CN105838647A - Shewanella frigidimarina strain and method for producing exopolysaccharide - Google Patents
Shewanella frigidimarina strain and method for producing exopolysaccharide Download PDFInfo
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Abstract
The invention discloses shewanella frigidimarina and a method for producing exopolysaccharide. The strain W32-2(shewanella frigidimarina) capable of being used for high-yield production of exopolysaccharide is separated and screened from gravel and sediment samples of the South Pole, and preserved in China General Microbiological Culture Collection Center on Feb 01, 2016 with the preservation number of CGMCC No.12129. A polar region source exopolysaccharide product with antioxidant activity and probiotic activity is prepared by means of the strain W32-2(shewanella frigidimarina), and the product is easy to prepare and has broad market prospects.
Description
Technical field
The present invention relates to a kind of microbial strains and application thereof, particularly relate to a kind of Mare Frigoris Shewanella bacterial strain (Shewanella frigidimarina) W32-2 and the production method of extracellular polysaccharide thereof.
Background technology
Along with the fast development of aquaculture, especially high-density breeding, the preventing and treating of aquiculture disease determines a key factor of economic well-being of workers and staff often.Along with the Ministry of Agriculture puts into effect relevant policies, antibiotic application on aquaculture will progressively be substituted, aquiculture disease prevention is the most gradually valued by the people, and " anti-overweighting is controlled " becomes aquaculture main flow idea.How aquiculture disease is effectively prevented, improve economic well-being of workers and staff simultaneously and realize green ecological cultivation, be the bottleneck problem of aquaculture industry.
Extracellular polysaccharide is the immunopotentiating agent of a kind of safety non-toxic; polar microorganism is because of extreme environment residing for it; big 5-50 times of the exopolysaccharide molecule amount of the secreted more general Marine microorganism of extracellular polysaccharide; it is effectively protected the ice crystal damage to cell under low temperature environment; and it imparts, compared to the specificity structure of Lu Yuan extracellular polysaccharide, the biological activity that source, polar region extracellular polysaccharide is special so that it is in occupation of irreplaceable critical role in extracellular polysaccharide researchs and develops.Generally believe whether health is to be directly connected to its resistance, the speed of growth and survival rate to the farming disease harms to aquatic animal intestinal in the world at present, using probiotic bacteria or prebiotics is the important method promoting cultivated animals intestinal health, in view of the biological activity that polar region extracellular polysaccharide is special so that it is the application in aquatic animal healthy aquaculture is possibly realized.But, there is the South Pole extracellular polysaccharide of antioxidation and prebiotic activity and be applied to the research of aquaculture and not yet have relevant report.So, those skilled in the art, in the urgent need to effectively preventing and treating aquaculture, promote preparation and the appearance of method of green health cultivation.
Summary of the invention
The technical problem to be solved be to provide a kind of Mare Frigoris Shewanella bacterial strain (Shewanella frigidimarina) W32-2, this Mare Frigoris Shewanella bacterial strain W32-2 was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on 02 01st, 2016, address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, deposit number is CGMCC No.12129, and uses this bacterial strain to prepare the production method of a kind of source, polar region extracellular polysaccharide goods with antioxidation and prebiotic activity.
The present invention is to solve above-mentioned technical problem by the following technical programs:
From South Pole sandy soil and sediment sample, separate and screen the bacterial strain obtaining high-yield extracellular polysaccharide, and use 16s rDNA method that this bacterial strain is carried out sequencing analysis, be finally identified as Mare Frigoris Shewanella (Shewanella frigidimarina) a bacterial strain.
