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.     

Protein–protein interactions of a whey–pea protein co-precipitate

The aim of this study was to investigate the mechanisms behind protein–protein interactions in a co-precipitate of whey protein isolate (WPI) and pea protein isolate (PPI). A co-precipitate and blend, consisting of 80% WPI and 20% PPI, were compared. Covalent disulphide interactions were studied by blocking of free thiols with N-Ethylmaleimide (NEM), while electrostatic interactions were studied in systems with 0.5 m NaCl and hydrophobic interactions with 0.2% SDS. Protein solubility, stability and secondary, tertiary and quaternary protein structures were analysed. Co-precipitation did not introduce different protein–protein interactions than the direct blending of proteins. SDS affected solubility (P < 0.05), secondary and tertiary structure. However, the effects of NEM and NaCl were significant greater (P < 0.05) on the same parameters and thermal stability, especially when combined (P < 0.01). Thus, the protein–protein interactions in a whey–pea co-precipitate and protein blend consisted of disulphide bonds and electrostatic interactions.     

Characterization of quinoa protein–chitosan blend edible films

Quinoa protein/chitosan films were obtained by solution casting of blends of quinoa protein extract (PE) and chitosan (CH). Films from a PE/CH blend were characterized by FTIR, X-ray diffraction, thermal analysis, and SEM. The tensile mechanical, barrier, and sorption properties of the films were also evaluated. The blend of PE with CH yielded mechanically resistant films without the use of a plasticizer. The film had large elongation at break, and its water barrier properties showed that they were more hydrophilic than CH film. The thickness and water-vapor permeability of PE/CH (v/v) 1/1 blend film increased significantly compared to pure CH films. CH films are translucent in appearance and yellowish in blend with PE. By blending anionic PE with cationic CH an interaction between biopolymers was established with different physicochemical properties from those of pure CH. Drying and sorption properties show significant differences between PE/CH blend film and CH film. The structural properties determined by XRD, FTIR and TGA showed a clear interaction between quinoa proteins and CH, forming a new material with enhanced mechanical properties.     

Protein–flavour interactions in relation to development of novel protein foods

Proteins are known to interact with relatively small molecules such as flavour compounds and saponins, and may thus influence the taste perception of food. In this study, the interactions of flavour volatiles with pea proteins, and the effects of heat on these interactions were investigated. The presence of saponins, which are non-volatile flavour compounds, was also explored. Saponins are known to contribute to the bitterness in pea and were found to interact with proteins. Pea proteins, legumin (11S) and vicilin (7S), were used for interaction studies with aldehydes and ketones using static headspace-gas chromatography (SH–GC). The binding of various flavour compounds as a function of concentration was studied at pH 7.6 and pH 3.8. Vicilin binds both aldehydes and ketones at pH 7.6 and pH 3.8. Legumin only showed binding to aldehydes at pH 7.6 and no binding to aldehydes or ketones at pH 3.8. The effect of heat on vicilin-flavour interactions was studied at pH 7.6. Heating of vicilin seemed to lead to a decrease in the binding of aldehydes and ketones to the protein. In addition, the presence of saponins in hulled pea flour was identified by high performance liquid chromatography and mass spectrometry (HPLC–MS) and three groups of saponins, A, B and DDMP saponins were found to be present, with group B saponins dominating.     

Effects of protein interactions on properties and microstructure of zein–gliadin composite films

Zein and gliadin are both readily dissolved in aqueous ethanol and have good film-forming property. This article describes an attempt to improve the flexibility of zein films by the addition of gliadin to the zein film-forming solution. The properties of zein–gliadin composite films, i.e., color, transparency, moisture content, water solubility, water vapor permeability, dynamic contact angle which in turn affected the mechanical property, water resistance and glass transition temperature of films were investigated. The contents of second structure were characterized via Fourier transform infrared spectroscopy (FTIR), whereas morphology of films was examined by scanning electron microscopy (SEM). It was observed that the addition of gliadin enhanced the strain at break of zein–gliadin composite films as a result of the increase in the content of α-helix, β-turn structures and decrease in the level of β-sheet structure. The water resistance of films decreased with the content of gliadin increasing. Morphology of composite films showed that gliadin and zein organized a homogeneous material. This work opens a new perspective for zein in flexible food package.     

Casein–whey protein interactions in heated milk: the influence of pH

Heat-treatment of milk causes denaturation of whey proteins, leading to a complex mixture of whey protein aggregates and whey protein coated casein micelles. In this paper we studied the effect of pH-adjustment of milk (6.9–6.35) prior to heat-treatment on the distribution of denatured whey proteins in aggregates and coating of casein micelles. Proteins were fractionated using an alternative fractionation technique based on renneting. Acid- and rennet-induced gelation of these milks were used to obtain more information on the characteristics of the milk. Acid-induced gelation appeared to be mainly influenced by the presence of whey protein aggregates. Rennet-induced gelation was determined by the whey protein coating of the casein micelles. Both the quantity of whey proteins present on the surface of the casein micelles and the homogeneity of the coating were determining the renneting properties. These results extend the current knowledge on pH dependent casein–whey protein interactions. In order to present a clear picture of the changes occuring during heat treatment of milk at various pH, the results are summarized in a model. In this model we propose that heating at a pH>6.6 lead to a partial coverage of the casein micelles and the formation of separate whey protein aggregates. Heating at a pH<6.6 lead to an attachment of all whey proteins to the casein micelles. At pH 6.55 the coverage is rather homogeneous but lowering the pH further lead to an inhomogeneus coverage of the casein micelles. Surprisingly small changes of the pH at which the milk was heated had considerable effects on the gelation behaviour both in renneting and in acid gelation.     

