Food Protection with Modified Atmospheres 353 Table 14–3 Some Examples of Gas and Water Vapor Transmission Properties of Film Used for Food Packaging∗ Transmission Properties OTR 7.8–9.3 ml/m2 /24 hours/37.8◦ C/70% RH a OTR ml/m2 /24 hours/4◦ C/100% RH b CO2 TR 124 ml/m2 /24 hours/100% RH c WVTR 18.6 g/m2 /24 hours/37◦ C/100% RH OTR 10 ml/m2 /24 hours/22.8◦ C/0% RH a OTR 32 ml/m2 /24 hours/23.9◦ C/50% RH b CO2 TR 47 ml/m2 /24 hours/23.9◦ C/70% RH c WVTR 0.8–1.8 g/m2 /24 hours/23.9◦ C/70% RH OTR 52 ml/m2 /24 hours/1 atm/25◦ C/75% RH OTR 154 ml/m2 /24 hours OTR 300 ml/m2 /24 hours/25◦ C/1 atm/100% RH OTR 1,000 ml/m2 /24 hours/25◦ C/1 atm/90% RH OTR 6,500 ml/m2 /24 hours/23◦ C/0% RH 10 OTR 7,800–13,900 ml/m2 /24 hours WVTR 240–419 g/m2 /24 hours 11 OTR 6,500 ml/m2 /24 hours/23◦ C/0% RH Comment Extremely high barrier Extremely high barrier High barrier High barrier Whirl-Pak bags Commonly used for vacuum packaging Essentially aerobic Highly permeable PVC film Stretch-wrapped film ∗ OTR = oxygen transmission rate; RH = relative humidity; WVTR = water vapor transmission; PVC = polyvinyl chloride 80–90◦ C water A method that is suitable for raw meats consists of simply squeezing out the excess air from a pouch followed by heat sealing In addition to retarding aerobic spoilage organisms, vacuum packaging minimizes product shrinkage, and retards both fat oxidation and discoloration Gas and water vapor transmission properties of some plastics used to vacuum-pack foods are listed in Table 14–3 In general, CO2 permeability of such films is always higher than for O2 by a factor of around to Modified Atmosphere Packaging Overall, MAP is a hyperbaric process that consists of altering the chamber or package atmosphere by flushing with varying mixtures of CO2 , N2 , and/or O2 The initial gas concentration cannot be readjusted during storage Two types of MAP are recognized.29 In high-O2 MAP, up to 70% of O2 along with about 20–30% CO2 and 0–20% N2 may be used Growth of aerobes is slowed but not suppressed by the moderate concentration of CO2 This method is suitable for the packaging of red meats, as the high level of O2 will aid in maintenance of the red color With time, the gas composition may be expected to change In a low-O2 MAP system, O2 levels may be as high as 10% while CO2 is maintained in the 20–30% range with N2 added as necessary Equilibrium-Modified Atmosphere Equilibrium-modified atmosphere (EMA) packaging is achieved by flushing a gas-permeable pack with gas, or sealing the pack without alteration EMA is used for fresh fruits and vegetables.76 354 Modern Food Microbiology Controlled-Atmosphere Packaging or Storage Although controlled-atmosphere packaging or storage (CAP, CAS) is regarded by some as being different from MAP, it may be considered a form of MAP While in a typical MAP the compositions may change upon storage, in CAP the gas compositions remain unchanged for the duration of the storage period While low- and high-O2 MAP systems may be prepared with high-barrier plastic films, CAP requires aluminum foil laminates, metal or glass containers, since single plastic film is not entirely impervious to gases Since vacuum, MAP, or CAP methods alter the concentrations of O2 and CO2 , albeit in different ways, the distinction between them is often obscured in studies on the effectiveness and mode of action of CO2 In the remaining sections of this chapter, the inhibitory effects of increased levels of CO2 on foodborne microorganisms and food quality are examined without regard to methodology PRIMARY EFFECTS OF CO2 ON MICROORGANISMS The following facts are well established following prolonged exposure of microorganisms to around 10% and above of CO2 The inhibitory activity increases as incubation or storage temperatures decrease This is due in part to the greater solubility of CO2 in water at the lower temperatures, and in part to the additive effect of a less than optimal growth temperature At atm, 100 ml of water will absorb 88 ml of CO2 at 20◦ C, but only 36 ml at 60◦ C Although concentrations from about 5–100% have been used, 20–30% seems optimal, with no additional benefits derived from higher levels This is especially true for fresh meats, where 20% is about ideal.30,31 Higher levels can be used for seafoods To maintain red meat color, they can first be exposed to carbon monoxide (CO) before CO2 , or be stored in a 20:80 mixture of CO2 + O2 Inhibition increases as pH is decreased into the acid range One practical effect of this is that CO2 is more effective for fresh red meats