Characterization of protein–polyphenol interactions

Dietary polyphenols have received attention for their biologically significant functions as antioxidants, anticarcinogens or antimutagens, which have led to their recognition as potential nutraceuticals. Polyphenols also characteristically possess a significant binding affinity for proteins, which can lead to the formation of soluble and insoluble protein–polyphenol complexes. Questions remain concerning whether and to what extent the protein–polyphenol interaction influences functionality. For example, is the formation of protein–polyphenol complexes an obstacle to the nutritional bioavailability of either species? This article discusses the development of suitable methodologies to investigate the physicochemical basis of protein–polyphenol interactions and the influence of structure–activity relationships on binding affinities.     

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.     

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.     

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.     

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.     

Water–water and water–macromolecule interactions in food dehydration and the effects of the pore structures of food on the energetics of the interactions

A molecular dynamics (MD) modeling and simulations approach has been rationally built and developed to study porous food systems constructed with amylose and dextran chains. The findings from our MD studies indicate that the presence of food macromolecules decreases the energetics of the water–water interactions for the nearby water molecules in the pore space, but provides additional water–macromolecule interactions that can significantly outweigh the partial loss of water–water interactions to make the adjacent water molecules strongly bound to the food macromolecules so that the water activity and water removal rate are decreased as dehydration proceeds and, thus, the dehydration energy requirement would be increased. The effects of pore structures are greater in systems with higher densities of food macromolecules, smaller in size pores, and stronger water–macromolecule interactions. Dehydration of food materials can thus be reasonably expected to start from the largest pores and from the middle of the pores, and to have non-uniform water removal rates and non-planar water–vapor interfaces inside individual pores as well as across sections of the food materials. The food porous structures are found to have good pore connectivity for water molecules. As dehydration proceeds, water content and the support from water–water and water–macromolecule interactions both decrease, causing the food porous structures to adopt more compact conformations and their main body to decrease in size. Dehydration in general also reduces pore sizes and the number of pore openings, increases the water–macromolecule interactions, and leads to the reduction of the overall thermal conductivity of the system, so that more energy (heat), longer times, and/or greater temperature gradients are needed in order to further dehydrate the porous materials. Our thermodynamic analysis also shows that the average minimum entropy requirement for food dehydration is greater when the water–macromolecule interactions are stronger and the food macromolecular density is higher. The importance of the physicochemical affinity of food molecules for water and of the compatibility of the resultant porous structures with water configurational structures in determining food properties and food processing through the water–macromolecule interactions, is clearly and fundamentally verified by the results and discussion presented in this work.     

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.     

The influence of protein–flavonoid interactions on protein digestibility in vitro and the antioxidant quality of breads enriched with onion skin

Different types of breads enriched with onion skin were studied. The objectives were twofold: to show and examine protein–phenolic interactions and to discuss results concerning phenolic content, antioxidant activity and protein digestibility in the light of in vitro bioaccessibility. Phenolic contents and antiradical abilities were linked with the level of onion skin supplement however, the amounts determined were significantly lower than expected. Fortification influenced protein digestibility (a reduction from 78.4% for control breads to 55% for breads with a 4% supplement). Electrophoretic and chromatographic studies showed the presence of indigestible protein–flavonoid complexes – with molecular weights about 25 kDa and 14.5 kDa; however, the reduction of free amino group levels and the increase in chromatogram areas suggest that flavonoids also bind to other bread proteins. The interaction of phenolics with proteins affects antioxidant efficacy and protein digestibility; thus, they have multiple effects on food quality and pro-health properties.     

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.     

Microassay for the quantitation of protein precipitable polyphenols: use of bovine serum albumin–benzidine conjugate as a protein probe

A simple, sensitive and indirect spectrophotometric method for the determination of protein precipitable polyphenols (tannins) has been developed, based on the ability of the polyphenols to precipitate the synthetic, brown coloured azo-protein, bovine serum albumin–benzidine conjugate (BSA–benzidine, mole ratio 1:7), which shows an absorption maxima at 405 nm. The amount of unprecipitated BSA–benzidine is measured directly at 405 nm, which is inversely related to the polyphenol concentration. Tannic acid was used as a reference standard. The microassay was performed in citrate/phosphate buffer (0.1 m), pH 4.8. The method was found to be linear in the range of 5–150 μg (3–88 nmol) of tannic acid (y=1.0+(−0.007)x; r=−0.989). Spiking studies carried out with various levels of tannic acid (0.01, 0.1 and 1.0%) indicated a recovery in the range of 94–101% and 94–98% in rice and sorghum samples, respectively. Free phenolics, when added in the range of 50–150 μg (catechin, chlorogenic acid, ferulic acid, caffeic acid and p-coumaric acid) had no influence on the protein precipitation in the microassay. Also spectral analysis of free phenolics and acid-methanolic sorghum extracts showed no interference in the present method. The conjugate was found to be stable over a period of 24 weeks in a freeze-dried condition and at 4°C, with <5% deterioration in aqueous condition. The microassay method developed has been used for the quantitation of protein precipitable polyphenols in various sorghum (Sorghum bicolor L. Moench) genotypes and compared with the widely used Folin–Denis chemical method of analysis.     

