A method for processing cigar tobacco leaves by using bacillus fermentation, a bacterial agent for fermenting processing cigar tobacco leaves and application thereof

Abstract

The application provides a method for processing cigar tobacco leaves by using bacillus fermentation, a bacterial agent for fermenting and processing cigar tobacco leaves and application thereof, and belongs to the technical field of tobacco fermentation. The bacillus is inoculated and cultured to obtain a seed liquid, the seed liquid is inoculated into a culture medium containing wine lees at an inoculation amount of 1-5% (v / v), and the culture is cultured at 30-40 DEG C for 12-14h to obtain a bacillus liquid; the bacillus liquid is sprayed on the cigar tobacco leaves, and the cigar tobacco leaves are subjected to temperature combination fermentation and then balanced in moisture. The bacillus is bacillus amyloliquefaciens SS0813, bacillus velezensis MZ1030 or bacillus aryabhattai GL0525. The strains are combined with temperature combination fermentation to process the cigar tobacco leaves, the content of various aroma components in the cigar tobacco leaves can be stably increased, the stimulating aroma components such as nicotine are obviously reduced, and the cigar tobacco is more mellow when smoked.

Description

Technical Field

[0001] This invention belongs to the field of tobacco fermentation technology, and particularly relates to a method for fermenting cigar tobacco leaves using Bacillus and a microbial agent for fermenting cigar tobacco leaves and its application. Background Technology

[0002] Domestically produced cigar tobacco leaves lag behind imported ones in sensory quality, primarily in terms of lower aroma richness, stronger off-flavors, more pronounced mouthfeel, and weaker sweetness. This has led to imported cigars consistently dominating the domestic and international cigar markets. To improve the smoking quality of domestically produced cigar tobacco leaves, cigar factories utilize fermentation technology to alter the physicochemical properties of the leaves, thereby enhancing their aroma and flavor while reducing harshness and off-flavors.

[0003] In the fermentation process of cigar tobacco leaves, oxidation, microbial activity, and enzymatic activity are the mechanisms by which cigar tobacco quality is altered. The introduction of fermentation media can affect the quality of cigar tobacco by influencing the growth and metabolism of microorganisms. However, current research on cigar tobacco fermentation processes, both domestically and internationally, mainly focuses on optimizing fermentation techniques. Research on microbial enhancement media for cigar tobacco leaves is limited, resulting in a scarcity of microbial strains suitable for practical cigar tobacco production.

[0004] Currently, the microbial enrichment fermentation of cigar tobacco leaves usually adopts the constant temperature fermentation method. However, during natural fermentation, the change in internal temperature is usually accompanied by the succession of the microbial community. The constant temperature fermentation method is not conducive to the reproduction, growth and metabolism of microorganisms.

[0005] As the main component of baijiu (Chinese white liquor) fermentation and the primary carrier of fermenting microorganisms, the microbial community diversity and composition within the fermentation mash are closely related to the flavor compounds in baijiu. During fermentation, the mash produces various volatile flavor components such as esters, alcohols, and acids, significantly contributing to the shaping of baijiu's aroma profile. Combining the flavor characteristics of baijiu with microbial fermentation technology to obtain baijiu-infused cigars is of great value in enriching the variety of cigars. However, research on enhancing the baijiu aroma of cigar tobacco using microbial fermentation is currently scarce. Therefore, providing microbial strains and fermentation methods that can enhance the baijiu aroma of cigar tobacco is a pressing technical challenge that needs to be addressed in this field. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide a method for fermenting cigar tobacco leaves using Bacillus and a microbial agent for fermenting cigar tobacco leaves and its application, so as to solve the problems of low aroma richness, heavy off-flavors, obvious oral residue and weak sweetness of existing cigar tobacco leaves.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides a method for treating cigar tobacco leaves using Bacillus fermentation, comprising the following steps:

[0009] 1) Inoculate the Bacillus seed culture into a culture medium containing fermented mash at an inoculation rate of 1% to 5% (v / v) and incubate at 30 to 40°C for 12 to 14 hours to obtain Bacillus culture.

[0010] 2) Spray Bacillus spores solution onto cigar tobacco leaves and balance the moisture content after variable temperature combined fermentation.

[0011] Preferably, the Bacillus is Bacillus amyloliquefaciens SS0813, Bacillus velezensis MZ1030, or Priestiaaryabhattai GL0525.

