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Interfacial and emulsifying properties of sucrose ester in coconut milk emulsions in comparison with Tween
Food Hydrocolloids 30 (2013) 358e367
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Interfacial and emulsifying properties of sucrose ester in coconut milk emulsions in comparison with Tween Suwimon Ariyaprakai a, *, Tanachote Limpachoti a, Pasawadee Pradipasena b a b
Department of Food Biotechnology, Faculty of Biotechnology, Assumption University, Ram Khamhaeng Rd. Soi 24, Hua Mak Campus, Bangkok 10240, Thailand Department of Food Technology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 January 2012 Accepted 6 June 2012
In this study, sucrose esters were presented as a promising alternative to petrochemically synthesized Tweens for application in coconut milk emulsions. The interfacial and emulsifier properties of sucrose ester (SE), mainly sucrose monostearate, had been investigated in comparison with Tween 60 (TW), an ethoxylate surfactant. The interfacial tension measurement showed that SE had a slightly better ability to lower the interfacial tension at coconut oilewater interface. These surfactants (0.25 wt%) were applied in coconut milk emulsions with 5 wt% fat content. The effects of changes in pH, salt concentration, and temperature on emulsion stability were analyzed from visual appearance, optical micrograph, droplet charges, particle size distributions, and creaming index. Oil droplets in both SE and TW coconut milk emulsions extensively flocculated at pH 4, or around the pI of the coconut proteins. Salt addition induced flocculation in both emulsions. The pH and salt dependence indicated polyelectrolyte nature of proteins, suggesting that the proteins on the surface of oil droplets were not completely displaced by either added nonionic SE or TW. TW coconut milk emulsions appeared to be thermally unstable with some coalesced oil drops after heating and some oil layers separated on top after freeze thawing. The change in temperature had much lesser influence on stability of SE coconut milk emulsions and, especially, it was found that SE emulsions were remarkably stable after the freeze thawing. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Sucrose esters, or sugar-based surfactants, are in current interest because they are produced from natural resources such as sucrose and vegetable oil. They are biodegradable and more biocompatible when compared to other petrochemically synthesized surfactants. Sucrose esters are non-toxic and safe for food and are approved as a food additive under food regulations and laws in several countries i.e. Japan, USA, and EC. It had been reported that sucrose esters have excellent emulsifying properties and be able to apply in various food products (Garti, 2001). Their wide range of hydrophilicelipophilic balance (HLB), depending on degree of esterification of fatty acids and sucrose, provides ultimate application of sucrose esters to each product type. Coconut milk is one of food emulsions that require additional surfactants or emulsifiers to improve emulsion stability. Several recent works have been carried out on physicochemical characterization of
* Corresponding author. Tel.: þ66 2 300 4553x3796; fax: þ66 2 300 4553x3792. E-mail address: [email protected] (S. Ariyaprakai). 0268-005X/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodhyd.2012.06.003
coconut milk employing other small molecule surfactants such as Tweens and sodium dodecyl sulfate (Tangsuphoom & Coupland, 2009a,b), however, data on sucrose esters remain fewer. In those studies, coconut milk emulsions after addition of surfactants were reported to smaller in average droplet size, decrease in total surface protein concentration, and change in droplet surface charges (Tangsuphoom & Coupland, 2008b, 2009a). This study aims to gain more understanding on the relationship between interfacial properties and stability of coconut milk emulsions with addition of sucrose ester. We described the comparative interfacial and emulsifier properties between sucrose ester (SE), mainly sucrose monostearate, and Tween 60, or polyoxyethylene sorbitan monostearate (TW). Their structures were displayed in Fig. 1. The difference between the carbohydrate and ethoxylate headgroups provided an interesting comparison. Their adsorption behaviors at the coconut oil and aqueous interface were investigated by the interfacial tension measurement. The coconut milk emulsions prepared with SE and with TW were processed at different temperatures (121 C, 100 C and 20 C) and their stability was investigated at different pH (2e8) and salt concentrations (0 and 20 mM CaCl2).
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359
CH OH O
O HO
HO
OH
O
O
O
O
O
O
HO HC
O
OH HO
O
O
O
O
O OH
HO O
O
O
O
O OH
O O
O
O
O
O
O O
Fig. 1. Chemical structures of (a) sucrose monosterate (SE) and (b) Tween 60 or ethoxylated sorbitan monostearate (TW).
2. Material and methods 2.1. Materials Ryoto sucrose esters (S1670) were supplied by MitsubishiKagaku Foods Corporation (Tokyo, Japan). Ryoto S1670 contained mainly of 50e53% sucrose monostearate with sucrose monopalmitate 18e20%, sucrose disterate 12e14 %, sucrose dipalmitate 5e6%, sucrose alkylate 5e10% and other ash and moisture. Tween 60 (Polyoxyethylene sorbitan monostearate), hydrochloric acid, sodium hydroxide, sodium azide (NaN3), calcium chloride (CaCl2), and Oil Red O were purchased from Sigma Chemical Company (St. Louis, MO, USA). Coconut oil and soybean oil were supplied by Tropicana Oil (Nakhonpathom, Thailand) and Thai Vegetable Oil Public Company Limited (Nakhonpathom, Thailand), respectively. Ground coconut meat was purchased locally. Distilled water was used for emulsion preparation throughout experiment. 2.2. Interfacial tension measurement Coconut oilewater interfacial tensions were measured according to Du Nouy ring method at 25 C using a digital tensiometer (K10, Kruss Scientific, Hamburg, Germany). A series of SE and TW aqueous solutions at different concentrations were prepared and kept in plastic bottles prior measurements. The SE solutions had to be preheated to 70 C due to its difficulty to dissolve in water. Before each measurement, the solution was poured into a measurement vessel and coconut oil was overlaid on top of the aqueous layer. The ring was cleaned with ethanol and distilled water and flamed with a Bunsen burner before each measurement. Each measured point was an average from at least two replicates. 2.3. Coconut milk emulsion preparation Coconut milk was produced by adding distilled water to ground coconut meat at a weight ratio of 2:1. The mixture was manually pressed and filtered through filter cloth to remove solid residues. The fat contents of the obtained coconut milk were determined by using Rose-Gottlieb method (AOAC, 2000). The extracted coconut milk was further diluted with either distilled water or surfactant aqueous solutions to a final fat content of 5 wt% and a final surfactant concentration of 0 or 0.25 wt%. The surfactant concentration of 0.25 wt% was chosen because this concentration had been proven to provide sufficient stability to coconut milk emulsions (Tangsuphoom & Coupland, 2009a). Sodium azide 0.02 wt% was added to prevent microbial growth. Each emulsion
was further homogenized by using a high speed homogenizer (Ultra-turrax, IKA Labortechnik, Germany) for 3 min at the speed of 11,200 rpm. 2.4. Measurement of emulsion properties 2.4.1. Droplet size measurement The particle size distributions of emulsions were measured by using a laser diffraction particle size analyzer (Mastersizer 2000; Malvern Instruments Ltd., Worcestershire, UK), with a dualwavelength detection system. Emulsion samples were dropped and diluted in the test chamber that filled with distilled water in order to prevent multiple light scattering effects. The size distributions of emulsions were obtained via a best fit using Mie theory. The refractive indices of 1.33 for water and 1.15 for coconut oil were employed as optical properties of emulsions. The particle sizes were reported as surface-volume average diameters, P P d32 ¼ ni d3i = ni d2i , and volume-weighted average diameters, P P d43 ¼ ni d4i = ni d3i , where di is the midpoint of the size interval i and ni is the number of particles in that interval. 2.4.2. z-potential measurement The electrical charges on emulsion droplets were determined by using a particle electrophoresis instrument (Zeta-Meter System 3.0þ, Zeta-Meter, Inc., Staunton, VA, USA). The emulsion sample after 1/100 dilution was put into an electrophoresis cell that had electrodes at both ends. The direction and velocity of emulsion droplets that move in the applied electrical field were observed under a microscope and the charge or z-potential on emulsion droplets was calculated. Each reported z-potential was an average from five replicates. 2.4.3. Optical microscopy The emulsion microstructure was examined by using a standard optical microscope (Alphaphot-2, YS2-H, Nikon Corporation, Japan) equipped with a microscope eyepiece camera (AM423 Dino-Eye USB, AnMo Electronics Corporation, Taipei, Taiwan) with a software (DinoCapture Software) installed on a computer. A few drops of emulsion were put on a glass slide and fully covered with a cover slid. The microscope magnification of 100 was employed. 2.4.4. Visual appearance and creaming index measurement The emulsion sample (10 g) was transferred into a standard test tube and tightly sealed with a cap. The appearance of the emulsion sample was closely captured by a digital camera. After gently mixed by inversion, the tube was left on a tube stand for 10 min. The height
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of aqueous layer left after the emulsion droplets creamed to the top and the height of initial total sample in the tube were measured. The creaming index was determined from the percentage of the height of the aqueous layer over the height of the total sample. 2.5. Coconut milk emulsion stability 2.5.1. Emulsion stability against pH and salt concentration The emulsion sample (10 g) was transferred into a standard test tube and adjusted to the specified pH values (2, 4, 6, and 8) by using 0.1 and 1 M HCl or 0.1 and 1 M NaOH. Noted that the normal pH of extracted coconut milk was w6 before the adjustment. For the salt experiment, CaCl2 salt was also added to a concentration of 20 mM. The charges on the emulsion droplets were determined from z-potential measurement and the stability of emulsion was analyzed by z-potential measurement, creaming index measurement, and optical micrograph.
