Method for preparing flower fragrance type tobacco flavor by using biological method

Abstract

The invention discloses a method for preparing flower fragrance type tobacco flavor by a biological method. The method comprises the following steps: preparing a Klebsiella seed solution; preparing tobacco extract liquid; peeling and denucleating snow pears, juicing, and separating pulp filter residues and pulp filtrate; adding ethanol into pulp filter residues, mixing and extracting to obtain re-extracted filtrate 1; removing ethanol from the re-extracted filtrate 1 to obtain a re-extracted filtrate 2; mixing the re-extracted filtrate 2 with the pulp filtrate to obtain a snow pear extract; uniformly mixing the tobacco extract with the snow pear extract to obtain a mixed solution; mixing and fermenting the seed solution and the mixed solution to obtain a fermented mixed solution; centrifuging the fermentation mixed solution to obtain supernate; and concentrating the supernate to obtain a concentrated solution, mixing the concentrated solution with absolute ethyl alcohol for alcohol precipitation, and separating to obtain a liquid concentrated solution, namely the flower fragrance type tobacco flavor. The method provided by the invention solves the problem of waste tobacco treatment, realizes efficient utilization of resources, and reduces environmental pollution; through fermentation conversion, the quality of the spice is improved, the fragrance is rich, the taste is coordinated, offensive odor can be effectively covered, and the aftertaste is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and particularly to a method for preparing floral tobacco flavor by a biological method. Background Art

[0002] With the trend of diversified cigarette styles, the development of tobacco flavors with different fragrance styles has received increasing attention. Floral tobacco flavors are an important type of tobacco flavor. They can not only enhance the floral style characteristics of cigarettes but also improve the taste. They mainly include fresh flower extracts, as well as monomeric flavors such as 2-phenylethanol, β-ionone, and terpenoids. However, there are currently some problems. For example, fresh flower extracts are limited by raw materials and processes and are expensive. Some monomeric flavors have a single effect and are prone to bringing miscellaneous odors and masking the tobacco aroma. Therefore, it is of great significance to develop high-quality tobacco flavors that can significantly enhance the aroma style of cigarettes, improve the taste of cigarettes, and at the same time do not bring negative effects.

[0003] Every year, a large amount of unusable tobacco such as low-grade tobacco leaves, tobacco stems, and tobacco dust forms waste, causing serious waste of resources and forming tobacco waste. Preparing tobacco flavors is an effective way to convert tobacco waste. Due to the high content of macromolecules and the small amount of aroma substances, the flavors directly prepared from waste tobacco as raw materials generally have low quality. By using the microbial fermentation method, macromolecular substances can be effectively degraded and converted into flavor components, improving the quality of the prepared flavors. To reduce the influence of the original impurities in waste tobacco, it can be mixed and fermented with fruit extracts that can enhance the tobacco aroma, thereby preparing higher-quality tobacco flavors. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a method for preparing floral tobacco flavor by a biological method. It uses waste tobacco as the core, supplemented with the aqueous extract of Sydney pears without obvious odor for compounding, and prepares a high-quality tobacco flavor with an obvious floral fragrance and capable of masking miscellaneous odors through the fermentation of aroma-producing bacteria.

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

[0006] A method for preparing floral tobacco flavor by a biological method, comprising the following steps:

[0007] (1) Inoculate Klebsiella sp. HNYJ-1 with the preservation number of CGMCC No. 24327 into an LB liquid medium and culture it on a shaker to obtain a seed solution;

[0008] (2) Mix the waste tobacco powder with 10% ethanol at a ratio of 1:20 to 1:2, reflux and extract at 30°C to 120°C for 0.5 h to 5 h, filter, and rotary evaporate the filtrate at a vacuum of 120 mbar and a temperature of 55°C for 2 h, then sterilize to obtain the tobacco extract;

[0009] (3) Peel and core the Sydney pears, juice them using a grinder, and separate the pulp residue and the pulp filtrate; add 50% ethanol to the pulp residue and mix well, extract at a constant temperature of 40°C with shaking for 1 - 6 h, filter to obtain the re-extracted filtrate 1; heat the re-extracted filtrate 1 with constant stirring at 80°C for 2 h to remove ethanol and obtain the re-extracted filtrate 2; mix the re-extracted filtrate 2 with the pulp filtrate to obtain the Sydney pear extract;

