冷冻食品（英文第二版）：34990wp_09

9.1 Introduction As the chilled foods market has expanded and become more competitive, so have the demands for diversity, quality and longer shelf-life. Meeting these demands in a responsible, safe and cost-effective manner requires the application of an understanding of the factors that affect product safety and quality. Many problems can be avoided by applying this knowledge to a formalised HACCP approach to identify critical control points relating to quality as well as safety and to make realistic predictions of shelf-life. Considering these issues early in the product development process offers the best chance of providing a product that meets the consumer’s expectations and delivers the desired market opportunities to the company. Food is probably the most chemically complex substance that most people encounter. There are over half a million naturally occurring compounds in fresh plant food and more are formed as a result of processing, cooking and storage. They are responsible for the appearance, flavour, texture and nutritional value of the food (quality), and for its physiological effects when consumed (safety). Non-microbiological factors that affect quality and safety of chilled foods can be broadly divided into chemical, biochemical and physico-chemical factors. Each of these is dependent on properties of the food (e.g. pH, water activity) and the conditions in which the food is held (e.g. temperature, gaseous atmosphere). Attention to the selection of raw materials in order to achieve high quality is paramount, since subsequent processing cannot compensate for poor-quality raw materials, particularly for chilled foods in which the perception of ‘freshness’ is one of the most important criteria for its purchase. 9 Non-microbiological factors affecting quality and safety H. M. Brown and M. N. Hall, Campden and Chorleywood Food Research Association 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 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 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 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 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 C018oC 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 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 Fig. 9.2. Nitrosylmyoglobin. Myoglobin with the nitrite ligand. Non-microbiological factors affecting quality and safety 229 atmosphere reduced the TBA values and sensory scores for WOF intensity as compared with those cooked in air and stored in either nitrogen or air (Fig. 9.3). Autooxidation or oxidative rancidity is by no means confined to meat and meat products. Dairy products and fatty fish are also highly susceptible. Migration of copper into cream on churning can initiate the oxidative sequence of reactions causing rapid flavour impairment. Buttermilk has a high proportion of unsaturated phospholipids, particularly phosphatidylethanolamine, that can bind metal ions in a prooxidative fashion, and the presence of a metal–phospholipid complex at an oil-water interface facilitates lipid hydroperoxide formation. Fish fats contain a high proportion of nC03 polyunsaturated fatty acids, which are vulnerable to oxidation by atmospheric oxygen leading to deteriorative changes. Despite this, rancid flavours only appear to affect the acceptability of fattier species such as trout, sardine, herring and mackerel; and even then, trout and gutted mackerel oxidize at temperatures above 0oC whereas herring remains relatively unaffected. Castell (1971) has suggested that in fish, oxidized lipids become bound in lipid-protein complexes rather than forming carbonyl compounds associated with rancid flavours. The lipid–protein complexes also contribute to the toughened texture of poorly stored fish. Competing demands for available oxygen from microorganisms and enzymes, which differ between species, may also influence whether oxygen is available for autooxidation. In trout, reports of lipoxygenase activity in the skin tissue have suggested the Fig. 9.3. Effect of cooking and storage atmospheres on WOF development in chicken breasts. (? — ?) cooked and stored in air; (C2—C2) cooked in nitrogen, stored in air; (a73 — a73) cooked in air, stored in nitrogen; ([C52] — [C52]) cooked and stored in nitrogen. 230 Chilled foods 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 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) 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 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 C04oC. 