than for seafoods The vacuum packaging of red meats with pH >6.0 is not effective The self-life of vacuum-packaged fish is shortened by the growth of Photobacterium phosphoreum16 and Shewanella putrefaciens.1 In general, the Gram-negative bacteria are more sensitive to CO2 inhibition than Gram positives, with pseudomonads being among the most sensitive and clostridia the most resistant (Table 14–4) Upon prolonged storage of meats, CO2 affects a rather dramatic shift in biota from one that is largely Gram negative in fresh products to one that is largely or exclusively Gram positive This can be seen in Table 14–5 for smoked pork loins and frankfurter sausage.6 Both lag and logrithmic phases of growth are retarded CO2 under pressure is considerably more antimicrobial than when not under pressure, and pressures of 6–30 megapascal (MPa) can destroy bacteria and fungi under varying conditions (see High Hydrostatic Pressure in Chapter 19) The destructive action is believed to occur when pressure is released suddenly Mode of Action As to the mechanism of CO2 inhibition of microorganisms, two explanations have been offered King and Nagel57 found that CO2 blocked the metabolism of Pseudomonas aeruginosa and appeared to affect a mass action on enzymatic decarboxylations Sears and Eisenberg79 found that CO2 affected Food Protection with Modified Atmospheres 355 Table 14–4 Relative Sensitivity of Microorganisms to CO2 Relative to Vacuum and Modified-Atmosphere Packaging Pseudomonas spp Aeromonas spp Bacillus spp Molds Enterobacteriaceae Enterococcus spp Brochothrix spp Lactobacillus spp Clostridium spp (Most sensitive) (Most resistant) Source: Adapted with permission from G Molin,68 The Resistance to Carbon Dioxide of Some Food Related Bacteria, European Journal of Applied Microbiology and Biotechnology, Vol 18, pp 214–217, Copyright c 1983, Springer-Verlag, New York Table 14–5 Effect of Storage on the Microbiota of Two Meats Held from 48 to 140 Days at 4◦ C Smoked Pork Loins Log APC/g pH Dominant biota (%) Day Vacuum 48 Days CO2 48 Days N2 48 Days 2.5 5.8 Flavo (20) Arthro (20) Yeasts (20) Pseudo (11) Coryne (10) 7.6 5.8 Lactos (52)a 6.9 5.9 7.2 5.9 Lactos (74)b Lactos (67)c Frankfurter Sausage Log APC/g pH Dominant biota (%) Day Vacuum 98 Days CO2 140 Days N2 140 Days 1.7 5.9 Bac (34) Coryne (34) Flavo (8) Broch (8) 9.0 5.4 Lactos (38) 2.4 5.6 Lactos (88)d 4.8 5.9 Lactos (88)e Note: Percentage biota represented by Weissella viridescens: a 40; b 72; c 50; d 22; e35 APC = aerobic plate count; Flavo = Flavobacterium; Arthro = Arthrobacter; Pseudo = Pseudomonas; Coryne = Corynebacterium; Bac = Bacillus; Broch = Brochothrix Source: Adapted from Blickstad and Molin.6 356 Modern Food Microbiology the permeability of cell membranes, and Enfors and Molin22 found support for the latter hypothesis in their studies on the germination of Clostridium sporogenes and C perfringens endospores At atm CO2 , spore germination of these two species was stimulated, whereas B cereus spore germination was inhibited As was shown by others, CO2 is more stimulatory at low pH than at high pH With 55 atm CO2 , only 4% germination of C sporogenes spores occurred, whereas with C perfringens, 50 atm reduced germination to 4%.22 These authors suggested that CO2 inhibition was due to its accumulation in the membrane lipid bilayer such that increased fluidity results An adverse effect on cell permeability has been suggested by others If CO2 is dissolved in the form of carbonic acid, HCO− would be present as a dissociation product, and thus can cause changes in cell permeability.17 When dissolved in water, CO2 products are as follows: 2− − CO2 + H2 O −− H2 CO3 −− H+ + HCO− − 2H + CO3 The antimicrobial spectrum of CO2 and diacetyl is quite similar, and while this per se does not mean they possess identical modes of action, the striking similarities seem worthy of note Diacetyl is an arginine antagonist and its mode of action along with some other α-dicarbonyl compounds has been discussed.51 The greater sensitivity of Gram-negative bacteria to α-dicarbonyl inhibitors appears to be due to their capacity to inactivate amino acid-binding proteins of the cell’s periplasm, especially the arginine-binding proteins Thus, it is not inconceivable that the site of action of CO2 is the periplasm, where it interferes with the normal functioning of amino acid-binding proteins Food Products The successful use of vacuum packaging, MAP, and CAS to extend the self-life of a wide variety of food products is well documented, and some of the specific