Method for curing tobacco leaves for mildew prevention using high-voltage electric field

Abstract

A method for curing tobacco leaves for mildew prevention, including: disposing a high-voltage electric field generator in a conventional curing barn including a tobacco loading chamber for accommodating tobacco leaves to generate a high-voltage electric field above the tobacco loading chamber; presetting a curing process of the curing barn into four stages: a pre-yellowing stage, a yellowing stage, a color setting stage, and a stem-drying stage; selecting healthy tobacco leaves free from diseases and pests; placing the healthy tobacco leaves in the curing barn; during the curing process, when the yellowing stage is completed, turning on the high-voltage electric field generator; and when the curing process is completed, turning off the high-voltage electric field generator; after the curing process is completed, collecting the tobacco leaves from the curing barn; and adjusting the curing barn to room temperature, and cleaning the curing barn to remove tobacco debris.

Description

BACKGROUND

[0001] The disclosure relates to the field of tobacco curing, and more particularly, to a method for curing tobacco leaves for mildew prevention using a high-voltage electric field.

[0002] Mature, harvested tobacco leaves are covered with a variety of microorganisms. During the curing process, which involves high heat and humidity, the microorganisms thrive and multiply rapidly. Research has shown that some of the microorganisms impact the levels of harmful substances in the tobacco leaves, and in more severe cases, the microorganisms lead to the leaves rotting. Conventional curing equipment inadvertently create ideal conditions for microorganisms to flourish. As a result, mold grows on the leaves, and excessive dry matter is consumed during the curing process, which ultimately reduces the quality of the tobacco. The compromised quality of the final product affects the tobacco's usability in industrial applications and causes financial losses for tobacco farmers. Currently, there are several methods employed to control microbial growth during the tobacco curing process. Chemical methods use antimicrobial chemical agents to inhibit mold growth during curing. For example, a 0.5% sodium dehydroacetate solution and a 0.2% natamycin solution have been identified as effective treatments against mold. Physical methods focus on optimizing the curing process. The physical methods involve techniques like oscillating internal circulation, oscillating movement, and staged unidirectional moisture release to control mold. The physical adjustments help manage the microbial growth to some extent. However, the use of antimicrobial chemical agents carries the risk of pesticide residues on the tobacco leaves, which raises safety concerns and reduces the tobacco's suitability for industrial use after curing. The physical methods do not fundamentally change the nature of the curing environment, which still relies on conventional hot air circulation. While the physical methods improve curing quality, the need for process adjustments and re-training can increase labor costs and complicate large-scale adoption, especially in the short term.

[0003] Recent studies have demonstrated that high-voltage electric fields are effective in inactivating pathogenic bacteria and spoilage microorganisms. Importantly, the inactivation occurs without compromising the original quality of the product, making it suitable for use in food and agricultural processes. The use of high-voltage electric fields is characterized by its high efficiency, low energy consumption, and environmentally friendly nature, which makes it an attractive solution for practical, large-scale applications.SUMMARY

[0004] To solve the aforesaid problems, the disclosure provides a method for curing tobacco leaves for mildew prevention using a high-voltage electric field.

[0005] The method comprises:

[0006] S1: disposing a curing barn within a curing barn to generate a high-voltage electric field above the tobacco leaves; where, the curing barn comprises a tobacco loading chamber; the high-voltage electric field generator comprises a grounding electrode plate and a pin electrode plate; and the pin electrode plate is connected to a high-voltage power supply and a control unit;

[0007] S2: presetting a curing process of the curing barn into four stages: pre-yellowing stage, yellowing stage, color setting stage, and stem-drying stage; and pre-setting curing conditions for the four stages as follows:

[0008] pre-yellowing stage: within 3-5 hours, uniformly raising a dry-bulb temperature from room temperature to 30° C.; maintaining 30° C. for 8-9 hours; then, uniformly raising the dry-bulb temperature to 35° C. within 3-5 hours; maintaining 35° C. for 7-8 hours; and finally, uniformly raising dry-bulb temperature to 38° C. within 3-5 hours;

[0009] yellowing stage: holding the dry-bulb temperature constant at 38° C.;

[0010] color setting stage: within 7-8 hours, uniformly raising the dry-bulb temperature from 35° C. to 40° C.; and

[0011] stem-drying stage: within 7-8 hours, uniformly increasing the dry-bulb temperature from 40° C. to 42° C.; maintaining at 42° C. for 20-23 hours; raising the dry-bulb temperature to 45° C. and holding for 2-3 hours; raising the dry-bulb temperature to 48° C. and holding for 9-10 hours; raising the dry-bulb temperature to 54° C. and holding for 4-5 hours; raising the dry-bulb temperature to 62° C. and holding for 4-5 hours; and finally raising the dry-bulb temperature to 67° C. and holding for 10-11 hours;

[0012] S3: selecting healthy tobacco leaves free from diseases and pests; placing the healthy tobacco leaves in the curing barn; during the curing process, when the yellowing stage is completed, turning on the high-voltage electric field generator; and when the curing process is completed, turning off the high-voltage electric field generator;

[0013] S4: after the curing process is completed, collecting the tobacco leaves from the curing barn; where, the cured tobacco leaves have a moisture content of 3%-5%; and

[0014] S5: adjusting the curing barn to room temperature, and cleaning the curing barn to remove tobacco debris.

[0015] In a class of this embodiment, a distance between the pin electrode plate and the grounding electrode plate is 100-120 cm. The pin electrode plate comprises a bottom surface provided with a plurality of pin electrodes. The plurality of pin electrodes are evenly spaced and oriented in a downward direction; and a distance between every two adjacent pin electrodes is 20 cm.

[0016] In a class of this embodiment, air flows from the bottom to the top within the curing barn.

[0017] In a class of this embodiment, ionized air ions generated by the high-voltage electric field act on tobacco stems of the tobacco leaves; and the high-voltage electric field enhances the diversity of fungal communities in tobacco leaves by reducing the competitive advantage of Aspergillus.

[0018] In a class of this embodiment, a wet-bulb temperature is adjusted according to local curing practices and baking conditions of the tobacco leaves being cured. The wet-bulb temperature is lower than the dry-bulb temperature. The wet-bulb temperature changes accordingly when the dry-bulb temperature changes.

[0019] In a class of this embodiment, the high-voltage power supply is selected from the group consisting of power-frequency alternating current (AC) power supply, high-frequency AC power supply, and direct current (DC) power supply, and operates within a voltage range of 30-50 kV.

[0020] In a class of this embodiment, both the control unit and the high-voltage power supply are grounded and disposed outside the curing barn.

[0021] In a class of this embodiment, during the curing process, a potential difference between the pin electrode plate and the grounding electrode plate is equal to a pin electrode voltage.

[0022] In a class of this embodiment, before starting the curing process, the high-voltage power supply is debugged to confirm a normal working condition thereof.

[0023] The following advantages are associated with the method for curing tobacco leaves for mildew prevention using a high-voltage electric field of the disclosure.

[0024] 1. The high-voltage electric field is specifically applied to the stem, which helps prevent the spread of mold downward along the tobacco leaves. Therefore, the method inhibits the growth and reproduction of Aspergillus species during the color setting stage and the stem-drying stage. The inhibition results in a more balanced and diverse microbial environment, as other fungi can compete more effectively, leading to a greater overall diversity within the fungal community.

