Reconstituted tobacco and preparation method thereof

Abstract

The invention discloses a preparation method of reconstituted tobacco. The preparation method comprises the following steps: (1) preparing a tobacco raw material mixture; (2) carrying out first microbial fermentation; (3) secondary microbial fermentation; (4) carrying out third microbial fermentation; and (5) preparing the reconstituted tobacco. The invention further discloses the reconstituted tobacco. The preparation method provided by the invention effectively solves the problem of improving the quality of the paper-making reconstituted tobacco.

Description

Technical Field

[0001] The invention belongs to the technical field of tobacco, and in particular relates to a reconstituted tobacco leaf and a preparation method thereof. Background Art

[0002] Reconstituted tobacco is a tobacco product with properties close to natural tobacco that is made from tobacco raw materials such as tobacco leaf fragments, tobacco dust, cigarette ash, tobacco stems or low-grade tobacco leaves produced during the tobacco production and processing process. As an important component of tobacco raw materials for cigarettes, reconstituted tobacco has obvious advantages in terms of 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, has become increasingly prominent in strengthening the style of cigarette products. However, the existing tobacco raw material resources for reconstituted tobacco are difficult to match the demand for high-quality reconstituted tobacco products for the improvement of cigarette product structure. Therefore, it is necessary to explore the potential for improving the quality of reconstituted tobacco raw materials from a technical level and break through technical bottlenecks.

[0003] In recent years, the tobacco industry's medium- and long-term science and technology development plan and the "14th Five-Year Plan" science and technology innovation plan have proposed using "tobacco microorganisms and biofermentation technology" to solve common problems in the industry. Microorganisms can produce a variety of highly active enzymes during proliferation, such as proteases, cellulases, esterases, oxidoreductases, etc. Under the synergistic effects of enzymatic action, chemical action, and complex metabolism in microorganisms, the substrate can be fully degraded, oxidized, reduced, polymerized, and coupled to form various small molecule compounds, including various flavor substances, such as alcohols, aldehydes, ketones, phenols, esters, furans, pyrazines, pyridines, and terpenes. For this reason, tobacco researchers have carried out a large number of studies on tobacco microbial fermentation technology. For example, patent CN2016107818049 discloses a comprehensive method for comprehensively improving the quality of reconstituted tobacco leaves. In the extraction process of reconstituted tobacco leaves, Aspergillus niger ferulic acid esterase, soil lignin peroxidase, Bacillus licheniformis powder, and freeze-dried lactic acid bacteria powder are added for composite fermentation, which effectively improves the quality of reconstituted tobacco leaves. Patent CN202310116566X discloses a reconstituted tobacco coating liquid and a preparation method thereof. Pseudomonas paraflavum bacterial agent is added to the reconstituted tobacco slurry for fermentation treatment. The reconstituted tobacco obtained has reduced impurities, increased aroma quality and aroma quantity, and reduced irritation.

[0004] However, the fermentation of the above-mentioned single or composite microorganisms cannot meet the quality requirements of reconstituted tobacco. Therefore, in order to solve the inherent quality defects of existing tobacco raw materials for reconstituted tobacco and effectively improve the quality of reconstituted tobacco, the present invention proposes a method for solid-state fermentation of reconstituted tobacco raw materials with composite microorganisms to improve the quality of reconstituted tobacco. Summary of the invention

[0005] Aiming at the problem that the existing tobacco raw materials of papermaking reconstituted tobacco are of low quality and cannot further meet the quality requirements of high-quality reconstituted tobacco, the present invention provides a method for improving the quality of reconstituted tobacco by solid-state fermentation of reconstituted tobacco raw materials with composite microorganisms.

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

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

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

[0009] (2) Spraying the first microbial suspension evenly on the surface of the tobacco raw material mixture for fermentation, stirring the mixture up and down every 20-24 hours, and fermenting 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 number of viable bacteria in the spore suspension is (1-5)×(10 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% of water, turn it upside down 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 of the second microbial powder is (1-8)×(10 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 fermenting for 6-8 hours, then add 5-7wt% water, turn it upside down every 20-24 hours, and terminate the 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 number of live bacteria of the third microbial powder is (1-7)×(10 8 ~10 9 )cFu / g;

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

[0013] Preferably, the first microbial suspension in step (2) is a spore suspension of Aspergillus tubingensis; the preparation method of the spore suspension of Aspergillus tubingensis is: inoculating Aspergillus tubingensis into MEA agar medium, culturing at 30° C. for 7 days, and washing with sterile saline to obtain the spore suspension of Aspergillus tubingensis.

[0014] Preferably, the second microbial powder in step (3) is Pichia pastoris powder; the preparation method of the Pichia pastoris powder is: inoculating Pichia pastoris into BMGY culture medium, fermenting at pH = 6 and temperature 30°C for 24 hours, centrifuging the bacterial liquid to remove the supernatant, adding 4wt% glycerol at a weight ratio of 1:1 to the solid matter, and freeze-drying to obtain the Pichia pastoris powder.

