《食品包装技术》（英文版）Chapter 7 The use of freshness indicators in packaging.pdf_大学文库

The use of freshness indicators in packaging 129 7.2.1 Glucose Glucose is an initial substrate for many spoilage bacteria in air,vacuum packages and modified atmosphere packages (Dainty, 996) growth takes place glucose is depleted from meat surface and it has been proposed by Kress-Rogers(1993)that the measurement of the glucose gradient could be utilised to predict the remaining shelf-life. However glucose is not among the most promising quality-indicating compounds since the concentration decreases during storage and it would be more beneficial to have a quality-indicating compound with non-existent or low intitial concentration 7. 2.2 Organic acids Organic acids like lactic acid and acetic acid are the major compounds having a role in glucose fermentation by lactic acid bacteria. The amount of L-lactic acid has generally been reported to decrease during storage of fish and meat(Kakouri et al., 1997; Drosinos and Nychas, 1997; Nychas et al., 1998). On the contrary, the concentration of D-lactate has been reported to increase during storage of meat and therefore D-lactate seems to be a more promising freshness indicator ( Shu et al, 1993) Acetate concentrations have been reported to increase during storage of fresh fish(Kakouri et al, 1997). In our studies with modified atmosphere packaged poultry meat(Smolander et al. in preparation) we have also found that the concentration of acetic acid in the tissue fluid and homogenised meat increased as a function of storage time and temperature. We also studied the formation of formic acid. At the beginning of storage some decrease in the concentration in meat was seen but later the concentration increased however the increase was not as clearly dependent on storage temperature as in the case of acetic acid 7.2.3 Ethanol In addition to lactic and acetic acid, ethanol is another major end product of fermentative metabolism of lactic acid bacteria. It has been postulated that an increase in ethanol concentration in meat and fish indicates an increase of total viable count of the product. For instance, Rehbein(1993)studied the formation of ethanol in iced fish. He found that on an average the concentration of ethanol average of 1. 77 mg ethanol was found in 100 g fish. Rehbein(1993)also studied the formation of ethanol in smoked, vacuum-packed salmon. The concentrations in stored fish samples were considerably higher than in fresh, iced fish. At the end of shelf-life concentrations as high as 10 mg/100 g fish were observed Randell et al.(1995)studied the effect of storage time and package integrity on the formation of ethanol in modified atmosphere packaged, marinated rainbow trout slices. They found that the amount of ethanol in package headspace was increased together with storage time and size of the package leakage. In our unpublished studies analogous to Randell et al.(1995) we found also that

7.2.1 Glucose Glucose is an initial substrate for many spoilage bacteria in air, vacuum packages and modified atmosphere packages (Dainty, 1996). As bacterial growth takes place glucose is depleted from meat surface and it has been proposed by Kress-Rogers (1993) that the measurement of the glucose gradient could be utilised to predict the remaining shelf-life. However glucose is not among the most promising quality-indicating compounds since the concentration decreases during storage and it would be more beneficial to have a quality-indicating compound with non-existent or low intitial concentration. 7.2.2 Organic acids Organic acids like lactic acid and acetic acid are the major compounds having a role in glucose fermentation by lactic acid bacteria. The amount of L-lactic acid has generally been reported to decrease during storage of fish and meat (Kakouri et al., 1997; Drosinos and Nychas, 1997; Nychas et al., 1998). On the contrary, the concentration of D-lactate has been reported to increase during storage of meat and therefore D-lactate seems to be a more promising freshness indicator (Shu et al., 1993). Acetate concentrations have been reported to increase during storage of fresh fish (Kakouri et al., 1997). In our studies with modified atmosphere packaged poultry meat (Smolander et al. in preparation) we have also found that the concentration of acetic acid in the tissue fluid and homogenised meat increased as a function of storage time and temperature. We also studied the formation of formic acid. At the beginning of storage some decrease in the concentration in meat was seen but later the concentration increased, however the increase was not as clearly dependent on storage temperature as in the case of acetic acid. 7.2.3 Ethanol In addition to lactic and acetic acid, ethanol is another major end product of fermentative metabolism