A method for producing a liquor-type tobacco flavoring agent by combining enzymatic treatment with liquid fermentation

Abstract

The application discloses a kind of enzyme processing combination liquid fermentation production brewing type tobacco flavor agent's method, comprising the following steps: S1, cut tobacco is mixed with sorghum powder, after adding distilled water obtains tobacco-sorghum liquid medium;S2, yeast strain is inoculated to YPD culture medium and is cultivated, obtains yeast bacterial liquid;And / or lactobacillus strain is inoculated to MRS culture medium and is cultivated, obtains lactobacillus bacterial liquid;S3, configuration enzyme preparation solution, enzyme includes one or more of amylase, flavourzyme and saccharifying enzyme;S4, enzyme preparation solution is added to tobacco-sorghum liquid culture medium;S5, the yeast bacterial liquid and / or lactobacillus bacterial liquid of S2 is inoculated to the culture medium of S4 and is cultivated, obtains fermentation liquid flavor medium;S6, fermentation liquid flavor medium is freeze-dried, obtains tobacco flavor agent.The brewing type flavor agent of the application is prepared based on microbial fermentation technology, process is simple, condition is mild, cost is low, green environmental protection, improve the economic benefit of low quality cigarette.

Description

Technical Field

[0001] This invention relates to the field of tobacco flavoring technology, and in particular to a method for producing aroma-enhancing tobacco flavorings by combining enzyme treatment with liquid fermentation. Background Technology

[0002] Low-quality tobacco leaves are raw materials that are of poor quality or do not meet standards for production and application. Currently, there is still a large inventory of low-quality tobacco leaves in China. These leaves account for the storage and management costs of enterprises and are generally scrapped or discarded, which is not economically efficient and pollutes the environment. Tobacco flavorings are important tobacco flavorings in cigarette processing, giving cigarettes a fuller aroma. Faced with the diverse needs of cigarette product development and the need to improve the economic efficiency of low-quality tobacco leaves, the development of new flavorings is becoming increasingly important.

[0003] Brewing-aroma tobacco flavoring agents are a new type of flavoring agent that combines the brewing aroma of baijiu (Chinese liquor) with the natural aroma of tobacco. Brewing-aroma flavor is common in Chinese baijiu, and its main flavor substances are a complex flavor composed of many esters, alcohols, acids, and other volatile components. For example, ester compounds can contribute fruity sweetness, higher fatty alcohols can contribute floral and earthy aromas, aldehydes and ketones can contribute fruity and fresh grassy aromas, and acid compounds such as acetic acid and lactic acid can provide a certain sourness, thus bringing a rich sense of layering. Therefore, the experience of brewing-aroma flavor comes from the synergistic effect of these components, which requires complex biochemical reactions to form a unique aroma and taste.

[0004] Therefore, there is an urgent need to develop a new type of tobacco flavoring agent that can meet the requirements. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for producing aroma-enhancing tobacco flavoring agents by combining enzyme treatment with liquid fermentation. This method overcomes the problems of traditional solid-state fermentation, which often requires a long time and results in insufficient flavor. This method produces aroma-enhancing flavoring agents and improves the economic benefits of low-quality cigarettes.

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

[0007] A method for producing aroma-enhancing tobacco flavoring agents by combining enzyme treatment with liquid fermentation includes the following steps:

[0008] S1. Mix tobacco shreds and sorghum powder raw materials, add distilled water and mix evenly. Sterilize the prepared sample at high temperature to obtain tobacco-sorghum liquid culture medium, and sterilize it for later use.

[0009] S2. Inoculate *Saccharomyces cerevisiae* JNC001.002, deposited at the China Center for Type Culture Collection (CCTCC NO: M2024636), into YPD medium and culture in a constant temperature shaker to obtain yeast culture; and / or inoculate *Lactobacillus plantarum* JNC003.001, deposited at the China Center for Type Culture Collection (CCTCC NO: M2024447), into MRS medium and culture in a constant temperature shaker to obtain *Lactobacillus* culture;

[0010] S3. Prepare an enzyme preparation solution, wherein the enzyme is one or two of amylase, flavor enzyme, or saccharifying enzyme;

[0011] S4. Add the enzyme preparation solution from S3 to the tobacco-sorghum liquid culture medium after step S1.

[0012] S5. Inoculate the yeast culture and / or lactobacillus culture from S2 into the culture medium of S4 and culture in a constant temperature shaker to obtain a fermentation liquid flavor culture medium.

