Yunnan red basidiospora, biological preparation, preparation method and application
By using Yunnan red basidiomycete yeast SC-9 to ferment tobacco leaves, the problems of unstable quality and insufficient aroma during the fermentation process of cigar tobacco leaves have been solved, resulting in improved aroma components and reduced nicotine, thus improving the sensory quality of cigars.
Patent Information
- Application Number
- CN202511239166.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-01
- Publication Date
- 2025-11-11
AI Technical Summary
The natural fermentation of cigar tobacco leaves leads to large batch-to-batch quality fluctuations, long fermentation cycles, low efficiency of carotenoid oxidation and decomposition, insufficient generation of aroma substances, and insufficient aroma complexity. Existing technologies, such as using Bacillus or Acinetobacter, have poor degradation specificity and may produce off-flavor byproducts.
Fermented tobacco leaves were prepared by mixing Erythrobasidium yunnanense SC-9 with tobacco leaves under specific fermentation conditions, which improved the content and richness of aroma components and reduced nicotine content.
It significantly enhances the aroma quantity and richness of fermented tobacco leaves, highlighting characteristic aromas such as bean, nut, and wood, while reducing nicotine content, improving combustibility and ash formation, and increasing the usability of tobacco leaves.
Smart Images
Figure SMS_1 
Figure SMS_2 
Figure SMS_3
Abstract
Description
Technical Field
[0001] This invention belongs to the field of microbial fermentation and tobacco processing technology, and relates to a Yunnan red basidiospore yeast, a biological agent, a preparation method, and its application. Background Technology
[0002] Fermentation of cigar tobacco leaves is a core process shaping their sensory quality, relying on microbial activity and biochemical reactions to degrade macromolecules and release aroma compounds. However, natural fermentation depends on the random effects of environmental temperature and humidity and the indigenous microbial community, resulting in large batch-to-batch quality fluctuations and a long fermentation cycle. Furthermore, carotenoids, as key aroma precursors, are widely present in tobacco leaves. Their degradation products during tobacco leaf maturation and processing have a significant impact on the flavor and aroma of cigars. For example, β-ionone and mesostigmine can impart bean, nutty, and woody aroma characteristics to cigars. However, the oxidative degradation process of carotenoids is limited by the activity of endogenous enzymes in the tobacco leaves and environmental oxidation, resulting in low degradation efficiency, insufficient formation of aroma compounds, and insufficient aroma complexity.
[0003] To overcome these problems, the industry has tried various technical means, but all of them have limitations. For example, although Bacillus or Acinetobacter can degrade macromolecules such as proteins, their specificity for degrading macromolecules such as nicotine, chlorophyll, carotenoids, lysine, and cephalosporins is poor, and they may produce odorous byproducts. Summary of the Invention
[0004] Based on this, one or more embodiments of this application provide a Yunnan red basidiomycete yeast, a biological agent, a preparation method, and an application to improve the aroma component content of cigar tobacco leaves.
[0005] In some embodiments, a yeast strain called Erythrobasidium yunnanense SC-9 is provided, with accession number CGMCC No. 34233.
[0006] In some embodiments, a method for preparing the Yunnan red basidiomycete SC-9 is provided, the method comprising the step of fermenting the Yunnan red basidiomycete SC-9 with accession number CGMCC No. 34233.
[0007] In some embodiments, the method for preparing Yunnan red basidiomycete SC-9 satisfies one or more of the following conditions:
[0008] (1) The fermentation culture medium includes water, peptone, yeast powder and glucose, and each 1000 mL of water contains 20 g to 30 g of the peptone, 10 g to 15 g of the yeast powder and 20 g to 30 g of the glucose;
[0009] (2) The fermentation temperature is 28℃~30℃;
[0010] (3) Fermentation is carried out under stirred conditions, with the stirring speed optionally ranging from 180 rpm to 220 rpm; and
[0011] (4) The fermentation time is 1 to 2 days.
[0012] In some embodiments, a biological agent is provided, the biological agent containing the Yunnan red basidiospore yeast SC-9.
