Biological opium paste as well as preparation method and application thereof

Abstract

The invention discloses biological tobacco paste and a preparation method thereof. The biological tobacco paste is prepared by mixing and fermenting tobaccos with Klebsiella variicola HNYJ-3 with the preservation number of CGMCC (China General Microbiological Culture Collection Center) No.29654 (China General Microbiological Culture Collection Center) and Enterobacter sp. HNYJ-4 with the preservation number of CGMCC No.29655 (China General Microbiological Culture Collection Center) No.29655 (China General Microbiological Culture Collection Center No.29655 (China General Microbiological Culture Collection Center No.29655). According to the biological cigarette paste, through the specific microbial fermentation process, low-boiling-point aroma components in tobacco are remarkably increased, and the aroma quality and release efficiency of heated cigarettes are improved. The heated cigarette added with the biological cigarette paste shows better fragrance, harmony and aftertaste in sensory evaluation, offensive odor and irritation are reduced, and therefore the overall sensory experience of smoking is improved.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to a biological cigarette paste and a preparation method and application thereof. Background Art

[0002] Heated cigarettes are a new type of tobacco that has emerged in recent years. They are heated by electronic devices to release aroma components and nicotine and other smoke to meet the needs of smoking. The temperature of traditional cigarettes can reach thousands of degrees after burning, and the aroma substances and harmful substances in the tobacco are fully transferred and cracked; while the heating temperature of heated cigarettes is generally between 200-300℃ when smoking. Although less harmful substances are transferred, most of the aroma substances in the tobacco cannot be transferred and cracked into the smoke. Therefore, the aroma of heated cigarettes can never be comparable to that of traditional cigarettes.

[0003] There are limited ways to improve the aroma of heated cigarettes, mainly optimizing the heating method, improving the tobacco leaf formula, and optimizing the spice formula. In the final analysis, the aroma components in the heated cigarette need to be released as much as possible under the temperature condition below 250°C. Usually, considering the volatilization loss of the spices, the aroma components with a boiling point between 100-250°C are better. Among them, spices are an extremely important component of heated cigarettes, which to a certain extent determine the type and amount of aroma released by heated cigarettes. In particular, the most used tobacco paste is the most important heated cigarette flavor, which provides the most important tobacco flavor for heated cigarettes. Therefore, the development of special tobacco paste for heated cigarettes is crucial to improve the quality of heated cigarettes.

[0004] At present, the optimization of the flavor and fragrance formula of heated cigarettes is mainly to increase the usage, but the effect is limited. There is also the use of traditional physical and chemical means to lock in low-boiling point flavor substances as much as possible to prepare special tobacco paste for heated cigarettes, but the cost-effectiveness is low and the cost is high.

[0005] The invention utilizes mixed bacteria to ferment tobacco, and enriches flavor substances with boiling points below 250° C., such as nootkatone, beta-ionone, 2-acetylpyrrole, etc., so as to obtain a biological tobacco paste specially used for heating cigarettes. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a biological tobacco paste and a preparation method and application thereof, so as to solve the problem that the aroma release of heated cigarettes is insufficient compared with traditional cigarettes and enhance the release of aroma components.

[0007] The technical problem to be solved by the present invention is achieved through the following technical solutions:

[0008] The invention discloses a biological tobacco paste, which is prepared by fermenting tobacco by mixing Klebsiella variicola HNYJ-3 with a preservation number of CGMCC No.29654 and Enterobacter sp. HNYJ-4 with a preservation number of CGMCC No.29655.

