foods Article Factors Influencing Sulforaphane Content in Broccoli Sprouts and Subsequent Sulforaphane Extraction Jan Tˇríska 1, Josef Balík 2, Milan Houška 3, Pavla Novotná 3, Martin Magner 4,5, NadˇeždaVrchotová 1, Pavel Híc 2, Ladislav Jílek 6, KateˇrinaThorová 7, Petr Šnurkoviˇc 2 and Ivo Soural 2,* 1 Global Change Research Institute CAS, 603 00 Brno, Czech Republic; [email protected] (J.T.); [email protected] (N.V.) 2 Faculty of Horticulture, Mendel University in Brno, 691 44 Lednice, Czech Republic; [email protected] (J.B.); [email protected] (P.H.); [email protected] (P.Š.) 3 Food Research Institute Prague (FRIP), 102 00 Prague 10, Czech Republic; [email protected] (M.H.); [email protected] (P.N.) 4 Department of Pediatrics, University Thomayer Hospital, First Faculty of Medicine, Charles University, 140 59 Prague 4, Czech Republic; [email protected] or [email protected] 5 Department of Pediatrics and Inherited Metabolic Disorders, First Faculty of Medicine, Charles University, General University Hospital, 128 08 Prague 2, Czech Republic 6 Pure Food Norway, 1400 Ski, Norway; [email protected] 7 National Institute for Autism, 182 00 Prague 8, Czech Republic; [email protected] * Correspondence: [email protected] Abstract: Broccoli sprouts contain 10–100 times higher levels of sulforaphane than mature plants, something that has been well known since 1997. Sulforaphane has a whole range of unique biological Citation: Tˇríska,J.; Balík, J.; Houška, properties, and it is especially an inducer of phase 2 detoxication enzymes. Therefore, its use has M.; Novotná, P.; Magner, M.; been intensively studied in the field of health and nutrition. The formation of sulforaphane is Vrchotová, N.; Híc, P.; Jílek, L.; controlled by the epithiospecifier protein, a myrosinase co-factor, which is temperature-specific. This Thorová, K.; Šnurkoviˇc,P.; Soural, I. paper studies the influence of temperature, heating time, the addition of myrosinase in the form of Factors Influencing Sulforaphane Content in Broccoli Sprouts and Raphanus sativus sprouts in constant ratio to broccoli sprouts, and other technological steps on the final Subsequent Sulforaphane Extraction. sulforaphane content in broccoli sprout homogenates. These technological steps are very important Foods 2021, 10, 1927. https://doi.org/ for preserving sulforaphane in broccoli sprouts, but there are some limitations concerning the amount 10.3390/foods10081927 of sulforaphane. We focused, therefore, on the extraction process, using suitable β-cyclodextrin, hexane and ethanol, with the goal of increasing the amount of sulforaphane in the final extract, thus Academic Editor: Charis stabilizing it and reducing the required amount sulforaphane needed, e.g., as a dietary supplement. M. Galanakis Keywords: broccoli and radish sprouts; sulforaphane; myrosinase; extraction; epithiospecifier protein Received: 6 August 2021 Accepted: 16 August 2021 Published: 19 August 2021 1. Introduction Publisher’s Note: MDPI stays neutral By monitoring quinone reductase induction in cultured murine hepatoma cells in with regard to jurisdictional claims in published maps and institutional affil- biological assays, Zhang et al. [1] were able to isolate sulforaphane (SR), a significant and iations. potent phase II enzyme inducer, from broccoli. Approximately 9 mg of SR was isolated from 640 g of fresh broccoli florets (ca. 14 µg/g of fresh weight (f.w.)). Later, Fahey et al. [2] found that three-day-old broccoli sprouts (cultivar Saga) had an inducer content ca. 15 times higher than adult plants. Significant differences in the SR content of both broccoli and broccoli sprouts have been published in the literature; these differences can be Copyright: © 2021 by the authors. explained in many ways, including the method of sample preparation. For example, Chiang Licensee MDPI, Basel, Switzerland. et al. [3] either homogenized one gram of freshly harvested broccoli in 10 mL of warm This article is an open access article (50 ◦C) distilled water for 5 min using a mixer or they used frozen broccoli, to which they distributed under the terms and conditions of the Creative Commons added 25 units of thioglucosidase before homogenization (to ensure complete hydrolysis). Attribution (CC BY) license (https:// They found the SR content of broccoli ranged from 1.89–3.7 mg/g, with the highest being creativecommons.org/licenses/by/ the Super Dome variety, which was more than two orders of magnitude higher than the 4.0/). Foods 2021, 10, 1927. https://doi.org/10.3390/foods10081927 https://www.mdpi.com/journal/foods Foods 2021, 10, 1927 2 of 12 values initially stated by Zhang et al. [1]. This shows that sample preparation plays a very important role, which is often overlooked. The glucosinolates (GL) content and its transformation, especially the transformation of glucoraphanine (glucoraphanin) (GR) and, therefore, the