Selenium-rich bacillus velezensis with strong tolerance and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and relates to a selenium-rich bacillus velezensis with high tolerance and application of the selenium-rich bacillus velezensis. The invention relates to a selenium-rich bacillus velezensis 915-1 with strong tolerance, which is preserved in Guangdong Microbial Culture Collection Center, and the preservation number is (GDMCC No: 64021). The bacillus velezensis 915-1 can effectively inhibit growth of escherichia coli, salmonella and staphylococcus aureus after inoculation as first inoculation culture bacteria, and can be used as probiotics or drugs for colonization in intestinal tracts to play roles in inhibiting pathogenic bacteria and benefiting life. The bacillus velezensis is applied to production of organic selenium. The bacillus velezensis can absorb, convert and utilize sodium selenite, the sodium selenite is enriched into thalli to be converted into organic selenium, the content of the organic selenium is increased along with increase of the number of cells, and the bacillus velezensis can be used for production and preparation of the organic selenium.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology and relates to a selenium-rich Bacillus velezensis with strong tolerance and its application. Background Art

[0002] Bacillus velezensis belongs to the phylum Firmicutes, order Bacillales, family Bacillaceae, and genus Bacillus. It is a spore-forming Gram-positive bacterium. Most Bacillus species have advantages such as a broad antibacterial spectrum, rapid growth, easy isolation and culture, strong stress resistance, and high biosafety, and thus have been widely studied as probiotics in agriculture, food, industry, medicine, metallurgy, forestry, environmental protection, and military.

[0003] In recent years, the research on Bacillus velezensis has mainly focused on fields such as feed, medicine, textile, aquaculture, sewage treatment, and plant protection. As a new biocontrol microbial factor, the role of Bacillus velezensis in various aspects has attracted much attention.

[0004] With the development of biology and biotechnology, researchers hope to find Bacillus velezensis that can pass through the gastrointestinal tract and colonize in the intestine to exert probiotic activity. At the same time, screening microorganisms with selenium-rich ability for the production of organic selenium is also one of the current key research directions. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art, the present invention provides a Bacillus velezensis that can pass through the gastrointestinal tract and colonize in the intestine to exert probiotic activity, and it has the ability to enrich selenium and can be used for the production of organic selenium. The specific content is as follows:

[0006] A Bacillus velezensis, its taxonomic name is Bacillus velezensis, it is preserved in the Guangdong Provincial Microbial Culture Collection Center (address: 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences), the preservation number is GDMCC No: 64021, and the preservation date is November 14, 2023.

[0007] Furthermore, the Bacillus velezensis is a selenium-rich Bacillus velezensis.

[0008] Furthermore, the Bacillus velezensis is a Bacillus velezensis with strong tolerance. The strong tolerance means having strong acid tolerance, good tolerance to bile salts, and also good tolerance to gastric juice and intestinal juice.

[0009] The application of the described Bacillus velezensis in the preparation of drugs. Especially antibacterial drugs or drugs for the intestine.

[0010] Application of the described Bacillus velezensis in antibacterial activity.

[0011] Furthermore, the antibacterial activity is to inhibit the growth of one or more of Escherichia coli, Salmonella, and Staphylococcus aureus.

[0012] Furthermore, the antibacterial activity is to first inoculate and culture bacteria to inhibit the growth of subsequently inoculated pathogenic bacteria.

[0013] Bacillus velezensis 915-1 in this application, as a first-inoculated culture bacterium, can effectively inhibit the growth of subsequently inoculated Escherichia coli, Salmonella, and Staphylococcus aureus, and can be colonized in the intestine as a probiotic or drug to exert antibacterial and probiotic activities.

[0014] Application of the described Bacillus velezensis in the production of organic selenium. This Bacillus velezensis can absorb, transform, and utilize sodium selenite, enrich it in the cells and convert it into organic selenium. As the number of cells increases, the content of organic selenium also increases, and it can be used in the production and preparation of organic selenium.

[0015] Advantages of the present invention:

[0016] (1) The present invention provides a newly isolated and successfully preserved highly tolerant selenium-rich Bacillus velezensis.

