CN105087450A - Marine bacterium and exopolysaccharide generated from marine bacterium - Google Patents
Marine bacterium and exopolysaccharide generated from marine bacterium Download PDFInfo
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- CN105087450A CN105087450A CN201510630852.3A CN201510630852A CN105087450A CN 105087450 A CN105087450 A CN 105087450A CN 201510630852 A CN201510630852 A CN 201510630852A CN 105087450 A CN105087450 A CN 105087450A
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Abstract
The invention discloses a marine bacterium and an exopolysaccharide generated from the marine bacterium, and relates to a marine bacterium. The marine bacterium, namely, Alteromonase marina P2-B12, is registered in the China General Microbiological Culture Collection Center with the number of CGMCC No.11180. A method for preparing the exopolysaccharide from the marine bacterium, namely, Alteromonase marina P2-B12, comprises the following steps: 1) bacterial strain culture; 2) polysaccharide extraction and desalination; 3) separation and purification. The molecular weight of the prepared marine bacterium exopolysaccharide is larger than 167 kDa, and the marine bacterium exopolysaccharide adopts a structural unit ([-3]-alpha-Rhap-(1->3)-alpha-Manp-(1->4)-alpha-GalAp-(1->)) formed from rhamnose, mannose and galacturonic acid through combination of alpha-1,3 and alpha-1,4 bonds.
Description
Technical field
The present invention relates to a strain marine bacteria, especially relate to the exocellular polysaccharide of a strain marine bacteria and generation thereof.
Background technology
The general synthesis secretion exocellular polysaccharide of bacterium.Exocellular polysaccharide is the secondary metabolite of bacterium, is to be secreted into cell walls in bacteria growth process to form the soluble polysaccharide mixture be separated with thalline outward.Marine bacteria is because of the living environment of its uniqueness, and the exocellular polysaccharide of secretion usually has special construction, and therefore, the exocellular polysaccharide that marine bacteria produces has great potential using value in biological technical field.It is separated from different ocean environment that more existing exocellular polysaccharide produces bacterium, such as, from deep-sea hydrothermal spring mouth, and volcano, ocean and hot fluid area, and bay hot spring has found that abundant exocellular polysaccharide produces bacterium (Poli, 2010).The polysaccharide that the bacterial strain belonged to from the isolated Bacillus of shallow sea hot spring produces can antiviral (Gugliandolo, 2014).The polysaccharide of the Pseudoalteromonas bacterium synthesis be separated from Sea Ice Model has antifreeze properties (GugliandoloC, Span ò A, LentiniV, ArenaA, MaugeriTL.2014, Antiviralandimmunomodulatoryeffectsofanovelbacterialexop olysaccharideofshallowmarineventorigin.JApplMicrobiol.11 6 (4): 1028-34).
In recent years, along with going deep into of studying microbial polysaccharide, the annual increment of microbial polysaccharide output is more than 10% in the world, and some novel polysaccharide annual increments are more than 30%, now in the world annual polysaccharide aggregate demand more than 1,000,000 tons, total value 50 ~ 10,000,000,000 dollars.Therefore, the production of polysaccharide has huge marketable value.But the current bacterial polysaccharides be applied in actual production is very limited.Therefore, be necessary screening, be separated new polysaccharide producing strains, and to they produce the structure of polysaccharide, physics-chem characteristic deeply probes into, to expand their Application Areas further.
Summary of the invention
The object of the present invention is to provide a strain marine bacteria AlteromonasemarinaP2-B12.
Another object of the present invention is to provide a strain marine bacteria AlteromonasemarinaP2-B12 preparation to have novel structure and the method for the exocellular polysaccharide of safety non-toxic.
A described strain marine bacteria AlteromonasemarinaP2-B12, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on 08 07th, 2015, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, postcode: 100101, register on the books numbering: CGMCCNo.11180 at preservation center.
Marine bacteria AlteromonasemarinaP2-B12 of the present invention is separated and obtains from seawater sample, and seawater is collected in August, 2012, longitude 115 ° of E, latitude 18 ° of N, depth of water 75m; The seawater 100 μ L of collection is drawn and coats flat board, place incubated at room temperature.Until cultivation after 3 days, the bacterium colony varied in size from picking form flat board at random carries out purifying cultivation.Use transfering loop picking colony during purifying, rule at planar surface, whether the color of the dull and stereotyped upper bacterial strain of observation, size, form be identical. the bacterium colony line separation that picking is different, until isolate the bacterium colony of single form.Whether produce polysaccharide by phend-sulphuric acid to obtained strains culture supernatant to detect, primary dcreening operation polysaccharide yield, more than 100mg/L, obtains this product polysaccharide strains.The feature of this bacterial strain is: AlteromonasemarinaP2-B12 is Gram-negative bacteria, elongated rod shape, and at 2216E solid medium bacterium colony comparatively large (diameter 2mm), oyster white, circular, protuberance, edge is rounding comparatively, opaque, nonmigratory.
