Preparation method of rose-flavor tobacco extract

Abstract

The preparation method comprises the following steps: respectively inoculating micrococcus, gluconacetobacter and klebsiella variicola into an LB liquid culture medium for culturing, centrifuging supernate, adding sterile water for redissolving to obtain three seed solutions, and mixing the three seed solutions to obtain a composite seed solution; sieving the tobacco powder, mixing the sieved tobacco powder with deionized water, and extracting to obtain a tobacco extract; mixing the composite seed liquid with the tobacco extract liquid, stirring and fermenting; and centrifuging the fermented tobacco extract to obtain a supernatant, separating, purifying and concentrating the supernatant to obtain a concentrate, and adding 1, 2-propylene glycol to obtain the rose-flavor tobacco extract. The method is simple in process and low in cost. The content of rose-flavor main flavor components in the tobacco extract is remarkably increased, so that the tobacco extract is rich in fragrance and obvious in characteristic. Through sensory evaluation, the aroma of the cigarettes added with the extract is obviously improved, the rose aroma style of the cigarettes is effectively improved, and the extract has a good market application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a method for preparing a rose-scented tobacco extract. Background Art

[0002] Tobacco extract is an extremely important tobacco flavor in cigarette processing, which can give cigarettes a more natural and full aroma. Tobacco extract is generally prepared by extracting, purifying and concentrating organic matter or water from tobacco. Refined tobacco extract has fewer impurities and higher quality, but the process is generally complex and the cost is high; while crude tobacco extract usually has more impurities and poor quality. During the extraction process of tobacco extract, there will be many macromolecules such as protein, starch, pectin, etc., and the content of flavor substances is basically determined by the tobacco raw materials. If it is not separated and purified by complex methods, the quality of the prepared tobacco extract is not ideal. The use of microbial fermentation to prepare tobacco extract can effectively degrade macromolecules and convert them into special flavor components. The prepared tobacco extract has fewer impurities and sufficient aroma.

[0003] The aroma components isolated from roses are relatively complex, mainly linalool, 2-phenylethanol, citronellol, geraniol, β-ionone, rose alcohol, etc. Some of these substances, such as 2-phenylethanol, linalool, β-ionone, etc., have obvious rose aroma characteristics and can be perceived in very small amounts. They can be effectively applied to tobacco to enhance the rose aroma style of cigarettes. On the one hand, microbial fermentation can degrade the glycosides composed of the original aroma components and sugars in tobacco, thereby releasing the aroma components. On the other hand, it can be transformed through its own metabolism, which is an effective way to enhance 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, wherein a tobacco extract is obtained by water extraction, and a tobacco extract having obvious rose-scented characteristics is prepared by fermentation with a composite aroma-producing fungus. The method has the advantages of simple process, low cost, good addition effect, etc.

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

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

[0007] (1) Preparation of composite aroma-producing fungus seeds: Micrococcus ZY-02 with a preservation number of CGMCC No. 21313, Gluconacetobacter ZT-01 with a preservation number of CGMCC No. 21544, and Klebsiella variegata HNYJ-3 with a preservation number of CGMCC No. 29654 were inoculated into LB liquid culture medium, respectively, and cultured in a shaking incubator at 25-40° C. and 100-200 r / min for 6-36 h, centrifuged, and the supernatant was re-dissolved with sterile water, and the OD600 was adjusted to 2.0 to obtain three seed solutions, and the three seed solutions were evenly mixed according to a proportion to obtain a composite seed solution;

[0008] (2) Preparation of tobacco extract: sieve tobacco dust and mix with deionized water, extract at 30°C to 100°C for 0.5h to 5h, filter and sterilize the filtrate to obtain tobacco extract;

[0009] (3) fermenting tobacco extract: mixing the composite seed solution of step (1) with the tobacco extract of step (2) at a volume ratio of 1:1000 to 1:50, and fermenting at a temperature of 25-40° C. and 50-300 r / min with stirring for 1 to 5 days;

[0010] (4) Preparation of fermented tobacco extract: The fermented tobacco extract is centrifuged to obtain a supernatant, the supernatant is separated and purified, and a concentrate is obtained after concentration. 1,2-propylene glycol is added to obtain a 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 the LB liquid culture medium are: 30°C, 150r / min in a shaking incubator for 12h; and the centrifugation conditions are: 4°C, 4000r / min for 20min.