[0025] 2. The method involves installing a high-voltage electric field generator within the conventional curing barn. The modification allows the conventional curing barn to continue operating without requiring the construction of a new production line or retraining workers. The method reduces both capital costs and training expenses. Additionally, the method can be widely implemented in a short period of time, suppressing mold growth during the curing process. The suppression ensures that the tobacco leaves remain safe and free from mold contamination, which could otherwise degrade the quality of the tobacco. As a result, the method increases the usability of the cured tobacco within the tobacco industry.BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIG. 1 is a schematic diagram of a curing barn according to one example of the disclosure;

[0027] FIG. 2 is a schematic diagram showing the arrangement of a plurality of pin electrodes according to one example of the disclosure;

[0028] FIG. 3 is a schematic diagram of curing curves according to one example of the disclosure;

[0029] FIG. 4 is a schematic diagram of rarefaction curves during a curing process in one example versus a comparative example of the disclosure;

[0030] FIG. 5 is a schematic diagram of α-diversity indices of fungi during a curing process in one example versus a comparative example of the disclosure;

[0031] FIG. 6 is a diagram showing the composition of the fungal community across different taxonomic levels during a curing process in one example versus a comparative example of the disclosure;

[0032] FIG. 7 is a Venn diagram illustrating the fungal community during a curing process in one example versus a comparative example of the disclosure;

[0033] FIGS. 8A-8B show schematic diagrams of an average relative abundance of fungal communities at the phylum level during a curing process in two curing barns of the disclosure; and

[0034] FIGS. 9A-9B are schematic diagrams of an average relative abundance of fungal communities at the genus level during a curing process in two curing barns of the disclosure.

[0035] In the drawings, the following reference numbers are used: 1. Airflow direction; 2. Pin electrode plate; 3. High-voltage power supply; 4. Control unit; 5. Air inlet; 6. Circulating fan; 7. Corona zone; 8. Carbon crystal heating plate; and 9. Grounding electrode plate.DETAILED DESCRIPTION

[0036] To further illustrate the disclosure, embodiments detailing the method for curing tobacco leaves for mildew prevention using the high-voltage electric field are described below. It should be noted that the following embodiments are intended to describe and not to limit the disclosure.

[0037] A method for curing tobacco leaves for mildew prevention using a high-voltage electric field, and the method comprises:

[0038] S1: disposing a high-voltage electric field generator in a conventional curing barn comprising a tobacco loading chamber to generate a high-voltage electric field above the tobacco loading chamber; where, the high-voltage electric field generator comprises a grounding electrode plate and a pin electrode plate; and the pin electrode plate is connected to a high-voltage power supply and a control unit; and airflow within the curing barn is directed from the bottom to the top;

[0039] S2: presetting a curing process of the curing barn into four stages: pre-yellowing stage, yellowing stage, color setting stage, and stem-drying stage; and pre-setting curing conditions for the four stages as follows:

[0040] pre-yellowing stage: within 3-5 hours, uniformly raising a dry-bulb temperature from room temperature to 30° C.; maintaining 30° C. for 8-9 hours; then, uniformly raising the dry-bulb temperature to 35° C. within 3-5 hours; maintaining 35° C. for 7-8 hours; and finally, uniformly raising dry-bulb temperature to 38° C. within 3-5 hours;

[0041] yellowing stage: holding the dry-bulb temperature constant at 38° C.;

[0042] color setting stage: within 7-8 hours, uniformly raising the dry-bulb temperature from 35° C. to 40° C.; and

[0043] stem-drying stage: within 7-8 hours, uniformly increasing the dry-bulb temperature from 40° C. to 42° C.; maintaining at 42° C. for 20-23 hours; raising the dry-bulb temperature to 45° C. and holding for 2-3 hours; raising the dry-bulb temperature to 48° C. and holding for 9-10 hours; raising the dry-bulb temperature to 54° C. and holding for 4-5 hours; raising the dry-bulb temperature to 62° C. and holding for 4-5 hours; and finally raising the dry-bulb temperature to 67° C. and holding for 10-11 hours;

[0044] S3: selecting healthy tobacco leaves free from diseases and pests; placing the healthy tobacco leaves in the curing barn; during the curing process, when the yellowing stage is completed, turning on the high-voltage electric field generator; and turning off the high-voltage electric field generator when the curing process is completed;

[0045] S4: after the curing process is completed, collecting the tobacco leaves from the curing barn; where, the cured tobacco leaves have a moisture content of 3%-5%; and

[0046] S5: adjusting the curing barn to room temperature, and cleaning the curing barn to remove tobacco debris.

[0047] In S1, the high-voltage electric field generator comprises a grounding electrode plate 9 and a pin electrode plate 2. The pin electrode plate is connected to a high-voltage power supply 3 and a control unit 4. The pin electrode plate comprises a bottom surface provided with a plurality of pin electrodes. The plurality of pin electrodes are evenly spaced and oriented in a downward direction. A distance between every two adjacent pin electrodes is 20 cm. The high-voltage power supply 3 is selected from the group consisting of power-frequency alternating current (AC) power supply, high-frequency AC power supply, and direct current (DC) power supply, and operates within a voltage range of 30-50 kV. Both the control unit 4 and the high-voltage power supply 3 are grounded and disposed outside the curing barn. To prevent physical contact between the plurality of pin electrodes and the tobacco leaves, the distance between the pin electrode plate 2 and the grounding electrode plate 9 is 100-120 cm, and a potential difference between the pin electrode plate and the grounding electrode plate is equal to the pin electrode voltage.

[0048] The positioning of the pin electrodes is configured such that the ionized air ions generated by the high-voltage electric field act on tobacco stems of the tobacco leaves. Fewer types of fungi inhabit the stem compared to the tobacco leaf. However, among the fungi present, Aspergillus species are particularly relevant, as the presence of Aspergillus species correlates with the mold level of the tobacco. Large-area injuries caused by harvesting the tobacco are typically more concentrated in the stem. The large-area injuries create an environment that is richer in moisture and nutrients compared to other parts of the leaf. The environment is ideal for fungal growth, providing the necessary resources for fungi to thrive. Therefore, applying the high-voltage electric field to the stem prevents mold from spreading downward along the tobacco leaves. By targeting the stem, the high-voltage electric field fosters beneficial competition within the fungal community, which helps lead to a more robust and diverse fungal population.

[0049] In S2, a wet-bulb temperature is adjusted according to local curing practices and conditions of the tobacco leaves being cured. The wet-bulb temperature is lower than the dry-bulb temperature. The wet-bulb temperature changes accordingly when the dry-bulb temperature changes.Example

[0050] In a comparative example, the curing barn A refers to a conventional electric curing oven (model HNND-45) that operates with upward airflow. The curing barn A comprises a heating chamber, six carbon crystal heating panels, a tobacco loading chamber, a controller, and a frequency-variable fan. The heating chamber has internal dimensions of 0.50 m×1.35 m×1.40 m. The six carbon crystal heating panels are used as a heat source. The total heating power of the six carbon crystal heating panels is 2 kW, with a thermal efficiency of 95%. The tobacco loading chamber has internal dimensions of 1.40 m×1.35 m×1.40 m, capable of holding 12-14 racks of tobacco, 150-200 kg of fresh tobacco leaves, or 1300-1500 individual leaves. The controller is configured to automatically adjust the dry-bulb temperature and the wet-bulb temperature according to the preset curing curves (as shown in FIG. 3). In an example, a high-voltage electric field generator is disposed in the curing barn A to form a modified version of the curing barn, referred to as curing barn B. To prevent physical contact between the plurality of pin electrodes and the tobacco leaves, a distance between the pin electrode plate 2 and the grounding electrode plate 9 is 110 cm. As shown in FIGS. 1, 1. Airflow direction; 2. Pin electrode plate; 3. High-voltage power supply; 4. Control unit; 5. Air inlet; 6. Circulating fan; 7. Corona zone; 8. Carbon crystal heating plate; and 9. Grounding electrode plate.

