Speciation of selenium in selenium-enriched seeds,buckwheat (<Emphasis Type="Italic">Fagopyrum esculentum</Emphasis> Moench) and quinoa (<Emphasis Type="Italic">Chenopodium quinoa</Emphasis> Willdenow)

Buckwheat (Fagopyrum esculentum Moench) and quinoa (Chenopodium quinoa Willdenow) are widely used as food ingredients. The nutritional characteristics of these plants, i.e., high contents of proteins and amino acids suggest that selenium (Se) is preserved as selenoamino acid derivatives, in particular, selenomethionine (SeMet) in proteins, similar to selenized yeast. Therefore, buckwheat and quinoa are expected to be a good nutritional source of Se. Selenized buckwheat and quinoa were cultivated on Se-fortified soil using sparingly soluble Se salts, such as barium selenate and barium selenite. Se concentration in the edible parts of these plants was determined, and Se extraction efficiency with enzyme or alkali was evaluated. In addition, the chemical species of Se in the low molecular weight fraction of these plants were determined by HPLC-ICP-MS. Total Se concentrations in the edible parts of selenized buckwheat and quinoa were 170.4 ± 2.9 μg/g and 102.7 ± 2.4 μg/g wet weight, respectively. Thus, these selenized seeds were found to be Se accumulators. The results indicate that Se in selenized buckwheat exists mainly as SeMet, while Se in selenized quinoa exists not only as SeMet but also as selenate (Se(VI)) and non-protein forms.     

Pb2+Inhibits Competitively Flocculation of Saccharomyces cerevisiae

Pb²⁺inhibited calcium‐induced flocculation in Flo1 (S646–1B) and NewFlo phenotype strains (NCYC 1190, NCYC 1195 and NCYC 1364) of Saccharomyces cerevisiae. Flocculation was restored after washing with water or EDTA, which suggests a reversible binding of Pb²⁺to yeast cell walls. Pb²⁺probably inhibited flocculation by competing with Ca²⁺, since Pb²⁺inhibition was alleviated by excess of Ca²⁺. Using a fluorescent avidinfluorescein isothiocyanate (Avidin‐FITC) probe, active cell surface flocculation lectins, in the presence of Ca²⁺ions, were visualized. Conversely, Avidin‐FITC was not fixed to yeast walls of flocculent cells, in the presence of Pb²⁺ions or in the simultaneous presence of 0.05 mM Ca²⁺and 0.4 mM Pb²⁺. These results suggest that Pb²⁺ions were not able to induce the correct conformation of the lectin‐like component and reinforces the hypothesis that Pb²⁺ions compete for the same “calcium site” of flocculation zymolectins.     

Characteristics of Aspergillus niger xylanases produced on rice husk and wheat bran in submerged culture and solid‐state fermentation for an applicability proposal

Xylanolytic rich filtrates were obtained by A. niger sp in both submerged and solid‐state culture using rice husk or wheat bran as the only carbon source. Filtrates obtained on rice husk showed the highest activities (~6500 and 5200 U g^?1, respectively). Independent of carbon source, these filtrates were very stable in an acidic pH range (4–7) and mild temperatures, with high half‐life time values (more than 7 h at 50 °C) in the corresponding inactivation kinetic models. Also the effect of different metallic ions and denaturing substances was verified finding that these enzymes are not metaloproteins, and metals as Hg²⁺ and Pb²⁺ caused the greatest loss of xylanolytic activity (not higher than 30%). Xylanases produced by this A. niger strain showed important features that make them potential candidates for applications on human and livestock food industries.     

Transport of bovine milk-derived opioid peptides across a Caco-2 monolayer

The transepithelial transport of μ-opioid (MOP) receptor agonists, i.e., bovine β-casomorphin-5 and -7 (derived from β-casein) and an antagonist, i.e., casoxin-6 (derived from κ-casein) were investigated using a human intestinal cell (Caco-2) monolayer. To determine the transport pathway of the peptides across Caco-2 monolayers, two directions of the transport (apical-to-basolateral and basolateral-to-apical) were studied. All investigated peptides were transported across Caco-2. The cumulative fractions of transported peptides were increased linearly in time in each case. The permeability coefficients (P_aap) calculated for all peptides in two transport directions ranged between 0.57 and 9.21 × 10^?6 cm min^?1. These data suggest the possibility of transport of opioid peptides derived from food across human intestinal mucosa.     

