Enhancing the Stability of Oil-in-Water Emulsions Emulsified by

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Enhancing the Stability of Oil-in-Water Emulsions Emulsified by Coconut Milk Protein with the Application of Acoustic Cavitation Virangkumar N. Lad and Zagabathuni Venkata Panchakshari Murthy* Department of Chemical Engineering, Sardar Vallabhbhai National Institute of Technology, Surat 395007, Gujarat, India ABSTRACT: Coconut milk protein (CMP) is a naturally derived protein recovered from the kernel of fresh coconut (Cocos nucifera L.) having a high nutrient value. With increasing demand of naturally available efficient emulsifiers and stabilizers for the production of food and health care emulsions with reasonable stability, many emulsifiers are being utilized for the commercial production of many products. Even though the CMP is reported as a poor emulsifier, we prepared very stable emulsions with CMP using sonication. The effects of ultrasound (250 W, 20 kHz and 120 W, 20 kHz) on the stability of sunflower oil-in-water emulsions made by CMP are studied. It is found that though the acoustic energy is responsible for further reduction of droplet size for CMP emulsions, energetic cavitations and high pressure shock waves, generated due to the collapsing bubble, are responsible for droplet breakup. The size of the dispersed droplets, in the case of sonication using an ultrasonic horn with all the concentrations of CMP, was smaller than that created using an ultrasonic bath. Emulsions sonicated by the ultrasonic horn were found to be very stable with variation of salt concentration.

1. INTRODUCTION Emulsions are liquid−liquid dispersions in which droplets of one of the liquid phases (known as the dispersed phase) are dispersed in the other immiscible liquid phase (known as the continuous phase). Depending on the type of dispersed phase and continuous phase, emulsions are categorized as oil-in-water (O/W) or water-in-oil (W/O) emulsions. The O/W emulsions consist of droplets of organic oil phases dispersed in aqueous media. The W/O emulsions contain aqueous liquid phase dispersed in immiscible organic oil phase. The process of emulsification is entropically unfavorable; hence emulsions are thermodynamically unstable systems. Emulsions find many applications in various industries such as the pharmaceutical, food, cosmetics, agriculture, paints, and polymeric chemicals. Surface active agents, having amphiphilic molecular structure, are capable of emulsifying and stabilizing such liquid dispersions. Proteins are a very important class of molecules involved in the formation and stabilization of many food and cosmetic emulsions.1 Soy protein,2,3 whey protein,4−6 sodium caseinate,2,7−9 casein,1,10 β-lactoglobulin,4,11−14 bovine serum albumin,15 etc. are found to be suitable candidates as emulsifying agents. However, high sensitivity to temperature, pH, and ionic strength of protein stabilized emulsions have offered a constraint to their use in many food products.16 Kong et al.17 studied the stability of emulsions prepared by protein and sucrose ester and discussed the micellar interaction in emulsion containing protein. Dunlap and Côté18 found that increasing the size of the polysaccharide conjugated with β-lactoglobulin resulted in more stable emulsions. Usages of protein hydrolysates,10,19 mixtures of protein and hydrocolloids,20−22 and blending of other emulsifiers with proteins23,24 have been found to be beneficial for protein stabilized emulsions against environmental stresses. Fresh coconut milk mainly contains water (about 54%), 35− 37% fat, 2−4% protein, 2−5% carbohydrates, and nonfat solid matters.25−28 Tangsuphoom and Coupland29 have reported © 2012 American Chemical Society

