CN113151071B

CN113151071B - Bacillus belgii and application thereof

Info

Publication number: CN113151071B
Application number: CN202110367374.7A
Authority: CN
Inventors: 高洁; 吴凯璋; 方芳; 陆星宇; 万楚; 赵裕民
Original assignee: Guangxi University
Current assignee: Guangxi University
Priority date: 2021-04-06
Filing date: 2021-04-06
Publication date: 2023-03-24
Anticipated expiration: 2041-04-06
Also published as: CN113151071A

Abstract

The invention discloses a strain of Bacillus belgii, named as Bacillus velezensis M76T11B, which is preserved in Guangdong province microorganism strain preservation center in 12 and 23 days in 2020, wherein the preservation address is No. 9 building 5 building of Jieli Middy No. 100 college in Guangzhou city, and the preservation number is GDMCC NO:61384. the screened secondary metabolite of Bacillus velezensis M76T11B is exopolysaccharide which mainly comprises rhamnose and galacturonic acid, the exopolysaccharide produced by the strain has the advantages of short production period, low cost, high yield, controllable process and wide application prospect in the fields of materials, cosmetics and medicines.

Description

Bacillus belgii and application thereof

Technical Field

The invention relates to the technical field of microorganisms, in particular to a bacillus beiLeisi and application thereof.

Background

The extracellular polysaccharide of the bacillus beleisis is a secondary metabolite which is generated by fermentation of the bacillus beleisis in a growth metabolic process and secreted outside a cell wall, and can be divided into two types according to the strength of the binding capacity of the bacillus beleisis and the cell wall, wherein one type loses the binding capacity with the cell wall and permeates a culture medium to form mucus, and is called as mucus polysaccharide; the other adheres to the cell wall to form a capsule, called capsular polysaccharide. The exopolysaccharide is polymerized by hundreds or even thousands of monosaccharide repeating units with the same structure and 3-8. In the natural environment, exopolysaccharides generally play a role in protecting microorganisms, and can prevent cells from desiccation and dehydration, infection by bacteriophage, damage by antibiotics or toxic substances, damage by protozoa, and also stabilize osmotic pressure, support cell structures, participate in cell information transmission and cell formation, and the like.

The exopolysaccharide has wide application value in many fields, such as thickening agent, suspending agent, emulsifying agent, biological flocculating agent, anticancer medicine, packaging material and the like. The extracellular polysaccharide has the effects of resisting tumor activity, activating immunity, reducing serum cholesterol, prebiotics and the like, has recognized safety, and has great application potential in the fields of food, materials, cosmetics and medical industry.

Disclosure of Invention

The invention aims to screen a Bacillus beiLeisi with high extracellular polysaccharide (rhamnogalacturonan), research the application of the strain, separate and extract the extracellular polysaccharide produced by the strain, and can be used in the fields of materials, cosmetics and medicines.

In order to achieve the purpose, the technical scheme provided by the invention is as follows:

a strain of Bacillus belgii is named as Bacillus velezensis M76T11B, is preserved in Guangdong province microorganism strain preservation center in 12 and 23 months in 2020, is preserved in Guangzhou No. 9 Lou 5 of Miehu No. 100 of Mieli Zhonglu, guangzhou city, and has the preservation number of GDMCC NO:61384, and the gene sequence of Bacillus velezensis M76T11B16S rDNA is shown in SEQ ID NO. 1.

The application of the Bacillus velezensis M76T11B in the production of exopolysaccharides.

Preferably, the exopolysaccharide is rhamnogalacturonan.

The method for producing the exopolysaccharide by the Bacillus velezensis M76T11B comprises the following steps: performing three-region streak activation on the Bacillus velezensis M76T11B on an MRS solid culture medium plate to activate strains, performing static culture at 37 ℃ for 48h, then inoculating the strains into an MRS liquid culture medium, continuing the static culture at 37 ℃ for 24h, then inoculating the strains after the first generation activation into a 1000 mM MRS liquid culture medium in an inoculation amount of 2% (v/v), and performing shake culture at 37 ℃ and 100rpm for 24h to obtain fermentation liquor.

