Method for improving cigar tobacco quality through mixed enzyme enhanced fermentation, cigar tobacco and application of cigar tobacco

Abstract

The invention relates to a method for improving cigar tobacco quality through mixed enzyme enhanced fermentation, cigar tobacco and application of the cigar tobacco, and belongs to the technical field of tobacco processing. According to the method, a cigar tobacco raw material is sequentially subjected to cellulase treatment and protease treatment, and chemical components such as cellulose, protein, saccharides and amino acid in cigar tobacco are regulated and controlled under mild conditions by combining solid state fermentation. Compared with a single enzyme treatment method or a traditional fermentation method, the method has the advantages that the content of unfavorable macromolecular substances in cigar tobacco can be reduced, and accumulation of aroma precursor substances is promoted, so that the aroma release characteristic and smoke comfort of the cigar tobacco under the condition of lower heating temperature are improved; the method is suitable for improving the quality of cigar tobacco raw materials for heat-not-burn tobacco products, and has good stability and industrial application prospects.

Description

A method for enhancing cigar tobacco quality through mixed enzyme-enhanced fermentation, cigar tobacco and its applications Technical Field

[0001] This invention belongs to the field of tobacco processing and bio-fermentation technology, specifically relating to a method for enhancing the quality of cigar tobacco through mixed enzyme-enhanced fermentation, cigar tobacco, and its applications. Background Technology

[0002] Cigar tobacco is renowned for its rich aroma, full-bodied smoke, and complex flavor profile, making it a valuable ingredient in high-end tobacco products. With the development of heated tobacco products, tobacco raw materials need to effectively release their aroma and maintain a pleasant smoke even at relatively low heating temperatures. Cigar tobacco, due to its natural aroma advantages, is considered to have significant application potential. However, compared to flue-cured tobacco and other tobacco types, cigar tobacco typically has a higher content of cellulose and protein and a lower content of soluble sugars. Cellulose and protein are prone to producing irritating and unpleasant odors during heating or thermal decomposition, while the insufficient content of aroma precursors such as sugars hinders the formation and release of aroma compounds, resulting in a lack of sweetness and harmony in the smoke, thus limiting its effectiveness in heated cigarettes.

[0003] Currently, improving the quality of cigar tobacco mainly relies on natural aging or traditional fermentation processes. These methods are time-consuming, inefficient, and have limited effectiveness in degrading undesirable macromolecules. Previous studies have attempted to treat tobacco with cellulase or protease to promote the degradation of cellulose or protein; however, single-enzyme treatments cannot simultaneously regulate multiple key chemical components, resulting in limited quality improvement and failing to meet the comprehensive requirements of aroma release and smoke comfort in heated tobacco products. Therefore, there is an urgent need for an improved method suitable for cigar tobacco, capable of efficiently regulating the structure of multiple chemical components under mild conditions, and significantly enhancing its quality in heated tobacco products. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides a method for improving the quality of cigar tobacco through mixed enzyme-enhanced fermentation, cigar tobacco and its application. The method utilizes a mixture of cellulase and protease for treatment combined with solid-state fermentation to improve the chemical composition and sensory quality of cigar tobacco. This method is particularly suitable for improving the quality of cigar tobacco raw materials used in heated tobacco products.

[0005] To achieve the above objectives, the present invention adopts the following technical solution.

[0006] In a first aspect, the present invention provides a method for improving the quality of cigar tobacco through mixed enzyme-enhanced fermentation, comprising the following steps: Step 1, removing the stems from cigar tobacco leaves, pulverizing them, and sieving them to obtain tobacco powder; Step 2, adding cellulase solution to the cigar tobacco raw material, and performing a first enzymatic hydrolysis at 40-45 ℃ for 3-5 h to obtain cigar tobacco raw material after one enzymatic hydrolysis; Step 3, adding protease solution to the cigar tobacco raw material after one enzymatic hydrolysis in Step 2, and performing a second enzymatic hydrolysis at 45-55 ℃ for 8-12 h to obtain cigar tobacco raw material after two enzymatic hydrolysis; Step 4, performing solid-state fermentation of the cigar tobacco raw material after two enzymatic hydrolysis at 20-25 ℃ and 60-75% relative humidity; Step 5, drying the cigar tobacco raw material after solid-state fermentation to obtain cigar-flavored heated cigarette tobacco segment raw material, and then processing it by rolling and shredding to obtain heated cigarettes, i.e., cigar tobacco products.

