CN111621448A - Bacillus belgii SN-1 and method for producing exopolysaccharides through fermentation of bacillus belgii SN-1 - Google Patents

Abstract

The invention discloses a strain of Bacillus velesi SN-1 and a method for fermenting and producing extracellular polysaccharide, belonging to the field of microbiology. The present invention is obtained from the traditional natural fermented soybean paste in Northeast China, and it is found that it can produce extracellular polysaccharide with excellent characteristics. Bacillus velezensis (Bacillus velezensis) SN-1 was inoculated into the LB basal medium containing 2% sucrose, and the initial medium pH value was 7, the temperature was 37 ° C, and the rotating speed of the shaker was 100-200rpm. 48-72h to obtain fermentation broth; purify crude polysaccharide to obtain exopolysaccharide. The strain and the fermentation production method of the invention have the advantages of short sugar production period, high sugar production, safety and no toxic side effects, and the extracellular polysaccharide has high emulsification and thermal stability, which provides a theoretical basis and basis for industrial application. Broad commercial application prospects.

Description

A strain of Bacillus velesi SN-1 and its fermentation method for producing exopolysaccharide

technical field

The invention relates to a strain of Bacillus velesi SN-1 and a method for fermenting and producing extracellular polysaccharide, belonging to the field of microbiology.

Background technique

Exopolysaccharide (EPS) is a mucopolysaccharide or capsular polysaccharide secreted outside the cell by microorganisms (bacteria, yeast, fungi) during their growth and metabolism. With the increasing research on plant, animal and fungal polysaccharides, there is an urgent need to develop microbial exopolysaccharides with novel properties and high yields from edible microorganisms. Microbial exopolysaccharides have always been the main source of exopolysaccharides in theoretical and practical research because of their wide range of sources, mild reaction conditions, and easy separation and purification.

To date, a large number of microbial-derived commercial EPSs (dextran, alginate, xanthan and gellan gum) have been developed as potential biomaterials. However, some of them are extracted from pathogenic bacteria, such as hyaluronic acid produced by Streptococcus Zooepidemics and xanthan gum produced by Xantho monascampestris, which may put humans at risk of contamination with bacterial endotoxins. Due to the diverse properties of exopolysaccharides and their wide application, more and more researchers are devoted to developing microorganisms with novel properties and high EPS production. At the same time, the development and utilization of food-grade EPS from edible microorganisms has become one of the hot spots in industrial microorganism research. Bacillus exopolysaccharides have been used as stabilizers and biological flocculants in the production of pharmaceuticals, cosmetics and other products. In addition, Bacillus exopolysaccharides have excellent physicochemical properties and biological properties, such as biological emulsifiers, heavy metal chelators, antiviral and immunomodulatory agents, and can also be used as prebiotics to promote the growth of beneficial bacteria in the gut and regulate the gut The flora balances and promotes the health of the host. Therefore, compared with other polysaccharides, the research on Bacillus exopolysaccharides has more practical application value. , more suitable for humans than other microbial polysaccharides. There have been a lot of research reports on the EPS-producing lactic acid bacteria strains found in nature, but the problem of low yield is common. Therefore, it has always been a research hotspot in the field of microbiology to fully exploit the abundant Bacillus resources in nature, discover bacterial strains with high exopolysaccharide production, and obtain more exopolysaccharides in a short cycle and at low cost.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a high-yield straight-chain mannose strain and a fermentation production method thereof.

The present invention provides a strain of Bacillus velezensis SN-1, which was deposited in the China General Microorganism Culture Collection and Management Center on June 8, 2020, and the deposit number is CGMCC No.1.18401.

A high-yielding straight-chain mannose strain, which is Bacillus velezensis SN-1, is isolated from the traditional natural fermented soybean paste in Northeast China, and it is found that it can produce extracellular polysaccharides with excellent characteristics. The morphological, physiological and biochemical experiments proved that the bacterium was Bacillus velezensis, and it was named Bacillus velezensis SN-1 (Bacillus velezensis SN-1). Microorganism Culture Collection and Management Center, the deposit number is CGMCC No.1.18401.

