《冷冻食品》（英文第二版） Part 9 Non-microbiological factors affecting.pdf_大学文库

226 Chilled foods he effects of chemical, biochemical and physio-chemical factors mutually exclusive but these categories provide a convenient framework for iscussion. The effects of these factors are not always detrimental and in some instances they are essential for the development of the desired characteristics of a product. In this chapter, some of the characteristics of chemical, biochemical and physico-chemical reactions are described, along with examples that are of significance to chilled food 9.2 Characteristics of chemical reactions Chemical reactions will proceed if reactants are available, if they are in a suitable form and if the activation energy threshold of the reaction is exceeded The presence of inorganic catalysts reduces the activation energy threshold and causes reactions to proceed that would otherwise not have done so. The reaction rate is dependent on the concentration of the reactants and on the temperature Increases in temperature speed up the random movement of reactant molecules increasing the probability of their coming into contact. A general assumption is that for every 10C rise in temperature the rate of reaction doubles 9.3 Chemical reactions of significance in chilled foods 9.3.1 Lipid oxidation Lipid oxidation is one of the major causes of deterioration in the quality of meat and meat products. Cooked meats and poultry rapidly develop a characteristic oxidized flavour, termedwarmed-over' flavour (WoF) by Tims and Watts (1958). The flavour is best described as that associated with reheated meat and has been described as such by sensory assessors during free profiling of precooked meat, reheated after chill storage( Churchill et al. 1988, Lyon 1987) Further descriptors have been defined for WOF in pork(Byrne et al. 1999a)and chicken meat (Byrne et al. 1999b) and have resulted in the development of sensory vocabularies containing 16 and 18 terms respectively. In cooked meats held at chill storage temperatures, this stale, oxidized flavour becomes apparent within a short time(48 hours) which contrasts with the slower onset of rancidity during frozen storage(weeks)(Pearson and Gray 1983). Although WoF has generally been recognized as affecting only cooked meat, there is evidence that it develops just as rapidly in raw meat that has been ground and exposed to the air (Greene 1969, Sato and Hegarty 1971) and in restructured fresh meat products as a consequence of disruption of the tissue membranes and exposure to oxygen( Gray and Pearson 1987). Nevertheless, the significance of the levelopment of this flavour to food processors has increased with the advent and f markets for cooked chilled ready meals such as TV dinners airline catering, and fast food outlets. The consumer expectation in these situations is for ' freshly prepared flavours. The continued development and

The effects of chemical, biochemical and physio-chemical factors are rarely mutually exclusive but these categories provide a convenient framework for discussion. The effects of these factors are not always detrimental and in some instances they are essential for the development of the desired characteristics of a product. In this chapter, some of the characteristics of chemical, biochemical and physico-chemical reactions are described, along with examples that are of significance to chilled foods. 9.2 Characteristics of chemical reactions Chemical reactions will proceed if reactants are available, if they are in a suitable form and if the activation energy threshold of the reaction is exceeded. The presence of inorganic catalysts reduces the activation energy threshold and causes reactions to proceed that would otherwise not have done so. The reaction rate is dependent on the concentration of the reactants and on the temperature. Increases in temperature speed up the random movement of reactant molecules, increasing the probability of their coming into contact. A general assumption is that for every 10ºC rise in temperature the rate of reaction doubles. 9.3 Chemical reactions of significance in chilled foods 9.3.1 Lipid oxidation Lipid oxidation is one of the major causes of deterioration in the quality of meat and meat products. Cooked meats and poultry rapidly develop a characteristic oxidized flavour, termed ‘warmed-over’ flavour (WOF) by Tims and Watts (1958). The flavour is best described as that associated with reheated meat and has been described as such by sensory assessors during free profiling of precooked meat, reheated after chill storage (Churchill et al. 1988, Lyon 1987). Further descriptors have been defined for WOF in pork (Byrne et al. 1999a) and chicken meat (Byrne et al. 1999b) and have resulted in the development of sensory vocabularies containing 16 and 18 terms respectively. In cooked meats held at chill storage temperatures, this stale, oxidized flavour becomes apparent within a short time (48 hours) which contrasts with the slower onset of rancidity during frozen storage (weeks) (Pearson and Gray 1983). Although WOF has generally been recognized as affecting only cooked meat, there is evidence that it develops just as rapidly in raw meat that has been ground and exposed to the air (Greene 1969, Sato and Hegarty 1971) and in restructured fresh meat products as a consequence of disruption of the tissue membranes and exposure to oxygen (Gray and Pearson 1987). Nevertheless, the significance of the development of this flavour to food processors has increased with the advent and expansion of markets for cooked chilled ready meals such as ‘TV dinners’, airline catering, and fast food outlets. The consumer expectation in these situations is for ‘freshly prepared’ flavours. The continued development and 226 Chilled foods

