Tobacco leaf fermentation and quality improvement method based on oxygen content regulation and application thereof
By using Klebsiella pneumoniae HNYJ-1 and Enterobacter HNYJ-4 fermentation strains and a two-stage fermentation method with oxygen content regulation, the aroma characteristics of tobacco leaves were improved, solving the problem of quality improvement in traditional tobacco processing technology and achieving a significant improvement in aroma quality and aroma quantity.
Patent Information
- Application Number
- CN202511331068.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-17
- Publication Date
- 2025-12-16
AI Technical Summary
Existing tobacco processing techniques are insufficient to effectively improve tobacco quality, especially aroma characteristics. Traditional methods have limited effectiveness and lack the ability to utilize the aroma-producing advantages of facultative anaerobic bacteria.
Klebsiella pneumoniae HNYJ-1 and Enterobacter erythrocyte HNYJ-4 were used as fermentation strains. A compound seed liquid was prepared through a specific process and sprayed on the surface of tobacco leaves after moistening and before re-drying during the tobacco re-drying stage. The fermentation was carried out in two stages, including aerobic and hypoxic fermentation, combined with oxygen content control. After fermentation, the bacteria were inactivated by high-temperature re-drying.
It significantly increases the content of aroma components such as linalool and 2,3,5,6-tetramethylpyrazine in tobacco leaves, improves aroma quality, aroma quantity and fineness, reduces off-flavors and irritation, and has strong process adaptability and compatibility with existing processing procedures.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of tobacco, in particular to a tobacco leaf fermentation quality improvement method based on oxygen content regulation and application thereof. BACKGROUND
[0002] With the increasing demand of the consumer market for cigarette style categories and quality, the supply and demand relationship between tobacco raw materials and cigarette products is increasingly unbalanced, especially the demand for high-quality tobacco is more urgent. Most of the current methods promote the conversion of aroma precursor substances through traditional tobacco processing technology, the main means of which is to control the curing temperature and curing time, and the effect is general. Through the means of microbial fermentation, macromolecular substances in tobacco leaves can be effectively converted to generate aroma, improve tobacco aroma and reduce tobacco impurities, which is an effective way to improve tobacco quality.
[0003] Most of the current methods for improving the quality of tobacco fermentation are aerobic fermentation, which mainly reduces macromolecular substances such as starch and protein, or increases the aroma of tobacco by degrading aroma precursors in tobacco leaves, all of which have certain effects. As a kind of microorganism with outstanding aroma-producing effect, facultative anaerobic aroma-producing bacteria are widely used in the liquor, condiment, daily chemical and other industries. This kind of microorganism such as aroma-producing yeast and lactic acid bacteria can produce rich and mellow volatile aroma substances in a specific low-oxygen environment, and the aroma is more natural and harmonious, containing pyrazine compounds, organic acid esters, alcohols and other aroma components beneficial to improving the quality of tobacco. Making full use of the aroma-producing advantage of facultative anaerobic bacteria can open up a new path for improving the quality of tobacco. SUMMARY
[0004] The technical problem to be solved by the present application is to provide a tobacco leaf fermentation quality improvement method based on oxygen content regulation and application thereof, and to obtain a high-quality tobacco flavor with outstanding aroma characteristics.
