14.1 Introduction Modified atmosphere packaging (MAP) is widely used for many food products and is now a commercial and economic reality. MAP is common in markets that have a well established and controlled cold chain and that can sustain a high- priced quality product. However, MAP is a mild preservation method and a major concern is that MAP storage may not provide a sufficient level of safety for the extended storage of fresh chilled food products with regard to pathogenic bacteria. Other preservation steps may be necessary, in addition to MA packaging and low temperatures, in order to delay outgrowth of pathogens or toxin production beyond their point of spoilage. One feasible solution can be to use a combination of different preservation factors or techniques. This approach provides reliable, yet mild, multi-targeted preservation of foods, thereby facilitating improvements in food safety, quality and economics. The topic of this chapter is to outline the significance of combining MAP with other preservative techniques. Food environments are generally stressful for bacteria because most nutrients are in the form of complex substrates whereby the conditions for bacterial growth are not optimal. The level of free moisture may be restricted and the presence of acids and other chemicals may be at stressful levels. In addition, there is often competition from other microorganisms which are present. Replacing the normal atmosphere with a modified atmosphere, i.e., other concentrations of O 2 ,CO 2 and N 2 , will add additional stress to microorganisms and change the composition of the initial microbial flora. From the early development of MAP (Coyne, 1932; Coyne, 1933), it has been shown that MA can, on its own, inhibit growth of microorganisms. Higher levels of CO 2 have a 14 Combining MAP with other preservation techniques J.T. Rosnes, M. Sivertsvik and T. Ska?ra, NORCONSERV, Norway bacteriostatic effect on microorganisms and properly designed MAP can double a product’s shelf-life (Davies, 1995). In spite of 70 years of knowledge about CO 2 inhibition it is only in the last two decades that MAP has become a widely commercially used technology for storage and distribution of foods. This trend is mainly driven by the demands of modern consumers for pre-processed products that have a fresh appearance and are convenient and easy to prepare. The main focus in this chapter will therefore be on chilled MA packaged products, where pronounced effects of modified atmosphere packaging combined with preserva- tion factors can be seen. The potential of MAP to extend shelf-life for many foods is well documented, e.g., fish (Dalgaard et al., 1993), sandwiches (Farber, 1991), salads and vegetables (Day, 1990), and meat (Gill, 1996). Several review articles outline the different aspects of MA packaging (Farber, 1991; Church and Parsons, 1995; Davies, 1995; Phillips, 1996; Sivertsvik et al., 2002). A major concern associated with the use of MAP is that of product safety. The desired suppression of spoilage microorganisms extends the shelf-life if compared to food products stored in a normal air environment, and this may create opportunities for slower growing pathogenic bacteria. In particular the growth of psychrotrophic pathogens in refrigerated ready-to-eat food may create a health risk before the product is overtly spoiled (Farber, 1991). Since some preservation procedures (e.g. chemical additives) used in food products act by inhibiting growth, instead of inactivating microorganisms, their contribution may be most beneficial when used against pathogens that form toxins in foods, or those that need to reach high numbers to cause foodborne illness, especially in healthy consumers. However, in order to protect consumers at risk from foodborne illness or against microbes with low infectious doses, there is a need for complete inactivation of pathogens and avoidance of recontamination of foods during processing, distribution, and preparation for consumption. For each specific MA-packaged product this must be done either before packaging or later by adjusting to correct preservation intensity in the product. 14.2 Combining MAP with other preservative techniques The preservation of almost all foods in industrialised and developing countries is based on combinations of several factors that secure microbial safety, stability and sensory quality. This is true not only for traditional foods, but also for more novel products. The most important preservative methods in common use for food preservation are high temperature (heat treatment), low temperature, water activity (a w ), acidity (pH), redox potential (Eh), some preservatives, and a competitive flora (Leistner, 1992). The application of some processes using the aforementioned preservation methods at low intensity or concentrations is still in the exploratory and developmental stages. Other methods have obtained regulatory approval and are being introduced in HACCP plans and in the marketplace for consumer evaluation and acceptance. 288 Novel food packaging techniques The principle of combined preservation has been well described by Leistner et al., and is often referred to as hurdle technology (Leistner, 1992; Leistner, 1995b; Leistner, 2002). Whilst the hurdle concept is widely accepted as a food preservation strategy, its potential, using MAP, has still to be fully realised. The intelligent selection of hurdles in terms of the number required, the intensity of each and the sequence of applications to achieve a specified outcome are expected to have significant potential for the future (McMeekin and Ross, 2002). Homeostatsis is the tendency towards uniformity and stability in the internal status of living organisms. For instance, the maintenance of a defined pH within narrow limits is a prerequisite and feature of all living cells, and this applies to higher organisms, as well as microorganisms. In food preservation the homeostasis of microorganisms is a key phenomenon because if homeostasis of these organisms is disturbed by some preservation methods in foods, they will not multiply, i.e. they will remain in the lag phase or may even die before their homeostasis is re-established. Therefore, in actual fact, the preservation of food is achieved by disturbing, temporarily or permanently, the homeostasis of microorganisms in the food. In most foods microorganisms are able to operate homeostatically in order to react to the environmental stresses imposed by the applied preservation procedures. Applying additional preservation will inhibit repair of disturbed homeostasis and this requires extra energy from the microorganisms concerned. In MA products energy depletion increases as the intensity or concentration of preservation is increased and the restriction of the energy supply will inhibit the repair mechanisms of the microbial cells’ factors and leads to growth inhibition or death. 14.2.1 Preservation focused on specific groups of microorganisms If the true potential of some of the emerging preservation technologies, combined with MAP is to be realised, it will be important to develop systematic, kinetic data describing their efficiency against key target microorganisms. The type and numbers of microorganisms in the raw material have a direct influence on the effectiveness of MAP in inhibiting both spoilage organisms and pathogens. When adding extra preservation to packaged food, it is therefore important to understand which part of the bacterial population is inhibited and which is not. The shelf-life extension obtained with MA does not always give the same extension in safety. Pathogenic bacteria may gain advantage when the competing flora is inhibited, e.g., Listeria monocytogenes increased in numbers on raw chicken in 72.5:22.5:5 (CO 2 :N 2 :O 2 ) atmosphere at 4oC, irrespective of a decrease in the aerobic spoilage flora (Wimpfheimer et al., 1990). Many MA packaged products of meat, vegetable and sea-food origin have common key target organisms. For chilled products psychrotrophic pathogens are the target, while in heat-treated ready meals spore-forming Clostridium and Bacillus species are the target organisms. There are five food-borne pathogenic bacteria known to be capable of growth below 5oC: Bacillus cereus, non-proteolytic Clostridium botulinum type E, B and F (group