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Stabilising frozen dairy mousses by low molecular weight gelatin peptides
Food Hydrocolloids 60 (2016) 317e323
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Food Hydrocolloids journal homepage: www.elsevier.com/locate/foodhyd
Stabilising frozen dairy mousses by low molecular weight gelatin peptides Barbara Duquenne a, *, Bjorn Vergauwen b, Claude Capdepon b, Marijn A. Boone c, e, Thomas De Schryver d, Luc Van Hoorebeke d, Stephanie Van Weyenberg f, Paul Stevens b, Jan De Block a a
Institute for Agricultural and Fisheries Research (ILVO), Technology and Food Science Unit, Brusselsesteenweg 370, Melle, Belgium Rousselot BVBA, Expertise Center, Meulestedekaai 81, Ghent, Belgium UGCT-Pore-scale Processes in Geomaterials Research Group, Department of Geology and Soil Science, Ghent University, Krijgslaan 281 S8, Ghent, Belgium d UGCT-Radiation Physics Research Group, Department of Physics and Astronomy, Ghent University, Proeftuinstraat 86, Ghent, Belgium e X-ray Engineering bvba, De Pintelaan 111, Ghent, Belgium f Institute for Agricultural and Fisheries Research (ILVO), Technology and Food Science Unit, Burg. Van Gansberghelaan 115 Bus 1, Merelbeke, Belgium b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 January 2016 Received in revised form 15 March 2016 Accepted 2 April 2016 Available online 4 April 2016
The effect of low molecular weight gelatin peptides on the shrinkage of thawed mousses was investigated. Changes promoted by freezing-thawing processes were evaluated through texture and volume measurements and through X-ray analysis of the bubble distribution. Freezing provoked collapse of the standard reference recipe mousse and of the reference recipe mousse with 2% milk powder added, but samples containing 2% gelatin peptides showed no shrinkage. The bubble size and bubble number distribution of the different mousses were measured based on high-resolution X-ray tomography. Results indicated that the volume losses experienced by the controls were almost entirely caused by the disappearance of air bubbles having a diameter smaller than 50 mm. Hence, this fraction of overpressurised air bubbles is extra stabilised by the matrix due to the additional presence of gelatin peptides. Moreover, gelatin peptides were found to inhibit ice crystal growth, which resulted in smaller ice crystals that are believed to be less destructive to the microstructure of the freeze-thawed mousses. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Gelatin peptides Dairy mousses Frozen stability Air bubbles
1. Introduction Many food products are frozen to provide long-term stability to the product. The process of freezing and thawing is highly destabilising food products, especially mousses, leading to foam collapse, loss of gas and loss of desired foam structure and texture (Ghosh & Coupland, 2008; Murray & Ettelaie, 2004). The stability and behaviour of food foams such as ice cream, marshmallow and mousses, is closely related to their microstructure, and more exactly, to the air bubble size, distribution and volume fraction (Miquelim, Lannes, & Mezzenga, 2010). Bamforth (1995) and Muller-Fischer and Windhab (2005) noticed that the foams with an even distribution of smaller bubbles were more stable, creamier and more attractive to consumers. Bubble mechanics can be used to
* Corresponding author. ILVO, Technology and Food Science Unit, Brusselsesteenweg 370, 9090 Melle, Belgium. E-mail address: [email protected] (B. Duquenne). http://dx.doi.org/10.1016/j.foodhyd.2016.04.001 0268-005X/© 2016 Elsevier Ltd. All rights reserved.
estimate shelf life of whipped dairy products, since the textural appearance and mouthfeel are a direct consequence of the complex interactions between bubble mechanics and our senses (Niranjan, 1999). High-resolution X-ray tomography (X-ray micro-CT) has recently been introduced as a non-destructive material evaluation technique for the microstructure of food products. The fact that micro-CT can provide information about the internal structure and properties of food, is a major advantage in the study of their conservation. With X-ray micro-CT (mCT) the internal structure of most food products can be visualised by measuring the different attenuations for X-rays of the materials in the product. As explained in Kak and Slaney (2001) and for food materials in particular in Verboven et al. (2008) and Herremans et al. (2013), the level of transmission of these rays depends mainly on the mass density and mass absorption coefficient of the material. Because absorption is different in gas and water, gas-filled spaces can be distinguished from the matrix material.
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Freezing of mousses induces thinning of the lamellae between air cells, due to the cryoconcentration during ice formation, which directly affects foam stability (Camacho, Martinez-Navarrete, & Chiralt, 2001; Dickinson, 1992). Cryoconcentration implies alteration in the aqueous environment of macromolecules because of the separation of the more concentrated phase from the initial solution. Mechanical damage caused by ice crystals on air cell films can be considerable (Berger, 1990). Studies on ice cream have shown that hydrocolloids (locust bean gum, guar gum, xanthan gum, gelatin and carrageenan) exert a cryoprotective effect mainly due to the inhibition of ice crystal growth in the freezer and also by inhibiting ice recrystallisation throughout frozen storage (Caldwell et al., 1992; Donhowe & Hartel, 1996a, 1996b; Flores & Goff, 1999; Hagiwara & Hartel, 1996; Hartel, 1998; Sutton & Wilcox, 1998a, 1998b). Hydrocolloids increase the viscosity of the aqueous phase, hence reducing the availability of free water, and inhibiting de novo and/or recrystallisation events (Stanley, Goff, & Smith, 1996). In contrast to these findings, peptides (2000e5000 Da) obtained from proteolytic hydrolysis of gelatin also prevented ice crystallisation in frozen ice cream mixes while not affecting the product's viscosity (Damodaran, 2007; Wang & Damodaran, 2009). In this work, we aim to investigate the efficacy of low molecular weight gelatin peptides on the shrinkage of frozen mousses. Changes promoted by freezing-thawing processes were evaluated through texture and volume measurements and through X-ray analysis of both air bubbles and ice crystals.
treatment and the interaction as categorical independent variables. Post hoc comparison was done with tukey post hoc test.
2. Material and methods
2.5. Overrun of whipped mousses
2.1. Gelatin peptides
The overrun is a parameter which gives information on the percentage of gas in the whipped mousse. Volume ratio of air incorporated in mousse during whipping was measured in newly whipped mousse. Overrun was measured for each sample using the following equation:
Commercial porcine gelatin peptides with an average molecular weight of 2000 Da were supplied by Rousselot BVBA.
