Strain of bacillus sorola Desert, compound bacteria thereof and application of compound bacteria in tobacco mellowing

Abstract

The invention provides a bacillus soralensis strain, a compound bacterium thereof and application of the bacillus soralensis strain in tobacco mellowing, the bacillus soralensis strain DQ-3-2-7 is preserved in the China General Microbiological Culture Collection Center on November 20, 2023, the preservation number is CGMCC No.29072, and the preservation address is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The bacillus sorola DQ-3-2-7 strain can alcoholize tobacco leaves, so that volatile compounds are remarkably changed, the flower fragrance, radix aucklandiae and ketone compounds related to sweet and fruit fragrance are remarkably increased, and meanwhile, the flower fragrance type compounds are increased; irritant substances such as acetic acid and o-cresol are reduced, so that the whole flavor is more coordinated, and the quality of the tobacco leaves is remarkably improved; meanwhile, when the strain containing the bacillus sorola DQ-3-2-7 and the pseudomonas Hunan are used for fermentation, the tobacco leaf quality improvement effect is better. The bacillus sorola DQ-3-2-7 strain and the compound bacteria can be applied to the surface of tobacco shreds to alcoholize tobacco leaves, so that the flavor of the tobacco leaves is improved, and the quality of the tobacco leaves is improved.

Description

Technical Field

[0001] This invention relates to the field of tobacco fermentation technology, and more specifically, to a strain of Sonora desert Bacillus, its complex strains, and their application in tobacco aging. Background Technology

[0002] Tobacco is one of the world's important economic crops. With the upgrading of consumer demand, the requirements for tobacco leaf quality in the mid-to-high-end cigarette market are increasing. Currently, the industry faces insufficient utilization of low-quality tobacco leaves and excessive demand for high-quality tobacco leaves. These problems can be addressed by using tobacco leaf aging technology to improve tobacco leaf quality and increase its utilization value.

[0003] The tobacco leaves harvested that year have varying degrees of defects in their inherent quality. Direct combustion of these leaves produces a strong, unpleasant odor, making them unsuitable for direct processing into cigarettes for sale. They require a certain degree of aging to enrich the flavor compounds, reduce irritants, and improve the overall smoking quality.

[0004] Aging is the core technology of tobacco fermentation, encompassing both natural and artificial aging processes. Natural aging relies on the slow action of environmental microorganisms and enzymes, involving 1-3 years of storage under controlled temperature and humidity. Through microbial metabolism and non-enzymatic chemical oxidation, substances such as cellulose and protein in tobacco leaves are gradually transformed into small-molecule aroma components such as ionone, damascone, and furfural, thereby improving the sensory quality of the tobacco leaves. Microorganisms play a dominant role in this process: their secreted extracellular enzymes can efficiently degrade macromolecules such as pectin and starch that negatively impact flavor, while simultaneously synthesizing key aroma substances. However, natural aging has serious drawbacks: its excessively long cycle leads to high storage costs and low capital turnover; the uncontrollable environment results in batch-to-batch quality variations; and the process is passive, relying on the natural environment and unable to target flavor. Therefore, developing highly controllable and shorter-cycle artificial aging technology has become an industry consensus. Its core is to accelerate the process of tobacco leaf quality improvement by inoculating functional microorganisms or optimizing fermentation parameters.

[0005] Current research on fermented tobacco leaves has revealed that the artificial addition of specific microorganisms can effectively accelerate the aging process and regulate the content of chemical components in tobacco leaves, such as increasing aroma compounds, reducing nicotine content, lowering the content of tobacco-specific nitrosamines (TSNA), and reducing the content of macromolecules such as protein and starch, thereby improving tobacco quality. Current research on microorganisms used in tobacco aging includes bacteria and fungi. For example, Chinese invention patent CN105543122A discloses a microorganism composed of Bacillus subtilis, Bacillus licheniformis, and Bacillus amylase-lysing to age tobacco leaves and improve aging quality. Therefore, isolating and screening more functional microorganisms and inoculating them to enhance the tobacco aging process is of great significance for enriching tobacco flavor and improving tobacco quality. Summary of the Invention

