Low-oxygen fermented sweet and fragrant biological spice and application thereof in cigarettes

Abstract

The invention discloses a low-oxygen fermented sweet and fragrant biological spice and application thereof in cigarettes, and a preparation method comprises the following steps: extracting by adopting microwave and ultrasonic assistance to obtain a compound water extract of liquorice and osmanthus fragrans, fermenting the water extract under a low-oxygen condition by adopting a bacillus altitudinis ZY02-1 and klebsiella variicola HNYJ-3 compound bacterial agent, and filtering to obtain the low-oxygen fermented sweet and fragrant biological spice. And then concentrating and extracting to obtain the product. Compared with an unfermented spice, the prepared sweet and fragrant biological spice has the advantages that fragrance components such as glycyrrhizin, isoliquiritigenin, linalool, bornyl acetate and citral are obviously increased, and fragrance components such as beta-ionone, alpha-terpilenol and beta-caryophyllene are added; when the spice is added into cigarettes, the sweetness, the aroma quality and the aroma amount of smoke are remarkably improved.

Description

Technical Field

[0001] This invention relates to the field of tobacco technology, and in particular to a sweet-smelling bio-flavoring produced by low-oxygen fermentation and its application in cigarettes. Background Technology

[0002] Licorice, also known as sweet grass, sweet root, and licorice, is a perennial herb belonging to the genus Glycyrrhiza in the legume family. Its root has a brown outer skin and a pale yellow interior. It is mainly distributed in Northeast China, Gansu, Qinghai, Shandong, and other regions. It is a tonic Chinese herbal medicine, named for its sweet taste. As a traditional spice, licorice is widely used in food, health-promoting Chinese medicine, beverages, and cosmetics. It is used as a deodorant, sweetener, flavor enhancer, antioxidant, and antibacterial agent. However, its use in the tobacco industry is less studied.

[0003] Licorice has a sweet aroma, with notes of herbs and vanilla. The overall aroma is mild, rich, long-lasting, and has a mellow aftertaste. When applied to tobacco products, licorice extract can enrich the aroma and sweeten the smoke.

[0004] Current licorice flavoring technology typically involves extracting licorice material with water or alcohol and then further concentrating it under reduced pressure. However, this often results in insufficient aroma compounds, failing to meet the needs of cigarette development. Utilizing aroma-producing microorganisms for fermentation can effectively degrade and transform precursors in licorice, generating aroma components, which is an effective means of improving the quality of licorice flavoring.

[0005] Meanwhile, relying solely on the material transformation of licorice itself has limited effects. Adding a small amount of osmanthus flowers to licorice can enhance the layers and depth of its sweet aroma, making the overall fragrance more mellow and the prepared bio-fragrance more abundant and harmonious. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a sweet-smelling bio-flavoring produced by low-oxygen fermentation and its application in cigarettes. When this flavoring is added to cigarettes, it significantly improves the sweetness, aroma quality and aroma quantity of the smoke.

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

[0008] A method for preparing a sweet-smelling bio-flavoring product through low-oxygen fermentation includes the following steps:

[0009] (1) Mix dried licorice and dried osmanthus flowers evenly at a mass ratio of 8:1, grind them to 50-100 mesh, add water to make the material-liquid ratio 1:5-1:10, first extract at 60-80℃ and ultrasonic power 350w for 1.5-2h, then extract at microwave power 450w for 180s, filter, obtain the extract, sterilize at 121℃ for 15min, and set aside for use;

[0010] (2) Bacillus altitudinis ZY02-1 and Klebsiella variicola HNYJ-3 were inoculated into LB medium and cultured at 30℃ and 180 rpm for 24-48 h with shaking. OD 600 Value 1.8-2.0, forming seed liquid;

[0011] (3) Mix the seed liquids of Bacillus hygroscopicus ZY02-1 and Klebsiella variegata HNYJ-3 at a mass ratio of 5:2 to prepare a mixed bacterial agent;

[0012] (4) Inoculate the mixed bacterial agent into the extract of step (1) at an inoculation rate of 0.5-3%, ferment at 30℃ and 150rpm for 8-12 hours, then put it into a fermentation bag with a liquid volume of 75%, and let it stand and seal for fermentation for 24-48 hours.

