An endophytic bacterium producing isoamyl alcohol and 3-hydroxy-2-butanone and its applications

Abstract

This invention belongs to the field of microbiology, and specifically relates to an endophytic bacterium producing isoamyl alcohol and 3-hydroxy-2-butanone, and its applications. The endophytic bacterium is named *Raoultella planticola* HuaYu-2, with accession number GDMCC No: 65269, and was deposited at the Guangdong Provincial Microbial Culture Collection Center on October 15, 2024. This endophytic bacterium was isolated from fragrant lemon and can produce the aroma compounds isoamyl alcohol and 3-hydroxy-2-butanone through fermentation. This invention enriches the strain library for the fermentation production of fragrance compounds and provides a basis for screening aroma-producing microorganisms from fragrance plants.

Description

Technical Field

[0001] This invention belongs to the field of microbiology, and specifically relates to an endophytic bacterium that produces isoamyl alcohol and 3-hydroxy-2-butanone and its applications. Background Technology

[0002] Fragrances can be categorized into natural and synthetic fragrances based on their raw material sources and production processes. Synthetic fragrances are produced using petrochemical raw materials through chemical synthesis, inevitably causing environmental pollution and contradicting the principles of sustainable development. As society and government pursue high-quality development models, the production model of chemically synthesized fragrances is gradually losing favor. Furthermore, because petrochemical raw material prices are heavily influenced by oil prices, fluctuations in international crude oil prices ultimately lead to uncontrollable fragrance production costs. Natural spice plant resources, on the other hand, exhibit strong regional characteristics, primarily distributed across different climate zones. my country's climate zones exhibit distinct regional environmental features, resulting in significant localized differences in spice crops produced in different regions. Moreover, many growers currently face issues such as traditional planting methods, primitive production models, and low production levels, hindering the effective utilization of natural spice plant resources.

[0003] In 1985, the U.S. Food and Drug Administration (FDA) defined fragrances produced from natural raw materials through fermentation or enzymatic technology as natural equivalent fragrances. Producing fragrance compounds through microbial fermentation aligns with the principles of green and sustainable development and is not limited by the geographical constraints of traditional natural fragrance plant resources, thus increasingly becoming an important method for fragrance production. Currently, researchers primarily screen for aroma-producing microorganisms using methods such as soil, aquatic, and fermentation environments. However, these methods suffer from problems such as overly complex microbial communities and low screening efficiency; therefore, there is a need to develop new methods for screening aroma-producing microorganisms.

[0004] Plant endophytes refer to fungi or bacteria that live within the tissues and organs of plants at certain or all stages of their life cycle. Endophytes are ubiquitous in higher plants, found in woody, herbaceous, monocotyledonous, and dicotyledonous plants. Increasing research indicates that endophytes in aromatic plants often produce fragrance compounds, and secondary metabolites from plant endophytes are gradually becoming a novel source of these compounds.

[0005] Isoamyl alcohol, with its apple brandy aroma and spicy flavor, is a permitted food flavoring agent according to GB 2760-86. 3-Hydroxy-2-butanone can be used as an aroma enhancer in cream, cheese, coffee, and fruit flavorings. It is also widely used in the formulation of flavorings for cream, dairy products, yogurt, and strawberry-flavored products, and has extremely high application value. GB 2760-86 in my country stipulates that it is a permitted food flavoring agent. Currently, these two aroma compounds are mainly produced through chemical synthesis, which presents industry challenges such as environmental pollution.

[0006] If we can screen out aroma-producing microorganisms from aromatic plants, we can avoid the problems existing in the production process of synthetic fragrances and natural plant fragrances, and provide a certain theoretical basis for developing new screening methods for aroma-producing microorganisms. Summary of the Invention

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

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

[0009] The objective of this invention is achieved through the following technical solution: an endophytic bacterium producing isoamyl alcohol and 3-hydroxy-2-butanone, named Raoultella planticola HuaYu-2, with accession number GDMCC No: 65269, was deposited on October 15, 2024, at the Guangdong Provincial Microbial Culture Collection Center of the Institute of Microbiology, Guangdong Academy of Sciences, located on the 5th floor of Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0010] The application of the above-mentioned endophytic bacteria producing isoamyl alcohol and 3-hydroxy-2-butanone in the production of isoamyl alcohol and 3-hydroxy-2-butanone preferably includes the following steps: inoculating the above-mentioned endophytic bacteria producing isoamyl alcohol and 3-hydroxy-2-butanone into potato dextrose aqueous medium for fermentation culture to obtain a fermentation broth containing isoamyl alcohol and 3-hydroxy-2-butanone; more preferably, it includes the following steps: activating and amplifying the above-mentioned endophytic bacteria producing isoamyl alcohol and 3-hydroxy-2-butanone, and then inoculating them into potato dextrose aqueous medium for fermentation culture to obtain a fermentation broth containing isoamyl alcohol and 3-hydroxy-2-butanone.