[0010] (4) Mix the tobacco extract from step (2) and the Sydney pear extract from step (3) evenly at a mass ratio of 1:20 to 20:1 to obtain a mixed solution; mix the seed solution from step (1) and the mixed solution at a volume ratio of 1:1000 to 1:10, culture at 25 - 40°C with a rotation speed of 50 - 300 r / min, and stir and ferment for 12 - 96 h. After fermentation, obtain the fermented mixed solution;

[0011] (5) Centrifuge the fermented mixed solution from step (4) to obtain the supernatant; concentrate the supernatant at a temperature of 60°C and a condition of 90 mbar to obtain a concentrated solution; mix the concentrated solution and absolute ethanol at a mass ratio of 1:10 to 10:1, let it stand for alcohol precipitation at -20°C for 24 h, and then separate to obtain the liquid concentrated solution, which is the floral tobacco flavor.

[0012] Preferably, in the above technical solution, in step (1), the culture conditions of Klebsiella sp. HNYJ-1 in the LB liquid medium are: culture at 25 - 40°C in a shaker at 100 - 200 r / min for 6 - 36 h.

[0013] Preferably, in the above technical solution, in step (1), the culture conditions of Klebsiella sp. HNYJ-1 in the LB liquid medium are: culture at 30°C in a shaker at 150 r / min for 12 h.

[0014] Preferably, in the above technical solution, in step (2), the waste tobacco powder is obtained by mixing waste low-grade tobacco leaves, tobacco stems, and tobacco powder at a mass ratio of 4:1:3, grinding, and passing through a 50-mesh sieve.

[0015] Preferably, in the above technical solution, step (2) is specifically: mix the waste tobacco powder with 10% ethanol at a ratio of 1:10, reflux and extract at 90°C for 2 h, filter through a 200-mesh sieve to obtain the filtrate, rotary evaporate the filtrate at a vacuum of 120 mbar and a temperature of 55°C for 2 h to fully remove the ethanol in the system, and then sterilize at 121°C for 10 min to obtain the tobacco extract.

[0016] Preferably, in the above technical solution, in step (3), a 200-mesh gauze is used to filter and separate the pulp residue and the pulp filtrate; the pulp residue is added with 5 times the mass of 50% ethanol, fully mixed, extracted by constant temperature oscillation, and then filtered with a 200-mesh gauze.

[0017] Preferably, in the above technical solution, step (4) is specifically as follows: the tobacco extract and the Sydney extract are mixed evenly at a mass ratio of 9:1 to obtain a mixed solution; the seed solution and the mixed solution are mixed at a volume ratio of 1:200, cultured at 30 °C and a rotation speed of 180 r / min, and stirred and fermented for 48 h to obtain a fermentation mixed solution after fermentation.

[0018] Preferably, in the above technical solution, in step (5), the centrifugation conditions of the fermentation mixed solution are: centrifugation at a rotation speed of 8000 r / min and a temperature of 4 °C for 30 min; the density of the supernatant after concentration is 1.10 mg / mL ± 0.005 mg / mL; the mass ratio of the concentrated solution to absolute ethanol is 1:3.

[0019] Application of a floral tobacco flavor prepared by a method for preparing a floral tobacco flavor by a biological method in tobacco.

[0020] The above technical solution of the present invention has the following beneficial effects:

[0021] The present invention uses waste tobacco as a raw material, combines Sydney extract and microbial fermentation technology, and successfully prepares a floral tobacco flavor. On the one hand, this method effectively solves the problem of waste tobacco treatment, realizes the efficient utilization of resources, and reduces environmental pollution; on the other hand, through fermentation conversion, the quality of the flavor is improved, making its aroma rich, taste coordinated, effectively covering up miscellaneous odors and improving aftertaste. Applying it to cigarettes can significantly improve the aroma style and sensory quality of cigarettes, while reducing production costs, and has good economic benefits and market application potential. Specific Embodiments

[0022] Now, various exemplary embodiments of the present invention will be described in detail. It should be noted that: unless otherwise specifically stated, the relative arrangements, numerical expressions and values of the components and steps set forth in these embodiments do not limit the scope of the present invention.

