CN102816716B - Extraction and application of exopolysaccharide metabolite of bacillus amyloliquefaciens strain - Google Patents
Extraction and application of exopolysaccharide metabolite of bacillus amyloliquefaciens strain Download PDFInfo
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Abstract
The invention discloses an extraction and an application of exopolysaccharide metabolite of a bacillus amyloliquefaciens C-1 strain. A 16s rRNA amplified fragment of the bacillus amyloliquefaciens C-1 strain is sequenced and compared with a 16s rRNA sequence of bacteria in a NCBI (national center of biotechnology information) database; a comparison result proves that the bacillus amyloliquefaciens C-1 strain belongs to B.amyloliquefaciens; and an NCBI register number of the 16s rRNA sequence of the bacillus amyloliquefaciens C-1 strain is JN974457. The bacillus amyloliquefaciens C-1 strain is collected in china center for type culture collection (CCTCC), has the effects of reducing exopolysaccharide, removing DPPH (1,1-diphenyl-2-picryl-hydrazyl) free radical, scavenging hydroxyl free radical (.OH), scavenging superoxide anion (O<2->), inhibiting lipid peroxidation and inhibiting human tumor cell proliferation, and opens up a new way and a new research field for exploring anti-tumor drugs.
Description
Technical field
The invention belongs to biological technical field, be specifically related to the exocellular polysaccharide meta-bolites extraction and application of a bacillus amyloliquefaciens Bacillus amyloliquefaciens C-1 bacterial strain.
Technical background
In recent years, the antibacterial substance of employing microorganism and generation thereof is prevented and treated farm crop fungus disease has become one of important research direction, Caldelra etc. are separated to a bacillus amyloliquefaciens CCMI 1051 (Bacillus amyloliquefaciens) to head mold L-122(Rhizopussp) and trichoderma harziarum CCMI 783 (Trichoderma harzianum) etc. there is strong restraining effect [Annals of micrbilology, 2007,57:29-33].The report bacillus amyloliquefaciens PPCB004 such as Arrebola can suppress the mycelia of the Penicillium fungies such as Penicillium crustosum Thom and extend, Lee etc. find that slow sick series bacillus WJ5 (Paenibacillus lentimorbus) bacterial strain suppresses the various plants pathogenic bacterium such as grey mold (Botrytis cinerea), its antifungus active substance is secreted in extracellular, and can be by n-butanol extraction.Research shows: the antibacterial substance of genus bacillus and generation thereof has huge application potential in biocontrol of plant disease.
Bacillus amyloliquefaciens is a kind of bacterium very high with subtilis affinity, in the process of growth of himself, can produce a series of meta-bolites, these meta-bolitess make bacillus amyloliquefaciens can have the activity of Antifungi and bacterium widely.The people such as Wang Yingguo are separated to a bacillus amyloliquefaciens from compost, and it all has very strong restraining effect for plant pathogenic fungis such as Fusarium oxysporum, strawberry snake germs.The people such as Li Lubin filter out the bacillus amyloliquefaciens ZL725 blue Pathogens Causing Root Rot Disease Fusarium oxysporum of great Hua favour to antagonistic activity from the soil sample of the blue planting site collection of various places great Hua favour.The people such as Wang Yiwen are separated to a bacillus amyloliquefaciens from different melon fruits surface first, to 8 kinds of postharvest fruit and vegetable pathogenic fungies such as Botrytis cinerea, chain lattice spore, Fusarium oxysporum, aspergillus niger and trichothecium roseum significantly and the antagonistic action of wide spectrum.The people such as Jiang Junpo filter out from the ight soil of healthy ox has inhibiting bacillus amyloliquefaciens BN-9 to intestinal bacteria.The people such as Sutyak isolate 1 bacillus amyloliquefaciens from the probiotic bacteria milk product of Yoghourt factory.The aseptic supernatant liquor of its cell carries out after incubated overnight, can produce bacteriostatic action to listeria bacteria (Listeria monocytogenes).Meanwhile, in clinical trial, gardnerella vaginalis and streptococcus agalactiae are also had to inhibition, its effective antibacterial substance is bacteriocin.The people such as W.S.Wu have studied the potential prevention effect of bacillus amyloliquefaciens to Qiu Yingke crop Micobial Disease, by the experiment from separating the antagonist for treating active bacterial strain borne causal agent that bacillus amyloliquefaciens carries out in 2 hosts (experimental plot) (C, T) and greenhouse and field test, find that 6 strains come from bacillus amyloliquefaciens that host C and 1 strain come from host T and can effectively reduce the seriousness of seed and blade disease.
