CN103255075A - Pantoea ananatis which generates exopolysaccharide and extraction and usage of polysaccharide thereof - Google Patents
Pantoea ananatis which generates exopolysaccharide and extraction and usage of polysaccharide thereof Download PDFInfo
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Abstract
The invention discloses Pantoea ananatis which generates exopolysaccharide and extraction and usage of polysaccharide thereof, and the classification name is Pantoea ananatis G-14, which is preserved in the China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC M2010107. The P. ananatis G-14 carries out extracellular secretion of polysaccharide in the growth process, and after cultivation on the medium, condensation, deposition, deproteinization and dialysis, the P. ananatis G-14 exopolysaccharide purified product is obtained, and the output is 380 mg/L, wherein, the glycoprotein concentration is 0.7%. The exopolysaccharide secreted by fermentation cultivation of Pantoea ananatis G-14 has antioxidation activity and prebiotics like effect.
Description
Technical field
The invention belongs to the microbial technology field of producing exocellular polysaccharide, relate to a kind of the produce general bacterium of pineapple of exocellular polysaccharide and extraction and the application of polysaccharide thereof.
Background technology
The antioxygenation of polysaccharide has become one of active subject the most.Usually plant and alga-derived polysaccharide are comparatively general, but in the last few years, microbe-derived polysaccharide (exocellular polysaccharide) causes extensive concern, and the exocellular polysaccharide of bacterium and originated from fungus especially is because it is easy to separation and purification and the output advantages of higher is quite favored in industrial production.
(Exopolysaccharide is the general name that bacterial cell contains sugared structure outward EPS) to exocellular polysaccharide, comprises that gram negative bacterium after birth outer polysaccharide component and gram-positive microorganism get peptidoglycan, is the polymer substance that bacterium produces under specific environment is regulated.As the secondary metabolite of bacterium, exocellular polysaccharide mainly contains the effect of bacterium: the clusters of bacteria opposing is engulfed; Protection bacterium discord antibodies; Formation interior replacing property of the bacterium growth of trooping; Help bacterial population to obtain enough nutrition towards periphery.As a class biopolymer that is closely related with the human lives, the bacterium exocellular polysaccharide of discovering in recent years also has anti-oxidant, radioprotective, immunomodulatory, hypoglycemic, reducing blood-fat, and special biologic activity such as antitumor, hepatitis virus resisting.
In recent years, microbial polysaccharide Study on Antioxidant Activities report is a lot, and it can remove free radical external, also has the ability of anti peroxidation of lipid; Then can improve the activity of antioxidase in vivo, also show the ability of anti peroxidation of lipid.Compare with other exogenous antioxidant, anti-oxidant polysaccharide has characteristics such as low toxicity, safety source be wide.As exogenous antioxidant, polysaccharide plays a role more complicated in vivo, and it can be participated in directly outside the approach of cancellation free radical, also may be with relevant by the activity of endogenous antioxidant in the adjusting body.Polysaccharide can be used as a kind of natural biological anti-oxidant and carries out compositely in addition, strengthens the provide protection to HUMAN HEALTH.
Summary of the invention
The problem that the present invention solves is to provide a kind of the produce general bacterium of pineapple of exocellular polysaccharide and extraction and the application of polysaccharide thereof, and the general bacterium of this pineapple is a kind of new microorganism, and the exocellular polysaccharide of its secretion has anti-oxidant activity.
The present invention is achieved through the following technical solutions:
A kind of general bacterium of pineapple that produces exocellular polysaccharide, its classification called after Pantoea ananatis G-14 is preserved in Chinese typical culture collection center (CCTCC), and preserving number is CCTCC M2010107.
The general bacterium of the pineapple of described product exocellular polysaccharide in process of growth to the exocytosis polysaccharide.
Described polysaccharide has anti-oxidant activity.
Method based on Pantoea ananatis G-14 fermentation culture extraction exocellular polysaccharide may further comprise the steps:
1) with the single colony inoculation of P.ananatis G-14 in the LB liquid nutrient medium, under 25～30 ℃, stir aerated culture 8～10h; Be forwarded to then in the fermention medium, under 25～30 ℃, stir aerated culture 48～65h;
Described fermention medium is to add the monose of massfraction 1～5% or disaccharide as additive carbon in the LB liquid nutrient medium;
2) after cultivation is finished, centrifugal, collect the nutrient solution supernatant;
3) the nutrient solution supernatant is concentrated after, add the ethanolic soln of the volume fraction 80～95% of 2～5 times of its volumes, abundant mixing, leave standstill 12～24h under 0～10 ℃ after, collect the precipitation after leaving standstill, obtain Crude polysaccharides;
4) separate to remove albumen in the Crude polysaccharides after, molecular weight cut-off be in 8000～14000 the dialysis tubing with sterilization pure water damping fluid dialysis 24h more than, changed 1 damping fluid in per 12 hours;
Polysaccharide soln frozen drying in the dialysis tubing is obtained exocellular polysaccharide dry powder.
