Bacillus velezensis and application thereof in co-production of 6-methyl-2-heptanone and 2, 4-di-tert-butylphenol

Abstract

The invention provides bacillus velezensis and application of the bacillus velezensis in co-production of 6-methyl-2-heptanone and 2, 4-di-tert-butylphenol, and belongs to the technical field of biological engineering. The bacillus velezensis is classified and named as bacillus velezensis LT-22, and is preserved in the China Center for Type Culture Collection (CCTCC), the preservation number is CCTCC NO: M2023779, and the preservation date is May 19, 2023. According to the bacillus velezensis, the 6-methyl-2-heptanone and the 2, 4-di-tert-butylphenol can be co-produced, and the concentrations of the synthesized 6-methyl-2-heptanone and the 2, 4-di-tert-butylphenol can reach 75.72 mg / L and 42.48 mg / L; compared with chemical synthesis, the method for preparing the 6-methyl-2-heptanone and the 2, 4-di-tert-butylphenol is low in production cost, simple to operate, high in safety, environment-friendly and suitable for industrial popularization.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and specifically relates to a strain of Bacillus Velezii and application thereof in the co-production of 6-methyl-2-heptanone and 2,4-di-tert-butylphenol. Background Art

[0002] 6-Methyl-2-heptanone (2-hexanone-6-methyl) is a 16 O, the structural formula is shown in the following formula (I). 6-methyl-2-heptanone is colorless with a special smell. It exists naturally in tobacco leaves and is harmless to the human body. In addition, 6-methyl-2-heptanone has ketone characteristics, low volatility and low solubility. It is a common organic solvent and is widely used in chemical experiments, industrial production and pharmaceuticals. The commonly used method for synthesizing 6-methyl-2-heptanone is chemical synthesis. The chemical synthesis method uses 6-methyl-3-heptene-2-one as a raw material for catalytic hydrogenation to obtain 6-methyl-2-heptanone. The whole process is cumbersome.

[0003]

[0004] (I) 2,4-Di-tert-butylphenol is an important intermediate of fine chemical products. Its structural formula is shown in the following formula (II). It is mainly used to prepare antioxidant 168, light stabilizer 120 or 2402 resin, etc. In addition, it is also used in synthetic fragrances, natural rubber, activator 460, ultraviolet absorber or pesticide emulsifier, etc.

[0005]

[0006] (II) At present, 2,4-di-tert-methylphenol is mostly synthesized by chemical methods using phenol and isobutylene as raw materials. However, the synthesis of isobutylene by chemical synthesis requires high equipment and instruments, which increases the production cost. In addition, the chemical synthesis method uses strong acid solutions such as sulfuric acid and hydrofluoric acid as catalysts, which have the disadvantages of strong corrosion and environmental pollution. At the same time, the ion exchange resin catalyst has the disadvantages of poor thermal stability and short life, which also increases the cost of chemical synthesis.

[0007] After searching, there is no prior art report on the co-production of 6-methyl-2-heptanone and 2,4-di-tert-butylphenol by biological methods. In order to solve the problems of high cost and safety risks in the separate chemical synthesis of the above compounds 6-methyl-2-heptanone and 2,4-di-tert-butylphenol, it is necessary to develop a method for producing 6-methyl-2-heptanone and 2,4-di-tert-butylphenol based on biological methods. Summary of the invention

[0008] The object of the present invention is to solve the above technical problems and provide a Velez subtilis strain for co-producing 6-methyl-2-heptanone and 2,4-di-tert-butylphenol; another object of the present invention is to provide the application of the Velez subtilis strain in the co-production of 6-methyl-2-heptanone and 2,4-di-tert-butylphenol.

[0009] To achieve the above object, the present invention adopts the following technical solutions: A strain of Bacillus velez for co-producing 6-methyl-2-heptanone and 2,4-di-tert-butylphenol, wherein the classification of the Bacillus velez is named Bacillus velezensis LT-22 has been deposited in China Center for Type Culture Collection with the deposit number CCTCC NO: M2023779 and the deposit date is May 19, 2023. The deposit address is Wuhan University, Wuhan, China, zip code: 430072.

