Reconstituted tobacco leaf and method of making same

Abstract

The application discloses a preparation method of reconstituted tobacco leaves, which comprises the following steps: (1) preparing a tobacco raw material mixture; (2) first microbial fermentation; (3) second microbial fermentation; (4) third microbial fermentation; and (5) preparing reconstituted tobacco leaves. The application further discloses the reconstituted tobacco leaves. The preparation method effectively solves the problem of improving the quality of papermaking reconstituted tobacco leaves.

Description

Technical Field

[0001] This invention belongs to the field of tobacco technology, specifically relating to a reconstituted tobacco leaf and its preparation method. Background Technology

[0002] Reconstituted tobacco is a tobacco product made from tobacco scraps, dust, ash, stems, or low-grade tobacco leaves processed during tobacco production and processing, resulting in a product whose properties closely resemble natural tobacco leaves. As a crucial component of cigarette raw materials, reconstituted tobacco offers significant advantages in harm reduction, tar reduction, and cost control. With the continuous improvement of cigarette structure, reconstituted tobacco, as a core component supporting the diversified design of cigarette style characteristics, plays an increasingly prominent role in enhancing the style of cigarette products. However, existing tobacco raw material resources for reconstituted tobacco are insufficient to meet the demand for high-quality reconstituted tobacco products driven by the upgrading of cigarette product structure. Therefore, it is necessary to deeply explore the potential for improving the quality of reconstituted tobacco raw materials and overcome technological bottlenecks from a technical perspective.

[0003] In recent years, the medium- and long-term science and technology development plan for the tobacco industry has proposed using "tobacco microorganisms and bio-fermentation technology" to solve common problems in the industry. During their proliferation, microorganisms can produce various highly active enzymes, such as proteases, cellulases, esterases, and oxidoreductases. Through the synergistic effects of enzymatic action, chemical action, and complex metabolism within the microorganisms, substrates can be fully degraded, oxidized, reduced, polymerized, and coupled to form various small molecule compounds, including various aroma substances such as alcohols, aldehydes, ketones, phenols, esters, furans, pyrazines, pyridines, and terpenes. To this end, tobacco researchers have conducted extensive research on tobacco microbial fermentation technology. For example, patent CN2016107818049 discloses a comprehensive method for improving the quality of reconstituted tobacco leaves. This method involves adding Aspergillus niger ferulic acid esterase, soil lignin peroxidase, Bacillus licheniformis powder, and freeze-dried lactic acid bacteria powder to the extraction process of reconstituted tobacco leaves for compound fermentation, effectively improving the quality of reconstituted tobacco leaves. Patent CN202310116566X discloses a reconstituted tobacco coating liquid and its preparation method. The reconstituted tobacco pulp is fermented by adding Pseudomonas paraxanthii inoculum, resulting in reconstituted tobacco with reduced off-flavors, increased aroma quality and aroma quantity, and reduced irritation.

[0004] However, the fermentation by individual or combined microorganisms mentioned above cannot meet the quality requirements of reconstituted tobacco leaves. Therefore, in order to solve the inherent quality defects of existing tobacco raw materials for reconstituted tobacco leaves and effectively improve the quality of reconstituted tobacco leaves, this invention proposes a method for solid-state fermentation of reconstituted tobacco raw materials by combined microorganisms, thereby improving the quality of reconstituted tobacco leaves. Summary of the Invention

[0005] This invention addresses the problem that existing tobacco raw materials used in papermaking reconstituted tobacco have low quality and cannot further meet the quality requirements of high-quality reconstituted tobacco. It provides a method for improving the quality of reconstituted tobacco by using compound microbial solid-state fermentation to reconstitute tobacco raw materials.