[0012] The Bacillus amyloliquefaciens SS0813 is deposited at the China Center for Type Culture Collection, Wuhan University, with accession number: CCTCC NO:M20232418;

[0013] The Bacillus belye MZ1030 is deposited at the China Center for Type Culture Collection, Wuhan University, with accession number: CCTCC NO:M20232416;

[0014] The Bacillus GL0525 described is deposited at the China Center for Type Culture Collection, Wuhan University, with accession number CCTCCNO:M20232417.

[0015] Preferably, the variable-temperature combined fermentation is carried out for 1 to 6 fermentation cycles. Each fermentation cycle is divided into three fermentation stages: the temperature of the first fermentation stage is 20 to 40°C, the humidity is 65 to 75%, and the time is 42 to 54 hours; the temperature of the second fermentation stage is 41 to 55°C, the humidity is 80 to 90%, and the time is 42 to 54 hours; and the temperature of the third fermentation stage is 20 to 40°C, the humidity is 65 to 75%, and the time is 42 to 54 hours.

[0016] Preferably, in the temperature-dependent combined fermentation process of Bacillus amyloliquefaciens SS0813 or Bacillus belyss MZ1030, the first fermentation stage is at a temperature of 25°C, a humidity of 70%, and a time of 48 hours; the second fermentation stage is at a temperature of 50°C, a humidity of 85%, and a time of 48 hours; and the third fermentation stage is at a temperature of 25°C, a humidity of 70%, and a time of 48 hours.

[0017] In the variable-temperature combined fermentation process of Bacillus GL0525, the first fermentation stage is at a temperature of 35℃ and a humidity of 70% for 48 hours; the second fermentation stage is at a temperature of 45℃ and a humidity of 85% for 48 hours; and the third fermentation stage is at a temperature of 35℃ and a humidity of 70% for 48 hours.

[0018] Preferably, the culture medium containing fermented mash comprises the following components: 15-25 g / L of fermented mash, 3-8 g / L of glucose, and the balance being water.

[0019] Preferably, the moisture content of cigar tobacco leaves after spraying with Bacillus spores solution is 25%–35%, and the moisture content of cigar tobacco leaves after moisture balancing is 19%–21%.

[0020] Preferably, the inoculation amount of the Bacillus bacterial solution is 20% to 30%.

[0021] Preferably, the OD value of the Bacillus bacterial solution is 0.5 to 1.5.

[0022] The present invention also provides a microbial agent for fermenting cigar tobacco leaves, the microbial agent comprising Bacillus amyloliquefaciens SS0813, Bacillus velezensis MZ1030 or Priestia aryabhattai GL0525.

[0023] The Bacillus amyloliquefaciens SS0813 is deposited at the China Center for Type Culture Collection, Wuhan University, with accession number: CCTCC NO:M20232418;

[0024] The Bacillus belye MZ1030 is deposited at the China Center for Type Culture Collection, Wuhan University, with accession number: CCTCC NO:M20232416;

[0025] The Bacillus GL0525 described is deposited at the China Center for Type Culture Collection, Wuhan University, with accession number CCTCCNO:M20232417.

[0026] The present invention also provides the application of the method or the microbial agent described herein in reducing the irritation and off-flavors of cigar tobacco and improving the aroma of cigar tobacco.

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

[0028] This invention utilizes alcohol-infused microorganisms—Bacillus amyloliquefaciens SS0813, Bacillus belyss MZ1030, or Bacillus GL0525—to ferment cigar tobacco leaves, resulting in cigar tobacco leaves with a distinct alcoholic aroma. Cigar tobacco leaves fermented with Bacillus amyloliquefaciens SS0813, Bacillus belyss MZ1030, or Bacillus GL0525 exhibit a pronounced alcoholic aroma, increased aroma-producing components, and a significant reduction in pungent aroma components such as nicotine, resulting in a smoother and more mellow smoking experience.

[0029] This invention, through variable-temperature combined fermentation compared to constant-temperature fermentation, significantly increases the content of aroma components in cigar tobacco leaves. Simultaneously, while preserving the richness and quantity of aroma to the greatest extent, it significantly improves irritation and off-flavors. In the early stage of fermentation, low-temperature fermentation promotes microbial growth and metabolism, providing precursor conditions for subsequent enzyme activity and aroma component generation. In the middle stage, high-temperature and high-humidity fermentation facilitates enzyme activity, degrading proteins, starches, and alkaloids to generate small-molecule sugars and volatile nitrogenous compounds, thereby reducing the irritation and off-flavors of the tobacco leaves. Simultaneously, high-temperature fermentation promotes the Maillard reaction, generating aroma components such as pyrazines and furans. The temperature and humidity are further reduced in the later stage of fermentation to prevent prolonged high-temperature fermentation, which could lead to burnt cores in the fermented tobacco pile and damage the quality of the cigar tobacco leaves. Attached Figure Description