of lactic acid bacteria. It has been postulated that an increase in ethanol concentration in meat and fish indicates an increase of total viable count of the product. For instance, Rehbein (1993) studied the formation of ethanol in iced fish. He found that on an average the concentration of ethanol was increased as a function of storage time. At sensory rejection point an average of 1.77 mg ethanol was found in 100 g fish. Rehbein (1993) also studied the formation of ethanol in smoked, vacuum-packed salmon. The concentrations in stored fish samples were considerably higher than in fresh, iced fish. At the end of shelf-life concentrations as high as 10 mg/100 g fish were observed. Randell et al. (1995) studied the effect of storage time and package integrity on the formation of ethanol in modified atmosphere packaged, marinated rainbow trout slices. They found that the amount of ethanol in package headspace was increased together with storage time and size of the package leakage. In our unpublished studies analogous to Randell et al. (1995) we found also that The use of freshness indicators in packaging 129

130 Novel food packaging techniques storage temperature had a remarkable effect on the ethanol concentration. The higher the storage temperature was, the higher was the concentration of ethanol Randell et al(1995)also studied the volatile compounds in packages containing marinated chicken pieces in modified atmosphere (40%CO 60%N2). They found that ethanol concentration in the package headspace increased as a function of storage time In our own unpublished studies we also found some correspondence between sensory quality of modified atmosphere (40%CO2+ 60%N2) packaged, unmarinated poultry meat and ethanol concentration in the package headspace. However, in unmarinated poultry meat packaged in MAP with higher(80%)CO2 concentration we did not observe a clear trend in the formation of ethanol as a function of storage time and temperature 7. 2. 4 Volatile nitrogen c It is well known that high levels of basic volatile nitrogen compounds like ammonia,dimethylamine and trimethylamine give an indication about microbiological spoilage of fish(Ohlenschlager, 1997). The European Commission has even fixed TVB-N(total volatile basic nitrogen contributed by ammonia, dimethylamine and trimethy lamine) limits for some fish species (95/149/EEC). Trimethylamine, formed by microbial actions in fish muscle is generally considered as a major metabolite responsible for the spoilage odours of seafood. a drawback of using trimethylamine as a quality indicator for seafood is the variation in the concentration of its precursor trimethylamine N oxide according to the species and season(Dainty, 1996; Rodriguez et al. 1999) 7.2.5 Biogenic amines Biogenic amines (e.g. tyramine, cadaverine, putrescine, histamine)are specially widely considered as indicators of hygienic quality of meat products In addition to this indicative nature, they can have pharmacological physiological and toxic effects. Due to the health risks, a tolerance level of 100 mg/kg of fish has been established for histamine by FDA(Kaniou, et al 2001). Even if biogenic amines indicate the quality of food products they do not themself contribute to the sensory quality of the product Putrescine and cadaverine are formed from ornithine and lysine, respectively n enzymatic decarboxylation. It has been widely suggested that these diamines are indicators of the initial stage of decomposition of meat products. Okuma et al. (2000)describe an increase in the diamine concentration together with the increase of the total viable counts in aerobically stored chicken. Kaniou et al (2001)reported the formation of putrescine and cadaverine in unpacked beef. In addition to putrescine and cadaverine, histamine was also produced during the torage of vacuum-packed beef. Also in our studies we have found a clear correspondence between the microbiological quality of modified atmosphere

storage temperature had a remarkable effect on the ethanol concentration. The higher the storage temperature was, the higher was the concentration of ethanol. Randell et al. (1995) also studied the volatile compounds in packages containing marinated chicken pieces in modified atmosphere (40%CO2 + 60%N2). They found that ethanol concentration in the package headspace increased as a function of storage time. In our own unpublished studies we also found some correspondence between sensory quality of modified atmosphere (40%CO2 + 60%N2) packaged, unmarinated poultry meat and ethanol concentration in the package headspace. However, in unmarinated poultry meat packaged in MAP with higher (80%) CO2 concentration we did not observe a clear trend in the formation of ethanol as a function of storage time and temperature. 