2.4. Volume of whipped mousses Volume measurements were performed at 2 C after thawing, 1, 3, 6, 9 and 12 months after frozen storage. Volume loss during frozen storage was calculated by measuring the new height and diameter of the product. Height loss was measured at 6 different locations by measuring the distance between the surface of the product and a ruler placed on top of the recipient. Loss in diameter was measured at 6 different points as the distance of the edge of the shrunk mousse to the wall of the recipient. From these measurements, the dimensions of the shrunk cylinder was calculated providing the best estimation of the remaining volume. Remaining volume for each sample was calculated using the following equation:
Remainingvolumeð%Þ ¼ 100
where V1 is the volume before freezing of whipped mousse and V2 the volume after freezing and thawing of whipped mousse. For every batch, 3 different samples were analysed. Volume was found not to be normally distributed. Therefore, non-parametric analyses was performed with time and treatment as categorical independent variables.
2.2. Preparation of whipped mousses Commercial mousse recipes containing whole milk, sugar, cream, gelatin, modified starch, mono- and diglycerides of fatty acids, lactic acid esters of mono- and diglycerides of fatty acids, sodium phosphates, carrageenan, vanilla flavour and beta carotene were prepared either or not including 2% gelatin peptides or 2% milk powder. The sample containing 2% milk powder was used as a control sample to have the same dry matter as the recipe with 2% gelatin peptides. After mixing the ingredients, the mixes were heated (5 s, 120e130 C) and homogenised (downstream, 2-steps, 85 bar/25 bar), cooled and whipped in a kitchen aid (Hobart; Ohio, US) during 30 s at stand 2 and 4 min 30 s at stand 3. Mousses were stored in the freezer at 24 C in 0.20 l plastic cups with lid. Cups were completely filled and remainders were scraped away with a knife. Mousses were analysed after 1, 3, 6, 9 and 12 months. Before analysis, samples were removed from the freezer and thawed for 24 h at 2 C. The test was repeated once. 2.3. Texture of whipped mousses Texture of the whipped mousses was performed at 2 C before freezing and after thawing, 1, 3, 6, 9 and 12 months after frozen storage. Hardness of the samples was measured by penetration of a vertical sheet with 2 grooves of 15 mm (stainless steel; 33 33 mm) into the mousse to a depth of 20 mm at a constant speed of 120 mm/min with a Texture Analyser (LF-Plus; Lloyd Instruments Ltd., Hants, UK). For every batch, minimum 3 different samples were analysed. In order to test the effect on hardness (dependent variable), a linear regression was performed with time,
V1 V2 $100 V1
Overrunð%Þ ¼
M1 M2 $100 M2
where M1 is the weight of a fixed volume of unwhipped mousse and M2 the weight of the same volume of whipped mousse. In order to test the effect of treatment on overrun (dependent variable), a linear regression was performed with treatment as categorical independent variable. Post hoc comparison was done with tukey post hoc test. 2.6. Air bubble distribution of whipped mousses The air bubble distribution was studied using optical microscopy and X-ray mCT. In the experiment with the optical microscope (Olympus BH-2, Tokyo, Japan), a small sample of mousse was placed on a microscope glass slide and covered with a glass coverslip. Images were acquired using an Olympus Camera (Camedia C-3040 Zoom). X-ray mCT was performed at the Centre for X-ray Tomography of the Ghent University (UGCT; www.ugct.ugent.be) using the Environmental CT-scanner (EMCT), a custom-built gantry-based laboratory mCT scanner (Dierick et al., 2014). Samples of 1 1 1 cm were used for the mCT experiments. The Hamamatsu L9181-2 X-ray tube was operated at 65 kV and a power of 11.7 W and 1500 projections were taken over 360 with an exposure of 500 ms using a Teledyne Dalsa Xineos-1313 high-speed flat-panel detector. The EMCT was upgraded with a custom-made freezing stage which can control and monitor a sample's temperature with an accuracy of ±0.4 C (De Schryver et al., 2015), making it possible to scan a
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Table 1 Hardness (N) versus storage period (months) for reference mousse, mousse with 2% gelatin peptides and mousse with 2% milk powder. Results are expressed as mean (±SD) of minimum 3 determinations. Storage period 0 Reference 2% gelatin peptides 2% milk powder
1 a
1.04 ± 0.08 1.54 ± 0.11b 1.24 ± 0.07c
3 a
1.05 ± 0.03 1.52 ± 0.07b 1.19 ± 0.07a
6 a
1.02 ± 0.08 1.44 ± 0.21b 1.19 ± 0.07a
9 a
1.12 ± 0.18 1.49 ± 0.09b 1.24 ± 0.16a
12 a
1.25 ± 0.11 1.45 ± 0.08a 0.85 ± 0.19b
1.07 ± 0.15a 1.53 ± 0.10b 1.31 ± 0.07ab
a,b,c
Values with a different letter in the same column significantly differ (p < 0.05).
Table 2 Remaining volume (%) versus storage period (months) for reference mousse, mousse with 2% gelatin peptides and mousse with 2% milk powder. Results are expressed as mean (±SD) of minimum 3 determinations. Storage period 0 Reference 2% gelatin peptides 2% milk powder
100 ± 0 100 ± 0 100 ± 0
1 100 ± 0 100 ± 0 100 ± 0
3 100 ± 0 100 ± 0 100 ± 0
6 73 ± 12 100 ± 0b 86 ± 8c
a
9
12
65 ± 7 100 ± 0 75 ± 16
67 ± 9a 100 ± 0b 76 ± 15a
a,b,c
Values with a different letter in the same column significantly differ (p < 0.05).
Fig. 1. Pictures of a: thawed reference mousse after 12 months frozen storage; b: thawed mousse with gelatin peptides after 12 months frozen storage; c: thawed mousse with milk powder after 12 months frozen storage.
Fig. 2. Tomographic image of the air bubbles in a: fresh reference mousse; b: fresh mousse with gelatin peptides; c: fresh mousse with milk powder; d: thawed reference mousse after 12 months frozen storage; e: thawed mousse with gelatin peptides after 12 months frozen storage; f: thawed mousse with milk powder after 12 months frozen storage.
