Coral hairy sporidium, fungicide, preparation method and application

Abstract

The invention provides coral hairy sporidium, a fungicide, a preparation method and application, and belongs to the technical field of microbial fermentation and tobacco processing. The preservation number of the coral coremiforme SC-11 provided by the invention is CGMCC (China General Microbiological Culture Collection Center) No. 34235, and the preservation number of the coral coremiforme SC-11 is CGMCC No. 34235. The provided strain can increase the content of carotenoid degradation products, Maillard reaction products, chlorophyll degradation products, terpenoids and the like, and increase the total content of aroma components of tobacco leaves; nicotine and derivatives (nornicotine, dehydroneonicotine and the like) of nicotine can be degraded, the content of alkaloid substances can be effectively reduced, and smoke irritation is reduced; the metabolite can reconstruct the aroma characteristics of cigar tobacco leaves, highlight the multiple aroma levels of honey sweet aroma, resin aroma, flower aroma, coffee aroma and the like, and remarkably improve the soft feeling and fineness of smoke at the same time.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microbial fermentation and tobacco processing, and relates to a Trichosporon coremiiforme, a microbial agent, a preparation method and application. BACKGROUND

[0002] Fermentation of cigar tobacco leaves is a key process link for determining the sensory quality and style characteristics. In recent years, the intensified fermentation technology based on the directional regulation of functional microorganisms has gradually become a research hotspot. By screening strains with specific metabolic activity, the degradation of macromolecular substances in tobacco leaves can be accelerated, the generation of aroma components can be promoted, and the content of harmful substances such as alkaloids can be reduced, so as to improve the usability and smoking quality of tobacco leaves. However, at present, the types of functional microorganisms that can be used for the fermentation of cigar tobacco leaves are limited, and there are generally problems such as insufficient increase of aroma components and low degradation efficiency of harmful substances, which restrict the development of high-quality cigar products. SUMMARY

[0003] Therefore, one or more embodiments of the application provide a Trichosporon coremiiforme, a microbial agent, a preparation method and application to improve the content of aroma components in cigar tobacco leaves and reduce the content of harmful substances in cigar tobacco leaves.

[0004] In some embodiments, a Trichosporon coremiiforme SC-11 is provided, and the preservation number is CGMCC No. 34235.

[0005] In some embodiments, a preparation method of the Trichosporon coremiiforme SC-11 is provided, and the preparation method comprises the step of fermenting the Trichosporon coremiiforme SC-11 with the preservation number of CGMCC No. 34235.

[0006] In some embodiments, the preparation method of the Trichosporon coremiiforme SC-11 meets one or more of the following conditions:

[0007] (1) The fermentation medium comprises water and peptone, yeast powder and glucose, and 20g-30g of peptone, 10g-15g of yeast powder and 20g-30g of glucose are contained in 1000mL of water;

[0008] (2) The fermentation temperature is 28°C-30°C;

[0009] (3) The fermentation is carried out under stirring, and the stirring speed is 180rpm-220rpm; and

[0010] (4) The fermentation time is 1-2 days.

[0011] In some embodiments, a bacterial agent containing the said Mortiella chesnei SC-11 is provided.

[0012] In some embodiments, a method for preparing fermented tobacco leaves is provided, comprising the steps of: mixing the said Mortiella chesnei SC-11 or the said bacterial agent with tobacco leaves, and performing fermentation treatment to prepare fermented tobacco leaves.

[0013] In some embodiments, in the said method for preparing fermented tobacco leaves, a bacterial solution containing the said Mortiella chesnei SC-11 is mixed with the said tobacco leaves, wherein the concentration of the said Mortiella chesnei SC-11 in the bacterial solution is 10 6 × 10 8 / mL, and the mass ratio of the bacterial solution to the tobacco leaves is (15-25): 100.

