The Effect of Incubation Temperature on Recovery of Mesophilic Bacteria from Broiler Chicken Carcasses Subjected to Temperature Abuse

The Effect of Incubation Temperature on Recovery of Mesophilic Bacteria from Broiler Chicken Carcasses Subjected to Temperature Abuse S. M. RUSSELL,1 D. L. FLETCHER,1'2 and N. A. COX3 Department of Poultry Science, University of Georgia, Athens, Georgia 30602 and USDA, Agricultural Research Service, Richard B. Russell Agricultural Research Center, P.O. Box 5677, Athens, Georgia 30613

1994 Poultry Science 73:1144-1148

INTRODUCTION Immediately after slaughter, the initial microbial flora of a broiler chicken carcass reflects the population of mesophilic bacteria on the bird prior to slaughter. A number of studies have been conducted to

Received for publication October 22, 1993. Accepted for publication March 2, 1994. HJniversity of Georgia. 2 To whom correspondence should be addressed. 3USDA.

compare the bacterial profile on carcasses immediately after slaughter to the profile after the carcasses have been stored at refrigerated temperatures. Barnes and Thornley (1966) reported that the initial microbial flora of the broiler carcass consisted of Micrococcus (50%), Gram-positive rods (14%), Flavobacterium (14%), Enterobacteriaceae (8%), Acinetobacter (7%), Pseudomonas (2%), and unidentified organisms (5%). After storage at 1 C for 10 to 11 d, the flora was primarily psychrotrophic bacteria: 90%

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ABSTRACT The optimum temperature to recover mesophilic organisms from temperature-abused broiler chicken carcasses was determined. Twenty broiler carcasses were collected from a commercial processing plant in each of three trials. Ten carcasses were sampled immediately (Day 0 control); 8 carcasses were sampled after holding at 3 C for 3 d, temperature abusing at 25 C for 12 h, and holding them at 3 C for an additional 3 d (temperature abused); and the remaining 2 carcasses were sampled after holding at 3 C for 7 d (Day 7 control). Whole carcass rinses were analyzed using total aerobic plate counts (TPC) and impedimetric detection times (DT) at incubation temperatures of 41, 42,43, and 44 C. Total plate counts decreased (P < .05) and DT increased (P < .05), at all incubation temperatures tested, for carcasses held under refrigeration (Day 7 control). This demonstrates that mesophilic bacteria, rather than psychrotrophic bacteria, are being enumerated, because psychrotrophs increase on chicken skin upon cold storage. Total plate count was greater (P < .05) and DT was significantly lower for temperature-abused vs Day 7 control birds (5.7 vs 3.6 log10 cfu/mL and 3.7 vs 6.5 h), respectively. These results indicate that temperature-abused carcasses can be microbiologically distinguished from nonabused controls at all incubation temperatures tested. Recovery of mesophiles was optimal at 41 and 42 C for TPC and 41, 42, and 43 C for DT. Because there were no differences between DT at 41,42, and 43 C, and because 42 C has been successfully used in the past, either 42 C or 43 C can be used when determining DT. (Key words: temperature abuse, incubation temperature, mesophiles, impedance, broiler)