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

[0013] Preferably, in the above technical scheme, step (2) is specifically as follows: grinding the middle tobacco leaves of flue-cured tobacco to obtain tobacco dust, passing through a 50-mesh sieve, mixing the sieved tobacco dust with deionized water in a ratio of 1:20 to 1:2, extracting at 30°C to 100°C for 0.5h to 5h, filtering through a 200-mesh sieve, and obtaining the filtrate, sterilizing at 121°C for 15min to obtain the tobacco extract.

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

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

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

[0017] (41) The fermented tobacco extract was centrifuged at a speed of 4000 r / min and a temperature of 4° C. for 30 min to obtain a supernatant;

[0018] (42) The supernatant was separated and purified using HD-20 macroporous resin and linearly eluted with ethanol of varying purity gradually increasing from 0 to 100%;

[0019] (43) The second half of the elution fractions were collected and concentrated to a density of 1.10 ± 0.005 g / mL under the conditions of 50 °C and a vacuum degree of 120 mbar to obtain a concentrate;

[0020] (44) Add 1,2-propylene glycol in an amount of 1% to 50% by mass of the concentrate according to a mass ratio to obtain a final product of a rose-flavored tobacco extract.

[0021] Preferably, in the above technical solution, in step (44), 1,2-propylene glycol is added in an amount of 10% by mass of the concentrate.

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

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

[0024] This application uses composite aroma-producing bacteria (Micrococcus ZY-02, Gluconacetobacter ZT-01 and Klebsiella variegata HNYJ-3) to ferment tobacco extract, which has a simple process and low cost. By optimizing the proportion of strains and fermentation conditions, the content of the main rose-scented aroma components in the tobacco extract, such as 2-phenylethanol, linalool, etc., is significantly increased, making the aroma rich and distinctive. According to sensory evaluation, the aroma of cigarettes added with this extract is significantly improved, the impurities and irritation are reduced, the aftertaste is improved, the quality is higher, and the rose-scented style of cigarettes can be effectively improved, which has good market application prospects. DETAILED DESCRIPTION

[0025] 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.

[0026] Gluconacetobacter sp. ZT-01 is deposited in the General Microbiology Center (CGMCC) of China Microorganism Culture Collection, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing (Institute of Microbiology, Chinese Academy of Sciences), with the deposit number: CGMCC No. 21544, and the deposit date: December 23, 2020.

[0027] Micrococcus Pri ZY-02 is deposited in the General Microbiology Center (CGMCC) of China Microorganism Culture Collection, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing (Institute of Microbiology, Chinese Academy of Sciences), with the deposit number: CGMCC No.21313, and the deposit date: December 7, 2020.

[0028] Klebsiella variicola HNYJ-3 is deposited in the General Microbiology Center (CGMCC) of China Microorganism Culture Collection Administration, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing (Institute of Microbiology, Chinese Academy of Sciences), with the deposit number: CGMCC No. 29654, and the deposit date: January 18, 2024.

[0029] The experimental methods used in the following examples are all conventional methods unless otherwise specified. The materials and reagents used are all commercially available unless otherwise specified. The equipment used in the experiments are all well known to those skilled in the art unless otherwise specified.

[0030] Example 1

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

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

[0033] The colonies of the three aroma-producing fungi were inoculated into LB liquid culture medium with an inoculation loop, and cultured in a shaker at 25-40°C (optimal 30°C) and 100-200r / min (optimal 150r / min) for 6-36h (optimal 12h). After the culture, they were centrifuged at 4000r / min and 4°C for 20min, the supernatant was removed, sterile water was added for re-dissolution, and OD600 was adjusted to 2.0 to obtain seed solutions of the three aroma-producing fungi.

