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Food Hydrocolloids 25 (2011) 707e715

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Food Hydrocolloids

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Freezeethaw stability of mayonnaise type oil-in-water emulsions

Emma Magnusson a,b,*, Christer Rosén a,b, Lars Nilsson a

aDivision of Food Technology, Faculty of Engineering LTH, Lund University, Po Box 124, S-221 00 Lund, SwedenbKällbergs Industri AB, Po Box 73, S-545 22 Töreboda, Sweden

a r t i c l e i n f o

Article history:Received 1 April 2010Accepted 17 August 2010

Keywords:MayonnaiseFreezeethaw stabilityOil crystallizationEgg yolkEmulsion

* Corresponding author. Division of Food TechnoLTH, Lund University, Po Box 124, S-221 00 Lund, Swfax: þ46 462224622.

E-mail addresses: emma.magnusson@food.lth.se (food.lth.se (L. Nilsson).

0268-005X/$ e see front matter � 2010 Elsevier Ltd.doi:10.1016/j.foodhyd.2010.08.024

a b s t r a c t

In this paper, the freezeethaw stability of mayonnaise type oil-in-water emulsions is studied. Theemphasis of the experiments have been on the properties of the dispersed oil phase as only small, or noeffects, were observed from initial experiments on changing the properties of the aqueous phase withinthe investigated ranges. Different vegetable oils are investigated in order to relate the composition of theoil phase to the stability of the corresponding emulsion. The crystallization behaviour of the oils isstudied with differential scanning calorimetry (DSC) and by freezeethaw experiments in bulk systems.The amounts of triacylglycerides in the oils that theoretically crystallize at different temperatures arealso calculated. Moreover, the impact of the freezing rate on the stability of emulsions is investigated.Large differences in freezeethaw stability of emulsion prepared with different oils are observed. Byprincipal component analysis (PCA) the stability of the emulsion could be correlated with the compo-sition and crystallization behaviour of the oils. Small/no effects of the addition of different substances(for example polyglycerol esters and trehalose) to both oil and water phase are observed. Moreover, theexperiments on the freezing conditions show that alteration of the freezing rate have a large impact onthe freezeethaw stability.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Oil-in-water emulsions are thermodynamically unstable systemsand sensitive to environmental changes, as for example cooling andfreezing (Guzey & McClements, 2006). When an oil-in-wateremulsion is stored at low temperature, crystallization of both the oiland water phase may occur. These phase transitions may lead todestabilization of the emulsion. A destabilizing mechanism causedby oil crystallization is partial coalescence (Palanuwech & Coupland,2003). If semi-crystalline oil droplets collide, crystals from onedroplet may penetrate the interface of the other. The residual liquidoil in the droplets will then flow out and wet the solid fat, therebymaintaining the linkage between the droplets, and when the oilmelts true coalescence will occur (Vanapalli & Coupland, 2001).

The amount of solid fat formed in the droplets, as well as the sizeand morphology of the crystals, may have a great impact on thedestabilization (Boode, Walstra, & Degrootmostert, 1993). Thesefactors are in turn affected by the triglyceride composition (Boodeet al., 1993), additives to the oil (Kalnin, Schafer, Amenitsch, &

logy, Faculty of Engineeringeden. Tel.: þ46 462228303;

E. Magnusson), lars.nilsson@

All rights reserved.

Ollivon, 2004; Sakamoto et al., 2004) and the freezing conditions,such as the cooling rate and temperature (Tippetts & Martini, 2009).

A slow cooling rate generally gives larger crystals than a fastcooling rate. The crystals formed on fast cooling are rigid butunstable, whereas slowly cooled fat generally gives more stablecrystals (Campos, Narine, & Marangoni, 2002). Tippets and Martiniobserved an increased stability by slow cooling (0.2 �C/mincompared to 30 �C/min for fast cooling) of an oil-in-water emulsioncontaining soybean oil and anhydrous milk fat (50:50), especially ata lower oil content (20 vol%) (Tippetts & Martini, 2009). Vanapalli,Palanuwech, and Coupland (2002) however, observed the oppositefor an oil-in-water emulsion containing a confectionary coating fat(with similar physical properties as cocoa butter). When theemulsion (20 vol% oil) was cooled at a slow rate (1.5 �C/min) itdestabilized whereas it remained stable when it was cooled ata faster rate (5 �C/min).

Liquids in general may show a great degree of super cooling, asan energy barrier must be overcome before nucleation and crys-tallization may occur (Cramp, Docking, Ghosh, & Coupland, 2004).In a pure liquid, the crystallization will be homogeneous and theenergy needed for the small starting crystals to grow to macro-scopic size is high (Coupland, 2002). Impurities in the oil phasemayhowever increase the crystallization rate by acting as sites fornucleation (i.e. heterogeneous nucleation) (Cramp et al., 2004).Increased nucleation, by for example waxes, tends to give an

Table 2The most common triacylglycerides in the oils.

Oil The three dominating triacylglycerides

Sunflower LLO, 26.7% LLL, 22.5% OLO, 12.0%HO Sunflower OOO, 64.1% POO, 9.1% OOS, 6.7%Rapeseed OOO, 29.1% OLO, 20.5% LLO, 9.9%Corn LLO, 22.7% LLL, 17.3% PLL, 14.0%Soy LLL, 20.4% LLO, 18.4% PLL, 15.2%

L ¼ Linoleic acid, O ¼ oleic acid, S ¼ stearic acid and P ¼ palmitic acid.

