Bacillus licheniformis SXZY-02 and application thereof

Abstract

The application belongs to the technical field of biological fermentation, and relates to a bacillus licheniformis SXZY-02 and application thereof, and comprises the following steps: a bacillus licheniformis (Bacillus licheniformis) SXZY-02 is separated from the surface of tobacco leaves, Lysinibacillus macroides ) and the strain preservation number is CCTCC NO:M 2025152; the bacillus licheniformis SXZY-02 is fermented to obtain a biological agent containing the bacillus licheniformis SXZY-02, and after fermentation optimization, the lipase activity is 8.99 U / mL; the biological agent containing the bacillus licheniformis SXZY-02 is applied to tobacco shred fermentation, fat is decomposed into free fatty acid, tobacco aroma quality and alcohol degree are effectively improved, and irritation is reduced. The technology provides a green and environment-friendly solution for tobacco quality upgrading, and meets the high-quality development direction of the industry.

Description

Technical Field

[0001] This invention belongs to the field of bio-fermentation technology and relates to a strain of long-shaped lysine-containing Bacillus SXZY-02 and its applications. Background Technology

[0002] Fatty substances in tobacco leaves serve as important flavor precursors, and their degradation products (such as aldehydes, ketones, and esters) directly contribute to the nutty, sweet, and mellow flavor of tobacco. However, excessively high fat content or incomplete conversion in tobacco leaves can lead to the accumulation of long-chain fatty acids, resulting in increased irritation of the smoke, unpleasant odors such as grassy or rancid tastes, and reduced storage stability of the product.

[0003] Currently, the regulation of tobacco leaf fat in tobacco processing mainly relies on traditional physical or chemical methods such as high-temperature treatment or organic solvent extraction. However, these methods generally suffer from drawbacks such as low regulation efficiency, easy destruction of natural flavor components in tobacco leaves, and poor environmental friendliness. Although microbial degradation technology is widely used in environmental protection, food and other industrial fields, its research on tobacco fat regulation is still in its early stages. Bottlenecks such as the scarcity of specific degrading strains, unclear metabolic pathways, and lack of systematic optimization of fermentation process parameters lead to unsatisfactory application results and make it difficult to achieve targeted degradation of tobacco fat and precise improvement of flavor quality.

[0004] Therefore, there is an urgent need to develop directional fermentation technology based on lipase-degrading bacteria and screen for highly efficient lipase-producing microbial strains to precisely degrade long-chain fatty acids in tobacco leaves and generate low-molecular-weight flavor substances, thereby effectively improving the sweetness and mellowness of tobacco while reducing irritating off-flavors. Summary of the Invention

[0005] The technical solution of this invention is: a strain of long-shaped lysine-containing Bacillus (… Lysinibacillus macroides SXZY-02, with accession number CCTCC NO: M 2025152, was deposited at the China Center for Type Culture Collection on January 15, 2025.

[0006] The present invention also provides the application of the above-mentioned long-shaped lysine-containing Bacillus or its fermentation products in tobacco processing.

[0007] Preferably, the fermentation product includes lipase.

[0008] This invention also provides a biological agent prepared from *Bacillus longiformis* or its fermentation products, which is used to: degrade fatty substances in tobacco; increase the content of free fatty acids in tobacco; improve the sensory quality of tobacco, characterized by at least one of aroma quality, aroma intensity, woody off-flavors, irritation, aftertaste, strength, concentration, and smoothness; enhance aroma harmony and reduce the harshness of smoke; and secrete or prepare lipase. This biological agent is an enzyme preparation for tobacco processing.

[0009] Preferably, the method for preparing the biological agent includes the following steps: Step 1, Activation of bacterial strain: The long-shaped lysine spore bacillus SXZY-02 was inoculated into NA solid medium and cultured at 25-35℃ for 24-72h; Step 2, Preparation of seed culture: Take the bacterial cells cultured in Step 1, inoculate them into NA liquid culture medium, and culture them in a shaker at 30-35℃ and 180-200 r / min for 10-14 h to obtain the seed culture; Step 3, scale-up culture: Inoculate the seed culture obtained in step 2 into NA liquid medium at 2% to 4% of the culture medium weight, and culture at 30℃ to 35℃ and shaking speed of 180 to 200 r / min for 24 to 48 hours to obtain fermentation broth; Step 4: Preparation of crude enzyme solution: Centrifuge the fermentation broth obtained in Step 3 at 3-5℃ and 8000-10000 rpm for 9-11 minutes, and collect the supernatant, which is the biological agent. The fermentation product is the supernatant obtained after centrifugation of its fermentation broth.

