Heating non-combustion tobacco product based on anaerobic fermentation and preparation method of heating non-combustion tobacco product

Abstract

The invention relates to the technical field of tobacco preparation, in particular to a heat-not-burn tobacco product based on anaerobic fermentation and a preparation method of the heat-not-burn tobacco product. According to the invention, a blending process is directionally developed according to HNB, based on a tobacco imitated tea blending technology and a tobacco homogenizing method blending technology, through homogenizing and anaerobic fermentation technologies, low-temperature heat volatile components in tobacco leaves are remarkably increased, the aroma quality and the aroma amount are increased, the characteristic aroma is manifested, and the usability is improved. According to the method, the aroma concentration and the aroma amount of the tobacco leaf product can be effectively controlled by controlling the temperature and the time of anaerobic fermentation, particularly when the fermentation temperature is 40 DEG C and 60 DEG C, accumulation of low-temperature volatile aroma components such as ketones and aldehydes in the tobacco leaves is facilitated, the irritation of the tobacco leaves is reduced, and the taste of the tobacco leaves is improved.

Description

Technical Field

[0001] This invention relates to the field of tobacco preparation technology, specifically to heated non-combustible tobacco products based on anaerobic fermentation and their preparation methods. Background Technology

[0002] The global trend towards tobacco control is becoming increasingly severe, and consumers are paying more and more attention to their health. People are beginning to rethink the harmful effects of smoking and secondhand smoke on health. The drawbacks of traditional tobacco products are becoming increasingly prominent. Therefore, consumers are paying more and more attention to new tobacco products that are less harmful to health. The global HNB (heated tobacco products) market is developing rapidly. In recent years, domestic tobacco companies have gradually entered the HNB field and have already accumulated certain technological reserves.

[0003] Unlike traditional cigarettes, the core of heated tobacco products (HNB) is the tobacco sheet, composed of nicotine, smoke-generating agents, flavorings, and other ingredients. Nicotine and aroma components are carried by an aerosol generated at high temperatures. Therefore, the quality of the tobacco sheet is crucial. Given the enormous future development potential and market for HNB, the development of core material raw materials plays a vital role. Currently, Philip Morris International, represented by IQOS and Glo, and British American Tobacco, hold a monopoly on the intellectual property rights of core material product design in this field. Domestic HNB raw material research is still in its early stages, with relatively few fundamental studies on raw material modulation and evaluation available for reference. For example, Zhang Lina et al. mixed "Jiuqu Hongmei" black tea with heated tobacco products in a certain proportion to support HNB raw materials. The results showed that the addition of "Jiuqu Hongmei" black tea enhanced the elegance of cigarette smoke, strengthened the fruity, sweet, and floral aromas, and also increased the aroma quantity and richness, resulting in a delicate, smooth, and sweet aftertaste. Huo Xiankuan et al. studied the aroma release characteristics of flue-cured tobacco, aromatic tobacco, and burley tobacco raw materials at heating temperatures ranging from 200℃ to 500℃. The results showed that when the heating temperature was between 300℃ and 400℃, the release of aroma components, as well as the release of aldehydes, ketones, nitrogenous substances, and aliphatic hydrocarbons, increased significantly. Wang Yi et al. studied the effect of temperature on the delayed release of different types of tobacco matrix under heated-to-non-combustion conditions, finding a linear correlation between the smoke release from different tobacco matrixes under heated-to-non-combustion turntable conditions and temperature. Zhu Guihua et al., by comparing the application effects of pre-cured tobacco leaves with different curing processes in heated tobacco products, found that the process significantly affects the aroma release and smoking quality of heated tobacco. However, these studies are all based on comparisons of existing raw materials under heating conditions or improvements to existing processes, and do not specifically develop modulation processes for heated tobacco products (HNB). Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a method for preparing heated non-combustible tobacco products based on anaerobic fermentation. The present invention provides a method for producing heated non-combustible tobacco products by means of anaerobic fermentation, which significantly increases the low-temperature volatile components in tobacco leaves, resulting in sufficient aroma quantity, concentration, and richness, and exhibiting obvious tobacco aromas of hay and roasting.

[0005] This invention provides a method for preparing heat-not-burn tobacco products, comprising the following steps:

[0006] Step 1: Take fresh tobacco leaves, wither and crush them to obtain tobacco leaf homogenate;

[0007] Step 2: After the tobacco leaf homogenate from Step 1 is subjected to anaerobic fermentation, the resulting fermented homogenate is dried in stages to obtain the heated non-combustible tobacco product.

[0008] In some embodiments, step 1, withering includes: taking the fresh tobacco leaves and withering them in an environment with a dry bulb temperature of 38~40℃ and a wet bulb temperature of 35~36℃, wherein the moisture content of the withered tobacco leaves is 60%~65%.

[0009] In some embodiments, the dry bulb temperature is 38°C, the wet bulb temperature is 36°C, and the moisture content is 60%.

[0010] In some embodiments, in step 1, the crushing includes crushing a mixture of withered tobacco leaves and water in a mass ratio of 1:(1~2), the crushing speed is 20000~40000 r / min, and the crushing time is 20~40 min.

[0011] In some embodiments, the mass ratio of withered tobacco leaves to water is 1:1.5, the crushing speed is 30,000 r / min, and the crushing time is 30 min.

[0012] In some embodiments, in step 2, the container for anaerobic fermentation is a sealed fermentation tank with a one-way valve, the fermentation tank being a glass fermentation tank or a ceramic fermentation tank, and the fermentation tank containing 60 vol% to 80 vol% of the tobacco leaf homogenate.

[0013] In some specific embodiments, the fermenter contains 80 vol% of the tobacco homogenate.

[0014] In some embodiments, in step 2, the temperature of the anaerobic fermentation is 40~60℃ and the time is 3~9 days.