[0015] Preferably, the third microbial powder in step (4) is Bacillus amyloliquefaciens powder; the preparation method of the Bacillus amyloliquefaciens powder is: inoculating Bacillus amyloliquefaciens into LB culture medium, culturing at a constant temperature of 37°C for 24 hours, centrifuging the bacterial liquid to remove the supernatant, adding glucose (4g / 100mL) to the solid matter at a weight ratio of 1:1, and freeze-drying to obtain the Bacillus amyloliquefaciens powder.

[0016] Preferably, the temperature at which the tobacco raw material mixture is fermented in step (2), (3) or (4) is 20-35°C.

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

[0018] Beneficial effects of the present invention:

[0019] 1. The preparation method of the present invention sequentially uses three microorganisms, namely, Aspergillus tubingensis, Pichia pastoris, and Bacillus amyloliquefaciens, to ferment the tobacco raw material mixture. The composite enzymes such as protease, pectinase, cellulase, and amylase synthesized during the fermentation process degrade macromolecular substances such as protein, starch, and pectin in the tobacco raw material mixture into small molecular substances such as amino acids, reducing sugars, and galacturonic acid that are easily soluble in water, wherein the protein degradation rate is as high as 19.02±0.22%, the pectin degradation rate is as high as 12.62±0.21%, and the starch degradation rate is as high as 32.64±0.13%; the residues of substances such as protein, pectin, and starch are reduced, and the impurities, irritation, and oral residues during the smoking of reconstituted tobacco leaves are reduced, and the comfort is significantly improved.

[0020] 2. The preparation method of the present invention sequentially uses three microorganisms, namely, Aspergillus tubingensis, Pichia pastoris, and Bacillus amyloliquefaciens, to ferment the tobacco raw materials of reconstituted tobacco leaves, and the types and contents of volatile aroma components are increased, especially the alcohol, ester, and lactone aroma components are significantly improved; and new aroma components, such as alcohols, esters, aldehydes, etc., appear; the aroma quantity and aroma quality of the reconstituted tobacco leaves when smoking are improved.

[0021] 3. The preparation method of the present invention uses a mixture of waste materials such as tobacco fragments, tobacco dust, and tobacco stems as a tobacco raw material mixture for reconstituted tobacco leaves, and sequentially ferments them with three microorganisms such as Aspergillus tubingensis, Pichia pastoris, and Bacillus amyloliquefaciens. The aroma quantity and aroma quality of the prepared reconstituted tobacco leaves are excellent, and are superior to tobacco products made from natural tobacco leaves. The method of the present invention utilizes waste and protects the environment.

[0022] Instruction Manual

[0023] Figure 1 This is the GC-MS graph of the reconstituted tobacco leaves prepared in Example 1.

[0024] Figure 2 This is the GC-MS graph of the reconstituted tobacco leaves prepared in Comparative Example 3. DETAILED DESCRIPTION

[0025] The present invention is described clearly and completely below in conjunction with the technical solutions in the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Other technical solutions obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0026] Example 1: A method for preparing reconstituted tobacco leaves by composite microbial fermentation, the steps are as follows:

[0027] (1) mixing tobacco scraps, tobacco dust, tobacco stems and other tobacco waste materials as tobacco raw materials in a ratio of 2:1:1 to form a tobacco raw material mixture;

[0028] (2) Spraying the Aspergillus tubingensis spore suspension evenly on the surface of the tobacco raw material mixture for fermentation, turning it upside down every 22 hours, fermenting it for 45 hours, and the fermentation temperature is 32° C. The amount of Aspergillus tubingensis spore suspension added is 1.5% of the total weight of the tobacco raw material mixture, and the number of viable bacteria in the spore suspension is 3×(10 7 ~10 8 ) cFu / mL; the preparation method of the Aspergillus tubingensis spore suspension is: inoculating Aspergillus tubingensis into MEA agar medium, culturing at 30° C. for 7 days, and washing with sterile saline to obtain the Aspergillus tubingensis spore suspension;

[0029] (3) After stirring for 1 hour, the Pichia yeast powder was evenly sprayed on the surface of the tobacco raw material mixture fermented in step (2) to continue fermentation. After 6 hours, the mixture was watered to 6 wt %, and the mixture was stirred up and down every 22 hours. The mixture was fermented at 32° C. for 40 hours. The amount of Pichia yeast powder added was 0.5% of the total weight of the tobacco raw material mixture, and the number of viable bacteria in the powder was 8×(10 7 ~10 8) cFu / g; the preparation method of the Pichia yeast powder is: inoculating Pichia yeast in BMGY medium, fermenting at pH = 6 and temperature 30°C for 24 hours, centrifuging the bacterial liquid to remove the supernatant, adding 4wt% glycerol to the solid matter at a weight ratio of 1:1, and freeze-drying to obtain the Pichia yeast powder;