Preferably, the MRS solid medium: 10g/L of peptone, 5g/L of beef extract, 4g/L of yeast powder, 20g/L of glucose, 2g/L of dipotassium phosphate, 2g/L of ammonium citrate tribasic, 5g/L of sodium acetate, 0.2g/L of magnesium sulfate, 80 g/L of tween, 0.05g/L of manganese sulfate, 15g/L of agar and 6.2 of pH value.

Preferably, the MRS liquid medium: 10g/L of peptone, 5g/L of beef extract, 4g/L of yeast powder, 20g/L of glucose, 2g/L of dipotassium phosphate, 2g/L of ammonium citrate tribasic, 5g/L of sodium acetate, 0.2g/L of magnesium sulfate, 80 g/L of tween, 0.05g/L of manganese sulfate and 6.2 of pH value.

Preferably, the MRS-S medium: 10g/L of peptone, 5g/L of beef extract, 4g/L of yeast powder, 5g/L of glucose, 2g/L of dipotassium phosphate, 2g/L of ammonium citrate tribasic, 5g/L of sodium acetate, 0.2g/L of magnesium sulfate, 80 g/L of tween, 0.05g/L of manganese sulfate, 50g/L of sucrose and 6.2 of pH value.

Compared with the prior art, the invention has the following beneficial technical effects:

the secondary metabolite of the screened Bacillus velezensis M76T11B is exopolysaccharide which is rhamnogalacturonan, and the rhamnogalacturonan mainly comprises rhamnose and galacturonan.

Description of preservation information

Bacillus velezensis M76T11B is preserved in Guangdong province microorganism strain collection center in 2020, 12 and 23 days, and the preservation number is GDMCC NO:61384.

drawings

FIG. 1 is a diagram of Bacillus velezensis M76T11B colony; wherein A is a top view and B is a side view.

FIG. 2 is a gram stain of Bacillus velezensis M76T11B; wherein, the A magnification is 400, and the B magnification is 1000.

FIG. 3 is a GPC chart of EPS of Bacillus velezensis M76T11B.

FIG. 4 shows the crude polysaccharide from Bacillus velezensis M76T11B.

FIG. 5 is a graph showing the results of measurement of monosaccharide standards.

FIG. 6 is a graph showing the results of the monosaccharide composition of EPS of Bacillus velezensis M76T11B.

Detailed Description

The following detailed description is to be read with reference to the accompanying drawings, but it is to be understood that the scope of the invention is not limited to the specific embodiments. The raw materials and reagents used in the examples were all commercially available unless otherwise specified. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. In the quantitative experiments in the following examples, three replicates were set up and the results averaged.

Bacillus velezensis M76T11B is Bacillus belief (Bacillus velezensis) M76T11B.

The media used in the examples and their composition were:

MRS solid medium: 10g/L of peptone, 5g/L of beef extract, 4g/L of yeast powder, 20g/L of glucose, 2g/L of dipotassium phosphate, 2g/L of ammonium citrate tribasic, 5g/L of sodium acetate, 0.2g/L of magnesium sulfate, 80 g/L of tween, 0.05g/L of manganese sulfate, 15g/L of agar and 6.2 of pH value.

MRS liquid medium: 10g/L of peptone, 5g/L of beef extract, 4g/L of yeast powder, 20g/L of glucose, 2g/L of dipotassium phosphate, 2g/L of ammonium citrate tribasic, 5g/L of sodium acetate, 0.2g/L of magnesium sulfate, 80 g/L of tween, 0.05g/L of manganese sulfate and 6.2 of pH value.

MRS-S medium: 10g/L of peptone, 5g/L of beef extract, 4g/L of yeast powder, 5g/L of glucose, 2g/L of dipotassium phosphate, 2g/L of ammonium citrate tribasic, 5g/L of sodium acetate, 0.2g/L of magnesium sulfate, 80 g/L of tween, 0.05g/L of manganese sulfate, 50g/L of sucrose and 6.2 of pH value.

