A tobacco-derived bacillus velezensis chq3-4 and its amylase and application

Abstract

The application discloses a tobacco source bacillus velezensis chq3-4 and its amylase and application, and belongs to the field of tobacco microorganisms. The bacillus velezensis chq3-4 strain belongs to Bacillus, is classified and named as Bacillus velezensis chq3-4, is preserved in the China Center for Type Culture Collection, the preservation date is June 19, 2024, and the preservation number is CCTCC NO: M 20241305. The amylase crude enzyme solution prepared from the strain has an enzyme activity of 119.28+ / -0.76 U / mL, and the enzyme has strong stability and can still maintain certain enzyme activity under high-temperature and alkaline conditions. The amylase crude enzyme solution is applied to tobacco products, can effectively reduce the content of tobacco starch in the tobacco products, improve the content of reducing sugar, and then improve the quality of the tobacco products.

Description

Technical Field

[0001] This application relates to the field of tobacco microbiology technology, and in particular to a tobacco-derived Bacillus berleis chq3-4, its amylase, and its applications. Background Technology

[0002] Starch, an important carbohydrate in tobacco leaves, is a crucial component of the chemical composition of tobacco. Changes in its content can affect the quality of tobacco leaves. Excessive starch in tobacco leaves can negatively impact combustion speed and completeness, and can also produce a burnt odor and various harmful substances during combustion. The starch content in tobacco leaves can be reduced through enzymatic and microbial treatments to improve tobacco quality and ultimately enhance the smoking experience of cigarettes.

[0003] Currently, most cigarette processing companies using enzyme preparations improve tobacco leaf quality by purchasing commercially available food enzymes. However, due to the significant differences in the form and composition of starch in tobacco leaves and food substrates, the unique substrate environment of tobacco leaves places higher demands on enzyme preparations. Food enzymes exhibit limited degradation activity and specificity for tobacco starch; for example, commercially available food-grade amylases show only 40% of the degradation activity of tobacco starch compared to edible starch, hindering the efficient application of bio-enzyme treatment technology in the tobacco industry. Furthermore, the unique processing environment of tobacco leaves and the wide pH range of the raw material (different parts and growth stages of tobacco leaves result in a pH between 5.0 and 8.5) place higher demands on the stability of enzyme preparations. Currently screened tobacco starch-degrading bacteria produce amylases with low activity and low temperature tolerance, making them unsuitable for high-temperature fermentation of tobacco products, and the screening process for these bacteria is lengthy. Summary of the Invention

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art or related art.

[0005] Therefore, in a first aspect, the present invention provides a tobacco-derived Bacillus velezensis chq3-4 strain, which belongs to the Bacillus genus and is classified as Bacillus velezensis chq3-4. The Bacillus velezensis chq3-4 strain was deposited at the China Center for Type Culture Collection on June 19, 2024, with accession number CCTCC NO: M 20241305.

[0006] Furthermore, the screening method for tobacco-derived Bacillus belye chq3-4 includes:

[0007] Sampling and enrichment screening: Weigh tobacco samples, cut them into small pieces, add buffer solution, homogenize, heat in an 80°C water bath, and transfer the supernatant after heating to liquid screening medium for directional enrichment culture.

[0008] Initial screening of strains: The enriched bacterial culture was heated in an 80°C water bath, serially diluted, inoculated onto a solid screening medium, and iodine solution was added. The ratio of the clear zone diameter to the colony diameter of a single colony was measured as an indicator for initial screening.

[0009] Secondary screening of strains: The strains obtained from the initial screening were secondary screened, streaked onto LB solid medium and cultured, and single colonies were picked and inoculated into LB liquid medium and cultured. After high-speed centrifugation and microfiltration, the supernatant of different single colonies was added to the perforated solid screening medium and incubated. Iodine solution was added and the diameter of the clear zone was observed to obtain the strain with the strongest starch production capacity.

[0010] In a second aspect, the present invention provides an amylase obtained by culturing and fermenting the aforementioned tobacco-derived Bacillus berleis chq3-4.

[0011] Furthermore, the crude enzyme solution of the amylase exhibits an enzyme activity as high as 119.28 ± 0.76 U / mL;

[0012] The crude amylase solution exhibits the following relative enzyme activity at pH 7.0: 60% at 40℃~90℃, 80% at 40℃~85℃, and 100% at 75℃.

[0013] The crude amylase solution has a relative enzyme activity of over 60% at a temperature of 75°C and a pH range of 6.0 to 9.0, a relative enzyme activity of over 80% at a pH range of 7.0 to 8.5, and a relative enzyme activity of 100% at a pH of 8.0.

