Endophytic bacterium for producing isoamyl alcohol and 3-hydroxy-2-butanone and application of endophytic bacterium

Abstract

The invention belongs to the field of microorganisms, and particularly relates to an endophytic bacterium for producing isoamyl alcohol and 3-hydroxy-2-butanone and application of the endophytic bacterium. The name of the endophytic bacterium is Raoultella planticola HuaYu-2, the preservation number of the endophytic bacterium is GDMCC No: 65269, and the endophytic bacterium is preserved in the Guangdong Microbial Culture Collection Center on October 15, 2024. The endophytic bacteria are separated from perfume lemons and can generate endophytic bacteria strains of aroma compounds isoamyl alcohol and 3-hydroxy-2-butanone through fermentation, on one hand, a strain library for fermentation production of perfume compounds is enriched, and on the other hand, a certain basis is provided for screening aroma-producing microorganisms from perfume plants.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms, and particularly relates to an endophytic bacterium producing isopentanol and 3-hydroxy-2-butanone and application thereof. Background Art

[0002] Spices can be divided into natural spices and synthetic spices according to their raw material sources and production processes. Synthetic spices are produced by chemical synthesis based on petrochemical raw materials. Therefore, environmental pollution problems will inevitably occur during the production process, which is not in line with the concept of sustainable development. With the pursuit of high-quality development models by society and government departments, the production model of chemical synthetic spices is gradually unpopular; at the same time, since the price of petrochemical raw materials is greatly affected by oil prices, international crude oil price fluctuations will eventually lead to uncontrollable spice production costs. Natural spice plant resources have strong regional characteristics. Natural spice plant resources are mainly distributed in different regional climate zones, and my country's climate zone distribution has obvious regional environmental characteristics. Therefore, spice crops produced in different regions have obvious local differences; and many growers currently have problems such as traditional planting methods, primitive production models, and low production levels, which makes it difficult to effectively utilize natural spice plant resources.

[0003] In 1985, the U.S. Food and Drug Administration (FDA) defined flavors produced from natural raw materials through fermentation technology or enzyme technology as natural equivalent flavors. The production of flavor compounds through microbial fermentation is in line with the concept of sustainable development of green environmental protection, and is not restricted by the regional problems of traditional natural flavor plant resources. Therefore, it is becoming an increasingly important way to produce flavors. At present, the methods used by researchers to screen aroma-producing microorganisms mainly include soil environment, water environment, fermentation environment, etc., and there are problems such as too complex microbial communities and too low screening efficiency when screening aroma-producing microorganisms through these methods; therefore, it is necessary to develop new methods for screening aroma-producing microorganisms.

[0004] Plant endophytes refer to fungi or bacteria that live inside the tissues and organs of plants at certain stages or all stages. Endophytes are commonly found in higher plants, including woody plants, herbaceous plants, monocots and dicots. To date, more and more studies have shown that endophytes in spice plants can often produce aroma compounds, and secondary metabolites of endophytes in plants have gradually become a new source of spice compounds.

[0005] Isoamyl alcohol has apple brandy aroma and spicy taste and is a permitted food flavoring in GB 2760-86. 3-Hydroxy-2-butanone can be used as a flavor enhancer for cream, cheese, coffee, and fruit. It is used as a flavoring for cream, dairy products, yogurt, and strawberry. It is also widely used in tobacco, medicine, etc. It has a very high application value and is a permitted food flavoring in my country's GB2760-86. At present, these two aroma compounds are mainly produced by chemical synthesis, which has industry problems such as environmental pollution.

[0006] If aroma-producing microorganisms can be screened out from spice plants, on the one hand, the difficulties in the production process of synthetic spices and natural plant spices can be avoided, and on the other hand, a certain theoretical basis can be provided for the development of new methods for screening aroma-producing microorganisms. Summary of the invention

[0007] The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide an endophytic bacterium that produces isopentanol and 3-hydroxy-2-butanone.

[0008] Another object of the present invention is to provide the application of the above-mentioned endophytic bacteria producing isopentanol and 3-hydroxy-2-butanone.

[0009] The purpose of the present invention is achieved by the following technical scheme: an endophytic bacterium producing isopentanol and 3-hydroxy-2-butanone, named Raoultella planticola HuaYu-2, with a preservation number of GDMCC No: 65269, was preserved on October 15, 2024 in the Guangdong Microbial Culture Collection Center of the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, Compound 100, Xianlie Middle Road, Guangzhou.

