Bacillus thuringiensis for producing flavonoid components and application of bacillus thuringiensis

Abstract

The invention discloses bacillus thuringiensis for producing flavonoid components and application thereof, relates to the field of microorganisms, and aims to provide bacillus thuringiensis which is separated from industrial hemp leaves and is preserved in the China Center for Type Culture Collection, the preservation address is Wuhan University, Wuhan, on June 17, 2024, and the preservation number is CGMCC NO. The preservation number of the strain is CCTCC NO: M 20241242. The strain is not only a biological insect-resist agent, but also contains flavonoid compounds, namely rutin and orientin, in metabolites. The strain can be used for producing flavonoid compounds through fermentation.

Description

Technical Field

[0001] The invention relates to the field of microorganisms, in particular to a bacillus thuringiensis strain producing flavonoid components and application thereof. Background Art

[0002] Bacillus thuringiensis is a Gram-positive bacterium found in nature. + ), which can be isolated from animals and soil. There are relatively few studies on the use of plant leaves as materials for the isolation of Bacillus thuringiensis. Rizali et al. isolated 2 strains of Bacillus thuringiensis from mulberry leaves; Mizuki et al. isolated 120 strains of Bacillus thuringiensis from 35 woody and herbaceous plants.

[0003] Bacillus thuringiensis has now become one of the main biological insecticides to replace chemical agriculture and is widely used in biological control and other aspects. As a biological insecticide, the production process of Bacillus thuringiensis mainly includes liquid fermentation process and solid fermentation process. The purpose of fermentation production is to efficiently extract the insecticidal protein it contains, while other secondary metabolites it contains have not been reported. Summary of the invention

[0004] The purpose of the present invention is to provide a strain of Bacillus thuringiensis isolated from industrial hemp leaves. The strain is not only a biological insecticide, but also contains flavonoid compounds in its secondary metabolites, thus laying a foundation for the comprehensive development and utilization of the strain.

[0005] The present invention provides a Bacillus thuringiensis strain producing flavonoid components, which is Bacillus thuringiensis HDBt09, which has been deposited in the China Center for Type Culture Collection, the deposit address is Wuhan University, Wuhan, the deposit date is June 17, 2024, and the deposit number is CCTCC NO: M 20241242.

[0006] The colony of the Bacillus thuringiensis HDBt09 of the present invention is round, light yellow, has irregular edges, a raised and opaque surface, and is wax-like. The bacterial body is rod-shaped, and the spores are oval.

[0007] The Bacillus thuringiensis HDBt09 of the present invention is a Gram-positive bacterium, an aerobic bacterium, has flagella, is positive in a starch hydrolysis test, a gelatin liquefaction test, a catalase test, a VP test, a methyl red test, an indole test, a nitrate reduction test, a lipid hydrolysis test, a motility test, a biofilm formation test, has an optimum growth temperature of 37°C, an optimum pH of 7.5, and a logarithmic growth phase of 12 to 28 hours.

[0008] The 16S rDNA sequencing results of the Bacillus thuringiensis HDBt09 of the present invention were submitted to the NCBI database, analyzed and compared using BLAST, and then a phylogenetic tree of Bacillus thuringiensis HDBt09 was constructed using MEGA 7 software. The strain HDBt09 and Bacillus thuringiensis (OP986993) were clustered on the same branch, and the sequence similarity between the two reached 100%. Based on the morphological observation results and physiological and biochemical results, Bacillus thuringiensis HDBt09 was identified as Bacillus thuringiensis.

[0009] The present invention also provides application of Bacillus thuringiensis HDBt09 in preparing drugs for inhibiting bacteria.

[0010] Furthermore, the bacteria are Escherichia coli, Bacillus subtilis, Staphylococcus aureus, Bacillus pumilus, Pseudomonas aeruginosa, Listeria monocytogenes, Klebsiella pneumoniae, Acinetobacter baumannii, Enterococcus faecium and Enterococcus faecalis.

[0011] The present invention also provides the use of Bacillus thuringiensis HDBt09 in preparing a drug for inhibiting fungi, wherein the fungus is Candida albicans.

[0012] The present invention also provides the use of Bacillus thuringiensis HDBt09 in in vitro anti-oxidation.

[0013] The Bacillus thuringiensis HDBt09 of the present invention is used for fermentation to produce flavonoid compounds.

