Interactions between whey proteins and salivary proteins as related to astringency of whey protein beverages at low pH

Whey protein beverages have been shown to be astringent at low pH. In the present study, the interactions between model whey proteins (β-lactoglobulin and lactoferrin) and human saliva in the pH range from 7 to 2 were investigated using particle size, turbidity, and ζ-potential measurements and sodium dodecyl sulfate-PAGE. The correlation between the sensory results of astringency and the physicochemical data was discussed. Strong interactions between β-lactoglobulin and salivary proteins led to an increase in the particle size and turbidity of mixtures of both unheated and heated β-lactoglobulin and human saliva at pH ∼3.4. However, the large particle size and high turbidity that occurred at pH 2.0 were the result of aggregation of human salivary proteins. The intense astringency in whey protein beverages may result from these increases in particle size and turbidity at these pH values and from the aggregation and precipitation of human salivary proteins alone at pH <3.0. The involvement of salivary proteins in the interaction is a key factor in the perception of astringency in whey protein beverages. At any pH, the increases in particle size and turbidity were much smaller in mixtures of lactoferrin and saliva, which suggests that aggregation and precipitation may not be the only mechanism linked to the perception of astringency in whey protein.     

Factors regulating astringency of whey protein beverages

A rapidly growing area of whey protein use is in beverages. There are 2 types of whey protein-containing beverages: those at neutral pH and those at low pH. Astringency is very pronounced at low pH. Astringency is thought to be caused by compounds in foods that bind with and precipitate salivary proteins; however, the mechanism of astringency of whey proteins is not understood. The effect of viscosity and pH on the astringency of a model beverage containing whey protein isolate was investigated. Trained sensory panelists (n = 8) evaluated the viscosity and pH effects on astringency and basic tastes of whey protein beverages containing 6% wt/vol protein. Unlike what has been shown for alum and polyphenols, increasing viscosity (1.6 to 7.7 mPa·s) did not decrease the perception of astringency. In contrast, the pH of the whey protein solution had a major effect on astringency. A pH 6.8 whey protein beverage had a maximum astringency intensity of 1.2 (15-point scale), whereas that of a pH 3.4 beverage was 8.8 (15-point scale). Astringency decreased between pH 3.4 and 2.6, coinciding with an increase in sourness. Decreases in astringency corresponded to decreases in protein aggregation as observed by turbidity. We propose that astringency is related to interactions between positively charged whey proteins and negatively charged saliva proteins. As the pH decreased between 3.4 and 2.6, the negative charge on the saliva proteins decreased, causing the interactions with whey proteins to decrease.     

Lubricating properties of human whole saliva as affected by β-lactoglobulin

The effect of β-lactoglobulin (β-LG) at pH 3.5 and 7.0 on lubricating property of saliva as related to astringency perception was investigated using tribology. Saliva was adsorbed onto surfaces of a rotating poly dimethylsiloxane (PDMS) ball and disc to form a film under conditions that mimic the rubbing contacts in the oral cavity (Bongaerts, Rossetti, & Stokes, 2007) and the lubricity of saliva films upon exposure to astringent compounds was measured. While addition of non-astringent β-LG at pH 7.0 slowly increased friction of saliva film between tribopair surfaces, β-LG at pH 3.5 rapidly increased the friction coefficients of saliva, similar to other astringent compounds (epigallocatechin gallate and alum). This supports the hypothesis that astringency of β-LG arises from the loss of lubrication of saliva which is in agreement with the well-accepted astringency model of polyphenols. Increasing β-LG concentration at pH 3.5 (0.5–10% w/w) caused a rapid increase in friction coefficient; however, at the highest protein concentration, the friction coefficient, although higher than observed for water, was below the values observed for the lower protein concentrations. This suggests that static tribology testing is different from the dynamic in-mouth system such that a simple relationship between friction and sensory astringency cannot be found for all conditions.     

