Method for producing tobacco flavor through solid state fermentation of waste tobacco leaf tray

Abstract

The invention discloses a method for producing tobacco flavor through solid state fermentation of a waste tobacco tray, and belongs to the technical field of tobacco production. According to the method, bacillus subtilis P7 and bacillus velezensis L8 with relatively high protease and amylase activity are screened from southern Anhui tobacco leaves, and the bacillus subtilis and aroma-producing yeast are mixed and sprayed on the surfaces of the waste tobacco leaves in a tray, so that good solid-state fermentation can be realized. When the process is used for treating the waste tobacco leaves, nicotine can be reduced by 60% or above, a large number of flavor substances capable of being used for cigarettes are generated, meanwhile, generated solid waste can be directly used for organic fertilizer, high-added-value comprehensive resource utilization of the waste tobacco leaves is achieved, and pollution to the environment is reduced.

Description

Technical Field

[0001] The present invention relates to a method for producing tobacco flavor by solid-state fermentation of waste tobacco leaves in shallow trays, belonging to the technical field of tobacco production. Background Art

[0002] China is a major tobacco-growing country in the world. The annual output of tobacco leaves is about 3 million tons, of which about 25% of tobacco leaves, tobacco dust and other scraps cannot be reasonably utilized and cannot be used for cigarette production. If waste tobacco leaves are directly discarded or burned, it will cause waste of resources and environmental pollution. At present, many studies have focused on extracting solanesol and nicotine from waste tobacco leaves. Solanesol, which accounts for 0.5%-1% in tobacco leaves, is an intermediate for synthesizing new drugs such as coenzyme Q10 and vitamin K2. Nicotine, commonly known as nicotine, is an important raw material for green pesticides and biological medicines and is also the main component of e-cigarettes. Currently, the methods for extracting nicotine from tobacco leaves include steam distillation, solvent extraction, ion exchange, ultrasonic-assisted extraction, microwave-assisted extraction, and supercritical CO2 extraction, etc., each having its own advantages and disadvantages. Many scholars have also studied the extraction of active ingredients such as chlorogenic acid, oxalic acid, protein, rutin, and polysaccharides from waste tobacco leaves. There are also studies on using waste and inferior tobacco leaves to prepare activated carbon by burning or producing tobacco sheets after pretreatment. Tobacco leaves are rich in sugars, so some scholars have studied using waste tobacco leaves to ferment fuel ethanol. In addition, some scholars have used waste tobacco leaves as substrates to produce products such as cellulase, bacterial cellulose, and 2,3-butanediol through microbial fermentation. In addition, due to the large amount of nutrients in waste tobacco leaves, they can be mixed and fermented with blue algae, kitchen waste, chicken manure, etc. to prepare organic fertilizers, or can be used to cultivate Pleurotus ostreatus instead of cottonseed hulls.

[0003] Flavoring and dosing are key processes in cigarette manufacturing. At present, tobacco flavorings are mainly classified into three categories according to their sources: First, flavorings derived from tobacco itself, such as tobacco essential oil, extract, etc.; second, natural plant flavorings from non-tobacco sources, such as essential oils, extracts, tinctures, etc. extracted from the flowers, fruits, roots, stems, and leaves of various plants; third, synthetic flavorings, such as monomeric flavorings like alcohols, aldehydes, and ketones. In addition, applying microbial fermentation engineering to the development of tobacco flavorings is a brand-new approach. For example, Zhengzhou University used discarded tobacco leaves as raw materials, inoculated them with aroma-producing microorganisms, and after fermentation, 63 aroma components were collected and identified. Hubei China Tobacco Company used the aroma-producing yeast WY803 to treat tobacco leaf fragments and obtained a characteristic tobacco extract, which can increase the smoke concentration, enrich the tobacco aroma, improve the fineness and sweetness of the tobacco aroma, and endow the cigarette aroma with a special mellow and sweet fragrance. Yunnan China Tobacco Company isolated a microorganism named Lodderomyces elongisporus, inoculated it in a culture medium with tobacco as the raw material for fermentation, and natural flavors could be obtained, containing flavor components such as phenethyl alcohol, 4-hydroxybutyrolactone, dibutyl phthalate, etc., which can increase the fresh aroma, improve comfort, reduce irritation, and mask off-flavors. In 2014, the Chinese Academy of Agricultural Sciences found that Saccharomyces cerevisiae can produce 2-phenylethanol from tobacco waste. In 2015, Guizhou University isolated food-grade microorganisms from the lees of Moutai liquor and Fuzhuan tea in Meitan, and developed natural tobacco flavorings by fermenting plant materials. It was found that fermenting tea powder with Saccharomyces cerevisiae and Eurotium cristatum can obtain a flavoring with tea aroma, wine aroma, honey-sweet aroma, and special aroma. In 2017, Aureobasidium pullulans OF01 screened by Yunnan China Tobacco could utilize discarded tobacco. The fermentation broth was extracted and concentrated to prepare tobacco-derived aroma flavorings. After fermentation, the aroma components increased by 28%, and the main flavor components were benzaldehyde, 3-hydroxy-2-butanone, solanone, etc. Recently, Zhengzhou University of Light Industry used solid-state fermentation technology, added exogenous aroma-producing strains, and fermented low-grade tobacco leaves under the condition of independently controlling temperature and humidity. The quality of low-grade tobacco leaves was improved, the aroma quality and aroma quantity were increased, the irritation was reduced, the off-odors were decreased, the smoke was made milder, the aftertaste was improved, and the overall sensory quality was enhanced.

