Tobacco ripening-resistant agent as well as preparation method and application thereof

Abstract

The invention relates to the technical field of tobacco planting, in particular to a tobacco ripening-resistant agent and a preparation method and application thereof. The tobacco ripening resistance agent is prepared from 0.06 mmol / L to 0.10 mmol / L of beta-mercaptoethanol, l-ascorbic acid with a concentration of 0.06 to 0.10 mmol / L; the concentration of the L-glutamic acid is 0.008 to 0.012 mmol / L; the concentration of the L-tryptophan is 0.008 to 0.012 mmol / L; the concentration of 5-aminolevulinic acid is 0.008 to 0.012 mmol / L; copper sulfate with a concentration of 0.02 to 0.05 mmol / L; polyethylene glycol 400 with a concentration of 0.05 to 0.10 mmol / L; tween 20 with the concentration of 0.002 to 0.004 mmol / L is added And the balance of water. Through the synergistic effect of multiple components, the tobacco ripening-resistant agent shows remarkable comprehensive benefits in the aspects of delaying tobacco ripening, improving tobacco quality, enhancing environmental adaptability, ensuring safety and the like, an efficient and environment-friendly novel solution is provided for tobacco planting, and the tobacco ripening-resistant agent has wide application prospects.

Description

Technical Field

[0001] The invention relates to the technical field of tobacco planting, and in particular to a tobacco ripening-resistant agent and a preparation method and application thereof. Background Art

[0002] With the continuous development of tobacco planting technology, improving tobacco leaf quality and yield has always been the focus of the industry. In the later stage of tobacco leaf growth, controlling the maturity rate of tobacco leaves is crucial to improving tobacco leaf quality. Traditional maturity-resistant technology mainly relies on agronomic measures, such as timely topping, reasonable pruning and controlled watering. However, these methods are often affected by environmental factors and the effects are not stable enough.

[0003] In recent years, the application of chemical anti-ripening agents has provided new ideas for tobacco leaf anti-ripening. Currently, the commonly used chemical anti-ripening agents mainly include growth regulators and anti-aging substances. Growth regulator anti-ripening agents, such as gibberellins and cytokinins, can delay tobacco leaf aging, but they often cause excessive growth of tobacco leaves, affecting the intrinsic quality of tobacco leaves. Anti-aging substances, such as salicylic acid and methyl jasmonic acid, can delay tobacco leaf aging to a certain extent, but the effect lasts for a short time and has limited effect on improving tobacco leaf quality.

[0004] In addition, existing chemical anti-ripening agents generally have the following problems: first, it is difficult to fully regulate the complex physiological changes during the aging process of tobacco leaves; second, they have high requirements for environmental conditions and their effects are unstable under extreme weather conditions; third, some anti-ripening agents may remain in the tobacco leaves, affecting their safety; finally, existing anti-ripening agents often only focus on delaying aging, while ignoring the overall improvement of tobacco leaf quality.

[0005] In view of the shortcomings of existing technologies, there is an urgent need to develop a new type of tobacco maturation-resistant agent that can not only effectively delay the maturation of tobacco leaves, but also comprehensively improve the quality of tobacco leaves, while having good environmental adaptability and safety. Summary of the invention

[0006] In order to solve or partially solve the problems existing in the related art, the present invention provides a tobacco ripening-resistant agent and a preparation method and application thereof.

[0007] The present invention provides a tobacco ripening-resistant agent, which comprises: 0.06-0.10 mmol / L β-mercaptoethanol; 0.06-0.10 mmol / L L-ascorbic acid; 0.008-0.012 mmol / L of L-glutamic acid; 0.008-0.012 mmol / L of L-tryptophan; 0.008-0.012 mmol / L 5-aminolevulinic acid; 0.02-0.05 mmol / L copper sulfate; 0.05-0.10 mmol / L polyethylene glycol 400; 0.002-0.004 mmol / L Tween 20; and the balance of water.

[0008] Furthermore, the molar ratio of the β-mercaptoethanol to L-ascorbic acid is 0.9-1.1:1.

[0009] Furthermore, the molar ratio of L-glutamic acid, L-tryptophan and 5-aminolevulinic acid is 0.9-1.1: 0.9-1.1:1.

[0010] Furthermore, the content of copper sulfate is 0.03-0.05 mmol / L.

[0011] Furthermore, the pH value of the tobacco ripening-resistant agent is 6.8-7.2, and / or the osmotic pressure is 280-320 mOsm / L.

[0012] The present invention also provides a method for preparing the above tobacco ripening-resistant agent, which comprises: Step 1), under an inert atmosphere, dissolve β-mercaptoethanol and L-ascorbic acid in deionized water, and stir at 18-22° C. for 15-20 minutes to obtain solution A; Step 2), L-glutamic acid, L-tryptophan and 5-aminolevulinic acid are added to solution A in sequence, and ultrasonically dispersed at 23-27° C. for 30-40 minutes to obtain solution B; Step 3), dissolving copper sulfate in deionized water, stirring at 28-32°C for 10-15 minutes, then dropping into solution B at a rate of 1-2 mL / min, and continuing to stir at 33-37°C for 30-40 minutes to obtain solution C; Step 4), polyethylene glycol 400 and Tween 20 are stirred at 38-42°C for 10-15 minutes until uniform, then slowly added to solution C, and stirred at 43-47°C for 45-60 minutes; Step 5), the solution obtained in step 4) is filtered through a sterile filter membrane with a filter diameter of 2-0.3 μm, and filled into a pre-sterilized high-barrier plastic container under sterile conditions.

[0013] Furthermore, the step 4) further comprises: adding an acid-base regulator to the stirred solution to adjust the pH value of the solution to 6.8-7.2.