Astringency of bovine milk whey protein

Whey protein solutions at pH 3.5 elicited an astringent taste sensation. The astringency of whey protein isolate (WPI), the process whey protein (PWP) that was prepared by heating WPI at pH 7.0, and the process whey protein prepared at pH 3.5 (aPWP) were adjusted to pH 3.5 and evaluated by 2 sensory analyses (the threshold method and the scalar scoring method) and an instrumental analysis (taste sensor method). The taste-stimulating effects of bovine and porcine gelatin were also evaluated. The threshold value of astringency of WPI, PWP, and aPWP was 1.5, 1.0, and 0.7 mg/mL, respectively, whereas the gelatins did not give definite astringency. It was confirmed by the scalar scoring method that the astringency of these proteins increased with the increase in protein concentration, and these proteins elicited strong astringency at 10 mg/mL under acidic conditions. On the other hand, the astringency was not elicited at pH 3.5 by 2 types of gelatin. A taste sensor gave specific values for whey proteins at pH 3.5, which corresponded well to those obtained by the sensory analysis. Elicitation of astringency induced by whey protein under acidic conditions would be caused by aggregation and precipitation of protein molecules in the mouth.     

Interactions of enological tannins with the protein fraction of saliva and astringency perception are affected by pH

The effects of pH on both tannin-induced astringency and tannin–salivary protein interactions were investigated. A trained sensory panel evaluated astringency perception. Tannin–salivary protein interactions were assessed in vitro by examining the effects of either a condensed enological tannin or an hydrolyzable enological tannin on two physicochemical properties of the protein fraction of saliva, namely, its mode of diffusion on cellulose membranes and its precipitation. Comparative assays mimicking the degree of dilution experienced by saliva during a tasting assay were performed at pH 3.5 and pH 7.0. Results indicated that both enological tannins were perceived as clearly more astringent at pH 3.5 compared with pH 7.0. In addition, the effects of tannins on protein diffusion and protein precipitation were markedly exacerbated at pH 3.5.     

Ionic strength and buffering capacity of emulsifying salts determine denaturation and gelation temperatures of whey proteins

Ionic conditions affect the denaturation and gelling of whey proteins, affecting the physical properties of foods in which proteins are used as ingredients. We comprehensively investigated the effect of the presence of commonly used emulsifying salts on the denaturation and gelling properties of concentrated solutions of β-lactoglobulin (β-LG) and whey protein isolate (WPI). The denaturation temperature in water was 73.5°C [coefficient of variation (CV) 0.49%], 71.8°C (CV 0.38%), and 69.9°C (CV 0.41%) for β-LG (14% wt/wt), β-LG (30% wt/wt), and WPI (30% wt/wt), respectively. Increasing the concentration of salts, except for sodium hexametaphosphate, resulted in a linear increase in the denaturation temperature of WPI (kosmotropic behavior) and an acceleration in its gelling rate. Sodium chloride and tartrate salts exhibited the strongest effect in protecting WPI against thermal denaturation. Despite the constant initial pH of all solutions, salts having buffering capacity (e.g., phosphate and citrate salts) prevented a decrease in pH as the temperature increased above 70°C, resulting in a decline in denaturation temperature at low salt concentrations (≤0.2 mol/g). When pH was kept constant at denaturation temperature, all salts except sodium hexametaphosphate, which exhibited chaotropic behavior, exhibited similar effects on denaturation temperature. At low salt concentration, gelation was the controlling step, occurring up to 10°C above denaturation temperature. At high salt concentration (>3% wt/wt), thermal denaturation was the controlling step, with gelation occurring immediately after. These results indicate that the ionic and buffering properties of salts added to milk will determine the native versus denatured state and gelation of whey proteins in systems subjected to high temperature, short time processing (72°C for 15 s).     