II), Listeria monocytogenes, Combining MAP with other preservation techniques 289 Yersinia enterocolotica, and Aeromonas hydrophila. Consequently the ability of modified atmospheres to inhibit the growth of these organisms in foods under refrigerated storage is of vital importance and additional preservation factors have therefore been combined with MAP (Table 14.1). The main cause of concern, however, is the possible growth of non-proteolytic C.botulinum, because it is both anaerobic and low-temperature tolerant. Of particular concern is the fact that it may grow and produce toxin on the product before spoilage is detectable to the consumer. Few non-thermal treatments can currently be relied upon to inactivate bacterial spores. Hence low-temperature storage must be combined with an additional preservation hurdle such as acidic formulation or salt to prevent spore Table 14.1 Preservatives used to inhibit specific psychrotropic pathogens in combination with MAP Organism Relevant food Preservative References Bacillus cereus Dairy food Ready-to-eat food (Koseki and Itoh, 2002) Non-proteolytic Clostridium botulinum Ready-to-eat food Dinner Irradiation Microbial inhibition by Bacillus species NaCl (Lambert et al., 1991) (Lyver et al., 1998) (Gibson et al., 2000) Listeria monocytogenes Fish, meat, vegetables, fresh produce Competitive microbial flora Nisin Na-lactate Irradiation pH Oregano essential oils High O 2 level (Liserre et al., 2002; Wimpfheimer et al., 1990; Francis and O’Beirne, 1998; Bennik et al., 1999) (Szabo and Cahill, 1998) (Fang and Lin, 1994) (Devlieghere et al., 2001; Pothuri et al., 1996) (Thayer and Boyd, 2000; Thayer and Boyd, 1999) (Francis and O’Beirne, 2001) (Tsigarida et al., 2000) (Jacxsens et al., 2001) Yersinia enterocolitica Pork Poultry Lactate Lactic acid (Barakat and Harris, 1999) (Grau, 1981) Background flora (Kleinlein and Untermann, 1990) Low temperature (Gill and Reichel, 1989) Aeromonas hydrophila Fish, shellfish Mussels Meat Heat pH (Devlieghere et al., 2000b) (Doherty et al., 1996) Salmonella Poultry Sorbate (Elliott and Gray, 1981) 290 Novel food packaging techniques outgrowth. Most food spoilage moulds species have an absolute requirement for oxygen and appear to be sensitive to high levels of CO 2 . Consequently foods with low a w values, such as bakery products, that are susceptible to spoilage by moulds can have their shelf-lives extended by MAP. Many yeasts are capable of growing in the complete absence of oxygen and most are comparatively resistant to CO 2 . Although MAP can inhibit the growth of bacterial and fungal spoilage microorganisms, its effect on the survival of enteric viruses, including hepatitis A viruses (HAV), has not been well investigated. Both mussels and lettuce that are packaged in MAP may be a vehicle in the transmission of HAV (due to contact with contaminated water) and therefore can contribute to hepatitis A outbreaks (Cliver, 1997). Experiments by Bidawid et al. (2001) indicated that MAP does not influence HAV survival when present on the surface of produce with high CO 2 levels. This may have been attributed to the inhibition of spoilage-causing enzymatic activities in the lettuce, which may have reduced exposure of viruses to potential toxic by-products. 14.2.2 Preventative techniques combined with MAP The main preservation techniques currently used act in one of three ways: (i) preventing the access of microorganisms to foods, (ii) inactivating them when they have gained access, or (iii) preventing or slowing down their growth when they have gained access and not been inactivated. During the past few years there has been increasing interest in modifying these approaches or in developing new ones, with the objective of reducing the severity of the more extreme techniques. Many such developments have involved new uses of existing techniques in new combinations to inhibit the growth of micro- organisms. Approaches where preservation techniques are used at lower intensity or at lower concentration, causes inactivation and bacterial growth inhibition to overlap. It is the safety level, the quality level or the outcome of inactivation or growth inhibition of target organisms that determines the final use of the chosen preservation method(s) (Table 14.2). 14.2.3 Hygienic conditions Hygienic production is not a preservation method, but ingredients or raw material used in MAP should always be of superior quality, i.e. low bacterial numbers and preferably without pathogenic bacteria. This is a prerequisite for fresh products with increased shelf-life, and preservation should never be used to compensate for inadequate hygiene or poor raw material quality. A strategy for the control of pathogens and, to a large extent, spoilage microorganisms is basically one of exclusion, which requires reducing or eliminating the initial microbial load or preventing or minimising further contamination. Since MA packaged products are hermetically sealed, recontamination is eliminated and the hygienic pre-packaging conditions are the most important steps. An appropriate design and construction of the pre-packaging premises is necessary Combining MAP with other preservation techniques 291 Table 14.2 Uses and limitations of preservation technologies combined with MAP (Adapted from Leistner, 2002) Preventing assess Inactivation or growth/activity inhibition Effect in MAP Heat Ionising NaCl pH Bacterio- Low Preser- Na- Essential SGS 2 treatment irradiation cins Temp 1 vatives lactate oils Killing spores + + Killing veg. cells + + ± ± ± ± + Preventing growth + + + + + + Solids + + + + + + + + + Liquids + + + + + + + + + + In-pack treatments + + + + In-line treatment + + + + 1 Low temperature (super chilling and freezing) may also kill bacteria 2 SGS = soluble gas stabilisation to limit entry, multiplication, and spread of microorganisms in the environment where MA packaged foods are being produced or manufactured, in order to prevent or minimise cross-contamination of the products. New and hygienic design of production facilities, with elements from clean room technology, are now more frequently adopted in the production of high-priced products. These techniques meet the requirements of freeing the products from microorganisms by cross-contamination, decontaminating the packaging material, and sterilising air in contact with the product. 14.3 Heat treatment and irradiation Refrigerated ready-to-eat meals and entre′es, prepared salads, sandwiches, pizza, fresh pasta, soups, whole meals, and sauces are commonly packaged in MA after heat treatment. These products have received some form of heat treatment, and are for the most part ‘low acid’. They are marketed refrigerated ( 1 to 4oC) and require little preparation before consumption. There has been a recent expansion in the use of the combination of mild heating of vacuum-packaged foods, e.g., sous vide, and cook-and-chill products with controlled chill storage, particularly for catering but also for retail. MA packaging of cook-and-chill foods is now commonly used for processed minimal heat-treated ready meals. Many nursing homes and canteens currently receive heat-treated MA packaged meals prepared in a central kitchen unit. With this method the risk of recontamination of microorganisms after cooking must be taken into account. These ready-to-eat meals have a shelf-life of 7–14 days, depending on the amount of heat used. The success of heat-treated ready meals results primarily from the inactivation of the vegetative microbial flora by mild heating. Another fact is that the spores of psychrotrophic bacteria, which can grow at low chill temperatures, are generally more heat sensitive than those of mesophiles and thermopiles, which cannot grow at these temperatures. The mild heating therefore destroys the cold-growing fraction of the potential spoilage flora, whilst the minimal thermal damage and conditions of low oxygen tension ensure high product quality. Shelf-lives at temperatures below about 3oC can therefore be very long, i.e., in excess of three weeks, with eventual spoilage resulting from the slow growth of psychrothropic strains of Bacillus and Clostridium. In order to ensure safety, heat processes equivalent to 90oC for 10 min. (ACMSF- Advisory Committee on the Microbiological Safety of Food, 1992) are generally regarded as sufficient