Freeze–thaw stability of lecithin and modified starch-based nanoemulsions

Food Hydrocolloids 25 (2011) 1327e1336

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Freezeethaw stability of lecithin and modified starch-based nanoemulsions Francesco Donsì a, b, Yuwen Wang a, Qingrong Huang a, * a b

Department of Food Science, Rutgers University, 65 Dudley Road, New Brunswick, NJ 08901, USA Department of Chemical and Food Engineering, University of Salerno, Fisciano (SA) 84084, Italy

a r t i c l e i n f o

a b s t r a c t

Article history: Received 29 August 2010 Accepted 16 December 2010

The impact of freezeethaw cycles on the physical stability of oil-in-water emulsions containing lecithin e coated and modified starch e coated droplets has been studied by combined dynamic light scattering (DLS) and differential scanning calorimetry (DSC) measurements. Emulsions prepared by high-pressure homogenization were within 200 nm size ranges. Lecithin-based emulsion systems were unstable to freezee thaw cycles, which was attributed to extensive droplet aggregation induced by the ice formation during emulsion freezing process. Instead, modified starch systems were highly stable due to the formation of a thick layer of emulsifier which prevented the coalescence of nanoemulsions. The addition of ice nucleating protein lowered the freezeethaw stability of lecithin-based emulsions, but had negligible effect on modified starchbased emulsions. In contrast, the addition of poly(ethylene glycol) improved the stability of lecithin-based emulsions but destabilized the modified starch-based emulsion systems. Ó 2010 Elsevier Ltd. All rights reserved.

Keywords: Nanoemulsion Freezeethaw stability Lecithin Modified starch Ice nucleating proteins High-pressure homogenization

1. Introduction Oil-in-water food emulsions are commonly subjected to freezing, either for product preservation, especially to limit microbial growth and chemical degradation reactions, or for product preparation (i.e., ice creams and frozen cocktails) (Ghosh & Coupland, 2008). Nevertheless, many oil-in-water emulsions are highly unstable when they are chilled and frozen, and, upon thawing, emulsions may eventually break down (Ghosh & Coupland, 2008; Thanasukarn, Pongsawatmanit, & McClements, 2004a). The instability of emulsions upon freezeethaw processes can be explained in terms of different physicochemical mechanisms including fat crystallization, ice formation, interfacial phase transitions, and interfacial layer conformational, chemical or electrostatic interaction changes (Thanasukarn et al., 2004a). Upon freezing, ice formation forces the oil droplets into close proximity within the remaining liquid phase volume (Ghosh & Coupland, 2008). Close proximity of the droplets may cause the draining of the film of water separating them, bringing the surfactant layers into semi-dry contact, leading to the formation of the so-called Newton black film, which is defined as the bilayer film formed of two surfactant monolayers mutually adsorbed on the oil surface, with a certain limited number of water molecules

* Corresponding author. Tel.: þ1 732 932 7193; fax: þ1 732 932 6776. E-mail address: [email protected] (Q. Huang). 0268-005X/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodhyd.2010.12.008

comprised in between. If repulsive forces between droplets cannot limit droplet approach and film drainage, the rupture of the Newton black film, arising from the contact of the oil droplets, allows the contents of the droplets to flow together, causing the consequent coalescence of the droplets (van Aken, 2003; van Aken & Zoet, 2000; McClements, 1999). In addition, if oil droplets also crystallize during freezing, upon melting of the ice a network of crystalline solid droplets may eventually form, depending on phase volume of the fat, unfrozen phase volume and surfactant layers, which, upon further heating, can facilitate rapid coalescence and oiling off. Therefore, depending on freezing conditions, coalescence may occur as a consequence of oil-to-oil contacts caused by the rupture of the membranes surrounding the emulsion droplets (Ghosh & Coupland, 2008). Droplet sizes may also affect the tendency to coalescence. In fact, coarse emulsions are prone to coalescence as the lower internal pressure allows significant deformation and large vulnerability to rupture, while finer emulsions act as hard spheres. Therefore, high external energy is required to overcome the disjoining pressure and to force droplets into close proximity separated by a Newton black film (Ghosh, Cramp, & Coupland, 2006). In fact, the tendency to form channels between adjacent oil droplets depends on the bending energy and surface curvature of the interfacial membrane (Kabalnov & Wennerstrom, 1996). The volume fraction of the dispersed phase plays a significant role in the freezeethaw stability of emulsions since ice formation might increase droplet concentration at or beyond close packing (van Aken & Zoet, 2000). In particular, under high-speed centrifugation, it was

