A method for producing aromatic substances through culturing rattan tea cells and an aroma capture system

Abstract

The present invention belongs to the field of plant cell culture technology, and specifically relates to a method for producing aroma substances through rattan tea cell culture and an aroma capture system. The method comprises the following steps: induction and breeding of aroma-producing rattan tea callus; establishment of an aroma-producing rattan tea cell suspension culture system and breeding of excellent cell lines; establishment of an aroma-producing rattan tea cell fermentation tank culture system and acquisition of aroma substances. The present invention obtains a unique aroma-producing rattan tea callus line through induction and screening, and establishes a stable cell suspension culture technology system for synthesizing aroma substances. At the same time, a "rattan tea cell culture coupled aroma substance capture system" is designed and manufactured, which can provide a basis for plant cell culture biosynthesis of aroma substances and preparation of flavors and fragrances, thereby saving natural resources. The aroma substances prepared by the present invention are of various types and high content, and can be further processed into microcapsule solid essences or emulsified essences for use in flavoring products such as juices, purees, and jams, significantly improving their flavor quality.

Description

Technical Field

[0001] The invention belongs to the technical field of plant cell culture, and particularly relates to a method for producing aroma substances through culturing camellia vine cells and an aroma capture system. Background Art

[0002] Flavors and fragrances are widely used in food, cosmetics, and pharmaceuticals, with enormous and rapidly growing market demand. Currently, the main methods for producing flavors and fragrances are natural raw material extraction and synthetic methods. Natural raw material extraction has a long history, resulting in high-quality, safe, and highly accepted products by consumers, but its development is limited by the availability of natural raw materials. Chemical synthesis offers low costs and a high degree of automation, but it places high demands on production equipment and is environmentally unfriendly. Consumer acceptance of products produced using this method remains low due to safety and health concerns. Currently, natural raw material extraction remains the primary method for producing flavors and fragrances.

[0003] Plants are one of the primary raw materials for flavor and fragrance production. There are over 3,300 aromatic plant species worldwide, of which approximately 200 have been developed and utilized. While natural aromatic plants are diverse and widely distributed, they have long growth cycles, are dependent on natural conditions, and are significantly affected by the environment and pests and diseases. Furthermore, due to the inherent characteristics of plant growth, it is difficult to significantly increase production in a short period of time. Therefore, the lack of natural raw material resources will become a major constraint on the development of the flavor and fragrance industry.

[0004] Plant cell culture has many advantages, such as short growth cycle, strong controllability, little influence by the natural environment, and easy extraction and purification of target substances. However, from the current situation, the theory and technology of plant aroma substance synthesis and accumulation are mostly based on the whole plant or a certain organ of the plant (such as flower, fruit, seed, leaf, root, stem, etc.). There is very little research on the production of aroma substances by plant cell suspension culture, and related theories and technologies are very lacking, especially the lack of "suspension culture cell lines" with excellent properties. "Provenance" is a key problem that is urgently needed to be solved in the field of plant cell culture engineering production of aroma substances. At present, the research and application of plant cell culture engineering production of aroma substances are also very few, and related technologies and equipment are very lacking. There is no report on the "aroma capture equipment" matched with cell culture. Summary of the Invention

[0005] In response to the deficiencies in the prior art, the present invention provides a method for producing aromatic substances through cell culture of Camellia ulmoides and an aroma capture system. Through long-term induction and screening, a unique aroma-producing Camellia ulmoides callus system is obtained. On this basis, a stable cell suspension culture technology system for synthesizing aromatic substances is established. At the same time, a "Camellia ulmoides cell culture coupled aroma substance capture system" is designed and manufactured, and a corresponding aroma capture technology system is established, which can provide a basis for producing spices through plant cell culture instead of extracting spices from natural raw materials, thereby saving natural resources.

[0006] To achieve the above object, the present invention first provides a method for producing aroma substances through culturing Tengcha cells, comprising the following steps:

[0007] (1) Induction and selection of aroma-producing callus of Camellia tengcha: The young leaves and stems of Camellia tengcha were pretreated to obtain explants, which were then inoculated into an induction medium for callus induction culture. The induced callus was subcultured under full illumination. The growth rate and aroma-producing ability of the callus were used as the main evaluation indicators for cultivation and screening to obtain aroma-producing callus lines with excellent traits.

