Baking flavor type biological spice prepared by utilizing facultative anaerobic bacteria and application of baking flavor type biological spice in cigarettes

Abstract

The invention discloses a baking flavor type biological spice prepared by facultative anaerobic bacteria and application of the baking flavor type biological spice in cigarettes. A preparation method of the baking flavor type biological spice comprises the following steps: grinding peas into powder, mixing with vanilla pod seeds in a ratio of 8: 1, baking at 180-200 DEG C for 20-30 minutes, mixing with water in a ratio of 1: 5-1: 10, and carrying out ultrasonic and microwave extraction and sterilization; culturing bacillus altitudinis ZY02-1 and bacillus cereus ZY51 into a seed solution, mixing the seed solution according to a ratio of 3: 1, inoculating the seed solution into the extracting solution according to an inoculum size of 2-6%, carrying out oscillation fermentation, and carrying out sealed standing fermentation; and finally, centrifuging, carrying out rotary evaporation, adding ethanol, refrigerating, centrifuging, and adding 10-30% propylene glycol to obtain the perfume. The baking aroma type biological spice prepared by utilizing facultative anaerobic bacteria is richer in aroma component, and can remarkably improve the aroma quality and the aroma amount of smoke, reduce offensive odor and irritation, improve aftertaste and improve the quality of cigarettes after being added into the cigarettes.

Description

Technical Field

[0001] This invention relates to the field of tobacco technology, and in particular to a roasting-flavored bio-flavoring prepared using facultative anaerobic bacteria and its application in cigarettes. Background Technology

[0002] The aroma of cigarette smoke is an important indicator for evaluating the quality of cigarette products. High-quality traditional 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 terms of cigarette manufacturing processes, technologies such as adding tobacco sheets, expanding tobacco stems, and perforating cigarette paper have been widely adopted. While this has reduced the tar content of traditional cigarettes, it has also reduced the amount of aroma transported in the smoke, resulting in insufficient aroma. Heated cigarettes, due to their low-temperature smoking characteristics, generally suffer from insufficient aroma richness and a lack of pronounced natural tobacco aroma. Therefore, in existing technologies, adding flavorings and fragrances to compensate for the loss of aroma caused by harm reduction and tar reduction is a commonly used technical means.

[0003] Currently, most of the tobacco flavorings added to tobacco in my country are obtained by extraction and blending using traditional methods. Although the production process is simple, it also has drawbacks such as monotonous aroma and insignificant improvement in cigarette smoking effect.

[0004] Fermenting raw materials with microorganisms can increase the content of various aroma substances in traditionally extracted flavorings, enhance the richness of aroma, and thus produce cigarettes with rich aroma and high quality, better meeting consumers' requirements for cigarette quality. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a roasted aroma bioflavoring prepared by facultative anaerobic bacteria and its application in cigarettes. The roasted aroma bioflavoring has a unique roasted pea aroma accompanied by cream, caramel and sweet aroma. When added to cigarettes, it can effectively improve the quality of smoke, increase the aroma quality and aroma quantity of smoke, make the smoke aroma rich and clean aftertaste.

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

[0007] A method for preparing a baking-flavored bio-flavoring using facultative anaerobic bacteria includes the following steps:

[0008] (1) Grind the peas into pea flour of 50-80 mesh, remove the vanilla pod seeds from the vanilla pod, mix the pea flour and vanilla pod seeds at a mass ratio of 8:1, and bake at 180-200℃ for 20-30 min; mix the baked mixture with water at a mass ratio of 1:5-1:10, extract at 60-80℃ with ultrasonic power of 350w for 1.5-2 h, and then extract at microwave power of 450w for 180s, filter, obtain the extract, sterilize at 121℃ for 15 min, and set aside for use;

[0009] (2) Select Bacillus cereus ZY02-1 and Bacillus cereus ZY51 and inoculate them into LB liquid medium, and culture at 33℃ and 180 rpm for 24-48 h with shaking, until OD is reached. 600 The values ​​ranged from 1.8 to 2.0, and seed cultures of the two strains were obtained respectively.

