Peptide-metal complexes: obtention and role in increasing bioavailability and decreasing the pro-oxidant effect of minerals.

Bioactive peptides derived from food protein sources have been widely studied in the last years, and scientific researchers have been proving their role in human health, beyond their nutritional value. Several bioactivities have been attributed to these peptides, such as immunomodulatory, antimicrobial, antioxidant, antihypertensive, and opioid. Among them, metal-binding capacity has gained prominence. Mineral chelating peptides have shown potential to be applied in food products so as to decrease mineral deficiencies since peptide-metal complexes could enhance their bioavailability. Furthermore, many studies have been investigating their potential to decrease the Fe pro-oxidant effect by forming a stable structure with the metal and avoiding its interaction with other food constituents. These complexes can be formed during gastrointestinal digestion or can be synthesized prior to intake, with the aim to protect the mineral through the gastrointestinal tract. This review addresses: (i) the amino acid residues for metal-binding peptides and their main protein sources, (ii) peptide-metal complexation prior to or during gastrointestinal digestion, (iii) the function of metal (especially Fe, Ca, and Zn)-binding peptides on the metal bioavailability and (iv) their reactivity and possible pro-oxidant and side effects.

Sterols in infant formulas: validation of a gas chromatographic method.

Sterols are components present in the fat fraction of infant formulas (IFs). Their characterization is therefore of interest, though there are no official reference methods for their analysis in these matrices. AIM: To validate a gas chromatographic method with flame ionization detection for the determination of animal (cholesterol and desmosterol) and plant sterols (brassicasterol, campesterol, stigmasterol, ß-sitosterol and sitostanol) found in IFs. All correlation coefficients obtained for the calibration curves of sterols studied were >0.99. Limits of detection (<1 µg/100 mL) and quantification (<4 µg/100 mL) are suitable for sterols determination in IFs. The within-assay precision ranged from 1.6% to 8.8%, while the between-assay precision was <10% for most of sterols. Accuracy was satisfactory and was calculated by recovery assays (ranging 93-108%). The analytical parameters obtained showed the suitability of the proposed method for the determination of sterols in IFs.

Phospholipids in Human Milk and Infant Formulas: Benefits and Needs for Correct Infant Nutrition.

The composition of human milk has served as a basis for the development of infant formulas, which are used when breastfeeding is not possible. Among the human milk nutrients, 50% of the total energetic value corresponds to fat, with a high level of fatty acids and 0.2-2.0% present in the form of phospholipids (PLs). The PL contents and fatty acid distribution in PL species have been investigated as bioactive elements for the production of infant formulas, since they offer potential benefits for the optimum growth and health of the newborn infant. The differences in the amount of PLs and in fatty acid distribution in PL species between human milk and infant formulas can imply biologically significant differences for newborn infants fed with infant formulas versus human milk-mainly due to the greater proportion of sphingomyelin with respect to phosphatidylcholine in infant formulas. The limited information referred to the characterization of fatty acid distribution in PL species in infant formulas or in ingredients used to enrich them merits further research in order to obtain products with benefits similar to those of human milk in terms of infant growth, visual acuity, and neurological development. The present review establishes the scientific basis for helping to adjust formulations to the requirements of infant nutrition.

Chemopreventive Effect of an In Vitro Digested and Fermented Plant Sterol-Enriched Wholemeal Rye Bread in Colon Cancer Cells.