[0023] Klebsiella sp. HNYJ-1 is preserved in the China General Microbiological Culture Collection Center (CGMCC), address: No. 3, Building 1, Beichen West Road, Chaoyang District, Beijing (Institute of Microbiology, Chinese Academy of Sciences), with the preservation number of CGMCC No. 24327 and the preservation date: January 17, 2022.

[0024] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods. The materials and reagents used, unless otherwise specified, are all commercially available. The equipment used in the experiments, unless otherwise specified, are all well-known to those skilled in the art.

[0025] Example 1

[0026] A method for preparing a floral tobacco flavor by a biological method, comprising the following steps:

[0027] (1) The culture conditions of Klebsiella sp. HNYJ-1 seed liquid are as follows: Inoculate a single colony of Klebsiella sp. HNYJ-1 onto an LB liquid medium with an inoculation loop, and culture it in a shaker at 25 - 40 °C (optimal 30 °C) and 100 - 200 r / min (optimal 150 r / min) for 6 - 36 h (optimal 12 h) to obtain the seed liquid.

[0028] (2) Preparation of tobacco extract: Discarded low-grade tobacco leaves, tobacco stems and tobacco dust are mixed and ground according to a mass ratio of 4:1:3, passed through a 50-mesh sieve to obtain discarded tobacco powder. The discarded tobacco powder is mixed with 10% ethanol (mass ratio of ethanol to water is 1:9) according to a ratio of 1:20 to 1:2 (optimal 1:10), and reflux-extracted at 30 °C - 120 °C (optimal 90 °C) for 0.5 h - 5 h (optimal 2 h). After filtering through a 200-mesh sieve, the filtrate is taken, and the filtrate is rotary evaporated at a vacuum of 120 mbar and a temperature of 55 °C for 2 h to fully remove ethanol in the system, and then sterilized at 121 °C for 10 min to obtain the tobacco extract.

[0029] (3) Preparation of Sydney extract: Peel and core the Sydney, juice it with a grinder, and filter and separate the pulp residue and pulp filtrate with a 200-mesh gauze. The pulp residue is added with 5 times the mass of 50% ethanol and mixed thoroughly, and extracted at a constant temperature of 40 °C with shaking for 1 - 6 h (optimal 2 h). After filtering with a 200-mesh gauze, the first re-extracted filtrate is obtained. The first re-extracted filtrate is stirred and heated at a constant temperature of 80 °C for 2 h to remove ethanol, and the second re-extracted filtrate is obtained. The second re-extracted filtrate is mixed with the pulp filtrate to obtain the Sydney extract.

[0030] (4) The tobacco extract and the Sydney extract are mixed evenly according to a mass ratio of 1:20 to 20:1 (optimal 9:1) to obtain a mixed solution. The Klebsiella sp. HNYJ-1 seed liquid in step 1 is mixed with the mixed solution according to a volume ratio of 1:1000 to 1:10 (optimal 1:200) (i.e., the inoculation amount is 0.1% - 10%, optimal 0.5%). Set the culture temperature at 25 - 40 °C (optimal 30 °C), the rotation speed at 50 - 300 r / min (optimal 180 r / min), and stir and ferment for 12 - 96 h (optimal 48 h). After fermentation, the fermented mixed solution is obtained.

[0031] (5) Spice preparation: In step (4), the fermentation mixture is centrifuged at 8000 r / min and 4 °C for 30 min to obtain the supernatant; the supernatant is concentrated at 60 °C and 90 mbar to a density of 1.10 mg / mL ± 0.005 mg / mL to obtain the concentrated solution; the concentrated solution is mixed with absolute ethanol in a mass ratio of 1:10 to 10:1 (optimally 1:3), allowed to stand at -20 °C for alcohol precipitation for 24 h, and then the liquid concentrated solution is separated to obtain the floral fragrance tobacco flavor.

