Preparation method and application of grapefruit tobacco extract

Abstract

The application discloses a preparation method and application of a pomelo tobacco extract. The preparation method comprises the following steps: mixing and dissolving neoxanthin and Tween 80 in dichloromethane, adding anhydrous ethanol after vacuum distillation; grinding tobacco leaves into tobacco powder, mixing the tobacco powder with water to sterilize, and adding a neoxanthin solution; inoculating klebsiella variicola HNYJ-3 and enterobacter HNYJ-4 in a liquid culture medium to culture seed liquids, mixing the two kinds of seed liquids to inoculate in a tobacco and neoxanthin mixed solution, and carrying out oscillation culture and fermentation; carrying out centrifugal extraction after fermentation, rotary evaporation, adding anhydrous ethanol and propylene glycol, and obtaining the pomelo tobacco extract. The application utilizes two strains to ferment a neoxanthin and tobacco mixture to generate aroma components such as naringanin, alpha-lavandulol, perillaketone, carvone and dihydrocarveol, and the pomelo tobacco extract can be applied to cigarette products to improve smoke aroma quality, aroma amount, reduce miscellaneous gas and improve smoke transmittance.

Description

Technical Field

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

[0002] The aroma of cigarette smoke is an important indicator for evaluating the quality of cigarette products. High-quality cigarettes produce a large quantity and pure quality of aroma during combustion, with a mellow taste. As smoking and health issues have attracted increasing attention, tobacco companies have actively researched and developed cigarettes with low tar content. In cigarette manufacturing processes, technologies such as adding tobacco sheets, expanding tobacco stems, and perforating cigarette paper have been widely adopted. While this reduces the tar content, it also reduces the amount of aroma transported in the smoke, resulting in insufficient aroma. Therefore, in existing technologies, compensating for the loss of aroma caused by harm reduction and tar reduction through the addition of flavorings and fragrances is a commonly used technical method.

[0003] Due to the direct inhalation of tobacco products, current technologies generally employ naturally derived flavorings and fragrances to enhance the aroma of tobacco products, ensuring their safety. Tobacco extracts are a common ingredient used to supplement and improve the aroma and inhalability of tobacco.

[0004] In existing technologies, tobacco extracts are generally obtained by extracting tobacco materials with water or alcohol and then further concentrating them under reduced pressure. However, some important aroma precursors in tobacco, such as carotenoids and chlorophyll, are insoluble in water and alcohol and require degradation and transformation to generate aroma compounds. Although microbial or enzymatic fermentation or enzymatic hydrolysis methods have been used to increase the aroma content of tobacco extracts, practical results show that the aroma types of tobacco extracts obtained by these improved methods are still relatively limited and cannot fully meet the purpose of enhancing and improving tobacco aroma. Therefore, further research on tobacco extracts is still necessary. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for preparing grapefruit tobacco extract and its application, which can improve the aroma quality and quantity of cigarette smoke, reduce off-flavors, and improve permeability when used in cigarette products.

[0006] The technical problem to be solved by the present invention is achieved through the following technical solution:

[0007] A method for preparing a grapefruit-scented tobacco extract includes the following steps:

[0008] (1) Take neoxanthin and Tween 80 and form a suspension at a mass ratio of 1:10. Dissolve the suspension in dichloromethane and distill off the dichloromethane under reduced pressure. Add anhydrous ethanol to make the concentration of neoxanthin 50 mg / mL.

[0009] (2) Grind the tobacco leaves into 200-mesh tobacco powder, mix it with water at a mass ratio of 1:7-1:15, sterilize at 121℃ for 15 minutes, add neoxanthin solution to make the neoxanthin concentration 2g / L;

[0010] (3) Select Klebsiella variegata HNYJ-3 and Enterobacter HNYJ-4 and inoculate them into LB liquid medium. Incubate at 35-40℃ and 120-200rpm for 24-48h with shaking to form seed culture.

[0011] (4) Mix the seed liquid of Klebsiella variegata HNYJ-3 and the seed liquid of Enterobacter HNYJ-4 at a mass ratio of (5-7):(3-5). The total inoculation amount is 1-6%. Inoculate the seed liquid into the mixture of tobacco and neoxanthin and culture at 35-40℃ and 120-200rpm for 48-96h with shaking.

