High-terpene biological spice based on composite flora fermentation and application thereof

Abstract

The invention discloses a high-terpene biological spice based on composite flora fermentation and application of the high-terpene biological spice. According to the preparation method, a tobacco extracting solution is obtained through assistance of microwaves and ultrasonic waves, and a small amount of tomato juice is added into the tobacco extracting solution; the preparation method comprises the following steps: firstly, carrying out aerobic fermentation degradation on macromolecular substances such as polysaccharides by using one microbial agent, then carrying out anaerobic fermentation degradation on components such as flavonoids and carotenoids by using another composite microbial agent, converting the components into fragrant components, and preparing the biological spice by adopting an aerobic-anaerobic fermentation mode. The perfume is richer in variety of fragrance components and can generate various terpene fragrance components, the terpene fragrance components such as limonene, linalool, alpha-pinene, beta-myrcene and geraniol are remarkably increased, and the terpene fragrance components such as alpha-santalene, geraniene, alpha-terpinene, thujopsene, alpha-cedrene, fennel ketone and ocimene are newly added, so that the fragrance of the perfume is enhanced, and the fragrance of the perfume is enhanced. When being used for traditional cigarettes or heated cigarettes, the cigarette essence can remarkably improve the aroma quality and permeability of the cigarettes, improve aftertaste and reduce irritation.

Description

Technical Field

[0001] This invention relates to the field of tobacco technology, and in particular to a high-terpene bioflavoring based on fermentation of a complex microbial community and its application. 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, they widely adopt perforated paper technology and add tobacco sheets and expanded stems. While this reduces the tar content, it also results in insufficient aroma in the smoke. Therefore, adding flavorings and fragrances to compensate for the loss of aroma caused by harm reduction and tar reduction is a commonly used technical method.

[0003] Due to the direct inhalation of tobacco products, flavorings for cigarettes generally use naturally derived flavorings to ensure the safety of tobacco products, with tobacco extracts being one of the most common choices.

[0004] As important components of tobacco aroma compounds, terpenes such as β-ionone and damascone can effectively enhance the aroma of cigarettes. Some terpenes with special aromas, such as limonene and geraniol, can impart floral and fruity characteristics to the smoke. In existing technologies, tobacco extracts are generally prepared by water or alcohol extraction of tobacco materials followed by vacuum concentration. However, the amount of terpenes in these extracts is often insufficient to meet the needs of cigarette research and development. Utilizing aroma-producing microorganisms for fermentation can effectively degrade and transform precursors in the extracts, specifically generating terpenoid aroma components, which is an effective means to improve the quality of tobacco extracts.

[0005] Meanwhile, relying solely on the material transformation and degradation of tobacco itself to generate terpenes has limited effectiveness, and most extracts require the addition of terpene precursors. Tomatoes contain various alcohols, aldehydes, esters, terpenes, and are rich in lycopene and limonene. Adding a small amount of tomato juice to tobacco extracts can enrich the aroma layers, increase sweetness, and effectively enhance the content of terpene aroma compounds in the extract. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a high-terpene bioflavoring based on the fermentation of a complex microbial community and its application. The flavoring has a richer variety of aroma components and can significantly improve the aroma and permeability of traditional cigarettes or heated cigarettes, as well as improve the aftertaste and reduce irritation.

[0007] The technical problem to be solved by the present invention is achieved through the following technical solution:

[0008] A method for preparing a high-terpene bio-fragrant based on fermentation of a complex microbial community includes the following steps:

[0009] (1) *Bacillus belye* HJ-04, *Klebsiella variegata* HNYJ-3, *Bacillus hygroscopicus* ZY02-1, and *Bacillus cereus* ZY51 were respectively inoculated into LB liquid medium and cultured at 30℃ and 180 rpm for 24 h with shaking. OD 600 Value 1.8-2.0, forming seed liquid;

[0010] (2) Grind the tobacco leaves into 100-200 mesh tobacco powder, extract it at 60-80℃ and ultrasonic power 350W for 1.5-2h, then extract it at microwave power 450W for 180s, filter it to obtain tobacco extract; juice fresh tomatoes, mix the tobacco extract and tomato juice at a volume ratio of 9:1, sterilize at 121℃ for 15min to obtain a mixture;

