A method for improving the quality of reconstituted tobacco leaf

Abstract

The application discloses a preparation method of reconstituted tobacco leaves, which comprises the following steps: (1) preparing tobacco raw materials; (2) sterilizing; (3) fermenting with Aspergillus niger seed liquid; (4) separating, and beating the solid part; fermenting the liquid part with Saccharomyces paradoxus; (5) preparing tobacco paste; and (6) preparing reconstituted tobacco leaves. The application also 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 method for improving the quality of reconstituted tobacco leaves. 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 an important component of cigarette raw materials, reconstituted tobacco has significant advantages in reducing harmful substances and tar content, as well as 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 explore the potential for improving the quality of reconstituted tobacco raw materials and overcome technical bottlenecks from a technological perspective.

[0003] In recent years, the medium- and long-term science and technology development plan for the tobacco industry has proposed to use "tobacco microorganisms and bio-fermentation technology" to solve common problems in the industry. It also proposes to apply cutting-edge technologies and interdisciplinary knowledge and theories such as microbiome, metabolic control, and biosynthesis to the research and development of flavorings and cigarette products and to improve their quality and aroma, so as to further promote the technological progress of my country's tobacco industry.

[0004] *Aspergillus niger* is one of the most widely used microorganisms in the *Aspergillus* genus and an important industrial fermentation strain. Due to its high yield, high safety, high secretion rate, and excellent metabolic diversity, *Aspergillus niger* is widely used in the industrial production of pharmaceuticals, organic acids, and proteases. Under natural growth conditions, *Aspergillus niger* secretes a large number of highly active enzymes, effectively degrading and utilizing various biomolecules (such as starch, cellulose, hemicellulose, pectin, xylan, and proteins). *Wickham's abnormal yeast* is an ascospore-producing yeast. Distributed in various fruits and traditional brewing koji, it is a widely present non-brewing yeast in brewing environments. This yeast can secrete various glycosidases such as β-D-glucosidase, β-D-xylosidase, and α-L-rhamnosidase, and produces a high amount of ethyl acetate, exhibiting certain aroma-producing and ester-producing capabilities, making it an important functional microorganism in food fermentation processes.

[0005] 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, this invention develops a method for reconstituted tobacco raw materials through compound microbial liquid fermentation, thereby improving the quality of reconstituted tobacco. Summary of the Invention

[0006] This invention addresses the issue that existing tobacco raw materials used in papermaking reconstituted tobacco have low quality and cannot meet the quality requirements of high-quality reconstituted tobacco. It provides a method for reconstituted tobacco raw materials through liquid fermentation of compound microorganisms, thereby improving the quality of reconstituted tobacco.

[0007] The technical solution of the present invention is as follows:

[0008] The first aspect of this invention discloses a method for improving the quality of reconstituted tobacco leaves, comprising the following steps:

[0009] (1) Mix the reconstituted tobacco raw materials evenly in a certain proportion to form reconstituted tobacco raw materials;

[0010] (2) Sterilize the reconstituted tobacco raw material with water at a weight ratio of 1:(6-8);

[0011] (3) Inoculate with Aspergillus niger seed liquid for the first stage of liquid fermentation for 36-48 hours, with an inoculum size of (1-7)×(10) 8 ~10 9 cFu / 1000mL fermentation broth; fermentation temperature 30-40℃, stirring speed 10-30 rpm, aeration rate 150-200 L / h;

[0012] (4) Solid-liquid separation yields a solid portion and a liquid portion; the solid portion and the added fiber are pulped separately and then mixed to form reconstituted tobacco pulp; the liquid portion undergoes a second stage of liquid fermentation, the fermentation steps are as follows: the liquid portion is inoculated with abnormal Wickham yeast seed liquid and fermentation nutrients are added, and fermentation continues for 48-50 hours; the inoculation amount is (3-8)×(10 7 ~10 8 cFu / 1000mL fermentation broth, fermentation temperature 25-35℃, stirring speed 5-10 rpm, aeration rate 5-10 L / h;

[0013] (5) After the second stage of liquid fermentation in step (4) is completed, the liquid part after fermentation is filtered by a ceramic membrane of 200-500nm, and then concentrated to obtain tobacco paste.

[0014] (6) The slurry obtained in step (4) is used to form a reconstituted tobacco leaf base. The tobacco paste obtained in step (5) is coated on the leaf base and dried to obtain the reconstituted tobacco leaf.

[0015] Preferably, step (2) involves first autoclaving and then high-temperature sterilization.

[0016] Preferably, the preparation method of the Aspergillus niger seed liquid in step (3) is as follows: cultured in PDA solid medium at 28°C for 5 days, rinsed with sterile physiological saline and prepared bacterial suspension.

