A method for improving enzyme activity and uses thereof

Abstract

The present application relates to the field of tobacco, in particular to a method for improving enzyme activity and its use. The present application provides a method for improving enzyme activity, comprising: adding tobacco raw materials into water and mixing uniformly, then constant temperature stirring, solid-liquid separation, obtaining tobacco water extract; dissolving pectinase and / or hemicellulase in the tobacco water extract to obtain enzyme dissolution solution; the present application finds that the tobacco water extract can effectively improve the enzyme activity, further effectively play the function of enzyme preparation in the tobacco baking, alcoholization and fermentation stage, so as to improve the tobacco and promote the quality of tobacco, release the softness and delicacy of smoke, reduce the irritation of smoke.

Description

Technical Field

[0001] This invention relates to the tobacco industry, and more specifically to a method for improving enzyme activity and its uses. Background Technology

[0002] While reducing tar content in tobacco leaves through "harm reduction and tar reduction" processes, the aroma and quality of the leaves also decline. Tobacco leaves generally suffer from poor industrial adaptability, particularly in terms of insufficient aroma quality and excessive grassy or off-flavors. Large inventories of tobacco leaves severely restrict their economic benefits. Enzymes play a crucial role in the aging and fermentation process of tobacco leaves. Enzymatic reactions can improve the appearance of tobacco leaves, catalyze the decomposition or synthesis of certain substances, thereby enhancing the harmony of the chemical composition of the leaves, bringing out the aroma, making the taste more mellow, increasing the aroma of cigarettes, reducing off-flavors and irritation, and lowering harm, ultimately improving the sensory quality of cigarettes and achieving full utilization of the tobacco raw materials.

[0003] Due to the limited content and variety of endogenous enzymes in tobacco leaves, it is difficult to coordinate the quality of tobacco leaves in a short period of time. During the later stages of high-temperature curing, the activities of major enzymes such as pectinase, hemicellulase, and protease decrease sharply, or even disappear entirely. Many experts and scholars have conducted extensive research to address this issue, exploring methods such as flue-curing technology, enzyme preparations, and microbial fermentation to improve tobacco leaf quality. Bioenzymes are green and natural catalysts with characteristics such as strong substrate specificity, mild catalytic conditions, high catalytic efficiency, high biosafety, and significant effects, and have been widely used in the tobacco industry. In tobacco processing, bioenzymes can degrade and transform various macromolecules in tobacco leaves, such as proteins, pectin, and hemicellulose, reducing off-flavors and irritation. They can also promote the accumulation of small-molecule aroma compounds or flavor enhancers in tobacco leaves, improving their aroma and character. In particular, enzyme preparation technology, through enzymatic action during the aging and fermentation stage, continuously consumes and decomposes macromolecules in tobacco leaves, playing a crucial role in the production and formation of aroma compounds, thereby rapidly promoting changes in the aroma and flavor of cigarettes. Exogenous single enzymes and complex enzyme preparations can be used to degrade undesirable components in tobacco leaves and control the transformation of the internal components of tobacco leaves in the desired direction. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is to provide a method for improving enzyme activity and its use. The present invention uses tobacco leaf water extract as an enzyme solvent, which can effectively reduce the odor that may be produced by other chemical solvents, and can further improve enzyme activity, making the internal quality of tobacco leaves more coordinated. It can also effectively solve the problems and defects caused by poor cultivation or insufficient fermentation of tobacco leaves, fundamentally improve the grade of tobacco leaves, save costs in industrial production, and promote the application of the product.

[0005] Therefore, the present invention provides the following technical solution:

[0006] A method for improving enzyme activity includes:

[0007] S1. After adding tobacco leaf raw materials to water and mixing evenly, stir at a constant temperature and separate the solid and liquid to obtain tobacco leaf water extract;

[0008] S2. Dissolve pectinase raw materials and / or hemicellulase raw materials in tobacco leaf water extract to obtain enzyme solution.

[0009] Optionally, in step S1, the ratio of the tobacco raw material to water is (1-10):200, with a ratio of g / ml.

[0010] Optionally, in step S1, the tobacco raw material is aging C3F tobacco.

[0011] Optionally, in step S1, the conditions for the constant temperature stirring are: shaking for 30±5 minutes at a constant temperature shaker temperature of 25℃~30℃ and a speed of 180-220r / min.