[0051] The tobacco variety used in the experiment was Yunnan Tobacco No. 87. Yunnan Tobacco No. 87 was cultivated on flat terrain with medium soil fertility, following practices in high-quality tobacco leaf cultivation techniques. The experiment used middle tobacco leaves that were uniformly ripened and showed normal yellowing.

[0052] In the experiment, middle tobacco leaves with high-quality and no diseases were selected. The selected middle tobacco leaves were uniformly tied to curing rods and placed into the two curing barns. The preset curing curves (as shown in FIG. 3) guided the curing process. During the curing process, samples were collected at the following stages:

[0053] T1: pre-yellowing stage (sampling times: 40 hours for the curing barn A, and 41 hours for the curing barn B);

[0054] T2: yellowing stage (sampling times: 61 hours for the curing barn A, and 63 hours for the curing barn B);

[0055] T3: yellowing of the main veins during the color-setting phase (sampling times: 77 hours for both the curing barn A and the curing barn B); and

[0056] T4: aromatic substance synthesis period (sampling times: 105 hours for the curing barn A, and 96 hours for the curing barn B).

[0057] At each stage, 50 g of samples were collected from the two curing barns. Each sample was repeated three times. After collecting, the samples were immediately frozen in liquid nitrogen. In the curing barn B, the high-voltage electric field generator was activated when the yellowing stage is completed, and the high-voltage electric field generator remained active throughout the curing process until completion. The high-voltage power supply operated at 45 kV throughout the curing. A total of 24 samples were collected.

[0058] Genomic DNA was extracted using the E.Z.N.A.® Soil Kit (Omega Bio-tek, USA). PCR amplification was performed with corresponding primers. PCR products from the same sample were pooled and analyzed using 2% agarose gel electrophoresis to verify the length and purity of the amplified sequences. Qualified PCR products were recovered using the AxyPrep DNA Gel Extraction Kit (Axygen, USA). The purified PCR products were sent to Shanghai Majorbio Bio-pharm Technology Co., Ltd. for Illumina MiSeq high-throughput sequencing.

[0059] The bioinformatics analysis workflow comprises the following steps: the raw sequencing data was subjected to quality control using Fastp software; the qualified sequences were assembled using Flash software; The assembled sequences were filtered using Usearch software to remove chimera sequences, resulting in a dataset of high-quality, valid sequences; using Uparse software, the filtered sequences were clustered into Operational Taxonomic Units (OTUs) at 97% similarity; the OTUs was annotated and identified using the Unite 8.0 fungal taxonomy database; α-diversity indices were calculated using Mothur software (Version 1.30.2); various visualizations were plotted using R Studio (Version 2.2.1), including Rarefaction curves, Venn diagrams, and intergroup difference heatmaps; abundance graphs at different taxonomic levels were generated using Qiime software; LEfSe (Linear Discriminant Analysis Effect Size) software was used to identify biomarkers and genomic features with significant differences between groups; in the LEfSe analysis, a rank-sum test is used to identify taxa with significant abundance differences, and then, Linear Discriminant Analysis (LDA) is used to quantify the effect size of each of these taxa; the fungal functional groups were analyzed using FUNGuild 1.0, categorizing fungi based on nutritional modes and resource utilization strategies. All of the bioinformatics analyses were conducted using the Majorbio Cloud Platform provided by Shanghai Majorbio Bio-Pharm Technology Co., Ltd. (Platform URL: https: / / cloud.majorbio.com / page / tools / ).

[0060] After processing the raw sequencing data, a total of 1,898,756 high-quality sequence fragments were obtained from 24 samples, covering 441,282,333 bases. The sequence count for each individual sample ranged from 54,680 to 105,674, with an average sequence length of 232 base pairs (bp). For the samples from the curing barn A: a total of 949,242 high-quality sequence fragments were obtained, covering 217,090,008 bases. The sequence count per sample ranged from 69,403 to 105,674, with an average length of 229 bp. For the samples from the curing barn B: a total of 949,514 high-quality sequence fragments were obtained, covering 224,192,325 bases. The sequence count per sample ranged from 54,680 to 88,683, with an average length of 237 bp.

[0061] The Sobs index represents the number of observed species within the samples. The OTUs were clustered at 97% sequence similarity. Random subsampling was performed to calculate the Sobs index. The Sobs index was plotted against the number of sequences. In the plot, the x-axis represents the sequence count, and the y-axis represents the Sobs index. As shown in FIG. 4, the rarefaction curves illustrates how the Sobs index changes with sequencing depth. All curves flattened out at a sequencing depth of 5,000 sequences, indicating that the species diversity in the samples had been fully captured at that point. Beyond the sequencing depth, additional sequencing would not identify more OTUs. Therefore, the sequencing depth was considered sufficient for analyzing the microbial community diversity. The results are reliable for accurately reflecting the structure of the microbial communities present in the samples.

[0062] The bacterial community structure of the 24 samples collected from the two curing barns was analyzed at four distinct time points using MiSeq sequencing technology. The time points included two early curing stages (T1 and T2) and two later curing stages (T3 and T4). At each time point, three replicates were taken. The microbial diversity indices for the two curing barns were calculated for each time point (as shown in FIG. 5). The sequencing results demonstrated a coverage rate of 0.999 across all samples, indicating that the data sufficiently captured the fungal diversity within the samples. In the curing barn A, the fungal abundance indices exhibited an initial increase followed by a decrease as the curing process advanced. In contrast, the fungal abundance indices in the curing barn B at time points T1 and T2 closely mirrored those observed in the curing barn A. However, at time points T3 and T4, the fungal abundance indices in the curing barn B did not exhibit a significant decline, unlike those in the curing barn A. The findings indicate a divergence in the fungal community structure between the curing barn A and the curing barn B at time points T3 and T4 of the curing process. Further analysis revealed that the high-voltage electric field applied in the curing barn B increased the fungal community richness. Trends observed in the Shannon index, which measures the fungal diversity, corresponds with the fungal abundance indices, suggesting that the high-voltage electric field improved the fungal diversity in the curing barn B. Additionally, at time points T3 and T4, the Simpson index values for the curing barn A were lower than those for the curing barn B, indicating a greater concentration of dominant fungi and reduced diversity in the curing barn A. The observation was consistent with the results obtained from the Shannon index.

[0063] OTU clustering at 97% similarity identified a total of 653 fungi species distributed across 7 phyla, 25 classes, 79 orders, 213 families, 433 genera from the 24 analyzed samples. The composition of the fungal community across different taxonomic levels during the curing process is illustrated in FIG. 6.