Diet is crucial for the prevention of colorectal cancer. Whole grains are the source of beneficial compounds for this, such as fiber. The enrichment of wholemeal rye bread with plant sterols (PSs) could increase its beneficial effects. This study aimed to assess the potential antiproliferative effect of this enriched food on colon adenocarcinoma cells (Caco-2) compared with a non-enriched one. After a human oral chewing, simulated semi-dynamic gastrointestinal digestion and colonic fermentation in a simgi® system, fermentation liquids (FLs) obtained were used as treatment for cells. Cytotoxicity assay showed that samples diluted 1/5 (v/v) with DMEM are not toxic for non-tumoral cells, whereas they damage tumoral cells. Samples with PS (FLPS) produced a higher chemopreventive effect (vs. blank) in MTT and apoptosis assays, as well as higher gene expression of TP53 and Casp8. Nevertheless, FL0 (without PS) produced a higher chemopreventive effect in a cell cycle and reduced glutathione and calcium assays, besides producing higher gene expression of Casp3 and lower CCND1. The distinct antiproliferative effect of both FLs is attributed to differences in PSs, short chain fatty acids (lower concentration in FLPS vs. FL0) and antioxidant compounds. These results may support wholemeal rye bread consumption as a way of reducing the risk of colorectal cancer development, although further research would be needed.

Ethylcoprostanol modulates colorectal cancer cell proliferation and mitigates cytotoxicity of cholesterol metabolites in non-tumor colon cells.

Sterols can be metabolized by gut microbiota. The cholesterol metabolites have been proposed as promoters of colorectal cancer (CRC), while the effect of plant sterol metabolites is unknown. This study aimed to evaluate the cytotoxicity of metabolites from cholesterol (coprostanol, cholestanol, coprostanone and cholestenone) and ß-sitosterol (ethylcoprostanol) on human colon tumor (Caco-2) and non-tumor (CCD-18Co) cells at physiological concentrations (9-300 µM) and exposure time (24 h). Ethylcoprostanol reduced the tumor cell proliferation (MTT), showing in flow cytometry assays induction of apoptosis via production of reactive oxygen species (ROS) and ceramide. Transcriptomic analysis (qPCR) showed activation of the intrinsic apoptosis pathway (BAX/BCL2 ratio and CASP9 increased), accompanied by downregulation of the p21 gene. Cholesterol metabolites, mainly the most hydrophobic, induced apoptosis and G0/G1 phase arrest in non-tumor cells through overproduction of ROS. Both the intrinsic and extrinsic (CASP8 increased) apoptosis pathways occurred. In turn, a reduction in the expression of the cyclin E1 gene confirmed the cell cycle arrest. In addition, ethylcoprostanol protected non-tumor cells from the most cytotoxic cholesterol metabolite (cholestenone). In conclusion, ethylcoprostanol is a promising candidate as a therapeutic adjuvant in CRC, while cholesterol metabolites could act as CRC promoters through their cytotoxicity.

Aging-related gastrointestinal conditions decrease the bioaccessibility of plant sterols in enriched wholemeal rye bread: in vitro static digestion.

The prevention of cardiovascular disease using foods fortified with plant sterols (PS), with a hypocholesterolemic effect, is important for the elderly population. This study aimed at identifying the different PS present in PS-enriched wholemeal rye bread (WRB) and in the ingredient source of PS, to evaluate their bioaccessibility in WRB by simulated static digestion. The gastrointestinal conditions of the elderly were adapted, and the results were compared with the adult population. Nine PS were identified, and a total amount of 2.18 g/100 g WRB was determined. Bioaccessibility was reduced in the elderly model with gastrointestinal adaptation vs. the adult model (11.2 vs. 20.3%), but no differences were observed when adapting only the gastric phase. Even though there was lower bioaccessibility of PS in the elderly, they could benefit from the consumption of WRB as it has a good nutritional profile. Further investigation including in vivo assays is needed to strengthen the results.

Sterol bioaccessibility in a plant sterol-enriched beverage using the INFOGEST digestion method: Influence of gastric lipase, bile salts and cholesterol esterase.