[0014] A third aspect of the present invention provides an application of amylase in improving the quality of tobacco products, wherein the amylase is used in tobacco product processing, and the applicable temperature range is 40℃~90℃, and the applicable pH range is 6.0~9.0; the improvement of tobacco product quality includes reducing the content of tobacco starch in tobacco products and increasing the content of reducing sugars in tobacco products.

[0015] A fourth aspect of the present invention provides the application of Bacillus vesiculus chq3-4 from tobacco source in improving the quality of tobacco products. The Bacillus vesiculus chq3-4 is used to improve the quality of tobacco products by reducing the content of tobacco starch in the tobacco products and increasing the content of reducing sugar in the tobacco products.

[0016] Furthermore, the application methods include:

[0017] The fermentation supernatant filtrate was prepared using the Bacillus berberis chq3-4;

[0018] The fermentation supernatant filtrate is evenly sprayed onto the surface of tobacco products for fermentation.

[0019] Furthermore, the fermentation supernatant filtrate is prepared by a method comprising:

[0020] The *Bacillus belye* chq3-4 was inoculated into the fermentation medium for fermentation. The culture was carried out at 35℃~40℃ and 150 r / min~200 r / min for 55 h~65 h. Then, the culture was centrifuged at 2℃~6℃ and 8000 r / min~12000 r / min for 8 min~12 min. The supernatant was collected and filtered through a sterile aqueous filter membrane to obtain the fermentation supernatant filtrate.

[0021] Preferably, the Bacillus berreatus chq3-4 is inoculated into the fermentation medium for fermentation and cultured at 37°C and 180 r / min for 60 h. Then, it is centrifuged at 4°C and 10,000 r / min for 10 min, the supernatant is collected, and filtered through a 0.22 μm sterile aqueous filter membrane to obtain the fermentation supernatant filtrate.

[0022] Further, the method of uniformly spraying the fermentation supernatant filtrate onto the surface of the tobacco product for fermentation includes:

[0023] The fermentation supernatant filtrate is applied and fermented as follows: the fermentation supernatant filtrate is evenly sprayed onto the tobacco leaves or shredded tobacco to be treated, and then placed in a fermentation environment of 42℃~45℃ and 70%~75% humidity for 45 h~50 h.

[0024] Enzyme inactivation treatment: After fermentation, the tobacco leaves or shredded tobacco are heated at 100℃~120℃ to inactivate the residual fermentation supernatant filtrate.

[0025] Preferably, the method of uniformly spraying the fermentation supernatant filtrate onto the surface of tobacco products for fermentation includes:

[0026] The fermentation supernatant filtrate was applied and fermented as follows: The fermentation supernatant filtrate was evenly sprayed onto the tobacco leaves or shredded tobacco to be treated, and then fermented at 45°C and 75% humidity for 48 hours.

[0027] Enzyme inactivation treatment: After fermentation, the tobacco products are heated at 100°C for 1 minute to inactivate the residual crude enzyme solution.

[0028] Further, for every 10 mL to 20 mL of the fermentation supernatant filtrate, 45 g to 55 g of tobacco leaves or shredded tobacco are sprayed. Preferably, for every 15 mL of the fermentation supernatant filtrate, 50 g of tobacco leaves or shredded tobacco are sprayed.

[0029] Compared with the prior art, the present invention has at least the following beneficial effects:

[0030] The *Bacillus bellsii* chq3-4 strain provided in this application produces a crude amylase solution with an activity as high as 119.28 ± 0.76 U / mL. Furthermore, this enzyme exhibits strong stability, maintaining a certain level of activity even under high temperature and alkaline conditions. Applying it to tobacco products can effectively reduce the starch content and increase the reducing sugar content, thereby improving the quality of tobacco products. This provides valuable microbial resources for the tobacco industry and promotes the high-quality development of tobacco industrial enterprises. Attached Figure Description

[0031] Figure 1 The Bacillus belyssus in Example 2 of this application ( Bacillus velezensis Colony morphology of strain chq3-4 on solid LB medium;

[0032] Figure 2 The Bacillus belyssus in Example 2 of this application ( Bacillus velezensis Single colony morphology of strain chq3-4 on solid LB medium;

[0033] Figure 3 The Bacillus belyssus in Example 2 of this application ( Bacillus velezensis Morphological image of strain chq3-4 after Gram staining under a 100x oil immersion microscope during identification;

[0034] Figure 4 In Example 2 of this application, molecular biology was used to investigate Bacillus belyssus (… Bacillus velezensis The phylogenetic tree for identifying strain chq3-4 was constructed based on the 16S rDNA gene sequence.