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 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 ‘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 (a71 — a71) and mushrooms (? — ?) (Gibbs and Williams 1990). Non-microbiological factors affecting quality and safety 233 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 0.5C01% 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 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 at higher temperatures. For beef, the conditioning rate increases with temperature up to 45oC(Q 10 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 C20-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 three positions in the triacylglycerol whilst others are restricted to positions 1 and 3. The activity of lipases of either endogenous or microbial origin is responsible for changes in functional properties of some dairy products such as a reduction in the skimming properties of skim milk and the churning capacity of cream, but particularly for the soapy and rancid flavours of foodstuffs. Long-chain fatty acids are usually associated with soapy flavours, and short-chain fatty acids with unpleasant rancid flavours; for example, the odour of valeric acid is described as being like ‘sweaty feet’, and hexanoic acid as ‘goat-like’. The flavour threshold of these compounds is generally low, e.g. 14 ppm for hexanoic acid, so even a very little lipolytic activity can have a marked effect on quality. In milk, the release of as little as 1-1.5% of the fatty acids from triacylglycerols can cause it to be unpalatable (Table 9.2). Endogenous milk lipases are most likely to be responsible if lipolysis occurs before the milk has been heat-treated and if the total viable count is less than 10 6 per ml. Flavour changes due to endogenous lipases in milk are a rare occurrence. Endogenous lipases are denatured by pasteurization, but extracellular microbial lipases released by psychrotrophic bacteria such as Pseudomonas spp are heat-stable, withstanding pasteurization and, in some cases, HTST treatments. As psychrotrophic organisms are able to grow at 2–4oC, the preferred holding temperature for milk or cream in bulk storage tanks, significant levels of lipase may be reached. Heat-resistant lipases may take weeks to have an effect on Fig. 9.5. Action of lipase on triacylglycerol. Table 9.2 Free fatty acid concentrations in dairy products and rancid flavour threshold values (Allen 1989) Product Free fatty acid values (meq/g fat) Normal Likely to cause problems Milk powder 0.3–1.0 1.5–2.0 Ice cream 0.5–1.2 1.7–2.1 Butter 0.5–1.0 2.0 Cheese: Cheddar 1.2 2.9 Brie 1.2 – Blue 40.0 – Non-microbiological factors affecting quality and safety 237 product quality and are usually of greater significance to the quality of ambient and long shelf-life products. In cheese-making, hydrolysis due to lipase activity in the rennet may be needed to develop the required flavour (Peppler and Reed 1987). Almost all strongly flavoured cheeses, such as Stilton, Roquefort, Gorgonzola and Parmesan, depend on free fatty acids for their flavour. With the advent of microbial proteases as rennet substitutes there is a need to add lipases with the appropriate specificity to achieve the precise mixture of fatty acids responsible for the desired flavour. The difficulties associated with achieving the appropriate specificity and amounts of enzyme needed have been demonstrated with Cheddar cheeses. Differences in fatty acid levels that give a normal Cheddar and a rancid Cheddar can be reached despite extremely small differences in the amount of lipase (Law and Wigmore 1985). 9.6 Characteristics of physico-chemical reactions Physico-chemical reactions that affect the quality of chilled foods occur as a result of physical changes to the product or the chemical or biochemical reactions that follow. Thus migration of components either by diffusion or osmosis and light absorption by natural or artificial pigments, fall within this category. 9.7 Physico-chemical reactions of significance in chilled foods 9.7.1 Migration In mayonnaise-based salads, such as coleslaw and potato-based salads, the major quality changes observed are sensory changes related to the distribution of oil and water between the mayonnaise and vegetable tissue (Tunaley and Brocklehurst 1982). In the case of coleslaw, a 13.5% increase in ether- extractable solids from the cabbage and a translucent appearance, indicated the uptake of oil from the mayonnaise by the cabbage within 6 hours of mixing (Tunaley et al. 1985). In the mayonnaise, the change in oil content was reflected by an increase in the polydispersity of the globule size. In addition, migration of water from the cabbage to the mayonnaise, owing to the difference in osmotic potential, caused the mayonnaise to become runny and ‘non-coating’ within the same timeframe as the cabbage becoming translucent. Investigations of differences between cabbage varieties with respect to oil absorption have shown that stored Dutch cabbage gave no change in the assessment of ‘creamy- oiliness’ of the mayonnaise, whereas fresh English cabbage gave a significant decrease. Other ingredients with a large difference in osmotic potential with respect to the mayonnaise, such as celery and raisins, may also present problems owing to moisture migration resulting in the formation of pools of water on the surface of the mayonnaise. 