A method for the preparation of a rose-flavoured tobacco extract

Abstract

The application discloses a preparation method of a rose-type tobacco extract, and comprises the following steps: inoculating Micrococcus, Gluconacetobacter and Klebsiella variicola into LB liquid culture medium respectively for culture, centrifuging supernatant and redissolving in sterile water to obtain three kinds of seed liquids, mixing the three kinds of seed liquids to obtain a composite seed liquid; screening tobacco powder, mixing the tobacco powder with deionized water to extract, and obtaining a tobacco extract; mixing the composite seed liquid with the tobacco extract, stirring and fermenting; centrifuging the fermented tobacco extract to obtain supernatant, separating and purifying the supernatant, concentrating to obtain a concentrate, adding 1,2-propanediol, and obtaining the rose-type tobacco extract. The application has the advantages of simple process and low cost. The content of main rose-type flavor components in the tobacco extract is significantly improved, the aroma is rich and obvious in characteristics. Through sensory evaluation, the aroma of cigarettes added with the extract is significantly improved, the rose flavor of the cigarettes is effectively improved, and the application has a good market application prospect.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a method for preparing a rose-scented tobacco extract. Background Technology

[0002] Tobacco extracts are crucial flavoring agents in cigarette processing, imparting a more natural and full-bodied aroma. Tobacco extracts are generally prepared by extracting, purifying, and concentrating organic matter or water from tobacco. Refined tobacco extracts have fewer impurities and higher quality, but the process is usually complex and costly; while crude tobacco extracts typically contain more impurities and are of lower quality. During the extraction process, many large molecules such as proteins, starches, and pectins are present, and the content of aroma compounds is largely determined by the raw tobacco. Without complex separation and purification methods, the quality of the prepared tobacco extract is not ideal. Using microbial fermentation to prepare tobacco extracts can effectively degrade large molecules, transforming them into special aroma components, resulting in tobacco extracts with fewer impurities and a richer aroma.

[0003] The aroma components isolated from roses are quite complex, mainly including linalool, 2-phenylethanol, citronellol, geraniol, β-ionone, and rose alcohol. Some substances, such as 2-phenylethanol, linalool, and β-ionone, possess distinct rose aroma characteristics and are perceptible in very small quantities. When applied to tobacco, they can effectively enhance the rose aroma profile of cigarettes. Microbial fermentation can degrade the original aroma components and glycosides composed of sugars in tobacco, thereby releasing aroma components. Furthermore, it can facilitate metabolic transformation, making it an effective way to enhance the aroma of tobacco extracts. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a method for preparing a rose-scented tobacco extract. The method involves obtaining a tobacco extract by water extraction, followed by fermentation with a compound aroma-producing fungus to prepare a tobacco extract with obvious rose-scented characteristics. This method has the advantages of simple process, low cost, and good additive effect.

[0005] The technical problem to be solved by the present invention is achieved through the following technical solution:

[0006] A method for preparing a rose-scented tobacco extract includes the following steps:

[0007] (1) Preparation of compound aroma-producing fungus seeds: Micrococcus ZY-02 (CGMCC No. 21313), Gluconobacterium tumefaciens ZT-01 (CGMCC No. 21544), and Klebsiella variegata HNYJ-3 (CGMCC No. 29654) were inoculated into LB liquid medium and cultured at 25-40℃ and 100-200r / min for 6-36h. After centrifugation, the supernatant was reconstituted with sterile water and the OD600 was adjusted to 2.0 to obtain three seed liquids. The three seed liquids were mixed evenly in proportion to obtain a compound seed liquid.

[0008] (2) Preparation of tobacco extract: After sieving tobacco powder, mix it with deionized water and extract at 30℃~100℃ for 0.5h~5h. Filter the filtrate, sterilize it, and obtain tobacco extract.

[0009] (3) Fermented tobacco extract: The compound seed liquid from step (1) and the tobacco extract from step (2) are mixed at a volume ratio of 1:1000 to 1:50 and fermented at a temperature of 25-40℃ and 50-300r / min for 1-5 days.

[0010] (4) Preparation of fermented tobacco extract: The supernatant was obtained by centrifugation of the fermented tobacco extract, the supernatant was separated, purified, concentrated and then 1,2-propanediol was added to obtain rose-scented tobacco extract.

[0011] Preferably, in the above technical solution, in step (1), the culture conditions after the three aroma-producing fungi are inoculated into LB liquid medium are: cultured in a shaker at 30℃ and 150r / min for 12h; the centrifugation conditions are: centrifuged at 4℃ and 4000r / min for 20min.

