CN110628734A - Application of trametes hirsuta LCCC50416 in production of laccase and tobacco leaf fermentation - Google Patents
Application of trametes hirsuta LCCC50416 in production of laccase and tobacco leaf fermentation Download PDFInfo
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- 238000000855 fermentation Methods 0.000 title claims abstract description 46
- 230000004151 fermentation Effects 0.000 title claims abstract description 46
- 235000002637 Nicotiana tabacum Nutrition 0.000 title claims abstract description 39
- 108010029541 Laccase Proteins 0.000 title claims abstract description 34
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 18
- 241000222357 Trametes hirsuta Species 0.000 title abstract description 18
- 244000061176 Nicotiana tabacum Species 0.000 title 1
- 241001290175 Coriolopsis trogii Species 0.000 claims abstract description 58
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- 235000011130 ammonium sulphate Nutrition 0.000 claims description 7
- 239000006228 supernatant Substances 0.000 claims description 7
- 239000007836 KH2PO4 Substances 0.000 claims description 6
- 240000008042 Zea mays Species 0.000 claims description 6
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 6
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 6
- 239000000872 buffer Substances 0.000 claims description 6
- 235000005822 corn Nutrition 0.000 claims description 6
- 235000013312 flour Nutrition 0.000 claims description 6
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 6
- CSNNHWWHGAXBCP-UHFFFAOYSA-L magnesium sulphate Substances [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 6
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 6
- 229920002271 DEAE-Sepharose Polymers 0.000 claims description 5
- 238000000502 dialysis Methods 0.000 claims description 5
- 239000002054 inoculum Substances 0.000 claims description 5
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
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- 238000005571 anion exchange chromatography Methods 0.000 claims description 4
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- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 3
- 244000046052 Phaseolus vulgaris Species 0.000 claims description 3
- 235000010627 Phaseolus vulgaris Nutrition 0.000 claims description 3
- 238000010612 desalination reaction Methods 0.000 claims description 3
- 239000008103 glucose Substances 0.000 claims description 3
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- 238000005119 centrifugation Methods 0.000 claims description 2
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- 238000011033 desalting Methods 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 12
- 229920005610 lignin Polymers 0.000 abstract description 3
- 238000007254 oxidation reaction Methods 0.000 abstract description 3
- 230000002255 enzymatic effect Effects 0.000 abstract 1
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- 238000002474 experimental method Methods 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- VOWZNBNDMFLQGM-UHFFFAOYSA-N 2,5-dimethylaniline Chemical compound CC1=CC=C(C)C(N)=C1 VOWZNBNDMFLQGM-UHFFFAOYSA-N 0.000 description 2
- 108090000854 Oxidoreductases Proteins 0.000 description 2
- 102000004316 Oxidoreductases Human genes 0.000 description 2
- 229920002538 Polyethylene Glycol 20000 Polymers 0.000 description 2
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- CWVRJTMFETXNAD-FWCWNIRPSA-N 3-O-Caffeoylquinic acid Natural products O[C@H]1[C@@H](O)C[C@@](O)(C(O)=O)C[C@H]1OC(=O)\C=C\C1=CC=C(O)C(O)=C1 CWVRJTMFETXNAD-FWCWNIRPSA-N 0.000 description 1
- PZIRUHCJZBGLDY-UHFFFAOYSA-N Caffeoylquinic acid Natural products CC(CCC(=O)C(C)C1C(=O)CC2C3CC(O)C4CC(O)CCC4(C)C3CCC12C)C(=O)O PZIRUHCJZBGLDY-UHFFFAOYSA-N 0.000 description 1
- 108010031396 Catechol oxidase Proteins 0.000 description 1
- 102000030523 Catechol oxidase Human genes 0.000 description 1
- JPVYNHNXODAKFH-UHFFFAOYSA-N Cu2+ Chemical compound [Cu+2] JPVYNHNXODAKFH-UHFFFAOYSA-N 0.000 description 1
