CN102816716A - Extraction and application of exopolysaccharide metabolite of bacillus amyloliquefaciens strain - Google Patents

Abstract

Extraction and application of exopolysaccharide metabolites from a strain of Bacillus amyloliquefaciens C-1. The 16s rRNA amplified fragment was sequenced and compared with the bacterial 16s rRNA sequence in the NCBI database, confirming that the isolate belongs to Bacillus amyloliquefaciens B. amyloliquefaciens, whose 16s rRNA sequence NCBI accession number is JN974457, is now preserved in the China Type Culture Collection (CCTCC), and has the ability to reduce extracellular polysaccharides, scavenge DPPH free radicals, The scavenging effect of superoxide anion O ₂ ^- , the inhibitory effect of lipid peroxidation, and the inhibitory effect on the proliferation of human tumor cells have opened up new ideas and new research fields for exploring anti-tumor drugs.

Description

Extraction and application of exopolysaccharide metabolites from a strain of Bacillus amyloliquefaciens

technical field

The invention belongs to the field of biotechnology, and in particular relates to the extraction and application of extracellular polysaccharide metabolites of a Bacillus amyloliquefaciens C-1 strain. the

technical background

In recent years, the use of microorganisms and their antibacterial substances to prevent and control fungal diseases of crops has become one of the important research directions. Caldelra et al. isolated a strain of Bacillus amyloliquefaciens CCMI 1051 (Bacillus amyloliquefaciens) against Rhizopus L-122 (Rhizopussp). And Trichoderma harzianum CCMI 783 (Trichoderma harzianum) etc. have a strong inhibitory effect [Annals of micrbilology, 2007,57:29-33]. Arrebola et al. reported that Bacillus amyloliquefaciens PPCB004 can inhibit the mycelium extension of Penicillium crustosum Thom and other fungi of the genus Penicillium. Lee et al. found that Paenibacillus lentimorbus strain WJ5 (Paenibacillus lentimorbus) inhibited the pathogenicity of various plants such as Botrytis cinerea Bacteria, its antifungal active substances are secreted extracellularly and can be extracted by n-butanol. Studies have shown that: Bacillus and its antibacterial substances have great application potential in the biological control of plant diseases. the

Bacillus amyloliquefaciens is a bacterium with a high affinity with Bacillus subtilis, which can produce a series of metabolites during its own growth, which enable Bacillus amyloliquefaciens to have a wide range of inhibitory fungi and bacteria active. Wang Yingying and others isolated a strain of Bacillus amyloliquefaciens from compost, which has a strong inhibitory effect on plant pathogenic fungi such as Fusarium oxysporum and Strawberry snake disease. Li Lubin and others screened Bacillus amyloliquefaciens ZL725, which has antagonistic activity against the pathogen Fusarium oxysporum of the root rot of Huilan dahualan, from the soil samples collected from the planting places of Huilan dahualan. For the first time, Wang Yiwen et al. isolated a strain of Bacillus amyloliquefaciens from the surface of different melon fruits, which was significantly effective against eight postharvest pathogenic fungi of fruits and vegetables, including Botrytis cinerea, Alternaria, Fusarium oxysporum, Aspergillus niger and Trichothecene pink. Broad-spectrum antagonism. Jiang Junpo and others screened Bacillus amyloliquefaciens BN-9, which has an inhibitory effect on Escherichia coli, from the feces of healthy cattle. Sutyak et al. isolated a strain of Bacillus amyloliquefaciens from probiotic dairy products in a yogurt factory. The sterile supernatant of its cells can produce antibacterial effect on Listeria monocytogenes after overnight culture. At the same time, it also has inhibitory effect on Gardnerella vaginalis and Streptococcus agalactiae in clinical trials, and its effective antibacterial substance is bacteriocin. W.S.Wu et al studied to understand the potential control effect of Bacillus amyloliquefaciens on the bacterial diseases of Qiu Yingaceae crops, and the antagonistic treatment of active strain-derived pathogens by isolating Bacillus amyloliquefaciens from 2 hosts (experimental fields) (C, T) Experiments, greenhouse and field trials found that 6 strains of Bacillus amyloliquefaciens from host C and 1 strain from host T were effective in reducing the severity of seed and leaf diseases. the

