Whey Protein Samples Research Articles

Whey Protein Analysis Using the Lowry Assay and 96-Well-Plate Digital Images Acquired Using Smartphones

Here, students determine the total protein content in whey protein samples using the Lowry assay. Quantitative analysis was carried out using absorbance measured at 750 nm (standard method) and 96-well-plate digital images obtained using smartphones (proposed method). The proposed method was carried out using two smartphones (1 and 2). Smartphone 1 had a higher resolution than smartphone 2. The statistical evaluation of the data set produced by the students was carried out using JASP (Jeffreys’s Amazing Statistics Program), and it was a valuable tool to teach descriptive statistics and hypothesis tests. We chose JASP because it is free and open-source software with a graphical user interface. The protein percentage found in five whey protein samples using the standard method during three lab classes was compared using one-way and two-way ANOVA. Five whey protein samples were analyzed using the standard method and the proposed methods (smartphones 1 and 2). The results were compared using one-way repeated measure ANOVA (RMANOVA) and mixed-factor ANOVA. During the statistical analysis of the data set, students identified outlier results using boxplots and checked data normality using the Shapiro–Wilk test, histograms, and boxplots. They learned the difference between dependent and independent variables and compared variances using Levene’s test (dependent variables) and Mauchly’s test (independent variables). The interpretation of statistical tests was also carried out using descriptives plots, boxplots, and raincloud plots.

Effects of concentration by block freezing and vacuum evaporation on the physicochemical properties and digestibility of whey

ABSTRACT Whey is a source of protein of high nutritional value. In this work, the effects of block cryoconcentration assisted by centrifugation and vacuum evaporation on the physicochemical properties and in vitro digestibility of whey were determined. In vitro digestibility analysis was performed by evaluating the degrees of protein hydrolysis of the fresh, evaporated and cryoconcentrated whey protein samples from the third cycle. The results showed that the samples after the second and third cryoconcentration cycles had no significant differences in protein or lactose content. The in vitro digestibility analysis indicated that the protein obtained by cryoconcentration showed better gastrointestinal digestibility than that obtained by evaporation, with values of 68.2% and 55.4%, respectively. This study demonstrates that block cryoconcentration assisted by centrifugation is an efficient and novel technique that enables the preparation of concentrated protein solutions of high nutritional and biological value.

Elemental bioavailability in whey protein supplements

Whey protein (WP) as a dietary supplement for athletes and gym-users is characterized by a content of high-quality amino acids. The benefits of their consumption are well-known and mainly related to the improvements of strength and body composition. Nevertheless, there is a scarce information about the risk associated to their elemental composition. The aim was to evaluate the total content and bioavailability of twenty-five elements in twenty whey protein powder samples, using Inductively Coupled Plasma Mass Spectrometry, and to evaluate the possible risk associated to the excessive daily intake of such products. Results showed that Na, K, Ca and Mg were the most predominant elements. After Hierarchical Cluster Analysis three different groups of whey protein supplements were observed. ANOVA analysis indicates that the concentration of the Na, K and Mg above mentioned elements is the key for the observed classification. The average elemental bioavailable fraction in the stomach is 45 % whereas in the intestine is 64 %. Most of the elements tested (nineteen) show gastric bioavailability higher than 60 %, being Al the lowest (37 %) and Co the highest one (76 %). According to our results, previously published data and recommendations of producers, WP samples can be considered save from the elemental composition point of view.

Whey protein supplement adulteration with rice flour quantification: A simple method using ATR-FT-MIR and iPLS

In this work, a method using ATR-FT-MIR and iPLS was developed to quantify whey protein supplement adulteration with rice flour. The original vanilla flavor commercial whey protein samples were adulterated with commercial rice flour with concentrations between 11.49% to 29.14% (w/w). After the adulteration, the ATR-FT-MIR spectra were obtained with no additional preparation procedure. The iPLS model analysis was performed using RStudio software with the mdatools package. The RMSEC was 1.26, the R2= 0.954 and the cross-validation error (RMSECV) was 3.31. The prediction error (RMSEP) for the validation set was equal 3.48 and the validation R2 was 0.610. These parameters, associated with the fact that the method does not require sample preparation, demonstrate the procedure viability as a tool to quantify adulterations of whey protein with rice flour.

