Bacillus subtilis as well as preparation and application based on bacillus subtilis

Abstract

The invention belongs to the technical field of microorganisms. The invention provides bacillus subtilis QXKL-3, a preparation based on the bacillus subtilis QXKL-3 and application of the bacillus subtilis QXKL-3 in inhibition of streptococcus suis type 2. The strain is preserved in the China General Microbiological Culture Collection Center, the preservation date is September 11, 2023, and the preservation number is CGMCC (China General Microbiological Culture Collection Center) NO.28419. The cell-free supernate of the bacillus subtilis QXKL-3 provided by the invention has efficient antibacterial activity on streptococcus suis type 2, and the antibacterial effect of the cell-free supernate is obviously higher than that of penicillin; meanwhile, even at the temperature of 100 DEG C, the efficient antibacterial property on the streptococcus suis type 2 can still be kept; and the antibacterial activity is still stable under long-time preservation.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a strain of Bacillus subtilis and a preparation and application based thereon. Background Art

[0002] Streptococcus suis (SS) is a pathogen that seriously endangers pig farming and is also an important zoonosis pathogen. Infection in pigs can cause sepsis, meningitis, arthritis, endocarditis and other diseases. S. suis type 2 is one of the pathogens. Problems such as meningitis, sepsis, and arthritis in pigs caused by S. suis type 2 infection have always been one of the problems plaguing the pig farming industry. In addition, the excessive use of antibiotics has led to the emergence of drug-resistant strains of S. suis type 2 and antibiotic residues. Therefore, actively promoting the reduction and replacement of antibiotics and finding efficient antibiotic substitutes have become urgent practical needs.

[0003] Bacillus subtilis is a type of Gram-positive bacteria that is widely present in nature. It has passed the safety certification of the European Food Safety Authority and the U.S. Food and Drug Administration. It is environmentally friendly and is the main production strain in microecological preparations.

[0004] At present, the Bacillus subtilis strains in the prior art are mainly isolated from soil, food, livestock and poultry feces, etc., and the research on the Bacillus strains colonizing in the intestines of local pigs and their functions is relatively limited. In particular, the prior art has relatively few studies on the screening of Bacillus subtilis strains with good antibacterial properties against Streptococcus suis type 2, and there is no report that the active substances in the strains can still maintain antibacterial activity for a long time at high temperature (100°C). Summary of the invention

[0005] In view of the defects of the prior art, the object of the present invention is to provide a strain of Bacillus subtilis, the active ingredients of which have a highly effective antibacterial effect on Streptococcus suis type 2 and can maintain the antibacterial activity at high temperature and for a long time.

[0006] To achieve the above-mentioned purpose, the present invention provides the following strains:

[0007] A strain of Bacillus subtilis QXKL-3, which was deposited in the China General Microbiological Culture Collection Center on September 11, 2023, and the deposit number is CGMCC NO.28419.

[0008] The invention also provides the use of Bacillus subtilis QXKL-3 in inhibiting bacteriostasis of Streptococcus suis type 2.

[0009] In particular, the application is to inhibit Streptococcus suis type 2 when pigs are infected with Streptococcus suis type 2.

[0010] The present invention also provides a preparation for inhibiting Streptococcus suis type 2, and the active ingredient of the preparation is the cells of Bacillus subtilis QXKL-3, the cell-free supernatant of Bacillus subtilis QXKL-3, or the extract of the cell-free supernatant of Bacillus subtilis QXKL-3.

[0011] Preferably, the cell-free supernatant of Bacillus subtilis QXKL-3 is obtained by culturing Bacillus subtilis QXKL-3 and then removing the cells.

[0012] Preferably, the extract of the cell-free supernatant of Bacillus subtilis QXKL-3 is obtained by extracting the cell-free supernatant of Bacillus subtilis QXKL-3 using the ammonium sulfate fractional precipitation method.

[0013] Preferably, when preparing the extract of the cell-free supernatant of Bacillus subtilis QXKL-3, the concentration of ammonium sulfate used is 60%.

[0014] Preferably, when preparing the extract of the cell-free supernatant of Bacillus subtilis QXKL-3, after adding ammonium sulfate, it is left overnight at 4°C and then centrifuged at 12000 r / min for 15 min, and the precipitate is taken.