[0007] The present invention provides a method for enhancing the quality of cigar tobacco through mixed enzyme-enhanced fermentation. This method combines exogenous enzyme treatment with solid-state fermentation to regulate the chemical composition and structure of cigar tobacco under mild conditions, thereby improving its sensory quality and performance. Specifically, a stepwise enzymatic hydrolysis method is employed. First, cellulase is used for a primary enzymatic hydrolysis to generate water-soluble cellulose degradation products (i.e., oligosaccharide and other sugar precursors). Then, neutral protease is used for a secondary enzymatic hydrolysis to generate free amino acids (i.e., aldehydes, ketones, esters, and heterocyclic aroma precursors). This avoids inter-enzyme interference and increases the concentration of target aroma precursors.

[0008] Furthermore, the primary enzymatic hydrolysis temperature of cellulase (40-45℃) and the secondary enzymatic hydrolysis temperature of neutral protease (45-50℃) were determined based on the results of systematic single-factor optimization experiments and orthogonal experiments, and were not arbitrarily selected. The cellulase hydrolysis temperature was chosen to avoid damaging the sugars and aroma precursors in tobacco; 40-45℃ is the high-activity zone of cellulase, within which it can effectively degrade cellulose without destroying the sugars and aroma precursors in tobacco.

[0009] Optionally, in step 1, the cigar tobacco leaves are selected from sun-dried cigar tobacco leaves.

[0010] Optionally, in step 1, after removing the stems from the cigar tobacco leaves, they are pulverized at 20℃-25℃ and then passed through a 200-mesh sieve to obtain tobacco powder.

[0011] To further improve the tobacco quality of high-quality cigars, this invention removes the stems from cigar tobacco leaves to reduce lignin and bitterness sources, thereby improving the purity and smoothness of the aroma.

[0012] Preferably, in step 2, the first enzymatic hydrolysis partially hydrolyzes the cellulose in the cigar tobacco raw material.

[0013] This application involves partial enzymatic hydrolysis of cellulose in cigar tobacco raw materials to reduce irritation while maintaining the tobacco structure. In contrast, complete enzymatic hydrolysis is insufficient to preserve the tobacco structure. This partial enzymatic hydrolysis method, which preserves the tobacco structure, results in hydrolysis products that primarily consist of cellulose and protein degradation products after secondary enzymatic hydrolysis of the cigar tobacco raw materials. The types of sugar components are relatively uniform, the fiber structure is well preserved, the system viscosity is relatively low, the particles are dispersed, and the physical state is loose and uniformly distributed. These structural characteristics are beneficial for improving permeability and aeration during solid-state fermentation, making aroma precursors more readily available, thereby promoting the formation of target aroma compounds and improving the stability and controllability of the final product's flavor.

[0014] Optionally, in step 2, the cellulase is derived from a cellulase preparation made from microorganisms.

[0015] Preferably, the cellulase and protease are industrial enzyme preparations obtained by microbial fermentation.

[0016] Preferably, in step 3, the protease includes a neutral protease.

[0017] In this invention, the primary enzymatic hydrolysis is a directed enzymatic hydrolysis of cellulose in cigar tobacco by cellulase. Its mechanism of action involves hydrolyzing β-1,4-glycosidic bonds, converting cellulose into oligosaccharides and soluble sugars. The secondary enzymatic hydrolysis is a protease hydrolysis of proteins in cigar tobacco, converting large protein molecules into small peptides and free amino acids. After enzymatic treatment with cellulase and protease, followed by solid-state fermentation, the total amino acid content is significantly increased, while the protein content is significantly decreased. Preferably, the secondary enzymatic hydrolysis uses a neutral protease. The purpose is to use a highly active neutral protease within the pH range of the tobacco system to generate amino acids in a gentle manner, avoiding the production of bitter and irritating substances during enzymatic hydrolysis, which would negatively impact tobacco quality. To avoid the formation of undesirable decomposition products from proteins in cigar tobacco raw materials during enzymatic hydrolysis, the enzymatic hydrolysis temperature of the neutral protease (i.e., the secondary enzymatic hydrolysis temperature) is selected as 45-50℃, converting proteins in the cigar tobacco raw materials into amino acids without producing undesirable decomposition products.

[0018] Optionally, in step 2, the amount of cellulase used in a single enzymatic hydrolysis is 150-210 U / g (e.g., 160 U / g, 180 U / g, or 200 U / g) relative to the amount of cigar tobacco raw material used. That is, the amount of cellulase used for enzymatic hydrolysis of each gram of cigar tobacco raw material is 150-210 U.

