Growth of Listeria monocytogenes Scott A, serotype 4 and competitive spoilage organisms in raw chicken packaged under modified atmospheres and in air

International Journal of Food Microbiology, 11 (1990) 205-214 Elsevier

205

FOOD 00325

Growth of Listeria monocytogenes Scott A, serotype 4 and competitive spoilage organisms in raw chicken packaged under modified atmospheres and in air * Linda Wimpfheimer 1, Naomi S. Altman 2 and Joseph

H. H o t c h k i s s 1

1 Department of Food Science, Institute of Food Science, Stocking Hall, Cornell University, Ithaca, NY, U.S.A., and 2 Department of Plant Breeding and Biometry, Stocking Hall, Cornell University, Ithaca, NY, U.S.A. (Received 17 October 1989; accepted 18 April 1990)

The development of Listeria monocytogenes Scott A, serotype 4 and aerobic plate counts on minced raw chicken were determined independently at 4, 10, and 27 ° C. Samples were packaged in flexible film under two modified atmospheres (one containing oxygen and one containing no oxygen) or air. The anaerobic modified atmosphere (75 : 25, CO 2 : N2) resulted in the failure of both the aerobic plate counts and L. monocytogenes to grow at all temperatures. Both the L. monocytogenes and aerobic plate counts grew in air at all temperatures. The aerobic modified atmosphere (72.5 : 22.5 : 5, CO 2 : N 2 : O2), which more closely duplicates commercial practice, inhibited the increase in aerobic plate counts by more than 4 log10 c f u / g compared to air at 4 ° C . However, the L. monocytogenes was not affected by this atmosphere and increased in numbers by nearly 6 lOgl0 c f u / g at 4 ° C in 21 days. Regression analysis of the log10 growth and 95% confidence intervals showed that the differences between aerobic plate counts and L. monocytogenes in modified atmosphere were large. The ability of L. monocytogenes to grow in the aerobic modified atmosphere was not affected by level of the L. monocytogenes inoculum nor by the initial level of aerobic plate counts. These data show that modified atmosphere packaging of raw chicken (and probably other meats) can substantially inhibit the aerobic spoilage flora while allowing pathogenic L. monocytogenes to increase. Key words: Listeria monocytogenes; Modified atmospheres; Packaged

Introduction

Controlled and modified atmosphere packaging are becomng an increasingly common approach to extend the shelf life of perishable refrigerated foods (NFPA, 1988). It has become widely practiced in the poultry industry for both raw and cooked products. Several mixed and homogeneous atmospheres have been proCorrespondence address: J.H. Hotchkiss, Department of Food Science, Institute of Food Science, Stocking Hall, Cornell University, Ithaca, NY 14853, U.S.A. * Preliminary reports of a portion of these data were made at the June 1989 meeting of the Institute of Food Technologists, Chicago, IL, U.S.A. 0168-1605/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)90

206 posed, but the most widely used atmospheres usually contain significantly higher concentrations of CO 2 with or without O2 and/or N 2 (Genigeorgis, 1985). Elevated CO 2 concentrations selectively inhibit the growth of Gram-negative bacteria such as Pseudomonas, Acinetobacter and Moraxella while allowing many Gram-positive organisms to proliferate (Silliker and Wolfe, 1980). As a result, the characteristic off-odors, off-colors and spoilage associated with rapid growth of Gram-negative psychrotrophs may be inhibited and product deterioration is slowed. The effects of controlled and modified atmosphere packaging on the microbiological safety of foods has not been extensively studied. In the few published reports, several different experimental approaches have been used (Hotchkiss, 1988). Direct inoculation studies as well as more complex studies which attempt to correlate pathogenicity with organoleptic spoilage have been undertaken. Few studies, with the exception of those published by Genigeorgis and co-workers (Lindroth and Genigeorgis, 1986; Garcia and Genigeorgis, 1987) have been designed to be predictive. These workers determined the probability of toxin formation in modified atmosphere packed fish products. While their regression analysis provides statistical estimates of the likelihood of toxin formation under different storage conditions, they do not consider the role of spoilage in the safety of such products. Listeria monocytogenes has been isolated from or epidemiologically associated with various foods for human consumption such as soft cheeses (Pini and Gilbert, 1988), uncooked meat (Kwantes and Isaac, 1971), cabbage, (Schlech et al., 1983), fresh lettuce, celery and tomatoes (Ho et al., 1986). In 1971, Kwantes and Isaac isolated L. monocytogenes from 57% of fresh and frozen poultry samples. Pini and Gilbert (1988) suggest that as many as 60% of raw chickens available in the U.K. may be contaminated with L. monocytogenes. Both fresh and processed meats, including poultry products, have been reported to be commonly contaminated with L. rnonocytogenes (Brackett, 1988). The behavior of L. monocytogenes is of interest in refrigerated, extended shelf life foods, such as those packaged in contolled and modified atmospheres, due to its ability to survive and proliferate under adverse conditions, even at refrigeration temperatures, and its ability to grow in microaerophilic environments (Kahn et al., 1975). The relationship between the growth of spoilage organisms and pathogens is a critical safety factor in controlled and modified atmosphere packaging (Hintlian and Hotchkiss, 1987). The objective of our work was to determine if controlled and modified atmosphere packaging would inhibit aerobic plate counts while allowing the growth of L. monocytogenes in raw chicken.

