Binding of retinyl acetate to whey proteins or phosphocasein micelles: Impact of pressure-processing on protein structural changes and ligand embedding

A whey protein isolate (WPI) and native phosphocaseins (PC) at pH 6.5–6.6 were processed with retinyl acetate (RAC) using pressure-assisted technological tools to improve RAC embedding through processing-induced protein structural changes. To this end, protein-RAC dispersions were submitted to ultra-high pressure homogenisation (UHPH) at 300 MPa and an initial fluid temperature (T_in) of 14 °C or 24 °C, or isostatic high-pressure at 300 MPa and 14 °C or 34 °C for 15 min. A short-time thermal treatment (STTT, 73 °C for 4 s) able to generate WPI aggregates was assessed for comparison. Processing effects were investigated in terms of protein particle sizes and molecular weights (M_w). M_w calculated using protein size determination obtained from light scattering measurements were in agreement with the known values. The amounts of RAC retained in WPI particles (unfolded and/or aggregated proteins) or in PC assemblies were quantitated after protein precipitation by ammonium sulphate. A 2.3–3.7 nmol RAC was carried per mg of pressure-denatured whey proteins, significantly less than after STTT (6.3 nmol RAC per mg of heat-denatured whey proteins) indicating that RAC embedding varied according to the technological tool, pressure or temperature. A 3.8–5.4 nmol RAC was carried per mg of PC assemblies through pressure-induced dissociation/reassociation of PC micelles. Combined pressure and mild temperature increased RAC embedding in PC assemblies.     

3D microstructure of supercritical fluid extrudates I: Melt rheology and microstructure formation

The influence of melt rheology and processing conditions on the expansion and 3D microstructure of biopolymeric foams produced by supercritical fluid extrusion (SCFX) were investigated. Starch-based SCFX extrudates with five whey protein isolate (WPI) concentrations (0–18 wt%) and four SC-CO₂ levels (0–0.75 wt%) were produced. Melt rheology was studied with an online slit die rheometer. The 3D microstructure of foams was determined using X-ray microtomography. The starch-based melt showed shear-thinning behavior, with a lower consistency coefficient and higher flow behavior index with the addition of SC-CO₂. Whey protein acted as a diluent, which resulted in reduced melt viscosity. SC-CO₂ increased the expansion of whey protein added starch-based extrudates. However, structural collapse was observed at the 0.75 wt% SC-CO₂ level during post-extrusion drying at 85 °C. The cross-sectional expansion ratio of SCFX extrudates decreased by 48.9% with the addition of 18 wt% WPI. The cell number densities per unit volume and average cell size of SCFX extrudates were 1.4 × 10³–1.9 × 10⁴ cells/cm³ and 310.0–724.4 μm, respectively, depending on WPI and SC-CO₂ levels. A decrease in melt viscosity due to the addition of whey protein might be responsible for the lower cell number density and related decrease in expansion. Processing parameters and whey protein levels were critical to controlling the microstructure of starch-based SCFX extrudates.     

Effect of fermentation on biochemical and sensory characteristics of sorghum flour supplemented with whey protein

Changes in pH, titrable acidity, protein, non-protein nitrogen, total soluble solids, protein fractions and in vitro protein digestibility were investigated during fermentation and/or after supplementation of sorghum flour with whey protein. The pH of the fermenting material decreased sharply with a concomitant increase in the titrable acidity. The total soluble solids increased with progressive fermentation time. The crude protein and non-protein nitrogen both increased with fermentation time. The protein content and fractions were significantly (p ⩽ 0.05) increased after supplementation with whey protein. The albumin plus globulin fraction increased significantly (p ⩽ 0.05) during the first 8 h of fermentation after supplementation with 5% whey protein. Other fraction contents were observed to fluctuate during the fermentation time. Supplementation of the cultivar flour with 10% whey protein greatly increased the protein content as well as the albumin plus globulin fraction while other fractions were significantly decreased. The in vitro protein digestibility was significantly (p ⩽ 0.05) improved during fermentation and even after supplementation. Sensory evaluation of locally processed sorghum products (Kisra, Asida and Nasha) before and after supplementation showed no difference between the supplemented samples and the control ones as judged by trained panellists.     

