Influence of Cross-linked Waxy Maize Starch on the Aggregation Behavior of Casein Micelles During Acid-induced Gelation

The influence of cross-linked waxy maize starch on the aggregation behavior of casein micelles was investigated using a combination of physico-chemical techniques. Milk was homogenized at two different temperatures (55 and 65 °C) and then heated at 95 °C for 5 min in a pilot scale system. The possible interactions between modified starch and milk proteins during lactic acid fermentation were evaluated. While 1% starch did not show differences in the whey protein complexes formed during heating compared to milk with no starch (as measured by size exclusion chromatography), a higher (2.5%) concentration of starch clearly showed an increased amount of heat-induced whey protein aggregates. The gelation pH also increased significantly with 2.5% starch compared to that of the control samples. The storage modulus (G′) increased with increasing levels of starch, and confocal microscopy confirmed that the microstructure of the casein gels was altered by the presence of modified starch. Milk-starch mixtures preheated and homogenized at 55 or 65 °C exhibited similar physico-chemical behavior during acidification. The results suggested a lack of interaction between starch granules and casein micelles during acidification, and scanning electron microscopy images collected with a self-assembled monolayer technique also confirmed that starch granules were not attached to milk caseins but only embedded in the protein gel matrix.     

Phase Separation Behavior of Caseins in Milk Containing Flaxseed Gum and κ-Carrageenan: A Light-Scattering and Ultrasonic Spectroscopy Study

The physico-chemical properties of skim milk containing κ-carrageenan (in the concentration range 0–0.06% w/v), flaxseed gum (in the concentration range 0–0.40% w/v), or a mixture of both polysaccharides were studied using dynamic light scattering, under diluted conditions, as well as in situ, undiluted, using diffusing wave spectroscopy (DWS) and ultrasonic spectroscopy. Flaxseed gum causes phase separation in milk mixtures, because of thermodynamic incompatibility between the casein micelles and the polysaccharide chains. Confocal microscopy and ultrasonic spectroscopy showed that while the addition of 0.01% κ-carrageenan was not sufficient to hinder phase separation, when 0.03% was added, the helix–helix interactions between κ-carrageenan molecules were sufficient to form a network and stabilize the system. DWS clearly demonstrated that clusters of casein micelles still form even at very low concentrations of polysaccharides (below the visible phase separation threshold) and that κ-carrageenan hinders visible phase separation by decreasing the mobility of the casein micelles.     

Effect of Soy Protein Subunit Composition on the Rheological Properties of Soymilk during Acidification

The effect of soy protein subunit composition on the acid-induced aggregation of soymilk was investigated by preparing soymilk from different soybean lines lacking specific glycinin and β-conglycinin subunits. Acid gelation was induced by glucono-δ-lactone (GDL) and analysis was done using diffusing wave spectroscopy and rheology. Aggregation occurred near pH 5.8 and the increase in radius corresponded to an increase in the elastic modulus measured by small deformation rheology. Diffusing wave spectroscopy was also employed to follow acid gelation, and data indicated that particle interactions start to occur at a higher pH than the pH of onset of gelation (corresponding to the start of the rapid increase in elastic modulus). The protein subunit composition significantly affected the development of structure during acidification. The onset of aggregation occurred at a higher pH for soymilk samples containing group IIb (the acidic subunit A₃) of glycinin, than for samples prepared from Harovinton (a commercial variety containing all subunits) or from genotypes null in glycinin. The gels made from lines containing group I (A₁, A₂) and group IIb (A₃) of glycinin resulted in stiffer acid gels compared to the lines containing only β-conglycinin. These results confirmed that the ratio of glycinin/β-conglycinin has a significant effect on gel structure, with an increase in glycinin causing an increase in gel stiffness. The type of glycinin subunits also affected the aggregation behavior of soymilk.     