[0014] In some embodiments, the said method for preparing fermented tobacco leaves satisfies one or more of the following conditions:

[0015] (1) the water content of the cigar tobacco leaves is 30wt%-35wt%;

[0016] (2) the fermentation treatment temperature is 30℃-35℃, and the fermentation treatment time is 15 days-30 days;

[0017] (3) the relative humidity of the fermentation treatment is 70%-75%; and

[0018] (4) the tobacco leaves include cigar tobacco leaves.

[0019] In some embodiments, a fermented tobacco leaf prepared by the said method is provided.

[0020] In some embodiments, a cigar containing the said fermented tobacco leaf is provided.

[0021] In some embodiments, a method for preparing a cigar is provided, comprising:

[0022] preparing fermented tobacco leaves by the said method; and,

[0023] preparing a cigar using raw materials containing the said fermented tobacco leaves.

[0024] The Trichosporon coremiiforme provided can improve the content of carotenoid degradation products, Maillard reaction products, chlorophyll degradation products, terpenes and the like in the fermentation process of cigar tobacco leaves, improve the total content of aroma components of the tobacco leaves, degrade nicotine and its derivatives (nornicotine, dehydronicotine, etc.), effectively reduce the content of alkaloid substances, reduce the irritability of smoke, and reconstruct the aroma characteristics of the cigar tobacco leaves, highlight the multi-aroma levels of honey sweet, resin, flower and coffee, and significantly improve the softness and delicacy of smoke. BRIEF DESCRIPTION OF DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present application, more completely understand the present application and its beneficial effects, the drawings needed to be used in the embodiment description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0026] Figure 1 The figure of sensory evaluation results of cigar of the cigar obtained from Example 2 and Comparative Example 1 is shown, wherein a is mass property; b is aroma characteristics.

[0027] The Trichosporon coremiiforme SC-11 provided in the present application has a taxonomic name of Trichosporon coremiiforme. The strain has been preserved in the China General Microbiological Culture Collection Center, located at No. 1, Beichen West Road, Yard 3, Chaoyang District, Beijing, on April 16, 2025, with a preservation number of CGMCC No. 34235. The strain was received and registered by the preservation center on April 16, 2025, and was detected by the preservation center on April 16, 2025 as a viable strain. DETAILED DESCRIPTION

[0028] The present application will be further described in detail below in combination with the drawings, embodiments and examples. It should be understood that these embodiments and examples are only used to illustrate the present application and not to limit the scope of the present application, and the purpose of providing these embodiments and examples is to make the understanding of the disclosed content of the present application more thorough and comprehensive. It should also be understood that the present application can be realized in many different forms and is not limited to the embodiments and examples described herein, and those skilled in the art can make various modifications or changes without departing from the spirit of the present application, and the equivalent forms obtained are also within the protection scope of the present application. In addition, in the following description, a large number of specific details are given in order to provide a more complete understanding of the present application, and it should be understood that the present application can be implemented without one or more of these details.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description herein is for the purpose of describing only the embodiments and examples and is not intended to be limiting of the application.

[0030] Unless otherwise indicated, or unless the context clearly indicates otherwise, terms or phrases used in this application have the following meanings:

[0031] The selection scope of the terms "and / or", "or / and", "and / or" used herein includes any one of two or more relevant listed items, and also includes any and all combinations of relevant listed items, which includes any two relevant listed items, any more relevant listed items, or a combination of all relevant listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", "and / or", it should be understood that in this application, the technical solution undoubtedly includes the technical solution connected by "logical and", and also undoubtedly includes the technical solution connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For another example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C and D (i.e. the technical solution connected by "logical or"), and also includes any and all combinations of A, B, C and D, i.e. includes the combination of any two or any three of A, B, C and D, and also includes the four-item combination of A, B, C and D (i.e. the technical solution connected by "logical and").

[0032] In this application, "multiple", "various", "multiple times", "multiple" and the like are used without specific limitation, which means greater than or equal to 2 in quantity. For example, "one or more" means one or greater than or equal to two.

[0033] The "combination thereof", "any combination thereof", "any combination manner thereof" and the like used herein include all suitable combination manners of any two or more listed items.

