Leifsonia nigranovum for producing green tea aroma substances by fermentation and fermentation method

Abstract

The application discloses a Leifsonia shinsuensis strain screened from a perfume lemon peel part and a fermentation method for producing green tea aroma substances, and belongs to the technical field of microbial fermentation.The Leifsonia shinsuensis strain is Leifsonia shinsuensis HuaYu-3, which is preserved in the Guangdong Microbial Culture Collection Center on October 15, 2024, and the preservation number is GDMCC No.65270.A fermentation liquid with green tea aroma substances is obtained by using the Leifsonia shinsuensis HuaYu-3 strain to ferment and produce green tea aroma substances.The Leifsonia shinsuensis HuaYu-3 strain can produce green tea aroma substances through biological fermentation, the process is environmentally friendly and controllable, and the complexity and level of natural fragrances can be reproduced.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology, and specifically relates to a strain of Rifops nanano strain selected from the peel of perfume lemons that produces green tea aroma substances through fermentation, and a method for fermentation and aroma production. Background Technology

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

[0003] The production of fragrances and flavorings through chemical synthesis generates harmful byproducts and waste during the process, causing environmental pollution. Some synthetic fragrances may contain chemicals harmful to the human body, and long-term intake may have health effects. Furthermore, synthetic fragrances often cannot fully replicate the complexity and nuances of natural fragrances. Natural fragrances, characterized by their naturalness, greenness, environmental friendliness, and health benefits, along with their unique aromas, are often more favored by consumers.

[0004] Aroma-producing microorganisms are a collective term for microorganisms that can produce various aroma compounds through their own fermentation. Producing fragrance compounds through microbial fermentation aligns with the principles of green and sustainable development, and avoids the problems of difficult storage and geographical limitations inherent in traditional natural fragrance plant resources. Therefore, it is increasingly attracting the attention of researchers as a novel approach to fragrance resource development. Summary of the Invention

[0005] The purpose of this invention is to overcome the environmental hazards of chemically synthesized fragrances in the prior art. It provides a strain of Riftenia nagano selected from the peel of perfume lemons to produce green tea aroma substances through fermentation. The green tea aroma substances are produced through biological fermentation, which is environmentally friendly and controllable, and can replicate the complexity and layering of natural fragrances.

[0006] To achieve the above objectives, the present invention provides a strain of Leifsonia shinshuensis, namely Leifsonia shinshuensis HuaYu-3, which was deposited at the Guangdong Provincial Center for Microbial Culture Collection on October 15, 2024, with the accession number GDMCC No. 65270.

[0007] As a preferred application, the *Rifkino* HuaYu-3 is used in the fermentation of green tea aroma compounds.

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

[0009] (1) The colonies are round, raised, opaque, moist and smooth, with neat edges and yellow color;

[0010] (2) Individual bacteria are short rods, with a width of about 0.1-0.3 μm and a length of 1-2 μm. They have no spores, no capsules, and no flagella.

[0011] Preferably, the *Rifonsonia Nagano* HuaYu-3 was obtained by screening from the peel of lemon citron and identified by 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 Rifonella Nagano 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: linalool, phenethyl alcohol, and acetic acid compounds with aroma.

[0014] This invention also provides a method for producing aroma substances in green tea through fermentation, comprising the following steps:

[0015] Step S1: The Nagano Rifsonella HuaYu-3 is streaked into a solid plate culture medium and cultured until the colony diameter exceeds 2 mm before it can be inoculated into a liquid culture medium.

[0016] Step S2: Inoculate colonies that have grown to a suitable size into shake tubes containing liquid culture medium and culture until the OD value of the bacterial suspension at a wavelength of 600 nm exceeds 0.5;

[0017] Step S3: Inoculate the bacterial suspension of a certain concentration into a shake flask containing liquid culture medium at a certain volume ratio and culture it to finally obtain a fermentation broth with green tea aroma substances.

