Determining the mechanism of pulsed electric field for improving meat quality : A thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy at Lincoln University

Pulsed electric field (PEF) is a novel non-thermal technology that has recently attracted the attention of meat scientists and technologists due to its ability to modify membrane structure and enhance mass transfer. Several studies have confirmed the potential of PEF for improving meat tenderness in both pre-rigor and post-rigor muscles during ageing. However, there is a high degree of variability between studies and the underlying mechanisms for the reported outcomes are not clearly understood. While some studies have suggested physical disruption as the main cause of PEF induced tenderness, enzymatic nature of the tenderization seems to be the most plausible mechanism. Several studies have suggested the potential of PEF to mediate the tenderization process due to its membrane altering properties causing early release of calcium ions and early activation of the calpain proteases. However, experimental research is yet to confirm this postulation. The changes in the calpain activity and protein profile of beef from older animals during ageing is poorly explored and further understanding of biochemical processes is needed to design useful approaches for tenderization. Relatively few experiments have examined the effects of ageing on the quality of beef from older animals. To elucidate the changes in the calpain activity and protein profile of beef from culled dairy cows during ageing, cold-boned Semimembranosus (n=6) and Biceps femoris (n=6) were vacuum packaged and stored for 14 days at 4±1 °C. A significant (P<0.05) effect of ageing was observed on the pH, shear force and myofibrillar fragmentation index of both the muscles. Casein zymography results indicated the presence of intact and autolyzed forms of calpain 1 and calpain 2. An increase in proteolysis was observed in both the muscles during ageing. Ageing for two weeks resulted in reduction (P<0.05) of the shear force of both muscles by 30%, however, the aged muscles were still excessively tough. To evaluate the impact of PEF on the quality of beef from older animals, cold-boned beef Semimembranosus (n=6) and Biceps femoris (n=6) were processed with two different PEF treatments viz. T1 (5 kV, 90 Hz, 20 µs) and T2 (10 kV, 20 Hz, 20 µs) and were vacuum packaged and stored for 14 days at 4±1 °C along with a non-treated control. Samples from venison Longissimus dorsi (n=6) were also treated with PEF [T1 (2.5 kV, 50 Hz, 20 µs) and T2 (10 kV, 90 Hz, 20 µs)] and were subjected to 21 days of ageing at 4±1 °C. An improvement was recorded in calpain activity of all the samples treated with PEF along with an early activation of calpain 2 in beef. Increased proteolysis of troponin-T and desmin was also recorded, however, no significant (P>0.05) impact was observed on the shear force and myofibrillar fragmentation index of any of the muscles. These results provided an experimental evidence for the enzymatic nature of PEF. PEF has been reported to affect the structural and functional properties of proteins, which suggests that it may influence the digestion of meat proteins. To evaluate the impact of PEF on the protein digestion kinetics, PEF-treated beef Semimembranosus and Biceps femoris [T1 (5 kV, 90 Hz, 20 µs) and T2 (10 kV, 20 Hz, 20 µs)] and venison Longissimus dorsi [T1 (2.5 kV, 50 Hz, 20 µs) and T2 (10 kV, 90 Hz, 20 µs)] were subjected to in vitro simulated gastrointestinal digestion. Both raw (n=3) and cooked (n=3) samples were used separately. Samples were collected at 0, 30, and 60 minutes of gastric digestion and 120 and 180 minutes of intestinal digestion. PEF processing affected the digestion kinetics of all the muscles by modifying the protein profile (SDS-PAGE) of the meat digests and significantly (P<0.05) increasing the protein digestibility (%) and soluble protein (%). Concentration of almost all the free amino acids in all muscles were numerically higher (P>0.05) for the PEF treated samples whereas no significant (P>0.05) impact was observed on the release of minerals such as Fe, Zn, Cr, Cu, Mg, Ni, Na or K. PEF processing improved the digestion kinetics of the beef and venison during gastrointestinal digestion simulation in both raw and cooked samples. PEF has been shown to accelerate salt diffusion and enhance mass transfer processes in meat indicating a possibility for its application in sodium reduction management. By influencing the cellular and membrane permeability and by affecting structural and functional properties of proteins, PEF is expected to improve the salt diffusion and possibly sodium perception during chewing. The potential use of PEF to reduce sodium in processed meat was investigated using beef jerky as a model system (n=6). Beef jerky was prepared using different levels of NaCl viz. 2.0% (control), 1.2% (T1) and 1.2% along with PEF-processing (T2, 0.52 kV/cm, 10 kV, 20 Hz, 20 µs). A significant (P<0.05) effect of PEF was observed on shear force (N), toughness (N/mm.sec) and firmness (N/mm) of the products, which was also reflected in the sensory scores. PEF-treated samples (T2) had significantly (P<0.05) lower sodium content then the control, however, the sensory scores were comparable (P>0.05) with control and more than 84% of the panellists preferred T2 samples over T1 for saltiness. No undesirable effects of PEF were observed on colour, yield (%) and oxidative and microbial stability. These results suggest that PEF has a sodium-reduction potential that can play a role in public health. The general scope of the present project was to elucidate how PEF affects the meat quality during ageing and to explore the possible application of PEF in protein digestion and sodium reduction. It was expected that this project will decipher the mechanisms of how PEF affects meat quality and protein digestion and elucidate a possible role of PEF in sodium reduction. Research on PEF-assisted sodium reduction and its role in protein digestion is unavailable in the literature.