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- 241000570275 Leiotrametes lactinea Species 0.000 description 1
- CWVRJTMFETXNAD-KLZCAUPSSA-N Neochlorogenin-saeure Natural products O[C@H]1C[C@@](O)(C[C@@H](OC(=O)C=Cc2ccc(O)c(O)c2)[C@@H]1O)C(=O)O CWVRJTMFETXNAD-KLZCAUPSSA-N 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
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- CWVRJTMFETXNAD-JUHZACGLSA-N chlorogenic acid Chemical compound O[C@@H]1[C@H](O)C[C@@](O)(C(O)=O)C[C@H]1OC(=O)\C=C\C1=CC=C(O)C(O)=C1 CWVRJTMFETXNAD-JUHZACGLSA-N 0.000 description 1
- 229940074393 chlorogenic acid Drugs 0.000 description 1
- FFQSDFBBSXGVKF-KHSQJDLVSA-N chlorogenic acid Natural products O[C@@H]1C[C@](O)(C[C@@H](CC(=O)C=Cc2ccc(O)c(O)c2)[C@@H]1O)C(=O)O FFQSDFBBSXGVKF-KHSQJDLVSA-N 0.000 description 1
- 235000001368 chlorogenic acid Nutrition 0.000 description 1
- BMRSEYFENKXDIS-KLZCAUPSSA-N cis-3-O-p-coumaroylquinic acid Natural products O[C@H]1C[C@@](O)(C[C@@H](OC(=O)C=Cc2ccc(O)cc2)[C@@H]1O)C(=O)O BMRSEYFENKXDIS-KLZCAUPSSA-N 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 229910001431 copper ion Inorganic materials 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000003205 fragrance Substances 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
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- 235000019698 starch Nutrition 0.000 description 1
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- 239000000758 substrate Substances 0.000 description 1
Classifications
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- A—HUMAN NECESSITIES
- A24—TOBACCO; CIGARS; CIGARETTES; SIMULATED SMOKING DEVICES; SMOKERS' REQUISITES
- A24B—MANUFACTURE OR PREPARATION OF TOBACCO FOR SMOKING OR CHEWING; TOBACCO; SNUFF
- A24B15/00—Chemical features or treatment of tobacco; Tobacco substitutes, e.g. in liquid form
- A24B15/18—Treatment of tobacco products or tobacco substitutes
- A24B15/20—Biochemical treatment
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0055—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10)
- C12N9/0057—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10) with oxygen as acceptor (1.10.3)
- C12N9/0061—Laccase (1.10.3.2)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y110/00—Oxidoreductases acting on diphenols and related substances as donors (1.10)
- C12Y110/03—Oxidoreductases acting on diphenols and related substances as donors (1.10) with an oxygen as acceptor (1.10.3)
- C12Y110/03002—Laccase (1.10.3.2)
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- Chemical & Material Sciences (AREA)
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- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
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- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
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- Biomedical Technology (AREA)
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- Molecular Biology (AREA)
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- General Chemical & Material Sciences (AREA)
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Abstract
The invention provides an application of Trametes trogii (Trametes trogii) LCCC50416 in laccase production and tobacco leaf fermentation, the Trametes trogii (Trametes trogii) LCCC50416 can produce laccase with high enzymatic activity, and the Trametes trogii (Trametes trogii) LCCC50416 is used for producing laccase, so that a new way is provided for the production source of the laccase, and the large-scale production of the laccase can be realized; the trametes hirsuta with laccase having high enzyme activity is applied to tobacco leaf fermentation, so that oxidation reaction in tobacco leaves can be efficiently catalyzed, lignin in the tobacco leaves can be accelerated and decomposed, the fermentation period of the tobacco leaves is obviously shortened, and the quality of the tobacco leaves is improved.
Description
Technical Field
The invention relates to application of Trametes trogii (LCCC 50416) in laccase production and tobacco leaf fermentation, and belongs to the technical field of microbial application.
