Staphylococcus xylosus, biological agent, tobacco product and method for preparing same

Abstract

The application relates to Staphylococcus xylosus, a biological agent, a tobacco product and a preparation method thereof. The Staphylococcus xylosus has a preservation number of CGMCC No. 36128. The strain can secrete cellulase, protease and amylase, and by applying the Staphylococcus xylosus to intensified fermentation of tobacco leaves, the aroma characteristics of the tobacco leaves are enhanced, the overall quality of the tobacco leaves is improved, and an effective biological technology means is provided for a tobacco fermentation process.

Description

Technical Field

[0001] This application relates to the field of microbial technology, and in particular to Staphylococcus xylose, biological agents, tobacco products and their preparation methods. Background Technology

[0002] Fermentation of cigar tobacco leaves is a crucial step in determining their flavor and quality. Traditional fermentation relies on indigenous microbial communities in the natural environment, which has the following limitations: long fermentation cycles, usually requiring months or even years, resulting in low efficiency; and unstable quality, easily affected by the environment, leading to large batch-to-batch variations in tobacco leaves.

[0003] Studies show that microorganisms play a crucial role in the fermentation process of cigar tobacco. Microbial fermentation promotes changes in the chemical composition and physical properties of tobacco leaves, including the degradation of macromolecules such as cellulose, protein, and starch, producing substances like glucose and amino acids. This enhances the sweetness of the tobacco while reducing off-flavors and harshness. Simultaneously, amino acids undergo Maillard reactions with sugars to produce aroma compounds such as pyrazines, aldehydes, and ketones, increasing the roasted and nutty aromas of the tobacco. This process is a key means of improving the industrial usability of cigar tobacco raw materials.

[0004] Therefore, using biotechnology to discover new microbial resources and applying them to enhance the fermentation process of cigar tobacco leaves is of great significance for improving the quality of cigar tobacco leaves. Summary of the Invention

[0005] This application mainly provides a strain of Staphylococcus xylose, which, when used in the fermentation of tobacco leaves, can significantly reduce irritation and off-flavors, improve the cleanliness of tobacco leaves, and thus enhance the usability of tobacco leaves.

[0006] This application achieves the above objectives through the following technical solutions:

[0007] The first aspect of this application provides a Staphylococcus xylosus strain, accession number CGMCC No. 36128.

[0008] A second aspect of this application provides a biological agent comprising one or more of the following: Staphylococcus xylose described above, a culture of Staphylococcus xylose described above, a lysate of Staphylococcus xylose described above, and an extract of Staphylococcus xylose described above.

[0009] A third aspect of this application provides a method for preparing a tobacco product, comprising the following steps:

[0010] Tobacco products are prepared by fermenting tobacco leaves using the aforementioned Staphylococcus xylose or the aforementioned biological agents.

[0011] In some embodiments, the tobacco product includes cigar tobacco.

[0012] In some embodiments, the fermentation process includes the following steps:

[0013] Culture the *Staphylococcus xylose*, collect the culture medium, and obtain a bacterial suspension; and

[0014] The bacterial solution and the tobacco leaves are mixed and fermented.

[0015] In some embodiments, the culture conditions include: placing the Staphylococcus xylose in a sterilized liquid culture medium and culturing it at 35°C to 37°C and 150 rpm to 200 rpm for 24 to 48 hours.

[0016] In some embodiments, the bacterial concentration in the bacterial solution is 10. 6 Cells / mL ~10 8 cells / mL;

[0017] And / or, the tobacco extract liquid culture medium comprises tobacco leaves and glucose, wherein the concentration of the tobacco leaves is 1 g / L to 100 g / L and the concentration of the glucose is 0.5 g / L to 15 g / L.

[0018] In some embodiments, the volume-to-mass ratio of the bacterial solution to the tobacco leaves is 0.5 mL to 5 mL: 100 g.

[0019] In some embodiments, the fermentation temperature is 30°C to 45°C, the relative humidity is 70% to 75%, and the fermentation time is 15 to 45 days.