[0010] The use of the above-mentioned endophytic bacteria producing isoamyl alcohol and 3-hydroxy-2-butanone in producing isoamyl alcohol and 3-hydroxy-2-butanone preferably comprises the following steps: inoculating the above-mentioned endophytic bacteria producing isoamyl alcohol and 3-hydroxy-2-butanone into a potato glucose water culture medium for fermentation culture to obtain a fermentation liquid containing isoamyl alcohol and 3-hydroxy-2-butanone; more preferably comprises the following steps: activating and amplifying the above-mentioned endophytic bacteria producing isoamyl alcohol and 3-hydroxy-2-butanone and then inoculating them into a potato glucose water culture medium for fermentation culture to obtain a fermentation liquid containing isoamyl alcohol and 3-hydroxy-2-butanone.

[0011] The fermentation culture conditions are preferably: temperature 30-37°C, rotation speed 100-250 rpm; more preferably, temperature 35-37°C, rotation speed 150-250 rpm.

[0012] The fermentation culture time is preferably 24 to 48 hours.

[0013] The activation step is preferably as follows: streak the endophytic bacteria of isopentanol and 3-hydroxy-2-butanone onto LB solid culture medium and culture them at 30-37° C. in an aerobic environment for 16 to 32 hours.

[0014] The conditions for the amplification culture are preferably as follows: the activated bacteria are inoculated into LB liquid culture medium and cultured in an aerobic environment at 30-37°C and 150-250rpm until OD 600 Values ​​over 0.5.

[0015] The inoculation is preferably carried out by inoculating the bacterial suspension obtained by amplification culture at 1-3% of the volume of the fermentation medium.

[0016] Compared with the prior art, the present invention has the following advantages and effects:

[0017] The present invention screens out an endophytic bacterial strain that can produce aroma compounds isoamyl alcohol and 3-hydroxy-2-butanone by fermentation from the spice plant lemon, which enriches the strain library for fermentation production of aroma compounds on the one hand, and provides a certain basis for screening aroma-producing microorganisms from spice plants on the other hand. Both isoamyl alcohol and 3-hydroxy-2-butanone are edible spices allowed for use according to GB2760-86; therefore, the strain that can produce these two substances has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the colony morphology of strain HuaYu-2 on the LB plate.

[0019] Figure 2 This is a scanning electron microscope (SEM) image of strain HuaYu-2.

[0020] Figure 3 This is a growth curve of strain HuaYu-2.

[0021] Figure 4 This is the Gram staining effect of strain Raoultella planticola HuaYu-2.

[0022] Figure 5 This is the phylogenetic tree of strain Raoultella planticola HuaYu-2.

[0023] Figure 6 This is the total ion current graph of the fermentation broth of strain Raoultella planticola HuaYu-2 detected by GC-MS. DETAILED DESCRIPTION

[0024] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0025] Example 1: Screening of aroma-producing strains

[0026] Select perfume lemons with intact skin and obvious aroma, rinse the skin with running water, and then use clean filter paper to absorb the surface moisture for later use. In a sterile clean bench, first rinse the surface of the peel with sterile water, then soak it in a sodium hypochlorite solution with a concentration of 3% by mass for 3 minutes to sterilize and disinfect, then rinse it with sterile water 3 times; then soak it in an ethanol solution with a concentration of 75% by volume for 1 minute, then rinse it with sterile water 3 times; after rinsing, wipe the surface moisture with sterile filter paper. Cut a 2cm×2cm green outer peel with a sterile knife, place it on an LB solid culture medium, and culture it in an incubator at 30-37℃ for 3-7 days. After the colony grows, use an inoculation loop to streak and inoculate it into a new LB solid culture medium. After streaking and inoculating 3 times, separate and purify to obtain a single colony.

[0027] Pick a single colony and inoculate it into a potato glucose water liquid culture medium, culture it at 37°C for 24 hours, and if obvious aroma is observed by olfactory method, it can be determined that an aroma-producing strain has been screened.

[0028] The strain was named Hua Yu-2, and was streaked on LB solid medium and cultured at 37°C for 24 h. Figure 1 As shown: colonies with a diameter of about 2 mm are formed; the colonies are milky white, opaque, with a smooth and moist surface, regular edges, a convex center, and no halo.

