Real-Time Determination of Structural Changes of Sodium Caseinate-Stabilized Emulsions Containing Pectin Using High Resolution Ultrasonic Spectroscopy

The interactions that lead to structure transitions in oil-in-water emulsions were investigated using high-resolution ultrasonic spectroscopy. High methoxyl pectin (HMP) was added to the emulsions at various concentrations and the dynamics of aggregation induced by changes in pH were observed. Two independent ultrasonic parameters, velocity and attenuation, were measured as a function of time or pH. At pH 6.8, both velocity and attenuation of sound changed as a function of HMP concentration. During acidification, caused by the addition of glucono-δ-lactone, there were small changes in the overall ultrasonic velocity, but it was possible to relate these changes to the structural changes in the emulsion. The values of ultrasonic attenuation decreased at high pH with increasing amount of HMP, indicating changes in the flocculation state of the oil droplets caused by depletion forces. During acidification at pH 5.4, emulsions containing HMP showed a steep increase in the ultrasonic attenuation, and this pH corresponds to the pH of association of HMP with the casein-covered oil droplets. The adsorption of HMP onto the interface causes a rearrangement of the oil droplets, and the emulsions containing sufficient amounts of HMP no longer gel at acid pH. This is well described by the ultrasonic attenuation changes in the various emulsions. This research demonstrated for the first time that ultrasonic spectroscopy can be employed for in situ monitoring and analysis of acid-induced destabilization of food emulsions.     

Emulsifier Dependent <Emphasis Type="Italic">in vitro</Emphasis> Digestion and Bioaccessibility of β-Carotene Loaded in Oil-in-Water Emulsions

This study was performed to examine the effect of emulsifiers used to coat emulsion droplets containing β-carotene on the behavior of lipid digestion and bioaccessibility. Different emulsifiers (whey protein isolate, soy protein isolate, sodium caseinate, Tween 20, and soy lecithin) were used to prepare emulsions with similar sized droplets (200–400 nm). Protein-stabilized emulsions showed a similar behavior of digestion, and morphological change in the simulated gastrointestinal conditions. Soy lecithin-stabilized emulsions showed the lowest rate and extent of lipid digestion probably due to the low emulsifying capability of soy lecithin, showing coalesced droplets occurring after exposure to the gastric phase. Tween 20-stabilized emulsions had a lower rate and extent of lipid digestion than that of protein-stabilized emulsions, even though Tween 20-stabilized emulsions had a more stable structure to resistant to aggregation in gastric phase. Even though the difference in the digestion rate and extent, β-carotene bioaccessibility was not significantly different among emulsions stabilized by different emulsifiers at p?<?0.05.     

The Influence of Emulsion Droplet Interactions on the Structural,Material and Functional Properties of a Model Mozzarella Cheese

We present findings on the influence of interfacial layer composition on the colloidal interactions and associated structural and material properties of oil-in-protein gel emulsions, as applied to a model Mozzarella cheese analogue. Model cheese samples were produced through thermal mixing of pre-prepared oil-in-water emulsions with a renneted casein gel. Sodium caseinate and Tween 20 were used as the emulsifiers. Microstructural analysis showed sodium caseinate stabilised droplets to be homogeneously dispersed within the cheese structure, whilst droplets stabilised by Tween 20 were phase concentrated into localised fat domains within the continuous protein network. Particle size measurements determined that, on chilled storage, the droplets in these localised regions underwent extensive partial coalescence, whilst the homogenously distributed caseinate droplets showed little change in droplet size. Small deformation rheology (4 to 80 °C) determined the sodium caseinate emulsion as providing a reinforcing effect on the protein network across the entire temperature range, while the Tween 20 emulsion was observed to mechanically strengthen the cheese structure at only at temperatures for which the fat phase was solid whilst serving to weaken the structure on transitioning to a molten state. Differences in droplet structure and stability were determined as influencing cheese melt and flow characteristics. During melting, no oiling-off observed for cheese samples comprising sodium caseinate stabilised droplets, compared to Tween 20 stabilised emulsions where extensive oiling-off was observed. Findings corroborate the hypothesis that caseinate coated droplets behave as active fillers within the protein network, whilst the Tween 20 stabilised emulsion are non-interactive.     

