Combined Effect of Organic Acids and Modified Atmosphere Packaging on Listeria monocytogenes in Chicken Legs

Simple Summary Chicken can be contaminated with pathogens such as Listeria monocytogenes and cause illness in consumers. The illness caused is called listeriosis, and it is a severe illness with high levels of mortality, particularly in susceptible individuals (pregnant women, neonates, the elderly and immunocompromised individuals). Consequently, it is important to control this pathogen in chicken meat. We evaluated the effect of packaging and natural compounds (citric, propionic and acetic acids) on this pathogen. We found that treatment of chicken with 2% propionic acid or 2% acetic acid can decrease L. monocytogenes counts in packaged chicken. Abstract The combined effect of organic acid (citric, propionic or acetic acid) treatment and modified atmosphere packaging (MAP) on the growth of L. monocytogenes in chicken legs kept at 4 °C for 10 days was evaluated. Chicken legs were inoculated with L. monocytogenes and washed with either 2% citric, 2% propionic or 2% acetic acid solution or distilled water (control). Legs were packaged under the following conditions: air, vacuum, 80% N2/20% CO2, 60% N2/40% CO2 or 40% N2/60% CO2. The greatest L. monocytogenes growth reductions after treatment were observed in chicken legs washed with propionic acid (2.14 log units lower compared to control legs). The lowest growth rates of L. monocytogenes were found in samples washed with acetic acid and packaged in atmospheres containing CO2. An extended shelf life was observed in legs packaged in 40% N2/60% CO2, but these packaging conditions did not reduce L. monocytogenes growth. Consequently, it is necessary to design measures in order to control this bacterial pathogen. Washing of chicken with 2% propionic acid or 2% acetic acid can decrease L. monocytogenes counts in chicken packaged in MAP.


Introduction
Poultry is often identified as the source of foodborne outbreaks [1]. Listeria monocytogenes is frequently found in chicken and can grow on chicken meat. Moreover, the shelf life of fresh chicken is very limited, and it would be of interest to extend it [2].
Organic acids are used in foods because they are generally recognized as safe (GRAS) [3,4]. In order to reduce bacterial populations, high organic acid concentrations are needed, but these levels can adversely affect food quality [5]. Several investigations have addressed the application of organic acids on chicken [6,7].
The effectiveness of acetic, citric and propionic acids in reducing L. monocytogenes in chicken has been evaluated in early studies [8][9][10]. It was found that chicken legs washed with 2% acetic acid showed a decrease in L. monocytogenes counts and an extended shelf life of the samples by at least two

Sensorial Analysis
On the sampling days, six legs from each batch were evaluated for overall acceptability by a panel of nine members. A structured hedonic scale with numerical scores ranging from 7 (I like it very much) to 1 (I dislike it very much) was used. A score of 3 was considered the borderline of acceptability.

Microbiological Analyses and pH Determination
Each sample was prepared by aseptically removing 10 g of skin. The samples were blended in a sterile bag containing 90 mL of 0.1% sterile peptone water (Oxoid, Hampshire, England) and homogenized for 2 min (IUL, Barcelona, Spain). Serial dilutions in 0.1% sterile peptone were made. The psychrotrophs were determined on Plate Count Agar (Merck, Damstadt, Germany) with an incubation temperature of 7 • C for 10 days, using the pour plate method [14]. Listeria spp. were determined following the surface plate method on Palcam Agar at an incubation temperature of 30 • C for 48 h. Suspected colonies were identified according to the method described by Gonzalez-Fandos et al. [8]. Growth parameters (maximum growth rate and lag phase) were calculated by using the ComBase application and entering the data into the DMFit tool. The goodness of fit was evaluated using the coefficient of determination (R 2 ).
A Crison model 2002 pH meter with a penetration electrode was used for measurements of pH (Crison, Barcelona, Spain).

Gas Determination
Determination of carbon dioxide and oxygen was carried out using an O 2 and CO 2 headspace gas analyzer (Check-mate model 9900, PBI-Dansensor, Ringsted, Denmark).
