Segregation Behavior of Polysaccharide⁻Polysaccharide Mixtures-A Feasibility Study.

The segregative phase separation behavior of biopolymer mixtures composed entirely of polysaccharides was investigated. First, the electrical, optical, and rheological properties of alginate, modified beet pectin, and unmodified beet pectin solutions were characterized to determine their electrical charge, molecular weight, solubility, and flow behavior. Second, suitable conditions for inducing phase segregation in biopolymer mixtures were established by measuring biopolymer concentrations and segregation times. Third, alginate⁻beet pectin mixtures were blended at pH 7 to promote segregation and the partitioning of the biopolymers between the upper and lower phases was determined using UV⁻visible spectrophotometry, colorimetry, and calcium sensitivity measurements. The results revealed that phase separation depended on the overall biopolymer concentration and the degree of biopolymer hydrophobicity. A two-phase system could be formed when modified beet pectins (DE 68%) were used but not when unmodified ones (DE 53%) were used. Our measurements demonstrated that the phase separated systems consisted of a pectin-rich lower phase and an alginate-rich upper phase. These results suggest that novel structures may be formed by utilization of polysaccharide⁻polysaccharide phase separation. By controlling the product formulation and processing conditions it may therefore be possible to fabricate biopolymer particles with specific dimensions, shapes, and internal structures.

Influence of spray drying on the stability of food-grade solid lipid nanoparticles.

This study investigated spray drying of food-grade solid lipid particles (SLN) and nanostructured lipid carriers (NLC) containing ω-3 fish oil. Stable SLN and NLC dispersions with tristearin as carrier lipid were formed by using a combination of Quillaja saponins and high-melting lecithin as emulsifiers. Our specific goal was to study the influence of four different spray drying inlet and outlet temperatures (Tinlet/outlet = 140-170 °C/65-95 °C) and two different maltodextrin types (DE 6 and DE 21) with different molecular weights as protective wall materials on the physical and polymorphic stability of the solid lipid particles. The results revealed that the low molecular weight maltodextrin DE 21 was a superior wall material in stabilizing the solid lipid particles. Moreover, the lipid particles spray dried at Tinlet/outlet of 140/65 °C exhibited the highest physical and polymorphic stability, whereas using higher Tinlet/outlet led to bigger particles which were more prone to polymorphic transition. This was also verified in a 71-day storage test. The findings were explained that by preventing the melting of the tristearin carrier lipid during spray drying, the crystallized lipid particles remained intact inside the amorphous maltodextrin layer and exhibited high physical and polymorphic stability. These findings are important for generating stable food-grade spray dried powders.

Modulation of the bitterness of pea and potato proteins by a complex coacervation method.

The incorporation of novel plant-based proteins into foods is often challenging due to an unacceptable bitter sensation. Typically, a combination of electrostatic and hydrophobic forces contributes to the proteins' bitterness. The current study therefore focuses on the development of electrical properties on cationic plant proteins to reduce their overall bitterness in order to improve the perceived sensorial acceptance. As such, we utilized a simple mixing process to induce complex coacervation of oppositely charged biopolymers under acidic conditions. Pea and potato protein stock solutions were mixed with apple pectin (DE 71%) solutions at various biopolymer ratios to modulate the electrical, rheological, and sensorial properties of the complexes. Whey protein hydrolyzate was used as a control sample. Surface charge measurements revealed a transition from positive to negative values as the pectin concentration was increased regardless of the plant protein, whereas stable dispersions without sedimentation were observed above a critical pectin : protein ratio of 1. Low and intermediate biopolymer ratios (<1) promoted aggregation and led to rapid sedimentation. Sensory evaluation showed that bitterness scores depended on protein type and decreased from pea protein > potato protein > whey protein. Moreover, bitter off-notes were increasingly reduced with increasing pectin : protein ratios; however, high dispersion viscosities above 0.05 Pa s led to undesirable texture and mouthfeel of the biopolymer dispersions. Our results might have important implications for the utilization of novel plant proteins in food and beverage applications.