[0010] This strain has the following properties: This strain is a rod-shaped, Gram-positive bacterium; and the VP test, starch hydrolysis test, gelatin liquefaction test, citrate test, catalase test, indole test and nitrate reduction test are all positive, and the H2S gas production test and methyl red test are negative.

[0011] The present invention has been found through research that Bacillus velezensis LT-22 can co-produce 6-methyl-2-heptanone and 2,4-di-tert-butylphenol, while other strains of the same species cannot produce these two compounds at the same time, and the production of the related compound 6-methyl-2-heptanone by other strains of the same species is much lower than that of this strain. Bacillus velezensis LT-22; therefore, this Bacillus velezensis LT-22 has the application prospect of co-production of 6-methyl-2-heptanone and 2,4-di-tert-butylphenol.

[0012] Based on this Bacillus velezensis The invention discloses the application of the above-mentioned Bacillus Velezii in the co-production of 6-methyl-2-heptanone and 2,4-di-tert-butylphenol.

[0013] Furthermore, the application is to inoculate the Bacillus Velez subtilis into a fermentation medium for aerobic culture to prepare 6-methyl-2-heptanone and 2,4-di-tert-butylphenol.

[0014] Furthermore, the fermentation medium comprises the following components: 10-50 g / L of carbon source, 4-20 g / L of nitrogen source, 0.3-1.5 g / L of metal salt, pH value 6.5-8.0, and the solvent is water.

[0015] Furthermore, the nitrogen source is an organic nitrogen source or an inorganic nitrogen source; the organic nitrogen source is any one or a combination of soy protein, soybean meal, soybean cake powder, peanut cake powder and urea; the inorganic nitrogen source is any one or a combination of sodium nitrate, magnesium nitrate, ammonium nitrate, ammonium sulfate and ammonium chloride.

[0016] Furthermore, the carbon source is any one or a combination of glycerol, maltose, glucose, lactose and sucrose.

[0017] Furthermore, the metal salt is one or a combination of magnesium sulfate, potassium dihydrogen phosphate, calcium chloride, ferrous sulfate and sodium chloride.

[0018] Furthermore, the components of the fermentation medium are as follows: 20-45 g / L lactose, 10-20 g / L soybean meal, 4-20 g / L ammonium nitrate, and 0.4-2.0 g / L magnesium sulfate, and the initial pH of the fermentation liquid is adjusted to 6.0-9.0 using aqueous ammonia.

[0019] Furthermore, the aerobic culture is shake flask culture or fermentation tank culture, and the culture conditions are: initial pH is 6.5-7.5, and the culture temperature is 22-38 ° C; when the aerobic culture is shake flask culture, the shake flask inoculation amount is 1-7% of the fermentation medium volume fraction, and the culture time is 10-24 h; when the aerobic culture is fermentation tank culture, the fermentation tank inoculation amount is 1-14% of the fermentation medium volume fraction, the fermentation method is batch feeding method, the ventilation ratio is 1.0-2.5 VVM, and the culture time is 20-48 h.

[0020] Furthermore, the batch feeding method is as follows: lactose is supplemented at the 12th hour of fermentation so that its mass volume concentration in the fermentation medium is 35 g / L.

[0021] The beneficial effects of the present invention are as follows: (1) The present invention discovered a strain of Bacillus velez, which is classified and named Bacillus velezensis LT-22, compared with the same strains, can co-produce 6-methyl-2-heptanone and 2,4-di-tert-butylphenol, and its 6-methyl-2-heptanone production is significantly higher than other similar strains, and has application prospects in the co-production of 6-methyl-2-heptanone and 2,4-di-tert-butylphenol.

[0022] (2) By further studying the composition and fermentation conditions of the fermentation medium, the present invention found that the concentration of 6-methyl-2-heptanone accumulated in the medium could reach up to 75.72 mg / L, and the concentration of 2,4-di-tert-butylphenol could reach up to 42.48 mg / L. Compared with traditional chemical synthesis, the co-production of 6-methyl-2-heptanone and 2,4-di-tert-butylphenol by Bacillus benvelez greatly reduces the production cost, and has the advantages of simple operation, strong safety and environmental friendliness, and has great prospects for industrial promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Bacillus velezensis LT-22 phylogenetic tree.

