A composite preparation for preventing and controlling enzymatic browning in tobacco curing process and application thereof

Abstract

The present application relates to a kind of complex preparation and its application for preventing and controlling enzymatic browning in tobacco curing process, and the active component in the complex preparation is composed of glycyrrhiza charlton A, brassinazole and melatonin.The present application creatively combines three active components of glycyrrhiza charlton A, brassinazole and melatonin, and is applied to tobacco before curing, and the three components form a closed-loop control network of "enzyme activity inhibition-cell structure protection-oxidative damage repair", and respectively act on the starting, structure, amplification of three key links of enzymatic browning, and the target points of the three are complementary, have excellent technical effects of preventing and controlling enzymatic browning in tobacco curing process, improving tobacco ash phenomenon, improving tobacco quality, and the effect is far more than single component or binary compound system.

Description

Technical Field

[0001] This invention belongs to the field of tobacco curing and processing technology, and relates to a compound preparation for preventing enzymatic browning during tobacco curing and its application, especially a compound preparation suitable for preventing enzymatic browning during tobacco curing in the high temperature and high humidity tobacco-growing areas of South China and its application. Background Technology

[0002] Curing is a core process that determines the appearance grade, internal chemical composition harmony, and sensory evaluation quality of tobacco leaves. Under the adverse conditions of high temperature and humidity and water loss stress during curing, the cell membrane structure of tobacco leaves is easily damaged, the cell compartmentalization balance is disrupted, and polyphenols stored in vacuoles come into large amounts of contact with polyphenol oxidase (PPO) in the cytoplasm, rapidly inducing an enzymatic browning reaction. This leads to problems such as graying on the tobacco leaf surface, darkening of color, increased off-flavors, and increased irritation, ultimately causing a significant drop in the grade of tobacco leaves and seriously affecting the economic benefits of tobacco farmers and the quality of raw materials for the cigarette industry.

[0003] In the high-temperature and high-humidity tobacco-growing areas of South China (represented by Baise, Hezhou, and Hechi in Guangxi), the peak harvesting and curing period for flue-cured tobacco is from May to August. This period is generally characterized by high temperatures, high humidity, and small diurnal temperature differences. In addition, the karst topography leads to large fluctuations in temperature and humidity inside the curing barns. K326, as the main variety cultivated locally, ages rapidly after field maturity and suffers from high levels of cell membrane damage. During the curing process, the enzymatic browning reaction is more intense, and the incidence of ash-covered tobacco leaves is consistently above 40%, far exceeding the national average. This is the core technical bottleneck restricting the improvement of tobacco quality and income in the local area.

[0004] Currently, the industry's control technologies for browning and ash formation in tobacco leaves during curing are mainly divided into three categories: First, physical process control methods, which control browning by fine-tuning the curing temperature and humidity and the rate of temperature increase. This method is highly dependent on the experience of operators, and the curing skills of farmers in Guangxi tobacco-growing areas vary greatly, resulting in poor stability of the effect and an inability to inhibit enzymatic browning reactions at the root. Second, single chemical inhibition methods, which inhibit PPO activity with only a single agent. This method has a single target and limited control effect, and some agents pose a risk of residue. Third, biological control methods, which use biological agents and plant extracts to regulate the state of tobacco leaves. However, the active substances are easily affected by high temperature and humidity environments, resulting in poor stability and an inability to adapt to the curing environment in Guangxi tobacco-growing areas.

[0005] Among existing publicly available technologies, CN105146706A discloses an intensive baking method to reduce ash buildup on the upper leaves of K326 corn, which promotes dehydration by controlling the temperature at the initial stage of yellowing; CN202011095777 discloses a method for preventing ash buildup on K326 corn caused by pollen using a complex enzyme preparation of cellulase, pectinase, and lysozyme. These technologies either rely on specialized baking processes or target specific ash-causing factors, and cannot fundamentally inhibit the enzymatic browning reaction.

[0006] Since existing technologies mostly rely on single-mechanism control modes, their effects on reducing ash buildup are limited, and the increase in the proportion of high-grade tobacco is not significant. Furthermore, they generally require specialized curing processes, which conflict with the traditional production habits of tobacco farmers in Guangxi. They are also unsuitable for the high-temperature and high-humidity curing environment and conventional curing methods in the Baise, Hezhou, and Hechi production areas, resulting in significant limitations in localization and promotion. Therefore, there is an urgent need to develop a multi-target synergistic, easy-to-operate, and no-modification curing process required technology that can address the problem of browning and ash buildup in tobacco leaves during curing in the high-temperature and high-humidity tobacco-growing areas of South China. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the present invention aims to provide a compound preparation for preventing enzymatic browning during tobacco curing and its application, particularly a compound preparation suitable for preventing enzymatic browning during tobacco curing in the high-temperature and high-humidity tobacco-growing region of South China and its application.

[0008] To achieve this objective, the present invention adopts the following technical solution:

[0009] In a first aspect, the present invention provides a compound preparation for preventing enzymatic browning during the curing process of tobacco leaves, wherein the active components of the compound preparation are composed of glycyrrhizin A, brassinosteroids and melatonin.

[0010] This invention creatively combines three active components—glycyrrhizin chalcone A, brassinosteroids, and melatonin—for application to tobacco leaves before curing. This combination effectively controls enzymatic browning during tobacco curing, improves ash formation, and enhances tobacco quality.

[0011] Licorice chalcone A is a highly efficient polyphenol oxidase inhibitor. It can competitively bind to the active site of PPO and block the substrate catechol from entering the catalytic center, thereby inhibiting the initiation of enzymatic browning reaction from the source. At the same time, the multiple phenolic hydroxyl groups in its molecular structure endow it with free radical scavenging ability, which can block the oxidation chain reaction.