[0006] The technical solution of the present invention is as follows:

[0007] The first aspect of this invention discloses a method for preparing reconstituted tobacco leaves, comprising the following steps:

[0008] (1) Tobacco raw materials are mixed evenly in proportion to form a tobacco raw material mixture for reconstituted tobacco leaves;

[0009] (2) The first microbial suspension is evenly sprayed onto the surface of the tobacco raw material mixture for fermentation. The mixture is turned over once every 20-24 hours, and fermentation lasts for 40-48 hours. The amount of the first microbial suspension added is 0.5-2% of the total weight of the tobacco raw material mixture, and the viable count of the spore suspension is (1-5)×(10^6)^2. 7 ~10 8 cFu / mL;

[0010] (3) Spray the second microbial powder evenly on the surface of the above tobacco raw material mixture and continue fermentation. After 6-8 hours, add 5-7 wt% water, turn it over once every 20-24 hours, and ferment for 40-48 hours. The amount of the second microbial powder added is 0.2-0.6% of the total weight of the tobacco raw material mixture, and the number of viable bacteria in the second microbial powder is (1-8)×(10^6)^2. 7 ~10 8 ) cFu / g;

[0011] (4) Spray the third microbial powder evenly on the surface of the above tobacco raw material mixture and continue fermentation for 6-8 hours, then add 5-7 wt% water. Turn the mixture over once every 20-24 hours. Terminate fermentation after 40-48 hours. The amount of the third microbial powder added is 1-1.5% of the total weight of the tobacco raw material mixture, and the viable count of the third microbial powder is (1-7) × (10⁻⁶)⁻¹⁰. 8 ~10 9 ) cFu / g;

[0012] (5) After fermentation is terminated, the fermented tobacco raw material mixture is prepared into reconstituted tobacco leaves according to the conventional papermaking process.

[0013] Preferably, the first microbial suspension in step (2) is a *Aspergillus tabinei* spore suspension; the preparation method of the *Aspergillus tabinei* spore suspension is as follows: *Aspergillus tabinei* is inoculated into MEA agar medium, cultured at 30°C for 7 days, and washed with sterile physiological saline to obtain the *Aspergillus tabinei* spore suspension.

[0014] Preferably, the second microbial powder in step (3) is Pichia pastoris powder; the preparation method of the Pichia pastoris powder is as follows: Pichia pastoris is inoculated into BMGY medium, fermented at pH=6 and temperature 30℃ for 24 h, the supernatant is removed by centrifugation of the bacterial liquid, 4 wt% glycerol is added to the solids at a weight ratio of 1:1, and the Pichia pastoris powder is obtained by freeze drying.

[0015] Preferably, the third microbial powder in step (4) is Bacillus amyloliquefaciens powder; the preparation method of the Bacillus amyloliquefaciens powder is as follows: Bacillus amyloliquefaciens is inoculated into LB medium, cultured at 37°C for 24 h, the supernatant is removed by centrifugation, glucose (4g / 100mL) is added to the solids at a weight ratio of 1:1, and the Bacillus amyloliquefaciens powder is obtained by freeze drying.

[0016] Preferably, the fermentation temperature of the tobacco raw material mixture in steps (2), (3) or (4) is 20-35°C.

[0017] The second aspect of this invention discloses reconstituted tobacco leaves prepared by the aforementioned preparation method.

[0018] The beneficial effects of this invention are:

[0019] 1. The preparation method of this invention involves sequentially fermenting a mixture of tobacco raw materials using three microorganisms: *Aspergillus tabineum*, *Pichia pastoris*, and *Bacillus amyloliquefaciens*. During fermentation, a complex of enzymes, including proteases, pectinases, cellulases, and amylases, is synthesized, degrading macromolecules such as proteins, starches, and pectins in the tobacco raw material mixture into water-soluble amino acids, reducing sugars, galacturonic acid, and other small molecules. The protein degradation rate reaches as high as 19.02±0.22%, the pectin degradation rate as high as 12.62±0.21%, and the starch degradation rate as high as 32.64±0.13%. This reduces the residue of proteins, pectins, and starches, lowering the off-flavors, irritation, and oral residue when smoking reconstituted tobacco, significantly improving comfort.

