1869

Magnetic Resonance Studies of Food Freezing

William L. Kerr
Department of Food Science and Technology, University of Georgia, Athens, Georgia 30602, USA

Part III

Introduction
Freezing is one of the fundamental operations used for food preservation, as the reduction in temperature reduces the rate of deleterious chemical reactions and microbial growth. In some cases, as with ice cream and sherbet, freezing imparts desirable coolness and texture. Many foods are frozen including fruits, vegetables, muscle foods, eggs, dairy products, and dough, as well as an assortment of frozen entrees. Freezing is a complex physicochemical process involving the development of crystalline, liquid, and glassy states. Some damage may occur as ice develops and causes migration of water out of cells, or from one region to another. Several questions regarding freezing are of interest to food engineers: How long does it take an item to freeze? What size are the ice crystals that develop? What is the nature of unfrozen water in foods? What effect does freezing have on quality? How are food constituents affected by freezing? How does freezing inuence post-thawing processes? A variety of tools have emerged to study frozen foods, including X-ray diffraction, calorimetry, microscopy, and mechanical spectroscopy. As most foods contain water or oil, the use of 1 H NMR seems a natural approach for studying the quantity and dynamics of these components. Proton NMR has also proven useful for studying food structure. Other magnetic nuclei, including 31 P, 23 Na, and 13 C have been monitored to better understand the chemistry of food molecules and biopolymers during freezing. The study of materials containing ice can prove problematic to NMR spectroscopists. Whereas liquid water has magnetic spin relaxation times up to 12 s, or in the millisecond range when inside foods, ice has a spin relaxation time of tens of microseconds. In addition, obtaining eld homogeneity can be difcult. This article reviews the several NMR methods for studying frozen systems, and is categorized into relaxometry techniques, pulsed eld gradient diffusion techniques, magnetic resonance imaging (MRI), chemical spectroscopy, and solid-state NMR.

Spin Relaxometry
One of the rst reports on the use of NMR in frozen biological material was by Wikefeldt in 1971 [1]. Using continuous wave 1 H NMR, he tested the hypothesis that the rate of ice growth in small blood-drops was controlled by the translational mobility of water. He used the line width v 1/2 = 1/ T2 as the measure of mobility, but recognized that mobility might be inuenced, and even dominated by rotational motion in addition to translation. In more recent times, Ablett et al. found that recrystallization in frozen sugar solutions is correlated with molecular mobility as measured by time domain NMR. In addition, they found that the glass transition temperature (Tg ), and the temperature difference (T Tg ), are correlated with ice recrystallization rates [2].

Ice, Unfrozen Water, and Glassy States

The quantity of ice that exists in a frozen food at a given temperature is of great interest to food scientists. Traditionally, this has been determined by adiabatic calorimetry, differential scanning calorimetry (DSC), or differential thermal analysis (DTA). 1 H NMR has been investigated as a means for quantifying liquid and solid states in frozen materials, as the solid ice protons have a much shorter decay than the liquid water protons [3,4]. Studies have been conducted on sucrose solutions [4], sucroseprotein solutions [5], NaCl solutions [6], water and oil emulsions [7], porous media [8], food gels [9,10], orange juice [11], and wheat dough [12,13]. Free induction decay (FID) experiments following a 90 pulse have been most useful, as measurement time is short and dynamics in the microsecond range can be captured. The initial post dead time signal, at around 10 s, is proportional to the number of liquid water, ice, and food constituent protons, while the signal at 70 s is proportional to the liquid state protons. In some cases, tting with a series of Gaussian curves has been used to extrapolate to the zero time amplitude [13]. The dependence of the magnetization magnitude on temperature must be accounted for, and

Graham A. Webb (ed.), Modern Magnetic Resonance, 18691878.