This study evaluates the influence of increasing bile salts and the addition of key enzymes of the lipidic metabolism in the INFOGEST digestion method on sterol bioaccessibility from a plant sterol (PS)-enriched beverage. The assayed modifications were increasing concentration of bovine bile salts (10 vs. 17.5 mM), and addition of gastric lipase (GL) (60U/mL), cholesterol esterase (CE) (0.075 or 2U/mL) or both. Compared to the original method (10 mM bile salts without enzymes), the assayed conditions significantly reduced bioaccessibility of individual (from 11.3 to 19.7 to 5.1-16.6%) and total PS (13.7 to 6.9-8.0%), and cholesterol (52.8 to 20.9-26.1%), except only when CE is added not allowing cholesterol quantification. The bioaccessibility achieved when lipolytic enzymes were tested was similar for all sterols. For a more physiological approach to in vivo conditions, incorporation of bile salts (10 mM), GL (60U/mL) and CE (0.075U/mL) to the INFOGEST method is proposed, although it increases the cost compared to the established method.

Elderly gastrointestinal conditions increase sterol bioaccessibility in a plant sterol-enriched beverage: adaptation of the INFOGEST method.

Elderly people suffer from a higher cardiovascular risk. Thus, the fortification of foods with plant sterols (PSs), which have a cholesterol-lowering function, could be of great interest for this target group. To date, no studies have analyzed how the gastrointestinal conditions of the elderly affect PS bioaccessibility. Therefore, this study evaluated the impact of the adaptation of the gastric phase alone and in combination with the intestinal phase on sterol bioaccessibility. For this purpose, the standardized INFOGEST 2.0 method previously adapted for sterol bioaccessibility evaluation in healthy adults was applied to PS-enriched milk-based fruit beverages, examining changes in enzyme activity, incubation time, agitation and pH, based on elderly physiology. The results suggest that the specific gastrointestinal conditions of the elderly could increase absorption of PSs, since their bioaccessibility (%) in a PS-enriched milk-based fruit beverage was significantly increased compared with that in adults (14.95 ± 0.33 vs. 7.96 ± 0.26), also indicating that these conditions increase the bioaccessibility of the beverage's own cholesterol (61.25 ± 2.91 vs. 20.86 ± 2.79). These data support the recommendation of foods of this type for the elderly who can benefit from the increase in bioaccessibility of PSs to have an improved potential cholesterol lowering effect, thus decreasing their risk of cardiovascular disease. However, the performance of subsequent in vivo tests to confirm these results is necessary.

Bioaccessibility of Plant Sterols in Wholemeal Rye Bread Using the INFOGEST Protocol: Influence of Oral Phase and Enzymes of Lipid Metabolism.

Bioaccessibility of plant sterols (PS) in an enriched wholemeal rye bread was evaluated, for the first time, using the INFOGEST protocol without gastric lipase (GL) and cholesterol esterase (CE), with GL or GL + CE. Moreover, human chewing and an in vitro oral phase (simulated salivary fluid and α-amylase) were evaluated for this purpose. The addition of GL decreased the bioaccessibility of total PS (from 23.8 to 18.5%), whereas the use of GL + CE does not significantly affect PS bioaccessibility. The in vitro oral phase resulted in an ineffective homogenization of the fresh vs partially dried and milled bread, reducing the bioaccessibility of total (from 20.2 to 12.8%) and individual PS. The INFOGEST digestion including the use of GL and CE, as well as an oral phase with human chewing, is proposed for the assessment of PS bioaccessibility in a solid matrix such as wholemeal rye bread since it more closely approximates the in vivo situation.

Influence of Galactooligosaccharides on the Positive Effect of Plant Sterol-Enriched Beverages on Cardiovascular Risk and Sterol Colon Metabolism.

In the present study, the impact of galactooligosaccharide (GOS) addition to a plant sterol (PS)-enriched beverage on the hypocholesterolemic effect and on the bioavailability and colonic metabolization of sterols was evaluated. A crossover trial was undertaken in postmenopausal women who intook a PS-enriched (2 g PS/day) or PS-GOS-enriched beverage (2 g PS/day and 4.3 g GOS/day) for 6 weeks. The presence of GOS did not modify the hypocholesterolemic effect of the PS-enriched beverage (total- and low-density lipoprotein-cholesterol reductions) or sterol bioavailability (increments of serum markers of dietary PS intake and of cholesterol synthesis). The consumption of both beverages led to an increase of sterol and metabolite excretion (with the exception of coprostanol, which decreased) and to slight changes in women's capacities for sterol conversion, regardless of the GOS presence. This study demonstrates the suitability of simultaneous enrichment with PS and GOS in milk-based fruit beverages, considering their hypocholesterolemic effect.

