Method for preparing active alkaloid through high-temperature short-range fermentation of tobacco stems and application of active alkaloid
The preparation of active alkaloids by high-temperature short-range fermentation of tobacco stalks and microwave extraction has solved the problems of the lack of tobacco stalk utilization and the long preparation cycle of alkaloids, realizing the efficient and low-consumption resource utilization of alkaloids and improving the vitality of tobacco roots and the quality of tobacco leaves.
Patent Information
- Application Number
- CN202511076664.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-01
- Publication Date
- 2025-11-21
Smart Images
Figure CN120989169A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of agricultural waste resource utilization technology, specifically to a method for preparing active alkaloids by high-temperature short-range fermentation of tobacco stalks and its application. Background Technology
[0002] Low root activity in tobacco plants restricts their absorption of water and nutrients from the soil, leading to slow growth, poor leaf development, and consequently, reduced yield. Furthermore, low root activity decreases the plant's resilience, making it more susceptible to damage from drought, diseases, and other adverse environmental conditions, thus affecting the chemical composition of the tobacco leaves and lowering their quality.
[0003] Alkaloids have great potential for improving plant root vitality. Alkaloids are a class of nitrogen-containing basic organic compounds widely found in nature. They possess base-like properties, most have complex ring structures, and nitrogen is mostly contained within the ring, exhibiting significant biological activity. In agricultural production, alkaloids can enhance plant adaptability to adversity and improve root vitality by regulating physiological processes within plants.
[0004] The large amount of tobacco stalks generated during tobacco cultivation is considered waste, and traditional treatment methods (such as incineration and landfill) not only waste resources but also pollute the environment. However, tobacco stalks are rich in various pyridine alkaloids and can be converted into active alkaloids in a short time through microbial metabolism, resulting in high production efficiency.
[0005] Currently, there are many methods for synthesizing alkaloids, but most focus on organic synthesis, which involves complex synthetic routes. Furthermore, the conformations of synthesized products may differ from those of natural products due to reaction conditions. In existing technologies, the source of active alkaloids mainly relies on extraction from medicinal plants (such as ephedra and clematis), but these medicinal plants are difficult to cultivate, costly, and have low scalability, making it difficult to meet the needs of large-scale agricultural applications. Many published patents or literature on the preparation of alkaloids are still in the laboratory stage, focusing mainly on alkaloid extraction without studying their targeted regulation of tobacco root vitality, making it difficult to directly guide field practices.
[0006] In summary, while existing technologies have reference value in the preparation of alkaloids, there is still a lack of an efficient, low-consumption technology for the resource utilization of tobacco stalks that can stably retain the activity of alkaloids, thus solving the problems of long cycle, high degradation rate, and serious pollution in existing methods. Summary of the Invention
[0007] In view of this, the present invention aims to solve the problems of the lack of tobacco stalk utilization, long preparation cycle of alkaloids, high cost and low conversion rate in the prior art, and provides a method for preparing active alkaloids by high temperature short-range fermentation of tobacco stalks, so as to realize the resource utilization of tobacco waste. At the same time, by optimizing the application process, the promoting effect of active alkaloids on tobacco root vitality is significantly improved.
[0008] To achieve the above objectives, the present invention adopts the following technical solution:
[0009] A method for preparing active alkaloids by high-temperature short-path fermentation of tobacco stalks includes the following steps:
[0010] (1) Pretreatment of tobacco stalks;
[0011] (2) Add regulatory components and inoculate with compound microbial agent, and adjust the initial pH of the fermentation system to 6.5-8.0 to obtain a mixture; the regulatory components consist of lime, wheat bran, magnesium sulfate and yeast extract, and the compound microbial agent consists of Bacillus subtilis, Bacillus zanthoxylum, Pseudomonas aeruginosa and Arthrobacter spheroidae.
[0012] (3) High-temperature short-range fermentation: Adjust the initial moisture content of the mixture to 55-65%, and ferment it in a semi-closed state to obtain fermented product; if necessary, turn the pile once during the fermentation process to promote uniform heat distribution and oxygen exchange.
