MAP, product safety and nutritional quality F. Devlieghere and J. Debevere, Ghent University, Belgium and M I Gil, CEBAS-CSIC, Spain 11.1 Introduction Modified atmosphere packaging(MAP) may be defined as the enclosure of food products in gas-barrier materials, in which the gaseous environment has been changed(Young et al, 1988). Because of its substantial shelf-life extending effect, MAP has been one of the most significant and innovative growth areas in retail food packaging. The potential advantages and disadvantages of mAP have been presented by Farber (1991), Parry (1993) and Davies(1995) Whilst there is considerable information available regarding suitable mixtures for different food products, there is still a lack of scientific detail regarding many aspects relating to MAP. These include mechanism of action of carbon dioxide(CO2)on microorganisms safety of MAP packaged food products effect of MAP on the nutritional quality of packaged food products Current research and gaps in knowledge are discussed in the following sections 11.2 Carbon dioxide as an antimicrobial gas The gases that are applied in MAP today are basically O2, CO2 and N2. The last has no specific preservative effect but functions mainly as a filler gas to avoid the collapse that takes place when CO2 dissolves in the food product. CO because of its antimicrobial actrviduyced into the package, it is partly dissolved in
11.1 Introduction Modified atmosphere packaging (MAP) may be defined as ‘the enclosure of food products in gas-barrier materials, in which the gaseous environment has been changed’ (Young et al., 1988). Because of its substantial shelf-life extending effect, MAP has been one of the most significant and innovative growth areas in retail food packaging. The potential advantages and disadvantages of MAP have been presented by Farber (1991), Parry (1993) and Davies (1995). Whilst there is considerable information available regarding suitable gas mixtures for different food products, there is still a lack of scientific detail regarding many aspects relating to MAP. These include: • mechanism of action of carbon dioxide (CO2) on microorganisms • safety of MAP packaged food products • effect of MAP on the nutritional quality of packaged food products. Current research and gaps in knowledge are discussed in the following sections. 11.2 Carbon dioxide as an antimicrobial gas The gases that are applied in MAP today are basically O2, CO2 and N2. The last has no specific preservative effect but functions mainly as a filler gas to avoid the collapse that takes place when CO2 dissolves in the food product. CO2, because of its antimicrobial activity, is the most important component in applied gas mixtures. When CO2 is introduced into the package, it is partly dissolved in 11 MAP, product safety and nutritional quality F. Devlieghere and J. Debevere, Ghent University, Belgium and M I Gil, CEBAS-CSIC, Spain
MAP, product safety and nutritional quality 209 the water phase and the fat phase of the food. This results, after equilibrium, in a certain concentration of dissolved CO2([CO2ldiss )in the water phase of the product. Devlieghere et al. (1998)have demonstrated that the growth inhibition of microorganisms in modified atmospheres is determined by the concentration of dissolved cO, in the water phase The effect of the gaseous environment on microorganisms in foods is not as well understood by microbiologists and food technologists as are other external factors, such as pH and aw. Despite numerous reports of the effects of COz on microbial growth and metabolism, the 'mechanism' of CO2 inhibition still remains unclear (Dixon and Kell, 1989; Day, 2000). The question of whether any specific metabolic pathway or cellular activity is critically sensitive to CO inhibition has been examined in several studies. The different proposed mechanisms of action are. 1. Lowering the pH of the food 2. Cellular penetration followed by a decrease in the cytoplasmic pH of the 3. Specific actions on cytoplasmic enzymes 4. Specific actions on biological membranes When gaseous CO2 is applied to a biological tissue, it first dissolves in the iquid phase, where hydration and dissociation lead to a rapid pH decrease in the tissue. This drop in pH, which depends on the buffering capacity of the medium Dixon and Kell, 1989), is not large in food products. In fact, the pH drop in cooked meat products only amounted to 0.3 pH units when 80% of CO2 was applied in the gas phase with a gas/product volume ration of 4: 1 Devlieghere et al., 2000b). Several studies have proved that the observed inhibitory effects of COz could not solely be explained by the acidification of the substrate( Becker, 1933 Coyne,1933) Many researchers have documented the rapidity with which CO2 in solution penetrates into the cell. Krogh(1919) discovered that this rate is 30 times faster than for oxygen(O2) under most circumstances. Wolfe(1980) suggested the inhibitory effects of CO2 are the result of internal acidification of the cytoplasm Eklund(1984) supported this idea by pointing out that the growth inhibition of four bacteria obtained with CO2 had the same general form as that obtained with weak organic acids(chemical preservatives), such as sorbic and benzoic acid. Tan and Gill (1982)also found that the intracellular pH of Pseudomonas fluorescens fell by approximately 0.03 units for each I mM rise in extracellular CO2 concentration CO2 may also exert its influence upon a cell by affecting the rate at which particular enzymatic reactions proceed. One way this may be brought about is to cause an alteration in the production of a specific enzyme, or enzymes, via induction or repression of enzyme synthesis(Dixon, 1988; Dixon and Kell 1989, Jones, 1989 ). It was also suggested ones and greenfield, 1982, Dixon and Kell, 1989)that the primary sites where CO2 exerts its effects are the enzymatic carboxylation and decarboxylation reactions, although inhibition of other enzymes has also been reported (Jones and Greenfield, 1982
