α-Lactalbumin (α-LA) and β-lactoglobulin (β-LG) are major whey proteins in bovine milk. We studied the effects of these molecules on the intestinal cell response by comparing the native form with the denatured form containing oligomers obtained by treatment with 2,2,2-trifluoroethanol (TFE). We previously reported that proteins in native and TFE-treated forms exhibited cell growth stimulation and cytotoxicity, respectively, in undifferentiated rat crypt IEC-6 and human colon Caco-2 cells. However, neither whey protein showed cytotoxicity even in the TFE-treated form in differentiated Caco-2 cells. Only undifferentiated immature intestinal cells can distinguish between these native and denatured proteins. Moreover, α-LA and β-LG exhibited different oligomer formation characteristics during the TFE treatment. In the present study, we compared the effects of native and TFE-treated whey proteins on IEC-6 cells in more detail. The native forms of both whey proteins exhibited cell proliferative effects in a concentration-dependent manner. For the TFE-treated forms, α-LA showed rapid and potent cytotoxicity, whereas β-LG altered cell responses depending on its concentration and exposure time; lower concentration/shorter exposure and higher concentration/longer exposure induced cell growth stimulation and cytotoxicity, respectively. Pre-treatment of the cell membrane with cholesterol suppressed the effects on the cell response only in TFE-treated β-LG (TFE-β-LG). In a preliminary examination using inhibitors of signal transduction, TFE-treated α-LA acted on the intrinsic apoptosis pathway via Bcl-2-associated X and p53, whereas the action of TFE-LG did not require this pathway. Tyrosine phosphorylation is necessary for the cell proliferation effect of both native whey proteins; however, native α-LA, but not native β-LG, also required activation of the pathway with selective epidermal growth factor receptor tyrosine kinase and Janus kinase 2/3. In summary, the two major bovine milk whey proteins induced similar yet discrete responses in undifferentiated intestinal cells. Even when oligomers are formed, β-LG may be much less hazardous to immature intestinal cells than α-LA.

Functions of mucin, as the major macromolecular component in saliva or gastric fluids, are drawing increasing attention in the context of understanding the oral processing or digestion of dairy foods at the molecular level. This study was designed to investigate the interactions between β-lactoglobulin (BLG)-lactose, mucin-lactose and BLG-lactose-mucin at the molecular level under different temperature and pH conditions using fluorescence spectroscopy in combination with scanning electron microscopes (sem). It is the first study of its kind. There was no lactose-dependent quenching on BLG fluorophore in the range of 0–10 mM lactose concentration. On the contrary, there was a continuous increase in the fluorescence intensity of the BLG protein when the lactose concentration increased, especially at 25°C. BLG-lactose complex became thermally unstable at 37 and 45°C. Moreover, BLG exhibited a pH dependent conformational change and had higher fluorescence intensity at pH 3 than pH 6.8. The fluorescence result was in correspondence with sem images where we observed lactose crystals gathering around and on the BLG molecule, but lactose molecules could not be seen in the presence of mucin. It was anticipated that mucin molecules interacted with BLG-lactose complex via electrostatic attraction and formed an extra protective layer around the BLG molecules to avoid solvent exposure.

In this study, the effects of the degree of thermal denaturation of whey protein (WP) added to milk on the dissociation of κ-casein from casein micelles were investigated, since they are related to the strength of acid milk gel and its factors. Acid milk gels were prepared by heating thermally denatured WP isolate (WPI) and undenatured milk mixtures and treating them with glucono-δ-lactone as a coagulant. The strength of these gels was negatively correlated with the WPI denaturation degree and strongly positively correlated with the extent of κ-casein dissociation from casein micelles. This behavior was ascribed to the fact that α-lactalbumin (α-La) and β-lactoglobulin (β-Lg) contained in WPI denatured after heating and engaged in disulfide bond formation with each other. With an increase in the degree of denaturation and disulfide bond formation, the bonding between β-lactoglobulin and κ-casein was suppressed to decrease the amount of κ-casein–WPI complexes. When β-Lg forms SS bonds with α-La, the number of highly reactive, free SH groups decreases, which complicates the formation of SS bridges between β-Lg and κ-casein. Thus, the denaturation degree of WPI largely determined the degree of κ-casein dissociation from casein micelles and, consequently, the strength of acid milk gels. Adding WP to milk increases the strength of acid milk gel, and it can be controlled by changing the degree of thermal denaturation of the WP. Furthermore, it was clarified for the first time that the dissociation of κ-casein from casein micelles influences this effect. Further studies are needed to elucidate the structural features of κ-casein-dissociated micelles.