It is the first research on the anti-human tumor of the metabolites of Bacillus amyloliquefaciens, especially its exopolysaccharide, and the research on the antioxidant effect is the first. The antioxidant effect of polysaccharides has become one of the most active research topics. Generally, polysaccharides derived from plants and algae are more common, but in recent years, polysaccharides derived from microorganisms (exopolysaccharides) have attracted widespread attention, especially those derived from bacteria and fungi. Exopolysaccharides are favored in industrial production because of their advantages of easy separation and purification and high yield. the

Exopolysaccharide (Exopolysaccharide, EPS) is a general term for bacterial extracellular sugar-containing structures, including the polysaccharide components outside the cell membrane of Gram-negative bacteria and the peptidoglycan of Gram-positive bacteria, which are produced by bacteria under the regulation of specific environments Polymer substances. As a secondary metabolite of bacteria, the effects of exopolysaccharides on bacteria mainly include: making bacterial colonies resistant to phagocytosis; protecting bacteria from binding to antibodies; forming alternate growth of bacteria in the colony; helping the bacterial colony to obtain sufficient nutrients from the surrounding. As a type of biopolymer closely related to human life, recent studies have found that bacterial exopolysaccharides also have special biological properties such as anti-oxidation, anti-radiation, immune regulation, lowering blood sugar, lowering blood lipids, and anti-tumor and anti-hepatitis viruses. learning activity. the

In recent years, there have been many research reports on the antioxidant activity of microbial polysaccharides, which can scavenge free radicals in vitro and have the ability to resist lipid peroxidation; in vivo, they can increase the activity of antioxidant enzymes and also show anti-lipid peroxidation It may also be related to regulating the activity of endogenous antioxidants in the body. In addition, polysaccharides can be compounded as a natural biological antioxidant to enhance the protective effect on human health. the

Microbial polysaccharides are rarely used in the treatment and/or inhibition of tumors or in the auxiliary diagnosis and prognosis of tumors. Gene recombination or compositions containing Bacillus amyloliquefaciens and their biological activities such as inhibiting fungi, bacteria, and antioxidant effects and their applications are rarely According to reports, this study provides new ideas and new research areas for exploring anti-tumor drugs. the

It has been reported that Bacillus amyloliquefaciens has antibacterial properties (including fungi and bacteria), and has great application potential in the biological control of plant diseases. Studies on this aspect have not been reported. the

Contents of the invention

In order to overcome the shortcomings of the above-mentioned prior art, the object of the present invention is to provide a strain of Bacillus amyloliquefaciens C-1 bacterial strain extracellular polysaccharide metabolites extraction and application, to explore new ideas and new research areas for anti-tumor drugs . the

In order to achieve the above object, the technical scheme that the present invention takes is:

The exopolysaccharide metabolites of a strain of Bacillus amyloliquefaciens C-1 were amplified by PCR for its 16s rRNA sequence. After sequencing, the bacterial 16s rRNA sequence comparison of the NCBI database confirmed that the isolate belonged to Bacillus amyloliquefaciens Bacillus amyloliquefaciens, named Bacillus amyloliquefaciens C-1, its 16s rRNA sequence NCBI accession number is JN974457. the

A strain of Bacillus amyloliquefaciens C-1 produces exopolysaccharide fermentation and polysaccharide extraction: the bacterial lawn of the C-1 strain on the solid medium of LB+1% glucose is white, moist and plump; after concentration, After precipitation, protein removal and dialysis, the purified product of B.amyloliquefaciens C-1 exopolysaccharide was obtained, with a yield of 380mg/L and a glycoprotein concentration of 0.7%. the

In vitro antioxidant activity of exopolysaccharide of Bacillus amyloliquefaciens C-1: B.amyloliquefaciens C-1 exopolysaccharide reducing ability is shown in Table 1

Table 1

The scavenging effect of B.amyloliquefaciens C-1 exopolysaccharide on DPPH free radicals is shown in Table 2

