Food Hydrocolloids 20 (2006) 596606 www.elsevier.

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Properties and stability of oil-in-water emulsions stabilized by sh gelatin

Jeonghee Surh, Eric A. Decker, D. Julian McClements*
Biopolymers and Colloids Research Laboratory, Department of Food Science, University of Massachusetts, Amherst, MA 01003, USA Received 13 March 2005; revised 25 May 2005

Abstract The inuence of molecular weight on the ability of sh gelatin (FG) to form and stabilize oil-in-water emulsions was examined. Low molecular weight sh gelatin (LMWFG, w55 kDa) and high molecular weight sh gelatin (HMWFG, 120 kDa) were used to prepare 20 wt% corn oil-in-water emulsions (pH 3.0, 10 mM imidazole-acetate buffer). Emulsions with monomodal particle size distributions and small mean droplet diameters (d43z0.35 mm for LMWFG and 0.71 mm for HMWFG) could be produced at protein concentrations R4.0 wt%. However, optical microscopy showed that there was always a small population of large droplets present in the emulsions after homogenization and some oil destabilization (R2 wt%). The presence of these large droplets was attributed to the relatively low surface activity (c1/2Z0.10.2 wt%) of sh gelatin compared to globular proteins such as b-lactoglobulin (c1/2Z0.004 wt%). Emulsions stabilized by LMWFG contained a bigger population of large droplets than HMWFG emulsions, but were more stable to creaming, which was attributed to depletion occulation. Fish gelatin stabilized emulsions remained moderately stable to droplet aggregation and creaming after they were subjected to changes in holding temperature (30 or 90 8C for 30 min), salt concentration (NaCl%250 mM) and pH (38). This study demonstrates that sh gelatin may have some limited use as a protein emulsier in oil-in-water emulsions. q 2005 Elsevier Ltd. All rights reserved.
Keywords: Emulsion; Gelatin; Molecular weight; Surface activity; Viscosity

1. Introduction Emulsiers are surface active materials that adsorb to interfaces and facilitate the production of small droplets by lowering the interfacial tension during homogenization (Walstra, 2003). Emulsiers also improve the long-term stability of emulsions to droplet aggregation by generating repulsive forces between droplets and/or by forming interfacial membranes around the droplets that are resistant to rupture (Friberg, Larsson, & Sjoblom, 2004; McClements, 2005; Stang, Karbstein, & Schubert, 1994; Walstra, 1993). A wide variety of synthetic and natural emulsiers are legally acceptable for utilization in food emulsions, including small molecule surfactants, phospholipids, and biopolymers (Charlambous & Doxastakis, 1989; Krog & Sparso, 2004; Stauffer, 1999). Nevertheless, there is
* Corresponding author. Tel.:C1 413 545 1019; fax: C1 413 545 1262. E-mail address: mcclements@foodsci.umass.edu (D.J. McClements).

0268-005X/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodhyd.2005.06.002

a growing trend within the food industry to replace synthetic emulsiers with more natural ones (Garti, 1999). Proteins extracted from a variety of natural sources can be used as emulsiers in foods because of their ability to facilitate the formation, improve the stability, and produce desirable physicochemical properties in oil-in-water (O/W) emulsions, e.g. soy, whey, casein, sh, meat and plant proteins (Dickinson, 2003; McClements, 2004). A major potential advantage of proteins as emulsiers in foods is their ability to protect polyunsaturated lipids from ironcatalyzed oxidation (Hu, McClements, & Decker, 2003). At pH values below their isoelectric point (pI), proteins form positively charged interfacial membranes around oil droplets that electrostatically repel any Fe2C and Fe3C ions present in the aqueous phase. Thus, iron is prevented from catalyzing oxidation of the polyunsaturated lipids contained within the droplets. One of the major limitations of using food proteins for this purpose is that most of them have isoelectric points (pI) in the pH range 4.55.5, and so cationic emulsion droplets can only be produced at relatively low pH values (Damodaran, 1996). On the

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other hand, the aqueous phase of many food products has a pH above 5, and therefore emulsions created using most protein emulsiers would contain anionic droplets that are prone to lipid oxidation because cationic iron ions would be attracted to the droplet surfaces (McClements & Decker, 2000). Gelatin is a relatively high molecular weight protein derived from animal collagen, e.g. pig, cow or sh (Ledward, 1986). It is prepared by hydrolyzing collagen by boiling in the presence of either acid (Type A gelatin) or alkaline (Type B gelatin). The isoelectric point of Type A gelatin (w79) tends be higher than that of Type B gelatin (w5). The relatively high isoelectric point (pIR7.0) of Type A gelatin means that it should be possible to create oil-in-water emulsions that have a positive charge over a wider range of pH values than is possible with conventional protein emulsiers, such as soy, casein or whey proteins (Dickinson & Lopez, 2001). Consequently, Type A gelatin may be suitable for creating oil-in-water food emulsions with high oxidative stability since it could repel iron ions from oil droplet surfaces over most of the pH range typically found in foods. Nevertheless, it is important to determine whether gelatin can be used to prepare emulsions that are also physically stable. Some previous studies have shown that gelatin is surface-active and that it is capable of acting as an emulsier in oil-inwater emulsions (Lobo, 2002; Muller & Hermel, 1994). Nevertheless, when used on its own gelatin often produces relatively large droplet sizes during homogenization (Dickinson & Lopez, 2001; Lobo, 2002), so that it has to be either hydrophobically modied by attachment of nonpolar side-groups (Toledano & Magdassi, 1998) or used in conjunction with anionic surfactants to improve its effectiveness as an emulsier (Muller & Hermel, 1994; Olijve, Mori, & Toda, 2001; Surh, Gu, Decker, & McClements, 2005). The purpose of this study was to investigate whether physically stable oil-in-water emulsions could be produced using sh gelatin, and to determine the inuence of gelatin molecular weight and environmental stresses (pH, salt and thermal processing) on their stability. We selected sh gelatin to study rather than mammalian gelatin because there are no concerns about contamination with bovine spongiform encephalitis (BSE), and because it can be consumed by ethnic groups that do not consume pig or cow products. In addition, sh gelatin can be extracted from low value byproducts of the shing industry (e.g. sh skin, bones, heads, and tails) that are currently discarded because they are considered unsuitable for human consumption. Thus, the economic value of these byproducts could be increased by nding new applications and markets for them (Huidobro, Montero, & Borderias, 1998; Liceaga-Gesualdo & Li-Chan, 1999; Kristinsson & Rasco, 2000; Sato, Katayama, Sawabe, & Saeki, 2003).

