Leifsonia longwild for producing aroma substances of green tea through fermentation and method for producing aroma through fermentation

Abstract

The invention discloses a Leifsonia longwild strain screened from pericarp parts of perfume lemons and used for producing green tea aroma substances through fermentation and a fermentation aroma production method, and belongs to the technical field of microbial fermentation. According to the present invention, the Leifsoniashuensis is Leifsoniashuensis HuaYu-3, the Leifsoniashuensis HuaYu-3 is preserved in Guangdong Province Microbial Culture Collection Center on October 15, 2024, and the preservation number is GDMCC No.65270, and the Leifsoniashuensis HuaYu-3 is the Leifsoniashuensis HuaYu-3, according to the method for producing the green tea aroma substance through fermentation of the leifsonia longwild HuaYu-3, fermentation liquor with the green tea aroma substance is obtained; green tea aroma substances are generated through biological fermentation, the process is environmentally friendly and controllable, and the complexity and layering sense of natural perfume can be copied.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation, and particularly relates to a strain of Revsonella nagano selected from the peel of perfume lemon to produce green tea aroma substances by fermentation, and a fermentation aroma production method. Background Art

[0002] Aroma compounds are a general term for compounds that have their own aroma, such as esters, aldehydes, alcohols, ketones, terpenes, organic acids, etc. As people's living standards continue to improve, consumers' demand for flavors and fragrances in different fields is gradually increasing, and the requirements for the sources of flavors and fragrances are becoming increasingly stringent.

[0003] The production of flavors and fragrances through chemical synthesis will produce harmful byproducts and waste during the production process, causing environmental pollution; some synthetic fragrances may contain chemicals that are harmful to the human body, and long-term intake may affect health; and synthetic fragrances often cannot completely replicate the complexity and layering of natural fragrances. Natural fragrances that are natural, green, environmentally friendly, healthy, and have unique aromas are often more popular with consumers.

[0004] Aroma-producing microorganisms are a general term for microorganisms that can produce various aroma compounds through their own fermentation. Producing aroma compounds through microbial fermentation is in line with the concept of sustainable development of green environmental protection, and is not subject to the problems of difficult storage and geographical restrictions of traditional natural aroma plant resources. Therefore, it is increasingly valued by researchers as a new way to develop aroma resources. Summary of the invention

[0005] The purpose of the present invention is to overcome the defect that chemically synthesized fragrances in the prior art will cause harm to the environment, and to provide a strain of Nagano Revsonella selected from the peel of perfume lemon to produce green tea aroma substances through fermentation. The green tea aroma substances are produced through biological fermentation, the process is environmentally friendly and controllable, and the complexity and layering of natural fragrances can be replicated.

[0006] To achieve the above-mentioned purpose, the present invention provides a strain of Leifsonia shinshuensis, which is Leifsonia shinshuensis HuaYu-3, which was deposited in Guangdong Provincial Microbiological Culture Collection Center on October 15, 2024, with the deposit number GDMCC No.65270.

[0007] Preferably, the Revsonella Nagano HuaYu-3 is used for fermentation to produce green tea aroma substances.

[0008] Preferably, the Nagano Revsonella HuaYu-3 has the following characteristics:

[0009] (1) The colonies are round, convex, opaque, moist and smooth on the surface, with neat edges and yellow in color;

[0010] (2) Individual bacteria are short rod-shaped, approximately 0.1-0.3 μm in width and 1-2 μm in length, without spores, capsules, or flagella.

[0011] Preferably, the Revsonella Nagano HuaYu-3 is screened and obtained from the peel of lemongrass, and the identification method is 16s rDNA molecular biological identification.

[0012] The present invention also provides a green tea aroma substance produced by fermentation, which utilizes the microbial fermentation of the Revsonella naganoensis HuaYu-3 to produce a substance with the characteristic aroma of green tea.

[0013] Preferably, the substances having the characteristic aroma of green tea include: aroma compounds such as phyllotoluol, linalool, phenylethyl alcohol and acetic acid.

