Tobacco leaf baking quality improving microbial agent and application thereof

Abstract

The application discloses a tobacco leaf baking quality improvement microbial agent and application thereof, and the active component of the improvement microbial agent is composed of bacillus amyloliquefaciens and bacillus subtilis, and the improvement microbial agent is sprayed on the surface of tobacco leaves before and after harvesting and before baking. Through application of the specific compound microbial agent, the leaf microorganism community in the tobacco leaf conditioning process can be effectively controlled, and beneficial metabolism can be directionally promoted. Application results show that the application can significantly increase the contents of total sugar, reducing sugar, potassium and chlorine of tobacco leaves, selectively increase polyphenol substances such as scopoletin and rutin, enrich aroma components such as linalool and spirorolandane which give woody and floral styles, and improve the softness of tobacco leaves, so that the characteristic quality of honey sweet type tobacco leaves is comprehensively improved.

Description

Technical Field

[0001] This invention relates to a microbial agent for improving the quality of tobacco curing and its application, belonging to the field of tobacco curing technology. Background Technology

[0002] Tobacco is an important economic crop, widely cultivated and consumed globally. Tobacco curing is a crucial step in transforming freshly harvested tobacco leaves into finished products with specific qualities and flavors. Different curing methods and process parameters can significantly impact the quality of tobacco leaves.

[0003] During the curing process of flue-cured tobacco, the interaction between microorganisms and environmental factors has a significant impact on the formation of tobacco leaf quality. Microbial communities significantly influence tobacco leaf quality by decomposing organic matter and producing volatile aroma components. Microorganisms can break down macromolecules in tobacco leaves, such as proteins, starches, and cellulose, producing smaller organic acids, amino acids, and sugars. For example, microorganisms can decompose proteins in tobacco leaves to produce amino acids, which can undergo Maillard reactions with sugars in the tobacco leaves to produce substances with distinctive aromas and flavors. Simultaneously, microorganisms can also affect the content and proportion of polyphenols, alkaloids, and other chemical components in tobacco leaves, thereby altering the color, aroma, and taste characteristics. Furthermore, microorganisms can promote the transformation of chemical components in tobacco leaves, such as the degradation of nicotine and the oxidation of polyphenols; these changes also affect the quality and flavor of the tobacco leaves. Microorganisms may also affect the safety of tobacco leaves during the curing process. Some microorganisms can produce toxins, such as aflatoxin and ochratoxin, which pose potential health risks to humans. Therefore, effective measures must be taken to control the growth and metabolism of microorganisms during tobacco processing to ensure the safety of tobacco leaves.

[0004] Currently, existing modulation technologies mostly focus on temperature and humidity control, while methods for targeted regulation of the microbial community structure on the surface of tobacco leaves and internal enzymatic reactions are relatively limited. Current research and practice largely focus on using exogenous enzyme preparations (such as mesophilic α-amylase, cellulase, and neutral protease) to target and regulate the biochemical processes of tobacco leaves in order to improve quality. Mesophilic α-amylase can efficiently degrade starch in tobacco leaves into soluble sugars such as maltose and glucose, directly increasing sugar content. This process not only provides substrates but also alters the nutrient environment of the leaf foliage. Rapid starch consumption significantly reduces the competitive advantage of indigenous microorganisms that rely on slow starch degradation (such as actinomycetes), thus creating ecological niches for the colonization of exogenous and beneficial microbial communities. Cellulase gently disrupts the cell wall structure of tobacco leaves, increasing permeability, which not only facilitates the exudation of intracellular sugars and aroma precursors but also releases signaling molecules such as oligosaccharides and cellobiose. Neutral proteases can degrade proteins to produce peptides and amino acids, providing essential aroma precursors for the Maillard reaction. On the other hand, the accumulation of amino acids can specifically stimulate the growth of beneficial microorganisms with an amino acid nutrient preference (such as Lactobacillus and certain Bacillus species).