[0034] In this application, "suitable combination manner", "suitable manner", "any suitable manner" and the like mean that the technical solution of this application can be implemented, the technical problem of this application can be solved, and the expected technical effect of this application can be achieved.

[0035] In this application, "preferably", "better", "better", "as appropriate" only describe the implementation manner or embodiment with better effect, and it should be understood that it does not constitute a limitation on the protection scope of this application.

[0036] In this application, terms such as "further," "even more," and "particularly" are used for descriptive purposes and to indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0037] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0038] In this application, the terms "first aspect," "second aspect," "third aspect," "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0039] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0040] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0041] Unless otherwise specified, the temperature parameters in this application are permitted to be either constant-temperature treatment or variations within a certain temperature range. It should be understood that the constant-temperature treatment allows temperature fluctuations within the precision range of the instrument control, such as ±5℃, ±4℃, ±3℃, ±2℃, or ±1℃.

[0042] In this application, %(w / w) and wt% both represent weight percentage, %(v / v) refers to volume percentage, and %(w / v) refers to mass-volume percentage.

[0043] All references to this application are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the purpose and / or technical solution of this application, all cited references are incorporated herein by reference in their entirety and for all purposes. When references are cited in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. Examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.

[0044] Trichosporon coremiiforme, a filamentous yeast with broad-spectrum metabolic capabilities, can secrete various enzymes such as lipase, amylase, and protease. It has demonstrated unique biodegradation and synthesis abilities in food and environmental fields, and is used in the production of polyols in baijiu (Chinese liquor) brewing. However, its application in tobacco processing, especially in cigar tobacco fermentation, has not yet been reported.

[0045] In a first aspect of the embodiments of this application, a strain of Trichosporoncoremiiforme SC-11 is provided, which is deposited at the China General Microbiological Culture Collection Center with accession number CGMCC No. 34235.

[0046] The provided *Trichosporium coralum* strain was isolated from the surface of tobacco leaves. It is an indigenous microorganism, safe, and can be used to prepare fermented tobacco leaves.

[0047] The provided *Trichosporium corallis* strain SC-11 can synergistically enhance the overall quality indicators of cigar tobacco, such as aroma richness, complexity, and smoke smoothness.

[0048] The provided strain, *Trichosporon coremiiforme* SC-11, enhances the content of carotenoid degradation products, Maillard reaction products, chlorophyll degradation products, and terpenoids during cigar tobacco fermentation, resulting in a 35.5% increase in the total aroma components of the tobacco. It also reduces the content of nicotine, nornicotine, dehydroneonic acid, cotinine, and N'-acetylnornicotine by 26.2%, 17.4%, 21.4%, 25.4%, and 15.5%, respectively. This significantly improves the aroma quantity, richness, and maturity of cigar tobacco, enhancing the harshness, smoothness, and delicacy of the smoke, and highlighting aroma characteristics such as honey sweetness, resinous notes, floral aromas, and coffee aromas.

[0049] The colony morphology of the provided *Trichosporium corallis* strain SC-11 is round, white, convex, and with irregular edges.

[0050] In some embodiments, a method for preparing *Trichosporium coraltum* SC-11 is provided, including a fermentation step of *Trichosporium coraltum* SC-11 with accession number CGMCC No. 34235.

[0051] In some embodiments, the fermentation culture medium for preparing Coral Polyporus SC-11 includes water, peptone, yeast powder, and glucose, with each 1000 mL of water containing 20 g to 30 g of peptone, 10 g to 15 g of yeast powder, and 20 g to 30 g of glucose.

[0052] In some embodiments, the fermentation temperature in the preparation method of Coral Polyporus Trichosporium SC-11 is 28°C to 30°C. For example, the fermentation temperature can be 28°C, 29°C, 30°C, etc., or it can be any range of two of the aforementioned values.

[0053] In some embodiments, in the preparation method of Coral Polyporus Trichosporium SC-11, fermentation is carried out under stirred conditions.