[0006] The purpose of this invention is to solve the aforementioned technical problems and provide a strain of Sonora desert Bacillus, which can enhance the aging process of tobacco leaves, enrich the flavor of tobacco leaves, and improve the quality of tobacco leaves. Another purpose of this invention is to provide a compound bacteria formed by combining Sonora desert Bacillus with other strains, which can further enhance the aging process of tobacco leaves, enrich the flavor of tobacco leaves, and improve the quality of tobacco leaves. A further purpose of this invention is to provide the application of Sonora desert Bacillus and the compound bacteria formed by combining Sonora desert Bacillus with other strains in the aging process of tobacco leaves.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a strain of Sonora desert Bacillus ( Bacillus sonorensis The Sonora desert Bacillus strain DQ-3-2-7 was deposited on November 20, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 29072, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0008] This invention isolates and preserves a novel strain, DQ-3-2-7, from the surface of aged tobacco leaves, which can accelerate the aging process and improve the quality of tobacco leaves. Sequencing analysis revealed that the 16S rRNA gene sequence of this strain is shown in SEQ ID No:1. Nucleic acid sequence alignment of the sequenced material in GenBank confirmed that the strain is *Bacillus desertica*.

[0009] This strain can significantly alter the volatile compounds in tobacco leaves inoculated with *Bacillus desertica*. After aging, the tobacco leaves show a significant increase in ketones related to floral, woody, and sweet fruit aromas (such as megastigmatrienone, damascene, and β-ionone), while also increasing the content of floral compounds such as phenylacetaldehyde, benzyl alcohol, dihydroactinol, and trans-3-hexen-1-ol. It also reduces irritating substances such as nicotine, acetic acid, and o-cresol, resulting in a more harmonious overall flavor and significantly improving the quality of the tobacco leaves.

[0010] This invention provides a compound bacteria comprising the above-mentioned Sonora desert Bacillus strain DQ-3-2-7 and Hunan Pseudomonas ( Pseudomonas hunanensis ).

[0011] Based on the potential interaction between the two strains in accelerating aging efficiency and improving tobacco quality, the effect of improving tobacco quality is better when both are present during fermentation.

[0012] The present invention provides a probiotic agent for tobacco aging, comprising the above-mentioned Sonora desert Bacillus strain DQ-3-2-7 and / or the above-mentioned compound bacteria.

[0013] Furthermore, the probiotic agent is a solid or liquid probiotic agent, and the number of live bacteria in the probiotic agent is not less than 1×10⁻⁶. 8 CFU / ml or 1×10 8 CFU / g.

[0014] Preferably, the probiotic agent has a live bacteria count of 1×10⁻⁶. 8 CFU / g (CFU / mL), 1×10 9 CFU / g (CFU / mL), 1×10 10 CFU / g (CFU / mL), 5×10 10 CFU / g (CFU / mL), 1×10 11 CFU / g (CFU / mL), 5×10 11 CFU / g (CFU / mL), 1×10 12 CFU / g (CFU / mL), 3×10 12 CFU / g (CFU / mL), 5×10 12 CFU / g (CFU / mL), 1×10 13 CFU / g (CFU / mL), etc., and other specific point values ​​within this range can be selected, which will not be elaborated here.

[0015] Furthermore, the ratio of viable bacteria of Sonora desert Bacillus and Hunan Pseudomonas in the compound bacteria is 1:(0.8~1.2).

[0016] Based on the potential interaction between the two strains in accelerating aging efficiency and improving tobacco quality, the effect is better when the two meet the above-mentioned specific ratio. Preferably, the ratio of viable bacteria of Sonora desertis and Pseudomonas hunanense in the compound bacteria is 1:0.8, 1:0.9, 1:1.0, 1:1.1, and 1:1.2.

[0017] Furthermore, the probiotic agent described herein may be in the form of a solution, lyophilized powder, capsule, tablet, or granule.