Example 1

(1) Screening of strains

Taking pickled Chinese cabbage fermentation liquor as sample liquid, and performing gradient dilution with sterile normal saline to prepare 10 ^-1 、10 ^-2 、 10 ^-3 、10 ^-4 、10 ^-5 Standing the sample solution with the concentration for later use; taking the above concentratesSpreading the sample solution in an MRS solid culture medium, culturing for 24-48h in a constant temperature incubator at 37 ℃, selecting a colony which is sticky on a flat plate, and performing repeated three-region streak culture on the MRS solid culture medium until the whole MRS solid culture medium presents white, opaque, neat-edged colonies and a colony which is sticky and polysaccharide-producing, wherein the colony graph is shown in figure 1; inoculating the purified strain to an MRS liquid culture medium, and culturing at 37 ℃ with the serial number of M76T11B;

(2) Identification of strains

Morphological Observation of the Strain

The strain M76T11B is scribed on a MRS solid culture medium plate, the strain grows well after being cultured for 24h at 37 ℃, the colony morphology (shown in figure 1) is observed, the colony morphology is flower-shaped, circular, uneven in edge, wrinkled and convex in surface, white, viscous polysaccharide substances are generated on the surface of the colony, the colony is easily picked up by an inoculating loop and pulled into filamentous, the strain is presumed to belong to extracellular polysaccharide, gram staining is positive (shown in figure 2), and cells are rod-shaped.

Physiological and biochemical test identification

Referring to the handbook of identifying common bacteria systems, the partial physiological and biochemical experiments of the strain M76T11B are studied, and the results are shown in tables 1 and 2.

TABLE 1 part of the physiological and biochemical Properties of the strains

TABLE 2 fermentation test results of sugar alcohol of strains

(3) Sequencing of strains

Extracting the genome of the strain M76T11B, amplifying and sequencing the 16SrDNA of the strain M76T11B (finished by Shanghai Bioengineering Co., ltd.), and comparing the 16SrDNA sequence of the strain M76T11B (the 16SrDNA sequence of the M76T11B is shown as SEQ ID NO. 1) obtained by sequencing analysis in GenBank, wherein the strain is confirmed to be Bacillus subtilis and is named as Bacillus velezensis M76T11B.

Example 2

(1) Preparation of crude polysaccharide

Performing three-region streak activation on a strain of Bacillus velezensis M76T11B stored in a refrigerator at 4 ℃ on an MRS plate, performing static culture at 37 ℃ for 48 hours, inoculating the strain into MRS broth, continuing static culture at 37 ℃ for 24 hours, inoculating the strain after the first generation activation into 1000 mM MRS-S liquid culture medium in an inoculation amount of 2% (v/v), and performing shake culture at 37 ℃ and 100rpm for 24 hours to obtain fermentation liquor; centrifuging the fermentation liquor at 4 ℃ for 20min by 10,000 Xg for 20min to remove thallus precipitate, concentrating the supernatant to 1/5 of the original volume, adding 4 times of 4 ℃ 95% ethanol (final concentration of 80%) by volume, stirring uniformly, standing overnight at 4 ℃ for precipitating polysaccharide, centrifuging at 4 ℃ for 20min by 10,000 Xg to obtain polysaccharide precipitate, dissolving the precipitate in deionized water, adding 80% (m/v) trichloroacetic acid to 4% (m/v) of final concentration, stirring uniformly, standing at 4 ℃ for 10h, centrifuging at 4 ℃ for 20min by 10,000 Xg, discarding protein precipitate, and collecting the supernatant; concentrating the supernatant, adding 4 times volume of 95% ethanol (final concentration of 80%) at 4 deg.C, standing overnight at 4 deg.C to precipitate polysaccharide, filtering, collecting polysaccharide precipitate, dissolving the polysaccharide precipitate in deionized water, placing into dialysis bag (molecular weight cut-off of 8000-14000 Da) for dialysis for 48h, changing water every 8h, and freeze drying the dialyzed sugar solution to obtain crude polysaccharide with crude polysaccharide content of 4.3g/L, wherein the crude polysaccharide picture is shown in FIG. 4.

(2) Determination of polysaccharide molecular weight

The molecular weight of Bacillus velezensis M76T11B exopolysaccharide is measured by a GPC method, and a standard curve is drawn by taking serial glucan as a standard substance.

Preparation of sample and standard solution: 1mg/mL of Bacillus velezensis M76T11B crude polysaccharide solution and standard solution are prepared.

Configuration of the mobile phase: ultrapure water was used as mobile phase and filtered through a 0.22 μm membrane with suction.

