Method for cultivating kale with increased antioxidant and glucosinolate content

Abstract

The present invention relates to a method for cultivating kale with increased antioxidant components and glucosinolate content, and more specifically, to a method for cultivating kale with increased antioxidant components and glucosinolate content comprising the step of treating a culture solution containing green tea extract for 5 to 15 days before harvest. In the present invention, it has been confirmed that it is possible to cultivate kale with increased content of glucosinolate, a secondary metabolite of cruciferous plants, as well as antioxidant components, through a cultivation method using green tea extract. Therefore, plants cultivated by the method of the present invention can be utilized in various ways as antioxidant or anticancer compositions.

Description

Technology Field

[0001] The present invention relates to a method for cultivating kale with increased antioxidant components and glucosinolate content, and more specifically, to a method for cultivating kale with increased antioxidant components and glucosinolate content comprising the step of treating a culture solution containing green tea extract for 5 to 15 days before harvest. Background Technology

[0003] As the elderly population and the proportion of patients suffering from chronic diseases increase, there is a growing demand for personalized nutritional diets that take into account individual health conditions. Plant-based foods are rich in nutrients and contain many bioactive components that regulate the body's metabolic pathways, and some functional herbs are used as food or dietary supplements. Artificial cultivation is being carried out to mass-produce these herbs.

[0004] Kale (Brassica oleracea var. acephala) is a cruciferous vegetable containing glucosinolates and is known to be high in fiber, vitamin C, flavonoids, α-tocopherol, and β-carotene. In particular, glucosinolates are sulfur-containing active compounds known to have antioxidant, stress-reducing, anti-inflammatory, and anticancer effects (Schmidt, S. et al., Food Chem., 119(4):1293-1299, 2010).

[0005] Vertical farming is an alternative cultivation system for growing crops indoors; it does not rely on sunlight or soil and is independent of external conditions. Generally, by installing multi-tiered shelves inside buildings, vertical farming can increase yields per unit area compared to conventional methods. Environmental conditions in vertical farming are artificially organized, and light, temperature, humidity, carbon dioxide concentration, and water can be precisely controlled using information and communication technologies. Research is being conducted on controlling light sources to enhance plant nutrition and functional activity, and it has been reported that the use of blue light in vertical farming increases the levels of glucosinolates and phenolic compounds in leafy vegetables. Additionally, it has been reported that treating kale with NaCl and Na2SeO3 increases isothiocyanate content, and that harvested kale upregulates Nrf2 expression in HepG2 cell lines (Al-Kodmany, K., Buildings, 8(2):24, 2018; Kim, SY et al., Sci. Rep., 8(1):3999, 2018).

[0007] Meanwhile, green tea is an essential source of catechins, which are polyphenol compounds belonging to flavan-3-ols (flavanols). Catechins possess high antioxidant activity, and numerous animal studies have revealed their anti-obesity and anti-diabetic effects (Chacko, SM et al., Chin. Med., 5(1):1-9, 2010). The problem to be solved

[0009] In the present invention, as a result of efforts to cultivate kale with increased biologically active components, it was confirmed that kale cultivated by treating with green tea extract showed a significant increase in not only antioxidant components but also glucosinolate components, and the present invention was completed.

[0011] Accordingly, the objective of the present invention is to provide a method for cultivating kale with increased antioxidant components and glucosinolate content. means of solving the problem

[0013] In order to achieve the aforementioned purpose,

[0014] The present invention provides a method for cultivating kale with increased antioxidant components and glucosinolate content, comprising the step of culturing by treating a culture solution containing green tea extract for 5 to 15 days before harvesting the plant.

[0015] In addition, the present invention provides kale with increased antioxidant component and glucosinolate content cultivated by the above cultivation method.

[0017] In a preferred embodiment of the present invention, the culture solution containing the green tea extract can be supplied separately during the kale cultivation process as a nutrient solution for hydroponic cultivation.