[0018] Furthermore, the probiotic agent is in the form of a solution, which can be prepared by the following method: the above-mentioned Sonora desert Bacillus strain or compound bacteria are inoculated into a culture medium and activated and fermented sequentially to obtain a fermentation broth; the fermentation broth is centrifuged, and the bacterial sludge is resuspended in a solvent to obtain a bacterial suspension, which is the solution probiotic agent; Furthermore, the probiotic agent is in the form of a freeze-dried powder, which can be prepared by the following method: the above-mentioned Sonora desert Bacillus strain or compound strain is inoculated into a culture medium and activated and fermented sequentially to obtain a fermentation broth; the fermentation broth is centrifuged, and the bacterial sludge is mixed with a protective agent and then freeze-dried to obtain bacterial powder; Preferably, the culture medium comprises LB medium containing tobacco powder; Preferably, the activation and fermentation cultures are each carried out independently at 35-39°C; Preferably, the step of obtaining the bacterial powder further includes mixing the two bacterial powders according to the ratio of live bacteria count.

[0019] This invention provides the application of the above-mentioned Sonora desert Bacillus strain DQ-3-2-7 or the above-mentioned compound bacteria in tobacco aging.

[0020] Furthermore, the method of application involves applying the aforementioned Sonora desert Bacillus strain DQ-3-2-7 or the aforementioned compound bacteria to the surface of tobacco shreds for fermentation to improve the flavor and / or enhance the quality of tobacco leaves.

[0021] Furthermore, the fermentation conditions are as follows: fermentation temperature 35~39℃, fermentation time 5-10 days.

[0022] Furthermore, the Sonora desert Bacillus strain DQ-3-2-7 or the compound strain used in the fermentation has a viable count of not less than 1 × 10⁻⁶ per unit mass. 8 CFU, and its weight percentage with tobacco leaves is 5-10%.

[0023] The beneficial effects of this invention are as follows: (1) This invention has isolated and preserved a new Sonora desert Bacillus strain DQ-3-2-7 from the surface of aged tobacco leaves, which can accelerate the aging efficiency of tobacco leaves and improve the quality of tobacco leaves. This strain has a good ability to produce pectinase, which can significantly change the volatile compounds of aged tobacco leaves inoculated with this Sonora desert Bacillus. After aging, the tobacco leaves significantly increase the content of ketones related to floral, woody and sweet fruit aromas (such as megastigmatrienone, damascene, β-ionone), while increasing the content of floral compounds such as phenylacetaldehyde, benzyl alcohol, dihydroactinol, and trans-3-hexen-1-ol; and reducing the content of irritating substances such as nicotine, acetic acid and o-cresol, making the overall flavor more harmonious.

[0024] (2) The present invention further provides a compound bacteria comprising the above-mentioned Sonora desert Bacillus strain DQ-3-2-7 and Hunan Pseudomonas ( Pseudomonas hunanensis Based on the potential interaction between the two strains in accelerating aging efficiency and improving tobacco quality, the effect of improving tobacco quality is better when both are present during fermentation.

[0025] (3) The Sonora desert Bacillus strain DQ-3-2-7 and the compound bacteria can be applied to the surface of tobacco shreds for fermentation to improve the flavor and / or enhance the quality of tobacco leaves. They can be applied to the aging of tobacco leaves and have the application prospect of improving the quality of tobacco leaves and enhancing the aging efficiency.

[0026] The DQ-3-2-7 strain involved in this invention is classified as Sonora desert bacillus (Bacillus desertis). Bacillus sonorensis The depositary institution is the China General Microbiological Culture Collection Center (CGMCC), the deposit date is November 20, 2023, the accession number is CGMCC No. 29072, and the address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0027] Figure 1 This is a plate colony morphology diagram of Bacillus desertis DQ-3-2-7 from Sonora.

[0028] Figure 2 This is a phylogenetic tree of Bacillus desertis DQ-3-2-7 from Sonora.

