Method for improving processing quality of tobacco sheet by using two strains in combination

Abstract

This invention discloses a method for improving the processing quality of tobacco sheets through the combined use of two bacterial strains, comprising the following steps: preparing two enzyme preparations, namely Bacillus subtilis CX-26 and Bacillus belye DF-08, through steps such as strain activation, seed culture preparation, and scale-up cultivation; adding the CX-26 enzyme preparation to the extraction solution in a specific ratio during the multi-stage extraction stage of tobacco stems or tobacco dust; adding the DF-08 enzyme preparation to the concentrated solution in a specific ratio after the low-temperature concentration stage and before the concentrated solution preparation stage; and then inactivating the enzyme preparations used. The process of this application is simple and efficient, with low processing costs, a short processing cycle, and is safe and reliable. It can degrade various macromolecular components such as pectin, cellulose, and starch during tobacco sheet processing, effectively improving the quality of tobacco sheets. Therefore, it has good application value in the field of cigarette manufacturing technology.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of cigarette raw material production and processing technology, in particular to a method for improving the processing quality of tobacco sheet by using two strains in combination. BACKGROUND

[0002] Tobacco sheet, also known as reconstituted tobacco or homogenized tobacco, is a product of comprehensive utilization and fine processing in tobacco industry production, mainly composed of tobacco dust, fragments, tobacco stems or low-grade tobacco leaves, adhesives and other additives, etc. Tobacco sheet is one of the effective technical means for reducing tar and harm, which on the one hand utilizes the waste materials generated in the process of cigarette industrial production, and on the other hand reduces the content of nicotine and tar to some extent, and reduces and improves the adverse ingredients of tobacco.

[0003] Since tobacco sheet is recombined from tobacco waste materials such as tobacco dust and tobacco stems, the relative content of cell wall materials such as pectin, cellulose, hemicellulose and starch macromolecules is significantly higher than that of general tobacco leaves, while the content of water-soluble total sugar is significantly lower, resulting in an imbalance of sugar and alkali ratio, and thus there is a certain gap in smoke characteristics, taste and aroma compared with traditional modulated tobacco leaves.

[0004] Pectin, cellulose, starch and other substances are the main macromolecular chemical components in tobacco sheet, among which pectin and cellulose are the main substances constituting plant cell walls, accounting for more than 50% of all plant polysaccharides, and even more than 1 / 3 of the total mass in tobacco stems and tobacco sheet, which is the main reason for the sharp pain and unpleasant odor in the throat during the burning process, and has a side effect on the sensory quality of tobacco. The content of starch is closely related to the burning property and taste of tobacco, and incomplete degradation of starch significantly restricts the burning property of tobacco products and produces unpleasant odor, affecting the overall quality of cigarettes. In view of these problems, the use of exogenous microbial preparations or enzyme preparations can accelerate the degradation of pectin, cellulose and starch and other polysaccharide macromolecules in tobacco sheet, so as to generate small molecule substances, which further participate in the Maillard reaction to produce aroma components and aroma precursors, thereby having a wide and practical significance for improving the overall quality of cigarette raw materials and enhancing the industrial value of tobacco sheet. SUMMARY

[0005] The technical problem to be solved by the present application is to provide a method for improving the processing quality of tobacco sheet by using two strains in combination.

[0006] The technical problem to be solved by the present application is solved by the following technical scheme:

[0007] A method for improving the processing quality of tobacco sheet by using two strains in combination, comprising the following steps:

[0008] (1) Bacillus subtilis CX-26 and Bacillus velezensis DF-08 enzyme preparations are respectively prepared through strain activation, seed liquid preparation and expansion culture to obtain bacterial liquid;

[0009] (2) The supernatant after expansion culture in step (1) is collected, and the supernatant is partially filtered through a ceramic membrane and then partially filtered through an ultrafiltration membrane to obtain a crude enzyme liquid, which is then diluted with sterile water at a dilution ratio of 1:9 to obtain three enzyme preparations;

[0010] (3) In the multi-stage extraction stage of tobacco stems or tobacco fines, the Bacillus subtilis CX-26 enzyme preparation is added to the extraction liquid in proportion, and the pH of the extraction liquid is adjusted to 6.0. The enzyme preparation is allowed to act at 40°C for 6 hours. After the low-temperature concentration stage, the Bacillus velezensis DF-08 enzyme preparation is added to the concentrated liquid in proportion before the concentrated liquid preparation stage. The enzyme preparation is allowed to act at a temperature of 40°C for 4 hours, and then the enzyme preparation is inactivated by using the high-temperature environment (80°C) of the drying stage.

[0011] Preferably, the Bacillus subtilis CX-26 enzyme preparation is prepared from a Bacillus subtilis CX-26 strain, which is preserved in the China Center for Type Culture Collection (CCTCC) with a preservation number of CCTCC NO: M2023459.