[0018] In another preferred embodiment of the present invention, the green tea extract may be included in the culture medium at a concentration of 0.1 (w / v) to 1.0% (w / v).

[0019] In another preferred embodiment of the present invention, the green tea extract can be prepared by hot water extraction at a temperature of 50 to 90°C for 60 to 150 minutes.

[0020] In another preferred embodiment of the present invention, the antioxidant component may include P-coumaric acid, epicatechin, and epigallocatechin gallate (EGCG).

[0021] In another preferred embodiment of the present invention, the glucosinolate may comprise one or more selected from the group consisting of sinigrin, gluconapin, glucobrassicin, gluconasturtiin, and neoglucobrassicin. Effects of the invention

[0023] In the present invention, it has been confirmed that it is possible to cultivate kale with increased content of glucosinolate, a secondary metabolite of cruciferous plants, as well as antioxidant components, through a cultivation method using green tea extract. Therefore, plants cultivated by the method of the present invention can be utilized in various ways as antioxidant or anticancer compositions. Brief explanation of the drawing

[0025] Figure 1 is a flowchart showing the kale cultivation process. Figure 2 shows data confirming the yield of kale grown with green tea extract, Figure 2a shows the fresh weight and dry weight of the kale, and Figure 2b shows the mineral content of the kale. Figure 3 shows data confirming the content of each phenolic compound contained in kale grown with green tea extract, Figure 3a shows the content of chlorogenic acid and p-coumaric acid, and Figure 3b shows the content of epicatechin and epigallocatechin gallate. Figure 4 shows data regarding the glucosinolate content contained in kale grown with green tea extract. Figure 5 is heatmap analysis data for variables of kale grown with green tea extract. In the figure, the values ​​are represented by their size from green (minimum) to red (maximum). Figure 6 shows the principal component analysis (PCA) plot data of kale grown with green tea extract, and the PCA plot was visualized using variables (Figure 6a) and sample groups (Figure 6b). Specific details for implementing the invention

[0026] The present invention will be described in detail below.

[0028] In one aspect, the present invention relates to a method for cultivating kale with increased antioxidant components and glucosinolate content, comprising the step of culturing the plant by treating it with a culture medium containing green tea extract for 5 to 15 days before harvesting.

[0030] In the present invention, it was hypothesized that supplementing with green tea extract during kale cultivation could increase the functional activity of kale by absorbing the active compounds of the green tea extract. If catechin can penetrate kale, the biological activities of catechin and glucosinolate can be combined in a single plant. Furthermore, green tea extract may act as an inducer that stimulates and alters the expression of metabolic pathways in kale, particularly to synthesize intrinsic secondary metabolites such as glucosinolate.

[0031] Household vertical farming equipment was adopted to accurately control environmental conditions as a cultivation system, and it was also anticipated that using such equipment would enable people to independently obtain individually customized harvests.

[0032] Therefore, in this invention, the possibility of producing functional kale at home was examined by verifying the characteristics (color, flavor, antioxidant capacity, and content of biologically active compounds) of kale cultivated by treating it with green tea extract.

[0034] In the present invention, the kale can be cultivated using a hydroponic cultivation system, and the culture solution containing the green tea extract can be supplied separately during the kale cultivation process as a nutrient solution for hydroponic cultivation.

[0035] In the present invention, the culture medium containing the green tea extract can preferably be treated for 10 to 15 days prior to plant harvesting, and the green tea extract can be included in the culture medium at a concentration of 0.1 (w / v) to 1.0% (w / v), preferably at a concentration of 0.2% (w / v) to 0.5% (w / v). The green tea extract can be prepared by hot water extraction at a temperature of 50 to 90°C for 60 to 150 minutes.

[0036] In the present invention, the antioxidant component may include P-coumaric acid, epicatechin, and epigallocatechin gallate (EGCG).

[0037] In the present invention, the glucosinolate may include one or more selected from the group consisting of sinigrin, gluconapin, glucobrassicin, gluconasturtiin, and neoglucobrassicin.

