Capillary electromigration methods for food analysis and Foodomics: Advances and applications in the period March 2021 to March 2023.

This work presents a revision of the main applications of capillary electromigration (CE) methods in food analysis and Foodomics. Papers that were published during the period March 2021 to March 2023 are included. The work shows the multiple CE methods that have been developed and applied to analyze different types of molecules in foods and beverages. Namely, CE methods have been applied to analyze amino acids, biogenic amines, heterocyclic amines, peptides, proteins, phenols, polyphenols, pigments, lipids, carbohydrates, vitamins, DNAs, contaminants, toxins, pesticides, additives, residues, small organic and inorganic compounds, and other minor compounds. In addition, new CE procedures to perform chiral separation and for evaluating the effects of food processing as well as the last developments of microchip CE and new applications in Foodomics will be also discussed. The new procedures of CE to investigate food quality and safety, nutritional value, storage, and bioactivity are also included in the present review work.

Capillary electromigration methods for food analysis and Foodomics: Advances and applications in the period February 2019-February 2021.

This work presents a revision of the main applications of capillary electromigration methods in food analysis and Foodomics. Articles that were published during the period February 2019-February 2021 are included. The work shows the multiple CE methods that have been developed and applied to analyze different types of molecules in foods. Namely, CE methods have been applied to analyze amino acids, biogenic amines, carbohydrates, chiral compounds, contaminants, DNAs, food additives, heterocyclic amines, lipids, secondary metabolites, peptides, pesticides, phenols, pigments, polyphenols, proteins, residues, toxins, vitamins, small organic and inorganic compounds, as well as other minor compounds. The last results on the use of CE for monitoring food interactions and food processing, including recent microchips developments and new applications of CE in Foodomics, are discussed too. The new procedures of CE to investigate food quality and safety, nutritional value, storage and bioactivity are also included in the present review work.

Study of the potential neuroprotective effect of Dunaliella salina extract in SH-SY5Y cell model.

Alzheimer's disease (AD) is the most common form of dementia caused by a progressive loss of neurons from different regions of the brain. This multifactorial pathophysiology has been widely characterized by neuroinflammation, extensive oxidative damage, synaptic loss, and neuronal cell death. In this sense, the design of multi-target strategies to prevent or delay its progression is a challenging goal. In the present work, different in vitro assays including antioxidant, anti-inflammatory, and anti-cholinergic activities of a carotenoid-enriched extract from Dunaliella salina microalgae obtained by supercritical fluid extraction are studied. Moreover, its potential neuroprotective effect in the human neuron-like SH-SY5Y cell model against remarkable hallmarks of AD was also evaluated. In parallel, a comprehensive metabolomics study based on the use of charged-surface hybrid chromatography (CSH) and hydrophilic interaction liquid chromatography (HILIC) coupled to high-resolution tandem mass spectrometry (Q-TOF MS/MS) was applied to evaluate the effects of the extract on the metabolism of the treated cells. The use of advanced bioinformatics and statistical tools allowed the identification of more than 314 metabolites in SH-SY5Y cells, of which a great number of phosphatidylcholines, triacylglycerols, and fatty acids were significantly increased, while several phosphatidylglycerols were decreased, compared to controls. These lipidomic changes in cells along with the possible role exerted by carotenoids and other minor compounds on the cell membrane might explain the observed neuroprotective effect of the D. salina extract. However, future experiments using in vivo models to corroborate this hypothesis must be carried out.

In vitro Neuroprotective Potential and Lipidomics Study of Olive Leaves Extracts Enriched in Triterpenoids.

