1636 JOURNAL OF FOOD SCIENCE—Vol. 67, Nr. 5, 2002 © 2002 Institute of Food Technologists Food Chemistry and Toxicology Influence of Some Protease Inhibitors on Gelation of Squid Muscle M.G. AYENSA, M.P. MONTERO, A.J. BORDERÍAS, AND J.L. HURTADO ABSTRACT: The effect of 4 food-grade protease inhibitors—bovine plasma, potato powder, ethylenediamine- tetraacetic acid, and sodium pyrophosphate—on heat gelation of squid (Todaropsis eblanae) muscle was stud- ied. The action of these ingredients was characterized by determining the proteolytic activity in batters contain- ing them and the rheological properties of gels made with them. The gelation profile showed that the setting effect was greatest with pyrophosphate (PPI) and that gel rigidity was highest with bovine plasma (BP). All 4 inhibited proteolytic activity to some extent at 65 °C. BP was the most effective. At 40 °C, only EDTA and PPI were active. Rheologically, the most effective combination was PPI plus bovine plasma in that this combined inhibi- tion of proteolytic activity with gel enhancement. Keywords: squid, thermal gelation, food-grade protease inhibitors Introduction G ROWING NUMBERS OF CONSUMERS ARE ACCEPTING SQUID AS A regular component of their diet. Because it is abundant in some areas, it can be used as a raw material for processed prod- ucts. Like other cephalopods, its life cycle is short and therefore growth is rapid. This involves a high rate of turnover of body pro- teins, which is associated with a far higher level of proteolytic ac- tivity than any other marine species (Sakai and Matsumoto 1981; Konno and Fukazawa 1993). In mantle of squid (Todaropsis ebla- nae) (Ayensa and others 1999) proteases have been found which are active at acidic, neutral, and alkaline pH, at both moderate (40 °C) and high (65 °C) temperatures; activity is slightly higher at 60 °C than at 40 °C. It is also reported that this proteolytic ac- tivity was largely caused by cysteine proteinases in the acidic and basic pH ranges, whereas at neutral pH serine proteinase predominated at either temperature and aspartic-proteinases predominated at 40 °C. Several authors (Sakai and Matsumoto 1981; Rodger and others 1984; Kolodziejska and others 1987) have reported that the existence of proteases in squid muscle can cause myosin degradation. Fish minces are usually washed to at least partially eliminate the influence of these enzymes. This is not advisable in cephalopods because of the solubility of their myofibrillar proteins (Matsumoto 1958; Tsuchiya and oth- ers 1978), which would reduce yield. Additives are therefore re- quired to inhibit the action of proteases. The incorporation of various food-grade additives has proven effective in inhibiting proteolytic activity in several surimi gels from fish species (Akazawa and others 1993; Liu and Xiong 1997; Seymour and others 1997). However, we have found no informa- tion on the study of proteolysis inhibitors during gelation of cephalopods, which is a very important issue given the high en- zyme content of their muscle. Such inhibitors include bovine plasma (BP), potato powder (PP), ethylenediamine-tetraacetic acid (EDTA) and sodium pyrophosphate (PPi). BP proteins con- tain a 2 -macroglobulin which is effective in controlling proteolytic activity (Barrett and Starkey 1973) and has been described as one of the most effective gel enhancers and protease inhibitors (Akazawa and others 1993; Morrissey and others 1993). Weeras- inghe and others (1996) detected numerous serine proteinase inhibitors as components of PP. The inhibitory effect of EDTA has been clearly shown in prevention of squid muscle degradation by acting as a chelating agent (Stanley and Hultin 1984; Bond and Butler 1987). Konno and Fukazawa (1993) suggested the use of pyrophosphate to prevent autolysis during squid (Todarodes pacificus) muscle processing. The object of this research was to study the effectiveness of selected food-grade additive inhibitors on the gel-forming ca- pacity of a batter. To that end, proteolytic activity in the batters was determined and the effect of the inhibitors was compared with respect to various different properties of the gels made from these batters. Materials and Methods Squid The squid used was Todaropsis eblanae, caught on the Gali- cian continental shelf (Northwest Spain) and landed within 24 h. Whole squid, approximately 60 kg (150 individual), were trans- ported to the laboratory in refrigerated containers with abun- dant ice, where they were gutted and skinned immediately upon arrival. Tentacles were removed and only mantle tissue was used for sample. Specimens were frozen in a plate freezer set at –40 °C and stored at –80 °C in vacuum packs to prevent alteration dur- ing storage. Batter and gel preparation Chopped squid mantle tissue (moisture: 86.1 ± 0.12, crude protein: 12.75 ± 0.04) was semi-thawed and placed in a refriger- ated vacuum homogenizer (Stephan model UM5; Stephan U. Söhne GmbH & Co., Hameln, Germany). The muscle was ground for 1 min at high speed. NaCl (0.15 M) was then added with suffi- cient crushed ice to give the required final gel moisture (81%) and the mixture was homogenized for 5 min at low speed in vacu- um. Starch (5% Clearam CH–20) and 1 of the food-grade inhibi- tors were then added and the sample homogenized again for 5 to 7 min. Starch (Clearam CH–20) was supplied by Laisa (Valencia, JFS: Food Chemistry and Toxicology