Effects of combined high pressure and enzymatic treatments on the hydrolysis and immunoreactivity of dairy whey proteins

The effect of high-pressure (HP) treatment on the hydrolysis of dairy whey proteins by trypsin, chymotrypsin and pepsin was analysed. Isostatic pressure (100–300 MPa for 15 min at 37 °C) was applied to the protein substrate prior to its enzymatic hydrolysis. Digestion was also conducted at atmospheric pressure (0.1 MPa) and under high pressure. The extent of hydrolysis was measured by the o-phthaldialdehyde method, the peptide profile was analysed by reverse-phase high performance liquid chromatography (RP-HPLC) and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and the residual immunochemical reactivity was assessed by an ELISA test using a pool of seven sera from children allergic to bovine milk, an individual serum also positive (positive control) and two sera from non-allergic children (negative controls). The high pressure increased the degree of hydrolysis by the three enzymes used. Chymotrypsin and trypsin showed the highest proteolysis at 100 and 200 MPa followed by pepsin at 300 MPa. The β-lactoglobulin was hydrolysed by trypsin and chymotrypsin at atmospheric and at high pressures, whereas the pepsin only hydrolysed this protein under high pressure. Pepsin and trypsin hydrolysed α-lactalbumin in all cases. In contrast, this protein was not digested by chymotrypsin, irrespective of the pressure applied. An important decrease of immunochemical reactivity was found for pepsin and trypsin hydrolysates obtained under high pressure. The pool of seven sera detected immunoreactivity in the products of chymotrypsin hydrolysis under high pressure, which was not detected when the serum of one patient was used. The results suggest that dairy whey hydrolysates obtained by pepsin and trypsin in combination with HP treatment could be used as a source of peptides in hypo-allergenic infant formulae.     

The dominating effect of ionic strength on the heat-induced denaturation and aggregation of β-lactoglobulin in simulated milk ultrafiltrate

The denaturation/aggregation behaviour of heated (78 °C, 10 min) β-lactoglobulin (1%, w/w) was examined as a function of heating pH (5.0–7.0), in the presence of different salts. Heating β-lactoglobulin in the presence of calcium (5 mm) significantly increased the level of aggregated protein at most heating pH values, compared to heating in water or sodium chloride (100 mm). Heating β-lactoglobulin in the presence of calcium (5 mm) and phosphate (5 mm), resulted in similar denaturation levels in the pH range 5.0–5.8 as in the presence of calcium (5 mm) alone but reduced denaturation in the pH range 6.0–7.0, probably due to the formation of insoluble calcium phosphate. The addition of NaCl (100 mm) counteracted the aggregation promoting properties of the calcium and calcium/phosphate systems. Heating β-lg in a simulated milk ultrafiltrate solution was similar to heating in NaCl alone. This suggested that Ca²⁺ effects alone may not explain the heat-induced denaturation/aggregation behaviour of β-lactoglobulin in milk whey systems.     

HPLC-MS/MS-Detection of Caseins and Whey Proteins in Meat Products

Screening methods for the mass spectrometric detection of caseins and whey proteins in meat products have been developed. After tryptic digestion, two α-S1-casein and two β-lactoglobulin marker peptides were measured by HPLC-MS/MS. For matrix calibrations, emulsion-type sausages with different concentrations of milk and whey protein (ppm level) were produced. The limits of detection (LODs) were below 1 ppm for milk protein and about 3 ppm for whey protein. The determination coefficients for the correlation between peak area of the marker peptides and the concentrations of milk and whey proteins were R²≥0.9899.     

