CN111621448A

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

Info

Publication number: CN111621448A
Application number: CN202010632553.4A
Authority: CN
Inventors: 乌日娜; 武俊瑞; 曹承旭
Original assignee: Shenyang Agricultural University
Current assignee: Shenyang Agricultural University
Priority date: 2020-07-03
Filing date: 2020-07-03
Publication date: 2020-09-04

Abstract

The invention discloses a Bacillus belgii SN-1 strain and a method for producing exopolysaccharide by fermenting the same, and belongs to the field of microbiology. The invention is separated from northeast traditional naturally fermented soybean paste and is found to produce extracellular polysaccharide with excellent characteristics. Inoculating Bacillus velezensis (Bacillus velezensis) SN-1 into LB basic culture medium containing 2% sucrose, and culturing for 48-72h under the conditions that the pH value of the initial culture medium is 7, the temperature is 37 ℃, and the rotating speed of a shaking table is 100 plus 200rpm to obtain fermentation liquor; purifying the crude polysaccharide to obtain extracellular polysaccharide. The strain and the fermentation production method have the advantages of short sugar production period, high sugar yield, safety and no toxic or side effect, and the extracellular polysaccharide has higher emulsibility and thermal stability, provides theoretical basis and basis for industrial application, and has wide commercial application prospect.

Description

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

Technical Field

The invention relates to a Bacillus belgii SN-1 strain and a method for producing exopolysaccharide by fermenting the same, and belongs to the field of microbiology.

Background

Exopolysaccharides (EPS) are mucopolysaccharides or capsular polysaccharides secreted by microorganisms (bacteria, yeasts, fungi) outside the cells during their growth metabolism. With the intensive research on plant, animal and fungal polysaccharides, there is an urgent need to develop microbial extracellular polysaccharides having novel properties and high yield from edible microorganisms. The microbial exopolysaccharide has the advantages of wide source, mild reaction conditions, easy separation and purification and the like, and is always a main source in the theoretical and practical research of exopolysaccharide.

To date, a large number of commercial EPS (dextran, alginate, xanthan and gellan) of microbial origin 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 monascampenostis, and may risk contamination with bacterial endotoxins. Since exopolysaccharides have various properties and are widely used, more and more researchers are working on developing microorganisms having novel characteristics and high EPS production. Meanwhile, the development and utilization of food grade EPS from food microorganisms has become one of the hot spots of industrial microorganism research. Bacillus exopolysaccharides have been used as stabilizers and bioflocculants in the production of pharmaceuticals, cosmetics, and the like. In addition, the exopolysaccharide of the bacillus has excellent physicochemical and biological properties, such as a biological emulsifier, a heavy metal chelating agent, an antiviral agent and an immunomodulator, and can be used as a prebiotic to promote the growth of beneficial bacteria in intestinal tracts, regulate the balance of intestinal flora and promote the health of hosts. Therefore, compared with other polysaccharides, the research of the bacillus exopolysaccharide has more practical application value, the bacillus has strong vitality and lower requirement on culture conditions, and the extraction process of the bacillus exopolysaccharide is simpler, has higher safety and low price, and is more suitable for human beings compared with other microbial polysaccharides. There have been many studies reporting on EPS producing lactic acid bacteria strains found in nature, but the problem of low yield is common. Therefore, the method fully excavates abundant bacillus resources in the nature, finds bacterial strains with high extracellular polysaccharide yield, obtains more extracellular polysaccharide with short period and low cost, and is a research hotspot in the field of microbiology.

Disclosure of Invention

The invention aims to provide a bacterial strain for high yield of linear chain mannose and a fermentation production method thereof.

The invention provides a Bacillus velezensis (Bacillus velezensis) SN-1 which is preserved in China general microbiological culture collection center at 08.06.2020, with the preservation number of CGMCC No. 1.18401.

A high-yield linear-chain mannose strain, which is Bacillus velezensis (Bacillus velezensis) SN-1, is separated from traditional naturally fermented soybean paste in northeast China and is found to produce exopolysaccharides with excellent characteristics. The Bacillus is proved to be the Bacillus belief by morphological and physiological biochemical experiments, is named as Bacillus belief SN-1(Bacillus velezensis SN-1), is preserved in China general microbiological culture collection center at 2020 and 08 months, and has the preservation number of CGMCC No. 1.18401.

