Native Whey Proteins Research Articles

High Hydrostatic Pressure Pretreatment of Whey Protein Isolates Improves Their Digestibility and Antioxidant Capacity.

Whey proteins have well-established antioxidant and anti-inflammatory bioactivities. High hydrostatic pressure processing of whey protein isolates increases their in vitro digestibility resulting in enhanced antioxidant and anti-inflammatory effects. This study compared the effects of different digestion protocols on the digestibility of pressurized (pWPI) and native (nWPI) whey protein isolates and the antioxidant and anti-inflammatory properties of the hydrolysates. The pepsin-pancreatin digestion protocol was modified to better simulate human digestion by adjusting temperature and pH conditions, incubation times, enzymes utilized, enzyme-to-substrate ratio and ultrafiltration membrane molecular weight cut-off. pWPI showed a significantly greater proteolysis rate and rate of peptide appearance regardless of digestion protocol. Both digestion methods generated a greater relative abundance of eluting peptides and the appearance of new peptide peaks in association with pWPI digestion in comparison to nWPI hydrolysates. Hydrolysates of pWPI from both digestion conditions showed enhanced ferric-reducing antioxidant power relative to nWPI hydrolysates. Likewise, pWPI hydrolysates from both digestion protocols showed similar enhanced antioxidant and anti-inflammatory effects in a respiratory epithelial cell line as compared to nWPI hydrolysates. These findings indicate that regardless of considerable variations of in vitro digestion protocols, pressurization of WPI leads to more efficient digestion that improves its antioxidant and anti-inflammatory properties.

The effect of dynamic heat treatments of native whey protein concentrate on its dispersion characteristics

The aggregation of native whey protein concentrate (WPC) was studied after a range of dynamic heat treatments performed at 0.5%, 3.5%, 5.0% and 8.8% protein concentrations. Overall, it was observed that the changes caused by heating were mainly physical, but higher heat treatments were also found to increase the furosine content, indicating Maillard reactions. Most of the whey proteins remained native after heat treatment at 75 °C for 15 s at all the concentrations studied. Heat treatment at 90 °C for 5 min caused whey proteins to reach the maximum degree of denaturation at 3.5% protein concentration. The size of the aggregates kept growing with increasing protein concentration. At 8.8% protein, essentially all whey protein monomers and low-molecular weight oligomers had reacted, forming insoluble polymers, and only β-casein was left in the solution. Further heat treatments extended shelf life and ultra-high temperature conditions mostly decreased the hydrophobicity.

Physical Stability of Octenyl Succinate-Modified Polysaccharides and Whey Proteins for Potential Use as Bioactive Carriers in Food Systems.

The high cost and potential toxicity of biodegradable polymers like poly(lactic-co-glycolic)acid (PLGA) has increased the interest in natural and modified biopolymers as bioactive carriers. This study characterized the physical stability (water sorption and state transition behavior) of selected starch and proteins: octenyl succinate-modified depolymerized waxy corn starch (DWxCn), waxy rice starch (DWxRc), phytoglycogen, whey protein concentrate (80%, WPC), whey protein isolate (WPI), and α-lactalbumin (α-L) to determine their potential as carriers of bioactive compounds under different environmental conditions. After enzyme modification and particle size characterization, glass transition temperature and moisture isotherms were used to characterize the systems. DWxCn and DWxRc had increased water sorption compared to native starch. The level of octenyl succinate anhydrate (OSA) modification (3% and 7%) did not reduce the water sorption of the DWxCn and phytoglycogen samples. The Guggenheim-Andersen-de Boer model indicated that native waxy corn had significantly (P < 0.05) higher water monolayer capacity followed by 3%-OSA-modified DWxCn, WPI, 3%-OSA-modified DWxRc, α-L, and native phytoglycogen. WPC had significantly lower water monolayer capacity. All Tg values matched with the solid-like appearance of the biopolymers. Native polysaccharides and whey proteins had higher glass transition temperature (Tg) values. On the other hand, depolymerized waxy starches at 7%-OSA modification had a "melted" appearance when exposed to environments with high relative humidity (above 70%) after 10 days at 23 °C. The use of depolymerized and OSA-modified polysaccharides blended with proteins created more stable blends of biopolymers. Hence, this biopolymer would be suitable for materials exposed to high humidity environments in food applications.