Consumer perception of astringency in clear acidic whey protein beverages

Acidic whey protein beverages are a growing component of the functional food and beverage market. These beverages are also astringent, but astringency is an expected and desirable attribute of many beverages (red wine, tea, coffee) and may not necessarily be a negative attribute of acidic whey protein beverages. The goal of this study was to define the consumer perception of astringency in clear acidic whey protein beverages. Six focus groups (n=49) were held to gain understanding of consumer knowledge of astringency. Consumers were presented with beverages and asked to map them based on astringent mouthfeel and liking. Orthonasal thresholds for whey protein isolate (WPI) in water and flavored model beverages were determined using a 7-series ascending forced choice method. Mouthfeel/basic taste thresholds were determined for WPI in water. Acceptance tests on model beverages were conducted using consumers (n=120) with and without wearing nose clips. Consumers in focus groups were able to identify astringency in beverages. Astringency intensity was not directly related to dislike. The orthonasal threshold for WPI in water was lower (P < 0.05) than the mouthfeel/basic taste threshold of WPI in water. Consumer acceptance of beverages containing WPI was lower (P < 0.05) when consumers were not wearing nose clips compared to acceptance scores of beverages when consumers were wearing nose clips. These results suggest that flavors contributed by WPI in acidic beverages are more objectionable than the astringent mouthfeel and that both flavor and astringency should be the focus of ongoing studies to improve the palatability of these products.     

Functional and Biological Properties of Peptides Obtained by Enzymatic Hydrolysis of Whey Proteins

The study of peptides released by enzymatic hydrolysis of whey proteins has been initially focusing on improving their functional properties in food model systems. Our first study showed that peptides 41 to 60 and 21 to 40 from β-lactoglobulin (β-LG) were responsible for improved emulsifying properties of a tryptic hydrolysate of whey protein concentrate (WPC). Further work showed that adding negatively charged peptides from tryptic hydrolysates of WPC could prevent phase separation of dairy-based concentrated liquid infant formula, as a replacement for carrageenan. Hydrolysis of whey proteins using a bacterial enzyme was also successful in improving heat stability of whey proteins in an acidic beverage. Some tryptic peptides demonstrated improvement in the heat stability and in modifying thermal aggregation of whey proteins. Recent research has shown that whey peptides could trigger some physiological functions. Within the scope of this research our work has led to the development of a whey protein enzymatic hydrolysate that has demonstrated antihypertensive properties when orally administered to spontaneously hypertensive rats and human subjects. Our work then focused on the fractionation of hydrolysates by nanofiltration to prepare specific peptidic fractions; however, peptide/peptide and peptide/protein interactions impaired membrane selectivity. The study of those interactions has lead to the demonstration of the occurrence of interactions between β-LG and its hydrophobic fragment 102–105 (opioid peptide), which probably binds in the central cavity of the protein. This latest result suggests that β-LG could be used as a carrier for the protection of bioactive peptides from gastric digestion. Our work therefore has shown that the enzymatic hydrolysis of whey proteins is not only improving their functional properties, but it is also providing powerful technology in the exploitation of their biological properties for functional foods and nutraceutical applications.     

Astringency of tea catechins: More than an oral lubrication tactile percept

Oral astringency is the dry sensation experienced in the mouth on consumption of plant-based polyphenols (catechins) found in wine and tea as well as certain fruits and vegetables. It is commonly explained as arising from the loss of lubricity owing to the precipitation of proteins from the salivary film that coats and lubricates the oral cavity. Here, we investigate this hypothesis directly by probing the impact of astringent compounds on the lubricating properties of saliva. By preadsorbing saliva onto an elastic hydrophobic substrate to form a highly lubricating and robust film under conditions designed to mimic the low pressure rubbing contacts experienced in the oral cavity (Bongaerts, Rossetti, & Stokes, 2007), we probe the interaction of this film on exposure to solutions containing tea catechins. We examine the response of the adsorbed salivary film to polyphenol structure, concentration and temperature, as well as the influence of astringency modulating solutions consisting of a thickener (maltodextrin) and milk.We find that a significant increase in friction coefficient occurs upon exposure with epigallocatechin gallate (EGCG) solutions due to a depletion of the lubricating proteins from the elastic substrates. The friction coefficient increases more rapidly with increasing EGCG concentration, this is in line with a corresponding increase in astringency perception. In addition, the inclusion of a hydrocolloid thickener in EGCG solutions caused a decrease in astringency perception probably due to specific EGCG–maltodextrin interaction and to an increase in viscosity, which lowers the friction coefficient between the elastic substrates. These findings show that the physical interaction of saliva proteins with EGCG molecules, which we probe through the loss of saliva lubricity, can be advantageously used to predict the astringent acuity of EGCG using our simple oral mimetic technique, supporting the hypothesis that astringency is related to a loss of lubrication. However, epicatechin (EC) did not alter the lubricating properties of the salivary film, although the EC solutions were perceived to be astringent, an observation which seems to question a simple causal dependence on oral lubrication. In addition to that, milk mitigated the astringency perception of EGCG solutions although considerably reducing saliva lubricity. We conclude that the depletion of saliva lubricating proteins is not necessary to obtain an astringent perception, and that astringency is unlikely to be a purely tactile percept.     