[0038] In a specific embodiment of the present invention, kale was cultivated by treating a culture solution containing a green tea extract, extracted with hot water at a temperature of 50 to 90°C for 60 to 150 minutes, at a concentration of 0.1 to 0.5% for two weeks prior to plant harvesting. Since the green tea extract corresponds to a stress treatment, the treatment was performed after the growth of the kale was completed (after securing the production yield).

[0039] As a result, no changes in kale yield, mineral content, color, texture, or sensory characteristics were observed due to treatment with green tea extract, but it was confirmed that antioxidant components such as P-coumaric acid, epicatechin, and epigallocatechin gallate (EGCG) in kale increased (Figs. 3a and 3b). In addition, it was confirmed that the total glucosinolate content, including sinigrin, increased (Fig. 4).

[0041] Accordingly, in another aspect, the present invention relates to a pharmaceutical composition or a health functional food composition comprising a kale grown by the above cultivation method with increased antioxidant component and glucosinolate content, or said kale extract.

[0042] Since it has been confirmed that kale cultivated by the cultivation method of the present invention has increased antioxidant components and glucosinolate content, it can be utilized as an antioxidant, anti-inflammatory, and anticancer composition.

[0043] The pharmaceutical compositions of the present invention may be formulated into various forms according to conventional methods and used. For example, they may be formulated into oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, and syrups, and may be formulated into external preparations, suppositories, and sterile injectable solutions. Depending on each formulation, they may further include pharmaceutically acceptable carriers, excipients, and diluents. Additionally, they may be formulated into external preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, and sterile injectable solutions according to conventional methods and used.

[0044] The health functional food composition of the present invention may be used as a health functional food, a food additive, or a dietary supplement. When the composition of the present invention is used as a food additive, it may be appropriately used according to conventional methods, such as by adding it as is or by mixing it with other foods or food ingredients.

[0046] The present invention will be explained in more detail below through examples.

[0047] These examples are solely for illustrating the invention, and it will be obvious to those skilled in the art that the scope of the invention is not to be interpreted as being limited by these examples.

[0049] Kale cultivation using green tea extract

[0050] 1-1 : Kale Growth Conditions

[0051] Kale seeds (Manchoo collard, a variety of Brassica oleracea L. var. acephala (DC.) Alef) were purchased from Asia Seed Co. (Korea), and kale was grown using a household vertical farming device (tiiun; LG Electronics, Seoul, Korea).

[0052] First, kale seeds were sown in Rockwool cubes (W × L × H, 25 × 25 × 40 mm, Grodan Co., Netherlands) and placed directly into a household vertical farm. The light / dark cycle was 14:10 h, and the daytime light intensity (08:00–22:00) was set to level 5 out of 5 in the tiiun program, with an actual light intensity of 8,540 ± 320 Lx. The temperature was controlled to 26°C during the day and 18°C ​​during the night. Thinning was performed in the first week, and kale was cultivated for 5 weeks before harvesting. The kale cultivation flowchart is shown in Figure 1.

[0054] 1-2: Preparation of Culture Solution

[0055] The basic nutrient solution for kale cultivation may contain 120 to 150 ppm of nitrogen (N), 45 to 60 ppm of phosphorus (P), 150 to 200 ppm of potassium (K), 50 to 90 ppm of calcium (Ca), 15 to 50 ppm of magnesium (Mg), and 15 to 50 ppm of sulfur (S).

[0056] Specifically, a nutrient solution containing 138.5 ± 0.50 ppm nitrogen, 53.61 ± 0.12 ppm phosphoric acid, 168.87 ± 0.52 ppm potassium, 85.88 ± 0.26 ppm calcium, 19.97 ± 0.10 ppm magnesium, and 33.38 ± 0.02 ppm sulfur, with an electrical conductivity of 1.42 ms / cm and a pH of 6.4, was used as the basic culture medium.

