《食品包装技术》（英文版）Chapter 18 Active packaging in practice: fish

Fresh fish products are usually more perishable than most other foodstuffs due to their high aw, neutral pH, and presence of autolytic enzymes. The spoilage of fish and shellfish results from changes caused by oxidation of lipids, reactions due to activities of the fishes'own enzymes, and the metabolic activities of microorganisms (Ashie et al., 1996). The rate of deterioration is highly temperature dependent and can be inhibited by the use of low storage

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Active packaging in practice: fish 385 like the pelagic species(e.g. mackerel and herring) have a very short shelf-life Fresh fish is very different from the various processed fish products that need packaging: heat-treated fish products(ready meals, fish pudding/balls), smoked, dried. or salted fish. We still have not taken into account the numerous different pecies of crustaceans and molluscs This chapter will cover active packaging of fish products including the use of atmosphere modifiers such as oxygen scavengers and carbon dioxide emitters, packaging that controls water or with anti-microbial and anti-oxidative properties, and indicator mechanisms. Modified atmosphere packaging(MAP) is regarded by some as an active packaging technology. This is by now a well established method to extend the shelf-life of foods, including fish products, and will not be covered in this chapter except for the MA methods different from traditional MAP using gas flushing Obviously, having such a broad spectrum of products, it is unlikely that one pecific novel or active packaging technology will be a success for all, just as not all fishery products benefit from MAP when compared to vacuum packaging (Sivertsvik et al., 2002a). So, the potential for an active packaging technology to be successful for a product would depend on the technology's ability to control and inhibit the shelf-life deteriorating spoilage reactions(e.g. bacterial growth of pecific bacteria, oxidative rancidity, colour changes) in the specific product 18.2 The microbiology of fish products As mentioned, the deterioration of fresh fish is usually microbial so controlling the microbial growth is usually the most important parameter for an active packaging technology to be successful. Fish normally have a particularly heavy microbial load owing to the method of capture and transport to shore slaughtering method, evisceration and retention of skin in retail portions. The microorganisms associated with most seafood reflect the microbial population in their aquatic environment(Colby et al, 1993; Liston 1980; Gram and Huss 1996). Microorganisms are found on all the outer surfaces(skin and gills)and in the intestines of live and newly caught fish. The total numbers of organisms vary enormously and Liston(1980) states a normal range of 10-10 cfu(colony forming units)/cm on the skin surface. The gills and the intestines both contain between 10 and 10 cfu/g(Huss 1995). The fish muscle is sterile at the time of slaughtering/catch, but quickly becomes contaminated by surface and intestinal bacteria, equipment, and humans during handling and processing. The microflora of temperate-water fish is dominated by psychrotrophic, aerobic or facultative anaerobic Gram-negative, rod-shaped bacteria, but Gram-positive organisms can also be found in varying proportions. The flora on tropical fish often carries a slightly higher load of Gram-positive and enteric bacteria. The composition of fresh fish flesh makes it favourable to microbial growth he muscle is composed of low collagen, low lipid, and high levels of soluble non-protein-nitrogen(NPN) compounds. Trimethylamine-oxide(TMAO), a part

like the pelagic species (e.g. mackerel and herring) have a very short shelf-life. Fresh fish is very different from the various processed fish products that need packaging: heat-treated fish products (ready meals, fish pudding/balls), smoked, dried, or salted fish. We still have not taken into account the numerous different species of crustaceans and molluscs. This chapter will cover active packaging of fish products including the use of atmosphere modifiers such as oxygen scavengers and carbon dioxide emitters, packaging that controls water or with anti-microbial and anti-oxidative properties, and indicator mechanisms. Modified atmosphere packaging (MAP) is regarded by some as an active packaging technology. This is by now a well established method to extend the shelf-life of foods, including fish products, and will not be covered in this chapter except for the MA methods different from traditional MAP using gas flushing. Obviously, having such a broad spectrum of products, it is unlikely that one specific novel or active packaging technology will be a success for all, just as not all fishery products benefit from MAP when compared to vacuum packaging (Sivertsvik et al., 2002a). So, the potential for an active packaging technology to be successful for a product would depend on the technology’s ability to control and inhibit the shelf-life deteriorating spoilage reactions (e.g. bacterial growth of specific bacteria, oxidative rancidity, colour changes) in the specific product. 18.2 The microbiology of fish products As mentioned, the deterioration of fresh fish is usually microbial so controlling the microbial growth is usually the most important parameter for an active packaging technology to be successful. Fish normally have a particularly heavy microbial load owing to the method of capture and transport to shore, slaughtering method, evisceration and retention of skin in retail portions. The microorganisms associated with most seafood reflect the microbial population in their aquatic environment (Colby et al., 1993; Liston 1980; Gram and Huss 1996). Microorganisms are found on all the outer surfaces (skin and gills) and in the intestines of live and newly caught fish. The total numbers of organisms vary enormously and Liston (1980) states a normal range of 102 –107 cfu (colony forming units)/cm2 on the skin surface. The gills and the intestines both contain between 103 and 109 cfu/g (Huss 1995). The fish muscle is sterile at the time of slaughtering/catch, but quickly becomes contaminated by surface and intestinal bacteria, equipment, and humans during handling and processing. The microflora of temperate-water fish is dominated by psychrotrophic, aerobic or facultative anaerobic Gram-negative, rod-shaped bacteria, but Gram-positive organisms can also be found in varying proportions. The flora on tropical fish often carries a slightly higher load of Gram-positive and enteric bacteria. The composition of fresh fish flesh makes it favourable to microbial growth. The muscle is composed of low collagen, low lipid, and high levels of soluble non-protein-nitrogen (NPN) compounds. Trimethylamine-oxide (TMAO), a part Active packaging in practice: fish 385