238 Chilled foods One of the most widely experienced quality changes involving the migration of water is sogginess in sandwiches. Moisture migration from the filling to the bread can be reduced by the use of fat-based spreads to provide a moisture barrier at the interface (McCarthy and Kauten 1990). In pastry- and crust-based products such as pies and pizzas, migration of moisture from fillings and toppings to the pastry and crust causes similar problems. The migration of moisture or oils may be accompanied by soluble colours; for example, in pizza toppings where cheese and salami come into contact red streaking of the cheese is seen, and in multilayered trifles migration of colour between layers can detract from the visual appearance unless an appropriate strategy for colouring is used. Migration of enzymes from one component to another, for example when sliced unblanched vegetables are placed in contact with dairy products, can lead to flavour, colour or texture problems depending on the enzymes and substrates available (Labuza 1985). 9.7.2 Evaporation A high volume of chilled foods are sold unwrapped from delicatessen counters, particularly cooked fresh meat, fish, pa?te′s and cheese. The shelf-life of such products differs markedly from the wrapped equivalent – six hours versus a few days to weeks. The most common cause for this reduction in shelf-life is evaporative losses. These result in a change in appearance, to such an extent that the consumer will select products which have been loaded into the cabinet most recently in preference to those which have been held in the display cabinet. The practical display-life of unwrapped meat products is determined by surface colour changes that may make the product seem unattractive. Changes in appearance are related to weight loss due to evaporation (Table 9.3). The direct cost of evaporative loss from unwrapped foods in chilled display cabinets was estimated to be in excess of ￡5 million per annum in 1986 (Swain and James 1986). In stores where the rate of turnover of product is high, the average weight loss will be greater because of the continual exposure of freshly wetted surfaces to the air stream. Weight losses from the surface of unwrapped foods are dependent on the rate of evaporation of moisture from the surface and the rate of diffusion of moisture from within the product. Temperature, relative humidity and air velocity are the most influential factors affecting weight loss. Weight loss during storage of fruit and vegetables is mainly due to transpiration. Most have an equilibrium humidity of 97–98% and will lose water if kept at humidities less than this. For practical reasons, the recommended range for storage humidities is 80–100% (Sharp 1986). The rate of water loss is dependent on the difference between the water vapour pressure exerted by the produce and the water vapour pressure in the air, and air speed over the product. Loss of as little as 5% moisture by weight causes fruit and vegetables to shrivel or wilt. As the temperature of air increases, the amount of water required to saturate it increases (approximately doubling for each 10oC rise in temperature). If placed in a sealed container, foods will lose or Non-microbiological factors affecting quality and safety 239 gain water until the humidity inside the container reaches a value characteristic of that food at that temperature. If the temperature is increased and the water vapour in the atmosphere remains constant, then the humidity of the air will fall. Minimizing temperature fluctuations is crucial for the prevention of moisture loss in this situation. 9.7.3 Chill injury Although low-temperature storage of fruit and vegetables is considered to be the most effective method for preserving the quality of perishable horticultural products, for chill-sensitive crops it may be more harmful than beneficial. Most fruit and vegetables of tropical and subtropical origin are injured by exposure to low but not freezing temperatures (10–15oC) (Couey 1982). Some temperate fruit and vegetables are also susceptible to injury, but at lower threshold temperatures (below 5oCto10oC) (Bramlage 1982). Chill injury is indicated by a range of different symptoms that adversely affect quality. Pitting, a general collapse of the tissue, is induced by dehydration and low temperatures. It is most evident in mangoes, avocados, grapefruit and limes, in which the outermost covering is harder and thicker than that underneath. Surface discoloration is common in fruits with thin soft peels, such as bell peppers, aubergines and tomatoes. Uneven or incomplete ripening is induced in tomatoes, melons and bananas. Most frequently