In order to further verify the species of the strain and further study the structure and the properties of the exopolysaccharide produced by the strain, 16s rDNA sequence analysis is carried out (SEQ ID NO. 2: upstream primer 8F: 5-AGAGTTTGATCCTGGCTCAG-3; SEQ ID NO. 3: downstream primer 1492: 5-CTACGGCTACCTTGTTACGA-3); the result of the 16s rDNA gene sequencing of the strain is shown in a sequence table SEQ ID NO.1.

A method for producing exopolysaccharide by Bacillus velezensis (Bacillus velezensis) SN-1 fermentation comprises the following steps:

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

(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.

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

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

Further, in the above technical scheme, the pH value of the sugar-containing LB liquid medium in step (1) is 6 to 8, and the culture conditions are: culturing at 25-37 deg.C and shaking table rotation speed of 100-.

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

Further, in the above technical solution, the volume percentage concentration of the ethanol in the step (3) is 95-100%, and the volume of the ethanol in the ethanol precipitation method is 3-5 times of the volume of the supernatant; standing at 4-8 deg.C for 12-14 h; the conditions of centrifugation were: centrifuging at 10000-12000rpm for 20-40min at 4-8 ℃.

Further, in the above technical solution, the cut-off molecular weight of the dialysis in step (3) is 14000Da, and the dialysis conditions are as follows: dialyzing with 4-8 deg.C distilled water for 2-3d, and changing distilled water every 5-8 h.

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

Further, in the technical scheme, the exopolysaccharide is linear mannose connected by alpha- (1 → 4) glycosidic bond.

By Fourier 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 invention shows that: the exopolysaccharide only contains one sugar unit, is mannose mainly connected by alpha- (1 → 4) glycosidic bond, and does not contain a branched chain. The research based on thermogravimetric analysis (TG) and physicochemical properties proves that the exopolysaccharide has higher emulsibility and thermal stability and strong heat resistance (the degradation temperature is 270.7 ℃).

The invention has the beneficial effects that: the invention obtains extracellular polysaccharide of a strain fermentation product by a series of biochemical techniques, and extracts and purifies fermentation liquor by adopting a modern extraction technique to obtain high-purity polysaccharide. A series of experiments prove that the obtained strain has the characteristics of bacillus and can produce a large amount of extracellular polysaccharide under the induction of a substrate sucrose. At present, the yield of the microbial EPS is about 4.5g/L, the yield of the Bacillus velezensis SN-1EPS can reach 12.7g/L, and is improved by 2.82 times compared with other microbial EPS. Therefore, the fermentation liquor obtained by fermenting B.velezensis SN-1 in the sugar-producing fermentation medium contains the exopolysaccharide, has the advantages of short sugar-producing period, high sugar-producing amount, safety and no toxic or side effect, and the exopolysaccharide has higher emulsibility and thermal stability, provides theoretical basis and basis for industrial application, and has wide commercial application prospect.

Drawings

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

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

FIG. 3 is a DEAE-52 anion exchange column chromatography (A) and G-100 gel column chromatography (B) of Bacillus velezensis SN-1 for exopolysaccharide production.

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

FIG. 5 is a high performance liquid chromatogram (B) of Bacillus velezensis (Bacillus velezensis) SN-1 for producing pure exopolysaccharides and a high performance liquid chromatogram (A) of monosaccharide standard.

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

FIG. 7 is a scanning electron micrograph of pure exopolysaccharides produced by Bacillus velezensis SN-1, with a scale of 100 μm (A); scale bar 10 μm (B).

FIG. 8 shows that Bacillus velezensis SN-1 produces pure exopolysaccharides¹H NMR spectra (A) and¹³C NMR(B)。

FIG. 9 is a thermogravimetric plot of pure exopolysaccharides produced by Bacillus velezensis (Bacillus velezensis) SN-1.

Detailed Description

The following non-limiting examples will allow one of ordinary skill in the art to more fully understand the present invention, but are not intended to limit the invention in any way.

