Casein molecular assembly affects the properties of milk fat emulsions encapsulated in lactose or trehalose matrices

ARTICLE IN PRESS

International Dairy Journal 17 (2007) 683–695 www.elsevier.com/locate/idairyj

Casein molecular assembly affects the properties of milk fat emulsions encapsulated in lactose or trehalose matrices Ce´sar Vegaa, H. Douglas Goffb, Yrjo¨ H. Roosa, a

Department of Food and Nutritional Sciences, University College Cork, Cork, Ireland b Department of Food Science, University of Guelph, Guelph, ON, Canada N1G 2W1 Received 7 March 2006; accepted 16 August 2006

Abstract Properties of spray-dried anhydrous milk fat emulsions stabilized by micellar casein (milk protein isolate—MPI) or non-micellar casein (sodium caseinate—Na-caseinate) with trehalose or lactose as encapsulants were studied. A lower concentration of Na-caseinate (0.33%) compared with MPI (1.26%) was sufficient to stabilize a 10% fat emulsion. Reconstituted emulsions showed larger droplet size than fresh emulsions, especially for MPI systems (fromo1 mm to around 14 mm), which was attributed to lower shear resistance during atomization. Creaming behavior reflected changes in particle size. Powder surface free fat was affected by protein type and concentration. Trehalose systems (regardless of protein type) released significantly lower amounts of encapsulated fat upon crystallization compared with those containing lactose. Individual and hence, more mobile and flexible casein molecules, as opposed to aggregated and less mobile casein micelles, appear to result in superior co-encapsulation properties of Na-caseinate compared with MPI. r 2006 Elsevier Ltd. All rights reserved. Keywords: Spray-drying; Emulsion; Trehalose; Lactose; Surface fat; Casein

1. Introduction Dehydrated emulsions are prepared by drying liquid oilin-water (o/w) emulsions containing dissolved substances in an aqueous phase; these form a continuous solid matrix which encapsulates the dispersed fat component in the emulsion. Ideally, encapsulation should preserve the original emulsion droplet size distribution (MillqvistFureby, 2003). Encapsulants are substances that have fat-encapsulating (but not necessarily emulsifying) properties. They improve product stability by forming a solid, amorphous continuous phase (glass) as a result of the removal of water (Roos, Karel, & Kokini, 1996). Glasses are not in a thermodynamic equilibrium and they often crystallize with a rate depending on temperature and water content. Lactose is a typical glass forming material in spray drying (Fa¨ldt & Bergensta˚hl, 1996) and it has been recognized as Corresponding author. Tel.: +353 21 490 2386; fax: +353 21 427 0001.

E-mail address: [email protected] (Y.H. Roos). 0958-6946/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2006.08.004

the main encapsulant of milk fat in whole milk powder and spray-dried dairy-like emulsions (Buma, 1971; Young, Sarda, & Rosenberg, 1993). Trehalose (a,a-trehalose) is a non-reducing disaccharide formed by a 1,1 linkage of two D-glucose molecules with anhydrous molecular weight similar to that of lactose. The natural functions, mechanisms of action and technical qualities of trehalose in cryo-preservation and desiccation protection lend this sugar to several possible applications in the food, cosmetic and medical industries. This, along with reduced production costs enables the use of trehalose in a wide variety of cost sensitive applications, including foods (Cerdeira, Martini, & Herrera, 2005; Mazzobre, Soto, Aguilera, & Buera, 2001). Nonetheless, the use of trehalose as encapsulant has been limited (Cerdeira et al., 2005; Elofsson & Millqvist-Fureby, 2003). We have commented on the misuse of the term ‘‘encapsulant’’ for proteins used as emulsifiers of spraydried emulsions (Vega & Roos, 2006a). Depending on the fat load and homogenization conditions (provided no other competitive surfactants are present), excess protein (if any)

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may interact with the components of the aqueous phase and have a co-encapsulating role in drying (Hogan, McNamee, O’Riordan, & O’Sullivan, 2001; Vega & Roos, 2006a). Several studies have focused on understanding the physicochemical properties of spray-dried emulsions (Dollo et al., 2003; Keogh & O’Kennedy, 1999; Landstrom, Alsins, & Bergensthal, 2000; Millqvist-Fureby, Elofsson, & Bergensthal, 2001; Pedersen, Faldt, Bergensthal, & Kristensen, 1998; Sliwinski et al., 2003). However, these studies have not explained thoroughly how the oil-to-protein ratio, protein structure, interfacial layer thickness and flocculation (bridging/depletion) may affect the physicochemical properties of emulsions during drying, storage and reconstitution. Sugars often act as encapsulants, although their crystallization kinetics in the context of emulsion encapsulation remains poorly understood. Elofsson and Millqvist-Fureby (2003) reported that sucrose and maltose were more effective encapsulants of rapeseed-oil emulsions stabilized with Na-caseinate than lactose or trehalose because of the smaller droplet sizes and lower surface fat after storage at 75% RVP for 7 days. No data on water sorption behavior were provided. Hence, it is not known if such results were linked to (a) absence (or delay) of crystallization of sugars; (b) differences in the types of crystals formed; (c) proteinsugar interactions; (d) differences in solubilities of sugars; or (e) a combination thereof. The aims of the present study were to investigate: (i) the effects of casein type (micellar or non-micellar) on the effective minimum protein concentration necessary to stabilize an emulsion, both in its liquid and dry/reconstituted forms; (ii) crystallization effects of lactose or trehalose on the extent and rate of fat release; (iii) the interactions between lactose or trehalose and casein and the relevance of casein type in such interactions. 2. Materials and methods 2.1. Materials Milk protein isolate (MPI) with 87% protein (92% casein micelles, 8% whey proteins), 2.65% lactose and 8.4% ash was purchased from Ingredia (Arras, France); sodium caseinate (Na-caseinate) containing 95% protein and 3.9% ash was provided by Fonterra (Mississauga, ON, Canada); anhydrous milk fat (AMF) was donated by