The Effect of Shear Rate on the Molecular Mass Distribution of Heat-Induced Aggregates of Mixtures Containing Whey Proteins and κ-Carrageenan

The present study attempts to characterize the effect of shear rate on the composition, size, and molecular weight of the protein aggregates present in the upper layer after phase separation of 5% whey protein isolate (WPI) mixed with 0.5% κ-carrageenan (κ-car) at pH 7.0. The mixtures were heated and sheared under different shearing rates. Size exclusion chromatography (SEC), dynamic light scattering, and static light scattering were employed to describe the effect of shear rate on the size and molecular mass of WPI aggregates. At the molecular level, the size of the aggregates increased with an increase in shear rate. Shear rate also caused a decrease in turbidity of the upper layer after centrifugation. SEC combined with multi-angle laser light scattering showed that the WPI aggregates molecular mass was between 10⁶and 10⁷ g/mol when the shear rate increased from 3.6 to 86.4 s⁻¹. Two empirical models described well the effect of shear rate on the size of WPI aggregates, and both models gave comparable results. By varying process parameters such as flow behavior and temperature, it is possible to control WPI aggregation and, thus, obtain aggregates with a range of different characteristics (size).     

Characterization of beta-lactoglobulin A gelation in the presence of sodium caprate by ultrasound spectroscopy and electron microscopy

The effect of sodium caprate (a fatty acid salt) on the formation of beta-lactoglobulin A gels was studied at constant temperature (30 or 35 degrees C) using ultrasonic spectroscopy. During incubation at these temperatures, ultrasonic attenuation increased with the addition of sodium caprate, and reached a plateau after 5-7 h of incubation. Comparing beta-lactoglobulin A with and without sodium caprate, a decrease in net ultrasonic velocity was observed. These results suggested that aggregation occurred during incubation with sodium caprate, and the sample showed an increase in compressibility. Transmission electron microscopy with negative staining showed the formation of filamentous aggregates of beta-lactoglobulin A at around 3-4.5 h of incubation with sodium caprate. These results demonstrated that sodium caprate induced the formation of structures with unique gel properties compared to those formed by heating beta-lactoglobulin in the presence of NaCl alone.     

Diffusing Wave and Ultrasonic Spectroscopy of Rennet-Induced Gelation of Milk in the Presence of High-Methoxyl Pectin

Rennet-induced aggregation was studied in milk systems containing high-methoxyl pectin (HMP) using ultrasonic and diffusing wave spectroscopy. These two techniques allow for in situ measurements of sol–gel transitions without the need for dilution. At low HMP concentrations, the casein micelles aggregation behavior was similar to that of skim milk, although changes could be noted in the microstructure of the renneted gels. At HMP concentrations between 0.1 and 0.15%, phase-separation kinetics were slower than the rennet-induced aggregation, and different microstructures formed caused by different dynamics of interactions between casein micelles present in HMP-depleted flocs. Higher amounts of HMP failed to create a continuous gel, as phase separation occurred at a faster rate than rennet aggregation. These results highlight the importance of non-invasive techniques in the study of concentration-dependent phase separating and aggregating systems, as only with observations in situ is it possible to determine new ways to control the structuring of protein–polysaccharide mixed systems.     

Varietal differences of carbohydrates in defatted soybean flour and soy protein isolate by-products

Soy protein isolates (SPI) were prepared from 12 soybean lines grown in Harrow, Ontario and by-products (fibers and wheys) from SPI making were saved. The identification and quantification of soluble sugars in defatted flours, fibers and wheys were carried out using high-performance anion-exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD) and with a colorimetric method for uronic acids. Defatted flours and fibers were acid hydrolyzed, then analyzed by HPAEC-PAD for monosaccharide composition. The results showed varietal differences in the carbohydrate composition suggesting different applications for these defatted flours and their SPI by-products.     

Downregulation of Salmonella Virulence Gene Expression During Invasion of Epithelial Cells Treated with Lactococcus lactis subsp. cremoris JFR1 Requires OppA

