Fermentation method for baking puffed river no. 2 sun-cured tobacco sheet

Abstract

The present application relates to the field of biotechnology, and more particularly to a fermentation method for baking Piaohe No. 2 sun-cured tobacco. The present application is aimed at sun-cured tobacco, in particular Piaohe No. 2 sun-cured tobacco, in order to further improve its smoking quality, especially the performance of aftertaste and combustion, so as to better meet the needs of subsequent formula, a fermentation method for Piaohe No. 2 sun-cured tobacco is provided. After fermentation, the smoking impurities of Piaohe No. 2 sun-cured tobacco are significantly reduced, the quality of Piaohe No. 2 sun-cured tobacco, the smoking quality and the adaptability of tobacco are improved.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more particularly to a fermentation method for roasting Piaohe No. 2 sun-cured tobacco flakes. Background Technology

[0002] With the tobacco industry's efforts to reduce tar and harmful substances, the decline in cigarette aroma quality has become increasingly prominent. Sun-cured tobacco differs from flue-cured tobacco; its "sun-curing" refers not only to the drying and curing process but also to the unique tobacco variety. These varieties of tobacco leaves inherently possess a high aroma, so increasing the application of sun-cured tobacco in cigarette blends can improve the problem of declining cigarette aroma quality. However, the curing environment of natural air-curing is relatively mild, resulting in the cured raw tobacco exhibiting various quality defects to varying degrees, such as green leaves, heavy off-flavors, high irritation leading to an unpleasant smoke, monotonous aroma, and insufficient aroma quantity. Therefore, sun-cured tobacco is increasingly unable to meet the quality requirements of modern cigarette industrial processing.

[0003] To address the aforementioned issues, existing technologies, such as the patent document with publication number CN117158616A, employ a baking process to modulate sun-cured tobacco leaves, thereby significantly improving the smoking quality of sun-cured tobacco leaves. However, specifically in cigarette formulations, the aftertaste and combustibility still require further improvement to enhance the compatibility and harmony of the corresponding tobacco leaves. The most crucial aspect is further reducing off-flavors during smoking. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a fermentation method for roasting Piaohe No. 2 sun-dried tobacco sheets.

[0005] This invention provides a fermentation method for sun-dried tobacco leaves, which involves initial curing, rehydration and softening, first leaf moistening, second leaf moistening, separation of leaf stems, re-curing, cooling, and second rehydration, and then spreading the resulting tobacco leaves flat in a fermentation tank for fermentation.

[0006] The density of the tobacco flakes in the fermentation tank is 0.34 g / cm³. 3 ~0.45g / cm 3 The preferred tobacco density is 0.334 g / cm³. 3 ;

[0007] The fermentation time is 6 to 10 days; preferably 8 to 10 days. Most preferably 10 days.

[0008] The fermentation temperature is 40°C.

[0009] The relative humidity for fermentation is 80±5%.

[0010] The moisture content of the tobacco leaves after re-drying and before fermentation is 18%~22%.

[0011] The fermentation process involves turning the pile over every two days.

[0012] The initial baking includes the following steps:

[0013] The first stage of baking of the sun-dried tobacco was carried out under the conditions of dry bulb temperature of 23℃~30℃ and wet bulb temperature of 23℃~30℃.

[0014] After adjusting the dry bulb temperature to 32℃~34℃, proceed with the second stage of baking until the leaves are completely yellow.

[0015] The third stage of baking was carried out by adjusting the dry bulb temperature to 38℃~40℃ and the wet bulb temperature to 36℃~38℃ until brown spots appeared on the leaves.

[0016] The wet-bulb temperature was adjusted to 34℃~36℃ for the fourth stage of baking until the browning area of ​​the leaves reached 30%~70%.

[0017] The fifth stage of baking was carried out by adjusting the dry bulb temperature to 45℃~47℃ and the wet bulb temperature to 31℃~33℃ until the leaves were dry.

[0018] The sixth stage of baking was carried out by adjusting the dry bulb temperature to 60℃~68℃ and the wet bulb temperature to 29℃~35℃ until the leaf veins were completely dry.

[0019] The baking time for the first stage is 13h~15h; the baking time for the second stage is 70h~71h; the baking time for the third stage is 11h~13h; the baking time for the fourth stage is 18h~26h; the baking time for the fifth stage is 20h~60h; and the baking time for the sixth stage is 47h~60h.

[0020] Furthermore, the dry bulb temperature during the first stage of baking is 23.7°C, and the wet bulb temperature is 23.3°C.

[0021] The temperature for baking the dry balls in the second stage is 33°C;

[0022] The dry bulb temperature during the third stage of baking is 39°C, and the wet bulb temperature is 37°C.