[0015] The present invention also provides the application of the above preparation in inhibiting Streptococcus suis type 2.

[0016] In particular, the application is to inhibit Streptococcus suis type 2 when pigs are infected with Streptococcus suis type 2.

[0017] In the prior art, there have been sporadic reports on screening some Bacillus subtilis strains with antibacterial activity against Streptococcus suis type 2. For example, Liu Ni et al. (Acta Microbiologica Sinica, 2024, 64(08): 2967-2985) found that the cell-free supernatant (CFS) produced by Bacillus subtilis subsp. siamensis Bspi2104 maintained good antibacterial activity at 60°C, but completely lost its antibacterial activity after being treated at 80°C for 30 min. Another example is that M.F. FANGIO et al. (Italian journal of food science, 2013, 25(2): 181) studied the storage of the crude antibacterial extract of Bacillus subtilis R1 and found that its activity remained above 80% after being stored at freezing and refrigeration temperatures for 30 days. However, the cell-free supernatant of B. Subtilis QXKL-3 obtained in the present invention still maintained good antibacterial activity after being treated under high pressure at 121°C for 30 min and still maintained stable antibacterial activity after being stored at -20°C for 120 days. This indicates that the cell-free supernatant produced by B. Subtilis QXKL-3 obtained in the present invention has good stability and significant advantages in production, transportation, and long-term storage, etc.

[0018] Advantages of the present invention:

[0019] The cell-free supernatant of Bacillus subtilis QXKL-3 provided by the present invention has high antibacterial activity against Streptococcus suis type 2, and the antibacterial effect is significantly higher than that of penicillin. At the same time, even at 100°C, it can still maintain high antibacterial activity against Streptococcus suis type 2. Moreover, the antibacterial activity remains stable during long-term preservation. Description of the drawings

[0020] Figure 1 It is the stability experiment result of QXKL-3 CFS of the present invention at different temperatures and storage times;

[0021] Figure 2 It is the antibacterial curve experiment result of B. Subtilis QXKL-3 strain of the present invention;

[0022] Figure 3This is the KEGG pathway statistical chart of the present invention; wherein, the vertical axis is the tertiary classification of KEGG metabolic pathways, and the horizontal axis is the number of metabolites annotated to this pathway; alpha-Linolenic acid metabolism: α-linolenic acid metabolism; Toluene degradation: Toluene degradation; Polycyclic aromatic hydrocarbon degradation: Polycyclic aromatic hydrocarbon degradation; Phenylalanine, tyrosine and tryptophan biosynthesis: Phenylalanine, tyrosine and tryptophan biosynthesis; Nonribosomal peptide structures: Nonribosomal peptide structures; Microbial metabolism in diverse environments: Microbial metabolism in different environments; Degradation of aromatic compounds: Degradation of aromatic compounds; Biosynthesis of siderophore group nonribosomal peptides: Biosynthesis of siderophore group nonribosomal peptides; Benzoate degradation: Benzoate degradation; Aminobenzoate degradation: Aminobenzoate degradation; Metabolic pathways: Metabolic pathways; Biosynthesis of secondary metabolites: Biosynthesis of secondary metabolites. Detailed implementation manners

[0023] The present invention will be specifically described below through examples. It is necessary to point out here that the following examples are only used to further illustrate the present invention and cannot be understood as limiting the protection scope of the present invention. Some non-essential improvements and adjustments made by those skilled in the art according to the above invention content still fall within the protection scope of the present invention.

[0024] Example 1

[0025] 1 Materials

[0026] 1.1 Strains

[0027] Bacillus subtilis QXKL-3, isolated from the feces of Kele pigs, a local breed in Guizhou Province, is preserved in the China General Microbiological Culture Collection Center, address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the preservation time is September 11, 2023; the preservation number is CGMCC NO. 28419; Streptococcus suis type 2: SS2 GYSS74 strain (Modern Animal Husbandry Science and Technology, 2023(08): 1-6.), isolated from the outer three-way crossbred commercial pigs suffering from Streptococcus suis disease.

