Enterococcus gallinarum producing 3,4-dimethylbenzaldehyde and its application

Abstract

The invention belongs to the field of biotechnology, and specifically relates to Enterococcus gallinarum producing 3,4-dimethylbenzaldehyde and its application. In order to enrich the microbial sources of 3,4-dimethylbenzaldehyde production, the present invention screens and obtains a strain of Enterococcus gallinarum with a strong ability to produce 3,4-dimethylbenzaldehyde from nature, and the deposit number is CGMCC NO.25148. The strain has potential application prospects and application value in preparing microbial agents, freeze-dried bacterial powder, pest attractants, etc. containing 3,4-dimethylbenzaldehyde.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to enterococci of Gallinarum that produce 3,4-dimethylbenzaldehyde and application thereof. Background Art

[0002] 3,4-dimethyl-benzaldehyde is a colorless or light yellow transparent liquid with an almond flavor. It is widely used in the synthesis of fine chemicals such as medicines, pesticides, and spices, especially in the synthesis of polypropylene nucleating and transparent agents. It is a new generation of non-toxic plastic additives and is widely used in food packaging materials.

[0003] Patent CN201911074432.6 discloses the use of 3,4-dimethylbenzaldehyde in the preparation of pest attractants. 3,4-dimethylbenzaldehyde was isolated from the headspace volatiles of Chinese roses. Through GC-EAD, EAG, olfactory behavior, oviposition behavior biological tests and field oviposition tests, it was verified that 3,4-dimethylbenzaldehyde has a good attractant effect on female moths of cotton bollworm, so as to achieve pollution-free control of pests.

[0004] The synthesis reaction of 3,4-dimethylbenzaldehyde was first studied by Gattermann Koch in 1897 on the introduction of formyl groups into aromatic rings. The main methods for the synthesis of 3,4-dimethylbenzaldehyde are: ① indirect electro-oxidation method using 1,2,4-trimethylbenzene as raw material; ② synthesis method using o-xylene as raw material and HF-BF3 as catalyst or AlCl3 as main catalyst. However, these synthesis methods have disadvantages such as current efficiency restriction, strong corrosion of catalyst or decreased reaction selectivity.

[0005] Patent CN201811605384.4 discloses an environmentally friendly synthesis method of 3,4-dimethylbenzaldehyde, which uses anhydrous aluminum chloride and boron trichloride as composite catalysts, and does not use HF-BF3 superacid as a catalyst. Boron trichloride can be recycled to reduce the generation of "three wastes" during production. However, there is a risk of leakage of boron trichloride.

[0006] The microbial preparation method has the advantages of environmentally friendly natural metabolites, mild conditions, few by-products, and low equipment requirements. However, there are few studies on the microbial sources of 3,4-dimethylbenzaldehyde in the existing literature. It is known that Lan Jianglin et al. (Lan Jianglin, Liu Bo, Chen Zheng, et al. GS-MS analysis of degradation substances during the degradation of pig manure by strain LPF-1 (Brevibacillus brevis LPF-1) [J]. Journal of Fujian Agricultural Sciences, 2011, 26(6):9.) detected 3,4-dimethylbenzaldehyde in the fermentation broth of strain LPF-1 (Brevibacillus brevis LPF-1), but the literature did not mention the production of 3,4-dimethylbenzaldehyde. Summary of the invention

[0007] In order to enrich the sources of microorganisms producing 3,4-dimethylbenzaldehyde, the present invention screens and obtains a strain of Enterococcus gallinarum with strong ability to produce 3,4-dimethylbenzaldehyde from nature, providing the possibility for preparing 3,4-dimethylbenzaldehyde by microbial method. The strain is a natural metabolite, which solves the technical problems of large pollution, high cost, high equipment requirements, toxicity of by-products and the like in the prior art, and provides an available microorganism for the production and preparation of 3,4-dimethylbenzaldehyde.