To bacillus amyloliquefaciens meta-bolites, especially the research of the anti-human tumour aspect of its exocellular polysaccharide belongs to first, and the research of antioxygenation belongs to first.Polysaccharide antioxygenation has become one of the most active research topic, conventionally plant and alga-derived polysaccharide are comparatively general, but in the last few years, microbe-derived polysaccharide (exocellular polysaccharide) causes extensive concern, especially the exocellular polysaccharide of bacterium and originated from fungus, because it is easy to separation and purification and output advantages of higher is quite favored in industrial production.
Exocellular polysaccharide (Exopolysaccharide, EPS) be the general name of the outer sugary structure of bacterial cell, comprising that polysaccharide component and gram-positive microorganism outside gram negative bacterium after birth obtain peptidoglycan, is the polymer substance that bacterium produces under specific environment regulates.As the secondary metabolite of bacterium, exocellular polysaccharide mainly contains the effect of bacterium: 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 towards periphery enough nutrition.As the class biopolymer being closely related with human lives, research in recent years finds that extracellular polysaccharide of bacteria also has anti-oxidant, radioprotective, immunomodulatory, hypoglycemic, reducing blood-fat, and the special biologic activity such as antitumor, hepatitis virus resisting.
In recent years, the research report of microbial polysaccharide anti-oxidant activity is a lot, and it can remove free radical in vitro, also has the ability of anti peroxidation of lipid; Can improve in vivo the activity of antioxidase, also show the ability of anti peroxidation of lipid, also may be to relevant by the activity that regulates endogenous antioxidant in body.Polysaccharide can be used as a kind of natural biological anti-oxidant and carries out compositely in addition, strengthens provide protection to HUMAN HEALTH.
Microbial polysaccharide is being treated and/or is being suppressed tumour or apply in tumour auxiliary diagnosis, prognosis, in the biological activitys such as the gene recombination that comprises bacillus amyloliquefaciens or composition and Antifungi thereof, bacterium, antioxygenation and application thereof, rarely have report, this research provides new approaches and recent studies on field for seeking antitumor drug.
Report that partial solution bacillus amyloliquefaciens has antibacterial character (comprising fungus and bacterium), in biocontrol of plant disease, have huge application potential, the research of the current anti-human Tumor-assaciated of the exocellular polysaccharide aspect about bacillus amyloliquefaciens Bacillus amyloliquefaciens C-1 has no report.
Summary of the invention
In order to overcome the shortcoming of above-mentioned prior art, the object of the present invention is to provide the exocellular polysaccharide meta-bolites extraction and application of a bacillus amyloliquefaciens Bacillus amyloliquefaciens C-1 bacterial strain, for seeking antitumor drug, opened up new approaches and recent studies on field.
In order to achieve the above object, the technical scheme that the present invention takes is:
The exocellular polysaccharide meta-bolites of one bacillus amyloliquefaciens Bacillus amyloliquefaciens C-1 bacterial strain, its 16s rRNA sequence is carried out to pcr amplified fragment, after order-checking, carry out the bacterium 16s rRNA sequence alignment of ncbi database, confirm that this strain isolated belongs to bacillus amyloliquefaciens Bacillus amyloliquefaciens, called after bacillus amyloliquefaciens C-1, its 16s rRNA sequence NCBI accession number is JN974457, this bacterium is now stored in Chinese Typical Representative culture collection center (CCTCC), and preserving number is CCTCC NO:M 2012177.
One bacillus amyloliquefaciens Bacillus amyloliquefaciens C-1 produces the extraction of Exopolysaccharide Production From The Fermentation, polysaccharide: the lawn of C-1 bacterial strain on the solid medium of LB+1% glucose be white in color,, moistening, full; Through concentrated, precipitation, except obtaining B.amyloliquefaciens C-1 exocellular polysaccharide purified product after albumen, dialysis, output is 380mg/L, and glycoprotein concentration is wherein 0.7%.
