Green method for obtaining isothiocyanates from cruciferous vegetables

Abstract

The presently disclosure relates to a method of obtaining isothiocyanates (ITC) from frozen cruciferous vegetables using microwave gravity hydrodiffusion (MHG) technique, a solvent-free extraction technique. The advantages of this method are obtaining two liquid fractions (PLF + WLF) that can be used in different applications and a solid fraction (plant biomass) that can be reused, contributing to the preservation of biodiversity.

Description

GREEN METHOD FOR OBTAINING ISOTHIOCYANATES FROM CRUCIFEROUS VEGETABLESTechnical field

[0001] The present disclosure relates to a green method of obtaining isothiocyanates from cruciferous vegetables. Specifically, the present disclosure relates to a solvent-free method of obtaining isothiocyanates from cruciferous vegetables using microwave hydrodiffusion and gravity.Background

[0002] Isothiocyanates (R-N-C-S) are the most abundant products of the natural enzymatic degradation of glucosinolates by the enzyme myrosinase (a-thioglucosidase enzyme), which coexists separately with glucosinolates in plants. Isothiocyanates ("ITCs") are strong electrophiles due to the central carbon atom of their chemical structure. Thus, they can react readily with sulphur-, nitrogen- and oxygen-based biological nucleophiles, including the thiol and amino groups of proteins (e.g., glutathione)[l],

[0003] Glucosinolates ("GLs") are found almost exclusively in cruciferous vegetables from the Brassicaceae family, such as watercress, cauliflower, broccoli, brussels sprouts, and cabbage [1,2], Each glucosinolate produces a specific isothiocyanate ("ITC"). Generally, plants naturally produce ITC in response to stress, such as when tissues are damaged (by chopping or chewing) or as a defence system against attack by pathogens [2-4], Additionally, ITC can be released through processing, cooking and consumption. ITCs from cruciferous vegetables have been extensively studied for their preventive and therapeutic effects on chronic diseases and cancer[5]. There is evidence that ITCs modulate the activity of enzymes involved in phases I and II of the detoxification pathway, the cell cycle, oxidative stress, inflammation, angiogenesis, and epigenetic effects[6],

[0004] One of the most interesting ITCs is phenethyl isothiocyanate ("PEITC"), and the naturally rich source of this compound is watercress (Nasturtium officinale). The most abundant GL found in watercress is gluconasturtiin, a phenethyl glucosinolate[7]. The final product resulting from the myrosinase hydrolysis of gluconasturtiin is an isothiocyanate with a phenethyl radical attached to a nitrogen atom: PEITC[2], The health-promoting effects of this ITC have beenextensively studied by clinical trials and several in vitro and in vivo models[8], The chemopreventive action of PEITC in humans has been attributed to its antioxidant and antiinflammatory effects and its ability to block the stages of carcinogenesis[4,9,10], Besides, PEITC exhibits antimicrobial effects, especially against gastrointestinal pathogenic bacteria [5,10],

[0005] Various extraction techniques have been devised to produce and extract ITC from cruciferous vegetables, owing to the potential benefits of these metabolites to human health.

[0006] Document US2003064131 describes a method to stably prepare a non-pungent isothiocyanate product.

[0007] Document US2020255872 describes a method for producing isothiocyanate containing products from Brassicaceae material and lactic acid bacteria for use in such methods.

[0008] Document US2021100751 describes a method to produce a composition comprising isothiocyanate and / or glucosinolate for pharmaceutical or cosmetic applications.

[0009] Document WO2022129618 describes a method for preparing at least one watercress extract. This allows the isolation of a liquid fraction to provide an aqueous watercress extract.

[0010] However, the applied methodologies, in general, often involve the use of large amounts of organic solvents, long extraction times and complex equipment. For example, the conventional extraction technique for obtaining ITCs uses n-hexane, dichloromethane, ethyl acetate, acetoneethyl acetate, and methyl-t-butyl ether as solvents. Generally, it requires long incubation time, the use of acidic water (with a specific range of pH), and even the use of additional myrosinase enzyme to optimise the production of ITCs[ll], These factors can cause environmental pollution, health hazards, difficulty in being used at the food industry level (especially the use of polluting organic solvents) and high costs. Therefore, there is great interest in replacing or improving the conventional extraction of ITCs by sustainable methodologies. High-pressure Processing, High Voltage Electrical Discharges, Ultrasound-Assisted Extraction, Supercritical Fluid Extraction and Pressurised Fluid Extraction and Aqueous Micellar Systems with non-ionic and biodegradable surfactants are examples of non-conventional extraction techniques[7,ll]. Nevertheless, these are complex techniques, expensive, and not easily scalable.

[0011] Solvent-free extraction processes are very interesting as an alternative to some of these methodologies. Of all the advantages of solvent-free extraction technology, it stands out: avoiding costs and risks of large volumes of solvent, facilitating scaling-up, greater purity of the extract, and reducing extraction time from hours to minutes

[0012] .

[0012] These facts are disclosed in order to illustrate the technical problem addressed by the present disclosure.General Description

[0013] The present disclosure relates to a green method of obtaining isothiocyanates from cruciferous vegetables. Specifically, the present disclosure relates to a solvent-free method of obtaining isothiocyanates (ITCs) from cruciferous vegetables using microwave hydrodiffusion and gravity method (MHG).

[0014] The present disclosure relates to a novel extraction method designed to rapidly obtain extracts with high amounts of ITC, phenethyl isothiocyanate (PEITC). The method of the present disclosure is focused on optimizing the efficiency, yield, and scalability of PEITC extraction while adhering to green chemistry principles and minimizing water usage.

[0015] Surprisingly, the extraction method achieved a better efficiency, namely an efficiency of 394 ± 104 to 2452 ± 492 PEITC (pg / g dry basis) from turnip and watercress. This process was completed in just 30 minutes, significantly reducing the time required for the extraction process of ITCs (the freezing time of vegetables is not included in the 30 minutes). The absence of longer pre-treatment of plant material (e.g. heating or maceration) and fermentation in the method of the present disclosure allows to be complete the extraction process much faster. MHG method is a specialized technique used to extract essential oils, volatile compounds, and other heatsensitive compounds from plant materials. This method combines microwave-assisted heating with the principles of hydrodiffusion and gravity to achieve efficient and gentle extraction

[0013] ,

[0016] PEITC is a bioactive compound found in various cruciferous vegetables, such as watercress, known for its potential health benefits, particularly in cancer prevention and treatment.