Example 1 isolation screening of Bacillus velezensis SN-1

(1) Culture medium

LB liquid medium: tryptone 10g, yeast powder 5g, KH₂PO₄1.6g，K₂HPO₄2g，MgSO₄·7H₂O0.25g，CaSO₄·2H₂O 1g，FeCl₃2g, 1000mL of distilled water, pH 7.0, and sterilization at 115 ℃ for 20 min.

LB solid agar Medium: 15-20 g of agar powder is added into an LB liquid culture medium, and the mixture is sterilized for 20min at the temperature of 115 ℃.

LB sugar-producing fermentation liquid culture medium: 50g of cane sugar, 10g of tryptone, 5g of yeast powder and KH₂PO₄1.6g，K₂HPO₄2g，MgSO₄·7H₂O 0.25g，CaSO₄·2H₂O 1g，FeCl₃2g, 1000mL of distilled water, pH 7.0, and sterilization at 115 ℃ for 20 min.

(2) Sample pretreatment

Weighing 1g of northeast natural fermented farmyard sauce (the northeast natural fermented farmyard sauce is prepared by cleaning and cooking soybeans, crushing and making into sauce blocks, naturally fermenting in a shady and ventilated place, cleaning and removing the fimbriae on the surfaces of the sauce blocks after fermentation, crushing into small blocks, exposing in the sun, then putting into a sauce jar, adding cold water and edible iodized salt, sealing, and naturally fermenting in a place with sufficient sunlight). Taking appropriate amount of solution from the above treatment to make into 10^-1Diluting the sample solution to 10 degrees^-2、10^-3Standing for later use.

(3) Primary screen for producing slime colonies

And (3) taking 100 mu L of the bacterial liquid with each dilution, coating the bacterial liquid with each dilution on an LB solid agar culture medium plate, and performing inverted culture in a constant temperature incubator at 37 ℃ for 24-48 h. Picking the colony area on the plate, repeatedly marking on the LB solid agar medium plate to obtain pure culture plate, and storing in 4 deg.C refrigerator.

(4) Bacterial strain rescreening

The pure culture obtained from the plate was inoculated into LB liquid medium for 18 hours, further inoculated into LB sugar-producing fermentation liquid medium in an amount of 2% (v/v), and cultured at 37 ℃ for 48 hours. Heating the fermentation liquid in 90 deg.C water bath for 10min, removing enzyme in the bacterial liquid which may degrade polysaccharide, and cooling to room temperature. Adding 80% trichloroacetic acid (TCA) solution into the fermentation broth to final concentration of 5% (m/v), stirring at room temperature for 2h, centrifuging at 10000 Xg at 4 deg.C for 20min, and removing cell and protein precipitate. The supernatant was put into dialysis bag (molecular weight cut-off 14000Da), dialyzed with ultrapure water in refrigerator at 4 ℃ for 2d, and water was changed every 8 h. And (4) after the volume is fixed, measuring the yield of the extracellular polysaccharide produced by the strain. And selecting the strain with higher polysaccharide yield by combining the mucus concentration of the mucus-producing colony.

Example 2 identification of Bacillus velezensis SN-1

(1) Morphological characterization of SN-1 strains

Inoculating SN-1 strain to LB plate, three-zone streaking, culturing at 37 deg.c for 24-48 hr, observing and recording the single colony characteristic of the strain on the plate. SN-1 colony morphology is shown in FIG. 1A, and the results of colony morphology observations are summarized in Table 1. A small amount of fresh cells were picked up with an inoculating needle and spread on a clean slide glass for gram staining, and then the individual morphology and arrangement of cells were observed under a microscope (FIG. 1B).

TABLE 1 summary of morphological observations of SN-1 strains

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

① is connected toAnd (3) catalase test: selecting a small amount of bacterial strains with positive gram staining results, streaking and inoculating the bacterial strains on an LB solid culture medium plate, culturing the bacterial strains for 24 hours at 37 ℃, and dropwise adding H with the mass fraction of 10%₂O₂And observing the bubble generation condition on the bacterial colony, wherein the bubble generation condition is positive for the catalase. As a result, the SN-1 strain was negative in the catalase reaction, as shown in Table 2.

