Development of antimicrobial nanoemulsion-based delivery systems against selected pathogenic bacteria using a thymol-rich Thymus daenensis essential oil.

AIMS: Thymol-rich medicinal plants have been used in traditional medicine to relieve infectious diseases. However, the application of essential oils as medicine is limited by its low water solubility and high vapour pressure. The objective of this study was to produce stable nanoemulsions of Thymus daenensis oil in water by preventing Ostwald ripening and phase separation. METHODS AND RESULTS: The antibacterial activity of bulk and emulsified essential oil against selected pathogenic bacteria including Gram-negative (Haemophilus influenzae, Pseudomonas aeruginosa) and Gram-positive (Streptococcus pneumoniae) were investigated in the liquid and vapour phase. The optimum formulation (L2) contained 2% Tween 80 (surfactant) and 0·1% lecithin (cosurfactant) had a mean droplet diameter of 131 nm. In the liquid phase, the optimized nanoemulsion exhibited good antibacterial activity against S. pneumonia with MIC value of 0·0039 mg mL-1 . In the vapour phase, the MIC values against S. pneumonia were similar (<7·35 µL L-1 ) for both bulk and emulsified essential oil. However, there was no antibacterial activity in the vapour phase against H. influenzae and P. aeruginosa. Analysis of thymol concentration in the head space indicated that the nanoemulsion retarded the release of thymol into the vapour phase. CONCLUSIONS: These findings highlight the potential applications of nanoemulsions containing essential oils as antibacterial products. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of the current study highlight the advantages of nanoemulsification for improvement of the physicochemical properties and the antibacterial activity of T. daenensis EOs in the liquid and vapour phase for therapeutic purposes.

Electrostatic interactions of cationic lauric arginate with anionic polysaccharides affect antimicrobial activity against spoilage yeasts.

AIMS: To investigate the effect of anionic polysaccharides often used in beverage applications (xanthan and λ-carrageenan) on the antimicrobial efficacy of the cationic surfactant lauric arginate (LAE) against typical spoilage yeasts. METHODS AND RESULTS: The antimicrobial efficacy of LAE against Saccharomyces cerevisiae, Candida albicans and Zygosaccharomyces bailii in the absence and presence of anionic polysaccharides was assessed by microtitre and macrobroth dilution assays. Combining LAE with either xanthan or λ-carrageenan caused a pronounced decrease in LAE's antimicrobial efficacy, with the minimal inhibitory and lethal concentrations (MIC and MLC) both increasing with increasing polysaccharide concentration. This reduction in antimicrobial efficacy was more pronounced for the addition of λ-carrageenan. To determine the cause of loss of activity, physical properties of solutions were examined. Turbidity and sedimentation measurements indicated that complexes between LAE and anionic polysaccharides had been formed. Electrophoresis measurements showed that complexes had varying electrical charges and dimensions depending on solution composition. CONCLUSION: Results suggest that electrostatic interactions between LAE and anionic polysaccharides play a major role in complex formation and loss of antimicrobial activity. SIGNIFICANCE AND IMPACT OF THE STUDY: Results have important implications for the utilization of LAE as an antimicrobial agent in beverage and food products containing anionic polysaccharides.

Fabrication, functionalization, and application of electrospun biopolymer nanofibers.

The use of novel nanostructured materials has attracted considerable interest in the food industry for their utilization as highly functional ingredients, high-performance packaging materials, processing aids, and food quality and safety sensors. Most previous application interest has focused on the development of nanoparticles. However, more recently, the ability to produce non-woven mats composed of nanofibers that can be used in food applications is beginning to be investigated. Electrospinning is a novel fabrication technique that can be used to produce fibers with diameters below 100 nm from (bio-) polymer solutions. These nanofibers have been shown to possess unique properties that distinguish them from non-woven fibers produced by other methods, e.g., melt-blowing. This is because first the process involved results in a high orientation of polymers within the fibers that leads to mechanically superior properties, e.g., increased tensile strengths. Second, during the spinning of the fibers from polymer solutions, the solvent is rapidly evaporated allowing the production of fibers composed of polymer blends that would typically phase separate if spun with other processes. Third, the small dimensions of the fibers lead to very high specific surface areas. Because of this the fiber properties may be greatly influenced by surface properties giving rise to fiber functionalities not found in fibers of larger sizes. For food applications, the fibers may find uses as ingredients if they are composed solely of edible polymers and GRAS ingredients, (e.g., fibers could contain functional ingredients such as nutraceuticals, antioxidants, antimicrobials, and flavors), as active packaging materials or as processing aids (e.g., catalytic reactors, membranes, filters (Lala et al., 2007), and sensors (Manesh et al., 2007; Ren et al., 2006; Sawicka et al., 2005). This review is therefore intended to introduce interested food and agricultural scientists to the concept of nano-fiber manufacturing with a particular emphasis on the use of biopolymers. We will review typical fabrication set-ups, discuss the influence of process conditions on nanofiber properties, and then review previous studies that describe the production of biopolymer-based nanofibers. Finally we briefly discuss emerging methods to further functionalize fibers and discuss potential applications in the area of food science and technology.

