Bacillus velezensis FZ19-4 with high yield of amylase and application of bacillus velezensis FZ19-4

Abstract

The invention discloses bacillus velezensis FZ19-4 with high yield of amylase and application, and belongs to the field of tobacco microorganisms. The classification name of the bacillus velezensis FZ19-4 is bacillus velezensis FZ19-4, the bacillus velezensis FZ19-4 is preserved in the China Center for Type Culture Collection (CCTCC), the preservation date is December 12, 2022, and the preservation number is CCTCC NO: M 20221925. The invention provides a tobacco-source starch degradation strain, the enzyme activity of amylase produced by the strain can reach 77.83 + / -0.34 U / mL at the high temperature of 70 DEG C, the content of tobacco starch in tobacco products can be effectively reduced, and the quality of the tobacco products is further improved; therefore, the quality, applicability and utilization rate of low-grade and low-grade tobacco leaves are improved, and the problem of shortage of high-quality tobacco leaves at present is solved; precious microbial resources are provided for the tobacco industry, and high-quality development of tobacco industry enterprises is promoted.

Description

Technical Field

[0001] This application belongs to the technical field of microbial applications, and particularly relates to a high-amylase-producing Bacillus berberis FZ19-4 and its applications. Background Technology

[0002] Starch, an important carbohydrate in tobacco leaves, is a crucial component of the chemical composition of tobacco leaves, and changes in its content can affect tobacco quality. Currently, methods such as enzymatic and microbial approaches can be used to reduce the starch content in tobacco leaves, thereby improving tobacco quality.

[0003] Currently, tobacco enzyme preparations are mostly produced by screening strains from microbial banks to produce natural enzymes or by purchasing commercially available food enzymes. Compared with internationally advanced molecular modification and high-efficiency expression technologies, these methods result in lower activity and yield, higher relative costs, and significant room for improvement. Furthermore, due to the substantial 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 weak 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 processing technology in the tobacco industry. Currently screened tobacco starch-degrading bacteria produce amylases with low activity and low temperature tolerance, making them unsuitable for the high-temperature fermentation of tobacco products, and the screening process for these bacteria is lengthy.

[0004] Therefore, it is necessary to discover specific enzymes suitable for tobacco starch that can enhance its degradation activity, with the aim of applying them to tobacco fermentation and aging to improve tobacco quality. Furthermore, it is necessary to optimize screening methods, shorten the screening cycle, and obtain new strains capable of producing amylases that meet the above requirements for tobacco aging, thereby obtaining high-quality tobacco leaves. Summary of the Invention

[0005] To address the aforementioned issues, this application provides a high-yield amylase-producing Bacillus baileyi FZ19-4 strain. This strain can prepare a tobacco-specific amylase, which exhibits extremely strong degradation activity and specificity in tobacco starch, solving the problem of poor specificity in existing biological enzyme treatments for tobacco leaves and meeting the requirements for large-scale production and use of biological enzymes in the tobacco industry. Furthermore, its application in tobacco aging treatment can significantly improve tobacco quality.

[0006] The specific technical solution of this application is as follows:

[0007] On the one hand, this application provides a tobacco-derived Bacillus velezensis that produces amylase, classified as Bacillus velezensis FZ19-4, which has been deposited at the China Center for Type Culture Collection (CCTCC) on December 12, 2022, with accession number CCTCC NO: M 20221925.

[0008] This strain solves the problem that existing technologies use tobacco-derived microorganisms with low amylase activity and are unsuitable for high-temperature fermentation of tobacco products.

[0009] On the other hand, this application provides the following applications:

[0010] The application of the above-mentioned Bacillus berberis FZ19-4 in the preparation of tobacco-specific amylase.

[0011] The tobacco-specific amylase prepared from Bacillus berberis FZ19-4 was described above.

[0012] The application of Bacillus berberis FZ19-4 and / or the amylase as described in claim 3 in the degradation of tobacco starch.

[0013] The application of Bacillus bellis FZ19-4 and / or the amylase as described in claim 3 in improving tobacco quality. Specifically, the application method involves spraying the fermentation supernatant filtrate of Bacillus bellis onto the surface of tobacco products for fermentation.

[0014] Optionally, the above fermentation supernatant is prepared by the following method: inoculating the Bacillus belye into the fermentation medium for fermentation, culturing at 35-40℃ and 150-200 r / min for 55-65 h, centrifuging at 8000-12000 r / min for 8-12 min, taking the supernatant, filtering, and obtaining the fermentation supernatant filtrate.