Parmalat (London, ON, Canada); spray dried lactose was purchased from Saputo (Montreal, QC, Canada) and a,atrehalose dihydrate was donated by Cargill (Minneapolis, MN, USA). 2.2. Emulsion preparation The proteins were pre-dissolved in de-ionized water at 50 1C and subsequently crystalline lactose or trehalose was added. AMF was incorporated and allowed to melt. Prehomogenization was performed with a Silverson highspeed agitator (Silverson Machines Ltd., Waterside, Chesham, UK) at 10,000 rpm for 5 min at 70 1C. Mixes were then homogenized using a two stage laboratory homogenizer (APV Gaulin, Everett, MA, USA) using pressures of 32 and 8 MPa for the first and second stage, respectively. Emulsion compositions are summarized in Table 1. 2.3. Spray drying of emulsions Emulsions were pre-heated to 65–70 1C and then spray dried in a pilot scale spray dryer (Mobile Minor 2000 Model H, Niro, Soeborg, Denmark). The drier was operated co-currently and equipped with a nozzle atomizer. The inlet and outlet temperatures were 175 and 70 1C, respectively. Emulsions were prepared and dried by triplicate. 2.4. Emulsion particle size Particle size distribution of fresh and reconstituted emulsions was measured using a Mastersizer Hydro 2000 SM model (Malvern Instruments Ltd., Malvern, Worcs., UK). The refractive index used for AMF was 1.46. Mean particle size diameter d4,3 (volume-weighted diameter), instead of surface-weighted mean diameter (d3,2) was used to characterize droplet size since it was observed that it was significantly more sensitive to small changes in composition. Results are given as an average of five determinations. 2.5. Emulsion creaming behavior The creaming behavior of the emulsions before spray drying and after reconstitution was followed by measurement of the backscattering of a pulsed near infrared light source (lZ850 nm) from an emulsion as a function of its

Table 1 Composition of fresh and dry emulsions (%, w/w) stabilized with milk protein isolate or Na-caseinate at different oil:protein ratios Na-caseinate Fat:protein ratio Protein Carbohydrate Fat a

30:1 0.33 (1.11)a 19.67 (65.56) 10 (33.3)

Milk protein isolate 15:1 0.76 (2.22) 19.63 (64.44) 10 (33.3)

1.25:1 8 (26.66) 12 (40) 10 (33.3)

The compositions of the emulsions after dehydration are enclosed in parentheses.

9:1 1.24 (3.70) 18.86 (62.66) 10 (33.3)

5:1 2.23 (6.66) 17.95 (60) 10 (33.3)

1.25:1 8 (26.66) 11.81 (40) 10 (33.3)

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height using a TurbiScan MA2000 instrument (Formulation, Toulouse, France) as described elsewhere (Mengual, Meunier, Cayre, Puech, & Snabre, 1999). Emulsions were placed into cylindrical glass tubes and stored at room temperature (25 1C). Sodium azide (0.01%, w/w) was added to inhibit bacterial growth. The backscattering of light across the filled length of the tube was then measured to follow the height of the cream (top) layer as a function of time for up to 72 h. The results are presented as a plot of the height of the top layer vs. time (Chanamai & McClements, 2000). Measurements were performed in duplicate for each of the emulsion triplicates.

685

Scientific Isotemps, model 281, Pittsburgh, PA, USA). After drying, vials were stored in evacuated desiccators over a saturated NaNO2 solution (Sigma Chemical Co., St. Louis, MO, USA) to obtain a relative vapor pressure (RVP) of 65% at room temperature (22–25 1C). Water activity (aw) of the NaNO2 solution was checked using an Aqualab water activity meter (Decagon Devices, Inc., Pullman, WA, USA). Weight gain and loss on the samples was followed every hour during the first 8 h of storage; and subsequently after 24, 48 and 72 h. Water sorption is reported as g water per 100 g dry solids as a function of time.