internal breakdown and a weakening of the tissues makes the fruit or vegetable susceptible to decay by post-harvest plant pathogens. Chill injury may occur within a short space of time if temperatures are considerably below the critical level. In some cases, symptoms may only develop and become detectable after removal from cold storage and on holding at warmer temperatures, making it difficult to determine immediately after exposure to low temperatures whether chill injury has occurred. Changes in physical structures occurring at the time of chill injury have been described; however, their association with the development of symptoms of chill injury has not been established in the majority of cases. Changes in membrane lipid structure and composition (Whitaker 1991), alterations of the cytoskeletal Table 9.3 Evaporative weight loss from, and the corresponding appearance of, sliced beef topside after 6 hours’ display (James 1985) Evaporative loss (g/cm 2 ) Change in appearance Up to 0.01 Red, attractive and still wet; may lose some brightness 0.015–0.025 Surface becoming drier; still attractive but darker 0.025–0.035 Distinct obvious darkening; becoming dry and leathery 0.05 Dry, blackening 0.05–0.10 Black 240 Chilled foods structure of cells and conformational changes in some regulatory enzymes and structural proteins leading to loss of compartmentalization within cells have been reported. Resulting changes in plant physiology include loss of membrane integrity, leakage of solutes, stimulation of ethylene production (Wang and Adams 1980), and bursts of respiration (Wang 1982). Approaches to alleviate chill injury are highly dependent on the fruit or vegetable in question (Jackman et al. 1988). The most obvious is to avoid exposure of chill-sensitive fruit and vegetables to low temperatures. However, as already stated, chilling provides a means of reducing respiration rate, evaporation and transpiration and therefore extends storage-life. Temperature treatments – such as pre-storage conditioning at temperatures just above the threshold (acclimation) (suitable for cucumber and bananas); intermittent warming during storage (suitable for apples and stone fruits); or holding at ambient temperatures for a short time prior to chill storage – are effective in some cases. Controlled-atmosphere storage has been shown to be beneficial in a limited number of cases, e.g. avocados (Spalding and Reeder 1975), peaches (Anderson 1982) and okra (Ilker and Morris 1975), but it is considered to aggravate chill injury by imposing the additional stresses of low oxygen and high carbon dioxide levels on the produce (Wade 1979). Chemical treatments have been shown to be effective on some fruit and vegetables. On the basis that changes in membrane structure lead to chill injury, treatments leading to an alteration or protection of components of cell membranes have been used. Treatment of tomato seedlings with ethanolamine increased the levels of unsaturated fatty acids incorporated into membrane phospholipids; this reduced damage to cellular components during chilling (Ilker et al. 1976). Free radical scavengers or antioxidants such as ethoxyquin and sodium benzoate, diphenylamine and butylated hydroxytoluene have been shown to be effective on cucumbers, bell peppers (Wang and Baker 1979) and apples (Huelin and Coggiola 1970). Coating of fruits in waxes or oils (provided they are approved for food use) prior to chilling are effective by preventing moisture loss and reducing oxygen available for oxidation. Incorporation of the fungicides benomyl or thiabendazole (TBZ) into this type of coating has been shown to have further advantages for peaches and nectarines (Schiffman-Nadel et al. 1975). The ultimate goal for alleviating chill injury is to select, breed or genetically engineer fruit and vegetable crops to prevent chill sensitivity. Plant breeding and selection have had varying degrees of success. A better understanding of the mechanisms responsible for chill injury should provide the insight required for targeted genetic engineering programmes to overcome this problem, though the varying causes of chill injury are unlikely to be overcome by universal solutions. 