[0015] Preferably, the culture is carried out at 37℃ and 180r / min for 60h, centrifuged at 10000r / min for 10min, the supernatant is collected, filtered, and the fermentation supernatant filtrate is obtained.

[0016] Optionally, each 15-25 mL of fermentation supernatant filtrate corresponds to 45-55 g of tobacco products.

[0017] Preferably, every 20 mL of fermentation supernatant filtrate corresponds to 50 g of tobacco products.

[0018] Optionally, the fermentation conditions are a constant temperature of 42-45℃, a constant humidity of 70-75%, and a fermentation time of 45-50 hours.

[0019] Preferably, the fermentation conditions are a constant temperature of 45°C, a constant humidity of 75%, and a fermentation time of 48 hours.

[0020] Optionally, the tobacco product is tobacco leaf or shredded tobacco.

[0021] The beneficial effects of this application include, but are not limited to:

[0022] 1. The *Bacillus belyssus* strain used in this application is a tobacco-derived strain. This strain has a strong enzyme production capacity, significantly higher than other strains screened at the same time. Furthermore, the *Bacillus belyssus* strain in this application exhibits high activity, surviving normally at 80℃. The crude enzyme solutions of the tested strains all showed some starch degradation activity. Within the temperature range of 40–90℃, the amylase produced by *Bacillus belyssus* in this application showed relatively high enzyme activity, generally above 60%. At 90℃, the relative enzyme activity still reached 69.98%. This indicates that the enzyme has good heat resistance after binding to the substrate, and possesses a wide temperature range adaptability and high-temperature resistance, reflecting the structural stability of the enzyme.

[0023] 2. The amylase produced by *Bacillus belycera*, a tobacco-derived amylase-producing bacterium, exhibits extremely strong degradation activity and specificity for tobacco starch. Adding crude enzyme solution FZ19-4 can reduce the starch content in tobacco leaves by 0.89% within 48 hours, a reduction of 18%; the reducing sugar content can increase by approximately 24%. The reduction in starch content in tobacco leaves can effectively reduce the burnt flavor of tobacco, thereby improving the quality of tobacco products. This solves the problem of poor specificity in existing biological enzyme treatments for tobacco leaves, meeting the needs of large-scale production and use of biological enzymes in the tobacco industry; its application in tobacco leaf aging treatment can greatly improve the quality of tobacco leaves.

[0024] 3. Tobacco products treated with *Bacillus belycera*, which produces tobacco amylase, showed a significant improvement in smoke quality after sensory evaluation, with reduced irritation, milder smoke, and a cleaner aftertaste.

[0025] This application also provides a screening method for tobacco-derived amylase-producing Bacillus belye, in order to solve the problems of long screening cycles and aimless screening in the prior art for tobacco-derived starch-degrading bacteria. Attached Figure Description

[0026] Figure 1 This is a colony morphology diagram of Bacillus velezensis FZ19-4 strain in solid LB medium in Example 2 of this application;

[0027] Figure 2 This is a 100× microscopic morphological image of Bacillus velezensis FZ19-4 strain in Example 2 of this application;

[0028] Figure 3The image shows the morphology of Bacillus velezensis FZ19-4 strain after Gram staining under a 100x oil immersion microscope during identification in Example 2 of this application.

[0029] Figure 4 This is a phylogenetic tree constructed based on the 16S rDNA gene sequence during the identification of Bacillus velezensis FZ19-4 strain using molecular biology in Example 2 of this application;

[0030] Figure 5 The growth curve of Bacillus velezensis FZ19-4 in Example 3 of this application was determined;

[0031] Figure 6 This study describes the determination of the enzyme activity curve of Bacillus velezensis FZ19-4 in Example 4 of this application.

[0032] Figure 7 This is a graph showing the changes in enzyme activity of the amylase prepared by strain FZ19-4 in Example 5 of this application at different reaction temperatures. Detailed Implementation

[0033] 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, etc. used in the embodiments are commercially available products.

[0034] 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:

[0035] Culture medium:

[0036] 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°C for 30 min.

[0037] 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°C for 30 min.

[0038] 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°C for 30 min.