Membrane fouling during ultra- and microfiltration of whey and whey proteins at different environmental conditions: The role of aggregated whey proteins as fouling initiators

For the concentration of whey proteins from whey by ultrafiltration (UF) or fractionation by microfiltration (MF), mass transfer through the membrane is limited by deposit formation which is variable and not well understood, yet. There is little knowledge on the predominant molecular mechanism responsible for membrane fouling during whey micro- and ultrafiltration. Some works indicate that whey protein aggregates induce or enhance membrane fouling. The impact of whey protein aggregates as well as the interaction of native whey proteins and whey protein aggregates during membrane fouling was studied in both dead-end lab scale and cross-flow pilot scale. By means of targeted heat-treatment, a defined amount of protein aggregates was formed in β-Lactoglobulin (β-Lg) model suspensions as well as in sweet whey. β-Lg aggregates were found to accelerate membrane fouling during MF and UF due to covalent thiol/disulfide reactions. For sweet whey cross-flow filtration, membrane fouling was accelerated by whey protein aggregates up to a certain degree of whey protein denaturation. Above a critical value of 30% protein denaturation, flux increased again. This effect is explained by an improved erosion of larger whey protein aggregates as well as a reduced reactivity of heat-aged whey protein aggregates.

Improving the structure and rheology of high protein, low fat yoghurt with undenatured whey proteins

The objective of this study was to investigate the effects of whey protein denaturation and whey protein:casein-ratio on the structural, rheological and sensory properties of high protein (8% true protein), low fat (<0.5% fat) yoghurt. Yoghurt milk bases were made by adding undenatured whey proteins from native whey protein concentrate (NWPC) to casein concentrate in different whey protein:casein-ratios. The degree of whey protein denaturation was then controlled by the temperature treatment of the yoghurt milk bases. Addition of NWPC in low (whey protein:casein-ratio 25:75) or medium levels (whey protein:casein-ratio 35:65) in combination with heat treatment at 75 °C for 5 min gave yoghurts with significantly lower firmness, lower storage modulus (G′), and better sensory properties (less coarse and granular and more smooth), compared with corresponding yoghurts produced from yoghurt milk bases heat-treated at 95 °C for 5 min or with control yoghurts (no addition of NWPC).

Current ways to modify the structure of whey proteins for specific functionalities—a review

The native whey proteins have been intensively used in a multitude of food applications due to their high nutritional, biological, and versatile technofunctional properties. The range of applications of whey proteins has further been extended in the last decades by the use of whey protein aggregates offering new technofunctional properties. These properties are directly dependent on the structure of whey protein aggregates, i.e., their size, shape, density, surface properties, and the type of bonds maintaining proteins together in the aggregates. In this review, after a brief description of the major whey proteins, we examine the most important advances reported to date pertaining to the available approaches to modify the structure of whey proteins for specific functionalities. Our laboratory, Science and Technology of Milk and Eggs, has contributed significantly to the advancement of knowledge on the structure-function relationships of whey proteins either in the native or denatured/aggregated forms. Our expertise and research approaches are highlighted throughout some selected results accumulated during the last decade in comparison with results from the literature.

Effect of microparticulated whey proteins on milk coagulation properties

The enhancement of milk coagulation properties (MCP) and the reuse of whey produced by the dairy industry are of great interest to improve the efficiency of the cheese-making process. Native whey proteins (WP) can be aggregated and denatured to obtain colloidal microparticulated WP (MWP). The objective of this study was to assess the effect of MWP on MCP; namely, rennet coagulation time (RCT), curd-firming time, and curd firmness 30 min after rennet addition. Six concentrations of MWP (vol/vol; 1.5, 3.0, 4.5, 6.0, 7.5, and 9.0%) were added to 3 bulk milk samples (collected and analyzed during 3 d), and a sample without MWP was used as control. Within each day of analysis, 6 replicates of MCP for each treatment were obtained, changing the position of the treatment in the rack. For control samples, 2 replicates per day were performed. In addition to MCP, WP fractions were measured on each treatment during the 3 d of analysis. Milk coagulation properties were measured on 144 samples by using a Formagraph (Foss Electric, Hillerød, Denmark). Increasing the amount of MWP added to milk led to a longer RCT. In particular, significant differences were found between RCT of the control samples (13.5 min) and RCT of samples with 3.0% (14.6 min) or more MWP. A similar trend was observed for curd-firming time, which was shortest in the control samples and longest in samples with 9.0% MWP (21.4 min). No significant differences were detected for curd firmness at 30 min across concentrations of MWP. Adjustments in cheese processing should be made when recycling MWP, in particular during the coagulation process, by prolonging the time of rennet activity before cutting the curd.

