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Sulforaphane, allyl isothiocyanate, HPLC-grade methanol and acetonitrile were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Bakerbond SPE silica gel (SiOH) 3 mL disposable columns were from J.T. Baker (Phillipsburg, NJ, USA). Dichoromethane, ethylacetate, methanol, methanol, and ammonium acetate were obtained from Merck (Darmstadt, Germany), unless mentioned otherwise.

Brussels sprouts were purchased from local market. They were washed with water and dried with paper towels. The impact on the levels of SFN and AITC in brussels sprouts after steaming and ultrasonication relative to untreated brussels sprouts were studied. For steaming, after adding 1 L of distilled water to the electric steamer (HD-9132, Philips Co, Seoul, Korea) and heated it until the internal temperature reached 100℃. Then, 1 kg of brussels sprouts was placed into the steamer for the heat treatment. The heat treatment was performed for various durations, ranging from 1 to 10 min. At one-minute intervals, 100 g portions of the brussels sprouts were removed and the content SFN and AITC were determined immediately after steaming treatment. For ultrasound treatment, after washing 1 kg of brussels sprouts with distilled water, it immersed them in an ultrasound equipment (powersonic 440, Hwashin Co, Seoul, Korea) containing 35 L of distilled water. The ultrasound equipment operated at a frequency of 40 kHz, while the temperature of the chamber was set to 40℃. The brussels sprouts underwent ultrasound treatment at different intensities (200 w, 400 w, 600 w, 800 w, or 1,000 w) and for varying durations (10, 30, or 60 min). To serve as a control group, another 1 kg of brussels sprouts was placed in a thermostatic water bath at 40℃ and treated for the same durations (10, 30, or 60 min). The content SFN and AITC were determined immediately after ultrasound treatment.

Samples were weighed out in a beaker of 50 mL (1 g fresh sample), then 4 mL of acidic water (pH 6) was added, and the mixture was incubated at 45±2℃ in water bath for 2.5 h. At this step, the evaporation of the water was observed to have decreased the extract by about 70% from the initial weight of the mixture (sample+acidic water). Sulforaphane was extracted with 20 mL of dichloromethane by vortexing for 1 min and stored at room temperature during 1 h. Then, the resulting solution was filtered through Whatman no. 41 paper. The sulforaphane was purified with the method designed by Bertelli D et al (1998), using Bakerbond SPE silica gel (SiOH) 3 mL disposable columns. Prior to use, the silica gel cartridge was conditioned with 3 mL of dichoromethane. Sulforaphane was extracted by passing 20 mL of organic extract through the cartridge, washing the cartridge with 3 mL of ethylacetate (which was then discarded) and eluting the sulforaphane with 3 mL of methanol. The methanol extract was evaporated to dryness in a vacuum oven at 45℃ for 2 h, and redissolved with 2 mL of acetonitrile. The resulting solution was vortexed for 30 s and filtered with a membrane of 0.45 μm. A 20 μL sample of this solution was injected onto the column of the HPLC system (Agilent 1260 Infinity Quaternary liquid chromatograph, Hewlett Packard, Wilmington, NC, USA). All samples were analyzed five times.

Analyses of SFN were performed using an Agilent 1260 infinity quaternary liquid chromatograph (Hewlett Packard, Wilmington, NC, USA) equipped with a quaternary gradient pump and multiple wavelength detector operating at 202 nm. The samples were separated on a Zorbax Eclipse XDB-C18 column (4.6 mm × 150 mm, 5 μm; Agilent Technologies, Inc., Santa Clara, CA, USA) at 36℃ with a sample injection volume of 10 μL. The analysis were carried out isocratically at a flow rate of 0.6 mL/min, employing as the mobile phase a mixture of acetonitrile : ultrapure water (30 : 70, v/v). Data analysis was performed using Chemstation software (Hewlett Packard, Wilmington, NC, USA).

The mixtures were homogenized on an orbital shaker (VWR Mini Shaker, VWR, Darmstadt, Germany) at 450 rpm for 10 min and then sonicated for 30 min. The mixtures were subsequently centrifuged at 20,000 rpm at 7℃ for 10 min. A 2 mL syringe was used to remove the supernatant and the extract was then filtered using a 0.45 µm RC membrane filter (Phenomenex, Aschaffenburg, Germany). Five replicates of each sample were analyzed.

Analyses of AITC were performed using an Agilent 1260 infinity quaternary liquid chromatograph (Hewlett Packard, Wilmington, NC, USA) with a multiple wavelength detector (MWD) operating at 242 nm. Chromatographic separations were performed on a Supelcosil LC-18 RP column (4.6 × 150 mm, 5 µm, Sigma-Aldrich, St. Louis, MO, USA) using methanol and 0.1 N ammonium acetate (NH₄OAc) (3:97; v/v) as the mobile phase. The column temperature was 30℃, and the flow rate was 1 mL/ min. Data analysis was performed using Chemstation software (Hewlett Packard, Wilmington, NC, USA).

