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Structure and ice recrystallization in frozen stabilized ice cream model systems
Food Hydrocolloids 17 (2003) 95–102 www.elsevier.com/locate/foodhyd
Structure and ice recrystallization in frozen stabilized ice cream model systems Alejandra Regand, H. Douglas Goff* Department of Food Science, University of Guelph, Guelph, Ont., Canada N1G 2W1 Received 15 November 2001; revised 12 March 2002; accepted 12 March 2002
Abstract Hydrocolloid stabilizers (carrageenan, carboxymethyl cellulose, xanthan gum, sodium alginate, locust bean gum (LBG) and gelatin) were labeled with rhodamine isothiocyanate and incorporated into solutions of sucrose with or without milk solids-not-fat (MSNF). Resultant solutions were quench frozen to 2 50 8C and cycled between 2 3.5 and 2 6 8C, five times. The location of the stabilizer was observed using fluorescence microscopy. Significant retardation of recrystallization was observed in alginate and xanthan sucrose solutions without MSNF. In the presence of proteins, all stabilizers were effective retarding recrystallization except for gelatin. After cycling, a gel-like structure was observed in solutions containing LBG without MSNF, and in LBG, carrageenan and gelatin with MSNF. The fact that some non-gelling stabilizers (i.e. xanthan) were more effective retarding recrystallization than gelling stabilizers (i.e. gelatin) suggests that steric blocking of the interface or inhibition of solute transport to and from the ice interface caused by gelation of the polymer is not the only mechanism of stabilizer action. Molecular interactions between polysaccharides and proteins appear to be key factors in retarding ice recrystallization. q 2002 Elsevier Science Ltd. All rights reserved. Keywords: Ice cream; Recrystallization; Stabilizers; Fluorescence microscopy
1. Introduction Hydrocolloid stabilizers in ice cream improve smoothness of body, give uniformity of product, desired resistance to melting and improve handling properties, but the major role is to hinder crystal growth as temperature fluctuates during storage. The mechanisms by which they affect the freezing properties or limit recrystallization have been extensively studied but are as yet not fully understood. Stabilizers have little (Caldwell, Goff, & Stanley, 1992a,b) or no (Sutton & Wilcox, 1998a,b) impact on the initial ice crystal size distribution in ice cream at the time of draw from the scraped surface heat exchanger and also little or no impact on initial ice growth during quiescent freezing and hardening (Flores & Goff, 1999a), but do limit the rate of growth of ice crystals during recrystallization (Caldwell et al., 1992b; Donhowe & Hartel, 1996a,b; Flores & Goff, 1999b; Goff, Ferdinando, & Schorsch, 1999; Hagiwara & Hartel, 1996; Sutton & Wilcox, 1998a,b; Sutton, Cooke, & Russell, 1997a; Sutton, Evans, & Crilly, 1994; Sutton, Lips, * Corresponding author. Tel.: þ1-519-824-4120x3878; fax: þ 1-519824-6631. E-mail address: [email protected] (H.D. Goff).
& Piccirillo, 1996a; Sutton, Lips, Piccirillo, & Sztehlo, 1996b; Sutton, Wilcox, & Bedford, 1997b). Several authors have found that the presence of stabilizers at low concentration (0.5 – 2%) in model solutions and ice cream do not significantly alter their phase/state transition properties: glass transition temperature (Budiaman & Fennema, 1987a,b; Goff, Caldwell, Stanley, & Maurice, 1993; Hagiwara & Hartel, 1996; Sahagian & Goff, 1995), amount of freezable water or enthalpy of melting (Buyong & Fennema, 1988; Muhr & Blanshard, 1986; Sahagian & Goff, 1995), or heterogeneous nucleation (Blond, 1986; Muhr & Blanshard, 1986), and thus may not be expected to affect the initial ice crystallization processes. Many studies have been conducted to correlate enhanced viscosity caused by stabilizers with better control of ice crystal growth (Budiaman & Fennema, 1987a,b; Cottrell, Pass, & Phillips, 1979; Hagiwara & Hartel, 1996; Harper & Shoemaker, 1983; Hartel, 1996; Miller-Livney & Hartel, 1997) but without definitive conclusions. The functionality of a given stabilizer may be enhanced as the polymer concentration is increased, but different stabilizers are not equally effective for retarding ice crystal growth at the same level of concentration or viscosity (Budiaman & Fennema, 1987b). A different approach taken by Bolliger, Wildmoser,
0268-005X/03/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 2 6 8 - 0 0 5 X ( 0 2 ) 0 0 0 4 2 - 5
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Goff, and Tharp, (2000) found a linear relationship (r 2 ¼ 0.97) between a normalized ‘breakpoint’ apparent viscosity (break point apparent viscosity divided by initial mix apparent viscosity) and recrystallization rate. They suggested that some aspects of stabilizer functionality with respect to recrystallization protection could come from structure, as measured by rheological properties, that results from freeze-concentration of the polysaccharide in the unfrozen phase of ice cream. This structure from the stabilizer would affect the rate at which water can diffuse to the surface of a growing crystal during temperature fluctuation or the rate at which solutes and macromolecules can diffuse away from the surface of a growing ice crystal, an elaboration of the mechanism proposed by Caldwell et al. (1992b) and Goff et al. (1993). Experiments done with NMR in which the increase in viscosity due to the stabilizer is not accompanied by a similar decrease in mobility of water molecules (Martin, Abblet, Darke, Sutton, & Sahagian, 1999) seem to be contrary to this mechanism. However, it is important to remember that by an NMR technique the water diffusion or translational displacement of water molecules is being measured at intermolecular distances, usually less than 10 nm, while the water migration from one crystal to another involved in melt-refreeze recrystallization mechanisms implies distances between ice crystals usually longer than 10 mm. Therefore, the question formulated by Miller-Livney and Hartel (1997) still remains as to whether stabilizers in freeze-concentrated low temperature systems have different effects on macroviscosity (bulk fluid mobility) compared with microviscosity (molecular mobility), and whether increased microviscosity retards recrystallization by altering long-scale diffusion of water and sugar molecules. A modification of the ice crystal – serum interface through surface adsorption to the crystal itself has been suggested by Sutton & Wilcox (1998a,b), Sutton et al. (1996a, 1997a,b). This group of researchers has found that as the concentration of locust bean gum (LBG) is increased the recrystallization rate is reduced until it reaches a plateau after which it remains constant. In the case of guar, recrystallization rate decreases also with the increase in polymer concentration, but in contrast to LBG, after it reaches the minimum it remarkably rises with the addition of further polysaccharide. Non-gelling methoxy pectin gave similar concentration dependence to guar. Both were not as effective as LBG. The rise in recrystallization rate was suggested to be caused by a phase separation or incomplete dissolution and therefore lack of homogeneity above a critical stabilizer concentration. Recrystallization has been also related to the capacity of the stabilizer to form cryo-gels or entangled networks during freezing – thawing cycles. Gel firmness has been associated to inhibition of ice crystal growth and to a change in ice crystal morphology (Blond, 1988; Muhr & Blanshard, 1986). Crystallization velocity has been reduced as maturation time of the gel and gel elasticity increased.