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 (SEQ ID NO.2: upstream primer 8F: 5-AGAGTTTGATCCTGGCTCAG-3; SEQ ID NO.3: Downstream primer 1492: 5-CTACGGCTACCTTGTTACGA-3); the results of the 16s rDNA gene sequencing of this strain are shown in SEQ ID NO.1 in the sequence table.

A method for producing exopolysaccharide by fermentation of Bacillus velezensis SN-1, comprising the following steps:

(1) Bacillus velezensis (Bacillus velezensis) SN-1 is inoculated in a sugar-containing LB liquid medium and cultivated for a period of time to obtain a fermentation broth;

(2) centrifuging the fermentation broth, removing the bacterial cells, and taking the supernatant;

(3) adopt the ethanol precipitation method to precipitate the supernatant after removing the bacterial cells by centrifugation, let stand, centrifuge, take the precipitate, after the precipitate is dissolved in water, adopt the savage method to remove the protein, and obtain the crude polysaccharide aqueous solution after dialysis;

(4) Purify the crude polysaccharide aqueous solution to obtain pure exopolysaccharide.

Further, in the above technical solution, the sugar content in the LB liquid medium in step (1) is 2-5wt%; the sugar includes sucrose, glucose or maltose.

Further, in the above technical solution, the inoculum amount of Bacillus velezensis SN-1 in step (1) is 5%-10% (v/v).

Further, in the above-mentioned technical scheme, the pH value of the sugar-containing LB liquid medium described in step (1) is 6-8, and the culture conditions are: the temperature is 25-37 ° C, and under the conditions of the shaking table rotating speed of 100-200 rpm, Culture for 48-72h.

Further, in the above technical solution, the centrifugation temperature in step (2) is 4-30° C., the centrifugation speed is 10000-12000rpm, and the centrifugation time is 15-20min.

Further, in the above-mentioned technical scheme, the volume percentage concentration of the ethanol described in step (3) is 95-100%, and the ethanol volume in the ethanol precipitation method is 3-5 times the volume of the supernatant; the standing temperature is 4 -8°C, the standing time is 12-14h; the centrifugation conditions are: 4-8°C, 10000-12000rpm centrifugation for 20-40min.

Further, in the above technical solution, the molecular weight cut-off of the dialysis in step (3) is 14000Da, and the dialysis conditions are: 4-8°C distilled water dialysis for 2-3 days, and distilled water is changed every 5-8h.

Further, in the above technical solution, the purification in step (4) is to purify the crude polysaccharide by gel filtration chromatography.

Further, in the above technical solution, the exopolysaccharide is a straight-chain mannose linked by α-(1→4) glycosidic bonds.

By Fourier transform infrared spectroscopy (FT-IR), high performance liquid chromatography (HPLC), gel permeation chromatography (GPC) and nuclear magnetic resonance techniques. The structure analysis of the exopolysaccharide produced by the present invention shows that the exopolysaccharide of the present invention contains only one sugar unit, which is a mannose mainly connected by α-(1→4) glycosidic bonds, and does not contain branched chains . The research based on thermogravimetric analysis (TG) and physicochemical properties proves that the exopolysaccharide of the present invention has high emulsifying property, thermal stability, and strong heat resistance (degradation temperature is 270.7° C.).

The beneficial effects of the invention are as follows: the invention obtains the extracellular polysaccharide from the fermentation product of the strain through a series of biochemical technologies, and uses modern extraction technology to extract and purify the fermentation liquid to obtain high-purity polysaccharide. After a series of experiments, it is proved that the obtained strain has the characteristics of Bacillus, and can produce a large amount of exopolysaccharide under the induction of the substrate sucrose. At present, the yield of microbial EPS is about 4.5 g/L, and the yield of Bacillus velezensis SN-1 EPS in the present invention can reach 12.7 g/L, which is 2.82 times higher than that of other microbial EPS. Therefore, the fermentation broth obtained by B. velezensis SN-1 fermented in the sugar-producing fermentation medium contains extracellular polysaccharides, which has the advantages of short sugar production cycle, high sugar production, safe and non-toxic side effects , and the extracellular polysaccharide has high emulsification and thermal stability, which provides a theoretical basis and basis for industrial application, and has a wide commercial application prospect.