[0015] In some embodiments, the anaerobic fermentation is performed at a temperature of 40°C for a duration of 9 days; or

[0016] The anaerobic fermentation was carried out at a temperature of 60°C for 9 days.

[0017] In some embodiments, in step 2, the segmented drying includes initial drying and secondary drying, wherein:

[0018] The initial drying process includes drying the fermented homogenate at 80-85°C for 100-120 minutes, and then letting it rest for 1.5-3 hours to rehydrate.

[0019] The re-drying process involves taking the rehydrated fermented homogenate and drying it completely at 100-105°C.

[0020] In some embodiments, the initial drying includes drying the fermentation homogenate at 85°C for 120 min, and then letting it rest for 2 h to rehydrate.

[0021] The re-drying process involves taking the rehydrated fermented homogenate and drying it completely at 105°C.

[0022] This invention provides a heated non-combustible tobacco product prepared by the aforementioned preparation method.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] (1) The present invention has developed a modulation process based on HNB, which is based on tobacco imitation tea modulation technology and tobacco leaf homogenization modulation technology. Through homogenization and anaerobic fermentation technology, the low-temperature heat volatile components in tobacco leaves are significantly increased, the aroma quality and aroma quantity are increased, the distinctive aroma is highlighted, and the usability is improved.

[0025] (2) By controlling the temperature and time of anaerobic fermentation, the present invention can effectively control the aroma concentration and aroma amount of tobacco products. In particular, when the fermentation temperature is 40℃ and 60℃, it is more conducive to the accumulation of low-temperature volatile aroma components such as ketones and aldehydes in tobacco leaves, reducing the irritation of tobacco leaves and increasing the taste of tobacco leaves. Attached Figure Description

[0026] Figure 1 The α diversity index of fungal communities in tobacco leaves under different treatments is shown.

[0027] Figure 2 The α diversity index of bacterial communities in tobacco leaves under different treatments is shown.

[0028] Figure 3 Principal component analysis of β-diversity of fungi (A) and bacteria (B) in tobacco leaves under different treatments;

[0029] Figure 4 The OTU index upset diagram of fungi (A) and bacteria (B) in tobacco leaves under different treatments is shown.

[0030] Figure 5 The community composition is shown at the fungal level (A), genus level (B), and bacterial level (C), genus level (D). Detailed Implementation

[0031] This invention provides a method for preparing heat-not-burn tobacco products based on anaerobic fermentation. 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.

[0032] To address the lack of a specific modulation process for HNB in ​​existing technologies, the present invention aims to provide a method for preparing heated tobacco products based on anaerobic fermentation. By using tobacco leaf homogenization to fully break down the tobacco leaves and promote anaerobic fermentation, the method has shown outstanding results in heated tobacco cigarette samples. It significantly increases the amount, concentration, and richness of low-temperature volatile components in the tobacco leaves, resulting in a distinct aroma with a distinct dry and roasted tobacco flavor.

[0033] To achieve the above-mentioned technical objectives, the present invention provides a method for preparing a heat-not-combustible tobacco product based on anaerobic fermentation. The method involves weaving fresh tobacco leaves into stalks and then withering them to obtain withered tobacco leaves; removing the main veins from the withered tobacco leaves and then crushing and homogenizing them to obtain tobacco leaf homogenate; and then subjecting the tobacco leaf homogenate to anaerobic fermentation and segmented drying to obtain the final product.

[0034] The technical solution of this invention, on the one hand, involves crushing and homogenizing tobacco leaves to ensure complete breakage, stimulating aroma release and promoting subsequent anaerobic fermentation. On the other hand, combining anaerobic fermentation with tobacco significantly enhances the inherent aroma of tobacco, particularly the hay and roasted notes. In the anaerobic fermentation process of this invention, the entire fermentation process exhibits a dynamic and complex ecological cycle with vigorous microbial activity. In the initial stage of fermentation, the endogenous hydrolytic enzymes of the tobacco leaves and the active aerobic microbial community (such as *Pseudomonas* and *Sphingomonas*) work together to reduce off-gassing substances and rapidly decompose the large-molecule sugars abundant in the tobacco leaves, converting them into easily absorbed and utilized small-molecule soluble sugars. This process directly drives a significant increase in the reducing sugar content in the fermentation system. As the fermentation process deepens, the proteins in the tobacco leaves are broken down into amino acids by microorganisms. Soluble reducing sugars combine with amino acids to generate heterocyclic substances with a special aroma, thereby consuming some of the reducing sugars. In the later stages of fermentation, the demand for reducing sugars by microorganisms surges to maintain their vigorous life activities. This disrupts the balance between conversion and consumption established in the early stages, causing the reducing sugar content to decline. At this point, anaerobic bacteria become dominant, with Bacillus and Weizmannii playing a key role in the continuous degradation of macromolecules (plant pigments, cellulose, lignin, etc.). Consequently, the proportion of ketones in the tobacco leaves increases significantly, while the proportion of olefins decreases significantly. The combination of tobacco leaf homogenization and anaerobic fermentation in this invention significantly increases the proportion of aroma components in the medium- and low-temperature thermal volatile components of tobacco leaves, improving the performance of heated, non-combustible tobacco leaves during smoking evaluation.

[0035] As a preferred embodiment, the withering conditions are: dry-bulb temperature of 38-40℃, wet-bulb temperature of 35-36℃, and moisture content of 60-65%. Controlling the dry-bulb temperature effectively regulates the yellowing degree and water loss rate of the tobacco leaves, while controlling the wet-bulb temperature ensures adequate moisture evaporation during withering, preventing quality degradation due to excessive drying. The combination of dry-bulb and wet-bulb temperatures in this invention maximizes the generation of aroma compounds within the tobacco leaves, thereby improving their sensory evaluation score.

