Effect of processing on muscle structure and protein digestibility in vitro : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology at Massey University, Palmerston North, New Zealand

Abstract

The objective of this thesis was to investigate the effect of processing on meat protein properties, muscle structure and 𝘪𝘯 𝘷𝘪𝘵𝘳𝘰 protein digestibility of beef. Meat processing techniques including pulsed electric field (PEF), shockwave (SW) processing, exogenous enzyme (actinidin) treatment, and sous vide (SV) cooking were explored, either alone or in combination, in this project. This thesis also aimed to study the diffusion of enzymes (actinidin from kiwifruit and pepsin in the gastric juice) into the meat.

The first experiment investigated the effect of PEF processing alone on the ultrastructure and 𝘪𝘯 𝘷𝘪𝘵𝘳𝘰 protein digestibility of bovine 𝘓𝘰𝘯𝘨𝘪𝘴𝘴𝘪𝘮𝘶𝘴 𝘵𝘩𝘰𝘳𝘢𝘤𝘪𝘴, a tender meat cut (Chapter 3). It was observed that the moisture content of the PEF-treated samples (specific energy of 48 ± 5 kJ/kg and 178 ± 11 kJ/kg) was significantly lower (p < 0.05) by 1.3 to 4.6 %, compared to the untreated samples. The pH, colour, and protein thermal profile of the PEF-treated muscles remained unchanged. Pulsed electric field treatment caused the weakening of the Z-disk and I-band junctions and sarcomere elongation (25 to 38 % longer) of the muscles. The treatment improved 𝘪𝘯 𝘷𝘪𝘵𝘳𝘰 meat protein digestibility by at least 18 %. In this thesis, the protein digestibility was determined in terms of the ninhydrin-reactive amino nitrogen released during simulated oral-gastro-small intestinal digestion. An enhanced proteolysis of the PEF-treated meat proteins (such as α-actinin and β-actinin subunit) during simulated digestion was also observed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The improvement in protein digestibility of the PEF-treated meat was supported by more severe disruption of Z-disks and I-bands observed in PEF-treated samples, at the end of simulated digestion.

In the second experiment, PEF treatment (specific energy of 99 ± 5 kJ/kg) was applied to bovine 𝘋𝘦𝘦𝘱 and 𝘚𝘶𝘱𝘦𝘳𝘧𝘪𝘤𝘪𝘢𝘭 𝘱𝘦𝘤𝘵𝘰𝘳𝘢𝘭 muscles in conjunction with SV cooking (60 ℃ for 24 h) (Chapter 4). This muscle cut was tested as it is a tough cut and requires slow cooking. There was no significant difference detected in the specific activities of the sarcoplasmic cathepsins present in the cytosol between the control and PEF-treated samples, both before and after cooking. In addition, similar micro- and ultrastructures were observed between the control SV-cooked and PEF-treated SV-cooked 𝘱𝘦𝘤𝘵𝘰𝘳𝘢𝘭 muscles. The combined PEF-SV treatment increased the 𝘪𝘯 𝘷𝘪𝘵𝘳𝘰 protein digestibility of the 𝘱𝘦𝘤𝘵𝘰𝘳𝘢𝘭 muscles by approximately 29 %. An improvement in proteolysis of the treated meat proteins (e.g. myosin heavy chains and C-protein) during simulated digestion was also observed using SDS-PAGE. More damaged muscle micro- and ultrastructures were detected in PEF-treated SV-cooked muscles at the end of 𝘪𝘯 𝘷𝘪𝘵𝘳𝘰 oral-gastro-small intestinal digestion, showing its enhanced proteolysis compared to the control cooked meat.

Next, the effect of SW processing and subsequent SV cooking on meat protein properties, muscle structure and 𝘪𝘯 𝘷𝘪𝘵𝘳𝘰 protein digestibility of bovine 𝘋𝘦𝘦𝘱 and 𝘚𝘶𝘱𝘦𝘳𝘧𝘪𝘤𝘪𝘢𝘭 𝘱𝘦𝘤𝘵𝘰𝘳𝘢𝘭 muscles were investigated (Chapter 5 and 6). Shockwave processing (11 kJ/pulse) alone decreased the enthalpy and thermal denaturation temperature of the collagen (𝘱 < 0.05) when compared to the raw control, studied using a differential scanning calorimeter. The purge loss, pH, colour, and the protein gel electrophoresis profile of the SW-treated raw muscles remained unaffected. Shockwave processing led to the disorganisation of the sarcomere structure and also modified the protein secondary structure of the myofibres. After subsequent SV cooking (60 ℃ for 12 h), more severe muscle fibre coagulation and denaturation were observed in the SW-treated cooked meat compared to the cooked control. An increase in cook loss and a decrease in the Warner-Bratzler shear force were detected in the SW-treated SV-cooked meat compared to the control cooked meat (𝘱 < 0.05). The 𝘪𝘯 𝘷𝘪𝘵𝘳𝘰 protein digestibility of the SW-treated SV-cooked meat was improved by approximately 22 %, with an enhanced proteolysis observed via SDS-PAGE, compared to the control SV-cooked meat. These results were supported by the observation of more destruction of the micro- and ultrastructures of SW-treated cooked muscles, observed at the end of the simulated digestion.

The effect of the kiwifruit enzyme actinidin on muscle microstructure was studied using Picro-Sirius Red staining (Chapter 7). Meat samples were subjected to two different conditions, simulating meat marination (pH 5.6) and gastric digestion in humans (pH 3). Actinidin was found to have a greater proteolytic effect on the myofibrillar proteins than the connective tissue under both conditions. When compared with pepsin under simulated gastric conditions, actinidin had a weaker proteolytic effect on the connective tissue of cooked meats. Nevertheless, incubating the cooked meat in a solution containing both actinidin and pepsin resulted in more severe muscle structure degradation, when compared to muscles incubated in a single enzyme system. Thus, the co-ingestion of kiwifruit and meat could promote protein digestion of meat in the stomach. In addition, both actinidin and pepsin were successfully located at the edges of the muscle cells and in the endomysium using immunohistofluorescence imaging. The observations suggest that the incubation solutions penetrate into the muscle through the extracellular matrix to the intracellular matrix, enabling the proteases to access their substrates.

Overall, the present work demonstrated that there were strong interactions between processing, muscle protein properties and structure, and 𝘪𝘯 𝘷𝘪𝘵𝘳𝘰 protein digestibility of the meat. Processing induces changes in meat protein properties and muscle structure, which in turn affects the digestion characteristics of muscle-based foods.