[0013] (4) Extraction and enrichment of alkaloids: The fermentation product and the ethanol aqueous solution were mixed at a solid-liquid ratio of 1:10-1:30 and microwaved to obtain an extract. The extract was then cooled naturally or cooled to room temperature with cold water. The extract was filtered and centrifuged to remove impurities and obtain the alkaloid extract.
[0014] Further, the tobacco straw pretreatment operation in step (1) is as follows: the tobacco straw is crushed to a particle size of 5-15mm, and then the moisture content is adjusted to 30-40% to obtain wet straw material.
[0015] The above-mentioned solution of the present invention can increase the specific surface area of straw and improve the fermentation reaction activity, which is conducive to the penetration, fixation and growth of microorganisms.
[0016] Furthermore, in step (2), the amount of the controlled component added is 4-8% of the mass of the wet straw, and the amount of the compound microbial agent added is 3-5% of the mass of the wet straw.
[0017] Furthermore, the mass ratio of lime, wheat bran, magnesium sulfate, and yeast extract in the regulating components is 2:3:1:1.
[0018] Furthermore, the mass ratio of Bacillus subtilis, Bacillus zanthoxylum, Pseudomonas aeruginosa, and Arthrobacter spheroidae in the compound microbial agent is 2:2:3:3;
[0019] The concentration of the compound microbial agent in bacterial solution is not less than 10%. 7 The strain can be purchased from commercial microbial strain suppliers or isolated from tobacco rhizosphere soil or decomposed tobacco stalks (the isolation method refers to conventional procedures in the field). This invention does not involve biological preservation.
[0020] In the above-mentioned scheme of the present invention, Bacillus subtilis and Bacillus zanthoxylum mainly secrete cellulase (optimal temperature 60°C) to degrade the lignocellulose of tobacco stalks; Pseudomonas aeruginosa and Arthrobacter spp. promote the release of alkaloids.
[0021] Furthermore, the fermentation temperature in step (3) is 60±2℃ and the fermentation time is 60-90min.
[0022] Furthermore, in step (4), the volume ratio of ethanol to water in the ethanol-water mixture is 7:3.
[0023] The microwave power is 300-800W, the temperature is 50-70℃, and the microwave processing time is 15-30min;
[0024] The method for filtering and centrifuging to remove impurities is as follows: filter with a 100μm filter cloth, and then centrifuge at 3000rpm for 8-10min.
[0025] This invention also provides the application of the active alkaloids prepared by the above method in promoting tobacco root vitality, comprising the following steps:
[0026] Dilute the alkaloid extract to 50-75 mg / L, then mix it with humic acid carriers to prepare a functional seedling substrate; apply 60-120 liters of substrate per acre, 7-10 days after tobacco transplanting, and then apply once every 10-15 days for 2-3 consecutive applications.
[0027] Furthermore, the functional seedling substrate is composed of the following raw materials by dry weight: 30-50% coconut coir, 20-40% peat, 0.2-0.5% potassium humate, and the balance being a diluted alkaloid extract; the coconut coir and peat are used to adjust the air permeability and water retention.
[0028] The preparation method of the functional seedling substrate is as follows: the diluted alkaloid extract is premixed with potassium humate, and then evenly sprayed into a dry substrate composed of coconut coir and peat and stirred thoroughly to adjust the moisture content to 45-55%. After standing for 24 hours, the functional seedling substrate is obtained.
[0029] The beneficial effects of this invention are as follows:
[0030] (1) This invention utilizes high-temperature short-range fermentation of tobacco stalks and the synergistic metabolism of various advantageous microorganisms to significantly improve the yield and activity of alkaloids, shorten the fermentation cycle, and enhance the economic efficiency of the process.
[0031] (2) The present invention uses microwave-assisted impregnation method to extract alkaloids, which significantly improves extraction efficiency, shortens extraction time, and is conducive to the enrichment and purification of alkaloids, thereby enhancing the activity of the extract.
[0032] (3) This invention combines alkaloids with humic acid carriers to improve the slow-release performance and bioavailability of alkaloids, promote the recovery of tobacco root vitality and new root growth, enhance stress resistance, and improve the overall quality of tobacco.