2. Materials and methods 2.1. Materials Food/pharmaceutical grade sh gelatin samples (solids: 85% min.) were kindly provided by Norland Products Inc. (Cranbury, NJ, USA). As stated by the manufacturer, Norland dried sh gelatin (LMWFG, Lot # 1253KD) and Norland high molecular weight sh gelatin (HMWFG, Lot # 1313HMWD) are type A gelatins produced from the skins of deep water sh such as cod, haddock and pollock, and contain approximately 60 hydroxyproline and 96 proline residues per 1000 total residues. Fish gelatins have similar characteristics to animal gelatin with the exception that they gel at lower temperatures (typically !10 8C). The average molecular weights were reported to be w55 and w120 kDa, and the pI values were reported to be 7 and 8 for LMWFG and HMWFG, respectively. Powdered b-lactoglobulin (b-Lg) was obtained from Davisco Foods International (Lot # JE 001-1-922, Le Sueur, MN, USA). As stated by the manufacturer, the b-Lg content of the powder was 98%, with the remainder being mostly globulins. Analytical grade sodium chloride (NaCl), hydrochloric acid (HCl), sodium hydroxide (NaOH), Oil Red O, imidazole, and sodium azide (NaN3) were purchased from the Sigma Chemical Company (St Louis, MO, USA). Acetic acid was purchased from Fisher Science (Chicago, IL, USA). Corn oil was purchased from a local supermarket and used without further purication. Distilled and deionized water was used for the preparation of all solutions. 2.2. Emulsion preparation A buffer solution was prepared by dispersing 10 mM imidazole and acetic acid into water and then adjusting the pH to 3.0 using 1 M HCl. Fish gelatin solutions were prepared by dispersing the desired amount (0.6257.5 wt%) of protein powder into buffer solution and stirring overnight at room temperature to ensure complete dissolution. The pH of the sh gelatin solutions was adjusted to pH 3.0 using 1 M HCl if required. Oil-in-water emulsions were prepared by blending 20 wt% corn oil and 80 wt% sh gelatin solutions together using a high-speed blender (M133/1281-0, Biospec Products, Inc., ESGC, Switzerland) for 2 min. These coarse emulsions were then passed through a two-stage high-pressure valve homogenizer (LAB 1000, APVGaulin, Wilmington, MA, USA) three times: 4500 psi the rst stage, 500 psi the second stage. 0.02 wt% sodium azide (NaN 3) was added to the emulsions as an antimicrobial agent. The emulsions (20 wt% corn oil, 0.56.0 (0.5, 1.0, 1.5, 2.0, 4.0, 6.0) wt% sh gelatin, pH 3.0) were then stored at ambient temperature for 24 h before being analyzed.

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2.3. Surface tension measurements A digital tensiometer with a Wilhelmy plate (K10ST, ss, Charlotte, NC, USA) was used to measure the surface Kru tensions of sh gelatin solutions and b-Lg solutions at 30. 0 8C. The protein solutions were prepared by dispersing each protein into 10 mM imidazole/acetate buffer (pH 3.0) and stirring overnight at room temperature to ensure that they were homogeneous. These solutions were then placed in the tensiometer measurement cell for 40 min to reach the correct temperature before measurements were performed. Surface tension measurements were represented as the averageGstandard deviation of measurements made on two freshly prepared samples, with two measurements per sample. The surface pressure (p) was calculated from the surface tension measurements: pZg0-g, where g0 is the surface tension of 10 mM acetate/imidazole bufferair interface and g is the surface tension in the presence of the protein. The saturation surface pressure (psat) was determined by averaging the measured surface pressure values over the range of protein concentrations where p remained relatively constant as the protein concentration was increased further, i.e. R2 wt% protein. 2.4. Viscosity measurements The viscosity of sh gelatin solutions (0.06.0 wt% sh gelatin in 10 mM imidazole/acetate buffer, pH 3.0) was measured using a dynamic shear rheometer with a temperature controlled concentric cylinder measurement cell (Constant Stress Rheometer, CS-10, Bohlin Instruments, Cranbury, NJ, USA). The diameter of the rotating inner cylinder was 25 mm, and the diameter of the static outer cylinder was 27.5 mm, so the gap between the two cylinders was 1.25 mm wide. Samples were contained in the temperature-controlled measurement vessel and allowed to equilibrate to the required temperature (30 8C) prior to making the measurements. The viscosity of the samples was determined from measurements of the shear rate as the shear stress was increased from 0.02 to 5.0 Pa. 2.5. Particle size measurements To avoid multiple scattering effects, emulsions were diluted to a droplet concentration of approximately w0.005 wt% using buffer solution at the pH and NaCl concentration of the sample and stirred continuously throughout the measurements to ensure the samples were homogenous. The particle size distribution of the emulsions was then measured using a laser light scattering instrument (Mastersizer MSS, Malvern Instruments, Worcestershire, UK). This instrument measures the angular dependence of the intensity of laser light (lZ632.8 nm) scattered by a dilute emulsion, and then nds the particle size distribution that gives the best t between experimental measurements and predictions based on light scattering theory. Particle