[0032] Application Example 1

[0033] (1) Prepare the floral fragrance tobacco flavor according to the optimal parameters of the above Example 1, denoted as T.

[0034] (2) The tobacco extract does not add the snow pear extract, only adds an equal mass of water, adds the strain for fermentation, and the remaining steps and parameters are the same as those of T, denoted as T1.

[0035] (3) The tobacco extract adds the snow pear extract, but does not add the seed liquid for fermentation, only adds an equal mass of water, and the remaining steps and parameters are the same as those of T, denoted as T2.

[0036] (4) The tobacco extract does not add the snow pear extract, only adds an equal mass of water, does not add the seed liquid for fermentation, and also adds an equal mass of water, and the remaining steps and parameters are the same as those of T, denoted as CK.

[0037] Use GC-MS to detect the content of the main flavor components of the tobacco flavor; the tobacco flavor is added to the Golden Leaf brand cigarettes at a dosage of 50 ppm, and professional sensory evaluation personnel are organized for sensory evaluation.

[0038] The results are as follows:

[0039] The content of the main flavor components related to floral fragrance in the tobacco flavor is shown in Table 1. It can be seen that in the flavor T with snow pear added and fermented, the content of the floral fragrance-related flavor components is the highest; in T1 without snow pear added but fermented, the content of the floral fragrance-related flavor components is also very high; while in T2 with snow pear added but not fermented, the content of the floral fragrance-related flavor components is less and is not much different from that of CK. It shows that there are no floral fragrance components in the snow pear, which are formed by fermentation conversion. The role of the snow pear extract is not to increase the fragrance, but to improve the taste of the cigarette.

[0040] Table 1. Content of the main flavor components of the tobacco flavor

[0041]

[0042] The tobacco flavor was added to cigarettes for sensory evaluation, and the results are shown in Table 2. After adding the flavor CK without fermentation and without adding Sydney extract, the aroma was slightly improved, but the off-odor and aftertaste indexes decreased; after adding flavor T, the aroma increased significantly, and the indexes such as coordination, off-odor, irritation and aftertaste were all significantly improved; after adding flavor T1 which was only fermented without adding Sydney extract, the aroma increased significantly, but the off-odor, irritation and aftertaste indexes decreased; after adding flavor T2 with Sydney extract but without fermentation, the change in the aroma of cigarettes was not significant, but the indexes such as off-odor, irritation and aftertaste increased. It shows that the floral fragrance components can be significantly improved through fermentation, and adding Sydney extract helps to improve the taste indexes such as off-odor, irritation and aftertaste.

[0043] Table 2 Results of Cigarette Sensory Evaluation

[0044] Sample Luster Aroma Harmony Off-odor Irritation Aftertaste Total score 0 5.0 27.5 5.0 10.0 17.5 22.0 87.0 CK 5.0 27.7 5.0 9.8 17.5 21.7 86.7 T 5.0 28.2 5.2 10.5 18.0 22.4 89.3 T1 5.0 28.2 5.0 9.8 17.2 21.6 86.8 T2 5.0 27.6 5.1 10.3 18.0 22.5 88.5

[0045] Note: "0" represents the cigarette sample without adding any substances.

[0046] Although the present invention has been disclosed above by way of examples, it is not intended to limit the present invention. Any person skilled in the art can make various different selections and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention is defined by the claims and their equivalent forms.