High-protein fat-free acid milk gels: Control of protein composition and heat treatment

Skim milk supplemented with calcium caseinate (Ca-Cas) and whey protein isolate (WPI) at different ratios was preheated at 72–90 °C for 7 min and subsequently acidified for the production of set-style and stirred acid gels. Upon heat-treatment of the suspensions, the native whey protein content decreased and particle size increased with increasing temperature and WPI content. Consequently, the pH of gelation, firmness of the set gel, viscosity and particle size of the stirred gel all increased as the content in denatured whey protein increased. However, for preheating temperatures of 80 or 90 °C, the viscosity and firmness of the gels reached a plateau, although almost all whey proteins were denatured and largest particles were formed. These results open perspectives for the production of high-protein yoghurts without unusual firmness properties.

Initial pasteurisation effects on the protein fractionation of skimmed milk by microfiltration

Microfiltration using 0.1–0.2 μm membranes enables protein fractionation of skimmed milk into a casein-rich fraction and a permeate that contains native whey proteins. High-temperature, short-time pasteurisation is often one of the first treatments in many dairy processes. The objective of this study was to investigate whether initial pasteurisation would affect the fractions that are obtained from the one-stage microfiltration of skimmed milk. Pasteurised and unpasteurised skimmed milk were fractionated at 55–58 °C using 0.2 μm ceramic membranes. Chemical analysis of the microfiltration fractions showed that both the nitrogen and mineral distributions were altered by the initial pasteurisation. The permeate that was obtained from microfiltration of the unpasteurised milk contained higher amounts of calcium, phosphorous and native whey proteins, in addition to a lower amount of casein fragments passing through the membrane. The retentate that was obtained from microfiltration of the unpasteurised milk contained less total protein and casein.

Effects of non-covalent interactions between the milk proteins on the rheological properties of acid gels

When skim milk (SM) and whey-protein-enriched skim milk (WPE-SM) were heated (80 °C, 30 min) with N-ethylmaleimide (NEM, 0–0.8 mm), the levels of residual native whey proteins increased to ∼70%, whereas the levels of disulphide-bonded whey proteins decreased to <10%. Acid gels prepared from heated SM with added NEM had slightly lower firmness than those made from control heated SM because the former gels lacked intermolecular disulphide bonds. In contrast, acid gels made from heated WPE-SM with added NEM had higher firmness than those prepared from control heated WPE-SM. This implies that, even without intermolecular disulphide bonds, non-covalent interactions can be sufficient to produce acid gels with firmness higher than gels with disulphide bonds. Nevertheless, disulphide interactions can be more important than non-covalent interactions in influencing the yield properties of the gels because acid gels without disulphide bonds can be fractured more easily than those with disulphide bonds.

Bulk self-aggregation drives foam stabilization properties of whey protein microgels

Whey protein microgels (WPM) produced by controlled heat treatment of native whey protein isolate were characterized by a spherical and compact shape as well as a low polydispersity index. They exhibited maximum self-aggregation close to their isoelectrical pH (IEP) of 4.0–5.5, leading to a size increase from about 270 nm to several microns. The bulk viscosity of the WPM was increasing linearly with the concentration in WPM until an exponential increase was visible for a protein concentration ranging between 9.5 and 13.4 wt%. The intrinsic viscosity (∼10 mL g−1) and specific volume (∼4 mL g−1) of whey protein microgels were very close to those reported for casein micelles. WPM dispersions were able to reduce surface tension, but likely this phenomenon was due to the release of some surface-active material from the WPM. Stable foams were produced at 5 wt% WPM content and pH 5.0. In these conditions, the liquid drainage rate was strongly reduced as well as the disproportionation of air bubbles. It was concluded that WPM were able to stabilize foams close to their IEP by in situ self-aggregation leading to an arrest of the drainage enabling to counteract the gas flux responsible for bubble disproportionation. To our knowledge, this the first time that such mechanism has been described for food-grade protein based particles.