Described step 1) be with the single colony inoculation of P.ananatis G-14 in the LB liquid nutrient medium, under 25～30 ℃, 100～300rpm stirs, aerated culture 8～10h; Be forwarded in the fermention medium with 1～5% inoculum size then, under 25～30 ℃, stir aerated culture 48～65h;
Described fermention medium is to add the glucose of massfraction 1～5% as additive carbon in the LB liquid nutrient medium.
Described step 2) centrifugal is 8000～12000rpm, 3～10min, 4 ℃ of centrifugal collection nutrient solution supernatants.
Described step 3) is: the nutrient solution supernatant is evaporated to 1/5～1/10 of original volume, add the volume fraction 80～95% of 2～5 times of its volumes, 4 ℃ of precooled ethanol solution then, abundant mixing, 0～4 ℃ fully precipitates exocellular polysaccharide behind placement 12～24h down;
8000～12000rpm, 3～10min, 4 ℃ of centrifugal collecting precipitations obtain Crude polysaccharides.
Described step 4) is: Crude polysaccharides adopts the Sevage method except behind the albumen, and the centrifuging and taking supernatant is dialysis 48 hours in the sterilization pure water in 8000～14000 the dialysis tubing at molecular weight cut-off, changes 1 damping fluid in per 12 hours;
With the lyophilize in using freeze drier of the polysaccharide soln in the dialysis tubing, condensing temperature＜-50 ℃, vacuum tightness＜20Pa, freezing time are 18～24h, obtain exocellular polysaccharide dry powder.
The exocellular polysaccharide of Pantoea ananatis G-14 fermentation culture secretion is in the application of preparation antioxidant.
Described antioxidant is:
In the antioxidant of the antioxidant of removing hydroxy radical qiao, the antioxidant of removing ultra-oxygen anion free radical, lipid peroxidation inhibitor, removing DPPH free radical one or more.
The exocellular polysaccharide of Pantoea ananatis G-14 fermentation culture secretion repairs, promotes the preparation of the medicine of beneficial bacteria of intestinal tract breeding and/or the breeding of inhibition enteric pathogenic bacteria in the loss of preparation promotion enteron aisle.
Compared with prior art, the present invention has following beneficial technical effects:
The invention provides a kind of new microorganism that can produce exocellular polysaccharide, this microorganism belongs to the general bacterium P.ananatis of pineapple, its classification called after P.ananatis G-14, it is yellow, smooth, moistening, full that lawn on the solid medium of LB+1% glucose is, and its 16s rRNA sequence NCBI accession number is JN974457.
P.ananatis G-14 provided by the invention in process of growth to the exocytosis polysaccharide, after substratum is cultivated, obtain P.ananatis G-14 exocellular polysaccharide purified product after concentrating, precipitate, remove albumen, dialysis, output is 380mg/L, and glycoprotein concentration wherein is 0.7%.
The concrete anti-oxidant activity of exocellular polysaccharide of Pantoea ananatis G-14 fermentation culture secretion:
A) remove hydroxy radical qiao (OH)
Hydroxyl radical free radical (OH) is generally acknowledged it is the activating oxide of tool activity in the biosystem, can cause the interior DNA of organism, the irreversible damage of protein and lipid oxidation etc.The hydroxy radical qiao clearance rate is the important indicator of reflection medicine antioxygenation.P.ananatis G-14 exocellular polysaccharide has certain scavenging(action) to hydroxyl radical free radical, and when slightly carrying polysaccharide and be 5mg/ml, its removing ability reaches with 40% of concentration Vc.
B) remove ultra-oxygen anion free radical (O
2-)
The ultra-oxygen anion free radical that in bio-oxidation, forms, character is active, can be by the anion channel of cytolemma, directly be combined with multiple macromolecular substance and it is lost activity.Reductibility hemiacetal hydroxyl on the polysaccharide molecule is with ultra-oxygen anion free radical generation redox reaction.Slightly carry polysaccharide ultra-oxygen anion free radical is had certain removing effect, when 5mg/ml, the removing ability only is 15.8%.