[0020] 2. The preparation method of the present invention, which sequentially uses three microorganisms, Aspergillus tabineum, Pichia pastoris and Bacillus amyloliquefaciens, to ferment reconstituted tobacco raw materials, improves the types and contents of volatile aroma components, especially alcohols, esters and lactones. New aroma components, such as alcohols, esters and aldehydes, also appear. This improves the aroma quantity and aroma quality when reconstituted tobacco is smoked.

[0021] 3. The preparation method of this invention uses a mixture of tobacco scraps, tobacco dust, tobacco stems, and other waste materials as the raw material mixture for reconstituted tobacco leaves. This mixture is then sequentially fermented by three microorganisms: *Aspergillus tabingensis*, *Pichia pastoris*, and *Bacillus amyloliquefaciens*. The resulting reconstituted tobacco leaves exhibit superior aroma quantity and quality compared to tobacco products made from natural tobacco leaves. This method utilizes waste materials and protects the environment. Attached Figure Description

[0022] Figure 1 The image shows the GC-MS pattern of the reconstituted tobacco leaves prepared in Example 1.

[0023] Figure 2 The image shows the GC-MS pattern of the reconstituted tobacco leaves prepared in Comparative Example 3. Detailed Implementation

[0024] The present invention will be clearly and completely described below with reference to the technical solutions in the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Other technical solutions obtained by those skilled in the art without creative effort are all within the scope of protection of the present invention.

[0025] Example 1: A method for preparing reconstituted tobacco leaves by compound microbial fermentation, the steps of which are as follows:

[0026] (1) Tobacco scraps, tobacco dust, tobacco stems and other tobacco waste are mixed evenly in a ratio of 2:1:1 to form a tobacco raw material mixture;

[0027] (2) Spray the Aspergillus tabineum spore suspension evenly onto the surface of the tobacco raw material mixture for fermentation. Turn the mixture over once every 22 hours, and ferment for 45 hours at a temperature of 32℃. The amount of Aspergillus tabineum spore suspension added is 1.5% of the total weight of the tobacco raw material mixture, and the viable count of the spore suspension is 3×(10⁻⁶). 7 ~10 8 The preparation method of the Aspergillus tabineum spore suspension is as follows: Aspergillus tabineum is inoculated into MEA agar medium, cultured at 30°C for 7 days, and washed with sterile physiological saline to obtain the Aspergillus tabineum spore suspension;

[0028] (3) After turning over for 1 hour, the Pichia pastoris powder is evenly sprayed onto the surface of the tobacco raw material mixture after fermentation in step (2) and fermentation continues. After 6 hours, 6 wt% water is added, and the mixture is turned over once every 22 hours. Fermentation continues at 32℃ for 40 hours. The amount of Pichia pastoris powder added is 0.5% of the total weight of the tobacco raw material mixture, and the number of live bacteria in the powder is 8 × (10⁻⁶). 7 ~10 8The preparation method of the Pichia pastoris powder is as follows: Pichia pastoris is inoculated into BMGY medium and fermented at pH=6 and temperature 30℃ for 24 h. The supernatant is removed by centrifugation of the bacterial solution, and 4 wt% glycerol is added to the solids at a weight ratio of 1:1. After freeze-drying, the Pichia pastoris powder is obtained.

[0029] (4) After turning over for 1 hour, spray the amyloliquefaciens powder evenly on the surface of the tobacco raw material mixture after fermentation (3). Continue fermentation for 6 hours, then add 5 wt% water. Turn over once every 20 hours. Terminate fermentation after 48 hours. The amount of amyloliquefaciens powder added is 1.2% of the total weight of the tobacco raw material mixture, and the number of live bacteria in the powder is 4 × (10⁻⁶). 8 ~10 9 The preparation method of Bacillus amyloliquefaciens powder is as follows: Bacillus amyloliquefaciens is inoculated into LB medium and cultured at 37℃ for 24 h. The supernatant is removed by centrifugation of the bacterial solution. Glucose (4g / 100mL) is added to the solids at a weight ratio of 1:1. The solids are then freeze-dried to obtain the Bacillus amyloliquefaciens powder.