[0054] In some embodiments, the stirring speed in the preparation method of Coral Polyporus Trichosporium SC-11 is 180 rpm to 220 rpm. For example, the stirring speed can be 180 rpm, 190 rpm, 200 rpm, 210 rpm, 220 rpm, etc., or it can be any range of two of the aforementioned values.

[0055] In some embodiments, the fermentation time for preparing Coral Polyporus SC-11 is 1 to 2 days.

[0056] In some embodiments, a microbial agent is provided containing *Trichosporium corallis* SC-11.

[0057] In some embodiments, a method for preparing fermented tobacco leaves is provided, comprising the following steps: mixing tobacco leaves with Trichosporium corallis SC-11 or a bacterial agent, and performing fermentation treatment to prepare fermented tobacco leaves.

[0058] In some embodiments, in the method for preparing fermented tobacco leaves, a bacterial solution containing *Trichoderma corali* SC-11 is mixed with tobacco leaves, and the concentration of *Trichoderma corali* SC-11 in the bacterial solution is 10. 6 Cells / mL ~10 8 The bacterial culture concentration is 10⁶ cells / mL, and the mass ratio of bacterial culture to tobacco leaves is (15~25):100. For example, the concentration of *Trichosporium coralli* SC-11 in the bacterial culture can be 10⁶ cells / mL. 6 cells / mL, 10 6cells / mL, 10 8 The ratio of bacteria / mL can be any of the two values ​​mentioned above. The mass ratio of bacterial culture to tobacco leaves can be 15:100, 20:100, 25:100, etc., or any of the two ratios mentioned above.

[0059] In some embodiments, the moisture content of the cigar tobacco leaves in the preparation method of fermented tobacco leaves is 30wt% to 35wt%. For example, the moisture content of the cigar tobacco leaves can be 30wt%, 31wt%, 32wt%, 33wt%, 34wt%, 35wt%, etc., or it can be a range of any two of the aforementioned values.

[0060] In some embodiments, the fermentation temperature in the method for preparing fermented tobacco leaves is 30℃~35℃, and the fermentation time is 15 days~30 days. For example, the fermentation temperature can be 30℃, 33℃, 35℃, etc., or it can be any range of two of the aforementioned values. For example, the fermentation time can be 15 days, 20 days, 25 days, 30 days, etc., or it can be any range of two of the aforementioned values.

[0061] In some embodiments, the relative humidity of the fermentation treatment in the preparation method of fermented tobacco leaves is 70% to 75%. For example, the relative humidity of the fermentation treatment can be 70%, 71%, 72%, 73%, 74%, 75%, etc., or it can be a range of any two of the aforementioned values.

[0062] In some embodiments, the tobacco leaves used in the preparation of fermented tobacco leaves include cigar tobacco leaves.

[0063] In some embodiments, a fermented tobacco leaf is provided, prepared by the aforementioned preparation method.

[0064] In some embodiments, a cigar is provided that contains fermented tobacco leaves.

[0065] In some embodiments, a method for preparing a cigar is provided, the method comprising:

[0066] Fermented tobacco leaves were prepared using the aforementioned preparation method; and,

[0067] Cigars are made from raw materials containing fermented tobacco leaves.

[0068] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0069] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0070] Example 1: Isolation and Identification of Strains

[0071] Take 5g of Dexue No. 1 cigar tobacco leaves from the Great Wall Cigar Factory (Shifang, Sichuan) of Sichuan Tobacco Industry Co., Ltd., and use sterile scissors in a clean bench to cut the tobacco leaves into small pieces. Place the pieces in sterile physiological saline and shake at 30℃ and 200rpm for 30 minutes to wash away bacterial cells. Then, dilute the supernatant and spread it on yeast extract peptone-glucose solid medium. The medium consists of 2% peptone, 1% yeast extract, 2% glucose, and 2% agar powder. Incubate at 28℃ for 24-48 hours. Based on morphological differences, pick single colonies and streak them in three zones on a new plate. Then incubate at 28℃ for 24-48 hours until single colonies appear on the plate.