[0057] To prepare green tea extract, 100 g of green tea powder (Dan Nong Won, Korea) was mixed with 1 L of distilled water and extracted twice at 80°C for 2 hours. Then, the extract was filtered using Whatman Grade 1 filter paper (Cytiva, USA) and freeze-dried. The yield of the green tea extract was 20.98%.

[0059] 1-3: Growing Kale

[0060] The basal culture medium was initially supplied to all kale groups for 3 weeks, and the green tea extract treatment groups were supplied with a culture medium containing 0.1 (0.1-GTE), 0.2 (0.2-GTE), 0.3 (0.3-GTE), or 0.5 (0.5-GTE) g / L of green tea extract for 2 weeks prior to harvest. The control group was supplied with the basal culture medium for 2 weeks prior to harvest.

[0062] Verification of yield and mineral content of kale grown with green tea extract

[0063] 2-1: Check Yield

[0064] To determine the effects of green tea extract treatment on the growth and yield of kale, the fresh weight and dry weight of the cultivated kale were measured after harvest.

[0065] The weight of the kale was measured using a digital scale (HS224S; HANSUNG Instrument Co., Korea). The fresh weight of the kale was measured immediately after harvest, and the dry weight was measured after freeze-drying. Additionally, the weight of the kale was determined using the outer skin after removing the underground portion.

[0067] As a result, as shown in Figure 2a, the fresh weight of the kale ranged from 9.07 to 13.29 g per plant, with no significant difference between the groups. This means that home vertical farming devices can effectively grow kale and that treatment with green tea extract does not hinder the growth of the kale or reduce the yield.

[0068] Regarding dry weight, the control group did not show a significant difference from other groups except for the 0.2-GTE group (p < 0.05), and it was confirmed that the 0.2-GTE group had a higher dry weight than the control group. In other words, it was confirmed that there was no change in the growth rate and yield of kale even when 0.1% (w / v) to 0.5% (w / v) of green tea extract was added to the culture medium.

[0070] 2-2: Measurement of Mineral Content

[0071] Potassium is an essential mineral that mediates photosynthesis, protein synthesis, and enzyme activity in plants, while sodium is another essential mineral that maintains balance with potassium and regulates the organism's osmotic pressure. In other words, the amounts of potassium and sodium can regulate plant growth, and their quantities at harvest are important nutrients in the human diet.

[0072] The potassium and sodium content of cultivated kale was analyzed according to a previously known method (Son, Y.-J. et al., Industrial Crops and Products, 163:113313, 2021). Specifically, freeze-dried kale powder (0.05 g) was placed in a beaker and 40 ml of 60% HNO3 solution was added. Then, the solution was boiled at 130°C for more than 2 hours until it became a clear liquid, after which the volume of the solution was adjusted to 50 ml with distilled water. The prepared sample solution was filtered using a 0.45 µm syringe filter, and the mineral content was analyzed using an inductively coupled plasma emission spectrometer (ICP-OES) (OPTIMA 5300 DV; PerkinElmer, USA) at the BT Research Facility Center of Chung-Ang University.

[0074] As a result, as shown in Figure 2b, the potassium and sodium content of the kale samples were 45.90 to 49.91 mg / g and 5.23 to 5.63 mg / g, respectively, on a dry weight basis (DW), and neither showed a significant difference between the groups. In the present invention, the K and Na content of the kale was slightly higher than that of kale grown with a conventional cultivation system, but no change in concentration was observed depending on whether or not green tea extract was applied.

[0076] Observation of chlorophyll content and color of kale grown with green tea extract

[0077] 3-1: Measurement of Chlorophyll Content

[0078] Chlorophyll is a pigment that plays an important role in plant photosynthesis and the production of primary carbon-based energy sources, and chlorophyll content can be used to indirectly estimate the nutritional status of plants.

