Characterization of glucosinolates in 80 broccoli genotypes and different organs using UHPLC-Triple-TOF-MS method.

We aimed to characterize and quantify glucosinolate compounds and contents in broccoli, and a total of 80 genotypes and eight developmental organs were analyzed with UHPLC-Triple-TOF-MS. The method was validated in terms of performance, and the coefficients of determination (R2) were 0.97 and 0.99 for glucoraphanin and gluconapin, respectively. In 80 genotypes, twelve glucosinolates were found in broccoli florets ranging from 0.467 to 57.156 µmol/g DW, with the highest glucosinolate content being approximately 122-fold higher than the lowest value. The principal component of glucobrassicin, neoglucobrassicin and glucoraphanin explained 60.53% of the total variance. There were positive correlations among hydroxyglucobrassicin, methoxyglucobrassicin, glucobrassicin, glucoerucin, gluconasturtiin, glucoraphanin, and glucotropaeolin (P < 0.05). The root contained 43% of total glucosinolates in 80 genotypes, and glucoraphanin represented 29% of the total glucosinolate content in different organs. The mutant broccoli genotypes were found by analysis of gluconapin contents in different organs.

Blanching impact on pigments, glucosinolates, and phenolics of dehydrated broccoli by-products.

Because of high water content, the valorisation of broccoli by-products requires dehydration that can preserve bioactive compounds. Blanching pre-treatment has been reported to improve the drying rate of broccoli. As a thermal treatment, it promotes also enzyme inactivation. Therefore, in this study, the impact of pre-dehydration blanching step, freeze-drying, air-drying at 40 °C, and microwave hydrodiffusion and gravity (MHG) dehydration on the levels of pigments, glucosinolates, and phenolics, was evaluated by UHPLC-DAD-ESI/MSn. When compared to freeze-drying, a technique known to preserve compounds, a pre-blanching step increased the extractability of both pigments and phenolics, while air-drying only retained 49% of the pigments and 70% of phenolics, both without affecting glucosinolates. However, when air-drying was preceded by blanching, less than 50% of compounds were retained. On the other hand, MHG dehydration increased the phenolics extractability by 26%, particularly that of kaempferol derivatives while also retaining the amount of the glucosinolate glucoraphanin, when compared to freeze-drying. Nevertheless, only 23% of indole glucosinolates were recovered and pigments were severely reduced, with lutein accounting only for 32% and only chlorophyll b was observed in trace amounts after MHG dehydration. Therefore, to valorise broccoli by-products as ingredients, different drying technologies may be used when targeting different composition richness: freeze-drying is suitable for pigments and glucosinolates, air-drying is suitable for glucosinolates, while MHG promotes the extractability of phenolic compounds.

Use of elicitation in the cultivation of Bimi® for food and ingredients.

BACKGROUND: Cruciferous foods rich in health-promoting metabolites are of particular interest to consumers as well as being a good source of bioactives-enriched ingredients. Several elicitors have been used to stimulate the biosynthesis and accumulation of secondary metabolites in foods; however, little is known about the response of new hybrid varieties, such as Bimi®, under field-crop production conditions. Therefore, this study was designed to evaluate the effect of salicylic acid (200 µmol L-1 , SA), methyl jasmonate (100 µmol L-1 , MeJA), and their combination on Bimi plant organs (inflorescences and aerial vegetative tissues - stems and leaves). For this, the composition of the glucosinolates present in the tissues was evaluated. Also, aqueous extracts of the plant material, obtained with different times of extraction with boiling water, were studied. RESULTS: The results indicate that the combined treatment (SA + MeJA) significantly increased the content of glucosinolates in the inflorescences and that MeJA was the most effective elicitor in leaves. Regarding the aqueous extracts, the greatest amount of glucosinolates was extracted at 30 min - except for the leaves elicited with MeJA, for which 15 min was optimal. CONCLUSION: The elicitation in the field enriched leaves in glucobrassicin (GB), 4-methoxyglucobrassicin (MGB), and neoglucobrassicin (NGB) and stems and inflorescences in glucoraphanin, 4-hydroxyglucobrassicin, GB, MGB, and NGB. In this way, this enhanced vegetable material favored the presence of bioactives in the extracts, which is of great interest regarding enriched foods and ingredients with added value obtained from them. © 2019 Society of Chemical Industry.