[0030] Figure 1 Photographs of colony morphology of strains SS0813, MZ1030 and GL0525 on plates;

[0031] Figure 2 Gram staining micrographs of strains SS0813, MZ1030, and GL0525;

[0032] Figure 3 Scanning electron microscope images of strains SS0813, MZ1030 and GL0525;

[0033] Figure 4 The content of carotenoid degradation products in cigar tobacco leaves under different treatments before and after fermentation;

[0034] Figure 5 The content of phenylalanine conversion products in cigar tobacco leaves under different treatments before and after fermentation;

[0035] Figure 6 The content of cephalan degradation products in cigar tobacco leaves under different treatments before and after fermentation;

[0036] Figure 7 The content of chlorophyll degradation products in cigar tobacco leaves under different treatments before and after fermentation;

[0037] Figure 8The content of volatile nicotine in cigar tobacco leaves under different treatments before and after fermentation;

[0038] Figure 9 The content of other volatile flavor metabolites in cigar tobacco leaves under different treatments before and after fermentation.

[0039] Biological Preservation Instructions

[0040] The Bacillus amyloliquefaciens SS0813 provided by this invention is deposited at the China Center for Type Culture Collection, Wuhan University, with accession number CCTCC NO: M20232418, deposited on December 1, 2023, and located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0041] The Bacillus velezensis MZ1030 provided by this invention is deposited at the China Center for Type Culture Collection, Wuhan University, with accession number CCTCC NO: M20232416, deposited on December 1, 2023, and located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0042] The *Priestia aryabhattai* GL0525 provided by this invention is deposited at the China Center for Type Culture Collection, Wuhan University, with accession number CCTCC No: M20232417, deposited on December 1, 2023, and located at No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. Detailed Implementation

[0043] This invention provides a method for treating cigar tobacco leaves using Bacillus fermentation, comprising the following steps:

[0044] 1) Inoculate the Bacillus seed culture into a culture medium containing fermented mash at an inoculation rate of 1% to 5% (v / v) and incubate at 30 to 40°C for 12 to 14 hours to obtain Bacillus culture.

[0045] 2) Spray Bacillus spores solution onto cigar tobacco leaves and balance the moisture content after variable temperature combined fermentation.

[0046] In this invention, Bacillus seed culture is inoculated into a culture medium containing fermented mash at an inoculation rate of 1%–5% (v / v) and cultured at 30–40°C for 12–14 hours to obtain Bacillus bacterial culture. The preferred Bacillus species are *Bacillus amyloliquefaciens* SS0813, *Bacillus velezensis* MZ1030, or *Priestia aryabhattai* GL0525. *Bacillus amyloliquefaciens* SS0813 is deposited at the China Center for Type Culture Collection, Wuhan University, with accession number CCTCC. NO: M20232418; the *Bacillus belye* MZ1030 is deposited at the China Center for Type Culture Collection, Wuhan University, with accession number CCTCC NO: M20232416; the *Bacillus glesei* GL0525 is deposited at the China Center for Type Culture Collection, Wuhan University, with accession number CCTCC No: M20232417; the seed culture is obtained by inoculating *Bacillus* onto LB medium, with the preferred culture conditions being 32–42℃ and a rotation speed of 200–240 rpm. The culture medium is incubated at 34-40°C for 10-15 hours, or more preferably at 210-230 rpm for 11-14 hours. The inoculation amount of the seed liquid is preferably 2-4%. The culture medium containing fermented mash comprises the following components: 15-25 g / L of fermented mash, 3-8 g / L of glucose, and the balance being water. The amount of fermented mash is preferably 18-22 g / L, and the amount of glucose is preferably 4-7 g / L. The culture temperature is preferably 32-38°C, and the culture time is preferably 12.5-13.5 hours.

