Sulforaphane content enhancement of red cabbage microgreens by using different planting media and nutrition solution

Red cabbage contains sulforaphane that have many health benefit. The highest sulforaphane content is found in young plant, known as microgreens. Media and natural plant growth regulator as nutrient solutions adjustments can be used to raise the sulforaphane content of microgreens. This study aims to investigated the best planting media and natural plant growth regulator as nutrient solution to enhanced the sulforaphane content in red cabbage microgreens. Completely randomized design (CRD) with two factors (nutrition solution and planting media) is used for the research. Research result was analyzed by ANOVA followed by Duncan Multiple Range Test. Planting media and nutrition solutions had significant effects on the height, fresh weight, and the content of sulforaphane in microgreens. The combination of vermiculite as planting media and young coconut water as nutrient solution could increase the growth of microgreens, furthermore the used of cocopeat in combination with young coconut water may raise the sulforaphane content of microgreens. The amount of sulforaphane increased by two-fold with the used of cocopeat and young coconut water.


Introduction
Red cabbage is commonly known as a vegetable that contains high levels of vitamins and minerals [1,2] that are good for human health.Moreover, these cabbages also contain antioxidants, such as sulforaphane.According to epidemiological research, these substances may aid in defending the body against free radicals [3,4].Sulforaphane plays an anti-cancer role [5,6] and also an anti-arthritis role [7].
The cultivation of microgreens needs the proper planting media and nutrition.The appropriate media and nutrition are the key factors to produce high production and improve the sulforaphane compound in microgreens.Microgreens are typically given liquid and natural (organic) nutrients.Young coconut water is one of organic nutrition sources that can be used as a nutrient solution.Young coconut water contains amino acids [19] such as methionine.Methionine is a precursor for the formation of 1302 (2024) 012016 IOP Publishing doi:10.1088/1755-1315/1302/1/012016 2 sulforaphane.The addition of methionine as a precursor to planting media can increase the sulforaphane production.[20] Sulforaphane is formed from glucoraphanin in cruciferous vegetables, and requires chovamyrosinase enzyme to convert it to sulforaphane.
The planting media serves as a container for the microgreens' nutrient solution and water.Microgreens can be grown in a variety of media or substrate, including tissue paper, rockwool, cocopeat, and vermiculite.The planting media chosen will affect the chemical composition and antioxidant activities of microgreens [19].Media selection has a significant effect on yield, dry weight percentage and nitrate concentration in microgreens [21].
The modification of planting media for red cabbage microgreens has been carried out previously [22], but no evaluation has been carried out on the status of the microgreen content contained in them.The combination between planting media and natural plant growth regulator be expected could increase more sulforaphane content on red cabbage microgreens.This study aims to investigated the best planting media and natural plant growth regulator as nutrient solution to enhanced the sulforaphane content in red cabbage microgreens.

Materials
Planting microgreens is done inside laboratory room in January 2022.The materials used were red cabbage seeds, rockwool, cocopeat, tissue paper, vermiculite, mineral water, ethyl acetate, phosphate buffer pH 7.0 solution and young coconut water (100 ml/planting container).Chemicals used for testing the sulforaphane content were acetonitrile/hydrogen dioxide (30:70), and solution containing a standardized amount of sulforaphane (2 mg sulforaphane per milliliters acetonitrile).The instrument used were a plastic container, cutter, spray bottle (sprayer), centrifuge, mortar, scissors, ruler, test tube, Erlenmeyer tube, aluminum foil, hot plate, rotary evaporator and HPLC unit equipment.
The study was structured using a Double-Factor Completely Randomized Design (CRD).The initial factor pertains to the planting media, encompassing rockwool, tissue paper, cocopeat, and vermiculite.The subsequent factor, the nutrition solution, comprises mineral water and young coconut water.There are eight treatment combinations with three replications, so that in this study we obtained 24 units.Each experimental unit was planted with 2 g red cabbage seeds in the same container.

Planting microgreens
Media was prepared based on each treatment.These media were put into planting containers.The planting containers that used in this mica plastic (16 x 12 x 5.5 cm).For cocopeat, vermiculite and rockwool, there were put into planting container 4 cm thickness.For tissue paper media, it was used 10 sheets of tissue paper.Red cabbage seeds were sown in the amount of 2 g on each container.During seeding, microgreens were watered using hand sprayer according to the treatment (mineral water, young coconut water) every day with a volume of 100 ml per planting container.Red cabbage seeds were sown on media for 13 days and harvested in 14 days after planting.