[0019] Furthermore, in step 3, the amount of protease used in the secondary enzymatic hydrolysis is 150-210 U / g (e.g., 160 U / g, 180 U / g, or 200 U / g) relative to the amount of cigar tobacco raw material used. That is, the amount of protease used for enzymatic hydrolysis of each gram of cigar tobacco raw material is 150-210 U.

[0020] In this application, the total amount of water used in step 2 (adding cellulase solution) and step 3 (adding protease solution) is 0.4-0.8 mL / g (e.g., 0.5 mL / g, 0.6 mL / g, or 0.7 mL / g).

[0021] Furthermore, in step 4, no additional exogenous microorganisms are inoculated during the solid-state fermentation process.

[0022] Furthermore, in step 4, the solid-state fermentation time is 5-10 days (e.g., 6 days, 7 days, or 8 days).

[0023] In this application, to ensure sufficient accumulation of sugars and amino acids during solid-state fermentation, a fermentation time of 5-10 days is selected. If the solid-state fermentation time is too short, the conversion of sugars and amino acids will be insufficient; if the solid-state fermentation time is too long, the converted sugars and amino acids will be easily consumed by microorganisms, and there is also a risk of mold growth.

[0024] Furthermore, in step 4, the solid-state fermentation is carried out in a constant temperature and humidity fermentation chamber with a humidification device to control the environment at a relative humidity of 60-75%.

[0025] Furthermore, in step 5, the drying temperature is 50-80°C (e.g., 60°C or 70°C).

[0026] The present invention provides a method for enhancing the quality of cigar tobacco through mixed enzyme-enhanced fermentation. This method involves sequentially treating the cigar tobacco raw material with cellulase, protease, and then performing solid-state fermentation. This effectively degrades cellulose and protein in the cigar tobacco while promoting the accumulation of aroma precursors such as sugars and amino acids. Furthermore, the synergistic action of cellulase and protease on the cigar tobacco raw material reduces cellulose and protein content while increasing sugar and amino acid content, achieving synergistic regulation of various undesirable macromolecules.

[0027] Preferably, compared to cigar tobacco obtained by sterile water fermentation, cigar tobacco obtained by mixed enzyme enhanced fermentation has a 15% to 16% lower cellulose content (e.g., 15.61%), a 11% to 12% lower protein content (e.g., 11.77%), a 1.5 to 1.6 times higher total soluble sugar content (e.g., 1.53 times), a 2 to 2.1 times higher reducing sugar content (e.g., 2.03 times), and a 0.1 to 0.2 times higher amino acid content (e.g., 0.13 times).

[0028] In this invention, the cellulose and protein content in the cigar tobacco is reduced, while the content of reducing sugar, total sugar, and amino acids is increased, resulting in enhanced aroma, harmony, and taste.

[0029] Secondly, the present invention also provides a cigar tobacco prepared by the above method.

[0030] Preferably, during the pyrolysis process at 350°C, the cigar tobacco exhibits the following characteristics in the pyrolysis smoke: 4,7,9-mega-stigmatrien-3-one, which imparts floral and woody aromas, is released in an amount >270 μg / g; 3-methylacetophenone, which imparts fruity and fresh aromas, is released in an amount >100 μg / g; citral is released in an amount >50 μg / g; nicotine is released in an amount >7100 μg / g; and dihydroactinolone, which enhances fruity aromas and masks off-flavors, is released in an amount greater than 200 μg / g.

[0031] Thirdly, the present invention also provides an application of the above-mentioned cigar tobacco in heated tobacco products.

[0032] In this invention, the above technical solutions can be freely combined to form new technical solutions, provided that they do not conflict with each other.