Stability of emulsions stabilised by two physiological surfactants: L-alpha-phosphatidylcholine and sodium taurocholate

The emulsion phase formed within the stomach and duodenum during digestion of a fatty meal has been modelled using two physiological surfactants, the phospholipid L-alpha-phosphatidylcholine (PC) and the bile salt sodium taurocholate (NaT). Upon dilution of the phospholipid stabilised emulsions with a solution of NaT the bile salt became incorporated into the oil/water interface imparting a negative charge to the droplet surface. The magnitude of the droplet microelectrophoretic mobility for the mixed PC and NaT system was 47% of that found for emulsion droplets stabilised by NaT alone. But the electrostatic repulsion between droplets was not sufficient to account for the observed improvement in emulsion stability to coalescence. It is suggested that a residual liquid crystalline phospholipid interface is present imparting a significant steric component to the stabilisation of the emulsions droplets.     

Effects of Modification of Encapsulant Materials on the Susceptibility of Fish Oil Microcapsules to Lipolysis

The aim of the study was to assess the effects of modification of encapsulant materials before emulsion formation on the viscosity and interfacial properties of the emulsions and their influence on the susceptibility of emulsions to in vitro lipolysis. Emulsions (oil/protein ratio 2:1) were prepared by homogenizing mixtures containing fish oil and non-heated or heated (100 °C/120 min) dispersions comprising (a) sodium caseinate (NaCas), (b) mixtures of NaCas and a high amylose-resistant starch (Hylon VII; 1:1 mass ratio), and (c) mixtures of NaCas and previously modified resistant starch (heat/microfluidized [MF] Hylon VII; 1:1 mass ratio), followed by freeze drying. Reconstituted emulsion containing heated mixture of NaCas and heat/MF Hylon VII was the most viscous. The extent of lipolysis was the same in all emulsions stabilized by non-heated NaCas or non-heated mixtures of NaCas with resistant starch. Heat treatment of NaCas increased lipolysis of emulsions stabilized with protein alone, but heating NaCas with Hylon VII or heat/MF Hylon VII before emulsion formation reduced lipolysis. The emulsion stabilized with the heated NaCas–heat/MF Hylon VII mixture was the most resistant to lipolysis. Overall, the resistance to lipolysis was considered to be primarily dependent on the interfacial properties of the microcapsules. These findings of in vitro lipolysis of NaCas-resistant starch formulated oil powders may be relevant to an understanding of in vivo digestibility of the oil powders. The insights may be used as a guide to formulate oil systems for altering the susceptibility to lipolysis of ingested oil emulsions. Delivery of Functionality in Complex Food Systems: Physically inspired Approaches from Nanoscale to Microscale, University of Massachusetts, Amherst, MA, USA, 8th–10th October 2007.     

Effect of Processing and Storage Parameters on the Oxidative Deterioration of Oil-in-Water Emulsions

The effect of storage temperature, pH, and homogenization pressure on the oxidative deterioration of Tween 20 and sodium caseinate sunflower oil-in-water emulsions was studied by monitoring conjugated dienes (CD), lipid hydroperoxides (LH), and thiobarbituric acid reactive substances (TBARs). CD increased linearly with storage time, and the rate constant was temperature dependent according to the Arrhenius equation with an activation energy equal to 37.5 kJ mol⁻¹. The increase in LH and TBARs with temperature (5–60°C) was in good agreement with CD variation. Tween-stabilized emulsions oxidized faster as pH increased from 3 to 7, whereas a different behavior was observed in emulsions stabilized with sodium caseinate or a mixture of both emulsifiers. A change of homogenization pressure (30–900 bars), reflecting variation of emulsion average droplet size, had no effect on the oxidative stability of the emulsions.     

Emulsification Capacity of Microgels Assembled from β-Lactoglobulin and Pectin

Microgels formed from beta-lactoglobulin were used to prepare oil-in-water emulsions in order to examine their emulsifying capacity. Corn oil emulsions prepared with microgels of pure beta-lactoglobulin at pH 5.8 were initially stable, but a fraction of the droplets quickly flocculated to form a creamed layer that could not be dispersed by shear, which was attributed to hydrophobic attractions between the microgels on adjoining droplets. Emulsions prepared from microgels of beta-lactoglobulin and pectin at pH 4.75 possessed greater droplet sizes at lower concentrations, yet all emulsions were relatively stable to irreversible flocculation. Increased stability of emulsions stabilized by BP-gels was attributed to the presence of pectin on the surface of microgels, which increased repulsions between adjoining droplets. Stable corn oil emulsions were still prepared from microgels that were previously dialyzed to remove non-aggregated protein, which verified that the microgels were responsible for stabilizing emulsion droplets. Equilibrium surface pressure of corn oil droplets was similar between microgels and the unheated beta-lactoglobulin and pectin, yet the dynamic surface pressure was reduced at intermediate times and indicated a slow relaxation and deformation of the microgels at the interface. Microgels formed with pectin stabilized emulsions containing 90 % limonene for up to 5 days of room temperature storage, demonstrating the capacity of such protein microgels to stabilize flavor oil emulsions.     

