Tobacco source bacillus velezensis chq3-4 and amylase and application thereof

Abstract

The invention discloses tobacco source bacillus velezensis chq3-4 and amylase and application thereof, and belongs to the field of tobacco microorganisms. The strain of the bacillus velezensis chq3-4 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 (China Center for Type Culture Collection) NO: M 20241305. The enzyme activity of crude amylase liquid prepared from the strain reaches up to 119.28 + / -0.76 U / mL, and the amylase has relatively high stability and can still keep certain enzyme activity under high-temperature and alkaline conditions; the crude amylase liquid is applied to tobacco products, so that the content of tobacco starch in the tobacco products can be effectively reduced, the content of reducing sugar is increased, and the quality of the tobacco products is further improved; precious microbial resources are provided for the tobacco industry, and high-quality development of tobacco industry enterprises is promoted.

Description

Technical Field

[0001] The present application relates to the technical field of tobacco microorganisms, and in particular to a tobacco-derived Bacillus Velez subtilis chq3-4 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. Too much starch in tobacco leaves will affect the speed and completeness of smoking, and will produce burnt odor and a variety of harmful components when burning. The starch content in tobacco leaves can be reduced and the quality of tobacco leaves can be improved through enzyme preparation and microbial treatment, so as to improve the smoking quality of cigarettes.

[0003] At present, most of the cigarette processing enterprises using enzyme preparations improve the quality of tobacco leaves by purchasing commercial food enzymes. However, due to the 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. 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. In addition, due to the special processing environment of tobacco leaves and the wide pH range of tobacco leaves (the pH of tobacco leaves is between 5.0 and 8.5 due to different parts and growth periods of tobacco leaves), the stability requirements of enzyme preparations are also higher. The tobacco starch degrading bacteria currently screened out have low amylase activity and low temperature tolerance, which are not suitable for high-temperature fermentation of tobacco products, and the screening cycle of degradation bacteria is long. 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 chq3-4, which belongs to Bacillus and is classified and named Bacillus velezensis chq3-4. The Bacillus velezensis chq3-4 was deposited in the China Center for Type Culture Collection on June 19, 2024, with a deposit number of CCTCC NO: M 20241305.

[0006] Furthermore, the screening method of tobacco-derived Bacillus Velezii chq3-4 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, after high-speed centrifugation and microfiltration, take the supernatant of different single colonies and add them to the punched solid screening culture medium for incubation, add iodine solution, observe the diameter of the transparent circle, and obtain the strain with the strongest starch production ability.

[0010] The second aspect of the present invention provides an amylase, which is obtained by using the tobacco-derived Bacillus Velezii chq3-4 through strain culture and fermentation.

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

[0012] The crude enzyme solution of the amylase has a relative enzyme activity of more than 60% at 40°C to 90°C, more than 80% at 40°C to 85°C, and 100% at 75°C under the condition of a pH value of 7.0;

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

[0014] 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, and the applicable temperature range is 40°C to 90°C, and the applicable pH range is 6.0 to 9.0; the improving the quality of tobacco products includes reducing the content of tobacco starch in the tobacco products, and increasing the content of reducing sugars in the tobacco products.

[0015] The fourth aspect of the present invention provides an application of tobacco-derived Bacillus Velezii chq3-4 in improving the quality of tobacco products. The Bacillus Velezii chq3-4 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 sugars in tobacco products.

[0016] Furthermore, the application method includes:

[0017] Using the Bacillus Velezii chq3-4 to prepare a fermentation supernatant filtrate;

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

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

[0020] The Bacillus Velezii chq3-4 is inoculated into the fermentation medium for fermentation, and cultured at 35°C to 40°C and 150r / min to 200r / min for 55h to 65h, and then centrifuged at 2°C to 6°C and 8000r / min to 12000r / min for 8min to 12min, and the supernatant is taken and filtered with a sterile water filter membrane to obtain the fermentation supernatant filtrate.

[0021] Preferably, the Bacillus Velezii chq3-4 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.

[0022] Furthermore, the fermentation supernatant filtrate is uniformly sprayed on the surface of the tobacco product for fermentation, and the method comprises:

[0023] 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 at 42°C to 45°C and 70% to 75% humidity for 45h to 50h;

[0024] Enzyme inactivation treatment: After the fermentation is completed, the tobacco leaves or tobacco shreds are heated at 100° C. to 120° C. to inactivate the remaining fermentation supernatant filtrate.