[0028] 1.2 Main reagents

[0029] Tryptic soy agar (TSA) medium and tryptic soy broth (TSB) medium are both purchased from Qingdao Haibo Biotechnology Co., Ltd.; Brain heart infusion (BHI) broth medium is purchased from Beijing Solarbio Science & Technology Co., Ltd.; Newborn bovine serum is purchased from Zhejiang Tianhang Biotechnology Co., Ltd.; Penicillin standard (batch number 130437-201707) is purchased from the National Institutes for Food and Drug Control.

[0030] 1.3 Main instruments

[0031] Electronic balance (model BSA223S) is purchased from Sartorius Scientific Instruments (Beijing) Co., Ltd.; Vertical autoclave (model YSQ-LS-70A) is purchased from Shanghai Boxun Industry Co., Ltd.; Constant temperature shaking incubator (model THZ-98A) is purchased from Shanghai Yiheng Scientific Instruments Co., Ltd.; Laminar flow hood (model SW-CJ-2FD) is purchased from Suzhou Purification Equipment Co., Ltd.; Micro high-speed refrigerated centrifuge (model SORVAL Legend Micro 21R) is purchased from Thermo Fisher Scientific; Vacuum freeze dryer (model FD-1B-80+) is purchased from Boyikang (Beijing) Instrument Co., Ltd.

[0032] 2 Methods

[0033] 2.1 Preparation of B. Subtilis QXKL-3 CFS

[0034] Take the glycerol stock solution of B. Subtilis QXKL-3 strain, after activation on TSA plate, transfer it into 20 mL of TSB medium, and incubate it at 37 °C and 150 r / min for 24 h to prepare the seed solution. Then transfer it to 100 mL of TSB medium at an inoculation amount of 2%, and incubate it at 37 °C and 150 r / min for 24 h to obtain a bacterial suspension (the viable cell count is about 1×10 8 ~5×10 8cfu / mL). Centrifuge at 4°C and 6000 r / min for 15 min to remove the bacteria, collect the supernatant, filter and sterilize it through a 0.22-μm hydrophilic polyethersulfone membrane, and obtain the CFS of B. Subtilis QXKL-3 after sterility testing.

[0035] 2.2 Determination of the antibacterial activity of B. Subtilis QXKL-3 CFS against SS2

[0036] The plate colony counting method was used to determine the antibacterial activity of QXKL-3 CFS against SS2. The frozen SS2 was activated on a TSA plate and then transferred to BHI broth medium for culturing until the logarithmic growth phase. The bacterial solution concentration was adjusted to 1×10 8 ~5×10 8 cfu / mL (at the same order of magnitude as the BS bacterial solution concentration for preparing CSF). Take 200 μL of the bacterial solution into a 1.5-mL centrifuge tube, centrifuge at 3500 r / min for 5 min, discard the supernatant, add 200 μL of QXKL-3 CFS to the bacterial pellet, use 1 mg / mL penicillin solution and sterile normal saline as controls. At 10 min, 20 min, 30 min, 60 min, 90 min, 120 min, 12 h, and 24 h after culturing, take 100 μL of samples from each treatment group for plate colony counting. After culturing on the plate for 24 h, count the remaining viable bacteria. With the time point of QXKL-3 CFS action as the abscissa and the remaining viable bacteria count of SS2 (lg cfu / mL) as the ordinate, draw a dynamic antibacterial curve to compare and analyze the antibacterial activity of QXKL-3 CFS against SS2. Each treatment was set with 3 replicates.

[0037] 2.3 Determination of the antibacterial activity of the crude extract of B. Subtilis QXKL-3 CFS against SS2 2.3.1 Extraction of the crude extract of B. Subtilis QXKL-3 CFS

[0038] The ammonium sulfate fractional precipitation method (refer to International Journal of Food Microbiology, 2018, 266: 60 - 68) was used to extract the CFS. Slowly add powdered ammonium sulfate to 50 mL of QXKL-3 CFS to make the ammonium sulfate concentration in the solution 20%, 40%, 60%, 80%, and 100% respectively. After standing overnight at 4°C, centrifuge at 12000 r / min for 15 min. Dissolve the precipitate in 2 mL of sterile water and use SS2 as the indicator bacterium for antibacterial tests to determine the optimal precipitation concentration.

