Encapsulation of Pediococcus acidilactici cells in corn and olive oil microcapsules emulsified by peptides and stabilized with xanthan in oil-in-water emulsions: Studies on cell viability under gastro-intestinal simulating conditions

Three different encapsulation systems were developed in the form of oil-in-water acidic emulsions (pH 3.0) with the oil phase in the form of microdroplets in which Pediococcus acidilactici cells were enclosed. The first emulsion contained corn oil microdroplets (mean diameter 1.5 μm) emulsified with peptides and stabilized with SDS. The other two, were food grade systems with microdroplets of corn or olive oil (m.d. 2.1 and 2.2 μm, respectively) emulsified with peptides and stabilized with xanthan. In all systems, meat peptone, a rich source of peptides and amino acids, was provided in aqueous solution in which the cultures were suspended. Peptone derived peptides acted as emulsifiers and at the same time as nutrient substrates and osmoprotectants for cells. Emulsions were stored for 30 days at 4 °C. During this period, samples were examined for physical stability and viability of the encapsulated and freely suspended microorganisms present in the emulsions. Examinations were made through phase contrast microscopy and subsequent image capture and analysis with an automatic image analysis system. Viable and non-viable cells were discriminated on the basis of color differences produced through staining with trypan blue. The method permitted extraction of a large amount of information and produced large amounts of data through automated measurements on images. Emulsion characteristics and cell viabilities were also examined under conditions simulating the gastro-intestinal environment. Encapsulation proved to be critical since, following successive treatments of samples with simulated gastric juice (pH 2.0) and intestinal juice (pH 7.4), it ensured viability rates of encapsulated cells as high as 85% and delivered as much as 92% of the initially encapsulated cells to the target point. The formulated emulsion systems may have a large number of applications in the food sector provided further studies on engineering properties and improvements of stability over a wide pH range are carried out.     

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.     

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.     

Changes in Colloidal Properties of Oil in Water Emulsions Stabilized with Sodium Caseinate Observed by Acoustic and Electroacoustic Spectroscopy

Sodium caseinate is a commonly used emulsifier in foods, as it adsorbs on the surface of oil droplets and stabilizes them via electrostatic and steric stabilization, forming a polyelectrolyte layer at the interface. Since the protein interface is affected by varying environmental conditions such as pH, ionic strength, concentration of unadsorbed polymers, these emulsions are prone to a variety of destabilization mechanisms. The objective of the present work was to observe the destabilization of sodium caseinate stabilized oil in water emulsions using electroacoustic spectroscopy. This technique can be utilized for the characterization of concentrated colloidal systems in situ, without dilution. The electroacoustic and ultrasonic properties of soy oil in water emulsions were determined for sodium caseinate stabilized emulsions under conditions known to cause destabilization. Ultrasonic attenuation and electrophoretic mobility (ζ-potential) could clearly follow the changes occurring in the emulsion droplets, under minimal sample disruption. This is critical for these systems in a very fragile, metastable state. The emulsions were stable to the addition of high methoxyl pectin (HMP) up to 0.1% HMP. Addition of free sodium caseinate induced depletion flocculation, causing a decrease in the attenuation and electrophoretic mobility measured. The presence of HMP limited depletion interactions. Acidification of the emulsion droplets resulted in a clear sol–gel transition, as shown by a steep increase in the particle size and a decrease in attenuation. Again, destabilization was limited by HMP addition. It was concluded that ultrasonics and electroacoustics are suitable techniques to understand the details of the destabilization processes occurring to food emulsions, measured in situ.     

Effect of Gum Arabic,Gum Ghatti and Sugar Beet Pectin as Interfacial Layer on Lipid Digestibility in Oil-in-Water Emulsions

The impact of gum arabic (GA), ghatti gum (GG), and sugar beet pectin (SBP) on the digestion rate of emulsified lipids is investigated in vitro under model duodenal digestion condition. The aim was to understand the role of the interfacial layer surrounding the lipid droplets on lipid hydrolysis in order to control lipid digestion. The emulsifier concentration required to provide the same emulsion droplet size decreased in the order: GA > GG > SBP, demonstrating the best emulsifying activity of SBP. The rate and extent of free fatty acid release during lipid digestion did not differ significantly among the three types of gums in emulsions with D[2,3] < 2 μm. However, considerable difference was observed in emulsions with D[2,3] > 2 μm, and the digestive rate decreased in the order: GA > SBP > GG. The difference in digestion rate was attributed to the stability of the emulsified lipid droplets in the stimulated intestinal juice and the resistance of interfacial layer against displacement by bile salts. The difference of resisting against displacement by bile salts for the interfacial layers was detected with bile salts concentration of 0.025 mg/mL, and all of the pre-adsorbed emulsifiers could be completely displaced from interface by bile salts at 5 mg/mL. Emulsions with SBP were susceptible to Ca²⁺ and Na⁺ in simulated intestinal juice, resulting in the flocculation and coalescence of emulsion droplets. A reduction of the surface area of lipids would contribute to a slow digestion. Emulsion stabilized by GG was very effective at retarding lipolysis mainly due to the affinity of linked protein moieties of GG and its hydrophobic binding with bile salts. The knowledge gained in the study has important implications in designing proper emulsion-based systems for controlling lipid digestibility at specific sites within the gastrointestinal tract.     

