PROCESSING, PRODUCTS, AND FOOD SAFETY Shelf-life extension of vacuum-packaged meat from pheasant (Phasianus colchicus) by lactic acid treatment Agathe Pfeifer, Frans J. M. Smulders, and Peter Paulsen1 Institute of Meat Hygiene, Meat Technology and Food Science, University of Veterinary Medicine Vienna, Veterinärplatz 1, 1210 Vienna, Austria of such treated and stored samples were on an average 1.5 to 1.7 log units lower than in non-acid-treated samples. Similar results were found for Enterobacteriaceae. A significant decrease in pH was measured at d 7 and 10 in the acid-treated samples in comparison with the untreated ones. In summary, the immersion of pheasant breast meat cuts in dilute lactic acid significantly reduced microbiota during vacuum-packed storage, even at slight temperature abuse conditions.
Key words: pheasant meat, vacuum package, lactic acid, microbial shelf life 2014 Poultry Science 93:1818–1824 http://dx.doi.org/10.3382/ps.2013-03481
INTRODUCTION In previous studies on the implementation of good hygiene practice during the processing of meat from wild birds, we established that meat cuts from pheasants can be stored vacuum-packed at 0 to 1°C for up to 7 d without significant changes in microbiota (El-Ghareeb et al., 2009). However, in the meat chain, it has been established that even strict adherence to measures of good hygiene practice will not guarantee the absence of undesired bacteria or sufficiently low levels of bacterial contaminants to compensate for deficiencies in the cool chain (Koutsoumanis and Angelidis, 2007). Among the additional lines of defense studied in laboratories or implemented in industry practice, treatment with dilute lactic acid has proven antibacterial efficacy on red meat (Paulsen and Smulders, 2004) and on poultry carcasses (Loretz et al., 2010), with no or minor effects on sensory meat properties. As described by Alakomi et al. (2000), lactic acid is able to penetrate into gram-negative bacteria and make them more sensitive for external influences. A number of studies on the application of lactic acid on beef carcasses, cuts, and trimmings have been critically reviewed recently in the European Union (European Food Safety Authority, 2011), and in
consideration of the generally positive assessment, the treatment of cattle carcasses or primals with sprays or dips of lactic acid is allowed in the European Union as an antimicrobial measure, under some provisions (e.g. an operational hazard analysis and critical control points system and strict observation of general hygiene rules; European Commission, 2013). Beneficial effects of lactic acid (alone or in combinations) in regard to the reduction of contaminant bacteria on fresh, skinon (Anang et al., 2010; Burfoot and Mulvey, 2011), or marinated (Smaoui et al., 2012) poultry meat have been reported. Likewise, lactic acid application effectuated reductions of the pathogens Listeria monocytogenes (González-Fandos and Dominguez, 2006) or Salmonella Enteritidis and Campylobacter jejuni (Chaine et al., 2013), which had been inoculated onto poultry skin. Acid dips are usually more effective in reducing numbers of pathogenic bacteria on meat surfaces than acid sprays (Okolocha and Ellerbroek, 2005). The purpose of this study was to assess if lactic acid treatment of pheasant meat (skin off) would substantially reduce contaminant bacteria to allow prolonged shelf life of vacuum-packed meat stored under conditions of moderate temperature abuse (+6°C).
MATERIALS AND METHODS ©2014 Poultry Science Association Inc. Received July 9, 2013. Accepted April 14, 2014. 1 Corresponding author: [email protected]
Overview of Trials The effect of lactic acid on the contaminant flora of meat cuts from slaughtered pheasants was studied in 4 1818
Downloaded from http://ps.oxfordjournals.org/ at Oakland University on April 6, 2015
ABSTRACT We investigated the influence of lactic acid treatment of pheasant meat before vacuum-packaged storage of 3, 7, and 10 d at +6°C on microbiota and pH. Breast muscle samples were collected from carcasses of slaughtered as well as from hunted (shot) wild pheasants. Immersion of meat samples in 3% (wt/wt) lactic acid for 60 s effectuated a significant drop in pH of approximately 0.5 to 0.7 units, which remained during the entire storage period. In parallel, total aerobic counts
1819
SHELF-LIFE EXTENSION OF VACUUM-PACKAGED MEAT
Acid Treatment: Use of a Lactic Acid Releasing Wrap Film
trials. Whereas 2 trials investigated the usefulness of a lactic acid releasing wrap film, the following 2 trials explored the antibacterial effect of 10-s to 120-s dips in dilute lactic acid. The most promising treatments derived from the latter trials were then applied on pheasant meat with higher pH (i.e., birds that had been killed by shooting during drive hunts).