Characterization of Rheological and Molecular Properties of Whey Protein Thickeners

The production of whey protein thickeners by drying protein gels to produce instant thickening powders has been previously investigated. The use of native whey proteins as a food thickener, however, has not been studied earlier. In this work, we characterized thermally treated whey protein solutions with improved thickening properties. Viscosity contour maps were constructed to outline the viscosity values at different protein and salt concentrations. Huggins-Kraemer constants revealed that at low ionic strengths, polymerized protein molecules behave as flexible coils, while at higher ionic strengths, molecules appear to be aggregated in a spherical like shape.

Simultaneous single-cell protein production and COD removal with characterization of residual protein and intermediate metabolites during whey fermentation by K. marxianus

Cheese whey fermentation with Kluyveromyces marxianus was carried out at 40 °C and pH 3.5 to examine simultaneous single-cell protein production and chemical oxygen demand (COD) removal, determine the fate of soluble whey protein and characterize intermediate metabolites. After 36 h of batch fermentation, the biomass concentration increased from 2.0 to 6.0 g/L with 55 % COD reduction (including protein), whereas soluble whey protein concentration decreased from 5.6 to 4.1 g/L. It was confirmed through electrophoresis (SDS-PAGE) that the fermented whey protein was different from native whey protein. HPLC and GC-MS analysis revealed a change in composition of organic compounds post-fermentation. High inoculum concentration in batch fermentation resulted in an increase in biomass concentration from 10.3 to 15.9 g/L with 80 % COD reduction (including protein) within 36 h with residual protein concentration of 4.5 g/L. In third batch fermentation, the biomass concentration increased from 7.3 to 12.4 g/L with 71 % of COD removal and residual protein concentration of 4.3 g/L after 22 h. After 22 h, the batch process was shifted to a continuous process with cell recycle, and the steady state was achieved after another 60 h with biomass yield of 0.19 g biomass/g lactose and productivity of 0.26 g/L h. COD removal efficiency was 78-79 % with residual protein concentration of 3.8-4.2 g/L. The aerobic continuous fermentation process with cell recycle could be applied to single-cell protein production with substantial COD removal at low pH and high temperature from cheese whey.

Comparing serum responses to acute feedings of an extensively hydrolyzed whey protein concentrate versus a native whey protein concentrate in rats: a metabolomics approach

We examined how gavage feeding extensively hydrolyzed whey protein (WPH) versus a native whey protein concentrate (WPC) transiently affected serum biochemical profiles in rodents. Male Wistar rats (250-300 g) were 8 h fasted and subsequently fed isonitrogenous amounts of WPH or WPC, or remained unfed (control). Animals were sacrificed 15 min, 30 min, and 60 min post-gavage for serum extraction, and serum was analyzed using untargeted global metabolic profiling via gas chromatography/mass spectrometry (MS) and liquid chromatography/MS/MS platforms. We detected 333 serum metabolites amongst the experimental and control groups. Both WPH and WPC generally increased amino acids (1.2-2.8-fold), branched-chain amino acids (1.2-1.7-fold), and serum di- and oligo-peptides (1.1-2.7-fold) over the 60 min time course compared with control (q < 0.05). However, WPH increased lysine (false discovery rate using a q-value <0.05) and tended to increase isoleucine and valine 15 min post-feeding (q < 0.10) as well as aspartylleucine 30 min post-feeding compared with WPC (q < 0.05). While both protein sources led to a dramatic increase in free fatty acids compared with control (up to 6-fold increases, q < 0.05), WPH also uniquely resulted in a 30 min post-feeding elevation in free fatty acids compared with WPC (q < 0.05), an effect which may be due to the robust 30 min postprandial increase in epinephrine in the WPH cohort. These data provide a unique postprandial time-course perspective on how WPH versus WPC feedings affect circulating biochemicals and will guide future research comparing these 2 protein sources.