[0012] Preferably, the Bacillus velezensis DF-08 enzyme preparation is prepared from a Bacillus velezensis DF-08 strain, which is preserved in the China Center for Type Culture Collection (CCTCC) with a preservation number of CCTCC NO: M2023456.

[0013] Preferably, the ceramic membrane has a filtration accuracy of 0.2 μm, an outer diameter of 12 mm, an inner diameter of 8 mm, and a length of 300 mm.

[0014] Preferably, the ultrafiltration membrane has a molecular weight cut-off of 5000 daltons and a filtration area of 0.4 m 2 .

[0015] Preferably, the Bacillus subtilis CX-26 enzyme preparation is added to the extraction liquid in a proportion of 6% of the mass of tobacco stems or tobacco fines, and the Bacillus velezensis DF-08 enzyme preparation is added to the concentrated liquid in a proportion of 5% of the mass of the concentrated liquid.

[0016] Preferably, the high-temperature environment of the drying stage has a temperature of 80°C.

[0017] Application of a composite enzyme preparation in a method for improving the quality of tobacco sheet processing products.

[0018] Preferably, in the tobacco stem or tobacco dust multi-stage extraction phase, the Bacillus subtilis CX-26 enzyme system preparation is applied to the extraction liquid at 6% of the mass proportion of the tobacco stem or tobacco dust, the pH of the extraction liquid is adjusted to 6.0, and the extraction liquid is acted on at 40°C for 6h; after the low-temperature concentration phase and before the concentrated liquid preparation phase, the Bacillus velezensis DF-08 enzyme preparation is added to the concentrated liquid at 5% of the mass proportion of the concentrated liquid, and is acted on at a temperature of 40°C for 4h, and then the enzyme preparation is inactivated by using the high-temperature environment of the sheet drying stage.

[0019] The above technical solutions of the present application have the following beneficial effects:

[0020] The process flow of the present application is simple, efficient, low in processing cost, safe and reliable, can realize the degradation of pectin, cellulose, starch and other macromolecular components in the tobacco sheet processing process, effectively improve the quality of the tobacco sheet and other technical advantages, and thus has good popularization and application value in the field of cigarette manufacturing technology. BRIEF DESCRIPTION OF DRAWINGS

[0021] The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate embodiments of the present application and, together with the description, serve to explain the principles of the present application.

[0022] Figure 1 A microstructure diagram of CX-26.

[0023] Figure 2 A strain gene phylogenetic tree of CX-26.

[0024] Figure 3 A microstructure diagram of DF-08.

[0025] Figure 4 A strain gene phylogenetic tree of DF-08.

[0026] Figure 5 A schematic diagram of the multi-strain combined method. DETAILED DESCRIPTION

[0027] Various exemplary embodiments of the present application will now be described in detail with reference to the accompanying drawings. It should be noted that the relative arrangement, numerical expressions, and values of the components and steps set forth in these embodiments do not limit the scope of the present application unless otherwise specifically stated.

[0028] Example 1 strain preparation

[0029] (1) Bacillus subtilis CX-26:

[0030] Bacillus subtilis CX-26, preserved in China Center for Type Culture Collection (CCTCC), Wuhan University, with the preservation number of CCTCC NO: M 2023459, and the preservation time of April 3, 2023. The microstructure diagram is shown in Figure 1 .

[0031] (2) Bacillus velezensis DF-08, preserved in China Center for Type Culture Collection (CCTCC), Wuhan University, with the preservation number of CCTCC NO: M 2023456, and the preservation time of April 3, 2023. The microstructure diagram is shown in Figure 3 .

[0032] Strain preparation of Example 2

[0033] (1) Strain activation:

[0034] The preserved Bacillus subtilis CX-26 strain was inoculated on NA solid medium by point inoculation method, and cultured at 37°C for 1-2d;

[0035] The Bacillus velezensis DF-08 strain was inoculated on LB solid medium by point inoculation method, and cultured at 37°C for 24h.

[0036] (2) Seed liquid preparation:

[0037] The CX-26 strain cultured in step (1) was scraped and inoculated in NA liquid medium, and cultured in a shaker at 30°C and 180r / min for 24h to obtain the CX-26 seed liquid;

[0038] The DF-08 strain cultured in step (1) was scraped and inoculated in LB liquid medium, and cultured in a shaker at 35°C and 180r / min for 24h to obtain the DF-08 seed liquid.