C

2008 Springer.

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is described by Curies law: M0 = Bo N 2 h 2 I (I + 1) 3kT (1)

For sucrose solutions, the proportion of protons in the solid phase was given as[4] F =1 M0,F TF M0,L TL (2)

where M0 is the initial signal amplitude, and T is the temperature of the frozen (F) and control liquid (L) samples. For a few sugar solutions, the phase diagrams determined by NMR have been compared with those determined calorimetrically, and these are typically in agreement [4]. FID experiments using both H2 O and D2 O have been used to study the effects of cryoprotectants on the eutectic crystallization of NaCl in frozen solutions [6]. As the temperature of frozen NaCl solutions was increased, a sharp increase in the number of liquid-phase protons occurred, which corresponded with the eutectic temperature as measured by DSC. When cryoprotectant sugars were added to NaCl solutions, the number of liquid protons increased, with no apparent change at the expected eutectic temperature. As a result, they concluded that the addition of sugar inhibits eutectic crystallization of NaCl through formation of an amorphous mixture of NaCl, sugar, and water between ice crystals. Few NMR measurements of the ice content in semisolid frozen foods have been reported, and there are even fewer cases where concurrent calorimetric data is present. The solid and liquid phases of frozen bread dough have been studied using single pulse techniques. Typically, the FID relaxation curves can be resolved into a single fast component (< 20 s), and one or two slower components [12,13]. In a previous study, the most dramatic decrease in liquid water occurred between 0 and 10 C. At 30 C, a precipitous increase in the solid component, and decrease in the liquid component, was observed and attributed to a glass transition. These results were consistent with CPMG experiments, and were highly correlated with dynamic mechanical thermal analysis (DMTA) and dielectric thermal analysis (DETA) measurements. These authors also found that the liquid signal may increase during frozen storage, which they attributed to the growth of ice crystals and redistribution of water. Cornillon et al. used FID experiments to measure the amount of water in different states of various food gels between 40 and 20 C, then used the data to more accurately predict thermophysical properties of the materials [14]. At 40 C, they observed some liquid-like signal, and attributed this to unfreezable, or bound, water. The

amount of unfreezable water ranged from 0.05 to 0.66 g/g dry matter, depending on the gel type and whether sucrose was incorporated in the gel. They also found that 90% of the water was frozen between 0 and 10 C. Kim et al. [15] studied cornstarch gels prepared with 10 or 30% various sugars and sugar alcohols. They found that for non-sugar gels, the onset of melting (Tm ) occurred at 10 C, while the addition of sugars decreased the onset of melting to lower temperatures. They also found evidence of the occurrence of non-equilibrium freezing. In particular, sugar addition and rapid cooling promoted the formation of a glassy state, with a concomitant reduction in the amount of ice formed. At high cooling rates, the temperature may decrease such that the water is incorporated in a glassy matrix and prevented from further crystallization [16]. Indeed, many measurements of glass transitions in frozen foods incorporate an annealing process in which the sample is held just above the glass transition temperature (Tg ), thereby allowing the maximum amount of ice to be formed. In addition to quantifying the amount of unfreezable water, NMR has been used to better understand the glassy state of frozen foods. Glassy materials are amorphous solids with little mobility on everyday time scales. As the temperature of a glassy material is increased, one or more glass transitions occur at characteristic temperatures, corresponding to the onset of coordinated molecular motions in the polymer chains [17]. Such transitions in foods, and particularly in frozen foods, are much more complex. First, foods are usually comprised of a variety of biopolymer carbohydrates, proteins, and a myriad of small and medium molecular weight substances. In addition, ice readily forms in high moisture foods; however, a glassy phase may coexist at temperatures below a characteristic temperature (Tg ). A noticeable change in the spinspin relaxation time, T2 , often occurs at the glass transition temperature [13]. Several studies have suggested that some mobile water remains, even at very low temperatures. For example, Ullmann et al. used spin spin relaxation to study the effects of freeze-concentration on proteinase-catalyzed peptide synthesis, and found that some mobile water existed even at 70 C [18]. In a series of malto-oligomer solutions, Ablett et al. observed a fast (low mobility) and slow (high mobility) relaxing component of the spinspin relaxation [19]. As the slow relaxing component disappeared when the experiment was conducted with D2 O, they associated this with water. They found that, upon freezing, there was a signicant reduction in the intensity of the slow component down to 10 C, at which point maximum ice was assumed to have formed. These authors were quick to note, however, that signal intensity was not completely due to mobile water, as chemical exchange with labile protons enhances the apparent signal. At lower temperatures,