[0023] The wet-bulb temperature for the fourth stage of baking is 35°C.

[0024] The dry bulb temperature during the fifth stage of baking is 46°C, and the wet bulb temperature is 32°C.

[0025] The dry bulb temperature during the sixth stage of baking is 60℃~68℃, and the wet bulb temperature is 34℃.

[0026] The selection criteria for sun-dried tobacco leaves during harvesting are: the main vein turns white in 1 / 3 to 2 / 3 of its length, the secondary veins turn white in 1 / 3 of their length, the leaf tips and edges turn yellow, and the leaf surface is 50% to 60% yellow.

[0027] The conditions for the first leaf moistening and / or the second leaf moistening are: temperature 65℃~85℃;

[0028] The moisture content after the second leaf rinsing is 18%~20%;

[0029] The conditions for re-baking are: 60℃~130℃;

[0030] The moisture content after the second rehydration is 11%~13%.

[0031] The baking time for the first stage is 13 hours; the baking time for the second stage is 70 hours; the baking time for the third stage is 11 hours; the baking time for the fourth stage is 26 hours; the baking time for the fifth stage is 59 hours; and the baking time for the sixth stage is 60 hours.

[0032] In the fermentation method described in this invention, the sun-dried tobacco leaves are Piaohe No. 2.

[0033] This invention obtains the fermentation method of the Piaohe No. 2 sun-dried tobacco leaves through process optimization during fermentation, which has an essential difference in raw materials from the existing public cigar fermentation methods: First, it uses tobacco leaves that have been processed by sun-drying and roasting as the fermentation substrate, which are in the form of dispersed and irregular tobacco leaves; while the existing technology generally uses whole tobacco leaves of cigars that have not been sun-dried and roasted as raw materials, which are only harvested and dried in the field, and fermented by bundling / piling whole leaves.

[0034] Secondly, the fermentation method of the present invention reduces the impurities and irritation of the sun-cured tobacco after roasting compared to the original sun-cured tobacco.

[0035] Third, this invention employs a constant temperature and humidity chamber to implement closed-loop precise temperature and humidity control. Figure 26 This invention allows for the setting and maintenance of temperature and humidity parameters within a narrow range based on the characteristics of sun-cured tobacco leaves. The fermentation rate is stable and controllable, with strong batch consistency, achieving refined micro-fermentation that targets and weakens off-flavors and irritation while preserving the inherent aroma and style of sun-cured tobacco. Existing public cigar tobacco leaf fermentation mainly relies on ambient temperature in the natural environment, using the heat generated by microorganisms in the tobacco leaf stacking to create internal high temperatures. This results in a large spatial gradient of temperature and humidity, fluctuations that vary with the environment and stacking operations, uneven fermentation levels, long cycles, and high quality dispersion. The core purpose of this invention is to deeply degrade and soften the whole tobacco leaf. The process logic, parameter range, and technical objectives are all unsuitable for the quality improvement requirements of the refined sun-cured tobacco leaves of this invention. The fermentation implementation methods of the two inventions are substantially different, resulting in a reproducible and refined quality improvement effect, which is not a simple replacement of conventional processes in existing technologies.

[0036] This invention targets sun-cured tobacco, particularly the Piaohe No. 2 sun-cured tobacco variety. To further improve its smoking quality, especially its aftertaste and combustibility, and thus better meet the requirements of subsequent formulations, a fermentation method for Piaohe No. 2 sun-cured tobacco leaves is provided. After fermentation, the smoking impurities of Piaohe No. 2 sun-cured tobacco leaves are significantly reduced, improving the quality, smoking quality, and compatibility of Piaohe No. 2 sun-cured tobacco leaves. Attached Figure Description

[0037] Figure 1 The sensory quality evaluation results of S2d60, S4d60, S6d60, S8d60, and S10d60 are shown.

[0038] Figure 2 The sensory quality evaluation results of S2d70, S4d70, S6d70, S8d70, and S10d70 are shown.

[0039] Figure 3 The sensory quality evaluation results of S2d80, S4d80, S6d80, S8d80, and S10d80 are shown.

[0040] Figure 4 PCoA shows the microbial (bacterial) communities among different samples;

[0041] Figure 5 PCoA shows the microbial (fungal) community among different samples;

[0042] Figure 6 Showing the bacterial community structure at the phylum level among different samples;

[0043] Figure 7 Showing the genus-level bacterial community structure among different samples;

[0044] Figure 8 Showing the phylum-level fungal community structure among different samples;

[0045] Figure 9 This shows the genus-level fungal community structure among different samples;

[0046] Figure 10 This shows a random forest analysis diagram of bacterial communities among different samples;

[0047] Figure 11 This shows a random forest analysis diagram of fungal communities among different samples;

[0048] Figure 12 Bacterial CCA-RDA analysis diagram;

[0049] Figure 13 This shows a CCA-RDA analysis diagram of fungi.