Background
Laccase is a polyphenol oxidase (rho-diphenol oxidase, EC1.10.3.2) containing four copper ions, belongs to ceruloblue oxidase, and exists in the form of monomer glycoprotein. Laccase can catalyze the oxidation reaction of a plurality of compounds, the substrates are wide, and the only product after the reaction is water, so the laccase is essentially an environment-friendly enzyme.
At present, most of laccase used for producing food grade is white rot fungi, trametes hirsuta belongs to one of the white rot fungi, and the strain is safe. CN105543110B in the prior art discloses Trametes hirsuta (Trametes hirsuta) ZLH237, the strain has high laccase production activity, after the Trametes hirsuta (Trametes hirsuta) ZLH237 is fermented in a liquid-based enzyme production culture medium, the laccase activity in each milliliter of fermentation liquid is 1242.22U/mL; the extracellular laccase yield of Trametes hirsuta (Trametes hirsuta) ZLH237 after Trametes hirsuta (Trametes hirsuta) ZLH237 is 2500-2900U/mL after fermentation in 2, 5-dimethylaniline fermentation medium. The bacterial strain with high laccase yield in the patent of the invention is trametes hirsuta, and the trametes hirsuta with high laccase yield is not found and is applied to tobacco leaves.
Disclosure of Invention
The invention aims to provide application of Trametes trogii (LCCC 50416) in production of laccase and tobacco fermentation, and the Trametes trogii (LCCC 50416) can produce the laccase with high yield and is very suitable for production of the laccase and the tobacco fermentation.
In one aspect, a method for producing laccase using Trametes trogii LCCC50416 comprises inoculating Trametes trogii LCCC50416 to a liquid medium for fermentation culture, and centrifuging the fermentation broth to obtain a supernatant.
The inoculum size of Trametes trogii LCCC50416 is 105～107cfu/ml, wherein the culture is shaking culture, the rotating speed is 100-200 r/min, the temperature is 20-30 ℃, and the time is 5-10 d;
preferably, the inoculum size is 106cfu/ml, the rotating speed is 140r/min, the temperature is 28 ℃, and the time is 7 d;
preferably, the rotating speed of the centrifugation is 5000-10000 rpm, and the time is 8-12 min;
preferably, the liquid medium contains: corn flour 30g/L, wheat bran 2.0g/L, KH2PO4 0.3g/L、MgSO40.15g/L, 1.0g/L glucose and pH of 4.5-5.5;
preferably, the pH is 5.0;
preferably, the Trametes pilosus (Trametes trogii) LCCC50416 is seed liquid of Trametes pilosus (Trametes trogii) LCCC 50416;
preferably, the seed liquid of Trametes trogii (Trametes trogii) LCCC50416 is obtained by inoculating activated Trametes trogii (Trametes trogii) LCCC50416 into seed liquid culture medium;
preferably, the seed liquid culture medium comprises: corn flour 2.0g, bean cake powder 2.0g, (NH)4)2SO40.01g、KH2PO40.2g、MgSO40.05g, VB10.05g and 100ml of distilled water.
Further, the method also comprises the steps of adding ammonium sulfate into the supernatant for precipitation, and obtaining crude enzyme liquid after dialysis and desalination;
preferably, the saturation degree of the ammonium sulfate is 70-90%, and preferably, the saturation degree of the ammonium sulfate is 80%;
preferably, 8-12 mmol/L, pH 5.8.5.8-6.2 PBS buffer solution is used for dialysis desalination;
preferably, 10mmol/L, pH 6 PBS buffer is used.
Further, the method comprises a step of purifying the crude enzyme solution by using a DEAE Sepharose Fast Flow anion exchange chromatography column.
Preferably, after the crude enzyme solution is loaded, eluting by using 0.1-1mol/L PBS buffer solution, wherein the flow rate is 1.5-2.5 mL/min;
preferably, 0.5mol/L PBS buffer is used at a flow rate of 2 mL/min.