to ensure inactivation of spores in the coldest-growing pathogenic sporeformers such as psychrotrophic strains of Clostridium botulinum (Notermans et al., 1990; Lund and Peck, 1994). For lower heat treatments, strict limitations of shelf-life, efficient control of storage temperatures below 3.0oC or some form of intrinsic preservation is necessary. During a three-year period, 2168 heat-treated, commercially available ready- made meals with a shelf-life of 3–5 weeks were examined for sporeforming Combining MAP with other preservation techniques 293 bacteria (Nissen et al., 2003). Three-quarters of the samples had less than ten bacteria/g the day after production, and none had more than 1000. Similar numbers were found at the end of the shelf-life. At abuse temperatures (20oC), the number of bacteria increased to 10 6 –10 7 cfu/g in seven days. Three hundred and fifty isolates of spore-forming bacteria (aerobic and anaerobic) were collected and characterised as Bacillus licheniformis, B. thuringiensis, B. megatherium, B. pumilis, B. subtilis, B. sphaeicus, and B.cereus, but no Clostridium strains were detected. Growth experiments of 113 strains from this work showed that only 11 strains were able to grow at 7oC. Furthermore, none of the psychotropic strains were able to produce substantial amounts of toxins. These experiments show that spore-formers, especially Bacillus strains, survive mild heat treatments and some of their members may be a health risk in products with long shelf-lives or if stored at high temperatures. Further research on germination, growth and toxin production at chilled temperatures in modified atmosphere is required. 14.3.1 Low temperature (freezing, partial freezing, super chilling) A low and stable temperature is a general prerequisite for many MA products and has a particular importance in fresh storage. Both enzymatic and microbiological activity are greatly influenced by temperature. Many bacteria are unable to grow at temperatures below 10oC and even psychrotrophic organisms grow very slowly, and with extended lag phases, at temperatures that approach 0oC. Temperature can, however, be used to achieve special effects in MA products. Guldager et al. (1998) and B?kn?s et al. (2000) have found that frozen ( 20oC) and thawed cod fillets in MA had longer shelf-life than raw cod in MA. This shelf-life extension was most likely due to the inactivation of the spoilage bacterium Photobacterium phosphoreum during frozen storage. The use of frozen fillets as a raw material not only provides a more stable MAP product but also allows much greater flexibility for production and distribution. A similar effect was found when frozen and thawed salmon was packaged in MA. Here also the freezing eliminated P. phosphoreum and extended the shelf- life of MAP salmon at 2oC by 1–2 weeks (Emborg et al., 2002). Earlier experiments with whole gutted salmon have shown that MAP can be combined with super-chilling to extend further the shelf life and safety of fresh fish (Rosnes et al., 1998; Rosnes et al., 2001; Sivertsvik et al., 1999). In this technique, also known as partial freezing, the temperature of the fish is reduced to between 1 or 2oC below the initial freezing point and some ice is formed inside the product (Gould and Peters, 1971). Under normal conditions, the gas atmosphere surrounding a MA product will insulate the product, leading to a longer time until it is satisfactorily chilled. Partial freezing eliminates this problem by reducing the temperature of the fish before packaging. These experiments showed that super-chilling can decrease the temperature before packaging and increase stored refrigeration capacity during storage, and thereby significantly decrease microbial growth at temperatures of 2–6oC, which is often 294 Novel food packaging techniques found in chilled retail counters. Sikorski and Sun (1994) found that super- chilling can store enough refrigeration capacity to keep a core temperature < 0oC during the first three weeks of chilled storage. A shelf-life extension of seven days has been obtained for super-chilled fish when compared to traditional ice stored fish of the same type (Leblanc and Leblanc, 1992). Untreated salmon steaks in MA, and partial frozen salmon steaks in MA, had an acceptable microbiological quality of 22 days at 0oC, but were rejected by odour after 17 days. Salmon steaks in air had and acceptable microbiological quality for only eight days (Rosnes et al., 2001). MAP is also being used to package products for frozen storage. The reasoning behind the use of MAP for ready-to-eat products is that they can be distributed frozen, then thawed and sold as chilled products but with an extended shelf-life (Morris, 1989). 14.3.2 Irradiation The attraction of combining irradiation with MAP is that the modified atmospheres are not lethal to spoilage organisms and pathogens. The possibility exists, therefore, of using irradiation below the ‘threshold’ dose, i.e., the level at which spoilage organisms and pathogens are killed and below the level where undesirable organoleptic changes are introduced, in order to enhance the attractiveness of MAP. The effects of MAP/irradiation on sensory properties, and its effect upon depletion of vitamin content during storage, compared to untreated items, have been examined in detail. Studies on the effects of MAP/ irradiation methods on nutritional quality showed that the deleterious effects of irradiation on vitamins can be removed by modifying storage atmospheres (Robins, 1991). For a radiation dose of 0.25 kGy and in an air atmosphere, 60% of the thiamine content was lost over the storage period, compared to a minimal loss in the non-irradiated control over the same period. The loss of -tocopherol, exposed to 1 kGy irradiation, was some 50% over this period, compared to a similar minimal loss in the non-irradiated control sample. In both cases there were much reduced loss rates in N 2 atmospheres, which demonstrated that the effects of irradiation on these vitamins could be removed by modifying storage atmospheres. The growth rate of surviving microorganisms was measured as a function of atmospheric composition for the irradiated and non-irradiated food samples, and the optimum lethal atmospheres were found to range from CO 2 /N 2 : 25/75 to CO 2 /N 2 : 50/50. Tests at 10oC showed a similar trend, although the effectiveness of high concentrations of CO 2 was reduced. The major surviving organisms even in the irradiated packs were lactobacilli, in accordance with general expectations on their resistance to radiation. A series of experiments on MAP/irradiation combination, for use with chicken and pork products, with the goal of optimising sensory quality have shown that each particular food item requires careful evaluation and that generalisation can lead to incorrect and inappropriate specifications for optimum storage. However, as one of several different treatment combinations aimed at Combining MAP with other preservation techniques 295 reducing mould in strawberries, the MAP/irradiation method gave the best results. Several studies have been carried out on the use of MAP/irradiation treatments in fish products, e.g., low dose irradiation extended the shelf-life of haddock fillets and cod fillets (Licciardello et al., 1984) more than either process achieved on its own. Przybylski et al. (1989) examined fresh catfish fillets, processed with low dose irradiation in combination with MAP, and demonstrated that irradiation treatments with or without elevated carbon dioxide-modified atmosphere packaging significantly reduced the bacterial load and extended shelf-life from 5–7 days to between 20 and 30 days. In an experiment, cod fillets were packaged in MA (80:20 CO 2 :N 2 ) and under vacuum before irradiation with 2.2 kGy, and subsequent storage at 4oC. The results (Fig. 14.1) showed a large inhibitory effect of irradiation on micro- organisms. The best results were observed when combining irradiation with MAP. The sensory shelf-life of irradiated MA cod and irradiated vacuum packaged cod was >24 days and 24 days accordingly. For non-irradiated MA cod the shelf-life was <14 days, and for vacuum packaged cod, <9 days. This should indicate a large potential for seafood product shelf-life extensions through the use of MAP combined with low-dose irradiation. However, before this method is widely accepted, several issues need to be resolved, such as legislative, scientific (food safety), and also consumer attitudes towards irradiated foods (Sivertsvik et al., 2001). Nevertheless, all studies have shown that the advantages of MAP/irradiation treatment methods must be determined for specific applications with a fair degree of caution and this requires the ascertainment of exact conditions for every product in terms of microbiological safety. Fig. 14.1 Effect of irradiation on MAP and vacuum packaged cod fillets. 