CN105087450A - Marine bacterium and exopolysaccharide generated from marine bacterium - Google Patents

Abstract

A strain of marine bacteria and the exopolysaccharide it produces relate to a strain of marine bacteria. Said a strain of marine bacteria Alteromonase? marina? P2-B12, registration number of the collection center: CGMCC? No. 11180. A strain of marine bacteria Alteromonase? marina? The method for preparing extracellular polysaccharides from P2-B12: 1) strain cultivation; 2) extraction and desalination of polysaccharides; 3) separation and purification. The molecular weight of the prepared marine bacterial exopolysaccharide is greater than 167kDa, and the marine bacterial exopolysaccharide is composed of rhamnose, mannose and galacturonic acid with α-1,3 and α-1,4 bonds to form structural units ([- 3] -α-Rhap-(1→3)-α-Manp-(1→4)-α-GalAp-(1→)).

Description

A marine bacterium and its exopolysaccharide

technical field

The invention relates to a strain of marine bacteria, in particular to a strain of marine bacteria and the exopolysaccharide produced therefrom.

Background technique

Bacteria generally synthesize and secrete exopolysaccharides. Exopolysaccharides are secondary metabolites of bacteria, which are soluble polysaccharide complexes that are secreted outside the cell wall during bacterial growth and separated from the bacteria. Due to their unique living environment, the exopolysaccharides secreted by marine bacteria often have special structures. Therefore, the exopolysaccharides produced by marine bacteria have great potential application value in the field of biotechnology. Some exopolysaccharide-producing bacteria have been isolated from different marine environments, for example, abundant exopolysaccharide-producing bacteria have been found from deep-sea hydrothermal vents, marine volcanoes and hydrothermal areas, and shallow seabed hot springs (Poli, 2010). Polysaccharides produced by strains of the Bacillus genus isolated from shallow marine hydrothermal vents are antiviral (Gugliandolo, 2014). Polysaccharides synthesized by Pseudoalteromonas bacteria isolated from Arctic sea ice have antifreeze properties (GugliandoloC, SpanòA, LentiniV, ArenaA, MaugeriTL. 2014, Antiviralandimmunomodulatoryeffectsofanovelbacterialexopolysaccharideofshallowmarineventorigin.JApplMicrobiol.116(4):1028-34).

In recent years, with the in-depth research on microbial polysaccharides, the annual growth rate of microbial polysaccharide production in the world is more than 10%, and the annual growth rate of some new polysaccharides is more than 30%. Now the world's total annual demand for polysaccharides is 1 million Tons or more, with a total value of 5 to 10 billion US dollars. Therefore, the production of polysaccharides has huge market value. However, bacterial polysaccharides currently used in practical production are very limited. Therefore, it is necessary to screen and isolate new polysaccharide-producing bacteria, and to further explore the structure and physical and chemical properties of the polysaccharides they produce, so as to further expand their application fields.

Contents of the invention

The object of the present invention is to provide a strain of marine bacteria Alteromonase marina P2-B12.

Another object of the present invention is to provide a method for preparing a safe and non-toxic exopolysaccharide with a novel structure by a strain of marine bacteria Alteromonase marina P2-B12.

The said strain of marine bacterium Alteromonase marina P2-B12 was preserved on August 07, 2015 in the General Microbiology Center of China Committee for the Collection of Microbial Cultures, address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing , Zip Code: 100101, Registration Number of the Collection Center: CGMCCNo.11180.

The marine bacterium Alteromonase marina P2-B12 of the present invention is isolated from the seawater sample, and the seawater was collected in August 2012, with a longitude of 115°E, a latitude of 18°N, and a water depth of 75m; Incubate at room temperature. After 3 days of culture, colonies with different shapes and sizes were randomly picked from the plate for purification and culture. When purifying, use an inoculation loop to pick colonies, and streak on the surface of the plate to observe whether the color, size, and shape of the strains on the plate are the same. Pick different colonies and streak until a colony of a single form is isolated. The polysaccharide-producing strain was detected by the phenol-sulfuric acid method to detect whether the culture supernatant of the obtained strains produced polysaccharides. The characteristics of the strain are: Alteromonase marina P2-B12 is a Gram-negative bacterium, long rod-shaped, large colony (2mm in diameter) in 2216E solid medium, milky white, round, raised, rounded edges, opaque, non-migratory .