Table 2

The scavenging effect of B.amyloliquefaciens C-1 exopolysaccharide on hydroxyl radical·OH is shown in Table 3

table 3

The scavenging effect of B.amyloliquefaciens C-1 exopolysaccharide on superoxide anion O ₂ ^- is shown in Table 4

Table 4

The inhibitory effect of B.amyloliquefaciens C-1 exopolysaccharide on lipid peroxidation is shown in Table 5

table 5

B.amyloliquefaciens C-1 extracellular polysaccharide inhibits human tumor cell proliferation

See Table 6 and Table 7

The exopolysaccharide 2.5mg/ml that table 6 bacillus amyloliquefaciens purifies is to the inhibitory rate % of different human tumor cell growth

Tumor cell type 7901 A549 7721 MCF7 7402 HELA Inhibition rate% 73.2 65.0 64.0 57.7 41.1 78.6

Table 7 Inhibitory rate of different concentrations of Bacillus amyloliquefaciens exopolysaccharide on tumor cell 7901 and HELA

Concentration of exopolysaccharide mg/ml 0.07 0.14 0.28 0.56 1.12 2.24 Inhibition rate of 7901% 12.99 29.47 30.32 31.32 45.11 64.60 Inhibition rate of HELA % 10.50 22.68 23.46 40.50 43.70 61.87

Description of drawings

Figure 1a is a microscope observation picture of the bacteria. the

Figure 1b is the NJ phylogenetic tree of the 16s rRNA sequence of the bacteria. the

Figure 2 is a graph showing the effect of exopolysaccharide purified from Bacillus amyloliquefaciens on the apoptosis of SGC7901 cells for 48 hours. In the figure, Q3: normal cells; Q4: early apoptotic cells; Q2: late apoptotic cells; Q1: necrotic cellsa : negative control group; b: 0.28 mg/mL; c: 0.56 mg/mL; d: 1.12 mg/mL. the

Figure 3 is a diagram of the effect of exopolysaccharide purified from Bacillus amyloliquefaciens on the cycle of SGC7901 cells for 48 hours. In the figure, a: negative control group; b: 0.28 mg/mL; c: 0.56 mg/mL; d: 1.12 mg/mL. the

Detailed ways

The present invention will be described in detail below in conjunction with examples and accompanying drawings. the

Isolation source: The no-clean vegetable assortment sample collected from a supermarket, homogenized in a sterile state, weighed 0.5 sample, added sterilized 0.1% peptone solution to the test tube, mixed well, stood at room temperature for 5 minutes, and took 1ml of the supernatant Mix with 25ml of sterilized melted beef extract-peptone solid medium, pour the plate, and incubate at 37°C for 48h. It was found that the bacterium grew rapidly on beef extract-peptone solid medium, and the colonies were smooth, moist, and viscous when touched. When cultured in LB liquid test tube medium, cell flocculation appeared after 48 hours. the

Medium for separation: beef extract-peptone solid medium (0.3% beef extract, 1% peptone, 0.5% sodium chloride, 1.5% agar, autoclaved, 121°C, 20min)

The exopolysaccharide metabolites of a strain of Bacillus amyloliquefaciens C-1 were amplified by PCR for its 16s rRNA sequence. After sequencing, the bacterial 16s rRNA sequence comparison of the NCBI database confirmed that the isolate belonged to amyloliquefaciens Bacillus amyloliquefaciens C-1, named Bacillus amyloliquefaciens C-1, its 16srRNA sequence NCBI accession number is JN974457, the bacterium is now preserved in the China Center for Type Culture Collection (CCTCC), the preservation number is CCTCC NO: M 2012177 , Depository unit address: Wuhan University, Wuhan, China, deposit date: May 22, 2012. The microscopic observation picture of the bacteria is shown in Figure 1a, and the NJ evolutionary tree of its 16s rRNA sequence is shown in Figure 1b. the

A strain of Bacillus amyloliquefaciens C-1 produces extracellular polysaccharides for fermentation and polysaccharide extraction: the bacterial lawn of the C-1 strain on the solid medium of LB+1% glucose is white, moist and plump, as shown in Figure 1a; After concentration, precipitation, protein removal and dialysis, the purified exopolysaccharide of Bacillus amyloliquefaciens B. amyloliquefaciens C-1 was obtained, with a yield of 380 mg/L and a glycoprotein concentration of 0.7%. the

