Food Chemistry and Toxicology Effect of Endogenous Transglutaminase on Threadfin Bream Surimi Gelation J. YONGSAWATDIGUL, A. WORRATAO, AND J.W. PARK ABSTRACT: Transglutaminase(TGase) activity of threadfin bream mince was 99.6 units/g of dry weight. After wash- ing and screw-pressed dewatering, 44% residual activity was retained. Covalent cross-linking of myosin heavy chain (MHC) was observed at both 25 and 40 °C and supported by increased gel strength. When pre-incubation at 40 °C was prolonged to 4 h, breaking force and MHC decreased due to endogenous proteinase(s). TGase activity towards MHC and synthetic substrates was effectively inhibited by iodoacetic acid (IAA). Autolytic activity and degradation of MHC was inhibited by phenylmethanesulfonyl fluoride (PMSF). Addition of 0.2% Ca 2+ significantly improved breaking force and increased MHC cross-linking of surimi gels pre-incubated at 40 °C for 2 h. Keywords: transglutaminase, myosin heavy chain, cross-linking, threadfin bream Introduction T HREADFIN BREAM (NEMIPTERUS SPP.) IS THE 2ND LARGEST RESOURCE used for surimi production, after Alaska pollock. Thailand is one of the major threadfin bream surimi producers in the world with an approximate annual production of over 80000 metric tons. Despite its large production quantity and value, scientific information re- lated to its gelation characteristics is limited. Utilization of threadfin bream surimi in the surimi-seafood industry has primarily relied on the technical information of other species, such as Alaska pol- lock. However, the intrinsic properties of cold water fish, such as Alaska pollock, are significantly different from the properties of warm water fish, such as threadfin bream. Setting or “suwari” is a phenomenon describing an increased gel strength after pre-incubation of surimi paste at a certain temper- ature, between 5 and 40 °C for a specific period of time (Lanier 2000). An improvement of textural properties is attributed to an endogenous transglutaminase (TGase) (R-glutaminyl-peptide: amine -glutamyltransferase; EC 2.3.2.13) that catalyzes the cross- linking reaction of muscle proteins, especially myosin (Kimura and others 1991; Kishi and others 1991). The -carboxyamide groups of peptide-bound glutamine residues act as acyl donors, while the primary amino groups, including the -amino of lysine can be acyl acceptors (Folk 1980). The resulting -(-glutamyl) lysyl isopeptide bonds are stronger than hydrogen and hydrophobic interactions. Such covalent cross-linkings of myosin formed inter- and intramo- lecularly, resulting in higher gel elasticity (Lee and others 1997). Optimum temperature of setting varies with fish species. Klesk and others (2000) reported that setting of Alaska pollock surimi was noticed at 5 and 25 °C for 22 and 3 h, respectively. In contrast, op- timal setting for subtropical tilapia surimi was at 40 °C. Maximum cross-linking of myosin heavy chain (MHC) of croaker surimi was also found at 40 °C (Kamath and others 1992). Differences in opti- mum setting temperature were attributed to conformational chang- es of myosin (Joseph and others 1994). Optimum setting condition of threadfin bream surimi has not been studied extensively. Setting conditions of surimi are also affected by calcium content since endogenous TGase is a Ca 2+ -dependent enzyme (Lee and Park 1998; Kimura and others 1991). The optimal CaCl 2 concentra- tion for TGase also varied with the source of the enzyme (Yasueda and others 1994; Kumazawa and others 1996). Therefore, enhance- ment of gel strength through TGase can be achieved by optimizing the Ca 2+ concentration to be used for the respective species. Better understanding of the setting phenomenon of threadfin bream surimi could lead to better quality control and possible en- hancement of gel quality. Objectives of our study were to investi- gate changes of TGase activity during threadfin bream surimi pro- cessing. In addition, the effects of temperature and CaCl 2 concentration on setting of threadfin bream surimi were examined. Materials and Methods Materials and reagents Fresh threadfin bream (Nemipterus spp), washed mince, screw- pressed mince, and threadfin bream surimi (Grade AA) of the same lot of raw fish were collected from a surimi plant at Samutsakorn, Thailand. Samples were packed in a polystyrene box, filled with ice, and immediately transported to the Suranaree University labora- tory. TGase activity of washed mince was determined upon arrival. Surimi was cut into 1 kg blocks, vacuum-packed, and kept at –18 °C during the study. N, N’-Dimethylated casein (DMC), monodansylcadaverine (MDC), iodoacetic acid (IAA), phenylmethanesulfonyl fluoride (PMSF), and N-ethylmaleimide (NEM) were purchased from Sigma Chemical Company (St. Louis, Mo., USA). Ethylene glycol-bis (2- aminoethyl ether)-N, N, N’, N’-tetraacetic acid (EGTA) and dithio- threitol (DTT) were purchased from Fluka (Buchs, Switzerland). Reagents used for gel electrophoresis were obtained from Bio-Rad (Hercules, Calif., U.S.A.). All other chemicals were of analytical grade. Preparation of crude TGase extracts TGase activities of mince, 1st washed, 2nd washed, 3rd washed, and screw-pressed minces were determined. Samples were homog- enized, upon arrival, in 4 volumes of extraction buffer (10 mM NaCl, 5 mM EDTA, 2 mM DTT, 10 mM Tris-HCl, pH 7.5). The homo- genates were centrifuged at 16000 × g for 20 min (RC 28S; Sorvall Co., Newtown, Conn., U.S.A.) and supernatants were subsequently centrifuged at 100000 × g for 60 min (Beckman Class S; Beckman Co., Palo Alto, Calif., U.S.A.). The supernatants, after ultracentrif- ugation, were used as crude extract.