Butterfly basidiospora, biological agent and application of biological agent in preparation of tobacco products

Abstract

The invention provides basidiospora butterfly, a biological preparation and application of the basidiospora butterfly and the biological preparation in preparation of tobacco products. The papiliotrema flavescens SC-14 is preserved in the China General Microbiological Culture Collection Center (CGMCC) on April 16, 2025, and the preservation number of the papiliotrema flavescens SC-14 is CGMCC No. 34238. The papiliotrema flavescens SC-14 can be used for preparing the papiliotrema flavescens SC-14. The strain is used for enhanced fermentation of tobacco leaves, the potassium-chlorine ratio can be remarkably increased, the total content of aroma components is increased, aroma substances related to cigar characteristic fragrances such as bean fragrance, burnt sweet fragrance, elecampane and honey sweet fragrance can be generated through metabolism, and meanwhile the nicotine content and harm to a human body are reduced.

Description

Technical Field

[0001] This application relates to the fields of microbial fermentation and tobacco processing technology, specifically to Papilionobasidiospora, biological agents and their application in the preparation of tobacco products, and further to Papilionobasidiospora SC-14, biological agents, methods for preparing tobacco products and tobacco products. Background Technology

[0002] Cigar tobacco fermentation is a core process for improving quality through the synergistic effect of microbial metabolism and enzymatic reactions. Its core mechanism lies in the degradation of macromolecules such as cellulose, chlorophyll, carotenoids, and proteins, transforming them into aroma components. However, traditional natural fermentation faces significant technical bottlenecks: First, it relies on passive regulation of environmental temperature and humidity and the random succession of indigenous microbial communities, leading to an uncontrollable fermentation process, characterized by a lengthy cycle (typically 12-24 months) and significant batch-to-batch quality variations. Second, carotenoids, as important terpene aroma precursors in tobacco leaves, play a crucial role in the formation of characteristic aromas such as bean, caramel, wood, and floral notes in cigars through their oxidative decomposition, producing aroma compounds like β-ionone and mesostigmineone. However, limited by the activity of endogenous peroxidases and the efficiency of abiotic oxidation in tobacco leaves, their degradation rate is low, resulting in insufficient conversion of aroma precursors and limiting the improvement of cigar sensory quality.

[0003] Currently, strains such as Rhodotorula spp. and Acinetobacter spp., while effectively degrading macromolecules like proteins, exhibit weak selective degradation capabilities for macromolecules such as nicotine, chlorophyll, carotenoids, lysine, and sipranones, and are prone to producing amine-based off-flavor byproducts. It is noteworthy that the genus Papiliotrema has demonstrated unique metabolic characteristics in the food industry: strains of this genus not only possess the ability to synthesize β-carotene, but their extracellular oxidase system can also efficiently degrade carotenoids to generate high-value-added products. This metabolic characteristic has been successfully applied in areas such as Pu-erh tea fermentation and winemaking, but its application in the tobacco industry is currently unexplored. Summary of the Invention

[0004] Based on this, one or more embodiments of this application provide Papilionobasidiomyces yeast, biological agents, and their application in the preparation of tobacco products.

[0005] The first aspect of this application provides Papiliotrema flavescens SC-14, which was deposited on April 16, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34238.

[0006] The second aspect of this application provides a biological agent comprising at least one of Papilionobasidiomyces yeast, its culture, its lysate, and its extract; wherein the Papilionobasidiomyces yeast comprises the Papilionobasidiomyces yeast SC-14 described in the first aspect of this application.

[0007] The third aspect of this application provides the use of Papilionobasidiomyces SC-14 described in the first aspect of this application or the biological agent described in the second aspect of this application in tobacco fermentation or the preparation of tobacco products.

[0008] The fourth aspect of this application provides a method for preparing a tobacco product, comprising the following steps:

[0009] Tobacco products are prepared by fermenting tobacco leaves using the Papilionobasidisporum SC-14 described in the first aspect of this application or the biological agent described in the second aspect of this application.

[0010] In some embodiments, the tobacco leaves include cigar tobacco leaves.

[0011] In some implementations, the fermentation temperature is 30°C to 35°C.

[0012] In some implementations, the relative humidity of the fermentation process is 70% to 75%.

[0013] In some implementations, the fermentation process takes 15 to 30 days.