Further, using this bacterial strain to prepare a kind of source, polar region extracellular polysaccharide goods with antioxidation and prebiotic activity, what it was prepared concretely comprises the following steps:
(1) activation of the Mare Frigoris Shewanella bacterial strain W32-2 described in: the Mare Frigoris Shewanella bacterial strain W32-2 described in taking also is seeded to inclined-plane 2216E culture medium, and cultivate 4-7 days at 8-15 DEG C；With inoculating loop, the Mare Frigoris Shewanella bacterial strain W32-2 after slant culture is seeded in 2216E culture medium, and in 8-15 DEG C, activation culture 4-7 days under the conditions of 150-200rpm；
(2) preparation of fermentation liquid: Mare Frigoris Shewanella W32-2 step (1) activated is seeded to fermentation culture in 2216E culture medium, inoculum concentration 1-10%, 150-200 rpm, and temperature is set as 8-15 DEG C；Ferment 4-7 days and then obtain required fermentation liquid；
(3) thalline is removed: take the fermentation liquid of step (2) gained and carry out 8000-10000rpm and be centrifuged 5-10min, draw fermented supernatant fluid standby；
(4) extracellular polysaccharide is isolated and purified: the fermented supernatant fluid of step (3) gained is carried out ethanol precipitation, add the dehydrated alcohol of 3 times of fermentation supernatant volumes, precipitation 3-6h, 5000-10000rpm is centrifuged 5-10min, incline supernatant, the polysaccharide precipitated by the deionized water dissolving of fermentation supernatant volume, gained solution Sevag method removes protein, repeat 3-5 time, after be centrifuged 5-10min in 5000-10000rpm, retain supernatant, supernatant adds 3 times of volume dehydrated alcohol, precipitation 3-6h, 5000-10000rpm is centrifuged 5-10min, incline supernatant, again with the polysaccharide of deionized water dissolution precipitation；
(5) extracellular polysaccharide finished product: the polysaccharide solution that step (4) is dissolved is carried out vacuum lyophilization, carry out afterwards subpackage pack described in there is the finished product of extracellular polysaccharide goods of antioxidation and prebiotic activity.
Further, the component of described 2216E culture medium is: peptone 5g, yeast extract 1g, high ferric phosphate 0.1g, Chen Haishui 1000 mL, pH 7.6；
Described Chen Haishui be never by land dirt contamination or sea water that the place that is seldom mixed with fresh water is fetched load the sea water formed after vial stores in the dark a few weeks longer.
The beneficial effects of the present invention is:
A kind of Mare Frigoris Shewanella bacterial strain of offer (Shewanella frigidimarina) W32-2, and utilize this Mare Frigoris Shewanella bacterial strain (Shewanella frigidimarina) W32-2 preparation has source, the polar region extracellular polysaccharide goods of antioxidation and prebiotic activity, and these goods can be applicable to aquatic green healthy aquaculture, can effectively reduce breeding process feed cost, promote aquatic animal intestinal health, improve aquatic animal premunition and survival rate has preferable market application foreground.
Detailed description of the invention
Heretofore described Mare Frigoris Shewanella bacterial strain (Shewanella frigidimarina) W32-2 was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on 02 01st, 2016, address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, and deposit number is CGMCC No.12129
The present invention a kind of Mare Frigoris Shewanella bacterial strain W32-2 is to separate and screen the bacterial strain obtaining high-yield extracellular polysaccharide from South Pole sandy soil and deposit, and uses 16s rDNA method that this bacterial strain is carried out sequencing analysis, be finally identified as Mare Frigoris Shewanella (Shewanella frigidimarina) a bacterial strain.
1. the separation of bacterial strain:
(1) primary dcreening operation: take 1g South Pole sandy soil and sediment sample, add in 10mL 2216E culture medium, take the turbid liquid in upper strata after concussion mixing and be placed in 2216E culture medium, and in 10 DEG C, enrichment culture under 180rpm, after 5 days, bacterium solution carries out gradient dilution to enrichment culture and spread plate carries out lock out operation, the antibacterial line of the different colonial morphologies grown on picking 2216E flat board afterwards is isolated and purified, saves backup.
(2) multiple sieve: the antibacterial obtained in picking step (1) primary dcreening operation respectively is placed in 2216E culture medium, in 10 DEG C, 150rpm cultivate 7 days, centrifugal 6min under 10000rpm afterwards, obtain fermented supernatant fluid, each fermentation supernatant takes 0.5mL and adds dehydrated alcohol by 3 times of volumes, precipitate 4 hours, after in 10000rpm, centrifugal 6min, abandon supernatant, precipitation is dissolved in 1mL deionized water the solution obtained and measures its polyoses content with Phenol sulfuric acid procedure respectively, and then filters out the bacterial strain that yield of extracellular polysaccharide is the highest, and this bacterial strain is labeled as bacterial strain W32-2.