296 Novel food packaging techniques 14.4 Preservatives The use of chemical preservatives (benzoic acid, sorbic acid) is often very efficient in inhibiting microbial growth. These molecules inhibit the outgrowth of both bacterial and fungal cells. Sorbic acid is also reported to inhibit the germination and outgrowth of bacterial spores. Their effect, however, is strongly dependent on the pH value, and their use is rarely recommended if pH exceeds 6. The effect of adding potassium sorbate to ice used for cooling of red hake and salmon, packaged in modified atmosphere was studied by Fey and Regenstein (1982). These authors found that a CO 2 –O 2 atmosphere combined with 1% potassium sorbate ice was most satisfactory. Also other studies conducted on the use of sorbates in fish and fish products suggest that sorbates in combination with other compounds or techniques can be used as an effective preservative tool for extending the shelf-life of fish products (Thakur and Patel, 1994). Elliott and Gray (1981) discovered growth inhibition of Salmonella enteritidis following exposure to a combination treatment of potassium sorbate (0.5, 1.5 or 2.5%) and modified atmospheres of 20, 60 and 100% CO 2 at pH 6.5, 6.0 or 5.5 at 10oC. Dalgaard et al. (1998) found that including potassium sorbate was effective in reducing the growth of the specific spoilage organisms P. phosphoreum in model substrates. This may have a practical use in extending the shelf-life of MA packaged seafood. Cooked and brined shrimps, including benzoic, citric and sorbic acids, packaged in modified atmosphere were stored at 0, 5, 8, and 25oC (Dalgaard and J?rgensen, 2000). The shrimps had a shelf-life of > 7 months at 0oC, but spoiled in 4–6 days at 25oC. This pronounced effect of temperature was explained by changes in spoilage at different storage temperatures. 14.4.1 Sodium chloride, a w Sodium chloride is an old preserving agent with antimicrobial importance that generally binds water and thus inhibits bacterial growth through reducing water activity. Recent focus, however, on the adverse effects of a high sodium intake on blood pressure, has led to a sharp decrease in salt consumption. Therefore, salt is less and less used in the preservation of food, and more only for taste purposes. Furthermore, addition of salt activated and stabilised proteolytic activity (alkaline proteases primarily) over a wider temperature and pH area in Atlantic salmon (Olsen et al., 2002), although conclusive data on the effects remain to be presented. A number of different fish species have been studied after being subjected to 5 minutes treatment in a brine solution (5% NaCl) and then packaged in different gas mixtures. Mitsuda et al. (1980) and Pastoriza et al. (1998) found good texture and greatly repressed colour change at 3oC. The effects of an optimum gas mixture on hake slices when combined with a sodium chloride dip were studied. A delay in chemical, microbiological and sensorial alteration was found and total volatile bases (TVB) and microbiological levels were significantly lower when MAP-stored samples had been previously dipped in Combining MAP with other preservation techniques 297 NaCl. Additional effects which are important for MA packaged fish were reduced exudation, higher water binding capacity and increased time before MAP stored samples were rejected due to off-odours. The antimicrobial contribution of NaCl in a food system may also be influenced by the presence of other preservatives, e.g. benzoate, sorbate, phosphates, antioxidants, spices and liquid smoke. 14.4.2 Alteration of pH pH influences spoilage due to its effect on the microorganism and enzyme activity (Ashie et al., 1996). Daniels et al. (1985) claimed that the CO 2 / bicarbonate ion has an observed effect on the permeability of the cell membranes, and that CO 2 is able to produce rapid acidification of the internal pH of the microbial cell, with possible ramifications relating to metabolic activities. Fey and Regenstein (1982) noted that CO 2 did not lower the pH of the fish. In most fish products, even though a reduced pH could be advantageous in order to reduce bacterial growth, with the iso-electric point of fish proteins being approx. 5.5, it will also lead to reduced water holding capacity as well as textural changes. Devlieghere et al. (1998) modelled the effect of pH on the solubility of CO 2 and found that higher amounts of CO 2 can be dissolved in aqueous foods with high pH levels. In spite of the fact that higher concentrations of CO 2 are dissolved at higher pH, the preservative effect seems to be larger at low pH levels when in combination with a modified atmosphere. Beef with pH 6.3 in 100% N 2 at 5oC supported the growth of E. cloacae but not at pH 5.4 (Grau, 1981). Growth of S. liquefaciens was inhibited on beef with pH 5.4 in 100% N 2 at 5oC but grew to levels of 10 8 cfu/g in eight days on meat with pH 6.3. Yersinia enterocolitica failed to grow on beef ranging in pH from 5.4 to 5.9 under 100% N 2 , but grew at pH 6.0–6.2. Under aerobic conditions pH had little effect on the growth of Y. enterocolitica. In muscle foods, the initial decline in pH is reversed during later stages of post-mortem changes as a result of decomposition of nitrogenous compounds (Ashie et al., 1996). This effect may be inhibited by addition of buffer- compounds like polyphosphates. These enhance the preservative role by (i) acting as metal ion chelators, (ii) acting as pH buffers, (iii) interacting with proteins to promote hydration and water binding capacity, and thus (iv) preventing lipid oxidation and microbial growth (Ellinger, 1972). 14.4.3 Organic acids The antimicrobial properties of acetic, lactic, citric and malic acid have been utilised by the food industry for food preservation. It is generally accepted that the undissociated molecule of the organic acid or ester is responsible for the antimicrobial activity. Many weak acids, in their undissociated form, can penetrate the cell membrane and accumulate in the cytoplasm and acidify its 298 Novel food packaging techniques interior. The activity of the lactate has also been attributed to its lowering of the water activity of the food product, but this can only partly explain its antimicrobial effects on meat products (Debevere, 1989; Houtsma et al., 1993). Salts of organic acids, such as sodium and potassium lactate, are fully dissociated in aqueous solutions, and at the pH of an unfermented meat product, which is typically 6.0 to 6.5, the concentration of the undissociated form of the added lactate is low. The increased permeability of cellular membranes for lactic acid at higher pH values may be an important factor in understanding the anti- microbial activity of Na-lactate, as observed in neutral food media and food products. Cooked meat products packaged in oxygen-free atmospheres will spoil due to psychrotrophic lactic acid bacteria (Borch et al., 1996) but with addition of Na-lactate the shelf-life will be prolonged (Debevere, 1989). Devlieghere et al. (2000a) examined the shelf-life of MA packaged cooked meat products after the addition of Na-lactate and found that a significant shelf-life extension was obtained through the use of Na-lactate, and this was more pronounced at low temperatures. A synergistic effect was reported between Na-lactate and CO 2 , which could partly be explained by the pH lowering effect of CO 2 . The use of buffered lactic acid systems on poultry enhanced the decontaminating effect and increased the shelf-life of poultry (Zeitoun and Debevere, 1990). Further studies on poultry showed that buffered lactic acid treatment and MAP had an inhibitory effect on Listeria monocytogenes and increased the shelf-life (Zeitoun and Debevere, 1991). 