[0029] Figure 3 This is the growth curve of Bacillus desertis DQ-3-2-7 from Sonora. Detailed Implementation

[0030] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. 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 should fall within the scope of protection of the present invention.

[0031] The following examples involve the routinely preserved strain of *Pseudomonas hunanense* (Hunan Pseudomonas). Pseudomonas hunanensis ) Example 1: Isolation, Screening, and Physiological and Biochemical Identification of Strains 1. Test Methods (1) Isolation and culture of strains Samples of mellowed tobacco leaves were placed in liquid culture medium for enrichment and cultured at 37°C with shaking for 48 h. The bacterial suspension was then serially diluted with sterile water to a concentration of 10-1. -3 ~10 -6Serially diluted samples were spread onto isolation medium plates and incubated at 37°C for 48 h. After purifying the colonies by streaking multiple times, single colonies were picked and inoculated into LB medium and incubated at 37°C with shaking for 48 h. The bacterial culture was then stored in glycerol tubes. Simultaneously, 1 wt% of the culture was inoculated into fermentation medium and incubated at 37°C with shaking for 72 h to obtain the fermentation broth.

[0032] The liquid culture medium was formulated as follows: 20 g / L glucose, 2 g / L peptone, 2 g / L yeast extract, 0.5 g / L magnesium sulfate heptahydrate, 1.0 g / L dipotassium hydrogen phosphate, 0.46 g / L potassium dihydrogen phosphate, and 2 g / L tobacco powder, and was sterilized at 115℃ for 20 min.

[0033] The formulation of the isolation culture medium plates is as follows: glucose 20 g / L, peptone 2 g / L, yeast extract 2 g / L, magnesium sulfate heptahydrate 0.5 g / L, dipotassium hydrogen phosphate 1.0 g / L, potassium dihydrogen phosphate 0.46 g / L, tobacco powder 2 g / L, agar 18 g / L, sterilized at 115℃ for 20 min.

[0034] The LB medium formula is as follows: 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L sodium chloride, sterilized at 121℃ for 20 min.

[0035] The fermentation medium was prepared by adding 20 g / L of tobacco powder to deionized water and sterilizing at 115℃ for 20 min.

[0036] (2) The flavor compounds in the fermentation broth of each tobacco leaf were detected by gas chromatography-mass spectrometry and aroma-producing strains were screened.

[0037] The analytical conditions for gas chromatography and mass spectrometry were set accordingly. The fermentation broth was pretreated before HP-HSME-GC-MS analysis was performed. The specific method is as follows: Pretreatment: Take 6 mL of fermentation broth and add it to a 20 mL gas chromatography-mass extraction flask. Add 2.88 g of sodium chloride and 10 μL of 20 mg / L 2-octanol as an internal standard.

[0038] GC-MS analysis was performed using a gas chromatography-mass spectrometry system manufactured by Agilent Technologies, USA, with automated sample injection. The extraction head was conditioned for 30 min and then inserted into the GC-MS bottle at a distance of 3 cm from the liquid surface to begin extraction (extraction temperature 50℃, extraction time 35 min). After extraction, the extraction head was inserted into the GC-MS system for analysis (extraction temperature 250℃, extraction time 5 min).

[0039] Chromatographic conditions: A DB-Wax capillary column (60 m length, 0.32 mm inner diameter, 0.25 μm film thickness) was used. The injection port temperature was 250 °C, and the temperature program was 50 °C, held for 2 min, then increased to 145 °C at 3 °C / min, and then increased to 230 °C at 15 °C / min, held for 3 min. Helium was used as the carrier gas at a flow rate of 1.0 mL / min, with a split ratio of 20:1.

[0040] Mass spectrometry conditions: EI ionization source, ion source temperature 260℃, electron bombardment energy: 70 eV, scanning mass range: 33-350 m / z.

