Preparation method and application of pomelo-flavor tobacco extract

Abstract

The invention discloses a preparation method and application of a pomelo-flavor tobacco extract, and the preparation method of the pomelo-flavor tobacco extract comprises the following steps: mixing and dissolving neoxanthine and Tween 80 in dichloromethane, and adding absolute ethyl alcohol after reduced pressure distillation; grinding tobacco leaves into tobacco powder, mixing the tobacco powder with water for sterilization, and adding a neoxanthine solution; klebsiella variicola HNYJ-3 and enterobacter HNYJ-4 are inoculated into a liquid culture medium to culture seed solutions, the two seed solutions are mixed and inoculated into a mixed solution of tobacco and neoxanthine, and shaking culture fermentation is performed; after fermentation, centrifugal extraction and rotary evaporation are performed, absolute ethyl alcohol and propylene glycol are added, and the pomelo-flavor tobacco extract is obtained. The two strains are used for fermenting a mixture of neoxanthine and tobacco to generate noottanone, alpha-truffle alcohol, perillone, carvone, dihydrocarvone and other aroma components, and when the two strains are applied to cigarette products, the aroma quality and the aroma amount of smoke can be improved, miscellaneous gas is reduced, and the permeability is improved.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a preparation method and application of a grapefruit-flavored tobacco extract. Background Art

[0002] Smoke aroma is an important indicator for evaluating the quality of cigarette products. High-quality cigarettes produce a large amount of pure aroma during the smoking process, and the taste is mellow. As smoking and health issues have attracted increasing attention, tobacco companies have actively developed cigarettes with low tar content. The tobacco manufacturing process widely uses technologies such as adding tobacco flakes, puffing stems, and perforating paper. As a result, while the tar content of cigarettes is reduced, the amount of aroma transmitted in the smoke is reduced, and the aroma is insufficient. Therefore, in the prior art, adding flavors and fragrances to make up for the loss of aroma caused by harm reduction and tar reduction is a relatively common technical means.

[0003] Due to the special nature of tobacco products that are directly smoked, the existing technology generally uses natural flavors and fragrances to supplement the aroma of tobacco products to ensure the safety of tobacco products. Among them, tobacco extract is a relatively common material component that supplements and improves the aroma of tobacco and its smokability.

[0004] In the prior art, tobacco extracts are generally extracted from tobacco materials by water or alcohol and then further concentrated under reduced pressure before they can be used. However, some important aroma precursors in tobacco, such as carotenoids and chlorophyll, are insoluble in water and alcohol on the one hand, and need to be degraded and transformed to generate aroma substances on the other hand. Although there are ways to increase the aroma of tobacco extracts by further fermentation or enzymatic treatment using microorganisms or enzyme preparations, the actual use results show that the aroma type of tobacco extracts obtained by this improved method is still relatively single, which cannot fully meet the purpose of improving 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 preparation method and application of a grapefruit-flavored tobacco extract, which can be used in cigarette products to improve the smoke aroma quality and aroma quantity, reduce impurities, and improve permeability.

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

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

[0008] (1) Take neoxanthin and Tween 80 at 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;

[0009] (2) grinding tobacco leaves into 200-mesh tobacco powder, mixing with water at a mass ratio of 1:7-1:15, sterilizing at 121°C for 15 min, adding neoxanthin solution to make the neoxanthin concentration 2 g / L;

[0010] (3) selecting Klebsiella mutans HNYJ-3 and Enterobacter HNYJ-4 and inoculating them into LB liquid culture medium respectively, and culturing them at 35-40° C. and 120-200 rpm with shaking for 24-48 h to form seed solution;

[0011] (4) Mix the seed solution of Klebsiella mutans HNYJ-3 and the seed solution of Enterobacter HNYJ-4 in a mass ratio of (5-7):(3-5), and inoculate the seed solution into the mixture of tobacco and neoxanthin at a total inoculation amount of 1-6%, and culture at 35-40° C. and 120-200 rpm with shaking for 48-96 h;

[0012] (5) The fermented mixed solution is centrifuged, the supernatant is taken, and a 2-fold volume of a mixed solution of ethyl acetate, n-hexane, and anhydrous ethanol is added, and the mixture is shaken and extracted for 1-2 hours. The supernatant is taken, and the mixture is rotary evaporated at 60° C., and anhydrous ethanol is added in a volume ratio of 1:1. The mixture is placed at -22° C. for 24 hours, and the supernatant is centrifuged to obtain the supernatant, and the mixture is rotary evaporated again, and 10-30% propylene glycol is added to obtain a grapefruit-flavored tobacco extract.