2.6. Fat globule surface protein coverage 2.9. X-ray diffraction (XRD) The surface coverage of protein on the emulsion droplets (before spray drying and after reconstitution) was determined by using the method described by Euston and Hirst (1999) with some modifications. Emulsions were centrifuged at 20,000 or 31,000  g for 30 min in Beckman Coulter Optima Ultracentrifuge model LE-80 K using a Rotor 45Ti (S/N 1105 Beckman Coulter, Fullerton, CA) and the cream layer was removed. The cream layer was redispersed in fresh de-ionized water and re-centrifuged. The washed emulsion droplet layer was then removed and allowed to dry on filter paper (Whatman 40). Protein content was determined by performing Dumas-based nitrogen analysis (Leco FP528, St. Joseph, MI, USA). Measurements were performed in triplicate and were recorded as mg protein m2 fat and/or as percentage of adsorbed protein from the total protein in the original emulsion. The surface load was calculated by multiplying the measured protein content (P)—as g protein g1 fat—by the reciprocal of the SSA (g fat m2 fat) to obtain g protein m2 fat and finally by the equivalency 1000 mg protein g1 protein to obtain mg protein m2 fat. Results were the average of at least three determinations. 2.7. Surface free fat Estimation of surface fat of spray-dried particles was carried out as reported elsewhere (Vega, Kim, Chen, & Roos, 2005b). Two grams of dehydrated emulsion were weighed on a cone made out of filter paper (No. 42, Whatman, Maidstone, Kent, UK), and washed with 10 mL of hexane. This operation was repeated four times. The filtrate was allowed to evaporate until the extracted fat residue achieved constant weight. Extracted fat was reported as g of surface free fat 100 g1 powder. The same protocol was followed for powders that were subjected to time-dependent crystallization. Analysis was performed five times per treatment and results were averaged. 2.8. Water sorption Spray-dried samples (about 0.25 g) were placed in small glass vials and vacuum dried (65 1C) overnight (Fisher

Crystallization was followed only in emulsions made with the highest protein content (both carbohydrates) and for treatments MPI 9:1 (oil:protein ratio) and Na-caseinate 15:1 as they had the very similar protein content (i.e. 3.7% and 2.2%, w/w dry basis, respectively). Samples (around 0.25 g) were mounted and pressed onto a glass slide with a shallow rectangular depression (0.5 mm deep) which was roughened at the bottom and vacuum dried overnight (Fisher Scientific Isotemps). Samples were then stored within evacuated desiccators equilibrated at a RVP of 65% for periods of 1, 4, 8, 12 and 36 h for samples MPI 9:1 and Na-caseinate 15:1 and for 4, 12, 24, 36 and 72 h for samples with MPI and Na-caseinate at 1:1 oil:protein ratio. After incubation, glass slides were immediately placed into a Rigaku/MSC Multiflex X-ray diffractometer (Tokyo, Japan). The X-ray diffractometer was operated with an anode current of 44 mA, an accelerating voltage of 40 kV and wattage of 1.76 kW as to expose the samples to CuKa radiation at diffraction angles (2y) from 101 to 301 (step size, 0.02; time per step, 2.4 s). The peak search program of the JADE software (version 6.1 Materials Data Inc., Livermore, CA) was used to locate the peaks in XRD patterns by detecting the minima from the second derivative of the diffractogram. Intensity maxima were given as Ka1 net peak height in counts at Ka1 position in degrees. Measurements were performed at least three times for each of the replicates.

2.10. Field-emission scanning electron microscopy (SEM) Milk powders were deposited onto carbon paint on aluminum pin type mounts. Excess powder was removed with a jet of compressed air. Samples were coated with 30 nm of gold in the Emitech K550 sputter coater (Ashford, Kent, UK). Samples were imaged using the Hitachi S-4500 field emission scanning electron microscope (Hitachi High Technologies, Tokyo, Japan) at 3 and 5 kV. Digital images were acquired through Quartz PCI (Quartz Imaging Corp. Vancouver, BC, Canada).

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3. Results and discussion 3.1. Emulsion characterization Various fat:protein ratios were used to find the lowest concentrations of caseins (as MPI or Na-caseinate) to stabilize a 10% AMF emulsion and their compositions are shown in Table 1. A ‘‘stable’’ emulsion had (a) an average particle size (d4,3)o1 mm; and (b) absence of macroscopic creaming, depletion or bridging flocculation within 24 h of manufacture. As shown in Fig. 1, emulsions made with Nacaseinate gave particle sizes that remained below 1 mm at fat:protein ratios as high as 60:1 (0.16%, w/w Nacaseinate); whereas for MPI this was true only at ratios of 9:1 (1.24%, w/w) and below. Considerable bridging flocculation occurred in MPI systems made at ratios as low as 15:1. Specific surface area (SSA) values were higher for Na-caseinate emulsions regardless of protein concentration. These results, demonstrating superior emulsifying properties of Na-caseinate, were consistent with previous reports (Euston & Hirst, 1999; Mulvihill & Murphy, 1991). Surface coverage (mg protein m2 fat) did not show any dependence on the original emulsion oil:protein ratios, but was significantly higher for MPI-stabilized emulsions than for Na-caseinate systems (up to seven times as much) (Table 2). Adsorbed protein values for reconstituted emulsions are reported only as the percentage of total protein since coalescence during drying (especially for high oil:protein ratios) caused a severe decrease in the SSA, making comparisons groundless. For MPI-stabilized emulsions, drying decreased protein coverage values for MPI 9:1 with lactose (L) or trehalose (T) added (66 to 61 and 72 to 54%, respectively). No changes were observed for 1:1 or 5:1 emulsions. Na-caseinate systems showed a slight increment in the amount of protein adsorbed after drying,