In another aspect, the invention also provides the use of Trametes trogii (Trametes trogii) LCCC50416 or said process in tobacco leaf fermentation. Trametes trogii (Trametes trogii) LCCC50416 has laccase-producing activity, and lignin in tobacco leaves can be rapidly decomposed by Trametes trogii (Trametes trogii) LCCC50416 or the method.
On the other hand, the invention also provides a tobacco leaf fermentation method, and the trametes lactinea strain LCCC50416 is inoculated in tobacco leaves for fermentation.
Preferably, the inoculum size of Trametes trogii LCCC50416 is 105～107cfu/g, the fermentation temperature is 20-30 ℃, the humidity is 40-70%, and the time is 7-14 d.
The invention has the beneficial effects that:
the invention discloses Trametes trogii (Trametes trogii) LCCC50416 which can produce laccase with high enzyme activity, and provides a new way for the production source of laccase; the laccase with high enzyme activity is applied to the tobacco leaf fermentation, so that the oxidation reaction in the tobacco leaves can be efficiently catalyzed, the lignin in the tobacco leaves can be accelerated and decomposed, the fermentation period of the tobacco leaves is obviously shortened, and the quality of the tobacco leaves is improved.
Detailed Description
The present invention is described in detail with reference to specific examples, which are provided to facilitate the understanding of the technical solutions of the present invention by those skilled in the art, and the implementation or use of the present invention is not limited by the description of the present invention.
In the present invention, the raw materials and equipment used are commercially available or commonly used in the art, if not specified. Trametes trogii (Trametes trogii) used in the invention is purchased from Liaoning province microorganism strain preservation center, and the serial number is as follows: LCCC 50416.
The methods in the examples are conventional in the art unless otherwise specified.
Example 1Production of laccase by Trametes trogii (Trametes trogii) LCCC50416 fermentation culture
The fermentation culture conditions were optimized for increasing the enzyme production of Trametes trogii LCCC 50416. Mainly inspects the influence of the inoculation amount, the most suitable liquid loading amount, the fermentation temperature and the initial pH of the culture medium on the enzyme production. The optimal culture conditions for the enzyme production of Trametes hirsuta (Trametes trogii) LCCC50416 were finally determined by experiments as follows: the culture temperature is 25-28 ℃, the initial pH value is 5.0, the inoculation amount is 10%, the liquid loading amount is 100mL, and the culture time is 7 d.
Trametes trogii (Trametes trogii) LCCC50416 fermentation process
The liquid enzyme production process of the trametes hirsuta LCCC50416 is determined through formula tests such as single-factor multi-level experiments, multi-factor multi-level orthogonal experiments and the like.
Activating and preserving strains: inoculating trametes hirsuta LCCC50416 slant strain to PDA culture medium, activating and culturing at 25 deg.C for 4 days, inoculating the activated strain to PDA culture medium plate, culturing at 28 deg.C for 4 days, and storing at 4 deg.C for use.
Preparing a seed solution: culturing the activated trametes hirsuta LCCC50416 on a PDA culture medium flat plate at 28 ℃ for 4 days, when hyphae are about to grow over the critical position of the flat plate, uniformly cutting 3 hypha slices with the diameter of 1cm by using a sterilized puncher under the aseptic condition, inoculating the hyphae slices to a seed culture medium, and carrying out constant temperature shaking culture at 140r/min and 28 ℃ for 7 days.
Seed liquid culture medium: corn flour 2.0g, bean cake powder 2.0g, (NH)4)2SO40.01g、KH2PO4 0.2g、MgSO40.05g, VB10.05g and 100ml of distilled water.
Culture medium of fermentation liquid: corn flour 3.0g, wheat bran 2.0g, KH2PO4 0.3g、MgSO40.15g, glucose 1.0g, and distilled water 100ml, and the pH was adjusted to 5.0 with NaOH.