size was reported as volume-surface mean diameter, d32 (a Z Sni di3 =Sni di2 , where ni is the number of particles with diameter di) or volume-weighted mean diameter, d43 (ZSni di4 =Sni di3 ). It should be noted that the d43 value is more sensitive to the presence of large particles than the d32 value, and therefore it can give a good indication of droplet aggregation. All measurements were made on two freshly prepared samples and results are reported as averages and standard deviations. 2.6. x-Potential measurements Prior to analysis, emulsions were diluted to a droplet concentration of approximately w0.006 wt% using buffer solution at the pH and NaCl concentration of the sample to avoid multiple scattering effects. The x-potential of the emulsions was determined using a particle electrophoresis instrument (ZEM5002, Zetamaster, Malvern Instruments, Worcestershire, UK) that measured the direction and velocity of droplet movement in an applied electric eld. The x-potential was reported as the average and standard deviation of measurements made on two freshly prepared samples, with ve readings taken per sample. 2.7. Optical microscopy Emulsions were gently agitated in a glass test tube before analysis to ensure that they were homogenous. A drop of emulsion was placed on a microscope slide and then covered with a cover slip. The microstructure of the emulsion was then observed using conventional optical microscopy (Nikon microscope Eclipse E400, Nikon Corporation, Japan). The images were acquired using a CCD camera (CCD-300T-RC, DAGE-MTI, Michigan City, IN, USA) connected to Digital Image Processing Software (Micro Video Instruments Inc., Avon, MA, USA) installed on a computer. 2.8. Destabilized oil measurements The amounts of destabilized oil present in the emulsions were determined using the dye dilution technique described in detail by Palanuwech, Potineni, Roberts, & Coupland, (2003). Emulsions were agitated in a glass test tube before analysis to ensure that they were homogenous. Three grams of corn oil containing a known amount of oil-soluble red dye (0.001 wt% Oil Red O) was added to the top of 5 g of the emulsion to be tested. Samples were mixed by vortexing for 30 s and then centrifuged at 20,000 rpm for 20 min at 25 8C (Sorvall Centrifuges T-1270, Du Pont Company, Wilmington, DE). The dyed free oil on top of the emulsion was removed with a pipette and placed into a 1.5 mL cuvette where its absorbance was measured at 517 nm using UV visible spectrophotometer (UV-2101 PC, Shimadzu Corporation, Japan). The percentage of destabilized oil ( f d) was calculated using the following equation:

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fd % Z 100mo a K 1=me f, where mo is the mass of added dye solution, me is the mass of emulsion, f is the mass fraction of oil in the emulsion, and a is the ratio of the absorbance of the standard dye solution to that of the dye solution extracted from the top of the emulsion sample. 2.9. Creaming stability measurements Ten grams of emulsion were transferred into a test tube (internal diameter 15 mm, height 125 mm), tightly sealed with a plastic cap, and then stored for 1 day and 7 days at room temperature. After storage, emulsions separated into an optically opaque layer (Op) at the top, a strongly turbid layer (Tu (st)) in the middle, and/or a slightly turbid layer (Tu (sl)) at the bottom. The total height of the emulsions (HE) and the height of each layer (HL) were measured. The creaming stability was characterized by each layer (%)Z 100(HL/HE). All measurements were made on two freshly prepared samples. 2.10. Free protein measurements The concentration of free protein in the continuous phase of the emulsions was determined by the Lowry method (Lowry, Rosebrough, Farr, & Randall, 1951). Emulsions were centrifuged at 45,000 rpm for 40 min at 25 8C (Sorvall Centrifuges T-1270, Du Pont Company, Wilmington, DE) to separate the oil droplets from the serum phase. The serum phase was collected using a syringe and then diluted with 10 mM imidazole/acetate buffer solution to the proper concentration for spectrophotometer measurement. 1 mL of Lowry stock reagent (2% Na2CO3, 1% CuSO4, 2% NaKC4H4O6; 49:0.5:0.5, v/v/v) was added to 100 mL of diluted serum phase and thoroughly mixed using a vortex, then left to stand for 30 min. 100 mL of folins reagent diluted in distilled water (1:1, v/v) was then added to the solution, which was then mixed and left to stand for 30 min. The absorbance was measured at 595 nm in a spectrophotometer (UV-2101 PC, Shimadzu Corporation, Japan). The protein concentration of the emulsion serum was then determined using a calibration curve of absorbance versus protein concentration produced using sh gelatin as the standard. 2.11. Emulsion environmental stresses We examined the inuence of environmental stresses (temperature, ionic strength, and pH) on the mean particle diameter, x-potential, microstructure, and creaming stability of the emulsions. An emulsion was prepared as described above (20 wt% corn oil, 4.0 wt% sh gelatin, pH 3.0) and then diluted to a droplet concentration of 2.0 wt% using 10 mM imidazole/acetate buffer solution. This emulsion was placed in a separatory funnel overnight to allow any large droplets or non-emulsied oil to rise to the top, and then only the lower portion of emulsion was collected