[0020] The fourth aspect of this application provides a tobacco product prepared by fermentation with Staphylococcus xylose and / or the above-mentioned biological agents, or by the above-mentioned preparation method.

[0021] This application provides a strain of *Staphylococcus xylose* capable of secreting cellulase, protease, and amylase. By applying *Staphylococcus xylose* to the enhanced fermentation of tobacco leaves, it was determined that the volatile components in the fermented tobacco leaves were significantly increased. The content of 30 volatile components in the fermented tobacco leaves was increased, including aroma-producing compounds such as 4-hydroxy-3,5,5-trimethyl-4-(3-oxo-1-butenyl)-2-cyclohexen-1-one, vitamin E, nicotine, podophyllin, dihydroactinol, and isovaleric acid. The content of 12 volatile components decreased in the fermented tobacco leaves, including 4,6-dimethyl-dodecane, styracil lactone, and 2,4-dimethyl-1-heptene. These results indicate that the application of *Staphylococcus xylose* not only enhances the aroma characteristics of tobacco leaves but also helps improve the overall quality of tobacco leaves, providing an effective biotechnological means for tobacco fermentation processes. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the specific embodiments of this application or the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0023] Figure 1 This is a colony morphology diagram of Staphylococcus xylose SCT-B7 in Example 1 of this application;

[0024] Figure 2 This is a microscopic image of Staphylococcus xylose SCT-B7 in Example 1 of this application;

[0025] Figure 3 This is a growth curve diagram of Staphylococcus xylose SCT-B7 in Example 1 of this application;

[0026] Figure 4 This is a growth curve of Staphylococcus aureus in Example 1 of this application;

[0027] Figure 5 This is a growth curve of Staphylococcus saprophyticus in Example 1 of this application.

[0028] The *Staphylococcus xylose* strain provided in this application was deposited on October 10, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 36128. Detailed Implementation

[0029] To make the above-mentioned objectives, features, and advantages of this application more apparent and understandable, a detailed description of specific embodiments of this application is provided. Many specific details are set forth in the following description to provide a thorough understanding of this application. However, this application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of this application. Therefore, this application is not limited to the specific embodiments disclosed below.

[0030] 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 is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. Unless otherwise specifically stated, all raw materials, reagents, instruments, and equipment used in this application are commercially available or can be prepared by existing methods.

[0031] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0032] 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").

[0033] 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.

[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 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.

[0037] 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.

[0038] 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℃.

[0039] 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.

[0040] In traditional techniques, Staphylococcus is an indispensable functional microorganism in the fermentation of sausages, cheeses, and other traditional fermented products. It can produce enzyme systems such as proteases and lipases, generating fatty acids and amino acids. Some strains can also inhibit the growth of spoilage microorganisms, playing a crucial role. However, the Staphylococcus species currently reported for use in tobacco are *Staphylococcus epidermidis*, *Staphylococcus capitella*, and *Staphylococcus nebdominis*. There are currently no reports of screening *Staphylococcus xylose* from cigar tobacco leaves and applying it to the enhanced fermentation of tobacco. As an important member of the *Staphylococcus* genus, the compatibility of *Staphylococcus xylose*'s enzyme secretion characteristics with tobacco fermentation has not yet been explored and utilized.

[0041] Therefore, how to screen Staphylococcus xylose strains with high fermentation efficiency from cigar tobacco leaves, apply them to the enhanced fermentation process of tobacco leaves, enrich the functional microbial resources for tobacco fermentation, solve the problems of single strain types and insufficient adaptability of existing strains, and achieve targeted improvement and enhancement of tobacco leaf quality has become an urgent technical problem to be solved in the field of tobacco bio-fermentation.

[0042] Based on this, one embodiment of this application provides a strain of Staphylococcus xylosus, which was deposited on October 10, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 36128.