7.2.4 Volatile nitrogen compounds It is well known that high levels of basic volatile nitrogen compounds like ammonia, dimethylamine and trimethylamine give an indication about microbiological spoilage of fish (Ohlenschla¨ger, 1997). The European Commission has even fixed TVB-N (total volatile basic nitrogen contributed by ammonia, dimethylamine and trimethylamine) limits for some fish species (95/149/EEC). Trimethylamine, formed by microbial actions in fish muscle is generally considered as a major metabolite responsible for the spoilage odours of seafood. A drawback of using trimethylamine as a quality indicator for seafood is the variation in the concentration of its precursor trimethylamine Noxide according to the species and season (Dainty, 1996; Rodrı´guez et al., 1999). 7.2.5 Biogenic amines Biogenic amines (e.g. tyramine, cadaverine, putrescine, histamine) are especially widely considered as indicators of hygienic quality of meat products. In addition to this indicative nature, they can have pharmacological, physiological and toxic effects. Due to the health risks, a tolerance level of 100 mg/kg of fish has been established for histamine by FDA (Kaniou, et al. 2001). Even if biogenic amines indicate the quality of food products they do not themself contribute to the sensory quality of the product. Putrescine and cadaverine are formed from ornithine and lysine, respectively in enzymatic decarboxylation. It has been widely suggested that these diamines are indicators of the initial stage of decomposition of meat products. Okuma et al. (2000) describe an increase in the diamine concentration together with the increase of the total viable counts in aerobically stored chicken. Kaniou et al. (2001) reported the formation of putrescine and cadaverine in unpacked beef. In addition to putrescine and cadaverine, histamine was also produced during the storage of vacuum-packed beef. Also in our studies we have found a clear correspondence between the microbiological quality of modified atmosphere 130 Novel food packaging techniques

The use of freshness indicators in packaging 131 packaged poultry and the total amount of biogenic amines. Tyramine formation took place evenly during the storage period, the formation being however dependent on temperature. Storage temperature had a more striking effect on the formation of putrescine and cadaverine which were accumulated especially at the end of storage period(Rokka et al, submitted) Ruiz-Capillas and moral(2002)studied the effect of controlled and modified atmosphere on the production of biogenic amines during storage of hake. They found that controlled and modified atmosphere enera formation of biogenic amines and that cadaverine was a major biogenic amine formed in most of the studied atmospheres. Rodriguez et al.(1999) proposed that biogenic amines cadaverine and putrescine could indicate the freshness of freshwater rainbow trout either stored in air or in a vacuum package 2.6 Carbon dioxide Carbon dioxide(CO2) is generally known to be produced during microbial growth. COz is also typically added as a protecting gas to modified atmosphere packages, together with inert nitrogen, since it has bacteriostatic effects. A modified atmosphere package for non-respiring food typically has a COz concentration as high as 20-80 and, e.g., Fu et al. (1992)reported a further increase of CO2 during storage in modified atmosphere packages containing beef. Even if the indication of microbial growth by CO2 may be difficult in these modified atmosphere packages already containing a high concentration of CO2, it is possible to use the increase in CO2 concentration as a means of determining microbial contamination in other types of product. For instance Mattila et al. (1990)found a correlation between CO2 concentration and the growth of microbes in pea and tomato soup which were packaged aseptically either in air or in a mixture of O2(5%)and nitrogen 7.2.7 ATP degradation products K-value is defined as the ratio of the sum of hypoxanthine and inosine and the total concentration of ATP-related compounds(Henehan et al., 1997). This value, indicating the extent of ATP-degradation, correlates with the sensory quality of fish and also other types of meat and has been used as a freshness indicating parameter(Watanabe et al. 1989, Yano et al., 1995a). For fresh meat the value is low since the concentration of ATP-degradation products is low as compared to the concentration of all ATP-related compounds. The correlation between ATP-degradation products and fish quality has been extensively studied, e.g., by Hattula(1997 7.2.8 Sulphuric compoun Some sulphuric compounds have a remarkable effect on the sensory quality of meat products due to their typical odour and low odour threshold. Hydroger