Impact of food structure on the compatibility of heated WPI-pectin-complexes in meat dispersions.

Process-stable complexes composed of whey protein isolate (WPI) and sugar beet pectin have great potential as structuring agents or fat replacers in foods. The current study investigates the compatibility of heated WPI : pectin complexes in different meat matrices. Spreadable raw-fermented sausages and sliceable emulsion-type sausages were therefore manufactured containing biopolymer complexes with various WPI : pectin ratios r (2 : 1, 8 : 1). Macro- and microstructural properties of the meat dispersions were analyzed in terms of colour, texture, rheometry, sensory, and confocal laser scanning microscopy (CLSM) measurements. Textural and sensorial results demonstrated that the meat products became increasingly soft and yellowish as the biopolymer ratio r was increased regardless of the meat matrix, whereas pH and water activity values were not affected. CLSM images revealed that the meat protein network became disrupted and loose in the presence of pectin, which was attributed to a thermodynamic incompatibility effect. The results obtained from this study highlight that biopolymer complexes might be suitable fat mimetics, particularly for spreadable meat products.

Impact of laccase on the colour stability of structured oil-in-water emulsions.

The optical properties of food emulsions play a key role in determining their perceived quality because they are the first sensory cue that many consumers receive. The purpose of the current study was to investigate the impact of a cross-linking enzyme (laccase) on the appearance of structured oil-in-water emulsions containing a lipophilic model colorant (Nile red). A layer-by-layer electrostatic deposition approach was used to prepare oil-in-water emulsions stabilized by interfacial protein-pectin complexes under acidic conditions (pH3.5, 10mM citrate buffer). Laccase (an oxidoreductase) was then added to the system, since this enzyme is often used to covalently cross-link interfacial biopolymer layers. The optical properties of the emulsions were monitored during storage using spectral reflectance to determine the L*a*b values, while the physical properties were monitored by measuring changes in droplet surface charge and particle size distribution. No changes in the size or charge of the droplets were observed during storage, indicating that the emulsions had good physical stability. In the absence of laccase, the emulsions were stable to colour fading, but in the presence of laccase rapid colour changes occurred (red to blue to white). These results have important implications for the formation of structured food emulsions containing certain types of food dyes.

Enzyme-Based Strategies for Structuring Foods for Improved Functionality.

Enzyme technologies can be used to create food dispersions with novel functional attributes using structural design principles. Enzymes that utilize food-grade proteins and/or polysaccharides as substrates have gained recent interest among food scientists. The utilization of enzymes for structuring foods is an ecologically and economically viable alternative to the utilization of chemical cross-linking and depolymerization agents. This review highlights recent progress in the use of enzymes to modify food structures, particularly the interfacial and/or bulk properties of food dispersions with special emphasis on commercially available enzymes. Cross-linking enzymes such as transglutaminase and laccase promote the formation of intra- and intermolecular bonds between biopolymers to improve stability and functionality, whereas various degrading enzymes such as proteases alter the native conformation of proteins, leading to self-assembly of hierarchically ordered colloids. Results of this bio-inspired approach show that rational use of structure-affecting enzymes may enable food manufacturers to produce food dispersions with improved physical, functional, textural, and optical properties.

Mixing behaviour of WPI-pectin-complexes in meat dispersions: impact of biopolymer ratios.