Milk enhances intestinal absorption of green tea catechins in in vitro digestion/Caco-2 cells model

The effect of milk on the absorption of polyphenols is still controversial so far. In order to determine the impact of milk addition on green tea catechins bioaccessibility and intestinal absorption an in vitro digestion/Caco-2 cell model was applied. Green tea extract (GTE) was solubilized in distilled water at 23 °C and 100 °C, combined with skimmed milk (GTE + 10% milk and GTE + 25% milk) and subjected to simulated gastric and intestinal digestion, followed by transepithelial absorption in Caco-2 cells monolayers. In the mixture with milk, gallated catechins: ECG and EGCG showed binding to milk proteins while EC and EGC seemed to have weaker affinity. Catechins were stable during gastric incubation and very sensitive to intestinal digestion. Bioaccessibility of green tea catechins brewed at 100 °C was higher than brewed at 23 °C. Catechins from digested GTE with 10% and 25% milk exhibited enhanced intestinal permeability in Caco-2 model in comparison to non-digested GTE and digested GTE without milk. Apparent permeability coefficients (P_app) of EGCG and ECG in digested GTE with 25% milk were significantly higher compared to those in GTE with 10% milk, and amounted to 2.41 × 10^− 6 cm/s and 1.39 × 10^− 6 cm/s. The recoveries of all catechins in GTE with milk in Caco-2 cells after 2 h incubation were significantly higher than that without milk. To summarize, these data suggest that milk addition may increase catechin bioavailability by enhancing their transepithelial absorption and uptake from green tea extract.     

Determination of 5 natural selenium species in selenium-enriched bamboo shoots using LC-ICP-MS

A method for simultaneous determination of the selenium species methylselenocysteine (MeSeCys), selenocysteine (SeCys₂), selenite (SeIV), selenomethionine (SeMet), and selenates (SeVI) in natural selenium-enriched bamboo shoots was developed. Samples were extracted using sonication with a solution of pepsin. Different selenium species were separated using an anion exchange column with isocratic elution and a citric acid mobile phase at pH 4.7. Separated selenium species were detected as ⁸⁰Se using inductively coupled plasma mass spectrometry (ICPMS) in dynamic reaction cell (DRC) gas mode. The method achieved acceptable quantitative recoveries of 73.9 to 113.4% with relative standard deviations of <5%. The limits of detection were 1, 2, 2, 3, and 3 μg/kg (fresh weight) for SeCys₂, Se, MeSeCys, SeMet, and Se, respectively. The method was simple, rapid, accurate, and useful for determination of selenium species in natural selenium enriched bamboo shoots.     

Polyphenols bioaccessibility and bioavailability assessment in ipecac infusion using a combined assay of simulated in vitro digestion and Caco-2 cell model

In this report, we investigated for the first time the total polyphenols content (TPC) and antioxidant activity before and after digestion of Carapichea ipecacuanha root infusion, better known as ipecac, prepared at different concentrations. An in vitro digestion system coupled to a Caco-2 cell model was applied to study the bioavailability of antioxidant compounds. The ability of ipecac bioaccessible fractions to inhibit reactive oxygen species (ROS) generation at cellular level was also evaluated. The findings revealed that water volume of 50 mL g⁻¹ of sample provided the maximum yield of extraction of TPC and antioxidant activity. Polyphenols increased in content and activity after digestion and they were highly bioavailable (75% of intestinal absorption). Polyphenols were also present in the residual parts which indicate a possible local activity. Results also suggest that ipecac infusion could represent a promising source of effective bioavailable antioxidants to be exploited in functional foods field.     

