CN109929778B - Efficient aroma-enhancing strain and application thereof in improving tobacco quality - Google Patents

Abstract

The invention provides a high-efficiency aroma-increasing strain and application thereof in improving the quality of tobacco, wherein the high-efficiency aroma-increasing strain is Bacillus sp.J3, and the preservation number is as follows: CCTCC NO: M2018707. The bacterial strain is obtained by separating the surface of Zimbabwe flue-cured tobacco for the first time, the amylase, catalase and protease activity of the bacterial strain are found to be very high, and the bacterial strain is also found to contain a plurality of sets of oxidative stress systems, high-activity amylase expression systems and a plurality of protease gene information through whole genome sequencing; besides, the strain also has a polyglutamic acid synthesis gene cluster, and can synthesize a large amount of polyglutamic acid in the extracellular space. The bacillus of the invention can shorten the time of artificially aging flue-cured tobacco and improve the fragrance and the smoking quality of the flue-cured tobacco on one hand, and can be used as a fertilizer synergist for tobacco planting to improve the planting quality of the tobacco from the aspect of tobacco planting on the other hand.

Description

Efficient aroma-enhancing strain and application thereof in improving tobacco quality

Technical Field

The invention relates to the field of microorganisms, in particular to a high-efficiency aroma-increasing strain and application thereof in improving the quality of tobacco.

Background

The planting of the flue-cured tobacco in China has a history of more than one hundred years, the flue-cured tobacco makes important contribution to the development of Chinese economy and society, and along with the improvement of the smoking quality of the tobacco by people, the planting and the production of the flue-cured tobacco also have a series of changes.

It is well known that the aging of tobacco leaves is an important process in the tobacco processing process. Because the tobacco leaves harvested in the current year have different defects in quality, such as coarse aroma, heavy soil impurity gas, high irritation, spicy taste and the like, the biochemical reaction of the tobacco leaves is promoted by an aging process, so that the defects are improved, and the requirements of the cigarette quality are finally met. The tobacco leaf aging process is a process for improving the tobacco leaf quality, microorganisms, enzymes and other chemical substances jointly participate in a series of reactions related to aroma substances in the tobacco leaf aging process, the activities of the microorganisms are continuous, and enzymatic action is derived from the plants or enzymes secreted by the microorganisms, so that the microorganisms are applied to the tobacco leaf aging process and are an important way for improving the tobacco leaf aroma and taste quality.

Many researchers have been focusing on and making some progress in studying the important roles of microorganisms and enzymes in the aging process, and they screened microorganisms from the surface of tobacco leaves and applied to artificial fermentation of the tobacco leaves. The results not only prove that a new bacillus has an efficient aroma-enhancing effect in the tobacco aging process, but also deeply analyze the advantages and molecular mechanism of the bacillus aroma production through whole genome sequencing and physiological and biochemical tests, excavate polyglutamic acid which can produce high yield under specific conditions, and the polyglutamic acid is used as a biopolymer flocculant to effectively improve the dissolution, storage, transportation and absorption of a fertilizer, balance the soil acidity and alkalinity, and can be used as a fertilizer synergist for tobacco planting, so that the bacillus has an outstanding effect in the tobacco aging process and also has potential value in the tobacco planting process.

Disclosure of Invention

Aiming at the problems of long tobacco leaf aging time, unobvious aroma enhancement effect, poor smoke quality, poor smoking effect and the like at present, the invention provides the efficient aroma enhancement type bacillus, the actual effect of tobacco leaf fermentation and aging is improved by utilizing the biochemical treatment characteristic of the microorganism, and the sensory quality of the tobacco leaf is improved.

The technical scheme provided by the invention is as follows: an efficient aroma-increasing strain is Bacillus sp.J3 with a preservation number: CCTCC NO: M2018707, deposited in China center for type culture Collection, address: wuhan university in China, preservation date: 2018-10-24, wherein the nucleotide sequence of the 16S rDNA is shown in a sequence table SEQ ID NO: 1 is shown.

Further, the efficient aroma-increasing strain is separated from the surface of Zimbabwe flue-cured tobacco leaves.

Further, the high-efficiency aroma-enhancing strain contains amylase activity, wherein an amino acid sequence of the alpha-amylase is shown in a sequence table SEQ ID NO: 2, respectively.

Furthermore, the high-efficiency aroma-enhancing strain contains protease activity, wherein 4 serine proteases exist, and the amino acid sequences of the serine proteases are shown in a sequence table SEQ ID NO: 3-6.

Furthermore, the efficient aroma-increasing strain has polyglutamic acid synthesis capacity, and polyglutamic acid synthase gene clusters comprise pgsE, pgsA, pgsC and pgsB, and amino acid sequences of the polyglutamic acid synthase gene clusters are respectively shown in a sequence table SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9. SEQ ID NO: shown at 10.

The application of the efficient aroma-increasing strain in improving the quality of tobacco is disclosed, wherein the efficient aroma-increasing strain is used in a tobacco leaf aging process to improve the smoking quality of tobacco.

An application of a high-efficiency aroma-enhancing strain in improving the quality of tobacco comprises the following specific steps: (1) taking out the strain from-80 ℃, streaking the strain on an LB solid culture medium for resuscitation, and then picking out a single clone and putting the single clone into a liquid LB culture medium for further activation; (2) adding the activated bacterial suspension into a fermentation medium according to the proportion of 1:100, and culturing for 16 hours at 37 ℃ and 220 rpm; (3) collecting thalli at 4 ℃ and 6000-8000rpm for 10 min; (4) resuspending the thalli by deionized water, preparing bacterial suspension, and spraying the bacterial suspension on about 50g of tobacco leaves; (5) aging the mixture at the temperature of 28-42 ℃ and the relative humidity of 40-80 percent for 3-7 days. The application of the efficient aroma-increasing strain is characterized in that the product polyglutamic acid of the efficient aroma-increasing strain is used as a fertilizer synergist for tobacco planting, so that the tobacco planting quality is improved.

The application of the efficient aroma-increasing strain is characterized in that the product polyglutamic acid of the efficient aroma-increasing strain is used as a fertilizer synergist for tobacco planting, so that the tobacco planting quality is improved.

The high-efficiency aroma-enhancing type strain Bacillus (Bacillus sp.J3) separating and screening method comprises the following steps: weighing about 1g of tobacco leaves, placing the tobacco leaves into 10ml of sterilized phosphate buffer solution, fully and uniformly mixing the tobacco leaves in an oscillator, gradually diluting the tobacco leaves to an appropriate concentration according to ten times of series, and taking 10^-2、10^-3、10^-4、10^-5、10^-6The five-concentration bacterial suspension is used as an object for coating plate culture bacteria, the object is coated on each plate culture medium, each plate has 3 gradients, 0.2mL of liquid is added into each plate, the plate is coated by a scraper, the plate is cultured in an incubator at 28-30 ℃ for 1-2 days, single clones which grow well and are full of colonies are picked out, and the single clones are streaked again, separated and purified.

Re-screening an amylase identification culture medium: and (3) the separated bacterial strain is dripped on the culture medium again, the culture medium is put into an incubator at the temperature of 28-30 ℃ for 24 hours, the bacterial growth condition is observed, the cover of the flat plate is opened, a small amount of iodine solution is dripped into the flat dish, the flat dish is slightly rotated, the iodine solution is fully paved on the flat plate, and the amylase activity is judged according to the size of the formed transparent ring.

Re-screening the protease identification culture medium: and (3) inoculating the separated strain onto the culture medium again, putting the culture medium into an incubator at 28-30 ℃ for 24 hours, observing the growth condition of bacteria, opening a flat plate cover, observing the size of a proteolytic loop, and judging the protease activity.

Identification of polyglutamic acid-producing strains: and (3) the separated bacterial strain is spotted on the culture medium again, the bacterial colony growth condition is observed after the bacterial strain is put into an incubator at 28-30 ℃ for culturing for 24 hours, the bacterial colony is picked up by a sterilized toothpick, and if the bacterial colony has high viscosity, the bacterial colony can be drawn, the drawing degree is stronger, and the capability of producing polyglutamic acid is stronger.

Luria-Bertani (LB) Medium composition (g/L): 10g tryptone, 5g yeast extract, 5g sodium chloride, 15g agar, and autoclaving at 121 ℃ for 20 minutes.

Zoom medium composition (g/L): 5g of yeast extract, 5g of acid hydrolyzed casein, 2g of potassium dihydrogen phosphate, 1g of potassium dihydrogen phosphate and 15g of agar are sterilized at the high temperature and the high pressure of 121 ℃ for 20 minutes and are used for separating microorganisms in tobacco leaves.

R2A Medium composition (g/L): 0.5g of yeast extract, 0.5g of trypsin, 0.5g of enzymatically hydrolyzed casein, 0.5g of starch, 0.5g of glucose, 0.3 g of sodium pyruvate, 0.3 g of dipotassium hydrogen phosphate, 0.024 g of magnesium sulfate, 15g of agar, and autoclaving at 121 ℃ for 20 minutes for separation of microorganisms in tobacco leaves.