9.7.4 Syneresis The weeping or slow spontaneous movement and separation of liquid from a colloidal semi-solid mass is termed syneresis. It occurs as a result of physico- Non-microbiological factors affecting quality and safety 241 chemical changes in carbohydrates or proteins which influence their ability to hold water. As a food ingredient, starch fulfils a number of essential functions – thickens, gels, stabilizes emulsions, controls moisture migration, and influences texture. An inherent limitation of native starches and flour is a lack of stability at low temperatures and at fluctuating temperatures. At low temperatures they become prone to weeping or syneresis. Native starch is a complex carbohydrate composed of the homopolymers, amylose and amylopectin. Amylose is a linear chain molecule composed of 1, 4- linked C11-D-glucopyranose building blocks. Amylopectin has a backbone structure like amylose but, in addition, 1, 6-linkages give it a branched structure that confers a greater water-holding capacity than amylose. The ratio of amylopectin to amylose therefore alters the properties and texture of a starch. For example, wheat flour, a traditional thickener used in gravies and sauces, provides desirable flavour and opacity, but has no low-temperature stability and chilling results in syneresis. When the starch grains are swollen, the linear amylose molecules tend to leach out into solution and reassociate into aggregates aligned by hydrogen bonding. The reassociated amylose tends to expel water resulting in opacity and syneresis. Cooling or freezing causes the overall structure to shrink, greatly accelerating the rate at which syneresis occurs. Problems of syneresis often occur as a result of improper selection of starch. Incorporation of stabilized waxy maize-based starches into products that are to be chilled resists retrogradation and syneresis. Alternatively, stabilized starches are available that have been modified specifically with monofunctional blocking groups to prevent associations between leached amylose molecules and thereby prevent syneresis. Use of a modified starch in conjunction with a wheat flour provides stability in the final product. Syneresis in milk is known as ‘wheying off’, the point at which the curds and whey separate. It is obviously desirable for cheese making, but not in milk-based products such as yoghurts. Homogenization of milk for yoghurt production decreases syneresis by increasing the hydrophilicity and water-binding capacity by enhancing casein and fat globule membrane interactions and other protein- protein interactions (Tamime and Deeth 1980). Heat processing for yoghurt manufacture (85oC for 30 minutes, or 90–95oC for 5–10 minutes) is unique in dairy processing. It is believed to bring about important changes in the physico- chemical structure of the proteins, which minimizes syneresis and results in maximal firmness of the yoghurt coagulum. 9.7.5 Staling The market for sandwiches containing a wide variety of fillings that need chill storage has grown considerably. However, the staling of bread is one of the few reactions that has a negative temperature coefficient (McWeeney 1968); that is, bread stales more rapidly at reduced temperatures (Meisner 1953). The term 242 Chilled foods ‘staling’ in relation to bread is used to describe an increase in crumb firmness and crumb-texture hardness, loss of crust crispness and increased toughness, and disappearance of the fresh bread flavour and emergence of a stale bread flavour. Despite extensive research into the mechanism of staling, most researchers are only prepared to agree that firmness changes are attributed to physico-chemical reactions of the starch component, mainly due to its amylopectin fraction, and some include involvement of flour proteins. The shelf-life of commercial bread is considered to be two days (Maga 1975), which will be reduced by holding at chill temperatures. The use of modified atmosphere packaging, particularly carbon dioxide, is believed to slow the rate of staling of bread (Avital et al. 1990). 9.8 Non-microbiological safety issues of significance in chilled foods Non-microbiological safety issues associated with chilled foods are rarely a result of, or exacerbated by, chilled storage temperature. Some arise as a consequence of the ingredient combinations or minimal processing that subsequent chill storage enables. In most instances, judicious selection of raw materials and a carefully tailored monitoring programme, based on an assessment of the risks posed by individual ingredients and the final product, contributes to the assurance of product safety. If possible, it is always preferable for shelf-life to be limited by changes in quality rather than safety because changes in quality can usually be discerned by the smell, taste or appearance of the product, but such changes cannot be relied upon to indicate when safety limits the shelf-life. 9.8.1 Natural toxicants There is a tendency to associate ‘natural’ with a wholesome and healthy image, yet in some cases there is an awareness that some naturally occurring chemical compounds in food may contribute to human illness. Such an example is greening of potatoes, which is commonly associated with the potential to cause harm. Glycoalkaloids, the group of toxic compounds that can be found in potatoes stored under stress conditions, accumulate just beneath the peel and at eye regions, so peeling reduces potential human exposure. Cooking is not thought to reduce glycoalkaloid concentrations (Bushway and Ponnampalam 