[0024] Figure 2 Carbon source type Bacillus velezensis Effect of LT-22 on the synthesis of 6-methyl-2-heptanone and 2,4-di-tert-butylphenol.

[0025] Figure 3 Carbon source concentration Bacillus velezensis Effect of LT-22 on the synthesis of 6-methyl-2-heptanone and 2,4-di-tert-butylphenol.

[0026] Figure 4 Types of organic nitrogen sources Bacillus velezensis Effect of LT-22 on the synthesis of 6-methyl-2-heptanone and 2,4-di-tert-butylphenol.

[0027] Figure 5 The concentration of organic nitrogen source Bacillus velezensis Effect of LT-22 on the synthesis of 6-methyl-2-heptanone and 2,4-di-tert-butylphenol.

[0028] Figure 6 Inorganic nitrogen source types Bacillus velezensis Effect of LT-22 on the synthesis of 6-methyl-2-heptanone and 2,4-di-tert-butylphenol.

[0029] Figure 7 Effect of inorganic nitrogen source concentration on Bacillus velezensis Effect of LT-22 on the synthesis of 6-methyl-2-heptanone and 2,4-di-tert-butylphenol.

[0030] Figure 8 Metal salt types Bacillus velezensis Effect of LT-22 on the synthesis of 6-methyl-2-heptanone and 2,4-di-tert-butylphenol.

[0031] Figure 9 The metal salt concentration Bacillus velezensis Effect of LT-22 on the synthesis of 6-methyl-2-heptanone and 2,4-di-tert-butylphenol.

[0032] Figure 10 Temperature Bacillus velezensis Effect of LT-22 on the synthesis of 6-methyl-2-heptanone and 2,4-di-tert-butylphenol.

[0033] Figure 11 pH Bacillus velezensis Effect of LT-22 on the synthesis of 6-methyl-2-heptanone and 2,4-di-tert-butylphenol.

[0034] Figure 12 Fermentation curves of 6-methyl-2-heptanone and 2,4-di-tert-butylphenol synthesized in a 5L fermenter with batch feeding time. DETAILED DESCRIPTION

[0035] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0036] Unless otherwise specified, the reagents or drugs in the embodiments of the present invention are commercially available.

[0037] Example 1 This embodiment is aimed at Bacillus velezensis The isolation and screening of LT-22 and the determination of its morphological characteristics and physiological and biochemical properties are as follows: (1) Experimental method: 10 g of each of the 38 soils was added to a flask containing 90 mL of sterile water and incubated at 30°C and 200 rpm for 1 h. The flask was placed in a constant temperature water bath at 80°C for 30 min. After the water bath, the flask was placed in a clean bench for 5 min, and then the soil was diluted with sterile water to a concentration of 10 -4 , 10 -5 100 uL of soil dilution was spread on the solid enrichment medium, and the culture dish was inverted in a 30 ℃ constant temperature incubator for 48 h. After observation, a single colony was picked for full streaking and inoculated into the fermentation medium for 16 h to test the strains that can produce 6-methyl-2-heptanone and 2,4-di-tert-butylphenol, and the screened strains were cultured to observe and test their physiological and biochemical characteristics.

[0038] The fermentation medium composition is: glucose 5 g / L, trypsin 10 g / L, sodium chloride 10 g / L.

[0039] The composition of the solid enrichment medium is: lactose 5 g / L, tryptone 10 g / L, glucose 10 g / L, agar 20 g / L, and the solvent is water.

[0040] (2) Experimental results: Through the above screening experiment, a strain that can produce 6-methyl-2-heptanone and 2,4-di-tert-butylphenol was screened out. The screened strain was cultured, the colony morphology was observed, and its physiological and biochemical characteristics were tested. The test results are shown in Table 1.

[0041] Table 1 Morphological characteristics and physiological and biochemical properties of bacterial colonies

[0042] Note: “+” indicates positive, “-” indicates negative.

