Preparation method and application of tobacco source aroma raw material rich in small molecule aroma components

Abstract

The invention discloses a preparation method and application of a tobacco source aroma raw material rich in micromolecular aroma components, and the preparation method comprises the following steps: crushing and sieving tobacco leaves, adopting a 40% ethanol solution as a solvent, and carrying out long-time extraction and vacuum concentration under proper conditions to prepare a tobacco extract with set baume degree; the method comprises the following steps: unfreezing, activating and culturing a Wickerham LY13 strain to obtain a high-activity bacterial solution; inoculating the bacterial liquid into the tobacco extract, fermenting under a controlled condition, and enhancing the aroma components in a biotransformation manner; after fermentation is completed, fragrance components in different molecular weight ranges are finely separated out through a high-temperature sterilization and multi-stage membrane separation technology; the four groups of separated permeate liquid are used as the tobacco source flavor raw materials to be added into cigarettes, sensory evaluation is carried out, and the result shows that after the tobacco source flavor raw materials prepared through the method are applied to the cigarettes, the sensory quality of the cigarettes can be remarkably improved, and the effects of coordinating the tobacco flavor, increasing the smoke concentration, improving the aftertaste and the like are achieved.

Description

Technical Field

[0001] This invention belongs to the field of refined preparation technology of tobacco flavorings and fragrances, specifically relating to a preparation method and application of tobacco flavoring raw materials rich in small molecule flavor components. Background Technology

[0002] In the tobacco industry, tobacco-derived flavorings, prepared from tobacco, play an indispensable role in enhancing the natural aroma and overall quality of cigarettes, and are widely used in cigarette processing. However, the tobacco-derived flavoring raw materials currently used in cigarette processing are mainly prepared through ethanol extraction, a process that inevitably introduces macromolecules such as starch, pectin, cellulose, and protein. These substances produce unpleasant odors and irritation during cigarette combustion, affecting not only the purity and taste of the cigarette but also potentially weakening the flavoring effect of the flavorings, negatively impacting the overall quality of the cigarette and making it difficult to fully showcase the natural flavor and subtle nuances of the tobacco-derived flavorings. Therefore, how to accurately capture the main aroma components in tobacco and prepare tobacco-derived flavoring raw materials with significant effects has become a critical problem that urgently needs to be solved in the tobacco industry. Summary of the Invention

[0003] The purpose of this invention is to provide a method for preparing tobacco aroma raw materials rich in small molecule aroma components and their application, so as to solve the problems existing in the above-mentioned background art.

[0004] To achieve the above objectives, this application employs the following technical solution:

[0005] A method for preparing tobacco aroma raw material rich in small molecule aroma components includes the following steps:

[0006] S1. After crushing the tobacco leaves, pass them through a 60-mesh sieve, add 40% ethanol solution to soak them, then heat and extract for a set time, and then concentrate under reduced pressure to obtain tobacco extract.

[0007] S2. The fully activated abnormal Wickham LY13 strain was inoculated into LB medium at a 1% inoculum and cultured in a shaker at 30°C and 150 rpm for 12 h to obtain the bacterial culture.

[0008] S3. The bacterial solution obtained in step S2 is inoculated into the tobacco extract in step S1 at an inoculation rate of 12%, and fermented at 32°C and 50~400 r / min for 24 h.

[0009] S4. The fermented tobacco extract was redissolved in 40% ethanol solution, filtered, and then separated sequentially using 0.22 μm, 300KD, 30KD, and 10KD filter membranes to obtain four permeate components, which are four tobacco aroma raw materials rich in small molecule aroma components.

[0010] Furthermore, in step S1, the mass ratio of 40% ethanol solution to tobacco leaves is 8:1-10:1, and the extraction is carried out at 80-85℃ for 10-12 hours.

[0011] Furthermore, the tobacco extract with a Baume degree of 3-5 was obtained by depressurization concentration.

[0012] Further, step S2 involves taking the LY13 strain out of the -80℃ freezer and thawing it in the -20℃ and 4℃ freezers in sequence. After the strain is fully activated, it is inoculated into LB medium at a 1% inoculation rate.

[0013] Furthermore, in S4, the fermented tobacco extract is sterilized at 121°C for 20 min and then reconstituted with 10-12 times its mass of 40% ethanol.

[0014] Furthermore, the study also included the determination of aroma component content in four permeate components by GC-MS.

[0015] Application of a tobacco aroma ingredient rich in small molecule flavor components, and application of the tobacco aroma ingredient prepared by any of the above methods in cigarettes.