[0017] MHG is a solvent-free extraction technique that can be used on laboratory and industrial scales. MHG technique is based on the in situ dielectric heating on plant cells' water, causing the structure to distend, leading to the rupture of the membrane and cell wall. The metabolites and in situ water are collected from plant material. One of the advantages of this solvent-free extraction technology is avoiding costs and risks associated with handling large volumes of solvent. Additionally, it is cleaner, facilitates scaling-up, improves the purity of the extract, reduces extraction time from hours to minutes, and reduces energy consumption. Thesubsequent carbon load is inferior to other techniques, thus being environmentally friendly. Moreover, MHG technique offers an excellent opportunity to reuse plant biomass after extraction, promoting efficient and sustainable use while preserving biodiversity

[0012] . Besides, since plant biomass moisture is reduced during the process, the dry product has a stable and limited microbial activity, which is an added value for its use in the food industry, such as a potential multifunctional flour. MHG is a combination of microwaves for hydrodiffusion from the inside to the exterior of biological material and uses earth gravity to collect and separate. MHG is performed at atmospheric pressure without adding any solvent or water.

[0018] An aspect of the present disclosure relates to the use of MHG to extract PEITC from cruciferous vegetables, preferably watercress. The MHG process can produce high-purity extracts with consistent quality and can be scaled-up easily, which are important parameters to proceed for in vivo assays or clinical trials.

[0019] An aspect of the present disclosure is that the freezing step optimizes the efficiency of ITC extraction, particularly PEITC, which is beneficial for maintaining the integrity of bioactive compounds.

[0020] The present disclosure relates to a method for obtaining isothiocyanate extract in less than 30 minutes, comprising the steps of: obtaining frozen cruciferous vegetable; subjectingthe frozen vegetable to microwave hydrodiffusion and gravity extraction to obtain a liquid extract and a solid plant biomass; collecting the liquid extract to obtain an isothiocyanate extract.The time of the method for obtaining isothiocyanate extract does not include the cruciferous vegetable freezing time.

[0021] In an embodiment for better results, the microwave hydrodiffusion and gravity extraction may comprise the steps of: heating the frozen vegetable in a microwave reactor to obtain a crude extract and a solid plant biomass; cooling and collecting the obtained crude extract in a condenser to obtain an isothiocyanate rich extract.

[0022] In an embodiment for better results, the extraction process time may range from 10 - 30 minutes; preferably 15 - 30 minutes; more preferably 20 - 30 minutes.

[0023] In an embodiment for better results, the isothiocyanate may be phenethyl isothiocyanate.

[0024] In an embodiment for better results, the extraction is performed at a temperature less than 97 °C; preferably less than 94 °C; more preferably less than 92 °C.

[0025] In an embodiment for better results, the extraction may be performed at a temperature ranging from 80 - 99 °C; preferably 92 - 96 °C.

[0026] In an embodiment for better results, the frozen cruciferous vegetable may be frozen watercress.

[0027] In an embodiment for better results, the isothiocyanate rich extract may comprise between 400 - 2400 pg phenethyl isothiocyanate / g watercress dry basis (WC DB) and 394 pg phenethyl isothiocyanate / g turnip dry basis; preferably 400 - 1500 pg phenethyl isothiocyanate / g (WC DB).

[0028] In an embodiment for better results, the isothiocynate rich extract may comprise two liquid fractions.

[0029] In an embodiment for better results, the two liquid fractions may be a purple liquid fraction and a white liquid fraction.

[0030] In an embodiment for better results, the purple liquid fraction may comprise at least 26.86 pmol trolox eq. / g watercress dry basis (WC DB) of oxygen radical absorbance capacity.

[0031] In an embodiment for better results, the purple liquid fraction may comprise at least 8.55 pmol trolox eq. / g WC DB of ABTS radical scavenging activity.

[0032] In an embodiment for better results, the white liquid fraction may comprise at least 10.01 pmol trolox eq. / g WC DB of oxygen radical absorbance capacity.

[0033] In an embodiment for better results, the white liquid fraction may comprise at least 4.71 pmol trolox eq. / g WC DB of ABTS radical scavenging activity.

[0034] In an embodiment for better results, the frozen cruciferous vegetable may be a cruciferous vegetable frozen at -18 °C.

[0035] In an embodiment for better results, the microwave reactor may be at a fixed microwave power ranging from 1.5 - 2.5 W / g.

[0036] In an embodiment for better results, the biomass may be a watercress biomass.

[0037] An aspect of the present disclosure relates to a phenethyl isothiocyanate extract obtained by the method of the present disclosure.

[0038] In an embodiment for better results, the isothiocyanate-rich extract may comprise from 400- 2400 pg phenyethyl isothiocyanate / g WC DB and 394 pg PEITC / g turnip DB; preferably 400 - 1500 pg phenethyl isothiocyanate / g watercress dry basis.

[0039] In an embodiment for better results, the isothiocyanate-rich extract comprises a purple liquid fraction and a white liquid fraction.

[0040] In an embodiment for better results, the purple liquid fraction comprises at least 26.86 pmol trolox eq. / g WC DB of oxygen radical absorbance capacity.

[0041] In an embodiment for better results, the purple liquid fraction comprises at least 8.55 pmol trolox eq. / g WC DB of ABTS radical scavenging activity.

[0042] In an embodiment for better results, the white liquid fraction comprises at least 10.01 pmol trolox eq. / g WC DB of oxygen radical absorbance capacity.

[0043] In an embodiment for better results, the white liquid fraction comprises at least 4.71 pmol trolox eq. / g WC DB of ABTS radical scavenging activity.

