Composite tobacco flavoring essence containing rose extract and preparation method thereof

Abstract

This invention relates to the field of tobacco flavoring technology, and discloses a compound tobacco flavoring containing rose extract and its preparation method; the method includes the following steps: 1) inoculating rose suspension cells into MS medium and culturing at a constant temperature, during which high-yield aroma precursors are induced in the rose suspension cells to obtain rose cells that produce high-yield aroma precursors; 2) extracting and purifying the rose cells that produce high-yield aroma precursors obtained in step 1) to obtain a concentrated aroma precursor solution; 3) adding the concentrated aroma precursor solution obtained in step 2) dropwise into a wall material solution, adding a crosslinking agent for crosslinking, and spray drying to obtain pH / temperature dual-responsive nanocapsules; 4) adding the pH / temperature dual-responsive nanocapsules obtained in step 3) and a synergist into a solvent, ultrasonicating, and homogenizing to obtain the compound tobacco flavoring containing rose extract. This invention has a high sensory evaluation score, prominent rose characteristic aroma, and a high aroma retention rate.

Description

Technical Field

[0001] This invention relates to the field of tobacco flavoring technology, and more specifically, to a compound tobacco flavoring containing rose extract and its preparation method. Background Technology

[0002] Tobacco flavorings are a core component of cigarette formulations, and their aroma characteristics and retention rates directly determine the sensory quality and consumer experience of cigarettes. Rose flavoring, a classic floral flavoring ingredient in tobacco flavoring, is fresh and mellow, effectively enriching the aroma layers of cigarettes and alleviating their spiciness, making it a commonly used ingredient in compound tobacco flavorings.

[0003] Currently, the preparation of rose extracts in the tobacco industry mostly employs traditional solvent extraction and steam distillation methods. These methods suffer from low extraction rates of rose aroma precursors and the easy destruction of heat-sensitive aroma components, resulting in rose extracts with weak aroma and limited characteristic characteristics, making it difficult to create a prominent rose fragrance when applied to cigarettes. Furthermore, existing rose-based cigarette flavorings are mostly prepared by directly mixing the extract with solvents and flavor enhancers without effective encapsulation and protection of aroma components. During cigarette production and storage, aroma components are prone to volatilization and oxidative decomposition due to changes in temperature and pH, leading to low aroma retention rates. After a period of storage, the rose aroma significantly diminishes, and even off-flavors may appear, affecting the stability of cigarette quality.

[0004] In addition, in the preparation of existing rose-flavored cigarette flavorings, the synthesis of aroma precursors from rose cells is relatively low. The industry often uses a single methyl jasmonate induction method to increase the synthesis amount, but the induction effect is limited. Moreover, the flavoring enhancement system often uses a single flavor enhancer, which has poor synergy with the rose aroma, resulting in low scores for aroma quality, aroma quantity, and harmony of the flavoring, and insufficient integration of the characteristic rose aroma with the base aroma of cigarettes.

[0005] Meanwhile, conventional tobacco flavoring encapsulation materials are mostly single materials such as chitosan and sodium alginate, which do not have environmental responsiveness. During the burning of cigarettes, they cannot achieve controlled release of aroma according to changes in temperature and pH inside the cigarette, which can easily lead to problems such as aroma release being too fast and having a single layer of flavor.

[0006] Therefore, developing a compound tobacco flavoring containing rose extract with prominent rose characteristics, excellent sensory quality, high aroma retention rate, and controllable release has become a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0007] The purpose of this invention is to provide a compound tobacco flavoring containing rose extract and its preparation method.

[0008] To achieve the above objectives, the present invention provides the following technical solution: One of the technical solutions of this invention: A method for preparing a compound tobacco flavoring containing rose extract includes the following steps: 1) Rose suspension cells were inoculated into MS medium and cultured at a constant temperature. During the culture, the rose suspension cells were induced to produce high-yield aroma precursors, resulting in rose cells that produce high-yield aroma precursors. 2) Extract and purify the high-yield rose cells of aroma precursor obtained in step 1) to obtain aroma precursor concentrate; 3) The aroma precursor concentrate obtained in step 2) is added dropwise to the wall material solution, and a crosslinking agent is added for crosslinking. After spray drying, pH / temperature dual-responsive nanocapsules are obtained. 4) Add the pH / temperature dual-response nanocapsules and synergist obtained in step 3) to the solvent, sonicate, homogenize, and obtain the composite tobacco flavoring containing rose extract.

[0009] Further, in step 1), the rose suspension cells are rose suspension cells transfected with the RrTPS1 gene and the RrAAAT1 gene.

[0010] Further, in step 1), the constant temperature culture specifically refers to: controlling the culture pH to 5.4-5.6, the dissolved oxygen to 25%-35%, and cultured at 25°C for 9 days.

[0011] Further, in step 1), the high-yield induction of aroma precursors specifically involves: using methyl jasmonate coupled with ultrasound to induce high-yield production of aroma precursors in rose suspension cells.