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shown that dilute (5%) emulsions were less prone to coalescence than the concentrated emulsions (20e40%) (Ghosh & Coupland, 2008). Obviously, surfactant composition is also of paramount importance on the freezeethaw stability of emulsions since when droplets come under pressure from an expanding ice phase, the main resistance to coalescence is the interfacial layer. It has been reported that proteins provide better protection against droplet coalescence than small molecular surfactants (Palanuwech & Coupland, 2003; Tangsuphoom & Coupland, 2009; Thanasukarn, Pongsawatmanit, & McClements, 2004b; Vanapalli, Palanuwech, & Coupland, 2002), also in the case of crystallization of both oil and water phases, where the resultant destabilization is often more severe (Cramp, Docking, Ghosh, & Coupland, 2004). The protective role of proteins against freezeethaw instability is often explained in terms of their ability to form thick interfacial membranes (Vanapalli et al., 2002). This hypothesis is supported by the experimental evidence that thicker layers provide better protection for emulsion droplets. Examples were reported for multilayers of surfactants and/or polymers, such as sodium dodecyl sulfate (SDS), chitosan and pectins (Thanasukarn, Pongsawatmanit, & McClements, 2006) or for polysaccharide layers of pectin or carrageenan around the protein-coated (b-lactoglobulin) lipid droplets (Mun, Cho, Decker, & McClements, 2008). A crucial role is also played by lipid phase. Even though alkanes are significantly stable upon freezeethaw processes, they are of limited practical relevance to food systems, where the lipids mainly consist of mixtures of triacyl glycerol molecules which have significantly lower freezeethaw stability than the alkane systems (Vanapalli et al., 2002). This can be explained in terms of crystallization of the lipid phase during freezing cycles: when the lipid droplets remain in a liquid form, a lower tendency to coalescence was observed than that in the fat crystal form (Relkin & Sourdet, 2005; Thanasukarn et al., 2004b). In fact, under the pressure of the ice, the liquid droplets were able to deform while the crystalline droplets focused the stress at the point of contact between the droplets, forcing the surfactants out of the intervening gap (Cramp et al., 2004). For example, using Tween 20 as a stabilizer with different alkanes, the emulsions, whose droplets remained liquid at freezer temperatures (i.e., decane), were stable, while the emulsions, whose droplets were crystalline (dodecane to octadecane), were partly destabilized (Cramp et al., 2004). A more chemically specific role of lipids was instead suggested by Saito et al. (1999), who showed that the freezeethaw stability of emulsions depended also on the penetration of the lipid phase in the surfactant layer. For example, triolein emulsions were more freezeethaw stable than tricaprylin emulsions, because the penetration of medium-chain triglyceride tricaprylin in the surface layers of egg phosphatidylcholine was deeper than the long-chain triglyceride triolein. If solutes are present in the aqueous phase, the increase in solute concentration changes the chemical environment of the interfacial proteins and surfactants. As a consequence of ice formation, all aqueous solutes are concentrated in the aqueous phase, causing the eventual dehydration of the interfacial emulsifiers by changing their conformation, surface activity, and spontaneous curvature (Ghosh & Coupland, 2008). Moreover, the concentrated sugars may weakly interact with co-solutes that change the properties of the adsorbed layers (Baier & McClements, 2005). For example, while the presence of sugar co-solutes typically stabilizes globular proteins against denaturation, it enhances the rate of aggregation of denatured proteins. High sugar concentrations can also dehydrate the aqueous portion of surfactants and change the bending energy of the interface, and hence the free energy of the coalescence transition state (Kabalnov & Wennerstrom, 1996). The protective role of sugar solutes can be also explained in terms of their incorporation into the hydrophilic portions of the

surfactant, preventing the spontaneous curvature needed to form a pore in the membrane separating two droplets in the frozen state that would lead to droplet coalescence (Saito et al., 1999). From a physical point of view, increasing the sugar concentration in the aqueous phase of the emulsion is a well known and industrially practiced way to reduce the amount of ice formed on freezing and hence the pressure exerted on the concentrated emulsion. The added salts may also change the amount of ice formed in a frozen emulsion. However, the concentrated salt solutions in the unfrozen phase can have a much greater effect than sugars on the inter-droplet interactions. Screening the electrostatic repulsion between droplets makes it easier for the ice to force emulsion droplets together to form a Newton black film (Ghosh & Coupland, 2008). Hence, some authors reported the increase of destabilization following the addition of 150 mmol/kg NaCl to the emulsions (Thanasukarn et al., 2004a). However, the stresses induced by freezing, and the capacity of different ingredient combinations to resist them is poorly understood. Nanoemulsions are considered as one of the most effective ways to improve the bioavailability of nutraceuticals (Huang, Yu, & Ru, 2010). However, the available thermodynamically-stable foodgrade emulsions are limited. The search for stable food-grade nanoemulsions is a new research frontier in food science research. Significant experimental efforts are still needed to understand how external stresses can affect the stability of these nanoemulsions. In the present work, we investigated the freezeethaw stability of nanoemulsions prepared by different combinations of two lipid phases with significantly different melting points. Two different emulsifiers, such as lecithin and modified starch, and two different additives to the aqueous phase, such as poly(ethylene glycol) (PEG) and ice nucleating proteins, have been used in this paper to help us better understand the dependence of freezeethaw stability of nanoemulsions on their compositions. Ice nucleating proteins have attracted a lot of interest recently due to their ability of influencing the growth of ice crystals (Crevel, Fedyk, & Spurgeon, 2002). In contrast to PEG and some antifreeze proteins, which usually depress the freezing point of the aqueous phase, ice nucleating proteins actually elevate the freezing point of water (Li & Lee, 1998). By using the Ca2þ ATPase activity of actomyosin protein from tilapia as a model, the incorporation of ice nucleating proteins was found to effectively increase the freezeethaw stability of actomyosin protein (Zhu & Lee, 2007). However, the effect of ice nucleating proteins on the freezeethaw stability of the emulsions is largely unknown. We expect that the results obtained will enable us to better design nanoemulsion formulations that are stable to chilling and freezing. 2. Materials and methods 2.1. Materials A high melting temperature lipid Neobee 1095 (96% C-10 and 4% C-8 fatty acids) and a low melting temperature lipid Neobee 1053 (44% C-10 and 56% C-8), characterized by a melting point of 32  C and of 5  C, respectively, as indicated in manufacturer’s specifications, were donated by Stepan Company (Maywood, New Jersey). The modified starch Purity Gum 2000, which is succinylated waxy maize starch, was donated by National Starch (Bridgewater, New Jersey). Lecithin Alcolec PC75, which has a phospho-choline (PC) content of 70%, was donated by American Lecithin Company (Oxford, Connecticut). Polyethylene Glycol 200 with an average molecular weight of 200 Da was purchased from SigmaeAldrich Chemical Company (St. Louis, Missouri). Milli-Q water was used in all experiments.

