Does protein deamidation enhance rice protein concentrate's ability to produce and stabilize high internal phase emulsions?

Rice is one of the most consumed grains in the world. Rice protein has great nutritional value as a hypoallergenic protein and due to its high lysine content, a limiting amino acid in several other plant protein sources. However, rice protein has low solubility, hampering its use in many applications in the food industry. In this context, alkaline deamidation (0.5 h, 343 K, and pH 11) was applied to modify the protein structure of rice protein concentrate (RPC). After deamidation, two protein powders were produced: (i) one containing the whole protein fraction recovered after RPC deamidation (DT) and (ii) another containing only the soluble fraction recovered after RPC deamidation (DS). Protein dispersions were characterized by SDS-PAGE, zeta potential, solubility, surface hydrophobicity, and capacity to hold water and oil. RPC could not structure canola oil into a high internal phase emulsion (HIPE) due to its low solubility. DT and DS dispersions displayed solubility much higher than RPC and enabled the structuration of HIPEs with 75 % (w/w) canola oil and 25 % of DT or DS dispersions (2, 4, and 6 % w/w). HIPEs were characterized regarding particle size, microstructure, Turbiscan and oil loss stabilities, and rheological behavior for 60 days. Turbiscan analysis and oil loss measurements showed high stability, and the thixotropy tests showed high recovery in all HIPEs. Higher protein concentrations and DS dispersions produced HIPEs with smaller particle sizes. However, rheological measurements indicate that HIPEs produced with DT dispersions had better results, maintaining their structure over the 60 days. Furthermore, DT is cheaper to produce; therefore, DT 4 and 6 % w/w were the most promising for producing HIPEs. The HIPEs produced in this study displayed great potential as fat replacers.

Compartmentalization of lutein in simple and double emulsions containing protein nanoparticles: Effects on stability and bioaccessibility.

Delivery systems designed through protein stabilized emulsions are promising for incorporating carotenoids in different products. Nevertheless, the versatility in structures of such systems raises questions regarding the effect of the bioactive compound localization on their bio-efficacy, in particular for double emulsions. In this context, the aims of this study were to determine the impact of the localization of lutein in different water/oil/water double emulsions versus a single oil/water emulsion on the stability and in vitro bioaccessibility of lutein, a lipophilic carotenoid. The inner aqueous phase, which contained whey protein isolate (WPI) nanoparticles obtained by desolvation, was emulsified in sunflower oil stabilized by polyglycerol polyricinoleate (PGPR). The primary emulsion was then emulsified in a continuous aqueous phase containing whey protein isolate (WPI) and xanthan gum, the latter to increase the viscosity of the outer phase and delay creaming. Lutein was incorporated using different strategies: (1) lutein entrapped by WPI nanoparticles within the inner water phase of a double emulsion (W-L/O/W); (2) lutein incorporated into the oil phase of the double emulsion (W/O-L/W); (3) lutein incorporated in the oil phase of a single emulsion (O-L/W). All systems contained similar whey protein concentrations, as well as all other stabilizers. W-L/O/W sample showed the lowest lutein stability against light exposure during storage, and the highest lutein bioaccessibility after in vitro digestion, for freshly made samples. Furthermore, the in vitro bioaccessibility of lutein incorporated into the single emulsion was considerably lower than those observed for the double emulsions. The results reinforce the importance of designing appropriate structures for delivering improved stability and bioaccessibility of bioactive compounds.

Thermo-induced changes in the structure of lentil protein isolate (Lens culinaris) to stabilize high internal phase emulsions.