Plate count data were transformed to logarithms before their statistical analysis. The experimental design was a factorial experiment (4 × 5) in which the factors were the washing treatment and the gas atmosphere composition with six repetitions. Analysis of variance was carried out using the SYSTAT program for Windows, Statistics version 5.0 (Evanston, IL, USA). Tukey's test for comparison of means was performed using the same program. The level of significance was determined at p < 0.05.

Modified Atmosphere Packaging
The effect of MAP on psychrotroph populations is shown in Figure 1. Significant differences (p < 0.05) in psychrotroph populations were found between the samples packaged in MAP and the samples packaged in air (control legs), except on day 0. Psychrotroph growth reductions of between 1.16 and 2.04 log units were observed in samples packaged in 40% N2/60% CO2 compared to the control samples. The growth parameters (maximum growth rate and lag phase) estimated by ComBase are summarized in Table 2. No lag phase for psychrotrophs was detected in legs packaged in air. The maximum growth rate decreased and the lag phase increased when the CO2 concentration increased. The legs packaged in vacuum showed a higher maximum growth rate and a shorter lag phase for psychrotroph growth compared to those packaged in 40% N2/60% CO2 or 60% N2/40% CO2. The lower maximum growth rate and longer lag phase were found for psychrotrophs in legs packaged in 40% N2/60% CO2. The growth parameters were adequately described, since R 2 values obtained were above 0.95.   Mean ± standard deviation, Means in the same column with no superscript letters in common are significantly different (p < 0.05). λ, lag phase (day); µ max , maximum growth rate (log cfu/g/day); R 2 , coefficient of determination. Packaging conditions: A0, air; A20, 80% N 2 /20% CO 2 ; A40, 60% N 2 /40% CO 2 ; A60, 40% N 2 /60% CO 2 ; AV, vacuum.

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The effect of MAP on the growth of L. monocytogenes inoculated into legs is shown in Figure 2. No significant differences (p > 0.05) in these pathogen counts were found between samples packaged in MAP and control legs. No lag phase was detected in L. monocytogenes growth on samples packaged in different conditions. However, the maximum growth rate decreased when CO 2 concentration increased ( Table 2). The growth parameters were adequately described, as indicated by R 2 values above 0.98.
The effect of MAP on the growth of L. monocytogenes inoculated into legs is shown in Figure 2. No significant differences (p > 0.05) in these pathogen counts were found between samples packaged in MAP and control legs. No lag phase was detected in L. monocytogenes growth on samples packaged in different conditions. However, the maximum growth rate decreased when CO2 concentration increased ( Table 2). The growth parameters were adequately described, as indicated by R 2 values above 0.98.
The effect of MAP on the growth of L. monocytogenes inoculated into legs is shown in Figure 2. No significant differences (p > 0.05) in these pathogen counts were found between samples packaged in MAP and control legs. No lag phase was detected in L. monocytogenes growth on samples packaged in different conditions. However, the maximum growth rate decreased when CO2 concentration increased ( Table 2). The growth parameters were adequately described, as indicated by R 2 values above 0.98.
The pH value decreased when the CO2 concentration increased ( Figure 3). Significant differences (p < 0.05) were observed in pH values between legs packaged in atmospheres containing CO2 and those packaged in vacuum or air, except on day 0. On day 1, pH in legs packaged in 40% N2/60% CO2 was 5.7 ± 0.01, while that in control legs was 6.45 ± 0.16.    Changes in package atmospheres (O 2 , CO 2 , N 2 ) were not significant (p > 0.05) during storage among samples packaged in the same conditions. After 3 days of storage, CO 2 and O 2 levels remained steady.
The sensorial acceptability of packaged legs is shown in Figure 4. Control legs were unacceptable on day 6, while those packaged in 80% N 2 /20% CO 2 and vacuum were rejected on day 10. The samples packaged in 60% N 2 /40% CO 2 and 40% N 2 /60% CO 2 remained acceptable on day 10. Changes in package atmospheres (O2, CO2, N2) were not significant (p > 0.05) during storage among samples packaged in the same conditions. After 3 days of storage, CO2 and O2 levels remained steady.
The sensorial acceptability of packaged legs is shown in Figure 4. Control legs were unacceptable on day 6, while those packaged in 80% N2/20% CO2 and vacuum were rejected on day 10. The samples packaged in 60% N2/40% CO2 and 40% N2/60% CO2 remained acceptable on day 10.

