New Aspergillus niger, biological agents, tobacco products and their preparation methods

Abstract

This application relates to a novel Aspergillus neoniger, biological agents, tobacco products, and methods for preparing the same. The novel Aspergillus neoniger has the accession number CGMCC No. 42429. This strain is used to selectively improve the flavor of cigar tobacco leaves during fermentation, effectively transferring the "mellow, sweet, and pronounced aftertaste" qualities of dark tea to the tobacco product. This significantly enhances the sweetness, smoothness, and aftertaste quality of the fermented cigar tobacco leaves while reducing their harshness.

Description

Technical Field

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

[0002] The fermentation process of cigar tobacco leaves is the core factor determining their sensory quality. The natural fermentation technology currently widely used in the industry is highly dependent on environmental microorganisms, which has inherent defects such as long fermentation cycles, uncontrollable microbial community structure, and poor product quality stability. This can easily lead to problems such as insufficient accumulation of aroma substances in tobacco leaves and incomplete degradation of irritating components.

[0003] Furthermore, exogenous microorganisms have difficulty colonizing in the tobacco leaf environment and their metabolic activity is unstable. They lack the ability to directionally shape the characteristic flavor of cigar tobacco leaves, and in particular, it is difficult to achieve a mellow and sweet flavor similar to that of black tea.

[0004] Therefore, it is necessary to discover new microbial strains that can be used for the fermentation of cigar tobacco leaves. Summary of the Invention

[0005] This application mainly provides a new strain of Aspergillus niger, which is used to achieve targeted improvement of the flavor of cigar tobacco leaves through fermentation. It effectively transfers the "mellow and sweet, with obvious aftertaste" quality characteristics of black tea to tobacco products, significantly improving the sweetness, delicacy and aftertaste quality of fermented cigar tobacco leaves, while reducing their irritation.

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

[0007] The first aspect of this application provides a novel Aspergillus neoniger, with accession number CGMCC No. 42429.

[0008] A second aspect of this application provides a biological agent comprising one or more of the following: *Aspergillus niger*, a culture of *Aspergillus niger*, a lysate of *Aspergillus niger*, and an extract of *Aspergillus niger*.

[0009] The 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 with the aforementioned new Aspergillus niger or the aforementioned biological agent.

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

[0012] In some embodiments, the preparation method includes the following steps:

[0013] Culture the novel *Aspergillus niger*, collect the culture medium, and prepare a bacterial culture; and

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

[0015] In some embodiments, the culture conditions include placing the new Aspergillus niger in a sterilized liquid culture medium and culturing it at 26°C to 37°C for 12 to 48 hours.

[0016] In some embodiments, the bacterial concentration in the bacterial solution is 10. 5 cfu / mL ~10 7 cfu / mL;

[0017] And / or, the liquid culture medium comprises bean sprouts and glucose, wherein the concentration of the bean sprouts is 100 g / L to 300 g / L and the concentration of the glucose is 10 g / L to 30 g / L; or, the liquid culture medium comprises 100 g / L to 200 g / L malt extract powder.

[0018] In some embodiments, the volume-to-mass ratio of the bacterial solution to the tobacco leaves is 10 mL to 30 mL: 100 g.

[0019] In some embodiments, the fermentation temperature is 25°C to 40°C, the relative humidity is 70% to 75%, and the fermentation time is 14 to 35 days.

[0020] The fourth aspect of this application provides a tobacco product prepared by fermentation of the aforementioned Aspergillus niger or the aforementioned biological agent, or by the preparation method described above.

