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Survival of Listeria monocytogenes in ground beef
International JournalofFoodMicrobiology, 6 (1988) 243-247
243
Elsevier
JFM00202
Survival of Listeria monocytogenes in ground beef J e n n i f e r L. J o h n s o n 1,2, M i c h a e l P. D o y l e 1 a n d R o b e r t G . C a s s e n s 2 l Food Research Institute, University of Wisconsin-Madison, Madison, WI, U.S.A., and 2 Muscle Biology Laboratory, University of Wisconsin-Madison, Madison, WI, U.S.A. (Received 24 November 1987; accepted 29 January 1988)
Listeria monocytogenes, due to its association with animals and animal products and its proven pathogenicity, is an organism of potential importance to the meat industry. The survival of L. monocytogenes in ground beef held at 4 ° C for 2 weeks was investigated. The ground beef was inoculated with L. monocytogenes type 1 or type 4 at a level of 5 x 1 0 5 to 7 x 1 0 6 C F U / g and then packaged in either oxygen-permeable or oxygen-impermeable bags. Packages were sampled at r a n d o m at 0, 2, 3, 5, 7, 11, and 14 days post-inoculation, and assayed for L. rnonocytogenes counts and pH. The n u m b e r of L. monocytogenes in ground beef remained constant throughout the sampling period, and survival was not affected by package permeabifity to oxygen. Listeriae were not isolated from the control ground beef. The p H of the meat increased slightly during storage, but was always in the range of p H 5.6 to 5.9. It appears that L. monocytogenes in ground beef can survive without any substantial increase or decrease in viable cell population during refrigerated storage for 14 days. Key words: Listeria monocytogenes; Meat; Beef, ground
Introduction
Recognition of Listeria monocytogenes as a foodborne pathogen has raised new concerns about the possible role of meat as a vehicle of foodborne listeriosis. Long known for its association with animals, L. monocytogenes may be a common contaminant of meat and meat products, but little has been published about its incidence in these foods. Nicolas (1985) isolated L. rnonocytogenes from 5 of 52 samples of frozen ground beef in France; later studies revealed that 26% of frozen ground beef-steak was contaminated with L. monocytogenes (Nicolas and Vidaud, 1987). Given numerous opportunities for meat to become contaminated with listeriae and the psychrotrophic nature of this organism, knowledge of the fate of L. monocytogenes in a meat product such as ground beef is important. The objectives of this study were to examine the fate of L. monocytogenes inoculated into ground beef and held at 4 ° C for 2 weeks and to determine if package permeability to oxygen affected bacterial survival.
Correspondence address: M.P. Doyle, Food Research Institute, University of Wisconsin-Madison, 1925 Willow Drive, Madison, WI 53706, U.S.A. 0168-1605/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
244 Materials and Methods
Inoculum preparation Inoculum was prepared from a culture of L. monocytogenes strain Scott A or V7 grown under microaerobic conditions (5% 02, 10% CO2, 85% N 2 ) in tryptose broth (Difco, Detroit, MI) for 24 h at 37 ° C. Absorbance of the cultures was determined at 500 nm, and the cultures were diluted appropriately in 0.01 M phosphate-buffered saline solution (PBS, p H 7.2). Enumeration was done by spread plating 0.1 ml of serial (1 : 10) dilutions of the inoculum onto duplicate plates of tryptose agar (Difco, Detroit, MI), and incubating the plates at 37 ° C for 48 h. The targeted inoculum level w a s 10 6 C F U / g meat, but actual inoculum levels varied between 5 × 105 C F U / g and 7 × 10 6 C F U / g .
Preparation of ground beef Boneless beef chuck meat (38 kg), obtained from the University of WisconsinMadison Muscle Biology Lab, was ground through a 0.95 cm-grinder plate on a Toledo grinder (model 5323, Toledo Scale Co., Toledo, OH). The meat was mixed for 4 min, and reground through a 0.48 cm grinder plate. Portions (500 g) of ground beef were inoculated with 5 ml of either L. monocytogenes strain Scott A or V7; control ground beef received 5 ml of PBS. Inoculum was distributed by hand-kneading the meat for 2 rain. Each meat portion was then vacuum packaged in either an oxygen-permeable (PER) pouch (Chub film; ethylene vinyl a l c o h o l / n y l o n / e t h y l e n e vinyl alcohol; O z transmission: 50 cm3/m2/24 h at 22.8°C; American Can Co., Neenah, WI) or an oxygen-impermeable (IMP) package (Z-4450 film; nylon/ethylene vinyl alcohol/Surlyn; 02 transmission: < 3 cm3/m2/24 h at 22.8 ° C; American Can Co.) using a PAC 24V packaging machine (Packaging Aids Corporation, San Francisco, CA). Packaged ground beef was then stored in an incubator at 4 ° C until sampled. Two trials were done for each treatment.