[0047] In this invention, Bacillus spp. bacterial solution is sprayed onto cigar tobacco leaves, followed by variable-temperature combined fermentation to balance the moisture content. The OD value of the Bacillus spp. bacterial solution is preferably 0.5–1.5, more preferably 0.8–1.2; the inoculation amount of the Bacillus spp. bacterial solution is preferably 20%–30% (v: volume of Bacillus spp. bacterial solution mL / m: mass of cigar tobacco leaves g), more preferably 22%–28%; the moisture content of the cigar tobacco leaves after spraying the Bacillus spp. bacterial solution is preferably 25%–35%, more preferably 28%–32%; the variable-temperature combined fermentation is carried out for 1–6 fermentation cycles, with each fermentation cycle divided into three fermentation stages, the first… The preferred temperature for the first fermentation stage is 20–40℃, more preferably 22–38℃; the preferred humidity is 65–75%, more preferably 67–72%; and the preferred time is 42–54 h, more preferably 45–51 h. The preferred temperature for the second fermentation stage is 41–55℃, more preferably 43–52℃; the preferred humidity is 80–90%, more preferably 82–88%; and the preferred time is 42–54 h, more preferably 45–51 h. The preferred temperature for the third fermentation stage is 20–40℃, more preferably 22–38℃; the preferred humidity is 65–75%, more preferably 67–72%; and the preferred time is 42–54 h, more preferably 45–51 h. In the variable-temperature combined fermentation process of *Bacillus amyloliquefaciens* SS0813 or *Bacillus belyssus* MZ1030, the first fermentation stage has a temperature of 25℃, a humidity of 70%, and a time of 48 h; the second fermentation stage has a temperature of 50℃, a humidity of 85%, and a time of 48 h; the third fermentation stage… The temperature is 25℃, the humidity is 70%, and the time is 48h; in the variable temperature combined fermentation process of Bacillus GL0525, the first fermentation stage is 35℃, the humidity is 70%, and the time is 48h; the second fermentation stage is 45℃, the humidity is 85%, and the time is 48h; the third fermentation stage is 35℃, the humidity is 70%, and the time is 48h; the moisture content of the cigar tobacco leaves after moisture balancing is preferably 19-21%, more preferably 19.5-20.5%.

[0048] This invention also provides a microbial agent for fermenting cigar tobacco leaves, the microbial agent comprising Bacillus amyloliquefaciens SS0813, Bacillus velezensis MZ1030, or Priestia aryabhattai GL0525; wherein Bacillus amyloliquefaciens SS0813 is deposited at the China Center for Type Culture Collection, Wuhan University, with accession number CCTCC NO: M20232418; wherein Bacillus velezensis MZ1030 is deposited at the China Center for Type Culture Collection, Wuhan University, with accession number CCTCC NO: M20232416; and wherein Priestia aryabhattai GL0525 is deposited at the China Center for Type Culture Collection, Wuhan University, with accession number CCTCC NO: M20232417.

[0049] The present invention also provides the application of the method or the microbial agent described herein in reducing the irritation and off-flavors of cigar tobacco and improving the aroma of cigar tobacco.

[0050] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0051] Example 1

[0052] Isolation and identification of bacterial strains:

[0053] 1. Isolation of bacterial strains: The *Bacillus amyloliquefaciens* SS0813, *Bacillus belye* MZ1030, or *Bacillus lasiosporus* GL0525 of this invention were isolated and purified from fermented mash. Details are as follows:

[0054] (1) Place 1-5g of fermented mash in 50-100mL of sterile water and shake for 20-40min at 30-40℃ and 120-220r / min to obtain a bacterial suspension. Take 1mL of the bacterial suspension and place it in a 9mL sterile water test tube to obtain 10 -1 Diluted bacterial suspensions were prepared in the same manner, and so on, to make 10... -2 10-3, 10 -4 10 -5 10 -6 Dilute the bacterial suspension. Take 0.1 mL of 10 -4 -10 -6 The diluted bacterial suspension was evenly spread on beef extract peptone medium and incubated upside down in a 30°C incubator for 24 hours.

[0055] (2) Based on the different morphological characteristics of the colonies on the diluted plating plates, select single colonies with good growth and dominant position for microscopic examination. After the colonies that have been examined under the microscope are purified by streaking multiple times, they are inoculated into the corresponding plate culture medium and stored at 4°C for identification and use.

[0056] 2. Morphological identification

[0057] Single colonies purified multiple times were picked and Gram-stained. Under the microscope, the microbial colonies showed a distinct blue-purple color and were short rod-shaped, initially identifying them as Gram-positive bacilli. Further single colonies purified multiple times were picked and cultured overnight for enrichment. The bacterial suspension was centrifuged to obtain precipitated colonies. After freeze-drying, the precipitated colonies were observed using a scanning electron microscope. The colonies under the scanning electron microscope showed a distinct short rod-shaped structure, confirming them as Gram-positive rod-shaped bacteria (e.g., Gram-positive rod-shaped bacteria). Figure 1 , Figure 2 , Figure 3 (As shown).

[0058] 3. Physiological and biochemical tests for identification

[0059] After multiple purifications, single colonies were selected for physiological and biochemical tests. Based on the results of the physiological and biochemical tests (Table 1), this strain was further identified as Bacillus.