E. Magnusson et al. / Food Hydrocolloids 25 (2011) 707e715708

increased amount of small crystals, rather than few larger ones(Hartel, 2001). The nucleation and growth rate may also be alteredby the addition of different substances to the oil (Coupland, 2002).For instance, polyglycerol esters have been shown to accelerate thenucleation but decrease the growth of triglyceride crystals(Sakamoto et al., 2004). Moreover, addition of propylene glycolmonostearate has been indicated to increase the crystallizationtemperature, decrease the enthalpy of the crystallization and alterthe polymorphism of the crystals (Kalnin et al., 2004).

The crystallization of the aqueous phase will give rise to anextensive volume expansion and thereby increase the pressureexerted on the droplets. Thus the freeze stability may be increasedby altering of the crystallization of the aqueous phase. Addition ofsalt and sugar may decrease the crystallization temperature of theaqueous phase and the amount of ice formed. However, theseingredients may also affect the conformation of the adsorbedspecies and the interactions between substances (Ghosh &Coupland, 2008).

The purpose of this paper was to describe how various factorsaffect the freezeethaw stability of mayonnaise type oil-in-wateremulsions with egg yolk as emulsifier and with a high dispersedphase volume fraction (>60%). Emulsions were prepared withdifferent oils in order to relate the composition of the oil phase tothe freezeethaw stability. The effect of different additives on thecrystallization of the oil and water phase was also investigated, aswell as the effect of the freezing rate.

2. Experimental section

The oils used in this paper were sunflower oil (de-waxed andnon de-waxed), high oleic (HO) sunflower oil, corn oil (de-waxedand non de-waxed), soy oil and rapeseed oil provided by AarhusKarlshamn AB, Karlshamn, Sweden (AAK). A medium chaintriglyceride oil (MCT), mainly consisting of caprylic acid (8:0) andcaprinic acid (10:0) (Miglyol 812 oil, Sasol, Witten, Germany) wasalso studied. Oils were analyzed for fatty acid and triglyceridecomposition (GC-FID) (Eurofins Food & Agro Sweden AB, Lidköping,Sweden). According to analysis results for MCToil, 53.5% of the fattyacids are caprylic acid and 45.9% caprinic acid. The dominating fattyacids and triglycerides for the other oils are shown in Tables 1 and 2.

The different oils were investigated by differential scanningcalorimetry (DSC) (Seiko DSC 6200, Seiko Instruments, Tokyo,Japan). Samples of 5e10 mg were sealed in aluminum pans andloaded into the DSC with an empty reference pan. The temperatureinterval investigated was 30 to �30 �C and the cooling and heatingrate was 5 �C min�1.

Moreover, the pure oils were frozen in a calcium chloride bath(40% (w/w)) with a temperature of �25 �C, to study the crystalli-zation of the oils in bulk systems. From each oil, 30 ml was trans-ferred to plastic tubes (40 ml) and placed in the cooling bath. Thevisual appearance of the oils was then studied after 15 min, 30 min,1 h, 2 h, 3 h and 24 h.

Calculations on the crystallized amounts of triglycerides(b0-form) in the oils at different temperatures were made from the

Table 1The most common fatty acids in the oils.

Oil Fatty acid composition

16:0 18:0 18:1 18:2 18:3

Sunflower 5.9% 3.9% 27.5% 60.7% 0.1%HO sunflower 3.5% 2.9% 82.5% 8.9% 0.2%Rapeseed 4.2% 1.7% 62.2% 18.6% 9.8%Corn 10.6% 1.9% 30.4% 54.6% 1.0%Soy 10.2% 3.1% 24.5% 54.1% 6.4%

triglyceride composition, the molar masses of the triglycerides(Johansson & Bergenståhl, 1995) and the melting enthalpies andmelting points of the triglycerides (Hagemann, 1988; Johansson &Bergenståhl, 1995; Larsson, 1986; O’Brien, 2009). For some of thetriglycerides, experimental values on the melting enthalpies werenot found. Therefore they were estimated according to equationsfor the heat of fusion by Timms (1978). The soluble amount of eachtriglyceride (mole fraction) at a certain temperature was calculatedaccording to equation (1). By calculation of the mole fraction ofeach triglyceride in the different oils, the insoluble amount could bedetermined by subtraction of the soluble mole fraction from thecalculated mole fraction in the oil.

XM ¼ exp���DHm

.R���1.T � 1=Tm

��(1)

Where XM is the solubility of the triglyceride in oil (mole fraction),ΔHm is the melting enthalpy of the triglyceride, R is the ideal gasconstant, T is the absolute temperature and Tm is the melting pointof the triglyceride.

All mayonnaise type oil-in-water emulsions described in thisarticle were prepared under stirring with a high efficiency paddleassembly (ColeeParmer, Vernon Hills, USA) under vacuum. Astandard mayonnaise type emulsion formulation was used:Foil ¼ 67%, 1.3 wt% heat stable egg yolk powder (Källbergs Industri,Töreboda, Sweden), 1 wt% NaCl (Merck, Darmstadt, Germany),1.5 wt% sucrose (Danisco, Copenhagen, Denmark), guar gum0.08 wt% (Edicol 40e70, Lucid Colloids Ltd.,Mumbai, India), 0.02 wt% xantan gum (X200MC, Wykefold Ltd., London, UK) and 0.98 wt%acetic acid (24% (v/v)) (Fluka Chemie GmbH, Buchs, Switzerland).To some of the emulsions different additions to the oil or waterphase were made (described later in this section).