[0012] Furthermore, the methyl jasmonate coupled ultrasound specifically refers to: Phase 1: On day 5 of culture, add methyl jasmonate to a final concentration of 50 μmol / L and induce culture for 24 h; Second stage: On the 6th day of culture, add methyl jasmonate with a final concentration of 100 μmol / L, while controlling the sonication frequency to 20-40 kHz, sonicating for 15 seconds and then pausing for 45 seconds, and inducing culture for 24 hours. Third stage: On the 7th day of culture, add methyl jasmonate with a final concentration of 100 μmol / L and induce culture for 72 h.

[0013] Further, in step 2), the extraction and purification process specifically involves: first, rapidly freezing and freeze-drying the rose cells that produce aroma precursors in liquid nitrogen; then, extracting the aroma precursors using ultrasound-assisted supercritical CO2 extraction; and finally, purifying them by molecular distillation.

[0014] Furthermore, the liquid nitrogen quick-freezing and freeze-drying specifically involve immersing rose cells that produce aroma precursors in liquid nitrogen for quick-freezing for 3-5 minutes, followed by freeze-drying to constant weight.

[0015] Furthermore, the ultrasound-assisted supercritical CO2 extraction specifically involves: pulverizing high-yield rose cells, which are freeze-dried to constant weight, and passing them through a 40-60 mesh sieve; controlling the ultrasound frequency at 40-50 kHz, the CO2 flow rate at 1-3 mL / min, the extraction pressure at 20-30 MPa, and the extraction temperature at 40-50 °C; and performing ultrasound-assisted supercritical CO2 extraction for 30-90 min.

[0016] Furthermore, the molecular distillation specifically involves: controlling the feed temperature at 50–60°C, the distillation temperature at 110–130°C, the pressure at 3–5 Pa, the scraper rotation speed at 200 rpm, and the feed rate at 5–8 mL / min; performing molecular distillation on the aroma precursor extract obtained by ultrasonic-assisted supercritical CO2 extraction; collecting the light phase component; and concentrating it under reduced pressure to a relative density of 1.10 (25°C).

[0017] Further, in step 3), the wall material solution is prepared as follows: The mass ratio of chitosan, sodium alginate, sodium carboxymethyl cellulose, poly(N-isopropylacrylamide), and acetic acid solution is (0.8–1.2):(1.0–1.4):(0.5–0.7):(0.7–0.9):100. First, chitosan is added to a 1 vol.% acetic acid solution, and the stirring speed is controlled at 300–400 rpm. The mixture is stirred at 35–45°C for 20–40 min. Then, sodium alginate, sodium carboxymethyl cellulose, and poly(N-isopropylacrylamide) are added, and the stirring speed is controlled at 300–400 rpm. The mixture is stirred at 20–30°C for 30–90 min, followed by ultrasonic degassing for 5–15 min to obtain the wall material solution.

[0018] Further, in step 3), the volume ratio of the aroma precursor concentrate to the wall material solution is 1:(4-5).

[0019] Further, in step 3), the crosslinking agent is a mixture of CaCl2 and genipin in a mass ratio of (7-9):1.

[0020] Further, in step 3), the amount of crosslinking agent added is 2-3 wt.%.

[0021] Further, in step 3), the crosslinking specifically involves controlling the stirring speed to 200-300 rpm and crosslinking at 30-40°C for 1-2 hours.

[0022] Further, in step 3), the spray drying specifically involves controlling the inlet air temperature to be 135–145°C, the outlet air temperature to be 65–75°C, the feed flow rate to be 10–14 mL / min, the atomization pressure to be 0.25–0.35 MPa, and drying until the moisture content is ≤3%.

[0023] Further, in step 4), the synergist is a mixture of solanesol, carotenoid phosphate, maltitol and vitamin E acetate in a mass ratio of (0.8-1.2):(0.3-0.5):(1.5-2.5):(0.4-0.6).

[0024] Further, in step 4), the solvent is a mixture of ethanol and 1,2-propanediol in a mass ratio of 7:3.

[0025] Further, in step 4), the mass-volume ratio of the pH / temperature dual-response nanocapsules, synergist, and solvent is: (8-12) g : (0.5-1.5) g : 100 mL.

[0026] Further, in step 4), the ultrasonic treatment specifically involves controlling the ultrasonic frequency to 30–50 kHz and performing ultrasonic treatment for 30–50 min.

[0027] Further, in step 4), the homogenization specifically involves: controlling the shearing speed to 12000 rpm, shearing for 10 to 20 minutes, and then controlling the pressure to 20 to 30 kPa for vacuum degassing for 20 minutes.

[0028] The second technical solution of this invention: The above-mentioned method for preparing a compound tobacco flavoring containing rose extract yields a compound tobacco flavoring containing rose extract.

[0029] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention provides a compound tobacco flavoring essence containing rose extract with an aroma quality score of 18.2, an aroma quantity score of 18.5, a harmony score of 18.3, and an aftertaste score of 17.8, which has a high sensory evaluation score and a prominent rose characteristic aroma. The present invention provides a compound tobacco flavoring containing rose extract with an aroma retention rate of up to 97.5% after 30 days, 94.3% after 60 days, and 90.1% after 90 days, which has a high aroma retention rate. Detailed Implementation

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.