[0021] This application provides a novel Aspergillus niger strain that utilizes its natural adaptability to the tobacco leaf ecological environment and its unique metabolic characteristics to efficiently complete material transformation while achieving targeted shaping of distinctive flavors. Compared to conventional Aspergillus niger strains, the novel Aspergillus niger exhibits higher β-glucosidase activity, enabling more effective degradation of cellulose. Furthermore, the enzyme system secreted by the novel Aspergillus niger can effectively hydrolyze tobacco starch, pectin, and other polysaccharides into soluble reducing sugars, while simultaneously degrading proteins to generate free amino acids, providing ample substrates for the synthesis of aroma compounds. Cigar tobacco leaves fermented with this novel Aspergillus niger show a significant increase in the content of alcohols and acids, a significant improvement in chlorophyll degradation efficiency, and a significant increase in the content of neophydiene, a representative aroma component. The quality of tobacco leaves fermented with A. neoniger is significantly improved, with a smoother, sweeter smoke and a higher degree of maturity. Compared to the pure water treatment group (which relied on the tobacco leaves' own microorganisms), the introduction of *A. neoniger* significantly improved the smoothness, sweetness, and aftertaste of cigar tobacco leaves, while noticeably reducing harshness. These results indicate that the application of *A. neoniger* not only enhances the aroma characteristics of tobacco leaves but also helps improve their overall quality, providing an effective biotechnological approach 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 diagram showing the colony morphology of *Aspergillus niger* in Example 1 of this application.

[0024] Figure 2 This is the ITS-based phylogenetic tree of *Aspergillus niger* in Example 1 of this application.

[0025] The novel Aspergillus niger provided in this application was deposited on January 14, 2026, 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. 42429. Detailed Implementation

[0026] 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.

[0027] 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.

[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] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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℃.

[0036] 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.

[0037] Based on this, one embodiment of this application provides a new strain of Aspergillus neoniger, which was deposited on January 14, 2026, 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. 42429.

[0038] This application provides a novel Aspergillus niger strain that utilizes its natural adaptability to the tobacco leaf ecological environment and its unique metabolic characteristics to efficiently complete material transformation while achieving targeted shaping of distinctive flavors. Compared to conventional Aspergillus niger strains, the novel Aspergillus niger exhibits higher β-glucosidase activity, enabling more effective degradation of cellulose. Furthermore, the enzyme system secreted by the novel Aspergillus niger can effectively hydrolyze tobacco starch, pectin, and other polysaccharides into soluble reducing sugars, while simultaneously degrading proteins to generate free amino acids, providing ample substrates for the synthesis of aroma compounds. Cigar tobacco leaves fermented with this novel Aspergillus niger show a significant increase in the content of alcohols and acids, a significant improvement in chlorophyll degradation efficiency, and a significant increase in the content of neophydiene, a representative aroma component. The quality of tobacco leaves fermented with A. neoniger is significantly improved, with a smoother, sweeter smoke and a higher degree of maturity. Compared to the pure water treatment group (which relied on the tobacco leaves' own microorganisms), the introduction of *A. neoniger* significantly improved the smoothness, sweetness, and aftertaste of cigar tobacco leaves, while noticeably reducing harshness. These results indicate that the application of *A. neoniger* not only enhances the aroma characteristics of tobacco leaves but also helps improve their overall quality, providing an effective biotechnological approach for tobacco fermentation processes.

[0039] In some of these embodiments, the aforementioned novel Aspergillus niger was obtained by screening from dark tea.

[0040] One embodiment of this application also provides a biological agent comprising one or more of the following: Aspergillus niger, a culture of Aspergillus niger, a lysate of Aspergillus niger, and an extract of Aspergillus niger.

[0041] Another embodiment of this application provides a method for preparing tobacco products, comprising the following steps: fermenting tobacco leaves with the aforementioned Aspergillus niger or the aforementioned biological agent to prepare tobacco products.

[0042] In some embodiments, the types of tobacco products mentioned above include cigar tobacco leaves.

[0043] The method for preparing tobacco products described in this application is simple, safe, and non-toxic, making it suitable for industrial application.

[0044] In some embodiments, the preparation method described above includes steps S10 to S20.

[0045] Step S10: Cultivate the above-mentioned new Aspergillus niger under suitable conditions, collect the culture medium, and prepare bacterial culture.