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Fig. 3. Tomographic 3D image of the air bubbles in a: fresh reference mousse; b: fresh mousse with gelatin peptides; c: fresh mousse with milk powder; d: thawed reference mousse after 12 months frozen storage; e: thawed mousse with gelatin peptides after 12 months frozen storage; f: thawed mousse with milk powder after 12 months frozen storage (green bubbles: 500e750 mm; red bubbles: 250e500 mm; dark blue bubbles: 100e250 mm; light blue bubbles: 50e100 mm). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
sample in a conditioned environment. In this work each sample was conditioned at 4 C with the freezing stage while the 12.5 min long mCT scans were acquired. A 11.49 mm voxel resolution was obtained after reconstruction with the Octopus Reconstruction software (InsideMatters, https://insidematters.eu/). To quantify the individual bubbles, the reconstructed images were analysed with Avizo 9.0 software (FEI, Saint-Aubain, France). 2.7. Ice crystal growth in unwhipped mousses Ice crystal growth was studied using X-ray mCT (EMCT at the Centre for X-ray Tomography of the Ghent University) with 5% CsI (Sigma Aldrich, Belgium) as a contrast agent. Each sample (1 1 1 cm) was frozen in the freezer at 20 C and subsequently conditioned at 19 C with the freezing stage during 15 min. The temperature of the sample was then increased from 19 to 12 C and then cycled between 12 and 6 C at a rate of 1 cycle/60 min. Three cycles were applied. Scans were reconstructed with the Octopus Reconstruction software (InsideMatters, https:// insidematters.eu/). 3. Results and discussion 3.1. Textural characteristics and overrun of whipped mousses Because textural properties are an important component of food quality perception and acceptability (Foegeding, 2006), this parameter was measured for fresh mousses and for thawed mousses after a frozen storage period of 1, 3, 6, 9 and 12 months (Table 1). Mousses with gelatin peptides were significantly harder after 0, 1, 3, 6 and 12 months storage in comparison with the reference mousse. The hardness of the mousses during storage remained constant. The higher hardness for the samples with gelatin peptides can be explained by the lower overrun of these samples (140 ± 10%) in comparison with the reference mousse (153 ± 7%) and samples with milk powder (159 ± 5%). The overrun of the different samples was significantly different (p < 0.01). Camacho, Martinez-Navarrete, and Chiralt (1998) also noticed that hydrocolloids (locust bean gum and carrageenan) conferred a greater foaming resistance to dairy cream increasing the whipping
time and reducing the overrun. 3.2. Frozen stability of the whipped mousses Frozen stability of the thawed whipped mousses has been evaluated through the changes in sample volume and microstructure during freezing and frozen storage. During the first 3 months frozen storage, no shrinkage of the mousses was observed (Table 2). Shrinkage was seen after 4 months frozen storage. Mousses with 2% gelatin peptides showed no volume loss after 12 months frozen storage in contrast to the reference mousse which showed approximately 33% volume loss and mousse with 2% milk powder which showed approximately 24% volume loss (Fig. 1). A comparison of the tomographic images shows different microstructures for the fresh and thawed mousses (Fig. 2). A less densely packed microstructure is observed for the fresh mousses of all tested conditions. Fig. 3 shows the corresponding 3-D renderings of the air bubbles that were further used for the analysis. The individual samples taken for X-ray mCT sized 45 mm3 and bubbles in the range of 11.5e500 mm were counted (Fig. 4). Considering the fresh mousses, 19,258 ± 1680, 13,940 ± 696 and 15,985 ± 2234 bubbles were counted for the reference sample, the sample with gelatin peptides and the sample with milk powder respectively. The 45 mm3 sized samples taken from the thawed mousses contained 12,827 ± 1499, 15,992 ± 1493 and 13,557 ± 1544 bubbles for the reference sample, the sample with gelatin peptides and the sample with milk powder respectively. To compare the number of bubbles in mousses before and after shrinkage, the loss of volume must be taken into account since the 45 mm3 sample taken from a freezethawed collapsed reference mousse represented a bigger volume before the freeze-thaw cycle. Considering a 33% volume loss for the reference mousse and a 24% volume loss for the mousse with milk powder, the 45 mm3 sized samples corresponds to 59.9 mm3 of the fresh reference mousse and to 55.8 mm3 of the fresh mousse with milk powder. Therefore the number of bubbles was corrected to 8594 ± 1004, and 10,303 ± 1173 pores for the reference sample, and the sample with milk powder, respectively, while the number of bubbles in the gelatin samples did not need any correction since no shrinkage was observed due to freeze-thawing. A 55%, and a 36%
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Fig. 6. Theoretic curve of bubble pressure as a function of bubble diameter (according to YoungeLaplace equation with g ¼ 40 mN/m).
Fig. 4. a: Number distribution of fresh reference mousse ( ), fresh mousse with gelatin peptides ( ) and fresh mousse with milk powder ( ); b: Number distribution of the bubble diameter of stored reference mousse ( ), stored mousse with gelatin peptides ( ) and stored mousse with milk powder ( ) (12 months frozen storage). The error bars are the standard deviation over 5 replicates.
Fig. 5. Microscopic image of the air bubbles in fresh whipped reference mousse.
reduction of porosity in the CT-range of 11.5e500 mm for the reference sample, and the sample with milk powder, respectively, was observed while an increase of 13% for the recipe containing the gelatin peptides was noted after freezing. When bubble sizes are separated in shares of defined dimensions (Fig. 4), X-ray mCT
revealed that during prolonged freezing, especially the 11.5e50 mm sized bubbles disappeared in the reference mousse and the mousse with milk powder, while these bubbles did not statistically change in number for the mousse including gelatin peptides. In contrast, the latter mousses showed higher numbers of 50e250 mm sized bubbles in response to prolonged freezing. With the present analysis, bubbles with diameters ranging from 11.49 to 500 mm were found to take up a cumulative volume corresponding to 66% and 100% of the overrun for fresh and thawed reference mousses respectively, 67% and 85% of the overrun for fresh and thawed mousses with milk powder respectively and 75% of the overrun for the fresh and thawed mousses with gelatin peptides. However, microscopic observations of the fresh mousses (Fig. 5) show a lot of air bubbles which are smaller than 11.49 mm (voxel resolution mCT-scan) making them invisible for mCT and explaining the less densely packed structure of the tomographic images. It is therefore tempting to speculate that the missing volume underlying the inconsistency between cumulative volume and overrun is taken up for a big part by the fraction of these <11.5 mm sized bubbles. These small sized pores disappear after freezingthawing, thereby explaining why a higher percentage of overrun for the freeze-thawed reference mousse and the freeze-thawed mousse with milk powder can be calculated by mCT. Hence, all bubbles or most bubbles smaller than 11.49 mm dissolved entirely in the matrix of the reference mousse, or the mousse including 2% milk powder, respectively. To image structures at micron resolution, smaller samples must be studied at controlled temperature conditions which is currently not technically feasible (Herremans et al., 2013). New developments in X-ray mCT and in-situ conditioning equipment are therefore necessary to improve the resolution of small sized mousses. As Jang, Nikolov, Wasan, Chen, and Campbell (2005) mentioned, the number of bubbles of a certain size can be increased by the inflow of the bubbles of that size, the breakage of larger bubbles, and the coalescence of smaller bubbles. The number of bubbles of a certain size can be decreased by the outflow of the bubbles of that size, breakage into smaller bubbles, and coalescence into larger bubbles. This system preserves the amount of air in the system. However, the volume of incorporated air in the reference sample and the sample with milk powder decreases with time. Because of the overpressure of the smallest air bubbles (<10 mm) due to the curvature and surface tension according to the YoungeLaplace equation Dp ¼ g (1/R1 þ 1/R2) where p is the bubble pressure, g the surface tension (herein measured as 40 mN/m for the studied mousses) and R the radius of the bubble (Fig. 6), gas diffuses from
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Fig. 7. Tomographic image of the ice crystal formation in unwhipped mousse containing a contrast agent (CsI) at 12 C after three cycles 12/6 C for a: reference recipe; b: reference recipe with 2% gelatin peptides; c: reference recipe with 2% milk powder.