This effect has been explained as a mechanical interference with the ice growth (Blond, 1988). However, a firm gel has not always been effective at retarding ice crystal growth, as found by Muhr & Blanshard (1986). They explained this behavior suggesting that a firm gel would be more fragile and be ruptured more easily by the ice front while a more flexible gel would probably exert a stronger opposing force for ice front propagation. It has been reported as well that stabilizers that did not form a gel still have retarded ice crystal growth (Sutton & Wilcox, 1998a). In another attempt to clarify the stabilizer’s mechanism in retarding recrystallization, Goff, et al. (1999) recently modified a method to covalently-bond a fluorescent marker to LBG and guar gum to visualize the location of these polysaccharides in frozen sucrose or sucrose/milk protein solutions after temperature cycling. They found that LBG was capable of forming a gel-like network after freezing, which became more distinct with temperature cycling. No such structure could be seen with guar gum. The rheological properties of these cryo-gels of LBG, with and without milk solids-not-fat (MSNF), and compared to guar gum have recently been studied by Patmore, Goff, and Fernandes (2002). In the work of Goff et al. (1999), image analysis of ice crystals after repeated temperature cycling showed that LBG was more effective at inhibiting recrystallization than was guar gum. The addition of milk proteins to the sucrose solutions led to a decrease in the rate of recrystallization for all samples when compared with the same solutions without protein. They also observed that both LBG and guar gum were responsible for the formation of a separated protein phase in solution. The combination of the formation of an LBG network and the presence of phase-separated protein was more effective at controlling ice recrystallization. Based on these observations, it was suggested that the ability to form cryo-gels, or gel-like intermolecular interactions, and to interact with proteins, perhaps causing localized increases in concentration through phase-separation, are prerequisites for stabilizer effectiveness against ice recrystallization. The present research was intended to be an extension of the earlier work of Goff et al. (1999). The objective was to analyze other commonly used stabilizers: carrageenan, carboxymethyl cellulose, xanthan gum, sodium alginate and gelatin, plus LBG studied earlier, to verify a correlation between their capacity to form cryo-gels and their ability to retard recrystallization when used as stabilizers in ice cream.
2. Materials and methods 2.1. Labeling of stabilizers The following stabilizers were labeled with rhodamine isothiocyanate (RITC) (Sigma Aldrich Canada), according to the method of De Belder and Granath (1973), as modified
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by Goff et al. (1999): LBG, xanthan gum, carrageenan, carboxymethyl cellulose (CMC) (Germantown Canada, Inc.), sodium alginate (Pronova Biopolymer, Inc., Canada) and gelatin (Cangel, Inc., Canada). 2.2. Preparation of solutions The following aqueous solutions were prepared: 24% w/w sucrose (Fisher Scientific, Canada), 24% w/w sucrose þ 0.3% w/w RITC-stabilizer, 16% w/w sucrose þ 14.7% w/w MSNF (from skim milk powder, 36% protein, Gay Lea Foods, Canada), 16% w/w sucrose þ 14.7% w/w MSNF þ 0.27% w/w stabilizer, for a total of 14 solutions. Solute concentrations were chosen to reflect typical stabilizer/sugar ratios or stabilizer/MSNF/sugar ratios in an ice cream mix. All solutions were calculated to have similar freezing curves, due to the effect of lactose and milk salts in the MSNF. The RITC-carrageenan concentration in sucrose and sucrose þ MSNF solutions was reduced to 0.05% w/w in sucrose solutions and 0.045% w/w in sucrose þ MSNF solutions to avoid gelation of the sample before cycling. Solutions were heated to 80 8C for 30 min, while stirring. Evaporated water was added back, and the solutions were cooled and stored at 4 8C overnight. For statistical purposes, the different ice cream solutions were prepared in triplicate. 2.3. Freezing and temperature cycling protocols A small drop of solution was placed between a slide and a cover slip and loaded on a cold stage (Linkam Scientific Instruments, UK) on a BX-60 Olympus light microscope. The cold stage was quench cooled to 2 50 8C and warmed to 2 10 8C at 5 8C/min to allow for irruptive crystallization of the solution. Ice crystals were matured to obtain the same initial crystal size for each sample ( p , 0.05). Different precycling treatments were required for samples with or without MSNF due to lower initial values of ice crystal size in the presence of proteins. The temperature program was started at 2 3.5 8C, samples were held for 10 min (t ¼ 0), cooled at 1 8C/min to 2 6 8C, held for 10 min, warmed at 1 8C/min to 2 3.5 8C, and repeated for five cycles. The samples were then melted by warming from 2 3.5 to 0 8C at 1 8C/min, and in the cases where a gel-like structure was observed, as identified by fluorescence microscopy, the samples were further warmed to a rate of 1 8C/min until the observed structure disappeared. 2.4. Microscopy Two images per field were acquired at t ¼ 0 and at 23.5 8C of each cycle using both transmitted yellow/green light and epifluorescence illumination with a rhodamine filter set (Olympus Part #U-M41002, cube label 41002; ExcBP535/50, Dichroic 565, EmissionBP 610/75). At the end of the five cycles, brightfield and fluorescence images
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were collected every 0.5 8C until the crystals were melted and at 10, 22, 30 and 40 8C in the cases where a gel-like structure was observed in the sample. Images were acquired using a Photometrics SenSys 1401E array camera (Roper Scientific). The Image Processing Tool Kit (IPTK) (ver. 3.0 ISBN #1-928808-00-X) was used for all image processing and image analysis of the brightfield images. The IPTK is a set of Photoshop Plugins that are implemented within Adobe Photoshop 5.5. Photoshop ‘actions’ were used to record and automate a significant number of image processing and analysis functions. Grey level image processing. Before any analysis, all images were resized from the original image size of 1317 £ 1035 pixels at 100 pixels per inch (ppi) to a final size of 2400 £ 1886 pixels at 300 ppi. Significant light imbalances were compensated by duplicating the image and applying a ranking/brightest filter. This image was set up as a second image and then subtracted from the original to create a more uniformly illuminated image, better suited to uniform extraction of feature data. Thresholding was manually applied to each image in order to isolate features of interest. Binary image processing. Final separation of the features was achieved using a Boolean and operator to combine a skeletonized inverted image of the background to the thresholded image of features. The pen tool was then used, as required, to separate features using a partially transparent overlay of the original grey level image (layer) as a guiding template. The criterion of separation was that when accretion was evident, if greater than two thirds of the periphery of the crystal was intact, the crystal was counted and measured singularly (Flores & Goff, 1999a; Goff et al., 1999; Montoya, 2001). The end result in each case was a binary image that closely paralled the original grey level image. Using the feature cut off tool, all features touching the edges were removed. Analysis. All images were calibrated using images of a 1 mm stage micrometer acquired using conditions identical to the image being analysed. Binary images (black and white images) were analysed using measure all to acquire separate *.xls data files for each image. Data was then pasted to a spreadsheet template described and available at http:// members.aol.com/ImagProcTK/update.htm#Data. In this spreadsheet template, values of equivalent circular diameter with adjusted count correction factor were calculated for each feature in the images analysed. The adjusted count considers the cut off of the features touching the edges in the image processing. It corrects for bias due to large features being more likely to intersect the edge of the field and be unmeasurable. An explanation of the various measured parameters and the use of the adjusted count factor has been detailed in the companion reference for the IPTK (Russ, 1999). 2.5. Determination of recrystallization rate Ice crystal size distributions were characterized by the logistic model with a cumulative distribution of equivalent
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Fig. 2. Comparison between cumulative distribution functions after temperature cycling conditions for sucrose solutions containing carrageenan (0.05% w/w): (a) t ¼ 0, (b) cycle 1, (c) cycle 2, (d) cycle 3, (e) cycle 4 and (f) cycle 5. X50 represents the equivalent diameter at 50% of the cumulative distribution function for each cycle.