Fig. 2. Coconut oilewater interfacial tensions vs. logarithm of concentration (log C) isotherms for (D) sucrose ester (SE) and (B) Tween 60 (TW).
2.5.2. Emulsion thermal stability The emulsion samples (10 g each) were transferred into standard test tubes and kept in an autoclave at 121 C for 10 min, or in a boiling water bath at 100 C for 20 min, or in a freezer at 20 C
Fig. 3. Visual appearance of phase separation of coconut milk emulsions without addition of any surfactant (Control), with addition of 0.25 wt% Tween 60 (TW), and with addition of 0.25 wt% sucrose ester (SE) after thermal treatments at different temperatures. (a) White creamy emulsion. (b) Coagulated solid particles. (c) Destabilized oil layer (free oil).
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for 14 h. After removal from each storage conditions, the emulsions were stored to reach room temperature prior further analysis. The stability of emulsion was analyzed from droplet size measurement, visual appearance, and optical micrograph. In addition, freeze thaw stability of emulsions was assessed by measuring amount of free oil (destabilized oil) after three freezethaw cycles (each cycle: 20 C for 22 h and 30 C for 2 h) using the principle of dye dilution method (Palanuwech, Potineni, Roberts, & Coupland, 2003; Thanasukarn, Pongsawatmanit, & McClements, 2004b). The employed Oil-Red O is colored oil that only dissolves in free oil but does not dissolve in emulsified oil droplets. A stock solution of 0.0015 wt% Oil-Red O in soybean oil was initially prepared. A series of coconut oil solutions were obtained by adding different amounts of coconut oil to the stock Oil-Red O solution. The absorbance of the solution series was measured by using an ultravioletevisible spectrophotometer (Spectronic Genesys 5, Milton Roy company, Rochester, NY, USA) at a wavelength of at 520 nm. The plot between the measured absorbance and the coconut oil concentrations was used as a calibration curve. To determine amount of free oil in emulsions, the emulsion sample (35 g) was added with 3 g of the stock Oil-Red O solution, vortexed for 30 s, and centrifuged at the speed of 5500 rpm for 5 min (Universal centrifuge, Model PLC-012, Gemmy Industrial Corporation, Taipei, Taiwan). The colored oil on the upper layer was transferred by a micro-pipette into a plastic cuvette and measured its absorbance. The measured absorbance was converted to weight percent of free oil by using the prepared calibration curve.
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3. Results and discussion 3.1. Coconut oilewater interfacial behavior The basic information about the interfacial properties of SE and TW at coconut oilewater interface was obtained from the interfacial tension measurement. Fig. 2 is the plot between the measured interfacial tensions of SE and TW aqueous solutions at various concentrations. The gradually decrease in the measured interfacial tension was observed as surfactant concentrations increased. After the interfacial tensions reduced to a minimum value, it approached a constant as surfactants started forming micellar aggregates. The minimum interfacial tensions for SE and TW were <2 mN/m and 8 mN/m, respectively. Unfortunately, the minimum interfacial tension for SE was too small to be measured precisely from our existing instrument. It had reported that the lowest interfacial tension of SE at the rapeseed oilewater interface was 1.5 mN/m (Soultani, Ognier, Engasser, & Ghoul, 2003). SE tended to have a slightly better ability than TW in reducing the oilewater interfacial tension. The critical aggregation concentrations (CAC) at those minimum interfacial tensions in Fig. 2 were approaching to 0.05 wt% for SE and around 0.11 wt % for TW. The lower value of CAC indicated more hydrophobicity of SE, most likely due to this commercial SE also contained some proportion of diesters. Our results agreed with the reported CAC value of 0.05 wt% SE at the liquid paraffinewater interface (Yanke, Shufen, Jinzong, & Qinghui, 2004).
Fig. 4. Optical micrographs of coconut milk emulsions without addition of any surfactant (Control), with addition of 0.25 wt% Tween 60 (TW), and with addition of 0.25 wt% sucrose ester (SE) after thermal treatments at different temperatures. (a) Extensively coalesced oil drops. (b) Coalesced oil droplets. (c) Coagulated solid particles.
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a
b
c
d
e
Fig. 5. Particle size distributions of coconut milk emulsions without addition of any surfactant (Control), with addition of 0.25 wt% Tween 60 (TW), and with addition of 0.25 wt% sucrose ester (SE) after thermal treatments at different temperatures. (a) At room temperature. (b) At 121 C for 10 min. (c) At 100 C for 20 min. (d) At 20 C for 14 h. (e) All thermal treatment results for coconut milk emulsions with addition of 0.25 wt% sucrose ester (SE). The arrows indicate extra peaks from coagulated solid particles.
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Table 1 The average particle sizes (d3,2 and d4,3) of coconut milk emulsions without addition of any surfactant (Control), with addition of 0.25 wt% Tween 60 (TW), and with addition of 0.25 wt% sucrose ester (SE) after thermal treatments at different temperatures. Thermal treatments
Control coconut milk
TW coconut milk
Temperature
Holding time
d3,2 (mm)
d3,2 (mm)
28 C (Room temp.) 121 C 100 C 20 C
e 10 min 20 min 14 h
10.001 22.057 16.103 7.285
d4,3 (mm) 1.228 3.866 1.839 0.964
33.327 114.155 72.344 42.272
5.624 6.522 16.996 3.222
5.535 12.746 7.336 3.533
SE coconut milk d4,3 (mm)
0.175 0.264 0.298 0.112
13.785 125.284 23.710 11.702
d3,2 (mm) 0.482 5.600 5.894 0.274
8.800 8.340 7.967 7.858
0.326 0.541 0.441 0.266
d4,3 (mm) 25.891 58.275 29.078 25.628
1.467 8.155 1.698 0.366
Means standard deviation of at least two replicates.
3.2. Emulsion thermal stability 3.2.1. Coconut milk emulsions at room temperature Coconut milk emulsions were prepared with and without addition of SE or TW and kept at room temperature (w28 C) before determining their characteristics from visual appearance (Fig. 3), optical micrographs (Fig. 4), and particle size measurements (Fig. 5a and Table 1). Fig. 3 shows that all three types of emulsions: Control coconut milk (without addition of any surfactant), TW coconut milk, and SE coconut milk, appeared as white creamy emulsions. Their average sizes (d3,2) were 10.0 microns, 5.5 microns, and 8.8 microns, respectively (Table 1). The results from the optical micrographs (Fig. 4) confirmed that the particle sizes of coconut milk emulsions containing SE or TW were smaller than Control coconut milk. More droplet flocculation in SE emulsions was suggested to be the reason of SE emulsions having larger average size than TW emulsions. The bulky headgroup of TW seemed to effectively provide steric barriers that prevented droplet flocculation in TW emulsions. Even though SE had a better ability than TW in reducing the interfacial tension between coconut oil and water interface, this was not enough to observe its effect on emulsion droplet size.
three coconut milk emulsions (Fig. 3). The presence of such solid aggregates had also been reported in other studies (Chiewchan, Phungamngoen, & Siriwattanayothin, 2006; Tangsuphoom & Coupland, 2009b). Those particles were denatured proteins which were detected as solid substance in the optical micrographs (arrows in Fig. 4) and as an extra population on the right to the size distribution of emulsion population (arrows in Fig. 5b). From the optical micrographs, some large coalesced oil drops were observed in Control coconut milk that were significantly larger than oil droplets in
a
3.2.2. Stability of coconut milk emulsions after heating Further investigation was assessing thermal stability of coconut milk emulsions by heating the emulsions at 121 C for 10 min. After the heating, the coagulated white solid particles were observed in all
Control coconut milk TW coconut milk SE coconut milk
b % Destabilized oil (free oil)
100
80
60
40
20
0 1
2 3 Number of freeze-thaw cycles
Fig. 6. Percentage of destabilized oil (free oil) in coconut milk emulsions without addition of any surfactant (Control), with addition of 0.25 wt% Tween 60 (TW), and with addition of 0.25 wt% sucrose ester (SE) after 1, 2 and 3 freeze-thaw cycles.
