*2.4. Splat-Cooling Assay*

In order to determine the ice recrystallization inhibition (IRI) activity of ice cream stabilizers, the capillary method or splat-cooling assay can be performed [44]. Although it is an older method, it is a simple and valuable capillary method for studying recrystallization inhibition which is usually based on loading the samples into 10 μL glass capillaries (51 mm long, 1 mm outer diameter) by capillary action. Capillaries containing dilution series (for example different AFP concentrations to determine at what level that IRI activity is lost) are later folded together exactly, with no space between adjacent capillaries. Subsequently, all the series were snap frozen in different organic compounds, for example, 2,2,4-trimethylpentane or 95% ethanol and cooled to approximately −60◦C with dry ice. After the snap freezing, samples were immersed in a jacketed beaker filled with 50% ethylene glycol in order to maintain the temperature at the level of −6 ◦C. The incubation took 16 to 20 h and later microscopy and image capture were done using polarizing light filters [45,46]. These methods used a simple set-up and allowed the analysis of the IRI activity for a series of samples within one field of view. Tomczak et al. (2003) [45] claimed that the capillary method allowed samples to be aligned and viewed simultaneously, which facilitated the determination of the IRI endpoint. They noted that after the samples have been prepared they could be archived in a freezer for future IRI activity analysis. In fact, sample preparation was not so easy and could be problematic.

The IRI activity of different substances such as AFP and some polysaccharides can be demonstrated using "splat" assay. This is not a new method and has been used for nearly twenty years. In these experiments, a small volume of sample was usually expelled from a height of 1.5 to 3 m onto a metal plate (for example an aluminum block) that had been cooled in liquid nitrogen or dry ice. The sample drop froze upon hitting the metal plate, forming a thin (splat) wafer of ice. This wafer was later transferred to a microscope stage at a high sub-zero temperature where the sample recrystallized over time and annealing temperature (for example: −10, −8, −6, −4 ◦C). Different modifications of this method are possible, for example, the sample could be snap frozen between two cover slips. This technique was complemented by crossed polarizers with a dissecting microscope and ice-binding activities of some substances that were examined by Raymond and Knight (2003) [47]. The splat cooling technique had the advantage that ice crystal growth was directly observed and hence it was easier to interpret. However, preparation of each individual coverslip was problematic, and in addition must be photographed separately and only after the photographs or images are assembled and analyzed [45,46,48].

#### *2.5. Microscopy and Image Analysis*

The most popular and universal method of testing and describing the recrystallization process is direct light microscopy observation followed by image analysis of the ice crystals. When the work was started with another technique such as oscillatory thermos-rheometry or splat cooling assay, to complete it the work was usually supported by an appropriate microscopy technique, a cryo-scanning electron microscope or a microscope with a polarizer [32,44]. Physicists studying polar ice structures proposed examining the structures by the method of direct observation using an optical microscopy with episcopic coaxial lighting [49], and it was later adapted by Faydi et al. (2001) [50]. Caillet et al. (2003) [51] recommended the direct microscopy method as a good technique for analysis of frozen food structure. They compared it with two other methods: a destructive method by dispersion and observation by light microscopy; and an indirect method, by scanning electron microscopy after freeze-drying the sample. The three methods examined led to the same conclusions for the examination under the same conditions of freezing.

Donhowe and Hartel (1996a, 1996b) [16,17] examined sizes of ice crystals in ice cream with an optical microscope placed in a refrigerated glove box. Samples of ice cream were stored at −14 ◦C for several hours. Photomicrographs of ice crystals were taken within 15 min of the sample preparation (no change in ice crystal size occurred over the time period of measurement). Negatives were enlarged and analyzed. This assay led to the conclusion that recrystallization in ice cream stored in bulk containers increased with mean storage temperature for both constant and varying temperatures. Ice crystal sizes in these samples increased linearly with time as well.