[0014] Furthermore, the step 4) further comprises: adding an osmotic pressure regulator to the solution after adjusting the pH value, to adjust the osmotic pressure of the solution to 280-320 mOsm / L.

[0015] Further, in the step 2), the ultrasonic dispersion frequency is 20 - 40 kHz and the power is 100 - 200 W.

[0016] The present invention also provides an application of the above tobacco ripening retardant in delaying the ripening of tobacco leaves, which includes: Dilute the tobacco ripening retardant 50 - 100 times with deionized water, and spray it on the tobacco leaves when the tips of the tobacco leaves start to turn yellow during the ripening period. The dosage per mu is 30 - 40 L, and the spraying time is in the morning or evening on sunny days.

[0017] The tobacco ripening retardant provided by the present invention may have the following beneficial effects: First, through the synergistic effect of β-mercaptoethanol and L-ascorbic acid, the antioxidant capacity of the tobacco leaves is significantly enhanced, effectively delaying the degradation of chlorophyll, extending the green retention time of the tobacco leaves by 2 - 3 days, and providing more flexible harvesting time for tobacco farmers. Second, the added amino acid components not only participate in the plant metabolism process but also promote the accumulation of soluble sugars, increasing the sugar content of the cured tobacco leaves by 20 - 25%, significantly improving the internal quality of the tobacco leaves.

[0018] The experimental results show that the treated tobacco leaves have significant improvements in sensory indicators such as aroma, taste, and irritation. This may be due to the complex physiological and biochemical reactions generated by various components in the ripening retardant in the tobacco leaves, promoting the synthesis and transformation of aromatic substances. This discovery provides a new idea for improving the quality of tobacco leaves.

[0019] In addition, the ripening retardant of the present invention also exhibits excellent environmental adaptability. By adding polyethylene glycol 400 and Tween 20, the stability of the active ingredients and the foliar absorption efficiency are significantly improved, enabling the ripening retardant to maintain good effects under different climate conditions. This characteristic is of great significance for coping with the increasingly complex climate change.

[0020] Finally, the ripening retardant of the present invention uses natural amino acids, vitamins and other ingredients, having good safety and environmental friendliness. This not only conforms to the concept of sustainable development of modern agriculture but also provides the possibility for producing high-quality, green and safe tobacco leaves.

[0021] In summary, through the synergistic effect of multiple components, the tobacco ripening retardant of the present invention shows significant comprehensive benefits in delaying the ripening of tobacco leaves, improving the quality of tobacco leaves, enhancing environmental adaptability and ensuring safety, providing an efficient and environmentally friendly new solution for tobacco cultivation and having broad application prospects.

[0022] It should be understood that the above general description and the following detailed description are only exemplary and explanatory, and cannot limit the present invention. Detailed implementation manners

[0023] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] The terms used in the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The singular forms "a", "the" and "the" used in the present invention and the appended claims are also intended to include plural forms unless the context clearly indicates other meanings. It should also be understood that the term "and / or" used herein refers to and includes any or all possible combinations of one or more associated listed items.

[0025] It should be understood that although the terms "first", "second", "third", etc. may be used to describe various information in the present invention, such information should not be limited to these terms. These terms are only used to distinguish the same type of information from each other. For example, without departing from the scope of the present invention, the first information may also be referred to as the second information, and similarly, the second information may also be referred to as the first information. Thus, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. In the description of the present invention, the meaning of "multiple" is two or more, unless otherwise clearly and specifically defined.

[0026] The present invention aims to solve the above technical problems and provide a multi-component synergistic tobacco ripening-resistant agent and a preparation method thereof. The ripening-resistant agent can not only effectively delay the ripening of tobacco leaves through a carefully designed formula, but also significantly improve the quality of tobacco leaves, and has excellent environmental adaptability and safety.

[0027] The embodiment of the present invention provides a tobacco ripening-resistant agent, which includes: 0.06-0.10 mmol / L β-mercaptoethanol; 0.06-0.10 mmol / L L-ascorbic acid; 0.008-0.012 mmol / L of L-glutamic acid; 0.008-0.012 mmol / L of L-tryptophan; 0.008-0.012 mmol / L 5-aminolevulinic acid; 0.02-0.05 mmol / L copper sulfate; 0.05-0.10 mmol / L polyethylene glycol 400; 0.002-0.004 mmol / L Tween 20; and the balance of water.

[0028] The tobacco ripening-resistant agent comprises: As a strong reducing agent, β-mercaptoethanol inhibits the activity of polyphenol oxidase (PPO) in tobacco leaves through thiol groups, blocks the oxidation of phenolic substances into quinones, and protects the integrity of the chloroplast membrane structure. In addition, the inventors of the present application have also found that β-mercaptoethanol can not only inhibit oxidation, but also promote the absorption of other active ingredients by regulating cell membrane permeability. The content of β-mercaptoethanol is 0.06-0.10 mmol / L, preferably 0.07-0.10 mmol / L.

[0029] L-ascorbic acid directly removes hydroxyl radicals and superoxide anions, blocks lipid peroxidation chain reactions, and delays cell membrane damage. The inventors of the present application also found that it may also have the effect of regulating sugar metabolism. However, its excessive content leads to a decrease in soluble sugar content. The content of L-ascorbic acid is 0.06-0.10 mmol / L, preferably 0.07-0.10 mmol / L.

[0030] In addition, the effect of selecting both β-mercaptoethanol and L-ascorbic acid in the ripening-resistant agent is that the inventors of the present application have found that β-mercaptoethanol and L-ascorbic acid work synergistically to significantly enhance the antioxidant capacity of tobacco leaves. Preferably, the molar ratio of β-mercaptoethanol to L-ascorbic acid is 0.9-1.1:1, and most preferably 1:1, which can significantly improve the aroma score of tobacco leaves.