[0079] The chlorophyll content of the kale samples was measured by slightly modifying a previously known method (Wellburn, AR, J. Plant Physiol., 144(3):307-313, 1994). Specifically, 1 g of freeze-dried kale powder was extracted with 5 ml of 80% acetone at 40°C for 1 hour, and then the absorbance was measured at 663 nm and 646 nm using a spectrophotometer (SpectraMax M2e; Molecular Devices, USA).

[0080] Chlorophyll content was determined using the following mathematical formula 1.

[0081] [Mathematical Formula 1]

[0082] Chlorophyll a concentration = 12.25 × A 663 - 2.79 × A 646

[0083] Chlorophyll b concentration = 21.5 × A 646 - 5.1 × A 663

[0084] ("A" indicates absorbance and the subscript indicates wavelength).

[0086] Chlorophyll content of kale grown with green tea extract Sample Chlorophyll Chlorophyll a Chlorophyll b Chlorophyll (a+b) CON 0.372±0.014 0.271±0.030 0.643±0.044 0.1-GTE 0.378±0.012 0.271±0.025 0.649±0.037 0.2-GTE 0.381±0.014 0.285±0.029 0.666±0.042 0.3-GTE 0.378±0.008 0.269±0.018 0.646±0.026 0.5-GTE 0.377±0.004 0.275±0.009 0.659±0.011

[0087] Table 1 above shows the chlorophyll a and b content and their sum of kale grown with green tea extract, and there was no statistical difference between the groups. Although green tea extract did not change the chlorophyll content of kale, the chlorophyll content of the kale grown in the present invention was much lower than the chlorophyll content of kale grown conventionally.

[0089] 3-2: Measuring Color Values

[0090] The color value of the kale was measured using a colorimeter (CM-36dG; KONICA MINOLTA, Japan). 4 g of freeze-dried kale powder was placed in a Petri dish (35Φ) and the color was determined using the Hunter Lab color system. A D65 light source with an observation angle of 10° was used as the light source.

[0092] Color value of kale grown with green tea extract Sample Color values L (lightness) a (redness) b(yellowness) CON 47.03±1.23 -6.74±0.33 12.81±0.59 0.1-GTE 48.28±0.39 -7.06±0.16 13.47±0.39 0.2-GTE 45.56±1.27 -6.34±0.15 12.16±0.58 0.3-GTE 47.43±0.59 -6.90±0.13 13.09±0.35 0.5-GTE 47.11±1.27 -6.69±0.24 12.98±0.42

[0093] As shown in Table 2 above, regarding color values, 0.1-GTE showed the highest L (brightness) and b (yellow) values ​​and the lowest a (red) value, while 0.2-GTE showed the opposite trend to 0.1-GTE. However, no changes were observed with green tea extract treatment.

[0095] Confirmation of texture and sensory characteristics of kale cultivated with green tea extract

[0096] 4-1: Measuring Kale Texture

[0097] The mechanical texture of fresh kale samples was verified using a TA-XT ExpressC texture analyzer (Stable Micro Systems, UK), and punching (leaf) and cutting (petiole) test methods were applied because the texture characteristics of leaves and petioles are different.

[0098] First, the leaf portion of the kale was inspected by a punching test using a P / 20 cylinder probe. The compression mode was set to a pre-test speed of 5 mm / s, a test speed of 1 mm / s, and a post-test speed of 10 mm / s.

[0099] The texture characteristics of the leaf stalks were analyzed through cutting tests using the HDP / BS Knife-Edge probe. The compression modes used were a pre-test speed of 5 mm / s, a test speed of 1 mm / s, and a post-test speed of 10 mm / s. For both tests, the trigger force was set to 20 g.

[0100] Mechanical texture characteristics of kale Sample Texture Cutting (g) Punching (g) CON 319.37±96.46 59.98±12.45 0.1-GTE 384.07±66.38 52.96±8.83 0.2-GTE 503.62±156.90 50.03±5.78 0.3-GTE 366.63±53.34 52.86±6.05 0.5-GTE 320.52±79.44 52.36±4.01

[0102] As shown in Table 3 above, the hardness of the kale leaves ranged from 50.03 to 59.98 g, and no significant differences were observed between the groups. For the petiole, the 0.2-GTE showed the hardest texture at 0.50 ± 0.16 kg, but no distinct trend was observed between hardness and green tea extract concentration.