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2.2. Preparation of ice nucleating proteins

2.5. Differential scanning calorimetry (DSC)

Erwinia herbicola subsp. ananas (Cat. No. 11530) was obtained from the American Type Culture Collection (ATCC; Rockville, MD, USA). Bacteria were routinely grown at 18  C in a yeast extract as previously described (Crevel et al., 2002). Ice nucleating proteins were purified by centrifugation, filtration and density gradient centrifugation (Zhu & Lee, 2007). Purified ice nucleating proteins were freeze-dried and stored at 20  C before use.

Thermal properties of water, the high melting temperature lipid Neobee 1095, the low melting temperature lipid Neobee 1053, all the four emulsions and the effects of the addition of PEG or INP were investigated using differential scanning calorimetry (Model 823, Mettler Toledo Instruments, OH). Samples were placed into perforated aluminum sealed pans with amount of 15e20 mg. The thermal analysis was performed by a two-cycle model with a scanning range of 50  C to þ50  C at 10  C/min. The sample was initially cooled at 50  C and annealed for 5 min before starting the measurement. Nitrogen was utilized as blanket gas. Measurements were carried out at least in duplicate.

2.3. Preparation of emulsions An aqueous solution of 16.7% modified starch was prepared by dispersing the modified starch-in-water at room temperature for 2 h to ensure complete hydration of the starch prior to homogenization. 23% lecithin solution was prepared by dissolving lecithin either in the high melting temperature lipid or in the low melting temperature lipid at 60  C. PG-Lip1 and PG-Lip2 emulsions were prepared from 90% modified starch aqueous solution and 10% high melting temperature lipid (PG-Lip1) or low melting temperature lipid (PG-Lip2). Lec-Lip1 and Lec-Lip2 emulsions were prepared from 13% lecithin solution in high melting temperature lipid (LecLip1) or low melting temperature lipid (Lec-Lip2) plus 87% water. The oil and water phases were blended using a high-speed blender for 1 min. The coarse emulsions were then passed 10 times through a high-pressure homogenizer at 150 MPa to reduce the mean droplet diameter to below 150 nm. Formula of Lec-Lip1 was used to obtain optimal processing conditions, such as the homogenization pressure and the number of passes. The final emulsions contained 10% oil and 3% lecithin for Lec-Lip1 and Lec-Lip2, while 10% oil and 15% modified starch for PG-Lip1 and PG-Lip2. All these procedures were carried out at 15  C for emulsions using the low melting temperature lipid as the organic phase (Lec-Lip2 and PG-Lip2), and at 50  C for emulsions using the high melting temperature lipid as the organic phase (Lec-Lip1 and PG-Lip1) so that the crystallization of the high melting temperature lipid was prohibited during emulsion processing. To study the impact of ice nucleating proteins (INP) and PEG 200 (PEG) on freezeethaw stability of emulsions, 0.1% INP or 10% PEG were incorporated respectively into water phase of each emulsion above, which makes a total of 12 emulsions with different formulations. All procedures of preparation were the same as above. The particle sizes and thermal behaviors of emulsions were analyzed using particle size analyzer and differential scanning calorimetry (DSC). 2.4. Particle size measurements Emulsions were kept in 15 mL glass jars and stored at 4  C refrigerator before measurements. Small volumes of emulsions were sampled every interval to undergo particle size measurement. The particle sizes and size distributions of emulsions were measured using photon correlation spectroscopy (PCS) e based BIC 90 plus particle size analyzer equipped with a Brookhaven BI9000AT digital correlator (Brookhaven Instrument Corporation, New York, NY, USA). The light source of the particle size analyzer is a solid state laser operating at 658 nm with 30 mW power, and the signals were detected by a high sensitivity avalanche photodiode detector. All measurements were made at a fixed scattering angle of 90 and temperature of 25  0.1  C. To avoid multiple scattering effects the emulsions were diluted with distilled H2O prior to the measurements. The mean diameter or z-diameters were determined by Cumulant analysis of the intensityeintensity autocorrelation functions, G(q,t).