Preparation of Meat Stamps Meat samples were taken from pheasant carcasses (Phasianus colchichus L.), of which 30 were purchased (in batches of 5 to 9 carcasses) from a slaughter unit at a pheasantry and arrived in uneviscerated condition at +2°C not later than 12 h postmortem. Eleven hunted pheasants (i.e., birds that had been killed by lead shot) were obtained in uneviscerated condition. Carcasses were stored at 0°C and were processed 2 d postmortem. For obtaining meat samples, carcasses were suspended from the neck, wings, tail, and distal legs (at tarsal joints) were cut off and the skin was removed under hygienic precautions as described previously (ElGhareeb et al., 2009). From the skinned carcass, meat samples of constant size (surface and thickness) were removed from breast muscles (M. pectoralis superficialis) by means of a sterile punch (cork borer with 2.5 cm diameter) and a surgical scalpel. Meat parts exhibiting hemorrhages or shot lesions were not sampled. Due to differences in size and geometry of the sampled muscles, numbers of breast muscle samples varied from bird to bird and number of meat stamps per trial varied slightly between trials (indicated in Table 1). Within each trial, meat stamps were randomly assigned to (acid-) treatment and storage time groups (see below); the origin from a particular carcass and left/right muscle was not considered (i.e., the meat stamp was the experimental unit).
Acid Treatment: Dipping of Meat Stamps for 10 to 120 s into 3% Lactic Acid In the following trials, treatment consisted of immersing meat samples for periods ranging from 10 to 120 s in 80 mL of 3% wt/wt food grade lactic acid (diluted from Purac FCC80, Purac, Rayong, Thailand) and allowing the samples to dry for 10 min before vacuum packing. In trial 3, 40 meat stamps from 5 pheasant carcasses were randomly assigned to 5 groups (8 stamps per group). Whereas group 1 was analyzed immediately, groups 2 and 3 were immersed into lactic acid for 120 s, vacuum-packed, and stored for 7 and 10 d at +6°C, respectively. Groups 4 and 5 consisted of samples that had been vacuum packed without lactic acid treatment,
Table 1. Numbers of meat stamps tested for development of the microbiota of pheasant breast meat stamps (2.5 cm diameter, 8–10 mm depth), after lactic acid (La) treatment and vacuum-packed storage at +6°C for 3, 7, and 10 d1 Origin2
Prepackaging, n
1
8, s
8
2
8, s
8
3
5, s
8
4
9, s
8
5
4, h
7
6
6, h
7
7
1, h
6
Trial
1n
No acid treatment,3 n 8 8 8 8 8 8 8 8 7 7 7 7 6 6
(d (d (d (d (d (d (d (d (d (d (d (d (d (d
3) 7) 3) 7) 7) 10) 7) 10) 7) 10) 7) 10) 7) 10)
La film, n 8 8 8 8
= number of meat stamps per group. number of carcasses of slaughtered (s) or hunted (h) pheasants. 3Untreated samples stored vacuum packed. 4In 3% La. 2Origin:
(d (d (d (d
3) 7) 3) 7)
Dip,4 120 s, n
Dip,4 60 s, n
Dip,4 30 s, n
Dip,4 10 s, n
8 8 8 8 7 7
(d (d (d (d (d (d
7) 10) 7) 10) 7) 10)
8 (d 7) 8 (d 10) 7 7 6 6
(d (d (d (d
7) 10) 7) 10)
8 (d 7) 8 (d 10)
8 (d 7) 8 (d 10)
Downloaded from http://ps.oxfordjournals.org/ at Oakland University on April 6, 2015
In the first 2 trials, the antibacterial effect of a polyamide film releasing lactic acid (typically 67 µg/cm2) (Wanda et al., 2013) was studied. In the first trial, 40 meat stamps were taken from carcasses of 8 slaughtered pheasants. Stamps were randomly assigned to 5 groups (8 stamps per group), where group 1 was analyzed immediately (pretreatment). Stamps in groups 2 and 3 were individually wrapped in 9 × 3 cm antibacterial polyamide films, and then vacuum-packed (Combivac PA/PE film, 90 µm thickness, Felzmann, Linz, Austria, in a packaging machine, Henkovac, HFE Vacuum Systems BV, ML ‘s-Hertogenbosch, the Netherlands). Samples were tested after 3 and 7 d of storage at +6°C. Groups 4 and 5 were vacuum-packed without the wrap film (control) and stored for 3 and 7 d at +6°C, respectively. The whole experiment was repeated (trial 2; Table 1).