From each emulsion, 30 ml was transferred to 40 ml plastictubes and frozen as described for bulk oil above. The emulsionswere then thawed in a water bath at room temperature. Thestability or instability was observed visually. The distinction wasusually obvious as demonstrated in Fig. 1. In experiments with less

Fig. 1. Example picture on emulsions subjected to freezing and thawing. To the left,emulsion prepared with sunflower oil and to the right emulsion prepared with HOsunflower oil.

20 10 0 -10 -20 -30

F

E

D

B

C wolf tae

H

Temperature (°C)

0.2 mW

A

Fig. 2. Crystallization of vegetable oils in the interval 30 to �30 �C, cooling rate 5 �C/min. A) HO sunflower oil, B) MCT oil, C) rapeseed oil, D) soy oil, E) corn oil and F)sunflower oil.

E. Magnusson et al. / Food Hydrocolloids 25 (2011) 707e715 709

developed coalescence/partial coalescence the droplet size and sizedistribution was measured. Microscopy (Olympus light microscopeBX60 with an Olympus U-CMAD-2 camera, Olympus, Tokyo, Japan)was performed and the peak value from volume-weighted dropletsize distribution was determined by static light scattering (SLS)using a Coulter LS130 (Beckman-Coulter Inc, Fullerton, CA). After4 h, when thawing and analysis had been done, the emulsion wasrefrozen. This freezing and thawing was repeated and the dropletsize was studied after different freezeethaw cycles. Results for allthese analyses are not shown in the paper. Tubes with emulsionfrozen for a longer period of time (15 days) were also analyzed.Experiments were performed at least in duplicate.

To be able to relate the stability of the emulsion to the oilcomposition, emulsionswere preparedwith different oils. Emulsionwas prepared twice with each oil and subjected to freezeethawcycling as previously described in this section. The emulsions werealso analyzed by DSC with cooling and heating rates approximatingthe freezing conditions. 5e10mg emulsionwas sealed in aluminumpans and kept in the refrigerator overnight prior to analysis. Thesamples were then analyzed in the DSC with a cooling rate of 3 �C/min between 30 to 0 �C and 0.15 �C/min between0 to�30 �C. For theheating, the rate was 1.5 �C/min (�30 to 10 �C).

The impact of impurities in the oil phase was also investigated.Emulsion was prepared with de-waxed sunflower and corn oil andthe freezeethaw stability was compared with the stability ofmayonnaise prepared with oils with the naturally occurring waxesretained. Emulsions were also prepared with sunflower oil withaddition of waxes (1 wt %). The waxes, bee wax and candelilla wax(Frank B. Ross Co. Inc., Rahway, New Jersey), were dispersed insunflower oil, heated to 80 �C in a water bath and thereafter mixedwith an Ultra Turrax (Ystral X10/25, Ystral, Ballrechten-Dottingen,Germany) for 3 min. The oils were cooled to room temperature andemulsions were prepared and exerted to freezing and thawing.

The addition of additives, previously indicated to have impact onthe crystallization of emulsified oil, was also studied. Some of thestudied ingredients were two polyglycerol esters (PGEs) withdifferent degrees of stearic acid esterfied; PGE 20 (ca 18% C18) andPGE 55 (ca 50% C18) and a propylene glycol ester with stearic acid(PGMS) (Danisco, Copenhagen, Denmark). The ingredients weredispersed in sunflower oil (1 wt %) in the same way as previouslydescribed for the waxes, though the temperature was 65 �C.

The effect on the freezeethaw stability by exchanging sucrose inthe water phase to other sugars such as glucose (SigmaeAldrichInc, St Louis, USA), trehalose (Cargill Inc., Minneapolis, USA) andglucose-fructose syrup (Cargill Inc., Minneapolis, USA) was inves-tigated. Furthermore, the effect of exchanging the sugar to glycerol(VWR International, West Chester, USA) was investigated. Emul-sions were prepared with 22 mmol/L sugar or glycerol (sugarconcentration in the standard recipe) in the water phase and sub-jected to freezeethaw cycling.

Finally, emulsions prepared with sunflower oil were committedto different freezing rates. Tubes with 30 ml emulsion were placedin liquid nitrogen and held there until the desired temperature wasreached (�20 �C, after approximately 30 s or �40 �C reached afterapproximately 60 s). Thereafter the tubes were rapidly placed inthe calcium chloride bath in the freezer (�25 �C). As a reference,tubes with emulsion were put directly into the calcium chloridebath (slow cooling, approx. 3 �C/min to 0 �C and 0.15 �C/minbetween 0 and �25 �C). The temperature was measured by inser-tion of a thermo couple in the center of the tubes.

Principal component analysis was performed with TheUnscrambler 9.0 (Camo ASA, Oslo, Norway). Variables for thedifferent oils, such as the fatty acid and triglyceride composition, aswell as the calculated amount of crystallized triglycerides (b0-form)at �25 �C was correlated with the amount of separation (both oil

and water separation) observed for emulsion prepared withdifferent oils after five freezeethaw cycles.

