Acinetobacter B-12 capable of degrading tannic acid and its application

Abstract

This application discloses a tannic acid-degradable Acinetobacter B‑12 and its application, aiming to solve the technical problems of the difficulty in degrading tannic acid in tobacco leaves and the lack of microorganisms with good effects on improving the quality of tobacco leaves. The Acinetobacter B‑12 strain was isolated and screened from cigars in this community. Its metabolite enzyme products can reduce the tannic acid content in tobacco leaves and comprehensively improve the style and quality of tobacco leaves (such as improving the hay aroma, reducing undesirable odors such as green impurities, and improving the taste such as bitterness and astringency); the process of treating tobacco leaves with the metabolic enzyme products of the B‑12 strain is simple, the processing cycle is short, the cost is low, and it is easy to promote and implement.

Description

Technical Field

[0001] The present application relates to the field of bioengineering technology, and in particular to a tannic acid-degradable Acinetobacter B-12 and its application. Background Art

[0002] Many plants (such as tobacco) synthesize a polyphenolic substance called tannic acid during their growth and development. For tobacco plants, the amount of tannic acid is closely related to the quality of commercial tobacco. Too high a tannin content will deteriorate the smoking quality of tobacco products (produce bitter taste). Therefore, in the tobacco processing process, it is necessary to select appropriate process measures to degrade the tannic acid in tobacco leaves and digest it into small molecular components. This is a necessary measure to improve tobacco quality.

[0003] Using biotechnology to degrade tannic acid in tobacco leaves to improve the quality of tobacco leaf processing is an economical and effective process. Existing biological methods for degrading tobacco leaf tannic acid mainly include enzyme preparation method and microbial method. Among them, the microbial method has the advantages of low cost and simple operation, and has become a research hotspot. Therefore, screening and domesticating microorganisms with good specificity and applying them to tobacco leaf fermentation production are of great practical significance for effectively degrading macromolecular substances in tobacco and improving tobacco leaf quality at low cost.

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

[0005] The inventors have found that a strain of Acinetobacter B-12 isolated and screened from cigars ( Acinetobacter sp . B-12), whose metabolites can reduce the tannic acid content in tobacco leaves and comprehensively improve the quality of tobacco leaves (such as enhancing the hay aroma, reducing undesirable odors such as green and mixed odors, and effectively reducing bitterness and astringency).

[0006] According to one aspect of the present disclosure, a strain of Acinetobacter B-12 was screened and named Acinetobacter sp. B-12, 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: M20221166.

[0007] According to another aspect of the present disclosure, a microbial preparation containing the Acinetobacter B-12 or / and its metabolic enzyme products is provided.

[0008] According to another aspect of the present disclosure, the Acinetobacter B-12 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 Acinetobacter B-12 or the microbial preparation is used to improve the quality of tobacco leaves.

[0010] According to another aspect of the present disclosure, the Acinetobacter B-12 or the microbial preparation is used in the preparation of tannase or xylanase.

[0011] According to another aspect of the present disclosure, a tobacco leaf processing method is provided, comprising the following steps:

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

[0013] (2) Spraying the metabolic enzyme product of Acinetobacter B-12 or the microbial preparation uniformly on the surface of the tobacco leaves at a ratio of 20% to 30% by mass of the tobacco leaves, and then fermenting the leaves at a temperature of 40 to 50° C. and a humidity of 75 to 85% for 6 to 8 days;

[0014] (3) After fermentation is completed, inactivate the product at 75-85°C for 15-25 minutes.

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

[0016] (1) Activation of bacterial strains: Inoculate the Acinetobacter B-12 strain onto NA solid medium and culture at 35-39°C for 24-72 hours;

[0017] (2) Preparation of seed solution: scrape the bacterial cells cultured in step (1), inoculate them into liquid NA medium, and culture them in a shaking incubator at 28-32°C;

[0018] (3) Expansion culture: Transfer the seed solution obtained in step (2) to expansion culture medium and culture until OD 600 =1.3~1.9;

[0019] (4) Preparation of crude enzyme: Centrifuge the fermentation broth in step (3) and take the supernatant.

[0020] In some embodiments of the present disclosure, the NA solid culture medium in step (1) is composed of: in g / L, 2.5-3.5 beef extract, 8.0-12.0 peptone, 4.5-5.5 NaCl, 18-22 agar, pH 6.5-7.0, and sterilized at 121°C for 15-25 min.