Observation
In this research, fresh weight, sulforaphane content, and microgreen height were the variables that were observed.The height of microgreen was measured just before harvesting in five plant per container randomly.Furthermore, the fresh weight was measured just after harvesting by totaling the fresh weight of the plants per container.
Analysis of sulforaphane levels was carried out by using a sample of 200 g of fresh microgreens in each experimental unit.Sample preparation and extraction of red cabbage microgreens was carried out based on [23] method.Sample freshly ground and homogenized with buffer phosphate 0.1 mol (pH 7) in a ratio of 1:4 (g/ml).After being mixed for two hours in a 100 ml beaker with 15 ml of homogenate solution, thirty milliliters of ethyl acetate were introduced, and the mixture underwent a 30-minute extraction process.Subsequently, centrifugation was performed for ten minutes.After gathering the supernatant, the procedure was carried out three times.The supernatant from three replicates is evaporated at 35 °C through a rotary evaporator under low pressure.After being dissolved and added back to methanol, the residue is filtered using a 0.22 µm nylon membrane filter and stored at -20°C for subsequent analysis.
2 mg of sulforaphane were dissolved in 1 ml of acetonitrile (2000 ppm) to create a standard stock solution and kept at 4 o C in dark conditions.Reference standard purity is ≥ 90% (HPLC) namely DL-Sulforaphane.Implementation of analysis with using an HPLC tool is carried out based on method implemented by [24].The analysis is performed at a flow rate of 0.6 ml/min, utilizing a mobile phase comprising a mixture of acetonitrile and ultrapure water (30:70, v/v).Thermostat column at 36 o C. Sulforaphane was identified at a wavelength of 202 nm with a total time between injections of 15 minute.The injection volume was 20 µl and repeated 3 injections with the column used is C18 as the stationary phase.The data research findings were subjected to analysis using Analysis of Variance (ANOVA).Significantly different data was examined through Duncan's multiple range test.SAS software was employed for the data analysis.

Result and Discussion
The yield, quality and sustainability of the cultivating process of microgreens are significantly impacted by growing media.Related to this, the nutrition application also plays to increase the growth and phytochemical content of microgreens.The used of cocopeat in combination with young coconut water and also the used of vermiculite combined with young coconut water on red cabbage cultivation could gave the best response on microgreens height compared with other treatments (Table 1).Cocopeat and vermiculite are the substrate that have their own excellence.[26] cocopeat contain 0.49% N and 44.25% C, whereas vermiculite contain 0.31% and 41.31% C. Cocopeat could increase water use efficiency and nutrient use efficiency [27,28].Vermiculite has a relatively high-water holding capacity, which makes it an excellent material for retaining moisture in soilless growing media.[26] the use of vermiculite is directly increasing the water holding capacity.Cocopeat and vermiculite can hold well the nutrient solution given to microgreens.[25] explain that young coconut water contains inorganic ions (calcium, iron, magnesium, etc.), Vitamins (C and B2), Amino acids (Alanine, Arginine, Aspartic acid, etc.).These various elements are beneficial for promoting plant growth.In line with [27], the optimum response to the microgreens' height was obtained when vermiculite was used as the planting medium for Pak choy microgreens.Fresh weight is the main factor in microgreens industry.For economic reason, fresh weight is very important.Based on this research, vermiculite as a planting medium that combined with coconut water as a source of nutrients yields the optimum microgreens fresh weight (Table 2).With the high-water holding capacity [26] and good aeration [21], vermiculite is able to keep the young coconut water that watering to the microgreens.The amount of coconut water in microgreens might increase the fresh weight.[29] providing additional nutrition to microgreens can increase the fresh weight of microgreens.The findings indicated that the interaction of coconut water as a nutrition solution and cocopeat as a growing medium might increase the amount of sulforaphane in red cabbage microgreens (Table 3).There is 988.69 ppm or 11.597% sulforaphane content in 8,667 ppm of red cabbage microgreen extract.There was a two-fold increase in sulforaphane content compared to plants that were not watered with young coconut water.As reported in previously [13,30], the application of cocopeat and young coconut water gave the highest sulforaphane rate in microgreens.Microgreens can be nourished by coconut water that can be bound by cocopeat.Red cabbage microgreens' sulforaphane level may rise due to the abundance of amino acids, including methionine, found in young coconut water [31].The building block for the synthesis of sulforaphane is methionine.

Conclusion
The used of cocopeat as planting media in combination with young coconut water as nutrition solution could enhance the amount of sulforaphane in red cabbage microgreens.There was a two-fold increase in sulforaphane content when using cocopeat with young coconut water in red cabbage microgreens.

Table 1 .
The height (cm) of red cabbage microgreens Note: As per Duncan's Multiple Range Test (5%), values sharing the same letter in both the column and row denote a lack of significant difference.

Table 2 .
The fresh weight (gr) of red cabbage microgreens Note: As per Duncan's Multiple Range Test (5%), values sharing the same letter in both the column and row denote a lack of significant difference.