(about 90% of the total volume of the emulsion). This separation step was carried out because we noticed that there was a small population (!3%) of large droplets in the emulsions after homogenization (see later), and we wanted to remove this population before carrying out our examination of the inuence of environmental stresses. Thermal processing. Emulsions were diluted to a droplet concentration of 1.0 wt% using 10 mM imidazole/acetate buffer solution. Emulsion samples (10 g) were transferred into glass test tubes (internal diameter 15 mm, height 125 mm) and then incubated in a water bath for 30 min at either 30 8C or 90 8C. After incubation, the emulsion samples were stored at 30 8C for 24 h prior to analysis. NaCl stability. Emulsion samples were diluted with salt solutions to obtain a nal droplet concentration of 1.0 wt% and NaCl concentrations of 0 to 250 mM. Emulsion samples (10 g) were then transferred into glass test tubes (internal diameter 15 mm, height 125 mm) and stored at room temperature for 24 h before being analyzed. pH stability. Emulsion samples were diluted with 10 mM imidazole/acetate buffer (pH 3.0) to a nal droplet concentration of 1.0 wt%. The pH of the diluted emulsions was then adjusted to 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0 using NaOH solutions. The emulsions were then stored at room temperature for 24 h before being analyzed. 2.12. Statistical analysis Experiments were performed twice using freshly prepared samples. Average and standard deviations were calculated from these duplicate measurements. Statistical analysis in this study was performed using t-test and presented with P-value.

3. Results and discussion 3.1. Inuence of sh gelatin concentration and molecular weight on emulsion formation and stability We examined the inuence of sh gelatin concentration (0.56.0 wt%) and molecular weight (w55 and 120 kDa) on the formation and stability of corn oil-in-water emulsions produced by high pressure valve homogenization. The mean particle diameter (Fig. 1), particle size distribution (Fig. 2ac), electrical charge, creaming stability, microstructure (Fig. 3a), and amount of destabilized oil (Fig. 3b) in the emulsions were measured. The electrical charge of the emulsion droplets was positive at pH 3.0 because of the relatively high pI values of the sh gelatins. There was no signicant change in xpotential with sh gelatin concentration (0.56.0 wt%) or molecular weight, with the average over all FG concentrations being xZ18.2G0.8 mV for LMWFG and 18.8G 0.4 mV for HMWFG stabilized emulsions. The fact that the emulsion droplets were coated by a biopolymer with an

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16.0 LMW HMW 12.0

8.0

4.0

0.0 0.0

2.0

4.0 FG (%)

6.0

Fig. 1. Inuence of sh gelatin concentration on mean particle diameter (d43 mm) of 20 wt% corn oil-in-water emulsions (10 mM acetate/imidazole buffer, pH 3). The average molecular weights of sh gelatin are w55 and 120 kDa for LMW- and HMWFG, respectively.

appreciable electrical charge suggests that electrostatic repulsion may play an important role in stabilizing them against droplet aggregation (McClements, 2005). Nevertheless, our studies on the inuence of pH and salt
(a) 16
LMW

concentration (see below) suggest that steric interactions may also play an important role. For both types of sh gelatin used, the volume-surface mean particle diameter (d32) of the emulsions was relatively small and independent of sh gelatin concentration: 0.27G 0.05 mm for LMWFG and 0.38G0.09 mm for HMWFG. On the other hand, there was a fairly steep decrease in the volume-weighted mean particle diameter (d43) when the sh gelatin concentration was increased from 0.5 to 4.0 wt%, after which the mean particle diameter reached a relatively constant value: 0.35 mm for LMWFG and 0.71 mm for HMWFG (Fig. 1). The reason for the different dependence of the mean particle diameter on sh gelatin concentration for d32 and d43 is because they are sensitive to different aspects of the particle size distribution (McClements, 2005): d32 is more sensitive to the presence of small particles, whereas d43 is more sensitive to the presence of large particles. This can be seen when the full particle size distributions of the emulsions are examined (Fig. 2). At relatively low sh gelatin concentrations (!4 wt%), the particle size distributions were either bimodal or multimodal, consisting of a major peak corresponding to a large fraction of relatively small droplets and minor peaks corresponding to small fractions of relatively large particles (Fig. 2). At higher sh gelatin concentrations (R4 wt%), the particle size distributions appeared mono-modal, regardless of the molecular weight of sh gelatin used (Fig. 2a and b).
(b) 10 Volume Fraction (%)

d43 (m)

Volume Fraction (%)

12

0.5% 1.0% 2.0% 4.0%

HMW 8 6 4 2 0 0.01

0.5% 1.0% 2.0% 4.0%

0 0.01

0.1

10

100

1000

0.1

10

100

1000

Particle Diameter (m) (c) 16 Volume Fraction (%)

Particle Diameter (m)

HMW LMW

12

0 0.01

0.1

10

100

1000

Particle Diameter ( m)
Fig. 2. Inuence of sh gelatin concentration on particle size (mm) distribution of 20 wt% corn oil-in-water emulsions (10 mM acetate/imidazole buffer, pH 3): (a) 0.5, 1.0, 2.0 and 4.0 wt% LMWFG; (b) 0.5, 1.0, 2.0 and 4.0 wt% HMWFG; (c) Comparison of full particle size distributions at 4.0 wt% sh gelatin.