Influence of chaperone-like activity of caseinomacropeptide on the gelation behaviour of whey proteins at pH 6.4 and 7.2.

The effect of caseinomacropeptide (CMP) on the heat-induced denaturation and gelation of whey proteins (2.5–10%, w/v) at pH 6.4 and 7.2, at a whey protein:CMP ratio of 1:0.9 (w/w), was investigated using differential scanning calorimetry (DSC), oscillatory rheology (90 °C for 20 min) and confocal microscopy. Greater frequency-dependence in the presence of CMP suggested that the repulsive interactions between CMP and the whey proteins affected the network generated by the non-heated whey protein samples. At pH 6.4 or 7.2, CMP increased the temperature of denaturation of β-lactoglobulin by up to 3 °C and increased the gelation temperature by up to 7 °C. The inclusion of CMP strongly affected the structure of the heat-induced whey protein gels, resulting in a finer stranded structure at pH 6.4 and 7.2. The presence of CMP combined with a lower heating rate (2 °C/min) prevented the formation of a solid gel of whey proteins after heating for 20 min at 90 °C and at pH 7.2. These results show the potential of CMP for control of whey protein denaturation and gelation.

Determination of heavy metal content in whey protein samples from markets in Giza, Egypt, using inductively coupled plasma optical emission spectrometry and graphite furnace atomic absorption spectrometry: A probabilistic risk assessment study

Whey protein is a by-product obtained from cheese manufacturing, contains high amounts of essential metals and amino acids. We assessed levels and health risk of Co, Ni, Sn, Cr, Cu, Mn, Zn, Fe, Al, Na, Ca, Mg, Pb, Cd, and Hg in different whey protein samples collected in Egypt using inductively coupled plasma optical emission spectrometry (ICP OES) and graphite furnace atomic absorption spectrometry (GF AAS). Large differences were found between the measured and defined values for some metals (such as Na, Mn, Cr, Cu, and Mg), whereas others showed only minor variation (Fe and Zn). The maximum concentration of Ca, Na, Mg, Zn, Fe, Cu, Al, Mn, Ni, Cr, and Sn was 12860, 5336, 3248, 66.11, 40.14, 16.78, 16.26, 14.37, 0.927, 0.685, and 0.502 mg/kg, respectively. The Co levels in all samples were found to be varied between N.D (not detectable) and lower than the limit of quantification (<LOQ). The concentration of Cd was found to range between 0.022 and 0.335 mg/kg, while Pb concentration was found to vary between 0.036 and 0.096 mg/kg. Also, all samples were found to be free of any detectable amount of Hg. The health risk assessment was evaluated for estimated daily intake (EDI), average body weight (70 kg), hazard quotient (HQ), and hazard index (HI) using the FAO/WHO and US EPA recommendations. Based on our results, the EDI and HQ values for most of elements in whey protein were lower than 1, which indicates no adverse effects may occur. The HI values for most of whey protein samples were lower than 1. Among the samples, only two brands had HI > 1, indicates that there is some possibility that non-cancer effects may occur through consumption in the future.

A Polyphenolic Extract from Olive Mill Wastewaters Encapsulated in Whey Protein and Maltodextrin Exerts Antioxidant Activity in Endothelial Cells.