[0013] Preferably, in the above technical scheme, the Klebsiella variicola HNYJ-3 was deposited in the General Biological Center of the China National Microbiological Culture Collection Administration on January 18, 2024, with a deposit number of CGMCC No. 29654; the Enterobacter sp. HNYJ-4 was deposited in the General Biological Center of the China National Microbiological Culture Collection Administration on January 18, 2024, with a deposit number of CGMCC No. 29655.

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

[0015] Preferably, in the above technical solution, in step (3), the culture conditions are: 35°C, 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 mutans HNYJ-3 seed solution to Enterobacter HNYJ-4 seed solution is 7:3, and the total inoculation amount is 4%. The seed solution is inoculated into the tobacco and neoxanthin mixture, and cultured with shaking at 35°C and 150rpm for 72h.

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

[0018] Application of a grapefruit-flavored tobacco extract in cigarettes. The grapefruit-flavored tobacco extract is prepared according to the above preparation method.

[0019] Preferably, in the above technical solution, the application is to dilute the grapefruit tobacco extract and water in a mass ratio of 1: (20-200), and then add 0.5-1uL / stick to conventional cigarettes or heated cigarettes; or, dilute the grapefruit tobacco extract and water in a mass ratio of 1: (2000-20000), spray it on the tobacco at 10-15%, leave it for 2-6 hours, and roll it into cigarettes.

[0020] Preferably, in the above technical solution, the obtained grapefruit-flavored 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 technical solution of the present invention has the following beneficial effects:

[0022] The tobacco extract of the present application is applied to cigarette products to improve the aroma quality and amount of smoke, reduce impurities, and improve the permeability. The present application uses Klebsiella mutans HNYJ-3 and Enterobacter HNYJ-4 to ferment neoxanthin and tobacco mixture to produce aroma components such as oleanone, α-truffle, perilla ketone, carvone, and dihydrocarvone. Oleanone has sweet grapefruit and woody aroma; α-truffle has sweet and floral aroma; perilla ketone has a fresh fragrance; carvone has a medicinal aroma and minty cool taste; it can enrich the smoke and improve the quality of tobacco extract. DETAILED DESCRIPTION

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

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

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

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

[0027] Neoxanthin was commercially available and purchased online. Tobacco leaves were obtained from Henan China Tobacco Industry Co., Ltd.

[0028] Example 1

[0029] (1) Neoxanthin and Tween 80 were prepared in a mass ratio of 1:10 to form a suspension, and the suspension was dissolved in dichloromethane. The dichloromethane was distilled off under reduced pressure, and anhydrous ethanol was added to make the concentration of neoxanthin 50 mg / mL.

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

[0031] (3) Klebsiella mutans HNYJ-3 with the number CGMCC No. 29654 and Enterobacterium HNYJ-4 with the number CGMCC No. 29655 were selected and inoculated into LB liquid culture medium respectively, and cultured at 35°C and 150 rpm with shaking for 24 h. The OD 600 The value is about 1.8, forming seed liquid.

[0032] LB medium liquid culture medium is composed of NaCl 10g / L, peptone 10g / L, yeast extract powder 5g / L, and sterilized at 121°C for 15min.

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

[0034] (5) The fermented mixed solution was centrifuged, the supernatant was taken, and 2 volumes of a mixed solution of ethyl acetate, n-hexane, and anhydrous ethanol were added, wherein the volume ratio of ethyl acetate, n-hexane, and anhydrous ethanol was 5:4:2, and the mixture was shaken and extracted for 2 hours. The supernatant was taken, and rotary evaporated at 60° C. and 60 mbar to a density of about 1.09, and anhydrous ethanol was added in a volume ratio of 1:1. The mixture was placed at -22° C. for 24 hours, and the supernatant was centrifuged to obtain a supernatant, and rotary evaporated again to a density of about 1.2, and 10% propylene glycol was added to obtain a grapefruit-flavored tobacco extract.

[0035] Application Example 1

[0036] The grapefruit-flavored tobacco extract prepared in Example 1 was diluted with water at a mass ratio of 1:100, and then 0.7 uL / stick was added to conventional cigarettes, which was recorded as T1.

[0037] Comparative Example 1

[0038] (1) Grind tobacco leaves into 200-mesh tobacco powder, mix with water at a mass ratio of 1:10, and sterilize at 121°C for 15 min.