2.6. Freezeethaw cycles Any freezeethaw cycle consisted of a freezing phase, where the freshly-prepared nanoemulsions were kept in glass jars maintained at 18  C (in freezer) for 20 h, and a thawing phase, where the jars were transferred to oven with a temperature fixed at 40  C for 2 h. If the emulsion was stable after the cycle, it was sent back to freezer for another cycle up to 10 cycles. Between each cycle, besides observation of physical stability of the emulsion by naked eyes, particle size of the emulsion was measured using particle size analyzer as an indicator of emulsion stability. Measurements were carried out in duplicates. 3. Results and discussion 3.1. Optimization of nanoemulsion production The effect of the number of passes through high-pressure homogenizer, homogenization pressure and emulsifier loading on emulsion stability has been investigated using the high melting temperature lipid as lipid phase and lecithin as emulsifier. The high melting temperature lipid is solid below 35  C, and hence requires a hot homogenization technique (Mehnert & Mader, 2001). The hot high-pressure homogenization process was conducted at 50  C, by using a jacketed inlet tank to stabilize the inlet fluid at 50  C, by cooling the homogenizer chamber by means of 50  C water-cooled copper coils and by using a built-in heat exchanger immediately downstream of the homogenizer chamber, in order to reduce the temperature of the processed fluid, which increased due to frictional heating in the homogenizing chamber, back to 50  C. The stability of emulsion was monitored by particle size analyzer. Fig. 1 showed that both pressure and the number of passes affected the mean droplet sizes (reported as z-diameters) of the emulsions. In the case of the emulsion tested (3% lecithine10% high melting temperature lipid), below 5 passes, the increase in homogenization passes significantly reduced the droplet sizes. Further increase in homogenization passes had negligible impact on the mean droplet size. The extent of droplet size reduction also depends on the pressure level. In particular, the asymptotic droplet sizes at 50 MPa, 100 MPa, and 150 MPa were ∼150 nm, ∼130 nm, and ∼120 nm respectively. The polydispersity index, which is a measure of how broad the size distribution is, was always around 0.3 after three homogenization passes (results not reported), and the polydispersity index was independent on the pressure level. A simple model (equation (1)) was applied to determine the asymptotic mean droplet diameter and the rate of droplet size reduction.

dm ¼ d0 

am bþm

(1)

F. Donsì et al. / Food Hydrocolloids 25 (2011) 1327e1336

500 50 MPa 100 MPa 150 MPa

z-diameter (nm)

400

300

200

100

0 1

2

3

4

5

6

7

8

9

10

Passes Fig. 1. Effect of operation pressure and the number of homogenization cycles on mean droplet sizes of 3% lecithin e 10% Neobee 1095 emulsions. The lines are fits to equation (1).

In equation (1), dm was the mean droplet diameter (or z-diameter), m the number of passes, d0 the initial diameter that was always set at 500 nm, and a and b were the fitting parameters. The value of (d0  a) represented the asymptotic mean droplet diameter for infinite passes (m / N), while b represented the rate of droplet size reduction, expressed as the number of passes required to reduce the mean droplet size from d0 to (d0  a/2). The fitting parameters were reported in Table 1 along with the regression coefficients R2, which showed that the data fitting was always quite precise in all cases. In particular, R2 coefficients were reported with a number of decimal places exceeding the physical significance to highlight the negligible differences among the regression fits. The model indicated that only minor difference existed between the asymptotic droplet size at 50 MPa (125 nm) and at 100 MPa and at 150 MPa (106 nm and 103 nm, respectively). Instead, the rate of droplet size reduction was quite different, as indicated by the b value that was reduced to half when increasing the pressure level from 50 to 150 MPa. In particular, b was 0.90 at 50 MPa, meaning that after 1 pass the mean droplet size was reduced 300 nm, slightly below (d0  a/2), while b was 0.64 at 100 MPa, meaning that after 1 pass the mean droplet size was reduced to 260 nm, quite below (d0  a/2), and b was 0.42 at 150 MPa, meaning that after 1 pass the mean droplet size was reduced to 220 nm, significantly below (d0  a/2). However, no matter how many homogenization passes were used, the smallest droplet sizes at a fixed pressure level depend on the emulsifier properties and their rate of adsorption onto the newly formed surfaces during droplet size reduction in the homogenizing valve. In fact, a slower adsorption rate favors the Table 1 Values of the parameters a and b (equation (1)) and the coefficient of regression R2 obtained from the fitting of the size-reduction data of the 3% lecithin e 10% Neobee 1095 emulsions reported in Fig. 1.

50 MPa 100 MPa 150 MPa

a (nm)

b (passes)

R2

375 394 397

0.90 0.64 0.42

0.9955 0.9801 0.9998

occurrence of coalescence phenomena among the smaller droplets (McClements, 1999). Also emulsifier concentration is an important factor in determining the droplet sizes. For example, on the same emulsion system, the effect of lecithin at three concentration levels (1%, 3%, and 5%) on the mean droplet diameter was evaluated after 10 passes at 150 MPa (Fig. 2). The z-diameter was larger for the emulsion sample prepared with 1% lecithin (about 150 nm), while the samples prepared with the higher lecithin concentrations of 3% and 5% exhibited smaller mean droplet sizes, of 120 nm and 110 nm respectively. The effect of the lipid phase properties was also evaluated. The high melting temperature lipid, which contains 4% C-8 and 96% C-10 fatty acids, has a higher melting temperature (32  C) than the low melting temperature lipid (5  C), which contains 56% C-8 and 44% C-10 Caprylic and Capric Triglycerides. Homogenization conditions differed only in processing temperature: “hot” homogenization at 50  C was required for the high melting temperature lipid-based emulsions while “cold” processing at 15  C was performed for the low melting temperature lipidbased emulsions. Interestingly, after 10 HPH passes at 150 MPa and employing 3% lecithin in the formulation, the mean droplet sizes obtained for the low melting temperature lipid-based emulsions was 120 nm, which is very close to that for the high melting temperature lipid-based emulsions. The stability of the emulsions prepared was evaluated over time upon storage at 30  C. Results, as reported in Fig. 2, showed that none of the z-diameters of the 4 emulsions tested changed appreciably for a period of time ranging from 12 to 17 days, suggesting that all the four emulsion systems were very stable. Another set of emulsion systems was prepared by using a combination of the modified starch Purity Gum (PG), a starchbased emulsifier, with either the high melting temperature lipid (PG-Lip1) or the low melting temperature lipid (PG-Lip2). The detailed emulsion compositions, preparation procedures, and sizes were described in Table 2. The evolutions of droplet sizes over time upon storage at 30  C of PG-Lip1 and PG-Lip2 are reported in Fig. 3. For both emulsion systems, the mean droplet sizes increased over

300 1% Lecithin + Neobee 1095 3% Lecithin + Neobee 1095 5% Lecithin + Neobee 1095 3% Lecithin + Neobee 1053

250

z-diameter (nm)

1330

200

150

100

50

0 0

4

8

12

16

Days Fig. 2. Evolution of mean droplet sizes of lecithin-based emulsions (10% lipid phase) prepared with different lecithin concentrations and by 10 homogenization passes at 150 MPa.