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Pfeifer et al.
and were stored and analyzed like groups 2 and 3. The following trial 4 aimed at studying the effect of shorter acid-immersion periods on the number of contaminant bacteria and differed from trial 3 just by including 60-, 30-, and 10-s acid dips. A total of 88 meat stamps from 9 pheasant carcasses were used in trial 4 (Table 1). The 2 most effective acid-immersion times were now tested on meat stamps obtained from hunted pheasants. Trial 5 was essentially a repetition of trial 3, but with a smaller number of meat stamps per group (7 instead of 8). In trials 6 and 7, acid-immersion time of 60 s was studied.
Artificial Contamination
Determination of pH Determination of pH was done in a watery suspension of the samples (pH-meter CG822 with electrode BlueLine pH27, both from Schott, Mainz, Germany).
Microbiological Examination At the day of packaging and after the defined storage intervals, meat stamps were removed from their packages and macerated with buffered peptone water (Oxoid CM0509, Oxoid, Basingstoke, UK) at a 1 + 9 ratio on a weight basis for 120 s (Stomacher 80, Seward Medical, Worthing, UK). Subsequently decimal dilutions were prepared on a volume basis with MRD (Oxoid CM0733). Total aerobic count (TAC) was determined on plate count agar (Merck 1.05463, Merck, Darmstadt, Germany) after incubation for 72 h at 30°C according to International Organisation for Standardization (ISO) method 4833 (ISO, 2003). Enterobacteriaceae were enumerated on violet red bile glucose agar (Merck 1.10275) after 24-h aerobic incubation at 37°C. Pink to red colonies of ≥1 mm diameter were subcultured on plate count agar and tested for oxidase reaction and fermentation of glucose according to ISO 21528–2 (ISO, 2004) to yield counts of confirmed Enterobacteriaceae. Media were inoculated by surface-spreading technique. Microbial numbers of the fecal slurry were determined in the same way.
Numbers of samples per group were defined a priori. It was assumed that a 1 log unit difference in bacterial numbers should be detected, with a sigma of 0.5 and α = 0.05 (G*Power software; Faul et al., 2007). Even sample sizes of 6 per group (≥4 groups) would ensure a power of 0.80, which should be considered sufficient (Cohen, 1992). In addition, it was proven post hoc that the power was not below 0.80, using the same software. Microbial numbers were transformed to log values, and results below the limit of detection (2 log cfu/g) were set to 1.9 log cfu/g. Within experimental series, a completely randomized design was used, with numbers of treatments, replication, and storage times as indicated in Table 1. Differences between treatments and storage days were assessed by 2-way ANOVA (StatGraphics 3.0, Statistical Graphics Corp., Princeton, NJ), with (acid-) treatment and storage time as independent, and pH and bacterial numbers as dependent factors. The least significant difference test was applied to discriminate among means (Sachs, 1992). In case data were not normally distributed or more than one of the results was below the limit of detection, the Kruskal-Wallis test was used (trials 1, 2, 5, and 6). Statistical significance was established at P < 0.05.