[0013] S6. Freeze-dry the fermentation liquid flavor medium from S5 to obtain freeze-dried tobacco flavoring agent.

[0014] Preferably, in the above technical solution, in step S1, the concentration of tobacco shreds is 0.01-0.2 g / ml and the concentration of sorghum powder is 0.01-0.2 g / ml.

[0015] Preferably, in the above technical solution, in step S2, the yeast culture conditions are: constant temperature shaker at 200 r / min for 24-48 h, temperature 30℃; YPD liquid culture medium formula: peptone 10 g / L, yeast extract 5 g / L, glucose 20 g / L, chloramphenicol 0.1 g / L.

[0016] Preferably, in the above technical solution, the culture conditions for Lactobacillus in step S2 are as follows: shaking at 200 r / min for 12–24 h at a constant temperature of 37°C; the MAS liquid culture medium formula is: peptone 10 g / L, beef extract 10 g / L, yeast extract 5 g / L, glucose 20 g / L, sodium citrate 2 g / L, anhydrous sodium acetate 2 g / L, magnesium sulfate 0.1 g / L, manganese sulfate 0.05 g / L, dipotassium hydrogen phosphate 2 g / L, and Tween 80 1 mL / L.

[0017] Preferably, in the above technical solution, in step S3, the enzyme activity of the enzyme preparation solution is 100-1000 u / ml.

[0018] Preferably, in the above technical solution, in step S4, the volume ratio of the enzyme preparation solution to the tobacco-sorghum liquid culture medium in step S1 is 1:10 (v / v).

[0019] Preferably, in the above technical solution, in step S5, the inoculation amount of yeast culture is 0.5%-1.5% (v / v), and in step S5, the inoculation amount of lactobacillus culture is 0.25%-0.75% (v / v).

[0020] Preferably, in the above technical solution, the cultivation conditions in step S6 are: shaking in a constant temperature shaker at 200 r / min for 1 to 10 days at a temperature of 30°C.

[0021] A brewed tobacco flavoring agent, wherein the brewed tobacco flavoring agent is prepared according to the method described above.

[0022] The application of a flavoring-type tobacco flavoring agent in improving cigarette quality, wherein the flavoring agent is added to low-quality cigarettes at a mass ratio of 1:5 to enhance the aroma of the cigarettes.

[0023] The above-described technical solution of the present invention has the following beneficial effects:

[0024] (1) Compared with conventional chemically synthesized flavor enhancers, the brewing-type flavor enhancer obtained by the present invention has a rich flavor and is green and healthy;

[0025] (2) The present invention uses enzyme treatment and fermentation to greatly promote substrate fermentation and shorten the fermentation cycle;

[0026] (3) The addition of yeast and lactobacillus in this invention can increase the content of flavor substances and make the flavor more uniform, improve the flavor of low-quality cigarettes, and enhance the quality and market competitiveness of cigarettes. Attached Figure Description

[0027] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments of the invention and, together with their description, serve to explain the principles of the invention.

[0028] Figure 1 The images show the flavor agent samples of Examples 1-5 and the comparative examples after fermentation and freeze-drying, as presented in this application.

[0029] Figure 2 This is a process flow diagram of the enzyme treatment combined with liquid fermentation to produce a flavoring agent for brewing tobacco, according to the present invention. Detailed Implementation

[0030] Various exemplary embodiments of the present invention will now be described in detail with reference to the accompanying drawings. 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.

[0031] This invention proposes the use of yeast and lactobacillus in synergistic liquid fermentation to provide abundant alcohols and acids. Sorghum, often used as a primary raw material for Chinese baijiu fermentation, can be used together with tobacco as a fermentation substrate. Enzyme treatment can accelerate the degradation of macromolecules in the substrate, such as proteins, starches, and pectins, greatly improving fermentation efficiency. Furthermore, there are currently few reports on the combined effect of these two methods to enhance the flavor of tobacco.

[0032] The equipment used in this embodiment of the invention includes: a ZQZY AF8ES constant temperature shaker incubator (Shanghai Zhichu Instrument Co., Ltd.); a PR2202ZH / E electronic balance (Ohaus Instruments (Changzhou) Co., Ltd.); a SW-CJ-1FD-II clean bench (Suzhou Antai Air Technology Co., Ltd.); a BXM-110VE vertical pressure steam sterilizer (Shanghai Boxun Medical Biological Instrument Co., Ltd.); a K9840 automatic Kjeldahl nitrogen analyzer (Jinan Haineng Instrument Co., Ltd.); a DGG-9240B electric thermostatic drying oven (Shanghai Senxin Experimental Instrument Co., Ltd.); and a gas chromatography-mass spectrometry (GC-MS) system (Shimadzu Corporation, Japan).