Effect of high-pressure treatment on in-vitro digestibility of β-lactoglobulin

The effect of high-pressure (HP)-treatment on β-lactoglobulin (β-Lg) was investigated using in-vitro pepsin digestion under simulated gastric conditions. HP-treatment of β-Lg at 400 MPa for 10 min only slightly increased its subsequent hydrolysis by pepsin. However, higher pressure treatments (600 and 800 MPa) resulted in rapid digestion of β-Lg. After these higher pressure treatments, β-Lg disappeared in less than 1 min of pepsin incubation as determined by SDS-PAGE analysis. Mass spectrometry analysis of the digestion products at corresponding incubation times revealed rapid and progressive degradation of β-Lg. Most (> 90%) of the peptide products following pepsin digestion of HP-treated β-Lg were less than 1500 Da in size. Peptide products from pepsin digestion were identified and mapped to β-strand regions (Leu₃₂–Leu₅₄ and Phe₈₂–Leu₁₀₄) and to the N- and C-terminals regions (Leu₁–Leu₁₀ and Ser₁₅₀–Leu₁₅₆) of β-Lg. While these regions corresponded to known IgE epitopes of β-Lg, the predominant peptides resulting from 60 s of incubation were short (7–10 residues) in length. These results demonstrate that HP-treatment increased the digestibility of β-Lg and represents a promising processing technology for reducing the allergenicity of known allergens in a wide variety of food materials.Industrial relevanceHigh-pressure treatment is widely used to enhance the functional attributes of food proteins. The potential for enhanced nutritional value of β-Lg was also demonstrated here by its increased digestibility. High-pressure treatment followed by incubation with proteases may represent a method for the commercial production of bioactive peptides such as inhibitors of angiotensin converting enzyme. More importantly, high-pressure-induced unfolding of milk proteins may reduce their allergenicity. Unfolded proteins are less likely to become agents of immunological sensitization because they are more readily hydrolyzed. Thus high-pressure treatment applied to food ingredients such as whey protein isolate may contribute to the development of hypoallergenic foods.     

Isolation and characterisation of a novel antibacterial peptide from bovine αS1-casein

Bovine casein was hydrolysed with a range of proteolytic enzymes including pepsin, trypsin, α-chymotrypsin and β-chymotrypsin, and assessed for antibacterial activity. The pepsin digest of bovine casein, which showed antibacterial activity, was fractionated using reverse phase high performance liquid chromatography and the antibacterial peptides isolated were characterised using electrospray ionisation mass spectrometry. Two antibacterial peptides were identified, a novel peptide (Cp1) which corresponded to residues 99–109 of bovine α_S1-casein and a previously reported peptide (Cp2) which corresponded to residues 183–207 of bovine α_S2-casein. The minimum inhibitory concentration (MIC) of Cp1 and Cp2 were determined against a range of bacterial cultures. Cp1 exhibited an MIC of 125 μg mL⁻¹ against all Gram-positive bacteria tested, and MIC ranging between 125 and >1000 μg mL⁻¹ against the Gram-negative bacteria tested. Cp2 was generally far more potent against the Gram-positive bacteria, exhibiting an MIC of 21 μg mL⁻¹, compared to MICs ranging from 332 to >664 μg mL⁻¹ against most of the Gram-negative bacteria tested.     

Release of multifunctional peptides by gastrointestinal digestion of sea cucumber (Isostichopus badionotus)

Sea cucumber is a benthic marine organism distributed worldwide and used as food in several Asian countries. The species Isostichopus badionotus is captured intensively off the Yucatan Peninsula, Mexico. Boiled I. badionotus was subjected to in vitro simulated gastrointestinal digestion using pepsin and a pepsin–Corolase PP^® mixture. ACE-inhibitory and radical scavenging activities, iron reducing capacity and cytotoxic effects against colorectal cancer cells were evaluated in the hydrolysates and their ultrafiltered fractions. ACE-inhibitory activity was potent in fractions containing peptides <3000 Da, an effect augmented with combined action of gastric (pepsin) and intestinal (Corolase PP^®) enzymes (IC₅₀ = 0.038 ± 0.004 mg/mL). Antioxidant activity was exerted by peptides with low and high molecular weights, depending on hydrolysis method. This is the first report of cytotoxic capacity against colorectal HT-29 cells in peptides from sea cucumber. Sea cucumber hydrolysates and ultrafiltered fractions are potential ingredients for development of functional foods.     

Skim milk protein distribution as a result of very high hydrostatic pressure

This work studies the micellar size and the distribution of caseins, major and minor whey proteins in different fractions of skim milk treated up to 900 MPa for 5 min. Transmission electron microscopy showed that the smallest casein micelles were formed around 450 MPa with no variations at higher pressures. The changes found in micellar size correlated with the concentration of soluble casein, because treatments at 250 MPa significantly enhanced the level of non-sedimentable casein while, between 700 and 900 MPa, there were no further increases with respect to lower pressures. There was a severe β-lactoglobulin (β-Lg) denaturation at pressures ≥ 700 MPa, which reached 77–87%. α-Lactalbumin (α-La) was stable up to 550 MPa, but it denatured at higher pressures. The content of soluble lactoferrin (Lf) decreased with pressure, particularly from 550 to 800 MPa, while that of secretory IgA (sIgA) progressively decreased from 250 up to 700 MPa. Our results indicated that treatment of milk at very high pressures, from 700 to 900 MPa, did not reduce micellar size nor released more soluble casein with respect to treatments at lower pressures (250–550 MPa). However, these treatments led to a severe denaturation of the whey proteins, in particular of β-Lg and the minor proteins Lf and sIgA. The possibility of using high hydrostatic pressure to obtain a soluble milk fraction with a casein and whey protein composition similar to that of human milk is discussed.     