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(a)
0.5 % LMW 4.0 % LMW 100 m

0.5 % HMW

4.0 % HMW

(b)

30 LMW 25 HMW

Destabilized oil (%)

20 15 10 5 0

0.5

1.0

1.5

2.0

4.0

6.0

FG (%)
Fig. 3. (a) Microstructures of emulsions coated by 0.5 and 4.0 wt% sh gelatin obtained by optical microscopy. (b) Inuence of sh gelatin concentration on the amount of destabilized oil in 20 wt% corn oil-in-water emulsions (10 mM acetate/imidazole buffer, pH 3).

At high gelatin concentrations (4 wt%), the emulsions stabilized by LMWFG had a narrower particle size distribution and a smaller mean particle size than those stabilized by HMWFG (Fig. 2c). There are a number of possible reasons to account for the observed decrease in mean droplet size with increasing protein concentration: (i) the total droplet surface area that could be stabilized by the protein increased; (ii) the rate at which the droplet surfaces were covered with protein increased; (iii) the frequency of droplet collisions decreased due to the increase in aqueous phase viscosity. All of these factors should facilitate droplet disruption and prevent droplet coalescence within the homogenizer, thereby leading to the formation of smaller droplet sizes (McClements, 2005). The laser diffraction measurements suggested that LMWFG was slightly more effective than HMWFG at producing small oil droplets within the homogenizer at

high protein concentrations (Figs. 1 and 2c). Nevertheless, we believe this may have been an artifact of the sampling procedure for the laser diffraction measurements (see below). Optical microscopy indicated that there were some large droplets (dO10 mm) in all sh gelatin stabilized emulsions regardless of protein concentration or molecular weight (Fig. 3a). Nevertheless, the amount of large droplets observed in the emulsions tended to decrease as the sh gelatin concentration increased. In addition, oil destabilization measurements conrmed that there was appreciable oiling-off in all of the emulsions, with the extent of destabilized oil decreasing with increasing sh gelatin concentration and molecular weight (Fig. 3b). At high gelatin concentrations (R4 wt%), there was about 2% destabilized oil in the HMWFG emulsions and about 3% in the LMWFG emulsions. Initially, these results seemed

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surprising because the laser diffraction measurements did not indicate the presence of any large droplets in the emulsions containing R4.0 wt% sh gelatin (Fig. 2c). We attribute the difference between the laser diffraction, optical microscopy and destabilized oil measurements to sampling errors associated with the laser diffraction instrument. To obtain an accurate measurement of the particle size distribution of an emulsion using laser diffraction a representative sample must pass through the laser beam during the data acquisition period. If an emulsion contains very large droplets, or if there is some oiling off, then this fraction of the oil may cream rapidly and remain on the surface of the emulsion, thereby not passing through the laser beam and not being detected. These observations underline the importance of using a range of different analytical techniques to accurately characterize the stability of emulsions prone to droplet aggregation. Our results indicate that oil-in-water emulsions can be prepared using a relatively high concentration of sh gelatin as an emulsier, but that these emulsions contain a small fraction (!3%) of relatively large droplets (dO10 mm). A number of possible reasons could account for the existence of a population of large droplets in the emulsions after homogenization. First, there may not have been sufcient gelatin present in the emulsions to completely cover all of the oil droplet surface created within the homogenizer, although this seems unlikely because there was little difference in mean particle size, microstructure or oiling off between 4 and 6 wt% sh gelatin. Second, the sh gelatin may have had a relatively low surface activity and therefore did not adsorb strongly to the droplet surfaces (see later). Third, the adsorption kinetics of the protein within the homogenizer may not have been fast enough to completely cover the freshly formed droplet surfaces before droplet collisions and fusion occurred. Fourth, some droplet coalescence may have occurred either in or immediately after the emulsions left the homogenizer. Our results, suggest that the number of large droplets and the amount of destabilized oil were less in the HMWFG emulsions than in the LMWFG emulsions (Fig. 3b). This effect may be attributed to the fact that the thickness of an adsorbed gelatin membrane increases with increasing molecular weight, which has previously been shown to lead to an increase in the stability of emulsions to coalescence during homogenization (Lobo, 2002; Lobo & Svereika, 2003). The creaming stability of sh gelatin stabilized oil-inwater emulsions (pH 3.0, 0 mM NaCl) depended on droplet concentration and protein molecular weight. No creaming was observed in any of the non-diluted emulsions (20 wt% droplet concentration, 4 wt% FG) after storage for 1 month at room temperature. This good creaming stability can be attributed to the fact that the sh gelatin formed a high viscosity solution that slowed down droplet movement and/or the droplets were aggregated into a particle network that prevented them from moving. Rheological measurements showed that the viscosity of aqueous solutions of sh

0.008
LMW HMW

0.006 Viscosity (Pas)

0.004

0.002

0.000 0.0

2.0 FG (%)

4.0

6.0

Fig. 4. Inuence of sh gelatin concentration on the viscosity of sh gelatin solutions (10 mM acetate/imidazole buffer solution) at a constant shear stress of 0.03 Pa and at 30 8C.

