Extraction method for separating aroma-producing fungi from litchis and application of aroma-producing fungi in tobaccos

Abstract

The invention relates to the technical field of aroma-producing bacteria extraction, and discloses an extraction method for separating aroma-producing bacteria from litchis and application of the aroma-producing bacteria in tobaccos, and the extraction method comprises the following steps: S1, selecting fresh litchis without plant diseases and insect pests, peeling off peels, weighing pulp, cutting the pulp into pieces, adding sterile water to prepare a pulp suspension diluted by 10 times, and putting into a shaking table, oscillating for 30 minutes under the conditions that the temperature is 30 DEG C and the speed is 200r / min, and standing for 10 minutes, so as to prepare the gradient diluent. According to the extraction method for separating the aroma-producing fungi from the litchis and the application of the aroma-producing fungi in tobaccos, the market competitiveness of products is enhanced, and the current situation of high-end cigar raw materials in China is relieved. And thirdly, the technology provides a new microbial aroma enhancement resource and technical direction for the tobacco industry, can be expanded to quality improvement of other tobacco varieties based on the principle of inducing conversion of organic matters in tobacco leaves by microbial fermentation, promotes technical innovation of improving the quality of raw materials through a biological engineering technology in the industry, and has a wide application prospect. The method has the potential of standardized production and large-scale application.

Description

Technical Field

[0001] This invention relates to the field of aroma-producing fungus extraction technology, specifically to a method for isolating aroma-producing fungi from litchi and its application in tobacco. Background Technology

[0002] Currently, my country's cigar tobacco industry is in a critical initial development stage, but it faces a series of severe challenges, such as a lack of variety and low content of aroma substances, as well as poor quality stability. These problems directly lead to insufficient competitiveness of domestically produced cigar tobacco in the high-end cigar raw material market, making it difficult to meet the stringent requirements of high-end products for the richness, permeability, and uniformity of raw material aroma. From a technical perspective, traditional cigar tobacco fermentation processes mainly rely on the role of natural microbial communities. However, this method has significant drawbacks. Not only is the fermentation cycle long, but the microbial community structure is also complex and difficult to control, resulting in a high degree of randomness in the generation of aroma substances. This makes it impossible to achieve precise improvement in tobacco quality. Furthermore, in existing technologies, using microbial fermentation technology to improve the aroma quality of tobacco has become a research hotspot, but most studies focus on the screening of traditional tobacco-related microorganisms, which suffers from problems such as low aroma production efficiency of strains and single aroma components.

[0003] Based on this, the present invention provides a method for extracting aroma-producing fungi from litchi and its application in tobacco. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a method for extracting aroma-producing bacteria from litchi and its application in tobacco. This method has the advantage of utilizing highly aroma-producing strains from litchi to ferment cigar tobacco leaves, thereby increasing the aroma and quality of cigar tobacco leaves and solving the problems mentioned in the background art.