[0025] Preferably, the method of uniformly spraying the fermentation supernatant filtrate on the surface of the tobacco product for fermentation comprises:

[0026] Application and fermentation of the fermentation supernatant filtrate: spray the fermentation supernatant filtrate evenly on the tobacco leaves or tobacco shreds to be processed, and then ferment 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 10-20 mL of the fermentation supernatant filtrate, 45-55 g of tobacco leaves or tobacco shreds are sprayed. Preferably, for every 15 mL of the fermentation supernatant filtrate, 50 g of tobacco leaves or tobacco shreds are sprayed.

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

[0030] The tobacco-derived Bacillus Velez subtilis chq3-4 provided in the present application has a crude amylase enzyme solution prepared by the strain, and the enzyme activity is as high as 119.28±0.76U / mL, and the enzyme has strong stability and can still maintain a certain enzyme activity under high temperature and alkaline conditions; applying it to tobacco products can effectively reduce the content of tobacco starch in tobacco products, increase the content of reducing sugars, and thus improve the quality of tobacco products; provide valuable microbial resources for the tobacco industry, and promote the high-quality development of tobacco industry enterprises. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a colony morphology diagram of the Bacillus velezensis chq3-4 strain in solid LB medium in Example 2 of the present application;

[0032] Figure 2 This is a single colony morphology diagram of the Bacillus velezensis chq3-4 strain in solid LB medium in Example 2 of the present application;

[0033] Figure 3 This is a morphological picture of the Bacillus velezensis chq3-4 strain identified in Example 2 of the present application under a 100x oil microscope after Gram staining;

[0034] Figure 4 This is a phylogenetic tree constructed based on the 16S rDNA gene sequence when using molecular biology to identify the Bacillus velezensis chq3-4 strain in Example 2 of the present application;

[0035] Figure 5 This is a graph showing the temperature tolerance of the amylase produced by Bacillus velezensis chq3-4 in Example 3 of the present application;

[0036] Figure 6 This is a graph showing the acid-base tolerance results of the amylase produced by Bacillus velezensis chq3-4 in Example 4 of the present application;

[0037] Figure 7 This is a graph showing the change in starch content of tobacco products before and after being treated with amylase prepared by Bacillus velezensis chq3-4 in Example 5 of the present application;

[0038] Figure 8This is a graph showing the change in reducing sugar content of tobacco products before and after being treated with amylase prepared by Bacillus velezensis chq3-4 in Example 6 of the present application;

[0039] Fig. 9 This is a radar chart of the sensory evaluation effect of tobacco shreds treated with amylase prepared by Bacillus velezensis chq3-4 in Example 7 of the present application. DETAILED DESCRIPTION

[0040] 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.

[0041] Some of the biological materials, experimental reagents, experimental equipment, etc. involved in the following embodiments and test examples are briefly introduced as follows:

[0042] Culture medium:

[0043] 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.

[0044] 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.

[0045] 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.

[0046] Experimental reagents:

[0047] Soluble starch solution (20g / L): Weigh 2.177g (accurate to 0.001g) soluble starch (on an absolute dry basis) in a beaker, add a small amount of water to make a slurry, slowly add 70mL 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 100mL. The solution should be prepared and used immediately.

[0048] Note: Soluble starch should be analyzed using enzyme preparations special starch.

[0049] 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.

[0050] 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.

[0051] 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 make up to 1000 mL. Use after calibration of pH meter.

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

[0053] 1. A tobacco-derived Bacillus Velezii chq3-4 and its screening method

[0054] A tobacco-derived Bacillus velezensis chq3-4 belongs to the genus Bacillus, is classified and named Bacillus velezensis chq3-4, Bacillus velezensis (Bacillus velezensis) chq3-4, with a preservation number of CCTCCNO: M 20241305; the preservation date is June 19, 2024; the preservation unit is the China Center for Type Culture Collection; the preservation address is Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0055] The Bacillus velezensis in this application is a tobacco starch-degrading bacterium that produces amylase and is isolated from a flue-cured tobacco sample of Yunyan 87 in Xunyang City, Ankang City, Shaanxi Province. It is identified as Bacillus velezensis, which has a strong enzyme production ability and an enzyme activity of 119.28±0.76U / mL. It has a wide range of temperature adaptability and tolerance to alkaline conditions (it still has strong enzyme activity at temperatures of 40 to 90°C and has strong enzyme activity at pH 6.0-9.0), which can solve the problem of poor adaptability of enzyme preparations in special tobacco processing environments. At the same time, the Bacillus velezensis in this application has strong vitality, and the strain can survive normally at 80°C.

[0056] The amylase produced by the tobacco-derived amylase-producing Bacillus Velez subsp. chq3-4 of the present application has extremely strong degradation activity and specificity for tobacco starch. Adding chq3-4 crude enzyme solution can reduce the starch content in tobacco leaves by 4.50% in 48 hours, a decrease of 8.46%; the reducing sugar content can increase by about 6.5%. 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.

