CN115944111A - Preparation method and application of burley tobacco fermented spice - Google Patents

Abstract

The invention provides a preparation method and application of burley tobacco fermented flavor, relates to a preparation and extraction method of tobacco flavor, and belongs to the technical field of tobacco. The preparation method comprises the following steps: (1) preparing a streptomyces nicotianae N35 microbial inoculum; (2) Taking burley tobacco powder as a substrate, and performing combined fermentation treatment on the burley tobacco powder by adopting an actinomycete streptomyces tabacum N35 microbial inoculum and a bacterium rhizobacteria ky 7-1 microbial inoculum to obtain fermented burley tobacco; (3) And (3) carrying out ethanol reflux extraction on the fermented burley tobacco, and filtering and concentrating to obtain the burley tobacco fermented spice. In the invention, the actinomycete streptomyces nicotianae N35 and the bacterium rhizosphere Qidun bacillus yh7-1 play a synergistic role, so that cellulose, protein and the like in burley tobacco can be effectively degraded, and special aroma substances are released. The spice prepared from the fermented burley tobacco is applied to the cut tobacco of the cigarette, so that the aroma is harmoniously rich, the irritation is reduced, the smoke is softened, the herbal aroma of the tobacco can be supplemented, and the style characteristics of the tobacco leaves can be enhanced.

Description

Preparation method and application of burley tobacco fermented spice

Technical Field

The invention belongs to the technical field of tobacco flavor and flavor, and particularly relates to a preparation method and application of a burley tobacco fermented tobacco flavor.

Background

The tobacco is derived from spices applied to cigarette products, has natural and harmonious fragrance, can supplement the herbal fragrance of the cigarettes, can strengthen the style characteristics of the tobacco leaves, and is an irreplaceable spice in the cigarette blending process. Tobacco extracts typically contain certain amounts of proteins, pectins, hemicelluloses, lignin, etc., which are cleaved during smoking of a cigarette to produce components which increase the irritation and offensive odor of the cigarette smoke. Therefore, the method has important significance for producing fragrance precursor substances by utilizing the microbial fermentation and degradation of protein and cellulose.

Actinomycetes are an important microbial resource, and can produce a plurality of secondary metabolites with novel chemical structures and remarkable biological activity due to a complex secondary metabolic system. At present, actinomycetes are more in the aspect of antibiotic research, and the research and the utilization of actinomycetes to degrade organic compounds such as cellulose and the like are also used for carrying out biological treatment on environmental pollution, so that the research and the development of soil actinomycetes resources have extremely high scientific value, economic value and social value.

The present invention has been made to solve the above problems.

Disclosure of Invention

The burley tobacco is one of the important raw materials of the blended type cigarette, has the characteristics of strong strength, rich and plump aroma, large irritation, heavy miscellaneous gas and the like, utilizes the degradation effect of actinomycetes on cellulose, lignin and the like, combines bacteria to ferment the burley tobacco, reduces the irritation of smoking by degrading macromolecular substances, and has special aroma on some small molecular substances, can soften smoke and has a promotion effect on the quality of cigarette products.

The invention aims to provide a preparation method of burley tobacco fermented spice with soft smoke and application of the burley tobacco fermented spice in cigarettes aiming at the characteristics of large stimulation, heavy miscellaneous gas and the like of burley tobacco spice, wherein burley tobacco is used as a raw material, streptomyces tabaci N35 and rhizosphere Qidun bacillus yh7-1 are utilized to be treated by a synergistic fermentation process, and then a solvent extraction process is used for obtaining the burley tobacco fermented spice with good safety. Provides a method for preparing the spice which can obviously improve the smoking quality of the cigarette.

* All percentages used in the present invention are mass percentages unless otherwise indicated.

The invention relates to a preparation method of a burley tobacco fermented spice, which comprises the following steps:

(1) Preparing a streptomyces nicotianae N35 microbial inoculum and a rhizosphere Qidun bacillus yh7-1 microbial inoculum;

(2) Taking burley tobacco powder as a substrate, mixing an actinomycete streptomyces tabacum N35 microbial inoculum and a rhizosphere kirton bacillus yh7-1 microbial inoculum, and carrying out combined solid state fermentation treatment on the mixture to obtain fermented burley tobacco;

(3) And (3) carrying out ethanol reflux extraction on the fermented burley tobacco, and filtering and concentrating to obtain the burley tobacco fermented spice.

