Bacillus coagulans for producing phosphorus tributylate and application thereof

Abstract

The present application belongs to the field of biology, and particularly relates to a Bacillus coagulans strain for producing tributyl phosphate and application thereof. The Bacillus coagulans zqw106 provided in the present application has the preservation number of CGMCC No. 25151, has the ability of fermenting to produce tributyl phosphate, and the content of tributyl phosphate in the fermentation liquor is higher than that of the existing reported microorganism. In an experiment, the content of tributyl phosphate produced by the strain is 3.449 mg / L. The strain can provide a strain resource for the research of microbial fermentation to produce tributyl phosphate. The strain has the characteristics of acid tolerance, heat tolerance, salt tolerance, easy cultivation and preservation, and the like.

Description

Technical Field

[0001] This invention belongs to the field of microbiology, specifically relating to a Bacillus coagulans strain that produces tributyl phosphate and its applications. Background Technology

[0002] Tributyl phosphate (TBP) is a high-quality flame retardant with advantages such as being non-toxic, smokeless, halogen-free, having good material compatibility, long-lasting flame retardant effect, water resistance, heat resistance, and migration resistance. It can also be used as a neutral extractant, offering advantages such as long service life, high selectivity, easy back-extraction, and low water solubility, and is widely used in the extraction and separation of cobalt, nickel, rare earth elements, and other metals, as well as esters. Furthermore, it can be used as a plasticizer for nitrocellulose, cellulose acetate, chlorinated rubber, and polyvinyl chloride, a solvent for coatings, adhesives, and inks, and a stationary phase in gas chromatography. In research, TBP has also been shown to be used in pesticides. CN113545357A discloses a highly effective disinfectant and insecticide for ultra-low temperature storage, prepared by dissolving trichloroisocyanuric acid effervescent tablets in 30% ethanol and then adding a mixture containing tributyl phosphate, which showed good killing effect on Bacillus subtilis var. niger spores.

[0003] Industrially, tributyl phosphate is typically synthesized by direct esterification of butanol and phosphorus oxychloride. This reaction generates hydrogen chloride, which further reacts with butanol and the phosphate ester to produce chlorobutane. To minimize side reactions, very low reaction temperatures and high vacuum levels are required, leading to high butanol consumption, demanding equipment, and high energy consumption. If a microbial method could be used for preparation, the high raw material consumption and environmental pollution problems could be reduced.

[0004] Hua Yuefei (Hua Yuefei. Study on the antibacterial activity of endophytic Bacillus licheniformis SYt1 in Dioscorea shield-leaf [D]. Northwest A&F University, 2014.) first discovered tributyl phosphate in the metabolites of microorganisms, and the extract concentrate containing tributyl phosphate showed strong inhibitory effects on a variety of pathogens. However, the types of microorganisms that produce tributyl phosphate are limited, and the yield is low, necessitating the screening of microorganisms that produce high levels of tributyl phosphate. Summary of the Invention

[0005] This invention provides a Bacillus coagulans strain that produces tributyl phosphate, with accession number CGMCC No. 25151. This strain was screened from the mud of Luzhou Laojiao cellar.

[0006] Therefore, the present invention provides Bacillus coagulans, which was deposited on June 21, 2022 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. CGMCC NO.25151 and classification name Bacillus coagulans.

[0007] Therefore, the present invention provides the use of the aforementioned Bacillus coagulans in the production of tributyl phosphate.

[0008] Furthermore, a method for producing tributyl phosphate using the aforementioned Bacillus coagulans is provided, which involves culturing the Bacillus coagulans in a fermentation medium to produce tributyl phosphate.

[0009] Specifically, the cultivation conditions are static incubation at 35-39℃ for 24-64 hours, with a pH value of 6.0-8.0.

[0010] Preferably, the culture conditions are static culture at 37°C for 48 hours and pH value of 7.0.

[0011] In one specific embodiment, the fermentation medium is composed of 16g ​​glucose, 10g tryptone, 2.5g yeast powder, 5g beef extract, 3.85g bovine brain, 4.9g bovine heart, 0.5g Tween 80, 1g ammonium citrate, 2.5g sodium chloride, 2.5g anhydrous sodium acetate, 0.05g magnesium sulfate, 0.025g manganese sulfate, 1.25g disodium hydrogen phosphate, and 1g dipotassium hydrogen phosphate per liter.

[0012] The present invention also provides the application of the aforementioned Bacillus coagulans in the preparation of antimicrobial products.

