Chitosan/Polyvinyl Alcohol-Based Biofilms Using Ternary Deep Eutectic Solvents towards Innovative Color-Stabilizing Systems for Anthocyanins.

Anthocyanins are amazing plant-derived colorants with highly valuable properties; however, their chemical and color instability issues limit their wide application in different food industry-related products such as active and intelligent packaging. In a previous study, it was demonstrated that anthocyanins could be stabilized into green plasticizers namely deep eutectic solvents (DESs). In this work, the fabrication of edible films by integrating anthocyanins along with DESs into biocompatible chitosan (CHT)-based formulations enriched with polyvinyl alcohol (PVA) and PVA nanoparticles was investigated. CHT/PVA-DES films' physical properties were characterized by scanning electron microscopy, water vapor permeability, swelling index, moisture sorption isotherm, and thermogravimetry analysis. Innovative red-to-blue formulation films were achieved for CHT/PVA nanoparticles (for 5 min of sonication) at a molar ratio 1:1, and with 10% of ternary DES (TDES)-containing malvidin-3-glucoside (0.1%) where the physical properties of films were enhanced. After immersion in solutions at different pH values, films submitted to pHs 5-8 were revealed to be more color stable and resistant with time than at acidic pH values.

Exploring the Potential of Vine Shoots as a Source of Valuable Extracts and Stable Lignin Nanoparticles for Multiple Applications.

Large amounts of vine shoots are generated every year during vine pruning. This residue still presents many of the compounds found in the original plant, including low molecular weight phenolic compounds and structural compounds such as cellulose, hemicellulose, and lignin. For wine-producing regions, the challenge is to develop alternatives that will increase the value of this residue. This work proposes the full valorization of vine shoots, focusing on the extraction of lignin by mild acidolysis for the preparation of nanoparticles. The effect of the pretreatment solvents (ethanol/toluene, E/T, and water/ethanol, W/E), on the chemical and structural features of lignin, was evaluated. The chemical analysis suggests similar composition and structure regardless of the pretreatment solvent, although lignin isolated after pretreatment of biomass with E/T showed a higher content of proanthocyanidins (11%) compared with W/E (5%). Lignin nanoparticles (LNPs) presented an average size ranging from 130-200 nm and showed good stability for 30 days. Lignin and LNPs showed excellent antioxidant properties (half maximal inhibitory concentration, IC50 0.016-0.031 mg/mL) when compared to commercial antioxidants. In addition, extracts resulting from biomass pretreatment showed antioxidant activity, with W/E presenting a lower IC50 (0.170 mg/mL) than E/T (0.270 mg/mL), correlated with the higher polyphenol content of W/E, with (+)-catechin and (-)-epicatechin being the main compounds detected. Overall, this work shows that the pre-treatment of vine shoots with green solvents can yield (i) the production of high-purity lignin samples with antioxidant properties and (ii) phenolic-rich extracts, promoting the integral reuse of this byproduct and contributing to sustainability.

Interactions of dietary polyphenols with epithelial lipids: advances from membrane and cell models in the study of polyphenol absorption, transport and delivery to the epithelium.

Currently, diet-related diseases such as diabetes, obesity, hypertension, and cardiovascular diseases account for 70% of all global deaths. To counteract the rising prevalence of non-communicable diseases governments are investing in persuasive educational campaigns toward the ingestion of fresh fruits and vegetables. The intake of dietary polyphenols abundant in Mediterranean and Nordic-type diets holds great potential as nutritional strategies in the management of diet-related diseases. However, the successful implementation of healthy nutritional strategies relies on a pleasant sensory perception in the mouth able to persuade consumers to adopt polyphenol-rich diets and on a deeper understanding on the chemical modifications, that affect not only their chemical properties but also their physical interaction with epithelial lipids and in turn their permeability, location within the lipid bilayer, toxicity and biological activity, and fate during absorption at the gastro-intestinal epithelium, transport in circulation and delivery to the endothelium. In this paper, we review the current knowledge on the interactions between polyphenols and their metabolites with membrane lipids in artificial membranes and epithelial cell models (oral, stomach, gut and endothelium) and the findings from polyphenol-lipid interactions to physiological processes such as oral taste perception, gastrointestinal absorption and endothelial health. Finally, we discuss the limitations and challenges associated with the current experimental approaches in membrane and cell model studies and the potential of polyphenol-rich diets in the quest for personalized nutritional strategies ("personalized nutrition") to assist in the prevention, treatment, and management of non-communicable diseases in an increasingly aged population.