Comparison of properties of oil-in-water emulsions stabilized by coconut cream proteins with those stabilized by whey protein isolate

Coconut cream protein (CCP) fractions were isolated from coconuts using two different isolation procedures: isoelectric precipitation (CCP1-fraction) and freeze–thaw treatment (CCP2-fraction). The ability of these protein fractions to form and stabilize oil-in-water emulsions was compared with that of whey protein isolate (WPI). Protein solubility was a minimum at ∼pH 4, 4.5 and 5 for CCP1, CCP2, and WPI, respectively, and decreased with increasing salt concentration (0–200 mM NaCl) for the coconut proteins. All of the proteins studied were surface active, but WPI was more surface active than the two coconut cream proteins. The two coconut cream proteins were used to prepare 10 wt% corn oil-in-water emulsions (pH 6.2, 5 mM phosphate buffer). CCP2 emulsions had smaller mean droplet diameters (d₃₂ ≈ 2 μm) than CCP1 emulsions (d₃₂ ≈ 5 μm). Corn oil-in-water emulsions (10 wt%) stabilized by 0.2 wt% CCP2 and WPI were prepared with different pH values (3–8), salt concentrations (0–500 mM NaCl) and thermal treatments (50–90 °C for 30 min). Considerable droplet flocculation occurred in the emulsions near the isoelectric point of the proteins: CCP2 (pH ∼ 4.3); WPI (pH ∼ 4.8). Emulsions with monomodal particle size distributions, small mean droplet diameters, and good creaming stability could be produced at pH 7 for WPI, but CCP2 produced bimodal distributions at this pH. The CCP2 and WPI emulsions remained relatively stable to droplet aggregation and creaming at NaCl concentrations ⩽50 and ⩽100 mM, respectively. In the absence of salt, both CCP2 and WPI emulsions were quite stable to thermal treatments (50–90 °C for 30 min).     

Gelation of single heated vs. double heated whey protein isolate

Gelation of single and double heated whey protein dispersions was investigated using Ca²⁺ as inducing agents. Whey protein isolate (WPI) dispersions (10% w/w) were single heated (30 min, 80 °C at pH 7.0) or double heated (30 min, 80 °C at pH 8.0 and 30 min, 80 °C at pH 7.0) and diluted to obtain the desired protein and/or calcium ions concentration (4–9% and 5–30 mm, respectively). Calcium ions were added directly or by using a dialysis method. Double-heated dispersions gelled faster at lower protein and calcium ion concentrations than single-heated dispersions. Gels obtained from double-heated dispersions had lower values of shear strain and shear stress at fracture than gels obtained from single-heated dispersions. Double heating caused a significant complex modulus (G*) increase at 4% WPI and 15 mm calcium ions in comparison with gels obtained from single-heated dispersion. Less significant differences between gels made from double and single-heated dispersions were observed at 6% WPI, however a higher value of complex modulus was obtained for 8% protein gels from the single-heated solution. Native and non-reduced SDS–PAGE did not show clearly the effect of different procedures of heating on the quantities of polymerised proteins. Proteins in double-heated dispersions had higher hydrophobicity. Increased calcium concentration caused decreased protein hydrophobicity for both single and double-heated solutions.     

Screening of whey protein isolate hydrolysates for their dual functionality: Influence of heat pre-treatment and enzyme specificity

Heat pre-treated and non heat pre-treated whey protein isolate (WPI) were hydrolysed using α-chymotrypsin (chymotrypsin), pepsin and trypsin. The in vitro antioxidant activity, ACE-inhibition activity and surface hydrophobicities of the hydrolysates were measured in order to determine if peptides with dual functionalities were present. Dual functional peptides have both biological (e.g. antioxidant, ACE-inhibition, opioid activities) and technological (e.g. nanoemulsification abilities) functions in food systems. Heat pre-treatment marginally enhanced the hydrolysis of WPI by pepsin and trypsin but had no effect on WPI hydrolysis with chymotrypsin. With the exception of the hydrolysis by trypsin, heat pre-treatment did not affect the peptide profile of the hydrolysates as analysed using size exclusion chromatography, or the antioxidant activity (P > 0.05). Heat pre-treatment significantly affected the ACE-inhibition activities and the surface hydrophobicities of the hydrolysates (P < 0.05), which was a function of the specificity of the hydrolysing enzyme. Extended hydrolysis (up to 24 h) had no significant effect on the DH and the molecular weight profiles (P > 0.05) but in some instances caused a reduction in the antioxidant activity of WPI hydrolysates. The chymotrypsin hydrolysate showed a broad MW size range, and was followed by pepsin and then trypsin. The bioactivities of the hydrolysates generally decreased in the order; chymotrypsin > trypsin > pepsin. This study showed that by manipulating protein conformation with pre-hydrolysis heat treatment, combined with careful enzyme selection, peptides with dual functionalities can be produced from WPI for use as functional ingredients in the manufacture of functional foods.     