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Pseudomonas spp., 7% Acinetobacter, andmedia held at temperatures less than 5 C, 3% Enterobacteriaceae. Regez et al. (1988) as determined by total aerobic plate count found similar results in that, immediately (IPC), and 6 C, as determined using after processing, broiler carcasses con- impedance detection time (DT). In another tained approximately 35% Micrococcus, study using whole broiler carcasses held 24% Coryneforms, 16% Lactobacillus, 16%at 4 C, the level of mesophilic bacteria Enterobacteriaceae, 3% Aerotnonas and remained constant or decreased when rinse samples were incubated at 42 C, Vibrio, 2% Flavobacterium, 2% Acinetobacter and Moraxella, 2% Brochothrix ther- using both TPC and DT (Russell et al, mosphacta, and 0% pseudomonads. How- 1992b). This study also showed that ever, after storage at 4 C for 13 d, temperature-abused carcasses held at 15 C psychrotrophic bacteria predominated, for 12 h or 20 C for as little as 4 h could be with: 72% Pseudomonas, 20% Brochothrixmicrobiologically distinguished from thermosphacta, and 8% Acinetobacter and those that were held at 4 C. Although some bacteria may be classiMoraxella. These studies show that mesophilic organisms such as the En- fied as both mesophilic and psychroterobacteriaceae decrease and psychro- trophic, such as Listeria, which has an trophic organisms such as Pseudomonas optimum growth temperature of 35 C and and Acinetobacter increase in number when will grow at refrigerated temperatures, carcasses are held at temperatures below 4 these bacteria have little practical importance on determining temperature abuse C. by separating the mesophilic and psychroBacteria that are found in high numbers initially on broiler carcasses generally trophic bacterial populations using selechave higher optimum growth temperature tive incubation temperatures. This has ranges than organisms responsible for been illustrated by the general decrease in spoilage and are considered to be total bacterial numbers of organisms remesophilic. Therefore, after storage of covered at 42 C from carcasses stored at carcasses at 4 C, these mesophiles cannot refrigerated temperatures (<3 C) for 7 d compete with the rapidly growing (Russell et al, 1992a). Buchanan et al. (1991) and Russell et al. psychrotrophs, such as the pseudomonads, whose lower optimum growth (1992a,b), using an incubation temperature temperatures allow them to multiply at a of 42 C, and Firstenberg-Eden and Tricarico (1983), using 35 and 37 C, were able to faster rate. Buchanan et al. (1991) showed, that by differentiate mesophiles from psychroincubating total plate counts at 42 C, trophs; however, no attempts were made mesophilic bacteria can be separated from to determine which temperature was oppsychrotrophic bacteria in mixed samples timal or whether higher or lower incubacontaining both groups of organisms. tion temperatures would be more apSamples of ground chicken that were propriate. The purpose of this study was stored at 5 C could be separated from to determine the optimal incubation temthose stored at 12 or 19 C by incubating perature to separate mesophilic and plates at 42 C. Bacteria recovered at 42 C psychrotrophic bacteria directly from incubation, on samples tfiat were stored at broiler carcasses subjected to controlled 5 C, remained the same, whereas bacteria temperature abuse. on samples held at 12 or 19 C reached levels of 8 to 10 logxo cfu/g. The number MATERIALS AND METHODS of bacteria on samples held at 12 or 19 C Twenty ready-to-cook whole broiler increased because the temperature threshold, below which mesophilic bacteria will chicken carcasses were collected from the not grow, was exceeded and the chiller exit of a commercial processing facility in each of three separate trials over mesophiles began to multiply. Russell et al. (1992a) found that the a 9-mo period. The carcasses were transnumber of mesophilic bacteria recovered ported on ice, maintained at 3 C, and by incubating samples at 42 C remained individually bagged in sterile polyethylconstant or decreased in carcass *inse ene bags within 1 h of collection. Car-

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4 3M Microbiology Products, St. Paul, MN 55144-1000. 5 Difco Laboratories, Detroit, MI 48232. 6 bioMerieux Vitek, Inc., Hazelwood, MO 63042.

[•1 M2 D 3 |

6

^

I

I 6

a

FIGURE 1. Total plate counts (TPC) and impedance Bactometer detection times (DT) (X ± SEM) over all incubation temperatures (41, 42, 43, and 44 C) for carcasses analyzed at Day 0 (Treatment 1), held for 3 d at 3 C, temperature-abused at 25 C for 12

h, held again for an additional 3 d, and then analyzed (Treatment 2), and carcasses held for 7 d at 3 C and then analyzed (Treatment 3). Treatments within TPC or DT with no common letter differ significantly (P < .05).

ature of incubation, and the specific growth kinetics of the organism or group of organisms in a particular medium (Firstenberg-Eden, 1983). Results were analyzed using analysis of variance of the General Linear Models (GLM) procedure of SAS® (SAS Institute, 1988). Treatment means were separated using Duncan's multiple range option. For TPC, all counts were transformed to log10 cfu/mL prior to analyses. Incubation temperature means were separated using the contrast option of the GLM procedure. For both TPC and DT, there was a trial by treatment interaction (P < .05). Therefore, effects were tested using trial by treatment as the error term. RESULTS AND DISCUSSION Results for TPC and DT for the three treatments averaged over the three trials and all incubation temperatures are presented in Figure 1. For the temperature-abused carcasses (Treatment 2), TPC values were greater (P < .05) and DT values were lower (P < .05) than the Day 0 controls (Treatment 1). Because DT is the time required for a particular organism or group of organisms to reach a level of 106, a low DT is indicative of a high TPC. Therefore, TPC and DT values are inversely proportional to each other. Mesophilic bacteria recovered from Day 7 control carcasses (Treatment 3) were lower