[0060] Table 1. Physiological and biochemical test results of strains SS0813, MZ1030, and GL0525

[0061]

[0062]

[0063]

[0064] 4. Molecular biological identification

[0065] Bacillus amyloliquefaciens strain SS0813, Bacillus bereaves strain MZ1030, and Bacillus GL0525 were cultured in beef extract peptone medium for 48 h. Bacterial DNA was extracted, and the bacterial genomic DNA was amplified by PCR. The PCR products were recovered using an AxyPrep DNA gel extraction kit. The purified PCR products of each strain were used for DNA sequencing using an ABI3730-XL sequencer (as shown in Table 2).

[0066] Table 2. 16S rDNA sequences of each strain

[0067]

[0068]

[0069]

[0070]

[0071] The assembled sequence file was compared with data in the NCBI 16S database using the NCBI Blast program to obtain the species information with the greatest similarity to the sequence of the species to be tested. The strains SS0813, MZ1030 and GL0525 were identified as Bacillus amyloliquefaciens, Bacillus belye, and Bacillus argentis, respectively.

[0072] Example 2

[0073] Bacillus amyloliquefaciens SS0813, Bacillus belye MZ1030, or Bacillus GL0525 were inoculated into LB broth medium and cultured overnight at 37°C and 220 rpm for 12 hours to obtain seed culture. The seed culture was then transferred to high-quality cigar tobacco fermentation medium (20 g / L mash, 5 g / L glucose, and the remainder water, sterilized at 115°C for 30 min) and cultured at 37°C for 12 hours to obtain bacterial culture.

[0074] The bacterial suspensions of *Bacillus amyloliquefaciens* SS0813, *Bacillus bereaves* MZ1030, or *Bacillus lasiocarpa* GL0525 were uniformly diluted to 1.0 OD, and then sprayed evenly onto the cigar tobacco leaves to be fermented at a 30% inoculum (mL / g). The constant temperature and humidity during fermentation were 35℃ and 75%, respectively, with each fermentation cycle lasting 6 days, for a total of 5 fermentation cycles.

[0075] The bacterial suspensions of Bacillus amyloliquefaciens SS0813, Bacillus vesicle MZ1030, or Bacillus gl0525 were uniformly diluted to 1.0 OD and then sprayed evenly onto cigar tobacco leaves at an inoculation rate of 30% (mL / g). In the variable-temperature fermentation, the first fermentation stage of *Bacillus amyloliquefaciens* SS0813 or *Bacillus belyssus* MZ1030 is carried out at a temperature of 25°C and a humidity of 70% for 48 hours; the second fermentation stage is carried out at a temperature of 50°C and a humidity of 85% for 48 hours; and the third fermentation stage is carried out at a temperature of 25°C and a humidity of 70% for 48 hours. Similarly, for *Bacillus globus* GL0525, the first fermentation stage is carried out at a temperature of 35°C and a humidity of 70% for 48 hours; the second fermentation stage is carried out at a temperature of 45°C and a humidity of 85% for 48 hours; and the third fermentation stage is carried out at a temperature of 35°C and a humidity of 70% for 48 hours. Each fermentation cycle lasts 6 days, and a total of 5 fermentation cycles are required.

[0076] After fermentation, the volatile aroma components of cigar tobacco leaves under constant temperature and variable temperature fermentation were analyzed by HS-SPME-GC-MS (headspace solid phase microextraction-gas chromatography-mass spectrometry). The results are shown in Table 3.

[0077] Table 3. Analysis results of volatile aroma components in cigar tobacco leaves fermented at different temperatures (constant and variable temperatures) under different treatments.

[0078]

[0079]

[0080] The analysis results of the volatile aroma components in the above cigar samples show that the enrichment and cultivation of *Bacillus amyloliquefaciens* SS0813, *Bacillus belyss* MZ1030, and *Bacillus amyloliquefaciens* GL0525 during the cigar tobacco fermentation process, using a variable-temperature fermentation mode, can significantly improve tobacco quality. The enrichment and cultivation of these bacteria can result in a higher aroma content, a significant reduction in harshness and off-flavors, and a richer variety of volatile aroma components. This further demonstrates that the combined application of enrichment and cultivation of *Bacillus amyloliquefaciens* SS0813, *Bacillus belyss* MZ1030, and *Bacillus amyloliquefaciens* GL0525 and variable-temperature fermentation has excellent technical effects.