The antioxidation activity in vitro of one bacillus amyloliquefaciens B.amyloliquefaciens C-1 exocellular polysaccharide: B.amyloliquefaciens C-1 exocellular polysaccharide reducing power is in Table 1
Table 1
B.amyloliquefaciens C-1 exocellular polysaccharide to the scavenging(action) of DPPH free radical in Table 2
Table 2
B.amyloliquefaciens C-1 exocellular polysaccharide to the scavenging(action) of hydroxy radical qiao OH in Table 3
Table 3
B.amyloliquefaciens C-1 exocellular polysaccharide is to superoxide anion O
2 -scavenging(action) in Table 4
Table 4
B.amyloliquefaciens C-1 exocellular polysaccharide to Lipid peroxidation in Table 5
Table 5
The inhibition result of B.amyloliquefaciens C-1 exocellular polysaccharide to human tumor cells propagation
In Table 6 and table 7
The inhibiting rate % of the exocellular polysaccharide 2.5mg/ml that table 6 bacillus amyloliquefaciens is purified to different people growth of tumour cell
| Tumour cell kind | 7901 | A549 | 7721 | MCF7 | 7402 | HELA |
| Inhibiting rate % | 73.2 | 65.0 | 64.0 | 57.7 | 41.1 | 78.6 |
The inhibiting rate of table 7 bacillus amyloliquefaciens exocellular polysaccharide different concns to tumour cell 7901 and HELA
| Exocellular polysaccharide concentration mg/ml | 0.07 | 0.14 | 0.28 | 0.56 | 1.12 | 2.24 |
| To 7901 inhibiting rate % | 12.99 | 29.47 | 30.32 | 31.32 | 45.11 | 64.60 |
| To the inhibiting rate % of HELA | 10.50 | 22.68 | 23.46 | 40.50 | 43.70 | 61.87 |
Accompanying drawing explanation
Fig. 1 a is the microscopic examination picture of this bacterium.
Fig. 1 b is the NJ evolutionary tree of this bacterium 16s rRNA sequence.
Fig. 2 is that the exocellular polysaccharide that bacillus amyloliquefaciens is purified acts on the affect figure of SGC7901 cell 48h on its apoptosis, in figure, and Q3: normal cell; Q4: cell early withers; Q2: cell withers evening; Q1: non-viable non-apoptotic cell a: negative control group; B:0.28mg/mL; C:0.56mg/mL; D:1.12mg/mL.
Fig. 3 is that the exocellular polysaccharide that bacillus amyloliquefaciens is purified acts on the affect figure of SGC7901 cell 48h on its cycle, in figure, and a: negative control group; B:0.28mg/mL; C:0.56mg/mL; D:1.12mg/mL.
Embodiment
Below in conjunction with example and accompanying drawing, the present invention is described in detail.
Separate source: the disposable vegetables assorted cold dishes sample gathering from certain supermarket, homogenate under sterile state, scale takes 0.5 sample, add in 0.1% peptone solution test tube of sterilizing and fully mix, the standing 5min of room temperature, extractum carnis-peptone the solid medium dissolving of getting supernatant 1ml and 25ml sterilizing mixes, and pour plate is cultivated 48h for 37 ℃.Find that this bacterium grows rapidly on extractum carnis-peptone solid medium, bacterium colony is smooth, moistening, and toughness during contact, while cultivating in LB liquid tube substratum, occurs cell flocculation phenomenon for 48 hours later.
Separate used medium: extractum carnis-peptone solid medium (0.3% extractum carnis, 1% peptone, 0.5% sodium-chlor, 1.5% agar, high pressure steam sterilization, 121 ℃, 20min)
The exocellular polysaccharide meta-bolites of one bacillus amyloliquefaciens Bacillus amyloliquefaciens C-1 bacterial strain, its 16s rRNA sequence is carried out to pcr amplified fragment, after order-checking, carry out the bacterium 16s rRNA sequence alignment of ncbi database, confirm that this strain isolated belongs to bacillus amyloliquefaciens Bacillus amyloliquefaciens C-1, called after bacillus amyloliquefaciens C-1, its 16srRNA sequence NCBI accession number is JN974457, this bacterium is now stored in Chinese Typical Representative culture collection center (CCTCC), preserving number is CCTCC NO:M 2012177, depositary institution address: Wuhan, China Wuhan University, preservation date: on May 22nd, 2012.Fig. 1 a is shown in by the microscopic examination picture of this bacterium, and the NJ evolutionary tree of its 16s rRNA sequence sees Fig. 1 b.
