Pantoea B-3 capable of degrading tannic acid and its applications

Abstract

The present application discloses a Pantoea sp. B-3 capable of degrading tannic acid and its application, aiming to solve the technical problems that tannic acid substances are difficult to degrade in tobacco leaf processing and there is a lack of special microorganisms with good effects on improving the quality of tobacco leaves. The Pantoea sp. B-3 strain was isolated and screened from cigars, and its preservation number is CCTCC NO: M20221165. Its metabolite enzymes can significantly reduce the tannic acid content in tobacco leaves and comprehensively improve the processing quality of tobacco leaves (such as enhancing the baking aroma, reducing unpleasant odors such as green and miscellaneous odors and earthy smell, and improving the taste such as bitterness and astringency). The technological process of treating tobacco leaves with the metabolite enzymes of Pantoea sp. B-3 is simple, the treatment cycle is short, and the processing cost of tobacco leaves is low.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, and in particular to Pantoea B-3 capable of degrading tannic acid and application thereof. Background Art

[0002] Tannic acid is a type of polyphenol produced during the growth of tobacco plants. Its content is related to the quality of tobacco. Too high a content of tannic acid in tobacco leaves is very detrimental to the quality of tobacco products when smoked, and it will produce a bitter taste when smoked. Therefore, choosing to use effective methods to degrade tannic acid in tobacco leaves and reduce it to small molecules is one of the effective ways to improve the quality of tobacco leaves.

[0003] How to use biotechnology to reduce the tannic acid content in tobacco leaves and thus improve the quality of tobacco leaves has become a common concern of scientific and technological workers in the industry; among them, the research on biological degradation of tobacco tannic acid mainly includes enzyme preparation method and microbial method, and the latter has become a research hotspot due to its advantages of low cost and simple operation. In view of this, screening specific microorganisms for application in tobacco leaf biofermentation treatment is of great practical significance for effectively degrading macromolecular substances in tobacco leaves and effectively improving the quality of tobacco products.

[0004] The information disclosed in this background technology section is only used to deepen the understanding of the background technology of the present disclosure, and should not be regarded as acknowledging or suggesting in any form that the information constitutes the prior art known to those skilled in the art. Summary of the invention

[0005] Through research, the inventors found that the metabolic enzyme products of the Pantoea B-3 strain isolated and screened from cigars can reduce the tannin content in tobacco leaves and comprehensively improve the quality of tobacco leaves (such as enhancing the roasted aroma, reducing unpleasant odors such as green and earthy odors, and improving bitterness and astringency).

[0006] According to one aspect of the present disclosure, a Pantoea B-3 was screened and named Pantoea sp. B-3, deposited on July 25, 2022 in the China Center for Type Culture Collection (address: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Postal Code: 430072), and its deposit number is CCTCC NO: M20221165.

[0007] According to another aspect of the present disclosure, a microbial preparation containing Pantoea B-3 and / or its metabolic enzyme products is provided.

[0008] According to another aspect of the present disclosure, the Pantoea B-3 or the microbial preparation is used to reduce the tannic acid content in tobacco leaves.

[0009] According to another aspect of the present disclosure, the Pantoea B-3 or the microbial preparation is applied to improving the quality of tobacco leaves.

[0010] According to another aspect of the present disclosure, the Pantoea B-3 or the microbial preparation is used in the preparation of tannase.

[0011] According to another aspect of the present disclosure, a method for treating tobacco leaves is provided, including the following steps:

[0012] (1) Adjust the moisture content of the tobacco leaves to be treated to 20-30%;

[0013] (2) According to 20-30% of the mass ratio of the tobacco leaves, uniformly spray the metabolite of the Pantoea B-3 or the microbial preparation on the surface of the tobacco leaves, and then carry out fermentation treatment for 6-8 d under the conditions of a temperature of 42-48 °C and a humidity of 75-85%;

[0014] (3) After the fermentation is completed, carry out inactivation treatment at 75-85 °C for 15-25 min, and that's it.

