Food Chemistry and Toxicology Functional and Thermal Gelation Properties of Squid Mantle Proteins Affected by Chilled and Frozen Storage M.C. GÓMEZ-GUILLÉN, O. MARTÍNEZ-ALVAREZ, AND P. MONTERO ABSTRACT: Fresh squid (Loligo vulgaris) mantles that underwent chilled and frozen storage were studied for their ability to form thermal gels as well as the effects of changes to the functional and chemical properties of the muscle proteins during storage. Assays of protein extractability in 5% NaCl, apparent viscosity, autolytic activity, and SDS- PAGE (of the soluble fraction) were carried out periodically. After 4 d of chilled storage, there was a significant drop in protein functionality that negatively affected the thermal gelation profile. The rate of proteolysis remained very high throughout frozen storage, however functional properties and thermal behavior remained very stable. Keywords: squid, storage, functional properties, thermal gelation, viscoelastic properties Introduction S QUIDS ARE AN ABUNDANT NATURAL RESOURCE IN MANY REGIONS of the world. They offer many advantages over other seafood, such as a high post-processing yield, very low fat content, bland fla- vor, and very white flesh. In addition, they have been shown to have high functionality such as gelation, an important property in food processing. By using this property, squids may be used to produce gel-based products, it could possibly enhance the consumption in the areas where squid as such is still unappreciated. Unlike fish species with excellent heat-set gel forming proper- ties, cephalopod muscle proteins aggregate by heating, but the resulting structures are highly disorganized, leading to poor elas- ticity and low gel strength, as shown by the low folding and pen- etration force values (Gómez-Guillén and others 1998). Cephalo- pods typically have a high level of proteolytic activity, much higher than most fish species (Stanley and Hultin 1984; Kolodzie- jska and others 1987; Hurtado and others 1999). It has been sug- gested that this activity is one of the main reasons for its poor gel- forming ability, because it results in degradation of myosin heavy chains in the mantle proteins (Nagashima and others 1992). Gómez-Guillén and others (2002) reported that one of the main proteolytic activities hindering thermal gelation in frozen mantle of squid (Loligo vulgaris) was the predominance of neutral serine proteases with peak activity at 40 °C. Similarly, Ebina and others (1995) associated a neutral trypsin-like proteinase whose optimal temperature is 40 °C with textural deterioration of thermally in- duced gel from spear squid (Loligo bleekeri). Because of this, ther- mal scanning rigidity monitor and dynamic viscoelastic studies of Dosidicus gigas and Loligo vulgaris, respectively, showed a marked decrease in rheological values at temperatures around 35 to 40 °C (Gómez-Guillén and others 1997, 2002). As with a number of fish species having poor gel-forming ability, protease inhibitors and microbial transglutaminase have been suc- cessfully used to improve gel properties of frozen squid mantle (Pérez-Mateos and others 2002). In this study, however, thermal gelation profiles from the short- and long-term frozen squid re- vealed notable differences as a consequence of frozen storage. Cephalopod myofibrillar proteins have been shown to be highly resistant to freeze-induced denaturation (Moral and others 1981). They can maintain high and relatively stable levels of salt soluble protein throughout frozen storage (Moral and others 2002). Paramyosin, which represents about 14% of squid myofibrillar pro- teins, has been shown to be involved in decreasing the rate of pro- tein denaturation in frozen stored squid (Iguchi and others 1981). On the other hand, Morales (1997) observed the pronounced dif- ferences in the viscosity of different squid muscle homogenates un- accompanied by any changes in percent salt-soluble proteins. In a recent work, Pérez-Mateos and others (2002) postulated that this apparent inconsistency between the protein functionality param- eters, that is, protein extractability and viscosity, could in fact reflect denaturation/aggregation of sarcoplasmic proteins. This protein fraction, which according to Sikorski and Kolodziejska (1986), rep- resents around 15% of total protein in squid, is accompanied by high proteinase activity. Thus, a possible denaturation of the sar- coplasmic protein fraction could explain the reduction of autolyt- ic activity in the squid muscle after 5 mo of frozen storage (Pérez- Mateos and others 2002). On the other hand, changes during the early chilled storage were observed by Hurtado and others (1999) in octopus muscle. The MHC was the primary myofibrillar protein followed by paramyosin, which was hydrolyzed during autolysis of octopus arm muscle proteins. Actin showed no signs of hydrolysis during incubation up to 8 h. Due to its high affinity for myosin, the enzyme activity should be controlled during processing of octopus to ensure the functionality of myosin. Changes during chilled storage are expected to be considerably different from those occurring in the frozen state, because the activ- ity of different groups of proteases is largely governed by the post mortem changes occurring during the rigor mortis resolution, and by the subsequent growth of microorganisms. Thus, the freshness of squid may affect the mantle’s ability to form gels/aggregates dur- ing chilled storage and may also be an important factor affecting the holding time for subsequent freezing. The object of the present study was to determine changes in functional and chemical properties of squid muscle proteins during chilled and short-term (30 d) frozen storage, and their influence on the behavior of proteins during thermal gelation.