Non-microbiological factors affecting quality and safety 227 success of fast food facilities and precooked chilled meals will depend to some extent on the ability of processors to overcome the development of WoF id oxidation has long been considered to be the primary cause of woF supported by studies correlating increases in WoF determined sensorily (Love 1988) with measurements of the thiobarbituric acid (TBa)number(an indicator of lipid oxidation)(Igene et al. 1979, Igene et al. 1985, Smith et al. 1987), and identification of the volatile compounds extracted from the headspace above meat samples(St Angelo et al. 1987, Ang and Lyon 1990, Churchill et al. 1990) As with other examples of oxidative rancidity, the process of lipid oxidation results in the formation of many different compounds, some of which are more significant than others to the undesirable odour and flavour associated with rancidity. This gives rise to a less than perfect relationship between measured chemical markers and sensory assessment of rancidity The reactivity of food lipids is influenced by the degree of unsaturation of constituent fatty acids, their availability and the presence of activators or nhibitors. The composition of fats in meat reflects a number of factors including the diet of the animal and the type of fat. Lipids are most abundant as either storage depot(adipose) fats or in cell membranes as phospholipids During cooking, the unsaturated phospholipids, as opposed to the storage triglycerides, are rendered more susceptible to oxidation by disruption and dehydration of cell membranes. The higher degree of unsaturation of fatty acid in the phospholipids contributes to their more rapid rate of oxidation(Igene et al. 1981). The role of phospholipids in the formation of WoF (Igene and Pearson 1979)and TBa reactive substances( Roozen 1987, Pikul and Kummerow 1991) has been demonstrated Autoxidation of lipids is generally accepted to involve a free radical chain reaction(Fig. 9.1), which is initiated when a labile hydrogen atom is abstracted from a site on the lipid(RH with the production of lipid radicals (R) (initiation). Reaction with oxygen yields peroxyl radicals(ROO") and this followed by abstraction of another hydrogen from a lipid molecule. A hydroperoxide(rooh) and another free radical(R") which is capable of perpetuating the chain reaction, are formed (propagation). Decomposition of the hydroperoxides involves further free radical mechanisms and the formation of non-radical products including volatile aroma compounds Despite much research effort, the mechanism of initiation leading to the formation of the lipid(alkyl or allyl) radical(r") in meat is still an area of confusion and debate. The involvement of iron has been established(minotti and Aust 1987), but beyond this various mechanisms have been suggested but not supported by conclusive evidence(Ashgar et al. 1988) The rate of formation of free radicals is increased by the presence of metal atalysts. In the case of warmed-over flavour development in cooked meats, both free ferrous ions and haemoproteins, including metmyoglobin in the presence of hydrogen peroxide(Asghar et al. 1988)have been shown to have a prooxidant effect. The availability of free iron is known to increase as a result of cooking(Igene et al. 1979)as haemoproteins are broken down and release free

success of fast food facilities and precooked chilled meals will depend to some extent on the ability of processors to overcome the development of WOF. Lipid oxidation has long been considered to be the primary cause of WOF, supported by studies correlating increases in WOF determined sensorily (Love 1988) with measurements of the thiobarbituric acid (TBA) number (an indicator of lipid oxidation) (Igene et al. 1979, Igene et al. 1985, Smith et al. 1987), and identification of the volatile compounds extracted from the headspace above meat samples (St Angelo et al. 1987, Ang and Lyon 1990, Churchill et al. 1990). As with other examples of oxidative rancidity, the process of lipid oxidation results in the formation of many different compounds, some of which are more significant than others to the undesirable odour and flavour associated with rancidity. This gives rise to a less than perfect relationship between measured chemical markers and sensory assessment of rancidity. The reactivity of food lipids is influenced by the degree of unsaturation of constituent fatty acids, their availability and the presence of activators or inhibitors. The composition of fats in meat reflects a number of factors, including the diet of the animal and the type of fat. Lipids are most abundant as either storage depot (adipose) fats or in cell membranes as phospholipids. During cooking, the unsaturated phospholipids, as opposed to the storage triglycerides, are rendered more susceptible to oxidation by disruption and dehydration of cell membranes. The higher degree of unsaturation of fatty acids in the phospholipids contributes to their more rapid rate of oxidation (Igene et al. 1981). The role of phospholipids in the formation of WOF (Igene and Pearson 1979) and TBA reactive substances (Roozen 1987, Pikul and Kummerow 1991) has been demonstrated. Autoxidation of lipids is generally accepted to involve a free radical chain reaction (Fig. 9.1), which is initiated when a labile hydrogen atom is abstracted from a site on the lipid (RH) with the production of lipid radicals (R• ) (initiation). Reaction with oxygen yields peroxyl radicals (ROO• ) and this is followed by abstraction of another hydrogen from a lipid molecule. A hydroperoxide (ROOH) and another free radical (R• ) which is capable of perpetuating the chain reaction, are formed (propagation). Decomposition of the hydroperoxides involves further free radical mechanisms and the formation of non-radical products including volatile aroma compounds. Despite much research effort, the mechanism of initiation leading to the formation of the lipid (alkyl or allyl) radical (R• ) in meat is still an area of confusion and debate. The involvement of iron has been established (Minotti and Aust 1987), but beyond this various mechanisms have been suggested but not supported by conclusive evidence (Ashgar et al. 1988). The rate of formation of free radicals is increased by the presence of metal catalysts. In the case of warmed-over flavour development in cooked meats, both free ferrous ions and haemoproteins, including metmyoglobin in the presence of hydrogen peroxide (Asghar et al. 1988) have been shown to have a prooxidant effect. The availability of free iron is known to increase as a result of cooking (Igene et al. 1979) as haemoproteins are broken down and release free Non-microbiological factors affecting quality and safety 227

