response surface becomes steeper, indicating that the response value is more affected by the fermentation factors. Therefore, it can be considered that the four factors of sucrose concentration, magnesium sulfate concentration, soy peptone concentration, and fermentation time have a strong interaction in affecting the fermentation production of extracellular polysaccharides by Bacillus amyloliquefaciens D189. According to the analysis of DesignExpert software, the optimal fermentation conditions of extracellular polysaccharides of Bacillus amyloliquefaciens D189 are: sucrose concentration 451.198g / L, magnesium sulfate concentration 8.786g / L, soy peptone concentration 11.667g / L, fermentation time 64.789h, and the theoretical EPS yield is 134.639g / L.

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

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

[0082] Take 100 mg of extracellular polysaccharide sample EPS-OD and dissolve it in 5 ml of distilled water. Centrifuge at a speed of 8000 r / min for 10 min, and take the supernatant to pass through a 0.45 μm water filter membrane. Use a dropper to draw the polysaccharide solution slowly and evenly along the inner wall of the chromatography column. Use distilled water, 0.1 mol / L NaCl, 0.3 mol / L NaCl, 0.5 mol / L NaCl and 0.7 mol / L NaCl solutions for elution in turn. The elution flow rate is 1 mL / min, 10 mL per tube. The polysaccharide content of each tube was determined by the phenol-sulfuric acid method, and the polysaccharide elution curve was drawn. The results are as follows Figure 6 As shown, a polysaccharide elution fraction EPS was mainly obtained. According to the elution curve, the polysaccharide elution fractions were 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 the components of extracellular polysaccharides 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 purification by DEAE-52 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 of EPS after purification by DEAE-52 decreased to 0.82±0.62%, indicating that the EPS obtained by extraction and purification contained almost no uronic acid, and it was speculated that it was a neutral sugar. The protein content of EPS-OD was 1.34±0.35%, and the protein content of EPS after purification by DEAE-52 decreased to 0.78±0.18%, indicating that EPS-OD contained a small amount of protein, and EPS contained almost no protein after purification.

[0084] Table 3 Polysaccharide composition analysis

[0085]

[0086] Example 3 Structural Identification of Bacillus amyloliquefaciens Polysaccharide

[0087] 3.1 UV spectrum analysis

[0088] The ultraviolet absorption spectrum of extracellular polysaccharide was scanned in the 200-400nm band by ultraviolet spectrophotometer. Figure 7 The results show that EPS has a gentle trend at 260nm and no ultraviolet absorption peak, indicating that there is no nucleic acid substance in EPS; EPS has no obvious characteristic absorption peak at 280nm, indicating that there is no protein in EPS.

[0089] 3.2 Fourier transform infrared spectroscopy (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 infrared spectrum of EPS was determined by FT-IR spectrometer at 4000 cm -1 -500cm -1 FT-IR spectra of exopolysaccharides were recorded in a range of wavelengths.

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

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

[0093] The molecular weight of EPS was determined by high performance gel permeation chromatography (HPGPC), mobile phase: 0.05 mol / L NaCl solution; flow rate: 0.6 ml / min, column temperature: 40 °C; injection volume: 20 uL; chromatographic column: BRT105-104-102 series gel column (8×300 mm); the peak curve was drawn with elution time as the horizontal axis and the logarithm of the polysaccharide molecular weight as the vertical axis ( Fig. 9 ). The molecular weight is shown in Table 4. The Mw of EPS is 5135Da.

[0094] Table 4 EPS molecular weight

[0095]

[0096] 3.4 Determination of monosaccharide composition by ion chromatography

[0097] The sample (5 mg) was hydrolyzed with trifluoroacetic acid (TFA, 3M, 2 mL) (3 h, 80 ° C) and dried with nitrogen to completely remove TFA. Then deionized water (5 mL) was vortexed and mixed, 100 uL was transferred, 900 uL deionized water was added, centrifuged (12000 rpm, 5 min), and 25 uL of the supernatant was loaded for IC analysis. The chromatographic method was: mobile phase A: H 2 O; B: 15mmol / LNaOH; C: 15mmol / LNaOH & 100mmol / LNaOAc; flow rate: 0.3mL / min; injection volume: 25uL; chromatographic column: Dionex CarbopacTMPA20 (3*150mm); column temperature: 30℃; detector: electrochemical detector. The external standard method was used for quantification, and the ion exchange spectra of 16 monosaccharide standards were obtained as shown below. Fig.10 shown.

[0098] The ion exchange spectrum of EPS is as follows Fig.11 As shown in the figure, no uronic acid monosaccharide was detected in the EPS monosaccharide composition test, which is consistent with the polysaccharide composition test results. EPS is mainly composed of glucose (Glc) and fructose (Fru), with a molar ratio of Glc:Fru=0.139:0.861, of which fructose accounts for a larger proportion, exceeding 85% by molar ratio, followed by glucose, which accounts for about 14%, thus confirming that the polysaccharide is fructo-glucan.