Microbiological sampling Ground beef was sampled after inoculation and after 2, 3, 5, 7, 11, and 14 days of refrigerated storage at 4°C. Two I M P / V 7 packages, two P E R / V 7 packages, two I M P / S c o t t A packages, two P E R / S c o t t A packages, one I M P / c o n t r o l package, and one P E g / c o n t r o l package were sampled at each time interval. A 25 g composite sample, aseptically taken from five locations within the meat was diluted (1 : 10) in PBS and macerated for 2 min in a Stomacher (model 400, Tekmar Co., Cincinnati, OH). Serial dilutions (1 : 10) of each homogenized sample were made in PBS and 0.1 ml portions were spread plated in duplicate onto McBride's Listeria agar (McBride and Girard, 1960). The plates were incubated at 37 ° C for 48 h under a microaerobic atmosphere of 5% 02, 10% CO 2, 85% N 2. Colonies which were 0.5 to 1.5 mm in diameter, bluish or blue-grey, and slightly beta-hemolytic were streaked onto tryptose agar (Difco). Isolates were tested for catalase activity, and confirmed
245
with the following tests: tumbling motility; umbrella motility when grown at 25 ° and 37 ° C; carbohydrate fermentation (maltose, rhamnose, dextrose, salicin, xylose, mannitol, and dulcitol); nitrate reduction; methyl red reaction; litmus milk reaction; Gram stain; and serology with type 1 and type 40-antisera (Difco).
p H determination The pH of each meat sample was determined by adding 90 ml of distilled, deionized water to 10 g of meat and macerating for 2 min in a Stomacher. pH was then determined by a Coming combination electrode (Corning Glassworks, Corning, NY).
Statistical analysis Data were analyzed by a SAS General Linear Models procedure (SAS Institute, Inc., Raleigh, NC).
Results
Microbiological results The fate of L. monocytogenes in ground beef during Trial 1 is shown in Fig. 1. Results of Trial 2 were very similar, hence these data are not shown. No significant difference (P > 0.05) in L. monocytogenes numbers was observed during the storage period, and both strains of the organism behaved similarly. In Trial 1, ground beef
o--o IMP./~/'7 • - - o PERZV7 z x - - t x IMP/SA A - - A PER/SA
~.
,?
7,
5 JI o
o, 4
0
I
I
I
I
I
I
I
l
I
1
2
3
4-
5
6
7
8
9
I
I
I
I
I
10 11 12 13 14
SAMPUNG TIME (days)
Fig. 1. Growth of Listeria monocytogenes strains Scott A (SA) and V7 at 4 ° C in ground beef in gas permeable (PER) and gas impermeable (IMP) packaging.
246 in PER packages had slightly higher levels of listeriae than meat packaged in I M P packages. This difference was not significant ( P > 0.05), and results of Trial 2 indicated no difference in numbers of L. monocytogenes due to package permeability to oxygen. No listeriae were detected in any control (uninoculated) ground beef samples.
p H determination p H values of the ground beef increased slightly during storage, but remained in the range of p H 5.6 to 5.9 (data not shown).
Discussion The results of this study indicate that the viable population of L. monocytogenes in inoculated ground beef remained relatively constant during a 14-day storage period at 4 ° C. These results are similar to those of K h a n et al. (1975) who found that numbers of L. monocytogenes on lamb shoulder remained constant during 24 days at 0 o C. Earlier studies by this research group (Khan et al., 1973) revealed that levels of listeriae decreased on lamb meat held at 0 o C, but there was no change in numbers when the meat was held at 8 ° C. Studies of inoculated minced meat and sausage meat revealed that L. monocytogenes could be isolated from these meats after 15 days at 8 ° C or 20 days at 4 ° C , and numbers of the organism may have increased (Khan et al., 1973). Using sterile ground beef inoculated with L. monocytogenes, Gouet et al. (1978) found that numbers of listeriae decreased by approximately 1 log10 C F U / g initially, then remained constant in meat held at 8°C. Perhaps meat lacks a nutrient required for the growth of L. monocytogenes. Gouet et al. (1978) have suggested that degradation of muscle by proteolytic bacteria such as Pseudomonas fluorescens may allow growth of listeriae to occur. K h a n et al. (1973) also reported that inoculated lamb meat packaged in oxygenimpermeable bags had consistently fewer surviving listeriae than did meat packaged in oxygen-permeable bags. These authors suggested that increased concentrations of CO 2 in impermeable packages may inactivate the organism. Microbiological examination of control ground beef did not detect any L. monocytogenes, indicating that the organism was not present or was present at very low levels only detectable by enrichment procedures. The frequent inability of direct plating, as was used in this study, to detect low numbers of listeriae in naturallycontaminated foods is well known. In conclusion, L. monocytogenes in ground beef m a y survive 2 weeks of refrigerated (4 ° C) storage without any significant change in the viable cell population. Because of the apparent likelihood that L. monocytogenes will be present in beef, individuals at risk of acquiring listeric infection should avoid eating raw or undercooked ground beef.