228 Chilled foods Initiation RH→R R+O ROO ROo+rh- ROoH+R Fig 9. 1. Free radical chain reaction. iron. The amount released is dependent on the rate of heating and the final temperature, and therefore on the method of heating. Slow heating releases more free iron than fast heating- roasting or braising of meat releases more than does microwave heating(Shricker and Miller 1983) Procedures for the prevention of woF were reviewed by Pearson and Gray (1983). The method used is very often restricted by the requirements of the final product Phenolic antioxidants such as BhT and BHA are of little value in intact meat cuts(Watts 1961), whereas they may be more suited to comminuted meat products since a more even distribution of the antioxidant can be achieved Overheating or retorting of meat to produce compounds that have antioxidant activity(Maillard Reaction products) may be suitable for canned products but tends to result in a product with characteristics contrary to the fresh perception that is a necessary part of many chilled foods. Alternatively, these compound can be added to meat, but they are then restricted by the same considerations that apply to artificial antioxidants. Reduction of woF has also been achieved by use of vitamin E. Kerry et al. (1999)demonstrated that addition of alpha-tocopherol to cooked pig meat reduced lipid oxidation and WOF. Difficulties in achieving adequate distribution of the antioxidant in the meat could be overcome by the incorporation of vitamin E supplements to the feed of the animals. Addition of alpha-tocopherol acetate to the diet of rabbits(Lopez-Bote et al. 1997) and broiler chicks(ONeill et al. 1998) has been shown to be reflected by an increase in the muscle tissue and result in reduced woF development. Investigations of natural antioxidants present in vegetables have shown some benefits for using extracts from green peppers, onions and potato peelings(Pratt and Watts 1964) and herbs and spices, particularly rosemary, sage, marjoram(Hermann et al. 981) and clove (ayathilakan et al. 1997. Reports of the effectiveness of rosemary oleoresin as an antioxidant in precooked meats are conflicting although Murphy et al.(1998)found rosemary oleoresin and sodium tripolyphosphate to be effective in the prevention of woF in precooked roast beef slices. Precooked pork balls processed with rosemary stored at 4C for 48 hours did not develop oxidized flavours as the controls did(Korczack et al 1988), whereas restructured beef steaks processed with oleoresin rosemary stored at refrigerated temperatures showed no significant improvement in comparison to the controls(Stoick et al. 1991) The addition of nitrite between 50 and 200 ppm is an effective inhibitor of the development of WoF (Sato and Hegarty 1971, Cho and rhee 1997). Nitrite and

iron. The amount released is dependent on the rate of heating and the final temperature, and therefore on the method of heating. Slow heating releases more free iron than fast heating – roasting or braising of meat releases more than does microwave heating (Shricker and Miller 1983). Procedures for the prevention of WOF were reviewed by Pearson and Gray (1983). The method used is very often restricted by the requirements of the final product. Phenolic antioxidants such as BHT and BHA are of little value in intact meat cuts (Watts 1961), whereas they may be more suited to comminuted meat products since a more even distribution of the antioxidant can be achieved. Overheating or retorting of meat to produce compounds that have antioxidant activity (Maillard Reaction products) may be suitable for canned products but tends to result in a product with characteristics contrary to the ‘fresh’ perception that is a necessary part of many chilled foods. Alternatively, these compounds can be added to meat, but they are then restricted by the same considerations that apply to artificial antioxidants. Reduction of WOF has also been achieved by use of vitamin E. Kerry et al. (1999) demonstrated that addition of alpha-tocopherol to cooked pig meat reduced lipid oxidation and WOF. Difficulties in achieving adequate distribution of the antioxidant in the meat could be overcome by the incorporation of vitamin E supplements to the feed of the animals. Addition of alpha-tocopherol acetate to the diet of rabbits (Lopez-Bote et al. 1997) and broiler chicks (O’Neill et al. 1998) has been shown to be reflected by an increase in the muscle tissue and result in reduced WOF development. Investigations of natural antioxidants present in vegetables have shown some benefits for using extracts from green peppers, onions and potato peelings (Pratt and Watts 1964) and herbs and spices, particularly rosemary, sage, marjoram (Hermann et al. 1981) and clove (Jayathilakan et al. 1997). Reports of the effectiveness of rosemary oleoresin as an antioxidant in precooked meats are conflicting although Murphy et al. (1998) found rosemary oleoresin and sodium tripolyphosphate to be effective in the prevention of WOF in precooked roast beef slices. Precooked pork balls processed with rosemary stored at 4ºC for 48 hours did not develop oxidized flavours as the controls did (Korczack et al. 1988), whereas restructured beef steaks processed with oleoresin rosemary stored at refrigerated temperatures showed no significant improvement in comparison to the controls (Stoick et al. 1991). The addition of nitrite between 50 and 200 ppm is an effective inhibitor of the development of WOF (Sato and Hegarty 1971, Cho and Rhee 1997). Nitrite and Fig. 9.1. Free radical chain reaction. 228 Chilled foods