Magnetic Resonance of Food Freezing

Spin Relaxometry 1871

some portion of the water signal still persists, suggesting that there are mobile water protons in the glassy state. Strictly speaking, this indicates only that proton rotational motion persists, and this was better quantied with spinlattice (T1 ) measurements. They also conducted pulsed eld gradient experiments, which suggested that some translational motion of water occurs in the glassy state. NMR studies have also been conducted to determine the ice and unfrozen water states in model systems. Le Botlan et al. measured T2 and T1 relaxation in water-in-oil and oil-in-water emulsions [20]. In emulsions held in the frozen state, up to four phases may be present. At 13 C, at least 99% of the water existed in the solid state. Several researchers have also studied ice formation in porous media. Rault et al. used 2 H FID to examine ice formation in porous glass with a variety of pore sizes [21]. They showed that only part of the conned water froze, and that a non-frozen interfacial layer approximately 0.5 nm in thickness existed, which explained why water conned to regions less than 1 nm did not freeze. Morishige and Kawano showed that hysteresis occurred between freezing and melting curves in cylindrical pores, becoming more pronounced in larger pores. They attributed this to size-dependent supercooling of water conned in mesopores [22]. In addition, the melting point of water in small spaces, and with high curvature, is expected to be lower than that for bulk water [23]. Watanabe and Mizoguchi also studied porous media, including glass powder, silty soil, and clay saturated with salt solutions. They found that the amount of unfrozen water increased with increasing solute concentration, in the order NaCl = KCl < MgCl2 . In addition, Valiullin and Furo used nuclear magnetic resonance magnetization transfer and NMR cryoporometry to study coexisting liquid and frozen phases in porous glasses [24].

where D is the self-diffusion coefcient of water, and is the volume sink parameter. It can be shown that the measured magnetization is given by M(t) =
V

Part III

(r, t) dV

(4)

Structural Studies
NMR relaxometry has been used to study a variety of structural features in frozen materials, including ice crystal size, ice and water distribution, pore size, and aspects of cellular structure. Most of these studies rely on the effect of relaxation sinks on the apparent relaxation time, and are most often described by the BrownsteinTarr theory [25]. The sinks may be paramagnetic surface impurities, mechanisms that limit molecular tumbling near the surface, or opportunities for chemical exchange. The rate of change of the magnetic moment per unit volume () is given by = D 2 t (3)

In general, M(t) is related to a sum of exponential terms that include the relaxation time (Tn ). If the diffusion of water is sufciently rapid, and the size of the pores is relatively small, then water in the pore can diffuse to a surface and interact with a sink in a time much smaller than that characteristic of the relaxation processes. In this case, M(t) is a single-exponential process, with an averaged T2 (or T1 ) less than that of bulk water and dependent upon the size of the pore. Equations relating Tn to pore size depend upon the particular geometry assumed for the system. In general, porous systems are heterogeneous in size; therefore, one expects a distribution in Tn characteristic of the distribution of pore sizes. In general, magnetization relaxation data can be deconvolved into a distribution of exponential decay processes. One common approach is through the CONTIN program of Provencher [26]. The size of ice crystals in frozen materials is a key determinant to the quality of the food. However, in situ measurement of ice crystal size is problematic. Conventional microscopy requires xing, thin-sectioning, and a well-controlled temperature stage or the ability to place the microscope in a cold environment. Indirect spin spin relaxation techniques have been developed to give a bulk measurement of ice crystal size distribution in some products [27,28]. In frozen starch gels, ice crystals were sublimated by freeze-drying, leaving an intact structure containing spaces where ice crystals once existed. These spaces were lled with acetone, presumably leaving the gel structure unaffected. The starch surface acts as a strong relaxation sink for acetone proton transverse magnetization. Pore sizes were determined by the BrownsteinTarr theory. A multitude of NMR studies have been conducted on the structure of porous materials. Much fewer, however, have been conducted on frozen porous materials. Hills and Le Floch studied transverse relaxation time distributions in randomly packed beds of Sephadex beads [29]. As these included beads of differing radius and cross-link density, this allowed investigation of a broad range of pore size distributions similar to those that might be found in foods. In addition, the dextran matrix provided hydroxyl groups with substantial proton exchange with water. They found that unfrozen Sephadex contained two water-lled compartments consisting of a region inside the beads characterized by T2in as well as a bulk water region outside the beads. Furthermore, diffusion of water between the compartments was sufciently rapid so that an averaged T2

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was observed, which depended on the bead pore size. Ice crystals were found to form inside the Sephadex beads, and with sizes less than the bead diameter. In a series of controlled experiments, they examined the temperature dependence of T2 , and found the existence of two phases, that is, ice surrounded by a concentrated dextran gel. An interesting twist on the theme is the study of porous materials through cryogenic NMR [30]. This approach relies on the fact that water in small pores has a lower equilibrium freezing point. Prepared samples are frozen, raised stepwise to a variety of subfreezing temperatures, and the FID signal intensity determined after the solid signal has decayed. The pore size (R) distribution is derived from the GibbsThompson relationship. The advantage of the cryogenic NMR approach to pore size distribution is that complex properties such as the magnetization sink strength do not need to be measured or assumed. However, materials that are readily deformed or fractured by the formation of ice would give rise to artifacts using this approach.