386 Novel food packaging techniques of the NPN compounds, can be broken down to trimethylamine (TMa) by endogenous enzymes. However, at chilled temperatures TMA is produced by the bacterial enzyme TMA oxidase. TMA is recognised as the characteristic fishy odour of spoiled fish. When the oxygen level is depleted, many of the spoilage bacteria can utilise TMAO as a terminal hydrogen acceptor, thus allowing them to grow under anoxic conditions. Towards the end of shelf-life. various malodorous low molecular-weight sulphur-compounds such as H2S and CH3 SH, ogether with volatile fatty acids and ammonia are produced because of bacterial During chilled storage, there is a shift in bacterial types. The part of the microflora, which will ultimately grow on the products, is determined by the intrinsic(e.g. post mortem pH in the flesh, the poikilothermic nature of fish, and presence of TMAO and other NPN components)and extrinsic parameters(e.g temperature, processing, and packaging atmosphere). When a product is microbial spoiled, the spoilage microflora will usually consist of a mixture of species, many of which can be completely harmless both in terms of health hazards and in terms of ability to produce off-odours and off-flavours. The bacterial group causing the important chemical changes during fish spoilage often consists of a single species; the specific spoilage organisms(SSO). Little is known of the SSOs of different fish from various aquatic environments under different packaging conditions. However, for many fish stored under aerobic conditions in ice, Shewanella putrefaciens has been identified as the main spoilage bacteria( Gram et al., 1987). S. putrefaciens produce very intense and unpleasant off-odours, reduce TMAO to TMA and produce H2S Under anaerobic conditions(MAP, vacuum packaging, active packaging technologies)the spoilage bacteria differ from aerobic spoilage. The Gram- negative organism Photobacterium phosphoreum has been identified as the organism responsible for spoilage in VP and in MA packs(Dalgaard 1995). The growth rate of this organism is increased under anaerobic conditions and in contrast to S. putrefaciens, P. phosphoreum is shown to be highly resistant to CO2. It was also shown that the growth of this bacteria corresponds very well with the shelf-life of packed fresh cod. P phosphoreum reduces TMAO to TMA at 10-100 times the amount per cell than S. putrefaciens probably due to the large size of the former( diameter 5um) while very little HS is produced during growth in fish substrates(Dalgaard et al, 1996). Spoiled MAP cod is characterised by high levels of TMA, but little or no development of the putrid or HiS odours typical for some aerobically stored spoiled fish. P. phosphoreum is widespread in the marine environment and it seems likely that this organism or other highly CO2 resistant microorganisms are responsible for spoilage of packed seafood products. Lactic acid bacteria and Brochothrix thermosphacta have been identified as the typical SSOs of freshwater fish and fish from warmer waters To obtain a longer shelf-life for fresh fish than obtained by ice or chilled MAP/vacuum, the approach is to inhibit the SSo limiting shelf-life for the technology chosen. For example P. phosphoreum is sensitive to freezing and is

of the NPN compounds, can be broken down to trimethylamine (TMA) by endogenous enzymes. However, at chilled temperatures TMA is produced by the bacterial enzyme TMA oxidase. TMA is recognised as the characteristic ‘fishy’ odour of spoiled fish. When the oxygen level is depleted, many of the spoilage bacteria can utilise TMAO as a terminal hydrogen acceptor, thus allowing them to grow under anoxic conditions. Towards the end of shelf-life, various malodorous low molecular-weight sulphur-compounds such as H2S and CH3SH, together with volatile fatty acids and ammonia are produced because of bacterial growth. During chilled storage, there is a shift in bacterial types. The part of the microflora, which will ultimately grow on the products, is determined by the intrinsic (e.g. post mortem pH in the flesh, the poikilothermic nature of fish, and presence of TMAO and other NPN components) and extrinsic parameters (e.g. temperature, processing, and packaging atmosphere). When a product is microbial spoiled, the spoilage microflora will usually consist of a mixture of species, many of which can be completely harmless both in terms of health hazards and in terms of ability to produce off-odours and off-flavours. The bacterial group causing the important chemical changes during fish spoilage often consists of a single species; the specific spoilage organisms (SSO). Little is known of the SSOs of different fish from various aquatic environments under different packaging conditions. However, for many fish stored under aerobic conditions in ice, Shewanella putrefaciens has been identified as the main spoilage bacteria (Gram et al., 1987). S. putrefaciens produce very intense and unpleasant off-odours, reduce TMAO to TMA and produce H2S. Under anaerobic conditions (MAP, vacuum packaging, active packaging technologies) the spoilage bacteria differ from aerobic spoilage. The Gramnegative organism Photobacterium phosphoreum has been identified as the organism responsible for spoilage in VP and in MA packs (Dalgaard 1995). The growth rate of this organism is increased under anaerobic conditions and in contrast to S. putrefaciens, P. phosphoreum is shown to be highly resistant to CO2. It was also shown that the growth of this bacteria corresponds very well with the shelf-life of packed fresh cod. P. phosphoreum reduces TMAO to TMA at 10–100 times the amount per cell than S. putrefaciens probably due to the large size of the former (diameter 5m) while very little H2S is produced during growth in fish substrates (Dalgaard et al., 1996). Spoiled MAP cod is characterised by high levels of TMA, but little or no development of the putrid or H2S odours typical for some aerobically stored spoiled fish. P. phosphoreum is widespread in the marine environment and it seems likely that this organism or other highly CO2 resistant microorganisms are responsible for spoilage of packed seafood products. Lactic acid bacteria and Brochothrix thermosphacta have been identified as the typical SSOs of freshwater fish and fish from warmer waters. To obtain a longer shelf-life for fresh fish than obtained by ice or chilled MAP/vacuum, the approach is to inhibit the SSO limiting shelf-life for the technology chosen. For example P. phosphoreum is sensitive to freezing and is 386 Novel food packaging techniques