[0031] L-ascorbic acid, L-glutamic acid and L-tryptophan are amino acid components, among which L-glutamic acid, as a precursor of α-ketoglutarate, participates in the tricarboxylic acid cycle (TCA cycle), provides ATP and carbon skeleton, and its content is 0.008-0.012 mmol / L, preferably 0.07-0.10 mmol / L; L-tryptophan is a precursor of indoleacetic acid (IAA) synthesis, regulates plant auxin levels, and delays abscission layer formation, and its content is 0.008-0.012 mmol / L, preferably 0.07-0.10 mmol / L; 5-aminolevulinic acid (ALA) is a direct precursor of chlorophyll synthesis, promotes the conversion of protochlorophyllide esters, and its content is 0.008-0.012 mmol / L, preferably 0.07-0.10 mmol / L. The above amino acid components work together, not only participating in the plant metabolism process, but also promoting the accumulation of soluble sugars; amino acids and precursor substances can participate in the plant metabolism process, maintain the synthesis of chlorophyll, and thus delay aging. The molar ratio of L-glutamic acid, L-tryptophan and 5-aminolevulinic acid is preferably 0.9-1.1: 0.9-1.1:1. The most preferred ratio is 1:1:1, and the amino acid system of this ratio can significantly improve the taste score of tobacco leaves.

[0032] Copper sulfate can provide copper ions as a cofactor for superoxide dismutase (SOD) and ascorbate peroxidase (APX), activate the antioxidant enzyme system, and enhance the stress resistance of plants. In addition, the inventors of the present application have also found that copper ions not only activate enzyme activity, but may also participate in the stabilization of magnesium ions in chlorophyll molecules. Its content is 0.02-0.05 mmol / L, preferably 0.035-0.05 mmol / L.

[0033] Polyethylene glycol 400 and Tween 20 are used as solubilizers and emulsifiers. Polyethylene glycol 400 encapsulates hydrophobic components (such as ALA) through hydrogen bonds to improve solubility stability. Tween 20 can reduce surface tension (<30 mN / m) and promote the diffusion of the drug solution in the wax layer. The common effect of the two can improve the stability of the active ingredients and the leaf absorption efficiency, and significantly improve the stability of the active ingredients and the leaf absorption efficiency. The content of polyethylene glycol 400 is 0.05-0.10 mmol / L, preferably 0.0625-0.10mmol / L. The content of Tween 20 is 0.002-0.004 mmol / L, more preferably 0.0025-0.004 mmol / L.

[0034] The tobacco ripening-resistant agent provided in this embodiment has a multi-component synergistic effect, which not only delays the ripening of tobacco leaves, but also improves the quality of tobacco leaves and enhances environmental adaptability. The content and proportion of each component have a regulating effect on the three-element balance of sugar, aroma and irritation of tobacco leaves. As a preferred embodiment of this embodiment, the composition of the tobacco ripening-resistant agent is as follows: 0.07-0.10 mmol / L β-mercaptoethanol; 0.07-0.10 mmol / L L-ascorbic acid; 0.009-0.012 mmol / L of L-glutamic acid; 0.009-0.012 mmol / L of L-tryptophan; 0.009-0.012 mmol / L 5-aminolevulinic acid; 0.035-0.05 mmol / L copper sulfate; 0.0625-0.10 mmol / L polyethylene glycol 400; 0.0025-0.004 mmol / L Tween 20; and the balance of water.

[0035] The following is the composition of the most preferred tobacco ripening-resistant agent: β-mercaptoethanol 0.10 mmol / L; L-ascorbic acid 0.10 mmol / L; L-glutamic acid 0.012 mmol / L; L-tryptophan 0.012 mmol / L; 5-aminolevulinic acid 0.012 mmol / L; copper sulfate 0.05 mmol / L; polyethylene glycol 400 0.10 mmol / L; Tween 20 0.004 mmol / L; the rest is water. The tobacco ripening agent with this ratio shows significant comprehensive benefits in extending the harvest period, improving the quality of tobacco leaves and enhancing stress resistance.

[0036] The pH value of the above tobacco ripening agent is preferably 6.8-7.2, which is conducive to maintaining the stability of the antioxidant system, optimizing the activity of metal ions, and matching the cell enzyme environment. It can be achieved by adding an acid-base regulator (such as an acidic solution such as hydrochloric acid solution, or an alkaline solution such as sodium hydroxide solution); those skilled in the art can understand that the composition of the tobacco ripening agent should also include an acid-base regulator. The osmotic pressure of the tobacco ripening agent is preferably 280-320 mOsm / L, which is conducive to maintaining the isotonicity of the solution and plant cells and facilitating the absorption of active ingredients. It can be achieved by adding an osmotic pressure regulator. The osmotic pressure regulator preferably uses a sorbitol solution, which has the advantages of stable chemical properties, not easy to react, and friendly to plant cells. Those skilled in the art can understand that the composition of the tobacco ripening agent should also include an osmotic pressure regulator.

[0037] Another embodiment of the present invention also provides a method for preparing the above tobacco ripening-resistant agent, which comprises the following steps: Step 1), under an inert atmosphere, dissolve β-mercaptoethanol and L-ascorbic acid in deionized water, and stir at 18-22° C. for 15-20 minutes to obtain solution A; Step 2), L-glutamic acid, L-tryptophan and 5-aminolevulinic acid are added to solution A in sequence, and ultrasonically dispersed at 23-27° C. for 30-40 minutes to obtain solution B; Step 3), dissolving copper sulfate in deionized water, stirring at 28-32°C for 10-15 minutes, then dropping into solution B at a rate of 1-2 mL / min, and continuing to stir at 33-37°C for 30-40 minutes to obtain solution C; Step 4), polyethylene glycol 400 and Tween 20 are stirred at 38-42°C for 10-15 minutes until uniform, then slowly added to solution C, and stirred at 43-47°C for 45-60 minutes; Step 5), the solution obtained in step 4) is filtered through a sterile filter membrane with a filter diameter of -0.3 μm, and filled into a pre-sterilized high-barrier plastic container under sterile conditions.