This study aims to assess the impact of heat treatment on the emulsifying properties of lentil protein isolate (LPI) dispersion to produce high internal phase emulsions (HIPEs). The heat-treated LPI dispersion was characterized by size, turbidity, solubility, zeta potential, free sulfhydryl group, electrophoresis, differential scanning calorimetry, circular dichroism, Fourier transforms infrared spectroscopy and intrinsic fluorescence. HIPEs were produced with 25% of LPI dispersion (2%, w/w) and soybean oil (75%) using a rotor-stator (15,500 rpm/1 min). HIPEs were evaluated for their droplet size, zeta potential, centrifugal stability, microscopy, appearance, Turbiscan stability, and rheology over 60 days (25 °C). Heat treatment reduced the size of LPI, resulting in increased turbidity, solubility, and exposure of hydrophobic groups. HIPEs produced with heat-treated LPI at 70 °C (HIPE70) and 80 °C (HIPE80) for 20 min exhibited lower droplet sizes, increased stability, reduced oil loss, and a homogeneous appearance compared to HIPE produced with untreated LPI (HIPEc). In addition, HIPE70 and HIPE80 displayed resistance to shear stress, higher apparent viscosity, and increased storage modulus than HIPEc. HIPEs produced with heat-treated LPI were stable, suggesting that the treatment was efficient for improving the functional properties of the protein and the possibility of future research focusing on fat substitutes in food applications.

Effects of processing technologies on the antioxidant properties of common bean (Phaseolus vulgaris L.) and lentil (Lens culinaris) proteins and their hydrolysates.

The effects of ultrasound (280 W, 5 min), heat treatment (75 °C and 90 °C for 10 min) and microfluidization (125 MPa, 4 cycles) as pre or post treatments and their combination with enzymatic hydrolysis on the antioxidant properties of common bean and lentil protein hydrolysates were investigated. In general, hydrolysis resulted in increases of antioxidant activity, both in the presence and absence of processing technologies. The increases reached maximum values of 158% (ABTS), 105% (DPPH), 279% (FRAP) and 107% (TAC) for the bean protein hydrolysates submitted to post-treatment with ultrasound (ABTS, FRAP and TAC) and pre-treatment with microfluidization (DPPH), compared to their respective controls (untreated samples). For lentil proteins, the increases reached 197% (ABTS), 170% (DPPH), 690% (FRAP) and 213% (TAC) for samples submitted to ultrasound post-treatment (ABTS), microfluidization pre-treatment (DPPH) and post-treatment (FRAP), and 75 °C pre-treatment (TAC) compared to their respective controls. Surface hydrophobicity and molecular weight profile by SEC-HPLC analysis indicated modifications in the structures of proteins in function of the different processing technologies. For both proteins, electrophoresis indicated a similar profile for all hydrolysates, regardless of the process applied as pre or post treatment. Solubility of bean and lentil protein concentrates was also improved. These results indicated that different processing technologies can be successfully used in association with enzymatic hydrolysis to improve the antioxidant properties of lentil and bean proteins.

Stability of milk proteins subjected to UHT treatments: challenges and future perspectives.

Ultra-high temperature (UHT) treatments are of high economic relevance for food industries because they contribute to extending the shelf life of food products and facilitating their distribution. In the dairy segment, UHT treatments are applied to a wide range of products containing variable protein amounts. In this sense, the changes in the molecular structure of milk proteins induced by the severity of UHT treatments may lead to fouling in equipment during processing or sedimentation and/or gelation during storage. Nowadays, these concerns are even more relevant due to the increasing demand for UHT-treated high-protein beverages. This review will discuss the two main strategies used by industries to increase the stability of milk proteins during and/or after UHT treatments: (i) addition of chelating agents and (ii) use of polysaccharides. Moreover, the challenges and opportunities associated with promising strategies to improve the stability of milk proteins during and/or after UHT treatments will be covered in this review. The information compiled will be useful to guide researchers and industries in developing more stable UHT dairy products in harmony with consumers' demands.

Co-aggregation between whey proteins and carotenoids from yellow mombin (Spondias mombin): Impact of carotenoids' self-aggregation.

The interaction between whey proteins and carotenoid is reported to improve carotenoid solubility and stability, however, the strong trend of carotenoids to aggregate when in polar systems is often neglected in papers addressing their molecular interaction. Therefore, this study focused on characterizing the aggregative behavior of the carotenoids from yellow mombin (Spondias mombin) and to understand how these carotenoids behave when added to aqueous dispersions of whey proteins. Carotenoids-rich extract, containing mainly ß-cryptoxanthin and lutein, was obtained from freeze-dried yellow mombin pulp and its aggregative behavior in ethanol/water medium was studied. By increasing the medium polarity, carotenoids trend to form J-aggregation, causing a drop in the color intensity of the solution. When added to whey protein aqueous dispersions, rather than a protein-carotenoid bimolecular interaction, the formation of co-aggregates between carotenoids and whey proteins was evidenced by preparative size exclusion chromatography. These results may contribute to the developing functional food products.