[0035] Figure 5 The Bacillus belyssus in Example 3 of this application ( Bacillus velezensis Figure 1 shows the temperature tolerance results of the amylase prepared by chq3-4.

[0036] Figure 6 The Bacillus belyssus in Example 4 of this application ( Bacillus velezensis Figure showing the acid-base tolerance of amylase prepared by chq3-4;

[0037] Figure 7 The Bacillus repens in Example 5 of this application ( Bacillus velezensis The graph shows the change in starch content of tobacco products before and after treatment with the amylase prepared by chq3-4.

[0038] Figure 8 The Bacillus repens in Example 6 of this application ( Bacillus velezensis The graph shows the change in reducing sugar content of tobacco products before and after treatment with the amylase prepared by chq3-4.

[0039] Figure 9 The Bacillus belyssus in Example 7 of this application ( Bacillus velezensis Radar chart showing the sensory evaluation effect of tobacco shreds treated with amylase prepared by chq3-4. Detailed Implementation

[0040] The present application will be further described below with reference to specific embodiments, but the scope of protection of the present application is not limited thereto; unless otherwise specified, all kinds of reagents, instruments and other items used in the embodiments are commercially available products.

[0041] The following is a brief introduction to some of the biological materials, experimental reagents, and experimental equipment involved in the following examples and experimental cases:

[0042] Culture medium:

[0043] Liquid screening medium: 1 g / L tobacco starch, 5 g / L sodium chloride, 2 g / L yeast extract; if preparing solid screening medium, add 15 g / L agar powder and autoclave at 121℃ for 30 min.

[0044] LB medium: 5 g / L yeast extract, 10 g / L peptone, 10 g / L NaCl; if preparing solid medium, add 15 g / L agar powder, adjust pH to 7.0, and autoclave at 121℃ for 30 min.

[0045] Fermentation medium: 80 g / L peptone, 25 g / L yeast extract, 5 g / L dipotassium hydrogen phosphate, 6 g / L ammonium chloride, autoclaved at 121℃ for 30 min.

[0046] Experimental reagents:

[0047] Soluble starch solution (20 g / L): Weigh 2.177 g (accurate to 0.001 g) of soluble starch (on an oven-dry basis) into a beaker, mix with a small amount of water to form a slurry, and slowly add it to 70 mL of boiling water while stirring. Then rinse the beaker containing the starch several times with water, pour the washings into the solution, stir and heat until completely transparent, cool and dilute to 100 mL. Prepare the solution immediately before use.

[0048] Note: Soluble starch should be analyzed using enzyme-specific starch.

[0049] Original iodine solution: Weigh 11.0 g of iodine and 22.0 g of potassium iodide, dissolve the iodine completely in a small amount of water, and bring the volume up to 500 mL. Store in a brown bottle.

[0050] Diluted iodine solution: Take 2.00 mL of the original iodine solution, add 20.0 g of potassium iodide, dissolve in water and make up to 500 mL, then store in a brown bottle.

[0051] Phosphate buffer (pH 6.0): Weigh 45.23 g disodium hydrogen phosphate (Na2HPO4·12H2O) and 8.07 g citric acid (C6H8O7·H2O), dissolve in water and bring the volume to 1000 mL. Use after calibrating the pH meter.

[0052] Hydrochloric acid solution c(HCl) = 0.1 mol / L: Take 1 ml of concentrated hydrochloric acid and add 119 ml of water.

[0053] I. A tobacco-derived Bacillus belye chq3-4 strain and its screening method

[0054] A type of tobacco-derived Bacillus velezensis chq3-4, belonging to the genus Bacillus, is classified as Bacillus velezensis chq3-4. Bacillus velezensis )chq3-4, accession number CCTCCNO: M 20241305; accession date June 19, 2024; depositary institution China Center for Type Culture Collection; depositary address Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0055] The *Bacillus velezensis* strain described in this application is a tobacco starch-degrading bacterium producing amylase, isolated from a sample of Yunyan 87 flue-cured tobacco from Xunyang City, Ankang City, Shaanxi Province. Identified as *Bacillus velezensis*, this strain exhibits strong enzyme production capacity (119.28 ± 0.76 U / mL), wide temperature adaptability, and tolerance to alkaline conditions (strong enzyme activity is maintained at temperatures between 40 and 90°C and at pH levels between 6.0 and 9.0), thus addressing the problem of poor adaptability of enzyme preparations to the special processing environment of tobacco. Furthermore, the *Bacillus velezensis* strain described in this application demonstrates high viability, surviving normally at 80°C.

