Impact of physicochemical changes in milk ultrafiltration permeate concentrated by reverse osmosis on calcium phosphate precipitation.

This study aimed to characterize and compare the mechanisms of calcium phosphate precipitation in skimmed milk ultrafiltration permeate (MP) and MP preconcentrated by reverse osmosis (ROMP). The effects of different physicochemical parameters such as the pH (8.0), the heating time (60 or 120 min at 60 °C) and the seeding of samples with dicalcium phosphate (DCP) were tested. The concentration of salts (K, Ca, Na, Mg, and P) in the freeze-dried precipitates was measured using inductively coupled plasma (ICP). The amount of remaining ionic calcium was also monitored. Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD) analysis were used to characterize the type of calcium phosphate precipitates that formed. The morphological structure of particles was determined using scanning electron microscopy (SEM). The chemical analyses showed that RO increased the rate of precipitated ions, especially Ca and P in MP, while alkalinization to pH 8.0 and heating at 60 °C significantly increased the precipitation of salts, with the calcium phosphate structure changing into complex forms such as hydroxyapatite (HAP) and whitlockite. MP preconcentration by RO paves the way for improving the precipitation yield of milk salts in the form of HAP for Ca fortification in various foods. It offers an original way to valorize the milk salts contained in the high volumes of MP generated by the cheese industry.

Molecular Structure of Lactoferrin Influences the Thermal Resistance of Lactococcal Phages.

The protective effect of whey proteins on phages of lactic acid bacteria during heat treatment limits the recycling of whey proteins into cheese. To investigate this protective effect, we used lactoferrin (LF) as a whey protein model as a result of its unique physicochemical properties and its antiviral activity. First, the thermal inactivation of lactococcal thermoresistant virulent phage P1532 was measured in LF at 95 °C and under different pH values. Phage inactivation results revealed a strong protective effect of LF on P1532 phage at pH 5 but none at pH 7. The structural conformational changes of LF were then monitored by Fourier transform infrared and circular dichroism spectroscopies. Spectroscopic analysis showed that LF was unfolded after heating at pH 7, while it preserved its tertiary and secondary structures when heated at pH 5. There is a direct correlation between the thermal stability of LF and its ability to protect P1532 phage from heat treatment.

Investigation of the protective effect of whey proteins on lactococcal phages during heat treatment at various pH.

The incorporation of whey protein concentrates (WPC) into cheese is a risky process due to the potential contamination with thermo-resistant phages of lactic acid bacteria (LAB). Furthermore, whey proteins can protect phages during heat treatment, thereby increasing the above risk. The main objective of this work was to understand this protective effect in order to better control LAB phages and maximize whey recycling in the cheese industry. First, the inactivation of a previously characterized thermo-resistant lactococcal virulent phage (P1532) was investigated at 95 °C in WPC, in individual whey components ß-lactoglobulin, α-lactalbumin, and bovine serum albumin as well as under different heat and pH conditions. The structural changes of the tested proteins were also monitored by transmission FTIR spectroscopy. Phage inactivation results indicated that the protective effect of whey proteins was pH and time dependent at 95 °C and was not restricted to one component. FTIR spectra suggest that the protection is related to protein molecular structures and to the level of protein aggregates, which was more pronounced in acidic conditions. Moreover, the molecular structure of the three proteins tested was differently influenced by pH and the duration of the heat treatment. This work confirms the protective effect of WPC on phages during heat treatment and offers the first hint to explain such phenomenon. Finding the appropriate treatment of WPC to reduce the phage risk is one of the keys to improving the cheese manufacturing process.

Predictive response surface model for heat-induced rheological changes and aggregation of whey protein concentrate.

Whey proteins are now far more than a by-product of cheese processing. In the last 2 decades, food manufacturers have developed them as ingredients, with the dairy industry remaining as a major user. For many applications, whey proteins are modified (denatured) to alter their structure and functional properties. The objective of this research was to study the influence of 85 to 100 °C, with protein concentration of 8% to 12%, and treatment times of 5 to 30 min, while measuring rheological properties (storage modulus, loss modulus, and complex viscosity) and aggregation (intermolecular beta-sheet formation) in dispersions of whey protein concentrate (WPC). A Box-Behnken Response Surface Methodology modeled the heat denaturation of liquid sweet WPC at 3 variables and 3 levels. The model revealed a very significant fit for viscoelastic properties, and a lesser fit for protein aggregation, at temperatures not previously studied. An exponential increase of rheological parameters was governed by protein concentration and temperature, while a modest linear relationship of aggregation was governed by temperature. Models such as these can serve as valuable guides to the ingredient and dairy industries to develop target products, as whey is a major ingredient in many functional foods.