Amylase Activity identification Medium composition (g/L): 10g of peptone, 5g of sodium chloride, 5g of beef extract, 2g of soluble starch, 15g of agar and autoclaving at 121 ℃ for 20 minutes.

Protease Activity identification Medium composition (g/L): 5g of beef extract, 10g of peptone, 5g of sodium chloride, 30g of skimmed milk powder, pH 7.2-7.4, and autoclaving at 115 ℃ for 20 minutes.

The components (g/L) of a solid culture medium for producing polyglutamic acid: 10g of citric acid, 10g of glutamic acid, 6g of ammonium chloride, 1g of dipotassium phosphate, 0.5g of magnesium sulfate, 0.02g of ferric chloride, 0.2g of calcium chloride, 0.05g of manganese sulfate, 15g of agar, pH 7.2, and autoclaving at 121 ℃ for 20 minutes.

Methods for measuring alpha-amylase and protease activities:

100ml of the cells were collected, and the collected bacterial solution was centrifuged at 4 ℃ and low temperature, washed once with PBS, suspended, disrupted with an ultrasonication apparatus (SCIENTZ-IID, Ningbo xingzhi biotechnology co., China), and centrifuged at 4 ℃ to collect supernatant. Then standing at normal temperature for 15min to obtain crude enzyme solution. Then the alpha-amylase activity was determined using the alpha-amylase activity assay kit from Solarbio. Alkaline protease activity was also determined using the alkaline protease activity detection kit of Solarbio.

The strain with higher amylase activity, protease activity and catalase activity is selected for further 16S rDNA sequence identification, the strain is found to be Bacillus (Bacillus sp.J3), and the 16S rDNA nucleotide sequence of the strain is shown as a sequence table SEQ ID NO: 1 is shown.

The cellular morphological characteristics and physiological and biochemical characteristics of the Bacillus (Bacillus sp.j3) strain are: gram-positive, rod-shaped, with a size of 0.5-0.8um 4um-7um, peritrichous, motile, without capsule, and the observation under 1000 times of oil microscope is shown in figure 1. The optimum growth temperature is 28-42 ℃, the pH value is 5.0-9.0, and the tolerance of sodium chloride is 1% -8%.

The high-efficiency aroma-increasing type Bacillus (Bacillus sp.J3) obtained by the invention is not only an excellent high-efficiency aroma-increasing type bacterial strain, but also has potential fertilizer synergistic function in the aspect of flue-cured tobacco planting. The result of smoke panel test shows that the aroma quality and the aroma quantity of the tobacco leaves treated by Bacillus sp.J3 are obviously improved, the miscellaneous gas and the irritation are reduced, and the aging time is greatly shortened.

Drawings

Fig. 1 is a micrograph (magnification 1000 times) of Bacillus (Bacillus sp.j3).

Fig. 2 is a diagram of extracted Bacillus (Bacillus sp.j3) whole genome DNA glue.

Fig. 3 shows the distribution of different species of functional genes in the annotation results of the Bacillus (Bacillus sp.j3) whole genome.

Fig. 4 shows a polyglutamic acid synthesis gene cluster of Bacillus (Bacillus sp.j3).

FIG. 5 is a graph showing the identification of the hydrogen peroxide resistance of different strains.

FIG. 6 is a transparent circle formed on an amylase activity assay medium plate and a protease activity assay medium plate of strains separated from tobacco.

FIG. 7 is the preparation of a standard curve for glucose in the alpha-amylase activity assay.

FIG. 8 is a graph of alpha-amylase activity assay.

FIG. 9 shows the measurement of alkaline protease activity.

Fig. 10 is a result analysis of a biologies test of Bacillus (Bacillus sp.j3), which shows the test of utilization of different carbon sources by the strain.

Fig. 11 is a prediction of the alpha-amylase signal peptide in Bacillus (Bacillus sp.j3).

Fig. 12 is a prediction of an alkaline protease signal peptide in Bacillus (Bacillus sp.j3).

Detailed Description

Separation and culture of microorganisms on flue-cured tobacco leaves

Cutting tobacco leaves naturally stored in different places (Yunnan, Sichuan, etc.) under aseptic condition, weighing about 1g, placing in 10ml sterilized phosphate buffer solution, mixing in oscillator, gradually diluting to appropriate concentration according to ten times series, and taking 10^-2、10^-3、10^-4、10^-5、10^-6The five-concentration bacterial suspension is used as an object for coating plate culture bacteria, the object is coated on each plate culture medium, each plate has 3 gradients, 0.2mL of liquid is added into each plate, the plate is coated by a scraper, the plate is cultured in an incubator at 28-30 ℃ for 1-2 days, single clones which grow well and are full of colonies are picked out, and the single clones are streaked again, separated and purified.

After primary screening, 39 well-growing strains are respectively obtained from the tobacco leaves of Yunnan flue-cured tobacco, Sichuan flue-cured tobacco and Zimbabwe flue-cured tobacco, wherein 9 strains are obtained from the Yunnan flue-cured tobacco, 12 strains are obtained from the Sichuan flue-cured tobacco and 18 strains are obtained from the Zimbabwe flue-cured tobacco.

Identification of microbial catalase, amylase and protease activities

Numbering the 39 strains, respectively inoculating into LB culture medium for resuscitation, coating bacterial liquid on LB solid culture medium, placing sterilized filter paper sheet of about 0.5cm into LB solid culture medium coated with bacterial liquid, and dripping about 1ul H₂O₂After incubation for 16-24h on filter paper sheets, the transparent circles around the filter paper were observed and J3 was found to have the strongest resistance to hydrogen peroxide (see FIG. 5).

On the other hand, the inoculating loop was spotted on the amylase-identifying medium and protease-identifying medium plates, respectively, after culturing for 16 to 24 hours, a small amount of iodine solution was dropped on the amylase-identifying medium plate into the plate, the plate was gently rotated to allow the iodine solution to spread over the plate, formation of a transparent circle was found (see FIG. 6), and the ratio of the transparent circle to the colony diameter was calculated (see Table 1). In addition, the hydrolysis circle of the protease identification medium plate was observed, and it was found that a type clear circle was also formed, and the ratio of the hydrolysis circle to the colony diameter was calculated (see Table 2). Finally obtaining 3-4 strains with high-efficiency enzyme activity, and in order to further verify the enzyme activities of alpha-amylase and alkaline protease, determining the enzyme activities of the strains by using an alpha-amylase activity determination kit and an alkaline protease activity determination kit respectively, and finding that the alpha-amylase activity and the alkaline protease activity of the J3 strain are the highest (as shown in figures 8 and 9).

TABLE 1 Amylase hydrolysis Ring vs. colony diameter ratio

TABLE 2 ratio of protease hydrolysis circles to colony diameters

Characteristics of Bacillus (Bacillus sp.J3) for producing polyglutamic acid

The separated bacterial strain is applied to the culture medium again, the culture medium is placed into an incubator at the temperature of 28-30 ℃ for 24 hours, the growth condition of bacterial colonies is observed, the bacterial colonies are picked up by using sterilized toothpicks, the viscosity of the bacterial colonies of the J3 bacterial strain is found to be the maximum, the bacterial colonies can be drawn, then the fermentation liquor is centrifuged at 12000rpm for 20 minutes at the temperature of 4 ℃, the supernatant is taken, 4 times of volume of absolute ethyl alcohol is added, precipitates are separated out, and the bacterial strain is preliminarily identified as polyglutamic acid.

Analysis of cell morphological characteristics and physiological and biochemical characteristics of Bacillus (Bacillus sp.J3)

We analyzed the cytomorphological characteristics and the physiobiochemical characteristics of the Bacillus (Bacillus sp.j3) strain by using microscopy, biologics analysis and biochemical tests:

bacillus (Bacillus sp.j3) was found to be gram positive, rod-shaped, 0.5-0.8um 4um-7um in size, peritrichous, motile, without capsule, and observed under 1000 fold oil lens as in fig. 1. The optimum growth temperature is 28-42 ℃, the pH value is 5.0-9.0, the tolerance of sodium chloride is 1% -8%, and the nitrate reductase test, the starch hydrolysis test, the gelatin liquefaction test, the methyl red test, the catalase test and the citrate utilization test are all positive.

The results of the biologies assay are shown simultaneously (fig. 10): the strain can utilize various saccharide substances, such as D-glucose, D-mannose, D-fructose, D-galactose, L-rhamnose, dextrin, D-maltose, D-trehalose, D-cellobiose, gentiobiose, sucrose, pindusan, D-raffinose, D, melibiose, D-salicin, N-acetyl-D-glucoside, sodium lactate and the like, can also utilize various amino acids, such as L-alanine, L-arginine, L-aspartic acid, L-glutamic acid and L-histidine, and can also utilize methyl pyruvate, L-lactic acid, citric acid, L-malic acid, bromosuccinic acid, acetic acid, formic acid, sodium butyrate, lithium chloride and potassium tellurite.