[0005] This invention provides the following technical solution: a method for extracting aroma-producing fungi from litchi and its application in tobacco, comprising the following steps: S1. Select fresh, disease-free lychees, peel off the skin, weigh the pulp, cut it into small pieces, add sterile water to make a pulp suspension diluted 10 times, place it in a shaker and shake for 30 minutes at 30℃ and 200r / min, then let it stand for 10 minutes to obtain a gradient dilution. S2. Inoculate the diluted solution onto LB and YEPD plates, incubate at 37°C for 1-2 days, and isolate and purify single colonies by streak plate method; S3. The purified strain was inoculated into tobacco solid culture medium and cultured at 30℃ for 24-48h. Then, it was inoculated with cigar tobacco extract (material-to-liquid ratio 1:10) and cultured at 28℃ and 170r / min for 48-72h. The strain with strong aroma-producing ability was screened by smelling. S4. Morphological identification was performed using scanning electron microscopy (SEM). DNA samples of the bacterial strain were extracted according to the detailed instructions of the bacterial DNA extraction kit. 27F and 1492R were used as PCR amplification primers. Specific primer sequences for the 16S rDNA V4 region were also used: 16S 515F (5'-GTGCCAGCMGCCGCGGTAA-3') and 16S 806R (5'-GGACTACHVGGGTWTCTAAT-3'). PCR amplification and sequencing of relevant genes were performed. The specific PCR reaction system was configured as follows: 4 μL of dNTP Mixture and 5 μL of 10×PCR Buffer (containing Mg). 2+ 1 μL each of forward and reverse primers, 5 μL of sample DNA, and 0.25 μL of Ex Taq enzyme were added to bring the total volume to 50 μL. The mixture was thoroughly shaken and mixed. The PCR amplification program was as follows: 95℃ pre-denaturation for 3 minutes, followed by 35 cycles, each cycle consisting of 95℃ denaturation for 45 seconds, 50℃ annealing for 30 seconds, 72℃ extension for 1 minute, and finally extension at 72℃ for 5 minutes. The amplified product was stored at -20℃ and sequenced to identify the aroma-producing strain by comparing it with the NCBI database. S5. The identified strain was prepared into a bacterial suspension with an OD600 of 0.7 and sprayed onto cigar tobacco leaves at an inoculum of 15%. Fermentation was carried out for 14 days at 36°C and 70% humidity, with the tobacco leaves turned over every 6 hours. Fermentation was terminated by ultraviolet irradiation.

[0006] Preferably, the sample pretreatment method in S4 selects the strain with the highest aroma-producing score, inoculates it into LB liquid medium, and incubates it at 28°C for 12 hours. After washing, fixing, rinsing, dehydration and freeze-drying, the sample preparation is completed. Scanning electron microscopy is performed to obtain its surface morphology image and perform subsequent analysis.

[0007] Preferably, in step S1, the lychees are rinsed with sterile water, the fruit peels are removed in a clean bench, 10g of lychee pulp is weighed and chopped with sterilized scissors, and then placed into an Erlenmeyer flask containing 90mL of sterile water and shaken evenly.

[0008] Preferably, the gradient dilution solution in S1 is prepared in sequence in 10... -1 Up to 10 -5 Fruit peel dilutions at various concentration gradients.

[0009] Preferably, in step S2, 0.1 mL of the corresponding concentration of fruit peel dilution is drawn using a 1 mL pipette.

[0010] Preferably, in step S3, this operation is repeated to prepare multiple groups of bacterial fermentation broths. The control group is an equal volume of cigar tobacco extract with 10% sterile water added. Three groups of fermentation broths for each strain and a blank control group are prepared for parallel experiments. During the cultivation process, the fermentation broth of the aroma-producing bacteria is sampled every 1-2 days in a clean bench. The aroma changes of the fermentation broth are observed and recorded by smelling. Strains that can produce obvious aroma after fermentation are screened for further research. The strains are purified by the streak plate method and preserved by the glycerol preservation method. The aroma-producing ability is indicated by "+". The most promising aroma-producing strains are identified. Each treatment is performed in triplicate, and a blank control is set up.

[0011] Preferably, in step S5, healthy, uniformly sized tobacco leaves are selected, the midrib is removed, and the leaves are torn in half. Six halves of tobacco leaves are placed in each of two fermentation bottles, labeled as the experimental group and the control group. Three parallel experiments are conducted. The bacterial strain with the best aroma-producing effect, cultured on a solid culture medium for 24–48 hours, has its upper colony layer scraped off and suspended in sterile water to prepare a bacterial suspension. The OD600 is adjusted to 0.7. Using a sterile sprayer, the bacterial suspension is evenly sprayed onto both sides of the experimental group tobacco leaves at a 15% inoculum. After the sprayed tobacco leaves have dried, they are returned to the fermentation bottles and placed in a constant temperature incubator. A suitable temperature (36°C) and humidity (70%) are set, and the tobacco leaves are turned every 6 hours to ensure uniform fermentation. The control group is sprayed with an equal amount of sterile water. After 14 days of fermentation, the fermentation process is terminated by ultraviolet irradiation, and samples are then taken. One portion of the samples is used to roll cigars, and the other portion is used to determine the physicochemical indicators of the cigar tobacco leaves.