[0072] Single colonies that are round, white, convex, and have irregular edges were selected, and the resulting strains were subjected to molecular biological identification. The ITS bacterial identification sequence is shown in SEQ ID NO:1.

[0073] SEQ ID NO:1

[0074] .

[0075] The sequencing results were compared with microbial information in the NCBI database using BLAST software to confirm that the obtained strain was Trichosporon coremiiforme.

[0076] The obtained strain was named Trichosporon coremiiforme SC-11 and was deposited on April 16, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 34235.

[0077] Example 2

[0078] (1) Culture of strains: The coral polyps preserved on the slant were inoculated onto yeast extract peptone glucose plate medium and activated at 28°C for 2 days; single colonies after activation were picked and inoculated into yeast extract peptone glucose liquid medium for fermentation culture at 28°C and 200 rpm for 2 days to obtain fermentation broth.

[0079] The formula for yeast extract peptone glucose medium includes: 20g peptone, 10g yeast powder, 20g glucose and 1000mL distilled water. After measuring each substance according to the above formula, mix them evenly and put them into Erlenmeyer flasks (the liquid volume occupies about 20% of the volume of the Erlenmeyer flask). Autoclave for 20 minutes and set aside.

[0080] (2) Preparation of bacterial culture: The fermentation broth was diluted with sterile water to make the concentration of the diluted bacteria 10. 6 The bacterial culture was obtained by measuring 100 cells / mL.

[0081] (3) Fermentation of cigar tobacco leaves: The bacterial solution prepared in step (2) above is inoculated onto the surface of the rehydrated cigar tobacco leaves at a mass ratio of 20% to make the initial moisture content about 30wt%; the tobacco leaves after inoculation are put into burlap bags and placed in a fermentation room for fermentation at a temperature of 35℃, a relative humidity of 75%, and a fermentation time of 15 days.

[0082] (4) After the fermentation is completed, the cigar tobacco sample is dried at 45°C, crushed, and passed through a 0.25mm sieve to obtain the fermented tobacco sample.

[0083] Comparative Example 1

[0084] (1) Fermentation of cigar tobacco leaves: Inoculate the surface of the rehydrated cigar tobacco leaves with pure water at a mass ratio of 20%; after inoculation, put the tobacco leaves into a burlap bag and place them in a fermentation room for fermentation. The fermentation temperature is 35℃, the relative humidity is 75%, and the fermentation time is 15 days.

[0085] (2) After the fermentation is completed, the cigar tobacco sample is dried at 45°C, crushed, and passed through a 0.25mm sieve to obtain the fermented tobacco sample.

[0086] Performance testing

[0087] 1. Detection of physicochemical index content

[0088] The contents of total sugar, reducing sugar, total alkaloids, total nitrogen, potassium ions and chloride ions were determined using an API continuous flow analyzer.

[0089] Table 1. Physicochemical index content of fortified fermented tobacco leaves

[0090]

[0091] As shown in Table 1, compared with the fermentation of cigar tobacco leaves using sterile water in Comparative Example 1, the fermentation of cigar tobacco leaves using *Trichoderma corali* SC-11 resulted in a 4.6% decrease in total nitrogen and a 5.8% decrease in total alkaloids, respectively. Protein directly affects the irritancy of tobacco leaves during combustion; excessively high alkaloid content not only affects human health but also directly impacts the bitterness, astringency, and irritancy of the tobacco leaves. Furthermore, the reducing sugar and total sugar contents decreased by 20.0% and 44.4%, respectively. This may be due to *Trichoderma corali* utilizing sugar compounds in the tobacco leaves, leading to a reduction in total sugar and reducing sugar content. + / Cl - The ratio directly affects the combustibility of tobacco leaves; after fermentation by *Trichosporium corallis*, the k... + / Cl - The ratio increased by 11.4%.