[0099] 3.5 Methylation analysis

[0100] 3.5.1 Methylation: Methylation reaction was performed using a methylation kit. Accurately weigh 3 mg of polysaccharide sample into a reaction bottle, add 1 mL of DMSO, and quickly add methylation reagent A solution (anhydrous alkaline solution). In a closed state, dissolve by ultrasonication, and then quickly add methylation reagent B solution (iodomethane solution). Stir magnetically for 60 minutes in a 30°C water bath. Add 2 mL of ultrapure water to the mixed system to terminate the methylation reaction.

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

[0102] The acetylated product was dissolved in 3 mL CH 2 Cl 2 , transfer to a separatory funnel, add appropriate amount of ultrapure water and shake well, remove the upper layer, and repeat the operation 5-6 times. Dry the organic layer with appropriate amount of anhydrous sodium sulfate and dilute to 10mL. Use Agilent GCMS 6890-5973 gas chromatography-mass spectrometer to measure the sample. Equipped with RXI-5SILMS column 30m×0.25mm×0.25um; starting temperature 120℃, increase the temperature to 250℃ / min at 3℃ / min, and maintain for 5min; the injection port temperature is 250℃, and the detector temperature is 250℃ / min.

[0103] After the polysaccharide sample is derivatized, its connection information can be obtained by GC-MS determination. The mass spectrum is shown in the appendix. The linkage pattern of EPS (Table 5) shows that the largest proportion of the four residual sugars of fructose and glucose is →1)-Fruf-(2→ residual sugar, which constitutes the main chain of glucan, followed by fructose terminal sugar, which accounts for about 10%. Another monosaccharide, glucose, has two residual sugars, which are →3,6)-Glcp-(1→ with a branched structure and terminal sugar residual sugar. Therefore, it can be inferred that EPS is fructan, and the main linkage mode is Fruf-(2→.

[0104] Table 5 EPS methylated 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 2O, and the measured values ​​were determined on a Bruker Avance III 600 MHz NMR spectrometer. 1 D NMR spectroscopy ( 1 H and 13 C) and 2 D nuclear magnetic resonance spectroscopy (COSY, HSQC, HMBC and NOESY), the refined polysaccharide component EPS-3 sample was placed in 0.5 mL 2 O and were analyzed on a Bruker Avance III 600 MHz NMR spectrometer. 1 H and 13 CNMR spectrum.

[0108] Nuclear magnetic resonance (NMR) reveals the precise structural information of EPS. Fig.12 (A), in H NMR spectrum 1 In the anomeric region of HNMR (4.3-5.5ppm), multiple groups of anomeric hydrogen weak signals were found to have their main peak positions at 5.30, 5.27, 5.12, and 4.53ppm, confirming that the polysaccharide is composed of multiple residual sugars. In the 3.0-4.2ppm range of the hydrogen spectrum, there are typical proton signals outside the anomeric hydrogen of the residual sugar ring, which is consistent with the general characteristics of the hydrogen spectrum of polysaccharides.

[0109] Depend on Fig.12 (B) It can be seen that the NMR carbon spectrum 13 In CNMR, no obvious uronic acid carbonyl carbon signal was found within 170-210ppm, and the polysaccharide was neutral. In the anomeric region of the carbon spectrum (90-110ppm), multiple groups of main anomeric carbon signals can be identified, namely 104.15, 104.05, 103.95, 103.74, 103.56, 103.45, 101.74ppm, and multiple anomeric carbon signals correspond to multiple anomeric hydrogen signals; in the range of 55-85ppm, there are multiple typical polysaccharide residual sugars excluding anomeric carbons and carbon peak information, among which the signals at 60.31 and 63.32ppm are attributed to the residual sugar ring exomethylene -CH 2 - secondary carbon, Fig.12 (C) The DEPT135 spectrum of EPS confirmed the signal attribution; the multiple signals at 74-82 ppm represent the carbon information of the ring C2-C4 of fructose residues and C2-C5 of glucose.