Non-microbiological factors affecting quality and safety 229 H3C H3C Pr C= cH Pr -NO Fig. 9. 2. Nitrosylmyoglobin. Myoglobin with the nitrite ligand haemoproteins form nitrosylmyochrome and nitrosylhaemochrome complexes in which the iron is stabilised by the linking of nitric oxide to the porphyrin ring (Fig. 9.2); however, the pink coloration of the meat may be undesirable; causes of pinking in uncured cooked meat is further considered later in this chapter. The effectiveness of pyrophosphate, tripolyphosphate and hexametaphosphate, which chelate metal ions, particularly prooxidative ferrous ions, was demon strated by Tims and Watts(1958)in pork. It has since been verified for ground beef (Sato and Hegarty 1971), for restructured beef steaks(Mann et al. 1989) and for battered and breaded chicken(Brotsky 1976). Phosphates in combination with ascorbic acid may exert a synergistic effect, such that cooked ground pork was protected against lipid oxidation for up to 35 days at 4 C( Shahidi et al. 1986) An alternative approach is to protect the meat from oxidation. This can be achieved by creating an oxygen barrier, using a sauce or a gravy that can be in place at the time of cooking and during subsequent storage. This principle has been demonstrated by comparing the shelf-life of frozen meat to that of the same meats cooked without gravy coverings(Dalhoff and Jul 1965). Cooked pork covered with gravy could be stored at -18C for more than 100 weeks, whereas pork stored without gravy was unacceptable after 22 weeks Modified-atmosphere packaging to reduce Wof has been applied to precooked turkey and pork and pork products. Although those stored in nitrogen and carbon dioxide atmospheres were less oxidized than those in air, packaging was the most effective(Nolan et al. 1989 and Juncher et al ( 1998). Shaw(1997)reviewed the potential benefits of the use of MAP for cook chill ready meals. Protection against oxidation at the time of cooking is also beneficial. Cooking and subsequent storage of chicken breasts in a nitrogen

haemoproteins form nitrosylmyochrome and nitrosylhaemochrome complexes in which the iron is stabilised by the linking of nitric oxide to the porphyrin ring (Fig. 9.2); however, the pink coloration of the meat may be undesirable; causes of pinking in uncured cooked meat is further considered later in this chapter. The effectiveness of pyrophosphate, tripolyphosphate and hexametaphosphate, which chelate metal ions, particularly prooxidative ferrous ions, was demonstrated by Tims and Watts (1958) in pork. It has since been verified for ground beef (Sato and Hegarty 1971), for restructured beef steaks (Mann et al. 1989), and for battered and breaded chicken (Brotsky 1976). Phosphates in combination with ascorbic acid may exert a synergistic effect, such that cooked ground pork was protected against lipid oxidation for up to 35 days at 4ºC (Shahidi et al. 1986). An alternative approach is to protect the meat from oxidation. This can be achieved by creating an oxygen barrier, using a sauce or a gravy that can be in place at the time of cooking and during subsequent storage. This principle has been demonstrated by comparing the shelf-life of frozen meat to that of the same meats cooked without gravy coverings (Dalhoff and Jul 1965). Cooked pork covered with gravy could be stored at 18ºC for more than 100 weeks, whereas pork stored without gravy was unacceptable after 22 weeks. Modified-atmosphere packaging to reduce WOF has been applied to precooked turkey and pork and pork products. Although those stored in nitrogen and carbon dioxide atmospheres were less ‘oxidized’ than those in air, vacuum packaging was the most effective (Nolan et al. 1989 and Juncher et al (1998). Shaw (1997) reviewed the potential benefits of the use of MAP for cookchill ready meals. Protection against oxidation at the time of cooking is also beneficial. Cooking and subsequent storage of chicken breasts in a nitrogen Fig. 9.2. Nitrosylmyoglobin. Myoglobin with the nitrite ligand. Non-microbiological factors affecting quality and safety 229