[0012] And / or, the conditions for solid-liquid separation are: centrifugation at 8000-10000 r / min for 3-5 min, and a temperature setting of 4-6℃;

[0013] And / or, in step S1, the obtained tobacco water extract is filtered using a microporous membrane with a pore size of 0.22-0.25 μm.

[0014] Optionally, in step S2, when pectinase raw material is added alone, the mass-to-volume ratio of pectinase raw material to tobacco water extract is (4 / 300 to 8 / 300):1, preferably 5 / 300:1, with a ratio of g:ml; the pectinase raw material is 30000U / g;

[0015] And / or, in step S2, when hemicellulase raw material is added alone, the mass-to-volume ratio of hemicellulase raw material to tobacco water extract is (4 / 60 to 8 / 60):1, preferably 5 / 60:1, with a ratio of g:ml; the hemicellulase raw material is 6000U / g;

[0016] And / or, in step S2, when pectinase raw material and hemicellulase raw material are added simultaneously, the mass-to-volume ratio of pectinase raw material to tobacco water extract is (2 / 300 to 4 / 300):1, preferably 2.5 / 300:1, with a ratio of g:ml; the pectinase raw material is 30000 U / g; the mass-to-volume ratio of hemicellulase raw material to tobacco water extract is (2 / 60 to 4 / 60):1, preferably 2.5 / 60:1, with a ratio of g:ml; the hemicellulase raw material is 6000 U / g.

[0017] An enzyme solution obtained by the method described above for improving enzyme activity.

[0018] A method for treating tobacco leaves includes: spraying the enzyme solution onto the tobacco leaves at a ratio of 2-6 wt% (enzyme solution: tobacco leaf mass ratio), thoroughly mixing the tobacco leaves, and placing them at a constant temperature and humidity for 20-28 hours, with a temperature of 30±2℃ and a humidity of 60%±2%.

[0019] Preferably, the enzyme solution is sprayed onto the tobacco leaves at a ratio of 4 wt%.

[0020] A method for preparing cigarettes, using the obtained tobacco leaves.

[0021] Optionally, the tobacco leaves can be shredded.

[0022] A cigarette prepared by the cigarette preparation method described above.

[0023] The technical solution of this invention has the following advantages:

[0024] 1. This invention provides a method for improving enzyme activity, comprising: adding tobacco leaf raw materials to water and mixing evenly, then stirring at a constant temperature, and separating the solid and liquid to obtain a tobacco leaf aqueous extract; dissolving pectinase raw materials and / or hemicellulase raw materials in the tobacco leaf aqueous extract to obtain an enzyme solution; this invention has found that the tobacco leaf aqueous extract can promote the release of enzyme activity, effectively improve enzyme activity, and further effectively exert the functional role of enzyme preparations in the aging stage, thereby further improving tobacco leaves and promoting tobacco leaf quality, releasing a milder and finer smoke, and reducing the irritation of the smoke. Compared with enzyme preparations obtained from other solvents, this invention utilizes tobacco leaf aqueous extract to improve the activity of exogenous enzymes, thereby increasing the degradation rate of macromolecular substances in tobacco leaves and improving the quality of tobacco leaves. It has higher safety, better effect, can effectively reduce the odor that may be generated by other chemical solvents, and the raw material source is sufficient, reducing the cost of purchasing chemical solvents, and is suitable for large-scale industrial production.

[0025] 2. The tobacco leaf processing method provided by the present invention includes: spraying the enzyme solution onto the tobacco leaves, mixing the tobacco leaves, and processing the tobacco leaves. The treatment with the enzyme solution dissolved from the water extract of tobacco leaves can improve the degradation rate of macromolecular substances in the tobacco leaves, improve the quality of the tobacco leaves, and make the prepared cigarettes release a milder and finer smoke, reducing the irritation of the smoke. Attached Figure Description

[0026] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0027] Figure 1 This is the effect of different enzyme solvents on enzyme activity in Experiment 1 of this invention; in the figure, A) is the group with pectinase raw material added alone; B) is the group with hemicellulase raw material added alone; C) is the group with pectinase raw material + hemicellulase raw material; a and b indicate significant differences, p < 0.5;

[0028] Figure 2 These are the degradation of biomacromolecules in tobacco leaves by different enzyme solutions in Experiment Example 1 of this invention; a and b indicate significant differences, p < 0.5. Detailed Implementation

[0029] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0030] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0031] The pectinase (30000 U / g, Shanghai Maclean Biochemical Technology Co., Ltd.) and hemicellulase (6000 U / g, Shanghai Maclean Biochemical Technology Co., Ltd.) used in the following examples and experimental cases.

