Method for producing fen-flavor tobacco leaves

Abstract

The invention relates to a method for producing fen-flavor tobacco leaves. The method comprises the following steps: 1) raising seedlings; 2, transplanting is conducted on December 25-31 days; (3) topping when flower buds of the tobacco plants do not completely extend out and are still wrapped by top leaves; and 4) harvesting when 70%-80% of leaf surfaces of the tobacco leaves in the second shed are yellow green and main veins become white and 66%-74% of the tobacco leaves are white. The method is simple, convenient and operable, the content of aroma substances in the tobacco leaves is increased, and the quality and stability of the tobacco leaves are improved.

Description

Technical Field

[0001] The invention belongs to the technical field of tobacco, and in particular relates to a method for producing light-fragrant style tobacco leaves. Background Art

[0002] One of the typical characteristics of light-flavor tobacco is that it contains rich aromatic substances, such as dimethyl succinate (Butanedioic acid, dimethyl ester), 2-hydroxy-2-methylpropyl benzoate (Benzoic acid, 2-hydroxy-, 2-methylpropyl ester), 1,1-diethoxynonane (Nonane, 1,1-diethoxy-), 6,6-dimethylbicyclo[3.1.1]hept-2-ene-2-carboxaldehyde, 6,6-dimethyl-), which give tobacco leaves a fresh and sweet aroma. The accumulation of these aroma substances is significantly affected by the tobacco growth environment, agronomic measures and harvest time.

[0003] Cui Bi No. 1 is a representative tobacco variety in Fujian tobacco area, known for its unique light fragrance style. Cui Bi No. 1 tobacco leaves are soft and tough, with a compact leaf structure and good ability to accumulate aroma substances, especially dimethyl succinate, 2-hydroxy-2-methylpropyl benzoate, 1,1-diethoxynonane, and 6,6-dimethylbicyclo[3.1.1]-2-heptene-2-carboxaldehyde. However, this variety is sensitive to light, temperature and water requirements, especially in the production process, the timing of cultivation and management measures is high.

[0004] The climate of Fujian tobacco-growing areas presents typical subtropical monsoon climate characteristics, with warm and humid weather throughout the year, sufficient sunshine, but concentrated rainy seasons, especially during the tobacco production season, with frequent rainfall, which affects the growth of tobacco leaves and the accumulation of aroma substances. In the traditional tobacco planting model, the impact of changes in climate conditions on the formation of aroma substances is rarely considered.

[0005] How to optimize cultivation measures to increase the content of these key aroma substances in high-quality varieties such as Cui Bi No. 1 has become the key to improving the quality of tobacco leaves. Summary of the invention

[0006] The present invention provides a method for producing light-flavor style tobacco leaves, comprising:

[0007] 1) Seedling cultivation;

[0008] 2) Transplanting is carried out from December 25 to 31;

[0009] 3) Topping the tobacco plant when the flower buds have not yet fully extended and are still wrapped by the top leaves;

[0010] 4) The tobacco leaves in the upper second shed are harvested when the leaf surface is 70% to 80% yellow-green and the main veins are 66% to 74% white.

[0011] In certain embodiments, the sowing time for raising seedlings is from November 1 to 7.

[0012] In some embodiments, the light-flavor style tobacco leaf is Cui Bi No. 1 tobacco leaf. In some embodiments, the light-flavor style tobacco leaf is Cui Bi No. 1 upper tobacco leaf.

[0013] In certain embodiments, transplanting is performed between December 26 and 30.

[0014] In certain embodiments, the seedlings are raised by sowing tobacco seeds onto seedling trays.

[0015] In certain embodiments, the seedling raising method is wet seedling raising.

[0016] In certain embodiments, the transplanting site is a tobacco-growing area in Fujian, preferably Youxi County, Sanming City, Fujian Province, and preferably Longyang Village, Yangzhong Town, Youxi County.

[0017] In certain embodiments, the transplanting soil texture is sandy loam.

[0018] In certain embodiments, the crop prior to transplanting is rice.

[0019] In certain embodiments, ridges are formed and holes are dug in the tobacco field, and the tobacco seedlings are transplanted into the holes on the ridges after being grown.

[0020] In certain embodiments, the method further comprises: 5) baking.

[0021] The present invention also provides a tobacco leaf, which is prepared by the method for producing a light-flavor style tobacco leaf according to any embodiment of the present invention.

[0022] The present invention also provides a tobacco leaf composition, which comprises the tobacco leaf as described above.

[0023] The present invention also provides a tobacco product, which comprises the tobacco leaf and / or tobacco leaf composition of the present invention as described above. In certain embodiments, the tobacco product is a cigarette.

[0024] The present invention also provides the use of the tobacco leaf and / or tobacco leaf composition as described above in tobacco products. In certain embodiments, the tobacco product is a cigarette.

[0025] Definition of terms

[0026] In the present invention, the term "upper tobacco leaves" is determined according to the national standard tobacco terminology (GB / T 18771.1-2015), and generally refers to several tobacco leaves growing on the upper part of the main stem of the tobacco plant, including upper two-shed leaves and top leaves. Among them, the upper two-shed leaves are several upper tobacco leaves growing on and adjacent to the middle leaves; the top leaves are several tobacco leaves growing on the uppermost part of the main stem of the tobacco plant.

[0027] In the present invention, the term "upper two-bay leaves" generally refers to a number of upper tobacco leaves growing above and adjacent to the middle leaves, and "top leaves" generally refers to a number of tobacco leaves growing at the top of the main stem of the tobacco plant. The top leaves and upper two-bay leaves are collectively referred to as upper tobacco leaves.

[0028] In the present invention, the term "the second leaf from the top" refers to the second to last tobacco leaf from the top of the main stem of the tobacco plant.

[0029] In the present invention, the term "the fifth leaf from the top" refers to the fifth leaf from the top of the main stem of the tobacco plant.

[0030] In the present invention, the term "topping" refers to an agronomic measure of removing the top of the tobacco plant to reduce the consumption of nutrients in the tobacco plant due to flowering and fruiting, promote the distribution of nutrients in the body to the leaves and improve the quality of tobacco leaves.

[0031] In the present invention, the term "main vein" refers to a stem vein in the center of a plant leaf from the petiole to the leaf tip.

[0032] In the present invention, the term "budding" refers to the appearance of green buds at the top of the tobacco main stem.

[0033] In the present invention, the term "heart-locking" refers to the tobacco plant buds still enclosed in the top leaflets.

[0034] In the present invention, the term "jointing" refers to the phenomenon that each node of the main stem of a stem crop grows very fast at a certain stage during the growth period.

[0035] In the present invention, the term "dry-bulb temperature" refers to the value read from a dry-bulb thermometer exposed to the air but not directly exposed to the sun, and is the actual temperature of the air contacting the surface of the sphere.

[0036] In the present invention, the term "wet-bulb temperature" refers to wrapping the temperature-sensing part of an ordinary thermometer with wet gauze, with the lower end of the gauze immersed in water to maintain the air humidity of the temperature-sensing part to saturation, and maintaining a certain amount of air circulation around the gauze so that the surrounding air is close to isenthalpy. After the reading reaches stability, the reading displayed by the thermometer at this time is approximately considered to be the wet-bulb temperature.

[0037] In the present invention, the term "ridge" refers to a strip of high ground above the ground made in a certain width and interval in a farmland, and this structure consists of a ridge platform and a ridge ditch. The ridge platform is located at the upper part of the ridge and is a part of the ridge, and the ridge ditch is a naturally formed gully between the ridges.

[0038] Beneficial technical effects of the present invention

[0039] The method for producing light-fragrance style tobacco leaves provided by the present invention improves the content of dimethyl succinate (Butanedioic acid, dimethyl ester), 2-hydroxy-2-methylpropyl benzoate (Benzoic acid, 2-hydroxy-, 2-methylpropyl ester), 1,1-diethoxynonane (Nonane, 1,1-diethoxy-), and 6,6-dimethylbicyclo[3.1.1]hept-2-ene-2-carboxaldehyde (Bicyclo[3.1.1]hept-2-ene-2-carboxaldehyde, 6,6-dimethyl-) in light-fragrance style tobacco leaves (such as Cui Bi No. 1 tobacco leaves) by optimizing cultivation and management measures. The method focuses on early transplanting, early topping, and early harvesting, and promotes the synthesis of tobacco aroma substances by accurately adjusting the time nodes of key agronomic operations, thereby significantly improving the light-fragrance style of tobacco leaves.

[0040] This method is applicable to tobacco-growing areas in Fujian and areas with similar climate and soil conditions. The climate characteristics of tobacco-growing areas in Fujian are: high humidity, simultaneous rain and heat, sufficient sunlight but a long rainy season, which provide necessary conditions for the implementation of the "three early" measures. Soil in tobacco-growing areas in Fujian: The soil has moderate fertility and is loose and breathable, which is suitable for the rapid development of tobacco roots in the early stage.

[0041] This method transplants tobacco seedlings from December 25 to 31, which can extend the tobacco growth period, make full use of light and heat resources in early spring, extend the metabolic period of tobacco leaves, increase the accumulation of amino acids and their derivatives, sugars and lipid compounds, and accumulate more nutrients and aroma substances during the period of intense photosynthesis. By avoiding the overlap of the transplanting period and the high precipitation period, the impact of excessive precipitation on tobacco root absorption and growth is reduced, which is conducive to the accumulation of aroma substances. At the same time, the negative impact of the rainy season on tobacco leaf maturity and harvesting is avoided.

[0042] The method involves topping the tobacco plant when the flower buds of the tobacco plant have not yet fully extended and are still wrapped by the top leaves. By topping in advance, the competition of the top buds for nutrients can be reduced during the vegetative growth stage of the tobacco, so that more energy can be used for the development of the leaves and the synthesis of aromatic substances, and the storage of aroma precursors can be further promoted. The present invention controls the plant height and leaf growth of the tobacco by topping before the traditional topping period, so that the tobacco leaves accumulate more dimethyl succinate (Butanedioic acid, dimethyl ester), 2-hydroxy-2-methylpropyl benzoate (Benzoic acid, 2-hydroxy-, 2-methylpropyl ester), 1,1-diethoxynonane (Nonane, 1,1-diethoxy-), 6,6-dimethylbicyclo[3.1.1]-2-heptene-2-carboxaldehyde (Bicyclo[3.1.1]hept-2-ene-2-carboxaldehyde, 6,6-dimethyl-), thereby improving the aroma of the tobacco leaves.