Combined Treatments of Organic Acids and MAP
The effects of different organic acids (citric, propionic and acetic acids) on psychrotroph populations in packaged legs are shown in Figures 5a, 6a and 7a. Significant growth reductions (p < 0.05) in psychrotrophs were observed between the samples treated with organic acids packaged in air or MAP and those not treated with organic acids. Significant growth reductions (p < 0.05) in psychrotrophs were also observed between the samples treated with organic acids packaged in MAP and those treated and packaged in air. The fastest increase in psychrotroph growth was observed in the air-packaged legs that were not treated with organic acids (Batch C). The samples packaged in 40% N2/60% CO2 and washed with 2% acetic acid (Batch AA60) showed the lowest psychrotroph counts. After treatment with citric, propionic, and acetic acids, psychrotroph growth reductions of 0.99, 1.11 and 0.91 log units, respectively, were observed. The treatment with 2% acetic acid and packaging in 40% N2/60% CO2 decreased psychrotroph growth by between 2.51 and 4.99 log units compared to the untreated samples. The growth parameters (maximum growth rate and lag phase) estimated by ComBase are summarized in Table 3. No lag phase was found in psychrotroph growth in samples washed with citric or propionic acid and packaged in air. However, the treatment with acetic acid and packaging in air extended the lag phase of psychrotrophs. The maximum growth rate decreased and the lag phase increased when the CO2 concentration increased. A significantly longer (p < 0.05) lag phase of psychrotrophs was found in legs washed with 2% acetic acid and packaged in 60% N2/40% CO2 or 40% N2/60% CO2 when compared to the other conditions tested. The lowest maximum growth rate of psychrotrophs was observed in legs washed with 2% propionic acid and packaged in 40% N2/60% CO2. The growth parameters were adequately described since R 2 values were above 0.95. Washing treatment and packaging in modified atmospheres significantly affected psychrotroph growth (p < 0.05).
Figures 5b, 6b and 7b show the effects of different organic acids on the growth of L. monocytogenes in poultry samples packaged in different conditions. After treatment (day 0) with citric, propionic and acetic acids, L. monocytogenes populations were observed to decrease by 1.06, 2.14 and 0.88 log units, respectively, compared to the control samples. Significant reductions (p < 0.05) in L.

Combined Treatments of Organic Acids and MAP
The effects of different organic acids (citric, propionic and acetic acids) on psychrotroph populations in packaged legs are shown in Figures 5a, 6a and 7a. Significant growth reductions (p < 0.05) in psychrotrophs were observed between the samples treated with organic acids packaged in air or MAP and those not treated with organic acids. Significant growth reductions (p < 0.05) in psychrotrophs were also observed between the samples treated with organic acids packaged in MAP and those treated and packaged in air. The fastest increase in psychrotroph growth was observed in the air-packaged legs that were not treated with organic acids (Batch C). The samples packaged in 40% N 2 /60% CO 2 and washed with 2% acetic acid (Batch AA60) showed the lowest psychrotroph counts. After treatment with citric, propionic, and acetic acids, psychrotroph growth reductions of 0.99, 1.11 and 0.91 log units, respectively, were observed. The treatment with 2% acetic acid and packaging in 40% N 2 /60% CO 2 decreased psychrotroph growth by between 2.51 and 4.99 log units compared to the untreated samples. The growth parameters (maximum growth rate and lag phase) estimated by ComBase are summarized in Table 3. No lag phase was found in psychrotroph growth in samples washed with citric or propionic acid and packaged in air. However, the treatment with acetic acid and packaging in air extended the lag phase of psychrotrophs. The maximum growth rate decreased and the lag phase increased when the CO 2 concentration increased. A significantly longer (p < 0.05) lag phase of psychrotrophs was found in legs washed with 2% acetic acid and packaged in 60% N 2 /40% CO 2 or 40% N 2 /60% CO 2 when compared to the other