Alpha-lactalbumin (α-LA) and β-lactoglobulin (β-LG) are contained in bovine milk whey. Chemical and physical treatments are known to alter the conformation of these proteins and we have previously reported that α-LA denatured with trifluoroethanol (TFE) and isolated from sterilized market milk inhibited the growth of rat crypt IEC-6 cells. In the present study, we aimed to evaluate the effects of TFE-treated α-LA and β-LG on cell growth using cultured intestinal cells and on their safety using a suckling mouse model. First, we investigated the effect of the TFE-treated whey proteins on human colonic Caco-2 cells at various differentiation stages. In the undifferentiated stage, we assessed cell growth by a water-soluble tetrazolium-1 method. The native whey proteins enhanced cell proliferation, whereas the TFE-treated whey proteins strongly inhibited cell growth. We investigated cell barrier function in the post-differentiated stage by measuring transepithelial electrical resistance (TER). Not only native but also the TFE-treated whey proteins increased TER. Next, we evaluated whether the TFE-treated α-LA and β-LG have adverse effects on healthy suckling mice. No mice given by the TFE-treated samples showed any adverse symptoms. We also performed a safety test using a human rotavirus infected gastrointestinal disease suckling mice model. Even the TFE-treated whey proteins appeared to prevent the development of diarrheal symptoms without any adverse effects. Although we cannot know the effect of long-term ingestion of denatured whey proteins, these results suggest that they have no adverse effects on differentiated intestinal cells and digestive tract, at least in short-term ingestion.

The prevalence of type 2 diabetes mellitus (T2DM) is increasing and it is estimated that by 2030 approximately 366 million people will be diagnosed with this condition. The use of dipeptidyl peptidase IV (DPP-IV) inhibitors is an emerging strategy for the treatment of T2DM. DPP-IV is a ubiquitous aminodipeptidase that cleaves incretins such as glucagon like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), resulting in a loss in their insulinotropic activity. Synthetic DPP-IV drug inhibitors are being used to increase the half-life of the active GLP-1 and GIP. Dietary intervention is accepted as a key component in the prevention and management of T2DM. Therefore, identification of natural food protein-derived DPP-IV inhibitors is desirable. Peptides with DPP-IV inhibitory activity have been identified in a variety of food proteins. This review aims to provide an overview of food protein hydrolysates as a source of the DPP-IV inhibitory peptides with particular focus on milk proteins. In addition, the proposed modes of inhibition and structure–activity relationship of peptide inhibitors are discussed. Milk proteins and associated peptides also display insulinotropic activity and help regulate blood glucose in healthy and diabetic subjects. Therefore, milk protein derived peptide inhibitors may be a unique multifunctional peptide approach for the management of T2DM.

The present study examines the resistance of the α-lactalbumin to α-chymotrypsin (EC 3.4.21.1) digestion under various experimental conditions. Whey protein isolate (WPI) was hydrolysed using randomised hydrolysis conditions (5 and 10% of WPI; pH 7·0, 7·8 and 8·5; temperature 25, 37 and 50 °C; enzyme-to-substrate ratio, E/S, of 0·1%, 0·5 and 1%). Reversed-phase high performance liquid chromatography (RP-HPLC) was used to analyse residual proteins. Heat, pH adjustment and two inhibitors (Bowman–Birk inhibitor and trypsin inhibitor from chicken egg white) were used to stop the enzyme reaction. While operating outside of the enzyme optimum it was observed that at pH 8·5 selective hydrolysis of β-lactoglobulin was improved because of a dimer-to-monomer transition while α-la remained relatively resistant. The best conditions for the recovery of native and pure α-la were at 25 °C, pH 8·5, 1% E/S ratio, 5% WPI (w/v) while the enzyme was inhibited using Bowman–Birk inhibitor with around 81% of original α-la in WPI was recovered with no more β-lg. Operating conditions for hydrolysis away from the chymotrypsin optimum conditions offers a great potential for selective WPI hydrolysis, and removal, of β-lg with production of whey protein concentrates containing low or no β-lg and pure native α-la. This method also offers the possibility for production of β-lg-depleted milk products for sensitive populations.