[0039] Experimental reagents:

[0040] Preparation of soluble starch solution (2g / L): Weigh 1.000g (accurate to 0.001g) of soluble starch (on an oven-dry basis) into a beaker, add a small amount of water to make a slurry, and slowly add 200mL of boiling water while stirring. Then rinse the beaker containing the starch with water several times, pour the washing liquid into the solution, stir and heat until completely transparent, cool and make up to 500mL. The solution should be prepared and used immediately.

[0041] Original iodine solution: Weigh 11.0g of iodine and 22.0g of potassium iodide, dissolve the iodine completely in a small amount of water, and bring the volume up to 500mL. Store in a brown bottle.

[0042] 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.

[0043] Preparation of phosphate buffer (pH 6.0): Weigh 45.23 g of disodium hydrogen phosphate (Na2HPO4·12H2O) and 8.07 g of citric acid (C6H8O7·H2O), dissolve in water and bring the volume to 1000 mL. Use after pH meter calibration.

[0044] I. A high-amylase-producing Bacillus belye and its screening method

[0045] A high-amylase-producing Bacillus velezensis, Bacillus velezensis FZ19-4, with accession number CCTCC NO: M 20221925, deposited on December 12, 2022, at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, China.

[0046] Example 1: Screening of strains that efficiently degrade tobacco starch

[0047] This embodiment screened a strain capable of efficiently degrading tobacco starch from fresh tobacco leaf samples collected in Jiangchuan District, Yuxi City, Yunnan Province in July 2022. The specific implementation process is as follows:

[0048] 1.1 Sampling and Enrichment Screening

[0049] Weigh 5g of fresh tobacco leaf sample, cut it into small pieces, add 45mL of sterilized phosphate buffer, homogenize on speed 2 for 10min, heat in an 80℃ water bath for 15min, and transfer 1mL of the supernatant after heating to 50mL of liquid screening medium. Incubate at 37℃ and 180r / min for directional enrichment for 48h.

[0050] 1.2 Initial screening of strains

[0051] After heating the above bacterial culture in an 80°C water bath for 15 minutes, 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°C for 48 h. 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 results are shown in Table 1.

[0052] Table 1. Results of initial screening of bacterial strains

[0053]

[0054]

[0055] As shown in the table above, strain FZ19-4, which was initially screened, had the highest HC value, indicating that its ability to produce amylase was greater than that of other tobacco starch-degrading strains screened at the same time.

[0056] 1.3 Secondary screening of bacterial strains

[0057] Four strains obtained from the initial screening (FZ19-4, FX7-6, FX10-1, and FX10-3) were streaked onto LB solid medium and incubated at 37°C for 24 hours. Single colonies were picked and inoculated into LB liquid medium and incubated at 37°C for 48 hours. After centrifugation at 10000 rpm for 10 minutes and filtration through a 0.22 μm microfiltration membrane, the supernatant from different single colonies was added to pre-drilled (6 mm diameter) solid screening medium. A control group was also set up, using a known amylase-producing Bacillus amyloliquefaciens strain MK10163. Fermentation supernatant filtrate was obtained using the same preparation method described above. On the four pre-drilled plates containing the fermentation supernatant filtrate (FZ19-4, FX7-6, FX10-1, and FX10-3), additional wells of the same diameter were drilled to add the fermentation supernatant filtrate of the control strain. The bacteria were incubated at 37℃ for 24 hours, then iodine solution was added, and the diameter of the clear zone was measured using a colony counter to preliminarily determine the amylase activity. The results of the secondary screening of the strains are shown in Table 2.

[0058] The aforementioned Bacillus amyloliquefaciens, classified as Bacillus amyloliquefaciens MK10163, has been deposited at the China Center for Type Culture Collection (CCTCC) on December 4, 2023, with accession number CCTCC NO: M 20232452.

[0059] Table 2 Results of strain rescreening

[0060]

[0061] The ratio of the transparent zone diameter of the selected strain to that of the MK10163 strain was greater than 1, indicating that the selected strain had a stronger amylase-producing ability than the MK10163 strain. Among them, the ratio of the transparent zone diameter of the FZ19-4 strain to that of the MK10163 strain was the largest, indicating that the FZ19-4 strain had the strongest amylase-producing ability.

[0062] Based on the above screening results, strain FZ19-4 was selected as the optimal strain for the next stage of the experiment.