[0034] The ZY-02, ZT-01 and HNYJ-3 seed solutions were uniformly mixed 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 of the middle part of the flue-cured tobacco were ground to obtain tobacco dust, which was passed through a 50-mesh sieve. After being sieved, the tobacco dust was mixed with deionized water in a ratio of 1:20 to 1:2 (optimal 1:10), extracted at 30°C to 100°C (optimal 70°C) for 0.5h to 5h (optimal 2h), filtered through a 200-mesh sieve, and the filtrate was sterilized at 121°C for 15min to obtain the tobacco extract.

[0037] (3) Fermented tobacco extract:

[0038] The composite seed liquid in step (1) and the tobacco extract in step (2) are mixed in a volume ratio of 1:1000-1:50 (optimally 1:200) (i.e., the inoculation amount is 0.1%-2%, optimally 0.5%), and the mixture is stirred and fermented for 1-5 days (optimally 2 days) at a temperature of 25-40° C. (optimally 30° C.) and 50-300 r / min (optimally 150 r / min).

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

[0040] The fermented tobacco extract was centrifuged at a speed of 4000 r / min and a temperature of 4° C. for 30 min to obtain a supernatant.

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

[0042] The second half elution components were collected and concentrated to a density of 1.10±0.005 g / mL at a temperature of 50° C. and a vacuum degree of 120 mbar to obtain a concentrate, and 1% to 50% of 1,2-propylene glycol (optimal 10%) of the concentrate mass was added according to a mass ratio to obtain a final rose-scented tobacco extract.

[0043] Application Example 1

[0044] (1) According to the optimal parameters described in the steps of Example 1 above, three aroma-producing fungi were used for fermentation to prepare tobacco extract, which was recorded as T0.

[0045] (2) According to the above steps (2) to (4), step (3) was replaced by inoculating an equal volume of sterile water to prepare a rose-scented tobacco extract, which was recorded as CK (due to the lack of fermentation, the effective ingredients were small and less liquid was collected after column purification, so it was only concentrated to 20% of the original volume and the density was less than 1.10 mg / mL).

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

[0047] According to the optimal parameters described in the above steps, only Gluconacetobacter ZT-01 is used for fermentation to prepare Gluconacetobacter ZT-01, which is recorded as T2.

[0048] According to the optimal parameters described in the above steps, only Klebsiella mutans HNYJ-3 was used for fermentation to prepare tobacco extract, which was recorded as T3.

[0049] The tobacco extract was tested for the main rose-flavored aroma components by GC-MS. The tobacco extract was added to cigarette shreds at a dosage of 10 ppm, and professional smoking testers were organized to conduct sensory evaluation.

[0050] The results are as follows:

[0051] The main rose-flavored aroma components of tobacco extracts are shown in Table 1. From the results, it can be seen that in the unfermented CK group, the content of the main rose-flavored aroma components is 32.10ug / mL, which is relatively low and generally not easily perceived; while in the T0 extract fermented with composite aroma-producing fungi, the content of the main rose-flavored aroma components is 510.80ug / mL, which is dozens of times higher, and has a very obvious rose-flavor characteristic; while the main rose-flavored aroma components of the extracts T1, T2, and T3 fermented with a single bacteria have all increased, but are lower than T0, and some individual components are less, so the rose-flavor characteristics are not strong enough.

[0052] Table 1 Main rose-flavored aroma components in tobacco extract (unit: ug / mL)

[0053]

[0054]

[0055] The results of cigarette evaluation are shown in Table 2. From the results, it can be seen that after adding the unfermented tobacco extract CK, the various indicators did not change much, there was a slight smell, and the total score decreased by 0.2 points; after adding T0, the aroma was significantly improved, and the scores of indicators such as smell, stimulation and aftertaste were improved, and the total score increased by 2.3 points; after adding T1, T2, and T3, the aroma and other indicators were improved to a certain extent, but none of them was as obvious as T0. It can be seen that the tobacco extract prepared by compound aroma-producing fungi fermentation is of higher quality.