The present invention determines its species relationship through DNA sequence analysis, and belongs to Alteromonas genus Alteromonase. The 16SrRNA sequence of the strain is shown in the last part of the description.

The method for preparing exopolysaccharide by said strain of marine bacterium Alteromonase marina P2-B12, the specific steps are as follows:

1) Strain culture

Add industrial glucose as a single carbon source to artificial seawater for cultivation. The specific formula of the medium is (1L): glucose 10.0g/L, sodium chloride 19.0g/L, ammonium chloride 0.3g/L, dihydrogen phosphate Potassium 0.4g/L, potassium chloride 0.3g/L, magnesium sulfate heptahydrate 0.5g/L, calcium chloride heptahydrate 0.03g/L, pH7.6;

Inoculate Alteromonase marina P2-B12 into the medium shaker culture to obtain seed culture solution;

Separately prepare a culture medium, put it in a fermenter, sterilize it, and when the temperature drops to 25°C, insert the seed culture medium for cultivation to obtain a fermentation liquid;

2) Extraction and desalination of polysaccharides

Centrifuge the fermentation broth, take the supernatant, filter the supernatant, add absolute ethanol to the filtrate, let it stand overnight, then transfer the viscous bacterial polysaccharide into a centrifuge tube, add frozen absolute ethanol to soak, and collect the precipitate. Freeze-dried to obtain the extract of bacterial polysaccharides, then dissolved in water, moved into a dialysis bag, hung the dialysis bag in water, and dialyzed overnight. The bacterial polysaccharide sample is the crude polysaccharide extract;

3) Separation and purification

The crude polysaccharide extract is purified by ion exchange chromatography, the main peak is collected, the collected main peak is concentrated, desalted, and freeze-dried to obtain the exopolysaccharide of marine bacteria.

In step 1), the condition of the shaker culture can be 25°C, 180rpm, shaker culture overnight; the condition of the sterilization can be 120°C for 30min; the inoculum size of the seed culture solution is by volume percentage It can be 1%; the culture solution of the inserted seeds can be cultured at 25° C. and 500 rpm/min for 94 hours.

In step 2), the centrifugal conditions can be centrifuged at 8000rpm for 20min; the filtration can be filtered with a 0.45 μm filter membrane (MilliporeHAWP04700); the filtrate can be added with anhydrous ethanol that is 3 times the filtrate by volume Ethanol; the temperature of standing overnight can be 4°C; the described transfer of viscous polysaccharides into centrifuge tubes can use glass rods to get viscous polysaccharides and move them into 50mL centrifuge tubes; Dip and wash 3 times with 75% frozen absolute ethanol; the dialysis bag can be a dialysis bag with a diameter of 16mm and a molecular weight cut-off of 3500 Daltons; the addition of frozen ethanol for reprecipitation can add bacteriopolysaccharide with a volume ratio of 3 times The solution volume was reprecipitated with ice-cold ethanol.

In step 3), the conditions of the ion exchange chromatography can be: the column size is 1.6cm×30cm, the gel type is DEAE-SepharoseFastFlow, gradient elution is performed with 0-2M NaCl, and the flow rate is 1mL/min.

The molecular weight of the marine bacterial exopolysaccharide prepared by the present invention is greater than 167kDa, and the marine bacterial exopolysaccharide consists of rhamnose (Rha), mannose (Man) and galacturonic acid (GalA) in the form of α-1, 3 and α- The 1, 4-bond combination constitutes a structural unit ([-3]-α-Rhap-(1→3)-α-Manp-(1→4)-α-GalAp-(1→)). The glycosyl composition and glycosidic bond connection mode of the marine bacterial exopolysaccharide are different from other reported polysaccharides, and it is a novel exopolysaccharide.

