Bacillus velezensis for producing alpha-amylase from tobacco source and application of bacillus velezensis

Abstract

The invention relates to bacillus velezensis for producing alpha-amylase from a tobacco source and application of the bacillus velezensis, and belongs to the technical field of microorganisms. The bacillus velezensis for producing the alpha-amylase from the tobacco source is A8-1, and the preservation number of the bacillus velezensis is CCTCC (China Center For Type Culture Collection) NO: M 20232632. The activity of the alpha-amylase produced by the bacillus velezensis A8-1 can reach up to 147.85 + / -0.05 U / mL at the high temperature of 70 DEG C through determination, and the content of tobacco starch in tobacco products can be effectively reduced when the alpha-amylase is applied to the tobacco products, so that the quality of the tobacco products is improved. On one hand, the quality, applicability and utilization rate of low-grade and low-grade tobacco leaves can be improved; on the other hand, the quality grade of tobacco leaves can be improved, and defects of high-quality tobacco leaves are overcome. Meanwhile, valuable microbial resources are provided for the tobacco industry, and a new technology is provided for promoting high-quality development of tobacco industry enterprises.

Description

Technical Field

[0001] The invention relates to tobacco-derived Bacillus Velezii producing alpha-amylase and application thereof, belonging to the technical field of microorganisms. Background Art

[0002] Tobacco belongs to the genus Nicotiana of the Solanaceae family and is an annual herb. It is mostly distributed in temperate and tropical regions. In my country, it is mainly produced in Shandong, Henan, Anhui, Fujian, Hunan, Yunnan, Guizhou, etc. The chemical composition of tobacco varies with genetic characteristics, environmental conditions, cultivation measures and processing. The types of chemical components contained in different types of tobacco such as flue-cured tobacco, burley tobacco, Maryland tobacco, and aromatic tobacco are mostly the same, and the difference is mainly in the difference in the content of chemical components.

[0003] Starch, as an important carbohydrate in tobacco leaves, is an important component of tobacco leaf chemical composition, and changes in its content can affect the quality of tobacco leaves. The large accumulation of starch in tobacco leaves will cause the leaves to become thicker and brittle, which will not only affect the speed and completeness of smoking, but also produce a burnt smell and a variety of harmful substances when the starch burns, which will have an adverse effect on the color, aroma and taste of tobacco products. The survey found that the starch content in my country's high-quality tobacco leaves (4%-6%) is significantly higher than that of foreign high-quality tobacco leaves (1%-2%), which seriously restricts the improvement of domestic tobacco leaf quality. Therefore, how to degrade the starch in tobacco leaves and decompose it into small molecules such as reducing sugars that can improve the quality of tobacco leaves has become an important research hotspot.

[0004] At present, the starch content in tobacco leaves can be reduced to improve tobacco leaf quality through tobacco variety selection, cultivation methods, fertilization levels, modulation and aging processes, enzyme preparation methods and microbial methods. Compared with other measures, microorganisms have the advantages of a wide variety, easy access, rapid metabolism, easy cultivation and regeneration, low cost, and simple operation; and bioenzyme preparations, as a type of green, safe and efficient biocatalyst, have shown great application potential in improving tobacco leaf quality.

[0005] The bio-enzyme method degrades starch into small-molecule oligosaccharides and glucose through the action of enzymes, which not only reduces the starch content in tobacco leaves, but also increases the content of water-soluble total glycosyl reducing sugars, thereby improving the intrinsic quality of tobacco leaves. The addition of enzyme preparations can also improve the sensory quality of tobacco leaves. Studies have also shown that the addition of starch enzymes during the baking process can effectively degrade the starch in flue-cured tobacco. With the increase in the amount of added starch, the starch content in the flue-cured tobacco leaves decreases, while the content of water-soluble sugars and reducing sugars increases, further proving the feasibility of reducing the starch content in tobacco by enzymatic methods.

[0006] Feng Yingjie et al. discovered a strain of Bacillus thuringiensis with high amylase production. The bacterium can produce amylase during growth and metabolism, degrading the starch in tobacco leaves into reducing sugars. The cleavage of reducing sugars is beneficial to balancing the acidity and alkalinity of smoke and coordinating aroma. Xu Haidong et al. screened out a strain of Bacillus Velezii with high enzyme production, with the highest enzyme activity of 16.21U / mL. After fermentation, it was sprayed on tobacco leaves, and its starch degradation rate was 11.49%. However, tobacco products require high-temperature fermentation during the preparation process. The tobacco starch-degrading bacteria currently screened out not only have the problem of low amylase activity, but also have the problem of low temperature tolerance, which limits the application scope of the bioenzyme preparation method in improving tobacco quality. Summary of the invention

[0007] The first object of the present invention is to provide a tobacco-derived Bacillus Velezii that produces α-amylase, so as to solve the problem that tobacco-derived microorganisms in the prior art have low α-amylase activity and are not suitable for high-temperature fermentation of tobacco products.