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.     

Influence of encapsulation on the survival of probiotics in food matrix under simulated stress conditions

The main objective of the present study was to evaluate the influence of encapsulation by extrusion technique using two hydrogels, namely; sodium alginate (Na-ALG) and whey protein isolate (WPI) on Bifidobacterium bifidium viability and stability of yoghurt under simulated gastrointestinal conditions. Probiotic bacteria (free or encapsulated) were added to yogurt for four weeks to test their viability and stability. Physicochemical and sensory analysis of yoghurt were conducted. Viability of B. bifidium in the simulated gastrointestinal conditions pH 2 and pH 7.5 was determined. Also, the efficiency of encapsulated final yield of the microcapsules was determined. With storage time, the pH of yoghurt containing encapsulated bacteria increased more than that of yoghurt containing free probiotic bacteria, resulting in a decrease in acidity. When compared to yoghurt containing encapsulated bacteria, the lactose level of yoghurt containing free probiotic bacteria decreased over time. The viscosity of yoghurt containing encapsulated WPI remained stable over the storage period, with syneresis remaining stable. The sensory properties of yoghurt containing free probiotics deteriorated over time. Cell viability was significantly reduced in yoghurt-containing free probiotics compared to other treated yoghurts. Cell viability in free probiotics yoghurt was lower than in encapsulated ones when exposed to simulated gastric and intestinal juice. In conclusion, WPI- encapsulated probiotics showed better stability over 28 days of storage in both yoghurt and gastrointestinal conditions, followed by sodium alginate.     

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.     

Organoclay-assisted interfacial polymerization for microfluidic production of monodisperse PEG-microdroplets and in situ encapsulation of E. coli

We developed a novel one-pot synthetic strategy for preparing monodisperse polyethylene glycol diacrylate (PEGDA) microdroplets via organoclay-assisted interfacial polymerization approach for Escherichia coli encapsulation. Based on the mechanism of spontaneous and rapid polymerization of PEGDA precursor solution with Mg-organoclay, the prepared PEGDA microdroplets have uniform size and fine round shape, with size range of 74-118 μm. The size of microdroplets can be controlled through the changing continuous phase flow rate. Organoclay-assisted polymerization method provides a unique environment to produce non-toxic ways of fabricating microorganism encapsulated microdroplets and to prohibit microdroplets merge during the processes. Furthermore, we successfully carried out to entrap E. coli inside of the PEGDA microdroplets. E. coli expressing a green fluorescent protein shows a good viability inside the PEGDA microdroplets. The in situ microfluidic synthetic method provides a novel approach for the preparation of monodisperse PEGDA microdroplets via a one-pot route.     

Emulsifying property of a viscous exopolysaccharide fromSphingomonas paucimobilis

A viscous exopolysaccharide fromSphingomonas paucimobilis-GS1, emulsified xylene, benzene, 2-methylnaphthalene, hexadecane, hexane, kerosene and paraffin oil as well as castor, coconut and olive oils when used at 1 mg/ml. It stabilized the emulsions more efficiently than commercial gums such as arabic, tragacanth, karaya and xanthan. Emulsions were stable for 6 months, from 4 to 40°C and pH 4 to 10 and in the presence of NaCl up to 50 g/l. The polysaccharide had no discernible toxicity towards mice when tested using World Health Organization guidelines.The authors are with the Department of Microbiology and Biotechnology Centre, M. S. University of Baroda, Baroda 390 002, India     

Emulsifying properties of a marine bacterial exopolysaccharide

Exopolysaccharide produced by a marine Enterobacter cloaceae (designated as EPS 71a) emulsified hexane, benzene, xylene, kerosene, paraffin oil, cottonseed oil, coconut oil, jojoba oil, castor oil, groundnut oil and sunflower oil. However, it could form stable emulsions with groundnut oil and hexane at optimal concentration of 1 mg ml⁻¹. Further increase in concentration of EPS 71a did not show noteworthy increase in emulsification indices. Emulsions with groundnut oil and hexane were stable up to 10 days between pH 2 and 10 and in the presence of sodium chloride in the range of 5–50 mg ml⁻¹ at 35–37 °C. EPS 71a formed stable emulsion with xylene as compared to commercial gums such as arabic, tragacanth, karaya and xanthan.     