Materials and Methods

Packaging materials Pouches (19 × 14 cm) were constructed from moderate barrier copolymer film (CVP Systems, Inc., Carol Stream, IL; 20/~M Nylon, 30/~M ethyl vinyl acetate, and 30 ~M Surlyn; gas transmission rates (cc/m 2 x 24 h) were 28 to 38 for 02, 4 to 7 for

207 and 108 to 128 for C02). The volume of the pouch was 880 ml. Two holes were made in the pouch surface and fitted with rubber serum bottle stoppers. The perimeter of the septa was caulked with silicone sealant. After the pouches were sealed, two hypodermic needles (18 gauge) were inserted into one of the septa as a gas outlet. Another needle, connected to a glas flowmeterblender (2-31B Series Gas Blender, Scott Specialty Gases, Plumsteadville, PA), was inserted into the second septum. After the pouch was flushed for 60 s with the desired gas mixture, the inlet and outlet needles were removed. The gas blending system was calibrated against commercial certified gas mixtures (Scott Specialty Gases Mix No. 58) by gas chromatography (Varian Aerograph 200; dual thermal conductivity detectors; He carrier gas, 25 ml/min; 2 m × 0.32 mm OD stainless steel columns packed with Molecular Sieve 5A or Chromosorb 102). The minimum flush time required to achieve the desired atmosphere was also determined by gas chromatography. The pouch head space was monitored by gas chromatography on randomly selected pouches throughout the experiment. N2

Sample preparation Freshly-killed and iced chickens were obtained from the Cornell University Poultry Farm. The skin was removed and all meat (white and dark) was removed from the bones. The meat was coarsely minced for 20 s (until homogeneous). Minced chicken was sealed in Cryovac pouches and frozen ( - 5 ° C ) until use (approximately 30 days). Aerobic plate counts were determined after freezing. Standard curve for L. monocytogenes in Trypticase Soy Broth L. monocytogenes strain Scott A, serotype 4 (clinical isolate obtained from Dr. Catherine Donnelly, University of Vermont) was maintained on Trypticase Soy Agar with 0.6% Yeast Extract (TSA-YE; BBL Microbiology Systems, Cockeysville, MD). Cultures were incubated at 37°C for 24 h and held for 14 days at 4 ° C. Colonies were collected and incubated in Trypticase Soy Broth with Yeast Extract (TSB-YE; BBL Microbiology Systems) at 37 °C in a shaker bath. Periodically, a small sample of the suspension was removed and centrifuged to concentrate the cells. The pellet was resuspended in Phosphate Buffered Saline (PBS). The optical density (OD) of the diluted sample was read at 640 nm against PBS. The culture was further diluted with PBS and plated on Modified McBride Agar prepared as described below, to determine the number of cfu per ml and a standard curve was constructed. Plating methods for enumeration The aerobic plate count (APC) was obtained by pour plate using Plate Count Agar (PCA; Difco, Detroit, MI). The plates were incubated at 37 °C for 48 h and colonies were counted (Busta et al., 1984). L. monocytogenes was enumerated on MMA prepared according to the FDA method (Lovett, 1988). The media consisted of 35.5 g phenylethanol agar (Difco), 10 g glycine anhydride (Sigma Chemical Co., St. Louis, MO), 0.5 g lithium chloride, and 200 mg cycloheximide (Sigma Chemical Co.) per liter of media. The cyclo-

208 heximide was filter sterilized and then added to the tempered agar just before pouring plates. The plates were incubated at 37 ° C for 48 h. Selected colonies of L. monocytogenes were identified and biochemically confirmed using the F D A procedure (Lovett, 1988). Plating of uninoculated samples resulted in < 101 c f u / g .