[0041] The samples were analyzed under the above GC-MS conditions to obtain the total ion chromatogram of the fermentation broth. Each chromatographic peak was analyzed by fragmentation and qualitative analysis using the NIST 2.0 mass spectrometry library to determine the chemical structure of the products. Using blank fermentation broth as a control, strains showing a significant increase in total volatile compounds (P<0.05) and a significant increase in the content of alcohols and ketones (P<0.05) were considered superior strains and their morphological characteristics were observed.

[0042] (2) Physiological and biochemical identification The aerobic characteristics, optimal growth temperature, and biochemical properties of this strain were detected, mainly referring to the "Handbook of Systematic Identification of Common Bacteria" (Dong Xiuzhu, Cai Miaoying. 2001. Handbook of Systematic Identification of Common Bacteria. Beijing: Science Press).

[0043] 2. Test Results (1) A superior strain was obtained through screening. The plate colony morphology of the strain *Bacillus desertica* was observed and recorded, such as... Figure 1 As shown, the bacterial colonies on the culture medium are round with neat edges, smooth and moist surface, and milky white in color. It is named DQ-3-2-7.

[0044] (2) The physiological and biochemical characteristics of this strain are as follows: protease activity is 55.3 U, cellulase activity is 69.4 U, and amylase activity is 49.3 U (enzyme activity determination method: protease activity unit definition: at 40℃ and pH 7.2, the amount of enzyme that decomposes casein to produce 1 μg of tyrosine in 1 mL of fermentation broth for 10 min is defined as 1 enzyme activity unit, expressed as U; amylase activity unit definition: at 35℃ and pH 7.2, the amount of enzyme that produces 1 μg of glucose in 1 mL of fermentation broth for 1 min is defined as 1 enzyme activity unit, expressed as U; cellulase activity unit definition: at 50℃ and pH 7.2, the amount of enzyme that decomposes substrate to produce 1 μg of glucose in 1 mL of fermentation broth for 1 h is defined as 1 enzyme activity unit, expressed as U); it has motility; in TSB liquid medium, shake flask at 37℃ and culture at 180 rpm, its growth curve is as follows. Figure 3 As shown.

[0045] Example 2 Molecular identification of the strain 1. Test Methods Collect bacterial culture of strain DQ-3-2-7 in its growth phase by centrifugation. Add 500 μL of DNA lysis buffer and 2.5 μL of proteinase K, and incubate in a metal bath at 55℃ and 1500 rpm for 3 h. Add DNA extraction buffer, mix well, centrifuge at 12000 rpm, and discard the supernatant. Wash twice with anhydrous ethanol and sterile water, dry, and add TE buffer to obtain the extracted DNA.

[0046] PCR amplification method: The bacterial 16S rRNA gene fragment was amplified using universal primers 27F and 1492R. The PCR program was set as follows: pre-denaturation at 95℃ for 7 min, template denaturation at 95℃ for 25 s, annealing at 56℃ for 30 s, extension at 72℃ for 20 s, for 35 cycles. Finally, the product was held at 72℃ for 7 min to ensure complete extension and stored at 4℃. The amplified product was sent to Shanghai Sangon Biotech Co., Ltd. for sequencing to obtain the sequence results.

[0047] 2. Test Results The 16S rRNA gene sequence of *Bacillus desertis* strain DQ-3-2-7 is shown in SEQ ID No:1. A BLAST comparison was performed in GenBank, and the results are shown in Table 1. Its phylogenetic tree is as follows: Figure 2 As shown, the strain was identified as Sonora desert bacillus (Bacillus desertis). Bacillus sonorensis ).

[0048] The DQ-3-2-7 strain was preserved and classified as Sonora desert bacillus. Bacillus sonorensisThe depositary institution is the China General Microbiological Culture Collection Center (CGMCC), the deposit date is November 20, 2023, the accession number is CGMCC No. 29072, and the address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0049] Table 1. Comparison results of 16S rRNA in strain QR-3-2-7

[0050] Example 3: Determination of Volatile Compound Content in Tobacco Leaf Alcoholization by Sonora desert Bacillus and its Complex 1. Test Methods (1) Experimental grouping: The experiment was divided into five groups, namely the control group, Sonora desert Bacillus group, Hunan Pseudomonas group, Sonora desert Bacillus + Hunan Pseudomonas group and commercial Sonora desert Bacillus group; among them, Sonora desert Bacillus group, Hunan Pseudomonas group, Sonora desert Bacillus + Hunan Pseudomonas group and commercial Sonora desert Bacillus group were respectively designated as experimental group 1, experimental group 2, experimental group 3 and experimental group 4.