[0017] Preferably, the preparation method of the abnormal Wickham yeast seed liquid in step (4) is as follows: fermentation in YPD medium at 30°C and 180 r / min for 48 h.

[0018] Preferably, the fermentation nutrients in step (4) are: glutamic acid (0.01-0.6) g / 1000 mL fermentation broth and sodium citrate (0.05-0.2) g / 1000 mL fermentation broth.

[0019] The second aspect of the present invention discloses the reconstituted tobacco prepared by the method.

[0020] The beneficial effects of this invention are:

[0021] 1. The method of the present invention uses Aspergillus niger and Wickham's yeast as a complex microbial community to ferment reconstituted tobacco raw materials. The two microorganisms work synergistically to promote the degradation of macromolecular substances in tobacco raw materials and use them as fermentation substrates to metabolize and produce aroma components with typical aroma characteristics.

[0022] 2. The fermentation nutrients involved in the method of the present invention are optimized and determined based on the clear understanding of the metabolic network and mechanism of action of abnormal Wickham yeast. Its focus is on regulating the corresponding coding genes to promote the metabolic production of alcohol and ester flavor compounds by abnormal Wickham yeast.

[0023] 3. The reconstituted tobacco obtained by the method of the present invention has significantly improved aroma components such as alcohols, esters, and lactones; at the same time, new aroma components are generated, such as TRANS-3-oxo-α-ionol, ethyl palmitate, and 2-azhexanecycloone; the aroma of the cigarette is significantly improved when smoked, the off-flavors and irritation are significantly reduced, and the comfort is significantly improved.

[0024] 4. The method of the present invention uses a mixture of tobacco scraps, tobacco dust, tobacco stems and other waste materials as a raw material mixture for reconstituted tobacco leaves. The mixture is then fermented sequentially by two microorganisms, Aspergillus niger and Wickham yeast, to produce reconstituted tobacco leaves with excellent aroma and flavor, which are superior to tobacco products made from natural tobacco leaves. Waste is utilized, and the environment is protected. Attached Figure Description

[0025] Figure 1 The image shows the GC-MS pattern of the reconstituted tobacco leaves prepared in Example 1.

[0026] Figure 2 The image shows the GC-MS pattern of the reconstituted tobacco leaves prepared in Comparative Example 4. Detailed Implementation

[0027] 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.

[0028] Example 1: A method for improving the quality of reconstituted tobacco leaves, comprising the following steps:

[0029] (1) Mix tobacco scraps, tobacco dust, tobacco stems and other tobacco by-products evenly as raw materials for reconstituted tobacco leaves;

[0030] (2) Mix the reconstituted tobacco raw material with water at a weight ratio of 1:6, autoclave for 15 min, and autoclave again at 115℃ for 15 min.

[0031] (3) After sterilization, inoculate with Aspergillus niger seed liquid for the first stage of liquid fermentation for 45 hours. The inoculation amount is 5×(10) 8 ~10 9 The fermentation broth was prepared at 1000 mL cFu / 1000 mL, with a fermentation temperature of 35℃, a stirring speed of 25 rpm, and an aeration rate of 180 L / h. The seed culture of Aspergillus niger was prepared by culturing it in PDA solid medium at 28℃ for 5 days, rinsing it with sterile physiological saline, and preparing a bacterial suspension.

[0032] (4) After the first stage of fermentation, solid and liquid components are separated to obtain solid and liquid components. The solid component and the added fiber are pulped separately and then mixed to form reconstituted tobacco pulp. The liquid component undergoes the second stage of liquid fermentation. The steps for the second stage of liquid fermentation of the liquid component are as follows: the liquid component is inoculated with abnormal Wickham yeast seed liquid and fermentation nutrients (glutamic acid 0.3g / 1000mL fermentation liquid, sodium citrate 0.15g / 1000mL fermentation liquid) are added, and fermentation continues for 48h; the inoculum size is 4×(10 7 ~10 8 The fermentation broth was prepared by using cFu / 1000mL fermentation broth at a fermentation temperature of 30℃, a stirring speed of 10 rpm, and an aeration rate of 7 L / h. The seed culture of *Wickhamia lanceolata* was prepared by fermenting in YPD medium at 30℃ and 180 r / min for 48 h.

[0033] (5) After the second stage of liquid fermentation in step (4) is completed, the liquid part after fermentation is filtered by a 300nm ceramic membrane and then concentrated to obtain tobacco paste.

[0034] (6) The slurry obtained in step (4) is used to form a reconstituted tobacco leaf base. The tobacco paste obtained in step (5) is coated on the leaf base and dried to obtain reconstituted tobacco leaves.