Polysaccharide–protein interactions in dairy matrices,control and design of structures

Protein–polysaccharide interactions are of great importance in the design of dairy formulations, as they play a key role in the formation of structure and texture in dairy products. With a detailed understanding of the factors affecting the interactions, the ability of charged polysaccharides to associate with the milk proteins is continuously exploited to create functional complexes, novel ingredients and delivery systems. In addition, formulations containing non-interacting polysaccharides also need to be carefully controlled, as these biopolymers may give rise to segregative phase separation, with important consequences to the stability and quality of the final matrix. As casein micelles play a major role in imparting structure to dairy products, emphasis in this review will be given to the molecular details of the interactions between polysaccharides with these protein particles. Some of the most researched polysaccharides will be highlighted in this context, and the progress made in understanding their role during structure formation of dairy matrices will be discussed. The opportunity of creating novel microstructures provided by association or/and incompatibility of milk proteins and different polysaccharides will be assessed.     

Co-precipitation of whey and pea protein – indication of interactions

The aim was to optimise the yield of co-precipitation of whey protein isolate (WPI) and pea protein isolate (PPI) and compare co-precipitates and protein blends with respect to solubility. The yield of co-precipitates was tested with different protein ratios of WPI and PPI in combination with different temperatures and acid precipitation (pH 4.6). The highest precipitation yield was obtained at protein ratios WPI < PPI, high temperature and alkaline protein solvation. The solubility was measured by an instability index and absorption spectroscopy of re-suspended precipitated proteins at pH 3, 7 and 11.5. Co-precipitates had significantly lower solubility than protein blends. Protein ratios WPI > PPI, low precipitation temperature and high pH showed the highest solubility. Differences in protein composition between co-precipitates and protein blends were observed with SDS-PAGE and matrix-assisted laser desorption ionisation time-of-flight, and indicated different protein–protein interaction in samples, which needs further investigations.     

Effect of protein–starch interactions on starch retrogradation and implications for food product quality

Starch retrogradation is a consequential part of food processing that greatly impacts the texture and acceptability of products containing both starch and proteins, but the effect of proteins on starch retrogradation has only recently been explored. With the increased popularity of plant-based proteins in recent years, incorporation of proteins into starch-based products is more commonplace. These formulation changes may have unforeseen effects on ingredient functionality and sensory outcomes of starch-containing products during storage, which makes the investigation of protein–starch interactions and subsequent impact on starch retrogradation and product quality essential. Protein can inhibit or promote starch retrogradation based on its exposed residues. Charged residues promote charge–dipole interactions between starch-bound phosphate and protein, hydrophobic groups restrict amylose release and reassociation, while hydrophilic groups impact water/molecular mobility. Covalent bonds (disulfide linkages) formed between proteins may enhance starch retrogradation, while glycosidic bonds formed between starch and protein during high-temperature processing may limit starch retrogradation. With these protein–starch interactions in mind, products can be formulated with proteins that enhance or delay textural changes in starch-containing products. Future work to understand the impact of starch–protein interactions on retrogradation should focus on integrating the fields of proteomics and carbohydrate chemistry. This interdisciplinary approach should result in better methods to characterize mechanisms of interaction between starch and proteins to optimize their food applications. This review provides useful interpretations of current literature characterizing the mechanistic effect of protein on starch retrogradation.     

Stability of antioxidants in an apple polyphenol–milk model system

The stability of antioxidants in an apple polyphenol–milk model system was examined. The model system consisted of skim milk fortified with pH-neutralised apple polyphenols (AP, 0–200 mg per 100 ml milk), with or without ascorbic acid (100 mg per 100 ml milk). Physical and chemical changes were evaluated after thermal treatment (120 °C, 5 min) and oxidative storage (20 °C and 38 °C, up to 12 weeks). Antioxidant capacity was determined using both oxygen radical absorbance capacity (ORAC) assay and ferric reducing antioxidant power (FRAP) assay. Significant antioxidant capacity was detected in the presence of milk. Antioxidant capacity was retained during thermal treatment but decreased slowly during storage.     