Amplification and determination of 16S rDNA nucleotide sequence of Bacillus sp.J3

The specific operation is as follows:

firstly, 1ml of cultured bacterial liquid is taken to be put into a centrifugal tube of 1.5ml, centrifuged for 1min at 1200rpm, and thalli are collected; adding 500 mu L of lysis buffer solution, continuously blowing or shaking for uniformly mixing, and adding lysozyme; adding 300 mu L of 3mol/L NaCl, mixing uniformly, and centrifuging at 1200rpm for 10 min; transferring the supernatant to another centrifuge tube, adding 5 μ l RNase (1mg/ml), and placing in 37 deg.C incubator for 30 min; adding phenol/chloroform (1:1v/v) with the same volume for extraction once; centrifuging in a centrifuge at 4 deg.C for 10min, collecting supernatant, and extracting with chloroform of the same volume until no intermediate layer exists; centrifuging at 12000rpm for 10min in a 4 deg.C centrifuge, and sucking the upper layer into a new EP centrifuge tube; after precipitating DNA by adding two volumes of absolute ethanol, the precipitate was washed with ice-cold 70% ethanol 1 time, dried and dissolved in 50. mu.l of deionized water. Mu.l of the gel was run on a 1% agarose gel and the purity and concentration were checked (see FIG. 2).

Using the extracted whole genome DNA as a template, 16S rRNA universal primers (forward F primer: 5'-AGAGTTTGATCCTGGCTCAG-3' and reverse R primer: 5'-GGTTACCTTGTTACGACTT-3') were used for PCR amplification, the PCR system was: 2 MIX: 25ul, deionized water: 22ul, F primer: 1ul, R primer: 1ul, DNA template: 1 ul. The PCR amplification conditions were: 5 minutes at 95 ℃, 30 seconds at 55 ℃, 45 seconds at 72 ℃, 30 cycles, 10 minutes at 72 ℃. The PCR length is 1544bp, the PCR product is cut and recovered and sent to a sequencing company for sequencing, and the comparison result shows that: the similarity of the nucleotide sequence of the mutant strain IAM 12118 to Bacillus subtilis strain IAM 12118 in NCBI database is 99%, and the nucleotide sequence is shown in a sequence table SEQ ID NO: 1.

SEQ ID NO：1

AGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTTTGAACCGCATGGTTCAAACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTACCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTAGGAGCCAGCCGCCGAAGGTGGGACAGATGATTGGGGTGAAGTCGTAACAAGGTAGCCGTATCGGAAGGTGCGGCTGGATCACCTCCTTTCTA

whole genome sequencing and analysis of Bacillus (Bacillus sp.j3)

We used the extracted genomic DNA with NanoDrop^TMThe 8000 spectrophotometer measures the purity of the sample, and the result shows that OD260/280 is 1.96, the DNA band is single, no impurity band is generated, and no impurities such as RNA, protein and the like are polluted; the concentration is 276ng/ul, the total amount is 13.8ug, and the requirement of bacterial genome sequencing is completely met. We sent to Beijing Nuo He genesis bioinformation science and technology Limited company to build a library for sequencing, the sequencing result is spliced and assembled by a SPAdes Genome Assembler on a Linux system,the assembled results were subjected to genome-wide annotation using rasannotation Server, and the functional gene annotation results in the genome of Bacillus (Bacillus sp.j3) strain are shown in fig. 3. The annotation results contained a total of 4195 sequences of encoded proteins, which were functionally classified into the following categories: cofactor, vitamin, prosthetic and pigment related (212, 5.05%), cell wall and capsule formation related (150, 3.57%), toxin, infection and protection related (71, 3.11%), potassium ion metabolism related (15, 0.35%), multiple function related (49, 1.16%), phage, prophage, transposable element and plasmid related (11, 0.26%), membrane transport related (76, 1.81%), iron ion uptake related to metabolic species (28, 0.67%), RNA metabolism related (165, 3.93%), nucleoside related to nucleotide (125, 2.98%), protein metabolism related (230, 5.48%), cell division cycle related (54, 1.29%), motility and chemotaxis related (88, 2.10%), cell regulation and signal transduction related (61, 1.45%), secondary metabolism related (4, 0.10%), DNA metabolism related (115, 2.74%), fatty acid, Adipose and isoprenoid related (116, 2.77%), nitrogen metabolism related (31, 0.74%), dormancy and sporulation related (121, 2.88%), respiration related (68, 1.62%), stress related (109, 2.60%), aromatic metabolism related (12, 0.29%), amino acids and their derivatives related (434, 10.35%), sulfur metabolism related (49, 1.17%), phosphorus metabolism related (34, 0.81%), carbohydrate related (524, 12.49%), unknown function (1243, 29.63%).

From the annotated results, we found that the strain contains multiple sets of Oxidative stress systems, 8 candidate genes for Protection from Reactive Oxygen Species, 24 candidate genes for Oxidative stress, 3 candidate genes for Non-Redox reactions, 6 candidate genes for Redox regulation of nuclear processes, 3 candidate genes for Glutathione, and 2 candidate genes for Cluster connecting Glutathione synthase. These genes play an important role in the process of bacterial strains resisting oxidative stress inside and outside cells.

We also found the amino acid sequence of α -amylase in this annotation, as shown in SEQ ID NO: 2, respectively. SignalP 4.1 predicts that the signal peptide of the protein has a signal peptide and a cleavage site between amino acids A33 and E34 (as shown in FIG. 11), and the results of the alpha-amylase activity test prove that the strain has high activity of alpha-amylase activity and the alpha-amylase is secreted to the outside of cells. Meanwhile, the amino acid sequences of a plurality of proteases are also found, wherein 4 serine proteases are provided, and the amino acid sequences of the proteases are shown in a sequence table SEQ ID NO: 3-6. Meanwhile, the signal peptide of SignalP 4.1 is predicted to show (as shown in figure 12), wherein one alkaline protease also has the signal peptide, and a cleavage site is arranged between amino acids A29 and A30, and the combination of the alkaline protease activity test results proves that the strain has high-activity protease activity and is secreted to the outside. The amylase and the protease can degrade starch and protein in the tobacco leaves, if the content of the starch and the protein in the tobacco leaves is too high, burnt flavor can be generated in the burning process, excessive harmful substances can be formed, and the quality and the safety of the tobacco leaf flavor are seriously influenced. Therefore, the Bacillus (Bacillus sp.J3) has the enzyme activity of efficiently degrading starch and protein, and branched chain lower fatty acid contained in the Bacillus can increase the fragrance of tobacco leaves.

SEQ ID NO：2

The amino acid sequence of alpha-amylase:

Alpha-amylase：

MFAKRFKTSLLPLFAGFLLLFHLVLAGPAAASAETANKSNELTAPSIKSGTILHAWNWSFNTLKHNMKDIHDAGYTAIQTSPINQVKEGNQGDKSMSNWYWLYQPTSYQIGNRYLGTEQEFKEMCAAAEEYGIKVIVDAVINHTTSDYAAISNEVKSIPNWTHGNTQIKNWSDRWDVTQNSLLGLYDWNTQNTQVQSYLKRFLERALNDGADGFRFDAAKHIELPDDGSYGSQFWPNITNTSAEFQYGEILQDSASRDAAYANYMDVTASNYGHSIRSALKNRNLGVSNISHYASDVSADKLVTWVESHDTYANDDEESTWMSDDDIRLGWAVIASRSGSTPLFFSRPEGGGNGVRFPGKSQIGDRGSALFEDQAITAVNRFHNVMAGQPEELSNPNGNNQIFMNQRGSHGVVLANAGSSSVSINTATKLPDGRYDNKAGAGSFQVNDGKLTGTINARSVAVLYPDDIAKAPHVFLENYKTGVTHSFNDQLTITLRADANTTKAVYQINNGPETAFKDGDQFTIGKGDPFGKTYTIMLKGTNSDGVTRTEEYSFVKRDPASAKTIGYQNPNHWSQVNAYIYKHDGGQAIELTGSWPGKPMTKNADGIYTLTLPADTDTTNAKVIFNNGSAQVPGQNQPGFDYVQNGLYNDSGLSGSLPY

four serine protease amino acid sequences

SEQ ID NO：3

MRSKKLWSSLLFALTLIFTMAFSNMSAQAAGKSSTEKKYIVGFKQTMSAMSSAKKKDVISEKGGKVQKQFKYVNAAAATLDEKAVKELKKDPSVAYVEEDHIAHEYAQSVPYGISQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLNVRGGASFVPSETNPYQDGSSHGTHVAGTIAALNNSIGVLGVAPSASLYAVKVLDSTGSGQYSWIINGIEWAISNNMDVINMSLGGPTGSTALKTVVDKAVSSGIVVAAAAGNEGSSGSSSTVGYPAKYPSTIAVGAVNSSNQRASFSSAGSELDVMAPGVSIQSTLPGGTYGAYNGTSMATPHVAGAAALILSKHPTWTNAQVRDRLESTATYLGNSFYYGKGLINVQAAAQ