improved stability of coconut milk emulsions homogenized with various surface active agents such as sodium caseinate, whey protein isolate (WPI), sodium dodecyl sulfate (SDS), and polyoxyethylene sorbitan monolaurate. Consequently, they29 have found that the homogenized coconut milk prepared without additives destabilized by freeze−thaw cycles between −20 and −10 °C but not by chilling up to 5 °C. They also found that the emulsions prepared using WPI exhibited appreciable coalescence and phase separation after being heat treated at 90 °C.30 Onsaard et al.31 have studied corn oil-in-water emulsions prepared by coconut milk protein (CMP) using high pressure valve homogenization and found that CMP can be effectively used for stabilizing fairly viscous emulsions such as sauces, desserts, and salad dressings. The fair stability of emulsions prepared using CMP for other less viscous food emulsions still remains a challenge. Li and Fogler32 have explained the concept of acoustic emulsification, mentioning that the ultrasonic waves cause an interfacial instability at oil−water interfaces. The transient cavitation bubbles produced during sonication collapse with generation of high pressure shock waves. This phenomenon is almost adiabatic and generates very high local temperatures (3000 K to more than 10 000 K) and pressures (more than 100 MPa) within bubbles for a short period of time.33−36 Cucheval and Chow37 have demonstrated the emulsion formation by power ultrasound incorporated by high speed cavitation for a soybean oil-in-water system using Tween-80 as a surfactant. Kentish et al.38 have successfully prepared flaxseed oil-in-water emulsions using Tween-40 surfactant with a mean droplet size as low as 135 ± 5 nm using ultrasonic power at 20−24 kHz. Abismail̈ et al.39 have prepared kerosene-in-water emulsions by sonication Received: Revised: Accepted: Published: 4222

November 28, 2011 February 11, 2012 February 19, 2012 February 21, 2012 dx.doi.org/10.1021/ie202764f | Ind. Eng. Chem. Res. 2012, 51, 4222−4229

Industrial & Engineering Chemistry Research

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2.2. Preparation of Emulsion Samples. Phosphate buffer (5 mM) was prepared and adjusted to pH 6.2 corresponding to the pH of the aqueous phase of many coconut milk based products.31,42 Protein solutions were prepared by dispersing the required quantity of protein in buffer solution to have final emulsion samples containing CMP concentration varying from 0.2 to 2 wt %. Sunflower oil was used as the oil phase and was purchased from a local supermarket. The O/W emulsions were prepared by dispersing sunflower oil in protein solutions of required amounts using a high speed Ultra Turrax homogenizer (T25 Basic, IKA WERKE, Germany) for 4 min at 6500 rpm. 2.3. Application of Ultrasound to the Emulsion Samples. Homogenized emulsions were subjected to sonication using an ultrasonic bath (Ultrasonic Cleaner, Aqua Scientific Instruments, Surat, India) or an ultrasonic horn (Sonicator, Aqua Scientific Instruments, Surat, India). Ultrasound was provided by (i) the stainless steel ultrasonic bath operated at 120 W with a frequency of 20 kHz, (ii) the ultrasonic bath operated at 250 W with a frequency of 20 kHz, (iii) the ultrasonic horn of stainless steel having 15 mm diameter operated at 120 W with a frequency of 20 kHz, or (iv) the ultrasonic horn of stainless steel having 15 mm diameter operated at 250 W with a frequency of 20 kHz as per the requirements. In all the experiments, the ultrasound was applied in three stages for 4 min on mode (total 12 min) with 1 min intervals between the successive stages of sonication. 2.4. Evaluation of Droplet Size. The droplet size distribution for the emulsion samples was found using the dynamic light scattering (DLS) technique (Malvern Instruments, U.K.). It measures the Brownian motion of particles in the dispersion and relates it to the particle size. The DLS technique gives the harmonic intensity-weighted average hydrodynamic diameter or cumulative mean. The hydrodynamic diameter is calculated by the well-known Stokes−Einstein equation:44,45

using polyethoxylated sorbitan monostearate as the surfactant and obtained droplet sizes much smaller than the emulsions prepared by mechanical agitation. Though ultrasound has been used for emulsification using a variety of surfactants for many decades,39−41 the effects of sonication on coconut milk protein are yet to be understood. Because coconut milk has high medicinal and nutrient value, it is advantageous to explore the possibility of replacing conventional emulsifiers and stabilizers for emulsions used in cosmetics, pharmaceuticals, and food emulsions with reasonable stability. The objective of the present work is to study the effects of acoustic energy on the stability of emulsions made by coconut milk protein. Also, the properties of sunflower oil-in-water emulsions, prepared by acoustic energy using coconut milk protein, have been evaluated.