14.4.4 Essential oils Essential oils are regarded as natural alternatives to chemical preservatives. Their practical application is limited due to flavour considerations, and their effectiveness is moderate due to their interaction with food ingredients and structures. The results obtained by Skandamis et al. (2002) showed that volatile compounds of oregano essential oil are capable of affecting both the growth and metabolic activity of the microbial association of meat stored at modified atmospheres. This inhibition was not as strong as that found in the contact of pure essential oil with microorganisms when added directly on the surface of meat (Skandamis and Nychas, 2001). These authors conclude that the volatile compounds of oregano essential oils improve the shelf-life of meat by (i) delaying the growth of specific spoilage organisms, (ii) inhibiting or restricting metabolic activities that cause spoilage through the production of spoilage microbial metabolites, and (iii) minimising the flavour concentration. 14.5 Other techniques 14.5.1 Na 2 CaEDTA Low levels of Na 2 CaEDTA (25 to 500 ppm) have been approved for use in some foods. This chelating agent has little effect on most of the microorganisms found Combining MAP with other preservation techniques 299 in seafood (Dalgaard et al., 1998), but more importantly does inhibit P. phosphoreum in MAP cod. In naturally contaminated MA packaged cod fillets, 500 Na 2 CaEDTA reduced the growth rate of P. phosphoreum by 40% and shelf- life was increased proportionally by 40%, from 15–17 days up to 21–23 days at 0oC. In aerobic stored cod fillets other microorganisms were responsible for spoilage and Na 2 CaEDTA had no influence on shelf-life. 14.5.2 Soluble gas stabilisation The mode of action of the different gases used in MAP (including high O 2 concentrations) is discussed in other chapters. CO 2 gas, however, has special preservative effects in the package. Many bacteria are inhibited by very high CO 2 concentrations, and keeping a high CO 2 concentration in the product during shelf-life demands special techniques. One possible approach is to create a modified atmosphere for a product by either generating the CO 2 inside the package after packaging, or to dissolve the CO 2 into the product prior to packaging. Both methods can provide appropriate packages with smaller gas/ product ratios, and thus decrease the package size. An example of the first method includes the use of either CO 2 generators or small amounts of dry ice (solid CO 2 ) inside the package. CO 2 generators are commercially available (Ageless, Tokyo, Japan) and could be used on their own in order to extend the shelf-life of foods (Sivertsvik, 1999). CO 2 could also be produced inside the packages by letting the exudates from the product react with a mixture of sodium carbonate and citric acid inside the drip pad, as described by Bjerkeng et al. (1995). The development of a 100% CO 2 atmosphere can be obtained by combining dry ice (approx. 1 g pr. kg product) and vacuum packaging. Care must be taken to avoid direct contact between the dry ice and the product, because of freeze burns. Whole salmon in plastic bags which have one-way valves to let excess CO 2 seep out, offered a superior quality product compared to ordinary MAP and traditional ice packaging methods using this dry ice. When CO 2 is dissolved in the package prior to packaging (soluble gas stabilisation – SGS) the CO 2 is dissolved into the food product at low temperature (~0oC) and elevated pressures (> 2 atm). This is in contrast to ordinary MAP, where CO 2 is introduced into the package atmosphere at the time of packaging. The latter method can extend the shelf-life of different fish products either alone, combined with traditional MAP, or vacuum packaging (Sivertsvik, 1999). The additional benefit of this method compared to MAP, in addition to the inhibition of microorganisms that are obtained by dissolving CO 2 , is that the possible degree of filling is significantly increased. 14.5.3 Protective microbes and their bacteriocins The application of bacteriocins, i.e., antibacterial proteins produced by lactic acid bacteria (LAB), in combination with traditional methods of preservation and proper, hygienic processing can be effective in controlling spoilage and 300 Novel food packaging techniques pathogenic bacteria. A wide range of bacteriocins is produced by LAB, and although these are found in fermented and non-fermented foods, nisin is currently the only bacteriocin widely used as a food preservative. Nisin is approved for use in over 40 countries and has been in use as a food preservative for over 50 years (Cleveland et al., 2001). Since bacteriocins are isolated from foods such as meat and dairy products, which normally contain lactic acid bacteria, they have unknowingly been consumed for centuries. Today there are many examples of the effective use of nisin in food systems, e.g., cottage cheese (Ferreira and Lund, 1996), ricotta cheese (Davies et al., 1997), skimmed milk (Wandling et al., 1999), Bologna-type sausages (Davies et al., 1999), lean beef (Cutter and Siragusa, 1998), and Kimchi (Choi and Parrish, 2000). In principle, there are two common ways to use bacteriocins; by the addition of a starter culture which produces a bacteriocin which has the necessary inhibitory spectrum (Stiles, 1996), or the bacteriocin itself may be added as an ingredient at an early stage of the production process. A third way is by immobilising the bacteriocins on the packaging materials. Fang and Lin (1994) found that the numbers of Pseudomonas fragi on cooked tenderloin pork were reduced by MA storage, but were unaffected by nisin. In contrast to this, the growth of L.monocytogenes was prevented when samples were treated with 1 10 4 nisin IU/ml. In addition, the MAP (100% CO 2 , 80% CO 2 + 20% air)/nisin (10 3 , 10 4 IU/ml) combination system used in this study decreased the growth of both organisms, and the inhibition was more pronounced at 4oC than at 20oC. In a cocktail of seven L. monocytogenes isolates of food, human and environmental in origin, Szabo and Cahill (1998) found an increase in lag phase in all atmospheres when nisin was used. Increasing the concentration of nisin to 1250 IU/ml inhibited the growth of L. monocytogenes in all atmosphere combinations at 4 and 12oC. The addition of nisin and/or a CO 2 atmosphere increased the shelf-life of cold smoked salmon from four weeks (5oC) to five or six weeks (Paludan-Muller et al., 1998). Scannell et al. (2000) developed bioactive food packaging materials using immobilised bacteriocins lacticin 3147 and nisaplin. They found antimicrobial activity against the indicator strains Lactococcus lactis, Listeria innocua and Staphylococcus aureus. Adsorption of lacticin 3147 into plastic film was unsuccessful, but nisin bound well and the resulting film maintained its activity for a three-month period, both at room temperature and under refrigeration. 14.6 Consumer attitudes The number of food types involved in carrying foodborne illness has increased, together with an increase in pathogenic micoorganisms documented as being transmitted through food. This makes it necessary to reconsider our approach to food preservation and pathogen control in order to meet these new challenges and to enhance food safety. However, MAP is regarded as a mild preservation method by most consumers, inducing minor changes of the inherent raw Combining MAP with other preservation techniques 301 material qualities. A development towards using more preservatives in combination with MAP, e.g., additives or preservatives, and in some cases technologies with less well recognised effects (e.g. irradiation), may lead to a lowering of consumer acceptance for MA packaged foods. Consumer demands for both fresh and safe food, provides the producer with a dilemma; should he produce a product with a modest shelf-life or use preservatives to enhance product safety. Most legislative authorities in Europe and the US aim at giving the consumer complete information about processes and packaging conditions. Therefore the producer must clearly state on the label which additives, preservatives or methods have been used and may therefore have an effect on the properties of the food. Some of the preservatives examined for use in combination with MAP, in products for daily use in households, may therefore be met with scepticism. Furthermore, the food additives benzoate and sorbate are often associated with a negative image. The food control authorities are also concerned because some preservation techniques may mask poor and improper raw material quality. Irradiation and to a certain degree preservatives, for example, used at harvest may decrease or delay the onset of microbial growth without delaying biochemical reactions. In the end this may provide for a long shelf-life in MA products measured by microbiological analysis without offering improvement in eating quality. Irradiation treatments have been a matter of debate for a long time. Despite the advantages of irradiation both for the processor, retailer and consumer, irradiation is not widely used because of uncertainty regarding consumer acceptance, particularly given that there is a requirement to label all irradiated food in most countries. Research on consumer attitudes and a marked response to irradiated foods have shown that the public’s knowledge is limited and that the acceptance of food in the fresh food category is limited. 