[0004] Although a large number of studies have reported that using various microorganisms to ferment tobacco leaves or discarded tobacco leaves can obtain good flavor substances. However, most of these studies were carried out in shake flasks, and it was impossible to consider that the discarded shredded tobacco leaves were piled up together and could not obtain good ventilation and stirring, so good practical applications were not obtained, resulting in a large amount of discarded tobacco leaves being directly burned, and only a small part was utilized. Summary of the Invention

[0005] To solve the above problems, the present invention provides a method for producing tobacco flavor by solid-state fermentation of waste tobacco leaves in shallow trays. Bacillus strains with high protease and amylase activities are screened from waste tobacco leaves produced in southern Anhui, and then sprayed on the surface of waste tobacco leaves together with yeast. Solid-state fermentation is carried out on a temperature- and humidity-controlled shallow tray. Microorganisms can make good use of the sugars and proteins in tobacco leaves, remove the unpleasant odors of tobacco leaves, and produce better flavor components. The concentrated extract of the flavor substances prepared by the present invention can be applied to cigarette production. At the same time, the nicotine content in the fermented tobacco leaf waste is reduced and can be used as organic fertilizer, thus realizing the comprehensive utilization of waste tobacco leaves.

[0006] To achieve the purpose, the present invention adopts the following technical solutions:

[0007] The first object of the present invention is to obtain microbial strains with high amylase and protease activities, and the strains are screened from waste tobacco leaves produced in southern Anhui. The screened strains include a first microbial strain and a second microbial strain.

[0008] The first microbial strain is deposited in the General Microbiological Center of the China National Center for Culture Collection of Microorganisms, classified and named as Bacillus velezensis, with the deposit number: CGMCC No. 32738, and the deposit date is November 22, 2024. Hereinafter, it is simply referred to as Bacillus velezensis L8, and the deposit address: Beijing, China.

[0009] The second microbial strain is deposited in the General Microbiological Center of the China National Center for Culture Collection of Microorganisms, classified and named as Bacillus subtilis, with the deposit number: CGMCC No. 32739, and the deposit date is November 22, 2024. Hereinafter, it is simply referred to as Bacillus subtilis P7, and the deposit address: Beijing, China.

[0010] The degradation rates of starch and protein in tobacco leaves by the said Bacillus velezensis L8 reach 17% and 14% respectively; the degradation rates of starch and protein in tobacco leaves by the said Bacillus subtilis P7 reach 15% and 10% respectively.

[0011] The second object of the present invention is to provide a method for producing tobacco flavor by solid-state fermentation of waste tobacco leaves in shallow trays. Specifically, the screened Bacillus strains are mixed with aroma-producing yeast, and the mixture is sprayed on the surface of waste tobacco leaves together, and mixed-bacteria solid-state fermentation with temperature and humidity control is carried out in a shallow tray to reduce the nicotine content in tobacco leaves and produce better flavor substances, so as to obtain tobacco flavor.

[0012] The aroma-producing yeast mentioned above is Pichia anomala or Hansenula anomala. When the first microbial strain Bacillus velezensis L8 is used, the aroma-producing yeast is Hansenula anomala; when the second microbial strain Bacillus subtilis P7 is used, the aroma-producing yeast is Pichia anomala.