Active packaging in practice: fish 387 totally inactivated in thawed chilled MAP cod fillets after frozen storage at 20C and -30.C for 6-8 weeks. This approach has been used to further extend the shelf-life of MAP cod(Guldager et al, 1998)and salmon(Emborg et al 2002)at 2C, and should also be the approach for successful use of active ackaging technologies to control microbial spoilage 18.3 Active packaging: atmosphere modifiers Many of the most used active packaging technologies are closely modified atmosphere packaging. Together with anaerobic condition dioxide is the active gas of MaP because it inhibits growth of man normal spoilage bacteria (Sivertsvik et al, 2002b). The effect of CO2 on bacterial growth is complex and four activity mechanisms of CO2on microorganisms has been identified(Farber 1991; Daniels et al., 1985; Dixon and Kell 1989; Parkin and Brown 1982): Alteration of cell membrane function includes effects on nutrient uptake and absorption; direct inhibition of enzymes or decreases in the rate of enzyme reactions; penetration of bacterial membranes leading to intracellular pH changes, and direct changes in the physico-chemical properties of proteins. Probably a combination of all these activities accounts for the bacteriostatic effect(Sivertsvik et al, 2002a). The CO2 is usually introduced into the MA-package by evacuating the air and flushing the appropriate gas mixture into the package prior to sealing, typically using automatic form-fill- seal or flow-packaging machines. Two other approaches to create a modified atmosphere for a product are either to generate the CO2 and/or remove O2 inside the package after packaging or to dissolve the CO2 into the product prior to packaging. Both methods can give appropriate packages with smaller gas/ roduct ratio, and thus decrease the package size that has been a disadvantage of MAP from the start The first approach involves the most commercialised active packaging technology, namely oxygen scavengers, that by now are available from several manufacturers (Mitsubishi Gas Chemical Co., ATCO, Bioka, Sealed Air/ Cryovac, Multisorb a.o. ), in different forms(sachets, packaging film, closures) with different active ingredients (iron, enzymes, dye). Some of the same companies have also developed COz emitters, using the O2 in the package headspace to produce CO2 and to develop a CO2/N2 atmosphere inside a kage without the use of gas flushing. Other methods for generating the cO2 gas inside the packages after closure include the use of dry ice(solid CO2) (Sivertsvik et al., 1999)or carbonate possibly mixed with weak acids(Bjerkeng etal.,1995) The second approach is to dissolve the CO2 into the product prior to packaging Since the solubility increases at lower temperatures and at higher CO, pressures, a sufficient amount of CO2 can be dissolved into the product during 1-2 hours prior to packaging using elevated pressures. This method is called soluble gas stabilisation(SGS)(Sivertsvik 2000). This is not an active packaging technology

totally inactivated in thawed chilled MAP cod fillets after frozen storage at ÿ20ºC and ÿ30ºC for 6–8 weeks. This approach has been used to further extend the shelf-life of MAP cod (Guldager et al., 1998) and salmon (Emborg et al., 2002) at 2ºC, and should also be the approach for successful use of active packaging technologies to control microbial spoilage. 18.3 Active packaging: atmosphere modifiers Many of the most used active packaging technologies are closely related to modified atmosphere packaging. Together with anaerobic conditions, carbon dioxide is the active gas of MAP because it inhibits growth of many of the normal spoilage bacteria (Sivertsvik et al., 2002b). The effect of CO2 on bacterial growth is complex and four activity mechanisms of CO2 on microorganisms has been identified (Farber 1991; Daniels et al., 1985; Dixon and Kell 1989; Parkin and Brown 1982): Alteration of cell membrane function includes effects on nutrient uptake and absorption; direct inhibition of enzymes or decreases in the rate of enzyme reactions; penetration of bacterial membranes, leading to intracellular pH changes; and direct changes in the physico-chemical properties of proteins. Probably a combination of all these activities accounts for the bacteriostatic effect (Sivertsvik et al., 2002a). The CO2 is usually introduced into the MA-package by evacuating the air and flushing the appropriate gas mixture into the package prior to sealing, typically using automatic form-fillseal or flow-packaging machines. Two other approaches to create a modified atmosphere for a product are either to generate the CO2 and/or remove O2 inside the package after packaging or to dissolve the CO2 into the product prior to packaging. Both methods can give appropriate packages with smaller gas/ product ratio, and thus decrease the package size that has been a disadvantage of MAP from the start. The first approach involves the most commercialised active packaging technology, namely oxygen scavengers, that by now are available from several manufacturers (Mitsubishi Gas Chemical Co., ATCO, Bioka, Sealed Air/ Cryovac, Multisorb a.o.), in different forms (sachets, packaging film, closures) with different active ingredients (iron, enzymes, dye). Some of the same companies have also developed CO2 emitters, using the O2 in the package headspace to produce CO2 and to develop a CO2/N2 atmosphere inside a package without the use of gas flushing. Other methods for generating the CO2 gas inside the packages after closure include the use of dry ice (solid CO2) (Sivertsvik et al., 1999) or carbonate possibly mixed with weak acids (Bjerkeng et al., 1995). The second approach is to dissolve the CO2 into the product prior to packaging. Since the solubility increases at lower temperatures and at higher CO2 pressures, a sufficient amount of CO2 can be dissolved into the product during 1–2 hours prior to packaging using elevated pressures. This method is called soluble gas stabilisation (SGS) (Sivertsvik 2000). This is not an active packaging technology Active packaging in practice: fish 387