the water phase and the fat phase of the food. This results, after equilibrium, in a certain concentration of dissolved CO2 ([CO2]diss) in the water phase of the product. Devlieghere et al. (1998) have demonstrated that the growth inhibition of microorganisms in modified atmospheres is determined by the concentration of dissolved CO2 in the water phase. The effect of the gaseous environment on microorganisms in foods is not as well understood by microbiologists and food technologists as are other external factors, such as pH and aw. Despite numerous reports of the effects of CO2 on microbial growth and metabolism, the ‘mechanism’ of CO2 inhibition still remains unclear (Dixon and Kell, 1989; Day, 2000). The question of whether any specific metabolic pathway or cellular activity is critically sensitive to CO2 inhibition has been examined in several studies. The different proposed mechanisms of action are: 1. Lowering the pH of the food. 2. Cellular penetration followed by a decrease in the cytoplasmic pH of the cell. 3. Specific actions on cytoplasmic enzymes. 4. Specific actions on biological membranes. When gaseous CO2 is applied to a biological tissue, it first dissolves in the liquid phase, where hydration and dissociation lead to a rapid pH decrease in the tissue. This drop in pH, which depends on the buffering capacity of the medium (Dixon and Kell, 1989), is not large in food products. In fact, the pH drop in cooked meat products only amounted to 0.3 pH units when 80% of CO2 was applied in the gas phase with a gas/product volume ration of 4:1 (Devlieghere et al., 2000b). Several studies have proved that the observed inhibitory effects of CO2 could not solely be explained by the acidification of the substrate (Becker, 1933; Coyne, 1933). Many researchers have documented the rapidity with which CO2 in solution penetrates into the cell. Krogh (1919) discovered that this rate is 30 times faster than for oxygen (O2) under most circumstances. Wolfe (1980) suggested the inhibitory effects of CO2 are the result of internal acidification of the cytoplasm. Eklund (1984) supported this idea by pointing out that the growth inhibition of four bacteria obtained with CO2 had the same general form as that obtained with weak organic acids (chemical preservatives), such as sorbic and benzoic acid. Tan and Gill (1982) also found that the intracellular pH of Pseudomonas fluorescens fell by approximately 0.03 units for each 1 mM rise in extracellular CO2 concentration. CO2 may also exert its influence upon a cell by affecting the rate at which particular enzymatic reactions proceed. One way this may be brought about is to cause an alteration in the production of a specific enzyme, or enzymes, via induction or repression of enzyme synthesis (Dixon, 1988; Dixon and Kell, 1989; Jones, 1989). It was also suggested (Jones and Greenfield, 1982; Dixon and Kell, 1989) that the primary sites where CO2 exerts its effects are the enzymatic carboxylation and decarboxylation reactions, although inhibition of other enzymes has also been reported (Jones and Greenfield, 1982). MAP, product safety and nutritional quality 209
210 Novel food packaging techniques Another possible factor contributing to the growth-inhibitory effect of Co could be an alteration of the membrane properties(Daniels et al, 1985, Dixon and Kell, 1989). It was suggested that CO2 interacts with lipids in the cell membrane, decreasing the ability of the cell wall to uptake various ions Moreover, perturbations in membrane fluidity, caused by the disordering of the lipid bilayer, are postulated to alter the function of membrane proteins( Chin et a.,1976;Roth,1980) Studies examining the effect of a CO2 enriched atmosphere on the growth of microorganisms are often difficult to compare because of the lack of information regarding the packaging configurations applied. The gas/product volume ratio and the permeability of the applied film for O and CO, will influence the mount of dissolved CO2 and thus the microbial inhibition of the atmosphere For this reason, the concentration of dissolved CO2 in the aqueous phase of the food should always be measured and mentioned in publications concerning MAP (Devlieghere et al, 1998) Only a few publications deal with the effect of MAP on specific spoilage microorganisms. Gill and Tan(1980) compared the effect of CO2 on the growth of some fresh meat spoilage bacteria at 30oC. Molin(1983)determined the resistance to COz of several food spoilage bacteria. Boskou and Debevere(1997 1998) investigated the effect of CO2 on the growth and trimethy lamine ocLc ion of Shewanella putrefaciens in marine fish, and Devlieghere and Debevere(2000) compared the sensitivity for dissolved CO2 of different ilage bacteria at 7C. In general, Gram-negative microorganisms such as Pseudomonas, Shewanella and Aeromonas are very sensitive to CO2. Gram positive bacteria show less sensitivity and lactic acid bacteria are the most resistant. Most yeasts and moulds are also sensitive to CO2. The effect of CO2 on psychrotrophic food pathogens is discussed in Section 11.3 11.3 The microbial safety of MAP: Clostridium botulinum and Listeria monocytogenes Modified atmospheres containing CO] are effective in extending the shelf-life of many food products. However, one major concern is the inhibition of normal aerobic spoilage bacteria and the possible growth of psychrotrophic food pathogens, which may result in the food becoming unsafe for consumption before it appears to be organoleptically unacceptable. Most of the pathogenic bacteria can be inhibited by low temperatures(<7C). At these conditions, only psychrotrophic pathogens can proliferate. The effect of CO2 on the different psychrotrophic foodborne pathogens is described below a particular concern is the possibility that psychrotrophic, non strains of C. botulinum types B, E and F are able to grow and prod under MAP conditions. Little is known about the effects of modified atmosphere storage conditions on toxin production by C. botulinum. The possibility of nhibiting C. botulinum by incorporating low levels of Oz in the package does
Another possible factor contributing to the growth-inhibitory effect of CO2 could be an alteration of the membrane properties (Daniels et al., 1985; Dixon and Kell, 1989). It was suggested that CO2 interacts with lipids in the cell membrane, decreasing the ability of the cell wall to uptake various ions. Moreover, perturbations in membrane fluidity, caused by the disordering of the lipid bilayer, are postulated to alter the function of membrane proteins (Chin et al., 1976; Roth, 1980). Studies examining the effect of a CO2 enriched atmosphere on the growth of microorganisms are often difficult to compare because of the lack of information regarding the packaging configurations applied. The gas/product volume ratio and the permeability of the applied film for O2 and CO2 will influence the amount of dissolved CO2 and thus the microbial inhibition of the atmosphere. For this reason, the concentration of dissolved CO2 in the aqueous phase of the food should always be measured and mentioned in publications concerning MAP (Devlieghere et al., 1998). Only a few publications deal with the effect of MAP on specific spoilage microorganisms. Gill and Tan (1980) compared the effect of CO2 on the growth of some fresh meat spoilage bacteria at 30ºC. Molin (1983) determined the resistance to CO2 of several food spoilage bacteria. Boskou and Debevere (1997; 1998) investigated the effect of CO2 on the growth and trimethylamine production of Shewanella putrefaciens in marine fish, and Devlieghere and Debevere (2000) compared the sensitivity for dissolved CO2 of different spoilage bacteria at 7ºC. In general, Gram-negative microorganisms such as Pseudomonas, Shewanella and Aeromonas are very sensitive to CO2. Grampositive bacteria show less sensitivity and lactic acid bacteria are the most resistant. Most yeasts and moulds are also sensitive to CO2. The effect of CO2 on psychrotrophic food pathogens is discussed in Section 11.3. 