The experimental data were subjected to analysis of variance (ANOVA). Significant differences between the mean values, as determined from measurements carried out in five replicate tests (i.e., p<0.05), were obtained by Duncan’s multiple-range test using statistical analysis software (SPSS 20.0, IBM Inc., NY, USA).

Linear regression analysis was conducted to assess the linearity of the obtained data. The response of SFN and AITC exhibited linearity within the concentration range of 2—10 mg/L. The linear regression equations for SFN and AITC were determined as y=7.5392x—0.2858 with an r² value of 0.9991 and y=2.012x—0.06 with an r² value of 0.9964, respectively. The calibration plot is illustrated in Fig. 1, and the chromatogram is presented in Fig. 2. The coefficient of determination's acceptance value should not fall below 0.99, and the determined values, ranging from 0.993 to 0.9998, satisfied this criterion. The detection limits for SFN and AITC were both established at 0.05 mg/L.

Fig. 1. 
Calibration curve measured at 202 nm with r²=0.0991 for SFN (A) and 242 nm with r²=0.0964 for AITC (B).

Fig. 2. 
HPLC chromatogram of SFN (solid line) and AITC (dotted line) standards at 2—10 mg/L concentration.

The effect of steaming treatment on SFN content from brussels sprouts was analyzed was studied for different processing times, ranging from 1 to 10 min (Fig. 3). The initial content of SFN in unprocessed samples was found to be 741.09±5.89 µg/100 g FW. The content of SFN increased up to 3 min of steaming time, but decreased thereafter, reaching a level of 946.85±10.76 µg/100 g FW at 10 min. However, this content still showed a significantly (p<0.05) higher amount compared to the raw sample. Moreover, the SFN content exhibited the lowest value of 894.65±1.32 µg/100 g FW at 1 min, and the highest value of 1,095.57±7.24 µg/100 g FW at 3 min of steaming time. The rise in sulforaphane content can be attributed to the application of high temperatures, which likely led to a considerable reduction in myrosinase activity. However, the remaining glucoraphanin can still be converted into sulforaphane due to the enzymatic activity of the microbiota (Fahey JW et al 2012). Remarkably, sulforaphane demonstrated greater stability than myrosinase (Wu Y et al 2018), allowing it to accumulate despite ongoing myrosinase hydrolysis and limited thermal degradation. As the cooking time increased, the content of glucoraphanin experienced a notable decrease, and elevated temperatures accelerated the generation of specific volatile compounds from sulforaphane (Jin Y et al 1999). This series of events led to the decline in sulforaphane content. Notably, the sulforaphane content reached its peak at the 3 min of the steaming process (Fig. 3). Therefore, this finding can play a crucial role in determining the optimal cooking time to retain a high level of sulforaphane.

Fig. 3. 
The change of SFN content from brussels sprouts according to the steaming treatment.

Data represent the mean values for each sample±standard deviation (n=5).

^a∼h Means with different superscripts on the graph are significantly different (p<0.05).

The effect of ultrasound treatment on the formation of sulforaphane in brussels sprouts was examined at different intensities of ultrasound treatment ranging from 200 to 1,000 w and three different processing durations (10, 30, or 60 min) (Fig. 4). The content of sulforaphane exhibited an increasing trend followed by a decrease in response to the increase of ultrasonic power intensity. The high content of SFN was observed at ultrasonic power intensities of 200 w (1,108.43—1,312.78 µg/100 g FW) and 400 w (1,166.00—1,274.48 µg/100 g FW), while it decreased for power intensities exceeding 600 w. Notably, even in comparison to both the raw and control groups, the content of sulforaphane remained notably higher. Additionally, in all treatment groups of different power intensities, the content of sulforaphane increased as processing time extended. The treatment group with the highest sulforaphane content was observed at an ultrasonic power intensity of 200 w and a processing time of 60 min (1,312.78±14.02 µg/100 g FW). However, no significant difference was found between this group and the sample treated with an ultrasonic power intensity of 400 w and a processing time of 60 min (1,274.48±17.08 µg/100 g FW). The application of ultrasound processing in brussels sprouts has been relatively underexplored until now. Briones-Labarca V et al (2015) found that ultrasound-assisted extraction led to a 5-fold increase in SFN recovery from Chilean papaya seeds in a methanol solution. Pongmalai P et al (2017) noted that ultrasound-assisted extraction significantly improved glucoraphanin extractability (by 1.8-fold) from steamed cabbage leaves compared to fresh cabbage when using methanol as a solvent. Addition, ultrasound treatment as a mild heating treatment might induce more cell lysis, resulting in the diffusion of greater amounts of glucosinolates and myrosinase, thus promoted the formation of sulforaphane (Nugrahedi PY et al 2015). In the present study, ultrasound treatment significantly improved sulforaphane content in brussels sprouts compared with the control (Fig. 4). Besides, Olivero T et al (2014) reported that ultrasound processing lowers the activation energy required for myrosinase inactivation. This could represent significant energy savings in an industrial process, constituting a more efficient blanching process in comparison with traditional blanching. Myrosinase inactivation may be useful to preserve glucoraphanin in the vegetable tissue if the objective is extracting the precursor of SFN, glucoraphanin, to conduct the hydrolysis exogenously under controlled conditions. This will prevent the formation of undesirable compounds such as nitriles and isothionitriles which compete with SFN formation, thus maximizing the conversion of glucoraphanin in SFN. Therefore, ultrasonic treatment not only affects the inactivation of myrosinase, leading to increased sulforaphane content in brussels sprouts, but also offers the potential to achieve higher levels than by thermal processing, thus reducing the temperature necessaries to achieve the desired denaturation.