Antimicrobial activity of whey protein based edible films incorporated with oregano,rosemary and garlic essential oils

The use of edible films to release antimicrobial constituents in food packaging is a form of active packaging. Antimicrobial properties of spice extracts are well known, however their application to edible films is limited. In this study, antimicrobial properties of whey protein isolate (WPI) films containing 1.0–4.0% (wt/vol) ratios of oregano, rosemary and garlic essential oils were tested against Escherichia coli O157:H7 (ATCC 35218), Staphylococcus aureus (ATCC 43300), Salmonella enteritidis (ATCC 13076), Listeria monocytogenes (NCTC 2167) and Lactobacillus plantarum (DSM 20174). Ten millilitres of molten hard agar was inoculated by 200 μl of bacterial cultures (colony count of 1 × 10⁸ CFU/ml) grown overnight in appropriate medium. Circular discs of WPI films containing spice extracts, prepared by casting method, were placed on a bacterial lawn. Zones of inhibition were measured after an incubation period. The film containing oregano essential oil was the most effective against these bacteria at 2% level than those containing garlic and rosemary extracts (P < 0.05). The use of rosemary essential oil incorporated into WPI films did not exhibit any antimicrobial activity whereas inhibitory effect of WPI film containing garlic essential oil was observed only at 3% and 4% level (P < 0.05). The results of this study suggested that the antimicrobial activity of some spice extracts were expressed in a WPI based edible film.     

Effect of heat and enzymatic treatment on the antihypertensive activity of whey protein hydrolysates

The influence of heat and enzymatic treatments on the hypotensive activity of hydrolysates derived from whey protein isolate was examined. The whey protein isolate (WPI) was previously denatured at 65 or 95 °C and hydrolyzed using the enzymes Alcalase, α-chymotrypsin or Proteomix. The hydrolysates thus obtained were characterized and studied with regard to their angiotensin converting enzyme (ACE) inhibitory activity and hypotensive activity in spontaneously hypertensive rats (SHR). The enzyme α-chymotrypsin was found to produce hydrolysates with the highest ACE inhibitory activity. The hydrolysate that most effectively reduced blood pressure in SHR was obtained from WPI previously denatured at 65 °C and treated with the enzyme Alcalase. The hydrolysate with the highest ACE inhibitory activity was able to reduce the arterial blood pressure of the animals only after intraperitoneal administration, suggesting an interference of gastrointestinal enzymes in the absorption of active peptides from this hydrolysate.     

Optimisation of a coupled enzymatic assay of glutathione peroxidase activity in bovine milk and whey

Glutathione peroxidase (GSHPx) may have important functions in milk but studies of its activity during dairy processing are hampered by the lack of suitable assays. Thus, a coupled enzymatic assay of GSHPx activity was developed for bovine milk and whey. The reaction mixture contained reduced glutathione, tert-butylhydroperoxide, glutathione reductase and NADPH in phosphate buffer. The effect of reaction conditions on enzymatic and non-enzymatic NADPH consumption was studied, and blanks without added GSHPx sample or complete incubations containing 4 mmol L⁻¹ mercaptosuccinate as an inhibitor of GSHPx were used. With increasing pH and glutathione concentration the reaction rates increased both in the complete incubation and the blank but optimal GSHPx activity was attained at a glutathione concentration of 0.63 mmol L⁻¹. A pH of 7.6 was chosen to attain near-optimal activity without reaching high blanks. Application of the new procedure showed that bulk whey samples had rather constant GSHPx activity with an average (SD) of 36.1 (2.9) U mL⁻¹, but whey samples prepared from individual cows showed a two-fold variation (range 24.5–50.6 U mL⁻¹). The procedure described was more reproducible and precise than previous methods and will be employed in further studies of dairy products.     