[0014] Furthermore, the flavonoid compounds are rutin and orientin.

[0015] Beneficial effects of the present invention:

[0016] The present invention isolates an endophytic bacterium HDBt09 from industrial hemp leaves, which is identified as Bacillus thuringiensis. Bacillus thuringiensis HDBt09 has antibacterial activity and in vitro antioxidant activity, and has a good inhibitory effect on Escherichia coli and Bacillus subtilis.

[0017] In addition, the secondary metabolites of Bacillus thuringiensis HDBt09 contain two flavonoids: rutin and orientin, which can be used to produce flavonoids by fermentation. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is the colony morphology of Bacillus thuringiensis HDBt09;

[0019] Figure 2This is the microscopic morphology of Bacillus thuringiensis HDBt09;

[0020] Figure 3 is the phylogenetic tree of Bacillus thuringiensis HDBt09;

[0021] Figure 4 The results of antioxidant activity analysis of Bacillus thuringiensis HDBt09 fermentation broth;

[0022] Figure 5 D 101 HPLC analysis results of the 30% ethanol elution portion of the macroporous adsorption resin column;

[0023] Figure 6 is the HPLC analysis result of compound A;

[0024] Figure 7 For compound A 1 H NMR spectroscopy;

[0025] Figure 8 is the HPLC analysis result of compound B;

[0026] Fig. 9 For compound B 1 H NMR spectrum. DETAILED DESCRIPTION

[0027] The embodiments of the present invention are described in detail below. The following embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation schemes and specific operation processes are given, but the protection scope of the present invention is not limited to the following embodiments.

[0028] Embodiment 1:

[0029] The Bacillus thuringiensis in this example is Bacillus thuringiensis HDBt09, which has been deposited in the China Center for Type Culture Collection, the deposit address is Wuhan University, Wuhan, the deposit date is June 17, 2024, and the deposit number is CCTCC NO: M 20241242.

[0030] The isolation method of Bacillus thuringiensis HDBt09 in this embodiment:

[0031] Take industrial hemp (Cannabis sativa L.) leaves (Longma No. 5, collected from the Science and Technology Innovation Farm of Suihua Branch of Heilongjiang Academy of Agricultural Sciences) and disinfect the surface according to the following procedures: 75% alcohol 30s-sterile water washing-NaClO 5min-sterile water washing-75% alcohol 30s-sterile water washing. Then grind the experimental materials into a homogenate, dilute with distilled water, spread and inoculate in NA medium, and culture at 37°C for 5d. The last distilled water wash in the disinfection process was removed and spread and inoculated on NA medium as a control. Finally, the industrial hemp endophyte, namely the strain HDBt09, was isolated from the industrial hemp leaves. No bacteria grew in the negative control plate culture medium, and this was repeated many times, thus proving that the bacteria isolated were industrial hemp endophytes.

[0032] Example 2: Identification of strain HDBt09

[0033] The colony morphology of strain HDBt09 is as follows Figure 1 As shown, the microscopic morphology of strain HDBt09 is as follows Figure 2 As shown, the colonies are round, light yellow, with irregular edges, a raised and opaque surface, and a waxy appearance. The bacteria are rod-shaped and the spores are oval.

[0034] The results of physiological and biochemical tests of strain HDBt09 are shown in Table 1.

[0035] Table 1 Physiological and biochemical test results of strain HDBt09

[0036]

[0037] The 16S rDNA fragment of strain HDBt09 was amplified by PCR, and the product was recovered and purified to obtain a specific band with a length of 1455 bp. The band was sequenced, and the 16S rDNA sequence is shown in SEQ ID NO: 1 in the sequence table.

[0038] The sequencing results were submitted to the NCBI database, analyzed and aligned using BLAST, and then the phylogenetic tree of the endophytic bacterium HDBt09 was constructed using MEGA 7 software, as shown in Figure 3 As shown in the figure, the strain HDBt09 and Bacillus thuringiensis (OP986993) were clustered on the same branch, and the sequence similarity between the two reached 100%. Based on the morphological observation results and physiological and biochemical results, Bacillus thuringiensis HDBt09 was identified as Bacillus thuringiensis.