Particulated complexes composed of oppositely charged biopolymers were incorporated into highly concentrated protein matrices as potential fat replacers and structuring agents. A multistep procedure was therefore utilized to generate process-stable complexes, which were subsequently embedded into emulsion-type sausages, whereas macro- and microstructural properties were then assessed. Firstly, stock WPI and sugar beet pectin solutions were mixed under neutral conditions (pH 7) at various biopolymer ratios r (2 : 1, 5 : 1, 8 : 1). Secondly, the pH of the biopolymer mixture was decreased to 3.5 to promote associative complexation. Thirdly, electrostatically attracted biopolymer particles were subjected to a heat treatment (ϑ = 85 °C, 20 min) to enhance their stability against superimposed stresses. Finally, fat-reduced emulsion-type sausages were fabricated containing stable WPI-pectin complexes. The results revealed that the heat treatment increased the pH-stability of the biopolymer complexes. In addition, textural and sensorial analysis demonstrated that the meat products became increasingly soft as the biopolymer ratio r increased. This effect was attributed to thermodynamic incompatibility between meat proteins and beet pectin. The results obtained from this study might have important implications for the fabrication of processed meat products with reduced fat levels.

Initial Droplet Size Impacts pH-Induced Structural Changes in Phase-Separated Polymer Dispersions.

The effect of pH change on the morphology of whey protein isolate (WPI)-pectin dispersions obtained from phase-separated systems after mild shear was studied. The purpose of this study was to examine the impact of mixing speed on the initial particle size of biopolymer complexes and their structure morphology after sequentially changing the pH. Therefore, solutions of WPI and pectin were combined at pH 6.1, allowed to phase separate and were then mildly homogenized at 50, 100, and 150 rpm, respectively, to form a dispersion containing differently sized WPI droplets in a surrounding pectin-rich phase. Each dispersion was then subjected to a pH change, such as 6.1 to 5.2 and 3.2, by slowly adding hydrochloric acid. The systems morphology, size, appearance, rheology, and storage stability was then characterized by optical microscopy, static light scattering, visual inspections, and steady shear rheometry to gain insights into the structural rearrangements. Results indicated substantial changes in the structure of the dispersion when the pH was changed. Formation of core-shell structures from the WPI droplets was observed at an intermediate pH. There, initial droplet size was found to affect structures formed, that is, core-shell type particles would only form if droplets were large (>1.5 µm) prior to pH change. Insights gained may be of importance to food manufacturers intending to create new structures from mixtures of proteins and carbohydrates.

Tuneable stability of nanoemulsions fabricated using spontaneous emulsification by biopolymer electrostatic deposition.

Nanoemulsions can be formed spontaneously from surfactant-oil-water systems using low energy methods. In this work, we showed that the droplets in oil-in-water nanoemulsions fabricated by spontaneous emulsification could be coated with an anionic biopolymer (beet pectin) using electrostatic deposition. Nanoemulsions were formed by titrating oil (medium chain triglycerides) and surfactant (polyoxyethylene sorbitan monostearate+lauric arginate) mixtures into an aqueous solution (10 mM citrate buffer, pH 4). Lauric arginate was used to generate a positive charge on the droplet surfaces, thereby enabling subsequent electrostatic deposition of anionic pectin. Extensive droplet aggregation occurred when intermediate pectin concentrations were used due to bridging flocculation. However, stable anionic pectin-coated lipid droplets could be formed at high pectin concentrations. These results demonstrate the possibility of tailoring the functionality of lipid nanodroplets produced by spontaneous emulsification.

Reprint of: Impact of alcohols on the formation and stability of protein-stabilized nanoemulsions.

Nanoemulsions are increasingly being used for encapsulation, protection, and delivery of bioactive lipids, however, their formation from natural emulsifiers is still challenging. We investigated the impact of alcohol on the formation and stability of protein-stabilized oil-in-water nanoemulsions prepared by high-pressure homogenization. The influence of different alcohols (ethanol, 1-propanol, and 1-butanol) at various concentrations (0-25% w/w) on the formation and stability of emulsions stabilized by sodium caseinate, whey protein isolate, and fish gelatin was investigated. The mean particle diameter decreased with increasing alcohol concentrations from 0 to 10%w/w, but extensive droplet aggregation occurred at higher levels. This phenomenon was attributed to enhanced protein-protein interactions between the adsorbed emulsifier molecules in the presence of alcohol leading to droplet flocculation. The smallest droplets (d<100 nm) were obtained when 10%w/w 1-butanol was added to sodium caseinate-stabilized nanoemulsions, but relatively small droplets (d<150 nm) could also be obtained in the presence of a food-grade alcohol (ethanol). This study demonstrated that alcohol addition might be a useful tool for producing protein-stabilized nanoemulsions suitable for use as delivery systems of lipophilic bioactive agents.

