Bacillus megaterium MZ-01 as well as microbial preparation and application thereof

Abstract

The invention relates to bacillus megaterium MZ-01 as well as a microbial preparation and application thereof. The strain (the preservation number is CCTCC NO: M 2025985) can efficiently degrade lignin, total sugar and nicotine in tobacco stems and remarkably improve sensory indexes such as aroma quality and aroma amount, and the laccase activity is greatly improved when the strain and sodium lignin sulfonate have a synergistic effect. The microbial preparation is obtained through the processes of strain activation, fermentation culture and enzyme preparation preparation, and when the microbial preparation is applied to tobacco stem treatment, the technical breakthrough that the lignin content is reduced, the filling value is increased, and the tar release amount is greatly reduced is achieved through the steps of water content adjustment, preparation spraying, sealed fermentation, low-temperature drying and the like. Compared with a traditional method, the technology has the advantages that the treatment period is shortened, the cost is remarkably reduced, the COD value of wastewater is reduced, the cigarette quality is remarkably improved (the additional value is improved by 25%), the production environmental protection property is remarkably improved, and the industrial application prospect is wide.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a Bacillus megaterium MZ-01, its microbial preparations, and their applications. Background Technology

[0002] In the tobacco industry, tobacco stems, a byproduct of tobacco leaf processing, are the tough, vein-like parts of tobacco leaves, accounting for approximately 25% to 30% of the total weight of tobacco leaves. However, current methods for processing tobacco stems are extremely simplistic and inefficient in resource utilization. Only a very small amount of tobacco stems enters the cigarette production process; the majority are either directly buried underground or centrally incinerated. This extensive processing method not only results in a significant waste of this valuable resource but also has the potential to have a substantial negative impact on the ecological environment due to landfilling and atmospheric pollution from incineration.

[0003] To improve the resource utilization of tobacco stems, they need to be processed. However, tobacco stems are hard and brittle due to their high lignin content, making them prone to breakage and brittleness during subsequent processing. This reduces the integrity and uniformity of the stem fibers, affecting their filling effect and combustion performance in cigarettes. Furthermore, the high lignin content alters the distribution and release patterns of aroma components in the tobacco stems, resulting in a less mellow aroma, a less refined taste, and even off-flavors, thus reducing the smoking experience. However, current lignin degradation technologies have significant limitations in practical applications. Some traditional methods, such as chemical oxidation, require large amounts of strong oxidants, leading to high costs and easily destroying beneficial components of the tobacco stems, thus damaging their flavor and quality. At the same time, this method requires stringent reaction conditions, demanding strict control over parameters such as temperature, time, and oxidant concentration, making operation complex and generating difficult-to-treat waste liquid and residue, causing environmental pollution.

[0004] Given the shortcomings of existing technologies, developing a novel lignin degradation technology is of significant practical importance. By precisely controlling the lignin degradation process and stabilizing the lignin content in tobacco stems within a suitable range, the physical and chemical quality of the stems can be effectively improved, enhancing their filling and combustion performance in cigarettes. This results in a richer aroma and a smoother taste, significantly improving the overall quality of cigarettes. Simultaneously, this new lignin degradation technology helps optimize cigarette production processes, reduce production costs, and minimize environmental impact, providing strong technical support for promoting quality upgrading and sustainable development across the entire cigarette industry chain.

[0005] The information disclosed in this background section is intended only to enhance the understanding of the background technology of this disclosure and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0006] The technical problem to be solved by this invention is to provide a tobacco-derived Bacillus megaterium MZ-01, its microbial preparations and their applications. This strain and its metabolites can reduce the content of lignin, total sugar or nicotine in tobacco stems and improve the quality of tobacco stems. Its application in the tobacco industry has broad and practical significance for improving cigarette quality and increasing the economic benefits of enterprises.

[0007] According to one aspect of this disclosure, a species of Bacillus megaterium ( ) was isolated and screened. Bacillus megaterium MZ-01 was deposited on May 7, 2025, at the China Center for Type Culture Collection (address: Wuhan University, Wuhan, China, postcode: 430072), with accession number CCTCC NO: M 2025985.