247
Acknowledgements We thank Keith Lind and American Can Company for donating the packaging materials, and Juan Arias of the University of Wisconsin for assistance with statistical analysis of the data. This work was supported by the College of Agriculture and Life Sciences, University of Wisconsin-Madison, and by contributions to the Food Research Institute.
References Gouet, Ph., Labadie, J. and Serratore, C. (1978) Development of Listeria monocytogenes in monoxenic and polyxenic beef minces. Zentralbl. Bakteriol. Hyg., I. Abt. Orig. B 166, 87-94. Khan, M.A., Palmas, C.V., Seaman, A. and Woodbine, M. (1973) Survival vs. growth of a facultative psychrotroph: meat and products of meat. Zentralbl. Bakteriol. Hyg., I. Abt. Orig. B 157, 277-282. Khan, M.A., Newton, I.A., Seaman, A. and Woodbine, M. (1975) The survival of Listeria monocytogenes inside and outside its host. In: M. Woodbine (Ed.), Problems of Listeriosis, Leicester University Press, Leicester, England, pp. 75-83. McBride, M.E. and Girard, K.F. (1960) A selective media for the isolation of Listeria monocytogenes from mixed bacterial populations. Lab Clin. Med. 55, 153-157. Nicolas, J.A. (1985) Contamination des viandes et des produits de charcuterie par Listeria monocytogenes en Haute-Vienne. Sci. Aliments 5, 175-180. Nicolas, J.A. and Vidaud, N. (1987) Contribution a l'rtude des Listeria prrsentes dans les denrres d'origine animale destinres h la consommation humaine. Rec. Med. Vet. 163, 283-285.
243
Elsevier
JFM00202
Survival of Listeria monocytogenes in ground beef J e n n i f e r L. J o h n s o n 1,2, M i c h a e l P. D o y l e 1 a n d R o b e r t G . C a s s e n s 2 l Food Research Institute, University of Wisconsin-Madison, Madison, WI, U.S.A., and 2 Muscle Biology Laboratory, University of Wisconsin-Madison, Madison, WI, U.S.A. (Received 24 November 1987; accepted 29 January 1988)
Listeria monocytogenes, due to its association with animals and animal products and its proven pathogenicity, is an organism of potential importance to the meat industry. The survival of L. monocytogenes in ground beef held at 4 ° C for 2 weeks was investigated. The ground beef was inoculated with L. monocytogenes type 1 or type 4 at a level of 5 x 1 0 5 to 7 x 1 0 6 C F U / g and then packaged in either oxygen-permeable or oxygen-impermeable bags. Packages were sampled at r a n d o m at 0, 2, 3, 5, 7, 11, and 14 days post-inoculation, and assayed for L. rnonocytogenes counts and pH. The n u m b e r of L. monocytogenes in ground beef remained constant throughout the sampling period, and survival was not affected by package permeabifity to oxygen. Listeriae were not isolated from the control ground beef. The p H of the meat increased slightly during storage, but was always in the range of p H 5.6 to 5.9. It appears that L. monocytogenes in ground beef can survive without any substantial increase or decrease in viable cell population during refrigerated storage for 14 days. Key words: Listeria monocytogenes; Meat; Beef, ground
Introduction
Recognition of Listeria monocytogenes as a foodborne pathogen has raised new concerns about the possible role of meat as a vehicle of foodborne listeriosis. Long known for its association with animals, L. monocytogenes may be a common contaminant of meat and meat products, but little has been published about its incidence in these foods. Nicolas (1985) isolated L. rnonocytogenes from 5 of 52 samples of frozen ground beef in France; later studies revealed that 26% of frozen ground beef-steak was contaminated with L. monocytogenes (Nicolas and Vidaud, 1987). Given numerous opportunities for meat to become contaminated with listeriae and the psychrotrophic nature of this organism, knowledge of the fate of L. monocytogenes in a meat product such as ground beef is important. The objectives of this study were to examine the fate of L. monocytogenes inoculated into ground beef and held at 4 ° C for 2 weeks and to determine if package permeability to oxygen affected bacterial survival.
