Investigation of the effects of emerging technologies on the kinetics of glycolytic and proteolytic enzymes relevant for meat quality
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Date
2025
Authors
Kent, Mary Ann
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Publisher
University College Cork
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Abstract
Tenderness, along with flavour and juiciness, are important factors that influence the overall consumer satisfaction of meat. These quality parameters are influenced by two key biochemical processes related to the conversion of muscle to meat: anaerobic glycolysis and proteolysis. Glycolysis involves a series of metabolic reactions, where glycogen is broken down to glucose and subsequently converted to lactic acid, which is concomitant with a decline in pH post-mortem. Glycolysis is regulated by three key rate limiting enzymes: glycogen phosphorylase, phosphofructokinase and pyruvate kinase. Proteolysis involves the degradation of key structural muscle proteins such as desmin and titin, which contributes to the tenderisation of muscle. Enzymes involved in proteolysis include calpains and cathepsins. Carcass interventions offer the potential to modulate these biochemical processes that are key to determining meat quality. Several interventions are currently used by the meat industry, including aging (wet and dry), hanging and hot boning. Electrical stimulation is another well established carcass intervention that has been applied to improve meat quality through its ability to accelerate the rate of glycolysis and modify proteolysis, post-mortem. Innovative technologies such as ultrasound and high pressure processing have also been investigated to determine their effects on meat quality, with varying results and further studies in this area are warranted. Therefore, the objectives of the research were to investigate the impact of innovative technologies on post-mortem biochemical processes in bovine Longissimus thoracis et lumborum. It examined the impact of ultrasound on glycolytic enzyme activity in pre-rigor muscle, in vivo and in vitro, as well as in muscle homogenates (prepared from pre-rigor muscle). Additionally, it evaluated the effects of a high pressure pre-treatment on weight loss, eating quality (Warner-Bratzler shear force values, colour, lipid oxidation), proteolysis (MFI, SDS-PAGE, calpain activity), and metabolite profiles during dry aging.
Ultrasound was applied to pre-rigor (2 to 4 h post-mortem) bovine Longissimus thoracis et lumborum, using varying treatment parameters such as treatment duration and frequency (intact steaks in Chapter 3: 25/45 kHz, 15/30/45 min; and homogenates in Chapter 4: 60/100% amp, 15/30 min). The impact of ultrasound on glycolytic enzyme activity was determined within the muscle and in vitro using a glycolytic buffer system, which allows determination of the inherent enzyme activity by monitoring changes in pH and the concentration of key metabolites, namely, glycogen, reducing sugars and lactic acid. When the effect of ultrasound (25/45 kHz, 15/30/45 min) was investigated on the rate of pH decline in pre-rigor bovine Longissimus thoracis et lumborum, it was found that ultrasound frequency (25/45 kHz) did not have a significant (P > 0.05) effect on the rate of pH. However, treatment duration (15/30/45 min) had a significant (P < 0.05) effect on the rate of pH decline, in particular the 30 min treatment where the pH decline in muscle was accelerated. However, no significant (P > 0.05) effect on the ultimate pH was observed between the treatments at 24 h post-treatment. Using the in vitro glycolytic buffer system, it was found that ultrasound frequency and treatment duration did not have a significant effect (P > 0.05) on the inherent glycolytic enzyme activity, measured via changes in pH and content of glycogen, reducing sugars and lactic acid over a 24 h incubation period. It was postulated that the change in pH decline observed in muscle due to the 30 min treatment was potentially due to environmental factors within the muscle. Overall, it was hypothesised that the lack of effect on glycolysis was due to the ultrasound treatments applied not being strong enough to affect enzymatic activity, or that the matrix of the muscle provided protection to the glycolytic enzymes. To examine this further in Chapter 4, the matrix of pre-rigor bovine Longissimus thoracis et lumborum was disrupted by powdering the muscle using liquid nitrogen, from which crude extracts were prepared for ultrasound treatment (60/100% amp (11/19 W/cm2), for 15/30 min). When analysed using the same in vitro glycolytic buffer system, it was found that the degradation of glycogen was not significantly (P > 0.05) affected by ultrasound treatment. However, the rate of pH decline and changes in the content of reducing sugars and lactic acid over time were significantly (P < 0.05) affected by the 100% amp, 30 min treatment. Following this treatment, there was an accumulation of reducing sugars and delayed production of lactic acid and decline in pH within the first 4 h of incubation compared to the other treatments. However, following 24 h of incubation, no significant (P > 0.05) differences in pH and the content of reducing sugars and lactic acid were observed between treatments or between treatments and controls. From the results of Chapter 3 and 4, it was concluded that ultrasound had limited potential to modify enzymatic activity within pre-rigor bovine muscle.