[0008] According to another aspect of this disclosure, a microbial preparation is provided comprising the Bacillus megaterium MZ-01 and / or its metabolic enzyme products, or comprising the metabolic enzyme products of the Bacillus megaterium MZ-01 induced by sodium lignosulfonate.

[0009] According to the third aspect of this disclosure, Bacillus megaterium MZ-01 or its biological agents are used in at least one of the following (1) to (6): (1) Reduce the lignin content in tobacco stems or prepare a reagent to reduce the lignin content in tobacco stems; (2) Reduce the total sugar and / or nicotine content of tobacco stems or prepare reagents to reduce the total sugar and / or nicotine content of tobacco stems; (3) Improve the sensory quality of tobacco stems or prepare a reagent to improve the sensory quality of tobacco stems, wherein the sensory quality includes at least one of aroma quality, aroma quantity, woody off-flavor, irritation, aftertaste, strength, concentration, and smoothness; (4) Use in combination with sodium lignosulfonate or prepare a reagent for use in combination with sodium lignosulfonate to improve laccase activity; (5) Improve cigarette quality or prepare reagents to improve cigarette quality; (6) Preparation of laccase.

[0010] According to a fourth aspect of this disclosure, a method for preparing a microbial preparation is provided, comprising the following steps: (1) Activation of the strain: The Bacillus megaterium MZ-01 was inoculated onto NA solid medium and cultured at 28-32 °C for 48-72 h; (2) Preparation of seed culture: scrape the bacterial cells obtained from the previous step of culture, inoculate them into NA liquid culture medium, and culture them in a shaker at 33~37 ℃ and 140~180 r / min for 10~15 h to obtain seed culture; (3) Scale-up culture: Inoculate the seed liquid into the fermentation medium at an inoculation rate of 2% and culture it in a shaker at a temperature of 33~37℃ and a speed of 140~180 r / min for 46~50 h; (4) Preparation of microbial agent: Centrifuge the fermentation broth from the previous step and take its supernatant.

[0011] In some embodiments of this disclosure, the fermentation medium contains: sodium lignosulfonate 3-20 g / L, peptone 8-12 g / L, beef extract 3-7 g / L, and NaCl 3-7 g / L.

[0012] In some embodiments of this disclosure, the method for processing tobacco stems includes the following steps: (1) Adjust the moisture content of the tobacco stems to be treated to 25-30%; (2) Spray the microbial preparation onto the tobacco stems at 20-30% of the weight of the tobacco stems to be treated, then seal them and ferment them at 43-47℃ and 70-80% humidity for 10-15 hours. (3) After fermentation, the product is dried and the moisture content is balanced.

[0013] The present invention provides Bacillus megaterium ( Bacillus megaterium MZ-01 and its metabolites have demonstrated significant technical advantages and economic value in tobacco industry applications, specifically in the following aspects: 1. Technological innovation and cost reduction / efficiency improvement ① Process optimization: Using sodium lignosulfonate as a directional inducer to activate strain MZ-01 to produce laccase, an integrated "induction-degradation" process was constructed, which shortens the treatment cycle by more than 40% and reduces the number of process steps by at least 30% compared with traditional chemical treatment methods, significantly improving production efficiency; ② Cost advantage: The raw material sodium lignosulfonate can be produced from industrial by-products such as papermaking waste liquid, which reduces the cost by more than 60% compared with traditional enzyme preparations, and does not require energy-consuming equipment such as high temperature and high pressure, resulting in a comprehensive reduction in treatment cost of more than 50%; ③ Field expansion: Laccase technology is applied to the directional degradation of tobacco stem lignin for the first time, opening up a new path for the bio-enzymatic treatment of tobacco raw materials and providing a technical demonstration for the high-value utilization of industrial waste.