States of Water
Although many studies exist on the use of NMR relaxometry to examine water states in solutions of protein, carbohydrates, or other solutes, few exist on the properties of frozen solutions. Lucas et al. characterized both spinspin and spinlattice relaxations of water in frozen sucroseprotein solutions between 13 and 20 C [31]. At 13 C, three relaxation components could be distinguished. A short T2 (16.2 s) component was associated with ice, while two longer ones, 818 ms and 103110 ms, were associated with non-exchangeable sucrose protons and cryo-concentrated water protons, respectively. These authors were able to calculate the fraction of ice at each temperature. They also noted that T1 measurements, as opposed to T2 measurements, gave more information on the ice molecular structure. For example, they determined that the ice phase in sucrose or casein solutions contained pure water, and speculated that incorporation of ions or small molecules could be detected. A substantial number of NMR relaxometry studies have focused on the effect of freezing and thawing on structural aspects of cellular materials. Most of these have relied on the observation of distinct multiple relaxation phenomena, which can be associated with magnetic nuclei in different states or regions. As with porous systems, these distinctions cannot be made if molecules of interest rapidly diffuse amongst regions. Cameron et al. investigated the use of T1 as a measure of ice crystal size in rat tissue, tendons, and lens material [32]. They found that the average ice crystal size, as determined by microscopy, had a signicant positive correlation with T1 .

When maize was dried at temperatures between 30 and 25 C, two T1 components were observed, with minimum values at 20 C [33]. They concluded that water mobility decreased with temperature. Hills and Le Floch [29] used spinspin relaxation to study nonfreezing water in potato tissue. In isolated starch granules, distributed exponential analysis showed two water domains, a slower relaxing one outside the granules and a fast relaxing one inside the granules. The relaxation time of the shorter component decreased with temperature, and it was concluded that most unfreezable water was associated with the granules and cell wall material. In whole tissue at 15 C, they identied three peaks, and tentatively associated them with non-freezing water in cytoplasmic macromolecules (1 ms), water in partly frozen starch granules and cell wall material (34 ms), and extracellular or cytoplasmic water or water in vacuoles (10 ms). They also reinforced the idea that unfreezable water protons are still mobile, even when the material is in the glassy state (T < Tg ). Hills and Remigereau used NMR to monitor the subcellular and intercellular redistribution of water in apple tissue during drying and freezing [34]. During freezing at 3 C, ice rst formed in the vacuoles, while the cytoplasmic and cell wall compartments froze at much lower temperatures (<10 C). Similar relaxation time distributions were observed upon thawing as were seen upon freezing. However, upon complete thawing, T2 of vacuole water was 250 ms, as compared to 650 ms of the fresh tissue. In addition, the cell wall and cytoplasmic compartments were less well resolved. These authors concluded that NMR relaxometry is a more useful tool for studying subcellular water distributions than are spatially resolved imaging techniques. Similar observations were found during freezing and thawing of carrot parenchyma cells [35]. In seaweed shoots at 10 C, two fractions of water were observed [36]. The slow (T2 50 ms) fraction was attributed to protoplasmic water and cell wall polysaccharides, and was completely frozen at 25 C. The fast (T2 < 10 ms) fraction was attributed to water-soluble proteins. Freezing of the slow component occurred more rapidly than that of the fast component. Interestingly, some water (2%) related to the fast component remained unfrozen at temperatures as low as 40 C. Muscle foods have also been studied using NMR relaxometry. Heated, pressure treated, and frozen stored cod was studied using spinspin relaxation [37]. In this case, however, T2 data were collected over time for samples held at 20 and 10 C, but thawed before analysis. In fresh cod, a single exponential decay was observed, with T2 = 65 ms. In addition, by plotting the relaxation rate (1/T2 ) vs. the 90 180 pulse spacing, a dispersion in the relaxation rate was seen. This suggested that, in fact, there was a second short relaxation (T2 1 ms) process present. After freezing and thawing, an additional longer