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

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

[0013] The preferred activation step is as follows: the endophytic bacteria containing isoamyl alcohol and 3-hydroxy-2-butanone are streaked into LB solid medium and cultured in an aerobic environment at 30-37°C for 16-32 hours.

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

[0015] The preferred method for inoculation is to inoculate the bacterial suspension obtained from the amplification culture at 1-3% of the fermentation medium volume.

[0016] The present invention has the following advantages and effects compared with the prior art:

[0017] This invention identifies an endophytic bacterial strain from the aromatic plant lemon (Citrus reticulata) that can produce the aroma compounds isoamyl alcohol and 3-hydroxy-2-butanone through fermentation. This enriches the strain library for fermentation-produced aroma compounds and provides a basis for screening aroma-producing microorganisms from aromatic plants. Both isoamyl alcohol and 3-hydroxy-2-butanone are permitted edible flavorings according to GB2760-86; therefore, strains capable of producing these two substances have promising application prospects. Attached Figure Description

[0018] Figure 1 This is a colony morphology diagram of strain HuaYu-2 on LB agar plates.

[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 a Gram staining result of strain Raoultella planticola HuaYu-2.

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

[0023] Figure 6 This is a GC-MS chromatogram of the total ion flow of the fermentation broth of strain Raoultella planticola HuaYu-2. Detailed Implementation

[0024] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

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

[0026] Select fragrant lemons with intact peels and a distinct aroma. Rinse the peels thoroughly with running water, then blot dry with clean filter paper. In a sterile laminar flow hood, first rinse the peel surface with sterile water, then soak in a 3% sodium hypochlorite solution for 3 minutes for sterilization, followed by rinsing three times with sterile water. Next, soak in a 75% ethanol solution for 1 minute, then rinse three times with sterile water. After rinsing, dry the surface with sterile filter paper. Cut 2cm × 2cm pieces of green outer peel using a sterile knife and place them on LB agar plates. Incubate at 30–37°C for 3–7 days. After colonies have grown, streak them onto fresh LB agar plates using an inoculation loop. Repeat this streaking process three times, then isolate and purify single colonies.

[0027] Select a single colony and inoculate it into potato dextrose aqueous liquid medium. Incubate at 37°C for 24 hours. If a distinct aroma is observed by smell, the aroma-producing strain can be identified.

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

[0029] Observe under a scanning electron microscope, such as Figure 2 As shown: a single bacterial cell is short rod-shaped, about 0.5 μm wide and 2 to 4 μm long. It has no spores, a capsule, and no flagella.

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

[0031] Example 2: Physiological and biochemical identification of the strain:

[0032] (1) Gram staining experiment:

[0033] A purified single colony of strain HuaYu-2 was picked from the plate and fixed on a glass slide; it was stained with ammonium oxalate crystal violet for 1 minute and then rinsed with distilled water; the smear was covered with iodine solution and stained for about 1 minute, then washed with distilled water and the water was blotted with absorbent paper; a few drops of 95% alcohol were added and the slide was gently shaken to decolorize, and after 30 seconds it was washed with distilled water and the water was blotted; it was stained with safranin solution for 1 minute and then rinsed with distilled water; after the water was blotted dry, it was examined under an oil immersion microscope.

[0034] like Figure 4 As shown: the Gram staining result of strain HuaYu-2 is red, indicating a negative result.

[0035] (2) Citrate utilization experiment:

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

[0037] The indicator changed from light green to dark blue, indicating that strain HuaYu-2 can decompose citrate, making the culture medium alkaline. Therefore, the citrate utilization experiment was positive.

[0038] (3) Experiment on the utilization of malonate:

[0039] Prepare malonate medium, then pick a purified single colony and inoculate it into 5 mL of malonate medium. Incubate at 37℃ and 180 rpm for 24–48 h and observe the results.