[0064] The Venn diagrams were constructed to visualize the overlap of fungal species between the curing barn A and the curing barn B at different time points during the curing process. The OTU count in the two curing barns initially increased and then decreased as the curing process progressed. At time points T1 and T2 (prior to the application of high-voltage electric field), the OTU counts and the number of shared species between the two curing barns were comparable, with a significant overlap in the composition of the fungal community. After the introduction of the high-voltage electric field in the curing barn B (at time points T3 and T4), the overlap of OTUs between the two curing barns decreased, while number of unique OTUs in the curing barn B increased. Both the unique OTU counts and total OTU counts in the curing barn B were higher than those observed in the curing barn A at time points T3 and T4. The results demonstrate that while the fungal community structures in the two curing barns were similar at time points T1 and T2, the high-voltage electric field in the curing barn B contributed to the development of a more complex and distinct fungal community structure at time points T3 and T4.

[0065] FIGS. 8A-8B show the average relative abundance of the fungal communities at the phylum level during the curing process in the two curing barns. The dominant fungal groups, each contributing more than 1% to the fungal community, were Ascomycota, Basidiomycota, and a small portion of unclassified fungal species (unclassified_k_Fungi). All other fungal groups were classified as non-dominant, collectively contributing less than 1% to the overall fungal population. In the two curing barns, Ascomycota was the most dominant phylum, followed by Basidiomycota. At time points T1 and T2, the average relative abundance of Ascomycota in the curing barn A was 50.98% and 55.39%, respectively, while Basidiomycota accounted for 34.33% and 37.99%, respectively. In the curing barn B, the average relative abundance of Ascomycota was 46.43% and 53.12%, while Basidiomycota accounted for 29.47% and 39.23%, respectively. The results showed that prior to the application of the high-voltage electric field in the curing barn B, the fungal community structure at the phylum level was similar in the two curing barns. However, at time points T3 and T4, significant changes in the fungal composition were observed. In the curing barn A, Ascomycota became the overwhelmingly dominant phylum, with the average relative abundances of 96.15% and 92.15%, respectively, whereas the average relative abundance of Basidiomycota initially decreased significantly before showing a slight increase. In contrast, the curing barn B exhibited a reciprocal relationship between Ascomycota and Basidiomycota, with no significant changes were observed in the overall abundance of either phylum. Additionally, the average relative abundance of unclassified_k_Fungi decreased in the two curing barns as the curing process progressed. In conclusion, the fungal community structure at the phylum level was largely similar between the two curing barns at time points T1 and T2. However, at time points T3 and T4, Ascomycota emerged as the dominant phylum in the curing barn A, while the distribution of fungal phyla in the curing barn B was more variable, with Ascomycota, Basidiomycota showing a reciprocal relationship. The findings suggest that the application of the high-voltage electric field in the curing barn B influenced the growth and reproductive dynamics of the dominant fungal phyla during the curing process.

[0066] FIGS. 9A-9B show the average relative abundance of fungal communities at the genus level during the curing process in the two curing barns. At time points T1 and T2, before the high-voltage electric field is applied in the curing barn B, the dominant fungal genera (with the average relative abundance greater than 0.1%) in the two curing barns were Aspergillus, Alternaria, Sampaiozyma, and Plectosphaerella. In the curing barn A, the average relative abundance of Aspergillus significantly increased at time points T3 and T4, with the average relative abundance of 72.41% and 71.57% at time points T3 and T4, respectively. The increase made Aspergillus the absolute dominant genus, while the average relative abundance of other genera decreased to varying extents. In the curing barn B, the average relative abundance of Aspergillus decreased at time points T3 and T4, with the average relative abundance of 34.46% and 21.17%, respectively, compared to the time points T1 and T2. Meanwhile, the average relative abundance of Alternaria and Sampaiozyma increased. At time point T3, the average relative abundance of non-dominant genera (those with average relative abundance less than 0.1%) saw a noticeable rise. The result suggests that the high-voltage electric field applied in the curing barn B influenced the fungal community structure by reducing the dominance of Aspergillus and promoting greater fungal diversity at time points T3 and T4. In contrast, in the curing barn A, the absence of the high-voltage electric field allowed Aspergillus to dominate the fungal community at time points T3 and T4, while other genera declined.

[0067] The high-voltage electric field significantly increased the fungal community diversity and fungal abundance indices at different taxonomic levels in the curing barn B compared to conventional curing process in the curing barn A. The high-voltage electric field reduced the growth and reproduction of Aspergillus at time points T3 and T4, weakening the competitive advantage of Aspergillus within the fungal community. Aspergillus is known for secreting enzymes, degrading tissue components, and releasing metabolic byproducts such as CO2, mycotoxins, and antimicrobial agents, all of which can contribute to mold formation and may suppress the growth of other organisms in the environment. As a result, saprotrophic fungi increase in abundance. The high-voltage electric field curbed the spread of mold and encouraged beneficial competition within the fungal community, improving overall diversity and stability of the fungal community in the curing barn B.

[0068] It will be obvious to those skilled in the art that changes and modifications may be made, and therefore, the aim in the appended claims is to cover all such changes and modifications.

Examples

example

[0050]In a comparative example, the curing barn A refers to a conventional electric curing oven (model HNND-45) that operates with upward airflow. The curing barn A comprises a heating chamber, six carbon crystal heating panels, a tobacco loading chamber, a controller, and a frequency-variable fan. The heating chamber has internal dimensions of 0.50 m×1.35 m×1.40 m. The six carbon crystal heating panels are used as a heat source. The total heating power of the six carbon crystal heating panels is 2 kW, with a thermal efficiency of 95%. The tobacco loading chamber has internal dimensions of 1.40 m×1.35 m×1.40 m, capable of holding 12-14 racks of tobacco, 150-200 kg of fresh tobacco leaves, or 1300-1500 individual leaves. The controller is configured to automatically adjust the dry-bulb temperature and the wet-bulb temperature according to the preset curing curves (as shown in FIG. 3). In an example, a high-voltage electric field generator is disposed in the curing barn A to form a mo...

BACKGROUND

[0001] The disclosure relates to the field of tobacco curing, and more particularly, to a method for curing tobacco leaves for mildew prevention using a high-voltage electric field.

[0002] Mature, harvested tobacco leaves are covered with a variety of microorganisms. During the curing process, which involves high heat and humidity, the microorganisms thrive and multiply rapidly. Research has shown that some of the microorganisms impact the levels of harmful substances in the tobacco leaves, and in more severe cases, the microorganisms lead to the leaves rotting. Conventional curing equipment inadvertently create ideal conditions for microorganisms to flourish. As a result, mold grows on the leaves, and excessive dry matter is consumed during the curing process, which ultimately reduces the quality of the tobacco. The compromised quality of the final product affects the tobacco's usability in industrial applications and causes financial losses for tobacco farmers. Currently, there are several methods employed to control microbial growth during the tobacco curing process. Chemical methods use antimicrobial chemical agents to inhibit mold growth during curing. For example, a 0.5% sodium dehydroacetate solution and a 0.2% natamycin solution have been identified as effective treatments against mold. Physical methods focus on optimizing the curing process. The physical methods involve techniques like oscillating internal circulation, oscillating movement, and staged unidirectional moisture release to control mold. The physical adjustments help manage the microbial growth to some extent. However, the use of antimicrobial chemical agents carries the risk of pesticide residues on the tobacco leaves, which raises safety concerns and reduces the tobacco's suitability for industrial use after curing. The physical methods do not fundamentally change the nature of the curing environment, which still relies on conventional hot air circulation. While the physical methods improve curing quality, the need for process adjustments and re-training can increase labor costs and complicate large-scale adoption, especially in the short term.