[0046] Step S20: Mix the above bacterial solution and tobacco leaves for fermentation.

[0047] In some embodiments, the culture conditions include: placing the above-mentioned new Aspergillus niger in a sterilized liquid culture medium and culturing it at 26°C to 37°C for 12 to 48 hours.

[0048] In one specific example, the above-mentioned culture conditions include: placing the above-mentioned new Aspergillus niger in a sterilized liquid culture medium and culturing it at 30°C for 48 hours.

[0049] In some embodiments, the liquid culture medium comprises bean sprouts and glucose, wherein the concentration of the bean sprouts is 100 g / L to 300 g / L and the concentration of the glucose is 10 g / L to 30 g / L.

[0050] In some embodiments, the liquid culture medium comprises 100 g / L to 200 g / L malt extract powder.

[0051] In some embodiments, the nutrients in the liquid culture medium include: 100 g / L bean sprouts and 20 g / L glucose.

[0052] In some embodiments, the bacterial concentration in the bacterial solution is 10. 5 cfu / mL ~10 7 cfu / mL.

[0053] 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. 5 cfu / mL ~10 7 cfu / mL.

[0054] Optionally, the dilution factor is 1 to 5 times.

[0055] In some embodiments, the volume-to-mass ratio of the bacterial solution to the tobacco leaves is 10 mL to 30 mL: 100 g.

[0056] In some embodiments, the temperature of the fermentation treatment is 25°C to 40°C. For example, the temperature of the fermentation treatment can be 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, or 40°C, or any value within the range formed by any two of the above points.

[0057] 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.

[0058] In some embodiments, the fermentation time is 14 to 35 days. For example, the fermentation time can be 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 days, or any value within the range formed by any two of the above points.

[0059] Another embodiment of this application provides a tobacco product prepared by fermentation with the aforementioned Aspergillus niger or the aforementioned biological agent, or by the aforementioned preparation method.

[0060] The aforementioned tobacco products exhibit significantly reduced harshness and irritation, while significantly improved smoothness, sweetness, and aftertaste.

[0061] 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.

[0062] Example 1

[0063] I. Isolation, Screening and Identification of Strains

[0064] (1) Sample pretreatment

[0065] *Aspergillus niger* was isolated from Sichuan Ya'an dark tea. The Sichuan Ya'an dark tea was chopped using sterile scissors, and sterile water was added and shaken for 5 minutes to mix thoroughly, forming a bacterial suspension. This suspension was diluted 100 times and spread onto YPD solid medium. The formulation was: YPD medium A – 10g yeast extract, 20g peptone, 20g agar powder, 900mL deionized water; YPD medium B – 20g glucose, 100mL deionized water. Both were sterilized separately, and then solutions A and B were mixed, with the pH set to natural. The medium was incubated at 37℃ for 12 days. For secondary screening, malt extract medium was used, with the formulation: 130g malt extract medium, 20g agar, 1L distilled water, and the pH adjusted to 6.0. The medium was incubated at 37℃ for 4 days, followed by 2-3 purification cycles. Single colonies were picked to obtain *Aspergillus niger*.

[0066] (2) Strain identification

[0067] On solid culture media, colonies grow rapidly. Initially white, they turn light brown, then dark brown. The colonies are round, slightly raised, with distinct radial grooves on the surface and a fluffy texture. No exudate is produced during cultivation, and no soluble pigments are secreted (e.g., ...). Figure 1As shown in Figure a). Under an optical microscope (640×), the mycelium of this strain is septate, with numerous branches at small angles, exhibiting regular dichotomous branching. The mycelium is transparent, with smooth, unadorned cell walls. Conidiophores arise from the mycelium, with smooth, brown stem walls that swell at the apex to form a flask-shaped apex, covered with a single layer of pedicels. Conidia are spherical, arranged singly or in short chains at the apex of the pedicels (e.g., ...). Figure 1 b and Figure 1 As shown in c). In the SEM 1000× magnified image (e.g. Figure 1 In the field of view shown in d), the hyphae are distributed in a network, the conidiophores grow upright, there are no creeping hyphae, and the aerial hyphae differentiate directly into conidiophores at their tips. (10000× magnified image, as shown) Figure 1 As shown in Figure e), the conidiophore walls are smooth, the apical vesicle is flask-shaped, and the pedicels are closely arranged on the surface of the vesicle; the conidia are spherical, and the surface spores are connected by short chains. The individual spores are regular in shape and have no protrusions or depressions on the surface.