[0039] Example 3: Analysis of antibacterial activity of Bacillus thuringiensis HDBt09:

[0040] 1. Activation of Bacillus thuringiensis HDBt09

[0041] Take Bacillus thuringiensis HDBt09, inoculate it onto the test surface containing NA culture medium under sterile conditions, and culture it at a constant temperature of 37°C for 3 days for later use.

[0042] 2. Preparation of Bacillus thuringiensis HDBt09 fermentation broth:

[0043] Take the activated Bacillus thuringiensis HDBt09, pick the mycelium under sterile conditions and inoculate it into 60 mL NB medium, and culture it in a shaking incubator at 37°C and 120 r / min for 3 days to make 1×10 7 CFU / mL of seed liquid; the seed liquid was transferred to 3L sterilized NB medium at a 5% (v / v) inoculation amount, cultured at 37°C, 120r / min shaking for 7 days, and the fermentation liquid was filtered.

[0044] 3. Preparation of test solution:

[0045] Take the fermentation liquid of Bacillus thuringiensis HDBt09 and concentrate it to 150mL under reduced pressure at 50℃, add 150mL of methanol, and ultrasonicate for 60min to obtain the extract, which is concentrated to dryness under reduced pressure, and dissolved with 10mL of methanol, and used as the test solution for later use. Take appropriate amounts of streptomycin and nystatin powder, add appropriate amounts of methanol to dissolve them, and prepare 200μg / mL positive control solution for later use.

[0046] 4. Preparation of plates containing test strains:

[0047] Take the activated test bacteria: Staphlococcus aureus, Bacillus pumilus, Bacillus subtilis, Listeria monocytogenes, Enterococcus facalis, Enterococcus faecium, Escherichia coli, Klebsiella pneumoniae, Acinetobacter baummanii, Pseudomonas aeruginosa, inoculate in NA test tube slant, culture at 37℃ for 1 day; take the activated test fungus Candida albicans, inoculate in PDA test tube slant, culture at 28℃ for 3 days, and set aside. The above test strains were purchased from Heilongjiang Institute of Microbiology.

[0048] Take the activated test strains mentioned above, add appropriate amount of sterile water, shake and dilute each strain to 1×10 7 Pour the bacterial suspension of 0.1 CFU / mL into the corresponding culture medium and dispense it into sterile plates with Oxford cups. After it solidifies, take out the Oxford cups and set aside.

[0049] 5. Antibacterial activity analysis:

[0050] Accurately add 150 μL of the test solution, 150 μL of the positive control solution, and 150 μL of the negative control solution to the two wells of the prepared bacteria-containing plate. The plate containing bacteria was incubated at 37°C for 1 day, and then the diameter of the inhibition zone was measured (n=3); the plate containing fungi was incubated at 28°C for 3 days, and then the diameter of the inhibition zone was measured (n=3).

[0051] The antibacterial activity results of Bacillus thuringiensis HDBt09 are shown in Table 2.

[0052] Table 2 Antibacterial activity results of fermentation broth of Bacillus thuringiensis HDBt09

[0053]

[0054] Note: A: Escherichia coli; B: Bacillus subtilis; C: Staphylococcus aureus; D: Bacillus pumilus; E: Pseudomonas aeruginosa; F: Listeria monocytogenes; G: Klebsiella pneumoniae; H: Acinetobacter baumannii; I: Enterococcus faecium; J: Enterococcus faecalis; K: Candida albicans; Str: Streptomycin; Nys: Nystatin.

[0055] From the results in Table 2, it can be seen that Bacillus thuringiensis HDBt09 has an inhibitory effect on 11 pathogenic bacteria, especially on Escherichia coli and Bacillus subtilis, with the diameters of the inhibition zones being 16.72±0.16 mm and 14.86±0.21 mm, respectively.

[0056] Example 4: Analysis of Antioxidant Activity of Bacillus thuringiensis HDBt09

[0057] 1. Activation of Bacillus thuringiensis HDBt09

[0058] Take Bacillus thuringiensis HDBt09, inoculate it onto the test surface containing NA culture medium under sterile conditions, and culture it at a constant temperature of 37°C for 3 days for later use.