[0009] A method for preparing a biological cigarette paste comprises the following steps:

[0010] (1) Preparing a flue-cured tobacco aqueous extract, a burley tobacco aqueous extract and an oriental tobacco aqueous extract: respectively crushing the middle tobacco leaves of flue-cured tobacco strips, burley tobacco and oriental tobacco, and passing through a 50-mesh sieve to obtain tobacco powder; and then mixing the tobacco powder with pure water to obtain aqueous extracts of flue-cured tobacco, burley tobacco and oriental tobacco;

[0011] (2) preparing seed solutions of Klebsiella mutans HNYJ-3 and Enterobacter HNYJ-4 respectively for later use;

[0012] (3) Preparation of a mixed slurry: mixing the flue-cured tobacco water extract, burley tobacco water extract and aromatic tobacco water extract of step (1) in a volume ratio of (5-10):(1-5):(1-5), and then mixing the mixture with coffee chlorogenic acid in a volume ratio of 1:10-1:100 to obtain a mixed slurry;

[0013] (4) Preparation of fermentation mixed slurry: mixing the Klebsiella mutans HNYJ-3 seed liquid prepared in step (2) with the Enterobacter HNYJ-4 seed liquid in a volume ratio of 1:10-10:1 to obtain a composite seed liquid;

[0014] (5) mixing the composite seed liquid of step (4) with the mixed slurry of step (2) at a volume ratio of 1:1000-1:100, fermenting at a constant temperature of 25-40° C. for 12-96 h at a speed of 180 rpm, and centrifuging to obtain a fermentation mixed slurry; concentrating the fermentation mixed slurry under reduced pressure at 60° C. to 1 / 10 of the original volume to obtain a concentrated slurry;

[0015] (6) The concentrated slurry is mixed with anhydrous ethanol in a mass ratio of 2:1-1:10, and the mixture is centrifuged to obtain a crude bio-cigarette paste, and the crude bio-cigarette paste is concentrated under reduced pressure to obtain a bio-cigarette paste.

[0016] Preferably, the preparation conditions of the water extract in step (1) are as follows: flue-cured tobacco powder, burley tobacco powder and oriental tobacco powder are mixed with pure water in a ratio of 1:20-1:5, respectively, and extracted at a constant temperature of 30-100° C. for 1-10 h, and filtered through 200 mesh gauze to obtain three water extracts.

[0017] Preferably, the seed solution of step (2) is prepared by inoculating Klebsiella mutans HNYJ-3 and Enterobacter HNYJ-4 into LB liquid culture medium, culturing at 30°C for 24h, and OD 600 When it reaches 1.0-3.0, it is seed liquid.

[0018] Preferably, step (3) comprises:

[0019] The flue-cured tobacco aqueous extract, burley tobacco aqueous extract and oriental tobacco aqueous extract prepared in step (1) are mixed in a volume ratio of 7:2:1 to obtain a mixed tobacco aqueous extract;

[0020] The tobacco water extract and coffee chlorogenic acid were mixed in a volume-to-mass ratio of 1:50 to obtain a mixed slurry.

[0021] Preferably, step (4) specifically comprises: mixing the Klebsiella mutans HNYJ-3 seed solution prepared in step (2) with the Enterobacter HNYJ-4 seed solution in a volume ratio of 2:1 to obtain a composite seed solution.

[0022] Preferably, in step (5), the composite seed liquid and the mixed slurry are mixed in a volume ratio of 1:200, and fermented at a constant temperature of 30° C. with shaking for 48 h.

[0023] Preferably, step (6) is specifically as follows: the concentrated slurry is mixed with anhydrous ethanol in a mass ratio of 1:2, allowed to stand at -20°C for 24 hours, and centrifuged to obtain a crude bio-cigarette paste, and the crude bio-cigarette paste is concentrated under reduced pressure at 60°C to 1 / 5 of the original volume to obtain a bio-cigarette paste.

[0024] The invention discloses an application of a biological tobacco paste in a heated cigarette, wherein the biological tobacco paste is used to improve the aroma quality and sensory experience of the heated cigarette.

[0025] A method for improving the sensory quality of heated cigarettes, the method comprising adding biological tobacco paste to heated cigarettes to improve their aroma, coordination, reduce impurities, reduce irritation and enhance aftertaste.

[0026] The above technical solution of the present invention has the following beneficial effects:

[0027] (1) Enhanced aroma components: Bio-tobacco paste significantly increases the low-boiling point aroma components in tobacco through a specific microbial fermentation process, thereby improving the aroma quality and release efficiency of heated cigarettes.