[0030] (5) After the fermentation of the tobacco raw material mixture is terminated, the fermented tobacco raw material mixture is prepared into reconstituted tobacco leaves according to the conventional papermaking process.

[0031] Example 2: Compound microbial fermentation improves the quality of reconstituted tobacco leaves. The steps are as follows:

[0032] (1) Same as Example 1.

[0033] (2) Spray the Aspergillus tabineum spore suspension evenly onto the surface of the tobacco raw material mixture for fermentation. Turn the mixture over once every 20 hours, and ferment for 40 hours at a temperature of 25°C. The amount of Aspergillus tabineum spore suspension added is 0.5% of the total weight of the tobacco raw material mixture, and the viable count of the spore suspension is 2×(10⁻⁶). 7 ~10 8 The preparation method of the Aspergillus tabineum spore suspension is as follows: Aspergillus tabineum is inoculated into MEA agar medium, cultured at 30°C for 7 days, and washed with sterile physiological saline to obtain the Aspergillus tabineum spore suspension;

[0034] (3) After turning over for 2 hours, the Pichia pastoris powder is evenly sprayed onto the surface of the tobacco raw material mixture after fermentation in step (2) and fermentation continues. After 8 hours, 5 wt% water is added, and the mixture is turned over once every 20 hours. Fermentation continues at 25℃ for 48 hours. The amount of Pichia pastoris powder added is 0.6% of the total weight of the tobacco raw material mixture, and the number of live bacteria in the powder is 1×(10⁻⁶). 7 ~10 8The preparation method of Pichia pastoris powder is as follows: Pichia pastoris is inoculated into BMGY medium and fermented for 24 h at pH=6 and temperature 30℃. The supernatant is removed by centrifugation of the bacterial solution, and 4 wt% glycerol is added to the solids at a weight ratio of 1:1. After freeze-drying, the Pichia pastoris powder is obtained.

[0035] (4) After turning over for 1 hour, spray the amyloliquefaciens powder evenly on the surface of the tobacco raw material mixture after fermentation in step (3). Continue fermentation for 8 hours, then add 7% water. Turn over once every 20 hours. Terminate fermentation after 40 hours. The amount of amyloliquefaciens powder added is 1% of the total weight after fermentation in step (2), and the number of viable bacteria in the powder is 2 × (10⁻⁶). 8 ~10 9 The preparation method of Bacillus amyloliquefaciens powder is as follows: Bacillus amyloliquefaciens is inoculated into LB medium and cultured at 37℃ for 24 h. The supernatant is removed by centrifugation of the bacterial solution. Glucose (4g / 100mL) is added to the solids at a weight ratio of 1:1. The solids are then freeze-dried to obtain the Bacillus amyloliquefaciens powder.

[0036] (5) Same as Example 1.

[0037] Comparative Example 1: The microbial strains used were only Aspergillus tabineum spore suspension and Bacillus amyloliquefaciens powder, and all other steps were the same as in Example 1; the steps are as follows:

[0038] (1) Same as Example 1;

[0039] (2) Same as Example 1;

[0040] (3) Same as step (4) in Example 1;

[0041] (4) Same as step (5) in Example 1.

[0042] Comparative Example 2: The microbial strains used were only Pichia pastoris powder and Bacillus amyloliquefaciens powder, and all other steps were the same as in Example 1; the steps are as follows:

[0043] (1) Same as Example 1;

[0044] (2) Same as step (3) in the embodiment;

[0045] (3) Same as step (4) in Example 1;

[0046] (4) Same as step (5) in Example 1.