[0033] Compared with the prior art, the technical solution of the present invention has the following beneficial technical effects: (1) The method of improving the quality of cigar tobacco by mixed enzyme enhanced fermentation provided by the present invention can effectively degrade cellulose and protein in cigar tobacco through the synergistic effect of cellulase and protease, which is better than the single enzyme treatment method; after mixed enzyme treatment and fermentation, the content of aroma precursors such as sugars and amino acids in cigar tobacco is increased, which is conducive to the formation and release of aroma substances; (2) The method of improving the quality of cigar tobacco by mixed enzyme enhanced fermentation provided by the present invention uses cellulase without α-amylase and pectinase for the first enzymatic hydrolysis, so no pectin and starch degradation products are produced; after the second enzymatic hydrolysis, the hydrolysis products mainly include cellulose degradation products and protein degradation products, the sugar composition is relatively simple, the fiber structure is well maintained, the system viscosity is relatively low, the particles are dispersed, and the physical state is loose and uniformly distributed; this structural characteristic is conducive to improving permeability and aeration in the solid fermentation process, making the aroma precursors easier to utilize, thereby promoting the generation of target aroma compounds. (3) The method of improving the quality of cigar tobacco by mixed enzyme enhanced fermentation provided by the present invention uses cellulase to partially hydrolyze the cellulose in cigar tobacco. While preserving the integrity of the tobacco structure (i.e., without destroying the structure of the cigar tobacco) and the aroma release channel, it improves the quality of cigar tobacco under heating and non-combustion conditions by directional regulation of unfavorable macromolecules and enrichment of aroma precursors. (4) The method of improving the quality of cigar tobacco by mixed enzyme enhanced fermentation provided by the present invention can significantly improve the fermentation efficiency and shorten the fermentation cycle under milder conditions, which is suitable for industrial application. The low-temperature solid-state fermentation of the present invention is conducive to promoting the further transformation of microbial metabolism and enzymatic hydrolysis products without consuming aroma precursors, avoiding the problem of excessive consumption of sugar and amino acids and the generation of irritating substances caused by traditional high-temperature fermentation. At the same time, room temperature fermentation is conducive to reducing energy consumption. (5) The cigar tobacco treated by the method of the present invention exhibits better aroma concentration, smoke comfort and overall sensory quality in heated and non-combustion cigarettes. Attached Figure Description

[0034] Figure 1 shows the effects of different treatments on the main chemical components in cigar tobacco powder; where A: cellulose; B: protein; C: total soluble sugar; D: reducing sugar; E: total amino acids; F: total alkaloids; UF in the figure is the untreated control sample in Comparative Example 4, CK is the sterile water fermentation control sample in Comparative Example 3, CT is the cellulase-treated sample in Comparative Example 1, PT is the protease-treated sample in Comparative Example 2, and CPT is the sample treated with a mixture of cellulase and protease in Example 1; different letters (lowercase letters a, b, c, d, etc.) indicate statistically significant differences (p < 0.05).

[0035] Figure 2 shows the distribution of aroma components in cigar tobacco powder obtained by different treatment methods in Example 1 and Comparative Examples 1-4 under heating conditions. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions in the embodiments of the invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0037] Example 1: A method for enhancing the quality of cigar tobacco through mixed enzyme-enhanced fermentation, comprising the following steps.

[0038] Step 101, tobacco raw material pretreatment: Select sun-dried cigar tobacco leaves, remove the tobacco stems and crush them at 20℃-25℃, pass them through a 200-mesh sieve, and take the sieve material to obtain cigar tobacco powder for later use.

[0039] Step 102, single enzymatic hydrolysis: The cigar tobacco powder obtained in Step 101 is placed in a clean container, and a cellulase solution is added via spray to a concentration of 210 U / g. The mixture is then left to stand at 40°C for 4 hours to partially hydrolyze the cellulose in the tobacco. Here, "partial hydrolysis" means that the cellulase treatment causes significant degradation of the cellulose in the cigar tobacco, but not complete decomposition. The final effect is a 10-15% reduction in cellulose and protein in the fermented cigar tobacco. This "partial enzymatic hydrolysis" can reduce the irritating macromolecules while preserving the structural integrity and aroma release pathways of the tobacco. Simultaneously, total sugars, reducing sugars, and amino acids significantly increase and are converted into furans, aldehydes, ketones, esters, and heterocyclic aroma compounds during heating and pyrolysis, resulting in a richer aroma and smoother smoke in the CPT group. The cellulase solution is prepared by dissolving a cellulase preparation (10000 U / g, Shanghai Maclean Biochemical Technology Co., Ltd.) in water.

[0040] Step 103, Secondary Enzymatic Hydrolysis: After completing Step 102, a protease solution is sprayed onto the tobacco powder to a neutral protease concentration of 210 U / g. The mixture is treated at 50°C for 10 h to hydrolyze the proteins in the tobacco into small peptides and amino acids, resulting in cigar tobacco powder treated with the mixed enzymes. The protease solution is prepared by dissolving a neutral protease preparation (50,000 U / g, Beijing Solarbio Science & Technology Co., Ltd.) in water.