of recrystallization were observed in microscopy analyses such as accretion (crystal fusion), isomass rounding off and melt-regrow recrystallization (in which smaller ice crystals either melt partially or completely during warming, and larger crystals regrow during cooling). Fig. 1 shows some of the brightfield images acquired from a xanthan/sucrose solution at 2 3.5 8C, at time 0 and at the second, fourth and fifth temperature cycles. Several regions of these images are magnified in order to accentuate the different types of ice recrystallization induced by temperature cycling. A general review of the recrystallization mechanisms in ice cream can be found in Hartel (1998). 3.2. Ice recrystallization rate Fig. 1. Brightfield images acquired from xanthan : sucrose solutions at 23.5 8C, at time 0 and at the second, fourth and fifth cycles. Circular images are magnifications of small portions of each image that indicate the different mechanisms of recrystallization present in the sample: meltdiffuse-regrow (A), accretion and isomass rounding off (B) and accretion and melt-diffusion (C).
diameters, as described earlier by Flores and Goff (1999a,b), obtaining the ice crystal diameter at 50% of the cumulative distribution of the sample (X50) and the slope of the cumulative distribution at X50. Recrystallization rate was calculated as the slope of the linear regression of the curve plotted with the values of X50 for each cycle. For statistical purposes, solutions were prepared for each of the treatments in triplicate. Statistical analysis of the data was carried out using Microsoft Excel 97, ANOVA single factor test. When significant effects were evidenced (a ¼ 0.05) between sample treatments, T test (LSD) was used to compare the means of each parameter.
3. Results 3.1. Recrystallization mechanisms During the temperature cycling protocol, different forms
The temperature cycling treatment produced an expected increase in the equivalent diameter (X50) and a broadening of the distribution of the population characterized by a decrease of slope at the inflection point. A graphical comparison of the distributions for samples containing carrageenan (0.05% w/w) in sucrose solution clearly depicts the changes in distributions that were a result of cycling temperature conditions (Fig. 2). Recrystallization curves obtained from plotting the X50 values of the ice crystal size distributions as a function of time (number of cycles) resulted in a linear response (r 2 . 0.95) for all triplicates of all treatments (Fig. 3, a representative graph). Recrystallization rate values for each stabilizer/sucrose solution in the presence or absence of MSNF are presented in Table 1. In sucrose solutions, only alginate and xanthan significantly reduced recrystallization in comparison to the control ( p , 0.05). In solutions containing MSNF, all stabilizers retarded recrystallization significantly, with exception of gelatin. The trend obseved earlier for stabilizer solutions containing milk proteins having lower recrystallization rates than their corresponding sucrose solutions (Goff et al., 1999) was not found. However, initially the samples containing MSNF showed smaller ice crystal sizes than their corresponding sucrose solutions.
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Table 1 Recrystallization rates of sucrose solutions in the absence and presence of skim milk powder with or without stabilizer No SMP
None LBG Carrageenan Gelatin Xanthan CMC Alginate Fig. 3. Recrystallization curves of three replicates of CMC in sucrose solutions (0.3% w/w) at the same experimental conditions. (V) run 1, (B) run 2, (O) run 3.
3.3. Microstructure The fluorescence images showed clear structural differences between the various formulations. The source of fluorescence is the labeled polysaccharide, which enables its location in the unfrozen phase to be observed. Figs. 4 and 5 show the structures observed by fluorescence microscopy in sucrose/stabilizer solutions resulting from temperature cycling and after melting the ice crystals, in the absence or presence of milk proteins. Of the sucrose solutions, only the LBG sample showed evidence of a gel-like network structure after cycling. Gel-like networks of stabilizers were identified after cycling in LBG, gelatin and carrageenan sucrose solutions containing protein.
4. Discussion As stated earlier, the main objective of this study was to determine the existence of a relationship between the
With SMP
Slope (%/mm)
SEa
Slope (%/mm)
SE
4.02a,A 4.05a,A 3.87a,A 3.65a,b,A 2.82b,A 3.32a,b,A 2.77b,A
0.19 0.28 0.42 0.44 0.05 0.16 0.30
4.59a,A 3.17c,A 3.02c,A 4.13a,b,A 3.58b.c,B 2.83c,A 2.78c,A
0.34 0.39 0.33 0.26 0.13 0.23 0.28
a,b,c Values for each parameter with the same letter in the same column do not differ (a ¼ 0.05). A,BValues for each parameter with the same letter in the same row do not differ (a ¼ 0.05). a Standard error (n ¼ 3).
capability of the stabilizer to form a gel-like network, either by itself or with milk proteins, and the ability of the stabilizer to retard ice recrystallization in sucrose and sucrose/MSNF solutions. This hypothesis was derived from the proposed mechanism of ice recrystallization of Goff et al. (1999), which suggests that during temperature fluctuations, ice melting and growth becomes more favourable within the pore of the network than water diffusion to larger crystals, thus resulting in a preservation of the initial ice crystal size distribution. In the present research it was observed that, after temperature cycling and in the absence of proteins, a gellike network was formed only in solutions containing LBG. However, the recrystallization rate of LBG was not significantly different from the control ( p . 0.05). Previous studies suggest that gelatin and k-carrageenan could also form fine gel structures in their respective sucrose solutions, which were not identified by the present technique. Nevertheless, these stabilizers were as well ineffective at retarding
Fig. 4. Fluorescent images from stabilizers in sucrose solutions at 0 8C after cycling and ice crystal melting: (1) no stabilizer, (2) LBG, (3) xanthan, (4) CMC, (5) gelatin, (6) carrageenan and (7) alginate.
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Fig. 5. Fluorescent images from stabilizers in sucrose plus milk protein solutions at 0 8C after cycling and ice crystal melting: (1) no stabilizer, (2) LBG, (3) xanthan, (4) CMC, (5) gelatin, (6) carrageenan and (7) alginate.