[0010] (3) Mix the seed liquid of Bacillus cereus ZY02-1 and the seed liquid of Bacillus cereus ZY51 at a mass ratio of 3:1 to obtain a composite seed liquid; inoculate the composite seed liquid into the extract of step (1) at an inoculation amount of 2-6%, and culture it under shaking conditions of 28-40℃ and 120-200rpm for 24-48h to obtain the fermentation broth;

[0011] (4) Transfer the fermentation liquid into a sterile fermentation bag, retain 20% of the air volume in the fermentation bag, and seal and let it ferment at 28-40℃ for 48-96 hours.

[0012] (5) Centrifuge the solution after fermentation in step (4), take the supernatant, and rotary evaporate it at 60℃ and 60-100mbar until the density is 1.05±0.01g / mL. Add anhydrous ethanol to the evaporated liquid at a volume ratio of 1:1 and place it at -22℃ for 24h. Centrifuge again to obtain the supernatant, and add 10-30% propylene glycol by mass to the supernatant to obtain the baking-flavored biological fragrance.

[0013] Preferably, in the above technical solution, in step (1), the pea flour has a mesh size of 80, the baking temperature is 180℃, the baking time is 25min, the mass ratio of the mixture to water after baking is 1:7, the ultrasonic extraction temperature is 60℃, and the ultrasonic extraction time is 1.5h.

[0014] Preferably, in the above technical solution, in step (2), Bacillus altitudinis ZY02-1 is deposited at the China General Microbiological Culture Collection Center with accession number CGMCC No. 33916; Bacillus cereus ZY51 is deposited at the China General Microbiological Culture Collection Center with accession number CGMCC No. 33917.

[0015] Preferably, in the above technical solution, in step (2), the shaking culture time is 24 hours, and the bacterial OD... 600 The value is 2.0.

[0016] Preferably, in the above technical solution, in step (3), the inoculation amount of the composite seed liquid is 3%, the shaking culture temperature is 35℃, the shaking culture speed is 150rpm, and the shaking culture time is 48h.

[0017] Preferably, in the above technical solution, in step (4), the temperature for sealed static fermentation is 35°C and the fermentation time is 72h.

[0018] Preferably, in the above technical solution, in step (5), the rotary evaporation pressure is 70 mbar and the mass fraction of propylene glycol added is 30%.

[0019] A baking-flavored bio-fragrance prepared according to the above method.

[0020] The application of a roasted aroma bioflavoring in cigarettes is as follows: the roasted aroma bioflavoring is diluted with water at a mass ratio of 1:(20-200) and added to conventional or heated cigarettes at a rate of 0.5-1 μL / cigarette; or it is sprayed onto tobacco at a rate of 2 g / kg of tobacco shreds, left for 2-4 hours, and then rolled into cigarettes.

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

[0022] This invention relates to a roasted aroma-type bio-flavoring prepared by fermentation with facultative anaerobic bacteria. First, a mixture of pea flour and vanilla pod seeds is roasted, allowing various aroma components to interact during the roasting process. Then, the mixture is extracted, and the extract is subjected to low-oxygen fermentation using a facultative anaerobic compound bacterial agent. This process enriches the aroma components, including alcohols, aldehydes, ketones, and pyrazines. Results show that compared to unfermented flavorings, the aroma components such as vanillin, p-hydroxyphenylethanol, ethyl vanillin, limonene, benzaldehyde, phenylacetaldehyde, 2-acetylfuran, and furfural are significantly increased. This product contains high levels of flavoring agents, including stigmasterone, 2,3-dimethylpyrazine, 2-ethyl-3,5-dimethylpyrazine, 2,3,5-trimethylpyrazine, phenethyl phenylacetate, acetylpyrrole, 2,2'-isopropylidene di(5-methylfuran), and 2,3,6-trimethyl-1,4-naphthyldione. This roasted flavoring has a unique roasted pea aroma accompanied by creamy, caramel, and sweet notes. When added to cigarettes, it effectively improves smoke quality, enhancing the aroma quality and intensity, resulting in a richer smoke aroma and a cleaner aftertaste. 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] 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.

[0025] 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.

[0026] 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.

[0027] A baking-flavored bio-flavoring prepared using facultative anaerobic bacteria includes the following steps:

[0028] 1. Grind peas into 50-80 mesh pea flour. Remove vanilla pod seeds from the vanilla pod. Mix pea flour and vanilla pod seeds at an 8:1 ratio and bake at 180-200℃ for 20-30 minutes. Mix the baked mixture with water at a mass ratio of 1:5-1:10 and extract at 60-80℃ with ultrasonic power of 350W for 1.5-2 hours. Then extract with microwave power of 450W for 180 seconds. Filter to obtain the extract and sterilize at 121℃ for 15 minutes.