packaged poultry and the total amount of biogenic amines. Tyramine formation took place evenly during the storage period, the formation being however dependent on temperature. Storage temperature had a more striking effect on the formation of putrescine and cadaverine which were accumulated especially at the end of storage period (Rokka et al., submitted). Ruiz-Capillas and Moral (2002) studied the effect of controlled and modified atmosphere on the production of biogenic amines during storage of hake. They found that controlled and modified atmosphere generally restricted the formation of biogenic amines and that cadaverine was a major biogenic amine formed in most of the studied atmospheres. Rodrı´guez et al. (1999) proposed that biogenic amines cadaverine and putrescine could indicate the freshness of freshwater rainbow trout either stored in air or in a vacuum package. 7.2.6 Carbon dioxide Carbon dioxide (CO2) is generally known to be produced during microbial growth. CO2 is also typically added as a protecting gas to modified atmosphere packages, together with inert nitrogen, since it has bacteriostatic effects. A modified atmosphere package for non-respiring food typically has a CO2 concentration as high as 20–80 % and, e.g., Fu et al. (1992) reported a further increase of CO2 during storage in modified atmosphere packages containing beef. Even if the indication of microbial growth by CO2 may be difficult in these modified atmosphere packages already containing a high concentration of CO2, it is possible to use the increase in CO2 concentration as a means of determining microbial contamination in other types of product. For instance Mattila et al. (1990) found a correlation between CO2 concentration and the growth of microbes in pea and tomato soup which were packaged aseptically either in air or in a mixture of O2 (5%) and nitrogen. 7.2.7 ATP degradation products K-value is defined as the ratio of the sum of hypoxanthine and inosine and the total concentration of ATP-related compounds (Henehan et al., 1997). This value, indicating the extent of ATP-degradation, correlates with the sensory quality of fish and also other types of meat and has been used as a freshness indicating parameter (Watanabe et al., 1989; Yano et al., 1995a). For fresh meat the value is low since the concentration of ATP-degradation products is low as compared to the concentration of all ATP-related compounds. The correlation between ATP-degradation products and fish quality has been extensively studied, e.g., by Hattula (1997). 7.2.8 Sulphuric compounds Some sulphuric compounds have a remarkable effect on the sensory quality of meat products due to their typical odour and low odour threshold. Hydrogen The use of freshness indicators in packaging 131

132 Novel food packaging techniques sulphide(H2s)is produced from cysteine and triggered by glucose limitation (Borch et al, 1996). H2S forms a green pigment, sulphmyoglobin, when it is bound to myoglobin(Paine and Paine, 1992, Egan et al, 1989). However, ulphmyoglobin is not formed in anaerobic conditions H2s and other sulphuric compounds have been found to be produced during the spoilage of poultry by pseudomonas, Alteromonas sp. and psycrotrophic anaerobic clostridia( Freeman et al, 1976; Lea et al, 1969 Russell et al, 1997 Vieshweg et al, 1989; Arnaut-Rollier et al, 1999; Kalinowski and Tompkin, 1999). According to Dainty(1996), production of H2s can be used as an indication of Enterobacteriacae and hence also of hygienic problems in aerobically stored meat. H2s production by Alteromonas putrefaciens Enterobacter liquefaciens and pseudomonas was discovered in high ultimate pH beef from stressed animals(Gill and Newton, 1979, Nicol et al., 1970). It has also been found that in vacuum packed meat His indicates the growth of particular strains of lactic acid bacteria(Egan et al., 1989). Also in fish the volatile sulphuric compounds have been suggested as the main cause of putrid spoilage aromas(Olafsdottir and Fleurence, 1997) 7.3 Freshness indicators a variety of different concepts for freshness indicators have been presented the scientific literature. Most of these concepts are based on a colour change of the indicator tag due to the presence of microbial metabolites produced during spoilage(e.g Smolander et al, 2002; Wallach and Novikov, 1998; Kahn, 1996 Namiki, 1996), but also concepts for indicators relying on more advanced technology have been presented. For instance, a miniaturised gas detector based on conducting polymers has been patented by Aromascan, a manufacturer of electronic nose equipment(Payne and Persaud, 1995). Fibre optics can also be used to construct indicators for the volatile compounds produced in microbial spoilage(Honeybourne, 1993, Wolfbeis and List, 1995). Freshness indicator or detector concepts have been proposed, for example, for CO2, diacetyl, amines, ammonia and hydrogen sulphide(see Table 7. 