A method for improving quality of tobacco stem by using ganoderma lucidum fermentation liquor and cellulase complex preparation
By treating tobacco stems with a compound preparation of Ganoderma lucidum fermentation broth and cellulase, the sensory defects of tobacco stems used in cigarettes have been resolved, the sensory quality and utilization rate of tobacco stems have been improved, the processing cost has been reduced, and the efficient utilization of biomass resources has been achieved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- CHINA TOBACCO SICHUAN IND CO LTD
- Filing Date
- 2023-05-25
- Publication Date
- 2026-05-19
AI Technical Summary
When tobacco stems are used in cigarettes, they have sensory defects such as a woody smell, irritation, and burning sensation, resulting in low utilization rate, waste of biomass resources, and environmental pollution.
The tobacco stems were fermented using a compound preparation of Ganoderma lucidum fermentation liquid and cellulase. The Ganoderma lucidum fermentation liquid degraded the lignin in the tobacco stems, and the cellulase improved the quality of the tobacco stems.
It significantly improves the sensory quality of tobacco stems and shreds, resulting in a delicate and sweet smoke with reduced woody and irritating properties, shorter fermentation time, lower processing costs, and increased fermentation efficiency.
Smart Images
Figure CN116725222B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of tobacco processing technology, and in particular to a method for improving the quality of tobacco stems using a compound preparation of Ganoderma lucidum fermentation liquid and cellulase. Background Technology
[0002] Tobacco stems, a natural component of tobacco leaves, account for approximately 25-30% of the total weight of tobacco leaves, with over 500,000 tons produced annually. Tobacco cell wall substances are important components of tobacco, with lignin being one of them. Lignin, along with cellulose, hemicellulose, and pectin, forms the backbone of tobacco. Processing tobacco stems into shreds for use in cigarettes improves filling performance, regulates smoke characteristics, and reduces tar content, making it an important part of cigarette formulation. However, because shredded tobacco produces certain sensory defects such as a woody aroma, irritation, and burning sensation when burned, its usage should not be too high, generally only added appropriately to lower-grade cigarettes. Currently, the utilization rate of tobacco stems is low, with a large amount being disposed of as waste, wasting significant biomass resources and causing environmental pollution.
[0003] Chemical analysis revealed that the lignin, pectin, cellulose, and hemicellulose in tobacco stems contribute to a woody, burnt appearance, resulting in insufficient aroma and a bland taste. These defects severely limit the blending ratio of tobacco stems in cigarettes and their suitability for high-end cigarettes, leading to the waste of a large quantity of stems. Therefore, processing tobacco stems is of significant practical importance for improving smoking quality and reducing production costs. Summary of the Invention
[0004] The purpose of this invention is to provide a method for improving the quality of tobacco stems using a compound preparation of Ganoderma lucidum fermentation liquid and cellulase to solve the aforementioned problems. The bio-enzyme compound preparation of this invention mainly affects the tobacco leaf aging process by shortening the aging time, increasing tobacco aroma substances, and reducing the raw, unpleasant odors and irritation to varying degrees, resulting in a more comfortable and clean taste, and effectively improving the sensory quality of cigarettes. Therefore, using fermentation liquid or enzyme preparations to improve the intrinsic quality of tobacco stems to enhance their usability is of great significance, as it can improve both the intrinsic quality and usability of tobacco stems.
[0005] The present invention achieves the above objectives through the following technical solutions:
[0006] A method for improving the quality of tobacco stems using a compound preparation of Ganoderma lucidum fermentation broth and cellulase includes the following steps:
[0007] Step 1: Prepare Ganoderma lucidum seed liquid, inoculate the Ganoderma lucidum seed liquid into a fermentation enzyme-producing medium to produce laccase, and measure the laccase activity;
[0008] Step 2: Determine the optimal temperature and moisture content for the degradation of tobacco stem lignin by Ganoderma lucidum fermentation broth;
[0009] Step 3: Determine the optimal amount of Ganoderma lucidum fermentation liquid to add;
[0010] Step 4: Determine the type and optimal amount of enzyme to be added;
[0011] Step 5: After mixing the Ganoderma lucidum fermentation liquid with the enzyme preparation, ferment the tobacco stems at an addition rate of 8.0% to 13.3%.
[0012] A further embodiment is that, in step 1, the fermentation enzyme-producing culture medium contains 20 g / L glucose, 5 g / L yeast extract, 10 g / L peptone, 1 g / L MgSO4·7H2O, 1 g / L KH2PO4, 0.1 g / L vitamin B, and 0.05% defoamer.