[0012] Brassinosteroids are a specific inhibitor of brassinosteroid (BR) biosynthesis, which can inhibit the activity of C-22 hydroxylase (CYP90B1). Under baking stress, excessive activation of the endogenous BR signaling pathway in tobacco leaves can accelerate cell metabolic turnover and membrane lipid degradation. Brassinosteroids can moderately reduce the endogenous BR level, regulate membrane lipid metabolism balance and cell osmotic homeostasis, maintain cell membrane integrity and cell compartmentalization, and reduce the material contact conditions that lead to browning.

[0013] Melatonin possesses extremely strong antioxidant activity, which can directly eliminate reactive oxygen free radicals generated during the baking process, upregulate the activity of antioxidant enzymes such as SOD and POD, maintain cellular oxygen metabolism balance, and reduce membrane lipid peroxidation damage.

[0014] The three components form a closed-loop regulatory network of "enzyme activity inhibition—cell structure protection—oxidative damage repair," acting on the three key stages of enzymatic browning: initiation, structural modification, and amplification. Glycyrrhizin A blocks the PPO-catalyzed reaction at the browning initiation stage; brassinosteroids protects the cell membrane and vacuoles during the structural maintenance stage, reducing the probability of spatial contact between the substrate and the enzyme; and melatonin clears existing ROS and quinone intermediates during the oxidative amplification stage, preventing the spread of the oxidative chain reaction. The three components exhibit complementary targets and significant synergistic effects (see schematic diagram of the three-in-one synergistic mechanism). Figure 1 As shown in the figure, the prevention and control effect is far superior to that of single-component or binary compound systems.

[0015] In some embodiments, the active components in the compound formulation consist of 5-20 parts by mass of glycyrrhizin A, 1-30 parts of brassinosteroids, and 6-18 parts of melatonin.

[0016] The mass fractions of glycyrrhizin A can be selected from 5, 8, 10, 12, 14, 15, 16, 18, 20, etc.; the mass fractions of brassinosteroids can be selected from 1, 5, 10, 12, 15, 20, 22, 25, 30, etc.; and the mass fractions of melatonin can be selected from 6, 8, 10, 12, 14, 15, 16, 18, etc.

[0017] In some embodiments, the composite formulation also includes a penetration enhancer.

[0018] Adding a penetration enhancer to a compound formulation can reduce the surface tension of the liquid, improve the permeability and absorption rate of the active ingredients on the surface of tobacco leaves, and suit the characteristics of tobacco leaves with a thicker cuticle in tobacco-growing areas such as Guangxi.

[0019] In some embodiments, the penetration enhancer is selected from any one or a combination of at least two of propylene glycol, glycerin, or ethanol, with propylene glycol being more preferred.

[0020] This invention has found that propylene glycol, glycerin, and ethanol can all significantly reduce the surface tension of the pesticide solution and improve the penetration and absorption efficiency of the active components on the surface of tobacco leaves. Their effects on controlling ash buildup are significantly better than those of the control group without additives. Among them, propylene glycol has the best overall effect, while glycerin and ethanol have comparable effects and all meet the requirements for production and use.

[0021] In some embodiments, the penetration enhancer has a mass percentage of 1%-5% in the compound formulation, such as 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, etc. More preferably, it is 2%-4%.

[0022] The effective and safe range for penetration enhancers is 1%-5%, which can significantly reduce the surface tension of the drug solution and improve the absorption efficiency of the drug. Among them, 2%-4% is the optimal addition amount, which takes into account both the control effect and the production requirements of no residue.

[0023] In some embodiments, the composite formulation also includes a solvent.

[0024] In some embodiments, the compound formulation also includes a cosolvent.

[0025] In some embodiments, the glycyrrhizin A has a mass concentration of 0.005%-0.02% (e.g., 0.005%, 0.008%, 0.01%, 0.012%, 0.015%, 0.018%, 0.02%, etc.) (w / v), the brassinosteroid has a mass concentration of 0.001%-0.03% (e.g., 0.001%, 0.005%, 0.01%, 0.015%, 0.02%, 0.025%, 0.03%, etc.) (w / v), and the melatonin has a mass concentration of 0.006%-0.018% (e.g., 0.006%, 0.008%, 0.01%, 0.012%, 0.014%, 0.016%, 0.018%, etc.) (w / v).

[0026] In some embodiments, the mass concentration of glycyrrhizin A is 0.008%-0.012% (w / v), the mass concentration of brassinosteroid is 0.002%-0.008% (w / v), and the mass concentration of melatonin is 0.01%-0.015% (w / v).

[0027] In some embodiments, the compound formulation also includes a pH buffer.

[0028] Adding a pH buffer to the compound formulation can stabilize the activity of the active component, help inhibit PPO enzyme activity, and improve the stability of prevention and control under high temperature and high humidity conditions.

[0029] In some embodiments, the pH buffer is selected from citrate-sodium citrate buffer or disodium hydrogen phosphate-citrate buffer.

[0030] In some embodiments, the amount of pH buffer added is such that the pH value of the compound preparation is adjusted to 5.5-6.5, for example, 5.5, 5.7, 5.8, 6.0, 6.2, 6.4, 6.5, etc.

[0031] The present invention has found that the pH range of 5.5-6.5 is the optimal stability range for the compound formulation. Within this range, the formulation system is uniform and transparent, has good storage stability, good inhibition effect on PPO enzyme activity, and excellent control effect on tobacco ash.