[0012] Compared with the prior art, the present invention has the following beneficial effects: This study describes a method for extracting aroma-producing fungi from litchi and its application in tobacco. The method utilizes microbial fermentation technology to screen a high-aroma-producing strain, L3, from litchi and apply it to the fermentation of cigar tobacco leaves. 16S rRNA gene sequencing identified the strain as *Acinetobacter*. Continuous flow analysis was used to determine the total sugar, total nitrogen, nicotine, and other conventional chemical components of the tobacco leaves before and after treatment. Simultaneous distillation-extraction-gas chromatography-mass spectrometry was used to analyze changes in aroma components. This method has several specific applications in the tobacco industry: Firstly, fermenting cigar tobacco leaves with the litchi-derived aroma-producing fungus L3 significantly increases the total amount of volatile aroma substances by 95.5%, adding sweet aroma components such as linalool and phenylacetaldehyde. Furthermore, the content of key aroma components such as megalotrienone, neophytadiene, and phytone is significantly higher than the control group, which can be directly used to optimize the aroma composition of cigar tobacco leaves, meeting the demand for rich aromas in high-end cigar raw materials. Secondly, the aroma richness and permeability of tobacco leaves fermented by this strain are significantly enhanced, improving sensory quality and contributing to a better smoking experience for domestically produced cigars, strengthening their market competitiveness and alleviating the current shortage of high-end cigar raw materials in my country. Thirdly, this technology provides the tobacco industry with new microbial flavoring resources and technological directions. Based on the principle of microbial fermentation inducing the transformation of organic matter in tobacco leaves, it can be extended to the quality improvement of other tobacco varieties, driving technological innovation in the industry to improve raw material quality through bioengineering technology, and has the potential for standardized production and large-scale application. Attached Figure Description

[0013] Figure 1 This is a schematic diagram showing the cell morphology of L3 under electron microscopy (a, b) and the colony morphology on LB medium (c). Figure 2 Table showing the aroma-producing characteristics of the strains of this invention; Figure 3 This is a phylogenetic tree of the 16S rDNA gene sequence of strain L3 constructed based on the neighbor-joining method in this invention; Figure 4 This is a schematic diagram showing the comparison of aroma components in cigar tobacco leaves before and after fermentation by strain L3 of the present invention; Figure 5 This is a continuation of the schematic diagram comparing the aroma components in cigar tobacco leaves before and after fermentation by strain L3 of this invention; Figure 6 Table 2 is a continuation of the schematic diagram comparing the analysis of aroma components in cigar tobacco leaves before and after fermentation by strain L3 of this invention; Figure 7 This is a schematic diagram comparing the types of volatile substances in cigar tobacco leaves in the L3 group and the CK group control group of this invention. Detailed Implementation

[0014] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments 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.

[0015] Please see Figure 1-7 A method for isolating aroma-producing fungi from litchi and its application in tobacco. Enrichment of aroma-producing strains: Select fresh lychees with no spots or pests on the surface. Rinse them thoroughly with sterile water. In a clean bench, peel the fruit and weigh 10g of lychee pulp. Chop the pulp with sterilized scissors and place it in an Erlenmeyer flask containing 90mL of sterile water. Shake well to obtain a 10-fold diluted pulp suspension. Place the suspension in a shaker and shake for 30 minutes at 30℃ and 200 r / min, then let it stand for 10 minutes. Take the obtained suspension and perform serial dilution to prepare peel dilutions with concentration gradients from 10⁻¹ to 10⁻⁵.

[0016] Isolation of aroma-producing strains: Using a 1 mL pipette, take 0.1 mL of the corresponding concentration of fruit peel dilution and aseptically inoculate it onto LB and YEPD plates, spreading it evenly. Incubate at 37°C for 1–2 days until single colonies appear on the plates. During the incubation process, observe the morphology of the colonies, select suitable strains, and isolate and purify them using the streak plate method.