C) reducing power
P.ananatis G-14 exocellular polysaccharide has certain reducing power, and reducing power increases the significant dose-effect relationship that exists between reducing power and the polysaccharide concentration with the increase of polysaccharide concentration.
D) lipid peroxidation restraining effect
Lipid peroxidation refers to the oxidation of unsaturated fatty acids, lipid, the biological function of film can directly be disturbed and destroy to this process, many factor (alcohol, plant poison albumen, fluorosis etc.), uv-radiations that cause hydroxy radical qiao to increase all can be induced lipid peroxidation, and then be brought out a series of diseases.The model LPO system experimental result that with the lipovitellinin is reaction substrate shows, P.ananatis G-14 exocellular polysaccharide has higher lipid peroxidation restraining effect, during from 0.15mg/ml to 0.6mg/ml, the lipid peroxidation restraining effect increases and significantly increases with the concentration of polysaccharide, when polysaccharide concentration is higher than 1.25mg/ml, restraining effect tends to be steady, and when 5mg/ml, restraining effect can reach 35.8%.
E) remove the DPPH free radical
DPPH(1,1-phenylbenzene-2-picryl hydrazine free radical) be a kind of very stable free radical centered by nitrogen-atoms, by detecting polysaccharide to the removing ability of DPPH, the size of polysaccharide resistance of oxidation can be described.Along with the increase of polysaccharide concentration, its removing ability to DPPH is also strengthening gradually, and when 2.5mg/ml, clearance rate reaches 28%, and when 5mg/ml, clearance rate reaches 46%.
P.ananatis G-14 exocellular polysaccharide has the class prebiotic effect:
The histopathology feature of typical inflammation such as oedema, hyperemia appears in the intestines wall of irritating the shigella dysenteriae infected rats of stomach without P.ananatis G-14 exocellular polysaccharide, and mucosal epithelium has in various degree to be destroyed; Irritate the intestines wall of shigella dysenteriae infected rats of stomach through P.ananatis G-14 exocellular polysaccharide than smoothly, the destruction of mucosal epithelium has obtained reparation to a certain degree.Show that P.ananatis G-14 exocellular polysaccharide has the effect that promotes that the enteron aisle loss is repaired, it may regulate the intestinal microflora balance simultaneously, promotes beneficial bacteria of intestinal tract, suppresses pathogenic bacteria and colibacillary breeding, can promote animal health, show the effect that is similar to prebiotics.
The preservation explanation
P.ananatis G-14 of the present invention has carried out following preservation:
The preservation time: on April 11st, 2012, preservation place: China. Wuhan. Wuhan University, Chinese typical culture collection center, CCTCC; Preserving number is CCTCC M2012107; Classification name Pantoea ananatis G-14.
Description of drawings
Fig. 1 is CCTCC M2010107 at the microscopy of microscopically figure (100 * 100) as a result;
Fig. 2 is the product exocellular polysaccharide of CCTCC M2010107 on solid medium;
Fig. 3 is the systematic evolution tree (NJ evolutionary tree) that 16s rRNA sequence alignment makes up;
Fig. 4 is the external scavenging(action) to hydroxy radical qiao of CCTCC M2010107 secretion exocellular polysaccharide;
Fig. 5 is the external scavenging(action) to ultra-oxygen anion free radical of CCTCC M2010107 secretion exocellular polysaccharide;
Fig. 6 is the external reducing power detected result figure of CCTCC M2010107 secretion exocellular polysaccharide;
Fig. 7 is the lipid peroxidation restraining effect of CCTCC M2010107 secretion exocellular polysaccharide;
Fig. 8 is external to the DPPH measured by esr technique for CCTCC M2010107 secretion exocellular polysaccharide;
Fig. 9-1～9-2 changes for the intestinal tissue pathology of the shigella dysenteriae chronic infection rat that CCTCC M2010107 secretion exocellular polysaccharide is handled; Wherein Fig. 9-1 is shigella dysenteriae chronic infection rat; The chronic infection rat that Fig. 9-2 intervenes for the G-14 exocellular polysaccharide.
Embodiment
The present invention is described in further detail below in conjunction with separation, cultivation, the extraction of polysaccharide and the embodiment of effect of Pantoea ananatis G-14, and the explanation of the invention is not limited.