Improved bioavailability of vitamin D3 using a ß-lactoglobulin-based coagulum.

Vitamin D3 (D3) was encapsulated within a water-soluble matrix, formed by promoting the ßlg/D3 complex by acidification. The capacity of the ßlg-based coagulum to increase the long term stability of D3 in cold storage, upon exposure to intensive UV-light, and in the presence and absence of intestinal proteases, was evaluated. Additionally, the impact of the sequestration of D3 within the matrix of ßlg-based coagulum on its bioavailability was determined in vivo with force-fed rats. The water solubility, long-term storage and UV-light stability of D3 were significantly increased (p < 0.0001) due to the high encapsulation efficiency (94.5 ± 1.8%). The ßlg-based coagulum was not rapidly disrupted by the proteases in the intestines, leading to a slow release of D3, increased uptake of D3 and subsequent enhancement of the bioavailability of D3 in rats.

Self-assembly of ß-lactoglobulin and egg white lysozyme as a potential carrier for nutraceuticals.

Self-assembly structures of ß-lactoglobulin (ßlg) and egg protein lysozyme (Lyso) were developed, using electrostatic interactions between the two oppositely charged proteins. Different ßlg/Lyso concentration ratios were essayed at pH 6.8 to select the optimal ratio for the proteins co-precipitation, which behaviour was then studied at varying pH values. Optimal ßlg/Lyso concentration ratio, prepared at pH 7.5, was selected for protein co-precipitation. As a result, a structure with a mean diameter of 7.1±2.5 µm was formed, as indicated by static light scattering. Furthermore, the SEM images showed that ßlg and Lyso self-assembled to form a microsphere. Vitamin D3, used as a model nutraceutical, was successfully entrapped in the ßlg/Lyso microspheres with an encapsulation efficiency of 90.8±4.8%. Therefore, the ßlg/Lyso microspheres can serve as a potential food-grade vehicle for bioactives in the formulation of food products and pharmaceuticals.

Food protein-based microspheres for increased uptake of vitamin D3.

To protect vitamin D3 during cold storage and exposure to UV-light, vitamin D3 has been entrapped in microspheres formed by bovine protein ß-lactoglobulin (ßlg) and lysozyme (Lyso) from egg white. The behaviour of the ßlg/Lyso microspheres in simulated intestinal fluid and their impact on the kinetic release of D3 were determined. The impact of the D3-loaded ßlg/Lyso microspheres on the bioavailability of D3 was evaluated in vivo by force-feeding rats. The data indicate that the ßlg/Lyso microspheres effectively improved the stability of D3, which was readily released in the intestines. The release kinetics were accelerated in the presence of proteolytic enzymes. The bioavailability of D3 was improved, as confirmed by the significant increase in the serum levels of 25-hydroxy-D3 in rats. The current work demonstrates that water soluble proteins were used to substantially increase the bioavailability of the lipophilic vitamin, and thus can serve in the oral delivery of D3.

Increased stability and protease resistance of the ß-lactoglobulin/vitamin D3 complex.

The stability of the ß-lactoglobulin (ßlg)/vitamin D3 (D3) complex at 4°C and upon exposure to UV-C light, and in simulated intestinal fluid, were studied in vitro. Caco-2 cells were used to demonstrate the passage of the ßlg/D3 complex across the monolayers. Furthermore, an in vivo experiment was conducted by force-feeding rats with the free D3 and ßlg/D3 complex, with subsequent determination of the plasma concentration of 25-hydroxy-D3. The ßlg/D3 complex significantly improved the stability of the vitamin at 4 °C and when exposed to UV-C light. The resistance of ßlg to proteases was increased, indicating a mutual protective effect. The ßlg/D3 complex crossed the monolayers, which was confirmed by the significant increase in the concentration of 25-hydroxy-D3 in rats fed the ßlg/D3 complex compared to the ones fed the free D3. Therefore, the current study suggests that the ßlg/D3 complex can effectively be used for the fortification of milk products and low-fat content foods to improve the intake and bioavailability of D3.

