Preparation method of fermented fig extract and application thereof in cigarettes

Abstract

The application discloses a preparation method of fermented fig extract and application of the fermented fig extract in cigarettes. The preparation method comprises the following steps: grinding and crushing dried figs, mixing and heating, sterilizing to obtain a fig solution; inoculating Pseudomonas paracitrea ZY-03, Enterobacter HNYJ-04, Saccharomyces cerevisiae and Gluconacetobacter ZT-01 into a culture medium to culture a seed liquid, inoculating the seed liquid into the fig solution to perform fermentation; mixing the fermentation liquor with an organic reagent to perform reflux extraction; finally, adding ethanol and propylene glycol to obtain the fermented fig extract with improved cigarette aroma quality and aftertaste. The specific microorganism combination is used to degrade coumarin in the figs into dihydrocoumarin, and to degrade potential aroma substances such as flavones in the figs, so that more aroma components are released, and the quality of the fig extract is improved.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a method for preparing fermented fig extract and its application in cigarettes. Background Technology

[0002] Figs are a plant with both medicinal and edible value, containing abundant polysaccharides, flavonoids, phenolic compounds, coumarins, anthocyanins, and more. Fig extract is an important flavoring agent for tobacco, imparting a sweet fruity aroma to cigarettes, improving the taste, reducing irritation, enriching the smoke, and harmonizing with the cigarette's fragrance.

[0003] Fig extract for tobacco is typically obtained through solvent extraction to obtain a crude extract, which is then selectively concentrated by precipitation separation to remove precipitates or large molecular components. However, fig extract has a complex composition, and simple processing often fails to fully release its aroma precursors and flavor characteristics, limiting its applications. Coumarins are natural phytoalexins from figs, possessing aromatic properties. Adding coumarins to tobacco can enhance the natural aroma of tobacco leaves, making the flavor more delicate and rich. However, due to being classified as carcinogenic in the 1950s, their use in the food and cosmetics industries is now limited. Nevertheless, the degradation of coumarins into dihydrocoumarins can be added to tobacco for flavor enhancement. In addition, flavonoids and anthocyanins in figs are active ingredients and also belong to polyphenols, which are important latent aroma substances. They have little or no aroma on their own, but they can release aroma components during the decomposition or degradation process when heated. Therefore, it is necessary to further study the degradation of coumarin in figs into dihydrocoumarin and the degradation of latent aroma substances to improve the quality of fig extracts. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a method for preparing fermented fig extract and its application in cigarettes. When the extract is applied to cigarette products, it can increase the aroma and permeability of the smoke and improve the aftertaste.

[0005] The technical problem to be solved by the present invention is achieved through the following technical solution:

[0006] A method for preparing fermented fig extract includes the following steps:

[0007] (1) Grind the dried figs into 40-100 mesh dry powder, mix the dry powder with water at a mass ratio of 1:(2-15), heat at 60℃ for 90 minutes, filter, and sterilize;

[0008] (2) Pseudomonas paraxanthizobium ZY-03, Enterobacter HNYJ-4, and Saccharomyces cerevisiae were inoculated into LB medium at 30℃ and fermented at 150 r / min for 24-48 h to prepare OD.600 Seed solution with a value of 1.2-2.0;

[0009] (3) Gram-Acetobacter ZT-01 was inoculated into HS medium and fermented at 30℃, 120r / min for 24-48h until particles were observed to be produced by the naked eye.

[0010] (4) Inoculate the seed liquid of Pseudomonas paraxanthizobium ZY-03, the seed liquid of Enterobacter HNYJ-4, the seed liquid of Saccharomyces cerevisiae, and the seed liquid of Acetobacter gluconate ZT-01 into the fig solution at a mass ratio of (35-55):(30-40):(15-30):(1-10) and a total inoculum of 1-3%. Ferment at 120-180 r / min for 48-96 h at 25-40℃.

[0011] (5) Mix the fermentation broth with organic reagents at a mass ratio of 1:1-1.5:1. The organic reagents are ethyl acetate, ethanol and n-hexane mixed at a mass ratio of (20-30):(50-65):(5-10). Reflux the mixture at 70-80℃ for 2-4 hours, centrifuge to obtain the supernatant, and rotary evaporate to a density of 1.09±0.01. Add ethanol at a volume ratio of 1:1, place at -22℃ for 24 hours, centrifuge to obtain the supernatant, and add 10%-30% propylene glycol to obtain the fermented fig extract.