② test for acid production and gas production of glucose in liquid fermentation culture medium (peptone 2.7g, sodium chloride 5.0g, 0.2% bromothymol blue 0.03g, agar 3g, KH)₂PO₄0.3g, 10.0g of glucose and 1000mL of water) is added with a bromocresol purple indicator and an inverted Duchen small tube, an SN-1 strain is inoculated into the culture medium according to the inoculation amount of 1% (v/v), the test tube is gently shaken to be uniform, and the inverted small tube is prevented from entering bubbles. After culturing for 48h at 37 ℃, the observation result shows that the indicator color in the culture medium changes from purple to yellow to indicate that SN-1 can produce acid by using glucose, if the indicator color is purple, the indicator color indicates that acid is not produced, and if bubbles are produced in the Du's tubule, the indicator color indicates that the SN-1 strain can produce gas by using glucose. The results are shown in Table 2, and indicate that SN-1 strain can produce acid and gas by using glucose.

③ producing H₂S test: the SN-1 strain was inoculated by puncturing with an inoculating needle into a lead acetate medium (peptone 10.0g/L, beef extract 3.0g/L, sodium chloride 5.0g/L, sodium thiosulfate 2.5g/L, agar 12.0g/L, pH 7.3. + -. 0.1(25 ℃ C.)), cultured at 37 ℃ for 48 hours, and the presence or absence of black lead sulfide was observed. The results are shown in Table 2 and indicate that SN-1 strain is unable to produce H₂S。

Starch hydrolysis test: line drawing of the SN-1 strain is inoculated to a starch culture medium (0.5 g of beef extract, 1g of peptone, 0.5g of sodium chloride, 0.2g of soluble starch, 100mL of water, pH 7.0-7.2, 2g of agar, sterilization at 115 ℃, 0.1Mpa and 20min), and the plate is placed in a 37 ℃ incubator for 24 hours. And observing the growth condition of the strain, and then, dripping a small amount of Lugol iodine solution on the flat plate, wherein a colorless transparent ring appears around the bacterial colony to indicate that the starch is completely hydrolyzed, and the strain has the capability of decomposing the starch. The results are shown in Table 2.

Producing indole experiment: inoculating the SN-1 strain into a peptone water culture medium (peptone 1g, sodium chloride 0.5g, water added to 100mL, pH adjusted to 7.8), adding 1-2mL of diethyl ether into the culture solution at 37 ℃, standing for a moment, floating the diethyl ether layer on the upper surface of the culture solution, slowly adding 5-10 drops of indole reagent along the tube wall, and if indole exists, the diethyl ether layer is rose red, which is a positive reaction of an indole test, otherwise, the diethyl ether layer is a negative reaction. The results are shown in Table 2.

Gel liquefaction test: taking a gelatin culture medium test tube (5 g of NaCl, 10g of peptone, 3g of beef extract, 120g of gelatin, 1000mL of distilled water and pH 7.2-7.4), inoculating the SN-1 strain by puncturing with an inoculating needle, culturing the inoculated test tube at 20 ℃ for 3-5 days, and observing the liquefaction condition of gelatin. The results are shown in Table 2.

Seventh, the motility is: using the hanging drop method, a drop of bacterial suspension was suspended on the cover glass of a groove slide, and its motility was observed under a weak light by a normal microscope. The results are shown in Table 2.

TABLE 2 physiological and biochemical identification results of SN-1 strains

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

Fermentation test of sugar: respectively replacing glucose in an LB (Luria Bertani) sugar-producing fermentation liquid culture medium with sucrose, L-arabinose, fructose, galactose, lactose, mannose, maltose and rhamnose to prepare different sugar culture media, adding a bromocresol purple indicator, inoculating 1% (v/v) SN-1 strain, marking, and performing no inoculation on a blank control. After culturing for 48h at 37 ℃, the observation result shows that the SN-1 strain can produce acid by utilizing the sugar and is a positive reaction if the indicator in the culture medium turns yellow. The test results are shown in Table 3, and the test shows that the SN-1 strain can produce acid by utilizing sucrose, mannose, maltose and rhamnose.

