Non-microbiological factors affecting quality and safety 235 Adenosine triphosphate(ATP) is consumed continuously by the living cell to naintain its structure and function. It is produced from the metabolism of glycogen via glycolysis and the Krebs citric acid cycle. At slaughter, the blood supply and therefore replenishment of oxygen to the muscles ceases, but glycolytic activity continues using the stores within muscle cells. Glycogen is metabolized to pyruvate, but, under anaerobic conditions, the Krebs citric acid cycle is no longer functional and the pyruvate is reduced by NADH to lactic acid. The supply of NADH is replenished by glycolysis allowing the conversion of glycogen to lactic acid to continue until the glycogen stores are depleted. The breakdown of each glucose unit in muscle glycogen results in the production of two molecules of lactic acid. The accumulation of lactic acid progressively lowers the pH in the muscles, this action finally ceasing when the muscle supply of glycogen is depleted and the pH is about 5.5-5.6. When aTP is no longer generated the muscle fibres go into a state of stiffness known as rigor. Provided that there is an adequate supply of glycogen at the time of slaughter, the rate and extent of pH fall is dependent on the activity of key enzymes in the glycolytic pathway, competing reactions for adenosine diphosphate(ADP), and the temperature. The lower the temperature the longer the time taken to reach the pH limit, as biochemical reactions are slowed down. The rate of fall and the final ph can have a profound effect on the quality of the meat (Marsh et al. 1987). Lowering of muscle pH leads to protein denaturation and release of a pink proteinaceous fluid called ' drip Reducing the rate at which lactic acid accumulates by rapid chilling of the carcass can dramatically reduce drip loss(Taylor 1972, Swain et al. 1986), however, rapid chilling to temperatures below 12c before anaerobic glycolysis has ceased Ices a condition called 'cold shortening,, resulting in tough meat Animals that were exhausted at the time of slaughter will have depleted glycogen reserves and produce less lactic acid during the attainment of rigor Pork that has a pH greater than 6.0-6.2 at rigor is dark, firm, dry meat(DFD), and spoils microbiologically within 3-5 days owing to the high pH. Animals that were stressed at the time of slaughter to such an extent that respiration was anaerobic may attain rigor pH within one hour of slaughter. Pork which falls to pH 5.8 within 45 minutes of slaughter is pale, soft, exudative meat(PSE). It is characterized by excessive drip loss and is pale as a result of membrane leakage and protein denaturation. The shelf-life of such meat is reduced owing to enhanced microbial growth and oxidation of phospholipids 9.5.3 Proteolysis Activity of proteases can have both beneficial and detrimental effects depending on the situation Proteases in meat are important in the loss of stiffness that takes place after rigor, known as conditioning. Traditionally, conditioning is allowed to occur at the slaughterhouse and should be allowed to proceed until the meat is tender and acceptable to the consumer. Ideally this takes 2-3 weeks holding at chill temperatures; but unchilled carcasses lose stiffness sooner, as proteases act faster
Adenosine triphosphate (ATP) is consumed continuously by the living cell to maintain its structure and function. It is produced from the metabolism of glycogen via glycolysis and the Krebs citric acid cycle. At slaughter, the blood supply and therefore replenishment of oxygen to the muscles ceases, but glycolytic activity continues using the stores within muscle cells. Glycogen is metabolized to pyruvate, but, under anaerobic conditions, the Krebs citric acid cycle is no longer functional and the pyruvate is reduced by NADH to lactic acid. The supply of NADH is replenished by glycolysis allowing the conversion of glycogen to lactic acid to continue until the glycogen stores are depleted. The breakdown of each glucose unit in muscle glycogen results in the production of two molecules of lactic acid. The accumulation of lactic acid progressively lowers the pH in the muscles, this action finally ceasing when the muscle supply of glycogen is depleted and the pH is about 5.5–5.6. When ATP is no longer generated the muscle fibres go into a state of stiffness known as ‘rigor’. Provided that there is an adequate supply of glycogen at the time of slaughter, the rate and extent of pH fall is dependent on the activity of key enzymes in the glycolytic pathway, competing reactions for adenosine diphosphate (ADP), and the temperature. The lower the temperature the longer the time taken to reach the pH limit, as biochemical reactions are slowed down. The rate of fall and the final pH can have a profound effect on the quality of the meat (Marsh et al. 1987). Lowering of muscle pH leads to protein denaturation and release of a pink proteinaceous fluid called ‘drip’. Reducing the rate at which lactic acid accumulates by rapid chilling of the carcass can dramatically reduce drip loss (Taylor 1972, Swain et al. 1986); however, rapid chilling to temperatures below 12ºC before anaerobic glycolysis has ceased produces a condition called ‘cold shortening’, resulting in tough meat. Animals that were exhausted at the time of slaughter will have depleted glycogen reserves and produce less lactic acid during the attainment of rigor. Pork that has a pH greater than 6.0–6.2 at rigor is dark, firm, dry meat (DFD), and spoils microbiologically within 3–5 days owing to the high pH. Animals that were stressed at the time of slaughter to such an extent that respiration was anaerobic may attain rigor pH within one hour of slaughter. Pork which falls to pH 5.8 within 45 minutes of slaughter is pale, soft, exudative meat (PSE). It is characterized by excessive drip loss and is pale as a result of membrane leakage and protein denaturation. The shelf-life of such meat is reduced owing to enhanced microbial growth and oxidation of phospholipids. 9.5.3 Proteolysis Activity of proteases can have both beneficial and detrimental effects depending on the situation. Proteases in meat are important in the loss of stiffness that takes place after rigor, known as ‘conditioning’. Traditionally, conditioning is allowed to occur at the slaughterhouse and should be allowed to proceed until the meat is tender and acceptable to the consumer. Ideally this takes 2–3 weeks holding at chill temperatures; but unchilled carcasses lose stiffness sooner, as proteases act faster Non-microbiological factors affecting quality and safety 235