Part III

Magnetic Resonance of Food Freezing

Magnetic Resonance Imaging 1873

Part III

T2 component appeared, and was attributed to the appearance of thaw exudates. Steen and Lambelet investigated NMR as a tool to study the texture of cod mince [38]. The longest T2 of three components increased with temperature and duration of frozen storage. Changes in this component were correlated with degradation of sh texture, as measured by instrumental and sensory techniques. Farouk and Swan used spinlattice (T1 ) relaxation to study the combined effects of rigor temperature, electrical stimulation, and frozen storage (1 month at 20 C) on hot-boned beef [39]. They reafrmed that most of the water in the muscle cell exists between the thick and thin laments. They found that T1 values were shorter for frozen/thawed vs. 24 h post mortem beef, which they attributed to changes in muscle proteins as a result of denaturation or aggregation. They also found that T1 after freezing decreased with initial rigor temperature, and argued that this was related to increased protein denaturation and decreased water holding capacity.

cryogels, containing bovine serum albumen, were formed by freezing at 20 C, followed by warming to 20 C [43]. They found that diffusion of PVA sidechains was higher for the gels compared to PVA solutions, and increased with temperature up to 45 C, but decreased at higher temperature. In contrast, diffusion of water in the gels increased with temperature but did not show a maximum point. Dimensions of the cryogel were analyzed through the effects on restricted diffusion. The authors determined that the compartmental spaces were determined largely by ice and polymer microcrystallites formed during freezing.

Magnetic Resonance Imaging

While spin relaxation and PFGSE measurements give important information on physical states and structural aspects of frozen systems, they do not provide spatial discrimination of properties. MRI allows the measurement of magnetization intensity, typically at a resolution of 100 500 m, but in the case of microimaging in the 10100 m range. Contrast may be provided by differences in 1 H density, spin relaxation rates, diffusion rates, or even chemical shift. MRI is subject to similar constraints as found in other NMR techniques, and in particular the fact proton relaxation in ice is quite rapid, thus making it invisible to imaging. Thus, experiments are conducted on thawed materials, or rely on signals emanating from unfrozen water during freezing, or unfrozen liquid in equilibrium with ice after the material is frozen.

PFGSE Diffusion Measurements

While spinspin and spinlattice relaxation are sensitive to rotational movement of paramagnetic nuclei, pulsed eld gradient stimulated echo-NMR (PFGSENMR) techniques can determine translational motion of nuclei. Only a few studies exist of PFGSE-NMR in frozen systems. Callaghan et al. designed an NMR spectrometer that uses the Earths magnetic eld to study restricted diffusion in extracted sea ice cores [40]. At 6 C, they found diffusion rates greater than that in pure water. For samples oriented with the sea ice axis parallel to the gradient, the echo attenuation was independent of the diffusion time , indicating brine pockets with dimensions along the axis sufciently large to allow unrestricted diffusion. For samples oriented perpendicular to the gradient, diffusion was restricted at = 200 ms, suggesting pores on the order of 40 m in the perpendicular direction. These authors concluded that water moves tortuously between connected pores with narrow throats. In order to limit artifacts caused by core extraction, a probe was developed that could be inserted directly into the ice sheet [41]. They conrmed the existence of two diffusive elements, and found that water diffusivity was strongly anisotropic, and on short time scales its rapid motion suggested convective transport. Menzel et al. studied salt water ice formed by shock freezing or by slow freezing over several hours [42]. For shock frozen ice, the ice structure contained streak-like porous channels with diameters up to 300 m, and had unrestricted diffusion along one axis. For slowly grown ice, restricted diffusion was observed on the scale of 30 m. NMR diffusion studies have also helped characterize the structure of frozen/thawed gels. Polyvinyl alcohol

Freeze/Thaw Effects
Gamble used MRI to study the effects of freezing on thawed blueberries [44]. The distribution of unmodied water (T2 14 ms) in fresh and frozen/thawed blueberries was obtained using a slice-selective spin-echo technique. The distribution of sugar and motion-modied water (T2 6.6 ms) was also attained, by including an additional hard 180 pulse to invert the 1 H magnetization, then collecting signal at the null point of water. By constructing difference images of blueberries before and after freeze/thawing, Gamble showed that freeze/thaw results in the rupture of water-containing membranes in particular locations in the berry. Several MRI studies have been done on muscle foods. Guiheneuf et al. investigated MRI as a means of authenticating fresh vs. frozen pork [45]. They found that the magnetization transfer (MT) rate increased after freezing/thawing of muscle from the same animal, and attributed this to a decrease in moisture content and denaturation of myobrillar proteins. Evans et al. looked at the effects of freezing and thawing on beef, lamb, and pork placed on 6 6 multi-well plates [46]. By this approach,