The fermentation broth is cultured under 0.1MPa and 121 ℃, and sterilized for 30min, wherein the content of a 500mL triangular flask is 100 mL.
Liquid culture conditions:
inoculation: sterile inoculation is carried out by utilizing a flame ring, and the inoculation amount is 10 percent (the concentration is 10 percent)6cfu/ml)
The culture conditions are as follows: culturing at 28 deg.C and shaking at 140r/min for 7 d.
The enzyme activity of the fermentation liquor is measured as follows: 1500U/ml.
The laccase separation and purification method specifically comprises the following steps:
(1) ammonium sulfate precipitation
Inoculating 3 fungus cakes (the fungus cakes with the diameter of 10mm and the thickness of 2mm are made by a puncher) into a 500mL triangular flask containing 200mL liquid culture medium, culturing for 7d in a constant-temperature incubator with the temperature of 28 ℃ and the temperature of 140r/min, centrifuging fermentation liquor 8000r/min for 10min, and precipitating obtained fermentation supernatant by using ammonium sulfate with the saturation of 80% to remove impurity proteins to obtain target protein precipitate. The precipitate was dissolved in 2-fold volume of 10mmol/L PBS (pH 6.0) buffer, centrifuged at 8000r/min for 10min at low temperature to remove insoluble contaminating proteins, and the supernatant was desalted overnight by dialysis against 10mmol/L PBS (pH 6.0) buffer to give a crude enzyme solution.
(2) DEAE Sepharose Fast Flow anion exchange chromatography
The crude enzyme solution was applied to a DEAE sepharose Fast Flow column (3.6 cm. times.40 cm) equilibrated with PBS, and the column was washed with 0.1 to 1mol/L at a Flow rate of 2mL/min and the eluate was collected in a collection amount of 2mL per tube. Measuring absorbance at 595nm and laccase activity at 420nm of the eluates, collecting peak components of combined enzyme activity, concentrating with PEG20000, and freeze-drying.
Trametes trogii (LCCC 50416) fermentation liquor is subjected to ammonium sulfate precipitation (80 percent), DEAE Sepharose Fast Flow anion exchange chromatography and PEG20000 concentration to obtain electrophoresis-grade pure enzyme, the specific activity of the pure enzyme is 110.85U/mg, the purification multiple is 12.88, and the enzyme activity recovery rate is 17.1 percent.
Example 2Application of Trametes trogii (Trametes trogii) LCCC50416 in tobacco leaf fermentation
In the tobacco leaf fermentation process, preferably, just before the tobacco leaf fermentation, Trametes trogii (LCCC 50416) is inoculated into the tobacco leaf, and the inoculation amount of Trametes trogii (LCCC 50416) is 105～107cfu/g, the fermentation temperature is 20-30 ℃, the humidity is 40-70%, and the time is 5-10 d.
Or adding fermentation supernatant enzyme solution or crude enzyme solution or pure enzyme of Trametes trogii LCCC50416 obtained in example 1 into tobacco leaves in the tobacco leaf fermentation process, preferably in the early stage of the tobacco leaf fermentation, so that the activity of laccase added into the tobacco leaves is 10-20U/mg, wherein the fermentation conditions are as follows: the temperature is 20-30 ℃, the humidity is 40-70%, and the time is 5-10 d.
The invention takes 2018 Chongqing B4F raw tobacco as a control group, and 10 is added6cfu/g Trametes trogii (Trametes trogii) LCCC50416 is used as a treatment group 1, or 15U/mg of prepared laccase enzyme powder is added as a treatment group 2 to ferment the tobacco leaves, the fermentation temperature is 28 ℃, the humidity is 55%, the time is 10d, the fermentation period of the tobacco leaves is obviously shortened, and the change of each chemical component in the tobacco leaves is shown in Table 1.
