Optimization of a Microencapsulation Process Using Oil-in-Water (O/W) Emulsion to Increase Thermal Stability of Sulforaphane.

Sulforaphane (SFN) is a bioactive compound widely studied for its potential applications in pharmaceutical, nutraceutical, and food industries since it offers health benefits due to its nature as a Phase 2 enzyme inducer. Its application in the food industry has been limited because SFN is unstable at high temperatures in an aqueous milieu. An option to increase SFN stability and protect it from thermal degradation is microencapsulation. The aim of this work was to optimize a microencapsulation process using oil-in-water emulsion to increase the thermal stability of SFN. The operation conditions that gave the highest entrapment efficiency were determined via experimental design and response surface methodology. Thermal degradation of microencapsulated SFN was studied at 37, 50, 60, and 70 °C. The optimum microencapsulation conditions were 8 min stirring, SFN/Gum Arabic ratio of 0.82, and surfactant/oil ratio of 1.0, resulting in an entrapment efficiency of 65%, which is the highest reported so far. The thermal stability of microencapsulated SFN was greatly enhanced compared with free SFN, with a 6-fold decrease in the degradation kinetic constant and a 41% increase in the activation energy. These results will contribute to a more efficient incorporation of SFN in various food matrices and explore new microencapsulation technologies to maximize the efficiency and stability of SFN.

Maximization of Sulforaphane Content in Broccoli Sprouts by Blanching.

Broccoli sprouts are a recognized source of health-promoting compounds, such as glucosinolates, glucoraphanin, and sulforaphane (SFN). Maximization of SFN content can be achieved by technological processing. We investigated the effect of blanching conditions to determine the optimal treatment that maximizes sulforaphane content in broccoli sprouts. Broccoli seeds (cv. Traditional) grown under controlled conditions were harvested after 11 days from germination and subjected to different blanching conditions based on a central composite design with temperature and time as experimental factors. Results were analyzed by ANOVA followed by a Tukey test. The optimum conditions were identified through response surface methodology. Blanching increased sulforaphane content compared with untreated sprouts, agreeing with a decrease in total glucosinolates and glucoraphanin content. Temperature significantly affected SFN content. Higher temperatures and shorter immersion times favor glucoraphanin hydrolysis, thus increasing SFN content. The optimum conditions were blanching at 61 °C for 4.8 min, resulting in 54.3 ± 0.20 µmol SFN/g dry weight, representing a 3.3-fold increase with respect to untreated sprouts. This is the highest SFN content reported for sprouts subjected to any treatment so far. The process described in this work may contribute to developing functional foods and nutraceuticals that provide sulforaphane as an active principle.

Broccoli Myrosinase cDNA Expression in Escherichia coli and Saccharomyces cerevisiae.

Myrosinases (EC 3.2.1.147) are enzymes known for the generation of hydrolysis products that have a potential beneficial effect on human health. Their reaction mechanisms are widely studied, in order to improve and optimize secondary metabolite production processes. In this work, kinetic and biochemical properties of the broccoli myrosinase enzyme produced from its cDNA cloned in Escherichia coli and Saccharomyces cerevisiae were investigated. The results revealed that the thermal stability of the enzyme produced in S. cerevisiae was slightly higher (30 to 60 °C) than that of myrosinase produced in E. coli (20 to 50 °C). The effect of pH on the enzymatic activity was similar in both enzymes, with pH 3 being the optimum value under the reaction conditions used. The kinetic behavior of both enzymes was adjusted to the Michaelis-Menten model. The catalytic efficiency was up to 4 times higher in myrosinase produced in S. cerevisiae, compared to myrosinase produced in E. coli. The glycosylations present in the enzyme would be related to the formation of a dimeric quaternary structure and would not play an essential role in enzymatic activity, since both enzymes were biologically active. These results will probably allow the development of strategies for the production of bioactive metabolites of medical interest.

Optimization of an Extraction Process to Obtain a Food-Grade Sulforaphane-Rich Extract from Broccoli (Brassica oleracea var. italica).