1). These concepts are discussed in detail in following chapters 7.3.1 Indicators sensitive to pH change he majority of concepts described in the literature are based on the use of dyes, which change colour in the presence of volatile compounds produced during spoilage. As early as the 1940s Clark (1949)filed a patent application describing ' an indicator which exhibits an irreversible change in visual appearance upon an appreciable multiplication of bacteria in the indicator The idea was that if microbiological growth inducing a pH change has been possible in the indicator, the conditions might have been such that the food product itself may also have been subject to deterioration. A direct

sulphide (H2S) is produced from cysteine and triggered by glucose limitation (Borch et al., 1996). H2S forms a green pigment, sulphmyoglobin, when it is bound to myoglobin (Paine and Paine, 1992, Egan et al., 1989). However, sulphmyoglobin is not formed in anaerobic conditions. H2S and other sulphuric compounds have been found to be produced during the spoilage of poultry by pseudomonas, Alteromonas sp. and psycrotrophic anaerobic clostridia (Freeman et al., 1976; Lea et al., 1969; Russell et al., 1997, Vieshweg et al., 1989; Arnaut-Rollier et al., 1999; Kalinowski and Tompkin, 1999). According to Dainty (1996), production of H2S can be used as an indication of Enterobacteriacae and hence also of hygienic problems in aerobically stored meat. H2S production by Alteromonas putrefaciens, Enterobacter liquefaciens and pseudomonas was discovered in high ultimate pH beef from stressed animals (Gill and Newton, 1979; Nicol et al., 1970). It has also been found that in vacuum packed meat H2S indicates the growth of particular strains of lactic acid bacteria (Egan et al., 1989). Also in fish the volatile sulphuric compounds have been suggested as the main cause of putrid spoilage aromas (Olafsdottir and Fleurence, 1997). 7.3 Freshness indicators A variety of different concepts for freshness indicators have been presented in the scientific literature. Most of these concepts are based on a colour change of the indicator tag due to the presence of microbial metabolites produced during spoilage (e.g. Smolander et al., 2002; Wallach and Novikov, 1998; Kahn, 1996; Namiki, 1996), but also concepts for indicators relying on more advanced technology have been presented. For instance, a miniaturised gas detector based on conducting polymers has been patented by Aromascan, a manufacturer of electronic nose equipment (Payne and Persaud, 1995). Fibre optics can also be used to construct indicators for the volatile compounds produced in microbial spoilage (Honeybourne, 1993; Wolfbeis and List, 1995). Freshness indicator or detector concepts have been proposed, for example, for CO2, diacetyl, amines, ammonia and hydrogen sulphide (see Table 7.1). These concepts are discussed in detail in following chapters. 7.3.1 Indicators sensitive to pH change The majority of concepts described in the literature are based on the use of pHdyes, which change colour in the presence of volatile compounds produced during spoilage. As early as the 1940s Clark (1949) filed a patent application describing ‘an indicator which exhibits an irreversible change in visual appearance upon an appreciable multiplication of bacteria in the indicator’. The idea was that if microbiological growth inducing a pH change has been possible in the indicator, the conditions might have been such that the food product itself may also have been subject to deterioration. A direct 132 Novel food packaging techniques

134 Novel food packaging techniques Table 7.1 (continued Author/ Patent applicant or Principle of the indicator holder/Trade name Namiki(1996) Micro- Degradation of lipid embrane by micro- Persaud, 1995) component with electrical perties affected by volatile mpound associated wi Path dicators Food Sentinel System(Sira arIous Bar code detector comprising Technologies); Goldsmi microbial toxins a toxin printed onto a (1994) substrate and indicator colour eversibly bound to the toxin, in the presence of toxin the bar code is illegible Toxin Guard(Toxin Alert): Various Formation of a coloured Bodenhamer(2000) pattern when a target analyte is first bound to labelled tibody and subsequently Lawrence berkeley National E. coli 0157 Colour change Laboratory( Quan and enterotoxin lydiacetylene-based Stevens, 1998) determination of COz from the microbiologically spoiling product itself with a pH-dye(e.g. fuchsine acid) based indicator was proposed by Lawdermilt(1962) The use of the pH-dye bromothymol blue as an indicator for the formation CO2 by microbial growth has been suggested in many studies(Holte, 1993 Mattila et al, 1990). As mentioned before, an se in o, can be used to determine microbial