L. monocytogenes in sterile chicken loaf in the presence of. Pseudomonas fragi at 3, 7, ... Research Board through a grant administrated by the California Dairy.
Journal of Food Protection, Vol. 55. No. 2, Pages 88-92 (February 1992) Copyright©, International Association of Milk, Food and Environmental Sanitarians
Interactive Effect of Temperature, Atmosphere and Storage Time on the Probability of Colony Formation on Blood Agar by Four Listeria Species VADOOD RAZAVILAR and CONSTANTIN GENIGEORGIS* Department of Epidemiology and Preventive Medicine, School of Veterinary Medicine, University of California, Davis, California 95616 (Received for publication July 1, 1991)
ABSTRACT
The probability (P) of one Listeria cell and the cells needed to initiate colony formation on sheep blood agar plates as affected by atmospheric conditions (AC), storage temperature (T), time (t), and Listeria species was evaluated. The factorial design experiments included Listeria monocytogenes (2 strains), Listeria seeligeri, Listeria ivanovii, and Listeria innocua, as test organisms, storage of the plates at 4, 8, 20, and 30°C under air (A), modified atmosphere (MA) of 5% O, + 10% C0 2 + 85% N„ 100% C0 2 (C02), vacuum (V), and candle jar (CJ) for 7, 14, 21, 42, and 56 d. Statistical analysis indicated the significant effect of AC (p20°C. L.MONOCYTOGENES VPH-2
LMONOCYTOGENES VPH-1
Time(days)
Figure l. Comparison of air (A), modified atmosphere (MA), consisting of 5% 02 + 10% C02 + 85% N2, 100% C02 (C02), vacuum (V), and microaerophilic atmosphere of candle jar (CJ) on the probability of colony formation by one L. monocytogenes cell on TSBA agar incubated at temperatures of 4 to 20°C for up to 56 d. L.SEEL1GER1
L.INNOCUA
L.IVANOVII
Time(days)
Figure 2. Comparison of effect of air (A), modified atmosphere (MA) consisting of 5% 02 + 10% C02 + 85% N2, 100% C02 (CO ), vacuum (V), and microaerophilic atmosphere of candle jar (CJ) on the probability of colony formation by one cell of L. seeligeri, L. innocua, and L. ivanovii on TSBA agar incubated at temperatures of 5 to 20°C for up to 56 d.
JOURNAL OF FOOD PROTECTION, VOL. 55, FEBRUARY 1992
RAZAVILAR AND GENIGEORGIS
90
inhibitory to growth initiation than any other AC (p0.2) among MA, A, V, and CJ atmospheres. The significantly increasing inhibitory effect of C 0 2 with decreasing temperature is probably due to the greater dissolution of C 0 2 in water with decreasing temperature (13,18,31). At 8°C, C 0 2 extended the lag phase (time from beginning until first change in P) of Listeria growth but did not affect the rate of growth (slope of the P curve) as compared to other AC's. At 4°C, C 0 2 extended the lag phase and decreased the rate of growth as compared to other AC's. It has been reported that the growth kinetics of L. monocytogenes in a broth medium stored at 5 to 37°C under aerobic and anaerobic atmospheres were similar (7,8). Sterile lamb meat inoculated with L. monocytogenes and then packaged in gas permeable and gas impermeable film and stored at 8°C showed no remarkably different effect of the type of packaging on the growth of Listeria (24). Beuchat et al. (4) did not notice any difference in the effect of ambient air and 3% O, + 97% N 2 atmosphere on the growth of L. monocytogenes strains on shredded lettuce stored at 5 and 10°C for up to 15 d. Kallander et al. (23) showed that a modified atmosphere containing 70% C 0 2 + 30% N , surrounding shredded cabbage at 5 and 25°C, had no major influence on the fate of L. monocytogenes. Our findings are in disagreement with these studies since we demonstrated that only at 20 and 30°C, there was no difference among the five atmospheres tested. At 20°C. At 8°C, formation of a colony within 7 d required 6.3 x 105, 40, 18, 9, and 4 cells of strain VPH-1 exposed to C0 2 , CJ, V, MA, and A atmospheres, respectively. The corresponding numbers of cells for the 4°C incubation (within 7 d), were 7.4 x 105 for all atmospheres. After 14 d incubation