[0008] The second object of the present invention is to provide an application of Bacillus Velezii producing α-amylase from tobacco in improving the quality of tobacco products, so as to solve the problem in the prior art that the quality of tobacco products is affected by the increased starch content in tobacco products.

[0009] In order to achieve the above object, the technical scheme of a tobacco-derived α-amylase-producing Bacillus Velezii in the present invention is:

[0010] The invention discloses a tobacco-derived Bacillus velezensis producing alpha-amylase. The Bacillus velezensis is A8-1 and has a deposit number of CCTCC NO: M 20232632.

[0011] The beneficial effect of the above technical scheme is that the present invention is a pioneering invention of a tobacco-derived α-amylase-producing Bacillus velezensis. The present invention uses α-amylase activity as an evaluation index, first uses a screening medium for directional enrichment culture, and then uses a screening medium for primary screening and rescreening twice, and directional screening out a strain of tobacco-derived α-amylase with high activity. After physiological, biochemical and molecular biological identification, the bacterium is Bacillus velezensis (Bacillus velezensis), named A8-1, and the α-amylase produced by the bacterium is measured at a high temperature of 70°C, and the activity can be as high as 147.85±0.05U / mL.

[0012] Furthermore, the present invention analyzes the growth characteristics of strain A8-1 and finds that strain A8-1 has a strong tolerance to the pH in the environment, and a higher concentration of salt ions and a higher concentration of nicotine have little effect on its growth, which provides a good basis for its subsequent application in tobacco products.

[0013] In order to achieve the above object, the technical solution of the application of tobacco-derived α-amylase-producing Bacillus Velezii in improving the quality of tobacco products in the present invention is:

[0014] Application of tobacco-derived Bacillus Velezii producing α-amylase in improving the quality of tobacco products.

[0015] The beneficial effect of the above technical solution is that: the present invention uses α-amylase activity as an evaluation index, screens strains through a specific tobacco starch culture medium, and applies the amylase produced by the strain to tobacco products, which effectively reduces the content of tobacco starch. The measurement results show that the α-amylase produced by the Bacillus velezensis A8-1 strain obtained by screening the tobacco starch culture medium has an activity of up to 147.85±0.05U / mL at a high temperature of 70°C. When applied to tobacco products, the starch content decreased by 0.64%, a decrease of 12.5%; the reducing sugar content increased by 0.9%, an increase of 6.4%, and the smoke quality was significantly improved, which improved the quality of tobacco products and showed a good application effect.

[0016] As a further improvement, the quality of the tobacco product is improved by reducing the starch content and increasing the reducing sugar content in the tobacco product.

[0017] As a further improvement, the fermentation supernatant filtrate of the Bacillus Velez subtilis is sprayed on the surface of the tobacco product for fermentation.

[0018] The beneficial effect of the above technical solution is that the present invention uses the fermentation supernatant filtrate of Bacillus Velezii A8-1 instead of the bacterial liquid to treat tobacco products, which can effectively avoid the high temperature and high humidity conditions affecting the activity of the bacteria during the tobacco leaf fermentation process, thereby affecting its ability to produce amylase and causing different quality fluctuations after different batches of tobacco leaves are processed.

[0019] As a further improvement, every 25 to 30 mL of fermentation supernatant filtrate corresponds to 100 g of tobacco product.

[0020] As a further improvement, the fermentation conditions are a constant temperature of 42-45° C. and a constant humidity of 70-75%.