[0029] Observed under a scanning electron microscope, Figure 2 As shown: A single bacterium is short rod-shaped, about 0.5 μm wide and 2 to 4 μm long, without spores, with capsules and no flagella.

[0030] The strain was purified and inoculated into a shake flask containing 50 mL of LB liquid medium, cultured at 37°C and 180 rpm, and the growth curve was observed. The results are as follows: Figure 3 As shown: the lag phase lasts about 4 hours, followed by the logarithmic growth phase, and the stable phase about 16 hours after inoculation.

[0031] Example 2: Physiological and biochemical experimental identification of strains:

[0032] (1) Gram staining test:

[0033] Pick a purified single colony of strain HuaYu-2 from the plate, smear and fix it on a glass slide; stain with ammonium oxalate crystal violet for 1 minute and then rinse with distilled water; cover the surface with iodine solution and stain for about 1 minute and then rinse with distilled water, and absorb the moisture with absorbent paper; add a few drops of 95% alcohol and shake gently for decolorization, wash with distilled water after 30 seconds, and absorb the moisture; stain with safranin stain solution for 1 minute and then rinse with distilled water; after absorbing the moisture, examine with an oil objective under an optical microscope.

[0034] like Figure 4 As shown: The Gram staining result of strain HuaYu-2 is red and negative.

[0035] (2) Citrate Utilization Experiment:

[0036] Add 1% of the volume of the culture medium, bromothymol blue indicator, to the citrate medium, pick out the colonies and inoculate them into the citrate medium, culture them at 37°C for 1 to 4 days, and observe the results.

[0037] The indicator changes from light green to dark blue, indicating that the strain HuaYu-2 can decompose citrate and make the culture medium alkaline. Therefore, the citrate utilization test is positive.

[0038] (3) Malonate Utilization Experiment:

[0039] Prepare malonate medium, pick out purified single colonies and inoculate them into 5 mL of malonate medium, culture at 37°C and 180 rpm for 24 to 48 hours, and observe the results.

[0040] The indicator changes from light green to dark blue, indicating that the strain HuaYu-2 can use malonate as a carbon source, decompose malonate to produce sodium carbonate, and make the culture medium alkaline, so the malonate utilization test is positive.

[0041] (4) Indole experiment:

[0042] Prepare Dunhen's peptone medium, pick a purified single colony and inoculate it into 5mL Dunhen's peptone medium, and culture it at 37℃ and 180rpm for 24-48h. Add 2-3mL of amyl alcohol or xylene to the culture medium, shake well, let it stand for a while, and then add 2mL of Ehrlich's indole reagent along the wall of the test tube. If the liquid below the amyl alcohol or xylene turns red, it is a positive reaction.

[0043] The indicator did not change color, indicating that the strain HuaYu-2 in the experiment could not decompose tryptophan in peptone to produce indole, and the result was negative.

[0044] (5) VP experiment:

[0045] Inoculate a purified single colony of strain HuaYu-2 into 5 mL of glucose-peptone water culture medium, and culture at 37°C and 180 rpm for 24 to 48 h. Add 1 mL of VP test solution A (6% α-naphthol alcohol solution) to the culture medium, shake well, then add 0.5 mL of VP test solution B (40% KOH solution), shake well, and observe the results.

[0046] The culture medium showed red color, indicating that the strain HuaYu-2 in the experiment could decompose glucose to produce pyruvate, and further decarboxylate pyruvate to form acetylmethylcarbinol. Acetylmethylcarbinol was oxidized to diacetyl in an alkaline environment, and then combined with the guanidine group contained in arginine and the like in the culture medium to form a red compound. Therefore, the VP test was a positive result.

[0047] (6) Lysine decarboxylase assay:

[0048] Prepare lysine decarboxylase broth medium, add 1% bromocresol purple as an indicator in the volume of the medium, pick a purified single colony and inoculate it into 5 mL of lysine decarboxylase broth medium, culture at 37° C. and 180 rpm for 18-24 h, and observe the results.

[0049] The indicator developed color, indicating that the strain HuaYu-2 can produce decarboxylase to remove the carboxyl group of lysine, generate amines and CO2, and increase the pH of the culture medium, so that the indicator develops color, and the result is positive.