[0104] 4-2: Sensory Evaluation

[0105] The sensory evaluation of kale was validated using quantitative descriptive analysis (QDA). Eight trained panelists evaluated the bitterness, astringency, and grassiness of the kale samples using a 15 cm line scale. The panelists were recruited from Chung-Ang University, consisting of four women and four men. The sensory evaluation was conducted in compliance with the ethical guidelines of the Institutional Review Board (IRB), and the entire study design was approved by the Chung-Ang University IRB Committee (1041078-202210-HR-246).

[0107] Sensory evaluation of kale Sample Sensory evaluation Bitterness Astringency Grassy flavor CON 8.65±3.12 5.63±2.68 8.74±1.44 0.1-GTE 7.66±3.66 4.60±2.45 7.96±2.83 0.2-GTE 8.46±2.53 8.56±2.67 8.08±3.76 0.3-GTE 7.80±2.75 7.63±2.20 6.94±2.93 0.5-GTE 8.11±3.54 5.51±3.01 6.65±2.86

[0108] As shown in Table 4 above, no significant difference was observed regarding bitterness and grassiness, and only 0.2-GTE showed a higher convergence score than 0.1-GTE.

[0109] The results of the present invention confirmed that kale quality, such as yield, mineral content, chlorophyll content, color, texture, and sensory characteristics, was not reduced even when treated with green tea extract during kale cultivation.

[0111] Analysis of phenolic compounds in kale grown with green tea extract

[0112] Freeze-dried kale powder (0.25 g) was mixed with 80% ethanol (5 ml) and extracted by sonication at 40°C for 40 minutes (JAC-5020; Kodo Co., Hwaseong, Korea). After centrifugation, the supernatant was collected, and the remaining residue was re-extracted twice using 80% ethanol. The collected supernatant was then filtered through a 0.2 µm syringe filter and evaporated using a vacuum concentrator (SPD 2030; Thermo Fisher Scientific, USA) to prepare an 80% ethanol kale extract.

[0113] The content of each phenolic compound in the above 80% ethanol kale extract was analyzed using reverse-phase high-performance liquid chromatography (HPLC) (Agilent infinity series 1260; Agilent Technologies, USA) equipped with a C18 ODS-AQ column (4.6 × 150 mm, 5 µm; YMC, USA) with reference to a previously known method (Wang, X. et al., Ultrason. Sonochem., 20(3):846-854, 2013). The column temperature was 40°C and the mobile phase conditions were as follows: water containing 0.1% formic acid, solvent A; acetonitrile containing 0.1% formic acid, B; 0 min, 95:5; 10 min, 90:10; 35 min, 87:13; 45 min, 85:15; 50 min, 82:18; 60 min, 81:19; 70 min, 78:22; 75-76 min, 20:80; 80-85 min, 95:5 (A:B). The flow rate was 1 ml / min and the injection volume was 10 µl. The absorbance spectrum was measured at 254 nm, and each peak was identified using an analytical standard for phenol compounds.

[0115] Catechin component contained in green tea extract Green tea extract Contents (mg / g dry basis) Gallic acid 0.06 (-)-EGC 2.69 (+)-Catechin 0.56 (-)-EC 0.68 (-)-EGCG 0.86 (-)-ECG 0.56 Caffeine 3.28

[0117] As shown in Table 5 above, it was confirmed that green tea extract contains various catechin components such as gallic acid, (+)-catechin, (-)-epigallocatechin (EGC), (-)-epicatechin (EC), (-)-epigallocatechin gallate (EGCG), and (-)-epicatechin gallate (ECG).