[0044] Another aspect of the present disclosure relates to a functional flour obtained by the method of the present disclosure.Brief Description of the Drawings

[0045] The following figures provide preferred embodiments for illustrating the description and should not be seen as limiting the scope of invention.

[0046] Figure 1: Representation of the flowchart of the phenethyl isothiocyanate (PEITC) microwave hydrodiffusion and gravity (MHG) extraction method.

[0047] Figure 2: Photographic representation of the microstructure of fresh (A) and frozen (B) watercress observed through scanning electron microscopy (SEM).

[0048] Figure 3: Illustration of results of the angiotensin-converting enzyme (ACE) inhibitory activity of MHG extracts.

[0049] Figure 4: Illustration of results of the a-Glucosidase inhibition assay half-maximal inhibitory concentration (IC50) of MHG extract.

[0050] Figure 5: Photographic representation of the MHG extracts: PLF - Purple Liquid Fraction; WLF - White Liquid Fraction.

[0051] Figure 6: Photographic representation of the watercress biomass / residue after MHG extraction.

[0052] Figure ?: Illustration of results of the zeta potential of watercress MHG extracts.

[0053] Figure 8: illustration of results of the growth curves of the extracts (not exposed to bacteria) to evaluate the sterility of the extracts.

[0054] Figure 9: Illustration of results of the colony counting of MHG extracts. No growth of microorganisms was observed in the WLF.

[0055] Figure 10.1-10.2: Illustration of results of the inhibition growth curves (measured by absorbance at 600 nm) of WLF tested at 331, 166 and 83 pM PEITC against pathogenic bacteria; Gram-negative: (A) — E. coli, (B) — S. enterica, and (C) — P. aeruginosa; and Gram-positive: (D) S. aureus.

[0056] Figure 11: Illustration of results of the chromatogram of PEITC from watercress extract

[0057] Figure 12.1-12.2: Illustration of results of the MHG extracts High-performance liquid chromatography (HPLC-DAD) chromatograms (X = 245 nm). A - Watercress MHG extract, B - Turnip MHG extract, C - Broccoli MHG extract, D - Turnip green MHG extract.Detailed Description

[0058] The present disclosure relates to a green method of obtaining isothiocyanates from cruciferous vegetables. Specifically, the present disclosure relates to a method of obtaining isothiocyanates from cruciferous vegetables using microwave hydrodiffusion and gravity method (MHG).

[0059] In an embodiment, fresh sprigs of watercress side streams, resulting from the washing process and selection of plant material for direct consumption, were supplied by the food processing company Vitacress Portugal SA (Odemira, Portugal). When received, the plant material was stored at -18 °C until further use.Example 1

[0060] In an embodiment, extraction procedure was performed. The extraction procedures for MHG used were illustrated in the flowchart as shown in Fig. 1.

[0061] In an embodiment, the MHG was carried out in an advanced microwave extraction system (ETHOS X, Milestone, Milan, Italy) consisting of a microwave reactor of 2.45 GHz, with a maximum delivering power of 1800 W, accomplished by the presence of two magnetrons of 900 W each and an infrared sensor to monitor the temperature. The extraction procedures were performed using a 2 L glass reactor (Pyrex, Charleroi, PA, USA) closed with a glass cover. The MHG system was configured using the "Flavors' setup" coupled with a Chiller (Smart H150-2100S, Labtech S.r.l., Sorisole, Italy) to maintain the water temperature at 8 °C.

[0062] In an embodiment, the plant material used in the extraction was frozen (without any processing before) at -18 °C prior to the MHG extraction. During freezing, water in the cellular vegetables is crystallised and converted into ice crystals. Although it is preferable that the material be warm to room temperature to allow efficient use of microwave energy, in the present disclosure, the watercress was still frozen in the glass reactor so as not to lose the bioactive compounds present in the frozen water and maximise the extraction of the compounds of interest. Previously, it was tested using fresh watercress (without freezing), and it was not possible to identify PEICT by High-performance liquid chromatography (HPLC-DAD). That is, without the matrix being previously frozen, the extraction of ITC by MHG was not verified.

[0063] In an embodiment, the MHG procedure performed at atmospheric pressure, 250 g of frozen watercress material was heated using a fixed microwaved power (1.5 - 2.5 W / g) and extraction time (10 - 30 minutes) without adding any solvent or water. The temperature inside the reactor was kept below 98 - 99 °C during all extractions, with distillation stopped if the temperature exceeded 99 °C (to prevent the sample from burning in the reactor). The direct interaction of microwave irradiations with the in situ water distends the plant cells and leads to their rupture. This physical phenomenon facilitates the diffusion of the molecules of interest to the outside of the cells of the plant tissues. Due to the earth's gravity, the crude extract (in situ water, frozen water, and metabolites) naturally drops out of the microwave reactor into the condenser outside the reactor that continuously cools the extract. The extract is collected continuously in a receiving flask and then stored at -80 °C prior to analysis.

[0064] During a previous extraction process, it was observed that when the extraction temperature reaches 92 - 94 °C, the crude extract changes colour from purple (purple liquid fraction "PLF") to colourless / white (white liquid fraction "WLF").

[0065] In an embodiment, to obtain an extract rich in PEITC, it was decided to collect each liquid fraction separately in two different flasks and analyse them. Therefore, the PLF is the first fractionto be collected, and when it reaches the temperature range mentioned above and a whitish liquid is visible leaving the condenser, the bottle containing the PLF is replaced by an empty bottle to collect the WLF until the end of the extraction. At the end of the extraction, two bottles corresponding to two fractions was obtained, which can be applied separately or as a single total extract (PLF + WLF). The two liquid fractions (PLF and WLF) result from the same initial matrix (from the 250g of watercress placed in the reactor). They do not result from two different extractions. There are two collections of in situ water from the watercress that drop through the condenser.

[0066] In an embodiment, the solid fraction (watercress biomass) can be reused after extraction as a potential functional flour. A functional flour is a flour obtained from various grains which does not have gluten protein in it. The functional flour helps to maintain the consistency of food products and reduces the processing time.

[0067] In an embodiment, HPLC-DAD was performed to analyse the resulting liquid fractions to identify and quantify PEITC and validate the extraction procedure's efficacy.

