Analytical techniques for the study of polyphenol-protein interactions.

This mini review focuses on advances in biophysical techniques to study polyphenol interactions with proteins. Polyphenols have many beneficial pharmacological properties, as a result of which they have been the subject of intensive studies. The most conventional techniques described here can be divided into three groups: (i) methods used for screening (in-situ methods); (ii) methods used to gain insight into the mechanisms of polyphenol-protein interactions; and (iii) methods used to study protein aggregation and precipitation. All of these methods used to study polyphenol-protein interactions are based on modifications to the physicochemical properties of the polyphenols or proteins after binding/complex formation in solution. To date, numerous review articles have been published in the field of polyphenols. This review will give a brief insight in computational methods and biosensors and cell-based methods, spectroscopic methods including fluorescence emission, UV-vis adsorption, circular dichroism, Fourier transform infrared and mass spectrometry, nuclear magnetic resonance, X-ray diffraction, and light scattering techniques including small-angle X-ray scattering and small-angle neutron scattering, and calorimetric techniques (isothermal titration calorimetry and differential scanning calorimetry), microscopy, the techniques which have been successfully used for polyphenol-protein interactions. At the end the new methods based on single molecule detection with high potential to study polyphenol-protein interactions will be presented. The advantages and disadvantages of each technique will be discussed as well as the thermodynamic, kinetic or structural parameters, which can be obtained. The other relevant biophysical experimental techniques that have proven to be valuable, such electrochemical methods, hydrodynamic techniques and chromatographic techniques will not be described here.

Wine Flavonoids in Health and Disease Prevention.

Wine, and particularly red wine, is a beverage with a great chemical complexity that is in continuous evolution. Chemically, wine is a hydroalcoholic solution (~78% water) that comprises a wide variety of chemical components, including aldehydes, esters, ketones, lipids, minerals, organic acids, phenolics, soluble proteins, sugars and vitamins. Flavonoids constitute a major group of polyphenolic compounds which are directly associated with the organoleptic and health-promoting properties of red wine. However, due to the insufficient epidemiological and in vivo evidences on this subject, the presence of a high number of variables such as human age, metabolism, the presence of alcohol, the complex wine chemistry, and the wide array of in vivo biological effects of these compounds suggest that only cautious conclusions may be drawn from studies focusing on the direct effect of wine and any specific health issue. Nevertheless, there are several reports on the health protective properties of wine phenolics for several diseases such as cardiovascular diseases, some cancers, obesity, neurodegenerative diseases, diabetes, allergies and osteoporosis. The different interactions that wine flavonoids may have with key biological targets are crucial for some of these health-promoting effects. The interaction between some wine flavonoids and some specific enzymes are one example. The way wine flavonoids may be absorbed and metabolized could interfere with their bioavailability and therefore in their health-promoting effect. Hence, some reports have focused on flavonoids absorption, metabolism, microbiota effect and overall on flavonoids bioavailability. This review summarizes some of these major issues which are directly related to the potential health-promoting effects of wine flavonoids. Reports related to flavonoids and health highlight some relevant scientific information. However, there is still a gap between the knowledge of wine flavonoids bioavailability and their health-promoting effects. More in vivo results as well as studies focused on flavonoid metabolites are still required. Moreover, it is also necessary to better understand how biological interactions (with microbiota and cells, enzymes or general biological systems) could interfere with flavonoid bioavailability.

Multianalytical Approach to Understand Polyphenol-Mal d 1 Interactions to Predict Their Impact on the Allergenic Potential of Apples.

Interactions between phenolic compounds and the allergen Mal d 1 are discussed to be the reason for better tolerance of apple cultivars, which are rich in polyphenols. Because Mal d 1 is susceptible to proteolytic digestion and allergenic symptoms are usually restricted to the mouth and throat area, the release of native Mal d 1 during the oral phase is of particular interest. Therefore, we studied the release of Mal d 1 under different in vitro oral digestion conditions and revealed that only 6-15% of the total Mal d 1 present in apples is released. To investigate proposed polyphenol-Mal d 1 interactions, various analytical methods, e.g., isothermal titration calorimetry, 1H-15N-HSQC NMR, and untargeted mass spectrometry, were applied. For monomeric polyphenols, only limited noncovalent interactions were observed, whereas oligomeric polyphenols and browning products caused aggregation. While covalent modifications were not detectable in apple samples, a Michael addition of epicatechin at cysteine 107 in r-Mal d 1.01 was observed.

One- and two-dimensional high-performance thin-layer chromatography as an alternative analytical tool for investigating polyphenol-protein interactions.