[0012] Preferably, in the above technical solution, the *Pseudomonas parafulva* ZY-03 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on February 23, 2022, with accession number CGMCC No. 24422; the *Enterobacter sp.* HNYJ-4 was deposited at the CGMCC on January 18, 2024, with accession number CGMCC No. 29655; the *Gluconacetobacter sp.* ZT-01 was deposited at the CGMCC on December 23, 2020, with accession number CGMCC No. 21544; and the *Saccharomyces cerevisiae* was purchased from the CGMCC with accession number [not provided in the original text]. NO.2.3868.

[0013] Preferably, in the above technical solution, in step (1), the mass ratio of the dried fig powder to water is 1:7, and the sterilization conditions are sterilization at 121℃ for 15 minutes.

[0014] Preferably, in the above technical solution, in step (2), the LB liquid culture medium includes: NaCl 10g / L, peptone 10g / L, and yeast extract 5g / L.

[0015] Preferably, in the above technical solution, in step (3), the HS culture medium includes: glucose 20g / L, peptone 5g / L, yeast powder 5g / L, citric acid monohydrate 1.2g / L, and disodium hydrogen phosphate 4g / L.

[0016] Preferably, in the above technical solution, in step (4), the seed liquid of Pseudomonas paraxanthizobium ZY-03, the seed liquid of Enterobacter HNYJ-4, the seed liquid of Saccharomyces cerevisiae, and the seed liquid of Acetobacter gluconate ZT-01 are mixed in a mass ratio of 10:6:3:1, and the total inoculation amount of 2% is inoculated into the fig solution and fermented at 30°C and 150r / min for 72h.

[0017] Preferably, in the above technical solution, in step (5), the organic reagent is a mixture of ethyl acetate, ethanol and n-hexane in a mass ratio of 30:65:5, and the rotary evaporation conditions are: 60℃, 60-100mbar.

[0018] An application of a fermented fig extract in cigarettes, wherein the fermented fig extract is prepared according to the above preparation method.

[0019] Preferably, in the above technical solution, the application involves diluting fermented fig extract with water at a mass ratio of 1:(50-500), and then adding 0.5-1uL / cigarette to conventional or heated cigarettes; or, the obtained fermented fig extract is diluted with water at a mass ratio of 1:(5000-50000), sprayed onto tobacco at 10-15%, left to stand for 2-6 hours, and then rolled into cigarettes.

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

[0021] This application utilizes a specific microbial combination to degrade coumarin in figs into dihydrocoumarin, while simultaneously degrading latent aroma substances such as flavonoids in figs, in order to release more aroma components and thus improve the quality of fig extract. 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] The strain information required in this application is as follows:

[0024] (1) Pseudomonas parafulva ZY-03 was deposited on February 23, 2022, 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. The accession number is CGMCC No. 24422.

[0025] (2) Enterobacter sp. HNYJ-4 was deposited on January 18, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. Accession number: CGMCC No. 29655.

[0026] (3) Gluconacetobacter sp. ZT-01 was deposited on December 23, 2020, 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. The accession number is CGMCC NO.21544.

[0027] (4) The brewing yeast was purchased from the China General Microbiological Culture Collection Center, with the accession number CGMCC NO.2.3868.

[0028] The dried figs were purchased online.

[0029] Example 1

[0030] (1) Grind the dried figs to 40-100 mesh, mix the dried fig powder with water at a mass ratio of 1:7, heat at 60℃ for 90 minutes, filter, and sterilize at 121℃ for 15 minutes.

[0031] (2) Pseudomonas paraxanthizobium ZY-03, Enterobacter HNYJ-4, and Saccharomyces cerevisiae were inoculated into LB medium at 30℃ and fermented at 150 r / min for 24 h to prepare OD. 600 Seed liquid with a value of approximately 1.9.

[0032] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride.

[0033] (3) Inoculate Gluconobacterium acetate ZT-01 into HS medium, ferment at 30℃ and 120r / min for 48h, and observe the production of particles with the naked eye.

[0034] HS medium: glucose 20 g / L, peptone 5 g / L, yeast extract 5 g / L, citric acid monohydrate 1.2 g / L, disodium hydrogen phosphate 4 g / L.

[0035] (4) The seed liquids of Pseudomonas paraxanthizobium ZY-03, Enterobacter HNYJ-4, Saccharomyces cerevisiae, and Acetobacter gluconate ZT-01 were inoculated into fig solution at a mass ratio of 50:30:15:5 and a total inoculation amount of 2%. Fermentation was carried out at 30℃ and 150r / min for 72h.