Use of high-performance size exclusion chromatography to characterize protein aggregation in commercial whey protein concentrates

Protein aggregation profiles of seven commercial whey protein concentrates (WPCs) with protein contents ranging from 32% to 81% were investigated by high-performance size-exclusion chromatography (HPSEC). Two protein fractions were identified: the aggregated protein fraction (APF) and the native protein fraction (NPF). Important differences were found in the proportions of APF ranging from 10% to 29% of the total protein. Centrifugation at 48,000×g for 45 min at 25 °C achieved nearly complete separation of the two fractions for all samples based on HPSEC analysis of both the supernatant and pellet. NPF was shown to contain multiple peaks separable by HPSEC. Electrophoresis (native-PAGE, reducing SDS-PAGE and non-reducing SDS-PAGE) demonstrated that aggregates were bound together by covalent and non-covalent bonds. This study shows that HPSEC provides a quantitative measurement of WPC protein aggregation, which is a useful parameter to be determined prior to WPCs application in food formulations.     

X-ray diffraction analysis of lactose crystallization in freeze-dried lactose–whey protein systems

Water sorption, time-dependent crystallization and XRD patterns of lactose and lactose–WPI mixtures were studied with glass transition data. The results indicated that the sorbed water of lactose–WPI mixtures was fractional and water content of individual amorphous components in lactose–WPI mixtures at each a_w from 25 °C to 45 °C could be calculated. Crystallization occurred in pure lactose whereas partial crystallization was typical of lactose–WPI mixtures (protein content ≤ 50%) at intermediate and high a_w (> 0.44 a_w) from 25 °C to 45 °C. The extents of crystallization were significantly delayed by WPI. The T_g values of lactose–WPI systems showed the composition-dependent property in systems and might indicate the occurrence of phase separation phenomena during 240 h storage. XRD showed no anhydrous β-lactose and mixed α-/β-lactose with molar ratios of 4:1 crystals in crystallized lactose–WPI systems (70:30 and 50:50 solids ratios). Reduced crystallization in the presence of WPI was more pronounced possibly because of reduced nucleation and diffusion during crystal-growth. The present study showed that WPI could present an important role in preventing sugar crystallization.     

Effect of composition and antioxidants on the oxidative stability of fluid milk supplemented with an algae oil emulsion

The oxidative stability of an algal oil emulsion dispersed in water, or fluid milk of varying fat contents, was assessed from measurements of lipid hydroperoxide and propanal concentration. All of the milk samples, independent of their milk fat content, were stable compared to the aqueous samples. The extent of oxidation was unaffected when sodium azide (200 ppm) was added to inhibit microbial growth. Added iron (100 ppm) accelerated the oxidation rate in the aqueous samples, but had no effect on the milk samples. The antioxidant properties of milk were ascribed to the iron binding of casein. Added protein antioxidants (0.8 wt%) [i.e. sodium caseinate, whey protein isolate (WPI) and thermally denatured WPI] had minimal effects whereas EDTA and ascorbic acid (160 ppm) were effective antioxidants.     

Hepatoprotective effect of peptic hydrolysate from salmon pectoral fin protein byproducts on ethanol-induced oxidative stress in Sprague–Dawley rats

Bioactive peptides were generated by pepsin hydrolysis from salmon pectoral fin protein byproduct, and the hepatoprotective effect of the peptic hydrolysate (PH) on ethanol-induced oxidative stress was investigated in Sprague–Dawley rats. Administration of ethanol for 4 weeks significantly (p < 0.05) increased serum markers of liver damage, such as alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and lactate dehydrogenase; however, these activities decreased significantly (p < 0.05) after PH administration. Elevated thiobarbituric acid reactive substance levels in liver tissue and serum decreased significantly (p < 0.05) following administration of PH. Ethanol exposure significantly (p < 0.05) decreased glutathione contents in liver and serum, and hepatic antioxidant enzyme activities such as superoxide dismutase and glutathione peroxidase, while the PH treatment significantly (p < 0.05) increased these altered parameters. These results indicate that the PH had a protective effect against ethanol-induced hepatotoxicity that was comparable to that of silymarin, which was supported by evaluating liver histopathology in the rats.     