[0008] (2) Establishment of a cell suspension culture system for fragrant tea vines and selection of excellent cell lines: Take the fragrant tea callus and inoculate it into the suspension culture medium, shake the suspension culture, and subculture it every 4-8 days. The cell growth rate and fragrance production ability are used as the main evaluation indicators for subculture and screening to obtain a suspension culture cell line with stable growth and characteristics;

[0009] (3) Establishment of a fermentation tank culture system for aromatic tea vine cells and acquisition of aromatic substances: The suspension culture cell line was inoculated into an airlift fermentation tank filled with culture medium for cultivation. After continuous stirring and cultivation for 7-9 days, an aromatic substance capture system was used to capture the aromatic substances, and then eluted with ethanol solvent, rotary evaporated, or desorbed with high-temperature steam to obtain essential oils.

[0010] Furthermore, in step (1) of the above technical solution, the pretreatment method of the young leaves and stems of the rattan tea is as follows: select the young leaves and stems of the rattan tea, trim them, rinse them with running water for 20-60 minutes, and wipe off the surface moisture; on a sterile workbench, immerse the leaves in 75% alcohol for disinfection for 15-60 seconds; immediately rinse them in sterile water for 2-6 times after alcohol disinfection, then immerse them in 0.1% mercuric chloride aqueous solution for sterilization for 4-10 minutes, take them out and rinse them in sterile water for 5-6 times, cut the edges of the leaves and cut them into 5mm×5mm fragments, and cut the stems into 0.5-4cm stem segments, thereby obtaining leaf explants and stem segment explants.

[0011] Furthermore, in step (1) of the above technical solution, the induction culture medium is based on MS, and 2.0 mg / L 2,4-D and 30 g / L sucrose are added; the induction culture conditions are: temperature of 28±2°C; dark culture in the dark; relative humidity of air is 40%-60%; during the subculture process, except for the lighting conditions, other culture conditions are the same as the induction culture conditions.

[0012] Furthermore, in step (2) of the above technical solution, the suspension culture medium is based on MS, and is further supplemented with 1.0 mg / L KT, 0.5 mg / L 2,4-D, 0.3 mg / L NAA and 30 g / L sucrose, with a pH of 5.8±0.4, a culture temperature of 28±2°C, and an oscillation speed of 100-140 rpm.

[0013] Furthermore, in step (2) of the above technical solution, the subculture method is: mixing the cell culture to be subcultured with fresh culture medium at a ratio of 1:1 (V / V), and then dividing it into two and culturing them separately.

[0014] Furthermore, in the above technical solution, in step (1) and step (2), the identification method of the aroma production ability is: weigh 5 g of aroma-producing callus tissue or suspension culture cells, put it into a 10 mL headspace bottle, then add 1.6 g of NaCl and 10 μL of internal standard ethyl decanoate, tighten the bottle cap to seal, and then use "headspace solid phase microextraction-gas chromatography-mass spectrometry" technology to detect the aroma components, and calculate the content of the detected aroma substances according to the internal standard method.

[0015] Furthermore, in step (3) of the above technical solution, the weight (fresh weight) volume ratio W / V of the suspension culture cell line to the culture medium is 5-25%.

[0016] Furthermore, in step (3) of the above technical solution, the culture medium is based on MS, and 1.0 mg / L KT, 0.5 mg / L 2,4-D, 0.3 mg / L NAA and 30 g / L sucrose are added, the pH is 5.8±0.4, the culture temperature is 28±2°C, the ventilation volume is 1 L / min, and the stirring speed is 100 rpm.

[0017] The present invention also provides an aroma capture system for capturing the aroma substances produced by the above-mentioned vine tea cell fermentation tank culture. The aroma capture system includes five components, which are connected in series in sequence as an air compressor, a gas flow controller, an air filter sterilizer, a biological fermentation tank, and an aroma capturer; the biological fermentation tank includes an airlift and / or stirring cell culture chamber and a temperature control system, the cell culture chamber vent is located at the bottom, middle or top, and is a single-port, double-port, multiple-port or dispersed vent, and the temperature control system uses a water jacket or ventilation type to control the temperature of the biological fermentation tank; the aroma capturer is composed of a multi-stage capture adsorption tank, and its filler is any one of a macroporous resin, activated carbon, and a liquid adsorbent. When used, single-stage adsorption, two-stage adsorption, or multi-stage adsorption can be adopted. Specifically, the liquid adsorbent can be an organic solvent such as n-hexane and ethanol.

