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Phase separation in soft-serve ice cream mixes: rheology and microstructure
ARTICLE IN PRESS
International Dairy Journal 15 (2005) 249–254 www.elsevier.com/locate/idairyj
Phase separation in soft-serve ice cream mixes: rheology and microstructure C. Vega, H.D. Goff Department of Food Science, University of Guelph, Gordon Street, Guelph, ON, N1G 2W1, Canada Received 30 January 2004; accepted 12 July 2004
Abstract Soft-serve ice cream mixes containing 4% milk protein and 0.14% locust bean gum (LBG) showed extensive visual phaseseparation after 21 days at 5 1C, unless k-carrageenan was added to the formulation. Addition of k-carrageenan (0–0.02%) did not inhibit microscopic phase separation between protein and polysaccharide. A higher degree of ‘‘emulsification’’ of the proteinenriched phase into the continuous serum phase was observed as the k-carrageenan concentration increased, which correlated with inhibition of macroscopic serum separation in the mix. After macroscopic separation, the serum phase of mixes with different kcarrageenan concentrations showed similar microstructural and rheological characteristics, implying no concentration of LBG and no presence of k-carrageenan in the serum phase. The rheological behaviour of the protein-enriched phases indicated k-carrageenan/ casein interactions. There was no evidence of ‘‘weak-gel’’ formation through the addition of k-carrageenan as a means for macroscopic stability in the system. r 2004 Elsevier Ltd. All rights reserved. Keywords: Soft-serve ice cream; k-Carrageenan; Locust bean gum; Phase separation; Confocal scanning laser microscopy
1. Introduction Milk proteins are found in ice cream formulations as part of the milk solids-not-fat component; the protein content of a mix is usually about 4% and is comprised of about 70–80% casein and 20–30% whey protein. Proteins contribute three very important functional roles to the development of structure in ice cream. They emulsify the fat and, through interaction with emulsifier at the fat interface, contribute to partial coalescence and fat structure formation. They adsorb at the air interface, leading to enhanced aeration and foam stability. The proteins not present at interfaces contribute to viscosity enhancement, and freeze-concentration of the proteins contributes to ice recrystallization inhibition and enhanced structural and textural properties (Goff, 1997; Walstra & Jonkman, 1998). Polysaccharide stabilizers, Corresponding author. Tel.: +1-519-824-4120x53878; fax: +1519-824-6631. E-mail address: [email protected] (H.D. Goff).
0958-6946/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2004.07.007
e.g., locust bean gum (LBG) and/or guar gum, are also used in frozen dessert formulations for desired structural and textural properties, notably viscosity enhancement and ice recrystallization inhibition. However, proteins and polysaccharides are generally incompatible in solution, leading to phase separation (Schorsch, Jones, & Norton, 1999; Bourriot, Garnier & Doublier, 1999; Thaiudom & Goff, 2003). This phenomenon is particularly apparent and problematic in soft-serve ice cream mixes during quiescent storage of up to 3 weeks at 5 1C (Vega, Andrew, & Goff, 2004). Phase separation should be understood, in the context herein, as the generation of two discernible and immiscible liquid phases, one of them being opaque and containing most of the colloidal protein and fat components of the original emulsion and the other being transparent or translucent and containing dissolved solutes but depleted in protein and fat. Whether the transparent phase sediments, creams or flocculates and appears mottled will depend on phase densities and overall mix formulation.
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The typical preventive action to avoid this serum separation is the inclusion of a second stabilizer in the formulation, k-carrageenan. The means by which kcarrageenan achieves stabilization in dairy systems containing polysaccharides are still inconclusive, although the phenomena have been extensively studied (Hermansson & Lundin, 1997; Schorsch et al., 1999; Schorsch, Jones, & Norton, 2000; Rodd, Davis, Dunstan, Forrest, & Boger, 2000; Thaiudom & Goff, 2003). Supportive data exist for the electrostatic interaction between k-casein and k-carrageenan, which would lead to complex formation (Snoeren, Payens, Jeunink, & Both, 1975; Snoeren, Both, & Schmidt, 1976; Dalgleish & Morris, 1988), but this interaction in the case of micellar casein has been questioned due to both electrostatic and steric considerations. Conversely, the formation of a weak k-carrageenan gel may entrap casein micelles, thus holding them in suspension (Bourriot et al., 1999). Recently, we studied the effect of k-carrageenan and LBG concentrations and casein-whey protein ratio on the stability of soft-serve ice cream mixes to phase separation during quiescent storage (Vega et al., 2004). We concluded that the minimum amount of k-carrageenan to stabilize the mix against serum separation fluctuated between 0.015% and 0.02% (w/w) regardless of LBG concentration in the range of 0.06–0.2% (w/w). Reduction of casein–whey protein ratio at constant protein concentration increased mix stability. Mix viscosity was not found to be predictive of mix stability since unstable and stable mixes showed similar apparent viscosities. The objective of the research presented here was to extend our previous work with soft-serve ice cream mixes by characterizing microstructure and rheology of the nonseparated mixes and separated phases in the absence and presence of k-carrageenan.