Sensorial properties of red wine polyphenols: Astringency and bitterness.

Polyphenols have been the subject of numerous research over the past years, being referred as the nutraceuticals of modern life. The healthy properties of these compounds have been associated to a natural chemoprevention of 21st century major diseases such as cancer and neurodegenerative diseases (e.g. Parkinson's and Alzheimer's). This association led to an increased consumption of foodstuffs rich in these compounds such as red wine. Related to the ingestion of polyphenols are the herein revised sensorial properties (astringency and bitterness) which are not still pleasant. This review intends to be an outline both at a sensory as a molecular level of the mechanisms underlying astringency and bitterness of polyphenols. Up-to-date knowledge of this matter is discussed in detail.

Study of Serial Exposures of an Astringent Green Tea Flavonoid Extract with Oral Cell-Based Models.

It is well known that repeated exposure to phenolic compounds (PCs) raises astringency perception. However, the link between this increase and the oral cavity's interactions with salivary proteins (SPs) and other oral constituents is unknown. To delve deeper into this connection, a flavonoid-rich green tea extract was tested in a series of exposures to two oral cell-based models using a tongue cell line (HSC3) and a buccal mucosa cell line (TR146). Serial exposures show cumulative PC binding to all oral models at all concentrations of the green tea extract; however, the contribution for the first and second exposures varies. The tongue mucosal pellicle (HSC3-Mu-SP) may contribute more to first-stage astringency (retaining 0.15 ± 0.01 mg mL-1 PCs at the first exposure), whereas the buccal mucosal pellicle (TR146-Mu-SP) retained significantly less (0.08 ± 0.02 mg mL-1). Additionally, increased salivary volume (SV+), which simulates the stimulation of salivary flow brought by a food stimulus, significantly enhances PC binding, particularly for TR146 cells: TR46-Mu-SP_SV+ bound significantly higher total PC concentration (0.17 ± 0.02 mg mL-1) than the model without increased salivary volume TR146-Mu-SP_SV- (0.09 ± 0.03 mg mL-1). This could be associated with a higher contribution of these oral cells for astringency perception during repeated exposures. Furthermore, PCs adsorbed in the first exposure to cell monolayer models (+TR146 and +HSC3) change the profile of PCs bound to these models in the second exposure. Regarding the structure binding activity, PCs with a total higher number of hydroxyl groups were more bound by the models containing SP. Regarding the SP, basic proline-rich proteins (bPRPs) may be involved in the increased perception of astringency upon repeated exposures. The extent of bPRP precipitation by PCs in mucosal pellicle models for both cell lines (HSC3 and TR146) in the second exposure (76 ± 13 and 83 ± 6%, respectively) was significantly higher than in the first one (25 ± 14 and 5 ± 6%, respectively).

Effect of condensed tannins addition on the astringency of red wines.

Astringency has been defined as a group of sensations involving dryness, tightening, and shrinking of the oral surface. It has been accepted that astringency is due to the tannin-induced interaction and/or precipitation of the salivary proline-rich proteins (PRPs) in the oral cavity, as a result of the ingestion of food products rich in tannins, for example, red wine. The sensory evaluation of astringency is difficult, and the existence of fast and reliable methods to its study in vitro is scarce. So, in this work, the astringency of red wine supplemented with oligomeric procyanidins (condensed tannins), and the salivary proteins (SP) involved in its development were evaluated by high-performance liquid chromatography analysis of human saliva after its interaction with red wine and by sensorial evaluation. The results show that for low concentration of tannins, the decrease of acidic PRPs and statherin is correlated with astringency intensity, with these families having a high relative complexation and precipitation toward condensed tannins comparatively to the other SP. However, for higher concentrations of tannins, the relative astringency between wines seems to correlate's to the glycosylated PRPs changes. This work shows for the first time that the several families of SP could be involved in different stages of the astringency development.