[0050] (7) Ornithine decarboxylase assay:

[0051] Prepare ornithine decarboxylase broth medium, pick out purified single colonies and inoculate them into 5 mL of lysine decarboxylase medium, culture at 37°C and 180 rpm for 18 to 24 hours, and observe the results.

[0052] In the experiment, the strain HuaYu-2 could not produce ornithine decarboxylase, the indicator did not change color, and the test result was negative.

[0053] (8) Phenylalanine deaminase test:

[0054] Pick out a purified single colony, inoculate it into 5 mL of phenylalanine medium, culture it at 37°C and 180 rpm for 18 to 24 hours, add 4 to 5 drops of ferric chloride solution, shake and mix, and observe the results.

[0055] In the experiment, the strain HuaYu-2 could not produce phenylalanine deaminase, the ferric chloride indicator did not change color, and the test result was negative.

[0056] Table 1 Experimental results of physiological and biochemical characteristics of aroma-producing strains

[0057]

[0058] Example 3: Molecular Biological Identification of Strain:

[0059] DNA was extracted from strain HuaYu-2, and 16s rDNA was amplified using bacterial universal primer pair 27F / 1492R. The PCR reaction conditions were: 94℃3min; 94℃40s, 56℃35s, 72℃80s, 30 cycles; 72℃10min. After good results were identified by 1% agarose gel electrophoresis, the amplified product was sent for sequencing, and the results were compared by BLAST sequence comparison on the NCBI website; combined with physiological and biochemical identification experiments, the strain was confirmed to be Raoultella planticola, and the phylogenetic tree constructed is shown in the figure. Figure 5 shown.

[0060] The 16s rDNA sequence of strain Raoultella planticola HuaYu-2 is as follows:

[0061] AGCTACCTACTTCTTTTGCAACCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCG

[0062] TAGCATTCTGATCTACGATTACTAGCGATTCCGACTTCATGGAGTCGAGTTTGCAGACTCCAATCCGGACTACGACATACT

[0063] TTATGAGGTCCGCTTGCTCTCGCGAGGTCGCTTCTCTTTGTATATGCCATTGTAGCACGTGTGTAGCCCTACTCGTAAGG

[0064] GCCATGATGACTTGACGTCATCCCCACCTTCCTCCAGTTTATCACTGGCAGTCTCCTTTGAGTTCCCGACCGAATCGCTG

[0065] GCAACAAAGGATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATTTCACAACACGAGCTGACGACAGCCATGCAGCA

[0066] CCTGTCTCAGAGTTCCCGAAGGCACCAAAGCATCTCTGCTAAGTTCTCTGGATGTCAAGAGTAGGTAAGGTTCTTCGCGT

[0067] TGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCATTTGAGTTTTAACCTTGCGGCCGTACT

[0068] CCCCAGGCGGTCGACTTAACGCGTTAGCTCCGGAAGCCACTCCTCAAGGGAACAACCTCCAAGTCGACATCGTTTACAGC

[0069] GTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCACCTGAGCGTCAGTCTTTGTCCAGGGGGCCGCCT

[0070] TCGCCACCGGTATTCCTCCAGATCTCTACGCATTTCACCGCTACACCTGGAATTCTACCCCCCTCTACAAGACTCAAGCC

[0071] TGCCAGTTTCAAATGCAGTTCCCAGGTTGAGCCCGGGGATTTCACATCTGACTTAACAGACCGCCTGCGTGCGCTTTACG

[0072] CCCAGTAATTCCGATTAACGCTTGCACCCTCCGTATTACCGCGGCTGCTGGCACGGAGTTAGCCGGTGCTTCTTCTGCGA

[0073] GTAACGTCAATCGACAAGGTTATTAACCTCAATGCCTTCCTCCTCGCTGAAAGTACTTTACAACCCGAAGGCCTTCTTCA

[0074] TACACGCGGCATGGCTGCATCAGGCTTGCGCCCATTGTGCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGACCGT

[0075] GTCTCAGTTCCAGTGTGGCTGGTCATCCTCTCAGACCAGCTAGGGATCGTCGCCTAGGTGAGCCATTACCCCACCTACTA

[0076] GCTAATCCCATCTGGGCACATCTGATGGCATGAGGCCCGAAGGTCCCCCACTTTGGTCTTGCGACGTTATGCGGTATTAG

[0077] CTACCGTTTCCAGTAGTTATCCCCTCCATCAGGCAGTTTCCCAGACATTACTCACCCGTCCGCCACTCGTCACCCGAGA

[0078] GCAAGCTCTCTGTGCTACCGTTCGACTGC.