Efficiency of removal of whey protein from sweet whey using polymeric microfiltration membranes

Our objective was to measure whey protein removal percentage from separated sweet whey using spiral-wound (SW) polymeric microfiltration (MF) membranes using a 3-stage, 3× process at 50°C and to compare the performance of polymeric membranes with ceramic membranes. Pasteurized, separated Cheddar cheese whey (1,080 kg) was microfiltered using a polymeric 0.3-μm polyvinylidene (PVDF) fluoride SW membrane and a 3×, 3-stage MF process. Cheese making and whey processing were replicated 3 times. There was no detectable level of lactoferrin and no intact α- or β-casein detected in the MF permeate from the 0.3-μm SW PVDF membranes used in this study. We found BSA and IgG in both the retentate and permeate. The β-lactoglobulin (β-LG) and α-lactalbumin (α-LA) partitioned between retentate and permeate, but β-LG passage through the membrane was retarded more than α-LA because the ratio of β-LG to α-LA was higher in the MF retentate than either in the sweet whey feed or the MF permeate. About 69% of the crude protein present in the pasteurized separated sweet whey was removed using a 3×, 3-stage, 0.3-μm SW PVDF MF process at 50°C compared with 0.1-μm ceramic graded permeability MF that removed about 85% of crude protein from sweet whey. The polymeric SW membranes used in this study achieve approximately 20% lower yield of whey protein isolate (WPI) and a 50% higher yield of whey protein phospholipid concentrate (WPPC) under the same MF processing conditions as ceramic MF membranes used in the comparison study. Total gross revenue from the sale of WPI plus WPPC produced with polymeric versus ceramic membranes is influenced by both the absolute market price for each product and the ratio of market price of these 2 products. The combination of the market price of WPPC versus WPI and the influence of difference in yield of WPPC and WPI produced with polymeric versus ceramic membranes yielded a price ratio of WPPC versus WPI of 0.556 as the cross over point that determined which membrane type achieves higher total gross revenue return from production of these 2 products from separated sweet whey. A complete economic engineering study comparison of the WPI and WPPC manufacturing costs for polymeric versus ceramic MF membranes is needed to determine the effect of membrane material selection on long-term processing costs, which will affect net revenue and profit when the same quantity of sweet whey is processed under various market price conditions.     

Comparative Study on Heat Stability and Functionality of Camel and Bovine Milk Whey Proteins

Heat stability, emulsifying, and foaming properties of camel whey have been investigated and compared with that of bovine whey. Camel whey is similar to bovine whey in composition, but is deficient in β-lactoglubulin (β-LG), a major component of bovine whey. Whether the deficiency in β-LG will affect stability and functional properties is not yet known. Substantial information on the functional properties of bovine milk whey proteins is available; however, there is little research done on functional properties of camel whey proteins. Therefore, the objective of this study was to investigate the heat stability, emulsifying, and foaming characteristics of camel whey proteins. Calorimetric studies showed no significant difference in heat stability between bovine and camel whey proteins in liquid form. Upon drying, thermograms indicated that the 2 proteins are different in composition and thermal stability. The difference is represented in the absence of β-LG and the occurrence of protein denaturation peak at a lesser temperature in camel whey. The first marginal thermal transition in bovine whey appeared at 81°C, followed by 2 other transitions at 146 and 198°C. For camel whey, the transitions appeared at 139, 180, and 207°C respectively. The first marginal denaturation peak in bovine whey is due to β-LG, which is essentially absent in camel whey, while the second peak is due to the mixture of α-lactalbumin, serum albumin, and possibly part of the partially stabilized β-LG structure during the denaturation process. Because camel whey is deficient in β-LG, the denaturation peak at 139 must be due to the mixture of α-lactalbumin and camel serum albumin. In both proteins, the highest thermal transition is due to sugars such as lactose. The solubility study has shown that camel whey is more sensitive to pH than bovine milk whey and that heat stability is lowest near the isoelectric point of the proteins at pH 4.5. The sensitivity to pH resulted in partial denaturation and increased tendency to aggregate, which caused poor and unstable emulsion at pH 5. Both bovine and camel whey proteins have demonstrated good foaming properties; however, the magnitudes of these properties were considerably greater in bovine milk for all of the conditions studied.     