Transepithelial transport of 6-O-caffeoylsophorose across Caco-2 cell monolayers

The aim of this study was to clarify the transport behaviour and mechanism of caffeic acid analogue bearing a sugar-moiety, 6-O-caffeoylsophorose (CS), in Caco-2 cells. The absorption of CS was investigated by its transport across Caco-2 cell monolayers using a high-performance liquid chromatography-time-of-flight-mass spectrometry (LC–TOF-MS). The permeation of CS was concentration-dependent and reached the plateau at >6 mM. The apparent permeability (P_app) of CS in the apical-to-basolateral direction was 5.4 × 10⁻⁷ cm/s, while in the reversed direction the P_app value was significantly reduced (1.9 × 10⁻⁷ cm/s). CS transport was competitively inhibited by phloretin, an inhibitor of monocarboxylic acid transporter (MCT). Benzoic acid, an MCT substrate, also reduced CS transport. A less significant change of CS transport was observed across Caco-2 cell monolayers pretreated with quercetin, a suppressor of tight-junction. These findings strongly indicate that CS, a caffeic acid analogue bearing sophorose moiety, can be transported across Caco-2 cell monolayers via the MCT pathway.     

Effect of phytate on the in-vitro solubility of Al+, Ca2+, Hg2+ and Pb2+ as a function of pH at 37°C

The effect of phytate on the solubility of Al³⁺ and Pb^{2 +} has been investigated in vitro at 37°C across the pH ranges 3–7 (Pb²⁺) and 2–5 (Al³⁺). For both ions minimum solubility was found when the initial metal ion: phytate ratio was in the range 5:1–3:1 across the whole pH range. For Al³⁺ the solubility rose sharply either side of these ratios whereas for Pb^{2 +} solubility rose only slowly as the metal ion: phytate ratio was reduced. Hg²⁺ was totally soluble under all conditions tested. At pH 7 Ca²⁺ solubility was found to be at a minimum when the initial Ca : phytate ratio was 5 :1 irrespective of the initial Ca²⁺ concentration.     

Tea components influencing bioavailability of fluoride and potential transport mechanism in the Caco-2 cell line model

Previous work showed that the bioavailability of fluoride in dark tea was lower than NaF solution. However, limited information is available indicating the effects of tea components on the fluoride bioavailability. In this study, the effects of the components in tea on the bioavailability of fluoride were evaluated in the Caco-2 cell line model. Additionally, the mechanism of effect of aluminium on fluoride transport was investigated. The result showed that 10–100 μm of epigallocatechin gallate (EGCG) could not influence fluoride transport. Al³⁺ significantly decreased fluoride transport in both apical-basolateral and basolateral-apical directions. Moreover, aluminium could form different forms of aluminium fluoride complexes, which were transported through Caco-2 cells by different pathways. F⁻ transport was mainly dependent on the paracellular pathway and active transport involving Cl⁻ channels. The paracellular pathway played a predominant role in transport of AlF₃. The paracellular pathway and active transport both participated in AlF₂⁺ transport.     

Bio-fortification and isotopic labelling of Se metabolites in onions and carrots following foliar application of Se and Se

The aims were to bio-fortify onions by foliar application of selenium (Se) and to intrinsically label bioactive Se-metabolites in onion and carrot by enriched, stable ⁷⁷Se for use in human physiological studies. Onion bulbs and leaves were enriched in Se by repeated foliar spraying of 10 or 100 μg Se ml⁻¹ solutions of sodium selenite (Se(IV)) or sodium selenate (Se(VI)). ICP-MS analysis of onion leaves and bulbs showed that the Se concentration was enhanced by up to a factor of approximately 50 and 200 in bulbs and leaves, respectively. HPLC–ICP-MS analysis of proteolytic plant extracts showed that foliar application of Se(IV) gave rise to bio-synthesis of a higher fraction of the desired organic Se species and was better tolerated by the plants than Se(VI). Based on these findings onions and carrots were bio-fortified by foliar application of a solution of ⁷⁷Se(IV) that was enriched to 99.7% as ⁷⁷Se. The ⁷⁷Se- labelled metabolites in onions were predominantly γ-glutamyl-⁷⁷Se-selenomethyl-selenocysteine (γ-glu-Me⁷⁷SeCys), ⁷⁷Se-methylselenocysteine (Me⁷⁷SeCys) and ⁷⁷Se-selenomethionine (⁷⁷SeMet). Furthermore, we report here for the first time the finding in carrots of the bioactive Me⁷⁷SeCys, the identity of which was verified by HPLC–ESI-MS/MS.     