[0043] Example 2 This example identifies and preserves the selected strains, as follows: (1) Experimental method: The genomic DNA of the strain screened in the embodiment was extracted using a bacterial genomic DNA extraction kit, and the upstream primer 27F and the downstream primer 1492R were used for gene amplification. The extracted genomic DNA was used as a template for PCR, and the PCR amplification product was gel-recovered and purified. The gel-recovered and purified product was transferred to Suzhou Jinweizhi Biotechnology Co., Ltd. for sequencing. The nucleotide sequence length of the 16S rRNA gene of the strain obtained by sequencing was 1429 bp, and its gene sequence is shown in SEQ ID No. 1. The 16S rRNA sequence of the strain of the present invention was compared with the 16S rRNA sequence of the strain included in the GenBank database for nucleotide sequence homology, and the phylogenetic tree was drawn using the software MEGA 6.0, and the identified strain was preserved.

[0044] (2) Experimental results: The phylogenetic tree of this strain is as follows Figure 1 As shown, this strain Bacillus velezensis The homology similarity of YH21 reached up to 92%. Combined with the morphological characteristics and the results of physiological and biochemical experiments, strain LT-22 was identified as Bacillus velez. Bacillus velezensis , which has been deposited in the China Center for Type Culture Collection and named Bacillus velezensis LT-22, the deposit number is CCTCC NO: M2023779, and the deposit date is May 19, 2023. The deposit address is Wuhan University, Wuhan, China, Postal Code: 430072.

[0045] Example 3 This example is aimed at the strains screened Bacillus velezensisLT-22, the effect of carbon source type on its products 6-methyl-2-heptanone and 2,4-di-tert-butylphenol is studied as follows: (1) Experimental method: Bacillus velezensis The LT-22 seed culture fluid was inoculated at an inoculum of 3.5% (v / v) into fermentation medium with glycerol, fructose, glucose, lactose and sucrose as the sole carbon source, respectively, and the concentration of each sole carbon source in the medium was 30 g / L. The fermentation medium referred to Example 1, except for the type of carbon source. The pH value was 7.0, and the culture was shaken at 30 °C and 200 rpm. The fermentation medium was filled in 100 mL / 500 mL triangular shake flask, and the fermentation culture was carried out for 24 h. After the culture, the yields of 6-methyl-2-heptanone and 2,4-di-tert-butylphenol in the production stage were measured by mass spectrometry.

[0046] (2) Experimental results: The experimental results are as follows Figure 2 As shown by Figure 2 It can be seen that when lactose is used as the carbon source, the yield of 6-methyl-2-heptanone and 2,4-di-tert-butylphenol is the highest. The measured results show that the contents of 6-methyl-2-heptanone and 2,4-di-tert-butylphenol are 39.74 mg / L and 16.43 mg / L, respectively. Therefore, lactose is selected for subsequent fermentation.

[0047] Note: The following Examples 4-13 were optimized with lactose as the carbon source, and the experiments were conducted with 6-methyl-2-heptanone and 2,4-di-tert-butylphenol as the main products.

[0048] Example 4 This example is aimed at the strains screened Bacillus velezensis LT-22, the effect of carbon source concentration on its products 6-methyl-2-heptanone and 2,4-di-tert-butylphenol was studied as follows: (1) Experimental method: Bacillus velezensis The LT-22 seed culture fluid was inoculated at an inoculum size of 3.5% (v / v) into fermentation medium with lactose concentrations of 20 g / L, 25 g / L, 30 g / L, 35 g / L, 40 g / L, and 45 g / L, respectively. The pH value was 7.0. The culture was shaken at 30 °C and 200 rpm. The fermentation medium was filled with 100 mL / 500 mL triangular shake flask. The fermentation culture was carried out for 24 h. After the culture, the yields of 6-methyl-2-heptanone and 2,4-di-tert-butylphenol in the production stage were measured by mass spectrometry.

[0049] (2) Experimental results: Figure 3As shown, when the concentration of lactose in the fermentation medium was 35 g / L, the yields of 6-methyl-2-heptanone and 2,4-di-tert-butylphenol were the highest, and the measured results of the yields of 6-methyl-2-heptanone and 2,4-di-tert-butylphenol were 45.23 mg / L and 18.72 mg / L, respectively.