[0050] Figure 14PCA scores of tobacco metabolites at 70% of the packaged tobacco volume and different fermentation times;

[0051] Figure 15 Dispersion plot of PLS-DA metabolites from tobacco leaves at 70% of the packaged tobacco volume and different fermentation times;

[0052] Figure 16 Thermographic analysis of significantly differentially identified metabolites in samples with a fermentation time of 4 days;

[0053] Figure 17 Thermographic analysis of significantly differentially identified metabolites in samples with a fermentation time of 6 days;

[0054] Figure 18 Thermographic analysis of significantly differentially identified metabolites in samples with a fermentation time of 8 days;

[0055] Figure 19 Thermographic analysis of significantly differentially identified metabolites in samples with a fermentation time of 10 days;

[0056] Figure 20 The KEGG pathway enrichment map is shown for a fermentation time of 4 days.

[0057] Figure 21 The KEGG pathway enrichment map is shown for a fermentation time of 6 days.

[0058] Figure 22 The KEGG pathway enrichment map is shown for a fermentation time of 8 days.

[0059] Figure 23 The KEGG pathway enrichment map is shown for a fermentation time of 10 days.

[0060] Figure 24 A heatmap showing the correlation between microorganisms (bacteria) and metabolomics at different fermentation times;

[0061] Figure 25 A heatmap showing the correlation between microorganisms (fungi) and metabolomics at different fermentation times;

[0062] Figure 26 This shows the placement of oak barrels in a constant temperature and humidity chamber during the fermentation process. Detailed Implementation

[0063] This invention provides a fermentation method for roasting Piaohe No. 2 sun-cured tobacco flakes. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0064] The testing methods for the indicators involved in this invention are listed below for reference:

[0065] 1. Conventional methods for determining chemical components

[0066] Tobacco samples were prepared according to the specifications in YC / T31—1996 "Preparation of Tobacco and Tobacco Products Samples and Determination of Moisture by Oven Method". The samples were ground and passed through a 60-mesh sieve before analysis. The chemical indicators to be measured—total sugar, reducing sugar, total alkaloids, total nitrogen, chlorine, and potassium—were determined using a PI continuous flow analyzer according to YC / T159-162 "Automated Chemical Analysis Methods for Tobacco".

[0067] 2. Sensory quality evaluation methods

[0068] Sensory quality evaluation of tobacco leaves includes indicators such as aroma quality, aroma intensity, off-flavors, aftertaste, irritation, and smoke concentration. 20g samples of tobacco leaves from each of the different treatments were selected and processed into shredded tobacco using a micro-shredder. The moisture content of the shredded tobacco was adjusted to 12% before being rolled into single-material cigarettes. The cigarettes were then equilibrated for moisture at 22±1℃ and 60±1% relative humidity for 24 hours. Sensory evaluation experts from the industry evaluated and scored the samples according to the requirements of the "Sensory Evaluation Methods for Tobacco Products" standard YC / T 415-2011 published in 2012.

[0069] 3. The results of microbial community detection and non-targeted metabolomics detection were provided by Shanghai E-Sequencing Co., Ltd.

[0070] This invention relates to a method for fermenting sheet tobacco in the production of Piaohe No. 2 sun-cured tobacco. Specifically, the sheet tobacco obtained after roasting and re-roasting the Piaohe No. 2 sun-cured tobacco is evenly spread in a sealed fermentation tank for fermentation. The temperature is 40℃, the relative humidity is 80±5%, and the density of the sheet tobacco in the fermentation tank is 0.34 g / cm³. 3 ~0.45g / cm 3 The fermentation time is 6 to 10 days.

[0071] Preferably, the initial moisture content of the tobacco leaves is 18% to 22%. Controlling the initial moisture content has a significant impact on the subsequent fermentation effect. If the moisture content is too high, the tobacco leaves are prone to mold, while if it is too low, insufficient moisture will affect the fermentation effect.

[0072] Preferably, the density of the tobacco sheet is 0.382 g / cm³. 3 The preferred fermentation time is 8 to 10 days, and more preferably, the fermentation time is 10 days.

[0073] For tobacco sheet density of 0.334 g / cm³ 3 For the samples, the preferred fermentation time is 6 or 8 days.

[0074] The compost pile is turned over every two days during the fermentation process.

[0075] The process of curing Piaohe No. 2 sun-dried tobacco can be carried out with reference to CN117158616A.