11.3 The microbial safety of MAP: Clostridium botulinum and Listeria monocytogenes Modified atmospheres containing CO2 are effective in extending the shelf-life of many food products. However, one major concern is the inhibition of normal aerobic spoilage bacteria and the possible growth of psychrotrophic food pathogens, which may result in the food becoming unsafe for consumption before it appears to be organoleptically unacceptable. Most of the pathogenic bacteria can be inhibited by low temperatures (<7ºC). At these conditions, only psychrotrophic pathogens can proliferate. The effect of CO2 on the different psychrotrophic foodborne pathogens is described below. A particular concern is the possibility that psychrotrophic, non-proteolytic strains of C. botulinum types B, E and F are able to grow and produce toxins under MAP conditions. Little is known about the effects of modified atmosphere storage conditions on toxin production by C. botulinum. The possibility of inhibiting C. botulinum by incorporating low levels of O2 in the package does 210 Novel food packaging techniques
MAP, product safety and nutritional quality 211 not appear to be feasible. Miller(1988, cited by Connor et al., 1989)reported that psychrotrophic strains of C. botulinum are able to produce toxins in an environment with up to 10%O2. Toxin production by C. botulinum type E,prior to spoilage, has been described in three types of fish, at O2 levels of 2% and 4% OConnor-Shaw and Reyes, 2000). Dufresne et al.(2000) also proposed that additional barriers, other than headspace O2 and film, need to be considered to ensure the safety of MAP trout fillets, particularly at moderate temperature The probability of one spore of non-proteolytic C. botulinum(types B, E and F) being toxicogenic in rock fish was outlined in a report by Ikawa and Genigeorgis(1987). The results showed that the toxigenicity was significantly affected(P<0.005)by temperature and storage time, but not by the used modified atmosphere (vacuum, 100%CO2, or 70% CO2/30% air). In Tilapia fillets, a modified atmosphere(75% CO2/25% N2), at 8C, delayed toxin formation by C. botulinum type E, from 17 to 40 days, when compared to vacuum packaged fillets(Reddy et al, 1996). Similar inhibiting effects were recorded for salmon fillets and catfish fillets, at 4C (Reddy et al., 1997a and 1997b). Toxin production from non-proteolytic C. botulinum type B spores was also retarded by a CO2 enriched atmosphere(30% Co2/70% N2)in cooked turkey at 4C but not at 10C nor at 15.C (Lawlor et al, 2000). Recent results in a study by gibson et al.(2000)also indicated that 100% CO2 slows the growth rate of C. botulinum, and that this inhibitory effect is further enhanced with appropriate NaCl concentrations and chilled temperatures Listeria monocytogenes is considered a psychrotrophic foodborne pathogen Growth is possible at 1oC (Varnam and Evans, 1991) and has even been reported at temperatures as low as-15C (Hudson et al, 1994 ) The growth of L. monocytogenes in food products, packaged under modified atmospheres, has of several, although in some cases contradicting, stu de fernando et al., 1995). In general, L. monocytogenes is not greatly inhibited by CO2 enriched atmospheres(zhao et al, 1992)although when combined with other factors such as low temperature, decreased water activity and the addition of Na lactate the inhibiting effect of CO2 is significant (Devlieghere et al 2001). Listeria growth in anaerobic CO2 enriched atmosphere has been demonstrated in lamb in an atmosphere of 50: 50 CO2/N2, at 5C (Nychas 1994): in frankfurter type sausages in atmospheres of distinct proportions of CO2/N2, at 4, 7 and 10oC(Kramer and Baumgart, 1992)and in pork in an atmosphere of 40: 60 CO2/N2, at 4C(Manu-Tawiah et al., 1993).However other authors have not detected growth in chicken anaerobically packaged in 30:70 CO /N2, at 6C(Hart et al., 1991); in 75: 25 CO et al, 1990)and at 4C in 100% CO, in raw minced meat(franco-abuin et al. 997)or in buffered tryptose broth (Szabo and Cahill, 1998). Several investigations demonstrated possible growth of L. monocytogenes on modified atmosphere packaged fresh-cut vegetables, although the results depended very much on the type of vegetables and the storage temperature( Berrang et al. 1989a, Beuchat and Brackett, 1990; Omary et al., 1993, Carlin et al, 1995
not appear to be feasible. Miller (1988, cited by Connor et al., 1989) reported that psychrotrophic strains of C. botulinum are able to produce toxins in an environment with up to 10% O2. Toxin production by C. botulinum type E, prior to spoilage, has been described in three types of fish, at O2 levels of 2% and 4% (O’Connor-Shaw and Reyes, 2000). Dufresne et al. (2000) also proposed that additional barriers, other than headspace O2 and film, need to be considered to ensure the safety of MAP trout fillets, particularly at moderate temperature abuse conditions. The probability of one spore of non-proteolytic C. botulinum (types B, E and F) being toxicogenic in rock fish was outlined in a report by Ikawa and Genigeorgis (1987). The results showed that the toxigenicity was significantly affected (P<0.005) by temperature and storage time, but not by the used modified atmosphere (vacuum, 100% CO2, or 70% CO2/30% air). In Tilapia fillets, a modified atmosphere (75% CO2/25% N2), at 8ºC, delayed toxin formation by C. botulinum type E, from 17 to 40 days, when compared to vacuum packaged fillets (Reddy et al., 1996). Similar inhibiting effects were recorded for salmon fillets and catfish fillets, at 4ºC (Reddy et al., 1997a and 1997b). Toxin production from non-proteolytic C. botulinum type B spores was also retarded by a CO2 enriched atmosphere (30% CO2/70% N2) in cooked turkey