[0038] In the above preparation method, adding β-mercaptoethanol, L-ascorbic acid and amino acids first can ensure that they are fully dissolved and stabilized in the solution. In step 2), the ultrasonic dispersion frequency is preferably 20-40 kHz, and the power is preferably 100-200 W. The slow dropwise addition of copper sulfate solution helps to form a stable complex and avoid precipitation. Finally, adding polyethylene glycol 400 and Tween 20 can form a protective layer to enhance the stability of the entire formulation. Considering the oxidizability of β-mercaptoethanol and L-ascorbic acid, step 1) is carried out in an inert atmosphere, and the inert atmosphere is preferably nitrogen or argon. In order to avoid oxidation of β-mercaptoethanol and L-ascorbic acid during subsequent feeding and stirring, steps 2) to 4) are also preferably carried out in an inert atmosphere. After adding polyethylene glycol 400 and Tween 20, the active ingredients in the solution have been protected by other components and can be carried out under conventional conditions. After all the raw materials are mixed according to steps 1 to 4), they need to be post-processed according to step 5), wherein the non-membrane filtration can remove microorganisms and particulate matter in the solution, ensure the sterility and purity of the product, extend the shelf life of the anti-ripening agent, and prevent microbial contamination from causing adverse effects on tobacco leaves. The filter diameter of the sterile filter membrane is more preferably 0.22 μm. High-barrier plastic containers have excellent barrier properties and chemical inertness to gas, moisture, and light. After membrane filtration, high-barrier plastic containers are used for canning, which can ensure the stability of the prepared tobacco anti-ripening agent during storage and transportation and extend the shelf life.

[0039] In order to make the tobacco ripening-resistant agent have a suitable pH value, after adding polyethylene glycol 400 and Tween 20 and stirring in step 4), it is preferred to further include: adding an acid-base regulator to the stirred solution to adjust the pH value of the solution to 6.8-7.2. Specifically, sodium hydroxide solution or hydrochloric acid solution can be used to adjust the pH value of the mixed solution to 6.8-7.2.

[0040] Furthermore, in order to make the tobacco ripening resistance agent have a suitable osmotic pressure, the step 4) preferably further comprises: adding an osmotic pressure regulator to the solution after adjusting the pH value, and adjusting the osmotic pressure of the solution to 280-320 mOsm / L. The osmotic pressure regulator is preferably a sorbitol solution, specifically a sorbitol stock solution with a mass volume ratio of 50%, and the specific operation can be: slowly adding the sorbitol stock solution to the solution after adjusting the pH value, and measuring the osmotic pressure while adding until the target value of 280-320 mOsm / L is reached.

[0041] The prepared tobacco ripening-resistant agent is preferably stored in the following manner: The tobacco aging resistant agent is stored in a cool place at 4-8°C away from light; the validity period of the tobacco aging resistant agent is 6 months from the preparation date; before use, the tobacco aging resistant agent is equilibrated at 18-22°C for 1-2 hours and vortex mixed for 30-60 seconds.

[0042] Another embodiment of the present invention also provides an application of the above-mentioned tobacco ripening-resistant agent in delaying the ripening of tobacco leaves, which comprises: diluting the tobacco ripening-resistant agent with deionized water by 50-100 times, spraying it on the tobacco leaves when the leaf tips begin to turn yellow during the ripening period of the tobacco leaves, with an amount of 30-40L per mu, and the spraying time is in the morning or evening on a sunny day.

[0043] From the above content, it can be seen that the tobacco ripening-resistant agent provided by the embodiment of the present invention has the following advantages: First, through the synergistic effect of β-mercaptoethanol and L-ascorbic acid, the antioxidant capacity of tobacco leaves is significantly enhanced, the degradation of chlorophyll is effectively delayed, and the green time of tobacco leaves is extended by 2-3 days, providing tobacco farmers with more flexible harvesting time. Secondly, the added amino acid components not only participate in the plant metabolism process, but also promote the accumulation of soluble sugars, which increases the sugar content of tobacco leaves after curing by 20-25%, significantly improving the intrinsic quality of tobacco leaves.

[0044] The experimental results show that the sensory indicators of the treated tobacco leaves, such as aroma, taste and irritation, have been significantly improved. This may be because the various components in the ripening agent produce complex physiological and biochemical reactions in the tobacco leaves, promoting the synthesis and transformation of aromatic substances. This discovery provides a new idea for improving the quality of tobacco leaves.

[0045] In addition, the anti-ripening agent of the present invention also exhibits excellent environmental adaptability. By adding polyethylene glycol 400 and Tween 20, the stability of the active ingredients and the leaf surface absorption efficiency are significantly improved, so that the anti-ripening agent can maintain good effects under different climatic conditions. This feature is of great significance for coping with increasingly complex climate changes.

[0046] Finally, the ripening-resistant agent of the present invention uses natural amino acids and vitamins and other ingredients, and has good safety and environmental friendliness. This not only conforms to the concept of sustainable development of modern agriculture, but also provides the possibility for producing high-quality, green and safe tobacco leaves.

[0047] In summary, the tobacco maturation-resistant agent of the present invention, through the synergistic effect of multiple components, shows significant comprehensive benefits in delaying tobacco leaf maturation, improving tobacco leaf quality, enhancing environmental adaptability and ensuring safety, etc., providing a new type of efficient and environmentally friendly solution for tobacco planting, and has broad application prospects.