[0015] The preparation method of the above-mentioned microbial preparation includes the following steps:

[0016] (1) Activate the strain: Inoculate the Pantoea B-3 strain on the NA solid medium and culture it for 24 h-72 h at 35-39 °C;

[0017] (2) Prepare the seed liquid: Scrape the cultured bacteria in step (1) and inoculate them into the liquid NA medium, and carry out shaking culture at 28-32 °C;

[0018] (3) Scale-up culture: Transfer the seed liquid obtained in step (2) to the scale-up medium and culture it until OD 600 = 1.4-1.6;

[0019] (4) Prepare the crude enzyme agent: Centrifuge the fermentation broth in step (3) and take the supernatant to obtain it.

[0020] In some embodiments of the present disclosure, the composition of the NA solid medium in step (1) is: calculated by g / L, containing beef extract 2.8-3.2, peptone 9.0-11.0, NaCl 4.5-5.5, agar 18-22, pH 6.5-7.0, sterilized at 110-125 °C for 15-25 min.

[0021] In some embodiments of the present disclosure, the composition of the liquid NA medium in step (2) is: calculated by g / L, containing beef extract 2.8-3.2, peptone 9.0-11.0, NaCl 4.5-5.5, pH 6.5-7.0, sterilized at 110-125 °C for 15-25 min.

[0022] In some embodiments of the present disclosure, the composition of the enlarged culture medium in step (3) is as follows: in g / L, sucrose 15.0 - 15.6, yeast extract 16.0 - 18.0, NaCl 4.94, pH 6.5 - 7.0, sterilized at 110 - 125 °C for 15 - 25 min.

[0023] One or more technical solutions provided in the embodiments of the present application have at least any one of the following technical effects or advantages:

[0024] 1. Pantoea Pantoea sp. B-3 is screened, and its metabolites can effectively reduce tannic acid in tobacco leaves, improve the smoking quality or taste of tobacco leaves, such as increasing the baking aroma, enhancing the aroma, improving the smoke, and reducing the bitterness.

[0025] 2. A relatively simple technical solution for treating tobacco leaves with enzyme agents is designed. Its process flow is relatively simple, the cost of treating tobacco leaves is low, and the treatment cycle is short. It can significantly reduce the tannic acid content in tobacco leaves and improve the quality of tobacco leaves and other technical advantages, so it has good promotion and application value in the field of cigar manufacturing technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is a morphological diagram of Pantoea B-3 strain under an electron microscope in an embodiment of the present application.

[0027] Figure 2 It is a phylogenetic tree diagram constructed based on the 16S rDNA gene sequence of Pantoea B-3 strain in an embodiment of the present application.

[0028] Figure 3 It is a morphological diagram of Pantoea B-3 strain in an embodiment of the present application.

[0029] Figure 4 It is a trend diagram of the effects of different temperatures on the enzyme activity and growth of Pantoea B-3 strain in an embodiment of the present application.

[0030] Figure 5 It is a trend diagram of the effects of different pH values on the enzyme activity and growth of Pantoea B-3 strain in an embodiment of the present application.

[0031] Figure 6 It is an enzyme activity fingerprint diagram of Pantoea B-3 strain in an embodiment of the present application.

[0032] Figure 7 It is a standard curve diagram of gallic acid solution in an embodiment of the present application.

[0033] Figure 8 It is a standard curve diagram of tannin solution in an embodiment of the present application. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0034] Unless otherwise specified, the instruments and equipment involved in the following examples are all conventional instruments and equipment; the reagents and culture media involved are all commercially available conventional reagents and culture media; the test methods involved are all conventional methods unless otherwise specified.

[0035] To better understand the technical solution of this application, the above technical solution will be described in detail below in conjunction with the accompanying drawings of the specification and specific embodiments.

[0036] Example 1: Screening and Identification of Pantoea sp. Strain B-3

[0037] The preliminary screening method for microorganisms is as follows: Take 5 g of tobacco leaves and dissolve them in 50 ml of sterile water. Enrich for 12 h at 160 r / min and 35 °C in a shaker. Dilute the obtained overnight culture of leaves to 10 -3 、10 -4 、10 -5 gradients. Take 50 μl of each concentration and spread it evenly on the surface of the tannin differential medium. Place the spread plates in an incubator at 30 °C for 3 d. After bacteria grow, pick the strains that turn the medium from blue to yellow and culture them at 30 °C for 3 d for subsequent tests. The composition of the tannin differential medium (g / L) is: beef extract 3.0, peptone 10.0, NaCl 5.0, tannic acid 10.0, agar powder 15.0, bromophenol blue 0.04, pH 6.8, sterilize at 121 °C for 20 min.