[0021] In some embodiments of the present disclosure, the composition of the liquid NA culture medium in step (2) is as follows: in g / L, it contains 2.5-3.5 beef extract, 8.0-12.0 peptone, 4.5-5.5 NaCl, pH 6.5-7.0, and is sterilized at 121°C for 15-25 min.

[0022] In some embodiments of the present disclosure, the expansion medium in step (3) comprises, in g / L, sucrose 14.5-15.5, yeast extract 14-15.00, NaCl 4-6, pH 6.5-7.0, and sterilization at 121°C for 15-25 min.

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

[0024] 1. Screening of a strain of Acinetobacter sp . B-12) B-12, its metabolites can effectively degrade tannic acid in tobacco leaves, improve the smoking quality and taste of tobacco leaves, increase the baking aroma, make the aroma transparent, improve the smoke, and reduce the bitterness.

[0025] 2. The processing process is relatively simple, with low processing cost and short cycle. It can significantly reduce the tannic acid content in tobacco leaves and greatly improve the quality of tobacco leaves. Therefore, it has good promotion and application value in the field of cigar manufacturing technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a morphological diagram of the B-12 strain in one embodiment of the present application.

[0027] Figure 2 This is a phylogenetic tree diagram constructed based on the 16S rDNA gene sequence of the B-12 strain in an embodiment of the present application.

[0028] Figure 3 This is a trend chart of the effect of different temperatures on the growth of B-12 strain in one embodiment of the present application.

[0029] Figure 4 This is a trend chart of the effect of different pH on the growth of B-12 strain in one embodiment of the present application.

[0030] Figure 5 This is the enzyme activity fingerprint of the B-12 strain in one embodiment of the present application.

[0031] Figure 6 This is a standard curve diagram of gallic acid solution in one embodiment of the present application.

[0032] Figure 7 This is a standard curve diagram of tannin solution in one embodiment of the present application. DETAILED DESCRIPTION

[0033] 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 unless otherwise specified; the tests and determination methods involved are all conventional methods unless otherwise specified.

[0034] In order to better understand the technical solution of the present application, the above technical solution will be described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0035] Example 1: Screening and identification of Acinetobacter B-12

[0036] The preliminary screening method for microorganisms is as follows: 5g tobacco leaves are dissolved in 50ml sterile water. The leaves are shaken at 160r / min and 35°C for 12h. The overnight culture solution of the leaves is diluted to 10 -3 , 10 -4 , 10 -5 Gradient, take 50% of each concentration and evenly spread it on the surface of tannin identification medium. Place the coated plate in a 30℃ constant temperature incubator and culture for 3 days. After the bacteria grow, pick the strain that makes the medium turn from blue to yellow, culture it at 30℃ for 3 days, and use it for subsequent experiments.

[0037] The composition of tannin identification 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 6.7, sterilized at 121℃ for 20 min.

[0038] The screened strains were inoculated into 50 mL of fermentation medium and cultured at 30°C and 160 r / min for 12 h for strain identification and enzyme activity determination.

[0039] The B-12 strain was isolated from Indonesian cigars in 2013 in 2021 (isolation and collection person: Fu Bo, isolation and collection location: Tobacco Processing Laboratory, Tobacco College, Wenhua Road Campus, Henan Agricultural University, Zhengzhou City, Henan Province). On the NA culture plate, it is round or irregular, milky white, slightly shiny, with a flat and smooth surface, and the colony is opaque. Figure 1 shown.

[0040] Using 16S rDNA gene fragments as universal primers, PCR and cloning sequencing were performed on the strain, and phylogenetic analysis showed that the B-12 strain was similar to Acinetobacter ( Acinetobacter ) have a homology of more than 99% (e.g. Figure 2 as shown).

[0041] Example 2: Verification of environmental tolerance of Acinetobacter B-12

[0042] To determine the tolerance of the screened Acinetobacter B-12 strain to environmental conditions, the following experimental operations were performed:

[0043] (1) Prepare NA medium with pH values ​​of 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, and 9 to determine the strain's tolerance to acid and alkaline conditions.

[0044] (2) Activation of bacterial strains: Place Acinetobacter B-12 on NA plates and culture in a biochemical climate chamber at 37°C for 2 days;

[0045] (3) Preparation of seed solution: scrape the cultured bacteria in step (2) in a clean bench, inoculate them into NA medium with an inoculation needle, and culture them in a shaking incubator at 30°C and 180 rpm overnight;

[0046] (4) inoculating the seed solution prepared in step (3) into the culture medium prepared in step (1) at an inoculum rate of 2%, and culturing overnight in a shaking incubator at 30°C and 180 rpm;

[0047] (5) The seed solution prepared in step (3) was inoculated into NA medium at a 2% inoculum size and cultured overnight in a shaking incubator at 20°C, 25°C, 30°C, 35°C, and 40°C at 180 rpm to determine the strain's tolerance to temperature.

