A kind of lactobacillus buchneri producing 2,4-di-tert-butylphenol

Abstract

The invention relates to a 2,4-di-tert-butylphenol-producing lactobacillus buchneri. Aiming at the problems that the current chemical method for synthesizing 2,4-di-tert-butylphenol has high energy consumption and high cost, and the microbial production of 2,4-di-tert-butylphenol has low yield, the invention provides a 2,4-di-tert-butylphenol-producing lactobacillus buchneri, which is named as 2,4-di-tert-butylphenol-producing lactobacillus buchneri zqw46 and has a deposit number of CGMCC NO.25142. The 2,4-di-tert-butylphenol-producing lactobacillus buchneri zqw46 provided by the invention has the advantages of simple culture medium components, low raw material cost, easy control of fermentation conditions, mild reaction conditions, short fermentation cycle, etc.; the 2,4-di-tert-butylphenol produced by fermentation is green and natural, and its yield is higher than that of the strains currently found, providing new strain resources for the research of microbial fermentation method.

Description

Technical Field

[0001] The invention relates to the technical field of microbial application, in particular to a Lactobacillus buchneri that produces 2,4-di-tert-butylphenol. Background Art

[0002] 2,4-Di-tert-butylphenol is an important intermediate for fine chemical products. It is mainly used in the preparation of antioxidant 168, coatings, flame retardants, 2402 resin, etc. It can also be used to manufacture natural rubber and synthetic rubber antioxidants, plastic antioxidants, fuel stabilizers, ultraviolet absorbers, pesticides, dye intermediates, etc.

[0003] At present, the industrial production of 2,4-di-tert-butylphenol mostly uses phenol and isobutylene as raw materials, and the catalysts are generally liquid acid and ion exchange resin. Isobutylene has the potential safety hazard of being flammable and explosive, and has high requirements for instruments; liquid acid catalysts such as HF, H3PO4, H2SO4 have problems such as serious pollution and strong corrosion; ion exchange resin catalysts have the disadvantages of poor heat resistance and short life. For example:

[0004] CN106975517A discloses a green and efficient multiphase catalyst for synthesizing 2,4-di-tert-butylphenol, wherein the catalyst type is a porous solid acid resin catalyst with a high specific surface area. The catalyst is used to produce 2,4-di-tert-butylphenol under the catalytic action of a non-toxic and harmless green solid acid resin catalyst using phenol and isobutylene as reaction raw materials. The catalyst of the invention replaces the existing kaolin catalyst in production, can be repeatedly recycled, and has the characteristics of long life, greatly reducing the production cost of the catalyst, and greatly reducing the solid waste generated by the one-time use of the kaolin catalyst, realizing the greening of the production process. At the same time, the separation and recovery of the multiphase catalyst for synthesizing 2,4-di-tert-butylphenol provided by the invention is relatively simple, greatly reducing the separation time of the existing kaolin catalyst, and significantly improving production efficiency.

[0005] CN111205172A discloses a clean production method of 2,4-di-tert-butylphenol, A) mixing phenol and a catalyst, stirring and heating, adding isobutylene, and reacting to obtain 2,4-di-tert-butylphenol; the catalyst is a molecular sieve doped with a transition metal oxide; the transition metal oxide is La2O3, CeO2; the molecular sieve is an X-type molecular sieve and / or a Y-type molecular sieve; the mass fraction of the transition metal oxide in the catalyst is 6-20%; B) mixing the used catalyst with a new catalyst, adding phenol and isobutylene, reacting again, repeating the process several times, extracting and recovering the catalyst with an organic solvent, and then drying and calcining to complete the regeneration and recycling of the catalyst. The invention uses a molecular sieve catalyst doped with a transition metal for the alkylation reaction of phenol and isobutylene, has high catalytic activity, can reduce byproducts produced by the reaction of the target product with the isobutylene molecule, and improves the reaction selectivity.