[0017] Screening of aroma-producing strains The purified strain was inoculated onto tobacco solid culture medium plates and incubated at 30℃ for 24–48 h. Colonies with good growth were selected for secondary screening experiments. The initially screened bacterial strains were inoculated into LB and YEPD liquid media and cultured at 28℃ and 170 rpm for 24 h. The cultured bacterial solution was then inoculated into pre-prepared cigar tobacco extract at a material-to-liquid ratio of 1:10 and cultured at 28℃ and 170 rpm for 48-72 h to prepare fermentation broth. This process was repeated to prepare multiple groups of bacterial fermentation broths. The control group consisted of an equal volume of cigar tobacco extract with 10% sterile water added. Three groups of fermentation broths for each strain and a blank control group were prepared for parallel experiments. During the culture process, samples of the aroma-producing bacteria's fermentation broth were taken every 1-2 days in a clean bench. Aroma changes were observed and recorded using the olfactory method, and strains that did not produce aroma or had weak aroma-producing ability were removed. Aroma identification was performed by three personnel, evaluating the fermentation product based on aroma characteristics, off-odors, and aroma quantity. Strains that produced a significant aroma after fermentation were selected for further research. The strains were purified using the streak plating method and preserved using the glycerol preservation method. Aroma-producing ability was indicated by "+". The most promising aroma-producing strains were identified. Each treatment was performed in triplicate, with a blank control included.

[0018] Morphological identification of aroma-producing bacteria: Morphological identification was performed using scanning electron microscopy (SEM). Sample pretreatment followed the method described by Ma Chao for SEM. The strain with the highest aroma-producing score was selected and inoculated into LB broth, then incubated statically at 28 °C for 12 h. After washing, fixation, rinsing, dehydration, and freeze-drying, the samples were prepared. They were then sent to a professional testing institution for SEM examination using a Hitachi Su8100 scanning electron microscope (Hitachi, Japan) to obtain surface morphology images for subsequent analysis.

[0019] Molecular identification of aroma-producing bacteria: DNA samples were extracted from the bacterial strain according to the detailed instructions of the bacterial DNA extraction kit. 27F and 1492R were used as PCR amplification primers, along with specific primer sequences for the 16S rDNA V4 region: 16S 515F (5'-GTGCCAGCMGCCGCGGTAA-3') and 16S 806R (5'-GGACTACHVGGGTWTCTAAT-3'). PCR amplification and sequencing were performed on the relevant genes. The specific PCR reaction system was configured as follows: 4 μL of dNTP Mixture and 5 μL of 10×PCR Buffer (containing Mg). 2+1 μL each of forward and reverse primers, 5 μL of sample DNA, and 0.25 μL of Ex Taq enzyme were added to bring the total volume to 50 μL. The mixture was thoroughly shaken and mixed. The PCR amplification program was as follows: pre-denaturation at 95℃ for 3 minutes, followed by 35 cycles, each cycle consisting of denaturation at 95℃ for 45 seconds, annealing at 50℃ for 30 seconds, extension at 72℃ for 1 minute, and a final extension at 72℃ for 5 minutes. The amplified product was stored at -20℃ and sequenced to identify the aroma-producing strain by comparing it with the NCBI database.

[0020] Fermentation of cigar tobacco leaves: Healthy, uniformly sized tobacco leaves were selected, the midrib was removed, and the leaves were torn in half. Six halves of each leaf were placed in two separate fermentation bottles, designated as the experimental group and the control group, respectively. Three parallel experiments were conducted. The bacterial strain with the best aroma-producing effect, cultured on solid culture medium for 24–48 hours, was scraped off the top layer of the colonies and suspended in sterile water to prepare a bacterial suspension, with an OD600 adjusted to 0.7. Using a sterile sprayer, the bacterial suspension was evenly sprayed onto both sides of the experimental group tobacco leaves at a 15% inoculum. After the sprayed tobacco leaves dried, they were returned to the fermentation bottles and placed in a constant temperature incubator with a suitable temperature (36℃) and humidity (70%). The tobacco leaves were turned every 6 hours to ensure uniform fermentation. The control group was sprayed with an equal amount of sterile water. After 14 days of fermentation, the fermentation process was terminated by ultraviolet irradiation, and samples were then taken. One portion of the samples was used to roll cigars, and the other portion was used to determine the physicochemical properties of the cigar tobacco leaves.