[0056] The amylase produced by *Bacillus belycera* chq3-4, a tobacco-derived amylase-producing bacterium, exhibits extremely strong degradation activity and specificity for tobacco starch. Adding crude chq3-4 enzyme solution can reduce the starch content in tobacco leaves by 4.50% within 48 hours, a reduction of 8.46%; the reducing sugar content can increase by approximately 6.5%. The reduction in starch content in tobacco leaves effectively reduces the burnt flavor of tobacco, while the increase in reducing sugar content increases the composition and content of aroma substances in the smoke, thereby improving the quality of tobacco products. This solution addresses the problem of poor specificity in existing biological enzyme treatments for tobacco leaves, meeting the needs for large-scale production and use of biological enzymes in the tobacco industry.

[0057] Tobacco products treated with the amylase produced by Bacillus berberis chq3-4, a tobacco-derived amylase-producing bacterium, showed improved aroma quality, aroma quantity, and smoothness in the smoke after sensory evaluation, reduced irritation and strength, and improved aftertaste comfort, significantly enhancing the overall quality of cigarette smoking.

[0058] The screening method for tobacco-derived Bacillus cylindrica chq3-4 that produces amylase solves the problems of long screening cycles and aimless screening in the prior art for tobacco-derived starch-degrading bacteria.

[0059] Example 1: Screening of strains that efficiently degrade tobacco starch

[0060] This embodiment screened a strain capable of efficiently degrading tobacco starch from Yunyan 87 flue-cured tobacco samples collected in Xunyang City, Ankang City, Shaanxi Province in July 2024. The specific implementation process is as follows:

[0061] 1.1 Sampling and Enrichment Screening

[0062] Weigh 5 g of flue-cured tobacco sample, cut it into small pieces, add 45 mL of sterilized phosphate buffer, homogenize on speed 2 for 10 min, heat in a water bath at 80℃ for 15 min, and transfer 1 mL of the supernatant after heating to 50 mL of liquid screening medium. Incubate at 37℃ and 180 r / min for directional enrichment for 48 h.

[0063] 1.2 Initial screening of strains

[0064] After heating the above bacterial culture in an 80°C water bath for 15 min, perform serial dilutions (initial dilution of 10). -1 ), respectively absorb 10 -4 -10 -8 Five dilutions of bacterial suspension, 100 μL each, were spread onto solid screening medium and incubated at 37℃ for 48 h in a constant temperature and humidity incubator. Iodine solution was added, and single colonies with large transparent zones were visually observed. The diameters of the transparent zones and colonies were measured using digital calipers, and their ratio (HC value) was calculated. Initial screening yielded four strains with strong tobacco starch degradation capabilities. The measurement results are shown in Table 1.

[0065] Table 1. Results of initial screening of bacterial strains

[0066]

[0067] As shown in the table above, the HC value of the initial screening strain chq3-4 is the highest, indicating that its ability to produce amylase is greater than that of other tobacco starch-degrading strains screened at the same time.

[0068] 1.3 Secondary screening of bacterial strains

[0069] Four strains (chq3-4, chq4-2, chq5-1, and chq5-2) obtained from the initial screening were streaked onto LB solid medium and incubated at 37°C for 12 h. Single colonies were picked and inoculated into LB liquid medium and incubated at 37°C for 48 h. After centrifugation at 10000 r / min for 10 min at 4°C and filtration through a 0.22 μm microfiltration membrane, 500 μL of the supernatant from each single colony was added to pre-drilled (6 mm diameter) solid screening medium and incubated at 37°C for 24 h. Iodine solution was added, and the diameter of the clear zone was measured using a colony counter to preliminarily determine the amylase-producing capacity of the strains. The results of the secondary screening are shown in Table 2. Chq3-4 had the largest clear zone diameter, indicating that chq3-4 had the strongest amylase-producing capacity.

[0070] Table 2 Results of strain rescreening measurement

[0071]

[0072] Based on the above screening results, strain chq3-4 was selected as the optimal strain for the next stage of the experiment.

