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Influence of modified atmospheric storage, lactic acid, and NaCl on survival of sublethally heat-injured Listeria monocytogenes
International Journal of Food Microbiology 64 (2001) 379–386 www.elsevier.nl / locate / ijfoodmicro
Short communication
Influence of modified atmospheric storage, lactic acid, and NaCl on survival of sublethally heat-injured Listeria monocytogenes Robert C. Williams, David A. Golden* The University of Tennessee, Agricultural Experiment Station, Department of Food Science and Technology, 2605 River Drive, Knoxville, TN 37996, USA Received 13 June 2000; received in revised form 25 September 2000; accepted 1 November 2000
Abstract The effect of package atmosphere on survival of uninjured and sublethally heat-injured Listeria monocytogenes, inoculated onto tryptose phosphate agar containing 0.85% lactic acid and 2% NaCl (TPALAS) was investigated. Inoculated TPALAS plates were packaged in air, 100% N 2 (N 2 ), 30% CO 2 –70% N 2 (CO 2 –N 2 ), and vacuum and stored at 4 and 208C for up to 31 days. Recovery of L. monocytogenes from TPALAS was influenced by the injury status (i.e., injured and uninjured) of the inoculum, storage atmosphere (air, N 2 , CO 2 –N 2 , and vacuum), storage temperature (4 and 208C), and recovery media [tryptose phosphate agar (TPA) and modified Oxford agar (MOX)] (P , 0.05). Overall, storage at 48C supported greater survival than storage at 208C (P , 0.05). Uninjured L. monocytogenes stored at 48C was recovered on TPA better than sublethally heat-injured L. monocytogenes stored at 48C (P , 0.05). Recovery of sublethally heat-injured L. monocytogenes stored at 48C followed the order N 2 . CO 2 –N 2 . air . vacuum (P , 0.05), whereas recovery of uninjured L. monocytogenes stored at 48C followed the order N 2 . CO 2 –N 2 . vacuum . air (P , 0.05). Air and vacuum atmospheres supported greater survival of uninjured and heat-injured L. monocytogenes than N 2 and CO 2 –N 2 atmospheres at 208C (P , 0.05). Recovery of sublethally heat-injured L. monocytogenes stored at 208C followed the order vacuum . air . CO 2 –N 2 5 N 2 (P , 0.05), whereas recovery of uninjured L. monocytogenes stored at 208C followed the order vacuum . air . CO 2 –N 2 . N 2 (P , 0.05). Uninjured L. monocytogenes stored under N 2 at 48C was recovered best, whereas sublethally heat-injured L. monocytogenes stored under N 2 at 208C was recovered poorest (P , 0.05). Factors such as package atmosphere and storage temperature, involved in the production, storage, and distribution of fermented foods must be thoroughly evaluated when determining strategies for control and detection of L. monocytogenes in such products. 2001 Elsevier Science B.V. All rights reserved. Keywords: Listeria monocytogenes; Heat injury; Storage atmosphere; Lactic acid; NaCl
1. Introduction *Corresponding author. Tel.: 1 1-865-974-7247; fax: 1 1-865974-2750. E-mail address: [email protected] (D.A. Golden).
Listeria monocytogenes has been recovered from many foods, including raw beef, raw pork, milk, and cabbage (Jay, 2000). These foods are commonly
0168-1605 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0168-1605( 00 )00479-7
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fermented for the purpose of preservation or to impart desired organoleptic characteristics. The aim of acid-type fermentations is to increase the level of acid (e.g., lactic acid) in the fermentation mixture, while decreasing pH to yield a product with desirable characteristics and increased inhibition of spoilage microorganisms (Booth and Kroll, 1989). Production of fermented foods includes acidification of raw or pasteurized products via starter cultures or naturally occurring microflora and may include further processing treatments such as heat (Jay, 2000). Food processing treatments, including heating and acidification, have been shown to induce sublethal injury in L. monocytogenes (Golden et al., 1988; Smith and Archer, 1988; Ahmad and Marth, 1990). Sublethally injured organisms may exhibit increased sensitivity to various components of selective / differential media (Ray, 1989). As a result, sublethally injured L. monocytogenes may grow poorly or not at all on the selective media used for its detection. The major concern with sublethally injured L. monocytogenes is that they may repair, grow, and present a health threat if placed in an appropriate environment (Ray, 1993). For example, heat-stressed L. monocytogenes has been shown to be less pathogenic than nonstressed L. monocytogenes, but regains pathogenicity equal to that of nonstressed cells upon resuscitation in an appropriate environment (McCarthy, 1991). Consequently, under routine testing, a food product may appear to be free of L. monocytogenes, but upon storage, may become unsafe for consumption. Modified atmosphere packaging (MAP) can enhance or reduce survival of L. monocytogenes in a variety of food products (Gill and Reichel, 1989; Chen and Hotchkiss, 1993; Pothuri et al., 1995; Francis and O’Beirne, 1997). L. monocytogenes was inhibited by packaging atmospheres comprised of high carbon dioxide levels (35 to 100%) in packaged cottage cheese (Chen and Hotchkiss, 1993), beef (Gill and Reichel, 1989), and crayfish tail meat (Pothuri et al., 1995) when stored at # 58C. Kallander et al. (1991) reported that an atmosphere containing 75% CO 2 did not effect survival of L. monocytogenes in packaged cabbage stored at 58C. L. monocytogenes survival was enhanced by an atmosphere comprised of 96% N 2 –4% O 2 in packaged lettuce stored at 38C (Francis and O’Beirne, 1997) but not influenced by 97% N 2 –3% O 2 atmos-
phere in packaged lettuce stored at 5 or 108C (Beuchat and Brackett, 1990a,b). L. monocytogenes may survive production and storage of fermented foods, including meat (Johnson et al., 1988; Glass and Doyle, 1989; Buncic et al., 1991; Farber et al., 1993), milk (Ashenafi, 1994; Dalu and Feresu, 1996), kimchi (Lee et al., 1995), and tempeh (Ashenafi, 1991). In general, the microorganism grows during the fermentation, but declines during storage, with low levels persisting. The exception was fermented sausages, where no growth occurred but low levels of the organism persisted even after lengthy storage. L. monocytogenes may undergo injury during production of fermented foods due to exposure to stresses such as acidification and heating. Sublethal injury of L. monocytogenes may complicate detection of this microorganism in fermented foods and may result in underestimation or missed detection of L. monocytogenes. The influence of packaging atmosphere and storage temperature on resuscitation of sublethally injured L. monocytogenes from fermented foods is not known. Therefore, the objective of this study was to determine the fate of sublethally heat-injured L. monocytogenes at 4 and 208C on media formulated to simulate fermented food products and exposed to four different modified atmospheres.