High pressure processing (200 MPa, 20 min, 8 °C) was explored as a pre-treatment to dry aging bovine Longissimus thoracis et lumborum (2 °C, 80% relative humidity, air flow 0.5-2.0 m/s) for up to 28 days. In Chapter 5, the effects of high pressure pre-treatment on weight loss, quality parameters (shear force values, colour, lipid oxidation) and aspects of proteolysis (calpain activity, myofibrillar fragmentation index (MFI)) were investigated, while Chapter 6 explored the impact of the same treatment on the protein (SDS-PAGE) and metabolomic profile (UPLC-Q-TOF/MS). High pressure pre-treatment significantly (P < 0.05) increased weight loss over the 28 day dry aging period, allowing for the potential concentration of flavour related compounds. When compared to the control, it was found that high pressure significantly (P < 0.05) affected quality parameters such as Warner-Bratzler shear force values, lipid oxidation and colour, in particular lightness (L*). Lightness (L*) was significantly (P < 0.05) higher at all time points examined, when compared to the control. However, differences in colour between control and high pressure pre-treatment could be minimised following cooking. Warner-Bratzler shear force values of the HPP pre-treated samples were significantly (P < 0.05) higher than the control for the first 21 days of dry aging, however, following 28 days of dry aging, no significant (P > 0.05) differences were present between control and treated samples. High pressure significantly (P < 0.05) increased lipid oxidation, although the TBARS values were within the consumer’s limit of acceptance. HPP pre-treatment did not significantly (P > 0.05) affect drip and cook loss. When its impact on aspects of proteolysis was investigated, high pressure pre-treatment was found to significantly (P < 0.05) decrease calpain activity, however, myofibrillar fragmentation index significantly (P < 0.05) increased with high pressure pre-treatment and over dry aging time. When examined further using SDS-PAGE, high pressure pre-treatment led to a significant (P < 0.05) reduction in the relative density (%) of myosin heavy chain (MHC) over time, with a corresponding increase (P < 0.05) in the 160 kDa band, identified in the literature as a degradation product of MHC; indicating that the high pressure pre-treatment altered the degradation profile of muscle over time. Metabolomics was used as a tool to examine the effects that the high pressure pre-treatment and dry aging over time have on the metabolomic profile of bovine muscle. When metabolic profiles were examined using PCA and PLS-DA analysis, no differences between control and HPP pre-treated muscle were observed immediately post-treatment; however differences were evident following 14, 21 and 28 days post-treatment. On the other hand, OPLS-DA was able to distinguish between all age time points within and between treatments. High pressure pre-treatment altered the metabolomic profile of bovine muscle at all aging time points (0, 14, 21 and 28 days post-treatment), resulting in an increase in the abundance of peptides and phospholipids, while dry aging time resulted in the increase in peptide content and altered nucleotide concentrations. Metabolites such as peptides, phospholipids and nucleotides may act as precursors to flavour compounds. Hence, alterations to the weight loss, lipid oxidation and metabolomic profile following high pressure pre-treatment have the potential to alter the flavour of dry aged beef.
In conclusion, ultrasound, under the experimental conditions used in this research, appears to have limited potential for altering the rate of glycolysis of pre-rigor bovine Longissimus thoracis et lumborum. However, the results indicate that high pressure processing pre-treatment can accelerate moisture loss of dry aged muscle, increase lipid oxidation and alter the metabolomic profile of bovine muscle, without having a detrimental effect on the shear force values following 28 days of aging, potentially enhancing the flavour of dry aged beef. Further detailed investigations are warranted to determine the impact of HPP pre-treatment on the flavour of dry aged bovine meat and potential consumer preferences.
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Keywords
Meat , Glycolysis , Proteolysis , Meat quality , Ultrasound , High pressure processing , Innovative technologies , Carcass interventions , Meat biochemistry
Citation
Kent, M. A. 2025. Investigation of the effects of emerging technologies on the kinetics of glycolytic and proteolytic enzymes relevant for meat quality. PhD Thesis, University College Cork.