[0076] Based on this, the present invention studied the advantages and disadvantages of this variety of sun-dried tobacco, and used fermentation to control the direction of raw material style and quality optimization, ultimately reducing its intake of impurities, improving the smoking quality of tobacco leaves, and enhancing the adaptability and coordination of tobacco leaves.

[0077] The subsequent leaf threshing and re-drying can be carried out in accordance with the following method: industry standard YC / T 146-2025 "Specification for Tobacco Leaf Threshing and Re-drying Process".

[0078] The test materials used in this invention are all common commercially available products. The invention is further illustrated below with reference to embodiments:

[0079] Example 1: Fermentation method for roasting Piaohe No. 2 sun-cured tobacco flakes

[0080] The raw material used was 2023 Jilin Jiaohe Piaohe No. 2 sun-dried tobacco, and three oak barrels were used (18cm in diameter, 21cm in belly, and 27cm in height). Distilled water was used in the process.

[0081] After initial curing, the Piaohe No. 2 sun-cured tobacco is vacuum rehydrated until soft, then subjected to one and two moistening processes (temperature 65℃~85℃) to achieve a moisture content of 18%~20%. The leaves and stems are then separated using a leaf-beating machine. The separated leaves are dried in a re-drying machine (temperature 60℃~130℃), cooled, and rehydrated to precisely control the moisture content of the tobacco leaves at 11%~13%. This yields destemmed tobacco leaves.

[0082] To balance the moisture content of the tested tobacco leaves to an initial moisture content of 18%–22%, the samples were divided into three groups (groups 1, 2, and 3). 2500g of tobacco leaves were weighed out of each group and evenly spread inside an oak barrel. The barrel lid was placed on top of the tobacco leaves, and sandbags of 450g, 350g, and 250g were placed on top respectively for compression. This resulted in the same mass of tobacco leaves occupying approximately 60%, 70%, and 80% of the oak barrel volume (hereinafter referred to as the tobacco volume), respectively, corresponding to tobacco leaf densities of 0.4457g / cm³. 30.3821 g / cm 3 0.3343 g / cm 3 The corresponding wooden barrels were labeled 1, 2, and 3 sequentially, and fermented at 40℃ and 80% relative humidity for 10 days, turning the barrels 4-5 times. 250g samples were taken on days 2, 4, 6, 8, and 10 after fermentation began. 20g of each sample was freeze-dried, sealed, and stored for the determination of microbial diversity and non-targeted metabolomics components. The remaining samples were used to detect other indicators, with unfermented Piaohe No. 2 sun-dried tobacco leaves serving as a control (denoted as S). The sample numbering rules for each treatment are as follows: S2d60 represents the sample taken on day 2 of fermentation in an oak barrel with 60% tobacco volume; S4d70 represents the sample taken on day 4 of fermentation in an oak barrel with 70% tobacco volume, and so on.

[0083] 1. Routine chemical composition testing

[0084] Table 1 below shows the results of the content of conventional chemical components in different samples.

[0085] Table 1

[0086]

[0087] As shown in Table 1, compared with the control sample before fermentation, the total nitrogen and nicotine content of tobacco leaves increased after fermentation, while the total sugar, reducing sugar, and disaccharide ratio decreased, the potassium content increased, the chlorine content decreased, and the sugar-alkali ratio and potassium-chlorine ratio decreased and increased, respectively.

[0088] The results of the significance analysis showed that, compared with before fermentation, as the volume of tobacco leaves occupying the oak barrel (i.e., the volume of tobacco filling) increased, the total sugar and reducing sugar showed a trend of first increasing, then decreasing, and finally increasing again. The treatment with 80% tobacco filling volume showed the most significant changes, with the total sugar and reducing sugar reaching their lowest levels on day 8 of fermentation, at 14.82% and 14.68%, respectively. The total nitrogen and nicotine contents of the tobacco leaves increased after fermentation, showing a trend of first increasing and then decreasing with the extension of fermentation time. The treatment with 70% tobacco filling volume showed the most significant changes compared with the control, with the highest total nitrogen content (3.98%) and the lowest nicotine content (4.01%) on day 6 of fermentation. The potassium content of the sun-dried tobacco increased after treatment, and the change was more stable in the treatment with 70% tobacco filling volume. The potassium content in this treatment was the highest on day 4 of fermentation, at 3.38%. The chlorine content decreased compared with before fermentation, with the treatment with 70% tobacco filling volume showing a relatively higher content, reaching its highest level of 0.53% on day 4 of fermentation.