2. Materials and methods Ingredients, mix manufacture and storage procedures were similar to those used by Vega et al. (2004). Briefly, emulsions were prepared to meet the following final softserve ice cream mix composition: 4% milk fat (from anhydrous milkfat), 13% sucrose, 13% milk solids-notfat (4% protein, 70:30 ratio of casein:whey protein, from skim milk powder and whey powder), 0.3% emulsifier (80% mono- and diglycerides, 20% polysorbate 80), 0.14% LBG and 0%, 0.0125%, 0.015% or 0.02% k-II carrageenan from Gigartina radula (all in w/w). Mix emulsions were blended at 30–35 1C, heated to 74 1C (with no holding time) and homogenized using a 2-stage single piston homogenizer (APV Gaulin, Everett, MA). After homogenization, mixes were cooled to 5 1C, poured into standard 100 mL plastic bags and held for 21 days at 5 1C. Three replicates of each treatment were
prepared. Protein analyses of the emulsion before phase separation and of the protein-enriched and the serum phases after phase separation were conducted by freezedrying samples and performing Dumas-based nitrogen analyses (Leco FP528, St. Joseph, MI) on the dry material. 2.1. Rheology Apparent viscosity and mechanical spectra of the separated phases were determined at 5 1C using a Haake Rheostress 1 Rheometer (Thermo-Haake, Karlsruhe, Germany) with titanium cone and plate geometry (60 mm diameter, 1 deg). For apparent viscosity measurements, a stress of 0.01–2 Pa was applied in log steps and data were collected at each stress after the instrument reached steady state (when 3 consecutives readings at 10 s intervals did not differ more than 5% from each other). Oscillatory measurements to generate the mechanical spectra consisted of a frequency sweep from 0.01 to 10 Hz at 5 1C with an oscillatory stress of 0.01 Pa (within the linear viscoelastic region). All measurements of each replicate were made in triplicate. Data were analyzed using protected least significant difference techniques (Microsoft Excel). 2.2. Confocal scanning laser microscopy (CSLM) Two-dimensional CSLM was used to observe the microstructure of the mixes (before and after separation) using a Biorad MRC 600 microscope (Hertfordshire, UK). Three drops of a Rhodamine B concentrated solution were added to 5 mL sample to stain the protein phase. Observations were made directly after emulsion manufacture and at different intervals during storage at 5 1C. Samples were placed on a microscope slide and covered with a glass coverslip. Three separate observations were made of each replicate. Image analysis of the phases was conducted by differentiating white and black areas of the image (Adobe Photoshop software).
3. Results and discussion 3.1. Non separated mixes Immediately after manufacture, before any visual separation had occurred, emulsions with no k-carrageenan showed (Fig. 1A) two well-differentiated domains that were protein-enriched (which appear white due to fluorescence from rhodamine staining of the protein) and protein-depleted (which appear black). The structures could be described as water-in-water emulsions or bicontinuous networks. As interfacial tension between these two aqueous domains is very low, discrete domains are typically not globular. This image resembles those
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Fig. 1. Soft-serve ice cream mixes after preparation containing 0 (A); 0.0125 (B); 0.015 (C); or 0.02% (D) k-carrageenan, as viewed by confocal scanning laser microscopy. White areas are the protein-enriched domains; dark areas are protein-depleted. Bar=40 mm.
generated by other authors in systems comprised of LBG or guar gum and micellar casein (Schorsch et al., 1999; Bourriot et al., 1999). As k-carrageenan concentration increased, the size of the individual protein domains decreased (Figs. 1B–D). Image analysis showed that the protein-depleted zone (serum phase) had the same image surface area regardless of the k-carrageenan content. These images show that k-carrageenan does not inhibit microscopic phase separation, as also shown by Thaiudom and Goff (2003), but rather emulsifies or disperses the protein-enriched domains into smaller droplets. The action of k-carrageenan could be described also as making it increasingly more difficult for the protein-enriched phase to aggregate or coalesce into distinct droplets and consequently to cream or sediment leading to macroscopic phase separation. 3.2. Serum phase characterization The serum phases from mixes with varying kcarrageenan concentrations were collected after 21 days quiescent storage at 5 1C. The serum phase volumes were different: 0.77, 0.65, 0.24 and 0, for 0%, 0.0125%, 0.015% and 0.02% k-carrageenan, respectively (Vega et al., 2004). Protein analysis of the separated phases suggested that both casein and whey protein were depleted from the serum phase (e.g., with no kcarrageenan, 13% of the total casein and 35% of the total whey protein were found in the 77% volume). All serum phase fractions showed identical CSLM images, with essentially no discernible structure and no staining (not shown). The apparent viscosities of the serum
Fig. 2. Apparent viscosity of the serum phase (filled circles) and the protein-enriched phase (open triangles) separated after 21 days from mixes containing different k-carrageenan concentrations, measured at 1 s1.