[0040] The indicator changed from light green to dark blue, indicating that strain HuaYu-2 can utilize malonate as a carbon source, decompose malonate to generate sodium carbonate, and make the culture medium alkaline. Therefore, the malonate utilization experiment was positive.

[0041] (4) Indole experiment:

[0042] Prepare Dunhen's peptone medium. Pick a purified single colony and inoculate it into 5 mL of Dunhen's peptone medium. Incubate at 37°C and 180 rpm for 24–48 h. Add 2–3 mL of pentanol or xylene to the culture medium, shake well, let stand for a moment, and then add 2 mL of Ehrlich's indole reagent along the wall of the test tube. A positive reaction is indicated by the liquid turning red below the pentanol or xylene.

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

[0044] (5) VP experiment:

[0045] A single purified colony of strain HuaYu-2 was inoculated into 5 mL of glucose peptone water medium and cultured at 37℃ and 180 rpm for 24-48 h. 1 mL of VP test solution A (6% α-naphthol alcohol solution) was added to the medium and shaken well. Then 0.5 mL of VP test solution B (40% KOH solution) was added and shaken well. The results were then observed.

[0046] The culture medium turned red, indicating that strain HuaYu-2 in the experiment could decompose glucose to produce pyruvate, and further decarboxylate pyruvate to acetylmethylmethanol. Acetylmethylmethanol was oxidized to diacetyl in an alkaline environment, and then combined with guanidine groups contained in arginine and other substances in the culture medium to form a red compound. Therefore, the VP experiment was a positive result.

[0047] (6) Lysine decarboxylase experiment:

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

[0049] The indicator color development indicates that strain HuaYu-2 can produce a decarboxylase that removes the carboxyl group of lysine, generating amine and CO2, which raises the pH of the culture medium, thus causing the indicator color development, resulting in a positive result.

[0050] (7) Ornithine decarboxylase experiment:

[0051] Prepare ornithine decarboxylase broth medium, pick a purified single colony and inoculate it into 5 mL of lysine decarboxylase medium, and incubate at 37℃ and 180 rpm for 18–24 h, and observe the results.

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

[0053] (8) Phenylalanine deaminase test:

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

[0055] In the experiment, strain HuaYu-2 could not produce phenylalanine deaminase, and the ferric chloride indicator did not change color, so the experimental 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 the strain:

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

[0077] CTACCGTTTCCAGTAGTTATCCCCTCCATCAGGCAGTTTCCCAGACATTACTCACCCGTCCGCCACTCGTCACCCGAGA

[0078] GCAAGCTCTCTGTGCTACCGTTCGACTGC.

[0079] The strain was deposited at the Guangdong Provincial Center for Microbial Culture Collection on October 15, 2024, with accession 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 hours until the colony diameter exceeded 2 mm.

[0082] Colonies of suitable size were inoculated into shake tubes containing 4 mL of LB liquid medium and incubated in an aerobic environment at 37°C and 200 rpm for 24 h, until the bacterial suspension showed an OD value of 600 nm. 600 The value exceeds 0.5.

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

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

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

[0086] The formula for potato glucose aqueous culture medium is: 200 g / L potato extract powder. -1 20g / L glucose -1 Natural pH value; solvent is deionized water.

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

[0088] After centrifuging the fermentation broth at 8000 rpm for 10 min, 5 mL of the supernatant was collected in a 20 mL headspace vial. After saturation with 2 g of sodium chloride, the volatile aroma compounds 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 is a DVB / CAR / PBDS composite fiber extraction head; the sample is equilibrated at 45℃ for 10 min and then extracted for 40 min. After extraction, the extraction head is placed in the gas phase inlet at 250℃ for 3 min for desorption.

[0091] GC conditions: HP-innowax gas chromatographic column (60m × 250μm × 0.25μm, Agilent). Sample port temperature was 250℃, and helium was used as the carrier gas (flow rate 1mL·min). -1 The sample was analyzed under the following conditions: a split ratio of 50:1. The temperature program was as follows: initial temperature of 50℃ held for 5 min, then increased by 3℃ / min. -1 Continue heating to 120℃, then reduce the temperature by 6℃ / min. -1 The temperature is increased to 230°C and held for 5 minutes.

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

[0093] The mass spectrometry analysis used the NIST14.L database, from the National Institute of Standards and Technology (NIST). The volatile aroma compounds were identified and confirmed by calculating the retention index (RI) using a mixture of n-alkane standards.