[0029] 2. Select Bacillus cereus ZY02-1 and Bacillus cereus ZY51 and inoculate them into LB liquid medium. Incubate at 33℃ and 180 rpm for 24-48 h with shaking. The OD600 value is about 1.8-2.0, forming a seed culture.

[0030] 3. The seed cultures of Bacillus cereus ZY02-1 and Bacillus cereus ZY51 were mixed at a mass ratio of 3:1 and inoculated into the extract at an inoculation amount of 2-6%. The mixture was cultured at 28-40℃ and 120-200rpm for 24-48 hours with shaking to form fermentation broth 1.

[0031] 4. Transfer the fermentation liquid 1 into a sterile fermentation bag, retain 20% air volume, and seal and let it ferment at 28-40℃ for 48-96 hours.

[0032] 5. Centrifuge the fermented solution, take the supernatant, and rotary evaporate it at 60℃ and 60-100mbar until the density is 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% propylene glycol by mass to obtain the baking-flavored biological fragrance.

[0033] Applications of baking-grade bio-flavorings:

[0034] The obtained roasted aroma bio-flavoring is diluted with water at a mass 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 rate of 2g / kg, left to stand for 2-4 hours, and then rolled into cigarettes.

[0035] Example

[0036] 1. Grind peas into 80-mesh pea flour. Remove vanilla pod seeds from the vanilla pod. Mix pea flour and vanilla pod seeds at an 8:1 ratio and bake at 180℃ for 25 minutes. Mix the baked mixture with water at a mass ratio of 1:7 and extract at 60℃ with ultrasonic power of 350W for 1.5 hours. Then extract with microwave power of 450W for 180 seconds. Filter to obtain the extract and sterilize at 121℃ for 15 minutes.

[0037] 2. Select *Bacillus cereus* ZY02-1 and *Bacillus cereus* ZY51 and inoculate them into LB liquid medium. Incubate at 33℃ and 180 rpm for 24 hours with shaking. OD 600 The value is approximately 2.0, forming seed liquid.

[0038] 3. The seed cultures of Bacillus cereus ZY02-1 and Bacillus cereus ZY51 were mixed at a mass ratio of 3:1 and inoculated into the extract at an inoculation amount of 3%. The mixture was cultured at 35℃ and 150 rpm for 48 hours with shaking to form fermentation broth 1.

[0039] 4. Transfer fermentation liquid 1 into a sterile fermentation bag, retain 20% air volume, and seal and let it ferment at 35℃ for 72 hours.

[0040] 5. Centrifuge the fermented solution, take the supernatant, and rotary evaporate it at 60℃ and 70mbar until the density is 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% propylene glycol to obtain the baking-flavored biological fragrance.

[0041] Compare with Example 1

[0042] 1. Grind peas into 80-mesh pea flour and bake at 180℃ for 25 minutes. Mix the baked pea flour with water at a ratio of 1:7, extract at 60℃ with ultrasonic power of 350W for 1.5 hours, and then extract with microwave power of 450W for 180 seconds. Filter to obtain the extract, and sterilize at 121℃ for 15 minutes.

[0043] 2. Centrifuge the solution and collect the supernatant. Evaporate the supernatant at 60℃ and 70mbar until the density is 1.05±0.01g / mL. Add anhydrous ethanol at a volume ratio of 1:1 and place at -22℃ for 24h. Centrifuge to obtain the supernatant and add 30% propylene glycol to obtain the fragrance CK1.

[0044] Compare with Example 2

[0045] 1. Grind peas into 80-mesh pea flour. Remove vanilla pod seeds from the vanilla pod. Mix pea flour and vanilla pod seeds at an 8:1 ratio and bake at 180℃ for 25 minutes. Mix the baked mixture with water at a mass ratio of 1:7 and extract at 60℃ with ultrasonic power of 350W for 1.5 hours. Then extract with microwave power of 450W for 180 seconds. Filter to obtain the extract and sterilize at 121℃ for 15 minutes.

[0046] 2. Centrifuge the solution and collect the supernatant. Evaporate the supernatant at 60℃ and 70mbar until the density is 1.05±0.01g / mL. Add anhydrous ethanol at a volume ratio of 1:1 and place at -22℃ for 24h. Centrifuge to obtain the supernatant and add 30% propylene glycol to obtain the fragrance CK2.