[0013] (5) Centrifuge the fermentation broth to obtain the supernatant, and rotary evaporate it to a density of 1.05±0.01g / mL at 60℃ and 60-100mbar. Add anhydrous ethanol at a volume ratio of 1:1, place it at -22℃ for 24h, centrifuge to obtain the supernatant, and add 10-30% of 1,2-propanediol to obtain a sweet-scented biological fragrance.

[0014] Preferably, in the above technical solution, in step (1), the particle size is 100 mesh, the material-liquid mass ratio is 1:7; the extraction is carried out at 60℃ and ultrasonic power of 350w for 1.5h, and then extracted for 180s under microwave power of 450w.

[0015] Preferably, in the above technical solution, in step (2), Bacillus saltitudinis ZY02-1 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on March 20, 2025, with accession number CGMCC No. 33916; Klebsiella variicola HNYJ-3 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on January 18, 2024, with accession number CGMCC No. 29654.

[0016] Preferably, in the above technical solution, in step (4), the amount of compound microbial agent inoculation is 1%; after fermentation at 30℃ and 150rpm for 8 hours, it is left to stand for 48 hours.

[0017] Preferably, in the above technical solution, in step (5), the rotary evaporation conditions are 60°C and 60 mbar; and finally, 30% 1,2-propanediol is added.

[0018] A low-oxygen fermentation sweet-scented bio-fragrant agent prepared according to the above preparation method.

[0019] The application of a low-oxygen fermentation sweet-scented bio-flavoring in cigarettes involves diluting the flavoring with water at a ratio of 1:20–1:200, adding it at a rate of 0.5–1 μL / cigarette to conventional or heated cigarettes, or spraying it evenly at a rate of 2 g / kg of tobacco shreds, and then rolling it into cigarettes after equilibration for 2–4 hours.

[0020] The above-described technical solution of the present invention has the following beneficial effects:

[0021] This invention relates to a hypoxic fermentation-based sweet-scented bio-flavoring. The flavoring is primarily prepared by microwave and ultrasonic-assisted extraction of a composite aqueous extract of licorice and osmanthus. The extract is then fermented under hypoxic conditions using a compound bacterial agent of *Bacillus hygroscopicus* ZY02-1 and *Klebsiella variegata* HNYJ-3, followed by concentration and extraction. Compared to unfermented flavoring, the prepared sweet-scented bio-flavoring exhibits significantly increased levels of aroma components such as glycyrrhizic acid, isoglycyrrhizin, linalool, bornyl acetate, and citral, and also includes newly added aroma components such as β-ionone, α-terpineol, and β-caryophyllene. When added to cigarettes, this flavoring significantly enhances the sweetness, aroma quality, and aroma intensity of the smoke. Detailed Implementation

[0022] 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.

[0023] 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.

[0024] Bacillus altitudinis ZY02-1 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. 33916.

[0025] 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.

[0026] A method for preparing a sweet-smelling bio-flavoring product through low-oxygen fermentation includes the following steps:

[0027] 1. Mix dried licorice root and dried osmanthus flowers evenly at a mass ratio of 8:1, grind them into a powder of 50-100 mesh, then mix them with water at a mass ratio of 1:5-1:10, extract at 60-80℃ and ultrasonic power of 350w for 1.5-2 hours, then extract at microwave power of 450w for 180s, filter, obtain the extract, and sterilize at 121℃ for 15 minutes.

[0028] 2. Select *Bacillus hygroscopicus* ZY02-1 and *Klebsiella variegata* HNYJ-3 and inoculate them separately onto LB medium. Incubate at 30℃ and 180 rpm with shaking for 24-48 hours. OD 600 The value is approximately 1.8-2.0, forming seed liquid.

[0029] 3. Prepare a mixed bacterial agent by mixing seed liquids of Bacillus hygroscopicus ZY02-1 and Klebsiella variegata HNYJ-3 in a mass ratio of 5:2.