The aim of the present study was to compare maltodextrin and whey protein as encapsulation carriers for olive mill wastewater (OMWW) phenolic extract for producing antioxidant powder, by using spray drying under 17 different conditions. In some samples, gelatin was also added in the encapsulation mixture. The antioxidant activity was assessed in vitro by using the DPPH•, ABTS•+, reducing power and DNA plasmid strand breakage assays. The results showed that both materials were equally effective for producing antioxidant powder, although by using different conditions. For example, inlet/outlet temperature of the spray drying did not seem to affect the maltodextrin samples’ antioxidant activity, but whey protein samples showed better antioxidant activity at lower temperatures. Gelatin use decreased antioxidant activity, especially in whey protein samples. The two most potent samples, one encapsulated in maltodextrin and the other in whey protein, were examined for their antioxidant effects in human endothelial cells by assessing glutathione (GSH) and reactive oxygen species (ROS) levels. Both samples significantly enhanced the antioxidant molecule of GSH, while maltodextrin sample also decreased ROS. The present findings suggested both materials for encapsulation of OMWW extract for producing antioxidant powder which may be used in food products, especially for the protection from ROS-induced endothelium pathologies.

Comparison of dry- and wet-heat induced changes in physicochemical properties of whey protein in absence or presence of inulin.

Changes in whey protein (10%, w/v) induced by dry-heating (60°C for 5days at a relative humidity of 63%), wet-heating (85°C for 30min) or the two-combined heating in absence or presence of inulin (8%, w/v) were studied. Mixture of whey protein and inulin showed significantly higher absorbance at 290nm than whey protein alone in all heating conditions while only dry-heated samples showed significantly increased absorbance value at 420nm (p < 0.05). Whey protein after heating showed significantly lower zeta potential and inulin decreased the value of all heated samples further (p < 0.05) except for samples after dry-heating. Heating decreased the free sulfhydryl group content of whey protein samples while presence of inulin decreased further (p < 0.05). Dry-heating decreased while wet-heating increased the surface hydrophobicity of whey protein. Inulin had no effect on the surface hydrophobicity of heated whey protein under dry-heating but decreased under wet-heating.

A kinetic model for whey protein denaturation at different moisture contents and temperatures

The denaturation of whey protein samples that had previously undergone heat-treatment for different times at different temperatures and moisture contents was analysed by differential scanning calorimetry (DSC), using the DSC enthalpy as a measure of residual undenatured protein. Data were fitted to first order irreversible or reversible kinetic expressions, and the resulting rate constants were found to increase with both temperature and moisture content. The whole data set was then fitted as a function of time, temperature and moisture content, with rate constants varying according to either Arrhenius or Williams-Landel-Ferry (WLF) kinetics and with selected fit parameters made empirical functions of moisture content. The best fits were obtained using reversible WLF kinetics, which could be further slightly simplified without loss of accuracy. The model provides a platform for single- and multi-objective drying trajectory optimisation with respect to protein denaturation in dairy products.

High hydrostatic pressure assisted enzymatic hydrolysis of whey proteins

Abstract Whey proteins, due to their high nutritional value, are generally hydrolyzed to reduce the allergenicity and used as ingredients in many special products, such as infant formulae, geriatric products, highly energetic supplements or dietetic foods or in foods produced to prevent nutrition related diseases, like food intolerances and allergies. The aim of this work was to assess the applicability of innovative technologies, such as high hydrostatic pressure (HHP) processes, to assist the enzymatic hydrolysis of target proteins, namely whey protein concentrate (WPC-80), in order to modify their antigenicity. Experiments were carried out to verify the effectiveness of HHP technology to accelerate whey protein hydrolysis reaction with selected enzymes (α-chymotrypsin, bromelain), and to affect the protein allergenic power. To this purpose, different HHP treatments were carried out at several pressure levels (100, 200, 300 and 400 MPa) and the untreated whey protein samples were used as control. A defined enzyme/substrate ratio of 1/10 w/w was used in the experiments, while the treatment time was changed from 0 to 30 min (0, 5, 15, or 30 min). The experimental data demonstrated that High Hydrostatic Pressure (HHP) induced WPC-80 unfolding at the highest value of the pressure applied (400 MPa) as indicated by the higher exposure of free sulfhydryl groups. When HHP was used in combination with enzymatic hydrolysis, the degree of hydrolysis increased not only with the pressure level applied but also with the processing time. These results suggested that, even if the exposure of hidden epitopes upon protein unfolding increased the antigenicity of whey proteins, further peptide bonds cleavage also took place after hydrolysis. This effect could modify whey proteins antigenic sequences, and thus their antigenic power.