2. Materials and methods
2.1. Culture maintenance L. monocytogenes LM103M (meat isolate) was used in this study because of its demonstrated tolerance to heat and lactic acid (Williams and Golden, 1998). The culture was maintained in tryptose phosphate broth (TPB; Difco, Detroit, MI, USA) through transfer at 24-h intervals and incubation at 378C.
2.2. Heat injury L. monocytogenes was heated to produce injury in . 90% of cells using protocols described by Golden et al. (1988). A 24-h, 378C culture of L. monocytogenes, grown in TPB, was thoroughly mixed and dispensed in 3-ml portions into 10 13 3 100 ml test
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tubes. These tubes were placed in a test tube rack with sufficient space between tubes to allow for water circulation and placed into a 548C shaker waterbath (Dubnoff Metabolic Shaking Incubator; Precision Scientific, Chicago, IL, USA) operated at 90 rpm. An electronic thermometer was placed into one 13 3 100 ml test tube to determine the time at which the temperature of the contents reached equilibrium with the temperature of the waterbath. Upon reaching 548C ( | 2 min equilibration time) cultures were heated at 548C for 20 min. After 20 min, the test tubes were immediately submerged in an ice-water bath to cool the contents to about 258C. After cooling, contents of the test tubes were recombined and centrifuged twice at 11 000 g for 10 min. Cell pellets were washed with and resuspended in 0.1% peptone water (PW). The purpose of dividing the test culture among 10 13 3 100 ml test tubes was to facilitate heat transfer during heating and cooling.
2.3. Preparation of test media A nutrient medium was formulated to simulate the pH, acidity (as lactic acid), and NaCl content of a fermented food product, such as salami. This medium was used to eliminate the influence of background microflora associated with fermented products. Buffered tryptose phosphate agar containing 0.85% lactic acid and 2.0% NaCl (TPALAS) was prepared by combining TPB (29.5 g), agar (15 g), NaCl (15 g), K 2 HPO 4 ? 3H 2 O (15.02 g), KH 2 PO 4 (4.76 g), and deionized water (1000 ml). The medium was thoroughly mixed and brought to boiling on a hot plate / stirrer, sterilized in an autoclave at 1218C for 15 min, and cooled to about 508C. Upon cooling, the medium was acidified with 85% lactic acid syrup ( DL-lactic acid; Acros, NJ, USA) and poured into 60 3 15 mm Petri dishes. The final medium (TPALAS, pH 4.860.1) contained 0.85% lactic acid and 2% NaCl (TPB inherently contains 0.5% NaCl).
2.4. Fate of sublethally-injured L. monocytogenes on TPALAS An uninjured L. monocytogenes control was prepared as described for the heat-injured culture, with the omission of the heating step. The uninjured,
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washed cell suspension was diluted 10-fold in 0.1% PW prior to inoculation of TPALAS. Uninjured and injured cell suspensions were dispensed separately (0.1 ml) onto TPALAS. Prior to packaging, three holes (approx. 0.4 cm in diameter) were burned into the lid of each TPALAS plate with a Creative Woodburner, Jr. (Walnut Hollow, Dodgeville, WI, USA) to facilitate gas exchange. Inoculated TPALAS plates were packaged in atmospheres of air, 100% nitrogen (N 2 ), 30% carbon dioxide–70% nitrogen (CO 2 –N 2 ), and vacuum in Cryovac B-Series high barrier bags (Sealed Air-Cryovac; Duncan, SC, USA) using a Multivac A300 Packaging Machine (Multivac, Wolfertschweden, Germany). Inoculated and packaged plates were incubated at 4 and 208C. Samples, representing a single plate in a package, were taken initially (day 0), after day 1, and then every other day through 31 days of storage for microbiological and gas analyses. At each sampling interval, headspace gases were withdrawn from each package through adhesive septum tape (Modern Controls, Minneapolis, MN, USA), using a 5 ml syringe and 20 gauge needle. Gas samples were analyzed for O 2 , CO 2 , and N 2 using a HewlettPackard 5890 Series II gas chromatograph fitted with an Alltech CTR-1 column (Alltech, Deerfield, IL, USA) and a thermal conductivity detector. Helium was used as the carrier gas at a flow rate of 64 ml / min. The column and detector were held at ambient temperature.