2. the qualification of bacterial strain:
Screening obtained strains W32-2 is seeded to 2216E solid medium, cultivates at 37 DEG C, observe colonial morphology, and do the mensuration of some physiological-biochemical characteristics, result is as follows: bacterium colony milky, and smooth surface is opaque, neat in edge, Gram-negative, D-Glucose fermentation feminine gender, PEARLITOL 25C fermentation feminine gender, trypsin is positive, and chymase is negative.This bacterial strain is carried out 16S rDNA mensuration simultaneously, obtains its 16s rDNA sequence as shown in SEQ ID NO:1.
The 16s rDNA sequence inputting NCBI recorded is carried out homology search, find its similarity the highest for Mare Frigoris Shewanella (Shewanella frigidimarina)；Then combine physiological and biochemical test result and 16s rDNA sequence library comparison result, determine this bacterial strain be Mare Frigoris Shewanella (Shewanella frigidimarina).
3. having source, polar region extracellular polysaccharide goods for antioxidation and prebiotic activity, what it was prepared concretely comprises the following steps:
(1) activation of the Mare Frigoris Shewanella bacterial strain W32-2 described in: the Mare Frigoris Shewanella bacterial strain W32-2 described in taking also is seeded to inclined-plane 2216E culture medium, and cultivate 4-7 days at 8-15 DEG C；With inoculating loop, the Mare Frigoris Shewanella bacterial strain W32-2 after slant culture is seeded in 2216E culture medium, and in 8-15 DEG C, activation culture 4-7 days under the conditions of 150-200rpm；
(2) preparation of fermentation liquid: Mare Frigoris Shewanella W32-2 step (1) activated is seeded to fermentation culture in 2216E culture medium, inoculum concentration 1-10%, 150-200 rpm, and temperature is set as 8-15 DEG C；Ferment 4-7 days and then obtain required fermentation liquid；
(3) thalline is removed: take the fermentation liquid of step (2) gained and carry out 8000-10000rpm and be centrifuged 5-10min, draw fermented supernatant fluid standby；
(4) extracellular polysaccharide is isolated and purified: the fermented supernatant fluid of step (3) gained is carried out ethanol precipitation, add the dehydrated alcohol of 3 times of fermentation supernatant volumes, precipitation 3-6h, 5000-10000rpm is centrifuged 5-10min, incline supernatant, the polysaccharide precipitated by the deionized water dissolving of fermentation supernatant volume, gained solution Sevag method removes protein, repeat 3-5 time, after be centrifuged 5-10min in 5000-10000rpm, retain supernatant, supernatant adds 3 times of volume dehydrated alcohol, precipitation 3-6h, 5000-10000rpm is centrifuged 5-10min, incline supernatant, again with the polysaccharide of deionized water dissolution precipitation；
(5) extracellular polysaccharide finished product: the polysaccharide solution that step (4) is dissolved is carried out vacuum lyophilization, carry out afterwards subpackage pack described in there is the finished product of extracellular polysaccharide goods of antioxidation and prebiotic activity.
Further, the component of described 2216E culture medium is: peptone 5g, yeast extract 1g, high ferric phosphate 0.1g, Chen Haishui 1000 mL, pH 7.6；
Described Chen Haishui be never by land dirt contamination or sea water that the place that is seldom mixed with fresh water is fetched load the sea water formed after vial stores in the dark a few weeks longer.
In order to preferably source, polar region extracellular polysaccharide goods in the present invention are further elaborated explanation, applicant illustrates below embodiment.