[0032] In a second aspect, the present invention provides the application of the compound formulation according to the first aspect in inhibiting enzymatic browning reaction during tobacco curing, improving ash accumulation after tobacco curing, and enhancing the sensory quality of tobacco.

[0033] Thirdly, the present invention provides a method for preventing enzymatic browning and ash accumulation during tobacco curing, the method comprising the following steps:

[0034] After the tobacco leaves are harvested and woven into poles, but before they are loaded into the drying kiln, the compound preparation described in the first aspect is applied to both sides of the tobacco leaves; after application, the leaves are left to stand to allow the compound preparation to penetrate and be absorbed; after standing, the leaves are loaded into the drying kiln as usual for drying.

[0035] The application standard is: the leaf surface is completely wet and there are no dripping liquids. The application operation must ensure that the entire tobacco leaf is sprayed evenly from top to bottom, and avoid missed spraying or double spraying.

[0036] In some implementations, the settling time is 1-2 hours, such as 1 hour, 1.5 hours, 2 hours, etc.

[0037] The process should be carried out in a cool, well-ventilated place, avoiding direct sunlight and rain during the resting period.

[0038] The baking process is completed directly using local conventional baking parameters, without the need to change process parameters such as temperature, humidity, and heating rate.

[0039] In some embodiments, the compound preparation is applied at a rate of 10-20 L per 1000 kg of fresh tobacco leaves, such as 10 L / 1000 kg, 13 L / 1000 kg, 15 L / 1000 kg, 18 L / 1000 kg, 20 L / 1000 kg, etc.

[0040] Those skilled in the art can flexibly adjust the application rate within the range of 10-20 L / 1000 kg based on the field maturity of tobacco leaves and the moisture content of fresh leaves.

[0041] In some embodiments, the preparation method of the compound formulation includes: taking glycyrrhizin chalcone A, brassinosteroids and melatonin in proportion, adding 3%-5% (e.g., 3%, 3.5%, 4%, 4.5%, 5%, etc.) of a co-solvent (e.g., ethanol) of the total volume, then adding a penetration enhancer and a pH buffer in sequence, adding deionized water to make up the volume, and stirring at 15-35℃ (e.g., 15℃, 20℃, 25℃, 30℃, 35℃, etc.) for 5-30 min (e.g., 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, etc.) to obtain the product.

[0042] All other specific point values ​​not listed in the numerical range involved in this invention can be selected and are all within the protection scope of this invention. For the sake of brevity, they will not be described in detail here.

[0043] Compared with the prior art, the present invention has the following beneficial effects:

[0044] (1) Three-in-one synergistic effect: This invention adopts a multi-target three-dimensional prevention and control system, which is different from the traditional single mechanism regulation technology. It targets the browning characteristics of K326 variety in Guangxi under high temperature and high humidity environment from June to August. The ash area of ​​the flue-cured tobacco leaves is reduced by more than 70%, and the proportion of high-grade tobacco is increased by more than 19 percentage points. The prevention and control effect is far superior to the existing similar technologies.

[0045] (2) The process is highly adaptable and applicable to multiple tobacco-growing areas: The process of spraying once before loading the kiln and letting it stand to absorb the residue does not require modification of the curing equipment or adjustment of any conventional curing parameters. It is fully compatible with the dense curing kilns and traditional curing habits in Baise, Hezhou and Hechi, and solves the stubborn problem of browning and ash accumulation in tobacco leaves during curing.

[0046] (3) Easy to operate and saves labor: The entire process of prevention and control can be completed with one spraying. No manual intervention or parameter adjustment is required during the baking stage, which significantly reduces labor costs and avoids temperature and humidity fluctuations caused by opening the baking room. It is suitable for the large-scale and household-based production model in the Guangxi tobacco area.

[0047] (4) High cost performance and outstanding localization promotion value: The use of industrial-grade plant extracts for large-scale feeding has resulted in an average input-output ratio of 10.9:1 in the tobacco-growing areas of Guangxi, which is far higher than the average return level of conventional agricultural inputs, resulting in significant economic benefits. Attached Figure Description

[0048] Figure 1 This is a schematic diagram illustrating the synergistic mechanism of the compound formulation of the present invention in preventing and controlling browning and ash accumulation in tobacco leaves. Detailed Implementation

[0049] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0050] In the following examples, all treatment groups were configured with 3 biological replicates and 3 technical replicates, and the data are expressed as mean ± standard deviation.

[0051] Testing standards and methods: Ash area ratio was determined by image analysis; browning index was calculated by visual grading from 0 to 4 levels; PPO relative activity was determined by spectrophotometry; MDA content was determined by the thiobarbituric acid method; SOD activity was determined by the nitroblue tetrazolium method; relative conductivity was determined by the conductivity meter method; tobacco leaf grading was based on the national standard GB 2635-1992 "Flue-cured Tobacco"; sensory evaluation was conducted in accordance with the industry standard YC / T 530-2015.

[0052] Uniform description of experimental materials: Unless otherwise specified, the fresh tobacco leaves used in all examples were normal mature upper and middle leaves of the main tobacco variety K326 cultivated in the corresponding tobacco-growing areas of Guangxi, corresponding to the 10th-14th leaves from the bottom of the tobacco plant (including the waist leaves and the upper two leaves). This part of the tobacco leaf has a high content of polyphenols and a high risk of cell membrane damage under curing stress, making it a high-incidence area for enzymatic browning and ash-forming defects. At the same time, it is also a core utilization part of the raw materials for the cigarette industry. Using this part as the experimental subject has sufficient industrial representativeness and technical verification value. The concentrated harvesting and curing period for this part of the tobacco leaf is from July to August each year, corresponding to the peak curing season in the Guangxi tobacco-growing areas.