Experimental protocol L. monocytogenes Scott A was incubated in 15 ml of TSB-YE overnight (37 ° C), the solution was centrifuged, the pellet was resuspended in 7 ml of PBS, and the O D was taken. The solution was diluted (PBS) to O D equal to cfu densities of 10 4 and 10: cfu/ml. 10 /~1 of each inoculum was directly plated on M M A to verify the accuracy of the dilutions; 10/~1 of PBS was plated as a control. Duplicate 1-g samples of raw minced chicken were placed in each of the six wells of sterile deep-well tissue culture trays (Falcon 3046). The surface was inoculated with 10 ~tl of one of the cell suspensions (or sterile PBS for APC determination) using a sterile micropipet. Water was added to the additional small center well of each culture tray to prevent drying. The six-well uncovered tray was placed in a pouch and the sides of the pouch were heat-sealed. The pouch was flushed with one of the atmospheres to be tested (75 : 25, CO 2 : N 2 or 72.5 : 22.5 : 5 CO 2 : N 2 : 02) or air and incubated at 4, 10, or 27°C. At sampling time, the entire chicken sample from each well was aseptically transferred and serially diluted in PBS and plated on M M A (inoculated samples) or PCA (uninoculated samples). Selected colonies of L. monocytogenes were identified and biochemically confirmed using the F D A procedure (Lovett, 1988). All plating was done in duplicate and each experiment was repeated twice. Statistical analysis A statistical analysis was performed over the linear portion of the log10 growth phase of the curves in order to estimate growth curves for L. monocytogenes and the aerobic colony forming organisms under each set of environmental conditions. The curves were then used to determine the differential growth of the organisms under each set of experimental conditions. Separate linear regression equations were computed for each set of conditions using the SAS package (SAS, 1985) and 95% confidence intervals (CI) were used to assess the accuracy of the equations.

Results and Discussion

L. monocytogenes failed to increase at an inoculation level of 102 c f u / g in the anaerobic atmosphere (75 : 25, CO 2 : N 2) at 4, 10 and 27 o C, respectectively (Table I). Growth of the aerobic spoilage organisms was also inhibited. L. monocytogenes is a facultative anaerobe (Holt, 1984); however, we failed to detect growth on chicken in the presence of the high CO 2 concentration. In air, both L. monocytogenes and aerobic spoilage organisms grew (Table I). At 27 ° C, there was immediate logarithmic growth, whereas at 10 and 4 ° C, there were increasing lag phases, respectively. L. monocytogenes proliferated at all temperatures in the presence of air.

209 TABLE I log10 cfu/g of L. monocytogenes and aerobic plate count (APC) from raw chicken packaged in air or 75:25, CO2:N2 (AnMa). Each point represents the mean of four values (duplicate platings of two independent experiments) Temperature ( ° C)