The high nutritional value of whey makes it an interesting substrate for the development of fermented foods. The aim of this work was to evaluate the growth and proteolytic activity of sixty-four strains of lactic acid bacteria in whey to further formulate a starter culture for the development of fermented whey-based beverages. Fermentations were performed at 37°C for 24 h in 10 and 16% (w/v) reconstituted whey powder. Cultivable populations, pH, and proteolytic activity (o-phthaldialdehyde test) were determined at 6 and 24 h incubation. Hydrolysis of whey proteins was analysed by Tricine SDS-PAGE. A principal component analysis (PCA) was applied to evaluate the behaviour of strains. Forty-six percent of the strains grew between 1 and 2 Δlog CFU/ml while 19% grew less than 0·9 Δlog CFU/ml in both reconstituted whey solutions. Regarding the proteolytic activity, most of the lactobacilli released amino acids and small peptides during the first 6 h incubation while streptococci consumed the amino acids initially present in whey to sustain growth. Whey proteins were degraded by the studied strains although to different extents. Special attention was paid to the main allergenic whey protein, β-lactoglobulin, which was degraded the most by Lactobacillus acidophilus CRL 636 and Lb. delbrueckii subsp. bulgaricus CRL 656. The strain variability observed and the PCA applied in this study allowed selecting appropriate strains able to improve the nutritional characteristics (through amino group release and protein degradation) and storage (decrease in pH) of whey.

The effect of copper(II) ions (Cu+2) on the structure of β-lactoglobulin (β-lg) was investigated spectroscopically using UV-visible, fluorescence and circular dichroism (CD) and calorimetrically using isothermal titration calorimetry (ITC), at different temperatures. Results of the UV-visible studies showed that adding Cu+2 to β-lg solution caused increasing turbidity, indicative of protein aggregation. It was noticeable that the rate of increasing turbidity was directly proportional to increasing temperature. The far-UV CD studies displayed that the Cu+2 cannot induce any significant changes in the secondary structures of β-lg at different temperatures. Also, the ITC data indicated that the binding process of Cu+2 to β-lg is mainly entropically driven. The results highlight that copper ions cause the tertiary structure of β-lg to change and induce a slightly open structure leading to the formation of supramolecular aggregates in β-lg which may result in the reduced allergenicity of β-lg and its increased use in industrial applications.

Addition of methylated β-lactoglobulin (Met-BLG) in the medium of MDCK cell lines infected with influenza virus subtype H1N1 reduced hemagglutination activity (HA) in a concentration dependent manner. Antiviral activity of Met-BLG depended on its concentration, viral load, and duration of infection. Using 17 μg/ml of Met-BLG inhibited 50% of HA of H1N1 grown in MDCK cells at 1 MOI after 24 h incubation at 37°C and in 5% CO2. Extension of incubation time enhanced antiviral action since the same concentration of Met-BLG inhibited about 61% of viral activity after 48 h. This viral inhibition was accompanied by a protection of MDCK cells as observed by using neutral red or by direct microscope examination. Reduction of viral RNA replication upon the addition of Met-BLG (50 μg/ml) was observed by real time-PCR showing a reduction of viral log value of about 0·9. When viral stock solution was mixed with 25 μg/ml Met-BLG in absence of cell lines, the morphology and viability of virus particles were significantly affected as observed by electron microscopy, and the number of intact virus particles was reduced by roughly 65%.