[0110] The structure of the polysaccharide was further analyzed by two-dimensional NMR spectroscopy. Fig.13They are the COSY, HSQC, HMBC, and NOESY spectra of EPS. In the HSQC two-dimensional NMR spectrum reflecting the connected hydrogen-carbon correlation, multiple groups of H1 / C1 related weak signals in the anomeric region were identified, which were 5.32 / 92.33, 5.28 / 91.89, 5.13 / 91.89, and 4.53 / 95.80 ppm, respectively. Combined with the methylation results and the integral intensity of the hydrogen spectrum 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 NMR spectra, the positions and structural correspondence information of the carbon-hydrogen signals of various residual sugars in the polysaccharide structure are assigned in turn. For example, it is determined that the anomeric carbon 104.15ppm belongs to the quaternary carbon C2 in the residual sugar →1)-Fruf-(2→. By identifying the HMBC spectrum of hydrogen-carbon remote correlation, the signals strongly correlated with it are 3.56 and 3.64ppm, confirming that the positions of H1a and H1b are 3.56 and 3.64ppm; combined with the HSQC spectrum, the signal of C1 is confirmed to be 59.80; according to the HMBC information, the signal related to H1a and H1b is 76.06, confirming that the hydrogen spectrum position of C3 is 76.0 6ppm, and then combined with HSQC to find 4.08 / 76.06, the H3 carbon spectrum position was determined to be 4.08ppm, and the H4 / H3 related signals were determined to be 4.08 / 3.98 by the same method and combined with COSY spectrum, and H4 was determined to be 3.98ppm, and then the H5 / C5 and H6 / C6 position information were determined to be 3.85 / 80.18, 3.46, 3.80 / 63.05. Based on this method, other main positions of residual sugars were found and the same signal positions were assigned. The analysis results are shown in Table 6.

[0112] Table 6 EPS main residual sugar attribution analysis results

[0113]

[0114] The two-dimensional nuclear magnetic resonance spectrum HMBC plays a greater role in determining the hydrogen-carbon correlation between 2-3 chemical bonds. In the analysis of polysaccharides, it is of certain significance for determining the connection of glycosidic bonds. For example, in the β-1)-Fruf-(2→ residual sugar with a large content, the anomeric carbon related signals were found in the HMBC spectrum: 3.56 / 104.04, 3.64 / 104.04, 4.08 / 104.04. Through the above assignment, it was confirmed that the anomeric hydrogen of the residual sugar was related to 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 related to H1 confirmed the existence of glycosidic bonds connecting the same residual sugar 1→2, and constituted most of the glycosidic bonds in the fructoglucan; the related signals of 3.56 / 103.74 and 3.64 / 103.74 confirmed the existence of the terminal sugar β-t-Fruf-(2→ and β-1)-Fruf-(2→ In addition, the signal of 5.27 / 104.04 indicates that α-3,6)-Glcp-(1→ and β-1)-Fruf-(2→ have a 1→2 glycosidic bond connection, while the signal of 3.81 / 104.15 indicates that β-1)-Fruf-(2→ and α-3,6)-Glcp-(1→ have a 2→3 glycosidic bond connection; for the terminal sugar of β-t-Glcp-(1→, due to the weak signal, the signal of 3.67 / 95.80 is found, and it is speculated that it has a 1→6 glycosidic bond connection with α-3,6)-Glcp-(1→. The signal of 3.66 / 103.74 indicates that the terminal sugar t-β-Fruf-(2→ and α-3,6)-Glcp-(1→ have a 2→6 branched chain connection.

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

[0116]

[0117] 3.7 Scanning electron microscopy analysis

[0118] The polysaccharide powder was adhered to the stage by conductive glue, the unfixed powder was blown away by nitrogen, the surface was sprayed with gold and then nitrogen was blown again, and finally it was placed under a scanning electron microscope for observation, and the image was magnified at 500×, 1000× and 2000×.

[0119] The apparent morphology of refined polysaccharides is Fig.14 As shown in Figure 1, EPS is in the shape of a regular sphere or ellipsoid with a smooth surface and an average pore size of about 30-50 microns. The spherical conformation of EPS may be due to the high degree of branching of the polysaccharide molecules, with the branches extending in all directions to form a dense spherical structure.

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

[0121] 4.1 Inhibitory activity of exopolysaccharides on pancreatic lipase and α-glucosidase in vitro

[0122] 4.1.1 In vitro pancreatic lipase inhibition ability

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

[0124] 4.1.2 In vitro α-glucosidase inhibitory activity

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

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

[0127] 4.2 Extracellular polysaccharide binding capacity for total bile acid and cholesterol micelles in vitro

[0128] 4.2.1 Total bile acid binding capacity in vitro

[0129] Weigh cholesterol, oleic acid, and lecithin in 5 ml methanol solution for ultrasonic treatment, fully dissolve and dry with nitrogen. Add sodium chloride, sodium taurocholate, and phosphate buffer solution (pH = 7.4), and treat at 37°C for 12 hours to prepare a cholesterol micelle solution (the cholesterol micelle solution contains 10 mM sodium taurocholate, 2 mM cholesterol, 5 mM oleic acid, 5 mM lecithin, 132 mM sodium chloride, and 15 mM phosphate buffer).