conditions tested. The lowest maximum growth rate of psychrotrophs was observed in legs washed with 2% propionic acid and packaged in 40% N 2 /60% CO 2 . The growth parameters were adequately described since R 2 values were above 0.95. Washing treatment and packaging in modified atmospheres significantly affected psychrotroph growth (p < 0.05). Figures 5b, 6b and 7b show the effects of different organic acids on the growth of L. monocytogenes in poultry samples packaged in different conditions. After treatment (day 0) with citric, propionic and acetic acids, L. monocytogenes populations were observed to decrease by 1.06, 2.14 and 0.88 log units, respectively, compared to the control samples. Significant reductions (p < 0.05) in L. monocytogenes growth were found in the samples washed with citric acid and packaged in 60% N 2 /40% CO 2 or 40% N 2 /60% CO 2 when compared to those washed with this organic acid and packaged in air, after 1 day of storage. Significant reductions (p < 0.05) in L. monocytogenes growth were found in the samples washed with propionic acid and packaged in 60% N 2 /40% CO 2 or 40% N 2 /60% CO 2 when compared to those treated with this organic acid and packaged in air, after 3 days of storage. Significant growth reductions (p < 0.05) in L. monocytogenes were found in the samples washed with acetic acid and packaged in 60% N 2 /40% CO 2 or 40% N 2 /60% CO 2 when compared to those washed with this organic acid and packaged in air, after 3 days of storage. Until day 6, the lowest L. monocytogenes populations were found Animals 2020, 10, 1818 7 of 17 in legs washed with 2% propionic acid and packaged in 40% N 2 /60% CO 2 . The treatment with 2% propionic acid and packaging in 40% N 2 /60% CO 2 reduced L. monocytogenes growth by between 2.14 and 3.3 log units compared to the control samples. The treatment with 2% acetic acid and packaging in 40% N 2 /60% CO 2 reduced L. monocytogenes growth by between 0.88 and 2.09 log units compared to the control samples throughout storage. The growth parameters (maximum growth rate and lag phase) estimated by ComBase are summarized in Table 3. No lag phase was found in L. monocytogenes growth in legs treated with citric or acetic acid and packaged in air. However, the treatment with propionic acid and packaging in air extended the lag phase of this pathogen. The maximum growth rate decreased and the lag phase increased when the CO 2 concentration increased. A significantly lower (p < 0.05) growth rate of L. monocytogenes was found in legs treated with 2% acetic acid and packaged in modified atmospheres containing CO 2 than in those treated with citric or acetic acid and packaged under MAP. The R 2 values obtained were above 0.89. Washing treatment and packaging in modified atmospheres significantly affected L. monocytogenes growth (p < 0.05).
in air, after 1 day of storage. Significant reductions (p < 0.05) in L. monocytogenes growth were found in the samples washed with propionic acid and packaged in 60% N2/40% CO2 or 40% N2/60% CO2 when compared to those treated with this organic acid and packaged in air, after 3 days of storage. Significant growth reductions (p < 0.05) in L. monocytogenes were found in the samples washed with acetic acid and packaged in 60% N2/40% CO2 or 40% N2/60% CO2 when compared to those washed with this organic acid and packaged in air, after 3 days of storage. Until day 6, the lowest L. monocytogenes populations were found in legs washed with 2% propionic acid and packaged in 40% N2/60% CO2. The treatment with 2% propionic acid and packaging in 40% N2/60% CO2 reduced L. monocytogenes growth by between 2.14 and 3.3 log units compared to the control samples. The treatment with 2% acetic acid and packaging in 40% N2/60% CO2 reduced L. monocytogenes growth by between 0.88 and 2.09 log units compared to the control samples throughout storage. The growth parameters (maximum growth rate and lag phase) estimated by ComBase are summarized in Table  3. No lag phase was found in L. monocytogenes growth in legs treated with citric or acetic acid and packaged in air. However, the treatment with propionic acid and packaging in air extended the lag phase of this pathogen. The maximum growth rate decreased and the lag phase increased when the CO2 concentration increased. A significantly lower (p < 0.05) growth rate of L. monocytogenes was found in legs treated with 2% acetic acid and packaged in modified atmospheres containing CO2 than in those treated with citric or acetic acid and packaged under MAP. The R 2 values obtained were above 0.89. Washing treatment and packaging in modified atmospheres significantly affected L. monocytogenes growth (p < 0.05).  