[0043] This *Staphylococcus xylosus* strain can secrete cellulase, protease, and amylase. By applying *Staphylococcus xylosus* to enhance the fermentation of tobacco leaves, the volatile components in the fermented tobacco were significantly increased. The content of 30 volatile components increased in the fermented tobacco, including aroma-producing compounds such as 4-hydroxy-3,5,5-trimethyl-4-(3-oxo-1-butenyl)-2-cyclohexen-1-one, vitamin E, nicotine, podophyllin, dihydroactinol, and isovaleric acid. The content of 12 volatile components decreased in the fermented tobacco, including 4,6-dimethyl-dodecane, succinate lactone, and 2,4-dimethyl-1-heptene. These results indicate that the application of *Staphylococcus xylosus* not only enhances the aroma characteristics of tobacco leaves but also helps improve the overall quality of tobacco, providing an effective biotechnological means for tobacco fermentation.

[0044] In some of these embodiments, the aforementioned Staphylococcus xylose was obtained by screening from cigar tobacco leaves.

[0045] One embodiment of this application also provides a biological agent comprising one or more of the following: Staphylococcus xylose, a culture of Staphylococcus xylose, lysate of Staphylococcus xylose, and an extract of Staphylococcus xylose.

[0046] Another embodiment of this application provides a method for preparing tobacco products, comprising the following steps: fermenting tobacco leaves with the above-mentioned Staphylococcus xylose or the above-mentioned biological agent to prepare tobacco products.

[0047] In some embodiments, the types of tobacco products mentioned above include cigar tobacco leaves.

[0048] In some embodiments, the tobacco products mentioned above include one or more of flue-cured and sun-cured tobacco.

[0049] The method for preparing tobacco products described in this application is simple, safe, and non-toxic, making it suitable for industrial application.

[0050] In some embodiments, the preparation method described above includes steps S10 to S20.

[0051] Step S10: Cultivate the above-mentioned Staphylococcus xylose under suitable conditions, collect the culture medium, and prepare bacterial solution.

[0052] Step S20: Mix the above bacterial solution and tobacco leaves for fermentation.

[0053] In some embodiments, the culture conditions include: placing the above-mentioned Staphylococcus xylose in a sterilized tobacco extract liquid culture medium and culturing it at 35°C to 37°C and 150 rpm to 200 rpm for 24 h to 48 h.

[0054] In one specific example, the above culture conditions include: placing the above Staphylococcus xylose in a sterilized liquid culture medium and culturing it at 37°C and 150 rpm for 48 h.

[0055] In some embodiments, the nutrients in the above-mentioned tobacco extract liquid culture medium include: 5% tobacco leaves and 1% glucose.

[0056] In some embodiments, the bacterial concentration in the bacterial solution is 10. 6 Cells / mL ~10 8 CFU / mL. For example, the concentration of the bacterial culture could be 10⁻⁶. 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 value is either per mL or any value within the range formed by any two of the above point values.

[0057] In some embodiments, after collecting the culture medium in step S10, the method further includes: diluting the culture medium so that the diluted bacterial concentration is 10. 6 Cells / mL ~10 8 per mL.

[0058] Optionally, the dilution factor is 5 to 50 times.

[0059] In some embodiments, the tobacco extract liquid culture medium comprises tobacco leaves and glucose, wherein the concentration of tobacco leaves is 1 g / L to 100 g / L and the concentration of glucose is 0.5 g / L to 15 g / L.

[0060] In some embodiments, the volume-to-mass ratio of the bacterial solution to the tobacco leaves is 0.5 mL to 5 mL: 100 g.

[0061] In some embodiments, the fermentation temperature is 30°C to 45°C. For example, the fermentation temperature can be 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, or 45°C, or any value within the range formed by any two of the above points.

[0062] In some embodiments, the relative humidity of the fermentation process is 70% to 75%. For example, the relative humidity of the fermentation process can be 70%, 71%, 72%, 73%, 74%, 75%, or any value within the range of any two of the above values.

[0063] In some embodiments, the fermentation time is 15 to 45 days. For example, the fermentation time can be 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 43 days, 44 days, or 45 days, or any value within the range formed by any two of the above point values.