[0031] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included within this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0032] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0033] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0034] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0035] The following embodiments illustrate a method for preparing a compound tobacco flavoring containing rose extract, comprising the following steps: 1) Rose suspension cells were inoculated into MS medium and cultured at a constant temperature. During the culture, the rose suspension cells were induced to produce high-yield aroma precursors, resulting in rose cells that produce high-yield aroma precursors. The rose suspension cells are rose suspension cells transfected with the RrTPS1 gene and the RrAAAT1 gene. Specifically, the isothermal culture involves controlling the culture pH at 5.4–5.6 and the dissolved oxygen at 25%–35% and incubating at 25°C for 9 days. Specifically, the high-yield induction of aroma precursors involves using methyl jasmonate coupled with ultrasound to induce high-yield production of aroma precursors in rose suspension cells. Specifically, the methyl jasmonate coupled with ultrasound is as follows: Phase 1: On day 5 of culture, add methyl jasmonate to a final concentration of 50 μmol / L and induce culture for 24 h; Second stage: On the 6th day of culture, add methyl jasmonate with a final concentration of 100 μmol / L, while controlling the sonication frequency to 20-40 kHz, sonicating for 15 seconds and then pausing for 45 seconds, and inducing culture for 24 hours. Third stage: On the 7th day of culture, methyl jasmonate with a final concentration of 100 μmol / L was added, and the culture was induced for 72 h. 2) Extract and purify the high-yield rose cells of aroma precursor obtained in step 1) to obtain aroma precursor concentrate; The extraction and purification process specifically involves: first, rapidly freezing and freeze-drying the rose cells that produce aroma precursors in liquid nitrogen; then, extracting the aroma precursors using ultrasound-assisted supercritical CO2 extraction; and finally, purifying them by molecular distillation. Specifically, the liquid nitrogen quick-freezing and freeze-drying process involves immersing rose cells that produce aroma precursors in liquid nitrogen for quick-freezing for 3-5 minutes, followed by freeze-drying to constant weight. Specifically, the ultrasound-assisted supercritical CO2 extraction involves: pulverizing high-yield rose cells, which are freeze-dried to constant weight, and passing them through a 40-60 mesh sieve; controlling the ultrasound frequency at 40-50 kHz, the CO2 flow rate at 1-3 mL / min, the extraction pressure at 20-30 MPa, and the extraction temperature at 40-50 °C; and performing ultrasound-assisted supercritical CO2 extraction for 30-90 min. Specifically, the molecular distillation involves controlling the feed temperature at 50–60°C, the distillation temperature at 110–130°C, the pressure at 3–5 Pa, the scraper rotation speed at 200 rpm, and the feed rate at 5–8 mL / min to perform molecular distillation on the aroma precursor extract obtained by ultrasonic-assisted supercritical CO2 extraction, collecting the light phase component, and concentrating it under reduced pressure to a relative density of 1.10 (25°C). 3) The aroma precursor concentrate obtained in step 2) is added dropwise to the wall material solution, and a crosslinking agent is added for crosslinking. After spray drying, pH / temperature dual-responsive nanocapsules are obtained. The wall material solution is prepared as follows: The mass ratio of chitosan, sodium alginate, sodium carboxymethyl cellulose, poly(N-isopropylacrylamide), and acetic acid solution is (0.8–1.2):(1.0–1.4):(0.5–0.7):(0.7–0.9):100. First, chitosan is added to a 1 vol.% acetic acid solution, and the stirring speed is controlled at 300–400 rpm. The mixture is stirred at 35–45°C for 20–40 min. Then, sodium alginate, sodium carboxymethyl cellulose, and poly(N-isopropylacrylamide) are added, and the stirring speed is controlled at 300–400 rpm. The mixture is stirred at 20–30°C for 30–90 min, and then ultrasonically degassed for 5–15 min to obtain the wall material solution. The volume ratio of the aroma precursor concentrate to the wall material solution is 1:(4-5).

[0036] The crosslinking agent is composed of CaCl2 and genipin mixed in a mass ratio of (7-9):1; The amount of the crosslinking agent added is 2-3 wt.%. Specifically, the crosslinking process involves controlling the stirring speed to be 200–300 rpm and crosslinking at 30–40°C for 1–2 hours. Specifically, the spray drying process involves controlling the inlet air temperature to be 135–145°C, the outlet air temperature to be 65–75°C, the feed flow rate to be 10–14 mL / min, the atomization pressure to be 0.25–0.35 MPa, and drying until the moisture content is ≤3%. 4) Add the pH / temperature dual-response nanocapsules and synergist obtained in step 3) to a solvent, sonicate, homogenize, and obtain the composite tobacco flavoring containing rose extract. The synergist is composed of solanesol, carotenoid phosphate, maltitol and vitamin E acetate in a mass ratio of (0.8-1.2):(0.3-0.5):(1.5-2.5):(0.4-0.6); The solvent is composed of ethanol and 1,2-propanediol in a mass ratio of 7:3. The mass-volume ratio of the pH / temperature dual-response nanocapsules, synergist, and solvent is (8-12) g : (0.5-1.5) g : 100 mL. Specifically, the ultrasonic treatment involves controlling the ultrasonic frequency to be 30–50 kHz and performing ultrasonic treatment for 30–50 minutes. The homogenization process specifically involves controlling the shearing speed at 12000 rpm, shearing for 10–20 min, followed by controlling the pressure at 20–30 kPa and vacuum degassing for 20 min.