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they could simultaneously measure six NMR parameters on 168 meat samples. They found that freeze-thawing caused signicant changes in moisture content, MT, and T1 . However, they did note that interanimal variation would make it difcult to use MRI for authentication. Similar experiments were also conducted on trout [47], cod, and mackerel [48]. They were able to clearly resolve anatomical features of the sh, and using a three-point Dixon technique, they were able to highlight either highwater or high-fat containing regions. They found no signicant changes in NMR parameters when samples were fast-frozen in liquid nitrogen, then thawed, but did observe differences in MT, T1 , and T1,sat when slow freezing was used. As repeated freeze-thaw cycles produced less change, the authors felt this could be an avenue to authentication. That is, a sample with unknown history could be subjected to a short freeze-thaw cycle. Changes observed from a fresh sample would be greater than those found for a previously frozen sample. Several studies have used MRI to study changes in the structure of frozen/thawed bread dough. Takano et al. prepared dough with freeze-sensitive and freeze-tolerant yeast [49]. Images were collected during fermentation following thawing, with each set of images taking 5 min to measure, with a resolution of 100 m and a slice thickness of 1 mm. They observed that small gas cells formed during mixing without yeast, and that larger nonuniform pores formed during fermentation. They found that prefermentation before freezing increased bread loaf 10110% for baked breads made with freeze-tolerant yeast, but decreased bread volume when bread was made with freeze-sensitive yeast. Esselink et al. used both MRI and cryoSEM to study structural damage incurred during freezing of bread dough [50]. Time-domain NMR showed two T2 regions, one they associated with water bound to starch and one associated with water rapidly sampling hydration sites in gluten.They surmised that during freezing, ice crystallization causes separation of water, and that this does not return to its original state upon thawing. T1 and T2 -weighted MR images showed that water is not equally distributed over the dough slice. After frozen storage, the water-rich regions became thicker, consistent with migration of water.

Transient Freezing
Magnetic resonance imaging has also been used to follow the formation of ice during transient freezing. As the T2 of ice is quite short, ice is relatively invisible to the spinecho pulse sequences used in imaging. However, the unfrozen phase absent of ice still provides sufcient signal. Kerr et al. mimicked commercial freezing by directing the air from an air-blast freezer into a PVC pipe leading through the bore of the MR imaging system, and which

contained the sample [51]. They studied potatoes freezing at 42 and 11 C, at air speeds between 210 m/s. Images showed the advance of the asymmetric freezing interface and loss of signal intensity as water turned to ice. Temperature proles were simultaneously monitored with thermocouples, and the amount of heat removed over time was measured by a differential calorimeter. A numerical model was used to predict temperature proles and freezing interface over time, and was veried using the temperature, calorimetry, and image data. This approach was patented as a means for on-line prediction of enthalpy changes during food freezing [52]. The dynamics of freezing were also studied in immature kiwifruit [53]. With the conventional spin-echo sequence, images took approximately 4.25 min. Thus, a FLASH sequence was attempted, which gave no signicant difference in image quality, and allowed image formation within 25 s. Ice formation commenced at the epidermis and gradually progressed to the core. Freezing was asymmetric, and in some cases ice moved rapidly through the core. Spinspin relaxation was faster, and translational diffusion coefcients greater, in freeze-thawed compared to fresh fruit. Kerr et al. also studied a variety of other foods during freezing, including potatoes, carrots, peas, corn, and chicken legs [54]. Internal structures were distinguished based on water and fat. The ice interface advanced uniformly, but asymmetrically, in all samples except for corn. Freezing in corn seemed to be governed by nucleation, as kernels froze individually. The time required for the total signal to disappear corresponded to the time to reach steady-state enthalpy. Kuo et al. studied the effects of freezing on mozzarella cheeses [55]. In addition to monitoring the formation of ice in the product, they found that there was a signicant change in T2 and the water self-diffusion coefcient following freeze-thawing that could be used to determine areas of freeze-damage. In order to achieve even shorter imaging times, Lee et al. applied a single-point ramp imaging with T1 enhancement (SPRITE) imaging to produce 1- and 2-D images of orange juice, lean ground beef, and bread dough [56]. For samples held between 20 and 40 C, distribution of the liquid-like components was well resolved. The authors noted that SPRITE images are able to show unfrozen water intermingled with ice, while spin-echo images do not. While traditional MRI has a resolution on the order of 100 m, it is possible to acquire MR images at the microscopic level. In theory, this should allow the observation of how freezing proceeds into microscopic entities such as cells and cell organelles. Samples of potato 8 mm in length were studied using fast radial microimaging [57]. They captured cross-sectional projections every 30 s for samples placed at 20 C. The apparently noisy freezing interface was attributed to heterogeneity in the size and structure of cells. Higher resolution imaging has been used