[0014] The present invention also provides a method for producing green tea aroma substances by fermentation, comprising the following steps:

[0015] Step S1: the HuaYu-3 bacterium is streaked and inoculated in a solid plate culture medium for growth and cultivation until the colony diameter exceeds 2 mm, and then it can be inoculated into a liquid culture medium;

[0016] Step S2: inoculating the colonies grown to a suitable size into a shaking tube filled with liquid culture medium and culturing until the OD value of the bacterial suspension at a wavelength of 600 nm exceeds 0.5;

[0017] Step S3: inoculating the bacterial suspension reaching a certain concentration into a shake flask containing a liquid culture medium at a certain volume ratio for cultivation, and finally obtaining a fermentation liquid with green tea aroma substances.

[0018] Preferably, the solid plate culture medium in step S1 is LB solid culture medium, the culture conditions are an aerobic environment, the temperature is 28-33° C., and the culture time is 48 h;

[0019] The liquid culture medium in step S2 is LB liquid culture medium, the volume of the shaking tube is 3-4 mL, the culture conditions are an aerobic environment, the temperature is 28-33° C., the rotation speed is 150-200 rpm, and the culture time is 24-32 h;

[0020] The inoculation volume ratio in step S3 is 2%-5%, the liquid culture medium is potato glucose water liquid culture medium, the culture conditions are aerobic environment, the temperature is 28-33° C., the rotation speed is 120-150rpm, and the culture time is 36-48h.

[0021] Preferably, the LB solid culture medium is formulated with 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, 20 g / L agar, and a pH of 7.0±0.2;

[0022] The LB liquid culture medium is formulated with 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, and 20 g / L agar, with a pH of 7.0±0.2;

[0023] The preparation method of the potato glucose water liquid culture medium is as follows: weigh 200g of potato, cut it into small pieces, add water and boil it until it is soft, filter it with gauze, heat it, stir and mix it evenly, add 20g of glucose, stir it evenly, add water to 1000ml after cooling it slightly, adjust the pH value to 7.0±0.2, pack it into conical bottles, sterilize it at 115°C for 20 minutes, take it out, cool it and store it for later use.

[0024] Preferably, the solid plate culture medium in step S1 is LB solid culture medium, the culture conditions are an aerobic environment, the temperature is 33° C., and the culture time is 48 h; the colony diameter must be greater than 2 mm before it can be inoculated into the liquid culture medium;

[0025] The liquid culture medium in step S2 is LB liquid culture medium, the volume of the shaking tube is 3-4 mL, the culture conditions are an aerobic environment, the temperature is 33° C., the rotation speed is 200 rpm, and the culture time is 32 h; the culture is carried out until the OD value of the bacterial suspension at a wavelength of 600 nm exceeds 0.5;

[0026] The inoculation volume ratio in step S3 is 5%, the liquid culture medium is a potato glucose water liquid culture medium, the culture conditions are an aerobic environment, the temperature is 33° C., the rotation speed is 120 rpm, the culture time is 48 hours, and finally a fermentation liquid with green tea aroma substances is obtained.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The present invention obtains a strain of Leifsonia shinshuensis from the peel of perfumed lemon, wherein the Leifsonia shinshuensis is Leifsonia shinshuensis HuaYu-3, which was deposited in Guangdong Provincial Microbiological Culture Collection Center on October 15, 2024, with a deposit number of GDMCC No.65270; the strain HuaYu-3 is used to prepare a fermentation liquid with green tea aroma substances; the present invention produces green tea aroma substances through biological fermentation, the process is environmentally friendly and controllable, and the complexity and layering of natural spices can be replicated. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0030] Figure 1 The colony morphology of Leifsonia shinshuensis HuaYu-3 provided by the present invention on the LB plate;

[0031] Figure 2 This is a scanning electron microscope (SEM) photo of Leifsonia shinshuensis HuaYu-3 provided by the present invention;

[0032] Figure 3 This is a Gram staining effect diagram of Leifsonia shinshuensis HuaYu-3 provided by the present invention;