14.7 Future trends It is likely that the consumer demand for high quality, nutritious and ready-to-eat products will last for many years. Therefore MA packaged food products which use minimal preservation, contain few artificial additives and show little alteration from the raw product will be preferred. Preservation techniques which meet these requirements include the optimisation of gases (combinations and SGS), the use of low temperatures and the utilisation of protective microbes and their bacteriocines. Active packaging is an emerging technology in which the food, package and environment interact. This technology also includes different kinds of gas emitters and absorbers resulting in an extended shelf-life of the product. For many food products more relevant are oxygen absorbers and carbon dioxide producers used either alone to develop a modified atmosphere or in combination with a gas mixture. A number of novel processes are now under development for microbial control of foods (Leistner and Gorris, 1997). Many of these processes can be 302 Novel food packaging techniques used in the category with the aim of preventing microorganisms’ access to foods, e.g., in improved heat processing like infra-red heating, electric volume heating, electric resistance/ohmic heating, high frequency (HF) or radio- frequency heating, microwave heating, inductive electric heating (Ohlsson, 2002b). There are also non-thermal methods like high pressure, pulsed electric fields, pulsed white light, ultra sound and ultraviolet radiation (Ohlsson, 2002a). These treatments are promising as pre-treatments to MA packaging and have been proposed for use as part of combinations in multiple hurdle systems. However, many of them are still at an experimental stage, with expensive and ineffective batch production. An important question producers should address is the purpose or the need for using additional preservations to MA products. One obvious and sensible reason is that of increased safety, as previously described in this chapter. By using preservations together with MAP it is possible to get a long and safe shelf-life where target pathogens are under control. The definition of shelf-life is, however, not obvious. Most chilled raw or partly processed food products packaged in MA will have a limited period of good quality, then chemical and biochemical processes together with microbiological spoilage will decrease the sensory quality. After the period of good quality, a period with regular or even poor quality may follow, without producing safety hazards. Future use of preservation, next to safeguarding safety, should focus on prolonging the good quality lifespan of MA products. For heat-treated products, new methods that allow a faster and more even heat penetration may improve eating quality and survival of nutrients. Most processes or preservations used together with MAP do not prolong the high-quality period. An exception to this is low temperature and superchilling treatments which may inhibit both microbial spoilage and biochemical reactions. 14.8 Sources of further information and advice Books on modified atmosphere packaging of foods Farber, J.M. and Dodds, K.L. 1995. Principles of modified atmosphere packaging and sous-vide packaging. Technomic Publishing Co., Basel. pp. 464. Brody, A.L. 1994. Modified Atmosphere Food Packaging. IoPPress. pp. 275. Parry, R.T. 1993. Principles and Applications of Modified Atmosphere Packaging of Foods. Blackie Academic & Professional Publishing, London. pp. 305. Ooraikul, B. and Stiles, M.E. 1991. Modified Atmosphere Packaging of Food. Ellis Horwood, New York, pp. 293. Brody, A.L. 1989. Controlled/Modified Atmosphere/Vacuum Packaging of Food. Food & Nutrition Press, Trumbull. pp. 179. Combining MAP with other preservation techniques 303 Books on preservation and shelf-life of foods Jujena, V.K. and Sofos, J.N. 2002. Control of foodborne mircoorganisms. Marcel Dekker Inc. pp. 535. Man, D. and Jones, A. 2000. Shelf-life evaluation of foods. Aspen Publishers, Inc. Maryland. pp. 272. Ohlsson, T. and Bengtsson, N. 2002. Minimal processing technologies in the food industry. CRC. Woodhead Publishing Limited, Cambridge, England. Proceedings concerning modified atmosphere packaging of foods Institute of Packaging Professionals. 1995. Proceedings from MAPack’95 the leading edge conference on modified atmosphere packaging. October 19– 20, 1995, Anaheim, Ca. Campden and Chorleywood Food Research Association. 1995. Proceedings from Modified Atmosphere Packaging (MAP) and related technologies. September 6–7, 1995, UK, Chipping Campden, UK. Campden Food and Drink Research Association. 1990. Proceedings from International Conference on Modified Atmosphere Packaging. Parts 1 and 2, October 15–17, 1990, Stratford-upon-Avon, UK. Guidelines on modified atmosphere packaging of foods Day, B.P.F. 1992. Guidelines for the good manufacturing and handling of modified atmosphere packed food products. Technical Manual No. 34, Campden Food and Drink Research Association, Chipping Campden, Gloucestershire, UK. 14.9 References ACMSF-Advisory commitee on the microbiological safety of food. (1992) Report on vacuum packaging and associated processes. London, UK, HMSO. ASHIE, I. N. A., SMITH, J. P. and SIMPSON, B. K. (1996) Spoilage and shelf-life extension of fresh fish and shellfish. Crit Rev Food Sci Nutr 36, 87–121. BARAKAT, R. K. and HARRIS, L. J. (1999) Growth of Listeria monocytogenes and Yersinia enterocolitica on cooked modified-atmosphere-packaged poultry in the presence and absence of a naturally occurring microbiota. Appl. Environ. Microbiol 65, 342–5. BENNIK, M. H., VAN OVERBEEK, W., SMID, E. J. and GORRIS, L. G. (1999) Biopreservation in modified atmosphere stored mungbean sprouts: the use of vegetable-associated bacteriocinogenic lactic acid bacteria to control the growth of Listeria monocytogenes. Lett. Appl. Microbiol 28, 226–32. BIDAWID, S., FARBER, J. M. and SATTAR, S. A. (2001) Survival of hepatitis A viruses on modified atmosphere-packaged (MAP) lettuce. Food Microbiology 18, 95–102. BJERKENG, B., SIVERTSVIK, M., ROSNES, J. T. and BERGSLIEN, H. (1995) Reducing package deformation and increasing filling degree in packages of cod 304 Novel food packaging techniques fillets in CO2-enriched atmospheres by adding sodium carbonate and citric acid to an exudate absorber. In Foods and Packaging materials – Chemical Interactions (Edited by Ackermann, P., Ja¨gerstad, M., and Ohlsson, T.) pp. 222–7. The Royal Society of Chemistry, Cambridge, UK. B?KN?S, N., OSTERBERG, C., NIELSEN, J. and DALGAARD, P. (2000) Influence of freshness and frozen storage temperature on quality of thawed cod fillets stored in modified atmosphere packaging. Lebensmittel Wissenschaft und Technologie 33, 244–8. BORCH, E., KANT-MEUERMANS, M. L. and BLIXT, Y. (1996) Bacterial spoilage of meat and cured meat products. Int. J Food Microbiol. 33, 103–20. CHOI, Y. M. AND PARRISH, F. C. (2000) Selective control of lactobacilli in kimchi with nisin. Lett. Appl. Microbiol 30, 173–7. CHURCH, I. J. and PARSONS, A. L. (1995) Modified atmosphere packaging technology: A review. J. Sci. Food Agric. 