[0043] The method harvests the tobacco leaves in the upper second shed when the leaf surface is 70% to 80% yellow-green and the main veins are 66% to 74% white, ensuring that the tobacco leaves are picked in time at the end of the optimal growing period, preventing the loss of aroma substances due to the excessively long growing period and the influence of the rainy season, and effectively reducing the risk of aroma substance degradation caused by enzymatic reactions in over-mature tobacco leaves in a high-humidity environment. At the same time, it can avoid the quality of tobacco leaves being affected by excessive moisture in the rainy season, and ensure that the best aroma components are obtained when photosynthesis and nutrient accumulation reach their peak, maximize the retention of aroma precursors, and provide high-quality raw materials for subsequent baking.

[0044] In the subsequent baking process, this method efficiently converts aroma precursors (such as amino acids and lipids), promotes the formation of aromatic alcohols, aldehydes and pyrazine compounds, and enhances the aroma level of tobacco leaves.

[0045] This method combines the three key agronomic measures of "early transplanting, early topping, and early harvesting" to enable tobacco plants to carry out photosynthesis and metabolism more fully under conditions of suitable light and temperature, and accumulate more aromatic substances. In particular, in Cui Bi No. 1 tobacco leaves, the synthesis of dimethyl succinate (Butanedioic acid, dimethyl ester), 2-hydroxy-, 2-methylpropyl benzoate (Benzoic acid, 2-hydroxy-, 2-methylpropyl ester), 1,1-diethoxynonane (Nonane, 1,1-diethoxy-), and 6,6-dimethylbicyclo[3.1.1]hept-2-ene-2-carboxaldehyde (Bicyclo[3.1.1]hept-2-ene-2-carboxaldehyde, 6,6-dimethyl-) is significantly promoted by these management measures, which increases the aroma component content of tobacco leaves, thereby enhancing the fragrance style of tobacco leaves and meeting the market's quality demand for high-end tobacco leaves.

[0046] This method not only increases the content of aroma substances in tobacco leaves by optimizing cultivation management, but also improves the quality and stability of tobacco leaves. Compared with traditional cultivation methods, this method not only improves the aroma, but also effectively controls the negative impact of unfavorable climate factors (such as excessive precipitation in the rainy season) on tobacco production, making the tobacco production process more scientific and controllable, and has broad application prospects.

[0047] This method is simple and easy to operate and does not impose any extra burden on farmers.

[0048] This method has strong adaptability and is suitable for Fujian tobacco-growing areas and similar ecological areas. It can effectively avoid the impact of adverse environments, such as high humidity in the rainy season, on the quality of tobacco leaves and improve tobacco leaf yield and quality.

[0049] This method optimizes tobacco growth management measures, fully utilizes the synergistic effects of natural resources (light, heat, etc.) and agronomic operations, improves planting efficiency, reduces resource waste, promotes the sustainable development of tobacco planting, and improves the economic benefits of planting. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 Shows the orthogonal randomized experiment setting scheme for different combinations of sowing period, topping period and harvest maturity;

[0051] Figure 2 Radar chart showing 10 sensory flavors of flue-cured tobacco leaves from different test groups. DETAILED DESCRIPTION

[0052] The substantive content of the present invention is further described below in conjunction with specific embodiments of the present invention. It should be understood that the following examples are only used to illustrate the present invention, but are not intended to limit the scope of protection of the present invention. In the following examples, if specific conditions are not specified, they are carried out according to conventional conditions or manufacturer recommendations. The raw materials used are not specified by the manufacturer, and are all conventional products that can be obtained commercially.

[0053] Although many materials and operating methods used in the following examples are well known in the art, the present invention is still described in as much detail as possible. It is clear to those skilled in the art that the materials and operating methods used in the following examples are well known in the art unless otherwise specified.

[0054] One embodiment of the present invention provides a method for producing a light-flavor style tobacco leaf, comprising:

[0055] 1) Seedling cultivation;

[0056] 2) Transplanting is carried out from December 25 to 31;

[0057] 3) Topping the tobacco plant when the flower buds have not yet fully extended and are still wrapped by the top leaves;

[0058] 4) The tobacco leaves in the upper second shed are harvested when the leaf surface is 70% to 80% yellow-green and the main veins are 66% to 74% white.

[0059] In one embodiment, the sowing time for raising seedlings is from November 1 to 7. In one embodiment, the sowing time for raising seedlings is November 1.

[0060] In one embodiment, the light-flavor style tobacco leaf is Cui Bi No. 1 tobacco leaf. In one embodiment, the light-flavor style tobacco leaf is Cui Bi No. 1 upper tobacco leaf.

[0061] In one embodiment, the seedlings are raised by sowing tobacco seeds on seedling trays.

[0062] In one embodiment, the seedling raising method is wet seedling raising.

[0063] In one embodiment, the transplanting site is a tobacco-growing area in Fujian, preferably Youxi County, Sanming City, Fujian Province, and preferably Longyang Village, Yangzhong Town, Youxi County.

[0064] In one embodiment, the seeds of Cui Bi No. 1 are sown on a seedling tray for seedling raising, and the sowing time is November 1. The seedling raising method is wet seedling raising, and when the tobacco seedlings grow to five leaves and one heart, the strong tobacco seedlings are selected and transplanted to the field.

[0065] In one embodiment, the transplanting is performed on December 26-30. In one embodiment, the transplanting is performed on December 26. The transplanting time is 15-20 days earlier than the traditional transplanting time. According to the climatic conditions of the tobacco-growing areas in Fujian, the conventional transplanting time is usually mid-to-late January, but the present invention recommends transplanting in advance to late December, so that the tobacco seedling stage overlaps with the rainy season and fully utilizes the light and temperature conditions of the seedling stage.

[0066] In one embodiment, the transplanting soil texture is sandy loam.

[0067] In one embodiment, the crop prior to transplanting is rice.

[0068] In one embodiment, ridges are formed and holes are dug in the tobacco field, and the tobacco seedlings are transplanted into the holes on the ridges.

[0069] In one embodiment, healthy tobacco seedlings with intact leaves and well-developed root systems are selected for transplanting. One tobacco seedling is planted in each hole, and the row and plant spacing is controlled within an appropriate range (about 1 meter × 0.5 meter) to ensure that the tobacco has enough space for photosynthesis and nutrient accumulation.

[0070] In one embodiment, before transplanting, fields with high soil fertility and good drainage are selected for deep plowing and careful cultivation, and sufficient base fertilizer (mainly including nitrogen, phosphorus and potassium fertilizers) is applied to ensure that the tobacco roots can quickly take root and absorb sufficient nutrients after transplanting.

[0071] In one embodiment, the flower bud of tobacco plant has not yet stretched out completely, and is still in the process of being wrapped by the top leaf and topping. The topping time is about about 90 days after transplanting, which is about 7-10 days ahead of schedule than the traditional topping time. At this moment, tobacco has entered the vigorous growth stage, and the top flower bud forms gradually, but has not yet opened, and is in the best nutritional growth and aroma substance synthesis period. When the tobacco plant reaches 16-18 effective leaves, artificial topping is carried out, and the leaves below the top are retained. After topping, the nutrient of tobacco will be concentratedly supplied to the remaining leaves, which helps to increase the thickness of the leaves and the accumulation of aroma substances. After topping, remove the lateral buds (pinching off the newborn lateral buds) in time to avoid the lateral buds from growing and dispersing nutrients. Combine fertilization and watering management, supplement the trace elements such as nitrogen, phosphorus, potassium required for tobacco, promote leaf growth, and strengthen the synthesis of aroma components.

[0072] In one embodiment, the tobacco leaves in the upper two sheds are harvested when 70% to 80% of the leaf surface is yellow-green and the main veins turn white by 66% to 74%. This harvesting time is 3-5 days earlier than the traditional harvesting time, and the tobacco leaves are picked when they are moderately changing color and alternating between yellow and green. Usually the harvesting period is about 130 days after transplanting, but due to the influence of early topping and other management measures, the harvesting period is brought forward in time to avoid excessive moisture or rot of the leaves due to the rainy season. When harvesting, the tobacco leaves are picked layer by layer in batches from bottom to top. Priority is given to picking the mature tobacco leaves in the lower part to ensure that the middle and upper leaves continue to fully absorb nutrients and light to achieve the optimal aroma content.

[0073] During the entire growth process, scientific fertilization is carried out in combination with the fertilizer requirements of different growth stages. Nitrogen fertilizer is mainly used in the early stage, and the proportion of phosphorus and potassium fertilizers is increased in the middle and late stages to promote the thickness of tobacco leaves and the formation of aroma substances. Appropriate drainage management is carried out before the rainy season arrives to prevent water accumulation from affecting the health of the root system.

[0074] During the entire growth process, conventional plant protection programs are adopted. Since early transplanting avoids the arrival of the rainy season, pests and diseases may be weakened. However, the field pest and disease situation should still be monitored in time, and preventive spraying or biological control measures should be taken, especially to strengthen prevention and control before and after the plum rain season to avoid affecting the quality of tobacco leaves.

[0075] In one embodiment, the method further comprises: 5) baking.

[0076] In one embodiment, the tobacco leaves are immediately roasted after harvesting to ensure that the aroma substances are stabilized under high temperature treatment. During roasting, the temperature and humidity are adjusted according to the maturity of the tobacco leaves, and a gentle drying process is usually used to retain the aroma components of the tobacco leaves.

[0077] After the tobacco leaves were baked, the sensory evaluation was carried out on the baked tobacco leaves, and the aroma purity and the light fragrance style were examined in particular. The contents of key aroma substances reflecting the characteristics of the light fragrance style, such as butanedioic acid, dimethyl ester, 2-hydroxy-, 2-methylpropyl ester, 1,1-diethoxynonane, and 6,6-dimethylbicyclo[3.1.1]hept-2-ene-2-carboxaldehyde, 6,6-dimethyl-, were detected, and it was found that the contents of the above key aroma substances were significantly increased compared with those of other test groups.