[0003] Recent studies have demonstrated that high-voltage electric fields are effective in inactivating pathogenic bacteria and spoilage microorganisms. Importantly, the inactivation occurs without compromising the original quality of the product, making it suitable for use in food and agricultural processes. The use of high-voltage electric fields is characterized by its high efficiency, low energy consumption, and environmentally friendly nature, which makes it an attractive solution for practical, large-scale applications.SUMMARY

[0004] To solve the aforesaid problems, the disclosure provides a method for curing tobacco leaves for mildew prevention using a high-voltage electric field.

[0005] The method comprises:

[0006] S1: disposing a curing barn within a curing barn to generate a high-voltage electric field above the tobacco leaves; where, the curing barn comprises a tobacco loading chamber; the high-voltage electric field generator comprises a grounding electrode plate and a pin electrode plate; and the pin electrode plate is connected to a high-voltage power supply and a control unit;

[0007] S2: presetting a curing process of the curing barn into four stages: pre-yellowing stage, yellowing stage, color setting stage, and stem-drying stage; and pre-setting curing conditions for the four stages as follows:

[0008] pre-yellowing stage: within 3-5 hours, uniformly raising a dry-bulb temperature from room temperature to 30° C.; maintaining 30° C. for 8-9 hours; then, uniformly raising the dry-bulb temperature to 35° C. within 3-5 hours; maintaining 35° C. for 7-8 hours; and finally, uniformly raising dry-bulb temperature to 38° C. within 3-5 hours;

[0009] yellowing stage: holding the dry-bulb temperature constant at 38° C.;

[0010] color setting stage: within 7-8 hours, uniformly raising the dry-bulb temperature from 35° C. to 40° C.; and

[0011] stem-drying stage: within 7-8 hours, uniformly increasing the dry-bulb temperature from 40° C. to 42° C.; maintaining at 42° C. for 20-23 hours; raising the dry-bulb temperature to 45° C. and holding for 2-3 hours; raising the dry-bulb temperature to 48° C. and holding for 9-10 hours; raising the dry-bulb temperature to 54° C. and holding for 4-5 hours; raising the dry-bulb temperature to 62° C. and holding for 4-5 hours; and finally raising the dry-bulb temperature to 67° C. and holding for 10-11 hours;

[0012] S3: selecting healthy tobacco leaves free from diseases and pests; placing the healthy tobacco leaves in the curing barn; during the curing process, when the yellowing stage is completed, turning on the high-voltage electric field generator; and when the curing process is completed, turning off the high-voltage electric field generator;

[0013] S4: after the curing process is completed, collecting the tobacco leaves from the curing barn; where, the cured tobacco leaves have a moisture content of 3%-5%; and

[0014] S5: adjusting the curing barn to room temperature, and cleaning the curing barn to remove tobacco debris.

[0015] In a class of this embodiment, a distance between the pin electrode plate and the grounding electrode plate is 100-120 cm. The pin electrode plate comprises a bottom surface provided with a plurality of pin electrodes. The plurality of pin electrodes are evenly spaced and oriented in a downward direction; and a distance between every two adjacent pin electrodes is 20 cm.

[0016] In a class of this embodiment, air flows from the bottom to the top within the curing barn.

[0017] In a class of this embodiment, ionized air ions generated by the high-voltage electric field act on tobacco stems of the tobacco leaves; and the high-voltage electric field enhances the diversity of fungal communities in tobacco leaves by reducing the competitive advantage of Aspergillus.

[0018] In a class of this embodiment, a wet-bulb temperature is adjusted according to local curing practices and baking conditions of the tobacco leaves being cured. The wet-bulb temperature is lower than the dry-bulb temperature. The wet-bulb temperature changes accordingly when the dry-bulb temperature changes.

[0019] In a class of this embodiment, the high-voltage power supply is selected from the group consisting of power-frequency alternating current (AC) power supply, high-frequency AC power supply, and direct current (DC) power supply, and operates within a voltage range of 30-50 kV.

[0020] In a class of this embodiment, both the control unit and the high-voltage power supply are grounded and disposed outside the curing barn.

[0021] In a class of this embodiment, during the curing process, a potential difference between the pin electrode plate and the grounding electrode plate is equal to a pin electrode voltage.

[0022] In a class of this embodiment, before starting the curing process, the high-voltage power supply is debugged to confirm a normal working condition thereof.

[0023] The following advantages are associated with the method for curing tobacco leaves for mildew prevention using a high-voltage electric field of the disclosure.

[0024] 1. The high-voltage electric field is specifically applied to the stem, which helps prevent the spread of mold downward along the tobacco leaves. Therefore, the method inhibits the growth and reproduction of Aspergillus species during the color setting stage and the stem-drying stage. The inhibition results in a more balanced and diverse microbial environment, as other fungi can compete more effectively, leading to a greater overall diversity within the fungal community.

[0025] 2. The method involves installing a high-voltage electric field generator within the conventional curing barn. The modification allows the conventional curing barn to continue operating without requiring the construction of a new production line or retraining workers. The method reduces both capital costs and training expenses. Additionally, the method can be widely implemented in a short period of time, suppressing mold growth during the curing process. The suppression ensures that the tobacco leaves remain safe and free from mold contamination, which could otherwise degrade the quality of the tobacco. As a result, the method increases the usability of the cured tobacco within the tobacco industry.BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIG. 1 is a schematic diagram of a curing barn according to one example of the disclosure;

[0027] FIG. 2 is a schematic diagram showing the arrangement of a plurality of pin electrodes according to one example of the disclosure;

[0028] FIG. 3 is a schematic diagram of curing curves according to one example of the disclosure;

[0029] FIG. 4 is a schematic diagram of rarefaction curves during a curing process in one example versus a comparative example of the disclosure;

[0030] FIG. 5 is a schematic diagram of α-diversity indices of fungi during a curing process in one example versus a comparative example of the disclosure;

[0031] FIG. 6 is a diagram showing the composition of the fungal community across different taxonomic levels during a curing process in one example versus a comparative example of the disclosure;

[0032] FIG. 7 is a Venn diagram illustrating the fungal community during a curing process in one example versus a comparative example of the disclosure;

[0033] FIGS. 8A-8B show schematic diagrams of an average relative abundance of fungal communities at the phylum level during a curing process in two curing barns of the disclosure; and

[0034] FIGS. 9A-9B are schematic diagrams of an average relative abundance of fungal communities at the genus level during a curing process in two curing barns of the disclosure.

[0035] In the drawings, the following reference numbers are used: 1. Airflow direction; 2. Pin electrode plate; 3. High-voltage power supply; 4. Control unit; 5. Air inlet; 6. Circulating fan; 7. Corona zone; 8. Carbon crystal heating plate; and 9. Grounding electrode plate.DETAILED DESCRIPTION

[0036] To further illustrate the disclosure, embodiments detailing the method for curing tobacco leaves for mildew prevention using the high-voltage electric field are described below. It should be noted that the following embodiments are intended to describe and not to limit the disclosure.