The objective of the present study was twofold: first to compare the degradation patterns of caprine whey proteins digested with either human digestive juices (gastric or duodenal) or commercial porcine enzymes (pepsin or pancreatic enzymes) and second to observe the effect of gastric pH on digestion. An in vitro two-step assay was performed at 37°C to simulate digestion in the stomach (pH 2, 4 or 6) and the duodenum (pH 8). The whey proteins were degraded more efficiently by porcine pepsin than by human gastric juice at all pH values. Irrespective of the enzyme source, gastric digestion at pH 2 followed by duodenal digestion resulted in the most efficient degradation. Lactoferrin, serum albumin and the Ig heavy chains were highly degraded with less than 6 % remaining after digestion. About 15, 56 and 50 % Ig light chains, β-lactoglobulin (β-LG) and α-lactalbumin remained intact, respectively, when digested with porcine enzymes compared with 25, 74 and 81 % with human digestive juices. For comparison, purified bovine β-LG was digested and the peptide profiles obtained were compared with those of the caprine β-LG in the digested whey. The bovine β-LG seemed to be more extensively cleaved than the caprine β-LG in the whey. Commercial enzymes appear to digest whey proteins more efficiently compared with human digestive juices when used at similar enzyme activities. This could lead to conflicting results when comparing human in vivo protein digestion with digestion using purified enzymes of non-human species. Consequently the use of human digestive juices might be preferred.

Impact of milk protein composition on casein (CN) retention in curd during the milk coagulation process was studied using a model cheese making system. Individual milk samples from 110 cows in mid lactation of the Swedish Red and Swedish Holstein breeds with known genotypes of β-casein, κ-casein and β-lactoglobulin were defatted, coagulated with chymosin, subjected to syneresis and subsequent pressing simulated by centrifugation. The results indicated that κ-casein concentration of milk plays an important role in the curd formation process and initial syneresis (whey after cutting), whereas an increased CN ratio was associated with less casein in whey after simulated pressing. Increased κ-casein concentration of milk also characterized the milk samples with no measurable loss of casein in whey, compared with milk samples with casein lost in whey, both after cutting and after simulated pressing. Concentrations of αs1-casein, β-casein, and total casein in milk were positively associated with fresh curd yield, which showed a strong correlation with amount of casein retained in curd. No effect of protein genotype on fresh curd yield or casein in whey was found. The β-lactoglobulin BB genotype was associated with increased casein retention in curd, most likely due to the association of this genotype with CN ratio.

The objective was to evaluate the effect of β-lactoglobulin (β-lg) polymorphism and seasonality on milk composition (fat, lactose, total solids, milk urea nitrogen, total protein, true protein, casein and somatic cell counts) of Holstein and Girolando cows. Milk and blood samples from 278 Holsteins cows and 156 Girolando cows were taken during two dry seasons and two rainy seasons, for milk composition analysis and to determine β-lg genotypes, respectively. BB genotype was the most frequent for both breeds, followed by AA genotype for Holstein (BB>AA>AB) and by AB for Girolando cows (BB>AB>AA). No differences were found in milk compositional characteristics among genetic variants of β-lg (AA, AB and BB) either between Holstein or Girolando cows. No association between milk composition and β-lg genetic polymorphism was observed. During the dry season, independently of the breed considered, higher contents of lactose, true protein, casein and casein:true protein ratio were found.

In the study presented in this article, the influence of added α-lactalbumin and β-lactoglobulin on the changes that occur in casein micelles at 250 and 300 MPa were investigated by in-situ measurement of light transmission. Light transmission of a serum protein-free casein micelle suspension initially increased with increasing treatment time, indicating disruption of micelles, but prolonged holding of micelles at high pressure partially reversed HP-induced increases in light transmission, suggesting reformation of micellar particles of colloidal dimensions. The presence of α-la and/or β-lg did not influence the rate and extent of micellar disruption and the rate and extent of reformation of casein particles. These data indicate that reformation of casein particles during prolonged HP treatment occurs as a result of a solvent-mediated association of the micellar fragments. During the final stages of reformation, κ-casein, with or without denatured whey proteins attached, associates on the surface of the reformed particle to provide steric stabilisation.