Non-microbiological factors affecting quality and safety 231 potential to initiate lipid oxidation by providing a source of initiating radicals German and Kinsella 1985). A complicating factor in the assessment of the significance of oxidation to the quality of fish is that many products distributed chilled have previously been frozen, particularly for example, herring, to sprea seasonal availability 9.3.2 Pink discoloration in meat products Discoloration in foods is a common problem which can take many forms and be ssociated with a wide range of chemical reactions: biochemical or enzymic browning is considered later in this chapter. Pink discoloration in cooked meats br is a long-standing and all too common problem affecting manufacturing, retailing food service and domestic sectors and is often interpreted as undercooking. The problem is particularly evident with sliced meats, reformed roast products, pasties and casseroles. Various causes of pinking have beer identified and these are indicated in Table 9. 1 on the basis of the pigment type thought to be involved. Maga(1994)has reviewed the causes and factors affecting pink discoloration in cooked white meats L Myoglobin is a monomeric globular haem-protein found in all vertebrates hich together with haemoglobin give rise to the red colour of meats. The amount of myoglobin varies from species to species, tissue to tissue and is affected by a wide range of environmental factors. As indicated in Table 9. 1 myoglobin can be present in several forms, some of which can impart a red or pink residual colour to the meat even after cooking. Recent work has indicated that over 80% of instances of pinking are due to nitrosomyoglobin arising from nitrate contami- nation and its subsequent bacterial reduction to nitrite( Brown et al. 1998) 9. 4 Characteristics of biochemical reactions Biochemical reactions are catalysed by specialized proteins called enzymes They are highly specific and efficient catalysts, lowering the activation threshold so that the rate of reaction of thermodynamically possible reactions is Table 9.1 Pigment types and causes giving rise to pink coloration in meat products Brown et al. 1998) Cause of pink discoloration Oxymyoglobin Low temperature cooking Nitrosomyoglobin Nitrite contamination directly or from reduced nitrate nitrogen oxides in ovens Carboxymyoglobin Carbon monoxide in ovens; gamma-irradiation Reduced denatured myoglobin High pH, slow cooking, high salt and availability of educing agents

potential to initiate lipid oxidation by providing a source of initiating radicals (German and Kinsella 1985). A complicating factor in the assessment of the significance of oxidation to the quality of fish is that many products distributed chilled have previously been frozen, particularly for example, herring, to spread seasonal availability. 9.3.2 Pink discoloration in meat products Discoloration in foods is a common problem which can take many forms and be associated with a wide range of chemical reactions: biochemical or enzymic browning is considered later in this chapter. Pink discoloration in cooked meats is a long-standing and all too common problem affecting manufacturing, retailing food service and domestic sectors and is often interpreted as undercooking. The problem is particularly evident with sliced meats, reformed roast products, pasties and casseroles. Various causes of pinking have been identified and these are indicated in Table 9.1 on the basis of the pigment type thought to be involved. Maga (1994) has reviewed the causes and factors affecting pink discoloration in cooked white meats. Myoglobin is a monomeric globular haem-protein found in all vertebrates which together with haemoglobin give rise to the red colour of meats. The amount of myoglobin varies from species to species, tissue to tissue and is affected by a wide range of environmental factors. As indicated in Table 9.1 myoglobin can be present in several forms, some of which can impart a red or pink residual colour to the meat even after cooking. Recent work has indicated that over 80% of instances of pinking are due to nitrosomyoglobin arising from nitrate contamination and its subsequent bacterial reduction to nitrite (Brown et al. 1998). 9.4 Characteristics of biochemical reactions Biochemical reactions are catalysed by specialized proteins called enzymes. They are highly specific and efficient catalysts, lowering the activation threshold so that the rate of reaction of thermodynamically possible reactions is Table 9.1 Pigment types and causes giving rise to pink coloration in meat products (Brown et al. 1998) Pigment type Cause of pink discoloration Oxymyoglobin Low temperature cooking Nitrosomyoglobin Nitrite contamination directly or from reduced nitrate; nitrogen oxides in ovens Carboxymyoglobin Carbon monoxide in ovens; gamma-irradiation Reduced denatured myoglobin High pH, slow cooking, high salt and availability of reducing agents Non-microbiological factors affecting quality and safety 231