[0014] In some embodiments, the fermentation process includes the following steps:

[0015] The *Papilionobasidium* SC-14 was cultured, and the culture broth was collected to obtain a bacterial suspension; and,

[0016] The bacterial solution and the tobacco leaves are mixed and fermented.

[0017] In some implementations, the cultivation conditions include: incubation at 28°C to 30°C for 1 to 2 days.

[0018] In some embodiments, the culture is carried out under stirring conditions, with a stirring speed of 180 rpm to 220 rpm.

[0019] In some embodiments, the concentration of the Papilionobasidiomyces SC-14 prior to mixing is 10. 6 ~10 cells / mL 8 per mL.

[0020] In some embodiments, the mass ratio of the bacterial solution to the tobacco leaves is (15~25):100.

[0021] In some embodiments, the Papilionobasidiomyces SC-14 can reduce nicotine content by 35% to 40%, increase the potassium-chlorine ratio by 120% to 125%, and increase the total content of aroma components by 35% to 40% during the fermentation of tobacco leaves; wherein the aroma components include lysine degradation products, sipranone degradation products, chlorophyll degradation products, and carotenoid degradation products, increasing the content of lysine degradation products by 90% to 95%, increasing the content of sipranone degradation products by 55% to 60%, increasing the content of chlorophyll degradation products by 35% to 40%, and increasing the content of carotenoid degradation products by 25% to 30%.

[0022] The fifth aspect of this application provides a tobacco product prepared using the method for preparing tobacco products described in the third aspect of this application.

[0023] Compared with traditional technologies, this application has the following advantages:

[0024] This application provides a strain of Papiliotrema flavescens SC-14, which can reduce nicotine content (e.g., a reduction of 37.1%), increase the potassium-chlorine ratio (e.g., an increase of 123.2%), and increase the total content of aroma components (e.g., an increase of 37.0%) during tobacco fermentation. The contents of lysine degradation products, sipranone degradation products, chlorophyll degradation products, and carotenoid degradation products are significantly increased (e.g., increases of 92.8%, 57.3%, 35.4%, and 29.8%, respectively). It can metabolize and generate aroma compounds related to the characteristic aromas of cigars, such as bean aroma, caramel aroma, woody aroma, and honey aroma (e.g., 5,6-epoxy-7-mega-stigmoene-3,9-diol, 3,3,4,6-tetramethyl-2,3-dihydrobenzofuran-2-one, mega-stigmotrienone, geraniol, farnesol, neryl propionate, cedrol, and sagerol), while reducing nicotine content and its harmfulness to the human body. Detailed Implementation

[0025] The present application will be further described in detail below with reference to the embodiments and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. Furthermore, numerous specific details are set forth in the following description to provide a fuller understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0026] 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 herein in the specification of this application is for descriptive purposes only and is not intended to be limiting of the application.

[0027] the term

[0028] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0029] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related 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 "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0030] In this application, the terms "multiple", "various", "multiple times", "multi-dimensional", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0031] The terms “combinations of,” “any combination of,” and “any combination of” used in this article include all suitable combinations of any two or more of the listed items.

[0032] In this document, the term "suitable" as used in phrases such as "suitable combination," "suitable method," and "any suitable method" refers to the ability to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0033] In this document, terms such as “preferred,” “better,” “more suitable,” and “ideal” are merely used to describe implementation methods or examples that achieve better results, and should be understood not to limit the scope of protection of this application.

[0034] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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℃.

[0040] 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.

[0041] 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.

[0042] Currently, the types of functional microorganisms developed for cigar tobacco leaves are relatively limited, failing to meet the diverse quality requirements of cigars. Existing technologies, such as using Rhodotorula spp. and Acinetobacter spp., can effectively degrade macromolecules like proteins, but their selective degradation ability for macromolecules such as nicotine, chlorophyll, carotenoids, lysine, and sipranones is weak, and they easily produce amine-based off-flavor byproducts.

[0043] Based on this, the embodiments of this application at least provide Papilionobasidiomyces yeast, biological agents, and their application in the preparation of tobacco products.

[0044] In the first aspect of this application, we provide *Papiliotrema flavescens* SC-14, which 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. 34238. The colony morphology of this strain is round, pale yellow, viscous, smooth, moist, and reflective.

[0045] The Papiliotrema flavescens SC-14 strain provided in this application, with the taxonomic name Papiliotrema flavescens, 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. 34238. The strain was received and registered by the collection center on April 16, 2025, and was confirmed to be a viable strain by the collection center on the same day.