[0039] 2.3.2 Determination of the antibacterial ability of the crude extract against SS2

[0040] After freeze-drying the antibacterial crude extract obtained at the optimal ammonium sulfate precipitation concentration, it was prepared into solutions with mass concentrations of 18.750, 9.375, 4.688, 2.344, and 1.173 mg / mL using sterile water by the two-fold dilution method. Using SS2 as the indicator bacterium and 1 mg / mL penicillin as the control, the antibacterial test was carried out by the hole punching method to determine the antibacterial effect, determine the minimum inhibitory concentration of the crude extract, and compare the difference in antibacterial ability between the crude extract and penicillin.

[0041] 2.4 B.Subtilis QXKL-3CFS Temperature Tolerance Test

[0042] Take B.Subtilis QXKL-3CFS and place it in an EP tube, 1 mL per tube. Treat it at 37, 40, 50, 60, 70, 80, 90, and 100 °C for 30 min respectively, and treat it at 121 °C under high pressure for 30 min. Take 6600 μL of SS2 bacterial solution and evenly add it to 33 EP tubes. After centrifuging at 3500 r / min for 5 min, discard the supernatant. Add 200 μL of CFS cooled to room temperature after different heat treatments to the bacterial cell precipitate respectively. Use the CFS treated at 37 °C for 30 min and sterile normal saline as controls. Detect the effect of temperature on the antibacterial activity of QXKL-3CFS using the method in 2.2. Set 3 replicates for each treatment.

[0043] 2.5 B.Subtilis QXKL-3CFS Stability Detection under Different Storage Conditions

[0044] After the antibacterial ability of QXKL-3CFS was determined, it was stored at -20 °C, 4 °C, and room temperature (from March to July 2024, location: Qianxi City, Guizhou Province). Referring to the method in 2.2, samples were taken at 0, 30, 60, 90, and 120 d to detect its killing effect on SS2, evaluate the stability of QXKL-3CFS, and repeat each experiment 3 times.

[0045] 2.6 LC-MS Component Analysis

[0046] 2.6.1 B.Subtilis QXKL-3 Antibacterial Curve Determination

[0047] Transfer the B.Subtilis QXKL-3 seed liquid to 100 mL of TSB medium at an inoculation amount of 2%. Take samples every 2 h to prepare B.Subtilis QXKL-3CFS. At the same time, refer to the method in 2.2 to determine its antibacterial activity, obtain the antibacterial curve, and set 3 parallel replicates.

[0048] 2.6.2 Metabolomics Detection

[0049] According to the antibacterial curve of B. Subtilis QXKL-3, the CFS cultured for 18 h was taken as the metabolome sample of QXKL-3, and it was detected by liquid chromatography-tandem mass spectrometry (LC-MS / MS) technology. Subsequently, differential metabolite analysis was carried out to screen the active substances that might mediate the antibacterial activity of Bacillus subtilis QXKL-3. The same batch of TSB medium was treated in the same way as a control. The samples were entrusted to Shanghai Majorbio Bio-Pharm Technology Co., Ltd. for detection.

[0050] 2.7 Data processing and analysis

[0051] The experimental data were sorted out using Excel 2016 software. The data were expressed as "mean ± standard deviation", and graphs were made using GraphPad Prism 8 software. P>0.05 indicates no significant difference, and P<0.05 indicates a significant difference.

[0052] 3 Results and analysis

[0053] 3.1 Comparison of antibacterial abilities between B. Subtilis QXKL-3 CFS and penicillin

[0054] The killing effects of B. subtilis QXKL-3 CFS and penicillin solution on SS2 at different times were determined by the plate colony counting method. The results are shown in Table 1. It can be seen from Table 1 that after treatment in a 37 °C constant temperature incubator for 10 min, there was no viable bacteria growth in the SS2 treated with B. subtilis QXKL-3 CFS; after treatment with penicillin, the number of viable SS2 bacteria did not change significantly within 2 h, and there were still viable bacteria after 24 h (the number of viable bacteria was about 4.80×10 3 cfu / mL). It can be seen that QXKL-3 CFS can quickly inhibit the growth of SS2, and its inhibitory effect on SS2 within 24 h is extremely significantly stronger than that of 1 mg / mL penicillin (P<0.001).