232 Chilled foods dramatically increased. The specificity of enzymes for a particular substrate is indicated in the name, usually by attachment of the suffix -ase' to the name of the substrate on which it acts: for example, lipase acts on lipids, protease on proteins. The catalytic activity of enzymes is highly dependent on the conformational structure of the protein, and many of the characteristics of enzyme-catalysed reactions result from the influence of the localized environment. Heat, extremes of acidity of alkalinity, and high may denature the enzyme, causing impairment or loss of activity. Enzyme inhibitors and activators that bind either reversibly or irreversibly may act by causing changes in conformational structure or acting directly at the active site The temperature at which denaturation takes place is often a reflection of nvironmental conditions that the enzyme naturally operates in. For most enzymes from warm-blooded animals, denaturation begins around 45.C, and by 55C rapid denaturation destroys the catalytic function of the enzyme protein; enzymes from fruit and vegetables are generally denatured at higher temperatures(70-80oC); and some microbial enzymes, e.g. lipases and proteases, can withstand temperatures in excess of 100C( Cogan 1977) In the living cell, enzymes catalyse a vast array of reactions that taken together constitute metabolism. In the cellular environment, control and coordination of enzyme activity is achieved by means of feedback mechanisms and compartmentalisation. Disruption which occurs at the time of slaughter or harvest may necessitate steps being taken to prevent the subsequent action of enzymes(blanching of vegetables is a good example); or the activity of enzymes may be enhanced if they improve product quality, as in the case of conditioning of meats, where protease activity is used to break down muscle fibres to develop full flavour and tenderness The rate of enzyme-catalysed reactions increases with substrate concentration but only up to a limit (maximal activity) at which the enzyme is saturated with substrate. Further increases in substrate concentration do not increase the rate of reaction. The rate of reaction increases with temperature in the same way as chemical reactions up to an optimum temperature for activity. At temperatures above this, denaturation of the enzyme protein takes place and activity is lost. At hill storage temperatures, the activity of enzymes in most foods is low, but there are notable exceptions. Enzymes in cold-blooded species may be adapted to be active at cold temperatures. In cod, lipase activity at ooC shows a marked lag phase before maximal activity is achieved and the rate of activity decreases to oc and increases to a maximum at -4C Enzymes from different sources, although catalysing conversion of the same substrates to the same reaction products, may have different characteristics in terms of rate of reaction, or pH or temperature optima, depending upon thei rigin.In a chilled pasta salad composed of cooked pasta, onion, red and green peppers, cucumber, sweetcorn, mushrooms and vinaigrette dressing, shelf-life was limited by browning of either the sweetcorn or the mushrooms depending on the holding temperature(Gibbs and williams 1990). Holding the salad at storage temperatures between 2C and 15C showed that the temperature characteristics

dramatically increased. The specificity of enzymes for a particular substrate is indicated in the name, usually by attachment of the suffix ‘-ase’ to the name of the substrate on which it acts: for example, lipase acts on lipids, protease on proteins. The catalytic activity of enzymes is highly dependent on the conformational structure of the protein, and many of the characteristics of enzyme-catalysed reactions result from the influence of the localized environment. Heat, extremes of acidity of alkalinity, and high ionic strength may denature the enzyme, causing impairment or loss of activity. Enzyme inhibitors and activators that bind either reversibly or irreversibly may act by causing changes in conformational structure or acting directly at the active site. The temperature at which denaturation takes place is often a reflection of the environmental conditions that the enzyme naturally operates in. For most enzymes from warm-blooded animals, denaturation begins around 45ºC, and by 55ºC rapid denaturation destroys the catalytic function of the enzyme protein; enzymes from fruit and vegetables are generally denatured at higher temperatures (70–80ºC); and some microbial enzymes, e.g. lipases and proteases, can withstand temperatures in excess of 100ºC (Cogan 1977). In the living cell, enzymes catalyse a vast array of reactions that taken together constitute metabolism. In the cellular environment, control and coordination of enzyme activity is achieved by means of feedback mechanisms and compartmentalisation. Disruption which occurs at the time of slaughter or harvest may necessitate steps being taken to prevent the subsequent action of enzymes (blanching of vegetables is a good example); or the activity of enzymes may be enhanced if they improve product quality, as in the case of ‘conditioning’ of meats, where protease activity is used to break down muscle fibres to develop full flavour and tenderness. The rate of enzyme-catalysed reactions increases with substrate concentration but only up to a limit (maximal activity) at which the enzyme is saturated with substrate. Further increases in substrate concentration do not increase the rate of reaction. The rate of reaction increases with temperature in the same way as chemical reactions up to an optimum temperature for activity. At temperatures above this, denaturation of the enzyme protein takes place and activity is lost. At chill storage temperatures, the activity of enzymes in most foods is low, but there are notable exceptions. Enzymes in cold-blooded species may be adapted to be active at cold temperatures. In cod, lipase activity at 0ºC shows a marked lag phase before maximal activity is achieved and the rate of activity decreases to 0ºC and increases to a maximum at 4ºC. Enzymes from different sources, although catalysing conversion of the same substrates to the same reaction products, may have different characteristics in terms of rate of reaction, or pH or temperature optima, depending upon their origin. In a chilled pasta salad composed of cooked pasta, onion, red and green peppers, cucumber, sweetcorn, mushrooms and vinaigrette dressing, shelf-life was limited by browning of either the sweetcorn or the mushrooms depending on the holding temperature (Gibbs and Williams 1990). Holding the salad at storage temperatures between 2ºC and 15ºC showed that the temperature characteristics 232 Chilled foods