water and packaging in air (□); Batch CA, 2% citric acid and packaging in air (▪); Batch CAM20, 2% citric acid and packaging in 80% N2/20% CO2 (•); Batch CAM40, 2% citric acid and packaging in 60% N2/40% CO2 (•); Batch CAM60, 2% citric acid and packaging in 40% N2/60% CO2 (▲); Batch CAV (2% citric acid and packaging under vacuum (Δ).     Mean ± standard deviation. Means in the same column with no superscript letters in common are significantly different (p < 0.05). λ, lag phase (day); µ max , maximum growth rate (log cfu/g/day); R 2 , coefficient of determination. Treatment conditions: Batch C, distilled water and packaging in air; Batch CA, 2% citric acid and packaging in air; Batch CAMA20, 2% citric acid and packaging in 80% N 2 /20% CO 2 ; Batch CAMA40, 2% citric acid and packaging in 60% N 2 /40% CO 2 ; Batch CAMA60, 2% citric acid and packaging in 40% N 2 /60% CO 2 ; Batch CAMAV, 2% citric acid and packaging under vacuum. Figure 8 shows the pH values of chicken legs treated with organic acid and packaged under MAP. Initial pH value in control samples was 6.4 ± 0.11, while pH values in legs washed with solutions containing 2% citric, propionic or acetic acid (day 0) were 5.19 ± 0.09, 4.78 ± 0.06 and 4.80 ± 0.10, respectively. Significant reductions (p < 0.05) in pH value were observed in samples washed with an organic acid and packaged in 60% N 2 /40% CO 2 or 40% N 2 /60% CO 2 when compared to those washed with the same organic acid and packaged in air or vacuum, except on day 0. CO 2 concentrations decreased by about 4% during storage in all packaged samples. After 3 days of storage, CO 2 concentrations remained unchanged Animals 2020, 10, x 11 of 17 Figure 8 shows the pH values of chicken legs treated with organic acid and packaged under MAP. Initial pH value in control samples was 6.4 ± 0.11, while pH values in legs washed with solutions containing 2% citric, propionic or acetic acid (day 0) were 5.19 ± 0.09, 4.78 ± 0.06 and 4.80 ± 0.10, respectively. Significant reductions (p < 0.05) in pH value were observed in samples washed with an organic acid and packaged in 60% N2/40% CO2 or 40% N2/60% CO2 when compared to those washed with the same organic acid and packaged in air or vacuum, except on day 0. CO2 concentrations decreased by about 4% during storage in all packaged samples. After 3 days of storage, CO2 concentrations remained unchanged  The sensorial acceptability of legs washed with organic acids and packaged under MAP is given in Figure 9. No significant (p > 0.05) effect on overall appearance were observed on days 0 and 1 between the different treatments. Legs that were packaged in air and not washed with organic acids were unacceptable on day 6, while those washed with organic acids were rejected on day 8 (citric acid) or 10 (acetic and propionic acid). The packaging in 60% N 2 /40% CO 2 or 40% N 2 /60% CO 2 in combination with citric, propionic or acetic acid resulted in a longer shelf life of chicken legs by at least 5 days compared to poultry legs washed with organic acids and packaged in air.
Animals 2020, 10, x 12 of 17 The sensorial acceptability of legs washed with organic acids and packaged under MAP is given in Figure 9. No significant (p > 0.05) effect on overall appearance were observed on days 0 and 1 between the different treatments. Legs that were packaged in air and not washed with organic acids were unacceptable on day 6, while those washed with organic acids were rejected on day 8 (citric acid) or 10 (acetic and propionic acid). The packaging in 60% N2/40% CO2 or 40% N2/60% CO2 in combination with citric, propionic or acetic acid resulted in a longer shelf life of chicken legs by at least 5 days compared to poultry legs washed with organic acids and packaged in air.