Preferably, step (1) specifically comprises: inoculating streptomyces nicotianae N35 liquid strain into a fermentation culture medium according to the inoculation amount of 5-35%, and performing shake culture at 15-55 ℃ for 3-15 days to obtain a culture solution; and (4) centrifugally separating the culture solution, washing the precipitate with sterile water, uniformly shaking with the sterile water, and diluting by 5-20 times to obtain the streptomyces nicotianae N35 microbial inoculum.

Inoculating rhizosphere Qidun bacillus yh7-1 liquid strain into a fermentation culture medium according to the inoculation amount of 5-30%, and performing shake culture at 10-50 ℃ for 3-10 days to obtain a culture solution; and (3) centrifugally separating the culture solution, washing the precipitate with sterile water, uniformly shaking the precipitate with sterile water, and diluting the precipitate by 3 to 15 times to obtain the rhizosphere Qidun bacillus yh7-1 microbial inoculum.

The streptomyces nicotianae N35 is classified and named as streptomyces nicotianae N35 in microbiology, and the Latin literature name is as follows: streptomyces tabaci N35 has been preserved in China general microbiological culture Collection center (CGMCC) at 10/13 of 2021 with the preservation number of CGMCC 4.7701. Address: the institute of microbiology, national academy of sciences, west road No. 1, north Chen, chao, chaoyang, beijing.

Rhizosphere kirton bacillus yh7-1, the microbiological classification of which is named rhizosphere kirton bacillus yh7-1, latin literature name: chthonobacter rhizophilae yh7-1 is preserved in China general microbiological culture Collection center (CGMCC) 1.17236 in 27/5/2020. For the isolation, culture and identification of rhizosphere kirton bacillus yh7-1, reference is made to a patent application, filed by the applicant at 3/1/2021, with application number 202110226320.9, entitled rhizosphere kirton bacillus and a method for isolation and use thereof, while the description of rhizosphere kirton bacillus yh7-1 in the patent is incorporated in its entirety into the present application.

The streptomyces nicotianae N35 strain obtained by separation has the main morphological characteristics and physiological and biochemical property characteristics as follows: the growth was good in most of the media, and branched intrabasal hyphae and aerial hyphae were formed on the R2A medium, and the colonies were white. Casein hydrolysis, cellulose hydrolysis, tween 20, tween 40, tween 60 and Tween 80 hydrolysis are positive, starch hydrolysis is negative, and no indole is produced. Can utilize alpha-lactose, maltose, cellobiose, trehalose, mannose, fructose, sorbitol, galactose, rhamnose, inositol. The polar lipid component of cell membrane mainly comprises phosphatidylethanolamine and phosphatidylInositol and an unknown phospholipid, the cellular respiratory quinone is menaquinone-9 (MK-9 (H) ₈ ))。

The nucleotide sequence of the 16S rDNA gene of the microorganism streptomyces nicotianae N35 obtained by separation is shown in a sequence table, the sequence is submitted to an international nucleotide sequence database (GenBank), and the sequence retrieval number is as follows: MT598006.

Preferably, step (2) specifically comprises: the dried burley tobacco leaves are crushed into powder with the grain diameter of 30 to 80 meshes, and the moisture is balanced to 10 to 13 percent. The streptomyces nicotianae N35 bacterium and rhizosphere Qidun bacillus yh7-1 bacterium agent are mixed according to the proportion of 1: the ratio of 1 is sprayed on the burley tobacco powder, 10-50 mL of microbial inoculum is sprayed in each 100g of the burley tobacco powder, the burley tobacco powder is evenly mixed, and the processed burley tobacco powder is placed in a constant temperature and humidity box with the temperature of 22 ℃ and the concentration of 60 percent for fermentation for 48-96 h, so as to obtain the fermented burley tobacco.

Preferably, step (3) specifically comprises: adding ethanol with the concentration of 80-95 percent into the mixture which is 5-10 times of the mass of the fermented burley tobacco, extracting for 1-3 h under the condition of hot reflux at 50-80 ℃, filtering the obtained clear liquid, and concentrating to obtain the burley tobacco fermented spice.

In a second aspect, the invention provides an application of the burley tobacco fermented spice obtained by the preparation method in the first aspect, and the burley tobacco fermented spice is applied to cut tobacco of cigarettes to obtain cigarettes containing the burley tobacco fermented spice.