[0068] The purified monoclonal colonies from step (1) were placed in sterile water, boiled at high temperature to extract DNA, amplified, and then sent for sequencing. Sequencing was performed using primers SEQ ID No. 1: 5'-TCCCGTAGGTGAACCTGCGG-3' and SEQ ID No. 2: 5'-TCCTCCGCTTATTGATATGC-3'. The ITS sequence is shown in SEQ ID No. 3. Further sequence comparison revealed that the analyzed strain showed 100% homology with *Aspergillus niger*, and could be identified as *Aspergillus niger*. This *Aspergillus niger* was deposited on January 14, 2026, 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. 42429. The ITS sequence shown in SEQ ID No. 3 is 3'--5'.

[0069] Secondly, through numerous calculations using MEGA 11, the branching position of the strain was further determined, and a phylogenetic tree was constructed. The results are as follows: Figure 2In the phylogenetic tree, the selected strain clustered closely with the type strain *Aspergillus neoniger* CBS 115656, forming a distinct branch from other species in the *Aspergillus* genus (such as *Aspergillus piperis* and *Aspergillus foetidus*), with significant genetic distances between the branches. Based on its morphological characteristics, this strain was accurately identified as *Aspergillus neoniger* of the *Aspergillus* genus.

[0070] II. Fermentation Methods of Cigar Tobacco Leaves

[0071] (1) Inoculate new Aspergillus niger into liquid culture medium (the specific preparation method is to add 100 g of soybean sprouts to 1000 mL of water, boil, filter to obtain soybean sprout juice, then add 20 g of glucose, sterilize at 121℃ and then use), place in a constant temperature device and culture at 30℃ for 2 days to obtain seed liquid.

[0072] (2) Mix the above seed liquid with pure water at a volume ratio of 2:1 to obtain fermentation liquid.

[0073] (3) Spray the fermentation liquid evenly onto the surface of the cigar tobacco leaves at 15% of the tobacco leaf weight.

[0074] (4) Ferment the inoculated tobacco leaves for 35 days at 35°C and 75% relative humidity.

[0075] Example 2

[0076] The fermentation method of tobacco leaves in Example 2 is basically the same as that in Example 1, except that the fermentation time is different. The specific steps are as follows:

[0077] (1) Inoculate new Aspergillus niger into liquid culture medium (the specific preparation method is to add 100 g of soybean sprouts to 1000 mL of water, boil, filter to obtain soybean sprout juice, then add 20 g of glucose, sterilize at 121℃ and then use), place in a constant temperature device at 30℃, and culture for 2 days to obtain seed liquid.

[0078] (2) Mix the above seed liquid with pure water at a volume ratio of 2:1 to obtain fermentation liquid.

[0079] (3) Spray the fermentation liquid evenly onto the surface of the cigar tobacco leaves at 15% of the tobacco leaf weight.

[0080] (4) Ferment the inoculated tobacco leaves for 28 days at 35°C and 75% relative humidity.

[0081] Example 3

[0082] The fermentation method of tobacco leaves in Example 3 is basically the same as that in Example 1, except that the fermentation time is different. The specific steps are as follows:

[0083] (1) Inoculate new Aspergillus niger into liquid culture medium (the specific preparation method is to add 100 g of soybean sprouts to 1000 mL of water, boil, filter to obtain soybean sprout juice, then add 20 g of glucose, sterilize at 121℃ and then use), place in a constant temperature device at 30℃, and culture for 2 days to obtain seed liquid.

