CN109777752A - The apex pulmonis Psychrobacter T-15 bacterial strain and application thereof of one plant of production polysaccharide - Google Patents
The apex pulmonis Psychrobacter T-15 bacterial strain and application thereof of one plant of production polysaccharide Download PDFInfo
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Abstract
The present invention provides a kind of T-15 bacterial strains for producing polysaccharide, belong to a kind of apex pulmonis Psychrobacter (Psychrobacter pulmonis strain), which is preserved in China typical culture collection center, address: China, Wuhan, Wuhan University；Postcode 430072, the deposit date is on January 17th, 2019, deposit numbers are as follows: CCTCC M 2019056；The 16S rDNA sequence of the bacterial strain is as shown in SEQ ID NO.1.Above-mentioned apex pulmonis Psychrobacter T-15 bacterial strain can be used in fermentation and prepare polysaccharide.Bacterial strain provided by the invention can carry out large scale fermentation culture in a short time, and fermentation costs are lower, it is not limited again by conditions such as region, seasons, therefore the increasingly deficient status of water shield natural resources can be solved by the approach of microbial fermentation, to realize higher economic benefit.
Description
Technical field
The present invention relates to a kind of apex pulmonis Psychrobacter bacterial strain, which can produce polysaccharide, belong to microorganisms technical field.
Background technique
The research of endophyte of plant is gradually taken seriously at present, and the various endophytes of different plants are found in succession.Interior life
The diversity of strain class also gives the diversity of its metabolite, this, which is also implied, excavates biology using microbial metabolism, changes
The new resources such as, medicine have huge potentiality.It is a large amount of studies have shown that endophyte of plant can produce it is identical as host plant or
Similar active material and precursor, this discovery have pushed directly on the especially economical endophyte of plant research of endophyte of plant
Expansion extensively.Gradually research with people to economical endophyte of plant generates corresponding active constituent using that can be metabolized
Endophyte produces novel, efficient, inexpensive active medicine, solves rare economical plant resources status in short supply, will
Emphasis as economical endophyte of plant research in the future.
Although the concern increasingly by numerous scholars in recent years of the especially economical endophyte of plant of endophyte of plant,
But the endophyte research of several hundred kinds of plants is still only carried out at present.Water shield (Brasenia schreberi) also known as horseshoe
Dish, lake dish, water certain herbaceous plants with big flowers, dew certain herbaceous plants with big flowers, water lotus leaf, flower case dish etc. are that perennial root floats leaf water plant, belong to Nymphaeceae water shield category.Water shield
The stickiness pectin main component on dish surface is stickiness polysaccharide, has high nutritive value.Water shield is a kind of important aquatic warp
Ji plant, the requirement to water quality are very high.It is worsening instantly in the wild border environment of water shield, find a kind of Polysaccharide of Brasenia Schreberi production
Alternative route be particularly important.
Water shield endophyte just starts to be concerned by people in recent years, on the whole, especially about water shield endophyte
Can be metabolized generate corresponding Polysaccharide of Brasenia Schreberi ingredient research it is still relatively fewer, rarely have about produce Polysaccharide of Brasenia Schreberi water shield endophyte
Report.
Summary of the invention
The present invention solves the problems in background technique, provides a kind of apex pulmonis Psychrobacter T-15 bacterial strain for producing polysaccharide,
The bacterial strain can produce Polysaccharide of Brasenia Schreberi.
The present inventor screens one plant of novel strain, which is named as T-15, belongs to a kind of apex pulmonis Psychrobacter
(Psychrobacter pulmonis strain), which is preserved in China typical culture collection center, address: China,
Wuhan, Wuhan University；Postcode 430072, the deposit date is on January 17th, 2019, deposit numbers are as follows: CCTCC M 2019056；
The 16S rDNA sequence of the bacterial strain is as shown in SEQ ID NO.1.
Above-mentioned apex pulmonis Psychrobacter T-15 bacterial strain can be used in fermentation and prepare polysaccharide.