2.5. Microbiological analysis For microbiological analysis, the entire block of agar from each sample plate was aseptically transferred to a stomacher bag, diluted in 50 ml PW, and pummeled for 2 min using a Stomacher 400 Lab Blender (Seward Medical, London, UK). Pummeled samples were serially diluted (1:10) in PW and surface plated (0.1 ml) onto tryptose phosphate agar (TPA; Difco, Detroit, MI, USA) and modified Oxford media (MOX; Oxoid, Ogdensberg, NY, USA). Inoculated media were incubated at 308C for 72 h before colony counts were obtained. Colony counts were reported as log 10 CFU / cm 2 of TPALAS surface. The limit of detection was 0.95 log 10 CFU / cm 2 using the plating scheme described. When L. monocytogenes was no longer recovered by direct plating, test samples were subjected to enrichment proce-
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dures. Test samples were pummeled, as described above, in 50 ml of Listeria enrichment broth base without selective supplements (UVM formulation; Oxoid,) and incubated at 308C for 48 h. Enriched samples were pummeled for 1 min and inoculated onto MOX using a 10-ml loop. Inoculated MOX plates were incubated at 308C for 72 h.
2.6. Statistical analysis This experiment was performed in triplicate. The design was a randomized block, split-plot, with a whole plot factorial treatment arrangement, blocked on replication. Data were analyzed using the mixed procedure (Proc Mixed) of SAS (SAS Institute, versions 6.12 and 7.00 beta; Cary, NC, USA).
3. Results It is possible that the storage conditions themselves used for this study could result in sublethal injury of L. monocytogenes. However, use of ‘‘uninjured’’ in the text that follows refers to the physiological state of L. monocytogenes prior to inoculation onto TPALAS, to differentiate this uninjured inoculum from that subjected to heat-injury. Neither uninjured nor sublethally heat-injured L. monocytogenes grew under any of the storage conditions used in this study. Overall, populations of uninjured L. monocytogenes were recovered better than populations of sublethally heat-injured cells (P , 0.05). Sublethally heat-injured L. monocytogenes stored at 48C (Fig. 1) and recovered on TPA, decreased in number (from 4.33 log 10 CFU / cm 2 ) by 2 2.99, 3.00, and 2.77 log 10 CFU / cm in air, N 2 , and CO 2 –N 2 , respectively, over 31 days of storage, but was not detected by direct plating from vacuum packaged samples after 23 days. Sublethally heatinjured L. monocytogenes stored at 48C (Fig. 1) was not detectable using MOX after 19, 29, and 19 days in air, N 2 , and vacuum, respectively, and decreased in number (from 3.51 log 10 CFU / cm 2 ) by 2.39 log 10 CFU / cm 2 in CO 2 –N 2 over 31 days of storage. Uninjured L. monocytogenes stored at 48C (Fig. 2) and recovered on TPA decreased (from 5.42 log 10 CFU / cm 2 ) by 2.11, 1.87, 2.08, and 2.88 log 10 CFU / cm 2 , in air, N 2 , CO 2 –N 2 , and vacuum, respectively,
Fig. 1. Survival of sublethally heat-injured L. monocytogenes on TPALAS stored under air, 100% N 2 , 30% CO 2 –70% N 2 , and vacuum at 48C for up to 31 days and recovered on TPA and MOX. E 5 Recovered after enrichment.
over 31 days of storage. Uninjured L. monocytogenes stored at 48C (Fig. 2) and recovered on MOX decreased (from 5.43 log 10 CFU / cm 2 ) by 3.44, 2.69, 3.44, and 4.42 log 10 CFU / cm 2 , in air, N 2 , CO 2 –N 2 , and vacuum, respectively, over 31 days of storage. Sublethally heat-injured L. monocytogenes stored at 208C (Fig. 3) and recovered on TPA decreased from 4.33 log 10 CFU / cm 2 to the limit of detection (0.95 log 10 CFU / cm 2 ) after 17, 5, 5, and 19 days, in air, N 2 , CO 2 –N 2 , and vacuum, respectively. Sublethally heat-injured L. monocytogenes stored at 208C decreased from 3.51 log 10 CFU / cm 2 to undetectable levels in 17, 5, 5, and 19 days, in air, N 2 , CO 2 –N 2 , and vacuum, respectively, as indicated by recovery on MOX (Fig. 3). Uninjured L. monocytogenes stored at 208C (Fig. 4) and recovered on TPA decreased from 5.42 log 10 CFU / cm 2 to levels not
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Fig. 2. Survival of uninjured L. monocytogenes on TPALAS stored under air, 100% N 2 , 30% CO 2 –70% N 2 , and vacuum at 48C for up to 31 days and recovered on TPA and MOX. E 5 Recovered after enrichment.
Fig. 3. Survival of sublethally heat-injured L. monocytogenes on TPALAS stored under air, 100% N 2 , 30% CO 2 –70% N 2 , and vacuum at 208C for up to 31 days and recovered on TPA and MOX. E 5 Recovered after enrichment.
detectable by direct plating after 25, 13, and 15 days in air, N 2 , and CO 2 –N 2 , respectively, and decreased from 3.91 log 10 CFU / cm 2 under vacuum over 31 days. Using MOX, counts for uninjured L. monocytogenes stored at 208C (Fig. 4) decreased from 5.43 log 10 CFU / cm 2 to non-detectable levels after 27, 13, 15, and 31 days in air, N 2 , CO 2 –N 2 , and vacuum, respectively. Regardless of storage temperature and package atmosphere, L. monocytogenes was detected after enrichment from all samples through 31 days of storage, with the exceptions that sublethally heat-injured L. monocytogenes stored at 208C under CO 2 –N 2 and N 2 were not recovered by enrichment after 23 and 21 days, respectively. Overall, storage at 48C supported greater survival than storage at 208C (P , 0.05). Uninjured L. monocytogenes stored at 48C was recovered on TPA better
than sublethally heat-injured L. monocytogenes stored at 48C (P , 0.05). Recovery of sublethally heat-injured L. monocytogenes stored at 48C followed the order N 2 . CO 2 –N 2 . air . vacuum (P , 0.05), whereas recovery of uninjured L. monocytogenes stored at 48C followed the order N 2 . CO 2 – N 2 . vacuum . air (P , 0.05). Air and vacuum atmospheres supported greater survival of uninjured and heat-injured L. monocytogenes than N 2 and CO 2 –N 2 atmospheres at 208C (P , 0.05). Recovery of sublethally heat-injured L. monocytogenes stored at 208C followed the order vacuum . air . CO 2 – N 2 5 N 2 (P , 0.05), whereas recovery of uninjured L. monocytogenes stored at 208C followed the order vacuum . air . CO 2 –N 2 . N 2 (P , 0.05). Uninjured L. monocytogenes stored under N 2 at 48C was recovered best, whereas sublethally heat-injured L.