[0005] The technical problem to be solved by the present application is solved by the following technical scheme:
[0006] A tobacco leaf fermentation quality improvement method based on oxygen content regulation, comprising the following steps:
[0007] (1) Seed liquid preparation:
[0008] (11) Preparation of culture medium: After the tobacco leaves are crushed, tobacco powder is obtained by passing through a 100-mesh screen. The tobacco powder is mixed with water at a mass ratio of 1:20-1:2, and stirred at a temperature of 30-100℃ for 0.5-12h at a stirring speed of 50rpm. After extraction, the tobacco water extract is filtered with a 200-mesh filter, and the tobacco culture medium is obtained by sterilization at 121℃ for 30min;
[0009] (12) Seed culture: single colonies of Klebsiella HNYJ-1 and Enterobacter HNYJ-4 are inoculated into different bottles of tobacco culture medium, cultured at 25-40℃, 100-200r / min in a shaker for 6-36h, to obtain seed liquid of the two strains, at this time OD 600 value is between 1.5-2.5;
[0010] (13) Preparation of composite seed liquid: Klebsiella HNYJ-1 and Enterobacter HNYJ-4 seed liquids are mixed according to a mass ratio of 1:10-10:1 (optimum 2:1) to obtain a composite seed liquid;
[0011] (2) Oxygen content regulation for tobacco fermentation:
[0012] In the process segment after leaf conditioning and before redrying in the tobacco redrying stage, the composite seed liquid is sprayed on the surface of the tobacco leaves in a quantitative manner, and the mass ratio of the composite seed liquid to the tobacco leaves is controlled to be 1:1000-1:50; the sprayed tobacco leaves are transferred to a closed temporary storage cabinet, the cabinet volume is 80%, the temperature is controlled to be 30℃±1℃, the humidity is controlled to be 60%±5%, and an oxygen content detection device is placed in the temporary storage cabinet; the cabinet door is opened to make the air circulate, and the tobacco leaves are left to ferment for 12h for aerobic fermentation; then the tobacco leaves are turned over, the cabinet door is closed, and the oxygen content in the cabinet is adjusted by filling nitrogen and sterile air, the adjustment is performed once per hour, and the oxygen content is maintained between 5%-20% for low-oxygen fermentation for 24h; after the fermentation is completed, the tobacco leaves are transferred to the redrying process to start high-temperature redrying, and the normal processing process, and the bacterial strain is inactivated.
[0013] Preferably, in the above technical solution, the Enterobacter HNYJ-4 is preserved in the China General Microbiological Culture Collection Center (CGMCC) with a preservation number of CGMCC No. 29655; and the Klebsiella HNYJ-1 is preserved in the China General Microbiological Culture Collection Center (CGMCC) with a preservation number of CGMCC No. 24327.
[0014] Preferably, in the above technical solution, in step (11), the mass ratio of the tobacco fines to water is 1:10, the extraction temperature is 60℃, and the extraction time is 2h.
[0015] Preferably, in the above technical solution, in step (12), the culture temperature of the shaker is 28℃, the rotation speed is 160r / min, and the culture time is 24h.
[0016] Preferably, in the above technical solution, in step (13), the mixed mass ratio of the Klebsiella HNYJ-1 seed liquid to the Enterobacter HNYJ-4 seed liquid is 2:1.
[0017] Preferably, in the above technical solution, in step (2), the mass ratio of the composite seed solution to the tobacco leaves is 1:200, and the oxygen content in the cabinet during the low-oxygen fermentation stage is 17% ± 1%.
[0018] An application of a tobacco leaf fermentation quality improvement method based on oxygen content regulation in improving the quality of tobacco leaves.
[0019] Preferably, in the above technical solution, the application is to increase the content of linalool, 2,3,5,6-tetramethylpyrazine, 2,6-dimethylpyrazine, phenethyl acetate, isoamyl acetate, ethyl lactate, and butyl acetate, and to improve the aroma quality, aroma quantity, and delicacy of the tobacco leaves, and to reduce the pungency and irritation.
[0020] The above technical solution of the present application has the following beneficial effects:
[0021] In the present application, Klebsiella HNYJ-1 and Enterobacter HNYJ-4 are used as fermentation strains. First, a tobacco culture medium is prepared through a specific process, and a composite seed solution is obtained by mixing and culturing. Then, the composite seed solution is sprayed on the surface of the tobacco leaves in a quantitative spraying manner after leaf conditioning and before re-drying in the tobacco re-drying process. Subsequently, a two-stage oxygen control process is used for fermentation, and the fermented tobacco leaves are subjected to high-temperature re-drying to inactivate the bacteria. The results show that, compared with non-fermentation and single aerobic / low-oxygen fermentation treatment, this technology can more significantly increase the content of key aroma components such as linalool and 2,3,5,6-tetramethylpyrazine in tobacco leaves, comprehensively improve the aroma quality, aroma quantity, and delicacy of tobacco leaves, and reduce the pungency and irritation. Moreover, the process is compatible with the existing tobacco re-drying processing flow, and does not require additional complex equipment. It has the characteristics of good bacteria compatibility, good quality improvement effect, and strong process compatibility. DETAILED DESCRIPTION
[0022] Various exemplary embodiments of the present application will now be described in detail. It should be noted that the relative arrangement, numerical expressions, and values of the components and steps set forth in these embodiments do not limit the scope of the present application unless otherwise specifically stated.