In vitro antioxidant activity of a Bacillus amyloliquefaciens C-1 exopolysaccharide: the reducing ability of Bacillus amyloliquefaciens C-1 exopolysaccharide is shown in Table 1

Table 1

The reducing power of a drug reflects the strength of its preventive antioxidant function to a certain extent. Antioxidants scavenge free radicals by donating electrons through their own reduction, and the antioxidant capacity increases with the increase of reducing power. Therefore, the strength of antioxidant activity can be reflected by measuring the reducing power. the

The scavenging effect of Bacillus amyloliquefaciens C-1 exopolysaccharide on DPPH free radicals is shown in Table 2

Table 2

DPPH free radical is a very stable nitrogen-centered free radical. If the sample can remove DPPH, it indicates that the sample has an effective concentration for removing hydroxyl free radicals, alkyl free radicals and peroxyl free radicals and interrupts lipid peroxidation. chain reaction effect. DPPH free radical has a single electron, and has a strong absorption peak at 517nm, and its ethanol solution is dark purple. After adding the sample, its scavenging effect on DPPH free radical is measured at 517nm. the

The scavenging effect of Bacillus amyloliquefaciens C-1 exopolysaccharide on hydroxyl radical·OH is shown in Table 3

table 3

Hydroxyl radical scavenging rate is an important index reflecting the antioxidant effect of drugs. In the reaction system, H2O2 and Fe2+ are mixed and undergo Fenton reaction to produce OH. It has high reactivity and can be effectively captured by salicylic acid to generate colored substances. However, if a substance with a scavenging effect is added to the reaction system, this substance will compete with salicylic acid, reducing the amount of colored products produced. the

The scavenging effect of exopolysaccharide on superoxide anion O ₂ ^- is shown in Table 4

Table 4

Under certain conditions, pyrogallol auto-oxidizes to produce O2-, superoxide anion radical is the first oxygen radical formed after ground state oxygen accepts an electron, and can generate other oxygen radicals through a series of reactions. There are reducing hemiacetal hydroxyl groups on polysaccharide molecules, which can undergo redox reactions with superoxide anion radicals, thereby terminating the free radical chain reaction. O2-clearance rate is one of the important indicators reflecting the antioxidant effect of drugs. the

Inhibitory effect of lipid peroxidation is shown in Table 5

table 5

Lipid peroxidation mainly refers to a free radical chain reaction that occurs in polyunsaturated fatty acids, and ·OH free radical is the main initiator of lipid peroxidation. the

The inhibition results of Bacillus amyloliquefaciens on human tumor cell proliferation are shown in Table 6 and Table 7

The exopolysaccharide 2.5mg/ml that table 6 bacillus amyloliquefaciens purifies is to the inhibitory rate % of different human tumor cell growth

Tumor cell type 7901 A549 7721 MCF7 7402 HELA Inhibition rate% 73.2 65.0 64.0 57.7 41.1 78.6

Table 7 Inhibitory rate of different concentrations of Bacillus amyloliquefaciens exopolysaccharide on tumor cell 7901 and HELA

Concentration of exopolysaccharide mg/ml 0.07 0.14 0.28 0.56 1.12 2.24 Inhibition rate of 7901% 12.99 29.47 30.32 31.32 45.11 64.60 Inhibition rate of HELA % 10.50 22.68 23.46 40.50 43.70 61.87

The present invention is described in detail below in conjunction with example. the

1. Extract C-1 total DNA and use it as a template to amplify 1.5kb 16S rRNA sequence. 16S rRNA PCR primers are 16S-F: 5'AGA GTT TGA TCC TGG CTC AG3', 16S-R: 5'GGT ACC T TG TTA CGA CTT3'. The amplification program was: 94°C, 4min, 1 cycle of pre-denaturation; 94°C denaturation, 30sec, 58°C annealing, 30sec, 72°C extension, 1min, 30 cycles; 72°C 7min. The PCR amplification system is 20 μl. Add 1.6 μl of 10mM dNTP, 2 μl of 10×Taq enzyme buffer, 1 μl of 1nM PCR primers, 2-3ng of G-14 total DNA template, and 0.2 μl of Taq DNA polymerase into the PCR system, and make up with MiniQ water 20 μl. After the PCR amplification product was verified by agarose gel electrophoresis, it was recovered with a PCR product recovery kit and sent to a sequencing company for sequencing. The sequencing results were compared with the bacterial 16S rRNA sequences published on NCBI. the