[0008] The present invention provides a strain of Enterococcus gallinarum producing 3,4-dimethylbenzaldehyde, with a deposit number of CGMCC NO. 25148. The deposit time is June 21, 2022, and the deposit center is the General Microbiological Center of China Microbiological Culture Collection Administration CGMCC, with an address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, zip code 100101.

[0009] The 16S rDNA sequence of the Enterococcus gallinarum producing 3,4-dimethylbenzaldehyde is shown in SEQ ID NO:1.

[0010] Among them, the amount of 3,4-dimethylbenzaldehyde produced by the above-mentioned Gallinaceous Enterococcus is 1.833 mg / L.

[0011] The present invention also provides a microbial agent, which contains the above-mentioned Enterococcus gallinarum that produces 3,4-dimethylbenzaldehyde.

[0012] Furthermore, the microbial agent is a liquid agent or a solid agent.

[0013] Furthermore, the liquid bacterial agent or solid bacterial agent contains at least one of the live cells of Enterococcus gallinarum, dry Enterococcus gallinarum obtained by freeze-drying, and immobilized Enterococcus gallinarum cells.

[0014] The invention also provides a freeze-dried bacterial powder, which contains the above-mentioned Enterococcus gallinarum producing 3,4-dimethylbenzaldehyde.

[0015] Furthermore, the freeze-dried bacterial powder is prepared according to the following method: the above-mentioned glycerol storage tube of chicken enterococcus producing 3,4-dimethylbenzaldehyde is activated and inoculated into a culture medium for enrichment culture, and cultured at 37°C for 24-48 hours. After the culture is completed, the bacteria are collected by centrifugation, and trehalose, vitamin C, resistant dextrin, sorbitol, and skim milk powder are added thereto as freeze-drying protective agents, and the mixture is evenly mixed and then vacuum freeze-dried to obtain the freeze-dried bacterial powder.

[0016] The invention also provides application of the Enterococcus gallinarum in producing 3,4-dimethylbenzaldehyde.

[0017] Specifically, the application is to culture the Enterococcus gallinarum in a fermentation medium at 37°C for 2 days, with a pH value of 7.0, and the inoculation amount of the Enterococcus gallinarum is 2% (×10 7 cfu / mL).

[0018] More specifically, the composition of the fermentation medium is 16g of glucose, 10g of tryptone, 2.5g of yeast powder, 5g of beef extract, 3.85g of bovine brain, 4.9g of bovine heart, 0.5g of Tween 80, 1g of ammonium citrate, 2.5g of sodium chloride, 2.5g of anhydrous sodium acetate, 0.05g of magnesium sulfate, 0.025g of manganese sulfate, 1.25g of disodium hydrogen phosphate, and 1g of dipotassium hydrogen phosphate per liter of medium.

[0019] Using the above fermentation process, the content of 3,4-dimethylbenzaldehyde produced by the strain was 1.833 mg / L.

[0020] The present invention also provides the use of the above-mentioned Enterococcus gallus strain, microbial agent or freeze-dried bacterial powder in preparing pest attractants.

[0021] Furthermore, the application is to add the above-mentioned Enterococcus gallus strain, microbial agent or freeze-dried bacterial powder into a fermentation medium for cultivation to prepare a pest attractant containing 3,4-dimethylbenzaldehyde and auxiliary materials.

[0022] Furthermore, the excipient is at least one of a solubilizer, a synergist, and other metabolites.

[0023] Furthermore, the solubilizing agent is at least one of ethanol, acetone, polysorbate, polyethylene glycol or methanol.

[0024] Furthermore, the synergist is at least one of 3-phenylpropanaldehyde, eucalyptol, α-pinene, 3-pyridinecarboxylic acid ethyl ester or 4-methoxybenzaldehyde.

[0025] Furthermore, the other metabolites are at least one of acetic acid, hexanoic acid, octanoic acid, nonanoic acid, isopentanol, n-hexanol or phenylethanol.

[0026] The present invention also provides the use of the Enterococcus gallinarum strain, microbial agent or freeze-dried bacterial powder in the preparation of nucleating transparent agents, pesticides, daily necessities, spices or pharmaceutical intermediates.