A study was carried out to determine genetic variants of β-lactoglobulin gene and to explore associations between these and milk composition traits in riverine buffalo. Single strand conformation polymorphism was employed to detect the genetic variants of the gene. Two fragments of this gene i.e. 119 bp of exon I and 400 bp spanning exon IV and intron IV were included in the study. For 119 bp fragment, three alleles namely, A, B and C were observed in all the buffalo breeds whereas four alleles (A, B, C and D) were detected for 400 bp fragment. The frequency distribution of alleles was different in different breeds of buffaloes for both the fragments. For exon I fragment, the milk composition traits such as total SNF, protein, solid, fat and whey protein yield were found to be significantly (P<0·05) associated with genotypes in Murrah and Bhadawari buffalo whereas in Mehsana breed genotypes were significantly (P<0·05) co-related with total SNF, solid and fat yield. Genotypes of 400 bp fragment, only total fat yield in Mehsana buffalo was found to be significantly (P<0·05) associated with genotypes.

The effects of high pressure treatments (100–300 MPa; 15 min; 9 °C or 20 °C) on the distribution of minerals and proteins of raw skim milk (RSM) and of a dispersion of industrial phosphocaseinate (PC) were studied after separation of the micellar and soluble phases by ultracentrifugation (UCF). Whatever the temperature of high pressure treatments, the pressure-induced dissociation of the casein micelles was accompanied by calcium (Ca), phosphorus (P) and casein release from the micelles. The released Ca and P were or became bound to soluble proteins since progressive increases in Ca and P concentrations were observed in the UCF supernatants of RSM and of the PC dispersion but not in the ultrafiltrates from these UCF supernatants (free of soluble proteins). Simultaneously, αS1-, αS2-, β- and κ-caseins were progressively released from the micelles, as seen by electrophoretic analysis. The pressure-induced solubilisation of αS1- and αS2-caseins, essentially located in the core of the micelles, suggests that high pressure destabilised micelles including their internal structure.

This work describes the effect of the hydrolysis time and pressure (0·1–400 MPa) on the proteolysis of β-lactoglobulin A (β-lg A) with trypsin, either conducting hydrolysis of β-lg under pressure or hydrolysing β-lg that was previously pressure treated. Pressurisation, before or during enzyme treatments, enhanced tryptic hydrolysis of β-lg. Trypsin degraded pressure-modified β-lg and pressure-induced β-lg aggregates, favouring proteolysis to the intermediate degradation products: (Val15-Arg40), (Val41-Lys69)S-S(Leu149-Ile162) and (Val41-Lys70)S-S(Leu149-Ile162). These were further cleaved at the later stages of proteolysis to yield: (Val15-Tyr20), (Ser21-Arg40), (Val41-Tyr60), (Trp61-Lys69)S-S(Leu149-Ile162) and (Trp61-Lys70)S-S(Leu149-Ile162). Particularly, in the tryptic hydrolysates of pre-pressurized β-lg, two other fragments linked by disulphide bonds: (Lys101-Arg124)S-S(Leu149-Ile162) and (Tyr102-Arg124)S-S(Leu149-Ile162), were found. These corresponded to rearrangement products induced by SH/SS exchange between the free thiol group of Cys121 and Cys160, that normally forms the disulphide bond Cys66-Cys160. In the light of these results, structural modifications of β-lg under high pressure are discussed.

The properties of commercial directly and indirectly heated UHT milks, both after heating and during storage at room temperature for 24 weeks, were studied. Thermally induced changes were examined by changes in lactulose, furosine and acid-soluble whey proteins. The results confirmed previous reports that directly heated UHT milks suffer less heat damage than indirectly heated milk. During storage, furosine increased and bovine serum albumin in directly heat-treated milks decreased significantly. The changes in lactulose, α-lactalbumin and β-lactoglobulin were not statistically significant. The data suggest that heat treatment indicators should be measured as soon as possible after processing to avoid any misinterpretations of the intensity of the heat treatment.