[0013] In some embodiments, a method for preparing fermented tobacco leaves is provided, comprising the following steps: mixing the Yunnan red basidiomycete yeast SC-9 or the biological agent with tobacco leaves and performing fermentation treatment to prepare fermented tobacco leaves.
[0014] In some embodiments, in the provided method for preparing fermented tobacco leaves, a bacterial solution containing *Rhizopus yunnanensis* SC-9 is mixed with the tobacco leaves, wherein the concentration of *Rhizopus yunnanensis* SC-9 in the bacterial solution is 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.
[0015] In some embodiments, the provided method for preparing fermented tobacco leaves is characterized by satisfying one or more of the following conditions:
[0016] (1) The moisture content of the tobacco leaves is 30wt%~35wt%;
[0017] (2) The fermentation temperature is 30℃~35℃;
[0018] (3) The fermentation time is 15 to 30 days;
[0019] (4) The relative humidity of the fermentation treatment is 70%~75%; and
[0020] (5) The tobacco leaves include cigar tobacco leaves.
[0021] In some embodiments, a fermented tobacco leaf is provided, prepared by the aforementioned preparation method.
[0022] In some embodiments, a cigarette is provided containing the fermented tobacco leaves.
[0023] In some embodiments, a method for preparing a cigarette is provided, the method comprising:
[0024] Fermented tobacco leaves are prepared using the aforementioned preparation method; and,
[0025] Cigarettes are prepared using raw materials containing the fermented tobacco leaves.
[0026] The provided *Erythrobasidium yunnanense* strain can directionally regulate the fermentation process of cigar tobacco leaves. Through its unique metabolic capabilities, it can metabolize and generate aroma substances related to the characteristic aromas of cigars, such as bean, nutty, and woody notes, including megastigmatrienone, 3,3,4,6-tetramethyl-2,3-dihydrobenzofuran-2-one, 5,6-epoxy-7-megastigne-3,9-diol, farnesol, iridophyllene, α-ionol, cedrol, sagerol, and betulinol, while simultaneously reducing nicotine content.
[0027] The Yunnan Erythrobasidium yunnanense SC-9 provided in this application, taxonomically named Erythrobasidium yunnanense, 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 CGMCCNo. 34233. This 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. Detailed Implementation
[0028] 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.
[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 herein in the specification of this application is for descriptive purposes only and is not intended to be limiting of the application.
[0030] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:
[0031] 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").
[0032] 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.
[0033] 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.
[0034] 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.
[0035] 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.
[0036] 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.
[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] Erythrobasidium species, due to their ability to synthesize or metabolize red pigments such as carotenoids, have been developed for use as natural pigments or antioxidants in the food, pharmaceutical, cosmetic, and textile industries. Their potential for highly efficient carotenoid degradation could address the problem of insufficient conversion of cigar aroma precursors.
[0045] In some embodiments, a *Erythrobasidium yunnanense* SC-9 is provided, with accession number CGMCC No. 34233. The provided *Erythrobasidium yunnanense* SC-9 was deposited on April 16, 2025, at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.
[0046] The colony morphology of the provided Yunnan red basidiospore yeast SC-9 is round, pink, viscous, smooth, moist, and reflective.
[0047] In some embodiments, a method for preparing the Yunnan red basidiomycete SC-9 is provided, the method comprising the step of fermenting the Yunnan red basidiomycete SC-9 with accession number CGMCC No. 34233.
[0048] In some embodiments, the method for preparing Yunnan red basidiomycete SC-9 includes a fermentation culture medium comprising water, peptone, yeast powder, and glucose, wherein 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.
[0049] In some embodiments, the fermentation temperature in the preparation method of Yunnan red basidiomycete SC-9 is 28℃~30℃.
[0050] In some embodiments, in the method for preparing Yunnan red basidiomycete SC-9, fermentation is carried out under stirring conditions, optionally with a stirring speed of 180 rpm to 220 rpm.
[0051] In some embodiments, the fermentation time for the preparation method of Yunnan red basidiospore yeast SC-9 is 1 to 2 days.