[0053] Example 1

[0054] Optimization experiments on the concentration range of each core component of the compound formulation:

[0055] All the following compound formulations were prepared using the following method: Glycyrrhizin chalcone A, brassinosteroids and melatonin were taken according to the requirements of the validation, and 5% anhydrous ethanol was added to aid dissolution. Then, 3% propylene glycol and disodium hydrogen phosphate-citric acid buffer were added sequentially (until the pH of the formulation was 6.0). Deionized water was added to make up the volume, and the mixture was stirred at 25°C for 15 min to obtain the final product.

[0056] The test subject was the K326 tobacco variety from the tobacco science and technology demonstration park in Huadong Town, Jingxi City, Baise City, Guangxi Province. Normal mature middle and upper leaves in the field were selected as test materials.

[0057] Each compound preparation was sprayed on both sides of the tobacco leaves, with the standard being that the leaf surface was completely wet and no liquid droplets were dripping. The application rate was 15 L per 1000 kg of fresh tobacco leaves. After application, the leaves were placed in a cool and ventilated place and left to stand for 1 hour to allow the compound preparation to penetrate and be absorbed. After standing, the leaves were loaded into the drying oven as usual and dried in a dense drying oven with rising airflow, using the local conventional drying process.

[0058] (1) Verification of the full concentration range of glycyrrhizin A

[0059] With the concentrations of brassinosteroids 0.005% (w / v) and melatonin 0.012% (w / v) kept constant, glycyrrhizin chalcone A concentrations (w / v) were set at a gradient of 0.002%, 0.005%, 0.01%, 0.02%, and 0.05%, with water as a blank control. The experimental results are shown in Table 1. Different lowercase letters after the data in the same column indicate significant differences between treatments (P < 0.05).

[0060] Table 1

[0061]

[0062] As shown in Table 1, licorice chalcone A has a significant effect on controlling enzymatic browning and ash-laden defects in K326 tobacco leaves within the range of 0.005%-0.02%. Beyond this range, the control effect is not significantly improved and leaf residue is more likely to appear. 0.01% is the optimal cost-effectiveness concentration.

[0063] (2) Validation of the full concentration range of brassinolide

[0064] With the concentrations of glycyrrhizin chalcone A (0.01% w / v) and melatonin (0.012% w / v) kept constant, a binary formulation with brassinoazole concentration (w / v) gradients of 0.001%, 0.003%, 0.005%, 0.010%, 0.016%, and 0.030% was set up, with water (without brassinoazole) serving as the control. The effective concentration range was verified. The experimental results are shown in Table 2. Different lowercase letters after the data in the same column indicate significant differences between treatments (P < 0.05).

[0065] Table 2

[0066]

[0067] As shown in Table 2, brassinolide can stably protect the cell membrane of K326 tobacco leaves and delay leaf senescence within the range of 0.001%-0.030%, and the browning control effect is stable. 0.005% is the optimal cost-effectiveness concentration.

[0068] (3) Validation of melatonin concentration range

[0069] With the concentrations of glycyrrhizin A (0.01% w / v) and brassinosteroids (0.005% w / v) kept constant, melatonin concentration (w / v) gradients of 0.003%, 0.006%, 0.012%, 0.018%, and 0.024% were set up. A binary formulation without melatonin (replaced with water) served as a control to verify the effective concentration range. The experimental results are shown in Table 3. Different lowercase letters after the data in the same column indicate significant differences between treatments (P < 0.05).

[0070] Table 3

[0071]

[0072] As shown in Table 3, melatonin has the best antioxidant and anti-browning effects in the range of 0.006% to 0.018%, which can be adapted to the baking adversity environment of high temperature and high humidity in Guangxi in summer and has a stable control effect. Among them, 0.012% is the optimal cost-effectiveness concentration.

[0073] Example 2

[0074] Optimization test of penetration aid:

[0075] All the following compound formulations were prepared using the following method: Glycyrrhizin chalcone A (final concentration 0.01% (w / v)), brassinoazole (final concentration 0.005% (w / v)), and melatonin (final concentration 0.012% (w / v)) were taken in proportion, and 5% anhydrous ethanol was added to aid dissolution. Then, 3% of each group of osmotic aids, or a gradient concentration of 0%, 1%, 3%, 5%, and 7% propylene glycol, was added. Disodium hydrogen phosphate-citrate buffer was then added (until the pH of the formulation was 6.0), and deionized water was added to bring the volume to a final level. The mixture was stirred at 25°C for 15 min to obtain the final product.

[0076] The test subject was the K326 tobacco variety from the tobacco science and technology demonstration park in Huadong Town, Jingxi City, Baise City, Guangxi Province. Normal mature middle and upper leaves in the field were selected as test materials.

[0077] Each compound preparation was sprayed on both sides of the tobacco leaves, with the standard being that the leaf surface was completely wet and no liquid droplets were dripping. The application rate was 20 L per 1000 kg of fresh tobacco leaves. After application, the leaves were placed in a cool and ventilated place and left to stand for 2 hours to allow the compound preparation to penetrate and be absorbed. After standing, the leaves were loaded into the drying oven as usual and dried in a dense drying oven with rising airflow, using the local conventional drying process.

[0078] (1) Validation of different types of permeation aids

[0079] Three adjuvants—propylene glycol, glycerin, and ethanol—were selected and compared at a 3% addition level. A control group without adjuvants was also included. The experimental results are shown in Table 4. Different lowercase letters after the data in the same column indicate significant differences between treatments (P < 0.05).