[0030] 4. Inoculate the extract with an inoculum of 0.5-3% and ferment at 30℃ and 150rpm for 8-12 hours. Then, transfer the extract to a fermentation bag with a liquid volume of 75% and allow it to stand and ferment in a sealed container for 24-48 hours.

[0031] 5. Centrifuge the fermented solution, take the supernatant, and rotary evaporate it at 60℃ and 60-100mbar until the density reaches 1.05±0.01g / mL. Add anhydrous ethanol at a volume ratio of 1:1, place at -22℃ for 24h, centrifuge to obtain the supernatant, and add 10-30% of 1,2-propanediol to obtain the sweet-smelling biological fragrance.

[0032] Spice application:

[0033] The obtained sweet-scented bio-fragrance is diluted with water at a ratio of 1:(20-200), and then 0.5-1uL / cigarette is added to regular cigarettes or heated cigarettes, or sprayed onto tobacco at a ratio of 2g / kg, left for 2-4 hours, and then rolled into cigarettes.

[0034] Example

[0035] 1. Mix dried licorice root and dried osmanthus flowers evenly at a mass ratio of 8:1, grind them into 100-mesh powder, then mix them with water at a mass ratio of 1:7, extract at 60℃ and ultrasonic power of 350w for 1.5h, then extract at microwave power of 450w for 180s, filter, obtain the extract, and sterilize at 121℃ for 15min.

[0036] 2. Select *Bacillus hygroscopicus* ZY02-1 and *Klebsiella variegata* HNYJ-3 and inoculate them separately onto LB medium. Incubate at 30℃ and 180 rpm with shaking for 24 h. OD 600 The value is approximately 2.0, forming seed liquid.

[0037] 3. Prepare a mixed bacterial agent by mixing Bacillus hygroscopicus ZY02-1 and Klebsiella variegata HNYJ-3 seed liquids in a mass ratio of 5:2.

[0038] 4. Inoculate the extract with 1% of the extract and ferment at 30℃ and 150rpm for 8 hours. Then, transfer the extract to a fermentation bag with a liquid volume of 75% and allow it to stand and ferment in a sealed container for 48 hours.

[0039] 5. Centrifuge the fermented solution, take the supernatant, and rotary evaporate it at 60℃ and 60mbar until the density reaches 1.05±0.01g / mL. Add anhydrous ethanol at a volume ratio of 1:1, place at -22℃ for 24h, centrifuge to obtain the supernatant, and add 30% 1,2-propanediol to obtain the sweet-scented bio-fragrance.

[0040] Compare with Example 1

[0041] 1. Grind dried licorice into 100-mesh powder, then mix it with water at a mass ratio of 1:7. Extract at 60℃ and ultrasonic power of 350W for 1.5 hours, then extract at microwave power of 450W for 180 seconds. Filter to obtain the extract, and sterilize at 121℃ for 15 minutes.

[0042] 2. Centrifuge the solution, take the supernatant, and rotary evaporate it at 60℃ and 60mbar until the density reaches 1.05±0.01g / mL. Add anhydrous ethanol at a volume ratio of 1:1, place at -22℃ for 24h, centrifuge to obtain the supernatant, and add 30% 1,2-propanediol to obtain Control Example 1.

[0043] Compare with Example 2

[0044] 1. Mix dried licorice root and dried osmanthus flowers evenly at a mass ratio of 8:1, grind them into 100-mesh powder, then mix them with water at a mass ratio of 1:7, extract at 60℃ and ultrasonic power of 350w for 1.5h, then extract at microwave power of 450w for 180s, filter, obtain the extract, and sterilize at 121℃ for 15min.

[0045] 2. Centrifuge the solution, take the supernatant, and rotary evaporate it at 60℃ and 60mbar until the density reaches 1.05±0.01g / mL. Add anhydrous ethanol at a volume ratio of 1:1, place at -22℃ for 24h, centrifuge to obtain the supernatant, and add 30% 1,2-propanediol to obtain Control Example 2.

[0046] Compare with Example 3

[0047] 1. Mix dried licorice root and dried osmanthus flowers evenly at a mass ratio of 8:1, grind them into 100-mesh powder, then mix them with water at a mass ratio of 1:7, extract at 60℃ and ultrasonic power of 350w for 1.5h, then extract at microwave power of 450w for 180s, filter, obtain the extract, and sterilize at 121℃ for 15min.