Using above-mentioned apex pulmonis Psychrobacter T-15 bacterial strain prepare polysaccharide method the following steps are included: first by apex pulmonis it is thermophilic cold
Bacillus T-15 strain inoculated is in LB liquid medium, the overnight shaking culture 2 days of 37 DEG C of 200r/min revolving speed, by zymocyte liquid in
12000r/min centrifugation, takes supernatant；Supernatant and chloroform-butanol solution are mixed in centrifuge tube, 20-30min is vibrated,
Supernatant is taken after being centrifuged again, by supernatant with twice of membrane filtration of 0.45 μm, heating is concentrated into original volume at 70 DEG C after filtering
1/2~2/3,95% ethyl alcohol of 4 times of volumes is added, be put into refrigerator freezing stay overnight, finally with 12000r/min revolving speed be centrifuged
10min, precipitating generated are polysaccharide.
Compared with prior art, the invention has the following advantages that apex pulmonis Psychrobacter T-15 bacterial strain provided by the present invention
Generation polysaccharide can be metabolized.It is metabolized the polysaccharide generated and the polysaccharide HPLC having the same isolated and purified from natural water shield
Wave spectrum peak and similar polysaccharide infrared spectrum characteristic peak and similar or identical chemical functional group.And the strain fermentation system
It is more that the biological activity of standby polysaccharide is significantly higher than natural water shield compared with natural Polysaccharide of Brasenia Schreberi, to the removing effect of hydroxy radical
Sugar has better antioxidant activity.
Bacterial strain provided by the invention can carry out large scale fermentation culture in a short time, and fermentation costs are lower, but not by
The limitation of the conditions such as region, season, therefore showing for water shield natural resources increasingly scarcity can be solved by the approach of microbial fermentation
Shape, to realize higher economic benefit.
Detailed description of the invention
Fig. 1 is the colonial morphology figure of bacterial strain provided by the invention in the medium；
Fig. 2 is efficient liquid phase (HPLC) analysis of Polysaccharide of Brasenia Schreberi extract and strain fermentation product polysaccharide provided by the invention
Figure；
Fig. 3 is the infrared spectrum analysis figure of strain fermentation product polysaccharide provided by the invention.
Specific embodiment
Detailed specific description done to the present invention combined with specific embodiments below, but protection scope of the present invention not office
It is limited to following embodiment.
The separation of water shield endophyte
Stem, the Ye Hegen for taking fresh water shield are first rinsed with distilled water with mercuric chloride disinfection several again under sterile environment
Time, it is inoculated on LB solid medium, is protected from light culture.Go out well-grown bacterium colony from culture medium picking and moves to new LB culture
In base plate, is purified, numbered respectively.The Strain Designation provided in the present embodiment is T-15 bacterial strain, by the bacterium of the bacterial strain
It falls and moves to well-grown in new LB culture medium flat plate, colonial morphology figure is as shown in Figure 1, Fig. 1 medium scale=2mm.From Fig. 1
In it can be seen that diameter is about 3-5mm, bacterium colony is in more regular circle, milky.
The identification of water shield endophyte
The endophyte isolated and purified (is extracted DNA or boil processing) after processing, expands bacterial 16 S rDNA sequence
(use 27F/1492R primer), amplified production carry out sanger sequencing, sequencing result the website NCBI (https: //
Blast.ncbi.nlm.nih.gov/Blast.cgi analysis) is compared, with homology 97% or more and the highest object of homology
Kind is judged as the kind of endophyte.
16S rDNA sequence amplification primer sequence:
1 27F:5 '-AGAGTTTGATCCTGGCTCAG-3 ' of primer
Primer 2 1492R:5 '-TACGGCTACCTTGTTACGACTT-3 '
Amplified fragments size about 1400-1700bp
According to the standard sequencing methods of bacterium, the sequence of above-mentioned water shield endosymbiosis bacterium is measured as shown in SEQ ID NO.1, is surveyed
Sequence result is compared on the website NCBI, the results showed that, the water shield endosymbiosis bacterium provided in the present embodiment is bacterium, interior
The Latin generic name of raw bacterium is apex pulmonis Psychrobacter category actinomyces (Psychrobacter pulmonis strain).