Non-microbiological factors affecting quality and safety 233 260 265 70 275 280 Fig. 9.4. Organoleptic changes in chill-stored pasta salad in vinaigrette. Temperature dependence of the rates of browning of sweetcorn(●-● and mushrooms((-○) Gibbs and williams 1990) of the browning reaction, likely to be catalysed by the enzyme polyphenolox idase, were quite different in the mushrooms and sweetcorn(Fig. 9.4). In mushrooms, the rate of browning reaction appeared to be less temperature- sensitive than was the reaction in sweetcorn, such that at higher temperatures the shelf-life of the salad was limited by browning of the sweetcorn, and at lower temperatures by browning of the mushrooms. To prevent such changes or to predict the shelf-life as a function of temperature, the subtleties of the reactions causing the changes in visual appearance need to be known Enzymes in food may be endogenous, that is, they are present naturally in the tissues of the plant or animal that comprises the food. Many hundreds of enzymes fall into this category, though not all will have a significant effect on food quality. Exogenous enzymes in food may be added by the manufacturer to perform a specific function, such as papain for the tenderization of meat proteases for cheese ripening, or naringinase for the debittering of citrus juices particularly grapefruit juice. Enzymes may be present as a result of tion from one food to another when they are in contact;an example would be the migration of lipases from unblanched peppers n a pizza topping to the cheese if the appropriate tracy glycerols are available, lipolysis will result in soapy flavours. Alternatively, there may be contamination by extracellular enzymes from microorganisms such as lipases and proteases, where the organism may be destroyed by heat processing but the enzyme which is resistant to the heat treatment remains 9.5 Biochemical reactions of significance in chilled foods 9.5.1 Enzymic browning In fruits and vegetables, enzymic browning occurs due to damage such as bruising and preparation procedures of cutting, peeling and slicing. The

of the browning reaction, likely to be catalysed by the enzyme polyphenoloxidase, were quite different in the mushrooms and sweetcorn (Fig. 9.4). In mushrooms, the rate of browning reaction appeared to be less temperaturesensitive than was the reaction in sweetcorn, such that at higher temperatures the shelf-life of the salad was limited by browning of the sweetcorn, and at lower temperatures by browning of the mushrooms. To prevent such changes or to predict the shelf-life as a function of temperature, the subtleties of the reactions causing the changes in visual appearance need to be known. Enzymes in food may be endogenous, that is, they are present naturally in the tissues of the plant or animal that comprises the food. Many hundreds of enzymes fall into this category, though not all will have a significant effect on food quality. Exogenous enzymes in food may be added by the manufacturer to perform a specific function, such as papain for the tenderization of meat, proteases for cheese ripening, or naringinase for the debittering of citrus juices particularly grapefruit juice. Enzymes may be present as a result of ‘contamination’ by migration from one food to another when they are in contact; an example would be the migration of lipases from unblanched peppers in a pizza topping to the cheese where, if the appropriate triacy1glycerols are available, lipolysis will result in soapy flavours. Alternatively, there may be ‘contamination’ by extracellular enzymes from microorganisms such as lipases and proteases, where the organism may be destroyed by heat processing but the enzyme which is resistant to the heat treatment remains. 9.5 Biochemical reactions of significance in chilled foods 9.5.1 Enzymic browning In fruits and vegetables, enzymic browning occurs due to damage such as bruising and preparation procedures of cutting, peeling and slicing. The Fig. 9.4. Organoleptic changes in chill-stored pasta salad in vinaigrette. Temperature dependence of the rates of browning of sweetcorn (●—●) and mushrooms (❍— ❍) (Gibbs and Williams 1990). Non-microbiological factors affecting quality and safety 233