[0030] (4) After stirring for 1 hour, the amylolytic spore powder is evenly sprayed on the surface of the tobacco raw material mixture fermented in step (3), and the mixture is continued to ferment for 6 hours, and then water is added to 5wt%, and the mixture is stirred up and down once every 20 hours. The fermentation is terminated after 48 hours; the amount of amylolytic spore 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 8 ~10 9 ) cFu / g; the preparation method of the amyloliquefaciens powder is as follows: inoculating Bacillus amyloliquefaciens in LB medium, culturing at 37°C for 24 hours, centrifuging the bacterial solution to remove the supernatant, adding glucose (4 g / 100 mL) at a weight ratio of 1:1 to the solid matter, and freeze-drying to obtain the amyloliquefaciens powder;

[0031] (5) After the fermentation of the tobacco raw material mixture is completed, the fermented tobacco raw material mixture is prepared into reconstituted tobacco leaves according to the conventional process of papermaking.

[0032] Example 2: Composite microbial fermentation improves the quality of reconstituted tobacco leaves, the steps are as follows:

[0033] (1) Same as Example 1.

[0034] (2) Spraying the Aspergillus tubingensis spore suspension evenly on the surface of the tobacco raw material mixture for fermentation, turning it upside down every 20 hours, fermenting it for 40 hours, and the fermentation temperature is 25° C. The amount of Aspergillus tubingensis spore suspension added is 0.5% of the total weight of the tobacco raw material mixture, and the number of viable bacteria in the spore suspension is 2×(10 7 ~10 8 ) cFu / mL; the preparation method of the Aspergillus tubingensis spore suspension is: inoculating Aspergillus tubingensis into MEA agar medium, culturing at 30° C. for 7 days, and washing with sterile saline to obtain the Aspergillus tubingensis spore suspension;

[0035] (3) After stirring for 2 hours, the Pichia yeast powder was evenly sprayed on the surface of the tobacco raw material mixture fermented in step (2) to continue fermentation. After 8 hours, 5 wt% of water was added, and the mixture was stirred up and down every 20 hours. The mixture was fermented at 25° C. for 48 hours. The amount of Pichia yeast powder added was 0.6% of the total weight of the tobacco raw material mixture, and the number of viable bacteria of the powder was 1×(10 7 ~10 8) cFu / g; the preparation method of Pichia pastoris powder is as follows: inoculating Pichia pastoris into BMGY medium, fermenting at pH=6 and temperature 30°C for 24 hours, centrifuging the bacterial solution to remove the supernatant, adding 4wt% glycerol to the solid matter at a weight ratio of 1:1, and freeze-drying to obtain the Pichia pastoris powder;

[0036] (4) After stirring for 1 hour, the amylolytic spore powder is evenly sprayed on the surface of the tobacco raw material mixture fermented in step (3), and the mixture is continued to ferment for 8 hours, and then 7% of water is added. The mixture is stirred up and down once every 20 hours, and the fermentation is terminated after 40 hours. The amount of amylolytic spore 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 8 ~10 9 ) cFu / g; the preparation method of the amyloliquefaciens powder is as follows: inoculating Bacillus amyloliquefaciens in LB medium, culturing at 37°C for 24 hours, centrifuging the bacterial solution to remove the supernatant, adding glucose (4 g / 100 mL) at a weight ratio of 1:1 to the solid matter, and freeze-drying to obtain the amyloliquefaciens powder;

[0037] (5) Same as Example 1.

[0038] Comparative Example 1: The microbial strains used were only Aspergillus tubingensis spore suspension and Bacillus amyloliquefaciens powder, and the rest was the same as in Example 1; the steps were as follows:

[0039] (1) Same as Example 1;

[0040] (2) Same as Example 1;

[0041] (3) Same as step (4) in Example 1;

[0042] (4) Same as step (5) in Example 1.

[0043] Comparative Example 2: The microbial strains used were only Pichia pastoris powder and Bacillus amyloliquefaciens powder, and the rest was the same as in Example 1; the steps were as follows:

[0044] (1) Same as Example 1;

[0045] (2) Same as step (3) in Example 1;

[0046] (3) Same as step (4) in Example 1;

[0047] (4) Same as step (5) in Example 1.

[0048] Comparative Example 3: No microbial strains were added, and the rest was the same as Example 1; the steps were as follows:

[0049] (1) Same as Example 1;

[0050] (2) spraying sterile water on the surface of the tobacco raw material module at an addition amount of 11 wt%, turning it upside down every 22 h, and fermenting it at 32° C. for 133 h;

[0051] (3) Same as step (5) in Example 1.