Correspondence address: M.P. Doyle, Food Research Institute, University of Wisconsin-Madison, 1925 Willow Drive, Madison, WI 53706, U.S.A. 0168-1605/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
244 Materials and Methods
Inoculum preparation Inoculum was prepared from a culture of L. monocytogenes strain Scott A or V7 grown under microaerobic conditions (5% 02, 10% CO2, 85% N 2 ) in tryptose broth (Difco, Detroit, MI) for 24 h at 37 ° C. Absorbance of the cultures was determined at 500 nm, and the cultures were diluted appropriately in 0.01 M phosphate-buffered saline solution (PBS, p H 7.2). Enumeration was done by spread plating 0.1 ml of serial (1 : 10) dilutions of the inoculum onto duplicate plates of tryptose agar (Difco, Detroit, MI), and incubating the plates at 37 ° C for 48 h. The targeted inoculum level w a s 10 6 C F U / g meat, but actual inoculum levels varied between 5 × 105 C F U / g and 7 × 10 6 C F U / g .
Preparation of ground beef Boneless beef chuck meat (38 kg), obtained from the University of WisconsinMadison Muscle Biology Lab, was ground through a 0.95 cm-grinder plate on a Toledo grinder (model 5323, Toledo Scale Co., Toledo, OH). The meat was mixed for 4 min, and reground through a 0.48 cm grinder plate. Portions (500 g) of ground beef were inoculated with 5 ml of either L. monocytogenes strain Scott A or V7; control ground beef received 5 ml of PBS. Inoculum was distributed by hand-kneading the meat for 2 rain. Each meat portion was then vacuum packaged in either an oxygen-permeable (PER) pouch (Chub film; ethylene vinyl a l c o h o l / n y l o n / e t h y l e n e vinyl alcohol; O z transmission: 50 cm3/m2/24 h at 22.8°C; American Can Co., Neenah, WI) or an oxygen-impermeable (IMP) package (Z-4450 film; nylon/ethylene vinyl alcohol/Surlyn; 02 transmission: < 3 cm3/m2/24 h at 22.8 ° C; American Can Co.) using a PAC 24V packaging machine (Packaging Aids Corporation, San Francisco, CA). Packaged ground beef was then stored in an incubator at 4 ° C until sampled. Two trials were done for each treatment.
Microbiological sampling Ground beef was sampled after inoculation and after 2, 3, 5, 7, 11, and 14 days of refrigerated storage at 4°C. Two I M P / V 7 packages, two P E R / V 7 packages, two I M P / S c o t t A packages, two P E R / S c o t t A packages, one I M P / c o n t r o l package, and one P E g / c o n t r o l package were sampled at each time interval. A 25 g composite sample, aseptically taken from five locations within the meat was diluted (1 : 10) in PBS and macerated for 2 min in a Stomacher (model 400, Tekmar Co., Cincinnati, OH). Serial dilutions (1 : 10) of each homogenized sample were made in PBS and 0.1 ml portions were spread plated in duplicate onto McBride's Listeria agar (McBride and Girard, 1960). The plates were incubated at 37 ° C for 48 h under a microaerobic atmosphere of 5% 02, 10% CO 2, 85% N 2. Colonies which were 0.5 to 1.5 mm in diameter, bluish or blue-grey, and slightly beta-hemolytic were streaked onto tryptose agar (Difco). Isolates were tested for catalase activity, and confirmed
245
with the following tests: tumbling motility; umbrella motility when grown at 25 ° and 37 ° C; carbohydrate fermentation (maltose, rhamnose, dextrose, salicin, xylose, mannitol, and dulcitol); nitrate reduction; methyl red reaction; litmus milk reaction; Gram stain; and serology with type 1 and type 40-antisera (Difco).
p H determination The pH of each meat sample was determined by adding 90 ml of distilled, deionized water to 10 g of meat and macerating for 2 min in a Stomacher. pH was then determined by a Coming combination electrode (Corning Glassworks, Corning, NY).
Statistical analysis Data were analyzed by a SAS General Linear Models procedure (SAS Institute, Inc., Raleigh, NC).
Results
Microbiological results The fate of L. monocytogenes in ground beef during Trial 1 is shown in Fig. 1. Results of Trial 2 were very similar, hence these data are not shown. No significant difference (P > 0.05) in L. monocytogenes numbers was observed during the storage period, and both strains of the organism behaved similarly. In Trial 1, ground beef
o--o IMP./~/'7 • - - o PERZV7 z x - - t x IMP/SA A - - A PER/SA
~.