[0005] However, with the increasing demand for diversified and distinctive tobacco leaf quality, especially in ecoregions with specific flavor profiles such as the "honey-sweet aroma," relying solely on enzyme treatment technology is no longer sufficient to meet the need for systematic and targeted improvement of the overall quality of tobacco leaves. Although the addition of exogenous enzyme preparations can effectively regulate specific biochemical reaction pathways, the final quality of tobacco leaves is the result of the combined action and dynamic balance of their complex internal chemical components, physical structure, and leaf surface microbial community. Further research and improvement in this area are necessary. Summary of the Invention

[0006] Based on the above, the present invention provides a tobacco curing quality improving microbial agent and its application, which can systematically and specifically increase the sugar content and characteristic aroma substance content of tobacco leaves in honey-sweet aroma ecological zones, and improve the appearance quality of tobacco leaves.

[0007] The technical solution of this invention is:

[0008] In a first aspect, the present invention provides a microbial agent for improving the quality of tobacco curing, the active ingredients of which are composed of Bacillus amyloliquefaciens and Bacillus subtilis.

[0009] Preferably, the bacterial agent is a sprayable bacterial suspension, and its OD value is [not specified]. 600 The value ranges from 0.5 to 1.0.

[0010] Preferably, the ratio of viable Bacillus amyloliquefaciens to Bacillus subtilis is 1:1.

[0011] Secondly, the present invention provides an application of the improved microbial agent in improving the curing quality of tobacco leaves in honey-sweet aroma ecological zones.

[0012] Preferably, the baking quality includes the following aspects:

[0013] 1) Increase the content of total sugar, reducing sugar, potassium, and chloride;

[0014] 2) Increase the content of hyoscyamine, rutin, and campheneol rutin;

[0015] 3) Enriched with linalool, spirotropane, and geraniol acetone to enhance woody and floral notes.

[0016] Thirdly, the present invention provides a method for promoting the sugar and aroma enhancement of tobacco leaves in honey-sweet aroma ecological zones, comprising:

[0017] S1 sprays exogenous bacterial solution onto tobacco leaves before harvesting;

[0018] S2 sprays the exogenous bacterial solution onto the tobacco leaves again after the tobacco leaves are harvested;

[0019] S3 involves drying and baking the sprayed tobacco leaves;

[0020] The exogenous bacterial solution is prepared by mixing Bacillus amyloliquefaciens and Bacillus subtilis bacterial solutions.

[0021] Preferably, the exogenous bacterial solution is prepared by mixing Bacillus amyloliquefaciens bacterial solution and Bacillus subtilis bacterial solution at a volume ratio of 1:1.

[0022] Preferably, the amount of exogenous bacterial solution applied is 2-5 ml per tablet each time.

[0023] The beneficial effects of this invention: The tobacco curing quality improving microbial agent and its application provided by this invention, through Bacillus amyloliquefaciens and Bacillus subtilis, achieve multi-dimensional and systematic quality improvement of tobacco leaves in the "honey-sweet aroma" ecological zone, specifically reflected in the following aspects:

[0024] 1. The microbial agent treatment provided by this invention can significantly and synergistically improve the chemical fundamental indicators that are crucial to the "honey-sweet aroma" style. Specifically, it significantly increases the total sugar and reducing sugar content in the roasted tobacco leaves, providing a rich base of sweet substances; at the same time, it effectively increases the content of mineral elements such as potassium (K) and chlorine (Cl), which helps to improve the combustibility and smoldering endurance of the tobacco leaves. In addition, it can selectively enrich characteristic polyphenolic substances such as hyoscyamine, rutin, and campheneol rutin. The increase of these substances not only enhances the antioxidant capacity of the tobacco leaves, but also endows them with unique quality potential.

[0025] 2. Compared with conventional treatment, this invention can specifically drive the transformation of the aroma composition of tobacco leaves towards a sweet and elegant style. In the treated tobacco leaves, the content of terpenes and their derivatives such as linalool, spirophyllodiphenyl ether, and geraniol is significantly enriched. These substances usually contribute prominent woody, floral, and sweet aromas.

[0026] 3. This invention can significantly reduce the hardness of tobacco leaves after curing and improve their softness. During the curing process, it helps the tobacco leaves maintain a uniform and full color, reduces ash accumulation and excessive degradation, and improves the appearance quality.