[0013] A further embodiment is that, in step 1, the inoculum size is 5-10%, the rotation speed is 120-180 rpm, and the temperature is 28-32℃, with continuous culture for 5-7 days.
[0014] A further method is as follows: In step 1, the enzyme activity assay is performed as follows: a 3 mL reaction system contains 0.5 mL of 2 mmol / L LABTS solution, 2 mL of 0.05 mol / L pH 3 disodium hydrogen phosphate-citric acid buffer, and 0.5 mL of crude enzyme solution (fermentation broth filtered through a 0.22 μm microporous membrane). The reaction is carried out at 45 °C for 5 min, and the OD is measured. 420 .
[0015] Laccase = 10 6 ×V 总 ×A 420 / ε×V 酶 ×Δt
[0016] In the formula:
[0017] ε — coefficient, with a value of 3.6 × 10 4 , L·(moL·cm) -1 ;
[0018] V 总 —Total volume of the reaction system for laccase activity assay, mL;
[0019] V 酶 —Volume of enzyme solution added, mL;
[0020] Δt — reaction time, min.
[0021] A further embodiment is that, in step 3, the fermentation broth is filtered using a plate and frame filter press with a microporous membrane of 30cm diameter and pore size of 100-1000um.
[0022] A further embodiment is that, in step 4, the enzyme preparations include cellulase and pectinase, wherein the cellulase is sourced from leaves (biotechnology grade, 400u / mg) and the pectinase is sourced from leaves (BR, 500u / mg).
[0023] A further proposed solution is that, in step 3, the amount of Ganoderma lucidum fermentation liquid added is 400u / kg.
[0024] A further embodiment is that, in step 2, the optimal temperature for the degradation of tobacco stem lignin by Ganoderma lucidum fermentation liquid is 50℃, the moisture content is 35%, and the treatment time is 6-8 hours.
[0025] In another aspect, the present invention provides a compound preparation of fermented tobacco leaves, which is prepared by the above-described method.
[0026] Another aspect of the present invention provides a compound preparation of fermented tobacco leaves, comprising Ganoderma lucidum fermentation liquid and cellulase, wherein the mass ratio of Ganoderma lucidum fermentation liquid to cellulase is 1:15-25; wherein the laccase activity of Ganoderma lucidum fermentation liquid is 100U / ml to 150U / ml, and the cellulase activity is 400U / mg.
[0027] A further embodiment is that the Ganoderma lucidum fermentation liquid in the compound preparation includes one or two of the following: (1) or (2):
[0028] (1) The microbial community and / or inoculants of Ganoderma lucidum strains;
[0029] (2) Cultures, lysates and / or extracts of Ganoderma lucidum strains.
[0030] The beneficial effects of this invention are as follows:
[0031] (1) The sensory quality of tobacco stems and shreds is significantly improved, the smoke is delicate and sweet, and the woody and irritating properties are significantly reduced.
[0032] (2) Compared with microbial fermentation of tobacco stems, the time is greatly shortened, the solid-state fermentation conditions are more relaxed, and the fermentation efficiency is effectively improved.
[0033] (3) Using Ganoderma lucidum fermentation liquid to degrade lignin greatly reduces processing costs and increases efficiency. Attached Figure Description
[0034] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0035] Figure 1This is a schematic diagram illustrating the effect of processing temperature on the degradation rate of lignin in tobacco stems according to the present invention.
[0036] Figure 2 This is a schematic diagram illustrating the effect of processing time on the degradation rate of lignin in tobacco stems according to the present invention. Detailed Implementation
[0037] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other implementation methods obtained by those skilled in the art without creative effort are within the scope of protection of this invention.
[0038] Example 1
[0039] In any embodiment, such as Figure 1-2 As shown, a method for improving the quality of tobacco stems using a compound preparation of Ganoderma lucidum fermentation broth and cellulase according to the present invention includes the following steps:
[0040] Step 1: Prepare Ganoderma lucidum seed liquid, inoculate the Ganoderma lucidum seed liquid into a fermentation enzyme-producing medium to produce laccase, and measure the laccase activity;
[0041] Step 2: Determine the optimal temperature and moisture content for the degradation of tobacco stem lignin by Ganoderma lucidum fermentation broth;
[0042] Step 3: Determine the optimal amount of Ganoderma lucidum fermentation liquid to add;
[0043] Step 4: Determine the type and optimal amount of enzyme to be added;
[0044] Step 5: Combine the Ganoderma lucidum fermentation liquid with enzyme preparations and then ferment the tobacco stems.