Description of drawings

Figure 1 shows the colony morphology (A) and individual cell morphology (B) of Bacillus velezensis SN-1.

Figure 2 is a phylogenetic tree of Bacillus velezensis SN-1 based on the 16S rDNA gene sequence.

3 is a DEAE-52 anion exchange column chromatogram (A) and a G-100 gel column chromatogram (B) of the exopolysaccharide produced by Bacillus velezensis SN-1.

Figure 4 is a UV spectrum of pure exopolysaccharide produced by Bacillus velezensis SN-1.

Figure 5 is a high performance liquid chromatogram (B) and a monosaccharide standard high performance liquid chromatogram (A) of pure exopolysaccharide produced by Bacillus velezensis SN-1.

Figure 6 is an infrared spectrum of pure exopolysaccharide produced by Bacillus velezensis SN-1.

Figure 7 is a scanning electron microscope image of pure exopolysaccharide produced by Bacillus velezensis SN-1, scale: 100 μm (A); scale: 10 μm (B).

Figure 8 is the ¹ H NMR spectrum (A) and ¹³ C NMR (B) of pure exopolysaccharide produced by Bacillus velezensis SN-1.

Figure 9 is a thermogravimetric spectrum of pure exopolysaccharide produced by Bacillus velezensis SN-1.

Detailed ways

The following non-limiting examples may enable those of ordinary skill in the art to more fully understand the present invention, but do not limit the present invention in any way.

Example 1 Isolation and screening of Bacillus velezensis SN-1

(1) Culture medium

LB liquid medium: tryptone 10g, yeast powder 5g, KH ₂ PO ₄ 1.6g, K ₂ HPO ₄ 2g, MgSO ₄ 7H ₂ O 0.25g, CaSO ₄ 2H ₂ O 1g, FeCl ₃ 2g, distilled water 1000mL, pH 7.0, sterilized at 115°C for 20min.

LB solid agar medium: add 15-20 g of agar powder to the LB liquid medium, and sterilize at 115°C for 20 minutes.

LB sugar-producing fermentation broth: sucrose 50g, tryptone 10g, yeast powder 5g, KH ₂ PO ₄ 1.6g, K ₂ HPO ₄ 2g, MgSO ₄ 7H ₂ O 0.25g, CaSO ₄ 2H ₂ O 1g, FeCl ₃ 2g, distilled water 1000mL, pH 7.0, sterilized at 115°C for 20min.

(2) Sample pretreatment

Take by weighing Northeast Natural Fermentation Farmhouse Sauce 1g (described Northeast Natural Fermentation Farmhouse Sauce is after the soybean is cleaned and cooked, pulverized and made into a sauce block, naturally fermented in a cool and ventilated place, cleaned and removed the fimbriae on the surface of the sauce block after fermentation, After being broken into small pieces, expose to the sun in the sun, then put into the sauce tank, add cold water and edible iodized salt, seal, and place the natural fermentation at the sunny place. The northeast natural fermentation farmhouse sauce of the present invention can be prepared in the above-mentioned manner, but it is not (limited to this method) was added to 9 mL of sterile saline, shaken and mixed, and water bathed at 80°C for 20min. Take an appropriate amount of solution from the above treatment to prepare a sample solution with a concentration of 10 ^-1 , and then dilute to 10 ^-2 and 10 ^-3 step by step and let stand for later use.

(3) Primary screening of myxogenic colonies

Take 100 μL of the above-mentioned bacterial solutions of each dilution and spread them on LB solid agar medium plates, and invert them in a constant temperature incubator at 37°C for 24-48h. Pick the colony area that produces stickiness on the plate, streak repeatedly on the LB solid agar medium plate, and store the plate of pure culture at 4°C.