[0027] 4. This invention can effectively regulate the microbial community structure during the tobacco curing process, promote the enrichment and metabolic activities of beneficial microorganisms, thereby optimizing the degradation efficiency of organic matter and enhancing the generation of aroma precursors. Attached Figure Description

[0028] Figure 1 1. Appearance and sensory indicators of flue-cured tobacco under different treatments; A. Control of flue-cured tobacco under different treatments; B. Comparison of color difference of flue-cured tobacco leaves under different treatments; C. Comparison of softness of flue-cured tobacco leaves under different treatments.

[0029] Figure 2 Changes in the content of compounds, polyphenols, and aroma substances in roasted samples under different treatments;

[0030] Figure 3 Stacked diagrams of intergroup species abundance at the phylum and genus levels under different treatments; A. Stacked diagram of intergroup species abundance at the phylum and genus levels of bacteria; B. Stacked diagram of intergroup species abundance at the phylum and genus levels of eukaryotes.

[0031] Figure 4 α-diversity analysis at the phylum and genus levels under different treatments; A. α-diversity analysis at the phylum and genus levels under different treatments in bacteria; B. α-diversity analysis at the phylum and genus levels under different treatments in eukaryotes.

[0032] Figure 5 Comparison of microbial community enrichment functions among different treatments: A. Bacterial community enrichment function of T2_MvsCK vs T2_JvsCK; B. Bacterial community enrichment function of T3_MvsCK vs T32_JvsCK; C. Bacterial community enrichment function of T4_MvsCK vs T4_JvsCK; D. Eukaryotic community enrichment function of T2_MvsCK vs T2_JvsCK; E3_MvsCK vs T3_JvsCK; F. Eukaryotic community enrichment function of T4_MvsCK vs T4_JvsCK. Detailed Implementation

[0033] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention can be practiced 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 the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0034] I. Materials and Methods

[0035] 1. Sampling location and sampling

[0036] In August 2024, normally grown, mature, and properly cured tobacco leaves were collected in Longchang Town, Fuquan City, Guizhou Province (107°14′E, 27°02′N). During the curing process, three treatments (microbial treatment, enzyme treatment, and blank control) were implemented, with four sampling points (T1: 25℃, 34.25RH; T2: 42℃, 36.5RH; T3: 54℃, 38RH; T4: 68℃, 41RH). At each sampling point, 50 plant leaves were rigorously selected, following the principle of approximately three leaves per replicate, resulting in a total of eight replicates. A total of 96 samples were collected, and the dry-bulb and wet-bulb temperatures were recorded at each sampling point.

[0037] 2. Experimental Design

[0038] (1) Treatment with microbial preparations

[0039] Bacterial treatments (Bacillus subtilis, Bacillus amyloliquefaciens); the bacterial treatment gradients were: J1:OD600=0.5, J2:OD600=0.75, J3:OD600=1. Solutions for each gradient were prepared at a 1:1 volume ratio.

[0040] (2) Enzyme treatment

[0041] Enzyme treatment (α-amylase, cellulase, neutral protease); treatment gradients were: M1: 50 U / g + 40 U / g + 80 U / g, M2: 80 U / g + 80 U / g + 120 U / g, M3: 110 U / g + 120 U / g + 160 U / g. Solutions for each gradient were prepared at a 1:1:1 volume ratio.

[0042] (3) Blank control treatment

[0043] Spraying with clean water served as a control.

[0044] Each treatment selected 87 varieties of Yunnan tobacco, with 100 sample plants selected for each treatment, totaling 700 plants. These were divided into three groups: enzyme-added group (M1, M2, M3), bacteria-added group (J1, J2, J3), and control group (CK). Groups were distinguished by labels. Five days before the normal harvest period, on a clear, rainless evening, each treatment solution was sprayed evenly on both sides of the tobacco leaves using a manual sprayer until each leaf was covered with a thin film without dripping. A second spray was applied five days after harvest, for a total of two sprays. Approximately 3 ml was sprayed per leaf each time, and labels were affixed after each treatment was completed.