[0064] Another embodiment of this application provides a tobacco product prepared by fermentation with Staphylococcus xylose and / or the above-described biological agents, or by the above-described preparation method.

[0065] The aforementioned tobacco products exhibit significantly reduced irritation from impurities and improved cleanliness.

[0066] The present application will be further described below with reference to specific embodiments and comparative examples, but these should not be construed as limiting the scope of protection of the present application. Unless otherwise specified, the raw materials involved in the following specific embodiments are all commercially available, the instruments used are all commercially available, and the processes involved are conventionally selected by those skilled in the art unless otherwise specified.

[0067] Example 1

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

[0069] (1) Sample pretreatment

[0070] Staphylococcus xylose SCT-B7 was isolated from the surface of high-quality cigar tobacco leaves. The cigar tobacco leaves were cut into small pieces using sterile scissors, added to sterile water and shaken for 5 minutes. After diluting 10 times, the mixture was spread onto a Staphylococcus selective agar plate (product number: HB4123-1, manufacturer: Qingdao Haibo Biotechnology), incubated at 37℃ for 48 hours, and purified 2-3 times. Single colonies were then picked to obtain Staphylococcus xylose.

[0071] (2) Strain identification

[0072] On selective agar medium, staphylococcal colonies are pale white and translucent, with regular edges and a raised center, and are Gram-positive. The strain grows well between 30℃ and 37℃, with an optimum growth temperature of 37℃. Growth is inhibited below 28℃ or above 40℃.

[0073]

[0074] (3) Investigate the utilization of carbon and nitrogen sources by Staphylococcus xylose SCT-B7.

[0075] Activate the OD strain of two generations 600 After adjusting to 1, 2% inoculum was added to different carbon and nitrogen source media, and the absorbance was measured after incubation at 37℃ and 200 rpm for 48 h. The carbon source liquid medium formula / L was: ammonium sulfate 2.0 g, magnesium sulfate 0.2 g, sodium dihydrogen phosphate 0.5 g, calcium chloride 0.1 g, sodium chloride 5.0 g, dipotassium hydrogen phosphate 0.5 g, with a carbon source addition of 10 g, sterilized at 115℃ for 30 min. The nitrogen source liquid medium formula / L was: calcium chloride 0.1 g, disodium hydrogen phosphate 2.13 g, glucose 10.0 g, magnesium sulfate 0.2 g, sodium chloride 5.0 g, ferrous sulfate 0.02 g, with a nitrogen source addition of 10 g, sterilized at 115℃ for 30 min. Staphylococcus aureus and Staphylococcus saprophyticus screened from tobacco leaves were used as controls. The results are shown in Table 1.

[0076] Table 1

[0077]

[0078] Note: "-" indicates no growth.

[0079] Table 1 shows that *Staphylococcus xylose* SCT-B7 can utilize eight carbon sources, including glucose, lactose, maltose, fructose, sucrose, cellobiose, trehalose, and xylan, and four nitrogen sources, including soybean meal, soybean peptone, tryptone, and yeast extract, and grows well in LB medium. *Staphylococcus nebula* utilizes three nitrogen sources, including soybean peptone, tryptone, and yeast extract. *Staphylococcus saprophyticus* can utilize five carbon sources, including maltose, fructose, sucrose, cellobiose, and trehalose, and four nitrogen sources, including soybean meal, soybean peptone, tryptone, and yeast extract.