[0027] Beneficial effects:

[0028] The present invention successfully isolated a new strain of Gallinarum enterococci that can produce 3,4-dimethylbenzaldehyde from nature. The strain has strong adaptability, short culture time and is environmentally friendly. In the fermentation medium, the content of 3,4-dimethylbenzaldehyde produced by the strain is 1.833 mg / L, which provides strain resources for studying the preparation of 3,4-dimethylbenzaldehyde by microorganisms and is conducive to promoting the transformation of green production technology.

[0029] The Enterococcus gallinarum strain provided by the present invention has a deposit number of CGMCC NO.25148. The deposit time is June 21, 2022, and the deposit center is the General Microbiological Center of the China Microbiological Culture Collection Administration CGMCC, the address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the zip code is 100101. The classification name is: Enterococcus gallinarum. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is the GC-MS molecular fragment mass spectrum of 3,4-dimethylbenzaldehyde produced by the Gallinarum enterococcus strain of the present invention;

[0031] Figure 2 This is the GC-MS molecular fragmentation mass spectrum of 3,4-dimethylbenzaldehyde standard. DETAILED DESCRIPTION

[0032] The present invention provides an Enterococcus gallinarum strain, with a deposit number of CGMCCNO.25148. The deposit time is June 21, 2022, and the deposit center is the General Microbiological Center of China Microbiological Culture Collection Administration CGMCC, with an address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, zip code 100101.

[0033] The 16S rDNA sequence of the Enterococcus gallinarum producing 3,4-dimethylbenzaldehyde is shown in SEQ ID NO:1.

[0034] SEQ ID NO:1 16S rDNA sequence of Enterococcus gallinarum producing 3,4-dimethylbenzaldehyde

[0035]

[0036] The biological characteristics of the above-mentioned 3,4-dimethylbenzaldehyde-producing Enterococcus graniformis are: spherical, single, paired or arranged in chains of different lengths, Gram-positive bacteria, without spores and flagella.

[0037] Among them, the growth conditions of the above-mentioned 3,4-dimethylbenzaldehyde-producing chicken enterococci are: growth temperature is 10°C-45°C, the optimum temperature is 37°C, tolerance pH is 5.0-9.6, growth cycle is 24-48h, and L-arabinose, D-ribose, D-mannitol, lactose, and D-tagatose can be utilized.

[0038] The present invention provides a method for screening and identifying the above-mentioned Enterococcus gallinarum producing 3,4-dimethylbenzaldehyde, comprising the following steps: breaking up and separating a Daqu sample, purifying by streaking, and preserving the strain, and then using the ability to produce 3,4-dimethylbenzaldehyde as a screening standard to obtain the Enterococcus gallinarum producing 3,4-dimethylbenzaldehyde.

[0039] Among them, the Daqu sample is broken up by oscillation, and then the suspension is diluted, and multiple gradient dilutions are spread on a solid culture medium plate for anaerobic culture.

[0040] The streak purification is to pick single colonies with different colony morphologies on the plate where the colonies grow, and purify all the single colonies by secondary streaking.

[0041] Furthermore, each purified strain was cultured anaerobically in a liquid medium in the form of a single colony.

[0042] Among them, different strains were fermented to obtain fermentation broth, and the target compounds were extracted and enriched by headspace solid phase microextraction, and then GC-MS was used for chromatographic detection and analysis, so as to screen out Gallinaceous Enterococcus with high production of 3,4-dimethylbenzaldehyde by comparing the content of volatile substances.

[0043] The invention also provides a microbial agent, which contains the above-mentioned Enterococcus gallinarum that produces 3,4-dimethylbenzaldehyde.

[0044] The microbial agent is a liquid agent or a solid agent containing one or more of live cells of Enterococcus gallinarum, dry Enterococcus gallinarum obtained by freeze-drying, and immobilized Enterococcus gallinarum cells.