[0089] 2. Changes in sensory quality

[0090] Figures 1-3The sensory quality evaluation results of sun-dried tobacco at different fermentation stages are shown. It can be seen that, compared to the control, tobacco leaves fermented in wooden barrels for 2 days showed a more pleasant aftertaste after all three treatments, with little change in aroma quality, aroma quantity, concentration, flammability, strength, and irritation. Tobacco leaves fermented in wooden barrels for 8 days showed significant differences in aftertaste and flammability scores among the three treatments compared to the control, but the overall aftertaste was more pleasant and flammability improved. Tobacco leaves fermented for 10 days showed reduced off-flavors and improved flammability compared to the unfermented tobacco leaves (control). Overall, the treatment with 70% tobacco volume and 10 days of fermentation showed the best overall sensory quality evaluation, followed by the treatment with 80% tobacco volume and 6th and 8th days of fermentation. In summary, the treatment with 70% tobacco volume showed the best improvement in sensory quality during 8-10 days of fermentation.

[0091] 3. The influence of tobacco leaf microbial community

[0092] 3.1 PCoA Analysis

[0093] To explain the reliability of the test data, a P=0.001 was used, and PCoA analysis was performed on the samples. PCoA, or Principal Coordinate Analysis, can visually display the similarity and differences of microbial communities in different environmental samples. The horizontal and vertical axes in the graph reflect the proportion of sample differences, and the position of the points can determine whether the community structures between samples are similar. Figure 4 and Figure 5 It can be seen that the bacterial and fungal community differences among samples with different flue gas volumes and fermentation times explained 78.89% and 91.44% of the data, respectively. Furthermore, points of the same color showed obvious clustering, while points of different colors were significantly separated from each other, proving that the samples were representative and the data could be further analyzed.

[0094] 3.2 Community Structure

[0095] Bacterial community structure at the phylum level for samples with different smoke volumes, such as Figure 6 As shown in the figure, a total of 23 bacterial phyla were detected in 16 samples, with Firmicutes, Proteobacteria, Bacteroidetes, and Actinobacteriota being the dominant phyla. Proteobacteria were detected in every sample and were the dominant phylum. The relative abundance of Proteobacteria reached 96.33% and 96.04% in the treatments with 70% and 80% of the smoke volume after 6 days of fermentation, respectively.

[0096] Bacterial community structure at the genus level for samples with different smoke volumes, as shown below Figure 7As shown in the figure, a total of 661 bacterial genera were detected in 16 samples. The dominant genera were Acinetobacter, Methylobacterium-Methylerythrobacter, Sphingomonas, and Aureimonas. These four dominant genera were detected in each sample. In the treatments with 4 days of fermentation and 70% and 80% of the smoke volume, the relative abundance of Acinetobacter reached 47.32% and 48.75%, respectively.

[0097] The fungal community structure at the phylum level in samples with different flue gas volumes and fermentation times is as follows: Figure 8 As shown in the figure, six fungal phyla were detected in 16 samples, with Ascomycota and Basidiomycota being the dominant phyla. After 2 days of fermentation, the relative abundance of Ascomycota in the 60% and 70% smoke volume treatments was higher than the 80.00% before fermentation, reaching 90.12% and 92.48%, respectively. The relative abundance of Basidiomycota in the 60% and 70% smoke volume treatments reached its highest values ​​at 6 and 10 days of fermentation, respectively, at 92.25% and 94.48%.

[0098] The fungal community structure at the genus level for samples with different flue gas volumes and fermentation times is as follows: Figure 9 As shown in the figure, a total of 205 fungal genera were detected in 16 samples, with *Sampayo* and *Alternaria* being the dominant genera. In the treatment with 60% of the tobacco volume and 10 days of fermentation, the relative abundance of *Sampayo* significantly increased from 11.58% before fermentation, reaching 89.62%; in the treatment with 4 days of fermentation, the relative abundance of *Alternaria* significantly increased from 19.66% before fermentation, reaching 60.71%.

[0099] 3.3 Random Forest Analysis

[0100] To determine the importance of a particular microorganism in the fermentation of sun-dried tobacco barrels, a random forest analysis was performed on the top 15 microorganisms by abundance. The higher the mean-decrease-accuracy and mean-decrease-Gini index, the greater the importance of the variable.

[0101] Bacterial community random forest analysis diagram as shown Figure 10As shown, genera such as *Acinetobacter*, *Panthera*, *Methylobacterium-methylerythrobium*, and *Aureomyces* exhibit high importance in distinguishing samples with different flue gas volumes. From an ecological niche perspective, *Acinetobacter* is widely distributed across various environments and exhibits strong adaptability to environmental changes. In sun-dried tobacco environments, changes in flue gas volume alter microenvironmental factors such as temperature, humidity, and oxygen content. Higher flue gas volumes result in lower oxygen content and higher humidity; under these conditions, the relative abundance of *Acinetobacter* changes significantly, indicating its sensitivity to such microenvironmental changes, and suggesting that its metabolic activity may be altered by flue gas volume.