One bacillus amyloliquefaciens Bacillus amyloliquefaciens C-1 produces the extraction of Exopolysaccharide Production From The Fermentation, polysaccharide: the lawn of C-1 bacterial strain on the solid medium of LB+1% glucose be white in color, moistening, full, sees Fig. 1 a; Through concentrated, precipitation, except obtaining bacillus amyloliquefaciens B.amyloliquefaciens C-1 exocellular polysaccharide purified product after albumen, dialysis, output is 380mg/L, and glycoprotein concentration is wherein 0.7%.
The antioxidation activity in vitro of one bacillus amyloliquefaciens Bacillus amyloliquefaciens C-1 exocellular polysaccharide: Bacillus amyloliquefaciens C-1 exocellular polysaccharide reducing power is in Table 1
Table 1
The size of medicine reducing power has reflected the power of its preventative anti-oxidant function to a certain extent.Antioxidant is by the reductive action of self, to provide electronics to remove free radical, and oxidation-resistance strengthens with the enhancing of reducing power.Therefore, can reflect by measuring the size of reducing power the power of anti-oxidant activity.
Bacillus amyloliquefaciens C-1 exocellular polysaccharide to the scavenging(action) of DPPH free radical in Table 2
Table 2
DPPH free radical is a kind of very stable free radical centered by nitrogen, if sample can be removed DPPH, points out sample to have and removes the effective concentration of hydroxy radical qiao, alkane free radical and peroxy radical and interrupt the effect of lipid peroxidation chain reaction.DPPH free radical has single electron, at 517nm place, has strong absorption peak, and its ethanolic soln is intense violet color, adds after sample, at 517nm place, measures it to DPPH free radical scavenging effect.
Bacillus amyloliquefaciens C-1 exocellular polysaccharide to the scavenging(action) of hydroxy radical qiao OH in Table 3
Table 3
Scavenging action to hydroxyl free radical is the important indicator of reflection antioxidation of drug.In reaction system, H202 and Fe2+ mix generation Fenton reaction, produce OH.It has very high reactive behavior, can effectively be caught by Whitfield's ointment, and then generate coloring matter.If but in reaction system, adding the material with scavenging(action), this material is competed with Whitfield's ointment, and the growing amount of coloured product is reduced.
Exocellular polysaccharide is to superoxide anion O
2 -scavenging(action) in Table 4
Table 4
Under certain condition, pyrogallol autoxidation produces O2-, and ultra-oxygen anion free radical is that ground state oxygen is accepted first oxyradical forming after an electronics, can generate through a serial response other oxyradical.On polysaccharide molecule, have the hemiacetal hydroxyl of reductibility, can with ultra-oxygen anion free radical generation redox reaction, thereby stop free chain reaction.O2-clearance rate is one of important indicator of reflection antioxidation of drug.
Lipid peroxidation is in Table 5
Table 5
Lipid peroxidation mainly refers to a kind of free chain reaction occurring in polyunsaturated fatty acid, and OH free radical is the main initiator of lipid peroxidation.
Bacillus amyloliquefaciens the results are shown in Table 6 and table 7 to the inhibition of human tumor cells propagation
The inhibiting rate % of the exocellular polysaccharide 2.5mg/ml that table 6 bacillus amyloliquefaciens is purified to different people growth of tumour cell
| Tumour cell kind | 7901 | A549 | 7721 | MCF7 | 7402 | HELA |
| Inhibiting rate % | 73.2 | 65.0 | 64.0 | 57.7 | 41.1 | 78.6 |
The inhibiting rate of table 7 bacillus amyloliquefaciens exocellular polysaccharide different concns to tumour cell 7901 and HELA
| Exocellular polysaccharide concentration mg/ml | 0.07 | 0.14 | 0.28 | 0.56 | 1.12 | 2.24 |
| To 7901 inhibiting rate % | 12.99 | 29.47 | 30.32 | 31.32 | 45.11 | 64.60 |
| To the inhibiting rate % of HELA | 10.50 | 22.68 | 23.46 | 40.50 | 43.70 | 61.87 |
Below in conjunction with example, the present invention is described in detail.