2. EXPERIMENTAL PROCEDURES 2.1. Recovery of Coconut Milk Protein. Coconuts (Cocos nucifera L.) were purchased from a local market. Mature coconuts were dehisced and cracked to get the kernels. The fresh kernels of mature coconuts were finely comminuted using a domestic electric grater. The coconut milk was obtained by pressing the grated kernel in a triple-layer cloth filter. The cream and skim milk were obtained from the coconut by centrifuging the coconut milk in a laboratory centrifuge at 4000g for 40 min. This coconut milk was refrigerated overnight at 6 ± 2 °C in order to separate a soluble supernatant phase from an insoluble precipitate phase. The pH of the supernatant phase was adjusted to 3.9 using 0.1 M HCl, and it was centrifuged at 5400g for 25 min twice with an interval of 3 min in between. Precipitates thus obtained were dispersed in Millipore water of pH 5.9 ± 0.2 and conductivity 1.0 μS/cm (Millipore, Bangalore, India), and the pH was adjusted to 7.0 using 0.1 M NaOH. It was then dried at room temperature to get powdered material which was used as coconut milk protein. The drying by atmospheric air at room temperature was done for approximately 15−16 h which avoided the exposure of CMP to elevated temperature and helped to prevent any possible thermal denaturation of the CMP at higher temperature. In order to achieve the microbiological purity of the O/W emulsion samples containing CMP, we used sodium benzoate (food grade) as an antimicrobial agent (a preservative) during preparation of protein solutions. The protein solubility was measured as per the method described by Onsaard et al.42 in principle. The CMP was added to Millipore water containing 10, 100, 200, and 400 mM NaCl and adjusted to pH values ranging from 3 to 8. The solutions were stirred with a magnetic stirrer (Remi Motor, Mumbai, India) at room temperature for 8 h and centrifuged thrice at 7500g for 20 min with 3 min of idling the centrifuge in between. The solution was filtered using Whatman No. 1 filter paper, and the filtered supernatant was analyzed for protein content using the Lowry method,43 with whey protein isolate as a standard. The protein solubility (% PS) was calculated using the following expression:

dh =

κT 3πηD

(2)

where dh is the hydrodynamic diameter, κ is the Boltzmann constant, T is the absolute temperature, η is the viscosity of the dispersion medium, and D is the translational diffusion coefficient. Detailed discussions of the DLS principle are available in the literature.44,46,47 The emulsion samples were diluted 100 times to prevent multiple scattering. The emulsion particle refractive index was taken as 1.456 as reported in the literature,45 and that for the dispersion medium was 1.33. The relative refractive index of 1.09 was used in the calculation of the droplet size distribution. It was then presented in terms of the volume-weighted mean diameter, also known as the De Brouker diameter. The volumeweighted mean diameter is calculated by the following formula:48 d4,3 =

∑ nidi 4 ∑ nidi 3

(3) th

P % PS = s ·100 Pt

where ni is the number of droplets in the i fraction and di is the diameter of a droplet in the ith fraction. 2.5. Microscopy. Samples of emulsions were placed on a microscope slide, gently covered with a coverslip and observed using an optical microscope (Labovision, Ambala Cantt, India) equipped with a digital video camera. Micrographs were taken from four different fields on each slide and representative

(1)

where Ps is the protein concentration in the supernatant after filtration and Pt is the total protein concentration present in the original solution. 4223

dx.doi.org/10.1021/ie202764f | Ind. Eng. Chem. Res. 2012, 51, 4222−4229


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micrographs were obtained. A few samples were also analyzed for the droplet size measurement by image analysis using the image analysis software Image J of NIH, USA. 2.6. Stability to Creaming. The creaming index was calculated by measuring the height of the lower aqueous layer HA and the initial height of the emulsion HE: % creaming index =

HA ·100 HE

(4)

The creaming index provides indirect information of droplet aggregation in an emulsion;29 in general, the more aggregation, the faster the creaming. 2.7. Analysis by Turbiscan. Turbiscan (Formulaction, France) was used to study the stability of samples using backscattering technique. The principle of Turbiscan can be found elsewhere.49 All measurements were repeated once with freshly prepared samples all the time.

Figure 1. Variation of solubility of coconut milk protein with pH.