[0050] Example 5 This example is aimed at the strains screened Bacillus velezensis LT-22, study the effect of organic nitrogen source type on its products 6-methyl-2-heptanone and 2,4-di-tert-butylphenol, as follows: (1) Experimental method: Bacillus velezensis The LT-22 seed culture fluid was inoculated at an inoculum rate of 3.5% (v / v) into fermentation media with soybean protein, soybean meal, soybean cake powder, peanut cake powder and urea as the sole organic nitrogen source, respectively. The concentration of each sole organic nitrogen source in the fermentation medium was 14 g / L, and the pH value was 7.0. The culture was shaken at 30 °C and 200 rpm. The fermentation medium was filled with 100 mL / 500 mL triangular shake flask for 24 h. After the culture, the yields of 6-methyl-2-heptanone and 2,4-di-tert-butylphenol in the production stage were measured by mass spectrometry.

[0051] (2) Experimental results: Figure 4 As shown in the figure, when soybean meal is used as the organic nitrogen source, the yield of 6-methyl-2-heptanone and 2,4-di-tert-butylphenol is the highest, and the measured results show that the contents of 6-methyl-2-heptanone and 2,4-di-tert-butylphenol are 46.53 mg / L and 19.68 mg / L, respectively. Therefore, soybean meal was selected for subsequent fermentation.

[0052] Example 6 This example is aimed at the strains screened Bacillus velezensis LT-22, study the effect of organic nitrogen source concentration on its products 6-methyl-2-heptanone and 2,4-di-tert-butylphenol, as follows: (1) Experimental method: Bacillus velezensis The LT-22 seed culture fluid was inoculated at an inoculum rate of 3.5% (v / v) into fermentation medium with soybean meal concentrations of 10 g / L, 12 g / L, 14 g / L, 16 g / L, 18 g / L, and 20 g / L, respectively. The pH value was 7.0. The culture was shaken at 30 °C and 200 rpm. The fermentation medium was filled with 100 mL / 500 mL triangular shake flask. The fermentation culture was carried out for 24 h. After the culture, the yields of 6-methyl-2-heptanone and 2,4-di-tert-butylphenol in the production stage were measured by mass spectrometry.

[0053] (2) Experimental results: Figure 5As shown, when the concentration of soybean meal in the fermentation medium is 16 g / L, the yields of 6-methyl-2-heptanone and 2,4-di-tert-butylphenol are the highest. The measured results show that the contents of 6-methyl-2-heptanone and 2,4-di-tert-butylphenol are 48.62 mg / L and 20.94 mg / L, respectively. Therefore, 16 g / L soybean meal is selected for subsequent fermentation.

[0054] Example 7 This example is aimed at the strains screened Bacillus velezensis LT-22, study the effect of inorganic nitrogen source type on its products 6-methyl-2-heptanone and 2,4-di-tert-butylphenol, as follows: (1) Experimental method: Bacillus velezensis The LT-22 seed culture fluid was inoculated at an inoculum size of 3.5% (v / v) into fermentation media with sodium nitrate, magnesium nitrate, ammonium nitrate, ammonium sulfate, and ammonium chloride as the sole inorganic nitrogen source, respectively. The concentration of each sole inorganic nitrogen source in the fermentation medium was 4 g / L, and the pH value was 7.0. The culture was shaken at 30 °C and 200 rpm. The fermentation medium was filled with 100 mL / 500 mL triangular shake flask for 24 h. After the culture, the yields of 6-methyl-2-heptanone and 2,4-di-tert-butylphenol in the production stage were measured by mass spectrometry.

[0055] (2) Experimental results: Figure 6 As shown, when ammonium nitrate is used as the inorganic nitrogen source, the yields of 6-methyl-2-heptanone and 2,4-di-tert-butylphenol are the highest. The measured results show that the contents of 6-methyl-2-heptanone and 2,4-di-tert-butylphenol are 49.12 mg / L and 21.76 mg / L, respectively. Therefore, ammonium nitrate is selected for subsequent fermentation.