Effects of phytosterol ester-enriched low-fat milk on serum lipoprotein profile in mildly hypercholesterolaemic patients are not related to dietary cholesterol or saturated fat intake.

Phytosterols (PS) are recommended to reduce LDL-cholesterol. However, the influence of cholesterol and fat intake on the lipid-lowering effect of PS in mildly hypercholesterolaemia is unclear. Thus, the aim of the present study was to evaluate whether the efficacy of PS is related to the composition of saturated fat and dietary cholesterol intake. Additionally, serum carotenoid content was analysed to evaluate to what extent it was undermined by PS. This was a 3-month randomised, parallel trial with a three-arm design. Patients were divided into three groups: healthy diet (n 24), healthy diet+PS (n 31) and free diet+PS (n 29), receiving 2 g/d of PS. Healthy and free diets were characterised by a daily ingestion of 6.8 % of saturated fat and 194.4 mg of cholesterol and 12.7 % of saturated fat and 268.1 mg of cholesterol, respectively. After PS therapy, patients receiving the healthy diet+PS or a free diet+PS exhibited a similar reduction in total cholesterol (6.7 and 5.5 %), LDL-cholesterol (9.6 and 7.0 %), non-HDL-cholesterol (12.2 and 8.9 %) and apo B-100/apo A-I ratio (11.5 and 11.6 %), respectively. In patients following the healthy diet, (ß-carotene concentration rose by 26.9 %, whereas the ß-carotene and lycopene levels dropped by 21.0 and 22.8 % in the group receiving the free diet+PS, respectively. No change was observed in carotenoid levels in healthy diet+PS group. In conclusion, the efficacy of PS in relation to lipoprotein profile is not influenced by saturated fat or dietary cholesterol intake, which confirms the positive effect of healthy diet therapy in improving the negative effects that PS exert on carotenoid levels.

Anti-Inflammatory and Cytoprotective Effect of Plant Sterol and Galactooligosaccharides-Enriched Beverages in Caco-2 Cells.

Plant sterol (PS) (1 g/100 mL) enriched milk-based fruit beverages with or without galactooligosaccharides (GOS) (1.8 g/100 mL) were used in differentiated Caco-2 cells. Their potential cytopreventive effect against oxidative stress induced by cholesterol oxidation products (COPs) and their anti-inflammatory properties were evaluated. Preincubation (24 h) with bioaccessible fractions from beverages without and with GOS (MfB and MfB-G) completely prevented the COPs (60 µM/4 h) induced oxidative stress independent to GOS presence with exception to calcium influx and GSH content, where a partial protective effect was observed. Besides, MfB produced a significant (p < 0.05) reduction of IL-8 (40%) and IL-6 (50%) after IL-1ß-induction (1 ng/mL/24 h) through the inhibition of NF-κB p65 translocation into the nucleus (16%) compared to control cells, while GOS presence compromised their anti-inflammatory effect. Therefore, PS-enriched milk-based fruit beverage could be an interesting strategy to prevent intestinal injury produced by COPs and to attenuate the pro-inflammatory process in intestinal human diseases. GOS addition had no extra beneficial antioxidant effect and even reduced their anti-inflammatory properties.

Impact of high-pressure processing on the stability and bioaccessibility of bioactive compounds in Clementine mandarin juice and its cytoprotective effect on Caco-2 cells.

