Aspergillus scherweri for cigar tobacco leaf fermentation and application thereof

Abstract

The invention discloses Aspergillus scherweri for cigar tobacco leaf fermentation and application of the Aspergillus scherweri. The aspergillus scherweri is preserved in Guangdong Microbial Culture Collection Center, and the number is GDMCC No: 66976. The microbial composition (Aspergillus scherweri C1, Trichoderma xerophyllum YMF1.09953 and Brevundimonas sp. 1.547-1) provided by the invention can form efficient microbial community synergy in a cigar tobacco leaf fermentation system. The fermentation inoculant prepared on the basis of the microbial composition shows an excellent effect in cigar tobacco leaf fermentation. After the cigar tobacco leaves are fermented for only 15 days, the total content and composition richness of various key aroma components (such as neophytadiene, geraniol and the like) in the cigar tobacco leaves and the degradation efficiency on cellulose and hemicellulose are obviously superior to those of control group tobacco leaves which are naturally fermented for 45 days, so that the microbial composition disclosed by the invention can be used for effectively improving the aroma quality of the cigar tobacco leaves; and the degradation of fibrous components is accelerated.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a type of Aspergillus chevaleri used in the fermentation of cigar tobacco leaves and its application. Background Technology

[0002] Cigars are cigarettes that are entirely hand-rolled from tobacco leaves. They possess a distinctive aroma, unique appeal, and are characterized by low tar and high nicotine content. Currently, because domestic cigar raw material suppliers are still developing and have not yet achieved economies of scale, most domestically produced cigar raw materials still rely on imports. This, to some extent, hinders the development of Chinese-style cigars and the overall growth of the Chinese cigar industry.

[0003] The filler tobacco is the core of a cigar, determining its flavor and the layers of aroma during smoking. Filler tobacco should possess a cigar aroma profile, a pleasant taste with a certain strength and appropriate potency, and burn well, appearing as a tightly rolled, grayish-white leaf. The world's finest filler tobacco comes from the Caribbean region; currently, most filler tobacco produced in my country lacks a rich aroma. The quality and style of cigar filler tobacco are inextricably linked to ecological conditions, cultivation techniques, harvesting, processing, and subsequent fermentation and aging.

[0004] Cigar tobacco fermentation is a continuation of the conditioning process, an artificially accelerated aging process. During fermentation, the substances contained in the tobacco leaves are further decomposed and transformed, which can make the various chemical components in the tobacco leaves more coordinated, the smoke more delicate, the irritation less, and thus improve the quality of the tobacco leaves.

[0005] Cigar filler tobacco can be fermented using different methods depending on its intended use (usually referring to artificial fermentation). Currently, the traditional heap fermentation method is mainly used for cigar filler tobacco in my country. Heap fermentation requires cigar tobacco leaves to be piled up while still moist, and then turned several times. Steam fermentation can significantly reduce the harshness and off-flavors of cigar filler tobacco leaves, resulting in leaves with a deep and uniform color and high oil content, thus significantly improving the industrial usability of the tobacco leaves. The fermentation method for cigar tobacco leaves should be selected based on the differences in the quality of the filler tobacco leaves and the desired style characteristics, in order to maximize the industrial usability of the tobacco leaves.

[0006] Whether through natural or artificial fermentation, microbial activity during cigar production is closely related to the quality of cigar tobacco leaves, as fermentation degrades large organic molecules in the leaves. Studies have shown that Bacillus cereus (… Bacillus cereus ) can reduce the total nitrogen content of cigar tobacco leaves, reduce off-flavors, and increase the aroma of the tobacco leaves; Pseudomonas spp. can effectively degrade nicotine; Pasteurella multocida ( Microbacterium barkeri ), Stenotrophomonas maltophilia ( Stenotrophomonas maltophiliaIt has an extremely strong ability to reduce nicotine. Bacillus species can produce small aromatic substances by decomposing large molecules, such as carotene.

[0007] Adding microorganisms during tobacco fermentation can shorten fermentation time, increase aroma, and improve tobacco quality. Adding microorganisms during fermentation has a more significant effect on improving tobacco quality and increasing the industrial usability of tobacco leaves.