[0013] Preferably, the antibacterial properties refer to resistance to black spot pathogens of Chinese cabbage, dry rot pathogens of potatoes, and curvature spore pathogens of corn. Further preferably, the product is, for example, a pesticide.

[0014] The *Bacillus coagulans* zqw106 provided by this invention has the ability to ferment and produce tributyl phosphate, and the tributyl phosphate content in its fermentation broth is higher than that of previously reported microorganisms (in one experiment, the tributyl phosphate content produced by this strain was 3.449 mg / L), which can provide strain resources for the study of microbial fermentation to produce tributyl phosphate. Furthermore, the strain exhibits characteristics such as acid resistance, heat resistance, salt resistance, and ease of cultivation and preservation. Attached Figure Description

[0015] Figure 1 GC-MS image of the strain producing tributyl phosphate.

[0016] Figure 2 This is a GC-MS molecular fragment mass spectrum of tributyl phosphate produced by the strain.

[0017] Figure 3 This is a GC-MS molecular fragment mass spectrum of tributyl phosphate standard.

[0018] Strain preservation information:

[0019] The *Bacillus coagulans* strain of this invention was deposited on June 21, 2022, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China. The accession number for this strain is CGMCC No. 25151, and its taxonomic name is *Bacillus coagulans*. Detailed Implementation

[0020] The present invention will be further illustrated below with specific embodiments in order to better understand the present invention, but these embodiments do not constitute a limitation thereof.

[0021] The culture medium formulations involved in the examples are as follows:

[0022] MRS liquid culture medium: glucose 20 g / L, peptone 10 g / L, yeast extract 4 g / L, beef extract 5 g / L, Tween 80 1 g / L, triammonium citrate 2 g / L, sodium acetate 5 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L, dipotassium hydrogen phosphate 1 g / L, pH 6.0, autoclaved at 115°C for 20 minutes.

[0023] MRS solid medium: Add 15 g / L agar to the MRS liquid medium and autoclave at 115°C for 20 minutes.

[0024] Fermentation medium: glucose 16g / L, tryptone 10g / L, yeast extract 2.5g / L, beef extract 5g / L, bovine brain 3.85g / L, bovine heart 4.9g / L, Tween 80 0.5g / L, ammonium citrate 1g / L, sodium chloride 2.5g / L, anhydrous sodium acetate 2.5g / L, magnesium sulfate 0.05g / L, manganese sulfate 0.025g / L, disodium hydrogen phosphate 1.25g / L, dipotassium hydrogen phosphate 1g / L, pH 6.0, autoclaved at 115℃ for 20 minutes.

[0025] Example 1: Isolation and purification of strains

[0026] 1) Separation method: Take 20g of Luzhou Laojiao pit mud sample and place it in an Erlenmeyer flask containing 180mL of sterile distilled water. Shake on a constant temperature shaker for 10min to thoroughly disperse and mix the sample. Take 1mL of the sample suspension and dilute it to 10 using the serial dilution method. -2 ~10 -7 Take 100 μL of each dilution and spread it evenly on an MRS solid medium plate. Prepare two parallel plates, invert them, and incubate them anaerobically at 37°C for 36-48 h, observing them regularly.

[0027] 2) Streak purification: Remove the plate from which colonies have grown, pick single colonies with different colony morphologies, and streak them a second time until all single colonies are purified.

[0028] 3) Preservation of bacterial strains: After purification, single colonies of each strain were picked into 5 mL of LMR broth and cultured anaerobically at 37°C for 20-24 h. 1 mL of the bacterial solution was then transferred to a preservation tube, 0.5 mL of 60% sterile glycerol solution was added, the culture was resuspended, and stored at -80°C.

[0029] Example 2: Molecular identification of strain zqw106

[0030] After amplification of the target strain, fresh bacterial culture in the logarithmic growth phase was collected by centrifugation, and genomic DNA was extracted using a bacterial genome extraction kit. The full-length 16S rDNA sequence was amplified using the universal primers 27F / 1541R for Bacillus, as follows:

[0031] 27F(5′-AGAGTTTGATCCTGGCTCAG-3′)

[0032] 1541R(5′-AAGGAGGTGATCCAGCC-3′)

[0033] ①Reaction system (50 μl)

[0034]

[0035]

[0036] ②Reaction Procedure

[0037]

[0038] PCR products were separated and tested by 1.0% agarose gel electrophoresis at a voltage of approximately 11 V / cm for 20 min.