Lutein bioaccessibility in casein-stabilized emulsions is influenced by the free to acylated carotenoid ratio, but not by the casein aggregation state.

Considering that carotenoids are found acylated to fatty acids in most edible fruits, the influence of the ratio of free to acylated lutein on the hydrolysis extent and bioaccessibility was evaluated by in vitro digestion. For this purpose, for the first time, esterified, free, or a mixture of both carotenoid forms was used in the lipid phase of emulsions stabilized by sodium caseinate (NaCas) and native phosphocaseinate (PPCN). Marigold petals was used as a source of lutein-rich extracts. The emulsions were characterized and the extent of ester hydrolysis, carotenoid recovery, and bioaccessibility were evaluated by LC-DAD-MS/MS. Besides low polydispersity, NaCas and PPCN stabilized emulsions exhibited a constant mean droplet diameter of about 260 and 330 nm, respectively, after 7 days. Caseins were completely digested after the gastric digestion step. Moreover, casein supramolecular structure did not significantly affect carotenoid bioaccessibility. Lutein was majorly found in its free form in all bioaccessible fractions. The carotenoid bioaccessibility increased from 3% to 40% by increasing the percentage of free carotenoids from 0.5 to 100% in the emulsions; but the carotenoid recovery and hydrolysis extent of lutein esters were not affected. In conclusion, emulsion-based systems for carotenoid delivery stabilized either by NaCas or PPCN provided similar carotenoid bioaccessibility. Furthermore, bioaccessibility was inversely dependent on the overall hydrophobicity of the carotenoid extract. Our results suggest that the low bioaccessibility of esterified carotenoids was a consequence of their limited hydrolysis extent. This study provides information that may help design emulsion-based systems stabilized by food protein as a vehicle for carotenoids.

Designing covalent sodium caseinate-quercetin complexes to improve emulsifying properties and oxidative stability.

The complexation of proteins with phenolic compounds has been considered a promising route to improve the oxidative stability of oil-in-water (O/W) emulsions. In this context, physicochemical and techno-functional properties of sodium caseinate (SC) chemically modified by the interaction with quercetin (Q) under alkaline conditions were evaluated using different molar ratios of the components (SC:Q). The formation of covalent complexes was analysed by the changes in SC structure and properties.. The results showed that the surface hydrophobicity of SC gradually decreased with increasing Q concentration. Whereas an increase in phenolic content and antioxidant activity by DPPH and FRAP was observed. The techno-functionality of the complexes was evaluated by their capacity of stabilizing oil-in-water emulsions. The emulsion stabilized by complexes and non-modified SC showed an average droplet size (D4.3) between 1,632 ± 0,068 and 1,945 ± 0,431 µm. After 21d of storage, no coalescence was observed, evidencing the formation of stable emulsions. Furthermore, the highest oxidative stability of the emulsion was observed at the highest concentration of Q, while the formation of hydroperoxides and aldehydes was greater in the emulsions stabilized with non-modified SC. The formation of primary lipid oxidation compounds was approximately 3x higher in emulsions stabilized only with SC compared to those stabilized with the SC:Q complex in a 1:1 ratio. These results suggest that modification of SC with Q is a potential approach tuning both chemical and techno-functional properties of SC.

High-pressure microfluidization of whey proteins: Impact on protein structure and ability to bind and protect lutein.

Dynamic high-pressure homogenization microfluidization (DHPM) is a versatile emerging technology that may be applied to food processing to achieve several goals. DHPM may, depending on nature of the molecules and the working parameters, induce changes in protein structure, which may improve or impair their techno-functional properties and ability to bind other molecules. In this context, DHPM (12 passes, 120 MPa), coupled or not to a cooling device, was applied to ß-lactoglobulin (ß-lg) and whey protein isolate (WPI) dispersions. Minor changes in the structure of whey proteins were induced by DHPM with sample cooling; although, when sample cooling was not applied, aggregation and increases of around 30% of protein surface hydrophobicity were noticeable for the WPI dispersion. The association constant between the proteins and lutein was in the magnitude of 104 M-1, and lutein photodegradation constant diminished about 3 times in the presence of proteins, compared to in their absence.