Fig. 7. Emulsion droplet charge (or z-potential) of () coconut milk emulsions without addition of any surfactant (Control), (B) with addition of 0.25 wt% Tween 60 (TW), and (D) with addition of 0.25 wt% sucrose ester (SE). (a) After only pH adjustment. (b) After pH adjustment and addition of 20 mM CaCl2 salt.
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either SE and TW emulsions (Fig. 4). Table 1 shows that the average particle size (d3,2) of Control coconut milk increased from 10.0 to 22.1 microns (or by a factor of 2.2) and TW coconut milk increased from 5.5 to 12.7 microns (or by a factor of 2.3). However, the average particle size of SE emulsion was still around 8e9 microns showing no significantly change (Table 1 and Fig. 5e). Noted that we chose the surface-volume average diameter (d3,2) instead of d4,3 to analyze our results of multi-modal distributions here. D3,2 was based on area distribution and better representing the average size of smaller particles (with more surface area) which supposed to be emulsion droplets in this case. D4,3,or volume-weighted average diameter, was calculated from volume distribution and more representing the average size of larger particles (with more volume) which could be the size of flocculated droplets or solid aggregates. Similarly, after coconut milk emulsions were kept at the temperature of 100 C for 20 min, the average sizes of Control coconut milk emulsions increased by a factor of 1.6 and the size of TW emulsions slightly increased by a factor of 1.3, but SE emulsions remained the same average size of 8e9 microns (Table 1 and Fig. 5c and e). This was accompanied by the results from the optical micrographs (Fig. 4) showing that SE and TW emulsions still appeared as small oil droplets but Control coconut milks became
large coalesced oil drops. No coagulated solid particles floated in TW or SE emulsions as observed in TW and SE coconut milk at 121 C (Fig. 3), suggesting that the degree of protein denaturation was lesser at this lower temperature (100 C). Coconut proteins, a natural coconut milk emulsifier, when denatured by heat lose their ability to stabilize emulsion droplets. We observed large coalesced oil drops in Control coconut milk after heated at 121 C or 100 C as oil droplets that come in close proximity undergo extensively coalesce. Lesser degrees of droplet coalescence in SE and TW emulsions when compared with Control coconut milk indicated that heating stability of coconut milk enhanced by addition of SE or TW. We found that SE stabilized emulsions seemed to be more heat stable than TW emulsions as the average sizes of SE emulsions (d3,2) did not significantly alter after the heating at both temperatures (Table 1 and Fig. 5e). The properties of SE had been reported to be lesser sensitive to temperature variation when compared to other nonionic surfactants with oxyethylene groups (Stubenrauch, 2001). 3.2.3. Stability of coconut milk emulsions after freeze thawing Coconut milk emulsions were kept at freezing temperature of 20 C for 14 h, and after being thawed at room temperature, their
Fig. 8. Optical micrographs of coconut milk emulsions without addition of any surfactant (Control), with addition of 0.25 wt% Tween 60 (TW), and with addition of 0.25 wt% sucrose ester (SE) after pH adjustment (a) Extensive flocculation. (b) Some flocculation.
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characteristics were examined. From their visual appearance (Fig. 3), some oil layers were accumulated on top of Control and TW coconut milk emulsions, but in a sharp contrast, no oil layer was observed in SE coconut milk. The optical micrographs in Fig. 4 show some coalesced oil droplets in the Control and TW samples. The particles size distributions (Fig. 5d) and average sizes (d3,2) (Table 1) of Control and TW emulsions were significantly altered from their original emulsions at room temperature. This deviation should be reasoned that some oil droplets in emulsions were already coalesced and separated out as oil layers and those oil droplets were not detected during the size measurement. Differently, SE emulsified droplets appeared the same as their originally prepared emulsions (Fig. 4) and its particle size distribution remained the same (Fig. 5e) with the average size w8 microns closed to its original size (Table 1), revealing good emulsion stability. To ensure our results, numbers of freeze-thaw cycles were increased up to three and the amounts of destabilized oils were determined after each cycle. Fig. 6 clearly demonstrated that the amounts of destabilized oil coming out from SE emulsions after each freezing cycle were much lesser than from Control and TW emulsions. After the first cycle, almost no destabilized oil was detected in SE emulsions, while 93% and 68% of destabilized oils were obtained from Control and TW emulsions, respectively. After the third cycle, the percentage of destabilized oils in SE emulsions was three times lesser than in Control or TW emulsions. Noted that even though this dye-dilution method (Palanuwech et al., 2003) might not be very precise to quantify amounts of oil, the results should be accurate enough for comparing relative amounts of oil coming out from each emulsion. Many other studies have shown that their emulsions also destabilized into oil layer after freeze thawing; for example, coconut milks prepared from Tween 20 (Tangsuphoom & Coupland, 2009b), palm oil emulsions prepared from Tween 20 (Thanasukarn et al., 2004b; Thanasukarn, Pongsawatmanit, & McClements, 2006), and n-alkanes emulsions prepared from SDS and Tween 20 (Cramp, Docking, Ghosh, & Coupland, 2004). We noticed that those emulsions that prepared from Tweens were prone to be unstable similarly to our TW result. Several researchers aim to understand the destabilization mechanism in order to improve freeze-thaw stability of emulsions (Cramp et al., 2004; Ghosh & Coupland, 2008). Thus, it is very interesting here to discover that emulsions prepared from SE were more freeze-thaw stable than TW emulsions. A number of studies have reported that the presence of sucrose increased freeze-thaw stability of emulsions (Ghosh, Cramp, & Coupland, 2006; Thanasukarn, Pongsawatmanit, & McClements, 2004a; Thanasukarn et al., 2006). Sucrose has been known to have a cryo-protective property coming from sucrose ability to lower the freezing point of water. Such unfrozen water protects other compounds dissolving in the aqueous phase from the freeze damage. The unfrozen water in emulsions protects oil droplets from penetration of growing ice crystal that would disrupt the interfacial membrane and cause droplet coalescence. We found that there was also a report about the presence of unfrozen water in the aqueous solution of sugar-based surfactant and its possible cryo-protective effect (Ogawa, Asakura, & Osanai, 2009). We suspected that our SE coconut milk contained some unfrozen water and this was the reason that SE coconut milk emulsions were still stable after the freeze thawing.