[0041] Step 104, solid-state fermentation: The cigar tobacco powder treated with mixed enzymes is placed in a solid-state fermentation environment of 20-25℃ and 60%-70% relative humidity for 7 days.

[0042] Step 105, Drying treatment: After fermentation, the tobacco is dried at 60-70℃ to obtain a cigar tobacco sample with mixed enzyme enhanced fermentation, denoted as CPT.

[0043] Comparative Example 1: A method for improving the quality of cigar tobacco, which involves treating cigar tobacco powder with a single cellulase, specifically including the following steps.

[0044] Step 101, tobacco raw material pretreatment: Select sun-dried cigar tobacco leaves, remove the tobacco stems and crush them at 20℃-25℃, pass them through a 200-mesh sieve, and take the sieve material to obtain cigar tobacco powder for later use.

[0045] Step 102, Cellulase treatment: Place the cigar tobacco powder obtained in step 101 in a clean container, add cellulase solution by spraying (same as in Example 1), so that the amount of cellulase is 210 U / g, and let it stand at 40°C for 4 h to allow partial hydrolysis of the cellulose in the tobacco.

[0046] Step 103, solid-state fermentation. The cigar tobacco powder treated with cellulase is placed in a solid-state fermentation environment at 20-25℃ and 60%-70% relative humidity for 7 days.

[0047] Step 104, Drying treatment. After fermentation, the tobacco is dried, and the resulting cigar tobacco powder sample is designated as CT, i.e., cellulase-treated sample.

[0048] Comparative Example 2: A method for improving the quality of cigar tobacco, which involves treating cigar tobacco powder with a single protease, specifically including the following steps.

[0049] Step 101, tobacco raw material pretreatment: Select sun-dried cigar tobacco leaves, remove the tobacco stems and crush them at 20℃-25℃, pass them through a 200-mesh sieve, and take the sieve material to obtain cigar tobacco powder for later use.

[0050] Step 102, Protease treatment: Place the cigar tobacco powder obtained in step 101 in a clean container, and add protease solution by spraying (same as in Example 1) to make the amount of neutral protease 210 U / g. Treat at 50°C for 10 hours to hydrolyze the proteins in the tobacco into small molecule peptides and amino acids; and obtain cigar tobacco powder treated with protease.

[0051] Step 103, solid-state fermentation: The cigar tobacco powder treated with protease is placed in a solid-state fermentation environment of 20-25℃ and 60%-70% relative humidity for 7 days.

[0052] Step 104, Drying treatment: After fermentation, the tobacco is dried at 60-70℃. The resulting cigar tobacco powder sample is denoted as PT, i.e., protease-treated sample.

[0053] Comparative Example 3 is a method for improving the quality of cigar tobacco, which differs from Example 1 in that no exogenous enzymes are added, that is, no cellulase and protease are added, only an equal volume of sterile water is sprayed on. The method specifically includes the following steps.

[0054] Step 101, tobacco raw material pretreatment: Select sun-dried cigar tobacco leaves, remove the tobacco stems and crush them at 20℃-25℃, pass them through a 200-mesh sieve, and take the sieve material to obtain cigar tobacco powder for later use.

[0055] Step 102, Sterile water treatment: Place the cigar tobacco powder obtained in step 101 in a clean container, add sterile water by spraying, and let it stand at 40°C for 4 hours.

[0056] Step 103, solid-state fermentation: The cigar tobacco powder treated with sterile water is placed in a solid-state fermentation environment of 20-25℃ and 60%-70% relative humidity for 7 days.

[0057] Step 105, Drying treatment: After fermentation, the tobacco is dried at 60-70℃. The resulting cigar tobacco powder sample is recorded as CK, i.e., the sterile water fermentation control sample.

[0058] Comparative Example 4: A method for preparing cigar tobacco, which, compared with Example 1, does not involve enzyme treatment or solid-state fermentation.

[0059] Specifically, in step 101, tobacco raw material pretreatment: select sun-dried cigar tobacco leaves, remove the tobacco stems and crush them at 20℃-25℃, pass them through a 200-mesh sieve, and take the sieve-passing material. The resulting cigar tobacco powder is used as an untreated control sample and is denoted as UF.