Yogurts with an increased protein content and physically modified starch: rheological, structural, oral digestion and sensory properties related to enhanced satiating capacity

Protein is the most effective food macronutrient providing a satiating effect. Thus, formulating dairy foods with increased protein contents can help to modulate food intake. Oral perception cues also contribute an increased perception of satiating capacity when the oral residence time and handling are longer and more laborious. In the present work, yogurts were prepared with double skimmed milk powder (MP) and whey protein (WP), as well as a control (C) without extra protein. Three more samples were prepared by adding 2% of a physically modified starch to each (CS, MPS and WPS, respectively), in order to increase the consistency and impart creaminess. Rheological tests were used to characterize the flow and viscoelastic properties of the samples before and after saliva treatment, and their microstructure was observed. Finally the differences in sensory perceptions elicited by the samples were related to consumers' expected satiating capacity and liking scores. Before in vitro oral digestion, MP showed denser areas than C; in WP, two protein networks could be distinguished. In the samples with added starch, starch granules were embedded in the protein networks. After in vitro oral digestion the protein tended to aggregate; the starch granules maintained their structure indicating that they were not broken down by the saliva. These observations were related to the samples' rheological behavior. The sensory graininess, lumpiness and grittiness detected in the WP samples could be linked to the aggregation of whey protein and the formation of two different protein networks. All the added-starch samples elicited creamier and denser sensations than their counterparts without starch. MP with starch was scored as the most satiating and best-liked yogurt sample.

Partial chemical and functional characterization of milk whey products obtained by different processes

Whey protein samples (S-1 to S-5) were tested in vivo and in vitro for nutritional properties and selected bioactivities. Weanling male Wistar rats fed modified AIN-93G (12 g protein.100 g-1) diets for 21 days were used the in vivo studies. The nutritional parameters did not differ among the protein diets tested. Erythrocyte glutathione content was considered high and was higher for S-3, but liver glutathione was the same for all dietary groups. For S-3, cytokine secretion (IL-10 and TNF-α) by human peripheral blood mononuclear cells (in RPMI-1640 medium) was higher in the absence of antigen than in the presence of BCG antigen. Interleukin-4 secretion was repressed in all treatments. The IC50, whey protein concentration required to inhibit 50% of the melanoma cell proliferation, was 2.68 mg.mL-1 of culture medium for the S-3 sample and 3.66 mg.mL-1 for the S-2 sample. Based on these results, it was concluded that S-3 (whey protein concentrate enriched with TGF-β and lactoferrin) produced better nutritional and immunological responses than the other products tested.

Comparison of Size-Exclusion Chromatography and Flow Field-Flow Fractionation for Separation of Whey Proteins

Whey protein (WP) is a mixture of proteins, and is of high nutritional values. WP has become an important source of functional ingredients in various health-promoting foods. In this study, size-exclusion chromatography (SEC) and asymmetrical flow field-flow fractionation (AsFlFFF) were used for separation and analysis of whey proteins. It was found that a lab-prepared WP from raw milk is mostly of <TEX>${\beta}$</TEX>-lactoglobulin with small amount of higher molecular weight components, while a commercial whey protein isolate (WPI) powder contains relatively larger amount of components other than <TEX>${\beta}$</TEX>-lactoglobulin, including IgG and protein aggregates. Results suggest that AsFlFFF provides higher resolution for the major whey proteins than SEC in their normal operation conditions. AsFlFFF could differentiate the BSA and Albumin, despite a small difference in their molecular weights, and also was able to separate much smaller amount of aggregates from monomers. It is noted that SEC was able to show the presence of low molecular weight components other than the major whey proteins in the WP samples, which AsFlFFF could not show, probably due to the partial loss of those low molecular weight species through the membrane.