[0014] 2. Quality leap and performance enhancement ① Optimized composition: After treatment with strain MZ-01, the lignin content of tobacco stems was significantly reduced, the total sugar content decreased significantly, the uniformity of nicotine distribution was improved, and the coordination of chemical components was significantly improved; ② Improved physical properties: The filling value of tobacco stems prepared after treatment increased by more than 20%, the tensile strength of reconstituted tobacco leaves was greatly improved, the cigarette combustion cone shedding rate was also greatly reduced, and the tar release during smoking was significantly reduced; ③ Breakthrough in sensory quality: Professional smoking evaluation results showed that the cigarette had less off-flavors, less irritation, and the aftertaste comfort was improved by 2 levels, increasing the product added value by more than 20%.

[0015] 3. Green Manufacturing and Industrial Empowerment ① Environmental benefits: No organic solvents are used throughout the entire process, the COD value of wastewater is significantly reduced compared to traditional processes, and the amount of solid waste generated is greatly reduced, meeting the requirements for carbon-neutral production; ② Application scalability: The established lignin biodegradation system can be extended to the pretreatment of biomass materials such as straw and bamboo, providing a general technology platform for the biomass refining industry; ③ Market adaptability: The technology modules can be seamlessly integrated with existing cigarette production lines, and the return on investment for equipment upgrades is less than 12 months. Calculations show that it can increase the overall gross profit margin of cigarette companies by 8-12 percentage points.

[0016] This invention, through cross-innovation of microbial enzyme engineering and tobacco processing technology, constructs a three-in-one technical solution integrating "cost-quality-environmental protection," providing key technical support for the transformation and upgrading of the tobacco industry and the high-value utilization of biomass resources, and has significant economic benefits and strategic value. Attached Figure Description

[0017] Figure 1 Phylogenetic tree of strain MZ-01 based on 16S rDNA gene sequence in one embodiment of the application.

[0018] Figure 2 This is a scanning electron microscope image of strain MZ-01 from one of the embodiments of the application.

[0019] Figure 3 The image shows the lignin content determination results of tobacco stems treated with a fungicide in one embodiment of the application.

[0020] Figure 4 The image shows the results of determining the total sugar content of tobacco stems treated with a microbial agent in one embodiment of the application.

[0021] Figure 5 The image shows the nicotine content determination results of tobacco stems treated with a microbial agent in one embodiment of the application.

[0022] Figure 6 This image shows the results of laccase activity assay in tobacco stems treated with a microbial agent in one embodiment of the application. Detailed Implementation

[0023] To better understand the technical solution of this application, the above technical solution will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0024] Unless otherwise specified, the instruments and equipment involved in the following embodiments are all conventional instruments and equipment; unless otherwise specified, the reagents and culture media involved are all commercially available conventional reagents and culture media; unless otherwise specified, the experimental methods involved are all conventional methods.

[0025] Example 1: Screening and identification of Bacillus megaterium strain MZ-01 (1) Strain screening: Weigh 1 g of tobacco leaves (from Yunnan province) and add them to a test tube containing 9 mL of sterile water. Shake at 250 r / min for 15 min, then let stand for 5 min. Filter using two layers of sterile lens paper. Take 1 mL of this bacterial suspension and add it to 9 mL of sterile water to prepare 10... -3 10 -4 and 10 -5 Diluted bacterial suspension; and aspirate 10 -3 10 -4 and 10 -5 100 μL of diluted bacterial suspension was spread onto NA agar plates and incubated overnight at 37°C. Alternatively, the bacterial suspension was spread onto NA plates using a triangular scraper and incubated upside down at 37°C for 48–72 h. Single colonies with different morphological characteristics were picked and inoculated onto NA slant agar. Bacteria isolated were picked with a bamboo stick and cross-shaped on BM agar containing aniline blue (Azure B). The samples were incubated at 30°C in the dark for at least 48 h to observe the results. Finally, 10 g of the collected sample was weighed and added to an Erlenmeyer flask containing 100 mL of sterile physiological saline. The flask was placed on a shaker and shaken at 180 rpm for 30 min. The suspension obtained by shaking was serially diluted, and the diluted solutions were spread onto functional plates PDA-AzureB and PDA-GU. The plates were incubated at 37°C for 3-4 days, with daily observations and records. The effect of colonies on the functional plates was observed: strains that produced degradation zones on PDA-AzureB plates may have the ability to produce LiP and MiP, and strains that produced chromogenic zones on PDA-GU plates may have the ability to produce Lac. Stranded cultures producing degradation clear zones or brown chromogenic zones were selected and continued until single colonies were obtained. The isolated and purified strains were inoculated into slant agar plates of the corresponding bacteria, fungi, and actinomycetes. After colony growth, the cultures were refrigerated at 4°C for later use. The bacteria obtained from the initial screening were inoculated into Erlenmeyer flasks containing 50 mL of NB medium and incubated at 37°C and 120 rpm for 48 hours. Centrifuge at 10000 r / min, 4℃ for 5 min, and spot the supernatant into microwells of BM medium containing aniline blue (Azure B), 75 μL per well. Incubate at 37℃ in the dark. After 48 h, observe and record the presence and size of the decolorization zone (screening was completed by Zhang Quanbin in April 2024 at the Biological Quality Improvement and Flavor Enhancement Laboratory of Tobacco College, Henan Agricultural University).