[0033] Figure 4 This is the growth curve of Leifsonia shinshuensis HuaYu-3 provided by the present invention;

[0034] Figure 5 It is a phylogenetic tree of Leifsonia shinshuensis HuaYu-3 provided by the present invention;

[0035] Figure 6 It is a radar chart of aroma characteristics of the fermentation liquid of Leifsonia shinshuensis HuaYu-3 provided by the present invention;

[0036] Figure 7 The present invention provides a total ion current diagram of the fermentation liquid of Leifsonia shinshuensis HuaYu-3 detected by HS-SPMEGC-MS. DETAILED DESCRIPTION

[0037] The technical scheme in this embodiment of the present invention will be clearly and completely described below in conjunction with the drawings in this embodiment of the present invention. Obviously, the described embodiment is one embodiment of the present invention, not all embodiments of the present invention. Based on this embodiment of the present invention, all other embodiments of the present invention obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0038] The invention provides a strain of Leifsonia shinshuensis HuaYu-3 which can produce green tea aroma substances through fermentation.

[0039] The described Leifsonia shinshuensis HuaYu-3 was deposited in the Guangdong Microbial Culture Collection Center of the Institute of Microbiology, Guangdong Academy of Sciences on October 15, 2024; address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou; Collection number: GDMCCNo.65270.

[0040] The biological characteristics of the Nagano Leifsonia leifsonia shinshuensis HuaYu-3 of the present invention are as follows:

[0041] (1) Colony characteristics: The colonies are round, convex, opaque, moist and smooth on the surface, with neat edges and yellow in color. After streaking on LB solid medium and culturing at 37°C for 48 hours, yellow colonies with a diameter of about 1 mm are formed. See Appendix. Figure 1 ; Gram staining shows purple, the identification result is positive, see Figure 3 .

[0042] (2) Morphological characteristics: A single bacterium is short rod-shaped, about 0.1-0.3 μm wide and 1-2 μm long, without spores, capsules, or flagella; see Appendix Figure 2 .

[0043] (3) Growth characteristics: After the strain is inoculated into a new liquid culture medium, the lag phase lasts about 8 hours, followed by a logarithmic growth phase, and a stable phase about 22 hours after inoculation; see Appendix Figure 4 .

[0044] The invention provides an activation method for the Leifsonia shinshuensis HuaYu-3.

[0045] The activation steps of Leifsonia shinshuensis HuaYu-3 of the present invention are as follows:

[0046] (1) streaking the Leifsonia shinshuensis HuaYu-3 of the present invention onto an LB solid plate medium and growing and culturing at 28-33° C. for 36-48 h;

[0047] More preferably, the culture is carried out at 33°C for 48 hours. The colonies can be inoculated into liquid culture medium only when the colony diameter exceeds 2 mm.

[0048] (2) Inoculate the colonies that have grown to an appropriate size into a shaking tube containing 3-4 mL of liquid LB medium and culture for 24-32 hours in an aerobic environment at a temperature of 28-33°C and a rotation speed of 150-200 rpm;

[0049] More preferably, the culture is carried out at 33° C. and 200 rpm for 32 h until the OD value of the bacterial suspension at a wavelength of 600 nm exceeds 0.5.

[0050] The invention provides a method for producing green tea aroma substances by fermenting the leifsonia shinshuensis HuaYu-3.

[0051] The activated bacterial suspension is transferred to a shake flask containing potato syrup liquid culture medium at an inoculation volume ratio of 2%-5%, and cultured and fermented for 36-48 hours in an aerobic environment at a temperature of 28-33°C and a rotation speed of 120-150rpm;

[0052] More preferably, the inoculation volume ratio is 5%; after transfer, fermentation is carried out at 33° C. and a rotation speed of 120 rpm for 48 hours to obtain a fermentation liquid with an elegant and refreshing green tea aroma.