[0018] The present invention also provides an application of the aroma substance prepared by the above method, wherein the aroma substance is processed into microcapsule solid flavor or emulsified flavor, which is used in fruit juice, fruit puree, and jam products.

[0019] Specifically, when essential oil is processed into microcapsule solid flavor, the preparation method is as follows: using gum arabic (25%), dextrin (27%) and corn syrup (48%) as wall materials, and the essential oil as core material, wherein the amount of the core material is 40% of the total weight of the wall material, emulsifying, homogenizing after emulsification, and then spray drying to obtain the microcapsule solid flavor.

[0020] When processing essential oil into emulsified flavor, the following ratios are used: 6% essential oil, 2.5% Tween-80, 4.5% polyglycerol ester, 62% co-emulsifier (ethanol: glycerol volume ratio 1:1), 0.4% NaCl, and deionized water added to 100%. The preparation method is as follows: After mixing the above ingredients, the oil phase and the aqueous phase are stirred at high speed to form a crude emulsion. The crude emulsion is then subjected to high-speed shearing and then subjected to two high-pressure homogenization processes (first pressure 0.3MPa-0.4MPa, second pressure 1.6MPa-3.0MPa) to obtain an emulsified flavor with a particle size of less than 2μm.

[0021] The present invention has the following beneficial effects:

[0022] The present invention provides for the first time a complete technical solution for "producing aromatic substances through suspension culture of rattan tea cells", which provides ideas, methods and technologies for solving the problem of "resource-constrained development"; through long-term induction, screening and cultivation, aromatic rattan tea callus lines and suspension culture cell lines with excellent traits are obtained, providing high-quality "seed sources" for plant cell culture production of aromatic substances; based on the growth and aroma-producing characteristics of rattan tea suspension culture cells, a "cell culture coupled aroma capture" solution is designed, and a rattan tea cell culture coupled aroma capture system is made by ourselves, establishing a "cell culture coupled aroma capture" technology system, which provides an alternative solution for extracting flavors and fragrances from natural raw materials and saves natural resources.

[0023] The aroma substances prepared by the method of the present invention are of various types and high content, and can be further processed into microcapsule solid flavors or emulsified flavors, which can be used for flavoring products such as fruit juice, puree, and jam, and can significantly improve their aroma quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a process flow chart for inducing and breeding the aroma-producing callus of Tengcha in Example 1 of the present invention;

[0025] Figure 2 This is a diagram of the fragrant tea callus line with excellent properties obtained in Example 1 of the present invention;

[0026] Figure 3 This is a process flow chart for establishing a cell suspension culture system for fragrant vine tea and selecting a superior cell line in Example 2 of the present invention;

[0027] Figure 4 This is a diagram of the suspension culture cell line of fragrant vine tea with excellent properties in Example 2 of the present invention;

[0028] Figure 5 This is a schematic diagram of the rattan tea cell suspension culture coupled aroma capture system in Example 3 of the present invention.

[0029] Among them, ① air compressor; ② gas flow controller; ③ air filter sterilizer; ④ biological fermentation tank; ⑤ aroma collector. DETAILED DESCRIPTION

[0030] The experimental methods in the following examples are conventional methods unless otherwise specified. The raw materials involved in the following examples are common commercial products and can be purchased from the market unless otherwise specified.

[0031] The present invention is described in further detail below in conjunction with the embodiments:

[0032] The young leaves and stems of the rattan tea used in the experiment of the present invention were obtained from the College of Food Science and Engineering of Jiangxi Agricultural University;

[0033] The MS basal medium used was formulated with 1900 mg / L potassium nitrate, 332.2 mg / L calcium chloride, 1650 mg / L ammonium nitrate, 180.7 mg / L magnesium sulfate, 170 mg / L potassium dihydrogen phosphate, 0.025 mg / L 1 It is composed of copper sulfate, 6.2 mg / L boric acid, 16.9 mg / L manganese sulfate, 0.25 mg / L sodium molybdate, 8.6 mg / L zinc sulfate, 0.025 mg / L cobalt chloride, 0.83 mg / L potassium iodide, 27.8 mg / LFeSO4·7H2O, 37.26 mg / L disodium edetate, 2.0 mg / L glycine, 100 mg / L inositol, 0.50 mg / L niacin, 0.1 mg / L vitamin B1 and 0.5 mg / L vitamin B6.