Mandarin juice is a rich source of antioxidant bioactive compounds. While the content and profile of bioactives are known, the impact of high-pressure processing (HPP) on their stability and bioaccessibility (BA) is unknown, but may allow obtaining safe, nutritious, and fresh-tasting juices with highly extractable bioactive compounds. The stability and BA of bioactive antioxidant compounds in untreated and HPP-treated (400 MPa/40 °C/1 min) Clementine mandarin juices, and the cytoprotective effect of its bioaccessible fractions (BF) obtained after simulated gastrointestinal digestion against H2O2-induced oxidative stress in differentiated Caco-2 cells were investigated. The BF of HPP-treated juices showed a better retention of carotenoids, flavonoids, ascorbic acid, total polyphenols and FRAP value, and slightly higher cytoprotection (mitochondrial membrane potential and ROS) than untreated juices. Therefore, HPP can be recommended as a suitable technology to retain or indeed increase antioxidant bioactives and their cytoprotective activity in mandarin juices after gastrointestinal digestion.

Antiproliferative Effect of Bioaccessible Fractions of Four Brassicaceae Microgreens on Human Colon Cancer Cells Linked to Their Phytochemical Composition.

The antiproliferative effect of the bioaccessible fractions (BFs) of four hydroponic Brassicaceae microgreens (broccoli, kale, mustard and radish) was evaluated on colon cancer Caco-2 cells vs. normal colon CCD18-Co cells after 24 h treatment with BFs diluted 1:10 v/v in cell culture medium. Their bioactivity was compared with the digestion blank, while the colon cancer chemotherapeutic drug 5-fluorouracil was used as a positive control. Cell viability (mitochondrial enzyme activity assay (MTT test) and Trypan blue test) and mechanisms related to antiproliferative activity (cell cycle, apoptosis/necrosis, mitochondrial membrane potential, reactive oxygen species (ROS) production, Ca2+ and glutathione (GSH) intracellular content) were studied. All microgreen BFs increased ROS and decreased GSH, altering the redox status and causing mitochondrial membrane dissipation followed by a general cell cycle arrest in G2/M and apoptotic cell death via a Ca2+-independent mechanism. As a result, the antioxidant bioactive compounds present in these microgreen species reduced the proliferation of tumoral cells (10 to 12.8% -MTT or 20 to 41.9% -Trypan blue), showing lesser effects with broccoli microgreens, in line with their lower ascorbic acid content and total antioxidant capacity. Therefore, the daily intake of microgreens within a balanced diet could be a preventive nutritional strategy to reduce the burden of chronic degenerative diseases such as colon cancer.

Optimization of the Red Tilapia (Oreochromis spp.) Viscera Hydrolysis for Obtaining Iron-Binding Peptides and Evaluation of In Vitro Iron Bioavailability.

Iron deficiencies continue to cause significant health problems in vulnerable populations. A good strategy to combat mineral deficiency includes fortification with iron-binding peptides. This research aims to determine the optimal conditions to hydrolyze red tilapia viscera (RTV) using Alcalase 2.4 L and recovery of iron-binding protein hydrolysate. The result showed that under the optimal hydrolysis condition including pH 10, 60 °C, E/S ratio of 0.306 U/g protein, and substrate concentration of 8 g protein/L, the obtained hydrolysate with 42.5% degree of hydrolysis (RTVH-B), displayed the maximal iron-binding capacity of 67.1 ± 1.9%. Peptide fractionation was performed using ultrafiltration and the <1 kDa fraction (FRTVH-V) expressed the highest iron-binding capacity of 95.8 ± 1.5%. Iron content of RTVH-B and its fraction was assessed, whereas iron uptake was measured indirectly as ferritin synthesis in a Caco-2 cell model and the result showed that bioavailability of bound minerals from protein complexes was significantly higher (p < 0.05) than iron salt in its free form, increased 4.7 times for the Fe2+-RTVH-B complex. This research suggests a potential application of RTVH-B as dietary supplements to improve iron absorption.

Impact of a Plant Sterol- and Galactooligosaccharide-Enriched Beverage on Colonic Metabolism and Gut Microbiota Composition Using an In Vitro Dynamic Model.