Ultrasonic assisted enzymatic digestion (USAED) coupled with high performance liquid chromatography and electrothermal atomic absorption spectrometry as a powerful tool for total selenium and selenium species control in Se-enriched food supplements

The suitability of hyphenated USAED with HPLC separation and ET-AAS determination as a new rapid methodology for Se control in Se-enriched food supplements is demonstrated. Total Se determination and Se speciation are accomplished in a single sample treatment using low sample amounts (ca. 10 mg), and low extracting volume (1 mL). The total Se content in seven of the 10 Se-enriched supplements studied was in agreement with the values obtained after microwave pressurized acid digestion, MW, (test t, p = 0.05). The Se species studied were Se(IV), Se(VI), SeMet, SeMeSeCys, and SeCys₂, being some of the most common found in the 10 supplements studied. Although SeMet was the Se species expected to be present at the highest concentration in most Se-enriched food supplements, we detected it in only three of the 10 samples studied. In the other seven samples, two of them had Se(IV) as the main Se species. The other five supplements had Se species that did not match with any of the five standards selected by us. We have also systematically demonstrated that ultrasonication does not alter the following Se species: Se(IV), Se(VI), SeMet, SeMeSeCys, and SeCys₂. The new procedure can be easily adapted to more Se species and can be routinely used for Se control in Se-enriched food supplements. Concerning the supplements studied, our results suggest that stricter control on the Se content in enriched food supplements in terms of Se species will need to become mandatory.     

The soluble fraction of soy protein peptic hydrolysate reduces cholesterol micellar solubility and uptake

Previous studies have demonstrated that the undigested or insoluble high molecular fraction of soy protein exerts a lowering effect on serum cholesterol by inhibiting the absorption of cholesterol in the intestinal tract. The aim of this study was to investigate the interaction between the soluble fraction of soy protein peptic hydrolysate and dietary mixed micelles (DMMs) for cholesterol micellar solubility and uptake. The collected soluble tofu peptic hydrolysate (STPH) components, generated from hard and lactone tofu were evaluated by two in vitro models and a Caco-2 cell model after tofu soy protein was digested, thus simulating the gastrointestinal tract. The results showed that the administration of STPH effectively decreased the cholesterol solubility and uptake by 37.6% and 18.99%, respectively, as detected by the in vitro models and the Caco-2 cell model. Current findings demonstrated the potential hypocholesterolaemic benefit of the STPH.     

Mineral composition,antioxidant and cytotoxic biopotentials of wild‐growing Ganoderma species (Serbia): G. lucidum (Curtis) P. Karst vs. G. applanatum (Pers.) Pat.

The aim of this work was to analyse mineral composition and chemical profile of two nonedible fungal species: Ganoderma lucidum and Ganoderma applanatum (Fru?ka Gora, Serbia) vs. their antioxidant (ABTS and A.E.A.C. assay) and cytotoxic biopotentials (MTT assay on MCF‐7). Both species were analysed for their content of macro‐ and microelements by atomic absorption spectrophotometry, while phenolic profile of EtOH and H₂O extracts was examined by LC‐MS/MS technique. Both species mostly contained the following ions: K⁺ > Ca²⁺ > Mg²⁺ > Mn²⁺ > Zn²⁺ > Cu²⁺ > Cr³⁺. Among nine phenolic compounds, the highest content of vanillic acid was detected in G. applanatum extracts while protocatechuic acid in EtOH extract and quinic acid in H₂O extract were mostly contained in G. lucidum. Ganoderma applanatum EtOH extract showed the best antioxidant activities related to its phenolic and flavonoid content. Further, the best cytotoxic effect after 72 h was observed in this extract as well.     