[0036] As a preferred embodiment, the solvent used in the crushing and homogenizing process is water, the mass ratio of tobacco leaves to water is 1:1.5~2, the crushing speed is 20000~30000 r / min, the crushing time is 20~30 min, and the homogenized slurry should be a brown paste that can be flowed down in streams, and there should be no obvious fibers and broken leaves visible to the naked eye.

[0037] As a preferred embodiment, the anaerobic fermentation employs a sealed glass or ceramic fermenter equipped with a one-way valve, and the volume of the tobacco homogenate filled into the fermenter occupies 60-80% of its total volume. During operation, the one-way valve is sealed with water to enhance airtightness and prevent gas backflow. The volume of tobacco homogenate in the fermenter should not be too high; an excessively high volume may lead to excessive pressure inside the fermenter, affecting the growth and metabolism of microorganisms. Conversely, an excessively low volume may deprive microorganisms of sufficient nutrients, impacting the efficiency of the fermentation process.

[0038] As a preferred embodiment, the anaerobic fermentation temperature is 40~60℃. The anaerobic fermentation temperature in this invention has a significant impact on the main chemical components of tobacco leaves. This is because under different temperature conditions, the microbial community, microbial activity, and chemical composition in the fermentation system will vary, thus affecting the fermentation effect and tobacco leaf quality.

[0039] As a preferred method, the anaerobic fermentation time is 3-9 days. The fermentation time directly affects the completeness of tobacco leaf fermentation. During fermentation, as time progresses, the proteins in the tobacco leaves are broken down into amino acids by microorganisms. Soluble reducing sugars combine with amino acids and participate in the Maillard reaction, generating aroma substances. For example, when fermenting at 40℃, the total sugar and reducing sugar content in the tobacco leaves shows a trend of first increasing and then decreasing. This may be due to the combined action of microorganisms and the tobacco leaves' own hydrolytic enzymes in the early stage of fermentation, followed by a decrease in reducing sugar content due to the weakening of microbial activity.

[0040] As a preferred embodiment, the anaerobic fermentation conditions are 40℃ for 9 days or 60℃ for 9 days. The inventors discovered through experiments that both 40℃ and 60℃ are conducive to the accumulation of low-temperature volatile aroma components such as organic acids and alcohols in tobacco leaves, and exhibit different characteristics under these two temperature conditions. When higher requirements are placed on aroma quantity, concentration, and roasting aroma, fermentation at 60℃ for 9 days can be further preferred; when a sweet aroma is desired, fermentation at 40℃ for 9 days can be further preferred.

[0041] As a preferred embodiment, the segmented drying process involves first drying at 80-85°C for 100-120 minutes, then removing the product and allowing it to rest for more than 2 hours to rehydrate, and finally placing it back into an aroma-enhancing machine at 100-105°C for complete drying.

[0042] The fresh tobacco leaves used in the embodiments and comparative examples of this invention were obtained using the following methods:

[0043] Variety: K326; Part: Middle leaves (18 leaves retained, with the 9th, 10th, and 11th leaves in the middle); Maturity: Suitable for maturity; Location: Longshan County, Xiangxi Prefecture. Sampling requirements: Tobacco leaves should be of good appearance and vigor, with consistent harvest maturity, from the same plot, with uniform growth, consistent fertilization plan, and the same field management measures, away from roads, without any cover, and free from diseases.

[0044] Sampling method: After removing the bottom leaves, select two consecutive plots of about 3 mu each (one as a backup to avoid diseases affecting tobacco quality). Mark the tobacco leaves in the field that meet the sampling standards. When the tobacco leaves are at the appropriate maturity, take 220 kg of tobacco leaves at once. 20 kg of the tobacco leaves will be stalked and cured in four stages. The curing process is consistent with the local technical plan (four-stage curing process of wilting-yellowing-color fixing-drying). 1 kg of tobacco leaf sample will be blanched at 105℃ and then subjected to routine physicochemical property tests. The rest will be used for curing experiments.

[0045] Precautions: Contact the local tobacco station technician in advance, select tobacco farmers with good planting and flue-curing techniques, and collect field management information after determining the sampling farmers and plots.

[0046] In the embodiments and comparative examples of this invention, the total sugar and reducing sugar content were determined according to YC / T 159—2002; the total alkali content was determined according to YC / T468—2013; the chloride ion content was determined according to YC / T 162—2002; the potassium ion content was determined according to YC / T 217—2007; the total nitrogen content was determined according to YC / T 161—2002; and the starch content was determined according to YC / T 216—2007. The smoking evaluation was organized and implemented by the Hunan Tobacco Technology Center, and the evaluation results were presented in written description, mainly including the amount of aroma, richness of aroma, concentration, strength, smoothness and delicacy, characteristic aroma, off-flavors, and irritation.

[0047] The experimental data were initially processed and tabulated using Microsoft Excel 2021, and analysis of variance was performed using SPSS 25.

[0048] The test materials used in this invention are all commercially available products. The invention will be further illustrated below with reference to specific embodiments.

[0049] Example 1

[0050] (1) Traditional baking

[0051] The baking samples were prepared according to local technical procedures, and the baking process was divided into four stages: yellowing, wilting, color fixing, and stem drying. The primary yellowing temperature was 38℃ for the dry bulb, with relative humidity controlled at 80%–85% to enhance the yellowing degree. Before reaching a dry bulb temperature of 43℃, the tobacco leaves were required to achieve a state of yellow leaves with green veins and full wilting and collapse. Slow heating and extended color fixing time were used. Upper and lower thresholds for wet bulb temperature were set throughout the baking process. Specifically, this included:

[0052] Yellowing stage

[0053] Smoke profile target: the lower tobacco leaves should turn yellow to 70-80%, the middle and upper tobacco leaves should turn yellow to 80-90%, and the leaves should be dry enough to soften.

[0054] Fan speed: The circulating fan operates at low speed.