RESULTS AND DISCUSSION Development of pH of Meat Stamps The pH of non-acid-treated breast meat stamps at the day of carcass cutting was 5.7 ± 0.1 for slaughtered pheasants and 6.8 ± 0.1 for hunted pheasants, and remained in that range during a 10-d storage period at +6°C. The pH values in hunted pheasants were roughly 1 unit higher than reported in previous studies (Paulsen et al., 2008; Hofbauer et al., 2010), presumably indicating ante mortem energy depletion due to a special mode of mass drive-hunting. Likewise, average pH in thighs (M. iliotibialis lateralis) was higher in hunted (6.8) than in slaughtered pheasants (6.1; A. Pfeifer, unpublished data). Inoculation of samples with feces slurry had no significant effect on pH. Immersion of meat samples in 3% lactic acid for 60 or 120 s effectuated a significant drop in pH of average 0.5 to 0.7 units, which remained during the entire storage period (7 or 10 d). Shorter immersion times of 10 and 30 s resulted in less pronounced pH reduction in the range of 0.3 to 0.4 units at best, whereas no significant changes in pH were observed for samples wrapped in lactic acid releasing film.
Development of Microbiota, Trials with Lactic Acid Releasing Film In trials 1 and 2, initial TAC were 3.1 ± 0.7 and 2.9 ± 1.3 log cfu/g for breast muscle stamps. Within these
Downloaded from http://ps.oxfordjournals.org/ at Oakland University on April 6, 2015
In trial 7, meat samples were contaminated with gut content from pheasants to simulate meat surfaces contaminated by deficiencies in evisceration technique. In brief, 0.5 g of fecal material from pheasants was suspended in 1.5 mL of distilled water, and 0.1-mL aliquots were spread on the surface of the meat stamp. The meat samples were left for 10 min at 25°C before acid treatment and packing or solely packing. A group of inoculated meat stamps was examined immediately after inoculation to define the initial contamination level. Storage and analyses were done as in trial 6.
Statistical Processing of Data
SHELF-LIFE EXTENSION OF VACUUM-PACKAGED MEAT
Microbiota in Meat from Slaughtered Pheasants, Effects of Dipping in Dilute Lactic Acid In trial 3, the immersion of breast meat stamps (120 s in 3% lactic acid) effectuated a statistically significant
reduction in total aerobic counts. Before treatment, microbial numbers were 3.1 ± 0.7 log cfu/g, and after treatment, numbers at d 3 and 7 were
Key words: pheasant meat, vacuum package, lactic acid, microbial shelf life 2014 Poultry Science 93:1818–1824 http://dx.doi.org/10.3382/ps.2013-03481
INTRODUCTION In previous studies on the implementation of good hygiene practice during the processing of meat from wild birds, we established that meat cuts from pheasants can be stored vacuum-packed at 0 to 1°C for up to 7 d without significant changes in microbiota (El-Ghareeb et al., 2009). However, in the meat chain, it has been established that even strict adherence to measures of good hygiene practice will not guarantee the absence of undesired bacteria or sufficiently low levels of bacterial contaminants to compensate for deficiencies in the cool chain (Koutsoumanis and Angelidis, 2007). Among the additional lines of defense studied in laboratories or implemented in industry practice, treatment with dilute lactic acid has proven antibacterial efficacy on red meat (Paulsen and Smulders, 2004) and on poultry carcasses (Loretz et al., 2010), with no or minor effects on sensory meat properties. As described by Alakomi et al. (2000), lactic acid is able to penetrate into gram-negative bacteria and make them more sensitive for external influences. A number of studies on the application of lactic acid on beef carcasses, cuts, and trimmings have been critically reviewed recently in the European Union (European Food Safety Authority, 2011), and in
consideration of the generally positive assessment, the treatment of cattle carcasses or primals with sprays or dips of lactic acid is allowed in the European Union as an antimicrobial measure, under some provisions (e.g. an operational hazard analysis and critical control points system and strict observation of general hygiene rules; European Commission, 2013). Beneficial effects of lactic acid (alone or in combinations) in regard to the reduction of contaminant bacteria on fresh, skinon (Anang et al., 2010; Burfoot and Mulvey, 2011), or marinated (Smaoui et al., 2012) poultry meat have been reported. Likewise, lactic acid application effectuated reductions of the pathogens Listeria monocytogenes (González-Fandos and Dominguez, 2006) or Salmonella Enteritidis and Campylobacter jejuni (Chaine et al., 2013), which had been inoculated onto poultry skin. Acid dips are usually more effective in reducing numbers of pathogenic bacteria on meat surfaces than acid sprays (Okolocha and Ellerbroek, 2005). The purpose of this study was to assess if lactic acid treatment of pheasant meat (skin off) would substantially reduce contaminant bacteria to allow prolonged shelf life of vacuum-packed meat stored under conditions of moderate temperature abuse (+6°C).