[0012] Preferably, in the above technical solution, in step (1), the seed liquids ZY-02, ZT-01 and HNYJ-3 are mixed evenly in a volume ratio of (1-5):(1-5):(1-5) to obtain a composite seed liquid, and the preferred volume ratio is 1:3:2.

[0013] Preferably, in the above technical solution, step (2) specifically involves: grinding the middle part of flue-cured tobacco leaves to obtain tobacco dust, passing it through a 50-mesh sieve, mixing the tobacco dust with deionized water at a ratio of 1:20 to 1:2, extracting at 30℃ to 100℃ for 0.5h to 5h, filtering through a 200-mesh sieve, taking the filtrate, sterilizing at 121℃ for 15min to obtain tobacco extract.

[0014] Preferably, in the above technical solution, in step (2), the sieved tobacco powder is mixed with deionized water at a ratio of 1:10 and extracted at 70°C for 2 hours.

[0015] Preferably, in the above technical solution, in step (3), the volume ratio of the compound seed liquid to the tobacco extract is 1:200, and the temperature is 30℃, with stirring and fermentation at 150r / min for 2 days.

[0016] Preferably, in the above technical solution, step (4) specifically includes:

[0017] (41) The fermented tobacco extract was centrifuged at 4000 r / min and 4℃ for 30 min to obtain the supernatant.

[0018] (42) The supernatant was separated and purified by HD-20 macroporous resin, using linear elution with ethanol of different purities gradually increased from 0% to 100%.

[0019] (43) Collect the second half of the elution fraction and concentrate it to a density of 1.10±0.005 g / mL under the conditions of 50℃ and 120 mbar to obtain the concentrate;

[0020] (44) Add 1% to 50% of the concentrate by mass of 1,2-propanediol to obtain the final product, a rose-scented tobacco extract.

[0021] Preferably, in the above technical solution, in step (44), 10% of the concentrate mass of 1,2-propanediol is added according to the mass ratio.

[0022] Application of a rose-scented tobacco extract in tobacco flavoring.

[0023] The above-described technical solution of the present invention has the following beneficial effects:

[0024] This application utilizes a compound aroma-producing microorganism (Micrococcus ZY-02, Gluconobacterium acetate-01, and Klebsiella variegata HNYJ-3) to ferment tobacco extract, resulting in a simple and low-cost process. By optimizing the microbial ratio and fermentation conditions, the content of key rose-scented aroma components in the tobacco extract, such as 2-phenylethanol and linalool, is significantly increased, resulting in a richer and more distinctive aroma. Sensory evaluation shows that adding this extract significantly enhances the aroma of cigarettes, reduces off-flavors and irritation, improves the aftertaste, and elevates the overall quality. It effectively enhances the rose-scented character of cigarettes and has promising market application prospects. Detailed Implementation

[0025] Various exemplary embodiments of the present invention will now be described in detail. It should be noted that, unless otherwise specifically stated, the relative arrangement, numerical expressions, and values ​​of the components and steps set forth in these embodiments do not limit the scope of the invention.

[0026] Gluconacetobacter sp. ZT-01 is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing (Institute of Microbiology, Chinese Academy of Sciences), with accession number CGMCC No. 21544, on December 23, 2020.

[0027] Micrococcus Pri ZY-02 is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing (Institute of Microbiology, Chinese Academy of Sciences), with accession number CGMCC No. 21313, on December 7, 2020.

[0028] Klebsiella variicola HNYJ-3 is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing (Institute of Microbiology, Chinese Academy of Sciences), with accession number CGMCC No. 29654, on January 18, 2024.

[0029] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and 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.

[0030] Example 1

[0031] A method for preparing a rose-scented tobacco extract includes the following steps:

[0032] (1) Preparation of compound aroma-producing fungus seeds:

[0033] Colonies of the three aroma-producing fungi were inoculated into LB liquid medium using an inoculation loop and cultured in a shaker at 25-40℃ (optimal 30℃) and 100-200 r / min (optimal 150 r / min) for 6-36 h (optimal 12 h). After the culture was completed, all were centrifuged at 4000 r / min and 4℃ for 20 min, the supernatant was removed, sterile water was added to reconstitute the solution, and the OD600 was adjusted to 2.0 to obtain the seed liquid of the three aroma-producing fungi.

[0034] The seed solutions ZY-02, ZT-01 and HNYJ-3 were mixed evenly in a volume ratio of (1-5):(1-5):(1-5) (optimal 1:3:2) to obtain a composite seed solution.