Solubilization of octane in cationic surfactant-anionic polymer complexes: effect of polymer concentration and temperature.

Polymers may alter the ability of oppositely charged surfactant micelles to solubilize hydrophobic molecules depending on surfactant-polymer interactions. This study was conducted to investigate the effects of polymer concentration and temperature on the solubilization thermodynamics of an octane oil-in-water emulsion in mixtures of an anionic polymer (carboxymethyl cellulose) and cationic cetyltrimethylammonium bromide (CTAB) surfactant micelles using isothermal titration calorimetry (ITC). Results showed that the CTAB binding capacity of carboxymethyl cellulose increased with increasing temperature from 301 to 323 K, and correspondingly the thermodynamic behavior of octane solubilization in CTAB micelles, either in the absence or presence of polymer, was found to depend on temperature. The addition of carboxymethyl cellulose caused the solubilization in CTAB micelles to be less endothermic, and increased the solubilization capacity. Based on the phase separation model, the solubilization was suggested to be mainly driven by enthalpy gains. Results suggest that increasing concentrations of the anionic polymer gave rise to a larger Gibbs energy decrease and a larger unfavorable entropy increase for octane solubilization in cationic surfactant micelles.

Electrostatic modulation and enzymatic cross-linking of interfacial layers impacts gastrointestinal fate of multilayer emulsions.

In this study, membrane properties were modulated using layer-by-layer electrostatic depositioning in combination with salt and/or enzyme treatment to control the gastrointestinal fate of emulsified oils. Lipid droplets coated by a single-layer of biopolymers (gelatin) were prepared by high pressure homogenization. Lipid droplets coated by a double-layer of biopolymers (gelatin-pectin) were prepared by electrostatically depositing sugar beet pectin on the gelatin-coated droplets. Laccase was added to the double-layer emulsions to covalently crosslink the adsorbed pectin molecules, whereas sodium chloride was added to modulate interfacial properties through electrostatic screening effects. Non-cross-linked and cross-linked double-layer emulsions (with and without salt) were then passed through a simulated gastrointestinal tract (GIT) that included mouth, gastric and intestinal phases. Free fatty acid release profiles suggested that the stability of the emulsified droplets within the GIT played a more important role in determining the rate and extent of lipid digestion than the initial interfacial layer properties.

Retention and release of oil-in-water emulsions from filled hydrogel beads composed of calcium alginate: impact of emulsifier type and pH.

Delivery systems based on filled hydrogel particles (microgels) can be fabricated from natural food-grade lipids and biopolymers. The potential for controlling release characteristics by modulating the electrostatic interactions between emulsifier-coated lipid droplets and the biopolymer matrix within hydrogel particles was investigated. A multistage procedure was used to fabricate calcium alginate beads filled with lipid droplets stabilized by non-ionic, cationic, anionic, or zwitterionic emulsifiers. Oil-in-water emulsions stabilized by Tween 60, DTAB, SDS, or whey protein were prepared by microfluidization, mixed with various alginate solutions, and then microgels were formed by simple extrusion into calcium solutions. The microgels were placed into a series of buffer solutions with different pH values (2 to 11). Lipid droplets remained encapsulated under acidic and neutral conditions, but were released under highly basic conditions (pH 11) due to hydrogel swelling when the alginate concentration was sufficiently high. Lipid droplet release increased with decreasing alginate concentration, which could be attributed to an increase in the pore size of the hydrogel matrix. These results have important implications for the design of delivery systems to entrap and control the release of lipophilic bioactive components within filled hydrogel particles.