[0094] The retention index (RI) is an important parameter used for qualitative analysis in gas chromatography (GC). Researchers can identify compounds by measuring the retention times (RT) of two closely spaced n-alkanes preceding and following them. The retention index is only related to the properties of the stationary phase and column temperature, and is independent of other experimental conditions. Therefore, it has excellent reproducibility and accuracy, and can be used in conjunction with GC-MS databases to identify unknown compounds.

[0095] The elution times of the n-alkane mixed standard under the above GC-MS conditions are shown in Table 2; the retention times of isoamyl alcohol and 3-hydroxy-2-butanone are shown in Table 3; the total ion chromatogram of the fermentation broth as determined by HS-SPME-GC-MS is shown in Table 3. Figure 6 As shown.

[0096] Table 2 Retention Schedule for n-Alkanes Mixed Standards

[0097]

[0098]

[0099] Note: The retention index (RI) is calculated as follows:

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

[0101] In the formula: RT(x) represents the retention time of target compound x, RT(z) represents the retention time of n-alkanes that elute before compound x, and z+1 represents the retention time of n-alkanes that elute after compound x.

[0102] Z and Z+1 represent the number of carbon atoms in the n-alkanes before and after the target compound (X) flows out, respectively.

[0103] Table 3. Retention times and retention indices of isoamyl alcohol and 3-hydroxy-2-butanone

[0104]

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

[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 results are consistent with the retention indices of isoamyl alcohol and 3-hydroxy-2-butanone in the literature, and are also consistent with the detection results of the NIST14.L mass spectrometry database. Therefore, it is preliminarily determined that the aroma compounds produced by the strain are isoamyl alcohol and 3-hydroxy-2-butanone.

[0109] Finally, the peak times were verified using standards, and the unknown compounds were ultimately identified as isoamyl alcohol and 3-hydroxy-2-butanone.

[0110] It is worth noting that although the preferred embodiments of the present invention have been disclosed above, the above content is not intended to limit the present invention. Any researcher skilled in the art can make various modifications and substitutions without departing from the spirit and scope of the present invention, and all such modifications or substitutions should fall within the protection scope of the appended claims.

Claims

1. An endophytic bacterium producing isopentanol and 3-hydroxy-2-butanone, characterized in that: The endogenous bacteria producing isopentanol and 3-hydroxy-2-butanone is named as Raoultella planticola Raoultella planticola ) HuaYu-2, with the preservation number of GDMCC No: 65269, preserved in the Guangdong Microbial Culture Collection Center, located at the 5th floor of No. 59 Building, Guangdong Academy of Microbiology, Guangzhou Military Middle Road 100, on October 15, 2024.

2. The use of the endogenous bacteria producing isopentanol and 3-hydroxy-2-butanone according to claim 1 in producing isopentanol and 3-hydroxy-2-butanone.

3. Use according to claim 2, characterized in that: The method comprises the following steps: inoculating the endogenous bacteria producing isopentanol and 3-hydroxy-2-butanone according to claim 1 into a potato glucose aqueous culture medium to carry out fermentation culture, so as to obtain a fermentation liquor containing isopentanol and 3-hydroxy-2-butanone.

4. Use according to claim 3, characterized in that: The method comprises the following steps: inoculating the endogenous bacteria producing isopentanol and 3-hydroxy-2-butanone according to claim 1 into a potato glucose aqueous culture medium to carry out fermentation culture, so as to obtain a fermentation liquor containing isopentanol and 3-hydroxy-2-butanone.

5. Use according to claim 3 or 4, characterized in that: The fermentation culture condition is that the temperature is 30-37 ℃, and the rotating speed is 100-250 rpm.

6. Use according to claim 5, characterized in that: The fermentation culture condition is that the temperature is 35-37 ℃, and the rotating speed is 150-250 rpm.

7. Use according to claim 5, characterized in that: The fermentation culture time is 24-48 h.

8. Use according to claim 4, characterized in that: The activation step is as follows: the endogenous bacteria producing isopentanol and 3-hydroxy-2-butanone according to claim 1 is streaked and inoculated into an LB solid culture medium, and cultured in an aerobic environment at 30-37 ℃ for 16-32 h.

9. Use according to claim 4, characterized in that: The conditions of the amplification culture are as follows: inoculate the activated bacteria into LB liquid medium, cultivate under aerobic environment, 30-37℃, 150-250 rpm until OD 600 value exceeds 0.

5.

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

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