[0038] Inoculate the screened strains into 50 mL of fermentation medium respectively, and culture them with shaking at 30 °C and 160 r / min for 12 h for strain identification and enzyme activity determination.

[0039] Strain B-3 was isolated from Indonesian cigars in 2013 in 2021 (isolated and collected by Fu Bo). Its colonies on the NA culture plate are round or irregular, light yellow, slightly shiny, the colony surface is flat and smooth, and the colonies are opaque ( Figure 3 ). Under the electron microscope, the morphology is as shown in Figure 1 , and it can be observed that the bacteria are evenly distributed, rod-shaped, and have folds on the surface.

[0040] Using the 16S rDNA gene fragment as a universal primer, PCR and cloning sequencing were performed on this strain to obtain a 1473 bp sequence, and phylogenetic analysis was carried out, showing that the homology between strain B-3 and Pantoea sp. ( Pantoea ) is over 99%, that is, it has the closest genetic relationship with Bacillus pumilus (as shown in Figure 2 ).

[0041] Further physiological and biochemical analysis of the strain was carried out, and the results are shown in Table 1.

[0042] Table 1 Physiological and Biochemical Test Results of Pantoea sp. Strain B-3

[0043]

[0044] Note: + indicates positive, yes or can utilize; - indicates negative, no or cannot utilize.

[0045] Therefore, it was identified that strain B-3 belongs to the genus Pantoea ( Pantoea ).

[0046] Example 2: Verification of Environmental Tolerance of Pantoea sp. Strain B-3

[0047] To determine the tolerance of the screened Pantoea sp. strain B-3 to environmental conditions, the following test operations were carried out:

[0048] (1) Prepare NA media with pH values of 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, and 9 respectively to determine the tolerance of the strain to acid-base conditions

[0049] (2) Activate the strain: Place strain B-3 on an NA plate and culture it in a biochemical incubator at 37 °C for 1 - 2 d;

[0050] (3) Prepare the seed solution: Scrape the cultured bacteria in step (2) in a laminar flow hood and inoculate them into NA medium with an inoculation needle, and culture them overnight on a shaker at 30 °C and 180 rpm;

[0051] (4) Inoculate the seed solution prepared in step (3) into the medium prepared in step (1) at an inoculation amount of 2%, and culture it overnight on a shaker at 30 °C and 180 rpm;

[0052] (5) Inoculate the seed solution prepared in step (3) into NA medium at an inoculation amount of 2%, and culture it overnight on a shaker at 20 °C, 25 °C, 30 °C, 35 °C, and 40 °C respectively at 180 rpm to determine the tolerance of the strain to temperature.

[0053] The test results are as shown in Figure 4 , 5 , indicating that too high or too low temperature and pH will affect the growth of the strain.

[0054] Example 3: Enzyme Solution Preparation, Enzyme Activity Determination and Optimization of Enzyme Production Conditions of Pantoea sp. Strain B-3

[0055] When using the screened strain B-3 to degrade tannic acid in tobacco leaves, it is necessary to first prepare the strain into an enzyme solution that is easy to use. The specific preparation process is as follows:

[0056] (1)Activation of bacterial strains: Place Pantoea sp. strain B-3 on an NA plate (calculated in g / L, containing 3.0 g of beef extract, 10.0 g of peptone, 5.0 g of NaCl, 20 g of agar, pH 6.5 - 7.0, sterilized at 121 °C for 20 min), and culture it in a biochemical incubator at 37 °C for 1 - 2 days;

[0057] (2)Preparation of seed solution: Scrape the cultured bacteria in step (1) in a laminar flow hood, and inoculate them into an 18×180 test tube containing 5 mL of liquid NA medium (calculated in g / L, containing 3.0 g of beef extract, 10.0 g of peptone, 5.0 g of NaCl, pH 6.8, sterilized at 121 °C for 20 min), and culture them overnight on a shaker at 30 °C and 180 rpm;