[0081] Example 3

[0082] Bacillus amyloliquefaciens SS0813, Bacillus bereaves MZ1030, or Bacillus argentea GL0525 were inoculated into LB broth medium and cultured overnight at 37°C and 220 rpm for 12 hours to obtain seed culture. The seed culture was then transferred to fermentation medium (fermentation mash 20 g / L, glucose 5 g / L, the remainder water, sterilized at 115°C for 30 min) and cultured at 37°C for 12 hours to obtain bacterial culture.

[0083] The bacterial suspensions of *Bacillus amyloliquefaciens* SS0813, *Bacillus belyss* MZ1030, or *Bacillus gleseri* GL0525 were uniformly diluted to 1.0 OD, and then sprayed evenly onto cigar tobacco leaves at a 30% inoculum rate (mL / g). In the variable-temperature fermentation, for the *Bacillus amyloliquefaciens* SS0813 or *Bacillus belyss* MZ1030, the first fermentation stage was at 25°C, 70% humidity, and 48 h; the second fermentation stage was at 50°C, 85% humidity, and 48 h; and the third fermentation stage was at 25°C, 70% humidity, and 48 h. For the *Bacillus gleseri* GL0525, the first fermentation stage was at 35°C, 70% humidity, and 48 h; the second fermentation stage was at 45°C, 85% humidity, and 48 h; and the third fermentation stage was at 35°C, 70% humidity, and 48 h. It requires one fermentation cycle.

[0084] Fermentation was carried out using a culture medium (FT, 20 g / L mash, 5 g / L glucose, and the remainder water, sterilized at 115 °C for 30 min) without the addition of Bacillus amyloliquefaciens SS0813, Bacillus bereaves MZ1030, or Bacillus lasiocarpa GL0525, and ultrapure water (WA) as controls. After fermentation, the volatile aroma components of cigar tobacco leaves under different fermentation treatments were analyzed by HS-SPME-GC-MS (headspace solid phase microextraction-gas chromatography-mass spectrometry). The results are shown in Table 4.

[0085] Table 4. Analysis results of volatile aroma components in cigar tobacco leaves under different fermentation treatments during one fermentation cycle.

[0086]

[0087] As shown in Table 4, the cigar leaves fermented by Bacillus amyloliquefaciens SS0813, Bacillus belye MZ1030 and Bacillus GL0525 have an increase in aldehydes and ketones with a light and licorice-like aroma, resulting in a more distinctive cigar tobacco style. The nicotine content is slightly reduced, the irritation of the obtained cigar tobacco is slightly reduced, and the balance of aroma components is increased.

[0088] Example 4

[0089] The fermentation cycle in Example 3 was replaced with a total of 2 fermentation cycles, while other treatments remained the same. The experimental results are shown in Table 5.

[0090] Table 5. Analysis results of volatile aroma components in cigar tobacco leaves under different fermentation treatments during two fermentation cycles.

[0091]

[0092] As shown in Table 5, the nicotine content of cigar leaves fermented by Bacillus amyloliquefaciens SS0813, Bacillus belye MZ1030 and Bacillus GL0525 was slightly reduced, the irritation of the obtained cigar tobacco leaves was slightly reduced, and the balance of aroma components was further increased.

[0093] Example 5

[0094] The fermentation cycle in Example 3 was replaced with a total of 3 fermentation cycles, while all other treatments remained the same. The experimental results are shown in Table 6.

[0095] Table 6. Analysis results of volatile aroma components in cigar tobacco leaves under different fermentation treatments during three fermentation cycles.

[0096]

[0097]

[0098] As shown in Table 6, the cigar leaves fermented by Bacillus amyloliquefaciens SS0813, Bacillus belye MZ1030 and Bacillus GL0525 showed a slight increase in aldehydes and ketones with a light and licorice-like aroma, a decrease in nicotine content, reduced irritation of the obtained cigar tobacco leaves, and a significant increase in the balance and harmony of aroma components.

[0099] Example 6

[0100] The fermentation cycle in Example 3 was replaced with a total of 4 fermentation cycles, while all other treatments remained the same. The experimental results are shown in Table 7.

[0101] Table 7. Analysis results of volatile aroma components in cigar tobacco leaves under different fermentation treatments during four fermentation cycles.

[0102]

[0103]

[0104] As shown in Table 7, the cigar leaves fermented by Bacillus amyloliquefaciens SS0813, Bacillus belye MZ1030, and Bacillus GL0525 showed a significant increase in aldehydes and ketones with a light and licorice-like aroma, resulting in a more prominent cigar tobacco style, lower nicotine content, reduced irritation, and a further significant increase in the balance and harmony of aroma components.

[0105] Example 7

[0106] The fermentation cycle in Example 3 was replaced with a total of 5 fermentation cycles, while all other treatments remained the same. The experimental results are shown in Table 8.