[0068] In an embodiment, potential bioactivities of the extracts, such as antioxidant activity (ABTS - Radical Scavenging Assay) and ORAC scavenging assays) and the quantification of total phenolic compounds, were also explored.

[0069] In an embodiment, PEITC chromatographic analysis was performed.

[0070] In an embodiment, to isolate PEITC for its identification and quantification, 5 ml of each PEITC-rich extract was mixed vigorously with 5 ml of n-hexane for 2h for 10 minutes at a constant temperature (37.0 °C) and centrifuged at 5000 rpm for 10 min. This process was carried out because of the greater affinity that PEITC has for the organic phase. Then 4 ml of the organic phase was evaporated till dryness in an RVC 2-18 speed-vacuum evaporator (Christ, Osterode am Harz, Germany) at 40 °C. The resulting residue was dissolved in 1 ml of methanol.

[0071] In an embodiment, HPLC-DAD analysis was performed to identify and quantify PEITC[7] . The separation was carried out by applying the operating conditions described by Coscueta et al. 2020[7], Briefly, the operating conditions were as follows: gradient elution starts at 50% mobile phase A (ultra-pure water + 0.1% formic acid) and 50% mobile phase B (methanol + 0.1% formic acid), with a phase B increment up to 80% for 20 min, remaining isocratic for 10 min, and returning to 50% of phase B the next 5 min, at a continuous flow of 0.60 mL / min. The procedure was performed in a reversed-phase column coupled with a guard column containing the samestationary phase (COSMOSIL 5C18-AR-II Packed Column — 4.6 mm ID x 250 mm). The separation analysis was performed using a Waters e2695 separation module system interfaced with a photodiode array UV / Vis detector (PDA 190 - 600 nm). The PEITC peak was identified by comparing the retention time and absorption spectrum with the corresponding commercial standard. The quantification was carried out by integrating the peak area with external standardisation.

[0072] In an embodiment, the antioxidant activity of the extracts obtained was determined.

[0073] In an embodiment, ABTS radical method and Oxygen radical absorbance capacity (ORAC) assays were performed following the methodology used by Coscueta et al. ( 2020) [7]. The results were expressed in pmol trolox eq. / g WC DB (watercress dry basis).

[0074] In an embodiment, quantification of total phenolic compounds was performed.

[0075] In an embodiment, Folin-Ciocalteu method was used to quantify the total phenolic compounds following the procedure reported by Vilas-Boas et al. (2020)

[0014] , The results were expressed in mg of gallic acid equivalents (GAEs) / g watercress dry basis.

[0076] In an embodiment, different extraction conditions (Table 1), fixing a microwaved power (1.5 - 2.5 W / g) and extraction time (10 - 30 minutes) were performed. The most preferable conditions found for the PEITC extraction from 250 g of watercress were 2 W / g of microwave power and 30 min of extraction, as a 1557 pg PEITC / g WC DB in the crude extract and is comparable to other extraction techniques, including the convention extraction with n-hexane and others non-conventional methods reported in the literature (Table 2). The amount obtained by MHG was almost half of that reported by Rodrigues et al. (2016)[8] (3346 pg PEITC / g WC DB) with the conventional extraction, but, on the other hand, it was similar to the amount obtained by Coscueta et al. (2020)[7] under similar conditions. It showed that the production of ITC can vary depending on the conditions to which the plant is subjected[7]. Besides that, the production also depends on the plant species, age, and other factors such as place of cultivation, climatic conditions, storage, and processing.

[0077] As such, to obtain extracts rich in PEICT, more than its extraction, the emphasis is on the production of PEITC itself. The process of the present disclosure to get PEITC is, therefore, innovative and effective. A fundamental step in this process is the freezing of the raw material. During freezing, water in the cellular foods is crystallised and converted into ice crystals. Consequently, the food cells experience a wide range of factors, such as osmotic pressure.mechanical damage, and thermal stresses, leading to the injury or even death of ce I ls

[0015] . This is essential because, as mentioned above, in intact plants, the GLs are stable compounds localised in vacuoles, being physically separated from myrosinase. Therefore, it is expected that the freezing process and the formation of ice crystals cause damage to cells, promoting contact between GLs and myrosinase and, consequently, leading to the formation of ITC. In this way, freezing (a simple, low-cost, and physical process that can be implemented at an industrial level) promotes the production of ITC, making their extraction more efficient. In the method of the present disclosure, there is no need for a step prior to extraction in which the matrix is macerated with acidified water and commercial enzyme for a prolonged time and controlled temperature. Therefore, ITC extraction by MHG was ineffective without the matrix being previously frozen. Furthermore, at an industrial level, cruciferous vegetable by-products can be stored in freezers, and the raw material can be used directly to extract ITC in the MHG, thus contributing to the circular economy.

[0078] In an embodiment, WLF generally has a considerable amount of PEICT than the PLF. This is interesting and innovative, as it allows a liquid fraction with a higher concentration of PEITC (m / v) to be obtained. As such, each liquid fraction can be used for different purposes. For example, a PLF, the first liquid fraction, is probably richer in more hydrophilic compounds with bioactive potential, such as antioxidant activity, including polyphenols (Table 3). On the other hand, a WFL is a fraction with a few less hydrophilic compounds, such as ITCs, being more concentrated in PEITC (m / v) than PLF.

[0079] In an embodiment, as an option, it is also possible to obtain only one liquid fraction, the total crude extract. Forthat, it just does not change the collection bottle for the liquid that comes out of the condenser during removal. This method allows two different liquid fractions or a single liquid fraction as desired to be obtained.

[0080] Furthermore, based on the antioxidant analysis (Table 3), both liquid fractions showed antioxidant power. In addition to the presence of ITCs / PEITC, this is very interesting for the potential application of these extracts as nutraceuticals orfood supplements. Another advantage of this method is the potential to reuse the solid fraction (plant biomass). This solid fraction is a dry product with stable and limited microbial activity (due to the extraction process, it was subjected to the microwave phenomenon, and at high temperatures, similar to a sterilisation process), an added value for its use in the food industry, such as a potential multifunctional flour.