[0092] 2. Aroma component content detection

[0093] The aroma component content of fermented tobacco leaf samples obtained in the examples and comparative examples was determined by gas chromatography-mass spectrometry (GC-MS).

[0094] Table 2. Aroma component content (mg / kg)

[0095]

[0096] Table 2 (continued) (1)

[0097]

[0098] Table 2 continues (2)

[0099]

[0100] The total amount of aroma components is an important indicator of cigar tobacco quality, enriching the aroma characteristics of the tobacco (such as floral, fruity, and woody aromas), and is positively correlated with the maturity of the tobacco leaves. As shown in Table 2 above, compared with the use of sterile water for fermentation of cigar tobacco leaves in Comparative Example 1, the use of *Trichoderma corallis* SC-11 for fermentation increased the total content of aroma components in the tobacco leaves by 35.5%. The aroma substances with significantly increased content mainly include cephalosporin degradation products (solanum ketone, isothiocycloenol), carotenoid degradation products (d-limonene, tetrahydrogeraniol, geraniol, terpene dioxide, dihydroactinol, citronellol, megastigmatrienone 1,4-oxo-β-isadamantine, (+)-(E)-limonene oxide, dihydrojasmone, 3-hydroxy-β-damascone, 3-hydroxy-7,8-dihydro-β-ionol, 9-hydroxy-4,7-dihydrojasmone). - Megastigmadiene-3-one, dihydrodehydro-β-ionone, 5,6-epoxy-7-megamane-3,9-diol, 5,6-epoxy-β-ionone, 6-hydroxy-4,4,7a-trimethyl-5,6,7,7a-tetrahydrophenylfuran-2(4H)-one, (6S)-6-hydroxy-3-oxo-α-ionone, α-ionone, 7,11-epoxymegamane-5(6)-ene-9-one, nerolidol, (E)-, acetal, 7, 8-Epoxy-α-ionone, ionone, nerol propionate, γ-iridinone, 6-methyl-α-ionone, 4-hydroxy-β-ionone, nerolidol, dihydro-β-ionone, farnesol, β-damascone, 7,8-dihydroionone), Maillard reaction products (tetrahydro-2,5-dimethyl-2H-pyranol), chlorophyll degradation products (phytols), terpenoids (3,4-dihydro-4,5,7-trimethylcoumarin, sesquirose), Aroma compounds including furan, lavender alcohol, terpinene, β-santalol, isolongifol, neosyringin, carvone, 8S,13-cedaranediol, (-)-globulin, cedrolone, β-longifine, 2,6-dimethyl-10-methylene-2,6,11-dodecanetrienal, eucalyptol, longifine, trans-carvone, eucalyptol oxide, cedarene diol, melaleuca oleracea alcohol, elemol, α-caryophyllene alcohol, eucalyptol oxide 1, elemol) and others.

[0101] 3. Detection of the content of alkaloids and their degradation products

[0102] The alkaloid content of fermented tobacco leaf samples obtained in the examples and comparative examples was determined by gas chromatography-mass spectrometry (GC-MS).

[0103] Table 3. Content of alkaloids and their degradation products (mg / kg)

[0104]

[0105] As shown in Table 3 above, compared with the use of sterile water to ferment cigar tobacco leaves in Comparative Example 1, the use of *Trichoderma repens* SC-11 to ferment cigar tobacco leaves in Example 2 resulted in a reduction of nicotine, nornicotine, dehydroneonicotinic acid, cotinine, and N'-acetylnornicotine contents by 26.2%, 17.4%, 21.4%, 25.4%, and 15.5%, respectively.

[0106] 4. Sensory evaluation

[0107] After fermentation, the tobacco leaf samples were rolled into single-material tobacco with a length of 110 mm and a diameter of 14 mm. They were then cured in a Binder constant temperature and humidity chamber at 18℃ and 65% relative humidity for one month before being used for sensory evaluation. A sensory quality evaluation team of 30 experts was organized to conduct the sensory quality evaluation.

