Jeremy Petit

Influence of calcium on β-lactoglobulin denaturation kinetics: Implications in unfolding and aggregation mechanisms

Much research dealing with the processing of milk by-products in heat exchangers has noted the key role of calcium in β-lactoglobulin (β-LG) fouling behavior. Nevertheless, the manner by which Ca affects β-LG denaturation has rarely been quantified using reliable kinetic and thermodynamic data. To this end, the influence of Ca on β-LG denaturation mechanisms in simulated lactoserum concentrates was studied on the laboratory-scale under 100°C by HPLC analysis. The heat-treated solutions were composed of 53.3g/L β-LG and were enriched in Ca at various concentrations (0, 66, 132, and 264 mg/kg). The kinetic parameters (reaction order, activation energy, and frequency factor) associated with β-LG denaturation, along with the unfolding and aggregation thermodynamic parameters were deduced from these experiments and discussed with respect to Ca content. We found that the multistage process characterizing β-LG thermal denaturation is not greatly affected by Ca addition. In fact, the general model subdividing β-LG denaturation mechanisms in 2 steps, namely, unfolding and aggregation, remained valid for all tested Ca concentrations. The change in the predominant mechanism from unfolding to aggregation was observed at 80°C across the entire Ca concentration range. Moreover, the classical 1.5 reaction order value was unaffected by the presence of Ca. Interpretation of the acquired kinetic data showed that Ca addition led to a significant increase in kinetic rate, and more so in the aggregation temperature range. This indicates that Ca principally catalyzes β-LG aggregation, by lowering the Coulombian repulsion between the negatively charged β-LG reactive species, bridging β-LG proteins, or via an ion-specific conformational change. To a lesser extent, Ca favors β-LG unfolding, probably by disturbing the noncovalent binding network of native β-LG. Simultaneously, Ca has a slight protective role on the native and unfolded β-LG species, as shown by the increase in activation energy with Ca concentration. The calculation of thermodynamic parameters related to β-LG denaturation confirmed this observation. A threshold effect in Ca influence was noted in this study: no further significant kinetic rate change was observed above 132 mg/kg of Ca; at this concentration, the studied solution was an almost equimolar mixture of β-LG and Ca. Finally, we simulated the temporal evolution of β-LG species concentrations at diverse Ca contents at 3 holding temperatures. The simulations were based on the acquired kinetic parameters. This permitted us to highlight the greater effect of Ca on β-LG denaturation at high Ca content or for short-time heat treatments at temperatures near 100°C, as in heat exchangers.

Predicting the distribution of whey protein fouling in a plate heat exchanger using the kinetic parameters of the thermal denaturation reaction of β-lactoglobulin and the bulk temperature profiles

Fouling of plate heat exchangers (PHE) is a severe problem in the dairy industry, notably because the relationship between the build-up of protein fouling deposits and the chemical reactions taking place in the fouling solution has not yet been fully elucidated. Experiments were conducted at pilot scale in a corrugated PHE, and fouling deposits were generated using a model β-lactoglobulin (β-LG) fouling solution for which the β-LG thermal denaturation reaction constants had been previously determined experimentally. Then 18 different bulk temperature profiles within the PHE were imposed. Analysis of the fouling runs shows that the dry deposit mass per channel versus the ratio R=kunf/kagg (with kunf and kagg representing, respectively, the unfolding and aggregation rate constants computed from both the identification of the β-LG thermal denaturation process and knowledge of the imposed bulk temperature profile into the PHE channel) is able to gather reasonably well the experimental fouling mass data into a unique master curve. This type of representation of the results clearly shows that the heat-induced reactions (unfolding and aggregation) of the various β-LG molecular species in the bulk fluid are essential to capture the trend of the fouling mass distribution inside a PHE. This investigation also illustrates unambiguously that the release of the unfolded β-LG (also called β-LG molten globule) within the bulk fluid (and the absence of its consumption in the form of aggregates) is a key phenomenon that controls the extent of protein fouling as well as its location inside the PHE.