recrystallization. Conversely, in samples containing proteins the only stabilizer that was not effective at retarding recrystallization was gelatin, which was observed to form a very distinctive composite gel with proteins. Therefore, steric blocking of the interface, or inhibition of solute transport to and from the ice interface caused by the gelation of the polymer, is not the only mechanism of stabilizer action. Given that we, as others, have shown that stabilizers do indeed inhibit recrystallization under some conditions, this implies that they have the ability to promote shrink-regrow mechanisms of recrystallization under those conditions, rather than melt-diffuse-grow. This in turn, implies that stabilizers are influencing water and/or solute migration to/from the surface of a growing/shrinking crystal during temperature fluctuations. Considering several mechanisms suggested by other researchers involving concepts of microviscosity (Miller-Livney & Hartel, 1997) and gelation (Blond, 1988; Goff et al., 1999; Muhr & Blanshard, 1986) of stabilizers, the following mechanism for stabilizer action in retarding ice recrystallization in sucrose and sucrose þ milk protein solutions, frozen by quiescent conditions and after temperature cycling, is proposed. During cooling, the solids are pushed from the growing crystal region, causing an actual increase in concentration in the immediate border between the ice crystal and the unfrozen phase. This increase in concentration will promote a microviscosity increase or the formation of a gel that is kinetically dependent. Either of these two results will retard (not avoid) the migration of water to a larger crystal in the surrounding area and the growth of the ice crystal. In the absence of stabilizer, this microviscosity or steric barrier is not present, so the water can diffuse faster and regrow on a bigger crystal, which is more thermodynamically favourable. No technique has been developed so far to prove the reduction of water mobility at appropriate scale distances
and temperatures by biopolymers such as stabilizers. However, there is definitely a biopolymer effect, as shown by the reduction of ice recrystallization. A general trend was observed in this study in which the expected gelling stabilizers, LBG, carrageenan and gelatin, showed higher recrystallization rates, both in the presence and absence of MSNF, in comparison to the non-gelling agents, xanthan, CMC and alginate. These differences in recrystallization rates can be attributed to the visco-elastic properties (Patmore et al., 2002) of the unfrozen phase imparted by the stabilizers. Using gelatin in sucrose/MSNF solution as an example for systems containing a gelling stabilizer and proteins, the formation of the stabilizer gellike network is created by the thermal history of the sample and the increase in concentration in the unfrozen phase during ice crystal growth (Fig. 6). Therefore, initially, a weak gel was formed, which retarded water mobility through basically two mechanisms: steric hindrance and water holding. During temperature cycling, the network around the ice crystal expanded as the temperature was decreased but did not collapse to match the shrinking crystal surface during warming suggesting the formation of a rigid structure of low flexibility. This gel-like structure became more evident with every cycle probably due to an increase in self-association. It is proposed that the continuous shrinking of the rigid gel-like structure at every cycle could promote some reduction in the water holding capacity of the stabilizer, causing some syneresis and increasing the water mobility around the ice crystal. With this, after extensive cycling, the inhibition of ice recrystallization by the stabilizer in gelling systems would be caused only by the steric hindrance of the gel-like network. Different freezing and cycling protocols and formulations could alter the number of cycles needed for a gelling stabilizer to start to loose its functionality in retarding recrystallization. In this study, all
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play a critical role in this process due to phase separation with polysaccharides, causing localized increases in concentration of each phase, and a further element of structure formation. Special attributes, such as the presence of small aggregates observed by TEM (Lundin & Hermansson, 1995), which confer a yield value to the solution, as well as the high visco-elasticity of the system, can be used to explain the remarkable ability of xanthan to retard recrystallization. This model is intended to explain some of the results found by earlier researchers in which non-gelling agents have been able to retard recrystallization (Bolliger et al., 2000; Sutton & Wilcox, 1998a), and other results in which the most effective stabilizer was not the firmest because it probably would be more fragile and be ruptured more easily by the ice front in comparison to a more flexible gel exerting a stronger opposing force for ice front propagation (Muhr & Blanshard, 1986).
5. Conclusions
Fig. 6. Brightfield (a) and fluorescence (b) images of gelatin in sucrose þ MSNF solution acquired: (1) before freezing, (2) during temperature cycling at cycle 1, (3) at cycle 3, (4) at cycle 5, and (5) after ice crystal melting.
the recrystallization curves showed linear relationships with high slopes for some of the gelling agents. This indicates that the freezing (before t ¼ 0) and precycling protocol at which the samples were subjected was enough to consolidate some strong gel-like structures of low flexibility that resulted in constant high recrystallization rates (i.e. gelatin with MSNF). For non-gelling stabilizers, the main mechanism by which they retard recrystallization is through the increase in microviscosity of the unfrozen phase. This microstructural arrangement gives a high flexibility to the unfrozen phase, and it is expected that it would not be affected by the temperature cycling protocol, thus resulting in a more effective reduction in water mobility of the system, and as a consequence lower recrystallization rates would be observed than in the gelling systems. Proteins may
The application of a fluorescence microscopy technique to the analysis of the microstructure of stabilizers (carrageenan, CMC, xanthan, alginate, LBG and gelatin) in sucrose and sucrose/MSNF solutions was successfully achieved. Gel-like structures in LBG with or without MSNF, carrageenan with MSNF and gelatin with MSNF in sucrose solutions that were quiescently frozen and temperature cycled (i.e. on a cold stage) were observed by this technique. Ice crystal size distributions were calculated from the acquired brightfield images with the aid of image processing and analysis software (Image Processing Tool Kit, IPTK). Recrystallization rates were obtained from the slopes of the curves plotted with the ice crystal mean size (equivalent diameter X50) values vs. number of cycles. Significant retardation in ice recrystallization was observed in the systems containing xanthan and alginate in sucrose solutions and in all the systems containing stabilizer (except gelatin) in sucrose plus MSNF solutions. The fact that non-gelling systems showed in some cases lower recrystallization rates than the systems where a gellike structure was observed, suggests that steric blocking of the interface, or inhibition of solute transport to and from the ice interface caused by the gelation of the polymer, is not the only mechanism of stabilizer action. The obtained results suggest that the kinetics of the recrystallization phenomena is restricted in some cases with the incorporation of a stabilizer in solution. Water holding by the stabilizer and proteins, and in some cases steric hindrance induced by a stabilizer gel-like network, probably caused a reduction in water mobility of the system, promoting ice recrystallization mechanisms of melt-regrow instead of melt-diffusegrow. These mechanisms result in the preservation of ice crystal size and in a small span of ice crystal size distribution. Several factors, like specific microviscosity,
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network pore size, ionic charges in the molecules, gel flexibility or other structural features, could influence both of the mechanisms for retarding water mobility: steric hindrance and water holding capability. The most efficient stabilizer would be the one giving sufficient microviscosity to the solution and steric hindrance to retard water diffusion to other crystals, but at the same time, it would need to have high flexibility properties that are not affected by heat shock.
Acknowledgments The authors wish to thank Sandy Smith and Ken Baker for their technical support and intellectual contributions to the present research. Thanks as well to CONACYT Mexico and the Natural Sciences and Engineering Research Council of Canada for the financial support that made possible this investigation.