Interpolymeric Complexes Formed Between Whey Proteins and Biopolymers: Delivery Systems of Bioactive Ingredients

Whey proteins are obtained from dairy industry waste. Studies involving the analysis of the bioactive compounds in whey show health benefits, as it is an excellent source of indispensable amino acids. Milk whey contains principally β‐lactoglobulin, α‐lactoglobulin, bovine serum albumin, and lactoferrin, proteins with innumerable functional and technological properties. One application of these proteins in food is the formation of interpolymer complexes, along with other proteins or anionic polysaccharides. The formation of complexes occurs mainly through electrostatic interactions between a negatively charged biopolymer and a positively charged biopolymer. This formation is influenced by factors such as pH, ionic strength, and biopolymer ratio. Because they do not use high temperatures and chemical reagents and have additional nutritional and functional value, these complexes have been used as encapsulating agents for bioactive ingredients. Recent studies on their training and applications are addressed in this review to boost new research and applications in the food industry, thus increasing opportunities for utilizing whey proteins.     

Study of the acid and thermal stability of β-lactoglobulin–ligand complexes using fluorescence quenching

The major protein in bovine milk whey, β-lactoglobulin (β-LG), has several binding sites for ligands. Its interactions with folic acid (a hydrophilic compound), resveratrol (amphiphilic) and α-tocopherol (hydrophobic) at neutral and acidic pH and after heating to 85 °C were studied using fluorescence quenching. Binding of folic acid occurs in a hydrophobic pocket in the groove between the α-helix and the β-barrel and is disturbed by decreasing the pH from 7.0 to 2.0. Resveratrol binds to the outer surface of β-LG near Trp19–Arg124 to form complexes that are stable at acidic pH. Acidification caused the release of α-tocopherol bound to the internal cavity but had no influence on that bound to a site at the surface of β-LG. The β-LG/folic acid complex was thermally stable. Thermal denaturing improved the affinity of the protein for resveratrol but decreased somewhat its affinity for α-tocopherol. These results should help guide the development of formulations based on β-LG as a carrier of a wide range of bioactive nutrients.     

乳铁蛋白与牛乳中其他蛋白质相互作用机制研究进展

牛乳蛋白分为酪蛋白和乳清蛋白,其中乳铁蛋白是乳清蛋白中的一种,具有多种生物学功能,被广泛应用于食品、医药、化妆品工业等领域。在牛乳中,乳铁蛋白带正电荷,会与一些带负电荷的蛋白质,如酪蛋白、骨桥蛋白、β-乳球蛋白、血清白蛋白、免疫球蛋白等发生相互作用。这种相互作用影响着这些蛋白的生物化学功能或者分离制备特性,尤其是后者更是工业化生产中需要关注的问题。本文阐述了乳铁蛋白与牛乳中其他蛋白质之间的相互作用机制及其应用现状,对于开发乳铁蛋白的工业化制备方法、以及系统理解牛乳中各种活性蛋白间的协同生物作用等方面都具有重要意义,为乳铁蛋白生物学特性的深入研究和工业化生产技术开发提供一定依据。     

Assessment of the interactions between pea and salivary proteins in aqueous dispersions

To understand and limit the unpleasant oral sensation of astringency felt during the consumption of pea-based drinks, we investigated the interaction between mixtures of salivary and pea proteins, as compared to mixtures where HEPES buffer (at pH 6.8) was used as a negative control for saliva. Since astringent compounds have the ability to bind with salivary proteins, mixes of freshly collected whole unstimulated saliva and a pea protein isolate (PPI) (a dispersion at 3.5% w/v) were prepared in ratios 95:5 and 1:1 saliva:PPI, to allow different stoichiometries to occur in the mouth. Samples were incubated at 37 °C during 30 min, after centrifugation at 16000g during 20 min to separate pellet from supernatant. Using techniques such as SEC, Native-PAGE and LC-MS, 7 pea proteins were identified as being capable of forming aggregates with at least 7 saliva proteins, some of which have been previously connected to astringency.     