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.     

Interfacial Behavior and Antioxidant Efficiency of Olive Phenolic Compounds in O/W Olive oil Emulsions as Affected by Surface Active Agent Type

The aim of this study was to evaluate the interfacial behavior of syringic acid, tyrosol and oleuropein, phenolic antioxidant compounds naturally present in olives and olive oils, and their ability to influence the stability to lipid oxidation of olive oil O/W emulsions. To test also the interactions of these molecules with other components and the effects on their activity, two different surfactants were used to prepare the olive oil emulsions, Tween 20, and a whey protein concentrate (WPC). All the antioxidants affected the olive oil/water interfacial tension; among them, oleuropein showed the highest interfacial activity and, thus, is supposed to locate at the interface. In emulsified state, the presence of the phenolic compounds in WPC emulsions did not cause any significant effect on the dispersion degree if compared to the control whilst a general improvement was observed in Tween 20-emulsions, in particular when oleuropein was added systems. The antioxidants were thus proven not to impair the dispersed structure but rather to improve it. As regards the oxidative stability, the antioxidants under investigation caused the occurrence of similar induction phases in the hydroperoxides production not observed in the control emulsions. In the case of secondary oxidation products, the highest inhibition was achieved in both the emulsified systems by oleuropein. In general, however, a lower amount of both primary and secondary oxidation products were observed in WPC emulsions than in Tween 20-emulsions likely due to the antioxidative effect of whey proteins.     

Preparation and Characterization of Water-in-Oil-in-Water Emulsions Containing a High Concentration of L-Ascorbic Acid

This study sought to encapsulate a high concentration of L-ascorbic acid, up to 30% (w/v), in the inner aqueous phase of water-in-oil-water (W/O/W) emulsions with soybean oil as the oil phase. Two-step homogenization was conducted to prepare W/O/W emulsions stabilized by a hydrophobic emulsifier and 30% (v/v) of W/O droplets stabilized by a hydrophilic emulsifier. First-step homogenization prepared W/O emulsions with an average aqueous droplet diameter of 2.0 to 3.0 μm. Second-step homogenization prepared W/O/W emulsions with an average W/O droplet diameter of 14 to 18 μm and coefficients of variation (CVs) of 18% to 25%. The results indicated that stable W/O/W emulsions containing a high concentration of L-ascorbic acid were obtained by adding gelatin and magnesium sulfate in the inner aqueous phase and glucose in both aqueous phases. L-Ascorbic acid retention in the W/O/W emulsions was 40% on day 30 and followed first-order kinetics.     

Increased heat tolerance afforded by oil-based conidial formulations of Metarhizium anisopliae and Metarhizium robertsii

The thermotolerance of oil-based conidial formulations of Metarhizium anisopliae s.l. (IP 46) and Metarhizium robertsii (ARSEF 2575) were investigated. Conidia of IP 46 or ARSEF 2575 were suspended in different adjuvants and exposed to 45?±?0.2°C for 4, 6, 8 or 24?h; their viability was then assessed after 48?h incubation at 27?±?1°C. Conidia heated in pure mineral or vegetable oil exhibited mean relative viability exceeding 70% after 8?h of heat exposure, whereas low germination (≤20%) was observed when conidia were heated in water (Tween 80^® 0.01%), carboxymethyl cellulose gel or emulsifiable oils (Graxol^® or Assist^®) and exposed to heat for 6 or 8?h. In addition, conidia of IP 46 suspended in either pure mineral or canola oil and exposed to heat for 48?h had moderate viability, 57% or 41%, respectively. Unstable oil-in-water emulsions showed a higher percentage of conidia incorporated into oil micellae, while the stable emulsions had higher percentage of conidia outside the oil micellae. The thermotolerance of conidia formulated in stable emulsions, however, did not differ from that of conidia formulated in unstable emulsions. The present study highlights possibilities to alleviate the deleterious effects of heat stress towards Metarhizium spp. conidia applied for controlling arthropod pests and vectors through oil-based formulations.     

Partition of lipids between emulsified oil and micellar phases of glyceride-bile salt dispersions

The composition of the emulsified oil and of the micellar phases obtained when a glyceride-fatty acid mixture is dispersed in bile salt solution has been defined. The micellar phase in equilibrium with the emulsified oil phase was obtained by filtration through Millipore filters. The behavior of different lipids in such systems was defined as the partition ratio, micellar/emulsified oil phase (m/o). Partition of fatty acids was found to be strongly dependent on the chain length of the fatty acid and the pH of the dispersion. The curve for partition against pH for oleic acid was interpreted to show a pK(a) for oleic acid in bile salt solution of approximately 7. The partition between micellar and oil phases is given for a series of lipids of different polarity. No significant difference in behavior was found for cholesterol and sitosterol. A relationship was found between the partition m/o and filtration rates through a Millipore filter in micellar solution. The lower the partition coefficient the lower was the rate of filtration. The results obtained are discussed in relation to the mechanism of absorption of fat from the small intestine.     