[0016] Furthermore, the amount of tobacco flavoring ingredients added is one ten-thousandth of the weight of the tobacco shreds.

[0017] The beneficial effects of this invention are:

[0018] This invention utilizes membrane separation technology to selectively retain macromolecular components, which can effectively improve the sensory quality of tobacco extracts and obtain tobacco aroma raw materials rich in small molecule aroma components. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the membrane separation process of the present invention.

[0020] Figure 2 This is a graph showing the changes in the content of the main aroma components between the original sample and each stage of the permeate. Detailed Implementation

[0021] The technical solution of the present invention will be described in detail below with reference to the accompanying drawings. The following embodiments are merely exemplary and can only be used to explain and illustrate the technical solution of the present invention, and should not be construed as limiting the technical solution of the present invention.

[0022] The Wickerhamomyces anomalus strain involved in this application is currently deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.28452, on September 13, 2023.

[0023] Example 1

[0024] 100 g of Yunnan Honghe tobacco leaves were weighed, pulverized, and passed through a 60-mesh sieve. 800 g of 40% ethanol solution was added, and the mixture was soaked overnight. Extraction was carried out at 85℃ for 12 h, and the extract was concentrated under reduced pressure to obtain a tobacco extract with a Baume degree of 3. The abnormal Wickham LY13 strain was removed from a -80℃ freezer and thawed sequentially at -20℃ and 4℃. After full activation, it was inoculated into LB medium at a 1% inoculum and cultured at 30℃ and 150 rpm for 12 h to obtain a bacterial culture. This bacterial culture was inoculated into the tobacco extract at a 12% inoculum and fermented at 32℃ and 200 r / min for 24 h. The fermented tobacco extract was sterilized at 121℃ for 20 min, reconstituted with 10 times its mass of 40% ethanol solution, filtered, and sample A was obtained. A 0.22... Sample A was sequentially separated using μm, 300KD, 30KD, and 10KD filter membranes, yielding four permeate components (C, E, G, and I) and four retentate components (B, D, F, and H). Components C, E, G, and I are four tobacco-derived aroma raw materials rich in small-molecule aroma components, such as... Figure 1 As shown, the aroma component content of samples A and the four permeate components C, E, G and I was determined by GC-MS. 10 mL of each component was placed in a centrifuge tube, 20 μL of internal standard solution (2,6-dichlorotoluene, 0.07025 g / 100 mL) was added, followed by 10 mL of dichloromethane. The mixture was vortexed at 250 rpm for 10 min. The dichloromethane phase was collected, concentrated to 1 mL by nitrogen blowing, and then loaded onto a membrane GC-MS.

[0025] Chromatographic conditions: Agilent 122-5562 DB-5MS (60 m × 250 μm × 0.25 μm); Injector temperature: 280 ℃; Carrier gas: 99.999% high-purity helium, flow rate 1.0 mL / min; Injection method: split injection, split ratio 5:1, injection volume 1 μL. Temperature program: Initial temperature 40 ℃, hold for 10 min, increase to 80 ℃ at a rate of 2.5 °C / min, then increase to 120 ℃ at a rate of 4 °C / min, then increase to 240 ℃ at a rate of 1.5 °C / min, and finally increase to 270 ℃ at a rate of 5 °C / min and hold for 5 min.

[0026] Mass spectrometry conditions: SCAN mode was used, with a transfer line temperature of 280 °C, an ion source temperature of 230 °C, a quadrupole temperature of 150 °C, an electron impact ionization (EI) source with an electron energy of 70 eV, a solvent delay time of 10 min, and a scan range of 10–800 amu.

[0027] The results of GC-MS determination of the main aroma components in the original sample and the four permeate components are shown in Table 1.

[0028] Depend on Figure 2 It is known that while semi-permeable membranes at each stage selectively separate macromolecular components, they also retain aroma components. The content of the main aroma components in the tobacco extract decreases progressively after processing through each stage of the membrane. Therefore, the aim is to determine the flavoring ratio for sensory evaluation by analyzing the change in damascone content.

[0029] Samples A and four permeate components (C, E, G, and I) were uniformly injected into blank Golden Leaf Golden Medium-Length cigarettes at a ratio of 1 / 10,000 of the tobacco shreds mass. After equilibration in a constant temperature and humidity chamber at 22±2℃ and 60±5% for 48 h, sensory effects were evaluated. The evaluation results are shown in Table 2.

[0030] Table 1. Comparison of the main aroma components content between the original sample and each stage of permeate.