[0021] As a further improvement, the tobacco product is tobacco leaves, tobacco shreds or tobacco flakes. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a colony morphology diagram of strain A8-1 in Example 2 of the present invention on solid LB medium;

[0023] Figure 2This is a 100× microscopic morphology of strain A8-1 in Example 2 of the present invention;

[0024] Figure 3 This is the spectrum of strain A8-1 identified in Example 2 of the present invention under a 100x oil microscope after Gram staining;

[0025] Figure 4 This is the phylogenetic tree constructed based on the 16S rDNA gene sequence when strain A8-1 was identified using molecular biology in Example 2 of the present invention;

[0026] Figure 5 Determination of the growth curve of Bacillus velezensis A8-1 in Example 3 of the present invention;

[0027] Figure 6 Determination of the enzyme activity curve of Bacillus velezensis A8-1 in Example 4 of the present invention;

[0028] Figure 7 The effect of different pH values ​​in the culture system on strain A8-1 in Example 5 of the present invention (wherein the abscissa is pH and the ordinate is OD value);

[0029] Figure 8 The different salt ions (Na + ) on strain A8-1 (wherein the abscissa is the concentration of salt ions and the ordinate is the OD value);

[0030] Fig. 9 The figure shows the effect of different concentrations of nicotine on the strain A8-1 in the culture system of Example 5 of the present invention (wherein the abscissa is the concentration of nicotine and the ordinate is the OD value). DETAILED DESCRIPTION

[0031] Existing technical research shows that the starch content in tobacco leaves is closely related to the quality of tobacco leaves. The starch content in high-quality tobacco leaves in my country (4%-6%) is significantly higher than that in foreign high-quality tobacco leaves (1%-2%), which seriously restricts the improvement of domestic tobacco leaf quality. Therefore, how to degrade the starch in tobacco leaves and decompose it into small molecules such as reducing sugars that can improve the quality of tobacco leaves has become an important research hotspot. As a type of green, safe and efficient biocatalyst, bioenzyme preparations have shown great application potential in improving tobacco leaf quality. However, the tobacco starch-degrading bacteria currently screened still have low amylase activity and low tolerance temperature, which are not suitable for high-temperature fermentation of tobacco products.

[0032] Furthermore, Zhang Mingzhu et al. (2022) and Zhao Dehou et al. (2021) used scanning electron microscopy to observe the morphology of starch particles of optimized extracted tobacco crude starch. The analysis found that there are certain differences in the morphology of tobacco crude starch and ordinary starch. Tobacco crude starch is single-granular, most of which are ellipsoidal or kidney-shaped, with small particles, and the particle size is mainly concentrated in 3μm~6μm; a small part of the starch particles are small and spherical, and some starch particles are adhered to and covered by fibers, resulting in a rough surface of starch particles. Ordinary starch particles are generally polygonal, elliptical and circular, with no damage or cracks on the surface, and the particle size is relatively uniform and the surface is relatively smooth, with a particle size of 2μm~110μm. It can be seen from the scanning electron microscope image that there is a big difference between the two.

[0033] Moreover, through XRD spectrum analysis of tobacco crude starch, the results show that there are typical absorption peaks of C-type crystalline starch near 15° and 23°, and there are two strong diffraction peaks at 26°. Therefore, tobacco crude starch belongs to C-type crystalline starch, and it is calculated that the starch crystallinity is about 12%. At present, the most common starch on the market is cereal starch, such as corn starch. According to literature reports (Zhao Dehou, 2021; Liu Lin, 2022), it has four strong diffraction peaks at diffraction angles 2θ=15.3°, 17.1°, 18.1° and 23.6°, indicating that the crystalline structure of corn starch is a typical A-type structure. Another starch is B-type crystalline starch. C-type crystals are composed of A-type and B-type, which are between the two crystal forms. By comparing XRD spectra, it can be seen that the two are significantly different. The ordered structure of starch molecules was determined by infrared spectroscopy analysis of tobacco crude starch. The results show that 707cm -1 The characteristic absorption peak of starch is 930cm -1 The absorption peak of 1,4-glycosidic bond of starch, 2930cm -1 is the characteristic stretching vibration of CH, 3000cm -1 ~3700cm -1 are characteristic peaks of different types of hydroxyl groups, among which 3400cm -1 Nearby is the -OH stretching vibration peak of starch, 610 cm -1 The characteristic peak of pyranose ring is 1080cm -1 The characteristic peak of COH is 1150cm -1 The peaks are CO and CC characteristic peaks. The literature reports (Zhao Dehou, 2021) that the hydroxyl stretching vibration peak of ordinary starch is at 3423.77 cm -1 2930cm -1 It is the characteristic stretching vibration of CH, 1651cm -1It is the characteristic stretching vibration of C=O. By comparison, we can know that the structure of tobacco crude starch is different from that of ordinary starch. In summary, the existing α-amylase suitable for degrading common ordinary starch may have a lower fitness in degrading tobacco starch. Therefore, screening out tobacco-derived strains with the ability to degrade tobacco starch is important for improving the quality of tobacco leaves.