Alzheimer's Disease (AD) is the most common form of dementia that is associated with extracellular amyloid beta (Aß) plaque formation. Genetic, environmental, and nutrition factors have been suggested as contributors to oxidative stress and neuroinflammation events that are connected to AD etiology, and secondary metabolites, such as triterpenes, have shown promising results in AD prevention. In this work, the neuroprotective and anti-inflammatory potential of an olive leaves fraction enriched in triterpenoid compounds obtained using supercritical fluid extraction (SFE) and dynamic adsorption/desorption using sea sand as adsorbent has been performed. In addition, a comprehensive lipidomics study of the response of SH-SY5Y neuroblastoma cell line to this fraction was carried out using advanced analytical methodologies, namely, charged-surface hybrid chromatography-quadrupole-time of flight mass spectrometry (CSH-Q-TOF MS/MS). The use of freely available lipidomic annotation tools and databases, and stringent cut-off filters allowed the annotation of more than 250 intracellular lipids. Advanced bioinformatics and statistical tools showed a number of phosphatidylcholines and phosphatidylethanolamines significantly increased, which could explain the protection against the cell death caused by Aß1-42. Moreover, several triacylglycerols were found decreased. These results suggest triterpenoids from olive leaves as good neuroprotective candidates, and open a new gate for future experiments using in vivo models to corroborate this hypothesis.

Chemometric optimisation of pressurised liquid extraction for the determination of alliin and S-allyl-cysteine in giant garlic (Allium ampeloprasum L.) by liquid chromatography tandem mass spectrometry.

INTRODUCTION: Giant garlic is a functional food that contains different kinds of bioactive molecules with beneficial effects on chronic noncommunicable diseases like diabetes and cardiovascular conditions. Considering biosynthesis pathways, abundance, and biological activity, alliin and S-allyl-cysteine were used as chemical markers of organosulphur compounds present in giant garlic. OBJECTIVE: To establish a chemometric optimisation of pressurised liquid extraction for the determination of alliin and S-allyl-cysteine in giant garlic by liquid chromatography tandem mass spectrometry (LC-MS/MS). METHODOLOGY: Samples were blanched (ca. 90°C for 10 min) to inactivate alliinase and γ-glutamyl transpeptidase enzymes and then freeze-dried. Chemometric optimisation was performed via response surface methodology based on central composite design (CCD). Organosulphur compound yields were determined applying a validated LC-MS/MS method in multiple reaction monitoring (MRM) mode using the following transitions: for alliin m/z 178 → 74 and for S-allyl-cysteine m/z 162 → 41. RESULTS: According to CCD results, under constant conditions of pressure (1500 psi) and time (20 min), the optimal conditions for pressurised liquid extraction of alliin and S-allyl-cysteine were 70.75 and 68.97% v/v of ethanol in extraction solvent and 76.45 and 98.88°C as extraction temperature, respectively. Multiple response optimisation for the simultaneous extraction of both organosulphur compounds was established via desirability function. Under these conditions, 2.70 ± 0.27 mg g-1 dry weight (DW) of alliin and 2.79 ± 0.22 mg g-1 DW of S-allyl-cysteine were extracted. CONCLUSIONS: These results clearly demonstrated that pressurised liquid extraction is an efficient green technique to extract bioactive organosulphur compounds from giant garlic. Extraction yields were significantly (p < 0.05) higher than those obtained with conventional ultra-turrax extraction.

Preparative Separation of Procyanidins from Cocoa Polyphenolic Extract: Comparative Study of Different Fractionation Techniques.

To provide further insight into the antioxidant potential of procyanidins (PCs) from cocoa beans, PC extract was fractionated by several methodologies, including solid phase extraction, Sephadex LH-20 gel permeation, and preparative HPLC using C18 and diol stationary phases. All the isolated fractions were analyzed by UHPLC-QTOF-MS to determine their relative composition. According to our results, classical techniques allowed good separation of alkaloids, catechins, dimers, and trimers, but were inefficient for oligomeric PCs. Preparative C18-HPLC method allowed the attainment of high relative composition of fractions enriched with alkaloids, catechins, and PCs with degree of polymerization (DP) < 4. However, the best results were obtained by preparative diol-HPLC, providing a separation according to the increasing DP. According to the mass spectrometry fragmentation pattern, the nine isolated fractions (Fractions II-X) consisted of exclusively individual PCs and their corresponding isomers (same DP). In summary, an efficient, robust, and fast method using a preparative diol column for the isolation of PCs is proposed. Regarding DPPH• and ABTS•+ scavenging activity, it increases according to the DP; therefore, the highest activity was for cocoa extract > PCs > monomers. Thereby, cocoa procyanidins might be of interest to be used as alternative antioxidants.