[0057] After sensory evaluation, tobacco products treated with amylase produced by the tobacco-derived amylase-producing Bacillus Velezii chq3-4 of the present application have improved smoke aroma quality, aroma volume and softness, reduced irritation and strength, improved aftertaste comfort, and significantly improved the smoking quality of cigarettes.

[0058] The screening method of tobacco-derived amylase-producing Bacillus Velezii chq3-4 of the present application solves the problems of long screening cycle and aimless screening of tobacco-derived starch-degrading bacteria in the prior art.

[0059] Example 1 Screening of strains capable of efficiently degrading tobacco starch

[0060] In this example, a strain that can efficiently degrade tobacco starch was screened out from the 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 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.

[0063] 1.2 Initial screening of strains

[0064] 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.

[0065] Table 1 Results of initial screening of bacterial species

[0066] Strain number Transparent ring diameter (mm) Colony diameter (mm) HC value chq3-4 16.92 5.19 3.26 chq4-2 14.50 5.62 2.58 chq5-1 14.27 4.82 2.96 chq5-2 15.74 5.23 3.01

[0067] As can be seen from the above table, the HC value of the initially screened strain chq3-4 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.

[0068] 1.3 Rescreening of strains

[0069] The four strains chq3-4, chq4-2, chq5-1, and chq5-2 obtained from the initial screening were streaked and inoculated on LB solid medium and cultured at 37°C for 12 hours. Single colonies were picked and inoculated into LB liquid medium and cultured at 37°C for 48 hours. After high-speed centrifugation at 10000r / min for 10 minutes at 4°C and filtration with a 0.22μm microfiltration membrane, 500μL of the supernatant of different single colonies was added to the punched (6mm diameter) solid screening medium, incubated at 37°C for 24 hours, iodine solution was added, and the diameter of the transparent circle was measured with a colony counter to preliminarily determine the strain's ability to produce amylase. The results of the strain re-screening measurement are shown in Table 2. The diameter of the transparent circle of chq3-4 is the largest, indicating that chq3-4 has the strongest ability to produce amylase.

[0070] Table 2 Strain rescreening measurement results

[0071] serial number chq3-4 chq4-2 chq5-1 chq5-2 Diameter of transparent zone of screening strains d(mm) 16.20 15.23 15.82 15.49

[0072] According to the above screening results, the strain chq3-4 was selected as the best strain for the next experiment.

[0073] 1.4 Determination of amylase activity of strains

[0074] 1.4.1 Preparation of crude enzyme solution

[0075] Use 1μL disposable sterile inoculation loop to dip the bacterial liquid of strain chq3-4, streak inoculate on LB solid medium, and incubate inverted in a biochemical incubator at 37℃ for about 12h. Pick the obtained single colony and inoculate it into 5mL liquid LB medium, 37℃, 180r / min, and culture for 12h, which is the seed liquid. With an inoculum 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 to obtain the crude enzyme liquid.

[0076] 1.4.2 Determination of amylase activity

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

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

[0079] The results of the determination of amylase activity in the crude enzyme solution of the strains 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.

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

[0081] Strain number Amylase activity (U / mL) chq3-4 119.28±0.76 FZ19-4 77.83±0.34 FYZ1-3 71.63±0.05

[0082] In summary, compared with the existing amylase-producing strains FZ19-4 and FYZ1-3, the amylase activity of strain chq3-4 is as high as 119.28±0.76 (U / mL), and the amylase activity produced is higher than other screened starch-degrading strains. The purified chq3-4 strain was frozen at -80℃ to maintain the original properties of the strain, to prevent bacterial death, degeneration or contamination, and to activate and rejuvenate regularly.

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

[0084] 2.1 Physiological and biochemical identification

[0085] Morphological observation: strain chq3-4 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 chq3-4 are milky white and opaque, the surface of the colonies is flat and highly moist, and they are mucus-like and have a certain fluidity. There is obvious stringing phenomenon when inoculated.

[0086] Gram staining: Take the chq3-4 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 away 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 chq3-4 is rod-shaped and is a Gram-positive bacterium.

[0087] 2.2 Molecular Biological Identification

[0088] The Bacillus velezensis chq3-4 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).

[0089] 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 chq3-4 obtained by sequencing had 1491 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 chq3-4 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 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 chq3-4 belongs to Bacillus velezensis. Combining morphological characteristics and 16S rDNA gene sequence analysis, the strain was identified as Bacillus velezensis and named Bacillus velezensis chq3-4. It was deposited in the China Center for Type Culture Collection (CCTCC) on June 19, 2024, with the deposit number CCTCC NO: M 20241305.