[0046] When the *Papilionobasidium* yeast obtained in this application is used in the fermentation of cigar tobacco leaves, it can reduce nicotine content (e.g., a reduction of 37.1%), increase the potassium-to-chlorine ratio (e.g., an increase of 123.2%), and increase the total content of aroma components (e.g., an increase of 37.0%). The contents of lysine degradation products, sipranine degradation products, chlorophyll degradation products, and carotenoid degradation products are significantly increased (e.g., increases of 92.8%, 57.3%, 35.4%, and 29.8%, respectively). It can metabolize and generate aroma substances related to the characteristic aromas of cigars, such as bean, caramel, wood, and honey (e.g., 5,6-epoxy-7-mega-stigmoben-3,9-diol, 3,3,4,6-tetramethyl-2,3-dihydrobenzofuran-2-one, mega-stigmotrienone, geraniol, farnesol, neryl propionate, cedrenediol, and stigmobenol), while simultaneously reducing nicotine content and its harmfulness to the human body.

[0047] In a second aspect of this application, a biological agent is provided, comprising at least one of Papilionobasidiomyces yeast, its culture, its lysate, and its extract; wherein the Papilionobasidiomyces yeast includes the aforementioned Papilionobasidiomyces yeast SC-14.

[0048] In a third aspect of this application, the use of the aforementioned Papilionobasidiomyces SC-14 or the aforementioned biological agents in tobacco fermentation or the preparation of tobacco products is provided.

[0049] In a fourth aspect of this application, a method for preparing a tobacco product is provided, comprising the following steps:

[0050] Tobacco products are prepared by fermenting tobacco leaves with the aforementioned Papilionobasidiomyces SC-14 or the aforementioned biological agents.

[0051] In some embodiments, the tobacco leaves include cigar tobacco leaves, and further, the tobacco leaves include one or more different varieties of cigar tobacco leaves or shredded leaves.

[0052] In some implementations, the fermentation process includes the following steps:

[0053] S100: Culture of *Papilionobasidium* SC-14, collection of the culture medium to obtain bacterial suspension; and,

[0054] S200: The bacterial solution and tobacco leaves are mixed and fermented.

[0055] In some embodiments, in step S100, the cultivation conditions include: cultivation at 28°C to 30°C and 180 rpm to 220 rpm for 1 to 2 days. Non-limitingly, the cultivation temperature can be, but is not limited to, 28°C, 29°C, 30°C, or any value or range between two of the above; the cultivation speed can be, but is not limited to, 180 rpm, 190 rpm, 200 rpm, 210 rpm, 220 rpm, or any value or range between two of the above; and the cultivation time can be, but is not limited to, 1 day, 2 days, or any value or range between two of the above.

[0056] In some embodiments, in step S100, the culture medium used for cultivation is yeast extract peptone glucose medium. Further, the yeast extract peptone glucose medium includes peptone, yeast extract, glucose, and distilled water. Even further, the yeast extract peptone glucose medium includes 2% (w / v) peptone, 1% (w / v) yeast extract, 2% (w / v) glucose, and distilled water.

[0057] In some embodiments, in step S200, the concentration of Papilionobasidiospora SC-14 in the bacterial culture is 10. 6 Cells / mL ~10 8 / mL. Without limitation, the concentration of *Papilionobasidium* SC-14 in the bacterial culture can be, but is not limited to, 10-10. 6 cells / mL, 2×10 6 cells / mL, 3×10 6 cells / mL, 4×10 6 cells / mL, 5×10 6 cells / mL, 6×10 6 cells / mL, 7×10 6 cells / mL, 8×10 6 cells / mL, 9×10 6 cells / mL, 10 7 cells / mL, 2×10 7 cells / mL, 3×10 7 cells / mL, 4×10 7 cells / mL, 5×10 7 cells / mL, 6×10 7 cells / mL, 7×10 7 cells / mL, 8×10 7 cells / mL, 9×10 7 cells / mL, 10 8 The number of cells / mL or any value or range between the two values ​​mentioned above.

[0058] In some embodiments, after culturing *Papilionobasidium* SC-14 in step S100, the following step is further included: diluting the culture product. Optionally, the dilution factor is 5 to 50 times. It should be noted that as long as the concentration of the diluted bacterial solution is 10... 6 ~10 8 The desired result is 1 / mL.