[0055] Table 1 Inhibitory effects of QXKL-3 CFS and penicillin on SS2

[0056]

[0057] Note: Completely different lowercase letters in the data superscript indicate significant differences (P<0.05), and the same lowercase letters indicate no significant differences (P>0.05).

[0058] 3.2 Antibacterial activity of the crude extract of B. Subtilis QXKL-3 CFS

[0059] 3.2.1 Crude extraction of antibacterial substances in B. Subtilis QXKL-3 CFS

[0060] B. Subtilis QXKL-3CFS was extracted with ammonium sulfate at different concentrations, and the antibacterial substance activity was determined. As can be seen from Table 2, when the ammonium sulfate concentration was 20%, the diameter of the antibacterial circle of the extract against SS2 was small. As the ammonium sulfate concentration increased, the diameter of the antibacterial circle of the extract gradually increased. When the ammonium sulfate concentration was 60%, the diameter of the antibacterial circle was the largest, and then gradually decreased. Compared with the antibacterial activity of the QXKL-3CFS without ammonium sulfate treatment, the diameter of the antibacterial circle of the extract with 60% ammonium sulfate concentration increased extremely significantly (P<0.001), indicating that the optimal ammonium sulfate concentration for extracting antibacterial active substances from QXKL-3CFS was 60%.

[0061] Table 2 Effects of ammonium sulfate concentration on antibacterial substance activity

[0062]

[0063] Note: NC is QXKL-3CFS without ammonium sulfate treatment; different lowercase letters in the data superscript indicate significant differences (P<0.05), and the same lowercase letters indicate no significant differences (P>0.05).

[0064] 3.2.2 Determination of the minimum inhibitory concentration of the crude extract

[0065] The antibacterial crude extract was obtained by precipitation with 60% ammonium sulfate and its MIC was determined. Using 1 mg / mL penicillin solution as a control, the results are shown in Table 3. As can be seen from Table 3, the diameter of the antibacterial circle of the crude extract decreased with the decrease of the mass concentration. The diameter of the antibacterial circle of the crude extract with a mass concentration of 18.750 mg / mL was the largest, which was (24.583±0.086) mm. When the mass concentration was 1.172 mg / mL, almost no antibacterial circle was produced, indicating that its MIC value was 2.344 mg / mL. The diameter of the antibacterial circle of 1 mg / mL penicillin solution against SS2 was (35.502±0.221) mm, which was significantly higher than the inhibition diameter of the CFS crude extract against SS2 (P<0.05).

[0066] Table 3 Minimum inhibitory concentration (MIC) of the crude extract

[0067]

[0068]

[0069] Note: Different lowercase letters in the same column of data superscript indicate significant differences (P<0.05).

[0070] 3.3 Effect of temperature on the antibacterial activity of B. Subtilis QXKL-3CFS

[0071] The inhibitory effect of B. Subtilis QXKL-3 CFS on SS2 after different temperature treatments is shown in Table 4. Compared with the 37 °C treatment group, after being treated at 40-100 °C for 30 min, QXKL-3 CFS still completely inhibited the growth of SS2, and its antibacterial activity remained basically stable; after being treated under high pressure at 121 °C for 30 min, the number of remaining viable SS2 bacteria increased, but was significantly lower than that of the normal saline control group. This indicates that the antibacterial active substances in QXKL-3 CFS have temperature tolerance and still have good stability during heat treatment.

[0072] Table 4 Effects of temperature on the antibacterial activity of QXKL-3 CFS

[0073]

[0074] Note: CK is the normal saline control; different lowercase letters in the data superscripts indicate significant differences (P < 0.05), and the same lowercase letters indicate no significant differences (P > 0.05).

[0075] 3.4 Stability of B. Subtilis QXKL-3 CFS under different storage conditions

[0076] The changes in the antibacterial activity of B. Subtilis QXKL-3 CFS stored at -20 °C, 4 °C and room temperature are shown in Figure 1 . As Figure 1 can be seen, compared with the activity of the initial sample, the activity of the samples stored under each condition was stable at 30 d; after 30 d, the activity of the samples stored at room temperature gradually decreased; after 60 d, the activity of the samples stored at 4 °C began to decrease; after 90 d, the activity of the samples stored at room temperature decreased significantly (P < 0.05); after 120 d, the activity of the samples stored at 4 °C decreased significantly (P < 0.05); at the same time, when the samples were stored at -20 °C for 120 d, their antibacterial activity did not change significantly and was basically the same as that of the initial sample.