antimicrobial aspects are outlined below Fresh and Processed Meats Among the first to demonstrate the effectiveness of high levels of CO2 in preserving cut-up meats was J Brooks in England, who in 1933 studied the effect of CO2 on lean meat; E Callow in England, who studied pork and bacon; R.B Haines, also in England, who was among the first to show the effect of CO2 on spoilage organisms; and W.A Empey in Australia, who in 1933 applied CO2 to beef.73 In general, the self-life of red meats can be extended for up to months if packaged in 75% O2 + 25% CO2 and stored at −1◦ C The high level of oxygen ensures that the red-meat color is maintained It has been shown that at least 15% CO2 is necessary to retard microbial growth on beef steaks, and that the mixture of 15% CO2 + 75% O2 + 10% N2 was more effective than vacuum both for red-meat color and microbial quality.4 The importance of the temperature of storage of MAP meats was shown early by Jaye et al.52 who found striking differences in quality when ground beef was stored at 30◦ C versus 38◦ They compared the use of the more gas-impermeable Saran to the gas-permeable cellophane packs Earlier, Halleck et al.38 showed the dramatic inhibitory effect of vacuum packaging and storage at 1.1–3.3◦ C The importance of temperature of storage was demonstrated in another study using the packaging system known as the Captech process, which combines hygienic processing, storage at −1.5◦ C, high CO2 , low O2 , and gas-impermeable packaging.36 The process was applied to pork loins, with the temperature of holding for simulated retail display being raised to 8◦ C Lactic acid bacteria grew without perceptible decrease in lag phase, and reached 107 /cm2 within weeks The behavior of the biota of smoked pork loins and frankfurter sausage stored under vacuum and CO2 is presented in Food Protection with Modified Atmospheres 357 Figure 14–1 Lack of increase in hydration capacity of fresh ground beef stored in high-barrier bags at 7◦ C for 13 days as measured by extract-release volume (ERV) The foil-wrapped samples underwent aerobic spoilage as evidenced by increased hydration and endotoxin titers Table 14–5 As is typical of MAP meats, the initial heterogeneous biota became homogeneous upon long-term storage under vacuum or MAP with pH being decreased due to predominance of lactic acid bacteria.6 The relative effectiveness of MAP/vacuum packaging of red meats can be assessed by determining changes that occur in hydration capacity When fresh ground beef was stored in high-barrier bags and held at 7◦ C for up to 13 days, the hydration capacity was essentially unchanged in comparison to the samples that were loosely wrapped in foil to allow for aerobic conditions (Figure 14–1) This is reflected by extract-release volume (ERV; see Chapter 4) Over the holding period, Gram-negative bacteria increased by about log10 but by only log10 for the aerobically stored foil-wrapped and highbarrier bag-stored meats, respectively Similar results can be obtained by using the filter-paper press method to measure hydration capacity.50 The increased hydration is brought about by the preferential growth of Gram-negative bacteria accompanied by increases in pH into the alkaline range In their study of beef and pork livers, and beef kidneys, packaged in high-barrier bags, Hanna et al.43 found that pH decreased in each product when held at 2◦ C for up to 28 days ERV has been used to assess the spoilage of vacuum-packaged meats.75 In a study of normal and DFD ground beef stored at 3◦ C in 100% CO2 for up to 11 days, self-life increased by ca 3—4 days with the predominant bacterial biota consisting of lactic acid bacteria and Brochothrix thermosphacta in contrast to samples stored in air where the pseudomonads predominated.72 In the normal low-pH meat under CO2 , lactate appeared to increase, while in air-stored samples it decreased over the 11-day holding period When Greek taverna sausage was stored under vacuum and ... contrast to samples stored in air where the pseudomonads predominated .72 In the normal low-pH meat under CO2 , lactate appeared to increase, while in air-stored samples it decreased over the 11-day... self-life of red meats can be extended for up to months if packaged in 75% O2 + 25% CO2 and stored at −1◦ C The high level of oxygen ensures that the red-meat color is maintained It has been...354 Modern Food Microbiology Controlled-Atmosphere Packaging or Storage Although controlled-atmosphere packaging or storage (CAP, CAS) is regarded by some as being different