[0026] (2) Strain identification: The screened strains were inoculated into NA solid medium and incubated at 30 ℃ for 24 h. The colony morphology was observed and the cell characteristics were observed under an electron microscope. The morphology and physiological and biochemical identification of the strains were performed with reference to Bergey's Manual of Bacteriological Identification and the Manual of Systematic Identification of Common Bacteria. The strains were inoculated into NA solid medium and incubated at 30 ℃ and 180 r / min for 24 h. After the incubation, 10 µL of bacterial solution was transferred into a PCR tube, heated at 95 ℃ for 7 min in a PCR instrument, and centrifuged at 10000 r / min at 4 ℃ for 10 min. The supernatant was then used as a DNA template. Using the genomic DNA of the strain as a template, the 16S rDNA sequence was amplified using universal primers 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′). The PCR amplification system consisted of 25 µL of 2×Taq Master Mix, 2 µL each of forward and reverse primers (10 µmol / L), 2 µL of DNA template, and 19 µL of ddH2O. The PCR reaction program was as follows: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 1 min, 30 cycles; 72℃ extension for 5 min; and finally, storage at 4℃. The PCR products were sequenced by Sangon Biotech (Shanghai) Co., Ltd. The sequencing results were compared with known sequences in the NCBI database using the NCBI BLAST online tool. A phylogenetic tree of the strain was constructed using Mega11.0 software (e.g., ...). Figure 1 (As shown).

[0027] The final identification determined that strain MZ-01 was Bacillus megaterium (Betaminae). Bacillus megaterium (Strain morphology as follows) Figure 2 (As shown), it has been deposited at the China Center for Type Culture Collection, address: Wuhan University, Wuhan, China, accession number CCTCCNO: M 2025985, deposit date: May 7, 2025.

[0028] Example 2: Preparation of Bacillus megaterium MZ-01 inoculum (1) Activation of strain: Bacillus megaterium MZ-01 strain was inoculated onto NA solid medium by spot inoculation and cultured at 30 °C for 48-72 h.

[0029] (2) Seed culture preparation: scrape the bacterial cells cultured in step (1), inoculate them into NA liquid culture medium, and culture them in a shaker at 35 ℃ and 160 r / min for 12 h to obtain the seed culture.

[0030] (3) Preparation of liquid fermentation medium for laccase production: T1: Peptone 10.0 g / L, Beef Extract 5.0 g / L, NaCl 5.0 g / L; T2: Sodium lignosulfonate 5.0 g / L, peptone 10.0 g / L, beef extract 5.0 g / L, NaCl 5.0 g / L; T3: Sodium lignosulfonate 10.0 g / L, peptone 10.0 g / L, beef extract 5.0 g / L, NaCl 5.0 g / L; T4: Sodium lignosulfonate 15.0 g / L, peptone 10.0 g / L, beef extract 5.0 g / L, NaCl 5.0 g / L; T5: Sodium lignosulfonate 20.0 g / L, peptone 10.0 g / L, beef extract 5.0 g / L, NaCl 5.0 g / L.