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

[0019] The liquid culture medium mentioned in step S2 is LB liquid culture medium, the volume of the shaker tube is 3-4 mL, the culture conditions are an aerobic environment, the temperature is 28-33℃, 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 an aerobic environment, the temperature is 28-33℃, 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 was 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 potato glucose aqueous liquid culture medium is prepared as follows: Weigh 200g of potatoes, cut them into small pieces, add water and boil until soft, filter with gauze, heat, stir and mix well, add 20g of glucose, stir evenly, cool slightly and then add water to 1000ml, adjust the pH value to 7.0±0.2, dispense into Erlenmeyer flasks, sterilize at 115℃ for 20 minutes, remove, cool and store for later use.

[0024] Preferably, the solid plate culture medium mentioned in step S1 is LB solid culture medium, and the culture conditions are an aerobic environment, a temperature of 33°C, and a culture time of 48 hours; colonies with a diameter of more than 2 mm can be inoculated into liquid culture medium.

[0025] The liquid culture medium mentioned in step S2 is LB liquid culture medium. The volume of the culture tube is 3-4 mL. The culture conditions are an aerobic environment, a temperature of 33℃, a rotation speed of 200 rpm, and a culture time of 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] In step S3, the inoculation volume ratio is 5%, the liquid culture medium is potato glucose water liquid culture medium, the culture conditions are an aerobic environment, the temperature is 33℃, the rotation speed is 120rpm, and the culture time is 48h, finally obtaining a fermentation broth with green tea aroma substances.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0028] This invention obtains a strain of Leifsonia shinshuensis from the peel of lemon, namely Leifsonia shinshuensis HuaYu-3, which was deposited at the Guangdong Provincial Center for Microbial Culture Collection on October 15, 2024, with accession number GDMCC No. 65270. A fermentation broth with green tea aroma compounds was prepared using this strain HuaYu-3. This invention produces green tea aroma compounds through biological fermentation, a process that is environmentally friendly and controllable, and can replicate the complexity and layering of natural fragrances. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0030] Figure 1 The colony morphology of Leifsonia shinshuensis HuaYu-3 on LB agar plates provided by this invention;

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

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

[0035] Figure 6 This is a radar image of the aroma characteristics of the fermentation broth of Leifsonia shinshuensis HuaYu-3 provided by the present invention.

[0036] Figure 7 The total ion chromatogram of the fermentation broth of Leifsonia shinshuensis HuaYu-3 provided by this invention is obtained by HS-SPME-GC-MS detection. Detailed Implementation

[0037] The technical solution of this embodiment of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiment is one embodiment of the present invention, and not all embodiments thereof. Based on this embodiment of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

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

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

[0040] The biological characteristics of *Leifsonia shinshuensis* HuaYu-3 in this invention are as follows:

[0041] (1) Colony characteristics: The colonies are round, raised, opaque, moist and smooth, with neat edges and a yellow color; after streaking on LB solid medium and incubating at 37℃ for 48 h, yellow colonies with a diameter of about 1 mm are formed. See Appendix Figure 1 Gram staining shows purple, indicating a positive identification result. See [link to relevant documentation]. Figure 3 .

[0042] (2) Morphological characteristics: Individual bacteria are short rod-shaped, with a width of about 0.1-0.3 μm and a length of 1-2 μm. They are non-spore-forming, non-capsulated, and non-flagellated; see Appendix Figure 2 .

[0043] (3) Growth characteristics: After inoculation into fresh liquid culture medium, the strain exhibits a lag phase lasting approximately 8 hours, followed by the logarithmic growth phase, and enters the stationary phase approximately 22 hours after inoculation; see Appendix Figure 4 .

[0044] This invention provides a method for activating Leifsonia shinshuensis HuaYu-3.

[0045] The activation steps of *Leifsonia shinshuensis* HuaYu-3 described in this invention are as follows:

[0046] (1) The *Leifsonia shinshuensis* HuaYu-3 of the present invention was streaked onto LB solid plate medium and cultured at 28-33℃ for 36-48 h.

[0047] More preferably, the culture should be carried out at 33°C for 48 h. Only colonies with a diameter exceeding 2 mm can be inoculated into liquid culture medium.