[0023] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used are commercially available. Unless otherwise specified, the equipment used in the experiments is well known to those skilled in the art.
[0024] Klebsiella sp. HNYJ-1 was deposited with the China General Microbiological Culture Collection Center (CGMCC) on January 17, 2022, at the address of No. 1, Beichen West Road, Yard 3, Beijing City, Chaoyang District, Institute of Microbiology of Chinese Academy of Sciences, with the preservation number of CGMCC No. 24327.
[0025] Enterobacter sp. HNYJ-4, deposited on January 18, 2024 at China General Microbiological Culture Collection Center (CGMCC), located at No. 1, Yihuangyuan, Beichenxi Road, Haidian District, Beijing, China, and having the accession number CGMCC No. 29655.
[0026] Embodiment
[0027] A tobacco leaf fermentation upgrading method based on oxygen content regulation, comprising the following steps:
[0028] 1. Seed liquid preparation
[0029] (1) Medium preparation: After the tobacco leaves are crushed, tobacco powder is obtained by passing through a 100-mesh sieve. The tobacco powder is mixed with water at a mass ratio of 1:20-1:2 (optimum 1:10), and stirred at a temperature of 30-100°C (optimum 60°C) for 0.5-12h (optimum 2h) at a stirring speed of 50rpm. After extraction, the tobacco water extract is filtered with a 200-mesh sieve, and sterilized at 121°C for 30min to obtain the tobacco culture medium.
[0030] (2) Seed culture: Single colonies of Klebsiella HNYJ-1 and Enterobacter HNYJ-4 are inoculated into different bottles of tobacco culture medium, respectively, and cultured in a shaker at a temperature of 25-40°C (optimum 28°C) and a speed of 100-200r / min (optimum 160r / min) for 6-36h (optimum 24h) to obtain seed liquids of the two strains. At this time, the OD value is between 1.5 and 2.5. 600
[0031] The seed liquids of Klebsiella HNYJ-1 and Enterobacter HNYJ-4 are mixed at a mass ratio of 1:10-10:1 (optimum 2:1) to obtain a composite seed liquid.
[0032] 2. Oxygen content regulated tobacco leaf fermentation
[0033] In the tobacco redrying stage, after leaf moistening and before redrying, a compound seed solution is sprayed on the surface of the tobacco leaves using a spray head in a quantitative spraying manner, the spraying flow is controlled so that the mass ratio of the compound seed solution to the tobacco leaves is 1:1000-1:50 (optimally 1:200). The sprayed tobacco leaves are transferred to a closed temporary storage cabinet (ordinary cubic box), the cabinet volume is 80%, the temperature is controlled at 30℃±1℃, the humidity is controlled at 60%±5%, and an oxygen content detection device is placed in the cabinet. The cabinet door is opened to make the air circulate, and the tobacco leaves are left to ferment for 12h for aerobic fermentation. Then the tobacco leaves are turned over, the cabinet door is closed, and the oxygen content in the cabinet is adjusted by filling nitrogen and sterile air, the adjustment is performed once per hour, the oxygen content is maintained between 5%-20% (optimally 17%±1%), and the fermentation is continued for 24h for low-oxygen fermentation. After the fermentation is completed, the tobacco leaves are transferred to the redrying process to start high-temperature redrying, and are transferred to the normal processing process, and the bacteria are inactivated. The aerobic fermentation is to adapt the bacteria to the tobacco leaf environment, and the low-oxygen fermentation is to increase the aroma. The present application is mainly to increase part of the organic acid esters and pyrazine aroma components in the tobacco leaves.
[0034] Application example
[0035] Material selection: The tobacco leaves for preparing the culture medium are selected as C3F tobacco leaves in the middle part after redrying in Xuchang, Henan, and the tobacco leaves for the tobacco leaf fermentation test are selected as DCFB tobacco leaves in Pingdingshan, Henan.