[0031] Main fragrance components Original concentration C0.22μm E300KD G30KD I10KD Phenylacetyl alcohol 0.320 0.226 0.182 0.162 0.107 Solanone 0.128 0.101 0.097 0.083 0.056 Damaskone 0.143 0.109 0.076 0.065 0.034 Dihydroactin 0.178 0.176 0.167 0.147 0.083 Megastigmatrienone 0.078 0.051 0.049 0.035 0.027 4-Hydroxy-β-dihydrodamascone 0.689 0.658 0.666 0.530 0.406 3-Oxo-α-ionol 0.597 0.623 0.477 0.471 0.365 3-Oxo-7,8-dihydro-α-ionol 0.335 0.328 0.305 0.226 0.174 Digoxin 0.291 0.286 0.262 0.219 0.205 Methyl palmitate 0.331 0.309 0.279 0.206 0.126 Sibertrienol 1.064 0.660 0.517 0.472 0.213

[0032] Note: The unit is μg / mL.

[0033] Table 2 Sensory rating table for original sample and permeate components at each stage

[0034] Sample type Fragrance Aroma Mixed gases translucency energy concentration Delicate and soft Stimulation level Dryness Aftertaste Aftertaste Total Score A 3.00 3.00 3.00 3.00 3.00 3.00 3.00 3.00 3.00 3.00 3.00 33.00 C 3.28 3.41 3.21 3.27 3.00 3.46 3.35 3.15 3.00 3.39 3.35 35.87 E 3.24 3.35 3.12 3.19 3.00 3.34 3.26 3.08 3.00 3.16 3.27 35.01 G 3.06 3.18 3.05 3.15 3.00 3.19 2.95 2.95 3.00 3.05 2.87 33.45 I 3.08 3.09 3.09 3.12 3.00 3.21 2.98 2.87 3.00 3.05 2.91 33.40

[0035] Table 2 shows that the sensory scores of all permeate components were higher than those of the original control group. However, the sensory quality scores gradually decreased with each stage of separation. Among them, C, the extract separated through a 0.22 μm pore size semi-permeable membrane, showed the best aroma absorption results, followed by E. G and I, on the other hand, had overall sensory scores similar to the original sample, with only minor improvements. Specific sensory quality descriptions are as follows:

[0036] C: The most significant improvement is in aroma, with a substantial increase in both aroma quantity and concentration. The smoke is delicate and smooth, with a sweet aroma, a noticeably improved aftertaste, and the best overall harmony.

[0037] E: The aroma has also improved significantly, and the smoke concentration has increased, but the penetration is slightly poor, there is a slight off-flavor, and the overall aroma is slightly unbalanced.

[0038] G: The irritation is significantly increased, and there are more impurities than in C and E. The smoke is cloudy and unclear, the aroma is not prominent, and although the smoke concentration is increased, the aftertaste becomes unpleasant.

[0039] I: The smoke is dull, the aroma is not prominent and is masked by other smells, the irritation is increased, the concentration is increased, but the overall effect is very uncoordinated and the restlessness is amplified.

[0040] The overall sensory evaluation results were: C > E > G > I > A. This indicates that selectively retaining macromolecular components with a particle size greater than 0.22 μm using membrane separation technology can effectively improve the sensory quality of tobacco extracts and obtain tobacco aroma raw materials rich in small molecule aroma components.

[0041] After membrane separation, large molecules were retained. Sensory quality evaluation of the retentate from the 0.22 μm pore size membrane revealed a lack of aroma, increased off-odors, unclear smoke, and poor aftertaste, significantly negatively impacting sensory performance. Therefore, selectively retaining large molecules with a particle size greater than 0.22 μm using membrane separation technology can effectively improve the sensory quality of tobacco extracts and obtain tobacco aroma raw materials rich in small molecule aroma components.

[0042] Example 2

[0043] 100 g of Yunnan Honghe tobacco leaves were weighed, pulverized, and passed through a 60-mesh sieve. 1000 g of 40% ethanol solution was added, and the mixture was soaked overnight. Extraction was carried out at 80℃ for 10 h, and the extract was concentrated under reduced pressure to obtain a tobacco extract with a Baume degree of 5. The abnormal Wickham LY13 strain was removed from a -80℃ freezer and thawed sequentially at -20℃ and 4℃. After full activation, it was inoculated into LB medium at a 1% inoculum and cultured at 30℃ and 150 rpm for 12 h to obtain a bacterial culture. This bacterial culture was inoculated into the tobacco extract at a 12% inoculum and fermented at 32℃ and 200 r / min for 24 h. The fermented tobacco extract was sterilized at 121℃ for 20 min, reconstituted with 12 times its mass of 40% ethanol solution, filtered, and sample J was obtained. 0.22 Sample J was sequentially separated using μm, 300KD, 30KD, and 10KD filter membranes to obtain four permeate components (K, M, O, and Q) and four retentate components (L, N, P, and R). Among them, the four permeate components (K, M, O, and Q) are four tobacco-derived aroma raw materials rich in small molecule aroma components.