Milk whey proteins and xanthan gum interactions in solution and at the air–water interface: A rheokinetic study

In this contribution, we present experimental information about the effect of xanthan gum (XG) on the adsorption behaviour of two milk whey protein samples (MWP), β-lactoglobulin (β-LG) and whey protein concentrate (WPC), at the air–water interface. The MWP concentration studied corresponded to the protein bulk concentration which is able to saturate the air–water interface (1.0 wt%). Temperature, pH and ionic strength of aqueous systems were kept constant at 20 °C, pH 7 and 0.05 M, respectively, while the XG bulk concentration varied in the range 0.00–0.25 wt%. Biopolymer interactions in solution were analyzed by extrinsic fluorescence spectroscopy using 1-anilino-8-naphtalene sulphonic acid (ANS) as a protein fluorescence probe. Interfacial biopolymer interactions were evaluated by dynamic tensiometry and surface dilatational rheology. Adsorption behaviour was discussed from a rheokinetic point of view in terms of molecular diffusion, penetration and conformational rearrangement of adsorbed protein residues at the air–water interface. Differences in the interaction magnitude, both in solution and at the interface vicinity, and in the adsorption rheokinetic parameters were observed in MWP/XG mixed systems depending on the protein type (β-LG or WPC) and biopolymer relative concentration. β-LG adsorption in XG presence could be promoted by mechanisms based on biopolymer segregative interactions and thermodynamic incompatibility in the interface vicinity, resulting in better surface and viscoelastic properties. The same mechanism could be responsible of WPC interfacial adsorption in the presence of XG. The interfacial functionality of WPC was improved by the synergistic interactions with XG, although WPC chemical complexity might complicate the elucidation of molecular events that govern adsorption dynamics of WPC/XG mixed systems at the air–water interface.

Indicators of non-enzymatic browning in the evaluation of heat damage of ingredient proteins used in manufactured infant formulas

Twelve infant formula ingredients of animal origin (caseinates, whey proteins and hydrolysates of casein and of whey proteins) and three of vegetable origin (soybean proteins) were analyzed. Furosine, hydroxymethylfurfural (HMF) and pyrraline were studied as indicators of thermal damage and available lysine as nutritional indicator, determined by HPLC in phase reverse and UV detector. The objectives were to evaluate heat damage to ingredients used in commercial infant formulas by measuring non-enzymatic browning indicators and to determine the nutritional value of these ingredients by available lysine determination. Very high furosine values were detected in whey proteins, ranging from 354 to 1,435 mg/100 g of protein. Lower furosine values were found in the remaining ingredients, ranging from 1.36 mg/100 g in hydrolyzed casein to 60.5 mg/100 g in sodium caseinate. Available lysine content ranged from 1.85 g/100 g of protein in hydrolyzed casein to 7.91 g/100 g in calcium caseinate. HMF was detected in whey protein samples between 0.16 and 2.47 mg/100 g of protein. Pyrraline was only detected in one sample of whey proteins at 41 mg/100 g of protein. Similar ingredients from different manufacturers show varied heat damage and nutritional values

Heat-induced whey protein isolate fibrils: Conversion, hydrolysis, and disulphide bond formation

Fibril formation of individual pure whey proteins and whey protein isolate (WPI) was studied. The heat-induced conversion of WPI monomers into fibrils at pH 2 and low ionic strength increased with heating time and protein concentration. Previous studies, using a precipitation method, size-exclusion method, or proton NMR spectroscopy, reported a wide range of values for the conversion. An alternative method was developed, namely centrifugal filtration, giving a consistent picture of the conversion. The present results help to explain the disparities reported in literature. No fibrils formed upon heating pure α-lactalbumin or pure BSA at pH 2, whereas fibrils formed in pure β-lactoglobulin ( β-lg) and WPI solutions. Experiments indicate that β-lg was the only whey protein involved in fibril formation. In all whey protein samples, hydrolysis occurred during heating at pH 2, as determined by HPLC and SDS-PAGE. When WPI fibrils formed at pH 2 were stored at pH 7 or 10, disulphide bonds were formed in the samples.