[0108] The sensory quality evaluation standard was based on the product technical standard of Great Wall Cigar Factory, "Sensory Evaluation Method for the 'Mellow and Sweet Aroma' Style Characteristics and Quality of Chinese Cigars" QJ / 08.J.6005-2020 A.

[0109] Table 4. Quality Characteristic Scoring Results of Cigars

[0110]

[0111] Table 5. Scoring results of cigar aroma characteristics

[0112]

[0113] From the above Figure 1 As shown in Tables 4 and 5, compared with the use of sterile water to ferment cigar tobacco leaves in Comparative Example 1, the use of Trichosporium corallidum SC-11 to ferment cigar tobacco leaves in Example 2 can significantly improve the aroma quantity, aroma richness, maturity, sweetness and combustibility of cigar tobacco leaves, reduce irritation and off-flavors, and highlight the aroma characteristics such as caramel sweetness, woody aroma and bean aroma, thereby improving the usability of tobacco leaves.

[0114] The technical features of the above-described embodiments and examples can be combined in any suitable manner. For the sake of brevity, not all possible combinations of the technical features in the above-described embodiments and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0115] The embodiments described above are merely illustrative of several implementation methods of this application, intended to facilitate a detailed understanding of the technical solutions of this application, but should not be construed as limiting the scope of protection of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the scope of protection of this application. Furthermore, it should be understood that after reading the above teachings of this application, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the scope of protection of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this patent application should be determined by the content of the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A type of Trichosporon coremiiforme SC-11, characterized in that, The accession number is CGMCC No. 34235.

2. The method for preparing *Trichosporium corallis* SC-11 according to claim 1, characterized in that, The preparation method includes a step of fermenting *Trichosporium corallis* SC-11 with accession number CGMCC No. 34235.

3. The method for preparing Coral Polyporus Trichosporium SC-11 according to claim 2, characterized in that, One or more of the following conditions must be met: (1) The fermentation medium includes water, peptone, yeast powder and glucose. Each 1000 mL of water contains 20 g to 30 g of peptone, 10 g to 15 g of yeast powder and 20 g to 30 g of glucose. (2) The fermentation temperature is 28℃~30℃; (3) Fermentation is carried out under stirred conditions, with the stirring speed optionally ranging from 180 rpm to 220 rpm; and (4) The fermentation time is 1 to 2 days.

4. A microbial agent, characterized in that, The bacterial agent contains *Trichosporium corallis* SC-11 as described in claim 1.

5. A method for preparing fermented tobacco leaves, characterized in that, The process includes the following steps: mixing tobacco leaves with the *Trichosporium corallis* SC-11 as described in claim 1 or the inoculum as described in claim 4, and then performing fermentation treatment to prepare fermented tobacco leaves.

6. The method for preparing fermented tobacco leaves according to claim 5, characterized in that, The bacterial solution containing *Trichoderma corali* SC-11 was mixed with the tobacco leaves, wherein the concentration of *Trichoderma corali* SC-11 in the bacterial solution was 10. 6 Cells / mL ~10 8 The bacterial culture volume is 15-25 cells / mL, and the mass ratio of the bacterial culture to the tobacco leaves is (15-25):

100.

7. The method for preparing fermented tobacco leaves according to claim 5 or 6, characterized in that, One or more of the following conditions must be met: (1) The moisture content of the cigar tobacco leaves is 30wt%~35wt%; (2) The fermentation temperature is 30℃~35℃ and the fermentation time is 15 days~30 days; (3) The relative humidity of the fermentation treatment is 70%~75%; and (4) The tobacco leaves include cigar tobacco leaves.

8. A fermented tobacco leaf, characterized in that, Prepared by the preparation method according to any one of claims 5 to 7.

9. A cigar, characterized in that, The cigar contains the fermented tobacco leaves as described in claim 8.

10. A method for preparing a cigar, characterized in that, The preparation method includes: Fermented tobacco leaves are prepared using the preparation method according to any one of claims 5 to 7; and, Cigars are prepared using raw materials containing the fermented tobacco leaves.

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