3.3. Effects of Ultrasound and Concentration of Coconut Milk Protein on the Droplet Size of Dispersed Phase in the Emulsions. Figure 2 shows the volume-weighted mean droplet size (d4,3) of the dispersed phase in 10% O/W emulsions prepared with different concentrations of CMP. The droplet sizes were measured within 30 min after preparation of emulsion samples, at the end of 7 days, and at the end of 14 days after preparation of samples. The samples were stored at 26−30 °C. The emulsions prepared with CMP concentration less than 1%, without the application of sonication, exhibited phase separation on the eighth or ninth day after preparation. The droplet size decreased for all the emulsions prepared without using ultrasound as well as for emulsions prepared using an ultrasonic bath up to 1.2% CMP concentration. No significant reduction in the droplet size was observed beyond 1.2% concentration of CMP. This shows that the concentration of 1.2% CMP was sufficient to produce almost stable emulsions in the case of emulsions prepared without ultrasound as well as for emulsions prepared using the ultrasonic bath. The minimum amount of CMP was 1.6%, which resulted in the formation of oil droplets of 3.2 μm in the case of emulsification with application of ultrasound at 120 W and 20 kHz using an ultrasonic horn. Further increase in the concentration of CMP did not alter the mean size of the droplets appreciably. This is due to the fact that sonication using the ultrasonic horn created vigorous cavitations and ultimately led to the formation of smaller droplets, resulting in more interfacial area. Hence, the minimum amount of CMP used to occupy this interfacial area was increased to 1.6%. Further, for all the range of CMP concentration, the smallest size of emulsion droplets was produced by the use of the ultrasonic horn. It is clear from Figure 2 that the power ultrasound has resulted in significant reduction in droplet size. For instance, with 1% CMP, the mean size of dispersed droplets in emulsions sonicated by the ultrasonic bath was 18.1 μm and that sonicated by the ultrasonic horn was 5.9 μm, whereas the mean size of droplets in the absence of ultrasound was 22.3 μm. A similar reduction in mean droplet size has also been reported by Cucheval and Chow37 for a soybean oil-in-water emulsion with Tween-80 where they obtained a droplet size of 0.7 μm using power ultrasound. The reduction of the mean droplet size due to the application of ultrasonic power has also been reported by Juang and Lin53 for water-in-oil emulsions prepared using Span80 as an emulsifier. Gaikwad and Pandit54 and Tal-Figiel55 also

3. RESULTS AND DISCUSSION 3.1. Composition of Coconut Milk Protein. The composition of the protein recovered from coconut milk is presented in Table 1 based on triplicate experimental runs. The amount of Table 1. Composition of Coconut Milk Protein Recovered from Fresh Coconuta component proteins (by nitrogen conversion factor 6.25) fat moisture ash carbohydrates (by difference)

% by weight 62.1 26.9 2.9 4.8 3.3

± ± ± ± ±

0.2 0.1 0.2 0.1 0.9

a

Deviations indicate the variation of the corresponding value during triplicate experimental runs.

protein found in the recovered material is comparable to that of the coconut skim milk protein isolate as reported by Onsaard et al.42 Detailed characterization of the protein recovered from coconut milk is out of the scope of the present paper. Kwon et al.50 have reported the characterization of coconut protein and showed that albumins and globulins were the predominant coconut protein fractions. They50 concluded that the coconut protein had relatively high levels of glutamic acid, arginine, and aspartic acid. They50 also reported that the total protein, globulins, and glutelins were composed of polypeptides linked via disulfide bond(s). 3.2. Solubility of Coconut Milk Protein. The effect of pH on the CMP solubility is shown in Figure 1. The minimum CMP solubility occurred between 4 and 4.5, which is in the near vicinity of the isoelectric point of protein as reported by various researchers.25,42,51,52 As shown in Figure 1, the solubility of CMP increases with the pH as the sample set farther from that corresponding to the isoelectric point. The reason for this behavior can be explained by the fact that, at the isoelectric point, there is negligible electrostatic repulsion between protein molecules due to the presence of almost zero net charge on their surfaces. This electrostatic repulsion becomes stronger due to the increase in net charge on the surfaces of molecules at pH values farther than that corresponding to the isoelectric point. The strong electrostatic repulsion prevents the aggregation of molecules and results in increased solubility.42 4224