[0006] In addition, there are also technical solutions for synthesizing 2,4-di-tert-butylphenol using other raw materials in the prior art, such as:

[0007] CN109081771A discloses a method for preparing 2,4-di-tert-butylphenol, comprising the following steps: 1) adding a catalyst and methyl tert-butyl ether under a certain pressure and in the presence of an organic solvent, and stirring; 2) dropping phenol into step 1), heating, and stirring; 3) cooling the reaction solution in step 2) to room temperature, filtering, washing the filtrate, and distilling off the solvent; 4) distilling and drying the crude 2,4-di-tert-butylphenol product in step 3) to obtain a 2,4-di-tert-butylphenol product. The preparation method of the invention has mild reaction conditions, high product selectivity, and the catalyst can be recycled.

[0008] CN1199923C discloses a purification method for synthesizing 2,4-di-tert-butylphenol by using methyl tert-butyl ether (MTBE) as an alkylating agent to react with phenol. The method is characterized in that a treatment must be performed before distillation and purification, i.e., an alkaline substance is added to the alkylating liquid for neutralization, and then distillation and purification are performed after simple distillation, so as to solve the problem that the dealkenyl reaction occurring during the intermittent distillation process makes it difficult to switch the distillation and the yield is low.

[0009] However, the chemical method for synthesizing 2,4-di-tert-butylphenol consumes a lot of energy and is costly. In comparison, the microbial fermentation method has the characteristics of retaining the biological activity of the product, short fermentation cycle, and low raw material cost, which provides a new idea for the synthesis of 2,4-di-tert-butylphenol. There are nearly 169 natural sources of 2,4-di-tert-butylphenol, including about 16 bacteria and 11 fungi. The bacteria include Bacillus licheniformis, Bacillus subtilis, Streptococcus, Pseudomonas, Vibrio alginolyticus, etc. Studies at home and abroad have found that the natural product 2,4-di-tert-butylphenol has certain biological functions, such as antioxidant, anti-inflammatory, antibacterial, etc. For example:

[0010] CN114410526A discloses a strain of Bacillus licheniformis XNRB-3 and its application, belonging to the field of agricultural microbial technology. The biological preservation number of the strain is: CGMCC NO.15308. The strain provided by the invention has the function of degrading phenolic acid substances such as phloridzin and antagonizing pathogens. Its fermentation metabolites contain antifungal active substances and substances that promote plant growth, among which α-bisabolol and 2,4-di-tert-butylphenol have significant inhibitory effects on many pathogenic fungi, and succinic acid, monomethyl ester and dibutyl phthalate can promote the root elongation and lateral root development of Arabidopsis. The strain can also produce protease, cellulase, chitosanase and β-1,3-glucanase, etc.; secrete iron carriers and auxins, and has certain phosphorus dissolving, potassium dissolving and nitrogen fixing capabilities. In potted and field tests, the addition of strain XNRB-3 significantly promoted the growth of apple rootstock Pingyi sweet tea seedlings and two-year-old apple saplings.

[0011] However, the content of 2,4-di-tert-butylphenol produced by microorganisms is currently only in the range of 2 to 4.3 mg / L, which limits the development of microbial production of 2,4-di-tert-butylphenol. Therefore, finding a microorganism with high production of 2,4-di-tert-butylphenol is a technical problem that urgently needs to be solved in this field.

[0012] In addition, on the one hand, there are differences in understanding among those skilled in the art; on the other hand, the applicant studied a large number of documents and patents when making the present invention, but due to space limitations, not all details and contents are listed in detail. However, this does not mean that the present invention does not have the characteristics of these prior arts. On the contrary, the present invention already has all the characteristics of the prior art, and the applicant reserves the right to add relevant prior art to the background technology. Summary of the invention

[0013] In view of the shortcomings of the prior art, the object of the present invention is to provide a lactobacillus buchneri that produces 2,4-di-tert-butylphenol. The lactobacillus buchneri can greatly increase the yield of 2,4-di-tert-butylphenol and prepare 2,4-di-tert-butylphenol in a relatively lower cost and lower energy consumption manner, thereby facilitating the application of the lactobacillus buchneri in the preparation of antioxidants 168, coatings, flame retardants, 2402 resins and other fields.