1, separation CCTCC M2010107(Pantoea ananatis G-14)
Report that the general bacterium P.ananatis of part pineapple has antibacterial character (comprising fungi and bacterium); Some bacterial strain has the ice nucleating bacteria characteristic, can be used as biotic additives and is used for Food Freezing Process; The part bacterial strain then is considered to a kind of phytopathogen, can cause rotten pathologies such as onion, paddy.Present research is not reported about the exocellular polysaccharide Study on Antioxidant Activities of the general bacterium P.ananatis of pineapple.
CCTCC M2010107 is separated into:
Separate the source: in April, 2011, the slicing fruits sample was gathered in ten thousand tame supermarkets from the China Resources, Xi'an, homogenate under the sterile state, scale takes by weighing the 0.5g sample, add in the 0.1% peptone solution test tube of sterilization fully mixing, room temperature leaves standstill 5min, get the extractum carnis that the dissolves-peptone solid medium mixing of supernatant 1ml and 25ml sterilization, pour plate is cultivated 48h for 37 ℃.
Screening has obtained the general bacterium P.ananatis of a strain pineapple from above-mentioned substratum, find this bacterium in extractum carnis-peptone solid medium growth rapidly, bacterium colony is smooth, moistening, toughness during contact, when cultivating in LB liquid tube substratum, the cell agglutination phenomenon appearred later in 48 hours.
Separate used substratum: extractum carnis-peptone solid medium (0.3% extractum carnis, 1% peptone, 0.5% sodium-chlor, 1.5% agar, high pressure steam sterilization, 121 ℃, 20min)
When this bacterium grows at the P.ananatis substratum, in the microscopy result of microscopically as shown in Figure 1, magnification 100 * 100.
P.ananatis substratum: TSB substratum (0.5% sodium-chlor, solid medium add 1.5% agar for 1.5% Tryptones, 0.5% soy peptone)
The lawn of this bacterial strain on the solid medium of LB+1% glucose is yellow, smooth, moistening, full, specifically as shown in Figure 2.
After finding the secretion exocellular polysaccharide in the substratum of this bacterium, filtered out the substratum of the product exocellular polysaccharide fermentation that is fit to it: LB substratum (1% peptone, 0.5% yeast powder, 1% sodium-chlor)+1-5% glucose; Its culture condition is: producing the exocellular polysaccharide fermentation condition is 30 ℃, isothermal vibration shaking table 200rpm, aerated culture 48-65h.
2, the evaluation of CCTCC M2010107 strain isolated
Extract total DNA of CCTCC M2010107, and be template with it, amplification 1.5kb16S rRNA sequence, 16S rRNAPCR primer is:
16S-F:5'AGA?GTT?TGA?TCC?TGG?CTC?AG3'，
16S-R:5‘GGT?ACC?T?TG?TTA?CGA?CTT3'。
Amplification program is: 94 ℃, and 4min, 1 pre-sex change of circulation; 94 ℃ of sex change, 30sec, 58 ℃ of annealing, 30sec, 72 ℃ of extensions, 1min, 30 circulations; 72 ℃ of 7min.
The pcr amplification system is to add 10mM dNTP 1.6 μ l respectively in the 20 μ l PCR systems, 10 * Taq enzyme buffer, 2 μ l, and each 1 μ l of 1nM PCR primer, the total dna profiling 2-3ng of G-14, Taq archaeal dna polymerase 0.2 μ l supplies 20 μ l with MiniQ water.
Pcr amplification product reclaims test kit with the PCR product and reclaims after the agarose gel electrophoresis checking, and send the order-checking of order-checking company.Sequencing result and NCBI go up the bacterial 16 S rRNA sequence alignment of announcing, confirm that this strain isolated belongs to the general bacterium P.ananatis of pineapple, and its 16s rRNA sequence NCBI accession number is JN974457.The N-J systematic evolution tree is seen Fig. 3, determines that this bacterial strain is the general bacterium of a kind of new pineapple.
3, CCTCC M2010107 produces the extraction of exocellular polysaccharide fermentation, polysaccharide
Method based on Pantoea ananatis G-14 fermentation culture extraction exocellular polysaccharide may further comprise the steps:
1) with the single colony inoculation of P.ananatis G-14 in the LB liquid nutrient medium, under 25～30 ℃, stir aerated culture 8～10h; Be forwarded to then in the fermention medium, under 25～30 ℃, stir aerated culture 48～65h;
Described fermention medium is to add the monose of massfraction 1～5% or disaccharide as additive carbon in the LB liquid nutrient medium;
Concrete: be with the single colony inoculation of P.ananatis G-14 in the LB liquid nutrient medium, under 25～30 ℃, 100～300rpm stirs, aerated culture 8～10h; Be forwarded in the fermention medium with 1～5% inoculum size then, under 25～30 ℃, stir aerated culture 48～65h;
Described fermention medium is to add the glucose of massfraction 1～5% as additive carbon in the LB liquid nutrient medium.