[0017] (2) The Bacillus velezensis provided by the present invention has strong tolerance. The strong tolerance means having strong acid resistance, good tolerance to bile salts, and also good tolerance to gastric juice and intestinal juice, and can survive in the gastrointestinal tract for a long time, having strong probiotic characteristics, providing necessary conditions for it to pass through the gastrointestinal tract and colonize in the intestine and exert probiotic activities.

[0018] (3) Bacillus velezensis 915-1 in this application, as a first-inoculated culture bacterium, can effectively inhibit the growth of subsequently inoculated Escherichia coli, Salmonella, and Staphylococcus aureus, and can be colonized in the intestine as a probiotic or drug to exert antibacterial and probiotic activities.

[0019] (4) Bacillus velezensis can absorb, transform, and utilize sodium selenite, enrich it in the cells and convert it into organic selenium. As the number of cells increases, the content of organic selenium also increases, and it can be used in the production and preparation of organic selenium. Description of the drawings

[0020] Figure 1 It is a growth curve of Bacillus velezensis 915-1.

[0021] Figure 2 It is a result graph of the effect of different pH values on the growth of Bacillus velezensis 915-1.

[0022] Figure 3 The figure shows the effect of different bile salt concentrations on the growth of Bacillus velezensis 915-1.

[0023] Figure 4 The figure shows the effect of artificial gastroenteritis on the growth of Bacillus velezensis 915-1.

[0024] Figure 5 The figure shows the effect of early colonization of Bacillus velezensis 915-1 on the growth of pathogenic bacteria. Detailed implementation manners

[0025] The following further elaborates on the present invention in conjunction with specific embodiments, which is an explanation rather than a limitation of the present invention.

[0026] Embodiment: Research on Bacillus velezensis 915-1

[0027] Materials and methods

[0028] 1.1 Samples

[0029] A strain of Bacillus velezensis isolated and preserved by Guangxi Veterinary Research Institute.

[0030] 1.2 Main reagents

[0031] MRS solid and liquid media, LB solid and liquid media, bacterial micro biochemical reaction tubes, DNA extraction kits.

[0032] 1.3 Plotting the growth curve of viable bacteria

[0033] Inoculate the purified bacterial liquid of each bacterium into 5 mL of LB broth medium at a dose of 3%, and culture it in a shaker at 37 °C and 160 r / min. Measure the OD value at 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 28 hours of culture, and plot the growth curve.

[0034] 1.4 Acid tolerance test

[0035] Use 0.1 mol / L HCl to adjust the pH values of 1×PBS buffer to 2, 2.5, 3, and 3.5 respectively, and sterilize it at 121 °C for 20 min for later use. After activating the frozen probiotics, inoculate them into LB liquid medium, culture them overnight at 37 °C, centrifuge at 12000 r / min for 10 min, discard the supernatant, resuspend the bacterial cells in the same volume of PBS buffer, inoculate them into the adjusted PBS buffer at an inoculation amount of 10%, culture them at 37 °C, take samples every 2 h, perform gradient dilution with PBS buffer, inoculate on the plate for colony counting, and measure the change in the number of viable bacteria from 0 h to 6 h.

[0036] 1.5 Bile salt tolerance test

[0037] The prepared probiotic bacterial solution was centrifuged at 12,000 r / min for 10 min, the supernatant was discarded, the bacterial pellet was resuspended in the same volume of PBS buffer, and inoculated into 0.03%, 0.1%, 0.2%, and 0.3% bile salt solutions at an inoculation amount of 10%. Incubate at a constant temperature of 37°C, sample every 2 h and perform gradient dilution with PBS buffer, inoculate the plate for colony counting, and determine the change in the viable count from 0 h to 6 h.