Preferably, the burley tobacco fermented flavor prepared by the preparation method of the first aspect of the invention is diluted by propylene glycol or ethanol by 3-5 times of the weight of the cigarette tobacco shred by 0.3-0.6% of the weight of the cigarette tobacco shred, and then is sprayed on the cigarette tobacco shred in a spraying manner to obtain the cigarette containing the burley tobacco fermented flavor.

In a third aspect, the invention provides a cigarette containing the burley tobacco fermented flavor prepared by the preparation method in the first aspect.

The invention has the following beneficial effects:

1. according to the invention, the burley tobacco fermented spice is prepared by performing combined fermentation treatment on the burley tobacco powder by utilizing the actinomycete streptomyces tabacum N35 microbial inoculum and the bacterium rhizosphere Kindobacillus yh7-1 microbial inoculum, and the actinomycete streptomyces tabacum N35 and the bacterium rhizosphere Kindobacillus yh7-1 exert a synergistic effect, so that cellulose, protein and the like in the burley tobacco can be effectively degraded, and special aroma substances are released. The spice prepared from the fermented burley tobacco is applied to the cut tobacco of the cigarette, so that the aroma is harmoniously rich, the irritation is reduced, the smoke is softened, the herbal aroma of the tobacco can be supplemented, and the style characteristics of the tobacco leaves can be enhanced.

2. The invention improves the quality of burley tobacco flavor, is applied to cigarette processing, enriches the sources and types of tobacco essence flavor, and meets the individual and diversified requirements of consumers. The whole preparation process has no introduction of unfavorable organic solvents, and the obtained essence and flavor and the processing technology are environment-friendly and safe, have easily obtained raw materials and low cost, are convenient to realize industrial production, and have good application value.

Drawings

FIG. 1 is an electron micrograph of Streptomyces nicotianae N35 of the invention on an R2A medium;

FIG. 2 is a phylogenetic tree constructed by Streptomyces nicotianae N35 and a part of related strains according to a 16SrDNA gene sequence.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

Example 1

And (3) separating, culturing and identifying the streptomyces nicotianae N35.

1. Isolation of Streptomyces nicotianae N35

Taking a tobacco soil sample from a Yunnan Kunming tobacco planting base, sealing the tobacco soil sample with a plastic bag, and storing the tobacco soil sample at 4 ℃ for later use. Accurately weighing 10g of soil sample, adding 90mL of sterile water, shaking for 30min at 30 ℃ and 180rpm by a shaking table; diluting the concentration to 10 with sterile water ^-1 、10 ^-2 、10 ^-3 、10 ^-4 、10 ^-5 The number of times of the total number of the parts,respectively taking 0.2mL of bacterial liquid with 5 concentrations, coating the bacterial liquid on an R2A plate culture medium, setting 3 parallel tests on the bacterial liquid with each concentration, culturing for 48h at 30 ℃, selecting different single bacterial colonies from the plate of each soil sample, purifying on an LB plate culture medium, adding glycerol according to 20% of the volume of the bacterial liquid, and storing in a refrigerator at-80 ℃ for later use.

Respectively inoculating the separated microorganisms into LB liquid culture medium, shaking and culturing at 30 ℃ and 160rpm on a shaking table until the concentration of the bacterial liquid is OD =2.0, and using the bacterial liquid as a seed liquid for later use. Inoculating 1% of seed liquid of each strain to be tested in a screening culture medium, shaking and culturing at 30 deg.C and 160rpm for 3-5 days, observing fermentation liquor appearance and fragrance change every day, and screening to obtain microorganism capable of producing fragrance, with number of L4-6.

2. Identification of Streptomyces nicotianae N35

The separated and purified strain N35 is analyzed by morphology, physiological and biochemical properties and 16SrDNA, and the identification result shows that the strain belongs to Streptomyces, and the strain is classified and named as Streptomyces tabaci N35 (Latin literature name: streptomyces tabaci N35) microbiologically.

An electron micrograph of Streptomyces nicotianae N35 on R2A medium is shown in figure 1.