YdfH Identified as a Repressor of rspA by the Use of Reduced Genome Escherichia coli MGF-01

The effects of carnauba wax addition on the physical state of palm kernel oil-in-water emulsions were investigated. The oil-in-water emulsion (40 wt% oil + 60 wt% aqueous phase) kept the liquid state at 25°C irrespective of the presence or absence of carnauba wax in the oil phase. The emulsion containing the wax transformed from the liquid state to the solid state by shearing after storage for 20 h at 4°C, although the liquid-solid transition was not observed for the emulsion not containing the wax upon the same treatment. The viscoelasticity of the solid emulsions was demonstrated by small-deformation mechanical testing. Analysis of flow behavior of the emulsions showed that the change in physical properties of the emulsion containing the wax at 4°C was caused by the shearing at a low shear rate, around 50 s^?1–100 s^?1. According to the transition from the liquid state to the solid state of the emulsion containing the wax, the aggregation of oil droplets was found to occur to a large extent. The results of differential scanning calorimetry and surface pressure–surface area isotherms suggested that triglyceride molecules of palm kernel oil were more oriented at the oil–water interfaces in the emulsions after the wax addition. Based on these results, it is thought that carnauba wax is important in destabilization of palm kernel oil-in-water emulsions by modifying the physical state of the oil triglyceride molecules at the interfaces.     

Rapid exchange of pancreatic lipase between triacylglycerol droplets

Two types of experiments were performed to study the reversibility of interfacial adsorption of pancreatic lipase (PL) to fat droplets during lipolysis. Lipolysis was measured in olive oil/gum arabic emulsions containing radiolabeled triolein in the presence of bile salts and lecithin at rate-limiting concentrations of porcine PL (PPL) or human PL (HPL). The lipolysis rate in a labeled emulsion, i.e. release of [(14)C]oleic acid, was immediately reduced by around 50% upon dilution with an equal amount of an unlabeled emulsion. Further, lipolysis was rapidly and completely suppressed when a non-exchanging lipase inhibitor was present in the second emulsion. These results indicate hopping of lipase between emulsion droplets. Alternative explanations were excluded. Hopping of PL between triolein droplets stabilized with gum arabic at supramicellar bile salt concentrations was observed only in the presence, not in the absence, of lecithin. Displacement from a trioctanoin-water interface of active HPL by an inactive mutant (S152G) was studied in the presence of bile salts by measuring HPL distribution between the water phase and the oil-water interface. Colipase was limiting for HPL binding to the oil-water interface (colipase to lipase molar ratio: 0.5) and, thus, for lipolysis. Upon adding S152G, which has the same affinity for colipase, inactive and active HPL were found to compete for binding at the oil-water interface. When equal amounts of HPL and HPL S152G were used, the lipolysis rate dropped to half the maximum rate recorded with HPL alone, suggesting that half the active HPL was rapidly desorbed from the oil-water interface. Therefore, under various conditions, PL does not remain irreversibly adsorbed to the oil-water interface, but can exchange rapidly between oil droplets, via an equilibrium between soluble and lipid-bound PL.     

Effect of Tween Emulsifiers on the Shear Stability of Partially Crystalline Oil-in-Water Emulsions Stabilized By Sodium Caseinate

We investigated the effects of Tween emulsifier fatty acid chain length on the shear stability and crystallization behavior of 35 wt% partially crystalline oil-in-water emulsions prepared with and without 1 wt% sodium caseinate. Emulsions containing sodium caseinate and Tween 20, 40, 60 or 80 varied in shear stability, degree of supercooling and crystallization behavior depending on the type and concentration of Tween as well as the presence of protein. Generally, emulsions containing the unsaturated emulsifier Tween 80 were the most shear sensitive followed by the saturated emulsifiers Tween 20, 40 and 60 in order of increasing fatty acid chain length. Long chain saturated Tween emulsifiers (40 and 60) improved shear stability regardless of whether sodium caseinate was present indicating that alone, these emulsifiers form more robust interfacial films compared to the saturated short chain length Tween 20 and Tween 80. In emulsions prepared with sodium caseinate, the degree of supercooling decreased and the crystallization rate diminished with increasing saturated fatty acid chain length but only negligible changes were found without sodium caseinate. Together, these findings indicate that long chain saturated Tween emulsifiers provide better emulsion stability regardless of the presence of sodium caseinate but with sodium caseinate, stability may also be affected by changes to fat crystallization. These novel findings provide guidance on how combinations of proteins and emulsifiers can be used to modify and control the stability of partially crystalline oil-in-water emulsions through their combined effects on the properties of the interfacial film and fat crystallization.     