which agreed with results reported by Sliwinski et al. (2003). The mean particle sizes (d4,3) for all liquid emulsions studied before and after drying are shown in Fig. 2. The d4,3 of MPI-stabilized emulsions increased from at least 7(5:1) to 15-fold (9:1 oil:protein ratio). For Na-caseinatestabilized emulsions, the increase in the d4,3 values was from 3- (15:1) to 10-fold (30:1) but overall, d4,3 values were considerably smaller than for MPI systems. Furthermore, lower concentrations of Na-caseinate than MPI (0.33% vs. 1.24% or 0.76% vs. 2.24%, w/w, respectively) were enough to provide better ‘‘protection’’ to the fat globules during drying. To isolate the influence of the atomizing step on droplet size, aliquots of atomized-only emulsions (15:1 Nacaseinate and 5:1 MPI) were analyzed. Samples were collected immediately after atomization (at the tip of the nozzle) in the drier, which was operated with the heater off. Results indicated that atomizing alone was accountable for almost 50% of the net increase in the mean particle size for emulsions stabilized by either protein (Table 3). The absolute effect of shear on the original d4,3 was significantly greater for MPI-stabilized emulsions. Micellar casein has been shown to have higher susceptibility to orthokinetic shear in comparison to Na-caseinate (Vega, Goff, & Roos, 2005a). This was attributed to higher flexibility (film stretching) and hydrophobicity of individual or slightly aggregated caseins (mainly b-casein) compared with a more rigid casein micelle. Large stresses can drag protein molecules along the interface, creating regions depleted of protein that increase the probability of coalescence during droplet–droplet encounters (van Aken, 2004). This process is likely to be important if the re-distribution of the adsorbed-protein layer is relatively slow compared with the duration of the applied stresses and droplet encounter frequency (McClements, 2004). The reasons behind the

90 75 60 45 30 15

50

40

30

3.0 2.5

20

2.0

SSA (m2g-1)

d4.3 (microns)

3.5

1.5 10

1.0 0.5 0.0

0 1

3

5

7

9

15 20 25 30 35 40 60 100

Oil to protein ratio (x:1) Fig. 1. Volume-weighted average diameter (d4,3) (circles) and specific surface area (SSA) (squares) as a function of oil-to-protein ratio for emulsions made with 10% fat and 30% total solids stabilized with Na-caseinate (J, &) or milk protein isolate (MPI) (K,’). Lines are a guide to the eye.

ARTICLE IN PRESS C. Vega et al. / International Dairy Journal 17 (2007) 683–695 Table 2 Fat globule protein surface coverage values (7 standard deviation) for fresh and reconstituted emulsions stabilized with milk protein isolate or Na-caseinate at different oil:protein ratios

MPI 1:1 T MPI 1:1 L MPI 5:1 T MPI 5:1 L MPI 9:1 T MPI 9:1 L NaCas 1:1 T NaCas 1:1 L NaCas 15:1 T NaCas 15:1 L NaCas 30:1 T NaCas 30:1 L

mg protein m2 fat

% of total protein

Fresh

Fresh

Reconstituted

3.0070.08 3.8270.49 3.1770.18 3.4370.10 3.9170.57 3.8670.41 0.5270.05 0.5370.10 0.4270.04 0.5070.04 0.3570.10 0.3070.07

15.0370.39 18.2572.35 41.4372.38 44.2771.23 71.55710.53 66.6077.09 3.1270.27 3.1770.61 25.6872.39 28.0972.05 30.6379.25 23.2175.59

NMb 17.8670.00 42.4571.21 45.7171.49 53.72722.88 61.3572.43 3.7370.37 4.0770.44 32.2075.87 32.2075.05 34.3375.95 41.34726.21

14 12 10 d[4.3] µm

Samplea

687

8 6 4 2 0 1:1L

1:1 T

1:1 L

1:1 T

(a)

5:1 L 5:1 T Treatment

9:1 L

9:1 T

4.5

a

MPI ¼ milk protein isolate; NaCas ¼ sodium caseinate; L ¼ lactose; T ¼ trehalose. b Not measured.

4.0 3.5

d[4.3] µm

3.0

post-atomizing change in droplet size (i.e. the net effect of drying) were not studied, but we hypothesize that drying experiments using a single pendant-drop set up (Adhikari, Bhandari, Howes, & Troung, 2004) could help to elucidate such contribution.

2.5 2.0 1.5 1.0

3.2. Creaming behavior 0.5

Creaming behavior is reported as the % volume fraction (CLf) of the cream layer as a function of time (Fig. 3). As expected, creaming increased with decreasing concentration of either protein, but not to the same extent nor at the same rate. Significant creaming was detected after 1 h for MPI emulsions at low protein contents and the final cream layer volume fractions correlated to their corresponding d4,3. Na-caseinate emulsions showed longer lag periods before creaming could be observed. No creaming was detected for MPI (1:1) after the 72 h incubation period. Except for the latter, all MPI systems showed higher CLf than the corresponding Na-caseinate emulsions. Similar creaming behavior was found regardless of the sugar used. As previously noted, for the protein concentrations studied, none of these emulsions were prone to any separation mechanism other than the creaming of individual droplets. As a result, creaming would be dependent solely on particle size. Such behavior was corroborated by the creaming profiles observed during the scanning of the backscattering intensity as a function of emulsion height (data not shown) and was consistent with results published elsewhere (Blijdenstein, van Winden, van Vliet, van der Linden, & van Aken, 2004). To note, systems with the highest protein concentration showed significantly greater (visually observed) viscosity, which also played a role on inhibiting creaming.