Kinetics of the breakdown of cross-linked soy protein films for drug delivery.

The aim of the present work was to investigate the potential of soy protein isolate (SPI) films as controlled release systems for active compounds. Mechanical properties, dissolution and compound release kinetics of SPI films prepared with different concentrations of formaldehyde were measured over time in the absence or presence of digestive enzymes at gastric or intestinal pH. The effect of formaldehyde on tensile strength, elastic modulus, % elongation and swelling suggested that increasing its concentration increased film cross-linking density. Film bulk erosion in the presence of digestive enzymes followed first-order kinetics. Methylene blue or rifampicin release followed variable kinetics depending on compound solubility during a 1-2h initial phase, followed by zero-order release. Cross-linking density appears to provide effective means of regulating the erosion and release rate of SPI films. SPI film networks displayed excellent compound binding capacity, especially for hydrophobic molecules, and hence potential for use in controlled release systems based on matrix erosion.

Tryptophan-mediated denaturation of beta-lactoglobulin A by UV irradiation.

beta-Lactoglobulin A, a genetic variant of one of the main whey proteins, was irradiated at 295 nm for 24 h. After irradiation, 18% of the protein was denatured (determined by reverse-phase chromatography). The fluorescence spectrum of the irradiated protein was red-shifted compared to that of the native protein, indicating a change in protein folding. Sulfhydryl groups, which are buried in native beta-lactoglobulin, were exposed following irradiation and became available for quantification using the Ellman assay. The quantity of exposed sulfhydryls increased, but the number of total sulfhydryl groups decreased. Gel permeation chromatography showed that some protein aggregation occurred during irradiation. Fourier transform infrared (FTIR) spectroscopy of irradiated beta-lactoglobulin revealed changes in the secondary structure, comparable to that of early events during heat-induced denaturation. There was evidence for some photo-oxidation of tryptophan.

Use of whey protein beads as a new carrier system for recombinant yeasts in human digestive tract.

A new immobilizing protocol using whey protein isolates was developed to entrap recombinant Saccharomyces cerevisiae. The model yeast strain expresses the heterologous P45073A1 that converts trans-cinnamic acid into p-coumaric acid. Beads resulted from a cold-induced gelation of a whey protein solution (10%) containing yeasts (7.5 x 10(7)cells ml(-1)) into 0.1M CaCl(2). The viability and growth capability of yeasts were not altered by our entrapment process. The release and activity of immobilized yeasts were studied in simulated human gastric conditions. During the first 60 min of digestion, 2.2+/-0.9% (n=3) of initial entrapped yeasts were recovered in the gastric medium suggesting that beads should cross the gastric barrier in human. The P45073A1 activity of entrapped yeasts remained significantly higher (p<0.05) than that of free ones throughout digestion (trans-cinnamic acid conversion rate of 63.4+/-1.6% versus 51.5+/-1.8% (n=3) at 120 min). The protein matrix seemed to create a microenvironment favoring the activity of yeasts in the stringent gastric conditions. These results open up new opportunities for the development of drug delivery system using recombinant yeasts entrapped in whey protein beads. The main potential medical applications include biodetoxication or the correction of digestive enzyme deficiencies.

Characterization and evaluation of whey protein-based biofilms as substrates for in vitro cell cultures.

Whey proteins-based biofilms were prepared using different plasticizers in order to obtain a biomaterial for the human keratinocytes and fibroblasts in vitro culture. The film properties were evaluated by Fourier Transform Infrared Spectroscopy (FTIR) technique and mechanical tests. A relationship was found between the decrease of intermolecular hydrogen bond strength and film mechanical behavior changes, expressed by a breaking stress and Young modulus values diminishing. These results allow stating that the film molecular configuration could induce dissimilarities in its mechanical properties. The films toxicity was assessed by evaluating the cutaneous cells adherence, growth, proliferation and structural stratification. Microscopic observation demonstrated that both keratinocytes and fibroblasts adhered to the biofilms. The trypan blue exclusion test showed that keratinocytes grew at a significantly high rate on all the biofilms. Structural analysis demonstrated that keratinocytes stratified when cultured on the whey protein-based biofilms and gave rise to multi-layered epidermal structures. The most organized epidermis was obtained with whey protein isolate/DEG biofilm. This structure had a well-organized basal layer under supra-basal and corneous layers. This study demonstrated that whey proteins, an inexpensive renewable resource which can be obtained readily, were non-toxic to cutaneous cells and thus they could be useful substrates for a variety of biomedical applications, including tissue engineering.