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Table 2 Composition, preparation procedures and sizes of the tested emulsions. Sample name

Oil

Lec-Lip1 Lec-Lip2 PG-Lip1 PG-Lip2

10% 10% 10% 10%

Neobee Neobee Neobee Neobee

1095 1053 1095 1053

Emulsifier

Preparation procedures

3%wt lecithin 3%wt lecithin 15%wt modified starch 15%wt modified starch

HSH, HSH, HSH, HSH,

5 5 5 5

min; min; min; min;

HPH, HPH, HPH, HPH,

z-diameter (nm)

10 cycles at 150 MPa, 50  C 10 cycles at 150 MPa, 15  C 10 cycles at 150 MPa, 50  C 5 cycles at 150 MPa, 15  C

110 120 130 140

HSH: High-speed homogenization; HPH: High-pressure homogenization.

time during the first 5 days, reaching a plateau value of 175 nm. After 5 days no significant variation was observed. The usage levels for lecithin and modified starch are very different. For the same fraction of lipid phase (10%) for lecithinbased emulsions, only 3% of emulsifier is needed to attain the desired droplet sizes (Fig. 2), while for modified starch-based emulsions, 15% emulsifiers was required in order to obtain a stable formulation. The difference in particle size can be explained in terms of higher surface activity of lecithin. For example, a recent work reported that lecithin had a higher surface activity than neutral branched polysaccharides, and that the increase of surface activity of the polysaccharides can be accelerated by increasing their concentrations (Mezdour, Lepine, Erazo-Majewicz, Ducept, & Michon, 2008). Since the increase of surface activity is related to the kinetics of adsorption to the surface, it is a measure of how quickly the emulsifier molecules can cover the oilewater interfaces formed during HPH, before coalescence phenomena occur. Hence, in comparison to the smaller but faster adsorption of lecithin molecules, the larger amount of modified starch was required to achieve the emulsions of similar mean droplet sizes (130 and 140 nm respectively for PG-Lip1 and PG-Lip2) due to the slower adsorption kinetics of the modified starch molecules.

of either PEG 200 (PEG) or ice-nucleating proteins (INP) on the melting and freezing was also evaluated. INP is a protein that enhances ice nucleation by providing seeds for the ice crystal formation, leading to the formation of smaller crystals at higher temperature (Crevel et al., 2002). On the other side, PEG is a polymer capable of lowering the freezing temperature of water, which is dependent upon solution properties such as viscosity and self-diffusion of solute molecules (Kimizuka, Viriyarattanasak, & Suzuki, 2008). Fig. 4 shows the DSC results of two lipids, the high melting temperature lipid Neobee 1095 and the low melting temperature lipid Neobee 1053, as well as water. The high melting temperature lipid exhibited a freezing peak at temperature of 16  C and a melting peak at temperature of 30  C. In contrast, the low melting temperature lipid is characterized by significantly lower transition freezing and melting temperatures, at 45  C and 2  C respectively. These data can be compared with water freezing and melting temperatures, measured at 30  C and 6  C respectively. Fig. 5 shows the DSC measurements of lecithin-based emulsions with and without the use of additives (i.e., PEG 200 and ice-nucleating proteins). In every DSC thermogram, two main

3.2. Effects of lipids and additives on thermal properties of nanoemulsions

-30°C

Water Neobee 1095 Neobee 1053

10

The thermal properties of emulsion ingredients and the four emulsion systems listed in Table 2 were characterized by differential scanning calorimetry (DSC) measurements. The effect of the addition

Heat flow (W/g)

300

250

z-diameter (nm)

200

0

-16°C 6°C

150

-10 -45°C

100

30°C

50 PG-Lip1 PG-Lip2

-3°C

-20 -40

0 0

4

8

12

16

-20

0

20

40

20

Temperature (°C) Days Fig. 3. Evolution of mean droplet sizes of modified starch-based emulsions upon storage at 30  C over time.

Fig. 4. Differential scanning calorimetry (DSC) measurements of the melting and freezing temperatures of water, Neobee 1095 and Neobee 1053 at temperature range between 50 and 50  C (rate 10  C/min).