388 Novel food packaging by definition, but it is a novel alternative to MA and it has been used successfully alone and in combination with O2 scavengers(see below) The commercial use of atmosphere modifiers, and O scavengers in particular, with fish products has been mostly limited to the Japanese market and to dried (seaweed, salmon jerky, sardines, shark's fin, rose mackerel, cod, squid)or smoked(salmon) products(Ashie et al., 1996). These ambient stored products have low aw (<0.85)so the microbial deterioration is not shelf-life limiting, therefore the effect of the O2 scavengers is to prevent oxidative reactions discolouration, and mould growth. Other commercial products are fresh yellow- tail, salmon roe, and sea urchin all stored at superchilling conditions packaged with O2 scavenger primarily to prevent oxidation and discolouration, but also to inhibit bacterial growth to a lesser degree(Ashie et al., 1996). Different O scavengers are chosen dependent on the amount of O2 to scavenge(pack size and material)and product aw. O2 scavengers for high aw foods react faster compared to scavengers for dry foods but in general the absorption is slow and exothermic Removal of oxygen from package interiors improves shelf-life by sub- ptimising the environment for aerobic microbiological growth and for adverse oxidative reactions such as rancidity. Ferrous ironbased oxygen scavengers rely on the presence of moisture for activation, with a water activity of at least 0.7 required, and 0.85-0.9 being preferred(Brody 2001). Oxygen absorbers are designed to reduce oxygen levels to less than 100 ppm in package head-space. In on-based en scavengers the oxygen is removed by oxidation(rusting) of powdered iron forming non-toxic iron oxide( Ashie et al., 1996). Oxygen absorbers could be used to create oxygen-free conditions in head-space of packages of medium barrier properties. The sachet will absorb residual oxygen and oxygen permeated through the packaging material during storage. More environmentally 'friendly' packaging materials with lower oxygen barriers could be used in combination with an oxygen absorber instead of high-cost barrier materials (Sivertsvik, 1997). However, not all oxygen absorbers can be combined with MAP. Some of them. like the iron-based Ageless SS-type from Mitsubishi meant for use in high aw foods, will unintentionally absorb some of the carbon dioxide present, and decrease some of the inhibitory effects of CO2 on bacterial growth. This is caused by a reaction of iron with CO2 to form ferrous carbonate, and secondarily this ferrous carbonate reacts with O This reaction will also slow down the O2 absorption(Sivertsvik, 1997 and 1999) The use of O2 absorbers(Ageless SS-100) had only a marginal effect on microbial growth in packages of fishcakes, fish pudding and mackerel fillets, and far less than the significant effect obtained by MAP(Sivertsvik 1997) However, a signficant effect of the O2 absorber was observed in packages with salmon fillets. The use of O2 absorbers inhibited development of rancidity TBARS)in both mackerel and salmon fillets (Sivertsvik, 1997), but in no higher degree than O2-free MAP. The effect of Sgs treatment. different O -absorbers/CO-emitters and combinations of these on growth of psychrotrophic bacteria in salmon fillets shown in Fig. 18.1(Sivertsvik, 1999). The best microbial quality was

by definition, but it is a novel alternative to MA and it has been used successfully alone and in combination with O2 scavengers (see below). The commercial use of atmosphere modifiers, and O2 scavengers in particular, with fish products has been mostly limited to the Japanese market and to dried (seaweed, salmon jerky, sardines, shark’s fin, rose mackerel, cod, squid) or smoked (salmon) products (Ashie et al., 1996). These ambient stored products have low aW (<0.85) so the microbial deterioration is not shelf-life limiting, therefore the effect of the O2 scavengers is to prevent oxidative reactions, discolouration, and mould growth. Other commercial products are fresh yellowtail, salmon roe, and sea urchin all stored at superchilling conditions packaged with O2 scavenger primarily to prevent oxidation and discolouration, but also to inhibit bacterial growth to a lesser degree (Ashie et al., 1996). Different O2 scavengers are chosen dependent on the amount of O2 to scavenge (pack size and material) and product aW. O2 scavengers for high aW foods react faster compared to scavengers for dry foods but in general the absorption is slow and exothermic. Removal of oxygen from package interiors improves shelf-life by suboptimising the environment for aerobic microbiological growth and for adverse oxidative reactions such as rancidity. Ferrous ironbased oxygen scavengers rely on the presence of moisture for activation, with a water activity of at least 0.7 required, and 0.85–0.9 being preferred (Brody 2001). Oxygen absorbers are designed to reduce oxygen levels to less than 100 ppm in package head-space. In iron-based oxygen scavengers the oxygen is removed by oxidation (rusting) of powdered iron forming non-toxic iron oxide (Ashie et al., 1996). Oxygen absorbers could be used to create oxygen-free conditions in head-space of packages of medium barrier properties. The sachet will absorb residual oxygen and oxygen permeated through the packaging material during storage. More inexpensive or environmentally ‘friendly’ packaging materials with lower oxygen barriers could be used in combination with an oxygen absorber instead of high-cost barrier materials (Sivertsvik, 1997). However, not all oxygen absorbers can be combined with MAP. Some of them, like the iron-based Ageless SS-type from Mitsubishi meant for use in high aW foods, will unintentionally absorb some of the carbon dioxide present, and decrease some of the inhibitory effects of CO2 on bacterial growth. This is caused by a reaction of iron with CO2 to form ferrous carbonate, and secondarily this ferrous carbonate reacts with O2. This reaction will also slow down the O2 absorption (Sivertsvik, 1997 and 1999). The use of O2 absorbers (Ageless SS-100) had only a marginal effect on microbial growth in packages of fishcakes, fish pudding and mackerel fillets, and far less than the significant effect obtained by MAP (Sivertsvik 1997). However, a signficant effect of the O2 absorber was observed in packages with salmon fillets. The use of O2 absorbers inhibited development of rancidity (TBARS) in both mackerel and salmon fillets (Sivertsvik, 1997), but in no higher degree than O2-free MAP. The effect of SGS treatment, different O2-absorbers/CO2-emitters and combinations of these on growth of psychrotrophic bacteria in salmon fillets is shown in Fig. 18.1 (Sivertsvik, 1999). The best microbial quality was 388 Novel food packaging techniques