Nanotechnology Approaches for Increasing Nutrient Bioavailability.

Health-promoting ingredients such as phenolic compounds, vitamins, and minerals are being increasingly introduced into foods and beverages to produce "functional foods" specifically designed to improve human health, well-being, and performance. However, it is often challenging to incorporate these nutraceuticals into foods because they have poor solubility characteristics, impart undesirable flavor profiles, are chemically unstable, or have low bioavailability. This problem can often be overcome by encapsulating the bioactive components in nanoparticle-based delivery systems. The bioavailability of encapsulated bioactive agents often increases when the size of the particles containing them decreases, due to their faster digestion, ability to penetrate the mucus layer, or direct uptake by cells. Nanoparticles can be formulated to survive passage through specific regions of the gastrointestinal tract and then release their payload at a specified point, thus maximizing their potential health benefits. Nutraceutical-loaded nanoparticles can be fabricated through lipid formulations, natural nanocarriers, specialized equipment, biopolymer nanoparticles, and miscellaneous techniques. Classification into these five groups is based on the main mechanism or ingredient used to fabricate the nanoparticles. This chapter focuses on the utilization of food-grade nanoparticles for improving the performance of nutraceuticals in functional foods and beverages.

The influence of lipid droplet size on the oral bioavailability of vitamin D2 encapsulated in emulsions: an in vitro and in vivo study.

Vitamin D deficiency is prevalent in some populations leading to adverse health effects, and therefore there is a need to supplement functional foods and beverages with this important micronutrient. In this study, we examined the influence of the initial lipid droplet size on the in vitro bioaccessibility and in vivo absorption of vitamin D2 encapsulated in oil-in-water emulsions. Changes in particle size, charge, and microstructure were measured as vitamin-loaded lipid droplets were passed through a simulated GIT (mouth, stomach, small intestine). The in vitro studies showed that smaller lipid droplets were digested more rapidly than larger ones, thereby leading to the more rapid formation of mixed micelles in the small intestine capable of solubilizing the lipophilic vitamins. This effect may account for the highest vitamin D2 bioaccessibility being observed for the emulsions containing the smallest droplets. In contrast, the in vivo rat feeding studies suggested that the absorption of vitamin D2 was the highest for the emulsions containing the largest droplets. The poor in vitro-in vivo correlation observed in our study may have occurred for a number of reasons: the simulated GIT did not accurately model the complexity of a real GIT; the in vivo approach used did not monitor changes in vitamin levels in the blood over time. Overall, this study suggests that particle size does influence the gastrointestinal fate of encapsulated oil-soluble vitamins, but that further work is needed to establish strong correlations between in vitro and in vivo methods.

Enhancement of lycopene bioaccessibility from tomato juice using excipient emulsions: Influence of lipid droplet size.

The use of excipient emulsions to increase the bioaccessibility of lycopene in tomato juice was studied by simulating gastrointestinal conditions. The influence of droplet diameter (d=0.17 or 19µm) and thermal treatment (90°C, 10min) on lycopene bioaccessibility was evaluated. Lycopene bioaccessibility was relatively low (<8%) in the absence of excipient emulsions due to the crystalline nature of the carotenoids and their entrapment within chromoplasts. Emulsions containing small droplets were fully digested within the small intestine phase, and led to a higher bioaccessibility (12.5%) than emulsions containing large droplets (10.0%) or emulsion-free samples (7.5%). The relatively modest increase in bioaccessibility was attributed to the high level of entrapment in crystalline form. Thermal processing did not appreciably disrupt tomato cells, and therefore only led to a slight increase in lycopene bioaccessibility. Overall, this study shows that excipient emulsions may increase the bioaccessibility of carotenoids in tomato juices.