[0063] 1.4 Determination of amylase activity in strains

[0064] The assay method is a modification of the α-amylase activity assay method in the national standard GB1886.174-2016. The main modification is the preparation of standard solutions, the specification of a standard curve, and the acquisition of a concentration-absorbance equation to determine amylase activity. The specific improved assay steps are as follows:

[0065] 1.4.1 Drawing the Standard Curve

[0066] The standard curve is plotted as follows:

[0067] Soluble starch standard solution: Prepare according to Table 3.

[0068] Table 3 Soluble starch standard solutions

[0069]

[0070] Pipette 1.00 mL of each of the above solutions (parallel experiments are required) into centrifuge tubes containing 0.5 mL of 0.1 mol / L hydrochloric acid solution and 5.00 mL of dilute iodine solution, respectively. Shake thoroughly. Using the 0.5 mL 0.1 mol / L hydrochloric acid solution and 5.00 mL dilute iodine solution as blanks, quickly measure the absorbance (A) at 660 nm using a spectrophotometer. Plot a standard curve with absorbance (A) as the abscissa (x) and the concentration of the soluble starch standard solution as the ordinate (y). The standard equation obtained is y = 1.2352x - 0.0017, R 2 =0.9992, and the slope K =1.2352 is obtained.

[0071] 1.4.2 Preparation of crude enzyme solution

[0072] Using a 1 μL sterile disposable inoculation loop, dip a 1 μL tube of bacterial suspension from strain FZ19-4 onto LB solid medium and incubate upside down at 37°C for approximately 12 hours. Pick a single colony and inoculate it into 5 mL of liquid LB medium, incubating at 37°C and 180 rpm for 12 hours 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 hours. Then centrifuge at 10000 rpm for 10 minutes, collect the supernatant, and filter through a 0.22 μm sterile aqueous filter to obtain the crude enzyme solution, i.e., amylase.

[0073] 1.4.3 Measurement

[0074] Pipette 20.0 mL of soluble starch solution (2 mg / mL) into a 50 mL centrifuge tube, add 5.00 mL of pH 6.0 phosphate buffer, shake well, and preheat in a 70 ± 0.2 °C water bath for 8 min.

[0075] Take 1 mL of crude enzyme solution and dilute it to a suitable concentration with phosphate buffer (pH 6.0). Add 1.00 mL of the diluted enzyme solution, start timing immediately, shake well, and maintain a constant temperature of 70 ± 0.2 °C for 10 min. Immediately use an automatic pipette to pipette 1.00 mL of the reaction solution into a centrifuge tube containing 0.5 mL of 0.1 mol / L hydrochloric acid solution and 5.00 mL of dilute iodine solution. Shake well, and using 0.5 mL of hydrochloric acid solution and 5.00 mL of dilute iodine solution as blanks, quickly measure the absorbance (A) at a wavelength of 660 nm using a spectrophotometer.

[0076] 1.4.4 Results: Calculation of amylase activity

[0077] The enzyme activity of amylases produced by different strains was detected under high temperature conditions (70℃). The specific detection methods and results are as follows:

[0078] Calculation method:

[0079] The enzyme activity X of amylase, expressed in U / mL or U / g, is calculated using the following formula:

[0080] X = (2 × 20 - 26 × AK) × n / 10

[0081] In the formula:

[0082] 2—Initial concentration of soluble starch solution;

[0083] 20 — Volume of soluble starch solution during the determination;

[0084] 26 — The sum of the volume of the enzyme solution to be tested, the volume of the phosphate buffer solution at pH 6.0, and the volume of the soluble starch solution used for the test;

[0085] A – The absorbance value of the enzyme solution being measured;

[0086] K—the slope of the obtained standard curve;

[0087] n—the dilution factor of the enzyme solution;

[0088] 10 — Reaction time of the enzyme solution during the assay.

[0089] The test results are based on the arithmetic mean of parallel measurements. The relative error between two independent measurements obtained under repeatability conditions shall not exceed 5%.

[0090] The results of the amylase activity assay for the strains are shown in Table 4. FX8-4 and FYZ3-1 are tobacco-derived amylase-producing strains previously screened by the applicant, and the screening method was the same as above.

[0091] Table 4 Results of amylase activity assay for strains

[0092] strain number Amylase activity (U / mL) at 70℃ FZ19-4 77.83±0.34 FX8-4 22.13±0.11 FYZ3-1 31.31±0.24

[0093] In summary, compared with existing amylase-producing strains FX8-4 and FYZ3-1, strain FZ19-4 exhibits an amylase activity as high as 77.83±0.34 (U / mL) at 70℃, which is significantly higher than that of other screened starch-degrading strains. The purified FZ19-4 strain was frozen at -80℃ to maintain its original characteristics and prevent cell death, degeneration, or contamination, and was periodically revitalized.