[0037] A method for curing tobacco leaves for mildew prevention using a high-voltage electric field, and the method comprises:

[0038] S1: disposing a high-voltage electric field generator in a conventional curing barn comprising a tobacco loading chamber to generate a high-voltage electric field above the tobacco loading chamber; where, the high-voltage electric field generator comprises a grounding electrode plate and a pin electrode plate; and the pin electrode plate is connected to a high-voltage power supply and a control unit; and airflow within the curing barn is directed from the bottom to the top;

[0039] S2: presetting a curing process of the curing barn into four stages: pre-yellowing stage, yellowing stage, color setting stage, and stem-drying stage; and pre-setting curing conditions for the four stages as follows:

[0040] pre-yellowing stage: within 3-5 hours, uniformly raising a dry-bulb temperature from room temperature to 30° C.; maintaining 30° C. for 8-9 hours; then, uniformly raising the dry-bulb temperature to 35° C. within 3-5 hours; maintaining 35° C. for 7-8 hours; and finally, uniformly raising dry-bulb temperature to 38° C. within 3-5 hours;

[0041] yellowing stage: holding the dry-bulb temperature constant at 38° C.;

[0042] color setting stage: within 7-8 hours, uniformly raising the dry-bulb temperature from 35° C. to 40° C.; and

[0043] stem-drying stage: within 7-8 hours, uniformly increasing the dry-bulb temperature from 40° C. to 42° C.; maintaining at 42° C. for 20-23 hours; raising the dry-bulb temperature to 45° C. and holding for 2-3 hours; raising the dry-bulb temperature to 48° C. and holding for 9-10 hours; raising the dry-bulb temperature to 54° C. and holding for 4-5 hours; raising the dry-bulb temperature to 62° C. and holding for 4-5 hours; and finally raising the dry-bulb temperature to 67° C. and holding for 10-11 hours;

[0044] S3: selecting healthy tobacco leaves free from diseases and pests; placing the healthy tobacco leaves in the curing barn; during the curing process, when the yellowing stage is completed, turning on the high-voltage electric field generator; and turning off the high-voltage electric field generator when the curing process is completed;

[0045] S4: after the curing process is completed, collecting the tobacco leaves from the curing barn; where, the cured tobacco leaves have a moisture content of 3%-5%; and

[0046] S5: adjusting the curing barn to room temperature, and cleaning the curing barn to remove tobacco debris.

[0047] In S1, the high-voltage electric field generator comprises a grounding electrode plate 9 and a pin electrode plate 2. The pin electrode plate is connected to a high-voltage power supply 3 and a control unit 4. The pin electrode plate comprises a bottom surface provided with a plurality of pin electrodes. The plurality of pin electrodes are evenly spaced and oriented in a downward direction. A distance between every two adjacent pin electrodes is 20 cm. The high-voltage power supply 3 is selected from the group consisting of power-frequency alternating current (AC) power supply, high-frequency AC power supply, and direct current (DC) power supply, and operates within a voltage range of 30-50 kV. Both the control unit 4 and the high-voltage power supply 3 are grounded and disposed outside the curing barn. To prevent physical contact between the plurality of pin electrodes and the tobacco leaves, the distance between the pin electrode plate 2 and the grounding electrode plate 9 is 100-120 cm, and a potential difference between the pin electrode plate and the grounding electrode plate is equal to the pin electrode voltage.

[0048] The positioning of the pin electrodes is configured such that the ionized air ions generated by the high-voltage electric field act on tobacco stems of the tobacco leaves. Fewer types of fungi inhabit the stem compared to the tobacco leaf. However, among the fungi present, Aspergillus species are particularly relevant, as the presence of Aspergillus species correlates with the mold level of the tobacco. Large-area injuries caused by harvesting the tobacco are typically more concentrated in the stem. The large-area injuries create an environment that is richer in moisture and nutrients compared to other parts of the leaf. The environment is ideal for fungal growth, providing the necessary resources for fungi to thrive. Therefore, applying the high-voltage electric field to the stem prevents mold from spreading downward along the tobacco leaves. By targeting the stem, the high-voltage electric field fosters beneficial competition within the fungal community, which helps lead to a more robust and diverse fungal population.

[0049] In S2, a wet-bulb temperature is adjusted according to local curing practices and conditions of the tobacco leaves being cured. The wet-bulb temperature is lower than the dry-bulb temperature. The wet-bulb temperature changes accordingly when the dry-bulb temperature changes.Example

[0050] In a comparative example, the curing barn A refers to a conventional electric curing oven (model HNND-45) that operates with upward airflow. The curing barn A comprises a heating chamber, six carbon crystal heating panels, a tobacco loading chamber, a controller, and a frequency-variable fan. The heating chamber has internal dimensions of 0.50 m×1.35 m×1.40 m. The six carbon crystal heating panels are used as a heat source. The total heating power of the six carbon crystal heating panels is 2 kW, with a thermal efficiency of 95%. The tobacco loading chamber has internal dimensions of 1.40 m×1.35 m×1.40 m, capable of holding 12-14 racks of tobacco, 150-200 kg of fresh tobacco leaves, or 1300-1500 individual leaves. The controller is configured to automatically adjust the dry-bulb temperature and the wet-bulb temperature according to the preset curing curves (as shown in FIG. 3). In an example, a high-voltage electric field generator is disposed in the curing barn A to form a modified version of the curing barn, referred to as curing barn B. To prevent physical contact between the plurality of pin electrodes and the tobacco leaves, a distance between the pin electrode plate 2 and the grounding electrode plate 9 is 110 cm. As shown in FIGS. 1, 1. Airflow direction; 2. Pin electrode plate; 3. High-voltage power supply; 4. Control unit; 5. Air inlet; 6. Circulating fan; 7. Corona zone; 8. Carbon crystal heating plate; and 9. Grounding electrode plate.

[0051] The tobacco variety used in the experiment was Yunnan Tobacco No. 87. Yunnan Tobacco No. 87 was cultivated on flat terrain with medium soil fertility, following practices in high-quality tobacco leaf cultivation techniques. The experiment used middle tobacco leaves that were uniformly ripened and showed normal yellowing.

[0052] In the experiment, middle tobacco leaves with high-quality and no diseases were selected. The selected middle tobacco leaves were uniformly tied to curing rods and placed into the two curing barns. The preset curing curves (as shown in FIG. 3) guided the curing process. During the curing process, samples were collected at the following stages:

[0053] T1: pre-yellowing stage (sampling times: 40 hours for the curing barn A, and 41 hours for the curing barn B);

[0054] T2: yellowing stage (sampling times: 61 hours for the curing barn A, and 63 hours for the curing barn B);

[0055] T3: yellowing of the main veins during the color-setting phase (sampling times: 77 hours for both the curing barn A and the curing barn B); and

[0056] T4: aromatic substance synthesis period (sampling times: 105 hours for the curing barn A, and 96 hours for the curing barn B).

[0057] At each stage, 50 g of samples were collected from the two curing barns. Each sample was repeated three times. After collecting, the samples were immediately frozen in liquid nitrogen. In the curing barn B, the high-voltage electric field generator was activated when the yellowing stage is completed, and the high-voltage electric field generator remained active throughout the curing process until completion. The high-voltage power supply operated at 45 kV throughout the curing. A total of 24 samples were collected.