[0034] Example 1: Induction and breeding of fragrant vine tea callus

[0035] The process flow chart of induction and selection of callus tissue of fragrant vine tea is as follows: Figure 1 shown.

[0036] (1) Preparation of explants: Select young leaves and stems of Tengcha, trim them appropriately, rinse them with running water for 20-60 minutes, and wipe off the surface moisture; immerse the leaves in 75% alcohol on a sterile workbench for 15-60 seconds; immediately rinse them in sterile water 2-6 times after alcohol disinfection, then immerse them in 0.1% mercuric chloride aqueous solution for 4-10 minutes, and immediately take them out and rinse them in sterile water 5-6 times; cut off the edges of the leaves and cut them into pieces of about 5mm×5mm, and cut the stem segments into pieces of about 1cm to make the corresponding leaf explants and stem segment explants.

[0037] (2) Callus induction: Drain the prepared leaf and stem explants and inoculate them into MS medium containing 2.0 mg / L 2,4-D and 30 g / L sucrose for callus induction. The culture conditions were: temperature 28 ± 1°C; dark culture with no light; and relative humidity 40%-60%.

[0038] (3) Subculture of callus tissue: The induced callus tissues of various phenotypes are picked out and continuously subcultured under full illumination. The subculture medium and other culture conditions are the same as those for the induction of callus tissue mentioned above.

[0039] (4) Selection of excellent aroma-producing callus: With the callus growth rate (with the increase in callus biomass (dry weight and wet weight) as the evaluation index) and aroma-producing ability (mainly based on sensory evaluation combined with GC-MS detection) as the main evaluation index, through continuous subculture and screening, we obtained aroma-producing callus lines with excellent traits, such as Figure 2 As shown. Figure 2 It can be seen that the callus tissue is light yellow, the tissue is soft, and the growth condition is good.

[0040] (5) Identification of aroma components of callus: Weigh 5 g of fresh callus tissue of the fragrant tea vine and place it in a 10 mL headspace vial. Then, add 1.6 g of NaCl and 10 μL of the internal standard ethyl decanoate. Immediately tighten the cap and seal the vial. The aroma components were detected using the headspace solid phase microextraction-gas chromatography-mass spectrometry (HSSPME-GC / Q-TOF) technique. 18 aroma components were identified, among which hexanal, hexanol, and benzyl alcohol had the highest contents, reaching 303.58 mg / L, 102.82 mg / L, and 374.41 mg / L, respectively.

[0041] Example 2: Establishment of a cell suspension culture system for fragrant vine tea and selection of superior cell lines

[0042] The specific process flow chart for the establishment of a cell suspension culture system for fragrant vine tea and the selection of excellent cell lines is as follows: Figure 3 shown.

[0043] (1) Establishment of a cell suspension culture system for the fragrant camellia vine: 4-12 g of the fresh excellent fragrant camellia vine callus tissue obtained in Example 1 was inoculated into 40 mL of culture medium for shaking suspension culture.

[0044] The main components of the suspension culture medium are as follows: MS medium supplemented with 1.0 mg / L KT, 0.5 mg / L 2,4-D, 0.3 mg / L NAA, and 30 g / L sucrose. The main culture conditions are as follows: pH 5.8±0.4, temperature 28±2°C, and rotation speed 100-140 rpm (revolutions per minute). Subculture every 4-8 days (the cycle depends on the cell growth situation) until a suspension culture cell line with stable growth and characteristics is obtained. The subculture method is as follows: the cell culture to be subcultured is mixed with fresh culture medium at a ratio of 1:1 (V / V), and then divided into two and cultured separately.

[0045] (2) Selection of excellent aroma-producing vine tea suspension culture cell lines: With the cell growth rate (with the growth of cell biomass (dry weight and wet weight) as the evaluation index) and aroma-producing ability (mainly based on sensory evaluation combined with GC-MS detection) as the main evaluation indicators, through continuous subculture and screening, excellent aroma-producing vine tea suspension culture cell lines were obtained, such as Figure 4 As shown, the cell line is light yellow in color, with uniform cell granules and a strong fruity aroma.