[0094] Example 2: Physiological, biochemical, and molecular biological identification of tobacco starch-degrading bacteria

[0095] 2.1 Physiological and Biochemical Identification

[0096] Morphological observation: Strain FZ19-4 was inoculated onto LB agar plates and incubated at 37°C. Colony morphology was observed after 12 hours. Results are as follows: Figure 1 , Figure 2 As shown, the bacterial cells of strain FZ19-4 are straight rods with irregular edges. On LB solid medium, the colonies are round, light yellow, with irregular edges, and have round raised bumps on the surface. They are opaque and can produce mucus.

[0097] Gram staining: Apply a thin, actively growing layer of FZ19-4 bacteria to a glass slide, allowing it to dry and fix. Add ammonium oxalate crystal violet stain to the smeared area, let it 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 it 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 an oil immersion microscope. The results are as follows: Figure 3 As shown, strain FZ19-4 stains purple with Gram stain, indicating it is a Gram-positive bacterium.

[0098] 2.2 Molecular biological identification

[0099] Bacillus belye FZ19-4 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 94℃ for 5 min (1 cycle); denaturation 94℃ for 45 s, annealing 55℃ for 45 s, extension 72℃ for 1 min (30 cycles); total extension 72℃ for 10 min (1 cycle).

[0100] The obtained 16S rDNA fragment was purified and recovered, and DNA sequencing was performed by Sangon Biotech (Shanghai) Co., Ltd. The 16S rDNA gene sequence of the high-amylase-producing strain FZ19-4, as shown in SEQ ID No. 1, contains 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 FZ19-4 was highly similar to that of 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 neighbor-joining method. The number of replicates for testing bootstrap support was 1000. The phylogenetic tree is shown below. Figure 4As shown, strain FZ19-4 belongs to Bacillus velezensis. Based on morphological characteristics and 16S rDNA gene sequence analysis, this strain was identified as Bacillus velezensis and named Bacillus velezensis FZ19-4. It was deposited at the China Center for Type Culture Collection (CCTCC) on December 12, 2022, with accession number CCTCC NO: M 20221925.

[0101] Example 3: Growth Analysis of Strain FZ19-4

[0102] Single colonies were picked and inoculated into 5 mL of LB liquid medium and cultured at 37°C with shaking at 200 rpm for 8 h to obtain the seed culture. The seed culture was then inoculated into 50 mL of fermentation medium at a 3% (v / v) inoculation rate and cultured continuously at 37°C with shaking at 180 rpm for 72 h. Samples were taken every 6 h in a biosafety cabinet, and the absorbance at 600 nm was measured using a spectrophotometer. If the bacterial culture was too concentrated, it was appropriately diluted with purified water, and the absorbance was controlled between 0.2 and 0.8. LB medium of the corresponding dilution factor was used as a control. A growth curve was constructed with the culture time (t) on the x-axis and the absorbance (OD600) on the y-axis.

[0103] The results are as follows Figure 5 As shown, strain FZ19-4, when cultured at 37℃, basically entered the stable growth phase from the 48th hour onwards, with an OD600 of 2.23. At this point, the total number of cells in the strain reached its maximum, making it suitable for subsequent applications.

[0104] Example 4: Amylase Activity Analysis of Strains FZ19-4 in Liquid Fermentation

[0105] The FZ19-4 strain was activated, and the seed culture was added to 50 mL of fermentation medium at an inoculation rate of 3% (v / v). Liquid fermentation was carried out at 37°C and 180 rpm, with the pH value kept relatively stable at around 8.0 during the process. Amylase activity in the fermentation broth was measured and analyzed every 6 hours from 0 to 72 hours, following the method described in Example 1.

[0106] The results are as follows Figure 6 As shown, the amylase activity was highest at 60 h, reaching a maximum of 77.83 ± 0.34 U / mL. As time went on, the amylase activity gradually decreased.

[0107] Example 5: Effect of different reaction temperatures on the activity of amylase prepared from FZ19-4

[0108] Prepare an appropriate amount of crude enzyme solution according to the method in Example 1. Adjust the pH of the reaction system to 6.0 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. 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 5.