TABLE 3 SN-1 Strain sugar fermentation test results

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

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

Extraction of bacterial genomic DNA

100 mu L of SN-1 strain suspension is sucked into sterilized LB liquid culture medium and cultured for 24h in a shaking incubator at 37 ℃. The genomic DNA of SN-14-3 was extracted according to the procedures on the bacterial genome kit (Solambio D1600).

② PCR amplification

The PCR primers were designed as follows:

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

SEQ ID NO. 3: a downstream primer 1492R: 5'-CTACGGCTACCTTGTTACGA-3', primers were synthesized and shipped by Shanghai Senno bioengineering.

The PCR reaction conditions are as follows: preheating at 95 deg.C for 5min, denaturation at 95 deg.C for 30s, annealing at 55 deg.C for 30s, extension at 72 deg.C for 90s, circulating for 35 times, maintaining at 72 deg.C for 7min, and keeping at 4 deg.C.

Recovery of PCR product

The PCR product was recovered using AxyPrep DNA gel recovery kit (Boyao ASJ0013), the detailed procedure was performed according to the kit instructions.

Fourthly, 16S rDNA sequencing and sequence comparison

And (3) feeding the positive PCR product to Shanghai Senno for sequencing, comparing and analyzing the sequencing result with the existing sequence of the GenBank database in an NCBI database by using a BLAST tool, analyzing the homology of the strain to be detected and the corresponding sequence of the known strain, and determining the species of the screened sugar-producing strain. The phylogenetic tree of SN-1 strains based on the 16S rDNA gene sequence is shown in FIG. 2.

And (3) confirming that the SN-1 is Bacillus velezensis (Bacillus velezensis) by combining the morphological, physiological and biochemical properties and the 16S rDNA sequence homology comparison result of the SN-1 strain.

The strain is preserved in the China general microbiological culture Collection center (CGMCC for short, the address: No.3 of Xilu No.1 of Beijing city morning area, microbial research institute of Chinese academy of sciences, zip code 100101). The preservation number of the Bacillus velezensis SN-1 is CGMCC No.1.18401, the preservation date is 2020, 06 and 08 days, the Bacillus velezensis is named by classification, and the strain name is SN-1.

Example 3 fermentation Process for the production of exopolysaccharides by fermentation of Bacillus velezensis SN-1 Strain

The fermentation process for fermenting the high-yield exopolysaccharide strain by using the Bacillus velezensis SN-1 strain comprises the following steps of:

(1) preparation of seed liquid

Inoculating Bacillus velezensis (Bacillus velezensis) SN-1 into LB liquid culture medium to make initial cell concentration in the culture medium 1.0 × 10⁸CFU/mL, namely the seed liquid.

(2) Sugar-producing fermentation conditions

Inoculating Bacillus velezensis (Bacillus velezensis) SN-1 into LB sugar-producing fermentation liquid culture medium with initial pH value of 7 in an inoculation amount of 0.5% (V/V), performing shake culture at constant temperature of 37 ℃ and 100rpm for 36-72h, and measuring polysaccharide content in fermentation supernatant by using phenol-sulfuric acid method.

Inoculating Bacillus velezensis (Bacillus velezensis) SN-1 into LB sugar-producing fermentation liquid culture medium with initial pH value of 7 in an inoculation amount of 0.5% (V/V), culturing for 48h at 37 ℃ and shaking table rotation speed of 100rpm to obtain fermentation liquid, centrifuging the fermentation liquid at 4 ℃ and 10000rpm for 20min, and removing thallus. The supernatant was added with 3 volumes of pre-cooled 95% ethanol and incubated overnight at 4 ℃ to precipitate the polysaccharide. Centrifuging at 4 deg.C and 10000rpm for 20min to collect polysaccharide precipitate, dissolving polysaccharide precipitate with ultrapure water, adding 10% trichloroacetic acid solution with the same volume as ultrapure water for dissolving polysaccharide to remove protein, standing at 4 deg.C for 10 hr, centrifuging at 4 deg.C and 10000rpm for 20min to collect supernatant. Add 3 volumes of pre-cooled 95% ethanol and incubate overnight at 4 ℃ to precipitate the polysaccharide. Centrifuging at 4 deg.C and 10000rpm for 20min to collect polysaccharide precipitate, redissolving in ultrapure water (about 200mL), filling into dialysis bag (molecular weight cut-off of 14000Da), dialyzing with 4 deg.C distilled water for 2d, and changing water every 8 h. And freeze-drying to obtain the exopolysaccharide. The content of exopolysaccharide obtained in the fermentation broth obtained in this example was 12.7 g/L.

