Protein cross-linking with oxidative enzymes and transglutaminase:Effects in meat protein systems: Dissertation

The oxidative enzymes tyrosinase and laccase, as well as the acyltransferase transglutaminase (TG), are capable of creating covalent cross-links in proteinaceous substrates. These enzymes differ from each other on the basis of their different reaction mechanisms and the amino acid residues with which they react. TG and tyrosinase are well-known protein cross-linkers, and TG is already used industrially in the food sector. The commercial feasibility of tyrosinase is under assessment, whereas the effectiveness of laccase is not known because the reaction mechanism of protein modification by laccase is still poorly understood. The effects of tyrosinase-, laccase- and TG-catalysed protein modification were studied in different meat protein systems from the myofibrillar proteins of chicken breast muscle to heated ground chicken breast meat systems containing different amounts of meat and salts. The study was focused on the effects of the enzymes on the thermal behaviour and gel formation properties of myofibrils, and on the textural and water-holding properties of the heated meat systems. The cross-linking efficiency of a novel tyrosinase from the fungus Trichoderma reesei was compared to that of the commercial tyrosinase from the mushroom Agaricus bisporus. Trichoderma tyrosinase was found to be superior compared to the Agaricus enzyme in its protein cross-linking efficiency and in the incorporation of a small molecule, dihydroxyphenylalanine, into a complex proteinaceous substrate, wool fibre. Agaricus tyrosinase was found to have a propensity towards oxidation of small tyrosine-containing substrates. All three enzymes, although having different reaction mechanisms, affected the same myofibrillar proteins. Myosin and troponin T were found to be most sensitive to enzymatic modification, whereas actin was clearly more resistant. Tyrosinase, laccase and TG all polymerised myofibrillar proteins, but only laccase was found to cause protein fragmentation. The fragmentation products originated from myosin and troponin T. In the differential scanning calorimetric (DSC) measurements alteration of the peak temperature of myosin or actin transitions was not observed. However, tyrosinase and TG decreased, whereas laccase increased, the heat of myosin transition. The heat of actin transition was increased with all three enzymes. Tyrosinase and TG improved the gel formation of a 4% myofibrillar suspension at 0.35 M (2%) NaCl. With both enzymes the gel formation increased along with increasing enzyme dosage, showing a positive connection between covalent cross-link and gel formation. Laccase was able to increase the gel formation slightly only when the NaCl concentration was increased to 0.60 M (3.5%). With an excessive laccase dosage the gel formation declined markedly due to protein fragmentation. Tyrosinase, laccase and TG had different effects on the texture and water-holding of the heated chicken breast meat homogenates. The homogenates were prepared free of phosphate (75% meat, 2% NaCl), with a low meat content (65% meat, 2% NaCl, 0.34% phosphate), with a low salt content (75% meat, 1% NaCl, 0.34% phosphate) or with low amounts of both NaCl and phosphate (75% meat, 1% NaCl, 0.17% phosphate). Tyrosinase improved the firmness of the homogenate gels free of phosphate and with a low amount of meat. TG improved the firmness of all studied homogenates. Laccase weakened the gel firmness of the low-meat, low-salt and low-salt/phosphate homogenates and maintained the firmness on the control level in the homogenate free of phosphate. Tyrosinase reduced the weight loss in the homogenates containing a low amount of meat and a low amount of NaCl, whereas TG and laccase were not able to decrease the weight loss of any homogenate. TG was the only enzyme that could positively affect the firmness of the homogenate gel containing both low NaCl (1%) and phosphate (0.17%) amounts. In pilot scale the test products were made of coarsely ground chicken breast fillet with a moderate amount of salt (1.2-1.8% NaCl), a meat amount 70-80% and a TG dosage 0-20 nkat/g protein (0-0.2% of the meat mass). All three factors, meat, salt and TG contents, favoured the development of firmness of the test products. The evaporation loss decreased slightly along with increasing TG and NaCl amounts in the experimental conditions used, indicating a positive interaction between these two factors. In this work it was shown that tyrosinase, laccase and TG affected the same myofibrillar proteins. However, these enzymes had distinguishable effects on the gel formation of a myofibril system as well as on the textural and water-holding properties of the finely ground meat homogenates, reflecting distinctions at least in the reaction mechanisms and target amino acid availability in the protein substrates for these enzymes