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net charge gradually reduced to zero when pH approached to the value of 4, corresponding to the PI of coconut protein reported in other literatures (Onsaard, Vittayanont, Srigam, & McClements, 2005; Tangsuphoom & Coupland, 2008a). When proteins lose their net charge at this pH, the Control emulsion droplets became extensively flocculated (Fig. 8) and creamed rapidly with the creaming index of 42% after ten-minute storage (Fig. 9). At other pH values, the electrostatic repulsion provided by coconut proteins was sufficient to prevent aggregation of droplets and no creaming was observed. The result of pH effect for SE and TW coconut milk was relatively invariant to Control coconut milk. The z-potential of SE and TW emulsions decreased from a positively charge at pH 2 to a negatively charge at pH 8 with a net zero charge at pH close to 4 (Fig. 7a). SE and TW emulsions only creamed at pH 4 with the creaming index after ten-minute storage of 8% for SE emulsions and of 50% for TW emulsions (Fig. 9). The much lower in creaming index of SE emulsions suggested that SE might also alter rheological property of emulsions and beneficial as a creaming stabilizer. The highest creaming rate was accompanied by the highest flocculation shown in the micrographs at pH 4 in both emulsions (Fig. 8). It was a little surprising that, after addition of nonionic surfactants such as SE and TW, the charges on droplets were still dominant and sensitive to the pH condition since nonionic surfactants would provide steric stability and remove electrostatic effect of proteins. If the proteins on emulsion droplets were completely displaced then the properties of the emulsion should be the same as the replacing surfactants (McClements, 2004). More investigation on the role of droplet charges was by addition of 20 mM CaCl2 salt. The micrographs from Fig. 10 show that salt addition induced droplet flocculation at pH of 6 and 8, similarly in Control, TW and SE emulsions. The flocculation was obvious when comparing the micrograph before (Fig. 8) and after salt addition (Fig. 10). The presence of flocculated droplets had been attributed to ability of salt to screen charges of coconut proteins (Fig. 7b) and
3.3. Emulsion stability against pH and salt concentrations The charge characteristics of coconut protein on oil droplets were investigated by exposing emulsions to different pH conditions. Fig. 7a shows that the z-potential of Control coconut milk droplets changed from a positively charge of þ16 mV to a negatively charge of 28 mV when increasing the pH from 2 to 8. From the graph, the
Fig. 9. Creaming index after ten-minute storage of () coconut milk emulsions without addition of any surfactant (Control), (B) with addition of 0.25 wt% Tween 60 (TW), and (D) with addition of 0.25 wt% sucrose ester (SE) after adjustment to different pH conditions.
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Fig. 10. Optical micrographs of coconut milk emulsions without addition of any surfactant (Control), with addition of 0.25 wt% Tween 60 (TW), and with addition of 0.25 wt% sucrose ester (SE) after pH adjustment and addition of 20 mM CaCl2 salt. (a) Extensive flocculation.
reduced electrostatic repulsion between oil droplets. We noticed that the oil droplets did not flocculate after addition of salt at pH 2 where amino acids were mostly protonated and proteins had highly net positively charge. It seemed that oil droplets were most repulsive at this low pH condition as also observed in emulsions stabilized by other protein (Kim, Decker, & McClements, 2004). The sensitivity to pH and salt confirmed the presence of proteins at interfacial droplets in SE and TW emulsions. Thus, in the present case, proteins on droplets of coconut milk emulsion were not completely displaced by the SE or TW content of 0.25 wt%. We postulated that SE or TW molecules stayed on oil droplet interface together with coconut protein stabilizing emulsions by both steric and electrostatic repulsion. Further investigation on interfacial behaviors when coconut proteins coexist with these surfactants is suggested. The interfacial characteristics are governed by structure and thickness of proteinesurfactant films, the interfacial ratio of protein to surfactant, and the proteinesurfactant interactions (Rodriguez Patino, Rodriguez Nino, & Carrera Sanchez, 2007). A previous study showed that addition of sucrose ester decreased the interfacial tension and the interfacial elasticity of milk protein membrane in
corresponding to the surfactant/protein ratio (Rouimi, Schorsch, Valentini, & Vaslin, 2005). Some literature also evidenced that Tween 60 only partially displaced sodium caseinate (Dalgleish, Srinivasan, & Singh, 1995) and albumin (Seta, Baldino, Gabriele, Lupi, & de Cindio, 2012) from the oilewater interface even at some high surfactant/protein ratios. 4. Conclusions This work clearly showed that sucrose ester (SE) exhibited interesting interfacial and emulsifying properties when compared with Tween 60 (TW). SE was slightly better in lowering the interfacial tension between coconut oil and water interface. The stability and surface charge characteristics of coconut milk emulsions emulsified with 0.25 wt% of nonionic SE or TW strongly depended on pH and salt environments. Thus, it was likely that the addition of SE or TW did not completely displace proteins on oil droplets to remove their electrostatic interaction. The presence of either SE or TW had a distinct effect on the thermal characteristics of coconut milk emulsions: coconut milk emulsions emulsified with SE were more thermally stable than TW emulsions. Especially, we found
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that SE coconut milk emulsions were much more stable after freeze thawing. The complex between coconut protein and SE seemed to be an emulsifying membrane that could better protect coconut milk emulsions from the heat and freeze damages. Acknowledgment This research was fully supported by the Research Grant for New Scholar (MRG53) provided by the Thailand Research Fund and Assumption University and the mentor of this project was Asst. Prof. Pasawadee Pradipasena from Department of Food Technology, Faculty of Science, Chulalongkorn University. Special thanks to Ms.Treuktongjai Saenghiruna and Ms. Rungchat Kladcharoen, undergraduate students from Assumption University who dedicated in some experimental works and to Ms. Martha Intan Budiati, a practical training student from Faculty of Agricultural Technology, Soegijapranata Catholic University, Semarang, Indonesia, who helped during sample preparation. Also thanks to Caltech Corp., Ltd. for kindly donating the sucrose ester used in this study. References AOAC. (2000). Official methods of analysis of AOAC international (17 ed.). Gaithersburg, MD: Association of Analytical Communities. Chiewchan, N., Phungamngoen, C., & Siriwattanayothin, S. (2006). Effect of homogenizing pressure and sterilizing condition on quality of canned high fat coconut milk. Journal of Food Engineering, 73(1), 38e44. Cramp, G. L., Docking, A. M., Ghosh, S., & Coupland, J. N. (2004). On the stability of oil-in-water emulsions to freezing. Food Hydrocolloids, 18(6), 899e905. Dalgleish, D. G., Srinivasan, M., & Singh, H. (1995). Surface properties of oil-in-water emulsion droplets containing casein and Tween 60. Journal of Agricultural and Food Chemistry, 43(9), 2351e2355. Garti, N. (2001). Food emulsifiers and stabilizers. In N. A. M. Eskin, & D. S. Robinson (Eds.), Food shelf life stability: Chemical, biochemical, and microbiological changes. Boca Raton: CRC Press. Ghosh, S., & Coupland, J. N. (2008). Factors affecting the freeze-thaw stability of emulsions. Food Hydrocolloids, 22(1), 105e111. Ghosh, S., Cramp, G. L., & Coupland, J. N. (2006). Effect of aqueous composition on the freeze-thaw stability of emulsions. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 272(1e2), 82e88. Kim, H. J., Decker, E. A., & McClements, D. J. (2004). Comparison of droplet flocculation in hexadecane oil-in-water emulsions stabilized by Î2-lactoglobulin at pH 3 and 7. Langmuir, 20(14), 5753e5758.