[0053] The LB solid medium formula is 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, 20 g / L agar, and the pH is 7.0±0.2;

[0054] The LB liquid culture medium is formulated with 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, and 20 g / L agar, with a pH of 7.0±0.2;

[0055] The preparation method of the potato glucose water liquid culture medium is as follows: weigh 200g of potato, cut it into small pieces, add water and boil it until it is soft, filter it with gauze, heat it, stir and mix it evenly, add 20g of glucose, stir it evenly, add water to 1000ml after cooling it slightly, adjust the pH value to 7.0±0.2, pack it into conical bottles, sterilize it at 115°C for 20 minutes, take it out, cool it and store it for later use.

[0056] Embodiment 1

[0057] Screening of bacterial strains

[0058] Step S21: Select a scented lemon with intact skin and obvious fragrance, rinse the skin with running water, and then use clean filter paper to absorb the surface moisture for later use.

[0059] Step S22: In a sterile clean bench, first rinse the surface of the peel with sterile water, then soak it in 3% sodium hypochlorite solution for 3 minutes for sterilization, and then rinse it with sterile water for 3 times; then soak it in 75% ethanol solution for 1 minute, and then rinse it with sterile water for 3 times; after rinsing, wipe the surface moisture with sterile filter paper.

[0060] Step S23: Cut the green outer peel into pieces of 2 cm×2 cm in size using a sterile knife, place it on LB solid culture medium, and culture it in a 30-37° C. incubator for 3-7 days.

[0061] Step S24: After the colonies grow, streak the culture medium with an inoculation loop onto a new LB solid medium. After streak inoculation three times, separate and purify to obtain a single colony.

[0062] Step S25: Pick a single colony and inoculate it into a potato glucose water liquid culture medium, and culture it at 28-37°C for 24-48 hours. If obvious aroma is observed by olfactory method, it can be determined that an aroma-producing strain has been screened.

[0063] Embodiment 2

[0064] Morphological observation of strains

[0065] Colony morphology observation:

[0066] The isolated and purified strains were streaked on LB solid plates using the three-zone streak method and cultured at 33°C in an aerobic environment for 48 hours to observe the morphological characteristics of the colonies; the results are shown in the attached Figure 1 .

[0067] Gram staining observation:

[0068] Pick colonies from the plate and fix them on a glass slide; stain with ammonium oxalate crystal violet for 1 minute and then rinse with distilled water; add iodine solution to cover the surface and stain for about 1 minute and then rinse with distilled water, and absorb the water with absorbent paper; add a few drops of 95% alcohol and shake gently to decolorize, wash with distilled water after 30 seconds, and absorb the water; stain with safranin dye for 1 minute and then rinse with distilled water; after absorbing the water, examine under an optical microscope with an oil lens. The Gram stain of strain HuaYu-3 shows purple, and the result is positive, see the attached Figure 3 .

[0069] Scanning electron microscopy observation:

[0070] Collect bacteria: Take 1 mL of the bacterial solution after 48 hours of activation culture, centrifuge at 4000 rpm, 4°C for 5 minutes, discard the supernatant, and wash three times with 0.01 M phosphate buffer.

[0071] Fixation: Add 25% glutaraldehyde fixative (25% glutaraldehyde, prepared in 0.01 M phosphate buffer) into the centrifuge tube, and then place it in a 4°C refrigerator for fixation overnight.

[0072] Ethanol gradient dehydration: The fixed bacterial suspension was centrifuged and washed three times with pH 7.2 phosphate buffer. The fixed cell samples were eluted with 30%, 50%, 70%, 80%, 90%, and 95% ethanol solutions once each, and 100% ethanol twice (each time for 20 minutes, each step required centrifugation at 4000r / min for 5 minutes, and the supernatant was discarded).

[0073] After freeze drying, the strain morphology was observed using a scanning electron microscope (SEM); see Appendix Figure 2 .