[0058] (3)Subculture: Transfer the seed solution in step (2) to a sterilized 250 mL conical flask containing 100 mL of liquid NA medium (calculated in g / L, containing 3.0 g of beef extract, 10.0 g of peptone, 5.0 g of NaCl, pH 6.8, sterilized at 121 °C for 20 min) at a ratio of 2 mL of bacterial suspension / 100 ml of NA medium (2% volume ratio), and culture it at 30 °C and 180 rpm until OD 600 reaches about 1.5;

[0059] (4)Preparation of bacterial agent: Centrifuge the bacterial liquid in step (3) at 8000 rpm for 10 min, and take the supernatant to obtain the bacterial agent;

[0060] (5)Measure cellulase, pectinase, amylase, xylanase, tannase, acid protease, neutral protease, alkaline protease, peroxidase, manganese peroxidase, laccase, nicotine enzyme, and β - carotene enzyme in the metabolites of Pantoea sp. strain B-3 respectively.

[0061] The determination of cellulase, pectinase, amylase and xylanase was carried out by the DNS method. Glucose, galacturonic acid, maltose and xylose were used as standard solutions respectively, and sodium carboxymethylcellulose solution, pectin solution, starch solution and xylan solution were used as substrates. After reacting precisely in a 50°C water bath for 30 min, DNS was added and then reacted in a boiling water bath for 5 min for color development. Then, the absorbance was measured at 540 nm, and the enzyme activity was calculated according to the formula. The determination of tannase was carried out by the circumtannin method, using propyl gallate as the substrate, reacting in a 30°C water bath for 5 min, and measuring the absorbance at 520 nm. The determination of protease was carried out by the Folin-Ciocalteu method. Acid protease, neutral protease and alkaline protease were determined respectively. A tyrosine solution was used as the standard curve, casein was used as the substrate, and the absorbance was measured at 680 nm. The determination of nicotine enzyme was carried out by the dichlorophenol indophenol method, and the absorbance of nicotine at 578 nm was measured using nicotine as the substrate. The determination of β-carotene enzyme: Using β-carotene as the substrate, reacting in a 37°C water bath for 10 min, and then measuring the absorbance with a microplate reader at 460 nm. The determination of lignin enzyme: Using ABTS, MnSO 4 and veratryl alcohol as substrates to determine the laccase, manganese peroxidase and peroxidase of this strain, and measuring the absorbance at 430 nm, 270 nm and 310 nm respectively.

[0062] The measurement results showed (see Figure 6 ), in addition to a high content of tannase in the metabolites of Pantoea sp. B-3 strain, it also contained certain amounts of acid protease, neutral protease, alkaline protease, peroxidase and laccase.

[0063] Further optimize the enzyme production conditions of B-3 strain: Carbon source, nitrogen source, inorganic salt: Using carbon source, nitrogen source and inorganic salt as influencing factors respectively, a 3-factor 3-level response surface optimization was carried out, and the specific design is shown in Table 2.

[0064] Table 2 Response surface optimization method

[0065] .

[0066] Preparation of gallic acid standard curve: Use citric acid buffer solution with pH = 5.0 to prepare 6 different concentration gradients of gallic acid standard solutions with concentrations of 10, 20, 40, 80, 160, 320 μmol / L respectively. Take 0.5 mL of gallic acid standard solution and 0.3 mL of methanol rhodanine solution (0.05 mol / L) and add them to all test tubes, and react at 30°C for 5 min. Add 0.2 mL (0.5 mol / L) of KOH aqueous solution to all test tubes, and then react in a 30°C constant temperature water bath for 5 min again. Finally, add 4 mL of distilled water to all test tubes for dilution. Using distilled water as the blank, measure the absorbance of the gallic acid standard solution with a microplate reader at a wavelength of 520 nm to obtain the standard curve of the gallic acid solution (Figure 7 )

[0067] Determination of tannase activity: The activity of tannase was determined according to the method of forming a chromogenic substance between gallic acid (produced by the hydrolysis of propyl gallate catalyzed by tannase) and methanol rhodanine. The amount of enzyme required to produce 1 μmol of gallic acid per minute at 30 °C was defined as one enzyme activity unit.