[0048] 2. Select *Bacillus hygroscopicus* ZY02-1 and inoculate it onto LB medium. Incubate at 30℃ and 180 rpm with shaking for 24 hours. OD 600 The value is approximately 2.0, forming seed liquid.

[0049] 3. Inoculate the extract with 1% of the extract and ferment at 30℃ and 150rpm for 8 hours. Then, transfer the extract to a fermentation bag with a liquid volume of 75% and allow it to stand and ferment in a sealed container for 48 hours.

[0050] 4. Centrifuge the fermented solution, take the supernatant, and rotary evaporate it at 60℃ and 60mbar until the density reaches 1.05±0.01g / mL. Add anhydrous ethanol at a volume ratio of 1:1, place at -22℃ for 24h, centrifuge to obtain the supernatant, and add 30% 1,2-propanediol to obtain Control Example 3.

[0051] Compare with Example 4

[0052] 1. Mix dried licorice root and dried osmanthus flowers evenly at a mass ratio of 8:1, grind them into 100-mesh powder, then mix them with water at a mass ratio of 1:7, extract at 60℃ and ultrasonic power of 350w for 1.5h, then extract at microwave power of 450w for 180s, filter, obtain the extract, and sterilize at 121℃ for 15min.

[0053] 2. Select Klebsiella variegata HNYJ-3 and inoculate it onto LB medium. Incubate at 30℃ and 180 rpm with shaking for 24 h. OD 600 The value is approximately 2.0, forming seed liquid.

[0054] 3. Inoculate the extract with 1% of the extract and ferment at 30℃ and 150rpm for 8 hours. Then, transfer the extract to a fermentation bag with a liquid volume of 75% and allow it to stand and ferment in a sealed container for 48 hours.

[0055] 4. Centrifuge the fermented solution, take the supernatant, and rotary evaporate it at 60℃ and 60mbar until the density reaches 1.05±0.01g / mL. Add anhydrous ethanol at a volume ratio of 1:1, place at -22℃ for 24h, centrifuge to obtain the supernatant, and add 30% 1,2-propanediol to obtain Control Example 4.

[0056] Compare with Example 5

[0057] 1. Grind dried licorice into 100-mesh powder, then mix it with water at a mass ratio of 1:7. Extract at 60℃ and ultrasonic power of 350W for 1.5 hours, then extract at microwave power of 450W for 180 seconds. Filter to obtain the extract, and sterilize at 121℃ for 15 minutes.

[0058] 2. Select Bacillus hygroscopicus ZY02-1 and Klebsiella variegata HNYJ-3 and inoculate them into LB medium. Incubate at 30℃ and 180 rpm for 24 h with shaking. The OD600 value is about 2.0, forming a seed culture.

[0059] 3. Prepare a mixed bacterial agent by mixing Bacillus hygroscopicus ZY02-1 and Klebsiella variegata HNYJ-3 seed liquids in a mass ratio of 5:2.

[0060] 4. Inoculate the extract with 1% of the extract and ferment at 30℃ and 150rpm for 8 hours. Then, transfer the extract to a fermentation bag with a liquid volume of 75% and allow it to stand and ferment in a sealed container for 48 hours.

[0061] 5. Centrifuge the fermented solution, take the supernatant, and rotary evaporate it at 60℃ and 60mbar until the density reaches 1.05±0.01g / mL. Add anhydrous ethanol at a volume ratio of 1:1, place at -22℃ for 24h, centrifuge to obtain the supernatant, and add 30% 1,2-propanediol to obtain Control Example 5.

[0062] The flavorings prepared in Examples 1-5 and Comparative Examples 1-5 were diluted 100 times with pure water and sprayed onto the moisture-balanced cigarette tobacco at a rate of 2 g / kg of tobacco. After standing for 3 hours, the tobacco was made into cigarettes, and sensory quality was evaluated. Professional judges assessed the 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, cigarettes with flavorings from Comparative Examples 1-5 were designated CK1-CK5, and cigarettes with flavorings from the examples were designated T.