[0081] Table 1 - MHG extractions carried out on frozen watercress: experimental conditions and PEITC content of the extracts obtained.

[0082] Table 2 - Different extraction techniques for phenethyl isothiocyanate found in literature.

[0083] Table 3 - Total phenolic compounds and antioxidant activity by oxygen radical absorbance capacity (ORAC) and ABTS (Radical Scavenging Assay).Abbreviations: WC DB - watercress dry basisExample 2Microwave hydrodiffusion and gravity (MHG) extraction

[0084] In an embodiment, the MHG was performed in an advanced microwave extraction system (ETHOS X, Milestone, Milan, Italy). The system consisted of a microwave reactor with a maximum delivering power of 1800 W. The MHG system was configured using the "Flavors' setup" and a chiller to maintain the water temperature at 8 °C.

[0085] In an embodiment, the plant material used for extraction was frozen at -18 °C before the MHG extraction process and was kept frozen in the glass reactor. All extractions were carried out with frozen watercress. In the MHG procedure performed at atmospheric pressure, 250 g of frozen watercress material was heated using a fixed microwave power (1.91 W / g) and extraction time (29 minutes) without adding any solvent or water. Extract falls into a condenser due to gravity, continuously cooled, and collected in a flask. Temperature is monitored by a sensor, and the final temperature is recorded.

[0086] In an embodiment, frozen watercress under MHG was found to produce two extract fractions: a colored fraction and a colorless / whiteness fraction. The crude extract changes color from purple to colorless / white at 92 - 94°C. During processing, green vegetables can be exposed to heat or acidic conditions. As a result, the chlorophylls are converted to pheophytins by losing magnesium ions (Mg2+), causing theirgreen color to shift to dark / brown

[0016] . Two liquid fractions (PLF and WLF) are obtained at the end of the extraction, which can be applied separately or as a single total extract. In an embodiment, after extraction, PEITC was quantified and identified using HPLC-DAD following the method by Coscueta et al. (2020)[7] and the results were expressed in micrograms of PEITC per gram of watercress on a dry basis (pg PEITC / g WC DB). Moreover, the total phenolic compounds (TPC) of MHG extracts were determined by the Folin-Ciocalteu method, following the procedure reported by Coscueta et al. (2018)

[0017] were expressed in milligrams of gallic acid equivalent per g of watercress dry basis (mg GAE / g WC DB).

[0087] The antioxidant activity was determined through ABTS scavenging and Oxygen radical absorbance capacity (ORAC) assays, following the procedures described by Coscueta et al. (2020) [7] and the results were expressed in micro mols of Trolox equivalents per g of watercress dry basis (pmol TE / g WC DB).

[0088] Table 4. Phenethyl isothiocyanate amount, total phenolic compounds content and antioxidant activity content (n=4).

[0089] An aspect of the present disclosure indicates that the MHG approach was highly effective in extracting PEITC. The MHG method was highly effective in extracting PEITC and requires less than 30 minutes of irradiation. Furthermore, it does not use solvents, eliminating the need for solvent evaporation during extract purification. MHG provides an excellent opportunity to reuse post-extraction biomass. Additionally, this biomass can be processed to obtain gluten-free flour, ensuring the use of plant biomass using a zero-waste strategy.Freezing and PEITC extraction

[0090] An aspect of the present disclosure relates to the watercress freezing before extraction, which was crucial to obtaining PEITC, resulting in higher amounts of phenolic compounds and antioxidant activity. The watercress leaf microstructure was studied using scanning electron microscopy (SEM) (Fig. 2). Freezing can damage the microstructure of cellular food by forming ice crystals that destroy cell membranes and walls. SEM was used to compare fresh and frozen watercress leaves and revealed that freezing decreased the uniformity of structure and enlarged or ruptured pores, indicating deformation of cell walls. Freezing promotes the natural enzymatic hydrolysis of gluconasturtiin into PEITC.MHG extraction - New batch

[0091] In an embodiment, a new batch of fresh watercress (not by-products) was used for the extraction process according to the optimal conditions previously mentioned (1.91 W / g and 29 min). A watercress methanolic extract was obtained, according to Gomez-Garcia et al. (2021)

[0018] , as a control to compare to the MHG extracts. PEITC amount, ABTS, and ORAC for antioxidant capacity, total flavonoid content

[0019] , vitamin C

[0020] , and protein content (Pierce BCA Protein Assay Kit) and the quantifications of the most abundant minerals in watercress

[0021] .

[0092] Table 5. Phenethyl isothiocyanate amount, antioxidant activity, phenolic compounds amount and protein and vitamin C content from a new batch of watercress (n=3).Abbreviations: (PLF) - purple liquid fraction, (WLF) - white liquid fraction.

[0093] In an embodiment, based on the findings in Table 4, it is possible to extract PEITC using the MHG method, even with a different batch. The difference in the amount of PEITC extracted could be due to biological variability and external factors that may affect the extraction of bioactives. The MHG extracts (PLF + WLF) had more antioxidant power than the control extract. Regarding the total flavonoid content, the MHG process was more efficient in extracting these compounds than methanolic extraction. The PLF also contains vitamin C, which is an essential natural antioxidant and preservative.Table 6. Quantification of the most representative minerals in the extracts.Abbreviations: (PLF) - purple liquid fraction, (WLF) - white liquid fraction, ND - not detected. *Due to matrix interference, It was impossible to carry out this analysis. Each value was expressed as mean + standard deviation (n=3).

[0094] Minerals are essential to human life and play crucial roles in metabolic functions. This category of elements can be classified into macrominerals, including sodium (Na), potassium (K), calcium (Ca), magnesium (Mg), and phosphorus (P), which are needed in more amounts by the human body, and trace elements, such as copper (Cu), iron (Fe), manganese (Mn), and zinc (Zn), which are required in lesser amounts

[0021] , Watercress is a good source of macro minerals such as K and Ca, a 100 g serving of watercress contributes 5.74% of the RDA of K and 15.10% of the RDA of Ca. Watercress also contains trace Cu, iron Fe, Mn, and Zn, which are important for the body's functions.