[0090] Example 3 Analysis of temperature stability of amylase produced by strain chq3-4

[0091] An appropriate amount of crude enzyme solution was prepared according to the method in Example 1, the pH value of the reaction system was adjusted to 7.0, the temperature was adjusted to 40°C ~ 90°C, the temperature of the reaction system was set at 5°C intervals, the enzyme activity of the crude enzyme solution at the corresponding temperature (maintained for 30 min) was measured, and the relationship between the enzyme activities at different temperatures was expressed by relative enzyme activity (the ratio of the amylase activity measured at each temperature to the maximum enzyme activity measured). The measurement results are shown in Table 4.

[0092] Table 4 Amylase activity test results at different temperatures

[0093]

[0094]

[0095] According to Table 4 and Figure 5 It can be seen that under the experimental conditions, the reaction temperature is in the range of 40-90°C, and the crude enzyme solution of the test strain has a certain starch degradation effect, and the relative enzyme activity is above 60%. The enzyme activity is the highest at 75°C, which is 125.33U / mL (the relative enzyme activity at this temperature is 100%), indicating that the optimal reaction temperature of the amylase in the crude enzyme solution of the strain chq3-4 is 75°C. When the temperature is <75°C, the enzyme activity increases with the increase of temperature; when the temperature is >75°C, the enzyme activity gradually decreases, but the decrease is not large. The relative enzyme activity at 85°C can still be maintained above 83.77%, which shows that the enzyme activity is relatively stable in the temperature range of 40-85°C; the relative enzyme activity can still reach 69.98% at a temperature of 90°C, indicating that the enzyme has good heat resistance after combining with the substrate, and the enzyme has a wide range of temperature adaptability and high temperature resistance, reflecting the structural stability of the enzyme. The use of amylase prepared by strain chq3-4 in the special processing environment of tobacco can solve the problem of temperature inadaptability of commercial enzymes.

[0096] Example 4 pH stability analysis of amylase produced by strain chq3-4

[0097] An appropriate amount of crude enzyme solution was prepared according to the method in Example 1, the temperature of the reaction system was adjusted to 75°C, the pH was adjusted to 5-9, the pH of the reaction system was set every 0.5, the enzyme activity of the crude enzyme solution at the corresponding pH (maintained for 30 min) was measured, and the relationship between the enzyme activities at different pH was expressed by relative enzyme activity (the ratio of the amylase activity measured at each pH to the maximum enzyme activity measured). The measurement results are shown in Table 5.

[0098] Table 5 Amylase activity test results at different pH

[0099] Different pH Amylase activity (U / mL) Relative enzyme activity (%) 5.0 21.03 16.14 5.5 29.65 22.76 6.0 89.65 68.82 6.5 97.89 75.15 7.0 108.63 83.39 7.5 124.36 95.47 8.0 130.26 100.00 8.5 105.63 81.09 9.0 85.65 65.75

[0100] According to Table 5 and Figure 6 It can be seen that under the experimental conditions, the crude enzyme solution of the test strain has a certain starch degradation effect in the pH range of 6.0-9.0. The enzyme activity is the highest at pH 8.0, which is 130.26U / mL (the relative enzyme activity at this pH value is 100%), indicating that the optimal reaction pH of the amylase in the crude enzyme solution of the strain chq3-4 is 8.0. When pH <8.0, the enzyme activity increases with the increase of pH; when pH>8.0, the enzyme activity gradually decreases, but the decrease is not large. The enzyme activity is relatively stable in the pH range of 6.0-9.0, indicating that the enzyme has a wide pH adaptability and good weak alkalinity resistance, reflecting the structural stability of the enzyme. The use of amylase prepared by strain chq3-4 in the special processing environment of tobacco can solve the pH inadaptability problem of commercial enzymes.

[0101] 2. Application of tobacco-derived amylase-producing Bacillus velez chq3-4 in improving tobacco leaf quality

[0102] In the present application, a tobacco-derived amylase-producing strain chq3-4 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 before and after the tobacco product treatment were detected by the tobacco industry standard method, and the improvement of the sensory quality of tobacco leaves by the chq3-4 crude enzyme solution was evaluated by sensory evaluation. The specific implementation operations are as follows:

[0103] Example 5 Degradation of starch content in tobacco leaves using Bacillus velezensis chq3-4

[0104] The crude enzyme solution was prepared according to the method of Example 1.

[0105] 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".