[0014] The invention provides a 2,4-di-tert-butylphenol-producing Lentilactobacillus buchneri, which is named as Lentilactobacillus buchneri zqw46 (Lentilactobacillus buchneri), and is preserved in the General Microbiological Center of China Microbiological Culture Collection Administration, with a preservation number of CGMCC NO.25142.

[0015] Preferably, the nucleotide sequence of the Lactobacillus buchneri zqw46 based on 16S rDNA is as shown in SEQ ID NO.1.

[0016] Further, the amplification primer sequences are:

[0017] 27F: 5′-AGAGTTTGATCCTGGCTCAG-3′ (SEQ ID NO. 2)

[0018] 1492R: 5′-GGTTACCTTGTTACGACTT-3′ (SEQ ID NO.3)

[0019] Preferably, the colony characteristics of the Lactobacillus buchneri zqw46 on MRS agar medium are: the colonies are raised, white, round, and have neat edges.

[0020] The present invention provides a screening and identification method for the aforementioned Lactobacillus buchneri, which comprises the following steps: breaking up and separating the fermented grains sample, purifying by streaking, and preserving the strain, and then using the ability to produce 2,4-di-tert-butylphenol as a screening standard to obtain Lactobacillus buchneri that produces 2,4-di-tert-butylphenol.

[0021] Preferably, the fermented grains sample is shaken and then dispersed, and then the suspension is diluted, and multiple gradient dilutions are spread on a solid culture medium plate for anaerobic culture.

[0022] Preferably, single colonies with different colony morphologies are picked from the plate on which the colonies grow, and all the single colonies are purified by secondary streaking.

[0023] Preferably, each purified strain is cultured anaerobically in a liquid medium in the form of a single colony.

[0024] Preferably, different strains are fermented separately to obtain fermentation broth, the target compound is extracted and enriched by headspace solid phase microextraction, and then chromatographic detection and analysis are performed by GC-MS, so as to screen out Lactobacillus buchneri with high production of 2,4-di-tert-butylphenol by comparing the content of volatile substances.

[0025] The present invention provides a microbial agent, which contains the aforementioned Lactobacillus brunneri. The agent is a liquid agent or a solid agent containing one or more of live cells of Lactobacillus brunneri, freeze-dried dry cells of Lactobacillus brunneri, and immobilized cells of Lactobacillus brunneri.

[0026] The invention provides a freeze-dried bacterial powder. The freeze-dried bacterial powder is prepared by the following method: a glycerol storage tube of Lactobacillus brunneri is activated and then inoculated into a culture medium for enrichment culture, and the culture is carried out at 37°C for 20-24 hours. After the culture is completed, the bacterial bodies are collected by centrifugation, trehalose, 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.

[0027] The present invention provides an application of the aforementioned Lactobacillus buchneri or the aforementioned microbial agent or the aforementioned freeze-dried bacterial powder in fermentation production of 2,4-di-tert-butylphenol.

[0028] Preferably, the application is to statically culture the aforementioned Lactobacillus buchneri or the aforementioned microbial agent or the aforementioned freeze-dried bacterial powder in a fermentation medium.

[0029] Furthermore, the culture conditions were static culture at 37°C for 3 days, the pH value was 7.0, and the inoculation amount of the Lactobacillus buchneri was 2% (×10 7 cfu / mL).

[0030] Preferably, the upper layer liquid after static culture is concentrated and evaporated to dryness and then fixed to volume, and the content of volatile substances can be detected by GC-MS.

[0031] Preferably, after continuously culturing Lactobacillus buchneri for 3 days using the above method, the yield of 2,4-di-tert-butylphenol produced can reach 10.77 mg / L.

[0032] The present invention provides an application of the aforementioned Lactobacillus buchneri, the aforementioned microbial agent or the aforementioned freeze-dried bacterial powder in sterilization.