[0102] Fungal community random forest analysis diagram as shown Figure 11 As shown, *Alternaria*, *Coccidioidomyces*, and *Sampayo* species exhibit significant importance in barrel fermentation.

[0103] 3.4 Correlation analysis between microorganisms and chemical composition and sensory quality

[0104] The CCA-RDA tool was used to analyze genus-level microorganisms and related data to present the correlation between microorganisms and other indicators in tobacco leaves after barrel fermentation. The results are as follows: Figure 12 and 13 As shown in the figure, blue rays represent microorganisms, and red rays represent chemical components or sensory quality. An acute angle between indicators indicates a positive correlation, while an obtuse angle indicates a negative correlation. The length of the ray indicates the degree of influence between the indicator and the microorganism. Among bacteria, *Pseudomonas*, *Pantotheca*, and *Methylobacterium* show significant correlations with other indicators. *Pantotheca* and *Pseudomonas* are positively correlated with potassium, chlorine, nicotine, and sensory quality (irritation, aftertaste, aroma quantity, and off-flavors); and negatively correlated with total sugar, smoothness, strength, and aroma quality. *Methylobacterium* shows the opposite correlation with chemical components and sensory quality. Among fungi, *Alternaria* and *Aspergillus* show significant correlations with other indicators. *Aspergillus* is positively correlated with total sugar, chlorine, and sensory quality (strength and smoothness), and negatively correlated with aroma quantity, aroma quality, aftertaste, and total nitrogen. *Alternaria*, in addition to a positive correlation with strength, is negatively correlated with aftertaste, off-flavors, total sugar, and several other indicators.

[0105] 4. Changes in metabolites

[0106] The following shows the changes in metabolites in samples obtained with 70% of the smoke volume and different fermentation times.

[0107] 4.1 Multivariate statistical analysis

[0108] Unsupervised, multi-factor principal component analysis (PCA) was used to analyze the metabolomics data of fermented tobacco leaves. The results are as follows: Figure 14As shown, the principal components of samples from different sampling times are significantly separated; the partial least squares regression model (PLS-DA) indicates that the scores of each sample from different sampling times are also significantly separated, such as... Figure 15 As shown, similar to the results of principal component analysis, the dispersion trend among samples within each sampling time group is not obvious and all samples are within the 95% confidence interval, indicating that there are significant differences in metabolites among tobacco leaves at different fermentation times under fermentation conditions of 70% tobacco volume.

[0109] 4.2 Isolation and Identification of Metabolites

[0110] A total of 5133 metabolites were detected in this study. All metabolites were screened and analyzed using thresholds of VIP>1, FC>2 or FC<0.5, P<0.05. The results showed that, compared with the control, tobacco leaves fermented for 4 days contained 524 differentially expressed metabolites, of which 330 were successfully annotated in the HMDB database, including 224 upregulated metabolites and 106 downregulated metabolites; tobacco leaves fermented for 6 days contained 835 differentially expressed metabolites, of which 494 were successfully annotated in the HMDB database, including 296 upregulated metabolites and 198 downregulated metabolites; tobacco leaves fermented for 8 days contained 954 differentially expressed metabolites, of which 607 were successfully annotated in the HMDB database, including 381 upregulated metabolites and 226 downregulated metabolites; and tobacco leaves fermented for 10 days contained 1255 differentially expressed metabolites, of which 756 were successfully annotated in the HMDB database, including 416 upregulated metabolites and 340 downregulated metabolites.

[0111] Figures 16-19 A heatmap analysis was conducted to identify significantly different metabolites in samples from different fermentation times (corresponding to days 4, 6, 8, and 10; S1, S2, and S3 represent three replicates of unfermented tobacco leaves (i.e., the control), and the corresponding 70% tobacco volume 1, 2, and 3 represent three replicates at the corresponding fermentation time). Red indicates that the relative content of metabolites is higher than the average, and blue indicates that the relative content of metabolites is lower than the average. The figure shows that most metabolites in samples from different fermentation times exhibit similar trends. This is similar to the results of principal component analysis, indicating significant differences in tobacco metabolites between samples from different fermentation times and unfermented sun-dried tobacco samples. After removing potentially non-plant-specific metabolites, using VIP < 1 and P < 0.0001 as the screening range, 4, 9, 27, and 44 significantly different metabolites were screened from samples fermented for 4, 6, 8, and 10 days, respectively. These mainly included lipids and lipid-like molecules, organic acids and their derivatives, organic heterocyclic compounds, phenylpropanoids, and polyketides.