gelatin only increased slightly with protein concentration and molecular weight, with the values being about two- to four-fold higher than that of the pure buffer solution for 4 wt% of LMWFG and HMWFG, respectively (Fig. 4). This relatively small increase in continuous phase viscosity with increasing protein concentration suggested that the relatively good creaming stability of the 20 wt% oil-inwater emulsions was due to aggregate network formation. When the original emulsions were diluted to 1 wt% droplet concentration (0.2 wt% FG), no creaming was observed in LMWFG emulsions after 1 day, but HMWFG emulsions separated into a thin (w1.5 vol%) opaque cream layer at the top and a thick (w98.5 vol%) highly turbid layer at the bottom. This result suggested that there was a small fraction of large droplets in the HMWFG emulsions that moved upward to form a cream layer, while the remainder of the droplets stayed evenly distributed. We would have expected the HMWFG emulsions to be more stable to creaming than the LMWFG emulsions because they had a higher aqueous phase viscosity, which should have retarded droplet movement more effectively (Fig. 4). The fact that the low molecular weight sh gelatin emulsions appeared to be more stable may have been because non-adsorbed gelatin promoted some depletion occulation in the HMWFG emulsions. It is known that the strength of the depletion attraction between emulsion droplets increases as the size of the droplets increases, the radius of gyration of nonadsorbed polymer increases, and the molecular weight of the non-adsorbed polymer increases (McClements, 2005). Hence, the HMWFG may have promoted some occulation and accelerated creaming of the larger droplets in the emulsion, whereas the LMWFG did not. Nevertheless, we are not able to show this quantitatively because we do not know the radius of gyration of the different types of gelatin. The above results indicate that at least 4 wt% sh gelatin is required to make emulsions where the majority of oil

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droplets are relatively small and there is little evidence of destabilized oil. Consequently, we used this concentration in the remainder of the study. 3.2. Surface activity and surface load of sh gelatins The ability of proteins to form and stabilize emulsions is dependent on their ability to adsorb to interfaces and on the amount of protein required to saturate the interface (McClements, 2005). In an attempt to explain the emulsifying ability of sh gelatins we therefore measured the surface activity and surface load of low and high molecular weight gelatins, and compared them with those of b-lactoglobulin (a widely used globular protein emulsier). The dependence of the surface pressure (pZg0Kg) on protein concentration for sh gelatin and b-lactoglobulin solutions (10 mM acetate/imidazole, pH 3.0) was measured (Fig. 5). As the protein concentration increased, the surface pressure increased from zero in the absence of protein to a constant value (psat) when the interface became saturated with protein. The measured values of psat were around 25G 1, 21G1, and 24G1 mN/m for LMWFG, HMWFG, and b-lactoglobulin, respectively. HMWFG therefore appeared to be slightly least effective at minimizing the thermodynamically unfavorable interactions between the air and water phases at saturation (HMWFG !b-LgzLMW FG). The surface activity of an emulsier (KZ1/c1/2) can be determined from a pversus concentration prole, where K is the equilibrium constant for adsorption and c1/2 is the concentration where pZpsat/2 (McClements, 2005). The higher the value of K, the greater the surface activity of the emulsier. We found that the surface activity of b-lactoglobulin (KZ1/0.004 wt%Z250 wt%K1) was more than an order of magnitude higher than LMWFG (KZ1/ 0.07 wt%Z14 wt%K1), which was in turn more surface active than HMWFG (KZ1/0.2 wt%Z5 wt%K1). The lower surface activity of sh gelatins compared to b-lactoglobulin can be attributed to its relatively high hydrophilic character. Indeed, previous studies have shown
30 25 (mN/m) 20 15 10 5 0 0 1 2 3 4 5 Concentration (%)
Fig. 5. Inuence of sh gelatin concentration on the surface tension of sh gelatin solutions (10 mM acetate/imidazole buffer solution) at 30 8C.

that hydrophobically modied gelatin is considerably more surface active than non-modied gelatin (Toledano & Magdassi, 1998; Djagny, Wang, & Xu, 2001; Werten et al., 2001). The fact that relatively high concentrations of gelatin are required to form emulsions with small droplets may therefore be due to the fact that it has a relatively low surface activity for the oil-water interface. The effectiveness of a protein as an emulsier also depends on how much is present at the oil-water interface when the droplets are completely saturated, i.e. the surface load at saturation (GSAT). Thus, we determined surface load on the emulsions stabilized with 4 wt% of LMWFG because LMWFG was relatively more surface active and more effective at minimizing thermodynamically unfavorable interactions than HMWFG. The surface load was calculated from knowledge of the disperse phase volume fraction (f) and from measurements of the free gelatin concentration and mean particle diameter (d32) (McClements, 2005): G Z Ci K Ceq d32 =6f (1)