[0033] Preferably, the application is to add the aforementioned Lactobacillus buchneri or the aforementioned microbial agent or the aforementioned freeze-dried bacterial powder to the fermentation medium to cultivate 2,4-di-tert-butylphenol and auxiliary materials to form a bactericide.

[0034] Preferably, the excipient is one or more of a solubilizer, a synergist, an emulsifier, and a penetrant.

[0035] Preferably, the solubilizer is one or more of sodium dodecylbenzene sulfonate, potassium dodecylbenzene sulfonate, polysorbate, polyethylene glycol, sorbitan polyoxyethylene ether, lignin sulfonate, tea dregs, saponins, and the like.

[0036] Preferably, the emulsifier is one or more of Spans, Tweens, ethylene glycol esters, propylene glycol esters and / or polyethylene glycol type nonionic surfactants.

[0037] Preferably, the synergist is one or more of tea saponin, polyoxyethylene ether, etc.

[0038] Preferably, the penetrant is azone or the like.

[0039] The present invention provides one or more applications of the aforementioned Lactobacillus buchneri or the aforementioned microbial agent or the aforementioned freeze-dried bacterial powder in the preparation of natural rubber and synthetic rubber antioxidants, plastic antioxidants, fuel stabilizers, ultraviolet absorbers, and pesticide and dye intermediates.

[0040] Compared with the prior art, the Lactobacillus buchneri zqw46 provided by the present invention has the advantages of simple culture medium components for fermenting 2,4-di-tert-butylphenol, low raw material cost, easy to control fermentation conditions, mild reaction conditions, short fermentation cycle, etc.; the 2,4-di-tert-butylphenol produced by fermentation is green and natural, and its output is higher than that of the strains discovered so far, which provides a new strain resource for the research of microbial fermentation method and has good application prospects.

[0041] Biological Deposit Description

[0042] The Lentilactobacillus buchneri provided by the present invention is deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration, with a deposit number of CGMCC NO.25142, a deposit date of June 21, 2022, and a deposit address of the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is a GC-MS graph of 2,4-di-tert-butylphenol produced by the strain provided by the present invention;

[0044] Figure 2 It is a GC-MS molecular fragment mass spectrum of 2,4-di-tert-butylphenol produced by the strain provided by the present invention;

[0045] Figure 3 The present invention provides a GC-MS molecular fragment mass spectrum of a 2,4-di-tert-butylphenol standard product. DETAILED DESCRIPTION

[0046] The following is a detailed description with reference to the accompanying drawings.

[0047] Figure 1 This is a GC-MS graph of 2,4-di-tert-butylphenol produced by the strain provided by the present invention; Figure 2 It is a GC-MS molecular fragment mass spectrum of 2,4-di-tert-butylphenol produced by the strain provided by the present invention; Figure 3 The present invention provides a GC-MS molecular fragment mass spectrum of a 2,4-di-tert-butylphenol standard product.

[0048] The present invention provides a 2,4-di-tert-butylphenol-producing Lactobacillus buchneri, which is named Lentilactobacillus buchneri zqw46 (Lentilactobacillus buchneri), and is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with a deposit number of CGMCC NO.25142, a deposit date of June 21, 2022, and a deposit address of Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code 100101.

[0049] The present invention also provides a screening and identification method for Lactobacillus buckerii, which is applicable to the aforementioned Lactobacillus buckerii that produces 2,4-di-tert-butylphenol, and comprises the following steps: breaking up and separating the fermented grains sample, purifying by streaking, and preserving the strain, and using the ability to produce 2,4-di-tert-butylphenol as a screening standard to obtain Lactobacillus buckerii that produces 2,4-di-tert-butylphenol. Preferably, the present invention can determine the ability of a microorganism to produce 2,4-di-tert-butylphenol by detecting the content of 2,4-di-tert-butylphenol in volatile substances.

[0050] The present invention also provides the use of the above-mentioned Lactobacillus brucei in fermenting 2,4-di-tert-butylphenol, wherein the use is to statically culture the Lactobacillus brucei in a fermentation medium. Further, the use is to statically culture the Lactobacillus brucei in a fermentation medium at 37°C for 3 days, with a pH value of 7.0, and the inoculation amount of the Lactobacillus brucei is 2% (×10 7 cfu / mL).