[0068] Take three clean test tubes and label them as blank tube, test tube, and control tube respectively. Preheat the substrate propyl gallate and the crude enzyme solution in a 30 °C water bath for 5 - 10 min before the reaction starts. Add 0.25 mL of propyl gallate solution to each of the three labeled test tubes. Then add 0.25 mL of citrate buffer to the blank tube, 0.25 mL of crude enzyme solution to the test tube, and place all three test tubes in a 30 °C water bath for 5 min. Then add 0.3 mL of methanol rhodanine (0.05 mol / L) solution to all test tubes, keep them in a 30 °C water bath for 5 min. After that, add 0.2 mL of 0.5 mol / L KOH aqueous solution to all test tubes, keep them in a 30 °C water bath for 5 min. Then add 0.25 mL of crude enzyme solution only to the reaction mixture in the control tube. Finally, dilute each test tube with 4 mL of distilled water, keep them at 30 °C for 10 min, use distilled water as the blank at a wavelength of 520 nm, and measure the absorbance value of the reaction mixture. All tests are performed in three parallel experiments and the arithmetic mean is taken. The activity of tannase is calculated from the change in absorbance value:

[0069] ΔA520=(A test -A blank )-(A control -A blank );

[0070] Performing regression analysis, the multiple regression equation of each factor on the enzyme activity of the strain is:

[0071] Enzyme activity = 55.69 + 1.36A + 2.22B - 0.31C - 0.48AB + 1.25AC + 0.067BC - 6.25A 2 -2.71B 2 -2.78C 2 .

[0072] Table 3 Response surface optimization design and results

[0073] .

[0074] The optimal medium formula for strain B-3 predicted by response surface analysis is sucrose 15.45 g / L, yeast extract 17.00 g / L, and NaCl 4.94 g / L, and the predicted enzyme activity is 56.21 U / mL. The enzyme activity measured through experiments under this medium formula is 55.83 U / mL, which is basically similar to the predicted value, indicating that this model is relatively reliable. The optimal enzyme-producing medium formula is obtained.

[0075] Example 4: Application of the enzyme solution of Pantoea sp. B-3 strain in the treatment of cigar tobacco leaves

[0076] Use the enzyme solution and enzyme production method prepared in Example 3 to treat Indonesian cigar tobacco leaves in 2013. The specific steps are as follows:

[0077] When treating the tobacco leaves, adjust the moisture content of the tobacco leaves to 25%; according to the tobacco leaf mass ratio of 25%, use a sprayer to evenly spray the optimized microbial agent (diluted 10 times with distilled water) on the surface of the tobacco leaves, and use spraying equal amounts of pure water, 0.2 mol / L Na 2 CO 3 and 0.2 mol / L NaHCO 3 as a comparison. Under the conditions of a temperature of 45 °C and a humidity of 80%, ferment for 7 days. After fermentation is completed, treat at 80 °C for 20 minutes to inactivate the bacteria and the protease they produce.

[0078] 1. Sensory quality evaluation of cigar tobacco leaves

[0079] (1) Rolling of samples: Before rolling, balance the moisture of the cigar tobacco leaves treated by various fermentation processes to 15 ± 1% according to the requirements of GB / T 16447-2004.

[0080] If there are no special requirements, uniformly make the following cigarette specifications, that is: length: 90 mm, diameter: 15 mm (or circumference: 47.1 mm); cigarette weight: 4.1 ± 0.5 g;

[0081] After the cigarette is rolled, balance the moisture of the sample to 13.0 - 15.0% according to the requirements of GB15269-2011.

[0082] (2) Scoring scale for sensory quality indicators of tobacco leaves, see Table 4.

[0083] Table 4 Scoring scale for sensory quality indicators of tobacco leaves

[0084] 。

[0085] (3) Evaluation requirements

[0086] ① Before sensory evaluation, the panelists should not eat spicy and other irritating foods and should not drink alcohol.

[0087] ② Ensure that the assessors are in good physical condition. The assessment environment should be quiet, well-ventilated, odor-free and undisturbed.

[0088] ③ Generally, a butane gas lighter or an alcohol lamp is required as the ignition source. Matches without wax and odor can also be selected as the ignition source.