[0058] Genomic DNA was extracted using the E.Z.N.A.® Soil Kit (Omega Bio-tek, USA). PCR amplification was performed with corresponding primers. PCR products from the same sample were pooled and analyzed using 2% agarose gel electrophoresis to verify the length and purity of the amplified sequences. Qualified PCR products were recovered using the AxyPrep DNA Gel Extraction Kit (Axygen, USA). The purified PCR products were sent to Shanghai Majorbio Bio-pharm Technology Co., Ltd. for Illumina MiSeq high-throughput sequencing.

[0059] The bioinformatics analysis workflow comprises the following steps: the raw sequencing data was subjected to quality control using Fastp software; the qualified sequences were assembled using Flash software; The assembled sequences were filtered using Usearch software to remove chimera sequences, resulting in a dataset of high-quality, valid sequences; using Uparse software, the filtered sequences were clustered into Operational Taxonomic Units (OTUs) at 97% similarity; the OTUs was annotated and identified using the Unite 8.0 fungal taxonomy database; α-diversity indices were calculated using Mothur software (Version 1.30.2); various visualizations were plotted using R Studio (Version 2.2.1), including Rarefaction curves, Venn diagrams, and intergroup difference heatmaps; abundance graphs at different taxonomic levels were generated using Qiime software; LEfSe (Linear Discriminant Analysis Effect Size) software was used to identify biomarkers and genomic features with significant differences between groups; in the LEfSe analysis, a rank-sum test is used to identify taxa with significant abundance differences, and then, Linear Discriminant Analysis (LDA) is used to quantify the effect size of each of these taxa; the fungal functional groups were analyzed using FUNGuild 1.0, categorizing fungi based on nutritional modes and resource utilization strategies. All of the bioinformatics analyses were conducted using the Majorbio Cloud Platform provided by Shanghai Majorbio Bio-Pharm Technology Co., Ltd. (Platform URL: https: / / cloud.majorbio.com / page / tools / ).

[0060] After processing the raw sequencing data, a total of 1,898,756 high-quality sequence fragments were obtained from 24 samples, covering 441,282,333 bases. The sequence count for each individual sample ranged from 54,680 to 105,674, with an average sequence length of 232 base pairs (bp). For the samples from the curing barn A: a total of 949,242 high-quality sequence fragments were obtained, covering 217,090,008 bases. The sequence count per sample ranged from 69,403 to 105,674, with an average length of 229 bp. For the samples from the curing barn B: a total of 949,514 high-quality sequence fragments were obtained, covering 224,192,325 bases. The sequence count per sample ranged from 54,680 to 88,683, with an average length of 237 bp.

[0061] The Sobs index represents the number of observed species within the samples. The OTUs were clustered at 97% sequence similarity. Random subsampling was performed to calculate the Sobs index. The Sobs index was plotted against the number of sequences. In the plot, the x-axis represents the sequence count, and the y-axis represents the Sobs index. As shown in FIG. 4, the rarefaction curves illustrates how the Sobs index changes with sequencing depth. All curves flattened out at a sequencing depth of 5,000 sequences, indicating that the species diversity in the samples had been fully captured at that point. Beyond the sequencing depth, additional sequencing would not identify more OTUs. Therefore, the sequencing depth was considered sufficient for analyzing the microbial community diversity. The results are reliable for accurately reflecting the structure of the microbial communities present in the samples.

[0062] The bacterial community structure of the 24 samples collected from the two curing barns was analyzed at four distinct time points using MiSeq sequencing technology. The time points included two early curing stages (T1 and T2) and two later curing stages (T3 and T4). At each time point, three replicates were taken. The microbial diversity indices for the two curing barns were calculated for each time point (as shown in FIG. 5). The sequencing results demonstrated a coverage rate of 0.999 across all samples, indicating that the data sufficiently captured the fungal diversity within the samples. In the curing barn A, the fungal abundance indices exhibited an initial increase followed by a decrease as the curing process advanced. In contrast, the fungal abundance indices in the curing barn B at time points T1 and T2 closely mirrored those observed in the curing barn A. However, at time points T3 and T4, the fungal abundance indices in the curing barn B did not exhibit a significant decline, unlike those in the curing barn A. The findings indicate a divergence in the fungal community structure between the curing barn A and the curing barn B at time points T3 and T4 of the curing process. Further analysis revealed that the high-voltage electric field applied in the curing barn B increased the fungal community richness. Trends observed in the Shannon index, which measures the fungal diversity, corresponds with the fungal abundance indices, suggesting that the high-voltage electric field improved the fungal diversity in the curing barn B. Additionally, at time points T3 and T4, the Simpson index values for the curing barn A were lower than those for the curing barn B, indicating a greater concentration of dominant fungi and reduced diversity in the curing barn A. The observation was consistent with the results obtained from the Shannon index.

[0063] OTU clustering at 97% similarity identified a total of 653 fungi species distributed across 7 phyla, 25 classes, 79 orders, 213 families, 433 genera from the 24 analyzed samples. The composition of the fungal community across different taxonomic levels during the curing process is illustrated in FIG. 6.

[0064] The Venn diagrams were constructed to visualize the overlap of fungal species between the curing barn A and the curing barn B at different time points during the curing process. The OTU count in the two curing barns initially increased and then decreased as the curing process progressed. At time points T1 and T2 (prior to the application of high-voltage electric field), the OTU counts and the number of shared species between the two curing barns were comparable, with a significant overlap in the composition of the fungal community. After the introduction of the high-voltage electric field in the curing barn B (at time points T3 and T4), the overlap of OTUs between the two curing barns decreased, while number of unique OTUs in the curing barn B increased. Both the unique OTU counts and total OTU counts in the curing barn B were higher than those observed in the curing barn A at time points T3 and T4. The results demonstrate that while the fungal community structures in the two curing barns were similar at time points T1 and T2, the high-voltage electric field in the curing barn B contributed to the development of a more complex and distinct fungal community structure at time points T3 and T4.

[0065] FIGS. 8A-8B show the average relative abundance of the fungal communities at the phylum level during the curing process in the two curing barns. The dominant fungal groups, each contributing more than 1% to the fungal community, were Ascomycota, Basidiomycota, and a small portion of unclassified fungal species (unclassified_k_Fungi). All other fungal groups were classified as non-dominant, collectively contributing less than 1% to the overall fungal population. In the two curing barns, Ascomycota was the most dominant phylum, followed by Basidiomycota. At time points T1 and T2, the average relative abundance of Ascomycota in the curing barn A was 50.98% and 55.39%, respectively, while Basidiomycota accounted for 34.33% and 37.99%, respectively. In the curing barn B, the average relative abundance of Ascomycota was 46.43% and 53.12%, while Basidiomycota accounted for 29.47% and 39.23%, respectively. The results showed that prior to the application of the high-voltage electric field in the curing barn B, the fungal community structure at the phylum level was similar in the two curing barns. However, at time points T3 and T4, significant changes in the fungal composition were observed. In the curing barn A, Ascomycota became the overwhelmingly dominant phylum, with the average relative abundances of 96.15% and 92.15%, respectively, whereas the average relative abundance of Basidiomycota initially decreased significantly before showing a slight increase. In contrast, the curing barn B exhibited a reciprocal relationship between Ascomycota and Basidiomycota, with no significant changes were observed in the overall abundance of either phylum. Additionally, the average relative abundance of unclassified_k_Fungi decreased in the two curing barns as the curing process progressed. In conclusion, the fungal community structure at the phylum level was largely similar between the two curing barns at time points T1 and T2. However, at time points T3 and T4, Ascomycota emerged as the dominant phylum in the curing barn A, while the distribution of fungal phyla in the curing barn B was more variable, with Ascomycota, Basidiomycota showing a reciprocal relationship. The findings suggest that the application of the high-voltage electric field in the curing barn B influenced the growth and reproductive dynamics of the dominant fungal phyla during the curing process.