[0008] Aspergillus chevaleri ( Aspergillus chevalieri Aspergillus chevallis is a common xerophytic fungus capable of growing in environments with water activity as low as 0.70, and is widely distributed in natural environments. Its typical morphological characteristics include yellow cleistothecia, a single conidiophore, and yellow-orange hyphal masses. When environmental moisture exceeds safe limits, Aspergillus chevallis readily proliferates and induces pyrogenic foci. This allows it to preferentially utilize raw materials during solid-state composting and effectively maintain the compost pile temperature, helping other microorganisms in the compost overcome the initial period of environmental maladaptation and providing them with normal growth temperatures to enter the normal fermentation phase. Notably, in storage environments, Aspergillus chevallis exhibits significant tolerance to low oxygen concentrations, enabling it to enter the fermentation state normally even under oxygen-deficient conditions during solid-state composting.

[0009] The present invention aims to provide the application of a strain of Aspergillus chevaleris C1 in the fermentation of cigar tobacco leaves, with the purpose of using this strain to improve the fermentation efficiency and enhance the fermentation quality and flavor of cigar tobacco leaves. Summary of the Invention

[0010] The first objective of this invention is to provide *Aspergillus chevaleri* (… Aspergillus chevalieri The application of C1 in cigar tobacco fermentation, and a second objective of the present invention is to provide a microbial composition for cigar tobacco fermentation.

[0011] The first objective of this invention is achieved as follows: Aspergillus chevaleri ( Aspergillus chevalieri The application of C1 in cigar tobacco fermentation: The Aspergillus chevaleri C1 is deposited at the Guangdong Provincial Microbial Culture Collection Center, with the number GDMCC No: 66976, deposit date: September 17, 2025; deposit address: No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province.

[0012] The second object of the present invention is achieved by providing a microbial composition for the fermentation of cigar tobacco leaves, comprising Aspergillus chevaleri C1 and Trichoderma xenograft (…). Trichoderma xerophilum YMF1.09953 (deposited at Guangdong Provincial Microbial Culture Collection Center, GDMCC No.: 66977, deposit date: September 17, 2025) and *Syntrophus spp.* Brevundimonas huaxiensis1.547-1 (deposited at Guangdong Provincial Center for Microbial Culture Collection, GDMCC No: 65867, deposited on January 16, 2025).

[0013] The beneficial effects of this invention are as follows: 1. The microbial composition provided by this invention (Aspergillus chevaleri C1, Trichoderma xerophyte YMF1.09953, and Shortwave Monoclonalella spp. 1.547-1) can form a highly efficient synergistic microbial community in the cigar tobacco fermentation system. Specifically, the Aspergillus chevaleri C1 strain can rapidly raise the temperature of the tobacco pile during the natural pile fermentation process, helping other microorganisms in the pile overcome the period of environmental maladaptation and shortening the pre-fermentation period. Simultaneously, C1's tolerance to low oxygen concentrations and its arid growth habit allow it to be the first among the many microorganisms in the natural tobacco pile to utilize the tobacco substrate and enter fermentation, thus shortening the overall fermentation period. The Trichoderma xerophyte YMF1.09953 strain can preferentially and rapidly utilize the fibrous components in the tobacco leaves, improving the fiber toughness of the cigar tobacco leaves and thus enhancing the quality of the tobacco. The Shortwave Monoclonalella spp. 1.547-1 strain can utilize the tobacco leaves and intermediate components of tobacco leaf degradation to produce pleasant flavor and aroma components, enriching the smoking experience of cigar tobacco.

[0014] 2. The fermentation agent prepared based on the microbial composition of this invention exhibits remarkable effects in the fermentation of cigar tobacco leaves. After only 15 days of fermentation, the total content and richness of various key aroma components (such as neophytadiene and geraniol) in cigar tobacco leaves, as well as the degradation efficiency of cellulose and hemicellulose, significantly surpass those of the control group tobacco leaves that underwent natural fermentation for 45 days. This demonstrates that the microbial composition of this invention effectively enhances the aroma quality of cigar tobacco leaves. Attached Figure Description