[0089] ④ Before the evaluation, use the standard sample to calibrate and unify the assessors' criteria.

[0090] ⑤ The evaluation method for cigars is the local circulation method, mainly evaluating from one-third to two-thirds of the cigar.

[0091] ⑥ During the sensory evaluation, light fruits or mineral water can be used to adjust and clean the mouth.

[0092] ⑦ There should be at least a 5-minute break between different assessment tasks to allow the mouth and nose to rest and ensure their sensitivity.

[0093] ⑧ During the assessment, if the assessor is unwell or the sensory function is abnormal, they should withdraw from the assessment.

[0094] ⑨ The organizer of the sensory evaluation meeting prepares the test samples, standard samples and "Sensory Evaluation Form for Cigar Tobacco" (see Table 1) for the assessors. The assessors record the data and fill in the evaluation opinions according to the requirements of the evaluation form. Record the scale value in the scale value column. The scale value is divided into five grades with a gradient of 0.5 points.

[0095] ⑩ When evaluating the aroma, off-odor and taste, if the characteristics of the sample exceed the given indicators in the table, describe the aroma and taste characteristics in other columns and record the scale value.

[0096] ⑪ For the overall evaluation of the sample, the team leader organizes a discussion based on the statistical results and makes a unified description.

[0097] ⑫ Samples with serious quality defects are not evaluated, including:

[0098] Having obvious off-odor or mildew;

[0099] Burning out.

[0100] (4) Terms and Definitions

[0101] Baked aroma: The aroma produced during the baking process of grains;

[0102] Hay aroma: The characteristic aromatic smell similar to that of straw cut and dried in the sun;

[0103] Green off-odor: The unpleasant smell emitted by immature green plants;

[0104] Earthy smell: The unpleasant smell emitted by moist soil;

[0105] Taste: The oral taste characteristics during the cigar smoking process;

[0106] Volatility: The degree of aroma revelation and diffusion power.

[0107] Process the tobacco leaves according to the above-mentioned scheme, and then evaluate the sensory quality of the tobacco leaves. The evaluation results are shown in Table 5.

[0108] Treatment Baked aroma Hay aroma Green and miscellaneous odor Earthy smell Bitterness Astringency Volatility Aroma quantity CK 1 2.5 2 1 3.5 3 4.5 4.5 <![CDATA[Sodium 2 Carbon monoxide 3 > 1 2.5 2 1 3 3 4.5 4.5 <![CDATA[NaHCO 3 > 1 2.5 2 1 3.5 2.5 4.5 4.5 B-3 1.5 2.5 1.5 1 2.5 2.5 5 5

[0109] It can be seen from the results (Table 5) that after spraying the crude enzyme solution, the smoking effect of the tobacco leaves has been improved, the baking aroma has been enhanced, the bad odors such as green and earthy odors have decreased, and the taste (such as bitterness and astringency) has been improved; the overall score has increased by 2.5 points compared with the control.

[0110] 2. Comparison of tannic acid content in cigar tobacco leaves

[0111] ① Preparation of tannin standard curve: Weigh 100 mg of tannin, dissolve it in a small amount of water, transfer it to a 100 ml volumetric flask and make up to the mark (this solution should be prepared freshly and not stored for a long time), and prepare a standard solution of 1 mg / ml. Sequentially obtain standard solutions of 0.1, 0.2, 0.3, 0.4, and 0.5 mg / ml. Take 5 test tubes, add 20 µl of the above dilution to each test tube in turn, then add 50 µl of Folin-Denis reagent and 100 µl of saturated Na 2 CO 3 solution, mix well, and finally make up to 1 ml with distilled water. After standing for 30 min, start colorimetry. Use the mixed solution without adding tannin as the blank, measure the absorbance at 756 nm, and plot the standard curve with the tannin content (mg) as the abscissa and the absorbance value as the ordinate ( Figure 8 ).