Simultaneous direct determination of 15 glucosinolates in eight Brassica species by UHPLC-Q-Orbitrap-MS.

Glucosinolates (GLS) have been reported to have significant anti-oxidative, antimicrobial, and anti-cancer activities. The current study was aimed to develop an analytical method for glucosinolate quantitation in eight Brassica species from Gwangju, Republic of Korea. For this purpose the UHPLC-Q-Orbitrap-MS technique was used and validated for optimal extraction conditions, detection and quantitation limits, linearity, precision, and accuracy. According to the results of GLS profiling, the total GLS concentration decreased in the order of cabbage > broccoli > cauliflower > mustard > kimchi cabbage > young radish ∼ kale. All Brassica species contained glucoerucin (GER) and glucobrassicin (GBR) as major GLS with the high levels in cabbage (5.913 µM/g) and broccoli (1.723 µM/g), respectively. The contents of minor GLS were species-dependent, and could therefore be used for Brassica species classification.

Influence of different light conditions and time of sprouting on harmful and beneficial aspects of rutabaga sprouts in comparison to their roots and seeds.

BACKGROUND: This study aimed to evaluate the presence and content of selected phytochemicals, namely glucosinolates, fatty acids and phenolic compounds, in rutabaga (Brassica napus L. var. napobrassica) sprouts grown under various light conditions, in comparison to rutabaga seeds and roots. As rutabaga sprouts are likely to become new functional food, special emphasis was placed on the related risks of progoitrin and erucic acid presence - compounds with proven antinutritive properties. RESULTS: Time of sprouting significantly decreased progoitrin content, especially after 10 days (by 91.5%) and 12 days (by 97.5%), as compared to 8 days. In addition, sprouts grown under dark conditions showed 27%, 60% and 17% reduction in progoitrin level in 8, 10 and 12 days after sowing, respectively, as compared to sprouts grown under natural conditions. Progoitrin was found to be the predominant glucosinolate in rutabaga seeds (804.07 ± 60.89 mg 100 g-1 dry weight (DW)), accompanied by glucoerucin (157.82 ± 21.04 mg 100 g-1 DW), also found in the roots (82.20 ± 16.53 mg 100 g-1 DW). Among the unsaturated fatty acids in rutabaga sprouts, erucic, linoleic, linolenic and gondoic acids decreased significantly, and only oleic acid increased as germination days progressed. The amount of harmful erucic acid in rutabaga sprouts was found to vary between 1.8% and 7%, depending on the day of seeding or light conditions, as compared to 42.5% in the seeds. CONCLUSION: The evaluated rutabaga products showed a wide content range of potentially antinutritive compounds, sprouts having the lowest amounts of erucic acid and progoitrin. © 2018 Society of Chemical Industry.

Slightly Acidic Electrolyzed Water Treatment Enhances the Main Bioactive Phytochemicals Content in Broccoli Sprouts via Changing Metabolism.

Changes in the content of bioactive phytochemicals in the broccoli sprouts subjected to different slightly acidic electrolyzed water (SAEW) treatments were investigated in the present study. The highest sulforaphane amount in broccoli sprouts treated with SAEW with an available chlorine concentration (ACC) of 50 mg/L was 11.49 mg/g in dry weight (DW), which increased by 61.2% compared to the control. SAEW treatment enhanced the sulforaphane content mainly by increasing the glucoraphanin (GRA) concentration due to the promotion of methionine metabolism and increased myrosinase activities. In addition, the relative anthocyanin contents of light-germinated broccoli under SAEW 50 treatment were 2.03 times that of broccoli sprouts with tap water treatment, and these contents were associated with an increase in phenylalanine ammonia lyase (PAL) activities and phenylalanine participation in biosynthesis. In summary, SAEW promotes metabolism to induce the accumulation of bioactive compounds in broccoli sprouts.

Screening and identification of major phytochemical compounds in seeds, sprouts and leaves of Tuscan black kale Brassica oleracea (L.) ssp acephala (DC) var. sabellica L.