[0011] (3) Inoculate the Bacillus vesiculosus HJ-04 seed liquid and Klebsiella variegata HNYJ-3 seed liquid prepared in step (1) into the mixture in step (2) at a volume ratio of 2:1. The total inoculation amount is 0.5-1% of the volume of the mixture. Aerobic fermentation is carried out at 35℃ and 150rpm for 24-48h. After fermentation, the fermentation liquid is put into a sealed fermentation bag with a liquid volume of 95% and sterilized at 121℃ for 20min to obtain aerobic fermentation product. Then, inoculate the Bacillus cereus ZY51 seed liquid and Bacillus glomeratus ZY02-1 seed liquid prepared in step (1) into the aerobic fermentation product at a volume ratio of 1:1. The total inoculation amount is 1%-1.5% of the volume of the aerobic fermentation product. Continue anaerobic fermentation for 48-60h to obtain anaerobic fermentation product.

[0012] (4) Centrifuge the anaerobic fermentation product from step (3), take the supernatant and rotary evaporate it at 55-60℃ and 60-80mbar until the density is 1.08±0.01g / mL, then add anhydrous ethanol of the same mass as the supernatant after rotary evaporation, let it stand at -22℃ for 24h, centrifuge at low temperature, take the supernatant and add 1,2-propanediol accounting for 30% of the mass of the supernatant to obtain high terpene bio-fragrance.

[0013] Preferably, in the above technical solution, in step (1), Bacillus velezensis HJ-04 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO:M 20231959; Klebsiella variicola HNYJ-3 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No.29654; Bacillus altitudinis ZY02-1 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No.33916; and Bacillus cereus ZY51 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No.33917.

[0014] Preferably, in the above technical solution, in step (2), the tobacco leaves are ground to a mesh size of 100, the ultrasonic extraction temperature is 60℃, and the extraction time is 1.5h.

[0015] Preferably, in the above technical solution, in step (3), the total inoculation amount of Bacillus belye HJ-04 seed liquid and Klebsiella variegata HNYJ-3 seed liquid is 1% of the mixed liquid volume, and the aerobic fermentation time is 48h; the total inoculation amount of Bacillus cereus ZY51 seed liquid and Bacillus hygroscopicus ZY02-1 seed liquid is 1.5% of the aerobic fermentation product volume, and the anaerobic fermentation time is 48h.

[0016] Preferably, in the above technical solution, in step (4), the rotary evaporation temperature is 60°C and the vacuum degree is 60 mbar.

[0017] A high-terpene bio-fragrance prepared according to the above preparation method.

[0018] The application of a high-terpene bio-flavor in cigarette manufacturing is as follows:

[0019] Method 1: Dilute the high-terpene bio-flavoring with water at a volume ratio of 1:(10-200), and add it to regular or heated cigarettes at a dosage of 0.5-1 μL / cigarette; or

[0020] Method 2: Dilute the high-terpene bio-fragrance with water at a volume ratio of 1:(1000-10000), spray it onto the tobacco at a spraying rate of 10-15%, stir it evenly and let it stand for 2-6 hours, dry it at 60-70℃ for 10-15 minutes, and then roll it into cigarettes.

[0021] Preferably, in the above technical solution, the dilution ratio of high terpene bio-fragrance to water in method 2 is 1:2000, the spraying amount is 100g / kg tobacco, the standing time is 4h, the drying temperature is 70℃, and the drying time is 15min.