2. Inoculate a single colony of Bacillus amyloliquefaciens C-1 into a 3ml LB liquid test tube culture medium, culture at 30°C and 200rpm aeration for 8-10 hours, and transfer 2% of the transfer amount to the fermentation medium for producing exopolysaccharides -- --Aeration culture in LB+1% glucose for 60 hours, 10000rpm, 3min, centrifuge at 4°C to collect the culture supernatant to extract exopolysaccharides. the

Concentrate the supernatant to 1/10 of the original volume at room temperature using a rotary evaporator, then add 3 times the volume of 95% ice ethanol, mix thoroughly, and place at 4°C for 18 hours to fully precipitate the exopolysaccharide. Centrifuge at 8000rpm for 10min at 4°C to collect the precipitate, which is crude polysaccharide at this time. After the crude polysaccharide was deproteinized by Sevage method, the supernatant was collected by centrifugation, and dialyzed in a dialysis bag with a molecular weight cut-off of 8000-14000 with sterile pure water buffer solution for 48 hours, and the buffer solution was changed every 12 hours. Finally, the polysaccharide solution was freeze-dried at low temperature to obtain Bacillus amyloliquefaciens C-1 exopolysaccharide dry powder. the

Bacillus amyloliquefaciens C-1 exopolysaccharide dry powder is dissolved in sterile water and prepared as a 70mg/L solution for later use. the

3. In vitro antioxidant test results of exopolysaccharide purified from Bacillus amyloliquefaciens

3-1.C-1 Extracellular polysaccharide reducing ability

test methods:

Weigh 20 mg of EPS powder on an electronic balance, and configure it into a 5.0 mg/mL EPS solution. Dilute the EPS solution with double distilled water to 6 concentrations: 5.0, 2.5, 1.25, 0.6, 0.3, 0.15mg/mL by doubling dilution method. Use vitamin C (Vc) as a control: use the same method to prepare 6 concentrations of Vc solutions. Take 12 test tubes with stoppers, add 1mL EPS solution and vitamin C solution of different concentrations respectively; add 2.5mL pH 6.6 phosphate buffer solution, 2.5mL 1% potassium ferricyanide solution; mix well; Then add 2.5mL of 10% trichloroacetic acid and mix thoroughly; centrifuge at 3000rpm for 10min; take 2.5mL of the supernatant in a test tube; then add 2.5mL of distilled water and 0.5mL of 0.1% FeCl3 solution , mix thoroughly; react at room temperature for 5 minutes. Take an appropriate amount of the solution in each test tube in a cuvette, and measure the absorbance at 700 nm with a UV spectrophotometer. the

3-2. Scavenging effect of exopolysaccharides on DPPH free radicals

test methods:

Weigh 20 mg of EPS powder on an electronic balance, and configure it into a 5.0 mg/mL EPS solution. Dilute the EPS solution with double distilled water to 6 concentrations: 5.0, 2.5, 1.25, 0.6, 0.3, 0.15mg/mL by doubling dilution method. Use vitamin C (Vc) as a control: use the same method to prepare 6 concentrations of Vc solutions. Weigh 20 mg of DPPH and dissolve it in absolute ethanol to 500 mL to prepare a 40 mg/L DPPH solution. The experiment is divided into sample group and blank control group: respectively take 2mL of 40mg/l DPPH solution and add them to 13 10mL test tubes with stoppers; add 2mL EPS solution and vitamin C solution of different concentrations to the sample group, and add 2mL double Distilled water; after fully mixing, avoid light, and react for 30min at room temperature. Take an appropriate amount of the solution in each test tube in a cuvette, and measure the absorbance at 517 nm with a UV spectrophotometer. The ability of EPS and vitamin C to scavenge DPPH free radicals is reflected by the scavenging rate, and its calculation formula is as (1), where A0 is the average absorbance value of the blank control, and A1 is the average absorbance value of the EPS and vitamin C samples. the