[0059] In some embodiments, in step S200, the mass ratio of bacterial solution to tobacco leaves is (15~25):100. Non-limitingly, the mass ratio of bacterial solution to tobacco leaves can be, but is not limited to, 15:100, 16:100, 17:100, 18:100, 19:100, 20:100, 21:100, 22:100, 23:100, 24:100, 25:100, or any ratio or range between two of the above.

[0060] In some embodiments, the fermentation temperature in step S200 is 30°C to 35°C. Non-limitingly, the fermentation temperature can be, but is not limited to, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, or any value or range between two of the above.

[0061] In some embodiments, in step S200, the relative humidity of the fermentation treatment is 70% to 75%. Non-limitingly, the relative humidity of the fermentation treatment can be, but is not limited to, 70%, 71%, 72%, 73%, 74%, 75%, or any value or range between two of the above.

[0062] In some embodiments, the fermentation treatment time in step S200 is 15 to 30 days. Non-limitingly, the fermentation treatment time can be, but is not limited to, 15 days, 20 days, 25 days, 30 days, or any value or range between two of the above.

[0063] In a fifth aspect of this application, a tobacco product is provided, which is prepared using the method for preparing the tobacco product described above.

[0064] 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.

[0065] 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.

[0066] In the following embodiments, the tobacco leaves for Dexue No. 1 cigars are sourced from the Great Wall Cigar Factory of Sichuan Tobacco Industry Co., Ltd., Shifang, Sichuan.

[0067] Example 1

[0068] I. Isolation, Screening and Identification of Strains

[0069] This embodiment provides a strain of Papiliotrema flavescens SC-14, which was isolated from the surface of Dexue No. 1 cigar tobacco leaves.

[0070] The specific separation steps are as follows:

[0071] Using sterile scissors, take 5g of cigar tobacco leaves in a clean bench, cut them into small pieces, and place them in sterile physiological saline. Shake at 30℃ and 200rpm for 30 minutes to elute bacterial cells. Then, dilute the supernatant and spread it onto yeast-extracted peptone-glucose plates, incubating at 28℃ for 24-48 hours. Based on morphological differences, pick single colonies and streak them in three zones on a new plate. Incubate at 28℃ for another 24-48 hours until single colonies appear on the plate. Colonies should be round, pale yellow, viscous, smooth, moist, and reflective.

[0072] Physiological and biochemical tests were performed on the strain:

[0073] (1) Lipase activity detection: Olive oil emulsion was used as the sole carbon source. The culture medium consisted of Rhodamine B 0.01%, agar 1.5%, MgSO4·7H2O 0.04%, KH2PO4 0.1%, (NH4)2SO4 0.1%, sodium chloride 0.4%, and olive oil 2%. After culturing on plates for 2-4 days, the presence of a clear zone was observed.

[0074] (2) Lipoxygenase activity assay: Bengal red was used as the substrate. The culture medium consisted of 0.5% peptone, 1% glucose, 0.1% potassium dihydrogen phosphate, 0.05% magnesium sulfate, 0.01% 1 / 3000 Bengal red solution, and 1.5% agar powder. After culturing on plates for 2-4 days, the presence of a clear zone was observed.

[0075] (3) Protease activity detection: Skim milk powder was used as the substrate. The culture medium consisted of 1% peptone, 0.5% yeast extract, 1% sodium chloride, 1% skim milk powder, and 1.5% agar powder. After the plates were cultured for 2-4 days, the presence of a clear zone was observed.

[0076] (4) Amylase activity detection: Starch was used as the substrate. The culture medium consisted of 1% peptone, 0.5% yeast extract, 1% sodium chloride, 0.2% soluble starch, and 1.5% agar powder. After the plates were cultured for 2-4 days, 5 ml of iodine solution was added for staining for 2 minutes, and then the presence of a clear zone was observed.

[0077] (5) Cellulase activity assay: Sodium carboxymethyl cellulose was used as the substrate. The culture medium consisted of 1% peptone, 0.5% yeast extract, 1% sodium chloride, 0.1% sodium carboxymethyl cellulose, and 1.5% agar powder. After culturing the plates for 2-4 days, they were stained with 1% Congo red solution prepared with 95% anhydrous ethanol for 10-15 min. After destaining with 1M NaCl solution, the size of the transparent zone was observed.

[0078] The specific results of the physiological and biochemical indicators are shown in Table 1.