(4) Rescreening of strains

The pure culture obtained from the plate was inoculated into LB liquid medium for 18 hours, and then inoculated into LB sugar-producing fermentation liquid medium at 2% (v/v) inoculation amount, and cultured at 37° C. for 48 hours. The fermentation broth was heated in a 90°C water bath for 10 min to remove enzymes that may degrade polysaccharides in the bacterial broth, and cooled to room temperature. Add 80% trichloroacetic acid (TCA) solution to the fermentation broth to a final concentration of 5% (m/v), stir at room temperature for 2 h, centrifuge at 10000 × g for 20 min at 4°C, and remove cells and protein precipitates. The supernatant was put into a dialysis bag (molecular weight cut-off 14000 Da), and dialyzed with ultrapure water in a 4°C refrigerator for 2 d, and the water was changed every 8 h. After constant volume, the yield of exopolysaccharide produced by the strain was determined. Combined with the mucus concentration of the mucilage-producing colonies, the strains with higher polysaccharide production were selected.

Example 2 Identification of Bacillus velezensis SN-1

(1) Morphological identification of SN-1 strain

The SN-1 strain was inoculated on the LB plate with three-district lines, and after culturing at 37°C for 24-48 hours, the single colony characteristics of the strain on the plate were observed and recorded. The colony morphology of SN-1 is shown in Figure 1A, and the observation results of colony morphology are summarized in Table 1. Pick a small amount of fresh bacterial cells with an inoculating needle and spread them on a clean glass slide for Gram staining, and then observe the individual cell morphology and arrangement under a microscope (Figure 1B).

Table 1 Summary of morphological observations of SN-1 strains

(2) Physiological and biochemical identification of SN-1 strain

①Contact enzyme test: select a small amount of Gram-positive strains and inoculate them on LB solid medium plates, incubate at 37°C for 24 hours, add 10% H ₂ O ₂ dropwise, and observe the generation of bubbles on the colonies In some cases, the production of air bubbles is positive for the contact enzyme. The results are shown in Table 2, and the contact enzyme reaction of the SN-1 strain was negative.

②Glucose acid production and gas production test: in glucose liquid fermentation medium (peptone 2.7g, sodium chloride 5.0g, 0.2% bromothymol blue 0.03g, agar 3g, KH ₂ PO ₄ 0.3g, glucose 10.0g, water 1000mL), add bromocresol purple indicator and inverted Dulbecco's tube, insert SN-1 strain in the medium according to 1% (v/v) inoculation amount, shake the test tube gently to make it uniform and prevent the inverted tube from entering bubble. After culturing at 37°C for 48 hours, observe the results. The color of the indicator in the medium changes from purple to yellow, indicating that SN-1 can use glucose to produce acid, if it is still purple, it means no acid production, and if there are bubbles in Duchenne tubules, it means SN-1. The -1 strain can use glucose to produce gas. The results are shown in Table 2. The results show that the SN-1 strain can utilize glucose to produce acid and gas.

③H ₂ S production test: use an inoculating needle to puncture the SN-1 strain into lead acetate medium (peptone 10.0g/L, beef extract powder 3.0g/L, sodium chloride 5.0g/L, sodium thiosulfate 2.5g /L, agar 12.0g/L, PH: 7.3±0.1 (25°C)), placed at 37°C for 48h, and observed whether black lead sulfide was produced. The results are shown in Table 2. The results show that the SN-1 strain cannot produce H ₂ S.

④ Starch hydrolysis test: inoculate SN-1 strain line drawing in starch medium (beef extract 0.5g, peptone 1g, sodium chloride 0.5g, soluble starch 0.2g, water 100mL, pH 7.0～7.2, agar 2g, sterilization 115°C, 0.1Mpa, 20min), the plate was incubated in a 37°C incubator for 24h. Observe the growth of the strain, and then drop a small amount of Lugol's iodine solution on the plate. A colorless and transparent circle appears around the colony, indicating that the starch is completely hydrolyzed, and the strain has the ability to decompose starch. The results are shown in Table 2.