[0073] 1.4 Determination of amylase activity in strains

[0074] 1.4.1 Preparation of crude enzyme solution

[0075] Using a 1 μL disposable sterile inoculation loop, dip a chq3-4 bacterial suspension into LB solid medium and streak it onto the medium. Incubate at 37°C inverted for approximately 12 h. Pick a single colony and inoculate it into 5 mL of liquid LB medium. Incubate at 37°C and 180 rpm for 12 h to obtain the seed culture. Add 1.5 mL of the seed culture (3% v / v) to 50 mL of fermentation medium and incubate at 37°C and 180 rpm for 60 h. Then centrifuge at 4°C and 10,000 rpm for 10 min. Collect the supernatant and filter it through a 0.22 μm sterile aqueous filter to obtain the crude enzyme solution.

[0076] 1.4.2 Amylase Activity Assay

[0077] The assay method refers to the α-amylase activity assay method in the national standard GB1886.174-2016.

[0078] The enzyme activity of amylase is defined as the amount of enzyme required to liquefy 1g of soluble starch in 1mL (or 1g of solid enzyme powder) at pH 6.0 and 60℃ for 1h. This amount is called one enzyme activity unit (U), expressed as U / mL (or U / g).

[0079] The results of amylase activity assay in the crude enzyme solution of the strains are shown in Table 4. Strains FZ19-4 and FYZ1-3 are tobacco-derived amylase-producing strains previously screened by the applicant, and the screening method is the same as above.

[0080] Table 3 Results of amylase activity assay of strains

[0081]

[0082] In summary, compared with existing amylase-producing strains FZ19-4 and FYZ1-3, strain chq3-4 exhibits an amylase activity as high as 119.28 ± 0.76 (U / mL), which is higher than that of other starch-degrading strains screened out. The purified chq3-4 strain was frozen at -80℃ to maintain its original characteristics and prevent cell death, degeneration, or contamination, and was periodically revitalized.

[0083] Example 2: Physiological, biochemical, and molecular biological identification of tobacco starch-degrading bacteria (Bacillus belye chq3-4)

[0084] 2.1 Physiological and biochemical identification

[0085] Morphological observation: Strain chq3-4 was inoculated onto LB agar plates and incubated at 37°C. Colony morphology was observed after 12 h. Results are as follows: Figure 1 , Figure 2 As shown, the colonies of strain chq3-4 are milky white, opaque, with a smooth and highly moist surface, a mucous-like consistency, and a certain degree of fluidity. They exhibit obvious stringing during inoculation.

[0086] Gram staining: Apply a thin, non-overly thick layer of actively growing Chq3-4 bacteria to a glass slide and allow it to dry and fix. Add ammonium oxalate crystal violet stain to the smeared area, let stand for 1-2 minutes, then carefully rinse with water until the effluent is almost colorless. After it has mostly dried, add iodine-potassium iodide solution to the smeared area, let stand for 1-2 minutes, then carefully rinse with water until the effluent is almost colorless. Add 95% ethanol solution to the smear (approximately 20-30 seconds), and when the effluent is almost colorless, immediately wash off the ethanol with water. Blot away any remaining water on the slide with absorbent paper, then counterstain with safranin for 2 minutes, then carefully rinse with water until the effluent is almost colorless, and blot away any remaining water on the slide with absorbent paper. Observe under a 100× oil immersion microscope. The results are as follows: Figure 3 As shown, the microstructure of strain chq3-4 is rod-shaped, indicating it is a Gram-positive bacterium.

[0087] 2.2 Molecular biological identification

[0088] Bacillus belye chq3-4 strain was inoculated into LB liquid medium and cultured at 37℃ for 12 h. The bacterial cells were collected by centrifugation, and their 16S rDNA was amplified by PCR using upstream primer 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and downstream primer 1492R (5'-GGTTACCTTGTTACGACTT-3'). The amplification conditions were as follows: pre-denaturation at 94℃ for 5 min (1 cycle); denaturation at 94℃ for 45 s, annealing at 55℃ for 45 s, extension at 72℃ for 1 min (30 cycles); and total extension at 72℃ for 10 min (1 cycle).

[0089] The obtained 16S rDNA fragment was purified and recovered, and DNA sequencing was performed by Sangon Biotech (Shanghai) Co., Ltd. The sequenced 16S rDNA gene sequence of the high-amylase-producing strain chq3-4 contained 1491 bases. The sequencing results were compared with nucleotide sequences uploaded to GenBank using Blast software. Homology analysis showed that the 16S rDNA gene sequence of strain chq3-4 was similar to that of Bacillus belye (…). Bacillus velezensis Sequences with high similarity were selected, and a phylogenetic tree was constructed using MEGA 6.06 software with a Kimura two-parameter model and the Neighbor-Joining method. The bootstrap support test was performed using 1000 repeated samplings. The phylogenetic tree is shown below. Figure 4 As shown, strain chq3-4 belongs to *Bacillus belyes*. Based on morphological characteristics and 16S rDNA gene sequence analysis, this strain was identified as *Bacillus belyes*. Bacillus velezensis The strain, named *Bacillus belyssus* chq3-4, was deposited at the China Center for Type Culture Collection (CCTCC) on June 19, 2024, with accession number CCTCC NO: M 20241305.