[0046] (3) Identification of aroma components of Tengcha suspension culture cells: Weigh 5 g of fresh aroma-producing Tengcha suspension culture cells and place them in a 10 mL headspace vial. Then, add 1.6 g of NaCl and 10 μL of internal standard. Immediately tighten the cap and seal the vial. The aroma components were detected using headspace solid phase microextraction-gas chromatography-mass spectrometry (HSSPME-GC×GC / Q-TOF) technology, from which 203 aroma components were identified, including 32 alcohols, 22 aldehydes, 9 carboxylic acids, 45 esters, 13 ketones, 44 hydrocarbons, and 38 other substances. In terms of relative concentrations, the total concentrations of alcohols, aldehydes, and esters were relatively high. For example, the relative concentrations of 1-pentanol, benzyl alcohol, isopentanol, 1-hexanol, trans-2-hexen-1-ol, hexanal, 2,4-nonadienal, benzaldehyde, phenylacetaldehyde, 2-hexenal, ethyl isobutyrate, ethyl benzoate, ethyl 2-methylbutyrate, ethyl isovalerate, ethyl butyrate, ethyl (2Z)-but-2-enoate, and ethyl 2-hydroxy-3-methyl-butyrate were all greater than 0.2%. Among them, the relative concentration of hexanal was the highest among all substances, reaching 5.788%. While alkanes and other substances were abundant in variety, their relative concentrations were generally low. These test data indicate that alcohols, aldehydes, and esters contribute significantly to the aroma of Tengcha suspension culture cells.

[0047] Example 3: Design of a rattan tea cell suspension culture coupled with an aroma capture system

[0048] Based on the growth and aroma production characteristics of Tengcha suspension culture cells, a "cell suspension culture coupled aroma capture system" was designed. The system mainly includes the following five components (such as Figure 5 As shown) are connected in series in sequence as ① air compressor; ② gas flow controller; ③ air filter sterilizer; ④ biological fermentation tank; ⑤ aroma collector. Among them, the air compressor and the gas flow controller constitute a ventilation system, and the ventilation system can set the gas flow and regulate the ventilation volume according to actual needs. The air filter sterilizer is an air sterilization system, which can work continuously for 20 days after it is turned on and meets the sterile culture requirements. The biological fermentation tank includes an air lift and / or stirring cell culture chamber and a temperature control system, which is a whole, wherein the air lift and / or stirring cell culture chamber vents can be set at the bottom, middle or top, and can be single-port, double-port, multi-port or dispersed ventilation; the temperature control system can use a water jacket or ventilation type to control the temperature of the biological fermentation tank, and can also control the temperature in the cell culture chamber by controlling the ambient temperature. The aroma collector is an aroma substance capture system, which consists of a multi-stage capture adsorption tank. Its filler is made of macroporous resin, activated carbon, organic solvent and other materials. When in use, single-stage adsorption, two-stage adsorption or multi-stage adsorption can be used to dynamically capture aroma substances through adsorption.

[0049] Example 4: Establishment of a fermentation tank culture system for fragrant tea leaves and acquisition of its fragrant substances

[0050] (1) Suspension culture of Camellia ulmoides cells: Take the fresh, subcultured, excellent aroma-producing Camellia ulmoides suspension culture cell line of Example 2 and inoculate it at a ratio of 5-25% (fresh cell weight / culture medium volume, W / V), and then culture it in an airlift fermenter with continuous stirring during the culture process.

[0051] The culture medium consisted of MS medium supplemented with 1.0 mg / L KT, 0.5 mg / L 2,4-D, 0.3 mg / L NAA, and 30 g / L sucrose. The main culture conditions were pH 5.8 ± 0.4, temperature 28 ± 2°C, aeration 1 L / min, and rotor speed 100 rpm. Under these conditions, the fresh weight of the cells reached 278.50 g / L on day 9, allowing for aroma capture.

[0052] (2) Aroma capture: Aroma capture is performed by macroporous resin or activated carbon adsorption. During the capture, one adsorbent can be used for single-stage, two-stage or multi-stage adsorption, or a combination of two adsorbents can be used for two-stage or multi-stage adsorption.