[0078] The present invention significantly improves the contents of aroma components of tobacco leaves, especially dimethyl succinate (Butanedioic acid, dimethyl ester), 2-hydroxy-2-methylpropyl benzoate (Benzoic acid, 2-hydroxy-, 2-methylpropyl ester), 1,1-diethoxynonane (Nonane, 1,1-diethoxy-), and 6,6-dimethylbicyclo[3.1.1]-2-heptene-2-carboxaldehyde (Bicyclo[3.1.1]hept-2-ene-2-carboxaldehyde, 6,6-dimethyl-) through measures such as early transplanting, early topping and early harvesting, and the fresh fragrance style of tobacco leaves is more prominent. At the same time, through measures such as early transplanting, early topping and early harvesting, the adverse effects of the rainy season on the quality of tobacco leaves were avoided. The total number of upper leaves of Cui Bi No. 1, total weight, and B2F and B3F grades after grading were significantly improved. The color, maturity, leaf structure, identity, oil content, chroma and other aspects of the flue-cured tobacco leaves were significantly improved. The fresh and sweet style, sweetness, aroma quality, aroma volume, permeability, smoke characteristics, taste characteristics and other indicators of the flue-cured tobacco leaves were significantly improved, which enhanced the overall market competitiveness of tobacco leaves.

[0079] Another embodiment of the present invention further provides a tobacco leaf, which is prepared by the method for producing a light-flavor style tobacco leaf according to any embodiment of the present invention.

[0080] Another embodiment of the present invention also provides a tobacco leaf composition, which comprises the tobacco leaf as described above.

[0081] Another embodiment of the present invention also provides a tobacco product, which comprises the tobacco leaf and / or tobacco leaf composition of the present invention as described above. In one embodiment, the tobacco product is a cigarette.

[0082] Another embodiment of the present invention also provides the use of the tobacco leaf and / or tobacco leaf composition as described above in tobacco products. In one embodiment, the tobacco product is a cigarette.

[0083] The present invention is further described below in conjunction with a specific embodiment.

[0084] Example

[0085] 1. Cultivation

[0086] In different experimental groups, the "three early" experimental groups of early transplanting, early topping and early harvesting and other orthogonal experiments designed according to two-factor experiments were set as control group combinations. Specifically, the experimental group adjusted the transplanting period according to temperature conditions, adjusted the topping period according to differences in growth stages, and adjusted the harvesting time according to maturity. A total of 4 experimental groups were obtained by designing orthogonal experiments with 3 variables and 2 factors. At the same time, considering the key role of early harvesting, early sowing, early topping and late harvesting were added, totaling 6 experimental groups (Table 1). In Table 1, bud topping refers to topping when the flower buds of the tobacco plants have not yet fully extended and are still wrapped by the top leaves.

[0087] Table 1 Orthogonal test settings for different combinations of sowing period, topping period and harvest maturity

[0088]

[0089] The cultivation experiment was carried out in Longyang Village, Yangzhong Town, Youxi County, Sanming City, Fujian Province from November 2022 to June 2024. The experimental site selected representative plots, with the previous crop being rice, sandy loam soil, medium fertility, uniform texture, flat ground, convenient drainage and irrigation, and no soil-borne diseases in recent years. Each experimental block group was repeated three times, and the random block arrangement (such as Figure 1 Seeds of Cui Bi No. 1 were sown on seedling trays for seedling raising on November 1. The seedling raising method was wet seedling raising. When the tobacco seedlings grew to five leaves and one heart, the strong tobacco seedlings were selected and transplanted to the field. Each experimental area was square in layout and had an area of ​​about 222.79m 2 , transplant about 350 tobacco seedlings, the row spacing of tobacco plants in the area is 120cm, the plant spacing is 50cm, and protective rows are set around. Sowing, topping and harvesting are carried out according to the time listed in Table 1, and other production measures are implemented according to local production technical standards.

[0090] 2. Baking

[0091] After harvesting, baking tests were carried out according to the local mature baking process. The specific process conditions are shown in Table 2.

[0092] Table 2 Baking process conditions

[0093]

[0094] 3. Test various indicators of tobacco leaves

[0095] The carotenoid substances and primary metabolites in the fresh tobacco leaves obtained above are detected, and the aroma components in the baked tobacco leaves are detected, and the appearance and sensory evaluation are performed, and the economic indicators are calculated.

[0096] (1) Determination of carotenoids in tobacco leaves:

[0097] Take out the fresh tobacco leaf sample stored at low temperature, grind it into powder with a ball mill (30Hz, 1min); weigh 50mg of the ground sample, and extract it with 0.5mL of a mixed solution of n-hexane / acetone / ethanol (1:1:1, v / v / v) containing 0.01% BHT (g / mL); vortex at room temperature for 20min, centrifuge at 12000r / min for 5min at 4℃, take the supernatant, repeat the extraction once, and then centrifuge and combine the supernatants; concentrate the obtained extract, re-dissolve it with 100μL of dichloromethane, filter it through a 0.22μm filter membrane, and store it in a brown injection bottle for LC-MS / MS analysis.

[0098] Ultra Performance Liquid Chromatography (UPLC) TM AD, https: / / sciex.com.cn / ) and tandem mass spectrometry (Tandem Mass Spectrometry, MS / MS) ( 6500+, https: / / sciex.com.cn / ) to detect carotenoids in samples. The liquid phase conditions mainly include: chromatographic column: YMC C30 (3μm, 100mm×2.0mm id); mobile phase: phase A, methanol / acetonitrile (1:3, v / v) with 0.01% BHT and 0.1% formic acid; phase B, methyl tert-butyl ether with 0.01% BHT; gradient elution program: 0min A / B is 100:0 (V / V), 3min is 100:0 (V / V), 5min is 30:70 (V / V), 9min is 5:95 (V / V), 10min is 100:0 (V / V), 11min is 100:0 (V / V); flow rate is 0.8mL / min; column temperature is 28℃; injection volume is 2μL. The mass spectrometry conditions mainly include: atmospheric pressure chemical ionization source (APCI) temperature 350°C, curtain gas (CUR) 25psi. In Q-Trap 6500+, each ion pair is scanned and detected according to the optimized declustering potential (DP) and collision energy (CE).

[0099] Based on the standard, the MWDB (Metware Database) database was constructed to qualitatively analyze the data of mass spectrometry detection. Quantification was completed by using the multiple reaction monitoring (MRM) analysis of the triple quadrupole mass spectrometer. In the MRM mode, the quadrupole first screens the precursor ions (parent ions) of the target substance, and excludes the ions corresponding to other molecular weight substances to preliminarily eliminate interference; the precursor ions are induced by the collision chamber and then broken to form multiple fragment ions, and the fragment ions are then filtered by the triple quadrupole to select the required characteristic fragment ions, eliminating the interference of non-target ions, making the quantification more accurate and repeatable. After obtaining the mass spectrometry analysis data of different samples, the chromatographic peaks of all targets were integrated and quantitatively analyzed by the standard curve. Analyst 1.6.3 software was used to process the mass spectrometry data. MultiQuant 3.0.3 software was used to process the mass spectrometry data, and the chromatographic peaks detected in different samples of the analyte were integrated and corrected with reference to the retention time and peak shape information of the standard to ensure the accuracy of qualitative and quantitative analysis.

[0100] Use methanol / methyl tert-butyl ether mixed solution (3:1, v / v) to prepare 0.001 μg / mL, 0.005 μg / mL, 0.01 μg / mL, 0.05 μg / mL, 0.1 μg / mL, 0.5 μg / mL, 1 μg / mL, 5 μg / mL, 10 μg / mL, 50 μg / mL, 100 μg / mL, 250 μg / mL, 400 μg / mL carotenoid standard solutions (carotenoid standards include: antheraxanthin, phytoene, 8'-β-apocarotene aldehyde , astaxanthin, canthaxanthin, capsanthin, capsanthin, echinenone, lutein, lycopene, neoxanthin, phytoene, violaxanthin, zeaxanthin, zeaxanthin dipalmitate, α-carotene, α-cryptoxanthin, β-carotene, β-leucine, β-cryptoxanthin, γ-carotene, ε-carotene), obtain the chromatographic peak intensity data of the corresponding quantitative signal of each concentration standard; use the standard concentration (Concentration) as the horizontal coordinate and the standard peak area (Area) as the vertical coordinate to draw the standard curve of different substances. Substitute the integrated peak area of ​​all samples detected into the linear equation of the standard curve for calculation, and further substitute it into the calculation formula for calculation, and finally obtain the content data of the substance in the actual sample.

[0101] (2) Determination of primary metabolites of tobacco leaves (including amino acids, sugars, lipids, nucleotides, etc.):

[0102] Fresh tobacco leaf samples were placed in a freeze dryer (Scientz-100F) for vacuum freeze drying; ground (30 Hz, 1.5 minutes) to powder using a grinder (MM400, Retsch); 50 mg of powder was weighed using an electronic balance (MS105DM), dissolved in 1000 μL of 70% methanol internal standard extract, 500 μL of petroleum ether was added, vortexed for 5 minutes, allowed to stand for stratification, and centrifuged at 12000 r / min for 10 minutes at 4°C. All the supernatant was transferred through a 0.22 μm PTFE filter membrane into a glass-lined tube in a brown injection bottle and stored at -20°C until LC-MS / MS analysis. The 70% methanol internal standard extract was prepared by dissolving 1 mg of L-2-phenylalanine standard (B&K) in 1 mL of 70% methanol water to prepare a 1000 μg / mL standard stock solution, and the 1000 μg / mL stock solution was further diluted with 70% methanol water to prepare a 250 μg / mL internal standard solution to obtain a 70% methanol internal standard extract.

[0103] Ultra Performance Liquid Chromatography (UPLC) TMAD, https: / / sciex.com.cn / ) and tandem mass spectrometry (MS / MS) were used to detect the primary metabolites in the samples. The liquid phase conditions mainly include: chromatographic column: Agilent SB-C18 1.8μm, 2.1mm*100mm; mobile phase: phase A is ultrapure water (added with 0.1% formic acid), phase B is acetonitrile (added with 0.1% formic acid); elution gradient: 0.00min B phase ratio is 5%, within 9.00min, the B phase ratio increases linearly to 95%, and maintains at 95% for 1min, 10.00-11.10min, the B phase ratio is reduced to 5%, and balanced at 5% to 14min; flow rate 0.35mL / min; column temperature 40℃; injection volume 2μL. The mass spectrometry conditions mainly include: electrospray ionization (ESI) temperature 500℃; ion spray voltage (IS) 5500V (positive ion mode) / -4500V (negative ion mode); ion source gas I (GSI), gas II (GSII) and curtain gas (CUR) are set to 50, 60 and 25psi respectively, and the collision induced ionization parameter is set to high. The QQQ scan uses the MRM mode and the collision gas (nitrogen) is set to medium. The DP and CE of each MRM ion pair are completed by further optimization of the declustering potential (DP) and collision energy (CE). According to the metabolites eluted in each period, a specific set of MRM ion pairs is monitored in each period.