[0055] Processing procedure: After ignition, raise the dry bulb temperature to 38℃ at a rate of 1℃ / h and stabilize it. Adjust the wet bulb temperature to 36±1℃. The wet bulb temperature should not be lower than 35℃ or higher than 37℃. The stabilization time should be determined based on the yellowing and dehydration of the tobacco leaves to meet the requirements of that stage.

[0056] wilting stage

[0057] Tobacco phase target: The tobacco leaves should reach a state of 90-100% yellowing and fully wilted and collapsed.

[0058] Fan speed: When the dry bulb temperature reaches 40℃, the circulating fan operates at high speed.

[0059] Processing: Steadily increase the dry-bulb temperature to 41℃~42℃ at a rate of 1℃ / 2h and maintain a stable temperature, while keeping the wet-bulb temperature at 37±1℃. Before the tobacco leaves reach the desired tobacco phase change, the dry-bulb temperature should not exceed 43℃, and the wet-bulb temperature should not fall below 36℃. Once the target tobacco phase is achieved, continue maintaining the temperature for 8~12h before proceeding to the color-fixing stage.

[0060] Color setting stage

[0061] Tobacco phase target: At the end of the 48℃ period, the tobacco leaves in the high-temperature layer should reach the stage of yellow leaves with yellow veins and small-scale rolling. At the end of the 54℃ period, the tobacco leaves in the entire chamber should reach the stage of 100% yellowing and large-scale rolling.

[0062] Fan speed: The circulating fan operates at high speed.

[0063] Process Operation: Raise the dry bulb temperature to 46℃~48℃ at a rate of 1℃ / 3-4h and stabilize, while maintaining the wet bulb temperature at 38±1℃. After achieving the target smoke phase, raise the dry bulb temperature to 54±1℃ at a rate of 1℃ / 1.5-2h and stabilize, while maintaining the wet bulb temperature at 39±1℃. After achieving the target smoke phase, continue stabilizing the temperature for 12h before proceeding to the drying stage.

[0064] During this stage, the wet-bulb temperature should not be lower than 37℃ or higher than 40℃, and should not fluctuate drastically. The heating rate should not be too fast or too slow, and there should be no significant drop in temperature or a sudden increase in temperature.

[0065] Dry tendon stage

[0066] Smoke phase target: At the end of 60℃, the tobacco leaves should exhibit significant dehydration of the main stems and show purple patches. At the end of 68℃, the main stems of the tobacco leaves in the entire chamber should be dry.

[0067] Fan speed: Before reaching 60℃, the circulating fan runs at high speed. After stabilizing at 60℃ until the baking is complete, the circulating fan runs at low speed.

[0068] Process operation: Increase the dry bulb temperature to 60℃ at a rate of 1℃ / h and maintain it at a stable temperature, while keeping the wet bulb temperature at 40℃~41℃. After the target smoke phase is achieved, increase the dry bulb temperature to 65℃~68℃ at a rate of 1℃ / h and maintain it at a stable temperature, while keeping the wet bulb temperature at 41℃~43℃. After the main stems of the tobacco leaves in the entire chamber are dry, the curing process is completed.

[0069] When the temperature is stable between 65℃ and 68℃, the wet-bulb temperature should not exceed 43℃ or fall below 40℃, and the dry-bulb temperature should not exceed 68℃.

[0070] (2) Homogenization method + anaerobic fermentation

[0071] Withering: The fresh tobacco leaves are woven into stalks and evenly hung in the withering machine. The desired temperature is then set for withering. During this process, the condition of the tobacco leaves and the withering process must be frequently observed. If uneven withering occurs, the position of the tobacco leaves on the stalks must be adjusted promptly. The withering temperature is set to 38℃ for the dry bulb and 36℃ for the wet bulb. The tobacco leaves are removed when the moisture content reaches 60%.

[0072] Homogenization: Place the tobacco leaves with the main veins removed into a high-speed blender for crushing, add an appropriate amount of distilled water, the ratio of tobacco leaves to distilled water is 1:1.5, set the high-speed blender to 30000r / min, and the crushing time is 30min.

[0073] Anaerobic fermentation: Prepare a sealable glass or ceramic fermentation tank with a one-way valve. Fill the tank with tobacco leaf homogenate, which should occupy about 80% of the total volume of the fermentation tank. Seal the tank with water through the one-way valve and place it in an environment with the appropriate temperature for fermentation.

[0074] Drying: Take out the fermented tobacco leaf homogenate and spread it evenly on a drying tray. After placing it, turn on the dryer to preheat it. After reaching the set temperature, put the drying tray into the dryer and dry it at 85℃ for 120 minutes. After the tobacco leaf homogenate is basically dry, take it out and let it rest for 2 hours to rehydrate. Set the temperature of the aroma enhancer to 105℃. After reaching the set temperature, put the rehydrated tobacco leaf homogenate into the dryer and take it out after it is completely dry.

[0075] Three fermentation temperatures were set: A1: 40℃; A2: 50℃; A3: 60℃; three fermentation times were set: B1: 3 days; B2: 6 days; B3: 9 days; a total of 9 treatments were set up. The withered tobacco sample was used as CK1, and the traditionally cured tobacco sample was used as CK2.

[0076] The microbial community structure of tobacco leaves changed significantly after homogenization. The results of microbial α-diversity analysis are as follows: Figure 1 , Figure 2 The results showed that the microbial coverage of the tobacco leaf samples after fermentation was close to 1, indicating that the sampling and sequencing results could accurately reflect the distribution of the microbial community. The Chao 1 index of fungi and bacteria in the experimental group was significantly lower than that in the control group, indicating that this processing method significantly reduced the diversity of the tobacco leaf microbial community.

[0077] β-diversity primarily describes the difference coefficients between samples, with specific results as follows: Figure 3 The results showed that the contributions of fungi and bacteria as the first principal components were 98.86% and 99.23%, respectively. The significant difference in distance between the two groups indicated that anaerobic homogenization fermentation had a substantial impact on the bacterial community results.