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Figure 2. Effect of ultrasound (120 W, 20 kHz) and concentration of coconut milk protein on the volume mean diameter of dispersed droplets in O/W emulsions. Solid filled bars represent mean droplet size of dispersed droplets in emulsions measured within 30 min after their preparation. Bars with slanted strips and horizontal strips represent the droplet size measured at the end of the 7th day and the 14th day, respectively, after emulsion preparation.

observed reduction in the droplet size of the dispersed phase due to acoustic power. It is observed that an increase in the power of ultrasound resulted in the formation of smaller droplets. This is shown in Figure 3a for a CMP concentration of 1.2% and in Figure 3b for a CMP concentration of 0.8%. This can be explained by the fact that at higher intensity the bubble collapse produces increasingly high pressure responsible for more droplet breakup and formation of smaller droplets. Further, as clear from Figures 2 and 3, the ultrasonic horn is more efficient in the formation of smaller droplets than is the ultrasonic bath. This is due to the presence of the predominant collapse of microbubbles in the case of the ultrasonic horn where the cavitational bubble cloud is focused in a comparatively small zone in the near vicinity adjacent to the horn transducer.45 In the case of sonication with the ultrasonic bath, there is a lack of such an intense collapse of microbubbles but the local intensity and amount of microjet streaming are the overall effect. Maximum local cavitational activity is more predominant in the case of the ultrasonic horn compared to that of the ultrasonic bath, because it depends on the total power dissipation per unit volume of liquid which is greater in the case of the horn.56 Different effects of sonication due to various types and geometries of ultrasonic probes are also reported by Atobe et al.57 and Faid et al.58 As revealed from Figure 3, the increase in the ultrasonic intensity has resulted in smaller droplets, which has been also reported by Djenouhat et al.59 and Juang and Lin,53 where they produced W/O emulsion using Span-80 as an emulsifier. The formation of smaller sizes of droplets due to high intensity ultrasound is attributed to the vigorous pressure waves transmitted through the ultrasound, which propagated by the vibrational motion of molecules comprising the liquid phase. Hence, the protein molecules experienced compression and stretching of their molecular structures alternately. This caused unfolding of the protein chains, which is the most responsible factor for enhanced surface activity of acoustically treated

Figure 3. Effect of ultrasound on change in mean droplet size of dispersed droplets of O/W emulsions stored at 26−30 °C: (a) for 1.2% coconut milk protein emulsion; (b) for emulsion containing 0.8% coconut milk protein The error bars represent deviations for three repetitions. Sonication was applied at 20 kHz.

proteins. This has resulted in the smaller sizes of droplets as shown in Figures 2 and 3. 4225



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prepared using an ultrasonic bath. Much more stable emulsions were produced by applying ultrasound through an ultrasonic horn. Figure 5 shows representative micrographs of 10% sunflower oil-in-water emulsions prepared using 1.2% CMP. 3.4. Stability to Creaming. The stability of emulsions against creaming is shown in Figure 6, which reflects poor

The physicochemical phenomena resulting due to sonication is very much sensitive to the mode of application of ultrasound.60,61 When ultrasound is supplied through a horn, this causes very intense pressure waves capable of deforming the molecular structure resulting in the formation of a cavity. This cavity in turn excites the droplet by this time-varying pressure, thus leading to experiencing transient cavitation resulting in fragmentation of smaller droplets. The reason for the larger mean droplet size of emulsions sonicated by the sonication bath is that in this case there is an absence of cavitation responsible for fragmentation of larger droplets to smaller droplets. Stable cavitation occurred due to oscillation of droplets around their equilibrium position which caused the stretching and compression of the protein chains. The reorientation of protein chains helped in increased stability of the emulsion by offering higher stabilizing capability. As presented in Figure 3, the stability of emulsions was increased remarkably by the application of ultrasound. The almost constant size of dispersed droplets (produced by sonication using the ultrasonic horn) in Figures 2 and 3 reveal that the emulsions sonicated by the ultrasonic horn were very stable even at the end of 14 days. Backscattering data presented in Figure 4 also confirm the increased stability of emulsions