[0036] (5) The fermentation broth after fermentation was mixed with organic reagents at a mass ratio of 1:1. The organic reagents were ethyl acetate, ethanol, and n-hexane mixed evenly at a mass ratio of 30:65:5. The mixture was extracted by reflux at 70°C for 4 hours. The supernatant was collected by centrifugation and then evaporated at 60°C and 80 mbar to a density of about 1.1. Ethanol was added at a volume ratio of 1:1. The mixture was placed at -22°C for 24 hours. The supernatant was collected by centrifugation and then 10% propylene glycol was added to obtain the fermented fig extract, denoted as T1.

[0037] Application Example 1

[0038] (1) The fig extract obtained in Example 1 was diluted with water at a mass ratio of 1:5000, sprayed onto tobacco at 10%, left for 6 hours, and rolled into cigarettes.

[0039] Comparative Example 1

[0040] (1) Grind the dried figs to 40-100 mesh, mix the dried fig powder with water at a mass ratio of 1:7, heat at 60℃ for 90 minutes, filter, and sterilize at 121℃ for 15 minutes.

[0041] (2) After sterilization, the solution was mixed with organic reagents at a mass ratio of 1:1. The organic reagents were ethyl acetate, ethanol, and n-hexane mixed at a mass ratio of 30:65:5. The mixture was extracted by reflux at 70°C for 4 hours. The supernatant was collected by centrifugation and then evaporated at 60°C and 80 mbar to a density of about 1.1. Ethanol was added at a volume ratio of 1:1. The mixture was placed at -22°C for 24 hours and then centrifuged to obtain the supernatant. 10% propylene glycol was added to obtain the fig extract, which was denoted as CK1.

[0042] (3) The obtained fig extract was diluted with water at a mass ratio of 1:5000, sprayed onto the tobacco at 10%, left for 6 hours, and rolled into cigarettes.

[0043] Comparative Example 2

[0044] (1) Grind the dried figs to 40-100 mesh, mix the dried fig powder with water at a mass ratio of 1:7, heat at 60℃ for 90 minutes, filter, and sterilize at 121℃ for 15 minutes.

[0045] (2) Gluconobacterium glutamicum ZT-01 was inoculated into HS medium and fermented at 30℃, 120r / min for 48h. Particles were observed to be produced by the naked eye.

[0046] HS medium: glucose 20 g / L, peptone 5 g / L, yeast extract 5 g / L, citric acid monohydrate 1.2 g / L, disodium hydrogen phosphate 4 g / L.

[0047] (3) Inoculate 2% of Gluconobacterium acetate ZT-01 into fig solution and ferment at 30℃ and 150r / min for 72h.

[0048] (4) The fermentation broth after fermentation was mixed with organic reagents at a mass ratio of 1:1. The organic reagents were ethyl acetate, ethanol, and n-hexane mixed evenly at a mass ratio of 30:65:5. The mixture was extracted by reflux at 70°C for 4 hours. The supernatant was collected by centrifugation and then evaporated at 60°C and 80 mbar to a density of about 1.1. Ethanol was added at a volume ratio of 1:1. The mixture was placed at -22°C for 24 hours. The supernatant was collected by centrifugation and then 10% propylene glycol was added to obtain the fermented fig extract, which was denoted as CK2.

[0049] (5) The obtained fig extract was diluted with water at a mass ratio of 1:5000, sprayed onto the tobacco at 10%, left for 6 hours, and rolled into cigarettes.

[0050] Comparative Example 3

[0051] (1) Grind the dried figs to 40-100 mesh, mix the dried fig powder with water at a mass ratio of 1:7, heat at 60℃ for 90 minutes, filter, and sterilize at 121℃ for 15 minutes.

[0052] (2) Saccharomyces cerevisiae was inoculated into LB medium at 30°C and fermented at 150 r / min for 24 h to prepare OD. 600 Seed culture with a pH of approximately 1.9. LB medium: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L.

[0053] (3) Gluconobacterium glutamicum ZT-01 was inoculated into HS medium and fermented at 30℃, 120r / min for 24-48h. Particles were observed to be produced by the naked eye.

[0054] HS medium: glucose 20 g / L, peptone 5 g / L, yeast extract 5 g / L, citric acid monohydrate 1.2 g / L, disodium hydrogen phosphate 4 g / L.

[0055] (4) Saccharomyces cerevisiae ZT-01 was inoculated into fig solution at a mass ratio of 15:5 and a total inoculation amount of 2%, and fermented at 150r / min for 72h at 25-40℃.

[0056] (5) The fermentation broth after fermentation was mixed with organic reagents at a mass ratio of 1:1. The organic reagents were ethyl acetate, ethanol, and n-hexane mixed at a mass ratio of 30:65:5. The mixture was extracted by reflux at 70°C for 4 hours. The supernatant was collected by centrifugation and then evaporated at 60°C and 80 mbar to a density of about 1.1. Ethanol was added at a volume ratio of 1:1. The mixture was placed at -22°C for 24 hours. The supernatant was collected by centrifugation and then 10% propylene glycol was added to obtain the fermented fig extract, which was denoted as CK3.