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Fig. 4. Survival of uninjured L. monocytogenes on TPALAS stored under air, 100% N 2 , 30% CO 2 –70% N 2 , and vacuum at 208C for up to 31 days and recovered on TPA and MOX. E 5 Recovered after enrichment.
monocytogenes stored under N 2 at 208C was recovered poorest (P , 0.05).
4. Discussion Survival of L. monocytogenes on TPALAS was influenced by the injury status (i.e., injured and uninjured) of the inoculum, storage atmosphere (air, N 2 , CO 2 –N 2 , and vacuum), storage temperature (4 and 208C), and recovery media (TPA and MOX) (P , 0.05). Research has shown that Escherichia coli O157:H7 in acidic environments survives better at temperatures near refrigeration than at higher temperatures. Zhao et al. (1993) reported that E. coli O157:H7 in apple cider stored at 88C survived for 31 days but survived for only 3 days in apple cider stored at 258C. Likewise, Fisher and Golden (1998)
reported that E. coli O157:H7 survived better in apple cider at 48C than 128C. In mayonnaise-based sauces (pH , 4.5), E. coli O157:H7 survived for 35 days when stored under refrigeration (Weagent et al., 1994). Results of the present study show that L. monocytogenes in an acidic environment (i.e., TPALAS) also survives better at 48C than 208C. Previous studies have shown the inhibitory effect of high levels of CO 2 in package headspace gases coupled with refrigerated storage on survival of L. monocytogenes. Pothuri et al. (1995) found that an atmosphere comprised of 74.8% CO 2 –10.4% O 2 – 14.8% N 2 inhibited L. monocytogenes on crayfish tail meat stored at 48C better than air or vacuum. L. monocytogenes was unable to grow in cottage cheese at 48C when stored under an atmosphere containing 35% CO 2 but grew under the same atmosphere at 78C (Chen and Hotchkiss, 1993). L. monocytogenes was inhibited in beef at 58C when packaged in a 100% CO 2 atmosphere (Gill and Reichel, 1989). Wimpfheimer et al. (1990) reported that L. monocytogenes growth was inhibited on raw chicken by an atmosphere of 75% CO 2 –25% O 2 at 4, 10, and 278C but was not inhibited by an atmosphere of 72.5% CO 2 –22.5% N 2 –5% O 2 . The latter modified atmosphere, while not inhibitory to L. monocytogenes, inhibited growth of the spoilage microflora. In the present study, storage in a CO 2 –N 2 atmosphere was more inhibitory to survival of uninjured L. monocytogenes stored at 208C than air or vacuum, but less inhibitory than N 2 (P , 0.05). However, when sublethally heat-injured L. monocytogenes was stored at 208C, air and vacuum atmospheres supported better recovery than CO 2 –N 2 and N 2 atmospheres (P , 0.05). At 48C, uninjured and sublethally heat-injured L. monocytogenes were recovered in highest numbers from samples packaged in N 2 followed by CO 2 –N 2 (P . 0.05). Vacuum-packaged fermented food products, such as salami or pepperoni, that are intended to be held at room temperature (20–258C), may support greater survival of L. monocytogenes than if packaged in a high N 2 or CO 2 atmospheres. However, vacuum packaging may be an appropriate choice for similar products intended to be stored at refrigeration temperatures ( | 48C). Furthermore, prevention of postprocessing contamination with L. monocytogenes that has not been exposed to processing steps common to production of fermented foods (i.e.,
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heating) is of particular concern due to the enhanced ability of uninjured populations to survive during modified atmosphere storage. While these data demonstrate the influence of injury status, temperature of storage, and gas atmosphere on the recovery of L. monocytogenes, media used for recovery are also critical. Recovery media intended for the selection of L. monocytogenes also influence recovery of L. monocytogenes. Overall, non-selective TPA allowed greater recovery of L. monocytogenes than the selective MOX (P , 0.05). This is likely due to increased sensitivity of L. monocytogenes to selective agents found in MOX as a result of previous sublethal heat-injury or possible acid injury induced during storage on TPALAS. The temperature at which samples were stored and the status of the inocula prior to inoculation of TPALAS affected recovery on TPA and MOX. After storage at 48C, uninjured cells were recovered better than sublethally heat-injured cells on TPA (P , 0.05). It should be noted that sublethally heat-injured and uninjured inocula were likely acid-injured as a result of the acidic TPALAS environment. The atmosphere under which samples were stored also affected recovery of L. monocytogenes. In support of numerous reports on sublethal injury of L. monocytogenes, results of this study demonstrate the variability in recovery of L. monocytogenes on selective vs. non selective media. Survival of L. monocytogenes on media formulated to simulate fermented food products is influenced by injury status, storage atmosphere, and storage temperature. It is concluded that factors involved in the production, storage, and distribution of fermented foods must be thoroughly evaluated when determining strategies for control and detection of L. monocytogenes in such products.