3. Results

In this paper, the crystallization behaviour of different vegetableoils was studied with differential scanning calorimetry (DSC) andby freezeethaw experiments in bulk systems. Moreover, mayon-naise type oil-in-water emulsions were prepared with the oils andsubjected to freezeethaw cycling and analyzed with DSC. Theamount of triglycerides in the oils that theoretically crystallize atdifferent temperatures was calculated. Furthermore, the impact ofadditions to the oil and water phase, as well as the impact of thefreezing rate, on the stability of emulsions was investigated.

The results from the thermal analysis of the oils indicate thatsoy, corn and sunflower oil begin to crystallize at a highertemperature, whereas the onset of crystallization of rapeseed oilshowed the lowest temperature for the oils studied (Fig. 2 and Table3). When analyzing the thawing of the oils with DSC, largeexothermal peaks were seen for HO sunflower and MCT oil. Themelting did also occur at a higher temperature for these oils (Fig. 3and Table 4).

Significant differences between the crystallization of differentoils in bulk systems were observed (Table 5 and Fig. 4). When theoils were frozen in tubes at �25 �C, sunflower, corn and soy oilappeared solid like (however still semitransparent) quite rapidlywhereas the other oils stayed in a pourable condition for more thanan hour. In MCT oil small crystals were visible already after 15 minof freezing. High oleic (HO) sunflower oil and rapeseed oil showedthe same crystallization behavior; however, the procedure seemedto go on more slowly for the rapeseed oil. These two oils initiallybecame very viscous and thereafter a layer of white crystals wasformed at the bottom of the tubes. The layer grew and was even-tually covering the whole tube. After 24 h of freezing, the tubeswith HO sunflower, MCT and rapeseed oil were very white andsolid. Also the corn oil was quite solid whereas the other oilsappeared solid-like but were easy to stir.

The theoretical solubility of the triglycerides is estimated usingequation (1), assuming full equilibrium, as a function of tempera-ture (Fig. 5). Although this is an approximate approach that doesnot take into account TAG interactions and polymorphic transi-tions, differences in SFC values were so significant that they sup-ported the conclusions of our study. From the estimation of the

Table 3Crystallization temperatures of vegetable oils. The temperature interval was 30e(�30) �C and the cooling rate 5 �C/min.

Oil Onset (Mean � SE, �C) Peak (Mean � SE, �C)

Sunflower �12.6 � 0.3 �17.4 � 0.5HO sunflower �14.7 � 0.2 �23.9 � 0.4Rapeseed �23.9 � 0.2 �27.7 � 0.2Corn �12.9 � 0.5 �16.1 � 0.1Soy �11.7 � 0.1 �19.6 � 0.1MCT �18.7 � 0.9 �29.3 � 0.2

Table 4Melting temperatures of vegetable oils. The temperature interval was (�30)e30 �Cand the heating rate 5 �C/min.

Oil Peak (Mean � SE, �C) Endset (Mean � SE, �C)

Sunflower �12.77 � 0.12 �5.4 � 1.3HO sunflower �6.54 � 0.21 �0.9 � 0.0Rapeseed �16.49 � 0.39 �11.5 � 0.4Corn �13.15 � 0.05 �7.3 � 0.3Soy �10.83 � 0.06 �5.6 � 0.6MCT �3.58 � 0.14 �0.6 � 0.3

E. Magnusson et al. / Food Hydrocolloids 25 (2011) 707e715710

amount of triglycerides that crystallize (b0-form) in the oils atdifferent temperatures, HO sunflower oil has the highest content ofcrystallized fat at �25 �C (81%), followed by rapeseed oil whereapproximately 64% of the oil crystallizes. The crystallization isindicated to be lower in corn, soy and sunflower oil (58, 52 and 53%respectively). Calculations could not be performed for the MCT oil,as the triglyceride composition was unknown.

Peak values, from the volume-weighted droplet size distribu-tion, of freshly prepared emulsions and emulsion subjected tofreezing and thawing are shown in Table 5. After repeated freeze-ethaw cycles, emulsions prepared with sunflower oil were themost coalescence stable (i.e. least increase in droplet size). A higherdegree of coalescence was observed in emulsions with soy or cornoil and for the latter considerable phase separation was observed.HO sunflower oil, rapeseed and MCT oil gave very unstable emul-sions. On thawing after 15 days in the freezer, all emulsions showedextensive destabilization and no significant differences could beobserved for emulsions prepared with different oils.

Analyses of emulsions with DSC showed only one peak, corre-sponding to crystallization of the water phase, for most emulsions.For HO sunflower oil and MCT oil, however, additional bulky peaksat�22.77 �C� 0.27 and�14.49 �C� 0.44 (Mean� SE), respectively,were observed (Fig. 6).