[0010] More preferably, in step 1, the composition of the NA solid culture medium includes: 2-4 g / L beef extract, 8-12 g / L peptone, 4-6 g / L sodium chloride, and 18-22 g / L agar powder; in steps 2 and 3, the composition of the NA liquid culture medium includes: 2-4 g / L beef extract, 8-12 g / L peptone, and 4-6 g / L sodium chloride.

[0011] More preferably, in steps 2 and 3, the culture temperature is 30°C, the culture time is 24 h, and the pH is 6.

[0012] Preferably, the biological agent is used to treat tobacco shreds, comprising the following steps: Step A: Spray the biological agent onto the tobacco shreds at 2% to 6% of the weight of the tobacco leaves to be treated, adjust the moisture content of the tobacco shreds to 18% to 22%, seal, and ferment at 40 to 50°C and 70% to 80% humidity for 20 to 30 hours. Step B: Dry the tobacco shreds at 80℃ for 10-15 minutes to balance the moisture content.

[0013] The beneficial effects of this invention are: 1. This invention possesses highly efficient fat degradation capabilities: The *Bacillus longiformis* strain SXZY-02 of this invention exhibits excellent lipase production capabilities, with an enzyme activity reaching 8.99 U / mL under optimized culture conditions, significantly higher than the 4.85 U / mL under conventional culture conditions. This strain can effectively promote the targeted degradation of fatty substances in tobacco leaves. Experimental studies show that the free fatty acid content in tobacco treated with strain SXZY-02 reaches 4256 nmol / g, an increase of 52.60% compared to the original tobacco (CK1) and 49.44% compared to the sterile water control group (CK2), thus avoiding the problem of long-chain fatty acid accumulation caused by incomplete fat degradation.

[0014] 2. This invention significantly improves the sensory quality of tobacco: By precisely controlling the fat degradation process, this invention directs the conversion of long-chain fatty acids in tobacco leaves into low-molecular-weight flavor compounds (such as ethyl acetate and nonanal), thereby significantly enhancing the sensory quality of tobacco. Sensory evaluation results show that tobacco treated with the strain of this invention exhibits significant improvements in key indicators such as aroma, irritation, aftertaste, and smoothness, achieving a total score of 45.5 points, which is 5.6%-5.9% higher than the control group (42.5-43.0 points). Specifically, this manifests as enhanced aroma, improved permeability, reduced burnt smell, increased roundness of smoke, prominent sweetness, and significant improvement in bitterness and lingering sensation in the aftertaste, effectively overcoming the shortcomings of existing physicochemical methods that easily destroy natural flavor components.

[0015] 3. This invention enables precise optimization of tobacco processing parameters: Through systematic multi-factor experimental research, this invention determined the optimal culture conditions for strain SXZY-02 (temperature 30℃, time 24 h, pH 6.0), maximizing lipase yield. This optimized process not only improves enzyme activity but also ensures process repeatability and industrial application feasibility, overcoming the technical bottleneck of lacking systematic optimization of process parameters in existing microbial technologies for tobacco applications.

[0016] 4. This invention uses a microbial enzymatic hydrolysis method to achieve targeted degradation of fats under mild conditions, avoiding the environmental shortcomings and flavor destruction problems of traditional physical or chemical methods. While efficiently improving the quality of tobacco, it fully preserves the natural flavor components of tobacco, which is in line with the tobacco industry's high-quality development direction of "reducing harm and tar, improving quality and enhancing aroma".

[0017] 5. This invention achieves precise regulation of fatty substances in tobacco by screening the highly efficient lipase-producing strain SXZY-02 and optimizing its cultivation process. It effectively solves the problems of incomplete fat degradation, destruction of flavor components, and unclear process parameters in the existing technology, and provides an innovative technical path for the green improvement of tobacco quality. Attached Figure Description

[0018] Figure 1 This is a streak plate diagram of a long-shaped lysine-containing Bacillus SXZY-02 strain of the present invention and its application. Figure 2 This is a phylogenetic tree diagram constructed based on the 16S rDNA gene sequence of the *Bacillus longissima* strain SXZY-02 in an embodiment of the present invention. Figure 3 This is a standard curve diagram based on lipase activity at 410 nm in an embodiment of the present invention. Detailed Implementation

[0019] The related technologies of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0020] like Figures 1-3 As shown, this invention addresses the technical problems of incomplete fat degradation leading to poor sensory quality in existing tobacco processing, and the low efficiency and flavor degradation of traditional physicochemical methods. It employs a strain of long, lysine-containing Bacillus with high lipase activity isolated from the surface of tobacco. Lysinibacillus macroides SXZY-02 was used, and its culture conditions were optimized to further enhance its enzyme activity. When applied to tobacco fermentation, it achieved remarkable technical results in efficiently degrading fatty substances, significantly improving the sensory quality of tobacco, and reducing irritating odors.