[0015] Figure 1 The colony morphology of Aspergillus schwanniferus strain C1 of this invention; Figure 2 This is the phylogenetic tree of the *Aspergillus schwanniferus* C1 strain of this invention; Figure 3 The colony and microscopic morphology of the *Trichoderma xerophyte* strain YMF1.09953 of this invention are shown below (AB represents the colony morphology of *Trichoderma xerophyte* strain YMF1.09953; DI represents the spore attachment morphology of *Trichoderma xerophyte* strain YMF1.09953; J and K represent the molecular spore morphology of *Trichoderma xerophyte* strain YMF1.09953). Figure 4 This is the phylogenetic tree of the *Trichoderma xerophyte* strain YMF1.09953 of the present invention; Figure 5 This is a phylogenetic tree of the *Shortwave Monoclonalella* strain 1.547-1 from West China, as described in this invention. Figure 6This is a flowchart of the tobacco leaf sample preparation process for determining cellulose content in Example 3 of the present invention. Detailed Implementation

[0016] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments, but this does not limit the present invention in any way. Any modifications or improvements made based on the teachings of the present invention shall fall within the protection scope of the present invention.

[0017] This invention provides *Aspergillus schwanniferus* ( Aspergillus chevalieri Application of C1 in cigar tobacco fermentation. Aspergillus chevalieri C1 is deposited at Guangdong Provincial Microbial Culture Collection Center, with the number GDMCC No: 66976, located at No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province.

[0018] The present invention also provides a microbial composition for cigar tobacco fermentation, comprising Aspergillus chevaleris strain C1 as described in claim 1, Trichoderma xerophyte YMF1.09953, and Shortwave monoclonal bacteria 1.547-1.

[0019] The present invention further provides a method for fermenting cigar tobacco leaves, specifically by inoculating the microbial composition into the cigar tobacco leaves for fermentation.

[0020] The microbial composition is used in the form of a liquid fermentation agent, and the preparation method of the liquid fermentation agent includes the following steps: 1) The *Aspergillus schavar* strain C1 and the *Trichoderma xerophyte* strain YMF1.09953 were respectively inoculated into the first seed culture medium and cultured at 28-30℃ and 130-160 r / min for 36-48 hours to obtain seed liquid; the fungal seed liquid was then inoculated into the first fermentation medium for further fermentation culture to obtain *Aspergillus schavar* strain C1 and *Trichoderma xerophyte* strain YMF1.09953, respectively. 2) Inoculate the *Syntrophus huaxilensis* 1.547-1 into the second seed culture medium and incubate at 33-35℃ and 150-180 r / min for 24-36 hours until OD (Organic Depth). 600 The value was 0.5-0.9, and the seed culture was obtained; the seed culture was inoculated into the second fermentation medium and fermentation culture was continued to obtain the bacterial culture of *Shortwave Monoclonalella spp.* 1.547-1. 3) Mix the Aspergillus spp. and Trichoderma xerophyte solution obtained in step 1) with the Shortwave Monoclonal bacteria solution obtained in step 2) in an equal volume ratio to obtain the liquid fermentation agent.

[0021] The liquid fermentation agent is further mixed with an edible adsorbent at a mass ratio of 1:1, and after drying, a solid fermentation agent is obtained.

[0022] In step 1), the first seed culture medium or the first fermentation culture medium is PDA culture medium.

[0023] In step 2), the second seed culture medium and / or the second fermentation culture medium is LB medium.

[0024] Example 1: Preparation of Fermentation Agent 1. Inoculate Aspergillus schwanniferus C1 strain and Trichoderma xerophyte YMF1.09953 strain into PDA liquid medium and culture them in a shaker at 28℃ and 145r / min for 36-48 hours (when many mycelia can be seen in the liquid). Inoculate the cultured seed liquid into PDA medium at an inoculation rate of 1% (v / v) and continue to ferment and culture in a shaker at 28℃ and 145r / min for 68 hours to obtain the bacterial culture of Aspergillus schwanniferus C1 and Trichoderma xerophyte YMF1.09953. 2. Inoculate *Syntrophus schreberi* 1.547-1 into LB medium and incubate at 34℃ and 135 rpm on a shaker for 24-36 hours until OD (Organic Depth). 600 When the nm value reaches 0.5-0.9, the cultured seed liquid is inoculated into LB medium at an inoculation rate of 1% (v / v), and fermented on a shaker at 34℃ and 135r / min for 68 hours to obtain the bacterial culture of *Syntropha huaxiensis* 1.547-1. 3. Mix the bacterial solutions of the three strains in a 1:1:1 ratio to obtain a mixed bacterial solution, which is the liquid fermentation agent.