[0045] The present invention also provides a freeze-dried bacterial powder, which is prepared by the following method: activating a glycerol preservation tube of Enterococcus gallus domesticus and then inoculating it into a culture medium for enrichment culture, culturing it at 37°C for 24-48 hours, collecting the bacterial bodies by centrifugation after the culture is completed, adding trehalose, vitamin C, resistant dextrin, sorbitol, and skim milk powder as freeze-drying protective agents, mixing them evenly, and performing vacuum freeze drying to obtain the freeze-dried bacterial powder.

[0046] The invention also provides application of the Enterococcus gallinarum in producing 3,4-dimethylbenzaldehyde.

[0047] Specifically, the application is to culture the Enterococcus gallinarum in a fermentation medium at 37°C for 2 days, with a pH value of 7.0, and the inoculation amount of the Enterococcus gallinarum is 2% (×10 7 cfu / mL).

[0048] More specifically, the composition of the fermentation medium is 16g of glucose, 10g of tryptone, 2.5g of yeast powder, 5g of beef extract, 3.85g of bovine brain, 4.9g of bovine heart, 0.5g of Tween 80, 1g of ammonium citrate, 2.5g of sodium chloride, 2.5g of anhydrous sodium acetate, 0.05g of magnesium sulfate, 0.025g of manganese sulfate, 1.25g of disodium hydrogen phosphate, and 1g of dipotassium hydrogen phosphate per liter of medium.

[0049] Using the above fermentation process, the content of 3,4-dimethylbenzaldehyde produced by the strain was 1.833 mg / L.

[0050] The present invention also provides the use of the Enterococcus gallinarum strain, the microbial agent or the freeze-dried bacterial powder in the preparation of pest attractants.

[0051] Preferably, the application is to add the above-mentioned Enterococcus gallinarum strain, the above-mentioned microbial agent or freeze-dried bacterial powder into a fermentation medium, and after culturing, to prepare a pest attractant composed of 3,4-dimethylbenzaldehyde and auxiliary materials.

[0052] Preferably, the excipient is at least one of a solubilizer, a synergist or other metabolites.

[0053] Preferably, the solubilizer is at least one of ethanol, acetone, polysorbate, polyethylene glycol or methanol.

[0054] Preferably, the synergist is at least one of 3-phenylpropanaldehyde, eucalyptol, α-pinene, ethyl 3-pyridinecarboxylate or 4-methoxybenzaldehyde.

[0055] Preferably, the other metabolites are at least one of acetic acid, hexanoic acid, octanoic acid, nonanoic acid, isopentanol, n-hexanol or phenylethanol.

[0056] The present invention also provides the use of the Enterococcus gallinarum strain, the microbial agent or the freeze-dried bacterial powder in the preparation of nucleating transparent agents, pesticides, daily necessities, spices or pharmaceutical intermediates.

[0057] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

[0058] The culture medium formula involved in the embodiment is:

[0059] MRS liquid culture medium: glucose 20g / L, peptone 10g / L, yeast powder 4g / L, beef extract powder 5g / L, Tween 801g / L, triammonium citrate 2g / L, sodium acetate 5g / L, magnesium sulfate 0.2g / L, manganese sulfate 0.05g / L, dipotassium hydrogen phosphate 1g / L, pH 7.0, high pressure steam sterilization at 115℃ for 20 min.

[0060] MRS solid medium: Add 15 g / L agar to MRS liquid medium and sterilize with high pressure steam at 115°C for 20 minutes.

[0061] Fermentation medium: glucose 16 g / L, tryptone 10 g / L, yeast powder 2.5 g / L, beef extract 5 g / L, bovine brain 3.85 g / L, bovine heart 4.9 g / L, Tween 80 0.5 g / L, ammonium citrate 1 g / L, sodium chloride 2.5 g / L, anhydrous sodium acetate 2.5 g / L, magnesium sulfate 0.05 g / L, manganese sulfate 0.025 g / L, disodium hydrogen phosphate 1.25 g / L, dipotassium hydrogen phosphate 1 g / L, pH 7.0, high-pressure steam sterilization at 115°C for 20 min.

