Tobacco source bacillus velezensis CK3-5 and amylase and application thereof

Abstract

The invention discloses tobacco source bacillus velezensis CK3-5 and application thereof, and belongs to the field of tobacco microorganisms. The strain belongs to bacillus, is classified and named as bacillus velezensis CK3-5, is preserved in the China Center for Type Culture Collection on June 19, 2024, and has the preservation number of CCTCC NO: M 20241306. The invention further discloses a method for preparing the bacillus velezensis strain. The enzyme activity of crude amylase liquid prepared from the strain is as high as 159.67 + / -2.35 U / mL, and a solid amylase preparation prepared from the strain can still keep more than 80% of relative enzyme activity after being preserved for 90 days at 4 DEG C, and has relatively strong stability; the crude amylase liquid is applied to tobacco products, the content of tobacco starch in the tobacco products can be effectively reduced, the content of reducing sugar is increased, meanwhile, the content of aroma substances is increased, and the smoking quality of cigarettes is remarkably improved.

Description

Technical Field

[0001] The present application relates to the technical field of tobacco microorganisms, and in particular to a tobacco-derived Bacillus Velez CK3-5 and its amylase and application. Background Art

[0002] Starch is an important carbohydrate in tobacco leaves and an important component of tobacco leaf chemical composition. Changes in its content can affect tobacco leaf quality. Currently, enzyme preparations and microbial methods can be used to reduce the starch content in tobacco leaves and improve tobacco leaf quality.

[0003] At present, tobacco enzyme preparations are mostly produced by screening strains from strain libraries to produce natural enzymes or purchasing commercial food enzymes. Compared with the international cutting-edge molecular modification and efficient expression technology, the activity and yield are low, the cost is relatively high, and there is still a huge room for improvement; and because there are huge differences in the existence form and composition structure of tobacco leaves and food matrix starch, the special matrix environment of tobacco leaves puts forward higher requirements for enzyme preparations, and the degradation activity and specificity of food enzymes on tobacco starch are not strong. For example, the degradation activity of commercial food-grade amylase on tobacco starch is only 40% of that on edible starch, which affects the efficient use of bio-enzyme treatment technology in the tobacco industry. The tobacco starch-degrading bacteria currently screened out have low amylase activity, poor storage stability of solid amylase preparations, and a long screening cycle for degradation bacteria. 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] To this end, the first aspect of the present invention provides a tobacco-derived Bacillus velezensis CK3-5, which belongs to Bacillus and is classified and named Bacillus velezensis CK3-5. It has been deposited in the China Center for Type Culture Collection on June 19, 2024, with a preservation number of CCTCC NO: M 20241306.

[0006] Further, the screening method of tobacco-derived Bacillus Velezii CK3-5 comprises:

[0007] Sampling and enrichment screening: Weigh the tobacco sample, cut it into pieces, add buffer, homogenize, heat it in a water bath at 80°C, and draw the heated supernatant into the liquid screening medium for directional enrichment culture;

[0008] Initial screening of strains: Heat the enriched culture solution in a water bath at 80°C, perform gradient dilution, inoculate on a solid screening medium for culture, add iodine solution, and measure the ratio of the transparent zone diameter of a single colony strain to the colony diameter as an indicator for initial screening;

[0009] Rescreening of strains: Rescreen the strains obtained in the initial screening, streak inoculate on LB solid culture medium for culture, pick single colonies and inoculate in LB liquid culture medium for culture, and after high-speed centrifugation and microfiltration, take the supernatants of different single colonies and add them to the punched solid screening culture medium respectively; and select a strain known to produce amylase as a control, use the same method to obtain the supernatant, and add the supernatant of the control strain to the punched solid screening culture medium of different initial screening strains; after incubation, add iodine solution, and obtain the strain with the strongest starch production ability according to the ratio of the transparent circle diameter of the initial screening strain to the transparent circle diameter of the control strain.

[0010] The second aspect of the present invention provides an amylase, which is obtained by using the tobacco-derived Bacillus Velez CK3-5 through strain culture and fermentation.

[0011] Furthermore, the enzymatic activity of the crude enzyme solution of the amylase is as high as 159.67±2.35 U / mL; and the solid preparation of the amylase maintains more than 80% of the relative enzyme activity after being stored at 4° C. for 90 days.

[0012] The third aspect of the present invention provides an application of an amylase in improving the quality of tobacco products. The amylase is used in tobacco product processing to reduce the content of tobacco starch in tobacco products, increase the content of reducing sugars in tobacco products, and increase the flavor components and precursors in tobacco products.

[0013] The fourth aspect of the present invention provides an application of tobacco-derived Bacillus Velez CK3-5 in improving the quality of tobacco products, wherein the Bacillus Velez CK3-5 is used to reduce the content of tobacco starch in tobacco products, increase the content of reducing sugars in tobacco products, and increase the flavor components and precursors in tobacco products.

[0014] Furthermore, the application method includes:

[0015] Using the Bacillus Velezii CK3-5 to prepare a fermentation supernatant filtrate;

[0016] The fermentation supernatant filtrate is evenly sprayed on the surface of the tobacco product for fermentation.

[0017] Furthermore, the fermentation supernatant filtrate is prepared by a method comprising:

[0018] The Bacillus Velez CK3-5 is inoculated into the fermentation medium for fermentation, cultured at 35°C-40°C and 150r / min-200r / min for 55h-65h, centrifuged at 8000-12000r / min for 8min-12min at 2°C-6°C, the supernatant is taken, and filtered with a sterile water filter membrane to obtain the fermentation supernatant filtrate.