[0056] Example 8 This example is aimed at the strains screened Bacillus velezensis LT-22, the effect of ammonium nitrate concentration on its products 6-methyl-2-heptanone and 2,4-di-tert-butylphenol was studied as follows: (1) Experimental method: Bacillus velezensis LT-22 seed culture fluid was inoculated at an inoculum size of 3.5% (v / v) into fermentation medium with ammonium nitrate concentrations of 4 g / L, 8 g / L, 12 g / L, 16 g / L, and 20 g / L, respectively. The initial pH value was 7.0. The culture was shaken at 30 °C and 200 rpm. The fermentation medium volume was 100 mL / 500 mL triangular shake flask, and the fermentation culture was carried out for 24 h. After the culture, mass spectrometry was used to measure the production stage of 6-methyl-2-heptanone and 2,4-di-tert-butylphenol.

[0057] (2) Experimental results:Figure 7 As shown in the figure, when the concentration of ammonium nitrate is 8 g / L, the yield of 6-methyl-2-heptanone and 2,4-di-tert-butylphenol is the highest. The measured results show that the contents of 6-methyl-2-heptanone and 2,4-di-tert-butylphenol are 52.43 mg / L and 24.68 mg / L, respectively. Therefore, 8 g / L ammonium nitrate is selected for subsequent fermentation.

[0058] Example 9 This example is aimed at the strains screened Bacillus velezensis LT-22, study the effect of metal salt types on the products 6-methyl-2-heptanone and 2,4-di-tert-butylphenol, as follows: (1) Experimental method: Bacillus velezensis The LT-22 seed culture fluid was inoculated at an inoculum of 3.5% (v / v) into fermentation medium containing magnesium sulfate, potassium hydrogen phosphate, calcium chloride, ferrous sulfate, and sodium chloride as the sole metal salt, respectively, and the concentration of each sole metal salt in the fermentation medium was 0.5 g / L. The control group was a fermentation medium without adding metal salts, with an initial pH of 7.0, and cultured at 30 °C, 200 rpm shaking, and the fermentation medium was filled with 100 mL / 500 mL triangular shake flask for 24 h. After the culture was completed, the yields of 6-methyl-2-heptanone and 2,4-di-tert-butylphenol in the production stage were measured by mass spectrometry.

[0059] (2) Experimental results: Figure 8 As shown in the figure, when magnesium sulfate is used as the metal salt, the yield of 6-methyl-2-heptanone and 2,4-di-tert-butylphenol is the highest, and the measured results show that the contents of 6-methyl-2-heptanone and 2,4-di-tert-butylphenol are 54.38 mg / L and 27.46 mg / L, respectively. Therefore, magnesium sulfate is selected as the most suitable metal salt.

[0060] Example 10 This example is aimed at the strains screened Bacillus velezensis LT-22, the effect of metal salt concentration on its products 6-methyl-2-heptanone and 2,4-di-tert-butylphenol was studied as follows: (1) Experimental method: Bacillus velezensis The LT-22 seed culture fluid was inoculated at an inoculum size of 3.5% (v / v) into fermentation medium with the concentration of potassium hydrogen phosphate in the fermentation medium being 0.4 g / L, 0.5 g / L, 0.6 g / L, 0.7 g / L, and 0.8 g / L, respectively. The initial pH value was 7.0. The culture was shaken at 30 °C and 200 rpm. The fermentation medium volume was 100 mL / 500 mL triangular shake flask. The fermentation culture was carried out for 24 h. After the culture was completed, the yields of 6-methyl-2-heptanone and 2,4-di-tert-butylphenol in the production stage were measured by mass spectrometry.

[0061] (2) Experimental results: Figure 9 As shown in the figure, when the concentration of magnesium sulfate in the fermentation medium is 0.6 g / L, the yield of 6-methyl-2-heptanone and 2,4-di-tert-butylphenol is the highest. The measured results show that the contents of 6-methyl-2-heptanone and 2,4-di-tert-butylphenol are 56.24 mg / L and 29.18 mg / L, respectively. Therefore, 0.6 g / L magnesium sulfate is selected as the optimal metal salt concentration.