Description of drawings

Figure 1 is a transmission electron microscope image of Alteromonase marina P2-B12. In Fig. 1, the scale bar of a is 200 nm, and the scale bar of b is 0.2 μm.

Figure 2 is the analysis of P2-B12 phylogenetic tree based on 16SrRNA.

Figure 3 is a schematic diagram of the main peak collected by gradient elution.

Figure 4 is a schematic diagram of the elution peaks obtained by elution.

Detailed ways

The present invention will be further described below in conjunction with embodiment.

The acquisition of embodiment 1 bacterial strain

Bacterial strain of the present invention obtains by following method screening: seawater is collected in August, 2012, longitude 115 ° E, latitude 18 ° N, depth of water 75m. Pipette 100 μL of the collected seawater onto the 2216E plate. 2216E medium: peptone 5.0g, yeast extract 1.0g, iron phosphate 0.01g, agar 20.0g, artificial seawater 1000ml, adjust ph to 7.6. Place at room temperature for incubation. After 3 days of culture, colonies with different shapes and sizes were randomly picked from the plate for purification and culture. When purifying, use an inoculation loop to pick colonies, and streak on the surface of the plate to observe whether the color, size, and shape of the strains on the plate are the same. Pick different colonies and streak until a colony of a single form is isolated. The polysaccharide-producing strain was detected by the phenol-sulfuric acid method to detect whether the culture supernatant of the obtained strains produced polysaccharides.

The characteristics of the strain are: Alteromonase marina P2-B12 is a Gram-negative bacterium, long rod-shaped (Figure 1), and the colony in the 2216E solid medium is relatively large (diameter 2mm), milky white, round, raised, with rounded edges, and opaque , no migration. 16SrRNA gene analysis.

Alteromonase marina P2-B12 genomic DNA was extracted for 16SrRNA gene amplification. 16SrRNA gene sequence amplification primers are: forward primer 27f (5'-AGAGTTTGATCMTGGCTCAG-3'), reverse primer 1492r (5'-TACGGYTACCTTGTTACGACTT-3'). 16SrRNA gene amplification PCR reaction system (50 μL): PremixExTaq enzyme (5U/μL) 25 μL, forward primer 1 μL (10 μmol/L), reverse primer 1 μL (10 μmol/L), template DNA 1 μL and sterile water 22 μL. The PCR target sequence amplification program was set as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 45 s, annealing at 55°C for 45 s, extension at 72°C for 1.5 min, 30 cycles; extension at 72°C for 10 min; incubation at 4°C.

This PCR product was then sequenced. Through the comparison analysis of the 16SrRNA gene sequence of the strain P2-B12, it was found that the strain had the highest similarity with Alteromonas marina SW-47 ^T (AF529060). Therefore, this strain was named Alteromonas marina P2-B12. The 16SrRNA sequence of the strain is shown in the last part of the description.

The extraction of embodiment 2 polysaccharides

Determination of polysaccharide molecular weight

The molecular weight of polysaccharides was determined by high performance liquid chromatography. Polysaccharides were analyzed using a size exclusion column. The chromatographic column is composed of TSKGelGS3000SW _XL gel chromatography column (7.8mmID×30cm) and TSKGelG2000SW _XL gel chromatography column (7.8mmID×30cm) in series, and the detector is a refraction detector. The mobile phase was 0.1 M ammonium acetate containing 0.2‰ sodium azide. The temperature of the column was set at 30° C., and the flow rate was set at 0.6 mL/min. Standard solutions include dextran (511, 167, 67, 40, 10, 5, 1 kDa) and maltotetraose (666.6 Da) with different molecular weights. Polysaccharides with different molecular weights have different retention times on size exclusion chromatography columns (see Table 1). According to the correlation between the molecular weight of the standard polysaccharide and the retention time in the column, the molecular weight of the P2-B12 polysaccharide was calculated to be greater than 167kDa.