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Fig. 5. DSC measurements of the melting and freezing temperatures of Lec-Lip1 (a) and Lec-Lip2 (b) in pure water (no additives), with the addition of 10% poly(ethylene glycol) (PEG), or 0.1% ice nucleating proteins (INP) at temperature range between 50 and 50  C (rate 10  C/min).

peaks attributed to the freezing and melting of the aqueous phase of emulsions could be observed. In particular, the positions of the freezing peaks were affected by the ice nuclei available in the systems, which were mainly constituted by the lipid droplets. When the freezing temperatures of Lec-Lip1 and Lec-Lip2 emulsions were compared, the latter exhibited higher temperatures and, without additives, also broader peaks, suggesting the onset of nucleation mechanisms upon freezing was not as efficient as for Lec-Lip1. In any case, the presence of the emulsion droplets increased the freezing temperatures of emulsions with respect to pure water by more than 10  C. In contrast, the melting temperatures of the emulsions, which were not dependent on the ice nuclei, were nearly the same as water (around 6  C), were affected only by the presence of PEG in the aqueous phase. From a theoretical point of view, the energy released during the emulsion freezing results in a symmetric and narrow peak if the droplet polydispersity is low. However, asymmetric and relatively broad peaks were observed, which may be due to the large distribution of droplet sizes. In particular, the apex temperature of the peak can be correlated to the mean droplet sizes on the basis of either theoretical considerations or empirical results (Clausse, Gomez, Pezron, Komunjer, & Dalmazzone, 2005; Dalmazzone, Noik, & Clausse, 2009). In particular, different authors found empirical correlations between droplet sizes and their crystallization temperatures for both W/O and O/W emulsions (Clausse, Gomez, Dalmazzone, & Noik, 2005; Clausse, Gomez, Pezron, et al., 2005; Montenegro, Antonietti, Mastai, & Landfester, 2003). On the contrary, as there was no delay in the melting phenomenon, all the emulsions were melted at nearly the same temperature, similar to the one observed for a bulk material (Clausse, Gomez, Dalmazzone, et al., 2005). The main difference between

Lec-Lip1 and Lec-Lip2 is the presence of additional freezing and melting peaks of smaller dimensions only for the Lec-Lip1 (Neobee 1095)-based emulsion systems. In the DSC thermograms of LecLip1, these extra peaks occurred during both cooling (lower than the freezing point of the aqueous phase) and heating (higher than the melting point of the aqueous phase). In particular, without and with the addition of 0.1% INP, the extra peaks all occurred at 19  C and 28  C respectively. When PEG was added, the extra freezing peak temperature was shifted to 28  C. In consideration of their positions, which were close but not exactly to the same as the positions of freezing and melting peaks of the pure high melting temperature lipid, the observed peaks could be attributed to the phase transition of the emulsified lipid phase: for Lec-Lip1, the lipid phase crystallized after ice crystals formed. Such phenomenon was not observed in Lec-Lip2-based emulsion systems. In DSC results of Purity Gum-based systems (Fig. 6), two peaks were also observed in the freezing and melting thermograms of PG-Lip1-based emulsion. Similarly, we interpret the additional smaller peaks as due to the phase transition of the emulsified high melting temperature lipid. Interestingly, for PG-Lip1 the lipid freezing peaks were located at around 35  C, significantly lower than the melting temperatures of both pure Neobee 1095 and LecLip1. In fact, it appears that the interfacial material can affect the lipid crystallization when emulsified, even though there is no observable effect on the bulk lipid (Palanuwech & Coupland, 2003). Through the comparison of the small peaks of Lec-Lip1 (Fig. 5) and of PG-Lip1 (Fig. 6), we found that they covered similar areas without and with the addition of either PEG or ice nucleation proteins, although they occurred at temperatures different from each other and different from the freezing temperature of the pure lipid. Our results further suggested that the extra peaks should be

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Fig. 6. DSC measurements of the melting and freezing temperatures of PG-Lip1 (a) and PG-Lip2 (b) in pure water (no additives), with the addition of 10% PEG, or 0.1% INP at temperature range between 50 and 50  C (rate 10  C/min).

attributed to the emulsified fat (it is always 10% for all emulsion systems) rather than the free fat present in the system since the freezing point of the high melting temperature lipid is 16  C (Fig. 4) (Clausse, Gomez, Dalmazzone, et al., 2005; Clausse, Gomez, Pezron, et al., 2005). The effect of modified starch and lecithin on the freezing temperatures of emulsion aqueous phase was also different. DSC results for lecithin-based emulsion systems showed a clear effect of the lipid phase on the observed freezing temperatures. However, the freezing temperatures of both PG-Lip1 and PG-Lip2-based emulsions without additives were 17  C. The difference can be explained in terms of the difference in waterelipid interfaces. Since modified starch is made of large molecules and used in large excess (15%), the freezing nuclei are just represented by the starch molecules completely covering the oil droplets and masking the lipid phase. However, the small lecithin molecules, when used in low amount (3%), may not be able to entirely cover the droplets surface, causing the water freezing and other macroscopic properties, such as viscosity, to be affected by the lipid phase used (Mezdour et al., 2008). More interestingly, our DSC results showed negligible differences between PG-Lip1 and PG-Lip2 upon the addition of INP and PEG. INP probably created new nuclei in the aqueous phase, which broadened and shifted the freezing peaks. In fact, the addition of INP affected only the freezing temperature, but did not affect the melting peak. We interpret this as due to the fact that INP enhanced the ice nucleation sites, resulting in the broadening of freezing peaks and shifting of freezing temperatures to higher values. On the other side, the addition of PEG affected the properties of the aqueous phase, reducing both freezing and melting temperatures. In both cases, the lipid identity appeared to be masked by the modified starch interface layer.