[0130] Add 1.0 mL of micellar solution to a test tube, add the sample to be tested and mix well, incubate at 37°C with shaking for 2 hours, and centrifuge to obtain the supernatant for testing. Cholestyramine is used as a positive control, and no sample is used as a negative control. The cholesterol concentration of the supernatant is determined using a total cholesterol kit.

[0131] 4.2.2 In vitro cholesterol micelle binding ability

[0132] Weigh cholesterol, oleic acid, and lecithin in 5 ml methanol solution for ultrasonic treatment, fully dissolve and dry with nitrogen. Add sodium chloride, sodium taurocholate, and phosphate buffer solution (pH = 7.4), and treat at 37°C for 12 hours to prepare a cholesterol micelle solution (the cholesterol micelle solution contains 10 mM sodium taurocholate, 2 mM cholesterol, 5 mM oleic acid, 5 mM lecithin, 132 mM sodium chloride, and 15 mM phosphate buffer).

[0133] Add 1.0 mL of micellar solution to a test tube, add the sample to be tested and mix well, incubate at 37°C with shaking for 2 hours, and centrifuge to obtain the supernatant for testing. Cholestyramine is used as a positive control, and no sample is used as a negative control. The cholesterol concentration of the supernatant is determined using a total cholesterol kit.

[0134] Test results: EPS-OD and EPS have good in vitro bile acid and cholesterol binding abilities. Using 10 mg of cholestyramine as a positive control, 10 mg of EPS-OD and EPS were tested for in vitro bile acid and cholesterol micelle binding abilities. As shown in Table 7, the binding abilities of EPS-OD to bile acid and cholesterol micelles were 34.35±3.60% and 42.47±1.21%, respectively, and the binding abilities of EPS to bile acid and cholesterol micelles were 33.35±2.54% and 37.23±0.65%, respectively. Compared with EPS-OD, the bile acid binding ability of EPS did not change much, while the cholesterol micelle binding ability decreased. The spatial configuration of the purified EPS changed, and the number of exposed binding sites decreased, which reduced the binding ability of polysaccharides to cholesterol micelles.

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

[0136]

[0137]

[0138] The embodiments of the present invention are described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions and variations of these embodiments are made without departing from the principles and spirit of the present invention, and still fall within the protection scope of the present invention.

Claims

1. A strain of Bacillus amyloliquefaciens, characterized in that The strain is Bacillus amyloliquefaciens D189, which was deposited in the China Center for Type Culture Collection on April 29, 2021, with the deposit number CCTCC NO: M2021484.

2. Use of the Bacillus amyloliquefaciens D189 according to claim 1 in the preparation of blood sugar and lipid lowering products.

3. The use according to claim 2, characterized in that: The crude polysaccharide was obtained by fermentation with Bacillus amyloliquefaciens D189 and used to inhibit α-glucose enzyme and pancreatic lipase.

4. Use of the Bacillus amyloliquefaciens D189 according to claim 1 in the preparation of exopolysaccharides.

5. A method for preparing exopolysaccharides using Bacillus amyloliquefaciens, characterized in that: The steps include: Step S1-strain activation and expansion culture: activating the Bacillus amyloliquefaciens D189 described in claim 1, and then inoculating the activated single colony into LB solid culture medium for continuous culture for 24 to 48 hours to obtain a seed fermentation liquid, adjusting the absorbance value of the bacterial liquid to about 1.0 at 600nm, and then inoculating it into a fermentation medium for expansion culture; Step S2-crude polysaccharide extraction: centrifuge the fermentation liquid after the expanded culture in step S1, remove the precipitated bacteria, retain the supernatant and add ethanol, let it stand and then centrifuge to collect the precipitate, and redissolve to obtain a crude polysaccharide solution; Step S3-deproteinization: adding sevage reagent to the polysaccharide solution of step S2, and collecting the upper aqueous phase; Step S4-dialysis: After evaporating the aqueous phase solution in step S3 to remove the organic solvent, the aqueous phase solution is transferred to a dialysis bag for dialysis, and the dialyzed polysaccharide solution is collected and dried to obtain crude extracellular polysaccharide of Bacillus amyloliquefaciens D189.

6. The preparation method according to claim 5, characterized in that: 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, soy peptone concentration 11.7±0.1 g / L.

7. The preparation method according to claim 5, characterized in that: The time for inoculating into the fermentation medium and carrying out expansion culture in step S1 is 60 to 72 hours.

8. The preparation method according to claim 5, characterized in that: The inoculation ratio in step S1 is 1-3%.

9. The preparation method according to claim 5, characterized in that: The molecular weight cut-off of the dialysis in step S4 is below 2000 Da.

10. A crude exopolysaccharide of Bacillus amyloliquefaciens D189 prepared by the preparation method according to any one of claims 5 to 9, characterized in that: The crude exopolysaccharide of Bacillus amyloliquefaciens D189 was further purified to obtain an exopolysaccharide having the following main structure:

Images