[0104] Based on the self-built database MWDB (metware database), the qualitative analysis of the substances was performed according to the secondary spectrum information. The isotope signals were removed during the analysis, including K + Ion, Na + Ion, NH 4+ions, and fragment ions of other substances with larger molecular weights. Metabolite quantification is completed using the multiple reaction monitoring (MRM) mode (as shown below) of the triple quadrupole mass spectrometer. In the MRM mode, the quadrupole first screens the precursor ions (parent ions) of the target substance, and excludes ions corresponding to other molecular weight substances to preliminarily eliminate interference; the precursor ions are induced by the collision chamber and then broken to form many fragment ions, which are then filtered by the triple quadrupole to select the required characteristic fragment ions, eliminating non-target ion interference, making quantification more accurate and more repeatable. After obtaining the metabolite mass spectrometry analysis data of different samples, the peak areas of all material chromatographic peaks are integrated, and the mass spectrometry peaks of the same metabolite in different samples are integrated and corrected. Based on the local metabolic database, the metabolites of the samples were qualitatively and quantitatively analyzed by mass spectrometry.

[0105] (3) Determination of aroma components of tobacco leaves after baking:

[0106] The flue-cured tobacco leaves were ground with liquid nitrogen and vortexed to mix evenly. About 500 mg of each sample was weighed into a headspace bottle. 2 ml of saturated NaCl solution (5.5 mol / L) and 20 μL (10 μg / mL) of internal standard solution (3-Hexanone-2,2,4,4-d4, dissolved in n-hexane and fixed to 10 mL, diluted 100 times with n-hexane) were added respectively. The samples were extracted by fully automatic headspace solid phase microextraction HS-SPME for GC-MS analysis. Under constant temperature of 60 ° C, the sample was shaken for 5 min, and the 120 μm DVB / CWR / PDMS extraction head was inserted into the sample headspace bottle. The headspace extraction was carried out for 15 min, and the sample was analyzed at 250 ° C for 5 min, and then GC-MS separation and identification were performed. The extraction head was aged at 250 ° C for 5 min in the Fiber Conditioning Station before sampling. Note: The new extraction head was aged in the Fiber Conditioning Station for 2 h before extraction. And it uses SPME Arrow, which has a sensitivity 10 times that of the traditional SPME fiber head. Chromatographic conditions: DB-5MS capillary column (30m×0.25mm×0.25μm, Agilent J&W Scientific, Folsom, CA, USA), carrier gas is high-purity helium (purity not less than 99.999%), constant flow rate 1.2mL / min, injection port temperature 250℃, non-split injection, solvent delay 3.5min. Program temperature rise: 40℃ for 3.5min, increase to 100℃ at 10℃ / min, then increase to 180℃ at 7℃ / min, and finally increase to 280℃ at 25℃ / min, and maintain for 5min. Mass spectrometry conditions: electron impact ion source (EI), ion source temperature 230°C, quadrupole temperature 150°C, mass spectrometry interface temperature 280°C, electron energy 70eV, scanning mode selected ion detection mode (SIM), qualitative and quantitative ion precise scanning (GB 23200.8-2016). The raw data after mass spectrometry analysis was processed by MassHunter software for qualitative and quantitative analysis.

[0107] (4) Evaluation of appearance indicators: The appearance standard grouping and grading of the cured tobacco leaves shall be evaluated in accordance with the National Standard of the People's Republic of China GB2635-92.

[0108] (5) Economic indicators: calculated based on the market price of the year and different grades of flue-cured tobacco.

[0109] (6) Sensory evaluation: The cured tobacco leaves are submitted to Fujian China Tobacco Industry Co., Ltd. for sensory evaluation and scoring in accordance with the Tobacco Leaf Quality Evaluation Technical Requirements Q / FJZYT0301-37-A / 1.

[0110] 4. Test results

[0111] OPLS-DA modeling was used to analyze the significance of differences in the contents of carotenoids, primary metabolites and aroma components among the experimental groups. The analysis results showed that there were significant differences in the contents of carotenoids, primary metabolites and aroma components between the Sanzao (A) experimental group and other experimental groups.

[0112] 4.1 Analysis of carotenoid test results

[0113] The results of comparative analysis of carotenoid substances in the three early (A) test group and the early early (B) test group are shown in Table 3. Compared with the early early (B) test group, the content of one substance in the tobacco leaves of the three early (A) test group was up-regulated, and the content of four substances was down-regulated. Among the up-regulated substances, there was one xanthophyll, which was violaxanthin; among the down-regulated substances, there were two carotenes, namely γ-carotene and lycopene; there were two xanthophylls, namely zeaxanthin and antheraxanthin.

[0114] Table 3 Comparative analysis results of carotenoid content in the three early (A) test group and the early early late (B) test group

[0115]

[0116] The results of comparative analysis of carotenoid substances in the Sanzao (A) test group and the Zaochichi (C) test group are shown in Table 4. Compared with the Zaochichi (C) test group, the content of one substance in the tobacco leaves of the Sanzao (A) test group was increased, and the content of three substances was decreased. Among the increased substances, there was one lutein, echinenone; among the decreased substances, there were two carotenes, namely γ-carotene and lycopene; and there was one lutein, namely zeaxanthin.

[0117] Table 4 Comparative analysis results of carotenoid content in the three early (A) test group and the early and late (C) test group

[0118]

[0119] The results of comparative analysis of carotenoid substances in the three early (A) test group and the late early (D) test group are shown in Table 5. Compared with the late early (D) test group, the content of one substance in the tobacco leaves of the three early (A) test group was increased, and the content of four substances was decreased. Among the increased substances, there was one lutein, echinenone; among the decreased substances, there were two carotenes, namely γ-carotene and lycopene; there was one lutein, zeaxanthin.

[0120] Table 5 Comparative analysis results of carotenoid content in the three early (A) test group and the late early (D) test group

[0121]

[0122] The results of comparative analysis of carotenoid substances in the Sanzao (A) experimental group and the Chichizao (E) experimental group are shown in Table 6. Compared with the Chichizao (E) experimental group, the contents of four substances in the tobacco leaves of the Sanzao (A) experimental group were reduced, including two carotenes, namely γ-carotene and lycopene; and two xanthophylls, namely zeaxanthin and antheraxanthin.

[0123] Table 6 Comparative analysis results of carotenoid content in the three early (A) test group and the late early (E) test group

[0124]

[0125] The results of comparative analysis of carotenoid substances in the three early (A) test group and the three late (CK) test group are shown in Table 7. Compared with the three late (CK) test group, the content of one substance in the tobacco leaves of the three early (A) test group was increased, and the content of two substances was decreased. Among the increased substances, there was one lutein, which was echinenone; among the decreased substances, there were two carotenes, namely γ-carotene and lycopene.

[0126] Table 7 Comparative analysis results of carotenoid content in the three early (A) test group and the three late (CK) test group

[0127]

[0128] 4.2 Analysis of primary metabolite detection results

[0129] The comparative analysis results of primary metabolites in the three-early (A) experimental group and the early-early-late (B) experimental group are shown in Table 8. Compared with the early-early-late (B) experimental group, the contents of 36 substances in the tobacco leaves of the three-early (A) experimental group were increased, and the contents of 28 substances were decreased. Among the up-regulated substances, there are 10 amino acids and their derivatives, namely cycloleucine, DL-tryptophan, cyclo(alanyl-phenylalanine), L-pyroglutamic acid, L-valine-L-proline, L-lysine-L-phenylalanine, N-monomethyl-L-arginine, L-tryptophan, N6-methyladenosine, and adenine; there are 4 other categories, namely glucaric acid-1-phosphate, D-sedoheptulose 7-phosphate, 2,4-di(2-methyl)butyryl-3-(isobutyryl)sucrose-6'-O-acetate, and rutin; there are 5 organic acids, namely L-piperidinic acid, isocitrate, phenylpyruvic acid, phosphoenolpyruvic acid, and α-ketoglutaric acid; there are 17 lipids, namely lysophosphatidylcholine 17:2, 2-α-linolenic acid glyceride-1-O-glucoside, 1- α-linolenic acid glyceryl ester-3-O-glucoside, dehydrosphingomyelin, 4-hydroxysphingosine (phytosphingosine), octadeca-9,12,15-trienoic acid, punicic acid (9Z,11E,13Z-octadecatrienoic acid), 5,8,11,14-pentadecanamide, 13-hydroxyoctadecyl-9,11-dienoic acid, 9S-hydroxy-10E,12Z-octadecadienoic acid, α-hydroxylinoleic acid, 1-(((2-(dimethylamino)ethoxy)(hydroxy)phosphoryl)oxy)-3-hydroxyprop-2-yl(9Z,12Z)-octadec-9,12-dienoate, (9S)-hydroxyoctadecadienoic acid, 17-hydroxylinolenic acid, α-linolenic acid, 9-hydroxy-12-oxo-10(E),15(Z)-octadecadienoic acid, pentadecanoic acid.Twenty-eight substances were down-regulated, including 14 amino acids and their derivatives, namely N(6),N(6)-dimethyl-L-lysine, alloisoleucine, L-arginine, L-asparagine, L-leucine, L-isoleucine, cis-L-3-hydroxyproline, L-homocysteine, N6-acetyl-L-lysine, N-acetyl-L-leucine, N-[3-(4-hydroxyphenyl)acryloyl]-L-tyrosine, jasmonoyl-L-isoleucine, uridine diphosphate acetylglucosamine, and 9-(arabinosyl) hypoxanthine; and 4 other categories, namely N -Acetyl-D-glucosamine, solanose, 1-(sn-glycero-3-phosphate)-1D-inositol, butyl-β-D-pyranoside; there are 2 organic acids, abscisic acid and jasmonic acid; there are 8 lipids, namely lysophosphatidylethanolamine 15:0 (2n isomer), lysophosphatidylethanolamine 15:0, lysophosphatidylethanolamine 18:1, lysophosphatidylethanolamine 18:1 (2n isomer), lysophosphatidylcholine 15:1, lysophosphatidylcholine 14:0, lysophosphatidylcholine 20:5, 5,8-dihydroxy-9,12-octadecadienoic acid.

[0130] Table 8 Comparative analysis results of the content of primary metabolites in the three early (A) test group and the early early late (B) test group

[0131]

[0132]

[0133] Note: The number before the colon represents the number of C atoms in the molecule, and the number after the colon represents the number of double bonds in the molecule; the 2n isomerism in the brackets represents that the fatty acid chain in the molecule has an n-isomer at position 2.