[0039] (3) Expansion culture:

[0040] 1) Pectinase production induction:

[0041] The Bacillus subtilis CX-26 seed liquid in step (2) was inoculated in a special liquid medium (medium composition: pectin 4g / L, K2HPO40.1g / L, MgSO4·7H2O 0.5g / L, NaNO3 3g / L, FeSO4·7H2O 0.01g / L, congo red 0.4g / L, pH=7.0) according to the inoculation amount of 2%, and cultured in a shaker at 38°C and 165r / min for 72h, at which time the OD 600 of the bacterial liquid was 1.8.

[0042] 2) Induction of amylase production:

[0043] The Bacillus DF08DF-08 seed culture from step 2 was inoculated at an inoculation rate of 2% into a selectively induced amylase-producing liquid medium (medium composition: beef extract 5.0 g / L, peptone 10.0 g / L, NaCl 5.0 g / L, soluble starch 20.0 g / L, pH 7.0–7.2). The medium was cultured at 36°C and 195 r / min on a shaker for 60 h. At this time, the OD600 of the bacterial culture was 1.7.

[0044] (4) Preparation of crude enzyme preparation:

[0045] After the strain in step (3) is expanded and cultured, the supernatant is collected. The supernatant is partially filtered through a ceramic membrane (filtration accuracy 0.2 μm, outer diameter 12 mm, inner diameter 8 mm, length 300 mm) and then partially filtered through an 1812 ultrafiltration membrane (molecular weight cutoff 5000 Daltons, filtration area 0.4 m²) to obtain crude enzyme mother liquor. Then, it is diluted with sterile water at a dilution ratio of 1:9 to obtain the enzyme preparation.

[0046] Example 4: Combination of multiple strains

[0047] During the multi-stage extraction of tobacco stems or tobacco dust, Bacillus subtilis CX-26 enzyme preparation was added to the extract at 6% of the mass ratio of tobacco stems or tobacco dust, the pH of the extract was adjusted to 6.0, and it was reacted at 40℃ for 6 hours. After the low-temperature concentration stage and before the concentrate preparation stage, Bacillus vesiculosus DF-08 enzyme preparation was added to the concentrate at 5% of the mass ratio of the concentrate, and it was reacted at 40℃ for 4 hours. Then, the enzyme preparation was inactivated by the high-temperature environment of the drying stage.

[0048] Effects of using two strains in combination:

[0049] Table 1. Changes in the material content of tobacco sheets before and after treatment.

[0050]

[0051] Sensory quality of tobacco sheets treated with a combination of two strains:

[0052] Table 2 Sensory quality evaluation scores for tobacco sheets treated with a combination of two strains

[0053]

[0054] The processed tobacco sheets have improved aroma quality, with increased aroma quantity and concentration. The off-flavors, irritation, and aftertaste are also improved, and the smoothness of the smoke is significantly enhanced, resulting in a significant improvement in the overall smoking score.

[0055] 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 improving the processing quality of tobacco sheets by using a combination of two bacterial strains, characterized in that, Includes the following steps: (1) The Bacillus subtilis CX-26 with preservation number CCTCC NO: M 2023459 and the Bacillus belys DF-08 enzyme preparation with preservation number CCTCC NO: M 2023456 were prepared into bacterial solutions through strain activation, seed liquid preparation and large-scale culture, respectively. (2) Collect the supernatant after the expansion culture in step (1) respectively, filter the supernatant through a ceramic membrane, and then filter it through an ultrafiltration membrane to obtain crude enzyme mother liquor. Then dilute it with sterile water at a dilution ratio of 1:9 to obtain two enzyme preparations. (3) In the multi-stage extraction stage of tobacco stems or tobacco dust, Bacillus subtilis CX-26 enzyme preparation was added to the extraction solution at 6% of the mass ratio of tobacco stems or tobacco dust, the pH of the extraction solution was adjusted to 6.0, and it was treated at 40℃ for 6 h; after the low temperature concentration stage and before the concentration preparation stage, Bacillus vesiculosus DF-08 enzyme preparation was added to the concentration solution at 5% of the mass ratio of the concentration solution, and it was treated at 40℃ for 4 h, and then the enzyme preparation was inactivated by the 80℃ high temperature environment during the drying stage. Bacillus subtilis CX-26 enzyme preparation was added to the extract at 6% of the mass ratio of tobacco stems or tobacco dust, and Bacillus vesiculosus DF-08 enzyme preparation was added to the concentrate at 5% of the mass ratio of the concentrate.

2. The method according to claim 1, characterized in that, The ceramic membrane has a filtration accuracy of 0.2 μm, an outer diameter of 12 mm, an inner diameter of 8 mm, and a length of 300 mm.

3. The method according to claim 1, characterized in that, The ultrafiltration membrane has a molecular weight cutoff of 5000 Daltons and a filtration area of ​​0.4 m². 2 .

4. The application of the method according to any one of claims 1-3 in improving the processing quality of tobacco sheets.

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