[0037] Example 1 A compound tobacco flavoring containing rose extract 1) Rose suspension cells were inoculated into MS medium and cultured at a constant temperature. During the culture, the rose suspension cells were induced to produce high-yield aroma precursors, resulting in rose cells that produce high-yield aroma precursors. The rose suspension cells are rose suspension cells transfected with the RrTPS1 gene and the RrAAAT1 gene. Specifically, the isothermal culture involves controlling the culture pH at 5.4 and dissolved oxygen at 25% and incubating at 25°C for 9 days. Specifically, the high-yield induction of aroma precursors involves using methyl jasmonate coupled with ultrasound to induce high-yield production of aroma precursors in rose suspension cells. Specifically, the methyl jasmonate coupled with ultrasound is as follows: Phase 1: On day 5 of culture, add methyl jasmonate to a final concentration of 50 μmol / L and induce culture for 24 h; Second stage: On the 6th day of culture, add methyl jasmonate with a final concentration of 100 μmol / L, while controlling the sonication frequency to 20 kHz, sonicating for 15 seconds and then intermittently for 45 seconds, and inducing culture for 24 hours. Third stage: On the 7th day of culture, methyl jasmonate with a final concentration of 100 μmol / L was added, and the culture was induced for 72 h. 2) Extract and purify the high-yield rose cells of aroma precursor obtained in step 1) to obtain aroma precursor concentrate; The extraction and purification process specifically involves: first, rapidly freezing and freeze-drying the rose cells that produce aroma precursors in liquid nitrogen; then, extracting the aroma precursors using ultrasound-assisted supercritical CO2 extraction; and finally, purifying them by molecular distillation. Specifically, the liquid nitrogen quick-freezing and freeze-drying process involves immersing rose cells that produce aroma precursors in liquid nitrogen for 3 minutes and then freeze-drying them to a constant weight. Specifically, the ultrasound-assisted supercritical CO2 extraction involves: pulverizing high-yield rose cells, which are freeze-dried to constant weight, and passing them through a 40-mesh sieve; controlling the ultrasound frequency at 40kHz, the CO2 flow rate at 1mL / min, the extraction pressure at 20MPa, the extraction temperature at 40℃, and performing ultrasound-assisted supercritical CO2 extraction for 30min. Specifically, the molecular distillation involves controlling the feed temperature at 50°C, the distillation temperature at 110°C, the pressure at 3Pa, the scraper rotation speed at 200rpm, and the feed rate at 5mL / min to perform molecular distillation on the aroma precursor extract obtained by ultrasonic-assisted supercritical CO2 extraction, collecting the light phase component, and concentrating it under reduced pressure to a relative density of 1.10 (25°C). 3) The aroma precursor concentrate obtained in step 2) is added dropwise to the wall material solution, and a crosslinking agent is added for crosslinking. After spray drying, pH / temperature dual-responsive nanocapsules are obtained. The wall material solution is prepared as follows: The mass ratio of chitosan, sodium alginate, sodium carboxymethyl cellulose, poly(N-isopropylacrylamide), and acetic acid solution is 0.8:1.0:0.5:0.7:100. First, chitosan is added to a 1 vol.% acetic acid solution, and the stirring speed is controlled at 300 rpm. The mixture is stirred at 35°C for 20 min. Then, sodium alginate, sodium carboxymethyl cellulose, and poly(N-isopropylacrylamide) are added, and the stirring speed is controlled at 300 rpm. The mixture is stirred at 20°C for 30 min, and then ultrasonically degassed for 5 min to obtain the wall material solution. The volume ratio of the aroma precursor concentrate to the wall material solution is 1:4.

[0038] The crosslinking agent is composed of CaCl2 and genipin mixed in a mass ratio of 7:1; The amount of the crosslinking agent added is 2 wt.%. Specifically, the crosslinking process involves controlling the stirring speed to 200 rpm and crosslinking at 30°C for 1 hour. Specifically, the spray drying process involves controlling the inlet air temperature to 135℃, the outlet air temperature to 65℃, the feed flow rate to 10mL / min, the atomization pressure to 0.25MPa, and drying until the moisture content is ≤3%. 4) Add the pH / temperature dual-response nanocapsules and synergist obtained in step 3) to a solvent, sonicate, homogenize, and obtain the composite tobacco flavoring containing rose extract. The synergist is composed of solanesol, carotenoid phosphate, maltitol and vitamin E acetate in a mass ratio of 0.8:0.3:1.5:0.4. The solvent is composed of ethanol and 1,2-propanediol in a mass ratio of 7:3. The mass-volume ratio of the pH / temperature dual-response nanocapsules, synergist, and solvent is 8g:0.5g:100mL. Specifically, the ultrasonic treatment involves controlling the ultrasonic frequency to 30 kHz and performing ultrasonic treatment for 30 minutes. The homogenization process specifically involves controlling the shearing speed at 12000 rpm, shearing for 10 minutes, and then controlling the pressure at 20 kPa for vacuum degassing for 20 minutes.