where Ci is the initial concentration of gelatin per unit volume of emulsion (kg/m3) and Ceq is the concentration of non-adsorbed gelatin per unit volume of emulsion (kg/m3). For the LMWFG containing 4 wt%, Ciw38.8 kg/m3 which was calculated by the fact that FG occupied 4 wt % of the emulsions and 1 mL of the emulsions weighed 0.97 g, Ceqw35.9G1.4 kg/m3, d32Z0.23!10K6 m and fZ0.2, hence GSATz 0.6! 10K6 kg/m2 (z 0.6 mg/m2). This value is relatively low compared to most other food proteins which have GSATZ110 mg/m2 (McClements, 2005). 3.3. Inuence of thermal processing on emulsions stability The purpose of these experiments was to examine the inuence of thermal processing on the stability of emulsions stabilized by sh gelatin. Emulsions (20 wt% corn oil, 4.0 wt% sh gelatin, pH 3.0) were diluted to a droplet concentration of 1.0 wt% as described earlier, held at either 30 or 90 8C for 30 min, and then stored at 30 8C for 1 day prior to analysis. There was no signicant effect of heating on the x-potential of the droplets in the emulsions, regardless of the molecular weight of sh gelatin used: 19. 5G1.0 mV and 22.4G1.3 mV for LMWFG at 30 and 90 8C; 19.5G0.6 mV and 21.4G1.2 mV for HMWFG at 30 and 90 8C, respectively. In addition, there was no statistically signicant difference in the volume-surface mean particle diameters (d32) of the LMWFG (0.25G 0.01 mm at 30 8C and 0.22G0.01 mm at 90 8C) and HMW FG emulsions (0.53G0.01 mm at 30 8C and 0.48G0.03 mm at 90 8C), and in the volume-weighted mean particle diameters (d43) of the LMWFG (1.90G1.21 mm at 30 8C and 2.97G0.33 mm at 90 8C) and HMWFG emulsions (3.09G1.25 mm at 30 8C and 3.26G2.16 mm at 90 8C) with heating temperature (pO0.05). Nevertheless, there were

LMW FG HMW FG beta Lg

604

J. Surh et al. / Food Hydrocolloids 20 (2006) 596606 Table 1 Inuence of NaCl concentration on mean droplet diameters of FG emulsions (20 wt% corn oil, 4.0 wt% FG, 10 mM acetate/imidazole buffer, pH 3.0) NaCl (mM) LMWFG d32 (mm) 0 50 100 150 200 250 0.21G0.00 0.21G0.01 0.21G0.01 0.21G0.00 0.22G0.00 0.23G0.00 d43 (mm) 0.75G0.35 0.83G0.54 1.92G2.10 1.95G1.91 2.57G2.10 2.38G1.87 HMWFG d32 (mm) 0.52G0.01 0.54G0.06 0.52G0.06 0.52G0.06 0.53G0.05 0.50G0.04 d43 (mm) 0.75G0.04 0.76G0.06 2.30G2.28 0.88G0.26 1.65G1.35 1.04G0.51

changes in particle size distributions from mono-modal before thermal treatment to bi-modal or multi-modal after thermal treatment (data not shown), which can be attributed to the formation of a small fraction of large droplets after thermal treatment. This suggested that the majority of the droplets in the emulsions were unaffected by heating, but that there was a small fraction of larger particles formed as a result of heating. The origin of this effect is unknown, but may have been due to some partial desorption of gelatin molecules from the droplet surfaces at elevated temperatures that led to occulation and/or coalescence. Thermal treatment had little inuence on the creaming stability of the emulsions stabilized by both types of gelatin. However, the LMWFG emulsion had a better creaming stability than the HMWFG emulsions. After storage for 1 week at room temperature, LMWFG emulsions separated into 2G1% of opaque layer at the top and 98G1% of a strongly turbid zone at the bottom, while HMWFG emulsions separated into opaque (4G1%), strongly turbid (79G3%), and slightly turbid (17G3%) layers from the top to the bottom. As mentioned earlier, we would have expected less creaming in the emulsion containing the HMWFG because it had the highest aqueous phase viscosity (Fig. 4). The fact that the HMWFG emulsion was the least stable was therefore attributed to a depletion effect (see earlier) (Klinkesorn, Sophanodora, Chinachoti, & McClements, 2004; McClements, 2000). 3.4. Inuence of ionic strength on emulsion stability The purpose of this experiment was to examine the inuence of NaCl (0250 mM) on the stability of sh gelatin emulsions. Studies have shown that the magnitude of the x-potential of emulsion droplets decreases as the concentration of NaCl is increased due to electrostatic screening effects (Hunter, 1986). Nevertheless, we found that it was not possible to obtain reliable x-potential measurements for the emulsion droplets coated by sh gelatin at high salt concentrations (i.e. the x-potential distribution was very wide and highly variable from measurement to measurement), presumably due to the high electrical conductivity of the aqueous solution. The volume-surface mean particle diameters (d32) remained constant as the salt concentration was increased from 0 to 250 mM NaCl (0.21G0.01 mm for LMWFG and 0.52 G 0.01 mm for HMWFG). In addition, d43 was relatively small at all NaCl concentrations studied, although the values were slightly increased at O50 mM NaCl (Table 1). Compared with d32 (which is more sensitive to the presence of small particles), d43 (which is more sensitive to the presence of large particles) had a relatively high standard deviation, which could be attributed to the sampling problem associated with the preparation of diluted emulsions (see Section 2.11) because optical microscopy showed there was a very small population of big oil droplets present intermittently in the diluted ones collected from

Measurements were conducted after storage at room temperature for 24 h.

the lower portion of the separatory funnel (data not shown). No signicant difference was observed in the creaming stability of emulsions containing different salt concentrations (0250 mM NaCl), however, the LMWFG emulsions were slightly more stable than the HMWFG emulsions as discussed earlier. These results suggest that the sh gelatin stabilized emulsions remained relatively stable to droplet aggregation even at high ionic strength where electrostatic interactions should be highly screened. Hence, we can conclude that polymeric steric repulsion (rather than electrostatic repulsion) plays a major role in preventing the droplets from aggregating. 3.5. Inuence of storage pH on emulsion stability The inuence of storage pH (38) on the x-potential, mean particle diameter, and creaming stability of diluted emulsions (1 wt% oil) was measured (Fig. 6, Table 2). The x-potential of the droplets in the LMWFG and HMWFG emulsions went from positive (C17.2 and C17.8 mV, respectively) to negative (K5.8 and K2.0 mV) as the
25
LMW

20 15 - Potential (mV) 10 5 0 5 10 15 2 3 4 5 pH 6 7

HMW

Fig. 6. Inuence of storage pH on particle electrical charge (x-potential) of sh gelatin stabilized corn oil-in-water emulsions.