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McClements, D. J. (2004). Protein-stabilized emulsions. Current Opinion in Colloid & Interface Science, 9(5), 305e313. Ogawa, S., Asakura, K., & Osanai, S. (2009). 117. Freeze-thawing behavior of theoctyl-b-D-glucoside/water system. Cryobiology, 59(3), 402e403. Onsaard, E., Vittayanont, M., Srigam, S., & McClements, D. J. (2005). Properties and stability of oil-in-water emulsions stabilized by coconut skim milk proteins. Journal of Agricultural and Food Chemistry, 53(14), 5747e5753. Palanuwech, J., Potineni, R., Roberts, R. F., & Coupland, J. N. (2003). A method to determine free fat in emulsions. Food Hydrocolloids, 17(1), 55e62. Rodriguez Patino, J. M., Rodriguez Nino, M. R., & Carrera Sanchez, C. (2007). Physico-chemical properties of surfactant and protein films. Current Opinion in Colloid & Interface Science, 12, 187e195. Rouimi, S., Schorsch, C., Valentini, C. l., & Vaslin, S. (2005). Foam stability and interfacial properties of milk protein-surfactant systems. Food Hydrocolloids, 19(3), 467e478. Seta, L., Baldino, N., Gabriele, D., Lupi, F. R., & de Cindio, B. (2012). The effect of surfactant type on the rheology of ovalbumin layers at the air/water and oil/ water interfaces. Food Hydrocolloids, 29(2), 247e257. Soultani, S., Ognier, S., Engasser, J. M., & Ghoul, M. (2003). Comparative study of some surface active properties of fructose esters and commercial sucrose esters. Colloids and Surfaces A-Physicochemical and Engineering Aspects, 227(1e3), 35e44. Stubenrauch, C. (2001). Sugar surfactants e aggregation, interfacial, and adsorption phenomena. Current Opinion in Colloid & Interface Science, 6(2), 160e170. Tangsuphoom, N., & Coupland, J. N. (2008a). Effect of pH and ionic strength on the physicochemical properties of coconut milk emulsions. Journal of Food Science, 73(6), E274eE280. Tangsuphoom, N., & Coupland, J. N. (2008b). Effect of surface-active stabilizers on the microstructure and stability of coconut milk emulsions. Food Hydrocolloids, 22(7), 1233e1242. Tangsuphoom, N., & Coupland, J. N. (2009a). Effect of surface-active stabilizers on the surface properties of coconut milk emulsions. Food Hydrocolloids, 23(7), 1801e1809. Tangsuphoom, N., & Coupland, J. N. (2009b). Effect of thermal treatments on the properties of coconut milk emulsions prepared with surface-active stabilizers. Food Hydrocolloids, 23(7), 1792e1800. Thanasukarn, P., Pongsawatmanit, R., & McClements, D. J. (2004a). Impact of fat and water crystallization on the stability of hydrogenated palm oil-in-water emulsions stabilized by whey protein isolate. Colloids and Surfaces A-Physicochemical and Engineering Aspects, 246(1e3), 49e59. Thanasukarn, P., Pongsawatmanit, R., & McClements, D. J. (2004b). Influence of emulsifier type on freeze-thaw stability of hydrogenated palm oil-in-water emulsions. Food Hydrocolloids, 18(6), 1033e1043. Thanasukarn, P., Pongsawatmanit, R., & McClements, D. J. (2006). Impact of fat and water crystallization on the stability of hydrogenated palm oil-in-water emulsions stabilized by a nonionic surfactant. Journal of Agricultural and Food Chemistry, 54(10), 3591e3597. Yanke, L., Shufen, Z., Jinzong, Y., & Qinghui, W. (2004). Relationship of solubility parameters to interfacial properties of sucrose esters. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 248(1e3), 127e133.
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Food Hydrocolloids journal homepage: www.elsevier.com/locate/foodhyd
Interfacial and emulsifying properties of sucrose ester in coconut milk emulsions in comparison with Tween Suwimon Ariyaprakai a, *, Tanachote Limpachoti a, Pasawadee Pradipasena b a b
Department of Food Biotechnology, Faculty of Biotechnology, Assumption University, Ram Khamhaeng Rd. Soi 24, Hua Mak Campus, Bangkok 10240, Thailand Department of Food Technology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 January 2012 Accepted 6 June 2012
In this study, sucrose esters were presented as a promising alternative to petrochemically synthesized Tweens for application in coconut milk emulsions. The interfacial and emulsifier properties of sucrose ester (SE), mainly sucrose monostearate, had been investigated in comparison with Tween 60 (TW), an ethoxylate surfactant. The interfacial tension measurement showed that SE had a slightly better ability to lower the interfacial tension at coconut oilewater interface. These surfactants (0.25 wt%) were applied in coconut milk emulsions with 5 wt% fat content. The effects of changes in pH, salt concentration, and temperature on emulsion stability were analyzed from visual appearance, optical micrograph, droplet charges, particle size distributions, and creaming index. Oil droplets in both SE and TW coconut milk emulsions extensively flocculated at pH 4, or around the pI of the coconut proteins. Salt addition induced flocculation in both emulsions. The pH and salt dependence indicated polyelectrolyte nature of proteins, suggesting that the proteins on the surface of oil droplets were not completely displaced by either added nonionic SE or TW. TW coconut milk emulsions appeared to be thermally unstable with some coalesced oil drops after heating and some oil layers separated on top after freeze thawing. The change in temperature had much lesser influence on stability of SE coconut milk emulsions and, especially, it was found that SE emulsions were remarkably stable after the freeze thawing. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Sucrose esters, or sugar-based surfactants, are in current interest because they are produced from natural resources such as sucrose and vegetable oil. They are biodegradable and more biocompatible when compared to other petrochemically synthesized surfactants. Sucrose esters are non-toxic and safe for food and are approved as a food additive under food regulations and laws in several countries i.e. Japan, USA, and EC. It had been reported that sucrose esters have excellent emulsifying properties and be able to apply in various food products (Garti, 2001). Their wide range of hydrophilicelipophilic balance (HLB), depending on degree of esterification of fatty acids and sucrose, provides ultimate application of sucrose esters to each product type. Coconut milk is one of food emulsions that require additional surfactants or emulsifiers to improve emulsion stability. Several recent works have been carried out on physicochemical characterization of
* Corresponding author. Tel.: þ66 2 300 4553x3796; fax: þ66 2 300 4553x3792. E-mail address: [email protected] (S. Ariyaprakai). 0268-005X/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodhyd.2012.06.003
coconut milk employing other small molecule surfactants such as Tweens and sodium dodecyl sulfate (Tangsuphoom & Coupland, 2009a,b), however, data on sucrose esters remain fewer. In those studies, coconut milk emulsions after addition of surfactants were reported to smaller in average droplet size, decrease in total surface protein concentration, and change in droplet surface charges (Tangsuphoom & Coupland, 2008b, 2009a). This study aims to gain more understanding on the relationship between interfacial properties and stability of coconut milk emulsions with addition of sucrose ester. We described the comparative interfacial and emulsifier properties between sucrose ester (SE), mainly sucrose monostearate, and Tween 60, or polyoxyethylene sorbitan monostearate (TW). Their structures were displayed in Fig. 1. The difference between the carbohydrate and ethoxylate headgroups provided an interesting comparison. Their adsorption behaviors at the coconut oil and aqueous interface were investigated by the interfacial tension measurement. The coconut milk emulsions prepared with SE and with TW were processed at different temperatures (121 C, 100 C and 20 C) and their stability was investigated at different pH (2e8) and salt concentrations (0 and 20 mM CaCl2).
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359
CH OH O
O HO
HO
OH
O
O
O
O
O
O
HO HC
O
OH HO
O
O
O
O
O OH
HO O
O
O
O
O OH
O O
O
O
O
O
O O
Fig. 1. Chemical structures of (a) sucrose monosterate (SE) and (b) Tween 60 or ethoxylated sorbitan monostearate (TW).