2) after cultivation is finished, centrifugal, collect the nutrient solution supernatant;
Concrete: centrifugal be 8000～12000rpm, 3～10min, 4 ℃ of centrifugal collection nutrient solution supernatants.
3) the nutrient solution supernatant is concentrated after, add the ethanolic soln of the volume fraction 80～95% of 2～5 times of its volumes, abundant mixing, leave standstill 12～24h under 0～10 ℃ after, collect the precipitation after leaving standstill, obtain Crude polysaccharides;
Concrete: the nutrient solution supernatant is evaporated to 1/5～1/10 of original volume, adds 95% ethanolic soln (4 ℃ of precoolings) of 2～3 times of its volumes then, abundant mixing, 0～4 ℃ fully precipitates exocellular polysaccharide behind placement 12～24h down;
8000～12000rpm, 3～10min, 4 ℃ of centrifugal collecting precipitations obtain Crude polysaccharides.
4) separate to remove albumen in the Crude polysaccharides after, be to be more than the dialyzate dialysis 24h with the sterilization pure water in 8000～14000 the dialysis tubing at molecular weight cut-off, changed 1 damping fluid in per 12 hours;
Polysaccharide soln frozen drying in the dialysis tubing is obtained exocellular polysaccharide dry powder.
Concrete: Crude polysaccharides adopts the Sevage method except behind the albumen, and the centrifuging and taking supernatant is to be dialyzate dialysis 48 hours with the sterilization pure water in 8000～14000 the dialysis tubing at molecular weight cut-off, changes 1 damping fluid in per 12 hours;
With the polysaccharide soln in the dialysis tubing carry out frozen drying (use freeze drier, condensing temperature＜-50 ℃, vacuum tightness＜20Pa 18-24h) obtains exocellular polysaccharide dry powder.
Obtain P.ananatis G-14 exocellular polysaccharide purified product after concentrating, precipitate, remove albumen, dialysis, output is 380mg/L, and glycoprotein concentration wherein is 0.7%.
4, the antioxidation activity in vitro of P.ananatis G-14 exocellular polysaccharide is measured:
4.1 remove hydroxy radical qiao (OH)
The exocellular polysaccharide solution (take by weighing lyophilized powder, be configured to 0.15mg/ml, 0.31mg/ml, 0.62mg/ml, 1.25mg/ml, 2.5mg/ml, 5.0mg/ml) that in the 10ml test tube, adds the 2ml different concns respectively, 2ml6mM FeSO
4, 2ml H
2O
2, mixing room temperature standing and reacting 30min measures OD
510Value.Vc solution with same concentrations replaces exocellular polysaccharide solution as positive controls, with aseptic ddH
2O replaces exocellular polysaccharide solution as negative control group.
Exo polysaccharides is removed hydroxy radical qiao (%)=(A
0-A
1)/A
0* 100%, A wherein
0Average light absorption value for the blank group; A
1Average light absorption value for sample sets
Hydroxyl radical free radical (OH) is generally acknowledged it is the activating oxide of tool activity in the biosystem, can cause the interior DNA of organism, the irreversible damage of protein and lipid oxidation etc.The hydroxy radical qiao clearance rate is the important indicator of reflection medicine antioxygenation.
P.ananatis G-14 exocellular polysaccharide has certain scavenging(action) to hydroxyl radical free radical, and when slightly carrying polysaccharide and be 5mg/ml, its removing ability reaches with 40% of concentration Vc, detected result as shown in Figure 4, wherein black represents the G-14 exocellular polysaccharide, grey represents Vc..
4.2 remove ultra-oxygen anion free radical (O
2 -)
Preheating 30min's is added with 4.5ml0.05M Tris-HCl(pH8.2 in 25 ℃ of water-baths) test tube in add the 1ml different concns respectively exocellular polysaccharide solution (take by weighing lyophilized powder, be configured to 0.15mg/ml, 0.31mg/ml, 0.62mg/ml, 1.25mg/ml, 2.5mg/ml, 5.0mg/ml), and 0.4ml25mM pyrogallol solution, Vc solution with same concentrations replaces exocellular polysaccharide solution as positive controls, with aseptic ddH
2O replaces exocellular polysaccharide solution as negative control group.Behind the mixing, place 25 ℃ of water-baths to react 5min respectively, add 1ml8M HCl stopped reaction, measure OD
299Value.