[0035] Example 2: A method for improving the quality of reconstituted tobacco leaves, comprising the following steps:

[0036] (1) Same as Example 1;

[0037] (2) Mix the reconstituted tobacco raw material with water at a weight ratio of 1:8, autoclave for 15 min, and autoclave again at 115℃ for 15 min;

[0038] (3) After sterilization, inoculate with Aspergillus niger seed liquid for the first stage of liquid fermentation for 40 hours. The inoculation amount is 2×(10) 8 ~10 9 The fermentation broth was prepared at 1000 mL cFu / 1000 mL, with a fermentation temperature of 30 °C, a stirring speed of 15 rpm, and an aeration rate of 150 L / h. The seed culture of Aspergillus niger was prepared by incubating it in PDA solid medium at 28 °C for 5 days, rinsing it with sterile physiological saline, and preparing a bacterial suspension.

[0039] (4) After the first stage of fermentation, solid-liquid separation is carried out to form the solid part of the tobacco raw material module and the tobacco extract. The solid part and the added fiber are pulped separately and then mixed to form reconstituted tobacco pulp. The tobacco extract is subjected to the second stage of liquid fermentation. The steps for the second stage of liquid fermentation of the liquid part are as follows: the liquid part is inoculated with abnormal Wickham yeast seed liquid and fermentation nutrients (glutamic acid 0.02g / 1000mL fermentation liquid, sodium citrate 0.06g / 1000mL fermentation liquid) are added, and fermentation is continued for 48h. The inoculation amount is 1×(10 7 ~10 8 The fermentation broth was prepared by using cFu / 1000mL fermentation broth at a fermentation temperature of 26℃, a stirring speed of 5 rpm, and an aeration rate of 5L / h. The seed culture of *Saccharomyces cerevisiae* was prepared by fermenting in YPD medium at 30℃ and 180 r / min for 48 h.

[0040] (5) Same as Example 1;

[0041] (6) Same as Example 1.

[0042] Comparative Example 1: Not inoculated with Aspergillus niger, otherwise the same as in Example 1. The steps are as follows:

[0043] (1) Same as Example 1;

[0044] (2) Same as Example 1;

[0045] (3) Solid-liquid separation was performed to obtain a solid part and a liquid part. The solid part and the added fiber were pulped separately and then mixed to form reconstituted tobacco pulp. The liquid part underwent a second stage of liquid fermentation. The steps for the second stage of liquid fermentation of the liquid part were as follows: the liquid part was inoculated with abnormal Wickham yeast seed liquid and fermentation nutrients (glutamic acid 0.3g / 1000mL fermentation liquid, sodium citrate 0.15g / 1000mL fermentation liquid) were added, and fermentation continued for 48h; the inoculum size was 4×(10 7 ~10 8 The fermentation broth was prepared by using cFu / 1000mL fermentation broth at a fermentation temperature of 30℃, a stirring speed of 10 rpm, and an aeration rate of 7 L / h. The seed culture of *Wickhamia lanceolata* was prepared by fermenting in YPD medium at 30℃ and 180 r / min for 48 h.

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

[0047] (5) Same as step (6) in Example 1.

[0048] Comparative Example 2: No abnormal Wickham yeast was inoculated; all other procedures were the same as in Example 1. The steps are as follows:

[0049] (1) Same as Example 1;

[0050] (2) Same as Example 1;

[0051] (3) Same as Example 1;

[0052] (4) After fermentation, solid-liquid separation is carried out to form solid and liquid parts of tobacco raw materials. The solid part and the added fiber are pulped separately and then mixed to form reconstituted tobacco pulp. The liquid part is fermented with Wickham yeast without any abnormalities.

[0053] (5) The liquid part is filtered by a 300nm ceramic membrane and then concentrated to obtain tobacco paste;

[0054] (6) Same as Example 1.

[0055] Comparative Example 3: No fermentation nutrients were added; all other steps were the same as in Example 1. The steps are as follows:

[0056] (1) Same as Example 1;

[0057] (2) Same as Example 1;

[0058] (3) Same as Example 1;

[0059] (4) After the first stage of fermentation, solid-liquid separation is carried out to form solid and liquid parts of tobacco raw materials. The solid part and the added fiber are pulped separately and then mixed to form reconstituted tobacco pulp. The liquid part of the tobacco is subjected to the second stage of liquid fermentation.