[0107] Table 8. Analysis results of volatile aroma components in cigar tobacco leaves under different fermentation treatments during five fermentation cycles.

[0108]

[0109]

[0110] As shown in Table 8, the cigar leaves fermented by Bacillus amyloliquefaciens SS0813, Bacillus belye MZ1030, and Bacillus GL0525 showed a significant increase in aldehydes and ketones with a light and licorice-like aroma, resulting in a more prominent cigar tobacco style, a significantly reduced nicotine content, a significantly reduced irritation, and a further significant increase in the balance and harmony of aroma components.

[0111] Example 8

[0112] The fermentation cycle in Example 3 was replaced with a total of 6 fermentation cycles, while all other treatments remained the same. The experimental results are shown in Table 9.

[0113] Table 9. Analysis results of volatile aroma components in cigar tobacco leaves under different fermentation treatments during six fermentation cycles.

[0114]

[0115] As shown in Table 9, the cigar leaves fermented by Bacillus amyloliquefaciens SS0813, Bacillus belye MZ1030, and Bacillus GL0525 showed a significant increase in aldehydes and ketones with a light and licorice-like aroma, resulting in a more prominent cigar tobacco style, a significantly reduced nicotine content, a significantly reduced irritation, and a further significant increase in the balance and harmony of aroma components.

[0116] Example 9

[0117] The aroma components of the cigar tobacco leaves in Example 8 after 6 fermentation cycles and before fermentation were qualitatively and quantitatively analyzed using an Agilent 8890-7000D gas chromatography-mass spectrometry (GC-MS) system.

[0118] The specific detection method is as follows: Headspace solid-phase microextraction (HPE) was used for the pretreatment of fermented cigar tobacco samples. 0.5 g of cigar tobacco sample was weighed and added to a 20 mL headspace vial containing 8 mL of saturated sodium chloride solution and 1 μL of phenylethyl 2-acetate internal standard solution. The headspace vial was then placed in a 70℃ water bath and continuously incubated with stirring for 20 minutes. After stabilization, the extraction needle was withdrawn, and the adsorbed volatiles were extracted for 35 minutes. Immediately afterwards, the samples were analyzed by GC-MS for 5 minutes. VOCs were separated in an HP-5MS capillary column (30 m × 0.25 mm × 0.25 μm) under the following GC conditions: injection port temperature, 250℃; carrier gas, helium (99.99% purity); flow rate, 0.8 mL / min. The sample was injected without splitting. The temperature program was performed under the following conditions: initial temperature of 60 °C, held for 2 min, then increased to 180 °C at a rate of 3 °C / min, held for 2 min, then increased to 260 °C at a rate of 6 °C / min, and held at 260 °C for 2 min. MS conditions were as follows: ionization energy of 70 eV; ion source temperature of 230 °C; quadrupole mass detector temperature of 150 °C; mass scan range of 35–450 m / z.

[0119] The qualitative and quantitative conditions for aroma compounds were as follows: GC-MS data files were deconvolved using Quantitative Analysis B.09.00 to identify the separated components of the compounds. The deconvolved components were then compared with the mass spectra of the target compounds, using the retention times of n-alkanes as a reference. These compounds were initially identified by referring to retention times provided in the NIST 20 database. Volatile compounds were quantified using the internal standard method with 128.75 μg / μL phenethyl acetate as an internal standard.

[0120] Experimental results: such as Figures 4-9 As shown. From Figures 4-9 Analysis revealed six major categories of aroma components in tobacco leaves from different treatment groups: carotenoid degradation products, phenylalanine conversion products, cephalosporin degradation products, chlorophyll degradation products, volatile alkaloids, and other volatile flavor metabolites. Among these, carotenoids or their degradation products can provide a sweet, fruity, floral, and woody aroma during tobacco combustion. Phenylalanine conversion products are an important component of aroma, enhancing the floral, nutty, and fruity notes of tobacco. Cephalosporin degradation products primarily produce solanone, which has a unique aroma. Volatile alkaloids significantly affect the flavor of cigar tobacco leaves and the smoking experience, playing a crucial role in the overall flavor, strength, and satisfaction of cigars. Chlorophyll and its degradation products have a refreshing aroma, reducing the irritation of the smoke, improving its flavor, and making it smoother.

[0121] As demonstrated by the above embodiments, when the present invention combines the addition of Bacillus amyloliquefaciens SS0813, Bacillus belycei MZ1030, or Bacillus GL0525 with temperature-controlled fermentation, it can stably increase the content of various aroma compounds in cigar tobacco leaves, while significantly reducing irritating aroma components such as nicotine, resulting in a smoother cigar smoke. This provides a technical reference for improving the overall quality and industrial usability of domestically produced cigar tobacco leaves.