[0038] 1.6 Intestinal juice and gastric juice tolerance test

[0039] Artificial gastric juice: 0.2 g of sodium chloride and 1.0 g of pepsin were diluted with 100 mL of hydrochloric acid aqueous solution with a pH of 2.0, fully dissolved, filtered and sterilized with a 0.22 μm filter, and stored at 4°C for later use. Artificial intestinal juice: 0.68 g of potassium dihydrogen phosphate was dissolved in distilled water, the pH was adjusted to 7.0 with 0.1 mol / L NaOH solution, and another 1.0 g of trypsin was dissolved in an appropriate amount of water. The two solutions were mixed and made up to 100 mL with water, filtered and sterilized with a 0.22 μm filter, and stored at 4°C for later use. The activated probiotics were inoculated into MRS liquid medium and incubated overnight at a constant temperature of 37°C, centrifuged at 12,000 r / min for 10 min, the supernatant was discarded, the bacterial pellet was resuspended in the same volume of PBS buffer, and inoculated into artificial intestinal juice or artificial gastric juice at an inoculation amount of 10%. Samples were taken at 0 min, 30 min, 60 min, 90 min, and 120 min for colony counting to observe the change in the viable count.

[0040] 1.7 Early colonization

[0041] The concentration of Bacillus velezensis was adjusted to the same concentration as Escherichia coli, Salmonella, and Staphylococcus aureus, and 10 μL of each Bacillus velezensis was added to the MHA solid plate. Incubate at 37°C for 24 h, take out the probiotic culture plate incubated for 24 h, and inoculate 10 μL of the pathogenic bacteria close to the early colonized probiotics on the culture plate. Incubate at 37°C for 24 h, and record the growth of Bacillus velezensis with each pathogenic bacteria.

[0042] 1.8 Determination of selenium addition amount

[0043] Sterilized sodium selenite (Na2SeO3) was added to 2 L of LB culture medium, inoculated at 5% and cultured at 37°C for 24 h, and then the OD value of the bacterial solution at different concentrations was measured to observe the growth and tolerance of Bacillus velezensis 915-1.

[0044] 1.9 Determination of selenium content

[0045] The sterilized sodium selenite (Na2Se03) solution was added to the LB culture medium at a concentration of 4 mmol / L, inoculated at 5%, and fermented at a constant temperature of 37°C for 100 L. Samples were taken at different time points to measure the selenium content at each growth stage.

[0046] 1.10 Statistical analysis

[0047] The above data were analyzed, processed, and graphed using GraphPad. The experimental data were the means of 3 replicates.

[0048] 2 Results

[0049] 2.1 Results of growth curve determination

[0050] See specifically Figure 1

[0051] 2.2 Results of acid tolerance test

[0052] From Figure 2 it can be seen that for Bacillus velezensis 915-1 in PBS with a pH of 2.0 - 3.5, the viable cell count decreased to varying degrees over time. Bacillus velezensis still had good tolerance after being treated with PSB solution with a pH of 2.5 - 3.5 for 6 h. Among them, the logarithm value was still 2.6 after being treated with PBS with a pH of 2 for 6 h. This indicates that Bacillus velezensis has strong acid tolerance.

[0053] 2.3 Results of bile salt tolerance test

[0054] From Figure 3 it can be seen that for Bacillus velezensis 915-1 in bile salts with a concentration of 0.03% - 0.3%, the concentration decreased to varying degrees over time; as the concentration increased, the viable cell count gradually decreased. When treated with 0.3% porcine bile salt, the logarithm value of the viable cell count of the strain decreased significantly from 9.70 to 6.11 in the first 2 h, and from 6.11 to 5.70 from 2 h to 6 h. The degree of decrease was lower than that in the first two hours, and there were still 5.70 logarithm values of viable cells at 6 h; this indicates that Bacillus velezensis 915-1 has good tolerance to bile salts and strong probiotic characteristics, providing the necessary conditions for it to pass through the gastrointestinal tract and colonize in the intestine and exert probiotic activity.

[0055] 2.4 Tolerance of Bacillus velezensis in simulated artificial intestinal fluid and artificial gastric juice

[0056] From Figure 4It can be seen that when Bacillus velezensis 915-1 was treated with simulated artificial gastric juice and intestinal juice for 2 h, the logarithmic values of the viable cell numbers of the strains decreased to varying degrees compared with the initial values, decreasing by 1.39 and 2.7 respectively compared with the initial logarithmic values. There were still viable bacteria with a logarithmic value of at least 4 after 2 h of treatment, indicating that Bacillus velezensis 915-1 had good tolerance to gastric juice and intestinal juice, and a large number of bacteria still survived after 2 h of treatment.