Days

27 27 27 27

0.00 0.17 0.33 0.75

2.90 2.93 2.67 2.57

2.96 3.55 5.85 7.82

4.39 4.58 4.45 4.28

4.24 5.94 6.84 9.52

10 10 10 10 10 10

0 1 2 3 4 5

2.77 2.83 2.30 2.17 2.00 1.89

2.08 3.48 4.18 4.69 5.23 6.30

4.16 4.74 4.85 3.62 3.05 2.47

4.30 4.47 5.42 5.94 6.74 7.00

4 4 4 4 4 4 4

0 1 2 3 4 5 6

2.91 2.57 2.27 2.27 1.81 1.69 < 1.00

2.19 2.21 2.33 2.34 2.66 2.87 3.62

4.29 3.64 2.88 2.00 1.49 1.40 < 1.00

4.18 4.27 4.35 4.53 4.54 4.79 4.94

L. monocytogenes

AnMA

Air

APC AnMA

Air

The arobic C O 2 atmosphere (72.5% CO2, 22.5% N 2, 5% 0 2 ) had an inhibitory effect on the A P C and this effect was more p r o n o u n c e d at the lower temperature (Table II). F o r example, at 4 ° C, the log10 A P C / g in air increased f r o m 4.63 to 9.27 in 21 days, while in the aerobic C O 2 atmosphere, it increased f r o m 4.42 to 5.16 in 21 days. The loga0 c f u / g for L. monocytogenes increased f r o m about 2.5 to about 8.5 in b o t h atmospheres during 21 days at 4 o C. F o r construction of growth curves linear regression was used to fit the linear portion of the loga0 growth phase. While this undercounts organisms during the initial lag phase, and overestimates in the asymptotic phase, it is a good fit to the growth curve in the area of most rapid growth. The regression plots and 95% confidence intervals of data f r o m a second set of experiments for the growth in air and aerobic M A (72.5% CO2, 22.5% N2, 5% 0 2 ) of A P C (Fig. 1) and L. monocytogenes (Fig. 2) were c o m p a r e d at 4 o C. The growth of A P C organisms was inhibited by the aerobic M A c o m p a r e d to air. After only 5 days of storage, the 95% CIs diverged. Organoleptic spoilage of raw chicken takes place at an A P C of approximately 108 c f u / g when stored aerobically (Baker et al., 1986). The regression plots (Fig. 1) indicated that in air the chicken would reach this point between day 13 and 14 which is what would be expected in commercial practice. The growth rate curves for A P C in the aerobic M A did not increase significantly f r o m initial levels b y d a y 14. This substantial increase in shelf life for poultry packed in modified atmosphere has been previously reported (Baker et al., 1986).

210 TABLE II log]0 c f u / g of L. monocytogenes and aerobic plate counts (APC) from raw chicken packaged in air or 72.5 : 22.5 : 5, CO 2 : N 2 : O z (AeMA). Each point represents the mean of four values (duplicate platings of two independent experiments) Temperature

Days

( o C)