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Short communication
Influence of modified atmospheric storage, lactic acid, and NaCl on survival of sublethally heat-injured Listeria monocytogenes Robert C. Williams, David A. Golden* The University of Tennessee, Agricultural Experiment Station, Department of Food Science and Technology, 2605 River Drive, Knoxville, TN 37996, USA Received 13 June 2000; received in revised form 25 September 2000; accepted 1 November 2000
Abstract The effect of package atmosphere on survival of uninjured and sublethally heat-injured Listeria monocytogenes, inoculated onto tryptose phosphate agar containing 0.85% lactic acid and 2% NaCl (TPALAS) was investigated. Inoculated TPALAS plates were packaged in air, 100% N 2 (N 2 ), 30% CO 2 –70% N 2 (CO 2 –N 2 ), and vacuum and stored at 4 and 208C for up to 31 days. Recovery of L. monocytogenes from TPALAS was influenced by the injury status (i.e., injured and uninjured) of the inoculum, storage atmosphere (air, N 2 , CO 2 –N 2 , and vacuum), storage temperature (4 and 208C), and recovery media [tryptose phosphate agar (TPA) and modified Oxford agar (MOX)] (P , 0.05). Overall, storage at 48C supported greater survival than storage at 208C (P , 0.05). Uninjured L. monocytogenes stored at 48C was recovered on TPA better than sublethally heat-injured L. monocytogenes stored at 48C (P , 0.05). Recovery of sublethally heat-injured L. monocytogenes stored at 48C followed the order N 2 . CO 2 –N 2 . air . vacuum (P , 0.05), whereas recovery of uninjured L. monocytogenes stored at 48C followed the order N 2 . CO 2 –N 2 . vacuum . air (P , 0.05). Air and vacuum atmospheres supported greater survival of uninjured and heat-injured L. monocytogenes than N 2 and CO 2 –N 2 atmospheres at 208C (P , 0.05). Recovery of sublethally heat-injured L. monocytogenes stored at 208C followed the order vacuum . air . CO 2 –N 2 5 N 2 (P , 0.05), whereas recovery of uninjured L. monocytogenes stored at 208C followed the order vacuum . air . CO 2 –N 2 . N 2 (P , 0.05). Uninjured L. monocytogenes stored under N 2 at 48C was recovered best, whereas sublethally heat-injured L. monocytogenes stored under N 2 at 208C was recovered poorest (P , 0.05). Factors such as package atmosphere and storage temperature, involved in the production, storage, and distribution of fermented foods must be thoroughly evaluated when determining strategies for control and detection of L. monocytogenes in such products. 2001 Elsevier Science B.V. All rights reserved. Keywords: Listeria monocytogenes; Heat injury; Storage atmosphere; Lactic acid; NaCl
1. Introduction *Corresponding author. Tel.: 1 1-865-974-7247; fax: 1 1-865974-2750. E-mail address: [email protected] (D.A. Golden).
Listeria monocytogenes has been recovered from many foods, including raw beef, raw pork, milk, and cabbage (Jay, 2000). These foods are commonly
0168-1605 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0168-1605( 00 )00479-7
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fermented for the purpose of preservation or to impart desired organoleptic characteristics. The aim of acid-type fermentations is to increase the level of acid (e.g., lactic acid) in the fermentation mixture, while decreasing pH to yield a product with desirable characteristics and increased inhibition of spoilage microorganisms (Booth and Kroll, 1989). Production of fermented foods includes acidification of raw or pasteurized products via starter cultures or naturally occurring microflora and may include further processing treatments such as heat (Jay, 2000). Food processing treatments, including heating and acidification, have been shown to induce sublethal injury in L. monocytogenes (Golden et al., 1988; Smith and Archer, 1988; Ahmad and Marth, 1990). Sublethally injured organisms may exhibit increased sensitivity to various components of selective / differential media (Ray, 1989). As a result, sublethally injured L. monocytogenes may grow poorly or not at all on the selective media used for its detection. The major concern with sublethally injured L. monocytogenes is that they may repair, grow, and present a health threat if placed in an appropriate environment (Ray, 1993). For example, heat-stressed L. monocytogenes has been shown to be less pathogenic than nonstressed L. monocytogenes, but regains pathogenicity equal to that of nonstressed cells upon resuscitation in an appropriate environment (McCarthy, 1991). Consequently, under routine testing, a food product may appear to be free of L. monocytogenes, but upon storage, may become unsafe for consumption. Modified atmosphere packaging (MAP) can enhance or reduce survival of L. monocytogenes in a variety of food products (Gill and Reichel, 1989; Chen and Hotchkiss, 1993; Pothuri et al., 1995; Francis and O’Beirne, 1997). L. monocytogenes was inhibited by packaging atmospheres comprised of high carbon dioxide levels (35 to 100%) in packaged cottage cheese (Chen and Hotchkiss, 1993), beef (Gill and Reichel, 1989), and crayfish tail meat (Pothuri et al., 1995) when stored at # 58C. Kallander et al. (1991) reported that an atmosphere containing 75% CO 2 did not effect survival of L. monocytogenes in packaged cabbage stored at 58C. L. monocytogenes survival was enhanced by an atmosphere comprised of 96% N 2 –4% O 2 in packaged lettuce stored at 38C (Francis and O’Beirne, 1997) but not influenced by 97% N 2 –3% O 2 atmos-
phere in packaged lettuce stored at 5 or 108C (Beuchat and Brackett, 1990a,b). L. monocytogenes may survive production and storage of fermented foods, including meat (Johnson et al., 1988; Glass and Doyle, 1989; Buncic et al., 1991; Farber et al., 1993), milk (Ashenafi, 1994; Dalu and Feresu, 1996), kimchi (Lee et al., 1995), and tempeh (Ashenafi, 1991). In general, the microorganism grows during the fermentation, but declines during storage, with low levels persisting. The exception was fermented sausages, where no growth occurred but low levels of the organism persisted even after lengthy storage. L. monocytogenes may undergo injury during production of fermented foods due to exposure to stresses such as acidification and heating. Sublethal injury of L. monocytogenes may complicate detection of this microorganism in fermented foods and may result in underestimation or missed detection of L. monocytogenes. The influence of packaging atmosphere and storage temperature on resuscitation of sublethally injured L. monocytogenes from fermented foods is not known. Therefore, the objective of this study was to determine the fate of sublethally heat-injured L. monocytogenes at 4 and 208C on media formulated to simulate fermented food products and exposed to four different modified atmospheres.