[0028] (2) Improved sensory experience: HTCs with added bio-tobacco paste showed better aroma, harmony, and aftertaste in sensory evaluation, reduced odor and irritation, and thus improved the overall sensory experience of smoking.

[0029] (3) Preparation process innovation: A cost-effective method for preparing bio-tobacillus paste was developed by using the mixed fermentation of Klebsiella mutans HNYJ-3 and Enterobacter HNYJ-4, providing a new technical path for improving the quality of heated cigarettes. DETAILED DESCRIPTION

[0030] Various exemplary embodiments of the present invention will now be described in detail. It should be noted that the relative arrangement of components and steps, numerical expressions and numerical values ​​set forth in these embodiments do not limit the scope of the present invention unless specifically stated otherwise.

[0031] A biological tobacco paste and a preparation method and application thereof. The biological tobacco paste is obtained by mixed fermentation of tobacco by Klebsiella variicola HNYJ-3 and Enterobacter sp. HNYJ-4.

[0032] Klebsiella variicola HNYJ-3 originates from the surface of tobacco leaves and is taxonomically named Klebsiella variicola HNYJ-3. It is deposited in the General Microbiology Center (CGMCC) of the China Microbiological Culture Collection Administration, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The deposit number is CGMCC No. 29654 and the deposit date is January 18, 2024.

[0033] Enterobacter sp. HNYJ-4 originates from the surface of tobacco leaves and is taxonomically named Enterobacter sp. HNYJ-4. It is deposited in the General Microbiology Center (CGMCC) of the China Microbiological Culture Collection Administration, located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The deposit number is CGMCC No. 29655 and the deposit date is January 18, 2024.

[0034] Example 1 Preparation method of biological cigarette paste

[0035] The biological cigarette paste and its preparation method include the following steps:

[0036] (1) Preparation methods of flue-cured tobacco water extract, burley tobacco water extract and oriental tobacco water extract:

[0037] Preparation of flue-cured tobacco water extract: crush the middle tobacco leaves of flue-cured tobacco sheets, pass through a 50-mesh sieve to obtain flue-cured tobacco powder. Mix the flue-cured tobacco powder with pure water in a ratio of 1:20-1:5, extract at a constant temperature of 30-100°C for 1-10h, and filter through a 200-mesh gauze to obtain the flue-cured tobacco water extract.

[0038] Preparation of burley tobacco water extract: grind the middle part of burley tobacco, and the other steps are the same as the preparation of tobacco water extract.

[0039] Preparation of oriental tobacco water extract: crush the middle tobacco leaves of oriental tobacco, and the other steps are the same as the preparation of tobacco water extract.

[0040] (2) Preparation of strain seed solution:

[0041] Klebsiella mutans HNYJ-3 and Enterobacter HNYJ-4 were inoculated into LB liquid medium and cultured at 30°C for 24 h. 600 When it reaches about 1.0-3.0, it is seed liquid.

[0042] LB liquid culture medium is a conventional formula and will not be described in detail here.

[0043] (3) Preparation of mixed slurry:

[0044] The flue-cured tobacco aqueous extract, burley tobacco aqueous extract and oriental tobacco aqueous extract prepared in Example 1 are mixed in a volume ratio of (5-10):(1-5):(1-5) to obtain a mixed tobacco aqueous extract;

[0045] The mixed tobacco water extract and coffee chlorogenic acid (25% purity) are mixed in a volume-to-mass ratio of 1:10-1:100 to obtain a mixed slurry.

[0046] (4) Preparation of fermentation mixed slurry:

[0047] The Klebsiella mutans HNYJ-3 seed solution and the Enterobacter HNYJ-4 seed solution are mixed in a volume ratio of 1:10-10:1 to obtain a composite seed solution.

[0048] (5) Composite seed solution and mixed slurry:

[0049] The mixture was mixed in a volume ratio of 1:1000-1:100, and fermented at 25-40°C with constant temperature shaking for 12-96h at a speed of 180rpm. After the fermentation, the supernatant was collected by centrifugation to obtain a fermentation mixed slurry.