⑤Indole production experiment: inoculate the SN-1 strain into peptone water medium (1g peptone, 0.5g sodium chloride, add water to 100mL, pH adjusted to 7.8), add ether 1- 2mL, after standing for a while, the ether layer floats on the top of the culture medium. At this time, slowly add 5-10 drops of indole reagent along the tube wall. If there is indole, the ether layer will appear rose red, which is a positive reaction of the indole test. Otherwise it is a negative reaction. The results are shown in Table 2.

⑥ Gelatin liquefaction test: take a test tube of gelatin medium (NaCl 5g, peptone 10g, beef extract 3g, gelatin 120g, distilled water 1000mL, pH 7.2-7.4), puncture the SN-1 strain with an inoculation needle, and place the inoculated test tube at 20 Cultivate at ℃ for 3-5 days, and observe the liquefaction of gelatin. The results are shown in Table 2.

⑦ Motility: Using the hanging drop method, a drop of bacterial suspension was suspended on the cover glass of the grooved glass slide, and then its motility was observed under weak light through an ordinary microscope. The results are shown in Table 2.

Table 2 Physiological and biochemical identification results of SN-1 strain

Note: "+" indicates positive reaction; "-" indicates negative reaction.

⑧ Sugar fermentation test: The glucose in the LB sugar-producing fermentation liquid medium was replaced with sucrose, L-arabinose, fructose, galactose, lactose, mannose, maltose, and rhamnose to prepare different sugar medium, and bromine was added. Cresyl violet indicator, inoculated with 1% (v/v) SN-1 strain and marked, and blank control was not inoculated. After culturing at 37°C for 48 hours, the observation results showed that if the indicator in the medium turned yellow, it indicated that the SN-1 strain could utilize the sugar to produce acid, which was a positive reaction. The test results are shown in Table 3. The test shows that the SN-1 strain can utilize sucrose, mannose, maltose and rhamnose to produce acid.

Table 3 Sugar fermentation test results of SN-1 strain

Note: "+" indicates positive reaction; "-" indicates negative reaction.

(3) 16S rDNA sequence analysis of SN-1 strain

①Extraction of bacterial genomic DNA

Pipette 100 μL of the SN-1 strain bacterial suspension into the sterilized LB liquid medium, and cultivate it in a shaking incubator at 37°C for 24 hours. The genomic DNA of SN-1 4-3 was extracted according to the procedure on the bacterial genome kit (Solarbio D1600).

②PCR amplification

The designed PCR primers are:

SEQ ID NO. 2: Upstream primer 27F: 5'-AGAGTTTGATCCTGGCTCAG-3'

SEQ ID NO.3: Downstream primer 1492R: 5'-CTACGGCTACCTTGTTACGA-3', the primer was synthesized and delivered by Shanghai Paisenuo Bioengineering Company.

PCR reaction conditions were: preheating at 95°C for 5 min, denaturation at 95°C for 30s, annealing at 55°C for 30s, extension at 72°C for 90s, cycle 35 times, hold at 72°C for 7 min, and incubate at 4°C.

③ Recovery of PCR products

PCR products were recovered with AxyPrep DNA gel recovery kit (Boyao ASJ0013), and the specific operations were carried out according to the kit instructions.

④16S rDNA sequencing and sequence alignment

The positive PCR products were sent to Shanghai Parsenno for sequencing, and the sequencing results were compared and analyzed in the NCBI database using the BLAST tool with the existing sequences in the GenBank database to analyze the homology of the strains to be tested and the corresponding sequences of known strains to determine The screened sugar-producing strains. Figure 2 shows the phylogenetic tree of the SN-1 strain based on the 16S rDNA gene sequence.

Combined with the morphological, physiological and biochemical properties of the SN-1 strain and the 16S rDNA sequence homology alignment results, it was confirmed that SN-1 was Bacillus velezensis.

The strains were preserved in the General Microbiology Center of the China Microorganism Culture Collection Management Committee (CGMCC for short, address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, zip code 100101). The strain collection number of Bacillus velezensis SN-1 is CGMCC No.1.18401, the preservation date is June 8, 2020, the classification name is Bacillus velezensis, and the strain name is SN- 1.