We report the spectrophotometric determination of total polyphenols, flavonoids, glucosinolates and antioxidant activity in seeds, seedlings and leaves of Tuscan black kale. The highest content of phytochemicals was observed in 10 days sprouts and antioxidant activity was maximum in 2, 4 days seedlings. Identification and characterisation of phytochemicals were performed by mass spectrometry (MS), high resolution and tandem MS with electrospray ionisation mode. Low-molecular-weight metabolites were evidenced in seeds while metabolites at high m/z range were detected in cotyledons and leaves. MS spectra evidenced different phenolic compounds (flavonoid caffeoyl glucose, hydroxycinnamic acid sinapine) and glucosinolates (glucoerucin, glucobrassicin and glucoraphanin) in function of developmental stage; galactolipids ω3 and ω6 were observed in leaves. Identification of stages with the highest phytochemicals content encourages the consumption of black kale sprouts and young leaves. Our research can support food and pharmaceutical industries for production of health promoting products from black kale.

Reconstructing Biosynthetic Pathway of the Plant-Derived Cancer Chemopreventive-Precursor Glucoraphanin in Escherichia coli.

Epidemiological data confirmed a strong correlation between regular consumption of cruciferous vegetables and lower cancer risk. This cancer preventive property is mainly attributed to the glucosinolate products, such as glucoraphanin found in broccoli that is derived from methionine. Here we report the first successful reconstruction of the complete biosynthetic pathway of glucoraphanin from methionine in Escherichia coli via gene selection, pathway design, and protein engineering. We used branched-chain amino transferase 3 to catalyze two transamination steps to ensure the purity of precursor molecules and used cysteine as a sulfur donor to simplify the synthesis pathway. Two chimeric cytochrome P450 enzymes were engineered and expressed in E. coli functionally. The original plant C-S lyase was replaced by the Neurospora crassa hercynylcysteine sulfoxide lyase. Other pathway enzymes were successfully mined from Arabidopsis thaliana, Brassica rapa, and Brassica oleracea. Biosynthesis of glucoraphanin upon coexpression of the optimized enzymes in vivo was confirmed by liquid chromatography-tandem mass spectrometry analysis. No other glucosinolate analogues (except for glucoiberin) were identified that could facilitate the downstream purification processes. Production of glucoraphanin in this study laid the foundation for microbial production of such health-beneficial glucosinolates in a large-scale.

Characterization of industrial broccoli discards (Brassica oleracea var. italica) for their glucosinolate, polyphenol and flavonoid contents using UPLC MS/MS and spectrophotometric methods.

The agrifood industry produces tons of waste and substandard products that are discarded at great expense. Valorization of industrial residues curbs issues related to food security and environmental problems. Broccoli (Brassica oleracea var. italica) is associated with varied beneficial health effects, but its production yields greater than 25% rejects. We aimed to characterize and quantify industrial broccoli by-products for their glucosinolate and polyphenol contents as a first step towards industrial bio-refining. Broccoli segments and rejected lots of 10 seed cultivars were analyzed using UPLC MS/MS. Variability in the contents of bioactive molecules was observed within and between the cultivars. Broccoli by-products were rich in glucosinolates (0.2-2% dry weight sample), predominantly glucoraphanin (32-64% of the total glucosinolates), whereas the polyphenolic content was less than 0.02% dry weight sample. Valorization of industrial residues facilitates the production of high value functional food ingredients along with socio-economic sustainability.

Chemopreventive glucosinolate accumulation in various broccoli and collard tissues: Microfluidic-based targeted transcriptomics for by-product valorization.

Floret, leaf, and root tissues were harvested from broccoli and collard cultivars and extracted to determine their glucosinolate and hydrolysis product profiles using high performance liquid chromatography and gas chromotography. Quinone reductase inducing bioactivity, an estimate of anti-cancer chemopreventive potential, of the extracts was measured using a hepa1c1c7 murine cell line. Extracts from root tissues were significantly different from other tissues and contained high levels of gluconasturtiin and glucoerucin. Targeted gene expression analysis on glucosinolate biosynthesis revealed that broccoli root tissue has elevated gene expression of AOP2 and low expression of FMOGS-OX homologs, essentially the opposite of what was observed in broccoli florets, which accumulated high levels of glucoraphanin. Broccoli floret tissue has significantly higher nitrile formation (%) and epithionitrile specifier protein gene expression than other tissues. This study provides basic information of the glucosinolate metabolome and transcriptome for various tissues of Brassica oleracea that maybe utilized as potential byproducts for the nutraceutical market.

Rapid and Cost-Effective Quantification of Glucosinolates and Total Phenolic Content in Rocket Leaves by Visible/Near-Infrared Spectroscopy.