Evaluation of the astringency of commercial tannins by means of the SDS–PAGE-based method

The astringency of wines enriched with commercial tannins (CTs) was evaluated by a method based on the SDS–PAGE electrophoresis of salivary proteins after the reaction of saliva with wine. Nineteen CTs tested in synthetic wine at the same pH (3.6) and concentration (1 g/l) gave different values of saliva precipitation index (SPI). The effect of CTs addition was investigated in four wines. Results showed that the wine matrix influenced the astringent capacity of CTs and that became less pronounced as wine polyphenolic complexity increased. For some types of wine, astringency was not affected, indicating that the effect of CTs utilisation is not easily predictable by classical methods. The ability to objectively evaluate the astringency provided by CTs with the SDS–PAGE-based method would supply producers and winemakers with a useful tool to manage the processing conditions and thus to improve the quality of wine.     

Whey protein aggregate formation during heating in the presence of κ-carrageenan

The effect of a negatively charged polymer, κ-carrageenan, on the aggregation behaviour of whey proteins during heating was studied. Aqueous solutions of whey protein isolate (WPI) at 0.5% were heated in the presence of κ-carrageenan (0.1%) at pH 7.0. This concentration was chosen as optimal in the detection of the intermediate aggregates during chromatographic analysis. The residual unaggregated protein, the intermediate aggregates and the soluble aggregates were all examined as a function of heating time and temperature, using size-exclusion chromatography coupled with light scattering detection. The presence of κ-carrageenan did not affect the aggregation of whey proteins heated at 75 °C; however, a change in the mechanism of aggregation seemed to occur at higher temperatures, and intermediates with higher molecular mass formed at 85 °C. At 90 °C, in the presence of κ-carrageenan, the extent of WPI aggregation was much larger, as soluble aggregates were no longer present and less residual protein was recovered in the unaggregated peak.     

The role of salivary proteins in the mechanism of astringency

Understanding astringency has focused on the interaction of tannins with the salivary proline-rich proteins (PRPs), although it remains unclear if other astringents precipitate the PRPs or how this interaction relates to sensory perceptions of astringency. We used 2 approaches to compare how distinct classes of astringent compounds interacted with the salivary PRPs and mucins. Using sodium dodecyl sulfate polyacrylamide gel electrophoresis, we evaluated protein patterns and characterized the salivary proteins present in the supernatants and pellets of pooled saliva assayed with tannin, alum, and hydrochloric acid solutions. Tannins and alum precipitated many of the PRPs, but acid did not. Mucins were precipitated by both the acid and alum, but not by the tannins. From our research, it appears that the precipitation of salivary proteins may be involved in the mechanism of astringency, but the precipitation of PRPs is not requisite for the development of astringency. We also measured mucin and deoxyribonucleic acid content of expectorated solutions of astringents that panelists swished in their mouths to determine if astringency was associated with a loss of oral lubricating films.     

Distinction of different heat-treated bovine milks by native-PAGE fingerprinting of their whey proteins

Native-PAGE (polyacrylamide gel electrophoresis) was used for the simultaneous qualitative and quantitative analysis of whey proteins of raw, commercial and laboratory heat-treated bovine milks. Four whey protein bands, including β-lactoglobulin variants (β-LG A and B), could be distinctively separated in the gel. The results showed that levels of the major whey proteins were reduced by less than 23% in the pasteurized milks and by more than 85% in the UHT milks as compared with raw milk. The α-lactalbumin (α-LA) exhibited the strongest heat-tolerance: about 32% of it remained in its native state after the milk was heated at 100 °C for 10 min. About 42% of β-LG A and 53% of β-LG B were lost after the milk was heated at 75 °C for 30 min. Blood serum albumin (BSA) was lost almost completely when the milk at pH 5.0 was heated at a temperature of 75 °C or higher. The β-LGA and β-LGB were much more stable at low pH than in neutral conditions.