[0050] The fermentation mixed slurry was concentrated under reduced pressure at 60°C to 1 / 10 of the original volume to obtain a concentrated slurry.

[0051] The concentrated slurry was mixed with anhydrous ethanol in a mass ratio of 2:1-1:10, allowed to stand at -20°C for 24 hours, and centrifuged to obtain a crude bio-cigarette paste. The crude bio-cigarette paste was concentrated under reduced pressure at 60°C to 1 / 5 of the original volume to obtain a bio-cigarette paste.

[0052] The tobacco paste contains more low-boiling point (below 100-250°C) aroma substances, and has a more obvious sweet and floral aroma with a slight ambergris flavor, and is suitable for use in heated cigarettes.

[0053] Example 2 Preparation method of biological cigarette paste

[0054] The biological cigarette paste and its preparation method include the following steps:

[0055] (1) Preparation methods of flue-cured tobacco water extract, burley tobacco water extract and oriental tobacco water extract:

[0056] Preparation of flue-cured tobacco water extract: The middle tobacco leaves of flue-cured tobacco strips were crushed and passed through a 50-mesh sieve to obtain flue-cured tobacco powder. The flue-cured tobacco powder was mixed with pure water in a ratio of 1:12, and the mixture was extracted at a constant temperature of 60°C for 2 hours, and filtered through a 200-mesh gauze to obtain the flue-cured tobacco water extract.

[0057] Preparation of burley tobacco water extract: grind the middle part of burley tobacco, and the other steps are the same as the preparation of tobacco water extract.

[0058] Preparation of oriental tobacco water extract: crush the middle tobacco leaves of oriental tobacco, and the other steps are the same as the preparation of tobacco water extract.

[0059] (2) Preparation of strain seed solution:

[0060] Klebsiella mutans HNYJ-3 and Enterobacter HNYJ-4 were inoculated into LB liquid medium and cultured at 30°C for 24 h. 600 When it reaches about 2.0, it is seed liquid.

[0061] LB liquid culture medium is a conventional formula and will not be described in detail here.

[0062] (3) Preparation of mixed slurry:

[0063] The flue-cured tobacco aqueous extract, burley tobacco aqueous extract and oriental tobacco aqueous extract prepared in Example 1 were mixed in a volume ratio of 7:2:1 to obtain a mixed tobacco aqueous extract;

[0064] The mixed tobacco water extract and coffee chlorogenic acid (25% purity) were mixed in a volume-to-mass ratio of 1:50 to obtain a mixed slurry.

[0065] (4) Preparation of fermentation mixed slurry:

[0066] The Klebsiella mutans HNYJ-3 seed solution and the Enterobacter HNYJ-4 seed solution were mixed at a volume ratio of 2:1 to obtain a composite seed solution.

[0067] (5) Composite seed solution and mixed slurry:

[0068] The mixture was mixed at a volume ratio of 1:200 and fermented at 30°C with constant temperature shaking for 48 hours at a rotation speed of 180 rpm. After the fermentation was completed, the supernatant was collected by centrifugation to obtain a fermentation mixed slurry.

[0069] The fermentation mixed slurry was concentrated under reduced pressure at 60°C to 1 / 10 of the original volume to obtain a concentrated slurry.

[0070] The concentrated slurry was mixed with anhydrous ethanol in a mass ratio of 1:2, allowed to stand at -20°C for 24 hours, and centrifuged to obtain a crude bio-cigarette paste. The crude bio-cigarette paste was concentrated under reduced pressure at 60°C to 1 / 5 of the original volume to obtain a bio-cigarette paste.

[0071] The tobacco paste contains more low-boiling point (below 100-250°C) aroma substances, and has a more obvious sweet and floral aroma with a slight ambergris flavor, and is suitable for use in heated cigarettes.