390 Novel food packaging techniques microbiologically spoiled after 13 days of storage. The effects of the absorbers and packaging method were not significant on the sensory evaluation scores but multiple comparisons confirmed the findings of the microbiological analyses The SGS samples were evaluated as better compared to air samples on cooked flavour, cooked odour and texture, but got slightly lower scores on raw odour evaluation Samples packaged with G-100 absorber/emitter gave the best cooked sensory scores, while samples without absorber got the lowest cooked sensory scores On raw odour the samples packaged with G-100 and without absorbers were evaluated as better than samples with SS-200 and ZPT-100 absorbers. No differences were observed in the colour of the samples, in contrast to the reddish colour change observed when packaging perch and pike perch fillets with the Ageless G-100 COz-emitters(Ahvenainen et al., 1997). They observed the same shelf-life for fresh perch and pike perch fillets packaged with G-100 as for traditional MAP using an anoxic high CO2 atmosphere, and 2-4 days longer shelf-life when compared with over-wrap or vacuum packaging. However colour change and a smell of raw liver in the raw fillets in the active packages was observed. This was not observed in the traditional MA-packages. No differences between the two packaging technologies were observed after cooking of the fish The commercial CO2 emitters usually contain ferrous carbonate and a metal ide catalyst alth though non-ferrous variants are available, absorbing the O2 and producing equal volumes of CO2. Carbon dioxide could also be produced inside the packages after packaging by allowing the exudates from the product to react with a mixture of sodium carbonate and citric acid inside the drip pad, an approach used successfully for cod fillets(Bjerkeng et al. 1995) increasing shelf-life as compared to traditional MAP, even when using a low gas head space in the package. The Verifrais package manufactured by Codimer, which has been used for extending the shelf-life of fresh meats and fish, is a similar concept(Day 1998). This package consists of a standard MAP tray but has a perforated false bottom under which a porous sachet containing sodium bicarbonate/ascorbate is positioned. When exudate from packed meat or fish drips onto the sachet, CO2 is emitted and counteracts package collapse due to the CO2 solubility in the food 18.4 Active packaging: water control Excess moisture is a major cause of food spoilage and different humidity absorbers are used to protect dried products from humidity damage. However, these absorbers have a limited effect on fish products. Several companie manufacture moisture drip absorbent pads, sheets and blankets for liquid water control in watery foods such as meat, fish, poultry, fruit and vegetables Moisture drip absorber pads or false-bottomed trays are commonly placed under packaged fresh meat, fish, poultry and prepared fruit to absorb unsightly tissue drip discharge. Larger sheets and blankets are used for absorption of melted ice

microbiologically spoiled after 13 days of storage. The effects of the absorbers and packaging method were not significant on the sensory evaluation scores but multiple comparisons confirmed the findings of the microbiological analyses. The SGS samples were evaluated as better compared to air samples on cooked flavour, cooked odour and texture, but got slightly lower scores on raw odour evaluation. Samples packaged with G-100 absorber/emitter gave the best cooked sensory scores, while samples without absorber got the lowest cooked sensory scores. On raw odour the samples packaged with G-100 and without absorbers were evaluated as better than samples with SS-200 and ZPT-100 absorbers. No differences were observed in the colour of the samples, in contrast to the reddish colour change observed when packaging perch and pike perch fillets with the Ageless G-100 CO2-emitters (Ahvenainen et al., 1997). They observed the same shelf-life for fresh perch and pike perch fillets packaged with G-100 as for traditional MAP using an anoxic high CO2 atmosphere, and 2–4 days longer shelf-life when compared with over-wrap or vacuum packaging. However, colour change and a smell of raw liver in the raw fillets in the active packages was observed. This was not observed in the traditional MA-packages. No differences between the two packaging technologies were observed after cooking of the fish. The commercial CO2 emitters usually contain ferrous carbonate and a metal halide catalyst although non-ferrous variants are available, absorbing the O2 and producing equal volumes of CO2. Carbon dioxide could also be produced inside the packages after packaging by allowing the exudates from the product to react with a mixture of sodium carbonate and citric acid inside the drip pad, an approach used successfully for cod fillets (Bjerkeng et al. 1995) increasing shelf-life as compared to traditional MAP, even when using a low gas headspace in the package. The Verifrais package manufactured by Codimer, which has been used for extending the shelf-life of fresh meats and fish, is a similar concept (Day 1998). This package consists of a standard MAP tray but has a perforated false bottom under which a porous sachet containing sodium bicarbonate/ascorbate is positioned. When exudate from packed meat or fish drips onto the sachet, CO2 is emitted and counteracts package collapse due to the CO2 solubility in the food. 18.4 Active packaging: water control Excess moisture is a major cause of food spoilage and different humidity absorbers are used to protect dried products from humidity damage. However, these absorbers have a limited effect on fish products. Several companies manufacture moisture drip absorbent pads, sheets and blankets for liquid water control in watery foods such as meat, fish, poultry, fruit and vegetables. Moisture drip absorber pads or false-bottomed trays are commonly placed under packaged fresh meat, fish, poultry and prepared fruit to absorb unsightly tissue drip discharge. Larger sheets and blankets are used for absorption of melted ice 390 Novel food packaging techniques