Encapsulation of Resveratrol Using Water-in-Oil-in-Water Double Emulsions

The suitability of water-in-oil-in-water multiple emulsions to encapsulate resveratrol was assessed. Multiple emulsions were prepared by emulsifying a primary emulsion (40 wt.%) in water containing 0.5 wt.% sodium caseinate and 0.1 M NaCl. Four primary emulsions of canola oil (20 wt.%) stabilized by 8 wt.% polyglycerol polyricinoleate were chosen. The dispersed phase of the primary emulsions contained 0.1 M NaCl and either water, 20 wt.% ethanol in water, 2.5 wt.% whey protein isolate (WPI) in water, or 2.5 wt.% WPI and 5 wt.% gelatine in water. Resveratrol was incorporated into these primary emulsions at 0.25 wt.% to give a final 0.02 wt.% resveratrol in the multiple emulsions. Slight increase in particle size with storage at 23 °C for up to 2 weeks was observed. Further, less than 10% of the total encapsulated resveratrol is released to the external, continuous, aqueous phase. This work demonstrates the potential of multiple emulsions to encapsulate resveratrol for food applications.     

Factors Affecting Foamed Emulsions Prepared with an Extract from <Emphasis Type="Italic">Quillaja saponaria</Emphasis> Molina: Oil Droplet Size,pH and Presence of Beta-Lactoglobulin

Oil is well-known to act as antifoam and to destabilize foam lamellae by bridging between two adjacent foam bubbles. It was hypothesized that an optimal oil droplet size exists with respect to the stability of a foamed emulsions, where the oil droplets are sufficiently small to postpone bridging and the amount of free surfactant is sufficient to stabilize the oil/water-interface and the air/water-interface. Emulsions with 0.3% Quillaja saponin and a median oil drop-let size between 0.2 and 2.0 μm were prepared under varying homogenization conditions and characterized in a dynamic foam analyzer. Results confirmed the above mentioned hypothesis. Stability of the foamed emulsions considerably increased with increasing pH, which was attributed to electrostatic repulsion between oil droplets and the effect on the balance between disjoining pressure and capillary pressure. In a binary system containing proteins and saponins, stability of foamed emulsions can be further increased when emulsifiers are added sequentially. When the emulsion is stabilized by β-LG and QS is added after emulsification stability of the foamed emulsion is distinctly higher compared to systems, where QS and β-LG are added prior to emulsification. Future studies should deepen our understanding of these complex dispersed systems by investigating the molecular interactions including other proteins and additional food constituents.     

Emulsions Based on the Interactions Between Lactoferrin and Chitosans

In this work, purification of lactoferrin from whey was performed with high recovery rate. Lactoferrin was then exploited in the preparation of food emulsions. Two tertiary emulsions, formed by olive oil, lecithin, chitosan, and lactoferrin, were compared: both the emulsions showed similar turbidity and stability. In the secondary emulsion formed by oil/lecithin/chitosan, the pH was increased to 9 before addition of lactoferrin. Then, lactoferrin was added, and the pH was stabilized above pH 9. Lactoferrin was found in amounts of 1 to 2.5 mg/ml in the multiple experiments. A fraction of the added lactoferrin was also present in a milky layer above the emulsion layer. This was, to our knowledge, the first study of emulsions made exploiting the interactions between lactoferrin and chitosan. It was noted that chitosan droplets remained soluble, although the hydrocolloid solubility occurs at pH lower than 5.9. These results showed the feasibility of manufacturing lactoferrin-based emulsions as functional foods.     