[0077] 3.5 Metabolomics analysis

[0078] 3.5.1 Antibacterial curve of B. Subtilis QXKL-3

[0079] Using the culture time of B. Subtilis QXKL-3 as the abscissa and the number of remaining viable SS2 bacteria as the ordinate, the antibacterial curve of the strain was plotted. As Figure 2 can be seen, QXKL-3 CFS showed an inhibitory effect on SS2 after 2 h of culture, and the antibacterial substances in QXKL-3 CFS could completely inhibit SS2 after 18 h of culture.

[0080] 3.5.2 Analysis of antibacterial components in B. Subtilis QXKL-3 CFS

[0081] According to the results in 3.5.1, the QXKL-3 CFS after 18 h of cultivation was taken for metabolomics detection and analysis. As shown in Table 5, B. Subtilis QXKL-3 contains various organic acids and their derivatives, peptide compounds, and lipids and lipid-like molecules, etc., which are substances related to antibacterial activity, including surfactin, surfactin A, surfactin B, bacillaene, protocatechuic acid, amikacin, spergualin and other substances. KEGG pathway analysis was performed on the above metabolites, and the results are as Figure 3 shown. The metabolites are involved in 12 metabolic pathways. Among them, the polyphenolic compound protocatechuic acid is involved in multiple metabolic pathways (10 / 12), including alpha-linolenic acid metabolism, biosynthesis of siderophore group non-ribosomal peptides, biosynthesis of secondary metabolites, and biosynthesis of phenylalanine, tyrosine and tryptophan and other metabolic pathways, indicating that the production of antibacterial active substances by B. Subtilis QXKL-3 strain may be closely related to the metabolism of various amino acids, biosynthesis of non-ribosomal peptides, and biosynthesis of secondary metabolites.

[0082] Table 5 Substances related to antibacterial activity in QXKL-3 CFS

[0083]

Claims

1. A Bacillus subtilis QXKL-3, characterized in that, It is preserved in the China General Microbiological Culture Collection Center. The preservation date is September 11, 2023, and the preservation number is CGMCC NO. 28419.

2. Application of the Bacillus subtilis QXKL-3 described in claim 1 in antibacterial activity against Streptococcus suis type 2.

3. The application according to claim 2, wherein The said application is to inhibit Streptococcus suis type 2 when pigs are infected with Streptococcus suis type 2.

4. A preparation for inhibiting Streptococcus suis type 2, characterized in that, The active ingredient of the said preparation is the cells of Bacillus subtilis QXKL-3, the cell-free supernatant of Bacillus subtilis QXKL-3, or the extract of the cell-free supernatant of Bacillus subtilis QXKL-3.

5. The preparation according to claim 4, characterized in that, The cell-free supernatant of Bacillus subtilis QXKL-3 is obtained by culturing Bacillus subtilis QXKL-3 and then removing the cells.

6. The preparation according to claim 4, wherein The extract of the cell-free supernatant of Bacillus subtilis QXKL-3 is obtained by extracting the cell-free supernatant of Bacillus subtilis QXKL-3 using the ammonium sulfate fractional precipitation method.

7. The preparation according to claim 6, characterized in that, When preparing the extract of the cell-free supernatant of Bacillus subtilis QXKL-3, the concentration of ammonium sulfate used is 60%.

8. The preparation according to claim 7, characterized in that, When preparing the extract of the cell-free supernatant of Bacillus subtilis QXKL-3, after adding ammonium sulfate, it is left overnight at 4°C and then centrifuged at 12,000 r / min for 15 min, and the precipitate is taken.

9. Application of the preparation according to any one of claims 4 to 8 in antibacterial activity against Streptococcus suis type 2.

10. The application according to claim 9, wherein The said application is to inhibit Streptococcus suis type 2 when pigs are infected with Streptococcus suis type 2.

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