[0019] Preferably, the Bacillus Velezii CK3-5 is inoculated into the fermentation medium for fermentation, cultured at 37°C and 180 r / min for 60 h, then centrifuged at 4°C and 10,000 r / min for 10 min, the supernatant is taken, and filtered with a 0.22 μm sterile water filter membrane to obtain the fermentation supernatant filtrate.

[0020] Furthermore, the fermentation supernatant filtrate is uniformly sprayed on the surface of the tobacco product for fermentation, and the method comprises:

[0021] Application of the fermentation supernatant filtrate and fermentation of tobacco products: spray the fermentation supernatant filtrate evenly on the tobacco leaves or tobacco shreds to be processed, and then ferment them at 42° C.-45° C. and 70%-75% humidity for 45h-50h.

[0022] Enzyme inactivation treatment: After fermentation, the tobacco leaves or tobacco shreds are heated at 100°C-120°C to inactivate the remaining fermentation supernatant.

[0023] Preferably, the method of uniformly spraying the fermentation supernatant filtrate on the surface of the tobacco product for fermentation comprises:

[0024] Applying and fermenting the fermentation supernatant filtrate: spraying the fermentation supernatant filtrate evenly on the tobacco leaves or tobacco shreds to be processed, and then fermenting them at 45° C. and 75% humidity for 48 hours;

[0025] Enzyme inactivation treatment: After fermentation, the tobacco products are heated at 100°C for 1 minute to inactivate the residual crude enzyme solution.

[0026] Furthermore, 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.

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

[0028] The tobacco-derived Bacillus Velez subtilis CK3-5 provided in the present application has a crude amylase enzyme liquid prepared by the strain, and the enzyme activity is as high as 159.67±2.35U / mL. The solid amylase preparation prepared by the strain can still maintain more than 80% of the relative enzyme activity after being stored at 4°C for 90 days, and has strong stability. The application of the crude amylase enzyme liquid to tobacco products can effectively reduce the content of tobacco starch in the tobacco products, increase the content of reducing sugars, and at the same time increase the content of aroma substances, thereby significantly improving the smoking quality of cigarettes. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1This is a colony morphology diagram of Bacillus velezensis CK3-5 strain in solid LB medium in Example 2 of the present application;

[0030] Figure 2 This is a single colony morphology diagram of Bacillus velezensis CK3-5 strain in solid LB medium in Example 2 of the present application;

[0031] Figure 3 This is a morphological picture of the Bacillus velezensis CK3-5 strain identified in Example 2 of the present application under a 100x oil microscope after Gram staining;

[0032] Figure 4 This is a phylogenetic tree constructed based on the 16S rDNA gene sequence when using molecular biology to identify the Bacillus velezensis CK3-5 strain in Example 2 of the present application;

[0033] Figure 5 Determination of the growth curve of Bacillus velezensis CK3-5 in Example 3 of the present application;

[0034] Figure 6 This is the determination of the enzyme activity curve of Bacillus velezensis CK3-5 in Example 4 of the present application.

[0035] Figure 7 This is a graph showing the results of a stability test of a solid amylase preparation prepared by Bacillus velezensis CK3-5 during storage in Example 5 of the present application.

[0036] Figure 8 This is a graph showing the change in starch content of tobacco products before and after being treated with amylase prepared by Bacillus velezensis CK3-5 in Example 6 of the present application.

[0037] Fig. 9 This is a graph showing the change in reducing sugar content of tobacco products before and after being treated with amylase prepared by Bacillus velezensis CK3-5 in Example 7 of the present application.

[0038] Fig.10 This is a radar chart of the sensory evaluation effect of tobacco shreds treated with amylase prepared by Bacillus velezensis CK3-5 in Example 8 of the present application. DETAILED DESCRIPTION

[0039] The present application is further described below in conjunction with specific implementation methods, but the protection scope of the present application is not limited thereto; unless otherwise specified, various reagents, instruments, etc. used in the examples are commercially available products.

[0040] Some of the biological materials, experimental reagents, experimental equipment, etc. involved in the following embodiments and test examples are briefly introduced as follows:

[0041] Culture medium:

[0042] Liquid screening medium: tobacco starch 1g / L, sodium chloride 5g / L, yeast powder 2g / L; if preparing solid screening medium, add 15g / L agar powder and sterilize at 121℃ for 30min.

[0043] LB medium: 5 g / L yeast powder, 10 g / L peptone, 10 g / L NaCl; if preparing solid culture medium, add 15 g / L agar powder, adjust the pH to 7.0, and sterilize at 121°C for 30 min.

[0044] Fermentation medium: peptone 80 g / L, yeast powder 25 g / L, dipotassium hydrogen phosphate 5 g / L, ammonium chloride 6 g / L, high pressure sterilization at 121°C for 30 min.

[0045] Experimental reagents:

[0046] Preparation of soluble starch solution (2g / L): Weigh 1.000g (accurate to 0.001g) soluble starch (on an absolute dry basis) into a beaker, add a small amount of water to make a slurry, slowly add it into 200mL of boiling water while stirring, then rinse the beaker containing starch with water several times, pour the washing liquid into it, stir and heat until completely transparent, cool and dilute to 500mL, and the solution is prepared and used immediately.

[0047] Original iodine solution: Weigh 11.0g iodine and 22.0g potassium iodide, use a small amount of water to completely dissolve the iodine, make up to 500mL, and store in a brown bottle.