the bubble to the unsaturated aqueous phase and to the atmosphere. As the bubble size decreases, the Laplace pressure inside the bubble increases and the gas loss from the mousse accelerates (Jang, Nikolov, & Wasan, 2006). This provokes volume loss and shrinkage of the mousses (Dutta et al., 2004). As observed in Table 2, the shrinkage of the mousses levelled off towards the end of frozen storage; the aqueous phase is believed to have reached equilibrium, hence the driving force for gas diffusion from the bubbles has been gradually undermined. The loss of air bubbles in the range of 11e50 mm might not be fully accounted for by the YoungeLaplace equation due to the relatively lower inner overpressures calculated to be present in these bubbles (Fig. 6). Since ice crystals are formed in frozen mousses, it might be that bubbles are perforated by these structures and/or that such bigger ice crystals form, upon thawing, watery channels that facilitate gas diffusion out of the affected bubbles saturating the matrix and/or going to the surface and into the atmosphere. Hence, the bigger the ice crystals, so the more damage is to be expected to the foam. The effect of gelatin peptides on ice crystal formation in frozen mousse mixes was therefore studied. Fig. 7 shows contrasted mCT-scans of frozen unwhipped mousses, stressed through repeated freeze-thaw cycling, which reveal that significantly smaller ice crystals developed in mousses containing additional gelatin peptides. This inhibitory effect of gelatin peptides was also shown by Damodaran (2007), who rationalised that gelatin peptides might bind to ice nuclei via hydrogen bonding thereby decreasing the ability of the ice crystal to grow. Our data suggest that freezing-initiated mousse foam damage is being the resultant of an interplay between bubble-wall strength and ice crystal size. Gelatin peptides, although not able to increase the viscosity by themselves, affect these both aspects in that gelatin peptides fortify the foam lamellae and reduce ice crystal size. Gelatin peptides, although too small to form a gel, largely retain the capacity to form triple helical structures with themselves or with bigger sized gelatin molecules also present in the recipe. On top of that, gelatin peptides have surface active properties and are known to stabilise watery foam structures (Djagny, Wang, & Xu, 2001). So, gelatin peptides cover the walls of the foam bubbles and at the same time make contact with immobilised (cfr. part of a rigid gel structure) gelatin molecules. This combined affect should clearly stabilise/fortify the bubble wall. This line of reasoning, combined with the ice crystal inhibiting activity of gelatin peptides likely explains the long term survival of the fraction of small overpressured bubbles in frozen mousses. 4. Conclusions The process of freezing and thawing is highly destabilising to
mousses. Freezing provoked collapse of the mousse, but samples containing gelatin peptides showed no shrinkage. Mousses were shown to have a complex 3-D microstructure using mCT. Micro-CT with a voxel resolution of 11.49 mm allowed measuring the larger bubbles of the matrix. The bubble size and bubble number distribution were measured and quantified and were demonstrated to be dependent on freezing-thawing. The sample with gelatin peptides had a more stable microstructure in comparison with the reference sample and the sample with milk powder. Its fraction smaller bubbles that suffer from increased internal overpressures is better stabilised. This is likely because the gelatin peptides work synergistically with gelatin to enhance the strength and flexibility of the films covering the bubbles and by providing a more rigid and stable matrix. Additionally, smaller ice crystals are observed in mousses with gelatin peptides. The combined effect as a bubble foam stabiliser and an inhibitor of ice crystal formation explains why gelatin peptides inhibit shrinkage of mousses. Acknowledgements This research was funded by the Agency for Innovation by Science and Technology in Flanders (IWT110644). References Bamforth, C. W. (1995). Beer and cider. In S. T. Beckett (Ed.), Physico-chemical aspects of food processing (pp. 417e439). London: Blackie Academic & Professional. Berger, K. G. (1990). Ice cream. In K. Larsson, & S. E. Friber (Eds.), Food emulsions (pp. 367e444). New York: Marcel Dekker Inc. Caldwell, K. B., Goff, H. D., Stanley, D. W., Martin, R. W., Lewis, D. F., & Johari, O. (1992). A low-temperature scanning electron-microscopy study of ice-cream .2. Influence of selected ingredients and processes. Food Structure, 11, 11e23. Camacho, M. M., Martinez-Navarrete, N., & Chiralt, A. (1998). Influence of locust bean gum/lambda-carrageenan mixtures on whipping and mechanical properties and stability of dairy creams. Food Research International, 31, 653e658. Camacho, M. M., Martinez-Navarrete, N., & Chiralt, A. (2001). Stability of whipped dairy creams containing locust bean gum/lambda-carrageenan mixtures during freezing-thawing processes. Food Research International, 34, 887e894. Damodaran, S. (2007). Inhibition of ice crystal growth in ice cream mix by gelatin hydrolysate. Journal of Agricultural and Food Chemistry, 55, 10918e10923. De Schryver, T., Boone, M. A., De Kock, T., Duquenne, B., Christaki, M., Masschaele, B., et al. (2015). A compact low cost cooling stage for lab based X-ray micro-CT setups. In 12th international conference on X-ray microscopy AIP conference proceedings. Dickinson, E. (1992). An introduction to food colloid. Oxford: Science Publications. Dierick, M., Van Loo, D., Masschaele, B., Van den Bulcke, J., Van Acker, J., Cnudde, V., et al. (2014). Recent micro-CT scanner developments at UGCT. Nuclear Instruments & Methods in Physics Research Section B-Beam Interactions with Materials and Atoms, 324, 35e40. Djagny, K. B., Wang, Z., & Xu, S. Y. (2001). Gelatin: a valuable protein for food and pharmaceutical industries: review. Critical Reviews in Food Science and Nutrition, 41, 481e492. Donhowe, D. P., & Hartel, R. W. (1996a). Recrystallization of ice during bulk storage of ice cream. International Dairy Journal, 6, 1209e1221. Donhowe, D. P., & Hartel, R. W. (1996b). Recrystallization of ice in ice cream during
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Food Hydrocolloids journal homepage: www.elsevier.com/locate/foodhyd
Stabilising frozen dairy mousses by low molecular weight gelatin peptides Barbara Duquenne a, *, Bjorn Vergauwen b, Claude Capdepon b, Marijn A. Boone c, e, Thomas De Schryver d, Luc Van Hoorebeke d, Stephanie Van Weyenberg f, Paul Stevens b, Jan De Block a a