The bacterial strain is preserved in China typical culture collection center by applicant, address: China, Wuhan, Wuhan University；Postal
430072 are compiled, the deposit date is on January 17th, 2019, patented strain deposit numbers are as follows: CCTCC M 2019056.
The preparation of water shield endophyte polysaccharide
The strain inoculated for being partially transferred to purifying is picked them separately in LB liquid medium from the bacterium in LB culture medium flat plate,
Zymocyte liquid is centrifuged 5 minutes in 12000r/min, takes supernatant by the overnight shaking culture 2 days of 37 DEG C of 200r/min revolving speed.Configuration
Supernatant is mixed by 3:1 with centrifuge tube with organic solution, is vibrated by chloroform-butanol solution (chloroform: n-butanol=4:1)
Supernatant is taken after 20-30min, 8000r/min centrifugation 10min, pays attention to the protein precipitation that cannot be drawn between two-phase.Continue by upper
It states operation and carries out 5 removing proteins.By supernatant with twice of membrane filtration of 0.45 μm, heating is concentrated into original at 70 DEG C after filtering
95% ethyl alcohol of 4 times of volumes is added in the 1/2-2/3 of volume, be put into refrigerator freezing stay overnight, second day with 12000r/min revolving speed from
Heart 10min, precipitating are endophyte polysaccharide.
Whether the fermentation liquid for water shield endophyte more provided by the invention contains polysaccharide, internally raw fermented liquid extract
Measurement of the polysaccharide content is carried out.
Thick many candies are extracted and isolated first from natural water shield as object of reference, the method is as follows: are taken water shield tender shoots, be put into
Extraction in NaOH (0.1mol/L), solid-liquid ratio 1:2 (w/v), 25 DEG C of lye adjust pH to 7.0 after extracting 1.5h.Choose water shield bud,
It is centrifugated residue with centrifuge, with magnetic stirring apparatus by supernatant in 60 DEG C of concentration 1h.Then it is handled repeatedly with sevage method
The crude extract of concentration removes free protein, is again concentrated to 2/3 of original volume or so.Four times of volume dehydrated alcohol precipitatings are added
Overnight, mixture is then centrifuged 10min with 4000r/min to obtain sediment, is concentrated again after being precipitated with the dissolution of a small amount of water
Four times of volume ethanol precipitations are added to stay overnight, repetitive operation is three times, finally dry to obtain Polysaccharide of Brasenia Schreberi (BSP).
Measuring method is as follows: by polysaccharide after sulphuric acid hydrolysis, measuring bacterium using DNS method (3- amino -5-NITROSALICYLIC ACID)
Secretion sugared content in liquid is tried by the glucose standard of various concentration and DNS first using D-Glucose aldehydic acid as standard
Absorbance after agent, sulfuric acid reaction makes standard curve.After measured, standard curve is y=0.0368x-0.0436 (R2=
0.9807).According to standard curve, the content of polysaccharide is measured.The experimental results showed that in BSP sample polysaccharide content be 0.30 ±
0.01mg/mL, the polyoses content in apex pulmonis Psychrobacter category actinomyces T-15 strain fermentation extract are 0.06 ± 0.001mg/
mL。
The Performance Testing of polysaccharide component
Whether to compare water shield endophyte polysaccharide and Polysaccharide of Brasenia Schreberi there are also identical polysaccharide component, the present embodiment is carried out
HPLC analysis.As a result as shown in Fig. 2, wherein upper figure is endophyte polysaccharide in the present embodiment, the following figure is the natural water shield extracted
Polysaccharide, from figure 2 it can be seen that apex pulmonis Psychrobacter category actinomyces polyoses extract has a wave spectrum peak in 19min,
There is a subwave spectral peak in 21min, it is notable that the HPLC appearance at secondary pop peak and Polysaccharide of Brasenia Schreberi that 21min occurs
Time is similar, shows water shield endosymbiosis bacterium in addition to generating specific polysaccharide peak value, it is similar with Polysaccharide of Brasenia Schreberi also to have generation
The ability of polysaccharide component.