[0051] Preferably, in the present invention, the "liquid culture medium" can be 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°C for 20 minutes.

[0052] Preferably, in the present invention, the "solid culture medium" may be an MRS solid culture medium: 15 g / L agar is added to the MRS liquid culture medium and sterilized by high pressure steam at 115° C. for 20 minutes.

[0053] Preferably, in the present invention, the composition of the "fermentation medium" can be: 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, and high-pressure steam sterilization at 115°C for 20 minutes.

[0054] Preferably, in the present invention, "headspace solid phase microextraction" can be a solid phase microextraction technology that uses headspace sampling, wherein the headspace is used as a sampling method, and the sample to be tested can be placed in a constant temperature closed container, and the volatile components are evaporated from the sample by heating. After the two phases in the headspace bottle reach thermodynamic equilibrium, the top gas is directly extracted and injected into a gas chromatography-mass spectrometer for separation and analysis, thereby detecting some volatile or odorous substances; and the role of solid phase microextraction technology is to use a fused quartz fiber coated with a stationary phase to adsorb and enrich when extracting gas samples, and then perform analysis and sampling.

[0055] Preferably, in the present invention, "GC-MS" can be a gas chromatography-mass spectrometer, which is a device that combines the characteristics of gas chromatography and mass spectrometry to identify different substances in a sample. When a multi-component mixed sample enters the chromatographic column, due to the different adsorption forces of the adsorbent on each component, after a certain period of time, the running speed of each component in the chromatographic column is also different. Components with weak adsorption forces are easily desorbed and leave the chromatographic column first and enter the detector, while components with the strongest adsorption forces are least likely to be desorbed and therefore leave the chromatographic column last. In this way, the components are separated from each other in the chromatographic column and sequentially enter the detector to be detected and recorded.

[0056] Preferably, in the present invention, the "head space bottle" can be a sample bottle used for head space analysis, wherein the head space analysis is to analyze the gas present in the head space sample bottle, and the volatile sample is heated and volatilized to form gas in the space at the top of the sample bottle and then diffuse, and finally the top gas above the volatile sample in the gas chromatography bottle enters the gas phase detection.

[0057] Example 1

[0058] The Lactobacillus buchneri of the present invention can be separated, purified and preserved in the following manner:

[0059] S1.1. Separation: After the mash sample is shaken and broken up, the suspension is diluted and multiple gradient dilutions are spread on a solid culture medium plate for anaerobic culture.

[0060] Specifically, take 20g of the fermented grains sample and put it into 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.

[0061] S1.2, Purification: Pick single colonies with different colony morphologies on the plate where colonies grow, and purify all single colonies by secondary streaking.

[0062] S1.3. Preservation: Each purified strain was cultured anaerobically in liquid culture medium in the form of a single colony.

[0063] Specifically, a single colony of each strain after purification was picked into 5 mL of MRS liquid culture medium, placed in anaerobic culture at 37°C for 20-24 h, 1 mL of bacterial solution was pipetted into a bacterial preservation tube, 0.5 mL of 60% sterile glycerol solution was added, resuspended, and stored at -80°C.

[0064] Example 2

[0065] This embodiment is a further improvement on Embodiment 1, and the repeated contents will not be repeated here.

[0066] The present invention can detect the ability of each strain to produce a target compound after separation and purification and preservation in a glycerol storage tube to screen out a strain with high production capacity, wherein the target compound in this embodiment is 2,4-di-tert-butylphenol. That is, in this embodiment, by detecting the content of 2,4-di-tert-butylphenol produced by each strain, a strain with high production capacity of 2,4-di-tert-butylphenol can be screened out.