[0013] The most preferred process conditions for the solid-state fermentation are as follows: the volume ratio of the microbial strain to the aroma-producing yeast broth is 1:1, the fermentation temperature is 30 °C, and the relative humidity is 65%.

[0014] The beneficial effects of the present invention are as follows:

[0015] In the present invention, the Bacillus strains with high amylase and protease activities screened from the waste tobacco leaves produced in southern Anhui are mixed with the aroma-producing yeast and applied to the shallow tray solid-state fermentation of waste tobacco leaves, which can reduce the nicotine content in the tobacco leaves. At the same time, good aroma substances are produced. The tobacco extract produced by concentration extraction can be applied to cigarette production, while the residual tobacco leaf solid waste can be used for organic fertilizer production, realizing the high-value comprehensive utilization of waste tobacco leaves. Specifically:

[0016] 1. The two Bacillus strains screened in the present invention have high protease and amylase activities, and can be mixed with the aroma-producing yeast and applied to the solid-state fermentation of waste tobacco leaves, reducing the nicotine content by more than 60%;

[0017] 2. Through the solid-state fermentation of the mixed bacteria of Bacillus strains - aroma-producing yeast in the present invention, the miscellaneous odors of the tobacco leaves can be reduced, and the contents of aroma components such as β-ionone, megastigmatrienone, and phenylethyl alcohol can be increased;

[0018] 3. The shallow tray solid-state fermentation adopted in the present invention can take into account the temperature control and humidity control of the tobacco leaf fermentation, and is convenient for large-scale production;

[0019] 4. The nicotine content of the solid waste finally formed by the process adopted in the present invention is relatively low and can be used as an organic fertilizer. Description of the Drawings

[0020] Figure 1 It is a circular diagram of the degradation index of different strains in Example 1;

[0021] Figure 2 It is the rescreening enzyme activity of different strains in Example 2;

[0022] Figure 3 It is a phylogenetic tree of strains based on the 16S rDNA sequence in Example 3;

[0023] Figure 4 It is a graph of the content change of substances in single-strain fermentation in Example 4;

[0024] Figure 5For the types and contents of various compounds under different fermentation conditions of Bacillus subtilis P7 and Pichia anomala in Example 5;

[0025] Figure 6 For the types and contents of various compounds under different fermentation conditions of Bacillus velezensis L8 and Hansenula anomala in Example 6. Detailed implementation manners

[0026] For a better understanding of the technical features, objectives and beneficial effects of the present invention, the present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, but the present invention is not limited to the following embodiments.

[0027] The waste tobacco leaves in the following embodiments are from the waste tobacco leaves in southern Anhui of China Tobacco Anhui Industrial Co., Ltd.

[0028] The Pichia anomala (preservation number: ACCC No. 20233) and Hansenula anomala (preservation number: CGMCC No. 2.302) in the following embodiments are purchased from the Guangdong Microbial Strain Preservation Center.

[0029] The method for measuring amylase activity in the following embodiments is: using the 3,5-dinitrosalicylic acid method. Take 1 mL of crude enzyme solution, preheat it in a water bath at 65 °C for 5 min, add 1 mL of a 1% starch-containing pH 6.0 phosphate-citrate buffer solution, keep it in a constant-temperature water bath at 65 °C for 30 min, add 2 mL of DNS to terminate the reaction, heat it in a boiling water bath for 5 min, immediately cool it with ice water, and make up the volume to 10 mL with water. Measure the absorbance at a wavelength of 540 nm. The amount of enzyme required to produce 1 μg of glucose per minute by 1 mL of crude enzyme solution under the conditions of 65 °C and pH 6.0 is defined as one enzyme activity unit, expressed as U / mL.