[0048] Dilute iodine solution: Take 2.00 mL of the original iodine solution, add 20.0 g of potassium iodide, dissolve it in water and make up to 500 mL, and store it in a brown bottle.

[0049] Preparation of phosphate buffer (pH 6.0): weigh 45.23 g of disodium hydrogen phosphate (Na 2 HPO 4 12H 2 O) and 8.07 g citric acid (C 6 H 8 O 7 ·H 2O), dissolve in water and make up to 1000 mL. Use after calibration with a pH meter.

[0050] 1. A tobacco-derived amylase-producing Bacillus Velezii and its screening method

[0051] A tobacco-derived amylase-producing Bacillus velezensis is Bacillus velezensis CK3-5, with a preservation number of CCTCC NO: M 20241306; the preservation date is June 19, 2024; the preservation unit is the China Center for Type Culture Collection; and the preservation address is Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0052] The Bacillus velezensis of the present application is a tobacco starch-degrading bacterium that produces amylase and is isolated from a Yunyan 93 flue-cured tobacco sample in Zhushan County, Shiyan City, Hubei Province. It has been identified as Bacillus velezensis, which has a strong enzyme-producing ability. The enzyme activity of amylase at 70°C is 159.67±2.35U / mL, which is significantly higher than other strains screened during the same period. At the same time, the Bacillus velezensis in the present application has strong vitality, and the strain can survive normally at 80°C.

[0053] The amylase produced by the tobacco-derived amylase-producing Bacillus Velez CK3-5 of the present application has extremely strong degradation activity and specificity for tobacco starch. Adding CK3-5 crude enzyme solution can reduce the starch content in tobacco leaves by 0.66% in 48 hours, a decrease of 12.4%; the reducing sugar content can increase by about 13.53%. The reduction of starch content in tobacco leaves can effectively reduce the burnt smell of tobacco, and the increase of reducing sugar content can increase the composition and content of flavor substances in smoke, thereby improving the quality of tobacco products. It can solve the problem of poor specificity of existing biological enzyme treatment of tobacco leaves and meet the large-scale production and use of biological enzymes in the tobacco industry.

[0054] The tobacco products treated with amylase produced by tobacco-derived amylase-producing Bacillus Velezii CK3-5 showed a significant increase in aroma components and precursors after GC-MS testing.

[0055] Tobacco products treated with amylase produced by tobacco-derived Bacillus Velezii CK3-5 showed, after sensory evaluation, improved smoke aroma quality, aroma volume and softness, reduced irritation and strength, increased aftertaste comfort, and significantly improved the smoking quality of cigarettes.

[0056] The present application also provides a method for screening tobacco-derived amylase-producing Bacillus Velezii CK3-5 to solve the problems of long screening cycle and aimless screening of tobacco-derived starch-degrading bacteria in the prior art.

[0057] Example 1 Screening of strains capable of efficiently degrading tobacco starch

[0058] In this example, a strain that can efficiently degrade tobacco starch was screened from the Yunyan 93 flue-cured tobacco samples collected in Zhushan County, Shiyan City, Hubei Province in October 2023. The specific implementation process is as follows:

[0059] 1.1 Sampling and enrichment screening

[0060] Weigh 5 g of flue-cured tobacco sample, cut it into pieces, add 45 mL of sterilized phosphate buffer, homogenize for 10 min at gear 2 of a homogenizer, heat in a water bath at 80°C for 15 min, pipette 1 mL of the heated supernatant into 50 mL of liquid screening medium, and carry out directed enrichment culture at 37°C and 180 r / min for 48 h.

[0061] 1.2 Initial screening of strains

[0062] The bacterial solution was heated in a water bath at 80°C for 15 min and then diluted in a gradient manner (the initial dilution was 10 -1 ), absorb 10 -4 -10 -8 100 μL of bacterial suspensions of five dilutions were inoculated on solid screening medium and cultured in a constant temperature and humidity chamber at 37°C for 48 hours; iodine solution was added, and the single colony strain with a larger transparent circle was visually observed, and the transparent circle diameter and colony diameter were measured with a digital vernier caliper, and the ratio of the two (HC value) was calculated. Four strains with strong ability to degrade tobacco starch were obtained in the initial screening of strains, and the measurement results are shown in Table 1.

[0063] Table 1 Results of initial screening of bacterial species

[0064] Strain number Transparent ring diameter (mm) Colony diameter (mm) HC value CK3-5 14.20 4.29 3.31 CK14-1 13.10 4.75 2.76 CK14-2 15.26 5.98 2.55 CK15-2 14.26 5.42 2.63

[0065] As can be seen from the above table, the HC value of the initially screened strain CK3-5 is the largest, indicating that its ability to produce amylase is greater than that of other tobacco starch-degrading strains screened during the same period.

[0066] 1.3 Rescreening of strains

[0067] The four strains CK3-5, CK14-1, CK14-2, and CK15-2 obtained from the initial screening were streaked and inoculated on LB solid culture medium and cultured at 37°C for 12h. Single colonies were picked and inoculated into LB liquid culture medium and cultured at 37°C for 48h. After high-speed centrifugation at 10000r / min for 10min and filtration with a 0.22μm microfiltration membrane at 4°C, 500μL of the supernatant of different single colonies was added to the punched (6mm diameter) solid screening culture medium; and a control group was set up, and a strain of Bacillus amyloliquefaciens MK10163 known to produce amylase was selected, and the fermentation supernatant filtrate was obtained using the same preparation method as above, and holes of the same diameter were punched on the four plates of CK3-5, CK14-1, CK14-2, and CK15-2 that had been punched and added with the fermentation supernatant filtrate, and 500μL of the fermentation supernatant filtrate of the control strain was added. Incubate at 37℃ for 24h, add iodine solution, and measure the diameter of the transparent zone with a colony counter to preliminarily determine the amylase activity. The results of the strain rescreening measurement are shown in Table 2.