[0109] Table 5 Results of amylase activity assay at different measurement temperatures

[0110]

[0111]

[0112] According to Table 5 above and Figure 7 It can be seen that under the experimental conditions, the crude enzyme solutions of the test strains all exhibited a certain starch degradation effect within the reaction temperature range of 40–90℃. The enzyme activity was highest at 70℃, reaching 77.83 U / mL (the relative enzyme activity at this temperature is 100%), indicating that the optimal reaction temperature for amylase in the crude enzyme solution of strain FZ19-4 is 70℃. At temperatures below 70℃, enzyme activity increases with increasing temperature. The increase is most pronounced at temperatures below 50℃, reaching a high level (relative activity 91.96%) at 50℃. Afterward, the activity increases slowly with increasing temperature. At temperatures above 70℃, enzyme activity gradually decreases, but the decrease is not significant. The relative activity at 80℃ remains above 88.63%, indicating relatively stable activity within the 50–80℃ temperature range. At temperatures above 80℃, enzyme activity begins to decrease significantly. At 90℃, the relative activity still reaches 69.98%, demonstrating good heat resistance after substrate binding and a wide temperature range, reflecting structural stability.

[0113] Based on the above experimental results, the optimal enzymatic reaction temperature for amylase produced by fermentation using strain FZ19-4 is 70℃.

[0114] II. Application of *Bacillus belyceta*, a tobacco-derived amylase-producing bacillus, in improving tobacco leaf quality.

[0115] This application screened a tobacco-derived amylase-producing strain, FZ19-4, from fresh tobacco leaf samples. After identification as *Bacillus velezensis*, the crude enzyme solution (i.e., fermentation supernatant filtrate) prepared by centrifuging and filtering the fermentation broth was used to treat tobacco products, and the starch and reducing sugar content in the tobacco products was then detected. The specific implementation procedures are as follows:

[0116] Example 6: Degradation of starch content in tobacco leaves using Bacillus belye FZ19-4

[0117] The crude enzyme solution was prepared according to the method in Example 1.

[0118] Measure 20 mL of crude enzyme solution (i.e., fermentation supernatant filtrate) and spray it evenly onto the surface of 50 g of 2019 Qujing Luliang C2F tobacco leaves. Shake thoroughly to mix evenly, then place in a constant temperature and humidity (temperature 45℃, humidity 75%) incubator for 48 h of fermentation. Samples are taken, with 3 replicates per group. A blank control group was prepared by spraying 20 mL of sterile water (equal volume to the crude enzyme solution) onto 2019 Qujing Luliang C2F tobacco leaves. After enzyme treatment, the tobacco leaves were placed in an 80℃ incubator for 20 min to inactivate the enzyme. Then, they were dried in a 40℃ incubator for 2 h, and the moisture content was measured. The leaves were then 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".

[0119] After fermentation, the starch content in the control group tobacco leaves was 4.83%, while the starch content in the experimental group tobacco leaves was 3.94%. Adding FZ19-4 crude enzyme solution reduced the starch content by 0.89% within 48 hours, a reduction of 18%. This reduction in starch content effectively decreases the burnt flavor of tobacco, thereby improving the quality of tobacco products.

[0120] Example 7: Using Bacillus belye FZ19-4 to increase the reducing sugar content in tobacco shreds

[0121] The crude enzyme solution was prepared according to the method in Example 1.

[0122] Measure 20 mL of crude enzyme solution (i.e., fermentation supernatant filtrate) and spray it evenly onto the surface of 50 g of 2019 Qujing Luliang C2F tobacco shreds. After thorough mixing, place the mixture in a constant temperature and humidity (45℃, 75%) incubator for 48 h of fermentation, and take samples, with 3 replicates per group. Use 2019 Qujing Luliang C2F tobacco shreds as a control group, spraying with an equal volume of sterile water (20 mL) to the crude enzyme solution. After enzyme treatment, place the tobacco shreds in an 80℃ incubator for 20 min to inactivate the enzyme. Then, place them in a 40℃ incubator to dry for 2 h and determine the moisture content. Grind and pass through a 40-mesh sieve. The reducing sugar content in the tobacco shreds is determined using YC / T 159-2019 "Determination of Water-Soluble Sugars in Tobacco and Tobacco Products - Continuous Flow Method".