[0048] The technical solution of the present invention is further described below in conjunction with specific embodiments: Example 1 The components and concentrations of the tobacco ripening-resistant agent provided in this embodiment are as follows: β-mercaptoethanol 0.06 mmol / L; L-ascorbic acid 0.06 mmol / L; L-glutamic acid 0.008 mmol / L; L-tryptophan 0.008 mmol / L; 5-aminolevulinic acid 0.008 mmol / L; copper sulfate 0.03 mmol / L; polyethylene glycol 400 0.05 mmol / L; Tween 20 0.002 mmol / L; the balance is deionized water.

[0049] The preparation method of the tobacco ripening-resistant agent comprises the following steps: (1) Under a nitrogen atmosphere (101 kPa), β-mercaptoethanol and L-ascorbic acid were dissolved in deionized water and stirred at 18°C ​​for 15 minutes to obtain solution A.

[0050] (2) L-glutamic acid, L-tryptophan and 5-aminolevulinic acid were added to solution A in sequence, and ultrasonic dispersion was performed at 20 kHz and 100 W at 23°C for 30 minutes to obtain solution B.

[0051] (3) Dissolve copper sulfate in deionized water, stir at 28°C for 10 minutes, then add it dropwise to solution B at a rate of 1 mL / min, and continue stirring at 33°C for 30 minutes to obtain solution C.

[0052] (4) Stir polyethylene glycol 400 and Tween 20 at 38°C for 10 minutes until uniform, then slowly add them to solution C and stir at 43°C for 45 minutes.

[0053] (5) Use 0.1M sodium hydroxide solution to adjust the pH value of the mixture to 6.8, add deionized water to the set volume, and stir at 20°C for 30 minutes. Prepare a 50% (w / v) sorbitol stock solution. Slowly add an appropriate amount of the stock solution to the ripening agent, measuring the osmotic pressure while adding until the target value of 280mOsm / L is reached.

[0054] (6) Filter the final solution through a 0.22 μm sterile filter membrane and fill it into a pre-sterilized high-barrier plastic container under aseptic conditions.

[0055] Example 2 The components and concentrations of the tobacco ripening-resistant agent provided in this embodiment are as follows: β-mercaptoethanol 0.08 mmol / L; L-ascorbic acid 0.08 mmol / L; L-glutamic acid 0.010 mmol / L; L-tryptophan 0.010 mmol / L; 5-aminolevulinic acid 0.010 mmol / L; copper sulfate 0.04 mmol / L; polyethylene glycol 400 0.075 mmol / L; Tween 20 0.003 mmol / L; deionized water 99.75 weight percent.

[0056] The preparation method of the tobacco ripening-resistant agent comprises the following steps: (1) Under argon atmosphere (102 kPa), β-mercaptoethanol and L-ascorbic acid were dissolved in deionized water and stirred at 20°C for 17 minutes to obtain solution A.

[0057] (2) L-glutamic acid, L-tryptophan and 5-aminolevulinic acid were added to solution A in sequence, and ultrasonic dispersion was performed at 30 kHz and 150 W for 35 minutes at 25°C to obtain solution B.

[0058] (3) Dissolve copper sulfate in deionized water, stir at 30°C for 12 minutes, then add it dropwise to solution B at a rate of 1.5 mL / min, and continue stirring at 35°C for 35 minutes to obtain solution C.

[0059] (4) Stir polyethylene glycol 400 and Tween 20 at 40°C for 12 minutes until uniform, then slowly add them to solution C and stir at 45°C for 52 minutes.

[0060] (5) Next, the pH value of the mixture was adjusted to 7.0 using 0.1 M hydrochloric acid solution, and deionized water was added to the set volume, and stirred at 22° C. for 35 minutes. At the same time, an appropriate amount of the stock solution (same as in Example 1) was slowly added to the ripening agent, and the osmotic pressure was measured while adding until the target value of 300 mOsm / L was reached.

[0061] (6) Filter the final solution through a 0.3-μm sterile filter membrane and fill it into a pre-sterilized high-barrier plastic container under sterile conditions.

[0062] Example 3 The components and concentrations of the tobacco ripening tolerance agent in this example are as follows: β-mercaptoethanol 0.10 mmol / L; L-ascorbic acid 0.10 mmol / L; L-glutamic acid 0.012 mmol / L; L-tryptophan 0.012 mmol / L; 5-aminolevulinic acid 0.012 mmol / L; copper sulfate 0.05 mmol / L; polyethylene glycol 400 0.10 mmol / L; Tween 20 0.004 mmol / L; deionized water 99.67 wt%.

[0063] The preparation method of this tobacco ripening tolerance agent includes the following steps: (1) Under a nitrogen atmosphere (103 kPa), dissolve β-mercaptoethanol and L-ascorbic acid in deionized water and stir at 22°C for 20 minutes to obtain solution A.

[0064] (2) Sequentially add L-glutamic acid, L-tryptophan, and 5-aminolevulinic acid to solution A, and perform ultrasonic dispersion at 40 kHz and 200 W at 27°C for 40 minutes to obtain solution B.

[0065] (3) Dissolve copper sulfate in deionized water, stir at 32°C for 15 minutes, and then add it dropwise to solution B at a rate of 2 mL / min and continue to stir at 37°C for 40 minutes to obtain solution C.

[0066] (4) Stir polyethylene glycol 400 and Tween 20 at 42°C for 15 minutes until homogeneous, and then slowly add them to solution C and stir at 47°C for 60 minutes.

[0067] (5) Use 0.1 M sodium hydroxide solution to adjust the pH value of the mixture to 7.2, add deionized water to the set volume, and stir at 25°C for 40 minutes. At the same time, slowly add an appropriate amount of stock solution (the same as in Example 1) to the ripening tolerance agent, and measure the osmotic pressure while adding until the target value of 320 mOsm / L is reached.