The present invention determines its race relation by DNA sequence analysis, belongs to Alteromonas Alteromonase.This bacterial strain 16SrRNA sequence is shown in specification sheets final section.
A described strain marine bacteria AlteromonasemarinaP2-B12 prepares the method for exocellular polysaccharide, and 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 (1L): 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.
In step 1) in, the condition that described shaking table is cultivated can be 25 DEG C, 180rpm, shaking table overnight incubation; The condition of described sterilizing can be 120 DEG C of sterilizing 30min; The inoculum size of described seed culture fluid by volume percentage ratio can be 1%; Described access seed culture fluid is cultivated and be can be 25 DEG C, and 500rpm/min cultivates 94h.
In step 2) in, described centrifugal condition can at the centrifugal 20min of 8000rpm; Described filtration can adopt the filtration of 0.45 μm of filter membrane (MilliporeHAWP04700); 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; The described centrifuge tube that moved into by the granulose of thickness can adopt glass stick to twine to get the granulose of thickness to move in 50mL centrifuge tube; Described embathe can add mass percentage concentration be 75% freezing dehydrated alcohol embathe 3 times; Described dialysis tubing can adopt 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.
In step 3) in, the condition of described ion exchange chromatography can be: chromatography column specification is 1.6cm × 30cm, and gel type is DEAE-SepharoseFastFlow, carries out gradient elution with 0 ~ 2MNaCl, and flow velocity is 1mL/min.
The molecular weight that the present invention prepares the amylovorin of sea bacteria of gained is greater than 167kDa, amylovorin of sea bacteria by rhamnosyl (Rha), seminose (Man) and galacturonic acid (GalA) with α-1,3 and α-Isosorbide-5-Nitrae key be bonded structural unit ([-3]-α-Rhap-(1 → 3)-α-Manp-(1 → 4)-α-GalAp-(1 →)).The glycosyl composition of this amylovorin of sea bacteria is not identical with the polysaccharide that glycosidic link mode of connection and other have been reported, is a kind of exocellular polysaccharide of novelty.
Accompanying drawing explanation
Fig. 1 is AlteromonasemarinaP2-B12 transmission electron microscope picture.In FIG, the scale of a is the scale of 200nm, b is 0.2 μm.
Fig. 2 is the P2-B12 phylogenetic analysis based on 16SrRNA.
Fig. 3 is that gradient elution collects the main peak schematic diagram obtained.
Fig. 4 is the elution peak schematic diagram that wash-out obtains.
Embodiment
Below in conjunction with embodiment, the present invention is further illustrated.
The acquisition of embodiment 1 bacterial strain
Bacterial strain of the present invention screens by the following method and obtains: seawater is collected in August, 2012, longitude 115 ° of E, latitude 18 ° of N, depth of water 75m.The seawater 100 μ L of collection is drawn and coats 2216E flat board.2216E substratum: peptone 5.0g, yeast extract paste 1.0g, tertiary iron phosphate 0.01g, agar 20.0g, artificial seawater 1000ml, adjust ph to 7.6.Place incubated at room temperature.Until cultivation after 3 days, the bacterium colony varied in size from picking form flat board at random carries out purifying cultivation.Use transfering loop picking colony during purifying, rule at planar surface, whether the color of the dull and stereotyped upper bacterial strain of observation, size, form be identical. the bacterium colony line separation that picking is different, until isolate the bacterium colony of single form.Whether produce polysaccharide by phend-sulphuric acid to obtained strains culture supernatant to detect, primary dcreening operation polysaccharide yield, more than 100mg/L, obtains this product polysaccharide strains.
The feature of this bacterial strain is: AlteromonasemarinaP2-B12 is Gram-negative bacteria, elongated rod shape (Fig. 1), at 2216E solid medium bacterium colony comparatively large (diameter 2mm), oyster white, circular, protuberance, edge is rounding comparatively, opaque, nonmigratory.16SrRNA genetic analysis.
Extract AlteromonasemarinaP2-B12 genomic dna, carry out 16SrRNA gene amplification.16SrRNA gene order amplimer is: forward primer 27f (5 '-AGAGTTTGATCMTGGCTCAG-3 '), reverse primer 1492r (5 '-TACGGYTACCTTGTTACGACTT-3 ').The PCR reaction system (50 μ L) of 16SrRNA gene amplification: PremixExTaq enzyme (5U/ μ L) 25 μ L, forward primer 1 μ L (10 μm of ol/L), reverse primer 1 μ L (10 μm of ol/L), template DNA 1 μ L and sterilized water 22 μ L.PCR target sequence amplification programming is: 95 DEG C of denaturation 5min; 95 DEG C of sex change 45s, 55 DEG C of annealing 45s, 72 DEG C extend 1.5min, 30 circulations; 72 DEG C extend 10min; 4 DEG C of insulations.