[0039] Example 2 A compound tobacco flavoring containing rose extract 1) Rose suspension cells were inoculated into MS medium and cultured at a constant temperature. During the culture, the rose suspension cells were induced to produce high-yield aroma precursors, resulting in rose cells that produce high-yield aroma precursors. The rose suspension cells are rose suspension cells transfected with the RrTPS1 gene and the RrAAAT1 gene. Specifically, the constant temperature culture involves controlling the culture pH at 5.5 and dissolved oxygen at 30% and then incubating at 25°C for 9 days. Specifically, the high-yield induction of aroma precursors involves using methyl jasmonate coupled with ultrasound to induce high-yield production of aroma precursors in rose suspension cells. Specifically, the methyl jasmonate coupled with ultrasound is as follows: Phase 1: On day 5 of culture, add methyl jasmonate to a final concentration of 50 μmol / L and induce culture for 24 h; Second stage: On the 6th day of culture, add methyl jasmonate with a final concentration of 100 μmol / L, while controlling the sonication frequency to 30 kHz, sonicating for 15 seconds and then intermittently for 45 seconds, and inducing culture for 24 hours. Third stage: On the 7th day of culture, methyl jasmonate with a final concentration of 100 μmol / L was added, and the culture was induced for 72 h. 2) Extract and purify the high-yield rose cells of aroma precursor obtained in step 1) to obtain aroma precursor concentrate; The extraction and purification process specifically involves: first, rapidly freezing and freeze-drying the rose cells that produce aroma precursors in liquid nitrogen; then, extracting the aroma precursors using ultrasound-assisted supercritical CO2 extraction; and finally, purifying them by molecular distillation. Specifically, the liquid nitrogen quick-freezing and freeze-drying process involves immersing rose cells that produce aroma precursors in liquid nitrogen for 4 minutes and then freeze-drying them to a constant weight. Specifically, the ultrasound-assisted supercritical CO2 extraction involves: pulverizing high-yield rose cells, which are freeze-dried to constant weight, and passing them through a 50-mesh sieve; controlling the ultrasound frequency at 45kHz, the CO2 flow rate at 2mL / min, the extraction pressure at 25MPa, the extraction temperature at 45℃, and performing ultrasound-assisted supercritical CO2 extraction for 60min. Specifically, the molecular distillation involves controlling the feed temperature at 55°C, the distillation temperature at 120°C, the pressure at 4Pa, the scraper rotation speed at 200rpm, and the feed rate at 6mL / min to perform molecular distillation on the aroma precursor extract obtained by ultrasonic-assisted supercritical CO2 extraction, collecting the light phase component, and concentrating it under reduced pressure to a relative density of 1.10 (25°C). 3) The aroma precursor concentrate obtained in step 2) is added dropwise to the wall material solution, and a crosslinking agent is added for crosslinking. After spray drying, pH / temperature dual-responsive nanocapsules are obtained. The wall material solution is prepared as follows: The chitosan, sodium alginate, sodium carboxymethyl cellulose, poly(N-isopropylacrylamide), and acetic acid solution are mixed in a mass ratio of 1:1.2:0.6:0.8:100. First, chitosan is added to a 1 vol.% acetic acid solution, and the stirring speed is controlled at 350 rpm. The mixture is stirred at 40°C for 30 min. Then, sodium alginate, sodium carboxymethyl cellulose, and poly(N-isopropylacrylamide) are added, and the stirring speed is controlled at 350 rpm. The mixture is stirred at 25°C for 60 min, and then ultrasonically degassed for 10 min to obtain the wall material solution. The volume ratio of the aroma precursor concentrate to the wall material solution is 1:4.

[0040] The crosslinking agent is composed of CaCl2 and genipin mixed in a mass ratio of 8:1; The amount of the crosslinking agent added is 2.2 wt.%. Specifically, the crosslinking process involves controlling the stirring speed at 250 rpm and crosslinking at 35°C for 1.5 hours. Specifically, the spray drying process involves controlling the inlet air temperature to 140℃, the outlet air temperature to 70℃, the feed flow rate to 12mL / min, the atomization pressure to 0.3MPa, and drying until the moisture content is ≤3%. 4) Add the pH / temperature dual-response nanocapsules and synergist obtained in step 3) to a solvent, sonicate, homogenize, and obtain the composite tobacco flavoring containing rose extract. The synergist is composed of solanesol, carotenoid phosphate, maltitol and vitamin E acetate in a mass ratio of 1:0.4:2:0.5. The solvent is composed of ethanol and 1,2-propanediol in a mass ratio of 7:3. The mass-volume ratio of the pH / temperature dual-response nanocapsules, synergist, and solvent is 10g:1g:100mL. Specifically, the ultrasonic treatment involves controlling the ultrasonic frequency to 40 kHz and performing ultrasonic treatment for 40 minutes. The homogenization process specifically involves controlling the shearing speed at 12,000 rpm, shearing for 15 minutes, and then controlling the pressure at 25 kPa for vacuum degassing for 20 minutes.