[0080] Table 4

[0081]

[0082] As shown in Table 4, the three adjuvants, propylene glycol, glycerin, and ethanol, can significantly reduce the surface tension of the drug solution and improve the penetration and absorption efficiency of active ingredients on the surface of K326 tobacco leaves. Their ash control effects are significantly better than those of the control group without adjuvants. Among them, propylene glycol has the best overall effect, while glycerin and ethanol have similar effects and all meet the requirements for production and use.

[0083] (2) Verification of the amount of penetrant added

[0084] Using propylene glycol, which showed the best overall effect, as a representative, we set up addition gradients of 0%, 1%, 3%, 5%, and 7% to verify the optimal addition range. The experimental results are shown in Table 5. Different lowercase letters after the data in the same column indicate significant differences between treatments (P < 0.05).

[0085] Table 5

[0086]

[0087] As shown in Table 5, the effective and safe range for the penetration enhancer is 1%-5%, which can significantly reduce the surface tension of the drug solution and improve the absorption efficiency of the drug. Among them, 3% is the optimal addition amount, which takes into account both the control effect and the production requirement of no residue.

[0088] Example 3

[0089] pH optimization test of formulation:

[0090] All the following compound preparations were prepared using the following method: Glycyrrhizin chalcone A (final concentration 0.01% (w / v)), brassinoazole (final concentration 0.005% (w / v)), and melatonin (final concentration 0.012% (w / v)) were taken in proportion, and 5% anhydrous ethanol was added to aid dissolution. Then, 3% propylene glycol was added, followed by disodium hydrogen phosphate-citric acid buffer (until the pH of the preparation was 5.0, 5.5, 6.0, 6.5, and 7.0). Deionized water was added to bring the volume to a final level, and the mixture was stirred at 25°C for 15 min to obtain the final product.

[0091] The test subject was the K326 tobacco variety from the tobacco science and technology demonstration park in Huadong Town, Jingxi City, Baise City, Guangxi Province. Normal mature middle and upper leaves in the field were selected as test materials.

[0092] Each compound preparation was sprayed on both sides of the tobacco leaves, with the standard being that the leaf surface was completely wet and no liquid droplets were dripping. The application rate was 20 L per 1000 kg of fresh tobacco leaves. After application, the leaves were placed in a cool and ventilated place and left to stand for 2 hours to allow the compound preparation to penetrate and be absorbed. After standing, the leaves were loaded into the drying oven as usual and dried in a dense drying oven with rising airflow, using the local conventional drying process.

[0093] The experimental results are shown in Table 6. Different lowercase letters after the data in the same column indicate significant differences between treatments (P < 0.05).

[0094] Table 6

[0095]

[0096] The data in Table 6 show that the optimal stability range for the formulation is pH 5.5-6.5. Within this range, the formulation system is uniform and transparent, has good storage stability, exhibits excellent inhibition of PPO enzyme activity, and achieves the best control effect on tobacco ash.

[0097] Example 4

[0098] Validation of synergistic effects of ternary components:

[0099] The test subject was the K326 tobacco variety from the tobacco science and technology demonstration park in Huadong Town, Jingxi City, Baise City, Guangxi Province. Normal mature middle and upper leaves in the field were selected as test materials.

[0100] Preparation method of the ternary complete formulation: Take glycyrrhizin chalcone A (final concentration of 0.01% (w / v)), brassinoazole (final concentration of 0.005% (w / v)) and melatonin (final concentration of 0.012% (w / v)) according to the proportion, add 5% anhydrous ethanol to aid dissolution, then add 3% propylene glycol, then add disodium hydrogen phosphate-citric acid buffer (to the pH of the preparation is 6.0), add deionized water to make up the volume, stir at 25℃ for 15 min, and the product is obtained.

[0101] Eight formulation treatment groups were set up: CK: water control group; T1: glycyrrhizin chalcone A group (lacking only brassinolide and melatonin compared to the full formulation); T2: brassinolide group (lacking only glycyrrhizin chalcone A and melatonin compared to the full formulation); T3: melatonin group (lacking only glycyrrhizin chalcone A and brassinolide compared to the full formulation); T4: glycyrrhizin chalcone A + brassinolide group (lacking only melatonin compared to the full formulation); T5: glycyrrhizin chalcone A + melatonin group (lacking only brassinolide compared to the full formulation); T6: brassinolide + melatonin group (lacking only glycyrrhizin chalcone A compared to the full formulation); T7: ternary full formulation.

[0102] Treatment method: Spray each group of preparations onto both sides of the tobacco leaves, with the standard being that the leaf surface is completely wet and there are no drips. The application amount is 10 L per 1000 kg of fresh tobacco leaves. After application, place the leaves in a cool and ventilated place and let them stand for 2 hours to allow the compound preparations to penetrate and be absorbed. After standing, load the leaves into the drying room as usual and use the local conventional drying process in a dense drying room with rising airflow.

[0103] (1) Verification using the Colby exponent method

[0104] Colby's calculation formula: Eexpected = EA + EB + EC - EA×EB - EA×EC - EB×EC + EA×EB×EC; CI = Eactual / Eexpected. Where E is the ash reduction rate, CI > 1.15 indicates a synergistic effect, 0.85 ≤ CI ≤ 1.15 indicates an additive effect, and CI < 0.85 indicates an antagonistic effect.

[0105] The comparison results of the control effect of different treatment groups on browning of tobacco leaves of K326 are shown in Table 7. Different lowercase letters after the data in the same column indicate significant differences between treatments (P<0.05).

[0106] Table 7

[0107]

[0108] (2) Validation of the Bliss independent action model

[0109] To further determine the synergistic effect, cross-validation was performed using the Bliss independent action model. The Bliss model assumes that each component acts independently, and the expected effect formula is:

[0110] E expected = 1 - (1 - EA)(1 - EB)(1 - EC).