[0068] The above-mentioned Bacillus amyloliquefaciens MK10163 is classified and named Bacillus amyloliquefaciens MK10163, and has been deposited in the China Center for Type Culture Collection (CCTCC) with a deposit date of December 4, 2023 and a deposit number of CCTCC NO: M 20232425.

[0069] Table 2 Strain rescreening measurement results

[0070] serial number CK3-5 CK14-1 CK14-2 CK15-2 Diameter of transparent zone of screening strains d(mm) 14.50 13.28 14.85 14.06 Diameter of transparent circle of Bacillus amyloliquefaciens MK10163 (mm) 14.05 14.25 14.58 13.98 Ratio (d / D) 1.03 0.93 1.02 1.01

[0071] The ratios of the transparent zone diameters of CK3-5, CK14-2 and CK15-2 to those of MK10163 were all greater than 1, indicating that the three strains had stronger amylase production abilities than MK10163. Among them, the ratio of the transparent zone diameter of CK3-5 to that of MK10163 was the largest, indicating that CK3-5 had the strongest amylase production ability.

[0072] According to the above screening results, strain CK3-5 was selected as the best strain for the next experiment.

[0073] 1.4 Determination of amylase activity of strains

[0074] The determination method is a modified method based on the α-amylase activity determination method in the national standard GB1886.174-2016. The main modification is to prepare the standard solution to specify the standard curve and obtain the concentration-absorbance equation to obtain the amylase activity. The specific improved determination steps are as follows:

[0075] 1.4.1 Drawing of standard curve

[0076] The standard curve is drawn as follows:

[0077] Soluble starch standard solution: prepare according to Table 3.

[0078] Table 3 Soluble starch standard solution

[0079]

[0080] 1.00 mL of each of the above solutions was taken (parallel tests were required) and added to centrifuge tubes containing 0.5 mL of 0.1 mol / L hydrochloric acid solution and 5.00 mL of dilute iodine solution, respectively. The tubes were shaken thoroughly, and the absorbance (A) was rapidly determined at a wavelength of 660 nm using a spectrophotometer with 0.5 mL of 0.1 mol / L hydrochloric acid solution and 5.00 mL of dilute iodine solution as blanks. The absorbance (A) was used as the abscissa (x) and the concentration of the soluble starch standard solution was used as the ordinate (y) to plot a standard curve, and the standard equation y=1.2349x-0.0016 was obtained, R 2 =0.9995, and the slope K=1.2348 is obtained.

[0081] 1.4.2 Preparation of crude enzyme solution

[0082] Use 1μL disposable sterile inoculation loop to dip the bacterial liquid of strain CK3-5, streak inoculate on LB solid culture medium, and invert and culture in a biochemical incubator at 37℃ for about 12h. Pick the obtained single colony and inoculate it into 5mL liquid LB culture medium, 37℃, 180r / min, culture for 12h, which is the seed liquid. With an inoculation amount of 3% (v / v), draw 1.5mL of seed liquid and add it to 50mL fermentation medium, culture at 37℃180r / min for 60h, then centrifuge at 4℃, 10000r / min for 10min, take the supernatant, and filter it with a 0.22μm sterile water filter membrane to obtain a crude enzyme liquid, i.e. amylase.

[0083] 1.4.3 Determination

[0084] 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℃ constant temperature water bath for 8 min.

[0085] Take 1mL of crude enzyme solution and dilute it to a suitable concentration with a phosphate buffer solution of pH 6.0. Add 1.00mL of the diluted enzyme solution, start the time immediately, shake well, and keep the reaction at 70±0.2℃ for 10 minutes; immediately use an automatic pipette to draw 1.00mL of the reaction solution, add it to a centrifuge tube pre-filled with 0.5mL of 0.1mol / L hydrochloric acid solution and 5.00mL of dilute iodine solution, shake well, and use 0.5mL of hydrochloric acid solution and 5.00mL of dilute iodine solution as blanks, and quickly measure its absorbance (A) at a wavelength of 660nm using a spectrophotometer.

[0086] 1.4.4 Results Calculation of amylase activity

[0087] The enzyme activity of amylase produced by different strains was tested under high temperature conditions (70°C). The specific test method and test results are as follows:

[0088] Calculation method:

[0089] The enzymatic activity X of amylase, expressed in U / mL or U / g, is calculated as follows:

[0090] X=(2×20-26×AK)×n / 10

[0091] Where:

[0092] 2——initial concentration of soluble starch solution;

[0093] 20 – the volume of soluble starch solution during the measurement;

[0094] 26——The sum of the volume of the enzyme solution to be tested, the volume of the pH 6.0 phosphate buffer and the volume of the soluble starch solution during the test;

[0095] A——absorbance value of the enzyme solution measured;

[0096] K——the slope of the obtained standard curve;

[0097] n——Dilution multiple of enzyme solution;

[0098] 10——Reaction time of enzyme solution during measurement.

[0099] The test result shall be based on the arithmetic mean of the parallel determination results. The relative error of two independent determination results obtained under repeatability conditions shall not exceed 5%.