Nopal cladode as a novel reinforcing and antioxidant agent for starch-based films: A comparison with lignin and propolis extract.

The potential use of nopal cladode flour (NC) as reinforcing/bioactive agent in cassava starch-based films was evaluated and compared with the use of propolis extract or lignin, which are commonly used for these purposes. Cassava starch-based films containing untreated NC (S-NC), NC treated at pH 12 (S-NC12), aqueous propolis extract at two different concentrations (SP1 or SP2), or lignin (S-L) were produced by the casting technique; glycerol was used as plasticizer. NC12 and NC affected the mechanical properties of the cassava starch-based film similarly as compared to propolis extract and lignin. Moreover, NC and NC12 had different performance as reinforcing and antioxidant agent in cassava starch-based film. Thus, S-NC12 film was more elongable (28.5 ± 6.5%), more hydrophobic (contact angle: 70.8° ± 0.1), less permeable to water vapor (0.8 ± 0.0 × 10-10 g·m-1·s-1·Pa-1) and had better antioxidant activity by ABTS•+ (44.70 ± 0.3 µM Trolox·g-1 of film) than the S-NC film. SEM and TGA analysis of films showed that NC12 was better incorporated into the cassava starch matrix than NC, lignin and propolis extract. Overall, nopal cladode flour has potential use in the production of active biodegradable packaging for the food preservation with high oxidation rate.

Interaction of a Procyanidin Mixture with Human Saliva and the Variations of Salivary Protein Profiles over a 1-Year Period.

Procyanidins are widely associated with astringency perception and promptly interact/precipitate salivary proteins (SPs). In this work, the SP profile of 17 volunteers was monitored for 1 year, focusing on the SP families most related to astringency [aPRPs (acidic proline-rich proteins), bPRPs (basic proline-rich proteins), gPRPs (glycosylated proline-rich proteins), cystatins, P-B peptide, and statherin]. Although the total SP content remained constant, bPRPs showed high variability. Saliva from 5 volunteers was selected, each individual's saliva presenting a prominence in one of the referred SP families; each was used to interact with grape seed procyanidin oligomeric fraction. Independent of the prominences, a total depletion in statherin and P-B peptide was observed. These subjects performed a sensory assay and the limit of detection for astringency was determined. Overall, the specificity of SP toward procyanidins seemed to be more important in the interactions than the total SP content. The highest reactivity toward SPs was observed for epicatechin gallate, procyanidin dimers B7, B2g, and trimer C1.

Oral interactions between a green tea flavanol extract and red wine anthocyanin extract using a new cell-based model: insights on the effect of different oral epithelia.

Phenolic compounds (PC) are linked to astringency sensation. Astringency studies typically use simple models, with pure PC and/or proteins, far from what is likely to occur in the oral cavity. Different oral models have been developed here, comprising different oral epithelia (buccal mucosa (TR146) and tongue (HSC-3)) and other main oral constituents (human saliva and mucosal pellicle). These models, were used to study the interaction with two PC extracts, one rich in flavanols (a green tea extract) and one rich in anthocyanins (a red wine extract). It was observed that within a family of PC, the PC seem to have a similar binding to both TR146 and HSC-3 cell lines. When the oral constituents occur altogether, flavanols showed a higher interaction, driven by the salivary proteins. Conversely, anthocyanins showed a lower interaction when the oral constituents occur altogether, having a higher interaction only with oral cells. Epigallocatechin gallate, epicatechin gallate, epigallocatechin-3-O(3-O-methyl) gallate were the flavanols with the highest interaction. For the studied anthocyanins (delphinidin-3-glucoside, peonidin-3-glucoside, petunidin-3-glucoside and malvidin-3-glucoside), there was not a marked difference on their interaction ability. Overall, the results support that the different oral constituents can have a different function at different phases of food (PC) intake. These differences can be related to the perception of different astringency sub-qualities.

Inhibition Mechanisms of Wine Polysaccharides on Salivary Protein Precipitation.