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Solid-State NMR 1875

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to study non-food systems including wintering leaf buds of Momi r and Japanese red pine [58], and both leaf and ower buds of fool-moon maple [59]. In-plane resolution of 39 m was attained with a slice thickness of 0.5 mm. MG images clearly showed internal structures including bark, crown tissue, pith, resin canals, leaf bud scales, vascular tissue, and xylem. Bud scales of Momi r froze quickly at 7 C, but supercooling down to 21 C was found in shoot primordial of March buds. In maple, scales and stem barked froze at 7 C, primordial inorescence and terminal primordial shoots remained supercooled to 14 C, and lateral primordial shoots remained unfrozen at 21 C.

Liquid Phase Chemical Spectroscopy

Chemical shift spectroscopy has been used to study a variety of frozen materials, with the aim of understanding basic chemical, structural and dynamics features as inuenced by freezing. Though few studies have dealt directly with foods, much of the results have direct application to food systems. As frozen foods are not readily studied by conventional NMR spectroscopy, studies have focused on the effects of freezing. Direct studies on frozen materials by solid-state NMR will be discussed in a subsequent section.

Phospholipid Membranes
As all foods of a cellular nature, including meat, fruit, and vegetables, contain phospholipid membranes, the study of the effects of freezing on membranes is of interest. Hsieh and Wu used both 2 H and 31 P NMR to study six phospholipid bilayers at temperatures between 20 and 70 C [60]. Three types of interbilayer water were observed: supercooled water in two of the bilayer systems at temperatures between 20 and 35 C which spontaneously nucleates at 35 C; perturbed water at 20 to 70 C; and bound water. 31 P NMR suggested that rotational motion of phospholipids groups was frozen at 40 C.

water. They also postulated that there was a transfer of water from loosely bound to tightly bound states. Lockett et al. used lactate-edited 1 H NMR difference spectra to study lactate production in isolated rat liver held on ice [62]. Water suppressed spectra were observed by an interleaved binomial sequence optimized for lactate. In addition, a spin-echo double resonance technique was used in which the lactate group was decoupled by lowpower RF energy on alternate scans. An initial peak at 1.26 ppm was assigned to lactate, and steadily increased in the liver tissue over a 24 h period. An additional peak, assigned to alanine, also increased with time. Both 1 H NMR and 31 P NMR were used to measure high energy phosphates in wood frogs frozen at 1.5 C for 7 days. Decreases in the water peaks were used to assess freezing. While most frogs survived slow freezing, no frogs survived the entire rapid freeze. Frogs maintained ATP and creatine phosphate (CP) levels during slow freeze, while rapidly frozen frogs had up to 80% reductions in ATP and CP. Changes in pH were monitored by slight shifts in ATP spectra. Intracellular pH remained between 7.2 and 7.6 for slowly frozen frogs, but decreased to 6.57 during fast freezing. NMR spectroscopy has also been used to assess cryopreservation of cells and tissues. Hubel et al. examined the inuence of preculture on the prefreeze and postthaw characteristics of hepatocytes [63]. Cells were processed in a cryopreservation medium containing 10% DMSO, and frozen in a complicated protocol to a nal temperature of 100 C. NMR spectroscopy of lipids showed that in vitro cultivation increased the relative levels of cholesterol as compared to membrane phospholipids, as well as phospholipid interconversion. 31 P NMR showed an increase in the net energy charge during culturing. Eugene et al. monitored the kinetics of permeation of a variety of cryoprotective agents used to preserve human arteries. They found that equilibration was achieved within 120 mins for DMSO, 2,3-butanediol, and glycerol, while polyethyleneglycol (MW 20,000) reached concentrations between 40 and 50% in extracellular spaces.