[0039] The PCR products were purified according to the instructions of the small-volume gel recovery PCR product purification kit from Shanghai Sangon Biotech Co., Ltd., and the sequencing was performed by Shanghai Sangon Biotech Co., Ltd.

[0040] The gene sequence of the 16S rDNA fragment obtained by sequencing was compared with NCBI's BLAST to determine the species information of the strain. It was identified as Bacillus coagulans and named Bacillus coagulans zqw106 (Bacillus coagulans).

[0041] Example 3: Detection of the ability of strain zqw106 to produce tributyl phosphate

[0042] (1) Prepare the bacterial suspension to be tested:

[0043] After the glycerol preservation tubes of the selected strains were dissolved, they were inoculated into MRS liquid medium and incubated at 37°C for 20 h to obtain the bacterial suspension to be tested.

[0044] (2) GC-MS method for detecting tributyl phosphate:

[0045] Headspace solid-phase microextraction (HSP) was employed: the supernatant was collected and added to a headspace vial, along with saturated NaCl solution. 2-Octanol (0.822 mg / ml) was used as an internal standard. The prepared sample was incubated at 60°C for 5 min to equilibrate, then extracted at 60°C for 50 min using a 50 / 30 μm DVB / CAR / PDMS extraction head. After extraction, desorption was performed at 250°C for 5 min at the GC inlet. Compound search results were matched with the NIST standard spectral library; compounds with a similarity of 80% or higher were confirmed as target compounds. A culture broth without added bacteria was used as a blank control group, and the content of each volatile substance was calculated.

[0046] GC-MS detection chromatographic conditions:

[0047] Gas chromatography conditions: HP-INNOWAX column (60m × 0.25mm × 0.25μm); Temperature program: initial temperature 40℃, hold for 5 min, increase to 100℃ at 4℃ / min, then increase to 230℃ at 6℃ / min.

[0048] Hold for 10 min with high-purity helium as the carrier gas (1.0 mL / min); injector temperature 250℃, no splitting.

[0049] Mass spectrometry conditions: electron ionization source, electron energy 70 eV; electron multiplier voltage 350 V; ion source temperature 230 °C; transfer line temperature 250 °C; mass range 40–450 m / z.

[0050] Using the above method, one of the isolated strains, strain zqw106, was found to have a high production capacity of tributyl phosphate. The GC-MS mass spectrum is shown below. Figure 1 As shown. This strain was selected for further research.

[0051] Example 4: Fermentation experiment of strain zqw106

[0052] Based on optimized fermentation conditions (inoculum size, culture temperature, culture time, etc.), the obtained Bacillus coagulans zqw106 glycerol preservation tube was dissolved and inoculated into 10 mL of MRS liquid medium at a 5% inoculum size. After static culture at 37°C for 24 h, it was inoculated into fermentation medium at a 2% inoculum size and cultured continuously for 2 days. The detection method was the same as that in Example 3. After continuous fermentation for 2 days, the yield of tributyl phosphate produced by Bacillus coagulans zqw106 was 3.449 mg / L.

[0053] Example 5: Morphological characteristics and physicochemical properties of Bacillus coagulans zqw106

[0054] 1. Experiments show that the culture characteristics of Bacillus coagulans zqw106 are as follows: the optimal growth temperature is 37℃, the pH value is 7, it is facultative anaerobic, and it can grow under aerobic, anaerobic, or microaerobic conditions. Regarding temperature tolerance: the bacterium grows well at 37-42℃, can grow at 45℃ but growth is somewhat poor, and cannot grow at 48℃.

[0055] 2. Under anaerobic conditions at 37℃ for 48 hours, this bacterium, as identified by the API 50CH kit, can produce acid from 27 single carbon sources, including L-arabinose, D-ribose, D-xylose, D-galactose, D-glucose, D-fructose, D-mannose, D-cellobiose, D-maltose, D-lactose, D-micobiose, D-sucrose, D-trehalose, D-melatoninose, D-raffinose, D-thulene; mannitol, sorbitol, N-acetylglucosamine, amygdalin, arbutin, salicin, D-gentiobiose; calendula alcohol / ribitol, potassium gluconate; methyl-α-D-mannopyranoside, and potassium 2-ketogluconate. Their utilization capacity, from strongest to weakest, is as follows: L-arabinose, D-ribose, D-xylose, D-galactose, D-glucose, D-fructose, D-mannose, D-cellobiose, D-maltose, D-lactose, D-micobiose, D-sucrose, D-trehalose, D-minotriose, D-raffinose, D-thulene; mannitol, sorbitol, N-acetylglucosamine, amygdalin, arbutin, salicin, D-gentiobiose; calendula alcohol / ribitol, potassium gluconate; methyl-α-D-mannopyranoside, potassium 2-ketogluconate. A relatively large number of single carbon sources are available, and their overall utilization capacity is quite strong.