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Flores, A. A., & Goff, H. D. (1999b). Recrystallization in ice cream after constant and cycling temperature storage conditions as affected by stabilizers. Journal of Dairy Science, 82, 1408–1415. Goff, H. D., Caldwell, K. B., Stanley, D. W., & Maurice, T. J. (1993). Influence of polysaccharides on the glass transition in frozen sucrose solutions and ice cream. Journal of Dairy Science, 76, 1268–1277. Goff, H. D., Ferdinando, D., & Schorsch, C. (1999). Fluorescence microscopy to study galactomannan structure in frozen sucrose and milk protein solutions. Food Hydrocolloids, 13, 353– 362. Hagiwara, Y., & Hartel, R. W. (1996). Effect of sweetener, stabilizer and storage temperature on ice recrystallization in ice cream. Journal of Dairy Science, 79, 735–744. Harper, E. K., & Shoemaker, C. F. (1983). Effects of locust bean gum and selected sweetening agents on ice recrystallization rates. Journal of Food Science, 48, 1801–1803. Hartel, R. W. (1996). Ice crystallization during the manufacture of ice cream. Trends in Food Science and Technology, 7, 315–321. Hartel, R. W. (1998). Mechanisms and kinetics of recrystallization in ice cream. In D. S. Reid (Ed.), The properties of water in foods (pp. 287 –319). London, UK: Blackie Academic and Professional. Lundin, L., & Hermansson, A. M. (1995). Supermolecular aspects of xanthan-locust bean gum gels based on rheology and electron microscopy. Carbohydrate Polymers, 26, 129– 140. Martin, D. R., Ablett, S., Darke, A., Sutton, R. L., & Sahagian, M. E. (1999). Diffusion of aqueous sugar solutions as affected by locust bean gum studied by NMR. Journal of Food Science, 64, 46–49. Miller-Livney, T., & Hartel, W. (1997). Ice recrystallization in ice cream: Interactions between sweeteners and stabilizers. Journal of Dairy Science, 80, 447–456. Montoya, K (2001). Effects of stabilizers and processing on the microstructure and stability of a model ice cream. MSc Thesis, University of Guelph, Guelph, Ont., Canada. Muhr, A. H., & Blanshard, J. M. (1986). Effect of polysaccharide stabilizers on the rate of growth of ice. Journal of Food Technology, 21, 683 –710. Patmore, J. V., Goff, H. D., & Fernandes, S (2002). Cryogelation of galactomannans on ice cream model systems. Food Hydrocoloids, in press. Russ, J. C. (1999). The image processing handbook (3rd ed). Boca Florida, USA: CRC Press/IEEE Press. pp. 1–763.. Sahagian, M. E., & Goff, H. D. (1995). Thermal, mechanical and molecular relaxation properties of stabilized sucrose solutions at sub-zero temperatures. Food Research International, 28, 1–8. Sutton, R. L., & Wilcox, J. (1998a). Recrystallization in ice cream as affected by stabilizers. Journal of Food Science, 63, 104 –107. Sutton, R. L., & Wilcox, J. (1998b). Recrystallization in model ice cream solutions as affected by stabilizer concentration. Journal of Food Science, 63, 9–11. Sutton, R. L., Cooke, D., & Russell, A. (1997a). Recrystallization in sugar/ stabilizer solutions as affected by molecular structure. Journal of Food Science, 62, 1145–1149. Sutton, R. L., Evans, I. D., & Crilly, J. F. (1994). Modelling ice crystal coarsening in concentrated dispersed food systems. Journal of Food Science, 59, 1227–1233. Sutton, R. L., Lips, A., & Piccirillo, G. (1996a). Recrystallization in aqueous fructose solutions as affected by locust bean gum. Journal of Food Science, 61, 746 –748. Sutton, R. L., Lips, A., Piccirillo, G., & Sztehlo, A. (1996b). Kinetics of ice recrystallization in aqueous fructose solutions. Journal of Food Science, 61, 741–745. Sutton, R. L., Wilcox, J., & Bedford, G. B. (1997b). Factors affecting recrystallization in ice cream products. European Dairy Magazine, 1, 17 –19.
Structure and ice recrystallization in frozen stabilized ice cream model systems Alejandra Regand, H. Douglas Goff* Department of Food Science, University of Guelph, Guelph, Ont., Canada N1G 2W1 Received 15 November 2001; revised 12 March 2002; accepted 12 March 2002
Abstract Hydrocolloid stabilizers (carrageenan, carboxymethyl cellulose, xanthan gum, sodium alginate, locust bean gum (LBG) and gelatin) were labeled with rhodamine isothiocyanate and incorporated into solutions of sucrose with or without milk solids-not-fat (MSNF). Resultant solutions were quench frozen to 2 50 8C and cycled between 2 3.5 and 2 6 8C, five times. The location of the stabilizer was observed using fluorescence microscopy. Significant retardation of recrystallization was observed in alginate and xanthan sucrose solutions without MSNF. In the presence of proteins, all stabilizers were effective retarding recrystallization except for gelatin. After cycling, a gel-like structure was observed in solutions containing LBG without MSNF, and in LBG, carrageenan and gelatin with MSNF. The fact that some non-gelling stabilizers (i.e. xanthan) were more effective retarding recrystallization than gelling stabilizers (i.e. gelatin) suggests that steric blocking of the interface or inhibition of solute transport to and from the ice interface caused by gelation of the polymer is not the only mechanism of stabilizer action. Molecular interactions between polysaccharides and proteins appear to be key factors in retarding ice recrystallization. q 2002 Elsevier Science Ltd. All rights reserved. Keywords: Ice cream; Recrystallization; Stabilizers; Fluorescence microscopy
1. Introduction Hydrocolloid stabilizers in ice cream improve smoothness of body, give uniformity of product, desired resistance to melting and improve handling properties, but the major role is to hinder crystal growth as temperature fluctuates during storage. The mechanisms by which they affect the freezing properties or limit recrystallization have been extensively studied but are as yet not fully understood. Stabilizers have little (Caldwell, Goff, & Stanley, 1992a,b) or no (Sutton & Wilcox, 1998a,b) impact on the initial ice crystal size distribution in ice cream at the time of draw from the scraped surface heat exchanger and also little or no impact on initial ice growth during quiescent freezing and hardening (Flores & Goff, 1999a), but do limit the rate of growth of ice crystals during recrystallization (Caldwell et al., 1992b; Donhowe & Hartel, 1996a,b; Flores & Goff, 1999b; Goff, Ferdinando, & Schorsch, 1999; Hagiwara & Hartel, 1996; Sutton & Wilcox, 1998a,b; Sutton, Cooke, & Russell, 1997a; Sutton, Evans, & Crilly, 1994; Sutton, Lips, * Corresponding author. Tel.: þ1-519-824-4120x3878; fax: þ 1-519824-6631. E-mail address: [email protected] (H.D. Goff).
& Piccirillo, 1996a; Sutton, Lips, Piccirillo, & Sztehlo, 1996b; Sutton, Wilcox, & Bedford, 1997b). Several authors have found that the presence of stabilizers at low concentration (0.5 – 2%) in model solutions and ice cream do not significantly alter their phase/state transition properties: glass transition temperature (Budiaman & Fennema, 1987a,b; Goff, Caldwell, Stanley, & Maurice, 1993; Hagiwara & Hartel, 1996; Sahagian & Goff, 1995), amount of freezable water or enthalpy of melting (Buyong & Fennema, 1988; Muhr & Blanshard, 1986; Sahagian & Goff, 1995), or heterogeneous nucleation (Blond, 1986; Muhr & Blanshard, 1986), and thus may not be expected to affect the initial ice crystallization processes. Many studies have been conducted to correlate enhanced viscosity caused by stabilizers with better control of ice crystal growth (Budiaman & Fennema, 1987a,b; Cottrell, Pass, & Phillips, 1979; Hagiwara & Hartel, 1996; Harper & Shoemaker, 1983; Hartel, 1996; Miller-Livney & Hartel, 1997) but without definitive conclusions. The functionality of a given stabilizer may be enhanced as the polymer concentration is increased, but different stabilizers are not equally effective for retarding ice crystal growth at the same level of concentration or viscosity (Budiaman & Fennema, 1987b). A different approach taken by Bolliger, Wildmoser,
0268-005X/03/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 2 6 8 - 0 0 5 X ( 0 2 ) 0 0 0 4 2 - 5
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Goff, and Tharp, (2000) found a linear relationship (r 2 ¼ 0.97) between a normalized ‘breakpoint’ apparent viscosity (break point apparent viscosity divided by initial mix apparent viscosity) and recrystallization rate. They suggested that some aspects of stabilizer functionality with respect to recrystallization protection could come from structure, as measured by rheological properties, that results from freeze-concentration of the polysaccharide in the unfrozen phase of ice cream. This structure from the stabilizer would affect the rate at which water can diffuse to the surface of a growing crystal during temperature fluctuation or the rate at which solutes and macromolecules can diffuse away from the surface of a growing ice crystal, an elaboration of the mechanism proposed by Caldwell et al. (1992b) and Goff et al. (1993). Experiments done with NMR in which the increase in viscosity due to the stabilizer is not accompanied by a similar decrease in mobility of water molecules (Martin, Abblet, Darke, Sutton, & Sahagian, 1999) seem to be contrary to this mechanism. However, it is important to remember that by an NMR technique the water diffusion or translational displacement of water molecules is being measured at intermolecular distances, usually less than 10 nm, while the water migration from one crystal to another involved in melt-refreeze recrystallization mechanisms implies distances between ice crystals usually longer than 10 mm. Therefore, the question formulated by Miller-Livney and Hartel (1997) still remains as to whether stabilizers in freeze-concentrated low temperature systems have different effects on macroviscosity (bulk fluid mobility) compared with microviscosity (molecular mobility), and whether increased microviscosity retards recrystallization by altering long-scale diffusion of water and sugar molecules. A modification of the ice crystal – serum interface through surface adsorption to the crystal itself has been suggested by Sutton & Wilcox (1998a,b), Sutton et al. (1996a, 1997a,b). This group of researchers has found that as the concentration of locust bean gum (LBG) is increased the recrystallization rate is reduced until it reaches a plateau after which it remains constant. In the case of guar, recrystallization rate decreases also with the increase in polymer concentration, but in contrast to LBG, after it reaches the minimum it remarkably rises with the addition of further polysaccharide. Non-gelling methoxy pectin gave similar concentration dependence to guar. Both were not as effective as LBG. The rise in recrystallization rate was suggested to be caused by a phase separation or incomplete dissolution and therefore lack of homogeneity above a critical stabilizer concentration. Recrystallization has been also related to the capacity of the stabilizer to form cryo-gels or entangled networks during freezing – thawing cycles. Gel firmness has been associated to inhibition of ice crystal growth and to a change in ice crystal morphology (Blond, 1988; Muhr & Blanshard, 1986). Crystallization velocity has been reduced as maturation time of the gel and gel elasticity increased.