[0052] In some embodiments, a biological agent is provided, the biological agent containing the Yunnan red basidiospore yeast SC-9.
[0053] In some embodiments, a method for preparing fermented tobacco leaves is provided, comprising the following steps: mixing the Yunnan red basidiomycete yeast SC-9 or the biological agent with tobacco leaves and performing fermentation treatment to prepare fermented tobacco leaves.
[0054] In some embodiments, in the provided method for preparing fermented tobacco leaves, a bacterial solution containing *Rhizopus yunnanensis* SC-9 is mixed with the tobacco leaves, wherein the concentration of *Rhizopus yunnanensis* SC-9 in the bacterial solution is 10. 6 Cells / mL ~10 8 The bacterial culture volume is [number] cells / mL, and the mass ratio of the bacterial culture to the tobacco leaves is (15~25):100. For example, the concentration of *Yunnan basidiospores* SC-9 in the bacterial culture volume can be 10 [units / mL]. 6 cells / mL, 10 7 cells / mL, 10 8 The ratio of bacteria / mL can be any of the two values mentioned above; the mass ratio of the bacterial solution to the tobacco leaf can be 15:100, 20:100, 15:100, etc., or any of the two ratios mentioned above.
[0055] In some embodiments, in the provided method for preparing fermented tobacco leaves, the moisture content of the tobacco leaves is 30wt%~35wt%, for example, 30wt%, 31wt%, 32wt%, 33wt%, 34wt%, 35wt%, etc., or it can be a range composed of any two of the aforementioned values.
[0056] In some embodiments, the fermentation temperature in the provided method for preparing fermented tobacco leaves is 30°C to 35°C, for example, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, etc., or it can be a range composed of any two of the aforementioned values.
[0057] In some embodiments, the fermentation time in the provided method for preparing fermented tobacco leaves is 15 to 30 days, for example, 15 days, 20 days, 25 days, 30 days, etc., or it can be a range composed of any two of the aforementioned values.
[0058] In some embodiments, the relative humidity of the fermentation treatment in the provided method for preparing fermented tobacco leaves is 70% to 75%, for example, 70%, 71%, 72%, 73%, 74%, 75%, etc., or it can be a range composed of any two of the aforementioned values.
[0059] In some embodiments, the provided method for preparing fermented tobacco leaves includes cigar tobacco leaves.
[0060] In some embodiments, a fermented tobacco leaf is provided, prepared by the aforementioned preparation method.
[0061] In some embodiments, a cigarette is provided containing the fermented tobacco leaves.
[0062] In some embodiments, a method for preparing a cigarette is provided, the method comprising:
[0063] Fermented tobacco leaves are prepared using the aforementioned preparation method; and,
[0064] Cigarettes are prepared using raw materials containing the fermented tobacco leaves.
[0065] The provided *Erythrobasidium yunnanense* SC-9 strain can reduce nicotine content by 14.1%, increase the potassium-chlorine ratio by 82.2%, and increase the total aroma component content by 29.4% during tobacco fermentation. Specifically, the contents of lysine degradation products, cephalosporin degradation products, chlorophyll degradation products, and carotenoid degradation products increased by 100.5%, 27.1%, 25.2%, and 24.1%, respectively. It can significantly enhance the aroma quantity and richness of fermented tobacco leaves, highlighting aroma characteristics such as caramel sweetness, woody aroma, and bean aroma, reducing irritation and off-flavors, improving permeability, producing a smooth and delicate smoke, improving combustibility and ash formation, and thus improving the usability of tobacco leaves. Simultaneously, it can reduce nicotine content.
[0066] 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.
[0067] 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.