[0112] ② Tannin determination: Accurately weigh 40 mg (accurate to 0.0001 g) of the fermented cigar tobacco leaves of each treatment, add 2 ml of extraction reagent (ethanol solution with a volume fraction of 20%), place it in a constant temperature water bath at 70 °C and heat for 40 min, centrifuge at 8000 rpm for 10 min, and the supernatant obtained is the tannin solution. Pipette 20 µl of the tannin solution into a 2 ml centrifuge tube, and sequentially add 50 µl of Folin-Denis reagent and 100 µl of saturated Na 2 CO 3 solution according to the order of preparing the standard curve above. After standing for 30 min, perform colorimetry at 756 nm; the measurement results are shown in Table 6.

[0113] Treatment Tannic acid content (mg / g) Reduction amount of tannic acid (%) CK 13.24 0 <![CDATA[Sodium 2 CO 3 > 12.19 7.93 <![CDATA[NaHCO 3 > 12.07 8.84 B-3 11.08 16.29

[0114] As can be seen from Table 6, compared with CK, all three treatments can reduce the tannic acid content in cigar tobacco leaves, and the effect of Pantoea sp. strain B-3 is the most obvious, with a reduction of 16.29% compared to CK.

[0115] Although some preferred embodiments of the present application have been described, those skilled in the art can make additional changes and modifications to these embodiments once they know the basic creative concept. Therefore, the appended claims are intended to be construed to include the preferred embodiments as well as all changes and modifications falling within the scope of the present invention.

[0116] Obviously, those skilled in the art can make various changes and modifications to the present application without departing from the spirit and scope of the present invention. Thus, if these modifications and variations are within the scope of the claims of the present application and their equivalent technologies, the present application is also intended to include these modifications and variations.

Claims

1. A Pantoea ( Pantoea sp.) B-3, Characterized in that, This microorganism has been deposited with the China Center for Type Culture Collection, and its deposit number is CCTCC NO: M20221165.

2. A microbial preparation, Characterized in that, It contains the Pantoea B-3 described in claim 1.

3. Use of the Pantoea B-3 described in claim 1 or the microbial preparation described in claim 2 in degrading tannic acid in tobacco leaves.

4. Use of the Pantoea B-3 described in claim 1 or the microbial preparation described in claim 2 in improving the quality of tobacco leaves.

5. Use of the Pantoea B-3 described in claim 1 or the microbial preparation described in claim 2 in the preparation of tannase.

6. A method for treating tobacco leaves, Characterized in that, It includes the following steps: (1) Adjust the moisture content of the tobacco leaves to be treated to 20-30%; (2) According to 20-30% of the mass ratio of the tobacco leaves, evenly spray the microbial preparation described in claim 2 on the surface of the tobacco leaves, and then ferment at a temperature of 42-48°C and a humidity of 75-85% for 6-8 days; (3) After fermentation is completed, inactivate at 75-85°C for 15-25 minutes to obtain the finished product.

7. The preparation method of the microbial preparation described in claim 2, including the following steps: (1) Activate the strain: Inoculate the Pantoea B-3 strain described in claim 1 on the NA solid medium and culture it at 35-39°C for 24 h-72 h; (2) Prepare the seed liquid: Scrape the cultured bacteria in step (1) and inoculate them into the liquid NA medium, and perform shaking culture at 28-32°C; (3) Subculture: Transfer the seed solution obtained in step (2) to a subculture medium and culture until OD 600 = 1.4 - 1.6; (4) Prepare the crude enzyme agent: Centrifuge the fermentation broth in step (3) and take the supernatant to obtain it.

8. According to the preparation method described in claim 7, Characterized in that, In the step (1), the composition of the NA solid medium is: calculated in g / L, beef extract 2.8-3.2, peptone 9.0-11.0, NaCl 4.5-5.5, agar 18-22, pH 6.5-7.0, sterilize at 110-125°C for 15-25 minutes.

9. According to the preparation method described in claim 7, Characterized in that, In the step (2), the composition of the liquid NA medium is: calculated in g / L, containing beef extract 2.8-3.2, peptone 9.0-11.0, NaCl 4.5-5.0, pH 6.5-7.0, sterilize at 110-125°C for 15-25 minutes.

10. According to the preparation method described in claim 7, Characterized in that, In the step (3), the composition of the enlarged medium is: calculated in g / L, sucrose 15.0-15.6, yeast extract 16.0-18.0, NaCl 4.5-5.5, pH 6.5-7.0, sterilize at 110-125°C for 15-25 minutes.

Images