[0067] Preferably, the Lactobacillus buchneri of the present invention can be screened by the following method:

[0068] S2.1. Prepare the bacterial solution to be tested

[0069] Specifically, the strain obtained after separation and purification was dissolved in a glycerol storage tube, inoculated into MRS liquid culture medium, and cultured at 37°C under anaerobic conditions for 20 hours. After activation for three generations, the strain was inoculated into 50 mL MRS liquid culture medium at a 2% (volume ratio) inoculation amount and cultured for 20 hours. After the fermentation was completed, the fermentation liquid to be tested was obtained.

[0070] S2.2. Detection of 2,4-di-tert-butylphenol content

[0071] Preferably, the fermentation broth to be tested is subjected to headspace solid phase microextraction to extract and enrich the target compound, and then chromatographic detection and analysis is performed by GC-MS.

[0072] Specifically, the supernatant was taken and added to the headspace bottle, and a saturated NaCl solution was added, with 0.822 mg / ml 2-octanol as the internal standard. The prepared sample was kept at 60°C for 5 minutes, and then extracted at 60°C for 50 minutes using a 50 / 30 μm DVB / CAR / PDMS extraction head. After the extraction, the sample was desorbed at 250°C for 5 minutes at the GC injection port. The compound search results were matched with the NIST standard spectral library, and the target compound was confirmed when the similarity reached more than 80%. The culture solution without the strain was used as the blank control group to calculate the content of each volatile substance.

[0073] Preferably, the GC-MS detection chromatographic conditions are as follows:

[0074] 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, hold for 10min, carrier gas is high-purity helium (1.0mL / min); injection port temperature 250°C, no split;

[0075] 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.

[0076] Preferably, the above screening method is to ferment different strains to obtain fermentation broth, extract and enrich the target compound by headspace solid phase microextraction, and then perform chromatographic detection and analysis by GC-MS, so as to screen out Lactobacillus buchneri with high production of 2,4-di-tert-butylphenol by comparing the content of volatile substances, and its GC-MS mass spectrum is as follows: Figure 1 shown.

[0077] Preferably, the above screening method can be used as a method for detecting the ability of microorganisms to produce 2,4-di-tert-butylphenol, and can also be used as a method for characterizing the yield of 2,4-di-tert-butylphenol.

[0078] Example 3

[0079] This embodiment is a further improvement of Embodiment 1 and / or 2, and the repeated contents will not be repeated here.

[0080] Preferably, the strains screened by the present invention can be identified by the following method:

[0081] S3.1, 16S rDNA sequence amplification: take 1mL of the culture solution of the strain and centrifuge at 10000rpm for 5min, remove the supernatant, add CTAB solution and phenol-chloroform-isoamyl alcohol (25:24:1) to extract nucleic acid DNA, centrifuge at 12500rpm for 5min to take the supernatant, add an equal volume of chloroform-isoamyl alcohol (24:1), mix well and centrifuge at 12000rpm for 5min to take the supernatant, rinse twice with ethanol solution, centrifuge at 12000rpm for 5min to remove the supernatant, blow dry the precipitate, add 50μL sterile water to resuspend, and obtain the strain DNA template. The primers are 27F: 5′-AGAGTTTGATCCTGGCTCAG-3′ and 1492R: 5′-GGTTACCTTGTTACGACTT-3′, and PCR amplification is performed.

[0082] Furthermore, 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, and it was identified as Lentilactobacillus buchneri and named Lentilactobacillus buchneri zqw46.

[0083] Example 4

[0084] This embodiment is a further improvement of Embodiments 1, 2 and / or 3, and the repeated contents will not be repeated here.

[0085] The present invention also discloses the application of the aforementioned embodiment of the slow-growing Lactobacillus brunneri zqw46 in fermentation to produce 2,4-di-tert-butylphenol. Preferably, the application is to statically culture the slow-growing Lactobacillus brunneri in a fermentation medium, wherein the culture conditions are static culture at 37°C for 3 days, the pH value is 7.0, and the inoculation amount of the slow-growing Lactobacillus brunneri is 2% (×10 7 cfu / mL).

[0086] 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.