[0025] Example 2 The liquid fermentation inoculum prepared in Example 1 was mixed with food-grade diatomaceous earth adsorbent at a mass ratio of 1:1 and stirred thoroughly to ensure uniform adsorption. The mixture was dried at 38°C to constant weight, pulverized, and sieved to obtain the solid fermentation inoculum.

[0026] Example 3: Fermentation of cigar tobacco leaves using liquid fermentation inoculant The liquid fermentation agent prepared in Example 1 was evenly sprayed onto the surface of the tobacco leaves at a ratio of 5% (V / W) by weight of the cigar tobacco leaves, while adjusting the moisture content of the tobacco leaves to 25-30%. Ten to twenty treated tobacco leaves were stacked in a constant temperature chamber, and the temperature of the tobacco leaves was monitored in real time during fermentation: when the temperature exceeded 45°C, the cooling device was turned on; when the temperature fell below room temperature, the cooling device was turned off. The humidity in the constant temperature chamber was maintained at 70%, and fermentation continued for 15 days.

[0027] Reference settings: Blank control A: Cigar tobacco leaves from the same batch were sprayed with an equal amount of sterile water and fermented for 15 days under the same temperature and humidity conditions as described above.

[0028] Blank control B: Cigar tobacco leaves from the same batch, without any inoculation, fermented for 45 days under normal natural conditions (temperature and humidity fluctuations).

[0029] After fermentation, the components of tobacco leaf samples from the treatment group (Example 3) and two control groups (blank controls A and B) of this invention were analyzed.

[0030] 1. Determination of volatile oil components in tobacco leaves The volatile aroma components in cigar tobacco leaves were determined using gas chromatography-mass spectrometry. The tobacco leaf samples were prepared using a simultaneous distillation-extraction method.

[0031] The GC-MS analysis conditions are as follows: Chromatographic column: HP-5MS (30m*0.25mm*0.25μm); Injector temperature: 250℃; Temperature program: The column temperature was 80℃ and held for 1 min, then increased to 200℃ at 5℃ / min and held for 3 min; finally, it was increased to 280℃ at 8℃ / min and held for 10 min; carrier gas: helium; flow rate: 1.0 mL / min; injection method: split injection, split ratio 50:1, injection volume: 0.2 μL.

[0032] Ion source: EI source, ion source temperature: 230℃, quadrupole temperature: 150℃; ionization energy: 70eV, full ion scan detection mass range: 50-550 amu, solvent delay: 3 min.

[0033] 2. Cellulose determination The determination was performed using a combination of the filament washing method and spectrophotometry.

[0034] (1) Sample preparation After crushing the tobacco leaf sample, sieve it through a 100-mesh standard sieve, and take the powder that passes through the sieve. Then process it according to the following steps to obtain the tobacco leaf sample ( Figure 6 ): Take 1g of 100-mesh tobacco leaf sample, wash it with neutral detergent to obtain neutral detergent fiber (W1) (containing hemicellulose, cellulose, and lignin). Neutral detergent fiber W1 is treated with 2M hydrochloric acid, and filtrate 1 is collected. Hemicellulose is determined by the lichenol method; simultaneously, acid detergent fiber (W2) (containing cellulose and lignin) is obtained. Acid detergent fiber W2 is treated with 72% sulfuric acid, and filtrate 2 is collected. Cellulose is determined by the anthrone method; fiber residue (W3) is obtained.

[0035] (2) Determination of cellulose content First, dry the anhydrous glucose standard to constant weight at 105℃, accurately weigh 100mg, and dilute to 100mL to obtain the standard stock solution. Take six clean test tubes, label them, and dilute them as shown in Table 1 to prepare standard working solutions of different concentrations. Add 4mL of anthrone reagent to each test tube, mix thoroughly, heat in a boiling water bath for 10min, remove and cool to room temperature with running water, and measure the absorbance at 620nm. Plot a standard curve with glucose concentration on the x-axis and absorbance on the y-axis. Dilute sample filtrate 2 to an appropriate factor, measure the absorbance following the same procedure as above for plotting the standard curve, substitute the measured value into the standard curve to calculate the concentration, and multiply the final result by a coefficient of 0.9 to obtain the cellulose content.