[0032] Example 1

[0033] This embodiment provides a method for improving enzyme activity, including the following steps:

[0034] (1) Weigh 1g of high-quality C3F tobacco leaves after alcoholization, cut them into small pieces with sterile scissors, and add them to an Erlenmeyer flask containing 200mL of sterile distilled water. Shake the flask at 30℃ and 200r / min for 30min on a constant temperature shaker (ZHWY211B, Shanghai Zhicheng Analytical Instruments Manufacturing Co., Ltd.). Centrifuge the crude liquid after shaking using a high-speed refrigerated centrifuge (D-37520 type, Thermo Fisher Scientific GmbH, Germany) at 4℃ and 8000r / min for 3min. Filter the centrifuged liquid through a PES 0.22μm microporous membrane into an Erlenmeyer flask. Use the filtrate from the Erlenmeyer flask as the tobacco leaf water extract with a concentration of 0.5%. Then, use a pipette to obtain the tobacco leaf water extract sample. The concentration of the tobacco leaf water extract sample = mass of tobacco leaf raw material (g) / volume of water added (ml) × 100%.

[0035] (2) Dissolve the pectinase raw material in the tobacco leaf water extract sample (the mass-volume ratio of pectinase raw material to tobacco leaf water extract is 5 / 300:1, and the ratio is g:ml). Then, put it into an ultrasonic oscillator (model SY-300, Shanghai Ningshang Ultrasonic Instrument Co., Ltd.) to fully dissolve it and obtain the enzyme solution.

[0036] Example 2

[0037] (1) Weigh 6g of high-quality C3F tobacco leaves after aging, cut them into small pieces with sterile scissors, and add them to an Erlenmeyer flask containing 200mL of sterile distilled water. Shake the flask at 25℃ and 220r / min for 35min. Centrifuge the crude liquid after shaking using a high-speed refrigerated centrifuge (D-37520 type, Thermo Fisher Scientific, Germany) at 4℃ and 9000r / min for 5min. Filter the centrifuged liquid through a PES 0.25μm microporous membrane into an Erlenmeyer flask. Use the filtrate from the Erlenmeyer flask as the tobacco leaf water extract with a concentration of 3%. Then, pipette the extract into a test tube to obtain the tobacco leaf water extract sample.

[0038] (2) Weigh the pectinase raw material and dissolve it in the tobacco leaf water extract sample (the mass-volume ratio of pectinase raw material and tobacco leaf water extract is 4 / 300:1, and the ratio is g:ml). Then put it into an ultrasonic oscillator (model SY-300, Shanghai Ningshang Ultrasonic Instrument Co., Ltd.) to fully dissolve it and obtain the enzyme solution.

[0039] Example 3

[0040] (1) Weigh 10g of high-quality C3F tobacco leaves after aging, cut them into small pieces with sterile scissors, and add them to an Erlenmeyer flask containing 200mL of sterile distilled water. Shake the flask at 28℃ and 180r / min for 35min on a constant temperature shaker (ZHWY211B, Shanghai Zhicheng Analytical Instruments Manufacturing Co., Ltd.). Centrifuge the crude liquid after shaking using a high-speed refrigerated centrifuge (D-37520 type, Thermo Fisher Scientific GmbH, Germany) at 4℃ and 10000r / min for 3min. Filter the centrifuged liquid through a PES 0.22μm microporous membrane into an Erlenmeyer flask. Use the filtrate from the Erlenmeyer flask as the tobacco leaf water extract with a concentration of 5%. Then, pipette the extract into a test tube to obtain the tobacco leaf water extract sample.

[0041] (2) Weigh the pectinase raw material and dissolve it in the tobacco leaf water extract sample (the mass-volume ratio of pectinase raw material and tobacco leaf water extract is 8 / 300:1, and the ratio is g:ml). Then put it into an ultrasonic oscillator (model SY-300, Shanghai Ningshang Ultrasonic Instrument Co., Ltd.) to fully dissolve it and obtain the enzyme solution.

[0042] Example 4

[0043] The difference between this embodiment and embodiment 1 is that the enzyme raw material in step (2) is hemicellulase raw material, and the mass-volume ratio of hemicellulase raw material and tobacco leaf water extract is 5 / 60:1, with a ratio of g:ml.

