1. Introduction
Spoilage is a problem for fresh meat products because fresh meat is
contaminated during slaughter and processing with bacteria from the
feces, hide, and hooves of the animal (Ayres, 1955). Meat also has a
favorable 5.4–6.4 pH, high water activity (0.99), and abundance of low
molecular weight compounds, such as glucose and amino acids, for
bacteria to utilize (Samelis, 2006). However, spoilage of fresh meat
may be managed and extended by lower storage temperature, reduced
initial bacterial populations, packaging conditions, and increased carbon
dioxide (CO2) concentrations (Gill, 1986). Lower storage temperature
can slow bacterial growth by increasing the lag phase duration and
generation time of the bacteria. The greatest increases in lag phase
duration and generation time were observed at a storage temperature
of −1.5 °C (Gill, 1986; Jeremiah, 1997), but lag phase duration and
generation times increased at refrigeration temperatures (less than
4 °C). Freezing the meat would greatly extend the shelf life, but the
meat could no longer be marketed as fresh meat.
Initial bacterial populations present at packaging can also influence
the length of shelf life for a fresh meat product. For example, fewer
than 2 colony forming units (CFU)/g present at packaging resulted in a
7 week shelf life of fresh pork (Holley, Pierson, Lam, & Tam, 2004)
Packaging also affects the shelf life of fresh meat, especially if it involves
altering the atmosphere by vacuum or modified atmosphere packaging.
Overwrap packages have a high oxygen (O2) content in their package so
the shelf life is, on average, 5–7 days (Delmore, 2009; Jeremiah, 1997).
The aerobic atmosphere preferentially selects for aerobic and facultatively
anaerobic genera such as Pseudomonas spp., Brochothrix thermosphacta,
and Enterobacteriaceae spp. (Doulgeraki, Ercolini, Villani, & Nychas,
2012; Gill, 1986). Modified-atmosphere packages (MAP) may be either
high or low oxygen. High oxygen MAP typically have 80% O2 and 20%
CO2 present and the meat has a similar spoilage microflora as aerobic
overwrap packages, with the high oxygen content preferentially selecting
for aerobic and facultatively anaerobic genera such as Pseudomonas spp.,
B. thermosphacta, and Enterobacteriaceae spp. (Borch, Kant-Muermans,
& Blixt, 1996). A typical low oxygen MAP will not be more than 10% O2,
with the remaining balance composed of CO2, and nitrogen (a typical
MAP might be 10% O2, 20% CO2 and 70% N2), and lactic acid bacteria
(LAB), Enterobacteriaceae spp., B. thermosphacta, and Pseudomonas spp.
dominate the spoilage microflora (Doulgeraki et al., 2012). Because of
the differences in atmosphere and the composition of the spoilage micro-
flora, meat packaged in high oxygen MAP has an average shelf life of
10–21 days, while low oxygen MAP packaged meat is 25–35 days
(Delmore, 2009; Jeremiah, 1997). Vacuum-packaging is similar to low
oxygen MAP, but it has an anaerobic environment that preferentially
selects for facultatively anaerobic bacteria such as lactic acid bacteria,
Enterobacteriaceae spp., B. thermosphacta, and a few Pseudomonas spp.
in the beginning of the shelf life. Vacuum-packaging, due to its anaerobic environment, can have a
shelf life of 45–90 days (Delmore, 2009; Jeremiah, 1997).
The shelf life of vacuum-packaged meats is also affected by the CO2
concentration present in the package, which is a metabolic by-product
of microbial metabolism. Carbon dioxide is known to inhibit bacteria
by affecting the cell membrane permeability, decarboxylating enzymes,
and acidifying the intracellular pH (Dixon & Kell, 1989; Gill, 1986).
Carbon dioxide can inhibit bacteria because it is soluble in water at
refrigeration temperatures, forming dissolved CO2, and carbonic acid
(Dixon & Kell, 1989; Gill, 1986). Carbon dioxide is also soluble in the
fresh meat tissue at a rate of 960 mL of CO2/kg of fresh meat at 1 atm,
0 °C, and pH 5.5 (Gill, 1988). This rate is similar for pork, beef, and
lamb and it can be affected by pH and storage temperature (Gill,
1988). CO2 will dissolve into the fresh meat product, forming carbonic
acid and inhibiting the spoilage bacteria by acidifying the intracellular
pH and affecting cell membrane permeability.
CO2 inhibits different types of bacteria at different rates. LAB have the
highest resistance to CO2 because they produce CO2 as a by-product
of cellular respiration, whereas Pseudomonas has the least resistance
(Dixon & Kell, 1989). B. thermosphacta and Enterobacteriaceae have
intermediate resistance to CO2 (Dixon & Kell, 1989; Nowak, Rygala,
Oltuszak-Walczak, & Walczak, 2012).
The standard method of determining microbial populations in
packaged meat is a method is time-consuming, destructive, and expensive
to conduct (Bruckner, Albrecht, Petersen, & Kreyenschmidt, 2013;
McDonald & Sun, 1999; McMeekin & Ross, 1996). A total mesophilic
aerobic bacterial enumeration requires three days to complete, and
delivers historical data on the microbial population which was in the
product 72 h earlier. Because of this, there is interest in developing a
method of estimating microbial populations based upon an more
rapid instrument measurement (Bruckner et al., 2013; McDonald &
Sun, 1999; McMeekin & Ross, 1996). This instrument measurement
would focus on a by-product of microbial metabolism (Hammes &
Hertel, 2006). Potentially, a microbial metabolic byproduct could
estimate microbial populations and be used to estimate shelf life of
packaged meats. There are very few studies conducted which determine
the interaction of microbial metabolic byproducts and microbial populations
using a meat system and in the context of shelf life. However,
previous work by Devlieghere and Debevere (2000) and Devlieghere,
Debevere, and Impe (1998) used Brain–Heart-Infusion media, or a
similar broth system, to determine how dissolved CO2 affected certain
types of spoilage bacteria.
The objectives of this study were to determine the dissolved CO2 and
O2 concentrations in the purge of vacuum-packaged pork chops during
storage, and to determine the relationship between dissolved CO2 and
O2 concentrations to the microbial populations and shelf life. The
hypothesis was that dissolved CO2 concentrations will increase and
dissolved O2 concentrations will decrease inside the vacuum-package,
and that the concentrations of the dissolved gases could be used to
estimate microbial populations. In addition, scanning electron microscopic
images were taken to document the development of the microflora
over time in the packaged meat.