[0048] The results showed that high or low temperature and pH would affect the growth of the strain (see Figure 3 , Figure 4 ).

[0049] Example 3: Preparation of B-12 strain enzyme solution and determination of various enzyme activities

[0050] First, prepare the B-12 strain into an easy-to-use enzyme solution:

[0051] (1) Activated bacteria: Take the Acinetobacter B-12 strain and place it on a NA plate (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 6.8, sterilized at 121°C for 20 min) and culture it in a biochemical climate chamber at 37°C for 2 days;

[0052] (2) Preparation of seed solution: scrape the bacterial cells obtained in step (1) in a clean bench, inoculate them into a 18×180 test tube containing 5 mL of liquid NA medium (in g / L, containing 3.0 beef extract, 10.0 peptone, 5.0 NaCl, pH 6.8, sterilized at 121°C for 20 min), and culture overnight in a shaking incubator at 30°C and 180 rpm;

[0053] (3) Expansion culture: The seed solution in step (2) was transferred to a sterilized 250 mL conical flask containing 100 mL of liquid NA medium (in g / L, sucrose 10, yeast extract 15.00, NaCl 5, pH 6.8, sterilized at 121°C for 20 min) at a ratio of 2 mL bacterial suspension / 100 mL NA medium (2% volume ratio), and cultured at 30°C and 180 rpm until the OD 600 = about 1.8;

[0054] (4) Preparation of bacterial agent: Centrifuge the bacterial solution in step (3) at 8000 rpm for 10 min and collect the supernatant.

[0055] (5) The activities of cellulase, pectinase, amylase, xylanase, tannase, acid protease, neutral protease, alkaline protease, peroxidase, manganese peroxidase, laccase, nicotinoids and β-carotene in the metabolites of Acinetobacter B-12 were determined respectively.

[0056] The DNS method was used to determine cellulase, pectinase, amylase and xylanase, with glucose, galacturonic acid, maltose and xylose as standard solutions, respectively, and sodium carboxymethyl cellulose solution, pectin solution, starch solution and xylan solution (all must be prepared before use) as substrates. After precise reaction at 50°C water bath for 30 minutes, DNS was added and reacted in boiling water bath for 5 minutes for color development. Then the absorbance was measured at 540nm and the enzyme activity was calculated according to the formula.

[0057] Tanninase was determined by the tannin method, with gallic acid solution as the standard curve. Figure 6 , using propyl gallate as substrate, incubate in a constant temperature water bath at 30°C for 5 min, and measure its absorbance at a wavelength of 520 nm.

[0058] The protease was determined by the Folin phenol method, and the acidic protease, neutral protease and alkaline protease were determined respectively. Tyrosine solution was used as the standard curve, casein was used as the substrate, and the absorbance was determined at 680nm.

[0059] The nicotinic acid enzyme was determined by the dichlorophenol indophenol method, with nicotine as substrate to determine its absorbance at a wavelength of 578 nm.

[0060] Determination of β-carotene enzyme: Use β-carotene as substrate and react in a water bath at 37°C for 10 minutes. Then measure its absorbance at a wavelength of 460nm using an enzyme marker.

[0061] Determination of ligninase: Laccase, manganese peroxidase and peroxidase of the strain were determined using ABTS, MnSO4 and resveratrol as substrates, and their absorbances were measured at wavelengths of 430 nm, 270 nm and 310 nm, respectively.

[0062] The results showed that (see Figure 5 ), in addition to a high content of tannase, the metabolites of the B-12 strain also contain certain amounts of cellulase, pectinase, xylanase and β-carotene enzyme.

[0063] Example 4: Verification of the treatment of cigar tobacco leaves with the enzyme solution of Acinetobacter B-12 strain

[0064] The enzyme solution and enzyme production method prepared in Example 3 were used to treat 2013 Indonesian cigar tobacco leaves. The specific steps are as follows:

[0065] When treating tobacco leaves, the moisture content of the tobacco leaves was adjusted to 25%; according to the mass ratio of tobacco leaves of 25%, the bacterial agent obtained in Example 3 (diluted 10 times with distilled water) was evenly sprayed on the surface of the tobacco leaves with a sprayer, and the same amount of pure water, 0.2 mol / L Na2CO3 and 0.2 mol / L NaHCO3 were sprayed for comparison. The fermentation treatment was carried out for 7 days at a temperature of 45°C and a humidity of 80%. After the fermentation was completed, the bacterial agent was treated at 80°C for 20 minutes for inactivation.