[0062] Example 1: Isolation and purification of strains

[0063] (1) Separation method: Take 20g of sample Luzhou Laojiao medium-temperature Daqu and place it in a conical flask filled with 180mL of sterile distilled water. Oscillate on a constant temperature shaker for 10min to fully disperse and mix the sample. Take 1mL of the sample suspension and dilute it to 10 by multiple dilution method. -2 ~10 -7 , take 100 μL from each gradient dilution and spread it evenly on the MRS solid culture medium plate, prepare two plates in parallel, invert them, and culture them at 37℃ under anaerobic conditions for 36-48h, and observe them in time.

[0064] (2) Streak purification: Take out the plate with colonies, pick out single colonies with different colony morphologies, and perform streaking a second time until all single colonies are purified.

[0065] (3) Preservation of bacterial strains: After purification, a single colony of each strain was picked into 5 mL of MRS liquid culture medium and cultured at 37°C under anaerobic conditions for 20-24 h. 1 mL of bacterial solution was transferred to a bacterial preservation tube, and 0.5 mL of 60% sterile glycerol solution was added, resuspended, and stored at -80°C.

[0066] Example 2: Detection of the ability to produce 3,4-dimethylbenzaldehyde

[0067] (1) Prepare the bacterial solution to be tested:

[0068] After the glycerol preservation tubes of the screened strains were dissolved, they were inoculated into MRS liquid culture medium and cultured at 37°C under anaerobic conditions for 24 hours. After activation for three generations, they were inoculated into 50 mL MRS liquid culture medium at a 2% (volume ratio) inoculation rate and cultured for 24 hours to obtain the bacterial solution to be tested.

[0069] (2) HS-SPME / GC-MS detection method for 3,4-dimethylbenzaldehyde:

[0070] Using headspace solid phase microextraction / gas chromatography-mass spectrometry technology: take the supernatant, add it to the headspace bottle, add saturated NaCl solution, use 0.822mg / ml 2-octanol as the internal standard, keep the prepared sample at 60℃ for 5min, extract it with 50 / 30μmDVB / CAR / PDMS extraction head at 60℃ for 50min, and desorb it at 250℃ at the GC injection port for 5min after extraction. The compound search results are matched with the NIST standard spectral library, and the similarity reaches more than 80% to confirm the target compound. The culture broth without bacterial fermentation is used as the blank control group to calculate the content of each volatile substance.

[0071] GC-MS detection chromatographic conditions:

[0072] Gas chromatography conditions: HP-INNOWAX column (60m×0.25mm×0.25μm); temperature program: starting temperature 40°C, hold for 5min, increase to 100°C at 4°C / min, then increase to 230°C at 6°C / min,

[0073] Maintain for 10 min, with high-purity helium as the carrier gas (1.0 mL / min); injection port temperature at 250 °C, without splitting.

[0074] 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.

[0075] Through the above detection method, a strain with a strong ability to produce 3,4-dimethylbenzaldehyde was screened out from the isolated strains, and this strain was selected for further research.

[0076] Example 3: Molecular identification of strains

[0077] After the target strain was expanded, fresh bacterial liquid in the logarithmic growth phase was taken, centrifuged to collect the bacteria, and genomic DNA was extracted using a bacterial genome extraction kit. The universal primers 27F / 1541R for lactic acid bacteria were used to amplify the full-length sequence of 16S rDNA, as follows:

[0078] SEQ ID NO: 2: 27F (5′-AGAGTTTGATCCTGGCTCAG-3′)

[0079] SEQ ID NO: 3: 1541R (5′-AAGGAGGTGATCCAGCC-3′)

[0080] ①Reaction system (50 μl)

[0081]

[0082] ②Reaction procedure

[0083]

[0084] The PCR products were separated and tested by 1.0% agarose gel electrophoresis with a voltage of about 11 V / cm and an electrophoresis time of 20 min.

[0085] The purification of PCR products was carried out according to the instructions of the mini-gel recovery PCR product purification kit of Shanghai Sangon Biotechnology Co., Ltd., and the sequencing was completed by Shanghai Sangon Biotechnology Co., Ltd.