SEQ ID NO：4

MFGYSMVQMVRANAHKLDWPLRETVLQLYKPFKWTPCFLHKFFETKLQNRKKMSVIIEFEEGCHETGFQMAGEVLQKEKRSKLKSRFNKINCCSAEVTPSALHSLLSECSNIRKVYLNREVKALLDTATEASHAKEVVRNRQTLTGKGVTVAVVDTGIYPHPDLEGRIIGFADMVNQKTEPYDDNGHGTHCAGDVASSGASSSGQYRGPAPEANLIGVKVLNKQGSGTLADIIEGVEWCIQYNEDNPDEPIDIISMSLGGDALRYDHEQEDPLVRAVEEAWSAGIVVCVAAGNSGPDSQTIASPGVSEKVITVGALDDNNTASSDDDTVASFSSRGPTVYGKEKPDILAPGVNIISLRSPNSYIDKLQKSSRVGSQYFTMSGTSMATPICAGIAALILQQNPDLTPDEVKELLKNGTDKWKDEDPNVYGAGAVNAENSVPGQ

SEQ ID NO：5

MKKGIIRFLLVSFVLFFALSTGITGVQAAPASSKTSADLEKAEVFGDIDMTTSKKTTVIVELKEKSLAEAKEAGESQSKSKLKTARTKAKNKAIKAVKNGKVNREYEQVFSGFSMKLPANEIPKLLAVKDVKAVYPNVTYKTDNMKDKDVTISEDAVSPQMDDSAPYIGANDAWDLGYTGKGIKVAIIDTGVEYNHPDLKKNFGQYKGYDFVDNDYDPKETPTGDPRGGATDHGTHVAGTVAANGTIKGVAPDATLLAYRVLGPGGSGTTENVIAGVERAVQDGADVMNLSLGNSLNNPDWATSTALDWAMSEGVVAVTSNGNSGPNGWTVGSPGTSREAISVGATQLPLNEYAVTFGSYSSAKVMGYNKEDDVKALNNKEIELVEAGIGEEKDFEGKDLTGKVAVVKRGSIAFVDKADNAKKAGAIGMVVYNNLSGEIEANVPGMSVPTIKLSLEDGEKLVSALKAGETKTTFKLTVSKALGEQVADFSSRGPVMDTWMIKPDISAPGVNIVSTIPTHDPDHPYGYGSKQGTSMASPHIAGAVAVIKQAKPKWSVEQIKAAIMNTAVTLKDSDGEVYPHNAQGAGSARIMNAIKADSLVSPGSYSYGTFLKENGNETKNETFTIENQSSIRKSYTLEYSFNGSGISTSGTSRVVIPAHQTGKATAKVKVNTKKTKAGTYEGTVIVREGGKTVAKVPTLLIVKEPDYPRVTSVSVSEGSVQGTYQIETYLPAGAEELAFLVYDSNLDFAGQAGIYKNQDKGYQYFDWDGTINGGTKLPAGEYYLLAYAANKGKSSQVLTEEPFTVE

SEQ ID NO：6

MFLLEYTYWKIAAHLVNNGYGVIQAGESDEIWLEAPDKSSHDLVRLYKHDLDFRQEMVRDIEEQAERVERVRHQLVRRRMKLLNVFFSTEAPVDDWEEIAKKTFEKGTVSVEPAIVRGTMLRDDLQAVFPSFRTEDCTEEHASFEDAQMARERFLSLVLKQEEQRKTEAAVFQNGKPIFTYLFIALQILMFFLLEINGGSTNTETLVAFGAKENSLIAAGEWWRLLTPIVLHIGIAHLAFNTLALWSVGTAVERMYGSGRFLLIYLAAGITGSIASFVFSPYPSAGASGAIFGCLGALLYVALSNRKMFLRTIGTNIIVIIIINLGFGFAVSNIDNSGHIGGLIGGFFAAAALGLPKAGAFGKRLLSAVLLIALAVGFLYYGLHSPSHQESALIQQASELYQEGKYEEVTELLNGEAAQKDASADLLKILAVSDIQIGEYDQAVFHLERAVKKEPKDHAAYYYLALLYAEKNELVQAEKAIRLALKLEPKEQRYKELQRQIENNKES

We have also found a gene cluster that produces a stringy viscous strand (polyglutamic acid), pgsacb, as shown in fig. 4, having an amino acid sequence as set forth in SEQ ID NO: 7-10. Experiments prove that the polyglutamic acid is well applied to the planting process of the flue-cured tobacco as a fertilizer synergist, and the results show that the yield, the medium and medium grade tobacco yield and the yield value of the tobacco are obviously improved by applying the tobacco fertilizer with different concentrations of polyglutamic acid, wherein the special fertilizer for the tobacco with the addition of 0.1 percent of the polyglutamic acid has the best effect, and the yield of the flue-cured tobacco is improved by 18.57 percent.

Polyglutamic acid synthetase gene cluster

SEQ ID NO：7

MKFVKAIWPFVAVAIVFMFMSAFKFNDQLTDQEKQKIDMEMNKIQQQEEPVNANK

SEQ ID NO：8

MKKELSFHEKLLKLTKQQKKKTNKHVFIAIPIVFVLMFAFMWAGKAETPKVKTYSDDVLSASFVGDIMMGRYVEKVTEQKGADSIFQYVEPIFRASDYVAGNFENPVTYQKNYKQADKEIHLQTNKESVKVLKDMNFTVLNSANNHAMDYGVQGMKDTLGEFAKQNLDIVGAGYSLSDAKKKISYQKVNGVTIATLGFTDVSGKGFAAKKNTPGVLPADPEIFIPMISEAKKHADIVVVQSHWGQEYDNDPNDRQRQLARAMSDAGADIIVGHHPHVLEPIEVYNGTVIFYSLGNFVFDQGWTRTRDSALVQYHLKKNGTGRFEVTPIDIHEATPAPVKKDSLKQKTIIRELTKDSNFAWKVEDGKLTFDIDHSDKLKSK

SEQ ID NO：9

MFGSDLYIALILGVLLSLIFAEKTGIVPAGLVVPGYLGLVFNQPVFILLVLLVSLLTYVIVKYGLSKFMILYGRRKFAAMLITGIVLKIAFDFLYPIVPFEIAEFRGIGIIVPGLIANTIQKQGLTITFGSTLLLSGATFAIMFVYYLI

SEQ ID NO：10

MWLLIIACAVILVIGILEKRRHQKNIDALPVRVNINGIRGKSTVTRLTTGILIEAGYKTVGKTTGTDARMIYWDTPEEKPIKRKPQGPNIGEQKEVMRETVERGANAIVSECMAVNPDYQIIFQEELLQANIGVIVNVLEDHMDVMGPTLDEIAEAFTATIPYNGHLVITDSEYTEFFKQKAKERNTKVIIADNSKITDEYLRKFEYMVFPDNASLALGVAQALGIDEETAFKGMLNAPPDPGAMRILPLISPSEPGHFVNGFAANDASSTLNIWKRVKEIGYPTDDPIIIMNCRADRVDRTQQFANDVLPYIEASELILIGETTEPIVKAYEEGKIPADKLHDLEYKSTDEIMELLKKRMHNRVIYGVGNIHGAAEPLIEKIHEYKVKQLVS

Fermentation culture of Bacillus (Bacillus sp.J3)

Firstly, taking out a Bacillus (Bacillus sp.J3) strain from minus 80 ℃, streaking the strain on an LB solid culture medium for resuscitation, then picking out a single clone, putting the single clone into 5ml of liquid LB culture medium for further activation, and finally adding the activated bacterial suspension into a fermentation culture medium according to the proportion of 1:100 for shake flask fermentation culture. The culture conditions are as follows: the temperature was 37 ℃ and the rotational speed 220rpm for 16 hours.

Artificial aging test of Bacillus (Bacillus sp.J3) on tobacco leaves

Taking the fermentation liquor, and collecting thalli at 6000-8000rpm for 10min at the temperature of 4 ℃; resuspending the thalli by deionized water, preparing bacterial suspension, spraying the bacterial suspension on about 50g of tobacco leaves, and using the same amount of deionized water as a reference; the comparison and the treatment are respectively carried out at the temperature of 28-42 ℃ and the relative humidity of 40-80 percent, and the aging time is 3-7 days. The tobacco leaves are dried for 6 to 12 hours at the temperature of between 60 and 90 ℃ after being aged, and then made into tobacco shreds which are evaluated in a sensory mode. The results of the smoke panel test are shown in Table 3, and the aged tobacco leaves have richer fragrance.

TABLE 3 evaluation of sensory Effect

The above description is only an embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention.