[0021] According to one aspect of this disclosure, a strain of long-shaped lysine-containing Bacillus was isolated through screening. Lysinibacillus macroides SXZY-02 was deposited on January 15, 2025 at the China Center for Type Culture Collection (Wuhan University, Wuhan, China, 430072, China) with accession number CCTCC NO: M 2025152.

[0022] According to one aspect of this disclosure, a biological agent is provided containing the *Bacillus longiformis* SXZY-02 and / or its fermentation products, said fermentation products including lipase.

[0023] According to one aspect of this disclosure, the use of the *Bacillus longiformis* SXZY-02 or the biological agent in at least one of the following or in the preparation of a product having at least one of the following functions: (1) Degrading fatty substances in tobacco; (2) Increase the content of free fatty acids in tobacco; (3) Improve the sensory quality of tobacco, wherein the sensory quality is characterized by at least one of aroma quality, aroma quantity, woody off-flavor, irritation, aftertaste, strength, concentration, and softness; (4) Enhance aroma harmony and reduce the harshness of smoke; (5) Secretion or preparation of lipase.

[0024] According to another aspect of this disclosure, a method for preparing the biological agent is provided, characterized by comprising the following steps: (1) Activation of bacterial strain: The long-shaped lysine spore bacillus SXZY-02 was inoculated into NA solid medium and cultured at 25-35℃ for 24-72h; (2) Preparation of seed culture: Pick the bacterial cells cultured in step (1), inoculate them into NA liquid culture medium, and culture them in a shaker at 30-35℃ and 180-200 r / min for 10-14 h to obtain seed culture; (3) Expanded culture: The seed culture obtained in step (2) is inoculated into NA liquid culture medium at 2% to 4% of the weight of the culture medium, and cultured at 30℃ to 35℃ and shaking speed of 180 to 200 r / min for 24 to 48 hours to obtain fermentation broth; (4) Preparation of crude enzyme solution: Centrifuge the obtained fermentation broth at 3-5℃ and 8000-10000rpm for 9-11min, and take the supernatant, which is the biological agent.

[0025] In some of the disclosed embodiments, the NA solid culture medium composition is as follows: beef extract 3.0 g / L, peptone 10.0 g / L, sodium chloride 5.0 g / L, and agar powder 20.0 g / L.

[0026] In some of the disclosed embodiments, the NA liquid culture medium is composed of: 3.0 g / L beef extract, 10.0 g / L peptone, and 5.0 g / L sodium chloride.

[0027] According to another aspect of this disclosure, a method for processing tobacco shreds is provided, characterized by comprising the following steps: (1) Spray the biological agent onto the tobacco shreds at 2% to 6% of the mass of the tobacco leaves to be treated, and adjust the moisture content of the tobacco shreds to 18% to 22% and then seal it. Ferment at 40 to 50°C and 70% to 80% humidity for 20 to 30 hours. (2) Dry the tobacco at 80°C for 10-15 minutes to balance the moisture content.

[0028] Example Example 1: Screening and identification of *Lysinus SXZY-02* In 2020, a long-shaped lysine-containing bacillus was isolated from the surface of tobacco leaves in Shangluo, Shaanxi Province, and named SXZY-02.

[0029] 2g of Shaanxi Shangluo tobacco shreds were placed in 30 mL of sterile water and cultured on a shaker at 30℃ and 180 r / min for 36 h. Oil-degrading bacteria were screened using sunflower oil as the sole nutrient source; strains capable of growing in this medium possessed the ability to degrade oils. Finally, strain SXZY-02 was purified by streaking on NA medium. Figure 1 ).

[0030] The strain sequence was amplified using universal 16S rDNA primers, and the PCR products were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The obtained sequences were compared with the NCBI database, showing 99.05% homology with *Bacillus longiformis*, indicating a close phylogenetic relationship between the two (e.g., *Bacillus longiformis*). Figure 2 (As shown).

[0031] Therefore, SXZY-02 was identified as a long-shaped lysine spore bacterium ( Lysinibacillus macroides It is currently deposited at the China Center for Type Culture Collection, accession number CCTCC NO:M 2025152.