[0068] Example 1
[0069] I. Isolation, Screening and Identification of Strains
[0070] (1) Sample pretreatment
[0071] Cigar tobacco leaves from the Great Wall Cigar Factory (Shifang, Sichuan) of Sichuan Tobacco Industry Co., Ltd. were used as the source for strain screening. Before the experiment, the samples were temporarily stored at -4℃. 5g of the sample was taken with sterile scissors in a clean bench, chopped, and placed in a pre-filled Erlenmeyer flask containing sterilized phosphate buffer. The phosphate buffer contained 8g of sodium chloride (NaCl), 0.2g of potassium chloride (KCl), 1.44g of disodium hydrogen phosphate (Na2HPO4), and 0.24g of potassium dihydrogen phosphate (KH2PO4) per 1L. The Erlenmeyer flask was placed in a shaker at 37℃ and 200rpm for 30min.
[0072] (2) Diluting the coating plate
[0073] Dilute the sample supernatant from step (1) to 10. -1 10 -2 and 10 -3 Then spread them separately on YEPD plates (the specific composition is 2% peptone, 1% yeast powder, 2% glucose, and 1.5% agar powder). Invert the spread plates and incubate them in a constant temperature and humidity incubator at 30°C for 24-28 hours until single colonies appear on the plates.
[0074] (3) Three-zone separation
[0075] Based on the differences in morphological characteristics, select different monoclonal colonies from step 2, and streak them in three zones on a new YEPD plate. Invert the streaked plate and incubate it at 30°C for 1 to 3 days until monoclonal colonies appear on the plate. The morphology of strain SC-9 is round, pink, viscous, smooth, moist, and reflective.
[0076] (4) Purification and preservation
[0077] Repeat step 3 for 2-3 purifications until only single-clone colonies with a single morphological characteristic are found on the plate. Then, take a single-clone colony and inoculate it into liquid culture medium. Place it on a shaker at 30°C and 200 rpm for 1-3 days. Then, take an equal proportion of the bacterial solution and mix it with sterilized 30% glycerol. Place the mixture in a cryovial and store it at -80°C.
[0078] (5) Strain identification
[0079] The purified single-clone colonies from step (4) were placed in sterile water, boiled at high temperature to extract DNA, amplified, and then sent for sequencing. Sequencing was performed using primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'). The ITS sequence is shown in SEQ ID No. 1:
[0080] SEQ ID No.1
[0081] .
[0082] The sequencing results were compared with microbial information in the NCBI database using BLAST software to confirm that the obtained strain was Erythrobasidium yunnanense.
[0083] The obtained strain was named *Erythrobasidium yunnanense* SC-9 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. 34233.
[0084] II. Fermentation Methods of Cigar Tobacco Leaves
[0085] (1) Culture of strains: The Yunnan red 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.
[0086] 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.
[0087] (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.
[0088] (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.
[0089] (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).
[0090] (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.
[0091] 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.
[0092] Comparative Example 1
[0093] (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.
[0094] (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).
[0095] (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.
[0096] Comparative Example 2
[0097] The Rhodotorula mucilaginosa used in Comparative Example 2 was deposited on March 18, 2013, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC No: M2013088.
[0098] (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.
[0099] (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.
[0100] (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.
[0101] (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).
[0102] (5) After fermentation, the tobacco leaf samples were rolled into single-material tobaccos with a length of 110 mm and a diameter of 14 mm. After curing in a Binder constant temperature and humidity chamber at 18℃ and 65% relative humidity for one month, the tobaccos were put into evaluation. A sensory quality evaluation was conducted by a group of 30 sensory quality evaluation experts. The sensory quality evaluation standard was based on the product technical standard of Great Wall Cigar Factory, "Sensory Evaluation Method of 'Mellow and Sweet Aroma' Style Characteristics and Quality of Chinese Cigars" QJ / 08.J.6005-2020 A.
[0103] 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 1.
[0104] Table 1. Content of physicochemical metabolic indicators after enhanced fermentation of tobacco leaves
[0105]
[0106] The results of the aroma component content of cigar tobacco leaves after fermentation in the above embodiments and comparative examples are shown in Table 2.
[0107] Table 2. Content of aroma components in fortified fermented cigar tobacco leaves (mg / kg)
[0108]
[0109] Table 2 (continued) (1)
[0110]
[0111] Table 2 continues (2)
[0112]
[0113] Table 2 continues (3)
[0114]
[0115] The sensory quality scores for the above embodiments and comparative examples are shown in Tables 3 and 4. Table 3 shows the cigar quality characteristic scores; Table 4 shows the aroma characteristic scores.