As shown in Table 1, the contents of chlorogenic acid, starch and pectin in tobacco leaves are reduced and the contents of total sugar and hyoscyami are increased after Trametes trogii (LCCC 50416) or laccase is added. The total sugar generates an acidic reaction to inhibit the alkalinity of alkaline substances in the smoke, and the acid-base balance of the smoke is moderate to reduce the irritation; saccharides are precursor materials for forming aroma substances; the proportion of the sugar and the alkali is coordinated to ensure that the smoke is mellow and mild, and the smoke has fragrance, taste and appropriate concentration strength.
TABLE 1 chemical composition of tobacco leaves in each treatment
The above description is only an example of the present application, and the protection scope of the present application is not limited by these specific examples, but is defined by the claims of the present application. Various modifications and changes may occur to those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the technical idea and principle of the present application should be included in the protection scope of the present application.
Claims (10)
1. Use of Trametes trogii (Trametes trogii) LCCC50416 for the production of laccase.
2. A method for producing laccase by using Trametes trogii LCCC50416 is characterized in that the method comprises the steps of inoculating Trametes trogii LCCC50416 in a liquid culture medium for fermentation culture, and centrifuging the fermentation liquid to obtain a supernatant.
3. The method of claim 2, wherein the amount of Trametes trogii LCCC50416 is 105～107cfu/ml, wherein the culture is shaking culture, the rotating speed is 100-200 r/min, the temperature is 20-30 ℃, and the time is 5-10 d;
preferably, the inoculum size is 106cfu/ml, the rotating speed is 140r/min, the temperature is 28 ℃, and the time is 7 d;
preferably, the rotating speed of the centrifugation is 5000-10000 rpm, and the time is 8-12 min;
preferably, the liquid medium contains: corn flour 30g/L, wheat bran 2.0g/L, KH2PO4 0.3g/L、MgSO40.15g/L, 1.0g/L glucose and pH of 4.5-5.5;
preferably, the pH is 5.0;
preferably, the Trametes pilosus (Trametes trogii) LCCC50416 is seed liquid of Trametes pilosus (Trametes trogii) LCCC 50416;
preferably, the seed liquid of Trametes trogii (Trametes trogii) LCCC50416 is obtained by inoculating activated Trametes trogii (Trametes trogii) LCCC50416 into seed liquid culture medium;
preferably, the seed liquid culture medium comprises: 2.0g of corn flour, 2.0g of bean cake powder,
(NH4)2SO40.01g、KH2PO4 0.2g、MgSO40.05g, VB10.05g and 100ml of distilled water.
4. The method of claim 2, further comprising the steps of adding ammonium sulfate to the supernatant for precipitation, and desalting by dialysis to obtain a crude enzyme solution.
5. The method according to claim 4, wherein the saturation degree of ammonium sulfate is 70-90%, preferably, the saturation degree of ammonium sulfate is 80%;
preferably, 8-12 mmol/L, pH 5.8.5.8-6.2 PBS buffer solution is used for dialysis desalination;
preferably, 10mmol/L, pH 6 PBS buffer is used.
6. The method of claim 4, further comprising the step of purifying the crude enzyme solution using a DEAE Sepharose Fast Flow anion exchange chromatography column.
7. The method of claim 6, wherein after the crude enzyme solution is loaded, the crude enzyme solution is eluted by using 0.1-1mol/L PBS buffer solution at a flow rate of 1.5-2.5 mL/min;
preferably, 0.5mol/L PBS buffer is used at a flow rate of 2 mL/min.
8. Use of Trametes trogii (LCCC 50416) or a method according to any of claims 2 to 7 in the fermentation of tobacco leaves.
9. A tobacco leaf fermentation method is characterized in that Trametes trogii (LCCC 50416) is inoculated into tobacco leaves for fermentation.
10. The tobacco leaf fermentation process of claim 9, wherein the inoculum size of Trametes trogii (LCCC 50416) is 105～107cfu/g, the fermentation temperature is 20-30 ℃, the humidity is 40-70%, and the time is 7-14 d.
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