Exocellular polysaccharide is removed superoxide anion (%)=(A
0-A
1)/A
0* 100%, A wherein
0Average light absorption value for the blank group; A
1Average light absorption value for sample sets
The ultra-oxygen anion free radical that in bio-oxidation, forms, character is active, can be by the anion channel of cytolemma, directly be combined with multiple macromolecular substance and it is lost activity.Reductibility hemiacetal hydroxyl on the polysaccharide molecule is with ultra-oxygen anion free radical generation redox reaction.
Slightly carry polysaccharide to the exercising result of ultra-oxygen anion free radical as shown in Figure 5, wherein black represents the G-14 exocellular polysaccharide, and grey represents Vc.; Its removing effect to ultra-oxygen anion free radical is relatively poor, and when 5mg/ml, the removing ability only is 15.8%.
4.3 reducing power
The exocellular polysaccharide solution that adds the 1ml different concns in the 10ml test tube respectively (takes by weighing lyophilized powder, be configured to 0.15mg/ml, 0.31mg/ml, 0.62mg/ml, 1.25mg/ml, 2.5mg/ml, 5.0mg/ml), 2.5ml phosphate solution (pH6.6), react 20min 2.5ml the Tripotassium iron hexacyanide, mixing are placed in 50 ℃ of water-baths, add 2.5ml10% trichoroacetic acid(TCA) solution again, mixing, the centrifugal 10min of 3000rpm gets supernatant 2.5ml in new test tube, adds the aseptic ddH of 2.5ml
2O, 0.5ml0.1%FeCl
3, room temperature is placed 3min, measures OD
700Value.Vc solution with same concentrations replaces exocellular polysaccharide solution as positive controls.
Detected result as shown in Figure 6, solid line represents the G-14 exocellular polysaccharide, dotted line represents Vc.; The P.ananatisG-14 exocellular polysaccharide has certain reducing power, and reducing power increases the significant dose-effect relationship that exists between reducing power and the polysaccharide concentration with the increase of polysaccharide concentration.
4.4 lipid peroxidation restraining effect
Get the yolk suspension (using pH7.45, the 0.1MPBS configuration) of 0.2ml1:40 dilution, 0.2ml25mMFeSO
4, the exocellular polysaccharide solution of 100 μ l different concns (take by weighing lyophilized powder, be configured to 0.15mg/ml, 0.31mg/ml, 0.62mg/ml, 1.25mg/ml, 2.5mg/ml, 5.0mg/ml) is with aseptic ddH
2O supplies 2ml, and mixing places 37 ℃ of water-baths to shake 15min, after the taking-up, sample hose adds 0.5ml20%TCA solution, leaves standstill 10min, and the centrifugal 10min of 3500rpm shifts supernatant, the thiobarbituricacid solution that adds 1ml0.8% again, airtight test tube, 100 ℃ are boiled 15min, naturally cooling.The negative control pipe replaces polysaccharide soln with 2mlPBS.Measure OD
532Value.
Exocellular polysaccharide is to restraining effect rate (%)=(1-A of lipid peroxidation
1/ A
0) * 100%; A wherein
0Average light absorption value for the blank group; A
1Average light absorption value for sample sets.
Lipid peroxidation refers to the oxidation of unsaturated fatty acids, lipid, the biological function of film can directly be disturbed and destroy to this process, many factor (alcohol, plant poison albumen, fluorosis etc.), uv-radiations that cause hydroxy radical qiao to increase all can be induced lipid peroxidation, and then be brought out a series of diseases.
With the lipovitellinin be the model LPO system experimental result of reaction substrate as shown in Figure 7, P.ananatis G-14 exocellular polysaccharide has higher lipid peroxidation restraining effect, during from 0.15mg/ml to 0.6mg/ml, the lipid peroxidation restraining effect increases and significantly increases with the concentration of polysaccharide, when polysaccharide concentration is higher than 1.25mg/ml, restraining effect tends to be steady, and when 5mg/ml, restraining effect can reach 35.8%.
4.5 remove the DPPH free radical
In the 10ml test tube, add 2ml40mg/ml DPPH respectively, (take by weighing lyophilized powder with the exocellular polysaccharide solution of 2ml different concns, be configured to 0.15mg/ml, 0.31mg/ml, 0.62mg/ml, 1.25mg/ml, 2.5mg/ml, 5.0mg/ml), abundant mixing, lucifuge reaction 30min measures OD under the room temperature
517Value.Vc solution with same concentrations replaces exocellular polysaccharide solution as positive controls, with aseptic ddH
2O replaces exocellular polysaccharide solution as negative control group.