[0030] The method for measuring protease activity in the following embodiments is: using the national standard Folin-phenol method. First, put the casein solution into a constant-temperature water bath at 40 °C ± 0.2 °C and preheat it for 5 min. Take 1 mL of crude enzyme solution, preheat it in a water bath at 40 °C for 2 min, add 1 mL of the preheated casein solution, and accurately incubate it for 10 min. After the time is up, immediately add 2 mL of 0.4 mol / L trichloroacetic acid to terminate the reaction, take it out and let it stand for 10 min, and then filter to obtain the filtrate. In the blank group, 0.4 mol / L trichloroacetic acid is added to inactivate the enzyme before adding the casein solution, and then the casein solution is added. The other operations are the same as those in the experimental group. Take 1 mL of the filtrate, add 5 mL of 0.4 mol / L Na2CO3, add 1 mL of the Folin reagent working solution, shake well, keep it at 40 °C for incubation and color development for 20 min, and measure the absorbance at a wavelength of 680 nm. The amount of enzyme required to hydrolyze casein to produce 1 μg of tyrosine per minute by 1 mL of crude enzyme solution at 40 °C is 1 enzyme activity unit, expressed as U / mL.

[0031] Example 1: Screening of tobacco leaf microorganisms

[0032] Weigh 5.0 g of tobacco leaves from different regions with a balance with an accuracy of 0.01 g. After cutting them into pieces with a sterilized scissors, add them to a conical flask containing 100 mL of sterilized distilled water, and place appropriate sterile glassware. Shake well at 37 °C and 200 r·min -1 Under the condition. After culturing for 12 h, filter with sterile gauze and take the filtrate as the bacterial suspension. Dilute the bacterial suspension according to the ten-fold gradient dilution method, and pipette 100 μL of the diluted bacterial suspension with dilution factors of 10 -2 、10 -3 、10 -4 、10 -5 、10 -6 onto the corresponding solid medium plates until dry, and then invert them in an incubator until single colonies appear on the plates. Pick single colonies with different morphologies for streak purification for subsequent experiments. A total of 72 strains were isolated from tobacco leaves in regions such as southern Anhui, Kunming, Yuxi, Zunyi, and Zimbabwe by gradient dilution combined with plate coating method. Use a sterilized toothpick to pick single colonies and spot them on protease, amylase, and pectinase screening plates respectively, observe whether there is a clear zone, and measure the diameters of the colonies and the clear zones respectively. The ratio of the two is the degradation index I. Select the enzyme-producing strains according to the degradation index for subsequent rescreening.

[0033] Plot the strains with the potential to degrade starch and protein among the microorganisms isolated and purified from the tobacco leaf surface and their degradation indices for starch and protein in the clear zone test on Figure 1 . Among the screened strains, about 48% of the strains can produce clear zones. Generally speaking, the degradation ability of the screened strains for protein is generally better than that for starch. The degradation abilities of different strains for the two substances vary greatly. The starch degradation index of strain D10P2 is nearly 4.9 times larger than that of strain D3P1, and the protein degradation index of strain D1P2 is about 8 times larger than that of strain D10P2. And it can be found that some strains only have the degradation ability for one of the substances, or the degradation abilities for the two substances are extremely different, such as D10P2. Considering that there are various factors affecting the growth and degradation ability of strains in the clear zone test, such as the difference in the agar content in the plates poured in the early and late stages during the medium pouring process, different strains have different growth and enzyme production rates, the size of the bacterial lawn and the stacking situation on the plate, etc. Therefore, it is very necessary to select relatively dominant strains from the strains that can produce clear zones for subsequent fermentation rescreening.

[0034] Example 2: Rescreening of fermentation microorganisms from waste tobacco leaves

[0035] The strains obtained from the primary screening were activated and inoculated into a liquid medium, and cultured in a shaker at 37 °C and 200 r·min -1 for 24 h to obtain a seed solution. The seed solution was inoculated into 50 mL of liquid medium at an inoculation amount of 5%, and cultured in a shaker at 37 °C and 200 r·min -1 for 48 h. After the culture was completed, centrifugation was carried out at 4 °C and 8000 r, and the supernatant was the crude enzyme solution. As Figure 2 can be seen, the enzyme activity rescreening results of most strains can correspond to the trends obtained from the clear zone test. However, it can be found that in the clear zone test, the degradation index of the strains for proteins is generally higher than that for starch. Theoretically, the strains should generally have higher protease enzyme activities. The results of the enzyme activity rescreening experiment showed that except for D0L5, D0L8, D1P2, D2P1, D9P2, and D9P3, the amylase enzyme activities of other strains were higher than the protease enzyme activities. Even for D10P2, D3P1, and D7P2, no protease enzyme activity was detected. And very few strains that showed good enzyme-producing ability in the clear zone test did not get good results in the fermentation rescreening. For example, for strain D10P2 with a starch degradation index as high as 6.042 ± 1.208 in the clear zone test, its amylase enzyme activity results were not dominant. This also indicates that the size of the clear zone may be able to reflect the enzyme concentration entering the screening plate, but it cannot fully represent the enzyme-producing ability of the strains. At the same time, the differences in the solid culture and liquid culture conditions may also have a certain impact on the enzyme-producing ability of the strains. Finally, five strains with good performance in both the clear zone primary screening and the fermentation rescreening experiments were selected as D0L5, D0L8, D0P7, D1P2, and D9P3.