[0041] Example 3 A compound tobacco flavoring containing rose extract 1) Rose suspension cells were inoculated into MS medium and cultured at a constant temperature. During the culture, the rose suspension cells were induced to produce high-yield aroma precursors, resulting in rose cells that produce high-yield aroma precursors. The rose suspension cells are rose suspension cells transfected with the RrTPS1 gene and the RrAAAT1 gene. Specifically, the constant temperature culture involves controlling the culture pH at 5.6 and dissolved oxygen at 35% and incubating at 25°C for 9 days. Specifically, the high-yield induction of aroma precursors involves using methyl jasmonate coupled with ultrasound to induce high-yield production of aroma precursors in rose suspension cells. Specifically, the methyl jasmonate coupled with ultrasound is as follows: Phase 1: On day 5 of culture, add methyl jasmonate to a final concentration of 50 μmol / L and induce culture for 24 h; Second stage: On the 6th day of culture, add methyl jasmonate with a final concentration of 100 μmol / L, while controlling the sonication frequency to 40 kHz, sonicating for 15 seconds and then intermittently for 45 seconds, and inducing culture for 24 hours. Third stage: On the 7th day of culture, methyl jasmonate with a final concentration of 100 μmol / L was added, and the culture was induced for 72 h. 2) Extract and purify the high-yield rose cells of aroma precursor obtained in step 1) to obtain aroma precursor concentrate; The extraction and purification process specifically involves: first, rapidly freezing and freeze-drying the rose cells that produce aroma precursors in liquid nitrogen; then, extracting the aroma precursors using ultrasound-assisted supercritical CO2 extraction; and finally, purifying them by molecular distillation. Specifically, the liquid nitrogen quick-freezing and freeze-drying process involves immersing rose cells that produce aroma precursors in liquid nitrogen for 5 minutes and then freeze-drying them to a constant weight. Specifically, the ultrasound-assisted supercritical CO2 extraction involves: pulverizing high-yield rose cells, which are freeze-dried to constant weight, and passing them through a 60-mesh sieve; controlling the ultrasound frequency at 50 kHz, the CO2 flow rate at 3 mL / min, the extraction pressure at 30 MPa, the extraction temperature at 50 ℃, and performing ultrasound-assisted supercritical CO2 extraction for 90 min. Specifically, the molecular distillation involves controlling the feed temperature at 60°C, the distillation temperature at 130°C, the pressure at 5Pa, the scraper rotation speed at 200rpm, and the feed rate at 8mL / min to perform molecular distillation on the aroma precursor extract obtained by ultrasonic-assisted supercritical CO2 extraction, collecting the light phase components, and concentrating them under reduced pressure to a relative density of 1.10 (25°C). 3) The aroma precursor concentrate obtained in step 2) is added dropwise to the wall material solution, and a crosslinking agent is added for crosslinking. After spray drying, pH / temperature dual-responsive nanocapsules are obtained. The wall material solution is prepared as follows: The mass ratio of chitosan, sodium alginate, sodium carboxymethyl cellulose, poly(N-isopropylacrylamide), and acetic acid solution is 1.2:1.4:0.7:0.9:100. First, chitosan is added to a 1 vol.% acetic acid solution, and the stirring speed is controlled at 400 rpm. The mixture is stirred at 45°C for 40 min. Then, sodium alginate, sodium carboxymethyl cellulose, and poly(N-isopropylacrylamide) are added, and the stirring speed is controlled at 400 rpm. The mixture is stirred at 30°C for 90 min, and then ultrasonically degassed for 15 min to obtain the wall material solution. The volume ratio of the aroma precursor concentrate to the wall material solution is 1:5.

[0042] The crosslinking agent is composed of CaCl2 and genipin mixed in a mass ratio of 9:1; The amount of the crosslinking agent added is 3 wt.%. Specifically, the crosslinking process involves controlling the stirring speed to 300 rpm and crosslinking at 40°C for 2 hours. Specifically, the spray drying process involves controlling the inlet air temperature to 145℃, the outlet air temperature to 75℃, the feed flow rate to 14mL / min, the atomization pressure to 0.35MPa, and drying until the moisture content is ≤3%. 4) Add the pH / temperature dual-response nanocapsules and synergist obtained in step 3) to a solvent, sonicate, homogenize, and obtain the composite tobacco flavoring containing rose extract. The synergist is composed of solanesol, carotenoid phosphate, maltitol and vitamin E acetate in a mass ratio of 1.2:0.5:2.5:0.6. The solvent is composed of ethanol and 1,2-propanediol in a mass ratio of 7:3. The mass-volume ratio of the pH / temperature dual-response nanocapsules, synergist, and solvent is 12g:1.5g:100mL. Specifically, the ultrasonic treatment involves controlling the ultrasonic frequency to 50 kHz and performing ultrasonic treatment for 50 minutes. The homogenization process specifically involves controlling the shearing speed at 12000 rpm, shearing for 20 minutes, and then controlling the pressure at 30 kPa for vacuum degassing for 20 minutes.

[0043] Comparative Example 1 A compound tobacco flavoring containing rose extract Same as Example 2, except that in step 1), ultrasound induction is omitted, and only methyl jasmonic acid gradient induction is performed, specifically: Phase 1: On day 5 of culture, add methyl jasmonate to a final concentration of 50 μmol / L and induce culture for 24 h; Second stage: On the 6th day of culture, methyl jasmonate with a final concentration of 100 μmol / L was added, and the culture was induced for 24 h. Third stage: On the 7th day of culture, add methyl jasmonate with a final concentration of 100 μmol / L and induce culture for 72 h.