[0033] (4) This invention utilizes tobacco straw waste resources, reduces environmental burden, and realizes high-value utilization of agricultural waste, which is in line with the concept of sustainable development. Attached Figure Description
[0034] Figure 1 This is a comparison diagram of the root systems of different transplanting schemes in Experiment Example 2 of the present invention. Detailed Implementation
[0035] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0036] Example 1
[0037] (1) Pretreatment of tobacco straw: Take dry tobacco straw, crush it to a particle size of 15mm, and adjust the moisture content to 35%.
[0038] (2) Inoculation with control components and microbial strains: The following control components were added to every 1000g of tobacco straw, accounting for 5.25% of the wet weight of the fermented product: lime (particle size <0.5cm): 1.5%; wheat bran: 2.25%; magnesium sulfate: 0.75%; yeast extract: 0.75%; and inoculation compound microbial agent mixture (total concentration 1×10⁻⁶). 8 The inoculum volume was 4% of the wet weight (CFU / mL).
[0039] (3) High-temperature short-range fermentation: After mixing evenly, the mixture is placed into a stainless steel reactor. The initial moisture content is adjusted to 60%, the fermentation temperature is 60℃, and the reaction time is 60min. The mixture is manually stirred once during the process.
[0040] (4) Extraction and enrichment of alkaloids: Take 200g of fermentation product, add 70% ethanol and water mixture at a solid-liquid ratio of 1:20, and process using a microwave reactor: power 500W, temperature set at 70℃, duration 20min, cool to room temperature and filter with 100μm filter cloth, and then centrifuge at 3000rpm for 10min to obtain alkaloid extract.
[0041] (5) Rhizosphere application method: Dilute the extract to a concentration of 50 mg / L, and irrigate 50 mL per hole, once on the 7th day and 21st day after tobacco transplanting.
[0042] Example 2
[0043] (1) Pretreatment of tobacco stalks: Take dry tobacco stalks, crush them to a particle size of 5 mm, and adjust the moisture content to 35%.
[0044] (2) Inoculation with control components and microbial strains: The following control components were added to every 1000g of tobacco straw, accounting for 5.25% of the wet weight of the fermented product: lime (particle size <0.5cm): 1.5%; wheat bran: 2.25%; magnesium sulfate: 0.75%; yeast extract: 0.75%; and inoculation compound microbial agent mixture (total concentration 1×10⁻⁶). 8 The inoculum volume was 4% of the wet weight (CFU / mL).
[0045] (3) High-temperature short-range fermentation: After mixing evenly, the mixture is placed into a stainless steel reactor. The initial moisture content is adjusted to 60%, the fermentation temperature is 60℃, and the reaction time is 75 minutes. The mixture is manually stirred once during the process.
[0046] (4) Extraction and enrichment of alkaloids: Take 200g of fermentation product, add 70% ethanol and water mixture at a solid-liquid ratio of 1:20, and process using a microwave reactor: power 500W, temperature set at 65℃, duration 20min; after cooling to room temperature, filter with 100μm filter cloth, and then centrifuge at 3000rpm for 10min to obtain a clear extract.
[0047] (5) Rhizosphere application: Dilute the alkaloid extract to 50 mg / L, mix with potassium humate, and spray evenly into the substrate. The mixture should be prepared as follows (dry weight percentage): peat 30%, coconut coir 40%, potassium humate 0.5%, with the remainder being the diluted alkaloid extract. Adjust the moisture content to 50%, let stand for 24 hours, then fill the trays with 150 mL of substrate per hole before transplanting. At the same transplanting time, apply again on days 7 and 15 (30 mL / hole).
[0048] Example 3
[0049] (1) Pretreatment of tobacco stalks: After the tobacco stalks are crushed, the particle size is controlled to be 5 mm and the initial moisture content is adjusted to 35%.
[0050] (2) Controlling components and inoculation: Add the following controlling components (total mass percentage of 7%) to every 1000g of tobacco straw: lime: 2%, wheat bran: 3%, magnesium sulfate: 1%, yeast extract: 1%; inoculate with a compound microbial agent mixture (concentration of 1x10⁻¹⁰). 8 The inoculum volume is 5% of the wet weight (CFU / mL).