Evaluation of pea protein–polysaccharide matrices for encapsulation of acid-sensitive bacteria

Protein–polysaccharide capsules containing Bifidobacterium adolescentis were produced and tested in a series of in vitro survival experiments to evaluate capsule protection of the bacterium to simulated stomach conditions, as well as their ability to release the encapsulated bacteria under conditions similar to those found in the lower gut. A protein fraction isolated from peas (pea protein isolate: PPI; 2.0%; w/v) was mixed with each of three different polysaccharides (0.5% (w/v) of either sodium alginate, iota-carrageenan and gellan gum) to produce capsules ranging in size from 2 to 3 mm diameter. All capsule formulations provided significant protection for cells exposed to synthetic stomach juice at 37 °C relative to non-encapsulated bacteria. In addition, PPI-alginate and PPI-iota-carrageenan capsules were found to dissolve in simulated intestinal fluid at 37 °C, releasing 70–79% of their bacteria “payload” within 3 h, with higher cell numbers being released from the freeze-dried capsules. PPI-gellan gum capsules did not dissolve to the same extent and the number of released cells was ~ 26–30% lower. Following a temporal rat feeding study with the test bacterium encapsulated in PPI-alginate, B. adolescentis-specific PCR and qPCR analyses confirmed the presence of DNA from this species in rat feces, but only during the period of capsule intake.     

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.     

Screening of the polyphenol content of tomato-based products through accurate-mass spectrometry (HPLC–ESI-QTOF)

Tomatoes, the second most important vegetable crop worldwide, are a key component in the so-called “Mediterranean diet” and its consumption has greatly increased worldwide over the past 2 decades, mostly due to a growing demand for tomato-based products such as ketchups, gazpachos and tomato juices.     

Feasibility of the SPME method for the determination of the aroma retention capacity of proteose–peptone milk protein fraction at different pH values

The work aimed to develop a “true headspace-SPME–GC” method for evaluating the aroma retention capacity of the proteose–peptone (PP) milk protein fraction. The SPME analytical conditions were optimized in aqueous solution to which was added a blend containing seven aroma compounds from different chemical classes. The following parameters were tested: sample equilibrium time, fibre coating and fibre exposure time. The optimal equilibrium time was found to be 4 h and the DVB/CAR/PDMS fibre offered the highest sensitivity and repeatability. Finally, a 1 min fibre exposure time was chosen in order to avoid aroma competition and extraction phenomena. This method was then employed to evaluate the flavour binding capacity of PP aqueous systems at different pH values (6.8, 5.0 and 3.8). For all the pH conditions the retention capacity of the PP fraction gradually increased with hydrophobic chain length or overall hydrophobicity of the aroma compounds. Higher aroma binding capacity was found at pH 3.8.     

Sweet–bitter interactions and the solution properties of chlorinated sugars

Among chlorinated sugars, some are intensely sweet, some are bitter and others are tasteless. Although chlorination of sugars provokes an increase in lipophilicity, a certain hydrophilic/lipophilic balance is needed for sweeteners to be perceived. Two chlorinated sugars, sucralose (trichlorogalactosucrose) and methyldichlorogalactoside, respectively known for their enhanced sweetness (650×) and inhibitory effect on the sweetness of sucrose, are studied. Their sapid properties are interpreted on the basis of their physicochemical properties (intrinsic viscosity, apparent specific volume, surface tension, contact angle and vibrational spectra). It is particularly shown that the perturbation of the structure of water by these molecules, compared with that by simple sugars, helps in understanding their taste mechanism.     

Determining the physical stability and water–solid interactions responsible for caking during storage of alpha-anhydrous glucose

Typically, a crystalline powder is considered reasonably stable below it deliquescence point (RH₀), however, caking has been reported for some materials below their RH₀. The critical relative humidity (RH) values for caking and hydrate formation in alpha-anhydrous glucose (α-AG) and α-AG partitioned into three particle sizes were assessed using saturated salt slurries ranging from 0 to 84 % RH at 25 °C for 20 weeks. The degree of caking was determined by a five-point visual physical stability scale, from free flowing with minimal clumping (1) to fully caked (5), and X-ray powder diffraction was used to determine the composition of the samples. Caking was observed in α-AG during storage at 68 % RH at 25 °C and the severity of caking increased with increasing RH. Fine particle α-AG caked during storage at 64 % RH, whereas medium and large particle α-AG caked at 68 and 75 % RH, respectively, at 25 °C. Caking was observed in the absence of deliquescence, amorphous content, and hydrate formation; therefore, it is proposed that capillary condensation leads to caking in α-AG below its RH₀. Capillary condensation caking occurs at a specific RH (termed RH_cc) where direct condensation of moisture into confined spaces, such as particle contact points or surface defects, causes the formation of liquid bridges, which may solidify over time without changes in RH or temperature. To avoid caking, α-AG should be stored below its RH_cc, which is highly dependent on particle size; and to avoid conversion to glucose monohydrate, α-AG, regardless of particle size, should be stored below 64 % RH at 25 °C.