at 4ºC but not at 10ºC nor at 15ºC (Lawlor et al., 2000). Recent results in a study by Gibson et al. (2000) also indicated that 100% CO2 slows the growth rate of C. botulinum, and that this inhibitory effect is further enhanced with appropriate NaC1 concentrations and chilled temperatures. Listeria monocytogenes is considered a psychrotrophic foodborne pathogen. Growth is possible at 1ºC (Varnam and Evans, 1991) and has even been reported at temperatures as low as ÿ1.5C (Hudson et al., 1994). The growth of L. monocytogenes in food products, packaged under modified atmospheres, has been the focus of several, although in some cases contradicting, studies (Garcia de Fernando et al., 1995). In general, L. monocytogenes is not greatly inhibited by CO2 enriched atmospheres (Zhao et al., 1992) although when combined with other factors such as low temperature, decreased water activity and the addition of Na lactate the inhibiting effect of CO2 is significant (Devlieghere et al., 2001). Listeria growth in anaerobic CO2 enriched atmosphere has been demonstrated in lamb in an atmosphere of 50:50 CO2/N2, at 5ºC (Nychas, 1994); in frankfurter type sausages in atmospheres of distinct proportions of CO2/N2, at 4, 7 and 10ºC (Kra¨mer and Baumgart, 1992) and in pork in an atmosphere of 40:60 CO2/N2, at 4ºC (Manu-Tawiah et al., 1993). However, other authors have not detected growth in chicken anaerobically packaged in 30:70 CO2/N2, at 6ºC (Hart et al., 1991); in 75:25 CO2/N2 at 4ºC (Wimpfheimer et al., 1990) and at 4ºC in 100% CO2 in raw minced meat (Franco-Abuin et al., 1997) or in buffered tryptose broth (Szabo and Cahill, 1998). Several investigations demonstrated possible growth of L. monocytogenes on modified atmosphere packaged fresh-cut vegetables, although the results depended very much on the type of vegetables and the storage temperature (Berrang et al., 1989a; Beuchat and Brackett, 1990; Omary et al., 1993; Carlin et al., 1995; MAP, product safety and nutritional quality 211
212 Novel food packaging techniques Carlin et al, 1996a and 1996b, Zhang and Farber, 1996, Juneja et al., 1998; Bennick et al, 1999, Jacxsens et al, 1999, Liao and Sapers, 1999: Thomas et 1., 1999; Castillejo-Rodriguez et al, 2000) There is no agreement about the effect of incorporating O2 in the atmosphere on the antimicrobial activity of CO2 on L. monocytogenes( Garcia de Fernando et al, 1995). However, this effect could be very important in practice, as the existence of residual O2 levels after packaging, and the diffusion of o2 through the packaging film, can result in substantial Oz levels during the storage of industrially anaerobically'modified atmosphere packaged food products. Most publications suggest there is a decrease in the inhibitory effect of CO2 on L monocytogenes when O2 is incorporated into the atmosphere. Experiments on raw chicken showed L. monocytogenes failed to grow at 4, 10 and 27 C, in an anaerobic atmosphere containing 75%CO2 and 25% N2(Wimpfheimer et al. 1990). However, an aerobic atmosphere containing 72.5%CO2, 22. 5% N2, and 5%O2 did not inhibit the growth of L. monocytogenes, even at 4C. L. monocytogenes was also only minimally inhibited on chicken legs, in an atmosphere containing 10% O2 and 90% CO2(Zeitoun and Debevere, 1991) There was no significant difference in the inhibitory effect of COz, between the range of o% and 50%, when 1.5%O2, or 21%O2 was present in the atmosphere of gas packaged brain heart infusion agar plates(Bennik et al. 1995). When L. monocytogenes was cultured in buffered nutrient broth, at 7.5C, in atmospheres containing 30%CO2, with four different O2 concentrations(0, 10, 20 and 40%) the results showed that bacterial growth increased with the increasing O concentrations(Hendricks and Hotchkiss, 1997) 11.4 The microbial safety of MAP: Yersinia enterocolitica and Aeromonas spp. Yersinia enterocolitica is generally regarded as one of the most psychrotrophic foodborne pathogens. Growth of y. enterocolitica was reported in vacuum packaged lamb at 0oC(Doherty et al, 1995, Sheridan and Doherty, 1994; Sheridan et aL., 1992), beef at -2C( Gill and Reichel, 1989), pork at 4C Bodnaruk and Draughon, 1998, Manu-Tawiah et al., 1993), fresh chicken breasts(Ozbas et al, 1997)and roast beef at 3C but not at -1 5C(Hudson et a.,1994) CO2 retards the growth of y. enterocolitica at refrigerated temperatures. The ffect of CO2 on the growth of Y. enterocolitica has been described by several luthors. Some of the results are shown in Table 11. 1. Oxygen also seems to play an inhibiting role on the growth of Y. enterocolitica( Garcia de fernando et al. 1995). To ensure total inhibition of Y. enterocolitica in O2 poor atmospheres and at realistic temperatures throughout the cooling chain, high CO2 concentrations Aeromonas species are able to multiply in food products stored in refrigerated conditions. Growth of A. hydrophila has been detected at low temperatures in a
Carlin et al., 1996a and 1996b; Zhang and Farber, 1996; Juneja et al., 1998; Bennick et al., 1999; Jacxsens et al., 1999; Liao and Sapers, 1999; Thomas et al., 1999; Castillejo-Rodriguez et al., 2000). There is no agreement about the effect of incorporating O2 in the atmosphere on the antimicrobial activity of CO2 on L. monocytogenes (Garcia de Fernando et al., 1995). However, this effect could be very important in practice, as the existence of residual O2 levels after packaging, and the diffusion of O2 through the packaging film, can result in substantial O2 levels during the storage of industrially ‘anaerobically’ modified atmosphere packaged food products. Most publications suggest there is a decrease in the inhibitory effect of CO2 on L. monocytogenes when O2 is incorporated into the atmosphere. Experiments on raw chicken showed L. monocytogenes failed to grow at 4, 10 and 27ºC, in an anaerobic atmosphere containing 75% CO2 and 25% N2 (Wimpfheimer et al., 1990). However, an aerobic atmosphere containing 72.5% CO2, 22.5% N2, and 5% O2 did not inhibit the growth of L. monocytogenes, even at 4ºC. L. monocytogenes was also only minimally inhibited on chicken legs, in an atmosphere containing 10% O2 and 90% CO2 (Zeitoun and Debevere, 1991). There was no significant difference in the inhibitory effect of CO2, between the range of 0% and 50%, when 1.5% O2, or 21% O2 was present in the atmosphere of gas packaged brain heart infusion agar plates (Bennik et al. 1995). When L. monocytogenes was cultured in buffered nutrient broth, at 7.5ºC, in atmospheres containing 30% CO2, with four different O2 concentrations (0, 10, 20 and 40%), the results showed that bacterial growth increased with the increasing O2 concentrations (Hendricks and Hotchkiss, 1997). 