Table 1

Structural Analysis of Polysaccharides

Through a series of nuclear magnetic resonance analysis, it is clear that the exopolysaccharide of P2-B12 is composed of three monosaccharides, rhamnose (Rha), mannose (Man) and galacturonic acid (GalA). , 3 and α-1, 4 are combined to form a[-3]-α-Rhap-(1→3)-α-Manp-(1→4)-α-3OAc-GalAp-(1→) structural unit.

16SrRNA sequence:

GAGTTTGATCCTGGCTCAGGTTGAACGCTGGCGGCAGGCCTAACACATGCA

AGTCGAACGGTAACATTTCTAGCTTGCTAGAAGATGACGAGTGGCGGACG

GGTGAGTAATGCTTGGGAACTTGCCTTTGCGAGGGGGATAACAGTTGGAA

ACGACTGCTAATACCGCATAATGTCTTCGGACCAAACGGGGCTTCGGCTC

CGGCGCAAAGAGAGGCCCAAGTGAGATTAGCTAGTTGGTAAGGTAACGGC

TTACCAAGGCGACGATCTCTAGCTGTTCTGAGAGGAAGATCAGCCACT

GGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATAT

TGCACAATGGGGGAAACCCTGATGCAGCCATGCCGCGTGTGTGAAGAAGG

CCTTCGGGTTGTAAAGCACTTTCAGTTGTGAGGAAAAGTTAGTAGTTAAT

ACCTGCTAGCTGTGACGTTAACAACAGAAGAAGCACCGGCTAACTCCGTG

CCAGCAGCCGCGGTAATACGGAGGGTGCGAGCGTTAATCGGAATTACTGG

GCGTAAAGCGCACGCAGGCGGTTTGTTAAGCTAGATGTGAAAGCCCCGGG

CTCAACCTGGGATGGTCATTTAGAACTGGCAGACTAGAGTCTTGGAGAGG

GGAGTGGAATTCCAGGTGTAGCGGTGAAATGCGTAGATATCTGGAGGAAC

ATCAGTGGCGAAGGCGACTCCCTGGCCAAAGACTGACGCTCATGTGCGAA

AGTGTGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCACACCGTAAAC

GCTGTCTACTAGCTGTGTGTGCCTTTAAGGCGTGCGTAGCGAAGCTAACG

CGCTAAGTAGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATG

AATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGC

AACGCGAAGAACCTTACCTACACTTGACATGCTGAGAAGTTACTAGAGAT

AGTTTCGTGCCTTCGGGAACTCAGACACAGGTGCTGCATGGCTGTCGTCA

GCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTG

TCCTTAGTTGCCAGCCTTAAGTTGGGCACTCTAAGGAGACTGCCGGTGAC

AAACCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGGCCCTTACGTGT

AGGGCTACACACGTGCTACAATGGCATTTACAGAGGGAAGCGAGACAGTG

ATGTGGAGCGGACCCCTTAAAGAATGTCGTAGTCCGGATTGGAGTCTGCA

ACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGCAGGTCAGAATACTG

CGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGA

GTGGGATGCAAAAGAAGTAGTTAGTCTAACCTTCGGGAGGACGATTACCA

CTTTGTGTTTCATGACTGGGGTGAAGTCGTAACAAGGTAACCA.

Claims (10)

1. a strain marine bacteria AlteromonasemarinaP2-B12, was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on 08 07th, 2015, and register on the books numbering: CGMCCNo.11180 at preservation center.