[0078] OTU analysis results are as follows Figure 4 The results showed that anaerobic homogenization fermentation reduced the OTU index, with a more significant reduction effect on bacteria. The A1B3 sample contained only 26 unique bacterial species, while the CK1 sample contained as many as 259 species. The difference in fungal species was smaller, with the A1B3 sample containing 258 unique fungal OTUs, while the CK1 sample contained 338. The difference in the number of unique OTU species between the two samples was significant.

[0079] The composition of tobacco communities under different treatments consists of Figure 5The results showed that, in terms of bacterial community structure, Pseudomonadota was the dominant phylum at the beginning of fermentation, while Bacillota became the new dominant phylum after fermentation. At the genus level, Enterobacter, Enterococcus, Weizmannia, Klebsiella, and Bacillus were the dominant genus in anaerobic homogenized tobacco leaves, while before fermentation, the dominant genus composition of tobacco leaves was Enterobacter, Sphingomonas, Pantoea, and Methylobacterium. The fungal community structure of the fermented tobacco leaves showed significant differences in dominant communities. At the phylum level, Basidiomycota, Ascomycota, and Anthophyta were all dominant communities, but there were significant differences in their relative proportions. At the genus level, at the beginning of fermentation, *Sampaiozyma*, *Alternaria*, *Moesziomyces*, and *Wallemia* were dominant genera, while after fermentation, *Moesziomyces*, *Sampaiozyma*, *Nigrospora*, and *Cladosporium* became new dominant genera.

[0080] In summary, the anaerobic homogenization method has a significant impact on the succession of tobacco microbial communities, exhibiting different effects on bacteria and fungi. After fermentation, some tobacco leaves transform from Pseudomonas (aerobic) to Bacillus (facultative anaerobic) and strict anaerobic bacteria, reflecting the oxygen consumption of the fermentation environment. The dominant fungi are mostly aerobic or facultative anaerobic, but yeasts may maintain metabolism through fermentation in the absence of oxygen.

[0081] Related studies have shown that microorganisms play a crucial role in the aging process of traditionally cured tobacco leaves, further degrading macromolecular substances and converting them into effective aroma components. During tobacco aging, aerobic bacteria, such as Bacillus and Pseudomonas, typically play a major role, improving the industrial usability and smoking quality of tobacco leaves by decomposing macromolecules like proteins and starches. This experiment encountered rare anaerobic and anaerobic fermentation environments, resulting in a significant reduction in microbial diversity. Enterobacteriaceae, Enterococci, Weizmannella, Klebsiella, and Bacillus were all facultative anaerobes. Enterobacteriaceae, Enterococci, and Weizmannella, among others, play key roles in fermented flavored products such as yogurt and fermented black beans, but their application in tobacco processing has not yet been systematically reported.

[0082] The differences in the content of volatile aroma compounds are shown in Table 1. Compared with flue-cured tobacco, the total volatile aroma component content of homogenized tobacco leaves is significantly reduced, with the most significant decreases in olefins and aromatic compounds. Conversely, the percentages of aldehydes, ketones, heterocyclic compounds, and esters increase in homogenized anaerobic fermented tobacco leaves, with ketones, an important aroma component, accounting for 46.45%. This tobacco processing method significantly depletes olefins, which often require incomplete combustion in traditional cigarettes to produce complex aromas. Ketones, being low-boiling-point compounds, are better able to impart a unique and elegant aroma to tobacco materials.

[0083] Table 1. Differences in aroma substance content between anaerobic fermented tobacco and flue-cured tobacco.

[0084]

[0085] Table 2 shows a comparison of the conventional chemical components in tobacco leaves (A1B1, A1B2 and A1B3) and tobacco leaves after withering (CK1) during fermentation.

[0086] Table 2. Conventional chemical composition of tobacco leaves and CK1 sample at different fermentation times at 40℃

[0087]

[0088] The experimental results in Table 2 show that the total sugar content of all three treatments was significantly lower than that of CK1. The order of the three treatments was A1B2 > A1B3 > A1B1, and the differences among the treatments were significant (the total sugar content first increased and then decreased with increasing fermentation time). The reducing sugar content of the A1B2 treatment was significantly higher than that of the other treatments, while the reducing sugar content of the CK1 and A1B1 treatments was the lowest. The total alkali content of the CK1 treatment was the highest, followed by A1B3, while the other two treatments were lower and the differences were not significant. The potassium ion content of the CK1 treatment was significantly higher than that of the other treatments, and the starch content was significantly lower than that of the other treatments. Except for CK1, the differences in potassium ion and starch content among the treatments were not significant. There were no significant differences in chloride ion and total nitrogen content among the treatments.

[0089] Table 3 shows a comparison of the conventional chemical components in tobacco leaves (A2B1, A2B2 and A2B3) fermented at 50℃ for different times and in the withered tobacco sample (CK1).

[0090] Table 3. Chemical composition of tobacco leaves and CK1 at different fermentation times at 50℃

[0091]

[0092] The experimental results in Table 3 show that the total sugar content of all three treatments was significantly lower than that of CK1. The order of the three treatments was A2B1 > A2B3 > A2B2, and the differences among the treatments were significant (the total sugar content first decreased and then increased with increasing fermentation time). The reducing sugar content of treatment A2B1 was significantly higher than that of the other treatments, while the reducing sugar content of treatments CK1 and A2B2 was the lowest. The total alkali content of treatment CK1 was the highest, while that of treatment A2B1 was the lowest, and the differences between the other two treatments were not significant. The potassium ion content of treatment CK1 was significantly higher than that of the other treatments, while the starch content was significantly lower than that of the other treatments. Except for CK1, the differences in potassium ion and starch content among the treatments were not significant. There were no significant differences in chloride ion and total nitrogen content among the treatments.

[0093] Table 4 shows a comparison of the conventional chemical components in tobacco leaves (A3B1, A3B2 and A3B3) fermented at 60℃ for different times and in the withered tobacco sample (CK1).