Structural and foaming properties of whey and soy protein isolates in mixed systems before and after heat treatment.

The partial replacement of proteins from animal sources by plant proteins in formulated food products has been proposed as useful to improve sustainability aspects of the products without dramatically changing their techno-functional properties. Although several research groups have published on the gelling properties of mixed systems containing whey and soy protein isolates (WPI and SPI), their foaming properties are much less described. In this context, the main objective of this paper was to evaluate the structural and foaming properties of samples containing different mass ratios of WPI:SPI (100:0, 75:25, 50:50, 25:75 and 0:100) before and after heat treatment. The samples were evaluated according to their solubility, foaming capacity (FC), foam microstructure and foam stability (FS). Before heat treatment, mixing SPI to WPI did not affect the solubility of whey proteins, but, after heat treatment, insoluble co-aggregates were formed. Similar FC was measured for all samples despite their WPI:SPI ratio and the applied heat treatment. The partial replacement of WPI by SPI changed the microstructure of the foams and had an antagonistic effect on the FS of the samples, due to the negative effect of insoluble soy protein aggregates and/or insoluble co-aggregates on the reinforcement of the air-water interfacial film.

Unraveling the molecular mechanisms underlying interactions between caseins and lutein.

Understanding the food protein binding to bioactive compounds is of utmost importance for the development of efficient protein-based delivery systems. The binding of lutein to sodium caseinate (NaCas) or native casein micelle (PPCN) was investigated at pH 7 to evaluate the effect of casein supramolecular structures on the interaction. Fluorescence quenching, UV-vis spectroscopy, and dynamic light scattering were carried out. Under the medium conditions of interaction analysis (DMSO-water and ethanol-water), lutein exists as H-type aggregates. The investigation of lutein/casein interaction showed a predominantly static mechanism of fluorescence quenching and the presence of two fluorophore populations on NaCas and PPCN, but only one accessible to lutein. Moreover, the Scatchard plot indicated that lutein interacted with both caseins in one binding site. The interaction of lutein with caseins occurred with binding constant Kb of 105 M-1, regardless of casein supramolecular structure.

Complexation of chitosan with gum Arabic, sodium alginate and κ-carrageenan: Effects of pH, polymer ratio and salt concentration.

The effects of pH, ionic strength and polymer ratio in the complexation of chitosan (CHI) with different anionic polysaccharides, namely gum Arabic (GA), sodium alginate (ALG) and κ-carrageenan (CRG), were investigated. This was made using titration techniques, which allowed the determination of stoichiometry and binding constant of complexes. The sulfated polysaccharide interacted more strongly with CHI than carboxylated polysaccharides. The increase of ionic strength (0-100 mM NaCl) in the polysaccharides complexation resulted in a significant reduction in the binding constant of GA:CHI and CRG:CHI, but did not influence the complexation of ALG with CHI. The pH and polymer ratio affected the formation and solubility of complexes GA:CHI, while for ALG:CHI and CRG:CHI, insoluble complexes were observed in all pH and polymer ratio evaluated. A phase transition of coacervate to gel was proposed to ALG:CHI and CRG:CHI, which can be related to the self-association of anionic polymers, when these are in excess.

Technological aspects of lactose-hydrolyzed milk powder.

Few reports describe the effect of lactose hydrolysis on the properties of milk powder during production and storage. Hence, the aim of this study was to evaluate the effects of five different levels of enzymatic lactose hydrolysis during the production and storage of milk powder. As the lactose hydrolysis rate increased, adhesion to the drying chamber also increased, due to higher levels of particle agglomeration. Additionally, more brown powder was obtained when the lactose hydrolysis rate was increased, which in turn negatively affected rehydration ability. Using Raman spectroscopy, crystallization of the lactose residues in various samples was assessed over 6weeks of accelerated aging at a room temperature environment with 75.5% of air moisture. Products with 25% or greater lactose hydrolysis showed no signs of crystallization, in contrast to the non-hydrolyzed sample.