Sulforaphane (SFN) is a powerful health-promoting compound found in broccoli in the form of its inactive precursor, glucoraphanin (GFN). SFN formation occurs through the enzymatic hydrolysis of glucoraphanin by myrosinase under specific chemical conditions. Its incorporation in food formulations has been hindered by the thermal instability of SFN and low concentration in Brassicaceae. Then, extracting SFN from broccoli at a temperature below 40 °C appears as an option to recover and stabilize SFN, aiming at delivering it as a nutraceutical. We studied an eco-friendly extraction process to obtain an SFN-rich extract from broccoli. The effect of the broccoli mass/solvent ratio, ethanol concentration in the extractant solution, and extraction time on the recovery of SFN, GFN, phenolic compounds, and antioxidant activity were studied through a Box-Behnken design. The regression models explained more than 70% of the variability in the responses, adequately representing the system. The experimental factors differently affected the bioactive compound recovery and antioxidant activity of the extracts. The extraction conditions that allowed the highest recovery of bioactive compounds and antioxidant activity were identified and experimentally validated. The results may provide the basis for the design of a process to produce a sulforaphane-rich food supplement or nutraceutical by using a GRAS extractant.

Comparative Analysis of Conventional and Emerging Technologies for Seawater Desalination: Northern Chile as A Case Study.

The aim of this work was to study different desalination technologies as alternatives to conventional reverse osmosis (RO) through a systematic literature review. An expert panel evaluated thermal and membrane processes considering their possible implementation at a pilot plant scale (100 m3/d of purified water) starting from seawater at 20 °C with an average salinity of 34,000 ppm. The desalination plant would be located in the Atacama Region (Chile), where the high solar radiation level justifies an off-grid installation using photovoltaic panels. We classified the collected information about conventional and emerging technologies for seawater desalination, and then an expert panel evaluated these technologies considering five categories: (1) technical characteristics, (2) scale-up potential, (3) temperature effect, (4) electrical supply options, and (5) economic viability. Further, the potential inclusion of graphene oxide and aquaporin-based biomimetic membranes in the desalinization processes was analyzed. The comparative analysis lets us conclude that nanomembranes represent a technically and economically competitive alternative versus RO membranes. Therefore, a profitable desalination process should consider nanomembranes, use of an energy recovery system, and mixed energy supply (non-conventional renewable energy + electrical network). This document presents an up-to-date overview of the impact of emerging technologies on desalinated quality water, process costs, productivity, renewable energy use, and separation efficiency.

Potential of Sulforaphane as a Natural Immune System Enhancer: A Review.

Brassicaceae are an outstanding source of bioactive compounds such as ascorbic acid, polyphenols, essential minerals, isothiocyanates and their precursors, glucosinolates (GSL). Recently, GSL gained great attention because of the health promoting properties of their hydrolysis products: isothiocyanates. Among them, sulforaphane (SFN) became the most attractive one owing to its remarkable health-promoting properties. SFN may prevent different types of cancer and has the ability to improve hypertensive states, to prevent type 2 diabetes-induced cardiomyopathy, and to protect against gastric ulcer. SFN may also help in schizophrenia treatment, and recently it was proposed that SFN has potential to help those who struggle with obesity. The mechanism underlying the health-promoting effect of SFN relates to its indirect action at cellular level by inducing antioxidant and Phase II detoxifying enzymes through the activation of transcription nuclear factor (erythroid-derived 2)-like (Nrf2). The effect of SFN on immune response is generating scientific interest, because of its bioavailability, which is much higher than other phytochemicals, and its capacity to induce Nrf2 target genes. Clinical trials suggest that sulforaphane produces favorable results in cases where pharmaceutical products fail. This article provides a revision about the relationship between sulforaphane and immune response in different diseases. Special attention is given to clinical trials related with immune system disorders.

Effect of Drum-Drying Conditions on the Content of Bioactive Compounds of Broccoli Pulp.

This work studied the effect of drum-rotation frequency, drum temperature, and water-to-pulp ratio in a double-drum drier on the content of sulforaphane, glucoraphanin, total phenolic compounds, ascorbic acid, and antioxidant activity of broccoli pulp through a multilevel factorial design with one replicate. Drum-drying conditions did not significantly affect sulforaphane content, unlike glucoraphanin, however the poor adherence of broccoli pulp resulted in a final product with undefined shape and heterogeneous color. On the other hand, antioxidant activity was unevenly affected by drying conditions; however, drum-rotation frequency affected it in the same way that phenolic compounds and ascorbic acid, showing a concordant behavior. The ascorbic acid content decreased significantly after drying, and it was highly dependent on the experimental factors, resulting in a regression model that explained 90% of its variability. Drum-rotation frequency of 5 Hz, drum temperature of 125 °C, and water-to-pulp ratio of 0.25 resulted in an apparent increase of sulforaphane and phenolic compounds content of 13.7% and 47.6%, respectively. Drum drying has great potential to fabricate dehydrated broccoli-based foods with functional properties. Besides, since drum drying has low investment and operation costs, it represents a very attractive option for the industrialization of broccoli derivatives.