Isothermal titration calorimetry study of pectin-ionic surfactant interactions.

Isothermal titration calorimetry (ITC) was used to measure enthalpy changes resulting from injection of anionic (sodium dodecyl sulfate, SDS) or cationic (dodecyl trimethylammonium bromide, DTAB) surfactants into aqueous 1 wt % pectin solutions (30, 60, or 90% methoxylated). In the absence of pectin, the critical micelle concentrations (cmc) determined by ITC were 14.7 mM for DTAB and 7.7 mM for SDS. Binding of DTAB to pectin was endothermic and was attributed to electrostatic attraction between the cationic surfactant and anionic biopolymer. Binding of SDS to pectin was exothermic and was attributed to hydrophobic interactions. Pectin reduced the cmc of SDS, probably because of long-range electrostatic repulsion between the molecules. Above a particular concentration, which depended on pectin and surfactant type, both ionic surfactants promoted pectin aggregation (monitored by turbidity increase). This study demonstrates the potential of ITC for providing valuable information about interactions between polysaccharides and amphiphiles.

Influence of EDTA and citrate on physicochemical properties of whey protein-stabilized oil-in-water emulsions containing CaCl2.

The influence of chelating agents (disodium ethylenediaminetetraacetate (EDTA) and sodium citrate) on the physicochemical properties of whey protein isolate (WPI)-stabilized oil-in-water emulsions containing calcium chloride was determined. The calcium-binding characteristics of EDTA and citrate at 30 degrees C were characterized in aqueous solutions (20 mM Tris buffer, pH 7.0) by isothermal titration calorimetry (ITC). EDTA and citrate both bound calcium ions in a 1:1 ratio, but EDTA had a much higher binding constant. Oil-in-water emulsions (pH 7.0) were prepared containing 6.94% (w/v) soybean oil, 0.35% (w/v) WPI, 0.02% (w/v) sodium azide, 20 mM Tris buffer, 10 mM CaCl(2), and 0-40 mM chelating agent. The particle size, apparent viscosity, creaming stability, free calcium concentration, and particle surface potential of the emulsions were measured. The chelating agents reduced or prevented droplet aggregation in the emulsions. When they were present above a certain concentration (>3.5 mM EDTA or >5 mM citrate), droplet aggregation was prevented. The reduction of aggregation was indicated by decreases in particle size, shear-thinning behavior, apparent viscosity, and creaming. Emulsions containing chelating agents had lower free calcium concentrations and more negatively charged droplets, indicating that the chelating agents improved emulsion stability by binding calcium ions. EDTA could be used at lower concentrations than citrate because of its higher calcium ion binding constant.

Color changes in hydrocarbon oil-in-water emulsions caused by Ostwald ripening.

The influence of Ostwald ripening on the optical properties of hydrocarbon oil-in-water emulsions stabilized by sodium dodecyl sulfate was investigated. The droplet size, spectral reflectance, and tristimulus color coordinates (L, a, and b) of a series of n-hexadecane and n-octadecane oil-in-water emulsions were measured in the presence and absence of a water-soluble red dye (FD&C Red No. 40). The droplets grew more rapidly in the emulsion containing n-hexadecane than in the emulsion containing n-octadecane because of the higher solubility of n-hexadecane molecules in the aqueous phase. Ostwald ripening caused appreciable changes in n-hexadecane emulsion spectral reflectance and color L, a, and b values due to the growth of emulsion droplets. L, a, and b color values and spectral reflectances of n-octadecane emulsions did not significantly change during the course of the experiment. The results were explained in terms of Ostwald ripening theory and a previously described light scattering theory. The model enables emulsion manufacturers to predict color changes in oil-in-water emulsions that exhibit transcondensational ripening.

Impact of weighting agents and sucrose on gravitational separation of beverage emulsions.