[0044] Example 3

[0045] 100 g of Yunnan Honghe tobacco leaves were weighed, pulverized, and passed through a 60-mesh sieve. 900 g of 40% ethanol solution was added, and the mixture was soaked overnight. Extraction was carried out at 85℃ for 12 h, and the extract was concentrated under reduced pressure to obtain a tobacco extract with a Baume degree of 4. The abnormal Wickham LY13 strain was removed from a -80℃ freezer and thawed sequentially at -20℃ and 4℃. After full activation, it was inoculated into LB medium at a 1% inoculum and cultured at 30℃ and 150 rpm for 12 h to obtain a bacterial culture. This bacterial culture was inoculated into the tobacco extract at a 12% inoculum and fermented at 32℃ and 200 r / min for 24 h. The fermented tobacco extract was sterilized at 121℃ for 20 min, reconstituted with 11 times its mass of 40% ethanol solution, filtered, and sample 3 was obtained. 0.22 Sample 3 was separated sequentially using μm, 300KD, 30KD, and 10KD filter membranes to obtain four permeate components (M1, M2, M3, and M4) and four retentate components (M5, M6, M7, and M8). Among them, the four permeate components (M1, M2, M3, and M4) are four tobacco-derived aroma raw materials rich in small molecule aroma components.

[0046] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for preparing tobacco aroma raw materials rich in small molecule aroma components, characterized in that, Includes the following steps: S1. After crushing the tobacco leaves, pass them through a 60-mesh sieve, add 40% ethanol solution to soak them, then heat and extract for a set time, and then concentrate under reduced pressure to obtain tobacco extract. S2. The fully activated abnormal Wickham LY13 strain was inoculated into LB medium at a 1% inoculum and cultured in a shaker at 30°C and 150 rpm for 12 h to obtain the bacterial culture. S3. The bacterial solution obtained in step S2 is inoculated into the tobacco extract in step S1 at an inoculation rate of 12%, and fermented at 32°C and 50~400 r / min for 24 h. S4. The fermented tobacco extract was redissolved in 40% ethanol solution, filtered, and then separated sequentially using 0.22 μm, 300KD, 30KD, and 10KD filter membranes to obtain four permeate components, which are four tobacco aroma raw materials rich in small molecule aroma components.

2. The method for preparing tobacco aroma raw material rich in small molecule aroma components according to claim 1, characterized in that, In step S1, the mass ratio of 40% ethanol solution to tobacco leaves is 8:1-10:1, and the extraction is carried out at 80-85℃ for 10-12 hours.

3. The method for preparing tobacco aroma raw material rich in small molecule aroma components according to claim 2, characterized in that, Tobacco extract with a Baume degree of 3-5 was obtained by vacuum concentration.

4. The method for preparing tobacco aroma raw material rich in small molecule aroma components according to claim 1, characterized in that, Wickerhamomyces anomalus LY13 is deposited at the China Center for Type Culture Collection (CCTCC) under accession number CCTCC NO.28452.

5. The method for preparing tobacco aroma raw material rich in small molecule aroma components according to claim 1, characterized in that, Step S2 involves taking the LY13 strain out of the -80℃ freezer and thawing it in the -20℃ and 4℃ freezers in sequence. After the strain is fully activated, it is inoculated into LB medium at a 1% inoculation rate.

6. The method for preparing tobacco aroma raw material rich in small molecule aroma components according to claim 1, characterized in that, In S4, the fermented tobacco extract was sterilized at 121°C for 20 min and then reconstituted with 10-12 times its weight of 40% ethanol.

7. The method for preparing tobacco aroma raw material rich in small molecule aroma components according to claim 1, characterized in that, It also includes the determination of aroma component content in four permeate components by GC-MS.

8. An application of a tobacco-derived aroma raw material rich in small molecule aroma components, characterized in that, The application of the tobacco aroma raw material prepared by the preparation method of any one of claims 1 to 7 in cigarettes.

9. The application of the tobacco aroma source raw material rich in small molecule aroma components according to claim 8, characterized in that, The amount of tobacco flavoring material added is one ten-thousandth of the weight of the tobacco shreds.

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