Institute for Agricultural and Fisheries Research (ILVO), Technology and Food Science Unit, Brusselsesteenweg 370, Melle, Belgium Rousselot BVBA, Expertise Center, Meulestedekaai 81, Ghent, Belgium UGCT-Pore-scale Processes in Geomaterials Research Group, Department of Geology and Soil Science, Ghent University, Krijgslaan 281 S8, Ghent, Belgium d UGCT-Radiation Physics Research Group, Department of Physics and Astronomy, Ghent University, Proeftuinstraat 86, Ghent, Belgium e X-ray Engineering bvba, De Pintelaan 111, Ghent, Belgium f Institute for Agricultural and Fisheries Research (ILVO), Technology and Food Science Unit, Burg. Van Gansberghelaan 115 Bus 1, Merelbeke, Belgium b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 January 2016 Received in revised form 15 March 2016 Accepted 2 April 2016 Available online 4 April 2016
The effect of low molecular weight gelatin peptides on the shrinkage of thawed mousses was investigated. Changes promoted by freezing-thawing processes were evaluated through texture and volume measurements and through X-ray analysis of the bubble distribution. Freezing provoked collapse of the standard reference recipe mousse and of the reference recipe mousse with 2% milk powder added, but samples containing 2% gelatin peptides showed no shrinkage. The bubble size and bubble number distribution of the different mousses were measured based on high-resolution X-ray tomography. Results indicated that the volume losses experienced by the controls were almost entirely caused by the disappearance of air bubbles having a diameter smaller than 50 mm. Hence, this fraction of overpressurised air bubbles is extra stabilised by the matrix due to the additional presence of gelatin peptides. Moreover, gelatin peptides were found to inhibit ice crystal growth, which resulted in smaller ice crystals that are believed to be less destructive to the microstructure of the freeze-thawed mousses. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Gelatin peptides Dairy mousses Frozen stability Air bubbles
1. Introduction Many food products are frozen to provide long-term stability to the product. The process of freezing and thawing is highly destabilising food products, especially mousses, leading to foam collapse, loss of gas and loss of desired foam structure and texture (Ghosh & Coupland, 2008; Murray & Ettelaie, 2004). The stability and behaviour of food foams such as ice cream, marshmallow and mousses, is closely related to their microstructure, and more exactly, to the air bubble size, distribution and volume fraction (Miquelim, Lannes, & Mezzenga, 2010). Bamforth (1995) and Muller-Fischer and Windhab (2005) noticed that the foams with an even distribution of smaller bubbles were more stable, creamier and more attractive to consumers. Bubble mechanics can be used to
* Corresponding author. ILVO, Technology and Food Science Unit, Brusselsesteenweg 370, 9090 Melle, Belgium. E-mail address: [email protected] (B. Duquenne). http://dx.doi.org/10.1016/j.foodhyd.2016.04.001 0268-005X/© 2016 Elsevier Ltd. All rights reserved.
estimate shelf life of whipped dairy products, since the textural appearance and mouthfeel are a direct consequence of the complex interactions between bubble mechanics and our senses (Niranjan, 1999). High-resolution X-ray tomography (X-ray micro-CT) has recently been introduced as a non-destructive material evaluation technique for the microstructure of food products. The fact that micro-CT can provide information about the internal structure and properties of food, is a major advantage in the study of their conservation. With X-ray micro-CT (mCT) the internal structure of most food products can be visualised by measuring the different attenuations for X-rays of the materials in the product. As explained in Kak and Slaney (2001) and for food materials in particular in Verboven et al. (2008) and Herremans et al. (2013), the level of transmission of these rays depends mainly on the mass density and mass absorption coefficient of the material. Because absorption is different in gas and water, gas-filled spaces can be distinguished from the matrix material.
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Freezing of mousses induces thinning of the lamellae between air cells, due to the cryoconcentration during ice formation, which directly affects foam stability (Camacho, Martinez-Navarrete, & Chiralt, 2001; Dickinson, 1992). Cryoconcentration implies alteration in the aqueous environment of macromolecules because of the separation of the more concentrated phase from the initial solution. Mechanical damage caused by ice crystals on air cell films can be considerable (Berger, 1990). Studies on ice cream have shown that hydrocolloids (locust bean gum, guar gum, xanthan gum, gelatin and carrageenan) exert a cryoprotective effect mainly due to the inhibition of ice crystal growth in the freezer and also by inhibiting ice recrystallisation throughout frozen storage (Caldwell et al., 1992; Donhowe & Hartel, 1996a, 1996b; Flores & Goff, 1999; Hagiwara & Hartel, 1996; Hartel, 1998; Sutton & Wilcox, 1998a, 1998b). Hydrocolloids increase the viscosity of the aqueous phase, hence reducing the availability of free water, and inhibiting de novo and/or recrystallisation events (Stanley, Goff, & Smith, 1996). In contrast to these findings, peptides (2000e5000 Da) obtained from proteolytic hydrolysis of gelatin also prevented ice crystallisation in frozen ice cream mixes while not affecting the product's viscosity (Damodaran, 2007; Wang & Damodaran, 2009). In this work, we aim to investigate the efficacy of low molecular weight gelatin peptides on the shrinkage of frozen mousses. Changes promoted by freezing-thawing processes were evaluated through texture and volume measurements and through X-ray analysis of both air bubbles and ice crystals.
treatment and the interaction as categorical independent variables. Post hoc comparison was done with tukey post hoc test.
2. Material and methods
2.5. Overrun of whipped mousses
2.1. Gelatin peptides
The overrun is a parameter which gives information on the percentage of gas in the whipped mousse. Volume ratio of air incorporated in mousse during whipping was measured in newly whipped mousse. Overrun was measured for each sample using the following equation:
Commercial porcine gelatin peptides with an average molecular weight of 2000 Da were supplied by Rousselot BVBA.