Synthesis and utilization of E. coli‐encapsulated PEG‐based microdroplet using a microfluidic chip for biological application

We report herein an effective strategy for encapsulating Escherichia coli in polyethylene glycol diacrylate (PEGDA) microdroplets using a microfluidic device and chemical polymerization. PEGDA was employed as a reactant due to the biocompatibility, high porosity, and hydrophilic property. The uniform size and shape of microdroplets are obtained in a single‐step process using microfluidic device. The size of microdroplets can be controlled through the changing continuous flow rate. The combination of microdroplet generation and chemical polymerization techniques provide unique environment to produce non‐toxic ways of fabricating microorganism‐encapsulated hydrogel microbeads. Due to these unique properties of micro‐sized hydrogel microbeads, the encapsulated E. coli can maintain viability inside of microbeads and green fluorescent protein (GFP) and red fluorescent protein (RFP) genes are efficiently expressed inside of microbeads after isopropyl‐β‐D ‐thiogalactopyranoside induction, suggesting that there is no low‐molecular weight substrate transfer limitation inside of microbeads. Furthermore, non‐toxic, gentle, and outstanding biocompatibility of microbeads, the encapsulated E. coli can be used in various applications including biotransformation, biosensing, bioremediation, and engineering of artificial cells. Biotechnol. Bioeng. 2010;107:747–751. © 2010 Wiley Periodicals, Inc.     

Effect of Polyglycerol Esters of Fatty Acids on the Physicochemical Properties and Stability of β-Carotene Emulsions during Digestion in Simulated Gastric Fluid

The effects of six different polyglycerol esters of fatty acids (PGEs) and two different particle sizes produced using various processing parameters on the physicochemical properties and stability of the β-carotene emulsions during digestion in simulated gastric fluid (SGF) were investigated. β-Carotene emulsions were prepared by high-pressure homogenization using β-carotene (0.1% w/w) in soybean oil as the oil phase and 1% (w/w) PGE in Milli-Q water as the water phase. The particle size of β-carotene emulsions was measured by a laser diffraction technique, and the stability of emulsions was interpreted in terms of the increase in particle size and span value of emulsion droplets and the retention of β-carotene during digestion in SGF. The average particle size ranges of emulsions were 0.17 to 0.27 μm for fine emulsions and 1.16 to 1.59 μm for coarse emulsions. In the prepared β-carotene emulsions, the particle size decreased with increasing polymerization of the glycerol in PGEs, and the higher polymerization of the glycerol also increased the stability of emulsions during digestion in SGF. Although the β-carotene content in the emulsions significantly decreased with increasing digestion period, loss of β-carotene was more severe in unstable emulsions than in stable emulsions, suggesting that the particles incorporated into droplets could provide some protective barrier for decreasing the β-carotene degradation. Therefore, β-carotene emulsions stabilized by PGEs with high polymerization of the glycerol may be useful for further applications in food and drug formulations. Decaglycerol monooleate (MO750) was demonstrated to be the most effective emulsifier in stabilizing β-carotene emulsions in this study.     

Microencapsulation of Linoleic Acid with Low- and High-molecular-weight Components of Soluble Soybean Polysaccharide and Its Oxidation Process

Soluble soybean polysaccharide (SSPS) was fractionated into its low- (LMW) and high-molecular-weight (HMW) components to test their antioxidative and emulsifying properties. Linoleic acid was emulsified with an aqueous solution of SSPS, HMW, a mixture of LMW or HMW with maltodextrin, or maltodextrin alone. The emulsions prepared with SSPS, HWM and the mixture of HMW with maltodextrin were stable. These emulsions were spay-dried to produce microcapsules. The encapsulated linoleic acid was oxidized at 37°C and at various levels of relative humidity. Linoleic acid encapsulated with the mixture of LMW with maltodextrin or HMW was stable to oxidation, and this stability increased as the weight fraction of LMW in the mixture was increased. The LMW components also had high DPPH-radical scavenging activity. These results indicate that LMW played an important role in suppressing or retarding the oxidation of linoleic acid encapsulated with SSPS. The oxidative stability of linoleic acid encapsulated with a mixture of the LMW and HMW components was high at low and high relative humidity, but not at intermediate levels of relative humidity.