[0095] In an embodiment, according to preliminary tests in Table 6, MHG can extract some mineral content. The largest amount of minerals was retained in the biomass, which is interesting because the biomass can be transformed into flour that is rich in minerals. These essential elements are important for the organism's good physiological functioning.Antihypertensive Activity and a-Glucosidase Inhibitory Activity

[0096] Beyond PEITC, cruciferous vegetables are a source of indole-3-carbinol. This phytochemical demonstrated cardioprotective, antioxidant, and anti-inflammatory effects against the cardiotoxicity of doxorubicin

[0022] ,

[0097] In an embodiment, the angiotensin-converting enzyme-1 inhibition (iACE) assay was conducted according to the procedure described by Coscueta et al. (2019)

[0023] and the iACE concentration required to inhibit 50% of the enzymatic activity, (half-maximal inhibitory concentration, IC50) was quantified (Fig. 3). Besides, natural food extracts like flavonoids, alkaloids, terpenoids, anthocyanins, glycosides, and phenolic compounds can enhance the nutritional value of food and prevent health problems. They can inhibit enzymes that turn carbohydrates into glucose and control postprandial hyperglycemia. The potential cr-glucosidase inhibitory activity of the extracts was also evaluated. The oc-glucosidase inhibition (IC50) was calculated according to the procedure described by Oliveira et al. (2022)

[0024] (Fig. 4). The results of both activities are expressed in GAE (mg / ml extract).

[0098] MHG extraction - Watercress by-products new batch

[0099] In an embodiment, a new batch of watercress by-products was used for the extraction process according to the optimal conditions previously mentioned (1.91 W / g and 29 minutes).Table 7. Phenethyl isothiocyanate amount, antioxidant activity, phenolic compounds amount from a new batch of watercress by-products (n=3).

[0100] Fig. 5 and Fig. 6 shows the MHG extracts (PLF - purple liquid fraction and WLF - white liquid fraction) and the watercress biomass / residue after MHG extraction.Zeta potential

[0101] In an embodiment, the zeta potential of slurry particles is used to gauge their stability in the colloidal sense, which can affect bioaccessibility. The potential zeta was determined according to Coscueta et al. (2021)

[0025] , It was observed that the zeta potential increased as the content of hydrophobic compounds, such as PEITC, increased, resulting in a better value for the white fraction compared to the purple (Fig. 7).Antimicrobial activity

[0102] In an embodiment, regarding the antimicrobial activity of MHG extracts, MHG extracts (PLF and WLF) were not subjected to any post -treatment to reduce microorganisms. Fig. 8 and Fig. 9 showed that no microorganism growth was observed in WLF, which is naturally sterile. Therefore, this extract was used for antimicrobial testing. The three Gram-negative food contaminants or pathogenic bacteria used were Escherichia coli ATCC® 8739™, Salmonella enterica serovar Enteritidis ATCC 13076, and Pseudomonas aeruginosa ATCC® 27853. The Gramnegative food contaminant or pathogenic bacteria used: Staphylococcus aureus ATCC 25923. According to the results (Fig. 10), the WLF extract also inhibited gastrointestinal pathogenic bacteria, including proteobacteria, which are involved in increased metabolic disorders and inflammatory bowel disease. The results are consistent with the conclusions reached by Abrantes et al. (2024)

[0010] , Thus, the extract of watercress leaves (WLF) contains phenethyl isothiocyanate (PEITC) and other bioactive compounds. This contributed to the reduction in growth of all tested microorganisms when exposed to a concentration of 331 pM PEITC.Cruciferous extracts

[0103] In an embodiment, the MHG extraction method was applied to other cruciferous species beyond the watercress: turnip, turnip greens, and broccoli since the literature mentions that broccoli and turnip also contain PEITC[11,26], The extract from each vegetable was collected in a single flask. PEITC was also identified and quantified in turnip extract. Broccoli and turnip greens possibly also have other isothiocyanates.Table 8. Phenethyl isothiocyanate amount, antioxidant activity, phenolic compounds amount, and protein of turnip, turnip greens, broccoli, and watercress (n-3).ND - not detected.

[0104] In an embodiment, each extracted sample was analysed, and the results obtained are presented in Fig.12. ITCs were identified based on UV spectra. The UV spectra of ITCs, in most cases, displayed similar shapes with three maxima of absorption at about 240, 250, and 270 nm. The UV spectra ITC in the range of 220-400 nm

[0027] .Microbiological quality

[0105] In an embodiment, the impact of this extraction method on the final products' microbiological quality was assessed by evaluating the biomass's microbiological quality and extracts before and after extraction. For the microbiological analyses, 10 grams of each sample were homogenized with 90 g of sterile peptone water (1 g / L) (Thermo Fisher Scientific, Massachusetts, EUA), using a stomacher (Seward, Worthing, UK) for 90 s. Plate Count Agar (PCA) was used to analyse the existence of total mesophilic aerobic bacteria; Violet Red Bile Glucose Agar (VRBGA) for the presence of Enterobacteriaceae; MacConkey for Escherichia coli; Bacillus Cereus Agar to see the presence of Bacillus cereus and, lastly, Rose Bengal Chloramphenicol Agar to see yeasts and molds. Serial dilutions of the homogenized samples from 10-2to 10-7were made and transferred to the culture mediums

[0020] . The results were expressed in log colony forming units (CFU) / g of cruciferous vegetable fresh weight (FW).Table 9. Results of microbiological quality of cruciferous before and after MHG extraction process.Each value was expressed as mean + standard deviation n = 8 before MHG; n = 3 after MHG). not detected.

[0106] Studies have indicated that microwaves are effective in inactivating microorganisms in different food products, with no pathogenic bacteria showing resistance to microwave heating

[0028] , The radiation heats the water in the vegetable cells, leading to cell rupture at high temperatures. This can reduce microorganisms since only a few can survive such conditions. Therefore, the products obtained through MHG, such as biomass and extracts, are safe from a microbiological perspective and can be used in products intended for human consumption.