[0060] (5) The second stage of liquid fermentation for the liquid portion is as follows: tobacco extract is inoculated with abnormal Wickham yeast and fermented for 48 hours. The inoculum size is 4×(10) 7 ~10 8 cFu / 1000mL fermentation broth, fermentation temperature 30℃, stirring speed 10 rpm, aeration rate 7 L / h; no added fermentation nutrients;

[0061] (6) Same as Example 1;

[0062] (7) Same as Example 1.

[0063] Comparative Example 4: No bacterial strains were added; other conditions were the same as in Example 1. The steps are as follows:

[0064] (1) Same as Example 1;

[0065] (2) Mix tobacco raw materials with water at a weight ratio of 1:6 and heat to extract. The extraction temperature is 60℃ and the extraction time is 30min.

[0066] (3) After extraction, solid-liquid separation is carried out to form solid and liquid parts of tobacco raw materials. The solid part and the added fiber are pulped separately and then mixed to form reconstituted tobacco pulp. The liquid part is concentrated to obtain tobacco paste.

[0067] (4) The slurry obtained in step (3) is used to reconstitute tobacco leaf base. The obtained tobacco paste is coated on the leaf base and dried to obtain reconstituted tobacco leaf.

[0068] Aroma components were analyzed in the reconstituted tobacco leaves prepared in the examples and comparative examples. The reconstituted tobacco leaf samples were extracted with dichloromethane solvent and concentrated under reduced pressure to prepare a concentrated sample solution, 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. Sensory quality was also evaluated for the reconstituted tobacco leaves prepared in the examples and comparative examples, and the effects of each sample were assessed. The results are shown in Tables 1 and 2.

[0069] Table 1. Detection results of aroma components in reconstituted tobacco samples

[0070] serial number Relative content of aroma-producing components (%) Example 1 147.25±4.37 Example 2 122.18±7.38 Comparative Example 1 110.12±5.34 Comparative Example 2 112.54±7.04 Comparative Example 3 109.27±6.31 Comparative Example 4 100.41±5.14

[0071] Table 2 Sensory quality evaluation results of reconstituted tobacco samples

[0072] serial number Sensory quality evaluation results Example 1 The aroma is moderately strong and concentrated, with a delicate and mellow fragrance, minimal woodiness and burnt notes, a pleasant mouthfeel, and a sweet aftertaste. Example 2 The aroma is of medium to high quantity and concentration, with good clarity, less woody and burnt notes, a pleasant mouthfeel, and minimal residue. Comparative Example 1 The aroma quantity and concentration are above average, but the aroma clarity is slightly lacking. The woody, burnt, and other off-flavors are quite noticeable, and the spiciness and irritation are quite pronounced, leaving a noticeable residue in the mouth. Comparative Example 2 The aroma is of medium quantity and concentration, but slightly rough, with noticeable woody and burnt notes. It is slightly spicy in the mouth, quite irritating to the nasal cavity, and only moderately comfortable in the mouth. Comparative Example 3 The aroma is moderate in quantity and slightly above average in concentration. The aroma is a bit rough, with a slightly noticeable woody and burnt smell. It is slightly spicy in the mouth, quite irritating to the nasal cavity, and has average oral comfort. Comparative Example 4 The aroma is of medium quantity and concentration, but the aroma is rather rough and lacks clarity. The woody and burnt notes are quite noticeable, and the mouthfeel is quite spicy and has a noticeable aftertaste.

[0073] Table 1 shows that the reconstituted tobacco prepared in Example 1 had the best sensory quality when smoked, followed by Example 2. The sensory quality of Comparative Examples 1-3 was significantly lower than that of Example 1. Although the sensory quality of Comparative Examples 1-3 was better than that of Comparative Example 4, it was difficult to achieve the effects of Examples 1 and 2. Meanwhile, the comparison results of the changes in aroma component content in Table 1 also confirm that Example 1 had the best effect. Both Table 1 (changes in aroma component content) and Table 2 (comparison of sensory quality evaluation) confirm that the reconstituted tobacco prepared in Example 1 had the best sensory quality and the highest content of aroma components when smoked.