0.0 (b)

15:1 L 15:1T Treatment

30:1L

30:1T

Fig. 2. Volume-weighted average diameter (d4,3) for emulsions stabilized with (a) milk protein isolate and (b) Na-caseinate before spray drying (black bars) and after drying and reconstitution (white bars). Suffixes L and T denote lactose or trehalose matrices, respectively. Error bars represent the standard deviation of five replicates.

After reconstitution, CLf increased for most treatments and creaming was immediately evident after incubation (Fig. 3). Compared to fresh emulsions, reconstituted MPI systems 9:1 and 5:1 with lactose showed a CLf increase of 100% and 40%, respectively. MPI 1:1 L showed creaming after 1 h and its CLf remained constant for the rest of the experiment (3%). Reconstituted 15:1 and 30:1 Na-caseinate systems showed a final CLf of 12% and 8%, respectively (with no lag period). Na-caseinate 1:1 L showed a similar lag period as observed in the fresh emulsion, but its final CLf increased by 100%. The type of carbohydrate seemed to have inconsistent effects on the initial creaming rate, but none on the final CLf (data not shown). The analysis of the backscattering profiles revealed that the separation mechanism after reconstitution remained unchanged for most of the systems under study.

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Table 3 Mean particle sizes (d4,3) and their relative changes for emulsions stabilized with either MPI or Na-caseinate before drying (fresh), atomized and after drying and reconstitution d4,3 (mm)

Emulsion

15:1 Lactose Na-caseinate 5:1 Lactose milk protein isolate a

Fresh

Atomized

Reconstituted

0.336 0.51

0.712 3.165

1.57 7.257

(A)a

(B)b

(A/B)  100

2.11 6.20

4.67 14.22

45.18 43.64

(A) Quotient obtained by dividing the d4,3 of the atomized emulsion by the d4,3 of the fresh emulsion. (B) Quotient obtained by dividing the d4,3 of the reconstituted emulsion by the d4,3 of the fresh emulsion.

b

3.3. Surface free fat

10

Cream layer % vol. fraction

9 8 7 6 5 4 3 2 1 0 0

2

4

6

8

30 40 50 60 70

Time (h)

(a)

Cream layer % vol. fraction

16 14 12 10 8 6 4 2 0 0 (b)

2

4

6

8

30 40 50 60 70

Time (h)

Fig. 3. Cream layer % volume fraction as a function of time for fresh (a) and reconstituted emulsions (b) stabilized with milk protein isolate (MPI) and Na-caseinate using lactose as encapsulant. MPI emulsions at 1:1 (’), 5:1 (&) and 9:1 (2) oil-to-protein ratio and Na-caseinate emulsions at 1:1 (K), 15:1 (J) and 30:1 () oil-to-protein ratios, respectively.

Some of the profiles of MPI emulsions 9:1 and 5:1 were characteristic of the creaming of small aggregates (not shown), indicative of flocculation (Blijdenstein et al., 2004), which was supported by the presence of droplets of around 8 and 14 mm for MPI-stabilized emulsions at 5 and 9:1 oilto-protein ratios, respectively.

The solvent extractable surface free fat values for spraydried emulsions are illustrated in Fig. 4. The values increased with decreasing protein concentration regardless of the protein type. These results suggested that encapsulation was only partially dependent on emulsion properties since (a) although MPI-stabilized emulsions showed significantly higher d4,3 values after reconstitution, their surface free fat values were only slightly higher than Nacaseinate systems; (b) this also implied that the majority of the de-emulsified or flocculated fat globules were preferentially located within the glassy carbohydrate matrix; and (c) even at the highest protein loads, fat was present on the powder surface. The tendency to observe higher values of surface fat through increased oil-to-protein ratios has been reported previously (Hogan et al., 2001; Onwulata, Smith, Craig, & Holsinger, 1994; Vega et al., 2005a). Trehalose-Na-caseinate systems showed slightly better encapsulation properties than lactose-based powders. There are only two published studies on the encapsulation properties of trehalose (Cerdeira et al., 2005; Elofsson & Millqvist-Fureby, 2003). Cerdeira et al. (2005) used trehalose, trehalose/lactose and trehalose/sucrose blends (70/30) to encapsulate emulsions (freeze-dried) of the low melting fraction of milk fat stabilized with palmitic sucrose esters. The trehalose/sucrose blend showed superior encapsulating properties (91% of initial fat load encapsulated) compared with trehalose alone (83%) and trehalose/ lactose (42%) blends after lyophilization. However, there was a significant shortcoming in their methodology— trehalose (and not its blends with sucrose, or lactose) crystallized during sample preparation. Successful freezedrying of low MW carbohydrate solutions (or emulsions) renders amorphous materials (Roos & Karel, 1990). This negatively affected their comparisons in terms of initial encapsulation efficiency, water sorption studies (and subsequent crystallization) and emulsion stability. Elofsson and Millqvist-Fureby (2003) reported that the surface fat values for rapeseed oil spray-dried emulsions stabilized with Na-caseinate (1:1 oil-to-protein ratio) using trehalose or lactose as encapsulants were around 1% (g fat g1 powder) regardless of the sugar used. Our results were in agreement with this report.