[0068] (6) Finally, filter the final solution through a 0.25-μm sterile filter membrane and fill it into a pre-sterilized high-barrier plastic container under sterile conditions.

[0069] Example 4 The components and concentrations of the tobacco ripening tolerance agent provided in this example are as follows: β-mercaptoethanol 0.07 mmol / L; L-ascorbic acid 0.07 mmol / L; L-glutamic acid 0.009 mmol / L; L-tryptophan 0.009 mmol / L; 5-aminolevulinic acid 0.009 mmol / L; copper sulfate 0.035 mmol / L; polyethylene glycol 400 0.0625 mmol / L; Tween 20 0.0025 mmol / L; the balance is water.

[0070] The preparation method of the tobacco ripening-resistant agent comprises the following steps: (1) Under an argon atmosphere (101.5 kPa), β-mercaptoethanol and L-ascorbic acid were dissolved in deionized water and stirred at 19°C for 16 minutes to obtain solution A.

[0071] (2) L-glutamic acid, L-tryptophan and 5-aminolevulinic acid were added to solution A in sequence, and ultrasonic dispersion was performed at 24°C, 25 kHz, 125 W for 32 minutes to obtain solution B.

[0072] (3) Dissolve copper sulfate in deionized water, stir at 29°C for 11 minutes, then add it dropwise to solution B at a rate of 1.25 mL / min, and continue stirring at 34°C for 32 minutes to obtain solution C.

[0073] (4) Stir polyethylene glycol 400 and Tween 20 at 39°C for 11 minutes until uniform, then slowly add them to solution C and stir at 44°C for 48 minutes.

[0074] (5) Use 0.1M hydrochloric acid solution to adjust the pH value of the mixed solution to 6.9, add deionized water to the set volume, and stir at 21°C for 32 minutes. At the same time, slowly add an appropriate amount of the stock solution (same as in Example 1) to the ripening agent, and measure the osmotic pressure while adding until the target value of 290mOsm / L is reached.

[0075] (6) Filter the final solution through a 0.22 μm sterile filter membrane and fill it into a pre-sterilized high-barrier plastic container under aseptic conditions.

[0076] The above embodiments fully demonstrate the various formulations and preparation methods of the tobacco ripening-resistant agent of the present invention, and provide abundant choices for practical applications.

[0077] Comparative Example 1: Formula lacking β-mercaptoethanol This comparative example is intended to verify the importance of β-mercaptoethanol in the ripening resistance agent, corresponding to Example 1. The formula composition is as follows: L-ascorbic acid 0.06 mmol / L; L-glutamic acid 0.008 mmol / L; L-tryptophan 0.008 mmol / L; 5-aminolevulinic acid 0.008 mmol / L; copper sulfate 0.03 mmol / L; polyethylene glycol 400 0.05 mmol / L; Tween 200 0.002 mmol / L; the balance is water.

[0078] The preparation method is the same as Example 1, but the step of adding β-mercaptoethanol is omitted. This comparative example will show that in the absence of β-mercaptoethanol, the effect of the anti-ripening agent in delaying chlorophyll degradation and inhibiting oxidation reactions is weakened.

[0079] Comparative Example 2: Formula with increased L-ascorbic acid concentration This comparative example is intended to verify the optimal concentration range of L-ascorbic acid, corresponding to Example 2. The formulation composition is as follows: β-Mercaptoethanol 0.08 mmol / L; L-ascorbic acid 0.15 mmol / L; L-glutamic acid 0.010 mmol / L; L-tryptophan 0.010 mmol / L; 5-aminolevulinic acid 0.010 mmol / L; copper sulfate 0.04 mmol / L; polyethylene glycol 400 0.075 mmol / L; Tween 20 0.003 mmol / L.

[0080] The preparation method is the same as that of Example 2, but the amount of L-ascorbic acid added in step (1) is increased. This comparative example will show the negative effects that excessive concentration of L-ascorbic acid may cause, such as excessive inhibition of normal physiological processes.

[0081] Comparative Example 3: Formula with adjusted amino acid ratio This comparative example is intended to verify the optimal ratio of L-glutamic acid, L-tryptophan and 5-aminolevulinic acid, corresponding to Example 3. The formula composition is as follows: β-Mercaptoethanol 0.10 mmol / L; L-ascorbic acid 0.10 mmol / L; L-glutamic acid 0.020 mmol / L; L-tryptophan 0.008 mmol / L; 5-aminolevulinic acid 0.008 mmol / L; copper sulfate 0.05 mmol / L; polyethylene glycol 400 0.10 mmol / L; Tween 20 0.004 mmol / L.

[0082] The preparation method is the same as that of Example 3, but the ratio of amino acid addition is adjusted in step (2). This comparative example will show that an imbalance in the ratio of amino acids may cause plant metabolic disorders, thereby affecting the ripening resistance effect.

[0083] Comparative Example 4: Formula for reducing copper sulfate concentration This comparative example is intended to verify the importance of copper sulfate, corresponding to Example 4. The formula composition is as follows: β-Mercaptoethanol 0.07 mmol / L; L-ascorbic acid 0.07 mmol / L; L-glutamic acid 0.009 mmol / L; L-tryptophan 0.009 mmol / L; 5-aminolevulinic acid 0.009 mmol / L; copper sulfate 0.01 mmol / L; polyethylene glycol 400 0.0625 mmol / L; Tween 20 0.0025 mmol / L.

[0084] The preparation method is the same as that of Example 4, but the amount of copper sulfate added in step (3) is reduced. This comparative example will illustrate the importance of an appropriate amount of copper ions for activating the antioxidant enzyme system, and the reduced stress resistance that may result from too low a concentration.