phases at 1 s1 were not significantly different (Fig. 2). They were also not significantly different from a solution containing 0.14% LBG. This implied that kcarrageenan was not present in the serum phase in sufficient concentrations to have an impact on its viscosity. It also implied that LBG was not being concentrated in the serum phase (hence, not depleted from the protein-enriched phase), as we expected. The mechanical spectra of the serum phases from emulsions made with 0%, 0.0125% and 0.015%
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Fig. 3. Mechanical spectra for serum phases from mixes containing different k-carrageenan concentrations (0%, circle; 0.0125%, triangle; 0.015%, square). G0 , open symbols; G00 , filled symbols.
k-carrageenan show that all three fractions have practically the same viscoelastic character: they all were dilute solutions (Fig. 3). Although there were slight differences in storage moduli, the loss moduli were identical for all three solutions. Thus, two different rheological protocols failed to demonstrate concentration of LBG in the serum phase. We expected phase separation to result in proeinenriched and LBG-enriched phases. Schorsch et al. (1999) presented a phase diagram for LBG and milk protein (micellar casein) showing a tie-line that depicts the system studied here (0.14% LBG/2.8% casein). Their phase diagram predicts the concentration of casein in the protein-enriched phase at 11.2%. In our phaseseparated system, the concentration of casein was 11.2%. The other extreme of the tie-line on their phase diagram predicts no concentration of LBG in the expelled phase, due to the particular shape of that tieline. This also corresponds to our rheological observations. Thus, these systems exhibited protein-enriched and protein-depleted phases, but not an LBG-enriched phase.
was observed for the sample with 0.0125% k-carrageenan (850 mPa s), despite a higher phase volume (i.e., dilution with serum). This was followed by a slight but significant decrease for the sample with 0.015% kcarrageenan (620 mPa s) and finally a non-significant decrease for the fraction containing 0.02% k-carrageenan (560 mPa s) (Fig. 2). The steep increase in viscosity from 0 to 0.0125% k-carrageenan must result from the presence of k-carrageenan in the protein-enriched phase. Subsequent viscosity decreases with increasing k-carrageenan concentration are due to the dilution effect of entrapped serum, which offset the increase expected due to the higher k-carrageenan concentration. The mechanical spectrum of the protein-enriched phase from the sample with no k-carrageenan showed behaviour characteristic of a dilute solution, with G00 higher than G0 along the whole frequency range (Fig. 4). Previous work by Bourriot et al. (1999) showed that the rheological profile of the protein-enriched phase after separation from a system comprised of micellar casein and guar gum was different than a suspension that was prepared to have the same composition as the separated phase. The protein-enriched phase was more viscous and showed a higher degree of thixotropy. They attributed this to a flocculated or aggregated state of the protein after phase separation. Similarly, Dickinson and Golding (1997a,b) showed that sodium caseinate-enriched separated phases have high shear thinning behaviour and hence concluded that they were highly flocculated. However, our results did not indicate rheological behaviour typical of aggregated protein. Addition of k-carrageenan modified the mechanical spectra of the protein-enriched phases (Fig. 4). Systems
3.3. Protein phase characterization Protein distribution results showed that 82.5% of the total protein (87% of the casein and 65% of the whey protein) resided in the protein-enriched phase after separation, e.g., in 23% of the volume in the absence of k-carrageenan. The protein-enriched fractions showed large differences in apparent viscosity due to the presence of k-carrageenan in the original system, unlike the serum phase fractions. Samples from emulsions with 0% k-carrageenan showed the lowest apparent viscosity (190 mPa s). A steep and significant increase in viscosity
Fig. 4. Mechanical spectra for protein-enriched phases from mixes containing different k-carrageenan concentrations (0%, circle; 0.0125%, triangle; 0.015%, square; 0.02%, diamond). G0 , open symbols; G00 , filled symbols.
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Fig. 5. Protein-enriched phases of soft-serve ice cream mixes after 21 days storage containing 0% (A); 0.0125% (B); 0.015% (C); or 0.02% (D) kcarrageenan, as viewed by confocal scanning laser microscopy. White areas are the protein-enriched domains; dark areas are protein-depleted. Bar=40 mm.