The potential of visible-near infrared spectroscopy to predict glucosinolates and total phenolic content in rocket (Eruca vesicaria) leaves has been evaluated. Accessions of the E. vesicaria species were scanned by NIRS as ground leaf, and their reference values regressed against different spectral transformations by modified partial least squares (MPLS) regression. The coefficients of determination in the external validation (R²VAL) for the different quality components analyzed in rocket ranged from 0.59 to 0.84, which characterize those equations as having from good to excellent quantitative information. These results show that the total glucosinolates, glucosativin and glucoerucin equations obtained, can be used to identify those samples with low and high contents. The glucoraphanin equation obtained can be used for rough predictions of samples and in case of total phenolic content, the equation showed good correlation. The standard deviation (SD) to standard error of prediction ratio (RPD) and SD to range (RER) were variable for the different quality compounds and showed values that were characteristic of equations suitable for screening purposes or to perform accurate analyses. From the study of the MPLS loadings of the first three terms of the different equations, it can be concluded that some major cell components such as protein and cellulose, highly participated in modelling the equations for glucosinolates.

Variation of glucosinolates and quinone reductase activity among different varieties of Chinese kale and improvement of glucoraphanin by metabolic engineering.

The variation of glucosinolates and quinone reductase (QR) activity in fourteen varieties of Chinese kale (Brassica oleracea var. alboglabra Bailey) was investigated in the present study. Results showed that gluconapin (GNA), instead of glucoraphanin (GRA), was the most predominant glucosinolate in all varieties, and QR activity was remarkably positively correlated with the glucoraphanin level. AOP2, a tandem 2-oxoglutarate-dependent dioxygenase, catalyzes the conversion of glucoraphanin to gluconapin in glucosinolate biosynthesis. Here, antisense AOP2 was transformed into Gailan-04, the variety with the highest gluconapin content and ratio of GNA/GRA. The glucoraphanin content and corresponding QR activity were notably increased in transgenic plants, while no significant difference at the level of other main nutritional compounds (total phenolics, vitamin C, carotenoids and chlorophyll) was observed between the transgenic lines and the wide-type plants. Taken together, metabolic engineering is a good practice for improvement of glucoraphanin in Chinese kale.

Optimization of pulsed electric field pre-treatments to enhance health-promoting glucosinolates in broccoli flowers and stalk.

BACKGROUND: The effect of pulsed electric field (PEF) treatment variables (electric field strength and treatment time) on the glucosinolate content of broccoli flowers and stalks was evaluated. Samples were subjected to electric field strengths from 1 to 4 kV cm(-1) and treatment times from 50 to 1000 µs at 5 Hz. RESULTS: Data fitted significantly (P < 0.0014) the proposed second-order response functions. The results showed that PEF combined treatment conditions of 4 kV cm(-1) for 525 and 1000 µs were optimal to maximize glucosinolate levels in broccoli flowers (ranging from 187.1 to 212.5%) and stalks (ranging from 110.6 to 203.0%) respectively. The predicted values from the developed quadratic polynomial equation were in close agreement with the actual experimental values, with low average mean deviations (E%) ranging from 0.59 to 8.80%. CONCLUSION: The use of PEF processing at moderate conditions could be a suitable method to stimulate production of broccoli with high health-promoting glucosinolate content.

Variations in the most abundant types of glucosinolates found in the leaves of baby leaf rocket under typical commercial conditions.