Molecular Modeling of Epithiospecifier and Nitrile-Specifier Proteins of Broccoli and Their Interaction with Aglycones.

Glucosinolates are secondary plant metabolites of Brassicaceae. They exert their effect after enzymatic hydrolysis to yield aglycones, which become nitriles and epithionitriles through the action of epithiospecifier (ESP) and nitrile-specifier proteins (NSP). The mechanism of action of broccoli ESP and NSP is poorly understood mainly because ESP and NSP structures have not been completely characterized and because aglycones are unstable, thus hindering experimental measurements. The aim of this work was to investigate the interaction of broccoli ESP and NSP with the aglycones derived from broccoli glucosinolates using molecular simulations. The three-dimensional structure of broccoli ESP was built based on its amino-acid sequence, and the NSP structure was constructed based on a consensus amino-acid sequence. The models obtained using Iterative Threading ASSEmbly Refinement (I-TASSER) were refined with the OPLS-AA/L all atom force field of GROMACS 5.0.7 and were validated by Veryfy3D and ERRAT. The structures were selected based on molecular dynamics simulations. Interactions between the proteins and aglycones were simulated with Autodock Vina at different pH. It was concluded that pH determines the stability of the complexes and that the aglycone derived from glucoraphanin has the highest affinity to both ESP and NSP. This agrees with the fact that glucoraphanin is the most abundant glucosinolate in broccoli florets.

Insights about stabilization of sulforaphane through microencapsulation.

The health-promoting properties of sulforaphane (SFN) are well known, however its instability is still a hurdle for its incorporation into food matrices. SFN can be stabilized by microencapsulation, technique sparingly explored for isothiocyanates so far. This review summarizes the advances in microencapsulation of SFN and other isothiocyanates. Encapsulation efficiency and degradation rate of sulforaphane in different systems are compared and discussed. Ionic gelation and complex coacervation seem more adequate for SFN, both underexplored until now. Drying conditions after chemical encapsulation are determinant, most likely related to thermal degradation of SFN. The current information is insufficient to identify the most adequate encapsulation system and the optimal process conditions to stabilize SFN aiming at its incorporation into food matrices. Accordingly, encapsulation conditions should be investigated, which arises as a new research line. Stability studies are encouraged since this information will help in designing SFN microencapsulation strategies that extend the industrial application of this promising health-promoting compound.

Kinetic study of sulforaphane stability in blanched and un-blanched broccoli (Brassica oleracea var. italica) florets during storage at low temperatures.

Sulforaphane is a health-promoting compound found in broccoli. Given its high thermo-lability, its preservation through high-temperature processes seems inconvenient. Accordingly, storage at low temperature is an alternative. There are no studies about the evolution of sulforaphane content during storage at low temperatures. The change of sulforaphane content in blanched and un-blanched broccoli florets during storage at 10, - 1, - 21 and - 45 °C for 83 days was studied. In blanched broccoli, sulforaphane content followed a first-order degradation kinetics (R2 ≥ 0.95). A two-consecutive irreversible reactions model described adequately the evolution of sulforaphane content in un-blanched broccoli (R2 ≥ 0.94). Activation energies from Arrhenius equation resulted in 19.4 kJ/mol for blanched and 30 kJ/mol (formation) and 58 kJ/mol (degradation) for un-blanched broccoli. Storage of un-blanched broccoli at - 45 °C for 40 days maximized sulforaphane content. These results could be useful to propose broccoli storage conditions that preserve or maximize sulforaphane content.

Kinetic and structural study of broccoli myrosinase and its interaction with different glucosinolates.

Myrosinase is a glycosylated enzyme present in the Brassicaceae family that catalyzes the hydrolysis of glucoraphanin to yield sulforaphane, recognized as a health-promoting compound found in cruciferous foods. Broccoli myrosinase has been poorly characterized. In this work, the enzyme was purified from broccoli florets and its kinetic behaviour was analyzed. The cDNA of broccoli myrosinase was isolated and sequenced to obtain the amino acids sequence of the enzyme. A three-dimensional structural model of a broccoli myrosinase subunit was built and used to perform molecular docking simulations with glucoraphanin and other glucosinolates. Kinetic data were adjusted to the Two-Binding Sites Model that describes substrate inhibition, obtaining R2 higher than 97%. The docking simulations confirmed the existence of two substrate-binding sites in the monomer, and allowed identifying the residues that interact with the substrate in each site. Our findings will help to design strategies to better exploit the health-promoting properties of broccoli.

Modelling of the effect of selenium fertilization on the content of bioactive compounds in broccoli heads.