DPPH free radical scavenging rate (%)=(A0-A1)/A0×100%(1) 

3-3. Scavenging effect of exopolysaccharide on hydroxyl radical·OH

test methods:

Weigh 20 mg of EPS powder on an electronic balance, and configure it into a 5.0 mg/mL EPS solution. Dilute the EPS solution with double distilled water to 6 concentrations: 5.0, 2.5, 1.25, 0.6, 0.3, 0.15mg/mL. Use vitamin C (Vc) as a control: use the same method to prepare 6 Various concentrations of Vc solution. The experiment was divided into a sample group and a blank control group: the sample group took 2 mL of EPS solution and vitamin solution in 12 stoppered test tubes, and the blank control group added 2 mL of double distilled water to 1 test tube; sequentially added 2 mL of 6 mmol/l FeSO4 solution, fully Mix well; add 2mL of 6mmol/L H2O2 solution respectively, mix well; stand at room temperature for 30min. Take an appropriate amount of the solution in each test tube in a cuvette, and measure the absorbance at 510 nm with a UV spectrophotometer. The ability of EPS and vitamin C to scavenge hydroxyl radicals is reflected by the scavenging rate, and its calculation formula is as (2), where A0 is the average absorbance value of the blank control, and A1 is the average absorbance value of EPS and vitamin C samples. the

Hydroxyl radical scavenging rate (%)=(A0-A1)/A0×100%(2) 

3-4. Scavenging effect of exopolysaccharide on superoxide anion O2-

test methods:

Weigh 20 mg of EPS powder on an electronic balance, and configure it into a 5.0 mg/mL EPS solution. Dilute the EPS solution with double distilled water to 6 concentrations: 5.0, 2.5, 1.25, 0.6, 0.3, 0.15mg/mL. Use vitamin C (Vc) as a control: use the same method to prepare 6 kinds Concentration of Vc solution. The experiment was divided into sample group and blank control group: take 4.5mL0.05mol/L Tris-HCl buffer solution (pH 8.2) in 13 test tubes with stoppers of 10mL, place them in a 25°C water bath to preheat for 25min; EPS solution and vitamin C solution with different concentrations, add 1mL double distilled water to the blank control group; then add 0.4mL 25mmol/L pyrogallol solution, mix well; /l of HCl 1mL to stop the reaction. Take an appropriate amount of the solution in each test tube in a cuvette, and measure the absorbance at 299 nm with a UV spectrophotometer. The ability of EPS and vitamin C to scavenge superoxide anion is reflected by the scavenging rate, and its calculation formula is as (3), where A0 is the average absorbance value of the blank control, and A1 is the average absorbance value of the EPS and vitamin C samples. the

Scavenging rate of superoxide anion (%)=(A0-A1)/A0×100%(3) 

3-5. Inhibition of lipid peroxidation

test methods:

The model LPO reaction system with yolk lipoprotein as the substrate includes: 0.2mL 1:40 diluted yolk suspension (the yolk is prepared with an equal volume of PBS with a pH of 7.450.1mol/L, magnetically stirred for 10min before use, and 0.2mL 25mmol/L FeSO4·7H2O solution, 100μL EPS solution of a certain concentration with PBS, and the solution is supplemented with 2.0mL. The control tube is the same as before except that EPS solution is not added, and 0.5mL 20% TCA solution is added in advance. The above two The test tubes were shaken in a water bath at 37°C for 15 min at the same time. After taking them out, 0.5 mL of 20% TCA solution was added to the sample tubes. After standing for 10 min, they were centrifuged with the control tubes at 3500 rpm for 10 min. Tauric acid (TBA 0.8%) solution, stoppered, placed in a water bath at 100°C for 15 minutes, taken out and cooled. Zeroed with a blank tube (the blank was replaced by 2.0mL PBS solution), determined A532, the inhibition rate of the sample EPS on vitellolipoprotein LPO (% ). Expressed as (4). 