Impact of whey protein isolates and concentrates on the formation of protein nanoparticles‐stabilised Pickering emulsions

Whey protein nanoparticles (NPs) were prepared by heat‐induced method. The influences of whey protein isolates (WPIs) and concentrates (WPCs) on the formation of NPs were first investigated. Then Pickering emulsions were produced by protein NPs and their properties were evaluated. After heat treatment, WPC NPs showed larger particle size, higher stability against NaCl, lower negative charge and contact angle between air and water. Dispersions of WPC NPs appeared as higher turbidity and viscosity than those of WPI NPs. The interfacial tension of WPC NPs (~7.9 mN/m at 3 wt% NPs) was greatly lower than that of WPI NPs (~12.1 mN/m at 3 wt% NPs). WPC NPs‐stabilised emulsions had smaller particle size and were more homogeneous than WPI NPs‐stabilised emulsions. WPC NPs‐stabilised emulsions had higher stability against NaCl, pH and coalescence during storage.     

Heat stability of oil-in-water emulsions formed with intact or hydrolysed whey proteins: influence of polysaccharides

The heat stability of emulsions (4 wt% corn oil) formed with whey protein isolate (WPI) or extensively hydrolysed whey protein (WPH) products and containing xanthan gum or guar gum was examined after a retort treatment at 121 °C for 16 min. At neutral pH and low ionic strength, emulsions stabilized with both 0.5 and 4 wt% WPI (intact whey protein) were stable against retorting. The amount of β-lactoglobulin (β-lg) at the droplet surface increased during retorting, especially in the emulsion containing 4 wt% protein, whereas the amount of adsorbed α-lactalbumin (α-la) decreased markedly. Addition of xanthan gum or guar gum caused depletion flocculation of the emulsion droplets, but this flocculation did not lead to their aggregation during heating. In contrast, the droplet size of emulsions formed with WPH increased during heat treatment, indicating that coalescence had occurred. The coalescence during heating was enhanced considerably with increasing concentration of polysaccharide in the emulsions, up to 0.12% and 0.2% for xanthan gum and guar gum, respectively; whey peptides in the WPH emulsions formed weaker and looser, mobile interfacial structures than those formed with intact whey proteins. Consequently, the lack of electrostatic and steric repulsion resulted in the coalescence of flocculated droplets during retort treatment. At higher levels of xanthan gum or guar gum addition, the extent of coalescence decreased gradually, apparently because of the high viscosity of the aqueous phase.     

Physicochemical changes in intermediate-moisture protein bars made with whey protein or calcium caseinate

This study examined model protein bars made with whey protein isolate (WPI) or calcium caseinate and stored at 20 °C for 50 days. WPI bars remained very soft and, throughout storage, confocal micrographs showed a continuous matrix containing soluble protein and increasing quantities of glucose crystals. In contrast, calcium caseinate bars had a firm texture within 1–5 days of manufacture (fracture stress 199 ± 16 Pa) and hardened progressively during storage (final fracture stress 301 ± 18 Pa). Electrophoresis showed no evidence of covalent protein aggregation, but there were substantial changes in microstructure over the first day of storage, resulting in segregation of a protein phase from a water–glucose–glycerol phase. Proton nuclear magnetic resonance (¹H-NMR) relaxometry and nuclear Overhauser effect spectroscopy (NOESY) experiments showed that water migration away from protein towards glucose and glycerol occurred 10–18 h after manufacture, lowering the molecular mobility of protein. Phase separation was probably driven by the high osmotic pressure generated by the glucose and glycerol. These results confirm that the hardening of protein bars is driven by migration of water from protein to glucose and glycerol, and microstructural phase separation of aggregated protein.     

Development of a fermented goat's milk containing probiotic bacteria

A set-type fermented milk manufactured from goat's milk was developed. Optimal curd tension was achieved by supplementation of milk with skim milk powder and whey protein concentrate (WPC). Milk was fermented employing a commercial probiotic starter culture (ABT-2), which contained Streptococcus thermophilus ST-20Y, Lactobacillus acidophilus LA-5, and Bifidobacterium BB-12. Supplementation of milk with 3% WPC reduced fermentation time by 2 h due to the increase in viable counts of S. thermophilus and Bifidobacterium by 0.3 and 0.7 log units, respectively. Addition of WPC increased the protein content (1%) as well as potassium and magnesium content (0.3 and 0.02 g kg⁻¹, respectively). Increase of the protein content led to an increase in the apparent viscosity and gel firmness of the product, and at the same time whey syneresis was reduced. As a consequence, the product received a high score for appearance, taste, aroma, texture and overall acceptance.     