Embodiment one
(1) activation of the Mare Frigoris Shewanella bacterial strain W32-2 described in: the Mare Frigoris Shewanella bacterial strain W32-2 described in taking also is seeded to inclined-plane 2216E culture medium, and cultivate 5 days at 10 DEG C；With inoculating loop, the Mare Frigoris Shewanella bacterial strain W32-2 after slant culture is seeded in 10mL 2216E culture medium, and in 10 DEG C, activation culture 5 days under the conditions of 150rpm；
(2) preparation of fermentation liquid: Mare Frigoris Shewanella W32-2 step (1) activated is seeded to fermentation culture in 1L 2216E culture medium, inoculum concentration 5%, 150 rpm, and temperature is set as 10 DEG C；Ferment and then obtain required fermentation liquid in 5 days；
(3) thalline is removed: take the fermentation liquid of step (2) gained and carry out 8000rpm and be centrifuged 10min, draw fermented supernatant fluid standby；
(4) extracellular polysaccharide is isolated and purified: the fermented supernatant fluid of step (3) gained is carried out ethanol precipitation, add the dehydrated alcohol of 3 times of fermentation supernatant volumes, precipitate 3 hours, 10000rpm is centrifuged 10min, incline supernatant, the polysaccharide precipitated by the deionized water dissolving of fermentation supernatant volume, gained solution with Sevag method remove protein, be repeated 3 times, after be centrifuged 10min in 5000rpm, retain supernatant, supernatant adds 3 times of volume dehydrated alcohol, precipitates 3 hours, and 5000rpm is centrifuged 10min, incline supernatant, then the polysaccharide with deionized water dissolution precipitation；
(5) extracellular polysaccharide finished product: the polysaccharide solution that step (4) is dissolved is carried out vacuum lyophilization, carry out afterwards subpackage pack described in there is the finished product of source, polar region extracellular polysaccharide goods of antioxidation and prebiotic activity.
Embodiment two
(1) activation of the Mare Frigoris Shewanella bacterial strain W32-2 described in: the Mare Frigoris Shewanella bacterial strain W32-2 described in taking also is seeded to inclined-plane 2216E culture medium, and cultivate 7 days at 15 DEG C；With inoculating loop, the Mare Frigoris Shewanella bacterial strain W32-2 after slant culture is seeded in 10mL 2216E culture medium, and in 15 DEG C, activation culture 7 days under the conditions of 180rpm；
(2) preparation of fermentation liquid: Mare Frigoris Shewanella W32-2 step (1) activated is seeded to fermentation culture in 1L 2216E culture medium, inoculum concentration 10%, 180 rpm, and temperature is set as 15 DEG C；Ferment and then obtain required fermentation liquid in 7 days；
(3) thalline is removed: take the fermentation liquid of step (2) gained and carry out 10000rpm and be centrifuged 5min, draw fermented supernatant fluid standby；
(4) extracellular polysaccharide is isolated and purified: the fermented supernatant fluid of step (3) gained is carried out ethanol precipitation, add the dehydrated alcohol of 3 times of fermentation supernatant volumes, precipitate 6 hours, 10000rpm is centrifuged 5min, incline supernatant, the polysaccharide precipitated by the deionized water dissolving of fermentation supernatant volume, gained solution with Sevag method remove protein, be repeated 5 times, after be centrifuged 5min in 10000rpm, retain supernatant, supernatant adds 3 times of volume dehydrated alcohol, precipitates 6 hours, and 10000rpm is centrifuged 5min, incline supernatant, then the polysaccharide with deionized water dissolution precipitation；
(5) extracellular polysaccharide finished product: the polysaccharide solution that step (4) is dissolved is carried out vacuum lyophilization, carry out afterwards subpackage pack described in there is the finished product of source, polar region extracellular polysaccharide goods of antioxidation and prebiotic activity.
4. the Oxidation Resistance Test of extracellular polysaccharide goods
Test one: DPPH clearance rate is measured by extracellular polysaccharide goods
(1) test method: the extracellular polysaccharide 0.1g of Example one preparation respectively, the extracellular polysaccharide 0.2g of embodiment two preparation, is all dissolved in 10mL deionized water, makes polysaccharide solution；Take solution 2mL to be measured, add 0.2mmol/L DPPH and dehydrated alcohol 1mL, at room temperature lucifuge reaction 30min after mixing, after at 5000rpm, centrifugal 10min takes supernatant and measures OD517 value and (remember A1), substitute polysaccharide solution with equal-volume deionized water and measure OD517 value (note A by above-mentioned same operation0), it is respectively provided with after three repetitions according to the following formula:
DPPH clearance rate=[1-(A0- A1)/A0】×100%
(2) result of the test: the polysaccharide solution that in 0.1g embodiment one, the extracellular polysaccharide of preparation is dissolved in 10mL deionized water is 36.22 ± 0.17% to DPPH free radical scavenging activity；The polysaccharide solution clearance rate free to DPPH that in 0.2g embodiment two, the extracellular polysaccharide of preparation is dissolved in 10mL deionized water is 56.54 ± 0.33%.