[0062] Embodiment 11 This example is aimed at the strains screened Bacillus velezensis LT-22, study the effect of temperature on its products 6-methyl-2-heptanone and 2,4-di-tert-butylphenol, as follows: (1) Experimental method: Bacillus velezensis The inoculation amount of LT-22 seed culture solution was 3.5% (v / v) inoculated into the fermentation medium, the initial pH value was 7.0, the shaking culture was carried out at 200 rpm, the culture temperature was 28 °C, 30 °C, 32 °C, 34 °C, 36 °C and 38 °C, the fermentation medium volume was 100 mL / 500 mL triangular shake flask, and the fermentation culture was carried out for 24 h. The fermentation broth at each temperature was taken and the yield of 6-methyl-2-heptanone and 2,4-di-tert-butylphenol in the production stage was measured by mass spectrometry.

[0063] (2) Experimental results: Figure 10 As shown in the results, the contents of 6-methyl-2-heptanone and 2,4-di-tert-butylphenol were 61.55 mg / L and 32.43 mg / L, respectively. When the temperature was 34 °C, the yield of 6-methyl-2-heptanone and 2,4-di-tert-butylphenol was the highest. Therefore, 34 °C was selected as the optimal fermentation temperature.

[0064] Example 12 This example is aimed at the strains screened Bacillus velezensis LT-22, study the effect of pH on its products 6-methyl-2-heptanone and 2,4-di-tert-butylphenol, as follows: (1) Experimental method: Bacillus velezensis The LT-22 seed culture fluid was inoculated into the fermentation medium at an inoculum size of 3.5% (v / v) and cultured at 34 °C with shaking at 200 rpm. The culture medium filling volume was 100 mL / 500 mL triangular shake flask. The culture medium was cultured at fermentation medium with pH values ​​of 6.7, 7.0, 7.3, 7.6 and 7.9, respectively. The fermentation broth at each pH was taken and mass spectrometry was used to measure the production stage of 6-methyl-2-heptanone and 2,4-di-tert-butylphenol.

[0065] (2) Experimental results:Figure 11 As shown in the results, the contents of 6-methyl-2-heptanone and 2,4-di-tert-butylphenol were 63.22 mg / L and 34.98 mg / L, respectively. When the pH value was 7.3, the yields of 6-methyl-2-heptanone and 2,4-di-tert-butylphenol were the highest. Therefore, the pH value of 7.7 was selected as the optimal fermentation pH.

[0066] Example 13 This example is aimed at the strains screened Bacillus velezensis LT-22, the effect of fed-batch feeding in a 5 L fermenter on the products 6-methyl-2-heptanone and 2,4-di-tert-butylphenol was studied as follows: (1) Experimental method: Use an inoculation loop to pick up Bacillus Velezii from a glycerol tube stored in a -80 ℃ refrigerator. Bacillus velezensis LT-22 strains were inoculated in LB solid medium, and colonies were selected from the primary screening medium plate and inoculated into the seed medium for shaking culture at 34 °C for 16-18 h, and then transferred to the fermentation medium (lactose 15 g / L, ammonium nitrate 8 g / L, soybean meal 16 g / L, magnesium sulfate 0.6 g / L, and the initial pH of the fermentation liquid was adjusted to 7.3 with ammonia water) at a ratio of 8%. The total liquid volume of the fermenter was 3 L, the fermentation temperature was 34 °C, the stirring speed was 200 rpm, and the ventilation volume was 1.5 VVM for fermentation; the initial pH value of the fermentation was 7.3, and the pH automatic control device was turned on during the fermentation process, and the pH value of the fermentation liquid was controlled at about 7.3 with ammonia water or hydrochloric acid; the fermentation time was 36 h, and the contents of 6-methyl-2-heptanone and 2,4-di-tert-butylphenol in the fermentation liquid were measured every 4 h. Lactose was supplemented at the 12th hour of fermentation to make its mass volume concentration in the fermentation medium 35 g / L.