Rheological Properties of Kefir as Affected by Heat Treatment and Whey Protein Addition

Abstract The effects of heat treatment and whey protein addition on the fermentation time and the rheological properties of kefir determined using a pneumatic tube viscometer of novel design, were investigated. Kefir samples were prepared with and without the addition of whey protein concentrates (WPC) at varying concentrations, while heat treatment of the milk was made prior or after WPC addition (resulted to native or denatured whey proteins, respectively). Increasing WPC concentration resulted to increased lactic acid concentration and reduced fermentation time. The flow curves of the samples suggested that kefir behaves as a pseudoplastic fluid. The apparent viscosity of kefir samples was increased with increasing WPC concentration. However, the “state” of added whey proteins greatly affected the rheological properties of kefir. Particularly, denatured whey proteins resulted to higher consistency index values and thus apparent viscosity and lower flow behavior index values, when compared with native whey proteins. Practical Applications Kefir is a fermented beverage, which is reported of being beneficial to health. The large number of different microorganisms found in it and the variety of bioactive compounds produced during fermentation distinguishes it from other probiotic products. The study of the rheological properties of kefir as well as the factors that have an effect on them is of great scientific and commercial interest, because they can affect the quality of the final product and thus its acceptance by the consumers, the promotion of its standardization, and allow the correct selection of equipments like pumps and filling machines in the industry. The addition of whey protein concentrates (WPC) can enhance rheological properties of kefir and thus improve the quality of the final product. Furthermore, the “state” of the added whey proteins greatly affects the impact degree on the rheological behavior of kefir.

The effect of pre-denatured whey proteins on the textural and micro-structural properties of model processed cheese spreads

Whey proteins (WP) were heated in a scraped-surface heat exchanger to produce products with different levels of denaturation. Model processed cheese spreads containing the WP were prepared. Rheological measurements showed that higher levels of denaturation in the WP produced softer (lower elastic modulus) cheeses. Temperature scans indicated that the cheeses prepared with high levels of native whey proteins did not melt (the elastic modulus was higher than the viscous modulus at all temperatures), whereas those prepared with high levels of denatured whey proteins melted on increasing the temperature. Microstructural examination of selected cheeses indicated that, when denatured whey proteins were used in processed cheeses, the whey proteins were incorporated as large aggregates dispersed within the cheese matrix. These aggregates did not contribute to, and may disrupt the structure of the cheese. When native whey proteins were used, they were incorporated into the cheese matrix, producing a denser network structure.

From acacia honey monosaccharide content to a new external binary plasticizer applicable in aqueous whey protein films

Plasticizer is an essential adjuvant in food and pharmaceutical film coatings affecting the appearance, mechanical and permeation properties of the final coat. In the present study, film formation and plasticization of native whey proteins (potential future “green” coating agents for pharmaceuticals and food products), were studied with free isolated films. Special attention was paid to the effects of plasticizer, preheating and film forming solution pH on the mechanical stress–strain and moisture permeation properties of whey protein films. Glycerol, binary mixtures of fructose and glucose, and non-crystalline acacia honey were studied as external plasticizers. The type and amount of plasticizer affected the mechanical stress–strain properties of the whey protein films. A short preheating treatment of whey proteins prior to film casting resulted in mechanically strong films with a reduced elongation. The film forming properties of aqueous whey proteins could be modified by adjusting the pH above the isoelectric point of β-lactoglobulin prior to film coating. For effective plasticization, whey protein films required a high amount of monosaccharide containing plasticizer ranging from 80% to 120% (calculated from the protein weight). A new external binary plasticizer having the same ratio of monosaccharides as non-crystalline acacia honey (fructose and glucose 1.67:1) was found to be applicable in aqueous whey protein films.

Post‐feeding serum metabolic responses to extensively hydrolyzed whey (WPH) or native whey concentrate (WPC) in rats