[0051] (3) High-temperature short-distance fermentation: Adjust the humidity of the mixture to 60%, and ferment in a sealed container at 60℃ for 90 minutes, stirring manually once during the process. After fermentation, cool to room temperature for later use.
[0052] (4) Extraction and enrichment of alkaloids: Take 200g of fermentation material, add 70% ethanol-water solution (solid-liquid ratio 1:20), and perform microwave-assisted extraction at a power of 600W, a temperature of 60℃, and a processing time of 30min. The resulting extract is cooled naturally or cooled with cold water. After filtration with a 100μm filter cloth, centrifuge and then centrifuge at 3000rpm for 10min.
[0053] (5) Rhizosphere application: Dilute the alkaloid extract to 50 mg / L, mix with potassium humate, and spray evenly into the substrate. The mixture should be prepared as follows (dry weight percentage): peat 30%, coconut coir 40%, potassium humate 0.5%, with the remainder being the diluted alkaloid extract. Adjust the moisture content to 50%, let stand for 24 hours, then fill the trays with 150 mL of substrate per hole before transplanting. At the same transplanting time, apply again on days 7 and 15 (30 mL / hole).
[0054] Comparative Example 1
[0055] After crushing, the tobacco stalks were controlled to have a particle size of 5 mm, and the initial moisture content was adjusted to 35%. No high-temperature fermentation was performed; the remaining pretreatment was the same as in Example 3, followed by microwave-assisted impregnation. Specifically, a 70% ethanol-water solution (solid-liquid ratio 1:20) was added, the power was 800 W, the temperature was 60°C, and the treatment time was 30 min. The resulting extract was allowed to cool naturally or with cold water. After filtration through a 100 μm filter cloth, the extract was centrifuged, and then centrifuged again at 3000 rpm for 10 min. The alkaloid content was determined.
[0056] Comparative Example 2
[0057] Prepare a commercial seedling substrate, adjusting the moisture content to 50%, pH 7.0, and potassium humate to 0.5%. Adjust the moisture content to 50%, let it stand for 24 hours, then fill the trays with 150 mL of substrate per hole before transplanting. At the same transplanting time, apply a supplemental application (30 mL / hole) on days 7 and 15.
[0058] Comparative Example 3
[0059] Commercially available alkaloids were diluted to 50 mg / L and mixed with potassium humate. The mixture was then sprayed evenly into the substrate, with the following formulation (dry weight percentage): peat moss 30%, coconut coir 40%, potassium humate 0.5%, and the remainder being the diluted commercially available alkaloids. The moisture content was adjusted to 50%, and the mixture was allowed to stand for 24 hours before being used in trays. Each seedling was filled with 150 mL of substrate and transplanted. Reapplication (30 mL / plant) was applied at 7 days and 15 days after transplanting.
[0060] Experimental Example 1: Determination of Alkaloid Content
[0061] The alkaloid content in the extract was determined using the bromocresol green (BCG) colorimetric method. The absorbance was measured and substituted into the standard curve equation to calculate the alkaloid concentration in the extract. The results were expressed as mg / g dry weight. Each sample was analyzed in triplicate, and the average value was taken.
[0062] As shown in Table 1, the alkaloid content in all three examples was higher than that in the control group, and the alkaloid accumulation was the best in Example 3, indicating that the fermentation and extraction process can effectively increase the alkaloid concentration in tobacco stalks, with a significant improvement effect.
[0063] Table 1 Comparison of alkaloid content under different treatments
[0064] Example 1 Example 2 Example 3 Comparative Example 1 Alkaloid content (mg / g) 5.96±0.13 6.64±0.26 7.22±0.21 2.81±0.11
[0065] Experiment Example 2: Root Vigor Assessment After Transplanting
[0066] Compared to the comparative example, the increased root volume and root dry weight in the examples indicate a more complete and robust root structure, providing a physical basis for nutrient and water absorption by the plants. The enhanced root vitality suggests more active root function, enabling more efficient respiration and nutrient absorption. Example 3 showed the most significant effect, demonstrating that the combination of alkaloids prepared from tobacco straw through short-range high-temperature fermentation and the substrate significantly enhanced root vitality in transplanted tobacco plants.