Antioxidant,immunomodulatory and antiproliferative effects of gelatin hydrolysates from seabass (Lates calcarifer) skins

This study investigated the antioxidant, immunomodulatory and antiproliferative potentials of gelatin hydrolysates from seabass skins in cell model systems. Gelatin hydrolysates were extracted from seabass skins using different processes and enzyme concentrations. The ability of the hydrolysates to protect against H₂O₂‐induced DNA damage was assessed on U937 cells using the Comet assay, and one of the samples showed DNA protective effects. All samples showed immunomodulatory potential by significantly (P < 0.05) reducing interleukin‐6 (IL‐6) and IL‐1β production in lipopolysaccharide (LPS)‐stimulated RAW 264.7 macrophage cells. Antiproliferative activities of seabass skin hydrolysates were measured using human colon cancer (Caco‐2) and liver cancer (HepG2) cell lines as the model cell cultures. The inhibition of cell proliferation of Caco‐2 and HepG2 cancer cells occurred in a dose‐dependent manner at concentrations of 1–25 mg mL^?1. Therefore, seabass skin hydrolysates prepared using an appropriate process could serve as a potential functional food ingredient with various health benefits.     

Probiotic potential of <Emphasis Type="Italic">L. sake</Emphasis> C2 isolated from traditional Chinese fermented cabbage

The probiotic properties of Lactobacillus sake C2 isolated from traditional Chinese fermentation cabbage were evaluated. L. sake C2 displayed good tolerance to the gastrointestinal environments including low pH, presence of bile salts, pepsin, and pancreatin. L. sake C2 not only could produce the bacteriocin with a broad inhibitory spectrum, but also secrete a large amount of lactic acid (11.26 mg/mL) and certain amount of H₂O₂ (3.14 mg/mL). L. sake C2 displayed a high adhesion rate of 15.2 ± 1.2% to Caco-2 cells. Moreover, viable cells of L. sake C2 have fairly strong ability to remove cholesterol (53.2 ± 1.4%). These results indicated that L. sake C2 has a potential as probiotic. Moreover, this study suggested that besides lactic acid bacteria originating from human, those isolated from fermented foods have potential to be used as probiotic and may be exploited in food industry and probiotic preparations.     

Effect of modifiers on thermal behaviour of Se in acid digestates and slurries of vegetables by graphite furnace atomic absorption spectrometry

The influence of sample preparation strategy of vegetables on the electrothermal behaviour of Se without and with chemical modifiers such as Pd(NO₃)₂, Pd(NO₃)₂ + Mg(NO₃)₂, Pd(NO₃)₂ + Cd(NO₃)₂, pre-reduced Pd, Mg(NO₃)₂, and Ni(NO₃)₂ was investigated. Acid digestates and slurries of vegetables (0.1% m/v in 1% v/v HNO₃ + 0.005% v/v of Triton X-100) were used to prepare reference solutions or slurries. For 10 μl of each modifier tested, pyrolysis and atomization temperatures were evaluated using pyrolysis and atomization curves, respectively. Best conditions, such as thermal stability, signal profile, repeatability and sensitivity were attained using Pd(NO₃)₂ as chemical modifier. The following heating program (temperature, ramp/hold time) of the graphite tube of the Varian SpectrAA-800Z atomic absorption spectrometer was used: dry step (85 °C, 5/0 s; 95 °C, 40/0 s; 120 °C, 10/5 s); pyrolysis step (1400 °C, 10/3s); atomization step (2200 °C, 1/2 s); clean step (2600 °C, 2/0 s). This pyrolysis temperature is 800 °C higher than when measuring without any modifier. For 20 μL sample volume and 10 μg Pd(NO₃)₂, analytical curves in the 3.0–30 μg Se l⁻¹ range were obtained. The method was applied for Se determination in acid digestates and slurries of 10 vegetable samples and one standard reference material (rice flower) and results were in agreement at 95% confidence level. Recoveries varied from 89 to 95% for spiked samples. The lifetime of the graphite tube was ca. 250 firings and the relative standard deviations (n=12) for a typical acid digestate and slurry containing 20 μg Se l⁻¹ were 3.8% and 8.3%, respectively. The limits of detection were 2.0 μg Se l⁻¹ and 0.6 μg Se l⁻¹ Se for digestates and slurries, respectively.     