[0134] The comparative analysis results of the primary metabolites in the three early (A) test group and the early and late (C) test group are shown in Table 9. Compared with the early and late (C) test group, the contents of 33 substances in the tobacco leaves of the three early (A) test group were upregulated, and the contents of 28 substances were downregulated. Among the upregulated substances, there were 15 amino acids and their derivatives, namely L-homomethionine, cycloleucine, DL-tryptophan, cyclo(alanyl-phenylalanine), 3-(pyrazol-1-yl)-L-alanine, L-histidine, L-pyroglutamic acid, 1-methylhistidine, glycyl-isoleucine, 1-aminocyclopropane-1-carboxylic acid, N-(carboxymethyl)glycine, L-tryptophan, N-(1-deoxy-1-fructose)glutamic acid, N6- Methyladenosine, 2-deoxyribose-1-phosphate; there are 2 other types, namely glucaric acid-1-phosphate, D-sedoheptulose 7-phosphate; there is 1 organic acid, which is L-piperidinic acid; there are 15 lipids, namely lysophosphatidylcholine 17:2, dehydrosphingomyelin, lysophosphatidylcholine 18:3, lysophosphatidylcholine 18:3 (2n isomer), lysophosphatidylethanolamine 18:2 (2n isomer), lysophosphatidylethanolamine 18:2, Lysophosphatidylcholine 18:2 (2n isomer), 2-aminotetradecane-1,4-diol, lysophosphatidylethanolamine 17:1 (2n isomer), 1-(2,3-dihydroxypropoxy)-3-(((2-(dimethylamino)ethoxy)(hydroxy)phosphoryl)oxy)propan-2-yl (8E,11Z,14Z)-octadecane-8,11,14-trienoic acid, 2-(2,3-dihydroxypropoxy)-3-(((2-( 2-(2,3-dihydroxypropoxy)-3-(((2-(dimethylamino)ethoxy)(hydroxy)phosphoryl)oxy)propyl(11Z,14Z)-octadeca-11,14-dienoic acid, 1-linoleoyl-2-lysophosphatidic acid monobutylamine ester, β-hydroxypalmitic acid, pentadecanoic acid.Among the down-regulated substances, there are 8 amino acids and their derivatives, namely L-valyl-L-leucine, L-isoleucine-L-phenylalanine, L-leucyl-L-phenylalanine, N6-acetyl-L-lysine, N-acetyl-L-leucine, oxidized glutathione, jasmonoyl-L-isoleucine, and uridine diphosphate acetylglucosamine; there are 5 other categories, namely N-glucosylnicotinic acid, D-panthenol, 1-(sn-glycero-3-phosphate)-1D-inositol, 2,4-di(isovaleryl)-3-(2-methylcrotonyl)sucrose-6'-O-acetate, butyl-β-D-pyridinium bromide, Fructofranosides; there are 3 organic acids, namely γ-aminobutyric acid, abscisic acid, and jasmonic acid, and 12 lipids, namely lysophosphatidylcholine 15:0 (2n isomerism), lysophosphatidylcholine 16:0, lysophosphatidylethanolamine 18:1 (2n isomerism), lysophosphatidylcholine 15:1, lysophosphatidylcholine 15:0, lysophosphatidylcholine 17:0, lysophosphatidylcholine 14:0, lysophosphatidylcholine 16:0 (2n isomerism), taurine, 9,12,13-trihydroxy-10,15-octadecadienoic acid, and 9,10,13-trihydroxy-11-octadecenoic acid.

[0135] Table 9 Comparative analysis results of the content of primary metabolites in the three early (A) test group and the early and late (C) test group

[0136]

[0137]

[0138] Note: The number before the colon represents the number of C atoms in the molecule, and the number after the colon represents the number of double bonds in the molecule; the 2n isomerism in the brackets represents that the fatty acid chain in the molecule has an n-isomer at position 2.

[0139] The comparative analysis results of primary metabolites in the three early (A) experimental group and the late early (D) experimental group are shown in Table 10. Compared with the late early (D) experimental group, the contents of 32 substances in the tobacco leaves of the three early (A) experimental group were increased, and the contents of 25 substances were decreased. Among the up-regulated substances, there are 15 amino acids and their derivatives, namely L-homomethionine, cycloleucine, L-glutamic acid-L-arginine, DL-tryptophan, cyclo(alanyl-phenylalanine), 3-(pyrazol-1-yl)-L-alanine, L-histidine, L-pyroglutamic acid, L-phenylalanine, 3-methyl-L-histidine, L-homoglutamic acid, L-alanyl-L-leucine, L-theanine, L-tryptophan, and N6-methyladenosine; there are 4 other categories, namely isoascorbic acid, glucaric acid-1-phosphate, rutin, and L-fucose; there are 4 organic acids, namely L-piperidinic acid, phenylpyruvic acid, DL-glyceric acid, and malic acid 1-O-α-D-pyranoglucopyranoside (I); and there are 9 lipids, namely lysophosphorus Acylcholine 17:2, dehydrosphingomyelin, lysophosphatidylcholine 18:3 (2n isomer), lysophosphatidylethanolamine 18:2 (2n isomer), lysophosphatidylethanolamine 17:1 (2n isomer), lysophosphatidylethanolamine 16:1 (2n isomer), α-hydroxylinoleic acid, 1-(2,3-dihydroxypropoxy)-3-(((2-(dimethylamino)ethoxy)(hydroxy)phosphoryl)oxy)prop-2-yl(8E,11Z,14Z)-octadeca-8,11,14-trienoic acid, 2-(2,3-dihydroxypropoxy)-3-(((2-(dimethylamino)ethoxy)(hydroxy)phosphoryl)oxy)propyl(8E,11Z,14Z)-octadeca-8,11,14-trienoic acid. Among the down-regulated substances, there are 9 amino acids and their derivatives, namely L-valyl-L-leucine, L-phenylalanine-L-isoleucine, L-leucyl-L-phenylalanine, N6-acetyl-L-lysine, N-acetyl-L-leucine, oxidized glutathione, jasmonic-L-isoleucine, uridine diphosphate acetylglucosamine, and 9-(arabinosyl) hypoxanthine; there is 1 other category, which is 1-(sn-glycerol-3-phosphate)-1D-inositol; there are 4 organic acids, namely γ-aminobutyric acid, 3-hydroxybutyric acid, jasmonic acid, and 4 -Hydroxy-2-oxopentanoic acid; there are 11 lipids, namely lysophosphatidylcholine 15:0 (2n isomer), lysophosphatidylethanolamine 18:1, lysophosphatidylethanolamine 18:1 (2n isomer), lysophosphatidylcholine 15:0, lysophosphatidylcholine 17:0, lysophosphatidylcholine 14:0, lysophosphatidylcholine 16:0 (2n isomer), lysophosphatidylcholine 20:5, tautomer acid, 9,12,13-trihydroxy-10,15-octadecadienoic acid, and 9,10,13-trihydroxy-11-octadecenoic acid.

[0140] Table 10 Comparative analysis results of the content of primary metabolites in the three early (A) test group and the late early (D) test group

[0141]

[0142]

[0143]

[0144] Note: The number before the colon represents the number of C atoms in the molecule, and the number after the colon represents the number of double bonds in the molecule; the 2n isomerism in the brackets represents that the fatty acid chain in the molecule has an n-isomer at position 2.

[0145] The comparative analysis results of primary metabolites in the Sanzao (A) experimental group and the Chichizao (E) experimental group are shown in Table 11. Compared with the Chichizao (E) experimental group, the contents of 13 substances in the tobacco leaves of the Sanzao (A) experimental group were increased and the contents of 42 substances were decreased. Among the up-regulated substances, there are 7 amino acids and their derivatives, namely cycloleucine, DL-leucine, L-homoglutamate, L-homocysteine, N6-methyladenosine, xanthosine, and 2-deoxyribose-1-phosphate; there is 1 other type, which is L-fucose; there is 1 organic acid, which is L-piperidinic acid; and there are 4 lipids, which are hemolytic phosphatidylethanolamine 16:1 (2n isomer), 1-(2,3-dihydroxypropoxy)-3-(((2-(dimethylamino)ethoxy)(hydroxy)phosphoryl)oxy)prop-2-yl(E)-9-hexadecenoic acid, pentadecanoic acid, and 13-hydroperoxy-6Z,9Z,11E-octadecatrienoic acid. Among the down-regulated substances, there are 14 amino acids and their derivatives, namely L-valyl-L-leucine, L-phenylalanine-L-isoleucine, 2,6-diamino-7-methyl-5-oxooctanoic acid, L-isoleucine-L-phenylalanine, L-leucyl-L-phenylalanine, N6-acetyl-L-lysine, N-acetyl-L-leucine, oxidized glutathione, jasmonoyl-L-isoleucine, 3-hydroxy-3-methylglutamic acid, 1-methyladenine, uridine diphosphate acetylglucosamine, adenosine-2'-phosphate, 9-(arabinosyl) hypoxanthine; there are 2 other categories, namely D-panthenol and sorbitol; there are 2 organic acids, namely γ-aminobutyric acid and jasmonic acid; there are 24 lipids, namely lysophosphatidylethanolamine 15:0 (2n isomer), lysophosphatidylcholine 15:0 (2n isomer), lysophosphatidylethanolamine 15:0, 9-Oxo-10,12-Octadecadienoic acid Acid, lysophosphatidylcholine 18:0, 14,15-dehydrogenated ginsenoic acid, lysophosphatidylcholine 16:0, lysophosphatidylethanolamine 18:1 (2n isomer), lysophosphatidylethanolamine 18:0, octadecadiene-6-ynoic acid, lysophosphatidylcholine 15:1, lysophosphatidylcholine 15:0, lysophosphatidylcholine 17:0, lysophosphatidylcholine 14:0, lysophosphatidylcholine 16:0 (2n isomer), lysophosphatidylcholine 20:5, Tianshi acid, 6,9,10-trihydroxydecanoic acid Octa-7-enoic acid; triolenic acid, 1-(2,3-dihydroxypropoxy)-3-(((2-(dimethylamino)ethoxy)(hydroxy)phosphoryl)oxy)prop-2-yl palmitate, (9S)-hydroxyoctadecadienoic acid, 2R-hydroxy-9Z,12Z,15Z-octadecadienoic acid, 9,12,13-trihydroxy-10,15-octadecadienoic acid, 9,10,13-trihydroxy-11-octadecadienoic acid, 9-hydroxy-12-oxo-10(E),15(Z)-octadecadienoic acid.