A beverage enriched with plant sterols (1 g/100 mL) and galactooligosaccharides (1.8 g/100 mL) was subjected to a dynamic gastrointestinal and colonic fermentation process to evaluate the effect on sterol metabolism, organic acid production, and microbiota composition. Production of sterol metabolites (coprostanol, methylcoprostanol, ethylcoprostenol, ethylcoprostanol, and sitostenone) was observed in the transverse colon (TC) and descending colon (DC) vessels in general, from 24 and 48 h, respectively. Microbial activity was assessed through the production of organic acids, mainly acetate in all colon vessels, lactate in the AC, and butyrate and propionate in the TC and DC. A higher diversity in the microbial community was found in the TC and DC, in accordance with a higher sterol metabolism and organic acid production. Although the prebiotic effect of galactooligosaccharides was not detected, changes in microbiota composition (an increase in the Parabacteroides genus and the Synergistaceae and Lachnospiraceae families) indicated an enhancement of sterol metabolism.

Effect of a Milk-Based Fruit Beverage Enriched with Plant Sterols and/or Galactooligosaccharides in a Murine Chronic Colitis Model.

The potential anti-inflammatory effect of plant sterols (PS) enriched milk-based fruit beverages (PS, 1 g/100 mL) (MfB) with/without galactooligosaccharides (GOS, 2 g/100 mL) (MfB-G) in an experimental mice model of chronic ulcerative colitis was evaluated. Beverages were orally administered to mice every day by gavage to achieve PS and GOS doses of 35 and 90 mg/kg, respectively, and experimental colitis was induced by giving mice drinking water ad libitum containing 2% (w/v) dextran sulphate sodium (DSS) for 7 days, alternating with periods without DSS up to the end of the study (56 days). MfB beverage showed significant reduction of symptoms associated to ulcerative colitis and improved the colon shortening and mucosal colonic damage, but it was not able to reduce the increase of myeloperoxidase levels produced by DSS. MfB-G showed higher incidence of bloody feces and loss of stool consistency than MfB, as well as high levels of immune cells infiltration in colon tissue and myeloperoxidase. Therefore, PS-enriched milk-based fruit beverage could be an interesting healthy food to extend the remission periods of the diseases and the need to evaluate, in a pre-clinical model, the anti-inflammatory effect of the combination of bioactive compounds in the context of a whole food matrix.

In-vitro antioxidant capacity and cytoprotective/cytotoxic effects upon Caco-2 cells of red tilapia (Oreochromis spp.) viscera hydrolysates.

The antioxidant capacity of red tilapia viscera hydrolysates (RTVH) with different degrees of hydrolysis (DH) as well as their ultrafiltration membrane fractions, were analyzed using different chemical assays. Their protective effects against oxidative stress were evaluated using H2O2-stressed human intestinal differentiated Caco-2. The highest antioxidant capacity was obtained with a DH of 42.5% (RTVH-A) and its <1 kDa fraction (FRTVH-V). RTVH-A and FRTVH-V did not show cytotoxic effects at a concentration of ≤0.5 mg/mL,prevented the decrease in cell viability, and suppressed intracellular reactive oxygen species (ROS) accumulation induced by H2O2. However, pretreatment with RTVH-A after adding H2O2, showed a greater decrease in glutathione levels. Moreover, FRTVH-V allowed for a recovery close to that of control levels of cell proportions in the G1 and G2/M cell cycle phases; and a decrease in the cell proportion in late apoptosis. These results suggest that RTVH-A and FRTVH-V can be beneficial ingredients with antioxidant properties and can have protective effects against ROS-mediated intestinal injuries.

Impact of colonic fermentation on sterols after the intake of a plant sterol-enriched beverage: A randomized, double-blind crossover trial.