67, 143–52. CLEVELAND, J., MONTVILLE, T. J., NES, I. F. and CHIKINDAS, M. L. (2001) Bacteriocins: safe, natural antimicrobials for food preservation. Int. J Food Microbiol. 71, 1–20. CLIVER, D. O. (1997) Virus transmission via food. Food Technology 51, 71–8. COYNE, F. P. (1932) The effect of carbon dioxide on bacterial growth with special reference to the preservation of fish. Part I. J. Soc. Chem. Ind. 51, 119T– 121T. COYNE, F. P. (1933) The effect of carbon dioxide on bacterial growth with special reference to the preservation of fish. Part II. J. Soc. Chem. Ind. 52, 19T– 24T. CUTTER, C. N. and SIRAGUSA, G. R. (1998) Incorporation of nisin into a meat binding system to inhibit bacteria on beef surfaces. Lett. Appl. Microbiol 27, 19–23. DALGAARD, P., GRAM, L. and HUSS, H. H. (1993) Spoilage and Shelf-Life of Cod Fillets Packed in Vacuum or Modified Atmospheres. International Journal of Food Microbiology 19, 283–94. DALGAARD, P., MUNOZ, L. G. and MEJLHOLM, O. (1998) Specific inhibition of Photobacterium phosphoreum extends the shelf-life of modified- atmosphere-packed cod fillets. Journal of Food Protection 61, 1191–4. DALGAARD, P. and J?RGENSEN, L. V. (2000) Cooked and brined shrimps packaged in a modified atmosphere have a shelf-life of >7 months at 0oC but spoil in 4–6 days at 25oC. International Journal of Food Science and Technology 35, 431–42. DANIELS, J. A., KRISHNAMURTHI, R. and RIZVI, S. S. H. (1985) A review of the effects of carbon dioxide on microbial growth and food quality. J Food Prot. 48, 532–7. DAVIES, A. R. (1995) Advances in Modified Atmosphere packaging. In New methods of food preservation (Edited by Gould, G. W.) pp. 304–20. Blackie Acedemic & Professional, Glasgow, UK. DAVIES, E. A., BEVIS, H. E. and DELVES-BROUGHTON, J. (1997) The use of the bacteriocin, nisin, as a preservative in ricotta-type cheeses to control the Combining MAP with other preservation techniques 305 food-borne pathogen Listeria monocytogenes. Lett. Appl. Microbiol 24, 343–6. DAVIES, E. A., MILNE, C. F., BEVIS, H. E., POTTER, R. W., HARRIS, J. M., WILLIAMS, G. C., THOMAS, L. V. and DELVES-BROUGHTON, J. (1999) Effective use of nisin to control lactic acid bacterial spoilage in vacuum-packaged Bologna-type sausage. J. Food Prot. 62, 1004–10. DAY, B. (1990) A Perspective of Modified Atmosphere Packaging of Fresh produce in Western Europe. Food Science and Technology Today 4, 215. DEBEVERE, J. M. (1989) The effect of sodium lactate on shelf-life of vacuum- packaged coarse liver pa?te′. Fleischwirtschaft 69, 223–24. DEVLIEGHERE, F., DEBEVERE, J. and VAN IMPE, J. (1998) Concentration of carbon dioxide in the water-phase as a parameter to model the effect of a modified atmosphere on microorganisms. Int. J Food Microbiol 43, 105–13. DEVLIEGHERE, F., GEERAERD, A. H., VERSYCK, K. J., BERNAERT, H., VAN IMPE, J. F. and DEBEVERE, J. (2000a) Shelf life of modified atmosphere packed cooked meat products: addition of Na-lactate as a fourth shelf-life determinative factor in a model and product validation. International Journal of Food Microbiology 58, 93–106. DEVLIEGHERE, F., LEFEVERE, I., MAGNIN, A. and DEBEVERE, J. (2000b) Growth of Aeromonas hydrophila in modified-atmosphere-packed cooked meat products. Food Microbiology 17, 185–96. DEVLIEGHERE, F., GEERAERD, A. H., VERSYCK, K. J., VANDEWAETERE, B., VAN IMPE, J. and DEBEVERE, J. (2001) Growth of Listeria monocytogenes in modified atmosphere packed cooked meat products: a predictive model. Food Microbiology 18, 53–66. DOHERTY, A., SHERIDAN, J. J., ALLEN, P., MCDOWELL, D. A., BLAIR, I. S. and HARRINGTON, D. (1996) Survival and growth of Aeromonas hydrophila on modified atmosphere packaged normal and high pH lamb. International Journal of Food Microbiology 28, 379–92. ELLINGER, R. H. (1972) Phosphates in food processing. In Handbook of Food Additives (Edited by Furia, T.) pp. 617–780. CRC Press, Boca Raton, FL. ELLIOTT, P. and GRAY, R. J. H. (1981) Salmonella sensitivity in a sorbate/modified atmosphere combination system. J. Food Protection 44, 903–8. EMBORG, J., LAURSEN, B. G., RATHJEN, T. and DALGAARD, P. (2002) Microbial spoilage and formation of biogenic amines in fresh and thawed modified atmosphere-packed salmon (Salmo salar) at 2 degrees C. Journal of Applied Microbiology 92, 790–9. FANG, T. J. and LIN, L.-W. (1994) Growth of Listeria monocytogenes and Pseudomonas fragi on cooked pork in a modified atmosphere packaging/ nisin combination. J. Food Protection 57, 485. FARBER, J. M. (1991) Microbiological Aspects of Modified-Atmosphere Packaging Technology – A review. J. Food Prot. 54, 58–70. FERREIRA, M. A. and LUND, B. M. (1996) The effect of nisin on Listeria monocytogenes in culture medium and long-life cottage cheese. Lett. Appl. Microbiol 22, 433–8. 306 Novel food packaging techniques FEY, M. S. and REGENSTEIN, J. M. (1982) Extending Shelf-Life of Fresh Red Hake and Salmon Using CO 2 –O 2 Modified Atmosphere and Potassium Sorbate Ice at 1oC. J Food Sci 47, 1048–54. FRANCIS, G. A. and O’BEIRNE, D. (1998) Effects of storage atmosphere on Listeria monocytogenes and competing microflora using a surface model system. International Journal of Food Science and Technology 33, 465–76. FRANCIS, G. A. and O’BEIRNE, D. (2001) Effects of acid adaptation on the survival of Listeria monocytogenes on modified atmosphere packaged vegetables. International Journal of Food Science and Technology 36, 477–87. GIBSON, A. M., ELLIS-BROWNLEE, R. C. L., CAHILL, M. E., SZABO, E. A., FLETCHER, G. C. and BREMER, P. J. (2000) The effect of 100% CO2 on the growth of nonproteolytic Clostridium botulinum at chill temperatures. International Journal of Food Microbiology 54, 39–48. GILL, C. O. (1996) Extending the storage life of raw chilled meats. Meat Sci. 43, S99–S109. GILL, C. O. and REICHEL, M. P. (1989) Growth of the cold-tolerant pathogens Yersinia enterocolitica, Aeromonas hydrophila and Listera monocytogenes on high-pH beef packaged under vacuum or carbon dioxide. Food Microbiology 6, 223–30. GOULD, E. and PETERS, J. A. (1971) On testing the freshness of frozen fish. In Fishing News Books Ltd. pp. 45–47. London. GRAU, R. D. (1981) Role of pH, lactate, and anaerobiosis in controlling the growth of some fermentative, gram-negative bacteria on beef. Appl. Environ. Microbiol 42, 1043–50. GULDAGER, H. S., B?KN?S, N., ?STERBERG, C., NIELSEN, J. and DALGAARD, P. (1998) Thawed Cod Fillets Spoil Less Rapidly Than Unfrozen Fillets When Stored under Modified Atmosphere at 2oC. J. Food Prot. 61, 1129–36. HOUTSMA, P. C., DE WIT, J. C. and ROMBOUTS, F. M. (1993) Minimum inhibitory concentration (MIC) of sodium lactate for pathogens and spoilage organisms occurring in meat products. Int. J Food Microbiol 20, 247–57. JACXSENS, L., DEVLIEGHERE, F., VAN DER STEEN, C. and DEBEVERE, J. (2001) Effect of high oxygen modified atmosphere packaging on microbial growth and sensorial qualities of fresh-cut produce. International Journal of Food Microbiology 71, 197–210. KLEINLEIN, N. and UNTERMANN, F. (1990) Growth of pathogenic Yersinia enterocolitica strains in minced meat with and without protective gas with consideration of the competitive background flora. Int. J Food Microbiol 10, 65–71. KOSEKI, S. and ITOH, K. (2002) Effect of nitrogen gas packaging on the quality and microbial growth of fresh-cut vegetables under low temperatures. J Food Prot. 65, 326–32. LAMBERT, A. D., SMITH, J. P. and DODDS, K. L. (1991) Effect of initial O 2 and CO 2 and low-dose irradiation on toxin production by Clostridium botulinum in MAP fresh pork. J. Food Prot. 54, 939–44. LEBLANC, E. L. and LEBLANC, R. J. (1992) Determination of Hydrophobicity and Combining MAP with other preservation techniques 307 Reactive Groups in Proteins of Cod (Gadus morhua) Muscle During Frozen Storage. Food Chemistry 43. LEISTNER, L. (1992) Food preservation by combined methods. Food Research International 25, 151–8. LEISTNER, L. (1995a) Principles and applications of hurdle technology. In New Methods of Food Preservation (edited by Gould, G. W.) pp. 1–21. Blackie Acedemic & Professional, Glasgow. LEISTNER, L. (1995b) Use of Hurdle Technology in Food Processing: Recent advances. In Food Preservation by Moisture Control (Edited by Barbosa- Ca′novas, G. V. and Welti-Chanes, J.) pp. 377–96. Technomic Publishing Company, Inc., Basel, Switzerland. LEISTNER, L. (2002) Hurdle technology. In Control of foodborne microorganisms (Edited by Juneja, V. K. and Sofos, J. N.) pp. 493–508. Marcel Dekker, Inc., Basel, Switzerland. LEISTNER, L. and GORRIS, L. G. M. Food preservation by combining processes. Final report – FLAIR Concerted Action No. 7, Subgroup, 1–100. 