Check order after this PCR primer.By finding the 16SrRNA gene order compare of analysis of bacterial strain P2-B12, this bacterial strain and AlteromonasmarinaSW-47
t(AF529060) similarity is the highest.Therefore, this Strain Designation is AlteromonasmarinaP2-B12.This bacterial strain 16SrRNA sequence is shown in specification sheets final section.
The extraction of embodiment 2 polysaccharide
The mensuration of polysaccharide molecular weight
The molecular weight of high performance liquid chromatography to polysaccharide is utilized to measure.Molecular-exclusion chromatography post is adopted to analyze polysaccharide.Column chromatography post is by TSKGelGS3000SW
xLgel chromatography column (7.8mmID × 30cm) and TSKGelG2000SW
xLgel chromatography column (7.8mmID × 30cm) is in series, and detector is refraction detectors.Moving phase is the ammonium acetate of the 0.1M containing 0.2 ‰ sodiumazide.Barrel temperatures is set to 30 DEG C, and flow velocity is set to 0.6mL/min.Standardized solution comprises dextran (511,167,67,40,10,5,1kDa) and the maltotetrose (666.6Da) of different molecular weight.The polysaccharide of different molecular weight is in the retention time difference (see table 1) of molecular-exclusion chromatography post.According to the correlationship of the molecular weight of standard polysaccharide with the retention time in post, the molecular weight calculating P2-B12 polysaccharide is greater than 167kDa.
Table 1
The structural analysis of polysaccharide
By a series of nuclear magnetic resonance spectroscopy, specify P2-B12 exocellular polysaccharide by three kinds of monosaccharides rhamnoses (Rha), seminose (Man) and galacturonic acid (GalA) composition, these three kinds of sugar are with α-1,3 and α-Isosorbide-5-Nitrae key be bonded a [-3]-α-Rhap-(1 → 3)-α-Manp-(1 → 4)-α-3OAc-GalAp-(1 →) structural unit.
16SrRNA sequence:
GAGTTTGATCCTGGCTCAGGTTGAACGCTGGCGGCAGGCCTAACACATGCA
AGTCGAACGGTAACATTTCTAGCTTGCTAGAAGATGACGAGTGGCGGACG
GGTGAGTAATGCTTGGGAACTTGCCTTTGCGAGGGGGATAACAGTTGGAA
ACGACTGCTAATACCGCATAATGTCTTCGGACCAAACGGGGCTTCGGCTC
CGGCGCAAAGAGAGGCCCAAGTGAGATTAGCTAGTTGGTAAGGTAACGGC
TTACCAAGGCGACGATCTCTAGCTGTTCTGAGAGGAAGATCAGCCACACT
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.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105647990A (en) * | 2016-03-14 | 2016-06-08 | 山东省食品发酵工业研究设计院 | Microbial extracellular polysaccharide and method for preparing same |
| CN106119153A (en) * | 2016-06-24 | 2016-11-16 | 厦门大学 | The extracellular polysaccharide that one strain marine bacteria produces application in heavy metal adsorption |
| CN106244718A (en) * | 2016-10-09 | 2016-12-21 | 齐鲁工业大学 | A kind of method whether quick detection antibacterial produces polysaccharide |
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| CN1102151C (en) * | 1997-12-25 | 2003-02-26 | 中国科学院水生生物研究所 | Method for extracting and separating filiform blue-green algae water-soluble polyose and ecto-polyose |
| FR2780063B1 (en) * | 1998-06-22 | 2001-07-13 | Ifremer | PURIFIED POLYSACCHARIDE OF ALTEROMONAS MACLEODII AND USES THEREOF |
| KR100481679B1 (en) * | 2003-02-07 | 2005-04-07 | 한국해양연구원 | Alteromonas sp. 00SS11568 and method for preparation of extracellular polysaccharide therefrom |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105647990A (en) * | 2016-03-14 | 2016-06-08 | 山东省食品发酵工业研究设计院 | Microbial extracellular polysaccharide and method for preparing same |
| CN105647990B (en) * | 2016-03-14 | 2019-07-02 | 山东省食品发酵工业研究设计院 | A kind of Microbial exopolysaccharides and preparation method thereof |
| CN106119153A (en) * | 2016-06-24 | 2016-11-16 | 厦门大学 | The extracellular polysaccharide that one strain marine bacteria produces application in heavy metal adsorption |
| CN106244718A (en) * | 2016-10-09 | 2016-12-21 | 齐鲁工业大学 | A kind of method whether quick detection antibacterial produces polysaccharide |
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