[0031] (4) Fermentation broth preparation: The Bacillus megaterium MZ-01 strain seed liquid from step (2) was inoculated into the special fermentation medium from step (3) at an inoculation rate of 2%, and cultured at 35℃ and a shaking speed of 160 r / min for 48 h. OD 600 =2.0; (5) Preparation of extracellular crude enzyme solution: Centrifuge the fermentation broth in step (4) at 4 ℃ and 8000 r / min for 10 min, and take the supernatant as the extracellular crude enzyme solution.

[0032] Example 3: Treatment of tobacco stems When treating tobacco stems with the microbial agent prepared in Example 2, the specific steps are as follows: The crude enzyme solution obtained at 20% was sprayed evenly at 25% of the weight of the stem fibers. The mixture was then sealed in a sealed bag and fermented for 12 hours at 45℃ and 75% humidity to obtain stem fibers treated with the extracellular crude enzyme solution. After fermentation, the stem fibers were placed in an oven at 80℃ for 10 minutes and then placed in a constant temperature and humidity chamber to balance the moisture content for 24 hours. Sensory quality evaluation, determination of lignin content, determination of total sugar content, and determination of nicotine content were then performed.

[0033] Laccase activity assay: Laccase activity was determined using the ABTS method.

[0034] Method for determining lignin content: Use the lignin content kit from Enzyme-Linked Biotechnology Co., Ltd. to determine the cellulose content according to its instructions.

[0035] Total sugar content was determined according to the tobacco industry standard YC / T 159-2002 / 2019 "Determination of water-soluble sugars in tobacco and tobacco products - continuous flow method".

[0036] Nicotine content determination method: Nicotine content was determined according to tobacco industry standard YC / T 217-2007 "Determination of potassium in tobacco and tobacco products - Continuous flow method".

[0037] Depend on Figure 3 Compared with the control group, the lignin content of tobacco stems treated with extracellular crude enzyme solution of Bacillus megaterium MZ-01 induced by sodium lignin sulfonate was reduced. Among them, the T5 treatment showed the best degradation effect on the stems, reducing the content by 43.2%. This indicates that the laccase produced by Bacillus megaterium MZ-01 induced by sodium lignin sulfonate can effectively reduce the lignin content in tobacco stems. Figure 4 and Figure 5 This indicates that the extracellular crude enzyme solution of Bacillus megaterium MZ-01 induced by sodium lignin sulfonate can slightly reduce the total sugar and nicotine content of the stems, which is beneficial to improving its safety.

[0038] Example 4: Optimization of induction conditions for laccase production by Bacillus megaterium MZ-01 The carbon source was beef extract (5 g / L), the nitrogen source was peptone (10 g / L), and the inorganic salt was NaCl (5 g / L). Different concentrations of the inducer sodium lignin sulfonate (5 g / L, 10 g / L, 15 g / L, and 20 g / L) were added sequentially. The laccase activity assay results are as follows: Figure 6 As shown.

[0039] As shown in the figure, the laccase activity of Bacillus megaterium MZ-01 gradually increased with the increase of sodium lignosulfonate concentration, reaching the highest value of 21.29 U / mL at a concentration of 25 g / L.

[0040] Example 5: Effect of tobacco stem filament fermentation on lignin degradation and sensory evaluation The tobacco stems were processed according to the method in Example 3. The tobacco stems treated with an equal amount of sterile water were denoted as CK. The extracellular crude enzyme solutions produced by induction culture in different liquid fermentation media (T1, T2, T3, T4, T5) in Example 4 were denoted as T1, T2, T3, T4 and T5 respectively.