Effect of Oil Content and Composition on the Gelling Properties of Egg-SPI Proteins Stabilized Emulsion Gels

Effects of different oils on the rheological properties, textural profile, water loss (WL), oil loss (OL) and microstructure of egg-soybean protein isolate (SPI) stabilized emulsion gels were investigated at neutral pH, wherein soybean oil, olive oil and menhaden oil were used to form emulsions. The results showed that viscosity of emulsions progressively increased with the increase of oil content. Similarly, analysis of the rheological behavior of the formulated emulsion gels revealed an increase in the mechanical strength (G’) with the increase in oil concentration, indicating that oil droplets played a significant role in the formation of the gel structure. In addition, at high levels of oil, the hardness and chewiness of emulsion gels were also high, while a slight decrease in springiness and cohesiveness were observed. A linear relationship between hardness and water/oil loss was found, whereas the Pearson correlation suggested that less drainage of water may slow down the outflow of oil. The microstructural images showed a more compact network as a result of the increase of oil content in the formulation. Scarce significant differences were found among emulsion gels formulated with different oil type, suggesting oil composition played a dispensable role on the gelling properties of emulsion gels.

     

Caseinate-Induced Competitive Displacement of Whey Protein from Interfaces

The caseinate-induced competitive displacement of whey protein from planar air-water interfaces was investigated based on atomic force microscopy (AFM) imaging and that from the surfaces of oil droplets immersed in aqueous solution based on AFM force spectroscopy. After the addition of sodium caseinate to the sub-phase, the surface pressure of planar interfacial films of pre-adsorbed whey protein increased from 8 mN/m to up to 21 mN/m. The thicknesses of interfacial films were uniform and remained to be approximately 2 nm at relatively low surface pressures up to 18 mN/m, while they became uneven at higher surface pressures and increased to up to 7.1 nm, presumably due to the compression of interfacial whey protein networks by adsorbed caseinate. The rigidity of oil droplets coated with protein adsorbed to their surfaces was then evaluated based on the slope of approximately linear force-distance curves obtained by pressing an oil droplet against another. The adsorption of whey protein to oil droplet surfaces increased droplets’ rigidity. The subsequent addition of caseinate to the bulk solution surrounding oil droplets coated with pre-adsorbed whey protein further increased droplets’ rigidity. The present results suggest that caseinate adsorbed to an interface to which whey protein had adsorbed in advance did not completely expel pre-adsorbed whey protein molecules into the aqueous phase but caused a compaction of interfacial whey protein networks and thereby strengthened the interfacial film.     

Impact of Heat and Laccase on the pH and Freeze-Thaw Stability of Oil-in-Water Emulsions Stabilized by Adsorbed Biopolymer Nanoparticles

The enzymatic cross-linking of adsorbed biopolymer nanoparticles formed between whey protein isolate (WPI) and sugar beet pectin using the complex coacervation method was investigated. A sequential electrostatic depositioning process was used to prepare emulsions containing oil droplets stabilized by WPI – nanoparticle – membranes. Firstly, a finely dispersed primary emulsion (10 % w/w miglyol oil, 1 % w/w WPI, 10 mM acetate buffer at pH 4) was produced using a high-pressure homogenizer. Secondly, a series of biopolymer particles were formed by mixing WPI (0.5 % w/w) and pectin (0.25 % w/w) solutions with subsequent heating above the thermal denaturation temperature (85 °C, 20 min) to prepare dispersions containing particles in the submicron range. Thirdly, nanoparticle-covered emulsions were formed by diluting the primary emulsion into coacervate solutions (0–0.675 % w/w) to coat the droplets. Oil droplets of stable emulsions with different interfacial membrane compositions were subjected to enzymatic cross-linking. We used cross-linked multilayered emulsions as a comparison. The pH stability of primary emulsions, biopolymer complexes and nanoparticle-coated base emulsions, as well as multilayered emulsions, was determined before and after enzyme addition. Freeze-thaw stability (?9 °C for 22 h, 25 °C for 2 h) of nanoparticle-coated emulsions was not affected by laccase. Results indicated that cross-linking occurred exclusively in the multilamellar layers and not between adsorbed biopolymer nanoparticles. Results suggest that the accessibility of distinct structures may play a key role for biopolymer-cross-linking enzymes.     