MATERIALS AND METHODS ©2014 Poultry Science Association Inc. Received July 9, 2013. Accepted April 14, 2014. 1 Corresponding author: [email protected]
Overview of Trials The effect of lactic acid on the contaminant flora of meat cuts from slaughtered pheasants was studied in 4 1818
Downloaded from http://ps.oxfordjournals.org/ at Oakland University on April 6, 2015
ABSTRACT We investigated the influence of lactic acid treatment of pheasant meat before vacuum-packaged storage of 3, 7, and 10 d at +6°C on microbiota and pH. Breast muscle samples were collected from carcasses of slaughtered as well as from hunted (shot) wild pheasants. Immersion of meat samples in 3% (wt/wt) lactic acid for 60 s effectuated a significant drop in pH of approximately 0.5 to 0.7 units, which remained during the entire storage period. In parallel, total aerobic counts
1819
SHELF-LIFE EXTENSION OF VACUUM-PACKAGED MEAT
Acid Treatment: Use of a Lactic Acid Releasing Wrap Film
trials. Whereas 2 trials investigated the usefulness of a lactic acid releasing wrap film, the following 2 trials explored the antibacterial effect of 10-s to 120-s dips in dilute lactic acid. The most promising treatments derived from the latter trials were then applied on pheasant meat with higher pH (i.e., birds that had been killed by shooting during drive hunts).
Preparation of Meat Stamps Meat samples were taken from pheasant carcasses (Phasianus colchichus L.), of which 30 were purchased (in batches of 5 to 9 carcasses) from a slaughter unit at a pheasantry and arrived in uneviscerated condition at +2°C not later than 12 h postmortem. Eleven hunted pheasants (i.e., birds that had been killed by lead shot) were obtained in uneviscerated condition. Carcasses were stored at 0°C and were processed 2 d postmortem. For obtaining meat samples, carcasses were suspended from the neck, wings, tail, and distal legs (at tarsal joints) were cut off and the skin was removed under hygienic precautions as described previously (ElGhareeb et al., 2009). From the skinned carcass, meat samples of constant size (surface and thickness) were removed from breast muscles (M. pectoralis superficialis) by means of a sterile punch (cork borer with 2.5 cm diameter) and a surgical scalpel. Meat parts exhibiting hemorrhages or shot lesions were not sampled. Due to differences in size and geometry of the sampled muscles, numbers of breast muscle samples varied from bird to bird and number of meat stamps per trial varied slightly between trials (indicated in Table 1). Within each trial, meat stamps were randomly assigned to (acid-) treatment and storage time groups (see below); the origin from a particular carcass and left/right muscle was not considered (i.e., the meat stamp was the experimental unit).