[0056] 3. Under microaerobic conditions at 37℃, this bacterium was cultured statically in MRS liquid medium for 48 hours. The non-volatile metabolites were detected by HPLC. It was found that the bacterium could metabolize and produce lactic acid 18.00 g / L, acetic acid 0.17 g / L, ethanol 0.08 g / L, and residual sugar 0.50 g / L.

[0057] Example 6: Experiment on the production of other volatile metabolites by fermentation of Bacillus coagulans zqw106

[0058] Fermentation of Bacillus coagulans zqw106 according to the method in Example 4 can produce 3.449 mg / L of tributyl phosphate, as well as 39 other volatile metabolites, namely: n-heptadecane 0.317 mg / L, isoamyl alcohol 0.759 mg / L, n-heptanol 1.081 mg / L, n-octanol 0.168 mg / L, 1-nonanol 0.406 mg / L, dodecanol 0.515 mg / L, benzyl alcohol 0.277 mg / L, phenethyl alcohol 1.233 mg / L, geraniol 0. 270 mg / L, Acetic acid 6.864 mg / L, Butyric acid 0.376 mg / L, Isovaleric acid 0.346 mg / L, Hexanoic acid 1.531 mg / L, Isooctanoic acid 0.427 mg / L, Octanoic acid 2.085 mg / L, Nonanoic acid 1.656 mg / L, Decanoic acid 0.518 mg / L, Lauric acid 0.328 mg / L, 2-octanone 0.033 mg / L, Acetophenone 0.031 mg / L, Geraniolacetone 0.476 mg / L, 2,3-Dihydro-2,2,6-Trimethylbenzene Formaldehyde 1.214 mg / L, phenylacetaldehyde 1.100 mg / L, (4E,8E)-5,9,13-trimethyltetradec-4,8,12-trienal 0.482 mg / L, 2-octyl acetate 0.212 mg / L, 3-hydroxy-2,2,4-trimethylpentyl isobutyrate 0.243 mg / L, 2,2,4-trimethyl-1,3-pentanediol diisobutyrate 0.252 mg / L, triisobutyl phosphate 2.312 mg / L, ethyl palmitate 0.335 mg / L, linoleic acid... Ethyl myristate 0.036 mg / L, ethyl myristate 0.266 mg / L, ethyl transoleate 1.426 mg / L, phenol 0.023 mg / L, 2,4-di-tert-butylphenol 3.150 mg / L, 4-chloro-3,5-dimethylphenol 0.829 mg / L, 2,6-di-tert-butyl-4-methylphenol 0.590 mg / L, 2-methyl-6-vinylpyrazine 0.034 mg / L, 2,6-dimethylpyrazine 0.369 mg / L, benzothiazole 0.034 mg / L.

Claims

1. A strain of Bacillus coagulans that produces tributyl phosphate (Bacillus coagulans) Bacillus coagulans zqw106, its collection number is CGMCC No. 25151.

2. The application of Bacillus coagulans zqw106 as described in claim 1 in the production of tributyl phosphate.

3. A method for producing tributyl phosphate using Bacillus coagulans zqw106 as described in claim 1, characterized in that, The Bacillus coagulans zqw106 was cultured in a fermentation medium to produce tributyl phosphate.

4. The method as described in claim 3, characterized in that, The cultivation conditions are: static incubation at 35-39℃ for 24-64 hours, with a pH of 6.0-8.

0.

5. The method as described in claim 4, characterized in that, The culture conditions were: static incubation at 37°C for 48 hours and pH 7.

0.

6. The method as described in claim 4, characterized in that, The fermentation medium is composed of 16g ​​glucose, 10g tryptone, 2.5g yeast powder, 5g beef extract, 3.85g bovine brain, 4.9g bovine heart, 0.5g Tween 80, 1g ammonium citrate, 2.5g sodium chloride, 2.5g anhydrous sodium acetate, 0.05g magnesium sulfate, 0.025g manganese sulfate, 1.25g disodium hydrogen phosphate and 1g dipotassium hydrogen phosphate per liter.

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