2.4. Volume of whipped mousses Volume measurements were performed at 2 C after thawing, 1, 3, 6, 9 and 12 months after frozen storage. Volume loss during frozen storage was calculated by measuring the new height and diameter of the product. Height loss was measured at 6 different locations by measuring the distance between the surface of the product and a ruler placed on top of the recipient. Loss in diameter was measured at 6 different points as the distance of the edge of the shrunk mousse to the wall of the recipient. From these measurements, the dimensions of the shrunk cylinder was calculated providing the best estimation of the remaining volume. Remaining volume for each sample was calculated using the following equation:
Remainingvolumeð%Þ ¼ 100
where V1 is the volume before freezing of whipped mousse and V2 the volume after freezing and thawing of whipped mousse. For every batch, 3 different samples were analysed. Volume was found not to be normally distributed. Therefore, non-parametric analyses was performed with time and treatment as categorical independent variables.
2.2. Preparation of whipped mousses Commercial mousse recipes containing whole milk, sugar, cream, gelatin, modified starch, mono- and diglycerides of fatty acids, lactic acid esters of mono- and diglycerides of fatty acids, sodium phosphates, carrageenan, vanilla flavour and beta carotene were prepared either or not including 2% gelatin peptides or 2% milk powder. The sample containing 2% milk powder was used as a control sample to have the same dry matter as the recipe with 2% gelatin peptides. After mixing the ingredients, the mixes were heated (5 s, 120e130 C) and homogenised (downstream, 2-steps, 85 bar/25 bar), cooled and whipped in a kitchen aid (Hobart; Ohio, US) during 30 s at stand 2 and 4 min 30 s at stand 3. Mousses were stored in the freezer at 24 C in 0.20 l plastic cups with lid. Cups were completely filled and remainders were scraped away with a knife. Mousses were analysed after 1, 3, 6, 9 and 12 months. Before analysis, samples were removed from the freezer and thawed for 24 h at 2 C. The test was repeated once. 2.3. Texture of whipped mousses Texture of the whipped mousses was performed at 2 C before freezing and after thawing, 1, 3, 6, 9 and 12 months after frozen storage. Hardness of the samples was measured by penetration of a vertical sheet with 2 grooves of 15 mm (stainless steel; 33 33 mm) into the mousse to a depth of 20 mm at a constant speed of 120 mm/min with a Texture Analyser (LF-Plus; Lloyd Instruments Ltd., Hants, UK). For every batch, minimum 3 different samples were analysed. In order to test the effect on hardness (dependent variable), a linear regression was performed with time,
V1 V2 $100 V1
Overrunð%Þ ¼
M1 M2 $100 M2
where M1 is the weight of a fixed volume of unwhipped mousse and M2 the weight of the same volume of whipped mousse. In order to test the effect of treatment on overrun (dependent variable), a linear regression was performed with treatment as categorical independent variable. Post hoc comparison was done with tukey post hoc test. 2.6. Air bubble distribution of whipped mousses The air bubble distribution was studied using optical microscopy and X-ray mCT. In the experiment with the optical microscope (Olympus BH-2, Tokyo, Japan), a small sample of mousse was placed on a microscope glass slide and covered with a glass coverslip. Images were acquired using an Olympus Camera (Camedia C-3040 Zoom). X-ray mCT was performed at the Centre for X-ray Tomography of the Ghent University (UGCT; www.ugct.ugent.be) using the Environmental CT-scanner (EMCT), a custom-built gantry-based laboratory mCT scanner (Dierick et al., 2014). Samples of 1 1 1 cm were used for the mCT experiments. The Hamamatsu L9181-2 X-ray tube was operated at 65 kV and a power of 11.7 W and 1500 projections were taken over 360 with an exposure of 500 ms using a Teledyne Dalsa Xineos-1313 high-speed flat-panel detector. The EMCT was upgraded with a custom-made freezing stage which can control and monitor a sample's temperature with an accuracy of ±0.4 C (De Schryver et al., 2015), making it possible to scan a
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Table 1 Hardness (N) versus storage period (months) for reference mousse, mousse with 2% gelatin peptides and mousse with 2% milk powder. Results are expressed as mean (±SD) of minimum 3 determinations. Storage period 0 Reference 2% gelatin peptides 2% milk powder
1 a
1.04 ± 0.08 1.54 ± 0.11b 1.24 ± 0.07c
3 a
1.05 ± 0.03 1.52 ± 0.07b 1.19 ± 0.07a
6 a
1.02 ± 0.08 1.44 ± 0.21b 1.19 ± 0.07a
9 a
1.12 ± 0.18 1.49 ± 0.09b 1.24 ± 0.16a
12 a
1.25 ± 0.11 1.45 ± 0.08a 0.85 ± 0.19b
1.07 ± 0.15a 1.53 ± 0.10b 1.31 ± 0.07ab
a,b,c
Values with a different letter in the same column significantly differ (p < 0.05).
Table 2 Remaining volume (%) versus storage period (months) for reference mousse, mousse with 2% gelatin peptides and mousse with 2% milk powder. Results are expressed as mean (±SD) of minimum 3 determinations. Storage period 0 Reference 2% gelatin peptides 2% milk powder
100 ± 0 100 ± 0 100 ± 0
1 100 ± 0 100 ± 0 100 ± 0
3 100 ± 0 100 ± 0 100 ± 0
6 73 ± 12 100 ± 0b 86 ± 8c
a
9
12
65 ± 7 100 ± 0 75 ± 16
67 ± 9a 100 ± 0b 76 ± 15a
a,b,c
Values with a different letter in the same column significantly differ (p < 0.05).
Fig. 1. Pictures of a: thawed reference mousse after 12 months frozen storage; b: thawed mousse with gelatin peptides after 12 months frozen storage; c: thawed mousse with milk powder after 12 months frozen storage.
Fig. 2. Tomographic image of the air bubbles in a: fresh reference mousse; b: fresh mousse with gelatin peptides; c: fresh mousse with milk powder; d: thawed reference mousse after 12 months frozen storage; e: thawed mousse with gelatin peptides after 12 months frozen storage; f: thawed mousse with milk powder after 12 months frozen storage.