Test two: extracellular polysaccharide goods are to hydroxy radical (HO·) clearance rate mensuration
(1) test method: the extracellular polysaccharide 0.1g of Example one preparation respectively, the extracellular polysaccharide 0.2g of embodiment two preparation, is all dissolved in 10mL deionized water, makes polysaccharide solution standby；It is sequentially added into 1mL 5mmol/L ferrous sulfate in tool plug test tube, 1mL 5mmol/L salicylic acid-ethanol solution, 1mL 3mmol/L hydrogen peroxide solution, 0.5mL extracellular polysaccharide solution is added after mixing, it is settled to 10mL with deionized water, in 37 DEG C of water bath with thermostatic control 15min, after be centrifuged 10min at 6000rpm, measure OD510 value and (remember Ai), substitute polysaccharide solution with equal-volume deionized water and measure OD510 value (note A by above-mentioned same operationj), it is respectively provided with after three repetitions according to the following formula:
Scavenging action to hydroxyl free radical=[1-(Aj- Ai)/Aj】×100%
(2) result of the test: the polysaccharide solution that in 0.1g embodiment one, the extracellular polysaccharide of preparation is dissolved in 10mL deionized water is 45.69 ± 0.85% to Scavenging action to hydroxyl free radical；The polysaccharide solution clearance rate free to hydroxyl that in 0.2g embodiment two, the extracellular polysaccharide of preparation is dissolved in 10mL deionized water is 75.26 ± 0.41%.
5. extracellular polysaccharide compound probiotic bacteria mix raise be applied to aquaculture test
Test one: extracellular polysaccharide compound probiotic bacteria mix raise be applied to breeding soft-shell turtle test
(1) test method: the extracellular polysaccharide that Example one prepares compounds, by 1:10, symphysis unit sample that probiotic powder is configured to and is used in cultivating in Trionyx sinensis Wiegmann pond: design one group of matched group and two groups of experimental grouies；Experimental group 1 adds, by the bait amount of mixing of 4g/kg, the symphysis unit sample prepared in soft-shelled turtle feed, mixes bait and once feed every day；Experimental group 2 adds probiotic powder by the bait amount of mixing of 4g/kg in soft-shelled turtle feed；Matched group is the identical bait of blank, i.e. feed but without probiotic bacteria/symphysis unit；After testing 5 months, calculate feed coefficient and the survival rate of Trionyx sinensis Wiegmann in Trionyx sinensis Wiegmann pond.
By calculating result of the test it is: blank group feed coefficient 1.53, survival rate 80.1%；And experimental group 1 feed coefficient 1.12, survival rate 92.9%；Experimental group 2 feed coefficient is 1.28, and survival rate is 88.6%.
Test two: extracellular polysaccharide compound probiotic bacteria mix raise be applied to culture of Penaeus vannamei test
(1) test method: the extracellular polysaccharide that Example two prepares compounds, by 1:5, symphysis unit sample that probiotic powder is configured to and is used in cultivating in Penaeus vannamei pond: design one group of matched group and two groups of experimental grouies；Experimental group 1 adds, by the bait amount of mixing of 6g/kg, the symphysis unit sample prepared in penaeus vannamei boone feed, mixes bait and once feed every day；Experimental group 2 adds probiotic powder by the bait amount of mixing of 6g/kg in penaeus vannamei boone feed；Matched group is the identical bait of blank, i.e. feed but without probiotic bacteria/symphysis unit；After testing 3 months, calculate feed coefficient and the survival rate of prawn in Penaeus vannamei pond.
(2) result of the test: be by calculating result of the test: blank group feed coefficient 1.34, survival rate 78.3%；And experimental group 1 feed coefficient 0.97, survival rate 90.4%；Experimental group 2 feed coefficient is 1.18, and survival rate is 87.1%.
In summary, the present invention utilizes Mare Frigoris Shewanella bacterial strain W32-2(Shewanella frigidimarina W32-2) prepare source, the polar region extracellular polysaccharide goods of gained, all have preferable Scavenging activity for DPPH free radical and hydroxy radical, have higher antioxidant activity；Source, polar region extracellular polysaccharide goods coordinate probiotic bacteria to use, and as feed additive in aquaculture process, can be effectively reduced feed coefficient, improve survival rate, have preferable production application prospect.