Iron availability from whey protein hydrogels: an in vitro study.

The influence of whey protein hydrogel microstructure, filamentous versus particulate, on iron delivery was studied under different conditions, including simulated gastrointestinal conditions. Experiments were initially conducted to determine the impact of pH and enzymes on iron release. The results show that different iron release profiles can be obtained from filamentous and particulate gels. Particulate gels released more iron than filamentous gels at acidic pH, but the opposite was observed at alkaline pH. In the presence of pepsin at pH 1.2 or pancreatin at pH 7.5, both gel types showed increased protein hydrolysis, but only filamentous gels showed increased iron release, suggesting that matrix structure plays an important role in iron delivery. A dissolution test was carried out under gastrointestinal conditions to mimic the in vivo dissolution process. Filamentous gel released most of its iron during the intestinal phase of a simulated digestion, hence protecting iron during its transit in the gastric zone. Absorption of iron by the Caco-2 system, used to estimate intestinal absorption, revealed that filamentous gels favored intracellular iron absorption. These results suggest that filamentous gels show promise as matrices for transporting iron and promoting its absorption and therefore should be of major interest in the development of innovative functional foods.

Molecular mechanisms of Fe2+-induced beta-lactoglobulin cold gelation.

To get more insight into the mechanisms of cold gelation of beta-lactoglobulin (beta-lg), macroscopic and molecular structural changes during Fe(2+)-induced gelation of beta-lg were investigated using Fourier transform-infrared (FTIR) spectroscopy and rheological methods. The FTIR spectroscopy results show that, upon the preheating treatment (first step of gel process), native globular proteins are denatured and aggregated molecules are found in solution. The spectra are similar to those of gels obtained in the second step of the process upon incorporation of Fe, which suggests that aggregated molecules formed during the preheating treatment constitute the structural basis of the aggregation. However, the rheological data show that the aggregation is achieved via two molecular mechanisms, both of which are modulated by the iron concentration. At 30 mM of iron, gel formation is essentially controlled by van der Waals interactions, while at 10 mM of iron, hydrophobic interactions predominate. At the two concentrations, disulfide bonds contribute to gel consolidation, the effect being more pronounced at 10 mM of iron. These mechanisms lead to the formation of gels of different microstructures. At the highest iron concentration, a strong and rapid decrease in the repulsion forces is produced, resulting in random aggregation. At the lowest iron concentration, the iron diminishes the superficial charge of both molecules and aggregated molecules, facilitating the interaction among hydrophobic regions and leading to the growth of the aggregation in the preferential direction and to filamentous gel formation. This study provides a comprehensive view of the different modes of gelation.

Hydration properties of soybean protein isolates

Estudaram-se as propriedades de hidrataçäo de isolados de soja com diferentes condiçöes de processamento (trratamentos térmicos, pH e concentraçäo de proteínas). Para diferentes amostras determinaram-se e correlacionaram-se o grau de desnaturalizaçäo, a solubilidade em água, em 0,2mol/L NaCl e em diferentes concentraçöes de dodecil sulfato de sódio, viscosidade e capacidade de absorçäo de água. Os tratamentos a temperaturas superiores aos 80§C desnaturaram as fracçöes 11S e 7S, provocando a exposiçäo de gruipos hidrofóbicos os que produziram agregados insolúveis, em água como em soluçäo com alta força ionica. Estes isolados possuíam alta capacidade de absorçäo de água e dispersöes com alta viscosidade. Achou-se uma correlaçäo significativa entre as propriedades de hidrataçäo e o coeficiente m, calculado através da funçäo de potência que relaciona a viscosidade com a concentraçäo proteica da dispersäo. Este coeficiente m também correlacionou com a entalpia dos isolados. Sobre a base destes resultados poderia-se sugerir que o coeficiente m dependente do comportamento hidrodinâmico das partículas foi um bom estimador do grau de desnaturaçäo proteica