Solid-State NMR Cellular and Tissue Systems

NMR spectroscopy has also been used to study the state of water and cellular metabolism in plant and animal tissue. Haranczyl et al. used 1 H NMR spectroscopy to study freezing of lichen thallus at temperatures down to 45 C [61]. They observed a narrow line, which they associated with bound water, and a broader peak associated with free water. A broadening line width and decrease in peak area below 0 C was associated with freezing. At 20 C, a rapid decrease in area for low moisture samples occurred and was attributed to freezing of loosely bound As frozen systems contain a high degree of solids, it would seem logical to apply solid-state NMR for their study.

Frozen Solutions
A variety of solutes and biomolecules have been studied by solid-state NMR. Appleyard and Evans used 13 C NMR with magic angle sample-spinning to study solutions of glycine under a variety of freezing conditions. To avoid crystallization of water, some samples were red through a ne nozzle into liquid propane to form an amorphous

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solid state. Others were subject to slow freezing rates. Although they predicted that the distribution of ice Ih and glycine would become more dispersive as the freezing rate increased, they did not observe differences in the NH3 + group rotations between fast and slow frozen samples. They felt that these results were encouraging, in that any formation of ice Ih in rapidly frozen samples should not be detrimental to the solute. In addition, they felt this was a good approach to trapping chemical intermediates from enzymatic reactions. Volke et al. studied solutions of -chymotrypsin frozen during magic angle spinning at 4000 Hz [64]. A very broad ice peak was observed, spanning a region of 160 ppm. To improve resolution, a sine-bell window function was applied to the FID, but this allowed only a qualitative interpretation of the resulting spectrum. However, resonances from protons of the mobile polypeptide chains could be observed. From the T2 values they estimated a non-frozen water content of 0.15 to 0.24 g H2 O/g protein. Dynamic nuclear polarization (DNP) techniques have proven useful to improving the sensitivity of spectra from frozen aqueous solutions. Here, the high-spin polarization of unpaired electrons is coupled to the nuclear spins through microwave irradiation near the electron paramagnetic resonance frequency, using a nitroxide radical as the paramagnetic polarizing agent. Gerfen et al. used this approach to study frozen solutions of water and glycerol [65]. The 1 H and 13 C NMR signal strengths were improved by a factor of 185. Hall et al. used this approach to study arginine and T4 lysozyme in frozen glycerol-water solutions [66]. Smith et al. extended the idea of studying biomolecules trapped in low-energy states by freezing them in a glassy matrix [67], using rotational-echo double resonance (REDOR) spectroscopy. Samples of 2-13 C,15 Nglycine and 13 C-methyl -D-15 N-acetylglucosamine were frozen at 80 C, and spun at 4.5 kHz. Low energy conformation states were developed for both molecules and corroborated with molecular dynamics computational methods.

in the muscles of stressed and electrically stunned animals, as compared to control CO2 -stunned animals.

Antifreeze Glycoproteins
Antifreeze proteins and glycoproteins are found in a variety of sh and insects subject to freezing conditions, and help protect the organisms from freezing. There have been some hopes that these could be used in food systems, particularly as they may affect ice nucleation processes. NMR has been used to study the dynamics of antifreeze glycoproteins (AFGP) in the presence of ice [70]. Solid-state 13 C NMR indicated that AFGP adopted similar three-dimensional folding in the presence of ice and in the freeze-dried state. A high number of conformers existed, indicative of a very exible molecule. AFGP-ice interactions were dominated by fast motions on the nanoto picosecond time scale.

Conclusion
A variety of NMR techniques are available to study frozen foods and food components. These include relatively simple relaxation techniques for measuring solid/liquid ratios, imaging approaches to monitor food during freezing, and chemical spectroscopy to measure unfreezable water and conformations of molecules.

References
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Whole Foods
In food systems, Li et al. used 2 H and 1 H solid-state NMR to study the mobility of unfreezable and freezable water in waxy corn starch [68]. They found that a large percentage of the unfreezable water was relatively mobile even at 32 C. Bertram et al. used 13 C cross-polarization MAS NMR to examine differences in pre-slaughter treatments on rapidly frozen pork longissimus muscle [69]. Resonance peaks from fatty acids, carboxylic groups, and carbons in lactate and glycogen were identied. They found a signicant difference in methylene carbons attributed to membrane lipids, suggesting altered membrane properties

Magnetic Resonance of Food Freezing

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