[0012] (5) Centrifuge the fermented mixture, take the supernatant, add 2 times the volume of ethyl acetate, n-hexane and anhydrous ethanol mixed solution, shake and extract for 1-2 hours, take the supernatant, evaporate at 60℃, add anhydrous ethanol at a volume ratio of 1:1, place at -22℃ for 24 hours, centrifuge to obtain the supernatant, evaporate again, add 10-30% propylene glycol to obtain grapefruit tobacco extract.

[0013] Preferably, in the above technical solution, the Klebsiella variicola HNYJ-3 was deposited at the China General Microbiological Culture Collection Center on January 18, 2024, with accession number CGMCC No. 29654; and the Enterobacter sp. HNYJ-4 was deposited at the China General Microbiological Culture Collection Center on January 18, 2024, with accession number CGMCC No. 29655.

[0014] Preferably, in the above technical solution, in step (3), the LB culture medium includes: NaCl 10g / L, peptone 10g / L, and yeast extract 5g / L.

[0015] Preferably, in the above technical solution, in step (3), the culture conditions are: 35℃, 150rpm, shaking culture for 24h, OD 600 Value 1.8.

[0016] Preferably, in the above technical solution, in step (4), the mass ratio of Klebsiella variegata HNYJ-3 seed liquid to Enterobacter HNYJ-4 seed liquid is 7:3, and the total inoculation amount is 4%. The seed liquid is inoculated into a mixture of tobacco and neoxanthin and cultured at 35°C and 150 rpm for 72 h with shaking.

[0017] Preferably, in the above technical solution, in step (5), the volume ratio of ethyl acetate, n-hexane, and anhydrous ethanol is 5:4:2. After shaking extraction for 2 hours, the supernatant is taken and evaporated at 60°C and 60 mbar to a density of 1.09. Anhydrous ethanol is added at a volume ratio of 1:1, and the mixture is placed at -22°C for 24 hours. The supernatant is obtained by centrifugation and evaporated again to a density of 1.2. 10% propylene glycol is added to obtain the grapefruit tobacco extract.

[0018] The application of a grapefruit-scented tobacco extract in cigarettes, wherein the grapefruit-scented tobacco extract is prepared according to the above-described preparation method.

[0019] Preferably, in the above technical solution, the application involves diluting the grapefruit tobacco extract with water at a mass ratio of 1:(20-200), and then adding 0.5-1 μL / cigarette to conventional or heated cigarettes; or, diluting the grapefruit tobacco extract with water at a mass ratio of 1:(2000-20000), spraying it onto tobacco shreds at 10-15%, letting it stand for 2-6 hours, and then rolling it into cigarettes.

[0020] Preferably, in the above technical solution, the obtained grapefruit tobacco extract is diluted with water at a mass ratio of 1:100, and then 0.7uL / cigarette is added to conventional cigarettes or heated cigarettes.

[0021] The above-described technical solution of the present invention has the following beneficial effects:

[0022] The tobacco extract of this application, when applied to cigarette products, can improve the aroma quality and quantity of cigarette smoke, reduce off-flavors, and enhance permeability. This application utilizes *Klebsiella variegata* HNYJ-3 and *Enterobacter oryzae* HNYJ-4 to ferment a mixture of neoxanthin and tobacco, producing aroma-enhancing components such as naringin, α-truffle alcohol, perillone, carvone, and dihydrocarvone. Naringin has a sweet grapefruit and woody aroma; α-truffle alcohol has a sweet and floral aroma; perillone has a refreshing aroma; and carvone has a medicinal and minty coolness. These components enrich the smoke and improve the quality of the tobacco extract. Detailed Implementation

[0023] Various exemplary embodiments of the present invention will now be described in detail. It should be noted that, unless otherwise specifically stated, the relative arrangement, numerical expressions, and values ​​of the components and steps set forth in these embodiments do not limit the scope of the invention.

[0024] The strain information required in this application is as follows:

[0025] (1) Klebsiella variicola HNYJ-3 was deposited on January 18, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. Accession number: CGMCC No. 29654.

[0026] (2) Enterobacter sp. HNYJ-4 was deposited on January 18, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. Accession number: CGMCC No. 29655.

[0027] The new yellow tobacco is commercially available and purchased online. The tobacco leaves are sourced from Henan China Tobacco Industry Co., Ltd.