[0080] (4) Analysis of enzyme production characteristics of strains

[0081] Staphylococcus xylose SCT-B7 was inoculated into a selective staphylococcal culture medium and cultured in a constant temperature shaker at 37°C and 150 rpm for 2 days. The fermentation broth was centrifuged to obtain the supernatant. The activities of cellulase, protease, and amylase in the supernatant were determined using the clear zone method. Specifically, 10 μL of the supernatant was transferred to cellulase-producing differential medium (1.0% sodium carboxymethyl cellulose, 0.5% yeast extract, 0.5% peptone, 1.0% sodium chloride, 0.15% potassium dihydrogen phosphate, 2% agar powder) and protease-producing differential medium (0.36 g / L KH₂PO₄, 0.5 g / L MgSO₄, 0.014 g / L ZnCl₂, 1.07 g / L Na₂HPO₄·7H₂O, 0.16 g / L NaCl, 0.002 g / L CaCl₂, 0.002 g / L FeSO₄, 4 g / L casein, 0.05 g / L tryptone, 20 g / L agar, pH 1.00). The cells were cultured in 6.5-7.0% amylase-producing differential medium (soluble starch 1.0%, peptone 0.5%, glucose 0.5%, beef extract 0.5%, agar 2.0%, sodium chloride 0.5%) at 37℃ for 24 h. The cellulase-producing differential medium was stained with 0.2% Congo red for 0.5 h, washed off with distilled water, washed again with 1 mol / L sodium chloride solution, and finally fixed with 5% acetic acid. The appearance of a white transparent zone around the well indicates that the strain produces cellulase. The presence of a hydrolysis zone was observed on the protease-producing differential medium to determine whether the strain produces protease. Gram's iodine solution was used to stain the amylase-producing differential medium; the appearance of a transparent zone around the well indicates that the strain produces amylase. The diameter of the hydrolysis zone or transparent zone was measured with calipers to determine the enzyme activity. The results are shown in Table 2.

[0082] Three strains were found to produce protease, amylase, and cellulase. However, Staphylococcus xylosus SCT-B7 showed the strongest ability to produce all three enzymes, specifically demonstrating a strong protease production capacity, as well as some cellulase and amylase production capacity.

[0083] Table 2

[0084]

[0085] (5) Determination of the growth curve of strain SCT-B7

[0086] The activated second-generation Staphylococcus xylosus SCT-B7, Staphylococcus nigra, and Staphylococcus saprophyticus were inoculated at a 2% inoculum into LB medium (10 g tryptone, 5 g yeast extract, 10 g NaCl, 1000 mL water, pH adjusted to 7.0, sterilized at 121℃ for 20 min) and tobacco powder medium (60 g tobacco powder added to 1200 mL of 100℃ hot water, soaked for 2 h, filtered through 4 layers of gauze, the supernatant was centrifuged again (10000 rpm, 10 min), 1000 mL of the supernatant was collected, 10 g glucose was added, and after the glucose was fully dissolved, it was dispensed into 100 mL bottles and sterilized at 115℃ for 30 min). The growth curves of the strains were determined using an online cell OD value detection bioreactor (ZWYF-290, Shanghai Zhicheng), and the graphs are shown below. Figures 3-5 As shown. By Figures 3-5 It can be seen that the strains grew better in LB medium than in tobacco ash medium. Furthermore, the growth curves of the three strains differed, and *Staphylococcus saprophyticus* grew better in tobacco ash medium.

[0087] II. Fermentation Methods of Cigar Tobacco Leaves

[0088] (1) Inoculate Staphylococcus xylose into tobacco extract medium (5% tobacco leaves, 1% glucose) and incubate for 2 days at 37°C and 150 rpm in a constant temperature shaker. Take seed liquid and inoculate it into tobacco extract medium (5% tobacco leaves, 1% glucose) at a volume ratio of 1% and incubate for 2 days at 37°C and 150 rpm in a constant temperature shaker.

[0089] (2) Take 10 mL of fermentation broth and dilute it with sterile water to a final concentration of 10 mL. 7 CFU / mL was used to obtain the fermentation broth.

[0090] (3) Spray the fermentation liquid at 0.5% of the tobacco leaf weight onto cigar tobacco leaves from Hainan with a moisture content of 20%.

[0091] (4) Ferment the inoculated tobacco leaves for 15 days at 35°C and 75% relative humidity.