[0100] The results of the strain amylase activity determination are shown in Table 4. The strains FZ19-4 and FYZ1-3 are tobacco-derived amylase-producing strains screened by the applicant in the early stage, and the screening method is the same as above.

[0101] Table 4 Results of amylase activity determination of strains

[0102]

[0103]

[0104] In summary, compared with the existing amylase-producing strains FZ19-4 and FYZ1-3, the amylase activity of strain CK3-5 at 70°C is as high as 159.67±2.35 (U / mL), which is much higher than other starch-degrading strains screened. The purified CK3-5 strain was frozen at -80°C to maintain the original properties of the strain, prevent bacterial death, degeneration or contamination, and activate and rejuvenate regularly.

[0105] Example 2 Physiological, biochemical and molecular biological identification of tobacco starch-degrading bacteria

[0106] 2.1 Physiological and biochemical identification

[0107] Morphological observation: strain CK3-5 was inoculated onto LB solid culture medium plates and cultured at 37°C. The colony morphology was observed after 12 hours. Figure 1 , Figure 2 As shown, it can be seen that the colonies of strain CK3-5 are milky white and opaque, the surface of the colonies is flat and highly moist, and they are mucus-like with a certain fluidity, and there is obvious stringing phenomenon during inoculation.

[0108] Gram staining: Take the CK3-5 bacteria in the active growth stage and smear it on the slide. The thickness should not be too thick, and it should be dried and fixed. Add ammonium oxalate crystal violet stain solution to the smeared area, let it stand for 1-2 minutes, and then rinse it carefully with water until the outflowing water is basically colorless. After it is basically dry, add iodine-potassium iodide solution to the smeared area, let it stand for 1-2 minutes, and then rinse it carefully with water until the outflowing water is basically colorless. Add 95% ethanol solution (about 20-30 seconds) on the smear, and when the outflowing liquid is basically colorless, immediately wash off the ethanol with water; use absorbent paper to absorb the remaining water stains on the slide, and then use safranin reagent to re-stain for 2 minutes, and then rinse it carefully with water until the outflowing water is basically colorless, and use absorbent paper to absorb the remaining water stains on the slide. Observe under a 100× oil microscope. The results are as follows: Figure 3 As shown, the microscopic structure of strain CK3-5 is rod-shaped and it is a Gram-positive bacterium.

[0109] 2.2 Molecular Biological Identification

[0110] The Bacillus velezensis CK3-5 strain was inoculated into LB liquid culture medium and cultured at 37°C for 12 h. The bacteria 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°C for 5 min (1 cycle); denaturation at 94°C for 45 s, annealing at 55°C for 45 s, extension at 72°C for 1 min (30 cycles); total extension at 72°C for 10 min (1 cycle).

[0111] After the 16S rDNA fragments were purified and recovered, DNA sequencing was completed by Sangon Biotech (Shanghai) Co., Ltd. The 16S rDNA gene sequence of the high-yield amylase strain CK3-5 obtained by sequencing had 1437 bases. The sequencing results were compared with the nucleotide sequences uploaded on GenBank using Blast software. The homology analysis results showed that the 16S rDNA gene sequence of strain CK3-5 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 using the Kimura two-parameter model and the neighbor-joining method. The number of repeated samplings for testing the bootstrap support rate was 1000. The phylogenetic tree is shown in the figure. Figure 4 As shown, strain CK3-5 belongs to Bacillus velezensis. Combining morphological characteristics and 16S rDNA gene sequence analysis, the strain was identified as Bacillus velezensis and named Bacillus velezensis CK3-5. It was deposited in the China Center for Type Culture Collection (CCTCC) on June 19, 2024, with the deposit number CCTCC NO: M20241306.

[0112] Example 3 Growth analysis of strain CK3-5

[0113] Pick a single colony of CK3-5 and inoculate it in 5mL LB liquid medium, and culture it at 37℃ and 200r / min for 12h as seed liquid. Inoculate the seed liquid into 50mL fermentation medium at 3% (v / v) inoculation amount, and culture it continuously at 37℃ and 180r / min for 64h. Take samples in the biosafety cabinet every 8h, and use a spectrophotometer to measure the absorbance at 600nm. If the bacterial solution is too concentrated, dilute it appropriately with pure water, and control the absorbance value at 0.2-0.8. Use LB medium with corresponding dilution multiples as a control. Use the culture time t as the horizontal axis and the absorbance value OD600 as the vertical axis to make a growth curve.

[0114] The results are as follows Figure 5 As shown, strain CK3-5 was cultured at 37°C and basically entered the stable growth phase from the 48th hour, with OD600 reaching 2.67, at which time the total number of cells reached the maximum, which was suitable for subsequent applications.

[0115] Example 4 Analysis of amylase activity in liquid fermentation of strain CK3-5

[0116] A single colony of CK3-5 was selected and inoculated into 5 mL LB liquid medium, and cultured at 37°C and 200 r / min for 12 h as seed liquid. The seed liquid was added to 50 mL fermentation medium at an inoculum of 3% (v / v), and liquid fermentation was carried out at 37°C and 180 r / min. During the process, the pH value remained relatively stable at about 8.0. Samples were taken at regular intervals of 6 h from 0 to 66 h to determine and analyze the amylase activity in the fermentation liquid. The amylase activity determination method was described in Example 1.

[0117] The results are as follows Figure 6 As shown, the amylase activity reaches a peak value between 56h and 66h and then decreases. In Example 1, the amylase activity at 60h is 159.67±2.35 (U / mL), which is exactly in this range.