Selenium (Se) exerts many effects beneficial to health. Broccoli is a Se-hyperaccumulator plant, with Se-fertilization increasing its potential as a functional food. We studied the effect of dose, and the developmental stage at the beginning of Se-fortification, on antioxidant capacity, phenolics, glucosinolates, sulphoraphane, Se-methyl selenocysteine and myrosinase in broccoli. Se-fortification decreased the antioxidant properties and sulphur-containing compounds, but increased Se-methyl-selenocysteine content. Regression models gave r>0.77 confirming that Se dose and developmental stage largely determine the behaviour of the system. Correlation models gave r>0.95, allowing estimation of saturation concentration of Se-methyl-selenocysteine in broccoli cv. Traditional (3.13µmolg-1DM) and the concentration (2-mmol sodium selenate) above which the content of phenolic compounds decreases significantly. Sulphoraphane and glucosinolates' dependence on total Se supply was consistent with myrosinase activity below 3.5-mmol sodium selenate. Our results would enable design of optimal fertilization strategies to enrich broccoli in Se with minimal impairment of antioxidants properties.

Optimization of an incubation step to maximize sulforaphane content in pre-processed broccoli.

Sulforaphane is a powerful anticancer compound, found naturally in food, which comes from the hydrolysis of glucoraphanin, the main glucosinolate of broccoli. The aim of this work was to maximize sulforaphane content in broccoli by designing an incubation step after subjecting broccoli pieces to an optimized blanching step. Incubation was optimized through a Box-Behnken design using ascorbic acid concentration, incubation temperature and incubation time as factors. The optimal incubation conditions were 38 °C for 3 h and 0.22 mg ascorbic acid per g fresh broccoli. The maximum sulforaphane concentration predicted by the model was 8.0 µmol g-1, which was confirmed experimentally yielding a value of 8.1 ± 0.3 µmol g-1. This represents a 585% increase with respect to fresh broccoli and a 119% increase in relation to blanched broccoli, equivalent to a conversion of 94% of glucoraphanin. The process proposed here allows maximizing sulforaphane content, thus avoiding artificial chemical synthesis. The compound could probably be isolated from broccoli, and may find application as nutraceutical or functional ingredient.

Purification and characterization of broccoli (Brassica oleracea var. italica) myrosinase (ß-thioglucosidase glucohydrolase).

Myrosinase (ß-thioglucosidase glucohydrolase, EC 3.2.1.147) from broccoli (Brassica oleracea var. italica) was purified by ammonium sulfate precipitation followed by concanavalin A affinity chromatography, with an intermediate dialysis step, resulting in 88% recovery and 1318-fold purification. These are the highest values reported for the purification of any myrosinase. The subunits of broccoli myrosinase have a molecular mass of 50-55 kDa. The native molecular mass of myrosinase was 157 kDa, and accordingly, it is composed of three subunits. The maximum activity was observed at 40 °C and at pH below 5.0. Kinetic assays demonstrated that broccoli myrosinase is subjected to substrate (sinigrin) inhibition. The Michaelis-Menten model, considering substrate inhibition, gave Vmax equal to 0.246 µmol min(-1), Km equal to 0.086 mM, and K(I) equal to 0.368 mM. This is the first study about purification and characterization of broccoli myrosinase.

Effect of methyl jasmonate, sodium selenate and chitosan as exogenous elicitors on the phenolic compounds profile of broccoli sprouts.

BACKGROUND: Broccoli sprouts are a good source of secondary metabolites, exhibiting biological activity, such as polyphenols, whose concentration is affected by the exposure to exogenous elicitors. The aim of this work was to investigate the effect of sodium selenate, chitosan and methyl jasmonate, applied directly to the seeds or through irrigation, on the content and profile of phenolic compounds in broccoli sprouts. The effect on antioxidant activity was also investigated. RESULTS: Methyl jasmonate and chitosan decreased antioxidant capacity. Methyl jasmonate significantly decreased total polyphenols content in comparison with control sprouts, while chitosan significantly increased it. Sodium selenate had no statistical effect on antioxidant capacity and total polyphenols concentration. The polyphenols profile in sprouts was composed by quercetin, morine, genisteine, luteoline and sinapic acid. Elicitor type and concentration affected the synthesis of these compounds. Chitosan stimulated the synthesis of quercetin, sinapic acid and morine, whereas methyl jasmonate stimulated the synthesis of luteoline. Sodium selenate had no effect on polyphenols synthesis. CONCLUSION: The exposure of broccoli to the elicitors produced changes in the phenolic compounds profile of broccoli sprouts. Besides, the stimulation of phenolic compounds synthesis was elicitor-specific, thus opening the possibility of managing culture conditions to increase the content of a specific phenolic compound.