contamination in certain types of products. On the other hand, Balderson and Whitwood(1994)proposed a CO2-sensitive packaging indicator for correct filling and subsequent opening of the package. In addition to the detection of spoilage and package filling, pH-dyes reacting to the presence of CO2 have also been used to construct intelligent packaging ndicating the ripeness of traditional fermented vegetable foods (kimchi)(hong and Park, 2000) In addition to the most frequently used pH-dye bromothymol blue, many other reagents e.g. xylenol blue, bromocresol purple, bromocresol green, cresol red phenol red, methyl red and alizarin, among others, have been proposed for the same purpose(Horan 2000). Besides COz, other metabolites like SO2, NH4, volatile amines and organic acids have been proposed to be suitable target molecules for H-sensitive indicators(Mattila and Auvinen, 1990a, b: Horan, 2000)

determination of CO2 from the microbiologically spoiling product itself with a pH-dye (e.g. fuchsine acid) based indicator was proposed by Lawdermilt (1962). The use of the pH-dye bromothymol blue as an indicator for the formation of CO2 by microbial growth has been suggested in many studies (Holte, 1993; Mattila et al., 1990). As mentioned before, an increase in CO2 can be used to determine microbial contamination in certain types of products. On the other hand, Balderson and Whitwood (1994) proposed a CO2-sensitive packaging indicator for correct filling and subsequent opening of the package. In addition to the detection of spoilage and package filling, pH-dyes reacting to the presence of CO2 have also been used to construct intelligent packaging concepts indicating the ripeness of traditional fermented vegetable foods in Korea (kimchi) (Hong and Park, 2000). In addition to the most frequently used pH-dye bromothymol blue, many other reagents e.g. xylenol blue, bromocresol purple, bromocresol green, cresol red, phenol red, methyl red and alizarin, among others, have been proposed for the same purpose (Horan 2000). Besides CO2, other metabolites like SO2, NH4, volatile amines and organic acids have been proposed to be suitable target molecules for pH-sensitive indicators (Mattila and Auvinen, 1990a, b; Horan, 2000). Table 7.1 (continued) Author/ Patent applicant or holder/Trade name Metabolite detected Principle of the indicator Namiki (1996) Microorganisms Degradation of lipid membrane by microorganisms and subsequent diffusion of coloured compound Aromascan (Payne and Persaud, 1995) Not specified Miniaturised electronic component with electrical properties affected by volatile compound associated with spoilage Pathogen indicators Food Sentinel System (Sira Technologies); Goldsmith (1994) Various microbial toxins Bar code detector comprising a toxin printed onto a substrate and indicator colour irreversibly bound to the toxin, in the presence of toxin the bar code is illegible Toxin Guard (Toxin Alert); Bodenhamer (2000) Various pathogens Formation of a coloured pattern when a target analyte is first bound to labelled antibody and subsequently to capture antibody Lawrence Berkeley National Laboratory (Quan and Stevens, 1998) E. coli 0157 enterotoxin Colour change of polydiacetylene-based polymer 134 Novel food packaging techniques

The use of freshness indicators in packaging 135 7.3.2 Indicators sensitive to volatile nitrogen compound A concept reacting to volatile amines with a colour change, hence indicating freshness of seafood, has been proposed by Miller et al.(1999). This concept was marketed by COX Recorders(USA)with the trade name FreshTag". The concept consisted of a plastic chip incorporating a reagent-containing wick. As the label was attached to the package the sharp barb on the back of the label penetrated the packaging film thus enabling contact between the package headspace gases and the reagent. As volatile amines passed through the wick a bright pink colour was developed along the wick Loughran and Diamond(2000) propose a simple method for the determination of volatile nitrogen compounds (NH3, DMA, TMA)with the aid of chromogenic dye calix[ 4 ]arene which has been impregnated on a paper disc. They show that the reflectance spectrum of the indicator disc is changed due to the volatile compounds emitted from cod stored on ice. They also propose that the system could form a base for an intelligent packaging concept 7.3.3 Indicators sensitive to hydrogen sulphide In our own studies we have utilised a reaction between hydrogen sulphide and myoglobin in a freshness indicator for the quality control of modified- atmosphere-packed poultry meat(Ahvenainen et al, 1997, Smolander et al 2002). Freshness indication is based on the colour change of myoglobin by hydrogen sulphide(H]S), which is produced in considerable amounts during the ageing of