1981). However, as a result of dietary advice to increase the intake of fibre, an increasing number of potato products incorporate or retain the skin, e.g. chilled, filled, baked potatoes and potato skins; such products could present a higher risk. It has been generally agreed that tubers for human consumption should not exceed 20 mg glycoalkaloid per 100 g fresh tuber weight. Monitoring of the levels of glycoalkaloids is advised, particularly in new cultivars and after changes in storage and processing procedures, to ensure that they do not exceed recommended limits. Non-microbiological factors affecting quality and safety 243 Pulses and grain legumes have long been known to contain highly toxic lectins (haemagglutinins) which agglutinate red blood cells. Haemagglutinins have been detected in a wide range of leguminous seeds including lentils, soyabeans, lima or butter beans, and red kidney beans (Liener 1974). During the last decade a number of incidents of food poisoning have been associated with red kidney beans, and in one case with butter beans (Bender and Reaidi 1982, Rodhouse et al. 1990). A tendency to partially cook pulses or to eat them raw, particularly red kidney beans in salads, led to numerous cases of gastrointestinal disturbances. Soaking of beans for at least 5 hours leaches out lectins and boiling in fresh water for at least 10 minutes heat-inactivates any that remain, preventing the possibility of food poisoning. The inclusion of nuts, figs and dates in exotic salads such as hosaf carries the associated risk of contamination by mycotoxins (fungal toxins). Mycotoxins are contaminants rather than natural toxicants, being secondary metabolites of the fungal species e.g. Aspergillus, Penicillium, and Fusarium. These fungal species grow on a wide variety of substrates, most notably cereals and ground nuts and other high carbohydrate seeds (e.g. figs) under environmental conditions ranging from tropical to domestic refrigeration temperatures. Unfortunately, mycotoxin production is associated with storage conditions designed to prevent fungal growth. Mycotoxins are chemically very diverse (they include groups such as aflatoxins, ochratoxins and trichothecenes), ranging in molecular complexity and toxicity (some are extremely toxic and others are carcinogenic). Control of mycotoxin contamination has focused on treatments to prevent mould growth or mycotoxin production during storage (Moss and Frank 1987), and on the development of improved analytical methods for their detection. Improved quality of raw materials and post harvest treatments, coupled with improved storage and distribution conditions, reduces the incidence of contamination. In keeping with the principles of HACCP awareness of the possibility of mycotoxin contamination should be accompanied by the implementation of a suitable monitoring programme, based on an assessment of the potential risks involved, and written into the raw material specifications. 9.8.2 Phycotoxins Toxic compounds produced by algae (phycotoxins) enter the food chain via seafood, usually either shellfish (shellfish toxins) or finfish (ciguatoxins). The growing awareness of the beneficial dietary effects associated with eating fish and seafood products and the availability of the chill chain to distribute these products has resulted in an increase in their geographic availability and consumption (Przybyla Wilkes 1991). Importation of seafoods means more exotic forms of phycotoxin are now potentially found on a global scale (Scoging 1991). Four different forms of shellfish poisoning are recognized: Paralytic Shellfish Poisons (PSP), Diarrhetic Shellfish Poisons (DSP), Amnesic Shellfish Poisons (ASP) and Neurotoxic Shellfish Poisons (NSP). Shellfish, particularly bivalve molluscs, e.g. mussels, clams and oysters, accumulate these toxins and are 244 Chilled foods unharmed by them. Subsequent consumption of shellfish by humans produces immediate and severe effects, depending on the type of toxin involved. Accumulation of toxins by shellfish coincides with high levels of particular algal species in coastal waters, so-called ‘algal blooms’. These result from increased availability of nutrients and light in surface waters associated with seasonal climate and hydrographic changes. In the UK, extensive monitoring is undertaken by the Ministry of Agriculture, Fisheries and Food during high-risk periods. Coastal waters, shellfish and some crustacea are analysed for PSP toxins. Prohibition orders on the collection of shellfish are put in place when toxins accumulate to levels which are regarded as unsafe for human consumption (West et al. 1985). This is currently believed to be the most effective control method, as the toxins which the shellfish accumulate are reduced, but not eliminated, by cooking (Krogh 1987) or by holding shellfish in purification tanks. PSP is linked