[0107] In an embodiment, the MHG extraction process significantly reduced the number of microorganisms in the biomass. Samples did not show any presence of Enterobacteriaceae, Escherichia coli, Bacillus cereus, and total yeasts and molds. After extraction, the biomass of turnip and turnip greens showed some growth of TMAB, but in reduced amounts compared to before processing. Regarding extracts, finding a few microorganisms after the MHG process is due to the liquid collected not having been in the reactor long enough to be properly sterilized, as it started being collected for the flask at 50°C in the first minutes of the extraction process. Additionally, some compounds, such as sugars, in the extracts can promote microbial growth. On the other hand, the biomass is outside the reactor and exposed to radiation and higher temperatures for a longer time, which helps prevent microbial growth. Therefore, the MHG process was efficient in reducing the formation of colonies in different samples.

[0108] The term "comprising" whenever used in this document is intended to indicate the presence of stated features, integers, steps, components, but not to preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

[0109] As used in the specification and claims, the singular forms "a", "an" and "the" include plural references unless the context clearly dictates otherwise. Forexample, the term "a sample" includes a plurality of samples, including mixtures thereof.

[0110] Furthermore, where the claims recite a composition, it is to be understood that methods of using the composition for any of the purposes disclosed herein are included, and methods of making the composition according to any of the methods of making disclosed herein or other methods known in the art are included, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise.

[0111] Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and / or the understandingof one of ordinary skill in the art, values that are expressed as ranges can assume any specific value within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. It is also to be understood that unless otherwise indicated or otherwise evident from the context and / or the understanding of one of ordinary skill in the art, values expressed as ranges can assume any subrange within the given range, wherein the endpoints of the subrange are expressed to the same degree of accuracy as the tenth of the unit of the lower limit of the range.

[0112] The disclosure should not be seen in any way restricted to the embodiments described and a person with ordinary skill in the art will foresee many possibilities to modifications thereof.

[0113] The embodiments described above are combinable.

[0114] The following claims further set out particular embodiments of the disclosure.References1. Palliyaguru DL, Yuan JM, Kensler TW, Fahey JW. Isothiocyanates: Translating the Power of Plants to People. Mol Nutr Food Res. 2018 Sep;62(18):1700965.2. National Center for Biotechnology Information. Phenethyl isothiocyanate [Internet], PubChemCompound Summary for CID 16741. 2005. Available from: https: / / pubchem.ncbi.nlm.nih.gov / compound / Phenethyl-isothiocyanate3. Troncoso-Rojas R, Tiznado-Hernandez ME. Alternaria alternata (Black Rot, Black Spot). Postharvest Decay: Control Strategies. 2014 Jan;147-87.4. Sundaram MK, R P, Haque S, Akhter N, Khan S, Ahmad S, et al. Dietary isothiocyanates inhibit cancer progression by modulation of epigenome. Semin Cancer Biol. 2022 Aug;83:353-76.5. Coscueta ER, Sousa AS, Reis CA, Pintado MM. Phenylethyl Isothiocyanate: A Bioactive Agent for Gastrointestinal Health. Molecules. 2022 Jan 25;27(3):794.6. Esteve M. Mechanisms Underlying Biological Effects of Cruciferous Glucosinolate-Derived Isothiocyanates / lndoles: A Focus on Metabolic Syndrome. Front Nutr. 2020 Sep 2;7.7. Coscueta ER, Reis CA, Pintado M. Phenylethyl Isothiocyanate Extracted from Watercress By- Products with Aqueous Micellar Systems: Development and Optimisation. Antioxidants. 2020 Aug;9(8):698.8. Rodrigues L, Silva I, Poejo J, Serra AT, Matias AA, Simph'cio AL, et al. Recovery of antioxidant and antiproliferative compounds from watercress using pressurized fluid extraction. RSC Adv. 2016;6(37):30905-18.9. Park J E, Sun Y, Lim SK, Tam JP, Dekker M, Chen H, et al. Dietary phytochemical PEITC restricts tumor development via modulation of epigenetic writers and erasers. Scientific Reports 2017 7:1. 2017 Jan;7(l):l-13.Abrantes R, Ramos CC, Coscueta ER, Costa J, Gomes J, Gomes C, et al. Effects of microencapsulated phenethyl isothiocyanate on gastrointestinal cancer cells and pathogenic bacteria. Food Biosci. 2024 Oct l;61:104950. Karanikolopoulou S, Revelou PK, Xagoraris M, Kokotou MG, Constantinou-Kokotou V. Current Methods for the Extraction and Analysis of Isothiocyanates and Indoles in Cruciferous Vegetables. Analytica. 2021 Sep 24;2(4):93-120. Chemat F, Abert-vian M, Fabiano-tixier AS, Strube J, Uhlenbrock L, Gunjevic V, et al. Green extraction of natural products. Origins, current status, and future challenges. Trends in Analytical Chemistry. 2019;118:248-63. Vian MA, Fernandez X, Visinoni F, Chemat F. Microwave hydrodiffusion and gravity, a new technique for extraction of essential oils. J Chromatogr A. 2008 May;1190(l-2):14-7. Vilas-Boas AA, Campos DA, Nunes C, Ribeiro S, Nunes J, Oliveira A, et al. Polyphenol Extraction by Different Techniques for Valorisation of Non-Compliant Portuguese Sweet Cherries towards a Novel Antioxidant Extract. Sustainability. 2020 Jul 10;12(14):5556. Li D, Zhu Z, Sun DW. Effects of freezing on cell structure of fresh cellular food materials: A review. Trends Food Sci Technol. 2018 May;75:46-55. Pither RJ. CANNING | Quality Changes During Canning. In: Encyclopedia of Food Sciences and Nutrition. Elsevier; 2003. p. 845-51. Coscueta ER, Pellegrini Malpiedi L, Nerli BB. Micellar systems of aliphatic alcohol ethoxylates as a sustainable alternative to extract soybean isoflavones. Food Chem. 2018 Oct;264:135-41. Gomez-Garcia R, Campos DA, Oliveira A, Aguilar CN, Madureira AR, Pintado M. A chemical valorisation of melon peels towards functional food ingredients: Bioactives profile and antioxidant properties. Food Chem. 2021 Jan 15;335:127579. Castro LMG, Ribeiro TB, Alexandre EMC, Saraiva JA, Pintado M. Unveiling the phytochemical nature of acorns: the relevance of dehusking. Food Funct. 2022;13(12):6636-47. Margal S, Sousa S, Araujo-Rodrigues H, Silva I V., Campos DA, Pintado M. Impact of washing and freezing on nutritional composition, bioactive compounds, antioxidant activity and microstructure of mango peels. Food Chem. 2024 Jun;442:138368. Pinela J, Carvalho AM, Ferreira ICFR. Watercress. In: Nutritional Composition and Antioxidant Properties of Fruits and Vegetables. Elsevier; 2020. p. 197-219. Prado NJ, Ramirez D, Mazzei L, Parra M, Casarotto M, Calvo JP, et al. Anti-inflammatory, antioxidant, antihypertensive, and antiarrhythmic effect of indole-3-carbinol, a phytochemical derived from cruciferous vegetables. Heliyon. 2022 Feb;8(2):e08989. Coscueta ER, Campos DA, Osorio H, Nerli BB, Pintado M. Enzymatic soy protein hydrolysis: A tool for biofunctional food ingredient production. Food Chem X. 2019 Mar;l:100006.Oliveira ALS, Carvalho MJ, Oliveira DL, Costa E, Pintado M, Madureira AR. Sugarcane Straw Polyphenols as Potential Food and Nutraceutical Ingredient. Foods. 2022 Dec 13;ll(24):4025. Coscueta ER, Sousa AS, Reis CA, Pintado M. Chitosan-olive oil microparticles for phenylethyl isothiocyanate delivery: Optimal formulation. PLoS One. 2021 May 6;15(5):e0248257. M. Ezzat S, M. 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Claims