[0045] After spraying, the tobacco leaves were air-dried in a cool, ventilated place, then stalked and left to dry for 6-8 hours before being cured using the corresponding curing process (Table 1). The tobacco leaf samples from each treatment were placed in the middle layer of a standard intensive curing barn. Four sampling points were set up: on the day of entry into the curing barn, at the end of the yellowing period, at the end of the color-fixing period, and at the end of the drying period. Disposable gloves were worn during sampling to avoid contaminating the tobacco samples. After collection, the main veins of the tobacco leaves were removed, and the leaves were wrapped in aluminum foil with labels placed on top. The outer layer of the aluminum foil was also labeled. The tobacco samples were then rapidly frozen with liquid nitrogen to maintain sample stability and prevent tissue damage. They were then placed in sealed bags (maintaining a semi-vacuum state by minimizing air inside the bags). The bags were then transported to the laboratory via dry ice and stored in an ultra-low temperature freezer. Changes in the appearance, sensory indicators, compound content, polyphenol content, and aroma substance content of the tobacco leaves under different treatments were analyzed.

[0046] Table 1 Matching Baking Process

[0047]

[0048] 3. Testing Methods

[0049] 1) Measurement of organic compounds

[0050] Based on concentration gradient treatment and appearance quality evaluation, treatments M2, J2, and CK were selected as subsequent test subjects. The collected samples were freeze-dried under vacuum and ground into powder using a grinder (30 Hz, 1.5 min). 0.25 g of sample was weighed and placed in a 50 mL bottle. The powder was mixed with 25 mL of 5% acetic acid, shaken for 30 minutes, and then filtered through a membrane filter. Flow analysis was performed on the filtrate to determine the contents of total sugar, reducing sugar, total nitrogen, nicotine, potassium, and chlorine.

[0051] 3) Metagenomic sequencing

[0052] a. Sample collection and sequencing

[0053] Based on concentration gradient treatment and appearance quality evaluation, treatments M2, J2, and CK were selected as subsequent experimental subjects. One μg of genomic DNA from samples M2, J2, and S was randomly fragmented into fragments of approximately 350 bp using a Covaris ultrasonic disruptor for library construction. The entire library preparation process included end repair, A-tailing, sequencing adapter addition, purification, and PCR amplification. After library construction, the integrity and insert size of the library fragments were first checked using AATI. If the results met expectations, the effective concentration of the library was accurately quantified using Q-PCR (effective concentration > 3 nM) to ensure library quality. After passing the library inspection, different libraries were pooled according to their effective concentration and the target data volume requirements before PE150 sequencing.

[0054] b. Sequencing result preprocessing

[0055] The raw data obtained from the NovaSeq sequencing platform was preprocessed using fastp (https: / / github.com / OpenGene / fastp) to obtain clean data for subsequent analysis. The specific processing steps are as follows: a) If any sequencing read contains an adapter sequence, remove the paired read; b) If the number of low-quality (Q<=5) bases in any sequencing read exceeds 50% of the total base count, remove the paired read; c) If the N content in any sequencing read exceeds 10% of the total base count, remove the paired read.

[0056] c. Metagenome assembly

[0057] The clean data was assembled and analyzed using MEGAHIT software with the following assembly parameters set: -- presetsmeta-large (--end-to-end, --sensitive, -I 200, -X 400 (Karlsson FH et al.,2013; Nielsen HB et al., 2014). The assembled scaffolds were then broken at the N-connections to obtain scaffolds without N.

[0058] d. Gene prediction and abundance analysis

[0059] MetaGeneMark (http: / / topaz.gatech.edu / GeneMark / ) was used to perform ORF prediction on the scaftigs (>=500bp) of each sample, and information with a length less than 100 nt in the prediction results was filtered out. Default parameters were used for all results. The ORF prediction results were deredundantd using CD-HIT software (http: / / www.bioinformatics.org / cd-hit / ) to obtain a non-redundant initial gene catalogue (here, the non-redundant continuous gene-encoding nucleic acid sequences are referred to as genes, with parameters set as: -c 0.95, -G 0, -aS 0.9, -g 1, -d 0). Bowtie2 was used to align the clean data of each sample to the initial gene catalogue, calculating the number of aligned reads for each gene in each sample. Alignment parameters were: --end-to-end, --sensitive, -I 200, -X 400 (Qin J et al., 2010; Li J et al., 2014). Genes with <=2 reads in each sample were filtered out to obtain the final gene catalogue (unigenes) for subsequent analysis. From the aligned reads... Starting from the number of reads and gene length, the abundance information of each gene in each sample was calculated (Cotillard A et al., 2013; Buchfink B et al., 2015; Villar E et al., 2015).