[0034] The present invention provides a tobacco-derived Bacillus velezensis that produces alpha-amylase. The present invention uses the alpha-amylase activity as an evaluation index, first uses a screening medium for directional enrichment culture, and then uses the screening medium for primary screening and rescreening twice, and directional screening out a strain of tobacco-derived bacteria with high alpha-amylase activity. After physiological, biochemical and molecular biological identification, the bacterium is Bacillus velezensis (Bacillus velezensis), named A8-1, and the alpha-amylase produced by the bacterium is measured to have an activity of up to 147.85±0.05U / mL at a high temperature of 70°C. Moreover, Bacillus velezensis is a biological safety strain, which can ensure its safety issues in subsequent applications.

[0035] The present invention is further described below in conjunction with specific implementation methods, but the protection scope of the present invention is not limited thereto; unless otherwise specified, various reagents, instruments, etc. used in the examples are commercially available products.

[0036] The culture medium and specific formula used in the following embodiments of the present invention are as follows:

[0037] Liquid screening medium: tobacco starch 1g / L, sodium chloride 5g / L, yeast powder 2g / L, adjust the pH to 7.0; if preparing solid screening medium, add 15g / L agar powder and adjust the pH to 7.0.

[0038] LB medium: 5 g / L yeast powder, 10 g / L peptone, 10 g / L NaCl, adjust the pH to 7.0; if preparing solid culture medium, add 15 g / L agar powder and adjust the pH to 7.0.

[0039] Fermentation medium: starch 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.

[0040] The experimental reagents and preparation methods used in the following embodiments of the present invention are as follows:

[0041] 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.

[0042] 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.

[0043] 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.

[0044] Preparation of phosphate buffer (pH 6.0): Weigh 45.23 g of disodium hydrogen phosphate (Na2HPO4·12H2O) and 8.07 g of citric acid (C6H8O7·H2O), dissolve them in water and make up to 1000 mL, and use after calibration with a pH meter.

[0045] 1. A specific embodiment of the tobacco-derived α-amylase-producing Bacillus Velezii of the present invention:

[0046] The tobacco-derived Bacillus velezensis producing α-amylase of the present invention is Bacillus velezensis A8-1, with a deposit number of CCTCC NO: M 20232632; the deposit date is December 22, 2023; the deposit unit is China Center for Type Culture Collection; the deposit address is Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. The specific implementation operation is as follows:

[0047] Example 1 Screening of highly efficient tobacco starch degradation strains

[0048] In this example, a strain that can efficiently degrade tobacco starch was screened from an alcoholized tobacco leaf sample. The α-amylase activity produced by the strain was found to be 147.85±0.05 U / mL. The specific process is as follows:

[0049] 1.1 Sampling and enrichment screening

[0050] Weigh 5 g of 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, 180 r / min for 48 h.

[0051] 1.2. Initial screening of strains

[0052] 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 -8100 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 single colony strains with larger transparent circles were 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. Several strains with the ability to degrade starch were selected in the initial screening, and the results of the initial screening of strains are shown in Table 1.

[0053] Table 1 Results of initial screening of bacterial species

[0054]

[0055] 1.3. Rescreening of strains

[0056] Select a single colony with a larger HC value, streak it on LB solid medium, and culture it at 37°C for 24h. Pick a single colony, inoculate it in LB liquid medium, and culture it at 37°C for 48h. After high-speed centrifugation at 10000r / min for 10min and filtration with a 0.22μm microfiltration membrane, take the supernatant and add it to the punched (6mm diameter) solid screening medium. The control is the fermentation supernatant filtrate of Bacillus amyloliquefaciens LCCC10163 strain (this strain is the strain that has been preserved in this experiment, and its α-amylase activity is 84.24U / mL). Incubate at 37°C for 24h, add iodine solution, and measure the diameter of the transparent circle with a colony counter to preliminarily determine the amylase activity. The results of the strain rescreening measurement are shown in Table 2.

[0057] Table 2 Strain rescreening measurement results

[0058]

[0059] 1.4. α-Amylase activity assay

[0060] The method for determining α-amylase activity in the national standard GB1886.174-2016 was modified. The main modification was to prepare the standard solution, specify the standard curve, and obtain the concentration-absorbance equation to obtain the amylase activity. The specific improvement method is as follows:

[0061] 1.4.1. Drawing of standard curve

[0062] The standard curve is drawn as follows:

[0063] Soluble starch standard solution: prepare according to Table 3.