EPS(%)=(1-sample A532/control A532)×100%

4. Tumor cell culture and inhibition rate detection:

1) Breast cancer MCF7, liver cancer 7721, 7402, gastric cancer 7901, lung cancer A549, and HELA cells were purchased from ATCC cell bank in the United States, and placed in 1640 culture medium containing 10% fetal bovine serum at 37°C and 5% CO2 cultured in an incubator. the

2) MCF7, 7721, 7402, 7901, A549, HELA cells in good growth state and growing in the logarithmic phase were inoculated in 96-well plates at 10 ⁵ , and the cells naturally adhered to the wall overnight. Add 0.5mg/ml Bacillus sphaericus crystal protein extract to the above cells, and then culture the cells at 37°C, 5% CO ₂ for 48h.

3) Determination of cell growth by MTT method: After culturing for 48 hours, add 20 μl of MTT solution (5 mg/ml) to each well to be tested, and continue culturing at 37°C for 4 hours. After terminating the culture, carefully absorb the culture medium in the wells to be tested, add 150 μl DMSO to each well, shake for 10 min to fully dissolve the crystals, and measure the absorbance at a wavelength of 490 nm on a microplate reader. Five parallel wells were set up in each group, and each experiment was repeated three times. the

Inhibition rate of cell growth (%) = (1- cell survival rate) × 100%

5. Using flow cytometry to detect the effect of exopolysaccharide purified from Bacillus amyloliquefaciens on tumor cell cycle and apoptosis

1) Collect cells by centrifugation at 1500rpm for 10min;

2) Discard the supernatant, rinse once with PBS, resuspend the cells in pre-cooled 80% ethanol, and fix at -20°C for more than 24 hours;

3) Before performing flow cytometry detection, centrifuge at 1500rpm for 10min to collect the fixed cells;

4) Wash once with PBS, centrifuge at 1500rpm for 10min to collect cells;

5) Resuspend the cells in PBS containing 100ug/ml RNaseA and 50ug/ml PI, and incubate at room temperature for 30min;

Analysis of cell apoptosis rate, using 0.28mg/mL, 0.56mg/mL, 1.12mg/mL exopolysaccharides purified from Bacillus amyloliquefaciens to act on SGC7901 cells for 48 hours, then using AnnexinV-FITC/PI double staining for apoptosis analysis , to examine the proportion of apoptotic cells. To investigate the effect of the extract on apoptosis induction of gastric cell line SGC7901. The results of flow cytometry show that each area of the scatter plot represents the following meanings: Q3 (Annexin-/PI-) is normal living cells; Q4 (Annexin+/PI-) is early apoptotic cells; Q1 (Annexin+/PI+) Late apoptotic cells; Q2 (Annexin-/PI+) mechanically damaged cells and necrotic cells. the

For cell cycle detection, use 0.28mg/mL, 0.56mg/mL, 1.12mg/mL exopolysaccharides purified from Bacillus amyloliquefaciens to act on SGC7901 cells for 48 hours, then use PI single staining for cycle analysis, and investigate the effect of extracts on cell cycle. The effect of the proportion of time period. the

The present invention finds for the first time that this type of polysaccharide has a certain inhibitory effect on human malignant tumor cells. Through the batch fermentation of wild-type Bacillus amyloliquefaciens, the cells were lysed and centrifuged to obtain its metabolite Bacillus amyloliquefaciens exopolysaccharide, which was first applied to the experiment of anti-human tumor cells. the

For the preliminary screening of human tumor cell lines, several human tumor cell lines were selected for preliminary screening at a certain concentration, and it was found that the protein had a high inhibitory rate on human liver cancer cell line 7901 and cervical cancer cell line HELA, which were 78% and 73% respectively. %. The tumor cells with higher inhibition rate were further detected, and the inhibition rate of tumor cells 7901 and HELA at different concentrations of Bacillus crystal protein was selected. The experiment found that 7901 and HELA tumor cells had a certain concentration dependence on the metabolite Bacillus crystal protein . the