Active packaging in practice: fish 391 from chilled seafood during airfreight transportation(Day 1998). Commercial moisture absorber sheets, blankets and trays include Toppan Sheet(Toppan Printing, Japan), Thermarite(Thermarite, Australia) and Fresh-R-Pax(Maxwell Chase, US) An approach to extending shelf-life of chilled fresh fish is to decrease the water activity at the surface. The Showa Denko Co. (Tokyo, Japan) has developed a film(Pichit film), which is in the form of a pillow with entrapped propylene glycol between layers of polyvinyl alcohol(PVA). The PVA-film is very permeable to water but is a barrier to the glycol. When placed in contact for several hours with the surface of meat or fish by wrapping the film around, it absorbs water and causes injury to spoilage bacteria. This technique can increase the shelf-life of ocean fish by 2-4 days Labuza 1993). The action is due to an a, difference between the fish(0.99)and the glycol (0.0); thus the water is rapidly drawn out of the fish surface. This surface dehydration not only inhibits son microbes but also may injure others without causing change in fish quality (Labuza and Breene 1989). It is most likely that some glycol also transfers to the food surface and slows microbial growth. The pichit film has been shown to maintain the colour of tuna, veal, pork and beef (Arakawa et al. 1990), since the colour in these products is related to the myoglobin content in the meat and a dehydration of the surface will lead to increased myoglobin concentration. The effect of the pichit film on the shelf-life of fish has been little exploited but for salmon fillets the effect of 2 and 4 hours of pichit pre-treatment on microbial growth and sensory spoilage was non-existent (Sivertsvik 2000) 18.5 Active packaging: anti-microbial and anti-oxidant applications Some commercial anti-microbial films and materials have been introduced again primarily in Japan. For example, one widely reported product is a synthetic silver zeolite which has been directly incorporated into food contact packaging film. The potential food applications for anti-microbial films include meat, fish, bread, cheese, fruit and vegetables. Several antimicrobial compounds might have potential to be incorporated into package structures to convert them into active packaging: chlorine dioxide, silver salts, bacteriocins ozone, and natural spices such as rosemary and its derivatives(Brody 2001)but few have been investigated to be used in or on packaging material of fish products. One exception is benzoic acid anhydride on PE-film used on fish fillets(Han 2000) One anti-microbial packaging application used commercially for semi-moist and dried fish products in Japan uses ethanol emitters(e.g. Ethicap, Antimold 102 and Negamold(Freund Industrial), Oitech (Nippon Kayaku), ET Pack (Ueno Seiyaku) and Ageless type SE(Mitsubishi Gas Chemical))(Day 1998) These films and sachets contain absorbed ethanol in a carrier material that allows the controlled release of ethanol vapour

from chilled seafood during airfreight transportation (Day 1998). Commercial moisture absorber sheets, blankets and trays include Toppan Sheet (Toppan Printing, Japan), Thermarite (Thermarite, Australia) and Fresh-R-Pax (Maxwell Chase, US). An approach to extending shelf-life of chilled fresh fish is to decrease the water activity at the surface. The Showa Denko Co. (Tokyo, Japan) has developed a film (Pichit film), which is in the form of a pillow with entrapped propylene glycol between layers of polyvinyl alcohol (PVA). The PVA-film is very permeable to water but is a barrier to the glycol. When placed in contact for several hours with the surface of meat or fish by wrapping the film around, it absorbs water and causes injury to spoilage bacteria. This technique can increase the shelf-life of ocean fish by 2–4 days (Labuza 1993). The action is due to an aw difference between the fish (0.99) and the glycol (0.0); thus the water is rapidly drawn out of the fish surface. This surface dehydration not only inhibits some microbes but also may injure others without causing change in fish quality (Labuza and Breene 1989). It is most likely that some glycol also transfers to the food surface and slows microbial growth. The pichit film has been shown to maintain the colour of tuna, veal, pork and beef (Arakawa et al. 1990), since the colour in these products is related to the myoglobin content in the meat and a dehydration of the surface will lead to increased myoglobin concentration. The effect of the pichit film on the shelf-life of fish has been little exploited but for salmon fillets the effect of 2 and 4 hours of pichit pre-treatment on microbial growth and sensory spoilage was non-existent (Sivertsvik 2000). 18.5 Active packaging: anti-microbial and anti-oxidant applications Some commercial anti-microbial films and materials have been introduced, again primarily in Japan. For example, one widely reported product is a synthetic silver zeolite which has been directly incorporated into food contact packaging film. The major potential food applications for anti-microbial films include meat, fish, bread, cheese, fruit and vegetables. Several antimicrobial compounds might have potential to be incorporated into package structures to convert them into active packaging: chlorine dioxide, silver salts, bacteriocins, ozone, and natural spices such as rosemary and its derivatives (Brody 2001) but few have been investigated to be used in or on packaging material of fish products. One exception is benzoic acid anhydride on PE-film used on fish fillets (Han 2000). One anti-microbial packaging application used commercially for semi-moist and dried fish products in Japan uses ethanol emitters (e.g. Ethicap, Antimold 102 and Negamold (Freund Industrial), Oitech (Nippon Kayaku), ET Pack (Ueno Seiyaku) and Ageless type SE (Mitsubishi Gas Chemical)) (Day 1998). These films and sachets contain absorbed ethanol in a carrier material that allows the controlled release of ethanol vapour. Active packaging in practice: fish 391