2. Materials and methods
2.1. Culture maintenance L. monocytogenes LM103M (meat isolate) was used in this study because of its demonstrated tolerance to heat and lactic acid (Williams and Golden, 1998). The culture was maintained in tryptose phosphate broth (TPB; Difco, Detroit, MI, USA) through transfer at 24-h intervals and incubation at 378C.
2.2. Heat injury L. monocytogenes was heated to produce injury in . 90% of cells using protocols described by Golden et al. (1988). A 24-h, 378C culture of L. monocytogenes, grown in TPB, was thoroughly mixed and dispensed in 3-ml portions into 10 13 3 100 ml test
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tubes. These tubes were placed in a test tube rack with sufficient space between tubes to allow for water circulation and placed into a 548C shaker waterbath (Dubnoff Metabolic Shaking Incubator; Precision Scientific, Chicago, IL, USA) operated at 90 rpm. An electronic thermometer was placed into one 13 3 100 ml test tube to determine the time at which the temperature of the contents reached equilibrium with the temperature of the waterbath. Upon reaching 548C ( | 2 min equilibration time) cultures were heated at 548C for 20 min. After 20 min, the test tubes were immediately submerged in an ice-water bath to cool the contents to about 258C. After cooling, contents of the test tubes were recombined and centrifuged twice at 11 000 g for 10 min. Cell pellets were washed with and resuspended in 0.1% peptone water (PW). The purpose of dividing the test culture among 10 13 3 100 ml test tubes was to facilitate heat transfer during heating and cooling.
2.3. Preparation of test media A nutrient medium was formulated to simulate the pH, acidity (as lactic acid), and NaCl content of a fermented food product, such as salami. This medium was used to eliminate the influence of background microflora associated with fermented products. Buffered tryptose phosphate agar containing 0.85% lactic acid and 2.0% NaCl (TPALAS) was prepared by combining TPB (29.5 g), agar (15 g), NaCl (15 g), K 2 HPO 4 ? 3H 2 O (15.02 g), KH 2 PO 4 (4.76 g), and deionized water (1000 ml). The medium was thoroughly mixed and brought to boiling on a hot plate / stirrer, sterilized in an autoclave at 1218C for 15 min, and cooled to about 508C. Upon cooling, the medium was acidified with 85% lactic acid syrup ( DL-lactic acid; Acros, NJ, USA) and poured into 60 3 15 mm Petri dishes. The final medium (TPALAS, pH 4.860.1) contained 0.85% lactic acid and 2% NaCl (TPB inherently contains 0.5% NaCl).
2.4. Fate of sublethally-injured L. monocytogenes on TPALAS An uninjured L. monocytogenes control was prepared as described for the heat-injured culture, with the omission of the heating step. The uninjured,
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washed cell suspension was diluted 10-fold in 0.1% PW prior to inoculation of TPALAS. Uninjured and injured cell suspensions were dispensed separately (0.1 ml) onto TPALAS. Prior to packaging, three holes (approx. 0.4 cm in diameter) were burned into the lid of each TPALAS plate with a Creative Woodburner, Jr. (Walnut Hollow, Dodgeville, WI, USA) to facilitate gas exchange. Inoculated TPALAS plates were packaged in atmospheres of air, 100% nitrogen (N 2 ), 30% carbon dioxide–70% nitrogen (CO 2 –N 2 ), and vacuum in Cryovac B-Series high barrier bags (Sealed Air-Cryovac; Duncan, SC, USA) using a Multivac A300 Packaging Machine (Multivac, Wolfertschweden, Germany). Inoculated and packaged plates were incubated at 4 and 208C. Samples, representing a single plate in a package, were taken initially (day 0), after day 1, and then every other day through 31 days of storage for microbiological and gas analyses. At each sampling interval, headspace gases were withdrawn from each package through adhesive septum tape (Modern Controls, Minneapolis, MN, USA), using a 5 ml syringe and 20 gauge needle. Gas samples were analyzed for O 2 , CO 2 , and N 2 using a HewlettPackard 5890 Series II gas chromatograph fitted with an Alltech CTR-1 column (Alltech, Deerfield, IL, USA) and a thermal conductivity detector. Helium was used as the carrier gas at a flow rate of 64 ml / min. The column and detector were held at ambient temperature.