[0022] The above-described technical solution of the present invention has the following beneficial effects:

[0023] This invention relates to a high-terpene bio-fragrant liquid based on fermentation with a complex microbial community. Tobacco extract is obtained using microwave and ultrasound assistance. A small amount of tomato juice is added to the tobacco extract. First, aerobic fermentation with one microbial agent degrades polysaccharides and other macromolecules. Then, anaerobic fermentation with another complex microbial agent degrades flavonoids, carotenoids, and other components, converting them into aroma components. This aerobic-anaerobic fermentation process yields a bio-fragrant liquid. This liquid exhibits a richer variety of aroma components and produces multiple terpene aroma components. Significant increases are observed in terpene aroma components such as limonene, linalool, α-pinene, β-myrcene, and geraniol. Newly added terpene aroma components include α-santalene, geraniene, α-terpinene, thujone, α-juniperene, fenugreekine, and ocimene. When used in traditional or heated cigarettes, it can significantly enhance the aroma and permeability of cigarettes, improve the aftertaste, and reduce irritation. Detailed Implementation

[0024] 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.

[0025] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are commercially available. Unless otherwise specified, the equipment used in the experiments is well known to those skilled in the art.

[0026] Bacillus cereus ZY51 was deposited on March 20, 2025, 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, with accession number CGMCC No. 33917.

[0027] Bacillus altitudinis ZY02-1 was deposited on March 20, 2025, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCCNo. 33916.

[0028] Klebsiella variicola HNYJ-3 was deposited on January 18, 2024, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 29654.

[0029] Bacillus velezensis HJ-04 was deposited on October 20, 2023, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO:M20231959.

[0030] A method for preparing a high-terpene bio-fragrant based on fermentation of a complex microbial community includes the following steps:

[0031] 1. Select Bacillus belye HJ-04, Klebsiella variegata HNYJ-3, Bacillus hygroscopicus ZY02-1, and Bacillus cereus ZY51 respectively and inoculate them into LB liquid medium. Incubate at 30℃ and 180rpm for 24h with shaking. The OD600 value is 1.8-2.0 to form seed culture.

[0032] 2. Grind the tobacco leaves into 100-200 mesh tobacco powder, extract it at 60-80℃ and ultrasonic power 350W for 1.5-2 hours, then extract it at microwave power 450W for 180 seconds, filter it to obtain tobacco extract; juice fresh tomatoes, mix the tobacco extract and tomato juice in a 9:1 ratio, and sterilize at 121℃ for 15 minutes.

[0033] 3. Inoculate *Bacillus belye* HJ-04 and *Klebsiella variegata* HNYJ-3 into the sterilized mixture at a ratio of 2:1, with a total inoculum of 0.5-1%. Aerobic fermentation is carried out at 35℃ and 150 rpm for 24-48 hours. After fermentation, the mixture is transferred to a sealed fermentation bag with a liquid volume of 95%, and sterilized at 121℃ for 20 minutes to obtain the aerobic fermentation product. Subsequently, *Bacillus cereus* ZY51 and *Bacillus pleuropneumoniae* ZY02-1 are inoculated into the aerobic fermentation product at a ratio of 1:1, with a total inoculum of 1%-1.5%. Anaerobic fermentation continues for 48-60 hours to obtain the anaerobic fermentation product.

[0034] 4. Centrifuge the anaerobic fermentation product, take the supernatant and rotary evaporate it at 55-60℃ and 60-80mbar until the density is 1.08±0.01g / mL, then add an equal mass of anhydrous ethanol, let it stand at -22℃ for 24h, then centrifuge it at low temperature, take the supernatant and add 30% by mass of 1,2-propanediol to obtain high terpene bio-fragrance.

[0035] Applications of high-terpene bio-fragrances:

[0036] The obtained fermented bio-flavoring is diluted with water at a ratio of 1:(10-200), and then 0.5-1uL / cigarette is added to regular cigarettes or heated cigarettes. Alternatively, the obtained fermented bio-flavoring is diluted with water at a ratio of 1:(1000-10000), sprayed onto tobacco at 10-15%, turned evenly and left to stand for 2-6 hours, dried at 60-70℃ for 10-15 minutes, and then rolled into cigarettes.

[0037] Example

[0038] 1. *Bacillus belye* HJ-04, *Klebsiella variegata* HNYJ-3, *Bacillus hygroscopicus* ZY02-1, and *Bacillus cereus* ZY51 were respectively inoculated into LB liquid medium and cultured at 30℃ and 180 rpm for 24 h with shaking. OD 600 Value 2.0, seed liquid is formed.