In this work, high-performance liquid chromatography, fluorescence quenching, nephelometry, and sodium dodecyl sulfate polyacrylamide gel electrophoresis were used to study the effect of polysaccharides naturally present in wine [rhamnogalacturonan II (RG II) and arabinogalactan proteins (AGPs)] on the interaction between salivary proteins (SP) together present in saliva and tannins (punicalagin (PNG) and procyanidin B2). In general, the RG II fraction was more efficient to inhibit SP precipitation by tannins, especially for acidic proline-rich proteins (aPRPs) and statherin/P-B peptide, than AGPs. The RG II fraction can act mainly by a competition mechanism in which polysaccharides compete by tannin binding. However, in the presence of Na+ ions in solution, no RG II effect was observed on SP-tannin interactions. On the other hand, dependent upon the saliva sample as well as the tannin studied, AGPs can act by both mechanisms, competition and ternary (formation of a ternary complex with SP-tannin aggregates enhancing their solubility).

Interaction of polyphenols with model membranes: Putative implications to mouthfeel perception.

Food polyphenols in fruits juices, tea, coffee, wine and beer confer sensory properties such as colour, astringency and bitterness. The development of functional healthy drinks without the unpleasant sensory feeling is boosting research for a clearer understanding on the interactions of polyphenols within the oral mucosa. In this study we investigated the interaction of astringent polyphenols, namely ECG, EGCG, procyanidin B4 and PGG, with lipids in model membranes by spectroscopic techniques. The membrane model was built varying the cholesterol content to mimic mouth regions and experiments were conducted at pH 5 to mimic the pH drop at the moment of beverage (e.g. green tea, red wine) intake. Fluorescence quenching results conducted on LUVs with cholesterol molar fractions ranging between 0.34 < χchol < 0.74 and similar size distributions (122.9 ± 3.7 nm) showed that interaction of polyphenols is structure- and concentration-dependent. Also, the decrease of partition constants (Kp) with increasing cholesterol content (χchol) suggest that the affinity of polyphenols is weaker in cholesterol-rich liposomes. STD results revealed that the interaction of EGCG and PGG with membrane lipids involved mainly galloyl residues. Overall, spectroscopic data show that polyphenols interact to higher extent with more polar regions found in buccal, flour of the mouth and gingiva regions than with more hydrophobic regions located in the palate and tongue supporting that lipid microenvironments play a role in oral sensory perception.

Effects of Futsal Demands on Serum and Salivary Levels of Trace Elements and Minerals Detected by Total Reflection X-Ray Fluorescence.

The aim of this study was to monitor the circulating and salivary ion concentrations by total reflection X-ray fluorescence (TXRF) in futsal players submitted to the futsal-specific intermittent shuttle protocol (FISP). TXRF may allow identification of changes in ion concentrations induced by physical efforts. Saliva and blood samples of 13 male futsal players were collected before (Pre) and after (Post) the FISP. Salivary and plasma ion levels were detected by TXRF, and differences from Pre to Post (paired t test or Wilcoxon test) and correlations between both biological fluids were determined (P < 0.05). All saliva samples presented phosphorus (P), sulfur (S), chlorine (Cl), potassium (K), calcium (Ca), iron (Fe), zinc (Zn), bromine (Br), and rubidium (Rb). S, Cl, Ca, Fe, Cu, Zn, Br, and Rb were detected in all blood samples. K, Cu, Br, and Rb presented reduced secretion rate from Pre to Post samples (P < 0.05). The salivary concentrations of K (r = - 0.53) and Zn (r = 0.54) were correlated with plasmatic concentrations. After FISP, salivary secretion of S (r = - 0.76), Cl (r = - 0.64), P (r = - 0.67), Mn (r = - 0.74), and Zn (r = 0.69) were correlated with plasma levels. We concluded that TXRF may be used to monitor salivary (P, S, Cl, K, Ca, Fe, Zn, Br, and Rb) and circulating (S, Cl, Ca, Fe, Cu, Zn, Br, and Rb) levels of several elements in futsal athletes. However, an acute bout of futsal-specific physical effort did not significantly imbalance ion concentrations in saliva or plasma.

Development of a New Cell-Based Oral Model To Study the Interaction of Oral Constituents with Food Polyphenols.