Invasion of Salmonella into host intestinal epithelial cells requires the expression of virulence genes. In this study, cell culture models of human intestinal cells (mucus-producing HT29-MTX cells, absorptive Caco-2 cells, and combined cocultures of the two) were used to determine the effects of Lactococcus lactis subsp. cremoris treatments (exopolysaccharide producing and nonproducing strains) on the virulence gene expression of Salmonella Typhimurium and its mutant lacking the oligopeptide permease subunit A (ΔoppA). During the course of epithelial cell (HT29-MTX, Caco-2, and combined) infection by Salmonella Typhimurium DT104, improved barrier function was reflected by increased transepithelial electrical resistance in cells treated with both strains of L. lactis subsp. cremoris. In addition, virulence gene expression was downregulated, accompanied with lower numbers of invasive bacteria into epithelial cells in the presence of L. lactis subsp. cremoris treatments. Similarly, virulence gene expression of Salmonella was also suppressed when coincubated with overnight cultures of both L. lactis subsp. cremoris strains in the absence of epithelial cells. However, in medium or in the presence of cell cultures, Salmonella lacking the OppA permease function remained virulent. HT29-MTX cells and combined cultures stimulated by Salmonella Typhimurium DT104 showed significantly lower secretion levels of pro-inflammatory cytokine IL-8 after treatment with L. lactis subsp. cremoris cell suspensions. Contrarily, these responses were not observed during infection with S. Typhimurium ΔoppA. Both the exopolysaccharide producing and nonproducing strains of L. lactis subsp. cremoris JFR1 exhibited an antivirulence effect against S. Typhimurium DT104 although no significant difference between the two strains was observed. Our results show that an intact peptide transporter is essential for the suppression of Salmonella virulence genes which leads to the protection of the barrier function in the cell culture models studied.

     

Changes in Colloidal Properties of Oil in Water Emulsions Stabilized with Sodium Caseinate Observed by Acoustic and Electroacoustic Spectroscopy

Sodium caseinate is a commonly used emulsifier in foods, as it adsorbs on the surface of oil droplets and stabilizes them via electrostatic and steric stabilization, forming a polyelectrolyte layer at the interface. Since the protein interface is affected by varying environmental conditions such as pH, ionic strength, concentration of unadsorbed polymers, these emulsions are prone to a variety of destabilization mechanisms. The objective of the present work was to observe the destabilization of sodium caseinate stabilized oil in water emulsions using electroacoustic spectroscopy. This technique can be utilized for the characterization of concentrated colloidal systems in situ, without dilution. The electroacoustic and ultrasonic properties of soy oil in water emulsions were determined for sodium caseinate stabilized emulsions under conditions known to cause destabilization. Ultrasonic attenuation and electrophoretic mobility (ζ-potential) could clearly follow the changes occurring in the emulsion droplets, under minimal sample disruption. This is critical for these systems in a very fragile, metastable state. The emulsions were stable to the addition of high methoxyl pectin (HMP) up to 0.1% HMP. Addition of free sodium caseinate induced depletion flocculation, causing a decrease in the attenuation and electrophoretic mobility measured. The presence of HMP limited depletion interactions. Acidification of the emulsion droplets resulted in a clear sol–gel transition, as shown by a steep increase in the particle size and a decrease in attenuation. Again, destabilization was limited by HMP addition. It was concluded that ultrasonics and electroacoustics are suitable techniques to understand the details of the destabilization processes occurring to food emulsions, measured in situ.     

Real-Time Determination of Structural Changes of Sodium Caseinate-Stabilized Emulsions Containing Pectin Using High Resolution Ultrasonic Spectroscopy

The interactions that lead to structure transitions in oil-in-water emulsions were investigated using high-resolution ultrasonic spectroscopy. High methoxyl pectin (HMP) was added to the emulsions at various concentrations and the dynamics of aggregation induced by changes in pH were observed. Two independent ultrasonic parameters, velocity and attenuation, were measured as a function of time or pH. At pH 6.8, both velocity and attenuation of sound changed as a function of HMP concentration. During acidification, caused by the addition of glucono-δ-lactone, there were small changes in the overall ultrasonic velocity, but it was possible to relate these changes to the structural changes in the emulsion. The values of ultrasonic attenuation decreased at high pH with increasing amount of HMP, indicating changes in the flocculation state of the oil droplets caused by depletion forces. During acidification at pH 5.4, emulsions containing HMP showed a steep increase in the ultrasonic attenuation, and this pH corresponds to the pH of association of HMP with the casein-covered oil droplets. The adsorption of HMP onto the interface causes a rearrangement of the oil droplets, and the emulsions containing sufficient amounts of HMP no longer gel at acid pH. This is well described by the ultrasonic attenuation changes in the various emulsions. This research demonstrated for the first time that ultrasonic spectroscopy can be employed for in situ monitoring and analysis of acid-induced destabilization of food emulsions.