[0044] Example 5

[0045] The difference between this embodiment and embodiment 2 is that the enzyme raw material in step (2) is hemicellulase raw material, and the mass-volume ratio of hemicellulase raw material and tobacco leaf water extract is 4 / 60:1, with a ratio of g:ml.

[0046] Example 6

[0047] The difference between this embodiment and embodiment 3 is that the enzyme raw material in step (2) is hemicellulase raw material, and the mass-volume ratio of hemicellulase raw material and tobacco leaf water extract is 8 / 60:1, with a ratio of g:ml.

[0048] Example 7

[0049] The difference between this embodiment and embodiment 1 is that the enzyme raw materials in step (2) are hemicellulase raw materials and pectinase raw materials. The mass-volume ratio of pectinase raw materials to tobacco water extract is 2.5 / 300:1, and the mass-volume ratio of hemicellulase raw materials to tobacco water extract is 2.5 / 60:1. The ratio is g:ml.

[0050] Example 8

[0051] The difference between this embodiment and embodiment 2 is that the enzyme raw materials in step (2) are hemicellulase raw materials and pectinase raw materials. The mass-volume ratio of pectinase raw materials to tobacco water extract is 2 / 300:1, and the mass-volume ratio of hemicellulase raw materials to tobacco water extract is 2 / 60:1. The ratio is g:ml.

[0052] Example 9

[0053] The difference between this embodiment and embodiment 3 is that the enzyme raw materials in step (2) are hemicellulase raw materials and pectinase raw materials. The mass-volume ratio of pectinase raw materials to tobacco water extract is 4 / 300:1, and the mass-volume ratio of hemicellulase raw materials to tobacco water extract is 4 / 60:1. The ratio is g:ml.

[0054] Example 10

[0055] This embodiment provides a method for preparing cigarettes using Example 1, including the following steps: spraying the enzyme solution of Example 1 onto tobacco leaves, wherein the mass ratio of the enzyme solution to tobacco leaves is 1g:25g, mixing the tobacco leaves, and placing them at a constant temperature and humidity for 24 hours, with a temperature of 30±2℃ and a humidity of 60%±2%.

[0056] Example 11

[0057] This embodiment provides a method for preparing cigarettes using Example 2, including the following steps: spraying the enzyme solution of Example 2 onto tobacco leaves, wherein the mass ratio of the enzyme solution to the tobacco leaves is 1g:50g, mixing the tobacco leaves, and placing them at a constant temperature and humidity for 28 hours, with a temperature of 30±2℃ and a humidity of 60%±2%.

[0058] Example 12

[0059] This embodiment provides a method for preparing cigarettes using Example 3, including the following steps: spraying the enzyme solution of Example 3 onto tobacco leaves, wherein the mass ratio of the enzyme solution to tobacco leaves is 3g:50g, mixing the tobacco leaves, and placing them at a constant temperature and humidity for 20 hours, with a temperature of 30±2℃ and a humidity of 60%±2%.

[0060] Example 13

[0061] This embodiment provides a method for preparing cigarettes using Example 4, including the following steps: spraying the enzyme solution of Example 4 onto tobacco leaves, wherein the mass ratio of the enzyme solution to the tobacco leaves is 1g:25g, mixing the tobacco leaves, and placing them at a constant temperature and humidity for 24 hours, with a temperature of 30±2℃ and a humidity of 60%±2%.

[0062] Example 14

[0063] This embodiment provides a method for preparing cigarettes using Example 5, including the following steps: spraying the enzyme solution of Example 5 onto tobacco leaves, wherein the mass ratio of the enzyme solution to the tobacco leaves is 1g:50g, mixing the tobacco leaves, and placing them at a constant temperature and humidity for 20h, with a temperature of 30±2℃ and a humidity of 60%±2%.

[0064] Example 15

[0065] This embodiment provides a method for preparing cigarettes using Example 6, including the following steps: spraying the enzyme solution of Example 6 onto tobacco leaves, wherein the mass ratio of the enzyme solution to the tobacco leaves is 3g:50g, mixing the tobacco leaves, and placing them at a constant temperature and humidity for 28 hours, with a temperature of 30±2℃ and a humidity of 60%±2%.

[0066] Example 16

[0067] This embodiment provides a method for preparing cigarettes using Example 7, including the following steps: spraying the enzyme solution of Example 7 onto tobacco leaves, wherein the mass ratio of the enzyme solution to tobacco leaves is 1g:25g, mixing the tobacco leaves, and placing them at a constant temperature and humidity for 24 hours, with a temperature of 30±2℃ and a humidity of 60%±2%.