The structural analysis of Polysaccharide of Brasenia Schreberi is compared with the carbohydrate of endophyte
To further determine that water shield endophyte polysaccharide and Polysaccharide of Brasenia Schreberi whether also containing identical chemical functional group, this implementation
Further progress infrared spectrum analysis in example.
The strain inoculated for being partially transferred to purifying is picked them separately in LB liquid medium from the bacterium in LB culture medium flat plate,
The overnight shaking culture 2 days of 37 DEG C of 200r/min revolving speed.Bacterium solution is centrifuged, supernatant removes deproteinized with sevage method, then with 4 times of bodies
Product alcohol chromatography, is separated by filtration precipitating, whether has glycosidic bond in infrared spectrum analysis precipitating or alcohol analysis liquid.Use infrared spectroscopy
The polysaccharide extracted from water shield is analyzed, is made comparisons with the map of the precipitating containing glycosidic bond or alcohol analysis liquid.
The polyoses content of BSP is measured using phend-sulphuric acid, uses D-Glucose as standard to measure.Firstly, using
D-Glucose prepares contrast solution (0.1mg/mL).Secondly, by reference solution be put into pipe (0.2mL, 0.4mL, 0.6mL,
0.8mL and 1.0mL) in, phenol solution, which is added, keeps its static.Then being rapidly added the concentrated sulfuric acid makes its concussion.Test tube is placed
Then 10min measures absorbance until they are cooled to room temperature at 490nm.Redistilled water is used as blank control.As a result it uses
Linear regression measurement.Then the level of absorbance of sample is measured to determine the content of uronic acid.
Infrared spectrum analysis figure is as shown in figure 3, show that water shield endophyte polysaccharide provided in this embodiment has 9 in Fig. 3
Characteristic absorption peak.It can be seen that from infrared spectrogram, 3392.79cm-1The absorption peak at place is the stretching vibration absworption peak of-OH,
2924.09cm-1It is the stretching vibration absworption peak of C-H, this is the typical wave spectrum peak of two polysaccharide, the typical wave of this and Polysaccharide of Brasenia Schreberi
Spectral peak is similar；2854.65cm-1、1558.48cm-1And 1458.18cm-The absorption peak at place is that the stretching vibration of the C-H on phenyl ring is inhaled
Peak is received, is water shield endosymbiosis bacterium distinctive polysaccharide wave spectrum peak (table 3)；1653.00cm-1The absorption peak at place is C=O bond (C=
O) absorption peak；1404.18cm-1, 551.64cm-1The absorption peak at place is the stretching vibration absworption peak of C-H.It is specifically intended that
1083.99cm-1The absorption peak at place is absorption peak associated with glycosidic inkage C-O-H, is the characteristic peak of beta glucan.
Compared with Polysaccharide of Brasenia Schreberi, water shield endosymbiosis bacterium produces polysaccharide in 3392.79cm-1And 2924.09cm-1It is having the same
Polysaccharide absorption peak, while having unique characteristic peak (in 2854.65cm again-1、1558.48cm-1And 1458.18cm-1).Water shield
Endosymbiosis bacterium and Polysaccharide of Brasenia Schreberi, in 1083.99cm-1There is the characteristic peak of identical beta glucan at place.Separate sources polysaccharide infrared light
Spectrum analysis and its characteristic peak parsing are as shown in the table:
The active measurement of polysaccharide anti-oxidative
The present embodiment detects Polysaccharide of Brasenia Schreberi extract using the hydroxy radical kit that biology offer is built up in Nanjing.
Polysaccharide sample is uniformly mixed with reagent, in 37 DEG C accurate response 1 minute, immediately be added color developing agent terminate reaction, be uniformly mixed
Afterwards, 20 minutes are placed at room temperature for.Spectrophotometer is returned to zero with distilled water, the absorbance value of each pipe is surveyed at 550nm.Pass through experiment
As the result is shown to the Scavenging activity of hydroxy radical, the Partial Antioxidation activity of Polysaccharide of Brasenia Schreberi is obtained.