Quantitative analysis of aqueous phases of bio-oils resulting from pyrolysis of different biomasses by two-dimensional comprehensive liquid chromatography.

Agrifood by-products are perfect candidates to be further processed under the concept of circular economy, in order to produce their valorization. Although significant amounts of food-related wastes that are discarded are produced worldwide, these might still be rich in valuable compounds. A strategy to further valorize agrifood-related by-products is based on pyrolysis processes. The result of this process is a liquid product termed bio-oil which is composed of an organic phase and an aqueous phase. This bio-oil is rich on a variety of components and its analysis implies several challenges. In this work, quantitative on-line comprehensive two-dimensional liquid chromatography (LC × LC) is proposed for the first time to characterize several aqueous phases of different bio-oils. Rice husk, peanut shell, spent coffee grounds, peach core and Eucalyptus sawdust biomasses were analyzed. The developed quantitative LC × LC method presented very good linearity, precision, reproducibility, recovery and LODs and LOQs as low as 0.05 µg mL-1 and 0.16 µg mL-1, respectively. As much as 28 components were simultaneously separated and quantified in those samples. Our results found that the composition of these bio-oils was different but strongly related to the agrifood by-product submitted to pyrolysis. The developed methodology is foreseen as a valuable tool for the quantitative study of other bio-oils, considering the great complexity and high dimensionality of these samples.

Insight of Stability of Procyanidins in Free and Liposomal Form under an in Vitro Digestion Model: Study of Bioaccessibility, Kinetic Release Profile, Degradation, and Antioxidant Activity.

Small unilamellar and multilayered liposomes loaded with polymeric (epi)catechins up to pentamers were produced. The bioaccessibility, kinetic release profile, and degradation under in vitro gastrointestinal conditions were monitored by UHPLC-DAD-QTOF-MS/MS. The results show that all of the procyanidins underwent depolymerization and epimerization into small molecular oligomers and mainly to (epi)catechin subunits. Moreover, all of the liposome formulations presented higher bioaccessibility and antioxidant activity in comparison to their respective counterparts in non-encapsulated form. Similar results were obtained with procyanidins from cocoa extract-loaded liposomes. Namely, the bioaccessibility of dimer, trimer, and tetramer fractions from cocoa-loaded liposomes were 4.5-, 2.1-, and 9.3-fold higher than those from the non-encapsulated cocoa extract. Overall, the procyanidin release profile was dependent on their chemical structure and physicochemical interaction with the lipid carrier. These results confirmed that liposomes are efficient carriers to stabilize and transport procyanidins with the aim of enhancing their bioaccessibility at a controlled release rate.

Design, Fabrication, Characterization, and In Vitro Digestion of Alkaloid-, Catechin-, and Cocoa Extract-Loaded Liposomes.

Liposomes containing theobromine, caffeine, catechin, epicatechin, and a cocoa extract were fabricated using microfluidization and sonication. A high encapsulation efficiency and good physicochemical stability were obtained by sonication (75% amplitude, 7 min). Liposomes produced at pH 5.0 had mean particle diameter ranging from 73.9 to 84.3 nm. The structural and physicochemical properties of the liposomes were characterized by transmission electron microscopy, confocal fluorescence microscopy, and antioxidant activity assays. The release profile was measured by ultra-high performance liquid chromatography coupled to diode array detection. The bioaccessibility of the bioactive compounds encapsulated in liposomes was determined after exposure to a simulated in vitro digestion model. Higher bioaccessibilities were measured for all catechins-loaded liposome formulations as compared to nonencapsulated counterparts. These results demonstrated that liposomes are capable of increasing the bioaccessibility of flavan-3-ols, which may be important for the development of nutraceutical-enriched functional foods.

Determination of phenolic compounds in ancient and modern durum wheat genotypes.