[0047] Comparative Example 3: No microbial strains were added; all other steps were the same as in Example 1. The steps are as follows:

[0048] (1) Same as Example 1;

[0049] (2) Spray sterile water onto the surface of the tobacco raw material module at an amount of 11 wt%, turn it over once every 22 hours, and ferment at 32°C for 133 hours;

[0050] (3) Same as step (5) in Example 1.

[0051] The aroma components of the reconstituted tobacco leaves prepared in each example and comparative example were analyzed. The reconstituted tobacco leaf samples were extracted with dichloromethane solvent and concentrated under reduced pressure to prepare a sample concentrate, which was then further analyzed by GC-MS. The relative content of flavor compounds was calculated based on the ratio of the peak area of ​​flavor substances to the peak area of ​​the internal standard. Simultaneously, the sensory quality of the reconstituted tobacco leaves prepared in the examples and comparative examples was evaluated to assess the performance of each sample. The results are shown in Tables 1 and 2.

[0052] Table 1. Detection results of aroma components in reconstituted tobacco samples

[0053] serial number Relative content of aroma-producing components (%) Example 1 139.81±6.14 Example 2 127.54±8.27 Comparative Example 1 109.76±6.54 Comparative Example 2 105.41±7.31 Comparative Example 3 100.21±3.58

[0054] Table 2 Sensory quality evaluation results of reconstituted tobacco leaves

[0055] serial number Sensory quality evaluation results Example 1 The aroma is above average in quantity and concentration, with a delicate and mellow fragrance, few off-flavors, a comfortable mouthfeel, and a pleasantly sweet aftertaste. Example 2 The aroma quantity and concentration are above average, the aroma is clear, there are few woody off-flavors, the mouth feels comfortable, and there is little residue. Comparative Example 1 The aroma quantity and concentration are above average, but the aroma clarity is slightly lacking. The woody and off-flavors are quite noticeable, the spiciness is quite pronounced, and the oral residue is quite noticeable. Comparative Example 2 The aroma is of medium quantity and concentration, slightly rough, with a slightly noticeable woody or off-flavor. It is slightly spicy in the mouth, quite irritating to the nasal cavity, and has only average oral comfort. Comparative Example 3 The aroma is of medium quantity and concentration, but it is rather rough and lacks clarity. The woody notes are quite noticeable, and the mouth feels spicy, irritating, and has a noticeable aftertaste.

[0056] Table 1 shows the changes in aroma component content, and Table 2 shows the comparison of sensory quality evaluation results. Both confirm that the reconstituted tobacco prepared in Example 1 has the best sensory quality when smoked, with the highest content of aroma components.

[0057] Table 3. Detection results of aroma components in reconstituted tobacco leaves prepared in Example 1 and Comparative Example 3.

[0058] name Comparative Example 3 / % Example 1 / % Decaethylene glycol 5.84 8.91 Carbidol 7.59 11.37 cis-1,4-cyclohexanediol - 25.64 Shark liver alcohol - 119.82 Phenylacetyl alcohol - 487.4 Phytosterols 10.78 43.46 4-Amino-1-butanol 7.16 19.43 Triethylene glycol 7.86 15.82 diethylene glycol 1.37 5.99 5-Hexenal - 4.7 15-Octadecanal - 17.45 Phenylacetyl acetate 1.67 9.12 9H-beta-carboline-4-carboxylic acid methyl ester 2.78 10.47 Dimethyl 3-hydroxyglutarate - 163.63 L-Ascorbic acid-2,6-dipalmitate - 1527.48 3-Methylquinoline-4-carboxylate - 37.99 Diethyl iminodiacetate 10.87 32.58 Phenylephrine acetate 10.87 85.02 Methyl cyanoacetate 6.13 8.49 2-Piperidinone 6.58 7.47 E-5-Isopropyl-8-methyl-6,8-nonadien-2-one 9.14 29.99 3-Hydroxy-2-Butanone (acetoin) - 138.43 Hydroxyacetone 10.78 87.47