1, extract the total DNA of C-1, and be template always, amplification 1.5kb 16S rRNA sequence, 16S rRNA PCR 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 circulation denaturation; 94 ℃ of sex change, 30sec, 58 ℃ of annealing, 30sec, 72 ℃ of extensions, 1min, 30 circulations; 72 ℃ of 7min.Pcr amplification system is to add respectively 10mM dNTP 1.6 μ l in 20 μ l PCR systems, 10 × Taq enzyme buffer, 2 μ l, and the 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, after agarose gel electrophoresis checking, reclaims test kit with PCR product and reclaims, and send the order-checking of order-checking company.The upper bacterial 16 S rRNA sequence alignment of announcing of sequencing result and NCBI.
2, by the mono-colony inoculation of Bacillus amyloliquefaciens C-1 to 3ml LB liquid tube substratum, 30 ℃, 200rpm aerated culture 8-10 hour, 2% switching amount is forwarded to fermention medium---the aerated culture 60 hours in-LB+1% glucose that produces exocellular polysaccharide, 10000rpm, 3min, 4 ℃ of centrifugal collection nutrient solution supernatants extract exocellular polysaccharide.
Adopt Rotary Evaporators that supernatant liquor is concentrated into 1/10 of original volume at ambient temperature, then add 95% ice ethanol of 3 times of volumes, fully mix, place at 4 ℃ and exocellular polysaccharide was fully precipitated in 18 hours.8000rpm, 10min, 4 ℃ of centrifugal collecting precipitations, are now Crude polysaccharides.Crude polysaccharides profit adopts Sevage method except after albumen, and centrifuging and taking supernatant with sterilizing pure water damping fluid dialysis 48 hours, is changed 1 damping fluid for every 12 hours in the dialysis tubing that is 8000-14000 at molecular weight cut-off.Finally polysaccharide soln is carried out to frozen drying and obtain Bacillus amyloliquefaciens C-1 exocellular polysaccharide dry powder.
Bacillus amyloliquefaciens C-1 exocellular polysaccharide dry powder is dissolved in sterilized water, is configured to the solution of 70mg/L, standby.
3, the exocellular polysaccharide antioxidation in vitro experimental result that bacillus amyloliquefaciens is purified
3-1.C-1 exocellular polysaccharide reducing power
Measuring method:
On electronic balance, take 20mg EPS powder, be configured to the EPS solution of 5.0mg/mL.5.0,2.5,1.25,0.6,0.3,0.15mg/mL adopt coubling dilution, with distilled water, this EPS solution dilution is become to 6 kinds of concentration:.With vitamins C (Vc) as contrast: the Vc solution that adopts 6 kinds of concentration of same method preparation.Get 12 brace plug test tubes, add respectively EPS solution and the vitamin c solution of 1mL different concns; Add phosphate buffered saline buffer, 2.5mL 1% potassium ferricyanide solution of 2.5mL pH 6.6; Fully mix; Under 50 ℃ of water bath condition, reaction 20min; The trichoroacetic acid(TCA) that adds again 2.5mL 10%, fully mixes; Under 3000rpm rotating speed, centrifugal 10min; Get supernatant liquor 2.5mL in test tube; Then add 2.5mL distilled water and 0.5mL 0.1%FeCl3 solution, fully mix; At ambient temperature, reaction 5min.Get solution in appropriate each test tube in cuvette, with uv-spectrophotometric instrument, survey the absorbancy at 700nm place.
The scavenging(action) of 3-2. exocellular polysaccharide to DPPH free radical
Measuring method:
On electronic balance, take 20mg EPS powder, be configured to the EPS solution of 5.0mg/mL.5.0,2.5,1.25,0.6,0.3,0.15mg/mL adopt coubling dilution, with distilled water, this EPS solution dilution is become to 6 kinds of concentration:.With vitamins C (Vc) as contrast: the Vc solution that adopts 6 kinds of concentration of same method preparation.The DPPH anhydrous alcohol solution that takes 20mg is settled to 500mL, is mixed with the DPPH solution of 40mg/L.Experiment point sample sets and blank group: get respectively the DPPH solution 2mL of 40mg/l, add in the test tube of 13 10mL tool plugs; Sample sets adds respectively EPS solution and the vitamin c solution of 2mL different concns, and blank group adds 2mL distilled water; After fully mixing, lucifuge, at ambient temperature, reaction 30min.Get solution in appropriate each test tube in cuvette, with uv-spectrophotometric instrument, survey the absorbancy at 517nm place.The ability that EPS and vitamins C are removed DPPH free radical reflects by clearance rate, and its calculation formula is as (1), the mean light absorbency value that in formula, A0 is blank, and A1 is the mean light absorbency value of EPS and vitamins C sample.