[0059] 2. Preparation of Bacillus thuringiensis HDBt09 fermentation broth:

[0060] Take the activated Bacillus thuringiensis HDBt09, pick the mycelium under sterile conditions and inoculate it into 60 mL NB medium, and culture it in a shaking incubator at 37°C and 120 r / min for 3 days to make 1×10 7 CFU / mL of seed liquid; the seed liquid was transferred to 3L sterilized NB medium at a 5% (v / v) inoculation amount, cultured at 37°C, 120r / min shaking for 7 days, and the fermentation liquid was filtered.

[0061] 3. Weigh an appropriate amount of DPPH and dissolve it in an appropriate amount of methanol to prepare a DPPH solution with a concentration of 0.15mmol / L. Weigh 2mg of vitamin C and dissolve it in methanol to prepare a reference solution with a concentration of 0.2mg / mL for later use.

[0062] The above-mentioned reference and test solutions were diluted in sequence, and the concentrations of each solution were diluted to a gradient of 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.5 μg / mL and 6.25 μg / mL. 2 mL of each solution was drawn into a test tube, and methanol was used as a blank control group. 4 mL of 0.15 mmol / mL DPPH solution was added to each test tube, placed in the dark for 30 minutes, and the absorbance of each sample was measured at 517 nm (n=3). The DPPH free radical scavenging rate was calculated as follows:

[0063]

[0064] In the formula, A i Indicates the absorbance value when the test product and DPPH solution are added simultaneously; A j A0 represents the absorbance value when the test sample and blank control are added at the same time; A1 represents the absorbance value when the blank control and DPPH solution are added at the same time.

[0065] Taking DPPH free radical scavenging ability as an indicator, the results of the antioxidant capacity determination of Bacillus thuringiensis HDBt09 fermentation broth are shown in Figure 4 .Depend on Figure 4 It can be seen that the scavenging ability of the fermentation broth on DPPH free radicals is positively correlated with the test sample concentration. When the concentration is 100 μg / mL, the scavenging rate of the fermentation broth on DPPH free radicals reaches a maximum value of 60.28%. After calculation, the half scavenging rate (IC50 value) of the fermentation broth on DPPH free radicals is 47.250±0.14 μg / mL, indicating that HDBt09 fermentation broth has good antioxidant activity.

[0066] Example 5: Separation of secondary metabolites from Bacillus thuringiensis HDBt09 fermentation broth

[0067] Take 10L of Bacillus thuringiensis HDBt09 fermentation liquid, slowly pass it through D101 macroporous resin column, use 30%, 50%, 70%, 90% ethanol gradient elution, collect each elution part, recover the solvent, take 30% ethanol elution part, add methanol to dissolve, and use HPLC analysis, conditions: Global Chromatography-C 18 (4.6 mm × 250 mm, 5 μm). Mobile phase: methanol: 0.1% glacial acetic acid (50: 50, v / v), detection wavelength: 254 nm, flow rate: 1.0 mL·min -1 , injection volume: 20μL / time.

[0068] Results A chromatographic peak with the same retention time as the rutin reference substance was detected in the 30% ethanol elution fraction, and there was no interference with the negative control, indicating that rutin may exist in the 30% ethanol elution fraction. Figure 5 , Figure 5 A is the 30% ethanol elution portion, B is the rutin reference substance, C is the methanol extract of industrial hemp leaves, and D is the blank control (NB culture medium).

[0069] According to the HPLC analysis results, the 30% ethanol elution portion was separated by silica gel column chromatography using petroleum ether: ethyl acetate = 7:1 as the eluent, and the eluate was collected (10 mL / bottle of collection volume), and then examined by thin layer chromatography, and the components from bottles 261 to 295 were combined and recrystallized to obtain compound A.

[0070] The 50% ethanol elution part of the D101 macroporous resin column was separated by polyamide column chromatography, and petroleum ether: ethyl acetate = 1:4 was used as the eluent for elution, and the eluate was collected (10 mL / bottle collection volume), and thin layer chromatography was used for inspection, and the components of the 34th to 61st bottles were combined to obtain a crude product, and then silica gel column chromatography was used again for separation, and petroleum ether: ethyl acetate = 7:1 was used as the eluent for elution, and the eluate was collected (10 mL / bottle collection volume), and thin layer chromatography was used for inspection, and the components of the 34th to 61st bottles were combined, and recrystallized to obtain compound B. Using orientin reference substance as a control, the thin layer behavior of compound B is basically similar to that of orientin.