[0066] 1. Sensory quality evaluation of cigar tobacco leaves

[0067] (1) Sample rolling: Before rolling, the moisture content of cigar tobacco leaves that have undergone various fermentation treatments shall be balanced to 15±1% in accordance with the requirements of GB / T 16447-2004.

[0068] The cigarettes are manufactured to the following specifications, namely: length: 90 mm; diameter: 15 mm (or circumference: 47.1 mm); cigarette weight: 4.1±0.5 g;

[0069] After the cigarettes are rolled, the moisture content of the samples is balanced to 13.0-15.0% according to the requirements of GB15269-2011.

[0070] (2) Scoring scale for tobacco leaf sensory quality indicators, see Table 1.

[0071] Table 1 Scoring scale for sensory quality indicators of tobacco leaves

[0072] .

[0073] (3) Evaluation requirements

[0074] ① Before sensory evaluation, the tasters should not eat spicy or other irritating foods, and should not drink alcohol.

[0075] ② Ensure that the assessors are in good physical condition. The assessment environment should be quiet, ventilated, odor-free and undisturbed.

[0076] ③ The fire source is generally required to be a butane gas lighter or an alcohol lamp. You can also use wax-free and odorless matches as the fire source.

[0077] ④ Before evaluation, use standard samples to calibrate and unify the evaluation standards.

[0078] ⑤ The evaluation method for cigars is the local circulation method, which mainly evaluates one-third to two-thirds of the cigarette.

[0079] ⑥ During the sensory evaluation process, light fruits or mineral water can be used to regulate and clean the oral cavity.

[0080] ⑦ Pause for at least 5 minutes between different inhalation assessment tasks to allow the oral and nasal cavities to rest and ensure their sensitivity.

[0081] ⑧ During the process of smoking assessment, if you feel unwell or your sensory function is abnormal, you should quit the smoking assessment.

[0082] ⑨ The organizer of the sensory evaluation meeting prepares test samples, standard samples and the "Cigar Tobacco Sensory Evaluation Form" (see Table 1) for the testers. The testers record the data and fill in the evaluation opinions according to the requirements of the evaluation form. The scale value is recorded in the scale value column. The scale value is divided into five levels, with a gradient of 0.5 points.

[0083] ⑩ When evaluating aroma, odor and taste, if the characteristics of the sample exceed the indicators given in the table, describe the aroma and taste characteristics in other columns and record the scale value.

[0084] ⑪ The overall evaluation of the samples will be described uniformly by the team leader after organizing a discussion based on the statistical results.

[0085] ⑫ Samples with serious quality defects will not be evaluated, including: obvious odor or mildew; combustion failure.

[0086] (4) Terms and definitions

[0087] Baking aroma: the aroma produced by the grain roasting process;

[0088] Hay aroma: similar to the characteristic aroma of straw after being cut and dried;

[0089] Green miscellaneous air: the unpleasant smell given off by immature green plants;

[0090] Earthy smell: the unpleasant smell of moist soil;

[0091] Taste: the taste characteristics of the mouth during the cigar smoking process;

[0092] Permeability: the degree of visibility and diffusion of aroma.

[0093] The tobacco leaves were treated according to the above scheme, and then the sensory quality of the tobacco leaves was evaluated. The results are shown in Table 2.

[0094] deal with Baking aroma Hay aroma Green mixed gas Earthy smell bitterness Astringency Translucent Aroma volume CK 1 2.5 2 1 3.5 3 4.5 4.5 <![CDATA[Na2CO3]]> 1 2.5 2 1 3 3 4.5 4.5 <![CDATA[NaHCO3]]> 1 2.5 2 1 3.5 2.5 4.5 4.5 B-12 1 3 1.5 1 3 2.5 5 5

[0095] From the results (Table 2), it can be seen that after spraying the crude enzyme solution, the smoking effect of tobacco leaves was improved, the hay aroma was enhanced, the unpleasant smell (such as green smell) and the taste (such as bitterness and astringency) were improved. The overall score was improved by 2 points compared with the control.

