Impact of two thermal processing routes on protein interactions and acid gelation properties of casein micelle-pea protein mixture compared to casein micelle-whey protein one.

The influence of two heating protocols (protocol 1 and 2) on protein interactions and acid-induced gelation properties of casein micelle-pea protein mixture (CM-PP) was investigated and then compared to casein micelle-whey protein mixture (CM-WP). The CM:PP and CM:WP protein weight ratio for mixtures was 7.5:2.5, for a total protein content of 4% (pH 6.7). Protocol 1 consisted of a heat treatment (85 °C for 1 h) of CM-PP and CM-WP mixtures, respectively. Regarding protocol 2, casein micelle, pea protein and whey protein stock dispersions were individually pretreated by heating (85 °C for 1 h) before the mixtures were made and heated in the same conditions of protocol 1 (85 °C for 1 h). Heat pretreatment carried out in the protocol 2 significantly increased PP hydrophobicity and reinforced weak interactions of the initial pea protein particles. This pretreatment on protein stock dispersions led to twofold smaller pea protein particles compared to whey protein aggregates. The hydrophobic interactions between pea proteins and casein micelles promoted by the two heating protocols have greatly contributed to improve acid gelation functionalities of CM. Regardless of the heating protocol, acid-induced gelation of the CM-PP mixtures led to the formation of gel networks with a significant increase in stiffness and firmness compared to casein micelle or CM-WP mixtures gels.

Effect of pH change on size and nanomechanical behavior of whey protein microgels.

Microgels specific structural and functional features are attracting high research interest in several applications such as bioactives and drug delivery or functional food ingredients. Whey protein microgels (WPM) are obtained by heat treatment of whey protein isolate (WPI) in order to promote intramolecular cross-linking. In the present work, atomic force microscopy (AFM) was used in contact mode and in liquid to investigate WPM particles topography and mechanical properties at the nanoscale at native pH (6.5) and acid pH (5.5 and 3.0). Prior to AFM, WPM particles were captured on a gold substrate via low energy interactions by means of specific monoclonal antibodies. AFM images clearly showed an increase in the size of WPM particles induced by pH decrease. AFM in force spectroscopy mode was employed to monitor the elasticity of WPMs. The obtained effective Young's modulus data showed a significant increase in stiffness at pH 5.5 and pH 3.0, over 15-fold compared to native pH. These findings indicate that the mechanical profile of the WPM network varied with the pH decrease. The WPM topographic and nanomechanical changes induced by acidification were most likely due to substantial changes in the shape and inner structure of WPM particles. Our results suggest that internally cross-linked structures, modified by acidification could display interesting functional properties when used as a food ingredient.

Binding analysis between monomeric ß-casein and hydrophobic bioactive compounds investigated by surface plasmon resonance and fluorescence spectroscopy.

ß-Casein, a phosphoprotein representing 37% of the bovine milk caseins, has specific features promoting its application as a nanocarrier for hydrophobic bioactives. In this study, the interactions of ß-casein with curcumin and vitamin D3 under the same physico-chemical conditions were investigated. The interaction kinetics have been studied by surface plasmon resonance (SPR) and fluorescence spectroscopy. The KD value for curcumin-ß-casein interaction has been successfully evaluated (4.1 ±â€¯0.7 × 10-4 M) using SPR by fitting data to a 1:1 Langmuir interaction model. Conversely, the SPR responses obtained for vitamin D3 show that the interactions between this hydrophobic compound and the ß-casein immobilized on the sensor chip were below the sensitivity of the SPR apparatus. Moreover, the fluorescence quenching data show that curcumin has higher affinity to ß-casein (KA = 23.5 ±â€¯1.9 × 104 M-1) than vitamin D3 (KA = 5.8 ±â€¯1.1 × 104 M-1).

Potentialities and Limits of Some Non-thermal Technologies to Improve Sustainability of Food Processing.

In the whole food production chain, from the farm to the fork, food manufacturing steps have a large environmental impact. Despite significant efforts made to optimize heat recovery or water consumption, conventional food processing remains poorly efficient in terms of energy requirements and waste management. Therefore, in the few last decades, much research has focused on the development of alternative non-thermal technologies. Some of them, such as membrane separation processes, hydrostatic or dynamic high pressure, dense phase or high-pressure carbon dioxide, and pulsed electric fields (PEFs) have been extensively studied for cold pasteurization, concentration, extraction, or food functionalization. However, it is still difficult to evaluate the actual advantages or limits of these innovative processing technologies to replace conventional processes. Thus, the overall aim of this paper is to present an overview of the most relevant studies dealing with the potentialities and limits of these non-thermal technologies to improve sustainability of food processing. After a brief presentation of the physical principles of these technologies, the paper illustrates how these technologies could play a decisive role for sustainable food preservation or valorization of raw materials and by-products.