[0028] Example 1

[0029] (1) Take neoxanthin and Tween 80 in a mass ratio of 1:10 to form a suspension. Dissolve the suspension in dichloromethane, distill off the dichloromethane under reduced pressure, and add anhydrous ethanol to make the concentration of neoxanthin 50 mg / mL.

[0030] (2) Grind the tobacco leaves to 200 mesh, mix with water at a mass ratio of 1:10, sterilize at 121℃ for 15 minutes, add neoxanthin solution to make the neoxanthin concentration 2g / L.

[0031] (3) Klebsiella variegata HNYJ-3 (CGMCC No. 29654) and Enterobacter HNYJ-4 (CGMCC No. 29655) were respectively inoculated into LB liquid medium and cultured at 35℃ and 150 rpm for 24 h with shaking. OD 600 The value is approximately 1.8, forming seed liquid.

[0032] The LB medium liquid medium consisted of 10 g / L NaCl, 10 g / L peptone, and 5 g / L yeast extract, and was sterilized at 121°C for 15 min.

[0033] (4) Klebsiella variegata HNYJ-3 and Enterobacter HNYJ-4 were inoculated into a mixture of tobacco and neoxanthin at a mass ratio of 7:3 and a total inoculation amount of 4%. The mixture was then cultured at 35°C and 150 rpm for 72 h with shaking.

[0034] (5) Centrifuge the fermented mixture, take the supernatant, add 2 times the volume of a mixed solution of ethyl acetate, n-hexane and anhydrous ethanol, wherein the volume ratio of ethyl acetate, n-hexane and anhydrous ethanol is 5:4:2, shake and extract for 2 hours, take the supernatant, rotary evaporate at 60℃ and 60mbar to a density of about 1.09, add anhydrous ethanol at a volume ratio of 1:1, place at -22℃ for 24 hours, centrifuge to obtain the supernatant, rotary evaporate again to a density of about 1.2, add 10% propylene glycol to obtain the grapefruit tobacco extract.

[0035] Application Example 1

[0036] The grapefruit-scented tobacco extract prepared in Example 1 was diluted with water at a mass ratio of 1:100, and then 0.7 μL / cigarette was added to a regular cigarette, denoted as T1.

[0037] Comparative Example 1

[0038] (1) Grind the tobacco leaves to 200 mesh tobacco powder, mix with water at a mass ratio of 1:10, and sterilize at 121℃ for 15 minutes.

[0039] (2) Centrifuge the sterilized mixture, take the supernatant, add 2 times the volume of a mixed solution of ethyl acetate, n-hexane and anhydrous ethanol, wherein the volume ratio of ethyl acetate, n-hexane and anhydrous ethanol is 5:4:2, shake and extract for 2 hours, take the supernatant, rotary evaporate at 60℃ and 60mbar to a density of about 1.09, add anhydrous ethanol at a volume ratio of 1:1, place at -22℃ for 24 hours, centrifuge to obtain the supernatant, rotary evaporate again to a density of about 1.2, add 10% propylene glycol to obtain the tobacco extract.

[0040] (3) The obtained tobacco extract was diluted with water at a mass ratio of 1:100, and then 0.7uL / cigarette was added to a regular cigarette, which was denoted as CK1.

[0041] Comparative Example 2

[0042] (1) Take neoxanthin and Tween 80 in a mass ratio of 1:10 to form a suspension. Dissolve the suspension in dichloromethane, distill off the dichloromethane under reduced pressure, and add anhydrous ethanol to make the concentration of neoxanthin 50 mg / mL.

[0043] (2) Grind the tobacco leaves to 200 mesh, mix with water at a mass ratio of 1:10, sterilize at 121℃ for 15 minutes, add neoxanthin solution to make the neoxanthin concentration 2g / L.

[0044] (3) Centrifuge the mixture, take the supernatant, add 2 times the volume of a mixed solution of ethyl acetate, n-hexane and anhydrous ethanol, wherein the volume ratio of ethyl acetate, n-hexane and anhydrous ethanol is 5:4:2, shake and extract for 2 hours, take the supernatant, rotary evaporate at 60℃ and 60mbar to a density of about 1.09, add anhydrous ethanol at a volume ratio of 1:1, place at -22℃ for 24 hours, centrifuge to obtain the supernatant, rotary evaporate again to a density of about 1.2, add 10% propylene glycol to obtain the grapefruit tobacco extract.