[0036] Table 1. Gradient Dilution Table of Glucose Standard Solution Concentration

[0037] (3) Determination of hemicellulose content Accurately weigh 10 mg of D-xylose standard and dilute to 1000 mL to prepare a standard stock solution with a concentration of 10 μg / mL. Take 6 clean test tubes and dilute to different concentrations as shown in Table 2. Add 4 mL of lichenin reagent to each test tube, mix thoroughly, seal with a rubber stopper, incubate in a boiling water bath for 20 min, remove and cool to room temperature with running water, and measure the absorbance at a wavelength of 660 nm. Plot a standard curve with D-xylose concentration as the x-axis and absorbance as the y-axis. Dilute sample filtrate 1 to an appropriate factor, measure the absorbance according to the above standard curve plotting procedure, substitute the measured value into the standard curve to calculate the concentration, and multiply the final result by a coefficient of 0.9 to obtain the hemicellulose content.

[0038] Table 2. Dilution gradient of D-xylose standard solution

[0039] 3. Results: As shown in Table 3.

[0040] Table 3 Comparison of component contents in tobacco leaves under different fermentation treatments

[0041] Results Analysis: Table 3 shows that cigar tobacco leaves fermented with the fermentation agent of this invention for only 15 days exhibited significantly higher levels of key aroma components (10 volatile oils identified by GC-MS) and higher degradation efficiency of cellulose and hemicellulose compared to tobacco leaves naturally fermented for 45 days. This indicates that the bacterial culture of this invention can effectively improve the aroma quality of cigar tobacco leaves and accelerate the degradation of their fibrous components.

[0042] Example 4: Fermentation of Cigar Tobacco Leaves Using Solid Fermentation Agents The solid fermentation inoculum prepared in Example 2 was reconstituted with sterile water to achieve a concentration comparable to that of the liquid inoculum in Example 1. Subsequent tobacco leaf treatment, inoculation ratio (5% V / W), and fermentation conditions (70% humidity, temperature management, 15 days) were the same as in Example 3.

Claims

1. Aspergillus sydowi (ATCC 201573) Aspergillus chevalieri ) C1 in the fermentation of cigar tobacco, characterized in that, Aspergillus chevalieri C1 is preserved in Guangdong Microbial Culture Collection Center, with the number of GDMCC No: 66976, and the address of preservation is No. 100, Martyrs Road, Yuexiu District, Guangzhou, Guangdong Province.

2. A microbial composition for use in the fermentation of cigar tobacco leaf, characterized in that, comprising the Aspergillus chevalieri strain C1 of claim 1, Trichoderma Trichoderma xerophilum harzianum (T. harzianum) YMF1.09953 and Parvibaculum Brevundimonas huaxiensis distasonium (P. distasonium) 1.547-1.

3. A method of fermentation of cigar tobacco leaf, characterized in that, The microbial composition of claim 2 is inoculated into cigar tobacco leaves for fermentation.

4. The fermentation process of claim 3, wherein, The microbial composition is used in the form of a liquid fermentation agent, and a preparation method of the liquid fermentation agent comprises the following steps: 1) Aspergillus chevalieri C1 and Trichoderma xerophilum YMF1.09953 of claim 1 are respectively inoculated into a first seed culture medium, and cultured at 28-30°C and 130-160 r / min for 36-48 hours to obtain a seed liquid; the fungal seed liquid is respectively inoculated into a first fermentation culture medium for further fermentation culture, and Aspergillus chevalieri C1 liquid and Trichoderma xerophilum YMF1.09953 liquid are respectively obtained; 2) inoculate the B. westlandica 1.547-1 into the second seed culture medium, and cultivate at 33-35°C and 150-180 r / min for 24-36 hours until the OD 600 value is 0.5-0.9, and obtain a seed liquid; inoculate the seed liquid into the second fermentation culture medium, and continue the fermentation cultivation to obtain the B. westlandica 1.547-1 bacterial liquid; 3) Aspergillus chevalieri liquid and Trichoderma xerophilum liquid obtained in step 1) are mixed with Brevundimonas huaxiensis liquid obtained in step 2) according to an equal volume ratio to obtain the liquid fermentation agent.

5. The fermentation process of claim 4, wherein, The liquid fermentation agent is further mixed with an edible adsorbent according to a mass ratio of 1:1, and after drying treatment, a solid fermentation agent is prepared.

6. The fermentation process of claim 4, wherein, In step 1), the first seed culture medium and the first fermentation culture medium are PDA culture media.

7. The fermentation process of claim 4, wherein, In step 2), the second seed culture medium and the second fermentation culture medium are LB culture media.

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