[0092] Tests: 1) Conduct sensory evaluation of the fermented cigar tobacco samples, specifically by referring to GB 15269.4-2011 Cigar Tobacco Part 4: Sensory Technical Requirements to evaluate the quality of the tobacco leaves.

[0093] 2) The chlorine, potassium, total nitrogen, total alkaloids, reducing sugar, and total sugar content of the fermented cigar tobacco samples were determined. Specifically, the chlorine (YC / T 162-2011 Determination of chlorine in tobacco and tobacco products by continuous flow method), potassium (YC / T 217-2007 Determination of potassium in tobacco and tobacco products by continuous flow method), total nitrogen (YC / T161-2002 Determination of total nitrogen in tobacco and tobacco products by continuous flow method), total alkaloids (YC / T 468-2021 Determination of total alkaloids in tobacco and tobacco products by continuous flow (potassium thiocyanate) method), reducing sugar, and total sugar (YC / T 159-2002 Determination of water-soluble sugars in tobacco and tobacco products by continuous flow method) were determined using continuous flow analysis.

[0094] The specific results are shown in Table 3 below.

[0095] Table 3

[0096]

[0097] According to professional tasting, the aroma of the tobacco leaves is significantly enhanced, with increased aroma volume and sweetness, reduced off-flavors and irritation, and a clean aftertaste.

[0098] The changes in conventional chemical components in tobacco leaves were determined using a flow analyzer, as shown in Table 4.

[0099] Table 4

[0100]

[0101] As shown in Table 4, the total sugar and reducing sugar content in tobacco leaves increased, while the total nitrogen and total alkaloid content decreased, which is consistent with the results of increased sweetness in the smoke in the sensory evaluation.

[0102] Example 2

[0103] The fermentation method of tobacco leaves in Example 2 is basically the same as that in Example 1, the difference being the type of tobacco leaves. The specific steps are as follows:

[0104] (1) Inoculate Staphylococcus xylose into tobacco extract medium and incubate for 2 days at 37°C and 150 rpm in a constant temperature shaker. Take seed liquid and inoculate it into tobacco extract medium (5% tobacco leaves, 1% glucose) at a volume ratio of 1% and incubate for 2 days at 37°C and 150 rpm in a constant temperature shaker.

[0105] (2) Take 10 mL of fermentation broth and dilute it with sterile water to a final concentration of 10 mL. 7 CFU / mL was used to obtain the fermentation broth.

[0106] (3) Spray the fermentation liquid at 1% of the weight of the tobacco leaves onto the Yunxue No. 2 cigar tobacco leaves from Yunnan with a moisture content of 17%.

[0107] (4) Ferment the inoculated tobacco leaves for 15 days at 35°C and 75% relative humidity.

[0108] Tests: 1) Sensory evaluation was conducted on the fermented cigar tobacco samples, specifically in accordance with GB 15269.4-2011 Cigar Tobacco Part 4: Sensory Technical Requirements, to evaluate the quality of the tobacco leaves. The specific results are shown in Table 5 below.

[0109] 2) The volatile components of the fermented cigar tobacco samples were tested. GC-MS was used to determine the volatile components in the tobacco leaves. Nicotine content was determined using the continuous flow method (YC / T160-2002, Determination of Total Alkaloids in Tobacco and Tobacco Products). Protein content was determined using the continuous flow method (YC / T249-2008, Determination of Protein in Tobacco and Tobacco Products). Starch content was determined using the continuous flow method (YC / T216-2013, Determination of Starch in Tobacco and Tobacco Products). The specific results are shown in Table 6 below.

[0110] 3) The fermented cigar tobacco samples were tested for chlorine, potassium, total nitrogen, total alkaloids, reducing sugar, and total sugar content. Specifically, continuous flow analysis was used to determine the chlorine (YC / T 162-2011 Determination of chlorine in tobacco and tobacco products by continuous flow method), potassium (YC / T 217-2007 Determination of potassium in tobacco and tobacco products by continuous flow method), total nitrogen (YC / T161-2002 Determination of total nitrogen in tobacco and tobacco products by continuous flow method), total alkaloids (YC / T 468-2021 Determination of total alkaloids in tobacco and tobacco products by continuous flow (potassium thiocyanate) method), reducing sugar, and total sugar (YC / T 159-2002 Determination of water-soluble sugars in tobacco and tobacco products by continuous flow method).