[0085] Comparative Example 5: Removal of polyethylene glycol 400 This comparative example is intended to verify the importance of polyethylene glycol 400 as a solubilizer, corresponding to Example 1. The formulation composition is as follows: β-Mercaptoethanol 0.06 mmol / L; L-ascorbic acid 0.06 mmol / L; L-glutamic acid 0.008 mmol / L; L-tryptophan 0.008 mmol / L; 5-aminolevulinic acid 0.008 mmol / L; copper sulfate 0.03 mmol / L; Tween 20 0.002 mmol / L.

[0086] The preparation method is the same as that of Example 1, but the addition of polyethylene glycol 400 in step (4) is omitted. This comparative example will show that in the absence of a suitable solubilizer, the stability and foliar absorption efficiency of the active ingredient may be reduced.

[0087] Comparative Example 6: Formula for adjusting pH and osmotic pressure This comparative example is intended to verify the influence of pH value and osmotic pressure on the effect of the anti-ripening agent, corresponding to Example 2. The formulation composition is the same as that of Example 2, but step (5) in the preparation method is adjusted as follows: The pH value of the mixture was adjusted to 8.0 using 0.1 M sodium hydroxide solution, and deionized water was added to the set volume, and stirred at 22°C for 35 minutes. At the same time, the osmotic pressure was adjusted to 400 mOsm / L.

[0088] This comparative example will illustrate the importance of pH value and osmotic pressure in ensuring the compatibility of the anti-ripening agent with plant cells and the absorption of the active ingredients. Inappropriate pH value and osmotic pressure may lead to reduced leaf absorption efficiency and even cause osmotic pressure damage to plant cells.

[0089] By comparing with the examples, it can be clearly seen that the present invention has the advantages of delaying the maturity of tobacco leaves and improving the quality of tobacco leaves.

[0090] In order to evaluate the effectiveness and superiority of the tobacco ripening-resistant agent of the present invention, a series of comprehensive experiments were designed to comprehensively evaluate the effect of the ripening-resistant agent on the tobacco leaf ripening process and its effect on improving the final tobacco leaf quality.

[0091] Experimental design and methods: 1. Field trial design The flue-cured tobacco planting base in Lanshan County, Yongzhou City, Hunan Province was selected for field experiments. The experiment adopted a randomized block design, with 3 replicates for each treatment and a plot area of ​​20 square meters. The tobacco variety tested was Yunyan 87. During the tobacco leaf maturity period (about 7-10 days after tobacco leaf harvest), different ripening agents were sprayed as follows: The tobacco ripening-resistant agent of the above-mentioned embodiment and comparative example was diluted 80 times with deionized water, and sprayed on tobacco leaves when the tips of the leaves began to turn yellow during the maturity period of the tobacco leaves. The dosage was 35L per mu, and the spraying time was in the morning or evening on a sunny day.

[0092] 2. Determination of chlorophyll content (SPAD value) Using a SPAD-502 chlorophyll meter, the SPAD value of the 15th tobacco leaf was measured on the 1st, 5th and 10th day after treatment, and 5 points were measured on each leaf to take the average value. The test results are shown in Table 1.

[0093] 3. Determination of malondialdehyde (MDA) content Thiobarbituric acid (TBA) colorimetric method was used to determine the MDA content. Samples were taken on the 10th day after treatment and stored at -80°C for testing. The test results are shown in Table 2.

[0094] 4. Antioxidant Enzyme Activity Assay The activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) were measured using a spectrophotometer and samples were taken on the 10th day after treatment. The test results are shown in Table 2.

[0095] 5. Determination of soluble sugar content The soluble sugar content of the flue-cured tobacco leaves was determined by anthrone colorimetry. The test results are shown in Table 3.

[0096] 6. Tobacco leaf quality evaluation Professional smokers evaluated the sensory quality of the flue-cured tobacco leaves, including aroma, taste, irritation, etc. The test results are shown in Table 3.

[0097] Experimental results: Table 1 Effects of different ripening resistance agents on the SPAD value of tobacco leaves

[0098] Table 2 Effects of different ripening agents on physiological and biochemical parameters of tobacco leaves (10 days after treatment)

[0099] Table 3 Effects of different ripening agents on the quality of flue-cured tobacco leaves

[0100] Result analysis: 1. Chlorophyll preservation effect: Examples 1-4 all showed obvious chlorophyll preservation effect, especially Example 3, where the SPAD value remained at 19.2 after 10 days of treatment, which was 82.9% higher than the clear water control. This shows that the ripening-resistant agent of the present invention can effectively delay the degradation of chlorophyll, thereby extending the harvest period of tobacco leaves.

[0101] 2. Improved antioxidant capacity: Compared with the comparative example and the clear water control, Examples 1-4 showed lower MDA content and higher antioxidant enzyme activity. Among them, the MDA content of Example 3 was 40.0% lower than that of the clear water control, and the SOD, POD and CAT activities were increased by 44.2%, 54.2% and 43.1%, respectively. This shows that the ripening-resistant agent of the present invention can significantly enhance the antioxidant capacity of tobacco leaves, reduce cell membrane lipid peroxidation, and thus delay the aging process.

[0102] 3. Improvement of tobacco leaf quality: Examples 1-4 are superior to the comparative example and the clear water control in terms of soluble sugar content and sensory quality scores. The soluble sugar content of Example 3 reached 25.8%, which was 41.0% higher than the clear water control. At the same time, the aroma, taste and irritation scores were also significantly improved, by 24.7%, 25.4% and 25.0% respectively. This shows that the ripening-resistant agent of the present invention not only delays the ripening of tobacco leaves, but also improves the intrinsic quality and sensory properties of tobacco leaves.