[0053] In this example, the cell culture volume in the culture tank was 800 mL, the ventilation rate was 1 L / min, HPD-100 macroporous resin was used for single-stage adsorption, the capture time was 30 min, elution was performed with ethanol, the eluate was concentrated to 5 mL, and detection was performed by GC-MS. After testing, 196 aroma substances were identified, including 4-ethylbenzaldehyde, p-ethylacetophenone, acetophenone, lauryl alcohol, ethyl 3-hydroxybutyrate, benzaldehyde, nonanal, 4-isopropylbenzyl alcohol, benzyl alcohol, isopentanol, 1-hexanol, hexanal, 2,4-nonadienal, phenylacetaldehyde, 2-hexenal, ethyl benzoate, ethyl isovalerate, and ethyl butyrate; according to the area normalization method, the content of 4-ethylbenzaldehyde, p-ethylacetophenone, acetophenone, lauryl alcohol, and ethyl 3-hydroxybutyrate reached 2.74%, 1.31%, 1.04%, 0.68%, and 0.37%, respectively.

[0054] Example 5: Application of aroma substances from suspension cultured cells of rattan tea

[0055] Aroma compounds are collected using a self-assembled "cell suspension culture coupled aroma capture system." After ethanol elution, they are then subjected to rotary evaporation to remove the ethanol, producing essential oils. These essential oils are then processed into microencapsulated solid flavors or emulsified flavors for use in flavoring products like juices, purees, and jams, significantly enhancing their flavor quality.

[0056] The preparation method of microcapsule solid flavor is as follows: using gum arabic (25%), dextrin (27%) and corn syrup (48%) as wall materials, using essential oil as core material, and adding the core material in an amount of 40% of the total weight of the wall materials, and then emulsifying, homogenizing after emulsification, and then spray drying to obtain the microcapsule solid flavor.

[0057] The preparation method of emulsified flavor is:

[0058] Formula: essential oil 6%; Tween-80 2.5%; polyglycerol ester 4.5%; co-emulsifier (ethanol and glycerol volume ratio 1:1) 62%; NaCl addition mass fraction: 0.4%, added to 100% with deionized water.

[0059] Preparation method: After mixing the above raw materials, the oil phase and the water phase are stirred at high speed to obtain a crude emulsion, the crude emulsion is sheared at high speed, and then homogenized twice under high pressure, wherein the first pressure is 0.3MPa-0.4MPa and the second pressure is 1.6MPa-3.0MPa, to obtain an emulsified flavor with a particle size of less than 2μm.

[0060] Based on the above analysis, the present invention obtained a unique aroma-producing camellia callus system through induction and screening, and on this basis established a stable cell suspension culture technology system for synthesizing aroma substances. At the same time, a "camellia tenghua cell culture coupled aroma capture system" was designed and self-made, and a corresponding aroma capture technology system was established, which can provide a basis for the production of flavors and fragrances by plant cell culture to replace natural raw material resources for the preparation of flavors and fragrances, thereby saving natural resources. The aroma substances prepared by the present invention are of many types and high content, and can be further processed into microcapsule solid flavors or emulsified flavors, which can be used for flavoring products such as fruit juice, fruit puree, and jam, and can significantly improve their flavor quality.

[0061] Finally, it should be emphasized that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various changes and modifications. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for producing aromatic substances through cell culture of Camellia tenghua, characterized in that: The following steps are involved: (1) Induction and selection of callus tissue of fragrant tea vines: The young leaves and stems of tea vines were pre-treated to obtain explants, which were then inoculated into induction medium for callus induction culture. The induced callus tissue was subcultured under full illumination. The growth rate and fragrance production ability of the callus were used as the main evaluation indicators for cultivation and screening to obtain fragrant callus lines with excellent traits. (2) Establishment of a cell suspension culture system for fragrant tea vines and selection of excellent cell lines: Take the fragrant tea callus and inoculate it into the suspension culture medium, shake the suspension culture, and subculture it every 4-8 days. The cell growth rate and fragrance production ability are used as the main evaluation indicators for subculture and screening to obtain a suspension culture cell line with stable growth and characteristics; (3) Establishment of a fermentation tank culture system for aromatic tea vines and the acquisition of aromatic substances: The suspension culture cell line was inoculated into an airlift fermentation tank filled with culture medium for cultivation. After continuous stirring and cultivation for 7-9 days, an aroma collector was used to capture the aromatic substances, and then eluted with ethanol solvent, rotary evaporated, or desorbed with high-temperature steam to obtain essential oils.