[0060] The effects of mixed enzyme-enhanced fermentation on the chemical composition regulation of cigar tobacco powder were investigated. The contents of cellulose, protein, total soluble sugar, reducing sugar, total amino acids, and total alkaloids in cigar tobacco samples obtained before and after solid-state fermentation in Example 1 and Comparative Examples 1-4 were determined. Specifically, the determination of total soluble sugar and reducing sugar content followed YC / T 159-2019; the determination of total free amino acids followed GB / T 8314-2013; the determination of cellulose content followed GB 5009.88-2014; the determination of protein content followed standard YC / T 249-2008; and the determination of total alkaloid content followed YC / T 468-2013. Figure 1 shows the effects of different treatment methods on the main chemical components in cigar tobacco powder, where 0 d represents the chemical composition content of the cigar tobacco sample before solid-state fermentation; and 7 d represents the chemical composition content of the cigar tobacco sample after solid-state fermentation.

[0061] As shown in Figure 1, compared with the untreated control sample UF in Comparative Example 4 and the sterile water fermentation control sample CK in Comparative Example 3 (i.e., the enzyme-free fermented cigar tobacco sample), the cellulase-treated cigar tobacco sample CT in Comparative Example 1, the protease-treated cigar tobacco sample PT in Comparative Example 2, and the mixed enzyme-treated cigar tobacco sample CPT in Example 1 all exhibited a regulatory effect on the chemical composition of the original cigar tobacco powder. Among them, the CPT group in Example 1 showed a comprehensive advantage in regulating the chemical composition of the cigar tobacco powder. Specifically, before solid-state fermentation, the content of cellulose and protein was significantly reduced, while the content of total soluble sugar, reducing sugar, and total amino acids was increased. After 7 days of solid-state fermentation, these changes were further enhanced, with the content of cellulose and protein continuing to decrease, the content of total soluble sugar, reducing sugar, and amino acids significantly increasing, while the content of total alkaloids only decreased slightly. Specifically, compared to the sterile water fermentation control (CK), the mixed enzyme fermentation of cigar tobacco powder resulted in a 15.61% decrease in cellulose content, an 11.77% decrease in protein content, a 1.53-fold increase in total soluble sugars, a 2.03-fold increase in reducing sugars, and a 0.13-fold increase in amino acid content. These results indicate that mixed enzyme treatment can effectively optimize the sugar-to-alkali ratio of tobacco (from 0.57 to 1.61) while maintaining tobacco characteristics, achieving comprehensive regulation of key chemical components.

[0062] Sensory quality comparison of heated tobacco cigarettes with different treated cigar tobacco powders: The cigar tobacco powders prepared in Example 1 and Comparative Examples 1-4 were used to prepare heated tobacco cigarettes. The product labeling of the heated tobacco cigarettes retained the sample labeling used for the cigar tobacco powders in each example and comparative example. Under the same heating conditions, five professional smokers conducted sensory evaluations of each group of samples. Evaluation indicators included smoke volume, aroma, strength, harmony, irritation, taste, and total score.

[0063] Sensory quality was calculated according to the Yunnan Tobacco Industry Standard "Sensory Evaluation Method for New Types of Cigarettes" (Q / YNZY.J04.022—2015). Specifically, the evaluation included indicators such as smoke volume (10 points), aroma and flavor (30 points), physiological intensity (10 points), harmony (10 points), irritation (15 points), and taste (25 points). Each score was the average of five experts. Table 1 shows the sensory evaluation results of heated tobacco cigarettes prepared from cigar tobacco powder processed using different methods.

[0064] Table 1. Sensory evaluation results of heated tobacco cigarettes prepared from cigar tobacco powder processed by different methods. Note: Different lowercase letters a, b, c, d under the same indicator represent significant differences, P ≤ 0.05. Specifically, this means that statistical analysis (such as ANOVA and post-hoc multiple comparisons) is performed on data from different samples under the same indicator (e.g., cellulose content, protein content, total sensory score, etc.). If two samples are labeled with different letters (e.g., a and b, b and c), it indicates that the difference between them on that indicator is statistically significant at a 95% confidence level (P ≤ 0.05); if any of the labeled letters are the same, it indicates that the difference between the samples is not significant.

[0065] As shown in Table 1, compared with the untreated control sample UF and the sterile water fermentation control sample CK, the cellulase-treated sample CT, the protease-treated sample PT, and the mixed enzyme-treated sample CPT all significantly improved the sensory quality of heated tobacco products. Among them, the mixed enzyme-treated sample CPT achieved the highest or higher scores in key indicators such as aroma, flavor, harmony, irritation, and taste, and had the best overall sensory quality. The sensory quality of the single enzyme-treated samples was between that of CPT and the control group.