[0045] (4) The obtained grapefruit tobacco extract was diluted with water at a mass ratio of 1:100, and then 0.7uL / cigarette was added to a regular cigarette, which was denoted as CK2.

[0046] Comparative Example 3

[0047] (1) Take neoxanthin and Tween 80 in a mass ratio of 1:10 to form a suspension. Dissolve the suspension in dichloromethane, distill off the dichloromethane under reduced pressure, and add anhydrous ethanol to make the concentration of neoxanthin 50 mg / mL.

[0048] (2) Grind the tobacco leaves to 200 mesh, mix with water at a mass ratio of 1:10, sterilize at 121℃ for 15 minutes, add neoxanthin solution to make the neoxanthin concentration 2g / L.

[0049] (3) Select Enterobacter HNYJ-4 (CGMCC NO.29655) and inoculate it into LB liquid medium. Incubate at 35℃ and 150 rpm for 24 h with shaking. OD 600 The value is approximately 1.8, forming seed liquid.

[0050] The LB medium liquid medium consisted of 10 g / L NaCl, 10 g / L peptone, and 5 g / L yeast extract, and was sterilized at 121°C for 15 min.

[0051] (4) Enterobacter HNYJ-4 inoculation amount 4% was inoculated into a mixture of tobacco and neoxanthin, and cultured at 35℃ and 150rpm for 72h with shaking.

[0052] (5) Centrifuge the fermented mixture, take the supernatant, add 2 times the volume of a mixed solution of ethyl acetate, n-hexane and anhydrous ethanol, wherein the volume ratio of ethyl acetate, n-hexane and anhydrous ethanol is 5:4:2, shake and extract for 2 hours, take the supernatant, rotary evaporate at 60℃ and 60mbar to a density of about 1.09, add anhydrous ethanol at a volume ratio of 1:1, place at -22℃ for 24 hours, centrifuge to obtain the supernatant, rotary evaporate again to a density of about 1.2, add 10% propylene glycol to obtain the grapefruit tobacco extract.

[0053] (6) The obtained grapefruit tobacco extract was diluted with water at a mass ratio of 1:100, and then 0.7uL / cigarette was added to a regular cigarette, which was denoted as CK3.

[0054] Comparative Example 4

[0055] Regular cigarettes without any added tobacco extracts are designated CK.

[0056] result:

[0057] When evaluating tobacco extracts using sensory quality assessment, 13 indicators are scored: aroma quality, aroma quantity, permeability, off-flavors, concentration, strength, fineness, cohesiveness, irritation, dryness, freshness, sweetness, and aftertaste, and the total score is calculated.

[0058] The sensory evaluation is shown in Table 1. Compared with CK (no tobacco extract added), CK1 (no neoxanthin added, unfermented tobacco extract) showed a certain degree of improvement in aroma quantity, off-flavors, fineness, and sweetness, but the improvement was smaller compared with T1. CK2 (neoxanthin added, unfermented tobacco extract) and CK3 (neoxanthin added, single strain fermentation) showed improvement in aroma quality, aroma quantity, and off-flavors between CK1 and T1. T1 showed significant improvement in aroma quality, aroma quantity, off-flavors, fineness, and sweetness, further enhancing the quality of the tobacco extract.

[0059] Table 1 Sensory evaluation results

[0060]

[0061] Table 2. Main aroma components (μg / g)

[0062] Aroma components CK1 T1 terpineone / 186.76 α-Truffle alcohol / 73.64 Perilla ketone / 34.85 carvone / 81.37 dihydrocarvone / 44.29

[0063] As shown in Table 2, compared with the tobacco extract of CK1, the grapefruit-scented tobacco extract of T1 contains aroma-producing components such as naringin, α-truffle alcohol, perillone, carvone, and dihydrocarvone. Naringin has a low odor threshold and has a sweet grapefruit and woody aroma; α-truffle alcohol has a sweet and floral aroma; perillone has a refreshing aroma; and carvone has a medicinal aroma and a minty coolness. These components can enrich the smoke and further improve the quality of the tobacco extract.

[0064] Although the present invention has been disclosed above with reference to embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various different choices and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention is defined by the claims and their equivalents.