The influence of weighting agents and sucrose on gravitational separation in 1 wt % oil-in-water emulsions was studied by measuring changes in the intensity of backscattered light from the emulsions with height. Emulsions with different droplet densities were prepared by mixing weighting agents [brominated vegetable oil (BVO), ester gum (EG), damar gum (DG), or sucrose acetate isobutyrate (SAIB)] with soybean oil prior to homogenization. Sedimentation or creaming occurred when the droplet density was greater than or lower than the aqueous phase density, respectively. The weighting agent concentrations required to match the oil and aqueous phase densities were 25 wt % BVO, 55 wt % EG, 55 wt % DG, and 45 wt % SAIB. The efficiency of droplet reduction during homogenization also depended on weighting agent type (BVO > SAIB > DG, EG) due to differences in oil phase viscosity. The influence of sucrose (0-13 wt %) on the creaming stability of 1 wt % soybean oil-in-water emulsions was also examined. Sucrose increased the aqueous phase viscosity (retarding creaming) and increased the density contrast between droplets and aqueous phase (accelerating creaming). These two effects largely canceled one another so that the creaming stability was relatively insensitive to sucrose concentration.

Impact of preferential interactions on thermal stability and gelation of bovine serum albumin in aqueous sucrose solutions.

The influence of sucrose (0--40 wt %) on the thermal denaturation and gelation of bovine serum albumin (BSA) in aqueous solution has been studied. The effect of sucrose on heat denaturation of 1 wt % BSA solutions (pH 6.9) was measured using ultrasensitive differential scanning calorimetry. The unfolding process was irreversible and could be characterized by a denaturation temperature (T(m)), activation energy (E(A)), and pre-exponential factor (A). As the sucrose concentration increased from 0 to 40 wt %, T(m) increased from 72.9 to 79.2 degrees C, E(A) decreased from 314 to 289 kJ mol(-1), and ln(A/s(-1)) decreased from 104 to 94. The rise in T(m) was attributed to the increased thermal stability of the globular state of BSA relative to its native state because of differences in their preferential interactions with sucrose. The change in preferential interaction coefficient (Delta Gamma(3,2)) associated with the native-to-denatured transition was estimated. The dynamic shear rheology of 2 wt % BSA solutions (pH 6.9, 100 mM NaCl) was monitored as they were heated from 30 to 90 degrees C, held at 90 degrees C for either 15 or 120 min, and then cooled to 30 degrees C. Sucrose increased the gelation temperature due to thermal stabilization of the native state of the protein. The complex shear modulus (G) of cooled gels decreased with sucrose concentration when they were held at 90 degrees C for 15 min because the fraction of irreversibly denatured protein decreased. On the other hand, G of cooled gels increased with sucrose concentration when they were held at 90 degrees C for 120 min because a greater fraction of irreversibly denatured protein was formed and the strength of the protein-protein interactions increased.

Ultrasonic determination of chicken composition.

An ultrasonic technique has been developed for measuring the composition of chicken meat. The relationship between the composition and ultrasonic velocity of chicken meat was determined using chicken analogues of different composition, prepared from dried chicken powder, corn oil, and distilled water. The ultrasonic velocity of chicken analogues was measured at temperatures from 5 to 35 degrees C using an ultrasonic spectrometer. The ultrasonic velocity increased with solids-nonfat (SNF) content at all temperatures but had a more complex dependence on fat content. Around 15 degrees C the ultrasonic velocity was independent of fat content; however, at lower temperatures it increased with fat content, and at higher temperatures it decreased. Semiempirical equations were developed to describe the relationship between ultrasonic velocity and chicken composition. To determine the usefulness of these equations, the ultrasonic velocities of various chicken meats were measured. The compositions of the chicken meats predicted on the basis of ultrasonic measurements were in good agreement with those determined by using standard methods (r(2) > 0. 97). The ultrasonic technique could also be used to measure the solid fat content of chicken fat. This study shows that ultrasonic velocity measurements can be used to characterize chicken composition. This method has great potential for application in the food industry because it is simple, fast, nondestructive, and reliable.

Influence of sucrose on the thermal denaturation, gelation, and emulsion stabilization of whey proteins.