containing 0.0125% k-carrageenan exhibited ‘‘weakgel’’-like behaviour, with G0 higher than G00 over the lower frequency range; frequency dependence; more liquid-like behaviour at the higher frequency range; and a cross-over near 10 Hz (Ross-Murphy, 1995; Rodd et al., 2000). When k-carrageenan was added at 0.015%, a slightly stronger structure appeared. The mechanical spectrum also indicated weak-gel behaviour, with G0 higher than that for the 0.0125% k-carrageenan system. Interestingly, the mechanical spectrum for the 0.02% kcarrageenan sample (which showed macroscopic stability) behaved as a dilute solution, showing a mechanical character almost identical to the 0% k-carrageenan sample. Both dynamic testing and apparent viscosity coincide in their characterization of the protein-enriched phase: 0% k-carrageenan samples showed less structured behaviour while the protein-enriched phase from the 0.0125% k-carrageenan sample showed a large viscosity increment and an increase in mechanical strength. At a level of 0.015% k-carrageenan, there was a slight discrepancy; apparent viscosity decreased while the mechanical spectrum showed a slightly more structured fraction. Finally, the presence of 0.02% k-carrageenan further decreased viscosity of the mix and caused it to behave as a dilute solution again. This rheological behaviour appears to be related to phase volume and microstructure. The protein phase volumes were 0.23, 0.35, 0.76 and 1.0, for 0%, 0.0125%, 0.015%, and 0.02% k-carrageenan, respectively (Vega et al., 2004). The microstructure of the protein-enriched phases showed an increase in
entrapped serum as the concentration of k-carrageenan increased (Fig. 5). In the absence of k-carrageenan (Fig. 5A), the higher concentration of protein in the proteinenriched phase compared to the mix before separation (Fig. 1A) led to a phase inversion, where the proteinenriched phase became continuous. Some serum was still detected. When the microstructure of the proteinenriched phase in the presence of k-carrageenan (0.0125%) was examined, a different structure was distinguished (Fig. 5B). There was more emulsified serum phase, similar to the results before separation (Fig. 1B) although again inverted. Further increases in k-carrageenan (Figs. 5C and D) led to more serum entrapped within the protein structure thus diluting the protein phase, which was directly reflected in the latter’s rheological character. Fig. 5D was similar in appearance to Fig. 1D, showing little change in the emulsified nature of the protein-enriched phase after 21 days of storage with the highest levels of k-carrageenan, when no macroscopic separation had occurred.
4. Conclusions Before macroscopic phase separation occurred, microscopy demonstrated that casein micelles and LBG in these systems were incompatible, leading to proteinenriched and protein-depleted domains in a water-inwater emulsion or bicontinuous network structure. Increasing concentrations of k-carrageenan led to smaller protein-enriched domains, suggesting a form of emulsification induced by the k-carrageenan. However,
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the systems with the highest concentration of kcarrageenan were still phase-separated. Analyses of the rheological properties of the serum and the proteinenriched phases after macroscopic separation suggested that k-carrageenan resided in the protein-enriched phase and was not evident in the protein-depleted phase. Rheological analyses also showed that LBG was not concentrated in the protein-depleted phase. The proteinenriched phases in the presence of low concentrations of k-carrageenan (hence, at the low phase volumes) could be described as weak gels, based on their mechanical spectra. However, with increasing concentration of kcarrageenan, the incorporation of more serum into the protein-enriched phases led to dilute solution behaviour, even though these systems were macroscopically stable. Thus, the formation of a weak gel does not seem to be the mechanism behind stabilization. It appeared that the protein-enriched domains were inhibited from coalescence and droplet growth by the k-carrageenan. Thus, kcarrageenan functionality in preventing or inhibiting macroscopic phase separation between casein and galactomannans in soft-serve ice cream mix resulted from direct interaction with casein micelles, thereby forming protein domains that cannot easily separate from the serum phase.
Acknowledgements The authors acknowledge financial support from Danisco Canada Inc. and the Natural Sciences and Engineering Research Council of Canada (NSERC). They are also thankful for the support from Unilever Research and Development Colworth in providing facilities to progress this work, especially to D. Ferdinando for assistance in the CSLM imaging and Drs. T. Foster and L. Lundin for their enlightening discussions.