The streptomyces nicotianae N35 grows well on most of the culture media, and forms branched intrabasal hyphae and aerial hyphae on the R2A culture medium, and colonies are white. Casein hydrolysis, cellulose hydrolysis, tween 20, tween 40, tween 60 and Tween 80 hydrolysis are positive, starch hydrolysis is negative, and no indole is produced. Can utilize alpha-lactose, maltose, cellobiose, trehalose, mannose, fructose, sorbitol, galactose, rhamnose, inositol. The polar lipid component of cell membrane mainly comprises phosphatidylethanolamine, phosphatidylinositol and an unknown phospholipid, and the respiratory quinone of cell is menaquinone-9 (MK-9 (H) ₈ ))。

The physiological and biochemical characteristics of streptomyces nicotianae N35 are shown in table 1:

TABLE 1 physiological and Biochemical characteristics of Streptomyces nicotianae N35

Experiment of	Growth reaction	Experiment of the invention	Growth reaction
				alpha-lactose	+ +	Tween 20,40,60 and 80	+ +
Indole production	- -	Casein as a food additive	+ +
				Hydrolysis of cellulose	+ +	Beta-cyclodextrin	- -
Starch hydrolysis	- -	D-mannose	+ +

Note: "+" indicates a positive result, and "-" indicates a negative result

The carbon and nitrogen utilization of streptomyces nicotianae N35 is shown in table 2:

TABLE 2 carbon and nitrogen source utilization of Streptomyces nicotianae N35

Carbon source utilization	Results	Carbon or nitrogen source utilization	Results
				Sucrose	-	Citric acid	-
Maltose	+	Benzoic acid	-
				Trehalose	+	Inositol	+
Cellobiose	+	L-aspartic acid	+
				Xylitol, its preparation method and use	-	L-histidine	+
Cotton seed candy	-	Tryptophan	-
				Melibiose	-	Proline	+

Note: "+" indicates a positive result, and "-" indicates a negative result

The 16S rDNA partial sequence of streptomyces nicotianae N35 is shown in the figure description, and the sequence is compared and analyzed with known sequences in GenBank database by BLAST, and 16SrDNA gene sequences of related species are obtained from the database, and a phylogenetic tree is constructed, which is shown in figure 2. Comparative analysis shows that the Streptomyces typhimurium N35 and the strain (Streptomyces caldifenitis CPCC 204147) of the invention ^T ) The genetic relationship is recent, independent branches are formed on the phylogenetic tree, and the characteristics of comprehensive morphology, physiology and biochemistry, cytochemistry, phylogenetic analysis and the like are obvious in difference, so that the Streptomyces nicotianae N35 is a new species and is named as Streptomyces tabaci N35.

The accession number of the nucleotide sequence of the 16S rDNA gene of the streptomyces nicotianae N35 in a GenBank database is MT598006, and the preservation number of the common microorganism center of the China Committee for culture Collection of microorganisms is CGMCC 4.7701. A phylogenetic tree constructed from Streptomyces nicotianae N35 and the 16S rDNA gene sequence of related species is shown in figure 2.

Example 2

1. Slant culture in test tubes

The culture medium is a slant preservation culture medium which is an R2A agar culture medium, and the formula of the culture medium is as follows: 0.5g of glucose, 0.5g of yeast extract, 0.5g of peptone, 0.5g of acid hydrolyzed casein, 0.5g of soluble starch, 0.3g of sodium pyruvate, 0.3g of dipotassium phosphate, 0.05g of magnesium sulfate, 15g of agar and distilled water to a constant volume of 1000mL, and the pH value of the solution is 7.2. Sterilizing the culture medium at 121 deg.C for 25 min, placing into slant, inoculating tobacco soil-borne bacterium L4-6 strain, and culturing at 28 deg.C for 1 week to obtain test tube strain.

2. Seed culture

The seed culture medium is adopted, and the formula of the seed culture medium is as follows: 120g of dextrin, 40g of soybean meal, 2g of yeast extract, 0.5g of tryptophan, 5g of beta-alanine, 0.5g of magnesium sulfate, 0.2g of ammonium phosphate and distilled water to 1000mL, wherein the pH value is 7.2. And (2) sterilizing the culture medium at 121 ℃ for 25 minutes, picking part of mycelia from the test tube inclined plane in the step (1), inoculating the mycelia into the seed solution, and performing shake culture at 28 ℃ for 36 hours to obtain a liquid strain.

3. Preparation of microbial inoculum

(1) Inoculating the streptomyces nicotianae N35 and the rhizosphere Qidun bacillus yh7-1 liquid strains into a fermentation culture medium according to the inoculation amount of 10%, and performing shake culture at 28 ℃ for 6 days to obtain fermentation liquor, thus obtaining the streptomyces nicotianae N35 and the rhizosphere Qidun bacillus yh7-1 microbial inoculum.