[0072] Application Example 1

[0073] (1) Materials:

[0074] The flue-cured tobacco is the central tobacco leaves from Kunming, Yunnan in 2022, the burley tobacco is the central tobacco leaves from Enshi, Hubei in 2022, and the aromatic tobacco is the central tobacco leaves from Baoshan, Yunnan in 2022.

[0075] Coffee chlorogenic acid (25% purity) is commercially available.

[0076] (2) Methods:

[0077] According to the method of Example 2, the mixed slurry was fermented separately by Klebsiella mutans HNYJ-3 and Enterobacter HNYJ-4 to prepare biological tobacco paste, which were recorded as T1 and T2; the mixed slurry was fermented with a composite strain of Klebsiella mutans HNYJ-3 and Enterobacter HNYJ-4 at the same time to prepare biological tobacco paste, which was recorded as T3; the mixed slurry was not fermented, and the subsequent steps were the same to prepare ordinary tobacco paste, which was recorded as CK.

[0078] The key aroma components in the tobacco paste were analyzed by dichloromethane extraction and GC-MS detection. 100ppm was added to the heated cigarettes of FAST, and professional judges were organized to conduct sensory evaluation, and the scores were averaged.

[0079] (3) Results:

[0080] The changes in the main aroma components of ordinary tobacco paste CK and biological tobacco paste are shown in Table 1.

[0081] Compared with ordinary tobacco paste, the content of important tobacco aroma components such as ambroxan, 6-methyl-2-heptanone, quinic acid-γ-lactone and norsolanidione in bio-tobacco paste T3 is significantly increased, which are aroma components that are easily transferred to smoke during the smoking process of heated cigarettes. The bio-tobacco paste has a more obvious sweet and floral aroma with a slight ambergris flavor.

[0082] Compared with T1 and T2 fermented by single bacteria, T3 biological tobacco paste fermented by mixed bacteria contains more aroma components. At the same time, it can be seen that ambroxan is mainly produced by fermentation of Enterobacter HNYJ-4, and 6-methyl-2-heptanone is mainly produced by fermentation of Klebsiella mutans HNYJ-3. Both bacteria can produce quinic acid-γ-lactone and norsolani dione. Only the combined fermentation of the two bacteria can produce the best effect.

[0083] Table 1 Main aroma components in tobacco paste with boiling points between 100-250℃ (μg / g)

[0084]

[0085] The bio-tobacco paste was added to the heated cigarettes, and the sensory results are shown in Table 2. The results show that the unfermented tobacco paste has a general effect, and the tobacco paste fermented by a single strain has a certain effect, which can improve the aroma and aftertaste, but the bio-tobacco paste fermented by the composite strain has a more obvious aroma, a more coordinated smoke, and the most obvious improvement in aftertaste, with the highest total score.

[0086] Table 2 Sensory evaluation of adding bio-tobacco paste into heated cigarettes

[0087]

[0088] The full marks for each indicator are: aroma 32, coordination 6, off-flavor 12, stimulation 20, aftertaste 25, and the total score is 95.

[0089] Although the present invention has been disclosed as above by the embodiments, it is not intended to limit the present invention. Any person skilled in the art may make various choices and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention is limited by the claims and their equivalents.

Claims

1. A biological tobacco paste, characterized in that: The biological tobacco paste is prepared by mixing and fermenting tobacco with Klebsiella variicola HNYJ-3 with a preservation number of CGMCC No. 29654 and Enterobacter sp. HNYJ-4 with a preservation number of CGMCC No. 29655.