at 4°C, the corresponding numbers were 8.7 x 104, 87, 87, 9, and 2 for C 0 2 , MA, A, CJ, and V, respectively. After 42 d storage at 4°C, even 1 -6 cells were able to form a colony under all AC for both L. monocytogenes strains. The growth profile of L. seeligeri, L. innocua, and L. ivanovii at 8 to 30°C under all atmospheric conditions was very similar (Fig. 2). At 4°C, under 100% C0 2 , L. ivanovii was the most sensitive and L. monocytogenes VPH-1 was the most resistant. Analysis of variance indicated that the log10P% was affected significantly by three main [AC (p0.65), and AC x temperature (p>0.46)]. A pairwise comparison of the mean logP% of growth of Listeria spp., in each of the five AC on TSBA plates (Table 2), indicated that CO, was significantly more
TABLE 1. Estimated number of cells of L. monocytogenes needed to initiate growth and colony formation on TSBA agar under five atmospheric conditions (AC) at 4, 8, and 20°C for 7 to 42 d. Storage time (d) 7 a
AC A
MA
CO,
CJ
14
Temp (°C)
42
14
7
L. monocytogenes VPH-1
L. monocytogenes VPH-2
20 8 4
2 1 7.4
20 8 4
2 6 7.4
20 8 4
2 6.3 7.4
20 8 4 20 8 4
105
2 1 13
2 1 1
2 4 7.4
105
2 1 3
2 1 1
1 9 7.4
105 105
2 6 7.4 x 10s
2 3 6
4 6.3 7.4
1 6 7.4 x 105
2 6 63
2 3 1
1 18 7.4
2 6.3 x 10s 7.4 X 105
2 1 3
2 1 1
4 40 7.4
X
X
X X
42
a A = air. MA = modified atmosphere of 5% 0 2 + 10% CO, + 85%N2, CO, = 100% CO, V = vacuum. CJ = candle jar atmosphere. JOURNAL OF FOOD PROTECTION, VOL. 55, FEBRUARY 1992
X
X
X X
X
X
105
2 2 87
2 1 4
10s
1 1 87
1 1 1
105 10s
4 2 8.7 x 104
4 1 4
10s
1 2 2
1 2 2
105
4 4 9
4 4 2
COLONY FORMATION ON BLOOD AGAR BY LISTERIA SPP. TABLE 2. p values for pairwise comparison of probability of colony formation of Listeria spp. on TSBA agar stored in five atmospheric conditions (AC) at 4 to 30°C for 7 to 56 d. AC»
CJ
A
V
MA
C02
0.0039
0.0006
0.0004
0.0000
0.5883
0.5188
0.2138
0.9171
0.4817
CJ A V
0.5488
a
A = air. MA = modified atmosphere of 5% 0 2 + 10% C02 + 85% N2, C02 = 100% C02. V = vacuum. CJ = candle jar atmosphere.
Seeliger (35) has stated that "Listeria thrives best at reduced oxygen tension" and that "after replacement of oxygen by carbon dioxide growth is excellent". This is contradictory to our findings, which indicated an increased inhibitory effect of 100% C0 2 with decreasing incubation temperature, and no statistically significant difference among the other four AC's. Ingham et al. (22) compared the effect of air and two COz containing modified atmospheres (C0 2 80% + 0 2 0%, and CO, 50% + On 10%) on the growth of 2
2
2
/
°
L. monocytogenes in sterile chicken loaf in the presence of Pseudomonas fragi at 3, 7, and 11°C. After 6 d storage at 3°C, inoculated L. monocytogenes increased by 3.14, 0.52, and 0.87 log|0 in air, 50%, and 80% C0 2 , atmospheres, respectively. At 7°C the corresponding log]f) increases were 4.8, 3.86, and 2.81 and at 11°C they were 5.18, 5.59, and 3.77. Hart et al. (21) did not observe growth of L. monocytogenes on chicken breast meat stored at 6°C under 30100% C0 2 atmospheres, although growth occurred under aerobic conditions. According to Gill and Reichel (19), L. monocytogenes grew in vacuum-packaged beef at >2°C, while under atmosphere of 100% C0 2 growth was observed when the storage temperature was >5°C. The extensive killing effect of high C0 2 pressure (61.2 atm) on L. monocytogenes in distilled water and chicken meat has been reported (38). In a recent paper, Buchanan and Klawitter (9) concluded that though L. monocytogenes generally grows better under aerobic conditions, limiting oxygen appeared to enhance survival and growth when other growth factors like pH and temperature were suboptimal. In our study, only at 4°C the inhibitory effect of air was similar to C0 2 for L. seeligeri but not for the other Listeria species. Overall, our study demonstrated quantitatively the increasing inhibitory effect of high concentrations of C0 2 on the growth of Listeria species with storage temperatures of