The foaming properties of camel and bovine whey: The impact of pH and heat treatment.

The effect of heat treatment (70°C or 90°C for 30min) on the foaming and interfacial properties of acid and sweet whey obtained from bovine and camel fresh milk was examined. The maximum foamability and foam stability were observed for acid whey when compared to sweet whey for both milks, with higher values for the camel whey. This behavior for acid whey was explained by the proximity of the pI of whey protein (4.9-5.2), where proteins were found to carry the lowest negative charge as confirmed by the zeta potential measurements. Interfacial properties of acid camel whey and acid bovine whey were preserved at air water interface even after a heat treatment at 90°C. These results confirmed the pronounced foaming and interfacial properties of acid camel whey, even if acid and sweet bovine whey exhibited the highest viscoelastic modulus after heating.

Performances of different protocols for exocellular polysaccharides extraction from milk acid gels: Application to yogurt.

Dextran or xanthan were used as model exocellular polysaccharides (EPS) to compare the extraction efficiency of EPS from skim milk acid gels using three different protocols. Extraction yields, residual protein concentrations and the macromolecular properties of extracted EPS were determined. For both model EPS, the highest extraction yield (∼80%) was obtained when samples were heated in acidic conditions at the first step of extraction (Protocol 1). Protocols that contained steps of acid/ethanol precipitation without heating (Protocols 2 and 3) show lower extraction yields (∼55%) but allow a better preservation of the EPS macromolecular properties. Changing the pH of acid gels up to 7 before extraction (Protocol 3) improved the extraction yield of anionic EPS without effect on the macromolecular properties of EPS. Protocol 1 was then applied for the quantification of EPS produced during the yogurt fermentation, while Protocol 3 was dedicated to their macromolecular characterization.

The effect of pH and heat treatments on the foaming properties of purified α-lactalbumin from camel milk.

The effect of pH (4.3 or 6.5) and heat treatment (70°C or 90°C for 30min) on the foaming and interfacial properties of α-lactalbumin extracted from camel milk were studied. The increased temperature treatment changed the foaming properties of camel α-lactalbumin solution and its ability to unfold at the air-water interface. At neutral pH, heat treatment was found to improve foamability, whereas at acid pH (4.3) this property decreased. Foams were more stable after a heat treatment at pH 4.3 than at 6.5, due to higher levels of protein aggregation at low pH. Heat treatment at 90°C for 30min affected the physicochemical properties of the camel α-lactalbumin by increasing free thiol group concentration at pH 6.5. Heat treatment also caused changes in α-lactalbumin's surface charge. These results also confirm the pronounced aggregation of heated camel α-lactalbumin solution at acid pH.

Atomic Force Microscopy Study of the Topography and Nanomechanics of Casein Micelles Captured by an Antibody.

Casein micelles (CMs) are colloidal phospho-protein-mineral complexes naturally present in milk. This study used atomic force microscopy (AFM) in a liquid environment to evaluate the topography and nanomechanics of single native CMs immobilized by a novel capture method. The proposed immobilization method involves weak interactions with the antiphospho-Ser/Thr/Tyr monoclonal antibody covalently bound to a carboxylic acid self-assembled monolayer (SAM) on a gold surface. This capture strategy was compared to the commonly used covalent immobilization method of CMs via carbodiimide chemistry. With this conventional method, CMs remained mainly mobile during AFM measurements in liquid, disturbing the evaluation of their average size and elastic properties. Conversely, when captured by the specific antibody, they were successfully immobilized and their integrity was preserved during the AFM measurement. The characterization of both CM topography and elastic properties was carried out in a liquid ionic environment at native pH 6.6. The CMs' capture efficiency via antibody was concurrently proved by surface plasmon resonance. The calculation of casein micelles' width, height, and contact angle was carried out from the recorded 2D AFM images. CMs were characterized by a mean width of 148 ± 8 nm and a mean height of 42 ± 1 nm. Weak forces were applied to single captured CMs. The obtained force versus indentation curves were fitted using the Hertz model in order to evaluate their elastic properties. The elasticity distribution of native CMs exhibited a unimodal trend with a peak centered at 269 ± 14 kPa.

Foaming and adsorption behavior of bovine and camel proteins mixed layers at the air/water interface.