[0079] Table 1

[0080]

[0081] In this case, "-" indicates a negative result.

[0082] This strain 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. 34238. The ITS sequence of the above-mentioned yeast is shown in SEQ ID NO:1. Further alignment with the NCBI database yielded a Per.Ident value of 99.6%, indicating that this strain can be classified as *Papilionobasidium*.

[0083] SEQ ID NO:1

[0084] .

[0085] II. Fermentation of Cigar Tobacco Leaves

[0086] (1) Culture of strains: The basidiospore yeast preserved on the slant was inoculated onto yeast extract peptone glucose plate medium and activated at 28℃ for 2 days; single colonies after activation were picked and inoculated into yeast extract peptone glucose liquid medium for fermentation culture at 28℃ and 200 rpm for 2 days to obtain fermentation broth.

[0087] 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.

[0088] (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.

[0089] (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 15%; the inoculated tobacco leaves are put into burlap bags and placed in a fermentation room for fermentation at a temperature of 35°C and a relative humidity of 75% for 20 days.

[0090] (4) After the fermentation is completed, the cigar tobacco samples are dried at 45°C, crushed, and passed through a 0.25 mm sieve. The aroma components are then determined by gas chromatography-mass spectrometry (GC-MS).

[0091] (5) The fermented tobacco 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°C ​​and 65% relative humidity for one month before being used for sensory evaluation. A sensory quality evaluation expert group was organized to conduct sensory quality evaluation.

[0092] 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.

[0093] Comparative Example 1

[0094] (1) Fermentation of cigar tobacco leaves: Inoculate the surface of the rehydrated cigar tobacco leaves with pure water at a mass ratio of 15%; 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 20 days.

[0095] (2) After the fermentation was completed, the cigar tobacco samples were dried at 45°C, crushed, passed through a 0.25 mm sieve, and the content of aroma components was determined by gas chromatography-mass spectrometry (GC-MS).

[0096] (3) The fermented tobacco 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 expert group was organized to conduct sensory quality evaluation.

[0097] Comparative Example 2

[0098] The Rhodotorula mucilaginosa used in Comparative Example 2 was deposited on March 18, 2013, at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with accession number M2013088.

[0099] (1) Culture of strains: The red yeast preserved on the slant was 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.

[0100] (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.

[0101] (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 15%; the inoculated tobacco leaves are put into burlap bags and placed in a fermentation room for fermentation at a temperature of 35°C and a relative humidity of 75% for 20 days.

[0102] (4) After the fermentation is completed, the cigar tobacco samples are dried at 45°C, crushed, and passed through a 0.25 mm sieve. The aroma components are then determined by gas chromatography-mass spectrometry (GC-MS).

[0103] (5) The fermented tobacco 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°C ​​and 65% relative humidity for one month before being used for sensory evaluation. A sensory quality evaluation expert group was organized to conduct sensory quality evaluation.

[0104] The results of the physicochemical metabolic index content of cigar tobacco leaves after fermentation in the above embodiments and comparative examples are shown in Table 2.

[0105] Table 2 Content of physicochemical metabolic indicators after enhanced fermentation of tobacco leaves

[0106]

[0107] The results of the aroma component content of cigar tobacco leaves after fermentation in the above embodiments and comparative examples are shown in Table 3.

[0108] Table 3. Content of aroma components in fortified fermented cigar tobacco leaves (mg / kg)

[0109]

[0110]

[0111]

[0112] The sensory quality scores for the above embodiments and comparative examples are shown in Tables 4 and 5. Table 4 shows the cigar quality characteristic scores; Table 5 shows the aroma characteristic scores.

[0113] Table 4. Quality Characteristic Scoring Results of Cigars

[0114]

[0115] Table 5. Scoring results of cigar aroma characteristics

[0116]

[0117] As shown in the table above, compared with the fermentation of cigar tobacco leaves using sterile water in Comparative Example 1, the fermentation of cigar tobacco leaves using *Papilionobasidium* SC-14 of Example 1 of this application resulted in a 37.1% decrease in nicotine content, a 123.2% increase in potassium-to-chlorine ratio, and a 37.0% increase in the total content of aroma components. Specifically, the contents of lysine degradation products, sipranone degradation products, chlorophyll degradation products, carotenoid degradation products, other aroma components, and Maillard reaction products increased by 92.8%, 57.3%, 35.4%, 29.8%, 54.4%, and 15.8%, respectively. It can metabolize and generate aroma compounds related to the characteristic aromas of cigars, such as bean aroma, caramel sweet aroma, and woody aroma (e.g., 5,6-epoxy-7-mega-stigmoene-3,9-diol, 3,3,4,6-tetramethyl-2,3-dihydrobenzofuran-2-one, mega-stigmotrienone, geraniol, farnesol, neryl propionate, cedrol, and sagerol).