Effect of feeding whey peptide fractions on the immune response in healthy and Escherichia coli infected mice

The antimicrobial and immunomodulatory activities of whey proteins and peptides make them promising candidates for protection against pathogens. To test this hypothesis, a whey protein isolate and three peptide fractions obtained from its trypsin/chymotrypsin digestion were given orally to mice for 7 days. Half the mice were then infected with Escherichia coli O157:H7 and serum cytokines and total immunoglobulin A (IgA) were measured over the next 7 days. All whey products strongly stimulated total IgA production in non-infected mice, suggesting a potential adjuvant role. The peptide fractions produced contrasting immunomodulatory effects: the neutral fraction (4.5 < pH < 7) stimulated serum interferon-gamma (IFN-γ), whereas the acidic fraction (pH < 4.5) inhibited it. In the infected model, only the basic fraction (pH > 7) induced a sustained serum IgA secretion, which coincided with increased transforming growth factor-beta 1 (TGF-β1) levels. These results indicate that some whey peptides modulate immune parameters in healthy mice, whereas the basic peptide fraction increased immune vigilance during the infection.     

Functional properties and Angiotensin-I converting enzyme inhibitory activity of soy–whey proteins and fractions

Soy proteins when prepared to high purity can confer good functional properties and the whey by-product is a potential source for bioactivity. In this study, we determined the protein, moisture, fiber, solubility, foaming, emulsion properties, as well as Angiotensin-I converting enzyme (ACE-I) inhibitory activity of prepared soy–whey proteins and its fractions. The soy–whey proteins were fractionated into < 5, > 5, > 10, and > 50 kDa using ultrafiltration. The expanded AACC methods were used to determine protein, moisture, and fiber analyses of the whey and its fractions. Solubility method was conducted to determine the protein solubility profile of the soy–whey and its fractions at varying pHs. Turbidimetric method was used to evaluate emulsifying activity (EA) and emulsion stability (ES). There were significant differences observed in moisture, protein and salt contents between unfractionated, > 50 kDa and smaller sized fractions. No significant differences were observed with phytic acid and total dietary fiber contents among all samples. The unfractionated whey protein and > 50 kDa fraction showed better solubility than other fractions. Unfractionated whey protein had the highest foam capacity (42.7 mL) while the fraction > 5 kDa showed the greatest foaming stability (46 min). The highest emulsion activity (0.33 ± 0.1) and stability (825.1 ± 45.1) was obtained with the > 50 kDa fraction while the unfractionated whey protein had the highest ACE-I inhibition activity. The findings indicate that soy–whey protein fraction (> 50 kDa) had good solubility, emulsion activity and stability, while the unfractionated whey protein exhibited the strongest ACE-I inhibition percentage (19%).     

Binding affinity between dietary polyphenols and β-lactoglobulin negatively correlates with the protein susceptibility to digestion and total antioxidant activity of complexes formed

Non-covalent interactions between β-lactoglobulin (BLG) and polyphenol extracts of teas, coffee and cocoa were studied by fluorescence and CD spectroscopy at pH values of the gastrointestinal tract (GIT). The biological implications of non-covalent binding of polyphenols to BLG were investigated by in vitro pepsin and pancreatin digestibility assay and ABTS radical scavenging activity of complexes formed. The polyphenol–BLG systems were stable at pH values of the GIT. The most profound effect of pH on binding affinity was observed for polyphenol extracts rich in phenolic acids. Stronger non-covalent interactions delayed pepsin and pancreatin digestion of BLG and induced β-sheet to α-helix transition at neutral pH. All polyphenols tested protected protein secondary structure at an extremely acidic pH of 1.2. A positive correlation was found between the strength of protein–polyphenol interactions and (a) half time of protein decay in gastric conditions (R² = 0.85), (b) masking of total antioxidant capacity of protein–polyphenol complexes (R² = 0.95).     