11.4 The microbial safety of MAP: Yersinia enterocolitica and Aeromonas spp. Yersinia enterocolitica is generally regarded as one of the most psychrotrophic foodborne pathogens. Growth of Y. enterocolitica was reported in vacuum packaged lamb at 0ºC (Doherty et al., 1995; Sheridan and Doherty, 1994; Sheridan et al., 1992), beef at ÿ2ºC (Gill and Reichel, 1989), pork at 4ºC (Bodnaruk and Draughon, 1998; Manu-Tawiah et al., 1993), fresh chicken breasts (O¨ zbas et al., 1997) and roast beef at 3ºC but not at ÿ1.5ºC (Hudson et al., 1994). CO2 retards the growth of Y. enterocolitica at refrigerated temperatures. The effect of CO2 on the growth of Y. enterocolitica has been described by several authors. Some of the results are shown in Table 11.1. Oxygen also seems to play an inhibiting role on the growth of Y. enterocolitica (Garcia de Fernando et al., 1995). To ensure total inhibition of Y. enterocolitica in O2 poor atmospheres and at realistic temperatures throughout the cooling chain, high CO2 concentrations in the headspace are necessary. Aeromonas species are able to multiply in food products stored in refrigerated conditions. Growth of A. hydrophila has been detected at low temperatures in a 212 Novel food packaging techniques
MAP, product safety and nutritional quality 213 Table 11.1 Growth of Yersina enterocolitica in different atmosphere Product pH Temp. Storage Atmosphere Increase Reference typ C time ( days) CO2/N2) Beef O/100/0 0/100/0 3 02040 4 (1989) 1195 Sliced 1.5112 100/00 Hudson et al (1994) 5.57 Bodnaruk and 4 Draughon 6.21 /100/0 2877761 vacuum 0/20/80 Manu- Tawiah et al 10/40/504.0 vacuum 4.1 Lamb 5.4-5.8 Doherty et al. 1995) 0/100/0 80/20/0 0/50/50 0/100/0 vacuum of vacuum packaged fresh products, such as chicken breasts et al, 1996), lamb at 0C under high pH conditions(Doherty 1996), and at-2.C(Gill and Reichel, 1989), and in sliced roast beef at 1.5C (Hudson et aL., 1994). Devlieghere et al.(2000a)developed a model, predicting the influence of temperature and CO2 on the growth of A. hydrophila Proliferation of A. hydrophila is greatly affected by CO2 enriched atmospheres Some reports regarding the effect of CO2 on the growth of A. hydrophila on meat are summarised in Table 11.2 In a study by Berrang et al.(1989b), regarding controlled atmosphere storage of broccoli, cauliflower and asparagus stored at 4C and 15.C, fast proliferation of A. hydrophila was observed at both temperatures, but growth was not significantly affected by gas atmosphere. Garcia-Gimeno et al.(1996)published the survival of A. hydrophila on mixed vegetable salads (lettuce, red cabbage and carrots)
variety of vacuum packaged fresh products, such as chicken breasts at 3ºC (O¨ zbas et al., 1996), lamb at 0ºC under high pH conditions (Doherty et al., 1996), and at ÿ2ºC (Gill and Reichel, 1989), and in sliced roast beef at 1.5ºC (Hudson et al., 1994). Devlieghere et al. (2000a) developed a model, predicting the influence of temperature and CO2 on the growth of A. hydrophila. Proliferation of A. hydrophila is greatly affected by CO2 enriched atmospheres. Some reports regarding the effect of CO2 on the growth of A. hydrophila on meat are summarised in Table 11.2. In a study by Berrang et al. (1989b), regarding controlled atmosphere storage of broccoli, cauliflower and asparagus stored at 4ºC and 15ºC, fast proliferation of A. hydrophila was observed at both temperatures, but growth was not significantly affected by gas atmosphere. Garcia-Gimeno et al. (1996) published the survival of A. hydrophila on mixed vegetable salads (lettuce, red cabbage and carrots) Table 11.1 Growth of Yersina enterocolitica in different atmospheres Product pH Temp. Storage Atmosphere Increase Reference type (ºC) time (%O2/ (log (days) CO2/N2) cfu/g) Beef >6.0 ÿ2 126 0/100/0 0 Gill and 63 vacuum 2.4 Reichel 0 98 0/100/0 0 (1989) 49 vacuum 4.1 2 42 0/100/0 0 35 vacuum 5.1 5 35 0/100/0 1.9 17 vacuum 5.5 10 10 0/100/0 3.4 5 vacuum 4.0 Sliced 6.1 ÿ1.5 112 0/100/0 0 Hudson et al. roast beef 56 vacuum 4.2 (1994) 3 70 0/100/0 3.8 21 vacuum 4.7 Pork 5.57 30 0/100/0 0 Bodnaruk and (normal) 4 25 vacuum 1.7 Draughon 6.21 30 0/100/0 1.7 (1998) (high) 25 vacuum 2.6 Pork 6.0 35 0/20/80 4.1 Manuchops 4 35 0/40/60 4.0 Tawiah et al. 35 10/40/50 4.0 (1993) 35 vacuum 4.1 Lamb 5.4–5.8 0 28 80/20/0 1.2 Doherty et al. 28 0/50/50 3.9 (1995) 28 0/100/0 1.6 28 vacuum 5.9 28 80/20/0 6.8 28 0/50/50 8.5 28 0/100/0 5.6 28 vacuum 8.1 MAP, product safety and nutritional quality 213
214 Novel food packaging techniques Table 11.2 Growth of Aeromonas hydrophila in different atmospheres Temp. Storage Atmosphere Increase Reference type (°) CO,N,) Beef o/100/0 Gill and (1989) 0/100/0 25370 vacuun 0/10003 008 Sliced. 0/10000 roast beef vacuun (1994) O/100/0 Lamb 54-5.8 34000000 Doherty et al 0/5050 1996 0/100/0 44444 555 vacuu Lamb >60 0 80/20/00 Doherty et al 0/50/500 0/100/00 vacuu 8020/04.2 0/100/00 acuum 4.0 packaged under MA (initial 10% of O2-10% CO2, after 48h 0%Ox-18%CO2)and stored at 4oC while at 15C a fast growth was noticed (5 log units in 24h). The combination of high CO2 concentration and low temperature was revealed as responsible for the inhibition of growth. Bennik et al.(1995)concluded from their solid-surface model that at MA-conditions, generally applied for minimally processed vegetables(1-5%O2 and 5-10% CO2), growth of A. hydrophila is possible. Growth was virtually the same under 1.5% and 21%O2. The behaviour of a cocktail of A caviae(HG4)and A bestiarum(hg2)in air or in low O2-low cO2 atmosphere was investigated in fresh-cut vegetables: no difference between both atmospheres was observed on grated carrots, a decreased growth on shredded Belgian endive and Brussels sprouts in Ma but an increased growth on shredded iceberg lettuce in MA storage (Jacxsens et al., 1999)