[0060] e. Species annotation

[0061] Using DIAMOND software (https: / / github.com / bbuchfink / diamond / ) (Buchfink B et al., 2015), unigene was compared with bacterial, fungal, archaea, and viral sequences extracted from the NCBI NR database (https: / / www.ncbi.nlm.nih.gov / ) (Karlsson FH et al., 2013). For each sequence alignment, results with an evalue <= 10e-5 were selected. Since each sequence may have multiple alignment results, the LCA algorithm (applied to the systematic classification of MEGAN software (https: / / en.wikipedia.org / wiki / Lowest_common_ancestor)) was used to determine the species annotation information of the sequence (Huson DH et al., 2011). Based on the LCA annotation results and gene abundance table, the abundance information and gene number table of each sample at each taxonomic level (kingdom, phylum, class, order, family, genus, species) were obtained. The abundance of a species in a sample is equal to the sum of the abundance of genes annotated as belonging to that species (Karlsson FH et al., 2012; Li J et al., 2014; Feng Q et al., 2015); the number of genes of a species in a sample is equal to the number of genes with a non-zero abundance among the genes annotated as belonging to that species.

[0062] 4) Metabolomics assay

[0063] 50 μg of sample was placed in a 1.5 mL Eppendorf tube and immersed in 800 μL of pre-chilled extraction buffer (methanol:H₂O = 7:3, v / v) and 20 μL of internal standard 1 (IS1). The sample was homogenized at 50 Hz for 10 min using a milling machine, followed by sonication in a 4°C water bath for 30 min. The extract was allowed to stand at -20°C for 1 h, followed by centrifugation at 14,000 rpm for 15 min at 4°C. 600 μL of the supernatant was filtered through a 0.22 μm membrane, and 20 μL of the filtered solution from each sample was mixed with the quality control sample to assess the repeatability and stability of the LC / MS analysis. The filtered sample and the mixed QC sample were transferred to 1.5 mL vials for instrument preparation. Metabolite determination was performed using a Hypersil GOLD aQ Dim column (1.9 μm, 2.1 × 100 mm, Thermo Fisher Scientific, USA). The mobile phase consisted of an aqueous solution containing 0.1% formic acid (solution A) and an acetonitrile solution containing 0.1% formic acid (solution B), eluted sequentially with the following gradient: 0–2 min, 5% solution B; 2–22 min, 5%–95% solution B; 22–27 min, 95% solution B; 27–27.1 min, 95% solution B–5% solution B; 27.1–2.7 min, 5% solution B. The flow rate was 0.3 mL / min, the column temperature was 40°C, and the injection volume was 5 μL (Bian, Sun et al., 2023). Downstream mass spectrometry data were imported into Compound Discoverer 3.3 (Thermo Fisher Scientific) software and analyzed using BMDB (BGI Metabolomics Database), mzCloud database, and ChemSpider online database to generate a data matrix containing metabolite peak areas and identification results. The tables were then further processed for information analysis. The metaX software was used to preprocess the exported data and to annotate it with detailed information using authoritative databases such as KEGG and HMDB, including KEGG ID, HMDB ID, classification information, and involvement in KEGG metabolic pathways (Pang, Lu et al., 2024).

[0064] 5) Statistical Analysis

[0065] The calculations and analyses were performed using R software (version 4.4.1, developed by the R Foundation for Statistical Computation in Vienna, Austria). Bar charts were created using the ggplot2 package (version 3.5.1). The α-diversity index was calculated to assess microbial community diversity, and the Bray-Curtis distance was used for similarity analysis, supplemented by a nonparametric test (ANOSIM) to determine the impact of different factors on community diversity. Heatmaps were generated using the R package "pheatmap". Differential metabolite analysis was performed using the BGI Dr. Tom multi-omics analysis platform [https: / / biosys.bgi.com / / report / login]. KEGG enrichment analysis was visualized using ggplot2.