[0067] Table 2. Effects of different treatment groups on root vigor after tobacco transplanting.
[0068]
[0069] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.
Claims
1. A method for preparing active alkaloids by high-temperature short-range fermentation of tobacco stalks, characterized in that, Includes the following steps: (1) Pretreatment of tobacco stalks; (2) Add regulatory components and inoculate with compound microbial agent to obtain a mixture; the regulatory components are composed of lime, wheat bran, magnesium sulfate and yeast extract, and the compound microbial agent is composed of Bacillus subtilis, Bacillus zanthoxylum, Pseudomonas aeruginosa and Arthrobacter spheroidae. (3) High-temperature short-distance fermentation: Adjust the initial moisture content of the mixture to 55-65% and carry out fermentation in a semi-closed state to obtain fermented product; (4) Extraction and enrichment of alkaloids: The fermentation product and ethanol aqueous solution were mixed at a solid-liquid ratio of 1:10-1:30 and microwaved to obtain an extract. The extract was then cooled to room temperature, filtered and centrifuged to remove impurities, and the alkaloid extract was obtained.
2. The method for preparing active alkaloids by high-temperature short-range fermentation of tobacco straw according to claim 1, characterized in that, The tobacco straw pretreatment operation in step (1) is as follows: the tobacco straw is crushed to a particle size of 5-15mm, and then the moisture content is adjusted to 30-40% to obtain wet straw material.
3. The method for preparing active alkaloids by high-temperature short-path fermentation of tobacco stalks according to claim 2, characterized in that, The amount of the regulating component added in step (2) is 4-8% of the mass of the wet straw, and the amount of the compound microbial agent added is 3-5% of the mass of the wet straw.
4. The method for preparing active alkaloids by high-temperature short-path fermentation of tobacco stalks according to claim 3, characterized in that, The mass ratio of lime, wheat bran, magnesium sulfate, and yeast extract in the regulating components is 2:3:1:
1.
5. The method for preparing active alkaloids by high-temperature short-path fermentation of tobacco stalks according to claim 4, characterized in that, The mass ratio of Bacillus subtilis, Bacillus zanthoxylum, Pseudomonas aeruginosa, and Arthroblastus in the compound microbial agent is 2:2:3:
3. The concentration of the compound microbial agent in bacterial solution is not less than 10%. 7 CFU / mL, the strain can be purchased from commercial microbial strain suppliers; or isolated from tobacco rhizosphere soil or decomposed tobacco stalks (the isolation method refers to conventional procedures in the art).
6. The method for preparing active alkaloids by high-temperature short-path fermentation of tobacco straw according to claim 1, characterized in that, The fermentation temperature in step (3) is 60±2℃ and the fermentation time is 60-90min.
7. The method for preparing active alkaloids by high-temperature short-path fermentation of tobacco straw according to claim 1, characterized in that, The microwave power in step (4) is 300-800W, the temperature is 50-70℃, and the microwave processing time is 15-30min; The method for filtering and centrifuging to remove impurities is as follows: filter with a 100μm filter cloth, and then centrifuge at 3000rpm for 8-10min.
8. The use of an active alkaloid prepared by the method of any one of claims 1-7 in promoting tobacco root vitality.
9. The application of an active alkaloid according to claim 8 in promoting tobacco root vitality, characterized in that, Includes the following steps: Dilute the alkaloid extract to 50-75 mg / L, then mix it with humic acid carriers to prepare a functional seedling substrate; apply 60-120 liters of substrate per acre, 7-10 days after tobacco transplanting, and then apply once every 10-15 days for 2-3 consecutive applications.
10. The application of an active alkaloid according to claim 9 in promoting tobacco root vitality, characterized in that, The functional seedling substrate is composed of the following raw materials by dry weight: 30-50% coconut coir, 20-40% peat, 0.2-0.5% potassium humate, and the remainder is a diluted alkaloid extract. The preparation method of the functional seedling substrate is as follows: the diluted alkaloid extract is premixed with potassium humate, and then evenly sprayed into a dry substrate composed of coconut coir and peat and stirred thoroughly. The moisture content is adjusted to 45-55%, and after standing for 24 hours, the functional seedling substrate is obtained.