2.5. Microbiological analysis For microbiological analysis, the entire block of agar from each sample plate was aseptically transferred to a stomacher bag, diluted in 50 ml PW, and pummeled for 2 min using a Stomacher 400 Lab Blender (Seward Medical, London, UK). Pummeled samples were serially diluted (1:10) in PW and surface plated (0.1 ml) onto tryptose phosphate agar (TPA; Difco, Detroit, MI, USA) and modified Oxford media (MOX; Oxoid, Ogdensberg, NY, USA). Inoculated media were incubated at 308C for 72 h before colony counts were obtained. Colony counts were reported as log 10 CFU / cm 2 of TPALAS surface. The limit of detection was 0.95 log 10 CFU / cm 2 using the plating scheme described. When L. monocytogenes was no longer recovered by direct plating, test samples were subjected to enrichment proce-
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dures. Test samples were pummeled, as described above, in 50 ml of Listeria enrichment broth base without selective supplements (UVM formulation; Oxoid,) and incubated at 308C for 48 h. Enriched samples were pummeled for 1 min and inoculated onto MOX using a 10-ml loop. Inoculated MOX plates were incubated at 308C for 72 h.
2.6. Statistical analysis This experiment was performed in triplicate. The design was a randomized block, split-plot, with a whole plot factorial treatment arrangement, blocked on replication. Data were analyzed using the mixed procedure (Proc Mixed) of SAS (SAS Institute, versions 6.12 and 7.00 beta; Cary, NC, USA).
3. Results It is possible that the storage conditions themselves used for this study could result in sublethal injury of L. monocytogenes. However, use of ‘‘uninjured’’ in the text that follows refers to the physiological state of L. monocytogenes prior to inoculation onto TPALAS, to differentiate this uninjured inoculum from that subjected to heat-injury. Neither uninjured nor sublethally heat-injured L. monocytogenes grew under any of the storage conditions used in this study. Overall, populations of uninjured L. monocytogenes were recovered better than populations of sublethally heat-injured cells (P , 0.05). Sublethally heat-injured L. monocytogenes stored at 48C (Fig. 1) and recovered on TPA, decreased in number (from 4.33 log 10 CFU / cm 2 ) by 2 2.99, 3.00, and 2.77 log 10 CFU / cm in air, N 2 , and CO 2 –N 2 , respectively, over 31 days of storage, but was not detected by direct plating from vacuum packaged samples after 23 days. Sublethally heatinjured L. monocytogenes stored at 48C (Fig. 1) was not detectable using MOX after 19, 29, and 19 days in air, N 2 , and vacuum, respectively, and decreased in number (from 3.51 log 10 CFU / cm 2 ) by 2.39 log 10 CFU / cm 2 in CO 2 –N 2 over 31 days of storage. Uninjured L. monocytogenes stored at 48C (Fig. 2) and recovered on TPA decreased (from 5.42 log 10 CFU / cm 2 ) by 2.11, 1.87, 2.08, and 2.88 log 10 CFU / cm 2 , in air, N 2 , CO 2 –N 2 , and vacuum, respectively,
Fig. 1. Survival of sublethally heat-injured L. monocytogenes on TPALAS stored under air, 100% N 2 , 30% CO 2 –70% N 2 , and vacuum at 48C for up to 31 days and recovered on TPA and MOX. E 5 Recovered after enrichment.
over 31 days of storage. Uninjured L. monocytogenes stored at 48C (Fig. 2) and recovered on MOX decreased (from 5.43 log 10 CFU / cm 2 ) by 3.44, 2.69, 3.44, and 4.42 log 10 CFU / cm 2 , in air, N 2 , CO 2 –N 2 , and vacuum, respectively, over 31 days of storage. Sublethally heat-injured L. monocytogenes stored at 208C (Fig. 3) and recovered on TPA decreased from 4.33 log 10 CFU / cm 2 to the limit of detection (0.95 log 10 CFU / cm 2 ) after 17, 5, 5, and 19 days, in air, N 2 , CO 2 –N 2 , and vacuum, respectively. Sublethally heat-injured L. monocytogenes stored at 208C decreased from 3.51 log 10 CFU / cm 2 to undetectable levels in 17, 5, 5, and 19 days, in air, N 2 , CO 2 –N 2 , and vacuum, respectively, as indicated by recovery on MOX (Fig. 3). Uninjured L. monocytogenes stored at 208C (Fig. 4) and recovered on TPA decreased from 5.42 log 10 CFU / cm 2 to levels not
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Fig. 2. Survival of uninjured L. monocytogenes on TPALAS stored under air, 100% N 2 , 30% CO 2 –70% N 2 , and vacuum at 48C for up to 31 days and recovered on TPA and MOX. E 5 Recovered after enrichment.
Fig. 3. Survival of sublethally heat-injured L. monocytogenes on TPALAS stored under air, 100% N 2 , 30% CO 2 –70% N 2 , and vacuum at 208C for up to 31 days and recovered on TPA and MOX. E 5 Recovered after enrichment.
detectable by direct plating after 25, 13, and 15 days in air, N 2 , and CO 2 –N 2 , respectively, and decreased from 3.91 log 10 CFU / cm 2 under vacuum over 31 days. Using MOX, counts for uninjured L. monocytogenes stored at 208C (Fig. 4) decreased from 5.43 log 10 CFU / cm 2 to non-detectable levels after 27, 13, 15, and 31 days in air, N 2 , CO 2 –N 2 , and vacuum, respectively. Regardless of storage temperature and package atmosphere, L. monocytogenes was detected after enrichment from all samples through 31 days of storage, with the exceptions that sublethally heat-injured L. monocytogenes stored at 208C under CO 2 –N 2 and N 2 were not recovered by enrichment after 23 and 21 days, respectively. Overall, storage at 48C supported greater survival than storage at 208C (P , 0.05). Uninjured L. monocytogenes stored at 48C was recovered on TPA better
than sublethally heat-injured L. monocytogenes stored at 48C (P , 0.05). Recovery of sublethally heat-injured L. monocytogenes stored at 48C followed the order N 2 . CO 2 –N 2 . air . vacuum (P , 0.05), whereas recovery of uninjured L. monocytogenes stored at 48C followed the order N 2 . CO 2 – N 2 . vacuum . air (P , 0.05). Air and vacuum atmospheres supported greater survival of uninjured and heat-injured L. monocytogenes than N 2 and CO 2 –N 2 atmospheres at 208C (P , 0.05). Recovery of sublethally heat-injured L. monocytogenes stored at 208C followed the order vacuum . air . CO 2 – N 2 5 N 2 (P , 0.05), whereas recovery of uninjured L. monocytogenes stored at 208C followed the order vacuum . air . CO 2 –N 2 . N 2 (P , 0.05). Uninjured L. monocytogenes stored under N 2 at 48C was recovered best, whereas sublethally heat-injured L.