DPPH free radical scavenging activity (%)=(A0-A1)/A0 × 100% (1)
The scavenging(action) of 3-3. exocellular polysaccharide to hydroxy radical qiao OH
Measuring method:
On electronic balance, take 20mg EPS powder, be configured to the EPS solution of 5.0mg/mL.Adopt coubling dilution, with distilled water, this EPS solution dilution is become to 6 kinds of concentration: 5.0,2.5,1.25,0.6,0.3,0.15mg/mL. for vitamins C (Vc) as contrast: adopt same method to prepare the Vc solution of 6 kinds of concentration.Experiment point sample sets and blank group: sample sets is got respectively EPS solution and vitamin solution 2mL in 12 brace plug test tubes, and blank group adds 2mL distilled water in 1 test tube; The FeSO4 solution that adds successively 2mL 6mmol/l, fully mixes; Adding respectively the H2O2 solution of 2mL 6mmol/L, fully mix; Under room temperature condition, standing and reacting 30min.Get solution in appropriate each test tube in cuvette, with uv-spectrophotometric instrument, survey the absorbancy at 510nm place.EPS and vitamins C are removed being reflected by clearance rate of ability of hydroxy radical qiao, and its calculation formula is as (2), the mean light absorbency value that in formula, A0 is blank, and A1 is the mean light absorbency value of EPS and vitamins C sample.
Scavenging action to hydroxyl free radical (%)=(A0-A1)/A0 × 100% (2)
The scavenging(action) of 3-4. exocellular polysaccharide to superoxide anion O2-
Measuring method:
On electronic balance, take 20mg EPS powder, be configured to the EPS solution of 5.0mg/mL.Adopt coubling dilution, with distilled water, this EPS solution dilution is become to 6 kinds of concentration: 5.0,2.5,1.25,0.6,0.3,0.15mg/mL. for vitamins C (Vc) as contrast: adopt same method to prepare the Vc solution of 6 kinds of concentration.Experiment point sample sets and blank group: get 4.5mL0.05mol/L Tris-HCl buffered soln (pH 8.2) in the test tube of 13 10mL tool plugs, be placed in 25 ℃ of water-bath preheating 25min; Sample sets adds respectively EPS solution and the vitamin c solution of 1mL different concns, and blank group adds 1mL distilled water; Add respectively again 0.4mL25mmol/L pyrogallol solution, fully mix; Under 25 ℃ of water bath condition, reaction 5min, then adds respectively the HCl 1mL of 8mol/l with termination reaction.Get solution in appropriate each test tube in cuvette, with uv-spectrophotometric instrument, survey the absorbancy at 299nm place.EPS and vitamins C are removed being reflected by clearance rate of ability of superoxide anion, and its calculation formula is as (3), the mean light absorbency value that in formula, A0 is blank, and A1 is the mean light absorbency value of EPS and vitamins C sample.
Clearance rate (%)=(A0-A1)/A0 × 100% (3) of superoxide anion
3-5. Lipid peroxidation
Measuring method:
Model LPO reaction system take lipovitellinin as substrate comprises: (the yolk PBS of isopyknic pH 7.450.1mol/L is made into the yolk suspension of 0.2mL 1:40 dilution, with FeSO47H2O solution, the certain density EPS solution of the 100 μ L PBS of front first magnetic agitation 10min, 0.2mL 25mmol/L, solution is supplied 2.0mL.The same 0.5mL 20%TCA solution that also adds in advance of control tube other reagent except not adding EPS solution.On-test, puts by above-mentioned two kinds of test tubes the 15min that vibrates in 37 ℃ of water-baths simultaneously, after taking-up, sample tube adds 20%TCA solution 0.5mL, after standing 10min, in the centrifugal 10min of 3500rpm, get supernatant liquor with control tube, add respectively 1.0mL thiobarbituricacidα-(TBA 0.8%) solution, jump a queue, in 100 ℃ of water-bath 15min, take out cooling.With blank tube zeroing (blank with the replacement of 2.0mL PBS solution), measure A532, the inhibiting rate (%) of sample EPS to lipovitellinin LPO.Be expressed as (4).