[0045] In step 1, the fermentation enzyme-producing culture medium contains 20 g / L glucose, 5 g / L yeast extract, 10 g / L peptone, 1 g / L MgSO4·7H2O, 1 g / L KH2PO4, 0.1 g / L vitamin B, and 0.05% defoamer.
[0046] In step 1, the inoculum size is 5-10%, the rotation speed is 120-180 rpm, and the temperature is 28-32℃, and the culture is carried out continuously for 5-7 days.
[0047] In step 1, the enzyme activity assay is performed as follows: a 3 mL reaction system contains 0.5 mL of 2 mmol / L ABTS solution, 2 mL of 0.05 mmol / L pH 3 disodium hydrogen phosphate-citric acid buffer, and 0.5 mL of crude enzyme solution (fermentation broth filtered through a 0.22 μm microporous membrane). The reaction is carried out at 45 °C for 5 min, and the OD is measured. 420 .
[0048] Laccase = 10 6 ×V 总 ×A 420 / ε×V 酶 ×Δt
[0049] In the formula:
[0050] ε — Coefficient, with a value of 3.6 × 10 4 , L·(moL·cm)- 1 ;
[0051] V 总 —Total volume of the reaction system for laccase activity assay, mL;
[0052] V 酶 —Volume of enzyme solution added, mL;
[0053] Δt — reaction time, min.
[0054] In step 3, the fermentation broth is filtered using a plate and frame filter press with a microporous membrane of 30cm diameter and 100-1000um pore size.
[0055] In step 4, the enzyme preparations include cellulase and pectinase, wherein the cellulase is source leaf extract (biotechnology grade, 400u / mg) and the pectinase is source leaf extract (BR, 500u / mg).
[0056] In step 3, the amount of Ganoderma lucidum fermentation liquid added is 400u / kg.
[0057] In step 2, the optimal temperature for the degradation of tobacco stem lignin by Ganoderma lucidum fermentation liquid is 50℃, the moisture content is 35%, and the treatment time is 6-8h.
[0058] In step 5, the compound preparation is added to tobacco stems at a rate of 8.0% to 13.3%, and can be diluted with water before spraying.
[0059] Application Example 1
[0060] (1) Effect of treatment temperature on lignin degradation
[0061] With the enzyme activity added to the Ganoderma lucidum fermentation broth at 4330 u / kg, the moisture content at 35%, and the treatment time at 6 h unchanged, the Ganoderma lucidum fermentation broth was mixed evenly with tobacco stems and then fermented at different temperatures. Five gradients were set: 30, 40, 50, 60, and 70 °C. After treatment, the tobacco stems were dried in an oven at 80 °C, and the lignin degradation rate of the tobacco stems was measured.
[0062] The Ganoderma lucidum fermentation broth was evenly sprayed onto the surface of tobacco stems at an addition rate of 4330 u / kg. After treatment in constant temperature and humidity chambers at different temperatures for 6 hours, the stems were dried in an 80℃ oven. The lignin content was determined using the acetyl bromide method. The results are shown below. Figure 1 The results show that reaction temperature has a significant impact on the degradation of lignin in tobacco stems by crude laccase solution. The degradation rate of lignin in tobacco stems gradually increases with increasing temperature; the degradation effect is best at 50℃; when the temperature exceeds 50℃, the degradation rate gradually decreases. Excessively high temperatures may denature the laccase, leading to poor lignin degradation.
[0063] (2) Effect of treatment time on lignin degradation
[0064] The enzyme activity of Ganoderma lucidum fermentation broth was kept constant at 4330 u / kg, moisture content at 35%, and treatment temperature at 50℃. After the Ganoderma lucidum fermentation broth was mixed evenly with tobacco stems, it was treated for different time periods, with five gradients set at 4, 6, 8, 10, and 12 hours. After the treatment was completed, the lignin degradation rate of the tobacco stems was measured.