234 Chilled foods yellowish brown through to black pigments that are formed can appear very rapidly and are unappetizing. In the intact tissue the enzymes responsible, are separated from the sul However, when they are brought into contact as a result of damage, naturally ccurring phenolic compounds are enzymically oxidized to form yellowish quinone compounds (vamos-Vigyazo 1981). A sequence of polymerization reactions follow, giving rise to brown products such as melanins The extent of browning is dependent on the activity and amount of the polyphenoloxidase in the specific fruit or vegetable and the availability of substrates which may be catechol, tyrosine or dopamine amongst others, but there is al ways a requirement for oxygen. A number of approaches have been taken to prevent or retard enzymic browning. Reduction of the available oxygen concentration has been achieved via various approaches: vacuum packaging which retarded enzymic browning in potato strips(O Beirne and Ballantyne 1987); modified atmosphere packaging, e.g. for shredded lettuce and cut carrots (McLachlan and Stark 1985); the addition of an oxygen scavenger to the pack, which retarded enzymic browning and textural changes in apricot and peach halves(Bolin and Huxsoll 1989); and restricting oxygen diffusion into tissues by immersion in water, brine or syrup solutions. In contrast, high levels of oxygen (70-100%)have also been shown to reduce ascorbic acid breakdown, lipid oxidation and enzymic browning in cut lettuce probably as a result of increasing the total antioxidant capacity of the material (Day 1998). A more direct method to prevent enzymic discoloration is to use enzyme inhibitors, though this may conflict with the ' fresh'image of the product or be restricted by legislation Traditionally, the use of sulphite in the form of metabisul phite dips provided an effective means of preventing enzymic browning in many instances. With restrictions on the use of sulphite, alternatives have been sought. The pH optimum for phenolase activity is generally between pH 5 and 7. Reduction of the ph to less than 4 by the use of edible acids inactivates the enzyme. Citric acid and ascorbic acid dips retard browning by both a reduction in pH and complexation of copper which is essential for the enzyme to function. Levels of 10% ascorbic acid were shown to be effective for potatoes, and 0.5-1% for apples(O Beirne 1988). Phenolases from most fruits and vegetables are inactivated by heat (vamos-Vigyazo, 1981)but for salads and pre-pre vegetables heat treatment may not be an acceptable option owing concomitant changes in colour and texture 9.5.2 Glycolysis This is a key metabolic pathway of intermediary metabolism found in almost all living organisms. Changes that take place at the time of slaughter and harvest nfluence the route that substrates metabolized via this pathway subsequently follow. Diversion of the pathway to produce end-products of lactic acid in meat and ethanol in vegetables have marked consequences for the subsequent quality

yellowish brown through to black pigments that are formed can appear very rapidly and are unappetizing. In the intact tissue the enzymes responsible, generically referred to as ‘phenolases’, are separated from the substrate. However, when they are brought into contact as a result of damage, naturally occurring phenolic compounds are enzymically oxidized to form yellowish quinone compounds (Va´mos-Vigya´zo´ 1981). A sequence of polymerization reactions follow, giving rise to brown products such as melanins. The extent of browning is dependent on the activity and amount of the polyphenoloxidase in the specific fruit or vegetable and the availability of substrates which may be catechol, tyrosine or dopamine amongst others, but there is always a requirement for oxygen. A number of approaches have been taken to prevent or retard enzymic browning. Reduction of the available oxygen concentration has been achieved via various approaches: vacuum packaging which retarded enzymic browning in potato strips (O’Beirne and Ballantyne 1987); modified atmosphere packaging, e.g. for shredded lettuce and cut carrots (McLachlan and Stark 1985); the addition of an oxygen scavenger to the pack, which retarded enzymic browning and textural changes in apricot and peach halves (Bolin and Huxsoll 1989); and restricting oxygen diffusion into tissues by immersion in water, brine or syrup solutions. In contrast, high levels of oxygen (70–100%) have also been shown to reduce ascorbic acid breakdown, lipid oxidation and enzymic browning in cut lettuce probably as a result of increasing the total antioxidant capacity of the material (Day 1998). A more direct method to prevent enzymic discoloration is to use enzyme inhibitors, though this may conflict with the ‘fresh’ image of the product or be restricted by legislation. Traditionally, the use of sulphite in the form of metabisulphite dips provided an effective means of preventing enzymic browning in many instances. With restrictions on the use of sulphite, alternatives have been sought. The pH optimum for phenolase activity is generally between pH 5 and 7. Reduction of the pH to less than 4 by the use of edible acids inactivates the enzyme. Citric acid and ascorbic acid dips retard browning by both a reduction in pH and complexation of copper which is essential for the enzyme to function. Levels of 10% ascorbic acid were shown to be effective for potatoes, and 0.51% for apples (O’Beirne 1988). Phenolases from most fruits and vegetables are readily inactivated by heat (Va´mos-Vigya´zo´, 1981) but for salads and pre-prepared vegetables heat treatment may not be an acceptable option owing to the concomitant changes in colour and texture. 9.5.2 Glycolysis This is a key metabolic pathway of intermediary metabolism found in almost all living organisms. Changes that take place at the time of slaughter and harvest influence the route that substrates metabolized via this pathway subsequently follow. Diversion of the pathway to produce end-products of lactic acid in meat and ethanol in vegetables have marked consequences for the subsequent quality of the food product. 234 Chilled foods