[0041] Sensory evaluation method: The stems treated with extracellular crude enzyme solution were rolled into cigarettes and equilibrated at 22±1℃ and 60±2% relative humidity for 48 h. Then, sensory evaluation experts were organized to assess the quality of the cigarettes in eight aspects: aroma quality, aroma quantity, off-flavors, irritation, aftertaste, strength, concentration, and smoothness. The evaluation standards or specifications referred to GB 5606.4-2005 "Sensory Quality Evaluation Standards for Cigarettes" and YC / T 138-1998 "Sensory Evaluation Methods for Tobacco and Tobacco Products". The evaluation results are shown in Table 1.

[0042] Table 1 Sensory evaluation scores of tobacco stems after fermentation .

[0043] As shown in Table 1, the stems treated with T5 exhibited excellent performance across multiple dimensions, including aroma intensity, reduction of off-flavors, reduction of irritation, improvement of aftertaste, and smoothness of texture, achieving the highest overall score among all treatment methods. This indicates that the T5 treatment process has significant advantages in improving the quality of stems, maximizing the optimization of their sensory quality and representing the most effective means of improving stem quality.

[0044] Although some preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the invention.

[0045] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from the spirit and scope of its inventive concept. Therefore, if these modifications and variations to this invention fall within the scope of the claims of this application and their equivalents, this invention is also intended to include these modifications and variations.

Claims

1. A type of Bacillus megaterium ( Bacillus megaterium MZ-01, its accession number is CCTCC NO: M2025985.

2. A microbial preparation comprising Bacillus megaterium MZ-01 of claim 1 and / or its metabolic enzyme products, or comprising Bacillus megaterium MZ-01 of claim 1 induced by sodium lignin sulfonate.

3. The use of Bacillus megaterium MZ-01 of claim 1 or the biological agent of claim 2 in at least one of the following (1) to (6): (1) Reduce the lignin content in tobacco stems or prepare a reagent to reduce the lignin content in tobacco stems; (2) Reduce the total sugar and / or nicotine content in tobacco stems or prepare reagents to reduce the total sugar and / or nicotine content in tobacco stems; (3) Improve the sensory quality of tobacco stems or prepare a reagent to improve the sensory quality of tobacco stems, wherein the sensory quality includes at least one of aroma quality, aroma quantity, woody off-flavor, irritation, aftertaste, strength, concentration, and smoothness; (4) Use in combination with sodium lignosulfonate or prepare a reagent for use in combination with sodium lignosulfonate to improve laccase activity; (5) Improve cigarette quality or prepare reagents to improve cigarette quality; (6) Preparation of laccase.

4. The method for preparing the microbial preparation according to claim 2, characterized in that, Includes the following steps: (1) Activation of strain: The Bacillus megaterium MZ-01 described in claim 1 is inoculated onto the corresponding NA solid medium and cultured at 28~32 ℃ for 48~72 h; (2) Preparation of seed culture: scrape the bacterial cells obtained from the previous step of culture, inoculate them into NA liquid culture medium, and culture them in a shaker at 33~37 ℃ and 140~180 r / min for 10~15 h to obtain seed culture; (3) Expanded culture: Inoculate the seed liquid into the fermentation medium at an inoculation rate of 2-4% and culture it in a shaker at a temperature of 33-37℃ and a speed of 140-180 r / min for 46-50 h; (4) Preparation of microbial agent: Centrifuge the fermentation broth from the previous step and take its supernatant.

5. The preparation method according to claim 4, characterized in that, The fermentation medium contains: sodium lignosulfonate 3~20 g / L, peptone 8~12 g / L, beef extract 3~7 g / L, and NaCl 3~7 g / L.

6. A method for processing tobacco stem shreds, characterized in that, Includes the following steps: (1) Adjust the moisture content of the tobacco stems to be treated to 25-30%; (2) Spray the microbial preparation described in claim 2 onto the tobacco stems at 20-30% of the mass of the tobacco stems to be treated, then seal and ferment at 43-47°C and 70-80% humidity for 10-15 hours; (3) After fermentation, the product is dried and the moisture content is balanced.

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