2. Material and methods 2.1. Materials Ryoto sucrose esters (S1670) were supplied by MitsubishiKagaku Foods Corporation (Tokyo, Japan). Ryoto S1670 contained mainly of 50e53% sucrose monostearate with sucrose monopalmitate 18e20%, sucrose disterate 12e14 %, sucrose dipalmitate 5e6%, sucrose alkylate 5e10% and other ash and moisture. Tween 60 (Polyoxyethylene sorbitan monostearate), hydrochloric acid, sodium hydroxide, sodium azide (NaN3), calcium chloride (CaCl2), and Oil Red O were purchased from Sigma Chemical Company (St. Louis, MO, USA). Coconut oil and soybean oil were supplied by Tropicana Oil (Nakhonpathom, Thailand) and Thai Vegetable Oil Public Company Limited (Nakhonpathom, Thailand), respectively. Ground coconut meat was purchased locally. Distilled water was used for emulsion preparation throughout experiment. 2.2. Interfacial tension measurement Coconut oilewater interfacial tensions were measured according to Du Nouy ring method at 25 C using a digital tensiometer (K10, Kruss Scientific, Hamburg, Germany). A series of SE and TW aqueous solutions at different concentrations were prepared and kept in plastic bottles prior measurements. The SE solutions had to be preheated to 70 C due to its difficulty to dissolve in water. Before each measurement, the solution was poured into a measurement vessel and coconut oil was overlaid on top of the aqueous layer. The ring was cleaned with ethanol and distilled water and flamed with a Bunsen burner before each measurement. Each measured point was an average from at least two replicates. 2.3. Coconut milk emulsion preparation Coconut milk was produced by adding distilled water to ground coconut meat at a weight ratio of 2:1. The mixture was manually pressed and filtered through filter cloth to remove solid residues. The fat contents of the obtained coconut milk were determined by using Rose-Gottlieb method (AOAC, 2000). The extracted coconut milk was further diluted with either distilled water or surfactant aqueous solutions to a final fat content of 5 wt% and a final surfactant concentration of 0 or 0.25 wt%. The surfactant concentration of 0.25 wt% was chosen because this concentration had been proven to provide sufficient stability to coconut milk emulsions (Tangsuphoom & Coupland, 2009a). Sodium azide 0.02 wt% was added to prevent microbial growth. Each emulsion
was further homogenized by using a high speed homogenizer (Ultra-turrax, IKA Labortechnik, Germany) for 3 min at the speed of 11,200 rpm. 2.4. Measurement of emulsion properties 2.4.1. Droplet size measurement The particle size distributions of emulsions were measured by using a laser diffraction particle size analyzer (Mastersizer 2000; Malvern Instruments Ltd., Worcestershire, UK), with a dualwavelength detection system. Emulsion samples were dropped and diluted in the test chamber that filled with distilled water in order to prevent multiple light scattering effects. The size distributions of emulsions were obtained via a best fit using Mie theory. The refractive indices of 1.33 for water and 1.15 for coconut oil were employed as optical properties of emulsions. The particle sizes were reported as surface-volume average diameters, P P d32 ¼ ni d3i = ni d2i , and volume-weighted average diameters, P P d43 ¼ ni d4i = ni d3i , where di is the midpoint of the size interval i and ni is the number of particles in that interval. 2.4.2. z-potential measurement The electrical charges on emulsion droplets were determined by using a particle electrophoresis instrument (Zeta-Meter System 3.0þ, Zeta-Meter, Inc., Staunton, VA, USA). The emulsion sample after 1/100 dilution was put into an electrophoresis cell that had electrodes at both ends. The direction and velocity of emulsion droplets that move in the applied electrical field were observed under a microscope and the charge or z-potential on emulsion droplets was calculated. Each reported z-potential was an average from five replicates. 2.4.3. Optical microscopy The emulsion microstructure was examined by using a standard optical microscope (Alphaphot-2, YS2-H, Nikon Corporation, Japan) equipped with a microscope eyepiece camera (AM423 Dino-Eye USB, AnMo Electronics Corporation, Taipei, Taiwan) with a software (DinoCapture Software) installed on a computer. A few drops of emulsion were put on a glass slide and fully covered with a cover slid. The microscope magnification of 100 was employed. 2.4.4. Visual appearance and creaming index measurement The emulsion sample (10 g) was transferred into a standard test tube and tightly sealed with a cap. The appearance of the emulsion sample was closely captured by a digital camera. After gently mixed by inversion, the tube was left on a tube stand for 10 min. The height
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of aqueous layer left after the emulsion droplets creamed to the top and the height of initial total sample in the tube were measured. The creaming index was determined from the percentage of the height of the aqueous layer over the height of the total sample. 2.5. Coconut milk emulsion stability 2.5.1. Emulsion stability against pH and salt concentration The emulsion sample (10 g) was transferred into a standard test tube and adjusted to the specified pH values (2, 4, 6, and 8) by using 0.1 and 1 M HCl or 0.1 and 1 M NaOH. Noted that the normal pH of extracted coconut milk was w6 before the adjustment. For the salt experiment, CaCl2 salt was also added to a concentration of 20 mM. The charges on the emulsion droplets were determined from z-potential measurement and the stability of emulsion was analyzed by z-potential measurement, creaming index measurement, and optical micrograph.
Fig. 2. Coconut oilewater interfacial tensions vs. logarithm of concentration (log C) isotherms for (D) sucrose ester (SE) and (B) Tween 60 (TW).
2.5.2. Emulsion thermal stability The emulsion samples (10 g each) were transferred into standard test tubes and kept in an autoclave at 121 C for 10 min, or in a boiling water bath at 100 C for 20 min, or in a freezer at 20 C
Fig. 3. Visual appearance of phase separation of coconut milk emulsions without addition of any surfactant (Control), with addition of 0.25 wt% Tween 60 (TW), and with addition of 0.25 wt% sucrose ester (SE) after thermal treatments at different temperatures. (a) White creamy emulsion. (b) Coagulated solid particles. (c) Destabilized oil layer (free oil).
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for 14 h. After removal from each storage conditions, the emulsions were stored to reach room temperature prior further analysis. The stability of emulsion was analyzed from droplet size measurement, visual appearance, and optical micrograph. In addition, freeze thaw stability of emulsions was assessed by measuring amount of free oil (destabilized oil) after three freezethaw cycles (each cycle: 20 C for 22 h and 30 C for 2 h) using the principle of dye dilution method (Palanuwech, Potineni, Roberts, & Coupland, 2003; Thanasukarn, Pongsawatmanit, & McClements, 2004b). The employed Oil-Red O is colored oil that only dissolves in free oil but does not dissolve in emulsified oil droplets. A stock solution of 0.0015 wt% Oil-Red O in soybean oil was initially prepared. A series of coconut oil solutions were obtained by adding different amounts of coconut oil to the stock Oil-Red O solution. The absorbance of the solution series was measured by using an ultravioletevisible spectrophotometer (Spectronic Genesys 5, Milton Roy company, Rochester, NY, USA) at a wavelength of at 520 nm. The plot between the measured absorbance and the coconut oil concentrations was used as a calibration curve. To determine amount of free oil in emulsions, the emulsion sample (35 g) was added with 3 g of the stock Oil-Red O solution, vortexed for 30 s, and centrifuged at the speed of 5500 rpm for 5 min (Universal centrifuge, Model PLC-012, Gemmy Industrial Corporation, Taipei, Taiwan). The colored oil on the upper layer was transferred by a micro-pipette into a plastic cuvette and measured its absorbance. The measured absorbance was converted to weight percent of free oil by using the prepared calibration curve.
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3. Results and discussion 3.1. Coconut oilewater interfacial behavior The basic information about the interfacial properties of SE and TW at coconut oilewater interface was obtained from the interfacial tension measurement. Fig. 2 is the plot between the measured interfacial tensions of SE and TW aqueous solutions at various concentrations. The gradually decrease in the measured interfacial tension was observed as surfactant concentrations increased. After the interfacial tensions reduced to a minimum value, it approached a constant as surfactants started forming micellar aggregates. The minimum interfacial tensions for SE and TW were <2 mN/m and 8 mN/m, respectively. Unfortunately, the minimum interfacial tension for SE was too small to be measured precisely from our existing instrument. It had reported that the lowest interfacial tension of SE at the rapeseed oilewater interface was 1.5 mN/m (Soultani, Ognier, Engasser, & Ghoul, 2003). SE tended to have a slightly better ability than TW in reducing the oilewater interfacial tension. The critical aggregation concentrations (CAC) at those minimum interfacial tensions in Fig. 2 were approaching to 0.05 wt% for SE and around 0.11 wt % for TW. The lower value of CAC indicated more hydrophobicity of SE, most likely due to this commercial SE also contained some proportion of diesters. Our results agreed with the reported CAC value of 0.05 wt% SE at the liquid paraffinewater interface (Yanke, Shufen, Jinzong, & Qinghui, 2004).
Fig. 4. Optical micrographs of coconut milk emulsions without addition of any surfactant (Control), with addition of 0.25 wt% Tween 60 (TW), and with addition of 0.25 wt% sucrose ester (SE) after thermal treatments at different temperatures. (a) Extensively coalesced oil drops. (b) Coalesced oil droplets. (c) Coagulated solid particles.
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a
b
c
d
e
Fig. 5. Particle size distributions of coconut milk emulsions without addition of any surfactant (Control), with addition of 0.25 wt% Tween 60 (TW), and with addition of 0.25 wt% sucrose ester (SE) after thermal treatments at different temperatures. (a) At room temperature. (b) At 121 C for 10 min. (c) At 100 C for 20 min. (d) At 20 C for 14 h. (e) All thermal treatment results for coconut milk emulsions with addition of 0.25 wt% sucrose ester (SE). The arrows indicate extra peaks from coagulated solid particles.
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Table 1 The average particle sizes (d3,2 and d4,3) of coconut milk emulsions without addition of any surfactant (Control), with addition of 0.25 wt% Tween 60 (TW), and with addition of 0.25 wt% sucrose ester (SE) after thermal treatments at different temperatures. Thermal treatments
Control coconut milk
TW coconut milk
Temperature
Holding time
d3,2 (mm)
d3,2 (mm)
28 C (Room temp.) 121 C 100 C 20 C
e 10 min 20 min 14 h
10.001 22.057 16.103 7.285
d4,3 (mm) 1.228 3.866 1.839 0.964
33.327 114.155 72.344 42.272
5.624 6.522 16.996 3.222
5.535 12.746 7.336 3.533
SE coconut milk d4,3 (mm)
0.175 0.264 0.298 0.112
13.785 125.284 23.710 11.702
d3,2 (mm) 0.482 5.600 5.894 0.274
8.800 8.340 7.967 7.858
0.326 0.541 0.441 0.266
d4,3 (mm) 25.891 58.275 29.078 25.628
1.467 8.155 1.698 0.366
Means standard deviation of at least two replicates.