Due to its significant amount of antioxidants, durum wheat (Triticum turgidum ssp. durum) could potentially contribute to the protection against a number of chronic diseases, such as diabetes, cardiovascular disease, and cancer. The increasing interest toward healthy food among both consumers and scientists has moved the focus toward the phytochemical content of whole wheat grains. The aim of this study was to identify the phytochemical composition of 22 cultivars belonging to old and modern durum wheat genotypes, including antioxidant capacity (DPPH and FRAP tests). In addition, five phenolic acids involved in the vanillin biosynthesis pathway and in the defence mechanism of plants were screened using UHPLC-MS/MS. Remarkable quantitative differences in the amount of the five phenolic acids analysed (p < 0.05) were detected among the wheat genotypes investigated. Results showed that among the investigated phenolic compounds, trans-ferulic acid was the most abundant, ranging from 13.28 to 324.69 µg/g; all the other identified compounds were present at lower concentrations. Moreover, significant differences on the antioxidant activity were observed. Collected data suggested possible differences between biosynthetic pathway of secondary metabolites among durum wheat genotypes.

Population mechanics: A mathematical framework to study T cell homeostasis.

Unlike other cell types, T cells do not form spatially arranged tissues, but move independently throughout the body. Accordingly, the number of T cells in the organism does not depend on physical constraints imposed by the shape or size of specific organs. Instead, it is determined by competition for interleukins. From the perspective of classical population dynamics, competition for resources seems to be at odds with the observed high clone diversity, leading to the so-called diversity paradox. In this work we make use of population mechanics, a non-standard theoretical approach to T cell homeostasis that accounts for clone diversity as arising from competition for interleukins. The proposed models show that carrying capacities of T cell populations naturally emerge from the balance between interleukins production and consumption. These models also suggest remarkable functional differences in the maintenance of diversity in naïve and memory pools. In particular, the distribution of memory clones would be biased towards clones activated more recently, or responding to more aggressive pathogenic threats. In contrast, permanence of naïve T cell clones would be determined by their affinity for cognate antigens. From this viewpoint, positive and negative selection can be understood as mechanisms to maximize naïve T cell diversity.

Dissipation kinetics of organophosphorus pesticides in milled toasted maize and wheat flour (gofio) during storage.

The dissipation/degradation of the pesticides dimethoate, terbufos, disulfoton, and pirimiphos-methyl were evaluated in milled toasted maize and wheat flour (gofio) during three months of storage. Their dissipation kinetics and residual levels were determined, as well as their possible decomposition into some of their main transformation products (disulfoton sulfoxide, terbufos sulfone and disulfoton sulfone). For this purpose, pesticide-free milled toasted maize and wheat samples were spiked with the pesticides, and they were then stored in the darkness at ambient temperature in a closed container to simulate current storage conditions of such packed food. A multiresidue analysis based on the QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) method was performed for the simultaneous determination of these pesticides and their metabolites. After three months of storage, the dissipation of residues ranged between 34% (pirimiphos-methyl) and 86% (disulfoton) for maize gofio and between 69% (terbufos) and 92% (disulfoton and pirimiphos-methyl) for wheat gofio. The results demonstrated that the degradation was slower in gofio than in wheat gofio and that none of the selected metabolites were detected in any of the samples. Dissipation curves of all studied pesticides fitted to a first-order decay curve in both types of cereals.

Comparative Study of Green Sub- and Supercritical Processes to Obtain Carnosic Acid and Carnosol-Enriched Rosemary Extracts with in Vitro Anti-Proliferative Activity on Colon Cancer Cells.