[0021] Determination of volatile aroma components: Sample pretreatment was optimized as follows: 10.0 g of sample was accurately weighed, and 600 mL of distilled water and 40 mL of dichloromethane were added. Extraction was carried out in a 60℃ water bath for 2.5 h. After extraction, anhydrous sodium sulfate was added for thorough drying. The resulting extract was concentrated by rotary evaporation and brought to a final volume of 1 mL. Phenylacetyl acetate (an internal standard) was added, and the solution was filtered through a 0.22 μm filter before analysis. GC-MS analysis was performed under the following conditions: a DB-WAX column (30.0 m × 250 μm, stationary phase thickness 0.25 μm) was used, with helium (He) as the carrier gas at a flow rate of 1.0 mL / min. The injection volume was 1 μL. The temperature program was set as follows: initial temperature 50℃ held for 2 min; ramped up to 100℃ at a rate of 5℃ / min; ramped up to 240℃ at a rate of 6℃ / min and held for 15 min; the transfer line temperature was set to 240℃. The ionization source used electron impact (EI) mode with an electron energy of 70 eV; the ion source temperature was 230℃, and the quadrupole temperature was 150℃. Mass spectrometry acquisition used Scan mode, covering the mass range of m / z 35~500, with a solvent delay time of 3.5 min.

[0022] Routine chemical composition determination of fermented cigar tobacco leaves: The reducing sugar and total sugar content in tobacco leaves were determined according to the "Determination of Water-Soluble Sugars in Tobacco and Tobacco Products - Continuous Flow Method" (YC / T 159—2019); the nicotine content was determined according to the "Determination of Total Alkaloids in Tobacco and Tobacco Products - Continuous Flow Method" (YC / T 160—2002); the total nitrogen content was determined according to the "Determination of Total Nitrogen in Tobacco and Tobacco Products - Continuous Flow Method" (YC / T161—2002); the chlorine content was determined according to the "Determination of Chlorine in Tobacco and Tobacco Products - Continuous Flow Method" (YC / T 162—2011); and the potassium content was determined according to the "Determination of Potassium in Tobacco and Tobacco Products - Continuous Flow Method" (YC / T 217-2007).

[0023] Sensory evaluation of cigar tobacco leaves: The fermented cigar tobacco leaves were rolled into cigar sticks and kept at 22°C and 60% relative humidity for 2 days to balance the moisture content. Tobacco leaves that were not sprayed with fermentation bacteria were used as a control group. The samples were then sent to the Sichuan Tobacco Key Laboratory for Cigars for sensory evaluation by experts.

[0024] Please see Figure 1 The colony morphology and cell morphology of various microorganisms exhibit specific differences, which are important criteria for identifying different microbial species. Morphological observation was used to identify strain L3. After culturing on LB medium for 2–3 days, as shown... Figure 1 As shown in (c), the colonies of strain L3 are round, raised, and moist, ranging in color from white to light yellow. The scanning electron microscope (SEM) observation results of strain L3 are as follows... Figure 1 As shown in (a, b), under electron microscopy, the cells exhibit a short rod-like morphology, a relatively rough cell surface, no flagella, and a relatively uniform texture, with a size of 2.00–5.00 micrometers. In summary, the colony and cell morphology of L3 are quite similar to those of Acinetobacter.

[0025] Please see Figure 2 After enrichment and isolation of the bacterial strains, 15 test strains were selected. These strains were inoculated onto tobacco solid agar plates and cultured in a constant temperature and humidity incubator for 2–3 days. Eight strains with larger colony morphology and better growth were selected: L3, C5, M9, Li1, Li2, M3, L5, and M1. These eight strains were then inoculated into cigar tobacco extract culture medium and cultured for another 2–3 days. After culture, three individuals separately performed olfactory identification on the cultured tobacco extract samples. The combined olfactory identification results from the three individuals were then analyzed. Figure 2 As shown in the table, the results indicate that strain L3 exhibits good aroma-producing ability, and its fermentation broth has unique fruity and sweet aroma characteristics, showing good potential for aroma enhancement applications.