Influence of droplet size on the antioxidant activity of rosemary extract loaded oil-in-water emulsions in mixed systems.

The influence of droplet size on the antioxidant activity of oil-in-water emulsions loaded with rosemary extract in mixed emulsion systems was investigated. Firstly, differently sized hexadecane-in-water model emulsions (10% (w/w) hexadecane, 2% (w/w) Tween 80, pH 5 or 7) containing 4000 ppm rosemary extract in the oil phase or without added antioxidant were prepared using a high shear blender and/or high-pressure homogenizer. Secondly, emulsions were mixed with fish oil-in-water emulsions (10% (w/w) fish oil, 2% (w/w) Tween 80, pH 5 or 7) at a mixing ratio of 1 : 1. Optical microscopy and static light scattering measurements indicated that emulsions were physically stable for 21 days, except for the slight aggregation of emulsions with a mean droplet size d43 of 4500 nm. The droplet size of hexadecane-in-water emulsions containing rosemary extract had no influence on the formation of lipid hydroperoxides at pH 5 and 7. Significantly lower concentrations of propanal were observed for the emulsions loaded with rosemary extract with a mean droplet size d43 of 4500 nm from day 12 to 16 at pH 7. Finally, hexadecane-in-water emulsions containing rosemary extract significantly retarded lipid oxidation of fish oil-in-water emulsions in mixed systems, but no differences in antioxidant efficacy between the differently sized emulsions were observed at pH 5.

Isothermal titration calorimetric analysis on solubilization of an octane oil-in-water emulsion in surfactant micelles and surfactant-anionic polymer complexes.

Polymers may alter the ability of surfactant micelles to solubilize hydrophobic molecules depending on surfactant-polymer interactions. In this study, isothermal titration calorimetry (ITC) was used to investigate the solubilization thermodynamics of an octane oil-in-water emulsion in anionic sodium dodecylsulphate (SDS), nonionic polyoxyethylene sorbitan monooleate (Tween 80), cationic cetyltrimethylammonium bromide (CTAB) surfactant micelles and respective complexes formed by these micelles and an anionic polymer (carboxymethyl cellulose). Results indicated that the oil solubilization in single ionic micelles was endothermic, while in nonionic micelles or mixed ionic/nonionic micelles it was exothermic. The addition of carboxymethyl cellulose did not influence the solubilization behavior in these micelles, but affected the solubilization capacities of these systems. The solubilization capacity of cationic micelles or mixed cationic/nonionic micelles was enhanced while that of nonionic or anionic micelles was decreased. Based on the phase separation model, a molecular pathway mechanism driven by enthalpy was proposed for octane solubilization in surfactant micelles and surfactant-polymer complexes.

Impact of alcohols on the formation and stability of protein-stabilized nanoemulsions.

Nanoemulsions are increasingly being used for encapsulation, protection, and delivery of bioactive lipids, however, their formation from natural emulsifiers is still challenging. We investigated the impact of alcohol on the formation and stability of protein-stabilized oil-in-water nanoemulsions prepared by high-pressure homogenization. The influence of different alcohols (ethanol, 1-propanol, and 1-butanol) at various concentrations (0-25% w/w) on the formation and stability of emulsions stabilized by sodium caseinate, whey protein isolate, and fish gelatin was investigated. The mean particle diameter decreased with increasing alcohol concentrations from 0 to 10%w/w, but extensive droplet aggregation occurred at higher levels. This phenomenon was attributed to enhanced protein-protein interactions between the adsorbed emulsifier molecules in the presence of alcohol leading to droplet flocculation. The smallest droplets (d<100nm) were obtained when 10%w/w 1-butanol was added to sodium caseinate-stabilized nanoemulsions, but relatively small droplets (d<150nm) could also be obtained in the presence of a food-grade alcohol (ethanol). This study demonstrated that alcohol addition might be a useful tool for producing protein-stabilized nanoemulsions suitable for use as delivery systems of lipophilic bioactive agents.