[0123] After fermentation, the reducing sugar content in the control group tobacco was 13.4%, while the reducing sugar content in the experimental group tobacco was 17.3%. The reducing sugar content increased by 3.9% over 48 hours, representing a 24% increase. This indicates that under the action of amylase, starch is hydrolyzed into small-molecule reducing sugars, leading to an increase in reducing sugar content, which can significantly improve the quality of tobacco products.

[0124] Example 8: Sensory evaluation of tobacco flakes treated with crude enzyme solution of Bacillus belyssus FZ19-4

[0125] The crude enzyme solution was prepared according to the method in Example 1.

[0126] Measure 20 mL of crude enzyme solution (i.e., fermentation supernatant filtrate) and spray it evenly onto the surface of 50 g of tobacco flakes. After thorough mixing, place the flakes in a constant temperature and humidity (45℃, 75%) incubator for 48 h of fermentation, with three replicates per group. The control group in this application was sprayed with an equal volume of sterile water (20 mL). After enzyme treatment, the tobacco flakes were placed in an 80℃ incubator for 20 min to inactivate the enzyme. After removal, the tobacco flakes were cut into shreds using a shredder, hand-rolled into cigarettes, and equilibrated in a balancing cabinet (22℃, 60%) for 48-72 hours. Based on the sensory technical requirements for cigarettes in the national standard GB5606.4-2005, sensory evaluation showed that treatment with the crude enzyme solution of strain FZ19-4 significantly improved the smoke quality, reduced irritation, resulted in a smoother smoke, and a clean aftertaste, thus significantly enhancing the smoke quality.

[0127] In summary, this application isolated and screened tobacco starch-degrading bacteria FZ19-4 from fresh tobacco leaf samples collected in Jiangchuan District, Yuxi City, Yunnan Province. Physiological, biochemical, and molecular biological identification confirmed that this strain is *Bacillus velezensis*. After fermentation, centrifugation, and filtration, strain FZ19-4 was used to prepare a crude enzyme solution, which was sprayed onto the surface of tobacco products. Following constant temperature and humidity fermentation, the starch content in the tobacco leaves significantly decreased, while the reducing sugar content in the tobacco shreds increased. Furthermore, after tasting the fermented tobacco products, the aroma quality of the cigarettes showed a significant improvement. This indicates that tobacco starch in tobacco products is decomposed into reducing sugars by amylase in the crude enzyme solution, thereby improving the quality of the tobacco products.

[0128] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.

Claims

1. A high-amylase-producing Bacillus belye FZ19-4, characterized in that: The Bacillus velezensis FZ19-4 described herein is classified as Bacillus velezensis FZ19-4 and has been deposited at the China Center for Type Culture Collection (CCTCC) on December 12, 2022, with accession number CCTCCNO: M 20221925.

2. The application of Bacillus berberis FZ19-4 as described in claim 1 in the preparation of tobacco-specific amylase.

3. A tobacco-specific amylase prepared using Bacillus berberis FZ19-4 as described in claim 1.

4. The use of Bacillus berberis FZ19-4 as described in claim 1 and / or the amylase as described in claim 3 in the degradation of tobacco starch.

5. The application of Bacillus berberis FZ19-4 as described in claim 1 and / or amylase as described in claim 3 in improving tobacco quality.

6. The application according to claim 5, characterized in that: The fermentation supernatant filtrate of the Bacillus vesiculosus was sprayed onto the surface of tobacco products for fermentation.

7. The application according to claim 6, characterized in that: The fermentation supernatant is prepared by the following method: Bacillus belye is inoculated into the fermentation medium for fermentation, and cultured at 35-40℃ and 150-200 r / min for 55-65 h, centrifuged at 8000-12000 r / min for 8-12 min, the supernatant is collected, filtered, and the fermentation supernatant filtrate is obtained; preferably, it is cultured at 37℃ and 180 r / min for 60 h, and centrifuged at 10000 r / min for 10 min.

8. The application according to claim 6, characterized in that: Each 15-25 mL of fermentation supernatant filtrate corresponds to 45-55 g of tobacco products; preferably, each 20 mL of fermentation supernatant filtrate corresponds to 50 g of tobacco products.

9. The application according to claim 7, characterized in that: The fermentation conditions are a constant temperature of 42-45℃, a constant humidity of 70-75%, and a fermentation time of 45-50 hours; preferably, the fermentation conditions are a constant temperature of 45℃, a constant humidity of 75%, and a fermentation time of 48 hours.

10. The application according to any one of claims 5-9, characterized in that: The tobacco product is tobacco leaf or shredded tobacco.

Images