[0065] Crude laccase solution was evenly sprayed onto the surface of tobacco stems at an addition rate of 4330 u / kg. The stems were then reacted in a 50℃ constant temperature and humidity chamber for different times (4, 6, 8, 10, 12 h), followed by drying in an 80℃ oven. The degradation rate of lignin was determined using the acetyl bromide method. The results are shown in [Figure number missing]. Figure 2 The results show that the degradation rate of lignin increases rapidly within 0–6 hours, gradually increases with time from 6–10 hours, and remains essentially unchanged from 10–12 hours. Considering the needs of industrial production, 6–8 hours is selected as the optimal time for treating tobacco stems.
[0066] (3) Effect of Ganoderma lucidum fermentation liquid addition on tobacco stem degradation effect
[0067] The treatment temperature was kept constant at 50℃, moisture content at 35%, and treatment time at 6h. The Ganoderma lucidum fermentation liquid was mixed evenly with tobacco stems at different addition amounts, with five gradients set: 0, 10, 200, 400, and 800 u / kg of tobacco stems. After the treatment was completed, experts were organized to conduct sensory evaluation.
[0068] The tobacco stems were evenly sprayed at dosages of 0, 10, 200, 400, 600, and 800 u / kg, and then placed in a 50℃ constant temperature and humidity chamber for 6 hours. After that, they were dried and inactivated in an 80℃ oven. Finally, the moisture content was adjusted to about 12% for sensory evaluation.
[0069] The results showed that 400 u / kg had the best sensory evaluation effect.
[0070] Ganoderma lucidum fermentation liquid addition amount u / kg Sensory evaluation results 0 As is 10 The burning sensation has been slightly reduced, the dryness and prickliness have been slightly improved, and the stalk flavor is stronger than before. 200 The burning sensation is slightly reduced, and the smoke is more moisturizing. 400 It has the lowest burning and dryness sensation, and the smoke is relatively sweet. 600 Compared to 400u / kg, the sensory experience deteriorates, and the smoke becomes unnatural and uncoordinated. 800 Compared to 400u / kg, the sensory quality deteriorates, the smoke becomes unnatural and uncoordinated, and the residue is heavy.
[0071] (4) Optimization of moisture content.
[0072] The processing temperature was kept constant at 50℃, the amount of Ganoderma lucidum fermentation liquid added was 400u / kg, and the processing time was kept constant at 6h. After the Ganoderma lucidum fermentation liquid was mixed evenly with the tobacco stems, it was treated at different moisture contents, with four gradients set at 25%, 30%, 35%, and 40%. After the treatment was completed, experts were organized to conduct sensory evaluation.
[0073] After the Ganoderma lucidum fermentation liquid was thoroughly mixed with tobacco stems, it was treated at different moisture contents, with four gradients set at 25%, 30%, 35%, and 40%. After treatment, experts conducted sensory evaluations. The results showed that the sensory evaluation effect was best at a moisture content of 35%.
[0074] Moisture content Sensory evaluation results 25％ The sweetness has increased a bit. 30％ The sweetness has increased a bit. 35％ The smoke is delicate, sweeter, and has a better taste. 40％ Sensations deteriorate.
[0075] (5) Optimization of cellulase compound.
[0076] The treatment temperature was kept constant at 50℃, the amount of Ganoderma lucidum fermentation broth added was 400u / kg, and the treatment time was 6h. Cellulase was mixed with Ganoderma lucidum fermentation broth at different dosages to prepare an aqueous solution, which was then mixed evenly with tobacco stems. Two gradients were set: 50u / g stems and 300u / g stems. After the treatment was completed, experts were organized to conduct sensory evaluation.
[0077] Sensory evaluation results showed that 50u / g of stem fibers had a good effect.
[0078] Cellulose compound addition amount u / g Sensory evaluation results 50 The smoke is natural and diffused, enhancing the sensory quality. 300 It has a good sweetness.
[0079] (6) Optimization of pectinase compound.
[0080] The treatment temperature was kept constant at 50℃, the amount of Ganoderma lucidum fermentation liquid added was 400u / kg, and the treatment time was kept constant at 6h. Pectinase was mixed evenly with tobacco stems at different addition levels, with two gradients set at 50u / g stems and 200u / g stems. After the treatment was completed, experts were organized to conduct sensory evaluation.
[0081] Sensory evaluation results showed that the addition of pectinase reduced the sensory quality.
[0082] Pectinase compound addition amount u / g Sensory evaluation results 50 It tastes good when paired with other ingredients, but its sensory quality is reduced after processing. 200 It has a strong burning sensation and a strong tingling sensation, and a good sweetness, but its sensory quality is reduced after processing.