[0026] Please see Figure 3 By determining the 16S rDNA sequence fragment of strain L3, a gene sequence of 1480 bp was obtained. The obtained sequence was submitted to the NCBI nucleic acid sequence database and compared with known homologous sequences. A phylogenetic tree was constructed using the neighbor-joining method. The results showed that the 16S rDNA gene sequence of strain L3 was most closely related to Acinetobacter seifertii strain AS71 (CP061568.1) in NCBI.

[0027] Please see Figure 4 , Figure 5 , Figure 6 and Figure 7 The aroma components of cigar tobacco leaves supplemented with strain L3 and those of the control group were determined by GC-MS. The internal standard method was used for quantitative analysis of the content of each aroma component. The results are shown in [Figure number missing]. Figure 4 , Figure 5 and Figure 6 There was a significant difference in the content of aroma components between the L3 group and the CK group. Figure 7 The L3 group yielded 39 aroma components, including 5 alcohols, 5 aromatic compounds, 5 aldehydes, 6 acids, 12 ketones, 2 esters, 3 alkenes, and 1 alkane. The CK group, on the other hand, detected 34 aroma components, primarily consisting of 6 aromatics, 2 alcohols, 4 aldehydes, 1 acid, 3 alkenes, 12 ketones, 4 alkanes, 1 ester, and 1 phenol. Analysis of aroma content changes revealed that the L3 strain significantly increased the variety and content of characteristic aroma components in cigar tobacco leaves. The increases in phytone, neophytadiene, megastigmatotrienone, phenylethanol, and palmitic acid were particularly significant. Compared to the CK group, the L3 group added 11 aroma components, including linalool, phenylacetaldehyde, pentadecaldehyde, and phytol. Meanwhile, the content of some aroma components in group L3 also decreased, such as ethylbenzene and trioxymethylene, while toluene, benzaldehyde, and four long-chain alkanes completely disappeared in group L3. Comprehensive analysis of the effects of adding strain L3 on aroma components during tobacco fermentation showed that this strain has a significant impact on the composition and content of volatile aroma substances in tobacco leaves during fermentation, which positively contributes to improving the quality of fermented tobacco leaves.

[0028] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0029] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for extracting aroma-producing fungi from litchi, characterized in that, Includes the following steps: S1. Select fresh, disease-free lychees, peel off the skin, weigh the pulp, cut it into small pieces, add sterile water to make a pulp suspension diluted 10 times, place it in a shaker and shake for 30 minutes at 30℃ and 200r / min, then let it stand for 10 minutes to obtain a gradient dilution. S2. Inoculate the diluted solution onto LB and YEPD plates, incubate at 37°C for 1-2 days, and isolate and purify single colonies by streak plate method; S3. The purified strain was inoculated into tobacco solid culture medium and cultured at 30℃ for 24-48h. Then, it was inoculated with cigar tobacco extract (material-to-liquid ratio 1:10) and cultured at 28℃ and 170r / min for 48-72h. The strain with strong aroma-producing ability was screened by smelling. S4. Morphological identification was performed using scanning electron microscopy (SEM). DNA samples of the bacterial strain were extracted according to the detailed instructions of the bacterial DNA extraction kit. 27F and 1492R were used as PCR amplification primers. Specific primer sequences for the 16S rDNA V4 region were also used: 16S 515F (5'-GTGCCAGCMGCCGCGGTAA-3') and 16S 806R (5'-GGACTACHVGGGTWTCTAAT-3'). PCR amplification and sequencing of relevant genes were performed. The specific PCR reaction system was configured as follows: 4 μL of dNTP Mixture and 5 μL of 10×PCR Buffer (containing Mg). 2+ 1 μL each of forward and reverse primers, 5 μL of sample DNA, and 0.25 μL of Ex Taq enzyme were added to bring the total volume to 50 μL. The mixture was thoroughly shaken and mixed. The PCR amplification program was as follows: 95℃ pre-denaturation for 3 minutes, followed by 35 cycles, each cycle consisting of 95℃ denaturation for 45 seconds, 50℃ annealing for 30 seconds, 72℃ extension for 1 minute, and finally extension at 72℃ for 5 minutes. The amplified product was stored at -20℃ and sequenced to identify the aroma-producing strain by comparing it with the NCBI database. S5. The identified strain was prepared into a bacterial suspension with an OD600 of 0.7 and sprayed onto cigar tobacco leaves at an inoculum of 15%. Fermentation was carried out for 14 days at 36°C and 70% humidity, with the tobacco leaves turned over every 6 hours. Fermentation was terminated by ultraviolet irradiation.