[0146] Table 11 Comparative analysis results of the content of primary metabolites in the three early (A) test group and the late early (E) test group

[0147]

[0148]

[0149]

[0150] Note: The number before the colon represents the number of C atoms in the molecule, and the number after the colon represents the number of double bonds in the molecule; the 2n isomerism in the brackets represents that the fatty acid chain in the molecule has an n-isomer at position 2.

[0151] The comparative analysis results of the primary metabolites in the three early (A) test group and the three late (CK) test group are shown in Table 12. Compared with the three late (CK) test group, the contents of 54 substances in the tobacco leaves of the three early (A) test group were up-regulated, and the contents of 21 substances were down-regulated. Among the up-regulated substances, there were 22 amino acids and their derivatives, namely cycloleucine, allo-isoleucine, DL-leucine, DL-tryptophan, cyclo(alanyl-phenylalanine), 3-(pyrazol-1-yl)-L-alanine, L-leucine, L-histidine, L-isoleucine, L-norleucine, L-phenylalanine, 3-methyl-L-histidine, L-allo-isoleucine, cis-L-3-hydroxyproline, DL-methionine, cis-4-hydroxy-D-proline, L-lysine-L-phenylalanine, L-theanine, glycyl-isoleucine, L-tryptophan, N6-methyladenosine, barbituric acid; malonylurea; 2 ,4,6-pyrimidinetrione; there are 4 other categories, namely D-glucose-6-phosphate, glucaric acid-1-phosphate, D-sedoheptulose 7-phosphate, sorbitol-6-phosphate; there are 4 organic acids, namely L-piperidinic acid, isocitric acid, phenylpyruvic acid, phosphoenolpyruvic acid; there are 24 lipids, namely 9,11,13-octadecatrienoylglycerol, lysophosphatidylethanolamine 18:3 (2n isomer), lysophosphatidylcholine 17:2, lysophosphatidylethanolamine 18:3, 2-α-linolenic acid glyceride-1-O-glucoside, 1-α-linolenic acid glyceride-3-O-glucoside, monogalactosylglycerol dihydrate ester, 1-linolenoylglycerol, lysophosphatidylcholine 18:3, lysophosphatidylcholine 18:3 (2n isomer), lysophosphatidylethanolamine 18:2 (2n isomer), lysophosphatidylethanolamine 18:2, lysophosphatidylcholine 18:2 (2n isomer), lysophosphatidylethanolamine 16:1 (2n isomer), 3-(((2-(dimethylamino)ethoxy)(hydroxy)phosphoryl)oxy)-2-hydroxypropyl (9Z,12Z,15E)-octadec-9,12,15-trienoate, 1-(((2-(dimethylamino)ethoxy)(hydroxy)phosphoryl)oxy)-3-hydroxyprop-2-yl (9Z, 1-(2,3-dihydroxypropoxy)-3-(((2-(dimethylamino)ethoxy)(hydroxy)phosphoryl)oxy)propyl(8E,11Z,14Z)-octadeca-8,11,14-trienoic acid, 1-linoleoyl-2-lysophosphatidic acid monobutylamine ester, α-linolenic acid, cis-octadeca-9-ene-12-ynoic acid, pentadecanoic acid.Among the down-regulated substances, there are 9 amino acids and their derivatives, namely N6-acetyl-L-lysine, N-acetyl-L-leucine, N-[3-(4-hydroxyphenyl)acryloyl]-L-tyrosine, N-acetyl-L-glutamate, jasmonic-L-isoleucine, aminomalonic acid, 1-methyladenine, uridine diphosphate acetylglucosamine, and 9-(arabinosyl)hypoxanthine; there are 6 other categories, namely N-acetyl-D-glucosamine, 1-(sn-glycero-3-phosphate)-1D-inositol, isoascorbic acid 2-O-glucoside, D-pinetol, D-threitol, and butyl-β-D-pyranofructose; there are 4 organic acids, namely succinic acid, methylmalonic acid, jasmonic acid, and 4-hydroxy-2-oxopentanoic acid; there are 2 lipids, namely lysophosphatidylcholine 14:0 and DL-2-hydroxystearic acid.

[0152] Table 12 Comparative analysis results of the contents of primary metabolites in the three early (A) test group and the three late (CK) test group

[0153]

[0154]

[0155] Note: The number before the colon represents the number of C atoms in the molecule, and the number after the colon represents the number of double bonds in the molecule; the 2n isomerism in the brackets represents that the fatty acid chain in the molecule has an n-isomer at position 2.

[0156] 4.3 Analysis of aroma component inspection results

[0157] Compared with the conventional three-late trial, the three-early (A) experimental group of early transplanting, early topping and early harvesting significantly increased the content of aroma components in Cui Bi No. 1 tobacco leaves.

[0158] Specifically, the results of the comparison of the content of aroma components showed that the content of 5 substances in tobacco leaves was increased and the content of 15 substances was decreased in the three early (A) test group and the early early (B) test group (see Table 13). Among the up-regulated substances, there were 3 heterocyclic compounds, namely 2-methyl-3-propylpyrazine, 2,3-dimethyl-5-n-propylpyrazine, and 2-methyl-6-propylpyrazine; there was 1 terpenoid, which was atractylodes alcohol; and there was 1 acid, which was 6-aminocaproic acid. Among the down-regulated substances, there is one ester, δ-decalactone; two heterocyclic compounds, 3-methoxy-2,5-dimethylpyrazine and 2-ethyl-3-methoxypyrazine; two ketones, 3-isopropylidene-5-methylhexenone and acetophenone; four terpenes, L-menthol, (1α,2α,5α)-2,6,6-trimethylbicyclo[3.1.1]heptane-3-one, dl-menthol, (1α,2α,5α)-5-methyl-2-(1-methylethyl)cyclohexanol; three aldehydes, (E)-2-nonenal, (Z)-2-nonenal and 2-nonenal; one phenol, 3-fluorophenol; and two alcohols, d-menthol and (1α,2α,5β)-5-methyl-2-(1-methylethyl)cyclohexanol.

[0159] Table 13 Comparative analysis results of aroma substance content of the three early (A) test group and the early early late (B) test group

[0160]

[0161] The results of the comparison of the content of aroma components showed that the content of 19 substances in tobacco leaves increased and the content of 34 substances decreased in the comparison between the Sanzao (A) test group and the Zaochichi (C) test group (Table 14). Among the up-regulated substances, there was one heterocyclic compound, 2,3-dimethyl-5-n-propylpyrazine; two ketones, 6-methyl-3,5-heptadien-2-one and 3-ethyl-2,6-piperidindione; one hydrocarbon, 4,5,9,10-dehydroisolongifolene; and 10 terpenes, (Z)-2,6-dimethyl-5,7-octadien-4-one, 6,6-dimethyl-bicyclo[3.1.1]-2-heptene-2-methanol, (-)-α-thujone, 3,3,6-trimethyl-1,5-heptadien-4-ol, 2-(4-methylcyclohexene)-1,4-diol, and 1,5-heptadien-2-ol. Hexa-2,4-dien-1-yl)-2-propanol, (-)-cis-isopiperidinol, 4-(1-methylethyl)-1,5-cyclohexadiene-1-methanol, (-)-trans-isopiperidinol, trans-carveol, carveol; there is 1 aldehyde, which is 2-methylundecanal; there is 1 aromatic hydrocarbon, which is 1-methyl-2-(1-ethylpropyl)benzene; there are 3 alcohols, namely 11,11-dimethyl-4,8-dimethylenebicyclo[7.2.0]undecan-3-ol, 2,4-decadien-1-ol, and 6,6-dimethylbicyclo[3.1.1]hept-2-ene-2-ethanol.Among the down-regulated substances, there are 4 esters, namely, 2-butylcarbyl, 2-octyl furanoate, eugenol acetate, and δ-decanoyl lactone; there are 3 heterocyclic compounds, namely, 5H-5-methyl-6,7-dihydrocyclopentylpyrazine, 3-methoxy-2,5-dimethylpyrazine, and 2-ethyl-3-methoxypyrazine; there are 4 ketones, namely, 3-isopropyl-5-methylhexenone, 1-(p-anisyl)-3-methyl-1-pentanone, (Z)-2, 6-dimethyl-2,5,7-octatrien-4-one, acetophenone; there is one hydrocarbon, 8-hexadecene; there are 15 terpenes, namely (E)-2,6-dimethyl-2,5,7-octatrien-4-one, L-menthol, trans-farnesol, 2,6-dimethyl-10-methylene-2,6,11-dodecatrienal, [2R-(2α,4aβ,8β)]-2,3,4,4a,5,6,7,8-octatrienal Hydrogen-α,α,4a,8-tetramethyl-2-naphthalenemethanol, (E,E)-3,7-dimethyl-10-(1-methylethylidene)-3,7-cyclododecyl-1-one, cis-β-santalol, bisabolol, 1-(2,6,6-trimethyl-2-cyclohexenyl)-1-penten-3-one, geranyl tiglate, (1α,2α,5α)-2,6,6-trimethylbicyclo[3.1.1]heptane-3-one, d l-Menthol, (1α,2α,5α)-5-methyl-2-(1-methylethyl)cyclohexanol, (Z)-epi-β-santalol, α-bisabolol; there are 4 aldehydes, namely (E)-2-nonenal, (Z)-2-nonenal, 2-nonenal, 3-fluorophenol; there are 2 alcohols, namely d-menthol, (1α,2α,5β)-5-methyl-2-(1-methylethyl)cyclohexanol; there is 1 amine, namely phenacetin.

[0162] Table 14 Comparative analysis results of aroma substance content between the three early (A) test group and the early and late (C) test group

[0163]

[0164]

[0165] The results of the comparison of the content of aroma components showed that the contents of 10 substances in tobacco leaves were increased and the contents of 6 substances were decreased in the three early (A) test group and the late early (D) test group (Table 15). Among the up-regulated substances, there was one ester, namely (4-methyl-1-propane-2-yl-3-bicyclo[3.1.0]hexyl) acetate; two heterocyclic compounds, namely 3,5-diethyl-2-methylpyrazine and 2,3,5-trimethyl-6-ethylpyrazine; one ketone, namely 1-(5-tert-butyl-2-pyrazinyl)ethanone; two terpenes, namely 2-methyl-5-(1-methylethyl)phenol and thymol; one ether, namely 1-methoxy-4-methyl-2-(1-methylethyl)benzene; and three phenols, namely guaiacol, p-tert-butylphenol, and m-tert-butylphenol. Among the down-regulated substances, there are three esters, namely 2-octyl furanoate, eugenyl acetate, and δ-decanoyl lactone; there is one heterocyclic compound, namely 5H-5-methyl-6,7-dihydrocyclopentylpyrazine; and there are two terpenes, namely (E)-2,6-dimethyl-2,5,7-octatrien-4-one and (1α,2α,5α)-2,6,6-trimethylbicyclo[3.1.1]heptane-3-one.