A principal component analysis was made to correlate variablesfor the different oils with the stability of the corresponding emul-sion (Fig. 7). In the plot TAG is triglycerides with different unsatu-ration in parenthesis. The letter combinations are triglycerides andfigures as for example 18 (1) are fatty acids with the length of thecarbon chain and the unsaturation in parenthesis. MonoU, PolyUand Sat are the amount (%) of mono unsaturated, polyunsaturatedand saturated fatty acids in the oil and SFC �25 is the estimated

-30 -20 -10 0 10 20 30

F

E

D

C

B

wolf taeH

Temperature (°C)

A

5 mW

Fig. 3. Thawing of vegetable oils (�30 to 30 �C) with a heating rate of 5 �C/min. A) HOsunflower oil, B) MCT oil, C) rapeseed oil, D) soy oil, E) corn oil and F) sunflower oil.

amount of triglycerides that crystallize at �25 �C. Separation is theamount of separation (both oil andwater) for emulsion subjected tofive freezeethaw cycles. All variables are specified in percentage. Astrong correlation between the amounts of mono unsaturated fattyacids (oleic acid) with the freezeethaw destabilization of theemulsions was indicated from PCA analysis, and also a correlationwith the percentage of triolein and the solid fat content at�25 �C. Anegative correlation was observed between the amount of sepa-ration and the amount of polyunsaturated fatty acids in the oil.

No significant differences in freezeethaw stability betweenemulsions prepared with de-waxed and non de-waxed oils wereobserved (Fig. 8). Emulsion prepared with oils with added waxeshad a larger initial droplet size and showed less coalescencestability as large droplets were seen on the microscopic images andoil separation was observed for the thawed emulsions.

Emulsion prepared with addition of polyglycerol esters, espe-cially with the higher degree of esterification (PGE 55), had a largerinitial droplet size, compared to the reference (Fig. 9). For emulsionwith PGE 20, the droplet size increased significantly after onefreezeethaw cycle whereas the droplet size remained more or lessthe same for emulsions with PGE 55. Emulsions with addition ofpropylene glycol monostearate (PGMS) showed comparabledroplet sizes and stability as the reference emulsion.

No significant differences in droplet size after freezing andthawing between emulsions prepared with different sugars couldbe observed. The droplet size did however increase substantially foremulsions with the sugar exchanged to glycerol (Fig. 10).

The effect of cooling rate on the freezeethaw stability was alsoinvestigated (Fig. 11). Emulsions subjected to rapid cooling to�40 �C before they were placed in calcium chloride solution(�25 �C) had poor stabilitywhen thawed. Also for emulsions rapidlycooled to�20 �C the oil separation upon thawing was pronounced.Emulsions cooled more slowly showed higher stability.

4. Discussion

When mayonnaise type oil-in-water emulsions are frozen to�25 �C, crystallization of both the oil andwater phase occur and theemulsion is unstable upon thawing, at least when frozen for 15days. In this paper we show that the composition of the oil phase

Table 5Visual perception of the solidification of the different oils by freezing to �25 �C.

Oil 15 min 30 min 1 h 2 h 3 h 24 h

Sunflower S S S S S SSunflower (DW) Vþþ S S S S SHO sunflower L V Vþþ S (C) S (C) S (C)þþRapeseed L V- V V V S (C)þþCorn V S S S S SþCorn (DW) V- Vþþ S S S SSoy V S S S S SMCT L (C) L (C (þ)) L (C(þþ)) L (C(þþ)) S (C) S (C)þþ

DW ¼ De-waxed, L ¼ liquid, V ¼ viscous, S ¼ Solid like behavior. C ¼ visible crystalformations and þ and � indicate an enhancement or diminishment of theproperties.

0 -5 -10 -15 -20 -25 -30 -35

C

B

wolf taeH

Temperature (°C)

2 mW

A

Fig. 6. Freezing and thawing of emulsion prepared with vegetable oils. The coolingrate was 3 �C/min between 30 to 0 �C and 0.15 �C/min between 0e(�30)�C. For theheating, the rate was 1.5 �C/min ((�30)e10 �C).

Fig. 4. Oils frozen for 24 h and then held at room temperature for 10 min. A) HOsunflower oil and B) sunflower oil.

E. Magnusson et al. / Food Hydrocolloids 25 (2011) 707e715 711

has a great impact on the freezeethaw stability of mayonnaise typeoil-in-water emulsions whereas a wide range of additives to boththe oil and water phase did not have any positive impact on thestability. The freezing rate is also shown to be of importance.

After repeated freezeethaw cycles, emulsions prepared withsunflower oil were themost coalescence stable (i.e. least increase indroplet size) whereas HO sunflower oil, rapeseed and MCT oil gavevery unstable emulsions. The differences in freezeethaw stabilitybetween emulsions prepared with different oils were dramatic and,thereby, the triglyceride composition seems to have a major impacton the freezeethaw stability (Table 6).

The oils that gave the least stable emulsions; HO sunflower, MCTand rapeseed oil showed the lowest crystallization temperatures inthe DSC analysis. However, the differences were small between theoils and as all oils crystallize when the temperature is lowered to�25 �C the crystallization temperature is, most likely, not the mostrelevant parameter. The crystal habit and the amount of crystalsformed do presumably have a much greater impact.