Regand and Goff (2003) [12] presented a study of stabilized ice cream model systems. Small drops of different solutions with the addition of some hydrocolloid stabilizers were placed between a slide and cover slips, frozen to −50 ◦C, then cycled between −3.5 ◦C and −6 ◦C on a cold stage of the light microscope, and then the images were acquired using a camera. The ice crystals were later counted and measured individually from the images (at least 200 crystals for the sample). They based the conclusion on a logistic model of ice crystal size distributions characterized earlier by Flores and Goff (1999a, 1999b) [52,53]. This method allowed them to obtain the ice crystal diameter at 50% of cumulative distribution of the sample (X50) and the slope of cumulative distribution at X50. The recrystallization rate was calculated as the slope of linear regression of the curve plotted with values of X50 for each cycle. Using this method, they proved there was significant retardation of recrystallization with the addition of sodium alginate and xanthan. The same technique (using different equipment and a different image analysis program) was first used to explain the IRI activity of kappa carrageenan hydrolysates in model sucrose solutions [4] (Table 1) and later in ice cream sorbet [1] (Table 2).


**Table 1.** X50 parameter value for model sucrose solutions with the addition of kappa carrageenan and its hydrolysates (value estimated from data presented by Kami ´nska-Dwórznicka et al., 2015 [4]).

Explanatory notes: suc: sucrose solutions, KK: kappa carrageenan, 3 h HCL: hydrolysates after 3 h of hydrolysis in HCL acid, 1.5 h H2SO4: hydrolysates after 1.5 h of hydrolysis in H2SO4 acid.

**Table 2.** X50 parameter value for strawberry sorbet with the addition of kappa carrageenan and its hydrolysates (value estimated from data presented by Kami ´nska-Dwórznicka et al., 2015 [1]).


Explanatory notes: KK: kappa carrageenan, 3 h HCL: hydrolysates after 3 h of hydrolysis in HCL acid, 1.5 h H2SO4: hydrolysates after 1.5 h of hydrolysis in H2SO4 acid.

The method described above makes it possible to analyze not only the changes in diameter of ice crystals but also their shape and location. On the basis of the images it is possible to analyze the mechanism of recrystallization. From the shape of the ice crystal, it is sometimes easy to read that accretion between adjacent crystals occurred (Figure 1). Numerous studies have shown that the shape of the ice crystal is influenced by the temperature cycle and by the addition of active substances. Gaukel et al. (2014) [8] focused on crystal morphology in model sucrose solutions with the addition of different types of AFPs. They constructed their theory based on microscope and image analysis. On the basis of the shape of the ice crystals, they claimed that the kappa carrageenan molecule interacted with the ice crystal surface similar to the AFP interactions. The AFPs (also called IBPs) were identified in the blood of Antarctic fish, and then they were found in different organisms. It was discovered [54] that by adsorption of AFP to the ice crystal surface, ice growth was only possible between proteins, leading to a micro curvature. The exact IRI mechanism of AFP is still not fully understood. In the case of pure sucrose solution (Figure 1), typical round ice crystals were formed. In contrast (Figure 3), the shape of ice crystals in sucrose with AFP III present was angular, elongated, and gave the illusion of a three-dimensional structure.

**Figure 3.** Microscopic image of ice crystals in strawberry sorbet with the addition of AFP III (0.000002%) after one month of storage at −18 ◦C (own work, not published).

When the growth rate of ice crystals in samples with the IRI active substances is small, there is no difference in morphology or size of ice crystals, as was observed by Kami ´nska-Dwórznicka et al. (2016) [5] for kappa carrageenan hydrolysates added to model sucrose solutions (Figure 4).

**Figure 4.** Microscopic images of ice crystals in model sucrose solutions with the addition of enzymatic hydrolysates of κ-carrageenan (after HCL hydrolysis), after 24 h (**a**), and 96 h (**b**) of storage at −8 ◦C (Kami ´nska-Dwórznicka et al., 2016 [5]).