[0048] (2) Inoculate colonies that have grown to a suitable size into shake tubes containing 3-4 mL of liquid LB medium and incubate them for 24-32 h in an aerobic environment at a temperature of 28-33℃ 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; the culture is continued until the OD value of the bacterial suspension at a wavelength of 600 nm exceeds 0.5.

[0050] This invention provides a method for producing green tea aroma substances by fermentation using *Leifsonia shinshuensis* HuaYu-3.

[0051] The activated bacterial suspension was transferred to a shake flask containing potato syrup liquid culture medium at an inoculation volume ratio of 2%-5%. The culture was fermented for 36-48 hours under aerobic conditions at a temperature of 28-33℃ and a rotation speed of 120-150 rpm.

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

[0053] The LB solid medium formulation consisted of 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.

[0054] The LB liquid culture medium was 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 potato glucose aqueous liquid culture medium is prepared as follows: Weigh 200g of potatoes, cut them into small pieces, add water and boil until soft, filter with gauze, heat, stir and mix well, add 20g of glucose, stir evenly, cool slightly and then add water to 1000ml, adjust the pH value to 7.0±0.2, dispense into Erlenmeyer flasks, sterilize at 115℃ for 20 minutes, remove, cool and store for later use.

[0056] Example 1

[0057] Strain screening

[0058] Step S21: Select a perfume lemon with an intact peel and a distinct aroma, rinse the peel with running water, and then use clean filter paper to absorb the surface moisture for later use.

[0059] Step S22: In a sterile ultra-clean workbench, first rinse the surface of the fruit peel with sterile water, then soak it in a 3% sodium hypochlorite solution for 3 minutes to sterilize and disinfect it, and then rinse it 3 times with sterile water; then soak it in a 75% ethanol solution for 1 minute, and then rinse it 3 times with sterile water; after rinsing, wipe the surface dry with sterile filter paper.

[0060] Step S23: Use a sterile knife to cut a 2 cm × 2 cm piece of green pericarp, place it on LB solid medium, and incubate in an incubator at 30-37℃ for 3-7 days.

[0061] Step S24: After the colonies have grown, streak them onto new LB solid medium using an inoculation loop. Repeat this streaking process three times, then isolate and purify to obtain single colonies.

[0062] Step S25: Select a single colony and inoculate it into potato glucose aqueous liquid medium. Incubate at 28-37℃ for 24-48 h. If a distinct aroma is observed by smelling, the aroma-producing strain can be identified.

[0063] Example 2

[0064] Morphological observation of strains

[0065] Colony morphology observation:

[0066] The isolated and purified bacterial strain was streaked onto LB agar plates using the three-zone streak method and incubated at 33℃ under aerobic conditions for 48 h. Colony morphology was observed. Results are attached. Figure 1 .

[0067] Gram staining observation:

[0068] Colonies were picked from agar plates and smeared onto a glass slide for fixation; stained with ammonium oxalate crystal violet for 1 minute, then rinsed with distilled water; iodine solution was added to cover the smear and stained for about 1 minute, then rinsed with distilled water and blotted dry with absorbent paper; a few drops of 95% alcohol were added and gently shaken to decolorize, followed by 30 seconds of rinsing with distilled water and blotting dry; safranin staining was applied for 1 minute, followed by rinsing with distilled water; after drying, the specimen was examined under an oil immersion microscope. The strain HuaYu-3 showed a purple Gram stain, indicating a positive result. (See Appendix) Figure 3 .

[0069] Scanning electron microscopy observation:

[0070] Collect bacterial cells: Take 1 mL of bacterial culture after 48 h of activation culture, centrifuge at 4000 rpm and 4℃ for 5 min, discard the supernatant, and wash three times with 0.01 M phosphate buffer.

[0071] Fixation: Add 25% glutaraldehyde fixative (25% glutaraldehyde, prepared with 0.01M phosphate buffer) to centrifuge tubes and then fix overnight at 4°C.