[0068] Example 17

[0069] This embodiment provides a method for preparing cigarettes using Example 8, including the following steps: spraying the enzyme solution of Example 8 onto tobacco leaves, wherein the ratio of the enzyme solution to tobacco leaves is 1g:50g by mass, mixing the tobacco leaves, and placing them at a constant temperature and humidity for 20 hours, with a temperature of 30±2℃ and a humidity of 60%±2%.

[0070] Example 18

[0071] This embodiment provides a method for preparing cigarettes using Example 9, including the following steps: spraying the enzyme solution of Example 9 onto tobacco leaves, wherein the ratio of the enzyme solution to tobacco leaves is 3g:50g, mixing the tobacco leaves, and placing them at a constant temperature and humidity for 28 hours, with a temperature of 30±2℃ and a humidity of 60%±2%.

[0072] Experimental Example

[0073] 1. Investigation on the effect of tobacco leaf water extract on enzyme activity

[0074] Experimental group: (1) Weigh 1.0g of high-quality C3F tobacco raw material after alcoholization using a balance with an accuracy of 0.01g. After cutting it into small pieces with sterile scissors, add it to a conical flask containing 200mL of sterile distilled water. Shake the flask at 30℃ and 200r / min for 30min. Centrifuge the crude original liquid after shaking using a high-speed refrigerated centrifuge (D-37520 type high-speed refrigerated centrifuge, Thermo Fisher Scientific GmbH, Germany). The temperature is set at 4℃ and centrifuged at 8000r / min for 3min. After centrifugation, filter the liquid through a PES 0.22μm microporous membrane into a conical flask. Use the filtrate from the conical flask as the tobacco water extract. The concentration of the tobacco water extract is 0.5%. Use a pipette to add it to a test tube to obtain the tobacco water extract sample. The concentration of water extract of tobacco leaves = weight of raw tobacco leaves (g) / volume of water added (ml) × 100%.

[0075] Control group: 4.202g of citric acid solution and 5.882g of sodium citrate solution were pipetted into an Erlenmeyer flask and brought to a final volume of 200ml. The mixture was shaken at 30℃ and 200r / min for 5min to obtain a citric acid-sodium citrate solution with pH 4.5.

[0076] Preparation of enzyme solutions (samples): Solutions from the experimental and control groups were used as enzyme solvents to dissolve pectinase raw materials, hemicellulase raw materials, or a mixture of both. For pectinase raw materials alone, the mass-to-volume ratio of pectinase raw materials to enzyme solvent was 5 / 300:1 (g:ml). For hemicellulase raw materials alone, the mass-to-volume ratio of hemicellulase raw materials to tobacco leaf water extract was 5 / 60:1 (g:ml). When pectinase raw materials and hemicellulase raw materials were mixed, the mass-to-volume ratio of pectinase raw materials to tobacco leaf water extract was 2.5 / 300:1, and the mass-to-volume ratio of hemicellulase raw materials to tobacco leaf water extract was 2.5 / 60:1 (g:ml). After adding the enzyme raw materials to the enzyme solvent, they were then placed in an ultrasonic vibrator (model SY-300, Shanghai Ningshang Ultrasonic Instrument Co., Ltd.) for complete dissolution (ultrasonic conditions: room temperature, ultrasonic time: 10 min).

[0077] Pectinase activity was detected using visible spectrophotometry. The spectrophotometer was preheated, the wavelength was adjusted to 540 nm, and the sample was zeroed with distilled water. A 125 μL sample was taken, and the absorbance at 540 nm was measured. The pectinase activity (U / mol) was calculated using the following formula: Pectinase activity (U / mol): X × n / t / V. Where: X - D-galacturonic acid produced by enzyme reaction (u / mol); n - sample dilution factor; t - reaction time (min); V - sample volume (ml).

[0078] Hemicellulase activity was detected using a hemicellulase kit. A standard curve was plotted using the following equation: y = 1.0584x - 0.0282, where x is the molar mass of the standard (μmol) and y is ΔA. Hemicellulase activity was calculated based on liquid volume using the following formula: Hemicellulase activity (nmol / min / mL) = [(ΔA + 0.0282) ÷ 1.0584 × 10⁻⁶] 3 ]÷V1÷T×D=157.5×(ΔA+0.0282)×D, where: T---reaction time, 60min; molecular weight of xylose---150.131; V1---volume of sample added, 0.1mL; D---dilution factor.