[0067] Adopting the Bacillus Velezii disclosed in the Chinese invention patent with publication number CN112143664A Bacillus velezensis As a comparison, LT-2 was cultured under the same culture medium conditions without lactose supplementation to test the content of 6-methyl-2-heptanone and 2,4-di-tert-butylphenol; and according to the above experimental method, only lactose was added to Bacillus velezensis LT-22 replaced by Bacillus velezensis LT-2, testing the content of 6-methyl-2-heptanone and 2,4-di-tert-butylphenol.

[0068] (2) Experimental results: Figure 12 As shown, the 6-methyl-2-heptanone and yield in the production stage were measured, and the measured results were 75.72 mg / L and 42.48 mg / L, respectively. Bacillus velezensis LT-2 was not supplemented with lactose and Bacillus velezensisUnder the same culture conditions as LT-22, the yield of synthesized 6-methyl-2-heptanone was 14.98 mg / ml, and 2,4-di-tert-butylphenol could not be synthesized. Bacillus velezensis The final yield of 6-methyl-2-heptanone synthesized by LT-2 was 25.38 mg / ml, which was much lower than that of the strain of the present invention. Bacillus velezensis LT-22.

[0069] Note: The fermentation medium in this example is an optimized medium, and the carbon source is supplemented, so a higher yield of 6-methyl-2-heptanone and 2,4-di-tert-butylphenol is obtained.

[0070] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the solution. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents based on the understanding of the present solution without departing from the purpose and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. Application of Bacillus Velez in the co-production of 6-methyl-2-heptanone and 2,4-di-tert-butylphenol, characterized in that: The classification of the Bacillus Velez Bacillus velezensis LT-22 has been deposited in the China Center for Type Culture Collection, with the deposit number being CCTCC NO: M2023779 and the deposit date being May 19, 2023.

2. The use according to claim 1, characterized in that: The Velez Bacillus is inoculated into a fermentation medium for aerobic culture to prepare 6-methyl-2-heptanone and 2,4-di-tert-butylphenol.

3. The use according to claim 2, characterized in that: The fermentation medium comprises the following components: 10-50 g / L of carbon source, 4-20 g / L of nitrogen source, 0.3-1.5 g / L of metal salt, pH value of 6.5-8.0, and the solvent is water.

4. The use according to claim 3, characterized in that: The nitrogen source is an organic nitrogen source or an inorganic nitrogen source; The organic nitrogen source is any one or a combination of soybean protein, soybean meal, soybean cake powder, peanut cake powder and urea; The inorganic nitrogen source is any one or a combination of sodium nitrate, magnesium nitrate, ammonium nitrate, ammonium sulfate and ammonium chloride.

5. The use according to claim 3, characterized in that: The carbon source is any one of glycerol, maltose, glucose, lactose and sucrose or a combination of several of them.

6. The use according to claim 3, characterized in that: The metal salt is one or a combination of magnesium sulfate, potassium dihydrogen phosphate, calcium chloride, ferrous sulfate and sodium chloride.

7. The use according to claim 3, characterized in that: The components of the fermentation medium are as follows: 20-45 g / L lactose, 10-20 g / L soybean meal, 4-20 g / L ammonium nitrate, and 0.4-2.0 g / L magnesium sulfate. The initial pH of the fermentation liquid is adjusted to 6.0-9.0 using ammonia water.

8. The use according to claim 2, characterized in that: The aerobic culture is shake flask culture or fermentation tank culture, and the culture conditions are: initial pH 6.5-7.5, culture temperature 22-38 ° C; When the aerobic culture is shake flask culture, the shake flask inoculation amount is 1-7% by volume of the fermentation medium, and the culture time is 10-24 h; When the aerobic culture is fermentation tank culture, the fermentation tank inoculation amount is 1-14% by volume fraction of the fermentation medium, the fermentation method is batch feeding method, the ventilation ratio is 1.0-2.5 VVM, and the culture time is 20-48 h.

9. The use according to claim 8, characterized in that: The batch feeding method is as follows: lactose is supplemented at the 12th hour of fermentation so that its mass volume concentration in the fermentation medium is 35 g / L.

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