[0084] (2) Mix the above seed liquid with pure water at a volume ratio of 2:1 to obtain fermentation liquid.

[0085] (3) Spray the fermentation liquid evenly onto the surface of the cigar tobacco leaves at 15% of the tobacco leaf weight.

[0086] (4) Ferment the inoculated tobacco leaves at 35°C and 75% relative humidity for 14 days.

[0087] Comparative Example 1

[0088] The fermentation method of tobacco leaves in Comparative Example 1 is basically the same as that in Example 1, except that pure water treatment without the addition of Aspergillus niger liquid is used as a control for fermentation.

[0089] The other steps and parameters are the same as in Example 1.

[0090] Comparative Example 2

[0091] The fermentation method of tobacco leaves in Comparative Example 2 is basically the same as that in Example 2, except that pure water treatment without adding Aspergillus niger liquid is used as a control for fermentation.

[0092] The other steps and parameters are the same as in Example 2.

[0093] Comparative Example 3

[0094] The fermentation method of tobacco leaves in Comparative Example 3 is basically the same as that in Example 3, except that pure water treatment without the addition of Aspergillus niger liquid is used as a control for fermentation.

[0095] The other steps and parameters are the same as in Example 3.

[0096] test:

[0097] 1) Sensory evaluation was conducted on the fermented cigar tobacco samples. The fermented tobacco samples were rolled into single-material tobaccos 110mm in length and 14mm in diameter, and equilibrated for 7 days in a Binder constant temperature and humidity chamber at 20℃ and 65% relative humidity before being used for evaluation. A 10-member evaluation panel, certified by the National Tobacco Quality Supervision and Inspection Center, conducted the sensory quality evaluation according to the Great Wall Cigar Factory's product technical standard, "Sensory Evaluation Method for the 'Mellow and Sweet Aroma' Style Characteristics of Chinese Cigars" QJ / 08.J.6005-2020 A.

[0098] 2) The physicochemical metabolic indicators of fermented cigar tobacco samples, including total nitrogen, alkaloids, total sugar, reducing sugar, potassium ions, chloride ions, potassium-chloride ratio, amino acids, lignin, and cellulose, were determined. Total nitrogen, alkaloids, total sugar, reducing sugar, potassium, and chloride were determined according to YC / T 161-2002, YC / T 160-2002, YC / T 159-2019, YC / T 217-2007, and YC / T162-2011, respectively. Total free amino acids were determined according to YC / T 448-2012 using the phorate colorimetric method. Crude fiber was determined according to YC / T347-2010 using the acetyl bromide method and the sodium chlorite-acetic acid method.

[0099] 3) The aroma compounds content of the fermented cigar tobacco samples was determined by GC-MS. GC-MS sample preparation: 2g of tobacco sample was crushed and treated with QuEChERS extraction reagent. The aroma components in the extract were analyzed by GC-MS, with phenethyl acetate as an internal standard.

[0100] GC-MS conditions: DB-5 MS column (60 m × 1.0 μm × 0.25 mm); initial temperature 60 °C, ramped to 250 °C at a rate of 2 °C / min, then ramped to 290 °C at a rate of 5 °C / min and held for 20 min; injection port temperature 290 °C; split ratio 10:1; carrier gas helium; flow rate 1.5 mL / min; ionization mode electron impact (EI); ion source temperature 230 °C; ionization energy 70 eV; quadrupole temperature 150 °C; mass spectrometry scan range 26–400 amu. Qualitative and quantitative analysis were performed by matching the mass spectrometry data with the NIST and Wiley databases.

[0101] The specific results are shown in Tables 1 to 4 below.