[0090] Example 3: Temperature stability analysis of amylase prepared from strain chq3-4

[0091] Prepare an appropriate amount of crude enzyme solution according to the method in Example 1. Adjust the pH of the reaction system to 7.0 and the temperature to 40℃~90℃. Set a reaction system temperature every 5℃ and measure the enzyme activity of the crude enzyme solution at the corresponding temperature (hold for 30 min). The relative enzyme activity (the ratio of the amylase activity measured at each temperature to the highest enzyme activity measured) is used to represent the enzyme activity relationship between different temperatures. The measurement results are shown in Table 4.

[0092] Table 4. Results of amylase activity assay at different temperatures

[0093]

[0094] According to Table 4 above and Figure 5 It was found that under the experimental conditions, within the reaction temperature range of 40-90℃, the crude enzyme solutions of the tested strains all exhibited a certain starch degradation effect, with relative enzyme activities all above 60%. The highest enzyme activity was observed at 75℃, reaching 125.33 U / mL (relative enzyme activity at this temperature is 100%), indicating that the optimal reaction temperature for amylase in the crude enzyme solution of strain chq3-4 is 75℃. At temperatures below 75℃, enzyme activity increased with increasing temperature; when the temperature exceeded 75℃, enzyme activity gradually decreased, but the decrease was not significant, and the relative enzyme activity at 85℃ still remained above 83.77%, demonstrating relative stability of enzyme activity within the temperature range of 40-85℃. At 90℃, the relative enzyme activity still reached 69.98%, indicating good heat resistance after binding to the substrate, and that the enzyme possesses a wide temperature range adaptability and high-temperature resistance, reflecting the structural stability of the enzyme. Using the amylase prepared by strain chq3-4 in the special processing environment of tobacco can solve the problem of temperature incompatibility of commercial enzymes.

[0095] Example 4: pH stability analysis of amylase prepared from strain chq3-4

[0096] Prepare an appropriate amount of crude enzyme solution according to the method in Example 1. Adjust the temperature of the reaction system to 75°C and the pH to 5-9. Set a pH of the reaction system every 0.5. Measure the enzyme activity of the crude enzyme solution at the corresponding pH (hold for 30 min). The enzyme activity relationship between different pH is expressed by relative enzyme activity (the ratio of the amylase activity measured at each pH to the highest enzyme activity measured). The measurement results are shown in Table 5.

[0097] Table 5. Results of amylase activity assay at different pH levels

[0098]

[0099] According to Table 5 above and Figure 6 It was found that under the experimental conditions, the crude enzyme solutions of the tested strains all exhibited some starch degradation activity within a pH range of 6.0-9.0. The enzyme activity was highest at pH 8.0, reaching 130.26 U / mL (relative enzyme activity at this pH value is 100%), indicating that the optimal reaction pH for amylase in the crude enzyme solution of strain chq3-4 is 8.0. At pH < 8.0, enzyme activity increased with increasing pH; when pH > 8.0, enzyme activity gradually decreased, but the decrease was not significant. Enzyme activity remained relatively stable within the pH range of 6.0-9.0, indicating that the enzyme has a wide pH adaptability and good tolerance to weak alkalis, reflecting the structural stability of the enzyme. Using the amylase prepared by strain chq3-4 in the special processing environment of tobacco can solve the pH incompatibility problem of commercial enzymes.

[0100] II. Application of Bacillus belyceta chq3-4, a tobacco-derived amylase-producing bacterium, in improving tobacco leaf quality.

[0101] This application screened a tobacco-derived amylase-producing strain, chq3-4, from flue-cured tobacco samples. After identification as *Bacillus velezensis*, the crude enzyme solution (fermentation supernatant filtrate) prepared by centrifuging and filtering the fermentation broth was used to treat tobacco products. Changes in starch and reducing sugar content before and after treatment were detected using standard tobacco industry methods. Sensory evaluation was conducted to assess the improvement of the sensory quality of tobacco leaves by the chq3-4 crude enzyme solution. The specific implementation procedures are as follows:

[0102] Example 5: Degradation of starch content in tobacco leaves using Bacillus belye chq3-4

[0103] The crude enzyme solution was prepared according to the method in Example 1.