[0079] The strain was deposited in the Guangdong Provincial Microbiological Culture Collection Center on October 15, 2024, with the collection number GDMCCNo: 65269.

[0080] Example 4: Production of isoamyl alcohol and 3-hydroxy-2-butanone by microbial fermentation of aroma-producing strain HuaYu-2

[0081] The aroma-producing strain HuaYu-2 was streaked onto LB solid medium and cultured in an aerobic environment at 37°C for 24 h until the colony diameter exceeded 2 mm.

[0082] The colonies that grew to a suitable size were inoculated into a shaking tube containing 4 mL of LB liquid medium and cultured in an aerobic environment at 37°C and 200 rpm for 24 h until the OD of the bacterial suspension at a wavelength of 600 nm was 600 Values ​​over 0.5.

[0083] The bacterial suspension reaching a certain concentration was inoculated into a shake flask containing 200 mL of potato glucose water culture medium at a volume ratio of 1%, and cultured for 36 hours at 37° C. and 120 rpm in an aerobic environment to finally obtain a fermentation broth containing isopentanol and 3-hydroxy-2-butanone.

[0084] The formula of LB solid medium is: tryptone 10g·L -1 , yeast extract 5g·L -1 、NaCl 10g·L -1 , agar 20 g·L -1 , pH is 7.0±0.2; the solvent is deionized water.

[0085] The formula of LB liquid culture medium is: tryptone 10g·L -1 , yeast extract 5g·L -1 、NaCl 10g·L -1 , agar 20 g·L -1 , pH is 7.0±0.2; the solvent is deionized water.

[0086] The formula of potato glucose water culture medium is: potato extract powder 200g·L -1 , Glucose 20g·L -1 , natural pH value; the solvent is deionized water.

[0087] Example 5: Detection of aroma compounds in fermentation broth

[0088] The fermentation broth was centrifuged at 8000 rpm for 10 min, and 5 mL of the supernatant was placed in a 20 mL headspace bottle. After saturation with 2 g of sodium chloride, the volatile aroma compounds therein were determined by headspace-solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS).

[0089] HS-SPME conditions:

[0090] The solid phase microextraction fiber head was a DVB / CAR / PBDS composite fiber extraction head; the sample was equilibrated at 45°C for 10 minutes and then extracted for 40 minutes. After the extraction was completed, the extraction head was placed in the gas phase injection port at 250°C for desorption for 3 minutes.

[0091] GC conditions: HP-innowax gas chromatography column (60m×250μm×0.25μm, Agilent) was used. The sample port temperature was 250°C, and helium was used as the carrier gas (flow rate was 1mL·min -1 The samples were analyzed under the conditions of 50:1 split ratio. The heating program was as follows: initial temperature 50°C for 5 min, then 3°C min -1 Continue to raise the temperature to 120℃, then at 6℃min -1 The temperature was raised to 230°C and maintained for 5 min.

[0092] MS conditions: EI ionization source, electron energy 70 eV, ion source temperature 230 °C; scanning speed 3.00 scans / s in the range of 35–500 m / z.

[0093] The database used for mass spectrometry analysis is NIST14.L, which comes from the National Institute of Standards and Technology (NIST). The retention index (RI) is calculated in combination with a mixed standard of normal alkanes to jointly identify and confirm volatile aroma compounds.

[0094] Retention index (RI) is an important parameter used for qualitative analysis in gas chromatography (GC) analysis. Researchers can calibrate compounds based on the retention time (RT) of two n-alkanes that are close to the component. The retention index is only related to the properties of the stationary phase and the column temperature, and has nothing to do with other experimental conditions. Therefore, it has good reproducibility and accuracy, and can be combined with the GC-MS database to determine unknown compounds.

[0095] The peak time of the normal alkane mixed standard under the above GC-MS conditions is shown in Table 2; the peak retention time of isopentanol and 3-hydroxy-2-butanone is shown in Table 3; the total ion current of the fermentation broth detected by HS-SPME-GC-MS is shown in Figure 6 shown.