[0074] Embodiment 3

[0075] Molecular biological identification of strains:

[0076] DNA was extracted from the strain leifsonia shinshuensis HuaYu-3, and 16s rDNA was amplified using the bacterial universal primer pair 27F / 1492R. The PCR reaction conditions were: 94°C pre-denaturation for 3 min followed by the following cycles: 94°C denaturation for 40 s, 56°C annealing for 35 s, 72°C extension for 80 s, 30 cycles; 72°C extension for 10 min; after good results were identified by 1% agarose gel electrophoresis, the amplified product was sent for sequencing, and the results were compared by BLAST on the NCBI website; the strain was confirmed to be leifsonia shinshuensis, and the phylogenetic tree constructed was as follows: Figure 5 shown.

[0077] The 16s rDNA sequence of strain leifsonia shinshuensis HuaYu-3 is as follows:

[0078] CGCCAGTGGCGGGGTGCTTACACATGCAGTCGAACGATGAACCTGGAGCTTGCTCTAGG

[0079] GAATTAGTGGCGAACGGGTGAGTAACACGTGAGTAACCTGCCCTTGACTCTGGGATAACCTCC

[0080] GGAAACGGAAGCTAATACCGGATATGACGCACGGAGGCATCTCCTGTGCGTGGAAAGAACTTC

[0081] GGTCAAGGATGGACTCGCGGCCTATCAGGTAGTTGGTGAGGTAACGGCCCACCAAGCCTACGA

[0082] CGGGTAGCCGGCCTGAGAGGGTGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTAC

[0083] GGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCAACGCCGCGTGAG

[0084] GGATGACGGCCTTCGGGTTGTAAACCTCTTTTAGTAGGGAAGAAGCGAAAGTGACGGTACCTG

[0085] CAGAAAAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTGT

[0086] CCGGAATTATTGGGCGTAAAGAGCTCGTAGGCGGTCTGTCGCGTCTGCTGTGAAAACCCGAGG

[0087] CTCAACCTCGGGCCTGCAGTGGGTACGGGCAGACTAGAGTGCGGTAGGGGAGAATGGAATTCC

[0088] TGGTGTAGCGGTGGAATGCGCAGATATCAGGAGGAACACCGATGGCGAAGGCAGTTCTCTGGG

[0089] CCGTAACTGACGCTGAGGAGCGAaAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCC

[0090] ACGCCGTAAACGTTGGGCGCTAGATGTGGGGACCATTCCACGGTTTCCGTGTCGCAGCTAACG

[0091] CATTAAGCGCCCCGCCTGGGGAGTACGGCCGCAAAGCTAAAACTCAAAGGAATTGACGGGGGC

[0092] CCGCACAAGCGGCGGAGCATGCGGATTAATTCGATGCAACGCGAAGAACCTTACCAAGCTTGA

[0093] CATACACGAGAACGCCCTAGAAATAGGGACTCTTTGGACACTCGTGAACAGGTGGTGCATGGT

[0094] TGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGTTCTA

[0095] TGTTGCCAGCGCGTAATGGCGGGAACTCATAGGAGACTGCCGGGGTCAACTCGGAGGAAGGTG

[0096] GGGATGACGTCAAATCATCATGCCCCTTATGTCTTGGGCTTCACGCATGCTACAATGGCCGGT

[0097] ACAAAGGGCTGCAATACCGTAAGGTGGAGCGAATCCCAAAAAGCCGGTCTCAGTTCGGATTGA

[0098] GGTCTGCAACTCGACCTCATGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGCGGT

[0099] GAATACGTTCCCGGGCCTTGTACACACCGCCCGTCAAGTCATGAAAGTCGGTAACACCCGAAG

[0100] CCGGTGGCCCAACCCTTGTGGAGGGAGCCGTCGAAGGTGATCAGTTT.

[0101] Embodiment 4

[0102] Strain HuaYu-3 produces characteristic aroma substances of green tea through microbial fermentation.

[0103] (1) The Leifsonia shinshuensis HuaYu-3 of the present invention was streaked onto an LB solid plate medium and cultured at 33° C. in an aerobic environment for 48 hours. The colony was inoculated into a liquid medium only when the colony diameter exceeded 2 mm.