Example 4 separation and purification Process for exopolysaccharide production by fermentation of Bacillus velezensis SN-1 Strain

(1) Crude exopolysaccharide preparation

Inoculating the activated Bacillus velezensis (Bacillus velezensis) SN-1 bacterial liquid into an LB sugar-producing fermentation liquid culture medium according to the inoculation amount of 6% (v/v), and culturing at 37 ℃ and 120rpm for 48h to obtain the extracellular polysaccharide fermentation liquid. 1000mL of the fermentation broth was centrifuged at 10000rpm at 4 ℃ for 20min to remove the cells. The supernatant was added with 3 volumes of pre-cooled 95% ethanol and incubated overnight at 4 ℃ to precipitate the polysaccharide. Centrifuging at 4 deg.C and 10000rpm for 20min to collect polysaccharide precipitate, dissolving the obtained precipitate with distilled water, removing protein by savage method (using protein denaturation in organic solvent such as trichloroethane, mixing the extractive solution with Sevage reagent [ chloroform: n-butanol ═ 5: 1(V/V) ] 5: 1, oscillating, centrifuging, and placing into dialysis bag (molecular weight cut-off of 14000Da) after denatured protein is at the junction of extractive solution and Sevage reagent), dialyzing with distilled water at 4 deg.C for 2d, and changing water every 8 h. Finally obtaining the water solution of crude polysaccharide, and freeze-drying and storing.

(2) Polysaccharide purification

Directly or moderately diluting the water solution of the crude polysaccharide, performing gel filtration chromatography, combining and collecting the polysaccharide solution of the pipe, and freeze-drying for 24h to obtain pure extracellular polysaccharide, wherein the purified extracellular polysaccharide content is 73.75% by using a phenol-sulfuric acid method. As shown in FIG. 3, it is an exopolysaccharide gel filtration chromatogram produced by Bacillus velezensis (Bacillus velezensis) SN-1 strain. First, purification was performed by passing through a DEAE-52 anion exchange column (FIG. 3A). Two peaks eluted. The first peak is strong indicating the presence of most EPS. The first peak of EPS was pooled, dialyzed, freeze-dried and further purified on a G-100 gel column. Finally, a purified homogeneous polysaccharide fraction was obtained (FIG. 3B).

(3) Purity identification of extracellular polysaccharide

Purity analysis by ultraviolet-visible spectroscopy:

accurately weighing 5mg of pure extracellular polysaccharide, dissolving the pure extracellular polysaccharide in 10mL of ultrapure water to prepare 0.5mg/mL of polysaccharide solution, and performing ultraviolet scanning by using an ultraviolet visible spectrometer at a wavelength of 200-400nm to detect the purity of the polysaccharide. The results are shown in FIG. 4. The uv spectrum shows that the exopolysaccharide shows lower absorbance below 190nm after deproteinization. Whereas amino acids typically show absorbance in the 200-280nm range. Indicating that the exopolysaccharide component is uniform and free from protein pollution.

(4) Extracellular polysaccharide structure identification

High performance liquid chromatography monosaccharide composition analysis: using K-501 high performance liquid chromatography, KS-805Shodex sugar column and differential detector pair at column temperature of 60 deg.C, flow rate of 1.0mL/min, 0.1mol/L, NaNO₃The test was carried out under the conditions of a mobile phase, a sample mass concentration of 5.0mg/mL, and a sample introduction amount of 10. mu.L. Comparison of the results with the monosaccharide standard (fig. 5A) shows that EPS (fig. 5B) is composed mainly of mannose.

Structural analysis by infrared spectroscopy: as shown in FIG. 6, a Fourier Infrared spectrometer was used at 800 and 4000cm^-1The EPS samples were scanned over the spectral range. The result showed that the absorption peak was 3387.19cm^-1、2928.24cm^-1、1640cm^-1、1400.91cm^-1And 1023.07cm^-1Which correspond to the structures O-H, C-H, C-O, C-H and C-O-C, respectively, thus confirming the presence of carbohydrates in EPS.