Some similarities between the oils that gave the least stablemayonnaises could be observed. In the thawing diagrams from theDSC analysis, an extra exothermal peak was observed for HOsunflower andMCToil, though not for rapeseed oil. However, whenthe oils were cooled to a lower temperature, a large peak in thethawing diagramwas observed also for the rapeseed oil but still notfor corn, soy or sunflower oil (measurement performed by AAK,results not shown in the paper). This peak could indicate additional

30 20 10 0 -10 -20 -300

10

20

30

40

50

60

70

80

90

100

)%(

sedirecylglycairtdezillatsyr

C

Temperature (°C)

Sunflower HO Sunflower Corn Rapeseed Soy

Fig. 5. The amount of crystallized triacylglycerides in vegetable oils at differenttemperatures according to calculations.

crystal growth and/or transformation to another polymorph. Thehigh enthalpy value of the peak and the high melting temperaturesobserved for HO sunflower and MCT oil indicate that anotherpolymorph is formed, as a lot of energy is released in a trans-formation and themelting point increasewith increased stability ofthe crystals (aeb0eb) (Hartel, 2001). However, in order to beconclusive regarding the polymorph of the oils, measurementswith x-ray diffraction could be performed.

From bulk oil experiments, large crystals were indicated to formin HO sunflower, MCT and rapeseed oil since white crystals wereformed and concentrated at the bottom of the tubes. HO sunflowerand MCT oil are quite homogeneous in fatty acid composition; HOsunflower oil is mainly consisting of oleic acid (82.5%) whereasMCToil is composed of two different fatty acids only (53.5% of 8:0 and45.9% 10:0). Rapeseed oil is not as homogeneous as HO sunflowerand MCT oil; however, it has a high content of oleic acid (62.2%).Homogeneous oils more readily form large crystals (Vaclavic &Christian, 2008) and the possibility of organization into a stablepolymorph is increased when the triglycerides are more homoge-neous (Campos et al., 2002). Formations of large crystals, possible

Fig. 7. Principal component analysis, PC1 against PC2. The degree of explanation is95.5% for PC1 and 98.71% when PC2 is included.

Fig. 8. Microscopic images of emulsions. A) Freshly made and B) after five freezeethaw cycles. 1) Emulsion with de-waxed sunflower oil, 2) emulsion with sunflower oil and 3)emulsion with added bee wax (1wt%). The scale bar is 40 mm.

E. Magnusson et al. / Food Hydrocolloids 25 (2011) 707e715712

in b-form as these crystals are larger and more stable than the a-and b0-forms, may thereby be one reason for the destabilizingeffect. b-crystals have an average of 25e50 mm in length andclusters of crystals can have a diameter of 1 mm or more, whereasb0-crystals and a-crystals are in the size range 1 and 5 mm respec-tively (Hoerr, 1960; Hoerr, Moncrieff, & Paulicka, 1966).

When analyzing the emulsions with DSC, peaks for the oilcrystallization could only be observed for HO sunflower and MCToil. Again this indicates that crystal formation is greater for theseoils and/or that another polymorphism is formed compared toemulsions containing the other oils studied. Interestingly, for HOsunflower oil the crystallization peak was observed at a lowertemperature for the emulsion than for the pure oil, whereas theopposite was observed for MCT oil i.e. the pure oil crystallized laterthan the dispersed oil. When the oil is emulsified, each droplet willcontain fewer impurities. Thus, the crystallization will be appar-ently homogeneous and occur at lower temperatures (Skoda &Vandentempel, 1963) which may explain the behavior of HOsunflower oil. MCT oil, however, is a purified oil and the differentbehavior of pure oil and emulsified oil is, thereby, more likely to bedetermined by the difference in cooling rate. The lower cooling ratein the analysis of the emulsions will result in the oil existing at eachtemperature for a longer period of time. This enables the organi-zation of triglycerides to take place more rapidly and may result in

Fig. 9. Microscopic images of emulsions, A) freshly prepared and B) after one freezeethaw c4) emulsion with PGE 55. The scale bar is 40 mm.

less time needed for the oil to crystallize when the crystallizationtemperature is reached (Campos et al., 2002). Less super coolingwill, thus, be needed before crystallization occurs. The fact that nopeaks were observed for the oil crystallization of the other emul-sions is most likely explained by less crystallization. Anotherdifference between the DSC diagrams for the oils and emulsionsrespectively was that the exothermal peak seen for HO sunflowerand MCT oil in the thawing diagram was missing when the oil wasemulsified. This is likely explained by the lower cooling rate used inthe analysis of the emulsions. As previously stated, less supercooling is needed before crystallization occurs when the coolingrate is lowered. This may result in that further crystal formationand/or transformation to another polymorph, which was indicatedfrom the DSC diagram upon thawing of the bulk oils, will occuralready upon cooling of the emulsions. From Fig. 6, differences inthe location of the water peak can be observed. The measurementwas, however, not reproducible concerning the water peak, largeerrors between measurements were observed. High energy releasecaused by rapid crystallization of under-cooled aqueous phasegenerated a torso in the peak as a function of the temperature.Therefore, the values of the water crystallization cannot be used forinterpretation, and the peaks are cut off in the diagram.

The bulk experiments indicate that the oils that were most solidafter 24 h of freezing gave themost unstable emulsions. The solidity

ycle. 1) Reference (sunflower oil), 2) emulsion with PGMS, 3) emulsion with PGE 20 and

Table 6Change in droplet size with freezing and thawing of emulsions prepared withdifferent oils. The droplet sizes are peak value from the volume-weighed droplet sizedistribution and the measurement was made twice for every emulsion and on twoemulsions prepared with the same oil i.e. 4 samples in total.