The influence of sucrose (0-40 wt %) on the thermal denaturation and functionality of whey protein isolate (WPI) solutions has been studied. The effect of sucrose on the heat denaturation of 0.2 wt % WPI solutions (pH 7.0) was measured using differential scanning calorimetry. Sucrose increased the temperature at which protein denaturation occurred, for example, by 6-8 degrees C for 40 wt % sucrose. The dynamic shear rheology of 10 wt % WPI solutions (pH 7.0, 100 mM NaCl) was monitored as they were heated from 30 to 90 degrees C and then cooled to 30 degrees C. Sucrose increased the gelation temperature and the final rigidity of the cooled gels. The degree of flocculation in 10 wt % oil-in-water emulsions stabilized by 1 wt % WPI (pH 7.0, 100 mM NaCl) was measured using a light scattering technique after they were heated at fixed temperatures from 30 to 90 degrees C for 15 min and then cooled to 30 degrees C. Sucrose increased the temperature at which maximum flocculation was observed and increased the extent of droplet flocculation. These results are interpreted in terms of the influence of sucrose on the thermal unfolding and aggregation of protein molecules.

Maltodextrin-anionic surfactant interactions: isothermal titration calorimetry and surface tension study.

Interactions between maltodextrin (DE = 10) and an anionic surfactant (sodium dodecyl sulfate, SDS) were studied in a buffer solution (pH 7.0, 10 mM NaCl, 20 mM Trizma, 30.0 degrees C) using isothermal titration calorimetry (ITC), surface tension, differential scanning calorimetry (DSC), and turbidity techniques. ITC measurements indicated that the binding of SDS to maltodextrin was exothermic and that, on average, one SDS monomer bound per 24 glucose units of maltodextrin at saturation. Surface tension measurements indicated that there was a critical surfactant concentration ( approximately 0.05 mM SDS) below which surfactant and maltodextrin did not interact and that the amount of surfactant bound to the maltodextrin above this concentration increased with increasing maltodextrin concentration. Turbidity measurements indicated that the solutions remained transparent at all maltodextrin (0-1 wt %) and SDS (0-20 mM) concentrations studied, which suggested that phase separation did not occur. DSC measurements indicated that no phase transitions occurred between 10 and 110 degrees C for maltodextrin solutions (0.5 wt %) in the presence or absence of surfactant. A phase diagram was developed to describe the interactions between SDS and maltodextrin.

Lipid oxidation in emulsions as affected by charge status of antioxidants and emulsion droplets.

The influence of charge status of both lipid emulsion droplets and phenolic antioxidants on lipid oxidation rates was evaluated using anionic sodium dodecyl sulfate (SDS) and nonionic polyoxyethylene 10 lauryl ether (Brij)-stabilized emulsion droplets and the structurally similar phenolic antioxidants gallamide, methyl gallate, and gallic acid. In nonionic, Brij-stabilized salmon oil emulsions at pH 7.0, gallyol derivatives (5 and 500 microM) inhibited lipid oxidation with methyl gallate > gallamide > gallic acid. In the Brij-stabilized salmon oil emulsions at pH 3.0, low concentrations of the galloyl derivatives were prooxidative or ineffective while high concentrations were antioxidative. In SDS-stabilized salmon oil emulsions, oxidation rates were faster and the galloyl derivatives were less effective compared to the Brij-stabilized emulsions. Differences in antioxidant activity were related to differences in the ability of the galloyl derivatives to partition into emulsion droplets and to increase the prooxidant activity of iron at low pH.

Impact of protein surface denaturation on droplet flocculation in hexadecane oil-in-water emulsions stabilized by beta-lactoglobulin.

The influence of globular protein denaturation after adsorption to the surface of hydrocarbon droplets on flocculation in oil-in-water emulsions was examined. n-Hexadecane oil-in-water emulsions (pH 7.0) stabilized by beta-lactoglobulin (1-wt % beta-Lg) were prepared by high-pressure valve homogenization. NaCl (0-150 mM) was added to these emulsions immediately after homogenization, and the evolution of the mean particle diameter (d) and particle size distribution (PSD) was measured by laser diffraction during storage at 30 degrees C for 48 h. No change in d or PSD was observed in the absence of added salt, which indicated that these emulsions were stable to flocculation. When 150 mM NaCl was added to emulsions immediately after homogenization, d increased rapidly during the following few hours until it reached a plateau value, while the PSD changed from monomodal to bimodal. Addition of N-ethylmaleimide, a sulfhydryl blocking agent, to the emulsions immediately after homogenization prevented (at 20 mM NaCl) or appreciably retarded (at 150 mM NaCl) droplet flocculation. These data suggests that protein unfolding occurred at the droplet interface, which increased the hydrophobic attraction and disulfide bond formation between droplets. In the absence of added salt, the electrostatic repulsion between droplets was sufficient to prevent flocculation, but in the presence of sufficient salt, the attractive interactions dominated, and flocculation occurred.