L. rnonocytogenes

APC

AeMA

Air

AeMA

Air

27 27 27 27

0.00 0.17 0.29 0.79

2.48 3.45 4.00 7.00

2.68 3.27 4.00 7.64

4.72 4.74 6.22 9.15

4.69 5.23 6.82 10.50

10 10 10 10 10 10 10 10

0 1 2 3 4 5 6 7

2.57 2.74 3.00 4.62 5.04 5.66 6.57 7.00

2.60 3.00 3.06 4.88 5.00 5.79 6.46 6.98

4.00 4.57 4.76 4.79 5.76 5.88 7.57 7.87

4.60 5.00 5.51 6.05 6.79 8.42 9.00 9.80

4 4 4 4 4 4 4 4

0 2 4 6 10 12 14 21

2.45 2.51 2.78 2.81 5.78 6.87 7.29 8.40

2.69 2.74 2.80 3.37 5.58 6.75 7.40 8.60

4.42 4.20 4.31 4.37 4.48 4.52 4.68 5.16

4.63 4.28 4.38 4.88 6.52 6.92 7.81 9.27

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In strong contrast to the APC regression plots, the plots for the growth of L. monocytogenes in the aerobic MA and air were not different at 4 ° C (Fig. 2). The ability of L. monocytogenes to grow at reduced 02 tensions and refrigeration temperatures in media and some foods is well documented (Brackett, 1988; Doyle, 1988). What is important about the present data is the comparison of APC and L. monocytogenes in the two tested types of MA (i.e., with and without the presence of oxygen). The data not shown from the same experiments conducted at 10 and 27 ° C were similar with two exceptions. First, both the APC and L. monocytogenes cfu increased at a much faster rate. Second the MA had a much smaller effect on the APC. The temperature dependence of MA on the inhibition of spoilage has been reviewed (Ogrydziak and Brown, 1982) and is due to a decrease in CO 2 solubility as the temperature increases. However, the 95% CIs for L. monocytogenes in air and aerobic MA overlapped while the CIs for APC diverged, even at the elevated temperature (data not included). We inoculated the samples with high (102 cfu/g) and low levels ( < 101 c f u / g ) levels of L. monocytogenes in order to determine the effect of initial level in both the aerobic MA and air at 27, 10 and 4°C. When stored at 4 ° C (Fig. 3), by day 20, both the low and high L. monocytogenes concentrations reached the same level, indicating that the initial inoculum level had very little, if any, effect on the final L. monocytogenes count. At 10 and 27 o C, low initial levels of L. monocytogenes also increased rapidly in both air and the aerobic MA (data not shown). The effect of the initial contents of aerobic spoilage organisms on poultry on the growth of L. monocytogenes was determined by inoculating two sets of samples which had either low ( 1 0 4 c f u / g ) or high (10 8 c f u / g ) initial levels of aerobic plate counts. The high APC were achieved by allowing lower count chicken to sit at room temperature for several hours prior to inoculation. Both sets were inoculated with low ( < 101 c f u / g ) levels of L. monocytogenes. The low APC chicken represents

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Fig. 3. Growth curves (4 ° C) of L. monocytogenes inoculated onto raw chicken at 103 (open circles) and < 10 +c f u / g (solid circles) and stored at 4 ° C under 72.5 : 22.5 : 5, CO 2 : N 2 : 02.

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Fig. 4. Growth curves (10 o C) of L. rnonocytogenes inoculated ( < 101 c f u / g ) onto raw chicken with low (10 4 c f u / g , solid circles) and high (10 8 c f u / g , open circles) aerobic plate counts.

acceptable product, while the high APC chicken is regarded as unacceptable. Low versus high initial poultry microflora levels had virtually no effect on L. monocytogenes growth (Fig. 4).

Conclusions

These data illustrate a specific combination of a product and modified atmosphere, where the growth of aerobic spoilage organisms is inhibited while the growth of a pathogen is uninhibited. As we have pointed out (Hintlian and Hotchkiss, 1986) this may be the most hazardous of all situations because the normal cues for

213 s p o i l a g e m a y b e a b s e n t w h e n t h e p r o d u c t h a s s u f f i c i e n t p a t h o g e n s to c a u s e disease. It s h o u l d b e p o i n t e d out, h o w e v e r , t h a t p o u l t r y is rarely, if ever, c o n s u m e d r a w a n d t h a t L . m o n o c y t o g e n e s w o u l d b e killed b y n o r m a l c o o k i n g p r o c e d u r e s . N o n e t h e l e s s , c o n s u m p t i o n of u n c o o k e d h o t d o g s o r u n d e r c o o k e d p o u l t r y m a y b e o n e o f t h e m o r e i m p o r t a n t i d e n t i f i a b l e risk f a c t o r s f o r listeriosis ( S c h w a r t z et al., 1988) a n d t h e r e h a s b e e n a r e c e n t c o n f i r m e d case o f listeriosis a t t r i b u t e d to L . m o n o c y t o g e n e s c o n t a m i n a t i o n o f a p r o c e s s e d p o u l t r y p r o d u c t t h a t was a p p a r e n t l y p r o p e r l y r e f r i g e r a t e d ( M M W R , 1989).

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214 Pini, P.N. and Gilbert, R.J. (1988) The occurrence in the U.K. of Listeria species in raw chickens and soft cheeses. Int. J. Food Microbiol. 6, 317-326. SAS (1985) SAS Users Guide: Statistics, Version 5 edition. SAS Institute Inc. Schlech, W.F., Lavigne, P.M., Bortolussi, R.A., Allen, A.C., Haldane, E.V., Hightower, A.W., Johnson, S.E., King, S.H., Nicholls, E.S. and Broome, C.V. (1983) Epidemic listeriosis--Evidence for transmission by food. New Engl. J. Med. 308, 203. Schwartz, B., Cielsielski, C., Broome, C., Gaventa, S., Brown, G.R., Gellin, B.G., Hightower, A.W. and Mascola, L. (1988) Dietary risk factors for sporadic listeriosis: association with consumption of uncooked hot dogs and undercooked chicken. Lancet ii, 779-782. Silliker, J.H. and Wolfe, S.K. (1980) Microbiological safety considerations in controlled atmosphere storage of meats. Food Technol. 34, 59-63.