Acid Treatment: Dipping of Meat Stamps for 10 to 120 s into 3% Lactic Acid In the following trials, treatment consisted of immersing meat samples for periods ranging from 10 to 120 s in 80 mL of 3% wt/wt food grade lactic acid (diluted from Purac FCC80, Purac, Rayong, Thailand) and allowing the samples to dry for 10 min before vacuum packing. In trial 3, 40 meat stamps from 5 pheasant carcasses were randomly assigned to 5 groups (8 stamps per group). Whereas group 1 was analyzed immediately, groups 2 and 3 were immersed into lactic acid for 120 s, vacuum-packed, and stored for 7 and 10 d at +6°C, respectively. Groups 4 and 5 consisted of samples that had been vacuum packed without lactic acid treatment,
Table 1. Numbers of meat stamps tested for development of the microbiota of pheasant breast meat stamps (2.5 cm diameter, 8–10 mm depth), after lactic acid (La) treatment and vacuum-packed storage at +6°C for 3, 7, and 10 d1 Origin2
Prepackaging, n
1
8, s
8
2
8, s
8
3
5, s
8
4
9, s
8
5
4, h
7
6
6, h
7
7
1, h
6
Trial
1n
No acid treatment,3 n 8 8 8 8 8 8 8 8 7 7 7 7 6 6
(d (d (d (d (d (d (d (d (d (d (d (d (d (d
3) 7) 3) 7) 7) 10) 7) 10) 7) 10) 7) 10) 7) 10)
La film, n 8 8 8 8
= number of meat stamps per group. number of carcasses of slaughtered (s) or hunted (h) pheasants. 3Untreated samples stored vacuum packed. 4In 3% La. 2Origin:
(d (d (d (d
3) 7) 3) 7)
Dip,4 120 s, n
Dip,4 60 s, n
Dip,4 30 s, n
Dip,4 10 s, n
8 8 8 8 7 7
(d (d (d (d (d (d
7) 10) 7) 10) 7) 10)
8 (d 7) 8 (d 10) 7 7 6 6
(d (d (d (d
7) 10) 7) 10)
8 (d 7) 8 (d 10)
8 (d 7) 8 (d 10)
Downloaded from http://ps.oxfordjournals.org/ at Oakland University on April 6, 2015
In the first 2 trials, the antibacterial effect of a polyamide film releasing lactic acid (typically 67 µg/cm2) (Wanda et al., 2013) was studied. In the first trial, 40 meat stamps were taken from carcasses of 8 slaughtered pheasants. Stamps were randomly assigned to 5 groups (8 stamps per group), where group 1 was analyzed immediately (pretreatment). Stamps in groups 2 and 3 were individually wrapped in 9 × 3 cm antibacterial polyamide films, and then vacuum-packed (Combivac PA/PE film, 90 µm thickness, Felzmann, Linz, Austria, in a packaging machine, Henkovac, HFE Vacuum Systems BV, ML ‘s-Hertogenbosch, the Netherlands). Samples were tested after 3 and 7 d of storage at +6°C. Groups 4 and 5 were vacuum-packed without the wrap film (control) and stored for 3 and 7 d at +6°C, respectively. The whole experiment was repeated (trial 2; Table 1).
1820
Pfeifer et al.
and were stored and analyzed like groups 2 and 3. The following trial 4 aimed at studying the effect of shorter acid-immersion periods on the number of contaminant bacteria and differed from trial 3 just by including 60-, 30-, and 10-s acid dips. A total of 88 meat stamps from 9 pheasant carcasses were used in trial 4 (Table 1). The 2 most effective acid-immersion times were now tested on meat stamps obtained from hunted pheasants. Trial 5 was essentially a repetition of trial 3, but with a smaller number of meat stamps per group (7 instead of 8). In trials 6 and 7, acid-immersion time of 60 s was studied.
Artificial Contamination
Determination of pH Determination of pH was done in a watery suspension of the samples (pH-meter CG822 with electrode BlueLine pH27, both from Schott, Mainz, Germany).
Microbiological Examination At the day of packaging and after the defined storage intervals, meat stamps were removed from their packages and macerated with buffered peptone water (Oxoid CM0509, Oxoid, Basingstoke, UK) at a 1 + 9 ratio on a weight basis for 120 s (Stomacher 80, Seward Medical, Worthing, UK). Subsequently decimal dilutions were prepared on a volume basis with MRD (Oxoid CM0733). Total aerobic count (TAC) was determined on plate count agar (Merck 1.05463, Merck, Darmstadt, Germany) after incubation for 72 h at 30°C according to International Organisation for Standardization (ISO) method 4833 (ISO, 2003). Enterobacteriaceae were enumerated on violet red bile glucose agar (Merck 1.10275) after 24-h aerobic incubation at 37°C. Pink to red colonies of ≥1 mm diameter were subcultured on plate count agar and tested for oxidase reaction and fermentation of glucose according to ISO 21528–2 (ISO, 2004) to yield counts of confirmed Enterobacteriaceae. Media were inoculated by surface-spreading technique. Microbial numbers of the fecal slurry were determined in the same way.