Experimental measurement conditions: agilent _1260 high performance liquid chromatograph; TSK-G2500 PWXL chromatographic column; the column temperature is 30 ℃; the mobile phase is ultrapure water; the flow rate is 0.5mL/min; the sample size was 20. Mu.L.

The experimental results are as follows: recording the peak time of the standard substance, and drawing a standard curve and an extracellular polysaccharide GPC spectrogram (shown in figure 3) by combining the molecular weight of the standard substance; obtaining the molecular weight of the exopolysaccharide according to the peak-appearing time of the exopolysaccharide sample; as can be seen from FIG. 3, the elution peak is a narrow single symmetrical peak, indicating that the polysaccharide composition is a homogeneous component, and the molecular weight of the exopolysaccharide is 1.89X 10 ⁵ Da。

(3) Analysis of monosaccharide composition

Measuring monosaccharide composition before and after enzymolysis of brown algae polysaccharide by derivatization method, hydrolyzing 200 μ L sample (10 mg/mL) with trifluoroacetic acid for 2 hr, adding 1-phenyl-3-methyl-5-pyrazolone (PMP) for reaction for 1 hr, and extracting with chloroform. Finally the aqueous phase was passed through a 0.22 μm microfiltration membrane and analyzed. HPLC analysis was performed using an Agilent 1260 HPLC system coupled to a VWD detector using a C18 column with a mobile phase of acetonitrile-phosphate buffer. The standard substance comprises D-mannose, L-rhamnose, D-glucose, D-arabinose, L-fucose, D-galactose, D-mannuronic acid, D-glucuronic acid and D-galacturonic acid. UV detection was carried out at 245 nm.

The experimental results are as follows: the monosaccharide composition of the strain is analyzed by using a high performance liquid chromatography, a standard spectrogram made by 9 monosaccharide standards is shown in fig. 5, and a high performance liquid chromatogram for the hydrolysis of the strain Bacillus velezensis M76T11B EPS of the invention is shown in fig. 6. Comparing the retention time of the sample with that of standard monosaccharide, the monosaccharide composition of the sample can be obtained, and extracellular polysaccharide monosaccharide composition produced by Bacillus velezensis M76T11B is rhamnose and galacturonic acid, wherein the rhamnose content is 62.11%, and the galacturonic acid content is 37.89%. Rhamnogalacturonan is a very special functional polysaccharide, mainly exists in primary cell walls of fruits and vegetables such as oranges, apples, beets, potatoes, okra, pumpkins and the like and some Chinese herbal medicines, and has potential effects of reducing blood sugar and blood fat, protecting intestines and stomach, regulating immunity, resisting tumors, preventing heart diseases and the like; rhamnogalacturonan is hardly digested and metabolized in the upper digestive tract, but can be fermented and utilized by intestinal flora to exert health effects.

The foregoing descriptions of specific exemplary embodiments of the present invention have been presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain certain principles of the invention and its practical application to enable one skilled in the art to make and use various exemplary embodiments of the invention and various alternatives and modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.