2. The method for extracting aroma-producing fungi from litchi according to claim 1, characterized in that: The sample pretreatment method of S4 selects the strain with the highest aroma-producing score, inoculates it into LB liquid medium, and incubates it at 28°C for 12 hours. After washing, fixing, rinsing, dehydration and freeze-drying, the sample preparation is completed. Scanning electron microscopy is performed to obtain its surface morphology image and perform subsequent analysis.

3. The method for extracting aroma-producing fungi from litchi according to claim 1, characterized in that: In step S1, the lychees need to be rinsed with sterile water, and the fruit peels are removed in a clean bench. 10g of lychee pulp is weighed and chopped with sterilized scissors. The pulp is then placed in an Erlenmeyer flask containing 90mL of sterile water and shaken well.

4. The method for extracting aroma-producing fungi from litchi according to claim 1, characterized in that: The gradient dilution solution in S1 is prepared sequentially in 10 steps. -1 Up to 10 -5 Fruit peel dilutions at various concentration gradients.

5. The method for extracting aroma-producing fungi from litchi according to claim 1, characterized in that: In step S2, 0.1 mL of the corresponding concentration of fruit peel dilution is taken using a 1 mL pipette.

6. The method for extracting aroma-producing fungi from litchi according to claim 1, characterized in that: In step S3, this operation was repeated to prepare multiple groups of fermentation broths for the bacterial strains. The control group consisted of an equal volume of cigar tobacco extract with 10% sterile water added. Three groups of fermentation broths for each strain and a blank control group were prepared for parallel experiments. During the cultivation process, the fermentation broth of the aroma-producing bacteria was sampled every 1-2 days in a clean bench. The aroma changes of the fermentation broth were observed and recorded by smelling. Strains that could produce obvious aromas after fermentation were screened for further research. The strains were purified using the streak plate method and preserved using the glycerol preservation method. The aroma-producing ability was indicated by "+". The most promising aroma-producing strains were identified. Each treatment was performed in triplicate, and a blank control was also included.

7. The method for extracting aroma-producing fungi from litchi according to claim 1, characterized in that: In step S5, healthy, uniformly sized tobacco leaves were selected, the midrib was removed, and the leaves were torn in half. Six halves of each leaf were placed in two separate fermentation bottles, designated as the experimental group and the control group. Three parallel experiments were conducted. The bacterial strain with the best aroma-producing effect, cultured on solid culture medium for 24–48 hours, was scraped off the top layer of the colonies and suspended in sterile water to prepare a bacterial suspension. The OD600 was adjusted to 0.

7. Using a sterile sprayer, the bacterial suspension was evenly sprayed onto both sides of the experimental group tobacco leaves at a 15% inoculum. After the sprayed tobacco leaves dried, they were returned to the fermentation bottles and placed in a constant temperature incubator. A suitable temperature (36℃) and humidity (70%) were set, and the tobacco leaves were turned every 6 hours to ensure uniform fermentation. The control group was sprayed with an equal amount of sterile water. After 14 days of fermentation, the fermentation process was terminated by ultraviolet irradiation, and samples were then taken. One portion of the samples was used to roll cigars, and the other portion was used to determine the physicochemical properties of the cigar tobacco leaves.

8. The application of the aroma-producing fungus prepared by the extraction method for isolating aroma-producing fungi from litchi according to any one of claims 1-7 in tobacco.

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