[0122] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for treating cigar tobacco leaves using Bacillus fermentation, characterized in that, Includes the following steps: 1) Inoculate the Bacillus seed culture into a culture medium containing fermented mash at an inoculation rate of 1%~5% v / v, and incubate at 30~40℃ for 12~14 h to obtain Bacillus bacterial culture; 2) Spray Bacillus spores solution onto cigar tobacco leaves and balance the moisture content after variable temperature combined fermentation; The Bacillus is Bacillus amyloliquefaciens (Bacillus amyloliquefaciens). Bacillus amyloliquefaciens SS0813, Bacillus belysinus ( Bacillus velezensis MZ1030 or Bacillus oryzae ( Priestia aryabhattai GL0525; The Bacillus amyloliquefaciens SS0813 is deposited at the China Center for Type Culture Collection, Wuhan University, with accession number: CCTCC NO:M20232418; The Bacillus belye MZ1030 is deposited at the China Center for Type Culture Collection, Wuhan University, with accession number: CCTCC NO:M20232416; The Bacillus GL0525 described is deposited at the China Center for Type Culture Collection, Wuhan University, with accession number CCTCC NO:M20232417.

2. The method according to claim 1, characterized in that, The variable-temperature combined fermentation is carried out in 1 to 6 fermentation cycles. Each fermentation cycle is divided into three fermentation stages. The temperature of the first fermentation stage is 20 to 40°C, the humidity is 65 to 75%, and the time is 42 to 54 hours. The temperature of the second fermentation stage is 41 to 55°C, the humidity is 80 to 90%, and the time is 42 to 54 hours. The temperature of the third fermentation stage is 20 to 40°C, the humidity is 65 to 75%, and the time is 42 to 54 hours.

3. The method according to claim 2, characterized in that, The first fermentation stage of the variable-temperature combined fermentation process of Bacillus amyloliquefaciens SS0813 or Bacillus belyss MZ1030 is characterized by a temperature of 25°C, a humidity of 70%, and a time of 48 hours; the second fermentation stage is characterized by a temperature of 50°C, a humidity of 85%, and a time of 48 hours; and the third fermentation stage is characterized by a temperature of 25°C, a humidity of 70%, and a time of 48 hours. In the variable-temperature combined fermentation process of Bacillus GL0525, the first fermentation stage is at a temperature of 35℃ and a humidity of 70% for 48 hours; the second fermentation stage is at a temperature of 45℃ and a humidity of 85% for 48 hours; and the third fermentation stage is at a temperature of 35℃ and a humidity of 70% for 48 hours.

4. The method according to claim 1, characterized in that, The culture medium containing fermented mash comprises the following components: 15-25 g / L of fermented mash, 3-8 g / L of glucose, and the remainder water.

5. The method according to claim 1, characterized in that, After spraying with Bacillus spores solution, the moisture content of cigar tobacco leaves was 25%~35%, and after balancing the moisture content, the moisture content of cigar tobacco leaves was 19%~21%.

6. The method according to claim 1, characterized in that, The inoculation amount of the Bacillus bacterial solution is expressed as the ratio of the volume of the Bacillus bacterial solution to the mass of the cigar tobacco leaves, and the ratio of the volume of the Bacillus bacterial solution to the mass of the cigar tobacco leaves is 0.2~0.3 mL / g.

7. The method according to claim 1, characterized in that, The OD value of the Bacillus bacterial solution is 0.5~1.

5.

8. A microbial agent for fermenting cigar tobacco leaves, characterized in that, The bacterial agent includes Bacillus amyloliquefaciens (Bacillus) Bacillus amyloliquefaciens SS0813, Bacillus belysinus ( Bacillus velezensis MZ1030 or Bacillus oryzae ( Priestia aryabhattai GL0525; The Bacillus amyloliquefaciens SS0813 is deposited at the China Center for Type Culture Collection, Wuhan University, with accession number: CCTCC NO:M20232418; The Bacillus belye MZ1030 is deposited at the China Center for Type Culture Collection, Wuhan University, with accession number: CCTCC NO:M20232416; The Bacillus GL0525 described is deposited at the China Center for Type Culture Collection, Wuhan University, with accession number CCTCC NO:M20232417.

9. The method according to any one of claims 1 to 7 or the microbial agent according to claim 8 in reducing the irritation and off-flavors of cigar tobacco and improving the aroma of cigar tobacco.

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