[0057] (6) The obtained fig extract was diluted with water at a mass ratio of 1:5000, sprayed onto tobacco at 10%, left for 6 hours, and rolled into cigarettes.

[0058] Compare with Example 4

[0059] (1) Grind the dried figs to 40-100 mesh, mix the dried fig powder with water at a mass ratio of 1:7, heat at 60℃ for 90 minutes, filter, and sterilize at 121℃ for 15 minutes.

[0060] (2) Enterobacter HNYJ-4 and Saccharomyces cerevisiae were inoculated into LB medium at 30℃ and fermented at 150 r / min for 24 h to prepare OD. 600 Seed liquid with a value of 1.9.

[0061] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride.

[0062] (3) Gluconobacterium glutamicum ZT-01 was inoculated into HS medium and fermented at 30℃, 120r / min for 48h. Particles were observed to be produced by the naked eye.

[0063] HS medium: glucose 20 g / L, peptone 5 g / L, yeast extract 5 g / L, citric acid monohydrate 1.2 g / L, disodium hydrogen phosphate 4 g / L.

[0064] (4) Enterobacter HNYJ-4, Saccharomyces cerevisiae, and Acetobacter gluconate ZT-01 were inoculated into fig solution at a mass ratio of 30:15:5 and a total inoculum of 2%, and fermented at 30℃ and 150r / min for 72h.

[0065] (5) The fermentation broth after fermentation was mixed with organic reagents at a mass ratio of 1:1. The organic reagents were ethyl acetate, ethanol, and n-hexane mixed at a mass ratio of 30:65:5. The mixture was extracted by reflux at 70°C for 4 hours. The supernatant was collected by centrifugation and then evaporated at 60°C and 80 mbar to a density of about 1.1. Ethanol was added at a volume ratio of 1:1. The mixture was placed at -22°C for 24 hours. The supernatant was collected by centrifugation and then 10% propylene glycol was added to obtain the fermented fig extract, which was designated as CK4.

[0066] (6) The obtained fig extract was diluted with water at a mass ratio of 1:5000, sprayed onto tobacco at 10%, left for 6 hours, and rolled into cigarettes.

[0067] Comparative Example 5

[0068] Cigarettes made from tobacco without the addition of any fig extract are designated CK0.

[0069] result:

[0070] When evaluating the sensory quality of fig extract, 13 indicators are scored, including aroma quality, aroma quantity, permeability, off-odors, concentration, strength, fineness, clumping, irritation, dryness, freshness, sweetness, and aftertaste, and the total score is calculated.

[0071] Table 1 Sensory evaluation results

[0072]

[0073]

[0074] Compared to cigarette CK0, which does not contain any fig extract, several fig extracts improved the sensory qualities of the cigarette, reducing off-flavors and increasing sweetness. CK1 showed improvements in aroma quality, aroma quantity, smoke concentration, and sweetness to some extent, but reduced fineness and clumping. CK2 showed increased transparency and sweetness, but no increase in aroma quality and aroma quantity, and reduced clumping. CK3 and CK4 showed improvements in aroma quality, aroma quantity, off-flavors, and sweetness, but the improvements were smaller compared to the examples. T1 significantly improved the aroma quality, aroma quantity, transparency, off-flavors, concentration, irritation, and sweetness of the smoke, greatly enhancing the sensory quality of the smoke and further improving the quality of the fig extract.

[0075] CK1 is a fig extract prepared without any fermentation treatment, with other steps being the same as in the example. It was analyzed by GC-MS with T1. The contents of coumarin and dihydrocoumarin are shown in Table 2, and the main aroma components are compared in Table 3.

[0076] Compared to CK1, the fig extract fermented with mixed cultures showed a significant decrease in coumarin content and a significant increase in dihydrocoumarin content, indicating that coumarin degraded into dihydrocoumarin during fermentation. The fermented fig extract also showed a significant increase in alcohols, acids, aldehydes, ketones, and esters. This is because, during fermentation, aroma precursors such as flavonoids and pigment glycosides degraded, generating aroma components such as pyranones and furans, enriching the smoke composition and increasing the aroma content. Simultaneously, the degradation of some macromolecules into alcohols and organic acids improved the quality of the fig extract.