[0079] When testing the pectinase activity or hemicellulase activity in the enzyme solution of a composite enzyme consisting of pectinase and hemicellulase, the pectinase activity or hemicellulase activity in the enzyme solution of the composite enzyme should be tested according to the method described above.

[0080] The results are shown in the table below, along with representative results. Figure 1 .

[0081] Table 1. Effects of different enzyme solvents on enzyme activity (n=3)

[0082]

[0083]

[0084] As shown in the table above, different solvents have a significant impact on enzyme activity. The activity of pectinase in the aqueous extract of tobacco leaves is 292.81 U / ml, which is significantly higher than that in the citrate-sodium citrate medium. The activity of hemicellulase in the aqueous extract of tobacco leaves is significantly higher than that in the citrate-sodium citrate environment. The complex enzyme composed of pectinase and hemicellulase has the highest activity in the aqueous extract of tobacco leaves, which is significantly different from that in the citrate-sodium citrate medium treatment.

[0085] 2. Degradation of biomacromolecules in tobacco leaves by different enzyme solutions

[0086] The enzyme solution of the complex enzyme dissolved in the tobacco leaf water extract or the enzyme solution of the complex enzyme dissolved in sodium citrate (as a control, the amount of complex enzyme is the same as that of the complex enzyme dissolved in the tobacco leaf water extract) was evenly sprayed onto the tobacco leaves, and the tobacco leaves were mixed well. The enzyme solution was sprayed onto the tobacco leaves at a ratio of 4 wt%. The experiment was repeated 3 times.

[0087] The contents of three types of biomolecules—pectin, hemicellulose, and protein—in the treated tobacco leaves were detected by visible spectrophotometry (using the Shanghai Sinobestbio kit).

[0088] The results are shown in the table below, along with representative results. Figure 2 (Figure a and b indicate significant differences, p < 0.5).

[0089] Table 2. Degradation of biomacromolecules in tobacco leaves by different enzyme solvents (n=3)

[0090]

[0091] As can be seen from the table above, compared with sodium citrate enzyme solvent, the aqueous extract of tobacco leaves has a better degradation effect on macromolecules. In particular, there are significant differences between the aqueous extract and the sodium citrate enzyme solvent in terms of protein and hemicellulose content. The protein and hemicellulose content are reduced by 1.97% and 2.02%, respectively. The pectin content of the sodium citrate enzyme solvent is 3.95%, which is 1.13% different from that of the aqueous extract of tobacco leaves.

[0092] 3. Effects of different enzyme solutions on the sensory quality of tobacco leaves

[0093] The enzyme solution of the complex enzyme dissolved in the tobacco leaf water extract or the enzyme solution of the complex enzyme dissolved in citric acid-sodium citrate (as a control, the amount of complex enzyme is the same as that of the complex enzyme dissolved in the tobacco leaf water extract) was evenly sprayed onto the tobacco leaves, and the tobacco leaves were mixed well. The enzyme solution or distilled water was sprayed onto the tobacco leaves at a ratio of 4 wt%.

[0094] The processed tobacco leaves were shredded and made into cigarettes for evaluation by the expert panel.

[0095] The scoring criteria for aroma quality, aroma intensity, off-flavors, irritation, transparency, smoothness, sweetness, aftertaste, concentration, and strength are as follows:

[0096] Table 3 Scoring Criteria

[0097]

[0098] The results are shown in the table below.

[0099] Table 4. Effects of different treatments on the sensory quality of tobacco leaves

[0100] CK 5.23b 5.21c 5.17b 5.13b 5.27a 5.16b M1-0.5％ 5.43a 5.46a 5.40a 5.37a 5.30a 5.40a M2 5.24b 5.31b 5.23b 5.08b 5.31a 5.37a

[0101] Table 4 shows the effects of different treatments on the sensory quality of tobacco leaves.

[0102] CK 5.17b 5.20b 5.63a 5.63a 52.8b M1-0.5％ 5.30a 5.37a 5.70a 5.73a 54.40a M2 5.27a 5.23b 5.67a 5.70a 53.41b

[0103] Note: M1 was treated with an enzyme solution containing 0.5% tobacco leaf water extract to dissolve the complex enzyme; M2 was treated with an enzyme solution containing citric acid-sodium citrate solution to dissolve the complex enzyme. a and b indicate significant differences (p < 0.5).