[0039] 2. Grind the tobacco leaves into 100-mesh tobacco powder, extract it at 60℃ and ultrasonic power of 350W for 1.5h, then extract it at microwave power of 450W for 180s, filter it to obtain tobacco extract; juice fresh tomatoes, mix the tobacco extract and tomato juice in a 9:1 ratio, and sterilize at 121℃ for 15min.

[0040] 3. *Bacillus belye* HJ-04 and *Klebsiella variegata* HNYJ-3 were inoculated into the sterilized mixture at a ratio of 2:1, with a total inoculum of 1%. Aerobic fermentation was carried out at 35℃ and 150 rpm for 48 hours. After fermentation, the mixture was transferred to a sealed fermentation bag with a liquid volume of 95% and sterilized at 121℃ for 20 minutes to obtain the aerobic fermentation product. Subsequently, *Bacillus cereus* ZY51 and *Bacillus pleuropneumoniae* ZY02-1 were inoculated into the aerobic fermentation product at a ratio of 1:1, with a total inoculum of 1.5%. Anaerobic fermentation was continued for 48 hours to obtain the anaerobic fermentation product.

[0041] 4. Centrifuge the anaerobic fermentation product, take the supernatant and rotary evaporate it at 60℃ and 60mbar until the density is 1.08±0.01g / mL, then add an equal mass of anhydrous ethanol, let it stand at -22℃ for 24h, then centrifuge it at low temperature, take the supernatant and add 30% by mass of 1,2-propanediol to obtain high terpene bio-fragrance.

[0042] Compare with Example 1

[0043] 1. Grind the tobacco leaves into 100-mesh tobacco powder, extract it at 60℃ and ultrasonic power of 350W for 1.5h, then extract it at microwave power of 450W for 180s, filter it to obtain tobacco extract, and sterilize it at 121℃ for 15min.

[0044] 2. Centrifuge the sterilized tobacco extract, take the supernatant and rotary evaporate it at -22℃ and 60mbar until the density is 1.08±0.01g / mL, then add an equal weight of anhydrous ethanol, let it stand at -22℃ for 24h, then centrifuge at low temperature, take the supernatant and add 30% by mass of 1,2-propanediol to obtain the flavoring CK1.

[0045] Compare with Example 2

[0046] 1. Grind tobacco leaves into 100-mesh tobacco powder, extract at 60℃ and ultrasonic power 350W for 1.5h, then extract at microwave power 450W for 180s, filter to obtain tobacco extract; juice fresh tomatoes, mix tobacco extract and tomato juice in a 9:1 ratio, sterilize at 121℃ for 15min.

[0047] 2. After sterilization, the solution is centrifuged, and the supernatant is rotary evaporated at 60℃ and 60mbar to a density of 1.08±0.01g / mL. Then, an equal weight of anhydrous ethanol is added, and the solution is allowed to stand at -22℃ for 24h. After centrifugation at low temperature, the supernatant is taken and 30% by mass of 1,2-propanediol is added to obtain the fragrance CK2.

[0048] Compare with Example 3

[0049] 1. Select Bacillus belye HJ-04 and Klebsiella variegata HNYJ-3 and inoculate them into LB liquid medium. Incubate at 30℃ and 180rpm for 24h with shaking. The OD600 value is about 2.0, which forms the seed culture.

[0050] 2. Grind the tobacco leaves into 100-mesh tobacco powder, extract it at 60℃ and ultrasonic power of 350W for 1.5h, then extract it at microwave power of 450W for 180s, filter it to obtain tobacco extract; juice fresh tomatoes, mix the tobacco extract and tomato juice in a 9:1 ratio, and sterilize at 121℃ for 15min.

[0051] 3. Inoculate Bacillus belye HJ-04 and Klebsiella variegata HNYJ-3 into the sterilized mixture at a ratio of 2:1, with a total inoculum of 1%. Ferment aerobically at 35°C and 150 rpm for 48 hours.