packaged poultry during storage. The indicators were prepared by applying commercial myoglobin dissolved in a sodium phosphate buffer on small squares of agarose. These indicators were tested in the quality control of MA-packaged fresh, unmarinated broiler cuts. It was found that the colour ange of the myoglobin-based indicators corresponded with the deterioration of the product quality, hence it could be concluded that the myoglobin-based indicators seem to be promising for the quality control of packaged poult 7.3.4 Indicators sensitive to miscellaneous microbial metabolites Cameron and Talasila (1995)have explored the potential of detecting the unacceptability of packaged, respiring products by measuring ethanol in the package headspace with the aid of alcohol oxidase, peroxidase and a chromogenic substrate. Honeybourne and co-workers(Honeybourne, 1993 Shiers and Honeybourne, 1993)have developed a diamine dye-based sensor system responding to the presence of diacetyl vapour. Diacetyl is a volatil compound evolving from meat as spoilage takes place and Honeybourne and workers have proposed that the dye could be printed onto the surface of a gas permeable meat package Diacetyl migrating through the packaging material would react with the dye and induce a colour change

7.3.2 Indicators sensitive to volatile nitrogen compounds A concept reacting to volatile amines with a colour change, hence indicating freshness of seafood, has been proposed by Miller et al. (1999). This concept was marketed by COX Recorders (USA) with the trade name FreshTagÕ. The concept consisted of a plastic chip incorporating a reagent-containing wick. As the label was attached to the package the sharp barb on the back of the label penetrated the packaging film thus enabling contact between the package headspace gases and the reagent. As volatile amines passed through the wick a bright pink colour was developed along the wick. Loughran and Diamond (2000) propose a simple method for the determination of volatile nitrogen compounds (NH3, DMA, TMA) with the aid of chromogenic dye calix[4]arene which has been impregnated on a paper disc. They show that the reflectance spectrum of the indicator disc is changed due to the volatile compounds emitted from cod stored on ice. They also propose that the system could form a base for an intelligent packaging concept. 7.3.3 Indicators sensitive to hydrogen sulphide In our own studies we have utilised a reaction between hydrogen sulphide and myoglobin in a freshness indicator for the quality control of modifiedatmosphere-packed poultry meat (Ahvenainen et al., 1997; Smolander et al., 2002). Freshness indication is based on the colour change of myoglobin by hydrogen sulphide (H2S), which is produced in considerable amounts during the ageing of packaged poultry during storage. The indicators were prepared by applying commercial myoglobin dissolved in a sodium phosphate buffer on small squares of agarose. These indicators were tested in the quality control of MA-packaged fresh, unmarinated broiler cuts. It was found that the colour change of the myoglobin-based indicators corresponded with the deterioration of the product quality, hence it could be concluded that the myoglobin-based indicators seem to be promising for the quality control of packaged poultry products. 7.3.4 Indicators sensitive to miscellaneous microbial metabolites Cameron and Talasila (1995) have explored the potential of detecting the unacceptability of packaged, respiring products by measuring ethanol in the package headspace with the aid of alcohol oxidase, peroxidase and a chromogenic substrate. Honeybourne and co-workers (Honeybourne, 1993; Shiers and Honeybourne, 1993) have developed a diamine dye-based sensor system responding to the presence of diacetyl vapour. Diacetyl is a volatile compound evolving from meat as spoilage takes place and Honeybourne and coworkers have proposed that the dye could be printed onto the surface of a gas permeable meat package. Diacetyl migrating through the packaging material would react with the dye and induce a colour change. The use of freshness indicators in packaging 135