In the present work, four green processes have been compared to evaluate their potential to obtain rosemary extracts with in vitro anti-proliferative activity against two colon cancer cell lines (HT-29 and HCT116). The processes, carried out under optimal conditions, were: (1) pressurized liquid extraction (PLE, using an hydroalcoholic mixture as solvent) at lab-scale; (2) Single-step supercritical fluid extraction (SFE) at pilot scale; (3) Intensified two-step sequential SFE at pilot scale; (4) Integrated PLE plus supercritical antisolvent fractionation (SAF) at pilot scale. Although higher extraction yields were achieved by using PLE (38.46% dry weight), this extract provided the lowest anti-proliferative activity with no observed cytotoxic effects at the assayed concentrations. On the other hand, extracts obtained using the PLE + SAF process provided the most active rosemary extracts against both colon cancer cell lines, with LC50 ranging from 11.2 to 12.4 µg/mL and from 21.8 to 31.9 µg/mL for HCT116 and HT-29, respectively. In general, active rosemary extracts were characterized by containing carnosic acid (CA) and carnosol (CS) at concentrations above 263.7 and 33.9 mg/g extract, respectively. Some distinct compounds have been identified in the SAF extracts (rosmaridiphenol and safficinolide), suggesting their possible role as additional contributors to the observed strong anti-proliferative activity of CA and CS in SAF extracts.

Anti-proliferative activity and chemical characterization by comprehensive two-dimensional liquid chromatography coupled to mass spectrometry of phlorotannins from the brown macroalga Sargassum muticum collected on North-Atlantic coasts.

In the present work, the phlorotannin composition of different Sargassum muticum samples collected at different locations along the North Atlantic coasts as well as the bioactivities related to these components were investigated. After pressurized liquid extraction, the samples collected at the extreme locations of a latitudinal gradient from Portugal and Norway, were found to be the richest on total phenols and, particularly, on phlorotannins, containing up to 148.97 and 5.12mg phloroglucinol equivalents g(-1), respectively. The extracts obtained from these locations were further purified and chemically characterized using a modified HILIC×RP-DAD-MS/MS method. The application of this methodology allowed the tentative identification of a great variability of phlorotannins with different degrees of polymerization (from 3 to 11) and structures, determined for the first time in S. muticum. The most-abundant phlorotannins on these samples were fuhalols, hydroxyfuhalols and phlorethols, showing also particularities and important differences depending on the geographical location. Afterwards, the antiproliferative activity of these extracts against HT-29 adenocarcinoma colon cancer cells was studied. Results revealed that the richest S. muticum samples in terms of total phlorotannins, i.e., those from Norway, presented the highest activity, showing a good cytotoxic potential at concentrations in the medium micromolar range.

An Emerging Allee Effect Is Critical for Tumor Initiation and Persistence.

Tumor cells develop different strategies to cope with changing microenvironmental conditions. A prominent example is the adaptive phenotypic switching between cell migration and proliferation. While it has been shown that the migration-proliferation plasticity influences tumor spread, it remains unclear how this particular phenotypic plasticity affects overall tumor growth, in particular initiation and persistence. To address this problem, we formulate and study a mathematical model of spatio-temporal tumor dynamics which incorporates the microenvironmental influence through a local cell density dependence. Our analysis reveals that two dynamic regimes can be distinguished. If cell motility is allowed to increase with local cell density, any tumor cell population will persist in time, irrespective of its initial size. On the contrary, if cell motility is assumed to decrease with respect to local cell density, any tumor population below a certain size threshold will eventually extinguish, a fact usually termed as Allee effect in ecology. These results suggest that strategies aimed at modulating migration are worth to be explored as alternatives to those mainly focused at keeping tumor proliferation under control.

Kojibiose ameliorates arachidic acid-induced metabolic alterations in hyperglycaemic rats.

Herein we hypothesise the positive effects of kojibiose (KJ), a prebiotic disaccharide, selected for reducing hepatic expression of inflammatory markers in vivo that could modulate the severity of saturated arachidic acid (ARa)-induced liver dysfunction in hyperglycaemic rats. Animals were fed daily (20 d) with ARa (0·3 mg) together or not with KJ (22 mg approximately 0·5 %, w/w diet). Glucose, total TAG and cholesterol contents and the phospholipid profile were determined in serum samples. Liver sections were collected for the expression (mRNA) of enzymes and innate biomarkers, and intrahepatic macrophage and T-cell populations were analysed by flow cytometry. ARa administration increased the proportion of liver to body weight that was associated with an increased (by 11 %) intrahepatic macrophage population. These effects were ameliorated when feeding with KJ, which also normalised the plasmatic levels of TAG and N-acyl-phosphatidylethenolamine in response to tissue damage. These results indicate that daily supplementation of KJ significantly improves the severity of ARa-induced hepatic alterations.