SEQUENCE LISTING

<110> Guangxi university

<120> Bacillus belgii and application thereof

<130> JC

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 1484

<212> DNA

<213> Bacillus velezensis

<400> 1

gctcaggacg aacgctggcg gcgtgcctaa tacatgcaag tcgagcggac agatgggagc 60

ttgctccctg atgttagcgg cggacgggtg agtaacacgt gggtaacctg cctgtaagac 120

tgggataact ccgggaaacc ggggctaata ccggatggtt gtttgaaccg catggttcag 180

acataaaagg tggcttcggc taccacttac agatggaccc gcggcgcatt agctagttgg 240

tgaggtaacg gctcaccaag gcgacgatgc gtagccgacc tgagagggtg atcggccaca 300

ctgggactga gacacggccc agactcctac gggaggcagc agtagggaat cttccgcaat 360

ggacgaaagt ctgacggagc aacgccgcgt gagtgatgaa ggttttcgga tcgtaaagct 420

ctgttgttag ggaagaacaa gtgccgttca aatagggcgg caccttgacg gtacctaacc 480

agaaagccac ggctaactac gtgccagcag ccgcggtaat acgtaggtgg caagcgttgt 540

ccggaattat tgggcgtaaa gggctcgcag gcggtttctt aagtctgatg tgaaagcccc 600

cggctcaacc ggggagggtc attggaaact ggggaacttg agtgcagaag aggagagtgg 660

aattccacgt gtagcggtga aatgcgtaga gatgtggagg aacaccagtg gcgaaggcga 720

ctctctggtc tgtaactgac gctgaggagc gaaagcgtgg ggagcgaaca ggattagata 780

ccctggtagt ccacgccgta aacgatgagt gctaagtgtt agggggtttc cgccccttag 840

tgctgcagct aacgcattaa gcactccgcc tggggagtac ggtcgcaaga ctgaaactca 900

aaggaattga cgggggcccg cacaagcggt ggagcatgtg gtttaattcg aagcaacgcg 960

aagaacctta ccaggtcttg acatcctctg acaatcctag agataggacg tccccttcgg 1020

gggcagagtg acaggtggtg catggttgtc gtcagctcgt gtcgtgagat gttgggttaa 1080

gtcccgcaac gagcgcaacc cttgatctta gttgccagca ttcagttggg cactctaagg 1140

tgactgccgg tgacaaaccg gaggaaggtg gggatgacgt caaatcatca tgccccttat 1200

gacctgggct acacacgtgc tacaatggac agaacaaagg gcagcgaaac cgcgaggtta 1260

agccaatccc acaaatctgt tctcagttcg gatcgcagtc tgcaactcga ctgcgtgaag 1320

ctggaatcgc tagtaatcgc ggatcagcat gccgcggtga atacgttccc gggccttgta 1380

cacaccgccc gtcacaccac gagagtttgt aacacccgaa gtcggtgagg taacctttta 1440

ggagccagcc gccgaaggtg ggacagatga ttggggtgaa gtcg 1484

Claims

1. The Bacillus belief is characterized by being named as Bacillus velezensis M76T11B, being preserved in Guangdong province microorganism strain preservation center in 12 and 23 days 2020, wherein the preservation address is No. 9 building 5 of Middleyao No. 100 Dazhou province in Guangzhou city, and the preservation number is GDMCC NO:61384, and the gene sequence of Bacillus velezensis M76T11B16S rDNA is shown in SEQ ID NO. 1.

2. The use of Bacillus velezensis M76T11B in the production of exopolysaccharides according to claim 1, wherein: the exopolysaccharide is rhamnogalacturonan.

3. The use according to claim 2, wherein the Bacillus velezensis M76T11B is used in a process for the production of exopolysaccharides: performing three-region streak activation on the Bacillus velezensis M76T11B on an MRS solid culture medium plate to activate strains, performing static culture at the constant temperature of 37 ℃ and inoculating the strains into an MRS liquid culture medium, continuing the static culture at the temperature of 37 ℃, inoculating the strains after the first generation activation into the MRS-S liquid culture medium by the inoculation amount of 2%, and performing shake culture for 24 hours to obtain fermentation liquor.

4. The use according to claim 3, wherein the MRS solid medium: 10g/L of peptone, 5g/L of beef extract, 4g/L of yeast powder, 20g/L of glucose, 2g/L of dipotassium phosphate, 2g/L of ammonium citrate tribasic, 5g/L of sodium acetate, 0.2g/L of magnesium sulfate, 80 g/L of Tween, 0.05g/L of manganese sulfate, 15g/L of agar and 6.2 of pH value.

5. The use according to claim 3, wherein the MRS liquid medium: 10g/L of peptone, 5g/L of beef extract, 4g/L of yeast powder, 20g/L of glucose, 2g/L of dipotassium phosphate, 2g/L of ammonium citrate tribasic, 5g/L of sodium acetate, 0.2g/L of magnesium sulfate, 80 g/L of tween, 0.05g/L of manganese sulfate and 6.2 of pH value.

6. The use according to claim 3, wherein the MRS-S medium: 10g/L of peptone, 5g/L of beef extract, 4g/L of yeast powder, 5g/L of glucose, 2g/L of dipotassium phosphate, 2g/L of ammonium citrate tribasic, 5g/L of sodium acetate, 0.2g/L of magnesium sulfate, 80 g/L of tween, 0.05g/L of manganese sulfate, 50g/L of sucrose and 6.2 of pH value.