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4.5

Free fat (g fat 100g-1 powder)

4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 1:1 (a)

5:1 Treatment

9:1

4.5

Free fat (g fat 100g-1 powder)

4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 1:1 (b)

15:1 Treatment

30:1

Fig. 4. Surface free fat (g fat 100 g1 powder) for spray-dried emulsions stabilized with (a) milk protein isolate and (b) Na-caseinate at different oil-to-protein ratios and using either trehalose (hashed bars) or lactose (white bars) as main encapsulant. Error bars are the standard deviation of five determinations.

3.4. Water plasticization and XRD Water sorption behavior as a function of time at 65% RVP of selected powdered emulsions is shown in Fig. 5. Water sorption was mainly dependent on carbohydrate composition, but the effects of protein type could also be discriminated. For 15:1 Na-caseinate and 9:1 MPI systems, lactose-containing powders were the first to crystallize as evidenced by the sudden loss of sorbed water (Miao & Roos, 2005). The equilibrium water content (EWC) of the Na-caseinate system was slightly higher than MPI [2.81 and 2.43 g water (g  100)1 of solids]. Crystallized trehalose powders showed substantially higher EWC compared with powders containing lactose [7.14 and 6.92 g water (g  100)1 of solids for Na-caseinate and MPI powders, respectively]. This difference was consistent with the formation of crystals of trehalose dihydrate, which

689

was also confirmed by XRD (data not shown). At high protein content (1:1) water sorption behavior was complex. For the MPI lactose-containing system, crystallization and equilibration were fast processes (3–8 h). In contrast, Nacaseinate system showed considerably delayed crystallization and very slow equilibration, especially for lactose-containing powders. EWC were higher than those corresponding to low protein systems [7.38 and 5.47 g water (g  100)1 of solids for Na-caseinate and MPI powders, respectively]. It has been reported that proteins, along with other polymeric materials, delay or inhibit sugar crystallization (Berggren & Alderbor, 2004; Biliaderis, Lazaridou, Mavropoulos, & Barbayiannis, 2002; Haque & Roos, 2004). Regardless of their larger molecular mass, 5  108 Da (Schorsch, Clark, Jones, & Norton, 1999) compared with that of Na-caseinate aggregates, 2.2  106 Da (Belyakova et al., 2003), micellar casein (as MPI) had a significantly less important effect on delaying both trehalose and lactose crystallization. We found similar results in systems comprised of sucrose, lactose and sucrose/lactose blends after addition of Na-caseinate at a 1:6 protein-to-carbohydrate ratio, while systems with added micellar casein behaved very similarly to those with no protein (Vega & Roos, 2006b). Lactose was found to crystallize mainly as a-lactose monohydrate with characteristic X-ray powder diffraction peak at 2y ¼ 20:91, whereas trehalose crystallization (as a dihydrate) was followed by the relative intensity of the peak located at 2y ¼ 23:81. Peak intensities were highly dependent on composition and showed that powders with low protein concentrations (regardless of carbohydrate type) showed significantly higher peak intensities than those with high protein levels. Peak intensity was timedependent only for the Na-caseinate 15:1 L systems and leveled off after 8 h of storage. For the rest of the systems, including those containing trehalose, crystallization was detected after 4 h and peak intensity remained constant. There was good agreement between water sorption kinetics and peak intensity by XRD. High-protein powders showed significantly lower peak intensities after equilibrium (72 h) than low-protein powders (Fig. 6); consistent with the lower amounts of carbohydrate in the matrix (40% instead of 64%). Peak intensities were measured after 12, 24, 36 and 72 h of storage, but crystals were only detected after 24 h. These results were not in agreement with water sorption data since samples MPI 1:1 with lactose and trehalose showed loss of sorbed water as early as 4 h after incubation. Corresponding powders containing Na-caseinate showed crystallization after 8 and between 8 and 24 h for system 1:1 with trehalose and lactose, respectively. The only consistent observations for high protein systems were that crystallization was severely reduced (in absolute number of crystal counts) and that Na-caseinate systems showed significantly reduced rates of crystallization compared with MPI, regardless of the carbohydrate used (Fig. 6).

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690

12

g H2O 100 g-1 solids

(a) 10 8 6 4 2 0 2

0

4 Time (h)

6

8

Fig. 5. Water sorption behavior at 65% RVP as a function of time of spray-dried emulsions stabilized with milk protein isolate (filled symbols) or Na-caseinate (open symbols) at different oil-to-protein ratios: 1:1 L (n,m), 1:1 T (&,’), 15:1 and 9:1 L ($,%), and 15:1 and 9:1 T (J,K). Inset (Fig. 5a) shows the whole time span of the experiment.

7500 6000 4500 3000 1500

Counts

0 9-1 T MPI

15-1 T NCN

9-1L MPI

1-1 T MPI

1-1 T NCN 1-1L MPI Treatment

15-1 L NCN

(a)

2400 2100 1800 1500 1200 900 600 300 0 (b)

1-1L NCN

Fig. 6. Extent of crystallization (counts) as a function of time. Low (a) and high (b) protein emulsions, respectively (4 h, diagonal patterned bars; 8 h, white bars; 12 h, black bars; 36 h, gray bars; 72 h, square patterned bars). MPI, NCN, L and T denote systems containing milk protein isolate, Na-caseinate, lactose and trehalose, respectively. Error bars are the standard deviation of at least three determinations.