Example 3 Fermentation process of Bacillus velezensis (Bacillus velezensis) SN-1 strain to produce exopolysaccharide by fermentation

The fermentation process of the Bacillus velezensis (Bacillus velezensis) SN-1 strain fermenting a high-yielding exopolysaccharide strain of the present embodiment is carried out according to the following steps:

(1) Preparation of seed solution

Bacillus velezensis SN-1 was inoculated into the LB liquid medium so that the initial cell concentration in the medium was 1.0×10 ⁸ CFU/mL, which was the seed solution.

(2) Fermentation conditions for sugar production

Bacillus velezensis (Bacillus velezensis) SN-1 was inoculated into the LB sugar-producing fermentation liquid medium with an initial pH value of 7 at an inoculum amount of 0.5% (V/V), and the temperature was kept at 37 ° C at a constant temperature of 100 rpm Shaking culture was performed for 36-72 h, and the polysaccharide content in the fermentation supernatant was determined by the phenol-sulfuric acid method.

Bacillus velezensis (Bacillus velezensis) SN-1 was inoculated into the LB sugar-producing fermentation liquid medium with an initial pH value of 7 at an inoculum amount of 0.5% (V/V), and the temperature was 37 ° C and a shaking table. Under the condition of rotating speed of 100rpm, culture for 48h to obtain fermentation broth, and centrifuge the fermentation broth at 4°C and 10000rpm for 20min to remove bacterial cells. The supernatant was added with 3 volumes of pre-chilled 95% ethanol and incubated overnight at 4°C to precipitate polysaccharides. 4°C, 10000rpm centrifugation for 20min to collect the polysaccharide precipitate, dissolve the polysaccharide precipitate with ultrapure water, add the same volume of 10% trichloroacetic acid solution as the ultrapure water used to dissolve the polysaccharide to remove the protein, stand at 4°C for 10h, 4°C, 10000rpm centrifugation for 20min Collect the supernatant. Add 3 volumes of pre-chilled 95% ethanol and incubate overnight at 4°C to precipitate polysaccharides. The polysaccharide precipitate was collected by centrifugation at 10,000 rpm for 20 min at 4°C, redissolved in ultrapure water (about 200 mL), put into a dialysis bag (molecular weight cut-off 14,000 Da), and dialyzed with distilled water at 4°C for 2 days, changing the water every 8 hours. , lyophilized to obtain extracellular polysaccharides. The content of extracellular polysaccharide obtained in the fermentation broth obtained in this example was 12.7 g/L.

Example 4 Separation and purification process of extracellular polysaccharide produced by fermentation of Bacillus velezensis SN-1 strain

(1) Preparation of crude exopolysaccharide

The activated Bacillus velezensis (Bacillus velezensis) SN-1 bacterial solution was inoculated into the LB sugar-producing fermentation liquid medium at an inoculum of 6% (v/v), and cultured at 37 ° C and 120 rpm for 48 h to obtain exopolysaccharide. fermentation broth. Centrifuge 1000 mL of the fermentation broth at 4°C and 10000 rpm for 20 min to remove the bacterial cells. The supernatant was added with 3 volumes of pre-chilled 95% ethanol and incubated overnight at 4°C to precipitate polysaccharides. The polysaccharide precipitate was collected by centrifugation at 10,000 rpm for 20 min at 4°C. The obtained precipitate was dissolved in distilled water, and the protein was removed by the savage method (using the characteristics of protein denaturation in organic solvents such as trichloroethane, the extract was mixed with Sevage reagent [chloroform: normal]. Butanol = 5:1 (V/V)] 5:1 mixing, shaking, centrifugation, the denatured protein is between the junction of the extract and the Sevage reagent), put into a dialysis bag (molecular weight cut-off 14000Da), 4 ℃ Distilled water was dialyzed for 2 d, and the water was changed every 8 h. Finally, an aqueous solution of crude polysaccharide is obtained, which is freeze-dried and stored.