EPS (%)=(1-sample A532/ contrasts A532) × 100%
4, tumor cell culture and inhibiting rate detect:
1) mammary cancer MCF7, liver cancer 7721,7402, cancer of the stomach 7901, lung cancer A549, six kinds of cells of HELA are all purchased from U.S. ATCC cell bank, in containing in the RPMI-1640 of 10% foetal calf serum, are placed in 37 ℃, 5%CO2 incubator and cultivate.
2) growth conditions is good, in MCF7,7721,7402,7901, the A549 of logarithmic phase growth, HELA cell with 10
5be inoculated in 96 orifice plates, spend the night and treat that cell is naturally adherent.By adding the Bacillus sphaericus crystallin extract of 0.5mg/ml concentration in above-mentioned cell, then cell is put to 37 ℃, 5%CO
2middle cultivation 48h.
3) mtt assay is measured Growth of Cells: cultivate after 48h, treat that the every hole of gaging hole adds MTT solution (5mg/ml) 20 μ l, 37 ℃ are continued to cultivate 4h.After stopping cultivating, careful exhaustion treats that nutrient solution in gaging hole, every hole add 150 μ l DMSO, and vibration 10min, fully dissolves xln, measures absorbancy in microplate reader 490nm wavelength place.Establish 5 parallel holes for every group, every group of experiment all in triplicate.
Inhibiting rate (%)=(survival rate of 1-cell) × 100% of Growth of Cells
5, the impact of the exocellular polysaccharide that employing Flow cytometry bacillus amyloliquefaciens is purified on tumour cell cycle, apoptosis
1) the centrifugal 10min collecting cell of 1500rpm;
2) supernatant discarded, PBS rinsing once, is resuspended in cell in 80% ethanol of precooling, more than-20 ℃ of fixing 24h;
3) carry out before flow cytometer detection, the centrifugal 10min of 1500rpm collects the cell after fixing;
4) PBS washs once, the centrifugal 10min collecting cell of 1500rpm;
5) cell is resuspended in the PBS containing 100ug/ml RNaseA and 50ug/ml PI to incubated at room 30min;
The analysis of apoptosis rate, after using exocellular polysaccharide effect SGC7901 cell that 0.28mg/mL, 0.56mg/mL, 1.12mg/mL bacillus amyloliquefaciens purify 48 hours, use two the dying of AnnexinV-FITC/PI to carry out apoptosis analysis, investigate the shared ratio of apoptotic cell.Investigating extract is the situation of SGC7901 apoptosis induction to gastric cells.The result of cells were tested by flow cytometry shows that the each district of scatter diagram represents respectively following implication: Q3(Annexin-/PI-) be normal viable cell; Q4(Annexin+/PI-) be viable apoptotic cell; Q1(Annexin+/PI+) be non-viable apoptotic cell; Q2(Annexin-/PI+) be mechanical injuries cell and downright bad cell.
Cell cycle is detected, after using exocellular polysaccharide effect SGC7901 cell that 0.28mg/mL, 0.56mg/mL, 1.12mg/mL bacillus amyloliquefaciens purify 48 hours, use PI mono-dying to carry out cycle analysis, investigate the impact of shared ratio in extract cell cycle each period.
The present invention finds that such polysaccharide has certain restraining effect to human malignant lesion's cell first.By the batch fermentation of wild-type bacillus amyloliquefaciens, its cell is carried out to the processing of the centrifugal grade of cracking, obtain its meta-bolites bacillus amyloliquefaciens exocellular polysaccharide, first this polysaccharide is applied in the experiment of anti-human tumour cell.
To the preliminary screening of human tumor cell line, select several human tumor cell lines, with finite concentration, carry out preliminary screening, find that this albumen has higher inhibiting rate to human liver cancer cell 7901 and cervical cancer cell HELA, is respectively 78%, 73%.The tumour cell higher to inhibiting rate further detects, select the inhibiting rate of genus bacillus crystallin different concns to tumour cell 7901 and HELA, experiment find, 7901 and HELA tumour cell its meta-bolites genus bacillus crystallin is had to certain concentration dependent.