[0066] II. Results and Analysis

[0067] 1. Appearance and sensory indicators of baked samples under different treatments

[0068] Figure 1 The figures show the appearance and sensory indicators of the baked samples under different treatments. As can be seen from the figures, in the T2 stage, treatments M1 and J2 performed better than other treatments; in the T3 stage, treatments M2 and J2 outperformed other treatments; and in the T4 stage, treatments M2 and J2 were even more outstanding compared to other treatments. Specifically, in the T2 stage, treatment M1 performed particularly well in terms of surface smoothness and color uniformity, while treatment J2 showed more saturated color. Entering the T3 stage, treatment M2 maintained the advantages of T2 while further preserving the appearance. Treatment J2 showed enhanced color saturation and layering in this stage. As for the T4 stage, the advantages of treatments M2 and J2 were even more pronounced. Treatments M2 and J2 maintained the original color uniformity without excessive degradation or graying.

[0069] Color difference analysis showed that the tobacco leaves in the enzyme-treated group M2 had the most vibrant color after curing, followed by M3. In the bacterial treatment groups, the color of the J3 treatment was brighter, while the color of the J2 treatment was deeper. In contrast, the color of the CK treatment (i.e., the control group CK) was closer to that of the J1 treatment.

[0070] Comparative analysis of softness revealed that treatments M2, J1, and J3 resulted in higher softness values ​​and poorer overall softness in the cured tobacco leaves, while treatments M1, M3, and J2 showed lower softness. Treatment M1 had the lowest softness, and the other treatments generally exhibited better softness. Compared to the control (CK) treatment, treatments M2, J1, and J3 reduced the softness of the tobacco leaves, while treatments M1, M3, and J2 improved it. In conclusion, both short-term and long-term effects showed that treatments M2 and J2 demonstrated better appearance quality. Therefore, treatments M2 and J2 were selected as the subsequent treatment materials.

[0071] 2. Changes in the content of compounds, polyphenols, and aroma substances in roasted samples under different treatments

[0072] Figure 2 The changes in the content of compounds, polyphenols, and aroma substances in the cured samples under different treatments are shown. Compared with the control (CK), the microbial preparation treatment significantly increased the content of total sugar, reducing sugar, potassium (K), and chlorine (Cl) in tobacco leaves, indicating its positive role in promoting carbohydrate accumulation and mineral absorption. Enzyme treatment more significantly increased the total nitrogen content, suggesting that this treatment may promote the synthesis and accumulation of nitrogen-containing compounds, which has an important impact on improving the strength and physiological properties of tobacco leaves. Furthermore, regarding polyphenols, enzyme treatment showed a broad-based enhancement effect, significantly increasing the content of various polyphenols. In contrast, the microbial preparation treatment was more selective, specifically increasing the content of only certain types of polyphenols such as hyoscyamine, rutin, and campheneol rutin, which may endow them with unique quality characteristics. The two treatments exhibited different regulatory tendencies in the composition of key aroma substances. Enzymatic treatment significantly promotes the formation of ketones such as megastigmatrienone and beta-dihydrodamascenone, as well as phenolic and aldehyde aroma compounds. These compounds typically contribute sweet, lipophilic, and caramel-sweet aromas, which are crucial for enhancing the aroma and richness of tobacco. Conversely, microbial treatment primarily enriches linalool, spirovetivane, and geranyl acetone compounds. It mainly increases woody and floral aromas, and the synergistic increase of these substances may make the tobacco aroma more refreshing and complex.