[0104] Measure 15 mL of crude enzyme solution (i.e., fermentation supernatant filtrate) and spray it evenly onto the surface of 50 g of 2020 Chongqing B4F tobacco leaves. Shake thoroughly to mix evenly, then place in a constant temperature and humidity (45℃, 75%) incubator for 48 h of fermentation. Samples are taken, with three replicates per group. A negative control group was prepared by spraying 2020 Chongqing B4F tobacco leaves with an equal volume of sterile water (equal to the crude enzyme solution), and a positive control group was prepared by spraying 2020 Chongqing B4F tobacco leaves with an equal volume of Novozymes α-amylase (product number: BAN480L, diluted 500 times) (equal to the crude enzyme solution). After enzyme treatment, the tobacco leaves were placed in a 100℃ oven for 1 min to inactivate the enzyme. Then, they were dried in a 40℃ oven for 2 h, ground, and passed through a 40-mesh sieve. The starch content was determined using the method in YC / T 216-2013 "Determination of Starch in Tobacco and Tobacco Products - Continuous Flow Method".

[0105] The results are shown in Figure 7. After fermentation, the starch content in the tobacco leaves of the negative control group (water treatment) was 5.32%, while the starch content in the experimental group (treated with chq3-4 crude enzyme solution) was 4.87%. Adding chq3-4 crude enzyme solution reduced the starch content by 4.50% in 48 hours, a reduction of 8.46%, which was superior to the positive control group (treated with Novozymes commercial amylase). This indicates that the crude enzyme solution of strain chq3-4 plays a role in degrading the starch content in tobacco leaves. The reduction in starch content can effectively reduce the burnt flavor of tobacco, thereby improving the quality of tobacco products.

[0106] Example 6: Using Bacillus belye chq3-4 to increase the reducing sugar content in tobacco shreds

[0107] The crude enzyme solution was prepared according to the method in Example 1.

[0108] Measure 15 mL of crude enzyme solution (i.e., fermentation supernatant filtrate) and spray it evenly onto the surface of 50 g of 2020 Chongqing B4F tobacco shreds. After thorough mixing, place the mixture in a constant temperature and humidity (45℃, 75%) incubator for 48 h of fermentation. Samples were taken, with three replicates per group. A negative control group was prepared by spraying 2020 Chongqing B4F tobacco shreds with an equal volume of sterile water (equal to the crude enzyme solution), and a positive control group was prepared by spraying 2020 Chongqing B4F tobacco shreds with an equal volume of Novozymes α-amylase (product number: BAN480L, diluted 500 times) (equal to the crude enzyme solution). After enzyme treatment, the tobacco shreds were placed in a 100℃ oven for 1 min to inactivate the enzyme. Then, they were dried in a 40℃ oven for 2 h, ground, and passed through a 40-mesh sieve. The reducing sugar content in the tobacco shreds was determined using YC / T 159-2019 "Determination of Water-Soluble Sugars in Tobacco and Tobacco Products - Continuous Flow Method".

[0109] As shown in Figure 8, after fermentation, the reducing sugar content in the negative control group tobacco was 14.63%, while the reducing sugar content in the experimental group tobacco was 15.58%. The reducing sugar content increased by 0.95% in 48 hours, an increase of 6.50%, which was better than that in the positive control group (treated with Novozymes commercial amylase). This indicates that under the action of amylase, starch is hydrolyzed into small molecule reducing sugars, leading to an increase in their content, thus significantly improving the quality of tobacco products.

[0110] Example 7 Sensory evaluation of tobacco flakes treated with crude enzyme solution of Bacillus belyssus chq3-4

[0111] The crude enzyme solution was prepared according to the method in Example 1.

[0112] Measure 15 mL of crude enzyme solution (i.e., fermentation supernatant filtrate) and spray it evenly onto the surface of 50 g of tobacco leaves. After thorough mixing, place the mixture in a constant temperature and humidity (45 ℃, 75% humidity) incubator for 48 h of fermentation, and take samples, with 3 replicates per group. A negative control group was prepared by spraying 2020 Chongqing B4F tobacco leaves with an equal volume of sterile water to the crude enzyme solution, and a positive control group was prepared by spraying 2020 Chongqing B4F tobacco leaves with an equal volume of Novozymes α-amylase (product number: BAN480L, diluted 500 times) to the crude enzyme solution. After enzyme treatment, the tobacco leaves were placed in a 100℃ oven for 1 min to inactivate the enzyme. After removal, the tobacco leaves were cut into shreds using a shredder, hand-rolled into cigarettes, and equilibrated in a balancing cabinet (22℃, 60% humidity) for 48 hours. Sensory evaluation was conducted according to the sensory technical requirements for cigarettes in the national standard GB5606.4-2005.