[0118] Compared with the fermentation of cigar tobacco leaves using Rhodotorula mucilaginosa in Comparative Example 2, the fermentation of cigar tobacco leaves using Papilionobasidisporum SC-14 of Example 1 of this application resulted in a 34.7% reduction in nicotine content, a 79.6% increase in the potassium-to-chlorine ratio, and a 34.0% increase in the total content of aroma components. Specifically, the contents of lysine degradation products, sipranone degradation products, chlorophyll degradation products, carotenoid degradation products, other aroma components, and Maillard reaction products increased by 111.3%, 41.6%, 17.2%, 42.5%, 19.1%, and 34.1%, respectively.

[0119] The evaluation results showed that the yeast-enhanced group could significantly improve the aroma quantity and richness of fermented tobacco leaves, highlighting aroma characteristics such as caramel sweetness, bean aroma, honey sweetness, and woody aroma, reducing irritation and off-flavors, improving permeability, increasing oral comfort, producing a smooth and delicate smoke, improving combustibility and ash formation, and thus improving the usability of tobacco leaves.

[0120] 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.

[0121] 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 can be used to interpret the content of the claims.

Claims

1. Papiliotrema flavescens SC-14, characterized in that, It was preserved in China General Microbiological Culture Collection Center on April 16, 2025, and the preservation number is CGMCC No. 34238.

2. A biological agent, characterized in that, The at least one of the Sabiea formosa yeast, a culture of the Sabiea formosa yeast, a lysate of the Sabiea formosa yeast, and an extract of the Sabiea formosa yeast; wherein the Sabiea formosa yeast comprises the Sabiea formosa yeast SC-14 of claim 1.

3. The Sabiea formosa yeast SC-14 of claim 1 or the biological agent of claim 2 for use in fermentation of tobacco leaves or preparation of tobacco products.

4. A method of making a tobacco product, characterized by, The method comprises the following steps: The tobacco leaves are fermented by using the Sabiea formosa yeast SC-14 of claim 1 or the biological agent of claim 2 to prepare the tobacco products. Optionally, the tobacco leaves comprise cigar tobacco leaves.

5. The method of making a tobacco product according to claim 4, wherein, The fermentation treatment satisfies one or more of the following conditions: The temperature of the fermentation treatment is 30-35℃; The relative humidity of the fermentation treatment is 70-75%; The time of the fermentation treatment is 15-30 days.

6. The method of making a tobacco product according to claim 5, wherein, The fermentation treatment comprises the following steps: The Sabiea formosa yeast SC-14 is cultured to obtain a culture solution; and The culture solution and the tobacco leaves are mixed for fermentation treatment.

7. The method of making a tobacco product according to claim 6, wherein, The culture conditions comprise: culturing at 28-30℃ for 1-2 days; optionally, culturing under stirring at a stirring speed of 180-220 rpm.

8. The method of making a tobacco product according to claim 6, wherein, The concentration of the Oospora formosana SC-14 in the bacterial solution before mixing was 10 6 × 10 8 / mL. Optionally, the mass ratio of the culture solution to the tobacco leaves is (15-25):

100.

9. A method of making a tobacco product according to any one of claims 4 to 8, wherein, In the fermentation process of the tobacco leaves, the Sabiea formosa yeast SC-14 can reduce the nicotine content by 35-40%, increase the potassium-chlorine ratio by 120-125%, and increase the total content of aroma components by 35-40%; wherein the aroma components comprise labdane degradation products, cembrane degradation products, chlorophyll degradation products, and carotenoid degradation products, and the content of the labdane degradation products is increased by 90-95%, the content of the cembrane degradation products is increased by 55-60%, the content of the chlorophyll degradation products is increased by 35-40%, and the content of the carotenoid degradation products is increased by 25-30%.

10. A tobacco product, characterised in that, The tobacco products are prepared by using the preparation method of any one of claims 4-9. The tobacco products are prepared by using the preparation method of any one of claims 4-9.

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