[0111] ① Using the rate of reduction in ash residue as an effect indicator:

[0112] Single-agent ash reduction rate: EA=50.2% (T1), EB=13.1% (T2), EC=17.0% (T3)

[0113] Bliss's expected reduction rate = 1 - (1 - 0.502)(1 - 0.131)(1 - 0.170) = 64.0%

[0114] Actual reduction rate = 76.9%

[0115] Actual / Expected = 1.20, indicating a significant level of synergy.

[0116] ②The increase rate of the above-mentioned tobacco ratio is an effect indicator:

[0117] Improvement rate formula: R = (Percentage of superior tobacco in the treatment group - Percentage of superior tobacco in the control group) / Percentage of superior tobacco in the control group × 100%

[0118] Single-dose improvement rates: RA = 26.78% (T1), RB = 8.42% (T2), RC = 12.53% (T3)

[0119] Bliss's expected improvement rate = 1 - (1 - 0.2678)(1 - 0.0842)(1 - 0.1253) = 41.72%

[0120] Actual improvement rate = (71.5 - 46.3) / 46.3 = 54.43%

[0121] The actual / expected ratio is 1.30, indicating a significant synergistic effect.

[0122] ③ Using the dual-indicator weighted composite index (WCI) as the effect indicator:

[0123] WCI = Ash Reduction Rate × 0.5 + High-Quality Smoke Proportion Increase Rate × 0.5

[0124] Single-dose WCI: T1=38.49%, T2=10.76%, T3=14.77%

[0125] Bliss's expected WCI = 1 - (1 - 0.3849)(1 - 0.1076)(1 - 0.1477) = 53.88%

[0126] Actual WCI = (76.9% + 54.43%) / 2 = 65.67%

[0127] The actual / expected ratio is 1.22, indicating a significant level of synergy.

[0128] The experimental results above show that the Colby index method is highly consistent with the Bliss independent action model: the Colby index of the single indicator of ash reduction rate reaches 1.20, which clearly exceeds the significant synergistic threshold of 1.15. Combined with the improvement rate of the proportion of high-grade tobacco (1.30) and the weighted comprehensive index of the two indicators (1.22), the three indicators jointly prove that the ternary formula has a significant synergistic effect.

[0129] This demonstrates that the three active components in the composite formulation of the present invention do not simply have an additive effect, but rather form a closed-loop regulatory network by acting on three key links: the initiation of browning, the maintenance of structure, and the amplification of oxidation. Under the special baking adversity of high temperature and high humidity in Guangxi, they produce technical effects that cannot be expected by those skilled in the art, and the evidence of synergistic effect is sufficient.

[0130] Example 5

[0131] Post-spray settling time gradient optimization experiment:

[0132] Preparation of the following formulation: Glycyrrhizin chalcone A (final concentration 0.01% (w / v)), brassinoazole (final concentration 0.005% (w / v)) and melatonin (final concentration 0.012% (w / v)) were taken in proportion, and 5% anhydrous ethanol was added to aid dissolution. Then 3% propylene glycol was added, followed by disodium hydrogen phosphate-citrate buffer (until the pH of the formulation was 6.0). Deionized water was added to make up the volume, and the mixture was stirred at 25°C for 15 min to obtain the final product.

[0133] The test subject was the K326 tobacco variety from Chashujiao Village, Fuyang Town, Fuchuan Yao Autonomous County, Hezhou, Guangxi. Normal mature middle and upper tobacco leaves in the field were selected as test materials.

[0134] The preparation was sprayed on both sides of the tobacco leaves, with the standard being that the leaf surface was completely wet and no liquid droplets were dripping. The application rate was 20 L per 1000 kg of fresh tobacco leaves. After application, the leaves were placed in a cool and ventilated place and allowed to stand for 0.5 h, 1 h, 1.5 h, 2 h, and 2.5 h respectively. After standing, the leaves were loaded into the drying ovens as usual and dried in a dense airflow drying oven using the local conventional drying process.

[0135] The experimental results are shown in Table 8. Different lowercase letters after the data in the same column indicate significant differences between treatments (P < 0.05).

[0136] Table 8

[0137]

[0138] The data in Table 8 show that the formulation is fully absorbed and the control effect is stable within the 1-2 hour standing period; insufficient standing leads to a decrease in effect, while excessive standing results in excessive water loss in the tobacco leaves, affecting quality. There was no significant difference between the water + standing control group and the group directly loaded into the drying kiln with water, proving that the control effect comes from the formulation itself, rather than the standing operation.

[0139] Example 6

[0140] Optimization experiment of spray dosage gradient:

[0141] Preparation of the following formulation: Glycyrrhizin chalcone A (final concentration 0.01% (w / v)), brassinoazole (final concentration 0.005% (w / v)) and melatonin (final concentration 0.012% (w / v)) were taken in proportion, and 5% anhydrous ethanol was added to aid dissolution. Then 3% propylene glycol was added, followed by disodium hydrogen phosphate-citrate buffer (until the pH of the formulation was 6.0). Deionized water was added to make up the volume, and the mixture was stirred at 25°C for 15 min to obtain the final product.

[0142] The test subject was the K326 tobacco variety from the Nandan tobacco region of Hechi City, Guangxi Province, and the middle and upper leaves of normally mature tobacco leaves in the field were selected as the test materials.

[0143] The preparation was sprayed on both sides of the tobacco leaves, with the standard being that the leaf surface was completely wet and no liquid droplets were dripping. The application amount was 5 L, 10 L, 15 L, 20 L, and 25 L per 1000 kg of fresh tobacco leaves, respectively. After application, the leaves were placed in a cool and ventilated place and allowed to stand for 2 hours. After standing, the leaves were loaded into the drying ovens as usual and dried in a dense airflow drying oven using the local conventional drying process.