Surface Tension of Whey and Whey Derivatives

The surface tension of various whole wheys, solutions of component whey proteins, UF fractions, and the effect of heating on the surface tensions of these solutions were determined using the Wilhemy plate method. The mean surface tension of three commercial cottage cheese wheys, a commercial cheddar cheese whey, and a laboratory rennet whey was found to be 41.7 ± 1.2 dyne/cm (25°C) and did not vary significantly with the type of whey despite differences in both pH and protein content. The surface tensions of aqueous solutions of individual pure protein fractions of whey (serum albumin, β-lactoglobulin, α-lactalbumin, and gammaglobulins), in concentrations approximating normal whey contents, were significantly different and greater than for the whole wheys.Heating of individual protein solutions at 80°C for 50 min produced insignificant changes in measured surface tension despite producing protein precipitation in some of the solutions. Similar heating of the whole whey solutions resulted in a significant decrease in surface tension and marked precipitation in most cases.The fractionation of the wheys into UF permeates and retentates resulted in a retentate fraction of significantly lower surface tension than for UF permeates. Heating increased the surface tension of retentate fractions while the permeate fractions showed a decrease.     

Use of an electrodialytic reactor for the simultaneous β-lactoglobulin enzymatic hydrolysis and fractionation of generated bioactive peptides

The enzymatic hydrolysis of β-lactoglobulin and the fractionation of peptides were performed in one step in an electrodialysis cell with ultrafiltration membranes stacked. After 240 min of treatment, 15 anionic and 4 cationic peptides were detected in the anionic and cationic peptide recovery compartments. Amongst these 15 anionic peptides, 2 hypocholesterolemic, 3 antihypertensive and 1 antibacterial peptides were recovered and concentrated with migration rates ranging from 5.5% and 81.7%. Amongst the 4 cationic peptides, the peptide sequence ALPMHIR, identified as lactokinin and known to exert an important antihypertensive effect, was recovered with an estimated 66% migration rate. To our knowledge, it was the first attempt to perform hydrolysis under an electric field and to simultaneously separate anionic and cationic peptides produced.     

Effects of controlled pepsin hydrolysis on antioxidant potential and fractional changes of chickpea proteins

This study investigated the effects of controlled pepsin hydrolysis on antioxidant potential and fractional changes of chickpea protein extracts (CPE). The enzyme hydrolysis increased soluble protein content (1.2 to 2-fold) and free radical scavenging activity (1.9 to 3-fold) of hydrolyzed chickpea protein extract (HCPE), but almost unaffected its antioxidant potential in oil-in-water emulsion system and reduced its iron chelating capacity (1.3-fold) and functional properties. The chromatographic fractions of CPE are mainly acidic, while those of HCPE are mainly basic and neutral. The majority of chickpea proteins had pI between 4.5 and 5.5, and molecular weight (MW) between 15 and 40 kDa, while MW of their pepsin hydrolysis products ranged between 6.5 and 14.2 kDa. The main antioxidant proteins in CPE and HCPE fractionated by ultrafiltration had MW greater than 30 kDa and between 2 and 10 kDa, respectively. The chickpea proteins and hydrolysates showed different potentials as functional food ingredients.     

Peptic hydrolysis of ovine β-lactoglobulin and α-lactalbumin Exceptional susceptibility of native ovine β-lactoglobulin to pepsinolysis

Ovine β-lactoglobulin (BLG, a mixture of variants A and B at a ratio of 46/54) and α-lactalbumin (ALA) were subjected to pepsin activity. The degree of peptic hydrolysis of native whole BLG reached 63%, 74%, 82% and 87% after 2, 4, 8 and 20 h hydrolysis, respectively. BLG variant B was degraded completely after 2 h of pepsin digestion while variant A was degraded gradually showing 19%, 44%, 61% and 73% hydrolysis after 2, 4, 8 and 20 h, respectively. The main factors responsible for the exceptional pepsin susceptibility of ovine BLG are the slightly different tertiary structure of ovine BLG (compared with bovine BLG) as perceived from near circular dichroism spectra at pH 2, and its higher surface hydrophobicity, as demonstrated by a higher binding activity to 1-anilinonaphthalene-8-sulphonate. Reversed phase-high performance liquid chromatograms (RP-HPLC) profiles of the peptic hydrolysates of BLG showed the production of hydrophobic peptides at the early stages of hydrolysis, while more hydrophilic peptides appeared only at a later stage of hydrolysis. Mass spectroscopy analysis allowed the characterisation of 17 and 13 peptides after 2 and 20 h hydrolysis, respectively. Most of the enzyme activity was oriented first towards the N-terminal part of the molecule and later towards the C-terminal part of the protein; little or no activity was observed in the central region of the molecule even after 20 h hydrolysis. Native ovine ALA was almost completely degraded by pepsin, yielding 93%, 94%, 95% and 98% hydrolysis after 2, 4, 8 and 24 h, respectively. The RP-HPLC profile of the ALA hydrolysate showed 5 major hydrophobic peptides and 7 minor more hydrophilic peptides, which did not change with the time of hydrolysis.     