<110>State Oceanic Administration Bureau The Third Oceanography Institute
<120>a kind of Mare Frigoris Shewanella bacterial strain and the production method of extracellular polysaccharide thereof
<160>1
<211>1447
<212>DNA
<213>Mare Frigoris Shewanella kind (Shewanella frigidimarina)
<400>
cccaatgccg gccgccctac
catgcagtcg agcggtaaca caagggagct tgctcctgag 60
gtgacgagcg gcggacgggt
gagtaatgcc tagggatctg cccagtcgat ggggataaca 120
gttggaaacg actgctaata
ccgcatacgc cctacggggg aaaggagggg accttcgggc 180
cttccgcgac aggatgaacc
taggtgggat tagctagttg gtgaggtaat ggctcaccaa 240
ggcgacgatc cctagctgtt
ctgagaggat gatcagccac actgggactg agacacggcc 300
cagactccta cgggaggcag
cagtggggaa tattgcacaa tgggggaaac cctgatgcag 360
ccatgccgcg tgtgtgaaga
aggccttcgg gttgtaaagc actttcagta gggaggaaag 420
gtagcgtgtt aatagcccgt
tactgtgacg ttacctacag aagaaggacc ggctaactcc 480
gtgccagcag ccgcggtaat
acggagggtc cgagcgttaa tcggaattac tgggcgtaaa 540
gcgtgcgcag gcggtttgtt
aagccagatg tgaaatcccc gggctcaacc tgggaattgc 600
atttggaact ggcgaactag
agtcttgtag aggggggtag aattccaggt gtagcggtga 660
aatgcgtaga tatctggagg
aataccggtg gcgaaggcgg ccccctggac aaagactgac 720
gctcatgcac gaaagcgtgg
ggagcaaaca ggattagata ccctggtagt ccacgccgta 780
aacgatgtct actcggagtt
tggtgactta gtcactgggc tcccaagcta acgcattaag 840
tagaccgcct ggggagtacg
gccgcaaggt taaaactcaa atgaattgac gggggcccgc 900
acaagcggtg gagcatgtgg
tttaattcga tgcaacgcga agaaccttac ctactcttga 960
catccacaga agagaccaga
gatggacttg tgccttcggg aactgtgaga caggtgctgc 1020
atggctgtcg tcagctcgtg
ttgtgaaatg ttgggttaag tcccgcaacg agcgcaaccc 1080
ctatccttat ttgccagcgc
gtaatggcgg gaactctagg gagactgccg gtgataaacc 1140
ggaggaaggt ggggacgacg
tcaagtcatc atggccctta cgagtagggc tacacacgtg 1200
ctacaatggc aagtacagag
ggttgcaaag ccgcgaggtg gagctaatct cacaaagctt 1260
gtcgtagtcc ggatcggagt
ctgcaactcg actccgtgaa gtcggaatcg ctagtaatcg 1320
tggatcagaa tgccacggtg
aatacgttcc cgggccttgt acacaccgcc cgtcacacca 1380
tgggagtggg ctgcaaaaga
agtgggtagt ttaaccttcg ggagaacgct caccactttt 1440
gtggcgg 1447
Claims (4)
1. a Mare Frigoris Shewanella bacterial strain, it is characterised in that described bacterial strain be Mare Frigoris Shewanella bacterial strain (Shewanella frigidimarina) W32-2, it being deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on 02 01st, 2016, deposit number is CGMCC No.