,?
7,
5 JI o
o, 4
0
I
I
I
I
I
I
I
l
I
1
2
3
4-
5
6
7
8
9
I
I
I
I
I
10 11 12 13 14
SAMPUNG TIME (days)
Fig. 1. Growth of Listeria monocytogenes strains Scott A (SA) and V7 at 4 ° C in ground beef in gas permeable (PER) and gas impermeable (IMP) packaging.
246 in PER packages had slightly higher levels of listeriae than meat packaged in I M P packages. This difference was not significant ( P > 0.05), and results of Trial 2 indicated no difference in numbers of L. monocytogenes due to package permeability to oxygen. No listeriae were detected in any control (uninoculated) ground beef samples.
p H determination p H values of the ground beef increased slightly during storage, but remained in the range of p H 5.6 to 5.9 (data not shown).
Discussion The results of this study indicate that the viable population of L. monocytogenes in inoculated ground beef remained relatively constant during a 14-day storage period at 4 ° C. These results are similar to those of K h a n et al. (1975) who found that numbers of L. monocytogenes on lamb shoulder remained constant during 24 days at 0 o C. Earlier studies by this research group (Khan et al., 1973) revealed that levels of listeriae decreased on lamb meat held at 0 o C, but there was no change in numbers when the meat was held at 8 ° C. Studies of inoculated minced meat and sausage meat revealed that L. monocytogenes could be isolated from these meats after 15 days at 8 ° C or 20 days at 4 ° C , and numbers of the organism may have increased (Khan et al., 1973). Using sterile ground beef inoculated with L. monocytogenes, Gouet et al. (1978) found that numbers of listeriae decreased by approximately 1 log10 C F U / g initially, then remained constant in meat held at 8°C. Perhaps meat lacks a nutrient required for the growth of L. monocytogenes. Gouet et al. (1978) have suggested that degradation of muscle by proteolytic bacteria such as Pseudomonas fluorescens may allow growth of listeriae to occur. K h a n et al. (1973) also reported that inoculated lamb meat packaged in oxygenimpermeable bags had consistently fewer surviving listeriae than did meat packaged in oxygen-permeable bags. These authors suggested that increased concentrations of CO 2 in impermeable packages may inactivate the organism. Microbiological examination of control ground beef did not detect any L. monocytogenes, indicating that the organism was not present or was present at very low levels only detectable by enrichment procedures. The frequent inability of direct plating, as was used in this study, to detect low numbers of listeriae in naturallycontaminated foods is well known. In conclusion, L. monocytogenes in ground beef m a y survive 2 weeks of refrigerated (4 ° C) storage without any significant change in the viable cell population. Because of the apparent likelihood that L. monocytogenes will be present in beef, individuals at risk of acquiring listeric infection should avoid eating raw or undercooked ground beef.
247
Acknowledgements We thank Keith Lind and American Can Company for donating the packaging materials, and Juan Arias of the University of Wisconsin for assistance with statistical analysis of the data. This work was supported by the College of Agriculture and Life Sciences, University of Wisconsin-Madison, and by contributions to the Food Research Institute.
References Gouet, Ph., Labadie, J. and Serratore, C. (1978) Development of Listeria monocytogenes in monoxenic and polyxenic beef minces. Zentralbl. Bakteriol. Hyg., I. Abt. Orig. B 166, 87-94. Khan, M.A., Palmas, C.V., Seaman, A. and Woodbine, M. (1973) Survival vs. growth of a facultative psychrotroph: meat and products of meat. Zentralbl. Bakteriol. Hyg., I. Abt. Orig. B 157, 277-282. Khan, M.A., Newton, I.A., Seaman, A. and Woodbine, M. (1975) The survival of Listeria monocytogenes inside and outside its host. In: M. Woodbine (Ed.), Problems of Listeriosis, Leicester University Press, Leicester, England, pp. 75-83. McBride, M.E. and Girard, K.F. (1960) A selective media for the isolation of Listeria monocytogenes from mixed bacterial populations. Lab Clin. Med. 55, 153-157. Nicolas, J.A. (1985) Contamination des viandes et des produits de charcuterie par Listeria monocytogenes en Haute-Vienne. Sci. Aliments 5, 175-180. Nicolas, J.A. and Vidaud, N. (1987) Contribution a l'rtude des Listeria prrsentes dans les denrres d'origine animale destinres h la consommation humaine. Rec. Med. Vet. 163, 283-285.