[0112] 4.3 Metabolic pathway analysis

[0113] Annotating differentially expressed metabolites selected at different fermentation times into the KEGG database provides insights into the differences in metabolic pathways among tobacco samples fermented at different times under 70% capacity conditions. Samples fermented for 4 days successfully annotated 79 metabolites, including 60 upregulated and 19 downregulated metabolites; samples fermented for 6 days successfully annotated 108 metabolites, including 60 upregulated and 48 downregulated metabolites; samples fermented for 8 days successfully annotated 121 metabolites, including 74 upregulated and 47 downregulated metabolites; and samples fermented for 10 days successfully annotated 171 metabolites, including 93 upregulated and 78 downregulated metabolites.

[0114] Metabolic pathway enrichment analysis was performed on the differentially metabolites screened above using KEGG, and the results are as follows: Figures 20-23 As shown, P < 0.05 was defined as a significant enrichment pathway. Two significantly differentially expressed metabolic pathways were obtained in samples fermented for 4 days: flavonoid biosynthesis and glutathione metabolism. Two significantly differentially expressed metabolic pathways were obtained in samples fermented for 6 days: flavonoid biosynthesis and linoleic acid metabolism. Three significantly differentially expressed metabolic pathways were obtained in samples fermented for 8 days: phenylalanine, tyrosine, and tryptophan biosynthesis, tryptophan metabolism, and glycine, serine, and threonine metabolism. Three significant metabolic pathways were obtained in samples fermented for 10 days: phenylalanine / tyrosine and tryptophan biosynthesis, glycine / serine and threonine metabolism, and β-alanine metabolism.

[0115] The most significant pathways enriched in tobacco samples from different fermentation times were flavonoid biosynthesis, glycine / serine and threonine metabolism, and phenylalanine / tyrosine and tryptophan biosynthesis. Flavonoid biosynthesis begins with the phenylalanine metabolic pathway, and some of the substances produced during this process are major aroma precursors in tobacco. The synthesized flavonoids can be degraded into phenols or aromatic compounds during tobacco combustion. The glycine / serine and threonine metabolic pathways refer to the biotransformation processes of glycine, serine, and threonine during tobacco fermentation, providing the necessary energy and substances for microbial growth and successful fermentation. Phenylalanine, tyrosine, and tryptophan are collectively known as aromatic amino acids. They are not only the basic raw materials for the synthesis of proteins, enzymes, and other essential substances in tobacco, but also the core precursors for the synthesis of important secondary metabolites such as flavonoids, alkaloids (e.g., nicotine), and lignin. During tobacco fermentation, phenylalanine / tyrosine and tryptophan biosynthesis provide the foundation for other metabolic pathways, collectively producing compounds beneficial to tobacco aroma.

[0116] The metabolites enriched in the three most significantly enriched pathways are chlorogenic acid, galangin, L-tryptophan, 5-aminolevulinic acid, and L-phenylalanine, as shown in Table 2:

[0117] Table 2

[0118]

[0119] Among them, the relative contents of chlorogenic acid, galangin and ginseng were significantly higher in the samples fermented for 4 days than in the control; the relative contents of 5-aminolevulinic acid were significantly higher in the samples fermented for 8 days and 10 days than in the control.

[0120] 4.4 Combined analysis of microorganisms and metabolomics

[0121] Correlation heatmap analysis was performed on microorganisms and metabolites that underwent significant changes during fermentation to explore the correlation between microorganisms and metabolites during barrel fermentation when the tobacco volume was 70%.

[0122] Depend on Figure 24 It can be seen that among bacteria, *Ureaplasma* is significantly positively correlated with multiple metabolites such as sphingosine and galangin; *Aureomyces* and *Methylobacterium* are extremely significantly positively correlated with L-tryptophan and phthalic acid, with *Aureomyces* being significantly negatively correlated with α-linolenic acid, nicotine, citric acid, and L-phenylalanine; *Sphingosomalmonella* and *Acinetobacter* show similar association patterns with metabolites, both being positively correlated with L-tryptophan, L-phenylalanine, and citric acid, and negatively correlated with metabolites such as oleamide, palmitic acid, and galangin.

[0123] Depend on Figure 25 It can be seen that the association patterns between fungi of the genera *Symplocos*, *Syneomyces*, and *Sampayo* and metabolites are similar, all showing significant positive correlations with multiple metabolites such as galangin, palmitic acid, palmitoleic acid, and oleamide. Among them, *Sampayo* shows extremely significant negative correlations with L-tryptophan, L-phenylalanine, and citric acid. The association patterns between *Aspergillus* and *Pseudomonas* are similar, and the associations between these two fungi and metabolites are exactly the opposite of those of the aforementioned fungi.