This effect has been explained as a mechanical interference with the ice growth (Blond, 1988). However, a firm gel has not always been effective at retarding ice crystal growth, as found by Muhr & Blanshard (1986). They explained this behavior suggesting that a firm gel would be more fragile and be ruptured more easily by the ice front while a more flexible gel would probably exert a stronger opposing force for ice front propagation. It has been reported as well that stabilizers that did not form a gel still have retarded ice crystal growth (Sutton & Wilcox, 1998a). In another attempt to clarify the stabilizer’s mechanism in retarding recrystallization, Goff, et al. (1999) recently modified a method to covalently-bond a fluorescent marker to LBG and guar gum to visualize the location of these polysaccharides in frozen sucrose or sucrose/milk protein solutions after temperature cycling. They found that LBG was capable of forming a gel-like network after freezing, which became more distinct with temperature cycling. No such structure could be seen with guar gum. The rheological properties of these cryo-gels of LBG, with and without milk solids-not-fat (MSNF), and compared to guar gum have recently been studied by Patmore, Goff, and Fernandes (2002). In the work of Goff et al. (1999), image analysis of ice crystals after repeated temperature cycling showed that LBG was more effective at inhibiting recrystallization than was guar gum. The addition of milk proteins to the sucrose solutions led to a decrease in the rate of recrystallization for all samples when compared with the same solutions without protein. They also observed that both LBG and guar gum were responsible for the formation of a separated protein phase in solution. The combination of the formation of an LBG network and the presence of phase-separated protein was more effective at controlling ice recrystallization. Based on these observations, it was suggested that the ability to form cryo-gels, or gel-like intermolecular interactions, and to interact with proteins, perhaps causing localized increases in concentration through phase-separation, are prerequisites for stabilizer effectiveness against ice recrystallization. The present research was intended to be an extension of the earlier work of Goff et al. (1999). The objective was to analyze other commonly used stabilizers: carrageenan, carboxymethyl cellulose, xanthan gum, sodium alginate and gelatin, plus LBG studied earlier, to verify a correlation between their capacity to form cryo-gels and their ability to retard recrystallization when used as stabilizers in ice cream.
2. Materials and methods 2.1. Labeling of stabilizers The following stabilizers were labeled with rhodamine isothiocyanate (RITC) (Sigma Aldrich Canada), according to the method of De Belder and Granath (1973), as modified
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by Goff et al. (1999): LBG, xanthan gum, carrageenan, carboxymethyl cellulose (CMC) (Germantown Canada, Inc.), sodium alginate (Pronova Biopolymer, Inc., Canada) and gelatin (Cangel, Inc., Canada). 2.2. Preparation of solutions The following aqueous solutions were prepared: 24% w/w sucrose (Fisher Scientific, Canada), 24% w/w sucrose þ 0.3% w/w RITC-stabilizer, 16% w/w sucrose þ 14.7% w/w MSNF (from skim milk powder, 36% protein, Gay Lea Foods, Canada), 16% w/w sucrose þ 14.7% w/w MSNF þ 0.27% w/w stabilizer, for a total of 14 solutions. Solute concentrations were chosen to reflect typical stabilizer/sugar ratios or stabilizer/MSNF/sugar ratios in an ice cream mix. All solutions were calculated to have similar freezing curves, due to the effect of lactose and milk salts in the MSNF. The RITC-carrageenan concentration in sucrose and sucrose þ MSNF solutions was reduced to 0.05% w/w in sucrose solutions and 0.045% w/w in sucrose þ MSNF solutions to avoid gelation of the sample before cycling. Solutions were heated to 80 8C for 30 min, while stirring. Evaporated water was added back, and the solutions were cooled and stored at 4 8C overnight. For statistical purposes, the different ice cream solutions were prepared in triplicate. 2.3. Freezing and temperature cycling protocols A small drop of solution was placed between a slide and a cover slip and loaded on a cold stage (Linkam Scientific Instruments, UK) on a BX-60 Olympus light microscope. The cold stage was quench cooled to 2 50 8C and warmed to 2 10 8C at 5 8C/min to allow for irruptive crystallization of the solution. Ice crystals were matured to obtain the same initial crystal size for each sample ( p , 0.05). Different precycling treatments were required for samples with or without MSNF due to lower initial values of ice crystal size in the presence of proteins. The temperature program was started at 2 3.5 8C, samples were held for 10 min (t ¼ 0), cooled at 1 8C/min to 2 6 8C, held for 10 min, warmed at 1 8C/min to 2 3.5 8C, and repeated for five cycles. The samples were then melted by warming from 2 3.5 to 0 8C at 1 8C/min, and in the cases where a gel-like structure was observed, as identified by fluorescence microscopy, the samples were further warmed to a rate of 1 8C/min until the observed structure disappeared. 2.4. Microscopy Two images per field were acquired at t ¼ 0 and at 23.5 8C of each cycle using both transmitted yellow/green light and epifluorescence illumination with a rhodamine filter set (Olympus Part #U-M41002, cube label 41002; ExcBP535/50, Dichroic 565, EmissionBP 610/75). At the end of the five cycles, brightfield and fluorescence images
97
were collected every 0.5 8C until the crystals were melted and at 10, 22, 30 and 40 8C in the cases where a gel-like structure was observed in the sample. Images were acquired using a Photometrics SenSys 1401E array camera (Roper Scientific). The Image Processing Tool Kit (IPTK) (ver. 3.0 ISBN #1-928808-00-X) was used for all image processing and image analysis of the brightfield images. The IPTK is a set of Photoshop Plugins that are implemented within Adobe Photoshop 5.5. Photoshop ‘actions’ were used to record and automate a significant number of image processing and analysis functions. Grey level image processing. Before any analysis, all images were resized from the original image size of 1317 £ 1035 pixels at 100 pixels per inch (ppi) to a final size of 2400 £ 1886 pixels at 300 ppi. Significant light imbalances were compensated by duplicating the image and applying a ranking/brightest filter. This image was set up as a second image and then subtracted from the original to create a more uniformly illuminated image, better suited to uniform extraction of feature data. Thresholding was manually applied to each image in order to isolate features of interest. Binary image processing. Final separation of the features was achieved using a Boolean and operator to combine a skeletonized inverted image of the background to the thresholded image of features. The pen tool was then used, as required, to separate features using a partially transparent overlay of the original grey level image (layer) as a guiding template. The criterion of separation was that when accretion was evident, if greater than two thirds of the periphery of the crystal was intact, the crystal was counted and measured singularly (Flores & Goff, 1999a; Goff et al., 1999; Montoya, 2001). The end result in each case was a binary image that closely paralled the original grey level image. Using the feature cut off tool, all features touching the edges were removed. Analysis. All images were calibrated using images of a 1 mm stage micrometer acquired using conditions identical to the image being analysed. Binary images (black and white images) were analysed using measure all to acquire separate *.xls data files for each image. Data was then pasted to a spreadsheet template described and available at http:// members.aol.com/ImagProcTK/update.htm#Data. In this spreadsheet template, values of equivalent circular diameter with adjusted count correction factor were calculated for each feature in the images analysed. The adjusted count considers the cut off of the features touching the edges in the image processing. It corrects for bias due to large features being more likely to intersect the edge of the field and be unmeasurable. An explanation of the various measured parameters and the use of the adjusted count factor has been detailed in the companion reference for the IPTK (Russ, 1999). 2.5. Determination of recrystallization rate Ice crystal size distributions were characterized by the logistic model with a cumulative distribution of equivalent
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Fig. 2. Comparison between cumulative distribution functions after temperature cycling conditions for sucrose solutions containing carrageenan (0.05% w/w): (a) t ¼ 0, (b) cycle 1, (c) cycle 2, (d) cycle 3, (e) cycle 4 and (f) cycle 5. X50 represents the equivalent diameter at 50% of the cumulative distribution function for each cycle.