[0118] As a result of analyzing the phenolic compounds contained in kale grown with green tea extract, as shown in Figure 3a, the chlorogenic acid content increased in the group treated with green tea extract at a concentration of 0.2% (w / v) or higher, but no significant results were observed. On the other hand, the p-coumaric acid content was found to have significantly increased in the group treated with 0.5% (w / v) green tea extract.

[0119] In addition, as shown in Figure 3b, EC and EGCG were not detected in the kale control group, but kale grown with green tea extract contained EC at 160.81 to 252.11 μg / g DW and EGCG at 64.97 to 173.26 μg / g DW.

[0120] These results suggest that EC and EGCG contained in green tea extract were transported to kale, implying that EC and EGCG may accumulate due to treatment with green tea extract during kale cultivation.

[0122] Phenolic compounds are secondary plant metabolites that are abundant in plant-based foods and have various biological activities. Long-term consumption of phenol-rich foods can help prevent or treat diabetes, cancer, and cardiovascular diseases.

[0123] Catechin-rich kale grown by the method of the present invention can be more effectively applied to inflammatory diseases, glucose and lipid metabolism disorders, coronary artery disease, and degenerative diseases.

[0125] As shown in Table 5 above, green tea extract generally contains five major catechin compounds, but only two types of catechins (EC and EGCG) were detected in kale grown with green tea extract. This means that EC and EGCG have excellent cell membrane penetration capabilities and were efficiently absorbed in kale.

[0127] Confirmation of glucosinolate content in kale grown with green tea extract

[0128] Glucosinolates are sulfur-rich anionic secondary metabolites primarily found in cruciferous plants, including kale. When ingested, glucosinolates are hydrolyzed into various biologically active forms, such as isothiocyanates and indole-3-carbinol, which can stimulate the activity of detoxification enzymes in the human body. Glucosinolates also possess anti-inflammatory and antioxidant activities and are known to have potent anticancer effects.

[0130] The glucosinolate content of kale was analyzed according to known methods (Kim, SY et al., Sci. Rep., 8(1):3999, 2018). Briefly, 0.1 g of freeze-dried kale powder was mixed with 2 ml of 70% methanol and boiled at 95°C for 10 minutes, then the solvent was immediately cooled on ice and centrifuged at 2,000 rpm at 4°C for 15 minutes. The supernatant was collected in a tube and the residue was extracted again, then Benzyl-GLS (1 mM, 0.1 ml) was added to the combined supernatant, and the total volume of the solution was adjusted to 4 ml using 70% methanol.

[0131] To precipitate the protein, 1.5 mL of the solution was transferred to a 2 mL microcentrifuge tube (MCT-200-C; Axygen, USA), and 0.15 mL of a mixture of 1 M lead acetate and 1 M barium acetate (1:1) was added. Next, the solution was centrifuged at 12,000 rpm for 5 minutes, and 1 mL of the supernatant was loaded onto a mini-prep column filled with diethyl-aminoethyl (DEAE) Sephadex A-25 anion exchange resin pre-activated with 0.1 M sodium acetate buffer. After adding 0.2 mL of 0.1% purified arylsulphatase, the column was covered and incubated at room temperature for 18 hours. Desulfo-glucosinolate was eluted twice with 0.5 ml of distilled water and filtered through a 0.2 µm syringe filter.

[0132] The prepared samples were analyzed using a reversed-phase HPLC (Agilent Technologies, USA) equipped with a C18 ODS-AQ column (YMC, USA), and the mobile phase gradient conditions were as follows: water, solvent A; acetonitrile, solvent B; 0 min, 99.5:0.5; 7 min, 98.5:1.5; 15 min, 90:10; 25 min, 80:20; 35-39 min, 70:30; 41-45 min, 99.5:0.5 (A:B). The flow rate was 1 ml / min, the column temperature was 35 ℃, and the injection volume was 20 µl.

[0133] The spectrum of glucosinolates was measured at 227 nm, and each peak was identified using an analytical glucosinolate standard. The glucosinolate content of kale was quantified by comparing the peak area with the peak area of ​​the internal standard, benzyl-glucosinolate.