392 Novel food packaging Essential oils have anti-microbial effects and oils of oregano and cinnamon have the strongest antimicrobial activity, followed by lemongrass, thyme, clove bay, marjoram, sage and basil oils(Mejlholm and Dalgaard 2002). Oregano oil (0.05% v/w) reduced growth of P. phosphoreum, the sso in naturally contaminated mAP cod fillets and extended shelf-life from 11-12 days to 21 26 days at 2C(Mejlholm and Dalgaard 2002). Obviously, essential oils can extend the shelf-life of map seafood but because of the volatile nature of these components incorporating them into an active packaging could be a challenge Another component with potential as an active packaging ingredient for fresh fish is acetate buffer that can extend the shelf-life of MAP packaged cod fillets by spraying it onto the fillets prior to packaging(Boskou and Debevere 2000) Production of total volatile bases and tma was inhibited in treated fillets for ten days storage under modified atmospheres. Inhibition of TMa production could be attributed to growth inhibition of H]S-producing bacteria, inhibition of the TMAO-dependent metabolism of TMAO-reducing bacteria and the stable ph during storage. The shelf-life, at 7C. of treated cod fillets based on cooked flavour score, was almost 12 days, approximately 8 days more than the shelf-life of the control fillets(Boskou and debevere 2000). Potassium sorbate is another preservative shown to increase shelf-life of fish products(Fey and Regenstein 1982; Drosinos et al., 1997) and could be an active ingredient in packaging materials for fishery products Incorporating anti-oxidants, such as vitamin C and E, in packaging film may potentially reduce oxidative reactions such as the development of rancid flavour and odour in fatty fish products. The degradation of texture, flavour, and odour of stored seafood is attributed to the oxidation of unsaturated lipids. Processing operations such as salting, cooking, and mincing promote oxidation while smoking, dehydration, and freezing retard oxidation. The rate and degree of lipid degradation in frozen fish depends upon the fish species and muscle type, dark or white. Lipid oxidation proceeds in the following decreasing order: skin, dark muscle, and white muscle. Lipid oxidation within a given species will vary with season and location within the tissue. Metal ions affect oxidation in the following decreasing order: Fe2+, haemin, Cu2+, and Fe3+ Oxidation can be reduced through the use of single or combined antioxidants however. vacuum packaging has a greater reduction on oxidation than the presence of additives (Flick et al, 1992) Lipid oxidation in fish fillets wrapped with butylated hydroxytoluene(BHT anti-oxidant incorporated PE-film was inhibited as compared to non-wrapped fish fillets(Huang and Weng 1998). The BHT-incorporated PE film was able to inhibit lipid oxidation in both fish muscle and o 18.6 Active packaging: edible coatings and films Edible films can be looked upon as an active packaging technology, and many of the anti-microbials or anti-oxidants mentioned above incorporated in the

Essential oils have anti-microbial effects and oils of oregano and cinnamon have the strongest antimicrobial activity, followed by lemongrass, thyme, clove, bay, marjoram, sage and basil oils (Mejlholm and Dalgaard 2002). Oregano oil (0.05% v/w) reduced growth of P. phosphoreum, the SSO in naturally contaminated MAP cod fillets and extended shelf-life from 11–12 days to 21– 26 days at 2ºC (Mejlholm and Dalgaard 2002). Obviously, essential oils can extend the shelf-life of MAP seafood but because of the volatile nature of these components incorporating them into an active packaging could be a challenge. Another component with potential as an active packaging ingredient for fresh fish is acetate buffer that can extend the shelf-life of MAP packaged cod fillets by spraying it onto the fillets prior to packaging (Boskou and Debevere 2000). Production of total volatile bases and TMA was inhibited in treated fillets for ten days’ storage under modified atmospheres. Inhibition of TMA production could be attributed to growth inhibition of H2S-producing bacteria, inhibition of the TMAO-dependent metabolism of TMAO-reducing bacteria and the stable pH during storage. The shelf-life, at 7ºC, of treated cod fillets, based on cooked flavour score, was almost 12 days, approximately 8 days more than the shelf-life of the control fillets (Boskou and Debevere 2000). Potassium sorbate is another preservative shown to increase shelf-life of fish products (Fey and Regenstein 1982; Drosinos et al., 1997) and could be an active ingredient in packaging materials for fishery products. Incorporating anti-oxidants, such as vitamin C and E, in packaging film may potentially reduce oxidative reactions such as the development of rancid flavour and odour in fatty fish products. The degradation of texture, flavour, and odour of stored seafood is attributed to the oxidation of unsaturated lipids. Processing operations such as salting, cooking, and mincing promote oxidation while smoking, dehydration, and freezing retard oxidation. The rate and degree of lipid degradation in frozen fish depends upon the fish species and muscle type, dark or white. Lipid oxidation proceeds in the following decreasing order: skin, dark muscle, and white muscle. Lipid oxidation within a given species will vary with season and location within the tissue. Metal ions affect oxidation in the following decreasing order: Fe2+, haemin, Cu2+, and Fe3+. Oxidation can be reduced through the use of single or combined antioxidants, however, vacuum packaging has a greater reduction on oxidation than the presence of additives (Flick et al., 1992). Lipid oxidation in fish fillets wrapped with butylated hydroxytoluene (BHT) anti-oxidant incorporated PE-film was inhibited as compared to non-wrapped fish fillets (Huang and Weng 1998). The BHT-incorporated PE film was able to inhibit lipid oxidation in both fish muscle and oil. 18.6 Active packaging: edible coatings and films Edible films can be looked upon as an active packaging technology, and many of the anti-microbials or anti-oxidants mentioned above incorporated in the 392 Novel food packaging techniques