References Bourriot, S., Garnier, C., & Doublier, J. L. (1999). Micellar casein-kcarrageenan mixtures I. Phase separation and ultrastructure. Carbohydrate Polymers, 40, 145–157. Dalgleish, D. G., & Morris, E. R. (1988). Interactions between carrageenans and casein micelles: electrophoretic and hydrodynamic properties of the particles. Food Hydrocolloids, 2, 311–320. Dickinson, E., & Golding, M. (1997a). Depletion flocculation of emulsions containing unadsorbed sodium caseinate. Food Hydrocolloids, 11, 13–18. Dickinson, E., & Golding, M. (1997b). Rheology of sodium caseinate stabilized oil-in-water emulsions. Journal of Colloid and Interface Science, 191, 166–176. Goff, H. D. (1997). Instability and partial coalescence in whippable dairy emulsions. Journal of Dairy Science, 80, 2620–2630. Hermansson, A. M., & Lundin, L. (1997). Multivariate analysis of the influences of locust bean gum, as-casein, k-casein on viscoelastic properties of Na–k-carrageenan gels. Food Hydrocolloids, 12, 175–187. Rodd, A. B., Davis, C. R., Dunstan, D. E., Forrest, B. A., & Boger, D. V. (2000). Rheological characterization of ‘weak gel’ carrageenan stabilized milks. Food Hydrocolloids, 14, 445–454. Ross-Murphy, S. B. (1995). Structure-property relationships in food biopolymer gels and solutions. Journal of Rheology, 39, 1451–1463. Schorsch, C., Jones, M., & Norton, I. T. (1999). Thermodynamic incompatibility and microstructure of milk protein/locust bean gum/sucrose systems. Food Hydrocolloids, 13, 89–99. Schorsch, C., Jones, M., & Norton, I. T. (2000). Phase behaviour of pure micellar casein/k-carrageenan systems in milk salt ultrafiltrate. Food Hydrocolloids, 14, 347–358. Snoeren, T. H. M., Both, P., & Schmidt, D. G. (1976). An electronmicroscopic study of carrageenan and its interaction with k-casein. Netherlands Milk Dairy Journal, 30, 132–141. Snoeren, T. H. M., Payens, T. A. J., Jeunink, J., & Both, P. (1975). Electrostatic interaction between k-carrageenan and k-casein. Milchwissenschaft, 30, 393–396. Thaiudom, S., & Goff, H. D. (2003). Effect of k-carrageenan on milk protein polysaccharide mixtures. International Dairy Journal, 13, 763–771. Vega, C., Andrew, R. A., & Goff, H. D. (2004). Serum separation in soft-serve ice cream mixes. Milchwissenchaft, 59, 284–287. Walstra, P., & Jonkman, M. (1998). The role of milkfat and protein in ice cream. In W. Buchheim (Ed.), Ice cream Special Issue (pp. 17–24). Brussels: International Dairy Federation.
International Dairy Journal 15 (2005) 249–254 www.elsevier.com/locate/idairyj
Phase separation in soft-serve ice cream mixes: rheology and microstructure C. Vega, H.D. Goff Department of Food Science, University of Guelph, Gordon Street, Guelph, ON, N1G 2W1, Canada Received 30 January 2004; accepted 12 July 2004
Abstract Soft-serve ice cream mixes containing 4% milk protein and 0.14% locust bean gum (LBG) showed extensive visual phaseseparation after 21 days at 5 1C, unless k-carrageenan was added to the formulation. Addition of k-carrageenan (0–0.02%) did not inhibit microscopic phase separation between protein and polysaccharide. A higher degree of ‘‘emulsification’’ of the proteinenriched phase into the continuous serum phase was observed as the k-carrageenan concentration increased, which correlated with inhibition of macroscopic serum separation in the mix. After macroscopic separation, the serum phase of mixes with different kcarrageenan concentrations showed similar microstructural and rheological characteristics, implying no concentration of LBG and no presence of k-carrageenan in the serum phase. The rheological behaviour of the protein-enriched phases indicated k-carrageenan/ casein interactions. There was no evidence of ‘‘weak-gel’’ formation through the addition of k-carrageenan as a means for macroscopic stability in the system. r 2004 Elsevier Ltd. All rights reserved. Keywords: Soft-serve ice cream; k-Carrageenan; Locust bean gum; Phase separation; Confocal scanning laser microscopy
1. Introduction Milk proteins are found in ice cream formulations as part of the milk solids-not-fat component; the protein content of a mix is usually about 4% and is comprised of about 70–80% casein and 20–30% whey protein. Proteins contribute three very important functional roles to the development of structure in ice cream. They emulsify the fat and, through interaction with emulsifier at the fat interface, contribute to partial coalescence and fat structure formation. They adsorb at the air interface, leading to enhanced aeration and foam stability. The proteins not present at interfaces contribute to viscosity enhancement, and freeze-concentration of the proteins contributes to ice recrystallization inhibition and enhanced structural and textural properties (Goff, 1997; Walstra & Jonkman, 1998). Polysaccharide stabilizers, Corresponding author. Tel.: +1-519-824-4120x53878; fax: +1519-824-6631. E-mail address: [email protected] (H.D. Goff).
0958-6946/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2004.07.007
e.g., locust bean gum (LBG) and/or guar gum, are also used in frozen dessert formulations for desired structural and textural properties, notably viscosity enhancement and ice recrystallization inhibition. However, proteins and polysaccharides are generally incompatible in solution, leading to phase separation (Schorsch, Jones, & Norton, 1999; Bourriot, Garnier & Doublier, 1999; Thaiudom & Goff, 2003). This phenomenon is particularly apparent and problematic in soft-serve ice cream mixes during quiescent storage of up to 3 weeks at 5 1C (Vega, Andrew, & Goff, 2004). Phase separation should be understood, in the context herein, as the generation of two discernible and immiscible liquid phases, one of them being opaque and containing most of the colloidal protein and fat components of the original emulsion and the other being transparent or translucent and containing dissolved solutes but depleted in protein and fat. Whether the transparent phase sediments, creams or flocculates and appears mottled will depend on phase densities and overall mix formulation.