Some polyphenols have unpleasant taste properties such as astringency, which could interfere with consumers' choices. The knowledge on astringency mechanisms points that astringency is a complex phenomenon probably related to more than one physical-chemical mechanism. Thus, this work aims to develop a new and more realistic cell-based model containing human saliva, mucosa pellicle, and an oral cell line (HSC-3) to understand the oral molecular events that could contribute to the overall astringency perception. This model was then used to study the interactions with a food procyanidin fraction (PF) by HPLC. In general, the results revealed higher interaction (synergism) for the model with all the referred oral constituents (mucosa pellicle, salivary proteins, and HSC-3 cell line, HSCMuSp) when compared to the interaction with individual constituents, the PF + cells or PF + saliva. Regarding the procyanidins, a significant interaction was observed for the procyanidin monomer EcG, procyanidin dimers B7 and B2G, and trimer C1.

Interaction between Ellagitannins and Salivary Proline-Rich Proteins.

The first contact of tannins with the human body occurs in the mouth, where some of these tannins are known to interact with salivary proteins, in particular with proline-rich proteins (PRPs). These interactions are important at a sensory level, especially for astringency development, but could also affect the biological activities of the tannins. This study gathers information on the relative affinity of the interaction, complex stoichiometry, and tannin molecular epitopes of binding for the interactions between the families of PRPs (bPRPs, gPRPs, and aPRPs) and three representative ellagitannins (castalagin, vescalagin, and punicalagin). These interactions were studied by saturation-tranfer difference NMR and microcalorimetry. The effect of the PRP-ellagitannin interaction on their antioxidant ability was also assessed by ferric reduction antioxidant power (FRAP) assays. The results support a significant interaction between the studied tannins and PRPs with binding affinities in the micromolar range. Punicalagin was always the ellagitannin with higher affinity. aPRPs were the salivary PRPs with higher affinity. Moreover, it was observed that when ellagitannins are present in low concentrations (5-50 µM), as occurs in food, the antioxidant ability of these tannins when complexed with salivary PRPs could be significantly impaired.

Impact of Lignosulfonates on the Thermodynamic and Kinetic Parameters of Malvidin-3- O-glucoside in Aqueous Solutions.

The interaction of malvidin-3- O-glucoside (1) and a lignosulfonate was studied by UV-visible spectroscopy, and the results obtained showed the formation of a complex between the negatively charged lignosulfonate and the flavylium cation form (AH+) of this anthocyanin at pH 1. The thermodynamic and kinetic parameters of 1 in the presence of a lignosulfonate were determined by UV-visible spectroscopy and stopped-flow techniques. The main differences were observed in the flavylium cation (AH+)/quinoidal base (A) equilibrium, the AH+ form being more stabilized than A (p Ka1 = 4.4 ± 0.1) compared with 1 in the absence of the lignosulfonate (p Ka1 = 3.9 ± 0.1). Furthermore, comparing the hydration ( kh = 0.028 s-1) and dehydration ( k-h = 40 M-1 s-1) processes of 1 in the presence of the lignosulfonate with the processes of 1 ( kh = 0.12 s-1 and k-h = 35 M-1 s-1) show that the hydration process is slower while the dehydration process is practically unaffected in the presence of the lignosulfonate.

A saliva molecular imprinted localized surface plasmon resonance biosensor for wine astringency estimation.

Wine astringency was evaluated based on the interaction of two complex matrices (red wine and saliva) by combining localized surface plasmon resonance (LSPR) and molecular imprinted polymers (MIP) at gold nanodisks as an alternative to sensorial analysis. The main objective of the work was to simulate wine astringency inside the mouth by mimicking this biological system. The LSPR/MIP sensor provided a linear response for astringency expressed in pentagalloyl glucose (PGG) units in concentrations ranging from 1 to 140µmol/L. The sensor was also applied to wine samples correlating well with sensorial analysis obtained by a trained panel. The correlation of astringency and wine composition was also evaluated showing that anthocyanins may have an important role, not only for pigmentation but also in astringency.

Procyanidins as antioxidants and tumor cell growth modulators.