[0063] As shown in Table 1, the sweet-scented bio-flavoring was evaluated using sensory quality assessment. The results showed that, compared with cigarettes without added flavoring, the addition of flavorings from Control Examples 1-5 could improve cigarette quality, but the improvement was less than that of the examples. The sweet-scented bio-flavoring of this application, when applied to cigarette products, increased sweetness, improved aftertaste, and reduced irritation. Compared with Control Example CK, it significantly improved the aroma quality and aroma quantity of the smoke, improved the aftertaste, and increased the total score by 1.13 points. Compared with flavorings without added osmanthus and without fermentation (CK1), the results are shown in Table 2. The aroma components such as glycyrrhizic acid, isoglycyrrhizin, linalool, bornyl acetate, and citral were significantly increased, and new aroma components such as β-ionone, α-terpineol, and β-caryophyllene were added.

[0064] Table 1. Sensory evaluation results of cigarettes

[0065]

[0066] Table 2. Main aroma components (μg / mL)

[0067]

[0068]

[0069] 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 sweet-smelling bio-flavoring through low-oxygen fermentation, characterized in that, Includes the following steps: (1) Mix dried licorice and dried osmanthus flowers evenly at a mass ratio of 8:1, grind them to 50-100 mesh, add water to make the material-liquid ratio 1:5-1:10, first extract at 60-80℃ and ultrasonic power 350w for 1.5-2h, then extract at microwave power 450w for 180s, filter, obtain the extract, sterilize at 121℃ for 15min, and set aside for use; (2) Bacillus altitudinis ZY02-1 and Klebsiella variicola HNYJ-3 were inoculated into LB medium and cultured at 30℃ and 180 rpm for 24-48 h with shaking. OD 600 Value 1.8-2.0, forming seed liquid; (3) Mix the seed liquids of Bacillus hygroscopicus ZY02-1 and Klebsiella variegata HNYJ-3 at a mass ratio of 5:2 to prepare a mixed bacterial agent; (4) Inoculate the mixed bacterial agent into the extract of step (1) at an inoculation rate of 0.5-3%, ferment at 30℃ and 150rpm for 8-12 hours, then put it into a fermentation bag with a liquid volume of 75%, and let it stand and seal for fermentation for 24-48 hours. (5) Centrifuge the fermentation broth to obtain the supernatant, and rotary evaporate it to a density of 1.05±0.01g / mL at 60℃ and 60-100mbar. Add anhydrous ethanol at a volume ratio of 1:1, place it at -22℃ for 24h, centrifuge to obtain the supernatant, and add 10-30% of 1,2-propanediol to obtain a sweet-scented biological fragrance.

2. The preparation method according to claim 1, characterized in that, In step (1), the particle size is 100 mesh and the material-to-liquid mass ratio is 1:7; the extraction is carried out at 60℃ and ultrasonic power of 350w for 1.5h, and then extracted for 180s at microwave power of 450w.

3. The preparation method according to claim 1, characterized in that, In step (2), Bacillus altitudinis ZY02-1 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on March 20, 2025, with accession number CGMCC No. 33916; Klebsiella variicola HNYJ-3 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on January 18, 2024, with accession number CGMCC No. 29654.

4. The preparation method according to claim 1, characterized in that, In step (4), the inoculum amount of compound microbial agent is 1%; after fermentation at 30℃ and 150rpm for 8 hours, it is allowed to stand for 48 hours.

5. The preparation method according to claim 1, characterized in that, In step (5), the rotary evaporation conditions are 60°C and 60 mbar; finally, 30% 1,2-propanediol is added.

6. A low-oxygen fermentation sweet-scented bio-flavoring prepared by the preparation method according to any one of claims 1-5.

7. The application of the low-oxygen fermentation sweet-scented bio-flavoring according to claim 6 in cigarettes, characterized in that, The flavoring is diluted with water at a ratio of 1:20–1:200 and added to regular or heated cigarettes at a rate of 0.5–1 μL / cigarette, or sprayed evenly at a rate of 2 g / kg of tobacco. After equilibration for 2–4 hours, the cigarettes are rolled.

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