[0035] (2) Preparation of tobacco extract:

[0036] The tobacco leaves from the middle part of flue-cured tobacco are ground to obtain tobacco dust, which is then passed through a 50-mesh sieve. After sieving, the tobacco dust is mixed with deionized water at a ratio of 1:20 to 1:2 (optimal 1:10) and extracted at 30℃ to 100℃ (optimal 70℃) for 0.5h to 5h (optimal 2h). After filtration through a 200-mesh sieve, the filtrate is collected and sterilized at 121℃ for 15min to obtain tobacco extract.

[0037] (3) Fermented tobacco extract:

[0038] In step (1), the compound seed liquid and the tobacco extract in step (2) are mixed at a volume ratio of 1:1000 to 1:50 (optimal 1:200) (i.e., the inoculum amount is 0.1% to 2%, optimal 0.5%), and fermented at a temperature of 25-40℃ (optimal 30℃) and a speed of 50-300r / min (optimal 150r / min) for 1-5 days (optimal 2 days).

[0039] (4) Preparation of fermented tobacco extract:

[0040] The fermented tobacco extract was centrifuged at 4000 r / min and 4℃ for 30 min to obtain the supernatant.

[0041] The supernatant was simply separated and purified using HD-20 macroporous resin, and linear elution was performed using ethanol of different purities gradually increased from 0% to 100%.

[0042] The elution fraction from the second half was collected and concentrated to a density of 1.10±0.005 g / mL under conditions of 50℃ and 120 mbar vacuum to obtain a concentrate. 1% to 50% (optimal 10%) of 1,2-propanediol was added according to the mass ratio of the concentrate to obtain the final product, a rose-scented tobacco extract.

[0043] Application Example 1

[0044] (1) Using the optimal parameters described in the steps of Example 1 above, three aroma-producing fungi were used to ferment and prepare a tobacco extract, denoted as T0.

[0045] (2) Following the above steps (2)-(4), step (3) is replaced with an equal volume of sterile water to prepare a rose-scented tobacco extract, denoted as CK (because it is not fermented, the effective ingredients are few, and the liquid collected after column purification is less, so it is concentrated to only 20% of the original volume, and the density is less than 1.10 mg / mL).

[0046] (3) Using the optimal parameters described in the above steps, only Micrococcus ZY-02 is used for fermentation to prepare tobacco extract, denoted as T1.

[0047] Following the optimal parameters described in the above steps, only Gluconobacterium Glucono-Acetic Acid Bacillus ZT-01 was used for fermentation to prepare Gluconobacterium Glucono-Acetic Acid Bacillus ZT-01, denoted as T2.

[0048] Following the optimal parameters described in the above steps, tobacco extract, denoted as T3, was prepared by fermentation using only Klebsiella variegata HNYJ-3.

[0049] The main aroma components of the rose-scented tobacco were detected by GC-MS. The tobacco extract was added to cigarette tobacco at a dosage of 10 ppm, and sensory evaluation was conducted by professional smokers.

[0050] The results are as follows:

[0051] The main aroma components of the rose-scented tobacco extracts are shown in Table 1. The results show that in the unfermented CK group, the main aroma component content of the rose-scented extract was 32.10 ug / mL, which is relatively low and generally not easily perceived. In contrast, the T0 extract, fermented with a complex aroma-producing fungus, had a main aroma component content of 510.80 ug / mL, an increase of several tens of times, exhibiting a very distinct rose aroma characteristic. While the main aroma components of the rose-scented extracts T1, T2, and T3, fermented with a single fungus, were all increased, they were all lower than those in T0, and some components were present in smaller quantities, resulting in a less intense rose aroma characteristic.

[0052] Table 1. Main rose-scented aroma components in tobacco extracts (unit: ug / mL)

[0053]

[0054]

[0055] The cigarette evaluation results are shown in Table 2. The results show that adding unfermented tobacco extract CK did not significantly change any of the indicators, but resulted in a slight off-flavor and a 0.2-point decrease in the total score. Adding T0 significantly improved the aroma, and the scores for off-flavors, irritation, and aftertaste improved, increasing the total score by 2.3 points. Adding T1, T2, and T3 all improved the aroma and other indicators to some extent, but none as significantly as T0. This indicates that the tobacco extract prepared by fermentation with compound aroma-producing fungi is of higher quality.