of recrystallization were observed in microscopy analyses such as accretion (crystal fusion), isomass rounding off and melt-regrow recrystallization (in which smaller ice crystals either melt partially or completely during warming, and larger crystals regrow during cooling). Fig. 1 shows some of the brightfield images acquired from a xanthan/sucrose solution at 2 3.5 8C, at time 0 and at the second, fourth and fifth temperature cycles. Several regions of these images are magnified in order to accentuate the different types of ice recrystallization induced by temperature cycling. A general review of the recrystallization mechanisms in ice cream can be found in Hartel (1998). 3.2. Ice recrystallization rate Fig. 1. Brightfield images acquired from xanthan : sucrose solutions at 23.5 8C, at time 0 and at the second, fourth and fifth cycles. Circular images are magnifications of small portions of each image that indicate the different mechanisms of recrystallization present in the sample: meltdiffuse-regrow (A), accretion and isomass rounding off (B) and accretion and melt-diffusion (C).
diameters, as described earlier by Flores and Goff (1999a,b), obtaining the ice crystal diameter at 50% of the cumulative distribution of the sample (X50) and the slope of the cumulative distribution at X50. Recrystallization rate was calculated as the slope of the linear regression of the curve plotted with the values of X50 for each cycle. For statistical purposes, solutions were prepared for each of the treatments in triplicate. Statistical analysis of the data was carried out using Microsoft Excel 97, ANOVA single factor test. When significant effects were evidenced (a ¼ 0.05) between sample treatments, T test (LSD) was used to compare the means of each parameter.
3. Results 3.1. Recrystallization mechanisms During the temperature cycling protocol, different forms
The temperature cycling treatment produced an expected increase in the equivalent diameter (X50) and a broadening of the distribution of the population characterized by a decrease of slope at the inflection point. A graphical comparison of the distributions for samples containing carrageenan (0.05% w/w) in sucrose solution clearly depicts the changes in distributions that were a result of cycling temperature conditions (Fig. 2). Recrystallization curves obtained from plotting the X50 values of the ice crystal size distributions as a function of time (number of cycles) resulted in a linear response (r 2 . 0.95) for all triplicates of all treatments (Fig. 3, a representative graph). Recrystallization rate values for each stabilizer/sucrose solution in the presence or absence of MSNF are presented in Table 1. In sucrose solutions, only alginate and xanthan significantly reduced recrystallization in comparison to the control ( p , 0.05). In solutions containing MSNF, all stabilizers retarded recrystallization significantly, with exception of gelatin. The trend obseved earlier for stabilizer solutions containing milk proteins having lower recrystallization rates than their corresponding sucrose solutions (Goff et al., 1999) was not found. However, initially the samples containing MSNF showed smaller ice crystal sizes than their corresponding sucrose solutions.
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Table 1 Recrystallization rates of sucrose solutions in the absence and presence of skim milk powder with or without stabilizer No SMP
None LBG Carrageenan Gelatin Xanthan CMC Alginate Fig. 3. Recrystallization curves of three replicates of CMC in sucrose solutions (0.3% w/w) at the same experimental conditions. (V) run 1, (B) run 2, (O) run 3.
3.3. Microstructure The fluorescence images showed clear structural differences between the various formulations. The source of fluorescence is the labeled polysaccharide, which enables its location in the unfrozen phase to be observed. Figs. 4 and 5 show the structures observed by fluorescence microscopy in sucrose/stabilizer solutions resulting from temperature cycling and after melting the ice crystals, in the absence or presence of milk proteins. Of the sucrose solutions, only the LBG sample showed evidence of a gel-like network structure after cycling. Gel-like networks of stabilizers were identified after cycling in LBG, gelatin and carrageenan sucrose solutions containing protein.
4. Discussion As stated earlier, the main objective of this study was to determine the existence of a relationship between the
With SMP
Slope (%/mm)
SEa
Slope (%/mm)
SE
4.02a,A 4.05a,A 3.87a,A 3.65a,b,A 2.82b,A 3.32a,b,A 2.77b,A
0.19 0.28 0.42 0.44 0.05 0.16 0.30
4.59a,A 3.17c,A 3.02c,A 4.13a,b,A 3.58b.c,B 2.83c,A 2.78c,A
0.34 0.39 0.33 0.26 0.13 0.23 0.28
a,b,c Values for each parameter with the same letter in the same column do not differ (a ¼ 0.05). A,BValues for each parameter with the same letter in the same row do not differ (a ¼ 0.05). a Standard error (n ¼ 3).
capability of the stabilizer to form a gel-like network, either by itself or with milk proteins, and the ability of the stabilizer to retard ice recrystallization in sucrose and sucrose/MSNF solutions. This hypothesis was derived from the proposed mechanism of ice recrystallization of Goff et al. (1999), which suggests that during temperature fluctuations, ice melting and growth becomes more favourable within the pore of the network than water diffusion to larger crystals, thus resulting in a preservation of the initial ice crystal size distribution. In the present research it was observed that, after temperature cycling and in the absence of proteins, a gellike network was formed only in solutions containing LBG. However, the recrystallization rate of LBG was not significantly different from the control ( p . 0.05). Previous studies suggest that gelatin and k-carrageenan could also form fine gel structures in their respective sucrose solutions, which were not identified by the present technique. Nevertheless, these stabilizers were as well ineffective at retarding
Fig. 4. Fluorescent images from stabilizers in sucrose solutions at 0 8C after cycling and ice crystal melting: (1) no stabilizer, (2) LBG, (3) xanthan, (4) CMC, (5) gelatin, (6) carrageenan and (7) alginate.