(2) Polysaccharide purification

The aqueous solution of the above crude polysaccharide was taken directly or moderately diluted and then subjected to gel filtration chromatography, and the polysaccharide solutions were combined and lyophilized for 24 hours to obtain pure exopolysaccharide. As shown in FIG. 3 , it is a gel filtration chromatogram of extracellular polysaccharide produced by Bacillus velezensis SN-1 strain. Purification was first performed by a DEAE-52 anion exchange column (Figure 3A). Two peaks were eluted. The first peak is strong, indicating the presence of most EPS. The first peak EPS was pooled, dialyzed, lyophilized and further purified on a G-100 gel column. A purified homogeneous polysaccharide fraction was finally obtained (Fig. 3B).

(3) Exopolysaccharide purity identification

Purity Analysis by UV-Vis Spectroscopy:

Accurately weigh 5 mg of pure exopolysaccharide, dissolve it in 10 mL of ultrapure water to prepare a 0.5 mg/mL polysaccharide solution, and perform ultraviolet scanning with an ultraviolet-visible spectrometer in the 200-400 nm band to detect the purity of the polysaccharide. The results are shown in Figure 4. Ultraviolet spectrum showed that exopolysaccharide showed lower absorbance below 190 nm after protein removal. While amino acids usually show absorbance in the range of 200-280 nm. It indicated that the extracted extracellular polysaccharide was uniform and free of protein contamination.

(4) Structure identification of exopolysaccharide

Monosaccharide composition analysis by high performance liquid chromatography: K-501 high performance liquid chromatography, KS-805Shodex sugar column and differential detector were used for the mobile phase at a column temperature of 60 °C, a flow rate of 1.0 mL/min, 0.1 mol/L, and NaNO ₃ , The sample mass concentration was 5.0 mg/mL and the injection volume was 10 μL. Comparison of the results with the monosaccharide standard (Fig. 5A) showed that EPS (Fig. 5B) was mainly composed of mannose.

Infrared Spectroscopic Structural Analysis: As shown in Figure 6, the EPS samples were scanned in the 800-4000 cm ^-1 spectral range using a Fourier transform infrared spectrometer. The results show that the absorption peaks at 3387.19cm ^-1 , 2928.24cm ^-1 , 1640cm ^-1 , 1400.91cm ^-1 and 1023.07cm ^-1 correspond to OH, CH, CO, CH and COC structures, respectively, thus confirming the carbon water in EPS the presence of compounds.

Nuclear Magnetic Resonance Structural Analysis: Dried exopolysaccharide samples were dissolved in _D2O at a concentration of 10 mg/mL, and an Ascend 500 instrument was used ^{for1H and13C} nuclear magnetic resonance (NMR) analysis. The exopolysaccharide can be deduced to contain α-glycosidic linkages by the ¹ H spectrum ( FIG. 8A ). The exopolysaccharide was inferred to be a polysaccharide with a branched structure of →4-Man-1→residues by ^the13C spectrum (Fig. 8B).

Scanning electron microscopy of purified exopolysaccharides is shown in Figure 7. The results show that EPS has an irregular structure of polysaccharides, and the smooth surface is conducive to the formation of biofilms, while EPS polymers with uniform surfaces can endow biofilms with mechanical stability.

Thermogravimetric analysis (TG) method: 3 mg of EPS was added to the Al ₂ O ₃ test box, placed in a thermal analyzer, and heated in the range of 25-700 °C at a rate of 10 °C/min. The results show that EPS has high thermal stability and strong heat resistance (Figure 9). In summary, through Fourier transform infrared spectroscopy (FT-IR), high performance liquid chromatography (HPLC), gel permeation chromatography (GPC) and nuclear magnetic resonance techniques. The structure analysis of the exopolysaccharide produced by the present invention shows that the exopolysaccharide of the present invention contains only one sugar unit, which is a mannose mainly connected by α-(1→4) glycosidic bonds, and does not contain branched chains . There is no protein and nucleic acid contamination, and the purity is 94.04%. The research based on thermogravimetric analysis (TG) found that exopolysaccharide has high thermal stability and strong heat resistance.