EFFECTS OF CALCIUM ALGINATE AND/OR WHEY PROTEIN ON PHYSICAL AND FUNCTIONAL PROPERTIES OF GROUND VENISON

This study used venison as a restructured meat because of its health benefits, and increased popularity throughout the world. Whey protein and calcium alginate binders were used at different concentrations on ground venison to mimic properties of a venison muscle cut. The pH, moisture content, water activity, water holding capacity, and texture profile analysis were methods used to identify these properties. Results conclude whey protein samples mimicked the control and ground venison for many tests, rather than calcium alginate. However, solubility of whey protein may be questionable due to similarities with ground venison. Calcium alginate was advantageous in increasing product yield because of moisture content, water holding capacity and cooking loss.

Heat‐induced gelation of whey protein at high pH studied by combined UV spectroscopy and refractive index measurement after size exclusion chromatography and byin‐situdynamic light scattering

SummaryThe interactions between β‐lactoglobulin and α‐lactalbumin involved in gelation at 67.5 °C at high pH and low salt concentration were studied by size exclusion chromatography, followed by UV and refractive index measurements, and byin‐situdynamic light scattering. This was achieved by choosing whey protein samples with different proportions of the two proteins. The ratio of absorbance at 280 nm to the refractive index was used to demonstrate that α‐lactalbumin was incorporated in aggregates and gels and drastically changed the properties of the gel, making them much more turbid than the transparent gels formed by β‐lactoglobulin alone at the same pH and ionic strength. At a ratio of 1:2 for α‐lactalbumin relative to β‐lactoglobulin in the samples, the gel consisted of a 1:1 mixture of the two proteins. The aggregates present after 10 min of heating at 67.5 °C had molar mass of about 6.106g/mol and a radius of gyration of about 40 nm. After gel formation the field autocorrelation function could be described as a power law over many decades of lag time for all samples, demonstrating selfsimilarity of the gel structure. The only exception to this was for the gel with high content of α‐lactalbumin which showed an oscillatory behaviour of the autocorrelation function. Significant amounts of glycosylated caseino‐macro‐peptide were observed in many of the samples at the position of β‐lactoglobulin. However it did not affect gelation as it remains in solution.

Rheological properties of commercial whey protein samples from the MADGELAS survey

Summary Thermally induced gelation of commercial whey protein samples from the Molecular Basis of the Aggregation, Denaturation, Gelation and Surface Activity of Whey Proteins (MADGELAS) survey has been studied in order to develop a current status report on their rheological properties. Solutions of 10% protein (w/v) were prepared in distilled water, 200 mm NaCl or 10 mm CaCl2 at neutral pH. Small‐scale deformation of the samples was measured by dynamic oscillatory rheometry using a Bohlin CS Rheometer. Large‐scale deformation at penetration mode was measured using a Texture Analyser (Stable Micro Systems, Surrey, UK). For solutions containing salts, there was a general trend for gel point to decrease and G′ values to increase, the effect being more marked in the presence of NaCl. Similarly, force values at failure also tended to increase in the presence of salts. Results obtained with samples of similar protein composition dissolved in water were highly scattered, these differences being reduced in the presence of added salts.

Effect of Contaminant on Adsorption of Whey Proteins at the Air−Water Interface

The dynamics of adsorption of commercial samples of α-lactalbumin and β-lactoglobulin was investigated through the measurement of dynamic surface pressure and surface concentration via a radiotracer method. An unusual two-step adsorption behavior was observed for both proteins at low concentration which was believed to be due to the presence of a surface active contaminant. This hypothesis was tested by comparing the native whey protein samples with those purified with charcoal extraction. Analysis of the charcoal extract by thin-layer chromatography revealed the presence of contaminating free fatty acid and triglycerides in the commercial samples. β-Lactoglobulin was found to contain a higher concentration of these contaminants than α-lactalbumin. Extraction by charcoal was found not to modify the protein structure with respect to their near-and far-UV circular dichroism spectra. The presence of contaminant resulted in a higher steady-state surface pressure for β-lactoglobulin, but the protein was not displaced from the interface. On the other hand, the presence of a low concentration of surfactant was sufficient to decrease the steady-state surface concentration of α-lactalbumin due to the displacement of the protein from the interface by the lipids.