Sequence listing

<110> tobacco industry Limited liability company in Hubei

<120> high-efficiency aroma-enhancing bacterial strain and application thereof in improving tobacco quality

<160> 10

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1544

<212> DNA

<213> Bacillus (Bacillus sp. J3)

<400> 1

agtttgatcc tggctcagga cgaacgctgg cggcgtgcct aatacatgca agtcgagcgg 60

acagatggga gcttgctccc tgatgttagc ggcggacggg tgagtaacac gtgggtaacc 120

tgcctgtaag actgggataa ctccgggaaa ccggggctaa taccggatgg ttgtttgaac 180

cgcatggttc aaacataaaa ggtggcttcg gctaccactt acagatggac ccgcggcgca 240

ttagctagtt ggtgaggtaa tggctcacca aggcaacgat gcgtagccga cctgagaggg 300

tgatcggcca cactgggact gagacacggc ccagactcct acgggaggca gcagtaggga 360

atcttccgca atggacgaaa gtctgacgga gcaacgccgc gtgagtgatg aaggttttcg 420

gatcgtaaag ctctgttgtt agggaagaac aagtaccgtt cgaatagggc ggtaccttga 480

cggtacctaa ccagaaagcc acggctaact acgtgccagc agccgcggta atacgtaggt 540

ggcaagcgtt gtccggaatt attgggcgta aagggctcgc aggcggtttc ttaagtctga 600

tgtgaaagcc cccggctcaa ccggggaggg tcattggaaa ctggggaact tgagtgcaga 660

agaggagagt ggaattccac gtgtagcggt gaaatgcgta gagatgtgga ggaacaccag 720

tggcgaaggc gactctctgg tctgtaactg acgctgagga gcgaaagcgt ggggagcgaa 780

caggattaga taccctggta gtccacgccg taaacgatga gtgctaagtg ttagggggtt 840

tccgcccctt agtgctgcag ctaacgcatt aagcactccg cctggggagt acggtcgcaa 900

gactgaaact caaaggaatt gacgggggcc cgcacaagcg gtggagcatg tggtttaatt 960

cgaagcaacg cgaagaacct taccaggtct tgacatcctc tgacaatcct agagatagga 1020

cgtccccttc gggggcagag tgacaggtgg tgcatggttg tcgtcagctc gtgtcgtgag 1080

atgttgggtt aagtcccgca acgagcgcaa cccttgatct tagttgccag cattcagttg 1140

ggcactctaa ggtgactgcc ggtgacaaac cggaggaagg tggggatgac gtcaaatcat 1200

catgcccctt atgacctggg ctacacacgt gctacaatgg acagaacaaa gggcagcgaa 1260

accgcgaggt taagccaatc ccacaaatct gttctcagtt cggatcgcag tctgcaactc 1320

gactgcgtga agctggaatc gctagtaatc gcggatcagc atgccgcggt gaatacgttc 1380

ccgggccttg tacacaccgc ccgtcacacc acgagagttt gtaacacccg aagtcggtga 1440

ggtaaccttt taggagccag ccgccgaagg tgggacagat gattggggtg aagtcgtaac 1500

aaggtagccg tatcggaagg tgcggctgga tcacctcctt tcta 1544

<210> 2

<211> 659

<212> PRT

<213> Bacillus (Bacillus sp. J3)

<400> 2

Met Phe Ala Lys Arg Phe Lys Thr Ser Leu Leu Pro Leu Phe Ala Gly

1 5 10 15

Phe Leu Leu Leu Phe His Leu Val Leu Ala Gly Pro Ala Ala Ala Ser

20 25 30

Ala Glu Thr Ala Asn Lys Ser Asn Glu Leu Thr Ala Pro Ser Ile Lys

35 40 45

Ser Gly Thr Ile Leu His Ala Trp Asn Trp Ser Phe Asn Thr Leu Lys

50 55 60

His Asn Met Lys Asp Ile His Asp Ala Gly Tyr Thr Ala Ile Gln Thr

65 70 75 80

Ser Pro Ile Asn Gln Val Lys Glu Gly Asn Gln Gly Asp Lys Ser Met

85 90 95

Ser Asn Trp Tyr Trp Leu Tyr Gln Pro Thr Ser Tyr Gln Ile Gly Asn

100 105 110

Arg Tyr Leu Gly Thr Glu Gln Glu Phe Lys Glu Met Cys Ala Ala Ala

115 120 125

Glu Glu Tyr Gly Ile Lys Val Ile Val Asp Ala Val Ile Asn His Thr

130 135 140

Thr Ser Asp Tyr Ala Ala Ile Ser Asn Glu Val Lys Ser Ile Pro Asn

145 150 155 160

Trp Thr His Gly Asn Thr Gln Ile Lys Asn Trp Ser Asp Arg Trp Asp

165 170 175

Val Thr Gln Asn Ser Leu Leu Gly Leu Tyr Asp Trp Asn Thr Gln Asn

180 185 190

Thr Gln Val Gln Ser Tyr Leu Lys Arg Phe Leu Glu Arg Ala Leu Asn

195 200 205

Asp Gly Ala Asp Gly Phe Arg Phe Asp Ala Ala Lys His Ile Glu Leu

210 215 220

Pro Asp Asp Gly Ser Tyr Gly Ser Gln Phe Trp Pro Asn Ile Thr Asn

225 230 235 240

Thr Ser Ala Glu Phe Gln Tyr Gly Glu Ile Leu Gln Asp Ser Ala Ser

245 250 255

Arg Asp Ala Ala Tyr Ala Asn Tyr Met Asp Val Thr Ala Ser Asn Tyr

260 265 270

Gly His Ser Ile Arg Ser Ala Leu Lys Asn Arg Asn Leu Gly Val Ser

275 280 285

Asn Ile Ser His Tyr Ala Ser Asp Val Ser Ala Asp Lys Leu Val Thr

290 295 300

Trp Val Glu Ser His Asp Thr Tyr Ala Asn Asp Asp Glu Glu Ser Thr

305 310 315 320

Trp Met Ser Asp Asp Asp Ile Arg Leu Gly Trp Ala Val Ile Ala Ser

325 330 335

Arg Ser Gly Ser Thr Pro Leu Phe Phe Ser Arg Pro Glu Gly Gly Gly

340 345 350

Asn Gly Val Arg Phe Pro Gly Lys Ser Gln Ile Gly Asp Arg Gly Ser

355 360 365

Ala Leu Phe Glu Asp Gln Ala Ile Thr Ala Val Asn Arg Phe His Asn

370 375 380

Val Met Ala Gly Gln Pro Glu Glu Leu Ser Asn Pro Asn Gly Asn Asn

385 390 395 400

Gln Ile Phe Met Asn Gln Arg Gly Ser His Gly Val Val Leu Ala Asn

405 410 415

Ala Gly Ser Ser Ser Val Ser Ile Asn Thr Ala Thr Lys Leu Pro Asp

420 425 430

Gly Arg Tyr Asp Asn Lys Ala Gly Ala Gly Ser Phe Gln Val Asn Asp

435 440 445

Gly Lys Leu Thr Gly Thr Ile Asn Ala Arg Ser Val Ala Val Leu Tyr

450 455 460

Pro Asp Asp Ile Ala Lys Ala Pro His Val Phe Leu Glu Asn Tyr Lys

465 470 475 480

Thr Gly Val Thr His Ser Phe Asn Asp Gln Leu Thr Ile Thr Leu Arg

485 490 495

Ala Asp Ala Asn Thr Thr Lys Ala Val Tyr Gln Ile Asn Asn Gly Pro

500 505 510

Glu Thr Ala Phe Lys Asp Gly Asp Gln Phe Thr Ile Gly Lys Gly Asp

515 520 525

Pro Phe Gly Lys Thr Tyr Thr Ile Met Leu Lys Gly Thr Asn Ser Asp

530 535 540

Gly Val Thr Arg Thr Glu Glu Tyr Ser Phe Val Lys Arg Asp Pro Ala

545 550 555 560

Ser Ala Lys Thr Ile Gly Tyr Gln Asn Pro Asn His Trp Ser Gln Val

565 570 575

Asn Ala Tyr Ile Tyr Lys His Asp Gly Gly Gln Ala Ile Glu Leu Thr

580 585 590

Gly Ser Trp Pro Gly Lys Pro Met Thr Lys Asn Ala Asp Gly Ile Tyr

595 600 605

Thr Leu Thr Leu Pro Ala Asp Thr Asp Thr Thr Asn Ala Lys Val Ile

610 615 620

Phe Asn Asn Gly Ser Ala Gln Val Pro Gly Gln Asn Gln Pro Gly Phe

625 630 635 640

Asp Tyr Val Gln Asn Gly Leu Tyr Asn Asp Ser Gly Leu Ser Gly Ser

645 650 655

Leu Pro Tyr

<210> 3

<211> 381

<212> PRT

<213> Bacillus (Bacillus sp. J3)