3.3. Effect of emulsifier types and lipids on freezeethaw stability The four emulsion systems described in Table 2, composed of the permutation of two lipid phases (high and low melting temperature lipids) and two emulsifiers (lecithin and modified starch) were compared in terms of freezeethaw stability. Results were reported in terms of the evolution of the mean droplet sizes (z-diameters) upon freezeethaw cycles in Figs. 7 and 8. Shown in Fig. 7 are the freezeethaw stability of lecithin-based emulsions with and without the addition of either PEG or INP. The z-diameters of Lec-Lip1 (without any additive) increased up to 500 nm only in the first 3 freezeethaw cycles, suggesting the occurrence of some destabilization. For Lec-Lip2 under similar conditions (no additive), one cycle was sufficient to attain complete destabilization of the system, with z-diameter well above the micrometric size and phase separation that was clearly observed. For modified starch-based emulsion systems, Fig. 8 shows that both PG-Lip1 and PG-Lip2 in pure water (no additives) were quite stable to freezeethaw cycles, with the z-diameter gradually increasing in the first 5 cycles, and then remaining at sizes lower than 300 nm after the fifth cycles in either system. Modified starch-based systems without additives resulted to be stable to freezeethaw cycles, while lecithin-based systems were not. From the DSC results (Figs. 5 and 6), the freezing temperatures for all 4 systems were increased probably due to the presence of lipid droplets as nuclei. However the freezing temperatures were increased more for lecithin systems (16  C for Lec-Lip1; 13  C for Lec-Lip2) than for Purity Gum systems (17  C for both systems), especially the Lec-Lip2, which could explain the stability of modified starch systems and the destabilization of lecithin systems. It was previously reported that the chemical structure of the

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emulsifier used could affect the crystallization temperature of emulsions (Cordiez, Grange, & Mutaftschiev, 1982; Grange, Lévis, & Mutaftschiev, 1986; Skoda & van den Tempel, 1963), since the hydrophobic tails of adsorbed surfactant molecules may act as template to cause some alignment of the oil molecules closest to the interface and initiate nucleation at higher temperature (Palanuwech & Coupland, 2003). For modified starch-based systems, due to the large amount of emulsifiers used, it appears that the emulsifier characteristics likely control the crystallization temperature, since no measurable differences were recorded in water and with PEG addition between PG-Lip1 and PG-Lip2 (Fig. 6). Instead, for lecithinbased systems, crystallization temperatures of Lec-Lip1 and Lec-Lip2 were always different (Fig. 5). In addition, the thick layer of modified starch can probably offer a better stability than lecithin. It was indeed observed that the thickness of the interfacial layer was the most important factor that granted emulsion stability, due to the complete coverage of droplet

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surfaces and the generation of steric repulsion forces (Mun et al., 2008; Palanuwech & Coupland, 2003). Such interfacial layer was considered as an important protection against partial coalescence. On the other hand, the incomplete coverage of the droplet surfaces by lecithin molecules, already postulated to explain the lipid effect on freezing temperatures of Lec-Lip1 and Lec-Lip2, could also be justified as the origin of the observed freezeethaw instability of lecithin-based systems. It can be observed that while PG-Lip1 and PG-Lip2 do not exhibit significantly different behaviors under freezeethaw cycles, notwithstanding the different composition, Lec-Lip1 is less destabilized than Lec-Lip2, with and without additives. This is even more interesting in consideration of the fact that during the freezing of the emulsions, the lipid droplets containing the high melting temperature lipid crystallized, while those containing the low melting temperature lipid remained liquid, as supported by DSC measurements of Figs. 5 and 6. In particular, lipid crystallization in Lec-Lip1 and PG-Lip1 systems

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occurs only after freezing of the aqueous phase. This observation excludes the onset of the partial coalescence mechanisms, which occurs when oil-in-water emulsions are cooled to temperatures where the fat phase becomes partially crystallized prior to the freezing of the water phase (Boode & Walstra, 1993; Boode, Walstra, & Degrootmostert, 1993; Fredrick, Walstra, & Dewettinck, 2010; Thanasukarn et al., 2004a; Thanasukarn et al., 2004b). Instead, the mobility of lecithin molecules on the droplet surface of the less viscous lipid (the low melting temperature lipid) could affect the coalescence extent, according to the coalescence transition state theory (Kabalnov & Wennerstrom, 1996; Saito et al., 1999). 3.4. Effect of PEG and INP on freezeethaw stability For the lecithin-based emulsion systems, the addition of 0.1% INP did not improve the stability of Lec-Lip2, while for Lec-Lip1, it caused extensive phase separation after 4 cycles, which is even worse than those without any additive (Fig. 7). In contrast, the addition of 10% PEG to the emulsions greatly stabilized the systems over freezeethaw cycles. For both Lec-Lip1 and Lec-Lip2, the mean droplet sizes did not change significantly for 10 freezeethaw cycles. Interestingly, the stability of the modified starch-based emulsions was impacted by the addition of PEG and INP in an opposite way compared to the lecithin-based systems (Fig. 8). The addition of 0.1% INP did not affect the behavior of PG-Lip1 and PG-Lip2, while the addition of PEG significantly destabilized modified starch-based emulsions. With the addition of PEG, the z-diameter of PG-Lip1 increased to about 1000 nm after 10 freezeethaw cycles, while the z-diameter of PG-Lip2 increased to 500 nm already after the first cycle and then it stayed the same in the remaining freezeethaw cycles. The extent of destabilization can be related to the freezing and melting temperatures of the emulsions, as evidenced by DSC measurement. We can observe that for lecithin-based systems, an increased destabilization was observed at higher freezing temperatures. The addition of PEG resulted in the complete stabilization of the emulsion to freezeethaw cycles, due to its ability to reduce the freezing temperature, and hence to increase the amount of unfrozen water and limit the total amount of ice crystals formed, by reducing crystallization temperature (Komatsu, Okada, & Handa, 1997; Ogawa, Decker, & McClements, 2003; Palanuwech & Coupland, 2003; Thanasukarn et al., 2004b). Moreover, it may be speculated that the addition of PEG could modify the ice crystal structure through the alteration of the ice crystal nucleation and growth kinetics, thereby altering their tendency to penetrate into the fat droplets and reduce their stability (Mun et al., 2008), as well as reduce the frequency of dropletedroplet collisions, and increase the aqueous phase viscosity, in analogy to the role attributed to sucrose (Thanasukarn et al., 2004a), maltodextrins, and cryoprotectants (Ogawa et al., 2003). In addition, PEG may form hydrogen bonds with lecithin molecules adsorbed to droplet surfaces, thereby reducing the tendency for interactions between droplet surfaces to occur, resulting in a decrease in the free water content induced by ice crystallization. This phenomenon would increase the volume fraction of unfrozen aqueous phase available to the oil droplets, thereby decrease the tendency for the lipid droplets to be forced into close proximity during freezing, and therefore increase the amount of unfrozen water available to hydrate the adsorbed proteins (Komatsu et al., 1997; Ogawa et al., 2003; Palanuwech & Coupland, 2003; Thanasukarn et al., 2004b). When present at high concentrations in the aqueous phase, lecithin may alter the thermal transition temperatures of certain types of emulsifiers, such as phospholipids and proteins (Ogawa et al., 2003). In the case of modified starch-based emulsions, PEG induced partial destabilization, notwithstanding the decrease in freezing