BACKGROUND: Changes in the concentration of the three most abundant glucosinolates were measured in the leaves of perennial wall rocket [Diplotaxis tenuifolia (L.) DC.], and annual garden rocket (Eruca sativa Mill.). HPLC-MS was used to identify glucoraphanin, 4-hydroxyglucobrassin and glucoerucin from perennial wall rocket, and glucoraphanin, glucobrassicin and 4-methoxyglucobrassicin from annual garden rocket. In separate experiments the responses of glucosinolates to harvest number, seasonal conditions, nitrogen supply and post-harvest storage conditions were measured. RESULTS: For perennial wall rocket, season influenced the concentration of glucoraphanin, which were highest for the spring [379 µg kg(-1) fresh weight (FW)] and summer (317 µg kg(-1) FW) plantings. The concentration of 4-hydroxyglucobrassin was higher in the leaves of first harvest crops. This response was due to this glucosinolate not being detected in the leaves of second harvest crops. Thus, the parent glucosinolate was altered between the first and second harvests in response to the abiotic stresses caused by harvesting. For annual garden rocket, there was an interaction between the harvest number and season for all glucosinolates measured. However, no clear response was observed between these factors. Higher concentrations of glucobrassicin and 4-methoxyglucobrassicin were measured for first harvest leaves when compared to the second harvest. This was due to the absence of detection of these glucosinolates in the leaves of second harvested plants; consequently higher total glucosinolate concentrations were measured for the first harvest winter (1224 µg kg(-1) FW) and summer (864 µg kg(-1) FW) crops. CONCLUSION: The concentrations of individual glucosinolates vary greatly over typical pre- and post-harvest commercial conditions. The absence of 4-hydroxyglucobrassin for perennial wall rocket, and glucobrassicin and 4-methoxyglucobrassicin for annual garden rocket between harvests, illustrates that abiotic stress from harvesting has the capacity to alter the types of glucosinolates in leaves. Concentrations do not generally decline during a typical storage period, indicating that the potential benefits of these compounds are not lost during post-harvest storage.

Isothiocyanate metabolism, distribution, and interconversion in mice following consumption of thermally processed broccoli sprouts or purified sulforaphane.

SCOPE: Broccoli sprouts are a rich source of glucosinolates, a group of phytochemicals that when hydrolyzed, are associated with cancer prevention. Our objectives were to investigate the metabolism, distribution, and interconversion of isothiocyanates (ITCs) in mice fed thermally processed broccoli sprout powders (BSPs) or the purified ITC sulforaphane. METHODS AND RESULTS: For 1 wk, mice were fed a control diet (n = 20) or one of four treatment diets (n = 10 each) containing nonheated BSP, 60°C mildly heated BSP, 5-min steamed BSP, or 3 mmol purified sulforaphane. Sulforaphane and erucin metabolite concentrations in skin, liver, kidney, bladder, lung, and plasma were quantified using HPLC-MS/MS. Thermal intensity of BSP processing had disparate effects on ITC metabolite concentrations upon consumption. Mild heating generally resulted in the greatest ITC metabolite concentrations in vivo, followed by the nonheated and steamed BSP diets. We observed interconversion between sulforaphane and erucin species or metabolites, and report that erucin is the favored form in liver, kidney, and bladder, even when only sulforaphane is consumed. CONCLUSION: ITC metabolites were distributed to all tissues analyzed, suggesting the potential for systemic benefits. We report for the first time tissue-dependent ratio of sulforaphane and erucin, though further investigation is warranted to assess biological activity of individual forms.

Glucosinolates from pak choi and broccoli induce enzymes and inhibit inflammation and colon cancer differently.

High consumption of Brassica vegetables is considered to prevent especially colon carcinogenesis. The content and pattern of glucosinolates (GSLs) can highly vary among different Brassica vegetables and may, thus, affect the outcome of Brassica intervention studies. Therefore, we aimed to feed mice with diets containing plant materials of the Brassica vegetables broccoli and pak choi. Further enrichment of the diets by adding GSL extracts allowed us to analyze the impact of different amounts (GSL-poor versus GSL-rich) and different patterns (broccoli versus pak choi) of GSLs on inflammation and tumor development in a model of inflammation-triggered colon carcinogenesis (AOM/DSS model). Serum albumin adducts were analyzed to confirm the up-take and bioactivation of GSLs after feeding the Brassica diets for four weeks. In agreement with their high glucoraphanin content, broccoli diets induced the formation of sulforaphane-lysine adducts. Levels of 1-methoxyindolyl-3-methyl-histidine adducts derived from neoglucobrassicin were the highest in the GSL-rich pak choi group. In the colon, the GSL-rich broccoli and the GSL-rich pak choi diet up-regulated the expression of different sets of typical Nrf2 target genes like Nqo1, Gstm1, Srxn1, and GPx2. GSL-rich pak choi induced the AhR target gene Cyp1a1 but did not affect Ugt1a1 expression. Both colitis and tumor number were drastically reduced after feeding the GSL-rich pak choi diet while the other three diets had no effect. GSLs can act anti-inflammatory and anti-carcinogenic but both effects depend on the specific amount and pattern of GSLs within a vegetable. Thus, a high Brassica consumption cannot be generally considered to be cancer-preventive.

In vivo formation and bioavailability of isothiocyanates from glucosinolates in broccoli as affected by processing conditions.