INTRODUCTION: Polyphenols and simple phenolic compounds are able to react with other food constituents during processing and storage. In the past, it has been shown that their reaction with proteins can lead to changes of the technofunctional or even physiological properties of both compound classes. However, identification of specific binding sites of small molecules within a protein sequence (and the corresponding conformational position) is still challenging. OBJECTIVE: Investigating the reaction between different food proteins and phenolic compounds in alkaline medium with one- and two-dimensional high-performance thin-layer chromatography (HPTLC) coupled to matrix-assisted laser desorption/ionisation (MALDI) with time-of-flight (TOF) MS for analysing the peptide profiles after tryptic digestion. METHODS: After modification with phenolic compounds, protein derivatives were digested and peptides were separated with one- and two-dimensional HPTLC. Peptide profiles were detected with visible and UV wavelengths as well as with fluorescamine, ninhydrin and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid staining. In order to perform mass spectrometric measurements, peptides separated in the first dimension were analysed by MALDI/TOF/MS. RESULTS: Results show that the phenolic acids applied in this study show different specificity and susceptibility when modifying proteins resulting in changes of the peptide profiles, peptide quantity, polarity, UV-activity, radical-scavenging activity and molecular mass. CONCLUSION: One- and two-dimensional HPTLC supported by mass spectrometric detection represents an innovative, alternative tool for investigating and understanding polyphenol-protein interactions. This approach enables the identification of binding sites inside the protein chain and contributes to understanding the mechanism of polyphenol-protein interactions in vitro and in vivo.

Structure dependent stability and antioxidant capacity of strawberry polyphenols in the presence of canola protein.

Polyphenol stability in processed food affects sensorial and health-promoting properties. Thus, understanding the effects of various food components on polyphenols degradation, as a function of their chemical structure, can contribute to optimal product engineering. The current study focuses on the impact of polyphenol structure on polyphenol-protein interactions in correlation with their stability and total antioxidant capacity (TAC) during shelf-life. A strawberry polyphenol extract (SPE) and canola protein extract (CPE) were used as multicomponent polyphenol and plant-based protein models. A non-covalent interaction of SPE and CPE was observed at pH = 3. Among CPE proteins cruciferin was the most involved in interactions, and the polyphenols with the highest relative binding were flavonols (45 ± 3%-68 ± 2%), while anthocyanins presented lower values (0 ± 0.4%-27 ± 1%). The presence of the proteins enhanced mostly the anthocyanins' stability, yet the extent of the impact was not correlated with the relative binding. TAC was not better preserved by the presence of CPE.

Bean phenolic compound changes during processing: Chemical interactions and identification.

The common bean (Phaseolus vulgaris L.) represents one of the main crops for human consumption, due to its nutritional and functional qualities. Phenolic compounds have beneficial health effects, and beans are an essential source of these molecules, being found mainly in the seed coat and its color depends on the concentration and type of phenolic compounds present. The bean during storage and processing, such as cooking, germination, extrusion, and fermentation, undergoes physical, chemical, and structural changes that affect the bioavailability of its nutrients; these changes are related to the interactions between phenolic compounds and other components of the food matrix. This review provides information about the identification and quantification of phenolic compounds present in beans and the changes they undergo during processing. It also includes information on the interactions between the phenolic compounds and the components of the bean's cell wall and the analytical methods used to identify the interactions of phenolic compounds with macromolecules.

Understanding the molecular interactions between a yeast protein extract and phenolic compounds.

Phenolic compounds are partially removed during fining, which may influence the organoleptic properties of beverages. Among phenolic compounds, tannins have been widely associated to the taste of beverages (namely astringency and bitterness). Furthermore, phenolic acids and anthocyanins may also influence bitterness and the latter are also responsible for beverages' color. Thus, it is necessary to perform molecular studies to better understand the effect of fining agents in the overall phenolic composition of beverages and the resulting organoleptic changes. The molecular interactions between these three classes of phenolic compounds and a yeast protein extract (YPE), designed as a new fining agent, was studied. The binding affinities were assessed by fluorescence quenching at two temperatures (21 °C and 37 °C) and in two reaction media (water and wine model solution). The size of aggregates formed was characterized by Dynamic Light Scattering and the selectivity of protein interaction was analyzed by electrophoresis. Overall, pentagalloylglucoside (tannin) showed the highest binding affinity for YPE, followed by malvidin 3-glucoside (anthocyanin), p-coumaric acid (phenolic acid) and gallic acid (phenolic acid). The studied temperatures and solvents affected the interaction affinities as well as the aggregates' size. Binding selectivity of proteins from YPE was not found. These results open new perspectives to control the fining process by using the YPE as a fining agent taking into account the further effect in the organoleptic properties of beverages.

A Critical Review of the Characterization of Polyphenol-Protein Interactions and of Their Potential Use for Improving Food Quality.

BACKGROUND: Interest in protein-phenol interactions in biological systems has grown substantially in recent decades. METHODS: The interest has focused largely on food systems in response to reports on the prominent roles of phenolic compounds in nutrition and health. RESULTS: Phenolic compounds can have both favourable and adverse nutritional effects. Polyphenols are widely known for their antioxidant, anti-inflammatory, anticancer and antiaging properties; however, they have also been ascribed anti-nutritional effects resulting from interactions with some proteins and enzymes. Interactions between proteins and polyphenols can additionally influence food quality by altering some physical-chemical and sensory properties of foods. These effects may be useful to develop new products in food science and technology provided the nature of physical-chemical interactions between proteins and phenols is accurately elucidated. In this paper, we review the different possible modes of interaction between selected food proteins and phenolic compounds. CONCLUSION: Existing knowledge on the mechanisms behind polyphenol-protein reactions, the structures of the resulting products and their potential uses is reviewed.