[0057] 2.5 Early colonization results

[0058] As Figure 5 shown, Bacillus velezensis 915-1, as the pre-inoculated culture bacterium, could effectively inhibit the growth of Escherichia coli, Salmonella and Staphylococcus aureus inoculated later.

[0059] 2.6 Determination of selenium addition amount

[0060] The results are shown in Table 1. When the concentration of Na2Se03 was in the range of 0 to 5 mmol, the change in the bacterial liquid concentration was not significant. This indicates that within this range, the bacteria showed selenium tolerance, and the addition of selenium did not affect the growth of the bacterial species. At the same time, the bacterial liquid gradually turned red, and the ability of the bacteria to convert inorganic selenium into organic selenium during metabolism was not inhibited. As the selenium concentration in the culture medium increased, the OD value of the bacterial liquid decreased and the viable cell number decreased, gradually showing selenium intolerance. When the selenium concentration was above 4 mmol, with the increase of the selenium addition amount, the growth of the bacterial cells was inhibited, and at the same time, the ability to convert selenium decreased. Therefore, 4 mmol was selected as the addition concentration of sodium selenite.

[0061] Table 1. OD values of Bacillus velezensis in different selenium contents

[0062]

[0063] 2.7 Determination of selenium content

[0064] The sterilized Na2Se03 solution was added to the LB culture medium at a concentration of 4 mmol / L, inoculated at 5%, and fermented at a constant temperature of 37 °C. Samples were taken at different time points to measure the selenium content at each growth stage. According to the method of GB5009.268-2016 for selenium determination, the detection results showed (specifically shown in Table 2) that the selenium content in the bacterial cells gradually increased from 0 to 24 h. When cultured for 24 h, the selenium enrichment amount of the bacterial cells reached the maximum. The sodium selenite in the culture medium was gradually absorbed and utilized by the bacteria, enriched in the bacteria and converted into organic selenium. As the number of cells increased, the organic selenium content increased. After culturing for 24 h, the selenium content in the bacterial cells gradually decreased. Considering the growth situation, as the culture time prolonged, the bacterial cells lysed, and a part of the selenium was released into the culture medium, resulting in a decrease in the selenium content in the cells. Considering comprehensively, 24 h of fermentation was selected as the fermentation culture time of selenium-enriched Bacillus velezensis 915-1.

[0065]

[0066] Finally, it was determined that the optimal conditions for selenium-enriched fermentation of Bacillus Velez 915-1 in the present invention were: LB medium, 37° C., inoculation with an inoculum size of 5%, a sodium selenite concentration of 4 Mm, and a fermentation time of 24 h.

[0067] Finally, it should be pointed out that the above embodiments are only representative examples of the present invention. Obviously, the technical solution of the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by ordinary technicians in this field should be considered as the protection scope of the present invention.

Claims

1. A Bacillus Velez, characterized in that It is deposited in Guangdong Microbiological Culture Collection Center with the deposit number GDMCC No: 64021.

2. The Bacillus Velezii according to claim 1, characterized in that The Bacillus Velez is a highly tolerant Bacillus Velez.

3. The Bacillus Velezii according to claim 1, characterized in that The strong tolerance is strong acid resistance.

4. The Bacillus Velezii according to claim 1, characterized in that The strong tolerance refers to good tolerance to bile salts.

5. The Bacillus Velezii according to claim 1, characterized in that The strong tolerance refers to good tolerance to gastric juice and intestinal juice.

6. The effect of the Bacillus Velezii described in claim 1 in preparing medicines.

7. Use of the Bacillus Velezii described in claim 1 in antibacterial applications.

8. The use according to claim 7, characterized in that: The antibacterial activity refers to inhibiting the growth of one or more of Escherichia coli, Salmonella and Staphylococcus aureus.

9. The use according to claim 7, characterized in that: The antibacterial method is to first inoculate cultured bacteria to inhibit the growth of later inoculated pathogenic bacteria.

10. Use of the Bacillus Velez subtilis according to claim 1 in producing organic selenium.

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