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The typical preventive action to avoid this serum separation is the inclusion of a second stabilizer in the formulation, k-carrageenan. The means by which kcarrageenan achieves stabilization in dairy systems containing polysaccharides are still inconclusive, although the phenomena have been extensively studied (Hermansson & Lundin, 1997; Schorsch et al., 1999; Schorsch, Jones, & Norton, 2000; Rodd, Davis, Dunstan, Forrest, & Boger, 2000; Thaiudom & Goff, 2003). Supportive data exist for the electrostatic interaction between k-casein and k-carrageenan, which would lead to complex formation (Snoeren, Payens, Jeunink, & Both, 1975; Snoeren, Both, & Schmidt, 1976; Dalgleish & Morris, 1988), but this interaction in the case of micellar casein has been questioned due to both electrostatic and steric considerations. Conversely, the formation of a weak k-carrageenan gel may entrap casein micelles, thus holding them in suspension (Bourriot et al., 1999). Recently, we studied the effect of k-carrageenan and LBG concentrations and casein-whey protein ratio on the stability of soft-serve ice cream mixes to phase separation during quiescent storage (Vega et al., 2004). We concluded that the minimum amount of k-carrageenan to stabilize the mix against serum separation fluctuated between 0.015% and 0.02% (w/w) regardless of LBG concentration in the range of 0.06–0.2% (w/w). Reduction of casein–whey protein ratio at constant protein concentration increased mix stability. Mix viscosity was not found to be predictive of mix stability since unstable and stable mixes showed similar apparent viscosities. The objective of the research presented here was to extend our previous work with soft-serve ice cream mixes by characterizing microstructure and rheology of the nonseparated mixes and separated phases in the absence and presence of k-carrageenan.
2. Materials and methods Ingredients, mix manufacture and storage procedures were similar to those used by Vega et al. (2004). Briefly, emulsions were prepared to meet the following final softserve ice cream mix composition: 4% milk fat (from anhydrous milkfat), 13% sucrose, 13% milk solids-notfat (4% protein, 70:30 ratio of casein:whey protein, from skim milk powder and whey powder), 0.3% emulsifier (80% mono- and diglycerides, 20% polysorbate 80), 0.14% LBG and 0%, 0.0125%, 0.015% or 0.02% k-II carrageenan from Gigartina radula (all in w/w). Mix emulsions were blended at 30–35 1C, heated to 74 1C (with no holding time) and homogenized using a 2-stage single piston homogenizer (APV Gaulin, Everett, MA). After homogenization, mixes were cooled to 5 1C, poured into standard 100 mL plastic bags and held for 21 days at 5 1C. Three replicates of each treatment were
prepared. Protein analyses of the emulsion before phase separation and of the protein-enriched and the serum phases after phase separation were conducted by freezedrying samples and performing Dumas-based nitrogen analyses (Leco FP528, St. Joseph, MI) on the dry material. 2.1. Rheology Apparent viscosity and mechanical spectra of the separated phases were determined at 5 1C using a Haake Rheostress 1 Rheometer (Thermo-Haake, Karlsruhe, Germany) with titanium cone and plate geometry (60 mm diameter, 1 deg). For apparent viscosity measurements, a stress of 0.01–2 Pa was applied in log steps and data were collected at each stress after the instrument reached steady state (when 3 consecutives readings at 10 s intervals did not differ more than 5% from each other). Oscillatory measurements to generate the mechanical spectra consisted of a frequency sweep from 0.01 to 10 Hz at 5 1C with an oscillatory stress of 0.01 Pa (within the linear viscoelastic region). All measurements of each replicate were made in triplicate. Data were analyzed using protected least significant difference techniques (Microsoft Excel). 2.2. Confocal scanning laser microscopy (CSLM) Two-dimensional CSLM was used to observe the microstructure of the mixes (before and after separation) using a Biorad MRC 600 microscope (Hertfordshire, UK). Three drops of a Rhodamine B concentrated solution were added to 5 mL sample to stain the protein phase. Observations were made directly after emulsion manufacture and at different intervals during storage at 5 1C. Samples were placed on a microscope slide and covered with a glass coverslip. Three separate observations were made of each replicate. Image analysis of the phases was conducted by differentiating white and black areas of the image (Adobe Photoshop software).
3. Results and discussion 3.1. Non separated mixes Immediately after manufacture, before any visual separation had occurred, emulsions with no k-carrageenan showed (Fig. 1A) two well-differentiated domains that were protein-enriched (which appear white due to fluorescence from rhodamine staining of the protein) and protein-depleted (which appear black). The structures could be described as water-in-water emulsions or bicontinuous networks. As interfacial tension between these two aqueous domains is very low, discrete domains are typically not globular. This image resembles those
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Fig. 1. Soft-serve ice cream mixes after preparation containing 0 (A); 0.0125 (B); 0.015 (C); or 0.02% (D) k-carrageenan, as viewed by confocal scanning laser microscopy. White areas are the protein-enriched domains; dark areas are protein-depleted. Bar=40 mm.