[0166] Table 15 Comparative analysis results of aroma substance content in the three early (A) test group and the late early (D) test group

[0167]

[0168] The results of the comparison of the content of aroma components showed that the content of 49 substances in tobacco leaves increased and the content of 51 substances decreased in the comparison of the three early (A) test group and the late early (D) test group (Table 16). Among the up-regulated substances, there was one amine, N,N-dimethylformamide; two alcohols, 1,5-pentanediol and 6,6-dimethylbicyclo[3.1.1]hept-2-ene-2-ethanol; one aromatic hydrocarbon, 1-methyl-2-(1-ethylpropyl)benzene; one phenol, 2-ethoxy-4-methylphenol; one ether, 1,1-diethoxynonane; and five aldehydes, 4-methylbenzaldehyde, (1R)-(-)-myrtle aldehyde, 5-propan-2-ylbicyclo[3.1.0]-2-hexene-2-carboxaldehyde, 2-methyl Benzaldehyde, 3-methylbenzaldehyde; there is one acid, which is 2-hydroxyoctanoic acid; there are 18 terpenes, namely 6,6-dimethylbicyclo[3.1.1]-2-heptene-2-carboxaldehyde, (Z)-2,6-dimethyl-5,7-octadien-4-one, 6,6-dimethyl-bicyclo[3.1.1]-2-heptene-2-methanol, p-cymene, (-)-α-thujone, 2-(4-methylcyclohexa-2,4-dien-1-yl)-2-propanol, citronellol, (R)-3,7-dimethyl-6-octenol, (-)-cis-isopiperidinol, 2-methyl-5-(1-methylvinyl)-cyclohexanone, (-)-verbenone, mabehenone, 4-(1-methylethyl)-1,5-cyclohexadiene-1-methanol, 1-methyl-3-(1-methylethyl)benzene, o-cymene, (-)-trans-isopiperidinol, trans-carveol, carveol; there are 3 hydrocarbons, namely 4,5,9,10-dehydroisolongifolene, 1,10-undecadiene, (1S,2S,6S,7R)-2,6-dimethyltricyclo[5.2.1.02,6]decatriene; there are 6 ketones, namely isoflavone There are 2-butylpyridine, (Z)-tetrahydro-6-(2-pentenyl)-2H-pyran-2-one, isomaltol, coumarin, 2,3-dimethyl-5-n-propylpyrazine; there are 5 esters, namely 2-methylpropionic acid-3-hydroxy-2,2,4-trimethylpentyl ester, 2-hydroxyglutaric acid-1,5-diethyl ester, 2-furoic acid methyl ester, isobornyl formate, and isobornyl formate.Among the down-regulated substances, there are 5 alcohols, namely α-2-cyclohexen-1-yl-benzyl alcohol, cyclooctane methanol, 1-undecene-4-ol, d-menthol, (1α,2α,5β)-5-methyl-2-(1-methylethyl) cyclohexanol; 2 phenols, namely 3-nitrophenol and 3-fluorophenol; 1 halogenated hydrocarbon, namely 1-[fluoro(methyl)phosphoryl]oxypentane; 6 aldehydes, namely 2-methyl-2-heptanal, nonanal, 4-(1,1-dimethylethyl)phenylpropanal, (E)-2-nonenal, (Z)-2-nonenal, 2-nonenal; and 3 There are 18 terpenoids, namely 6,6-dimethylbicyclo[3.1.1]-2-heptene-2-carboxaldehyde, (Z)-2,6-dimethyl-5,7-octadien-4-one, 6,6-dimethyl-bicyclo[3.1.1]-2-heptene-2-methanol, p-cymene, (-)-α-thujone, 2-(4-methylcyclohexa-2,4-dien-1-yl)-2-propanol, citronellol, (R)-3,7-dimethyl-6-octenol, (-)-cis- -isopiperidinol, 2-methyl-5-(1-methylvinyl)-cyclohexanone, (-)-verbenone, mabenone, 4-(1-methylethyl)-1,5-cyclohexadiene-1-methanol, 1-methyl-3-(1-methylethyl)benzene, o-cymene, (-)-trans-isopiperidinol, trans-carveol, carveol; there are 2 hydrocarbons, namely 4,6,8-trimethyl-1-nonene and 3-ethyl-3-methyldecane; there are 8 ketones, namely 3-isopropylidene-5-methylhexenone, 1-nonen-3-one, (Z)-2,6-dimethyl-2,5, 7-octatriene-4-one, 6-methyl-5-heptene-2-one, 3-ethyl-4-methyl-3-pentene-2-one, 2-octanone, acetophenone, 2-methyl-1-heptene-6-one; there are 5 heterocyclic compounds, namely 3-methoxy-2,5-dimethylpyrazine, 2,3'-octahydrofuran, 2-methylpiperazine, 2-ethyl-3-methoxypyrazine, 2,2'-octahydrofuran; there are 6 esters, namely, fenbutyl, eugenyl acetate, neryl butyrate, 2-methylpropyl 2-butenoate, 3,4-methylenedioxybenzyl acetate, and δ-decanoic acid.

[0169] Table 16 Comparative analysis results of aroma substance content between the three early (A) test group and the late early (E) test group

[0170]

[0171]

[0172]

[0173] The comparison results of the aroma component contents showed that the contents of 13 substances in tobacco leaves increased and the contents of 30 substances decreased in the three early (A) test group and the three late (CK) test group (Table 17). Among the up-regulated substances, there are 2 esters, namely dimethyl succinate and 2-hydroxy-2-methylpropyl benzoate; 2 heterocyclic compounds, namely 2-methyl-3-propylpyrazine and 2-methyl-6-propylpyrazine; 1 hydrocarbon, namely 1-undecyne; 3 terpenes, namely (Z)-2,6-dimethyl-5,7-octadien-4-one, 6,6-dimethyl-bicyclo[3.1.1]-2-heptene-2-methanol, and 4-(1-methylethyl)-1,5-cyclohexadiene-1-methanol; 3 phenols, namely guaiacol, 2,4,5-trimethylphenol, and 2,3,6-trimethylphenol; and 2 aromatic hydrocarbons, namely 1,2-diethylbenzene and 1-methyl-2-(1-ethylpropyl)benzene. Among the down-regulated substances, there are 3 esters, namely 2-furancarboxylic acid octyl ester, (5-methyl-2-prop-1-en-yl-2-hexen-4-yl) hexanoate, and δ-decanoic acid lactone; there are 3 heterocyclic compounds, namely 3-methoxy-2,5-dimethylpyrazine, isomaltol, and 2-ethyl-3-methoxypyrazine; there are 6 ketones, namely 3-isopropyl-5-methylhexenone, 6-methyl-5-hepten-2-one, 2-octanone, acetophenone, 1,3-diphenyl-2-propanone, and 2-methyl-1-hepten-6-one; there are 9 terpenes, namely 2-methyl-5-prop-2-ylbicyclo[3.1.0]-2-hexene, L-menthol, 2,3-dehydro-1,8-cineole, 1-(2,6,6-trimethyl-2 -cyclohexenyl)-1-penten-3-one, β-pinene, (1α,2α,5α)-2,6,6-trimethylbicyclo[3.1.1]heptane-3-one, dl-menthol, (1α,2α,5α)-5-methyl-2-(1-methylethyl)cyclohexanol, (1S)-6,6-dimethyl-2-methylene-bicyclo[3.1.1]heptane; there are 2 acids, namely 4-methyl-2-pentenoic acid and 4-methyl-3-pentenoic acid; there are 2 aldehydes, namely (Z)-2-nonenal and 2-nonenal; there is 1 phenol, namely 3-fluorophenol; there are 3 alcohols, namely α,α,4-trimethylcyclohexanemethanol, d-menthol, and (1α,2α,5β)-5-methyl-2-(1-methylethyl)cyclohexanol.

[0174] Table 17 Comparative analysis results of aroma substance content between the three early (A) test group and the three late (CK) test group

[0175]

[0176]

[0177] 4.4 Appearance index evaluation results

[0178] The evaluation results of appearance indexes are shown in Table 18. The results showed that the flue-cured tobacco leaves of the three early (A) test group were higher than those of other test groups in terms of color, maturity, leaf structure, identity, oil content, chroma, etc., and the weighted total score was 80.89 points, the highest among all test groups.

[0179] Table 18 Appearance evaluation summary

[0180]

[0181] 4.5 Statistical results of tobacco leaf economic indicators analysis

[0182] The statistical results of tobacco economic index analysis are shown in Table 19. The results show that the total number of upper leaves, total weight, B2F and B3F grades after classification of Cui Bi No. 1 in the San Zao (A) test group are higher than those in other test groups, and the total weight and total output value are higher than those in other test groups.

[0183] Table 19 Statistical analysis of economic traits of upper leaves

[0184]

[0185] 4.6 Sensory evaluation results

[0186] The sensory evaluation scores are shown in Table 20 and Table 21. The results show that the sensory evaluation quality of the three early (A) test group is much better than that of the three late (CK) test group. Among the six test groups, the three late (CK) to three early (A) showed an obvious gradient upward trend, and the indicators such as sweet and sweet style, sweetness, aroma quality, aroma volume, permeability, smoke characteristics, and taste characteristics all showed an obvious upward trend. The results also showed that the time of transplanting, topping and harvesting during the cultivation process had an impact on the sensory evaluation quality, transplanting> topping> harvesting.

[0187] Table 20 Effects of different sowing, topping and harvesting times on sensory evaluation of upper leaves of flue-cured tobacco, style characteristics

[0188]

[0189] Table 21 Effects of different sowing, topping and harvesting times on sensory evaluation of upper leaves of flue-cured tobacco, quality characteristics and comprehensive scores

[0190]

[0191] A radar chart was drawn for the 10 sensory flavors of the flue-cured tobacco leaves of each test group, such as Figure 2As shown. The results showed that compared with the conventional three-late (CK) test group, the three-early (A) test group, i.e. the combination of early transplanting, early topping and early harvesting, significantly improved the woody, green, fruit and sweet flavor characteristics of Cui Bi No. 1 tobacco leaves, highlighting the sweet and refreshing style of Cui Bi No. 1. Compared with the early-late (B) test group, the three-early (A) test group improved the cucumber, fatty, earthy and green style characteristics. Compared with the early-late (C) test group, the three-early (A) test group improved the sweet, waxy, floral and fruity style characteristics. Compared with the late-late (D) test group, the three-early (A) test group significantly increased the nutty flavor characteristics. Compared with the late-late (E) test group, the three-early (A) test group significantly improved the sweet, woody, fruit and green sweet style characteristics. In summary, the three early (A) experimental group significantly improved the fresh and sweet style represented by flavors such as cucumber, green, fruit, floral, and sweet.