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.

Changes in SeMSC, glucosinolates and sulforaphane levels, and in proteome profile in broccoli (Brassica oleracea var. Italica) fertilized with sodium selenate.

The aim of this work was to analyze the effect of sodium selenate fortification on the content of selenomethyl selenocysteine (SeMSC), total glucosinolates and sulforaphane, as well as the changes in protein profile of the inflorescences of broccoli (Brassica oleracea var. Italica). Two experimental groups were considered: plants treated with 100 µmol/L sodium selenate (final concentration in the pot) and control plants treated with water. Fortification began 2 weeks after transplantation and was repeated once a week during 10 weeks. Broccoli florets were harvested when they reached appropriate size. SeMSC content in broccoli florets increased significantly with sodium selenate fortification; but total glucosinolates and sulforaphane content as well as myrosinase activity were not affected. The protein profile of broccoli florets changed due to fortification with sodium selenate. Some proteins involved in general stress-responses were up-regulated, whereas down-regulated proteins were identified as proteins involved in protection against pathogens. This is the first attempt to evaluate the physiological effect of fortification with sodium selenate on broccoli at protein level. The results of this work will contribute to better understanding the metabolic processes related with selenium uptake and accumulation in broccoli.

An overview of health-promoting compounds of broccoli (Brassica oleracea var. italica) and the effect of processing.

Broccoli offers many heath-promoting properties owing to its content of antioxidant and anticarcinogenic compounds. The concentration and bioavailability of polyphenols, glucosinolates, sulforaphane and selenium depend on plant biochemistry, cultivation strategy and type of processing. In this article, the main biochemical properties of broccoli are reviewed regarding their health-promoting effects. Additionally, the way these properties are affected by processing is discussed. Steaming and drying result in an apparent increment of sulforaphane content as well as antioxidant activity, most likely due to an increase of the extractability of antioxidants and sulforaphane. Freezing and boiling diminish polyphenols concentration, mainly due to volatilization and leaching into the cooking water. In view of these results, the optimization of broccoli processing in order to maximize the content of bioactive compounds should be possible. The effect of processing on selenium compounds has been poorly studied so far, and therefore this topic should be investigated in the future. Finally, the effect of operating conditions in different drying processes on the content of bioactive compounds in broccoli should be investigated in a greater depth.

Purification of transthyretin as nutritional biomarker of selenium status.

Transthyretin has been proposed as nutritional biomarker of selenium intake. Previous transthyretin purification methods used different procedures to isolate transthyretin either from plasma or from pathological urine of humans. In general, the procedure for purification of transthyretin is laborious and expensive, and extensive sample recycling is necessary for purification in appreciable amounts. This work proposes a new, promissory, and cheap two-step process to purify transthyretin from blood plasma, composed by a first aqueous two-phase system fractionation followed by affinity chromatography, using thyroxine-immobilized on epoxy-activated Sepharose CL-6B. The aqueous two-phase system fractionation was demonstrated to perform better than commercial immunoaffinity-based kits for albumin depletion in blood plasma samples and is an effective first step for transthyretin purification. Thyroxine affinity chromatography was designed to bind transthyretin with high affinity, and was demonstrated to be useful to purify transthyretin, but was unable to completely resolve transthyretin from thyroxine-binding globulin and serum albumin, although the relative amount of albumin was lowered in the eluates. This purification process could be used in nutritional diagnosis tools or as a first step in structural and functional studies.

Depletion of highly abundant proteins in blood plasma by ammonium sulfate precipitation for 2D-PAGE analysis.

Ammonium sulfate precipitation (ASP) was explored as a method for depleting some highly abundant proteins from blood plasma, in order to reduce the dynamic range of protein concentration and to improve the detection of low abundance proteins by 2D-PAGE. 40% ammonium sulfate saturation was chosen since it allowed depleting 39% albumin and 82% α-1-antitrypsin. ASP-depletion showed high reproducibility in 2D-PAGE analysis (4.2% variation in relative abundance of albumin), similar to that offered by commercial affinity-depletion columns. Besides, it allowed detecting 59 spots per gel, very close to the number of spots detected in immuno-affinity-depleted plasma. Thus, ASP at 40% saturation is a reliable depletion method that may help in proteomic analysis of blood plasma. Finally, ASP-depletion seems to be complementary to hydrophobic interaction chromatography (HIC)-depletion, and therefore an ASP-step followed by a HIC-step could probably deplete the most highly abundant plasma proteins, thus improving the detection of low abundance proteins by 2D-PAGE.