The role of Saccharomyces cerevisiae in stabilizing emulsions of hexadecane in aqueous media.

During downstream operations involved in the purification of hydrophobic biofuels produced by microorganisms, undesired stable emulsions may be formed. Understanding the mechanisms behind this stability is a pre-requisite for designing cost-effective strategies to break these emulsions. In this work, we aimed at increasing our knowledge on the mechanisms responsible for stabilizing yeast-containing oil-in-water emulsions. For this purpose, emulsions containing hexadecane and different yeast-based aqueous phases were prepared and analyzed for phase separation, surface charge density, particle size, and rheology. First, we observed that compounds present in fresh tablet baker's yeast contribute to emulsion stability. In order to eliminate this effect, we generated stocks with this yeast in the laboratory, and compared its performance with an industrial fuel ethanol strain, namely Saccharomyces cerevisiae PE-2. We confirmed that the presence of yeast cells enhances emulsion stability. The cultivation medium (complex or defined) in which cells are grown, as well as the physiological state of the cells (pre- or post-diauxic), prior to emulsion preparation, influenced emulsion stability. The smaller cell size of tablet yeast probably also contributes to more stable emulsions, when compared to those prepared with yeast cells grown in the laboratory. Baker's and fuel ethanol yeast cells in post-diauxic phase promote the formation of more stable emulsions than those with cells in the pre-diauxic physiological state. Finally, we propose a mechanism to explain the enhanced emulsion stability due to the presence of yeast cells, with electrostatic repulsion between emulsion droplets having the prevailing effect.

Protein-based strategies for fat replacement: Approaching different protein colloidal types, structured systems and food applications.

In the last decade, food structuring has received considerable attention due to the concern of replacing trans and saturated fats with healthier alternatives without compromising neither technological nor sensorial aspects of food products. Moreover, sustainability topics, consumers' preference for natural ingredients and the molecular architecture displaying a myriad of techno-functionalities embolden the use of proteins. Therefore, a promising approach is to explore this biopolymer as a texture promoter in lipid-based systems, conveying an extra edge in nutritional, sustainable and technological values. A more in-depth comprehension should be cemented to fully harness the potential of proteins in developing soft matter intended for use as fat mimetic. High Internal Phase Emulsion (HIPE), High Internal Phase Pickering Emulsion (HIPPEs), emulgels, oleogels or even bigels can be used in such strategies. Essentially, the formation of such systems relies on the amphiphilic character of proteins. In this sense, the question that arises is how to optimize their solubility in oils to form oil-structured systems? Thus, for oleogel formation the challenge is to overcome the limited dispersibility of proteins in a hydrophobic environment. Therefore, face the growing interest and untapped potential in applying proteins in lipid media, a more wide-ranging picture of their colloidal form (e.g. native, microgels and protein-polysaccharide complexes or conjugates) affecting the structure-function relationship of proteins must be investigated. This review covers different strategies using proteins as building blocks to manufacture different structured systems. Finally, an outlook over the use of protein-based soft matter on an industrial basis is discussed, considering the challenges and perspectives.

Understanding the Performance of Plant Protein Concentrates as Partial Meat Substitutes in Hybrid Meat Emulsions.

Hybrid meat products are an excellent strategy to incorporate plant proteins into traditional meat formulations considering recent market trends focusing on the partial reduction in red meat content. In this work, we evaluated the effects of different concentrated plant proteins (soy, pea, fava bean, rice, and sunflower) in partially replacing meat in meat emulsion model systems. Soy, pea, and sunflower proteins showed great compatibility with the meat matrix, giving excellent emulsion stability and a cohesive protein network with good fat distribution. Otherwise, adding rice and fava bean proteins resulted in poor emulsion stability. Color parameters were affected by the intrinsic color of plant proteins and due to the reduction in myoglobin content. Both viscoelastic moduli, G' and G″ decreased with the incorporation of plant proteins, especially for rice and fava bean. The temperature sweep showed that myosin denaturation was the dominant effect on the G' increase. The water mobility was affected by plant proteins and the proportion between immobilized and intermyofibrillar water was quite different among treatments, especially those with fava bean and rice proteins. In vitro protein digestibility was lower for hybrid meat emulsion elaborated with rice protein. It is concluded that soy, pea, and mainly sunflower proteins have suitable compatibility with the meat matrix in emulsified products.