[0144] The experimental results are shown in Table 9. Different lowercase letters after the data in the same column indicate significant differences between treatments (P < 0.05).

[0145] Table 9

[0146]

[0147] The data in Table 9 show that the control effect is stable when the spraying dosage is in the range of 10-20 L / 1000kg, with 15 L being the optimal dosage. Insufficient dosage will result in incomplete coverage and reduced effectiveness, while excessive dosage will lead to waste of pesticide solution and residue on the leaves.

[0148] Example 7

[0149] Compatibility verification of conventional baking processes in three locations in Guangxi:

[0150] Preparation of the following formulation: Glycyrrhizin chalcone A (final concentration 0.01% (w / v)), brassinoazole (final concentration 0.005% (w / v)) and melatonin (final concentration 0.012% (w / v)) were taken in proportion, and 5% anhydrous ethanol was added to aid dissolution. Then 3% propylene glycol was added, followed by disodium hydrogen phosphate-citrate buffer (until the pH of the formulation was 6.0). Deionized water was added to make up the volume, and the mixture was stirred at 25°C for 15 min to obtain the final product.

[0151] This experiment was conducted in Baise, Hezhou, and Hechi, using mature tobacco leaves from the middle and upper parts of the locally grown K326 variety. The compound preparation (the control group (CK) used water) was sprayed onto both sides of the tobacco leaves, with the standard being complete wetting of the leaf surface without dripping. The application rate was 15 L / 1000 kg. After application, the leaves were placed in a cool, ventilated place and allowed to stand for 2 hours. After standing, the leaves were loaded into the drying kiln as usual and dried according to the local conventional drying process in each experimental location.

[0152] Baise process: Jingxi conventional baking process;

[0153] Hezhou process: Fuchuan conventional intensive drying room process;

[0154] Hechi process: Nandan conventional baking process.

[0155] The experimental results are shown in Table 10. Different lowercase letters after the data in the same column indicate significant differences between treatments (P < 0.05).

[0156] Table 10

[0157]

[0158] As shown in Table 10, the compound formulation of the present invention exhibits stable control effects under conventional baking processes in three locations in Guangxi, reducing the ash-covered area rate by more than 73% and increasing the proportion of high-grade tobacco by more than 22 percentage points. It does not rely on a special baking process and has extremely strong adaptability.

[0159] Example 8

[0160] Multi-site production verification test in three locations in Guangxi:

[0161] To verify the stability under field production conditions, in the 2025 harvesting and drying season, 10-mu contiguous demonstration fields were set up in Jingxi, Baise, Fuchuan, Hezhou, and Nandan, Hechi, Guangxi. The variety used was K326. The compound preparation formula and application process described in Example 7 were used uniformly. The same plot was sprayed with water as a control. After maturity, the crops were harvested and dried in the same drying pit.

[0162] The field production verification results in three locations in Guangxi are shown in Table 11. Different lowercase letters after the data in the same column indicate significant differences between treatments (P < 0.05).

[0163] Table 11

[0164]

[0165] As shown in Table 11, under field production conditions, the invention has a stable control effect on the K326 variety in the three major tobacco-growing areas of Guangxi, reducing the ash-covered area rate by more than 71%, increasing the proportion of high-quality tobacco by more than 21 percentage points, and increasing the average income per mu by more than 520 yuan, demonstrating extremely high production and promotion value.

[0166] Example 9

[0167] Sensory quality evaluation:

[0168] Tobacco leaves from the three locations in Example 8 were selected and processed into cigarette samples using the same process. Five smoking experts conducted sensory evaluations using a blind tasting method. The results are shown in Table 12. Different lowercase letters after the data in the same column indicate significant differences between treatments (P < 0.05).

[0169] Table 12

[0170]

[0171] As shown in Table 12, the compound formulation of the present invention is significantly superior to the blank control in all sensory indicators, especially in terms of aroma enhancement, reduction of off-flavors and irritation, and comprehensive improvement of the intrinsic quality of K326 tobacco leaves.

[0172] Example 10

[0173] Safety and residue testing:

[0174] The pesticide residue test was conducted on the upper part of the K326 tobacco leaves from the three curing locations in Example 8, completed by the Tobacco Quality and Safety Risk Assessment Laboratory of the Ministry of Agriculture and Rural Affairs. The test method followed GB2763-2021 and the tobacco industry residue testing standards. The results are shown in Table 13.

[0175] Table 13

[0176]

[0177] As shown in Table 13, none of the active components in the compound formulation of the present invention were detected as residues in the cured tobacco leaves, which meets the requirements for safe production of green tobacco.

[0178] Example 11

[0179] Validation of effectiveness across the entire concentration range:

[0180] Using the preparation process of the compound formulation in Example 1, the experimental materials and baking process in Example 1, and setting up three concentration combinations of the active component (low, medium, and high), the control effect at the boundary and within the region was verified:

[0181] Low-concentration combination: Glycyrrhizin A 0.005% + Brassinolide 0.001% + Melatonin 0.006%;

[0182] Medium concentration combination: Glycyrrhizin chalcone A 0.01% + Brassica oleracea 0.005% + Melatonin 0.012%;

[0183] High-concentration combination: Glycyrrhizin chalcone A 0.02% + Brassica oleracea 0.03% + Melatonin 0.018%.

[0184] The experimental results are shown in Table 14. Different lowercase letters after the data in the same column indicate significant differences between treatments (P < 0.05).