C L A I M S1. Method for obtaining isothiocyanate extract in less than 30 minutes comprising the steps of: obtaining frozen cruciferous vegetable; subjecting the frozen vegetable to microwave hydrodiffusion and gravity extraction to obtain a liquid extract and a solid plant biomass; collecting the liquid extract to obtain an isothiocyanate extract.

2. The method according to the previous claim, wherein the microwave hydrodiffusion and gravity extraction comprises the steps of: heating the frozen vegetable in a microwave reactor to obtain a crude extract and a solid plant biomass; cooling and collecting the obtained crude extract in a condenser in order to obtain an isothiocyanate rich extract.

3. The method according to any of the previous claims, wherein the extraction process time ranges from 10 - 30 minutes; preferably 15 - 30 minutes; more preferably 20 - 30 minutes.

4. The method according to any of the previous claims, wherein the isothiocyanate is phenethyl isothiocyanate.

5. The method according to any of the previous claims, wherein the extraction is performed at a temperature less than 97 °C; preferably less than 94 °C; more preferably less than 92 °C.

6. The method according to any of the previous claims, wherein the extraction is performed at a temperature ranging from 80 - 99 °C; preferably 92 - 96 °C.

7. The method according to any of the previous claims, wherein the frozen cruciferous vegetable is frozen watercress.

8. The method according to any of the previous claims, wherein the isothiocyanate rich extract comprises between 400 - 2400 pg phenethyl isothiocyanate / g watercress dry basis; preferably 400 - 1500 pg phenethyl isothiocyanate / g watercress dry basis.

9. The method according to any of the previous claims, wherein the isothiocynate-rich extract comprises two liquid fractions.

10. The method according to the previous claim, wherein the two liquid fractions are a purple liquid fraction and a white liquid fraction.

11. The method according to the previous claim 7, wherein the purple liquid fraction comprises at least 26.86 pmol trolox eq. / g watercress dry basis of oxygen radical absorbance capacity.

12. The method according to the previous claim 7, wherein the purple liquid fraction comprises at least 8.55 pmol trolox eq. / g watercress dry basis of ABTS radical scavenging activity.

13. The method according to the previous claim 7, wherein the white liquid fraction comprises at least 10.01 pmol trolox eq. / g watercress dry basis of oxygen radical absorbance capacity.

14. The method according to the previous claim 7, wherein the white liquid fraction comprises at least 4.71 pmol trolox eq. / g watercress dry basis of ABTS radical scavenging activity.

15. The method according to any of the previous claims, wherein the frozen cruciferous vegetable is a cruciferous vegetable frozen at -18 °C.

16. The method according to any of the previous claims, wherein the microwave reactor is at a fixed microwave power ranging from 1.5 - 2.5 W / g.

17. The method according to any of the previous claims, wherein the biomass is a watercress biomass.

18. A phenethyl isothiocyanate extract obtained by the method according to any of the previous claims.

19. The phenethyl isothiocyanate extract according to the previous claim, wherein the isothiocyanate-rich extract comprises between 400 - 2400 pg phenethyl isothiocyanate / g watercress dry basis and 394 pg phenethyl isothiocyanate / g turnip dry basis; preferably 400 - 1500 pg phenethyl isothiocyanate / g watercress dry basis.

20. The phenethyl isothiocyanate extract according to any of the previous claims 18-19, wherein the extract comprises a purple liquid fraction and a white liquid fraction.

21. The phenethyl isothiocyanate extract according to the previous claim, wherein the purple liquid fraction comprises at least 26.86 pmol trolox eq. / g watercress dry basis of oxygen radical absorbance capacity.

22. The phenethyl isothiocyanate extract according to the previous claim, wherein the purple liquid fraction comprises at least 8.55 pmol trolox eq. / g watercress dry basis of ABTS radical scavenging activity.

23. The phenethyl isothiocyanate extract according to the previous claim, wherein the white liquid fraction comprises at least 10.01 pmol trolox eq. / g watercress dry basis of oxygen radical absorbance capacity.

24. The phenethyl isothiocyanate extract according to the previous claim, wherein the white liquid fraction comprises at least 4.71 pmol trolox eq , / g watercress dry basis of ABTS radical scavenging activity.

25. A functional flour obtained by the method according to any of the previous claims 1-17.

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