packaged under MA (initial 10% of O2-10% CO2, after 48h 0% O2-18% CO2) and stored at 4ºC while at 15ºC a fast growth was noticed (5 log units in 24h). The combination of high CO2 concentration and low temperature was revealed as responsible for the inhibition of growth. Bennik et al. (1995) concluded from their solid-surface model that at MA-conditions, generally applied for minimally processed vegetables (1–5% O2 and 5–10% CO2), growth of A. hydrophila is possible. Growth was virtually the same under 1.5% and 21% O2. The behaviour of a cocktail of A. caviae (HG4) and A. bestiarum (HG2) in air or in low O2-low CO2 atmosphere was investigated in fresh-cut vegetables: no difference between both atmospheres was observed on grated carrots, a decreased growth on shredded Belgian endive and Brussels sprouts in MA but an increased growth on shredded iceberg lettuce in MA storage (Jacxsens et al., 1999). Table 11.2 Growth of Aeromonas hydrophila in different atmospheres Product pH Temp. Storage Atmosphere Increase Reference type (ºC) time (%O2/ (log (days) CO2/N2) cfu/g) Beef >6.0 ÿ2 126 0/100/0 0 Gill and 63 vacuum 1.0 Reichel 0 98 0/100/0 0 (1989) 49 vacuum 3.1 2 42 0/100/0 0 35 vacuum 3.0 5 35 0/100/0 0 17 vacuum 3.0 10 10 0/100/0 3.8 5 vacuum 5.8 Sliced 6.1 ÿ1.5 112 0/100/0 0 Hudson et al. roast beef 56 vacuum 4.3 (1994) 3 70 0/100/0 3.1 21 vacuum 4.6 Lamb 5.4–5.8 0 45 80/20/0 0 Doherty et al. 45 0/50/50 0 (1996) 45 0/100/0 0 45 vacuum 0 5 45 80/20/0 0 45 0/50/50 0 45 0/100/0 0 45 vacuum 0 Lamb >6.0 0 42 80/20/0 0 Doherty et al. 42 0/50/50 0 (1996) 42 0/100/0 0 42 vacuum 4.1 5 42 80/20/0 4.2 42 0/50/50 1.7 42 0/100/0 0 42 vacuum 4.0 214 Novel food packaging techniques
AAP, product safety and nutritional quality 215 11. 5 The effect of MAP on the nutritional quality of non respiring food products By using modified atmosphere packaging, the shelf-life of the packaged products can be extended by 50-200%, however, questions could arise regarding the nutritional consequences of MAP on the packaged food products. This section will discuss the effect of MAP on the nutritional quality of non-respiring food products while the effect of MAP on the nutritional value of respiring products, such as fresh fruits and vegetables, will be discussed in detail in the following sections Very little information is available about the influence of MAP on the nutritional quality of non-respiring food products. In most cases, for packaging non-respiring food products, oxygen is excluded from the atmosphere and therefore one should expect a retardation of oxidative degradation reactions Moreover, modified atmosphere packaged food products should be stored under refrigeration to allow CO2 to dissolve and perform its antimicrobial action. At these chilled conditions, chemical degradation reactions have only a limited No information is available regarding the nutritional consequences of enriched oxygen concentrations in modified atmospheres which can be applied for packaging fresh meat and marine fish. Some oxidative reactions can occur with nutritionally important compounds such as vitamins and polyunsaturated fatty acids. However, no quantitative information is available about these degradation reactions in products packaged in O2 enriched atmospheres 11.6 The effect of MAP on the nutritional quality of fresh fruits and vegetables: vitamin C and carotenoids During the last few years many studies have demonstrated that fruit and egetables are rich sources of micronutrients and dietary fibre. They also contain an immense variety of biologically active secondary metabolites that provide the plant with colour, flavour and sometimes antinutritional or toxic properties (Johnson et al., 1994). Among the most important classes of such substances are vitamin C, carotenoids, folates, flavonoids and more complex phenolics aponins, phytosterols, glycoalkaloids and the glucosinolates The nutrient content of fruit and vegetables can be influenced by various factors such as genetic and agronomic factors, maturity and harvesting methods, and postharvest handling procedures. There are some postharvest treatments which undoubtedly improve food quality by inhibiting the action of oxidative enzymes and slowing down deleterious processes. Storage of fresh fruits and vegetables within the optimum range of low O2 and/or elevated CO2 atmospheres for each commodity reduces their respiration and C2H4 production rates(Kader, 1986 Kader, 1997). Optimum CA retards loss of chlorophyll, biosynthesis of carotenoids and anthocyanins, and biosyntheses and oxidation of phenolic compounds
11.5 The effect of MAP on the nutritional quality of nonrespiring food products By using modified atmosphere packaging, the shelf-life of the packaged products can be extended by 50–200%, however, questions could arise regarding the nutritional consequences of MAP on the packaged food products. This section will discuss the effect of MAP on the nutritional quality of non-respiring food products while the effect of MAP on the nutritional value of respiring products, such as fresh fruits and vegetables, will be discussed in detail in the following sections. Very little information is available about the influence of MAP on the nutritional quality of non-respiring food products. In most cases, for packaging non-respiring food products, oxygen is excluded from the atmosphere and therefore one should expect a retardation of oxidative degradation reactions. Moreover, modified atmosphere packaged food products should be stored under refrigeration to allow CO2 to dissolve and perform its antimicrobial action. At these chilled conditions, chemical degradation reactions have only a limited importance. No information is available regarding the nutritional consequences of enriched oxygen concentrations in modified atmospheres which can be applied for packaging fresh meat and marine fish. Some oxidative reactions can occur with nutritionally important compounds such as vitamins and polyunsaturated fatty acids. However, no quantitative information is available about these degradation reactions in products packaged in O2 enriched atmospheres. 11.6 The effect of MAP on the nutritional quality of fresh fruits and vegetables: vitamin C and carotenoids During the last few years many studies have demonstrated that fruit and vegetables are rich sources of micronutrients and dietary fibre. They also contain an immense variety of biologically active secondary metabolites that provide the plant with colour, flavour and sometimes antinutritional or toxic properties (Johnson et al., 1994). Among the most important classes of such substances are vitamin C, carotenoids, folates, flavonoids and more complex phenolics, saponins, phytosterols, glycoalkaloids and the glucosinolates. The nutrient content of fruit and vegetables can be influenced by various factors such as genetic and agronomic factors, maturity and harvesting methods, and postharvest handling procedures. There are some postharvest treatments which undoubtedly improve food quality by inhibiting the action of oxidative enzymes and slowing down deleterious processes. Storage of fresh fruits and vegetables within the optimum range of low O2 and/or elevated CO2 atmospheres for each commodity reduces their respiration and C2H4 production rates (Kader, 1986; Kader, 1997). Optimum CA retards loss of chlorophyll, biosynthesis of carotenoids and anthocyanins, and biosyntheses and oxidation of phenolic compounds. MAP, product safety and nutritional quality 215