[0192] 4.7 Correlation analysis between omics and sensory evaluation

[0193] In order to identify the characteristic volatile aroma substances of the sweet and refreshing style, a joint analysis of sensory evaluation and volatile aroma substance omics was carried out. At the same time, in order to identify the tobacco leaf metabolites that cause the increase of characteristic aroma substances, a joint analysis of primary metabolomics and volatile aroma substance omics was carried out.

[0194] The joint analysis of volatile aroma omics and sensory evaluation and the joint analysis of volatile aroma omics and primary metabolomics were performed using the inter-group Pearson analysis method, using R language version 3.5.0, with the correlation filter threshold set to 0.8 and the Pvalue filter value set to 0.05.

[0195] After correlation analysis, 15 volatile aroma substances related to the sweet and refreshing style were obtained, namely (Z)-2,6-dimethyl-5,7-octadien-4-one, guaiacol, 6,6-dimethyl-bicyclo[3.1.1]-2-heptene-2-methanol, dimethyl succinate, 2,4,5-trimethylphenol, 1,2-diethylbenzene, 2-hydroxy-2-methylpropyl benzoate, 1-methyl-2-(1-ethylpropyl)benzene, 1-undecyne, 2-methyl-3-propylpyrazine, 4-(1-methylethyl)-1,5-cyclohexadiene-1-methanol, 2,3,6-trimethylphenol, 2-methyl-6-propylpyrazine, 1,1-diethoxynonane, and 6,6-dimethylbicyclo[3.1.1]-2-heptene-2-carbaldehyde. Among them, dimethyl succinate (Butanedioic acid, dimethyl ester), 2-hydroxy-2-methylpropyl benzoate (Benzoic acid, 2-hydroxy-, 2-methylpropyl ester), 1,1-diethoxynonane (Nonane, 1,1-diethoxy-), 6,6-dimethylbicyclo[3.1.1]-2-heptene-2-carboxaldehyde (Bicyclo[3.1.1]hept-2-ene-2-carboxaldehyde, 6,6-dimethyl-) and other four compounds have sweet, floral and honey-like aroma characteristics.

[0196] 4.8 Analysis of action mechanism

[0197] 1. Early transplanting, early topping and early harvesting promote the accumulation of aroma precursors

[0198] In Fujian tobacco-growing areas, early transplanting prolongs the tobacco growth period, increases leaf photosynthesis and metabolic activity, and promotes the accumulation of aroma precursors. In particular, the accumulation of amino acids and their derivatives in primary metabolites is an important aroma precursor. The combined effect of topping and harvesting: early topping reduces nutrient consumption, and early harvesting avoids the degradation of aroma substances caused by over-maturity. The synergistic effect of the two significantly increases the accumulation of aroma substance precursors, which include: cycloleucine, allo-isoleucine, DL-leucine, DL-tryptophan, cyclo(alanyl-phenylalanine), 3-(pyrazol-1-yl)-L-alanine, L-leucine, L-histidine, L-isoleucine, L-norleucine, L-phenylalanine, 3-methyl-L-histidine, L-allo-isoleucine, cis-L-3-hydroxyproline, DL-methionine, cis-4-hydroxy-D-proline, L-lysine-L-phenylalanine, L-theanine, glycyl-isoleucine, L-tryptophan, N6-methyladenosine, barbituric acid; malonylurea;2,4,6-pyrimidinetrione, D-glucose-6-phosphate, glucaric acid-1-phosphate, D-sedoheptulose 7-phosphate, sorbitol-6-phosphate, L-piperidinic acid, isocitric acid, phenylpyruvic acid, phosphoenolpyruvic acid, 9,11,13-octadecatrienoylglycerol, lysophosphatidylethanolamine 18:3 (2n isomer), lysophosphatidylcholine 17:2, lysophosphatidylethanolamine 18:3, 2-α-linolenic acid glyceride-1-O-glucoside, 1-α-linolenic acid glyceride-3-O-glucoside, monogalactosyl diglyceride, 1-linolenoylglycerol, lysophosphatidylcholine 18:3, lysophosphatidylcholine 18:3 (2n isomer), lysophosphatidylethanolamine 18:2 (2n isomer), lysophosphatidylethanolamine 18:2, lysophosphatidylcholine 18:2 (2n isomer), lysophosphatidylethanolamine 16:1 (2n isomer), 3-(((2-(dimethylamino)ethoxy)(hydroxy)phosphoryl)oxy)-2-hydroxypropyl (9Z,12Z,15E)-octadec-9,12,15-trienoate, 1-(((2-(dimethylamino)ethoxy)( 1-(2,3-dihydroxypropoxy)-3-(((2-(dimethylamino)ethoxy)(hydroxy)phosphoryl)oxy)prop-2-yl(8E,11Z,14Z)-octadeca-8,11,14-triene, 2-(2,3-dihydroxypropoxy)-3-(((2-(dimethylamino)ethoxy)(hydroxy)phosphoryl)oxy)propyl(8E,11Z,14Z)-octadeca-8,11,14-triene enoic acid, 1-(2,3-dihydroxypropoxy)-3-(((2-(dimethylamino)ethoxy)(hydroxy)phosphoryl)oxy)prop-2-yl(11Z,14Z)-octadec-11,14-dienoic acid, 2-(2,3-dihydroxypropoxy)-3-(((2-(dimethylamino)ethoxy)(hydroxy)phosphoryl)oxy)propyl(11Z,14Z)-octadec-11,14-dienoic acid, 1-linoleoyl-2-lysophosphatidic acid monobutylamine ester, α-linolenic acid, cis-octadec-9-ene-12-ynoic acid, pentadecanoic acid. ;

[0199] 2. Mechanism of action of primary metabolites

[0200] Amino acids and their derivatives: During the metabolism of leaves, the above amino acids can form a variety of intermediates with aroma potential through conversion reactions with organic acids or sugars. For example, leucine, isoleucine, phenylalanine, etc. can generate volatile flavor compounds such as alcohols, aldehydes and ketones through metabolic pathways such as deamination and carboxylation. These substances are further cracked during the roasting process to generate volatile compounds closely related to aroma.

[0201] Sugar substances: such as D-glucose-6-phosphate, accumulate through photosynthesis during the tobacco growth period, participate in the Maillard reaction and carbonyl-amino reaction, promote the formation of sugar compounds (such as pyrazines), and increase the complexity of aroma.

[0202] Contribution of lipid compounds: Lysophospholipids (such as lysophosphatidylethanolamine 18:3 and lysophosphatidylcholine 17:2) can generate short-chain fatty aldehydes and ketones through lipid peroxidation, enhancing the fruity and fragrant characteristics of tobacco.

[0203] During the tobacco curing process, α-linolenic acid and its derivatives are cleaved by lipoxygenase to produce aromatic aldehydes and alcohols.

[0204] 3. Synergistic effect of topping and harvesting

[0205] Early topping: By reducing the inhibition of apical preferential growth, the distribution efficiency of photosynthetic products by leaves is enhanced, which helps the accumulation of metabolites such as sugars and amino acids in leaves. Early harvesting: It avoids the degradation or conversion of photosynthetic products into secondary metabolites due to over-maturity. By reducing the increase in cellulose and lignin content in leaves, the stability of aroma precursor components is maintained, ensuring their conversion efficiency in subsequent baking.

[0206] 4. Chemical mechanism of baking: During the baking process, aroma precursors are effectively converted into aroma substances, promoting the dominant formation of four compounds rich in typical aroma substances such as fresh fragrance, fruity fragrance, cucumber fragrance, and floral fragrance, including Cui Bi No. 1 dimethyl succinate (Butanedioic acid, dimethyl ester), 2-hydroxy-2-methylpropyl benzoate (Benzoicacid, 2-hydroxy-, 2-methylpropyl ester), 1,1-diethoxynonane (Nonane, 1,1-diethoxy-), and 6,6-dimethylbicyclo[3.1.1]hept-2-ene-2-carboxaldehyde (Bicyclo[3.1.1]hept-2-ene-2-carboxaldehyde, 6,6-dimethyl-).

[0207] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and substitutions may be made to those details based on all the teachings disclosed, and these changes are within the scope of protection of the present invention. The full scope of the present invention is given by the attached claims and any equivalents thereof.

Claims

1. A method for producing a light-flavored tobacco leaf, comprising: 1) Seedling cultivation; 2) Transplanting is carried out from December 25 to 31; 3) Topping the tobacco plant when the flower buds have not yet fully extended and are still wrapped by the top leaves; 4) The tobacco leaves in the upper second shed are harvested when the leaf surface is 70% to 80% yellow-green and the main veins are 66% to 74% white.

2. The method according to claim 1, wherein the sowing time for raising seedlings is from November 7 to 15.

3. The method according to claim 1 or 2, wherein the light-fragrance style tobacco leaf is Cui Bi No. 1 tobacco leaf, preferably Cui Bi No. 1 upper tobacco leaf.

4. The method according to any one of claims 1 to 3, wherein the transplanting is carried out between December 26 and 30.

5. The method according to any one of claims 1 to 4, comprising one or more of the following: i) raising tobacco seeds by sowing them on seedling trays; ii) The seedling raising method is wet seedling raising; iii) The transplanting location is the tobacco-growing area in Fujian, preferably Youxi County, Sanming City, Fujian Province, preferably Longyang Village, Yangzhong Town, Youxi County; iv) The soil texture for transplanting is sandy loam; v) The previous crop before transplanting is rice; vi) Make ridges and holes in the tobacco field, and transplant the tobacco seedlings into the holes on the ridges.

6. The method according to any one of claims 1 to 5, further comprising: 5) Bake.

7. A tobacco leaf prepared by the method according to any one of claims 1 to 6. A tobacco leaf composition comprising the tobacco leaf according to claim 7.

9. A tobacco product comprising the tobacco leaf according to claim 7 and / or the tobacco leaf composition according to claim 8, preferably, the tobacco product is a cigarette.

10. Use of the tobacco leaf according to claim 7 and / or the tobacco leaf composition according to claim 8 in tobacco products, preferably, the tobacco product is a cigarette.

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