[0036] Example 3: Strain identification

[0037] The identification of the strains was carried out by the method of 16S rDNA sequence analysis. The bacterial universal primers 27F and 1492R were used to amplify 16S rDNA, and the amplification products were sequenced by Wuxi Tianlin Biotechnology Co., Ltd. After comparison, similar sequences with high homology were found, and the Mega 11 software was used to perform phylogenetic analysis by the neighbor-joining method to draw a phylogenetic tree, as Figure 3 shown. The 16S rDNA sequence homology similarity of D1P2 and Bacillus amyloliquefaciens Kt7-3 reached 93%, and it was identified as Bacillus amyloliquefaciens; the 16S rDNA sequence homology similarity of L8 (i.e., the above-mentioned D0L8) and Bacillus velezensis JS27A reached 94%, and it was identified as Bacillus velezensis; the 16S rDNA sequence homology similarity of L5 (i.e., the above-mentioned D0L5) and Bacillus siamensis YJ15 reached 94%, and it was identified as Bacillus siamensis; the 16S rDNA sequence homology similarity of D9P3 and P7 (i.e., the above-mentioned D0P7) with Bacillus subtilis G-13 and Bacillus subtilis CS10 reached 99%, and they were identified as Bacillus subtilis.

[0038] Example 4: Solid-state fermentation of tobacco leaves with a single strain in a shallow tray

[0039] Activate the strains D0L5, D0L8, D1P2, D9P3, and D0P7, and obtain the secondary seed liquid through cultivation. The initial moisture content of the tobacco leaf fermentation raw material will affect the growth of the bacteria. Excessive moisture content will cause the medium to cake and be not easy to ventilate, while too low moisture content cannot meet the growth of the bacteria. Research shows that a moisture content of 30% is more appropriate. Therefore, weigh 50 g of tobacco leaves in advance and place them in a self-sealing bag, and add an appropriate amount of sterile water to pre-equilibrate for 48 h. After the equilibration, stack them about 5 cm on a 45-mesh stainless steel screen, and the inoculation amount is 30% (OD 600 ≈0.8), and place them in a constant temperature and humidity incubator at 30 °C and a relative humidity of 75% for fermentation for 3 days, and turn them over every 8 - 10 h. The degradation of protein and starch was investigated. As Figure 4 can be seen, there are significant differences in the starch degradation rates of the tobacco leaf samples fermented by different strains in solid state. L8 and P7 can reach more than 15%, and the degradation rates of protein are basically around 10%.

[0040] Example 5: Solid-state fermentation with mixed bacteria in a shallow tray and on a screen

[0041] Culture Bacillus subtilis P7 and Pichia anomala separately with PDA liquid medium, and spray the bacterial liquid on the surface of the waste tobacco leaves. Stack the waste tobacco leaves in a shallow tray with a diameter of about 20 cm, and the stacking thickness is 3 cm. Mix the two different strains and pour them into a sprayer for spraying. Then put them into a constant temperature and humidity incubator for cultivation for 3 - 5 days. Explore the effects of the mixed bacteria ratio (CP21, CP11, CP12, that is, the volume ratios of the bacterial liquid of Pichia anomala to Bacillus subtilis P7 are 2:1, 1:1, and 2:1 respectively), fermentation temperature (25 °C, 30 °C, 35 °C), and fermentation humidity (55%, 65%, 75%). Use GC-MS to determine the aroma components. The aroma components of the mixed bacteria fermentation at different humidities are shown in Table 1, the aroma components of the mixed bacteria fermentation at different temperatures are shown in Table 2, and the aroma components of the mixed bacteria fermentation at different mixed bacteria ratios are shown in Table 3. The types and contents of various compounds under different fermentation conditions are as Figure 5 shown.