Structural differences of prebiotic oligosaccharides influence their capability to enhance iron absorption in deficient rats.

This study evaluates the influence of novel galacto-oligosaccharides derived from lactulose (GOS-Lu), kojibiose or 4'-galactosyl-kojibiose in hematological parameters of Fe homeostasis using Fe-deficient animals. Liver TfR-2, IL-6, NFκB and PPAR-γ expression (mRNA) were also determined by RT-qPCR analyses, and active hepcidin peptide production and short chain fatty acids by LC coupled to MS/MS or UV detection. Feeding animals with GOS-Lu or kojibiose together with FeCl3 increased hemoglobin (Hb) production (by 17%) and mean Hb concentration into erythrocytes relative to animals administered with FeCl3 alone (14.1% and 19.7%, respectively). Animals administered with prebiotics showed decreased plasmatic hepcidin levels, contributing to a higher intestinal absorption of the micronutrient. These data indicate that concurrent administration of these potentially prebiotic oligosaccharides together with a supplement of Fe ameliorates inflammation-mediated perturbations in the liver, according to the particular structure of the prebiotic compound, and result an attractive strategy to improve Fe absorption.

Separation and characterization of phlorotannins from brown algae Cystoseira abies-marina by comprehensive two-dimensional liquid chromatography.

Phlorotannins are an important class of polyphenolic compounds only found in brown algae. The chemical analysis of these bioactive polyphenols is rather difficult due to the great chemical variability and complexity of the natural composition of these components in algae, forming large phloroglucinol polymers. In the present work, a new approach based on the use of comprehensive 2D LC (LC × LC) is shown to analyze this complex family of compounds. The developed LC × LC methodology is based on the coupling of a hydrophilic-interaction LC (HILIC)-based separation in the first dimension and an RP-based separation in the second dimension. The employment of this online coupling together with DAD and MS/MS allowed the separation and identification of more than 50 compounds in a Cystoseira abies-marina brown alga extract. Phlorotannins containing from 5 to 17 phloroglucinol units were identified in this sample by HILIC × RP-DAD-MS/MS. Besides, using the 2,4-dimethoxybenzaldehyde assay, it was possible to determine that the total amount of phlorotannins present in the extract was 40.2 mg phloroglucinol equivalents per gram of extract. To our knowledge, this work is the first demonstration of the usefulness of HILIC × RP-DAD-MS/MS for the determination of phlorotannins.

Estimating dose painting effects in radiotherapy: a mathematical model.

Tumor heterogeneity is widely considered to be a determinant factor in tumor progression and in particular in its recurrence after therapy. Unfortunately, current medical techniques are unable to deduce clinically relevant information about tumor heterogeneity by means of non-invasive methods. As a consequence, when radiotherapy is used as a treatment of choice, radiation dosimetries are prescribed under the assumption that the malignancy targeted is of a homogeneous nature. In this work we discuss the effects of different radiation dose distributions on heterogeneous tumors by means of an individual cell-based model. To that end, a case is considered where two tumor cell phenotypes are present, which we assume to strongly differ in their respective cell cycle duration and radiosensitivity properties. We show herein that, as a result of such differences, the spatial distribution of the corresponding phenotypes, whence the resulting tumor heterogeneity can be predicted as growth proceeds. In particular, we show that if we start from a situation where a majority of ordinary cancer cells (CCs) and a minority of cancer stem cells (CSCs) are randomly distributed, and we assume that the length of CSC cycle is significantly longer than that of CCs, then CSCs become concentrated at an inner region as tumor grows. As a consequence we obtain that if CSCs are assumed to be more resistant to radiation than CCs, heterogeneous dosimetries can be selected to enhance tumor control by boosting radiation in the region occupied by the more radioresistant tumor cell phenotype. It is also shown that, when compared with homogeneous dose distributions as those being currently delivered in clinical practice, such heterogeneous radiation dosimetries fare always better than their homogeneous counterparts. Finally, limitations to our assumptions and their resulting clinical implications will be discussed.