[0094] Table 4. Chemical composition of tobacco leaves and CK1 at different fermentation times at 60℃

[0095]

[0096] The experimental results in Table 4 show that the total sugar content of all three treatments was significantly lower than that of CK1. The order of the three treatments was A3B1 > A3B2 > A3B1, and the differences among the treatments were significant (total sugar content decreased with increasing fermentation time). The reducing sugar content of treatment A3B1 was significantly higher than that of the other treatments, while CK1 had the lowest content. The order of the treatments was the same as that of total sugar, and the differences among the treatments were significant. The potassium ion content of treatment CK1 was significantly higher than that of the other treatments, while A3B2 had the highest content. The starch content was significantly higher than that of the other treatments. Except for CK1, there were no significant differences in potassium ion content among the treatments. There were no significant differences in chloride ion, total alkali, and total nitrogen content among the treatments.

[0097] Table 5 compares the conventional chemical components in tobacco leaves (A1B1, A2B1, and A3B1) fermented for 3 days at different fermentation temperatures and in the withered tobacco sample (CK1).

[0098] Table 5. Chemical composition of tobacco leaves and CK1 at different temperatures after 3 days of fermentation

[0099]

[0100] The experimental results in Table 5 show that the total sugar content of all three treatments was significantly lower than that of the control (CK). The order of the three treatments was A3B1 > A2B1 > A1B1 (total sugar content increased significantly with increasing fermentation temperature). Both A3B1 and A2B1 were significantly higher than A1B1, but the difference between the two was not significant. The reducing sugar content of the A3B1 treatment was significantly higher than that of the other treatments. The CK1 and A1B1 treatments had the lowest reducing sugar content, but the difference between the two was not significant. The order of the three treatments was the same as that of the total sugar content. The total alkali content of the CK1 and A3B1 treatments was significantly higher than that of the other treatments, with A1B1 having the lowest. The potassium ion content of the CK1 treatment was significantly higher than that of the other treatments. There were no significant differences in chloride ion, total nitrogen, and starch content among the treatments.

[0101] Table 6 compares the conventional chemical components in tobacco leaves (A1B2, A2B2, A3B2) fermented at different fermentation temperatures for 6 days and in the withered tobacco sample (CK1).

[0102] Table 6. Chemical composition of tobacco leaves and CK1 at different temperatures after 6 days of fermentation.

[0103]

[0104] The experimental results in Table 6 show that the total sugar content of all three treatments was significantly lower than that of CK1. The order of the three treatments was A3B2 > A1B2 > A2B2 (the total sugar content first decreased and then increased with increasing fermentation temperature). Both A3B2 and A1B2 were significantly higher than A2B2, but the difference between them was not significant. The reducing sugar content of A3B2 was significantly higher than that of the other treatments. The CK1 and A2B2 treatments had the lowest reducing sugar content, but the difference between them was not significant. The order of the three treatments was the same as that of the total sugar. The total alkali content of CK1 and A3B2 treatments was significantly higher than that of the other treatments, and A1B2 had the lowest. The potassium ion content of CK1 treatment was significantly higher than that of the other treatments. The starch content of CK1 treatment was significantly lower than that of the other three treatments. There was no significant difference in chloride ion and total nitrogen content among the treatments.

[0105] Table 7 compares the conventional chemical components in tobacco leaves (A1B3, A2B3, A3B3) fermented at different fermentation temperatures for 9 days with the samples after withering (CK1).

[0106] Table 7. Chemical composition of tobacco leaves fermented at different temperatures for 9 days and CK1 (conventional chemical composition)

[0107]

[0108] The experimental results in Table 7 show that the total sugar content of all three treatments was significantly lower than that of CK1; there was no significant difference among the three treatments; the reducing sugar content of treatment A3B3 was significantly higher than that of the other treatments (the reducing sugar content first decreased and then increased with increasing fermentation temperature), and treatment CK1 had the lowest content; the total alkali content of treatments CK1 and A3B3 was significantly higher than that of the other treatments; the potassium ion content of treatment CK1 was significantly higher than that of the other treatments, and the starch content was significantly lower than that of the other three treatments; there was no significant difference in chloride ion and total nitrogen content among the treatments.

[0109] Table 8 compares the conventional chemical composition of CK2 tobacco leaves under all treatment conditions and those under conventional flue-cured tobacco treatment. Table 9 shows the results of its application to the evaluation of heated tobacco products.

[0110] Table 8. Conventional chemical components of tobacco leaves under different treatments

[0111]

[0112] Table 9. Textual evaluation of upper tobacco leaves under different treatment methods

[0113]

[0114] As shown in Table 7, the total sugar content of the tobacco leaves was significantly reduced after homogenization, and the reducing sugar content was significantly lower than that of CK2 except for treatment A3B1. The total alkali and total nitrogen content of each treatment were significantly lower than those of CK2. The starch content was highest in treatment A2B3 and lowest in treatment A3B3.

[0115] As shown in Table 8, the A3B3 treatment has a sufficient amount of aroma, a high concentration, and a medium to high richness, with a hint of hay and roasted aroma. It is slightly stronger. Compared with the CK2 treatment, the amount, concentration, and richness of aroma are improved, and the original aroma of flue-cured tobacco, such as hay and roasted aroma, is highlighted, while the raw and impure smells disappear. The A1B3 treatment has a medium to high amount of aroma and concentration, and a medium richness. It has a sweet aroma, hay aroma, and roasted aroma. The evaluation results of the other treatments all have certain defects, such as less aroma and concentration, poor richness, or rough smoke with raw and impure smells.