[0185] Table 14

[0186]

[0187] As shown in Table 14, the low, medium, and high concentration combinations were all significantly better than the water control, with a reduction of ash area by more than 62% and an increase of more than 14 percentage points in the proportion of high-quality tobacco. After the concentration was increased to the upper limit of the range, the control effect remained stable, proving that the concentration range specified in the claims was reasonable and effective, and could cover the effective control range of the Guangxi K326 variety.

[0188] Example 12

[0189] Industrial practicality evaluation:

[0190] (1) Accurate calculation of raw material costs

[0191] Taking 1 ton of formulation as an example, the calculation based on the industrial-grade bulk purchase price is shown in Table 15:

[0192] Table 15

[0193]

[0194] (2) Calculation of application costs and economic benefits

[0195] Calculation logic: Increase in net profit per mu = Increase in output value per mu - Total input cost per mu;

[0196] Input-output ratio = Increase in net profit per acre ÷ Total input cost per acre.

[0197] Formulation cost calculation: Based on the optimized formula and large-scale production, the formulation production cost is approximately RMB 1.54 / L; in production applications, 15L of formulation is sprayed per 1000kg of fresh tobacco leaves. In the tobacco-growing areas of Guangxi, the average yield of fresh tobacco leaves per mu is 1600kg, corresponding to a formulation usage of 24L. The formulation cost per mu is calculated as: RMB 1.54 / L × 24L = RMB 36.96.

[0198] Labor cost calculation: The seasonal daily wage for tobacco harvesting and curing in Guangxi is about 170 yuan / person / day (8-hour work system). The spraying operation before loading the tobacco barn is carried out simultaneously with the pole weaving process. A skilled sprayer can complete the spraying operation of about 21 mu per day in conjunction with the pole weaving production line, which translates to a labor cost of about 8 yuan per mu.

[0199] Total input per mu: The total cost of pesticides and labor for spraying per mu is 36.96 yuan + 8 yuan = 44.96 yuan. The production calculation is based on 45 yuan / mu.

[0200] The economic benefits of different production areas in Guangxi were calculated using the above method, and the results are shown in Table 16.

[0201] Table 16

[0202]

[0203] As shown in Table 16, the compound formulation involved in this invention has good economic benefits and promotion feasibility in the three major tobacco-producing areas of Guangxi. The average input-output ratio is 10.9:1, which is much higher than the average return level of about 3:1 for conventional agricultural production materials. The returns in the three areas are stable, with the input-output ratio in Baise reaching 11.1:1. The economic and social benefits are significant, and it has the value for large-scale promotion and application.

[0204] The applicant declares that the technical solution of this invention is illustrated by the above embodiments, but this invention is not limited to the above embodiments, that is, it does not mean that this invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the products of this invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of this invention.

[0205] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0206] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. A compound preparation for preventing enzymatic browning during tobacco curing, characterized in that, The active components in the compound preparation consist of glycyrrhizin A, brassinosteroids, and melatonin.

2. The compound formulation according to claim 1, characterized in that, The active components in the compound preparation consist of 5-20 parts by mass of glycyrrhizin A, 1-30 parts of brassinosteroids, and 6-18 parts of melatonin.

3. The compound formulation according to claim 1, characterized in that, The compound formulation also includes a penetration enhancer; Preferably, the penetration enhancer is selected from any one or a combination of at least two of propylene glycol, glycerin, or ethanol, more preferably propylene glycol; Preferably, the permeation aid has a mass percentage of 1%-5% in the compound formulation, more preferably 2%-4%.

4. The compound formulation according to claim 1, characterized in that, The compound formulation also includes a solvent; Preferably, the compound formulation further includes a cosolvent; Preferably, the mass concentration of glycyrrhizin A is 0.005%-0.02% (w / v), the mass concentration of brassinosteroid is 0.001%-0.03% (w / v), and the mass concentration of melatonin is 0.006%-0.018% (w / v). Preferably, the mass concentration of glycyrrhizin A is 0.008%-0.012% (w / v), the mass concentration of brassinosteroid is 0.002%-0.008% (w / v), and the mass concentration of melatonin is 0.01%-0.015% (w / v).

5. The compound formulation according to claim 1, characterized in that, The compound formulation also includes a pH buffer; Preferably, the pH buffer is selected from citrate-sodium citrate buffer or disodium hydrogen phosphate-citrate buffer; Preferably, the amount of pH buffer added is such that the pH value of the compound preparation is adjusted to 5.5-6.

5.

6. The application of the compound formulation according to any one of claims 1-5 in inhibiting enzymatic browning reaction during tobacco curing, improving ash accumulation after tobacco curing, and enhancing the sensory quality of tobacco.

7. A method for preventing enzymatic browning and ash accumulation during tobacco leaf curing, characterized in that, The method includes the following steps: After the tobacco leaves are harvested and woven into poles, and before they are loaded into the drying kiln, the compound preparation as described in any one of claims 1-5 is applied to both sides of the tobacco leaves; after application, the leaves are left to stand to allow the compound preparation to penetrate and be absorbed; after standing, the leaves are loaded into the drying kiln normally for drying.

8. The method according to claim 7, characterized in that, The settling time is 1-2 hours.

9. The method according to claim 7, characterized in that, The compound preparation is applied at a rate of 10-20 L per 1000 kg of fresh tobacco leaves.

10. The method according to claim 7, characterized in that, The preparation method of the compound preparation includes: taking glycyrrhizin chalcone A, brassinosteroid and melatonin in proportion, adding 3%-5% of the total volume of solubilizer, then adding penetration enhancer and pH buffer in sequence, adding deionized water to make up the volume, and stirring at 15-35℃ for 5-30 min to obtain the product.

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