216 Novel food packaging techniques In general, CA influences flavour quality by reducing loss of acidity, starch to sugar conversion, and biosynthesis of aroma volatiles, especially esters Retention of ascorbic acid and other vitamins results in better nutritional quality, including antioxidant activity, of fruits and vegetables when kept in their optimum CA(Kader, 2001). However, little information is available on the effectiveness of controlled atmospheres or modified atmosphere packaging(C MAP)on nutrient retention during storage. The influence of CA/MAP on the antioxidant constituents related to nutritional quality of fruits and vegetables including vitamin C, carotenoids, phenolic compounds, as well as glucosinolates will be reviewed here 11.6.1 Vitamin C Vitamin C is one of the most important vitamins in fruits and vegetables for human nutrition. More than 90% of the vitamin C in human diets is supplied by the intake of fresh fruits and vegetables. Vitamin C is required for the prevention of scurvy and maintenance of healthy skin, gums and blood vessels. Vitamin C, as an antioxidant. reduces the risk of arteriosclerosis. cardiovascular diseases and some forms of cancer(Simon, 1992). Ascorbic oxidase has been proposed as he major enzyme responsible for enzymatic degradation of L-ascorbic acid (AA). The oxidation of AA, the active form of vitamin C, to dehydroascorbic acid (dha)does not result in loss of biological activity since dha is readily re- converted to l-AA in vivo. However, DHA is less stable than AA and may be hydrolysed to 2, 3-diketogulonic acid, which does not have physiological activity (Klein, 1987)and it has therefore been suggested that measurements of vitamin C in fruits and vegetables in relation to their nutritional value should include both aa and dha The vulnerability of different fruits and vegetables to oxidative loss of AA varies greatly, as indeed do general quality changes. Low pH fruits(citrus fruits are relatively stable, whereas soft fruits(strawberries, raspberries)undergo more rapid changes. Leafy vegetables(e.g. spinach) are very vulnerable to spoilage and AA loss, whereas root vegetables(e.g. potatoes) retain quality and AA for many months(Davey et al., 2000). Fruits and vegetables undergo changes from the moment of harvest and since l-aa is one of the more reactive compounds it is particularly vulnerable to treatment and storage conditions. In broad terms, the milder the treatment and the lower the temperature the better the retention vitamin C, but there are several interacting factors that affect Aa retention (Davey et al., 2000). The rate of postharvest oxidation of AA in plant tissues has been reported to depend upon several factors such as temperature, water content, storage atmosphere and storage time(Lee and Kader, 2002) The effect of controlled atmospheres on the ascorbate content of intact fruit has not been extensively studied. The results vary among fruit species and cultivars, but the tendency is for reduced O2 and/or elevated CO2 levels to enhance the retention of ascorbate(Weichmann, 1986; Kader et al., 1989).A reduction in temperature and of O2 concentration in the storage atmosphere have
In general, CA influences flavour quality by reducing loss of acidity, starch to sugar conversion, and biosynthesis of aroma volatiles, especially esters. Retention of ascorbic acid and other vitamins results in better nutritional quality, including antioxidant activity, of fruits and vegetables when kept in their optimum CA (Kader, 2001). However, little information is available on the effectiveness of controlled atmospheres or modified atmosphere packaging (CA/ MAP) on nutrient retention during storage. The influence of CA/MAP on the antioxidant constituents related to nutritional quality of fruits and vegetables, including vitamin C, carotenoids, phenolic compounds, as well as glucosinolates will be reviewed here. 11.6.1 Vitamin C Vitamin C is one of the most important vitamins in fruits and vegetables for human nutrition. More than 90% of the vitamin C in human diets is supplied by the intake of fresh fruits and vegetables. Vitamin C is required for the prevention of scurvy and maintenance of healthy skin, gums and blood vessels. Vitamin C, as an antioxidant, reduces the risk of arteriosclerosis, cardiovascular diseases and some forms of cancer (Simon, 1992). Ascorbic oxidase has been proposed as the major enzyme responsible for enzymatic degradation of L-ascorbic acid (AA). The oxidation of AA, the active form of vitamin C, to dehydroascorbic acid (DHA) does not result in loss of biological activity since DHA is readily reconverted to L-AA in vivo. However, DHA is less stable than AA and may be hydrolysed to 2,3-diketogulonic acid, which does not have physiological activity (Klein, 1987) and it has therefore been suggested that measurements of vitamin C in fruits and vegetables in relation to their nutritional value should include both AA and DHA. The vulnerability of different fruits and vegetables to oxidative loss of AA varies greatly, as indeed do general quality changes. Low pH fruits (citrus fruits) are relatively stable, whereas soft fruits (strawberries, raspberries) undergo more rapid changes. Leafy vegetables (e.g. spinach) are very vulnerable to spoilage and AA loss, whereas root vegetables (e.g. potatoes) retain quality and AA for many months (Davey et al., 2000). Fruits and vegetables undergo changes from the moment of harvest and since L-AA is one of the more reactive compounds it is particularly vulnerable to treatment and storage conditions. In broad terms, the milder the treatment and the lower the temperature the better the retention of vitamin C, but there are several interacting factors that affect AA retention (Davey et al., 2000). The rate of postharvest oxidation of AA in plant tissues has been reported to depend upon several factors such as temperature, water content, storage atmosphere and storage time (Lee and Kader, 2002). The effect of controlled atmospheres on the ascorbate content of intact fruit has not been extensively studied. The results vary among fruit species and cultivars, but the tendency is for reduced O2 and/or elevated CO2 levels to enhance the retention of ascorbate (Weichmann, 1986; Kader et al., 1989). A reduction in temperature and of O2 concentration in the storage atmosphere have 216 Novel food packaging techniques