[0066] A comparative analysis of aroma component release from cigar tobacco powder under different treatments was conducted. Cigar tobacco powder samples from Example 1 (CPT) and Comparative Examples 1-3 (CT, PT, CK) were subjected to thermal pyrolysis at 350 °C. The composition and relative release amounts of various aroma components in the pyrolyzed smoke were analyzed by GC-MS. The results showed that after stepwise enzymatic hydrolysis and natural fermentation at 25 °C for 7 days (Example 1), the pyrolysis (350 °C) of the cigar tobacco powder released 61 aroma substances, significantly more than the 44 aroma substances released by the untreated control group (i.e., the sterile water treatment control group of Comparative Example 3). Furthermore, the release amounts and proportions of alkaloids, alkenes, aldehydes and ketones, esters, alcohols, phenols, heterocyclic compounds, and organic acids all showed significant changes.

[0067] Table 2. Release amounts of major compounds in pyrolysis flue gas Table 2 shows the release amounts of major compounds in the pyrolysis flue gas. Figure 2 shows the distribution of aroma components in the pyrolysis flue gas of cigar tobacco powder obtained by different treatments in Example 1 and Comparative Examples 1-3.

[0068] As shown in Table 2, compared with the aseptic water fermentation control group (CK), the cellulase-treated group (CT), and the protease-treated group (PT), the mixed enzyme-treated group (CPT) exhibited a significant synergistic advantage in the release of multiple key aroma compounds. Particularly in terms of aldehydes and ketones, the release of 4,7,9-macrostigmatrien-3-one, which imparts floral and woody aroma characteristics, reached 271.72 μg / g in the CPT group, more than double that of the control group (CK), and significantly higher than the single enzyme treatment groups (CT and PT). This indicates that mixed enzyme treatment is more conducive to the conversion of carotenoid aroma precursors.

[0069] In addition, compounds with fruity and fresh aroma characteristics, such as 3-methylacetophenone and citral, were significantly higher in the CPT group than in the CK group and superior to single enzyme treatment, further enriching the aroma layers of the smoke.

[0070] In terms of alkaloids, the nicotine release in the CPT group (7127.65 μg / g) was significantly higher than that in the CK group and comparable to that in the single enzyme treatment groups (i.e., the CT group and the PT group), which can maintain the strength that cigar tobacco should have.

[0071] Meanwhile, the CPT group maintained a high level of dihydroactinol (204.53 μg / g) in esters, which is beneficial for enhancing fruit aroma and masking off-flavors, while phenolic substances in the CPT group were maintained at an appropriate level, avoiding the potential increase in irritation that may occur with single enzyme treatment.

[0072] Overall, the mixed enzyme-enhanced fermentation enhances the value of key aroma compounds such as aldehydes, ketones, and olefins while maintaining the smoothness and harmony of the smoke, resulting in cigar tobacco exhibiting richer, more balanced, and higher-quality aroma characteristics under heating conditions.

[0073] Figure 2 shows the overall proportion of different pyrolysis substances. Compared with the control (CK), the CT, PT, and CPT samples released more aroma components under heating conditions and changed the composition ratio of different categories of aroma substances. Among them, the CPT group showed a comprehensive advantage in the proportion of pyrolysis aroma components. Specifically, the proportions of aldehydes, ketones, and alkaloids were higher than those of other treatment groups, while the proportions of alcohols, phenols, and heterocyclic compounds were moderately reduced, making the main aroma components more prominent and the category distribution more balanced. This shows that the synergistic effect of cellulase and protease optimized the aroma structure and improved the overall aroma level and harmony of cigar tobacco powder.

[0074] In summary, this invention addresses the problems of high cellulose and protein content, low sugar and amino acid content, and limited aroma release under heat-not-burn conditions in cigar tobacco. It proposes a method for improving cigar tobacco by combining cellulase and protease treatment with solid-state fermentation. Through stepwise enzymatic hydrolysis and enzyme-enhanced fermentation, this method not only alters the composition and structure of the hydrolysis products and increases the content of aroma precursors, but also significantly affects the types, release amounts, and proportions of aroma substances in the fermentation products. Compared to single-enzyme treatment or traditional fermentation methods, this invention, through the synergistic effect of multiple enzymes, simultaneously reduces the content of unfavorable macromolecular substances in cigar tobacco and promotes the generation of aroma precursors such as sugars and amino acids during the same process, thereby significantly improving the chemical composition structure of the tobacco. Cigar tobacco treated with this method can release richer and more harmonious aroma components at lower heating temperatures, exhibiting significant advantages in aroma, flavor, harmony, and smoke comfort in heat-not-burn cigarettes, demonstrating good stability and industrial application value.