[0039] (2) The sterilized mixed solution was centrifuged, the supernatant was taken, and 2 volumes of a mixed solution of ethyl acetate, n-hexane, and anhydrous ethanol were added, wherein the volume ratio of ethyl acetate, n-hexane, and anhydrous ethanol was 5:4:2, and the mixture was extracted by shaking for 2 h. The supernatant was taken, and rotary evaporated at 60° C. and 60 mbar to a density of about 1.09, and anhydrous ethanol was added in a volume ratio of 1:1. The mixture was placed at -22° C. for 24 h, and the supernatant was obtained by centrifugation. The mixture was rotary evaporated again to a density of about 1.2, and 10% propylene glycol was added to obtain the tobacco extract.

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

[0041] Comparative Example 2

[0042] (1) Neoxanthin and Tween 80 were prepared in a mass ratio of 1:10 to form a suspension, and the suspension was dissolved in dichloromethane. The dichloromethane was distilled off under reduced pressure, and anhydrous ethanol was added to make the concentration of neoxanthin 50 mg / mL.

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

[0044] (3) The mixed solution was centrifuged, the supernatant was taken, and 2 volumes of a mixed solution of ethyl acetate, n-hexane, and anhydrous ethanol were added, wherein the volume ratio of ethyl acetate, n-hexane, and anhydrous ethanol was 5:4:2, and the mixture was extracted by shaking for 2 hours. The supernatant was taken, and the mixture was rotary evaporated at 60° C. and 60 mbar to a density of about 1.09, and anhydrous ethanol was added in a volume ratio of 1:1. The mixture was placed at -22° C. for 24 hours, and the supernatant was centrifuged to obtain a supernatant, and the mixture was rotary evaporated again to a density of about 1.2, and 10% propylene glycol was added to obtain a grapefruit-flavored tobacco extract.

[0045] (4) The obtained grapefruit-flavored tobacco extract was diluted with water at a mass ratio of 1:100, and then 0.7 uL / stick was added to conventional cigarettes, which was recorded as CK2.

[0046] Comparative Example 3

[0047] (1) Neoxanthin and Tween 80 were prepared in a mass ratio of 1:10 to form a suspension, and the suspension was dissolved in dichloromethane. The dichloromethane was distilled off under reduced pressure, and anhydrous ethanol was added to make the concentration of neoxanthin 50 mg / mL.

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

[0049] (3) Enterobacterium HNYJ-4 with the number CGMCC NO.29655 was selected and inoculated into LB liquid medium. The medium was cultured at 35°C and 150 rpm with shaking for 24 h. The OD 600 The value is about 1.8, forming seed liquid.

[0050] LB medium liquid culture medium is composed of NaCl 10g / L, peptone 10g / L, yeast extract powder 5g / L, and sterilized at 121°C for 15min.

[0051] (4) Inoculation amount of Enterobacter HNYJ-4: 4% Seed liquid was inoculated into the mixture of tobacco and neoxanthin and cultured with shaking at 35°C and 150 rpm for 72 h.

[0052] (5) The fermented mixed solution was centrifuged, the supernatant was taken, and 2 volumes of a mixed solution of ethyl acetate, n-hexane, and anhydrous ethanol were added, wherein the volume ratio of ethyl acetate, n-hexane, and anhydrous ethanol was 5:4:2, and the mixture was shaken and extracted for 2 hours. The supernatant was taken, and rotary evaporated at 60° C. and 60 mbar to a density of about 1.09, and anhydrous ethanol was added in a volume ratio of 1:1. The mixture was placed at -22° C. for 24 hours, and the supernatant was centrifuged to obtain a supernatant, and rotary evaporated again to a density of about 1.2, and 10% propylene glycol was added to obtain a grapefruit-flavored tobacco extract.

[0053] (6) The obtained grapefruit-flavored tobacco extract was diluted with water at a mass ratio of 1:100, and then 0.7 uL / stick was added to conventional cigarettes, which was recorded as CK3.

[0054] Comparative Example 4

[0055] Conventional cigarettes without any tobacco extracts are denoted as CK.

[0056] result:

[0057] When evaluating tobacco extracts using the sensory quality evaluation method, 13 indicators including aroma quality, aroma quantity, permeability, impurities, concentration, strength, fineness, clumping, irritation, dryness, freshness, sweetness, and aftertaste are scored, 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, non-fermented tobacco extract) has a certain degree of improvement in aroma quantity, assorted smells, delicateness and sweetness, but compared with T1, the improvement is smaller; CK2 (addition of neoxanthin, non-fermented tobacco extract) and CK3 (addition of neoxanthin, single strain fermentation) have an improvement in aroma quality, aroma quantity and assorted smells between CK1 and T1; T1 has obvious improvements in aroma quality, aroma quantity, assorted smells, delicateness and sweetness, further improving the quality of tobacco extract.