2. The method for preparing the biological cigarette paste according to claim 1, characterized in that: The following steps are involved: (1) Preparing a flue-cured tobacco aqueous extract, a burley tobacco aqueous extract and an oriental tobacco aqueous extract: respectively crushing the middle tobacco leaves of flue-cured tobacco strips, burley tobacco and oriental tobacco, and passing through a 50-mesh sieve to obtain tobacco powder; and then mixing the tobacco powder with pure water to obtain aqueous extracts of flue-cured tobacco, burley tobacco and oriental tobacco; (2) preparing seed solutions of Klebsiella mutans HNYJ-3 and Enterobacter HNYJ-4 respectively for later use; (3) Preparation of a mixed slurry: mixing the flue-cured tobacco water extract, burley tobacco water extract and aromatic tobacco water extract of step (1) in a volume ratio of (5-10):(1-5):(1-5), and then mixing the mixture with coffee chlorogenic acid in a volume ratio of 1:10-1:100 to obtain a mixed slurry; (4) Preparation of fermentation mixed slurry: mixing the Klebsiella mutans HNYJ-3 seed liquid prepared in step (2) with the Enterobacter HNYJ-4 seed liquid in a volume ratio of 1:10-10:1 to obtain a composite seed liquid; (5) mixing the composite seed liquid of step (4) with the mixed slurry of step (2) at a volume ratio of 1:1000-1:100, fermenting at a constant temperature of 25-40° C. for 12-96 h at a speed of 180 rpm, and centrifuging to obtain a fermentation mixed slurry; concentrating the fermentation mixed slurry under reduced pressure at 60° C. to 1 / 10 of the original volume to obtain a concentrated slurry; (6) The concentrated slurry is mixed with anhydrous ethanol in a mass ratio of 2:1-1:10, and the mixture is centrifuged to obtain a crude bio-cigarette paste, and the crude bio-cigarette paste is concentrated under reduced pressure to obtain a bio-cigarette paste.

3. The method for preparing the biological cigarette paste according to claim 2, characterized in that: The preparation conditions of the water extract in step (1) are as follows: flue-cured tobacco powder, burley tobacco powder and oriental tobacco powder are mixed with pure water in a ratio of 1:20-1:5, respectively, and extracted at a constant temperature of 30-100° C. for 1-10 hours, and filtered through 200-mesh gauze to obtain three water extracts.

4. The method for preparing the biological cigarette paste according to claim 2, characterized in that: The seed solution of step (2) is prepared by inoculating Klebsiella mutans HNYJ-3 and Enterobacter HNYJ-4 into LB liquid culture medium, culturing at 30°C for 24 hours, and OD 600 When it reaches 1.0-3.0, it is seed liquid.

5. The method for preparing the biological cigarette paste according to claim 2, characterized in that: Step (3) includes: The flue-cured tobacco aqueous extract, burley tobacco aqueous extract and oriental tobacco aqueous extract prepared in step (1) are mixed in a volume ratio of 7:2:1 to obtain a mixed tobacco aqueous extract; The tobacco water extract and coffee chlorogenic acid were mixed in a volume-to-mass ratio of 1:50 to obtain a mixed slurry.

6. The method for preparing the biological cigarette paste according to claim 2, characterized in that: Step (4) specifically comprises: mixing the Klebsiella mutans HNYJ-3 seed solution prepared in step (2) with the Enterobacter HNYJ-4 seed solution in a volume ratio of 2:1 to obtain a composite seed solution.

7. The method for preparing the biological cigarette paste according to claim 2, characterized in that: In step (5), the composite seed liquid and the mixed slurry are mixed at a volume ratio of 1:200, and the mixture is fermented at a constant temperature of 30° C. with shaking for 48 h.

8. The method for preparing the biological cigarette paste according to claim 2, characterized in that: Step (6) is specifically as follows: the concentrated slurry is mixed with anhydrous ethanol in a mass ratio of 1:2, and allowed to stand at -20°C for 24 hours, and a crude bio-cigarette paste is obtained after centrifugation, and the crude bio-cigarette paste is concentrated under reduced pressure at 60°C to 1 / 5 of the original volume to obtain the bio-cigarette paste.

9. The use of the biological tobacco paste according to claim 1 or the biological tobacco paste prepared according to the preparation method according to claims 2-8 in heating cigarettes, characterized in that: The biological tobacco paste is used to improve the aroma quality and sensory experience of heated cigarettes.

10. A method for improving the sensory quality of heated cigarettes, characterized in that: The method is to add the biological tobacco paste as claimed in claim 1 to the heated cigarette to improve its aroma, coordination, reduce impurities, reduce irritation and enhance aftertaste.

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