The aim of this work was to examine foaming and interfacial behavior of three milk protein mixtures, bovine α-lactalbumin-ß-casein (M1), camel α-lactalbumin-ß-casein (M2) and ß-lactoglobulin-ß-casein (M3), alone and in binary mixtures, at the air/water interface in order to better understand the foaming properties of bovine and camel milks. Different mixture ratios (100:0; 75:25; 50:50; 25:75; 0:100) were used during foaming tests and interfacial protein interactions were studied with a pendant drop tensiometer. Experimental results evidenced that the greatest foam was obtained with a higher ß-casein amount in all camel and bovine mixtures. Good correlation was observed with the adsorption and the interfacial rheological properties of camel and bovine protein mixtures. The proteins adsorbed layers are mainly affected by the presence of ß-casein molecules, which are probably the most abundant protein at interface and the most efficient in reducing the interfacial properties. In contrast of, the globular proteins, α-lactalbumin and ß-lactoglobulin that are involved in the protein layer composition, but could not compact well at the interface to ensure foams creation and stabilization because of their rigid molecular structure.

Heat treatment effect on polyphosphate chain length in aqueous and calcium solutions.

Polyphosphate blends are used in food such as meat and dairy products to improve their texture and stability by sequestering metal ions. This study aims to analyse the impact of high temperature treatments on the composition of polyphosphates with regards to phosphate chain length in aqueous solutions with or without calcium. Temperature treatments of 120°C for 10 min led to the hydrolytic degradation of long-chain phosphates into orthophosphate and trimetaphosphate whereas heating the salts to 100°C in aqueous solutions had little effect on their composition. The presence of calcium increased the hydrolysis rate of long-chain phosphates and led to more trimetaphosphate and pyrophosphate as end products. The evolution of emulsifying salts' composition under heat treatment may lead to a modification of their chelating properties since short-chain phosphates are less efficient to chelate calcium than long-chain phosphates.

Topography of the casein micelle surface by surface plasmon resonance (SPR) using a selection of specific monoclonal antibodies.

Several theoretical models of the casein micelle structure have been proposed in the past, but the exact organization of the four individual caseins (α(s1), α(s2), ß, and κ) within this supramolecular structure remains unknown. The present study aims at determining the topography of the casein micelle surface by following the interaction between 44 monoclonal antibodies specific for different epitopes of α(s1)-, α(s2)-, ß-, and κ-casein and the casein micelle in real time and no labeling using a surface plasmon resonance (SPR)-based biosensor. Although the four individual caseins were found to be accessible for antibody binding, data confirmed that the C-terminal extremity of κ-casein was highly accessible and located at the periphery of the structure. When casein micelles were submitted to proteolysis, the C-terminal extremity of κ-casein was rapidly hydrolyzed. Disintegration of the micellar structure resulted in an increased access for antibodies to hydrophobic areas of α(s1)- and α(s2)-casein.

Epitope characterization of a supramolecular protein assembly with a collection of monoclonal antibodies: the case of casein micelle.

In milk, kappa-, beta-, alphas(1)- and alphas(2)-casein (CN) are associated into a supramolecular assembly, the micelle. In this work, CN micelles contained in fresh skim milk were used to produce over 100 monoclonal antibodies. The specificity of these probes was determined using libraries of synthetic peptides and peptides fractionated from tryptic hydrolysis of purified CNs. Although kappa-CN and alphas(2)-CN are minor proteins in the micelle (ratio 1:1:4:4 for kappa, alphas(2), alphas(1), beta) a proportionally high number of clones were produced towards these two proteins (32 for each), compared to 9 and 29 for alphas(1)-CN and beta-CN, respectively. Most of the beta-CN and kappa-CN epitopes were identified, while about 50% of alphas(1)-CN and alphas(2)-CN antibodies were suspected to react to conformational linear or discontinuous epitopes, since no peptide binding could be identified. Antibody binding to the phosphoserine rich regions of the three calcium sensitive CNs was weak or non-existing, suggesting them to be hidden in the micelle structure together with alphas(1)-CN. The C-terminal glycomacropeptide of kappa-CN and the C-terminal moiety of beta-CN were well exposed generating the majority of the antibodies specific for these two proteins. The two major antigenic sites of alphas(2) were alphas(2)-CN (f96-114) and (f16-35). Cross-reaction between alphas(2)-CN specific antibodies with alphas(1)-CN illustrated the tangled structure between the two proteins. Immuno-dominant epitopes identified in the present study totally differ from those known for the purified caseins suggesting they were specific for the micelle supramolecular structure.