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Fig. 3. Tomographic 3D image of the air bubbles in a: fresh reference mousse; b: fresh mousse with gelatin peptides; c: fresh mousse with milk powder; d: thawed reference mousse after 12 months frozen storage; e: thawed mousse with gelatin peptides after 12 months frozen storage; f: thawed mousse with milk powder after 12 months frozen storage (green bubbles: 500e750 mm; red bubbles: 250e500 mm; dark blue bubbles: 100e250 mm; light blue bubbles: 50e100 mm). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
sample in a conditioned environment. In this work each sample was conditioned at 4 C with the freezing stage while the 12.5 min long mCT scans were acquired. A 11.49 mm voxel resolution was obtained after reconstruction with the Octopus Reconstruction software (InsideMatters, https://insidematters.eu/). To quantify the individual bubbles, the reconstructed images were analysed with Avizo 9.0 software (FEI, Saint-Aubain, France). 2.7. Ice crystal growth in unwhipped mousses Ice crystal growth was studied using X-ray mCT (EMCT at the Centre for X-ray Tomography of the Ghent University) with 5% CsI (Sigma Aldrich, Belgium) as a contrast agent. Each sample (1 1 1 cm) was frozen in the freezer at 20 C and subsequently conditioned at 19 C with the freezing stage during 15 min. The temperature of the sample was then increased from 19 to 12 C and then cycled between 12 and 6 C at a rate of 1 cycle/60 min. Three cycles were applied. Scans were reconstructed with the Octopus Reconstruction software (InsideMatters, https:// insidematters.eu/). 3. Results and discussion 3.1. Textural characteristics and overrun of whipped mousses Because textural properties are an important component of food quality perception and acceptability (Foegeding, 2006), this parameter was measured for fresh mousses and for thawed mousses after a frozen storage period of 1, 3, 6, 9 and 12 months (Table 1). Mousses with gelatin peptides were significantly harder after 0, 1, 3, 6 and 12 months storage in comparison with the reference mousse. The hardness of the mousses during storage remained constant. The higher hardness for the samples with gelatin peptides can be explained by the lower overrun of these samples (140 ± 10%) in comparison with the reference mousse (153 ± 7%) and samples with milk powder (159 ± 5%). The overrun of the different samples was significantly different (p < 0.01). Camacho, Martinez-Navarrete, and Chiralt (1998) also noticed that hydrocolloids (locust bean gum and carrageenan) conferred a greater foaming resistance to dairy cream increasing the whipping
time and reducing the overrun. 3.2. Frozen stability of the whipped mousses Frozen stability of the thawed whipped mousses has been evaluated through the changes in sample volume and microstructure during freezing and frozen storage. During the first 3 months frozen storage, no shrinkage of the mousses was observed (Table 2). Shrinkage was seen after 4 months frozen storage. Mousses with 2% gelatin peptides showed no volume loss after 12 months frozen storage in contrast to the reference mousse which showed approximately 33% volume loss and mousse with 2% milk powder which showed approximately 24% volume loss (Fig. 1). A comparison of the tomographic images shows different microstructures for the fresh and thawed mousses (Fig. 2). A less densely packed microstructure is observed for the fresh mousses of all tested conditions. Fig. 3 shows the corresponding 3-D renderings of the air bubbles that were further used for the analysis. The individual samples taken for X-ray mCT sized 45 mm3 and bubbles in the range of 11.5e500 mm were counted (Fig. 4). Considering the fresh mousses, 19,258 ± 1680, 13,940 ± 696 and 15,985 ± 2234 bubbles were counted for the reference sample, the sample with gelatin peptides and the sample with milk powder respectively. The 45 mm3 sized samples taken from the thawed mousses contained 12,827 ± 1499, 15,992 ± 1493 and 13,557 ± 1544 bubbles for the reference sample, the sample with gelatin peptides and the sample with milk powder respectively. To compare the number of bubbles in mousses before and after shrinkage, the loss of volume must be taken into account since the 45 mm3 sample taken from a freezethawed collapsed reference mousse represented a bigger volume before the freeze-thaw cycle. Considering a 33% volume loss for the reference mousse and a 24% volume loss for the mousse with milk powder, the 45 mm3 sized samples corresponds to 59.9 mm3 of the fresh reference mousse and to 55.8 mm3 of the fresh mousse with milk powder. Therefore the number of bubbles was corrected to 8594 ± 1004, and 10,303 ± 1173 pores for the reference sample, and the sample with milk powder, respectively, while the number of bubbles in the gelatin samples did not need any correction since no shrinkage was observed due to freeze-thawing. A 55%, and a 36%
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Fig. 6. Theoretic curve of bubble pressure as a function of bubble diameter (according to YoungeLaplace equation with g ¼ 40 mN/m).
Fig. 4. a: Number distribution of fresh reference mousse ( ), fresh mousse with gelatin peptides ( ) and fresh mousse with milk powder ( ); b: Number distribution of the bubble diameter of stored reference mousse ( ), stored mousse with gelatin peptides ( ) and stored mousse with milk powder ( ) (12 months frozen storage). The error bars are the standard deviation over 5 replicates.
Fig. 5. Microscopic image of the air bubbles in fresh whipped reference mousse.
reduction of porosity in the CT-range of 11.5e500 mm for the reference sample, and the sample with milk powder, respectively, was observed while an increase of 13% for the recipe containing the gelatin peptides was noted after freezing. When bubble sizes are separated in shares of defined dimensions (Fig. 4), X-ray mCT
revealed that during prolonged freezing, especially the 11.5e50 mm sized bubbles disappeared in the reference mousse and the mousse with milk powder, while these bubbles did not statistically change in number for the mousse including gelatin peptides. In contrast, the latter mousses showed higher numbers of 50e250 mm sized bubbles in response to prolonged freezing. With the present analysis, bubbles with diameters ranging from 11.49 to 500 mm were found to take up a cumulative volume corresponding to 66% and 100% of the overrun for fresh and thawed reference mousses respectively, 67% and 85% of the overrun for fresh and thawed mousses with milk powder respectively and 75% of the overrun for the fresh and thawed mousses with gelatin peptides. However, microscopic observations of the fresh mousses (Fig. 5) show a lot of air bubbles which are smaller than 11.49 mm (voxel resolution mCT-scan) making them invisible for mCT and explaining the less densely packed structure of the tomographic images. It is therefore tempting to speculate that the missing volume underlying the inconsistency between cumulative volume and overrun is taken up for a big part by the fraction of these <11.5 mm sized bubbles. These small sized pores disappear after freezingthawing, thereby explaining why a higher percentage of overrun for the freeze-thawed reference mousse and the freeze-thawed mousse with milk powder can be calculated by mCT. Hence, all bubbles or most bubbles smaller than 11.49 mm dissolved entirely in the matrix of the reference mousse, or the mousse including 2% milk powder, respectively. To image structures at micron resolution, smaller samples must be studied at controlled temperature conditions which is currently not technically feasible (Herremans et al., 2013). New developments in X-ray mCT and in-situ conditioning equipment are therefore necessary to improve the resolution of small sized mousses. As Jang, Nikolov, Wasan, Chen, and Campbell (2005) mentioned, the number of bubbles of a certain size can be increased by the inflow of the bubbles of that size, the breakage of larger bubbles, and the coalescence of smaller bubbles. The number of bubbles of a certain size can be decreased by the outflow of the bubbles of that size, breakage into smaller bubbles, and coalescence into larger bubbles. This system preserves the amount of air in the system. However, the volume of incorporated air in the reference sample and the sample with milk powder decreases with time. Because of the overpressure of the smallest air bubbles (<10 mm) due to the curvature and surface tension according to the YoungeLaplace equation Dp ¼ g (1/R1 þ 1/R2) where p is the bubble pressure, g the surface tension (herein measured as 40 mN/m for the studied mousses) and R the radius of the bubble (Fig. 6), gas diffuses from
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Fig. 7. Tomographic image of the ice crystal formation in unwhipped mousse containing a contrast agent (CsI) at 12 C after three cycles 12/6 C for a: reference recipe; b: reference recipe with 2% gelatin peptides; c: reference recipe with 2% milk powder.