[0073] 3. Species abundance at the phylum and genus levels under different treatments

[0074] Figure 3This is a stacked diagram of species abundance at the phylum and genus levels under different treatments. Species abundance analysis revealed that enzyme and inoculant treatments significantly altered the dominant microbial community structure during tobacco fermentation, primarily occurring during the yellowing stage, crucial for quality formation. At the phylum level, both treatments consistently increased the relative abundance of Pseudomonadota while decreasing the abundance of Bacillota. Pseudomonadota contains a large number of species with strong organic matter degradation capabilities; its increased abundance indicates an accelerated metabolic transformation of macromolecules in tobacco leaves, providing more precursors for subsequent aroma compound formation. At the genus level, the two treatments exhibited specific regulatory effects, directly related to their different application values. Enzyme treatment specifically and significantly increased the abundance of *Sphingomonas*, a bacterium capable of efficiently degrading complex organic compounds such as polycyclic aromatic hydrocarbons. The enrichment of these compounds during the yellowing stage suggests that enzyme treatment may synergistically enhance the biodegradation efficiency of tobacco leaves, helping to reduce undesirable residues, highlight the natural aroma of tobacco, and potentially produce unique aroma precursors. This aligns with the observed significant increase in aroma compounds such as ketones and phenols in the enzyme-treated group. Conversely, microbial treatment increased the abundance of *Escherichia* and *Enterobacter*. *Enterobacter*, as facultative anaerobes, possess extremely strong glycolysis and organic acid metabolism capabilities. In actual fermentation, their enrichment implies a more intense and rapid fermentation process, efficiently converting soluble sugars into metabolic products such as organic acids, thereby rapidly lowering the pH, inhibiting the growth of harmful microorganisms, and forming a unique aroma.

[0075] 4. Analysis of alpha diversity within and between groups at the phylum and genus levels under different treatments

[0076] To investigate the effects of different treatments on the structure and composition of the microbial community during tobacco curing, the Alpha diversity analysis method was used to systematically evaluate the dynamic changes of the microbial community. Figure 4The results of α-diversity analysis at the phylum and genus levels under different treatments are presented. The results showed that both enzyme and bacterial treatments significantly affected the diversity and richness of the microbial community, with the enzyme-treated group exhibiting higher overall diversity and richness than the other two groups. In the bacterial community, compared to the control group (CK), enzyme treatment significantly increased the Shannon diversity index during the yellowing stage. Chao1 index analysis further indicated that enzyme treatment also increased community richness at this stage; however, during the dry vegetative stage, enzyme treatment led to a decrease in community richness. In the eukaryotic community, none of the treatments caused significant changes in community diversity; only during the yellowing stage did the enzyme-treated group show an increase in community richness. In conclusion, this study demonstrates that applying enzyme and bacterial treatments before flue-cured tobacco significantly affects the structure and diversity of the phyllosphere microbial community, and this effect varies across different curing stages and biological groups.

[0077] 5. Comparison of the enrichment function of microbial communities among different treatments

[0078] Figure 5To compare the enrichment functions of microbial communities among different treatments, comparative analysis of different treatment groups (MT: enzyme treatment, JT: bacterial treatment) and the control group (CK) at different modulation stages revealed that the metabolic responses of the microbial communities and the eukaryotic host exhibited significantly different temporal characteristics and functional tendencies. Regarding microbial community function, the number of enriched pathways gradually decreased with the modulation process. In terms of the bacterial community, the number of enriched pathways in the MT vs CK comparison group was significantly greater than that in the JT vs CK group during the yellowing stage, indicating that MT treatment induced more intense microbial functional reorganization in the early stages of modulation. Under yellowing conditions, the MT-treated microbial community responded rapidly, exhibiting strong carbohydrate and protein metabolic activity, including key pathways such as the pentose phosphate pathway, arginine and proline metabolism, and tryptophan metabolism, demonstrating its ability to rapidly consume basic nutrients (carbohydrates and proteins) and generate flavor precursors. In contrast, JT treatment enriched pathways related to energy maintenance and cellular function, such as peroxisome and ubiquinone biosynthesis. During the color-fixing stage, the vast majority of enriched pathways were concentrated in the JTvsCK group, indicating that JT treatment had a more significant impact on microbial function at this stage. Enriched pathways included arachidonic acid metabolism and propionic acid metabolism, which are closely related to the generation of fatty acid derivatives and branched-chain ester precursors, contributing to the improvement of tobacco aroma and flavor from a metabolic perspective. However, the number of enriched pathways decreased further during the dry-rib stage. The enrichment of fat digestion and absorption pathways in MT treatment may accelerate the consumption of oils and fatty acid aroma precursors in tobacco, leading to the loss of aroma substances. Conversely, the JT treatment enriched the biosynthesis of various plant secondary metabolites, the degradation of flavonoids, and the biosynthesis of other types of O-glycans, indicating that it is more conducive to the comprehensive activation of the plant secondary metabolic network and promotes the transformation and synthesis of flavor substances.