[0077] Table 2. Coumarin degradation rate (μg / g)

[0078] sample Coumarin Dihydrocoumarin CK1 135.4 8.6 T1 11.5 85.8

[0079] Table 3. Main aroma components (content μg / g)

[0080]

[0081] In conclusion, the fermented fig extract of this application has significant advantages in improving the sensory quality and aroma components of cigarettes, and has good application prospects.

[0082] 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 fermented fig extract, characterized in that, Includes the following steps: (1) Grind the dried figs into 40-100 mesh dry powder, mix the dry powder with water at a mass ratio of 1:(2-15), heat at 60℃ for 90 minutes, filter, and sterilize; (2) Pseudomonas paraxanthizobium ZY-03, Enterobacter HNYJ-4, and Saccharomyces cerevisiae were inoculated into LB liquid medium at 30℃ and fermented at 150 r / min for 24-48 h to prepare OD. 600 Seed solution with a value of 1.2-2.0; (3) Inoculate Gluconobacterium Gluconate ZT-01 into HS medium and ferment at 30℃ and 120r / min for 24-48h; (4) Inoculate the seed culture of Pseudomonas paraxanthizobium ZY-03, Enterobacter HNYJ-4, Saccharomyces cerevisiae, and Acetobacter gluconate ZT-01 into fig solution at a mass ratio of (35-55):(30-40):(15-30):(1-10) and a total inoculum of 1-3%. Ferment at 120-180 r / min for 48-96 h at 25-40℃. (5) Mix the fermented broth with organic reagents at a mass ratio of 1:1-1.5:

1. The organic reagents are ethyl acetate, ethanol, and n-hexane mixed at a mass ratio of (20-30):(50-65):(5-10). Reflux the mixture at 70-80℃ for 2-4 hours, centrifuge to obtain the supernatant, and rotary evaporate to a density of 1.09±0.

01. Add ethanol at a volume ratio of 1:1, place at -22℃ for 24 hours, centrifuge to obtain the supernatant, and add 10%-30% propylene glycol to obtain the fermented fig extract. The aforementioned Pseudomonas paraxanthii ( Pseudomonas parafulva ZY-03 was deposited on February 23, 2022, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 24422; the Enterobacter (… Enterobacter sp. HNYJ-4, was deposited on January 18, 2024, at the China General Microbiological Culture Collection Center (CGMCC), accession number CGMCC No. 29655; the described *Acetobacter glucosinolates* (HNYJ-4) is... Gluconacetobacter sp. ZT-01, deposited on December 23, 2020 at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC NO.21544; the described brewer's yeast is *Saccharomyces cerevisiae* (Saccharomyces cerevisiae). Saccharomyces cerevisiae Purchased from China General Microbiological Culture Collection Center, accession number CGMCC NO. 2.3868.

2. The method for preparing fermented fig extract according to claim 1, characterized in that, In step (1), the mass ratio of the dried fig powder to water is 1:7, and the sterilization conditions are 121℃ for 15 minutes.

3. The method for preparing fermented fig extract according to claim 1, characterized in that, In step (2), the LB liquid culture medium includes: NaCl 10 g / L, peptone 10 g / L, and yeast extract 5 g / L.

4. The method for preparing fermented fig extract according to claim 1, characterized in that, In step (3), the HS culture medium includes: 20 g / L glucose, 5 g / L peptone, 5 g / L yeast extract, 1.2 g / L citric acid monohydrate, and 4 g / L disodium hydrogen phosphate.

5. The method for preparing fermented fig extract according to claim 1, characterized in that, In step (4), the seed liquid of Pseudomonas paraxanthizobium ZY-03, the seed liquid of Enterobacter HNYJ-4, the seed liquid of Saccharomyces cerevisiae, and the seed liquid of Acetobacter gluconate ZT-01 were mixed in a mass ratio of 10:6:3:1, and the total inoculum amount of 2% was inoculated into the fig solution and fermented at 30℃ and 150r / min for 72h.

6. The method for preparing fermented fig extract according to claim 1, characterized in that, In step (5), the organic reagent is a mixture of ethyl acetate, ethanol and n-hexane in a mass ratio of 30:65:5, and the rotary evaporation conditions are: 60℃, 60-100mbar.

7. The application of a fermented fig extract in cigarettes, characterized in that, The fermented fig extract is prepared by any one of the preparation methods according to claims 1-6.

8. The application according to claim 7, characterized in that, The application involves diluting fermented fig extract with water at a mass ratio of 1:(50-500), and then adding 0.5-1 μL / cigarette to conventional or heated cigarettes; or, diluting the obtained fermented fig extract with water at a mass ratio of 1:(5000-50000), spraying it onto tobacco at 10-15%, letting it stand for 2-6 hours, and then rolling it into cigarettes.

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