Discussion
Atmospheres with 60-80% N 2 and 20-40% CO 2 are usually used when packaging chicken [12]. In the current work, the effects of four atmospheres, namely 80% N 2 /20% CO 2 , 60% N 2 /40% CO 2 , 40% N 2 /60% CO 2 and vacuum, were evaluated. The use of atmospheres containing CO 2 or vacuum retarded the period to reach psychrotroph populations of 9 log cfu/g. Our data indicate that the atmosphere with the highest CO 2 concentration (40% N 2 /60% CO 2 ) was the most effective in decreasing the growth of psychrotrophs and extending the shelf life of chicken legs. Carbon dioxide has a more marked antimicrobial effect against psychrotrophic Gram-negative bacteria such as Pseudomonas, resulting in retarding the beginning of microbial spoilage [13]. Our data are in line with those presented by Arvanitoyannis and Stratakos [15], who reported that CO 2 affects microorganisms by causing a decrease in the growth rate and an extension of the lag phase. Other researchers have also shown the reduction of psychrotrophs in poultry and meat storage under modified atmospheres [16,17].
The ability of L. monocytogenes to grow on chicken packaged in enriched CO 2 atmospheres has also been pointed out by other authors [18,19]. In the current work, it was observed that the maximum growth rate of psychrotrophs in chicken legs packaged in MAP was lower than that of L. monocytogenes. Moreover, no lag phase for L. monocytogenes was detected in chicken samples packaged in modified atmospheres, while an extended lag phase was observed for psychrotrophs. Moreover, Marshall et al. [20] reported that L. monocytogenes can grow faster than spoilage bacteria in poultry meat packaged in MAP. Franco-Abuin et al. [19] found that an atmosphere containing 100% CO 2 is more effective for the inhibition of growth of L. monocytogenes than other atmospheres with lower CO 2 content. However, none of the gas mixtures was bactericidal. These findings suggest that strategies should be designed in order to control the growth of L. monocytogenes in poultry meat packaged in MAP.
Other authors have also observed a similar development of gas atmosphere in chicken filets packaged under MAP and kept at 4 • C [16,21]. Due to the high solubility of CO 2 in tissues, a small reduction of CO 2 was detected in all the packages over the first 2 days [22]. Our findings are in line with those of García de Fernando et al. [23], who reported a decrease in pH in meat packaged in atmospheres containing a high CO 2 concentration (40-60% CO 2 ).
Combinations of citric, acetic or propionic acid with packaging in modified atmospheres were effective in decreasing the growth of psychrotrophs, reducing their maximum growth rate and prolonging their lag phase in chicken meat. These treatments were more effective than treatments with these organic acids in nonpackaged chicken meat. In an earlier study, it was found that washing legs with 2% citric acid for 5 min decreased psychrotroph growth by between 0.58 and 1.1 log units compared with the control samples [8]. Cutter and Siragusa [24] also reported the efficacy of citric acid in reducing psychrotroph growth in beef. In the current study, the washing with 2% citric acid and packaging in 40% N 2 /60% CO 2 decreased psychrotroph growth by between 0.99 and 3.12 log units compared to the control samples (nontreated and packaged in air), while the reductions in legs washed with citric acid and packaged in air were between 0.74 and 1.1 log units. In another study, it was found that a treatment with 2% propionic acid for 5 min decreased psychrotroph growth by between 1.27 and 2.13 log units compared with the control samples [9]. In the current study, the washing with 2% propionic acid and packaging in 40% N 2 /60% CO 2 decreased psychrotroph growth by between 1.11 and 2.9 log units compared to the control samples, while the reductions in legs washed with propionic acid and packaged in air were between 1.1 and 1.29 log units. Jimenez et al. [13] also found that the immersion of chicken breast in acetic acid reduced psychrotroph growth. In an earlier study, it was found that washing with 2% acetic acid for 5 min decreased psychrotroph growth by between 0.49 and 2.52 log units compared with the control samples throughout storage [10]. In the current study, nontreated legs packaged in 40% N 2 /60% CO 2 showed a psychrotroph growth reduction of between 1.16 and 2.04 log units compared with the control samples, while reductions of between 0.91 and 2.19 log units were found in legs treated with acetic acid and packaged in air. The treatment with 2% acetic acid and packaging in 40% N 2 /60% CO 2 decreased psychrotroph growth by between 2.51 and 4.99 log units compared to the control samples, representing an additional 0.76 unit reduction when compared to the separate treatments. It seems that treatment with acetic acid and packaging in 40% N 2 /60% CO 2 had a synergistic effect in reducing psychrotroph growth in chicken meat.