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Fig. 5. Fluorescent images from stabilizers in sucrose plus milk protein solutions at 0 8C after cycling and ice crystal melting: (1) no stabilizer, (2) LBG, (3) xanthan, (4) CMC, (5) gelatin, (6) carrageenan and (7) alginate.
recrystallization. Conversely, in samples containing proteins the only stabilizer that was not effective at retarding recrystallization was gelatin, which was observed to form a very distinctive composite gel with proteins. Therefore, steric blocking of the interface, or inhibition of solute transport to and from the ice interface caused by the gelation of the polymer, is not the only mechanism of stabilizer action. Given that we, as others, have shown that stabilizers do indeed inhibit recrystallization under some conditions, this implies that they have the ability to promote shrink-regrow mechanisms of recrystallization under those conditions, rather than melt-diffuse-grow. This in turn, implies that stabilizers are influencing water and/or solute migration to/from the surface of a growing/shrinking crystal during temperature fluctuations. Considering several mechanisms suggested by other researchers involving concepts of microviscosity (Miller-Livney & Hartel, 1997) and gelation (Blond, 1988; Goff et al., 1999; Muhr & Blanshard, 1986) of stabilizers, the following mechanism for stabilizer action in retarding ice recrystallization in sucrose and sucrose þ milk protein solutions, frozen by quiescent conditions and after temperature cycling, is proposed. During cooling, the solids are pushed from the growing crystal region, causing an actual increase in concentration in the immediate border between the ice crystal and the unfrozen phase. This increase in concentration will promote a microviscosity increase or the formation of a gel that is kinetically dependent. Either of these two results will retard (not avoid) the migration of water to a larger crystal in the surrounding area and the growth of the ice crystal. In the absence of stabilizer, this microviscosity or steric barrier is not present, so the water can diffuse faster and regrow on a bigger crystal, which is more thermodynamically favourable. No technique has been developed so far to prove the reduction of water mobility at appropriate scale distances
and temperatures by biopolymers such as stabilizers. However, there is definitely a biopolymer effect, as shown by the reduction of ice recrystallization. A general trend was observed in this study in which the expected gelling stabilizers, LBG, carrageenan and gelatin, showed higher recrystallization rates, both in the presence and absence of MSNF, in comparison to the non-gelling agents, xanthan, CMC and alginate. These differences in recrystallization rates can be attributed to the visco-elastic properties (Patmore et al., 2002) of the unfrozen phase imparted by the stabilizers. Using gelatin in sucrose/MSNF solution as an example for systems containing a gelling stabilizer and proteins, the formation of the stabilizer gellike network is created by the thermal history of the sample and the increase in concentration in the unfrozen phase during ice crystal growth (Fig. 6). Therefore, initially, a weak gel was formed, which retarded water mobility through basically two mechanisms: steric hindrance and water holding. During temperature cycling, the network around the ice crystal expanded as the temperature was decreased but did not collapse to match the shrinking crystal surface during warming suggesting the formation of a rigid structure of low flexibility. This gel-like structure became more evident with every cycle probably due to an increase in self-association. It is proposed that the continuous shrinking of the rigid gel-like structure at every cycle could promote some reduction in the water holding capacity of the stabilizer, causing some syneresis and increasing the water mobility around the ice crystal. With this, after extensive cycling, the inhibition of ice recrystallization by the stabilizer in gelling systems would be caused only by the steric hindrance of the gel-like network. Different freezing and cycling protocols and formulations could alter the number of cycles needed for a gelling stabilizer to start to loose its functionality in retarding recrystallization. In this study, all
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play a critical role in this process due to phase separation with polysaccharides, causing localized increases in concentration of each phase, and a further element of structure formation. Special attributes, such as the presence of small aggregates observed by TEM (Lundin & Hermansson, 1995), which confer a yield value to the solution, as well as the high visco-elasticity of the system, can be used to explain the remarkable ability of xanthan to retard recrystallization. This model is intended to explain some of the results found by earlier researchers in which non-gelling agents have been able to retard recrystallization (Bolliger et al., 2000; Sutton & Wilcox, 1998a), and other results in which the most effective stabilizer was not the firmest because it probably would be more fragile and be ruptured more easily by the ice front in comparison to a more flexible gel exerting a stronger opposing force for ice front propagation (Muhr & Blanshard, 1986).
5. Conclusions
Fig. 6. Brightfield (a) and fluorescence (b) images of gelatin in sucrose þ MSNF solution acquired: (1) before freezing, (2) during temperature cycling at cycle 1, (3) at cycle 3, (4) at cycle 5, and (5) after ice crystal melting.
the recrystallization curves showed linear relationships with high slopes for some of the gelling agents. This indicates that the freezing (before t ¼ 0) and precycling protocol at which the samples were subjected was enough to consolidate some strong gel-like structures of low flexibility that resulted in constant high recrystallization rates (i.e. gelatin with MSNF). For non-gelling stabilizers, the main mechanism by which they retard recrystallization is through the increase in microviscosity of the unfrozen phase. This microstructural arrangement gives a high flexibility to the unfrozen phase, and it is expected that it would not be affected by the temperature cycling protocol, thus resulting in a more effective reduction in water mobility of the system, and as a consequence lower recrystallization rates would be observed than in the gelling systems. Proteins may
The application of a fluorescence microscopy technique to the analysis of the microstructure of stabilizers (carrageenan, CMC, xanthan, alginate, LBG and gelatin) in sucrose and sucrose/MSNF solutions was successfully achieved. Gel-like structures in LBG with or without MSNF, carrageenan with MSNF and gelatin with MSNF in sucrose solutions that were quiescently frozen and temperature cycled (i.e. on a cold stage) were observed by this technique. Ice crystal size distributions were calculated from the acquired brightfield images with the aid of image processing and analysis software (Image Processing Tool Kit, IPTK). Recrystallization rates were obtained from the slopes of the curves plotted with the ice crystal mean size (equivalent diameter X50) values vs. number of cycles. Significant retardation in ice recrystallization was observed in the systems containing xanthan and alginate in sucrose solutions and in all the systems containing stabilizer (except gelatin) in sucrose plus MSNF solutions. The fact that non-gelling systems showed in some cases lower recrystallization rates than the systems where a gellike structure was observed, suggests that steric blocking of the interface, or inhibition of solute transport to and from the ice interface caused by the gelation of the polymer, is not the only mechanism of stabilizer action. The obtained results suggest that the kinetics of the recrystallization phenomena is restricted in some cases with the incorporation of a stabilizer in solution. Water holding by the stabilizer and proteins, and in some cases steric hindrance induced by a stabilizer gel-like network, probably caused a reduction in water mobility of the system, promoting ice recrystallization mechanisms of melt-regrow instead of melt-diffusegrow. These mechanisms result in the preservation of ice crystal size and in a small span of ice crystal size distribution. Several factors, like specific microviscosity,
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network pore size, ionic charges in the molecules, gel flexibility or other structural features, could influence both of the mechanisms for retarding water mobility: steric hindrance and water holding capability. The most efficient stabilizer would be the one giving sufficient microviscosity to the solution and steric hindrance to retard water diffusion to other crystals, but at the same time, it would need to have high flexibility properties that are not affected by heat shock.
Acknowledgments The authors wish to thank Sandy Smith and Ken Baker for their technical support and intellectual contributions to the present research. Thanks as well to CONACYT Mexico and the Natural Sciences and Engineering Research Council of Canada for the financial support that made possible this investigation.
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