[0064] Table 3 Soluble starch standard solution

[0065]

[0066]

[0067] 1.00 mL of each of the above solutions was taken (three parallel tests) 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 quickly 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 standard curve was plotted with the absorbance (A) as the abscissa and the concentration of the soluble starch standard solution as the ordinate. 2 The closer it is to 1, the more effective it is, and the slope K is obtained.

[0068] 1.4.2 Preparation of enzyme solution to be tested

[0069] Pick a single colony and inoculate it into 50mL LB liquid medium, and culture it at 37℃, 180r / min for 9h. Inoculate it into 50mL fermentation medium with 3% (v / v) inoculation, and culture it at 37℃, 180r / min for 60h. Take the fermented bacterial liquid, centrifuge it at 10000r for 10min, and filter it with a 0.22μm aqueous filter membrane to obtain the crude enzyme liquid. Take 1mL of the crude enzyme liquid and dilute it to the appropriate concentration with phosphate buffer at pH 6.0.

[0070] 1.4.3 Determination

[0071] 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.

[0072] Add 1.00mL of the enzyme solution to be tested, start the time immediately, shake well, and maintain a constant temperature of 70±0.2℃ for accurate reaction 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.

[0073] 1.4.4. Result calculation

[0074] Enzyme activity of thermostable α-amylase

[0075] The enzyme activity X of thermostable α-amylase, expressed in U / mL or U / g, is calculated as follows:

[0076] X=(2×20-26×AK)×n / 10

[0077] Where:

[0078] 2—initial concentration of soluble starch solution;

[0079] 20—volume of soluble starch solution during measurement;

[0080] 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;

[0081] A—absorbance value of the enzyme solution measured;

[0082] K—the slope of the obtained standard curve;

[0083] n—dilution multiple of enzyme solution;

[0084] 10—Reaction time of enzyme solution during determination.

[0085] The test result shall be based on the arithmetic mean of the parallel measurement results. The relative error of two independent measurement results obtained under repeatability conditions shall not exceed 5%.

[0086] The results of the α-amylase activity test of the strains are shown in Table 4.

[0087] Table 4 Results of strain α-amylase activity determination

[0088]

[0089] In summary, compared with the existing α-amylase-producing strain in the laboratory (Bacillus amyloliquefaciens LCCC10163 strain), the α-amylase activity of strain A8-1 at 70°C is as high as 147.85±0.05 (U / mL), and the α-amylase activity produced is much higher than that of the control strain. The purified A8-1 strain was frozen and stored in a -80°C refrigerator to maintain the original properties of the strain, to prevent the bacteria from dying, degenerating or being contaminated, and to activate and rejuvenate regularly.

[0090] Example 2 Physiological, biochemical and molecular biological identification of tobacco starch-degrading bacteria

[0091] In this example, the strain A8-1 with the highest α-amylase activity isolated and identified in the example was subjected to physiological, biochemical and molecular biological identification. The specific process is as follows:

[0092] 2.1 Physiological and biochemical identification

[0093] Morphological observation: strain A8-1 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 bacteria of strain A8-1 are rod-shaped, and the colony surface on LB solid culture medium is round, smooth, slightly convex, milky white, and produces mucus.

[0094] Gram staining: Take the A8-1 bacteria in the active growth stage and smear it on the slide. The thickness should not be too thick. Dry and fix it. 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 the oil microscope, the results are as follows Figure 3 As shown, the Gram stain of strain A8-1 is purple, which is a Gram-positive bacterium.

[0095] 2.2 Molecular Biological Identification

[0096] The strain A8-1 was inoculated into LB liquid medium and cultured at 37°C for 12 hours. The cells were collected by centrifugation and 16S rDNA was amplified by PCR using the upstream primer 27F and the downstream primer 1492R. The amplification conditions were as follows: pre-denaturation at 94°C for 5 minutes (1 cycle); denaturation at 94°C for 45 seconds, annealing at 55°C for 45 seconds, extension at 72°C for 1 minute (30 cycles); total extension at 72°C for 10 minutes (1 cycle). The PCR product was 1377 bp in length.

[0097] Among them, the upstream primer 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (shown in SEQ ID NO.1);

[0098] Downstream primer 1492R: 5'-GGTTACCTTGTTACGACTT-3' (shown in SEQ ID NO. 2).

[0099] The obtained 16S rDNA fragment was amplified by PCR, purified and recovered, and then DNA sequencing was completed by Sangon Biotech (Shanghai) Co., Ltd. The 16S rDNA gene sequence of the high-yield amylase strain A8-1 obtained by sequencing has 1377 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 A8-1 was highly similar to that of Bacillus velezensis. Sequences with high similarity were selected, and a phylogenetic tree was constructed using MEGA6.06 software with 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, it was shown that strain A8-1 belonged to Bacillus velez.