Claims

1. A method of preparing a naringenated tobacco extract, characterized by, The method comprises the following steps: (1) Take neoxanthin and Tween 80, form a suspension with a mass ratio of 1:10, dissolve the suspension in dichloromethane, distill dichloromethane under reduced pressure, add anhydrous ethanol to make the concentration of neoxanthin 50 mg / mL; (2) Grind the tobacco to 200-mesh tobacco powder, mix with water at a mass ratio of 1:7-1:15, sterilize at 121℃ for 15 min, and add the neoxanthin solution to make the concentration of neoxanthin 2 g / L; (3) Pick Klebsiella variicola HNYJ-3 and Enterobacter sp. HNYJ-4, inoculate them in LB liquid medium respectively, and cultivate at 35-40℃ and 120-200 rpm for 24-48 h to form seed liquid; Klebsiella variicola (Klebsiella variicola) Klebsiella variicola ) HNYJ-3, which was preserved in the China General Microbiological Culture Collection Center on January 18, 2024, and has the preservation number CGMCC No. 29654; and the Enterobacter (Enterobacter cloacae) Enterobacter sp. ) HNYJ-4, which was preserved in the China General Microbiological Culture Collection Center on January 18, 2024, and has the preservation number CGMCC No. 29655. (4) Mix the Klebsiella variicola HNYJ-3 seed liquid and the Enterobacter sp. HNYJ-4 seed liquid at a mass ratio of (5-7):(3-5), inoculate the seed liquid in the tobacco and neoxanthin mixture at a total inoculation amount of 1-6%, cultivate at 35-40℃ and 120-200 rpm for 48-96 h; (5) Centrifuge the fermented mixture, take the supernatant, add a mixed solution of 2 volumes of ethyl acetate, n-hexane and anhydrous ethanol, shake and extract for 1-2 h, take the supernatant, rotary evaporate at 60℃, add anhydrous ethanol at a volume ratio of 1:1, place at -22℃ for 24 h, centrifuge to obtain the supernatant, rotary evaporate again, and add 10-30% propylene glycol to obtain the grapefruit tobacco extract.

2. The method of claim 1, wherein the grapefruit tobacco extract is prepared by the steps of: In step (3), the LB medium comprises: NaCl 10 g / L, protein peptone 10 g / L, and yeast extract powder 5 g / L.

3. The method for preparing the grapefruit-scented tobacco extract according to claim 1, characterized in that, In step (3), the culture conditions were: 35 °C, 150 rpm, and shaking culture for 24 h, OD 600 value 1.

8.

4. The method of preparing grapefruit tobacco extract according to claim 1, characterized by, In step (4), the Klebsiella variicola HNYJ-3 seed liquid and the Enterobacter sp. HNYJ-4 seed liquid are mixed at a mass ratio of 7:3, the seed liquid is inoculated in the tobacco and neoxanthin mixture at a total inoculation amount of 4%, and the cultivation is carried out at 35℃ and 150 rpm for 72 h.

5. The method for preparing the grapefruit-scented tobacco extract according to claim 1, characterized in that, In step (5), the volume ratio of ethyl acetate, n-hexane and anhydrous ethanol is 5:4:2, the shaking and extraction is carried out for 2 h, the supernatant is taken, rotary evaporated at 60℃ and 60 mbar to a density of 1.09, anhydrous ethanol is added at a volume ratio of 1:1, placed at -22℃ for 24 h, centrifuged to obtain the supernatant, rotary evaporated again to a density of 1.2, and 10% propylene glycol is added to obtain the grapefruit tobacco extract.

6. Use of a grapefruit tobacco extract in a cigarette, characterized in that, The grapefruit tobacco extract is prepared by the preparation method according to any one of claims 1-5.

7. Use according to claim 6, characterized in that, The application is to dilute the grapefruit tobacco extract with water at a mass ratio of 1:(20-200), and then add 0.5-1 uL / branch to conventional cigarettes or heat-not-burn cigarettes; or dilute the grapefruit tobacco extract with water at a mass ratio of 1:(2000-20000), and then spray 10-15% on the tobacco, and place for 2-6 h to roll into cigarettes.

8. Use according to claim 7, characterized in that, The obtained grapefruit tobacco extract is diluted with water at a mass ratio of 1:100, and then added to 0.7 uL / branch of conventional cigarettes or heat-not-burn cigarettes.

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