J. Surh et al. / Food Hydrocolloids 20 (2006) 596606 Table 2 Inuence of storage pH on mean droplet diameters of FG emulsions (20 wt% corn oil, 4.0 wt% FG, 10 mM acetate/imidazole buffer, pH 3.0) pH LMWFG d32 (mm) 3 4 5 6 7 8 0.21G0.00 0.22G0.00 0.21G0.01 0.21G0.00 0.21G0.00 0.21G0.01 d43 (mm) 0.75G0.35 2.51G0.53 1.12G0.82 1.77G0.40 1.25G0.28 0.88G0.24 HMWFG d32 (mm) 0.52G0.01 0.50G0.03 0.52G0.04 0.51G0.00 0.52G0.01 0.51G0.04 d43 (mm) 0.75G0.04 0.73G0.04 0.74G0.05 0.74G0.02 0.80G0.11 3.11G3.41

605

Measurements were conducted after storage at room temperature for 24 h.

storage pH was increased from 3 to 8 (Fig. 6). The droplets in protein-stabilized emulsions have zero net charge around the isoelectric point of the adsorbed proteins. Our x-potential versus pH measurements suggested that the pI values of the sh gelatins were at pH 6 and 6.5 for LMW FG and HMWFG, respectively. These values are appreciably lower than the pI values reported by the sh gelatin supplier (pH 7 and 8, respectively). This apparent difference may be because the actual pI of the proteins is different from the reported values, or because of ionic impurities in the samples that adsorbed to the surface of the oil droplets and changed their apparent isoelectric point, e.g. multivalent anions, free fatty acids or phospholipids. In addition, it should be noted that the absolute value of the x-potential across the whole pH range from 3 to 8 remained relatively low !20 mV compared to other types of protein. The low net charge on the droplets can be explained in terms of the relatively low surface load of sh gelatin and/or the fact that each gelatin molecule had a relatively low number of ionizable amino acid side groups. The mean particle diameter remained relatively small from pH 3 to 8 for LMWFG and HMWFG emulsions, indicating that little droplet aggregation occurred during storage (Table 2). The creaming stability of the emulsions was similar at all pH values, although the LMWFG emulsions were slightly more stable than the HMWFG emulsions as discussed earlier. These results suggest that sh gelatin stabilized emulsions remain relatively stable to droplet aggregation even at pH values where the electrical charge on the droplets is relatively low. Hence, we conclude that polymeric steric repulsion (rather than electrostatic repulsion) between the droplets must play a major role in preventing them from aggregating.

concentrations R4.0 wt% for both sh gelatins. However, the presence of a small fraction of relatively large droplets (O10 mm) was observed in the emulsions, even at the relatively high protein concentrations. This fact could be explained by the relatively low surface activity (KZ5 14 wt%K1) of sh gelatins compared with b-lactoglobulin (KZ250 wt%K1), a representative of good protein emulsier. LMWFG emulsions contained more large droplets and exhibited more oil destabilization than HMWFG emulsions, even though laser diffraction indicated that the LMWFG emulsions had a narrower particle size distribution and smaller mean particle size. This effect was attributed to problems associated with representative sampling of the emulsions in the laser diffraction instrument. Fish gelatin emulsions were moderately stable to creaming, with the majority of particles being evenly distributed throughout the volume of the sample and only a small fraction (of presumably large droplets) creaming. LMWFG emulsions had a better creaming stability than HMWFG emulsions, even though the continuous phase viscosity was less, which was attributed to a depletion occulation effect. The emulsions remained fairly stable when they were subjected to thermal treatments (30 and 90 8C for 30 min), high salt concentrations (250 mM NaCl) and various pH values (3 to 8). This study has provided valuable information about the potential application of sh gelatins as emulsiers in food products.

Acknowledgements This material is based upon work supported by the Cooperative State Research, Extension, Education Service, United State Department of Agriculture, Massachusetts Agricultural Experiment Station (project No. 831), by an United States Department of Agriculture, CREES, IFAFS Grant (Award Number 2001-4526) and an United States Department of Agriculture, CREES, NRI Grant (Award Number 2002-01655). This work was supported by the Korea Research Foundation Grant funded by Korea Government (MOEHRD, Basic Research Promotion Fund) (KRF-2004-214-M01-2004-000-20018-0). We greatly thank Norland Products Inc. and Davisco Food International for donating sh gelatins and b-lactoglobulin used in this study, respectively.

4. Conclusions The objective of this study was to examine the inuence of sh gelatin molecular weight and concentration on the formation and stability of emulsions. Emulsions with monomodal particle size distributions and small mean droplet diameters ( d43z 0.35 mm for LMWFG and 0.71 mm for HMWFG) could be produced at protein

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