[0087] Preferably, the obtained Lactobacillus buchneri zqw46 can be cultured in the following manner:

[0088] S4.1. Fermentation culture

[0089] Specifically, the obtained glycerol preservation tube of Lactobacillus buchneri zqw46 was dissolved and inoculated into 10 mL of MRS liquid culture medium at a 5% inoculation rate. After static culture at 37°C for 24 hours, it was inoculated into 400 mL of fermentation medium containing 40 mL of ethyl acetate at a 2% inoculation rate for two-phase fermentation and continuous culture for 3 days.

[0090] Preferably, the detection method described in Example 2 can be used to obtain the content of 2,4-di-tert-butylphenol produced after fermentation culture to determine the production capacity of the above-mentioned Lactobacillus buchneri zqw46 in application.

[0091] Specifically, 20 mL of the upper organic phase was concentrated and evaporated to dryness, and then the volume was made up to 5 mL with chromatography-grade methanol, and the content of volatile substances was detected by GC-MS.

[0092] Furthermore, after continuous cultivation of Lactobacillus buchneri for 3 days, the production of 2,4-di-tert-butylphenol can reach 10.77 mg / L.

[0093] It should be noted that the above-mentioned specific embodiments are exemplary, and those skilled in the art can come up with various solutions inspired by the disclosure of the present invention, and these solutions also belong to the disclosure scope of the present invention and fall within the protection scope of the present invention. Those skilled in the art should understand that the present invention specification and its drawings are illustrative and do not constitute a limitation of the claims. The scope of protection of the present invention is defined by the claims and their equivalents. The present invention specification contains multiple inventive concepts, such as "preferably", "according to a preferred embodiment" or "optionally", all of which indicate that the corresponding paragraph discloses an independent concept, and the applicant reserves the right to file a divisional application based on each inventive concept. Throughout the text, the feature guided by "preferably" is only an optional method and should not be understood as a must-have setting. Therefore, the applicant reserves the right to abandon or delete the relevant preferred features at any time.

Claims

1. A 2,4-di-tert-butylphenol-producing Lactobacillus buchneri strain zqw46 (Lentilactobacillus buchneri), characterized in that: The deposit number is CGMCC NO.25142.

2. The Lactobacillus buchneri of claim 1, characterized in that The biological characteristics of the Lactobacillus buchneri are that the colonies are raised, white, round, and have neat edges.

3. A microbial agent, characterized in that: Contains the lactobacillus buchneri described in claim 1.

4. The bacterial agent according to claim 3, characterized in that The bacterial agent is a liquid bacterial agent or a solid bacterial agent containing one or more of live cells of Lactobacillus brunneri, dry cells of Lactobacillus brunneri obtained by freeze drying, and immobilized cells of Lactobacillus brunneri.

5. A freeze-dried bacterial powder, characterized in that: The freeze-dried bacterial powder is prepared by the following method: the Lactobacillus buchneri described in claim 1 is activated and inoculated into a culture medium for enrichment culture, the bacterial bodies are collected by centrifugation after the culture is completed, trehalose, sorbitol, and skim milk powder are added thereto as freeze-drying protective agents, the mixture is evenly mixed, and vacuum freeze-dried to obtain freeze-dried bacterial powder.

6. Use of the Lactobacillus buchneri described in claim 1 or 2, or the microbial agent described in claim 3 or 4, or the freeze-dried bacterial powder described in claim 5 in the fermentation production of 2,4-di-tert-butylphenol.

7. The use according to claim 6, characterized in that The Lactobacillus buchneri described in claim 1 or 2, or the microbial agent described in claim 3 or 4, or the freeze-dried bacterial powder described in claim 5 is added to the fermentation medium and statically cultured.

8. One or more uses of the Lactobacillus buchneri described in claim 1 or 2, or the microbial agent described in claim 3 or 4, or the freeze-dried bacterial powder described in claim 5 in the preparation of natural rubber and synthetic rubber antioxidants, plastic antioxidants, fuel stabilizers, ultraviolet absorbers, and pesticide and dye intermediates.

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