Numbers of samples per group were defined a priori. It was assumed that a 1 log unit difference in bacterial numbers should be detected, with a sigma of 0.5 and α = 0.05 (G*Power software; Faul et al., 2007). Even sample sizes of 6 per group (≥4 groups) would ensure a power of 0.80, which should be considered sufficient (Cohen, 1992). In addition, it was proven post hoc that the power was not below 0.80, using the same software. Microbial numbers were transformed to log values, and results below the limit of detection (2 log cfu/g) were set to 1.9 log cfu/g. Within experimental series, a completely randomized design was used, with numbers of treatments, replication, and storage times as indicated in Table 1. Differences between treatments and storage days were assessed by 2-way ANOVA (StatGraphics 3.0, Statistical Graphics Corp., Princeton, NJ), with (acid-) treatment and storage time as independent, and pH and bacterial numbers as dependent factors. The least significant difference test was applied to discriminate among means (Sachs, 1992). In case data were not normally distributed or more than one of the results was below the limit of detection, the Kruskal-Wallis test was used (trials 1, 2, 5, and 6). Statistical significance was established at P < 0.05.
RESULTS AND DISCUSSION Development of pH of Meat Stamps The pH of non-acid-treated breast meat stamps at the day of carcass cutting was 5.7 ± 0.1 for slaughtered pheasants and 6.8 ± 0.1 for hunted pheasants, and remained in that range during a 10-d storage period at +6°C. The pH values in hunted pheasants were roughly 1 unit higher than reported in previous studies (Paulsen et al., 2008; Hofbauer et al., 2010), presumably indicating ante mortem energy depletion due to a special mode of mass drive-hunting. Likewise, average pH in thighs (M. iliotibialis lateralis) was higher in hunted (6.8) than in slaughtered pheasants (6.1; A. Pfeifer, unpublished data). Inoculation of samples with feces slurry had no significant effect on pH. Immersion of meat samples in 3% lactic acid for 60 or 120 s effectuated a significant drop in pH of average 0.5 to 0.7 units, which remained during the entire storage period (7 or 10 d). Shorter immersion times of 10 and 30 s resulted in less pronounced pH reduction in the range of 0.3 to 0.4 units at best, whereas no significant changes in pH were observed for samples wrapped in lactic acid releasing film.
Development of Microbiota, Trials with Lactic Acid Releasing Film In trials 1 and 2, initial TAC were 3.1 ± 0.7 and 2.9 ± 1.3 log cfu/g for breast muscle stamps. Within these
Downloaded from http://ps.oxfordjournals.org/ at Oakland University on April 6, 2015
In trial 7, meat samples were contaminated with gut content from pheasants to simulate meat surfaces contaminated by deficiencies in evisceration technique. In brief, 0.5 g of fecal material from pheasants was suspended in 1.5 mL of distilled water, and 0.1-mL aliquots were spread on the surface of the meat stamp. The meat samples were left for 10 min at 25°C before acid treatment and packing or solely packing. A group of inoculated meat stamps was examined immediately after inoculation to define the initial contamination level. Storage and analyses were done as in trial 6.
Statistical Processing of Data
SHELF-LIFE EXTENSION OF VACUUM-PACKAGED MEAT
Microbiota in Meat from Slaughtered Pheasants, Effects of Dipping in Dilute Lactic Acid In trial 3, the immersion of breast meat stamps (120 s in 3% lactic acid) effectuated a statistically significant
reduction in total aerobic counts. Before treatment, microbial numbers were 3.1 ± 0.7 log cfu/g, and after treatment, numbers at d 3 and 7 were