SEQUENCE LISTING

<110> Shenyang Agricultural University

<120> A strain of Bacillus velesi SN-1 and its fermentation method for producing exopolysaccharide

<130> 2020

<160> 3

<170> PatentIn version 3.5

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attactagcg attccagctt cacgcagtca agttgcaaac tgcgatccga actgaaaaca 180

gatttgtggg attggcttaa cctcgcggtt tcgctgccct ttgttctgtc cattgtagca 240

cgtgtgtagc ccaggtcata aggggcatga tgatttgacg tcatccccac cttcctccgg 300

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gccccccgtca attcctttga gtttcagtct tgcgaccgta ctccccaggc ggagtgctta 600

atgcgttagc tgcagcacta aggggcggaa accccctaac acttagcact catcgtttac 660

ggcgtggact accagggtat ctaatcctgt tcgctcccca cgctttcgct cctcagcgtc 720

agttacagac cagagagtcg ccttcgccac tggtgttcct ccacatctct acgcatttca 780

ccgctacacg tggaattcca ctctcctctt ctgcactcaa gttccccagt ttccaatgac 840

cctccccggt tgagccgggg gctttcacat cagacttaag aaaccgcctg cgagcccttt 900

acgcccaata attccggaca acgcttgcca cctacgtatt accgcggctg ctggcacgta 960

gttagccgtg gctttctggt taggtaccgt caaggtgccg ccctatttga acggcacttg 1020

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gaagccacct tttatgtctg aaccatgcgg ttcaaacaac catccggtat tagccccggt 1320

ttcccggagt tatcccagtc ttacaggcag gttacccacg tgttactcac ccgtccgccg 1380

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Claims (10)

1. Bacillus velezensis SN-1 is preserved in China general microbiological culture collection center at 08.06.2020 with the preservation number of CGMCC No. 1.18401.

2. A method for producing exopolysaccharide by Bacillus velezensis (Bacillus velezensis) SN-1 fermentation is characterized by comprising the following steps:

(1) inoculating Bacillus subtilis SN-1 of claim 1 into LB liquid medium containing sugar, and culturing for a certain period of time to obtain a fermentation liquid;

(2) centrifuging the fermentation liquor, removing thalli, and taking supernatant;

(3) precipitating the supernatant after removing the thalli by an ethanol precipitation method, standing, centrifuging, taking the precipitate, dissolving the precipitate in water, removing protein by a savage method, and dialyzing to obtain a crude polysaccharide aqueous solution;

(4) and purifying the crude polysaccharide water solution to obtain pure exopolysaccharide.

3. The method according to claim 2, wherein the LB liquid medium in step (1) contains 2 to 5 wt% of sugar; the sugar comprises sucrose, glucose or maltose.

4. The method according to claim 2, wherein the inoculation amount of Bacillus subtilis SN-1 in step (1) is 5 to 10% (v/v).

5. The method according to claim 2, wherein the sugar-containing LB liquid medium in step (1) has a pH of 6 to 8 and is cultured under the conditions: culturing at 25-37 deg.C and shaking table rotation speed of 100-.

6. The method as claimed in claim 2, wherein the centrifugation temperature in step (2) is 4-30 ℃, the centrifugation speed is 10000-12000rpm, and the centrifugation time is 15-20 min.

7. The method according to claim 2, wherein the ethanol concentration in step (3) is 95-100% by volume, and the ethanol precipitation method is performed in which the volume of ethanol is 3-5 times that of the supernatant; standing at 4-8 deg.C for 12-24 hr; the conditions of centrifugation were: centrifuging at 10000-12000rpm for 20-40min at 4-8 ℃.

8. The method of claim 2, wherein the dialysis of step (3) has a molecular weight cut-off of 14000Da and the dialysis conditions are: dialyzing with 4-8 deg.C distilled water for 2-3d, and changing distilled water every 5-8 h.

9. The method of claim 2, wherein the purification of step (4) is performed by gel filtration chromatography to purify the crude polysaccharide.

10. The method according to claim 2, wherein the exopolysaccharide is a linear mannose linked by α - (1 → 4) glycosidic bonds.

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