Toward a better determination of dairy powders surface composition through XPS matrices development

The surface composition of dairy powders prepared by mixing various amounts of micellar casein (MC), whey proteins isolate (WPI), lactose, and anhydrous milk fat (AMF) was investigated by XPS measurements. The use of matrices are generally accepted to transform surface atomic composition (i.e., C, O, N contents) into surface component composition (i.e., lactose, proteins, lipids). These atomic-based matrices were revisited and two new matrices based on the surface bond composition were developed. Surface compositions obtained from atomic and bond-based matrices were compared. A successful matrix allowing good correlations between XPS predicted and theoretical surface composition for powders free from fat was identified. Nevertheless, samples containing milk fat were found to present a possible segregation of components owing to the AMF overrepresentation on the surface. Supplementary analyses (FTIR, SEM) were carried out in order to investigate the homogeneity of the mixtures.

Recent Advances on Lactose Intolerance: Tolerance Thresholds and Currently Available Solutions.

ABSTRACT The genetically programmed reduction in lactase activity during adulthood affects 70% of the world adult population and can cause severe digestive disorders, which are the sign of lactose intolerance. Lactose intolerance symptoms vary depending on the residual lactase activity, the small bowel transit time, and especially the amount of ingested lactose. To formulate dairy products suitable for the vast majority of lactose intolerants, it is essential to define lactose intolerance threshold. A recent meta-analysis permitted to show that almost all lactose intolerants tolerate 12 g of lactose in one intake and approximately 18 g of lactose spread over the day. The prevalence and severity of lactose intolerance are probably overestimated by the general public. This misconception usually leads to an unnecessary reduction of dairy foodstuff consumption. Nevertheless, dairy products are essential for health mainly due to their calcium content and the positive influence of probiotic bacteria. The formulation of dairy products suitable for most intolerant and suspicious subjects seems necessary. The use of exogenous enzyme preparations, as well as the consumption of lactose-free products or products rich in probiotic bacteria are proposed as symptom-reducing strategies.

Impact of Spray-Drying Process Parameters on Dairy Powder Surface Composition and Properties

To design dairy powder properties according to their future use, it is necessary to know how to identify the main process parameters that influence the drying mechanisms and to optimize them on the basis of the desired powder properties. The impacts of atomization process parameters on powder physicochemical properties and surface composition were investigated and correlated. The analytical results permitted us to sort the produced powders into groups according to their dependency to process parameters. This study shows the feasibility of developing a model for the prediction of the physicochemical properties of dairy powders via a reverse engineering approach.

Advances in food powder agglomeration engineering.

Food powders are used in everyday life in many ways and offer technological solutions to the problem of food production. The natural origin of food powders, diversity in their chemical composition, variability of the raw materials, heterogeneity of the native structures, and physicochemical reactivity under hydrothermal stresses contribute to the complexity in their behavior. Food powder agglomeration has recently been considered according to a multiscale approach, which is followed in the chapter layout: (i) at the particle scale, by a presentation of particle properties and surface reactivity in connection with the agglomeration mechanisms, (ii) at the mechanisms scale, by describing the structuration dynamics of agglomerates, (iii) at the process scale, by a presentation of agglomeration technologies and sensors and by studying the stress transmission mode in the powder bed, and finally (iv) by an integration of the acquired knowledge, thanks to a dimensional analysis carried out at each scale.