[0056] Table 2 Sensory Evaluation Results of Cigarettes

[0057] sample luster aroma Harmony Mixed gases Irritating Aftertaste Total Score 0 5.0 27.5 5.0 10.0 17.5 22.0 87.0 CK 5.0 27.5 5.0 9.8 17.5 22.0 86.8 T0 5.0 28.5 5.0 10.5 18.0 22.3 89.3 T1 5.0 27.7 5.0 10.0 17.6 22.2 87.5 T2 5.0 27.9 5.0 10.3 17.7 22.0 87.9 T3 5.0 27.6 5.0 10.3 17.7 22.0 87.6

[0058] Note: "0" represents a cigarette sample without any added substances.

[0059] Although the present invention has been disclosed above with reference to embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various different choices and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention is defined by the claims and their equivalents.

Claims

1. A method for preparing a rose-scented tobacco extract, characterized in that, Includes the following steps: (1) Preparation of compound aroma-producing fungus seeds: Micrococcus ZY-02 (CGMCC No. 21313), Gluconobacterium tumefaciens ZT-01 (CGMCC No. 21544), and Klebsiella variegata HNYJ-3 (CGMCC No. 29654) were inoculated into LB liquid medium and cultured at 25-40℃ and 100-200r / min for 6-36h. After centrifugation, the supernatant was reconstituted with sterile water and the OD600 was adjusted to 2.0 to obtain ZY-02 seed liquid, ZT-01 seed liquid and HNYJ-3 seed liquid. The above three seed liquids were mixed evenly in a volume ratio of (1-5):(1-5):(1-5) to obtain compound seed liquid; (2) Preparation of tobacco extract: After sieving tobacco powder, mix it with deionized water and extract at 30℃~100℃ for 0.5h~5h. Filter the filtrate, sterilize it, and obtain tobacco extract. (3) Fermented tobacco extract: The compound seed liquid from step (1) and the tobacco extract from step (2) are mixed at a volume ratio of 1:1000 to 1:50 and fermented at a temperature of 25-40℃ and 50-300r / min for 1-5 days. (4) Preparation of fermented tobacco extract: The supernatant was obtained by centrifugation of the fermented tobacco extract. The supernatant was separated and purified by HD-20 macroporous resin. The extract was linearly eluted with ethanol of different purities gradually increased from 0% to 100%. The second half of the elution fraction was collected and concentrated to obtain the concentrate. 1,2-propanediol was added to obtain the rose-scented tobacco extract.

2. The method for preparing the rose-scented tobacco extract according to claim 1, characterized in that, In step (1), the culture conditions after the three aroma-producing fungi were inoculated into LB liquid medium were: 30℃, 150r / min in a shaker for 12h; centrifugation conditions were: 4℃, 4000r / min for 20min.

3. The method for preparing the rose-scented tobacco extract according to claim 1, characterized in that, Step (2) is as follows: the tobacco leaves in the middle part of the flue-cured tobacco are ground to obtain tobacco dust, which is then passed through a 50-mesh sieve. After passing through the sieve, the tobacco dust is mixed with deionized water at a ratio of 1:20 to 1:2, and extracted at 30℃ to 100℃ for 0.5h to 5h. After filtering through a 200-mesh sieve, the filtrate is collected and sterilized at 121℃ for 15min to obtain tobacco extract.

4. The method for preparing the rose-scented tobacco extract according to claim 3, characterized in that, In step (2), the sieved tobacco powder is mixed with deionized water at a ratio of 1:10 and extracted at 70°C for 2 hours.

5. The method for preparing the rose-scented tobacco extract according to claim 1, characterized in that, In step (3), the volume ratio of the compound seed liquid to the tobacco extract is 1:200, and the temperature is 30℃ with stirring and fermentation at 150r / min for 2 days.

6. The method for preparing the rose-scented tobacco extract according to claim 1, characterized in that, Step (4) is as follows: (41) The fermented tobacco extract was centrifuged at 4000 r / min and 4℃ for 30 min to obtain the supernatant. (42) The supernatant was separated and purified by HD-20 macroporous resin, using linear elution with ethanol of different purities gradually increasing from 0% to 100%. (43) Collect the second half of the elution fraction and concentrate it to a density of 1.10±0.005 g / mL under the conditions of 50℃ and 120 mbar to obtain the concentrate; (44) Add 1% to 50% of the concentrate by mass ratio of 1,2-propanediol to obtain the final product, a rose-scented tobacco extract.

7. The method for preparing the rose-scented tobacco extract according to claim 6, characterized in that, In step (44), 10% of the concentrate mass of 1,2-propanediol is added according to the mass ratio.

8. The application of the rose-scented tobacco extract prepared by any one of the preparation methods described in claims 1-7 in tobacco flavoring.

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