[0033] Strain origin: Two strains were obtained through experimental screening. Colony morphology was observed during the culture process, ultimately resulting in strains JNC001.002 and JNC003.001. 16S rDNA analysis confirmed that JNC001.002 is a *Saccharomyces cerevisiae*, named *Saccharomyces cerevisiae* JNC001.002, with accession number CCTCC NO: M2024636, deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, China, on April 7, 2024. JNC003.001 is a *Lactobacillus plantarum*, named *Lactobacillus plantarum* JNC003.001, with accession number CCTCC NO: M2024447, deposited at the CCTCC, Wuhan University, China, on March 11, 2024.

[0034] Amylase, flavor enzyme, and saccharifying enzyme were purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0035] Flavor compound detection method: Accurately weigh 8.0 g of sample into a headspace vial, add 1.0 g NaCl and 1 μL of 118.2 mg / L 2-methyl-3-heptanone as internal standard, and seal quickly. Then expose the solid-phase microextraction fiber head above the headspace vial. Place the headspace vial in a heating mantle at 70°C and extract the odor components using a simultaneous distillation extraction apparatus for 30 min. Subsequently, desorb at the gas chromatography (GC) inlet for 7 min.

[0036] Chromatographic conditions: Gas chromatography column was a DB-WAX capillary column (30 mm × 0.25 mm × 0.25 μm), carrier gas (He) flow rate was 0.8 mL / min, and inlet (split) temperature was 250 °C. Column temperature was set at 40 °C for 3 min, then increased to 90 °C at a rate of 5 °C / min, and then to 250 °C at a rate of 10 °C / min. Mass spectrometry conditions: Electron impact (EI) mode, ion energy 70 eV, detector voltage 350 V, scan range 33–450 m / z, scan frequency 3.00 scans / second. Qualitative and quantitative analyses were performed using the NIST08 mass library and an Agilent ChemWorkstation.

[0037] Protein content determination methods: The nitrogen determination method was used; the anthrone colorimetric method was used to determine the total soluble sugars; and the 3,5-dinitrosalicylic acid method was used to determine the reducing sugars.

[0038] Sensory evaluation form: See Table 1:

[0039] Table 1

[0040]

[0041]

[0042] Example 1

[0043] A method for producing aroma-enhancing tobacco flavoring agents by combining enzyme treatment with liquid fermentation includes the following steps:

[0044] (1) Prepare YPD liquid culture medium:

[0045] Formula: 10 g / L peptone, 5 g / L yeast extract, 20 g / L glucose, 0.1 g / L chloramphenicol, autoclaved at 121°C for 20 minutes before use.

[0046] (2) Prepare MAS liquid culture medium:

[0047] Formula: 10 g / L peptone, 10 g / L beef extract, 5 g / L yeast extract, 20 g / L glucose, 2 g / L sodium citrate, 2 g / L anhydrous sodium acetate, 0.1 g / L magnesium sulfate, 0.05 g / L manganese sulfate, 2 g / L dipotassium hydrogen phosphate, 1 mL / L Tween 80. Autoclave at 121°C for 20 minutes before use.

[0048] (3) Yeast culture:

[0049] The yeast strain was Saccharomyces cerevisiae JNC001.002. The yeast strain streaked on the plate was inoculated into the YPD liquid medium in step (1) and cultured in a shaker at 30°C and 200 r / min for 48 h.

[0050] (4) Lactobacillus culture:

[0051] Lactobacillus plantarum JNC003.001 was used. The yeast cells streaked on the plate were inoculated into the MAS liquid medium in step (2) and cultured in a shaker at 37°C and 200 r / min for 24 h.

[0052] (5) Preparation of liquid culture medium:

[0053] Prepare a liquid culture medium with a tobacco concentration of 0.05 g / ml and a sorghum powder concentration of 0.05 g / ml, and autoclave it at 121℃ for 20 minutes before use.

[0054] (6) Enzyme preparation:

[0055] Prepare an amylase solution with an enzyme activity of 500 u / ml and a flavor enzyme solution with an enzyme activity of 500 u / ml.