[0113] The results are as follows Figure 9As shown, sensory evaluation indicates that the crude amylase solution prepared by chq3-4 can increase the content of aroma substances, improve the aroma quality, aroma quantity and smoothness of tobacco products, reduce irritation and strength, and improve the aftertaste comfort. It has the same effect as commercial enzymes, but is superior to commercial enzymes in terms of reducing irritation and improving aroma quality, and significantly improves the quality of cigarette smoking.

[0114] In summary, this application isolated and screened tobacco starch-degrading bacteria chq3-4 from Yunyan 87 flue-cured tobacco samples from Xunyang City, Ankang City, Shaanxi Province. Physiological, biochemical, and molecular biological identification confirmed that this strain is *Bacillus belye* (*B. belye*). Bacillus velezensis After fermentation, the chq3-4 strain was centrifuged and filtered to prepare a crude enzyme solution, which was then evenly sprayed onto the surface of tobacco products. After constant temperature and humidity fermentation, the starch content in the tobacco leaves was significantly reduced, while the reducing sugar content in the tobacco shreds was increased. In addition, after smoking evaluation of the fermented tobacco products, the aroma quality, aroma quantity, and smoothness of the cigarettes were improved, the irritation and strength were reduced, and the aftertaste comfort was improved, significantly enhancing the smoking quality of cigarettes.

[0115] It will be readily understood by those skilled in the art that the above-described advantageous methods can be freely combined and superimposed without conflict. The above are merely preferred embodiments of this application and are not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application. The above are merely preferred embodiments of this application. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of this application, and these improvements and modifications should also be considered within the protection scope of this application.

Claims

1. A tobacco-derived Bacillus belye chq3-4, characterized in that, The Bacillus belesi ( Bacillus velezensis )chq3-4 was deposited at the China Center for Type Culture Collection on June 19, 2024, with accession number CCTCCNO: M 20241305; Amylase was obtained by culturing and fermenting the *Bacillus belye* chq3-4 strain. The crude enzyme solution of the amylase had an enzyme activity as high as 119.28 ± 0.76 U / mL. Under conditions of pH 7.0, the relative enzyme activity of the crude enzyme solution was higher than 60% at 40℃~90℃, higher than 80% at 40℃~85℃, and 100% at 75℃. Under conditions of 75℃ and pH 6.0~9.0, the relative enzyme activity of the crude enzyme solution was higher than 60% at 75℃, higher than 80% at 7.0~8.5, and 100% at pH 8.

0.

2. The application of *Bacillus vesiculosus* chq3-4 from tobacco source in improving the quality of tobacco products, characterized in that... The Bacillus berberis chq3-4 of claim 1 is used to improve the quality of tobacco products, including reducing the content of tobacco starch in tobacco products and increasing the content of reducing sugar in tobacco products.

3. The application according to claim 2, characterized in that, Application methods include: The fermentation supernatant filtrate was prepared using the Bacillus berberis chq3-4; The fermentation supernatant filtrate is evenly sprayed onto the surface of tobacco products for fermentation.

4. The application according to claim 3, characterized in that, The fermentation supernatant filtrate is prepared by the following method: The *Bacillus belye* chq3-4 was inoculated into the fermentation medium for fermentation. The culture was carried out at 35℃~40℃ and 150 r / min~200 r / min for 55 h~65 h. Then, the culture was centrifuged at 2℃~6℃ and 8000 r / min~12000 r / min for 8 min~12 min. The supernatant was collected and filtered through a sterile aqueous filter membrane to obtain the fermentation supernatant filtrate.

5. The application according to claim 3, characterized in that, The method of uniformly spraying the fermentation supernatant filtrate onto the surface of tobacco products for fermentation includes: The fermentation supernatant filtrate is applied and fermented as follows: the fermentation supernatant filtrate is evenly sprayed onto the tobacco leaves or shredded tobacco to be treated, and then placed in a fermentation environment of 42℃~45℃ and 70%~75% humidity for 45 h~50 h. Enzyme inactivation treatment: After fermentation, the tobacco leaves or shredded tobacco are heated at 100℃~120℃ to inactivate the residual fermentation supernatant filtrate.

6. The application according to claim 5, characterized in that, For every 10 mL to 20 mL of the fermentation supernatant filtrate, spray 45 g to 55 g of tobacco leaves or shredded tobacco.

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