[0044] Comparative Example 2 A compound tobacco flavoring containing rose extract Same as Example 2, except that in step 1), the concentration of methyl jasmonate gradient induction is changed, specifically: Phase 1: On day 5 of culture, add methyl jasmonate to a final concentration of 50 μmol / L and induce culture for 24 h; Second stage: On the 6th day of culture, methyl jasmonate with a final concentration of 50 μmol / L was added, and the ultrasonic frequency was controlled at 30 kHz, with ultrasonication for 15 seconds and an interval of 45 seconds, and induction culture for 24 hours. Third stage: On the 7th day of culture, add methyl jasmonate with a final concentration of 100 μmol / L and induce culture for 72 h.

[0045] Comparative Example 3 A compound tobacco flavoring containing rose extract Same as Example 2, except that in step 1), the concentration of methyl jasmonate gradient induction is changed, specifically: Phase 1: On day 5 of culture, add methyl jasmonate to a final concentration of 50 μmol / L and induce culture for 24 h; Second stage: On the 6th day of culture, methyl jasmonate with a final concentration of 80 μmol / L was added, and the ultrasonic frequency was controlled at 30 kHz, with ultrasonication for 15 seconds and an interval of 45 seconds, and induction culture for 24 hours. Third stage: On the 7th day of culture, add methyl jasmonate with a final concentration of 100 μmol / L and induce culture for 72 h.

[0046] Comparative Example 4 A compound tobacco flavoring containing rose extract Same as Example 2, except that in step 1), the gradient induction with methyl jasmonic acid is omitted, and only ultrasonic induction is performed, specifically: On day 6 of culture, the sonication frequency was controlled at 30kHz, with sonication for 15s followed by a 45s interval, and the culture was induced for 24h.

[0047] Comparative Example 5 A compound tobacco flavoring containing rose extract Same as Example 2, except that step 3) is omitted, and the aroma precursor concentrate obtained in step 2) is directly used for the subsequent preparation of a compound tobacco flavoring containing rose extract.

[0048] Comparative Example 6 A compound tobacco flavoring containing rose extract Same as Example 2, except that in step 3), the addition of poly-N-isopropylacrylamide is omitted.

[0049] Comparative Example 7 A compound tobacco flavoring containing rose extract Same as Example 2, except that in step 4), the synergist is replaced with propylene glycol methyl ether (a conventional tobacco flavoring agent).

[0050] Comparative Example 8 A compound tobacco flavoring containing rose extract Same as Example 2, except that in step 4), the solvent is replaced with ethanol.

[0051] Effect verification I. Sensory Evaluation of Cigarette Flavoring The composite tobacco flavorings containing rose extract prepared in Examples 1-3 and Comparative Examples 1-8 were added to the base cigarette tobacco at an addition amount of 0.1%. After tobacco processing and rolling, finished cigarettes were obtained. The aroma quality, aroma quantity, harmony and aftertaste of the finished cigarettes were scored according to the "Sensory Evaluation Methods for Tobacco and Tobacco Products" (each item has a full score of 20 points). At the same time, the prominence of the rose characteristic aroma was evaluated. The sensory evaluation results are shown in Table 1 below. Table 1 Sensory evaluation results

[0052] As shown in Table 1, the composite tobacco flavoring essence containing rose extract provided by this invention has a high sensory evaluation score of 18.2, an aroma quantity of 18.5, a harmony score of 18.3, and an aftertaste score of 17.8, and the rose characteristic aroma is prominent.

[0053] II. Aroma Stability Testing The composite tobacco flavorings containing rose extract prepared in Examples 1-3 and Comparative Examples 1-8 were added to the base cigarette tobacco at an addition amount of 0.1%. After tobacco processing and rolling, finished cigarettes were obtained. The finished cigarettes were then sealed and stored in an environment with a temperature of 25°C and a relative humidity of 60%. Sensory evaluation was carried out after 30 days, 60 days and 90 days of storage. The aroma stability was evaluated by the aroma retention rate (total score after storage / total score after 0 days of storage × 100%). The aroma stability test results are shown in Table 2. Table 2 Aroma stability test results

[0054] As shown in Table 2, the compound tobacco flavoring containing rose extract provided by this invention has an aroma retention rate of 97.5% after 30 days, 94.3% after 60 days, and 90.1% after 90 days, demonstrating a high aroma retention rate.

[0055] 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 it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the specific implementation of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing a compound tobacco flavoring containing rose extract, characterized in that, Includes the following steps: 1) Rose suspension cells were inoculated into MS medium and cultured at a constant temperature. During the culture, the rose suspension cells were induced to produce high-yield aroma precursors, resulting in rose cells that produce high-yield aroma precursors. 2) Extract and purify the high-yield rose cells of aroma precursor obtained in step 1) to obtain aroma precursor concentrate; 3) The aroma precursor concentrate obtained in step 2) is added dropwise to the wall material solution, and a crosslinking agent is added for crosslinking. After spray drying, pH / temperature dual-responsive nanocapsules are obtained. 4) Add the pH / temperature dual-response nanocapsules and synergist obtained in step 3) to the solvent, sonicate, homogenize, and obtain the composite tobacco flavoring containing rose extract.

