A method for improving the processing quality of stems by using multiple strains in combination

Abstract

This invention discloses a method for improving the processing quality of tobacco stems through the combined use of multiple strains, comprising the following steps: Bacillus subtilis CX-26, Priestella megaterium CX17 enzyme preparation, and Bacillus belyssus DF-08 enzyme preparation are prepared through steps such as strain activation, seed culture preparation, and large-scale cultivation to obtain three enzyme preparations. The CX-26 and CX17 enzyme preparations are mixed at a 2:1 mass ratio. During the stem soaking stage, the mixed enzyme preparation is added to the soaking aqueous solution, and the high-temperature environment during the stem steaming stage inactivates the mixed enzyme preparation. After the tobacco stems are added, the DF-08 enzyme preparation is applied to the stems, and the high-temperature environment during the stem drying stage inactivates the enzyme preparation. This application offers advantages such as low tobacco stem processing cost, short processing cycle, safety and reliability, and the ability to degrade various macromolecular components such as pectin, cellulose, protein, and starch during tobacco stem processing, effectively improving tobacco stem quality.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of raw material production and processing of cigarettes, and particularly relates to a method for improving processing quality of cut stem by using multiple strains in combination. BACKGROUND

[0002] Tobacco stem is the coarse and hard vein of tobacco leaf, and about 25% of the total weight of tobacco leaf can be obtained by separating stem and leaf. In the existing cigarette preparation, the application of appropriate content of tobacco stem can not only improve the filling property of tobacco, reduce the consumption of tobacco raw material, but also effectively reduce the release amount of harmful substances such as tar, nicotine and CO in cigarettes, and has become one of the important components in cigarette formula. However, the content of cell wall substances in tobacco stem, including pectin, cellulose, hemicellulose and lignin, is relatively high, while the content of total sugar and nicotine is relatively low, which leads to the imbalance of sugar and alkali ratio, and generates irritating odor such as choking, pungent and burning, and woody odor during smoking, thereby reducing the sensory quality of tobacco. In addition, pectin and other cell wall substances produce harmful substances such as various low aldehydes, polycyclic aromatic hydrocarbons (PAH), catechol and methanol during the combustion and pyrolysis process, which is not conducive to health.

[0003] Protein is the main nitrogen-containing compound in tobacco, which widely exists in the stems and leaves of tobacco and is closely related to the quality of tobacco. The excessive content of protein in tobacco stem will cause the generation of odor similar to burning feathers during smoking, and produce pungent and bitter taste, which seriously affects the overall quality of tobacco stem. In addition, excessive starch in tobacco stem is also not conducive to the quality of smoking, on the one hand, it will affect the speed and completeness of smoking, and on the other hand, starch will produce burnt odor and various harmful components during combustion, which will have adverse effects on the color, aroma and taste of cut stem.

[0004] Therefore, effectively degrading the excessive cellulose, pectin and other macromolecular cell wall substances, and protein and other macromolecular compounds in tobacco stem has important practical significance for improving the internal quality of tobacco stem and its products, and improving the safety of cut stem. SUMMARY

[0005] The technical problem to be solved by the present application is to provide a method for improving the processing quality of cut stem by using multiple strains in combination, and the total score of treated tobacco is obviously improved.

[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 cut stem by using multiple strains in combination, comprising the following steps:

[0008] (1) preparing bacterial liquid by activating, preparing seed liquid and expanding culture of Bacillus subtilis CX-26, Pristinamycetin CX17 enzyme preparation and Bacillus velezensis DF-08 enzyme preparation respectively;

[0009] (2) Collect the supernatant after step (1) of expansion culture, and then dilute the supernatant with sterile water at a dilution ratio of 1:9 to obtain three enzyme preparations;

[0010] (3) The Bacillus subtilis CX-26 enzyme preparation and the Priestia megaterium CX17 enzyme preparation in step (2) are mixed at a mass ratio of 2:1. During the stem soaking stage, the mixed enzyme preparation is added to the stem soaking solution at a tobacco stem mass ratio of 6%, and the mixed enzyme preparation is inactivated at a temperature of 35°C for 4 hours. After the stem soaking stage, the Bacillus velezensis DF-08 enzyme preparation is applied to the stem at a mass ratio of 4% after the stem is cut into strips, and the enzyme preparation is inactivated at a temperature of 37°C and a humidity of 75% for 6 hours. Then, the enzyme preparation is inactivated in a high-temperature environment (200°C-220°C) during the stem drying stage, and then enters the subsequent normal stem processing process.

[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 Priestia megaterium CX17 enzyme preparation is prepared from a Priestia megaterium CX17 strain, which is preserved in the China Center for Type Culture Collection (CCTCC) with a preservation number of CCTCC NO: M2023458.