[0079] In eukaryotic metabolism, the response pattern is the opposite of that of microorganisms. Metabolic diversity gradually increases with the modulation process, and the enrichment of pathways is more abundant in the later stages, including starch and sucrose metabolism, sulfur metabolism, and biotin / pantothenate and CoA biosynthesis. Specifically, the yellowing stage is dominated by the rapid initiation of metabolic processes, and the enrichment pathways include lysine degradation and ribosome biogenesis in eukaryotes. During the color fixation stage, both treatments continuously enrich amino acid metabolic pathways, such as histidine metabolism. The MT treatment also specifically enriches biotin metabolism and protein processing in the endoplasmic reticulum. During the gluten development phase, both treatments enriched key pathways such as starch and sucrose metabolism and sulfur metabolism, and activated multiple regulatory pathways such as the PI3K-Akt signaling pathway, estrogen signaling pathway, thyroid hormone signaling pathway, and T cell receptor signaling pathway to maintain cell signal transduction and metabolic homeostasis.

[0080] In summary, MT treatment strongly activates microbial primary metabolism in the early stages of modulation, potentially accelerating substrate consumption; while JT treatment more significantly affects microbial and plant secondary metabolism in the later stages, promoting the accumulation and transformation of aroma precursors. Eukaryotic host metabolism exhibits a gradually increasing and more diverse response, maintaining high metabolic and signaling regulatory activity, especially in the later stages of modulation.

[0081] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A microbial agent for improving the curing quality of tobacco leaves, characterized in that, Its active ingredients consist of Bacillus amyloliquefaciens and Bacillus subtilis.

2. The tobacco leaf curing quality improving microbial agent according to claim 1, characterized in that, The bacterial agent is a sprayable bacterial suspension, and its OD value is [not specified]. 600 The value ranges from 0.5 to 1.

0.

3. The tobacco curing quality improving microbial agent according to claim 1, characterized in that, The ratio of viable Bacillus amyloliquefaciens to Bacillus subtilis is 1:

1.

4. The application of the improved microbial agent according to any one of claims 1 to 3 in improving the curing quality of tobacco leaves in the honey-sweet aroma ecological zone.

5. The application according to claim 4, characterized in that, The baking quality includes the following aspects: 1) Increase the content of total sugar, reducing sugar, potassium, and chloride; 2) Increase the content of hyoscyamine, rutin, and campheneol rutin; 3) Enriched with linalool, spirotropane, and geraniol acetone to enhance woody and floral notes.

6. A method for promoting sugar and aroma enhancement in tobacco leaves from honey-sweet aroma ecological zones, characterized in that, include: S1 sprays exogenous bacterial solution onto tobacco leaves before harvesting; S2 sprays the exogenous bacterial solution onto the tobacco leaves again after the tobacco leaves are harvested; S3 involves drying and baking the sprayed tobacco leaves; The exogenous bacterial solution is prepared by mixing Bacillus amyloliquefaciens and Bacillus subtilis bacterial solutions.

7. The method for promoting sugar and aroma enhancement in tobacco leaves of honey-sweet aroma ecological zones according to claim 5, characterized in that, The exogenous treatment bacterial solution is prepared by mixing Bacillus amyloliquefaciens bacterial solution and Bacillus subtilis bacterial solution at a volume ratio of 1:

1.

8. The method for promoting sugar and aroma enhancement in tobacco leaves of honey-sweet aroma ecological zones according to claim 5, characterized in that, The amount of exogenous bacterial solution applied is 2-5 ml per tablet each time.

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