3.5. Surface free fat (post-crystallization) Surface free fat was also measured after exposing the materials to a 65% RVP for 4, 12 and 36 h (9:1 MPI and 15:1 Na-caseinate) and for 12, 36 and 72 h (1:1 MPI and

Na-caseinate). Regardless of the composition, surface free fat values increased as a result of lactose and trehalose crystallization and were consistent with previous reports (Fa¨ldt & Bergensta˚hl, 1995). Powders containing lactose showed significantly higher surface free fat values in

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4

Free fat (g fat 100g-1 powder)

MPI-stabilized emulsions (Fig. 7). High protein levels decreased the surface free fat from 20.5 too4 and from 24.5 to 17 g fat 100 g1 powder for Na-caseinate and MPI systems, respectively. This was an interesting observation since Fa¨ldt and Bergensta˚hl (1995, 1996) suggested that powders containing low mass fractions of lactose do not show high levels of surface fat upon crystallization. These results also suggested a preferential ‘‘interaction’’ (if any) between non-micellar casein with lactose. Powders containing trehalose showed surface free fat values of 10.2, 10.2, 2.0 and 2.1 g fat 100 g1 powder for 9:1 MPI, 15:1 Nacaseinate, 1:1 MPI, and 1:1 Na-caseinate, respectively. Regardless of protein type, powders with trehalose showed a significantly lower amount of surface free fat. Normalized differences in the amount of absolute fat released during crystallization (Table 4) showed that for MPI systems, lactose crystallization caused the release of 3.40 and 18 times more fat onto the powder surface than trehalose did, in 9:1 and 1:1 powders, respectively. For Nacaseinate systems, this relative value was independent of protein concentration (2.1  ) and was significantly lower than when MPI was used suggesting that only Na-caseinate played an active encapsulating/protecting role during powder storage.

28 26 24 22 20 18 16 14 12 10 8 6 4 2 0 12 Time (h)

(a)

691

36

20 18 16

4

2

3.6. Microstructure 0 12

36 Time (h)

(b)

72

Fig. 7. Surface free fat (g fat g1 powder) for spray-dried emulsions stabilized with milk protein isolate or Na-caseinate at 9:1 or 15:1 (a) and 1:1 (b) oil:protein ratios, respectively, after exposure to a 65% RVP for different periods of time. Powders were made using lactose (J,K) or trehalose (&,’). Filled and open symbols correspond to Na-caseinate and milk protein isolate systems, respectively. Error bars are the standard deviation of five determinations.

Fig. 8 shows representative micrographs of spray-dried emulsions stabilized with MPI (9:1) or Na-caseinate (15:1) containing either lactose or trehalose, taken before and after crystallization. Spray drying rendered mostly discrete and spherical particles regardless of composition. The outer surface appearance depended mostly on protein type with Na-caseinate-stabilized emulsions showing smooth surfaces along with very distinct indentations, whereas

Table 4 Analysis of the net and relative amounts of encapsulated fat released after sugar crystallization as affected by carbohydrate and protein type and concentration Samplea

MPI 9:1 L MPI 9: 1 T NaCas 15:1 L NaCas 15:1 T MPI 1:1 L MPI 1:1 T NaCas 1:1 L NaCas 1:1 T a

% Surface free-fat (g fat 100 g1 powder) Pre-crystallization

Post-crystallizationd

Net amount of fat released (from total fat load)b (%)

3.065 3.88 2.19 1.47 0.856 1.091 0.658 0.686

24.56 10.24 20.55 10.18 16.80 1.981 3.66 2.133

64.54 19.09 55.13 26.13 47.87 2.67 9.015 4.34

Lactose/trehalosec

3.38 2.10 18.0 2.07

MPI ¼ milk protein isolate; NaCas ¼ sodium caseinate; L ¼ lactose; T ¼ trehalose. Obtained by subtracting the pre-crystallization value from the post-crystallization value, and the result divided by 0.333, which represents the mass fraction of fat per gram of powder. c The quotient of the net amounts of fat released from powders with the same composition but different carbohydrate. d Values from the longest incubation times (see Fig. 7). b

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Fig. 8. Microstructural features of spray-dried emulsions before (A–D) and after (E–H) sugar crystallization by exposure to 65% RVP. Na-caseinate 15:1 lactose (A, E), Na-caseinate 15:1 trehalose (B, F), milk protein isolate 9:1 lactose (C, G), and milk protein isloate 9:1 trehalose (D, H). Bar is 6 mm.