[0047] Compare with Example 3

[0048] 1. Grind peas into 80-mesh pea flour. Remove vanilla pod seeds from the vanilla pod. Mix pea flour and vanilla pod seeds at an 8:1 ratio and bake at 180℃ for 25 minutes. Mix the baked mixture with water at a mass ratio of 1:7 and extract at 60℃ with ultrasonic power of 350W for 1.5 hours. Then extract with microwave power of 450W for 180 seconds. Filter to obtain the extract and sterilize at 121℃ for 15 minutes.

[0049] 2. Select *Bacillus hygroscopicus* ZY02-1 and inoculate it into LB liquid medium. Incubate at 33℃ and 180 rpm for 24 hours with shaking. OD 600 The value is approximately 2.0, forming seed liquid.

[0050] 3. Inoculate Bacillus hygroscopicus ZY02-1 into the extract at an inoculum size of 3%, and culture at 35℃ and 150 rpm for 48 hours with shaking to form fermentation broth 1.

[0051] 4. Transfer fermentation liquid 1 into a sterile fermentation bag, retain 20% air volume, and seal and let it ferment at 35℃ for 72 hours.

[0052] 5. Centrifuge the fermented solution, take the supernatant, and rotary evaporate it at 60℃ and 70mbar until the density is 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% propylene glycol to obtain CK3.

[0053] Compare with Example 4

[0054] 1. Grind peas into 80-mesh pea flour. Remove vanilla pod seeds from the vanilla pod. Mix pea flour and vanilla pod seeds at an 8:1 ratio and bake at 180℃ for 25 minutes. Mix the baked mixture with water at a mass ratio of 1:7 and extract at 60℃ with ultrasonic power of 350W for 1.5 hours. Then extract with microwave power of 450W for 180 seconds. Filter to obtain the extract and sterilize at 121℃ for 15 minutes.

[0055] 2. Select *Bacillus hygroscopicus* ZY02-1 and inoculate it into LB liquid medium. Incubate at 33℃ and 180 rpm for 24 hours with shaking. OD 600 The value is approximately 2.0, forming seed liquid.

[0056] 3. Bacillus cereus ZY51 was inoculated into the extract at an inoculum size of 3%, and cultured with shaking at 35℃ and 150 rpm for 48 hours to form fermentation broth 1.

[0057] 4. Transfer fermentation liquid 1 into a sterile fermentation bag, retain 20% air volume, and seal and let it ferment at 35℃ for 72 hours.

[0058] 5. Centrifuge the fermented solution, take the supernatant, and rotary evaporate it at 60℃ and 70mbar until the density is 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% propylene glycol to obtain CK4.

[0059] The flavorings prepared in Examples 1-4 and Comparative Examples 1-4 were diluted 100 times with pure water and then 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 as CK.

[0060] As shown in Table 1, the roasted aroma bio-flavoring was evaluated using a sensory quality evaluation method. The results showed that compared with cigarettes without added flavoring, the addition of flavorings from Control Examples 1-4 could improve the quality of cigarettes, but the improvement was less than that of the examples. The roasted aroma bio-flavoring of this application, when applied to cigarette products, can make the smoke richer and more abundant, improve the aftertaste, and reduce irritation. Compared with Control Example CK, it significantly improved the aroma quality, aroma quantity, concentration, fineness, and aftertaste of the smoke, and the total score was increased by 2.74 points. Compared with the non-fermented flavoring (CK2), the results are shown in Table 2. The aroma components such as vanillin, p-hydroxyphenylethanol, ethyl vanillin, limonene, benzaldehyde, phenylacetaldehyde, 2-acetylfuran, and furfural were significantly increased, and the aroma components such as megalotrienone, 2,3-dimethylpyrazine, 2-ethyl-3,5-dimethylpyrazine, 2,3,5-trimethylpyrazine, phenethyl phenylacetate, acetylpyrrole, 2,2'-isopropylidene di(5-methylfuran), and 2,3,6-trimethyl-1,4-naphthyldione were newly added.