<400> 3

Met Arg Ser Lys Lys Leu Trp Ser Ser Leu Leu Phe Ala Leu Thr Leu

1 5 10 15

Ile Phe Thr Met Ala Phe Ser Asn Met Ser Ala Gln Ala Ala Gly Lys

20 25 30

Ser Ser Thr Glu Lys Lys Tyr Ile Val Gly Phe Lys Gln Thr Met Ser

35 40 45

Ala Met Ser Ser Ala Lys Lys Lys Asp Val Ile Ser Glu Lys Gly Gly

50 55 60

Lys Val Gln Lys Gln Phe Lys Tyr Val Asn Ala Ala Ala Ala Thr Leu

65 70 75 80

Asp Glu Lys Ala Val Lys Glu Leu Lys Lys Asp Pro Ser Val Ala Tyr

85 90 95

Val Glu Glu Asp His Ile Ala His Glu Tyr Ala Gln Ser Val Pro Tyr

100 105 110

Gly Ile Ser Gln Ile Lys Ala Pro Ala Leu His Ser Gln Gly Tyr Thr

115 120 125

Gly Ser Asn Val Lys Val Ala Val Ile Asp Ser Gly Ile Asp Ser Ser

130 135 140

His Pro Asp Leu Asn Val Arg Gly Gly Ala Ser Phe Val Pro Ser Glu

145 150 155 160

Thr Asn Pro Tyr Gln Asp Gly Ser Ser His Gly Thr His Val Ala Gly

165 170 175

Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly Val Leu Gly Val Ala Pro

180 185 190

Ser Ala Ser Leu Tyr Ala Val Lys Val Leu Asp Ser Thr Gly Ser Gly

195 200 205

Gln Tyr Ser Trp Ile Ile Asn Gly Ile Glu Trp Ala Ile Ser Asn Asn

210 215 220

Met Asp Val Ile Asn Met Ser Leu Gly Gly Pro Thr Gly Ser Thr Ala

225 230 235 240

Leu Lys Thr Val Val Asp Lys Ala Val Ser Ser Gly Ile Val Val Ala

245 250 255

Ala Ala Ala Gly Asn Glu Gly Ser Ser Gly Ser Ser Ser Thr Val Gly

260 265 270

Tyr Pro Ala Lys Tyr Pro Ser Thr Ile Ala Val Gly Ala Val Asn Ser

275 280 285

Ser Asn Gln Arg Ala Ser Phe Ser Ser Ala Gly Ser Glu Leu Asp Val

290 295 300

Met Ala Pro Gly Val Ser Ile Gln Ser Thr Leu Pro Gly Gly Thr Tyr

305 310 315 320

Gly Ala Tyr Asn Gly Thr Ser Met Ala Thr Pro His Val Ala Gly Ala

325 330 335

Ala Ala Leu Ile Leu Ser Lys His Pro Thr Trp Thr Asn Ala Gln Val

340 345 350

Arg Asp Arg Leu Glu Ser Thr Ala Thr Tyr Leu Gly Asn Ser Phe Tyr

355 360 365

Tyr Gly Lys Gly Leu Ile Asn Val Gln Ala Ala Ala Gln

370 375 380

<210> 4

<211> 442

<212> PRT

<213> Bacillus (Bacillus sp. J3)

<400> 4

Met Phe Gly Tyr Ser Met Val Gln Met Val Arg Ala Asn Ala His Lys

1 5 10 15

Leu Asp Trp Pro Leu Arg Glu Thr Val Leu Gln Leu Tyr Lys Pro Phe

20 25 30

Lys Trp Thr Pro Cys Phe Leu His Lys Phe Phe Glu Thr Lys Leu Gln

35 40 45

Asn Arg Lys Lys Met Ser Val Ile Ile Glu Phe Glu Glu Gly Cys His

50 55 60

Glu Thr Gly Phe Gln Met Ala Gly Glu Val Leu Gln Lys Glu Lys Arg

65 70 75 80

Ser Lys Leu Lys Ser Arg Phe Asn Lys Ile Asn Cys Cys Ser Ala Glu

85 90 95

Val Thr Pro Ser Ala Leu His Ser Leu Leu Ser Glu Cys Ser Asn Ile

100 105 110

Arg Lys Val Tyr Leu Asn Arg Glu Val Lys Ala Leu Leu Asp Thr Ala

115 120 125

Thr Glu Ala Ser His Ala Lys Glu Val Val Arg Asn Arg Gln Thr Leu

130 135 140

Thr Gly Lys Gly Val Thr Val Ala Val Val Asp Thr Gly Ile Tyr Pro

145 150 155 160

His Pro Asp Leu Glu Gly Arg Ile Ile Gly Phe Ala Asp Met Val Asn

165 170 175

Gln Lys Thr Glu Pro Tyr Asp Asp Asn Gly His Gly Thr His Cys Ala

180 185 190

Gly Asp Val Ala Ser Ser Gly Ala Ser Ser Ser Gly Gln Tyr Arg Gly

195 200 205

Pro Ala Pro Glu Ala Asn Leu Ile Gly Val Lys Val Leu Asn Lys Gln

210 215 220

Gly Ser Gly Thr Leu Ala Asp Ile Ile Glu Gly Val Glu Trp Cys Ile

225 230 235 240

Gln Tyr Asn Glu Asp Asn Pro Asp Glu Pro Ile Asp Ile Ile Ser Met

245 250 255

Ser Leu Gly Gly Asp Ala Leu Arg Tyr Asp His Glu Gln Glu Asp Pro

260 265 270

Leu Val Arg Ala Val Glu Glu Ala Trp Ser Ala Gly Ile Val Val Cys

275 280 285

Val Ala Ala Gly Asn Ser Gly Pro Asp Ser Gln Thr Ile Ala Ser Pro

290 295 300

Gly Val Ser Glu Lys Val Ile Thr Val Gly Ala Leu Asp Asp Asn Asn

305 310 315 320

Thr Ala Ser Ser Asp Asp Asp Thr Val Ala Ser Phe Ser Ser Arg Gly

325 330 335

Pro Thr Val Tyr Gly Lys Glu Lys Pro Asp Ile Leu Ala Pro Gly Val

340 345 350

Asn Ile Ile Ser Leu Arg Ser Pro Asn Ser Tyr Ile Asp Lys Leu Gln

355 360 365

Lys Ser Ser Arg Val Gly Ser Gln Tyr Phe Thr Met Ser Gly Thr Ser

370 375 380

Met Ala Thr Pro Ile Cys Ala Gly Ile Ala Ala Leu Ile Leu Gln Gln

385 390 395 400

Asn Pro Asp Leu Thr Pro Asp Glu Val Lys Glu Leu Leu Lys Asn Gly

405 410 415

Thr Asp Lys Trp Lys Asp Glu Asp Pro Asn Val Tyr Gly Ala Gly Ala

420 425 430

Val Asn Ala Glu Asn Ser Val Pro Gly Gln

435 440

<210> 5

<211> 806

<212> PRT

<213> Bacillus (Bacillus sp. J3)