[0116] Table 3. Quality Characteristic Scoring Results of Cigars
[0117]
[0118] Table 4. Scoring Results of Cigar Aroma Characteristics
[0119]
[0120] As shown in Tables 1-4 above, compared with the fermentation of cigar tobacco leaves using sterile water in Comparative Example 1, the fermentation of cigar tobacco leaves using the *Yunnan basidiospores* SC-9 of this application resulted in a 14.1% decrease in nicotine content, an 82.2% increase in the potassium-to-chlorine ratio, and a 29.4% increase in the total content of aroma components. Specifically, the contents of lysine degradation products, cephalosine degradation products, chlorophyll degradation products, carotenoid degradation products, other aroma components, and Maillard reaction products increased by 100.5%, 27.1%, 25.2%, 24.1%, 60.7%, and 9.4%, respectively. By enhancing the fermentation of *Yunnan basidiospora* SC-9, it can metabolize and generate aroma substances related to bean aroma, nut aroma, floral aroma, woody aroma, such as megalotrienone, 3,3,4,6-tetramethyl-2,3-dihydrobenzofuran-2-one, 5,6-epoxy-7-megalotrien-3,9-diol, farnesol, calomel, α-ionol, cedrol, perillaldehyde, betulinol, and other aroma compounds.
[0121] Compared with the fermentation of cigar tobacco leaves using *Rhodotorula glutinis* in Comparative Example 2, the fermentation of cigar tobacco leaves using *Yunnan basidiospores* SC-9 (as described in this application) resulted in a 10.9% decrease in nicotine content, a 46.6% increase in the potassium-to-chlorine ratio, and a 26.6% increase in the total content of aroma components. Specifically, the contents of lysine degradation products, cephalosine degradation products, chlorophyll degradation products, carotenoid degradation products, other aroma components, and Maillard reaction products increased by 111.3%, 119.7%, 14.4%, 8.4%, 36.3%, 24.0%, and 26.7%, respectively.
[0122] 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 bean aroma, caramel sweet aroma, nutty aroma, and woody aroma, reducing irritation and off-flavors, producing a smooth and delicate smoke, improving combustibility and ash formation, and thus improving the usability of tobacco leaves.
[0123] 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.
[0124] 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. A type of Erythrobasidium yunnanense SC-9, characterized in that, The accession number is CGMCC No. 34233.
2. The method for preparing Yunnan red basidiomycete SC-9 according to claim 1, characterized in that, The preparation method includes the step of fermenting Yunnan red basidiomycetes SC-9 with accession number CGMCC No. 34233.
3. The method for preparing Yunnan red basidiospore yeast SC-9 according to claim 2, characterized in that, One or more of the following conditions must be met: (1) The fermentation culture medium includes water, peptone, yeast powder and glucose, and each 1000 mL of water contains 20 g to 30 g of the peptone, 10 g to 15 g of the yeast powder and 20 g to 30 g of the 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 biological agent, characterized in that, The biological agent contains Yunnan red basidiospore yeast SC-9 as described in claim 1.
5. A method for preparing fermented tobacco leaves, characterized in that, The process includes the following steps: mixing Yunnan red basidiomycete yeast SC-9 as described in claim 1 or the biological agent as described in claim 4 with tobacco leaves and 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 culture containing *Rhizopus yunnanensis* SC-9 was mixed with the tobacco leaves, wherein the concentration of *Rhizopus yunnanensis* SC-9 in the bacterial culture 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 tobacco leaves is 30wt%~35wt%; (2) The fermentation temperature is 30℃~35℃; (3) The fermentation time is 15 to 30 days; (4) The relative humidity of the fermentation treatment is 70%~75%; and (5) 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 cigarette, characterized in that, The cigarette contains the fermented tobacco leaves as described in claim 8.
10. A method for preparing a cigarette, 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, Cigarettes are prepared using raw materials containing the fermented tobacco leaves.