Exocellular polysaccharide is removed DPPH free radical (%)=(A
0-A
1)/A
0* 100%; A wherein
0Average light absorption value for the blank group; A
1Average light absorption value for sample sets
DPPH(1,1-phenylbenzene-2-picryl hydrazine free radical) be a kind of very stable free radical centered by nitrogen-atoms, by detecting polysaccharide to the removing ability of DPPH, the size of polysaccharide resistance of oxidation can be described.Along with the increase of polysaccharide concentration, its removing ability to DPPH is also strengthening gradually, and when 2.5mg/ml, clearance rate reaches 28%, and when 5mg/ml, clearance rate reaches 46%, and specifically as shown in Figure 8, wherein black represents the G-14 exocellular polysaccharide, and grey represents Vc..
5, P.ananatis G-14 exocellular polysaccharide is handled the physiologically active to the shigella dysenteriae infected rats
Irritate hello SD rat with infectious shigella dysenteriae, set up the dysentery bacterium chronic infection model of rat.The P.ananatis G-14 polysaccharide of purifying is carried out the infection model rat oral gavage according to the 10mg/kg body weight, every day 1 time, carried out for 4 weeks.Irritate the stomach processing with physiological saline and do negative control.Dissect rat after 4 days, get intestinal tissue, preparation paraffin section, HE dyeing, the intestinal tissue pathology situation of microscopic examination comparison process and contrast.
The histopathology feature of typical inflammation such as oedema, hyperemia appears in the intestines wall of irritating the shigella dysenteriae infected rats of stomach without P.ananatis G-14 exocellular polysaccharide, and mucosal epithelium has in various degree to be destroyed; Irritate the intestines wall of shigella dysenteriae infected rats of stomach through P.ananatis G-14 exocellular polysaccharide than smoothly, the destruction of mucosal epithelium has obtained reparation to a certain degree, the result is (HE * 40) shown in Fig. 9-1,9-2, and wherein Fig. 9-1 is shigella dysenteriae chronic infection rat; The chronic infection rat that Fig. 9-2 intervenes for the G-14 exocellular polysaccharide.
This shows that P.ananatis G-14 exocellular polysaccharide has the effect that promotes that the enteron aisle loss is repaired.It may regulate the intestinal microflora balance simultaneously, promotes beneficial bacteria of intestinal tract, suppresses pathogenic bacteria and colibacillary breeding, can promote animal health, shows the effect that is similar to prebiotics.
To sum up detect and show: P.ananatis G-14 polysaccharide has tangible anti-oxidant activity, radioprotective, strengthening immunity, anti-tumor activity, can be used for protective foods, foodstuff additive, makeup, fodder additives, veterinary drug etc.
Further, also can carry out the molecular biology transformation to P.ananatis G-14 bacterial strain, further improve the polysaccharide ability of producing on the original basis, through scale fermentation and extraction active polysaccharide, as antioxidant, prebiotics etc., be used for healthy food material, foodstuff additive, fodder additives, radioprotective makeup, veterinary drug injection, immunological adjuvant etc.
Also but development of new bacterium exocellular polysaccharide functional product also can act synergistically with other active substances (polyose, Polyphenols etc.), obtains the composite reactive additive.
Claims (10)
1. the general bacterium of pineapple that produces exocellular polysaccharide is characterized in that, its classification called after Pantoea ananatis G-14 is preserved in Chinese typical culture collection center (CCTCC), and preserving number is CCTCCM2010107.
2. the general bacterium of the pineapple of product exocellular polysaccharide as claimed in claim 1 is characterized in that, its in process of growth to the exocytosis polysaccharide.
3. extract the method for exocellular polysaccharide based on Pantoea ananatis G-14 fermentation culture, it is characterized in that, may further comprise the steps:
1) with the single colony inoculation of P.ananatis G-14 in the LB liquid nutrient medium, under 25～30 ℃, stir aerated culture 8～10h; Be forwarded to then in the fermention medium, under 25～30 ℃, stir aerated culture 48～65h;
Described fermention medium is to add the monose of massfraction 1～5% or disaccharide as additive carbon in the LB liquid nutrient medium;
2) after cultivation is finished, centrifugal, collect the nutrient solution supernatant;
3) the nutrient solution supernatant is concentrated after, add the ethanolic soln of the volume fraction 80～95% of 2～5 times of its volumes, abundant mixing, leave standstill 12～24h under 0～10 ℃ after, collect the precipitation after leaving standstill, obtain Crude polysaccharides;
4) separate to remove albumen in the Crude polysaccharides after, molecular weight cut-off be in 8000～14000 the dialysis tubing with sterilization pure water damping fluid dialysis 24h more than, changed 1 damping fluid in per 12 hours;
Polysaccharide soln frozen drying in the dialysis tubing is obtained exocellular polysaccharide dry powder.