[0056] (7) Enzyme culture:

[0057] The enzyme solution and fermentation broth were added to the liquid culture medium in step (5) at a volume ratio of 1:10 (v / v).

[0058] (8) Mixed culture:

[0059] In step (7), 10% (v / v) yeast and 5% (v / v) lactobacillus culture were inoculated into the liquid culture medium and cultured for 7 days in a shaker at 200 r / min at 30°C.

[0060] (9) Flavoring agent preparation:

[0061] The liquid culture medium was freeze-dried to obtain the flavoring agent.

[0062] Application Example 1

[0063] Flavoring agents were added to low-quality cigarettes at a mass ratio of 1:5, rolled into commercial cigarettes, and subjected to sensory evaluation. Five qualified tobacco sensory evaluation personnel were invited to conduct the evaluation, and the average score was taken.

[0064] Example 2

[0065] A method for producing aroma-enhancing tobacco flavoring agents by combining enzyme treatment with liquid fermentation, the specific operation steps are the same as in Example 1, the difference being:

[0066] In step (7), only amylase solution is added.

[0067] Example 3

[0068] A method for producing aroma-enhancing tobacco flavoring agents by combining enzyme treatment with liquid fermentation, the specific operation steps are the same as in Example 1, the difference being:

[0069] In step (7), only the flavor enzyme solution is added.

[0070] Example 4

[0071] A method for producing aroma-enhancing tobacco flavoring agents by combining enzyme treatment with liquid fermentation, the specific operation steps are the same as in Example 1, the difference being:

[0072] Step (7) replace the flavor enzyme with a saccharifying enzyme solution.

[0073] Example 5

[0074] A method for producing aroma-enhancing tobacco flavoring agents by combining enzyme treatment with liquid fermentation, the specific operation steps are the same as in Example 1, the difference being:

[0075] In step (7), only the saccharifying enzyme solution is added.

[0076] Comparative Example 1

[0077] A method for producing aroma-enhancing tobacco flavoring agents by combining enzyme treatment with liquid fermentation, the specific operation steps are the same as in Example 1, the difference being:

[0078] No enzyme solution is added, i.e. steps (6)-(7) are omitted.

[0079] The basic indicators are shown in Table 2 below:

[0080] Table 2

[0081]

[0082] The main aroma components of the flavor enzyme combination are shown in Table 3 below:

[0083] Table 3

[0084]

[0085]

[0086] The main aroma components of the saccharifying enzyme combination are shown in Table 4 below:

[0087] Table 4

[0088]

[0089]

[0090] The sensory evaluation of the flavor enzyme combination is shown in Table 5 below:

[0091] Table 5

[0092]

[0093] The sensory evaluation of the saccharifying enzyme combination is shown in Table 6 below:

[0094] Table 6

[0095]

[0096] The above results indicate that enzyme treatment can effectively degrade carbohydrates in the substrate, providing energy for the growth and reproduction of microorganisms, which is conducive to rapid fermentation and the production of more flavor compounds. The relatively small change in protein content may be due to the fact that the protein produced by the growth and reproduction of microorganisms replaces the protein content of the substrate itself, thus the measured protein content does not change much.

[0097] GC-MS results showed that the content of flavor compounds such as esters, alcohols, aldehydes, ketones, acids, and long-chain alkanes in the examples was significantly increased. Esters such as phenethyl acetate, phenethyl propionate, and dodecyl acetate had a rich floral and fruity aroma, alcohols such as isoamyl alcohol and phenethyl alcohol had a rich brewing aroma, and organic acids such as acetic acid and lactic acid provided a certain sourness, increasing the richness. In addition, substances that are inherent to tobacco aroma, such as megalotrienone, guaiacol, and neophytadiene, were also significantly increased.

[0098] Sensory evaluation results also showed that the enzyme-treated samples scored higher.

[0099] In this embodiment, yeast and lactobacillus are used as fermentation strains for mixed fermentation of tobacco and sorghum. Based on the technology of mixed-strain synergistic enzyme treatment fermentation and metabolic control, the resulting flavoring agent has a rich aroma with a dual flavor of brewing aroma and tobacco aroma. It has a significant aroma-enhancing effect, strong smokeability, and significantly improves the economic value of low-quality cigarettes.

[0100] The method of producing aroma-enhancing tobacco flavoring agents by combining enzyme treatment with liquid fermentation of the present invention can efficiently obtain flavoring agents with rich and uniformly distributed flavor substances, and has broad application prospects.