[0013] 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.

[0014] 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.

[0015] Preferably, the ultrafiltration membrane has a molecular weight cut-off of 5000 daltons and a filtration area of 0.4 m 2 .

[0016] Preferably, the high temperature in the stem steaming stage is 80°C-100°C.

[0017] Preferably, the high temperature environment in the stem drying stage is 200°C-220°C.

[0018] Application of a composite enzyme preparation in improving the processing quality of cut stem.

[0019] Preferably, the process applied is as follows: the prepared Bacillus subtilis CX-26 enzyme preparation is mixed with the Pasteuria megaspermatophora CX17 enzyme preparation according to a mass ratio of 2:1, in the stem soaking stage, the mixed enzyme preparation is added to the stem soaking aqueous solution according to 6% of the mass ratio of tobacco stem, and acts for 4h at a temperature of 35 DEG C, and then the mixed enzyme preparation is inactivated by using a high-temperature environment of 80 DEG C-100 DEG C in the stem steaming stage; after the cut stem is added, the Bacillus velezensis DF-08 enzyme preparation is applied on the cut stem according to 4% of the mass ratio of the cut stem, and is placed for 6h under the condition of a temperature of 37 DEG C and a humidity of 75%, and then the enzyme preparation is inactivated by using a high-temperature environment of 200 DEG C-220 DEG C in the cut stem drying stage, and then enters the subsequent normal cut stem processing process.

[0020] The above technical scheme of the present application has the following beneficial effects:

[0021] The process flow of the present application is simple and efficient, the cost of tobacco stem treatment is low, and the treatment period is short, safe and reliable, can realize the degradation of pectin, cellulose, protein, starch and other macromolecular material components in the tobacco stem processing process, effectively improve the quality of tobacco stem and other technical advantages, thus has good popularization and application value in the field of cigarette manufacturing technology. BRIEF DESCRIPTION OF DRAWINGS

[0022] 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.

[0023] Figure 1 The microstructure diagram of CX-26.

[0024] Figure 2 The strain gene phylogenetic tree of CX-26.

[0025] Figure 3 The microstructure diagram of CX-17.

[0026] Figure 4 The strain gene phylogenetic tree of CX-17.

[0027] Figure 5 The microstructure diagram of DF-08.

[0028] Figure 6 The strain gene phylogenetic tree of DF-08.

[0029] Figure 7 The schematic diagram of the multi-strain combined method. DETAILED DESCRIPTION

[0030] Various exemplary embodiments of the present application will now be described in detail with reference to the accompanying drawings. Note that the relative arrangement of the components and steps set forth in these embodiments, numerical expressions, and numerical values are not limiting to the scope of the present application unless specifically stated otherwise.

[0031] Example 1 Strain Preparation

[0032] (1) Bacillus subtilis CX-26:

[0033] Bacillus subtilis CX-26, deposited at China Center for Type Culture Collection (CCTCC), Wuhan University, with the accession number CCTCC NO: M 2023459, on April 3, 2023. The microstructure diagram is shown in Figure 1 .

[0034] (2) Priestia megaterium CX-17:

[0035] Priestia megaterium CX-17, deposited at China Center for Type Culture Collection (CCTCC), Wuhan University, with the accession number CCTCC NO: M 2023458, on April 3, 2023. The microstructure diagram is shown in Figure 3 .

[0036] (3) Bacillus velezensis DF-08, deposited at China Center for Type Culture Collection (CCTCC), Wuhan University, with the accession number CCTCC NO: M 2023456, on April 3, 2023. The microstructure diagram is shown in Figure 5 .

[0037] Example 2 Strain Preparation

[0038] (1) Strain Activation:

[0039] The preserved Bacillus subtilis CX-26 strain was inoculated on NA solid medium by point inoculation method and cultured at 37°C for 1-2 days;

[0040] The preserved Priestia megaterium CX-17 strain was inoculated on NA solid medium by point inoculation method and cultured at 37°C for 48-72 hours;

[0041] The Bacillus velezensis DF-08 strain was inoculated on LB solid medium by point inoculation method and cultured at 37°C for 24 hours.

[0042] (2) Seed liquid preparation:

[0043] Scrape the CX-26 cells cultured in step (1), inoculate them into NA liquid medium, and culture them in a shaker at 30℃ and 180r / min for 24h to obtain CX-26 seed liquid.

[0044] Scrape the CX-17 cells cultured in step (1), inoculate them into NA liquid medium, and culture them in a shaker at 45℃ and 160r / min for 48h to obtain CX-17 seed liquid.