[0118] Example 5 Stability analysis of solid amylase preparation prepared by strain CK3-5

[0119] The crude amylase solution of the screening strain CK3-5 was prepared according to the steps of 1.4.2 of Example 1, and the following treatments were performed:

[0120] ① Take 100 mL of the crude enzyme solution after the initial centrifugation (8000 r / min for 20 min at 4°C) for clarification. The clarification method is as follows: add 1% calcium chloride and 1% disodium hydrogen phosphate to the crude enzyme solution, mix well, let stand at 4°C for 4 h, and keep the supernatant;

[0121] ② Take 100 mL of the supernatant, place it in an ice bath at 10-15°C, slowly add 200 mL (twice the volume of the supernatant) of pre-cooled 95% ethanol, stir while adding, let it stand at 4°C for 30 minutes, centrifuge at 4°C, 5000 r / min for 5 minutes, and collect the precipitate;

[0122] ③ The precipitate was redissolved with PBS solution and dispensed into glass dishes with a diameter of 15 cm, about 30 mL per dish, and placed in an ultra-low temperature refrigerator for overnight freezing. The next day, the sample was placed in a freeze dryer. After freeze drying for 36 hours, the sample was taken out and ground through an 80-mesh sieve, bottled, and stored at 4°C. The amylase activity of the samples was measured on the day of preparation, 15 days, 30 days, 45 days, 60 days, 75 days, and 90 days after preparation, and the relative enzyme activity was calculated.

[0123] The relative enzyme activity was calculated as follows:

[0124]

[0125] The results are as follows Figure 7 As shown, the solid amylase preparation still has a relative enzyme activity of (91.12±1.16)% after being stored for 90 days, indicating that it has strong stability and can be better preserved and used in tobacco industry applications.

[0126] 2. Application of tobacco-derived amylase-producing Bacillus Velezii CK3-5 in improving tobacco leaf quality

[0127] In this application, a tobacco-derived amylase-producing strain CK3-5 was screened out from flue-cured tobacco samples and identified as Bacillus velezensis. The crude enzyme solution (i.e., fermentation supernatant filtrate) prepared by centrifugal filtration of the fermentation culture solution was used to treat tobacco products. The changes in starch and reducing sugar content of tobacco products before and after treatment were detected by the tobacco industry standard method, and the changes in flavor components and precursors of tobacco products before and after treatment were detected by simultaneous distillation extraction (SDE)-gas chromatography-mass spectrometry. The specific implementation operations are as follows:

[0128] Example 6 Degradation of starch content in tobacco leaves using Bacillus velezensis CK3-5

[0129] The crude enzyme solution was prepared according to the method of Example 1.

[0130] Measure 15mL of crude enzyme solution (i.e., fermentation supernatant filtrate) and spray it evenly on the surface of 50g 2020 Chongqing B4F tobacco leaves, shake it thoroughly to mix it evenly, and then place it in a constant temperature and humidity incubator (temperature 45°C, humidity 75%) for treatment. Ferment for 48 hours and take samples, with 3 parallels in each group. Spray 2020 Chongqing B4F tobacco leaves with sterile water equal to the volume of crude enzyme solution as the negative control group, and spray 2020 Chongqing B4F tobacco leaves with Novozymes α-amylase (item number: BAN480L, diluted 500 times) equal to the volume of crude enzyme solution as the positive control group. After the enzyme treatment, the tobacco leaves were placed in a 100°C oven for 1min to kill the enzyme. Then place it in a 40°C oven to dry for 2h, grind it, and pass it through a 40-mesh sieve. The starch content was detected using the method in YC / T 216-2013 "Continuous Flow Method for Determination of Starch in Tobacco and Tobacco Products".

[0131] The results are as follows Figure 8As shown, after fermentation, the starch content in tobacco leaves of the negative control group (water treatment) was 5.32%, and the starch content in tobacco leaves of the experimental group (CK3-5 crude enzyme solution treatment) was 4.66%. Adding CK3-5 crude enzyme solution can reduce the starch content by 0.66% in 48 hours, a decrease of 12.4%, and this result is better than the positive control group (Novozymes commercial amylase treatment), indicating that the crude enzyme solution of CK3-5 strain plays a role in degrading the starch content in tobacco leaves. The reduction of starch content can effectively reduce the burnt smell of tobacco, thereby improving the quality of tobacco products.

[0132] Example 7: Improving the reducing sugar content in tobacco shreds using Bacillus velezensis CK3-5

[0133] The crude enzyme solution was prepared according to the method of Example 1.

[0134] Measure 15 mL of crude enzyme solution (i.e., fermentation supernatant filtrate) and spray it evenly on the surface of 50 g 2020 Chongqing B4F tobacco shreds. After fully mixing, place it in a constant temperature and humidity incubator (temperature 45°C, humidity 75%) for treatment. Ferment for 48 hours and take samples, with 3 parallels in each group. Spray 2020 Chongqing B4F tobacco shreds with sterile water equal to the volume of crude enzyme solution as the negative control group, and spray 2020 Chongqing B4F tobacco shreds with Novozymes α-amylase (item number: BAN480L, diluted 500 times) equal to the volume of crude enzyme solution as the positive control group. After the enzyme treatment, the tobacco shreds were placed in a 100°C oven for 1 min to inactivate the enzyme. Then place it in a 40°C oven to dry for 2 hours, grind it, and pass it through a 40-mesh sieve. The reducing sugar content in tobacco shreds was detected using YC / T 159-2019 "Continuous Flow Method for Determination of Water-soluble Sugars in Tobacco and Tobacco Products".