[0051] (2) The Sonora desert bacillus strain, Hunan Pseudomonas strain and commercial Sonora desert bacillus were inoculated into LB liquid medium and cultured at 37℃ and 200 rpm for 24 h with shaking. The obtained bacterial suspension was centrifuged at 4℃ and 10000 rpm for 10 min. After removing the supernatant, the bacterial cells were washed twice with sterile water. The washed bacterial cells were diluted and resuspended with sterile water to prepare Sonora desert bacillus suspension, Hunan Pseudomonas suspension, and mixed bacterial suspension 1 of Sonora desert bacillus and Hunan Pseudomonas (the ratio of viable bacteria was 1:1) (the total viable bacteria count of each group of bacterial suspensions was 1×10⁻⁶). 8 (CFU / mL).

[0052] (3) The bacterial suspension of each group was sprayed evenly on the surface of tobacco shreds at an inoculation amount of 10 wt%. The tobacco leaves of the control group were not inoculated with bacterial suspension. All were fermented at 37℃ for 7 days. The volatile compound components of the treated tobacco leaf samples were detected by gas chromatography-mass spectrometry and compared.

[0053] The samples were analyzed according to the GC-MS conditions in Example 1 to obtain the total ion chromatogram of the fermented tobacco leaves. Each chromatographic peak was analyzed by fragment analysis and by consulting the NIST2.0 mass spectrometry library to determine the chemical structure of the product and obtain the types and relative contents of volatile compounds in the tobacco leaves.

[0054] 2. Test Results The changes in volatile compounds were statistically analyzed separately, and the results are shown in Tables 2-5.

[0055] Table 2. Newly generated volatile compounds and their relative contents in experimental groups 1 and 3 compared to the control group.

[0056] Note: NA indicates that the compound was not detected.

[0057] Table 3. Volatile compounds whose content increased in experimental groups 1 and 3 compared to the control group.

[0058] Table 4. Volatile compounds in experimental groups 1 and 3 showing inconsistent increases or decreases relative to the control group.

[0059] Note: NA indicates that the compound was not detected.

[0060] Table 5. Volatile compounds whose content decreased in experimental groups 1 and 3 compared to the control group.

[0061] Note: NA indicates that the compound was not detected.

[0062] As shown in Tables 2-5, overall, experimental group 3 exhibited the best treatment effect, with the most outstanding comprehensive aroma enhancement and quality improvement capabilities. This group not only generated the most diverse and comprehensive range of beneficial aroma components (such as phenylacetaldehyde, which provides a high-end floral aroma, and furfural, which provides caramel sweetness), but more importantly, it greatly promoted the synergistic accumulation of core tobacco aroma substances (such as neophytadiene, megastigmatrienone, benzyl alcohol, and benzaldehyde), with the most significant increase. At the same time, it effectively eliminated undesirable compounds such as nonanal and acetic acid, which bring off-flavors and irritation, achieving the best balance between aroma richness, smoothness, and purity.

[0063] Experimental group 1 showed significantly better results than the control group, experimental group 2, and experimental group 4, ranking second. Experimental group 1 demonstrated superior aroma intensity, with its newly generated beneficial compound 3-hydroxy-2-butanone content being 2.1 times that of experimental group 4, and it uniquely produced the important aroma compound 2-n-pentylfuran. In enhancing the core aroma, it significantly increased the levels of almost all key aroma-producing substances (neoptiadiene, megastigmatrienone, benzaldehyde, etc.) compared to experimental group 4, proving its fermentation aroma-producing performance far surpasses that of commercial strains. Although slightly inferior to experimental group 3 in the breadth of beneficial component production, its effect on removing harmful components was comparable to that of experimental group 4. Therefore, the overall quality improvement effect of experimental group 1 was clearly superior to that of experimental groups 2 and 4.