SCOPE: To study the effect of residual myrosinase (MYR) activity in differently processed broccoli on sulforaphane (SR) and iberin (IB) formation, bioavailability, and excretion in human volunteers. METHODS AND RESULTS: Five different broccoli products were obtained with similar glucoraphanin (GR) and glucoiberin (GI) content, yet different MYR activity. Excretion of SR and IB conjugates in urine were determined in 15 participants after ingestion of the broccoli products. A reduction of 80% of MYR in the product did not cause differences in the total amount of SR and IB found in urine compared to the product with 100% MYR. Complete inactivation of MYR gave the lowest total amount of SR and IB in urine (10 and 19%). A residual MYR of only 2% in the product gave an intermediate amount (17 and 29%). The excretion half-lives of SR and IB conjugates were comparable for all the products (2.5 h on average), although the maximum excretion peak times were clearly shorter when the residual MYR was higher (2.3-6.1 h). CONCLUSION: For the first time, the effect of residual MYR activity on isothiocyanate bioavailability was systematically and quantitatively studied. Processing conditions have a large effect on the kinetics and bioavailability of isothiocyanates from broccoli.

Effect of NaCl stress on health-promoting compounds and antioxidant activity in the sprouts of three broccoli cultivars.

Health-promoting compounds, antioxidant and myrosinase activity in the sprouts of three broccoli cultivars under 40 mM, 80 mM and 160 mM NaCl were investigated. LangYan (LY) sprouts had the richest health-promoting compounds among the evaluated cultivars. Treatment of 40 mM and 80 mM NaCl significantly decreased the content of ascorbic acid and total phenolic as well as antioxidant activity, but did not affect glucoraphanin, sulforaphane and myrosinase activity compared to the control. However, 160 mM NaCl treatment significantly enhanced the level of total phenolic, glucoraphanin, sulforaphane, antioxidant and myrosinase activity, while significantly decreased ascorbic acid content. The results suggest that 160 mM NaCl treatment would enhance antioxidant activity and sulforaphane yield in broccoli sprouts. The health-promoting value of broccoli sprouts depends on plant genotype and could be affected by NaCl stress.

Quantification of glucosinolates, anthocyanins, free amino acids, and vitamin C in inbred lines of cabbage (Brassica oleracea L.).

We profiled and quantified glucosinolates (GSLs), anthocyanins, free amino acids, and vitamin C metabolites in forty-five lines of green and red cabbages. Analysis of these distinct cabbages revealed the presence of 11 GSLs, 13 anthocyanins, 22 free amino acids, and vitamin C. GSL contents were varied amongst the different lines of cabbage. The total GSL content was mean 10.6 µmol/g DW, and sinigrin was the predominant GSL accounted mean 4.0 µmol/g DW (37.7% of the total) followed by glucoraphanin (1.9) and glucobrassicin (2.4). Amongst the 13 anthocyanins, cyanidin 3-(sinapoyl) diglucoside-5-glucoside levels were the highest. The amounts of total free amino acids in green cabbage lines ranged 365.9 mg/100g fresh weight (FW) to 1089.1mg/100g FW. Vitamin C levels were much higher in red cabbage line (129.9 mg/100g FW). Thus, the amounts of GSLs, anthocyanins, free amino acids, and vitamin C varied widely, and the variations in these compounds between the lines of cabbage were significant.

Optimization of a blanching step to maximize sulforaphane synthesis in broccoli florets.

A blanching step was designed to favor sulforaphane synthesis in broccoli. Blanching was optimised through a central composite design, and the effects of temperature (50-70 °C) and immersion time in water (5-15 min) on the content of total glucosinolates, glucoraphanin, sulforaphane, and myrosinase activity were determined. Results were analysed by ANOVA and the optimal condition was determined through response surface methodology. Temperature between 50 and 60 °C significantly increased sulforaphane content (p<0.05), whilst blanching at 70 and 74 °C diminished significantly this content, compared to fresh broccoli. The optimal blanching conditions given by the statistical model were immersion in water at 57 °C for 13 min; coinciding with the minimum glucosinolates and glucoraphanin content, and with the maximum myrosinase activity. In the optimal conditions, the predicted response of 4.0 µmol sulforaphane/g dry matter was confirmed experimentally. This value represents a 237% increase with respect to the fresh vegetable.