236 Chilled foods at higher temperatures. For beef, the conditioning rate increases with temperature up to 45C(Q10 2.4), then at a slower rate to 60C (Davey and Gilbert 1976).The role of proteases in conditioning has been reviewed( Goll et al. 1989, Quali and Talmant 1990). Meat proteases can be classified on the basis of preferred pH for functional activity. Proteases active at acid pH, e.g. the cathepsins, are found small organelles, lysosomes, located at the periphery of muscle cells. The stability of lysosomes decreases with a fall in pH, allowing leakage of proteases into the cell and eventually extracellular spaces. A protease active at neutral pH and thought to be involved in conditioning is calpain I which requires free calcium ions for activity. In meat, during the onset of rigor, the lack of ATP as an energy source to pump calcium ions out of cells leads to a rise in the levels of free calcium, and conditions suitable for protease activity. The duration of rigor stiffness is dependent on the species, being about one day for beef, half a day for pork and 24 hours for chicken. The reasons for these differences are not fully understood. Cathepsin levels are higher in chicken and pork which condition quickly(Etherington et al. 1987), and in beef the myofibrillar structure is more resistant to the action of cathepsin enzymes than it is in chicken(Mikami et al. 1987). More precise details of the proteases responsible and the conditions that control their activity have yet to be fully understood In cheese making, the addition to the milk of proteases in rennin and the microbial starter culture causes the development of characteristic flavour and texture during ripening. Chymosin, an aspartyl protease in rennin, splits a single peptide bond in K-casein, a milk protein, which results in clotting. A combination of the action of chymosin and proteases from the starter culture degrade casein to peptides. Many of these peptides can have bitter or sour flavours or no flavour at all, but intracellular proteases from the starter culture break the peptides down further to amino acids and small peptides which have flavour-enhancing properties Bitter flavours in dairy products may be an adverse effect of protease activ Peptides that are composed of predominantly non-polar amino acids tend to be bitter. In fermented dairy products, conditions that favour proteolysis and the accumulation of peptide intermediates are likely to have a bitter flavour In fish, proteases are responsible for the condition known as belly burst Heavy feeding prior to capture enhances the concentration and activity of gut enzymes. Unless the fish is gutted or cooled soon after capture, protease activity weakens the gut wall, allowing leakage of the contents to surrounding tissues Herring and mackerel are notably more susceptible to belly burst; herring can become unsuitable for smoking in one day. In crustacea such as lobster and lawns the process is even more rapid, with gut enzymes attacking the flesh within hours of death. Rapid chilling and processing after catching is required 9.5. 4 Lipolysis The hydrolysis of triacylglycerols at an oil-water interface is catalysed by lipase (Fig. 9.5). The specificity of lipases varies, some being able to attack esters at all
at higher temperatures. For beef, the conditioning rate increases with temperature up to 45ºC (Q10 2.4), then at a slower rate to 60ºC (Davey and Gilbert 1976). The role of proteases in conditioning has been reviewed (Goll et al. 1989, Quali and Talmant 1990). Meat proteases can be classified on the basis of preferred pH for functional activity. Proteases active at acid pH, e.g. the cathepsins, are found in small organelles, lysosomes, located at the periphery of muscle cells. The stability of lysosomes decreases with a fall in pH, allowing leakage of proteases into the cell and eventually extracellular spaces. A protease active at neutral pH and thought to be involved in conditioning is calpain I which requires free calcium ions for activity. In meat, during the onset of rigor, the lack of ATP as an energy source to pump calcium ions out of cells leads to a rise in the levels of free calcium, and conditions suitable for protease activity. The duration of rigor stiffness is dependent on the species, being about one day for beef, half a day for pork and 2–4 hours for chicken. The reasons for these differences are not fully understood. Cathepsin levels are higher in chicken and pork which condition quickly (Etherington et al. 1987), and in beef the myofibrillar structure is more resistant to the action of cathepsin enzymes than it is in chicken (Mikami et al. 1987). More precise details of the proteases responsible and the conditions that control their activity have yet to be fully understood. In cheese making, the addition to the milk of proteases in rennin and the microbial starter culture causes the development of characteristic flavour and texture during ripening. Chymosin, an aspartyl protease in rennin, splits a single peptide bond in -casein, a milk protein, which results in clotting. A combination of the action of chymosin and proteases from the starter culture degrade casein to peptides. Many of these peptides can have bitter or sour flavours or no flavour at all, but intracellular proteases from the starter culture break the peptides down further to amino acids and small peptides which have flavour-enhancing properties. Bitter flavours in dairy products may be an adverse effect of protease activity. Peptides that are composed of predominantly non-polar amino acids tend to be bitter. In fermented dairy products, conditions that favour proteolysis and the accumulation of peptide intermediates are likely to have a bitter flavour. In fish, proteases are responsible for the condition known as ‘belly burst’. Heavy feeding prior to capture enhances the concentration and activity of gut enzymes. Unless the fish is gutted or cooled soon after capture, protease activity weakens the gut wall, allowing leakage of the contents to surrounding tissues. Herring and mackerel are notably more susceptible to belly burst; herring can become unsuitable for smoking in one day. In crustacea such as lobster and prawns the process is even more rapid, with gut enzymes attacking the flesh within hours of death. Rapid chilling and processing after catching is required. 9.5.4 Lipolysis The hydrolysis of triacylglycerols at an oil-water interface is catalysed by lipase (Fig. 9.5). The specificity of lipases varies, some being able to attack esters at all 236 Chilled foods