Five procyanidin fractions with different structural complexities were obtained after fractionation of a grape seed extract. The procyanidin fraction's abilities to inhibit lipid peroxidation induced by 2,2'-azobis-2-methyl-propanimidamide dihydrochloride in a liposomal membrane system were examined. The antioxidant capacities of all fractions were evaluated through monitoring oxygen consumption and by measuring the formation of conjugated dienes. All tested fractions provided protection of membranes against peroxyl radicals by increasing the induction time of oxidation. This effect increased up to fraction II but decreased with the increase of the structural complexity of further procyanidin fractions, possibly due to steric hindrance effects exhibited by the more complex fractions. In addition, the antiradical properties and the reducing power of these fractions were determined by using 2,2-diphenyl-1-picrylhydrazyl and ferric reducing/antioxidant power methods, respectively. Moreover, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide reduction and DNA synthesis were measured in Michigan Cancer Foundation 7 (MCF-7), a human breast cancer cell line, treated with catechin or procyanidin fractions in order to evaluate the effect of these compounds on cell viability and proliferation. The results obtained showed that at 30 microg/mL, fractions I and II decreased cell viability and proliferation, which was not observed with 60 microg/mL of the same fractions. Catechin was also able to decrease cell viability and proliferation at 30 and 60 microg/mL. It is interesting to notice that the procyanidin fractions that exhibited higher antioxidant activity were the same to affect cell viability and proliferation.

Effect of flavonols on wine astringency and their interaction with human saliva.

The addition of external phenolic compounds to wines in order to improve their sensory quality is an established winemaking practice. This study was aimed at evaluating the effect of the addition of quercetin 3-O-glucoside on the astringency and bitterness of wines. Sensory results showed that the addition of this flavonol to wines results in an increase in astringency and bitterness. Additionally, flavonol-human salivary protein interactions were studied using fluorescence spectroscopy, dynamic light scattering and molecular dynamic simulations (MD). The apparent Stern-Volmer (KsvApp) and the apparent bimolecular quenching constants (kqApp) were calculated from fluorescence spectra. The KsvApp was 12620±390M(-1), and the apparent biomolecular constant was 3.94×10(12)M(-1)s(-1), which suggests that a complex was formed between the human salivary proteins and quercetin 3-O-glucoside. MD simulations showed that the quercetin 3-O-glucoside molecules have the ability to bind to the IB937 model peptide.

New procyanidin B3-human salivary protein complexes by mass spectrometry. Effect of salivary protein profile, tannin concentration, and time stability.

Several factors could influence the tannin-protein interaction such as the human salivary protein profile, the tannin tested, and the tannin/protein ratio. The goal of this study aims to study the effect of different salivas (A, B, and C) and different tannin concentrations (0.5 and 1 mg/mL) on the interaction process as well as the complex's stability over time. This study is focused on the identification of new procyanidin B3-human salivary protein complexes. Thus, 48 major B3-human salivary protein aggregates were identified regardless of the saliva and tannin concentration tested. A higher number of aggregates was found at lower tannin concentration. Moreover, the number of protein moieties involved in the aggregation process was higher when the tannin concentration was also higher. The selectivity of the different groups of proteins to bind tannin was also confirmed. It was also verified that the B3-human salivary protein complexes formed evolved over time.

In vivo interactions between procyanidins and human saliva proteins: effect of repeated exposures to procyanidins solution.

The general accepted mechanism for astringency arises from the interaction between tannins and salivary proteins (SP) resulting in (in)soluble aggregates. By HPLC analysis, it was observed that repeated sips of procyanidins (PC) solution practically depleted aPRPs (∼14%) and statherin (∼2%), and significantly reduced the amount of gPRPs. On the other hand, bPRPs were not significantly affected. In the analysis performed after the last exposure to PC solution, it was seen a significant recovering of the chromatographic peaks corresponding especially to aPRPs (∼74%) and statherin (∼80%). In vitro interaction between SP and PC results in the decrease of the chromatographic peaks of aPRPs and statherin, suggesting that these proteins were involved in the formation of a significant quantity of insoluble complexes. In general, the results suggest that the different families of SP can be involved in different stages of the development of astringency sensation.