[0103] 4. Component synergy: Through the results of Comparative Examples 1-6, it can be seen that there is an obvious synergistic effect between the components. The lack of β-mercaptoethanol (Comparative Example 1) or the reduction of copper sulfate content (Comparative Example 4) will lead to a significant reduction in the antioxidant effect. This shows that β-mercaptoethanol and copper ions play a key role in activating the antioxidant system. At the same time, adjusting the amino acid ratio (Comparative Example 3) will affect the accumulation of soluble sugars, indicating that the amino acid component has an important influence on the metabolic process of tobacco leaves.

[0104] 5. Unexpected technical effects: a) Significant improvement in aroma quality: Although the present invention is mainly aimed at delaying the maturity of tobacco leaves, the experimental results show that tobacco leaves treated with a maturation-resistant agent have a significant improvement in aroma scores. This may be due to the extension of the tobacco leaf maturity period, which allows more time for the aromatic precursors to accumulate and transform.

[0105] b) Harmony of irritation: Example 3 not only improves the aroma and taste, but also moderately improves the irritation score. This balance improvement may be due to the synergistic effect between the components, especially the amino acid component may be involved in the biosynthesis process of certain irritating substances.

[0106] c) Enhanced stress resistance: Although the experiment did not directly test stress resistance, it can be inferred from the significant increase in antioxidant enzyme activity that tobacco leaves treated with the ripening-resistant agent of the present invention may have stronger stress resistance. This is of great significance for coping with unpredictable climate change.

[0107] In summary, the tobacco ripening-resistant agent of the present invention not only achieves the expected delayed ripening effect through the synergistic effect of multiple components, but also shows unexpected superiority in improving tobacco leaf quality and antioxidant capacity. Among them, Example 3 (β-mercaptoethanol 0.10 mmol / L; L-ascorbic acid 0.10 mmol / L; L-glutamic acid 0.012 mmol / L; L-tryptophan 0.012 mmol / L; 5-aminolevulinic acid 0.012 mmol / L; copper sulfate 0.05 mmol / L; polyethylene glycol 4000.10 mmol / L; Tween 20 0.004 mmol / L) performs most prominently and can be regarded as the best embodiment of the present invention. This formula shows significant comprehensive benefits in extending the harvest period, improving tobacco leaf quality and enhancing stress resistance, providing a new type of efficient and environmentally friendly ripening-resistant agent solution for tobacco planting.

[0108] The embodiments of the present invention have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The selection of terms used herein is intended to best explain the principles of the embodiments, practical applications, or improvements to the technology in the market, or to enable other persons of ordinary skill in the art to understand the embodiments disclosed herein.

Claims

1. A tobacco ripening-resistant agent, characterized in that: It includes: 0.06-0.10 mmol / L β-mercaptoethanol; 0.06-0.10 mmol / L L-ascorbic acid; 0.008-0.012 mmol / L of L-glutamic acid; 0.008-0.012 mmol / L of L-tryptophan; 0.008-0.012 mmol / L 5-aminolevulinic acid; 0.02-0.05 mmol / L copper sulfate; 0.05-0.10 mmol / L polyethylene glycol 400; 0.002-0.004 mmol / L Tween 20; and the balance of water.

2. The tobacco ripening-resistant agent according to claim 1, characterized in that: The molar ratio of the β-mercaptoethanol to L-ascorbic acid is 0.9-1.1:

1.

3. The tobacco ripening-resistant agent according to claim 1, characterized in that The molar ratio of L-glutamic acid, L-tryptophan and 5-aminolevulinic acid is 0.9-1.1: 0.9-1.1:

1.

4. The tobacco ripening-resistant agent according to claim 1, characterized in that The content of copper sulfate is 0.03-0.05 mmol / L.

5. The tobacco ripening-resistant agent according to claim 1, characterized in that The pH value of the tobacco ripening-resistant agent is 6.8-7.2, and / or the osmotic pressure is 280-320 mOsm / L.

6. The method for preparing the tobacco ripening-resistant agent according to any one of claims 1 to 5, characterized in that: include: Step 1), under an inert atmosphere, dissolve β-mercaptoethanol and L-ascorbic acid in deionized water, and stir at 18-22° C. for 15-20 minutes to obtain solution A; Step 2), L-glutamic acid, L-tryptophan and 5-aminolevulinic acid are added to solution A in sequence, and ultrasonically dispersed at 23-27° C. for 30-40 minutes to obtain solution B; Step 3), dissolving copper sulfate in deionized water, stirring at 28-32°C for 10-15 minutes, then adding dropwise to solution B at a rate of 1-2 mL / min, and continuing stirring at 33-37°C for 30-40 minutes to obtain solution C; Step 4), polyethylene glycol 400 and Tween 20 are stirred at 38-42°C for 10-15 minutes until uniform, then slowly added to solution C, and stirred at 43-47°C for 45-60 minutes; Step 5), the solution obtained in step 4) is filtered through a sterile filter membrane with a filter diameter of 2-0.3 μm, and filled into a pre-sterilized high-barrier plastic container under sterile conditions.

7. The preparation method according to claim 6, characterized in that The step 4) further comprises: adding an acid-base regulator to the stirred solution to adjust the pH value of the solution to 6.8-7.

2.

8. The preparation method according to claim 7, characterized in that The step 4) further comprises: adding an osmotic pressure regulator to the solution after adjusting the pH value, to adjust the osmotic pressure of the solution to 280-320 mOsm / L.

9. The preparation method according to claim 6, characterized in that In the step 2), the ultrasonic dispersion frequency is 20-40 kHz and the power is 100-200 W.

10. Use of the tobacco maturation-resistant agent according to any one of claims 1 to 5 in delaying the maturation of tobacco leaves, characterized in that: include: The tobacco ripening-resistant agent is diluted 50-100 times with deionized water, and sprayed on tobacco leaves when the tips of the leaves begin to turn yellow during the maturity period of the leaves. The dosage is 30-40 L per mu, and the spraying time is in the morning or evening on a sunny day.

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