[0111] The specific results are shown in Tables 5 to 7 below.

[0112] Table 5

[0113]

[0114] Table 6

[0115]

[0116]

[0117] According to professional tasting, the aroma of the tobacco leaves was significantly enhanced, while off-flavors were reduced. Measurements showed a significant increase in volatile components in the fermented tobacco leaves, from the original 13161.81 μg / g to 15825.54 μg / g. The content of 30 volatile components increased in the fermented tobacco leaves, including aroma-producing compounds such as 4-hydroxy-3,5,5-trimethyl-4-(3-oxo-1-butenyl)-2-cyclohexen-1-one, vitamin E, nicotine, podophyllin, dihydroactinol, and isovaleric acid. The content of 12 volatile components decreased in the fermented tobacco leaves, including 4,6-dimethyl-dodecane, succinate lactone, and 2,4-dimethyl-1-heptene.

[0118] The changes in conventional chemical components in tobacco leaves were determined using a flow analyzer, as shown in Table 7.

[0119] Table 7

[0120]

[0121] As shown in Table 7, the content of irritating total nitrogen and total alkaloids in tobacco leaves decreased, while the content of total sugar and reducing sugar increased, which is consistent with the results of increased sweetness of smoke in sensory evaluation.

[0122] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments 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.

[0123] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. 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 protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims.

Claims

1. A Staphylococcus xylosus strain, characterized in that, The accession number is CGMCCNo.36128.

2. A biological agent, characterized in that, The biological agent comprises one or more of the following: Staphylococcus xylose as described in claim 1, a culture of Staphylococcus xylose as described in claim 1, a lysate of Staphylococcus xylose as described in claim 1, and an extract of Staphylococcus xylose as described in claim 1.

3. A method for preparing a tobacco product, characterized in that, Includes the following steps: Tobacco products are prepared by fermenting tobacco leaves using Staphylococcus xylose as described in claim 1 or the biological agent as described in claim 2.

4. The method for preparing tobacco products as described in claim 3, characterized in that, The types of tobacco products mentioned include cigar tobacco leaves.

5. The method for preparing a tobacco product according to any one of claims 3 to 4, characterized in that, The preparation method includes the following steps: The *Staphylococcus xylose* is cultured, the culture broth is collected, and a bacterial solution is prepared; the bacterial solution and the tobacco leaves are then mixed and fermented.

6. The method for preparing tobacco products as described in claim 5, characterized in that, The cultivation conditions include: placing the *Staphylococcus xylose* in sterilized tobacco extract liquid culture medium and culturing it at 35℃~37℃ and 150rpm~200rpm for 24h~48h.

7. The method for preparing tobacco products as described in claim 6, characterized in that, The bacterial concentration in the bacterial solution was 10. 6 Cells / mL ~10 8 cells / mL; And / or, the tobacco extract liquid culture medium comprises tobacco leaves and glucose, wherein the concentration of the tobacco leaves is 1 g / L to 100 g / L and the concentration of the glucose is 0.5 g / L to 15 g / L.

8. The method for preparing tobacco products as described in claim 5, characterized in that, The volume-to-mass ratio of the bacterial solution to the tobacco leaves is 0.5 mL to 5 mL: 100 g.

9. The method for preparing a tobacco product according to any one of claims 3-4 and 6-8, characterized in that, The fermentation temperature is 30℃~45℃, the relative humidity is 70%~75%, and the fermentation time is 15 days~45 days.

10. A tobacco product, characterized in that, It is prepared by fermentation of Staphylococcus xylose as described in claim 1 and / or the biological agent as described in claim 2, or by the preparation method described in any one of claims 3 to 9.

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