[0071] Compound A (6.213 mg), light yellow powder, easily soluble in hot water, alkaline water, methanol, pyridine, dimethyl sulfoxide, etc. HPLC analysis of compound A was performed with rutin as a control. The results are shown in Figure 6 , Figure 6 Where A is compound A, B is compound A + rutin reference substance, and C is rutin reference substance. Figure 6 It can be seen that compound A and the reference substance rutin have the same chromatographic peak at the same retention time, so it is preliminarily speculated that compound A may be rutin.

[0072] use 1 H NMR spectroscopy ( 1 H-NMR) to identify the chemical structure of compound A. The results are shown in Figure 7 .Depend on Figure 7 It can be seen that compound A 1 H-NMR: 1 H NMR (400MHz, DMSO) δ: 12.57 (s, 1H), 7.49 (s, 2H), 6.80 (d, J = 8.2Hz, 1H), 6.35 (d, J = 2.0Hz, 1H), 6.16 (d, J = 2.1Hz, 1H), 5 .34 (d, J=7.2Hz, 1H), 5.07 (d, J=15.9Hz, 2H), 4.34 (s, 2H), 3.67 (d, J=10.6Hz, 1H), 3.02 (s, 3H), 0.95 (d, J=6.2Hz, 3H).

[0073] The above NMR data are basically consistent with the relevant data of rutin reported in the literature, and through co-thin layer analysis with three solvent systems of rutin reference substance, they have the same Rf value, so compound A is identified as rutin, and its chemical formula is C 27 H 30 O 16 .

[0074] Compound B (5.766 mg), yellow powder, was dissolved in methanol, ethanol, ethyl acetate, etc., and the isolated compound B was analyzed by HPLC with orientin reference substance as reference. The results are shown in Figure 8 , Figure 8 A is compound B, B is orientin reference substance, C is compound B + orientin reference substance, and D is NB culture medium. Figure 8 It can be seen that compound B has the same chromatographic peak at the same retention time as the orientin reference substance, and the blank control (NB medium) has no interference, indicating that compound B is a secondary metabolite of Bacillus thuringiensis HDBt09.

[0075] Depend on Figure 8 It can be seen that the purity of compound B can reach 96.13% after HPLC analysis. In order to further confirm its structure, 1 H NMR spectroscopy ( 1 H-NMR) was used for identification, and the results are shown in Fig. 9 .

[0076] Depend on Fig. 9 It can be seen that compound B 1 H-NMR: 1H NMR (400MHz, DMSO) δ: 13.15 (1H, s, 5-OH), 7.45 (1H, s), 6.83 (1H, d, J = 8.3Hz), 6.62 (1H, s, H-6), 6.24 (1H, s, H-3), 4.64 (1H, d, J = 9.9Hz, H-1″).

[0077] Comprehensive HPLC of compound B, 1 The results of H-NMR identification, comparing and analyzing the data of compound B and orientin monomer compounds in the literature, show that compound B is consistent with orientin in all data. And through co-thin layer with orientin reference substance in three solvent systems, it has the same Rf value. Therefore, compound B is identified as orientin, and its chemical formula is C 21 H 20 O 11 .

Claims

1. A Bacillus thuringiensis strain producing flavonoids, characterized in that: It is Bacillus thuringiensis HDBt09, which has been deposited in the China Center for Type Culture Collection, with the deposit address at Wuhan University, Wuhan. The deposit date is June 17, 2024, and the deposit number is CCTCC NO: M 20241242.

2. Use of the Bacillus thuringiensis as claimed in claim 1 in the preparation of a drug for inhibiting bacteria.

3. The use according to claim 2, characterized in that: The bacteria are Escherichia coli, Bacillus subtilis, Staphylococcus aureus, Bacillus pumilus, Pseudomonas aeruginosa, Listeria monocytogenes, Klebsiella pneumoniae, Acinetobacter baumannii, Enterococcus faecium and Enterococcus faecalis.

4. Use of the Bacillus thuringiensis according to claim 1 in the preparation of a drug for inhibiting fungi, wherein the fungus is Candida albicans.

5. Use of the Bacillus thuringiensis as claimed in claim 1 in in vitro antioxidant activity.

6. Use of the Bacillus thuringiensis as claimed in claim 1 in fermentation to produce flavonoids.

7. The use according to claim 6, characterized in that: The flavonoid compounds are rutin and orientin.

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