[0045] Scrape the DF-08 cells cultured in step (1), inoculate them into LB liquid medium, and culture them in a shaker at 35℃ and 180r / min for 24h to obtain DF-08 seed liquid.

[0046] (3) Expand cultivation:

[0047] 1) Induction of pectinase production:

[0048] The Bacillus subtilis CX-26 seed culture from step (2) was inoculated at an inoculation rate of 2% into a special liquid culture medium (culture medium composition: pectin 4 g / L, K2HPO4 0.1 g / L, MgSO4·7H2O 0.5 g / L, NaNO3 3 g / L, FeSO4·7H2O 0.01 g / L, Congo red 0.4 g / L, pH = 7.0). The culture was incubated at 38℃ with shaking at 165 r / min for 72 h. At this time, the OD of the bacterial culture was... 600 =1.8.

[0049] 2) Induction of cellulase production:

[0050] The Priestella megaterium CX-17 seed culture from step (2) was inoculated into a special liquid culture medium at an inoculation rate of 3% (culture medium composition: sodium carboxymethyl cellulose (CMC-Na) 10.0 g / L, peptone 10.0 g / L, yeast extract 5.0 g / L, KH2PO4 1.0 g / L, MgSO4·7H2O 0.2 g / L, C6H 14 O6 2.0 g / L, NaCl 10.0 g / L, pH = 7.0) were cultured at 32℃ and shaken on a shaker at 195 r / min for 72 h, at which point OD600 = 2.0.

[0051] 3) Induction of amylase production:

[0052] 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.

[0053] (4) Preparation of crude enzyme preparation:

[0054] 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.

[0055] Example 4: Combination of multiple strains

[0056] (1) The Bacillus subtilis CX-26 enzyme preparation and Priestella magna CX17 enzyme preparation prepared in Example 3 were mixed at a mass ratio of 2:1. During the stalk soaking stage, the mixed enzyme preparation was added to the stalk soaking water at a mass ratio of 6% of the tobacco stalks. The mixture was then inactivated at a temperature of 35°C for 4 hours. After that, the mixed enzyme preparation was inactivated by the high temperature environment (80°C-100°C) during the stalk steaming stage.

[0057] (2) After the stems are added, the Bacillus DF-08 enzyme preparation is applied to the stems at 4% of the stem mass ratio. The stems are placed at 37°C and 75% humidity for 6 hours. Then, the enzyme preparation is inactivated by the high temperature environment (200°C-220°C) during the stem drying stage. After that, the stems are processed in the normal stem processing flow.

[0058] Effects of enzyme treatment during the stem soaking stage:

[0059] Table 1. Changes in the content of major cell wall substances before and after treatment.

[0060]

[0061] Effect of enzyme treatment on shredded stems:

[0062] Table 2. Changes in protein content of stem fibers before and after treatment.

[0063]

[0064] Sensory evaluation of stem fibers after multi-strain combination:

[0065] Table 3 Sensory quality evaluation scores of tobacco stems after treatment with a combination of multi-microbial agents

[0066]

[0067] As shown in Table 3, the aroma of the treated stems improved, with an increase in aroma quantity and concentration. It also improved the off-flavors, irritation, and aftertaste, significantly enhanced the smoothness of the smoke, and resulted in a significant increase in the overall smoking score.

[0068] 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 stems and filaments through the combined use of multiple strains, characterized in that, Includes the following steps: (1) The enzyme preparations of Bacillus subtilis CX-26, Priestella megaterium CX17 and Bacillus belyss DF-08 with preservation numbers CCTCC NO: M 2023458 were prepared by strain activation, seed culture 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 three enzyme preparations. (3) In step (2), the Bacillus subtilis CX-26 enzyme preparation and the Priestella giantiflora CX17 enzyme preparation with preservation number CCTCC NO: M 2023458 are mixed at a mass ratio of 2:

1. During the soaking stage, the mixed enzyme preparation is added to the soaking water at a mass ratio of 6% of the tobacco stems and acted at a temperature of 35℃ for 4 h. Then, the mixed enzyme preparation is inactivated by using a high temperature environment of 80℃-100℃ during the steaming stage. After the tobacco shreds are added, the Bacillus belys DF-08 enzyme preparation with preservation number CCTCC NO: M 2023456 is applied to the tobacco shreds at a mass ratio of 4% of the tobacco shreds and placed at a temperature of 37℃ and a humidity of 75% for 6 h. Then, the enzyme preparation is inactivated by using a high temperature environment of 200℃-220℃ during the drying stage of the tobacco shreds. After that, the tobacco shreds are processed into the subsequent normal tobacco shreds processing flow.

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 stems and fibers.

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