The purpose of this study was to compare how feeding extensively hydrolyzed whey protein (WPH) versus its native whey protein concentrate (WPC) in 8‐h fasted rats transiently affects serum metabolic profiles. Male rats (~300 g) were fed an isonitrogenous amounts of WPH or WPC and sacrificed 15 min‐, 30 min‐and 60 min post‐gavage for serum extraction. Rats fasted for 8–9 h were used as controls (CTL). Serum biochemicals were detected using GC/MS and LC/MS/MS. 333 serum metabolites were detected amongst all groups. Both WPH and WPC increased several amino acids (1.2–2.8‐fold), branched chain amino acids (BCAAs, 1.2–1.7‐fold), and serum peptides (1.1–2.7‐fold) over the 60 min time course compared to CTL rats (p &lt; 0.05). However, WPH feeding rapidly (at 15‐min post) increased select amino acids (including BCAAs) and peptides compared to WPC and CTL feedings (p &lt; 0.05). WPH and WPC led to increases in numerous free fatty acids (FFAs) over the post‐feeding time course compared to CTL rats (up to 6‐fold increases, p &lt; 0.05). However, WPH feeding uniquely resulted in a 30‐min post‐feeding elevation in numerous FFAs compared to WPC feeding (p &lt; 0.05), which may be due the WPH‐induced increase in circulating epinephrine compared to CTL and WPC (p &lt; 0.05). These data provide a unique postprandial time‐course perspective as to how WPH versus WPC feedings affect circulating biochemicals.Supported by Bionutritional Research Group

Process Optimization for the Production of Bio-functional Whey Protein Hydrolysates: Adopting Response Surface Methodology

Whey is a protein complex derived from milk, exhibit highest protein quality rating among other proteins, being touted as a functional food with number of health benefits. In the present investigation, whey proteins hydrolysates produced using trypsin enzyme to augment antioxidant activity and to assess angiotensin converting enzyme (ACE) inhibition activity. Hydrolysis parameters were standardized applying response surface methodology. The response antioxidant activity in terms of Trolox equivalent antioxidant capacity (TEAC) values was deter- mined by radical scavenging assay method. Optimum conditions for maximum antioxidant activity were stan- dardized at 88 � C of preheating, 7.3 pH, 0.05 enzymes to substrate ratio and hydrolysis was carried up to 8 h at 36.5 � C. Resulting peptide fractions obtained at 11.8 % of degree of hydrolysis displayed antioxidant capacity with TEAC values of 1.37 ± 0.12. The designed model found to be significant with R 2 value of 0.9972 for antioxidant activity and lack of fit test-as non significant, indicating that the optimized conditions were best suited. The hydrolysate further investigated for antihypertensive activity. The out- come indicate that to affect decrease in ACE inhibition activity 4,166.72 lg of native whey protein is required when compared to 229.96 lg of hydrolysates. These results indicate hydrolysate produced under these conditions could be an effective nutraceutical.

Whey protein hydrolysates decrease IL-8 secretion in lipopolysaccharide (LPS)-stimulated respiratory epithelial cells by affecting LPS binding to Toll-like receptor 4

Whey proteins (WP) exert anti-inflammatory and antioxidant effects. Hyperbaric pressurisation of whey increases its digestibility and changes the spectrum of peptides released during digestion. We have shown that dietary supplementation with pressurised whey improves nutritional status and systemic inflammation in patients with cystic fibrosis (CF). Both clinical indices are largely affected by airway processes, to which respiratory epithelial cells actively contribute. Here, we tested whether peptides released from the digestion of pressurised whey can attenuate the inflammatory responses of CF respiratory epithelial cells. Hydrolysates of pressurised WP (pWP) and native WP (nWP, control) were generated in vitro and tested for anti-inflammatory properties judged by the suppression of IL-8 production in CF and non-CF respiratory epithelial cell lines (CFTE29o- and 1HAEo-, respectively). We observed that, in both cell lines, pWP hydrolysate suppressed IL-8 production stimulated by lipopolysaccharide (LPS) to a greater magnitude compared with nWP hydrolysate. Neither hydrolysate suppressed IL-8 production induced by TNF-α or IL-1β, suggesting an effect on the Toll-like receptor (TLR) 4 pathway, the cellular sensor for LPS. Further, neither hydrolysate affected TLR4 expression or neutralised LPS. Both pWP and nWP hydrolysates similarly reduced LPS binding to surface TLR4, while pWP tended to more potently increase extracellular antioxidant capacity. (1) anti-inflammatory properties of whey are enhanced by pressurisation; (2) suppression of IL-8 production may contribute to the clinical effects of pressurised whey supplementation on CF; (3) this effect may be partly explained by a combination of reduced LPS binding to TLR4 and enhanced extracellular antioxidant capacity.