[0061] Table 1. Sensory evaluation results of cigarettes

[0062]

[0063]

[0064] Table 2. Main aroma components (μg / mL)

[0065] Aroma components CK2 T Vanillin 236.7 389.6 p-Hydroxyphenylethanol 66.7 84.2 Ethyl vanillin 71.6 91.3 benzaldehyde 103.9 121.5 phenylacetaldehyde 113.5 126.7 2-Acetylfuran 59.2 67.3 furfural 64.7 79.8 Megastigmatrienone 35.4 2,3-Dimethylpyrazine 100.7 2-Ethyl-3,5-dimethylpyrazine 84.6 2,3,5-Trimethylpyrazine 89.2 Phenylacetyl phenylacetate 55.1 2,2'-Isopropylidene di(5-methylfuran) 48.9 Acetylpyrrole 23.4 2,3,6-Trimethyl-1,4-Naphthyldione 35.1

[0066] 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 baking-flavored bio-flavoring using facultative anaerobic bacteria, characterized in that, Includes the following steps: (1) Grind the peas into pea flour of 50-80 mesh, remove the vanilla pod seeds from the vanilla pod, mix the pea flour and vanilla pod seeds at a mass ratio of 8:1, and bake at 180-200℃ for 20-30 min; mix the baked mixture with water at a mass ratio of 1:5-1:10, extract at 60-80℃ with ultrasonic power of 350w for 1.5-2 h, and then extract at microwave power of 450w for 180s, filter, obtain the extract, sterilize at 121℃ for 15 min, and set aside for use; (2) Select Bacillus cereus ZY02-1 and Bacillus cereus ZY51 and inoculate them into LB liquid medium, and culture at 33℃ and 180 rpm for 24-48 h with shaking, until OD is reached. 600 The values ​​ranged from 1.8 to 2.0, and seed cultures of the two strains were obtained respectively. (3) Mix the seed liquid of Bacillus cereus ZY02-1 and the seed liquid of Bacillus cereus ZY51 at a mass ratio of 3:1 to obtain a composite seed liquid; inoculate the composite seed liquid into the extract of step (1) at an inoculation amount of 2-6%, and culture it under shaking conditions of 28-40℃ and 120-200rpm for 24-48h to obtain the fermentation broth; (4) Transfer the fermentation liquid into a sterile fermentation bag, retain 20% of the air volume in the fermentation bag, and seal and let it ferment at 28-40℃ for 48-96 hours. (5) Centrifuge the solution after fermentation in step (4), take the supernatant, and rotary evaporate it at 60℃ and 60-100mbar until the density is 1.05±0.01g / mL. Add anhydrous ethanol to the evaporated liquid at a volume ratio of 1:1 and place it at -22℃ for 24h. Centrifuge again to obtain the supernatant, and add 10-30% propylene glycol by mass to the supernatant to obtain the baking-flavored biological fragrance.

2. The method according to claim 1, characterized in that, In step (1), the pea flour has a mesh size of 80, the baking temperature is 180℃, the baking time is 25min, the mass ratio of the mixture to water after baking is 1:7, the ultrasonic extraction temperature is 60℃, and the ultrasonic extraction time is 1.5h.

3. The method according to claim 1, characterized in that, In step (2), Bacillus saltitudinis ZY02-1 was deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33916; Bacillus cereus ZY51 was deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33917.

4. The method according to claim 1, characterized in that, In step (2), the shaking culture time is 24 hours, and the OD of the bacterial culture is... 600 The value is 2.

0.

5. The method according to claim 1, characterized in that, In step (3), the inoculation amount of the composite seed liquid is 3%, the shaking culture temperature is 35℃, the shaking culture speed is 150rpm, and the shaking culture time is 48h.

6. The method according to claim 1, characterized in that, In step (4), the sealed static fermentation temperature is 35℃ and the fermentation time is 72h.

7. The method according to claim 1, characterized in that, In step (5), the rotary evaporation pressure is 70 mbar and the mass fraction of propylene glycol added is 30%.

8. A roasted aroma bio-flavoring prepared by the method according to any one of claims 1-7.

9. The application of the roasted aroma-type bio-flavoring according to claim 8 in cigarettes, characterized in that, The application method is as follows: dilute the roasted aroma bio-flavoring with water at a mass ratio of 1:(20-200), and add it to regular cigarettes or heated cigarettes at a rate of 0.5-1μL / cigarette; or spray it onto the tobacco at a rate of 2g / kg of tobacco, and roll it into cigarettes after 2-4 hours.

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