[0052] The aroma components of the reconstituted tobacco leaves prepared in each embodiment and comparative example were analyzed and tested, and the reconstituted tobacco leaf samples were extracted with dichloromethane solvent and concentrated under reduced pressure to prepare a sample concentrate, which was further analyzed by GC-MS, and the relative content of the flavor compounds was calculated according to the ratio of the peak area of ​​the flavor substance to the peak area of ​​the internal standard; at the same time, the sensory quality of the reconstituted tobacco leaves prepared in the embodiments and comparative examples was evaluated to evaluate the effects of each sample. The results are shown in Tables 1 and 2.

[0053] Table 1 Detection results of aroma components of reconstituted tobacco samples

[0054] serial number Relative content of aroma 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

[0055] Table 2 Sensory quality evaluation results of reconstituted tobacco leaves

[0056]

[0057] The comparison results of the changes in the content of aroma components in Table 1 and the evaluation of the sensory quality effects in Table 2 both confirm that the reconstituted tobacco leaves prepared in Example 1 have the best sensory quality effect when smoked and have the highest content of aroma components.

[0058] Table 3 Detection results of aroma components of reconstituted tobacco leaves prepared in Example 1 and Comparative Example 3

[0059]

[0060]

[0061] Figure 1 This is the GC-MS graph of the reconstituted tobacco leaves prepared in Example 1; Figure 2 The GC-MS graph of the reconstituted tobacco prepared in Comparative Example 3. Table 3 is the detection results of some aroma components of the reconstituted tobacco prepared in Example 1 and Comparative Example 3. It can be seen from Table 3 that after the tobacco raw material mixture of the reconstituted tobacco was fermented in sequence using Aspergillus tubingensis, Pichia pastoris, and Bacillus amyloliquefaciens microorganisms, the types and contents of volatile aroma components were increased, especially the alcohol, ester, and lactone aroma components were significantly improved. It can also be seen from Table 3 that new aroma components appear, such as squalane, 15-octadecanal, L-ascorbic acid-2,6-dipalmitate, etc. This significantly increases the aroma amount and aroma quality of the reconstituted tobacco when it is smoked.

[0062] 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 they were all significantly better than the fermentation results of the comparative example.

[0063] 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 principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing reconstituted tobacco, characterized in that: The steps include: (1) mixing tobacco raw materials in proportion to form a tobacco raw material mixture for reconstituted tobacco leaves; (2) Spraying the first microbial suspension evenly on the surface of the tobacco raw material mixture for fermentation, stirring the mixture up and down every 20-24 hours, and fermenting 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 number of viable bacteria in the spore suspension is (1-5)×(10 7 ~10 8 )cFu / mL; (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% of water, turn it upside down 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 of the second microbial powder is (1-8)×(10 7 ~10 8 )cFu / g; (4) Spray the third microbial powder evenly on the surface of the above tobacco raw material mixture and continue fermenting for 6-8 hours, then add 5-7wt% water, turn it upside down every 20-24 hours, and terminate the 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 number of live bacteria of the third microbial powder is (1-7)×(10 8 ~10 9 )cFu / g; (5) After the fermentation is terminated, the fermented tobacco raw material mixture is prepared into reconstituted tobacco leaves according to the conventional papermaking process.

2. The preparation method according to claim 1, characterized in that: The first microbial suspension in step (2) is a spore suspension of Aspergillus tubingensis; the preparation method of the spore suspension of Aspergillus tubingensis is as follows: inoculating Aspergillus tubingensis into MEA agar medium, culturing at 30° C. for 7 days, and washing with sterile physiological saline to obtain the spore suspension of Aspergillus tubingensis.

3. The preparation method according to claim 1, characterized in that: The second microbial powder in step (3) is Pichia pastoris powder; the preparation method of the Pichia pastoris powder is as follows: inoculate Pichia pastoris into BMGY culture medium, ferment at pH=6 and temperature 30°C for 24 hours, centrifuge the culture liquid to remove the supernatant, add 4wt% glycerol at a weight ratio of 1:1 to the solid matter, and freeze-dry to obtain the Pichia pastoris powder.

4. The preparation method according to claim 1, characterized in that: The third microbial powder in step (4) is Bacillus amyloliquefaciens powder; the preparation method of the Bacillus amyloliquefaciens powder is: inoculating Bacillus amyloliquefaciens into LB culture medium, culturing at 37°C for 24 hours, centrifuging the bacterial liquid to remove the supernatant, adding glucose (4g / 100mL) to the solid matter at a weight ratio of 1:1, and freeze-drying to obtain the Bacillus amyloliquefaciens powder.

5. The preparation method according to claim 1, characterized in that: The temperature for fermenting the tobacco raw material mixture in step (2), (3) or (4) is 20-35°C.

6. The reconstituted tobacco leaf prepared according to the preparation method according to claims 1-5.

Images