with algal species which occur in waters where ambient temperatures are around 15–17oC. Initial symptoms, seen within 30 minutes of consumption, are tingling and numbness in the mouth and fingertips which spreads throughout the body, causing impaired muscle coordination and, in severe cases, paralysis. The major toxin is saxitoxin, though 18 other toxic derivatives have been identified which are either natural algal toxins or metabolized derivatives found in shellfish. DSP intoxications are common in Japan, but outbreaks have also been recorded in France, Italy and The Netherlands. Symptoms occurring within 30 minutes of consumption are vomiting, abdominal pain and diarrhoea. The major toxic components are okadaic acid and dinophysic toxins found in mussels, clams and scallops. Denaturation of these toxins only occurs after processing at 100oC for 163 minutes; therefore monitoring and prohibition orders are the only real safeguard. ASP is believed to be caused by a toxic amino acid, domoic acid, produced by a diatom occurring in USA, Japanese and Canadian coastal waters. Symptoms include nausea, diarrhoea and confusion/disorientation headaches and, in severe cases, memory loss. NSP intoxications have been mainly associated with the consumption of oysters, clams and other bivalve molluscs in North America. Symptoms occur within 3 hours of consumption and include gastrointestinal disturbances, numbness of the mouth, muscular aches and dizziness. The dinoflagellate responsible for NSP, Ptychodiscus brevis, is notorious for the massive fish kills that occur every 3–4 years off the west coast of Florida. The lack of availability of analytical standards has hampered the development of suitable chemical methods for the determination of these toxins. Most monitoring programmes rely on the use of a mouse bioassay to detect levels of toxicants. Restriction of harvesting of shellfish at those times of the year when algal blooms occur is currently the safest method of prevention. Ciguatera toxins are the largest global public health non-microbial problem associated with seafood. Most incidents occur in the USA, danger areas being the Pacific, Caribbean and Indian Oceans. To date, three incidents have been Non-microbiological factors affecting quality and safety 245 recorded in the UK (Scoging 1991). Finfish that harbour the toxin include the barracuda, red snapper, grouper, amberjack, surgeon fish and sea bass. Ciguatoxin is a neuromuscular toxin that affects the membrane potential of neural cells. Symptoms vary widely with the dose ingested but include vomiting, abdominal plain, dizziness, blurred vision, and reversal of the sensations of hot and cold. Onset is usually within a few hours of consumption and the effects can persist for several months. The toxins are heatstable and unaffected by processing methods. The appearance of the fish gives no indication of the toxin. Development of a dipstick immunoassay to detect ciguatoxins has facilitated sampling in the field (Hokama et al. 1989). 9.8.3 Scombroid fish poisoning Scombroid fish poisoning occurs throughout the world, though most incidents are recorded in the USA, Japan and the UK. In the USA, scombrotoxicosis was the cause of 29% of food-poisoning incidents caused by chemical agents between 1973 and 1987 (Hughes and Potter 1991), and in the UK, 348 suspected incidents were reported between 1976 and 1986 (Bartholomew et al. 1987). Scombridae and Scomberesocidae families (tuna, mackerel, saury, bonito and seerfish), but incidents have also been associated with non-scombroid fish (sardines, herring, pilchards, anchovies and marlin) (Bartholomew et al. 1987, Morrow et al. 1991). Scombrotoxicosis is characterized by the rapid onset (within a few minutes to 2–3 hours of eating the fish) of symptoms which can include flushing, headache, cardiac palpitations, dizziness, itching, burning of the mouth and throat, rapid and weak pulses, rashes on the face and neck, swelling of the face and tongue, abdominal cramp, nausea, vomiting and diarrhoea. The similarity of these symptoms with those related to food allergy has often resulted in misdiagnosis. Histamine has been considered to be the cause of scombrotoxic poisoning for a number of reasons. Analysis of the fish remaining ‘on the plate’ usually reveals it contains high levels of histamine; metabolites of histamine have been detected in the urine of victims; symptoms resemble those of known histamine responses; and administration of antihistamine drugs reduces the severity of symptoms. Scoging (1998) of the Food Hygiene Laboratory (Public Health Laboratory Service) proposed guidelines with respect to histamine levels and the potential for illness. Histamine is a spoilage product resulting from decarboxylation of the amino acid L-histidine which is abundant in scombroid fish flesh. Formation of histamine requires the enzyme