[0072] Ethanol gradient dehydration: Centrifuge the fixed bacterial suspension, wash three times with pH 7.2 phosphate buffer, and wash the fixed cell samples once each with 30%, 50%, 70%, 80%, 90%, and 95% ethanol solutions, and twice with 100% ethanol (20 min each time, centrifuge at 4000 r / min for 5 min at each step, and discard the supernatant).

[0073] After freeze-drying, the morphology of the strain was observed using scanning electron microscopy (SEM); see appendix. Figure 2 .

[0074] Example 3

[0075] Molecular biological identification of the strain:

[0076] DNA was extracted from strain *Leifsonia shinshuensis* HuaYu-3, and 16S rDNA was amplified using the universal bacterial primer pair 27F / 1492R. The PCR reaction conditions were: 94℃ pre-denaturation for 3 min, followed by the following cycles: 94℃ denaturation for 40 s, 56℃ annealing for 35 s, 72℃ extension for 80 s, for 30 cycles; followed by a final extension at 72℃ for 10 min. After confirming good results by 1% agarose gel electrophoresis, the amplified product was sequenced, and the results were compared using BLAST on the NCBI website. The strain was confirmed as *Leifsonia shinshuensis*, and the phylogenetic tree was constructed as follows: Figure 5 As shown.

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

[0078]

[0079] Example 4

[0080] The strain HuaYu-3 produces characteristic aroma compounds of green tea through microbial fermentation.

[0081] (1) The *Leifsonia shinshuensis* HuaYu-3 described in this invention was streaked onto LB agar plates and cultured at 33°C for 48 h in an aerobic environment. The colonies were inoculated into liquid culture medium only after their diameter exceeded 2 mm.

[0082] (2) Inoculate colonies of appropriate size into shake tubes containing 3-4 mL of liquid LB medium and culture them for 32 h in an aerobic environment at 33°C and 200 rpm. Culture until the OD value of the bacterial suspension at 600 nm exceeds 0.5.

[0083] The bacterial suspension was transferred at a 5% inoculation volume ratio to a shake flask containing potato syrup liquid culture medium. Fermentation was carried out for 48 hours in an aerobic environment at 33°C and 120 rpm to obtain a fermentation broth with an elegant and refreshing green tea aroma.

[0084] Example 5

[0085] Sensory evaluation analysis of the fermentation broth of aroma-producing strains.

[0086] This embodiment utilizes the microbial fermentation of *Rifsonia nagano* HuaYu-3 to produce substances with the characteristic aroma of green tea; the substances with the characteristic aroma of green tea include: linalool, linalool, phenylethyl alcohol, and acetic acid, which are aroma compounds.

[0087] To determine the overall aroma characteristics of the fermentation broth from strain HuaYu-3, a sensory evaluation was conducted.

[0088] Aroma analysis was conducted by a team of well-trained experts (5 men and 5 women) at the Sino-Singapore International Joint Research Institute's South China Medicinal Herbs International R&D Center laboratory. After three months of training and testing, the team members demonstrated excellent abilities in aroma memory and the accuracy of their scores for each aroma descriptor.

[0089] The samples were evaluated by a trained panel of assessors. Ten assessors were asked to rate the fermentation broth on six aroma profiles, from 0 (imperceptible) to 3 (strongly perceptible), based on aroma intensity. The six aroma profiles were: green tea, grass, fruit, floral, cooked beans, and potato. The fermentation broth (50 mL) was poured into a 25°C glass. The data in the figure represents the average score from all trained panel members.

[0090] The results showed that the fermentation broth of strain HuaYu-3 had a high intensity of aroma properties of green tea and grass; see appendix. Figure 6 .

[0091] Example 6

[0092] HS-SPME-GC-MS detection of aroma-producing strain fermentation broth

[0093] Detection of aroma compounds in the fermentation broth of the strain: After centrifuging the fermentation broth at 8000 rpm for 10 min, 5 mL of the supernatant was taken into a 20 mL headspace vial, saturated with 2 g of sodium chloride, and the volatile aroma compounds were determined by headspace-solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS).