[0064] Example 4 Sensory evaluation of tobacco leaves after aging 1. Test Methods Sensory quality evaluation of cigarettes in the control group and experimental groups 1-4 of Example 3 was conducted. The evaluation criteria referred to Part 4 of Cigarette Standard: Sensory Technical Requirements GB 5606.4-2005. The sensory quality evaluation criteria for cigarettes are shown in Table 6. The sensory quality evaluation categories of cigarettes are divided into Class I, Class II and Class III, and Class IV and Class V cigarettes, and the requirements are shown in Table 7. The sensory quality evaluation of cigarettes in the control group and experimental groups 1-4 was statistically analyzed to characterize the quality of the mellowed tobacco leaves in each probiotic intervention group.

[0065] Table 6. Sensory Quality Evaluation Criteria for Cigarettes

[0066] Table 7 Minimum Sensory Quality Scores for Category I, II & III, IV & V Cigarettes

[0067] 2. Test Results The experimental results are shown in Table 8, and the analysis is as follows: According to the data in Table 8, experimental group 3 was the highest-rated group, significantly better than all other groups. Experimental group 1 performed second best, and was significantly better than the control group, experimental group 2, and experimental group 4. In conclusion, experimental group 3 was the best, and experimental group 1 was second best, and significantly better than all other experimental groups except experimental group 3.

[0068] Table 8 Sensory quality evaluation scores and total scores for each group of cigarettes

[0069] Note: P<0.05 is marked as *; P<0.01 is marked as **; P<0.001 is marked as ***; P<0.0001 is marked as ****; the markings indicate a significant difference from the control group.

[0070] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit the solutions. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention based on the understanding of the present invention, without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A strain of Sonora desert Bacillus ( Bacillus sonorensis DQ-3-2-7 strain, characterized in that, The Sonora desert Bacillus strain DQ-3-2-7 was deposited on November 20, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 29072, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

2. A compound bacteria, characterized in that, Contains the Sonora desert Bacillus strain DQ-3-2-7 as described in claim 1 and Hunan Pseudomonas ( Pseudomonas hunanensis ).

3. A probiotic agent for tobacco aging, characterized in that, It contains the Sonora desert Bacillus strain DQ-3-2-7 as described in claim 1 and / or the complex bacteria as described in claim 2.

4. The probiotic agent according to claim 3, characterized in that, The probiotic agent is a solid or liquid probiotic agent, and the number of live bacteria in the probiotic agent is not less than 1×10⁻⁶. 8 CFU / ml or 1×10 8 CFU / g.

5. The probiotic agent according to claim 4, characterized in that, The ratio of viable Sonora desert Bacillus and Hunan Pseudomonas in the compound bacteria is 1:(0.8~1.2).

6. The probiotic agent according to claim 5, characterized in that, Its dosage forms include solutions, lyophilized powders, capsules, tablets, or granules.

7. The application of the Sonora desert Bacillus strain DQ-3-2-7 of claim 1 or the compound bacteria of claim 2 in tobacco aging.

8. The application according to claim 7, characterized in that, The Sonora desert Bacillus strain DQ-3-2-7 of claim 1 or the compound bacteria of claim 3 are applied to the surface of tobacco shreds for fermentation to improve the flavor and / or enhance the quality of tobacco leaves.

9. The application according to claim 8, characterized in that, The fermentation conditions are: fermentation temperature 35~39℃, fermentation time 5-10 days.

10. The application according to claim 8, characterized in that, The Sonora desert Bacillus strain DQ-3-2-7 or the compound bacteria used in the fermentation have a viable count of not less than 1 × 10⁻⁶ per unit mass. 8 CFU, and its weight percentage with tobacco leaves is 5-10%.

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