Incompatibility between sodium caseinate - locust bean gum induced by NaCl and yerba mate extract.

Aqueous two-phase system (ATPS) is a technique used for the separation of biopolymers in two aqueous phases. Some combinations of biopolymers can form a water-in-water (W/W) emulsion due to steric exclusion and thermodynamic incompatibility between these biopolymers under some specific conditions. In this work, the formation of W/W emulsions composed of sodium caseinate (SCN) and locust bean gum (LBG) was evaluated, using NaCl or yerba mate extract as the driving force for the phase separation, which was described by phase's diagrams. Phase diagrams are like fingerprints of ATPS systems, which demonstrate the specific conditions to develop separate phases. Phase diagrams of the two systems show that at the same concentrations of protein and carbohydrate, the addition of NaCl or extract induced the separation of the compounds differently. Salt promotes phase separation by steric exclusion, each phase being rich in one of the polymers. Since extract may also induce other effects, such as the formation of a SCN-extract-LBG complex, migration of LBG to the SCN-rich phase was promoted, modifying the characteristics of the tie lines in the phase diagrams. However, it was feasible to separate the protein in systems containing concentrated phenolic extract, whose incorporation is relevant considering its antioxidant activity.

Rheological behavior of cellulose nanofibers from cassava peel obtained by combination of chemical and physical processes.

This work aimed to produce and characterize cellulose nanofibers obtained from cassava peel with a combination of pre-treatments with acid hydrolysis or TEMPO-mediated oxidation and ultrasonic disintegration. All nanofibers presented nanometric diameter (5-16 nm) and high negative zeta potential values (around -30 mV). Oscillatory rheology showed a gel-like behavior of the aqueous suspensions of nanofibers (1.0-1.8 % w/w), indicating their use as reinforcement for nanocomposite or as a thickening agent. Additionally aqueous suspensions of nanofibers obtained by acid hydrolysis presented higher gel strength than those produced by TEMPO-mediated oxidation. However, ultrasound application increased even more viscoelastic properties. Flow curves showed that suspensions of nanofibers obtained by acid hydrolysis presented a thixotropy behavior and viscosity profile with three regions. Therefore our results showed that it is possible to tune mechanical properties of cellulose nanofibers choosing and modifying chemical and physical process conditions in order to allow a number of applications.

Effect of Ohmic heating on functionality of sodium caseinate - A relationship with protein gelation.

Sodium caseinate (NaCAS) is widely used in the food industry to provide nutritional and functional benefits. This work deals with the effects of applying moderate electric fields (MEF) of different intensity - ranging from 2 V·cm-1 to 17 V·cm-1 - on the physical and functional properties of NaCAS solutions during Ohmic heating (OH) at 95 °C. Self-standing gels were produced regardless the heating technique applied (i.e. conventional or OH), and these gels were much more prone to physical rupture when compared with the ones produced from unheated NaCAS. Interestingly, OH treatment formed gels with lower values of strain at rupture and water holding capacity than unheated samples; this pattern was not observed for gels obtained through the conventional heating treatment (at 0 V·cm-1). These effects may be linked with disturbances of the distribution of random coil structures and enhanced solubility of NaCAS at its isoelectric point, reducing aggregation and impairing the development of a more compact protein network. Results show that OH presents potential to be used as volumetric heating tool for NaCAS solubilization and for the production of distinctive acidified systems.

High solids emulsions produced by ultrasound as a function of energy density.

The use of emulsifying methods is frequently required before spray drying food ingredients, where using high concentration of solids increases the drying process yield. In this work, we used ultrasound to obtain kinetically stable palm oil-in-water emulsions with 30g solids/100g of emulsion. Sodium caseinate, maltodextrin and dried glucose syrup were used as stabilizing agents. Sonication time of 3, 7 and 11min were evaluated at power of 72, 105 and 148W (which represents 50%, 75% and 100% of power amplitude in relation to the nominal power of the equipment). Energy density required for each assay was calculated. Emulsions were characterized for droplets mean diameter and size distribution, optical microscopy, confocal microscopy, ζ-potential, creaming index (CI) and rheological behavior. Emulsions presented bimodal size distribution, with D[3,2] ranging from 0.7 to 1.4µm and CI between 5% and 12%, being these parameters inversely proportional to sonication time and power, but with a visual kinetically stabilization after the treatment at 148W at 7min sonication. D[3,2] showed to depend of energy density as a power function. Sonication presented as an effective method to be integrated to spray drying when emulsification is needed before the drying process.