[0102] Table 1

[0103]

[0104] As shown in Table 1, the alkaloid content decreased significantly in Example 3 after fermentation with *Aspergillus niger*, indicating that *Aspergillus niger* has a strong alkaloid degradation ability even with short-term fermentation, which helps reduce the irritation and strength of the smoke. Furthermore, the total sugar, reducing sugar, and amino acid contents in the examples were generally higher than those in the comparative example (except for the reducing sugar content, which was similar between Example 3 and Comparative Example 3). This indicates that the enzyme system secreted by *Aspergillus niger* can hydrolyze polysaccharides such as tobacco starch and pectin into soluble reducing sugars, while simultaneously degrading proteins to generate free amino acids, providing sufficient substrates for the synthesis of aroma compounds.

[0105] Neophytadiene, a degradation product of chlorophyll, is a representative aroma component in cigar tobacco. During the preparation and fermentation of tobacco, it participates in various biochemical reactions, directly contributing to the sensory quality of cigars and transforming into other flavor compounds through metabolic pathways, further influencing the overall aroma characteristics. The accumulation of neophytadiene significantly increased after the addition of *Aspergillus niger*, as shown in Table 2. The highest accumulation was observed in Example 3, at 306 μg / g, representing a 138% increase compared to Comparative Example 3. The accumulation levels in Examples 1 and 2 were also higher than those in the corresponding pure water treatment groups, increasing by 21% and 40%, respectively.

[0106] Table 2

[0107]

[0108] Besides neophytadiene, nicotine, and their derivatives, the aroma components in tobacco leaves were classified into seven categories, including ketones, phenols, esters, aldehydes, acids, alcohols, and nitrogen-containing heterocyclic compounds. In Example 1, fermentation with *Aspergillus niger* significantly increased the total content of the seven aroma components in tobacco leaves to 2680 μg / g, a 17% increase compared to 2286 μg / g in Comparative Example 1. The content and proportion of key aroma components such as esters, acids, and alcohols were significantly increased. The specific proportions of different aroma components in the total amount are shown in Table 3 below.

[0109] Table 3

[0110]

[0111] Table 4

[0112]

[0113] As shown in Table 4 above, the quality of the tobacco leaves in both Example 1 and Comparative Example 1 was improved to varying degrees after fermentation. Among them, the cigar tobacco leaves fermented with *A. neoniger* showed the best overall performance, with a smoother smoke, enhanced sweetness, and higher maturity. Compared to Comparative Example 1 (which relied on the tobacco leaves' own microorganisms), the introduction of *A. neoniger* significantly improved the smoothness, sweetness, and aftertaste of the cigar tobacco leaves, while noticeably reducing harshness.

[0114] 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.

[0115] 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 novel Aspergillus neoniger, characterized in that, The accession number is CGMCC No.42429.

2. A biological agent, characterized in that, The biological agent comprises one or more of the following: Aspergillus niger as described in claim 1, a culture of Aspergillus niger as described in claim 1, a lysate of Aspergillus niger as described in claim 1, and an extract of Aspergillus niger 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 the new Aspergillus niger 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: Culture the novel *Aspergillus niger*, collect the culture medium, and prepare a bacterial culture; and The bacterial solution and the tobacco leaves are mixed and fermented.

6. The method for preparing tobacco products as described in claim 5, characterized in that, The cultivation conditions include: placing the new Aspergillus niger in a sterilized liquid culture medium and culturing it at 26℃~37℃ for 12h~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. 5 cfu / mL ~10 7 cfu / mL; And / or, the liquid culture medium comprises bean sprouts and glucose, wherein the concentration of the bean sprouts is 100 g / L to 300 g / L and the concentration of the glucose is 10 g / L to 30 g / L; or, the liquid culture medium comprises 100 g / L to 200 g / L malt extract powder.

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 10 mL to 30 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 25℃~40℃, the relative humidity is 70%~75%, and the fermentation time is 14 days~35 days.

10. A tobacco product, characterized in that, It is prepared by fermentation of the new Aspergillus niger as described in claim 1 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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