The formula of the culture medium is as follows: 10g of soybean meal; 10g of glucose; 3g of peptone; 2.5g of sodium chloride; 2g of calcium carbonate; 500mL of distilled water, pH7.0, to obtain the fermentation medium.

2. Fermentation of burley tobacco

Balancing the moisture of the burley tobacco powder to 10%, spraying 20mL of streptomyces tabacum N35 and rhizosphere Qidun bacillus yh7-1 microbial inoculum to every 100g of gentiana pseudomacrophylla powder, uniformly mixing, and fermenting the treated burley tobacco powder in a constant-temperature and constant-humidity box with the temperature of 22 ℃ and the concentration of 60% for 24 hours to obtain the fermented burley tobacco.

3. Preparation of burley tobacco fermented spice

Taking the fermented burley tobacco, adding 3kg of 95% ethanol, and performing hot reflux extraction at 70 ℃ for 3h; the hot reflux extracting solution is filtered while being hot, and the filtering mesh number is 200 meshes; continuously filtering the obtained clear liquid, and concentrating at 45 deg.C under 80kPa to density of 1.3g/cm ³ The fermentation extract is the burley tobacco fermentation spice.

4. Application of flavor for burley tobacco

Taking burley tobacco flavor accounting for 0.4 percent of the mass of the tobacco shreds of the cigarettes, dissolving the burley tobacco flavor by 4 times of propylene glycol, spraying the mixture onto the tobacco shreds of the cigarettes in a spraying mode, placing the mixture into a constant-temperature constant-humidity box, balancing for 48 hours under the conditions of humidity of 60 percent +/-2 and temperature of 22 ℃ +/-2, and rolling into cigarette cigarettes, namely the test cigarettes containing the burley tobacco fermented flavor.

5. Evaluation of flavor for Burley tobacco

The test cigarettes and the blank cigarettes are handed to professional smokers, the cigarette aroma style is evaluated according to YC/T497-2014, namely, the cigarette Chinese style sensory evaluation method, evaluation comparison is carried out on the aspects of aroma style, comfort characteristics and smoke characteristics, and the results are shown in Table 3.

It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.

TABLE 3 sensory evaluation of Burley tobacco fermented flavors

A sequence table is attached:

the 16S rDNA partial sequence of the streptomyces nicotianae N35 is as follows:

AGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAACGATGAACCGCTTTCGGGCGGGGATTAGTGGCGAACGGGTGAGTAACACGTGGGCAATCTGCCCTTCACTCTGGGACAAGCCCTGGAAACGGGGTCTAATACCGGATAACACCTGCTGCCTCATGGCGGCAGGTTAAAAGCTCCGGCGGTGAAGGATGAGCCCGCGGCCTATCAGCTTGTTGGTGGGGTAATGGCCTACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGAAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCAGCAGGGAAGAAGCGAAAGTGACGGTACCTGCAGAAGAAGCGCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGCGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGAGCTCGTAGGCGGCCAGTCGCGTCgGGtGTGAAAGACCGGGGcTTAACCCCGGTTcTGCAtTCGATACgGGCTgGCTAGAGTgTGGTAgGGGAGATCgGAAtTCCTgGTGTAGCgGTGAAATGCGCAGAtATCAgGAGGAACACCgGTgGCGAAGGCGGaTCTCTgGGCCATTaCTGACGCTGAGGAgCGAAAGCGTGGGGAGCGAACAGGATTAGATaCCCTGGTAGTCCACGCCGTAAACGGTGGGAACTAGGTGTTGGCGACATTCCACGTCGTCGgTGCCGCAGCTAACGCATTAAGTtCCCCGCCTGGGGAGTACGgCCGCAAGGCTaAAACTCaAAGGAATTGACGGGGGCCCGCACAaGCAGCgGAGCATGTGGCTTAATTCGACGCAaCGCGAAGAACCTTACCAAGGCTTGACATCACCCGGAAAGCATTAGAGATAGTGCCCCCCTTGTGGTCGGGTGACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCCCGTGTTGCCAGCAACTTCTTCGGAAGGTTGGGGACTCACGGGAGACCGCCGGGGTCAACTCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGCCCCTTATGTCTTGGGCTGCACACGTGCTACAATGGCCGGTACAATGAGCTGCGATACCGCAAGGTGGAGCGAATCTCAAAAAGCCGGTCTCAGTTCGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTTGCTAGTAATCGCAGATCAGCATTGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCACGAAAGTCGGTAACACCCGAAGCCGGTGGCCCAACCCCTTGTGGGAGGGAGCTGTCGAAGGTGGGACCAGCGATTGGGACGAAGTCGTAACAAGGTAGCCGTACCGGAAGGTGCGGCTGGATCACCTCCT。