Oil Droplet size(mm) of freshemulsions

Droplet size(mm) after onefreezeethaw cycle

Droplet size (mm)after five freezeethaw cycles

Sunflower 5.26 � 0 5.92 � 0.67 9.07 � 0HO Sunflower 6.99 � 0.58 a a

Rapeseed 6.14 � 0.73 a a

Corn 5.26 � 0 5.83 � 0.58 a

Soy 5.26 � 0 9.08 � 0.80 44.70 � 2.04b

MCT 4.38 � 0 a a

a A high degree of phase separation was observed for thesemayonnaises wherebythey were not analyzed with the light scattering equipment.

b One of the emulsions prepared with soy oil separated after five freezeethawcycles, whereby this value is based only on results for two measurements on one ofthe prepared emulsions.

Freshly made After one freeze-thaw cycle0

20

100

120

(ezistelpor

Dµ

)m

Sucrose Glucose Trehalose Syrup Glycerol

Fig. 10. Change in droplet size after one freezeethaw cycle for emulsion prepared withdifferent sugars or glycerol.

E. Magnusson et al. / Food Hydrocolloids 25 (2011) 707e715 713

of the fat haspreviouslybeen indicated to stronglycorrelatewith theSFC (Campos et al., 2002). HO sunfloweroil and rapeseedoil have thehighest degree of solid fat at�25 �C, both according to experimentsand calculations. It was not possible to calculate the amount of solidfat in the MCT oil, as the triglyceride composition was not known.However, this oil is likely to have a high degree of solid fat at lowtemperatures as it is fully saturated (freezing point approx. �14 �C(Personal communication with the manufacturer, Plum, 2009-20-15)). A high amount of solid fat increases partial coalescence(Vanboekel & Walstra, 1981) and may contribute to the lowerfreezeethaw stability of emulsions prepared with HO sunflower,MCT and rapeseed oil. Nevertheless, all oils had quite high theoret-ical amounts of crystallized triglycerides at �25 �C according to thecalculations. The calculation, however, assumes isothermal andequilibrium conditions, which is far from the case in DSC measure-ments and the freezing at �25 �C. The results in this paper indicatethat the crystals are slowlygrowing in sunflower, soyand corn oil; asthese oils showedquite lowcrystallization/meltingenthalpies in theDSCanalysis and stillwerepossible to stir after storageof thepureoil24 h at�25 �C. These oils gavemore freezeethaw stable emulsions.

Fig. 11. Emulsions prepared with sunflower oil and subjected to different cooling. A)Emulsion cooled to �40 �C in liquid nitrogen, B) emulsion cooled to �20 �C in liquidnitrogen and D) emulsion cooled to �25 �C in a calcium chloride solution.

Furthermore, when the emulsion was stored at �25 �C for 15 days,destabilization was observed for all the emulsions, independent ofthe oil used. As isothermal, equilibrium conditions will be lessdistant when the emulsion is stored for a longer period of time;destabilization ismost likely explainedby further growthof crystals.The higher amount of solid fat, indicated to be formed according tocalculationsmay thenbe reachedalso for corn, soyandsunfloweroil.

Interestingly, a strong correlation between the amounts ofmono unsaturated fatty acids (oleic acid) with the freezeethawdestabilization of the emulsions was indicated from PCA analysis,and also a correlation with the percentage of triolein and the solidfat content at �25 �C. A high content of oleic acid in the oil therebyseems to be negative for the freezeethaw stability. This may beexplained by a large increase in SFC by crystallization of triglycer-ides with oleic acid, which is indicated from the SFC calculations.Differences in polymorphism between oils dominated by oleic acid(HO sunflower and rapeseed oil) or linoleic acid (corn, soy andsunflower oil) may possible be another contributing factor,however, no conclusion regarding this may be drawn from theresults in this paper. A negative correlation was observed betweenthe amount of separation and the amount of polyunsaturated fattyacids in the oil. Polyunsaturated acids have a lower crystallizationtemperature (Larsson, 1986) and will thereby generally decreasethe SFC in the emulsions. This may be one reason for their positiveimpact on the freezeethaw stability.

The delayed crystallization of de-waxed oils, observed byfreezing of oils in tubes (Table 5), in comparison to oils with waxesmay indicate that the impurities crystallize early and may act assites for nucleation. No significant differences in freezeethawstability of emulsion prepared with de-waxed and normalsunflower or corn oil respectively was observed (Fig. 8). A largerdroplet size was observed for the freshly made emulsions withadded waxes, which is probably explained by an increase in theviscosity of the oil. Moreover, these emulsions showed less coa-lescence stability. This may be explained by a large increase in thenumber of crystals in each droplet, which will increase the inter-actions and thereby the hardness of the fat (Campos et al., 2002).The crystallization of the waxes will also increase the solid contentin the droplets, which further may contribute to the destabilization.