[0094] HS-SPME conditions:

[0095] The solid-phase microextraction fiber head was a DVB / CAR / PBDS composite fiber extraction head; the sample was equilibrated at 45 °C for 10 min and then extracted for 40 min. After extraction, the extraction head was placed in the gas phase inlet at 250 °C for 3 min for desorption.

[0096] GC conditions:

[0097] An HP-innowax gas chromatographic column (60 m × 250 μm × 0.25 μm, Agilent) was used. The sample port temperature was 250℃, and helium was used as the carrier gas (flow rate 1 mL·min). -1 The sample was analyzed under the following conditions, with a split ratio of 50:1.

[0098] The heating program is as follows: hold at an initial temperature of 40 °C for 5 min, then increase the temperature by 3 °C per minute. -1 Continue heating to 120 °C, hold for 3 minutes, then reduce the temperature by 10 °C / min. -1 The temperature was increased to 230 °C and held for 5 min.

[0099] MS conditions: EI ionization source, electron energy 70 eV, ion source temperature 230 °C; scan at 3.00 scans / s in the range of 35–500 m / z.

[0100] The mass spectrometry analysis database used was NIST14.L, which comes from the National Institute of Standards and Technology (NIST).

[0101] The total ion chromatogram of the fermentation broth of the aroma-producing strain described in this invention is shown in the HS-SPME-GC-MS analysis. Figure 6 .

[0102] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A strain of Rifkinella Nagano ( Leifsonia shinshuensis HuaYu-3, characterized in that, The bacteria were deposited at the Guangdong Provincial Center for Microbial Culture Collection on October 15, 2024, with accession number GDMCC No. 65270.

2. A method for producing aroma substances in green tea through fermentation, characterized in that: Includes the following steps: Step S1: The *Rifons naganoense* strain described in claim 1 (… Leifsonia shinshuensis HuaYu-3 should be streaked onto solid agar plates and cultured until the colony diameter exceeds 2 mm before being inoculated into liquid culture medium. Step S2: Inoculate colonies that have grown to a suitable size into shake tubes containing liquid culture medium and culture until the OD value of the bacterial suspension at a wavelength of 600 nm exceeds 0.5; Step S3: Inoculate the bacterial suspension of a certain concentration into a shake flask containing liquid culture medium at a certain volume ratio and culture it to finally obtain a fermentation broth with green tea aroma substances; the green tea aroma substances are linalool, linalool or geraniol.

3. The method for producing green tea aroma substances by fermentation according to claim 2, characterized in that: The solid plate culture medium mentioned in step S1 is LB solid culture medium, and the culture conditions are an aerobic environment, a temperature of 28-33℃, and a culture time of 48h. The liquid culture medium mentioned in step S2 is LB liquid culture medium, the volume of the shaker tube is 3-4 mL, the culture conditions are an aerobic environment, the temperature is 28-33℃, 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 an aerobic environment, the temperature is 28-33℃, the rotation speed is 120-150rpm, and the culture time is 36-48h.

4. The method for producing green tea aroma substances by fermentation according to claim 3, characterized in that: The solid plate medium mentioned in step S1 is LB solid medium, and the culture conditions are an aerobic environment, a temperature of 33°C, and a culture time of 48 hours; colonies with a diameter of more than 2 mm can be inoculated into liquid medium. The liquid culture medium mentioned in step S2 is LB liquid culture medium. The volume of the culture tube is 3-4 mL. The culture conditions are an aerobic environment, a temperature of 33℃, a rotation speed of 200 rpm, and a culture time of 32 h. The culture is carried out until the OD value of the bacterial suspension at a wavelength of 600 nm exceeds 0.

5. In step S3, the inoculation volume ratio is 5%, the liquid culture medium is potato glucose water liquid culture medium, the culture conditions are an aerobic environment, the temperature is 33℃, the rotation speed is 120rpm, and the culture time is 48h, finally obtaining a fermentation broth with green tea aroma substances.

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