the bubble to the unsaturated aqueous phase and to the atmosphere. As the bubble size decreases, the Laplace pressure inside the bubble increases and the gas loss from the mousse accelerates (Jang, Nikolov, & Wasan, 2006). This provokes volume loss and shrinkage of the mousses (Dutta et al., 2004). As observed in Table 2, the shrinkage of the mousses levelled off towards the end of frozen storage; the aqueous phase is believed to have reached equilibrium, hence the driving force for gas diffusion from the bubbles has been gradually undermined. The loss of air bubbles in the range of 11e50 mm might not be fully accounted for by the YoungeLaplace equation due to the relatively lower inner overpressures calculated to be present in these bubbles (Fig. 6). Since ice crystals are formed in frozen mousses, it might be that bubbles are perforated by these structures and/or that such bigger ice crystals form, upon thawing, watery channels that facilitate gas diffusion out of the affected bubbles saturating the matrix and/or going to the surface and into the atmosphere. Hence, the bigger the ice crystals, so the more damage is to be expected to the foam. The effect of gelatin peptides on ice crystal formation in frozen mousse mixes was therefore studied. Fig. 7 shows contrasted mCT-scans of frozen unwhipped mousses, stressed through repeated freeze-thaw cycling, which reveal that significantly smaller ice crystals developed in mousses containing additional gelatin peptides. This inhibitory effect of gelatin peptides was also shown by Damodaran (2007), who rationalised that gelatin peptides might bind to ice nuclei via hydrogen bonding thereby decreasing the ability of the ice crystal to grow. Our data suggest that freezing-initiated mousse foam damage is being the resultant of an interplay between bubble-wall strength and ice crystal size. Gelatin peptides, although not able to increase the viscosity by themselves, affect these both aspects in that gelatin peptides fortify the foam lamellae and reduce ice crystal size. Gelatin peptides, although too small to form a gel, largely retain the capacity to form triple helical structures with themselves or with bigger sized gelatin molecules also present in the recipe. On top of that, gelatin peptides have surface active properties and are known to stabilise watery foam structures (Djagny, Wang, & Xu, 2001). So, gelatin peptides cover the walls of the foam bubbles and at the same time make contact with immobilised (cfr. part of a rigid gel structure) gelatin molecules. This combined affect should clearly stabilise/fortify the bubble wall. This line of reasoning, combined with the ice crystal inhibiting activity of gelatin peptides likely explains the long term survival of the fraction of small overpressured bubbles in frozen mousses. 4. Conclusions The process of freezing and thawing is highly destabilising to
mousses. Freezing provoked collapse of the mousse, but samples containing gelatin peptides showed no shrinkage. Mousses were shown to have a complex 3-D microstructure using mCT. Micro-CT with a voxel resolution of 11.49 mm allowed measuring the larger bubbles of the matrix. The bubble size and bubble number distribution were measured and quantified and were demonstrated to be dependent on freezing-thawing. The sample with gelatin peptides had a more stable microstructure in comparison with the reference sample and the sample with milk powder. Its fraction smaller bubbles that suffer from increased internal overpressures is better stabilised. This is likely because the gelatin peptides work synergistically with gelatin to enhance the strength and flexibility of the films covering the bubbles and by providing a more rigid and stable matrix. Additionally, smaller ice crystals are observed in mousses with gelatin peptides. The combined effect as a bubble foam stabiliser and an inhibitor of ice crystal formation explains why gelatin peptides inhibit shrinkage of mousses. Acknowledgements This research was funded by the Agency for Innovation by Science and Technology in Flanders (IWT110644). References Bamforth, C. W. (1995). Beer and cider. In S. T. Beckett (Ed.), Physico-chemical aspects of food processing (pp. 417e439). London: Blackie Academic & Professional. Berger, K. G. (1990). Ice cream. In K. Larsson, & S. E. Friber (Eds.), Food emulsions (pp. 367e444). New York: Marcel Dekker Inc. Caldwell, K. B., Goff, H. D., Stanley, D. W., Martin, R. W., Lewis, D. F., & Johari, O. (1992). A low-temperature scanning electron-microscopy study of ice-cream .2. Influence of selected ingredients and processes. Food Structure, 11, 11e23. Camacho, M. M., Martinez-Navarrete, N., & Chiralt, A. (1998). Influence of locust bean gum/lambda-carrageenan mixtures on whipping and mechanical properties and stability of dairy creams. Food Research International, 31, 653e658. Camacho, M. M., Martinez-Navarrete, N., & Chiralt, A. (2001). Stability of whipped dairy creams containing locust bean gum/lambda-carrageenan mixtures during freezing-thawing processes. Food Research International, 34, 887e894. Damodaran, S. (2007). Inhibition of ice crystal growth in ice cream mix by gelatin hydrolysate. Journal of Agricultural and Food Chemistry, 55, 10918e10923. De Schryver, T., Boone, M. A., De Kock, T., Duquenne, B., Christaki, M., Masschaele, B., et al. (2015). A compact low cost cooling stage for lab based X-ray micro-CT setups. In 12th international conference on X-ray microscopy AIP conference proceedings. Dickinson, E. (1992). An introduction to food colloid. Oxford: Science Publications. Dierick, M., Van Loo, D., Masschaele, B., Van den Bulcke, J., Van Acker, J., Cnudde, V., et al. (2014). Recent micro-CT scanner developments at UGCT. Nuclear Instruments & Methods in Physics Research Section B-Beam Interactions with Materials and Atoms, 324, 35e40. Djagny, K. B., Wang, Z., & Xu, S. Y. (2001). Gelatin: a valuable protein for food and pharmaceutical industries: review. Critical Reviews in Food Science and Nutrition, 41, 481e492. Donhowe, D. P., & Hartel, R. W. (1996a). Recrystallization of ice during bulk storage of ice cream. International Dairy Journal, 6, 1209e1221. Donhowe, D. P., & Hartel, R. W. (1996b). Recrystallization of ice in ice cream during
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