MPI powders had an irregular/coarse surface and no indentations (Fig. 8A–D) in agreement with previous reports (Vega et al., 2005b). After crystallization, distinct lactose crystals could be observed regardless of the protein used (Fig. 8E and G). In contrast, crystallized trehalose powders showed a slight overall change: powder

particles lost some of their individual identity as they fused with adjacent particles and the presence of a very small quantity of crystal could be observed (Fig. 8F and H). Similar results have been reported for lactose-containing powders (Fa¨ldt & Bergensta˚hl, 1995), whereas this appears to be the first time that observations of this

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kind are reported for trehalose-containing spray-dried emulsions. Lactose systems, regardless of protein level and type, showed the highest levels of surface free fat that in some cases represented as much as nine times more than its corresponding trehalose systems. We believe that the radical differences on ‘‘capsule’’ integrity after crystallization were related to the type of crystal formed during crystallization (a-lactose monohydrate and trehalose dihydrate) as well as in differences in the solubility of the sugars. It is well known that a-lactose monohydrate crystals can adopt the shape of a tomahawk (Norgaard, Hahn, Knudsen, Farhat, & Engelsen, 2005), and have been found after lactose crystallization in the presence of sodium caseinate (Fa¨ldt & Bergensta˚hl, 1995). In this study we observed, aside from the tomahawk-shaped crystals, the occurrence of needle-like lactose crystals, which were believed to exert an even greater mechanical stress over the system (Fig. 8E). Similar observations have been reported for bovine serum albumin/mannitol mixtures (Millqvist-Fureby, Malmsten, & Bergenstahl, 1999). Welldefined crystals were not observed in the crystallized trehalose systems and were in conformity with results by Cerdeira et al. (2005).

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et al., 2002; Shamblin, Huang, & Zografi, 1996). These observations were consistent with the fact that the presence of protein molecules reduced the ability of trehalose molecules to form intermolecular sugar-sugar hydrogen bonds, thereby retarding crystal formation (Lopez-Diez & Bone, 2000). Also, it has been found that the presence of sucrose or lactose (200–300 mM) in non-micellar casein solutions caused protein stabilization through preferential hydration of the protein and not by binding to the protein (Mora-Gutierrez & Farrell Jr., 2000). That is, the water layer around the protein is enriched in water relative to the sugar-water solvent, and as the concentration of the sugar increases, so does the preferential hydration of the protein. We propose that the enhanced delay in crystallization of lactose in Na-caseinate systems compared with MPI was due to (i) preferential hydration of casein; (ii) hindered nucleation process in the presence of Na-caseinate aggregates of significantly smaller size (8–50 nm) compared with that of casein micelles (50–600 nm) (Farrer & Lips, 1999; Huppertz & Fox, 2006; Radford & Dickinson, 2004); and (iii) the ‘‘open’’ structure of non-micellar casein that allows more interactions (hydrogen bonding) between its hydrophilic groups and the hydroxyl groups (–OH) of the sugars. 4. Conclusions

3.7. Stabilization mechanism Time-dependent lactose crystallization was delayed during humid storage in the presence of sodium caseinate, albumin, whey protein isolate (WPI) and gelatin (Haque & Roos, 2004). Instant crystallization temperatures of the same composites (1:3) were approx. 25 1C higher than those of lactose alone. Pullulan also retarded lactose crystallization when present at weight fraction between 0.25 and 0.33 and had only marginal influence on the glass transition temperature (Tg) over the entire water content range examined (Biliaderis et al., 2002). We have demonstrated that for lactose composites with micellar and non-micellar casein (6 to 1 lactose-to-casein ratio) there was: (i) an equally marginal increase of Tg of the composite compared with lactose alone; (ii) identical plasticization behavior (Tg depression) over the Aw ranged studied; and (iii) an enhanced delay on crystallization when Na-caseinate was used (Vega & Roos, 2006b). In the present study, results showed that casein type had a significant effect on the amount of surface fat upon crystallization. Also, the Tg (onset) of the powdered emulsions was slightly changed compared to that of pure lactose (107 1C) or trehalose (114 1C). Based on the above, we can rule out that the enhanced ‘‘protection’’ provided by Na-caseinate in systems containing lactose is explained by Tg driven mechanism. It has been proposed that the presence of proteins or polysaccharides exerts a significant effect on the mass transfer rate of sucrose and lactose molecules reducing the occurrence of nucleation as well as the positioning of sugar molecules onto the growing crystal interface (Biliaderis

We have demonstrated that the molecular conformation of casein greatly affects initial emulsion stability, resistance to shear (during atomizing), but more importantly, sugar crystallization kinetics during powder storage. Non-micellar casein showed superior properties contributing to emulsion stabilization and encapsulation compared with micellar casein. The mechanism is not driven by Tg of the system nor by MW of the protein, but seems to respond to the spatial arrangement of the protein molecules. Trehalose offers a new alternative for the formulation of powdered emulsions. Acknowledgments We thank Gay Lea Dairy Cooperative (Guelph, Canada), in particular to Anita Usas, for facilitating the use of the Niro Spray Drier. The technical support of Anthony P. Whelan in the atomizing and creaming experiments, Edita Verespej in the water sorption studies and Dr. Alexandra Smith for the FE-SEM micrographs is appreciated. Author Vega is grateful to the Mexican Consejo Nacional de Ciencia y Tecnologia (CONACyT) and to Dippin’ Dots Inc. for providing financial support to undertake this research. References Adhikari, B., Bhandari, B. R., Howes, T., & Troung, V. (2004). Effect of addition of maltodextrin in drying kinetics and stickiness of sugar and acid-rich foods during convective drying: Experiments and modelling. Journal of Food Engineering, 62, 53–68.

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