High acyl gellan as an emulsion stabilizer.

High acyl gellan (0.01-0.2% w/w) was used as stabilizer in oil in water emulsions containing 30% (w/w) of sunflower oil and prepared under different process conditions. Stable emulsions to phase separation could be obtained using high acyl gellan (HA) content above 0.05% (w/w), while low acyl gellan (LA) prepared at the same conditions could not stabilize emulsions. Emulsions properties depended on the process used to mix the oil and gellan dispersion since high pressure homogenization favored stabilization while very high energy density applied by ultrasound led to systems destabilization. Emulsions prepared using high pressure homogenization showed zeta potential values ranging from -50 up to -59 mV, suggesting that electrostatic repulsion could be contributing to the systems stability. Rheological properties of continuous phase were also responsible for emulsions stabilization, since HA gellan dispersions showed high viscosity and gel-like behavior. The high viscosity of the continuous phase could be associated to the presence of high acyl gellan microgels/aggregates. Disentanglement of these aggregates performed by ultrasound strongly decreased the viscosity and consequently affected the emulsions behavior, reducing the stability to phase separation.

Stabilization mechanisms of oil-in-water emulsions by Saccharomyces cerevisiae.

A multiphase system is commonly formed during the oil production by microbial route, which can lead to stable emulsions hindering product recovery. Thus, this study aimed to investigate the mechanisms of emulsion stabilization by the yeast Saccharomyces cerevisiae in order to contribute with processes development of oil production by fermentation. A model system using hexadecane as oil phase and yeast suspension as aqueous phase was used to prepare O/W emulsions. The yeast was subjected to different treatments as inactivation (autoclaving) and washing before to be resuspended in water. The washing water (water from the first washing) and suspension of commercial yeast (active) were also used as aqueous phase. After 24h of preparation, the emulsions separated into three phases: top (cream), intermediate, and bottom phase. The top or cream phase was a concentrated emulsion that kept stable during seven days, except for those prepared from washed yeast that were stable only for a short period of time. Emulsions prepared with washed yeast showed higher cell adhesion to the droplets interface, which implied in a higher amount of yeast into the cream phase in comparison to other formulations. Therefore, yeast cells adhesion plays a role on emulsion stability, but the greater contribution was provided by cell material dispersed into the aqueous phase, regardless of cell viability.

Heteroaggregation of lipid droplets coated with sodium caseinate and lactoferrin.

Formation and characterization of droplet heteroaggregates were investigated by mixing two emulsions previously stabilized by proteins oppositely charged. Emulsions were composed of 5vol.% of sunflower oil and 95vol.% of sodium caseinate or lactoferrin aqueous dispersions. They were produced using ultrasound with fixed power (300W) and sonication time (6min). Different volume ratios (0-100%) of sodium caseinate-stabilized emulsion (droplet diameter around 1.75µm) to lactoferrin-stabilized emulsion (droplet diameter around 1.55µm) were mixed under conditions that both proteins showed opposite charges (pH7). Influence of ionic strength (0-400mM NaCl) on the heteroaggregates stability was also evaluated. Creaming stability, zeta potential, microstructure, mean particle diameter and rheological properties of the heteroaggregates were measured. These properties depended on the volume ratio (0-100%) of sodium caseinate to lactoferrin-stabilized emulsion (C:L) and the ionic strength. In the absence of salt, different zeta potential values were obtained, rheological properties (viscosity and elastic moduli) were improved and the largest heteroaggregates were formed at higher content of lactoferrin-stabilized emulsion (60-80%). The system containing 40 and 60vol.% of sodium caseinate and lactoferrin stabilized emulsion, respectively, presented good stability against phase separation besides showing enhanced rheological and size properties due to extensive droplets aggregation. Phase separation was observed only in the absence of sodium caseinate, demonstrating the higher susceptibility of lactoferrin to NaCl. The heteroaggregates produced may be useful functional agents for texture modification and controlled release since different rheological properties and sizes can be achieved depending on protein concentrations.