[0036] Test process: The above steps are followed, the optimal parameters are selected in the brackets, the treated tobacco leaves are marked as T1, the tobacco leaves without fermentation treatment are marked as CK, the tobacco leaves are not subjected to two-stage fermentation, the same parameters are used, the closed temporary storage cabinet door is opened to keep the air circulating, the tobacco leaves are turned over after 12h, and the tobacco leaves obtained after 36h of fermentation are marked as T2, the tobacco leaves are not subjected to two-stage fermentation, the same parameters are used, the closed temporary storage cabinet door is closed to keep a 17% low-oxygen environment, the tobacco leaves are turned over after 12h, and the tobacco leaves obtained after 36h of fermentation are marked as T3.
[0037] After the tobacco leaves are crushed, dichloromethane is used for extraction, the main aroma components of the four kinds of tobacco leaves are analyzed by GC-MS, the treated and untreated tobacco leaves are cut into shreds and are rolled into cigarettes, and the sensory evaluation is performed by professional judges, and the scores are the average values of the judges.
[0038] Results: There were more aroma components in tobacco leaves. Table 1 only lists the components that changed greatly before and after fermentation and are beneficial to the quality of tobacco. Compared with CK tobacco leaves without fermentation, the aroma components of T1, T2 and T3 tobacco leaves after fermentation increased, especially linalool, 2,3,5,6-tetramethylpyrazine and 2,6-dimethylpyrazine increased by several times. Compared with T2 and T3 tobacco leaves fermented by oxygen or low oxygen alone, the aroma components of T1 tobacco leaves fermented by oxygen content regulation were higher, and the aroma components of T3 tobacco leaves fermented by low oxygen were higher than those of T2 tobacco leaves fermented by oxygen, fully illustrating that low oxygen environment is conducive to the generation of aroma components. At the same time, for two-stage fermentation, the aroma components of the treated tobacco leaves are more efficient after the first aerobic fermentation to promote bacterial growth and adaptation, and then low oxygen fermentation.
[0039] Specifically, the generation efficiency of alcohol and ketone substances such as furfuryl alcohol, 3-hexen-1-ol, linalool, dihydrodamascenone, 4,7,9-megastigmatrien-3-one is higher during ventilation fermentation, and the generation efficiency is low during pure low oxygen fermentation; the accumulation of ester and pyrazine substances such as phenethyl acetate, isoamyl acetate, 2,3,5,6-tetramethylpyrazine, 2-methylpyrazine, 2,6-dimethylpyrazine, 2-ethyl-6-methylpyrazine, ethyl lactate, dibutyl phthalate, and ethyl butyrate is less during ventilation fermentation, and the generation efficiency is higher in low oxygen environment. Except for individual aroma components, the content of most aroma components under two-stage fermentation conditions is better than that under one-stage fermentation.
[0040] Table 1 Change in content of important aroma substances in tobacco leaves before and after treatment (unit: μg / g)
[0041]
[0042] The sensory evaluation results of tobacco leaves before and after treatment are shown in Table 2. The scores of untreated tobacco leaves CK are taken as the benchmark, and the scores of treated tobacco leaves are added or subtracted on this basis. The sensory scores of T1, T2 and T3 tobacco leaves after fermentation treatment are higher than those of CK tobacco leaves without treatment, and the score of T1 tobacco leaves fermented by oxygen content regulation is the highest.
[0043] Specifically, only after the ventilation fermentation of T2 tobacco leaves, the aroma quality is slightly improved, the aroma amount is slightly increased, but the improvement of the indicators such as odor, irritation and aftertaste is obvious; only after the closed low-oxygen fermentation treatment of T3 tobacco leaves, the improvement of the indicators such as aroma quality, aroma amount and delicacy is obvious, but the improvement of the indicators such as odor, irritation and aftertaste is not obvious; and after the oxygen content control fermentation treatment of T1 tobacco leaves, all the indicators have obvious improvement except the concentration index. The indicators of aroma quality and aroma amount are consistent with the change rule of the content of the flavor components in Table 1. The indicators such as odor, irritation and aftertaste are related to the degradation of macromolecules such as protein, starch and pectin in tobacco leaves, and under aerobic conditions, the macromolecule degradation ability of the strain is better, the present application does not focus on the indicators, and mainly focuses on the aroma production ability.