3.2. Emulsion thermal stability 3.2.1. Coconut milk emulsions at room temperature Coconut milk emulsions were prepared with and without addition of SE or TW and kept at room temperature (w28 C) before determining their characteristics from visual appearance (Fig. 3), optical micrographs (Fig. 4), and particle size measurements (Fig. 5a and Table 1). Fig. 3 shows that all three types of emulsions: Control coconut milk (without addition of any surfactant), TW coconut milk, and SE coconut milk, appeared as white creamy emulsions. Their average sizes (d3,2) were 10.0 microns, 5.5 microns, and 8.8 microns, respectively (Table 1). The results from the optical micrographs (Fig. 4) confirmed that the particle sizes of coconut milk emulsions containing SE or TW were smaller than Control coconut milk. More droplet flocculation in SE emulsions was suggested to be the reason of SE emulsions having larger average size than TW emulsions. The bulky headgroup of TW seemed to effectively provide steric barriers that prevented droplet flocculation in TW emulsions. Even though SE had a better ability than TW in reducing the interfacial tension between coconut oil and water interface, this was not enough to observe its effect on emulsion droplet size.
three coconut milk emulsions (Fig. 3). The presence of such solid aggregates had also been reported in other studies (Chiewchan, Phungamngoen, & Siriwattanayothin, 2006; Tangsuphoom & Coupland, 2009b). Those particles were denatured proteins which were detected as solid substance in the optical micrographs (arrows in Fig. 4) and as an extra population on the right to the size distribution of emulsion population (arrows in Fig. 5b). From the optical micrographs, some large coalesced oil drops were observed in Control coconut milk that were significantly larger than oil droplets in
a
3.2.2. Stability of coconut milk emulsions after heating Further investigation was assessing thermal stability of coconut milk emulsions by heating the emulsions at 121 C for 10 min. After the heating, the coagulated white solid particles were observed in all
Control coconut milk TW coconut milk SE coconut milk
b % Destabilized oil (free oil)
100
80
60
40
20
0 1
2 3 Number of freeze-thaw cycles
Fig. 6. Percentage of destabilized oil (free oil) in coconut milk emulsions without addition of any surfactant (Control), with addition of 0.25 wt% Tween 60 (TW), and with addition of 0.25 wt% sucrose ester (SE) after 1, 2 and 3 freeze-thaw cycles.
Fig. 7. Emulsion droplet charge (or z-potential) of () coconut milk emulsions without addition of any surfactant (Control), (B) with addition of 0.25 wt% Tween 60 (TW), and (D) with addition of 0.25 wt% sucrose ester (SE). (a) After only pH adjustment. (b) After pH adjustment and addition of 20 mM CaCl2 salt.
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either SE and TW emulsions (Fig. 4). Table 1 shows that the average particle size (d3,2) of Control coconut milk increased from 10.0 to 22.1 microns (or by a factor of 2.2) and TW coconut milk increased from 5.5 to 12.7 microns (or by a factor of 2.3). However, the average particle size of SE emulsion was still around 8e9 microns showing no significantly change (Table 1 and Fig. 5e). Noted that we chose the surface-volume average diameter (d3,2) instead of d4,3 to analyze our results of multi-modal distributions here. D3,2 was based on area distribution and better representing the average size of smaller particles (with more surface area) which supposed to be emulsion droplets in this case. D4,3,or volume-weighted average diameter, was calculated from volume distribution and more representing the average size of larger particles (with more volume) which could be the size of flocculated droplets or solid aggregates. Similarly, after coconut milk emulsions were kept at the temperature of 100 C for 20 min, the average sizes of Control coconut milk emulsions increased by a factor of 1.6 and the size of TW emulsions slightly increased by a factor of 1.3, but SE emulsions remained the same average size of 8e9 microns (Table 1 and Fig. 5c and e). This was accompanied by the results from the optical micrographs (Fig. 4) showing that SE and TW emulsions still appeared as small oil droplets but Control coconut milks became
large coalesced oil drops. No coagulated solid particles floated in TW or SE emulsions as observed in TW and SE coconut milk at 121 C (Fig. 3), suggesting that the degree of protein denaturation was lesser at this lower temperature (100 C). Coconut proteins, a natural coconut milk emulsifier, when denatured by heat lose their ability to stabilize emulsion droplets. We observed large coalesced oil drops in Control coconut milk after heated at 121 C or 100 C as oil droplets that come in close proximity undergo extensively coalesce. Lesser degrees of droplet coalescence in SE and TW emulsions when compared with Control coconut milk indicated that heating stability of coconut milk enhanced by addition of SE or TW. We found that SE stabilized emulsions seemed to be more heat stable than TW emulsions as the average sizes of SE emulsions (d3,2) did not significantly alter after the heating at both temperatures (Table 1 and Fig. 5e). The properties of SE had been reported to be lesser sensitive to temperature variation when compared to other nonionic surfactants with oxyethylene groups (Stubenrauch, 2001). 3.2.3. Stability of coconut milk emulsions after freeze thawing Coconut milk emulsions were kept at freezing temperature of 20 C for 14 h, and after being thawed at room temperature, their
Fig. 8. Optical micrographs of coconut milk emulsions without addition of any surfactant (Control), with addition of 0.25 wt% Tween 60 (TW), and with addition of 0.25 wt% sucrose ester (SE) after pH adjustment (a) Extensive flocculation. (b) Some flocculation.
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characteristics were examined. From their visual appearance (Fig. 3), some oil layers were accumulated on top of Control and TW coconut milk emulsions, but in a sharp contrast, no oil layer was observed in SE coconut milk. The optical micrographs in Fig. 4 show some coalesced oil droplets in the Control and TW samples. The particles size distributions (Fig. 5d) and average sizes (d3,2) (Table 1) of Control and TW emulsions were significantly altered from their original emulsions at room temperature. This deviation should be reasoned that some oil droplets in emulsions were already coalesced and separated out as oil layers and those oil droplets were not detected during the size measurement. Differently, SE emulsified droplets appeared the same as their originally prepared emulsions (Fig. 4) and its particle size distribution remained the same (Fig. 5e) with the average size w8 microns closed to its original size (Table 1), revealing good emulsion stability. To ensure our results, numbers of freeze-thaw cycles were increased up to three and the amounts of destabilized oils were determined after each cycle. Fig. 6 clearly demonstrated that the amounts of destabilized oil coming out from SE emulsions after each freezing cycle were much lesser than from Control and TW emulsions. After the first cycle, almost no destabilized oil was detected in SE emulsions, while 93% and 68% of destabilized oils were obtained from Control and TW emulsions, respectively. After the third cycle, the percentage of destabilized oils in SE emulsions was three times lesser than in Control or TW emulsions. Noted that even though this dye-dilution method (Palanuwech et al., 2003) might not be very precise to quantify amounts of oil, the results should be accurate enough for comparing relative amounts of oil coming out from each emulsion. Many other studies have shown that their emulsions also destabilized into oil layer after freeze thawing; for example, coconut milks prepared from Tween 20 (Tangsuphoom & Coupland, 2009b), palm oil emulsions prepared from Tween 20 (Thanasukarn et al., 2004b; Thanasukarn, Pongsawatmanit, & McClements, 2006), and n-alkanes emulsions prepared from SDS and Tween 20 (Cramp, Docking, Ghosh, & Coupland, 2004). We noticed that those emulsions that prepared from Tweens were prone to be unstable similarly to our TW result. Several researchers aim to understand the destabilization mechanism in order to improve freeze-thaw stability of emulsions (Cramp et al., 2004; Ghosh & Coupland, 2008). Thus, it is very interesting here to discover that emulsions prepared from SE were more freeze-thaw stable than TW emulsions. A number of studies have reported that the presence of sucrose increased freeze-thaw stability of emulsions (Ghosh, Cramp, & Coupland, 2006; Thanasukarn, Pongsawatmanit, & McClements, 2004a; Thanasukarn et al., 2006). Sucrose has been known to have a cryo-protective property coming from sucrose ability to lower the freezing point of water. Such unfrozen water protects other compounds dissolving in the aqueous phase from the freeze damage. The unfrozen water in emulsions protects oil droplets from penetration of growing ice crystal that would disrupt the interfacial membrane and cause droplet coalescence. We found that there was also a report about the presence of unfrozen water in the aqueous solution of sugar-based surfactant and its possible cryo-protective effect (Ogawa, Asakura, & Osanai, 2009). We suspected that our SE coconut milk contained some unfrozen water and this was the reason that SE coconut milk emulsions were still stable after the freeze thawing.