1997. DG XII Flair, European Commission. LICCIARDELLO, J., RAVESI, E., TUHKUNEN, B. and RACICOT, L. (1984) Effects of some potentially synergistic treatments in combination with 100 krad irradiation on the shelf-life of cod fillets. J. Food Technol. 49, 1341. LISERRE, A. M., LANDGRAF, M., DESTRO, M. T. and FRANCO, B. D. G. M. (2002) Inhibition of Listeria monocytogenes by a bacteriocinogenic Lactobacillus sake strain in modified atmosphere-packaged Brazilian sausage. Meat Sci. 61, 449–55. LUND, B. M. and PECK, M. W. (1994) Heat resistance and recovery of spores of non-proteolytic Clostridium botulinum in relation to refrigerated, processed foods with an extended shelf-life. Soc. Appl. Bacteriol. Symp. Ser. 23, 115S–128S. LYVER, A., SMITH, J. P., AUSTIN, J. and BLANCHFIELD, B. (1998) Competitive inhibition of Clostridium botulinum type E by Bacillus species in a value- added seafood product packaged under a modified atmosphere. Food Research International 31, 311–19. MCMEEKIN, T. A. and Ross, T. (2002) Predictive microbiology: providing a knowledge-based framework for change management. Int. J. Food Microbiol. 78, 133–53. MITSUDA, H., NAKAJIMA, K., MIZUNO, H. and KAWAI, F. (1980) Use of sodium chloride solution and carbon dioxide for extending shelf-life of fish fillets. J. Food Sci. 45, 661–6. MORRIS, C. E. (1989) Convenience for supermarket delis with CAP pizza. Food Eng. 61, 53–54. NISSEN, H., ROSNES, J. T., BRENDEHAUG, J. and KLEIBERG, G. H. (2003) Safety evaluations of sous vide ready-meals. Letters Appl. Microbiol. In press. NOTERMANS, S., DUFRENNE, J. and LUND, B. M. (1990) Botulism risk of refrigerated, processed foods of extended durability. J. Food Prot. 53, 1020. 308 Novel food packaging techniques OHLSSON, T. (2002a) Minimal processing of foods with non-thermal methods. In Minimal processing technologies in the food industry (edited by Ohlsson, T. and Bengtsson, N.) pp. 34–60. Woodhead Publishing Limited, Cambridge, England. OHLSSON, T. (2002b) Minimal processing of foods with thermal methods. In Minimal processing technologies in the food industry (Edited by Ohlsson, T. and Bengtsson, N.) pp. 4–33. Woodhead Publishing Limited, Cambridge, England. OLSEN, S. O., STEINSB?, E. E., AND SKA ? RA, T. (2002) The Influence of Potential Pre- Treatment and Processing Parameters on General Proteolytic Activity Characteristics in Atlantic salmon (Salmo salar), studied in a Model System. Journal of Aquatic Food Product Technology 11, No. 3–4, 65–85. PALUDAN-MULLER, C., DALGAARD, P., HUSS, H. H. and GRAM, L. (1998) Evaluation of the role of Carnobacterium piscicola in spoilage of vacuum- and modified-atmosphere-packed cold-smoked salmon stored at 5 degrees C. Int. J Food Microbiol 39, 155–66. PASTORIZA, L., SAMPEDRO, G., HERRERA, J. J. and CABO, M. L. (1998) Influence of sodium chloride and modified atmosphere packaging on microbiological, chemical and sensorial properties in ice storage of slices of hake (Merluccius merluccius). Food Chem. 61, 23–28. PHILLIPS, C. A. (1996) Review: Modified atmosphere packaging and its effects on the microbiological quality and safety of produce. Int. J. Food Sci. & Technol. 31, 463–79. POTHURI, P., MARSHALL, D. L. and MCMILLIN, K. W. (1996) Combined effects of packaging atmosphere and lactic acid on growth and survival of Listeria monocytogenes in crayfish tail meat at 4 degrees C. J. Food Prot. 59, 253– 6. PRZYBYLSKI, L. A., FINERTY, M. W., GRODNER, R. M. and GERDES, D. L. (1989) Extension of shelf-life of iced fresh channel catfish fillets using modified atmosphere packaging and low dose irradiation. J. Food Sci. 54, 269–73. ROBINS, D. (1991) Combination treatments with food irradiation. In The preservation of food by irradiation. A factual guide to the process and its effect on food pp. 53–61. Dotesios Limited, Trowbridge, Wiltshire. ROSNES, J. T., SIVERTSVIK, M., SKIPNES, D., NORDTVEDT, T. S., CORNELIUSSEM, C. and JAKOBSEN, ?. (1998) Transport of superchilled salmon in modified atmosphere. International Insitute of Refrigeration – IIF/IIR, Nantes, France. ROSNES, J. T., KLEIBERG, G. H. and FOLKVORD, L. (2001) Increased shelf-life of salmon (Salmo salar) steaks using partial freezing (liquid nitrogen) and modified atmosphere packaging (MAP). Annales Societatis Scientiarum F?roensis Supplementum XXVIII, 83–91. SCANNELL, A. G., HILL, C., ROSS, R. P., MARX, S., HARTMEIER, W., ELKE and ARENDT, K. (2000) Development of bioactive food packaging materials using immobilised bacteriocins lacticin 3147 and nisaplin. Int. J Food Microbiol 60, 241–9. Combining MAP with other preservation techniques 309 SIKORSKI, Z. E. and SUN, P. (1994) Preservation of seafood quality. In Seafoods: Chemistry, Processing, Technology and Quality (Edited by Shahidi, F. and Botta, J. R.) pp. 168. Blackie Academic and Professional, Glasgow, UK. SIVERTSVIK, M. (1999) Use of soluble gas stabilisation to extend shelf-life of fish. Norconserv., WEFTA, 29th annual meeting, Oct. 10–14, 1999, Thessaloniki, Greece. SIVERTSVIK, M., NORDTVEDT, T. S., AUNE, E. J. and ROSNES, J. T. (1999) Storage quality of superchilled and modified atmosphere packaged whole salmon. 20th International Congress of Refrigeration, International Institute of Refrigeration, Sydney, Australia. SIVERTSVIK, M., BERGSLIEN, H. and ROSNES, J. T. (2001) Shelf-life extensions of seafood under modified atmospheres, Warsaw, Poland. SIVERTSVIK, M., JEKSRUD, W. K. and ROSNES, J. T. (2002) A review of modified atmosphere packaging of fish and fishery products – significance of microbial growth, activities and safety. Int. J. Food Science and Technol. 37, 107–27. SKANDAMIS, P. N. and NYCHAS, G. J. E. (2001) Effect of oregano essential oil on microbiological and physico-chemical attributes of minced meat stored in air and modified atmospheres. Journal of Applied Microbiology 91, 1011– 22. SKANDAMIS, P., TSIGARIDA, E. and NYCHAS, G. J. E. (2002) The effect of oregano essential oil on survival/death of Salmonella typhimurium in meat stored at 5 degrees C under aerobic, VP/MAP conditions. Food Microbiology 19, 97–103. STILES, M. E. (1996) Biopreservation by lactic acid bacteria. Antonie Van Leeuwenhoek 70, 331–45. SZABO, E. and CAHILL, M. E. (1998) The combined effect of modified atmosphere, temperature, nisin and ALTA 2341 on the growth of Listeria monocytogenes. Int. J Food Microbiol 43, 21–31. THAKUR, B. R. and PATEL, T. R. (1994) Sorbates in fish and fish products – A review. Food Reviews International 10, 93–107. THAYER, D. W. and BOYD, G. (1999) Irradiation and modified atmosphere packaging for the control of Listeria monocytogenes on turkey meat. J. Food Prot. 62, 1136–42. THAYER, D. W. and BOYD, G. (2000) Reduction of normal flora by irradiation and its effect on the ability of Listeria monocytogenes to multiply on ground turkey stored at PC when packaged under a modified atmosphere. J. Food Prot. 63, 1702–6. TSIGARIDA, E., SKANDAMIS, P. and NYCHAS, G. J. E. (2000) Behaviour of Listeria monocytogenes and autochthonous flora on meat stored under aerobic, vacuum and modified atmosphere packaging conditions with or without the presence of oregano essential oil at 5 degrees C. Journal of Applied Microbiology 89, 901–9. WANDLING, L. R., SHELDON, B. W. and FOEGEDING, P. M. (1999) Nisin in milk sensitizes Bacillus spores to heat and prevents recovery of survivors. J 310 Novel food packaging techniques Food Prot. 62, 492–8. WIMPFHEIMER, L., ALTMAN, N. S. and HOTCHKISS, J. H. (1990) Growth of Listeria monocytogenes Scott A, serotype 4 and competitive spoilage organisms in raw chicken packaged under modified atmospheres and in air. Int. J Food Microbiol 11, 205–14. ZEITOUN, A.A.M. and DEBEVERE, J. M. (1991) The effect of treatment with buffered lactic acid on microbial decontaminating on the shelf-life of poultry. Int. J. Food Microbiol 11, 305–12. ZEITOUN, A.A.M. and DEBEVERE, J. M. (1990) Inhibition, survival and growth of Listeria monocytogens on poultry as influenced by buffered lactic acid treatment and modified atmosphere packaging. Int. J. Food Microbiol 14, 161–70. Combining MAP with other preservation techniques 311