[0059] Figure 1 GC-MS image of reconstituted tobacco leaves prepared in Example 1; Figure 2 Table 3 shows the GC-MS image of the reconstituted tobacco prepared in Comparative Example 3. Table 3 presents the detection results of some aroma components in the reconstituted tobacco prepared in Example 1 and Comparative Example 3. As shown in Table 3, the types and contents of volatile aroma components were increased after sequential fermentation of the tobacco raw material mixture with Aspergillus tabingensis, Pichia pastoris, and Bacillus amyloliquefaciens, especially alcohols, esters, and lactones. Table 3 also shows the appearance of new aroma components, such as shark liver oil, 15-octadecaldehyde, and L-ascorbic acid-2,6-dipalmitate. This significantly improved the aroma quantity and quality when smoking the reconstituted tobacco.

[0060] Based on the above results, under the conditions of Example 1, the fermentation effect of the three microorganisms was the best; the fermentation effect of the three microorganisms in Example 2 was slightly worse; but all were significantly better than the fermentation results of the comparative example.

[0061] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing reconstituted tobacco leaves, characterized in that, Includes the following steps: (1) Mix tobacco fragments, tobacco dust and tobacco stems in a ratio of 2:1:1 to form a tobacco raw material mixture; (2) Spray the Aspergillus tabineum spore suspension evenly onto the surface of the tobacco raw material mixture for fermentation. Turn the mixture over once every 22 hours, and ferment for 45 hours at a temperature of 32℃. The amount of Aspergillus tabineum spore suspension added is 1.5% of the total weight of the tobacco raw material mixture, and the viable count of the spore suspension is 3×(10⁻⁶). 7 ~10 8 The preparation method of the Aspergillus tabineum spore suspension is as follows: Aspergillus tabineum is inoculated into MEA agar medium, cultured at 30°C for 7 days, and washed with sterile physiological saline to obtain the Aspergillus tabineum spore suspension; (3) After turning over for 1 hour, the Pichia pastoris powder is evenly sprayed onto the surface of the tobacco raw material mixture after fermentation in step (2) and fermentation continues. After 6 hours, 6 wt% water is added, and the mixture is turned over once every 22 hours. Fermentation continues at 32℃ for 40 hours. The amount of Pichia pastoris powder added is 0.5% of the total weight of the tobacco raw material mixture, and the number of live bacteria in the powder is 8 × (10⁻⁶). 7 ~10 8 The preparation method of the Pichia pastoris powder is as follows: Pichia pastoris is inoculated into BMGY medium and fermented at pH=6 and temperature 30℃ for 24 h. The supernatant is removed by centrifugation of the bacterial solution, and 4 wt% glycerol is added to the solids at a weight ratio of 1:

1. After freeze-drying, the Pichia pastoris powder is obtained. (4) After turning over for 1 hour, spray the amyloliquefaciens powder evenly on the surface of the tobacco raw material mixture after fermentation (3). Continue fermentation for 6 hours, then add 5 wt% water. Turn over once every 20 hours. Terminate fermentation after 48 hours. The amount of amyloliquefaciens powder added is 1.2% of the total weight of the tobacco raw material mixture, and the number of live bacteria in the powder is 4 × (10⁻⁶). 8 ~10 9 The preparation method of Bacillus amyloliquefaciens powder is as follows: Bacillus amyloliquefaciens is inoculated into LB medium and cultured at 37℃ for 24 h. The supernatant is removed by centrifugation of the bacterial solution. 4 g / 100 mL of glucose is added to the solids at a weight ratio of 1:

1. The mixture is then freeze-dried to obtain the Bacillus amyloliquefaciens powder. (5) After fermentation is terminated, the fermented tobacco raw material mixture is prepared into reconstituted tobacco leaves according to the conventional papermaking process.

2. Reconstituted tobacco leaves prepared by the preparation method according to claim 1.

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