As shown in Figure 2, the total number of early and late withered cells in the negative control group was 4.8%. In the small concentration administration group (0.28mg/mL), the total number of early and late withered cells was 28.4%, which was increased compared with the negative control group. In the middle concentration administration group (0.56mg/mL), the apoptosis rate was 38.0%. The apoptotic rate of the high-concentration administration group (1.12mg/mL) was 52.8%, which was significantly different from that of the negative control group. the

As shown in Figure 3, after the exopolysaccharide purified by Bacillus amyloliquefaciens acted on SGC7901 cells for 48 hours, the cycle analysis was performed using PI single staining to investigate the effect of the extract on the proportion of each phase of the cell cycle. The purified Bacillus amyloliquefaciens The extracellular polysaccharides had no significant effect on the gastric cancer cell line SGC7901. the

The toxin protein produced by Bacillus sphaericus was used for the first time in a new field of research against human tumor cells. With the continuous increase of tumor mortality, cancer has seriously threatened human health and life. The search and discovery of anti-tumor drugs is an important problem to be solved urgently. In recent years, the acquisition of anti-tumor components from natural substances has attracted much attention. This study explored, analyzed and discovered anti-tumor substances from the metabolite crystal protein of wild Bacillus sphaericus strains, expanded the scope of seeking and discovering anti-tumor drugs, and proposed new research ideas and new research areas. the

Claims (8)

1. A strain of Bacillus amyloliquefaciens C-1, which is characterized in that: its 16s rRNA sequence is subjected to PCR amplification fragments, and after sequencing, the bacterial 16s rRNA sequence comparison of the NCBI database is carried out to confirm that the isolate belongs to amyloliquefaciens Bacillus amyloliquefaciens, named Bacillus amyloliquefaciens C-1, its 16s rRNA sequence NCBI accession number is JN974457. 2. a strain Bacillus amyloliquefaciens Bacillus amyloliquefaciens C-1 according to claim 1, is characterized in that: produce the extraction of extracellular polysaccharide fermentation, polysaccharide, G-14 bacterial strain on the solid medium of LB+1% glucose The bacterial lawn is white, moist, and full; after concentration, precipitation, protein removal, and dialysis, the purified product of B.amyloliquefaciens C-1 exopolysaccharide is obtained, with a yield of 380mg/L and a glycoprotein concentration of 0.7%. 3. A strain of Bacillus amyloliquefaciens C-1 according to claim 1, characterized in that: B. amyloliquefaciens C-1 exopolysaccharide reducing ability is shown in Table 1 Table 1

4. according to claim 1 described a Bacillus amyloliquefaciens C-1, it is characterized in that: B.amyloliquefaciens C-1 extracellular polysaccharide is shown in Table 2 to the scavenging action of DPPH free radical Table 2

5. A strain of Bacillus amyloliquefaciens C-1 according to claim 1, characterized in that: the scavenging effect of B. amyloliquefaciens C-1 exopolysaccharide on hydroxyl radical OH is shown in Table 3 table 3

6. A strain of Bacillus amyloliquefaciens C-1 according to claim 1, characterized in that: the scavenging effect of exopolysaccharide on superoxide anion O ₂ ^- is shown in Table 4 Table 4

7. A strain of Bacillus amyloliquefaciens C-1 according to claim 1, characterized in that: lipid peroxidation inhibition is shown in Table 5 table 5

8. A strain of Bacillus amyloliquefaciens Bacillus amyloliquefaciens C-1 according to claim 1, characterized in that: the inhibition results of Bacillus amyloliquefaciens to human tumor cell proliferation are shown in Table 6 and Table 7 The exopolysaccharide 2.5mg/ml that table 6 bacillus amyloliquefaciens purifies is to the inhibitory rate % of different human tumor cell growth Tumor cell type 7901 A549 7721 MCF7 7402 HELA Inhibition rate% 73.2 65.0 64.0 57.7 41.1 78.6 Table 7 Inhibitory rate of different concentrations of Bacillus amyloliquefaciens exopolysaccharide on tumor cell 7901 and HELA Concentration of exopolysaccharide mg/ml 0.07 0.14 0.28 0.56 1.12 2.24 Inhibition rate of 7901% 12.99 29.47 30.32 31.32 45.11 64.60 Inhibition rate of HELA % 10.50 22.68 23.46 40.50 43.70 61.87

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