[0104] The effects of different treatments on the sensory quality of tobacco leaves are shown in Table 4. Compared with the control, the M1 treatment significantly increased the quality indicators of tobacco leaves, such as aroma quality, aroma quantity, permeability, smoothness, sweetness and concentration. Moreover, there were significant differences among the treatments. Compared with the M2 treatment, the M1 treatment significantly reduced off-odors and irritation, and the M1 treatment had the highest total score.

[0105] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for improving enzyme activity, characterized in that, include: S1. After mixing the tobacco leaf raw material with water until uniform, the mixture is stirred at a constant temperature and then the solid and liquid are separated to obtain a water extract of tobacco leaves. The tobacco leaf raw material is ethanolated C3F tobacco leaves. The ratio of tobacco leaf raw material to water is (1-10):200, with a ratio of g / ml. The conditions for constant temperature stirring are: shaking at 180-220 r / min for 30±5 min at a constant temperature of 25℃~30℃. The conditions for solid-liquid separation are: centrifugation at 8000-10000 r / min for 3-5 min at a temperature of 4-6℃. The obtained water extract of tobacco leaves is filtered through a microporous membrane with a pore size of 0.22-0.25μm. S2. Dissolve pectinase raw material and / or hemicellulase raw material in tobacco leaf aqueous extract to obtain an enzyme solution; the tobacco leaf aqueous extract can promote the release of enzyme activity. Spraying the enzyme solution onto tobacco leaves, mixing the tobacco leaves, and treating the tobacco leaves can improve the degradation rate of macromolecular substances in the tobacco leaves; when pectinase raw material is added alone, the mass-volume ratio of pectinase raw material to tobacco leaf aqueous extract is (4 / 300~8 / 300):1, with a ratio of g:ml; the pectinase raw material is 30000 U / g; Alternatively, when hemicellulase raw material is added alone, the mass-to-volume ratio of hemicellulase raw material to tobacco water extract is (4 / 60~8 / 60):1, with a ratio of g:ml; the hemicellulase raw material is 6000 U / g; Alternatively, when pectinase raw material and hemicellulase raw material are added simultaneously, the mass-to-volume ratio of pectinase raw material to tobacco water extract is (2 / 300~4 / 300):1, with a ratio of g:ml; the pectinase raw material is 30000 U / g; the mass-to-volume ratio of hemicellulase raw material to tobacco water extract is (2 / 60~4 / 60):1, with a ratio of g:ml; the hemicellulase raw material is 6000 U / g.

2. The method for improving enzyme activity according to claim 1, characterized in that, In step S2, when pectinase raw material is added alone, the mass-to-volume ratio of pectinase raw material to tobacco water extract is 5 / 300:1, with a ratio of g:ml; the pectinase raw material is 30000 U / g. And / or, in step S2, when the hemicellulase raw material is added alone, the mass-to-volume ratio of the hemicellulase raw material to the tobacco water extract is 5 / 60:1, with a ratio of g:ml; the hemicellulase raw material is 6000 U / g; And / or, in step S2, when pectinase raw material and hemicellulase raw material are added simultaneously, the mass-to-volume ratio of pectinase raw material to tobacco water extract is 2.5 / 300:1, with a ratio of g:ml; the pectinase raw material is 30000 U / g; the mass-to-volume ratio of hemicellulase raw material to tobacco water extract is 2.5 / 60:1, with a ratio of g:ml; the hemicellulase raw material is 6000 U / g.

3. An enzyme solution obtained by the method for improving enzyme activity as described in any one of claims 1-2.

4. A method for processing tobacco leaves, characterized in that, include: The enzyme solution described in claim 3 is sprayed onto tobacco leaves at a ratio of 2-6 wt%, the tobacco leaves are thoroughly mixed, and the mixture is placed at a constant temperature and humidity for 20-28 hours (temperature 30±2℃, humidity 60%±2%).

5. The tobacco leaf processing method according to claim 4, characterized in that, The enzyme solution was sprayed onto the tobacco leaves at a ratio of 4 wt%.

6. A method for preparing cigarettes, characterized in that, The tobacco leaves obtained according to claim 4 or 5 are used.

7. The cigarette manufacturing method according to claim 6, characterized in that, The tobacco leaves are shredded.

8. A cigarette prepared by the cigarette preparation method according to claim 6 or 7.

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