[0052] 4. After fermentation, centrifuge the fermentation broth, take the supernatant and rotary evaporate it at 60℃ and 60mbar until the density is 1.08±0.01g / mL. Then add an equal weight of anhydrous ethanol, let it stand at -22℃ for 24h, centrifuge it at low temperature, take the supernatant and add 30% by weight of 1,2-propanediol to obtain the fragrance CK3.

[0053] Compare with Example 4

[0054] 1. Select Bacillus cereus ZY02-1 and Bacillus cereus ZY51 respectively and inoculate them into LB liquid medium. Incubate at 30℃ and 180rpm for 24h with shaking. The OD600 value is about 2.0, which forms the seed culture.

[0055] 2. Grind the tobacco leaves into 100-mesh tobacco powder, extract it at 60℃ and ultrasonic power of 350W for 1.5h, and then extract it at microwave power of 450W for 180s. Filter to obtain tobacco extract. Juice fresh tomatoes, mix the tobacco extract with tomato juice at a ratio of 9:1, put the solution into a sealed fermentation bag with a volume of 95%, and sterilize at 21℃ for 15min.

[0056] 3. Inoculate Bacillus cereus ZY51 and Bacillus hygroscopicus ZY02-1 into the sterilized mixture at a ratio of 1:1, with a total inoculation amount of 1.5%, and carry out anaerobic fermentation for 48 hours.

[0057] 4. After fermentation, centrifuge the fermentation broth, take the supernatant and rotary evaporate it at 60℃ and 60mbar until the density is 1.08±0.01g / mL. Then add an equal weight of anhydrous ethanol, let it stand at -22℃ for 24h, centrifuge it under low temperature conditions, take the supernatant and add 30% by weight of 1,2-propanediol to obtain the fragrance CK4.

[0058] The flavorings prepared in Examples 1-4 and Comparative Examples 1-4 were diluted 2000 times with pure water and sprayed onto cigarette tobacco at a rate of 100g / kg. After being evenly mixed and left to stand for 4 hours, the tobacco was dried at 70℃ for 15 minutes and then rolled into cigarettes. Sensory quality evaluation was conducted, with professional judges assessing indicators such as aroma quality, aroma quantity, concentration, smoothness, aftertaste, off-flavors, and irritation. The total sensory evaluation score was the sum of the scores for each sub-item. Cigarettes without added flavorings were designated CK (Control Group).

[0059] As shown in Table 1, the results indicate that the addition of flavorings from Control Examples 1-4 can improve cigarette quality compared to cigarettes without added flavorings, but the improvement is less compared to the examples. The fermented bio-flavorings of this application, when applied to cigarette products, enrich the smoke, enhance the natural aroma of tobacco, and improve the permeability of the smoke. Compared to Control Example CK, it significantly improves the aroma quality, aroma quantity, permeability, reduces off-flavors, and improves the aftertaste, with a total score increase of 1.47 points. Compared to the non-fermented tobacco extract (CK1), the results are shown in Table 2. The flavorings prepared in the examples contain abundant terpene aroma substances, with significant increases in limonene, linalool, α-pinene, β-myrcene, geraniol, etc., and newly added aroma components such as α-santalene, geraniol, α-terpinene, thujone, α-juniperene, fenugreekine, and ocimene.

[0060] Table 1 Sensory evaluation results

[0061]

[0062] Table 2. Main aroma components (μg / mL)

[0063] Aroma components CK1 T Limonene 27.32 84.61 Linalool 23.41 36.26 α-pinene 10.83 16.94 β-Myrcene 2.28 8.71 Geraniol 12.43 18.50 α-Santalene 6.48 Myrcene 8.44 α-Pine 51.15 Rohanbium 4.26 α-Cupressene 44.10 Anethole 2.58 ocimene 2.08