Claims (8)

1. a preparation method of a burley tobacco fermented spice is characterized by comprising the following steps:

(1) Preparing a streptomyces nicotianae N35 microbial inoculum and a rhizosphere Qidun bacillus yh7-1 microbial inoculum;

(2) Taking burley tobacco powder as a substrate, and performing combined fermentation treatment on the burley tobacco powder by adopting an actinomycete streptomyces tabacum N35 microbial inoculum and a bacterium rhizobacteria ky 7-1 microbial inoculum to obtain fermented burley tobacco;

(3) And (3) carrying out ethanol reflux extraction on the fermented burley tobacco, and filtering and concentrating to obtain the burley tobacco fermented spice.

2. The method for preparing a burley tobacco fermented flavor according to claim 1, wherein the step (1) specifically comprises: inoculating streptomyces nicotianae N35 liquid strain into a fermentation culture medium according to the inoculation amount of 5-35%, and performing shake culture at 15-55 ℃ for 3-15 days to obtain a culture solution; centrifugally separating the culture solution, washing the precipitate with sterile water, and finally uniformly shaking the precipitate with sterile water to dilute the precipitate by 5 to 20 times to obtain the streptomyces nicotianae N35 microbial inoculum;

inoculating rhizosphere Qidun bacillus yh7-1 liquid strain into a fermentation culture medium according to the inoculation amount of 5-30%, and performing shake culture at 10-50 ℃ for 3-10 days to obtain a culture solution; and (3) centrifugally separating the culture solution, washing the precipitate with sterile water, uniformly shaking the precipitate with sterile water, and diluting the precipitate by 3 to 15 times to obtain the rhizosphere Qidun bacillus yh7-1 microbial inoculum.

3. The method for preparing a burley tobacco fermented flavor according to claim 1, wherein the streptomyces nicotianae N35 in the step (1) is classified and named as streptomyces nicotianae N35, latin literature name: streptomyces tabaciN35 which has been preserved in China general microbiological culture Collection center (CGMCC) at 10/13 of 2021 with the preservation number of CGMCC4.7701;

rhizosphere kirton bacillus yh7-1, the microbiological classification of which is named rhizosphere kirton bacillus yh7-1, latin literature name: chthonobactridiosphaeraeyh 7-1 is preserved in China general microbiological culture Collection center (CGMCC) at 27.5.2020, with the preservation number of CGMCC1.17236.

4. The method for preparing a burley tobacco fermented flavor according to claim 1, wherein the step (2) specifically comprises: the dried burley tobacco leaves are crushed into powder with the grain diameter of 30-80 meshes, and the moisture is balanced to 10-13%. The streptomyces nicotianae N35 bacteria and rhizosphere Qidun bacillus yh7-1 bacteria agent are mixed according to the proportion of 1:1, spraying 10-50 mL of microbial inoculum in every 100g of burley tobacco powder, uniformly mixing, and fermenting the treated burley tobacco powder in a constant-temperature and constant-humidity box with the temperature of 22 ℃ and the concentration of 60% for 48-96 h to obtain the fermented burley tobacco.

5. The method for preparing a burley tobacco fermented flavor according to claim 1, wherein the step (3) specifically comprises: adding ethanol with the concentration of 80-95 percent into the burley tobacco which is 5-10 times of the mass of the fermented burley tobacco, extracting for 1-3 hours under the condition of hot reflux at the temperature of 50-80 ℃, filtering the obtained clear liquid and concentrating to obtain the burley tobacco fermented spice.

6. Use of a burley tobacco fermented flavor obtained by the preparation method according to any one of claims 1 to 5, wherein the burley tobacco fermented flavor is applied to cut tobacco of a cigarette to obtain a cigarette containing the burley tobacco fermented flavor.

7. Use according to claim 1, characterized in that step (4) comprises in particular: the burley tobacco fermented spice is diluted by 3 to 5 times of propylene glycol or ethanol by 0.3 to 0.6 percent of the mass of the cigarette tobacco, and then is sprayed on the cigarette tobacco in a spraying way to obtain the cigarette containing the burley tobacco fermented spice.

8. A cigarette comprising a burley tobacco fermented flavor, which is produced by the production method according to any one of claims 1 to 5.

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