[0096] 2. Comparison of tannic acid content in cigar tobacco leaves

[0097] ① Preparation of tannin standard curve: Weigh 100 mg of tannin, dissolve it with a small amount of water, and transfer it to a 100 ml volumetric flask to make up the volume (this solution should be prepared immediately before use and should not be stored for a long time), and prepare a 1 mg / ml standard solution. Obtain 0.1, 0.2, 0.3, 0.4, and 0.5 mg / ml standard solutions in turn, take 5 test tubes, add 20 µl of the above dilutions to each test tube in turn, then add 50 µl of Folin-Denis reagent and 100 µl of saturated Na2CO3 solution, mix well, and finally make up the volume to 1 ml with distilled water. After leaving it for 30 minutes, start colorimetry, use the mixed solution without tannin as a blank, measure the absorbance at 756 nm, and draw a standard curve with tannin content (mg) as the horizontal axis and absorbance value as the vertical axis (see Figure 7 ).

[0098] ②Tannin determination

[0099] Weigh 40 mg (accurate to 0.0001 g) of fermented cigar tobacco leaves of each treatment accurately, add 2 ml of extraction reagent (20% ethanol solution by volume), heat in a constant temperature water bath at 70°C for 40 min, centrifuge at 8000 rpm for 10 min, and the supernatant obtained is the tannin solution; measure 20 µl of tannin solution in a 2 ml centrifuge tube, add 50 µl of Folin-Denis reagent and 100 µl of saturated Na2CO3 solution in the order of preparing the standard curve above, let it stand for 30 min, and then perform colorimetric determination at 756 nm; the determination results are shown in Table 3.

[0100] deal with Tannic acid content (mg / g) Tannic acid reduction (%) CK 13.24 0 <![CDATA[Na2CO3]]> 12.19 7.93 <![CDATA[NaHCO3]]> 12.07 8.84 B-12 11.37 14.12

[0101] As can be seen from Table 3, compared with CK, the three treatments can reduce the content of tannic acid in cigar tobacco leaves, and the treatment effect of strain B-12 is the most obvious, with a reduction of 14.12% compared with CK.

[0102] Although some preferred embodiments of the present application have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0103] 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 fall within the scope of the claims of the present application and their equivalents, the present application is also intended to include these modifications and variations.

Claims

1. An Acinetobacter species ( Acinetobacter sp .) B-12, characterized in that The microorganism has been deposited in China Center for Type Culture Collection, and its deposit number is CCTCC NO: M20221166.

2. A microbial preparation, characterized in that: The preparation method of the microbial preparation comprises the following steps: (1) Activated bacteria: inoculate the Acinetobacter B-12 strain described in claim 1 onto NA solid culture medium and culture at 35-39°C for 24-72 hours; (2) Preparation of seed solution: scrape the bacterial cells cultured in step (1), inoculate them into liquid NA medium, and culture them in a shaking incubator at 28-32°C; (3) Expansion culture: Transfer the seed solution obtained in step (2) to expansion culture medium and culture until OD 600 =1.3~1.9; (4) Preparation of crude enzyme: centrifuge the fermentation liquid in step (3) and take the supernatant.

3. The microbial preparation according to claim 2, characterized in that In step (1), the composition of the NA solid culture medium is as follows: in g / L, beef extract 2.5-3.5, peptone 8.0-12.0, NaCl 4.5-5.5, agar 18-22, pH 6.5-7.0, and sterilized at 121°C for 15-25 min.

4. The microbial preparation according to claim 2, characterized in that In step (2), the liquid NA culture medium is composed of: in g / L, 2.5-3.5 beef extract, 8.0-12.0 peptone, 4.5-5.5 NaCl, pH 6.5-7.0, and sterilized at 121°C for 15-25 min.

5. The microbial preparation according to claim 2, characterized in that: In step (3), the composition of the expansion medium is as follows: in g / L, sucrose 14.5-15.5, yeast extract 14-15.00, NaCl 4-6, pH 6.5-7.0, and sterilized at 121°C for 15-25 min.

6. Use of the Acinetobacter B-12 described in claim 1 or the microbial preparation described in claim 2 in degrading tannic acid in tobacco leaves.

7. Use of the Acinetobacter B-12 described in claim 1 or the microbial preparation described in claim 2 in improving the quality of tobacco leaves.

8. Use of the Acinetobacter B-12 according to claim 1 or the microbial preparation according to claim 2 in the preparation of tannase or xylanase.

9. A tobacco leaf processing method, characterized in that: The steps include: (1) Adjust the moisture content of the tobacco leaves to be processed to 20-30%; (2) Spraying the microbial preparation according to claim 2 uniformly on the surface of tobacco leaves at a ratio of 20% to 30% by mass of tobacco leaves, and then fermenting for 6 to 8 days at a temperature of 40 to 50°C and a humidity of 75 to 85%; (3) After fermentation is completed, inactivate the product at 75-85°C for 15-25 minutes.

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