[0106] The results are as follows Figure 7 As 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 (CHQ3-4 crude enzyme solution treatment) was 4.87%. Adding CHQ3-4 crude enzyme solution can reduce the starch content by 4.50% in 48 hours, and the reduction rate reaches 8.46%. This result is better than the positive control group (Novozymes commercial amylase treatment), indicating that the crude enzyme solution of CHQ3-4 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.

[0107] Example 6: Improving the reducing sugar content in tobacco shreds using Bacillus velezensis chq3-4

[0108] The crude enzyme solution was prepared according to the method of Example 1.

[0109] 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".

[0110] The results are as follows Figure 8 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 15.58%. The reducing sugar content increased by 0.95% in 48 hours, with an increase of 6.50%. 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.

[0111] Example 7 Sensory Evaluation of Tobacco Strips Treated with Crude Enzyme Solution of Bacillus velezine chq3-4

[0112] The crude enzyme solution was prepared according to the method of Example 1.

[0113] 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.

[0114] The results are as follows Fig. 9As shown, after sensory evaluation, it is believed that the crude amylase solution prepared by chq3-4 can increase the content of aroma substances, improve the aroma quality, aroma volume and softness of smoke, reduce irritation and strength, and improve the comfort of aftertaste after treating tobacco products. It has the same effect as commercial enzymes, and is superior to commercial enzymes in reducing irritation and improving aroma quality, and significantly improves the smoking quality of cigarettes.

[0115] In summary, the present application isolates and screens tobacco starch-degrading bacteria chq3-4 from Yunyan 87 flue-cured tobacco samples in Xunyang City, Ankang City, Shaanxi Province. The strain is identified as Bacillus velezensis through physiological, biochemical and molecular biological identification. The strain chq3-4 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; in addition, after the fermented tobacco products are evaluated for smoking, 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.

[0116] It is easy for those skilled in the art to understand that, under the premise of no conflict, the above-mentioned advantageous methods can be freely combined and superimposed. The above are only preferred embodiments of the present application and are not intended to limit the present application. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application should be included in the protection scope of the present application. The above are only preferred implementation methods of the present application. It should be pointed out that for ordinary technicians in this technical field, several improvements and variations can be made without departing from the technical principles of the present application, and these improvements and variations should also be regarded as the protection scope of the present application.

Claims

1. A tobacco-derived Bacillus Velezii chq3-4, characterized in that: The Bacillus velezensis chq3-4 was deposited in the China Center for Type Culture Collection on June 19, 2024, with a deposit number of CCTCC NO: M 20241305.

2. The tobacco-derived Bacillus Velezii chq3-4 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, after high-speed centrifugation and microfiltration, take the supernatant of different single colonies and add them to the punched solid screening culture medium for incubation, add iodine solution, observe the diameter of the transparent circle, and obtain the strain with the strongest starch production ability.

3. An amylase, characterized in that The amylase is obtained by using the tobacco-derived Bacillus Velezii chq3-4 described in claim 1 through strain culture and fermentation.

4. The amylase according to claim 3, characterized in that The crude enzyme activity of the amylase is as high as 119.28±0.76U / mL; The crude enzyme solution of the amylase has a relative enzyme activity of more than 60% at 40°C to 90°C, more than 80% at 40°C to 85°C, and 100% at 75°C under the condition of a pH value of 7.0; The crude enzyme solution of the amylase has a relative enzyme activity higher than 60% at a temperature of 75°C and a pH range of 6.0-9.0, a relative enzyme activity higher than 80% at a pH range of 7.0-8.5, and a relative enzyme activity of 100% at a pH of 8.

0.

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 for tobacco product processing, with an applicable temperature range of 40°C to 90°C and an applicable pH range of 6.0 to 9.0; the improving the quality of tobacco products includes reducing the content of tobacco starch in the tobacco products and increasing the content of reducing sugars in the tobacco products.

6. Application of tobacco-derived Bacillus Velezii chq3-4 in improving the quality of tobacco products, characterized in that: The Bacillus velezensis chq3-4 described in 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 sugars in tobacco products.

7. The use according to claim 6, characterized in that: Application methods include: Using the Bacillus Velezii chq3-4 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 Velezii chq3-4 is inoculated into the fermentation medium for fermentation, and cultured at 35°C to 40°C and 150r / min to 200r / min for 55h to 65h, and then centrifuged at 2°C to 6°C and 8000r / min to 12000r / min for 8min to 12min, and 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: 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 at 42°C to 45°C and 70% to 75% humidity for 45h to 50h; Enzyme inactivation treatment: After the fermentation is completed, the tobacco leaves or tobacco shreds are heated at 100° C. to 120° C. to inactivate the remaining fermentation supernatant filtrate.

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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