Foods Under the Microscope

Alejandra Regand, Ph.D.

Alejandra Regand is currently a Post-doctoral fellow at Ryerson University, Toronto, Canada. Information about H. D. Goff may be found in his earlier contributions in this series of Guest Food Microscopists 1 
Ken Baker owns his company, Microscopy, Imaging and Analysis in Acton, Ontario, Canada.

Effects of stabilizers on ice recrystallization in sucrose and sucrose-skim milk solutions: A light microscopy study

Alejandra Regand1, Douglas Goff1, Ken Baker2
1Department of Food Science, University of Guelph, Guelph, ON, Canada N1G 2W1 
2Ken Baker, Microscopy, Imaging and Analysis, 4943 Fourth Line, Acton, ON, Canada, L7J 2L8

Polysaccharide or protein hydrocolloids are known to retard ice recrystallization in frozen systems during storage at fluctuating temperatures, but the mechanism is not clear. Hydrocolloid stabilizers were labeled with rhodamine isothiocyanate (RITC) and incorporated into solutions of sucrose (24%) and sucrose (16%) with skim milk powder (SMP) (14.7%). Solutions contained either no stabilizer or 0.3% of carrageenan, carboxymethyl cellulose (CMC), xanthan gum, sodium alginate, locust bean gum (LBG), or gelatin. For light microscopy, a small drop of the solution was placed on a glass microscope slide, cover slipped and secured within a cold stage (Linkam Scientific Instruments, UK) mounted on an Olympus BX-60 microscope. The solutions were quench frozen to -50°C, precycled in order to get similar ice crystal size at t=0 (p<0.05) and cycled between -3.5°C and -6°C, 5 times. Samples were observed using either transmitted yellow/green light or epifluorescence illumination with an Olympus rhodamine filter set. Two images per field, one transmitted and one epifluorescence, were acquired at t=0 and at -3.5°C of each cycle. Images were acquired using a Photometrics SenSys 1401E monochrome camera. Adobe PhotoShop and the Image Processing Tool Kit (1) were used for all image processing and image analysis. Quantitative image analysis was used to measure the equivalent circular diameter (2) of ice crystals in all brightfield images. Recrystallization rate was then calculated as the slope of the linear regression of the ice crystal median diameters obtained from the brightfield data.

Figure 1 Brightfield (a) and fluorescence (b) images collected from the same field for locust bean gum in sucrose solution

Fig. 1. Brightfield (a) and fluorescence (b) images collected from the same field for locust bean gum in sucrose solution. 
1: Before freezing at 22°C, 2: t=0, 3: Cycle 1, 4: Cycle 3, 5: Cycle 4, 6: Cycle 5, 7: After cycling at 0°C.

Figure 1 shows both the brightfield and fluorescence microscopy images collected from the same field for the LBG + sucrose sample. The source of fluorescence is the labeled polysaccharide, which enables its location in the unfrozen phase to be seen. After cycling, the formation of a gel-like fluorescent structure surrounding the ice crystals was observed. Once the crystals were melted (-2°C), the LBG network remained intact.

Figure 2 Comparison between fluorescent images at 0°C from stabilizers in sucrose solutions after cycling and ice crystal melting, in the absence (a) or presence (b) of skim milk solids.

Fig. 2. Comparison between fluorescent images at 0°C from stabilizers in sucrose solutions after cycling and ice crystal melting, in the absence (a) or presence (b) of skim milk solids.  
1: No stabilizer, 2: Locust bean gum, 3: Xanthan gum, 4: Carboxymethyl cellulose, 5: Gelatin, 6:
Carrageenan, 7: Alginate.

Figure 2 shows the structures from stabilizer-sucrose solutions resulting from cycling, in the presence or absence of SMP. The only sucrose solutions in the absence of milk protein that developed a gel-like structure after cycling were those that contained locust bean gum. The recrystallization rate in these solutions was, however, similar to that in the control samples. In contrast, the recrystallization rate was significantly reduced by alginate and xanthan (p>0.05) (Table 1). Conversely, gelatin was the only stabilizer tested which did not retard ice recrystallization in sucrose-skim milk solutions (Table 1). It was observed to form distinctive gels with milk proteins. Similar gels were also formed in the presence of locust bean gum or carrageenan in sucrose solutions which contained milk solids (Figure 2).

Table 1. Ice recrystallization rates in sucrose solutions and in sucrose solutions containing skim milk powder (SMP) solids as affected by various stabilizers

Table 1
Stabilizer1 Sucrose solutions Sucrose solutions with SMP 
None 4.02a 4.59a
LBG 4.05a 3.17c
Carrageenan 3.87a 3.02c
CMC 3.32a,b 2.83c
Gelatin 3.65a,b 4.13a,b
Alginate 2.77b 2.78c
Xanthan 2.82b 3.58b,c

1Stabilizer concentrations used: Carrageenan at 0.05% w/w. All others at 0.3% w/w. 
a,b,c Values with the same letter in the same column do not differ (a=0.05).

It is therefore suggested 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. However, a structural molecular arrangement that keeps the melting water in close proximity to the ice crystal surface and ensures that this water refreezes onto the original crystal rather than migrating to the surface of a larger crystal elsewhere, must be present. The molecular interactions between polysaccharides and proteins appear to be key factors in retarding ice recrystallization.


  1. Russ, C. and Russ, J. C., Image Processing ToolKit,
  2. Russ, J. C. 1998. In "The Image Processing Handbook" 3rd edition, CRC Press, Boca Raton, FL, US

© Alejandra Regand 2001