*2.3. Nuclear Magnetic Resonance (NMR) Technique*

Content, purity, and molecular structure of a sample can be determined using an analytical chemistry technique called nuclear magnetic resonance (NMR). When the NMR technique is used it is possible to quantitatively analyze mixtures containing known compounds. For unknown compounds, NMR can either be used to match against spectral libraries or to infer the basic structure directly. The NMR technique can be used to determine molecular conformation in solution and to study physical properties at the molecular level such as conformational exchange, phase changes, solubility, and diffusion. In order to achieve the desired results a variety of NMR techniques are available. In addition, NMR is versatile and has the potential to be nondestructive, which makes it a potential tool in quality control of various products, including milk-based desserts [35].

The principle of the method relates to the fact that many nuclei possess spin and all nuclei are electrically charged. If an external magnetic field is applied, energy transfer is possible from the base energy to a higher energy level (generally a single energy gap). The energy transfer takes place at a wavelength that corresponds to the radiofrequencies, and when the spin returns to its base level, energy is emitted at the same frequency. The signal that matches this transfer is measured in many ways and processed in order to obtain an NMR spectrum for the charged nucleus [36,37].

The NMR technique has been shown to be an appropriate method to calculate the amount of unfrozen water in a food sample. Lucas et al. (2004) [38] examined liquid from the solid water in aqueous sucrose solutions (sucrose and/or casein). They considered spin-spin relaxation measurements that were usually used and also spin-lattice ones. They showed that spin-lattice relaxation provides information about the ice molecular structure. This work confirmed that the ice phase in the case of sucrose solutions is composed of pure water.

Hagiwara et al. (2006) [39] investigated the relationship between the recrystallization rate of ice crystals in sugar solutions (sucrose, maltose, glucose and fructose) and the water mobility in a freeze-concentrated matrix. They observed ice crystals during the recrystallization process using the cryomicroscope system. Their study was complemented with an NMR study in order to examine water mobility via the self-diffusion coefficient of the water component. They found that the recrystallization rate in a variety of sugar solutions depended significantly on the water mobility in the freeze-concentrated matrix and that the self-diffusion coefficient of the water component was a useful parameter to predict and control the recrystallization rate. Brown et al. (2014) [40] used NMR relaxation and time-dependent self-diffusion measurements to monitor the three-dimensional changes to the vein network in ices with and without addition of the ice binding proteins (IBP called antifreeze protein and AFP). They found that the NMR technique was useful in evaluation of the impact of IBPs (among other things) on the vein network structure and the recrystallization process. The IBPs were found useful to inhibit recrystallization and to modify the three-dimensional ice structures, resulting in persistent small size of ice crystals and shorter diffusion of distances along the vein.

It has been discovered that NMR could be used to determine fat globule size in ice cream and to determine the effect of the formulation on hard ice creams' structure [36,41,42]. Lucas et al. (2005) [43] presented the NMR technique as a nondestructive method to characterize the behavior of both fat and water in ice cream mixtures in the frozen state. They proved that the NMR technique described the crystallized and liquid phases separately, and that they could be applied to determine the amount of unfreezable water and mobility of the freeze-concentrated phase. The NMR technique was also used to determine the impact of the quantity of crystals and their organization on the mechanical properties and textures of ice cream mixes [35,43].

The NMR technique does not involve any thermal processes to assess the amount of ice and thus can be performed at a stable temperature. It is also important that the relaxation parameters of water were used to provide information on the water/non-water molecule interactions. This technique may be a valuable tool for understanding how various stabilizers affect the three-dimensional vein network and recrystallization processes.