[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 for preparing a high-terpene bio-fragrant liquid based on fermentation of a complex microbial community, characterized in that, Includes the following steps: (1) *Bacillus belye* HJ-04, *Klebsiella variegata* HNYJ-3, *Bacillus hygroscopicus* ZY02-1, and *Bacillus cereus* ZY51 were respectively inoculated into LB liquid medium and cultured at 30℃ and 180 rpm for 24 h with shaking. OD 600 Value 1.8-2.0, forming seed liquid; (2) Grind the tobacco leaves into 100-200 mesh tobacco powder, extract it at 60-80℃ and ultrasonic power 350W for 1.5-2h, then extract it at microwave power 450W for 180s, filter it to obtain tobacco extract; juice fresh tomatoes, mix the tobacco extract and tomato juice at a volume ratio of 9:1, sterilize at 121℃ for 15min to obtain a mixture; (3) Inoculate the Bacillus vesiculosus HJ-04 seed liquid and Klebsiella variegata HNYJ-3 seed liquid prepared in step (1) into the mixture in step (2) at a volume ratio of 2:

1. The total inoculation amount is 0.5-1% of the volume of the mixture. Aerobic fermentation is carried out at 35℃ and 150rpm for 24-48h. After fermentation, the fermentation liquid is put into a sealed fermentation bag with a liquid volume of 95% and sterilized at 121℃ for 20min to obtain aerobic fermentation product. Then, inoculate the Bacillus cereus ZY51 seed liquid and Bacillus glomeratus ZY02-1 seed liquid prepared in step (1) into the aerobic fermentation product at a volume ratio of 1:

1. The total inoculation amount is 1%-1.5% of the volume of the aerobic fermentation product. Continue anaerobic fermentation for 48-60h to obtain anaerobic fermentation product. (4) Centrifuge the anaerobic fermentation product from step (3), take the supernatant and rotary evaporate it at 55-60℃ and 60-80mbar until the density is 1.08±0.01g / mL, then add anhydrous ethanol of the same mass as the supernatant after rotary evaporation, let it stand at -22℃ for 24h, centrifuge at low temperature, take the supernatant and add 1,2-propanediol accounting for 30% of the mass of the supernatant to obtain high terpene bio-fragrance.

2. The preparation method according to claim 1, characterized in that, In step (1), Bacillus velezensis HJ-04 was deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M20231959; Klebsiella variicola HNYJ-3 was deposited at the China General Microbiological Culture Collection (CGMCC) with accession number CGMCC No. 29654; Bacillus altitudinis ZY02-1 was deposited at the China General Microbiological Culture Collection (CGMCC) with accession number CGMCC No. 33916; and Bacillus cereus ZY51 was deposited at the China General Microbiological Culture Collection (CGMCC) with accession number CGMCC No. 33917.

3. The preparation method according to claim 1, characterized in that, In step (2), the tobacco leaves are ground to a mesh size of 100, the ultrasonic extraction temperature is 60℃, and the extraction time is 1.5h.

4. The preparation method according to claim 1, characterized in that, In step (3), the total inoculum amount of Bacillus belye HJ-04 seed liquid and Klebsiella variegata HNYJ-3 seed liquid is 1% of the mixed liquid volume, and the aerobic fermentation time is 48h; the total inoculum amount of Bacillus cereus ZY51 seed liquid and Bacillus hygroscopicus ZY02-1 seed liquid is 1.5% of the aerobic fermentation product volume, and the anaerobic fermentation time is 48h.

5. The preparation method according to claim 1, characterized in that, In step (4), the rotary evaporation temperature is 60°C and the vacuum degree is 60 mbar.

6. A high-terpene bio-fragrant obtained by the preparation method according to any one of claims 1-5.

7. The application of the high-terpene bio-flavoring according to claim 6 in cigarette preparation, characterized in that, The application method is as follows: Method 1: Dilute the high-terpene bio-flavoring with water at a volume ratio of 1:(10-200), and add it to regular or heated cigarettes at a dosage of 0.5-1 μL / cigarette; or Method 2: Dilute the high-terpene bio-fragrance with water at a volume ratio of 1:(1000-10000), spray it onto the tobacco at a spraying rate of 10-15%, stir it evenly and let it stand for 2-6 hours, dry it at 60-70℃ for 10-15 minutes, and then roll it into cigarettes.

8. The application according to claim 7, characterized in that, In Method 2, the dilution ratio of high-terpene bio-fragrant to water is 1:2000, the spraying amount is 100g / kg of tobacco, the standing time is 4h, the drying temperature is 70℃, and the drying time is 15min.

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