[0044] Table 2 Sensory evaluation before and after treatment of tobacco leaves
[0045]
[0046] Although the present application has been disclosed as above with examples, it is not intended to limit the present application, and any person skilled in the art can make various selections and modifications without departing from the spirit and scope of the present application, therefore the protection scope of the present application is defined by the claims and equivalent forms thereof.
Claims
1. A method for improving the quality of tobacco leaves through fermentation based on oxygen content regulation, characterized in that, Includes the following steps: (1) Seed liquid preparation: (11) Culture medium preparation: tobacco leaves are crushed and passed through a 100-mesh sieve to obtain tobacco dust. The tobacco dust and water are mixed in a mass ratio of 1:20-1:
2. The mixture is stirred and extracted at 30-100℃ for 0.5-12h at a stirring speed of 50rpm. After extraction, the mixture is filtered through a 200-mesh sieve to obtain tobacco water extract. The extract is sterilized at 121℃ for 30min to obtain tobacco culture medium. (12) Seed culture: Single colonies of Klebsiella pneumoniae HNYJ-1 and Enterobacter hNYJ-4 were inoculated into different bottles of tobacco culture medium and cultured in a shaker at 25-40℃ and 100-200 r / min for 6-36 h to obtain seed cultures of the two strains. At this time, the OD 600 The value is between 1.5 and 2.5; (13) Preparation of composite seed liquid: Klebsiella pneumoniae HNYJ-1 and Enterobacter HNYJ-4 seed liquids were mixed at a mass ratio of 1:10-10:1 (optimal 2:1) to obtain composite seed liquid; (2) Regulating tobacco fermentation by oxygen content: In the process between moistening the leaves and before re-drying during the tobacco re-drying stage, a compound seed liquid is sprayed onto the surface of the tobacco leaves using a quantitative spraying method, controlling the mass ratio of the compound seed liquid to the tobacco leaves at 1:1000-1:
50. The sprayed tobacco leaves are then transferred to a closed temporary storage cabinet, filling the cabinet to 80% capacity, with the temperature controlled at 30℃±1℃ and the humidity at 60%±5%. An oxygen content detection device is placed inside the temporary storage cabinet. The cabinet door is opened to allow air circulation, and the leaves are left to ferment aerobically for 12 hours. Subsequently, the tobacco leaves are turned over, the cabinet door is closed, and the oxygen content inside the cabinet is regulated by introducing nitrogen and sterile air. The oxygen content is monitored and adjusted every hour, maintaining it between 5% and 20%, and fermentation continues for 24 hours for hypoxic fermentation. After fermentation, the tobacco leaves are transferred to the re-drying process for high-temperature re-drying, and then transferred to the normal processing process, where the inoculum is inactivated.
2. The method according to claim 1, characterized in that, The Enterobacter HNYJ-4 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 29655; the Klebsiella HNYJ-1 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 24327.
3. The method according to claim 1, characterized in that, In step (11), the mass ratio of tobacco dust to water is 1:10, the extraction temperature is 60℃, and the extraction time is 2h.
4. The method according to claim 1, characterized in that, In step (12), the culture temperature of the shaker is 28℃, the rotation speed is 160r / min, and the culture time is 24h.
5. The method according to claim 1, characterized in that, In step (13), the mass ratio of Klebsiella pneumoniae HNYJ-1 seed culture to Enterobacter HNYJ-4 seed culture is 2:
1.
6. The method according to claim 1, characterized in that, In step (2), the mass ratio of compound seed liquid to tobacco leaves is 1:200, and the oxygen content in the cabinet during the low-oxygen fermentation stage is 17%±1%.
7. The application of the tobacco fermentation quality improvement method based on oxygen content regulation according to any one of claims 1-6 in improving tobacco quality.
8. The application according to claim 7, characterized in that, The application aims to increase the content of aroma components such as linalool, 2,3,5,6-tetramethylpyrazine, 2,6-dimethylpyrazine, phenethyl acetate, isoamyl acetate, ethyl lactate, and ethyl butyrate in tobacco leaves, thereby improving the aroma quality, aroma quantity, and fineness of tobacco leaves, and reducing off-flavors and irritation.