[0100] Combined with morphological characteristics and 16S rDNA gene sequence analysis, the strain was identified as Bacillus velezensis and named Bacillus velezensis A8-1. It was deposited in the China Center for Type Culture Collection (CCTCC) on December 22, 2023, with the deposit number CCTCC NO: M 20232632.

[0101] Example 3 Growth analysis of strain A8-1

[0102] In this example, A8-1 was cultured, a growth curve was prepared, and its growth characteristics were analyzed. The specific process is as follows:

[0103] A single colony of A8-1 was selected and inoculated into 50 mL LB liquid medium, and cultured at 37°C and 200 r / min for 8 hours as seed liquid. The seed liquid was inoculated into 50 mL fermentation medium at a 3% (v / v) inoculation amount, and cultured continuously at 37°C and 180 r / min for 72 hours. Samples were taken in a biosafety cabinet every 12 hours, and the absorbance at 600 nm was measured by a spectrophotometer. If the bacterial solution was too concentrated, it was appropriately diluted with purified water, and the absorbance was controlled at 0.2-0.8. The LB medium with the corresponding dilution multiple was used as a control. The growth curve was prepared with the culture time t as the horizontal axis and the absorbance value OD600 as the vertical axis.

[0104] The results are as follows Figure 5 As shown, strain A8-1 was cultured at 37°C and basically entered the stable growth phase from the 6th hour, with OD600 reaching 1.20. At this time, the total number of cells of the strain reached the maximum, which was suitable for subsequent applications.

[0105] Example 4 Analysis of amylase activity in liquid fermentation of strain A8-1

[0106] This example analyzes the changes in the amylase activity of A8-1 during the fermentation process, and the specific process is as follows:

[0107] Take strain A8-1 for activation, add the seed liquid to 50mL fermentation medium (80g / L starch peptone, 25g / L yeast powder, 5g / L potassium dihydrogen phosphate, 6g / L ammonium chloride) at an inoculum of 3% (v / v), and carry out liquid fermentation at 37°C and 180r / min. During the process, the pH value remains relatively stable at about 8.0. Samples are taken at regular intervals within 0-72h to determine and analyze the amylase activity in the fermentation broth. The amylase activity determination method is modified according to the α-amylase activity determination method in the national standard GB1886.174-2016.

[0108] The results are as follows Figure 6 As shown in the figure, the enzyme activity of the strain increased slowly from 0 to 12 hours, and the enzyme activity was 18.7U / mL at 12 hours. In the early stage of fermentation, the strain mainly used nutrients to promote its own growth and reproduction, so the enzyme activity did not change significantly. The enzyme activity in the fermentation broth increased rapidly from 16 to 28 hours, and the nutrients in the culture medium were rapidly consumed at this time. The enzyme activity was maintained at a relatively stable state from 28 to 40 hours, and the enzyme activity reached the highest level at 126.5U / mL at 32 hours. With the continuous consumption of nutrients in the culture medium and the influence of other metabolites during the fermentation process, the enzyme activity in the fermentation broth from 40 to 60 hours continued to decrease, and the enzyme activity of the fermentation broth was 27.6U / mL at 60 hours. It can be seen that strain A8-1 can obtain a higher enzyme yield within 32 hours.

[0109] Example 5 Analysis of growth characteristics of strain A8-1

[0110] In this example, the pH of the culture system was changed or the culture system (liquid LB medium, culturing for 48 hours to detect OD 600 ) + ), nicotine, etc., to evaluate the growth characteristics of strain A8-1. The specific process is as follows:

[0111] Effects of different pH values ​​in the culture system on strain A8-1 Figure 7 As shown in the figure, it can be seen that it has strong tolerance to strong alkaline environment and can still survive in a culture environment with a pH of 10.

[0112] Effects of different salt ions (Na+) in the culture system on strain A8-1 Figure 8 As shown in the figure, it can be seen that it can still survive in high salt ion concentration, OD 600 Around 1.5, indicating that it has strong tolerance to high osmotic pressure.

[0113] Effects of different nicotine concentrations on strain A8-1 in the culture system Fig. 9 As shown in the figure, it can be seen that the strain has a strong tolerance to nicotine. When the nicotine content in the culture medium reaches 0.9%, the strain A8-1 still has strong activity, OD 600 Can reach 1.1.