generated by other authors in systems comprised of LBG or guar gum and micellar casein (Schorsch et al., 1999; Bourriot et al., 1999). As k-carrageenan concentration increased, the size of the individual protein domains decreased (Figs. 1B–D). Image analysis showed that the protein-depleted zone (serum phase) had the same image surface area regardless of the k-carrageenan content. These images show that k-carrageenan does not inhibit microscopic phase separation, as also shown by Thaiudom and Goff (2003), but rather emulsifies or disperses the protein-enriched domains into smaller droplets. The action of k-carrageenan could be described also as making it increasingly more difficult for the protein-enriched phase to aggregate or coalesce into distinct droplets and consequently to cream or sediment leading to macroscopic phase separation. 3.2. Serum phase characterization The serum phases from mixes with varying kcarrageenan concentrations were collected after 21 days quiescent storage at 5 1C. The serum phase volumes were different: 0.77, 0.65, 0.24 and 0, for 0%, 0.0125%, 0.015% and 0.02% k-carrageenan, respectively (Vega et al., 2004). Protein analysis of the separated phases suggested that both casein and whey protein were depleted from the serum phase (e.g., with no kcarrageenan, 13% of the total casein and 35% of the total whey protein were found in the 77% volume). All serum phase fractions showed identical CSLM images, with essentially no discernible structure and no staining (not shown). The apparent viscosities of the serum
Fig. 2. Apparent viscosity of the serum phase (filled circles) and the protein-enriched phase (open triangles) separated after 21 days from mixes containing different k-carrageenan concentrations, measured at 1 s1.
phases at 1 s1 were not significantly different (Fig. 2). They were also not significantly different from a solution containing 0.14% LBG. This implied that kcarrageenan was not present in the serum phase in sufficient concentrations to have an impact on its viscosity. It also implied that LBG was not being concentrated in the serum phase (hence, not depleted from the protein-enriched phase), as we expected. The mechanical spectra of the serum phases from emulsions made with 0%, 0.0125% and 0.015%
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Fig. 3. Mechanical spectra for serum phases from mixes containing different k-carrageenan concentrations (0%, circle; 0.0125%, triangle; 0.015%, square). G0 , open symbols; G00 , filled symbols.
k-carrageenan show that all three fractions have practically the same viscoelastic character: they all were dilute solutions (Fig. 3). Although there were slight differences in storage moduli, the loss moduli were identical for all three solutions. Thus, two different rheological protocols failed to demonstrate concentration of LBG in the serum phase. We expected phase separation to result in proeinenriched and LBG-enriched phases. Schorsch et al. (1999) presented a phase diagram for LBG and milk protein (micellar casein) showing a tie-line that depicts the system studied here (0.14% LBG/2.8% casein). Their phase diagram predicts the concentration of casein in the protein-enriched phase at 11.2%. In our phaseseparated system, the concentration of casein was 11.2%. The other extreme of the tie-line on their phase diagram predicts no concentration of LBG in the expelled phase, due to the particular shape of that tieline. This also corresponds to our rheological observations. Thus, these systems exhibited protein-enriched and protein-depleted phases, but not an LBG-enriched phase.
was observed for the sample with 0.0125% k-carrageenan (850 mPa s), despite a higher phase volume (i.e., dilution with serum). This was followed by a slight but significant decrease for the sample with 0.015% kcarrageenan (620 mPa s) and finally a non-significant decrease for the fraction containing 0.02% k-carrageenan (560 mPa s) (Fig. 2). The steep increase in viscosity from 0 to 0.0125% k-carrageenan must result from the presence of k-carrageenan in the protein-enriched phase. Subsequent viscosity decreases with increasing k-carrageenan concentration are due to the dilution effect of entrapped serum, which offset the increase expected due to the higher k-carrageenan concentration. The mechanical spectrum of the protein-enriched phase from the sample with no k-carrageenan showed behaviour characteristic of a dilute solution, with G00 higher than G0 along the whole frequency range (Fig. 4). Previous work by Bourriot et al. (1999) showed that the rheological profile of the protein-enriched phase after separation from a system comprised of micellar casein and guar gum was different than a suspension that was prepared to have the same composition as the separated phase. The protein-enriched phase was more viscous and showed a higher degree of thixotropy. They attributed this to a flocculated or aggregated state of the protein after phase separation. Similarly, Dickinson and Golding (1997a,b) showed that sodium caseinate-enriched separated phases have high shear thinning behaviour and hence concluded that they were highly flocculated. However, our results did not indicate rheological behaviour typical of aggregated protein. Addition of k-carrageenan modified the mechanical spectra of the protein-enriched phases (Fig. 4). Systems
3.3. Protein phase characterization Protein distribution results showed that 82.5% of the total protein (87% of the casein and 65% of the whey protein) resided in the protein-enriched phase after separation, e.g., in 23% of the volume in the absence of k-carrageenan. The protein-enriched fractions showed large differences in apparent viscosity due to the presence of k-carrageenan in the original system, unlike the serum phase fractions. Samples from emulsions with 0% k-carrageenan showed the lowest apparent viscosity (190 mPa s). A steep and significant increase in viscosity
Fig. 4. Mechanical spectra for protein-enriched phases from mixes containing different k-carrageenan concentrations (0%, circle; 0.0125%, triangle; 0.015%, square; 0.02%, diamond). G0 , open symbols; G00 , filled symbols.