[0056] Table 2 Cigarette sensory evaluation results

[0057] sample luster aroma Harmony Miscellaneous gas Irritant 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” refers to cigarette samples without any added substances.

[0059] 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 can 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 method for preparing a rose-scented tobacco extract, characterized in that: The following steps are involved: (1) Preparation of composite aroma-producing fungus seeds: Micrococcus ZY-02 with a preservation number of CGMCC No. 21313, Gluconacetobacter ZT-01 with a preservation number of CGMCC No. 21544, and Klebsiella variegata HNYJ-3 with a preservation number of CGMCC No. 29654 were inoculated into LB liquid culture medium, respectively, and cultured in a shaking incubator at 25-40° C. and 100-200 r / min for 6-36 h, centrifuged, and the supernatant was re-dissolved with sterile water, and the OD600 was adjusted to 2.0 to obtain three seed solutions, and the three seed solutions were evenly mixed according to a proportion to obtain a composite seed solution; (2) Preparation of tobacco extract: sieve tobacco dust and mix with deionized water, extract at 30°C to 100°C for 0.5h to 5h, filter and sterilize the filtrate to obtain tobacco extract; (3) fermenting tobacco extract: mixing the composite seed solution of step (1) with the tobacco extract of step (2) at a volume ratio of 1:1000 to 1:50, and fermenting at a temperature of 25-40° C. and 50-300 r / min with stirring for 1 to 5 days; (4) Preparation of fermented tobacco extract: The fermented tobacco extract is centrifuged to obtain a supernatant, the supernatant is separated and purified, and a concentrate is obtained after concentration. 1,2-propylene glycol is added to obtain a 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 are inoculated into the LB liquid culture medium are: 30° C., 150 r / min in a shaking incubator for 12 h; and the centrifugation conditions are: 4° C., 4000 r / min for 20 min.

3. The method for preparing the rose-scented tobacco extract according to claim 1, characterized in that: In step (1), the ZY-02 seed solution, the ZT-01 seed solution and the HNYJ-3 seed solution are uniformly mixed in a volume ratio of (1-5):(1-5):(1-5) to obtain a composite seed solution.

4. The method for preparing the rose-scented tobacco extract according to claim 1, characterized in that: Step (2) is specifically as follows: grinding the flue-cured tobacco leaves to obtain tobacco dust, passing the tobacco dust through a 50-mesh sieve, mixing the sieved tobacco dust with deionized water in a ratio of 1:20 to 1:2, extracting at 30° C. to 100° C. for 0.5 h to 5 h, filtering through a 200-mesh sieve, obtaining the filtrate, and sterilizing at 121° C. for 15 min to obtain a tobacco extract.

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

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

7. The method for preparing the rose-scented tobacco extract according to claim 1, characterized in that: Step (4) is specifically as follows: (41) The fermented tobacco extract was centrifuged at a speed of 4000 r / min and a temperature of 4° C. for 30 min to obtain a supernatant; (42) The supernatant was separated and purified using HD-20 macroporous resin and linearly eluted with ethanol of varying purity gradually increasing from 0 to 100%; (43) The second half of the elution fractions were collected and concentrated to a density of 1.10 ± 0.005 g / mL under the conditions of 50 °C and a vacuum degree of 120 mbar to obtain a concentrate; (44) Add 1,2-propylene glycol in an amount of 1% to 50% by mass of the concentrate to obtain a final rose-flavored tobacco extract.

8. The method for preparing the rose-scented tobacco extract according to claim 7, characterized in that: In step (44), 1,2-propylene glycol is added in an amount of 10% by mass based on the mass of the concentrate.

9. Use of the rose-scented tobacco extract prepared according to any one of the preparation methods of claims 1 to 8 in tobacco flavoring.

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