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net charge gradually reduced to zero when pH approached to the value of 4, corresponding to the PI of coconut protein reported in other literatures (Onsaard, Vittayanont, Srigam, & McClements, 2005; Tangsuphoom & Coupland, 2008a). When proteins lose their net charge at this pH, the Control emulsion droplets became extensively flocculated (Fig. 8) and creamed rapidly with the creaming index of 42% after ten-minute storage (Fig. 9). At other pH values, the electrostatic repulsion provided by coconut proteins was sufficient to prevent aggregation of droplets and no creaming was observed. The result of pH effect for SE and TW coconut milk was relatively invariant to Control coconut milk. The z-potential of SE and TW emulsions decreased from a positively charge at pH 2 to a negatively charge at pH 8 with a net zero charge at pH close to 4 (Fig. 7a). SE and TW emulsions only creamed at pH 4 with the creaming index after ten-minute storage of 8% for SE emulsions and of 50% for TW emulsions (Fig. 9). The much lower in creaming index of SE emulsions suggested that SE might also alter rheological property of emulsions and beneficial as a creaming stabilizer. The highest creaming rate was accompanied by the highest flocculation shown in the micrographs at pH 4 in both emulsions (Fig. 8). It was a little surprising that, after addition of nonionic surfactants such as SE and TW, the charges on droplets were still dominant and sensitive to the pH condition since nonionic surfactants would provide steric stability and remove electrostatic effect of proteins. If the proteins on emulsion droplets were completely displaced then the properties of the emulsion should be the same as the replacing surfactants (McClements, 2004). More investigation on the role of droplet charges was by addition of 20 mM CaCl2 salt. The micrographs from Fig. 10 show that salt addition induced droplet flocculation at pH of 6 and 8, similarly in Control, TW and SE emulsions. The flocculation was obvious when comparing the micrograph before (Fig. 8) and after salt addition (Fig. 10). The presence of flocculated droplets had been attributed to ability of salt to screen charges of coconut proteins (Fig. 7b) and
3.3. Emulsion stability against pH and salt concentrations The charge characteristics of coconut protein on oil droplets were investigated by exposing emulsions to different pH conditions. Fig. 7a shows that the z-potential of Control coconut milk droplets changed from a positively charge of þ16 mV to a negatively charge of 28 mV when increasing the pH from 2 to 8. From the graph, the
Fig. 9. Creaming index after ten-minute storage of () coconut milk emulsions without addition of any surfactant (Control), (B) with addition of 0.25 wt% Tween 60 (TW), and (D) with addition of 0.25 wt% sucrose ester (SE) after adjustment to different pH conditions.
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Fig. 10. Optical micrographs of coconut milk emulsions without addition of any surfactant (Control), with addition of 0.25 wt% Tween 60 (TW), and with addition of 0.25 wt% sucrose ester (SE) after pH adjustment and addition of 20 mM CaCl2 salt. (a) Extensive flocculation.
reduced electrostatic repulsion between oil droplets. We noticed that the oil droplets did not flocculate after addition of salt at pH 2 where amino acids were mostly protonated and proteins had highly net positively charge. It seemed that oil droplets were most repulsive at this low pH condition as also observed in emulsions stabilized by other protein (Kim, Decker, & McClements, 2004). The sensitivity to pH and salt confirmed the presence of proteins at interfacial droplets in SE and TW emulsions. Thus, in the present case, proteins on droplets of coconut milk emulsion were not completely displaced by the SE or TW content of 0.25 wt%. We postulated that SE or TW molecules stayed on oil droplet interface together with coconut protein stabilizing emulsions by both steric and electrostatic repulsion. Further investigation on interfacial behaviors when coconut proteins coexist with these surfactants is suggested. The interfacial characteristics are governed by structure and thickness of proteinesurfactant films, the interfacial ratio of protein to surfactant, and the proteinesurfactant interactions (Rodriguez Patino, Rodriguez Nino, & Carrera Sanchez, 2007). A previous study showed that addition of sucrose ester decreased the interfacial tension and the interfacial elasticity of milk protein membrane in
corresponding to the surfactant/protein ratio (Rouimi, Schorsch, Valentini, & Vaslin, 2005). Some literature also evidenced that Tween 60 only partially displaced sodium caseinate (Dalgleish, Srinivasan, & Singh, 1995) and albumin (Seta, Baldino, Gabriele, Lupi, & de Cindio, 2012) from the oilewater interface even at some high surfactant/protein ratios. 4. Conclusions This work clearly showed that sucrose ester (SE) exhibited interesting interfacial and emulsifying properties when compared with Tween 60 (TW). SE was slightly better in lowering the interfacial tension between coconut oil and water interface. The stability and surface charge characteristics of coconut milk emulsions emulsified with 0.25 wt% of nonionic SE or TW strongly depended on pH and salt environments. Thus, it was likely that the addition of SE or TW did not completely displace proteins on oil droplets to remove their electrostatic interaction. The presence of either SE or TW had a distinct effect on the thermal characteristics of coconut milk emulsions: coconut milk emulsions emulsified with SE were more thermally stable than TW emulsions. Especially, we found
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that SE coconut milk emulsions were much more stable after freeze thawing. The complex between coconut protein and SE seemed to be an emulsifying membrane that could better protect coconut milk emulsions from the heat and freeze damages. Acknowledgment This research was fully supported by the Research Grant for New Scholar (MRG53) provided by the Thailand Research Fund and Assumption University and the mentor of this project was Asst. Prof. Pasawadee Pradipasena from Department of Food Technology, Faculty of Science, Chulalongkorn University. Special thanks to Ms.Treuktongjai Saenghiruna and Ms. Rungchat Kladcharoen, undergraduate students from Assumption University who dedicated in some experimental works and to Ms. Martha Intan Budiati, a practical training student from Faculty of Agricultural Technology, Soegijapranata Catholic University, Semarang, Indonesia, who helped during sample preparation. Also thanks to Caltech Corp., Ltd. for kindly donating the sucrose ester used in this study. References AOAC. (2000). Official methods of analysis of AOAC international (17 ed.). Gaithersburg, MD: Association of Analytical Communities. Chiewchan, N., Phungamngoen, C., & Siriwattanayothin, S. (2006). Effect of homogenizing pressure and sterilizing condition on quality of canned high fat coconut milk. Journal of Food Engineering, 73(1), 38e44. Cramp, G. L., Docking, A. M., Ghosh, S., & Coupland, J. N. (2004). On the stability of oil-in-water emulsions to freezing. Food Hydrocolloids, 18(6), 899e905. Dalgleish, D. G., Srinivasan, M., & Singh, H. (1995). Surface properties of oil-in-water emulsion droplets containing casein and Tween 60. Journal of Agricultural and Food Chemistry, 43(9), 2351e2355. Garti, N. (2001). Food emulsifiers and stabilizers. In N. A. M. Eskin, & D. S. Robinson (Eds.), Food shelf life stability: Chemical, biochemical, and microbiological changes. Boca Raton: CRC Press. Ghosh, S., & Coupland, J. N. (2008). Factors affecting the freeze-thaw stability of emulsions. Food Hydrocolloids, 22(1), 105e111. Ghosh, S., Cramp, G. L., & Coupland, J. N. (2006). Effect of aqueous composition on the freeze-thaw stability of emulsions. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 272(1e2), 82e88. Kim, H. J., Decker, E. A., & McClements, D. J. (2004). Comparison of droplet flocculation in hexadecane oil-in-water emulsions stabilized by Î2-lactoglobulin at pH 3 and 7. Langmuir, 20(14), 5753e5758.
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