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(A)
(B)
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Fig. 5. Protein-enriched phases of soft-serve ice cream mixes after 21 days storage containing 0% (A); 0.0125% (B); 0.015% (C); or 0.02% (D) kcarrageenan, as viewed by confocal scanning laser microscopy. White areas are the protein-enriched domains; dark areas are protein-depleted. Bar=40 mm.
containing 0.0125% k-carrageenan exhibited ‘‘weakgel’’-like behaviour, with G0 higher than G00 over the lower frequency range; frequency dependence; more liquid-like behaviour at the higher frequency range; and a cross-over near 10 Hz (Ross-Murphy, 1995; Rodd et al., 2000). When k-carrageenan was added at 0.015%, a slightly stronger structure appeared. The mechanical spectrum also indicated weak-gel behaviour, with G0 higher than that for the 0.0125% k-carrageenan system. Interestingly, the mechanical spectrum for the 0.02% kcarrageenan sample (which showed macroscopic stability) behaved as a dilute solution, showing a mechanical character almost identical to the 0% k-carrageenan sample. Both dynamic testing and apparent viscosity coincide in their characterization of the protein-enriched phase: 0% k-carrageenan samples showed less structured behaviour while the protein-enriched phase from the 0.0125% k-carrageenan sample showed a large viscosity increment and an increase in mechanical strength. At a level of 0.015% k-carrageenan, there was a slight discrepancy; apparent viscosity decreased while the mechanical spectrum showed a slightly more structured fraction. Finally, the presence of 0.02% k-carrageenan further decreased viscosity of the mix and caused it to behave as a dilute solution again. This rheological behaviour appears to be related to phase volume and microstructure. The protein phase volumes were 0.23, 0.35, 0.76 and 1.0, for 0%, 0.0125%, 0.015%, and 0.02% k-carrageenan, respectively (Vega et al., 2004). The microstructure of the protein-enriched phases showed an increase in
entrapped serum as the concentration of k-carrageenan increased (Fig. 5). In the absence of k-carrageenan (Fig. 5A), the higher concentration of protein in the proteinenriched phase compared to the mix before separation (Fig. 1A) led to a phase inversion, where the proteinenriched phase became continuous. Some serum was still detected. When the microstructure of the proteinenriched phase in the presence of k-carrageenan (0.0125%) was examined, a different structure was distinguished (Fig. 5B). There was more emulsified serum phase, similar to the results before separation (Fig. 1B) although again inverted. Further increases in k-carrageenan (Figs. 5C and D) led to more serum entrapped within the protein structure thus diluting the protein phase, which was directly reflected in the latter’s rheological character. Fig. 5D was similar in appearance to Fig. 1D, showing little change in the emulsified nature of the protein-enriched phase after 21 days of storage with the highest levels of k-carrageenan, when no macroscopic separation had occurred.
4. Conclusions Before macroscopic phase separation occurred, microscopy demonstrated that casein micelles and LBG in these systems were incompatible, leading to proteinenriched and protein-depleted domains in a water-inwater emulsion or bicontinuous network structure. Increasing concentrations of k-carrageenan led to smaller protein-enriched domains, suggesting a form of emulsification induced by the k-carrageenan. However,
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the systems with the highest concentration of kcarrageenan were still phase-separated. Analyses of the rheological properties of the serum and the proteinenriched phases after macroscopic separation suggested that k-carrageenan resided in the protein-enriched phase and was not evident in the protein-depleted phase. Rheological analyses also showed that LBG was not concentrated in the protein-depleted phase. The proteinenriched phases in the presence of low concentrations of k-carrageenan (hence, at the low phase volumes) could be described as weak gels, based on their mechanical spectra. However, with increasing concentration of kcarrageenan, the incorporation of more serum into the protein-enriched phases led to dilute solution behaviour, even though these systems were macroscopically stable. Thus, the formation of a weak gel does not seem to be the mechanism behind stabilization. It appeared that the protein-enriched domains were inhibited from coalescence and droplet growth by the k-carrageenan. Thus, kcarrageenan functionality in preventing or inhibiting macroscopic phase separation between casein and galactomannans in soft-serve ice cream mix resulted from direct interaction with casein micelles, thereby forming protein domains that cannot easily separate from the serum phase.
Acknowledgements The authors acknowledge financial support from Danisco Canada Inc. and the Natural Sciences and Engineering Research Council of Canada (NSERC). They are also thankful for the support from Unilever Research and Development Colworth in providing facilities to progress this work, especially to D. Ferdinando for assistance in the CSLM imaging and Drs. T. Foster and L. Lundin for their enlightening discussions.
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