[0032] Example 2: Preparation of Biological Agents (1) Activation of bacterial strain: Long-shaped lysine spores SXZY-02 were inoculated on NA solid medium (beef extract 3.0 g / L, peptone 10.0 g / L, sodium chloride 5.0 g / L, agar powder 20.0 g / L) and cultured at 25-35℃ for 24-72 h; (2) Seed culture preparation: Pick the bacterial cells cultured in step (1) and inoculate them into NA liquid medium (beef extract 3g / L, peptone 10g / L, sodium chloride 5g / L). Culture at 30-35℃ and shaking at 180-200r / min for 10-14h to obtain seed culture. (3) Expanded culture: The seed culture of long-shaped lysine spores SXZY-02 obtained in step (2) was inoculated into NA liquid medium at 2% to 4% of the weight of the medium. The culture was carried out at 30℃ to 35℃ and at a shaking speed of 180 to 200 r / min for 24 to 48 hours to obtain the fermentation broth. (4) Preparation of crude enzyme solution: Centrifuge the obtained fermentation broth at 3-5℃ and 8000-10000rpm for 9-11min, and take the supernatant, which is the biological agent.

[0033] Example 3: Optimization of culture conditions for *Bacillus longiformis* strain SXZY-02 The culture medium was NA liquid medium, the shaking speed was 180 r / min, and the inoculum size was 3%. A three-factor, four-level combination experiment was set up, as shown in Table 1 (Experimental Design and Enzyme Activity Results), specifically: culture temperature (25℃, 30℃, 35℃, 40℃), culture time (24h, 36h, 48h, 60h), and pH (4, 6, 8, 10).

[0034]

[0035] Under the above culture conditions, strain SXZY-02 was cultured, and an enzyme activity standard curve was established using p-NP. Figure 3 The activity of the strain's lipase was measured, and the amount of enzyme required to catalyze the production of 1 μmol of p-NP per minute at 40℃ was defined as one activity unit (U). The results are shown in Table 1. When the culture temperature was 30℃, the culture time was 24h, and the pH was 6, the strain produced the highest lipase activity, which was 8.99 U / mL.

[0036] Example 4: Results of Fat Degradation Effect and Sensory Evaluation During Tobacco Fermentation The biological agent from Example 3 was sprayed onto the tobacco shreds at 4% of the weight of the tobacco leaves to be treated. After adjusting the moisture content of the tobacco shreds to 18%–22%, the mixture was sealed and fermented at 40°C and 70% humidity for 24 hours. The tobacco shreds were then dried at 80°C for 10 minutes to balance the moisture content, and the product was obtained.

[0037] The original tobacco shreds are denoted as CK1; the tobacco shreds sprayed with an equal amount of sterile water during the tobacco shred processing are denoted as CK2; and the tobacco shreds sprayed with an equal amount of biological agent cultured under optimal culture conditions (30℃ / 24 h / pH=6) are denoted as T.

[0038] The dried tobacco shreds were ground into powder, passed through a 40-mesh sieve, and the free fatty acid content in the tobacco leaves was determined using a free fatty acid content detection kit (manufactured by Shanghai Enzyme-Linked Biotechnology Co., Ltd., model number ml076656). The sensory quality of the tobacco shreds was evaluated in accordance with the YC / T 138-1998 Tobacco Sensory Evaluation Method.

[0039] The results are as follows: As shown in Table 2 (Free fatty acid content in different tobacco shreds), the free fatty acid content in treatment T is significantly higher than that in CK1 and CK2. The degradation rate of fatty substances in tobacco shreds reached 52.60% compared with CK1 and 49.44% compared with CK2. This indicates that the addition of Bacillus longiformis SXZY-02 bacterial agent with optimized culture conditions can effectively increase the degradation of fatty substances in tobacco shreds.

[0040]

[0041] Table 3 (Sensory Quality Evaluation Table for Treated Tobacco) shows that the sensory qualities of groups CK1 and CK2 are almost identical, indicating that spraying an equal amount of sterile water for fermentation has no significant impact on tobacco quality. However, the T-treatment group, sprayed with the microbial agent, exhibits significantly improved sensory quality compared to CK1 and CK2 after fermentation. Specifically, the aroma is significantly improved, the irritation is reduced, the smoke smoothness increases, and the bitterness and lingering sensation in the aftertaste are significantly reduced, with a slight increase in fineness. This indicates that treatment with *Bacillus longiformis* strain SXZY-02 can promote the directional conversion of fatty substances in tobacco leaves into free fatty acids. Through the positive regulatory effect of free fatty acids on tobacco quality, the overall sensory quality of tobacco leaves is significantly improved, thereby effectively enhancing the industrial usability and utilization value of low-grade tobacco leaves.