Investigations into aggregate formation with oppositely charged oil-in-water emulsions at different pH values.

The pH-dependent formation and stability of food-grade heteroaggregates from oppositely charged oil-in-water (O/W) emulsions was investigated. After screening suitable emulsifiers, 10% (w/w) oil in-water emulsions (d32≈1 µm) were prepared at pH 3-7 using a positively charged emulsifier (Na-lauroyl-l-arginine ethyl ester; LAE) and four negatively charged ones (citric esters of mono- and diglycerides, soy lecithin, sugar beet pectin, and Quillaja saponin). The oppositely charged emulsions were then combined at constant pH values at a volume flow rate ratio of 1:1. Emulsions and heteroaggregates were characterized by their surface charge, particle size distribution and microstructure using dynamic and static light scattering as well as confocal laser scanning microscopy. The emulsifier type was found to greatly influence the type of heteroaggregates formed, as well as the pH value, specifically in combined LAE/Quillaja saponin emulsions. Larger aggregates particularly were formed with increasing pH values (2.71±1.21 to 46.53±4.30 µm from pH 3 to 7, respectively), while LAE/pectin aggregates appeared not to be affected by pH over the full pH range investigated (3.80±2.89 to 3.94±2.78 µm from pH 3 to 7, respectively). Our study thus provides valuable first insights into the mechanism of the formation of food-grade heteroaggregates for later use in food systems.

Solubilization of octane in electrostatically-formed surfactant-polymer complexes.

Polymers can be used to modulate the stability and functionality of surfactant micelles. The purpose of this study was to investigate the solubilization of an octane oil-in-water emulsion in mixtures of an anionic polymer (carboxymethyl cellulose) and anionic sodium dodecylsulphate (SDS), nonionic polyoxyethylene sorbitan monooleate (Tween 80) and cationic cetyltrimethylammonium bromide (CTAB) surfactant micelles using dynamic light scattering, microelectrophoresis and turbidity measurements. The results showed that the addition of anionic carboxymethyl cellulose accelerated octane solubilization in cationic CTAB and CTAB-Tween 80 micelles, but did not affect the solubilization behaviors of micelles that were nonionic and anionic. The surfactant-polymer interactions were also studied using isothermal titration calorimetry (ITC) to characterize different physiochemical interaction regions depending on surfactant concentration in surfactant-polymer systems. Upon octane solubilization in CTAB-carboxymethyl cellulose mixtures, shape transitions of polymer-micelle complexes may have taken place that altered light scattering behavior. Based on these results, we suggest a mechanism for oil solubilization in electrostatically-formed surfactant-polymer complexes.

Stabilization of food dispersions by enzymes.

Food dispersions have become essential vehicles to carry and deliver functional ingredients such as bioactive compounds, flavors, antimicrobials, antioxidants, colors and vitamins. Most of these systems are thermodynamically unstable tending to break down over time. Much research has therefore been carried out to develop methodologies to improve their long-term stability. In this review, we will introduce readers to a new approach that has been developed over the past years to stabilize food dispersions, i.e. by use of various enzymes. First, basic design principles of modern food dispersions including conventional emulsions, multiple emulsions, multilayered emulsions, solid lipid particle suspensions, and liposomes are discussed. Enzymes able to generate intra- and intermolecular crosslinks between proteins and/or polysaccharides will be reviewed and specific reactions catalyzed by, e.g., transglutaminase, laccase, tyrosinase, sulfhydryl oxidase, glucose oxidase, lipoxygenase, polyphenol oxidase, peroxidase, and lysyl oxidase will be highlighted. Finally, potential applications of this enzymatic approach in the food industry will be critically discussed.