[0059] Table 1 Sensory evaluation results

[0060]

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

[0062] Aroma ingredients CK1 T1 Grapefruit ketone / 186.76 α-Treatol / 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-flavored tobacco extract of T1 contains aroma components such as oleander ketone, α-truffle, perilla ketone, carvone, and dihydrocarvone. The odor threshold of oleander ketone is low, with sweet grapefruit and woody aromas; α-truffle has sweet and floral aromas; perilla ketone has a fresh fragrance; carvone has a medicinal aroma and a minty cool taste; it can enrich the smoke and further improve the quality of tobacco extracts.

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

Claims

1. A method for preparing a grapefruit-flavored tobacco extract, characterized in that: The following steps are involved: (1) Take neoxanthin and Tween 80 at 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; (2) grinding tobacco leaves into 200-mesh tobacco powder, mixing with water at a mass ratio of 1:7-1:15, sterilizing at 121°C for 15 min, adding neoxanthin solution to make the neoxanthin concentration 2 g / L; (3) selecting Klebsiella mutans HNYJ-3 and Enterobacter HNYJ-4 and inoculating them into LB liquid culture medium respectively, and culturing them at 35-40° C. and 120-200 rpm with shaking for 24-48 h to form seed solution; (4) Mix the seed solution of Klebsiella mutans HNYJ-3 and the seed solution of Enterobacter HNYJ-4 in a mass ratio of (5-7):(3-5), and inoculate the seed solution into the mixture of tobacco and neoxanthin at a total inoculation amount of 1-6%, and culture at 35-40° C. and 120-200 rpm with shaking for 48-96 h; (5) The fermented mixed solution is centrifuged, the supernatant is taken, and a mixed solution of 2 times the volume of ethyl acetate, n-hexane and anhydrous ethanol is added, and the mixture is shaken and extracted for 1-2 hours. The supernatant is taken, and it is rotary evaporated at 60° C., and anhydrous ethanol is added in a volume ratio of 1:

1. The mixture is placed at -22° C. for 24 hours, and the supernatant is centrifuged to obtain the supernatant, and the mixture is rotary evaporated again, and 10-30% propylene glycol is added to obtain a grapefruit-flavored tobacco extract.

2. The method for preparing the grapefruit-flavored tobacco extract according to claim 1, characterized in that: The Klebsiella variicola HNYJ-3 was deposited in the General Biological Center of the China Microorganism Culture Collection Administration on January 18, 2024, with the deposit number CGMCC No. 29654; the Enterobacter sp. HNYJ-4 was deposited in the General Biological Center of the China Microorganism Culture Collection Administration on January 18, 2024, with the deposit number CGMCC No. 29655.

3. The method for preparing the grapefruit-flavored tobacco extract according to claim 1, characterized in that: In step (3), the LB culture medium includes: 10 g / L NaCl, 10 g / L peptone, and 5 g / L yeast extract powder.

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

8.

5. The method for preparing the grapefruit-flavored tobacco extract according to claim 1, characterized in that: In step (4), the mass ratio of Klebsiella mutans HNYJ-3 seed solution to Enterobacter HNYJ-4 seed solution is 7:3, and the total inoculation amount is 4%. The seed solution is inoculated into the tobacco and neoxanthin mixture, and cultured with shaking at 35° C. and 150 rpm for 72 hours.

6. The method for preparing the grapefruit-flavored 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, and the extraction is shaken for 2 hours. The supernatant is taken and rotary evaporated at 60° C. and 60 mbar to a density of 1.

09. Anhydrous ethanol is added in a volume ratio of 1:1, and the mixture is placed at -22° C. for 24 hours. The supernatant is obtained by centrifugation, and the mixture is rotary evaporated again to a density of 1.

2. 10% propylene glycol is added to obtain a grapefruit-flavored tobacco extract.

7. Application of a grapefruit-flavored tobacco extract in cigarettes, characterized in that: The grapefruit-flavored tobacco extract is prepared according to any one of the preparation methods described in claims 1-6.

8. The use according to claim 7, 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 0.5-1uL / stick to conventional cigarettes or heated cigarettes; or, dilute the grapefruit tobacco extract with water at a mass ratio of 1: (2000-20000), spray it on tobacco at 10-15%, leave it for 2-6 hours, and roll it into cigarettes.

9. The use according to claim 8, characterized in that: The obtained grapefruit-flavored tobacco extract was diluted with water at a mass ratio of 1:100, and then 0.7uL / stick was added to conventional cigarettes or heated cigarettes.

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