Granulomorphometry: A suitable tool for identifying hydrophobic and disulfide bonds in β-lactoglobulin aggregates. Application to the study of β-lactoglobulin aggregation mechanism between 70 and 95°C

This work deals with the investigation of β-lactoglobulin (β-LG) aggregation by granulomorphometry. In the first part of this study, we showed that the binding interactions involved in aggregate structure could be identified by their appearance in granulomorphometric pictures. The reliability of this analytical approach was demonstrated by comparing the appearance of β-LG aggregates in the presence and absence of a thiol-blocking agent (N-ethylmaleimide). The translucency of the aggregates was associated with hydrophobic interactions and their opacity was associated with disulfide bonds. We state, based on the morphology of the aggregates, along with the color of protein aggregates and insoluble materials, that hydrophobic interactions had a better water-holding capacity than disulfide bonds. Additionally, our results suggest that disulfide and hydrophobic bonds compete for β-LG aggregate shaping. In the second part of this work, interesting features of granulomorphometry useful for identifying aggregate binding interactions were highlighted to clarify the effect of temperature on the aggregation mechanisms occurring in a β-LG concentrate with a moderate calcium content (6.6mmol·L(-1)). Heat treatment experiments were performed between 70 and 95°C, and granulomorphometric measurements (aggregate size, aggregate number, and gray level of the picture) were conducted at different sampling times up to 4h. Results, which were interpreted in light of calculated β-LG denaturation levels, revealed that the aggregation mechanism could be split into 2 steps. Initially, β-LG denatured quickly, leading to fast β-LG aggregation by disulfide bonds. The denaturation rate then declined, which drastically slowed the disulfide aggregation mechanism. From that point on, a second aggregation path became preponderant. It consisted of the agglomeration of small aggregates by hydrophobic interactions and resulted in the formation of large aggregates containing both interaction types. This second aggregation mechanism was clearly favored at high temperatures because it was not detected in our experiments at temperatures below 85°C.

Characterisation of flow properties of foutou and foufou flours, staple foods in West Africa, using the FT4 powder rheometer

Flowability and fluidisation of plantain-based flours from three varieties (Orishele, Corne 1, and French 2) were assessed using FT4 powder rheometer. Studied powders exhibited good flowing and fluidisation properties with little differences attributable to variety type. Foutou flours constituted by larger particles with a low proportion of fine particles were more compressible than foufou flours. Foufou flour of Orishele had the better flowability and was the most fluidisable because of its high content in large particles of relatively convex and angular shape. Foutou flour of Corne 1, containing more small particles, was slightly less flowing and fluidisable than other flours.

Inline high frequency ultrasonic particle sizer

This paper reports the development of a new method of particle sizing in a liquid. This method uses high frequency focused ultrasounds to detect particles crossing the focal zone of an ultrasonic sensor and to determine their size distribution by processing the reflected echoes. The major advantage of this technique compared to optical sizing methods is its ability to measure the size of particles suspended in an opaque liquid without any dedicated sample preparation. Validations of ultrasonic measurements were achieved on suspensions of polymethyl methacrylate beads in a size range extending from a few micrometer to several hundred micrometer with a temporal resolution of 1 s. The inline detection of aggregate formation was also demonstrated.

High-Throughput Identification of Candidate Strains for Biopreservation by Using Bioluminescent Listeria monocytogenes

This article describes a method for high-throughput competition assays using a bioluminescent strain of L. monocytogenes. This method is based on the use of the luminescent indicator strain L. monocytogenes EGDelux. The luminescence of this strain is correlated to growth, which make it suitable to monitor the growth of L. monocytogenes in mixed cultures. To this aim, luminescence kinetics were converted into a single numerical value, called the Luminescence Disturbance Indicator (LDI), which takes into account growth inhibition phenomena resulting in latency increase, decrease in the luminescence rate, or reduction of the maximum luminescence. The LDI allows to automatically and simultaneously handle multiple competition assays which are required for high-throughput screening (HTS) approaches. The method was applied to screen a collection of 1810 strains isolated from raw cow’s milk in order to identify non-acidifying strains with anti-L. monocytogenes bioprotection properties. This method was also successfully used to identify anti-L. monocytogenes candidates within a collection of Lactococcus piscium, a species where antagonism was previously described as non-diffusible and requiring cell-to-cell contact. In conclusion, bioluminescent L. monocytogenes can be used in HTS to identify strains with anti-L. monocytogenes bioprotection properties, irrespectively of the inhibition mechanism.