The importance of polysaccharide is embodied on its physiological activity.Hydroxy radical (OH) is a kind of very strong freedom of oxidability
Base, property is very active, and the rate for aoxidizing various organic matters and inorganic matter is exceedingly fast, and is to cause tissue lipid peroxidating, nucleic acid disconnected
It splits, protein and how glycolytic principal element, it is related with body aging, tumour, radiation injury and cell phagocytic activity.For into
One step determines the biological activity of water shield endosymbiosis bacterium polysaccharide, generates system in the present embodiment with hydroxy radical and compares in water shield
The antioxidant activity of symbiosis granulose.Result of study shows that the supression of generation of the water shield endosymbiosis bacterium polysaccharide to hydroxy radical is made
Reach in the case of 1.0mg/mL most strong (as shown in the table), and the inhibition that Polysaccharide of Brasenia Schreberi generates hydroxy radical with
The raising of concentration and increase (as shown in the table).Experimental result shows that water shield endosymbiosis bacterium polysaccharide imitates the removing of hydroxy radical
Polysaccharide of Brasenia Schreberi should be significantly higher than.
Sequence table
<110>South-Center University For Nationalities
The apex pulmonis Psychrobacter T-15 bacterial strain and application thereof of<120>one plants of production polysaccharide
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1459
<212> DNA
<213>apex pulmonis Psychrobacter (Psychrobacter pulmonis strain)
<400> 1
gaatggcggg ccgctataat gctagtcgag cgaacagata aggagcttgc tcctttgacg 60
ttagcggcgg acgggtgagt aacacgtgga taacctacct ataagactgg gataacttcg 120
ggaaaccgga gctaataccg gataacatat tgaaccgcat ggttcaatag tgaaaggcgg 180
ctttgctgtc acttatagat ggatccgcgc cgtattagct agttggtaag gtaacggctt 240
accaaggcaa cgatacgtag ccgacctgag agggtgatcg gccacactgg aactgagaca 300
cggtccagac tcctacggga ggcagcagta gggaatcttc cgcaatgggc gaaagcctga 360
cggagcaacg ccgcgtgagt gatgaaggtc ttcggatcgt aaaactctgt tatcagggaa 420
gaacaaatgt gtaagtaact gtgcacatct tgacggtacc tgatcagaaa gccacggcta 480
actacgtgcc agcagccgcg gtaatacgta ggtggcaagc gttatccgga attattgggc 540
gtaaagcgcg cgtaggcggt tttttaagtc tgatgtgaaa gcccacggct caaccgtgga 600
gggtcattgg aaactggaaa acttgagtgc agaagaggaa agtggaattc catgtgtagc 660
ggtgaaatgc gcagagatat ggaggaacac cagtggcgaa ggcgactttc tggtctgtaa 720
ctgacgctga tgtgcgaaag cgtggggatc aaacaggatt agataccctg gtagtccacg 780
ccgtaaacga tgagtgctaa gtgttagggg gtttccgccc cttagtgctg cagctaacgc 840
attaagcact ccgcctgggg agtacgaccg caaggttgaa actcaaagga attgacgggg 900
acccgcacaa gcggtggagc atgtggttta attcgaagca acgcgaagaa ccttaccaaa 960
tcttgacatc ctttgaccgc tctagagata gagtcttccc cttcggggga caaagtgaca 1020
ggtggtgcat ggttgtcgtc agctcgtgtc gtgagatgtt gggttaagtc ccgcaacgag 1080
cgcaaccctt aagcttagtt gccatcatta agttgggcac tctaagttga ctgccggtga 1140
caaaccggag gaaggtgggg atgacgtcaa atcatcatgc cccttatgat ttgggctaca 1200
cacgtgctac aatggacaat acaaagggca gctaaaccgc gaggtcaagc aaatcccata 1260
aagttgttct cagttcggat tgtagtctgc aactcgacta catgaagctg gaatcgctag 1320
taatcgtaga tcagcatgct acggtgaata cgttcccggg tcttgtacac accgcccgtc 1380
acaccacgag agtttgtaac acccgaagcc ggtggagtaa ccatttatgg agctagccgt 1440
cgagtggaga gctcagtct 1459
Claims (3)
1. apex pulmonis Psychrobacter (Psychrobacter pulmonis strain) T-15 bacterial strain of one plant of production polysaccharide, the bacterial strain
It is preserved in China typical culture collection center, deposit number are as follows: CCTCC M 2019056, the 16S rDNA sequence of the bacterial strain
As shown in SEQ ID NO.1.