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Fig. 4. Survival of uninjured L. monocytogenes on TPALAS stored under air, 100% N 2 , 30% CO 2 –70% N 2 , and vacuum at 208C for up to 31 days and recovered on TPA and MOX. E 5 Recovered after enrichment.
monocytogenes stored under N 2 at 208C was recovered poorest (P , 0.05).
4. Discussion Survival of L. monocytogenes on TPALAS was influenced by the injury status (i.e., injured and uninjured) of the inoculum, storage atmosphere (air, N 2 , CO 2 –N 2 , and vacuum), storage temperature (4 and 208C), and recovery media (TPA and MOX) (P , 0.05). Research has shown that Escherichia coli O157:H7 in acidic environments survives better at temperatures near refrigeration than at higher temperatures. Zhao et al. (1993) reported that E. coli O157:H7 in apple cider stored at 88C survived for 31 days but survived for only 3 days in apple cider stored at 258C. Likewise, Fisher and Golden (1998)
reported that E. coli O157:H7 survived better in apple cider at 48C than 128C. In mayonnaise-based sauces (pH , 4.5), E. coli O157:H7 survived for 35 days when stored under refrigeration (Weagent et al., 1994). Results of the present study show that L. monocytogenes in an acidic environment (i.e., TPALAS) also survives better at 48C than 208C. Previous studies have shown the inhibitory effect of high levels of CO 2 in package headspace gases coupled with refrigerated storage on survival of L. monocytogenes. Pothuri et al. (1995) found that an atmosphere comprised of 74.8% CO 2 –10.4% O 2 – 14.8% N 2 inhibited L. monocytogenes on crayfish tail meat stored at 48C better than air or vacuum. L. monocytogenes was unable to grow in cottage cheese at 48C when stored under an atmosphere containing 35% CO 2 but grew under the same atmosphere at 78C (Chen and Hotchkiss, 1993). L. monocytogenes was inhibited in beef at 58C when packaged in a 100% CO 2 atmosphere (Gill and Reichel, 1989). Wimpfheimer et al. (1990) reported that L. monocytogenes growth was inhibited on raw chicken by an atmosphere of 75% CO 2 –25% O 2 at 4, 10, and 278C but was not inhibited by an atmosphere of 72.5% CO 2 –22.5% N 2 –5% O 2 . The latter modified atmosphere, while not inhibitory to L. monocytogenes, inhibited growth of the spoilage microflora. In the present study, storage in a CO 2 –N 2 atmosphere was more inhibitory to survival of uninjured L. monocytogenes stored at 208C than air or vacuum, but less inhibitory than N 2 (P , 0.05). However, when sublethally heat-injured L. monocytogenes was stored at 208C, air and vacuum atmospheres supported better recovery than CO 2 –N 2 and N 2 atmospheres (P , 0.05). At 48C, uninjured and sublethally heat-injured L. monocytogenes were recovered in highest numbers from samples packaged in N 2 followed by CO 2 –N 2 (P . 0.05). Vacuum-packaged fermented food products, such as salami or pepperoni, that are intended to be held at room temperature (20–258C), may support greater survival of L. monocytogenes than if packaged in a high N 2 or CO 2 atmospheres. However, vacuum packaging may be an appropriate choice for similar products intended to be stored at refrigeration temperatures ( | 48C). Furthermore, prevention of postprocessing contamination with L. monocytogenes that has not been exposed to processing steps common to production of fermented foods (i.e.,
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heating) is of particular concern due to the enhanced ability of uninjured populations to survive during modified atmosphere storage. While these data demonstrate the influence of injury status, temperature of storage, and gas atmosphere on the recovery of L. monocytogenes, media used for recovery are also critical. Recovery media intended for the selection of L. monocytogenes also influence recovery of L. monocytogenes. Overall, non-selective TPA allowed greater recovery of L. monocytogenes than the selective MOX (P , 0.05). This is likely due to increased sensitivity of L. monocytogenes to selective agents found in MOX as a result of previous sublethal heat-injury or possible acid injury induced during storage on TPALAS. The temperature at which samples were stored and the status of the inocula prior to inoculation of TPALAS affected recovery on TPA and MOX. After storage at 48C, uninjured cells were recovered better than sublethally heat-injured cells on TPA (P , 0.05). It should be noted that sublethally heat-injured and uninjured inocula were likely acid-injured as a result of the acidic TPALAS environment. The atmosphere under which samples were stored also affected recovery of L. monocytogenes. In support of numerous reports on sublethal injury of L. monocytogenes, results of this study demonstrate the variability in recovery of L. monocytogenes on selective vs. non selective media. Survival of L. monocytogenes on media formulated to simulate fermented food products is influenced by injury status, storage atmosphere, and storage temperature. It is concluded that factors involved in the production, storage, and distribution of fermented foods must be thoroughly evaluated when determining strategies for control and detection of L. monocytogenes in such products.
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