Mechanisms of the antioxidant activity of a high molecular weight fraction of whey.

The antioxidant mechanisms of whey proteins in a Tween 20-stabilized salmon oil-in-water emulsion were investigated. The antioxidant activity of the high molecular weight (HMW) fraction of whey from pasteurized milk was found to increase with concentration, as determined by its ability to inhibit TBARS and lipid peroxide formation. The ability of sulfhydryl-blocked whey to inhibit TBARS formation was reduced 60% compared to the HMW fraction alone at 7 days of storage. HMW fraction was able to scavenge peroxyl radicals, with scavenging decreasing approximately 20% when sulfhydryls were blocked. HMW fraction was able to chelate iron away from the surface of negatively charged BSA-stabilized emulsion droplets, indicating that the whey proteins were able to chelate iron. A better understanding of the mechanisms by which whey proteins inhibit lipid oxidation could increase the use of whey proteins as food antioxidants.

Impact of tween 20 hydroperoxides and iron on the oxidation of methyl linoleate and salmon oil dispersions.

To determine the role of surfactant hydroperoxides on the oxidative stability of fatty acids, the oxidation of methyl linoleate micelles and salmon oil-in-water emulsions was measured as a function of varying Tween 20 hydroperoxide concentrations. Increasing Tween 20 hydroperoxide concentrations from 3.5 to 14.7 micromol hydroperoxide/g Tween 20 decreased the lag phase of headspace hexanal formation but did not increase the total amount of hexanal formed in methyl linoleate/Tween 20 micelles. In the micelle system, Fe(2+) decreased the lag phase of hexanal formation but increased total hexanal concentrations only in micelles with the highest Tween 20 hydroperoxide concentrations (14.7 micromol hydroperoxide/g surfactant). Increasing Tween 20 surfactant hydroperoxide concentrations also increased the oxidation of salmon oil-in-water emulsions as determined by lipid hydroperoxides and headspace propanal. In both the micelle and emulsion systems, the prooxidant effect of Fe(2+) decreased with increasing Tween 20 hydroperoxide concentrations. These data show that surfactant hydroperoxides such as those in Tween 20 could decrease the oxidative stability of lipids in food emulsions.

Evidence that homogenization of BSA-stabilized hexadecane-in-water emulsions induces structure modification of the nonadsorbed protein.

The structural modification of globular proteins (bovine serum albumin, BSA) in the aqueous phase of emulsions produced by homogenization was studied using front-face fluorescence spectroscopy (FFFS). A series of hydrocarbon oil-in-water emulsions (30 wt % n-hexadecane, 0.35 wt % BSA, pH 7.0) were homogenized to differing degrees with a high-speed blender and a high-pressure valve homogenizer. The wavelength of the maximum in the tryptophan emission spectrum (lambda(max)) of serum phases collected from the emulsions by centrifugation was measured and compared to lambda(max) values of BSA solutions subjected to the same homogenization conditions. There was no significant (p < 0.05) change in lambda(max) with homogenization conditions for BSA solutions. In contrast, lambda(max) of serum phases from emulsions blended for 2 min in a high-speed blender was significantly smaller (p < 0.05) than nontreated BSA solutions (Deltalambda(max) = 2 nm). In addition, there was a further significant decrease in lambda(max) of the serum phases with an increasing number of passes of the emulsion through the high-pressure valve homogenizer (e.g., Deltalambda(max) = 4 nm for 12 passes). This study shows that globular proteins present in the aqueous phase of a hexadecane-in-water emulsion after homogenization could be altered, which is probably caused by surface modification of the protein structure during temporary adsorption to emulsion droplet surfaces during homogenization.