Figure 6. Effect of sonication on the creaming stability of O/W emulsions containing 1.2% coconut milk protein. Stability was measured after the emulsions were stored for 7 days between 26 and 30 °C.

stability to creaming for emulsions due to the deprived surface activity of CMP as reported by Tangsuphoom and Coupland.29 On the other hand, the emulsions prepared using sonication were comparatively more stable to creaming at pH >5. This is due to prevention of flocculation and creaming of oil droplets by sonication. Pongsawatmanit et al.12 have also reported that sonication breaks the flocs and imparts stability to creaming and flocculation by providing sufficient repulsive interaction between individual oil droplets. The reduction in the creaming index with application of ultrasound through the ultrasonic horn is due to the fact that the exaggerated cavitation resulting in the formation of smaller droplets caused the increase in depletion attraction between droplets. 3.5. Effect of Salt. The purpose of this series of experiments was to identify the effect of salt (which is most common in many food emulsions) on sonicated emulsions. The presence of NaCl resulted in increased protein solubility at all corresponding pH values as seen from Figure 1. This is due to the high interaction of ions with charged groups leading to more protein solvation as explained by Onsaard et al.42 Despite the increase in the protein solubility with NaCl, the emulsion samples prepared with NaCl concentration higher than 100 mM were relatively unstable due to flocculation caused by the addition of salt which was responsible for the reduction of the thickness of electrical double layer, which diminished the interdroplet repulsion through electrostatic screening.1

Figure 4. Backscattering data for 10% sunflower oil-in-water emulsions prepared using 1.2% coconut milk protein: (a) emulsion prepared without sonication; (b) emulsion prepared with sonication through an ultrasonic bath operated at 120 W and 20 kHz; (c) emulsion prepared with sonication through an ultrasonic horn operated at 120 W and 20 kHz.

Figure 5. Micrographs of 10% sunflower oil-in-water emulsions prepared using 1.2% coconut milk protein: (left) emulsion prepared without sonication; (middle) emulsion prepared with sonication through ultrasonic bath operated at 120 W and 20 kHz; (right) emulsion prepared with sonication through ultrasonic horn operated at 120 W and 20 kHz. 4226



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due to the much higher power dissipation per unit of liquid volume. The mean droplet size increased by the presence of NaCl up to 200 mM, above which there was no appreciable change in droplet size for emulsions prepared using the the ultrasonic bath. By the presence of NaCl, the mean droplet size of emulsions treated using the ultrasonic horn was not affected at all. Hence, it is concluded that the destabilization of emulsions with the addition of salt can be controlled by power ultrasound. Coconut milk protein along with power ultrasound is found to be capable of producing emulsions with smaller droplet size and better stability which can be effectively used for the production of many emulsions of high commercial value. Having the presence of beneficial nutrient ingredients and easy recovery from the natural material, coconut milk protein may offer a suitable alternative to many conventional emulsifiers and/or stabilizers used in a variety of cosmetics and food emulsions.

Figure 7. Influence of NaCl and ultrasound on mean droplet size of dispersed phase in O/W emulsions containing 1.2% coconut milk protein. Droplet sizes were measured at the end of 30 min after preparation of emulsions.

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AUTHOR INFORMATION

Corresponding Author

The mean droplet size of emulsion increased significantly with increasing NaCl concentration for emulsion prepared without sonication (see Figure 7). The concentration of NaCl has not influenced the mean droplet size of dispersed oil phases in the emulsions prepared using ultrasound through the ultrasonic horn. The increase in the droplet size of emulsions prepared using the ultrasonic bath was limited until a plateau region was reached, which is due to bridging flocculation of droplets.12 The droplet size of the dispersed phase increased from 15.4 to 19.5 μm in the presence of 400 mM NaCl for emulsification with the ultrasonic bath operated at 120 W and 20 kHz. This exhibited that the ultrasonicated emulsions were relatively more stable to droplet aggregation and creaming for various concentrations of NaCl. For emulsion samples prepared without application of ultrasound, droplet aggregation was more predominant with increasing salt concentration. This is explained by the fact that the effective energy supplied during emulsification by sonication has overcome the effect of electrostatic interactions among the dispersed droplets in the presence of ions (produced by ionization of NaCl in emulsions).