Interaction of gum arabic, maltodextrin and pullulan with lipids in emulsions

The interaction of gum arabic, maltodextrin and pullulan with lipids in emulsion systems was investigated. Interfacial tension and interfacial viscosity measurements revealed that only gum arabic could adsorb and form a viscoelastic film at the oil-water interface. Good emulsifying activity was demonstrated for gum arabic, whereas fine emulsions could not be produced from the other polysaccharide solutions and oil. Frequency-dependent increases in the storage and loss moduli were observed for all the polysaccharide solutions. Such rheological behavior did not substantially change when maltodextrin and pullulan were mixed with oil to form emulsions. However, the frequency-dependence of the dynamic moduli disappeared in the gum arabic-stabilized emulsion, suggesting the formation of a network structure in which oil droplets could form junctions with gum arabic chains. The results on the inhibition of lipid oxidation by polysaccharides suggest that gum arabic protected lipids from the attack of lipoxygenase and free radicals by adsorbing at the oil droplet surface.     

Evaluation of Electrostatic Interaction between Lysolecithin and Chitosan in Two-Layer Tuna Oil Emulsions by Nuclear Magnetic Resonance (NMR) Spectroscopy

The main objective of this work was to investigate the electrostatic interaction between lysolecithin and chitosan in two-layer tuna oil-in-water emulsions using nuclear magnetic resonance (NMR) spectroscopy. The influence of chitosan concentration on the stability and properties of these emulsions was also evaluated. The 5 wt% tuna oil one-layer emulsion (lysolecithin-stabilized oil droplets without chitosan) and two-layer emulsions (lysolecithin-chitosan stabilized oil droplets) containing 5 wt% tuna oil, 1 wt% lysolecithin and various chitosan concentrations (0.025–0.40 wt%) were prepared. The one-dimensional (1D) ³¹P and ¹H NMR spectra of emulsions were then recorded at 25 °C. The results showed that addition of chitosan affected the stability and properties of lysolecithin-stabilized one-layer emulsions. The ³¹P NMR peak of the choline head group on lysolecithin molecules disappeared when chitosan was added at concentrations above neutralization concentration (> 0.05 wt%). The ¹H NMR peak intensity monitoring free amino groups (?NH ₃ ⁺) of chitosan showed a strong positive linear relationship to the chitosan concentration with a high correlation coefficient (R² ≈ 0.99). This ¹H NMR peak in emulsions could not be detected for chitosan in emulsions lower than saturation concentration (< 0.15 wt%). These phenomena indicate an electrostatic interaction between lysolecithin and chitosan at droplet surface in emulsion and were consistent with the results from zeta-potential measurements. The T ₂* relaxation time of the choline head group (N-(CH ₃)₃) signal of lysolecithin also confirmed that lysolecithin-chitosan electrostatic interaction occurs at the surface of oil droplets in two-layer emulsions. The results suggest that NMR spectroscopy can be used as an alternative method for monitoring the electrostatic interaction between surfactant and oppositely charged electrolytes or biopolymers in two-layer emulsions.     

Effect of oil droplet size on the oxidative stability of spray-dried flaxseed oil powders

The effect of the size of oil droplets on the oxidative stability of flaxseed oil in spray-dried powders was investigated. Maltodextrin with a dextrose equivalent of 25 was used as a wall material, and sodium caseinate and transglutaminase-polymerized sodium caseinate were used as emulsifiers. The oxidative stability of flaxseed oil encapsulated in the spray-dried powders was evaluated using lipid oxidation and conductometric determination tests at 105 °C. The powders containing larger oil droplets exhibited higher surface oil content after spray drying, and higher peroxide value and conductivity after storage at 105 °C. Removal of the surface oil from the powders by washing with hexane significantly decreased the conductivity. The results indicated that the surface oil of the spray-dried flaxseed oil powders affected the oxidation stability.     

Stabilization of water-in-oil emulsion by <Emphasis Type="Italic">Rhodococcus opacus</Emphasis> B-4 and its application to biotransformation

Rhodococcus opacus B-4, which has recently been isolated as an organic solvent-tolerant bacterium, stabilized water-in-oil (w/o) emulsions by inhibition of droplet coalescence when the cells were dispersed in 90% (v/v) organic solvents. Confocal microscopy revealed that many bacterial cells assembled at the interface between oil and water droplets, though free cells were also detectable at the inside of water droplets. Bacterial cells in the w/o emulsion were capable of utilizing both a water-soluble (glucose) and an oil-soluble substrate (oleic acid) as an energy source. Availability of the w/o emulsion as an immobilized cell system in organic solvents was demonstrated using production of indigo from indole and production of o-cresol from toluene as model conversions. When glucose and oleic acid were simultaneously supplied as energy sources, the w/o emulsion culture of R. opacus B-4 produced indigo and o-cresol at levels of 0.217 and 2.12 mg ml⁻¹, respectively, by 12 h.