[0083] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims. It should also be noted that the various specific technical features described in the above embodiments can be combined in any suitable manner without contradiction. To avoid unnecessary repetition, the present invention will not describe the various possible combinations separately. Furthermore, various different embodiments of the present invention can also be arbitrarily combined, as long as they do not violate the spirit of the present invention, they should also be considered as the content disclosed in the present invention.
Claims
1. A method for improving the quality of tobacco stems using a compound preparation of Ganoderma lucidum fermentation broth and cellulase, characterized in that, Includes the following steps: Step 1: Prepare Ganoderma lucidum seed liquid, inoculate the Ganoderma lucidum seed liquid into a fermentation enzyme-producing medium to produce laccase, and measure the laccase activity; The fermentation enzyme-producing medium consists of 20 g / L glucose, 5 g / L yeast extract, 10 g / L peptone, 1 g / L MgSO4•7H2O, 1 g / L KH2PO4, 0.1 g / L vitamin B, and 0.05% defoamer. The Ganoderma lucidum fermentation broth was prepared by filtering a microporous membrane with a diameter of 30cm and a pore size of 100~1000um using a plate and frame filter press. The laccase activity of the prepared Ganoderma lucidum fermentation broth was 100U / ml~150U / ml. Step 2: Determine the optimal temperature and moisture content for the degradation of tobacco stem lignin by Ganoderma lucidum fermentation broth; the optimal temperature for degradation of tobacco stem lignin by Ganoderma lucidum fermentation broth is 50℃, the optimal moisture content is 35%, and the optimal treatment time is 6~8h. Step 3: Determine the optimal amount of Ganoderma lucidum fermentation liquid to be added; the amount of Ganoderma lucidum fermentation liquid to be added is 400u / kg; Step 4: Determine the type and optimal amount of enzyme to be added; Step 5: Ferment tobacco stems by adding the compound preparation of Ganoderma lucidum fermentation liquid and enzyme preparation at an addition amount of 8.0%~13.3%; the compound preparation includes Ganoderma lucidum fermentation liquid and cellulase, wherein the mass ratio of Ganoderma lucidum fermentation liquid to cellulase is 1:15-25; wherein the laccase activity of Ganoderma lucidum fermentation liquid is 100U / ml~150U / ml, and the cellulase activity is 400U / mg.
2. The method for improving tobacco stem quality using a compound preparation of Ganoderma lucidum fermentation broth and cellulase as described in claim 1, characterized in that, In step 1, the inoculum size is 5-10%, the rotation speed is 120-180 rpm, and the temperature is 28-32℃, and the culture is carried out continuously for 5-7 days.
3. The method for improving tobacco stem quality using a compound preparation of Ganoderma lucidum fermentation broth and cellulase as described in claim 1, characterized in that, In step 1, the enzyme activity assay method is as follows: a 3 mL reaction system contains 0.5 mL of 2 mmol / L ABTS solution, 2 mL of 0.05 mmol / L disodium hydrogen phosphate-citric acid buffer solution with pH=3, and 0.5 mL of crude enzyme solution. The crude enzyme solution is filtered through a 0.22 µm microporous membrane using fermentation broth, and reacted at 45 °C for 5 min. The OD is then measured. 420 ; Laccase = 10 6 ×V 总 ×OD 420 / ε×V 酶 ×∆t; In the formula: ε—the value of the coefficient is 3.6 × 10 4 L•(moL•cm) -1 V 总 —Total volume of the reaction system for laccase activity assay, in mL; OD 420 —Measure the absorbance of the sample at a wavelength of 420 nm using a spectrophotometer; V 酶 —Volume of enzyme solution added, in mL; ∆t —Reaction time, in min.
4. The method for improving tobacco stem quality using a compound preparation of Ganoderma lucidum fermentation broth and cellulase as described in claim 1, characterized in that, In step 4, the enzyme preparations include cellulase and pectinase. The cellulase and pectinase are derived from the source leaves. The cellulase is a solid powder with an enzyme activity of 400 U / mg.
5. A compound preparation of fermented tobacco leaves, characterized in that, Made by the method comprising claim 1.
6. The compound formulation according to claim 5, characterized in that, The Ganoderma lucidum fermentation liquid in the compound preparation includes one or both of the following: (1) or (2): (1) The microbial community and / or inoculants of Ganoderma lucidum strains; (2) Cultures, lysates and / or extracts of Ganoderma lucidum strains.