When polyglycerol esters were added to sunflower oil, a largerinitial droplet size was observed. Moreover, emulsions preparedwith PGE 20 were less freezeethaw stable than the reference,whereas PGE 55 showed the same stability as the reference. Poly-glycerol esters are surface active and may thereby adsorb at theinterface and affect the droplet size and interfacial layer. PGE 20 isless substituted than PGE 55 and, thus, less soluble in the oil. This

E. Magnusson et al. / Food Hydrocolloids 25 (2011) 707e715714

substance is thereby likely to adsorb at the oil/water interface toa greater extent than PGE 55 and thereby affect the properties ofthe emulsion more extensively. The adsorption of polyglycerolesters, resulting in thinner interfacial layers, may give rise to lessstable emulsions compared to emulsions with thicker interfaciallayers (Ghosh & Coupland, 2008; Palanuwech & Coupland, 2003), asthose created by proteins from egg yolk (Nilsson, Osmark,Fernandez, Andersson, & Bergenståhl, 2006).

Propylene glycol monostearate have previously been observedto change the crystallization behavior of the oil in emulsions,however, no effect on the freezeethaw stability of the substancewas observed in this study. This may be explained by the differencein triglyceride composition between the previous study and thisone, as the effect was suggested to originate from interactionbetween propylene glycol monostearate and certain triglycerides.

Sugars and polyols (such as glycerol) are generally attributed tofunction as cryoprotectants (Crowe et al., 2001; Strarnbini,Balestreri, Galli, & Gonnelli, 2008). Among the sugars, trehalosehas been indicated to be an extraordinarily good cryoprotectant(Crowe et al., 2001; Varga et al., 2010). However, at a temperaturebelow�15 �C, the effect of trehalose has been observed to decrease,whereas the effect of sucrose remained (Strarnbini et al., 2008). Inemulsions, only small differences between sugars have beenobserved (Ghosh, Cramp, & Coupland, 2006). The mechanism forthe cryoprotective effect of sugars is not fully understood and itseems to be very system dependent. For the mayonnaise type oil-in-water emulsion studied in this paper, no significant differencesin freezeethaw stability for emulsion with different sugars wereseen. Unexpectedly, glycerol decreased the stability of the emulsionsubstantially. Glycerol has, for example, been indicated to increasethe stability of proteins in solution by preventing the unfolding ofthe protein (Sousa & Lafer, 1990). This is often desirable in proteinsolutions, however, this effect may be negative for the stability of anemulsion as it might affect the surface activity of the protein andthereby the properties of the interfacial layer. Glycerol, in thepresence of low sodium chloride concentrations, has also beenindicated to destabilize red blood cells subjected to freezing (Pegg,1987).

In this paper, a higher cooling rate was indicated to decrease thestability of the emulsions. In 1964, deMan proposed that a highcooling rate generated a higher degree of solid fat, an increasedhardness and an increased network formation between crystals(deMan, 1964). Campos et al (2002) have also showed that crys-tallization of lard and anhydrous milk fat at a fast rate of coolingcompared to a lower rate resulted in a higher SFC and a harder fat.They explained the increased hardness by an increase in thepossible number of interactions between crystal particles at fastcooling, as these interactions increase with the number of particles(Campos et al., 2002). Nevertheless, the effect of the cooling rateseems to be more complex in emulsions as different effects on thestability have been observed for different systems (Tippetts &Martini, 2009; Vanapalli et al., 2002). The triglyceride composi-tion of the oil phase may however have an impact on the effect ofthe cooling rate. A homogeneous fat that are prone to form largecrystals, which tend to destabilize emulsions, may be favored by anincrease in the cooling rate as the crystal size decrease. On the otherhand, if the crystals get to small the number of possible interactionsincreases dramatically and the increased hardness and SFC maydestabilize the emulsion. A heterogeneous fat, that generally formssmall crystals, may thereby be destabilized if cooled rapidly. Theoptimal cooling rate may thus vary depending on the triglyceridecomposition. As shown in this paper, a cooling rate of 20 �C/min isnegative for the stability of mayonnaise type oil-in-water emul-sions prepared with sunflower oil, most likely because of the largeincrease in the number of crystals.

5. Conclusion

In this paper, HO sunflower, rapeseed and MCT oil wereobserved to give emulsions with very poor freeze stability at�25 �C, as significant oil separation was observed already after onefreezeethaw cycle. The instability was correlated with a highcontent of mono unsaturated fatty acids (oleic acid) in the oil anda high percentage of crystallized triglycerides at�25 �C (theoreticalamounts from calculations). In bulk experiments, larger crystalswere also indicated to be formed in HO sunflower, rapeseed andMCT oil which may be contributing to the destabilization. Emul-sions prepared with sunflower oil were most stable to freezeethawcycling and no significant increase in droplet size was observedeven after five freezeethaw cycles. However, high instability wasobserved for all emulsions, independently of the oil used, when theemulsions were frozen for 15 days at �25 �C. Most probably, this isexplained by further growth of crystals. Moreover, a high freezingrate was shown to decrease the freezeethaw stability of theemulsion. Various substances were added to both oil and waterphase in order to modify the crystallization of the two phases,however, no positive impact of the additives was observed. Fromthe results in this paper, the oil composition and the freezingconditions are shown to be two main factors affecting the freeze-ethaw stability of emulsions with a high volume fraction ofdispersed oil phase. However, the subject is still much unexploredand further investigations should be performed in order to fullyunderstand and control the freezeethaw stability of emulsions.

Acknowledgment

Financial support from the Faculty of Engineering LTH, LundUniversity and Källbergs Industri AB is gratefully acknowledged.

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