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temperature. This can be explained in terms of the surface activity of the PEG molecules, due to their amphilic characteristics that make them suitable to be used as co-surfactant, especially in selfassembled microemulsions (Azeem et al., 2009). In particular, some authors reported that PEG may induce a topological transition of the surfactant layers through the modification of the membrane Gaussian modulus due to polymer adsorption and the entropy gain of the adsorbed polymer when the membrane bends to form a vesicle (Iniguez-Palomares & Maldonado, 2009). In addition, from a kinetic point of view, we proposed that PEG might reduce the rate of modified starch adsorption at the liquid/liquid interface, in analogy with what was observed for hen egg-white lysozyme (Malzert-Freon, Benoit, & Boury, 2008), thus enhancing coalescence phenomena during the freezeethaw cycles. 4. Conclusions In summary, our work showed that lecithin-based nanoemulsions were unstable to freezeethaw cycles, due to the extensive droplet aggregation induced by the ice formation during emulsion freezing process. In contrast, Purity Gum-based nanoemulsions were highly stable to freezeethaw cycles due to the formation of thick emulsifier layer, which prevented the coalescence of the lipid droplets. The use of additives, such as ice nucleating proteins or PEG 200, had a very different effect on the stability upon freezeethaw cycles of lecithin and modified starch-based nanoemulsions. The stability of lecithin-based nanoemulsions was lowered by the addition of ice nucleating proteins but significantly improved by the addition of PEG 200. In contrast, the stability of the modified starch-based nanoemulsions was not affected by the addition of ice nucleating proteins and lowered by the addition of PEG 200. Acknowledgment We thank Dr. Tung-Ching Lee for providing INP. Francesco Donsì acknowledges Salerno Province and the University of Salerno for supporting his stay at Rutgers University (International Mobility Scholarship 2008). This work was supported by United States Department of Agriculture, Agriculture and Food Research Initiative (USDAeAFRI) grant (2009-65503-05793). References van Aken, G. A. (2003). Coalescence mechanisms in protein-stabilized emulsions. In S. E. Larsson, K. Sjoblom, & J. Friberg (Eds.), Foods. New York (US): Marcel Dekker, Inc. van Aken, G. A., & Zoet, F. D. (2000). Coalescence in highly concentrated coarse emulsions. Langmuir, 16(18), 7131e7138. Azeem, A., Rizwan, M., Ahmad, F. J., Khar, R. K., Iqbal, Z., & Talegaonkar, S. (2009). Components screening and influence of surfactant and cosurfactant on nanoemulsion formation. Current Nanoscience, 5(2), 220e226. Baier, S. K., & McClements, D. J. (2005). Influence of cosolvent systems on the gelation mechanism of globular protein: thermodynamic, kinetic, and structural aspects of globular protein gelation. Comprehensive Reviews in Food Science and Food Safety, 4(3), 43e54. Boode, K., & Walstra, P. (1993). Partial coalescence in oil-in-water emulsions 1. Nature of the aggregation. Colloids and Surfaces A-Physicochemical and Engineering Aspects, 81, 121e137. Boode, K., Walstra, P., & Degrootmostert, A. E. A. (1993). Partial coalescence in oil-inwater emulsions 2. Influence of the properties of the fat. Colloids and Surfaces A-Physicochemical and Engineering Aspects, 81, 139e151. Clausse, D., Gomez, E., Dalmazzone, C., & Noik, C. (2005). A method for the characterization of emulsions, thermogranulometry: application to water-in-crude oil emulsion. Journal of Colloid and Interface Science, 287(2), 694e703. Clausse, D., Gomez, F., Pezron, I., Komunjer, L., & Dalmazzone, C. (2005). Morphology characterization of emulsions by differential scanning calorimetry. Advances in Colloid and Interface Science, 117(1e3), 59e74. Cordiez, J. P., Grange, G., & Mutaftschiev, B. (1982). Droplet freezing experiments in stearic acidewater emulsions: role of the droplet-medium interface. Journal of Colloid and Interface Science, 85(2), 431e441.

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