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casses in each trial were subjected to one of three storage treatments as follows: 1) 10 carcasses were sampled immediately (Day 0 control); 2) 8 carcasses were sampled after holding at 3 C for 3 d, temperature abusing at 25 C for 12 h, then holding again at 3 C for 3 d (temperature abused); and 3) 2 carcasses were sampled after holding at 3 C until Day 7 (Day 7 control). Three replicate trials were conducted using incubation temperatures of 42 and 44 C and two replicate trials were conducted using incubation temperatures of 41 and 43 C. All carcasses were sampled using 100 mL sterile deionized water according to the rinse procedure described by Cox et al. (1981). Carcass rinse fluid was used for the determination of both TPC and DT. Total plate counts were performed using Petrifilm®4 Aerobic Count Plates according to the procedure described by Swanson et al. (1992) except that plates were incubated at 41 to 44 C instead of 35 C for 48 h. Carcass rinses were diluted to 102, 103, and 104 (Day 0 and Day 7 rinses) or 104, 105, and 106 (temperature-abused carcass rinses) using sterile BactoPeptone.5 One milliliter of diluent was placed onto one Petrifilm Aerobic Count Plate in duplicate. Petrifilm® plates were incubated at 41, 42, 43, and 44 C for 48 h. Impedance measurements were conducted using the Bactometer Microbial Monitoring System M128,6 using the procedure described by Russell et al. (1991b). One milliliter of carcass rinse fluid was diluted tenfold using sterile Brain Heart Infusion Broth.5 One milliliter of the diluent was placed into one Bactometer module sample well in duplicate. Modules were incubated at the same temperatures as used for TPC. The number of bacteria required for impedance changes to be detected is 106 to 107 cells/mL (Firstenberg-Eden, 1983). The time required for bacterial growth to reach the threshold level is defined as the DT and is a function of initial concentration of bacteria, temper-

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TABLE 1. Total plate counts (TPC) and Bactometei detection times (DT) at 41, 42, 43, and 44 C for carcasses analyzed at Day 0 (Treatment 1), carcasses held for 3 d at 3 C, temperature-abused at 25 C for 12 h, held again for an additional 3 d, and then analyzed (Treatment 2), and carcasses held for 7 d at 3 C and then analyzed (Treatment 3) Treatment temperature (Q 41

1 3.9 20 1 4.0 30 3.9 20 3.9 30

42 43 44

2

1

2

± .06

4.9 ± .10b 20

± .24

4.8 ± .07b 30 5.1 ± .lib 20 5.4 ± .10* 30

3.5 16 3.4 24 3.6 16 3.8 24

3

TPC (log 10 cfu/mL) 3.2 5.7 ± .11* 16 4 3.2 ± .07 5.9 ± .11" 24 6 ± .07 5.3 ± . l l b 3.1 16 4 ± .07 5.3 ± .14b 2.9 6 24

± .07

3 ryr (hi .

± .12 ± .31

± ,llb ± .10b ± .12b ± .ll a

6.2 4 5.9 6 6.3 4 6.6 6

± .11 ± .24 ± .05 ± .27

ab

(P < .05) than those recovered from Day 0 carcasses (Treatment 1) in all three trials, using TPC, and significantly higher, using DT. These results verify that mesophilic bacteria recovered at 41, 42, 43, or 44 C decrease on carcasses held at proper refrigeration and that the technique for determining temperature abuse described by Russell et al. (1992b) was able to microbiologically distinguish carcasses that had been temperature abused from those held at proper storage temperatures. Results for TPC and DT at 41, 42, 43, and 44 C for the three separate treatment groups are presented in Table 1. For Day 0 carcasses (Treatment 1) and Day 7 carcasses (Treatment 3) there were no differences (P > .05) in TPC, using different incubation temperatures. However, for Day 0 carcasses (Treatment 1), DT were higher (P < .05) at 44 C than samples incubated at lower temperatures. The DT for Day 7 carcasses (Treatment 3) were not different (P > .05). These results indicate that, at relatively low numbers, mesophilic bacteria are recovered equally well at any of the incubation temperatures tested, using TPC. However, the DT values indicate that, above 43 C, DT increases, demonstrating that the organisms are no longer able to proliferate as rapidly due to the high temperature. For the temperature-abused carcasses (Treatment 2), TPC was lower (P < .05) at 43 C and 44 C, respectively, than at