Structural analysis by nuclear magnetic resonance method: dissolving the dried exopolysaccharide sample in D at a concentration of 10mg/mL₂In O, using an Ascend 500 instrument¹H and¹³c Nuclear Magnetic Resonance (NMR) analysis. By passing¹The H spectrum can infer that the exopolysaccharide contains α -glycosidic bond (FIG. 8A)¹³The C spectrum suggests that the exopolysaccharide is a polysaccharide having a → 4-Man-1 → residue branch structure (FIG. 8B).

The scanning electron microscope of the purified exopolysaccharide is shown in fig. 7, and the result shows that the EPS has an irregular structure of the polysaccharide, the surface is smooth, the formation of a biological membrane is facilitated, and the EPS polymer with a uniform surface can endow the biological membrane with mechanical stability.

Thermogravimetric analysis (TG) method: 3mg EPS was added to Al₂O₃The test cell was placed in a thermal analyzer and heated at a rate of 10 ℃/min in the range of 25-700 ℃. The results show that EPS has high thermal stability and high heat resistance (FIG. 9). To sum upThe extracellular polysaccharide produced by the method is subjected to structural analysis through Fourier infrared spectroscopy (FT-IR), High Performance Liquid Chromatography (HPLC), Gel Permeation Chromatography (GPC) and nuclear magnetic resonance technology, and the result shows that the extracellular polysaccharide only contains one sugar unit, is mannose mainly connected by α - (1 → 4) glycosidic bonds, does not contain branched chains, has no protein and nucleic acid pollution, and has purity of 94.04%.

SEQUENCE LISTING

<110> Shenyang agriculture university

<120> Bacillus belgii SN-1 and method for producing exopolysaccharide by fermentation of Bacillus belgii SN-1

<130>2020

<160>3

<170>PatentIn version 3.5

<210>1

<211>1467

<212>DNA

<213> Bacillus belgii SN-1(Bacillus velezensis SN-1)

<400>1

ccttcggcgg ctggctccta aaggttacct caccgacttc gggtgttaca aactctcgtg 60

gggtgacggg cggggtgtac aaggcccggg aacgtattca ccgcggcatg ctgatccgcg 120

attactagcg attccagctt cacgcagtca agttgcaaac tgcgatccga actgaaaaca 180

gatttgtggg attggcttaa cctcgcggtt tcgctgccct ttgttctgtc cattgtagca 240

cgtgtgtagc ccaggtcata aggggcatga tgatttgacg tcatccccac cttcctccgg 300

tttgtcaccg gcagtcacct taaagtgccc aactgaatgc tggcaactaa aatcaagggt 360

tgcgctcgtt gcggaactta acccaacatc tcacgacacg agctgacaac aaccatgcac 420

cacctgtcac tctgcccccg aaggggacgt cctatctcta ggattgtcaa aggatgtcaa 480

gacctggtaa ggttcttcgc gttgcttcaa attaaaccac atgctccacc gcttgtgcgg 540

gcccccgtca 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

ttcttcccta acaacagagc tttacgatcc gaaaaccttc atcactcacg cggcgttgct 1080

ccgtcagact ttcgtccatt gcggaagatt ccctactgct gcctcccgta ggagtctggg 1140

ccgtgtctca gtcccagtgt ggccgatcac cctctcaggt cggctacgca tcgtcgcctt 1200

ggtgagccgt tacctcacca actagctaat gcgccgcggg tccatctgta agtggtagcc 1260

gaagccacct tttatgtctg aaccatgcgg ttcaaacaac catccggtat tagccccggt 1320

ttcccggagt tatcccagtc ttacaggcag gttacccacg tgttactcac ccgtccgccg 1380

ctaacatcag ggagcaagct cccatctgtc cgctcgactt gcatgtatta agcacgccgc 1440

cagcgttcgt cctgagccag gatcaaa 1467

<210>2

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>2

agagtttgat cctggctcag 20

<210>3

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>3

ctacggctac cttgttacga 20

Claims

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;

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.