Other researchers have also pointed out that the combination of MAP with organic acids can increase the inhibitory effect against bacteria. According to Jimenez et al. [13], 1% acetic acid decontamination and packaging in 30% N 2 /70% CO 2 can reduce the growth of Pseudomonas and prolong the lag phase of lactic acid bacteria in chicken breasts. Sawaya et al. [25] pointed out that a pretreatment of poultry with lactic acid and packaging in 70% CO 2 reduced psychrotroph growth and prolonged the shelf life of poultry. Djenane et al. [26] studied the effect of different modified atmospheres (40% CO 2 /60% O 2 and 10% N 2 /20% CO 2 /70% O 2 ) in combination with antioxidants (0.05% ascorbic acid and 0.1% rosemary) and lactic acid treatment on beef steaks. These authors observed that Pseudomonas spp. was inhibited by the treatment with lactic acid and the higher concentrations of CO 2 . The combination of organic acid treatments and CO 2 packaging seems to be more effective against spoilage bacteria than the combination with vacuum packaging [27]. Sawaya et al. [25] showed that a combination of sorbate treatment and vacuum packaging decreased microbial growth and prolonged the shelf life of chicken carcasses. Other decontaminating agents in combination with MAP can have an enhanced inhibitory effect against bacteria in chicken meat [28,29].
The reduction of pH values of samples washed with organic acids has also been reported by other authors [28]. Sensory quality was not negatively affected by 2% citric, propionic or acetic acid washing or vacuum packaging [8][9][10]13,30,31].
The combination of modified atmosphere packaging (MAP) and organic acid treatment of chicken meat seems to be more effective in reducing L. monocytogenes than treatment of nonpackaged chicken with organic acids. In an earlier study, it was found that legs washed with 2% citric acid for 5 min showed a significant growth reduction in L. monocytogenes (between 0.22 and 1.12 log units compared with the control samples) [8]. Moreover, Menconi et al. [32] showed the efficacy of citric acid against L. monocytogenes in chicken skin. In the current study, the washing with 2% citric acid and packaging in 40% N 2 /60% CO 2 reduced L. monocytogenes growth by between 1.13 and 2.15 log units compared to the control samples (nontreated and packaged in air) while no reduction of L. monocytogenes growth was found in legs packaged in 40% N 2 /60% CO 2 , and reductions of between 0.67 and 1.06 log units were found in legs treated with citric acid and packaged in air (1.09 units more than the separate treatments). In an earlier study, it was found that samples washed with 2% propionic acid for 5 min showed a significant decrease in L. monocytogenes growth, with reductions between of 1.89 and 2.72 log units compared with the control samples [9]. In the current study, the washing with 2% propionic acid and packaging in 40% N 2 /60% CO 2 reduced L. monocytogenes growth by between 2.14 and 3.3 log units compared to the control samples (nontreated and packaged in air), while no reduction of L. monocytogenes growth was observed in legs packaged in 40% N 2 /60% CO 2 , and reductions of between 2.14 and 2.81 log units were found in legs washed with propionic acid and packaged in air (0.49 units more than the separate treatments). In an earlier study, it was found that legs treated with 2% acetic acid for 5 min showed a significant decrease in L. monocytogenes growth, with reductions of between 0.65 and 1.66 log units compared with the control samples [10]. Dorsa et al. [33] also observed that the treatment of beef with 1.5-3% acetic acid reduced Listeria growth. In the current study, the treatment with 2% acetic acid and packaging in 40% N 2 /60% CO 2 decreased L. monocytogenes growth by between 0.88 and 2.09 log units compared to the control samples (nontreated and packaged in air), while no reduction in L. monocytogenes growth was observed in legs packaged in 40% N 2 /60% CO 2 , and growth reductions of between 0.88 and 1.87 log units were found in legs treated with acetic acid and packaged in air (0.22 units more than the separate treatments). It seems that treatment with citric, propionic and acetic acids and packaging in 40% N 2 /60% CO 2 had a synergistic effect in reducing L. monocytogenes growth in chicken meat.
After treatment with organic acids (day 0), the most effective treatment against L. monocytogenes was the propionic acid. Greater growth reductions of L. monocytogenes counts were obtained in legs washed