136 Novel food packaging techniques 7.3.5 Other principles for freshness indicating systems In addition to indicators based on reactions caused by microbial metabolites other concepts for contamination indicators have been proposed. DeCicco and Keeven(1995)describe an indicator based on a colour change of chromogenic substrates of enzymes produced by contaminating microbes. The indicator can be applied for contamination detection in liquid health care products Besides the products of microbial metabolism, the consumption of certain nutrients could also be used as a measure of freshness. Kress-Rogers(1993) has developed a knife-type freshness probe for meat. Freshness detection is based on the formation of a glucose gradient as glucose from the surface of meat is consumed during microbial growth and the bulk concentration of glucose remains stable. The detection of microorganisms proposed by Namiki(1996). The principle of the indicator is the degradation of lipid membrane by microorganisms and subsequent diffusion of coloured compound 7. 4 Pathogen indicators In addition to the above-mentioned indicators reacting to the normal spoilage of food products, systems for the detection of a certain contaminant in the food product have also been presented. Commercially available Toxin Guard by Toxin Alert Inc. (Ontario, Canada) is a system to buile polyethylene-based packaging material, which is able to detect the presence of pathogenic bacteria Salmonella, Campylobacter, Escherichia coli o157 and Listeria) with the aid of immobilised antibodies. As the analyte(toxin, micro- organism)is in contact with the material it will be bound first to a specific, belled antibody and then to a capturing antibody printed as a certain pattern (Bodenhamer, 2000). The method could also be applied for the detection of pesticide residues or proteins resulting from genetic modifications. Another commercial system for the detection of specific microorganisms like Salmonella sp, Listeria sp. and E. coli is Food Sentinel SystemM.This system is also based on immunochemical reaction, the reaction taking place in a bar code( Goldsmith, 1994). If the particular microorganism is present the bar code is converted unreadable Specific indicator material for the detection of Escherichia coli o157 enterotoxin has been developed at Lawrence Berkeley National Laboratory Kahn, 1996: Quan and Stevens, 1998). This sensor material, which can be incorporated in the packaging material, is composed of cross-polymerised polydiacetylene molecules and has a deep blue colour. The molecules pecifically binding the toxin are trapped in this polydiacetylene matrix and as the toxin is bound to the film the colour of the film changes from blue to red

7.3.5 Other principles for freshness indicating systems In addition to indicators based on reactions caused by microbial metabolites, other concepts for contamination indicators have been proposed. DeCicco and Keeven (1995) describe an indicator based on a colour change of chromogenic substrates of enzymes produced by contaminating microbes. The indicator can be applied for contamination detection in liquid health care products. Besides the products of microbial metabolism, the consumption of certain nutrients could also be used as a measure of freshness. Kress-Rogers (1993) has developed a knife-type freshness probe for meat. Freshness detection is based on the formation of a glucose gradient as glucose from the surface of meat is consumed during microbial growth and the bulk concentration of glucose remains stable. The detection of microorganisms as such was proposed by Namiki (1996). The principle of the indicator is the degradation of lipid membrane by microorganisms and subsequent diffusion of coloured compound. 7.4 Pathogen indicators In addition to the above-mentioned indicators reacting to the normal spoilage of food products, systems for the detection of a certain contaminant in the food product have also been presented. Commercially available Toxin GuardTM by Toxin Alert Inc. (Ontario, Canada) is a system to build polyethylene-based packaging material, which is able to detect the presence of pathogenic bacteria (Salmonella, Campylobacter, Escherichia coli O157 and Listeria) with the aid of immobilised antibodies. As the analyte (toxin, microorganism) is in contact with the material it will be bound first to a specific, labelled antibody and then to a capturing antibody printed as a certain pattern (Bodenhamer, 2000). The method could also be applied for the detection of pesticide residues or proteins resulting from genetic modifications. Another commercial system for the detection of specific microorganisms like Salmonella sp., Listeria sp. and E. coli is Food Sentinel SystemTM. This system is also based on immunochemical reaction, the reaction taking place in a bar code (Goldsmith, 1994). If the particular microorganism is present the bar code is converted unreadable. Specific indicator material for the detection of Escherichia coli O157 enterotoxin has been developed at Lawrence Berkeley National Laboratory (Kahn, 1996; Quan and Stevens, 1998). This sensor material, which can be incorporated in the packaging material, is composed of cross-polymerised polydiacetylene molecules and has a deep blue colour. The molecules specifically binding the toxin are trapped in this polydiacetylene matrix and as the toxin is bound to the film, the colour of the film changes from blue to red. 136 Novel food packaging techniques