Influence of milk pre-heating conditions on casein–whey protein interactions and skim milk concentrate viscosity

The viscosity of concentrates (50–55% total solids) prepared from skim milk heated (5 min at 80 or 90 °C) at pH 6.5 and 6.7 was examined. The extent of heat-induced whey protein denaturation increased with increasing temperature and pH. More denatured whey protein and κ-casein were found in the serum phase of milk heated at higher pH. The viscosity of milk concentrates increased considerably with increasing pH at concentration and increasing heating temperature, whereas the distribution of denatured whey proteins and κ-casein between the serum and micellar phase only marginally influenced concentrate viscosity. Skim milk concentrate viscosity thus appears to be governed primarily by volume fraction and interactions of particles, which are governed primarily by concentration factor, the extent of whey protein denaturation and pH. Control and optimization of these factors can facilitate control over skim milk concentrate viscosity and energy efficiency in spray-drying.     

Olive oil-in-water emulsions stabilized with caseinate: Elucidation of protein–lipid interactions by infrared spectroscopy

This paper reports a FT-IR study for probing lipid and protein structural changes and their interactions in various oil-in-water emulsions. Two different emulsions were prepared using sodium caseinate, as stabilizer system, without and with microbial transglutaminase (MTG), denominated E/SC and E/SC + MTG respectively. Proximate composition, fat and water binding properties and textural characteristics were also evaluated in the emulsions. Penetration force and gel strength values were used to distinguish different (P < 0.05) textural behaviours depending on the formulation of emulsifying system. E/SC + MTG emulsion showed gel textural behaviour while E/SC lack of this property. The spectral results showed frequency upshifting of the amide I band in going from protein stabilizer systems isolate (the solution used as reference) to their corresponding emulsions, what is attributable to greater protein structural order upon emulsion formation. Enzymatic action of MTG in the sodium caseinate stabilizing system induces structural changes, in terms of lipid chain disorder or lipid–protein interactions and protein secondary modifications, which may reflect the formation of a gel structure in the emulsion. These results could help to choose the stabilizing system that is most suitable and effective for its use in the formulation of food products.     

Characterization of supercritical fluid extrusion processed rice–soy crisps fortified with micronutrients and soy protein

Protein and micro-nutrients enriched rice–soy crisps (RSC) were prepared using supercritical fluid extrusion and their impact on quality attributes was determined. A low-shear, twin screw, co-rotating extruder was used to produce puffed RSC using supercritical CO₂ (SC-CO₂), which served as an expansion agent during the process carried out at lower temperatures (∼100 °C) compared to conventional steam based extrusion (∼130–180 °C). The fortified RSC contained 25–40 g/100 g soy protein and four micronutrients (iron, zinc, vitamin A and C) at the recommended daily values in 100 g product. The RSC were analyzed for physical characteristics and nutrient composition. The increasing soy protein fortification from 25 to 40 g/100 g reduced the crisps expansion ratio (4.27–2.95), crispiness (15.0–9.5), and increased piece density (0.21–0.27 g/cm³), bulk density (0.17–0.22 g/cm³) and hardness (76.39–129.05 N). The nutrient fortification improved protein (334–568%) and dietary fiber (571–901%) and the extrusion process retained all of the added minerals and about 50% retention of vitamin A and C in the final products. The SC-CO₂ assisted extrusion is an effective process-based approach to produce low-moisture, fortified crispy products. These products are appropriate for consumption as nutribars especially for school lunch programs in developing countries to reduce malnutrition through process based nutrient fortification approaches.     

An overview of binary taste–taste interactions

The human gustatory system is capable of identifying five major taste qualities: sweet, sour, bitter, salty and savory (umami), and perhaps several sub-qualities. This is a relatively small number of qualities given the vast number and structural diversity of chemical compounds that elicit taste. When we consume a food, our taste receptor cells are activated by numerous stimuli via several transduction pathways. An important food-related taste question which remains largely unanswered is: How do taste perceptions change when multiple taste stimuli are presented together in a food or beverage rather than when presented alone? The interactions among taste compounds is a large research area that has interested electrophysiologists, psychophysicists, biochemists, and food scientists alike. On a practical level, taste interactions are important in the development and modification of foods, beverages or oral care products. Is there enhancement or suppression of intensity when adding stimuli of the same or different qualities together? Relevant psychophysical literature on taste–taste interactions along with selected psychophysical theory is reviewed. We suggest that the position of the individual taste stimuli on the concentration-intensity psychophysical curve (expansive, linear, or compressive phase of the curve) predicts important interactions when reporting enhancement or suppression of taste mixtures.     

Characterization of chitosan–oleic acid composite films

Edible films based on high molecular weight chitosan (CH) and different concentrations of oleic acid (OA) were prepared. Film-forming dispersions (FFD) were characterized in terms of rheological properties, surface tension, particle size distribution and ζ-potential. In order to study the impact of the incorporation of OA into the CH matrix, the water sorption isotherms, water vapour permeability (WVP), mechanical properties, and optical properties of the dry films were evaluated. Results showed that the increase in OA promoted changes in the size and surface charge of the FFD particles, which had an impact on the rheological properties of the FFD. As regards the film properties, the higher the OA content, the lower the WVP and the moisture sorption capacity. In general, the addition of OA into the CH matrix leads to a significant increase in gloss and translucency and a decrease in the tensile strength, elongation at break and elastic modulus of the composite films. The mechanical and optical properties of the films were related with their microstructure, which was observed by SEM.