incubation temperatures of 41 and 42 C. However, DT were found to be higher (P < .05) at 44 C than when samples were incubated at 43 C or below. At high levels, as is the case with temperature-abused carcasses, mesophilic bacteria are recovered best at incubation temperatures of 41 and 42 C using TPC and 41, 42, and 43 C using DT. Mesophilic bacteria have a maximum growth temperature above which they multiply more slowly, are sublethally injured, or are destroyed. The ideal temperature to enumerate mesophiles from a mixed sample must be low enough so as not to reduce their recovery as a result of heat stress and high enough such that the psychrotrophic bacteria, which normally multiply on refrigerated poultry, are inhibited. Recovery of mesophilic bacteria from temperature-abused carcasses (Treatment 2), using TPC, appears to be most efficient at incubation temperatures 41 or 42 C, because the number of bacteria recovered did not decrease until samples were incubated at 43 or 44 C. The recovery of organisms using DT appears to be most efficient at incubation temperatures of 41, 42, and 43 C because, above 43 C, the DT significantly increased, which indicates a decrease in microbial growth. However, because 42 C has been used to separate mesophiles from psychrotrophs in broiler carcass rinses in previous studies, does not produce results significantly different

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' Means ± SEM within columns with no common superscript differ significantly (P < .05). 'Number of carcasses sampled per mean.

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ACKNOWLEDGMENTS

This study was supported in part by state and Hatch funds allocated to the Georgia Agricultural Experiment Station. Appreciation is extended to bioMerieux Vitek, Inc. for providing the Bactometer Microbial Monitoring System M128, technical assistance, and supplies.

REFERENCES Barnes, E. M, and M. J. Thornley, 1966. The spoilage flora of eviscerated chickens stored at different temperatures. J. Food Technol. 1:113-119.

Buchanan, R. L., F. J. Schultz, and L. A. Klawitter, 1991. Effectiveness of various indicators for detection of temperature abuse in refrigerated meat, poultry, and seafood. Presented at the Institute of Food Technologists. Abstract Number 135.(Abstr.) Cox, N. A., J. E. Thomson, and J. S. Bailey, 1981. Sampling of broiler carcasses for Salmonella with low volume water rinse. Poultry Sci. 60:768-770. Firstenberg-Eden, R., 1983. Rapid estimation of the number of microorganisms in raw meat by impedance measurement. Food Technol. 37: 64-70. Firstenberg-Eden, R., and M. K. Tricarico, 1983. Impedimetric determination of total, mesophilic and psychrotrophic counts in raw milk. J. Food Sci. 48:1750-1754. Regez, P., L. Gallo, R. E. Schmitt, and W. SchmidtLorenz, 1988. Microbial spoilage of refrigerated fresh broilers. HI. Effect of storage temperature on the microbial association of poultry carcasses. Lebensm.-Wiss. & Technol. 21:229-233. Russell, S. M., D. L. Fletcher, and N. A. Cox, 1992a. A model for determining differential growth at 18 and 42 C of bacteria removed from broiler chicken carcasses using traditional plate counts and impedance microbiology. J. Food Prot. 55(3):167-170. Russell, S. M, D. L. Fletcher, and N. A. Cox, 1992b. A rapid method for the determination of temperature abuse of fresh broiler chicken. Poultry Sci. 71:1391-1395. SAS Institute, 1988. SAS®/STAT Guide for Personal Computers. Version 6.03 Edition. SAS Institute Inc., Cary, NC. Swanson, K.M.J., F. F. Busta, E. H. Peterson, and M. G. Johnson, 1992. Colony count methods. Pages 88-89 in: Compendium of Methods for the Microbiological Examination of Foods. C. Vanderzant and D. Splittstoesser, ed. American Public Health Association, Washington, DC.

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from 41 or 43 C, and is also appropriate for conducting TPC, it may be more practical to use for DT. The differences found between the two microbiological enumeration methods are most likely due to the fact that TPC measures all genera of organisms that proliferate at 42 C (Firstenberg-Eden et at, 1983), whereas above 42 C not as many genera of organisms are able to multiply. At incubation temperatures above 43 C, using impedance, perhaps the organism or group of organisms that was previously being measured in the sample well is no longer able to multiply or multiplies at a slower rate. A greater understanding of the optimum temperature to enumerate mesophiles will lead to more accurate and reproducible results when conducting studies on temperature abuse.