How the presence of a small molecule affects the complex coacervation between lactoferrin and ß-lactoglobulin.

Heteroprotein complex coacervation corresponds to the formation of two liquid phases in equilibrium induced by the interaction of two oppositely charged proteins. The more concentrated phase known as coacervate phase, has attracted interest from several fields of science due to its potential applications for example for encapsulation and delivery of bioactives. Prior such application, it is necessary to understand how the presence of small ligands affects the complex coacervation. In this work, we report on the interaction of small ligand with individual proteins ß-lactoglobulin (ß-LG) and lactoferrin (LF) and consequences on their complex coacervation. ANS (8-Anilinonaphthalene-1-sulfonic acid), a fluorescent probe, was used as model ligand. While ANS did not interact with ß-LG, it presented two sets of binding sites with LF inducing its self-aggregation. Depending on its concentration, ANS modulated the shape of ß-LG-LF macromolecular assembly. Coacervates were observed for ANS/LF molar ratio <25 against amorphous aggregates for higher ANS/LF molar ratios. A maximum loading capacity of around 40mg of ANS per gram of LF in the formed heteroprotein coacervates was reached.

Heteroprotein complex coacervation: A generic process.

Proteins exhibit a rich diversity of functional, physico-chemical and biodegradable properties which makes them appealing for various applications in the food and non-food sectors. Such properties are attributed to their ability to interact and assemble into a diversity of supramolecular structures. The present review addresses the updated research progress in the recent field of complex coacervation made from mixtures of oppositely charged proteins (i.e. heteroprotein systems). First, we describe briefly the main proteins used for heteroprotein coacervation. Then, through some selected examples, we illustrate the particularity and specificity of each heteroprotein system and the requirements that drive optimal assembly into coacervates. Finally, possible and promising applications of heteroprotein coacervates are mentioned.

Structure and Dynamics of Heteroprotein Coacervates.

Under specific conditions, mixing two oppositely charged proteins induces liquid-liquid phase separation. The denser phase, or coacervate phase, can be potentially applied as a system to protect or encapsulate different bioactive molecules with a broad range of food and/or medical applications. The optimization of the design and efficiency of such systems requires a precise understanding of the structure and the equilibrium of the nanocomplexes formed within the coacervate. Here, we report on the nanocomplexes and the dynamics of the coacervates formed by two well-known, oppositely charged proteins ß-lactoglobulin (ß-LG, pI ≈ 5.2) and lactoferrin (LF, pI ≈ 8.5). Fluorescence recovery after photobleaching (FRAP) and solid-state nuclear magnetic resonance (NMR) experiments indicate the coexistence of several nanocomplexes as the primary units for the coacervation. To our knowledge, this is the first evidence of the occurrence of an equilibrium between quite unstable nanocomplexes in the coacervate phase. Combined with in silico docking experiments, these data support the fact that coacervation in the present heteroprotein system depends not only on the structural composition of the coacervates but also on the association rates of the proteins forming the nanocomplexes.

Binding of Folic Acid Induces Specific Self-Aggregation of Lactoferrin: Thermodynamic Characterization.

In the study presented here, we investigated the interaction at pH 5.5 between folic acid (FA) and lactoferrin (LF), a positively charged protein. We found a binding constant Ka of 10(5) M(-1) and a high stoichiometry of 10 mol of FA/mol of LF. The size and charge of the complexes formed evolved during titration experiments. Increasing the ionic strength to 50 mM completely abolished the isothermal titration calorimetry (ITC) signal, suggesting the predominance of electrostatic interactions in the exothermic binding obtained. We developed a theoretical model that explains the complex triphasic ITC profile. Our results revealed a two-step mechanism: FA/LF interaction followed by self-association of the complexes thus formed. We suggest that 10 FA molecules bind to LF to form saturated reactive complexes (FA10/LF) that further self-associate into aggregates with a finite size of around 15 nm. There is thus a critical saturation degree of the protein, above which the self-association can take place. We present here the first results that provide comprehensive details of the thermodynamics of FA/LF complexation-association. Given the high stoichiometry, allowing a load of 55 mg of FA/g of LF, we suggest that FA/LF aggregates would be an effective vehicle for FA in fortified drinks.