Selenium species in leaves of chicory,dandelion, lamb’s lettuce and parsley

Lamb’s lettuce, dandelion, parsley and four cultivars of chicory were cultivated aeroponically for 41 days with nutrient solution containing 7  mg Se/L in the form of Na₂SeO₄. Se compounds were determined by high performance liquid chromatography–ultraviolet treatment–hydride generation atomic fluorescence spectrometry (HPLC–UV–HG-AFS) in the green parts of the selected plants. Se species were extracted by water and by enzymatic hydrolysis with Protease XIV. Separation of Se^IV, Se^VI, SeMet, SeMeSeCys and SeCys₂ was made by a combination of anion and cation exchange chromatography in which the columns were connected on-line to a UV–HG-AFS detection system. Se accumulated efficiently in plant leaves up to 480 μg/g dry mass, mostly as Se^VI, i.e. the form of Se in the nutrient solution. Beside inorganic Se, selenomethionine (6–21%), selenomethylselenocysteine (0.5–4.4%) and selenocistine (<DL-0.8%) were determined in the extracts after enzymatic hydrolysis. Some unidentified peaks were also observed in the chromatograms.     

Prenylation preserves antioxidant properties and effect on cell viability of the natural dietary phenol curcumin

Curcumin (CRM) is a naturally occurring phenolic compound with a variety of biological and pharmacological activities. We have investigated the effect of aromatic C-prenylation on the antioxidant activity of this natural compound. The protective effect of CRM and its diprenyl derivative (PCRM) was investigated against neat cholesterol degradation (at 140 °C) and Cu^2 +-induced oxidation (at 37 °C) of liposomes and human low density lipoproteins. The activity of two simplified vanilloid analogs (vanillin and vanillyl alcohol) was also compared in the same systems. Cytotoxicity and cell permeation of both curcuminoids were also assessed using differentiated Caco-2 cell monolayers. PCRM, like CRM, significantly inhibited the oxidative degradation of polyunsaturated fatty acids and cholesterol, and the formation of their oxidation products in the oxidative stress systems, acting as scavenger of peroxyl radicals, without toxic effect (in the range 10–100 μM) on differentiated Caco-2 cell viability. Nevertheless, the structural modification of the lead compound severely affected membrane permeation through the Caco-2 monolayers, with apparent permeability coefficient (P_app) values in the apical-to-basolateral direction (2-h incubation) of 2.93 ± 0.94 × 10^− 6 cm/s and < 10^− 7 cm/s for CRM and PCRM, respectively. Taken together, our observations reveal a surprising bioactivity of PCRM, and qualify this compound as an interesting probe to explore the antioxidant pharmacophore of curcuminoids.     

Impact of magnesium and mannose in the cultivation media on the magnesium biosorption, the biomass yield and on the cell wall structure of Candida utilis yeast

Binding capacity of Mg²⁺ ions, the biomass yield and the cell wall structure of Candida utilis ATCC 9950 cultivated in the media containing magnesium and mannose were evaluated in the study. Mannose has been added to two types of culture media: not enriched and enriched with Mg²⁺ ions. The YPD medium was used as a control while the experimental media (without or with Mg²⁺ ions) have been prepared by replacing glucose in the YPD medium by 1% (YPDM) or 2% (YPM) of mannose. The highest content of magnesium (5.39 and 5.42 mg/g_d.w.) as well as the highest biomass yield (15.22 and 14.03 g_d.w./L) were observed after 24 h of cultivation in the media enriched with magnesium and supplemented with mannose. The yeast cultivated for 48 h in those media were also characterized by thicker cell walls (74.3 and 67.7 nm). Introduction of mannose to the cultivation media was the factor that has influenced biosorption of magnesium ions as the result of twofold thickening of the mannoprotein layer of the cell wall of Candida utilis ATCC 9950 yeast.