histidine decarboxylase, which is produced by the normal bacterial microflora of fish skin, gut and gills. If fish is stored above 4oC, these organisms proliferate and levels of histamine in the flesh increase. Prevention of scombroidfish poisoning would therefore appear to be highly dependent on good handling practices – rapid chilling of the catch, and adequate chilling of the fish prior to preparation for eating. However, in medically supervised feeding studies, deliberately spoiled mackerel and mackerel with added histamine, fed to volunteers, failed to 246 Chilled foods reproduce scornbrotoxic symptoms (Clifford et al. 1989). These workers suggested that histamine alone is unlikely to be the causative agent. Other amines, such as cadaverine, have been suggested as potentiators or as synergists to histamine (Bjeldanes et al. 1978). Further feeding studies, using mackerel implicated in a scombroid-fish poisoning outbreak which reproduced symptoms in volunteers, showed the potency of the mackerel was not related to the histamine dose (Ijomah et al. 1991), or the content of other amines (cadaverine, putrescine, spermidine, spermine, tyramine), or any relationship between the levels of these amines (Clifford et al. 1991). Vomiting and diarrhoea were abolished by administration of antihistamine drugs. It has been suggested that histamine, released by the human body as a part of the natural defence mechanism, is responsible for the observed symptoms, that dietary histamine has a minor role in scombroid-fish poisoning, and that, as yet, the agent in fish which is responsible for triggering the release of histamine by the body is unidentified. 9.8.4 Allergens Food allergy, as opposed to food intolerance, is an immunological reaction to some component of the food. This component or antigen can stimulate the body to release specific immunoglobulin E (IgE) antibodies that give rise to anaphylaxis. Such a reaction can range from a trivial event such as a sneeze to a life-threatening incident. Many food types have been associated with allergic reactions but perhaps best known are those involving milk, soya, shellfish and nuts, particularly peanuts. These issues are not specific to chilled foods but with the increase in formulated chilled food products and the severity of the potential hazards associated with the use of peanuts mention is appropriate here. Studies have indicated that peanut allergy has been reported by 0.5% of the adult population in the UK (Emmett et al. 1999) and that those with sensitivity to peanuts commonly show reaction to other nut types, i.e. Hazel nut and Brazil nut (Pumphrey et al. 1999). The allergenicity of peanut residues is heat stable and Ara h 1, a major peanut allergen, has been shown to retain its IgE binding characteristics despite significant structural denaturation (Koppelman et al. 1999). The possible carry over of allergenic material from product to product or production line to production line, therefore, necessitates stringent hygiene practices with associated quality assurance measures. Wherever possible, products containing peanut residues should be prepared and processed in separate areas, away from products that consumers do not expect to contain peanut residues. HACCP procedures should be used to identify all potential sources of cross contamination. Where there is potential for cross contamination, product scheduling and appropriate cleaning regimes are essential. 9.8.5 Products of lipid oxidation Lipid oxidation products are of great significance to the sensory properties of food, but, in addition, attention has been given to the health risks that they may Non-microbiological factors affecting quality and safety 247 pose, and to their role in reduction of nutrient availability via free radical production and destruction of fat-soluble vitamins A and E. Lipid hydroperoxides and their decomposition products may bind and polymerize proteins, and cause damage to membranes and biological components, thus affecting vital cell functions (Halliwell and Gutteridge 1986, Frankel 1984). Lipid peroxides and oxidized cholesterol may be involved in tumour promotion and in atherosclerosis. Malonaldehyde, a secondary product of lipid oxidation, has been implicated as a catalyst in the formation of N-nitrosamines and as a mutagen (Pearson et al. 1983, Jurdi-Haldernan et al. 1987, Sanders 1987). The significance to human health of eating foods which contain high levels of lipid hydroperoxides and their decomposition products is still to be established, particularly as the rate of formation of lipid peroxides in vivo is much greater than that arising from dietary intake. Nevertheless, whilst possible health risks associated with lipid oxidation products remain controversial, high levels of lipid peroxides are undesirable in the diet. 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