final SR content in broccoli sprouts, is affected by various factors, including pre-harvest factors, which can be very important. Although plant species and genotype significantly affect GR content and distribution, the effects of pre-harvest factors, including light, temperature, nutrition (i.e., sulfur (S) and nitrogen (N)), water, and phenological stage, cannot be ignored. There is limited literature regarding the effect of pre-harvest conditions on broccoli sprouts. It is quite clear that light is needed to achieve higher GR contents in broccoli sprouts. Pérez- Balibrea et al. [4] reported 33% higher concentrations of total GL relative to broccoli sprouts grown in the dark. GR is a sulfur-rich compound found in Brassicaceae plants; thus, its accumulation depends largely on the sulfur status of the whole plant and the N:S ratio. Schonhof et al. [5] found that an N:S ratio between 7:1 and 10:1 could promote plant yield and enhance GL accumulation in broccoli. Foliar application of a 5 mM and 10 mM methionine (a sulfur-containing amino acid) solution resulted in increased GL concentrations in 7-day- old broccoli sprouts, obtaining significantly higher amounts of GL (by 23% and 21%, respectively) than the non-treated sprouts on the same sampling date (Pérez-Balibrea et al. [6]). The same authors also showed that broccoli sprouts treated the standard elicitors, such as salicylic acid, methyl jasmonate, and chitosan, were highly effective, since every dose, on each sampling day, gave rise to a higher total GL content than that obtained in the control sprouts. Natella et al. [7] extended the study of the abovementioned elicitors to include sucrose, mannitol, and 1-aminocyclopropane-1-carboxylic acid. They identified sucrose as the most potent elicitor, which induced a significant increase in total and specific GL, in contrary to other studied elicitors. Guo et al. [8] evaluated GL metabolism in broccoli sprouts under NaCl treatment and found the highest SR content (14 mg/g f. w. against control—10 mg/g f.w.) in 3-day-old broccoli sprouts using 100 mmol/L of sodium chloride solution, which decreased with the age of the broccoli sprouts. When the broccoli seeds were soaked in slightly acidic electrolyzed water (SAEW) with chlorine concentrations of 50.33 mg/L at a pH = 5.52, for 3 h at 25 ◦C, relative humidity of 80%, and in the dark, the final SR concentration increased to 11.49 mg/g d.w. (a 61.2% increase compared to controls) [9]. The authors harvested broccoli sprouts with roots after eight days, stored them at −80 ◦C, and then freeze-dried them before analysis. The issue of global changes is also reflected in studies examining the ability to influence sulforaphane content in broccoli sprouts. Almuhayawia et al. [10] grew three varieties of broccoli sprouts (Southern Star, Prominence, and Monotop) in a growth chamber at 25 ◦C, under a 16 h light/8 h light cycle, relative humidity of 60%, ambient −1 carbon dioxide concentration of 400 ± 27 µmol CO2 mol , and elevated concentration −1 carbon dioxide levels of 620 ± 42 µmol CO2 mol air for nine days. The Monotop variety yielded an approximately threefold increase in sulforaphane (approx. 6 µmol/g f.w.) with half the content of sulforaphane nitrile (ca. 1.8 µmol/g f.w.), and with increased myrosinase activity and decrease in ESP activity. Glucosinolates formed and accumulated in broccoli sprouts, especially GR, must undergo a somewhat complicated enzymolysis that results in isothiocyanates, especially SR. To achieve an optimal yield of SR, we would have to know the exact content of its precursor, GR, the amount and activity of myrosinase, the amount and activity of epithiospecifier protein (ESP), as well as the ratio of sprout biomass to water, the pH of the reaction mixture, the temperature, etc. Several authors have conducted studies on the optimal enzymolysis conditions for sulforaphane production recently, e.g., Hanschen et al. [11] and Tian et al. [12]. Tian et al. [12] found that the maximum sulforaphane content (246.95 µg/g d.w.) was reached using a solid–liquid ratio of 1:30, a hydrolysis time of 1.5 h, an ascorbic acid content of 3.95 mg/g d.w., and a temperature of 65 ◦C. The highest content of SR in broccoli sprouts Foods 2021, 10, 1927 3 of 12 was 233.80 µg/g d.w. in 5-day-old sprouts and 1555.95 µg/g d.w. after four days of storage. During the preparation of broccoli sprout juice, Bello et al. [13] found that the yield of SR and sulforaphane nitrile (SRN) was only approximately 25% calculated based on the amount of GR in the original material. The loss during juice preparation was explained as (1) spontaneous conversion to sulforaphane-amine or (2) conjugation of the glutathione and proteins naturally present in the juice. The total amount of SRN extracted in the juice from 100 g of fresh sprouts was about 7 ± 3 µmol (0.12 ± 0.04 µmol/mL juice).