[0124] Based on the above indicators, 70% of the tobacco volume is filled and fermentation takes about 10 days, which are the optimal conditions for the initial fermentation of Jilin sun-cured tobacco leaves in wooden barrels. As fermentation conditions or time change, the microbial community in the tobacco leaves will also undergo corresponding changes.

[0125] Microbiological tests were conducted on samples with different fermentation times and different tobacco volumes in wooden barrels. The results showed that the dominant bacterial genera in the tobacco leaves were Acinetobacter, Methylobacterium-Methylerythrobacter, Sphingomonas, and Aureomyces; the dominant fungal genera were mainly Sampylomycin and Alternaria.

[0126] As fermentation time increased, the abundance of Acinetobacter and Methylbacterium-Methylerythrobacter, the dominant bacterial genera, showed a trend of increasing-decreasing-increasing, consistent with the trend of total sugar changes. Studies have shown that these two bacteria also make important contributions to the synthesis of aroma substances.

[0127] Among the dominant fungal genera, the abundance of *Sampayo* gradually increased with prolonged fermentation time, consistent with the trend of nicotine changes. In contrast, the abundance of *Alternaria* first increased and then gradually decreased, reaching a lower level than the control at 10 days of fermentation.

[0128] It is worth noting that *Filobasidium*, along with *Aspergillus* and *Staphylococcus*, directly or indirectly affects the production of total sugars and reducing sugars, providing metabolic precursors for other microorganisms. This plays a crucial role in the microbial community composition during tobacco fermentation. While its abundance was extremely low in the control group, it gradually increased as fermentation progressed. This pattern of change only occurred in the treatment with 70% tobacco volume, fully validating the sensory result that barrel fermentation with 70% tobacco volume had the best effect on improving Jilin sun-cured tobacco.

[0129] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A fermentation method for sun-dried tobacco leaves, characterized in that, The process involves initial curing, rehydration and softening, first leaf moistening, second leaf moistening, separation of leaf stems, re-curing, cooling, and second rehydration of the tobacco leaves. The resulting tobacco leaves are then spread out in a fermentation tank for fermentation. The density of the tobacco flakes in the fermentation tank is 0.34 g / cm³. 3 ~0.45g / cm 3 ; The fermentation time is 6 to 10 days.

2. The fermentation method according to claim 1, characterized in that, The fermentation temperature is 40°C.

3. The fermentation method according to claim 1, characterized in that, The relative humidity during fermentation is 75%~85%.

4. The fermentation method according to claim 1, characterized in that, The moisture content of the tobacco leaves after re-drying and before fermentation is 18%~22%.

5. The fermentation method according to claim 1, characterized in that, The fermentation process involves turning the pile over every two days.

6. The fermentation method according to claim 1, characterized in that, The initial baking includes the following steps: The first stage of baking of the sun-dried tobacco was carried out under the conditions of dry bulb temperature of 23℃~30℃ and wet bulb temperature of 23℃~30℃. After adjusting the dry bulb temperature to 32℃~34℃, proceed with the second stage of baking until the leaves are completely yellow. The third stage of baking was carried out by adjusting the dry bulb temperature to 38℃~40℃ and the wet bulb temperature to 36℃~38℃ until brown spots appeared on the leaves. The wet-bulb temperature was adjusted to 34℃~36℃ for the fourth stage of baking until the browning area of ​​the leaves reached 30%~70%. The fifth stage of baking was carried out by adjusting the dry bulb temperature to 45℃~47℃ and the wet bulb temperature to 31℃~33℃ until the leaves were dry. The sixth stage of baking is carried out by adjusting the dry bulb temperature to 60℃~68℃ and the wet bulb temperature to 29℃~35℃ until the leaf veins are completely dry.

7. The fermentation method according to claim 1, characterized in that, The conditions for harvesting sun-dried tobacco leaves are as follows: the main veins turn white in 1 / 3 to 2 / 3 of their length, the secondary veins turn white in 1 / 3 of their length, the leaf tips and edges turn yellow, and the leaf surface is 50-60% yellow.

8. The fermentation method according to claim 1, characterized in that, The conditions for the first leaf moistening and / or the second leaf moistening are: temperature 65℃~85℃; The moisture content after the second leaf rinsing is 18%~20%; The conditions for re-baking are: 60℃~130℃; The moisture content after the second rehydration is 11%~13%.

9. The fermentation method according to claim 1, characterized in that, The fermentation time is 8 to 10 days.

10. The fermentation method according to any one of claims 1 to 9, characterized in that, The sun-dried tobacco leaves mentioned are Piaohe No. 2.

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