[0135] The results are as follows Fig. 9 As shown, after the fermentation, the reducing sugar content in the tobacco of the negative control group was 14.63%, and the reducing sugar content in the tobacco of the experimental group was 16.61%. The reducing sugar content increased by 1.98% in 48 hours, with an increase of 13.53%. This result was better than that of the positive control group (treated with Novozymes commercial amylase), indicating that under the action of amylase, starch was hydrolyzed into small molecule reducing sugars, resulting in an increase in its content, thus significantly improving the quality of tobacco products.

[0136] Example 8: Using Bacillus Velezii CK3-5 to increase the aroma components and precursors in tobacco leaves

[0137] The tobacco shreds of the experimental group (CK3-5 treatment) and the negative control group (sterile water treatment) in Example 6 were ground into tobacco dust, passed through a 40-mesh sieve, and subjected to the following treatment and analysis:

[0138] (1) At the same time, one end of the distillation extraction device is connected to a 1000mL flat-bottom flask containing 25.0g of tobacco powder, 30.0g of sodium chloride and 400mL of water, as well as 65mL of phosphate buffer (1:4), the pH is adjusted to 1.5, a small amount of zeolite is added, and it is heated in an electric furnace with controllable voltage. The other end is connected to a 100mL flask containing 60mL of dichloromethane and heated in a 60℃ water bath. The distillation extraction time is 2.5h. After the extraction, 1mL of the internal standard n-heptadecane (0.199mg / mL) is added to the collected 60mL dichloromethane solution, and GC-MS analysis is performed after drying and concentration.

[0139] (2) Gas chromatography-mass spectrometry analysis

[0140] The analysis was mainly carried out using GC-MS (7890C GC-MS chromatograph, Agilent, USA), which was manually analyzed and compared with the standard mass spectra and searched, and finally compared with the standard known compounds in the laboratory for chromatographic analysis and proofreading with the standard material library including retention time to determine the chemical structure of each separated component.

[0141] Chromatographic conditions: HP-5MS capillary column (30m×250μm, 0.25μm), initial temperature 100°C, maintained for 3min, increased to 250°C at 15°C / min, then increased to 300°C at 4°C / min, and maintained for 5min; vaporization chamber temperature 280°C; carrier gas is high-purity helium (99.999%); flow rate 1.0mL / min; injection volume is 1.0μL, split ratio 5:1.

[0142] Mass spectrometry conditions: ionization mode: electron bombardment; electron energy: 70 eV; transfer line temperature: 280°C; quadrupole temperature: 150°C; electron multiplier voltage: 1.34 kV; scanning range: 35-500 u; full scan mode was used.

[0143] The results are shown in Table 5. For aroma components and precursors, the total amount increased from 130.66 μg / g to 214.17 μg / g, with an increase rate of 63.9%. The contents of 80 aroma components and precursors increased significantly, such as phenylacetaldehyde increased by 473.1%, damascenone increased by 535.1%, 4,7,9-megatrien-3-one increased by 346.4%, (-)-rubotrylene increased by 253.3%, (1S,3S)-trans-4-carene increased by 241.8%, 3-(4,8,12-trimethyltridecyl)furan increased by 338.1%, and geranyl acetone increased by 268%. There are 59 components that were not detected in the original sample of Chongqing B4F 2020 but were found in high levels after treatment, including 4-vinylguaiacol, (1S,2E,4R,7E,11E)-2,7,11-cepatrien-4-ol, 5-methyl-2,4-diisopropylphenol, isophorone, 2,6,6-trimethyl-2-cyclohexyl-1,4-dione, pineneol, 2,3,6-trimethylnaphthalene, nerolidol, 3-(but-3-enyl)-cyclohexanone, etc.

[0144] Table 5 Analysis results of aroma components of Chongqing B4F tobacco original sample and sample 2020

[0145]

[0146]

[0147]

[0148] Example 9 Sensory Evaluation of Tobacco Strips Treated with Crude Enzyme Solution of Bacillus Velezii CK3-5

[0149] The crude enzyme solution was prepared according to the method of Example 1.

[0150] Measure 15 mL of crude enzyme solution (i.e., fermentation supernatant filtrate) and spray it evenly on the surface of 50 g of tobacco leaves. After fully mixing, place it in a constant temperature and humidity incubator (temperature 45°C, humidity 75%) for treatment. Ferment for 48 hours and take samples, with 3 parallels in each group. Spray 2020 Chongqing B4F tobacco leaves with sterile water equal to the volume of crude enzyme solution as the negative control group, and spray 2020 Chongqing B4F tobacco leaves with Novozymes α-amylase (item number: BAN480L, diluted 500 times) equal to the volume of crude enzyme solution as the positive control group. After the enzyme treatment, place the tobacco leaves in a 100°C oven for 1 minute to kill the enzyme. After taking it out, cut it into shredded tobacco with a shredder, make it into cigarettes by hand, balance it in a balance cabinet (temperature 22°C, humidity 60%) for 48 hours, and perform sensory evaluation according to the sensory technical requirements of cigarettes in the national standard GB5606.4-2005.

[0151] The results are as follows Fig.10As shown in the figure, after sensory evaluation, it is believed that the crude amylase solution prepared by CK3-5 can increase the content of aroma substances, improve the quality, amount and softness of smoke aroma, reduce irritation and strength, and improve the comfort of aftertaste after treating tobacco products. It has the same effect as commercial enzymes, is superior to commercial enzymes in reducing impurities, and significantly improves the smoking quality of cigarettes.