Fig. 4. 
The change of SFN content from brussels sprouts according to the ultrasound treatment.

Data represent the mean values for each sample±standard deviation (n=5).

^a∼d Different small superscripts indicate significant differences according to the samples in the same treatment time at p<0.05 level.

The effect of steaming treatment on AITC content from brussels sprouts was analyzed was studied for different processing times, ranging from 1 to 10 min (Fig. 5). The initial AITC content in the raw sample was found to be 279.80±2.13 µg/100 g FW. As the steaming progressed, The AITC content increased up to 3 min of steaming time (466.31±3.34 µg/100 g FW). Subsequently, there was a decrease in AITC content, while the level remained consistent from 4 to 7 min of steaming time. However, AITC was not detectable after 8 min of steaming time. AITC is a compound found in plants, stored in a stable form as its precursor called sinigrin (allyl glucosinolate or 2-propenyl glucosinolate), which belongs to the aliphatic glucosinolate category. It’s notably abundant in vegetables like brussels sprouts, broccoli, and mustard seeds derived from Brassica nigra. These plants owe their characteristic pungency to AITC (Ishida M et al 2014). Myrosinase, an enzyme, acts on sinigrin by breaking down its glucose component, forming an intermediate aglycone. This intermediate structure is inherently unstable and rearranges itself spontaneously to yield AITC (Krul C et al 2002). The isothiocyanates formed through glucosinolate hydrolysis are unstable and can easily transform into thiocyanate ions and indole3-carbinol. Moreover, steaming has the potential to preserve some level of myrosinase activity, with myrosinase remaining active for up to 2 min of heating, but showing a significant 90% decline after 7 min (Fan Y et al 2016). Opting for shorter steaming durations could result in a greater formation of ITCs compared to nitriles. This effect is due to the inactivation of the epithiospecifier protein, which occurs at a lower temperature than the inactivation of myrosinase, as reported in the case of broccoli (Green DR & Llambi F 2015). Therefore, the complete impact of vegetables containing glucosinolates can be accurately evaluated by specifically examining how different cooking conditions affect both the glucosinolate content and the activity of myrosinase.

Fig. 5. 
The change of AITC content from brussels sprouts according to the steaming treatment.

Data represent the mean values for each sample±standard deviation (n=5).

^a∼d Means with different superscripts on the graph are significantly different (p<0.05).

The effect of ultrasound treatment on the formation of AITC in brussels sprouts was examined at different intensities of ultrasound treatment ranging from 200 to 1,000 w and three different processing durations (10, 30, or 60 min) (Fig. 6). The initial AITC content measured 279.80±2.13 µg/100 g FW in the raw sample, while the control group exhibited levels ranging from 375.63±12.13 µg/100 g FW to 416.32±5.23 µg/100 g FW, dependent on processing time. Within the ultrasonic treatment groups, the AITC content varied with ultrasonic power intensity. It demonstrated an ascending pattern until reaching an ultrasonic intensity of 400 w, after which it declined beyond 600 w. The treatment condition yielding the highest content was identified in the 200 w, 60 min treatment group, showing a content of 595.74±14.79 µg/100 g FW. Furthermore, the AITC content increased in all treatment groups with increasing ultrasonic treatment time. As ultrasound treatment induces cell membrane disruption (Nowacka M & Wedzik M 2016), it was expected that higher levels of AITC would result from ultrasound application due to the decompartmentalization of enzymes (myrosinase) and substrates (glucosinolates) from cell organelles, allowing their interaction. However, in this study, no significant difference was observed in the AITC content according to the ultrasonic power intensity. These findings suggest that myrosinase released from ultrasound-treated brussels sprouts was partially inactivated by the applied ultrasound conditions. It is established that ultrasound can deactivate enzymes by modifying the protein’s chemical structure, with its efficacy contingent upon various factors such as ultrasound power, temperature, time, frequency, and pH (Islam MN et al 2014). Therefore, it is possible that the ultrasound treatment applied in this study partially inactivated myrosinase, thereby impeding the hydrolysis of glucosinolates.

Fig. 6. 
The change of AITC content from brussels sprouts according to the ultrasound treatment.

Data represent the mean values for each sample±standard deviation (n=5).

^a∼f Different small superscripts indicate significant differences according to the samples in the same treatment time at p<0.05 level.