[0074] Table 3. Detection results of main aroma components in reconstituted tobacco leaves prepared in Example 1 and Comparative Example 4

[0075] Chinese name Comparative Example 4 / % Example 1 / % 4-Methoxy-1-butanol 2.26 4.78 Isoamyl alcohol 3.87 7.95 2,4-Dimethylheptane-4-ol 1.74 4.37 Iso-thormocycloenol 2.43 15.74 hexadecimal 2.78 9.67 2,3-Butanediol - 58.74 Eucalyptol - 16.47 Ginsenoyl cyclooxygenate - 9.36 benzyl alcohol 10.78 18.65 Non-ethylene glycol 4.31 6.91 Adonitol 3.16 6.54 Phenylacetyl alcohol - 148.32 furfuryl alcohol 3.67 5.92 TRANS-3-O-α-ionol - 86.34 methyl 3-hydroxyoctadecanoate - 42.97 Diethyl iminodiacetate 1.78 5.38 Octylethylene glycol mono-n-dodecyl ester 2.67 6.17 Dodecyl butyrate 1.64 4.06 Ethyl palmitate - 67.57 DL-pantolactone 10.67 18.64 Stearyl acetate - 43.97 3-(1H-benzimidazole-3-Om-2-yl)-3-hydroxypropionate 1.93 2.13 Dihydroactin 7.24 8.34 Dihydro-3-hydroxy-4,4-dimethyl-2(3H)furanone - 37.64 progesterone 27.34 31.93 15-Cyclohexanone - 51.67 2-Zahexanecycloone - 101.34 4-Methoxybut-2-one 0.91 1.63 E-5-Isopropyl-8-methyl-6,8-nonadien-2-one 6.24 7.64 2-Pyrrolidone 10.98 11.68 flavonoids from lacquer trees - 107.31

[0076] 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 4. Table 3 presents the detection results of some aroma components in the reconstituted tobacco prepared in Example 1 and Comparative Example 4. As shown in Table 3, after sequentially using *Aspergillus niger* and *Wickham's aberrant yeast* as a complex microbial community to ferment the reconstituted tobacco raw materials, the types and contents of volatile aroma components were increased, especially alcohols, esters, and ketones. Table 3 also shows that new aroma components, such as RANS-3-oxo-α-ionol, ethyl palmitate, and lacquer flavonoids, appeared after sequentially fermenting the reconstituted tobacco raw materials with *Aspergillus niger* and *Wickham's aberrant yeast*. This significantly improved the aroma quantity and quality when smoking the reconstituted tobacco.

[0077] The results of various embodiments and comparative examples confirm that Example 1 has the best effect. The two microorganisms, Aspergillus niger and Wickham yeast, can synergistically enhance each other, increasing the content of aroma components in the reconstituted tobacco sample and introducing new aroma components. At the same time, fermentation nutrients play a key role in this process.

[0078] 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 improving the quality of reconstituted tobacco leaves, characterized in that, Includes the following steps: (1) Mix the reconstituted tobacco raw materials evenly in a certain proportion to form reconstituted tobacco raw materials; (2) Sterilize the reconstituted tobacco raw material with water at a weight ratio of 1:(6-8); (3) Inoculate with Aspergillus niger seed liquid for the first stage of liquid fermentation for 36-48 hours, with an inoculum size of (1-7)×(10) 8 ~10 9 )cFu / 1000mL fermentation broth; fermentation temperature 30-40℃, stirring speed 10-30 rpm, aeration rate 150-200 L / h; (4) Solid-liquid separation yields a solid portion and a liquid portion; the solid portion and the added fiber are pulped separately and then mixed to form reconstituted tobacco pulp; the liquid portion undergoes a second stage of liquid fermentation, the fermentation steps are as follows: the liquid portion is inoculated with abnormal Wickham yeast seed liquid and fermentation nutrients are added, and fermentation continues for 48-50 hours; the inoculation amount is (3-8)×(10 7 ~10 8 The fermentation broth was prepared with cFU / 1000mL fermentation broth at a temperature of 25-35℃, a stirring speed of 5-10 rpm, and an aeration rate of 5-10 L / h. The fermentation nutrients were: glutamic acid (0.01-0.6) g / 1000mL fermentation broth and sodium citrate (0.05-0.2) g / 1000mL fermentation broth. (5) After the second stage of liquid fermentation in step (4) is completed, the liquid part after fermentation is filtered by a ceramic membrane of 200-500nm, and then concentrated to obtain tobacco paste. (6) The slurry obtained in step (4) is used to form a reconstituted tobacco leaf base. The tobacco paste obtained in step (5) is coated on the leaf base and dried to obtain the reconstituted tobacco leaf.

2. The method according to claim 1, characterized in that, Step (2) First autoclave, then autoclave.

3. The method according to claim 1, characterized in that, The method for preparing the Aspergillus niger seed liquid in step (3) is as follows: culture it in PDA solid medium at 28°C for 5 days, rinse it with sterile physiological saline and prepare a bacterial suspension.

4. The method according to claim 1, characterized in that, The preparation method of abnormal Wickham yeast seed liquid in step (4) is as follows: fermentation in YPD medium at 30°C and 180 r / min for 48 h.

5. Reconstituted tobacco leaves prepared by the method according to any one of claims 1-4.

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