[0116] The experimental results in Tables 1-7 show that different fermentation conditions have a significant impact on the main chemical components of tobacco leaves, as follows: At fermentation temperature of 40℃, the contents of total sugar, reducing sugar, and total alkali in tobacco leaves change drastically. The total sugar content decreases compared to after withering, while the reducing sugar content increases, showing a trend of first increasing and then decreasing. This indicates that at fermentation temperature of 40℃, the microbial activity in the entire fermentation system is intense. In the early stage of fermentation, the hydrolytic enzymes of the tobacco leaves and the metabolism of microorganisms work together to decompose the macromolecular sugars in the homogenized tobacco leaves and convert them into soluble sugars, leading to an increase in the reducing sugar content. The subsequent decrease is because in the later stage of fermentation, microbial activity is the main factor, requiring more reducing sugars to provide energy. Therefore, the balance between conversion and consumption is broken, leading to a decrease in the reducing sugar content. On the other hand, as fermentation progresses, the proteins in the tobacco leaves are decomposed into amino acids by microorganisms. Soluble reducing sugars combine with amino acids to generate Maillard reaction products, which consume some of the reducing sugars. Under this temperature condition, conversion, consumption, and combination occur simultaneously, thus showing a fluctuating trend of decreasing, increasing, and then decreasing again. At 50℃, the inflection point appears after 6 days of fermentation. This may be due to differences in enzyme activity and microbial community at different temperatures. At 60℃, the total sugar content shows a decreasing trend, while the reducing sugar content shows a trend of first increasing and then decreasing.

[0117] Under the same fermentation time, the total sugar and reducing sugar content in tobacco leaves changed differently under different temperature conditions. At 3 days of fermentation, the reduction sugar increased more significantly at higher temperatures, while the total sugar consumption decreased less. At 6 days of fermentation, both total sugar and reducing sugar were lowest at a fermentation temperature of 50℃. At 9 days of fermentation, there was no significant difference in the total sugar content at the three fermentation temperatures, but the reducing sugar content was 60℃ > 50℃ > 40℃. This indicates that different temperature conditions led to differences in the sugar content in tobacco leaves.

[0118] Under all three temperature conditions, the total alkali content showed an increasing trend with the extension of fermentation time. Moreover, under the same fermentation time, the total alkali content increased with the increase of fermentation temperature. This is similar to tobacco curing, where the total alkali content is affected by factors such as nitrogen application rate, moisture, light intensity, and part of the plant. Different curing processes can also lead to changes in the total alkali content.

[0119] Total nitrogen content can reflect the total amount of total alkali and protein in tobacco leaves. The total nitrogen content of each treatment was lower than that of withered tobacco leaves and conventional flue-cured tobacco to varying degrees, indicating that during the fermentation process, the activity of microorganisms decomposed the protein in tobacco leaves into amino acids, which further enhanced the flavor of tobacco leaves in heated cigarettes.

[0120] Smoking evaluation is the most direct reflection of the usability of tobacco raw materials. The evaluation results of A1B3 and A3B3 treatments were better, indicating that 40℃ and 60℃ are both conducive to the accumulation of low-temperature volatile aroma components in tobacco leaves, such as organic acids and alcohols. Moreover, different characteristics were shown under these two temperature conditions. The aroma quantity and concentration of A1B3 treatment were slightly less than those of A3B3 treatment, but it added a sweet aroma. The aroma quantity and concentration of A3B3 treatment were more sufficient, and the aromas of roasting and hay were prominent.

[0121] In summary, the tobacco samples fermented at 40℃ for 9 days and at 60℃ for 9 days using the anaerobic conditioning process are the best.

[0122] Example 2

[0123] (1) Traditional baking

[0124] Same as Example 1.

[0125] (2) Homogenization method + anaerobic fermentation

[0126] Withering: The fresh tobacco leaves are woven into stalks and evenly hung in the withering machine. The desired temperature is then set for withering. During this process, the condition of the tobacco leaves and the withering process must be frequently observed. If uneven withering occurs, the position of the tobacco leaves on the stalks must be adjusted promptly. The withering temperature is set to 38℃ for the dry bulb and 36℃ for the wet bulb. The tobacco leaves are removed when the moisture content reaches 60%.

[0127] Homogenization: Place the tobacco leaves with the main veins removed into a high-speed blender for crushing, add an appropriate amount of distilled water, the ratio of tobacco leaves to distilled water is 1:1.5, set the high-speed blender to 30000r / min, and the crushing time is 30min.

[0128] Anaerobic fermentation: Prepare a sealable glass or ceramic fermentation tank with a one-way valve. Fill the tank with tobacco leaf homogenate, which should occupy about 80% of the total volume of the fermentation tank. Seal the tank with water through the one-way valve and place it in an environment with the appropriate temperature for fermentation.

[0129] Drying: Take out the fermented tobacco leaf homogenate and spread it evenly on a drying tray. After placing it, turn on the dryer to preheat it. After reaching the set temperature, put the drying tray into the dryer and dry it at 85℃ for 120 minutes. After the tobacco leaf homogenate is basically dry, take it out and let it rest for 2 hours to rehydrate. Set the temperature of the aroma enhancer to 105℃. After reaching the set temperature, put the rehydrated tobacco leaf homogenate into the dryer and take it out after it is completely dry.

[0130] (3) Crushed leaves + anaerobic fermentation

[0131] Withering: The fresh tobacco leaves are woven into stalks and evenly hung in the withering machine. The desired temperature is then set for withering. During this process, the condition of the tobacco leaves and the withering process must be frequently observed. If uneven withering occurs, the position of the tobacco leaves on the stalks must be adjusted promptly. The withering temperature is set to 38℃ for the dry bulb and 36℃ for the wet bulb. The tobacco leaves are removed when the moisture content reaches 60%.

[0132] Shredded tobacco leaves: Cut the tobacco leaves (with the main vein removed) into 5*5cm pieces using scissors or other tools, and soak them in an equal amount of distilled water for subsequent anaerobic fermentation.