[0042] There are differences in the types and contents of the aroma components detected in the tobacco leaves fermented by solid state under different conditions. Whether from the perspective of types or quantities, the fermentation temperature has the greatest impact on the fermentation effect. At 30 °C, both the types and contents are the highest. From the results of the fermentation with different mixed bacteria ratios, it can be seen that the total types of the aroma components do not differ much, but it can be found that there are still significant differences in the specific contents of a certain type of substance. When fermenting at different relative humidities, the contents of various substances also have differences, especially for ketones and aldehydes.

[0043] Table 1. Contents of Main Aroma Components in Solid-State Fermentation Samples with Different Humidities

[0044]

[0045] Table 2. Contents of Main Aroma Components in Solid-State Fermentation Samples with Different Temperatures

[0046]

[0047] Table 3. Contents of Main Aroma Components in Solid-State Fermentation Samples with Different Mixed Bacteria Ratios

[0048]

[0049] Example 6: Solid-State Fermentation with Mixed Bacteria in Shallow Trays with Sieve Nets

[0050] Bacillus velezensis L8 and Hansenula anomala were cultured separately in PDA liquid medium, and then sprayed on the surface of waste tobacco leaves in different proportions. The waste tobacco leaves were piled up in shallow trays with a diameter of about 20 cm and a stacking thickness of 3 cm. The two different strains were mixed and poured into a sprayer for spraying. Then it was placed in a constant temperature and humidity incubator for culturing for 3 - 5 days. The effects of the mixed bacteria ratio (CL21, CL11, CL12, that is, the volume ratios of Hansenula anomala to Bacillus velezensis L8 bacterial liquid are 2:1, 1:1, 1:2 respectively), fermentation temperature (25 °C, 30 °C, 35 °C), and fermentation humidity (55%, 65%, 75%) were explored. The aroma components were determined by GC-MS. The aroma components of solid-state fermentation with different humidities are shown in Table 4, the aroma components of solid-state fermentation with different temperatures are shown in Table 5, the aroma components of solid-state fermentation with different mixed bacteria ratios are shown in Table 6, and the types and contents of various compounds under different fermentation conditions are as Figure 6 shown

[0051] There are differences in the types and contents of aroma components detected in tobacco leaves after solid-state fermentation under different conditions. Whether from the perspective of types or quantities, the temperature still has the greatest influence on the fermentation effect. At 30 °C, both the types and contents are the highest

[0052] Table 4. Contents of Main Aroma Components in Solid-State Fermentation Samples with Different Humidities

[0053]

[0054] Table 5. Contents of Main Aroma Components in Solid-State Fermentation Samples with Different Temperatures

[0055]

[0056] Table 6. Contents of Main Aroma Components in Solid-State Fermentation Samples with Different Mixed Bacteria Ratios

[0057]

[0058] Although the present invention has been disclosed above in preferred embodiments, it is not intended to limit the present invention. Anyone skilled in this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A microbial strain having high amylase and protease activity, characterized in that: The microbial strain is a first microbial strain or a second microbial strain; The first microbial strain is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, and is classified and named Bacillus velezensis, with a deposit number of CGMCC No.32738 and a deposit date of November 22, 2024; The second microbial strain is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, classified and named Bacillus subtilis, with the deposit number: CGMCC No.32739, and the deposit date is November 22, 2024.

2. Use of the microbial strain according to claim 1 in the shallow tray solid-state fermentation of discarded tobacco leaves.

3. A method for producing tobacco flavorings by solid-state fermentation of discarded tobacco leaves in shallow trays, characterized in that: The microbial strain described in claim 1 is mixed with aroma-producing yeast, and the mixture is sprayed on the surface of waste tobacco leaves, and mixed bacteria solid-state fermentation is carried out in a shallow dish to prepare tobacco flavoring.

4. The method for producing tobacco flavorings by solid-state fermentation of discarded tobacco leaves in shallow trays according to claim 3, characterized in that: When the first microbial strain is used, the aroma-producing yeast is Hansenula anomala; when the second microbial strain is used, the aroma-producing yeast is Pichia anomala.

5. The method for producing tobacco flavoring by solid-state fermentation of discarded tobacco leaves in shallow trays according to claim 3 or 4, characterized in that: The solid-state fermentation conditions are as follows: the volume ratio of the microbial strain to the aroma-producing yeast is 1:1, the fermentation temperature is 30° C., and the relative humidity is 65%.

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