Heat-induced inactivation of bovine plasmin, denaturation of β-lactoglobulin (β-lg), the interactions between both species and casein micelles and the subsequent net effect on proteolysis of β-casein was studied in a model system consisting of phosphocasein and β-lg in synthetic milk ultrafiltrate. The inactivation of plasmin and denaturation of β-lg were first order reactions, with the rate of inactivation of plasmin being greater than the rate of denaturation of β-lg. The predominant mechanism involved in the denaturation of plasmin in the temperature range 65–80 °C was its interaction with β-lg (kr at 60 °C, 0·0526; Ea, 176 KJ/mol). At the point of complete inactivation of plasmin ~45% of the β-lg remained undenatured. Thermal inactivation of plasmin through other mechanisms was negligible. The association of β-lg with the casein micelles at 60 °C had a rate constant of 3·71×10−5 min−1 and an Ea of 259 KJ/mol; thermal denaturation of β-lg was of much less importance, with a rate constant at 60 °C of the order of 1×10−10 min−1 and an Ea of 250 KJ/mol. On denaturation of all β-lg in the system, a maximum of ~55% was associated with the casein micelles. The effect of heating on the subsequent hydrolysis of β-casein indicated that the level of plasmin activity was the most important factor affecting proteolysis, while the interaction of β-lg with the casein micelles had limited effect. Overall, thermal stability of plasmin in milk is very much dependent upon its interaction with β-lg.

Pressure processing of foodstuff has been applied to produce or modify proteinaceous gel structures. In real pressure processing the treatment is non-isothermal, due to the adiabatic nature of the process and the heat loss from the product to the vessel. In order to estimate the effect of pressurization on milk constituents pressure and temperature dependent kinetics were determined separately from each other. In a detailed kinetic study whey protein isolate was treated under isobaric (200 to 800 MPa) and isothermal conditions (−2 to 70 °C), and the resulting degree of denaturation of β-lactoglobulin A and B and α-lactalbumin was analysed. Kinetic parameters of denaturation were estimated using a one step non-linear regression method which allowed a global fit of the whole data set. The isobaric isothermal denaturation of β-lactoglobulin and α-lactalbumin was found to follow third and second order kinetics, respectively. Isothermal pressure denaturation of both β-lactoglobulin fractions do not differ significantly and were characterized by an activation volume decreasing with increasing temperature from −10 to about −30 ml mol−1, which demonstrates that the denaturation rate is accelerated with increasing temperature. The activation energy of about 70 to 100 kJ mol−1 obtained for β-lactoglobulin A and B is not dependent to a great extent on the pressure which indicates that above 200 MPa denaturation rate is limited by the aggregation rate while pressure forces unfolding of the molecule.

In this study, high pressure (HP)-induced denaturation of α-lactalbumin (α-la) and β-lactoglobulin (β-lg) in dairy systems was examined. In both milk and whey, β-lg was less baroresistant than α-la; both proteins were considerably more resistant to HP-induced denaturation in whey than in milk. HP-induced denaturation of α-la and β-lg increased with increasing proportion of milk in mixtures of milk and whey. Addition of a sulphydryl-oxidising agent, KIO3, to milk or whey increased HP-induced denaturation of β-lg, but reduced the denaturation of α-la. Denaturation of both α-la and β-lg was prevented by adding a sulphydryl-blocking agent, N-ethylmaleimide, to milk or whey prior to HP treatment, highlighting the crucial role of sulphydryl-disulphide interchange reactions in HP-induced denaturation of α-la and β-lg. Removal of colloidal calcium phosphate from milk also reduced HP-induced denaturation of α-la and β-lg significantly. The higher level of HP-induced denaturation of α-la and β-lg in milk than in whey may be the result of the abscence of the casein micelles and colloidal calcium phosphate from whey, which facilitate HP-induced denaturation of α-la and β-lg in milk.