<400> 5

Met Lys Lys Gly Ile Ile Arg Phe Leu Leu Val Ser Phe Val Leu Phe

1 5 10 15

Phe Ala Leu Ser Thr Gly Ile Thr Gly Val Gln Ala Ala Pro Ala Ser

20 25 30

Ser Lys Thr Ser Ala Asp Leu Glu Lys Ala Glu Val Phe Gly Asp Ile

35 40 45

Asp Met Thr Thr Ser Lys Lys Thr Thr Val Ile Val Glu Leu Lys Glu

50 55 60

Lys Ser Leu Ala Glu Ala Lys Glu Ala Gly Glu Ser Gln Ser Lys Ser

65 70 75 80

Lys Leu Lys Thr Ala Arg Thr Lys Ala Lys Asn Lys Ala Ile Lys Ala

85 90 95

Val Lys Asn Gly Lys Val Asn Arg Glu Tyr Glu Gln Val Phe Ser Gly

100 105 110

Phe Ser Met Lys Leu Pro Ala Asn Glu Ile Pro Lys Leu Leu Ala Val

115 120 125

Lys Asp Val Lys Ala Val Tyr Pro Asn Val Thr Tyr Lys Thr Asp Asn

130 135 140

Met Lys Asp Lys Asp Val Thr Ile Ser Glu Asp Ala Val Ser Pro Gln

145 150 155 160

Met Asp Asp Ser Ala Pro Tyr Ile Gly Ala Asn Asp Ala Trp Asp Leu

165 170 175

Gly Tyr Thr Gly Lys Gly Ile Lys Val Ala Ile Ile Asp Thr Gly Val

180 185 190

Glu Tyr Asn His Pro Asp Leu Lys Lys Asn Phe Gly Gln Tyr Lys Gly

195 200 205

Tyr Asp Phe Val Asp Asn Asp Tyr Asp Pro Lys Glu Thr Pro Thr Gly

210 215 220

Asp Pro Arg Gly Gly Ala Thr Asp His Gly Thr His Val Ala Gly Thr

225 230 235 240

Val Ala Ala Asn Gly Thr Ile Lys Gly Val Ala Pro Asp Ala Thr Leu

245 250 255

Leu Ala Tyr Arg Val Leu Gly Pro Gly Gly Ser Gly Thr Thr Glu Asn

260 265 270

Val Ile Ala Gly Val Glu Arg Ala Val Gln Asp Gly Ala Asp Val Met

275 280 285

Asn Leu Ser Leu Gly Asn Ser Leu Asn Asn Pro Asp Trp Ala Thr Ser

290 295 300

Thr Ala Leu Asp Trp Ala Met Ser Glu Gly Val Val Ala Val Thr Ser

305 310 315 320

Asn Gly Asn Ser Gly Pro Asn Gly Trp Thr Val Gly Ser Pro Gly Thr

325 330 335

Ser Arg Glu Ala Ile Ser Val Gly Ala Thr Gln Leu Pro Leu Asn Glu

340 345 350

Tyr Ala Val Thr Phe Gly Ser Tyr Ser Ser Ala Lys Val Met Gly Tyr

355 360 365

Asn Lys Glu Asp Asp Val Lys Ala Leu Asn Asn Lys Glu Ile Glu Leu

370 375 380

Val Glu Ala Gly Ile Gly Glu Glu Lys Asp Phe Glu Gly Lys Asp Leu

385 390 395 400

Thr Gly Lys Val Ala Val Val Lys Arg Gly Ser Ile Ala Phe Val Asp

405 410 415

Lys Ala Asp Asn Ala Lys Lys Ala Gly Ala Ile Gly Met Val Val Tyr

420 425 430

Asn Asn Leu Ser Gly Glu Ile Glu Ala Asn Val Pro Gly Met Ser Val

435 440 445

Pro Thr Ile Lys Leu Ser Leu Glu Asp Gly Glu Lys Leu Val Ser Ala

450 455 460

Leu Lys Ala Gly Glu Thr Lys Thr Thr Phe Lys Leu Thr Val Ser Lys

465 470 475 480

Ala Leu Gly Glu Gln Val Ala Asp Phe Ser Ser Arg Gly Pro Val Met

485 490 495

Asp Thr Trp Met Ile Lys Pro Asp Ile Ser Ala Pro Gly Val Asn Ile

500 505 510

Val Ser Thr Ile Pro Thr His Asp Pro Asp His Pro Tyr Gly Tyr Gly

515 520 525

Ser Lys Gln Gly Thr Ser Met Ala Ser Pro His Ile Ala Gly Ala Val

530 535 540

Ala Val Ile Lys Gln Ala Lys Pro Lys Trp Ser Val Glu Gln Ile Lys

545 550 555 560

Ala Ala Ile Met Asn Thr Ala Val Thr Leu Lys Asp Ser Asp Gly Glu

565 570 575

Val Tyr Pro His Asn Ala Gln Gly Ala Gly Ser Ala Arg Ile Met Asn

580 585 590

Ala Ile Lys Ala Asp Ser Leu Val Ser Pro Gly Ser Tyr Ser Tyr Gly

595 600 605

Thr Phe Leu Lys Glu Asn Gly Asn Glu Thr Lys Asn Glu Thr Phe Thr

610 615 620

Ile Glu Asn Gln Ser Ser Ile Arg Lys Ser Tyr Thr Leu Glu Tyr Ser

625 630 635 640

Phe Asn Gly Ser Gly Ile Ser Thr Ser Gly Thr Ser Arg Val Val Ile

645 650 655

Pro Ala His Gln Thr Gly Lys Ala Thr Ala Lys Val Lys Val Asn Thr

660 665 670

Lys Lys Thr Lys Ala Gly Thr Tyr Glu Gly Thr Val Ile Val Arg Glu

675 680 685

Gly Gly Lys Thr Val Ala Lys Val Pro Thr Leu Leu Ile Val Lys Glu

690 695 700

Pro Asp Tyr Pro Arg Val Thr Ser Val Ser Val Ser Glu Gly Ser Val

705 710 715 720

Gln Gly Thr Tyr Gln Ile Glu Thr Tyr Leu Pro Ala Gly Ala Glu Glu

725 730 735

Leu Ala Phe Leu Val Tyr Asp Ser Asn Leu Asp Phe Ala Gly Gln Ala

740 745 750

Gly Ile Tyr Lys Asn Gln Asp Lys Gly Tyr Gln Tyr Phe Asp Trp Asp

755 760 765

Gly Thr Ile Asn Gly Gly Thr Lys Leu Pro Ala Gly Glu Tyr Tyr Leu

770 775 780

Leu Ala Tyr Ala Ala Asn Lys Gly Lys Ser Ser Gln Val Leu Thr Glu

785 790 795 800

Glu Pro Phe Thr Val Glu

805

<210> 6

<211> 507

<212> PRT

<213> Bacillus (Bacillus sp. J3)

<400> 6

Met Phe Leu Leu Glu Tyr Thr Tyr Trp Lys Ile Ala Ala His Leu Val

1 5 10 15

Asn Asn Gly Tyr Gly Val Ile Gln Ala Gly Glu Ser Asp Glu Ile Trp

20 25 30

Leu Glu Ala Pro Asp Lys Ser Ser His Asp Leu Val Arg Leu Tyr Lys

35 40 45

His Asp Leu Asp Phe Arg Gln Glu Met Val Arg Asp Ile Glu Glu Gln

50 55 60

Ala Glu Arg Val Glu Arg Val Arg His Gln Leu Val Arg Arg Arg Met

65 70 75 80

Lys Leu Leu Asn Val Phe Phe Ser Thr Glu Ala Pro Val Asp Asp Trp

85 90 95

Glu Glu Ile Ala Lys Lys Thr Phe Glu Lys Gly Thr Val Ser Val Glu

100 105 110

Pro Ala Ile Val Arg Gly Thr Met Leu Arg Asp Asp Leu Gln Ala Val

115 120 125

Phe Pro Ser Phe Arg Thr Glu Asp Cys Thr Glu Glu His Ala Ser Phe

130 135 140

Glu Asp Ala Gln Met Ala Arg Glu Arg Phe Leu Ser Leu Val Leu Lys

145 150 155 160

Gln Glu Glu Gln Arg Lys Thr Glu Ala Ala Val Phe Gln Asn Gly Lys

165 170 175

Pro Ile Phe Thr Tyr Leu Phe Ile Ala Leu Gln Ile Leu Met Phe Phe

180 185 190

Leu Leu Glu Ile Asn Gly Gly Ser Thr Asn Thr Glu Thr Leu Val Ala

195 200 205

Phe Gly Ala Lys Glu Asn Ser Leu Ile Ala Ala Gly Glu Trp Trp Arg

210 215 220

Leu Leu Thr Pro Ile Val Leu His Ile Gly Ile Ala His Leu Ala Phe

225 230 235 240

Asn Thr Leu Ala Leu Trp Ser Val Gly Thr Ala Val Glu Arg Met Tyr

245 250 255

Gly Ser Gly Arg Phe Leu Leu Ile Tyr Leu Ala Ala Gly Ile Thr Gly

260 265 270

Ser Ile Ala Ser Phe Val Phe Ser Pro Tyr Pro Ser Ala Gly Ala Ser

275 280 285

Gly Ala Ile Phe Gly Cys Leu Gly Ala Leu Leu Tyr Val Ala Leu Ser

290 295 300

Asn Arg Lys Met Phe Leu Arg Thr Ile Gly Thr Asn Ile Ile Val Ile

305 310 315 320

Ile Ile Ile Asn Leu Gly Phe Gly Phe Ala Val Ser Asn Ile Asp Asn

325 330 335

Ser Gly His Ile Gly Gly Leu Ile Gly Gly Phe Phe Ala Ala Ala Ala

340 345 350

Leu Gly Leu Pro Lys Ala Gly Ala Phe Gly Lys Arg Leu Leu Ser Ala

355 360 365

Val Leu Leu Ile Ala Leu Ala Val Gly Phe Leu Tyr Tyr Gly Leu His

370 375 380

Ser Pro Ser His Gln Glu Ser Ala Leu Ile Gln Gln Ala Ser Glu Leu

385 390 395 400

Tyr Gln Glu Gly Lys Tyr Glu Glu Val Thr Glu Leu Leu Asn Gly Glu

405 410 415

Ala Ala Gln Lys Asp Ala Ser Ala Asp Leu Leu Lys Ile Leu Ala Val

420 425 430

Ser Asp Ile Gln Ile Gly Glu Tyr Asp Gln Ala Val Phe His Leu Glu

435 440 445

Arg Ala Val Lys Lys Glu Pro Lys Asp His Ala Ala Tyr Tyr Tyr Leu

450 455 460

Ala Leu Leu Tyr Ala Glu Lys Asn Glu Leu Val Gln Ala Glu Lys Ala

465 470 475 480

Ile Arg Leu Ala Leu Lys Leu Glu Pro Lys Glu Gln Arg Tyr Lys Glu

485 490 495

Leu Gln Arg Gln Ile Glu Asn Asn Lys Glu Ser

500 505

<210> 7

<211> 55

<212> PRT

<213> Bacillus (Bacillus sp. J3)

<400> 7

Met Lys Phe Val Lys Ala Ile Trp Pro Phe Val Ala Val Ala Ile Val

1 5 10 15

Phe Met Phe Met Ser Ala Phe Lys Phe Asn Asp Gln Leu Thr Asp Gln

20 25 30

Glu Lys Gln Lys Ile Asp Met Glu Met Asn Lys Ile Gln Gln Gln Glu

35 40 45

Glu Pro Val Asn Ala Asn Lys

50 55

<210> 8

<211> 380

<212> PRT

<213> Bacillus (Bacillus sp. J3)