[0104] (2) Inoculate the colonies that have grown to an appropriate size into a shaking tube containing 3-4 mL of liquid LB medium and culture them in an aerobic environment at 33°C and 200 rpm for 32 h until the OD value of the bacterial suspension at a wavelength of 600 nm exceeds 0.5.

[0105] The bacterial suspension was transferred to a shake flask filled with potato syrup liquid culture medium at an inoculation volume ratio of 5%, and fermented for 48 hours in an aerobic environment at a temperature of 33° C. and a rotation speed of 120 rpm to obtain a fermentation liquid with an elegant and refreshing green tea aroma.

[0106] Embodiment 5

[0107] Sensory evaluation and analysis of fermentation broth of aroma-producing strains.

[0108] In this embodiment, the microbial fermentation of the Revsonella naganoense HuaYu-3 is used to produce a substance with a characteristic aroma of green tea; the substance with a characteristic aroma of green tea includes aroma compounds such as linalool, linalool, phenylethyl alcohol and acetic acid.

[0109] In order to determine the overall aroma characteristics of the fermentation broth of strain HuaYu-3, a sensory evaluation was conducted.

[0110] Aroma analysis was performed by well-trained experts (5 men and 5 women) from the laboratory of the Southern Medicinal Products International Research and Development Center of the Sino-Singapore International Joint Research Institute. After 3 months of training and testing, these panelists showed good abilities in aroma memory and scoring accuracy for each aroma descriptor.

[0111] The samples were evaluated by trained panelists. Ten assessors were asked to rate the six aroma descriptions of the fermented broth by aroma intensity, from 0 (not perceptible) to 3 (strongly perceptible). The six aroma characteristics were: green tea, grass, fruit, floral, cooked beans, and potato. The fermented broth (50 mL) was poured into a glass at 25°C. The data in the figure are the average of the scores of all trained panelists.

[0112] The results showed that the aroma properties of green tea and grass in the fermentation liquid of strain HuaYu-3 were relatively strong; see Appendix Figure 6 .

[0113] Embodiment 6

[0114] HS-SPME-GC-MS Detection of Fermentation Broth of Aroma-producing Strains

[0115] Detection of aroma compounds in the fermentation broth of the strain: After the fermentation broth was centrifuged at 8000 rpm for 10 min, 5 mL of the supernatant was taken into a 20 mL headspace bottle, saturated with 2 g of sodium chloride, and the volatile aroma compounds therein were determined by headspace-solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS).

[0116] HS-SPME conditions:

[0117] 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.

[0118] GC conditions:

[0119] HP-innowax gas chromatography column (60m×250μm×0.25μm, Agilent) was used. The sample port temperature was 250℃, and helium was used as the carrier gas (flow rate was 1mL·min -1 ) with a split ratio of 50:1.

[0120] The heating program was as follows: initial temperature was maintained at 40°C for 5 min, then at 3°C ​​min -1 Continue to raise the temperature to 120℃, maintain for 3 minutes, and then increase the temperature by 10℃min -1 The temperature was raised to 230°C and maintained for 5 min.

[0121] 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.

[0122] The database used for mass spectrometry analysis is NIST14.L, which comes from the National Institute of Standards and Technology (NIST).

[0123] The total ion current diagram of the HS-SPME-GC-MS detection of the aroma-producing strain fermentation broth of the present invention is shown in Figure 6 .

[0124] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A strain of Raifunesporium nagano, characterized in that The Leifsonia shinshuensis is Leifsonia shinshuensis HuaYu-3, which was deposited in the Guangdong Provincial Microbiological Culture Collection Center on October 15, 2024, with the collection number GDMCC No.65270.

2. The strain of Raifunesporium nagano according to claim 1, characterized in that: The application of the Revsonella Nagano HuaYu-3 in fermentation to produce green tea aroma substances.