2. The method for preparing a compound tobacco flavoring containing rose extract according to claim 1, characterized in that, In step 1), The rose suspension cells were rose suspension cells transfected with the RrTPS1 gene and the RrAAAT1 gene. The constant temperature culture specifically involves controlling the culture pH at 5.4–5.6 and the dissolved oxygen at 25%–35% and incubating at 25°C for 9 days. The high-yield induction of aroma precursors specifically involves using methyl jasmonate coupled with ultrasound to induce high-yield production of aroma precursors in rose suspension cells.

3. The method for preparing a compound tobacco flavoring containing rose extract according to claim 2, characterized in that, The methyl jasmonate coupled ultrasound specifically refers to: Phase 1: On day 5 of culture, add methyl jasmonate to a final concentration of 50 μmol / L and induce culture for 24 h; Second stage: On the 6th day of culture, add methyl jasmonate with a final concentration of 100 μmol / L, while controlling the sonication frequency to 20-40 kHz, sonicating for 15 seconds and then pausing for 45 seconds, and inducing culture for 24 hours. Third stage: On the 7th day of culture, add methyl jasmonate with a final concentration of 100 μmol / L and induce culture for 72 h.

4. The method for preparing a compound tobacco flavoring containing rose extract according to claim 1, characterized in that, In step 2), the extraction and purification process specifically involves: first, rapidly freezing and freeze-drying the rose cells that produce aroma precursors in liquid nitrogen; then, extracting the aroma precursors using ultrasound-assisted supercritical CO2 extraction; and finally, purifying them by molecular distillation.

5. The method for preparing a compound tobacco flavoring containing rose extract according to claim 4, characterized in that, The liquid nitrogen quick-freezing and freeze-drying process specifically involves: immersing rose cells that produce aroma precursors in liquid nitrogen for quick-freezing for 3-5 minutes, followed by freeze-drying to constant weight; The ultrasound-assisted supercritical CO2 extraction specifically involves: pulverizing high-yield rose cells, which are freeze-dried to constant weight, and passing them through a 40-60 mesh sieve; controlling the ultrasound frequency at 40-50 kHz, the CO2 flow rate at 1-3 mL / min, the extraction pressure at 20-30 MPa, the extraction temperature at 40-50 °C, and performing ultrasound-assisted supercritical CO2 extraction for 30-90 min. The molecular distillation specifically involves controlling the feed temperature at 50–60°C, the distillation temperature at 110–130°C, the pressure at 3–5 Pa, the scraper rotation speed at 200 rpm, and the feed rate at 5–8 mL / min. The aroma precursor extract obtained by ultrasonic-assisted supercritical CO2 extraction is subjected to molecular distillation, the light phase component is collected, and the mixture is concentrated under reduced pressure to a relative density of 1.

10.

6. The method for preparing a compound tobacco flavoring containing rose extract according to claim 1, characterized in that, In step 3), The wall material solution is prepared as follows: The mass ratio of chitosan, sodium alginate, sodium carboxymethyl cellulose, poly(N-isopropylacrylamide), and acetic acid solution is (0.8–1.2):(1.0–1.4):(0.5–0.7):(0.7–0.9):

100. First, chitosan is added to a 1 vol.% acetic acid solution, and the stirring speed is controlled at 300–400 rpm. The mixture is stirred at 35–45°C for 20–40 min. Then, sodium alginate, sodium carboxymethyl cellulose, and poly(N-isopropylacrylamide) are added, and the stirring speed is controlled at 300–400 rpm. The mixture is stirred at 20–30°C for 30–90 min, and then ultrasonically degassed for 5–15 min to obtain the wall material solution. The volume ratio of the aroma precursor concentrate to the wall material solution is 1:(4-5). The crosslinking agent is composed of CaCl2 and genipin in a mass ratio of (7-9):1; The amount of the crosslinking agent added is 2-3 wt.%; The crosslinking process specifically involves controlling the stirring speed to be 200–300 rpm and crosslinking at 30–40°C for 1–2 hours. The spray drying process specifically involves controlling the inlet air temperature to be 135–145°C, the outlet air temperature to be 65–75°C, the feed flow rate to be 10–14 mL / min, the atomization pressure to be 0.25–0.35 MPa, and drying until the moisture content is ≤3%.

7. The method for preparing a compound tobacco flavoring containing rose extract according to claim 1, characterized in that, In step 4), The synergist is composed of solanine alcohol, carotenoid phosphate, maltitol and vitamin E acetate in a mass ratio of (0.8-1.2):(0.3-0.5):(1.5-2.5):(0.4-0.6); The solvent is a mixture of ethanol and 1,2-propanediol in a mass ratio of 7:3; The mass-to-volume ratio of the pH / temperature dual-response nanocapsules, synergist, and solvent is (8-12) g : (0.5-1.5) g : 100 mL; The ultrasonic treatment specifically involves controlling the ultrasonic frequency to 30–50 kHz and performing ultrasonic treatment for 30–50 minutes. The homogenization process specifically involves controlling the shearing speed at 12000 rpm, shearing for 10–20 min, and then controlling the pressure at 20–30 kPa for vacuum degassing for 20 min.

8. A compound tobacco flavoring containing rose extract prepared by the method for preparing a compound tobacco flavoring containing rose extract as described in any one of claims 1 to 7.

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