2. a strain marine bacteria prepares the method for exocellular polysaccharide as claimed in claim 1, it is characterized in that its concrete steps are as follows:

1) strain culturing

Be that single carbon source is added in artificial seawater and cultivated with cerelose, the concrete formula of substratum is: glucose 10.0g/L, sodium-chlor 19.0g/L, ammonia chloride 0.3g/L, dipotassium hydrogen phosphate 0.4g/L, Repone K 0.3g/L, bitter salt 0.5g/L, seven calcium chloride hydrate 0.03g/L, pH7.6;

AlteromonasemarinaP2-B12 is inoculated into substratum shaking table to cultivate, obtains seed culture fluid;

Another preparation substratum, puts into fermentor tank, sterilizing, treats that temperature drops to 25 DEG C, and access seed culture fluid is cultivated, and obtains fermented liquid;

2) extraction of polysaccharide and desalination

Fermented liquid is centrifugal, get supernatant liquor, supernatant liquid filtering, add dehydrated alcohol in filtrate, hold over night, then the granulose of thickness is moved in centrifuge tube, add freezing dehydrated alcohol to embathe, precipitation is collected, freeze-drying, obtain the extract of granulose, then be dissolved in water, move into dialysis tubing, be hanging to by dialysis tubing in water, dialysed overnight, the granulose solution of dialysing adds ice-cold ethanol redeposition, freeze-drying, the granulose sample after dialysis desalination is polysaccharide crude extract;

3) separation and purification

Utilize ion exchange chromatography to carry out purifying to polysaccharide crude extract, collect main peak, by collect main peak concentrating and desalinating, lyophilize, obtain amylovorin of sea bacteria.

3. a strain marine bacteria prepares the method for exocellular polysaccharide as claimed in claim 2, it is characterized in that in step 1) in, the condition that described shaking table is cultivated is 25 DEG C, 180rpm, shaking table overnight incubation.

4. a strain marine bacteria prepares the method for exocellular polysaccharide as claimed in claim 2, it is characterized in that in step 1) in, the condition of described sterilizing is 120 DEG C of sterilizing 30min.

5. a strain marine bacteria prepares the method for exocellular polysaccharide as claimed in claim 2, it is characterized in that in step 1) in, the inoculum size of described seed culture fluid by volume percentage ratio is 1%.

6. a strain marine bacteria prepares the method for exocellular polysaccharide as claimed in claim 2, it is characterized in that in step 1) in, it is 25 DEG C that described access seed culture fluid is cultivated, and 500rpm/min cultivates 94h.

7. a strain marine bacteria prepares the method for exocellular polysaccharide as claimed in claim 2, it is characterized in that in step 2) in, described centrifugal condition is at the centrifugal 20min of 8000rpm; Described filtration can adopt 0.45 μm of membrane filtration; The dehydrated alcohol that dehydrated alcohol can add 3 times of filtrates is by volume added in described filtrate; The temperature of described hold over night can be 4 DEG C.

8. a strain marine bacteria prepares the method for exocellular polysaccharide as claimed in claim 2, it is characterized in that in step 2) in, the described centrifuge tube employing glass stick that moved into by the granulose of thickness twines in the granulose immigration 50mL centrifuge tube getting thickness; Described embathe can add mass percentage concentration be 75% freezing dehydrated alcohol embathe 3 times.

9. a strain marine bacteria prepares the method for exocellular polysaccharide as claimed in claim 2, it is characterized in that in step 2) in, described dialysis tubing adopts diameter 16mm, the daltonian dialysis tubing of molecular weight cut-off 3500; The described ice-cold ethanol redeposition adding ice-cold ethanol redeposition and can add 3 times of granulose liquor capacities by volume.

10. a strain marine bacteria prepares the method for exocellular polysaccharide as claimed in claim 2, it is characterized in that in step 3) in, the condition of described ion exchange chromatography is: chromatography column specification is 1.6cm × 30cm, gel type is DEAE-SepharoseFastFlow, carry out gradient elution with 0 ~ 2MNaCl, flow velocity is 1mL/min.