Effect of high-pressure treatment on the electrospray ionization mass spectrometry (ESI-MS) profiles of whey proteins

The effects of different high-pressure (HP) treatments (pressures ranging between 450 and 650 MPa) on β-lactoglobulin and α-lactalbumin and on two commercial whey protein isolates (WPI) were examined by electrospray ionization mass spectrometry (ESI-MS). HP treatment resulted in substantial changes in the charge-state distribution (CSD) of pure β-lactoglobulin, attributable to exposure of side chains of buried amino acids to solvent, whereas the CSD of pure α-lactalbumin was largely insensitive to HP treatment. The changes in the CSD of these proteins effected by HP treatment differed between the two commercial WPI samples examined, perhaps due to the different production methodologies employed by the two suppliers. These findings illustrate the important role of interactions between the various components in WPI in the response of whey proteins to HP treatment and underscore the difficulty of obtaining consistent rheological and nutraceutical properties by HP treatment of WPI, particularly when different sources of WPI are employed. The information provided by ESI-MS may be useful in monitoring the effects of HP treatment to verify that such consistency is achieved.     

Influence of emulsifier type on in vitro digestibility of lipid droplets by pancreatic lipase

The objective of this study was to investigate the influence of interfacial composition on the in vitro digestion of emulsified lipids coated by various emulsifiers by pancreatic lipase. Sodium caseinate, whey protein isolate (WPI), lecithin and Tween 20 were used to prepare corn oil-in-water emulsions (3 wt% oil). Pancreatic lipase (1.6 mg/mL) and/or bile extract (5.0 mg/mL) were added to each emulsion and the particle charge, droplet aggregation, microstructure and free fatty acids released were measured. In the absence of bile extract, the amount of free fatty acids released per unit volume of emulsion was much lower for lipid droplets coated by Tween 20 (13 ± 16 μmol ml⁻¹) than those coated by lecithin (75 ± 20 μmol ml⁻¹), sodium caseinate (220 ± 24 μmol ml⁻¹) or WPI (212 ± 6 μmol ml⁻¹). In the presence of bile extract, there was an appreciable increase in the amount of free fatty acids released in all the emulsions, with the most appreciable effects being observed in the Tween 20-stabilized emulsions. The stability of the emulsions to droplet flocculation and coalescence during hydrolysis was also strongly dependent on emulsifier type, with the WPI emulsions being the least stable and the Tween 20 emulsions being the most stable. Our results suggest that the access of pancreatic lipase to emulsified fats decreases in the following order: proteins (caseinate and WPI) > phospholipids (lecithin) > non-ionic surfactants (Tween 20). These results may have important consequences for the design of foods with either increased or decreased lipid bioavailability.     

Ability of conventional and nutritionally-modified whey protein concentrates to stabilize oil-in-water emulsions

The ability of a modified whey protein concentrate (MWPC), which contains relatively high proportions of phospholipid and high molecular weight protein fractions, to form and stabilize 10 wt% corn oil-in-water emulsions (pH 7.0, 5 mM phosphate buffer) was compared with that of a conventional whey protein concentrate (CWPC). The MWPC stabilized emulsions required less protein to prepare stable emulsions with monomodal particle size distributions and small mean droplet diameters (d₄₃ ≈ 0.3 μm at [WPC] ⩾ 0.5 wt%) than CWPC stabilized emulsions (d₄₃ ≈ 0.4 μm at [WPC] ⩾ 0.9 wt%) under similar homogenization conditions (5 passes at 5000 psi). In addition, the emulsions stabilized by 0.9 wt% MWPC were more stable to high salt concentration (NaCl ⩽ 200 mM), thermal processing (30–90 °C for 30 min) and pH (3, 6 and 7) than those stabilized by the same concentration of CWPC, which was attributed to polymeric steric repulsion rather than electrostatic repulsion. This study has important implications for the wide application of WPC as a natural emulsifier in food products.