2. The method for producing aroma substances by culturing cells of Camellia oleifera according to claim 1, characterized in that: In step (1), the pretreatment method of the young leaves and stems of the rattan tea is as follows: select the young leaves and stems of the rattan tea, trim them, rinse them with running water for 20-60 minutes, and wipe off the surface moisture; on a sterile workbench, immerse the leaves in 75% alcohol for disinfection for 15-60 seconds; immediately rinse them in sterile water for 2-6 times after alcohol disinfection, then immerse them in 0.1% mercuric chloride aqueous solution for sterilization for 4-10 minutes, take them out and rinse them in sterile water for 5-6 times, cut the edges of the leaves and cut them into 5 mm×5 mm fragments, and cut the stems into 0.5-4 cm stem segments, thereby obtaining leaf explants and stem segment explants.

3. The method for producing aroma substances by culturing cells of Camellia oleifera according to claim 1, characterized in that: In step (1), the induction culture medium is based on MS, and 2.0 mg / L 2,4-D and 30 g / L sucrose are added; the induction culture conditions are: temperature of 28±2°C; dark culture; relative humidity of 40%-60%; during the subculture process, except for the lighting conditions, other culture conditions are the same as the induction culture conditions.

4. The method for producing aroma substances by culturing cells of Camellia oleifera according to claim 1, characterized in that: In step (2), the suspension culture medium is based on MS, and 1.0 mg / L KT, 0.5 mg / L 2,4-D, 0.3 mg / L NAA and 30 g / L sucrose are added. The pH is 5.8±0.4, the culture temperature is 28±2°C, and the shaking speed is 100-140 rpm.

5. The method for producing aroma substances by culturing cells of Camellia oleifera according to claim 1, characterized in that: In step (2), the subculture method is: mixing the cell culture to be subcultured with fresh culture medium in a volume ratio of 1:1, and then dividing it into two and culturing them separately.

6. The method for producing aroma substances by culturing cells of Camellia oleifera according to claim 1, characterized in that: In step (1), the method for identifying the aroma-producing ability is as follows: weigh 5 g of the aroma-producing callus line, put it into a 10 mL headspace bottle, then add 1.6 g of NaCl and 10 μL of the internal standard ethyl decanoate, tighten the bottle cap to seal it, and then use the "headspace solid phase microextraction-gas chromatography-mass spectrometry" technology to detect the aroma components, and calculate the content of the detected aroma substances according to the internal standard method; in step (2), the method for identifying the aroma-producing ability is as follows: weigh 5 g of the suspension culture cell line, put it into a 10 mL headspace bottle, then add 1.6 g of NaCl and 10 μL of the internal standard ethyl decanoate, tighten the bottle cap to seal it, and then use the "headspace solid phase microextraction-gas chromatography-mass spectrometry" technology to detect the aroma components, and calculate the content of the detected aroma substances according to the internal standard method.

7. The method for producing aroma substances by culturing cells of Camellia oleifera according to claim 1, characterized in that: In step (3), the weight-to-volume ratio (W / V) of the suspension culture cell line to the culture medium is 5-25%.

8. The method for producing aroma substances by culturing cells of Camellia oleifera according to claim 1, characterized in that: In step (3), the culture medium is based on MS, and 1.0 mg / L KT, 0.5 mg / L 2,4-D, 0.3 mg / L NAA and 30 g / L sucrose are added. The pH is 5.8±0.4, the culture temperature is 28±2°C, the ventilation volume is 1 L / min, and the stirring speed is 100 rpm.

9. The method for producing aroma substances by culturing cells of Camellia oleifera according to claim 1, characterized in that: In step (3), the front of the aroma trap is connected in series with a biological fermentation tank, an air filter sterilizer, a gas flow controller, and an air compressor, and these five parts together constitute a coupled aroma capture system; the biological fermentation tank includes an airlift and / or stirring cell culture chamber and a temperature control system, the air vent of the cell culture chamber is arranged at the bottom, middle or top, and is a single port or a dispersed multiple ports, and the temperature control system adopts a water jacket type or a ventilation type to control the temperature of the biological fermentation tank; the aroma trap is composed of a multi-stage capture adsorption tank, and its filler is any one of a macroporous resin, activated carbon, and a liquid adsorbent. When in use, single-stage adsorption or multi-stage adsorption is adopted.

10. Use of an aroma substance prepared by the method according to any one of claims 1 to 9, characterized in that: Process aroma substances into microencapsulated solid flavors or emulsified flavors for use in juice, puree, and jam products.

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