[0075] The above description is merely a specific embodiment of this application, enabling those skilled in the art to understand or implement this application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of this application. Therefore, this application is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A method for enhancing the quality of cigar tobacco through mixed enzyme-enhanced fermentation, characterized in that, The process includes the following steps: Step 1: After removing the stems from the cigar tobacco leaves, crush them and sieve them to obtain tobacco powder; Step 2: Add cellulase solution to the cigar tobacco raw material and perform a first enzymatic hydrolysis at 40-45 ℃ for 3-5 h to obtain cigar tobacco raw material after one enzymatic hydrolysis; Step 3: Add protease solution to the cigar tobacco raw material after one enzymatic hydrolysis in Step 2 and perform a second enzymatic hydrolysis at 45-55 ℃ for 8-12 h to obtain cigar tobacco raw material after two enzymatic hydrolysis; Step 4: Perform solid-state fermentation on the cigar tobacco raw material after two enzymatic hydrolysis at 20-25 ℃ and 60-75% relative humidity; Step 5: Dry the solid-state fermented cigar tobacco raw material to obtain cigar-flavored heated cigarette tobacco segment raw material, and then process it by rolling and shredding to obtain cigar tobacco products.

2. The method for enhancing cigar tobacco quality through mixed enzyme-enhanced fermentation according to claim 1, characterized in that, In step 1, the cigar tobacco leaves are selected from sun-dried cigar tobacco leaves; in step 1, after removing the tobacco stems, the cigar tobacco leaves are pulverized at 20℃-25℃ and then passed through a 200-mesh sieve to obtain tobacco powder.

3. The method for enhancing cigar tobacco quality through mixed enzyme-enhanced fermentation according to claim 1, characterized in that, In step 2, the enzymatic hydrolysis partially hydrolyzes the cellulose in the cigar tobacco raw material; in step 2, the cellulase is derived from a cellulase preparation of microorganisms; in step 3, the protease includes neutral protease.

4. The method for enhancing cigar tobacco quality through mixed enzyme-enhanced fermentation according to claim 1, characterized in that, In step 2, the amount of cellulase used in the first enzymatic hydrolysis is 150-210 U / g relative to the amount of cigar tobacco raw material used; in step 3, the amount of protease used in the second enzymatic hydrolysis is 150-210 U / g relative to the amount of cigar tobacco raw material used; and / or the total amount of water used in the cellulase solution added in step 2 and the protease solution added in step 3 is 0.4-0.8 mL / g.

5. The method for enhancing cigar tobacco quality through mixed enzyme-enhanced fermentation according to any one of claims 1-4, characterized in that, In step 4, no additional exogenous microorganisms are inoculated during the solid-state fermentation process.

6. The method for enhancing cigar tobacco quality through mixed enzyme-enhanced fermentation according to any one of claims 1-4, characterized in that, In step 4, the solid-state fermentation time is 5-10 days; and / or in step 4, the solid-state fermentation is carried out in a constant temperature and humidity fermentation chamber with a humidification device to control the environment at a relative humidity of 60-75%; and / or in step 5, the drying temperature is 50-80℃.

7. The method for enhancing cigar tobacco quality through mixed enzyme-enhanced fermentation according to claim 6, characterized in that, Compared to cigar tobacco obtained through sterile water fermentation, cigar tobacco obtained through enzyme-enhanced fermentation has a 15%–16% lower cellulose content, a 11%–12% lower protein content, a 1.5–1.6 times higher total soluble sugar content, a 2–2.1 times higher reducing sugar content, and a 0.1–0.2 times higher amino acid content.

8. A type of cigar tobacco, characterized in that, The cigar tobacco is prepared by the method of enhancing cigar tobacco quality through mixed enzyme fermentation according to any one of claims 1-7.

9. The cigar tobacco according to claim 8, characterized in that, During the pyrolysis process at 350℃, the cigar tobacco exhibits the following release levels in the pyrolysis smoke: 4,7,9-mega-stigmatrien-3-one, which imparts floral and woody aromas, >270 μg / g; 3-methylacetophenone, which imparts fruity and fresh aromas, >100 μg / g; citral, >50 μg / g; nicotine, >7100 μg / g; and dihydroactinolone, which enhances fruity aromas and masks off-flavors, >200 μg / g.

10. The use of cigar tobacco according to claim 8 or 9 in the preparation of heated tobacco products.

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