[0133] Anaerobic fermentation: Prepare a sealable glass or ceramic fermentation tank with a one-way valve. Put the crushed tobacco leaves into the tank. The tobacco leaf homogenate should account for about 80% of the total volume of the fermentation tank. Fill the one-way valve with water to seal it and place it in the appropriate temperature environment for fermentation.

[0134] Drying: Take out the fermented tobacco leaf homogenate and spread it evenly on a drying tray. After placing it, turn on the dryer to preheat it. After reaching the set temperature, put the drying tray into the dryer and dry it at 85℃ for 20 minutes. After the tobacco leaf homogenate is basically dry, take it out and let it rest for 2 hours to rehydrate. Set the temperature of the aroma enhancer to 105℃. After reaching the set temperature, put the rehydrated tobacco leaf homogenate into the dryer and take it out after it is completely dry.

[0135] Test treatment

[0136] Using the optimal process parameters A1B3 from Example 1 as experimental conditions, the differences between flue-cured tobacco, anaerobic fermentation of crushed leaves, and anaerobic fermentation of homogenized tobacco were compared. CK is the raw material for heated non-combustible cigarettes made from traditionally flue-cured tobacco leaves; YJ is the raw material for tobacco leaves after anaerobic fermentation following homogenization; and SY is the raw material for tobacco leaves after anaerobic fermentation following crushing.

[0137] According to the results in Table 10, apart from total chlorine, which showed no significant differences in the main chemical components of tobacco leaves among the three treatments, all other component indicators showed significant differences. It is noteworthy that the YJ treatment had the lowest values ​​among the three treatments for total sugar, reducing sugar, total alkali, total nitrogen, total potassium, and starch, and the order of content was CK > SY > YJ. This indicates that homogenization fermentation consumed the main chemical components of tobacco leaves more strongly. Compared to the SY treatment, its sugar-to-saccharide ratio was 0.99, indicating that almost all the main sugars in the tobacco leaves were converted into reducing sugars at this stage.

[0138] Table 10. Conventional Chemical Composition of Different Treatment Methods

[0139]

[0140] According to the results in Table 11, YJ showed the best overall evaluation among the three treatments, significantly outperforming the others in aroma and taste characteristics. It is noteworthy that this anaerobic fermentation method using crushed leaves did not improve the smoking quality of the tobacco leaves; instead, it caused a severe loss of quality. The textual evaluation descriptions reveal that the YJ treatment had the best aroma intensity, exhibiting a sweet, hay-like, and smoky aroma distinct from that of HNB cigarettes made from flue-cured tobacco. Furthermore, the crushed leaf fermentation method also led to the loss of the natural tobacco aroma.

[0141] Table 11 Sensory evaluation scores and assessments for different treatment methods

[0142]

[0143] Under the same fermentation conditions, the composition and smoking quality of tobacco leaves changed significantly after the fermentation morphology was altered. The experimental results indicate that the crushed leaf fermentation method is merely a means of depleting the original quality of the tobacco leaves. Based on the aroma composition analysis in Example 1, it is speculated that anaerobic fermentation does indeed alter the aroma composition of tobacco leaves. The effective aroma components of the tobacco leaves can be retained in the solid-liquid mixture after homogenization. It is presumed that water-soluble aroma components are lost during the drying process as the fermentation liquid is discharged, while the anaerobic homogenized fermentation process retains the original moisture system during drying, allowing the effective volatile aroma components to be locked in the dried tobacco raw material.

[0144] 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 method of making a heat-not-burn tobacco product, characterised in that, The preparation method comprises the following steps: Step 1, obtaining tobacco homogenate by wilting and crushing fresh tobacco leaves; Step 2, obtaining the heated but not combusted tobacco product by segment drying the fermentation homogenate obtained by anaerobic fermentation of the tobacco homogenate in step 1.

2. The production method according to claim 1, characterized by, In step 1, the wilting comprises wilting the fresh tobacco leaves in an environment with a dry ball temperature of 38-40℃ and a wet ball temperature of 35-36℃, and the moisture content of the wilted tobacco leaves is 60%-65%.

3. The preparation method according to claim 1, characterized in that, In step 1, the crushing comprises crushing the wilted tobacco leaves mixed with water at a mass ratio of 1: (1-2), the rotation speed of the crushing is 20,000-40,000 r / min, and the crushing time is 20-40 min.

4. The production method according to claim 3, characterized by, The mass ratio of the wilted tobacco leaves to water is 1:1.5, the rotation speed of the crushing is 30,000 r / min, and the crushing time is 30 min.

5. The preparation method according to claim 1, characterized in that, In step 2, the container for anaerobic fermentation is a fermentation tank with a sealed and one-way valve, the fermentation tank is a glass fermentation tank or a ceramic fermentation tank, and the fermentation tank contains 60vol%-80vol% of the tobacco homogenate.

6. The method of claim 1, wherein, In step 2, the temperature for anaerobic fermentation is 40-60℃, and the time is 3-9 days.

7. The preparation method according to claim 6, characterized in that, the temperature for anaerobic fermentation is 40℃, and the time is 9 days; or the temperature for anaerobic fermentation is 60℃, and the time is 9 days.

8. The method of claim 1, wherein, In step 2, the segment drying comprises primary drying and secondary drying, wherein: the primary drying comprises drying the fermentation homogenate at 80-85℃ for 100-120 min, and placing the dried fermentation homogenate for 1.5-3 h for moisture recovery; the secondary drying comprises drying the moisture-recovered fermentation homogenate at 100-105℃ until completely dry.

9. The preparation method according to claim 8, characterized in that, the primary drying comprises drying the fermentation homogenate at 85℃ for 120 min, and placing the dried fermentation homogenate for 2 h for moisture recovery; the secondary drying comprises drying the moisture-recovered fermentation homogenate at 105℃ until completely dry.

10. The heated but not combusted tobacco product prepared by the preparation method according to any one of claims 1-9.

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