3. The strain of Raifunesporium nagano according to claim 1, characterized in that: The Nagano Revsonella HuaYu-3 has the following characteristics: (1) The colonies are round, convex, opaque, moist and smooth on the surface, with neat edges and yellow in color; (2) Individual bacteria are short rod-shaped, approximately 0.1-0.3 μm in width and 1-2 μm in length, without spores, capsules, or flagella.

4. The strain of Raifunesporium nagano according to claim 1, characterized in that: The Nagano Revsonella HuaYu-3 is screened and obtained from the peel of the fragrant lemon, and the identification method is 16srDNA molecular biological identification.

5. A green tea aroma substance produced by fermentation, characterized in that: The microbial fermentation of the Nagano Revsonella HuaYu-3 is used to produce a substance with a characteristic aroma of green tea.

6. The green tea aroma substance produced by fermentation according to claim 5, characterized in that: The substances with characteristic green tea aroma include: phyllotoluol, linalool, phenylethyl alcohol and acetic acid, which are aroma compounds.

7. A method for producing green tea aroma substances by fermentation, characterized in that: The following steps are involved: Step S1: the HuaYu-3 bacterium is streaked and inoculated in a solid plate culture medium for growth and cultivation until the colony diameter exceeds 2 mm, and then it can be inoculated into a liquid culture medium; Step S2: inoculating the colonies grown to a suitable size into a shaking tube filled with liquid culture medium and culturing until the OD value of the bacterial suspension at a wavelength of 600 nm exceeds 0.5; Step S3: inoculating the bacterial suspension reaching a certain concentration into a shake flask containing a liquid culture medium at a certain volume ratio for cultivation, and finally obtaining a fermentation liquid with green tea aroma substances.

8. The method for producing green tea aroma substances by fermentation according to claim 7, characterized in that: The solid plate culture medium in step S1 is LB solid culture medium, the culture conditions are an aerobic environment, the temperature is 28-33° C., and the culture time is 48 h; The liquid culture medium in step S2 is LB liquid culture medium, the volume of the shaking tube is 3-4 mL, the culture conditions are an aerobic environment, the temperature is 28-33° C., the rotation speed is 150-200 rpm, and the culture time is 24-32 h; The inoculation volume ratio in step S3 is 2%-5%, the liquid culture medium is potato glucose water liquid culture medium, the culture conditions are aerobic environment, the temperature is 28-33° C., the rotation speed is 120-150rpm, and the culture time is 36-48h.

9. The method for producing green tea aroma substances by fermentation according to claim 8, characterized in that: The LB solid medium formula is 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, 20 g / L agar, and the pH is 7.0±0.2; The LB liquid culture medium formula is 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, 20 g / L agar, and the pH is 7.0±0.2; The preparation method of the potato glucose water liquid culture medium is as follows: weigh 200g of potato, cut it into small pieces, add water and boil it until it is soft, filter it with gauze, heat it, stir and mix it evenly, add 20g of glucose, stir it evenly, add water to 1000ml after cooling it slightly, adjust the pH value to 7.0±0.2, pack it into conical bottles, sterilize it at 115°C for 20 minutes, take it out, cool it and store it for later use.

10. The method for producing green tea aroma substances by fermentation according to claim 8, characterized in that: The solid plate culture medium in step S1 is LB solid culture medium, the culture conditions are aerobic environment, the temperature is 33° C., and the culture time is 48 h; the colony diameter exceeds 2 mm before it can be inoculated into the liquid culture medium; The liquid culture medium in step S2 is LB liquid culture medium, the volume of the shaking tube is 3-4 mL, the culture conditions are an aerobic environment, the temperature is 33° C., the rotation speed is 200 rpm, and the culture time is 32 h; the culture is carried out until the OD value of the bacterial suspension at a wavelength of 600 nm exceeds 0.5; The inoculation volume ratio in step S3 is 5%, the liquid culture medium is a potato glucose water liquid culture medium, the culture conditions are an aerobic environment, the temperature is 33° C., the rotation speed is 120 rpm, the culture time is 48 hours, and finally a fermentation liquid with green tea aroma substances is obtained.

Images