MAP, product safety and nutritional quality 217 been described as the two treatments which contribute to preserve vitamin Cin fruits and vegetables(Watada, 1987). Delaporte(1971)and others observed that loss of AA can be reduced by storing apples in a reduced oxygen atmosphere However, Haffner et al.(1997) have shown that AA levels in various apple cultivars decreased more under ultra low oxygen (ULO) compared to air storage On the other hand. increasing cO, concentration above a certain threshold seems to have an adverse effect on vitamin c content in some fruits and vegetables. It has been reported that the effect of elevated co2 level and storage temperature and duration (Weichmann, 1986). Bangerth(1977)observed accelerated AA losses in apples and red currants stored in elevated CO atmospheres. Vitamin C content was reduced by high CO2 concentrations (10- 30%CO2)in strawberries and blackberries and only a moderate to negligible effect was found for black currants, red currants and raspberries(Agar et al 1997) torage of sweet pepper for six days at 13.C in CO2 enriched atmospheres resulted in a reduction in AA content(Wang, 1977). Wang(1983)noted that 1% O2 retarded aa degradation in Chinese cabbage stored for three months at 0C He observed that treatments with 10 or 20%CO2 for five or ten days produced no effect, and 30 or 40% CO2 increased AA decomposition. Veltman et al. (1999)have observed a 60% loss in AA content of "Conference pears after storage in 2%O2+10%CO2. There were no data available to show whether a parallel reduction in O2 concentration alleviates the negative CO2 effect. Agar et al.(1997) proposed that reducing O2 concentration in the storage atmosphere in the present of high CO2 had little effect on the vitamin C preservation. The only beneficial effect of low O, alleviating the co, effect could be observed when applying CO2 concentrations lower than 10% In fresh-cut products, high CO2 concentration in the storage atmosphere has also been described to cause degradation of vitamin C. Thus, concentrations of 5, 10 or 20% CO2 caused degradation of vitamin C in fresh-cut kiwifruit slices (Agar et al, 1999). Enhanced losses of vitamin C in response to CO2 higher than 10% may be due to the stimulating effects on oxidation of AA and/or inhibition of Dha reduction to AA(Agar et al., 1999). In addition, vitamin C content decreased in MAP-stored Swiss chard (Gil et al., 1998a)as well as in potato strips(Tudela et al, 2002). In contrast, MAP retarded the conversion of Aa to DHA that occurred in air-stored jalapeno pepper rings(Howard et al., 1994 Howard and Hernandez-Brenes 1998). Wright and Kader(1997a)found no significant losses of vitamin C occurred during the post-cutting life of fresh-cut strawberries and persimmons for eight days in CA (2%O2, air 12%CO2,or 2%O2+ 12%CO2at 0C In studies of cut broccoli florets and intact heads of broccoli CA/maP resulted in greater AA retention and shelf-life extension in contrast to air-stored samples(Barth et al, 1993; Paradis et al, 1996). Retention of Aa was found in fresh-cut lettuce packaged with nitrogen(Barry-Ryan and O Beirne, 1999) They suggest that high levels of CO2(30-40%) increased AA losses by conversion into Dha due to availability of oxygen in lettuce(Barry-Ryan and
been described as the two treatments which contribute to preserve vitamin C in fruits and vegetables (Watada, 1987). Delaporte (1971) and others observed that loss of AA can be reduced by storing apples in a reduced oxygen atmosphere. However, Haffner et al. (1997) have shown that AA levels in various apple cultivars decreased more under ultra low oxygen (ULO) compared to air storage. On the other hand, increasing CO2 concentration above a certain threshold seems to have an adverse effect on vitamin C content in some fruits and vegetables. It has been reported that the effect of elevated CO2 level and storage temperature and duration (Weichmann, 1986). Bangerth (1977) observed accelerated AA losses in apples and red currants stored in elevated CO2 atmospheres. Vitamin C content was reduced by high CO2 concentrations (10– 30% CO2) in strawberries and blackberries and only a moderate to negligible effect was found for black currants, red currants and raspberries (Agar et al., 1997). Storage of sweet pepper for six days at 13ºC in CO2 enriched atmospheres resulted in a reduction in AA content (Wang, 1977). Wang (1983) noted that 1% O2 retarded AA degradation in Chinese cabbage stored for three months at 0ºC. He observed that treatments with 10 or 20% CO2 for five or ten days produced no effect, and 30 or 40% CO2 increased AA decomposition. Veltman et al. (1999) have observed a 60% loss in AA content of ‘Conference’ pears after storage in 2% O2 + 10% CO2. There were no data available to show whether a parallel reduction in O2 concentration alleviates the negative CO2 effect. Agar et al. (1997) proposed that reducing O2 concentration in the storage atmosphere in the present of high CO2 had little effect on the vitamin C preservation. The only beneficial effect of low O2 alleviating the CO2 effect could be observed when applying CO2 concentrations lower than 10%. In fresh-cut products, high CO2 concentration in the storage atmosphere has also been described to cause degradation of vitamin C. Thus, concentrations of 5, 10 or 20% CO2 caused degradation of vitamin C in fresh-cut kiwifruit slices (Agar et al., 1999). Enhanced losses of vitamin C in response to CO2 higher than 10% may be due to the stimulating effects on oxidation of AA and/or inhibition of DHA reduction to AA (Agar et al., 1999). In addition, vitamin C content decreased in MAP-stored Swiss chard (Gil et al., 1998a) as well as in potato strips (Tudela et al., 2002). In contrast, MAP retarded the conversion of AA to DHA that occurred in air-stored jalapeno pepper rings (Howard et al., 1994; Howard and Hernandez-Brenes 1998). Wright and Kader (1997a) found no significant losses of vitamin C occurred during the post-cutting life of fresh-cut strawberries and persimmons for eight days in CA (2% O2, air + 12% CO2, or 2% O2 + 12% CO2) at 0ºC. In studies of cut broccoli florets and intact heads of broccoli CA/MAP resulted in greater AA retention and shelf-life extension in contrast to air-stored samples (Barth et al., 1993; Paradis et al., 1996). Retention of AA was found in fresh-cut lettuce packaged with nitrogen (Barry-Ryan and O’Beirne, 1999). They suggest that high levels of CO2 (30–40%) increased AA losses by conversion into DHA due to availability of oxygen in lettuce (Barry-Ryan and MAP, product safety and nutritional quality 217