[0066] FIGS. 9A-9B show the average relative abundance of fungal communities at the genus level during the curing process in the two curing barns. At time points T1 and T2, before the high-voltage electric field is applied in the curing barn B, the dominant fungal genera (with the average relative abundance greater than 0.1%) in the two curing barns were Aspergillus, Alternaria, Sampaiozyma, and Plectosphaerella. In the curing barn A, the average relative abundance of Aspergillus significantly increased at time points T3 and T4, with the average relative abundance of 72.41% and 71.57% at time points T3 and T4, respectively. The increase made Aspergillus the absolute dominant genus, while the average relative abundance of other genera decreased to varying extents. In the curing barn B, the average relative abundance of Aspergillus decreased at time points T3 and T4, with the average relative abundance of 34.46% and 21.17%, respectively, compared to the time points T1 and T2. Meanwhile, the average relative abundance of Alternaria and Sampaiozyma increased. At time point T3, the average relative abundance of non-dominant genera (those with average relative abundance less than 0.1%) saw a noticeable rise. The result suggests that the high-voltage electric field applied in the curing barn B influenced the fungal community structure by reducing the dominance of Aspergillus and promoting greater fungal diversity at time points T3 and T4. In contrast, in the curing barn A, the absence of the high-voltage electric field allowed Aspergillus to dominate the fungal community at time points T3 and T4, while other genera declined.

[0067] The high-voltage electric field significantly increased the fungal community diversity and fungal abundance indices at different taxonomic levels in the curing barn B compared to conventional curing process in the curing barn A. The high-voltage electric field reduced the growth and reproduction of Aspergillus at time points T3 and T4, weakening the competitive advantage of Aspergillus within the fungal community. Aspergillus is known for secreting enzymes, degrading tissue components, and releasing metabolic byproducts such as CO2, mycotoxins, and antimicrobial agents, all of which can contribute to mold formation and may suppress the growth of other organisms in the environment. As a result, saprotrophic fungi increase in abundance. The high-voltage electric field curbed the spread of mold and encouraged beneficial competition within the fungal community, improving overall diversity and stability of the fungal community in the curing barn B.

[0068] It will be obvious to those skilled in the art that changes and modifications may be made, and therefore, the aim in the appended claims is to cover all such changes and modifications.

Examples

example

[0050]In a comparative example, the curing barn A refers to a conventional electric curing oven (model HNND-45) that operates with upward airflow. The curing barn A comprises a heating chamber, six carbon crystal heating panels, a tobacco loading chamber, a controller, and a frequency-variable fan. The heating chamber has internal dimensions of 0.50 m×1.35 m×1.40 m. The six carbon crystal heating panels are used as a heat source. The total heating power of the six carbon crystal heating panels is 2 kW, with a thermal efficiency of 95%. The tobacco loading chamber has internal dimensions of 1.40 m×1.35 m×1.40 m, capable of holding 12-14 racks of tobacco, 150-200 kg of fresh tobacco leaves, or 1300-1500 individual leaves. The controller is configured to automatically adjust the dry-bulb temperature and the wet-bulb temperature according to the preset curing curves (as shown in FIG. 3). In an example, a high-voltage electric field generator is disposed in the curing barn A to form a mo...

Claims

1. A method for curing tobacco leaves for mildew prevention using a high-voltage electric field, comprising:S1: disposing a high-voltage electric field generator in a conventional curing barn comprising a tobacco loading chamber for accommodating tobacco leaves to generate a high-voltage electric field above the tobacco loading chamber; wherein, the high-voltage electric field generator comprises a grounding electrode plate and a pin electrode plate; and the pin electrode plate is connected to a high-voltage power supply and a control unit;S2: presetting a curing process of the curing barn into four stages: a pre-yellowing stage, a yellowing stage, a color setting stage, and a stem-drying stage; and pre-setting curing conditions for the four stages as follows:pre-yellowing stage: within 3-5 hours, uniformly raising a dry-bulb temperature from room temperature to 30° C.; maintaining 30° C. for 8-9 hours; then, uniformly raising the dry-bulb temperature to 35° C. within 3-5 hours; maintaining 35° C. for 7-8 hours; and finally, uniformly raising the dry-bulb temperature to 38° C. within 3-5 hours;yellowing stage: holding the dry-bulb temperature constant at 38° C.;color setting stage: within 7-8 hours, uniformly raising the dry-bulb temperature from 35° C. to 40° C.; andstem-drying stage: within 7-8 hours, uniformly increasing the dry-bulb temperature from 40° C. to 42° C.; maintaining at 42° C. for 20-23 hours; raising the dry-bulb temperature to 45°C and holding for 2-3 hours; raising the dry-bulb temperature to 48° C. and holding for 9-10 hours; raising the dry-bulb temperature to 54° C. and holding for 4-5 hours; raising the dry-bulb temperature to 62° C. and holding for 4-5 hours; and finally raising the dry-bulb temperature to 67° C. and holding for 10-11 hours;S3: selecting healthy tobacco leaves free from diseases and pests; placing the healthy tobacco leaves in the curing barn; during the curing process, when the yellowing stage is completed, turning on the high-voltage electric field generator; and when the curing process is completed, turning off the high-voltage electric field generator;S4: after the curing process is completed, collecting the tobacco leaves from the curing barn; wherein, the cured tobacco leaves have a moisture content of 3%-5%; andS5: adjusting the curing barn to room temperature, and cleaning the curing barn to remove tobacco debris.

2. The method of claim 1, wherein a distance between the pin electrode plate and the grounding electrode plate is 100-120 cm; the pin electrode plate comprises a bottom surface provided with a plurality of pin electrodes; the plurality of pin electrodes are evenly spaced and oriented in a downward direction; and a distance between every two adjacent pin electrodes is 20 cm.

3. The method of claim 1, wherein air flows from bottom to top within the curing barn.

4. The method of claim 1, wherein ionized air ions generated by the high-voltage electric field act on tobacco stems of the tobacco leaves; and the high-voltage electric field enhances the diversity of fungal communities in the tobacco leaves by reducing the competitive advantage of Aspergillus.

5. The method of claim 1, wherein a wet-bulb temperature is adjusted according to local curing practices and conditions of the tobacco leaves being cured; the wet-bulb temperature is lower than the dry-bulb temperature during the curing process; and the wet-bulb temperature changes accordingly when the dry-bulb temperature changes.

6. The method of claim 1, wherein the high-voltage power supply is selected from the group consisting of power-frequency alternating current (AC) power supply, high-frequency AC power supply, and direct current (DC) power supply, and operates within a voltage range of 30-50 kV.

7. The method of claim 1, wherein both the control unit and the high-voltage power supply are grounded and disposed outside the curing barn.

8. The method of claim 1, wherein during the curing process, a potential difference between the pin electrode plate and the grounding electrode plate is equal to a pin electrode voltage.

9. The method of claim 1, wherein before starting the curing process, the high-voltage power supply is debugged to confirm a normal working condition thereof.

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