2. the purposes of apex pulmonis Psychrobacter T-15 bacterial strain described in claim 1, it is characterised in that: prepare polysaccharide for fermentation.
3. utilizing the method that apex pulmonis Psychrobacter T-15 bacterial strain prepares polysaccharide described in claim 1, it is characterised in that including following
Step: first by apex pulmonis Psychrobacter T-15 strain inoculated in LB liquid medium, 37 DEG C of overnight shakings of 200r/min revolving speed
Zymocyte liquid is centrifuged in 12000r/min, takes supernatant by culture 2 days；By supernatant and chloroform-butanol solution be mixed in from
In heart pipe, 20-30min is vibrated, then takes supernatant after being centrifuged, by supernatant with twice of membrane filtration of 0.45 μm, 70 after filtering
Heating is concentrated into the 1/2-2/3 of original volume at DEG C, and 95% ethyl alcohol of 4 times of volumes is added, and is put into refrigerator freezing and stays overnight, finally uses
12000r/min revolving speed is centrifuged 10min, and precipitating generated is polysaccharide.
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Citations (4)
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|---|---|---|---|---|
| CN1118810A (en) * | 1994-09-13 | 1996-03-20 | 许维加 | Water shield polysaccharide gum producing bacteria and producing technology for water shield polysaccharide gum |
| CN102060933A (en) * | 2010-12-06 | 2011-05-18 | 湖北民族学院 | Method for extracting and separating in-vivo water-soluble polysaccharide from old water shield leaves |
| CN107904177A (en) * | 2017-07-26 | 2018-04-13 | 华南理工大学 | A kind of extracting method and application of dendrobium candidum endogenetic fungal bacterial strain and its exocellular polysaccharide of generation and the exocellular polysaccharide |
| CN108484784A (en) * | 2018-01-11 | 2018-09-04 | 浙江工业大学 | A kind of physical separation method of the external polysaccharide gum of water shield |
-
2019
- 2019-01-24 CN CN201910068679.0A patent/CN109777752B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1118810A (en) * | 1994-09-13 | 1996-03-20 | 许维加 | Water shield polysaccharide gum producing bacteria and producing technology for water shield polysaccharide gum |
| CN102060933A (en) * | 2010-12-06 | 2011-05-18 | 湖北民族学院 | Method for extracting and separating in-vivo water-soluble polysaccharide from old water shield leaves |
| CN107904177A (en) * | 2017-07-26 | 2018-04-13 | 华南理工大学 | A kind of extracting method and application of dendrobium candidum endogenetic fungal bacterial strain and its exocellular polysaccharide of generation and the exocellular polysaccharide |
| CN108484784A (en) * | 2018-01-11 | 2018-09-04 | 浙江工业大学 | A kind of physical separation method of the external polysaccharide gum of water shield |
Non-Patent Citations (3)
| Title |
|---|
| VELA AI ET AL.: "Psychrobacter pulmonis sp. nov., isolated from the lungs of lambs", 《INT J SYST EVOL MICROBIOL》 * |
| 徐光辉等: "莼菜叶面微生物生态分布及产粘胶细菌5242菌株形成多糖发酵条件的研究", 《武夷科学》 * |
| 秦娟秀等: "血培养检出嗜冷杆菌属亚种Psychrobacter sanguinis一例", 《检验医学》 * |
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