[0096] Table 2 Retention schedule of normal alkane mixed standard

[0097]

[0098]

[0099] Note: The calculation formula of retention index (RI) is as follows:

[0100] RI(x)=100Z+100[RT(x)-RT(z)] / [RT(z+1)-RT(z)]

[0101] Wherein: RT(x) represents the retention time of target compound x, RT(z) represents the retention time of normal alkanes that elute before compound x, and z+1 represents the retention time of normal alkanes that elute after compound x;

[0102] Z and Z+1 are the numbers of carbon atoms contained in the normal alkanes before and after the target compound (X) is eluted, respectively.

[0103] Table 3 Peak retention time and retention index of isoamyl alcohol and 3-hydroxy-2-butanone

[0104]

[0105] In the GC-MS detection, the elution times of unknown 1 and unknown 2 were 21.316 and 25.511 respectively. The retention indices of the two unknowns were calculated based on the elution times of the normal alkanes before and after the two unknowns:

[0106] RI(21.316)=12*100+100*(21.311-20.776) / (25.532-20.776)=1211;

[0107] RI(25.511)=12*100+100*(25.511-20.776) / (25.532-25.511)=1278.

[0108] The result is consistent with the retention index of isopentanol and 3-hydroxy-2-butanone in the literature, and is consistent with the detection results of the mass spectrometry database NIST14.L. Therefore, it is preliminarily determined that the aroma compounds produced by the strain are isopentanol and 3-hydroxy-2-butanone.

[0109] Finally, the peak time was verified using standard samples, and the unknown compounds were ultimately determined to be isopentanol and 3-hydroxy-2-butanone.

[0110] It is worth noting that although the present invention has disclosed the preferred embodiments as described above, the above contents are not intended to limit the present invention. Any researcher familiar with this technology may make various changes and substitutions without departing from the spirit and scope of the present invention, and all such changes or substitutions shall fall within the scope of protection of the claims attached to the present invention.

Claims

1. An endophytic bacterium producing isoamyl alcohol and 3-hydroxy-2-butanone, characterized in that: The name of the endophytic bacteria producing isopentanol and 3-hydroxy-2-butanone is Raoultella planticola HuaYu-2, the preservation number is GDMCC No: 65269, and it was preserved on October 15, 2024 at the Guangdong Microbial Culture Collection Center of the Institute of Microbiology, Guangdong Academy of Sciences, located on the 5th floor, Building 59, Compound 100, Xianlie Middle Road, Guangzhou.

2. Use of the endophytic bacteria producing isoamyl alcohol and 3-hydroxy-2-butanone according to claim 1 in producing isoamyl alcohol and 3-hydroxy-2-butanone.

3. The use according to claim 2, characterized in that The method comprises the following steps: inoculating the endophytic bacteria of isopentanol and 3-hydroxy-2-butanone according to claim 1 into a potato glucose water culture medium for fermentation culture to obtain a fermentation liquid containing isopentanol and 3-hydroxy-2-butanone.

4. The use according to claim 3, characterized in that The method comprises the following steps: activating and amplifying the endophytic bacteria of isopentanol and 3-hydroxy-2-butanone according to claim 1, and then inoculating the bacteria into a potato glucose water culture medium for fermentation to obtain a fermentation liquid containing isopentanol and 3-hydroxy-2-butanone.

5. The use according to claim 3 or 4, characterized in that: The fermentation culture conditions are: temperature 30-37°C, rotation speed 100-250rpm.

6. The use according to claim 5, characterized in that: The fermentation culture conditions are: temperature 35-37°C, rotation speed 150-250rpm.

7. The use according to claim 5, characterized in that: The fermentation culture time is 24 to 48 hours.

8. The use according to claim 4, characterized in that: The activation step is as follows: streak the endogenous bacteria of isopentanol and 3-hydroxy-2-butanone described in claim 1 into LB solid culture medium, and culture them in an aerobic environment at 30-37° C. for 16-32 hours.

9. The use according to claim 4, characterized in that: The conditions of the amplification culture are as follows: the activated bacteria are inoculated into LB liquid culture medium and cultured in an aerobic environment at 30-37°C and 150-250rpm until the OD 600 Values ​​over 0.

5.

10. The use according to claim 9, characterized in that: The inoculation is carried out by inoculating the bacterial suspension obtained by amplification culture at 1-3% of the volume of the fermentation medium.

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