[0101] 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 producing aroma-enhancing tobacco flavoring agents through enzyme treatment combined with liquid fermentation, characterized in that, Includes the following steps: S1. Mix tobacco shreds and sorghum powder raw materials, add distilled water and mix evenly. Sterilize the prepared sample at high temperature to obtain tobacco-sorghum liquid culture medium, and sterilize it for later use. S2. Inoculate *Saccharomyces cerevisiae* JNC001.002, deposited at the China Center for Type Culture Collection (CCTCC NO: M2024636), into YPD medium and culture in a constant temperature shaker to obtain yeast culture; and / or inoculate *Lactobacillus plantarum* JNC003.001, deposited at the China Center for Type Culture Collection (CCTCC NO: M2024447), into MRS medium and culture in a constant temperature shaker to obtain *Lactobacillus* culture; S3. Prepare an enzyme preparation solution, wherein the enzyme is one or two of amylase, flavor enzyme, or saccharifying enzyme; S4. Add the enzyme preparation solution from S3 to the tobacco-sorghum liquid culture medium after step S1. S5. Inoculate the yeast culture and / or lactobacillus culture from S2 into the culture medium of S4 and culture in a constant temperature shaker to obtain a fermentation liquid flavor culture medium. S6. Freeze-dry the fermentation liquid flavor medium from S5 to obtain freeze-dried tobacco flavoring agent.

2. The method for producing aroma-enhancing tobacco flavoring agents by enzyme treatment combined with liquid fermentation according to claim 1, characterized in that, In step S1, the concentration of tobacco shreds is 0.01–0.2 g / ml and the concentration of sorghum flour is 0.01–0.2 g / ml.

3. The method for producing aroma-enhancing tobacco flavoring agents by combining enzyme treatment with liquid fermentation according to claim 1, characterized in that, In step S2, the culture conditions for Saccharomyces cerevisiae JNC001.002 are as follows: constant temperature shaker at 200 r / min for 24–48 h at 30℃; YPD liquid culture medium formula: peptone 10 g / L, yeast extract 5 g / L, glucose 20 g / L, chloramphenicol 0.1 g / L.

4. The method for producing aroma-enhancing tobacco flavoring agents by combining enzyme treatment with liquid fermentation according to claim 1, characterized in that, In step S2, the culture conditions for Lactobacillus are as follows: shake at 200 r / min for 12–24 h at a constant temperature of 37°C; the MAS liquid culture medium formula is: peptone 10 g / L, beef extract 10 g / L, yeast extract 5 g / L, glucose 20 g / L, sodium citrate 2 g / L, anhydrous sodium acetate 2 g / L, magnesium sulfate 0.1 g / L, manganese sulfate 0.05 g / L, dipotassium hydrogen phosphate 2 g / L, and Tween 80 1 mL / L.

5. The method for producing aroma-enhancing tobacco flavoring agents by combining enzyme treatment with liquid fermentation according to claim 1, characterized in that, In step S3, the enzyme activity of the enzyme preparation solution is 100-1000 u / ml.

6. The method for producing aroma-enhancing tobacco flavoring agents by combining enzyme treatment with liquid fermentation according to claim 1, characterized in that, In step S4, the volume ratio of the enzyme preparation solution to the tobacco-sorghum liquid culture medium in step S1 is 1:10 (v / v).

7. The method for producing aroma-enhancing tobacco flavoring agents by combining enzyme treatment with liquid fermentation according to claim 1, characterized in that, In step S5, the inoculation amount of yeast culture is 0.5%-1.5% (v / v), and / or in step S5, the inoculation amount of lactobacillus culture is 0.25%-0.75% (v / v).

8. The method for producing aroma-type tobacco flavoring agents by combining enzyme treatment with liquid fermentation according to claim 1, characterized in that, In step S6, the cultivation conditions are as follows: the constant temperature shaker is used to shake at 200 r / min for 1 to 10 days at a temperature of 30℃.

9. A brewing-type tobacco flavoring agent, characterized in that, The aroma-enhancing tobacco flavoring agent is prepared according to any one of claims 1-8.

10. The application of the aroma-enhancing tobacco flavoring agent according to claim 9 in improving cigarette quality, characterized in that, The application involves adding flavoring agents to low-quality cigarettes at a mass ratio of 1:5 to enhance the aroma of the cigarettes.

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