Claims

1. A method for preparing floral tobacco flavoring by biological method, characterized in that: The following steps are involved: (1) inoculating Klebsiella HNYJ-1 with a deposit number of CGMCC No. 24327 into LB liquid culture medium and culturing on a shaking table to obtain a seed solution; (2) mixing the waste tobacco dust with 10% ethanol in a ratio of 1:20 to 1:2, extracting under reflux at 30° C. to 120° C. for 0.5 h to 5 h, filtering, and rotary evaporating the filtrate at a vacuum degree of 120 mbar and a temperature of 55° C. for 2 h to sterilize, thereby obtaining a tobacco extract; (3) Peeling and core-removing the snow pear, using a grinder to squeeze the juice, separating the pulp residue and the pulp filtrate; adding 50% ethanol to the pulp residue and mixing thoroughly, extracting at a constant temperature of 40° C. with shaking for 1-6 hours, filtering to obtain a re-extraction filtrate 1; heating the re-extraction filtrate 1 at a constant temperature of 80° C. with stirring for 2 hours, removing the ethanol, and obtaining a re-extraction filtrate 2; mixing the re-extraction filtrate 2 with the pulp filtrate to obtain a snow pear extract; (4) mixing the tobacco extract of step (2) and the snow pear extract of step (3) in a mass ratio of 1:20 to 20:1 to obtain a mixed solution; mixing the seed solution of step (1) with the mixed solution in a volume ratio of 1:1000 to 1:10, culturing at 25-40° C. and a rotation speed of 50 to 300 r / min, stirring and fermenting for 12 to 96 hours, and obtaining a fermentation mixed solution after fermentation; (5) centrifuging the fermentation mixture of step (4) to obtain a supernatant; concentrating the supernatant at 60° C. and 90 mbar to obtain a concentrated solution; mixing the concentrated solution with anhydrous ethanol at a mass ratio of 1:10 to 10:1, allowing the mixture to stand at -20° C. for 24 hours, and separating to obtain a liquid concentrated solution, i.e., a floral tobacco flavoring.

2. The method for preparing flower-scented tobacco flavoring by biological method according to claim 1, characterized in that: In step (1), the culture conditions of Klebsiella HNYJ-1 in LB liquid culture medium are: 25-40° C., 100-200 r / min in a shaking incubator for 6-36 hours.

3. The method for preparing flower-scented tobacco flavoring by biological method according to claim 2, characterized in that: In step (1), the culture conditions of Klebsiella HNYJ-1 in LB liquid culture medium are: culture at 30° C. and 150 r / min in a shaking incubator for 12 h.

4. The method for preparing flower-scented tobacco flavoring by biological method according to claim 1, characterized in that: In step (2), the waste tobacco dust is obtained by mixing and grinding waste low-grade tobacco leaves, tobacco stems and tobacco dust in a mass ratio of 4:1:3 and passing through a 50-mesh sieve.

5. The method for preparing flower-scented tobacco flavoring by biological method according to claim 4, characterized in that: Step (2) is specifically as follows: waste tobacco dust is mixed with 10% ethanol in a ratio of 1:10, refluxed and extracted at 90°C for 2h, filtered through a 200-mesh sieve to obtain a filtrate, and the filtrate is rotary evaporated at a vacuum degree of 120mbar and a temperature of 55°C for 2h to fully remove the ethanol in the system, and then sterilized at 121°C for 10min to obtain a tobacco extract.

6. The method for preparing floral tobacco flavoring by biological method according to claim 1, characterized in that: In step (3), the pulp residue and the pulp filtrate are separated by filtering with 200-mesh gauze; 5 times the mass of 50% ethanol is added to the pulp residue and mixed thoroughly, and the mixture is extracted by constant temperature oscillation and filtered with 200-mesh gauze.

7. The method for preparing flower-scented tobacco flavoring by biological method according to claim 1, characterized in that: Step (4) is specifically as follows: the tobacco extract and the snow pear extract are mixed evenly in a mass ratio of 9:1 to obtain a mixed solution; the seed solution and the mixed solution are mixed in a volume ratio of 1:200, cultured at 30° C. and a rotation speed of 180 r / min, stirred and fermented for 48 hours, and a fermentation mixed solution is obtained after the fermentation is completed.

8. The method for preparing flower-scented tobacco flavoring by biological method according to claim 1, characterized in that: In step (5), the centrifugal conditions of the fermentation mixture are: a rotation speed of 8000 r / min and a temperature of 4° C. for 30 min; the density of the supernatant after concentration is 1.10 mg / mL±0.005 mg / mL; and the mass ratio of the concentrate to anhydrous ethanol is 1:

3.

9. Use of the floral tobacco flavoring prepared by the method for preparing floral tobacco flavoring by biological method according to any one of claims 1 to 8 in tobacco.

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