4. the method for extracting exocellular polysaccharide based on Pantoea ananatis G-14 fermentation culture as claimed in claim 3, it is characterized in that, described step 1) be with the single colony inoculation of P.ananatis G-14 in the LB liquid nutrient medium, under 25～30 ℃, 100～300rpm stirs, aerated culture 8～10h; Be forwarded in the fermention medium with 1～5% inoculum size then, under 25～30 ℃, stir aerated culture 48～65h;
Described fermention medium is to add the glucose of massfraction 1～5% as additive carbon in the LB liquid nutrient medium.
5. as claimed in claim 3ly extract the method for exocellular polysaccharide based on Pantoea ananatis G-14 fermentation culture, it is characterized in that described step 2) centrifugal be 8000～12000rpm, 3～10min, 4 ℃ of centrifugal collection nutrient solution supernatants.
6. the method for extracting exocellular polysaccharide based on Pantoea ananatis G-14 fermentation culture as claimed in claim 3, it is characterized in that, described step 3) is: the nutrient solution supernatant is evaporated to 1/5～1/10 of original volume, add the volume fraction 80～95% of 2～5 times of its volumes, 4 ℃ of precooled ethanol solution then, abundant mixing, 0～4 ℃ fully precipitates exocellular polysaccharide behind placement 12～24h down;
8000～12000rpm, 3～10min, 4 ℃ of centrifugal collecting precipitations obtain Crude polysaccharides.
7. the method for extracting exocellular polysaccharide based on Pantoea ananatis G-14 fermentation culture as claimed in claim 3, it is characterized in that, described step 4) is: Crude polysaccharides adopts the Sevage method except behind the albumen, the centrifuging and taking supernatant, be dialysis 48 hours in the sterilization pure water in 8000～14000 the dialysis tubing at molecular weight cut-off, changed 1 damping fluid in per 12 hours;
With the lyophilize in using freeze drier of the polysaccharide soln in the dialysis tubing, condensing temperature＜-50 ℃, vacuum tightness＜20Pa, freezing time are 18～24h, obtain exocellular polysaccharide dry powder.
8.Pantoea the exocellular polysaccharide of ananatis G-14 fermentation culture secretion is in the application of preparation antioxidant.
9. application as claimed in claim 8 is characterized in that, described antioxidant is:
In the antioxidant of the antioxidant of removing hydroxy radical qiao, the antioxidant of removing ultra-oxygen anion free radical, lipid peroxidation inhibitor, removing DPPH free radical one or more.
10.Pantoea the exocellular polysaccharide of ananatis G-14 fermentation culture secretion repairs, promotes the preparation of the medicine of beneficial bacteria of intestinal tract breeding and/or the breeding of inhibition enteric pathogenic bacteria in the loss of preparation promotion enteron aisle.
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| CN104531553A (en) * | 2014-11-27 | 2015-04-22 | 安徽农业大学 | Pantoea ananatis Z1 having molluscicidal activity and application thereof |
| CN105147715A (en) * | 2015-06-15 | 2015-12-16 | 河南科技学院 | Novel application of neutral extracellular polysaccharides of paecilomyces hepiali |
| CN105530953A (en) * | 2013-10-03 | 2016-04-27 | 日东电工株式会社 | Injectable vaccine composition |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105530953A (en) * | 2013-10-03 | 2016-04-27 | 日东电工株式会社 | Injectable vaccine composition |
| CN104531553A (en) * | 2014-11-27 | 2015-04-22 | 安徽农业大学 | Pantoea ananatis Z1 having molluscicidal activity and application thereof |
| CN104531553B (en) * | 2014-11-27 | 2017-05-10 | 安徽农业大学 | Pantoea ananatis Z1 having molluscicidal activity and application thereof |
| CN105147715A (en) * | 2015-06-15 | 2015-12-16 | 河南科技学院 | Novel application of neutral extracellular polysaccharides of paecilomyces hepiali |
| CN105147715B (en) * | 2015-06-15 | 2019-01-01 | 河南科技学院 | A kind of new application of Paecilomyces hepiali chen neutrality exocellular polysaccharide |
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