[0114] 2. Specific examples of the application of the tobacco-derived α-amylase-producing Bacillus Velezii in improving the quality of tobacco products of the present invention:

[0115] The present invention uses the fermentation culture liquid of Bacillus Velez A8-1 producing α-amylase from tobacco to prepare a crude enzyme liquid by centrifugal filtration, and then uses the crude enzyme liquid to treat tobacco products, and then detects the content of starch and reducing sugar in the treated tobacco products. The specific implementation operation is as follows:

[0116] Example 6 Degradation of starch content in tobacco leaves by Bacillus Velezii A8-1

[0117] In this embodiment, the fermentation culture liquid of Bacillus Velez A8-1 producing α-amylase from tobacco was centrifuged and filtered to prepare a crude enzyme solution, and then the crude enzyme solution was used to treat tobacco leaves to reduce the starch content in the tobacco leaves. The specific process is as follows:

[0118] Use 1μL disposable sterile inoculation loop to dip the bacterial solution of strain A8-1, streak inoculate on LB solid culture medium, and invert and culture at 37℃ in a constant temperature and humidity incubator for 12-16h. Pick the obtained single colony and inoculate it into 50mL liquid LB culture medium, 37℃, 180r / min, culture for 12h, which is the seed solution. With an inoculum of 3% (v / v), draw 1.5mL of seed solution and add it to 50mL fermentation medium, culture at 37℃180r / min for 60h, then centrifuge at 10000r / min for 10min, take the supernatant, and filter it with a 0.22μm sterile water filter to obtain the crude enzyme solution.

[0119] Measure 30mL of crude enzyme solution and spray it evenly on the surface of 100g tobacco leaves (2020 Chongqing tobacco leaves B4F), shake it thoroughly to mix it evenly, and then place it in a constant temperature and humidity incubator (temperature 45℃, humidity 75%) for treatment. Ferment for 48h and take samples, with 3 parallels in each group. 2020 Chongqing tobacco leaves B4F was used as the control group, and the control group was inoculated with sterile water of the same volume as the crude enzyme solution. After the enzyme treatment, the tobacco leaves were placed in an 80℃ incubator for 20min to inactivate the enzyme. Then place it in a 40℃ incubator to dry for 2h, and measure the moisture. Grind and pass 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".

[0120] After fermentation, the starch content in tobacco leaves of the blank control group was 5.10%, and the starch content in tobacco leaves of the experimental group was 4.46%. Adding A8-1 crude enzyme solution can reduce the starch content by 0.64% in 48 hours, a decrease of 12.5%. The reduction of starch content can effectively reduce the burnt smell of tobacco, thereby improving the quality of tobacco products.

[0121] Example 7: Bacillus Velezii A8-1 increases the reducing sugar content in tobacco leaves

[0122] In this example, the fermentation culture fluid of Bacillus Velez A8-1 producing α-amylase from tobacco was centrifuged and filtered to prepare a crude enzyme solution, and then the crude enzyme solution was used to treat tobacco leaves to increase the content of reducing sugars in the tobacco leaves. The specific process is as follows:

[0123] Use 1μL disposable sterile inoculation loop to dip the bacterial solution of strain A8-1, streak inoculate on LB solid culture medium, and invert and culture at 37℃ in a constant temperature and humidity incubator for 12-16h. Pick the obtained single colony and inoculate it into 50mL liquid LB culture medium, 37℃, 180r / min, culture for 12h, which is the seed solution. With an inoculum of 3% (v / v), draw 1.5mL of seed solution and add it to 50mL fermentation medium, culture at 37℃180r / min for 60h, then centrifuge at 10000r / min for 10min, take the supernatant, and filter it with a 0.22μm sterile water filter to obtain the crude enzyme solution.

[0124] Measure 30 mL of crude enzyme solution and spray it evenly on the surface of 100 g of tobacco leaves (2020 Chongqing tobacco leaves B4F). 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. The control group was inoculated with sterile water of the same volume as the crude enzyme solution. After the enzyme treatment, the shredded tobacco was placed in an incubator at 80°C for 20 minutes to inactivate the enzyme. Then place it in an incubator at 40°C for 2 hours to dry it, and measure the moisture. Grind it and pass it through a 40-mesh sieve. The reducing sugar content in tobacco leaves was detected using YC / T159-2019 "Continuous Flow Method for the Determination of Water-soluble Sugars in Tobacco and Tobacco Products".