12129。
2. the Mare Frigoris Shewanella bacterial strain preparation that a kind utilizes described in claim 1 has the production method of the extracellular polysaccharide of antioxidant activity and prebiotic activity, it is characterised in that comprise the following steps:
(1) activation of the Mare Frigoris Shewanella bacterial strain W32-2 described in claim 1: take the Mare Frigoris Shewanella bacterial strain W32-2 described in claim 1 and be seeded to inclined-plane 2216E culture medium, and cultivate 4-7 days at 8-15 DEG C；With inoculating loop, the Mare Frigoris Shewanella bacterial strain W32-2 after slant culture is seeded in 2216E culture medium, and in 8-15 DEG C, activation culture 4-7 days under the conditions of 150-200rpm；
(2) preparation of fermentation liquid: Mare Frigoris Shewanella W32-2 step (1) activated is seeded to fermentation culture in 2216E culture medium, inoculum concentration 1-10%, 150-200 rpm, and temperature is set as 8-15 DEG C；Ferment 4-7 days and then obtain required fermentation liquid；
(3) thalline is removed: take the fermentation liquid of step (2) gained and carry out 8000-10000rpm and be centrifuged 5-10min, draw fermented supernatant fluid standby；
(4) extracellular polysaccharide is isolated and purified: the fermented supernatant fluid of step (3) gained is carried out ethanol precipitation, add the dehydrated alcohol of 3 times of fermentation supernatant volumes, precipitation 3-6h, 5000-10000rpm is centrifuged 5-10min, incline supernatant, the polysaccharide precipitated by the deionized water dissolving of fermentation supernatant volume, gained solution Sevag method removes protein, repeat 3-5 time, after be centrifuged 5-10min in 5000-10000rpm, retain supernatant, supernatant adds 3 times of volume dehydrated alcohol, precipitation 3-6h, 5000-10000rpm is centrifuged 5-10min, incline supernatant, again with the polysaccharide of deionized water dissolution precipitation；
(5) extracellular polysaccharide finished product: the polysaccharide solution that step (4) is dissolved is carried out vacuum lyophilization, carry out afterwards subpackage pack described in there is the finished product of extracellular polysaccharide goods of antioxidation and prebiotic activity.
The production method of a kind of extracellular polysaccharide the most as claimed in claim 2, it is characterised in that: the component of described 2216E culture medium is: peptone 5g, yeast extract 1g, high ferric phosphate 0.1g, Chen Haishui 1000 mL, pH 7.6.
The application in aquaculture feed of the production method gained extracellular polysaccharide of a kind of extracellular polysaccharide the most as claimed in claim 2.
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| CN201610299913.7A CN105838647B (en) | 2016-05-09 | 2016-05-09 | A kind of production method of Mare Frigoris Shewanella bacterial strain and its exocellular polysaccharide |
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| CN201610299913.7A CN105838647B (en) | 2016-05-09 | 2016-05-09 | A kind of production method of Mare Frigoris Shewanella bacterial strain and its exocellular polysaccharide |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111154701A (en) * | 2020-02-24 | 2020-05-15 | 荣成市泓派海洋生物科技有限公司 | Bacterial strain for producing alginate lyase and cellulase and application of bacterial strain in kelp fermentation |
| CN111440807A (en) * | 2020-04-07 | 2020-07-24 | 上海海洋大学 | Shewanella WP3 mutant strain with high yield of low-temperature catalase as well as construction method and application thereof |
| CN115772488A (en) * | 2022-12-20 | 2023-03-10 | 中国水产科学研究院黄海水产研究所 | Shewanella decolorationis for producing tetrodotoxin and application thereof |
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| CN102715357A (en) * | 2011-12-28 | 2012-10-10 | 大连海宝渔业有限公司 | Use of Shewanella as feed additive in abalone culture |
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| CN102715357A (en) * | 2011-12-28 | 2012-10-10 | 大连海宝渔业有限公司 | Use of Shewanella as feed additive in abalone culture |
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| MIAOMIAO SHI ET AL.: "Exoprotein production correlates with Morphotype changes of nonomotie shewanella oneidensis", 《JOURNAL OF BACTERIOLOGY》 * |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111154701A (en) * | 2020-02-24 | 2020-05-15 | 荣成市泓派海洋生物科技有限公司 | Bacterial strain for producing alginate lyase and cellulase and application of bacterial strain in kelp fermentation |
| CN111440807A (en) * | 2020-04-07 | 2020-07-24 | 上海海洋大学 | Shewanella WP3 mutant strain with high yield of low-temperature catalase as well as construction method and application thereof |
| CN115772488A (en) * | 2022-12-20 | 2023-03-10 | 中国水产科学研究院黄海水产研究所 | Shewanella decolorationis for producing tetrodotoxin and application thereof |
| CN115772488B (en) * | 2022-12-20 | 2023-05-09 | 中国水产科学研究院黄海水产研究所 | Shewanella decolorationis producing tetrodotoxin and application thereof |
| US12116568B2 (en) | 2022-12-20 | 2024-10-15 | Yellow Sea Fisheries Research Institute, Chinese Academy Of Fishery Sciences | Shewanella decolorationis producing tetrodotoxin and application thereof |
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