As shown in Figure 2, negative control group is early withered and is amounted to 4.8% with the cell withering evening.Small concentration administration group (0.28mg/mL) is early withered and is amounted to 28.4% with the cell withering evening, increases to some extent compared with negative control group.After middle concentration administration group (0.56mg/mL) administration is processed, apoptosis rate is 38.0%.Large concentration administration group (1.12mg/mL) apoptosis rate is 52.8%, all has significant difference compared with negative control group.
As shown in Figure 3, after the exocellular polysaccharide effect SGC7901 cell 48 hours that bacillus amyloliquefaciens is purified, use mono-the dying of PI to carry out cycle analysis, investigate the impact of shared ratio in extract cell cycle each period, the exocellular polysaccharide that bacillus amyloliquefaciens is purified does not have a significant impact gastric carcinoma cell lines SGC7901.
Toxin protein first Application i.e. research for anti-human tumour cell in new field that spherical bud bar spore bacterium is produced.Along with the continuous rising of tumor mortality rate, the serious health and lives that has threatened the mankind of cancer, the seeking and find it is major issue urgently to be resolved hurrily of antitumor drug.In recent years, from crude substance, obtaining antineoplastic component gets most of the attention, this research is inquired into and is analyzed and find antitumorigenic substance from the meta-bolites crystallin of wild spherical bud bar spore bacteria strain, expands the scope of seeking to find antitumor drug, proposes new Research Thinking and new research field.
Claims (1)
1. a bacillus amyloliquefaciens (Bacillus amyloliquefaciens) C-1, is characterized in that: this bacterium is now stored in Chinese Typical Representative culture collection center (CCTCC), and preserving number is CCTCC NO:M2012177.
Priority Applications (1)
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| CN103224898B (en) * | 2013-03-05 | 2014-10-29 | 中国水产科学研究院黄海水产研究所 | Marine bacillus and its polypeptide with antitumor activity |
| CN104031960B (en) * | 2014-06-12 | 2017-01-18 | 西安交通大学 | Extracting method and application of extracellular antimicrobial lipopeptides of bacillus amyloliquefaciens |
| TWI562780B (en) * | 2015-10-07 | 2016-12-21 | Univ Nat Pingtung Sci & Tech | Use of exopolysaccharides from bacillus amyloliquenfaciens in manufacturing compositions with hypoglycemic effect and composistions for ameliorating diabetes and complications thereof |
| CN107574129B (en) * | 2017-07-18 | 2020-05-05 | 大连交通大学 | Radix cynanchi bungei endophytic bacterial strain Bacillus amyloliquefaciens BSW-7 and application thereof in antitumor activity |
| CN107988105B (en) * | 2017-12-15 | 2021-06-18 | 中国科学院海洋研究所 | Marine bacillus and application thereof |
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| CN110373356B (en) * | 2019-07-30 | 2021-01-29 | 江南大学 | Bacillus amyloliquefaciens exopolysaccharide for inhibiting growth of enterotoxigenic escherichia coli |
| CN110607254B (en) * | 2019-08-27 | 2022-06-14 | 华南理工大学 | Bacillus amyloliquefaciens and preparation method of extracellular polysaccharide thereof |
| CN111073838B (en) * | 2020-01-08 | 2021-12-28 | 阜阳师范大学 | Bacillus amyloliquefaciens for producing extracellular fructan and application thereof |
| CN111647531B (en) * | 2020-06-03 | 2022-03-11 | 青岛北方茶仓茶文化有限公司 | Siamese bacillus LBP for increasing antioxidant activity, and fermentation product and application thereof |
| CN112043723B (en) * | 2020-08-21 | 2022-05-24 | 华南理工大学 | Application of bacillus amyloliquefaciens exopolysaccharide |
| CN115247141B (en) * | 2022-07-15 | 2023-04-18 | 广西科学院 | Bacillus amyloliquefaciens strain A9 and application thereof |
| CN115011531B (en) * | 2022-07-21 | 2023-07-14 | 北京工商大学 | Bacillus amyloliquefaciens BA-1, lysate and preparation method thereof, application thereof in cosmetics and face cream |
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