Effect of Temperature and Glassy States on the Molecular Mobility of Solutes in Frozen Tuna Muscle As Studied by Electron Spin Resonance Spectroscopy with Spin Probe Detection VIBEKE ORLIEN,MOGENS L. ANDERSEN,* SAARA JOUHTIMA ¨ KI,JENS RISBO, AND LEIF H. SKIBSTED Food Chemistry, Department of Dairy and Food Science, Royal Veterinary and Agricultural University, Rolighedsvej 30, DK-1958 Frederiksberg C, Denmark The mobility of solutes in frozen food systems (tuna muscle, sarcoplasmic protein fraction of tuna muscle, and carbohydrate-water) has been studied using the temperature dependence of the shape of electron spin resonance (ESR) spectra of the spin probe 4-hydroxy-2,2,6,6-tetramethylpiperidine- N-oxyl (TEMPOL). The spin probe was incorporated into the tuna meat from an aqueous solution of TEMPOL or by contact with a layer of TEMPOL crystals. The melting/freezing of freeze-concentrated solutes in frozen tuna meat was observed to take place over a range of temperatures from -25 to -10 °C. Lower temperatures gave ESR powder spectra due to the decreased mobility of the spin probe, and the temperature dependence of the mobility of the spin probe did not show abrupt changes at the glass transition temperatures of the systems. The mobility of nonglass forming solutes is concluded to be decoupled from the glass forming components. Similar behavior was also observed for TEMPOL in frozen, aqueous carbohydrate systems. The temperature dependence of the mobility of TEMPOL in the frozen systems was analyzed using the Arrhenius equation, and the logarithm of the Arrhenius preexponential factor τ a was found to be linearly correlated with the activation energy for all of the tuna and carbohydrate samples, indicating a common molecular mechanism for the observed mobility of TEMPOL in all of the systems. The linear correlation also suggests that the observed mobility of TEMPOL in the frozen aqueous systems is dominated by enthalpy-entropy compensation effects, where the mobility of TEMPOL is thermodynamically strongly coupled to the closest surrounding molecules. KEYWORDS: Frozen tuna; ESR; solute mobility; glass transition; melting INTRODUCTION Freezing is a common method for long-term storage of foods, and the subzero temperatures are assumed to suppress the deteriorating reactions, primarily by thermodynamic restrain on the reaction rates. However, storage of food at the normal freezing temperature (-18 °C) does not completely hinder the degradation of quality, and deteriorative reactions proceed, albeit very slowly, at temperatures below the freezing point of the aqueous phase. Foods that are susceptible to oxidation, such as fish meat, have been reported to degrade during frozen storage (1-3). The macroscopic properties of frozen systems have been studied intensively, mainly by differential scanning calorimetry (DSC), often leading to the construction of state diagrams that summarize the thermodynamic characteristics such as melting temperatures and the glass transition temperatures, T g (4-7). Particularly the temperature of formation of the maximally freeze-concentrated glass (T g ′) is expected to be of crucial importance for the food stability in relation to storage temper- ature. It has been suggested that improved long-term storage stability of food can be achieved by storing food in a frozen amorphous glassy state, where the molecules form a nonperiodic and nonsymmetric network presumably resulting in an extremely high viscosity and thus immobilized molecules. Because of this very limited molecular motion, a food product in a glassy state is assumed not to deteriorate during storage (8-12). However, the possibility that transition of the food system into a glassy state may not be sufficient to limit molecular motions has recently been discussed (13-15). Information about structural and dynamic properties at a microscopic (molecular) level is becoming of increasing interest in the study of molecular motions in frozen biological systems. Characterization of molecular mobility at subzero temperatures of compounds similar in sizes to compounds that are involved in degradation reactions is expected to give a better understand- * To whom correspondence should be addressed. Tel: +45 3528 3262. Fax: +45 3528 3344. E-mail: mola@kvl.dk.