BACKGROUND: Cholesterol microbial transformation has been widely studied using in vitro fermentation assays, but less information is available on the biotransformation of plant sterols (PS). The excretion percentage of animal sterols (AS) (67-73%) is considerably greater than that of PS (27-33%) in feces from healthy humans following a Western diet. However, a lower content of AS in feces from subjects following a vegetarian, vegan or low-fat animal diet has been seen when compared to omnivorous subjects. Although only one human study has reported fecal sterol excretion after the consumption of PS-enriched food (8.6 g PS/day), it was found that the target group showed an increase in the excretion of cholesterol and a 57% decrease in its metabolites compared to the control group. OBJECTIVE: Evaluation of the impact of a PS-enriched milk based fruit beverage intake on fecal sterol excretion and the microbial conversion of sterols in postmenopausal women with mild hypercholesterolemia. METHODS: Forty postmenopausal women participated in a randomized, double-blind, crossover study with two beverages, with a PS-enriched (2 g PS/day) or without. The women were divided in two groups: 20 women consumed the PS-enriched beverage and the other 20 women consumed a placebo (without PS) beverage for 6 weeks. After a four-week washout period, the type of beverage was exchanged and consumed for another 6 weeks. Feces were collected at the start (0 and 10 weeks) and end of each intervention period (6 and 16 weeks), and fecal sterols were determined by capillary gas chromatography with mass spectrometry. RESULTS: The intake of the PS-enriched beverage modified the fecal sterol excretion profile. A significant increase mainly in PS and their metabolites versus the placebo intervention period was observed. Although the same effect was not observed in the case of AS, a tendency towards increased cholesterol and decreased coprostanol (the main metabolite of cholesterol) was recorded after PS-enriched beverage intake versus placebo. Furthermore, the PS-enriched beverage also modified the microbial conversion of sterols. In this context, an important decrease in the conversion percentage of cholesterol in 16 women (between 11% and 50%) and of sitosterol in 24 women (between 15% and 61%) was observed. CONCLUSIONS: The results obtained suggest that the microbiota could preferably use PS as a substrate, when present in a greater proportion compared with cholesterol. Besides, a lower sitosterol and cholesterol conversion trend would mean that intake of the PS-enriched beverage could modulate the metabolic activity of the gut microbiota. Therefore, further studies on the impact of PS-enriched foods upon gut microbiota modulation are needed. Clinical Trial Registry Number: NCT02065024 listed on the NIH website: ClinicalTrials.gov. Clinical Trial Registry Name: Food Matrix and Genetic Variability as Determinants of Bioavailability and Biological Effects of Beta-cryptoxanthin and Phytosterols (foodmagenpol). The full trial protocol is available upon request to the corresponding author.

Evaluation of the Bioaccessibility of Antioxidant Bioactive Compounds and Minerals of Four Genotypes of Brassicaceae Microgreens.

Microgreens constitute an emerging class of fresh, healthy foods due to their nutritional composition. In this study the content of minerals and antioxidant bioactive compounds, and for the first time bioaccessibility, were evaluated in broccoli (Brassica oleracea L. var. italica Plenck), green curly kale (Brassica oleracea var. sabellica L.), red mustard (Brassica juncea (L.) Czern.) and radish (Raphanus sativus L.) hydroponic microgreens. Macro- (K, Ca, Mg) and oligo-elements (Fe, Zn), ascorbic acid, total soluble polyphenols, total carotenoids, total anthocyanins, total isothiocyanates and total antioxidant capacity (Trolox Equivalent Antioxidant Capacity and Oxygen Radical Absorbance Capacity) were determined before and after the standardized simulated gastrointestinal digestion process. All microgreens provided relevant amounts of vitamin C (31-56 mg/100 g fresh weight) and total carotenoids (162-224 mg ß-carotene/100 g dry weight). Mineral content was comparable to that normally found in hydroponic microgreens and the low potassium levels observed would allow their dietetic recommendation for patients with impaired kidney function. Both total soluble polyphenols and total isothiocyanates were the greatest contributors to the total antioxidant capacity after digestion (43-70% and 31-63% bioaccessibility, respectively) while macroelements showed an important bioaccessibility (34-90%). In general, radish and mustard presented the highest bioaccessibility of bioactive compounds and minerals. Overall, the four hydroponic Brassicaceae microgreens present a wide array of antioxidant bioactive compounds.