[0125] After fermentation, the reducing sugar content in the tobacco of the blank control group was 14.1%, and the reducing sugar content in the tobacco of the experimental group was 15.0%. The reducing sugar content increased by 0.9% in 48 hours, an increase of 6.4%. This shows that under the action of amylase, starch is hydrolyzed into small molecular reducing sugars, resulting in an increase in reducing sugar content, which can significantly improve the quality of tobacco products.

[0126] Example 8 Sensory Evaluation of Tobacco Strips Treated with Crude Enzyme Solution of Bacillus Velezii A8-1

[0127] In this example, the fermentation culture fluid of Bacillus Velez A8-1 producing α-amylase from tobacco was centrifuged and filtered to prepare a crude enzyme solution, and then the crude enzyme solution was used to treat tobacco leaves, and then the treated tobacco leaves were made into cigarettes for sensory evaluation. The specific process is as follows:

[0128] Use 1μL disposable sterile inoculation loop to dip the bacterial solution of strain A8-1, streak inoculate on LB solid culture medium, and invert and culture at 37℃ in a constant temperature and humidity incubator for 12-16h. Pick the obtained single colony and inoculate it into 50mL liquid LB culture medium, 37℃, 180r / min, culture for 12h, which is the seed solution. With an inoculum of 3% (v / v), draw 1.5mL of seed solution and add it to 50mL fermentation medium, culture at 37℃180r / min for 60h, then centrifuge at 10000r / min for 10min, take the supernatant, and filter it with a 0.22μm sterile water filter to obtain the crude enzyme solution.

[0129] Measure 30mL of crude enzyme solution and spray it evenly on the surface of 100g 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. The control group was inoculated with sterile water of the same volume as the crude enzyme solution. After the enzyme treatment, the tobacco leaves were placed in an 80°C incubator for 20 minutes for enzyme inactivation. After taking it out, it was cut into shredded tobacco with a shredder, made into cigarettes by hand, balanced in a balance cabinet (temperature 22°C, humidity 60%) for 48-72 hours, and then evaluated using the "Biological Enzyme Fermentation Tobacco Leaf Sensory Quality Evaluation Form" of the tobacco company. The specific results are shown in Table 5. After sensory evaluation, it is believed that after the crude enzyme solution of strain A8-1 is treated, the smoke quality is significantly improved, the irritation is reduced, the smoke is soft, the aftertaste is clean, and the smoke quality is significantly improved.

[0130] Table 5 Sensory quality evaluation table of enzyme fermented tobacco leaves

[0131]

[0132]

[0133] In summary, the present invention separates and screens strain A8-1 with strong starch degradation ability from aged tobacco leaf samples, and the strain is identified as Bacillus velezensis through physiological, biochemical and molecular biological identification. After fermentation and centrifugal filtration of strain A8-1, a crude enzyme solution is prepared, which is sprayed on the surface of tobacco products. After constant temperature and humidity fermentation, it is detected that the starch content in tobacco leaves is significantly reduced, and the reducing sugar content in tobacco shreds is increased; in addition, after smoking the fermented tobacco products, the aroma quality of the cigarettes is significantly improved.

[0134] Finally, it is noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A tobacco-derived α-amylase-producing Bacillus Velezii, characterized in that: The Bacillus Velez Bacillus velezensis ) is A8-1, and the deposit number is CCTCC NO: M 20232632.

2. Use of the tobacco-derived α-amylase-producing Bacillus Velezii as claimed in claim 1 in improving the quality of tobacco products.

3. The use of the tobacco-derived α-amylase-producing Bacillus Velezii in improving the quality of tobacco products according to claim 2, characterized in that: The quality of the tobacco products is improved by reducing the starch content and increasing the reducing sugar content in the tobacco products.

4. The use of the tobacco-derived α-amylase-producing Bacillus Velezii in improving the quality of tobacco products according to claim 2 or 3, characterized in that: The fermentation supernatant filtrate of the Bacillus Velez is sprayed on the surface of the tobacco product for fermentation.

5. The use of the tobacco-derived α-amylase-producing Bacillus Velezii in improving the quality of tobacco products according to claim 4, characterized in that: Every 25-30 mL of fermentation supernatant filtrate corresponds to 100 g of tobacco products.

6. The use of the tobacco-derived α-amylase-producing Bacillus Velezii in improving the quality of tobacco products according to claim 4, characterized in that: The fermentation conditions are a constant temperature of 42-45° C. and a constant humidity of 70-75%.

7. Use of the tobacco-derived α-amylase-producing Bacillus Velezii in improving the quality of tobacco products according to claim 2 or 3, characterized in that: The tobacco product is tobacco leaves, tobacco shreds or tobacco flakes.

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