[0152] In summary, the present application isolates and screens tobacco starch-degrading bacteria CK3-5 from Yunyan 93 flue-cured tobacco samples in Zhushan County, Shiyan City, Hubei Province. The strain is identified as Bacillus velezensis through physiological, biochemical and molecular biological identification. The strain CK3-5 is fermented and centrifuged to make a crude enzyme solution, which is evenly sprayed on the surface of tobacco products. After constant temperature and humidity fermentation, it is found that the starch content in tobacco leaves is significantly reduced and the reducing sugar content in tobacco shreds is increased; after using simultaneous distillation extraction (SDE)-gas chromatography-mass spectrometry to detect the changes in flavor components and precursor substances before and after treatment of tobacco products, it is found that after treatment with CK3-5 crude enzyme solution, the contents of 80 flavor components and precursors are significantly increased, with an increase rate of 63.9%; in addition, after evaluating the fermented tobacco products, the aroma quality, aroma volume and softness of the cigarettes are improved, the irritation and strength are reduced, and the comfort of the aftertaste is improved, which significantly improves the smoking quality of the cigarettes.

[0153] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present application, rather than to limit it. Although the present application has been described in detail with reference to the aforementioned embodiments, a person of ordinary skill in the art should understand that the technical solutions described in the aforementioned embodiments can still be modified, or some or all of the technical features therein can be replaced by equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solution to deviate from the scope of the technical solution of the embodiments of the present application.

Claims

1. A tobacco-derived Bacillus Velezii CK3-5, characterized in that: The Bacillus velezensis is classified and named Bacillus velezensis CK3-5, and was deposited in the China Center for Type Culture Collection on June 19, 2024, with a deposit number of CCTCC NO: M 20241306.

2. The tobacco-derived Bacillus Velezii CK3-5 according to claim 1, characterized in that: Screening methods include: Sampling and enrichment screening: Weigh the tobacco sample, cut it into pieces, add buffer, homogenize, heat it in a water bath at 80°C, and draw the heated supernatant into the liquid screening medium for directional enrichment culture; Initial screening of strains: Heat the enriched culture solution in a water bath at 80°C, perform gradient dilution, inoculate on a solid screening medium for culture, add iodine solution, and measure the ratio of the transparent zone diameter of a single colony strain to the colony diameter as an indicator for initial screening; Rescreening of strains: Rescreen the strains obtained in the initial screening, streak inoculate on LB solid culture medium for culture, pick single colonies and inoculate in LB liquid culture medium for culture, and after high-speed centrifugation and microfiltration, take the supernatants of different single colonies and add them to the punched solid screening culture medium respectively; and select a strain known to produce amylase as a control, use the same method to obtain the supernatant, and add the supernatant of the control strain to the punched solid screening culture medium of different initial screening strains; after incubation, add iodine solution, and obtain the strain with the strongest starch production ability according to the ratio of the transparent circle diameter of the initial screening strain to the transparent circle diameter of the control strain.

3. An amylase, characterized in that The amylase is obtained by using the tobacco-derived Bacillus Velez CK3-5 of claim 1 through strain culture and fermentation.

4. The amylase according to claim 3, characterized in that The enzymatic activity of the crude enzyme solution of the amylase is as high as 159.67±2.35 U / mL; and the solid preparation of the amylase maintains a relative enzyme activity of more than 80% after being stored at 4° C. for 90 days.

5. An application of amylase in improving the quality of tobacco products, characterized in that: The amylase described in claim 3 or 4 is used in tobacco product processing to reduce the content of tobacco starch in tobacco products, increase the content of reducing sugars in tobacco products, and increase the flavor components and precursors in tobacco products.

6. An application of Bacillus Velezii CK3-5 in improving the quality of tobacco products, characterized in that: The Bacillus Velezii CK3-5 described in claim 1 is used to reduce the content of tobacco starch in tobacco products, increase the content of reducing sugars in tobacco products, and increase the flavor components and precursors in tobacco products.

7. The use according to claim 6, characterized in that: Application methods include: Using the Bacillus Velezii CK3-5 to prepare a fermentation supernatant filtrate; The fermentation supernatant filtrate is evenly sprayed on the surface of the tobacco product for fermentation.

8. The use according to claim 7, characterized in that: The fermentation supernatant filtrate is prepared by a method comprising: The Bacillus Velez CK3-5 is inoculated into the fermentation medium for fermentation, cultured at 35°C-40°C and 150r / min-200r / min for 55h-65h, centrifuged at 8000-12000r / min for 8min-12min at 2°C-6°C, the supernatant is taken, and filtered with a sterile water filter membrane to obtain the fermentation supernatant filtrate.

9. The use according to claim 7, characterized in that: The method of uniformly spraying the fermentation supernatant filtrate on the surface of the tobacco product for fermentation comprises: Application of the fermentation supernatant filtrate and fermentation of tobacco products: spraying the fermentation supernatant filtrate evenly on the tobacco leaves or tobacco shreds to be processed, and then fermenting them at 42°C-45°C and 70%-75% humidity for 45h-50h; Enzyme inactivation treatment: After fermentation, the tobacco leaves or tobacco shreds are heated at 100°C-120°C to inactivate the remaining fermentation supernatant.

10. The use according to claim 9, characterized in that: 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.

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