[0135] As a result, as shown in Figure 4, five types of glucosinolates were detected in kale, including sinigrin, gluconapin, glucobrassicin, gluconasturtiin, and neoglucobrassicin. Of the five glucosinolates, two (sinigrin and gluconapin) were aliphatic, two (glucobrassicin and neoglucobrassicin) were indole, and only neoglucobrassicin was an aromatic glucosinolate.

[0136] The most abundant glucosinolate compound in the control group was gluconafine (4.80 ± 0.58 μmol / g DW), followed by sinigrin, gluconasturtin, glucobrassicin, and neoglucobrassicin.

[0137] Kale grown with green tea extract showed a significant increase in sinigrin content, and it was found that the 0.5-GTE group was about four times higher than the control group. Glucobrassicin also increased significantly in the green tea extract-grown group. Consequently, the total glucosinolate content in the 0.5-GTE group was found to be very high at 16.81 ± 2.38 μmol / g DW, which was nearly double that of the control group (9.56 ± 0.90 μmol / g DW).

[0138] That is, it was confirmed that the catechin content in kale grown by the method of the present invention increased, and the content of glucosinolate, a unique physiologically active compound of kale, was enhanced.

[0140] Heatmap Analysis and PCA Results of Kale Grown with Green Tea Extract

[0141] In the present invention, the synthesis of glucosinolates in kale was successfully increased by applying a green tea extract treatment technology to a home-use vertical farming device, and in particular, the content of aliphatic glucosinolates was significantly increased by green tea treatment. Therefore, it was confirmed whether green tea extract treatment promotes the expression of a series of metabolic pathways related to the biosynthesis of aliphatic glucosinolates.

[0142] Heatmap analysis and PCA were performed to analyze and visualize the relationship between variables and sample groups, and the heatmap was visualized using IBM SPSS Statistics version 28 (IBM, USA).

[0143] As shown in Figures 5 and 6, PC1 and PC2 showed low explained variance ratios (39.1% and 30.1%, respectively) in the PCA results, which can be attributed to the fact that the variables that did not change due to green tea extract treatment were mainly related to food quality factors such as yield, color, texture, and sensory characteristics.

[0144] Differences in overall characteristics between the control and 0.5-GTE groups were prominent in the PCA plots and heatmaps, and among the treatment groups, 0.3-GTE and 0.5-GTE showed the most distinct characteristics compared to the control group in terms of EC, EGCG, caffeine, sinigrin, and total glucosinolate content. Therefore, the use of green tea extract in kale cultivation was found to enhance some secondary metabolites, and it can be usefully applied to produce customized kale with enhanced health benefits.

Claims

Claim 1 A method for cultivating kale with increased antioxidant content, comprising the step of culturing by treating a culture solution containing green tea extract for 5 to 15 days prior to plant harvest, wherein the antioxidant component is one or more selected from the group consisting of glucosinolate, P-coumaric acid, epicatechin, and epigallocatechin gallate (EGCG). Claim 2 A method for cultivating kale with increased antioxidant content, characterized in that, in claim 1, the culture solution containing the green tea extract is supplied separately during the kale cultivation process as a nutrient solution for hydroponic cultivation. Claim 3 A method for cultivating kale with increased antioxidant content, characterized in that, in claim 1, the green tea extract is included in the culture medium at a concentration of 0.1 (w / v) to 1.0% (w / v). Claim 4 A method for cultivating kale with increased antioxidant content, characterized in that, in claim 1, the green tea extract is prepared by hot water extraction at a temperature of 50 to 90°C for 60 to 150 minutes. Claim 5 delete Claim 6 A method for cultivating kale with increased antioxidant content, wherein, in claim 1, the glucosinolate is one or more selected from the group consisting of sinigrin, gluconapin, glucobrassicin, gluconasturtiin, and neoglucobrassicin. Claim 7 delete

Images