Active packaging in practice: fish 393 packaging material could as well be incorporated in an edible film meant to be eaten together with the product. An edible film might be a better approach to ensure good contact between the active component and the food Different edible coatings have been developed to be used on fish products Methyl cellulose and hydroxypropyl cellulose reduce uptake of fat during frying, important to the preparation of many seafood products. Alginates reduce moisture loss from fresh fish, while palmitates reduce moisture loss from frozen fish. Other edible films demonstrated to reduce moisture transfer, especially out of frozen fish, include whey protein isolates, coconut oil, and acetylated mono diglycerides The chitosan coating of fresh fillets of cod( Gadus morhua) and herring (Clupea harengus)reduced moisture losses, lipid oxidation, headspace volatiles (total volatile basic nitrogen, TMA, and hypoxanthine), and growth of microorganisms as compared to uncoated samples. The preservative efficacy and the viscosity of chitosan were interrelated, the efficacy of chitosans with higher viscosities superior to that of lower viscosity. Thus, chitosan as an edible coating could enhance the quality of seafoods during storage (Jeon et al., 2002) Skinless tilapia( Oreochromis niloticus x D. aureus) fillets were covered with a gelatin coating containing benzoic acid as an antimicrobial agent. After seven days of storage under refrigeration, tilapia fillets coated with gelatin containing benzoic acid had acceptable VBn contents, increased moderately in microbial loads, and showed no significant sensory difference(P<0.05)from fresh fillets The results indicate that an antimicrobial gelatin coating is suitable for preservation of tilapia fillets(Ou et al, 2002). Glucose oxidase in a alginate coating extended shelf-life of winter flounder as compared to coating without enzyme (field et al, 1986) 18.7 Active packaging: taint removal During storage of packaged fish microbial metabolites and protein breakdown products, such as amines and aldehydes, accumulate in the head-space of the package, leading to, for example, putrid(H2S) and fishy (TMA)odours Removal of these components therefore often enhance the initial perception of the products upon pening, and also to some degree increase sensory shelf-life The effectiveness of an innovative foam plastic tray provided with absorbers for volatile amines and liquids on the shelf- life of different fish products packed under a modified atmosphere(40%CO2: 60%N2), was evaluated in comparison with a standard tray( Franzetti et al., 2001) Fillets of sole Solea solea), steaks of hake(Merluccius merluccius)and whole cuttlefish(Sepia fillouxi), placed in the two different kinds of tray, were kept at 3C. The novel packaging associated with a rigorous control of storage temperature, increased the shelf-life up to ten days. In fact, the innovative tray sequestrated the greater part of TMa from the headspace, and led to delayed microbial growth, especially of Gram-negative

packaging material could as well be incorporated in an edible film meant to be eaten together with the product. An edible film might be a better approach to ensure good contact between the active component and the food. Different edible coatings have been developed to be used on fish products. Methyl cellulose and hydroxypropyl cellulose reduce uptake of fat during frying, important to the preparation of many seafood products. Alginates reduce moisture loss from fresh fish, while palmitates reduce moisture loss from frozen fish. Other edible films demonstrated to reduce moisture transfer, especially out of frozen fish, include whey protein isolates, coconut oil, and acetylated mono and diglycerides (Brody 2001). The chitosan coating of fresh fillets of cod (Gadus morhua) and herring (Clupea harengus) reduced moisture losses, lipid oxidation, headspace volatiles (total volatile basic nitrogen, TMA, and hypoxanthine), and growth of microorganisms as compared to uncoated samples. The preservative efficacy and the viscosity of chitosan were interrelated, the efficacy of chitosans with higher viscosities superior to that of lower viscosity. Thus, chitosan as an edible coating could enhance the quality of seafoods during storage (Jeon et al., 2002). Skinless tilapia (Dreochromis niloticus x D. aureus) fillets were covered with a gelatin coating containing benzoic acid as an antimicrobial agent. After seven days of storage under refrigeration, tilapia fillets coated with gelatin containing benzoic acid had acceptable VBN contents, increased moderately in microbial loads, and showed no significant sensory difference (P < 0.05) from fresh fillets. The results indicate that an antimicrobial gelatin coating is suitable for preservation of tilapia fillets (Ou et al., 2002). Glucose oxidase in a alginate coating extended shelf-life of winter flounder as compared to coating without enzyme (Field et al., 1986). 18.7 Active packaging: taint removal During storage of packaged fish microbial metabolites and protein breakdown products, such as amines and aldehydes, accumulate in the head-space of the package, leading to, for example, putrid (H2S) and fishy (TMA) odours. Removal of these components would therefore often enhance the initial perception of the products upon package opening, and also to some degree increase sensory shelf-life. The effectiveness of an innovative foam plastic tray provided with absorbers for volatile amines and liquids on the shelf- life of different fish products packed under a modified atmosphere (40%CO2:60%N2), was evaluated in comparison with a standard tray (Franzetti et al., 2001). Fillets of sole (Solea solea), steaks of hake (Merluccius merluccius) and whole cuttlefish (Sepia fillouxi), placed in the two different kinds of tray, were kept at 3ºC. The novel packaging associated with a rigorous control of storage temperature, increased the shelf-life up to ten days. In fact, the innovative tray sequestrated the greater part of TMA from the headspace, and led to delayed microbial growth, especially of Gram-negative Active packaging in practice: fish 393

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