*E-mail: [email protected] or [email protected]. Tel.: +91 261 2201641 or +91 261 2201642. Fax: +91 261 2227334. Notes

The authors declare no competing financial interest.

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REFERENCES

(1) Dickinson, E. Flocculation of Protein-Stabilized Oil-in-Water Emulsions. Colloids Surf., B 2010, 81, 130. (2) Jang, W.; Nikolov, A.; Wasan, D. T.; Chen, K.; Campbell, B. Effect of Protein on the Texture of Food Emulsions Under Steady Flow. Ind. Eng. Chem. Res. 2005, 44, 4855. (3) Tang, C. H.; Chen, L.; Foegeding, E. A. Mechanical and WaterHolding Properties and Microstructures of Soy Protein Isolate Emulsion Gels Induced by CaCl2, Glucono-δ-Lactone (GDL), and Transglutaminase: Influence of Thermal Treatments Before and/or After Emulsification. J. Agric. Food Chem. 2011, 59, 4071. (4) Ma, H.; Forssell, P.; Partanen, R.; Buchert, J.; Boer, H. Improving Laccase Catalyzed Cross-Linking of Whey Protein Isolate and Their Application as Emulsifiers. J. Agric. Food Chem. 2011, 59, 1406. (5) Damodaran, S.; Anand, K. Sulfhydryl−disulfide InterchangeInduced Interparticle Protein Polymerization in Whey ProteinStabilized Emulsions and Its Relation to Emulsion Stability. J. Agric. Food Chem. 1997, 45, 3813. (6) Viljanen, K.; Kylli, P.; Hubbermann, E. M.; Schwarz, K.; Heinonen, M. Anthocyanin Antioxidant Activity and Partition Behavior in Whey Protein Emulsion. J. Agric. Food Chem. 2005, 53, 2022. (7) Dickinson, E.; Evison, J.; Owusu, R. K. Preparation of Fine Protein-Stabilized Water-in-Oil-in-Water Emulsions. Food Hydrocolloids 1991, 5, 481. (8) Hu, M.; Li, Y.; Decker, E. A.; Xiao, H.; McClements, D. J. Impact of Layer Structure on Physical Stability and Lipase Digestibility of Lipid Droplets Coated by Biopolymer Nanolaminated Coatings. Food Biophys. 2011, 6, 37. (9) Kellerby, S. S.; Gu, Y. S.; McClements, D. J.; Decker, E. A. Lipid Oxidation in a Menhaden Oil-in-Water Emulsion Stabilized by Sodium Caseinate Cross-Linked with Transglutaminase. J. Agric. Food Chem. 2006, 54, 10222. (10) van der Ven, C.; Gruppen, H.; de Bont, D. B. A.; Voragen, A. G. J. Emulsion Properties of Casein and Whey Protein Hydrolysates and the Relation with Other Hydrolysate Characteristics. J. Agric. Food Chem. 2001, 49, 5005. (11) Das, K. P.; Kinsella, J. E. pH Dependent Emulsifying Properties of β-Lactoglobulin. J. Dispersion Sci. Technol. 1989, 10, 77.

4. CONCLUSIONS Sunflower oil-in-water emulsions with remarkably improved stability were prepared with coconut milk protein by the application of acoustic energy for the first time. The mean droplet size and stability of sunflower oil-in-water emulsions prepared using CMP are found to be highly affected by the sonication. Further, the mode of application of ultrasound has a profound effect on the emulsification and stabilization of these emulsions. A 1.2% coconut milk protein concentration was found to be sufficient for emulsification with ultrasound. Sonication resulted in reducing the mean droplet size of the dispersed oil phase and imparted better stability against pH. Sonication with the ultrasonic horn is found to be more effective in all the experiments for producing emulsions with smaller droplet size and higher stability over the time period in turn. This is due to the exaggerated cavitation and collapse of bubbles associated with high energetic interaction with emulsion droplets in the case of direct sonication using the ultrasound horn. Moreover, the maximum local cavitational activity in the case of the ultrasonic horn is more predominant 4227



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