<400> 8

Met Lys Lys Glu Leu Ser Phe His Glu Lys Leu Leu Lys Leu Thr Lys

1 5 10 15

Gln Gln Lys Lys Lys Thr Asn Lys His Val Phe Ile Ala Ile Pro Ile

20 25 30

Val Phe Val Leu Met Phe Ala Phe Met Trp Ala Gly Lys Ala Glu Thr

35 40 45

Pro Lys Val Lys Thr Tyr Ser Asp Asp Val Leu Ser Ala Ser Phe Val

50 55 60

Gly Asp Ile Met Met Gly Arg Tyr Val Glu Lys Val Thr Glu Gln Lys

65 70 75 80

Gly Ala Asp Ser Ile Phe Gln Tyr Val Glu Pro Ile Phe Arg Ala Ser

85 90 95

Asp Tyr Val Ala Gly Asn Phe Glu Asn Pro Val Thr Tyr Gln Lys Asn

100 105 110

Tyr Lys Gln Ala Asp Lys Glu Ile His Leu Gln Thr Asn Lys Glu Ser

115 120 125

Val Lys Val Leu Lys Asp Met Asn Phe Thr Val Leu Asn Ser Ala Asn

130 135 140

Asn His Ala Met Asp Tyr Gly Val Gln Gly Met Lys Asp Thr Leu Gly

145 150 155 160

Glu Phe Ala Lys Gln Asn Leu Asp Ile Val Gly Ala Gly Tyr Ser Leu

165 170 175

Ser Asp Ala Lys Lys Lys Ile Ser Tyr Gln Lys Val Asn Gly Val Thr

180 185 190

Ile Ala Thr Leu Gly Phe Thr Asp Val Ser Gly Lys Gly Phe Ala Ala

195 200 205

Lys Lys Asn Thr Pro Gly Val Leu Pro Ala Asp Pro Glu Ile Phe Ile

210 215 220

Pro Met Ile Ser Glu Ala Lys Lys His Ala Asp Ile Val Val Val Gln

225 230 235 240

Ser His Trp Gly Gln Glu Tyr Asp Asn Asp Pro Asn Asp Arg Gln Arg

245 250 255

Gln Leu Ala Arg Ala Met Ser Asp Ala Gly Ala Asp Ile Ile Val Gly

260 265 270

His His Pro His Val Leu Glu Pro Ile Glu Val Tyr Asn Gly Thr Val

275 280 285

Ile Phe Tyr Ser Leu Gly Asn Phe Val Phe Asp Gln Gly Trp Thr Arg

290 295 300

Thr Arg Asp Ser Ala Leu Val Gln Tyr His Leu Lys Lys Asn Gly Thr

305 310 315 320

Gly Arg Phe Glu Val Thr Pro Ile Asp Ile His Glu Ala Thr Pro Ala

325 330 335

Pro Val Lys Lys Asp Ser Leu Lys Gln Lys Thr Ile Ile Arg Glu Leu

340 345 350

Thr Lys Asp Ser Asn Phe Ala Trp Lys Val Glu Asp Gly Lys Leu Thr

355 360 365

Phe Asp Ile Asp His Ser Asp Lys Leu Lys Ser Lys

370 375 380

<210> 9

<211> 149

<212> PRT

<213> Bacillus (Bacillus sp. J3)

<400> 9

Met Phe Gly Ser Asp Leu Tyr Ile Ala Leu Ile Leu Gly Val Leu Leu

1 5 10 15

Ser Leu Ile Phe Ala Glu Lys Thr Gly Ile Val Pro Ala Gly Leu Val

20 25 30

Val Pro Gly Tyr Leu Gly Leu Val Phe Asn Gln Pro Val Phe Ile Leu

35 40 45

Leu Val Leu Leu Val Ser Leu Leu Thr Tyr Val Ile Val Lys Tyr Gly

50 55 60

Leu Ser Lys Phe Met Ile Leu Tyr Gly Arg Arg Lys Phe Ala Ala Met

65 70 75 80

Leu Ile Thr Gly Ile Val Leu Lys Ile Ala Phe Asp Phe Leu Tyr Pro

85 90 95

Ile Val Pro Phe Glu Ile Ala Glu Phe Arg Gly Ile Gly Ile Ile Val

100 105 110

Pro Gly Leu Ile Ala Asn Thr Ile Gln Lys Gln Gly Leu Thr Ile Thr

115 120 125

Phe Gly Ser Thr Leu Leu Leu Ser Gly Ala Thr Phe Ala Ile Met Phe

130 135 140

Val Tyr Tyr Leu Ile

145

<210> 10

<211> 393

<212> PRT

<213> Bacillus (Bacillus sp. J3)

<400> 10

Met Trp Leu Leu Ile Ile Ala Cys Ala Val Ile Leu Val Ile Gly Ile

1 5 10 15

Leu Glu Lys Arg Arg His Gln Lys Asn Ile Asp Ala Leu Pro Val Arg

20 25 30

Val Asn Ile Asn Gly Ile Arg Gly Lys Ser Thr Val Thr Arg Leu Thr

35 40 45

Thr Gly Ile Leu Ile Glu Ala Gly Tyr Lys Thr Val Gly Lys Thr Thr

50 55 60

Gly Thr Asp Ala Arg Met Ile Tyr Trp Asp Thr Pro Glu Glu Lys Pro

65 70 75 80

Ile Lys Arg Lys Pro Gln Gly Pro Asn Ile Gly Glu Gln Lys Glu Val

85 90 95

Met Arg Glu Thr Val Glu Arg Gly Ala Asn Ala Ile Val Ser Glu Cys

100 105 110

Met Ala Val Asn Pro Asp Tyr Gln Ile Ile Phe Gln Glu Glu Leu Leu

115 120 125

Gln Ala Asn Ile Gly Val Ile Val Asn Val Leu Glu Asp His Met Asp

130 135 140

Val Met Gly Pro Thr Leu Asp Glu Ile Ala Glu Ala Phe Thr Ala Thr

145 150 155 160

Ile Pro Tyr Asn Gly His Leu Val Ile Thr Asp Ser Glu Tyr Thr Glu

165 170 175

Phe Phe Lys Gln Lys Ala Lys Glu Arg Asn Thr Lys Val Ile Ile Ala

180 185 190

Asp Asn Ser Lys Ile Thr Asp Glu Tyr Leu Arg Lys Phe Glu Tyr Met

195 200 205

Val Phe Pro Asp Asn Ala Ser Leu Ala Leu Gly Val Ala Gln Ala Leu

210 215 220

Gly Ile Asp Glu Glu Thr Ala Phe Lys Gly Met Leu Asn Ala Pro Pro

225 230 235 240

Asp Pro Gly Ala Met Arg Ile Leu Pro Leu Ile Ser Pro Ser Glu Pro

245 250 255

Gly His Phe Val Asn Gly Phe Ala Ala Asn Asp Ala Ser Ser Thr Leu

260 265 270

Asn Ile Trp Lys Arg Val Lys Glu Ile Gly Tyr Pro Thr Asp Asp Pro

275 280 285

Ile Ile Ile Met Asn Cys Arg Ala Asp Arg Val Asp Arg Thr Gln Gln

290 295 300

Phe Ala Asn Asp Val Leu Pro Tyr Ile Glu Ala Ser Glu Leu Ile Leu

305 310 315 320

Ile Gly Glu Thr Thr Glu Pro Ile Val Lys Ala Tyr Glu Glu Gly Lys

325 330 335

Ile Pro Ala Asp Lys Leu His Asp Leu Glu Tyr Lys Ser Thr Asp Glu

340 345 350

Ile Met Glu Leu Leu Lys Lys Arg Met His Asn Arg Val Ile Tyr Gly

355 360 365

Val Gly Asn Ile His Gly Ala Ala Glu Pro Leu Ile Glu Lys Ile His

370 375 380

Glu Tyr Lys Val Lys Gln Leu Val Ser

385 390

Claims (4)

1. An efficient aroma-enhancing strain is characterized in that: the efficient aroma-enhancing strain is bacillusBacillus sp.J3, accession number: m2018707, the nucleotide sequence of the 16S rDNA is shown in the sequence table SEQ ID NO: 1 is shown in the specification;

the efficient aroma-enhancing strain contains amylase activity, wherein an amino acid sequence of alpha-amylase is shown in a sequence table SEQ ID NO: 2 is shown in the specification;

the efficient aroma-increasing strain contains protease activity, wherein 4 serine proteases are contained, and the amino acid sequences of the serine proteases are shown in a sequence table SEQ ID NO: 3-6;

the high-efficiency aroma-enhancing strain has polyglutamic acid synthesis capacity, and a polyglutamic acid synthetase gene cluster comprisespgsE、pgsA、pgsCAndpgsBthe amino acid sequences are respectively shown in a sequence table SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9. SEQ ID NO: shown at 10.

2. The highly potent aromatizing strain of claim 1, wherein: the efficient aroma-increasing strain is separated from the surface of Zimbabwe flue-cured tobacco leaves.

3. Use of a highly potent flavoured strain according to claim 1 or 2 for improving tobacco quality, characterized in that: the efficient aroma-increasing strain is used in a tobacco leaf aging process, and the smoking quality of flue-cured tobacco is improved.

4. The application of the high-efficiency flavor-enhancing strain in improving the quality of tobacco according to claim 3 is characterized by comprising the following specific steps: (1) streaking the strain on an LB solid culture medium for resuscitation, then picking out a single clone and putting the single clone into a liquid LB culture medium for further activation; (2) adding the activated bacterial suspension into an LB culture medium according to the mass ratio of 1:100, and culturing for 16 hours at 37 ℃ and 220 rpm; (3) centrifuging at 6000-; (4) resuspending the thalli by deionized water, preparing bacterial suspension, and spraying the bacterial suspension on the tobacco leaves; (5) aging the mixture at the temperature of 28-42 ℃ and the relative humidity of 40-80% for 3-7 days.

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