[0042]

[0043] In summary, this invention, through screening and optimizing the culture process of *Bacillus longiformis* SXZY-02, has achieved significant results in the degradation of fatty substances in tobacco, improvement of sensory quality, and optimization of processing technology. The microbial enzymatic hydrolysis pathway employed in this invention is not only highly efficient and environmentally friendly but also preserves the natural flavor components of tobacco completely, providing a new technical approach and solution for the high-quality development of the tobacco industry. In the future, further in-depth research can be conducted on the application effects of this strain under different tobacco varieties and processing environments to explore its broader application scenarios and potential. Simultaneously, research on the mechanism of action of the strain can be strengthened to provide a more solid theoretical foundation for the continuous improvement of tobacco quality. Furthermore, combining this technology with other tobacco processing technologies can be considered to achieve complementary advantages and further promote the tobacco industry towards the goal of "reducing harm and tar, improving quality and enhancing aroma."

[0044] It should be emphasized that the above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any way. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention shall still fall within the scope of the technical solution of the present invention.

Claims

1. A strain of long-shaped lysine-containing Bacillus ( Lysinibacillus macroides SXZY-02, with accession number CCTCC NO: M 2025152, was deposited at the China Center for Type Culture Collection on January 15, 2025.

2. The application of the *Lysinus lysine-producing* bacillus or its fermentation product as described in claim 1 in tobacco processing.

3. The application of the long-shaped lysine-producing Bacillus or its fermentation product according to claim 2 in tobacco processing, characterized in that, The fermentation products include lipase.

4. A biological agent prepared from *Bacillus longiformis* or its fermentation product as described in claim 1, wherein the biological agent is used for: Degrades fatty substances in tobacco; Increase the content of free fatty acids in tobacco; Improving the sensory quality of tobacco, wherein the sensory quality is characterized by at least one of aroma quality, aroma quantity, woody off-flavors, irritation, aftertaste, strength, concentration, and smoothness. Enhance aroma harmony and reduce the harshness of smoke; Secretion or preparation of lipase.

5. The biological agent prepared from *Bacillus longiformis* or its fermentation product according to claim 4, characterized in that, The preparation method of the biological agent includes the following steps: Step 1, Activation of bacterial strain: The long-shaped lysine spore bacillus SXZY-02 was inoculated into NA solid medium and cultured at 25-35℃ for 24-72h; Step 2, Preparation of seed culture: Take the bacterial cells cultured in Step 1, inoculate them into NA liquid culture medium, and culture them in a shaker at 30-35℃ and 180-200 r / min for 10-14 h to obtain the seed culture; Step 3, scale-up culture: Inoculate the seed culture obtained in step 2 into NA liquid medium at 2% to 4% of the culture medium weight, and culture at 30℃ to 35℃ and shaking speed of 180 to 200 r / min for 24 to 48 hours to obtain fermentation broth; Step 4: Prepare crude enzyme solution: Centrifuge the fermentation broth obtained in step 3 at 3-5℃ and 8000-10000rpm for 9-11 minutes, and take the supernatant, which is the biological agent.

6. The biological agent prepared from *Bacillus longiformis* or its fermentation product according to claim 5, characterized in that, In step 1, the composition of the NA solid culture medium includes, in g / L: 2-4 g of beef extract, 8-12 g of peptone, 4-6 g of sodium chloride, and 18-22 g of agar powder. In steps 2 and 3, the NA liquid culture medium consists of: 2-4 g / L beef extract, 8-12 g / L peptone, and 4-6 g / L sodium chloride.

7. The biological agent prepared from *Bacillus longiformis* or its fermentation product according to claim 5, characterized in that, In steps 2 and 3, the culture temperature is 30℃, the culture time is 24 h, and the pH is 6.

8. The biological agent prepared from *Bacillus longiformis* or its fermentation product according to claim 4, characterized in that, When the biological agent is used to process tobacco, the following steps are included: Step A: Spray the biological agent onto the tobacco shreds at 2% to 6% of the weight of the tobacco leaves to be treated, adjust the moisture content of the tobacco shreds to 18% to 22%, seal, and ferment at 40 to 50°C and 70% to 80% humidity for 20 to 30 hours. Step B: Dry the tobacco shreds at 80℃ for 10-15 minutes to balance the moisture content.

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