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.     

Effects of Milk Composition and Genetic Polymorphism on Coagulation Properties of Milk

Milk samples from 31 Holstein cows of different phenotypes for β-casein, κ-casein, and β-lactoglobulin were collected monthly over the entire lactation. These samples were analyzed for total solids, fat, protein, casein fractions, lactose, urea, citric acid, somatic cell count, and pH. Rennet clotting time, rate of firming, and curd firmness as measured by a Formagraph were not significantly influenced by phenotypes for β-casein and κ-casein. Phenotype AA for β-lactoglobulin gave the best clotting time (3.91 min) and firmness of curd (36.30 mm) when compared with AB and BB phenotypes. Relative percentages of the different caseins and α-lactalbumin affected significantly rate of firming and curd firmness at cutting. Amount of κ-casein in milk was the most significant factor that affected curd firmness with a coefficient of regression of 15.96.     

Pepsin treatment of whey proteins under high pressure produces hypoallergenic hydrolysates

BALB/c mice were used to assess the ability of a whey protein hydrolysate obtained by pepsin treatment under high pressure (400 MPa, 37 °C, 30 min, HWP), to induce anaphylaxis, antibody production and cytokine responses in comparison with the whey protein isolate (WP) from which it is derived. HWP did not contain intact allergens and 50% of its peptides ranged between 10 and 3 kDa. Challenge with HWP did not induce clinical signs, body temperature changes or release of mast cell proteinase-1 in mice sensitized to WP. Immunization of mice with HWP did not produce WP-specific antibodies or allergic reactions upon HWP or WP challenge and thus, it can be considered hypoallergenic. However, HWP stimulated Th2 responses in splenocytes from sensitized mice. These characteristics make HWP a good candidate to be used in the management of milk allergy in diagnosed patients or to induce tolerance to whey proteins.Industrial relevanceHydrolysis with pepsin under high pressure produces in minutes a whey protein hydrolysate that complies with the health claims of the European guidelines on infant and follow-on formulas related to the reduction of risk to allergy to milk proteins. This process constitutes an alternative to the exhaustive enzymatic hydrolysis treatments used in the processing of hypoallergenic formulas that release short peptides and free amino acids to adversely affect organoleptic properties and technological value.     

Iron-binding peptides from whey protein hydrolysates: Evaluation,isolation and sequencing by LC–MS/MS

Iron–peptide complexes have been considered a promising source of more bioavailable iron, with reduced side effects as compared to iron salts. Whey protein isolate (WPI) hydrolyzed by alcalase, pancreatin or flavourzyme was ultrafiltered (cut off 5 kDa) and their fractions – retentates and filtrates – were evaluated for iron-binding capacity. The Fe–hydrolysate complexation reaction resulted in a dramatic increase in iron solubility at pH 7.0, from 0% to almost 100%. This result was obtained regardless of the molecular mass profile or the enzyme used to obtain the samples. Fractions from hydrolysate obtained with pancreatin (HP) were chosen to continue the study. The complexes formed with both fractions from HP were stable under simulated gastric digestion (50.8–89.4%). To identify the peptides with iron-binding capacity, the HP fractions were isolated by IMAC-Fe^3 +, and the retentate showed higher relative concentrations of iron-binding peptides than the filtrate. Iron-binding peptide sequencing, accomplished by LC–MS/MS, showed Glu and/or Asp in all the sequences, and their carboxylic groups were amongst the main iron-binding sites. WPI hydrolysis with pancreatin yields peptides that can form iron complexes with the potential to increase iron bioavailability and reduce its pro-oxidant effect.     

Production of novel ACE inhibitory peptides from β-lactoglobulin using Protease N Amano

Potent angiotensin I-converting enzyme (ACE) inhibitory peptide mixtures were obtained from the hydrolysis of β-lactoglobulin (βLg) using Protease N Amano, a food-grade commercial proteolytic preparation. Hydrolysis experiments were carried out for 8 h at two different temperatures and neutral pH. Based on their ACE inhibitory activity, samples of 6 h of digestion were chosen for further analysis. The temperature used for the hydrolysis had a marked influence on the type of peptides produced and their concentration in the hydrolysate. Protease N Amano was found to produce very complex peptide mixtures; however, the partially fractionated hydrolysates had already very potent ACE inhibitory activity. The novel heptapeptide SAPLRVY was isolated and characterised. It corresponded to βLg f(36–42) and had an IC₅₀ value of 8 μm, which is considerably lower than the most potent ACE inhibitory peptides derived from bovine βLg reported so far.     

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.