[0086] The gene sequence of the 16S rDNA fragment obtained by sequencing was compared with NCBI's BLAST to determine the strain species information. Its sequence is shown in SEQ ID NO: 1. It was identified as Enterococcus gallinarum and named Enterococcus gallinarum zqw82.

[0087] Example 4: Physiological and biochemical characteristics of Enterococcus gallinarum zqw82

[0088] Enterococcus gallinarum zqw82 was inoculated into MRS solid medium and cultured at 37°C for 2 days, and then the colonies and cell morphology of the strain were observed. Enterococcus gallinarum zqw82 was inoculated into MRS liquid medium and the OD of the culture medium was measured. 600 The temperature and pH tolerance ranges suitable for the growth of the strain were detected, and the carbon source available to the strain was analyzed by API RAPID ID 32STREP test. The above characteristics of Enterococcus gallinarum zqw82 are shown in Table 1.

[0089] Table 1 Physiological and biochemical characteristics of Enterococcus avium zqw82

[0090] Experimental Project Enterococcus gallinarum zqw82 oxygen Facultative anaerobic Colony morphology White round smooth surface Cell shape (size, μm) Spherical (0.6-2.0μm) flagellum none Growth temperature range (℃) 10-45 Optimum temperature (℃) 37 pH Range 5.0-9.6 Available carbon sources L-arabinose, D-ribose, D-mannitol, lactose, D-tagatose

[0091] Example 5: Fermentation culture of Enterococcus gallinarum zqw82

[0092] After dissolving the obtained chicken enterococcus zqw82 glycerol storage tube, inoculate it into 10mL MRS liquid culture medium at a 5% inoculum, and inoculate it into 400mL fermentation medium containing 40mL ethyl acetate at a 2% inoculum at 37°C for 24h, and perform two-phase fermentation for 2 consecutive days. Take 20mL of the upper organic phase, concentrate and evaporate to dryness, and then make it up to 5mL with chromatographic grade methanol. The detection method is the same as the volatile component determination method in Example 2. After continuous cultivation of chicken enterococcus for 2 days, the production of 3,4-dimethylbenzaldehyde can reach 1.833mg / L.

Claims

1. Enterococcus gallinarum producing 3,4-dimethylbenzaldehyde ( Enterococcus gallinarum ) strain, characterized in that: The deposit number is CGMCC NO. 25148.

2. The strain according to claim 1, characterized in that: The amount of 3,4-dimethylbenzaldehyde produced is 1.833 mg / L.

3. A microbial agent, characterized in that: Contains the Enterococcus gallinarum producing 3,4-dimethylbenzaldehyde as claimed in claim 1.

4. The microbial agent according to claim 3, characterized in that: The microbial agent is a liquid agent or a solid agent.

5. The microbial agent according to claim 4, characterized in that: The liquid bacterial agent or solid bacterial agent contains at least one of the following: live cells of Enterococcus gallinarum producing 3,4-dimethylbenzaldehyde as claimed in claim 1, dry Enterococcus gallinarum obtained by freeze drying, and immobilized Enterococcus gallinarum cells.

6. A freeze-dried bacterial powder, characterized in that: Contains the Enterococcus gallinarum producing 3,4-dimethylbenzaldehyde as claimed in claim 1.

7. Use of the 3,4-dimethylbenzaldehyde-producing Enterococcus gallinarum according to claim 1 in producing 3,4-dimethylbenzaldehyde.

8. Use of the Enterococcus gallinarum strain according to any one of claims 1 to 2, the microbial agent according to any one of claims 3 to 5, or the freeze-dried bacterial powder according to claim 6 in the preparation of a pest attractant containing 3,4-dimethylbenzaldehyde.

9. The use according to claim 8, characterized in that: The Enterococcus gallinarum strain described in any one of claims 1-2, the microbial agent described in any one of claims 3-5 or the freeze-dried bacterial powder described in claim 6 is added to a fermentation medium for cultivation to prepare a pest attractant containing 3,4-dimethylbenzaldehyde and auxiliary materials.

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