CN105087450B - The exocellular polysaccharide of one plant of marine bacteria and its generation - Google Patents

Abstract

The exocellular polysaccharide of one plant of marine bacteria and its generation is related to one plant of marine bacteria.One plant of marine bacteria Alteromonase marina P2 B12, collection are registered on the books number：CGMCC No.11180.The method that one plant of marine bacteria Alteromonase marina P2 B12 prepares exocellular polysaccharide：1) strain culturing；2) extraction and desalination of polysaccharide；3) it isolates and purifies.The molecular weight for preparing the amylovorin of sea bacteria of gained is more than 167kDa, amylovorin of sea bacteria is by rhamnose, mannose and galacturonic acid with α 1,3 and α Isosorbide-5-Nitrae keys are bonded structural unit ([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). Antiviral and immunomodulatory effects of a novel bacterial exopolysaccharide of shallowmarine vent origin. J Appl Microbiol. 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 bacteria Alteromonase marina P2-B12 was preserved in the General Microbiology Center of China Committee for the Collection of Microbial Cultures on August 07, 2015, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Chinese Academy of Sciences Microbiology Research Institute, Zip Code: 100101, Registration Number of the Collection Center: CGMCC No.11180.

The marine bacterium Alteromonase marina P2-B12 of the present invention is isolated from seawater samples, and the seawater was collected in August 2012 at a longitude of 115°E, a latitude of 18°N, and a water depth of 75m; 100 μL of the collected seawater was sucked and coated on a plate , placed 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, large colony (2mm in diameter) in 2216E solid medium, milky white, round, raised, rounded edges, opaque, no Migration.

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

The method for preparing exopolysaccharide by said strain of marine bacteria 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, pH 7.6;

Alteromonase marina P2-B12 was inoculated into the medium for shaker culture to obtain the 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 condition can be centrifuged at 8000rpm for 20min; the filtration can be filtered with a 0.45 μm filter membrane (Millipore HAWP04700); adding dehydrated alcohol to the filtrate can add 3 times the volume ratio of the filtrate without water ethanol; the temperature of standing overnight can be 4°C; the described moving the viscous polysaccharide into the centrifuge tube can be wrapped with a glass rod to get the viscous polysaccharide and moved into the 50mL centrifuge tube; the immersion can add mass The percentage concentration is 75% freezing absolute ethanol soaking 3 times; The dialysis bag can adopt the dialysis bag of diameter 16mm, molecular weight cut-off 3500 Dalton; The volume of the polysaccharide solution 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-Sepharose Fast Flow, gradient elution is carried out 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 16S rRNA.

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 colonies of a single form are isolated. The polysaccharide-producing strain was detected by the phenol-sulfuric acid method to detect whether the culture supernatant of the obtained strain produced polysaccharide.

The characteristics of the strain are: Alteromonase marina P2-B12 is a Gram-negative bacterium with a long rod shape (Figure 1), and the colony in the 2216E solid medium is relatively large (diameter 2mm), milky white, round, raised, with rounded edges , Opaque, non-migratory. 16S rRNA gene analysis.

Genomic DNA of Alteromonase marina P2-B12 was extracted for 16S rRNA gene amplification. The 16S rRNA gene sequence amplification primers are: forward primer 27f (5'-AGA GTT TGA TCM TGG CTC AG-3'), reverse primer 1492r (5'-TAC GGY TAC CTT GTT ACG ACT T-3'). 16S rRNA gene amplification PCR reaction system (50 μL): Premix Ex Taq 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 16S rRNA 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 16S rRNA 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 TSK Gel GS3000SW _XL gel chromatography column (7.8mmID×30cm) and TSK GelG2000SW _XL gel chromatography column (7.8mmID×30cm) connected 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.

16S rRNA 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 (7)

1. A marine bacterium, Alteromonase marina P2-B12, was deposited in the General Microbiology Center of China Committee for the Collection of Microbial Cultures on August 7, 2015. The registration number of the collection center is: CGMCCNo.11180. 2. the method that a strain of marine bacterium prepares exopolysaccharide as claimed in claim 1 is characterized in that its concrete 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: glucose 10.0g/L, sodium chloride 19.0g/L, ammonium chloride 0.3g/L, dipotassium hydrogen phosphate 0.4g /L, potassium chloride 0.3g/L, magnesium sulfate heptahydrate 0.5g/L, calcium chloride heptahydrate 0.03g/L, pH7.6; Alteromonase marina P2-B12 was inoculated into the medium for shaker culture, the condition of the shaker culture was 25°C, 180rpm, shaker culture overnight to obtain a seed culture solution; Separately prepare a culture medium, put it into a fermenter, and sterilize it. After the temperature drops to 25°C, insert the seed culture solution with a volume percentage of 1% for cultivation. Cultivate for 94h to obtain fermented 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 for soaking, 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 Use ion exchange chromatography to purify the polysaccharide crude extract, collect the main peak, concentrate and desalt the collected main peak, and freeze-dry to obtain the marine bacterial exopolysaccharide; the molecular weight of the prepared marine bacterial exopolysaccharide is greater than 167kDa, and the marine bacterial exopolysaccharide The exopolysaccharide is composed of rhamnose Rha, mannose Man and galacturonic acid GalA combined with α-1,3 and α-1,4 bonds to form structural units. 3. The method for preparing exopolysaccharide from a strain of marine bacteria according to claim 2, characterized in that in step 1), the sterilization condition is 120° C. for 30 minutes. 4. The method for preparing exopolysaccharide by a strain of marine bacteria as claimed in claim 2 is characterized in that in step 2), the centrifugal condition is centrifugal 20min at 8000rpm; the filtration adopts 0.45 μm membrane filtration; the Add absolute ethanol to the filtrate and add absolute ethanol that is 3 times the volume of the filtrate; the temperature of standing overnight is 4°C. 5. as claimed in claim 2, a strain of marine bacteria prepares the method for exopolysaccharide, it is characterized in that in step 2) in, described viscous mycopolysaccharide is moved into centrifuge tube and adopts glass rod to get viscous mycopolysaccharide to move into 50mL centrifuge tube; add frozen absolute ethanol with a concentration of 75% by mass to soak for 3 times. 6. as claimed in claim 2, a strain of marine bacterium prepares the method for exopolysaccharide, it is characterized in that in step 2) in, described dialysis bag adopts diameter 16mm, the dialysis bag of molecular weight cut-off 3500 Daltons; Described adding freezing For ethanol reprecipitation, add frozen ethanol with a volume ratio of 3 times the volume of the bacterial polysaccharide solution for reprecipitation. 7. The method for preparing exopolysaccharides from a strain of marine bacteria as claimed in claim 2 is characterized in that in step 3), the conditions of the ion exchange chromatography are: the chromatography column specification is 1.6cm × 30cm, and the gel The type is DEAE-Sepharose FastFlow, with 0-2M NaCl for gradient elution, and the flow rate is 1mL/min.